fumarates and fumaric-acid

Bioorthogonal reactions are rapid, specific and high yield reactions that can be performed in in vivo microenvironments or simulated microenvironments. At present, the main biorthogonal reactions include Staudinger ligation, copper-catalyzed azide alkyne cycloaddition, strain-promoted [3 + 2] reaction, tetrazine ligation, metal-catalyzed coupling reaction and photo-induced biorthogonal reactions. To date, many reviews have reported that bioorthogonal reactions have been used widely as a powerful tool in the field of life sciences, such as in target recognition, drug discovery, drug activation, omics research, visualization of life processes or exogenous bacterial infection processes, signal transduction pathway research, chemical reaction dynamics analysis, disease diagnosis and treatment. In contrast, to date, few studies have investigated the application of bioorthogonal reactions in the analysis of biomacromolecules in vivo. Therefore, the application of bioorthogonal reactions in the analysis of proteins, nucleic acids, metabolites, enzyme activities and other endogenous molecules, and the determination of disease-related targets is reviewed. In addition, this review discusses the future development opportunities and challenges of biorthogonal reactions. This review presents an overview of recent advances for application in biomolecular analysis and disease diagnosis, with a focus on proteins, metabolites and RNA detection.

Topics: Biomarkers; Cycloaddition Reaction; Feces; Fluorescent Dyes; Fumarates; Humans; Neoplasms; Proteins; RNA

Fumaric acid is a valuable compound used in foods, beverages, detergents, animal feed, pharmaceuticals and miscellaneous industrial products. It is produced on a large scale by the petrochemical route but the current tendency is towards implementing "green production" and environmental friendly technologies like biotechnological production of fumaric acid using low-cost raw materials. In this context, numerous studies focus on improving the fermentation process not only by using renewable raw material and genetically modified microorganisms, but also by developing and applying different downstream techniques for easy recovery of fumaric acid from the fermented broth. This review presents the main methods for production and separation of fumaric acid, highlighting the advantages and disadvantages of these and the potential economic impact in industry.

Topics: Biotechnology; Fumarates; Microorganisms, Genetically-Modified

Fumaric acid (FA), a metabolic intermediate, has been identified as an important carbohydrate derived platform chemical. Currently, it is commercially sourced from petrochemicals by chemical conversion. The shift to biochemical synthesis has become essential for sustainable development and for the transition to a biobased economy from a petroleum-based economy. The main limitation is that the concentrations of FA achieved during bioproduction are lower than that from a chemical process. Moreover, the high cost associated with bioproduction necessitates a higher yield to improve the feasibility of the process. To this effect, genetic modification of microorganism can be considered as an important tool to improve FA yield. This review discusses various genetic modifications strategies that have been studied in order to improve FA production. These strategies include the development of recombinant strains of

Topics: CRISPR-Cas Systems; Escherichia coli; Fumarates; Metabolic Engineering; Rhizopus; Saccharomyces cerevisiae

Diabetic kidney disease (DKD) is the leading cause of morbidity and mortality in diabetic patients. Defining risk factors for DKD using a reductionist approach has proven challenging. Integrative omics-based systems biology tools have shed new insights in our understanding of DKD and have provided several key breakthroughs for identifying novel predictive and diagnostic biomarkers. In this review, we highlight the role of the Warburg effect in DKD and potential regulating factors such as sphingomyelin, fumarate, and pyruvate kinase muscle isozyme M2 in shifting glucose flux from complete oxidation in mitochondria to the glycolytic pathway and its principal branches. With the development of highly sensitive instruments and more advanced automatic bioinformatics tools, we believe that omics analyses and imaging techniques will focus more on singular-cell-level studies, which will allow in-depth understanding of DKD and pave the path for personalized kidney precision medicine.

Topics: Animals; Carrier Proteins; Diabetic Nephropathies; Fumarates; Glycolysis; Humans; Membrane Proteins; Metabolomics; NADPH Oxidase 4; Reactive Oxygen Species; Sphingomyelins; Systems Biology; Thyroid Hormone-Binding Proteins; Thyroid Hormones

Several lines of evidence have demonstrated the potential biomedical applications of fumaric acid (FA) and its ester derivatives against many human disease conditions. Fumaric acid esters (FAEs) have been licensed for the systemic treatment of the immune-mediated disease psoriasis. Biogen Idec Inc. announced about the safety and efficacy of the formulation FAE (BG-12) for treating RRMS (relapsing-remitting multiple sclerosis). Another FAE formulation DMF (dimethyl fumarate) was found to be capable of reduction in inflammatory cardiac conditions, such as autoimmune myocarditis and ischemia and reperfusion. DMF has also been reported to be effective as a potential neuroprotectant against the HIV-associated neurocognitive disorders (HAND). Many in vivo studies carried out on rat and mice models indicated inhibitory effects of fumaric acid on carcinogenesis of different origins. Moreover, FAEs has emerged as an important matrix ingredient in the fabrication of biodegradable scaffolds for tissue engineering applications. Drug delivery vehicles composed of FAEs have shown promising results in delivering some leading drug molecules. Apart from these specific applications and findings, many more studies on FAEs have revealed new therapeutic potentials with the scope of clinical applications. However, until now, this scattered vital information has not been written into a collective account and analyzed for minute details. The aim of this paper is to review the advancement made in the biomedical application of FA and FAEs and to focus on the clinical investigation and molecular interpretation of the beneficial effects of FA and FAEs.

Topics: Animals; Dimethyl Fumarate; Esters; Fumarates; Humans; Multiple Sclerosis, Relapsing-Remitting; Neuroprotective Agents; Psoriasis

The review describes efforts toward metabolic engineering of production of organic acids. One aspect of the strategy involves the generation of an appropriate amount and type of reduced cofactor needed for the designed pathway. The ability to capture reducing power in the proper form, NADH or NADPH for the biosynthetic reactions leading to the organic acid, requires specific attention in designing the host and also depends on the feedstock used and cell energetic requirements for efficient metabolism during production. Recent work on the formation and commercial uses of a number of small mono- and diacids is discussed with redox differences, major biosynthetic precursors and engineering strategies outlined. Specific attention is given to those acids that are used in balancing cell redox or providing reduction equivalents for the cell, such as formate, which can be used in conjunction with metabolic engineering of other products to improve yields. Since a number of widely studied acids derived from oxaloacetate as an important precursor, several of these acids are covered with the general strategies and particular components summarized, including succinate, fumarate and malate. Since malate and fumarate are less reduced than succinate, the availability of reduction equivalents and level of aerobiosis are important parameters in optimizing production of these compounds in various hosts. Several other more oxidized acids are also discussed as in some cases, they may be desired products or their formation is minimized to afford higher yields of more reduced products. The placement and connections among acids in the typical central metabolic network are presented along with the use of a number of specific non-native enzymes to enhance routes to high production, where available alternative pathways and strategies are discussed. While many organic acids are derived from a few precursors within central metabolism, each organic acid has its own special requirements for high production and best compatibility with host physiology.

Topics: Carbon; Formates; Fumarates; Malates; Metabolic Engineering; Metabolic Networks and Pathways; Oxidation-Reduction; Propionates; Succinic Acid

Various economic and environmental sustainability concerns as well as consumer preference for bio-based products from natural sources have paved the way for the development and expansion of biorefining technologies. These involve the conversion of renewable biomass feedstock to fuels and chemicals using biological systems as alternatives to petroleum-based products. Filamentous fungi possess an expansive portfolio of products including the multifunctional organic acids itaconic, fumaric, and malic acids that have wide-ranging current applications and potentially addressable markets as platform chemicals. However, current bioprocessing technologies for the production of these compounds are mostly based on submerged fermentation, which necessitates physicochemical pretreatment and hydrolysis of lignocellulose biomass to soluble fermentable sugars in liquid media. This review will focus on current research work on fungal production of itaconic, fumaric, and malic acids and perspectives on the potential application of solid-state fungal cultivation techniques for the consolidated hydrolysis and organic acid fermentation of lignocellulosic biomass.

Topics: Biomass; Carbohydrate Metabolism; Fermentation; Fumarates; Fungi; Hydrolysis; Lignin; Malates; Succinates

Topics: Administration, Oral; Adult; Dose-Response Relationship, Drug; Drug Administration Schedule; Female; Follow-Up Studies; Fumarates; Humans; Multiple Sclerosis; Psoriasis; Severity of Illness Index; Treatment Outcome

After the approval of fumaric acid in February 2014 another first line agent is now available for the treatment of multiple sclerosis (MS). Along with the various beta interferon preparations, glatiramer acetate, teriflunomide and fumaric acid add to the repertoire of oral therapeutics for the initial treatment of relapsing remitting MS in daily practice. In order to employ these drugs in an individualized and precise medical manner and considering their efficacy and side effects, it seems worthwhile to learn the so far known mode of action and background history. Fumaric acid, as one of the newest drugs approved for MS, reveals the longest history as it was in use for decades as a treatment in psoriasis patients. Furthermore, fumaric acid is a good example for so far not extensively exploited option of drug reposition in medicine in general. The current review summarizes the outcomes of the clinical approval studies of fumaric acid in MS and discusses the dual mode of action, the immunomodulatory and tissue protective effect, as well as the reported adverse events under fumaric acid treatment. This review aims to serve an aid in the daily decision-making practice when choosing the baseline therapy for MS patients.

Topics: Dose-Response Relationship, Drug; Drug Administration Schedule; Evidence-Based Medicine; Flushing; Fumarates; Gastrointestinal Diseases; Humans; Immunologic Factors; Kidney Diseases; Multiple Sclerosis, Relapsing-Remitting; Neuroprotective Agents; Treatment Outcome

Sarcoidosis is a rare, systemic disease that is characterized by the formation of granulomas in various organs, including the skin. As the etiology remains unknown, the treatment of sarcoidosis is challenging. We present a 47-year-old female patient with progressive, multi-organ sarcoidosis who had a complete clinical improvement of the skin lesions, a moderate reduction in pulmonary opacities on chest X-ray, a marked subjective improvement in general status and pulmonary efficiency and a marked reduction in serum angiotensin-converting enzyme and soluble interleukin-2 receptor after 6 months of therapy with fumaric acid esters. The present case and similar reports in the literature highlight the probable efficacy of fumaric acid esters in the treatment of sarcoidosis and other non-infectious, granulomatous diseases.

Topics: Administration, Oral; Dose-Response Relationship, Drug; Drug Administration Schedule; Female; Follow-Up Studies; Fumarates; Humans; Middle Aged; Radiography, Thoracic; Rare Diseases; Sarcoidosis; Sarcoidosis, Pulmonary; Severity of Illness Index; Skin Diseases; Treatment Outcome

Fumaric acid was originally therapeutically used in psoriasis. Several lines of evidence have demonstrated immunomodulatory but also neuroprotective effects for FAE. Clinical studies in psoriasis showed a reduction of peripheral CD4+ and CD8+ T-lymphocytes due to the ability of FAE to induce apoptosis. In vitro studies with the ester dimethylfumarate (DMF) described an inhibitory effect on nuclear factor kappa B (NF-κB)-dependent transcription of tumor necrosis factor-alpha (TNF-α) induced genes in human endothelial cells. Animal experiments in the mouse model of central nervous system demyelination, MOG-induced experimental autoimmune encephalomyelitis, revealed a clear preservation of myelin and axonal density in the plaque. Molecular studies showed that this is based on the antioxidative mechanism of action via induction of the transcription factor Nrf-2. A phase II clinical trial in relapsing-remitting multiple sclerosis (RRMS) patients with dimethylfumarate showed a significant reduction in the number of gadolinium enhancing lesions after 24weeks.

Topics: Animals; Clinical Trials as Topic; Fumarates; Humans; Immunosuppressive Agents; Multiple Sclerosis, Relapsing-Remitting

Rhizopus oryzae is a filamentous fungus belonging to the Zygomycetes. It is among others known for its ability to produce the sustainable platform chemicals L: -(+)-lactic acid, fumaric acid, and ethanol. During glycolysis, all fermentable carbon sources are metabolized to pyruvate and subsequently distributed over the pathways leading to the formation of these products. These platform chemicals are produced in high yields on a wide range of carbon sources. The yields are in excess of 85 % of the theoretical yield for L: -(+)-lactic acid and ethanol and over 65 % for fumaric acid. The study and optimization of the metabolic pathways involved in the production of these compounds requires well-developed metabolic engineering tools and knowledge of the genetic makeup of this organism. This review focuses on the current metabolic engineering techniques available for R. oryzae and their application on the metabolic pathways of the main fermentation products.

Topics: Biotransformation; Carbon; Ethanol; Fumarates; Lactic Acid; Metabolic Engineering; Metabolic Networks and Pathways; Rhizopus

Fermentative fumaric acid production from renewable resources may become competitive with petrochemical production. This will require very efficient processes. So far, using Rhizopus strains, the best fermentations reported have achieved a fumaric acid titer of 126 g/L with a productivity of 1.38 g L(-1) h(-1) and a yield on glucose of 0.97 g/g. This requires pH control, aeration, and carbonate/CO(2) supply. Limitations of the used strains are their pH tolerance, morphology, accessibility for genetic engineering, and partly, versatility to alternative carbon sources. Understanding of the mechanism and energetics of fumaric acid export by Rhizopus strains will be a success factor for metabolic engineering of other hosts for fumaric acid production. So far, metabolic engineering has been described for Escherichia coli and Saccharomyces cerevisiae.

Topics: Biological Transport; Carbon; Escherichia coli; Fermentation; Fumarates; Metabolic Engineering; Metabolic Networks and Pathways; Rhizopus; Saccharomyces cerevisiae

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system and the most common cause of neurological disability in young adults, along with a considerable clinical and pathological heterogeneity. Since, current therapies appear to be modest in the magnitude of their treatment effects, particularly in the progressive phase of this disease, thus novel promising therapeutic strategies might open a light horizon in approaching to an efficient treatment in MS. In this review, we will discuss about relevant patents and novel designed immunosuppressive and anti-inflammatory oral drugs promising for treatment of multiple sclerosis.

Topics: Administration, Oral; Cladribine; Crotonates; Diterpenes; Epoxy Compounds; Fingolimod Hydrochloride; Fumarates; Humans; Hydroxybutyrates; Multiple Sclerosis; Nitriles; Patents as Topic; Phenanthrenes; Propylene Glycols; Quinolones; Sphingosine; Toluidines

Stomata, functionally specialized small pores on the surfaces of leaves, regulate the flow of gases in and out of plants. The pore is opened by an increase in osmotic pressure in the guard cells, resulting in the uptake of water. The subsequent increase in cell volume inflates the guard cell and culminates with the opening of the pore. Although guard cells can be regarded as one of the most thoroughly investigated cell types, our knowledge of the signaling pathways which regulate guard cell function remains fragmented. Recent research in guard cells has led to several new hypotheses, however, it is still a matter of debate as to whether guard cells function autonomously or are subject to regulation by their neighboring mesophyll cells.This review synthesizes what is known about the mechanisms and genes critical for modulating stomatal movement. Recent progress on the regulation of guard cell function is reviewed here including the involvement of environmental signals such as light, the concentration of atmospheric CO2 and endogenous plant hormones. In addition we re-evaluate the important role of organic acids such as malate and fumarate play in guard cell metabolism in this process.

Topics: Carbon Dioxide; Fumarates; Light; Malates; Plant Stomata; Signal Transduction

Fumaric aciduria is a rare metabolic disease, with 40 cases reported so far. Fumarase deficiency leads mainly to brain abnormalities, developmental delay, and great accumulation of fumaric acid in urine. This work presents the first case of fumaric aciduria described in Brazil, which presented with some interesting clinical and biochemical findings such as colpocephaly, hepatic alterations, and marked metabolic acidosis since birth. Common findings were ventriculomegaly, hypotonia, and microcephaly. Biochemically, besides the high urinary fumaric acid excretion, atypical elevation of plasma citrulline, tyrosine and methionine levels were also observed. In order to show all features and variants of fumaric aciduria, literature data of 40 patients was reviewed and compared with the case reported here. Findings in all these patients demonstrate that this disorder does not yet have its phenotype completely defined; it is important that more patients be described.

Topics: Brazil; Family Health; Female; Fumarate Hydratase; Fumarates; Humans; Infant; Metabolism, Inborn Errors

Disease-modifying treatments are now available in relapsing-remitting and secondary progressive multiple sclerosis (MS), and their beneficial effects have been shown in several clinical studies. However, as these treatments are only partially effective in halting the MS disease process and are frequently associated with side effects and suboptimal patient adherence, new oral therapeutic approaches are warranted. This review focuses on advances in current and novel oral treatment approaches for MS. Several pivotal reports have provided promising results for new oral therapies evaluating the safety and efficacy of new agents including fingolimod, fumaric acid, cladribine, teriflunomide and laquinimod.

Topics: Cladribine; Clinical Trials, Phase III as Topic; Crotonates; Fingolimod Hydrochloride; Fumarates; Humans; Hydroxybutyrates; Immunosuppressive Agents; Multiple Sclerosis; Nitriles; Propylene Glycols; Quinolones; Sphingosine; Toluidines

Topics: Animals; Dihydroorotate Dehydrogenase; Drug Design; Energy Metabolism; Fumarates; Nucleotides; Orotic Acid; Oxidation-Reduction; Oxidoreductases Acting on CH-CH Group Donors; Oxonic Acid; Protein Conformation; Succinic Acid; Trypanocidal Agents; Trypanosoma cruzi

Therapy for multiple sclerosis (MS) has changed dramatically over the past decade, yielding significant progress in the treatment of relapsing/remitting and secondary progressive MS. Disease-modifying treatments are now widely available, and their beneficial effects on relapse rates, MRI outcomes and, in some cases, relapse-related disability have been shown in several clinical studies. However, as these treatments are only partially effective in halting the MS disease process and in clinical practice are frequently associated with injection-related side effects and suboptimal patient adherence, new oral therapeutic approaches are warranted.. The aim of the present paper is to present new promising results from emerging oral drugs for multiple sclerosis.. This review focuses on advances in current and novel oral treatment approaches for MS. Nevertheless, most of the data were obtained from Phase I/II clinical trials, we need further confirmation of their safety and efficacy profile from longer Phase III clinical trials.. Several pivotal reports have provided promising results for new oral therapies evaluating the safety and efficacy of new agents including fingolimod, fumaric acid, cladribine, teriflunomide and laquinomid.. It is unknown whether these oral drugs could be used as first-line treatment for MS; this will depend mostly on their safety profile. Alternatively, these drugs could be used as add-on treatment for failed first-line therapy, or as an effective induction agent.

Topics: Administration, Oral; Cladribine; Crotonates; Fingolimod Hydrochloride; Fumarates; Humans; Hydroxybutyrates; Immunosuppressive Agents; Multiple Sclerosis; Nitriles; Propylene Glycols; Quinolones; Sphingosine; Toluidines

The potential of fumaric acid as a raw material in the polymer industry and the increment of cost of petroleum-based fumaric acid raises interest in fermentation processes for production of this compound from renewable resources. Although the chemical process yields 112% w/w fumaric acid from maleic anhydride and the fermentation process yields only 85% w/w from glucose, the latter raw material is three times cheaper. Besides, the fermentation fixes CO2. Production of fumaric acid by Rhizopus species and the involved metabolic pathways are reviewed. Submerged fermentation systems coupled with product recovery techniques seem to have achieved economically attractive yields and productivities. Future prospects for improvement of fumaric acid production include metabolic engineering approaches to achieve low pH fermentations.

Topics: Bacteria; Fermentation; Fumarates; Industrial Microbiology; Maleates; Oxygen

Poly (propylene fumarate) is an unsaturated linear polyester, which degrades, in the presence of water, into propylene glycol and fumaric acid, and the degradation products can be cleared from the human body by normal metabolic processes. In this paper, different methods of the synthesis of PPF are listed, the crosslinking characteristics of polymerizing composite as well as the mechanical properties of cross-linked composite are discussed, and the potentialities of PPF composite used as particularly degradable polymeric cement for orthopedic application are reviewed.

Topics: Biocompatible Materials; Biodegradation, Environmental; Cross-Linking Reagents; Fumarates; Humans; Polypropylenes

Topics: Adalimumab; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Biological Products; Cyclosporine; Dermatologic Agents; Forecasting; Fumarates; Humans; Infliximab; Nicotinic Acids; Psoriasis; Receptors, Tumor Necrosis Factor; Receptors, Tumor Necrosis Factor, Type I; Tacrolimus; Tumor Necrosis Factor Decoy Receptors; Ustekinumab

Over the last two decades significant advances have been made in our understanding of the anaerobic biodegradability of monoaromatic hydrocarbons. It is now known that compounds such as benzene, toluene, ethylbenzene, and all three xylene isomers can be biodegraded in the absence of oxygen by a broad diversity of organisms. These compounds have been shown to serve as carbon and energy sources for bacteria growing phototrophically, or respiratorily with nitrate, manganese, ferric iron, sulfate, or carbon dioxide as the sole electron acceptor. In addition, it has also been recently shown that complete degradation of monoaromatic hydrocarbons can also be coupled to the respiration of oxyanions of chlorine such as perchlorate or chlorate, or to the reduction of the quinone moieties of humic substances. Many pure cultures of hydrocarbon-degrading anaerobes now exist and some novel biochemical and genetic pathways have been identified. In general, a fumarate addition reaction is used as the initial activation step of the catabolic process of the corresponding monoaromatic hydrocarbon compounds. However, other reactions may alternatively be involved depending on the electron acceptor utilized or the compound being degraded. In the case of toluene, fumarate addition to the methyl group mediated by benzylsuccinate synthase appears to be the universal mechanism of activation and is now known to be utilized by anoxygenic phototrophs, nitrate-reducing, Fe(III)-reducing, sulfate-reducing, and methanogenic cultures. Many of these biochemical pathways produce unique extracellular intermediates that can be utilized as biomarkers for the monitoring of hydrocarbon degradation in anaerobic natural environments.

Topics: Anaerobiosis; Benzene; Benzene Derivatives; Biodegradation, Environmental; Biomarkers; Environmental Microbiology; Environmental Pollutants; Fumarates; Hydrocarbons, Aromatic; Oxidation-Reduction; Toluene; Xylenes

Topics: Dermatologic Agents; Dimethyl Fumarate; Esters; Fumarates; Humans; Psoriasis; T-Lymphocytes

Topics: Bacteria; Benzoquinones; Electron Transport Complex II; Fumarates; Hydroquinones; Intracellular Membranes; Models, Chemical; Models, Molecular; Multienzyme Complexes; Oxidoreductases; Succinate Dehydrogenase

Bacteria contain secondary carriers for the uptake, exchange or efflux of C4-dicarboxylates. In aerobic bacteria, dicarboxylate transport (Dct)A carriers catalyze uptake of C4-dicarboxylates in a H(+)- or Na(+)-C4-dicarboxylate symport. Carriers of the dicarboxylate uptake (Dcu)AB family are used for electroneutral fumarate:succinate antiport which is required in anaerobic fumarate respiration. The DcuC carriers apparently function in succinate efflux during fermentation. The tripartite ATP-independent periplasmic (TRAP) transporter carriers are secondary uptake carriers requiring a periplasmic solute binding protein. For heterologous exchange of C4-dicarboxylates with other carboxylic acids (such as citrate:succinate by CitT) further types of carriers are used. The different families of C4-dicarboxylate carriers, the biochemistry of the transport reactions, and their metabolic functions are described. Many bacteria contain membraneous C4-dicarboxylate sensors which control the synthesis of enzymes for C4-dicarboxylate metabolism. The C4-dicarboxylate sensors DcuS, DctB, and DctS are histidine protein kinases and belong to different families of two-component systems. They contain periplasmic domains presumably involved in C4-dicarboxylate sensing. In DcuS the periplasmic domain seems to be essential for direct interaction with the C4-dicarboxylates. In signal perception by DctB, interaction of the C4-dicarboxylates with DctB and the DctA carrier plays an important role.

Topics: Amino Acid Sequence; Bacteria, Aerobic; Bacterial Proteins; Biological Transport; Dicarboxylic Acid Transporters; Escherichia coli; Escherichia coli Proteins; Fumarates; Models, Chemical; Organic Anion Transporters; Phylogeny; Protein Kinases; Receptors, Cell Surface; Signal Transduction; Succinic Acid

Reversible succinate dehydrogenase (SDH) activities have been ubiquitously detected in organisms from the three domains of life. They represent constituents either of respiratory complexes II in aerobes, or of fumarate dehydrogenase complexes in anaerobes. The present review gives a survey on archaeal succinate:quinone oxidoreductases (SQRs) analyzed so far. Though some of these could be studied in detail enzymologically and spectroscopically, the existence of others has been deduced only from published genome sequences. Interestingly, two groups of enzyme complexes can be distinguished in Archaea. One group resembles the properties of SDHs known from bacteria and mitochondria. The other represents a novel class with an unusual iron-sulfur cluster in subunit B and atypical sequence motifs in subunit C which may influence electron transport mechanisms and pathways. This novel class of SQRs is discussed in comparison to the so-called 'classical' complexes. A phylogenetic analysis is presented suggesting a co-evolution of the flavoprotein-binding subunit A and subunit B containing the three iron-sulfur clusters.

Topics: Amino Acid Sequence; Archaea; Electron Transport Complex II; Evolution, Molecular; Fumarates; Iron-Sulfur Proteins; Molecular Sequence Data; Multienzyme Complexes; Operon; Oxidation-Reduction; Oxidoreductases; Phylogeny; Sequence Alignment; Succinate Dehydrogenase; Succinic Acid

The epsilon-proteobacteria form a subdivision of the Proteobacteria including the genera Wolinella, Campylobacter, Helicobacter, Sulfurospirillum, Arcobacter and Dehalospirillum. The majority of these bacteria are oxidase-positive microaerophiles indicating an electron transport chain with molecular oxygen as terminal electron acceptor. However, numerous members of the epsilon-proteobacteria also grow in the absence of oxygen. The common presence of menaquinone and fumarate reduction activity suggests anaerobic fumarate respiration which was demonstrated for the rumen bacterium Wolinella succinogenes as well as for Sulfurospirillum deleyianum, Campylobacter fetus, Campylobacter rectus and Dehalospirillum multivorans. To date, complete genome sequences of Helicobacter pylori and Campylobacter jejuni are available. These bacteria and W. succinogenes contain the genes frdC, A and B encoding highly similar heterotrimeric enzyme complexes belonging to the family of succinate:quinone oxidoreductases. The crystal structure of the W. succinogenes quinol:fumarate reductase complex (FrdCAB) was solved recently, thus providing a model of succinate:quinone oxidoreductases from epsilon-proteobacteria. Succinate:quinone oxidoreductases are being discussed as possible therapeutic targets in the treatment of several pathogenic epsilon-proteobacteria.

Topics: Citric Acid Cycle; Electron Transport; Electron Transport Complex II; Energy Metabolism; Fumarates; Models, Chemical; Models, Molecular; Multienzyme Complexes; Operon; Oxidation-Reduction; Oxidoreductases; Proteobacteria; Succinate Dehydrogenase; Succinic Acid; Wolinella

The succinate dehydrogenase consists of only four subunits, all nuclearly encoded, and is part of both the respiratory chain and the Krebs cycle. Mutations in the four genes encoding the subunits of the mitochondrial respiratory chain succinate dehydrogenase have been recently reported in human and shown to be associated with a wide spectrum of clinical presentations. Although a comparatively rare deficiency in human, molecularly defined succinate dehydrogenase deficiency has already been found to cause encephalomyopathy in childhood, optic atrophy or tumor in adulthood. Because none of the typical housekeeping genes encoding this respiratory chain complex is known to present tissue-specific isoforms, the tissue-specific involvement represents a quite intriguing question, which is mostly addressed in this review. A differential impairment of electron flow through the respiratory chain, handling of oxygen, and/or metabolic blockade possibly associated with defects in the different subunits that can be advocated to account for tissue-specific involvement is discussed.

Topics: Citric Acid Cycle; Electron Transport Complex II; Flavoproteins; Fumarates; Humans; Metabolism, Inborn Errors; Mitochondrial Diseases; Models, Chemical; Multienzyme Complexes; Mutation; Oxidoreductases; Succinate Dehydrogenase; Succinic Acid

Parasites have developed a variety of physiological functions necessary for existence within the specialized environment of the host. Regarding energy metabolism, which is an essential factor for survival, parasites adapt to low oxygen tension in host mammals using metabolic systems that are very different from that of the host. The majority of parasites do not use the oxygen available within the host, but employ systems other than oxidative phosphorylation for ATP synthesis. In addition, all parasites have a life cycle. In many cases, the parasite employs aerobic metabolism during their free-living stage outside the host. In such systems, parasite mitochondria play diverse roles. In particular, marked changes in the morphology and components of the mitochondria during the life cycle are very interesting elements of biological processes such as developmental control and environmental adaptation. Recent research has shown that the mitochondrial complex II plays an important role in the anaerobic energy metabolism of parasites inhabiting hosts, by acting as quinol-fumarate reductase.

Topics: Amino Acid Sequence; Anaerobiosis; Animals; Ascaris suum; Electron Transport Complex II; Energy Metabolism; Fumarates; Life Cycle Stages; Mitochondria; Models, Chemical; Molecular Sequence Data; Multienzyme Complexes; Oxidoreductases; Oxidoreductases Acting on CH-CH Group Donors; Phylogeny; Plasmodium falciparum; Sequence Alignment; Succinate Dehydrogenase; Succinic Acid

A comprehensive phylogenetic analysis of the core subunits of succinate:quinone oxidoreductases and quinol:fumarate oxidoreductases is performed, showing that the classification of the enzymes as type A to E based on the type of the membrane anchor fully correlates with the specific characteristics of the two core subunits. A special emphasis is given to the type E enzymes, which have an atypical association to the membrane, possibly involving anchor subunits with amphipathic helices. Furthermore, the redox properties of the SQR/QFR proteins are also reviewed, stressing out the recent observation of redox-Bohr effect upon haem reduction, observed for the Desulfovibrio gigas and Rhodothermus marinus enzymes, which indicates a direct protonation event at the haems or at a nearby residue. Finally, the possible contribution of these enzymes to the formation/dissipation of a transmembrane proton gradient is discussed, considering recent experimental and structural data.

Topics: Amino Acid Sequence; Electron Transport Complex II; Flavoproteins; Fumarates; Heme; Iron-Sulfur Proteins; Metals; Models, Molecular; Molecular Sequence Data; Multienzyme Complexes; Oxidation-Reduction; Oxidoreductases; Phylogeny; Quinone Reductases; Sequence Alignment; Sequence Homology, Amino Acid; Succinate Dehydrogenase; Succinic Acid

Topics: Animals; Ascaris suum; Electron Transport; Energy Metabolism; Fumarates; Mitochondria; Mitochondrial Proteins; NAD; Oxidoreductases; Oxygen Consumption; Plant Proteins; Trypanosoma brucei brucei

Topics: Citric Acid Cycle; Fumarate Hydratase; Fumarates; Humans; Isoenzymes; Maleates; Metabolism, Inborn Errors; Mitochondria; Substrate Specificity

The treatment of psoriasis vulgaris with fumaric acid esters has been controversial for more than 30 years. Recently the fumaric acid derivatives are marketed antipsoriatics in many European countries. In this paper the clinical efficacy, the side effects as well as the mode of action of these highly potent substances are summarized.

Topics: Dermatologic Agents; Esters; Fumarates; Humans; Immunotherapy; Psoriasis

To assess the effects of salazopyrin, auranofin, etretinate, fumaric acid, IMI gold, azathioprine, and methotrexate, in psoriatic arthritis.. We searched Medline up to 1995, and Excerpta Medica (June 1974-95). Search terms were psoriasis, arthritis, therapy and/or controlled trial. This was supplemented by manually searching bibliographies of previously published reviews, conference proceedings and contacting drug companies. All languages were included in the initial search.. All randomized trials comparing salazopyrin, auranofin, etretinate, fumaric acid, IMI gold, azathioprine, and methotrexate, in psoriatic arthritis. The main outcome measures included individual component variables derived from Outcome Measures in Rheumatology Clinical Trials (OMERACT). These include Acute Phase Reactants, Disability, Pain, Patient Global Assessment, Physician Global Assessment, Swollen joint count, Tender joint count and radiographic changes of joints in any trial of 1 year or longer [Tugwell 1993], and the change in pooled disease index. Only English trials were included in the review.. Data were independently extracted from the published reports by two of the reviewers. An independent blinded quality assessment was also performed.. Nineteen randomized trials were identified of which eleven were included in the quantitative analysis with data from 777 subjects. Although all agents were better than placebo, parenteral high dose methotrexate (not included), salazopyrin, azathioprine and etretinate were the agents that achieved statistical significance in a global index of disease activity (although it should be noted that only one component variable was available for azathioprine and only one trial was available for etretinate suggesting some caution is necessary in interpreting these results). Analysis of response in individual disease activity markers was more variable with considerable differences between different medications and responses. In all trials the placebo group improved over baseline (pooled improvement 0.43 DI units, 95% CI 0. 28-0.59). There was insufficient data to examine toxicity.. Parenteral high dose methotrexate and salazopyrin are the only two agents with well demonstrated published efficacy in psoriatic arthritis. The magnitude of the effect seen with azathioprine, etretinate, oral low dose methotrexate and perhaps colchicine suggests that they may be effective but that further multicentre clinical trials are required to establish their efficacy. Furthermore, the magnitude of the improvement observed in the placebo group strongly suggests that uncontrolled trials should not be used to guide management decisions in this condition.

Topics: Antirheumatic Agents; Arthritis, Psoriatic; Auranofin; Azathioprine; Dermatologic Agents; Etretinate; Fumarates; Humans; Immunosuppressive Agents; Methotrexate; Sulfasalazine

In the absence of oxygen many bacteria are able to utilise fumarate as a terminal oxidant for respiration. In most known organisms the fumarate reductases are membrane-bound iron-sulfur flavoproteins but Shewanella species produce a soluble, periplasmic flavocytochrome c(3) that catalyses this reaction. The active sites of all fumarate reductases are clearly conserved at the structural level, indicating a common mechanism. The structures of fumarate reductases from two Shewanella species have been determined. Fumarate, succinate and a partially hydrated fumarate ligand are found in equivalent locations in different crystals, tightly bound in the active site and close to N5 of the FAD cofactor, allowing identification of amino acid residues that are involved in substrate binding and catalysis. Conversion of fumarate to succinate requires hydride transfer from FAD and protonation by an active site acid. The identity of the proton donor has been open to question but we have used structural considerations to suggest that this function is provided by an arginine side chain. We have confirmed this experimentally by analysing the effects of site-directed mutations on enzyme activity. Substitutions of Arg402 lead to a dramatic loss of activity whereas neither of the two active site histidine residues is required for catalysis.

Topics: Binding Sites; Catalysis; Cytochrome c Group; Electron Transport; Fumarates; Models, Chemical; Models, Molecular; Molecular Structure; Mutagenesis, Site-Directed; Shewanella; Substrate Specificity; Succinate Dehydrogenase

Recently, the microbial production of multifunctional organic acid has received interest due to their increased use in the food industry and their potential as raw materials for the manufacture of biodegradable polymers. Certain species of microorganisms produce significant quantities of organic acids in high yields under specific cultivation conditions from biomass-derived carbohydrates. The accumulation of some acids, such as fumaric, malic and succinic acid, are believed to involve CO2-fixation which gives high yields of products. The application of special fermentation techniques and the methods for downstream processing of products are described. Techniques such as simultaneous fermentation and product recovery and downstream processing are likely to occupy an important role in the reduction of production costs. Finally, some aspects of process design and current industrial production processes are discussed.

Topics: Aspartic Acid; Carboxylic Acids; Citric Acid; Conservation of Natural Resources; Fermentation; Fumarates; Lactic Acid; Malates; Succinates

Topics: Atrophy; Biomarkers; Brain; Cytoplasm; Diagnosis, Differential; Fumarate Hydratase; Fumarates; Humans; Metabolism, Inborn Errors; Mitochondria; Mutation; Prognosis

Topics: Acetylation; Bacterial Proteins; Calcium; Chemotaxis; Escherichia coli; Escherichia coli Proteins; Flagella; Fumarates; Membrane Proteins; Methyl-Accepting Chemotaxis Proteins; Models, Biological; Phosphorylation; Protein Kinases; Protein Processing, Post-Translational; Rotation; Salmonella typhimurium; Signal Transduction

A family having two boys with progressive encephalomyopathy and fumaric aciduria due to fumarase deficiency is described. Both patients initially presented with polyhydramnios and enlarged cerebral ventricles in utero, with subsequent cerebral atrophy, severe developmental delay, infantile spasms, and hypsarythmia on electroencephalogram. Fumarase activity in blood mononuclear cells and in the mitochondrial and cytosolic fractions of cultured skin fibroblasts was less than 0.5% of the control mean or undetectable. The older boy died at the age of 5 years and 4 months and the younger one is now 2 years and 10 months. The unrelated parents are symptomless and the other three children in the family are clinically healthy. Fumarase activities in the blood mononuclear cells of the father, mother, sister, and two brothers were 59%, 52%, 52%, 120%, and 44% of the control mean, respectively. The results strongly support autosomal recessive inheritance of fumarase deficiency and suggest its consideration in children with congenital hydrocephalus, progressive brain atrophy, and infantile spasms.

Topics: Cells, Cultured; Cerebral Ventriculography; Child, Preschool; Electroencephalography; Fibroblasts; Fumarate Hydratase; Fumarates; Humans; Hydrocephalus; Male; Polyhydramnios; Skin; Tomography, X-Ray Computed

For the past two decades fumaric acid (FA) therapy has become an increasingly popular treatment in Western Europe for psoriasis. FA therapy originally was developed by Schweckendiek and subsequently standardized by Schäfer. Schäfer's fumaric acid compound therapy (FACT) consists of the oral intake of dimethylfumaric acid ester (DMFAE) and several salts of monoethylfumaric acid ester (MEFAE) in combination with topical fumaric acid therapy (1% to 3% MEFAE in an ointment or FA in bathing oils) and a diet. Schäfer claimed excellent results in a large number of patients. Preliminary studies by German dermatologists, however, revealed contradictory therapeutic results and serious side effects, and FA treatment was soon abandoned by dermatologists. To assess the value of FA therapy we conducted an open pilot study of 36 patients in which FACT therapy appeared to be rather effective. Thereafter, several controlled studies with MEFAE sodium in two different dosages versus placebo, and DMFAE versus placebo, were done. The results indicated that MEFAE sodium in dosages up to 240 mg daily was ineffective, whereas daily dosages of 720 mg resulted in a significant decrease in scaling and itching but did not affect extension of the eruption. DMFAE, 240 mg daily, produced a significant amelioration and prevented extension. Side effects of FA treatment were nausea, diarrhea, general malaise, and severe stomachache. Mild disturbances of liver and kidney function during treatment were observed with the 720 mg dosage of MEFAE and with the 240 mg dosage of DMFAE. Moreover, a relative lymphopenia with a selective decrease of suppressor T lymphocytes occurred in about 50% of the patients treated with DMFAE.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Administration, Oral; Clinical Trials as Topic; Double-Blind Method; Female; Fumarates; Humans; Male; Pilot Projects; Psoriasis; Random Allocation

Filamentous fungi are well known for their potential to accumulate organic acids in the medium when supplied with large amounts of sugar. Commercial applications of this are the production of citric and itaconic acids. The present review attempts to present the current state of knowledge on the biochemical basis of organic acid accumulation by filamentous fungi (citric, itaconic, fumaric and oxalic acids), particularly with respect to the role of citric acid cycle reactions. The explanations offered are based on recent advances in understanding the compartmentation of the fungal cell, and regulation of some key enzymes. The general conclusion is that fungi accumulate organic acids by mechanisms which avoid the channeling of substrates into the citric acid cycle under conditions of strongly active glycolysis.

Topics: Carboxylic Acids; Citric Acid Cycle; Fumarates; Fungi; Oxalates; Oxalic Acid; Succinates

Topics: Anti-Infective Agents; Anti-Inflammatory Agents, Non-Steroidal; Fumarates; Gallic Acid; Humans; Medicine, Chinese Traditional; Medicine, East Asian Traditional; Oleanolic Acid; Plant Extracts; Plants, Medicinal; Succinates; Succinic Acid

Electron transport-coupled phosphorylation with fumarate as terminal acceptor in Wolinella succinogenes yields less than 1 ATP/2 electrons. The delta mu H generated by the electron transport is 0.18 V and the H+/electron ratio is 1. The electron transport chain is made up of two dehydrogenases (hydrogenase and formate dehydrogenase) that catalyze the reduction of menaquinone, and fumarate reductase which catalyzes the oxidation of menaquinol. C-type cytochromes are not involved. The phosphorylative electron transport with sulfur as terminal acceptor in W. succinogenes or Desulfuromonas acetoxidans does not involve known quinones. The ATP yields should be even smaller than those with fumarate. Succinate oxidation by sulfur, which is a catabolic reaction in D. acetoxidans, is accomplished by reversed electron transport.

Topics: Electron Transport; Electron Transport Complex III; Fumarates; Gram-Negative Anaerobic Bacteria; Multienzyme Complexes; Oxidation-Reduction; Oxidoreductases; Phosphorylation; Quinone Reductases; Succinates; Succinic Acid; Vibrio

Topics: Animals; Citrates; Citric Acid; Food Additives; Fumarates; Humans; Hydrogen-Ion Concentration; Swine

Topics: Amino Acid Sequence; Binding Sites; Biological Transport, Active; Escherichia coli; Fluorescence; Fumarates; Macromolecular Substances; Microscopy, Electron; Models, Molecular; Operon; Protein Conformation; Solubility; Structure-Activity Relationship; Succinate Dehydrogenase; X-Ray Diffraction

Topics: Alanine; Aspartate Ammonia-Lyase; Biotechnology; Carboxy-Lyases; Escherichia coli; Fumarates; Pseudomonas

To investigate the effects of dietary fumaric acid (FUA) on performance, carcasses, nutrient digestibility, blood metabolites, digestive enzymes, and cecal microbiota in Japanese quail chicks. Three hundred unsexed Japanese quail (1-wk-old) were randomly assigned to 5 groups. Supplementation of FUA in the diet of Japanese quail chicks exhibited a significant improvement in growth performance through the different experimental periods studied compared with those receiving unsupplemented one. The digestibility of crude protein (CP) and metabolizable energy (ME) were improved with 10 and 15 g/kg FUA, respectively. Apart from lipase enzyme, birds fed 5 and 15 g/kg FUA recorded higher activity of amylase. There were no significant changes among experimental groups on the relative weights of carcass, gizzard, heart, and dressing. Dietary supplementation of FUA at different levels (P> 0.05) increased total protein (TP) and globulin (GLB) concentrations and A/G % compared with control group. A significant (P < 0.01) decrease in plasma low density lipoprotein (LDL) and total cholesterol (TC) levels and increase in high density lipoprotein (HDL) concentrations were observed in chicks fed with FUA containing diets. Immunoglobulin G (IgG) (P = 0.0026) and M (IgM) (P = 0.0007) levels were greater in groups treated with either 10 or 15 g FUA/kg diet. A significant increase in plasma Ca concentration was noticed in chicks received 15 g FUA/ kg compared with the other groups. Quail chicks received diets containing FUA at different levels exhibited reduced cecal count of coliform, E. coli, and Salmonella as compared with control group. In conclusion, supplementation of fumaric acid (especially 15 g/kg diet) in quail chick diets improved their growth, digestibility of nutrients, immune response, antioxidant status, digestive enzyme, and intestinal health.

Topics: Animal Feed; Animal Nutritional Physiological Phenomena; Animals; Chickens; Coturnix; Diet; Dietary Supplements; Digestion; Escherichia coli; Fumarates; Gastrointestinal Microbiome; Nutrients; Quail

A novel orotic acid salt form of tenofovir disoproxil (DA-2802) was developed and is expected to replace the fumaric acid salt form. The pharmacokinetic (PK) characteristics and tolerability profiles of DA-2802 were compared to those of tenofovir disoproxil fumarate (TDF, Viread

Topics: Adult; Cross-Over Studies; Dose-Response Relationship, Drug; Female; Fumarates; Healthy Volunteers; Humans; Male; Orotic Acid; Salts; Tenofovir; Young Adult

Two studies in healthy subjects assessed the absorption of edoxaban when delivered to specific locations within the gastrointestinal tract using Enterion capsules. In study 1 (single-dose, 4-way crossover), 8 participants received edoxaban 60 mg as immediate-release (IR) tablets (treatment A), as powder formulation delivered to the distal small bowel (treatment B) or ascending colon (treatment C), or as an aqueous suspension delivered to the ascending colon (treatment D). In study 2 (single-dose, 2-way crossover), 10 participants received edoxaban 30 mg as IR tablets (treatment E) or in granulate formulation with fumaric acid 50 mg, added to acidify the local gastrointestinal tract and enhance solubility, delivered to the ascending colon (treatment F). Peak and total exposure following targeted drug delivery to the distal gastrointestinal tract were significantly lower than with IR tablet delivery. In study 1, total exposure ratios of treatments B, C, and D compared with A were 14.9%, 7.9%, and 6.1%, respectively. In study 2, relative total exposure was 12.6% for treatment F despite the fumaric acid. Time to peak concentration was longer with higher variability for edoxaban delivered to the distal gastrointestinal tract compared with the IR tablet. These data indicate that edoxaban absorption occurs predominantly in the proximal small intestine.

Topics: Adolescent; Adult; Aged; Capsules; Colon; Cross-Over Studies; Factor Xa Inhibitors; Fumarates; Gastrointestinal Absorption; Humans; Intestine, Small; Male; Middle Aged; Pyridines; Tablets; Thiazoles; Young Adult

The aim of this study was to determine the influence of fumaric acid (FA) on ruminal fermentation and its effects on the acid-base balance of seven ruminally and duodenally fistulated multiparous German Holstein cows. The experiment was conducted in a change-over design with three periods in which the animals were randomly arranged in one of three treatments: Control (C; without FA), 300 or 600 g FA per day. The diets consisted of 7.4 kg DM grass silage, 4.2 kg concentrate mixture and 0, 300 or 600 g FA or wheat starch as isocaloric compensation per day and cow. FA supplementation decreased the rumen pH, acetic acid and butyric acid and increased propionic acid in rumen fluid. The results of the single-strand conformation polymorphism analysis (SSCP) did not show an influence of FA on the microbial population in the rumen. The beta-hydroxybutyrate (BHB) concentration in blood and the pH of the urine decreased, while the blood gases were unaffected by supplementation of the acid. The microbial protein per MJ ME decreased in the duodenum with FA supplementation. The milk fat concentration decreased after addition of FA. We conclude that in this study feeding of up to 600 g FA per day did not result in an acidosis. It seems that up to 600 g FA per day did not have a significant influence on the acid-base balance of dairy cows.

Topics: Animal Feed; Animal Nutritional Physiological Phenomena; Animals; Archaea; Bacteria; Cattle; Cross-Over Studies; Diet; Energy Metabolism; Female; Fermentation; Fumarates; Lactation; Milk; Rumen

Hand eczema is a common distressing skin problem. It is an immune reaction to haptens. Thus, substances that inhibit Immune system can be effective in the treatment of hand eczema. In this study, topical fumaric acid 5% cream is compared with topical steroid in the treatment of hand eczema. Patients with hand eczema were randomly divided into two groups. One group received fumaric acid 5% in a cream base, and the other received triamcinolone 0.1% in the same cream base. Both groups used creams twice daily for one month. Patients were checked for erythema, excoriation, population and lichenification, EASI score, and pruritus before and after treatment. In both groups, the mean of all signs of the disease and EASI score decreased after one month of treatment. There was no significant difference between the two treatments in decreasing erythema, but excoriation, population, lichenification, EASI score and itching were all decreased more in triamcinolone 0.1 % group. Although fumaric acid can inhibit the immune system; it was less effective for the treatment of all signs of hand eczema except erythema in comparison to triamcinolone. These results may be justified for two reasons: low penetration of topical fumaric acid through the skin or a low concentration used in this study.

Topics: Administration, Topical; Adult; Dose-Response Relationship, Drug; Double-Blind Method; Eczema; Female; Follow-Up Studies; Fumarates; Glucocorticoids; Hand; Humans; Male; Skin Cream; Skin Diseases; Treatment Outcome; Triamcinolone

To evaluate the efficacy of the combined drug furamag (furasidine potassium and magnesium hydroxycarbonate) in combination with the third-generation cephalosporin cefotaxime versus cephalosporin monotherapy for nosocomial urinary tract infections (NUTI).. The randomized open-label comparative parallel group clinical trial enrolled 52 male and female patients aged over 18 years with a documented diagnosis of NUTI. Group 1 (a study group) took oral furamag 300 mg/day in combination with intravenous cefotaxime 6 g/day; Group 2 (a control group) received cefotaxime monotherapy. The duration of therapy in both groups was 7 to 10 days until the efficiency levels were achieved.. A final efficiency analysis was made in 24 and 25 patients from Groups 1 and 2 who had different forms of NUTI (catheter-associated NUTI, cystitis, pyelonephritis). On day 3 of treatment, most patients were noted to have a decreased systemic inflammatory response; lower C-reactive protein and procalcitonin levels being in the study group patients. The clinical efficiency of antibacterial therapy, which had been evaluated both immediately after treatment termination and during further control, did not substantially differ in the furamag/cefotaxime combination and control groups although there was an obvious tendency towards the more marked effect of combined therapy 7-14 days after treatment (11.8% efficiency differences; p>0.05). Analysis of bacteriological efficacy revealed its most pronounced and clinically significant differences between the groups: the cefotaxime/furamag combination led to higher pathogen eradication in all follow-up periods: after 3 days of treatment (82.6%) and following a complete therapy cycle (95.8%) versus the cefotaxime monotherapy group (43.5 and 70.8%, respectively; p<0.01). Microbiological results showed that the major NUTI pathogens (Escherichia coli, enterococci) were more susceptible to potassium furasidine (furamag) versus cefotaxime. The in vitro higher activity of furamag versus cefotaxime was attended by the significantly higher eradication of one of the two important NUTI pathogens - Enterococcus faecalis.. Furamag used in combination with the third-generation cephalosporin cefotaxime showed a higher bacteriological efficacy and a rapider reduction in the symptoms of a systemic inflammatory response in patients with NUTI. On the basis of the findings, the above combination of furamag and cefotaxime may be recommended as first-line therapy for NUTI when multidrug- resistant nosocomial infection pathogens are widely distributed now.

Topics: Adult; Aged; Aged, 80 and over; Anti-Bacterial Agents; Cephalosporins; Cross Infection; Drug Therapy, Combination; Female; Fumarates; Humans; Male; Middle Aged; Treatment Outcome; Urinary Tract Infections

To evaluate the efficacy of nitrofurans in children and adolescents with pyelonephritis in the presence of crystalluria.. The study included 50 patients aged 4-14 years with chronic pyelonephritis in the presence of dysmetabolism. The patients underwent general blood test, general urinalysis with an urocytogram, bacteriological examination of urine, biochemical test of serum (uric acid, calcium, phosphorus, magnesium, urea, and creatinine) and 24-hour urinary excretion (uric acid, oxalates, calcium, phosphorus, and magnesium) at hospital admission and over time. The treatment regimen for Group 1 patients after antibiotic therapy involved furamag, Group 2 received furagin. The drugs were used in a dosage of 2 mg/kg/day in 2 divided doses for 14 days. Complaints, major clinical manifestations, crystalluria patterns, and a number of laboratory findings were analyzed over time.. The urinary sediment showed leukocyturia and bacteriuria in all the patients, oxaluria in 70% of the patients, uraturia in 10%, and mixed crystalluria in 20%. The main etiological agent of pyelonephritis was Escherichia coli (48.4%). Increased serum uric acid concentrations were revealed in 14% of the patients. Daily urine tests revealed hyperoxaluria, hyperuricosuria, and hypercalciuria in 86, 18, and 8% of the patients, respectively; urinary magnesium excretion was reduced in 86%. After treatment, Group 1 patients showed a more marked therapeutic effect in terms of a number of indicators (leukocyturia, crystalluria, uricosuria, magnesuria).. The results of the study showed that the antibacterial therapy involving antibiotics and nitrofurans for an exacerbation of chronic pyelonephritis in the presence of crystalluria not only provides an anti-inflammatory effect, but also leads to reductions in the level of crystalluria and the urinary content of uric acid and calcium. There was a significantly marked reduction in crystalluria, serum uric acid, and urinary oxalates and calcium in the children taking furamag. Out of nitrofurans, furamag may be recommended as the drug of choice to treat urinary tract infections in the presence of crystalluria.

Topics: Adolescent; Anti-Bacterial Agents; Child; Child, Preschool; Chronic Disease; Crystallization; Female; Fumarates; Furagin; Humans; Male; Pyelonephritis; Treatment Outcome; Urinary Calculi

To study the clinical aspects of using the furasidine potassium in combination with basic magnesium carbonate (furamag) and phosphomycin trometamol (monural) as antimicrobial agents most frequently used in outpatient practice during combination therapy for acute and chronic urinary tract (UT) diseases.. To study the specific features of therapy for UT infections, 60 patients were randomized to 2 groups: 1) 30 patients received a course therapy with furasidine potassium (furamag) in a dose of 50 mg t.i.d. for 7 days (a study group) and 2) 30 had phosphomycin trometamol (monural) in a single dose of 3 g for pulse therapy (a comparison group). The clinical efficacy of the drugs, symptom disappearance rates, bacterial changes, and laboratory and instrumental findings were assessed. The patient's opinion was mainly used to evaluate outpatient pharmacoeconomic efficiency. Patient compliance with the given therapy was estimated by taking into account the specific features of prehospital care.. During therapy, both groups showed positive clinical changes. In the study group, the symptoms of dysuria resolved 0.5 days more quickly and a complete clinical remission was achieved 0.8 days more promptly; the latter within the first 72 hours was achieved by 7.5% more of the patients; the symptoms of bacteriuria resolved 0.6 days more rapidly. With the similar average price of the packs of furasidine potassium (furamag) 50 mg (30 capsules) and phosphomycin trometamol (monural) 1 g (a sachet) being 350 and 370 rubles, the average costs of required treatment were 482 and 546 rubles, respectively. No case of adverse reactions was recorded during the study.. Patients with infectious and inflammatory diseases of UT should be given furasidine potassium in the standard dose of 50 mg t.i.d for 7 days.

Topics: Adult; Ambulatory Care; Anti-Infective Agents; Attitude of Health Personnel; Bacteria; Bacteriological Techniques; Drug Costs; Drug Therapy, Combination; Female; Fosfomycin; Fumarates; Humans; Male; Medication Adherence; Microbial Sensitivity Tests; Outcome Assessment, Health Care; Reproductive Tract Infections; Treatment Outcome; Urinary Tract Infections; Urogenital System

The aim of the present study was to determine the effects of the potential methane reducer fumaric acid on the fattening performance and acid-base balance of growing bulls fed two different silage types as roughage (maize and grass silage). A total of 62 fattening bulls (German Holstein breed, initial body weight: 266 +/- 42 kg), randomly assigned to eight feeding groups, received four levels of fumaric acid (0, 100, 200 or 300 g/d) at each silage type. The daily feed and water intake and the live weight were measured over the whole testing period of 280 days. In blood samples, blood cells and blood gases as a parameter of acid-base status were analysed. Feed and faeces were collected to determine the apparent nutrient digestibility. Five animals from each group fed maize or grass silage, supplemented with 0 g or 300 g fumaric acid, were slaughtered at 580 kg body weight. After slaughter, rumen fluid pH was measured and dressing percentage was calculated. Neither the total feed intake (8.81 +/- 0.07 kg/d) nor the daily weight gain (1277 +/- 24 g/d) was influenced significantly by treatments. Fumaric acid supplementation did not influence the erythrocyte count or the blood gas concentration. The silage type significantly influenced the apparent digestibility of the whole diet. The dressing percentage was slightly higher (p < 0.1) after fumaric acid supplementation. No signs of an incompatibility to fumaric acid on the animals were observed over the whole experimental period. However, it seems to be necessary to conduct more long-term studies with different silage types and addition of organic acids combined with direct measurements of methane.

Topics: Acid-Base Equilibrium; Animals; Cattle; Dietary Supplements; Fumarates; Hydrogen-Ion Concentration; Male; Poaceae; Silage; Weight Gain; Zea mays

It is a common belief among women that iron compounds have unpleasant gastrointestinal side effects.. To assess the gastrointestinal side effects of iron prophylaxis in pregnancy.. A randomized, double-blind study comprising 404 healthy pregnant women allocated to four groups taking ferrous iron supplement (as fumarate) in doses of 20 (n = 99), 40 (n = 100), 60 (n = 102) and 80 mg (n = 103) daily from 18 weeks of gestation to delivery. Iron supplement was predominantly taken at bedtime. Gastrointestinal symptoms (nausea, vomiting, epigastric pain, eructation, pyrosis, meteorism, borborygmi, colic pain, flatulence, constipation, thin feces, diarrhea), black feces, and use of laxatives were recorded by interview at 18, 32 and 39 weeks of gestation.. The frequencies of gastrointestinal symptoms were not significantly different in the four iron supplement groups either at inclusion or at 32 and 39 weeks of gestation and thus not related to the iron dose.. This study shows that a supplement of 20-80 mg ferrous iron (as fumarate), taken between meals, has no clinically significant gastrointestinal side effects. The implementation of iron prophylaxis to pregnant women should not be compromised by undue concern of non-existing side effects.

Topics: Administration, Oral; Adult; Anemia, Iron-Deficiency; Anticarcinogenic Agents; Dietary Supplements; Dose-Response Relationship, Drug; Double-Blind Method; Female; Fumarates; Gastrointestinal Diseases; Humans; Iron, Dietary; Pregnancy; Pregnancy Complications, Hematologic; Pregnancy Trimesters

The objective of this study was to identify feed additives that reduce enteric methane emissions from cattle. We measured methane emissions, total tract digestibility (using chromic oxide), and ruminal fermentation (4 h after feeding) in growing beef cattle fed a diet supplemented with various additives. The experiment was designed as a replicated 4 x 4 Latin square with 21-d periods and was conducted using 16 Angus heifers (initial BW of 260 +/- 32 kg). Treatments were: control (no additive), fumaric acid (175 g/d) with sodium bicarbonate (75 g/d), essential oil and spice extract (1 g/d), or canola oil (4.6% of DMI). The basal diet consisted of 75% whole-crop barley silage, 19% steam-rolled barley, and 6% supplement (DM basis). Four large chambers (2 animals fed the same diet per chamber) were equipped to measure methane emissions for 3 d each period. Adding canola oil to the diet decreased (P = 0.009) total daily methane emissions by 32% and tended (P = 0.09) to decrease methane emissions as a percentage of gross energy intake by 21%. However, much of the reduction in methane emissions was due to decreased (P < 0.05) feed intake and lower (P < 0.05) total tract digestibility of DM and fiber. Digestibility of all nutrients was also lowered (P < 0.05) by feeding essential oil, but there were no effects on ruminal fermentation or methane emissions. In contrast, adding fumaric acid to the diet increased total VFA concentration (P = 0.03), increased propionate proportions (P = 0.01), and decreased the acetate:propionate ratio (P = 0.002), but there was no measurable effect on methane emissions. The study demonstrates that canola oil can be used to reduce methane losses from cattle, but animal performance may be compromised due to lower feed intake and decreased fiber digestibility. Essential oils had no effect on methane emissions, whereas fumaric acid caused potentially beneficial changes in ruminal fermentation but no measurable reductions in methane emissions.

Topics: Animal Feed; Animal Nutritional Physiological Phenomena; Animals; Cattle; Diet; Digestion; Fatty Acids, Monounsaturated; Female; Fermentation; Fumarates; Methane; Oils, Volatile; Rapeseed Oil

Noninfectious granulomatous skin diseases are inflammatory disorders of unknown aetiology which are often recalcitrant to common anti-inflammatory treatment regimens. Recently, in several case reports, fumaric acid esters (FAE) have proved beneficial in granulomatous skin diseases, but studies on a larger collection of consecutive patients have not yet been performed.. To investigate the therapeutic efficacy of FAE for the treatment of granulomatous skin diseases.. The therapeutic efficacy and side-effects of FAE were analysed retrospectively in 32 patients with disseminated granuloma annulare (n = 13), annular elastolytic giant cell granuloma (n = 3), sarcoidosis (n = 11), necrobiosis lipoidica (n = 4), or granulomatous cheilitis (n = 1).. Three patients discontinued treatment within 4 weeks because of side-effects. Of the remaining 29 patients, 18 patients responded to treatment with FAE. Marked improvement or complete clearance was seen in seven patients. We observed a slight to moderate improvement in 11 patients, and 11 patients did not respond. In patients showing a complete remission, the maximum effect was observed after 8.5 months (SD +/-6 months, range 3-20 months). In two patients with systemic sarcoidosis, the pulmonary changes improved in parallel with the skin. Side-effects were usually mild and resolved spontaneously upon dose reduction or discontinuation of the therapy.. The data presented here indicate that FAE may be considered for the treatment of recalcitrant granulomatous skin disease.

Topics: Adolescent; Adult; Aged; Cheilitis; Drug Administration Schedule; Esters; Female; Fumarates; Granuloma Annulare; Granuloma, Giant Cell; Humans; Male; Middle Aged; Necrobiosis Lipoidica; Retrospective Studies; Sarcoidosis; Skin Diseases

Clinical placebo-controlled study of Enerlit-Clima (bioactive succinate-based food additive) a showed positive effect of the preparation on general clinical and psychoemotional manifestations of the climacteric syndrome. A trend to an increase in estradiol level in early pathological climacteric and normalization of the endometrial status were observed.

Topics: Adult; alpha-Tocopherol; Anxiety; Blood Glucose; Climacteric; Depression; Female; Food Additives; Fumarates; Glutamine; Glycine; Gonadal Steroid Hormones; Hot Flashes; Humans; Lipids; Middle Aged; Sleep Initiation and Maintenance Disorders; Succinates; Tocopherols

The effects of dietary levels of manganese (Mn) in inorganic (MnO) and organic (Mn fumarate) forms were evaluated on cockerel chicks. A basal corn-soybean diet with 23 mg/kg Mn was supplemented with levels of 0, 30, 60 and 240 ppm Mn from both Mn sources. Each treatment was replicated in five pens of 10 chicks. The chicks were fed diets ad libitum from 14 to 49 days of age, after which five birds per treatment were sacrificed for pathomorphological examinations and analysis. The treatments did not exert significant effects on the body weight (BW), the feed/gain (F/G) ratio or the mortality rate. According to the necropsy findings, no growth retardation or emaciation occurred in either of the groups and the differences in the average absolute and relative organ weights were not significant (P > 0.05). Tissue analysis indicated that the tibia showed the greatest response to Mn, followed by the liver and kidney. Accumulation in the tibia was higher (P < 0.05) with supplements of 30, 60 and 240 mg/kg from both Mn sources (3.71, 3.78, 4.44, and 3.68, 4.00, 4.36 mg/kg DM, MnO and Mn fumarate, respectively) compared to the control group (3.21 mg/kg). Accumulation in the liver increased significantly (P < 0.05) only with supplements of 60 and 240 ppm independently of the Mn source (12.7, 14.2, and 14.0, 14.9 mg/kg, respectively) compared to the control (9.8 mg/kg). Similarly, kidney tissue Mn was higher (P < 0.05) only with supplements of 60 and 240 ppm (12.8, 12.8, and 13.1, 12.5 mg/kg, respectively) compared to the control (10.2 mg/kg). At the same level of supplementation of the two Mn sources there were no significant differences (P > 0.05) between the Mn concentrations of organs and tissues. Droppings sensitively reflected the intake, whereas blood plasma and feathers showed only the extreme Mn loading.

Topics: Animal Feed; Animals; Chickens; Dietary Supplements; Fumarates; Kidney; Liver; Manganese; Manganese Compounds; Oxides; Tissue Distribution

To characterize the pharmacokinetics of fumarates in healthy subjects.. Ten subjects received a single fumarate tablet (containing 120 mg of dimethylfumarate and 95 mg of calcium-monoethylfumarate) in the fasted state and after a standardized breakfast in randomized order. Prior to and at fixed intervals after the dose, blood samples were drawn and the concentrations of monomethylfumarate, the biologically active metabolite, as well as dimethylfumarate and fumaric acid were measured using high-performance liquid chromatography.. After a lag time, a transient increase in serum monomethylfumarate concentrations in the blood was observed, whereas dimethylfumarate and fumaric acid concentrations remained below the detection limit. The tlag was 240 min [range 60-603 min; 95% confidence interval (CI) 139, 471] shorter when the tablet was taken after an overnight fast (90 min; range 60-120 min; 95% CI 66, 107) than when taken with breakfast (300 min; range 180-723 min; 95% CI 0, 1002). The tmax was 241 min (range 60-1189 min, 95% CI 53, 781) shorter when the tablet was taken after an overnight fast (182 min; range 120-240 min; 95% CI 146, 211) than when taken with breakfast (361 min; range 240-1429 min; 95% CI 0, 1062). The mean Cmax for monomethylfumarate in the blood of fasting subjects was to 0.84 mg l(-1) (range 0.37-1.29 mg l(-1); 95% CI 0.52, 1.07) and did not differ from that in fed subjects (0.48 mg l(-1); range 0-1.22 mg l(-1); 95% CI 0, 5.55).. The pharmacokinetics of monomethylfumarate in healthy subjects after a single tablet of fumarate are highly variable, particularly after food intake. Further experiments exploring the pharmacokinetics of oral fumarates are warranted in order to elucidate the mechanisms underlying variability in response in patients.

Topics: Administration, Oral; Adult; Anticarcinogenic Agents; Female; Fumarates; Humans; Male; Tablets

Methane emitted from the livestock sector contributes to greenhouse gas (GHG) emissions. Understanding the effects of diet on enteric methane production can help refine GHG emission inventories and identify viable GHG reduction strategies. Our study focused on measuring methane and carbon dioxide emissions, total-tract digestibility, and ruminal fermentation in growing beef cattle fed a diet supplemented with various additives or ingredients. Two experiments, each designed as a 4 x 4 Latin square with 21-d periods, were conducted using 16 Holstein steers (initial BW 311.6 +/- 12.3 kg). In Exp. 1, treatments were control (no additive), monensin (Rumensin, Elanco Animal Health, Indianapolis, IN; 33 mg/kg DM), sunflower oil (400 g/d, approximately 5% of DMI), and proteolytic enzyme (Protex 6-L, Genencor Int., Inc., CA; 1 mL/kg DM). In Exp. 2, treatments were control (no additive), Procreatin-7 yeast (Prince Agri Products, Inc., Quincy, IL; 4 g/d), Levucell SC yeast (Lallemand, Inc., Rexdale, Ontario, Canada; 1 g/d), and fumaric acid (Bartek Ingredients Inc., Stoney Creek, Ontario, Canada; 80 g/d). The basal diet consisted of 75% barley silage, 19% steam-rolled barley grain, and 6% supplement (DM basis). Four large chambers (two animals per chamber) were equipped with lasers and infrared gas analyzers to measure methane and carbon dioxide, respectively, for 3 d each period. Total-tract digestibility was determined using chromic oxide. Approximately 6.5% of the GE consumed was lost in the form of methane emissions from animals fed the control diet. In Exp. 1, sunflower oil decreased methane emissions by 22% (P = 0.001) compared with the control, whereas monensin (P = 0.44) and enzyme had no effect (P = 0.82). However, oil decreased (P = 0.03) the total-tract digestibility of NDF by 20%. When CH(4) emissions were corrected for differences in energy intake, the loss of GE to methane was decreased by 21% (P = 0.002) using oil and by 9% (P = 0.09) using monensin. In Exp. 2, Procreatin-7 yeast (P = 0.72), Levucell SC yeast (P = 0.28), and fumaric acid (P = 0.21) had no effect on methane emissions, although emissions as a percentage of GE intake were 3% (non-significant, P = 0.39) less for steers fed Procreatin-7 yeast compared with the control. This study demonstrates that sunflower oil, ionophores, and possibly some yeast products can be used to decrease the GE lost as methane from cattle, but fiber digestibility is impaired with oil supplementation.

Topics: Animal Feed; Animals; Carbon Dioxide; Cattle; Diet; Dietary Supplements; Digestion; Fermentation; Fumarates; Ionophores; Male; Methane; Monensin; Peptide Hydrolases; Plant Oils; Rumen; Sunflower Oil; Yeasts

To evaluate the efficacy and safety of administering quetiapine once vs twice daily.. Utilizing a double-blind design, 21 hospitalized adult men or women with DSM-IV schizophrenia or schizoaffective disorder, who had received unchanged doses (for 2 weeks) of either 400 or 600 mg daily of quetiapine administered in 2 doses, were randomly assigned to once- or twice-daily administration for 4 weeks and then crossed over to the opposite dosing regimen for an additional 4 weeks. Standard psychopathology and safety measures were used in the study.. Nearly 70% (15/21) of the subjects met the a priori efficacy responder criteria with no statistical differences in response between those assigned to once- or twice-daily quetiapine administration. Statistical analyses confirmed that most subjects maintained efficacy during the switch to once- or twice-daily administration with quetiapine. A minority (15%) did experience worsening of symptoms or orthostatic hypotension during the crossover. Quetiapine was generally well tolerated at either twice- or once-daily administration.. These pilot data suggest that it is clinically feasible to switch most quetiapine-treated subjects receiving a therapeutic twice-daily dosing schedule to a once-daily regimen. A minority may experience worsening of symptoms or orthostatic hypotension during the switch. This strategy of administering quetiapine entirely at bedtime may promote improved adherence to treatment.

Topics: Adolescent; Adult; Aged; Anticarcinogenic Agents; Antipsychotic Agents; Cross-Over Studies; Dibenzothiazepines; Double-Blind Method; Drug Administration Schedule; Female; Fumarates; Humans; Male; Middle Aged; Pilot Projects; Psychotic Disorders; Quetiapine Fumarate; Schizophrenia; Schizophrenic Psychology

Bioavibility and pharmacokinetics of magnesium after magnesium fumarate administration in humans. The volunteers were given a single oral dose of 530 mg of magnesium on an empty stomach. The preparation was a magnesium fumarate tablet. The simplest formule to describe the course of blood magnesium concentration changes was a two exponential equation for an open monocompartmental model.

Topics: Adult; Area Under Curve; Biological Availability; Female; Fumarates; Humans; Magnesium; Male; Middle Aged

In this study the authors examine the effects of acute hypoxia due to extracorporeal circulation (ECC) and the role played by L-carnitine treatment on some plasmatic metabolites linked to glycolytic cellular metabolism. To obtain biochemical data, 120 patients in extracorporeal circulation during aortopulmonary bypass surgery were evaluated. The patients received either sodium bicarbonate (40 patients), or L-carnitine during ECC (40 patients) or before and during ECC (40 patients), and plasma samples were collected before ECC, during ECC and after ECC. The levels of lactate and pyruvate showed significant alterations in sodium bicarbonate-treated patients, and there was also a considerable imbalance in the succinate/fumarate ratio. This means that tissue hypoxia due to ECC leads to cellular oxidative damage and to a considerable decrease in the intracellular energy pools. The use of L-carnitine antagonizes the oxidative stress, as is well documented by the levels of plasmatic metabolites which remain confined to normal amounts.

Topics: Bicarbonates; Carnitine; Double-Blind Method; Extracorporeal Circulation; Female; Fumarates; Humans; Hypoxia; Lactates; Lactic Acid; Male; Middle Aged; Pyruvates; Random Allocation; Sodium; Sodium Bicarbonate; Succinates; Succinic Acid

Topics: Adult; Arthritis, Psoriatic; Double-Blind Method; Female; Fumarates; Humans; Male; Middle Aged

Thirty-nine patients with psoriasis (12 females, 27 males) entered a randomised, double-blind, placebo-controlled study on the efficacy of fumaric acid therapy in an outpatient setting. During 16 weeks the patients were treated with tablets containing a combination of dimethylfumarate and different salts of monoethylfumarate, with octylhydrogen fumarate or with placebo tablets. All patients were treated with identical indifferent topical therapy and followed an elimination diet (avoidance of spices, wine and nuts). Thirty-four patients completed the study. Five patients dropped out because of side effects or aggravation of the skin lesions. The patients treated with the combination of monoethyl- and dimethylfumarate showed a significantly better therapeutic response compared with those who were treated with placebo or octylhydrogen fumarate. Side effects of the fumarate containing tablets were flushing, diarrhoea, a reversible elevation of transaminases, lymphocytopenia and eosinophilia. One patient developed a disturbance of the kidney function which normalised after discontinuation of the therapy.

Topics: Adult; Aged; Double-Blind Method; Female; Fumarates; Humans; Male; Middle Aged; Psoriasis

Topics: Administration, Oral; Adult; Aged; Double-Blind Method; Drug Administration Schedule; Female; Fumarates; Humans; Male; Middle Aged; Psoriasis; Random Allocation

In a 4-month double-blind study the effects of dimethylfumaric acid esters (DMFAE-EC) and DMFAE plus salts of monoethylfumaric acid esters (fumaric acid combination, FAC-EC) in enteric-coated tablets were compared in 22 respectively 23 patients with psoriasis. In both groups about 50% showed a considerable improvement, i.e. the initial score was more than halved. The therapeutic effects showed no significant differences in both groups with respect to the total psoriasis score or the different parameters. In the FAC-EC group the effects were obtained more rapidly. Most frequently observed side effects in both groups were flushings, stomachache and diarrhea. Due to these complaints 3 respectively 8 patients discontinued therapy. Eosinophilia, leukopenia and lymphopenia were the most frequently observed differences in lab tests. It was concluded that FAC-EC had no significantly better effect than monotherapy with DMFAE-EC. Moreover, enteric coating of the tablets did not prevent stomach complaints. Until more information has been obtained about the pharmacokinetics, the toxicity and optimal composition of the drug, the fumaric acid therapy in psoriasis should be seen as experimental.

Topics: Adolescent; Adult; Aged; Dimethyl Fumarate; Double-Blind Method; Drug Therapy, Combination; Esters; Female; Fumarates; Humans; Male; Middle Aged; Psoriasis; Random Allocation

For the past two decades fumaric acid (FA) therapy has become an increasingly popular treatment in Western Europe for psoriasis. FA therapy originally was developed by Schweckendiek and subsequently standardized by Schäfer. Schäfer's fumaric acid compound therapy (FACT) consists of the oral intake of dimethylfumaric acid ester (DMFAE) and several salts of monoethylfumaric acid ester (MEFAE) in combination with topical fumaric acid therapy (1% to 3% MEFAE in an ointment or FA in bathing oils) and a diet. Schäfer claimed excellent results in a large number of patients. Preliminary studies by German dermatologists, however, revealed contradictory therapeutic results and serious side effects, and FA treatment was soon abandoned by dermatologists. To assess the value of FA therapy we conducted an open pilot study of 36 patients in which FACT therapy appeared to be rather effective. Thereafter, several controlled studies with MEFAE sodium in two different dosages versus placebo, and DMFAE versus placebo, were done. The results indicated that MEFAE sodium in dosages up to 240 mg daily was ineffective, whereas daily dosages of 720 mg resulted in a significant decrease in scaling and itching but did not affect extension of the eruption. DMFAE, 240 mg daily, produced a significant amelioration and prevented extension. Side effects of FA treatment were nausea, diarrhea, general malaise, and severe stomachache. Mild disturbances of liver and kidney function during treatment were observed with the 720 mg dosage of MEFAE and with the 240 mg dosage of DMFAE. Moreover, a relative lymphopenia with a selective decrease of suppressor T lymphocytes occurred in about 50% of the patients treated with DMFAE.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Administration, Oral; Clinical Trials as Topic; Double-Blind Method; Female; Fumarates; Humans; Male; Pilot Projects; Psoriasis; Random Allocation

3-Aminopropionic acid (3-APA) has wide applications in food, cosmetics, pharmaceuticals, chemical, and polymer industries. This present study aimed to develop an eco-friendly whole-cell biocatalytic process for the bio-production of 3-APA from fumaric acid (FA) using Bacillus megaterium. A dual-enzyme cascade route with aspartate-1-decarboxylases (ADC) from Bacillus subtilis and native aspartate ammonia-lyase (AspA) was developed. Divergent catalytic efficiencies between these two enzymes led to an imbalance between both enzyme reactions. In order to coordinate AspA and ADC expression levels, gene mining, optimization, and duplication strategies were employed. Additionally, culture cultivation conditions and biocatalysis process parameters were optimized. A maximum 3-APA titer was obtained (11.68 ± 0.26 g/L) with a yield of 0.78 g/g under the following optimal conditions: 45 °C, pH 6.0, and 15 g/L FA. This study established a biocatalysis process for the production of 3-APA from FA using the whole cells of the recombinant B. megaterium.

Topics: Aspartate Ammonia-Lyase; Bacillus megaterium; beta-Alanine; Escherichia coli; Fumarates

Fumaric, malic, and succinic acids have been selectively separated from their mixture obtained by Rhizopus oryzae fermentation using reactive extraction with Amberlite LA-2 dissolved in three solvents with different dielectric constants (n-heptane, n-butyl acetate, and dichloromethane). This technique allows recovering preferentially fumaric acid from the mixture, the raffinate containing only malic and succinic acids. The extractant concentration and organic phase polarity control the efficiency and selectivity of acids extraction. The increase of aqueous phase viscosity reduces the extraction yield for all studied acids, but exhibits a positively effect on separation selectivity. By using Amberlite LA-2 concentration equal to that stoichiometrically required for interfacial reaction with fumaric acid and mixing intensity which does not allow higher diffusion rates for larger molecules (malic and succinic acids), the maximum value of fumaric acid extraction rate exceeds 90%, while the selectivity factor value becomes 20. Regardless of the extraction system, the complete separation of fumaric acid from their mixture is possible by multi-stage extraction process, adjusting the extractant concentration in each stage. At higher values of aqueous phase viscosity, more extraction stages are required, while the increase of solvent polarity reduce the required number of stages for total recovery of fumaric acid.

Topics: Acetates; Amines; Chemical Fractionation; Dicarboxylic Acids; Fermentation; Fumarates; Heptanes; Liquid-Liquid Extraction; Methylene Chloride; Rhizopus oryzae; Solvents; Succinic Acid; Viscosity; Water

Pirfenidone (PFD) is the first pharmacological agent approved by the US Food and Drug Administration (FDA) in 2014 for the treatment of idiopathic pulmonary fibrosis (IPF). The recommended daily dosage of PFD in patients with IPF is very high (2403 mg/day) and must be mitigated through additives. In the present work, sustained-release (SR) formulations of the PFD-FA cocrystal of two different strengths such as 200 and 600 mg were prepared and its comparative bioavailability in healthy human volunteers was studied against the reference formulation PIRFENEX (200 mg). A single-dose pharmacokinetic study (200 mg IR

Topics: Administration, Oral; Area Under Curve; Biological Availability; Cross-Over Studies; Delayed-Action Preparations; Fumarates; Healthy Volunteers; Humans; Pyridones; Solubility; Tablets; Therapeutic Equivalency

The base-controlled dearomative [3 + 2] cycloaddition reaction between 3-nitroindoles and fumaric acid amide esters has been disclosed by using the dearomatization and aromatization strategy. Three kinds of diverse functionalized pyrrolo[2,3-

Topics: Amides; Cycloaddition Reaction; Esters; Fumarates; Indoles

The control of cyanobacteria blooms is a global challenge. Here, we reported the efficient inhibition of M. aeruginosa by fumaric acid (FA), an intermediate metabolite of the tricarboxylic acid cycle. FA showed strong algicidal activity with an inhibition rate of 90.5% on the 8th day at a dose of 40 mg/L. The presence of FA caused severe membrane damage, as suggested by the fluorescence flow cytometry and morphology analysis. FA inhibited the formation of chlorophyll a, interrupting the photosynthesis system. It also induced oxidative stress in cells. Principal component analysis of the indicators suggested that the FA-treated sample had a significantly different inhibitory pattern than the acid-treated sample. Thus, the inhibitory effect was not solely caused by the pH effect. Untargeted metabolomic analysis revealed that 31 metabolites were differentially expressed in response to FA stress, which were mainly involved in the metabolite processes and the membranes. A commercial food-grade FA was able to inhibit the growth of M. aeruginosa similar to the analytical-grade FA. Our results suggest that FA can be potentially an efficient and low-risk chemical for inhibiting M. aeruginosa growth, which may find future applications in cyanobacteria bloom control.

Topics: Chlorophyll A; Cyanobacteria; Fumarates; Microcystis; Pseudomonas aeruginosa

While performing surgical treatment of the localized form of renal cell cancer by means of open or laparoscopic partial nephrectomy, renal warm ischemia is an important issue. Using renal warm ischemia allows to prevent parenchymal bleeding, to optimize conditions for resection of the tumor and to increase significantly the efficiency of hemostasis. However, an important problem is the probability of ischemic hypoxic damage of the remaining part of the kidney tissue during renal warm ischemia and renal functional impairment in the postoperative period.. To compare nephroprotective activity of sodium fumarate, mannitol and furosemide using experimental model of 30- and 60-minute renal warm ischemia in rabbits.. The experiments were carried out on 360 conventional male-rabbits of the "Chinchilla" breed weighed 2,6+/-0,3 kg which were allocated into 10 groups. The control group No1 included intact animals, the control group No2 included the rabbits in which renal artery was not clamped. For the animals from the trial groups (No3-No10) the experimental model of 30- and 60-minute renal warm ischemia was used. In groups No3 and No4 no drugs were provided. Other rabbits undergone renal warm ischemia with a protection by sodium fumarate (groups No5 and No6 - 1,5 ml/kg IV), lasix (groups No7 and No8 - 3,0 mg/kg IV) and mannitol (No9 and No10 - 1,0 g/kg IV). The influence of renal warm ischemia on the renal tissue ultrastructure and the levels of NGAL, Cystatin-C and creatinine in blood and urine were studied.. During experimental pharmacologically uncorrected 30-minute renal warm ischemia in animals, edema of the terminal part of microvilli of the proximal tubules epithelium, an increase of lysosome number in the hyaloplasm of epithelial cells, appearance of flaky content of medium electronic density in the lumens of distal tubules and collecting tubules, as well as sharp peak-like increase of NGAL and cystatin-C in blood and urine were observed. Increasing the time of ischemia up to 60 minutes was accompanied by more severe disturbances. In groups where sodium fumarate, lasix and mannitol were used the observed ultrastructural disturbances were expressed to lesser extent, whereas sodium fumarate demonstrated the best nephroprotective activity. After using mannitol the severity of disturbances was less than in the groups where mannitol, lasix or sodium fumarate were not given. Lasix and sodium salt of fumaric acid showed a higher nephroprotective activity. The best results were received in the animals received sodium fumarate.. The studied drugs provided a nephroprotective effect regarding ischemia of rabbit kidney. The effect of sodium fumarate was the most pronounced, followed by furosemide and, to a lesser extent, mannitol. Use of sodium fumarate allows to protect and stimulate the kidney tissue effectively during oxygen deprivation under ischemic state.

Topics: Animals; Female; Fumarates; Furosemide; Humans; Ischemia; Kidney; Kidney Neoplasms; Lipocalin-2; Male; Mannitol; Rabbits; Warm Ischemia

Two particular fumaric acid bridging lanthanide-encapsulated selenotungstates [H

Topics: Anions; Crystallography, X-Ray; Electrochemical Techniques; Fumarates; Lanthanoid Series Elements; Ligands; Models, Molecular; Mycotoxins; Polyelectrolytes; Reproducibility of Results

Self-propelled droplets are of considerable interest as an appropriate model for understanding the self-propulsion of objects in the fields of nonequilibrium physics and nonlinear science. Several research groups have reported the monodirectional motion of droplets, that is, chemotaxis, using stimuli-responsive materials. However, the precise control of chemotaxis remains challenging from the perspective of synthetic chemistry because chemotactic motion is primarily induced by the consumption of reactive oil or surfactants. Herein, we report a chemical system containing pH-responsive fumaric acid derivatives, in which the oil droplet exhibited positive chemotaxis over a wide pH range-from basic to acidic conditions. From the measurements of the interfacial tension between the oil and aqueous phases, it was deduced that the positive chemotaxis was due to heterogeneity in the interfacial tension of the droplet surface, which was accompanied by the production of surface-active compounds in the pH gradient in a linear-type channel.

Topics: Fumarates; Hydrogen-Ion Concentration; Motion; Surface Tension; Surface-Active Agents

Carbon nitride (CN) has a wide range of applications in photocatalytic treatment of environmental pollution. One of key challenges in the field is to conveniently prepare CN with tunable band gap towards efficient pollution degradation, which can be overcome by regulating the polymerization degree of its heptazines. Herein, a facile and green strategy to construct CN through co-firing urea, melamine and fumaric acid was reported. By simply inducing appropriate amount of fumaric acid during amidation reaction between fumaric acid and amino groups, the distance between heptazines of CN could be modified to obtain optimized polymerization degree and morphology. Among the considered CN systems, the modulated CN sample with the doped ratio of 2.50: 0.50: 0.03 m urea/m melamine/m fumaric acid (CNF30) displayed remarkable photocatalytic ability due to the largest specific surface area, the lowest photoluminescence emission intensity, and narrowest band gap, which led to the highest 98.0% methyl orange degradation within 60 min under a 10 W lamp and room temperature with the harmless and valuable carboxylic acids products. This study provides a new sight for the design of photocatalysts with tunable band structure towards green and efficient photocatalytic degradation of environmental pollution.

Topics: Catalysis; Fumarates; Heterocyclic Compounds, 3-Ring; Nitriles; Polymerization; Triazines; Urea

The objective of the study was to evaluate the physicochemical characteristics and shelf-life of corn tortilla stored at room temperature (25 °C) using a. Control, calcium hydroxide, calcium hydroxide + fumaric acid, calcium hydroxide + glycerol, calcium hydroxide + propylene glycol, and calcium hydroxide + sodium benzoate treatments had rounded half-lives of 1, 2, 1, 2, 2, and 2 days respectively. Glycerol combined with calcium hydroxide resulted in tortillas with lower pH variations over time. Mold presence was the critical attribute causing tortilla rejection.. The use of a

Topics: Bread; Calcium Hydroxide; Flour; Food Handling; Food Preservation; Food Preservatives; Food Storage; Fumarates; Humans; Sodium Benzoate; Taste; Zea mays

Improvement of astaxanthin yield is a continuing objective in Haematococcus pluvialis cultivation. In this study, a new strategy combining exogenous sodium fumarate (SF) with nitrogen deficiency was demonstrated to promote astaxanthin accumulation in H. pluvialis. SF significantly accelerated astaxanthin synthesis over cell growth. Notably, under nitrogen deficiency, 10 mM SF increased the cellular astaxanthin content by 1.75-fold and 1.47-fold on day 7 and 12, respectively. Further studies indicated that SF increased the level of respiratory metabolites in the Embden-Meyerhof-Parnas pathway, tricarboxylic acid cycle and pentose phosphate pathway. An enhanced substrate pool due to the acceleration of respiratory metabolic pathways directly induced astaxanthin synthesis. Meanwhile, the accumulation of C18:1, C18:2 and C18:3 fatty acids enhanced astaxanthin synthesis indirectly by promoting the process of esterification. This study not only helps to elucidate the role of respiratory metabolic pathways in astaxanthin synthesis but also provides a new effective technique to improve astaxanthin production.

Topics: Chlorophyceae; Fumarates; Metabolic Networks and Pathways; Xanthophylls

The tricarboxylic acid (TCA) cycle is one of the most important pathways of energy metabolism, and the profiles of its components are influenced by factors such as diseases and diets. Therefore, the differences in metabolic profile of TCA cycle between healthy and cancer cells have been the focus of studies to understand pathological conditions. In this study, we developed a quantitative method to measure TCA cycle metabolites using LC-MS/MS to obtain useful metabolic profiles for development of diagnostic and therapeutic methods for cancer. We successfully analyzed 11 TCA cycle metabolites by LC MS/MS with high reproducibility by using a PFP column with 0.5% formic acid as a mobile phase. Next, we analyzed the concentration of TCA cycle metabolites in human cell lines (HaCaT: normal skin keratinocytes; A431: skin squamous carcinoma cells; SW480: colorectal cancer cells). We observed reduced concentration of succinate and increased concentration of citrate, 2-hydroxyglutarate, and glutamine in A431 cells as compared with HaCaT cells. On the other hand, decreased concentration of isocitrate, fumarate, and α-ketoglutarate and increased concentration of malate, glutamine, and glutamate in A431 cells were observed in comparison with SW480 cells. These findings suggested the possibility of identifying disease-specific metabolites and/or organ-specific metabolites by using this targeted metabolomic analysis.

Topics: Cell Line, Tumor; Cells, Cultured; Chromatography, Liquid; Citric Acid Cycle; Energy Metabolism; Fumarates; Humans; Isocitrates; Ketoglutaric Acids; Malates; Metabolome; Metabolomics; Neoplasms; Reproducibility of Results; Tandem Mass Spectrometry

Experimental studies on the reactive extraction of fumaric acid with Amberlite LA-2 from

Topics: Fermentation; Fumarates; Rhizopus oryzae

The chicken eggshells and their subcrustal membranes are a valuable source of calcium, but they are not further processed but disposed of as waste from the food industry. Chicken eggshells have high content (>95%) of calcium carbonate. Some properties suggest that eggshells may be a promising alternative to the present calcium sources used in the pharmaceutical industry.. The effect of roasting chicken eggshells with a selected organic acid (citric or fumaric or lactic acid) on microbiological purity, including the presence of fungi and bacteria Salmonella spp., Staphylococcus aureus, Escherichia coli of obtained calcium salts, was investigated. In this study, chicken eggshells were subjected to chemical reactions with organic acids (citric, fumaric or lactic acid) at two different calcium-acid molar ratios (1:1 or 1:3) and the mixture was heat-treated for 1 or 3 hours at a temperature of 100°C or 120°C.. It was found that lactic acid was 100% effective against fungi, and the remaining citric and fumaric acids were -50% (regardless of the other examined conditions). The type of acid used has a significant effect on fungal growth inhibition (p<0.05). Fumaric acid and lactic acid will be nearly 100% effective against bacteria (100% fumaric acid and 97% lactic acid effectiveness), regardless of other factors.. Lactic acid is the most effective against pathogenic flora - fungi and bacteria. The transformation of chicken eggshells into calcium lactate can provide us with sterile calcium salt, free of 100% fungi and 97% of all bacteria.

Topics: Animals; Bacterial Physiological Phenomena; Calcium; Calcium Compounds; Chickens; Citric Acid; Egg Shell; Fumarates; Fungi; Lactic Acid; Salts

Fumaric acid production from the fermentation process by Rhizopus was considered a potential method. But poor conversion efficiency and low space-time productivity greatly hampered industrial production. Here, we reported improving these problems through carbon-nitrogen sources coordination optimization strategy. Five commonly used nitrogen sources were selected to conduct element analysis and fermentation efficiency comparison. Casein was proven to be the optimum nitrogen source and further investigated in a stirred-tank reactor. It showed that the fermentation cycle was significantly shortened by the application of casein. Combined with optimization of glucose content, the space-time productivity of fumaric acid reached 0.76 g/L h with a yield to 0.31 g/g glucose, which was the highest among the results gotten in the stirred-tank reactor. It illustrated that carbon-nitrogen sources coordination optimization strategy was in favor of the improvement of the fermentation process and laid a promising foundation for the development of fumaric acid industrial production.

Topics: Fumarates; Glucose; Rhizopus oryzae

We describe a method for the analysis of organic acids, including those of the tricarboxylic acid cycle (TCA cycle), by mixed-mode reversed-phase chromatography, on a CSH Phenyl-Hexyl column, to accomplish mixed-mode anion-exchange separations, which results in increased retention for acids without the need for ion-pairing reagents or other mobile phase additives. The developed method exhibited good retention time reproducibility for over 650 injections or more than 5 days of continuous operation. Additionally, it showed excellent resolution of the critical pairs, isocitric acid and citric acid as well as malic acid and fumaric acid, among others. The use of hybrid organic-inorganic surface technology incorporated into the hardware of the column not only improved the mass spectral quality and subsequent database match scoring but also increased the recovery of the analytes, showing particular benefit for low concentrations of phosphorylated species. The method was applied to the comparative metabolomic analysis of urine samples from healthy controls and breast cancer positive subjects. Unsupervised PCA analysis showed distinct grouping of samples from healthy and diseased subjects, with excellent reproducibility of respective injection clusters. Finally, abundance plots of selected analytes from the tricarboxylic acid cycle revealed differences between healthy control and disease groups.

Topics: Body Fluids; Chromatography, High Pressure Liquid; Citric Acid; Citric Acid Cycle; Fumarates; Humans; Isocitrates; Malates; Mass Spectrometry; Molecular Structure

Significant advances have been made in unknown metabolite identification and expansion of the number of quantifiable metabolites in human plasma, serum, and whole blood using NMR spectroscopy. However, reliable quantitation of metabolites is still a challenge. A major bottleneck is the lack of a suitable internal standard that does not interact with the complex blood sample matrix and also does not overlap with metabolite peaks apart from exhibiting other favorable characteristics. With the goal of addressing this challenge, a comprehensive investigation of fumaric and maleic acids as potential internal standards was made along with a comparison with the conventional standards, TSP (trimethylsilylpropionic acid) and DSS (trimethylsilylpropanesulfonic acid). Both fumaric acid and maleic acid exhibited a surprisingly high performance with a quantitation error <1%, while the TSP and DSS caused an average error of up to 35% in plasma, serum, and whole blood. Further, the results indicate that while fumaric acid is a robust standard for all three biospecimens, maleic acid is suitable for only plasma and serum. Maleic acid is not suited for the analysis of whole blood due to its overlap with coenzyme peaks. These findings provide new opportunities for improved and accurate quantitation of metabolites in human plasma, serum, and whole blood using NMR spectroscopy. Moreover, the use of protein precipitation prior to NMR analysis mirrors the sample preparation commonly used for mass spectrometry based metabolomics, such that these findings further strengthen efforts to combine and compare NMR and MS based metabolite data of human plasma, serum, and whole blood for metabolomics based research.

Topics: Fumarates; Humans; Magnetic Resonance Spectroscopy; Maleates; Metabolomics; Plasma; Serum

Naftopidil (NAF), an α

Topics: Chitosan; Fumarates; Naphthalenes; Piperazines; Solubility

Red mature calyces of

Topics: Acetic Acid; Betaine; Citrates; Correlation of Data; Desiccation; Flowers; Fumarates; gamma-Aminobutyric Acid; Hibiscus; India; Lipids; Magnetic Resonance Spectroscopy; Metabolome; Metabolomics; Methanol; Plant Extracts; Principal Component Analysis; Succinic Acid; Sugars

The current work investigates the impact of using immobilized Rhizopus oryzae NRRL 1526 for bioproduction of fumaric acid using agro-industrial residues as feedstock. This use of agro-industrial residues, a renewable feedstock, for the production of bio-based platform chemical makes the process cost-competitive as well as greener by preventing the release of assimilable organic carbon to the environment, thereby reducing the generation of greenhouse gases. Immobilization of R. oryzae has been proposed previously to alleviate operational difficulties confronted during free mycelial fungal fermentation. To this effect, three synthetic refuse materials namely polystyrene foam, polyester sponge and polyurethane foam were investigated for their suitability towards fumaric acid bioproduction. Polystyrene foam was identified as the most suitable support material for immobilization as well as fumaric acid production. In addition to the considerable reduction in the lag-phase (from 48 to 24 h) the reduction in the size of the support material from cubes of 1 cm to beads of 0.1-0.3 cm led to a 42% improvement in fumaric acid production (27 g/L against 19 g/L). Growing the polystyrene foam bead immobilized R. oryzae on apple pomace ultrafiltration sludge as sole feedstock yielded a final fumaric acid titer of 7.9 g/L whereas free mycelial fermentation yielded 6.3 g/L. Moreover, upon operating the fermentation with intermittent feeding, a three-fold increase (1.7 g/L to 5.1 g/L) in fumaric acid production was obtained upon supplementation of the apple pomace sludge media with molasses, an agro-industrial residue, as feed.

Topics: Fermentation; Fumarates; Rhizopus; Rhizopus oryzae

Class-II fumarases (fumarate hydratase, FH) are dual-targeted enzymes occurring in the mitochondria and cytosol of all eukaryotes. They are essential components in the DNA damage response (DDR) and, more specifically, protect cells from DNA double-strand breaks. Similarly, the gram-positive bacterium

Topics: AlkB Enzymes; Bacillus subtilis; Bacterial Proteins; Citric Acid Cycle; DNA Breaks, Double-Stranded; DNA Damage; DNA, Bacterial; Escherichia coli; Fumarate Hydratase; Fumarates; Genes, Bacterial; Ketoglutaric Acids

Yogurt is traditionally fermented by a symbiotic starter culture of Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus. These bacteria exchange metabolites with each other to meet their nutritional demands during protocooperation, resulting in a shorter fermentation time. In this study, we investigated whether fumaric acid functions as a symbiotic agent to promote the growth of Lb. bulgaricus by evaluating 8 strains of Lb. bulgaricus and 7 strains of Strep. thermophilus. All the tested Lb. bulgaricus strains metabolized the added fumaric acid into succinic acid during monoculture in milk, and 6 strains (75%) showed shorter fermentation time compared with the control. The addition of malic acid showed similar trends as that of fumaric acid, indicating that the reverse tricarboxylic acid cycle was functioning in Lb. bulgaricus. All 7 Strep. thermophilus strains tested produced fumaric acid during monoculture in milk. Further, in Lb. bulgaricus 2038, the gene expression of fumarate reductase that converts fumaric acid to succinic acid, was higher in the coculture with Strep. thermophilus 1131 than in the monoculture. These findings indicate that fumaric acid produced by Strep. thermophilus can function as a symbiotic substance during yogurt fermentation to stimulate the growth of Lb. bulgaricus.

Topics: Animals; Fermentation; Fumarates; Lactobacillus delbrueckii; Streptococcus thermophilus; Yogurt

Orally dosed drugs must dissolve in the gastrointestinal (GI) tract before being absorbed through the epithelial cell membrane.

Topics: Administration, Oral; Betaine; Biological Availability; Chemistry, Pharmaceutical; Computer Simulation; Drug Design; Drug Liberation; Excipients; Fumarates; Gastrointestinal Absorption; Humans; Hydrogen-Ion Concentration; Models, Biological; Solubility; Tartrates

Several therapies are available for psoriasis, including in some countries oral fumaric acid derivatives (FADs). Even if FADs are not available in the Italian market, they can be prescribed and reimbursed by the National Health Service, on request from the treating physician, when considered as a valuable option in selected patient.. We performed a retrospective analysis of the PsoReal registry data, restricted to adult psoriatic patients enrolled between 2009 and 2017. Demographic and clinical data were collected together with information on systemic therapies prescribed for psoriasis, drug shifts and adverse effects. We focused our analysis on FADs compared with other systemic drugs.. From the registry data, a total of 17,064 patients were extracted, and 11,592 patients (67.9%), fulfilled inclusion criteria. The majority of them had chronic plaque psoriasis, the mean disease duration was 17.1±12.6 years, and the mean PASI was 17.8±10.9, with 51.5% presenting a moderate Ps (PASI between 10 and 20). A total of 36 patients (0.3%) were treated by FADs. The average treatment duration of conventional (9.0±10.0 months) and biological agents (13.7±11.6 months) was lower compared to the duration of FADs (28.1±20.1, P value<0.001). FADs were used at an average dosage of 361.0±146.3 mg/day and FADs treated patients displayed an overall lower healthcare cost compared with other drugs.. The current study confirms previous European data about efficacy and safety of FADs and suggests a decrease of healthcare costs for FADs treated patients as compared to other treatments.

Topics: Adult; Fumarates; Humans; Italy; Psoriasis; Retrospective Studies; State Medicine

ST segment elevation myocardial infarction (STEMI) is one of the most common global causes of cardiovascular disease-related death. Several metabolites may change during STEMI. Hence, analysis of metabolites in body fluid may be considered as a rapid and accurate test for initial diagnosis. This study has therefore attempted to determine the variation in metabolites identified in the serum of STEMI patients (n = 20) and 15 controls. Samples collected from the Cardiology Department, Medical Faculty, Ataturk University, were extracted by liquid-liquid extraction and analysed using liquid chromatography quadrupole time-of-flight mass spectrometry. The METLIN database was used for the identification and characterization of metabolites. According to Q-TOF/MS measurements, 231 m/z values, which were significantly different between groups (P < 0.01 and fold analysis >1.5) were detected. Metabolite identification was achieved via the Human Metabolome database. According to the multivariate data analysis, leucine, isoleucine, l-proline, l-alanine, glycine, fumaric acid, citrate, succinate and carnitine levels were decreased, whereas levels of propionic acid, maleic acid, butyric acid, urea, oleic acid, palmitic acid, lysoPC [18:2(9Z)], glycerol, phoshpatidylethanolamine, caffeine and l-lactic acid were increased in STEMI patients compared with controls. In conclusion, malonic acid, maleic acid, fumaric acid and palmitic acid can be used as biomarkers for early risk stratification of patients with STEMI.

Topics: Amino Acids; Chromatography, Liquid; Female; Fumarates; Humans; Male; Maleates; Malonates; Mass Spectrometry; Metabolome; Metabolomics; Middle Aged; ST Elevation Myocardial Infarction

We evaluated the bactericidal efficacy of the simultaneous application of ultraviolet-A (UV-A) irradiation and fumaric acid (FA) against Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium, and Listeria monocytogenes in apple juice and as well as investigated the effects of this treatment on product quality. Further, we elucidated the mechanisms underlying their synergistic bactericidal action. Simultaneous UV-A light irradiation and 0.1% FA treatment for 30 min resulted in 6.65-, 6.27-, and 6.49-log CFU/ml reductions in E. coli O157:H7, S. Typhimurium, and L. monocytogenes, respectively, which involved 3.15, 2.21, and 3.43 log CFU reductions, respectively, and these were attributed to the synergistic action of the combined treatments. Mechanistic investigations suggested that the combined UVA-FA treatment resulted in significantly greater bacterial cell membrane damage and intracellular reactive oxygen species (ROS) generation. UVA-FA treatment for 30 min did not cause significant changes to the color, nonenzymatic browning index, pH, and total phenolic content of apple juice. These results suggest that combined UVA-FA treatment can be effectively used to control foodborne pathogens in apple juice without affecting its quality.

Topics: Anti-Bacterial Agents; Escherichia coli O157; Food Preservation; Fruit and Vegetable Juices; Fumarates; Listeria monocytogenes; Malus; Microbial Viability; Reactive Oxygen Species; Salmonella typhimurium; Ultraviolet Rays

The bioproduction of high-value chemicals such as itaconic and fumaric acids (IA and FA, respectively) from renewable resources via solid-state fermentation (SSF) represents an alternative to the current bioprocesses of submerged fermentation using refined sugars. Both acids are excellent platform chemicals with a wide range of applications in different market, such as plastics, coating, or cosmetics. The use of lignocellulosic biomass instead of food resources (starch or grains) in the frame of a sustainable development for IA and FA bioproduction is of prime importance. Filamentous fungi, especially belonging to the

Topics: Aspergillus oryzae; Biomass; Bioreactors; Biotechnology; Fermentation; Fumarates; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Lignin; Succinates

Agrobacterium tumefaciens strain SCUEC1 is a nicotine-degrading bacterium, which has been recently isolated from the tobacco waste-contaminated field soil. However, the mechanism for nicotine degradation in this strain remains unclear. Here, we analyze the function and biological properties of the agnH gene in the strain SCUEC1. The overexpression of the AgnH protein was detected by SDS-PAGE analysis, and functional insight of the AgnH protein was carried out with monitoring the changes of maleic acid into fumaric acid by high performance liquid chromatography (HPLC). Moreover, the effects of temperature, pH and metal ions on the enzymatic activities of the AgnH protein were also analyzed. The results demonstrated that the agnH gene was successfully ligated to the plasmid pET28a. The optimal condition for the enzymatic activities for the AgnH, approximately 28.0 kDa, was determined as 37 °C, pH 8.0 and 25 µM Mg2+. Conclusively, the agnH gene fulfils an important role in the conversion of maleic acid into fumaric acid involved in nicotine-degradation pathways in Agrobacterium tumefaciens strain SCUEC1.

Topics: Agrobacterium tumefaciens; Bacterial Proteins; Fumarates; Hydrogen-Ion Concentration; Maleates; Nicotine; Temperature

Wound and its treatment is one of the major health concerns throughout the globe. Various extrinsic and intrinsic factors can influence the dynamics of healing mechanism. One such extrinsic factor is moist environment in wound healing. The advantages of optimum hydration in wound healing are enhanced autolytic debridement, angiogenesis and accelerated cell proliferation and collagen formation. But hydrated wounds often end up with patient's uncomfortability, associated infection, and tissue lipid peroxidation. Healing process prefers antimicrobial, anti-inflammatory and optimum moist microenvironment. Here, we have synthesized fumaric acid incorporated agar-silver hydrogel (AA-Ag-FA); characterized by UV-Visible spectroscopy, FTIR spectroscopy and TEM. The surface morphology is evaluated through SEM. The size of the silver nanoparticles (Ag NPs) was found to be 10-15 nm. The hydrogel shows potential antibacterial effect against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa which are predominantly responsible for wound infection. The gel shows reasonable antioxidant property evaluated through 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Topical application of the gel on the wound site heals the wound at much faster rate even compared to standard (Mega heal, Composition: Colloidal silver 32 ppm hydrogel) gel. Histological analysis reveals better tissue proliferation (i.e. epithelialization), more granulation tissue formation, neovascularisation, fibroblast and mature collagen bundles. The lipid peroxidation of wound tissue estimated through malondialdehyde (MDA) assay was found to be reasonably less when treated with AA-Ag-FA hydrogel compared to standard (Mega heal). Cytotoxicity of the samples tested through MTT assay and live-dead cell staining shows its nontoxic biocompatibility nature. In our hydrogel scaffold, the bio-degradable agar-agar provides the moist environment; the Ag NPs inside the gel acts as bactericidal agent and fumaric acid facilities the antioxidant and angiogenesis path implicitly.

Topics: Agar; Animals; Anti-Bacterial Agents; Antioxidants; Apoptosis; Biocompatible Materials; Cell Line; Fumarates; Humans; Hydrogels; Male; Metal Nanoparticles; Mice; Neovascularization, Physiologic; Pseudomonas aeruginosa; Rats; Rats, Wistar; Silver; Staphylococcus aureus; Wound Healing

Carbamazepine cocrystals with nicotinamide, saccharin and fumaric acid were synthesized and characterized by time-domain terahertz spectroscopy. Lattice vibrations of cocrystals with their individual constituents were investigated by means of the dispersion-corrected density functional theory with and without cell parameter constraints. The simulated THz spectra successfully reproduce the features of all the crystals in their experimental spectra. A better agreement between experimental and theoretical THz spectra is achieved when the cell parameter constraints are applied in geometry optimization. Some intensive modes of neat carbamazepine and cocrystals were discussed in terms of the motions of hydrogen bonds. The effect of lattice vibration on these cocrystallizations was further examined to gain insights into the thermodynamics. It is found that lattice vibration is favorable for all these cocrystal formations.

Topics: Carbamazepine; Crystallization; Density Functional Theory; Fumarates; Hydrogen Bonding; Niacinamide; Powders; Saccharin; Terahertz Spectroscopy; Thermodynamics; Vibration; X-Ray Diffraction

Topics: Animals; Anti-Inflammatory Agents; Antioxidants; Cadmium; Chemical and Drug Induced Liver Injury; Female; Fumarates; Liver; Male; Oxidative Stress; Rats; Rats, Wistar

β-Alanine (β-Ala) is an important intermediate with numerous applications in food and feed additives, pharmaceuticals, polymeric materials, and electroplating industries. Its biological production routes that employ L-aspartate-α-decarboxylase (ADC) as the key enzyme are attractive. In this study, we developed an efficient and environmentally safe method for β-Ala production by co-expressing two different subtypes of ADC. A bacterial ADC from Bacillus subtilis (BSADC) and an insect ADC from Tribolium castaneum (TCADC) use pyruvoyl and pyridoxal-5'-phosphate (PLP) as cofactor, respectively. 3050 mM (271.5 g/L) β-Ala was achieved from L-aspartic acid by using the whole-cell biocatalyst co-expressing BSADC and TCADC, corresponding to a conversion rate of 92.4%. Meanwhile, one-pot synthesis of β-Ala from fumaric acid through using a tri-enzyme cascade route with two different subtypes of ADC and L-aspartase (AspA) from Escherichia coli was established. 2250 mM (200.3 g/L) β-Ala was obtained from fumaric acid with a conversion rate of 90.0%. This work proposes a novel strategy that improves β-Ala production in the decarboxylation pathway of L-aspartic acid.

Topics: Animals; Aspartate Ammonia-Lyase; Aspartic Acid; Bacillus subtilis; beta-Alanine; Biotechnology; Biotransformation; Carboxy-Lyases; Catalysis; Culture Media; Escherichia coli; Fumarates; Glutamate Decarboxylase; Hydrogen-Ion Concentration; Temperature; Tribolium

Limited knowledge is currently available on the biochemical basis for the development of dark-cutting beef. The objective of this research was to determine the metabolite profile and mitochondrial content differences between normal-pH and dark-cutting beef. A gas chromatography-mass spectrometer-based nontargeted metabolomic approach indicated downregulation of glycolytic metabolites, including glucose-1- and 6-phosphate and upregulation of tricarboxylic substrates such as malic and fumaric acids occurred in dark-cutting beef when compared to normal-pH beef. Neurotransmitters such as 4-aminobutyric acid and succinate semialdehyde were upregulated in dark-cutting beef than normal-pH beef. Immunohistochemistry indicated a more oxidative fiber type in dark-cutting beef than normal-pH beef. In support, the mitochondrial protein and DNA content were greater in dark-cutting beef. This increased mitochondrial content, in part, could influence oxygen consumption and myoglobin oxygenation/appearance of dark-cutting beef. The current results demonstrate that the more tricarboxylic metabolites and mitochondrial content in dark-cutting beef impact muscle pH and color.

Topics: Animals; Cattle; Color; Fumarates; Glucosephosphates; Hydrogen-Ion Concentration; Malates; Meat; Mitochondria; Muscle, Skeletal; Myoglobin; Oxidation-Reduction

Food waste is considered a serious global societal problem. How to degrade of food waste in a green and effective way has been to a hot topic. In this work, a method with hot water extraction pretreatment of food waste was investigated and optimized. Under the optimal conditions, more than half of the solid food waste could be transferred to soluble sugars. Meanwhile, in order to improve the tolerance of Rhizopus arrhizus on the food waste hydrolysate, UV combined with chemical mutagenesis were carried out, and a mutant of Rhizopus RH-7-13-807 was obtained. With the mutant strain, the yield of fumaric acid fermented from food waste increased to 1.8 times compared with the original strain, and 23.94 g/L fumaric acid was obtained from the fermentation. Besides, the COD of food waste was evaluated for the degradation of food waste by the Rhizopus RH-7-13-807. The process would decrease the quantity of food waste to be disposed of, and benefit the environment.

Topics: Fermentation; Food; Fumarates; Hydrolysis; Refuse Disposal; Rhizopus

This study investigated the effects of fumaric acid on push-out bond strength when applied to dentin surfaces and fiber posts. The root canals of 60 mandibular premolar teeth were instrumented and obturated. After removing two thirds of filling material, teeth were prepared according to six randomized groups (n = 10/group) defined by two fiber post surface treatments (0.7% fumaric acid or 9% hydrofluoric acid) and three dentin conditioning treatments [control (no conditioning); 17% ethylenediamine tetraacetic acid (EDTA); or 0.7% fumaric acid]. After fiber post-cementation, three 1-mm thick discs were obtained from each tooth by transverse sectioning, and each disc underwent push-out bond strength testing. Data were analyzed with a one-way analyses of variance (anova) and t tests; p < .05 was considered statistically significant. Failure modes were determined by stereomicroscopy, and the surface characteristics of dentin and fiber posts were observed by scanning electron microscopy. Push-out bond strength was greater for the group in which the post surface treated with hydrofluoric acid and the dentin surface treated with fumaric acid than the nontreated dentin and hydrofluoric acid-treated post group (p < .05). There were no significant differences between other comparison pairs (p > .05). A combination of fumaric acid dentin conditioning and hydrofluoric acid fiber post treatment strengthened the bonding ability of fiber posts.

Topics: Dental Bonding; Dental Pulp Cavity; Dentin; Fumarates; Humans; Materials Testing; Post and Core Technique; Resin Cements

Topical administration is a promising clinical strategy to avoid serious gastrointestinal adverse reactions of loxoprofen sodium (LOX), a new non-steroidal anti-inflammatory drug. Small molecule organic acids had been reported with the ability of promoting transdermal rate of several drugs. In this article, the effect of small molecule organic acids on the transdermal delivery of LOX was studied, and the possible mechanism was also explored by Fourier infrared spectroscopy, differential scanning calorimetry, tape stripping, etc. The results showed that lactic acid and fumaric acid could significantly increase the penetration rate of LOX and reduce time lag even without the help of acidic environment. The preliminary mechanism investigation inferred that fumaric acid could increase LOX's distribution in stratum corneum and might change its complexation state, but had little effect on the drug structure and skin's lipids and proteins configuration. The topical LOX gel using fumaric acid as penetration enhancer had higher transdermal rate, significant anti-inflammatory effect and no obvious skin irritation. This study proved the promising application of small molecule organic acids in transdermal enhancing and provided a potential strategy for transdermal delivery of LOX combined with fumaric acid.

Topics: Administration, Cutaneous; Animals; Anti-Inflammatory Agents, Non-Steroidal; Carrageenan; Disease Models, Animal; Female; Fumarates; Gels; Inflammation; Lactic Acid; Male; Phenylpropionates; Rabbits; Rats, Sprague-Dawley; Skin; Skin Absorption; Swine; Swine, Miniature; Time Factors

Hepatocellular carcinoma (HCC) is among the leading causes of cancer-related mortality worldwide. Our previous study identified a novel alternative splicing variant of prenyl diphosphate synthase subunit 2 (PDSS2) in HCC characterized by a deletion of exon 2, named PDSS2-Del2, which is devoid of the tumor-suppressive function of full-length PDSS2 (PDSS2-FL). To better understand the clinical significance of PDSS2-Del2, we performed a BaseScope™ assay on an HCC tissue microarray and found that positive staining for PDSS2-Del2 predicted a worse overall survival in patients with HCC (P = 0.02). PDSS2-Del2 levels correlated significantly with microvessel counts in HCC tumor tissues. Importantly, PDSS2-Del2 overexpression functionally promoted HCC metastasis, as demonstrated by in vitro and in vivo migration assays. In vivo assays also demonstrated that PDSS2-Del2 increased angiogenesis in xenografts. Furthermore, we discovered that elevated PDSS2-Del2 expression in HCC tumor cells decreased fumarate levels and activated the canonical nuclear factor-κB pathway. The epithelial-to-mesenchymal transition (EMT) and WNT/β-catenin signaling pathways were also activated by overexpression. Dimethyl fumarate (DMF), a fumaric acid ester, effectively reduced the metastasis induced by PDSS2-Del2 as observed with in vivo spleen-liver metastasis animal experiments. DMF is a prescribed oral therapy for multiple sclerosis and it might be a potential treatment for metastasis of patients with HCC. Early clinical trials are needed to validate its potential in this context.

Topics: Adult; Aged; Alkyl and Aryl Transferases; Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Dietary Supplements; Epithelial-Mesenchymal Transition; Female; Fumarates; Humans; Liver Neoplasms; Male; Mice, Inbred BALB C; Mice, Nude; Microvessels; Middle Aged; Neoplasm Metastasis; Neovascularization, Pathologic; NF-kappa B; Survival Analysis; Wnt Signaling Pathway; Young Adult

Topics: Adaptive Immunity; Adult; Antigen Presentation; Arsenic; Cells, Cultured; Cytokines; Dendritic Cells; Fumarates; Humans; Immunity, Innate; Inflammation; Male; Monocytes; Phagocytosis; Toll-Like Receptor 4; Up-Regulation; Young Adult

The purpose of this study was to evaluate the synergistic bactericidal efficacy of combining ultrasound (US) and fumaric acid (FA) treatment against Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes in apple juice and to identify the synergistic bactericidal mechanisms. Additionally, the effect of combination treatment on juice quality was determined by measuring the changes in color, pH, non-enzymatic browning index, and total phenolic content. A mixed cocktail of the three pathogens was inoculated into apple juice, followed by treatment with US (40 kHz) alone, FA (0.05, 0.1, and 0.15%) alone, and a combination of US and FA for 1, 2, 3, 4, and 5 min. Combined US and 0.15% FA treatment for 5 min achieved 5.67, 6.35, and 3.47 log reductions in E. coli O157:H7, S. Typhimurium, and L. monocytogenes, respectively, with the 1.55, 2.37, and 0.57 log CFU reductions attributed to the synergistic effect. Although the pH value slightly decreased as FA increased, there were no significant (P > 0.05) differences in color values, browning indices, and phenolic content between untreated and treated samples. To identify the mechanism of this synergistic bactericidal action, membrane integrity, malfunctions in the membrane efflux pump, and intracellular enzyme activity were measured. The analyses confirmed that damage to the cell envelope (membrane integrity and efflux pump) was strongly related to the synergistic microbial inactivation. These results suggest that simultaneous application of US treatment and FA is a novel method for ensuring the microbial safety of apple juice.

Topics: Anti-Bacterial Agents; Bacteria; Colony Count, Microbial; Escherichia coli O157; Food Microbiology; Food Preservation; Fruit and Vegetable Juices; Fumarates; Listeria monocytogenes; Malus; Microbial Viability; Salmonella typhimurium; Ultrasonic Waves

Application of Quercetin (Quer) as a natural flavonoid is confined because of limited solubility in water and stability in the body. Herein, we prepared a biodegradable super paramagnetic starch-based hydrogel grafted onto fumaric acid for increasing Quer bioavailability and controlling its release. The molecular structure of starch was modified by using fumaric acid to increase hydrophilicity of hydrogel, and iron oxide nanoparticles is used to strengthen its physical and mechanical properties. The structural, morphological and magnetic properties of the optimized hydrogel were characterized. Application of the synthesized hydrogel was assessed in the in vitro and in vivo studies. In vitro release curve was nicely fitted to the Higuchi model. Stability and bioavailability of Quer were significantly increased at the plasma and liver of rats which received 100 mg kg

Topics: Animals; Antioxidants; Biological Availability; Drug Carriers; Drug Liberation; Fumarates; Hydrogels; Magnetic Phenomena; Male; Microscopy, Electron, Scanning; Quercetin; Rats, Wistar; Solubility; Starch

Accumulation of succinate as a fermentation product of Fusobacterium varium was enhanced when the anaerobic bacterium was grown on complex peptone medium supplemented with fumarate. Residual substrates and fermentation products were determined by proton NMR spectroscopy. Cells collected from the fumarate-supplemented medium (8-10 h after inoculation) supported the conversion of fumarate to succinate when suspended with fumarate and a co-substrate (glucose, sorbitol, or glycerol). Succinate production was limited by the availability of fumarate or reducing equivalents supplied by catabolism of a co-substrate via the Embden-Meyerhof-Parnas (EMP) pathway. The choice of reducing co-substrate influenced the yield of acetate and lactate as side products. High conversions of fumarate to succinate were achieved over pH 6.6-8.2 and initial fumarate concentrations up to 300 mM. However, at high substrate concentrations, intracellular retention of succinate reduced extracellular yields. Overall, the efficient utilization of fumarate (≤ 400 mM) combined with the significant extracellular accumulation of succinate (corresponding to ≥ 70% conversion) indicated the effective utilization of fumarate as a terminal electron acceptor by F. varium and the potential of the methodology for the bioproduction of succinate.

Topics: Fumarates; Fusobacterium; Oxidation-Reduction; Succinic Acid

The dissolution behavior of a dibasic drug ketoconazole under the influence of pH has been evaluated and compared to its three 1:1 cocrystals with diacidic coformers, fumaric acid, succinic acid (SUC), and adipic acid. Mass transport models were developed by applying Fick's law of diffusion to dissolution with simultaneous chemical reactions in the hydrodynamic boundary layer adjacent to the dissolving surface to predict the interfacial pH and flux of the parent drug and cocrystals. All 3 cocrystals have the ability to modulate the interfacial pH to different extents compared to the parent drug due to the acidity of the coformers. Dissolution pH dependence of ketoconazole is significantly reduced by the cocrystallization with acidic coformers. Due to the different dissolution pH dependence, there exists a transition pH where the flux of the cocrystal is the same as the parent drug. Below this transition pH, the drug flux is higher, but above it, the cocrystal flux is higher. The development of these mass transport models provide a mechanistic understanding of the dissolution behavior and help identify cocrystalline solids with optimal dissolution characteristics.

Topics: Adipates; Crystallization; Drug Compounding; Fumarates; Hydrogen-Ion Concentration; Ketoconazole; Models, Chemical; Solubility; Succinic Acid; Surface Properties

This article presents the preparation of matrices from two new families of fumaric copolymers and the effect of structural differences on their physicochemical and biological behavior. Diisopropyl fumarate (DIPF) and poly(ethylene glycol) methyl ether methacrylate (OEGMA) or N-isopropylacrylamide (NIPAM) were copolymerized by conventional radical and RAFT polymerization to obtain lineal or start architectures, respectively. These copolymers were characterized by spectroscopic (FTIR and

Topics: Animals; Fumarates; Macrophages; Materials Testing; Methacrylates; Mice; Polyethylene Glycols; Porosity; RAW 264.7 Cells; Tissue Engineering; Tissue Scaffolds

Plaque psoriasis has significant impact on patients' quality of life. Topical therapy is considered the treatment mainstay for mild-to-moderate disease according to guidelines. Calcipotriol/betamethasone dipropionate (Cal/BD) [0.005%/0.05%] aerosol foam is indicated for psoriasis vulgaris treatment in adults. Cal/BD foam trials demonstrated improved efficacy and similar safety in this population. Psoriasis treatment is complicated by the broad range of disease presentation, variability and therapeutic options; particularly decisions on transition from topical to non-biologic systemic treatment are difficult. Assessing comparative effectiveness of treatment options provides meaningful value to treatment decisions.. To compare efficacy of Cal/BD foam individual patient data from pooled trials with efficacy of non-biologic systemic treatments based on aggregated patient characteristics and treatment outcomes.. Individual data from four Cal/BD foam trials in 749 psoriasis patients were pooled to conduct matching-adjusted indirect comparisons. Literature review identified non-biologic systemic treatment trials where methods, populations and outcomes align with Cal/BD foam trials. Of 3090 screened publications, four studies of apremilast, methotrexate, acitretin or fumaric acid esters (FAE) were included.. After baseline matching, patients treated with 4 weeks of Cal/BD foam had greater Physician's Global Assessment 0/1 response compared to those treated with 16 weeks of apremilast (52.7% vs. 30.4%; P < 0.001). Patients treated with Cal/BD foam had significantly greater Psoriasis Area and Severity Index (PASI) 75 response at Week 4 compared to 16 weeks of apremilast treatment (51.1% vs. 21.6%; P < 0.001). Cal/BD foam patients demonstrated significantly greater PASI 75 response improvements at Week 4 vs. 12 weeks of methotrexate (50.8% vs. 33.5%; P < 0.001) or acitretin (50.9% vs. 31.7%; P = 0.009), and comparable response to FAE (42.4% vs. 47.0%; P = 0.451).. Despite recent treatment advances, unmet needs for psoriasis patients remain. Cal/BD foam offers improved efficacy in baseline matched psoriasis patients compared to apremilast, methotrexate or acitretin, and comparable efficacy to FAE.

Topics: Acitretin; Administration, Cutaneous; Aerosols; Betamethasone; Calcitriol; Dermatologic Agents; Drug Therapy, Combination; Esters; Female; Fumarates; Humans; Male; Methotrexate; Middle Aged; Psoriasis; Thalidomide; Treatment Outcome

The aim of this study was to improve the dissolution rate and oral absorption of naftopidil (NAF) in rats via solid dispersion (SD) with a weak acid and copolymer. We hypothesized that the dissolution rate and oral bioavailability of NAF would increase through hydrogen bonding between NAF and weak acids/hydrophilic polymers. d‑α‑Tocopherol polyethylene glycol 1000 succinate (TPGS) and fumaric acid were selected as the hydrophilic polymer and weak acid based on their apparent solubility and pre-dissolution test results, respectively. The optimal formulation (SD5) comprised NAF: fumaric acid: TPGS: Aerosil® 200 at a ratio of 1:1:1:1. The dissolution rate of SD5 in distilled water and pH 1.2 media was significantly higher than that of a commercial product (Flivas®). The chemical interaction between NAF and fumaric acid was confirmed using attenuated total reflectance Fourier transform infrared spectroscopy. The SD5 formulation was stable for 12 months. The oral bioavailability and peak plasma concentration (C

Topics: Animals; Fumarates; Male; Naphthalenes; Piperazines; Rats; Rats, Sprague-Dawley; Solubility; Spectroscopy, Fourier Transform Infrared; X-Ray Diffraction

Dimethylfumarate (DMF) has been approved the for treatment of relapsing-remitting multiple sclerosis. The mode of action of DMF and its assumed active primary metabolite monomethylfumarate (MMF) is still not fully understood, notably for brain resident cells. Therefore we investigated potential direct effects of DMF and MMF on microglia and indirect effects on oligodendrocytes. Primary rat microglia were differentiated into M1-like, M2-like and M0 phenotypes and treated in vitro with DMF or MMF. The gene expression of pro-inflammatory and anti-inflammatory factors such as growth factors (IGF-1), interleukins (IL-10, IL-1β), chemokines (CCl3, CXCL-10) as well as cytokines (TGF-1β, TNFα), iNOS, and the mannose receptor (MRC1) was examined by determining their transcription level with qPCR, and on the protein level by ELISA and FACS analysis. Furthermore, microglia function was determined by phagocytosis assays and indirect effects on oligodendroglial proliferation and differentiation. DMF treatment of M0 and M1-like polarized microglia demonstrated an upregulation of gene expression for IGF-1 and MRC1, but not on the protein level. While the phagocytic activity remained unchanged, DMF and MMF treated microglia supernatants led to an enhanced proliferation of oligodendrocyte precursor cells (OPC). These results suggest that DMF has anti-inflammatory effects on microglia which may result in enhanced proliferation of OPC.

Topics: Animals; Cell Differentiation; Cell Proliferation; Dimethyl Fumarate; Fumarates; Gene Expression Regulation; Insulin-Like Growth Factor I; Maleates; Microglia; Neuroprotective Agents; Oligodendroglia; Phagocytosis; Rats, Sprague-Dawley; Stem Cells

Fumaric acid esters (FAEs) are used to treat psoriasis and are known to cause lymphopenia in roughly 60% of the patients. Much remains to be elucidated about the biological effects of FAEs on lymphocytes.. To evaluate the influence of long-term FAE (Fumaderm. In this single-centre retrospective observational subcohort study, we obtained leucocyte and lymphocyte subset counts before initiating FAE therapy in 371 psoriasis patients (mean age, 47.8 years; 63.3% males) and monitored them during treatment (mean treatment duration, 2.9 years). Multiparametric flow cytometry was used for immunophenotyping.. FAEs significantly reduced the numbers of CD4. Monitoring distinct T-cell subsets rather than just absolute lymphocyte counts may provide more meaningful insights into both the FAE treatment safety and efficacy. We therefore suggest optimizing pharmacovigilance by additionally monitoring CD4

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Child; Dermatologic Agents; Esters; Female; Fumarates; Humans; Immunophenotyping; Lymphopenia; Male; Middle Aged; Psoriasis; Retrospective Studies; T-Lymphocytes; Young Adult

Cocrystallization and salt formation were used to produce new multicomponent forms of a novel antimalarial imidazopyridazine drug lead (MMV652103) that displayed improved physicochemical properties. The drug lead had earlier shown good in vitro potency against multidrug resistant (K1) and sensitive (NF54) strains of the human malaria parasite Plasmodium falciparum, and high in vivo efficacy in both Plasmodium berghei and Plasmodium falciparum mouse models. A major drawback of MMV652103 is its limited aqueous solubility. Various new supramolecular products, including several multicomponent solid forms, are reported here, namely 3 cocrystal forms with the dicarboxylic acid coformers adipic acid, glutaric acid, and fumaric acid, and a salt form with malonic acid. These were characterized by thermal methods and their structures elucidated by single-crystal X-ray diffraction. A customized solubility experiment was performed in fasted-state simulated intestinal fluid for comparison of the solubility behavior of each new form of the drug lead with that of the untreated starting material. All of the multicomponent forms showed an improvement in the maximum concentrations (C

Topics: Adipates; Antimalarials; Crystallization; Crystallography, X-Ray; Dicarboxylic Acids; Fumarates; Glutarates; Imidazoles; Malonates; Powder Diffraction; Pyridines; Sodium Chloride; Solubility; X-Ray Diffraction

The Geobacter species evolved respiratory versatility to utilize a wide range of terminal electron acceptors. To explore this adaptive mechanism, Fe(III) citrate, hydrous ferric oxide, and fumarate were selected as electron acceptors, and the methylome and metabolome of Geobacter sulfurreducens PCA grown on each electron acceptor were investigated via third-generation, single-molecule real-time DNA sequencing and gas chromatography/time-of-flight mass spectrometry-based metabolomics, respectively. Results showed that the patterns of 4-methylcytosine (m4C) and 6-methyladenine (m6A) modification, the concentrations of fatty acids (e.g., caprylic acid, capric acid, and squalene), and the activity of antioxidant enzymes (e.g., superoxide dismutase, catalase, and glutathione reductase) were all varied in different electron acceptor cultures. Moreover, genes (e.g., GSU0466 and GSU1467) with low expression levels generally had high methylation levels. These findings suggest that m4C and m6A modifications, fatty acids, and antioxidant enzymes all play a role in the adaptation of G. sulfurreducens to diverse electron acceptors, and DNA methylation may be involved in the adaptation mainly via gene expression regulation.

Topics: Adaptation, Biological; DNA Methylation; Electron Transport; Ferric Compounds; Fumarates; Gene Expression; Geobacter; Metabolome; Metabolomics

Topics: Dermatologic Agents; Dimethyl Fumarate; Esters; Fumarates; Humans; Psoriasis

Ammonia is toxic to aquatic animal. Currently, only limited works were reported on the responses of aquatic animals after ammonia exposure using "omics" technologies. Tilapia suffers from the stress of ammonia-nitrogen during intensive recirculating aquaculture. Optimizing ammonia stress tolerance has become an important issue in tilapia breeding. The molecular and biochemical mechanisms of ammonia-nitrogen toxicity have not been understood comprehensively in tilapia yet. In this study, using RNA-seq and gas chromatograph system coupled with a Pegasus HT time-of-flight mass spectrometer (GC-TOF-MS) techniques, we investigated differential expressed genes (DEGs) and metabolomes in the liver at 6 h post-challenges (6 hpc) and 24 h post-challenges (24 hpc) under high concentration of ammonia-nitrogen treatment. We detected 2258 DEGs at 6 hpc and 315 DEGs at 24 hpc. Functional enrichment analysis indicated that DEGs were significantly associated with cholesterol biosynthesis, steroid and lipid metabolism, energy conservation, and mitochondrial tissue organization. Metabolomic analysis detected 31 and 36 metabolites showing significant responses to ammonia-nitrogen stress at 6 and 24 hpc, respectively. D-(Glycerol 1-phosphate), fumaric acid, and L-malic acid were found significantly down-regulated at both 6 and 24 hpc. The integrative analysis of transcriptomics and metabolomics suggested considerable alterations and precise control of gene expression at both physiological and molecular levels in response to the stress of ammonia-nitrogen in tilapia.

Topics: Ammonia; Animals; Cholesterol; Environmental Exposure; Fish Proteins; Fumarates; Gene Expression Profiling; Gene Expression Regulation; Gene Ontology; Glycerophosphates; Lipid Metabolism; Liver; Malates; Metabolome; Molecular Sequence Annotation; Stress, Physiological; Tilapia; Transcriptome; Water Pollutants, Chemical

EBV infection is a recognized epigenetic driver of carcinogenesis. We previously showed that EBV could protect cancer cells from TNF-induced necroptosis. This study aims to explore the epigenetic mechanisms allowing cancer cells with EBV infection to escape from RIP3-dependent necroptosis.

Topics: Aged; Animals; Carcinogenesis; Carcinoma; Cell Line, Tumor; Cellular Reprogramming; DNA Methylation; Epithelial Cells; Female; Fumarates; Gene Expression Regulation, Neoplastic; Herpesvirus 4, Human; Heterografts; Host-Pathogen Interactions; Humans; Ketoglutaric Acids; Male; Mice; Middle Aged; Nasopharyngeal Neoplasms; Nasopharynx; Necroptosis; Promoter Regions, Genetic; Receptor-Interacting Protein Serine-Threonine Kinases; Signal Transduction; Survival Analysis; Viral Matrix Proteins

Interest in the therapeutic effects of carbon monoxide (CO), a product of heme degradation catalyzed by the enzyme heme oxygenase-1 (HO-1), has led to the development of CO-releasing molecules (CO-RMs) for the controlled delivery of this gas in vivo. We recently proposed conjugating a cobalt-based CO-RM with various activators of nuclear factor erythroid 2-related factor 2 (Nrf2), the transcription factor that regulates HO-1 expression, in order to exploit the beneficial effects of exogenous and endogenous CO. In this study, we describe the preparation of hybrid molecules (termed HYCOs) conjugating a fumaric acid derivative as an Nrf2 activator to a Mn- or a Ru-based CO-RM known to be pharmacologically active. With the exception of an acyl-manganese complex, these hybrids were obtained by associating the two bioactive entities by means of a linker of variable structure. X-ray diffraction analyses and preliminary biological investigations are also presented.

Topics: Animals; Carbon Monoxide; Cell Line; Crystallography, X-Ray; Dose-Response Relationship, Drug; Drug Design; Fumarates; Manganese; Mice; Models, Molecular; Molecular Structure; NF-E2-Related Factor 2; Organometallic Compounds; Ruthenium; Structure-Activity Relationship

In the last decade, carbon monoxide-releasing molecules (CORMs) have been shown to act against several pathogens and to be promising antimicrobials. However, the understanding of the mode of action and reactivity of these compounds on bacterial cells is still deficient. In this work, we used a metabolomics approach to probe the toxicity of the ruthenium(II) complex Ru(CO)

Topics: Aconitate Hydratase; Anti-Bacterial Agents; Carbon Monoxide; Citric Acid Cycle; Escherichia coli; Fumarate Hydratase; Fumarates; Gene Expression Regulation, Bacterial; Glutamate Synthase; Glutamic Acid; Glycolysis; Ketoglutaric Acids; Magnetic Resonance Spectroscopy; Metabolomics; Nitrogen; Organometallic Compounds; Oxidation-Reduction

The use of microorganism fermentation for production of fumaric acid (FA), which is widely used in food, medicine, and other fields, can provide technical support for the FA industry. In this study, we aimed to increase the titer of FA production by using an improved Rhizopus oryzae WHT5, which was domesticated to obtain a furfural-resistant strain in corncob hydrolytes. The metabolic pathways and metabolic network of this strain were investigated, and the related enzymes and metabolic flux were analyzed. Metabolic pathway analysis showed that the R. oryzae WHT5 strain produced FA mainly through two pathways. One occurred in the cytoplasm and the other was a mitochondrial pathway. The key parameters of the fermentation process were analyzed. The FA titer was 49.05 g/L from corncob hydrolytes using R. oryzae WHT5 in a 7-L bioreactor. The use of a furfural-resistant strain developed through domestication effectively increased the titer of FA. This capacity of the microorganisms to produce high amounts of FA by bioconverting corncob hydrolyte can be further applied for industrial production of FA.

Topics: Bioreactors; Fumarates; Hydrolysis; Rhizopus; Zea mays

Biologics have greatly improved psoriasis management. However, primary and secondary non-response to treatment requires innovative strategies to optimize outcomes.. To describe the use of combined treatment of biologics with conventional systemic agents or phototherapy in daily clinical practice.. We collected data on frequency of use, demographics, treatment characteristics and drug survival of biologics combined with conventional systemic agents or phototherapy in five PSONET registries.. Of 9922 biologic treatment cycles, 982 (9.9%) were identified as combination treatment. 72.9% of treatment cycles concerned concomitant use of methotrexate, 25.3% concerned concomitant UVB therapy, acitretin or cyclosporin and 1.8% concerned combined treatment with PUVA, fumaric acids or a second biologic. Substantial variation was detected in type and frequency of combination treatments prescribed across registries. Patients initiated on combined treatment had generally severe disease and were affected with psoriasis for many years. The extent to which patients had been priory treated with biologic monotherapy and the proportion of patients affected with psoriatic arthritis differed between registries. Survival rates for etanercept, adalimumab, infliximab and ustekinumab with methotrexate ranged between 43 and 92%, 28 and 83%, 65 and 87% and 53 and 77%, respectively, across registries after one year with no consistent superior survival for a particular biologic. Longest survival on a biologic combined with methotrexate, acitretin or cyclosporin was 103, 78 and 34 months, respectively.. Methotrexate was the most commonly used concomitant treatment for patients on a biologic. Wide geographical variations in treatment selection and persistence of combination treatment exist. Data derived from ongoing studies may help to determine whether combined treatment is superior to biologic monotherapy.

Topics: Acitretin; Adalimumab; Austria; Biological Products; Combined Modality Therapy; Cyclosporine; Czech Republic; Dermatologic Agents; Drug Therapy, Combination; Etanercept; Female; Fumarates; Humans; Infliximab; Israel; Italy; Kaplan-Meier Estimate; Male; Methotrexate; Middle Aged; Netherlands; Psoriasis; PUVA Therapy; Registries; Severity of Illness Index; Ustekinumab

The pretreatment of softwood and hardwood samples (spruce and hornbeam wood) with 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) was undertaken for further simultaneous enzymatic saccharification of renewable non-food lignocellulosic biomass and microbial fermentation of obtained sugars to ethanol and fumaric acid. A multienzyme cocktail based on cellulases and yeast or fungus cells producing ethanol and fumaric acid were the main objects of [Bmim]Cl influence studies. A complex effect of lignocellulosic biomass pretreatment with [Bmim]Cl on various aspects of the process (both action of cellulases and microbial conversion of hydrolysates to target products) was revealed. Positive effects of the pretreatment with [Bmim]Cl included decreasing the lignin content in the biomass, and increasing the effectiveness of enzymatic hydrolysis and microbial transformation of pretreated biomass. Immobilized cells of both yeasts and fungi possessed improved productive characteristics in the biotransformation of biomass pretreated with [Bmim]Cl to ethanol and fumaric acid.

Topics: Biomass; Cells, Immobilized; Ethanol; Fermentation; Fumarates; Hydrolysis; Imidazoles; Ionic Liquids; Lignin

Consolidated bioprocessing (CBP) is a potential breakthrough technology for reducing costs of biochemical production from lignocellulosic biomass. Production of cellulase enzymes, saccharification of lignocellulose, and conversion of the resulting sugars into a chemical of interest occur simultaneously within a single bioreactor. In this study, synthetic fungal consortia composed of the cellulolytic fungus Trichoderma reesei and the production specialist Rhizopus delemar demonstrated conversion of microcrystalline cellulose (MCC) and alkaline pre-treated corn stover (CS) to fumaric acid in a fully consolidated manner without addition of cellulase enzymes or expensive supplements such as yeast extract. A titer of 6.87 g/L of fumaric acid, representing 0.17 w/w yield, were produced from 40 g/L MCC with a productivity of 31.8 mg/L/hr. In addition, lactic acid was produced from MCC using a fungal consortium with Rhizopus oryzae as the production specialist. These results are proof-of-concept demonstration of engineering synthetic microbial consortia for CBP production of naturally occurring biomolecules.

Topics: Cellulose; Coculture Techniques; Fermentation; Fumarates; Glucans; Glucose; Lactic Acid; Microbial Consortia; Rhizopus; Trichoderma; Xylans; Zea mays

Multiple Sclerosis (MS) is an autoimmune-neurodegenerative disorder managed therapeutically by modulating lymphocytes activity which has potential in disease management. Prohibitin 1(PHB) that controls the reactive oxygen species (ROS) and present on the activated lymphocytes have significance in the therapy of MS as esters of fumaric acid that regulates ROS is in phase II/III clinical trials. Thus, we evaluated the expression levels of PHB1 in experimental autoimmune encephalomyelitis (EAE), the animal model of MS and on MS patient's lymphocytes. PHB levels in brain tissue of EAE animals were determined by immunoblotting and on blood lymphocytes from MS relapse, Remission, Optic Neuritis, Neurological controls and Healthy volunteers by FACS using anti-PHB and anti-CD45 antibodies. We observed significant elevation of PHB in EAE brains (91.0 ± 17.59%) vs controls (29.8 ± 12.9%) (p = 0.01) and on lymphocytes of MS patients in acute (73.5 ± 11.20%) or relapsing (69.3 ± 17.33%) phase compared to remission (45.9 ± 8.08%) [p = 0.034 acute vs remission; p = 0.004 relapse vs remission]. Up regulation of PHB in relapsing vs remission MS patients imply the potential use of PHB to clinically evaluate subclinical disease status towards prognosis of an oncoming relapse. Elevated PHB levels in EAE brains signify the role of PHB in regulating ROS and implies PHB's role in oxidative stress.

Topics: Adolescent; Adult; Animals; Brain; Clinical Trials, Phase II as Topic; Clinical Trials, Phase III as Topic; Encephalomyelitis, Autoimmune, Experimental; Female; Fumarates; Humans; Lymphocytes; Male; Mice; Middle Aged; Multiple Sclerosis; Prohibitins; Repressor Proteins; Up-Regulation

Nisin-loaded chitosan-monomethyl fumaric acid (CM-N) nanoparticles were evaluated as a novel, direct food additive. Chitosan (CS) was modified with monomethyl fumaric acid (MFA) in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). CS-loaded nisin (CS-N) and CM-N nanoparticles were produced through ionic interactions between the positive amino group of CS and CS-MFA and negative tripolyphosphate ions. The resultant materials were characterized by TNBS assay, X-ray diffraction, Fourier-transform infrared spectroscopy, scanning electron microscopy, thermogravimetric analysis, and zeta potential analysis. CS-MFA was successfully synthesized with 8.38 ± 0.02% substitution of the amino groups, as confirmed by TNBS assay. The percentage yield of CS-N and CM-N nanoparticles was 81.64 and 76.83% and nisin encapsulation efficiency was 71.48 ± 0.48 and 60.32 ± 0.63%, respectively. The average particle size of CS-N and CM-N nanoparticles was 134.3 and 207.9 nm, while the zeta potential of CS-N and CM-N nanoparticles was +39.4 mV and +31.5 mV, respectively. Upon antibacterial activity against foodborne pathogens, CM-N significantly reduced bacterial counts compared to the other tested samples in orange juice after 48 h of incubation. Based on the preliminarily results, CM-N nanoparticles have shown impressive properties and can be used in the food industry as carriers and direct antimicrobial agents.

Topics: Anti-Bacterial Agents; Chitosan; Food Additives; Fumarates; Nanoparticles; Nisin; Spectroscopy, Fourier Transform Infrared; Staphylococcus aureus

Resistance to oncolytic virotherapy is frequently associated with failure of tumor cells to get infected by the virus. Dimethyl fumarate (DMF), a common treatment for psoriasis and multiple sclerosis, also has anticancer properties. We show that DMF and various fumaric and maleic acid esters (FMAEs) enhance viral infection of cancer cell lines as well as human tumor biopsies with several oncolytic viruses (OVs), improving therapeutic outcomes in resistant syngeneic and xenograft tumor models. This results in durable responses, even in models otherwise refractory to OV and drug monotherapies. The ability of DMF to enhance viral spread results from its ability to inhibit type I interferon (IFN) production and response, which is associated with its blockade of nuclear translocation of the transcription factor nuclear factor κB (NF-κB). This study demonstrates that unconventional application of U.S. Food and Drug Administration-approved drugs and biological agents can result in improved anticancer therapeutic outcomes.

Topics: Animals; Cell Line, Tumor; Cytokines; Dimethyl Fumarate; Esters; Fumarates; Glutathione; Humans; Interferon Type I; Maleates; Mice, Inbred BALB C; Mice, Inbred C57BL; NF-kappa B; Oncolytic Virotherapy; Oncolytic Viruses; Xenograft Model Antitumor Assays

Phenolics play a key role in communication between plants and microbes in the rhizosphere. In this study, shikimic, gallic, fumaric, ferulic, vanillic acid and quercetin in root exudates of Abelmoschus esculentus act as chemoattractants of endophytic Alcaligenes faecalis strains, BHU 12, BHU 16 and BHU M7. In vitro chemotaxis assay showed that BHU 12 expressed highest chemotactic movement (CFU ∼50×10

Topics: Abelmoschus; Alcaligenes faecalis; Biofilms; Chemotaxis; Coumaric Acids; Fumarates; Gallic Acid; Plant Exudates; Plant Roots; Quercetin; Rhizosphere; Shikimic Acid; Vanillic Acid

A novel fermentation system was employed whereby the mycelial mat of Rhizopus oryzae was attached to a polypropylene tube. Batch operation was used for growth, while continuous operation was employed during the fumaric acid production phase. A clear decrease in respiration, fumaric acid (FA) and ethanol production was observed when zero nitrogen was fed in the production phase, with FA productivity decreasing from an initial 0.7 g L

Topics: Cells, Immobilized; Ethanol; Fumarates; Nitrogen; Rhizopus

Fumarate adduction to cysteines has been implicated in the pathogenesis of several disorders. Its role, however, still remains elusive, and the need of predictive methods has not yet been met. The reactivity of cysteines found in fumarate-sensitive proteins was predicted when the collected data for eight network-type features were analyzed using classification models. Therefore, methods for evaluating the likelihood of a cysteine site to be modified by fumarate could be developed by combining concepts of network theory and machine learning.

Topics: Cysteine; Databases, Protein; Fumarates; Proteins; Sequence Analysis, Protein

Revascularization of natural and synthetic scaffolds is a critical part of the scaffold's incorporation and tissue ingrowth. Our goals were to create a biocompatible polymer scaffold with 3D-printing technology, capable of sustaining vascularization and tissue ingrowth.. We synthesized biodegradable polycaprolactone fumarate (PCLF) scaffolds to allow tissue ingrowth via large interconnected pores. The scaffolds were prepared with Poly(lactic-co-glycolic acid)(PLGA) microspheres seeded with or without different growth factors including VEGF,FGF-2, and/or BMP-2. Scaffolds were implanted into the subcutaneous tissues of rats before undergoing histologic and microCT angiographic analysis.. At harvest after 12 weeks, scaffolds had tissue infiltrating into their pores without signs of scar tissue formation, fibrous capsule formation, or immune responses against PCLF. Histology for M1/M2 macrophage phenotypes confirmed that there were no overt signs of immune responses. Both microCT angiography and histologic analysis demonstrated marked tissue and vessel ingrowth throughout the pores traversing the body of the scaffolds. Scaffolds seeded with microspheres containing VEGF or VEGF with either BMP-2 or FGF-2 had significantly higher vascular ingrowth and vessel penetration than controls. All VEGF-augmented scaffolds were positive for Factor-VIII and exhibited collagen tissue infiltration throughout the pores. Furthermore, scaffolds with VEGF and BMP-2 had high levels of mineral deposition throughout the scaffold that are attributable to BMP-2.. PCLF polymer scaffold can be utilized as a framework for vascular ingrowth and regeneration of multiple types of tissues. This novel scaffold material has promise in tissue regeneration across all types of tissues from soft tissue to bone.

Topics: Animals; Bone Morphogenetic Protein 2; Cross-Linking Reagents; Fumarates; Neovascularization, Physiologic; Polyesters; Printing, Three-Dimensional; Rats; Tissue Scaffolds; Vascular Endothelial Growth Factor A

The microbial production of fumaric acid by Rhizopus arrhizus NRRL 2582 has been evaluated using soybean cake from biodiesel production processes and very high polarity (VHP) sugar from sugarcane mills. Soybean cake was converted into a nutrient-rich hydrolysate via a two-stage bioprocess involving crude enzyme production via solid state fermentations (SSF) of either Aspergillus oryzae or R. arrhizus cultivated on soybean cake followed by enzymatic hydrolysis of soybean cake. The soybean cake hydrolysate produced using crude enzymes derived via SSF of R. arrhizus was supplemented with VHP sugar and evaluated using different initial free amino nitrogen (FAN) concentrations (100, 200, and 400 mg/L) in fed-batch cultures for fumaric acid production. The highest fumaric acid concentration (27.3 g/L) and yield (0.7 g/g of total consumed sugars) were achieved when the initial FAN concentration was 200 mg/L. The combination of VHP sugar with soybean cake hydrolysate derived from crude enzymes produced by SSF of A. oryzae at 200 mg/L initial FAN concentration led to the production of 40 g/L fumaric acid with a yield of 0.86 g/g of total consumed sugars. The utilization of sugarcane molasses led to low fumaric acid production by R. arrhizus, probably due to the presence of various minerals and phenolic compounds. The promising results achieved through the valorization of VHP sugar and soybean cake suggest that a focused study on molasses pretreatment could lead to enhanced fumaric acid production.

Topics: Aspergillus oryzae; Batch Cell Culture Techniques; Biofuels; Chemical Industry; Conservation of Natural Resources; Fermentation; Food Industry; Fumarates; Glycine max; Hydrolysis; Industrial Waste; Rhizopus; Saccharum; Sugars

Arabidopsis thaliana possesses two fumarase genes (FUM), AtFUM1 (At2g47510) encoding for the mitochondrial Krebs cycle-associated enzyme and AtFUM2 (At5g50950) for the cytosolic isoform required for fumarate massive accumulation. Here, the comprehensive biochemical studies of AtFUM1 and AtFUM2 shows that they are active enzymes with similar kinetic parameters but differential regulation. For both enzymes, fumarate hydratase (FH) activity is favored over the malate dehydratase (MD) activity; however, MD is the most regulated activity with several allosteric activators. Oxalacetate, glutamine, and/or asparagine are modulators causing the MD reaction to become preferred over the FH reaction. Activity profiles as a function of pH suggest a suboptimal FUM activity in Arabidopsis cells; moreover, the direction of the FUM reaction is sensitive to pH changes. Under mild oxidation conditions, AtFUMs form high mass molecular aggregates, which present both FUM activities decreased to a different extent. The biochemical properties of oxidized AtFUMs (oxAtFUMs) were completely reversed by NADPH-supplied Arabidopsis leaf extracts, suggesting that the AtFUMs redox regulation can be accomplished in vivo. Mass spectrometry analyses indicate the presence of an active site-associated intermolecular disulfide bridge in oxAtFUMs. Finally, a phylogenetic approach points out that other plant species may also possess cytosolic FUM2 enzymes mainly encoded by paralogous genes, indicating that the evolutionary history of this trait has been drawn through a process of parallel evolution. Overall, according to our results, a multilevel regulatory pattern of FUM activities emerges, supporting the role of this enzyme as a carbon flow monitoring point through the organic acid metabolism in plants.

Topics: Allosteric Regulation; Arabidopsis; Arabidopsis Proteins; Asparagine; Binding Sites; Evolution, Molecular; Fumarate Hydratase; Fumarates; Gene Expression; Gene Expression Regulation, Plant; Glutamine; Hydrogen-Ion Concentration; Kinetics; Malate Dehydrogenase; Models, Molecular; NADP; Oxaloacetic Acid; Oxidation-Reduction; Phylogeny; Protein Aggregates; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Protein Structure, Quaternary; Recombinant Proteins; Substrate Specificity

During compatibility study of the AZD7986 project, a peak of 3 area% at the tail (RRT 1.03) of the active pharmaceutical ingredient (API) was discovered for all tablets containing sodium stearyl fumarate (PRUV) under humid condition (e.g. 50 °C/75% RH), regardless of choice of disintegrant or filler combination. The degradant was needed to be identified to understand the corresponding reaction mechanism and help the final formulation design. Structure elucidation was therefore done by analysis using high resolution mass spectrometry. The degradant was found to be a Michael addition product of the API and fumaric acid. Reaction between deuterated fumaric acid and the API was carried to confirm the proposed structure and reaction mechanism. Fumaric acid was a degradant product of PRUV in the presence of other excipients, revealed by the stability study. The Michael addition reaction needs facilitation by water and basic conditions. The result from this study should serve as a precaution note for projects using PRUV as one of excipients where the API could act as a nucleophile. In such cases the microenvironment should be optimised to minimize the reaction, such as pH adjustment and incorporating protection from moisture.

Topics: Benzoxazoles; Chemistry, Pharmaceutical; Drug Incompatibility; Drug Stability; Excipients; Fumarates; Hydrogen-Ion Concentration; Mass Spectrometry; Oxazepines; Stearates; Tablets

The stoichiometry, X-ray structures and stability of four pharmaceutical cocrystals previously identified from liquid-assisted grinding (LAG) of 11-azaartemisinin (11-Aza; systematic name: 1,5,9-trimethyl-14,15,16-trioxa-11-azatetracyclo[10.3.1.0

Topics: Antimalarials; Crystallization; Crystallography, X-Ray; Fumarates; Hydrogen Bonding; Isomerism; Maleates; Molecular Conformation

The human gut microbiota is a crucial factor for the host's physiology with respect to health and disease. Metagenomic shotgun sequencing of microbial gut communities revealed that Prevotella copri is one of the most important players in the gastrointestinal tract of many individuals. Because of the importance of this bacterium we analyzed the growth behavior and the central metabolic pathways of P. copri. Bioinformatic data, transcriptome profiling and enzyme activity measurements indicated that the major pathways are based on glycolysis and succinate production from fumarate. In addition, pyruvate can be degraded to acetate and formate. Electron transport phosphorylation depends on fumarate respiration with NADH and reduced ferredoxin as electron donors. In contrast to Bacteroides vulgatus, P. copri showed a more pronounced dependency on the addition of CO

Topics: Acetate-CoA Ligase; Carbon Dioxide; Energy Metabolism; Formates; Fumarates; Gastrointestinal Microbiome; Gastrointestinal Tract; Glycolysis; Humans; Metabolic Networks and Pathways; Prevotella; Pyruvic Acid; Succinic Acid

An electroactive artificial biofilm has been optimized for the cathodic reduction of fumarate by Shewanella oneidensis. The system is based on the self-assembly of multi-walled carbon nanotubes with bacterial cells in the presence of a c-type cytochrome. The aggregates are then deposited on an electrode to form the electroactive artificial biofilm. Six c-type cytochromes have been studied, from bovine heart or Desulfuromonas and Desulfuvibrio strains. The isoelectric point of the cytochrome controls the self-assembly process that occurs only with positively-charged cytochromes. The redox potential of the cytochrome is critical for electron transfer reactions with membrane cytochromes of the Mtr pathway. Optimal results have been obtained with c

Topics: Biofilms; Catalysis; Cytochromes c; Desulfovibrio vulgaris; Electrodes; Electron Transport; Fumarates; Isoelectric Point; Oxidation-Reduction; Static Electricity; Succinic Acid

Fumaric acid is one of the top 12-biomass building-block chemicals. In this study, we reported manipulation of E. coli central carbon metabolism with the aim to decrease the by-products and improve fumaric acid production. PEP-dependent glucose phosphotransferase system was replaced with a galactose translocation system to minimize the consumption of phosphoenolpyruvate. Engineering anaplerotic pathway (phosphoenolpyruvate carboxylase) was employed to redistribute carbon flux from glycolysis to Krebs cycle. Deletion of malate dehydrogenase and overexpression of acetyl-CoA synthase could decrease the byproducts malic acid and acetic acid. The combined strategies led to fumaric acid yield up to 1.53 g/g dry cell weight, a 50% increase compared with the parental strain. The result demonstrated that these genetic modifications were effective strategies for improving the production of fumaric acid and the engineered strain may serve a platform microbial cell factory for efficient production of fumaric acid or other dicarboxylic acids.

Topics: Carbon; Carbon Cycle; Citric Acid Cycle; Escherichia coli; Fumarates; Glycolysis; Malate Dehydrogenase; Phosphoenolpyruvate Carboxylase

This study was aimed to evaluate the effects of organic acid (OA) and medium-chain fatty acid (MCFA) blends on production performance of sows and their litters. A total of 36 sows (Landrace × Yorkshire, average parity is 3.3, SE = 0.2) were randomly allocated to three treatments with 12 replicates. Dietary treatments were as follows: CON, basal diet; MC1, CON + 0.1% OA, and MCFA blends; MC2, CON + 0.2% OA, and MCFA blends. During lactation, no differences were observed in body weight (BW) loss, average daily feed intake, backfat thickness, digestibility of dry matter, nitrogen, or energy of sows. There were linear increase (p < 0.05) in BW and average daily gain of sucking piglets. On parturition and weaning day, there was a linear increase (p < 0.05) in fecal Lactobacillus counts, as well as a linear decrease (p < 0.05) in fecal Escherichia coli counts of sows on weaning day. The sucking piglets also had a linear increase (p < 0.05) in fecal Lactobacillus counts and a linear decrease (p < 0.05) in fecal E. coli counts. In conclusion, dietary supplementation of OA and MCFA blends in sows exerts beneficial effects to sows shifted fecal microbiota by increasing Lactobacillus and decreased E. coli counts. It also improved the performance of piglets.

Topics: Animal Feed; Animals; Bacterial Load; Citric Acid; Diet; Dietary Supplements; Digestion; Escherichia coli; Fatty Acids; Feces; Female; Fumarates; Lactation; Lactobacillus; Malates; Male; Nutrients; Swine

Hydrogen-producing bacteria are of environmental importance, since hydrogen is a major electron donor for prokaryotes in anoxic ecosystems. Epsilonproteobacteria are currently considered to be hydrogen-oxidizing bacteria exclusively. Here, we report hydrogen production upon pyruvate fermentation for free-living Epsilonproteobacteria, Sulfurospirillum spp. The amount of hydrogen produced is different in two subgroups of Sulfurospirillum spp., represented by S. cavolei and S. multivorans. The former produces more hydrogen and excretes acetate as sole organic acid, while the latter additionally produces lactate and succinate. Hydrogen production can be assigned by differential proteomics to a hydrogenase (similar to hydrogenase 4 from E. coli) that is more abundant during fermentation. A syntrophic interaction is established between Sulfurospirillum multivorans and Methanococcus voltae when cocultured with lactate as sole substrate, as the former cannot grow fermentatively on lactate alone and the latter relies on hydrogen for growth. This might hint to a yet unrecognized role of Epsilonproteobacteria as hydrogen producers in anoxic microbial communities.

Topics: Acetic Acid; Anaerobiosis; Campylobacteraceae; Coculture Techniques; Fermentation; Fumarates; Hydrogen; Kinetics; Lactic Acid; Methanococcus; Oxidation-Reduction; Pyruvic Acid; Succinic Acid; Symbiosis

The influence of the potential methane reducer, fumaric acid (FA), on ruminal parameters, the rumen wall and organ weights was investigated in a long-term study with growing bulls. In all, 20 bulls were fed with maize or grass silage as roughage, and with concentrate with or without 300 g FA per animal and day during the whole fattening period. After slaughtering, the organs were weighed and blood serum was analysed for glucose, β-hydroxybutyric acid (BHB) and non-esterified fatty acid concentration. The ruminal fluid was analysed for short-chain fatty acids, ammonia-N and the microbial community via single strand conformation polymorphism analysis. The rumen wall was examined histopathologically and results were graded as 'no visible lesions', 'few inflammatory infiltrates', 'some inflammatory infiltrates' or 'several inflammatory infiltrates'. In addition, the dimensions of the rumen villi were measured. The FA supplementation decreased the serum BHB concentration and the butyric acid concentration in the ruminal fluid. The microbial community in the ruminal fluid was not influenced by FA. An interaction between FA and silage type was observed for the inflammation centres counted in the villous area of rumen papillae. This interaction was also observed in the length and surface of the rumen villi. Rumen villi results show that the influence of FA depends on the roughage used in the diet.

Topics: 3-Hydroxybutyric Acid; Animals; Cattle; Diet; Dietary Fiber; Edible Grain; Fatty Acids, Volatile; Fumarates; Hydrogen-Ion Concentration; Male; Methane; Organ Size; Poaceae; Rumen; Silage; Zea mays

The vibrational spectra of solid-state acyclovir, fumaric acid and their cocrystal have been investigated by using terahertz time-domain spectroscopy (THz-TDS) and Raman spectroscopy at room temperature. In experimental THz spectra, the cocrystal has absorption peaks in 0.65, 0.94 and 1.10THz respectively, while the raw materials are absolutely different in this region. Raman spectra also show similar results about differences between the cocrystal and raw materials. Density functional theory (DFT) was performed to simulate vibrational modes of different theoretical forms between acyclovir and fumaric acid. The calculation of theoretical THz spectra shows that O8C7N1H27 and the carboxyl group COOH establish a dimer theoretical cocrystal form by the hydrogen bonding effect, which makes contributions to the formation of absorption peaks in 0.70, 1.01 and 1.34THz, and agrees well with experimental observations. The theoretical Raman result also indicates that this dimer form matches with experimental results. The characteristic bands of the cocrystal between acyclovir and fumaric acid are also assigned based on the simulation results from the DFT calculation.

Topics: Acyclovir; Crystallization; Fumarates; Spectrum Analysis, Raman; Terahertz Spectroscopy; Vibration

Effect of sequential combination of slightly acidic electrolyzed water (SAEW) with chemical and physical treatments on bacterial decontamination on fruits was investigated in this study. Effect of treatments on microbial and sensory quality was also analyzed after subsequent storage at 4 °C and room temperature (RT, 23 ± 0.15 °C). Whole apple and tomato fruits were inoculated with cocktail strains of Escherichia coli O157:H7 and Listeria monocytogenes. Uninoculated and inoculated fruits were washed first with distilled water (DW), calcium oxide (CaO), fumaric acid (FA), and SAEW at RT for 3 min. Combinations were performed by adding treatment one at a time to SAEW as following FA + SAEW, CaO + FA + SAEW, and CaO + FA + SAEW + ultrasonication (US) or microbubbles (MB). All the sanitizer treatments resulted in significant (p < 0.05) bacterial reduction compared to DW used as control. Increasing the treatments in combination from FA + SAEW to CaO + FA + SAEW + US resulted in an increased bacterial decontamination. The cavitation induced by ultrasonication in FA + SAEW solution resulted in a higher additive effect in decontamination of Escherichia coli O157:H7 and Listeria monocytogenes compare to the agitation generated by microbubble generator in FA + SAEW solution. CaO + FA + SAEW and CaO + FA + SAEW + US were effective in improving the microbial safety and quality of apple fruits. However, additional treatment of US impacted on the quality of tomato fruits during storage at RT. Therefore, a combination of SAEW with sanitizers (CaO and FA) and mechanical force (Ultrasonication) has the potential to be used in postharvest sanitation processing in the fresh fruit industry.

Topics: Calcium Compounds; Decontamination; Electrolysis; Escherichia coli O157; Food Preservation; Fruit; Fumarates; Listeria monocytogenes; Malus; Oxides; Solanum lycopersicum; Water

Topics: Fermentation; Fumarates; Rhizopus; Xylose

Fumaric acid is an important building-block chemical. The production of fumaric acid by fermentation is possible. Loofah fiber is a natural, biodegradable, renewable polymer material with highly sophisticated and pore structure. This work investigated a new immobilization method using loofah fiber as carrier to produce fumaric acid in a stirred-tank reactor. Compared with other carriers, loofah fiber was proven to be efficiently and successfully used in the reactor. After the optimization process, 20g addition of loofah fiber and 400rpm agitation speed were chosen as the most suitable process conditions. 30.3g/L fumaric acid in the broth as well as 19.16g fumaric acid in the precipitation of solid was achieved, while the yield from glucose reached 0.211g/g. Three batches of fermentation using the same loofah fiber carrier were conducted successfully, which meant it provided a new method to produce fumaric acid in a stirred-tank reactor.

Topics: Bioreactors; Fermentation; Fumarates; Luffa; Rhizopus

n-3 PUFA are lipids that play crucial roles in immune-regulation, cardio-protection and neurodevelopment. However, little is known about the role that these essential dietary fats play in modulating caecal microbiota composition and the subsequent production of functional metabolites. To investigate this, female C57BL/6 mice were assigned to one of three diets (control (CON), n-3 supplemented (n3+) or n-3 deficient (n3-)) during gestation, following which their male offspring were continued on the same diets for 12 weeks. Caecal content of mothers and offspring were collected for 16S sequencing and metabolic phenotyping. n3- male offspring displayed significantly less % fat mass than n3+ and CON. n-3 Status also induced a number of changes to gut microbiota composition such that n3- offspring had greater abundance of Tenericutes, Anaeroplasma and Coriobacteriaceae. Metabolomics analysis revealed an increase in caecal metabolites involved in energy metabolism in n3+ including α-ketoglutaric acid, malic acid and fumaric acid. n3- animals displayed significantly reduced acetate, butyrate and total caecal SCFA production. These results demonstrate that dietary n-3 PUFA regulate gut microbiota homoeostasis whereby n-3 deficiency may induce a state of disturbance. Further studies are warranted to examine whether these microbial and metabolic disturbances are causally related to changes in metabolic health outcomes.

Topics: Animal Nutritional Physiological Phenomena; Animals; Body Composition; Cecum; Diet; Dietary Supplements; DNA, Bacterial; Fatty Acids; Fatty Acids, Omega-3; Female; Fumarates; Gastrointestinal Microbiome; Ketoglutaric Acids; Malates; Male; Metabolome; Metabolomics; Mice; Mice, Inbred C57BL; RNA, Ribosomal, 16S; Sequence Analysis, DNA

The article describes some characteristics of temperature homeostasis regulation while intraoperative period and its correction methods by infusions of balanced crystalloid solutions on the basis amino acids and the Krebs cycle substrates.. 107 children of different ages were included into the study. All of them underwent surgery on thoracic or abdominal organs. The average age was 13 (7-16) years. All the operations were performed with total intravenous anesthesia and artificial lung ventilation. 0,9% sodium chloride solution, "Mafusol" "Infezol-40" and "Reamberin" were used in order to correct perioperative hypothermia. Results of the study. It was found that solutions based onfumarate (mafusol) and succinate (reamberin) have a significant positive effect on the temperature homeostasis. This fact means they can be recommendfor a broad usage in clinical practice for the purpose ofprevention and elimination of intraoperative hypothermia.

Topics: Adolescent; Body Temperature Regulation; Child; Citric Acid Cycle; Female; Fluid Therapy; Fumarates; Humans; Infusions, Intravenous; Intraoperative Care; Male; Malignant Hyperthermia; Meglumine; Random Allocation; Succinates; Treatment Outcome

We report novel pharmaceutical salts of an anti-hypertensive drug carvedilol (CVD) with pharmaceutically acceptable salt formers, including oxalic acid (OXA), fumaric acid (FUMA), benzoic acid (BZA), and mandelic acid (MDA) via conventional solvent evaporation technique. The pKa difference between CVD and selected acids was greater than 3, thus suggesting salt formation. Two polymorphic forms of CVD/MDA salts and one p-Dioxane solvate of CVD/FUMA salt were also reported in this paper. The salts were characterized by powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). Stability of the salts was assessed by storage at 40°C/75% RH for 1 month. All CVD salts exhibited higher solubility in phosphate buffer solution pH 6.8 compared to the parent drug CVD and showed good stability in accelerated ICH conditions at 40°C/75% RH for 1 month. CVD/FUMA salt showed the highest solubility (1.78 times). Based on thermal analysis and slurry experiment, it was found that CVD/MDA polymorphs were related monotropically with Form 1 as the stable form. The results suggested that salt formation could be an alternative method to improve CVD solubility.

Topics: Antihypertensive Agents; Carbazoles; Carvedilol; Fumarates; Mandelic Acids; Oxalic Acid; Propanolamines; Salts; Solubility

Production of fumaric acid from alkali-pretreated corncob (APC) at high solids loading was investigated using a combination of separated hydrolysis and fermentation (SHF) and fed-batch simultaneous saccharification and fermentation (SSF) by Rhizopus oryzae. Four different fermentation modes were tested to maximize fumaric acid concentration at high solids loading. The highest concentration of 41.32 g/L fumaric acid was obtained from 20 % (w/v) APC at 38 °C in the combined SHF and fed-batch SSF process, compared with 19.13 g/L fumaric acid in batch SSF alone. The results indicated that a combination of SHF and fed-batch SSF significantly improved production of fumaric acid from lignocellulose by R. oryzae than that achieved with batch SSF at high solids loading.

Topics: Alkalies; Batch Cell Culture Techniques; Bioreactors; Carbohydrate Metabolism; Fumarates; Glucose; Hydrolysis; Industrial Waste; Lignin; Plant Components, Aerial; Rhizopus; Sodium Hydroxide; Zea mays

Herring spermatozoa exhibit a high activity of NAD-preferring malic enzyme (NAD-ME). This enzyme is involved in the generation of NADH or NADPH in the decarboxylation of malate to form pyruvate and requires some divalent cations to express its activity. In order to confirm that NAD-ME isolated from herring sperm cells is localized in mitochondria, we performed immunofluorescent analysis and assayed spectrophotometrically the malic enzyme reaction. Production of polyclonal rabbit antibodies against NAD-ME from herring spermatozoa enabled identification of mitochondrial localization of this enzyme inside herring spermatozoa. The kinetic studies revealed that NAD-ME was competitively inhibited by ATP up to tenfold. Addition of fumarate reversed ATP-dependent inhibition of NAD-ME to 55 % of its maximum activity. The pH-dependent regulation of malic enzyme activity was also examined. Malic enzyme showed maximum activity at pH near 7.0 in all studied conditions. Finally, the role of malic enzyme activity regulation in mitochondria of herring sperm cells was discussed.

Topics: Adenosine Triphosphate; Animals; Fish Proteins; Fishes; Fumarates; Hydrogen-Ion Concentration; Malate Dehydrogenase; Male; Mitochondria; Muscle, Skeletal; Phosphocreatine; Spermatozoa

Hypothermic machine perfusion (HMP) and static cold storage (SCS) are the two methods used to preserve deceased donor kidneys prior to transplant. This study seeks to characterise the metabolic profile of HMP and SCS porcine kidneys in a cardiac death donor model. Twenty kidneys were cold flushed and stored for two hours following retrieval. Paired kidneys then underwent 24 h of HMP or SCS or served as time zero controls. Metabolite quantification in both storage fluid and kidney tissue was performed using one dimensional

Topics: Acetic Acid; Animals; Aspartic Acid; Cryopreservation; Energy Metabolism; Fumarates; Glutamic Acid; Hypothermia, Induced; Kidney; Lactic Acid; Magnetic Resonance Imaging; Male; Models, Animal; Organ Preservation; Perfusion; Renal Dialysis; Swine

Fumaric acid (FA), which is naturally found in organisms, is a well known intermediate of the citric acid cycle. We evaluated the effects of FA on tyrosinase activity and structure via enzyme kinetics and computational simulations. FA was found to be a reversible inhibitor of tyrosinase and its induced mechanism was the parabolic non-competitive inhibition type with IC

Topics: Agaricales; Enzyme Inhibitors; Fumarates; Kinetics; Molecular Docking Simulation; Monophenol Monooxygenase

MDH2 encodes mitochondrial malate dehydrogenase (MDH), which is essential for the conversion of malate to oxaloacetate as part of the proper functioning of the Krebs cycle. We report bi-allelic pathogenic mutations in MDH2 in three unrelated subjects presenting with early-onset generalized hypotonia, psychomotor delay, refractory epilepsy, and elevated lactate in the blood and cerebrospinal fluid. Functional studies in fibroblasts from affected subjects showed both an apparently complete loss of MDH2 levels and MDH2 enzymatic activity close to null. Metabolomics analyses demonstrated a significant concomitant accumulation of the MDH substrate, malate, and fumarate, its immediate precursor in the Krebs cycle, in affected subjects' fibroblasts. Lentiviral complementation with wild-type MDH2 cDNA restored MDH2 levels and mitochondrial MDH activity. Additionally, introduction of the three missense mutations from the affected subjects into Saccharomyces cerevisiae provided functional evidence to support their pathogenicity. Disruption of the Krebs cycle is a hallmark of cancer, and MDH2 has been recently identified as a novel pheochromocytoma and paraganglioma susceptibility gene. We show that loss-of-function mutations in MDH2 are also associated with severe neurological clinical presentations in children.

Topics: Age of Onset; Alleles; Amino Acid Sequence; Brain Diseases; Child; Child, Preschool; Citric Acid Cycle; Fibroblasts; Fumarates; Genetic Complementation Test; Humans; Infant; Infant, Newborn; Malate Dehydrogenase; Malates; Male; Metabolomics; Models, Molecular; Mutation

The production of fumaric acid through a biotechnological pathway has grown in importance because of its potential value in related industries. This has sparked an interest in developing an economically-efficient process for separation of fumaric acid (product of interest) from acetic acid (by-product). This study aimed to develop a simulated moving bed (SMB) chromatographic process for such separation in a systematic way. As a first step for this work, commercially available adsorbents were screened for their applicability to the considered separation, which revealed that an Amberchrom-CG71C resin had a sufficient potential to become an adsorbent of the targeted SMB. Using this adsorbent, the intrinsic parameters of fumaric and acetic acids were determined and then applied to optimizing the SMB process under consideration. The optimized SMB process was tested experimentally, from which the yield of fumaric-acid product was found to become lower than expected in the design. An investigation about the reason for such problem revealed that it was attributed to a fronting phenomenon occurring in the solute band of fumaric acid. To resolve this issue, the extent of the fronting was evaluated quantitatively using an experimental axial dispersion coefficient for fumaric acid, which was then considered in the design of the SMB of interest. The SMB experimental results showed that the SMB design based on the consideration of the fumaric-acid fronting could guarantee the attainment of both high purity (>99%) and high yield (>99%) for fumaric-acid product under the desorbent consumption of 2.6 and the throughput of 0.36L/L/h.

Topics: Acetic Acid; Chromatography, Liquid; Fumarates; Research Design

Microbial fumarate production from renewable feedstock is a promising and sustainable alternative to petroleum-based chemical synthesis. Here, we report a modular engineering approach that systematically removed metabolic pathway bottlenecks and led to significant titer improvements in a multi-gene fumarate metabolic pathway. On the basis of central pathway architecture, yeast fumarate biosynthesis was re-cast into three modules: reduction module, oxidation module, and byproduct module. We targeted reduction module and oxidation module to the cytoplasm and the mitochondria, respectively. Combinatorially tuning pathway efficiency by constructing protein fusions RoMDH-P160A and KGD2-SUCLG2 and optimizing metabolic balance by controlling genes RoPYC, RoMDH-P160A, KGD2-SUCLG2 and SDH1 expression strengths led to significantly improved fumarate production (20.46 g/L). In byproduct module, synthetizing DNA-guided scaffolds and designing sRNA switchs enabled further production improvement up to 33.13 g/L. These results suggest that modular pathway engineering can systematically optimize biosynthesis pathways to enable an efficient production of fumarate.

Topics: Escherichia coli; Escherichia coli Proteins; Fumarates; Fungal Proteins; Genetic Enhancement; Metabolic Engineering; Metabolic Flux Analysis; Multigene Family; Signal Transduction

As an important component of double-stranded DNA, adenine has powerful hydrogen-bond capability, due to rich hydrogen bond donors and acceptors existing within its molecular structure. Therefore, it is easy to form cocrystal between adenine and other small molecules with intermolecular hydrogen-bond effect. In this work, cocrystal of adenine and fumaric acid has been characterized as model system by FT-IR and FT-Raman spectral techniques. The experimental results show that the cocrystal formed between adenine and fumaric acid possesses unique spectroscopical characteristic compared with that of starting materials. Density functional theory (DFT) calculation has been performed to optimize the molecular structures and simulate vibrational modes of adenine, fumaric acid and the corresponding cocrystal. Combining the theoretical and experimental vibrational results, the characteristic bands corresponding to bending and stretching vibrations of amino and carbonyl groups within cocrystal are shifted into lower frequencies upon cocrystal formation, and the corresponding bond lengths show some increase due to the effect of intermolecular hydrogen bonding. Different vibrational modes shown in the experimental spectra have been assigned based on the simulation DFT results. The study could provide experimental and theoretical benchmarks to characterize cocrystal formed between active ingredients and cocrystal formers and also the intermolecular hydrogen-bond effect within cocrystal formation process by vibrational spectroscopic techniques.

Topics: Adenine; Crystallization; Fumarates; Molecular Conformation; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis, Raman; Vibration

The adduction of fumaric acid to the sulfhydryl group of certain cysteine (Cys) residues in proteins via a Michael-like reaction leads to the formation of S-(2-succino)cysteine (2SC) sites. Although its role remains to be fully understood, this post-translational Cys modification (protein succination) has been implicated in the pathogenesis of diabetes/obesity and fumarate hydratase-related diseases. In this study, theoretical approaches to address sequence- and 3D-structure-based features possibly underlying the specificity of protein succination have been applied to perform the first analysis of the available data on the succinate proteome. A total of 182 succinated proteins, 205 modifiable, and 1750 non-modifiable sites have been examined. The rate of 2SC sites per protein ranged from 1 to 3, and the overall relative abundance of modifiable sites was 10.8%. Modifiable and non-modifiable sites were not distinguishable when the hydrophobicity of the Cys-flaking peptides, the acid dissociation constant value of the sulfhydryl groups, and the secondary structure of the Cys-containing segments were compared. By contrast, significant differences were determined when the accessibility of the sulphur atoms and the amino acid composition of the Cys-flaking peptides were analysed. Based on these findings, a sequence-based score function has been evaluated as a descriptor for Cys residues. In conclusion, our results indicate that modifiable and non-modifiable sites form heterogeneous subsets when features often discussed to describe Cys reactivity are examined. However, they also suggest that some differences exist, which may constitute the baseline for further investigations aimed at the development of predictive methods for 2SC sites in proteins.

Topics: Amino Acids; Computational Biology; Cysteine; Fumarates; Humans; Models, Theoretical; Molecular Conformation; Protein Processing, Post-Translational; Proteins; Proteome; Sequence Analysis, Protein; Succinates

Currently, research is being focused on the industrial-scale production of fumaric acid and other relevant organic acids from renewable feedstocks via fermentation, preferably at low pH for better product recovery. However, at low pH a large fraction of the extracellular acid is present in the undissociated form, which is lipophilic and can diffuse into the cell. There have been no studies done on the impact of high extracellular concentrations of fumaric acid under aerobic conditions in S. cerevisiae, which is a relevant issue to study for industrial-scale production. In this work we studied the uptake and metabolism of fumaric acid in S. cerevisiae in glucose-limited chemostat cultures at a cultivation pH of 3.0 (pH < pK). Steady states were achieved with different extracellular levels of fumaric acid, obtained by adding different amounts of fumaric acid to the feed medium. The experiments were carried out with the wild-type S. cerevisiae CEN.PK 113-7D and an engineered S. cerevisiae ADIS 244 expressing a heterologous dicarboxylic acid transporter (DCT-02) from Aspergillus niger, to examine whether it would be capable of exporting fumaric acid. We observed that fumaric acid entered the cells most likely via passive diffusion of the undissociated form. Approximately two-thirds of the fumaric acid in the feed was metabolized together with glucose. From metabolic flux analysis, an increased ATP dissipation was observed only at high intracellular concentrations of fumarate, possibly due to the export of fumarate via an ABC transporter. The implications of our results for the industrial-scale production of fumaric acid are discussed.

Topics: Aerobiosis; Animal Feed; Anti-Bacterial Agents; Aspergillus niger; Biomass; Bioreactors; Cell Membrane; Dicarboxylic Acid Transporters; Dicarboxylic Acids; Fumarates; Glucose; Hydrogen-Ion Concentration; Malates; Oxygen; Permeability; Saccharomyces cerevisiae; Succinic Acid

The TET enzymes are members of the 2-oxoglutarate-dependent dioxygenase family and comprise three isoenzymes in humans: TETs 1-3. These TETs convert 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC) in DNA, and high 5-hmC levels are associated with active transcription. The importance of the balance in these modified cytosines is emphasized by the fact that TET2 is mutated in several human cancers, including myeloid malignancies such as acute myeloid leukemia (AML). We characterize here the kinetic and inhibitory properties of Tets and show that the Km value of Tets 1 and 2 for O2 is 30 μm, indicating that they retain high activity even under hypoxic conditions. The AML-associated mutations in the Fe(2+) and 2-oxoglutarate-binding residues increased the Km values for these factors 30-80-fold and reduced the Vmax values. Fumarate and succinate, which can accumulate to millimolar levels in succinate dehydrogenase and fumarate hydratase-mutant tumors, were identified as potent Tet inhibitors in vitro, with IC50 values ∼400-500 μm. Fumarate and succinate also down-regulated global 5-hmC levels in neuroblastoma cells and the expression levels of some hypoxia-inducible factor (HIF) target genes via TET inhibition, despite simultaneous HIFα stabilization. The combination of fumarate or succinate treatment with TET1 or TET3 silencing caused differential effects on the expression of specific HIF target genes. Altogether these data show that hypoxia-inducible genes are regulated in a multilayered manner that includes epigenetic regulation via TETs and 5-hmC levels in addition to HIF stabilization.

Topics: Animals; Cell Hypoxia; Cell Line, Tumor; Dioxygenases; DNA-Binding Proteins; Fumarates; Gene Expression Regulation, Neoplastic; Humans; Leukemia, Myeloid, Acute; Mice; Mixed Function Oxygenases; Mutation; Neuroblastoma; Proto-Oncogene Proteins; Succinic Acid

The wild mushroom Leucopaxillus candidus (Bres.) Singer was studied for the first time to obtain information about its chemical composition, nutritional value and bioactivity. Free sugars, fatty acids, tocopherols, organic and phenolic acids were analysed by chromatographic techniques coupled to different detectors. L. candidus methanolic extract was tested regarding antioxidant potential (reducing power, radical scavenging activity and lipid peroxidation inhibition). L. candidus was shown to be an interesting species in terms of nutritional value, with high content in proteins and carbohydrates, but low fat levels, with the prevalence of polyunsaturated fatty acids. Mannitol was the most abundant free sugar and β-tocopherol was the main tocopherol isoform. Other compounds detected were oxalic and fumaric acids, p-hydroxybenzoic and cinnamic acids. The methanolic extract revealed antioxidant activity and did not show hepatoxicity in porcine liver primary cells. The present study provides new information about L. candidus.

Topics: Agaricales; Animals; Antioxidants; beta-Tocopherol; Biphenyl Compounds; Cell Survival; Cinnamates; Complex Mixtures; Fatty Acids, Unsaturated; Fumarates; Hepatocytes; Mannitol; Metabolome; Nutritive Value; Oxalates; Parabens; Picrates; Primary Cell Culture; Swine

Bacterial persisters are a small fraction of quiescent cells that survive in the presence of lethal concentrations of antibiotics. They can regrow to give rise to a new population that has the same vulnerability to the antibiotics as did the parental population. Although formation of bacterial persisters in the presence of various antibiotics has been documented, the molecular mechanisms by which these persisters tolerate the antibiotics are still controversial. We found that amplification of the fumarate reductase operon (FRD) inEscherichia coliled to a higher frequency of persister formation. The persister frequency ofE. coliwas increased when the cells contained elevated levels of intracellular fumarate. Genetic perturbations of the electron transport chain (ETC), a metabolite supplementation assay, and even the toxin-antitoxin-relatedhipA7mutation indicated that surplus fumarate markedly elevated theE. colipersister frequency. AnE. colistrain lacking succinate dehydrogenase (SDH), thereby showing a lower intracellular fumarate concentration, was killed ∼1,000-fold more effectively than the wild-type strain in the stationary phase. It appears thatSDHandFRDrepresent a paired system that gives rise to and maintainsE. colipersisters by producing and utilizing fumarate, respectively.

Topics: Ampicillin; Anti-Bacterial Agents; Bacterial Proteins; Citric Acid Cycle; Drug Resistance, Multiple, Bacterial; Electron Transport; Escherichia coli; Fumarates; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Gene Library; Kanamycin; Microbial Sensitivity Tests; Norfloxacin; Operon; Succinate Dehydrogenase

Plant roots exude numerous metabolites into the soil that influence nutrient availability. Although root exudate composition is hypothesized to be under selection in low fertility soils, few studies have tested this hypothesis in a phylogenetic framework. In this study, we examined root exudates of three pairs of Helianthus species chosen as phylogenetically-independent contrasts with respect to native soil nutrient availability. Under controlled environmental conditions, seedlings were grown to the three-leaf-pair stage, then transferred to either high or low nutrient treatments. After five days of nutrient treatments, we used gas chromatography-mass spectrometry for analysis of root exudates, and detected 37 metabolites across species. When compared in the high nutrient treatment, species native to low nutrient soils exhibited overall higher exudation than their sister species native to high nutrient soils in all three species pairs, providing support for repeated evolutionary shifts in response to native soil fertility. Species native to low nutrient soils and those native to high nutrient soils responded similarly to low nutrient treatments with increased exudation of organic acids (fumaric, citric, malic acids) and glucose, potentially as a mechanism to enhance nutrition acquisition. However, species native to low nutrient soils also responded to low nutrient treatments with a larger decrease in exudation of amino acids than species native to high nutrient soils in all three species pairs. This indicates that species native to low nutrient soils have evolved a unique sensitivity to changes in nutrient availability for some, but not all, root exudates. Overall, these repeated evolutionary divergences between species native to low nutrient soils and those native to high nutrient soils provide evidence for the adaptive value of root exudation, and its plasticity, in contrasting soil environments.

Topics: Adaptation, Physiological; Biological Evolution; Citric Acid; Ecosystem; Fumarates; Gas Chromatography-Mass Spectrometry; Glucose; Helianthus; Malates; Phylogeny; Plant Growth Regulators; Plant Roots; Principal Component Analysis; Soil

With the aim of improving the solubility of ciprofloxacin, polybasic organic acids were utilized to react with ciprofloxacin in different stoichiometric proportions. The use of the solvent drop grinding (SDG) method, as well as the solvent evaporation method, resulted in the crystalline salts ciprofloxacin/fumaric acid (1:1, 2:1), ciprofloxacin/maleic acid (1:1) and ciprofloxacin/citric acid (2:1). The solubilities of these salts in pure water (pH 7.0) were determined using high-performance liquid chromatography (HPLC) at 310 K, with the salts showing considerably greater solubility than ciprofloxacin itself and, interestingly, ciprofloxacin/fumaric acid (2:1) being more soluble than ciprofloxacin/fumaric acid (1:1). Intrigued by this phenomenon, we undertook a comparison of the crystal structures of the salts: the three-dimensional sandwich-like structure observed in the 2:1 salt indicates that the preferred stacking may be a factor in increasing the solubility of ciprofloxacin.

Topics: Calorimetry, Differential Scanning; Ciprofloxacin; Citric Acid; Fumarates; Maleates; Organic Chemicals; Sodium Chloride; Solubility

Japanese ginjo sake is a premium refined sake characterized by a pleasant fruity apple-like flavor and a sophisticated taste. Because of technical difficulties inherent in brewing ginjo sake, off-flavors sometimes occur. However, the metabolites responsible for off-flavors as well as those present or absent in higher quality ginjo sake remain uncertain. Here, the relationship between 202 polar chemical compounds in sake identified using capillary electrophoresis coupled with time-of-flight mass spectrometry and its organoleptic properties, such as quality and off-flavor, was examined. First, we found that some off-flavored sakes contained higher total amounts of metabolites than other sake samples. The results also identified that levels of 2-oxoglutaric acid and fumaric acid, metabolites in the tricarboxylic acid cycle, were highly but oppositely correlated with ginjo sake quality. Similarly, pyridoxine and pyridoxamine, co-enzymes for amino transferase, were also highly but oppositely correlated with ginjo sake quality. Additionally, pyruvic acid levels were associated with good quality as well. Compounds involved in the methionine salvage cycle, oxidative glutathione derivatives, and amino acid catabolites were correlated with low quality. Among off-flavors, an inharmonious bitter taste appeared attributable to polyamines. Furthermore, protein analysis displayed that a diversity of protein components and yeast protein (triosephosphate isomerase, TPI) leakage was linked to the overall metabolite intensity in ginjo sake. This research provides insight into the relationship between sake components and organoleptic properties.

Topics: Alcoholic Beverages; Fatty Acids; Fumarates; Glutathione; Humans; Ketoglutaric Acids; Metabolomics; Pyruvic Acid; Saccharomyces cerevisiae; Sugar Acids; Taste

Fumaric acid is a commonly used excipient in pharmaceutical products. It is not known if its presence may lead to fluctuation of endogenous fumarate levels. An LC-MS/MS method was developed and validated to quantify fumarate in support of a toxicokinetics study.. Stability evaluation showed that endogenous fumarate was stable for 6 h at room temperature, while exogenously added fumaric acid was converted to malate within 1 h due to the presence of fumarase. Citric acid, a fumarase inhibitor, prevented the conversion of added fumaric acid in rat plasma.. The method was validated in citric acid stabilized rat plasma using a surrogate matrix approach. A discrepancy in stability was observed between endogenous fumarate and exogenously added fumaric acid.

Topics: Animals; Carbon Radioisotopes; Chromatography, High Pressure Liquid; Citric Acid; Drug Stability; Fumarate Hydratase; Fumarates; Isotope Labeling; Malates; Quality Control; Rats; Tandem Mass Spectrometry; Temperature

To develop a montelukast sodium-loaded stable oral suspension bioequivalent to the commercial granules in rats, several montelukast sodium-loaded suspensions were prepared with a suspending agent, stabilizers and anti-aggregation agents, and their stabilities were investigated by visually observing the sedimentation phenomenon and determining the concentration of the degradation product. Moreover, dissolution and pharmacokinetic studies of the optimized formulation were examined in rats compared to commercial montelukast sodium-loaded granules. Avicel RC-591 (Avicel), a suspending agent, prevented the sedimentation of these suspensions at >2.496 (w/v) per cent composition. Amongst the stabilizers tested, fumaric acid provided the lowest concentration of montelukast sulphoxide (a degradation product) in these suspensions at 40 °C, demonstrating its excellent stabilizing activity. Furthermore, as an anti-aggregation agent, glycerin gave lower amounts of degradation product than those with poloxamer 407 and Tween 80. In particular, montelukast-loaded oral suspension, an aqueous suspension containing montelukast sodium/Avicel/fumaric acid/glycerin at a concentration of 312/2496/15.6/62.4 (mg/100 ml), and the commercial granules exhibited similar dissolution profiles in 0.5% (w/v) aqueous solution of sodium lauryl sulphate. Moreover, the pharmacokinetics in rats provided by this suspension was comparable to that of the commercial granules, suggesting that they were bioequivalent. In addition, it was physically and chemically stable at 40 °C for at least 6 months. Thus, this montelukast sodium-loaded oral suspension, with bioequivalence to the commercial granules and excellent stability, could be a prospective dosage form for the treatment of asthma.

Topics: Acetates; Administration, Oral; Animals; Anti-Asthmatic Agents; Cellulose; Cyclopropanes; Drug Stability; Excipients; Fumarates; Glycerol; Male; Quinolines; Rats, Sprague-Dawley; Solubility; Sulfides; Suspensions; Technology, Pharmaceutical; Therapeutic Equivalency

Xylose is one of the most abundant lignocellulosic components, but it cannot be used by R. oryzae for fumaric acid production. Here, we applied high-throughput RNA sequencing to generate two transcriptional maps of R. oryzae following fermentation in glucose or xylose. The differential expression analysis showed that, genes involved in amino acid metabolism, fatty acid metabolism, and gluconeogenesis, were up-regulated in response to xylose. Moreover, we discovered the potential presence of oxidative stress in R. oryzae during xylose fermentation. To adapt to this unfavorable condition, R. oryzae displayed reduced growth and induce of a number of antioxidant enzymes, including genes involved in glutathione, trehalose synthesis, and the proteasomal pathway. These responses might divert the flow of carbon required for the accumulation of fumaric acid. Furthermore, using high-throughput RNA sequencing, we identified a large number of novel transcripts and a substantial number of genes that underwent alternative splicing. Our analysis provides remarkable insight into the mechanisms underlying xylose fermentation by R. oryzae. These results may reveal potential target genes or strategies to improve xylose fermentation.

Topics: Alternative Splicing; Biomass; Fermentation; Fumarates; Genome, Bacterial; Glucose; Rhizopus; RNA, Bacterial; Transcriptome; Xylose

Coffee is one of the most consumed beverages in the world, being a source of bioactive compounds as well as flavors. Hydroxycinnamic acids, flavonols, and carboxylic acids have been studied in the samples of instant coffee commercialized in Spain. The studies about contents of food components should be complemented with either in vitro or in vivo bioaccessibility studies to know the amount of food components effectively available for functions in the human body. In this sense, a widely used in vitro model has been applied to assess the potential intestinal absorption of phenolic compounds and organic acids. The contents of hydroxycinnamic acids and flavonols were higher in instant regular coffee samples than in the decaffeinated ones. Bioaccessible phenolic compounds in most analyzed samples account for 20-25% of hydroxycinnamic acids and 17-26% of flavonols. This could mean that a great part of them can remain in the gut, acting as potential in situ antioxidants. Quinic, acetic, pyroglutamic, citric and fumaric acids were identified in commercial instant coffee samples. Succinic acid was found in the coffee blend containing chicory. All carboxylic acids showed a very high bioaccessibility. Particularly, acetic acid and quinic acid were found in higher contents in the samples treated with the in vitro simulation of gastrointestinal processes, compared to the original ones, which can be explained by their cleavage from chlorogenic acid during digestion. This is considered as a positive effect, since quinic acid is considered as an antioxidant inducer.

Topics: Acetic Acid; Antioxidants; Biological Availability; Chlorogenic Acid; Citric Acid; Coffee; Coumaric Acids; Flavonols; Fumarates; Hydrogen-Ion Concentration; Intestinal Absorption; Models, Biological; Phenol; Pyrrolidonecarboxylic Acid; Quinic Acid; Spain

The fungal strain EML-DML3PNa1 isolated from leaf of white dogwood (Cornus alba L.) showed strong nematicidal activity with juvenile mortality of 87.6% at a concentration of 20% fermentation broth filtrate at 3 days after treatment. The active fungal strain was identified as Aspergillus oryzae, which belongs to section Flavi, based on the morphological characteristics and sequence analysis of the ITS rDNA, calmodulin (CaM), and β-tubulin (BenA) genes. The strain reduced the pH value to 5.62 after 7 days of incubation. Organic acid analysis revealed the presence of citric acid (515.0 mg/kg), malic acid (506.6 mg/kg), and fumaric acid (21.7 mg/kg). The three organic acids showed moderate nematicidal activities, but the mixture of citric acid, malic acid, and fumaric acid did not exhibit the full nematicidal activity of the culture filtrate of EML- DML3PNa1. Bioassay-guided fractionation coupled with (1)H- and (13)C-NMR and EI-MS analyses led to identification of kojic acid as the major nematicidal metabolite. Kojic acid exhibited dose-dependent mortality and inhibited the hatchability of M. incognita, showing EC50 values of 195.2 µg/ml and 238.3 µg/ml, respectively, at 72 h postexposure. These results suggest that A. oryzae EML-DML3PNa1 and kojic acid have potential as a biological control agent against M. incognita.

Topics: Animals; Antinematodal Agents; Antioxidants; Aspergillus oryzae; Calmodulin; Citric Acid; Cornus; Culture Media; Fermentation; Fumarates; Malates; Polymerase Chain Reaction; Pyrones; Sequence Analysis, DNA; Tubulin; Tylenchoidea

Bioavailability of heavy metals can be modified by different root exudates. Among them, low molecular weight organic acids (LMWOAs) play an important role in this process. Three plant species (Poa annua, Medicago polymorpha and Malva sylvestris), potentially used for phytoremediation, have been assessed for both metal uptake and LMWOAs excretion in contaminated environments with different concentrations of Cd, Cu and Zn. The experiments have been carried out in washed sand and in three contaminated soils where two organic amendments were added (biosolid compost and alperujo compost). The most abundant LMWOAs excreted by all studied plants were oxalic and malic acids, although citric and fumaric acids were also detected. The general tendency was that plants responded to an increase of heavy metal stress releasing higher amounts of LMWOAs. This is an efficient exclusion mechanism reducing the metal uptake and allowing the plant growth at high levels of contamination. In the experiment using wash sand as substrate, the organic acids composition and quantity depended mainly on plant species and metal contamination. M. polymorpha was the species that released the highest concentrations of LMWOAs, both in sand and in soils with no amendment addition, whereas a decrease of these acids was observed with the addition of amendments. Our results established a clear effect of organic matter on the composition and total amount of LMWOAs released. The increase of organic matter and nutrients, through amendments, improved the soil quality reducing phytotoxicity. As a result, organic acids exudates decreased and were solely composed of oxalic acid (except for M. polymorpha). The release of LMWOAs has proved to be an important mechanism against heavy metal stress, unique to each species and modifiable by means of organic amendment addition.

Topics: Biodegradation, Environmental; Citric Acid; Fumarates; Magnoliopsida; Malates; Malva; Medicago; Metals, Heavy; Oxalic Acid; Plant Roots; Poaceae; Silicon Dioxide; Soil; Soil Pollutants

Itaconic acid (IA) is a dicarboxylic acid included in the US Department of Energy's (DOE) 2004 list of the most promising chemical platforms derived from sugars. IA is produced industrially using liquid-state fermentation (LSF) by Aspergillus terreus with glucose as the carbon source. To utilize IA production in renewable resource-based biorefinery, the present study investigated the use of lignocellulosic biomass as a carbon source for LSF. We also investigated the production of fumaric acid (FA), which is also on the DOE's list. FA is a primary metabolite, whereas IA is a secondary metabolite and requires the enzyme cis-aconitate decarboxylase for its production. Two lignocellulosic biomasses (wheat bran and corn cobs) were tested for fungal fermentation. Liquid hydrolysates obtained after acid or enzymatic treatment were used in LSF. We show that each treatment resulted in different concentrations of sugars, metals, or inhibitors. Furthermore, different acid yields (IA and FA) were obtained depending on which of the four Aspergillus strains tested were employed. The maximum FA yield was obtained when A. terreus was used for LSF of corn cob hydrolysate (1.9% total glucose); whereas an IA yield of 0.14% was obtained by LSF of corn cob hydrolysates by A. oryzae.

Topics: Aspergillus niger; Biofuels; Biomass; Fermentation; Fumarates; Lignin; Succinates

Fumaric acid as a four-carbon unsaturated dicarboxylic acid is widely used in the food and chemical industries. Food waste (FW), rich in carbohydrates and protein, is a promising potential feedstock for renewable bio-based chemicals. In this research, we investigated the capability of Rhizopus arrhizus RH7-13 in producing fumaric acid from FW. The liquid fraction of the FW (L-FW) was proven to be the best seed culture medium in our research. When it was however used to be fermentation medium, the yield of fumaric acid reached 32.68 g/L, at a volumetric production of 0.34 g/L h. The solid fraction of FW mixed with water (S-FW) could also be used as fermentation medium when a certain amount of glucose was added, and the yield of fumaric acid reached 31.26 g/L. The results indicated that both fractions of FW could be well utilized in fermentation process and it could replace a part of common carbon, nitrogen, and nutrient. The process has an application potential since reducing the costs of raw materials.

Topics: Biodegradation, Environmental; Biomass; Fermentation; Food; Fumarates; Glucose; Rhizopus; Waste Products

To examine whether NMR analysis is a suitable method for the quantitative determination of wine components, an international collaborative trial was organized to evaluate the method according to the international regulations and guidelines of the German Institute for Standardization/International Organization for Standardization, AOAC INTERNATIONAL, the International Union of Pure and Applied Chemistry, and the International Organization of Vine and Wine. Sugars such as glucose; acids such as malic, acetic, fumaric, and shikimic acids (the latter two as minor components); and sorbic acid, a preservative, were selected for the exemplary quantitative determination of substances in wine. Selection criteria for the examination of sample material included different NMR spectral signal types (singlet and multiplet), as well as the suitability of the proposed substances for manual integration at different levels of challenge (e.g., interference as a result of the necessary suppression of a water signal or the coverage of different typical wine concentration ranges for a selection of major components, minor components, and additives). To show that this method can be universally applied, NMR measurement and the method of evaluation were not strictly elucidated. Fifteen international laboratories participated in the collaborative trial and determined six parameters in 10 samples. The values, in particular the reproducibility SD (SR), were compared with the expected Horwitz SD (SH) by forming the quotient SR/SH (i.e., the HorRat value). The resulting HorRat values of most parameters were predominantly between 0.6 and 1.5, and thus of an acceptable range.

Topics: Acetates; Fumarates; Glucose; Laboratories; Malates; Proton Magnetic Resonance Spectroscopy; Shikimic Acid; Sorbic Acid; Wine

S-adenosyl-l-methionine (SAM)-dependent methyltransfer is a common biosynthetic strategy to modify natural products. We investigated the previously uncharacterized Aspergillus fumigatus methyltransferase FtpM, which is encoded next to the bimodular fumaric acid amide synthetase FtpA. Structure elucidation of two new A. fumigatus natural products, the 1,11-dimethyl esters of fumaryl-l-tyrosine and fumaryl-l-phenylalanine, together with ftpM gene disruption suggested that FtpM catalyzes iterative methylation. Final evidence that a single enzyme repeatedly acts on fumaric acid amides came from an in vitro biochemical investigation with recombinantly produced FtpM. Size-exclusion chromatography indicated that this methyltransferase is active as a dimer. As ftpA and ftpM homologues are found clustered in other fungi, we expect our work will help to identify and annotate natural product biosynthesis genes in various species.

Topics: Amides; Aspergillus fumigatus; Biocatalysis; Fumarates; Methylation; Methyltransferases; Molecular Structure

The aim of the study was to (i) investigate the potential of edible mushroom Boletus badius (Fr.) Fr. to accumulate 53 elements from unpolluted acidic sandy soil and polluted alkaline flotation tailing sites in Poland, (ii) to estimate the low-molecular-weight organic acid (LMWOA) profile and contents in fruit bodies, and finally (iii) to explore the possible relationship between elements and LMWOA content in mushrooms. The content of most elements in fruiting bodies collected from the flotation tailings was significantly higher than in mushrooms from the unpolluted soils. The occurrence of elements determined in fruiting bodies of B. badius has been varied (from 0.01 mg kg

Topics: Agaricales; Fruiting Bodies, Fungal; Fumarates; Lactic Acid; Poland; Soil; Soil Pollutants; Succinic Acid

The present study aims at in vitro antibacterial and antioxidant activity evaluation of chitosan modified with monomethyl fumaric acid (MFA) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as mediator. Three different kinds of chitosan derivatives Ch-Ds-1,Ch-Ds-2 and Ch-Ds-3 were synthesized by feeding different concentration of MFA. The chemical structures of resulting materials were characterized by (1)H NMR, (13)C NMR, HR-XRD, FT-IR and TNBS assay. The results showed that Ch-Ds-1, Ch-Ds-2 and Ch-Ds-3 were successfully synthesized. The % amino groups of chitosan modified by MFA were evaluated by TNBS assay and ranging from 1.82±0.05% to 7.88±0.04%. All the chitosan derivatives are readily soluble in water and swelled by dimethyl sulfoxide (DMSO), toluene and dimethyl formamide (DMF). The antioxidant activity for all the chitosan derivatives have been significantly improved (P<0.05) compared to the chitosan. Upon antibacterial activity at pH 4.0, all the chitosan derivatives showed significant (P<0.05) antibacterial activity against Gram positive Staphylococcus aureus, Listeria monocytogenes strains and Gram negative Escherichia coli and Salmonella enteritidis strains compared to chitosan. In conclusion, MFA modified chitosan has shown enhanced activities along with solubility, and could be used as a novel food preservative and packaging material for long time food safety and security.

Topics: Anti-Bacterial Agents; Bacteria; Chitosan; Food Preservatives; Food Safety; Fumarates

Mutations of the tricarboxylic acid cycle enzyme fumarate hydratase cause hereditary leiomyomatosis and renal cell cancer. Fumarate hydratase-deficient renal cancers are highly aggressive and metastasize even when small, leading to a very poor clinical outcome. Fumarate, a small molecule metabolite that accumulates in fumarate hydratase-deficient cells, plays a key role in cell transformation, making it a bona fide oncometabolite. Fumarate has been shown to inhibit α-ketoglutarate-dependent dioxygenases that are involved in DNA and histone demethylation. However, the link between fumarate accumulation, epigenetic changes, and tumorigenesis is unclear. Here we show that loss of fumarate hydratase and the subsequent accumulation of fumarate in mouse and human cells elicits an epithelial-to-mesenchymal-transition (EMT), a phenotypic switch associated with cancer initiation, invasion, and metastasis. We demonstrate that fumarate inhibits Tet-mediated demethylation of a regulatory region of the antimetastatic miRNA cluster mir-200ba429, leading to the expression of EMT-related transcription factors and enhanced migratory properties. These epigenetic and phenotypic changes are recapitulated by the incubation of fumarate hydratase-proficient cells with cell-permeable fumarate. Loss of fumarate hydratase is associated with suppression of miR-200 and the EMT signature in renal cancer and is associated with poor clinical outcome. These results imply that loss of fumarate hydratase and fumarate accumulation contribute to the aggressive features of fumarate hydratase-deficient tumours.

Topics: Animals; Cell Movement; Cells, Cultured; Epigenesis, Genetic; Epithelial-Mesenchymal Transition; Fumarate Hydratase; Fumarates; HEK293 Cells; Humans; Kidney Neoplasms; Mesoderm; Mice; MicroRNAs; Transcription Factors; Transcriptome

Motility is a feature of many pathogens that contributes to the migration and dispersion of the infectious agent. Whether gentamycin has a post-antibiotic effect (PAE) on the swarming and swimming motility of Escherichia coli (E. coli) remains unknown. In this study, we aimed to examine whether short-term pretreatment of sub-inhibitory concentrations of gentamycin alter motility of E. coli and the mechanisms involved therein.. After exposure to sub-inhibitory concentrations (0.8 μg/ml) of gentamicin, the swarming and swimming motility of E. coli was tested in semi-solid media. Real-time PCR was used to detect the gene expression of succinate dehydrogenase (SDH). The production of SDH and fumarate by E. coli pretreated with or without gentamycin was measured. Fumarate was added to swarming agar to determine whether fumarate could restore the swarming motility of E. coli.. After pretreatment of E. coli with sub-inhibitory concentrations of gentamycin, swarming motility was repressed in the absence of growth inhibition. The expression of all four subunits of SDH was down-regulated, and the intracellular concentration of SDH and fumarate, produced by E. coli, were both decreased. Supplementary fumarate could restore the swarming motility inhibited by gentamycin. A selective inhibitor of SDH (propanedioic acid) could strongly repress the swarming motility.. Sub-inhibitory concentrations of gentamycin inhibits the swarming motility of E. coli. This effect is mediated by a reduction in cellular fumarate caused by down-regulation of SDH. Gentamycin may be advantageous for treatment of E. coli infections.

Topics: Anti-Bacterial Agents; Bacterial Proteins; Escherichia coli; Flavoproteins; Fumarates; Gene Expression Regulation, Bacterial; Gentamicins; Malonates; Microbial Sensitivity Tests; Movement; Protein Subunits; Signal Transduction; Succinic Acid; Time Factors

In order to relieve the toxicity of furfural on Rhizopus oryzae fermentation, the molecular mechanism of R. oryzae responding to furfural stress for fumaric acid-production was investigated by omics-based approaches. In metabolomics analysis, 29 metabolites including amino acid, sugars, polyols and fatty acids showed significant changes for maintaining the basic cell metabolism at the cost of lowering fumaric acid production. To further uncover the survival mechanism, lipidomics was carried out, revealing that phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol and polyunsaturated acyl chains might be closely correlated with R. oryzae's adapting to furfural stress. Based on the above omics analysis, lecithin, inositol and soybean oil were exogenously supplemented separately with an optimized concentration in the presence of furfural, which increased fumaric acid titer from 5.78g/L to 10.03g/L, 10.05g/L and 12.13g/L (increased by 73.5%, 73.8% and 110%, respectively). These findings provide a methodological guidance for hemicellulose-fumaric acid development.

Topics: Discriminant Analysis; Fermentation; Fumarates; Furaldehyde; Intracellular Space; Least-Squares Analysis; Metabolome; Metabolomics; Phospholipids; Rhizopus; Stress, Physiological; Time Factors; Xylose

To judge the developability and analyze functional mechanism of co-amorphouses, we investigated the physicochemical properties of co-amorphouses and compare the properties with the co-crystals having the same drug and counters. Co-amorphous compounds are a novel approach to improve the physicochemical properties of drugs. A co-amorphous is in an amorphous solid state allowing non-ionic interactions between drug molecules and counter molecules. The co-amorphous compounds composed of itraconazole (ITZ) with the organic carboxyl acid, fumaric acid (FA) or L-tartaric acid (TA), were prepared by mechanical grinding. Potential interactions within ITZ-FA co-amorphous were assessed by Raman spectroscopy. ITZ-FA co-amorphous was not crystallized as the co-crystal or as a single ITZ crystal, suggesting that the amorphous state, like the amorphous solid dispersion, was physically stable and that ITZ-FA co-amorphous was also chemically stable. In contrast, no clear interactions were observed within ITZ-TA co-amorphous, and the co-amorphous was physically stable but chemically unstable. The solubility of the co-amorphous state was much higher than those of ITZ crystal and the co-crystals and was almost identical to that of amorphous ITZ. A co-amorphous compound like ITZ-FA co-amorphous might be feasible to implement in the development of solid drug products and bring some merits compared to the co-crystals, and the function is governed by the interaction between a drug and a counter. The co-amorphous approach may be an effective strategy for drug development and can contribute to the production of novel drugs with improved functions.

Topics: Catalysis; Chemistry, Physical; Cobalt; Crystallization; Fumarates; Itraconazole; Molecular Structure; Solubility; Tartrates

Tomatoes have been described as a functional food because of their particular composition of different bioactive compounds. In this study, the proximate composition, minerals and trace elements, and antioxidant compounds were determined in two tomato cultivars (Mariana and Dunkan) that were grown in Gran Canaria (Spain) either conventionally or hydroponically. Although compositional data of this type require being subjected to the specific statistical techniques of compositional analysis, this approach has not usually been considered in this context. In the present case, a compositional Mann-Whitney U test of the data showed significant differences for each factor (cultivar and cultivation system) in several of the compositional variables studied. For the differences between cultivars, these parameters were the protein, Mg, lycopene, ascorbic acid, citric acid, and fumaric acid contents. For the differences between cultivation systems, they were mainly those of the mineral and trace elements group. Although one-year data are insufficient to make clear relationship among compounds because more repetitions in several localities and years are necessary, the compositional HJ-biplot (in which the links provide estimates of the linear relationship among variables) results agreed with other scientific results about linear relationship among some compounds analyzed.

Topics: Antioxidants; Ascorbic Acid; Carotenoids; Cations, Divalent; Citric Acid; Computer Graphics; Fruit; Fumarates; Hydroponics; Magnesium; Plant Breeding; Plant Proteins; Solanum lycopersicum; Species Specificity; Statistics, Nonparametric

In the years after the discovery of this pathogen, Escherichia coli O157:H7 has become increasingly prominent, and outbreaks have been reported in many areas.. In the current study, we determined the expressions of virulent factor genes shiga-like toxin II (stxII) and E. coli attaching and effacing (eaeA), in strains that were isolated from cattle feces and a clinical case in Taiwan. In addition, the effects of chemical and low-temperature stresses and adaptions on the expressions of virulent factor genes were investigated. Furthermore, the outer membrane proteins of acid-adapted E. coli O157:H7 TWC01 (TWC01) was separated using two-dimensional electrophoresis, and proteins were identified using mass spectrometry in order to illustrate the changes in protein expression after adaption.. Expressions of stxII and eaeA in the TWC01 isolated from a clinical case were higher than those in two strains isolated from cattle feces, and both organic and inorganic acid stresses and adaptions enhance the expression of genes encoding virulent factors in strains. In addition, the outer membrane proteins of TWC01 were regulated under hydrochloric acid adaption, indicating induction of acid tolerance and enhancement adhesion in TWC01. Lactic acid treatment of TWC01 resulted in downregulation of channel protein and adherence-related protein expressions.. The results of this study are helpful in understanding the resistance of locally isolated TWC01 to chemical and low-temperature stresses, and improving the control of this pathogen.

Topics: Adhesins, Bacterial; Animals; Bacterial Outer Membrane Proteins; Cattle; Cold Temperature; Electrophoresis, Gel, Two-Dimensional; Escherichia coli O157; Escherichia coli Proteins; Feces; Fumarates; Humans; Hydrochloric Acid; Lactic Acid; Shiga Toxin 2; Sodium Hypochlorite; Virulence Factors

To introduce a direct method for estimating relaxation and kinetic parameter values from rapid dissolution dynamic nuclear polarization (RD-DNP) NMR time courses.. The analysis relied on a kinetic model that is often used to analyze data in these studies-a unidirectional (bio)chemical reaction with rate constant k1 , coupled to longitudinal relaxation of the magnetization of substrate and product that is characterized by the time constant T1 . The latter value was estimated from the width of the product curve (peak) at the height α relative to the maximum height. We showed α ∼ 0.8 under most conditions, so we measured the interval between the falling and rising parts of the curve at the relative height 0.8. We called this the "fall-minus-rise time at height α," or FmRα , and found that FmR0.8 ∼ T1 . The ratio β = (product signal/substrate signal) when the product is maximal was shown to be equal to k1 T1 . Therefore, k1 = β/FmR0.8 .. FmRα analysis was demonstrated with (13) C NMR RD-DNP data recorded from hemolysates and from previously published data.. FmRα analysis enables immediate estimates of kinetic and relaxation parameters from (13) C NMR RD-DNP data. The values can be used as initial estimates in more extensive computer-based data-regression analysis.

Topics: Animals; Ascorbic Acid; Carbon Isotopes; Fumarates; Glutathione; Humans; Kinetics; Lactic Acid; Magnetic Resonance Spectroscopy; Malates; Mice; Models, Chemical; Models, Statistical; Pyruvic Acid

A multi-vitamin auxotrophic Torulopsis glabrata strain, a pyruvate producer, was further engineered to produce fumaric acid. Using the genome-scale metabolic model iNX804 of T. glabrata, four fumaric acid biosynthetic pathways, involving the four cytosolic enzymes, argininosuccinate lyase (ASL), adenylosuccinate lyase (ADSL), fumarylacetoacetase (FAA), and fumarase (FUM1), were found. Athough single overexpression of each of the four enzymes in the cytosol improved fumaric acid production, the highest fumaric acid titer (5.62 g L(-1) ) was obtained with strain T.G-ASL(H) -ADSL(L) by controlling the strength of ASL at a high level and ADSL at a low level. In order to further improve the production of fumaric acid, the SpMAE1 gene encoding the C4 -dicarboxylic acids transporter was overexpressed in strain T.G-ASL(H) -ADSL(L) -SpMAE1 and the final fumaric acid titer increased to 8.83 g L(-1) . This study provides a novel strategy for fumaric acid biosynthesis by utilizing the urea cycle and the purine nucleotide cycle to enhance the bridge between carbon metabolism and nitrogen metabolism.

Topics: Adenylosuccinate Lyase; Argininosuccinate Lyase; Candida glabrata; Fumarates; Fungal Proteins; Metabolic Engineering; Metabolic Networks and Pathways; Purine Nucleotides; Urea

Ensuring a suitable pH in the culture broth is a major problem in microorganism-assisted industrial fermentation of organic acids. To address this issue, we investigated the physiological changes in Rhizopus oryzae at different extracellular pH levels and attempted to solve the issue of cell shortage under low pH conditions. We compared various parameters, such as membrane fatty acids' composition, intracellular pH, and adenosine triphosphate (ATP) concentration. It was found that the shortage of intracellular ATP might be the main reason for the low rate of fumaric acid production by R. oryzae under low pH conditions. When 1 g/l citrate was added to the culture medium at pH 3.0, the intracellular ATP concentration increased from 0.4 to 0.7 µmol/mg, and the fumaric acid titer was enhanced by 63% compared with the control (pH 3.0 without citrate addition). The final fumaric acid concentration at pH 3.0 reached 21.9 g/l after 96 h of fermentation. This strategy is simple and feasible for industrial fumaric acid production under low pH conditions.

Topics: Acids; Culture Media; Fumarates; Hydrogen-Ion Concentration; Rhizopus

The bacterial strains of the genus Nocardia were used for the bioconversion of fumaric acid to L-malic acid. The ability of the bacterial strain Nocardia sp. CCM 4837/A to produce L-malic acid from fumaric acid was investigated under various conditions. The optimal temperature for the bioconversion was approximately 37 °C, and the optimal pH was around 8.0. The addition of an inductor (fumarate salt) to the fermentation medium was necessary to enhance enzyme activity. The presence of detergent Triton X-100 (0.02-0.1 %) in the reaction mixture rapidly increased the enzyme activity of fumarase. The specific fumarase activity of intact cells Nocardia sp. CCM 4837/A increased from 2.8 to 75 U/mg after optimising the experimental conditions described here. Pretreatment of the Nocardia cells with malonate was not necessary because succinate was not detected as a by-product under our experimental conditions.

Topics: Fumarate Hydratase; Fumarates; Malates; Nocardia

Cocrystal forming ability of antiviral drug acyclovir (ACV) with different coformers was studied. Three cocrystals containing ACV with fumaric acid, malonic acid, and DL-tartaric acid were isolated. Methods of cocrystallization included grinding with dropwise solvent addition and solvent evaporation. The cocrystals were characterized by powder X-ray diffraction, differential scanning calorimetry, and thermogravimetric analysis. The crystal structure of the cocrystal with fumaric acid as conformer was determined by single crystal X-ray diffraction. Formation of supramolecular synthon was observed in the cocrystal. Stability with respect to relative humidity for the three cocrystals was evaluated. The aqueous solubility of the ACV-cocrystal materials was significantly improved with a maximum of malonic acid cocrystal, which was about six times more soluble at 35°C compared with that of parent ACV. The dissolution profile indicates that at any particular dissolution time, the concentration of cocrystals in the solution was higher than that of the parent ACV, and malonic acid cocrystals had a maximum release of about twice than the hydrated ACV.

Topics: Acyclovir; Antiviral Agents; Calorimetry, Differential Scanning; Chemical Phenomena; Crystallization; Crystallography, X-Ray; Dicarboxylic Acids; Drug Compounding; Drug Stability; Fumarates; Hydrogen Bonding; Kinetics; Malonates; Models, Molecular; Molecular Conformation; Pharmaceutic Aids; Powder Diffraction; Solubility; Tartrates; Thermogravimetry

Potent compounds do not necessarily make the best drugs in the market. Consequently, with the aim to describe tools that may be fundamental for refining the screening of candidates for animal and preclinical studies and further development, molecules of different structural classes synthesized within the frame of a broad screening platform were evaluated for their trypanocidal activities, cytotoxicities against murine macrophages J774.1 and selectivity indices, as well as for their ligand efficiencies and structural chemical properties. To advance into their modes of action, we also describe the morphological and ultrastructural changes exerted by selected members of each compound class on the parasite Trypanosoma brucei. Our data suggest that the potential organelles targeted are either the flagellar pocket (compound 77, N-Arylpyridinium salt; 15, amino acid derivative with piperazine moieties), the endoplasmic reticulum membrane systems (37, bisquaternary bisnaphthalimide; 77, N-Arylpyridinium salt; 68, piperidine derivative), or mitochondria and kinetoplasts (88, N-Arylpyridinium salt; 68, piperidine derivative). Amino acid derivatives with fumaric acid and piperazine moieties (4, 15) weakly inhibiting cysteine proteases seem to preferentially target acidic compartments. Our results suggest that ligand efficiency indices may be helpful to learn about the relationship between potency and chemical characteristics of the compounds. Interestingly, the correlations found between the physico-chemical parameters of the selected compounds and those of commercial molecules that target specific organelles indicate that our rationale might be helpful to drive compound design toward high activities and acceptable pharmacokinetic properties for all compound families.

Topics: Animals; Cell Line; Cysteine Proteases; Fumarates; Hydrogen-Ion Concentration; Macrophages; Mice; Mitochondria; Organelles; Piperazine; Piperazines; Piperidines; Trypanocidal Agents; Trypanosoma brucei brucei

Antibacterial and antioxidant monomethyl fumaric acid (MFA) was selected to modify chitosan, using aqueous solution of an ionic liquid as a homogeneous and green reaction media. The chemical structures of resulting polymers were systematically characterized by (1)H NMR, diffusion ordered spectroscopy, solid (13)C NMR and wide-angle X-ray diffraction. The results show that two kinds of MFA modified chitosan materials with totally different chemical structures have been synthesized. One product was a MF-chitosan salt composed of chitosan cation and MFA anion, which was obtained with the mediation of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide. The other one synthesized with the mediation of EDC was a MF-chitosan amide in which MFA and chitosan are covalently attached. Solubility of chitosan has been improved, and MF-chitosan salt can be readily dissolved in water. The antioxidant activity has been enhanced with the introduction of MFA, irrespective of the chemical structure.

Topics: Biphenyl Compounds; Chitosan; Free Radical Scavengers; Fumarates; Ionic Liquids; Picrates; Solubility; Solutions

The Aspergillus fumigatus nonribosomal peptide synthetase FtpA is among the few of this species whose natural product has remained unknown. Both FtpA adenylation domains were characterized in vitro. Fumaric acid was identified as preferred substrate of the first and both l-tyrosine and l-phenylalanine as preferred substrates of the second adenylation domain. Genetically engineered A. fumigatus strains expressed either ftpA or the regulator gene ftpR, encoded in the same cluster of genes, under the control of the doxycycline-inducible tetracycline-induced transcriptional activation (tet-on) cassette. These strains produced fumaryl-l-tyrosine and fumaryl-l-phenylalanine which were identified by liquid chromatography and high-resolution mass spectrometry. Modeling of the first adenylation domain in silico provided insight into the structural requirements to bind fumaric acid as peptide synthetase substrate. This work adds aromatic fumaric acid amides to the secondary metabolome of the important human pathogen A. fumigatus which was previously not known as a producer of these compounds.

Topics: Amides; Aspergillus fumigatus; Chromatography, Liquid; Fumarates; Gene Expression; Mass Spectrometry; Metabolic Engineering; Metabolic Networks and Pathways; Multigene Family; Promoter Regions, Genetic; Recombinant Proteins; Transcription, Genetic

Currently, fumaric acid is produced by catalytic isomerization of maleic acid in aqueous solutions at low pH. Being petroleum based, requiring catalyst, and producing vast amounts of by-products and wastewater, the production of fumaric acid from renewable resources by a "green" process is increasingly attractive. In an aqueous solution, the reaction equilibrium constant of the fumarase-mediated conversion of L-malic acid to fumaric acid is 1:4.2 (fumaric acid to L-malic acid). To shift the reaction equilibrium to fumaric acid, solvent engineering was carried out by varying hydrophilic solvents and their concentrations. Generally, organic solvents may denature fumarase. Therefore, fumarase from Thermus thermophilus was employed to overcome this problem. Ethylene glycol was found more suitable than other solvents. This fumarase was shown to be more stable in 50% than in 70% ethylene glycol. Therefore, a preparation was carried out in 50% ethylene glycol. Under this condition, 54.7% conversion was observed using fumarase for transforming 1 mmol L-malic acid. After precipitation by adapting the pH, and washing to remove residual solvent and substrate, 27% total yield was obtained with 99% purity. The results demonstrated that the alternative green route to produce bio-based fumaric acid via L-malic acid is feasible and viable.

Topics: Bacterial Proteins; Biocatalysis; Enzyme Stability; Fumarate Hydratase; Fumarates; Malates; Solvents; Thermus thermophilus

The fumarate addition reaction mechanism is central to the anaerobic biodegradation pathway of various hydrocarbons, both aromatic (e.g., toluene, ethyl benzene) and aliphatic (e.g., n-hexane, dodecane). Succinate synthase enzymes, which belong to the glycyl radical enzyme family, are the main facilitators of these biochemical reactions. The overall catalytic mechanism that converts hydrocarbons to a succinate molecule involves three steps: (1) initial H-abstraction from the hydrocarbon by the radical enzyme, (2) addition of the resulting hydrocarbon radical to fumarate, and (3) hydrogen abstraction by the addition product to regenerate the radical enzyme. Since the biodegradation of hydrocarbon fuels via the fumarate addition mechanism is linked to bio-corrosion, an improved understanding of this reaction is imperative to our efforts of predicting the susceptibility of proposed alternative fuels to biodegradation. An improved understanding of the fuel biodegradation process also has the potential to benefit bioremediation. In this study, we consider model aromatic (toluene) and aliphatic (butane) compounds to evaluate the impact of hydrocarbon structure on the energetics and kinetics of the fumarate addition mechanism by means of high level ab initio gas-phase calculations. We predict that the rate of toluene degradation is ∼100 times faster than butane at 298 K, and that the first abstraction step is kinetically significant for both hydrocarbons, which is consistent with deuterium isotope effect studies on toluene degradation. The detailed computations also show that the predicted stereo-chemical preference of the succinate products for both toluene and butane are due to the differences in the radical addition rate constants for the various isomers. The computational and kinetic modeling work presented here demonstrates the importance of considering pre-reaction and product complexes in order to accurately treat gas phase systems that involve intra and inter-molecular non-covalent interactions.

Topics: Biocatalysis; Butanes; Enzymes; Free Radicals; Fumarates; Kinetics; Quantum Theory; Stereoisomerism; Succinates; Temperature; Thermodynamics; Toluene

In this work, wheat bran (WB) was utilized as feedstock to synthesize fumaric acid by Rhizopus oryzae. Firstly, the pretreatment process of WB by dilute sulfuric acid hydrolysis undertaken at 100°C for 30min offered the best performance for fumaric acid production. Subsequently, through optimizing the seed culture medium, a suitable morphology (0.55mm pellets diameter) of R. oryzae was obtained. Furthermore, a metabolic-based approach was developed to profile the differences of intracellular metabolites concentration of R. oryzae between xylose (the abundant sugar in wheat bran hydrolysate (WBH)) and glucose metabolism. The xylitol, sedoheptulose 7-phosphate, ribulose 5-phosphate, glucose 6-phosphate, proline and serine were responsible for fumaric acid biosynthesis limitation in xylose fermentation. Consequently, regulation strategies were proposed, leading to a 149% increase in titer (up to 15.4g/L). Finally, by combinatorial regulation strategies the highest production was 20.2g/L from WBH, 477% higher than that of initial medium.

Topics: Biotechnology; Culture Media; Dietary Fiber; Fumarates; Hydrogen-Ion Concentration; Hydrolysis; Rhizopus; Ribulosephosphates; Sugar Phosphates; Xylitol; Xylose

The devastating plant pathogen Sclerotinia sclerotiorum produces copious (up to 50 mM) amounts of oxalic acid, which, for over a quarter century, has been claimed as the pathogenicity determinant based on UV-induced mutants that concomitantly lost oxalate production and pathogenicity. Such a claim was made without fulfilling the molecular Koch's postulates because the UV mutants are genetically undefined and harbour a developmental defect in sclerotial production. Here, we generated oxalate-minus mutants of S. sclerotiorum using two independent mutagenesis techniques, and tested the resulting mutants for growth at different pHs and for pathogenicity on four host plants. The oxalate-minus mutants accumulated fumaric acid, produced functional sclerotia and have reduced ability to acidify the environment. The oxalate-minus mutants retained pathogenicity on plants, but their virulence varied depending on the pH and buffering capacity of host tissue. Acidifying the host tissue enhanced virulence of the oxalate-minus mutants, whereas supplementing with oxalate did not. These results suggest that it is low pH, not oxalic acid itself, that establishes the optimum conditions for growth, reproduction, pathogenicity and virulence expression of S. sclerotiorum. Exonerating oxalic acid as the primary pathogenicity determinant will stimulate research into identifying additional candidates as pathogenicity factors towards better understanding and managing Sclerotinia diseases.

Topics: Ascomycota; Fumarates; Hydrogen-Ion Concentration; Mutagenesis; Oxalic Acid; Plant Diseases; Virulence; Virulence Factors

Disruption of the Krebs cycle is a hallmark of cancer. IDH1 and IDH2 mutations are found in many neoplasms, and germline alterations in SDH genes and FH predispose to pheochromocytoma/paraganglioma and other cancers. We describe a paraganglioma family carrying a germline mutation in MDH2, which encodes a Krebs cycle enzyme. Whole-exome sequencing was applied to tumor DNA obtained from a man age 55 years diagnosed with multiple malignant paragangliomas. Data were analyzed with the two-sided Student's t and Mann-Whitney U tests with Bonferroni correction for multiple comparisons. Between six- and 14-fold lower levels of MDH2 expression were observed in MDH2-mutated tumors compared with control patients. Knockdown (KD) of MDH2 in HeLa cells by shRNA triggered the accumulation of both malate (mean ± SD: wild-type [WT] = 1±0.18; KD = 2.24±0.17, P = .043) and fumarate (WT = 1±0.06; KD = 2.6±0.25, P = .033), which was reversed by transient introduction of WT MDH2 cDNA. Segregation of the mutation with disease and absence of MDH2 in mutated tumors revealed MDH2 as a novel pheochromocytoma/paraganglioma susceptibility gene.

Topics: Citric Acid Cycle; DNA, Neoplasm; Down-Regulation; Exome; Fumarates; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Genetic Predisposition to Disease; Germ-Line Mutation; HeLa Cells; Humans; Malate Dehydrogenase; Malates; Male; Middle Aged; Paraganglioma; Pheochromocytoma; Sequence Analysis, DNA

Geobacter sulfurreducens is one of the dominant bacterial species found in biofilms growing on anodes in bioelectrochemical systems. The intracellular concentrations of reduced and oxidized forms of nicotinamide-adenine dinucleotide (NADH and NAD(+), respectively) and nicotinamide-adenine dinucleotide phosphate (NADPH and NADP(+), respectively) as well as adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) were measured in G. sulfurreducens using fumarate, Fe(III)-citrate, or anodes poised at different potentials (110, 10, -90, and -190 mV (vs. SHE)) as the electron acceptor. The ratios of CNADH/CNAD+ (0.088±0.022) and CNADPH/CNADP+ (0.268±0.098) were similar under all anode potentials tested and with Fe(III)-citrate (reduced extracellularly). Both ratios significantly increased with fumarate as the electron acceptor (0.331±0.094 for NAD and 1.96±0.37 for NADP). The adenylate energy charge (the fraction of phosphorylation in intracellular adenosine phosphates) was maintained near 0.47 under almost all conditions. Anode-growing biofilms demonstrated a significantly higher molar ratio of ATP/ADP relative to suspended cultures grown on fumarate or Fe(III)-citrate. These results provide evidence that the cellular location of reduction and not the redox potential of the electron acceptor controls the intracellular redox potential in G. sulfurreducens and that biofilm growth alters adenylate phosphorylation.

Topics: Adenine Nucleotides; Adenosine Monophosphate; Biofilms; Electrodes; Electron Transport; Ferric Compounds; Fumarates; Geobacter; NADP; Phosphorylation

Fumaric acid, as an important material for polymerization, is highly expected to be produced by fermentation of lignocellulosic biomass which is composed of cellulose, hemicellulose and lignin. Xylose as the main component of hemicellulose cannot be efficiently utilized by most of the common fermentation. In this study, a new strain Rhizopus arrhizus RH 7-13-9# was selected from the R. arrhizus RH 7-13 through a novel convenient and efficient selection method. Efficient production of fumaric acid (45.31 g/L) from xylose was achieved by the new strain, and the volumetric productivity was still 0.472 g/L h. Moreover, the conversion of xylose reached 73% which is close to the theoretic yield (77%). The production of fumaric acid was increased approximate by 172%, compared with the initial strain counterpart. These results indicated that xylose, as the main component of hemicellulose, has a promising application for the production of fumaric acid on an industrial-scale.

Topics: Cells, Immobilized; Chromatography, High Pressure Liquid; Fermentation; Fumarates; Rhizopus; Xylose

In this work, Scheffersomyces stipitis, the yeast with excellent xylose-utilizing ability, was firstly engineered for fumaric acid production from xylose with the heterologous reductive pathway from Rhizopus oryzae FM19, and 1.86g/L fumaric acid was produced by the initial strain PSRPMF under the oxygen-limited condition. Furthermore, three strategies were performed to improve the fumaric acid production, including increasing the reductive pathway activity by codon optimization, blocking the fumaric acid conversion in tricarboxylic acid cycle by knocking out the native fumarases, and improving the fumaric acid transportation by overexpressing heterologous transporter. Finally, the strain PSYPMFfS was obtained and the fumaric acid titer reached to 4.67g/L, significantly increased by 37.92-fold than that of the control strain PSPYSS. It was indicated that the S. stipitis was a promising platform for fumaric acid production from xylose.

Topics: Ascomycota; Fumarates; Genetic Enhancement; Recombinant Proteins; Rhizopus; Species Specificity; Xylose

Shewanella oneidensis MR-1 is capable of reducing extracellular electron acceptors, such as metals and electrodes, through the Mtr respiratory pathway, which consists of the outer membrane cytochromes OmcA and MtrC and associated proteins MtrA and MtrB. These proteins are encoded in the mtr gene cluster (omcA-mtrCAB) in the MR-1 chromosome.. Here, we investigated the transcriptional mechanisms for the mtr genes and demonstrated that omcA and mtrC are transcribed from two upstream promoters, P omcA and P mtrC, respectively. In vivo transcription and in vitro electrophoretic mobility shift assays revealed that a cAMP receptor protein (CRP) positively regulates the expression of the mtr genes by binding to the upstream regions of P omcA and P mtrC. However, the expression of omcA and mtrC was differentially regulated in response to culture conditions; specifically, the expression from P mtrC was higher under aerobic conditions than that under anaerobic conditions with fumarate as an electron acceptor, whereas expression from P omcA exhibited the opposite trend. Deletion of the region upstream of the CRP-binding site of P omcA resulted in a significant increase in promoter activity under aerobic conditions, demonstrating that the deleted region is involved in the negative regulation of P omcA.. Taken together, the present results indicate that transcription of the mtr genes is regulated by multiple promoters and regulatory systems, including the CRP/cAMP-dependent regulatory system and yet-unidentified negative regulators.

Topics: Aerobiosis; Anaerobiosis; ATP-Binding Cassette Transporters; Bacterial Outer Membrane Proteins; Bacterial Proteins; Base Sequence; Chromosomes, Bacterial; Cyclic AMP Receptor Protein; Cytochrome c Group; Electron Transport; Fumarates; Gene Expression Regulation, Bacterial; Molecular Sequence Data; Promoter Regions, Genetic; Shewanella; Transcription, Genetic

Fumaric acid (FA) is an important raw material in the chemical and pharmaceutical industries. In this work, Escherichia coli was metabolically engineered for the production of FA. The fumA, fumB, fumC, and frdABCD genes were deleted to cut off the downstream pathway of FA. In addition, the iclR and arcA genes were also deleted to activate the glyoxylate shunt and to reinforce the oxidative Krebs cycle. To increase the FA yield, this base strain was further engineered to be pulled in the urea cycle by overexpressing the native carAB, argI, and heterologous rocF genes. The metabolites and the proteins of the Krebs cycle and the urea cycle were analyzed to confirm that the induced urea cycle improved the FA accumulation. With the induced urea cycle, the resulting strain ABCDIA-RAC was able to produce 11.38 mmol/L of FA from 83.33 mmol/L of glucose in a flask culture during 24 h of incubation.

Topics: Escherichia coli; Escherichia coli Proteins; Fumarates; Gene Expression Regulation, Bacterial; Metabolic Engineering; Urea

In this study, we have demonstrated a targeted metabolomics method for analysis of cancer cells, based on high-performance ion chromatography (IC) separation, Q Exactive HF MS for high-resolution and accurate-mass (HR/AM) measurement and the use of stable isotope-labeled internal standards for absolute quantitation. Our method offers great technical advantages for metabolite analysis, including exquisite sensitivity, high speed and reproducibility, and wide dynamic range. The high-performance IC provided fast separation of cellular metabolites within 20 min and excellent resolving power for polar molecules including many isobaric metabolites. The IC/Q Exactive HF MS achieved wide dynamic ranges of 5 orders of magnitude for six targeted metabolites, pyruvate, succinic acid, malic acid, citric acid, fumaric acid, and alpha-ketoglutaric acid, with R(2) ≈ 0.99. Using this platform, metabolites can be simultaneously quantified from low fmol/μL to nmol/μL levels in cellular samples. The high flow rate IC at 380 μL/min has shown excellent reproducibility for a large set of samples (150 injections), with minimal variations of retention time (SD < ± 0.03 min). In addition, the IC-MS-based approach acquires targeted and global metabolomic data in a same analytical run, and the use of stable isotope-labeled standards facilitates accurate quantitation of targeted metabolites in large-scale metabolomics analysis. This metabolomics approach has been successfully applied to analysis of targeted metabolites in head and neck cancer cells as well as cancer stem-like cells (CSCs), and the findings indicate that the metabolic phenotypes may be distinct between high and low invasive head and neck cancer cells and between CSCs and non-SCCs.

Topics: Chromatography, High Pressure Liquid; Citric Acid; Fumarates; Head and Neck Neoplasms; Humans; Ketoglutaric Acids; Malates; Mass Spectrometry; Metabolomics; Pyruvic Acid; Succinic Acid

Fumaric acid is an important C4-dicarboxylic acid widely used in chemical, food, and pharmaceutical industries. Rational metabolic engineering together with flux optimization were performed for the development of an Escherichia coli strain capable of efficiently producing fumaric acid. The initial engineered strain, CWF4N overexpressing phosphoenolpyruvate carboxylase (PPC), produced 5.30 g/L of fumaric acid. Optimization of PPC flux by examining 24 types of synthetic PPC expression vectors further increased the titer up to 5.72 g/L with a yield of 0.432 g/g·glucose. Overexpression of the succinate dehydrogenase complex (sdhCDAB) led to an increase in carbon yield up to 0.493 g/g·glucose. Based on this mutant strain, citrate synthase (CS) was combinatorially overexpressed and balanced with PPC using 48 types of synthetic expression vectors. As a result, 6.24 g/L of fumaric acid was produced with a yield of 0.500 g/g·glucose. Fed-batch culture of this final strain allowed production of 25.5 g/L of fumaric acid with a yield of 0.366 g/g·glucose. Deletion of the aspA gene encoding aspartase and supplementation of aspartic acid further increased the fumaric acid titer to 35.1 g/L with a yield of 0.490 g/g·glucose.

Topics: Escherichia coli; Fumarates; Gene Deletion; Gene Expression; Genetic Vectors; Glucose; Metabolic Engineering; Metabolic Networks and Pathways

Heterobis imine Schiff base probe L is able to discriminate geometrical isomers (maleic acid vs fumaric acid) through sharp colorimetric as well as fluorogenic responses even conspicuous with the naked eye. Colorimetric as well as fluorogenic sensing of maleic acid among various carboxylic acids was also demonstrated in ethanol-buffer medium. Sensing behavior of L was corroborated by (1)H NMR spectra, mass spectrometry, and theoretical calculations. Subsequently sensing behavior of L was used to probe maleic acid in starch rich food samples.

Topics: Colorimetry; Fluorescent Dyes; Fluorometry; Food Additives; Fumarates; Imines; Isomerism; Maleates; Molecular Structure; Schiff Bases; Solutions; Time Factors

Rhizopus oryzae is strictly inhibited by biodiesel-based by-product crude glycerol, which results in low fumaric acid production. In this study, evolutionary engineering was employed to activate the glycerol utilization pathway for fumaric acid production. An evolved strain G80 was selected, which could tolerate and utilize high concentrations of crude glycerol to produce 14.9g/L fumaric acid with a yield of 0.248g/g glycerol. Key enzymes activity analysis revealed that the evolved strain displayed a significant upregulation in glycerol dissimilation, pyruvate consumption and reductive tricarboxylic acid pathways, compared with the parent strain. Subsequently, intracellular metabolic profiling analysis showed that amino acid biosynthesis, tricarboxylic acid cycle, fatty acid and stress response metabolites accounted for metabolic difference between two strains. Moreover, a glycerol fed-batch strategy was optimized to obtain the highest fumaric acid production of 25.5g/L, significantly increased by 20.9-fold than that of the parent strain of 1.2g/L.

Topics: Biofuels; Biological Evolution; Fermentation; Fumarates; Glycerol; Metabolic Networks and Pathways; Metabolome; Rhizopus

We previously constructed a Psychrophile-based Simple bioCatalyst (PSCat) reaction system, in which psychrophilic metabolic enzymes are inactivated by heat treatment, and used it here to study the conversion of aspartic acid from fumaric acid mediated by the activity of aspartate ammonia-lyase (aspartase). In Escherichia coli, the biosynthesis of aspartic acid competes with that of L-malic acid produced from fumaric acid by fumarase. In this study, E. coli aspartase was expressed in psychrophilic Shewanella livingstonensis Ac10 heat treated at 50 °C for 15 min. The resultant PSCat could convert fumaric acid to aspartic acid without the formation of L-malic acid because of heat inactivation of psychrophilic fumarase activity. Furthermore, alginate-immobilized PSCat produced high yields of aspartic acid and could be re-used nine times. The results of our study suggest that PSCat can be applied in biotechnological production as a new approach to increase the yield of target compounds.

Topics: Aspartate Ammonia-Lyase; Aspartic Acid; Biocatalysis; Biotechnology; Enzyme Stability; Equipment Reuse; Escherichia coli; Fumarate Hydratase; Fumarates; Hot Temperature; Malates; Shewanella

Ensuring a suitable pH is a major problem in industrial organic acid fermentation. To circumvent this problem, we used a metabolic profiling approach to analyze metabolite changes in Rhizopus oryzae under different pH conditions. A correlation between fumaric acid production and intracellular metabolic characteristics of R. oryzae was revealed by principal component analysis. The results showed that to help cell survival in the presence of low pH, R. oryzae altered amino acid and fatty acid metabolism and promoted sugar or sugar alcohol synthesis, corresponding with a suppressing of energy metabolism, phenylalanine, and tyrosine synthesis and finally resulting in the low performance of fumaric acid production. Based on this observation, 1 % linoleic acid was added to the culture medium in pH 3.0 to decrease the carbon demand for cell survival, and the fumaric acid titer was enhanced by 39.7 % compared with the control (pH 3.0 without linoleic acid addition), reaching 18.3 g/L after 84 h of fermentation. These findings provide new insights into the mechanism by which R. oryzae responds to acidic stress and would be helpful for the development of efficient strategies for fumaric acid production at low pH.

Topics: Amino Acids; Carbohydrate Metabolism; Fumarates; Hydrogen-Ion Concentration; Linoleic Acid; Metabolomics; Rhizopus

The present research account deals with the bioproduction of fumaric acid (FA) from apple pomace ultrafiltration sludge (APUS) and apple pomace (AP) through fermentation. The filamentous fungus Rhizopus oryzae 1526 was used as a biocatalyst and its morphological impact on FA production was analysed in detail. For submerged fermentation, 40 g L(-1) of total solids concentration of APUS, pH 6.0, 30 °C, 200 rpm flask shaking speed and 72 h of incubation were found to be optimum for FA production (25.2 ± 1.0 g L(-1), 0.350 g (L(-1) h(-1))). Broth viscosity (cP), residual reducing sugar (g L(-1)) and ethanol (g L(-1)) produced as by-product, were also analysed. Plastic trays were used for solid state fermentation and at optimized level of moisture and incubation period, 52 ± 2.67 g FA per kg dry weight of AP was obtained. Changes in the total phenolic content (mg g(-1) dry weight of AP) were monitored at regular intervals. Utilization of APUS and AP for the directed synthesis of the high-value platform chemical FA by the fungal strain R. oryzae 1526 was an excellent display of fungal physiological and morphological control over a fermentative product.

Topics: Biomass; Ethanol; Fermentation; Fumarates; Industrial Microbiology; Industrial Waste; Malus; Rhizopus; Waste Products

Glutathione S-transferases (GSTs) are involved in detoxification of xenobiotics such as fumaric acid esters (FAE).. To perform GSTT1 geno- and phenotyping in psoriasis patients treated with FAE to find out whether the responder status and/or occurrence of side-effects are associated with allelic variants and enzymatic activity of GSTT1.. We treated 106 psoriasis patients with FAE. GSTT1 genotyping was performed using PCR, phenotyping was carried out by means of a validated high performance liquid chromatography assay at baseline and under treatment.. The distribution of GSTT1 genotypes was as follows: 31% *A/*A; 49% *A/*0; 20% *0/*0. GSTT1 phenotypes as expressed in enzyme activity significantly differed between conjugators classes. (P < 0.001). GSTT1 activity under treatment was significantly (P = 0.0001) increased when compared with baseline. There were no significant associations between the aforementioned GSTT1 pheno- and genotypes and clinical parameters such as psoriasis area and severity index (PASI)50, adverse effects and FAE dosage (P > 0.05), except for the frequent occurrence of reduction (>50%) of circulating lymphocytes in patients with *0/*0 GSTT1 status (P = 0.036; odds ratio: 6, 95% CI: 1.1-32).. GSTT1 geno- and phenotypes significantly correlate in psoriasis patients and do not substantially differ from healthy controls. Response to FAE does likely not depend on GSTT1. However, *0/*0 GSTT1 status is a predictor for the occurrence of marked reduction of lymphocyte counts under FAE therapy. Notably, FAE seem to enhance GSTT1 enzyme activity in high and low conjugators.

Topics: Administration, Oral; Adult; Aged; Chromatography, High Pressure Liquid; Esters; Female; Fumarates; Genotype; Glutathione Transferase; Humans; Male; Middle Aged; Molecular Sequence Data; Phenotype; Psoriasis

The classical internal standard quantitative NMR (qNMR) method determines the purity of an analyte by the determination of a solution containing the analyte and a standard. Therefore, the standard must meet the requirements of chemical compatibility and lack of resonance interference with the analyte as well as a known purity. The identification of such a standard can be time consuming and must be repeated for each analyte. In contrast, the external standard qNMR method utilizes a standard with a known purity to calibrate the NMR instrument. The external standard and the analyte are measured separately, thereby eliminating the matter of chemical compatibility and resonance interference between the standard and the analyte. However, the instrumental factors, including the quality of NMR tubes, must be kept the same. Any deviations will compromise the accuracy of the results. An innovative qNMR method reported herein utilizes an internal reference substance along with an external standard to assume the role of the standard used in the traditional internal standard qNMR method. In this new method, the internal reference substance must only be chemically compatible and be free of resonance-interference with the analyte or external standard whereas the external standard must only be of a known purity. The exact purity or concentration of the internal reference substance is not required as long as the same quantity is added to the external standard and the analyte. The new method reduces the burden of searching for an appropriate standard for each analyte significantly. Therefore the efficiency of the qNMR purity assay increases while the precision of the internal standard method is retained.

Topics: Aspirin; Calibration; Dioxanes; Drug Industry; Fumarates; Magnetic Resonance Spectroscopy; Reference Standards; Reproducibility of Results; Siloxanes; Technology, Pharmaceutical

The aim of the research was to determine the composition of organic acids in fruit of different cultivars of three pumpkin species. The amount of acids immediately after fruit harvest and after 3 months of storage was compared. The content of organic acids in the examined pumpkin cultivars was assayed using the method of high performance liquid chromatography (HPLC). Three organic acids (citric acid, malic acid, and fumaric acid) were identified in the cultivars, whose content considerably varied depending on a cultivar. Three-month storage resulted in decreased content of the acids in the case of cultivars belonging to Cucurbita maxima and Cucurbita pepo species, while a slight increase was recorded for Cucurbita moschata species.

Topics: Citric Acid; Cucurbita; Fruit; Fumarates; Malates; Plant Extracts

The properties of nitrogen centres acting either as hydrogen-bond or Brønsted acceptors in solid molecular acid-base complexes have been probed by N 1s X-ray photoelectron spectroscopy (XPS) as well as (15)N solid-state nuclear magnetic resonance (ssNMR) spectroscopy and are interpreted with reference to local crystallographic structure information provided by X-ray diffraction (XRD). We have previously shown that the strong chemical shift of the N 1s binding energy associated with the protonation of nitrogen centres unequivocally distinguishes protonated (salt) from hydrogen-bonded (co-crystal) nitrogen species. This result is further supported by significant ssNMR shifts to low frequency, which occur with proton transfer from the acid to the base component. Generally, only minor chemical shifts occur upon co-crystal formation, unless a strong hydrogen bond is formed. CASTEP density functional theory (DFT) calculations of (15)N ssNMR isotropic chemical shifts correlate well with the experimental data, confirming that computational predictions of H-bond strengths and associated ssNMR chemical shifts allow the identification of salt and co-crystal structures (NMR crystallography). The excellent agreement between the conclusions drawn by XPS and the combined CASTEP/ssNMR investigations opens up a reliable avenue for local structure characterization in molecular systems even in the absence of crystal structure information, for example for non-crystalline or amorphous matter. The range of 17 different systems investigated in this study demonstrates the generic nature of this approach, which will be applicable to many other molecular materials in organic, physical, and materials chemistry.

Topics: Benzenesulfonates; Benzoates; Citric Acid; Crystallography, X-Ray; Fumarates; Glutarates; Hydrochloric Acid; Hydrogen Bonding; Malonates; Models, Molecular; Molecular Structure; Oxalic Acid; Protons; Quantum Theory; Salicylates; Spectrophotometry; X-Rays

Leishmania major and Trypanosoma cruzi are the main causes of leishmaniasis and Chagas disease, two endemic parasitosis identified as neglected diseases by the World Health Organization. Fumarate reductase (FR) is a central enzyme in the conversion of fumarate to succinate, an energy releasing path essential for the survival of these protozoans which is also absent in their mammalian hosts. FR can thus be considered as a good candidate for targeting specific inhibition by new drugs designed against L. major and T. cruzi. The lack of tertiary structures available for LmFR and TcFR has limited until now the possibility of performing structure-based drug design. Here we used homology modeling combined with enzyme-cofactor docking to propose tertiary structures for NADH-dependent LmFR and TcFR using an homologous X-ray crystallographic structure of flavine-adenine dinucleotide (FAD) dependent FR from Shewanella frigidimarina (PDB ID: 1QO8) as template. These models were refined and stabilized with/without substrate in the active site using classical molecular dynamics simulations under quasi-physiological conditions. Structural features relevant for understanding the mechanism of action of the enzyme were also analyzed, with special attention to the hydrogen bond network involving the cofactor and water molecules present at the binding sites. A small set of compounds previously synthesized and assayed for their inhibitory capacity against TcFR ([M(mpo)₂] metal complexes with M=Pt(II), Pd(II) and V(IV)O and mpo=2-mercaptopyridine N-oxide) and LmFR (licochalcone A) were screened by protein-ligand docking using the NADH-LmFR and NADH-TcFR models here proposed and validated, gaining insight into their binding modes in each enzyme.

Topics: Amino Acid Sequence; Catalytic Domain; Conserved Sequence; Fumarates; Hydrogen Bonding; Leishmania major; Molecular Docking Simulation; Molecular Sequence Data; NAD; Oxidoreductases Acting on CH-CH Group Donors; Protein Binding; Protein Structure, Secondary; Protozoan Proteins; Sequence Homology, Amino Acid; Structural Homology, Protein; Succinate Dehydrogenase; Succinic Acid; Trypanosoma cruzi

The present work explores brewery wastewater as a novel substrate for fumaric acid production employing the filamentous fungal strain Rhizopus oryzae 1526 through submerged fermentation. The effects of different parameters such as substrate total solid concentrations, fermentation pH, incubation temperature, flask shaking speed, and inoculum size on the fungal morphologies were investigated. Different morphological forms (mycelium clumps, suspended mycelium, and solid/hairy pellets) of R. oryzae 1526 were obtained at different applied fermentation pH, incubation temperature, flask shaking speed, and inoculum size. Among all the obtained morphologies, pellet morphology was found to be the most favorable for enhanced production of fumaric acid for different studied parameters. Scanning electron microscopic investigation was done to reveal the detailed morphologies of the pellets formed under all optimized conditions. With all the optimized growth conditions (pH 6, 25 °C, 200 rpm, 5% (v/v) inoculum size, 25 g/L total solid concentration, and pellet diameter of 0.465 ± 0.04 mm), the highest concentration of fumaric acid achieved was 31.3 ± 2.77 g/L. The results demonstrated that brewery wastewater could be used as a good substrate for the fungal strain R. oryzae 1526 in submerged fermentation for the production of fumaric acid.

Topics: Beer; Bioreactors; Fermentation; Food Industry; Fumarates; Humans; Hydrogen-Ion Concentration; Lactic Acid; Microscopy, Electron, Scanning; Mycelium; Rhizopus; Temperature; Wastewater

The characterization of the metabolites accumulated in the grapes of specific cultivars grown in different climates is of particular importance for viticulturists and enologists. In the present study, the metabolite profiling of grapes from the cultivars, Alvarinho, Arinto and Padeiro de Basto, of two Portuguese Controlled Denomination of Origin (DOC) regions (Vinho Verde and Lisboa) was investigated by gas chromatography-coupled time-of-flight mass spectrometry (GC-TOF-MS) and an amino acid analyzer. Primary metabolites, including sugars, organic acids and amino acids, and some secondary metabolites were identified. Tartaric and malic acids and free amino acids accumulated more in grapes from vines of the DOC region of Vinho Verde than DOC Lisboa, but a principal component analysis (PCA) plot showed that besides the DOC region, the grape cultivar also accounted for the variance in the relative abundance of metabolites. Grapes from the cultivar, Alvarinho, were particularly rich in malic acid and tartaric acids in both DOC regions, but sucrose accumulated more in the DOC region of Vinho Verde.

Topics: Amino Acids; Chromatography, Gas; Citric Acid; Fructose; Fruit; Fumarates; Geography; Glucose; Malates; Maleates; Mass Spectrometry; Metabolome; Metabolomics; Portugal; Principal Component Analysis; Species Specificity; Succinic Acid; Sucrose; Tartrates; Vitis

Under the conditions of fumaric acid fermentation, Rhizopus oryzae ME-F14 possessed at least two respiratory systems. The respiration of mycelia was partially inhibited by the cytochrome respiration inhibitor antimycin A or the alternative respiration inhibitor salicylhydroxamic acid and was completely inhibited in the presence of both antimycin A and salicylhydroxamic acid. During fumaric acid fermentation process, the activity of alternative respiration had a great correlation with fumaric acid productivity; both of them reached peak at the same time. The alternative oxidase gene, which encoded the mitochondrial alternative oxidase responsible for alternative respiration in R. oryzae ME-F14, was cloned and characterized in Escherichia coli. The activity of alternative respiration, the alternative oxidase gene transcription level, as well as the fumaric acid titer were measured under different carbon sources and different carbon-nitrogen ratios. The activity of alternative respiration was found to be comparable to the transcription level of the alternative oxidase gene and the fumaric acid titer. These results indicated that the activity of the alternative oxidase was regulated at the transcription stage under the conditions tested for R. oryzae ME-F14.

Topics: Cloning, Molecular; Cytochromes; Escherichia coli; Fermentation; Fumarates; Gene Expression; Mitochondrial Proteins; Oxidation-Reduction; Oxidoreductases; Plant Proteins; Rhizopus

Water soluble extract (WSE) is an important index for the quality evaluation of Astragali Radix (AR). In this study, the WSE of the wild AR from Shanxi province (SX) and the cultivated AR from Gansu Province (GS) were compared. The WSEs of two types of AR were determined according to the appendix of Chinese pharmacopoeia. Then the WSEs were subjected to NMR analysis, and the obtained data were analyzed using HCA, PCA, OPLS-DA, microarray analysis, and Spearman rank analysis. In addition, the Pearson correlation of differential metabolites were also calculated. The results showed that the WSE content of GS-AR (37.80%) was higher than that of SX-AR (32.13%). The main constituent of WSE was sucrose, and other 18 compounds, including amino acids, organic acids, were also detected. Multivariate analysis revealed that SX-AR contained more choline, succinic acid, citric acid, glutamate, taurine and aspartate, while GS samples contained more sucrose, arginine and fumaric acid. In addition, the Pearson correlations between different metabolites of the two types of AR also showed apparent differences. The results suggested that the WSE of two types of AR differs not only in the content, but also in the chemical compositions. Thus, the cultivation way is important to the quality of AR. This study supplied a new method for the comparison of extract of herbal drugs.

Topics: Arginine; Aspartic Acid; Astragalus propinquus; Choline; Citric Acid; Drugs, Chinese Herbal; Fumarates; Glutamic Acid; Magnetic Resonance Spectroscopy; Multivariate Analysis; Phylogeography; Plant Roots; Plants, Medicinal; Succinic Acid; Sucrose; Taurine

This work investigated the capability of Rhizopus arrhizus to assimilate biodiesel-derived crude glycerol and convert it into fumaric acid. After optimizing the initial glycerol concentration, spore inoculum and yeast extract concentration, smaller pellets (0.7 mm) and higher biomass (3.11 g/L) were obtained when R. arrhizus grew on crude glycerol. It was found that crude glycerol was more suitable than glucose for smaller R. arrhizus pellet forming. When 80 g/L crude glycerol was used as carbon source, the fumaric acid production of 4.37 g/L was obtained at 192 h. With a highest concentration of 22.81 g/L achieved in the co-fermentation of crude glycerol (40 g/L) and glucose (40 g/L) at 144 h, the fumaric acid production was enhanced by 553.6%, compared to the fermentation using glycerol (80 g/L) as sole carbon source. Moreover, the production cost of fumaric acid in co-fermentation was reduced by approximately 14% compared to glucose fermentation.

Topics: Biofuels; Biomass; Culture Media; Fermentation; Fumarates; Glucose; Glycerol; Rhizopus

Human mitochondrial NAD(P)+-dependent malic enzyme (m-NAD(P)-ME) is allosterically activated by the four-carbon trans dicarboxylic acid, fumarate. Previous studies have suggested that the dicarboxylic acid in a trans conformation around the carbon-carbon double bond is required for the allosteric activation of the enzyme. In this paper, the allosteric effects of fumarate analogs on m-NAD(P)-ME are investigated. Two fumarate-insensitive mutants, m-NAD(P)-ME_R67A/R91A and m-NAD(P)-ME_K57S/E59N/K73E/D102S, as well as c-NADP-ME, were used as the negative controls. Among these analogs, mesaconate, trans-aconitate, monomethyl fumarate and monoethyl fumarate were allosteric activators of the enzyme, while oxaloacetate, diethyl oxalacetate, and dimethyl fumarate were found to be allosteric inhibitors of human m-NAD(P)-ME. The IC50 value for diethyl oxalacetate was approximately 2.5 mM. This paper suggests that the allosteric inhibitors may impede the conformational change from open form to closed form and therefore inhibit m-NAD(P)-ME enzyme activity.

Topics: Allosteric Regulation; Cytosol; Enzyme Inhibitors; Fumarates; Humans; Malate Dehydrogenase; Mitochondria

A novel approach based on a hybrid system allowing nanofiltration, bipolar electrodialysis and reactive extraction, was proposed to remove fumaric acid from fermentation broth left after bioconversion of glycerol. The fumaric salts can be concentrated in the nanofiltration process to a high yield (80-95% depending on pressure), fumaric acid can be selectively separated from other fermentation components, as well as sodium fumarate can be conversed into the acid form in bipolar electrodialysis process (stack consists of bipolar and anion-exchange membranes). Reactive extraction with quaternary ammonium chloride (Aliquat 336) or alkylphosphine oxides (Cyanex 923) solutions (yield between 60% and 98%) was applied as the final step for fumaric acid recovery from aqueous streams after the membrane techniques. The hybrid system permitting nanofiltration, bipolar electrodialysis and reactive extraction was found effective for recovery of fumaric acid from the fermentation broth.

Topics: Chromatography, High Pressure Liquid; Dialysis; Electricity; Fermentation; Filtration; Fumarates; Hydrogen-Ion Concentration; Nanotechnology; Permeability; Pressure; Solutions

An ion-exclusion chromatographic method for the simultaneous determination of organic acids in rice wine was developed. An IC-Pak Ion Exclusion column (300 mm x 7.8 mm, 7 microm) was used at 50 degrees C. The mobile phases were H2SO4 (phase A) and acetonitrile (phase B) (98:2, v/v) at a flow rate of 0.5 mL/min. The gradient elution program was as follows: 0-40 min, 0.01 mol/L H2SO4 to 0.02 mol/L H2SO4; 40-50 min, 0.01 mol/L H2SO4. The injection volume was 10 microL. The detection wavelength was set at 210 nm. The results showed that oxalic acid, maleic acid, citric acid, tartaric acid, malic acid, ascorbic acid, succinic acid, lactic, fumaric acid, acetic acid, propionic acid, isobutyric acid and butyric acid were completely separated and determined in 30 min. The linear correlation coefficients were above 0.999 7 in the range of 0.001- 1.000 g/L. Under the optimized conditions, the recoveries of organic acids in rice wine were in the range of 93.4% - 103.8% with the relative standard deviations (RSDs, n = 5) of 0.1% - 1.5%. This method is feasible, convenient, fast, accurate and applicable for the quantitative analysis of the organic acids in rice wine.

Topics: Acids; Chromatography, Gel; Fumarates; Malates; Maleates; Oryza; Oxalic Acid; Tartrates; Wine

Inactivating mutations of the gene encoding the tricarboxylic acid cycle enzyme fumarate hydratase (FH) have been linked to an aggressive variant of hereditary kidney cancer (hereditary leiomyomatosis and renal cell cancer). These tumors accumulate markedly elevated levels of fumarate. Fumarate is among a growing list of oncometabolites identified in cancers with mutations of genes involved in intermediary metabolism. FH-deficient tumors are notable for their pronounced accumulation of the transcription factor hypoxia inducible factor-1α (HIF-1α) and aggressive behavior. To date, HIF-1α accumulation in hereditary leiomyomatosis and renal cell cancer tumors is thought to result from fumarate-dependent inhibition of prolyl hydroxylases and subsequent evasion from von Hippel-Lindau-dependent degradation. Here, we demonstrate a novel mechanism by which fumarate promotes HIF-1α mRNA and protein accumulation independent of the von Hippel-Lindau pathway. Here we demonstrate that fumarate promotes p65 phosphorylation and p65 accumulation at the HIF-1α promoter through non-canonical signaling via the upstream Tank binding kinase 1 (TBK1). Consistent with these data, inhibition of the TBK1/p65 axis blocks HIF-1α accumulation in cellular models of FH loss and markedly reduces cell invasion of FH-deficient RCC cancer cells. Collectively, our data demonstrate a novel mechanism by which pseudohypoxia is promoted in FH-deficient tumors and identifies TBK1 as a novel putative therapeutic target for the treatment of aggressive fumarate-driven tumors.

Topics: Base Sequence; Cell Hypoxia; Cell Line; Chromatin Immunoprecipitation; DNA Primers; Fumarates; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; NF-kappa B; Phosphorylation; Real-Time Polymerase Chain Reaction; RNA, Messenger; Signal Transduction

Productivity of recombinant bovine trypsin using a rice amylase 3D promoter has been studied in transgenic rice suspension culture. Alternative carbon sources were added to rice cell suspension cultures in order to improve the production of recombinant bovine trypsin. It was demonstrated that addition of alternative carbon sources such as succinic acid, fumaric acid and malic acid in the culture medium could increase the productivity of recombinant bovine trypsin 3.8-4.3-fold compared to those in the control medium without carbon sources. The highest accumulated trypsin reached 68.2 mg/L on day 5 in the culture medium with 40 mM fumaric acid. The feasibility of repeated use of the cells for recombinant trypsin production was tested in transgenic rice cell suspension culture with the culture medium containing the combination of variable sucrose concentration and 40 mM fumaric acid. Among the used combinations, the combination of 1% sucrose and 40 mM fumaric acid resulted in a yield of up to 53 mg/L five days after incubation. It also increased 31% (W/W) of dry cell weight and improved 43% of cell viability compared to that in control medium without sucrose. Based on these data, recycling of the trypsin production process with repeated 1% sucrose and 40 mM fumaric acid supplying-harvesting cycles was developed in flask scale culture. Recombinant bovine trypsin could be stably produced with a yield of up to 53-39 mg/L per cycle during five recycling cycles.

Topics: Amylases; Animals; Carbon; Cattle; Cell Culture Techniques; Cells, Cultured; Culture Media; Fumarates; Industrial Microbiology; Malates; Oryza; Plant Proteins; Plants, Genetically Modified; Promoter Regions, Genetic; Recombinant Fusion Proteins; Succinic Acid; Sucrose; Suspensions; Trypsin

The aim of the present work was to design a pH-modified solid dispersion (pH(M)-SD) that can improve the dissolution and bioavailability of poorly water-soluble weakly basic GT0918, a developing anti-prostate cancer drug. To select the appropriate acidifiers, a solubility test was carried out first. Solid dispersions (SDs) containing GT0918 and polyvinylpyrrolidone (PVP) were prepared using a solvent evaporation method and were characterized using dissolution studies in different media. The solid states of the SDs were investigated using scanning electron microscopy (SEM), powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC) and Fourier transformed infrared spectroscopy (FTIR). The in vivo pharmacokinetics of the pH(M)-SDs tablets were also studied in beagle dogs compared to the conventional tablets. The optimized pH(M)-SD (GT0918/PVP/citric acid, 1:2:2 weight ratio) exhibited a significant improvement in the dissolution behavior compared to both the physical mixture and the binary SDs. Solid-state characterization revealed that the amorphous formation of GT0918 in the SDs and the strong H-bonding were only found in the pH(M)-SDs containing citric acid. Furthermore, the GT0918-loaded pH(M)-SD tablets showed a higher AUC and a lower tmax compared to the conventional tablets. Accordingly, the pH(M)-SD might be an efficient route for enhancing the dissolution and bioavailability of poorly water-soluble GT0918.

Topics: Animals; Animals, Inbred Strains; Antineoplastic Agents; Biological Availability; Cinnamates; Citric Acid; Dogs; Drug Compounding; Drugs, Investigational; Excipients; Fumarates; Hydrogen-Ion Concentration; Imidazoles; Male; Nitriles; Oxazoles; Povidone; Prostatic Neoplasms; Random Allocation; Solubility; Succinic Acid; Suspensions; Tablets; Thiohydantoins

To evaluate synergetic effect of slight acidic electrolysed water (SAEW) and fumaric acid (FA) on inactivation of total viable count (TVC) and Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella Typhimurium in fresh beef and to study shelf life and sensory quality of beef.. Inoculated samples was dipped for 1, 3 and 5 min and immersed at 25, 40 and 60°C in SAEW, strong acidic electrolysed water (StAEW) and SAWE + FA. Treated meat was air-packaged and stored at 4 or 10°C. During storage, sampling was performed at 2-day intervals for microbiological and sensory changes. TVC was decontaminated at 40°C for 3 min by more than 3·70 log CFU g(-1) , and examined pathogens were reduced by more than 2·60 log CFU g(-1) with SAEW + FA treatment. This treatment prolonged shelf life of beef meat up to 9 and 7 days when stored at 4 and 10°C, respectively.. The combined treatment of SAEW + FA showed greater bactericidal effect and prolonged shelf life compared with individual treatments.. Combined treatment of SAEW and FA can be a suitable hurdle technology reducing bacteria in fresh beef, substantially enhancing their microbial safety and decreasing pathogens growth during storage.

Topics: Animals; Anti-Bacterial Agents; Cattle; Colony Count, Microbial; Drug Synergism; Electrolysis; Escherichia coli O157; Food Microbiology; Fumarates; Listeria monocytogenes; Meat; Salmonella typhimurium; Staphylococcus aureus; Water

Ischaemia-reperfusion injury occurs when the blood supply to an organ is disrupted and then restored, and underlies many disorders, notably heart attack and stroke. While reperfusion of ischaemic tissue is essential for survival, it also initiates oxidative damage, cell death and aberrant immune responses through the generation of mitochondrial reactive oxygen species (ROS). Although mitochondrial ROS production in ischaemia reperfusion is established, it has generally been considered a nonspecific response to reperfusion. Here we develop a comparative in vivo metabolomic analysis, and unexpectedly identify widely conserved metabolic pathways responsible for mitochondrial ROS production during ischaemia reperfusion. We show that selective accumulation of the citric acid cycle intermediate succinate is a universal metabolic signature of ischaemia in a range of tissues and is responsible for mitochondrial ROS production during reperfusion. Ischaemic succinate accumulation arises from reversal of succinate dehydrogenase, which in turn is driven by fumarate overflow from purine nucleotide breakdown and partial reversal of the malate/aspartate shuttle. After reperfusion, the accumulated succinate is rapidly re-oxidized by succinate dehydrogenase, driving extensive ROS generation by reverse electron transport at mitochondrial complex I. Decreasing ischaemic succinate accumulation by pharmacological inhibition is sufficient to ameliorate in vivo ischaemia-reperfusion injury in murine models of heart attack and stroke. Thus, we have identified a conserved metabolic response of tissues to ischaemia and reperfusion that unifies many hitherto unconnected aspects of ischaemia-reperfusion injury. Furthermore, these findings reveal a new pathway for metabolic control of ROS production in vivo, while demonstrating that inhibition of ischaemic succinate accumulation and its oxidation after subsequent reperfusion is a potential therapeutic target to decrease ischaemia-reperfusion injury in a range of pathologies.

Topics: Adenosine Monophosphate; Animals; Aspartic Acid; Citric Acid Cycle; Disease Models, Animal; Electron Transport; Electron Transport Complex I; Fumarates; Ischemia; Malates; Male; Metabolomics; Mice; Mitochondria; Myocardial Infarction; Myocardium; Myocytes, Cardiac; NAD; Reactive Oxygen Species; Reperfusion Injury; Stroke; Succinate Dehydrogenase; Succinic Acid

The evolved mutant Escherichia coli E2 previously developed for succinate production from glycerol was engineered in this study for fumaric acid production under aerobic conditions. Through deletion of three fumarases, 3.65g/L fumaric acid was produced with the yield of 0.25mol/mol glycerol and a large amount of acetate was accumulated as the main byproduct. In order to reduce acetate production several strategies were attempted, among which increasing the flux of the anaplerotic pathways through overexpression of phosphoenolpyruvate carboxylase gene ppc or the glyoxylate shunt operon aceBA effectively reduced acetate and improved fumaric acid production. In fed-batch culture, the resulting strain EF02(pSCppc) produced 41.5g/L fumaric acid from glycerol with 70% of the maximum theoretical yield and an overall productivity of 0.51g/L/h.

Topics: Acetates; Bacillus subtilis; Batch Cell Culture Techniques; Escherichia coli; Fermentation; Fumarate Hydratase; Fumarates; Gene Deletion; Gene Expression Regulation, Bacterial; Genes, Bacterial; Glycerol; Isocitrate Lyase; Metabolic Engineering; Multienzyme Complexes; Mutation; NADH, NADPH Oxidoreductases; Phosphoenolpyruvate Carboxylase

Topics: Aspirin; Flushing; Fumarates; Germany; Guideline Adherence; Humans; Psoriasis

Fumaric acid is a naturally occurring organic acid that is an intermediate of the tricarboxylic acid cycle. Fungal species belonging to Rhizopus have traditionally been employed for the production of fumaric acid. In this study, Escherichia coli was metabolically engineered for the production of fumaric acid under aerobic condition. For the aerobic production of fumaric acid, the iclR gene was deleted to redirect the carbon flux through the glyoxylate shunt. In addition, the fumA, fumB, and fumC genes were also deleted to enhance fumaric acid formation. The resulting strain was able to produce 1.45 g/L of fumaric acid from 15 g/L of glucose in flask culture. Based on in silico flux response analysis, this base strain was further engineered by plasmid-based overexpression of the native ppc gene, encoding phosphoenolpyruvate carboxylase (PPC), from the strong tac promoter, which resulted in the production of 4.09 g/L of fumaric acid. Additionally, the arcA and ptsG genes were deleted to reinforce the oxidative TCA cycle flux, and the aspA gene was deleted to block the conversion of fumaric acid into L-aspartic acid. Since it is desirable to avoid the use of inducer, the lacI gene was also deleted. To increase glucose uptake rate and fumaric acid productivity, the native promoter of the galP gene was replaced with the strong trc promoter. Fed-batch culture of the final strain CWF812 allowed production of 28.2 g/L fumaric acid in 63 h with the overall yield and productivity of 0.389 g fumaric acid/g glucose and 0.448 g/L/h, respectively. This study demonstrates the possibility for the efficient production of fumaric acid by metabolically engineered E. coli.

Topics: Aerobiosis; Biotechnology; Escherichia coli; Fumarates; Gene Deletion; Gene Expression; Genetic Vectors; Glucose; Metabolic Engineering; Metabolic Flux Analysis; Metabolic Networks and Pathways; Plasmids; Promoter Regions, Genetic

The gene encoding the Krebs cycle enzyme fumarate hydratase (FH) is mutated in hereditary leiomyomatosis and renal cell cancer (HLRCC). Loss of FH activity causes accumulation of intracellular fumarate, which can directly modify cysteine residues to form 2-succinocysteine through succination. We undertook a proteomic-based screen in cells and renal cysts from Fh1 (murine FH)-deficient mice and identified 94 protein succination targets. Notably, we identified the succination of three cysteine residues in mitochondrial Aconitase2 (ACO2) crucial for iron-sulfur cluster binding. We show that fumarate exerts a dose-dependent inhibition of ACO2 activity, which correlates with increased succination as determined by mass spectrometry, possibly by interfering with iron chelation. Importantly, we show that aconitase activity is impaired in FH-deficient cells. Our data provide evidence that succination, resulting from FH deficiency, targets and potentially alters the function of multiple proteins and may contribute to the dysregulated metabolism observed in HLRCC.

Topics: Aconitate Hydratase; Animals; Cell Line; Cysteine; Fumarate Hydratase; Fumarates; Humans; Iron; Kidney Neoplasms; Leiomyomatosis; Mice; Mice, Transgenic; Mitochondria; Neoplastic Syndromes, Hereditary; Proteome; Skin Neoplasms; Succinic Acid; Uterine Neoplasms

To rationally guide fumaric acid production improvement, metabolic profiling approach was performed to analyze metabolite changes of Rhizopus oryzae FM19 under different fermentation conditions. A correlation between the metabolic profiling and fumaric acid production was revealed by principal component analysis as well as partial least squares. Citric acid, oxaloacetic acid, 2-oxoglutarate, lactic acid, proline, alanine, valine, leucine were identified to be mainly responsible for the metabolism difference, which were involved in the Embden-Meyerhof-Parnas, tricarboxylic acid cycle, amino acid metabolism and fatty acid metabolism. Through the further analysis of metabolites changes together with the above pathways, exogenous addition strategies were developed, which resulted in 14% increase of fumaric acid (up to 56.5 g/L) and less by-products. These results demonstrated that metabolic profiling analysis could be successfully applied to the rational guidance of medium optimization and the productivity improvement of value-added compounds.

Topics: Amino Acids; Carbon; Culture Media; Fatty Acids; Fermentation; Fumarates; Gas Chromatography-Mass Spectrometry; Hydrogen-Ion Concentration; Least-Squares Analysis; Metabolome; Metabolomics; Principal Component Analysis; Rhizopus

Topics: Aged; Brain; Dermatologic Agents; Fumarates; Humans; Immune Reconstitution Inflammatory Syndrome; Immunologic Deficiency Syndromes; Leukoencephalopathy, Progressive Multifocal; Male; Psoriasis

Topics: Dermatologic Agents; Dimethyl Fumarate; Female; Fumarates; Humans; Immunologic Deficiency Syndromes; Leukoencephalopathy, Progressive Multifocal; Male

Intensification of the surfactant synthesis by Rhodococcus erythropolis IMV Ac-5017 on different substrates, including industrial waste, as well as the use of surfactant preparations for oil degradation were studied. It was established that the addition of fumarate (0.2 %) and citrate (0.1 %) into the medium with ethanol, n-hexadecane, or glycerol (1-2 %) was accompanied by an increase of conditional surfactant concentration by 1.5-1.7 times compared to the indexes in the medium without organic acids. The intensification of surfactant synthesis in the presence of fumarate and citrate is caused by the increased activity of isocitrate lyase (by 1.2-15-fold) and enzymes of the surfactant biosynthesis (by 2-4.8-fold) compared to their activity in the medium without precursors. The possibility of surfactant synthesis intensification (by 3-4-fold) while cultivating of R. erythropolis IMV Ac-5017 in the medium with oil containing substrates (2 %) and glucose (0.1 %) was shown. The introduction of 0.01 mM Cu(2+) in the exponential growth phase of strain IMV Ac-5017 in the medium with ethanol accompanied by the increasing conditional surfactant concentration by 1.9 times. The highly efficient remediation (92-95 %) of oil (2-2.6 g/L) and Cu(2+) polluted water after treatment with surfactant preparations (native cultural liquid) at low concentrations (5 %) was determined.

Topics: Bacterial Proteins; Biodegradation, Environmental; Citric Acid; Copper; Culture Media; Ethanol; Fumarates; Glycerol; Petroleum; Rhodococcus; Species Specificity; Surface-Active Agents; Water Pollutants, Chemical; Water Pollution; Water Purification

Gene knockout experiments are frequently performed for both fundamental and applied biological research. We developed an integration helper plasmid-based knockout system for more efficient and rapid engineering of Escherichia coli. The integration helper plasmid, pCW611, contains two recombinases that are expressed in the reverse direction by two independent inducible systems. One is Red recombinase under the control of the arabinose-inducible system to induce a recombination event by using the linear gene knockout DNA fragment, while the other is Cre recombinase, which is controlled by the isopropyl β-D-1-thiogalactopyranoside-inducible system to obtain markerless mutant strains. The time and effort required can be reduced with this system because iterative transformation and curing steps are not required. We could delete one target gene in three days by using pCW611. To verify the usefulness of this system, deletion experiments were performed to knock out four target genes individually (adhE, sfcA, frdABCD, and ackA) and two genes simultaneously for two cases (adhE-aspA and sfcA-aspA). Also, sequential deletion of four target genes (fumB, iclR, fumA, and fumC) was successfully performed to make a fumaric acid producing strain. This successfully developed and validated rapid and efficient gene manipulation system should be useful for the metabolic engineering of E. coli.

Topics: Drug Resistance, Bacterial; Escherichia coli; Escherichia coli Proteins; Fumarates; Gene Knockout Techniques; Genes, Bacterial; Genetic Markers; Metabolic Engineering; Plasmids; Recombinases; Reproducibility of Results

Eotaxin-1 is a potent chemoattractant for eosinophils and a critical mediator during the development of eosinophilic inflammation. Fumaric acid is an intermediate product of the citric acid cycle, which is source of intracellular energy. Although fumaric acid ameliorates psoriasis and multiple sclerosis, its involvement in eotaxin-1-mediated effects has not been assessed. In this study, we investigated the effects of fumaric acid on eotaxin-1 expression in a mouse fibroblast cell line. We found that fumaric acid significantly inhibited tumor necrosis factor-α (TNF-α-induced eotaxin-1 expression. This fumaric acid effect was mediated through the inhibition of p38 mitogen-activated protein kinase (MAPK)-dependent nuclear factor (NF)-κB signaling. We also found that fumaric acid operates downstream of MEKK3 during TNF-α-induced NF-κB signaling, which upregulated eotaxin-1 expression. In addition, fumaric acid attenuated expression of CC-chemokine receptor 3 (CCR3), an eotaxin-1 receptor, and adhesion molecules that play important roles in eosinophil binding to induce allergic inflammation. Taken together, these findings indicate that inhibiting TNF-α-induced eotaxin-1 expression by fumaric acid occurs primarily through suppression of NF-κB signaling, which is mediated by inhibiting p38 MAPK and suggest that fumaric acid may be used as a complementary treatment option for eotaxin-1-mediated diseases.

Topics: Animals; Chemokine CCL11; Enzyme-Linked Immunosorbent Assay; Fibroblasts; Fumarates; MAP Kinase Kinase Kinase 3; Mice; NF-kappa B; NIH 3T3 Cells; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Signal Transduction; Tumor Necrosis Factor-alpha

Silver based antimicrobial agents with TiO2 as a host support (AgCl/TiO2) are increasingly being evaluated for disinfection and therapeutic applications. In this work TiO2 supported silver chloride nanocomposites were synthesized and functionalized with fumaric acid. X-ray diffraction results showed that the TiO2 was in anatase phase and AgCI was in the cubic chalcoargyrite phase. The functionalization was confirmed by FTIR analysis. The fumaric acid functionalized AgCl/TiO2 (Fu-AgCl/TiO2) showed uniform particle size distribution in the range of 4-5 nm. The intensity size distribution of Fu-AgCl/TiO2 nanocomposite by DLS showed an average particle diameter of -290 nm with a polydispersity index (P. I.) of 0.47. Further, a high BET surface area of -320.7 m2/g was observed for Fu-AgCl/TiO2 with an average pore size distribution of 3.8 nm. The antimicrobial activity of Fu-AgCl/TiO2 and AgCl/TiO2 was evaluated by determining the MIC and MBC values, and it was observed that Fu-AgCl/TiO2 exhibited better antimicrobial activity as compared to the unfunctionalized nanocomposite. The antimicrobial activity of Fu-AgCl/TiO2 is mainly attributed to its superior physicochemical properties coupled with the synergistic action of both fumaric acid and silver ions. The changes in the cytoplasmic membrane of the cells due to the formation of intracellular ROS were observed by SEM imaging.

Topics: Anti-Bacterial Agents; Fumarates; Microscopy, Electron, Scanning; Nanocomposites; Silver Compounds; Spectroscopy, Fourier Transform Infrared; Titanium; X-Ray Diffraction

It was hypothesized that saliva from patients with erosion exhibits lower protective efficacy compared to saliva from patients without erosion, based on in vitro enamel softening studies. A total of 645 enamel specimens were distributed among seven experimental groups. Saliva was gathered from each of 10 volunteers without clinical signs of dental erosion and from 10 patients exhibiting severe erosive defects. Aliquots of 50 ml of saliva from each patient were mixed with sour drops or citric acid, respectively. Pooled saliva, sour drops and citric acid mixed with water served as controls. The enamel specimens were soaked in the respective mixture for 5 min and were subsequently incubated in pure saliva for 2 min. This cycle was repeated three times, then the specimens were kept in 100 ml of saliva for 8 h. Surface microhardness was evaluated at the beginning of the experiment and after each cycle. During the experiments, microhardness decreased significantly in all groups except for the pure saliva group. For sour drops and citric acid mixed with saliva from patients without erosion, the final microhardness was higher compared to the mixture of the two erosive compounds with saliva from patients with erosion. The storage of saliva for 8 h resulted in a certain amount of rehardening, with the highest level of rehardening being observed in the group that was least demineralized (sour drops plus saliva from patients without erosion). It is concluded that salivary components play a crucial role in the development of dental erosion.

Topics: Animals; Buffers; Candy; Cattle; Citric Acid; Dental Enamel; Fumarates; Gelatin; Glucose; Hardness; Humans; Hydrogen-Ion Concentration; Saliva; Time Factors; Tooth Erosion; Tooth Remineralization

The fumarate addition reaction, catalyzed by the enzyme benzylsuccinate synthase (BSS), is considered to be one of the most intriguing and energetically challenging reactions in biology. BSS belongs to the glycyl radical enzyme family and catalyzes the fumarate addition reaction, which enables microorganisms to utilize hydrocarbons as an energy source under anaerobic conditions. Unfortunately, the extreme sensitivity of the glycyl radical to oxygen has hampered the structural and kinetic characterization of BSS, thereby limiting our knowledge on this enzyme. To enhance our molecular-level understanding of BSS, a computational approach involving homology modeling, docking studies, and molecular dynamics (MD) simulations has been used to deduce the structure of BSS's catalytic subunit (BSSα) and illuminate the molecular basis for the fumarate addition reaction. We have identified two conserved and distinct binding pockets at the BSSα active site: a hydrophobic pocket for toluene binding and a polar pocket for fumaric acid binding. Subsequent dynamical and energetic evaluations have identified Glu509, Ser827, Leu390, and Phe384 as active site residues critical for substrate binding. The orientation of substrates at the active site observed in MD simulations is consistent with experimental observations of the syn addition of toluene to fumaric acid. It is also found that substrate binding tightens the active site and restricts the conformational flexibility of the thiyl radical, leading to hydrogen transfer distances conducive to the proposed reaction mechanism. The stability of substrates at the active site and the occurrence of feasible radical transfer distances between the thiyl radical, substrates, and the active site glycine indicate a substrate-assisted radical transfer pathway governing fumarate addition.

Topics: Amino Acid Sequence; Carbon-Carbon Lyases; Catalytic Domain; Fumarates; Glycine; Molecular Dynamics Simulation; Molecular Sequence Data; Mutation; Sequence Homology; Succinates; Thauera; Toluene

Cenicriviroc mesylate (CVC) is a potent dual antagonist of C-C chemokine receptor type 5 (CCR5) and C-C chemokine receptor type 2 (CCR2) in phase 2b development as an entry inhibitor for HIV-1 infection treatment.1,2 CVC is a weak base exhibiting BCS IV characteristics with a highly pH dependent solubility profile (>100 mg/mL for pH < 2 and <0.2 μg/mL for pH > 4) and low Caco-2 cell line permeability. Previous tablet formulations of CVC, including spray-dried dispersion and a wet granulation with citric acid, had been found unacceptable for commercial use due to chemical and physical instability or unacceptably high excipient loading precluding fixed-dose combinability. A high drug loading, 26% (w/w), acidic microenvironment tablet formulation with fumaric acid solubilizer (1:1 CVC/fumaric acid) and a dry granulation process was developed iteratively through a sequence of prototypes characterized by beagle dog absorption studies, focused beam reflectance measurement (FBRM), dynamic vapor sorption (DVS), and accelerated stability testing. The fumaric acid based dry granulated product demonstrated a mean bioavailability comparable to an oral solution dose in a dog model. Stability and moisture sensitivity of the formulation were improved via the dry granulation process technique and the use of fumaric acid. It is hypothesized that the observed slow dissolution kinetics of fumaric acid prolongs an acidic microenvironment around the agglomerated CVC crystals and excipients leading to increased CVC dissolution and thereby absorption. The fumaric acid formulation also demonstrated absorption resilience to gastric pH extremes in a dog model. This optimized formulation and process enables CVC to be a viable candidate for current HIV treatment paradigms of single once daily fixed-dose combination products.

Topics: Aconitic Acid; Chemistry, Pharmaceutical; Excipients; Fumarates; Solubility; Tablets

The greenhouse gas methane (CH4) contributes substantially to global climate change. As a potential approach to decrease ruminal methanogenesis, the effects of different dosages of fumaric acid (FA) on ruminal microbial metabolism and on the microbial community (archaea, bacteria) were studied using a rumen simulation technique (RUSITEC). FA acts as alternative hydrogen acceptor diverting 2H from methanogenesis of archaea towards propionate formation of bacteria. Three identical trials were conducted with 12 fermentation vessels over a period of 14 days. In each trial, four fermentation vessels were assigned to one of the three treatment groups differing in FA dosage: low fumaric acid (LFA), high fumaric acid (HFA) and without FA (control). FA was continuously infused with the buffer. Grass silage and concentrate served as substrate. FA led to decreases in pH and to higher production rates of total short chain fatty acids (SCFA) mediated by increases in propionate for LFA of 1.69 mmol d(-1) and in propionate and acetate production for HFA of 4.49 and 1.10 mmol d(-1), respectively. Concentrations of NH3-N, microbial crude protein synthesis, their efficiency, degradation of crude nutrients and detergent fibre fraction were unchanged. Total gas and CH4 production were not affected by FA. Effects of FA on structure of microbial community by means of single strand conformation polymorphism (SSCP) analyses could not be detected. Given the observed increase in propionate production and the unaffected CH4 production it can be supposed that the availability of reduction equivalents like 2H was not limited by the addition of FA in this study. It has to be concluded from the present study that the application of FA is not an appropriate approach to decrease the ruminal CH4 production.

Topics: Animals; Bacteria; Eukaryota; Fermentation; Fumarates; Hydrogen; Methane; Rumen

Preferential binding of a new reagent to fumaric acid could be utilized for its estimation in aqueous medium and in commercial fruit juice.

Topics: Beverages; Fluorescence; Fluorescence Resonance Energy Transfer; Fruit; Fumarates; Maleates; Molecular Structure; Quantum Theory; Water

Acyclovir is a well-known antiviral agent. It can be administered in very high doses (from 200 to 1000 mg even three-four times daily). It has absorption problems mainly due to its poor solubility in water (about 0.2 g/100 mL at 25°C) and its oral bioavailability is approximately 15%-20% with a half-life of about 3 h. To improve acyclovir solubility and/or its dissolution properties, two cocrystals of this drug were successfully produced with glutaric acid (AGA1:1) and fumaric acid (AFA1:1) as conformers, using a cogrinding method. Their effective formation was investigated by a broad range of techniques: thermal analysis, Fourier transform infrared spectroscopy, X-ray powder diffraction, solid state nuclear magnetic resonance, and scanning electron microscopy coupled with energy dispersive X-ray spectrometry. The water solubility of the AGA1:1 cocrystal was not improved in comparison to acyclovir, while AFA1:1 showed a slight increased solubility at equilibrium. The main difference was detected in terms of intrinsic dissolution rates (IDR). The IDR of the new phases were much faster compared with acyclovir, particularly at neutral pH. AFA1:1 showed the most rapid dissolution behavior in water; within 10 min, the drug was released completely, while just 60% of acyclovir was dissolved in 1 h.

Topics: Acyclovir; Antiviral Agents; Calorimetry, Differential Scanning; Fumarates; Glutarates; Magnetic Resonance Spectroscopy; Powder Diffraction; Solubility; X-Ray Diffraction

In this study, the simultaneous use of reductive and oxidative routes to produce fumaric acid was explored. The strain FMME003 (Saccharomyces cerevisiae CEN.PK2-1CΔTHI2) exhibited capability to accumulate pyruvate and was used for fumaric acid production. The fum1 mutant FMME004 could produce fumaric acid via oxidative route, but the introduction of reductive route derived from Rhizopus oryzae NRRL 1526 led to lower fumaric acid production. Analysis of the key factors associated with fumaric acid production revealed that pyruvate carboxylase had a low degree of control over the carbon flow to malic acid. The fumaric acid titer was improved dramatically when the heterologous gene RoPYC was overexpressed and 32 μg/L of biotin was added. Furthermore, under the optimal carbon/nitrogen ratio, the engineered strain FMME004-6 could produce up to 5.64 ± 0.16 g/L of fumaric acid. These results demonstrated that the proposed fermentative method is efficient for fumaric acid production.

Topics: Biotin; Carbon; Fermentation; Fumarates; Genetic Engineering; Malates; Nitrogen; Oxidation-Reduction; Pyruvate Carboxylase; Saccharomyces cerevisiae; Urea

While the toxicological effects of mercury (Hg) are well studied in mammals, little is known about the mechanisms of toxicity to bacterial cells lacking an Hg resistance (mer) operon. We determined that Shewanella oneidensis MR-1 is more sensitive to ionic mercury [Hg(II)] under aerobic conditions than in fumarate reducing conditions, with minimum inhibitory concentrations of 0.25 and 2 μM respectively. This increased sensitivity in aerobic conditions is not due to increased import, as more Hg is associated with cellular material in fumarate reducing conditions than in aerobic conditions. In fumarate reducing conditions, glutathione may provide protection, as glutathione levels decrease in a dose-dependent manner, but this does not occur in aerobic conditions. Hg(II) does not change the redox state of thioredoxin in MR1 in either fumarate reducing conditions or aerobic conditions, although thioredoxin is oxidized in Geobacter sulfurreducens PCA in response to Hg(II) treatment. However, treatment with 0.5 μM Hg(II) increases lipid peroxidation in aerobic conditions but not in fumarate reducing conditions in MR-1. We conclude that the enhanced sensitivity of MR-1 to Hg(II) in aerobic conditions is not due to differences in intracellular responses, but due to damage at the cell envelope.

Topics: Antioxidants; Cations, Divalent; Cell Wall; Culture Media; Fumarates; Geobacter; Glutathione; Lipid Peroxidation; Mercury; Oxidation-Reduction; Oxygen; Shewanella; Species Specificity; Thioredoxins

Sildenafil is a drug used to treat erectile dysfunction and pulmonary arterial hypertension. Because of poor aqueous solubility of the drug, the citrate salt, with improved solubility and pharmacokinetics, has been marketed. However, the citrate salt requires an hour to reach its peak plasma concentration. Thus, to improve solubility and bioavailability characteristics, cocrystals and salts of the drug have been prepared by treating aliphatic dicarboxylic acids with sildenafil; the N-methylated piperazine of the drug molecule interacts with the carboxyl group of the acid to form a heterosynthon. Salts are formed with oxalic and fumaric acid; salt monoanions are formed with succinic and glutaric acid. Sildenafil forms cocrystals with longer chain dicarboxylic acids such as adipic, pimelic, suberic, and sebacic acids. Auxiliary stabilization via C-H···O interactions is also present in these cocrystals and salts. Solubility experiments of sildenafil cocrystal/salts were carried out in 0.1N HCl aqueous medium and compared with the solubility of the citrate salt. The glutarate salt and pimelic acid cocrystal dissolve faster than the citrate salt in a two hour dissolution experiment. The glutarate salt exhibits improved solubility (3.2-fold) compared to the citrate salt in water. Solubilities of the binary salts follow an inverse correlation with their melting points, while the solubilities of the cocrystals follow solubilities of the coformer. Pharmacokinetic studies on rats showed that the glutarate salt exhibits doubled plasma AUC values in a single dose within an hour compared to the citrate salt. The high solubility of glutaric acid, in part originating from the strained conformation of the molecule and its high permeability, may be the reason for higher plasma levels of the drug.

Topics: Animals; Area Under Curve; Biological Availability; Crystallization; Dicarboxylic Acids; Fumarates; Glutarates; Male; Oxalic Acid; Permeability; Pimelic Acids; Piperazine; Piperazines; Purines; Rats; Rats, Sprague-Dawley; Salts; Sildenafil Citrate; Solubility; Succinic Acid; Sulfones; Water

We studied the effects of nitrogen concentration on xylose metabolism and organic acid production in Rhizopus oryzae.. We studied the effect of different nitrogen concentration in media on biomass, organic acids accumulation, activity of key enzymes (xylose reductase, XR, glucose-6-phosphate dehydrogenase, G6PD) involved in the metabolism and intracellular redox ratios of R. oryzae.. Both intracellular redox ratios and key enzymes activities stayed low, resulting in 4.02 g/L biomass, 6.55 g/L fumaric acid production and high concentration of residual xylose under nitrogen-limitation conditions (0.15 g/L urea). Intracellular redox ratios and activity of key enzymes increased under rich nitrogen culture (2.4 g/L urea), leading to high biomass 18.01 g/L, high xylose uptake speed 2.03 g/(L x h) but low fumaric acid production (0.27 g/L). When concentration of urea was 0.6 g/L, biomass 9.11 g/L and fumaric acid 12.28 g/L, NADPH/NADP(+), XR and G6PD activities were medium whereas NADH/NAD(+) reached the highest compared to those in rich nitrogen or nitrogen limitation.. Sufficient nitrogen source strengthened the xylose metabolic activity by promoting the key enzymes activity and intracellular redox ratios in R. oryzae. Optimized nitrogen concentration will enhance fumaric acid production with R. oryzae.

Topics: Biomass; Fumarates; Glucosephosphate Dehydrogenase; Nitrogen; Oxidation-Reduction; Rhizopus; Xylose

Two trials were conducted to identify the optimal levels of essential oil active components (EOAC) and their combination with fumarate on in vitro rumen fermentation. In trial 1, eugenol, carvacrol, citral and cinnamaldehyde were mixed at ratios of 1:2:3:4, 2:1:4:3, 3:4:1:2, 4:3:2:1 and 1:1:1:1 to make up five combinations (EOAC1, EOAC2, EOAC3, EOAC4 and EOAC5 respectively). The mixtures were supplied at levels of 0, 50, 200 or 500 mg/l to identify the optimal combination for methane reduction. Methane production and ammonia nitrogen were decreased by adding EOAC, irrespective of component compounds, but the production of gas and total volatile fatty acids (VFA) were also decreased. Hydrogen balance analysis indicated that the ratio of hydrogen consumed via methane to hydrogen consumed via VFA was lowest at 200 mg/l of EOAC5 treatment, from which the proportional change in methane was more than the change in VFA, with 31.5% of methane reduction and 12.9% of VFA reduction. In trial 2, 200 mg/l of EOAC5 was added with 0, 5, 10 and 15 mm monosodium fumarate to see whether fumarate had a further effect on rumen fermentation. The addition of fumarate had no influence on gas production, but it further decreased methane and increased the total VFA in comparison with EOAC added solely, with the greatest decrease occurring in methane (78.1%) from 10 mm of fumarate. Quantification of the microbial populations in rumen fluids by RT-PCR showed that methanogen, protozoa, fungi, Fibrobacter succinogenes and Ruminococcus flavefaciens populations were significantly decreased by EOAC5, but were not influenced by fumarate. In summary, the addition of EOAC had consistent effects on rumen fermentation parameters, but high levels of EOAC would induce the inhibition of rumen fermentation. Adding fumarate can enhance the methane-inhibiting effect of EOAC, and the decrease was higher than that calculated stoichiometrically.

Topics: Animals; Fermentation; Fumarates; Methane; Models, Biological; Oils, Volatile; Reverse Transcriptase Polymerase Chain Reaction; Rumen

The implication of organic acids in Pb translocation was studied in two species varying in shoot lead accumulation, Sesuvium portulacastrum and Brassica juncea. Citric, fumaric, malic and α-cetoglutaric acids were separated and determined by HPLC technique in shoots, roots and xylem saps of the both species grown in nutrient solutions added with 200 and 400 μM of Pb(II). The lead content of the xylem saps was determined by ICP-MS. Results showed that S. portulacastrum is more tolerant to Pb than B. juncea. Lead concentration in xylem sap of the S. portulacastrum was significantly greater than in that of B. juncea. For both species, a positive correlation was established between lead and citrate concentrations in xylem sap. However minor relationship was observed for fumaric, malic and α-cetoglutaric acids. In the shoots lead treatment also induced a significant increase in citric acid concentration. Both observations suggest the implication of citric acid in lead translocation and shoot accumulation in S. portulacastrum and B. juncea. The relatively high accumulation of citric acid in xylem sap and shoot of S. portulacastrum could explain its high potential to translocate and accumulate this metal in shoot suggesting their possible use to remediate Pb polluted soils.

Topics: Aizoaceae; Biological Transport; Citric Acid; Fumarates; Lead; Mustard Plant; Plant Roots; Plant Shoots; Soil Pollutants; Xylem

We investigated the effect of Al (400μM) on organic acids secretion, accumulation and metabolism in Plantago almogravensis Franco and Plantago algarbiensis Samp. Al induced a significant reduction on root elongation only in P. algarbiensis. Both species accumulated considerable amounts of Al (>120μgg(-1)) in their tissues, roots exhibiting the highest contents (>900μgg(-1)). Al stimulated malonic acid secretion in P. algarbiensis, while citric, succinic and malic acids were secreted by P. almogravensis. Moreover, Al uptake was accompanied by substantial increases of citric, oxalic, malonic and fumaric acids contents in the plantlets of either species. Overall, the acid metabolizing enzymes were not directly involved in the Al induced organic acid secretion and accumulation. Our data suggest that Al detoxification in P. almogravensis implies both secretion of organic acids from roots and tolerance to high Al tissue concentrations, while in P. algarbiensis only the tolerance mechanism seems to be involved.

Topics: Adaptation, Physiological; Aluminum; Biological Transport; Carboxylic Acids; Citric Acid; Fumarates; Inactivation, Metabolic; Malates; Malonates; Oxalic Acid; Plant Growth Regulators; Plant Roots; Plantago; Stress, Physiological; Succinic Acid

Refrigerated cucumber pickle products cannot be heat processed due to the loss of characteristic sensory attributes. Typically brined refrigerated pickles contain less than 100 mM acetic acid with pH values of 3.7 to 4.0. Refrigeration (4 to 10 °C) helps to inhibit the growth of spoilage bacteria and maintain flavor, texture, and appearance of the pickles. Previous research has shown that pathogenic Escherichia coli strains are unusually acid resistant and survive better in refrigerated acid solutions than at higher temperatures. We found that E. coli O157:H7 can survive for 1 mo or longer at 4 °C in brines typical of commercial refrigerated pickles. Our objective was to develop methods to assure a 5-log reduction of pathogenic E. coli in these types of products, while maintaining the sensory characteristics. A novel brine formulation was developed, based on current commercial refrigerated pickle brines, which contained 25 mM fumaric acid, 5 mM benzoic acid, 70 mM acetic acid, and 342 mM (2%) sodium chloride, with a pH of 3.8. Sensory data indicate that this formulation did not affect flavor or other sensory attributes of the product, compared to traditional formulations. We achieved a 5-log reduction of E. coli O157:H7 at 30 °C for 1.52 ± 0.15 d, at 20 °C for 3.12 ± 0.34 d, or at 10 °C for 8.83 ± 0.56 d. Growth of lactic acid bacteria was also inhibited. These results can be used by manufacturers to assure a 5-log reduction in cell numbers of E. coli O157:H7 and Salmonella without a heat process during the manufacture of refrigerated pickle products.. While refrigerated acidified vegetable products are exempt from the acidified foods regulations, we have shown that the vegetative microbial pathogens E. coli O157:H7 can survive for up to 1 mo in these products, given current commercial production practices. To improve the safety of refrigerated pickle products, a brine formulation with reduced acetic acid, but containing fumaric acid, was developed to assure a 5-log reduction in cell numbers of E. coli O157:H7 without a heat process. The formulation can be used to assure the safety of refrigerated pickled vegetables without altering sensory characteristics.

Topics: Acetic Acid; Colony Count, Microbial; Consumer Behavior; Consumer Product Safety; Cucumis sativus; Escherichia coli O157; Fermentation; Food Contamination; Food Handling; Food Microbiology; Food Preservation; Fumarates; Humans; Hydrogen-Ion Concentration; Lactic Acid; Nonlinear Dynamics; Refrigeration; Salmonella; Salts

An immobilization method using net was developed for fumaric acid fermentation by Rhizopus arrhizus RH-07-13. The large surface of the net immobilized enough filamentous mycelia which produced fumaric acid rapidly. Net size and spore concentration were optimized to enhance fermentation performance and 150cm(2) of net size, 0.5×10(6)per ml of spore concentration were selected finally. Compared to free-cell fermentation, fumaric acid production was flat (32.03 vs. 31.23g/L) but fermentation time reduced 83.3% (24 vs. 144h).

Topics: Batch Cell Culture Techniques; Bioreactors; Cells, Immobilized; Equipment Design; Equipment Failure Analysis; Fumarates; Glucose; Rhizopus; Ultrafiltration

AZD1305 is a novel, water-soluble investigational antiarrhythmic agent for restoration and maintenance of sinus rhythm in atrial fibrillation patients. The present studies were performed to evaluate the possibility for further development of the compound.. A set of technical approaches were used, including X-ray powder diffractometry, differential scanning calorimetry, thermogravimetrical analysis, dynamic vapor sorption, scanning electron microscopy, salt screen, and liquid chromatography.. AZD1305 is a crystalline oxabispidine and its neutral form is a base with a pK(a) of 9.9. The substance degrades with higher temperature and lower pH. The free base of the solid substance is stable at 25°C (closed container), 40°C/75% relative humidity (open container), and at 50°C (closed container) for at least 3 months. The free base of AZD1305 is polymorphic with two known forms. Both forms are non-hygroscopic ansolvates with melting points of approximately 90°C. No salt was found with overall improved properties. The substance had a strong odor, which was reduced by increased particle size.. The free base of AZD1305 seemed to be the most suitable agent for product development even though it has a fairly low melting point and occurred as two different crystal forms. Form B was the most stable thermodynamically in the temperature interval of interest.

Topics: Administration, Oral; Azabicyclo Compounds; Calorimetry, Differential Scanning; Carbamates; Chemistry, Pharmaceutical; Chromatography, Liquid; Crystallization; Drug Stability; Fumarates; Humidity; Hydrogen-Ion Concentration; Infusions, Intravenous; Maleates; Microscopy, Electron, Scanning; Particle Size; Powders; Salts; Solubility; Solutions; Temperature; Thermogravimetry; Transition Temperature; Wettability; X-Ray Diffraction

The effect of organic acids and mannanoligosaccharide addition to the diet was assessed in pigs orally inoculated with Salmonella typhimurium. Forty-six growers were distributed among four treatments: Basal Diet (BD); BD+encapsulated organic acids; BD+free organic acids; BD+mannanoligosaccharide. Seroconversion was monitored, and feces and tissue samples were tested for Salmonella isolation. No treatment prevented the carrier state, but a tendency of lower fecal excretion was observed in the group treated with mannanoligosaccharide.

Topics: Animals; Carrier State; Citric Acid; Diet; Dietary Supplements; Feces; Formates; Fumarates; Malates; Mannans; Oligosaccharides; Phosphoric Acids; Propionates; Salmonella Infections, Animal; Salmonella typhimurium; Swine; Swine Diseases

Topics: Administration, Topical; Adolescent; Adult; Aged; Aged, 80 and over; Child; Esters; Female; Fumarates; Granuloma Annulare; Humans; Male; Middle Aged; Photochemotherapy; Retrospective Studies; Young Adult

The plasticizer di (2-ethylhexyl) phthalate (DEHP) and its metabolites are considered ubiquitous contaminants, which have a range of implications on the environment and human health. This work considered several alternative compounds with structural features similar to DEHP. This added to the understanding of why DEHP is so poorly biodegraded once it enters the environment. These alternative compounds were based on 2-ethylhexyl diesters of maleic acid (cis-isomer), fumaric acid (trans-isomer) and succinic acid (saturated analogue). The rates of biodegradation by the common soil bacterium Rhodococcus rhodocrous were shown to be dependent on the structure of the central unit derived from the diacid used to make the ester. The diacid components of DEHP and the maleate both had a cis orientation and they were the two that were slow to biodegrade. Plasticizing properties were also compared and, because the ester of the saturated succinic acid was degraded quickly and also had good plasticizing properties, it was concluded that the succinic esters of straight chain alcohols would make the best green plasticizers. The maleate ester had excellent plasticizing properties but this is mitigated by a significant resistance to biodegradation.

Topics: Biodegradation, Environmental; Diethylhexyl Phthalate; Fumarates; Maleates; Plasticizers; Rhodococcus; Succinic Acid

Microbial production of C(4) dicarboxylic acids from renewable resources has gained renewed interest. The yeast Saccharomyces cerevisiae is known as a robust microorganism and is able to grow at low pH, which makes it a suitable candidate for biological production of organic acids. However, a successful metabolic engineering approach for overproduction of organic acids requires an incorporation of a proper exporter to increase the productivity. Moreover, low-pH fermentations, which are desirable for facilitating the downstream processing, may cause back diffusion of the undissociated acid into the cells with simultaneous active export, thereby creating an ATP-dissipating futile cycle. In this work, we have studied the uptake of fumaric acid in S. cerevisiae in carbon-limited chemostat cultures under anaerobic conditions. The effect of the presence of fumaric acid at different pH values (3 to 5) has been investigated in order to obtain more knowledge about possible uptake mechanisms. The experimental results showed that at a cultivation pH of 5.0 and an external fumaric acid concentration of approximately 0.8 mmol · liter(-1), the fumaric acid uptake rate was unexpectedly high and could not be explained by diffusion of the undissociated form across the plasma membrane alone. This could indicate the presence of protein-mediated import. At decreasing pH levels, the fumaric acid uptake rate was found to increase asymptotically to a maximum level. Although this observation is in accordance with protein-mediated import, the presence of a metabolic bottleneck for fumaric acid conversion under anaerobic conditions could not be excluded.

Topics: Anaerobiosis; Biological Transport; Fumarates; Hydrogen-Ion Concentration; Saccharomyces cerevisiae

A mutant strain with high glucoamylase activity and insensitive to catabolite repression was developed to produce fumaric acid by simultaneous saccharification and fermentation (SSF) of starch without additional commercial glucoamylase supplementation. A series of mutant strains resistant to the non-metabolizable and toxic glucose analog 2-deoxyglucose (2-DG) were obtained by implanting nitrogen ion (N(+)) into Rhizopus oryzae ME-F12. Among them, the best mutant strain DG-3 produced 39.80 g/L fumaric acid, which is 1.28-fold of that produced by ME-F12, and exhibited higher glucoamylase activity during SSF. Higher fumaric acid production (44.10 g/L) was achieved when the initial total sugar concentration of cornstarch was 100g/L. During SSF of cheap, raw bioresource-degermed corn powder (100g/L total sugar) by DG-3, the maximum fumaric acid concentration and productivity were 32.18 g/L and 0.44 g/(Lh), respectively.

Topics: Carbohydrate Metabolism; Deoxyglucose; Drug Resistance, Microbial; Fermentation; Fumarates; Mutation; Rhizopus

The present study aimed to develop and characterize new formulations of dipyridamole (DP), a pH-dependent poorly soluble drug, employing an acidic pH-modifier for improving dissolution and absorption under hypochlorhydric condition. Granule formulations of DP (DPG) with and without fumaric acid (FA) were prepared with wet granulation, physicochemical properties of which were characterized focusing on morphology, dissolution and stability. Pharmacokinetic profiling of orally dosed DPG or DPG with 60% loading of FA (DPG/FA60) was carried out in omeprazole-treated rats as a hypochlorhydric model. Although pH-dependent dissolution behavior was observed in DPG, DPG/FA exhibited high rate and extent of dissolution in both acidic and neutral media. Complete supersaturation was achieved with a 2 h testing period in pH6.8 medium, and co-existing fumaric acid had no impact on the chemical/photochemical stability of DP in solid-state. After oral administration of DPG or DPG/FA60 (10 mg-DP/kg), there was ca. 40% reduction of AUC(0-3) for DPG in omeprazole-treated rats as compared to that in normal rats; however, AUC(0-3) for DPG/FA60 under hypochlorhydria was almost identical to that of DPG in normal rats. Given the improved systemic exposure early after oral administration in hypochlorhydric rats, the DPG/FA might provide better clinical outcomes in hypochlorhydric patients.

Topics: Achlorhydria; Administration, Oral; Animals; Area Under Curve; Biological Availability; Chemistry, Pharmaceutical; Chromatography, Liquid; Crystallization; Crystallography, X-Ray; Dipyridamole; Disease Models, Animal; Drug Compounding; Drug Stability; Fumarates; Half-Life; Hydrogen-Ion Concentration; Male; Metabolic Clearance Rate; Microscopy, Electron, Scanning; Omeprazole; Phosphodiesterase Inhibitors; Powder Diffraction; Rats; Rats, Sprague-Dawley; Solubility; Spectrometry, Mass, Electrospray Ionization; Technology, Pharmaceutical

Fumaric acid is a commercially important component of foodstuffs, pharmaceuticals and industrial materials, yet the current methods of production are unsustainable and ecologically destructive.. In this study, the fumarate biosynthetic pathway involving reductive reactions of the tricarboxylic acid cycle was exogenously introduced in S. cerevisiae by a series of simple genetic modifications. First, the Rhizopus oryzae genes for malate dehydrogenase (RoMDH) and fumarase (RoFUM1) were heterologously expressed. Then, expression of the endogenous pyruvate carboxylase (PYC2) was up-regulated. The resultant yeast strain, FMME-001 ↑PYC2 + ↑RoMDH, was capable of producing significantly higher yields of fumarate in the glucose medium (3.18 ± 0.15 g liter-1) than the control strain FMME-001 empty vector.. The results presented here provide a novel strategy for fumarate biosynthesis, which represents an important advancement in producing high yields of fumarate in a sustainable and ecologically-friendly manner.

Topics: Cytosol; Fumarate Hydratase; Fumarates; Fungal Proteins; Malate Dehydrogenase; Pyruvate Carboxylase; Rhizopus; Saccharomyces cerevisiae

Mutation of the APC gene occurs during the early stages of colorectal cancer development. To obtain new insights into the mechanisms underlying the aberrant activation of the Wnt pathway that accompanies APC mutation, we carried out a gas chromatography-mass spectrometry-based semiquantitative metabolome analysis. In vitro experiments comparing SW480 cells expressing normal APC and truncated APC indicated that the levels of metabolites involved in the latter stages of the intracellular tricarboxylic acid cycle, including succinic acid, fumaric acid, and malic acid, were significantly higher in the SW480 cells expressing the truncated APC. In an in vivo study, we found that the levels of most amino acids were higher in the non-polyp tissues of APC(min/+) mice than in the normal tissues of the control mice and the polyp tissues of APC(min/+) mice. Ribitol, the levels of which were decreased in the polyp lesions of the APC(min/+) mice and the SW480 cells expressing the truncated APC, reduced the growth of SW480 cells with the APC mutation, but did not affect the growth of SW480 transfectants expressing full-length APC. The level of sarcosine was found to be significantly higher in the polyp tissues of APC(min/+) mice than in their non-polyp tissues and the normal tissues of the control mice, and the treatment of SW480 cells with 50 μM sarcosine resulted in a significant increase in their growth rate. These findings suggest that APC mutation causes changes in energetic metabolite pathways and that these alterations might be involved in the development of colorectal cancer.

Topics: Adenomatous Polyposis Coli Protein; Amino Acids; Animals; Cell Line, Tumor; Citric Acid Cycle; Colorectal Neoplasms; Fumarates; Gene Expression Regulation, Neoplastic; Genes, APC; Humans; Malates; Metabolomics; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Nude; Mutation; Sarcosine; Succinic Acid; Wnt Signaling Pathway

Femtosecond laser irradiation was employed to induce mutations in Rhizopus oryzae, leading to increases in fumaric acid production. Compared to the parental strain, mutant strain FM19 exhibited an increase in titer and yield of 56.3% and 36.6%, respectively, corresponding to a titer of 49.4 g/L and a yield of 0.56 g fumaric acid per g glucose. Metabolic profiling by gas chromatography-mass spectrometry revealed that higher levels of carbon (Embden-Meyerhof-Parnas and tricarboxylic acid cycle) and amino acid metabolism were operating in the high-yielding strain; particularly, 4-aminobutyric acid and 5-aminolevulinic acid were increased 10.33- and 7.22-fold, respectively, compared with parental strain during stationary phase. These findings provided new insights into metabolic characterization of high-yielding fumaric acid R. oryzae.

Topics: Aspartic Acid Endopeptidases; Fumarates; Mutation

Dihydroorotate dehydrogenase (DHODH) is the fourth enzyme in the de novo pyrimidine biosynthetic pathway and has been exploited as the target for therapy against proliferative and parasitic diseases. In this study, we report the crystal structures of DHODH from Leishmania major, the species of Leishmania associated with zoonotic cutaneous leishmaniasis, in its apo form and in complex with orotate and fumarate molecules. Both orotate and fumarate were found to bind to the same active site and exploit similar interactions, consistent with a ping-pong mechanism described for class 1A DHODHs. Analysis of LmDHODH structures reveals that rearrangements in the conformation of the catalytic loop have direct influence on the dimeric interface. This is the first structural evidence of a relationship between the dimeric form and the catalytic mechanism. According to our analysis, the high sequence and structural similarity observed among trypanosomatid DHODH suggest that a single strategy of structure-based inhibitor design can be used to validate DHODH as a druggable target against multiple neglected tropical diseases such as Leishmaniasis, Sleeping sickness and Chagas' diseases.

Topics: Amino Acid Sequence; Catalytic Domain; Crystallography, X-Ray; Dihydroorotate Dehydrogenase; Fumarates; Humans; Leishmania major; Leishmaniasis; Molecular Sequence Data; Orotic Acid; Oxidoreductases Acting on CH-CH Group Donors; Protein Conformation; Substrate Specificity

Types of nettles (Urtica dioica) were collected from different regions to analyze phenolic compounds in this research. Nettles are specially grown in the coastal part. According to this kind of properties, nettle samples were collected from coastal part of (Mediterranean, Aegean, Black sea, and Marmara) Turkey. Phenolic profile, total phenol compounds, and antioxidant activities of nettle samples were analyzed. Nettles were separated to the part of root, stalk, and leaves. Then, these parts of nettle were analyzed to understand the difference of phenolic compounds and amount of them. Nettle (root, stalk and leaves) samples were analyzed by using High-Performance Liquid Chromatography with Diode-Array Detection (HPLC-DAD) to qualitative and quantitative determination of the phenolic compounds. Total phenolic components were measured by using Folin-Ciocalteu method. The antioxidant activity was measured by using DPPH (2,2-diphenyl-1-picrylhydrazyl) which is generally used for herbal samples and based on single electron transfer (SET).

Topics: Antioxidants; Catechin; Chromatography, High Pressure Liquid; Fumarates; Gallic Acid; Phenols; Plant Leaves; Plant Roots; Plant Stems; Urtica dioica

Alkaline phosphatase (ALP), an enzyme involved in mineralization of bone, is incorporated into three hydrogel biomaterials to induce their mineralization with calcium phosphate (CaP). These are collagen type I, a mussel-protein-inspired adhesive consisting of PEG substituted with catechol groups, cPEG, and the PEG/fumaric acid copolymer OPF. After incubation in Ca-GP solution, FTIR, EDS, SEM, XRD, SAED, ICP-OES, and von Kossa staining confirm CaP formation. The amount of mineral formed decreases in the order cPEG > collagen > OPF. The mineral:polymer ratio decreases in the order collagen > cPEG > OPF. Mineralization increases Young's modulus, most profoundly for cPEG. Such enzymatically mineralized hydrogel/CaP composites may find application as bone regeneration materials.

Topics: Alkaline Phosphatase; Biocompatible Materials; Bone and Bones; Calcification, Physiologic; Calcium Phosphates; Collagen Type I; Elastic Modulus; Fumarates; Humans; Hydrogels; Materials Testing; Microscopy, Electron, Scanning; Polyethylene Glycols; Polymerization; Spectroscopy, Fourier Transform Infrared; Tissue Engineering; Tissue Scaffolds; X-Ray Diffraction

Abstract: A high performance liquid chromatographic method for the simultaneous determination of organic acids and saccharides in lactic acid fermentation broth from biomass was developed. A Bio-Rad Aminex HPX-87H column was used at 55 degrees C. The mobile phase was 5 mmol/L sulfuric acid solution at a flow rate of 0.6 mL/min. The samples were detected by a refractive index detector (RID). The results showed that six organic acids and three saccharides in fermentation broth were completely separated and determined in 17 min. The linear correlation coefficients were above 0.999 8 in the range of 0.15-5.19 g/L. Under the optimized conditions, the recoveries of the organic acids and saccharides in Rhizopus oryzae fermentation broth at two spiked levels were in the range of 96.91%-103.11% with the relative standard deviations (RSDs, n = 6) of 0.81%-4.61%. This method is fast and accurate for the quantitative analysis of the organic acids and saccharides in microbial fermentation broths.

Topics: Acids; Carbohydrates; Chromatography, High Pressure Liquid; Fermentation; Fumarates; Glucose; Lactic Acid; Oxalic Acid; Rhizopus

Fumaric acid, a dicarboxylic acid used as a food acidulant and in manufacturing synthetic resins, can be produced from glucose in fermentation by Rhizopus oryzae. However, the fumaric acid yield is limited by the co-production of ethanol and other byproducts. To increase fumaric acid production, overexpressing endogenous pyruvate carboxylase (PYC) and exogenous phosphoenolpyruvate carboxylase (PEPC) to increase the carbon flux toward oxaloacetate were investigated. Compared to the wild type, the PYC activity in the pyc transformants increased 56%-83%, whereas pepc transformants exhibited significant PEPC activity (3-6 mU/mg) that was absent in the wild type. Fumaric acid production by the pepc transformant increased 26% (0.78 g/g glucose vs. 0.62 g/g for the wild type). However, the pyc transformants grew poorly and had low fumaric acid yields (<0.05 g/g glucose) due to the formation of large cell pellets that limited oxygen supply and resulted in the accumulation of ethanol with a high yield of 0.13-0.36 g/g glucose. This study is the first attempt to use metabolic engineering to modify the fumaric acid biosynthesis pathway to increase fumaric acid production in R. oryzae.

Topics: Bacterial Proteins; Fumarates; Glucose; Metabolic Engineering; Phosphoenolpyruvate Carboxylase; Pyruvate Carboxylase; Rhizopus

Sugars, organic acids, and total phenolic content in fruit of 25 wild and cultivated berry species were identified and quantified with high-performance liquid chromatograph. The composition of sugars, organic acids, and total phenolic compounds in various species of Vaccinium, Rubus, Ribes, and Fragaria genus was evaluated. Additonally, total phenolics of less known berry species of the Morus, Amelanchier, Sorbus, Sambucus, Rosa, Lycium, Actinidia, and Aronia genus were determined in wild growing as well as in cultivated fruits. Significant differences in the concentration of sugars and organic acids were detected among the berry species. Glucose and fructose were the most abundant sugars in berry fruits and the major organic acids were malic and citric acid. However, in kiwi fruit, sucrose represented as much as 71.9% of total sugars. Sorbitol has been detected and quantified in chokeberry, rowanberry, and eastern shadbush fruit. The highest content of total analyzed sugars was determined in rowanberry fruit, followed by dog rose, eastern shadbush, hardy kiwifruit, American cranberry, chokeberry, and jostaberry fruit. Rowanberry stands out as the fruit with the highest content of total analyzed organic acids, followed by jostaberry, lingonberry, red gooseberry, hardy kiwifruit, and black currant. The berries of white gooseberry, black currant, red currant, and white currant had the lowest sugar/organic acid ratio and were thus perceptively the sourest species analyzed. On the other hand, the species with highest sugar/organic acid ratio were goji berry, eastern shadbush, black mulberry, and wild grown blackberry. The highest amounts of total phenols were quantified in chokeberry fruit. Wild strawberry, raspberry, and blackberry had 2- to 5-fold more total phenolics compared to cultivated plants.. The fruit of analyzed berry species contained different levels of sugars, organic acids, and total phenolics. Moreover, it has been demonstrated that wild grown species generally contain more phenolics than cultivated ones. This information is interesting for nutritionists as well as berry growers and breeders who can promote the cultivation of species and new cultivars with higher phenolic content.

Topics: Actinidia; Carbohydrates; Chromatography, High Pressure Liquid; Citric Acid; Fragaria; Fruit; Fumarates; Malates; Phenols; Photinia; Plant Extracts; Ribes; Rosaceae; Sambucus; Shikimic Acid; Sorbus; Tartrates; Vaccinium macrocarpon

A novel biodegradable sodium carboxymethyl cellulose (NaCMC)-based hydrogel was synthesized by using fumaric acid (FA) as a crosslinking agent at various ratios. Hydrogels (CMCF) were characterized using Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), X-ray diffraction analysis (XRD), scanning electron microscopy (SEM), and atomic force microscopy (AFM). Swelling behaviors of hydrogels were investigated in distilled water, various salt, and pH solutions. The FTIR results indicated the crosslinking between carboxyl groups of FA with hydroxyl group of NaCMC through ester formation. AFM analyses showed that roughness of hydrogel surface decreased with increasing crosslinker concentration. The swelling capacity decreased with an increase in charge of the metal cation (Al(3+)

Topics: Calorimetry, Differential Scanning; Carboxymethylcellulose Sodium; Cross-Linking Reagents; Fumarates; Hydrogels; Hydrogen-Ion Concentration; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Osmolar Concentration; Spectroscopy, Fourier Transform Infrared; Thermogravimetry; X-Ray Diffraction

Together with vitamin C, zinc, selenium, manganese, and magnesium play a vital role in the preservation of organs scheduled for transplantation. In the present study, it is shown that addition of 1 mg/l of these elements influences the stability of 0.3 mM ascorbic acid solutions. The solution's stability was estimated using an accelerated stability test. The concentration of vitamin C was measured using a validated spectrophotometric method, which uses the reduction of 2,6-dichlorophenoloindophenol by ascorbic acid. Elevated temperatures, the factor accelerating substances' decomposition reaction rate, were used in the tests. The research was conducted at two temperatures at intervals of 10 °C: 80 ± 0.1 and 90 ± 0.1 °C. It was stated that the studied substances' decomposition occurred in accordance with the equation for first-order reactions. The function of the logarithmic concentration (log%C) over time was revealed to be rectilinear. This dependence was used to determine the kinetics of decomposition reaction rate parameters. The stabilization of vitamin C solutions was measured as the time in which 10 % of the substance decomposed at 20 and 0 °C. Addition of Se(IV) or Mg(II) ions significantly increase the stability of ascorbic acid solution (∼34 and ∼16 %, respectively), but Zn(II) causes a significant decrease in stability by ∼23 %. Addition of Mn(II) has no significant influence on vitamin C stability.

Topics: Antioxidants; Ascorbic Acid; Drug Stability; Fumarates; Hot Temperature; Kinetics; Magnesium; Manganese; Manganese Compounds; Organ Preservation Solutions; Osmolar Concentration; Oxidation-Reduction; Preservatives, Pharmaceutical; Selenium; Sodium Selenite; Sulfates; Trace Elements; Zinc; Zinc Acetate

To improve surgical results in patients with benign prostatic hyperplasia (BPH) and urolithiasis (UL) and to evaluate the efficacy of Furamag used as an agent to prevent infectious and inflammatory complications.. Seventy-two patients with BPH (n = 36; Group 1) and UL (n = 36; Group 2) were examined. Within each group, the patients were divided into two subgroups: A) those in whom no preventive measures were taken during endoscopic operations; B) those who received Furamag as a preventive agent. The preventive efficacy was evaluated from the urine microbial spectrum and renal microcirculatory values.. The preventive use of Furamag could achieve better urine sanitation, normalize renal microcirculatory values, and reduce the incidence of postoperative complications.. The use of Furamag to prevent intravesical obstruction (IVO) during transurethral prostatic resection and UL reduces the incidence of IVO, results in less noticeable renal microcirculatory disorders, and accordingly assists in lowering the incidence of postoperative complications.

Topics: Endoscopy; Fumarates; Humans; Inflammation; Male; Middle Aged; Postoperative Complications; Prostatic Hyperplasia; Treatment Outcome; Urolithiasis

Fumaric acid (FA) is a promising biomass-derived building-block chemical. Bio-based FA production from renewable feedstock is a promising and sustainable alternative to petroleum-based chemical synthesis. Here we report on FA production by direct fermentation using metabolically engineered Saccharomyces cerevisiae with the aid of in silico analysis of a genome-scale metabolic model. First, FUM1 was selected as the target gene on the basis of extensive literature mining. Flux balance analysis (FBA) revealed that FUM1 deletion can lead to FA production and slightly lower growth of S. cerevisiae. The engineered S. cerevisiae strain obtained by deleting FUM1 can produce FA up to a concentration of 610±31 mg L(-1) without any apparent change in growth in fed-batch culture. FT-IR and (1)H and (13)C NMR spectra confirmed that FA was synthesized by the engineered S. cerevisiae strain. FBA identified pyruvate carboxylase as one of the factors limiting higher FA production. When the RoPYC gene was introduced, S. cerevisiae produced 1134±48 mg L(-1) FA. Furthermore, the final engineered S. cerevisiae strain was able to produce 1675±52 mg L(-1) FA in batch culture when the SFC1 gene encoding a succinate-fumarate transporter was introduced. These results demonstrate that the model shows great predictive capability for metabolic engineering. Moreover, FA production in S. cerevisiae can be efficiently developed with the aid of in silico metabolic engineering.

Topics: Biomass; Computer Simulation; Fumarates; Gene Deletion; Magnetic Resonance Spectroscopy; Metabolic Engineering; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Spectroscopy, Fourier Transform Infrared

Nano-structured three-dimensional lead(II) coordination polymer, [Pb(μ(6)-Fum)](n) (1); {Fum=Fumarate}, has been synthesized by sonochemical method and characterized by scanning electron microscopy, X-ray powder diffraction, elemental analyses and IR spectroscopy. Solvent effects on size and morphology of nano-structured compound 1 have been studied. Calcination of the nano-sized compound 1 at 600°C under air atmosphere yields PbO nanoparticles. The thermal stability of compound 1 both its bulk and nano-size has been studied by thermal gravimetric (TG) and differential thermal (DTA) analyses and compared each other.

Topics: Fumarates; Lead; Nanostructures; Organometallic Compounds; Particle Size; Polymers; Surface Properties; Ultrasonics

Plant health and fitness widely depend on interactions with soil microorganisms. Some bacteria such as pseudomonads can inhibit pathogens by producing antibiotics, and controlling these bacteria could help improve plant fitness. In the present study, we tested whether plants induce changes in the antifungal activity of root-associated bacteria as a response to root pathogens. We grew barley plants in a split-root system with one side of the root system challenged by the pathogen Pythium ultimum and the other side inoculated with the biocontrol strain Pseudomonas fluorescens CHA0. We used reporter genes to follow the expression of ribosomal RNA indicative of the metabolic state and of the gene phlA, required for production of 2,4-diacetylphloroglucinol, a key component of antifungal activity. Infection increased the expression of the antifungal gene phlA. No contact with the pathogen was required, indicating that barley influenced gene expression by the bacteria in a systemic way. This effect relied on increased exudation of diffusible molecules increasing phlA expression, suggesting that communication with rhizosphere bacteria is part of the pathogen response of plants. Tripartite interactions among plants, pathogens, and bacteria appear as a novel determinant of plant response to root pathogens.

Topics: Antifungal Agents; Coumaric Acids; Fumarates; Gene Expression; Gene Expression Regulation, Bacterial; Genes, Bacterial; Genes, rRNA; Hordeum; Pest Control, Biological; Phloroglucinol; Plant Diseases; Plant Roots; Propionates; Pseudomonas fluorescens; Pythium; Rhizosphere; Species Specificity; Vanillic Acid

Fumaric acid esters are considered efficacious and safe drugs for the treatment of psoriasis. Renal damage, caused either by acute renal injury or Fanconi syndrome, is a recognized side-effect of this therapy.. To investigate whether the measurement of urinary excretion of β2-microglobulin, a marker of renal proximal tubular dysfunction, allows early detection of kidney damage before an increase in serum creatinine or significant proteinuria occurs.. Urinary β2-microglobulin excretion was measured regularly in 23 patients undergoing fumaric acid ester therapy.. Urinary β2-microglobulin remained normal in all 10 male patients. Three (23%) out of 13 female patients experienced an increase in urinary β2-microglobulin excretion. In two of these patients a sharp increase was observed in association with high doses. One further patient had moderately elevated levels on rather low doses of fumaric acid esters. After discontinuing treatment, urinary β2-microglobulin levels returned to normal within a few weeks.. Determination of urinary β2-microglobulin possibly allows early detection of renal damage by fumaric acid esters. Female patients seem to be prone to this side-effect, especially when taking high doses.

Topics: Acute Kidney Injury; Adult; beta 2-Microglobulin; Biomarkers; Early Diagnosis; Female; Fumarates; Humans; Kidney Function Tests; Male; Middle Aged; Psoriasis

The aim was to design a pH-sensitive pulsatile drug delivery system that allows for an on-off pulsed release of a drug using polyacrylic acid (PAA) blended with ethyl cellulose (EC) in different ratios. PAA, a polyelectrolyte polymer, exhibits a highly coiled conformation at low pH but a highly extended structure at high pH. Fumaric acid, which is an internal acidifying agent, was incorporated into the hydroxypropyl methylcellulose-based core tablets to create an acidic microenvironmental pH (pH(M)). The concentration of fumaric acid inside the core tablet and the ratio of PAA/EC in the coating layer were very crucial in modulating drug release behaviors. When the fumaric acid was retained in the core tablet, it gave a more acidic pH(M), so that the PAA was kept in a highly coiled state in the coated film, which hindered drug release ("off" release pattern). Interestingly, the release profiles of the drug and fumaric acid from coated tablets showed the on-off pulsed pattern upon dissolution. Imaging analyses using scanning electron microscopy, near-infrared imaging, confocal laser scanning microscopy, and Fourier transform infrared spectroscopy confirmed this on-off release behavior of the drug and fumaric acid from coated tablets.

Topics: Acrylic Resins; Bronchodilator Agents; Cellulose; Delayed-Action Preparations; Drug Compounding; Excipients; Fumarates; Hydrogen-Ion Concentration; Hypromellose Derivatives; Methylcellulose; Microscopy, Confocal; Polymers; Solubility; Spectroscopy, Fourier Transform Infrared; Spectroscopy, Near-Infrared; Tablets; Terbutaline

Despite the fact that the organic acid content of a fruit is regarded as one of its most commercially important quality traits when assessed by the consumer, relatively little is known concerning the physiological importance of organic acid metabolism for the fruit itself. Here, we evaluate the effect of modifying malate metabolism in a fruit-specific manner, by reduction of the activities of either mitochondrial malate dehydrogenase or fumarase, via targeted antisense approaches in tomato (Solanum lycopersicum). While these genetic perturbations had relatively little effect on the total fruit yield, they had dramatic consequences for fruit metabolism, as well as unanticipated changes in postharvest shelf life and susceptibility to bacterial infection. Detailed characterization suggested that the rate of ripening was essentially unaltered but that lines containing higher malate were characterized by lower levels of transitory starch and a lower soluble sugars content at harvest, whereas those with lower malate contained higher levels of these carbohydrates. Analysis of the activation state of ADP-glucose pyrophosphorylase revealed that it correlated with the accumulation of transitory starch. Taken together with the altered activation state of the plastidial malate dehydrogenase and the modified pigment biosynthesis of the transgenic lines, these results suggest that the phenotypes are due to an altered cellular redox status. The combined data reveal the importance of malate metabolism in tomato fruit metabolism and development and confirm the importance of transitory starch in the determination of agronomic yield in this species.

Topics: Antisense Elements (Genetics); Fruit; Fumarate Hydratase; Fumarates; Glucose-1-Phosphate Adenylyltransferase; Malate Dehydrogenase; Malates; Phenotype; Plant Proteins; Plants, Genetically Modified; RNA, Plant; Solanum lycopersicum; Starch

Bioelectrochemical systems rely on microorganisms to link complex oxidation/reduction reactions to electrodes. For example, in Shewanella oneidensis strain MR-1, an electron transfer conduit consisting of cytochromes and structural proteins, known as the Mtr respiratory pathway, catalyzes electron flow from cytoplasmic oxidative reactions to electrodes. Reversing this electron flow to drive microbial reductive metabolism offers a possible route for electrosynthesis of high value fuels and chemicals. We examined electron flow from electrodes into Shewanella to determine the feasibility of this process, the molecular components of reductive electron flow, and what driving forces were required. Addition of fumarate to a film of S. oneidensis adhering to a graphite electrode poised at -0.36 V versus standard hydrogen electrode (SHE) immediately led to electron uptake, while a mutant lacking the periplasmic fumarate reductase FccA was unable to utilize electrodes for fumarate reduction. Deletion of the gene encoding the outer membrane cytochrome-anchoring protein MtrB eliminated 88% of fumarate reduction. A mutant lacking the periplasmic cytochrome MtrA demonstrated more severe defects. Surprisingly, disruption of menC, which prevents menaquinone biosynthesis, eliminated 85% of electron flux. Deletion of the gene encoding the quinone-linked cytochrome CymA had a similar negative effect, which showed that electrons primarily flowed from outer membrane cytochromes into the quinone pool, and back to periplasmic FccA. Soluble redox mediators only partially restored electron transfer in mutants, suggesting that soluble shuttles could not replace periplasmic protein-protein interactions. This work demonstrates that the Mtr pathway can power reductive reactions, shows this conduit is functionally reversible, and provides new evidence for distinct CymA:MtrA and CymA:FccA respiratory units.

Topics: ATP-Binding Cassette Transporters; Bacterial Outer Membrane Proteins; Bacterial Proteins; Biofilms; Biosensing Techniques; Biotechnology; Cell Respiration; Electricity; Electrodes; Electron Transport; Energy Metabolism; Feasibility Studies; Fumarates; Metabolic Networks and Pathways; Organisms, Genetically Modified; Oxidation-Reduction; Shewanella

Folate coenzymes and dependent enzymes introduce one carbon units at positions 2 (C(2)) and 8 (C(8)) of the purine ring during de novo biosynthesis. Formate is one source of one-carbon units. Although much is known about lower organisms, little data exists describing formate utilization for purine biosynthesis in humans.. Mass-spectrometric analysis of urinary uric acid, the final purine catabolite, following 1.0 g oral doses of sodium [(13)C] formate was performed and detected (13)C enrichment at C(2) and C(8) separately.. Three phenotypes were suggested. One incorporates (13)C 0.72 to 2.0% into C(2) versus only 0 to 0.07% into C(8). Another incorporates only 0 to 0.05% (13)C into C(2) or C(8). A third phenotype incorporates (13)C into C(8) (0.15%) but C(2) incorporation (0.44%) is still greater. In subjects who incorporated (13)C formate into C(2), peak enrichment occurred in voids from 8-12 h (24 h clock) suggesting a circadian rhythm.. Evidence that mammalian liver introduces C(8) and that C(2) is introduced in a non-hepatic site would explain our results. Our data are not similar to those in non-mammalian organisms or cells in culture and are not consistent with the hypothesis that formate from folate-dependent metabolism in mitochondria is a major one carbon source for purine biosynthesis. Timing of peak (13)C enrichment at C(2) corresponds to maximal DNA synthesis in human bone marrow. Phenotypes may explain the efficacy (or lack of) of certain anticancer and immunosuppressive drugs.

Topics: Adult; Carbon Isotopes; Chromatography, Liquid; Circadian Rhythm; Fumarates; Humans; Liver; Male; Mitochondria, Liver; Phenotype; Purine Nucleotides; Tandem Mass Spectrometry

Cytosolic fumarase, a key enzyme for the accumulation of fumaric acid in Rhizopus oryzae, catalyzes the dehydration of L-malic acid to fumaric acid. The effects of carbon-nitrogen ratio on the acid production and activity of cytosolic fumarase were investigated. Under nitrogen limitation stress, the cytosolic fumarase could keep high activity. With the urea concentration decreased from 2.0 to 0.1 g l⁻¹, the cytosolic fumarase activity increased by 300% and the production of fumaric acid increased from 14.4 to 40.3 g l⁻¹ and L-malic acid decreased from 2.1 to 0.3 g l⁻¹. Cytosolic fumarase could be inhibited by substrate analog 3-hydroxybutyric acid. With the addition of 3-hydroxybutyric acid (50 mM) in the fermentation culture, fumaric acid production decreased from 40.3 to 14.1 g l⁻¹ and L-malic acid increased from 0.3 to 5.4 g l⁻¹.

Topics: 3-Hydroxybutyric Acid; Aspartic Acid; Carbon; Cytosol; Enzyme Inhibitors; Fumarate Hydratase; Fumarates; Nitrogen; Rhizopus; Succinic Acid

Fumarase deficiency (FD), caused by biallelic alteration of the Fumarase Hydratase gene (FH), and a rare metabolic disorder that affects the Krebs cycle, causes severe neurological impairment and fumaric aciduria. Less than 30 unrelated cases are known to date. In addition, heterozygous mutations of the FH gene are responsible for hereditary leiomyomatosis and renal cell cancer (HLRCC). We report three additional patients with dramatically different clinical presentations of FD and novel missense mutations in the FH gene. One patient had severe neonatal encephalopathy, polymicrogyria, <1% enzyme activity, and mildly increased levels of urinary fumarate. The second patient had microcephaly, mental retardation, 20% of fumarase activity, and intermediate levels of urinary fumarate. The third patient had mild mental retardation, polymicrogyria, 42-61% enzyme activity in different cell types and massive amounts of urinary fumarate. In silico analysis predicted minor yet significant structural changes in the encoded proteins. The nuclear translocation of hypoxia-inducible factor (HIF)-1alpha (HIF1A) in cultured fibroblasts was similar to controls. These results extend the range of clinical and biochemical variation associated with FD, supporting the notion that patients with moderate increases in fumarate excretion should be investigated for this disease. The tumoral risk in the patients and their relatives requires adequate screening protocols.

Topics: Cell Hypoxia; Child; Child, Preschool; Computer Simulation; Female; Fumarate Hydratase; Fumarates; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Malformations of Cortical Development; Mutation; Signal Transduction

The paper presents an on-line transient moving chemical reaction boundary (MCRB) method for simply but efficiently stacking analytes in capillary electrophoresis (CE). The CE technique was developed for a rapid determination of fumaric and maleic acid. Based on the theory of MCRB, Effects of several important factors such as the pH and concentration of running buffer and the conditions of stacking analytes were investigated to acquire the optimum conditions. The optimized separations were carried out in a 20 mmol/L sulphate neutralized with ethylenediamine to pH 6.0 electrolytes using a capillary coated with poly (diallyldimethylammonium chloride) and direct UV detection at 214 nm. The optimized preconcentrations were carried out in 50 mmol/L borax (pH 9.0). The calibration curves were linear in the concentration range of 1.0×10⁻⁷-1.0×10⁻⁴ mol/L and 5.0×10⁻⁷-1.0×10⁻⁴ mol/L for fumaric and maleic acid with correlation coefficients higher than 0.9991. The detection limits were 5.34×10⁻⁸ mol/L for fumaric acid and 1.92×10⁻⁷ mol/L for maleic acid. This method was applied for determination of fumaric acid in apple juice and of fumaric and maleic acid in dl-malic, the recovery tests established for real samples were within the range 95-105%. This work provided a valid and simple approach to detect fumaric and maleic acid.

Topics: Alkalies; Borates; Electrophoresis, Capillary; Ethylenediamines; Fumarates; Hydrogen-Ion Concentration; Linear Models; Maleates; Reproducibility of Results; Sensitivity and Specificity; Sulfuric Acids

(rac)-1,1'-Binaphthyl-based simple receptors 1 and 2 have been designed, synthesized and studied theoretically. The receptors utilize naphthyridine as the binding motifs for complexation of dicarboxylic acids in CHCl(3). The emission of the BINOL moiety was monitored experimentally to ascertain the selectivity and sensitivity of the receptors. Receptor 1 distinguishes maleic acid from isomeric fumaric acid by exhibiting different fluorescence behavior and demonstrates stronger binding in the excited state. Modulation of the binding sites of 1 leads to a new receptor structure 2, which was found to be less efficient in distinguishing maleic from fumaric acid, fluorometrically. Both 1 and 2 also recognize other hydroxy di- and tricarboxylic acids. The binding interactions were monitored by (1)H NMR, fluorescence and UV-vis spectroscopic methods. Structures of apo-hosts, guests and host-guest complexes were determined using force-field based conformational searching. Low energy ensembles were grouped into geometrically similar families, and low energy structures from each family were verified using B3LYP/6-31G*/PB-SCRF(CHCl(3)) calculations. The atomistic calculations provide insight into the differential dicarboxylic acid binding behavior of receptors 1 and 2.

Topics: Computer Simulation; Fluorometry; Fumarates; Magnetic Resonance Spectroscopy; Maleates; Molecular Structure; Naphthalenes

Aspartate ammonia lyases (or aspartases) catalyze the reversible deamination of L-aspartate into fumarate and ammonia. The lack of crystal structures of complexes with substrate, product, or substrate analogues so far precluded determination of their precise mechanism of catalysis. Here, we report crystal structures of AspB, the aspartase from Bacillus sp. YM55-1, in an unliganded state and in complex with L-aspartate at 2.4 and 2.6 Å resolution, respectively. AspB forces the bound substrate to adopt a high-energy, enediolate-like conformation that is stabilized, in part, by an extensive network of hydrogen bonds between residues Thr101, Ser140, Thr141, and Ser319 and the substrate's β-carboxylate group. Furthermore, substrate binding induces a large conformational change in the SS loop (residues G(317)SSIMPGKVN(326)) from an open conformation to one that closes over the active site. In the closed conformation, the strictly conserved SS loop residue Ser318 is at a suitable position to act as a catalytic base, abstracting the Cβ proton of the substrate in the first step of the reaction mechanism. The catalytic importance of Ser318 was confirmed by site-directed mutagenesis. Site-directed mutagenesis of SS loop residues, combined with structural and kinetic analysis of a stable proteolytic AspB fragment, further suggests an important role for the small C-terminal domain of AspB in controlling the conformation of the SS loop and, hence, in regulating catalytic activity. Our results provide evidence supporting the notion that members of the aspartase/fumarase superfamily use a common catalytic mechanism involving general base-catalyzed formation of a stabilized enediolate intermediate.

Topics: Ammonia; Aspartate Ammonia-Lyase; Bacillus; Bacterial Proteins; Catalytic Domain; Crystallography, X-Ray; Fumarates; Ligands; Multigene Family; Protein Binding; Protein Conformation; Quantitative Structure-Activity Relationship; Serine; Substrate Specificity

Quantification of fumaric acid, an endogenous dicarboxylic acid with interesting biomedical applications either through its own biological activity or as a linker constitutive of the porous iron(III) fumarate metal organic framework (MOF) MIL-88A based drug nanocarrier (MIL stands for Material from Institut Lavoisier), has been developed in different rat biological complex media (liver, spleen and urine). After a liquid-liquid extraction procedure, fumaric acid concentration was determined by a simple and accurate high-performance liquid chromatography (HPLC) method coupled to a photodiode-array detector (PDA) using aminosalicylic acid as internal standard (IS) and a gradient elution. The recovery of fumaric acid reaches 89% and 92% for urine (for concentrations of 0.05 and 1μgml(-1), respectively) and 90% for liver and spleen tissues, exceeding 89% in all instances in comparison with the IS. Linearity has been kept from 0.05 to 1μgml(-1) and from 0.5 to 10μgg(-1) of fumaric acid in urine and tissues, respectively. The limit of detection of the method was 0.01μg per injection. This method has finally allowed the quantification of fumaric acid in rat urine and tissue samples after the intravenous administration of MIL-88A nanoparticles.

Topics: Animals; Calibration; Chromatography, High Pressure Liquid; Drug Monitoring; Female; Fumarates; Liver; Rats; Rats, Wistar; Reference Standards; Reproducibility of Results; Spleen

The naturally occurring compound, fumaric acid, was evaluated as a potential preservative for the long-term storage of cucumbers. Fumaric acid inhibited growth of lactic acid bacteria (LAB) in an acidified cucumber juice medium model system resembling conditions that could allow preservation of cucumbers in the presence of sodium benzoate. Forty millimolars of fumaric acid were required to inhibit growth of an extremely aciduric Lactobacillus plantarum LA0445 strain at pH 3.8. Half of this concentration was required to achieve inhibition of L. plantarum LA0445 at pH 3.5. As expected growth of the spoilage yeasts Zygosaccharomyces globiformis and Z. bailii was not inhibited by fumaric acid at near saturation concentrations in the same cucumber juice medium. To usefully apply fumaric acid as a preservative in acidified foods it will be necessary to combine it with a food grade yeast inhibitor. The antimicrobial agent, cinnamaldehyde (3.8 mM) prevented growth of Z. globiformis as well as the yeasts that were present on fresh cucumbers. Acidified cucumbers were successfully preserved, as indicated by lack of yeasts or LAB growth and microbial lactic acid or ethanol production by a combination of fumaric acid and cinnamaldehyde during storage at 30 °C for 2 mo. This combination of 2 naturally occurring preservative compounds may serve as an alternative approach to the use of sodium benzoate, potassium sorbate, or sodium metabisulfite for preservation of acidified vegetables without a thermal process.. This study evaluates the potential application of alternative preservatives for the long-term storage of cucumbers in a reduced NaCl cover brine solution and without a thermal process.

Topics: Acrolein; Anti-Infective Agents; Cucumis sativus; Fermentation; Food Preservation; Food Preservatives; Fumarates; Hydrogen-Ion Concentration; Lactobacillus plantarum; Zygosaccharomyces

Fumarate hydratase (FH) is an enzyme of the tricarboxylic acid cycle (TCA cycle) that catalyses the hydration of fumarate into malate. Germline mutations of FH are responsible for hereditary leiomyomatosis and renal-cell cancer (HLRCC). It has previously been demonstrated that the absence of FH leads to the accumulation of fumarate, which activates hypoxia-inducible factors (HIFs) at normal oxygen tensions. However, so far no mechanism that explains the ability of cells to survive without a functional TCA cycle has been provided. Here we use newly characterized genetically modified kidney mouse cells in which Fh1 has been deleted, and apply a newly developed computer model of the metabolism of these cells to predict and experimentally validate a linear metabolic pathway beginning with glutamine uptake and ending with bilirubin excretion from Fh1-deficient cells. This pathway, which involves the biosynthesis and degradation of haem, enables Fh1-deficient cells to use the accumulated TCA cycle metabolites and permits partial mitochondrial NADH production. We predicted and confirmed that targeting this pathway would render Fh1-deficient cells non-viable, while sparing wild-type Fh1-containing cells. This work goes beyond identifying a metabolic pathway that is induced in Fh1-deficient cells to demonstrate that inhibition of haem oxygenation is synthetically lethal when combined with Fh1 deficiency, providing a new potential target for treating HLRCC patients.

Topics: Animals; Bilirubin; Cell Line; Cells, Cultured; Citric Acid Cycle; Computer Simulation; Fumarate Hydratase; Fumarates; Genes, Lethal; Genes, Tumor Suppressor; Glutamine; Heme; Heme Oxygenase (Decyclizing); Kidney Neoplasms; Leiomyomatosis; Mice; Mitochondria; Mutation; NAD; Neoplastic Syndromes, Hereditary; Skin Neoplasms; Uterine Neoplasms

The effects of temperature, agitation rate and medium composition, including concentrations of glucose, soybean peptone, and inorganic ions, on pellet formation and pellet diameter of Rhizopus delemar (Rhizopus oryzae) NRRL1526 during pre-culture were studied. Inorganic ions and soybean peptone had negative and positive effects on pellet formation, respectively. The initial glucose and soybean peptone concentrations directly affected pellet diameter. Within a certain range, pellet diameter decreased with increased initial substrate concentrations; however, above this range there was an opposite trend. Thus, optimal concentrations of substrate during pre-culture were beneficial for producing small pellets of R. delemar. Furthermore, dry cell mass and yield of fumaric acid tended to increase with decreased pellet diameter. Based on the pellet morphology optimization, the final fumaric acid concentration was improved by 46.13% when fermented in a flask and 31.82% in stirred bioreactor tank fermentation.

Topics: Chromatography, High Pressure Liquid; Culture Media; Fumarates; Rhizopus; Temperature

The objective of the present study was to investigate the hypothesis that 9,10-anthraquinone (AQ) in combination with fumaric acid (FMA) may provide complementary effects to inhibit methanogens and enhance rumen's capacity for better utilisation of FMA towards propionate production. Three levels of AQ and four levels of FMA were tested in a 3 x 4 factorial design using in vitro gas production technique. AQ reduced the total gas and methane production significantly. The combination of 4 ppm AQ with FMA had additive effect on concentration of propionate. Supplementation of AQ alone resulted in hydrogen accumulation (p < 0.001), whereas presence of FMA (up to 6.5 mM) along with AQ declined hydrogen concentration (p < 0.001). The level of 4 ppm AQ did not affect in vitro digestibility, however, a reduction of organic matter digestibility was caused by 8 ppm AQ (p < 0.001), which was partially compensated by the addition of FMA (p = 0.06). The optimum FMA level depended on the AQ concentration. At 4 ppm AQ, a FMA level of 3.5 mM had best possible effect on partitioning factor and microbial biomass production (p < 0.001), though, at 8 ppm AQ the higher level of FMA (6.5 mM) responded better. Overall, FMA in combination with AQ provided an alternative hydrogen sink and might be introduced as a novel strategy for mitigation of enteric methane emission. Nevertheless, the result should be proved by in vivo experiments.

Topics: Animals; Anthraquinones; Bioreactors; Fermentation; Fumarates; Methane; Rumen

For L-aspartic acid biosynthesis, high production cells of Escherichia coli mutant B-715 and P1 were immobilized in chitosan gel using a technique developed in our laboratory. The immobilization process reduced initial activity of the intact cells, however, the biocatalyst produced was very stabile for long-term use in multi-repeated batch or continuous processes. Temperature influence on the conversion of ammonium fumarate to L-aspartic acid was investigated. In long-term experiments, over 603 hours, the temperature 40 degrees C was found to be the best for both biocatalyst stability and high conversion rate. The optimum substrate concentration was 1.0 M. Continuous production of L-aspartic acid was investigated in three types of column bioreactors characterized by different volumes as well as different high to biocatalyst bed volume rations (Hz/Vz). The highest conversion rate, 99.8%, and the productivity 6 g/g/h (mass of L-aspartic acid per dry mass of cells in biocatalyst per time unit) was achieved in the bioreactor with the highest value Hz/Vz = 3.1, and liquid hour space velocity value of 5.2, defined as the volume of feeding substrate passed per volume of catalyst in bioreactor per one hour.

Topics: Aspartic Acid; Biocatalysis; Bioreactors; Cells, Immobilized; Chitosan; Culture Media; Escherichia coli; Fumarates; Mutation; Temperature; Time Factors

Dicarboxylic acids that are produced from renewable resources are becoming attractive building blocks for the polymers industry. In this respect, fumaric acid is very interesting. Its low aqueous solubility facilitates product recovery. To avoid excessive waste salt production during downstream processing, a low pH for fumaric acid fermentation will be beneficial. Studying the influence of pH, working volume and shaking frequency on cell cultivation helped us to identify the best conditions to obtain appropriate pellet morphologies of a wild type strain of Rhizopus oryzae. Using these pellets, the effects of pH and CO(2) addition were studied to determine the best conditions to produce fumaric acid in batch fermentations under nitrogen-limited conditions with glucose as carbon source. Decreasing either the fermentation pH below 5 or increasing the CO(2) content of the inlet air above 10% was unfavourable for the cell-specific productivity, fumaric acid yield, and fumaric acid titer. However, switching off the pH control late in the batch phase did not affect these performance parameters and allowed achieving pH of 3.6. A concentration of 20 gL(-1) of fumaric acid was obtained at pH 3.6 while the average cell mass specific productivity and fumaric acid yield were the same as at pH 5.0. Consequently, relatively modest amounts of inorganic base were required for pH control, while recovery of the acid should be relatively easy at pH 3.6.

Topics: Bioreactors; Fermentation; Fumarates; Hydrogen-Ion Concentration; Industrial Microbiology; Oryza; Rhizopus

To study the chemical constituents from the ethyl acetate extracts of Cremastra appendiculata.. The compounds from the ethyl acetate extracts were isolated by the silica gel column and Sephadex LH-20 column chromatography, their structures were elucidated by the spectral analysis and chemical evidence.. Seven componds were separated and identified as fumaric acid (1), dimethylhexyl phthalate (2), L-pyroglutamic acid (3), 2-furoic acid (4), vanillic acid (5), p-coumaric acid (6), protocatechuic acid (7).. Compounds 1 to 6 are obtained from this plant for the first time.

Topics: Acetates; Coumaric Acids; Fumarates; Furans; Molecular Structure; Orchidaceae; Phthalic Acids; Plant Roots; Plants, Medicinal; Propionates; Pyrrolidonecarboxylic Acid; Vanillic Acid

A blended material composed of beta-tricalcium phosphate (beta-TCP) and fumaric acid (FA) was prepared using a mechanochemical process. The structure and properties of the TCP-FA material was probed using particle size analysis, infrared, (31)P and (13)C solid-state nuclear magnetic resonance (NMR) spectroscopy, powder X-ray diffraction and calcium bioavailability. NMR studies showed that orthophosphate environments within beta-TCP remain largely unaffected in the presence of FA during mechanochemical processing; alternately, (13)C data indicated the carboxylic groups of FA are strongly affected during processing with beta-TCP. X-ray results reveal beta-TCP diffraction plane shifting with lattice contractions likely arising at the C(3) symmetry site. While milled beta-TCP (mTCP) produces a higher flux of bioavailable calcium relative to native beta-TCP, the mechanochemical conditioning of TCP-FA generates more than seven times the level of ionic calcium relative to mTCP. Collectively, the results from these studies indicate FA interfaces with calcium oxide polyhedra of the beta-TCP hexagonal crystal lattice, especially with the underbonded CaO(3) cluster manifested within the C(3) symmetry site of the beta-TCP motif. An in vitro remineralization/demineralization pH cycling dental model was then used to assess the potential of the TCP-FA material in reversing early stage non-cavitated enamel lesions. Characterization of the remineralization via surface and longitudinal microhardness measurements demonstrated that the TCP-FA material provides statistically superior remineralization relative to milled and native beta-TCP.

Topics: Bone Substitutes; Calcium Phosphates; Crystallization; Fumarates; Materials Testing; Surface Properties; Tooth Remineralization

Batch fermentative production of fumaric acid by Rhizopus oryzae ME-F12 was investigated in a 7-l stirred tank fermentor under different dissolved oxygen (DO) concentrations. High fumaric acid yield on glucose (0.56 g/g) was achieved under high DO concentration (80%), but the glucose consumption rate and fumaric acid productivity were rather low (0.91 and 0.51 g/l/h). Fumaric acid productivity was enhanced under low DO concentration (30%), but the fuamric acid yield on glucose decreased to 0.52 g/g. In order to achieve the high fumaric acid yield and productivity simultaneously, a two-stage dissolved oxygen control strategy was proposed, in which the DO concentration was controlled at 80% in the first 18 h and then switched to 30%. This was experimentally proven to be successful. Relatively high fumaric acid production (56.2 g/l), high fumaric acid yield on glucose (0.54 g/g), and high glucose consumption rate (1.3 g/l/h) were achieved by applying this strategy. The productivity (0.7 g/l/h) was improved by 37%, 21%, and 9%, respectively, compared with fermentations in which DO concentrations were kept constant at 80%, 60%, and 30%.

Topics: Bioreactors; Culture Techniques; Fermentation; Fumarates; Glucose; Oxygen; Rhizopus

To better understand the behavior of low molecular weight organic acids in subsurface environments, the transport of three dicarboxylic acids (phthalic, maleic, and fumaric acid) in water-saturated columns packed with a hematite-coated sand was investigated in single and binary organic acid systems. Experiments were conducted at a single ionic strength (0.1M) and at two pH values (4.1 and 5.3). In single-acid systems, the order of breakthrough at both pH values was fumaric acid, followed by maleic acid, and then phthalic acid. The shape of the breakthrough curves for the acids at the two pH values were similar except at pH 5.3 phthalic acid showed two adsorption fronts. The initial front only partially broke through, whereas the second front proceeded to complete breakthrough. This behavior resulted from a marked pH increase during phthalic acid adsorption and suggests that the single-acid systems behaved as dual-component systems with the organic acid and hydrogen ion as variables. The breakthrough curves for the binary organic acid systems showed organic acids with a higher adsorption affinity (e.g., phthalic acid) competitively displace organic acids with a lower adsorption affinity (e.g., fumaric acid). The dual-component effect observed for phthalic acid in the single-acid systems was suppressed in the mixed acid systems, perhaps reflecting pH changes that accompanied the desorption of the weakly-binding acids. These results may provide an important step toward further elucidating the processes controlling organic acid fractionation in the subsurface.

Topics: Adsorption; Chromatography; Fumarates; Hydrogen-Ion Concentration; Maleates; Phthalic Acids; Porosity; Silicon Dioxide

Erlotinib [systematic name: N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine], a small-molecule epidermal growth factor receptor inhibitor, useful for the treatment of non-small-cell lung cancer, has been crystallized as erlotinib monohydrate, C(22)H(23)N(3)O(4).H(2)O, (I), the erlotinib hemioxalate salt [systematic name: 4-amino-N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-1-ium hemioxalate], C(22)H(24)N(3)O(4)(+).0.5C(2)O(4)(2-), (II), and the cocrystal erlotinib fumaric acid hemisolvate dihydrate, C(22)H(23)N(3)O(4).0.5C(4)H(4)O(4).2H(2)O, (III). In (II) and (III), the oxalate anion and the fumaric acid molecule are located across inversion centres. The water molecules in (I) and (III) play an active role in hydrogen-bonding interactions which lead to the formation of tetrameric and hexameric hydrogen-bonded networks, while in (II) the cations and anions form a tetrameric hydrogen-bonded network in the crystal packing. The title multicomponent crystals of erlotinib have been elucidated to study the assembly of molecules through intermolecular interactions, such as hydrogen bonds and aromatic pi-pi stacking.

Topics: Crystallography, X-Ray; Erlotinib Hydrochloride; Fumarates; Hydrogen Bonding; Models, Molecular; Oxalates; Quinazolines

While malate and fumarate participate in a multiplicity of pathways in plant metabolism, the function of these organic acids as carbon stores in C(3) plants has not been deeply addressed. Here, Arabidopsis (Arabidopsis thaliana) plants overexpressing a maize (Zea mays) plastidic NADP-malic enzyme (MEm plants) were used to analyze the consequences of sustained low malate and fumarate levels on the physiology of this C(3) plant. When grown in short days (sd), MEm plants developed a pale-green phenotype with decreased biomass and increased specific leaf area, with thin leaves having lower photosynthetic performance. These features were absent in plants growing in long days. The analysis of metabolite levels of rosettes from transgenic plants indicated similar disturbances in both sd and long days, with very low levels of malate and fumarate. Determinations of the respiratory quotient by the end of the night indicated a shift from carbohydrates to organic acids as the main substrates for respiration in the wild type, while MEm plants use more reduced compounds, like fatty acids and proteins, to fuel respiration. It is concluded that the alterations observed in sd MEm plants are a consequence of impairment in the supply of carbon skeletons during a long dark period. This carbon starvation phenotype observed at the end of the night demonstrates a physiological role of the C(4) acids, which may be a constitutive function in plants.

Topics: Arabidopsis; Carbon; Carbon Dioxide; Chlorophyll; Chloroplasts; Fluorescence; Fumarates; Gas Chromatography-Mass Spectrometry; Malates; Microscopy, Electron, Transmission; Phenotype; Plants, Genetically Modified

The Arabidopsis thaliana genome contains two genes encoding the mitochondrial NAD-malic enzyme (NAD-ME), NAD-ME1 (At2g13560) and NAD-ME2 (At4g00570). The characterization of recombinant NAD-ME1 and -2 indicated that both enzymes assemble as active homodimers; however, a heterodimeric enzyme (NAD-MEH) can also be detected by electrophoretic studies. To analyze the metabolic contribution of each enzymatic entity, NAD-MEH was obtained by a co-expression-based recombinant approach, and its kinetic and regulatory properties were analyzed. The three NAD-MEs show similar kinetic properties, although they differ in the regulation by several metabolic effectors. In this regard, whereas fumarate activates NAD-ME1 and CoA activates NAD-ME2, both compounds act synergistically on NAD-MEH activity. The characterization of two chimeric enzymes between NAD-ME1 and -2 allowed specific domains of the primary structure, which are involved in the differential allosteric regulation, to be identified. NAD-ME1 and -2 subunits showed a distinct pattern of accumulation in the separate components of the floral organ. In sepals, the NAD-ME1 subunit is present at a slightly higher proportion than the NAD-ME2 subunit, and thus, NAD-MEH and NAD-ME1 act in concert in this tissue. On the other hand, NAD-ME2 is the only isoform present in anthers. In view of the different properties of NAD-ME1, -2, and -H, we suggest that mitochondrial NAD-ME activity may be regulated by varying native association in vivo, rendering enzymatic entities with distinct allosteric regulation to fulfill specific roles. The presence of three different NAD-ME entities, which originate by alternative associations of two subunits, is suggested to be a novel phenomenon unique to plant mitochondria.

Topics: Allosteric Regulation; Amino Acid Sequence; Arabidopsis; Arabidopsis Proteins; Base Sequence; Coenzyme A; DNA Primers; Enzyme Activation; Fumarates; Genes, Plant; Isoenzymes; Kinetics; Malate Dehydrogenase; Mitochondria; Models, Biological; Molecular Sequence Data; Protein Multimerization; Protein Subunits; Recombinant Proteins; Sequence Homology, Amino Acid; Tissue Distribution

The Arabidopsis genome has two fumarase genes, one of which encodes a protein with mitochondrial targeting information (FUM1) while the other (FUM2) does not. We show that a FUM1-green fluorescent protein fusion is directed to mitochondria while FUM2-red fluorescent protein remains in the cytosol. While mitochondrial FUM1 is an essential gene, cytosolic FUM2 is not required for plant growth. However FUM2 is required for the massive accumulation of carbon into fumarate that occurs in Arabidopsis leaves during the day. In fum2 knock-out mutants, fumarate levels remain low while malate increases, and these changes can be reversed with a FUM2 transgene. The fum2 mutant has lower levels of many amino acids in leaves during the day compared with the wild type, but higher levels at night, consistent with a link between fumarate and amino acid metabolism. To further test this relationship we grew plants in the absence or presence of nitrogen fertilizer. The amount of fumarate in leaves increased several fold in response to nitrogen in wild-type plants, but not in fum2. Malate increased to a small extent in the wild type but to a greater extent in fum2. Growth of fum2 plants was similar to that of the wild type in low nitrogen but much slower in the presence of high nitrogen. Activities of key enzymes of nitrogen assimilation were similar in both genotypes. We conclude that FUM2 is required for the accumulation of fumarate in leaves, which is in turn required for rapid nitrogen assimilation and growth on high nitrogen.

Topics: Arabidopsis; Arabidopsis Proteins; DNA, Bacterial; Fumarate Hydratase; Fumarates; Gene Expression Regulation, Plant; Gene Knockout Techniques; Metabolome; Mutagenesis, Insertional; Mutation; Nitrogen; Plant Leaves; RNA, Plant

Low molecular weight organic acids comprise an important pool of reactive ligands in aquatic systems. These acids readily bind to nano-sized mineral particles and thereby strongly influence a particle's physicochemical behavior. Predicting this influence requires the integration of molecular-level details that control surface complexation mechanisms and structures with macro-scale observations of mineral colloid behavior. We report on the aggregation kinetics of nano-sized hematite in the presence of fumaric acid and maleic acid, which are naturally occurring dicarboxylic acids of similar size and structure. Our results indicate that the structure and orientation of the adsorbed dianion at the hematite surface, not the adsorption mechanism, defines the resulting effect. Maleate, which directs both carboxyl groups to the surface in the form of inner- and outer-sphere surface complexes, enhances colloidal stability. Fumarate, however, which binds to the hematite surface as an outer-sphere complex with just one carboxyl group only slightly influenced particle stability. This outcome suggests that subtle differences in the structure of adsorbed acids produce important differences in the physicochemical behavior of particles in dilute aquatic systems.

Topics: Adsorption; Colloids; Ferric Compounds; Fumarates; Magnetite Nanoparticles; Maleates; Surface Properties

Due to the abundant, low price characteristic, lots of efforts have been put into producing bulk chemical from lignocellulose biomass, but the low utility of xylose, which is the second main component in lignocellulose, becomes a bottleneck for efficient lignocellulose utilization. This study investigated a novel two-stage corn straw utilization strategy for fumaric acid production by Rhizopus oryzae. Fungal growth was approached in hydrolysates from acid hydrolysis of corn straw, contained 30 g/l xylose; and fumaric acid production was occurred in hydrolysates from enzymatic hydrolysis of the residue corn straw after acid hydrolysis, contained 80 g/l glucose. Under the optimal condition using this two-stage corn straw utilization strategy, the fumaric acid production, was up to 27.79 g/l, with the yield of 0.35 g/g, productivity of 0.33 g/l/h.

Topics: Bioreactors; Fumarates; Plant Components, Aerial; Rhizopus; Zea mays

Bacteria coordinate expression of virulence determinants in response to localized microenvironments in their hosts. Here we show that Shigella flexneri, which causes dysentery, encounters varying oxygen concentrations in the gastrointestinal tract, which govern activity of its type three secretion system (T3SS). The T3SS is essential for cell invasion and virulence. In anaerobic environments (for example, the gastrointestinal tract lumen), Shigella is primed for invasion and expresses extended T3SS needles while reducing Ipa (invasion plasmid antigen) effector secretion. This is mediated by FNR (fumarate and nitrate reduction), a regulator of anaerobic metabolism that represses transcription of spa32 and spa33, virulence genes that regulate secretion through the T3SS. We demonstrate there is a zone of relative oxygenation adjacent to the gastrointestinal tract mucosa, caused by diffusion from the capillary network at the tips of villi. This would reverse the anaerobic block of Ipa secretion, allowing T3SS activation at its precise site of action, enhancing invasion and virulence.

Topics: Anaerobiosis; Animals; Cells, Cultured; Epithelial Cells; Fumarates; Gastrointestinal Tract; Gene Expression Regulation, Bacterial; HeLa Cells; Host-Pathogen Interactions; Humans; Mice; Nitrates; Oxidation-Reduction; Oxygen; Rabbits; Shigella flexneri; Virulence

Methods for the simultaneous polarization of multiple 13C-enriched metabolites were developed to probe several enzymatic pathways and other physiologic properties in vivo, using a single intravenous bolus. A new method for polarization of 13C sodium bicarbonate suitable for use in patients was developed, and the co-polarization of 13C sodium bicarbonate and [1-(13)C] pyruvate in the same sample was achieved, resulting in high solution-state polarizations (15.7% and 17.6%, respectively) and long spin-lattice relaxation times (T1) (46.7 s and 47.7 s respectively at 3 T). Consistent with chemical shift anisotropy dominating the T1 relaxation of carbonyls, T1 values for 13C bicarbonate and [1-(13)C] pyruvate were even longer at 3 T (49.7s and 67.3s, respectively). Co-polarized 13C bicarbonate and [1-(13)C] pyruvate were injected into normal mice and a murine prostate tumor model at 3T. Rapid equilibration of injected hyperpolarized 13C sodium bicarbonate with 13C CO2 allowed calculation of pH on a voxel by voxel basis, and simultaneous assessment of pyruvate metabolism with cellular uptake and conversion of [1-(13)C] pyruvate to its metabolic products. Initial studies in a Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model demonstrated higher levels of hyperpolarized lactate and lower pH within tumor, relative to surrounding benign tissues and to the abdominal viscera of normal controls. There was no significant difference observed in the tumor lactate/pyruvate ratio obtained after the injection of co-polarized 13C bicarbonate and [1-(13)C] pyruvate or polarized [1-(13)C] pyruvate alone. The technique was extended to polarize four 13C labelled substrates potentially providing information on pH, metabolism, necrosis and perfusion, namely [1-(13)C]pyruvic acid, 13C sodium bicarbonate, [1,4-(13)C]fumaric acid, and 13C urea with high levels of solution polarization (17.5%, 10.3%, 15.6% and 11.6%, respectively) and spin-lattice relaxation values similar to those recorded for the individual metabolites. These studies demonstrated the feasibility of simultaneously measuring in vivo pH and tumor metabolism using nontoxic, endogenous species, and the potential to extend the multi-polarization approach to include up to four hyperpolarized probes providing multiple metabolic and physiologic measures in a single MR acquisition.

Topics: Animals; Biomarkers, Tumor; Enzymes; Fumarates; Gadolinium; Hydrogen-Ion Concentration; Indicators and Reagents; Injections, Intravenous; Isotope Labeling; Magnetic Resonance Spectroscopy; Male; Mice; Necrosis; Neoplasm Transplantation; Prostatic Neoplasms; Pyruvic Acid; Sodium Bicarbonate; Solubility; Urea

In this work, we evaluated the chemical stability profiles of UC781 based solutions to identify excipients that stabilize the microbicidal agent UC781. When different antioxidants were added to UC781 in sulfobutylether-beta-cyclodextrin (SBE-beta-CD) solutions and subjected to a 50 degrees C stability study, it was observed that EDTA was a better stabilizing agent than sodium metabisulfite, glutathione or ascorbic acid. Some antioxidants accelerated the degradation of UC781, suggesting metal-catalyzed degradation of UC781. Furthermore, we observed substantial degradation of UC781 when stored in 1% Tween 80 and 1% DMSO solutions alone or in those with 10mM EDTA. On the other hand, improved stability of UC781 in the presence of 100 and 200mM of EDTA was observed in these solutions. The addition of both EDTA and citric acid in the stock solutions resulted in recovery of more than 60% of UC781 after 12 weeks. Generally, 10% SBE-beta-CD in the presence of EDTA and citric acid stabilized UC781 solutions: the amount of UC781 recovered approaching 95% after 12 weeks of storage at 40 degrees C. We also showed that the desulfuration reaction of the UC781 thioamide involves oxygen by running solution stability studies in deoxygenated media. Improved stability of UC781 in the present study indicates that the incorporation of EDTA, citric acid and SBE-beta-CD and the removal of oxygen in formulations of this drug will aid in increasing the stability of UC781 where solutions of the drug are required.

Topics: 2-Hydroxypropyl-beta-cyclodextrin; Anilides; Antioxidants; Antiviral Agents; Ascorbic Acid; beta-Cyclodextrins; Chemistry, Pharmaceutical; Dimethyl Sulfoxide; Drug Compounding; Drug Stability; Edetic Acid; Excipients; Fumarates; Furans; Glutathione; Hot Temperature; Models, Chemical; Oxidation-Reduction; Polyethylene Glycols; Polysorbates; Solubility; Sulfites; Technology, Pharmaceutical; Thioamides; Time Factors; Vitamin E

Nitrogen (N) and phosphorus (P) are the most limiting factors for plant growth. Some microorganisms improve the uptake and availability of N and P, minimizing chemical fertilizer dependence. It has been published that the RD64 strain, a Sinorhizobium meliloti 1021 strain engineered to overproduce indole-3-acetic acid (IAA), showed improved nitrogen fixation ability compared to the wild-type 1021 strain. Here, we present data showing that RD64 is also highly effective in mobilizing P from insoluble sources, such as phosphate rock (PR). Under P-limiting conditions, the higher level of P-mobilizing activity of RD64 than of the 1021 wild-type strain is connected with the upregulation of genes coding for the high-affinity P transport system, the induction of acid phosphatase activity, and the increased secretion into the growth medium of malic, succinic, and fumaric acids. Medicago truncatula plants nodulated by RD64 (Mt-RD64), when grown under P-deficient conditions, released larger amounts of another P-solubilizing organic acid, 2-hydroxyglutaric acid, than plants nodulated by the wild-type strain (Mt-1021). It has already been shown that Mt-RD64 plants exhibited higher levels of dry-weight production than Mt-1021 plants. Here, we also report that P-starved Mt-RD64 plants show significant increases in both shoot and root fresh weights when compared to P-starved Mt-1021 plants. We discuss how, in a Rhizobium-legume model system, a balanced interplay of different factors linked to bacterial IAA overproduction rather than IAA production per se stimulates plant growth under stressful environmental conditions and, in particular, under P starvation.

Topics: Biomass; Fumarates; Indoleacetic Acids; Malates; Medicago truncatula; Phosphates; Root Nodules, Plant; Sinorhizobium meliloti; Succinic Acid

Without the addition of preservative compounds cucumbers acidified with 150 mM acetic acid with pH adjusted to 3.5 typically undergo fermentation by lactic acid bacteria. Fumaric acid (20 mM) inhibited growth of Lactobacillus plantarum and the lactic acid bacteria present on fresh cucumbers, but spoilage then occurred due to growth of fermentative yeasts, which produced ethanol in the cucumbers. Allyl isothiocyanate (2 mM) prevented growth of Zygosaccharomyces globiformis, which has been responsible for commercial pickle spoilage, as well as the yeasts that were present on fresh cucumbers. However, allyl isothiocyanate did not prevent growth of Lactobacillus plantarum. When these compounds were added in combination to acidified cucumbers, the cucumbers were successfully preserved as indicated by the fact that neither yeasts or lactic acid bacteria increased in numbers nor were lactic acid or ethanol produced by microorganisms when cucumbers were stored at 30 degrees C for at least 2 mo. This combination of 2 naturally occurring preservative compounds may serve as an alternative approach to the use of sodium benzoate or sodium metabisulfite for preservation of acidified vegetables without a thermal process.

Topics: Acetic Acid; Anti-Bacterial Agents; Antifungal Agents; Cucumis sativus; Ethanol; Fermentation; Food Handling; Food Microbiology; Food Preservation; Food Preservatives; Fumarates; Hexoses; Hydrogen-Ion Concentration; Isothiocyanates; Lactic Acid; Lactobacillales; Lactobacillus plantarum; Microbial Sensitivity Tests; Salts; Yeasts; Zygosaccharomyces

Sedoheptulose, arabitol, ribitol, and erythritol have been identified as key diagnostic metabolites in TALDO deficiency.. Urine from 6 TALDO-deficient patients and TALDO-deficient knock-out mice were analyzed using ¹H-NMR spectroscopy and GC-mass spectrometry.. Our data confirm the known metabolic characteristics in TALDO-deficient patients. The β-furanose form was the major sedoheptulose anomer in TALDO-deficient patients. Erythronic acid was identified as a major abnormal metabolite in all patients and in knock-out TALDO mice implicating an as yet unknown biochemical pathway in this disease. A putative sequence of enzymatic reactions leading to the formation of erythronic acid is presented. The urinary concentration of the citric acid cycle intermediates 2-oxoglutaric acid and fumaric acid was increased in the majority of TALDO-deficient patients but not in the knock-out mice.. Erythronic acid is a novel and major hallmark in TALDO deficiency. The pathway leading to its production may play a role in healthy humans as well. In TALDO-deficient patients, there is an increased flux through this pathway. The finding of increased citric acid cycle intermediates hints toward a disturbed mitochondrial metabolism in TALDO deficiency.

Topics: Adolescent; Animals; Biomarkers; Butyrates; Child, Preschool; Fumarates; Gas Chromatography-Mass Spectrometry; Heptoses; Humans; Infant; Infant, Newborn; Ketoglutaric Acids; Magnetic Resonance Spectroscopy; Mice; Mice, Knockout; Mitochondria; Molecular Structure; Pentose Phosphate Pathway; Ribitol; Sugar Alcohols; Transaldolase

Maleic acid and fumaric acid, the Z and E isomers of butenedioic acid, form 1:1 adducts with 2-amino-1,3-thiazole, namely 2-amino-1,3-thiazolium hydrogen maleate (2ATHM), C(3)H(5)N(2)S(+) x C(4)H(3)O(4)(-), and 2-amino-1,3-thiazolium hydrogen fumarate (2ATHF), C(3)H(5)N(2)S(+) x C(4)H(3)O(4)(-), respectively. In both compounds, protonation of the ring N atom of the 2-amino-1,3-thiazole and deprotonation of one of the carboxyl groups are observed. The asymmetric unit of 2ATHF contains three independent ion pairs. The hydrogen maleate ion of 2ATHM shows a short intramolecular O-H...O hydrogen bond with an O...O distance of 2.4663 (19) A. An extensive hydrogen-bonded network is observed in both compounds, involving N-H...O and O-H...O hydrogen bonds. 2ATHM forms two-dimensional sheets parallel to the ab plane, extending as independent parallel sheets along the c axis, whereas 2ATHF forms two-dimensional zigzag layers parallel to the bc plane, extending as independent parallel layers along the a axis.

Topics: Butyric Acid; Crystallography, X-Ray; Fumarates; Hydrogen Bonding; Isomerism; Maleates; Models, Molecular; Molecular Structure; Thiazoles

Geobacter species in a phylogenetic cluster known as subsurface clade 1 are often the predominant microorganisms in subsurface environments in which Fe(III) reduction is the primary electron-accepting process. Geobacter bemidjiensis, a member of this clade, was isolated from hydrocarbon-contaminated subsurface sediments in Bemidji, Minnesota, and is closely related to Geobacter species found to be abundant at other subsurface sites. This study examines whether there are significant differences in the metabolism and physiology of G. bemidjiensis compared to non-subsurface Geobacter species.. Annotation of the genome sequence of G. bemidjiensis indicates several differences in metabolism compared to previously sequenced non-subsurface Geobacteraceae, which will be useful for in silico metabolic modeling of subsurface bioremediation processes involving Geobacter species. Pathways can now be predicted for the use of various carbon sources such as propionate by G. bemidjiensis. Additional metabolic capabilities such as carbon dioxide fixation and growth on glucose were predicted from the genome annotation. The presence of different dicarboxylic acid transporters and two oxaloacetate decarboxylases in G. bemidjiensis may explain its ability to grow by disproportionation of fumarate. Although benzoate is the only aromatic compound that G. bemidjiensis is known or predicted to utilize as an electron donor and carbon source, the genome suggests that this species may be able to detoxify other aromatic pollutants without degrading them. Furthermore, G. bemidjiensis is auxotrophic for 4-aminobenzoate, which makes it the first Geobacter species identified as having a vitamin requirement. Several features of the genome indicated that G. bemidjiensis has enhanced abilities to respire, detoxify and avoid oxygen.. Overall, the genome sequence of G. bemidjiensis offers surprising insights into the metabolism and physiology of Geobacteraceae in subsurface environments, compared to non-subsurface Geobacter species, such as the ability to disproportionate fumarate, more efficient oxidation of propionate, enhanced responses to oxygen stress, and dependence on the environment for a vitamin requirement. Therefore, an understanding of the activity of Geobacter species in the subsurface is more likely to benefit from studies of subsurface isolates such as G. bemidjiensis than from the non-subsurface model species studied so far.

Topics: Aldehyde Oxidoreductases; Biodegradation, Environmental; Carbohydrate Metabolism; Carbon Dioxide; Cell Wall; Electrons; Environmental Microbiology; Fatty Acids; Frameshift Mutation; Fumarates; Genes, Bacterial; Genome, Bacterial; Geobacter; Glucose; Iron; Metabolic Networks and Pathways; Multienzyme Complexes; Multigene Family; Osmosis; Oxidation-Reduction; Oxo-Acid-Lyases; Propionates; Pyruvic Acid; Species Specificity; Surface Properties

Dental caries is initiated by demineralization of the tooth surface through acid production by sugar metabolism of supragingival plaque microflora. To elucidate the sugar metabolic system, we used CE-MS to perform metabolomics of the central carbon metabolism, the EMP pathway, the pentose-phosphate pathway, and the TCA cycle in supra- gingival plaque and representative oral bacteria, Streptococcus and Actinomyces. Supragingival plaque contained all the targeted metabolites in the central carbon metabolism, except erythrose 4-phosphate in the pentose-phosphate pathway. After glucose rinse, glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, dihydroxyacetone phosphate, and pyruvate in the EMP pathway and 6-phosphogluconate, ribulose 5-phosphate, and sedoheptulose 7-phosphate in the pentose-phosphate pathway, and acetyl CoA were increased. Meanwhile, 3-phosphoglycerate and phosphoenolpyruvate in the EMP pathway and succinate, fumarate, and malate in the TCA cycle were decreased. These pathways and changes in metabolites observed in supragingival plaque were similar to the integration of metabolite profiles in Streptococcus and Actinomyces.

Topics: Acetyl Coenzyme A; Actinomyces; Adult; Bacteriological Techniques; Carbon; Citric Acid Cycle; Dental Plaque; Dihydroxyacetone Phosphate; Female; Fructosediphosphates; Fructosephosphates; Fumarates; Gluconates; Glucose; Glucose-6-Phosphate; Glyceric Acids; Glycolysis; Humans; Malates; Male; Metabolomics; Pentose Phosphate Pathway; Phosphoenolpyruvate; Pyruvic Acid; Ribulosephosphates; Streptococcus; Streptococcus mutans; Succinic Acid; Sugar Phosphates

Aluminium toxicity is one of the major limiting factors of crop productivity on acid soils. High levels of available aluminium in soil may induce phosphorus deficiency in plants. This study investigates the influence of Aluminium (Al) on the phosphate (P(i)) uptake of two Phaseolus species, Phaseolus vulgaris L. var. Red Kidney and Phaseolus lunatus L. The two bean species were treated first with solutions of Al at different concentrations (0, 25, 50 and 100microM, pH 4.50) and second with solutions of P(i) (150microM) at pH 4.50. The higher the Al concentration the higher the Al concentration sorbed but P. vulgaris L var. Red Kidney adsorbed significantly more Al than P. lunatus L. Both species released organic acids: P. vulgaris L var. Red Kidney released fumaric acid and P. lunatus L. fumaric and oxalic acids which could have hindered further Al uptake. The two bean species showed a sigmoid P(i) uptake trend but with two different mechanisms. P. vulgaris L var. Red Kidney showed a starting point of 3h whereas P. lunatus L. adsorbed P(i) immediately within the first minutes. In addition, P. vulgaris L var. Red Kidney presented significantly higher P(i) uptake (higher uptake rate 'k' and higher maximum adsorption 'a' of the kinetic uptake model). The Al treatments did not significantly influence P(i) uptake. Results suggest that P. lunatus L. might adopt an external Al detoxification mechanism by the release of oxalic acid. P. vulgaris L var. Red Kidney on the other hand seemed to adopt an internal detoxification mechanism even if the Al sorbed is poorly translocated into the shoots. More detailed studies will be necessary to better define Al tolerance and/or resistance of Phaseolus spp.

Topics: Adsorption; Aluminum; Biological Transport; Fumarates; Oxalic Acid; Phaseolus; Phosphates; Phosphorus

Both maleic and fumaric acid readily form adducts or complexes with other organic molecules. The 1:1 adduct formed by quinolin-8-ol (oxine) with maleic and fumaric acid are salts, namely 8-hydroxyquinolinium hydrogen maleate, C(9)H(8)NO(+).C(4)H(3)O(4)(-), (I), and 8-hydroxyquinolinium hydrogen fumarate, C(9)H(8)NO(+).C(4)H(3)O(4)(-), (II). The cations and anions of both salts are linked by ionic N(+)-H...O(-) hydrogen bonds. The maleate salt crystallizes in the space group P2(1)2(1)2(1), while the fumarate salt crystallizes in P\\overline{1}. The maleic and fumaric acids in their complex forms exist as semimaleate and semifumarate ions (mono-ionized state), respectively. Classical N-H...O and O-H...O hydrogen bonds, together with short C-H...O contacts, generate an extensive hydrogen-bonding network. The crystal structures of the maleate and fumarate salts of oxine have been elucidated to study the importance of noncovalent interactions in the aggregation and interaction patterns of biological molecules. The structures of the salts of the Z and E isomers of butenedioic acid (maleic and fumaric acid, respectively) with quinolin-8-ol are compared.

Topics: Crystallography, X-Ray; Fumarates; Hydrogen Bonding; Isomerism; Maleates; Models, Molecular; Oxyquinoline

The purpose of the following research was to improve the original Celsior solution in order to obtain a higher degree of stability and effectiveness. The solution was modified by the addition of selected antioxidants such as vitamin C, cysteine, and fumaric acid in the following concentrations: 0.1, 0.3, and 0.5 mmol/l. The solution's stability was estimated using an accelerated stability test based on changes in histidine concentrations in the solution using Pauly's method for determining concentrations. Elevated temperatures, the factor accelerating substances' decomposition reaction rate, were used in the tests. The research was conducted at four temperatures at intervals of 10 degrees C: 60 +/- 0.2 degrees C, 70 +/- 0.2 degrees C, 80 +/- 0.2 degrees C, and 90 +/- 0.2 degrees C. It was stated that the studied substances' decomposition occurred in accordance with the equation for first-order reactions. The function of the logarithmic concentration (log%C) over time was revealed to be rectilinear. This dependence was used to determine the kinetics of decomposition reaction rate parameters (the rate constant of decomposition k, activation energy E (a), and frequency factor A). On the basis of these parameters, the stability of the modified solution was estimated at +5 degrees C. The results obtained show that the proposed antioxidants have a significant effect on lengthening the Celsior solution's stability. The best results were reached when combining two antioxidants: vitamin C and cysteine in 0.5 mmol/l concentrations. As a result, the Celsior solution's stability was lengthened from 22 to 299 days, which is 13.5 times. Vitamin C at a concentration of 0.5 mmol/l increased the solution's stability by 5.2 times (t(90) = 115 days), cysteine at a concentration of 0.5 mmol/l caused a 4.4 times stability increase (t(90) = 96 days), and fumaric acid at a concentration of 0.5 mmol/l extended the stability by 2.1 times (t(90) = 48 days) in relation to the original solution.

Topics: Antioxidants; Ascorbic Acid; Cysteine; Disaccharides; Drug Stability; Electrolytes; Fumarates; Glutamates; Glutathione; Histidine; Mannitol; Organ Preservation Solutions; Perfusion; Temperature

Crystal structure of fumaric acid was investigated by Car-Parrinello molecular dynamics and Path Integral molecular dynamics. We propose a mechanism of isomerization by proton transfer in the solid state. It is shown that the three conformers of fumaric acid observed in cryogenic Ar matrix are also present in the solid. Standard ab initio Car-Parrinello dynamics of the studied solid at 100 K indicates that barrier height for proton transfer is too high to enable thermal jump over the barrier. Path Integral method in this particular case significantly changes proton behavior in the hydrogen bridge, and the proton tunneling process is observed. Vibrational spectra of investigated system HOOC-CH=CH-COOH and its deuterated analog DOOC-CH=CH-COOD were calculated and compared with experimental data.

Topics: Computer Simulation; Crystallization; Fumarates; Isomerism; Models, Molecular; Molecular Structure; Protons

Campylobacter jejuni encodes all the enzymes necessary for a complete oxidative tricarboxylic acid (TCA) cycle. Because of its inability to utilize glucose, C. jejuni relies exclusively on amino acids as the source of reduced carbon, and they are incorporated into central carbon metabolism. The oxidation of succinate to fumarate is a key step in the oxidative TCA cycle. C. jejuni encodes enzymes annotated as a fumarate reductase (Cj0408 to Cj0410) and a succinate dehydrogenase (Cj0437 to Cj0439). Null alleles in the genes encoding each enzyme were constructed. Both enzymes contributed to the total fumarate reductase activity in vitro. The frdA::cat(+) strain was completely deficient in succinate dehydrogenase activity in vitro and was unable to perform whole-cell succinate-dependent respiration. The sdhA::cat(+) strain exhibited wild-type levels of succinate dehydrogenase activity both in vivo and in vitro. These data indicate that Frd is the only succinate dehydrogenase in C. jejuni and that the protein annotated as a succinate dehydrogenase has been misannotated. The frdA::cat(+) strain was also unable to grow with the characteristic wild-type biphasic growth pattern and exhibited only the first growth phase, which is marked by the consumption of aspartate, serine, and associated organic acids. Substrates consumed in the second growth phase (glutamate, proline, and associated organic acids) were not catabolized by the the frdA::cat(+) strain, indicating that the oxidation of succinate is a crucial step in metabolism of these substrates. Chicken colonization trials confirmed the in vivo importance of succinate oxidation, as the frdA::cat(+) strain colonized chickens at significantly lower levels than the wild type, while the sdhA::cat(+) strain colonized chickens at wild-type levels.

Topics: Animals; Bacterial Proteins; Campylobacter jejuni; Chickens; Citric Acid Cycle; Fumarates; Models, Genetic; Oxygen; Reverse Transcriptase Polymerase Chain Reaction; Succinate Dehydrogenase; Succinic Acid

Fumarate, a four-carbon trans dicarboxylic acid, is the allosteric activator of the human mitochondrial NAD(P)(+)-dependent malic enzyme (m-NAD(P)-ME). In this paper, we discuss the effects of the structural analogues of fumarate on human m-NAD(P)-ME. Succinate, a dicarboxylic acid with a carbon-carbon single bond, can also activate the enzyme, but the activating effect of succinate is less than that of fumarate. Succinamide, a diamide of succinate, cannot activate the enzyme and is a poor active-site inhibitor. The cis isomer of fumarate, maleic acid, significantly inhibits the ME activity, suggesting that the trans configuration of fumarate is crucial for operating the allosteric regulation of the enzyme. Other dicarboxylic acids, including glutaconic acid, malonic acid and alpha-ketoglutarate, cannot activate the enzyme and inversely inhibit enzyme activity. Our data suggest that these structural analogues are mainly active-site inhibitors, although they may enter the allosteric site to inhibit the enzyme. Furthermore, these data also suggest that the dicarboxylic acid must be in a trans conformation for allosteric activation of the enzyme.

Topics: Allosteric Regulation; Allosteric Site; Crystallography, X-Ray; Dicarboxylic Acids; Fumarates; Humans; Malate Dehydrogenase; Malates; Models, Molecular; Molecular Structure; Mutagenesis, Site-Directed; Protein Binding; Succinic Acid

A new class of cysteine protease inhibitors based on fumaric acid derived oligopeptides was successfully identified from a high-throughput screening of a solid-phase bound combinatorial library. As target enzymes falcipain and rhodesain were used, which play important roles in the life cycles of the parasites which cause malaria (Plasmodium falciparum) and African sleeping sickness (Trypanosoma brucei rhodesiense). The best inhibitors with unusual amino acid sequences not reported before for this type of enzyme were also fully analyzed in detail in solution. K(i) values in the lower micromolar and even nanomolar region were found. Some inhibitors are even active against plasmodia and show good selectivity relative to other enzymes. Also the mechanism of action was studied and could be shown to be irreversible inhibition.

Topics: Amino Acid Sequence; Animals; Antiprotozoal Agents; Cathepsin L; Cathepsins; Cattle; Combinatorial Chemistry Techniques; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Diamide; Drug Evaluation, Preclinical; Fumarates; Humans; Peptide Library; Plasmodium falciparum

88 g/L lactic acid was produced from waste potato starch (equivalent to 100 g/L glucose) in a bubble column reactor using appropriate acid-adapted precultures of Rhizopus arrhizus. Further experiment showed that repeated dilution of cultures caused the decrease of lactic acid concentration and productivity due to formation of large fungal pellets.

Topics: Bioreactors; Ethanol; Fumarates; Glucose; Hydrogen-Ion Concentration; Lactic Acid; Mycelium; Rhizopus; Waste Products

Multiple sclerosis (MS) represents the prototypic inflammatory autoimmune disorder of the central nervous system and the most common cause of neurological disability in young adults, exhibiting considerable clinical, radiological and pathological heterogeneity. A better understanding of the immunopathological processes underlying this disease have recently led to the design of numerous novel therapeutical approaches. Perhaps most importantly, therapy has changed dramatically over the past decade in that all relapsing forms of MS, including early forms of MS are now being treated relatively aggressively. However, there are still unmet needs in the management of this disease, especially since all of the currently available disease-modifying drugs are only partially effective. Most of the clinically relevant therapeutic agents are not yet available as oral formulations. A substantial number of pivotal and preliminary reports provide encouraging new evidence on advances being made in the development of oral therapies for MS. A different strategy is the development of very potent monoclonal antibodies, given intravenously or subcutaneously, many of which are being examined for clinical efficacy. These agents are potentially more effective, but may carry more serious side effects. Finally, drugs with a known good safety profile are being developed further. These advances are critically reviewed and put into perspective.

Topics: Administration, Oral; Alemtuzumab; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antibodies, Neoplasm; Cladribine; Crotonates; Fingolimod Hydrochloride; Fumarates; Humans; Hydroxybutyrates; Immunologic Factors; Injections, Intravenous; Injections, Subcutaneous; Interferon-beta; Multiple Sclerosis; Nitriles; Propylene Glycols; Quinolones; Sphingosine; Toluidines

The Arabidopsis thaliana genome contains four genes encoding NADP-malic enzymes (NADP-ME1-4). Two isoenzymes, NADP-ME2 and NADP-ME3, which are shown to be located in the cytosol, share a remarkably high degree of identity (90%). However, they display different expression patterns and show distinct kinetic properties, especially with regard to their regulation by effectors, in both the forward (malate oxidative decarboxylation) and reverse (pyruvate reductive carboxylation) reactions. In order to identify the domains in the primary structure that could be responsible for the regulatory differences, four chimeras between these isoenzymes were constructed and analysed. All chimeric versions exhibited the same native structures as the parental proteins. Analysis of the chimeras constructed indicated that the region from amino acid residue 303 to the C-terminal end of NADP-ME2 is critical for fumarate activation. However, the region flanked by amino acid residues 303 and 500 of NADP-ME3 is involved in the pH-dependent inhibition by high malate concentration. Furthermore, the N-terminal region of NADP-ME2 is necessary for the activation by succinate of the reverse reaction. Overall, the results show that NADP-ME2 and NADP-ME3 are able to distinguish and interact differently with similar C(4) acids as a result of minimal structural differences. Therefore, although the active sites of NADP-ME2 and NADP-ME3 are highly conserved, both isoenzymes acquire different allosteric sites, leading to the creation of proteins with unique regulatory mechanisms, probably best suited to the specific organ and developmental pattern of expression of each isoenzyme.

Topics: Allosteric Regulation; Allosteric Site; Amino Acid Sequence; Arabidopsis; Arabidopsis Proteins; Base Sequence; Catalytic Domain; Coenzyme A; Cytosol; DNA Primers; Enzyme Activation; Fumarates; Genes, Plant; Green Fluorescent Proteins; Hydrogen-Ion Concentration; Isoenzymes; Kinetics; Malate Dehydrogenase; Malate Dehydrogenase (NADP+); Molecular Sequence Data; Protein Structure, Tertiary; Recombinant Fusion Proteins; Sequence Homology, Amino Acid

Polycarboxylate-type green surfactants with either sulfide- (S-) or imino- (N-) linkages were prepared in high yields by a single addition reaction of fatty mercaptan or fatty amine with unsaturated polycarboxylic acids such as fumaric, maleic, itaconic and aconitic acids. They exhibited surfactant properties and excellent biodegradabilities. Also, green surfactants with S-linkages showed better calcium ion sequestration abilities compared to the corresponding surfactant having an N-linkage. Among these surfactants, aconitic acid-derived polycarboxylate with an S-linkage exhibited calcium ion sequestration capacities similar to that of disodium 3-oxapentanedioate (ODA), a conventional calcium ion sequestrant on a molar basis of the surfactant.

Topics: Aconitic Acid; Biodegradation, Environmental; Calcium; Carboxylic Acids; Cations; Fumarates; Imines; Maleates; Propofol; Succinates; Sulfhydryl Compounds; Sulfides; Surface Properties; Surface-Active Agents

The use of fumaric acid esters in the treatment of psoriasis was first proposed in 1959. In the 1980s, more standardized oral preparations of fumaric acid esters were developed, containing dimethylfumarate and monoethylfumarate as the main compounds. In 1994, the drug was approved for the treatment of psoriasis in Germany, and since then it has become the most commonly used systemic therapy in this country. In the last few years, an oral integrator containing dimethylfumarate and monoethylfumarate (Psocaps, Dermatika s.r.l., Padua) has also been available in Italy for the treatment of psoriasis. In this paper we report on the history of treatment using fumaric acid esters and we describe our own experience during and following the treatment with such drugs in 41 patients affected by mild vulgar psoriasis. In our trial, an improvement in cutaneous psoriasis was observed in 46% of treated patients, while side effects were noticed in 52% of patients; only three patients dropped out due to gastrointestinal problems. Our results are comparable to literature data in terms of efficacy, safety and side effects.

Topics: Administration, Oral; Adult; Aged; Aged, 80 and over; Esters; Female; Fumarates; Humans; Male; Middle Aged; Psoriasis; Treatment Outcome

The thermodynamics and kinetics of the reaction DeoxyHb-Fe(2+)<-->MetHb-Fe(3+) for human hemoglobin A (HbA), alpha- and beta-fumarate crosslinked hemoglobins were investigated by spectroelectrochemistry. Information from this study is used to determine what structural features and experimental conditions stabilize ferrous vs. ferric form of hemoglobin, and what implications this stabilization may have on the autoxidation reaction. Alpha- and beta-fumarate crosslinked hemoglobins, alphaXL-HbA and betaXL-HbA, were obtained by crosslinking deoxyhemoglobin and oxyhemoglobin, respectively, with bis(3,5-dibromosalicyl) fumarate (DBSF). Formal redox potentials, E(0), and reduction/oxidation rates were measured in the presence of mediator, hexammineruthenium(III) chloride. It was found that E(0) shifted positive for the alpha-, and negative for the beta-fumarate crosslinked hemoglobin compared to HbA for all experimental conditions investigated. This shift was consistent with stabilization of the tense (positive shift) or relaxed conformation (negative shift) conferred by crosslinking. Formal redox potentials shifted positive with addition of nitrate and chloride ions for alphaXL-HbA, indicating additional stabilization of the T quaternary. The slopes of the Nernst plots showed evidence of cooperativity as expressed by n(max). The data points (E(0), n(max)) were fitted by the MWC model which states that the electron transfer and the addition/removal of water are concerted. The set of K(R) and c values, where the parameter c is the ratio K(R)/K(T) and K(R) and K(T) are the ligand (water molecule and an electron-hole) dissociation constants for the R and T states, for the beta-crosslinked hemoglobin compared to that of HbA and alpha-crosslinked hemoglobin indicated that crosslinking of oxyhemoglobin affected differently the inner-coordination sphere at the heme site. By modulating the electrolyte concentration the reduction rates were measured as a function of DeltaE(0), the difference in E(0) between hemoglobin molecules and mediator. Linearization of the Marcus cross-relationship (based on the concerted water and electron transfer) was good for HbA, and poor for alphaXL-HbA and betaXL-HbA, consistent with results obtained by the MWC analysis. This may imply that the reduction of HbA is controlled by the driving force, DeltaE(0), whereas the reduction of alphaXL-HbA and betaXL-HbA occurs by a non-concerted mechanism controlled by structural features brought about

Topics: Cross-Linking Reagents; Electrochemistry; Fumarates; Hemoglobin A; Humans; Kinetics; Oxidation-Reduction; Spectrum Analysis

The Corynebacterium glutamicum R genome contains a total of eight genes encoding proteins with sequence similarity to C4-dicarboxylate transporters identified from other bacteria. Three of the genes encode proteins within the dicarboxylate/amino acid:cation symporter (DAACS) family, another three encode proteins within the tripartite ATP-independent periplasmic transporter family, and two encode proteins within the divalent anion:Na+ symporter (DASS) family. We observed that a mutant strain deficient in one of these genes, designated dcsT, of the DASS family did not aerobically grow on the C4 dicarboxylates succinate, fumarate, and malate as the sole carbon sources. Mutant strains deficient in each of the other seven genes grew as well as the wild-type strain under the same conditions, although one of these genes is a homologue of dctA of the DAACS family, involved in aerobic growth on C4 dicarboxylates in various bacteria. The utilization of C4 dicarboxylates was markedly enhanced by overexpression of the dcsT gene. We confirmed that the uptake of [13C]labeled succinate observed for the wild-type cells was hardly detected in the dcsT-deficient mutant but was markedly enhanced in a dcsT-overexpressing strain. These results suggested that in C. glutamicum, the uptake of C4 dicarboxylates for aerobic growth was mainly mediated by the DASS transporter encoded by dcsT. The expression level of the dcsT gene transiently increased in the early exponential phase during growth on nutrient-rich medium. This expression was enhanced by the addition of succinate in the mid-exponential phase and was repressed by the addition of glucose in the early exponential phase.

Topics: Bacterial Proteins; Chromosomes, Bacterial; Corynebacterium glutamicum; Dicarboxylic Acid Transporters; DNA, Bacterial; Fumarates; Gene Expression Regulation, Bacterial; Genes, Bacterial; Glucose; Malates; Membrane Transport Proteins; Plasmids; Reverse Transcriptase Polymerase Chain Reaction; Succinic Acid

The possibility that graphite electrodes can serve as the direct electron donor for microbially catalyzed reductive dechlorination was investigated with Geobacter lovleyi. In an initial evaluation of whether G. lovleyi could interact electronically with graphite electrodes, cells were provided with acetate as the electron donor and an electrode as the sole electron acceptor. Current was produced at levels that were ca. 10-fold lower than those previously reported for Geobacter sulfurreducens under similar conditions, and G. lovleyi anode biofilms were correspondingly thinner. When an electrode poised at -300 mV (versus a standard hydrogen electrode) was provided as the electron donor, G. lovleyi effectively reduced fumarate to succinate. The stoichiometry of electrons consumed to succinate produced was 2:1, the ratio expected if the electrode served as the sole electron donor for fumarate reduction. G. lovleyi effectively reduced tetrachloroethene (PCE) to cis-dichloroethene with a poised electrode as the sole electron donor at rates comparable to those obtained when acetate serves as the electron donor. Cells were less abundant on the electrodes when the electrodes served as an electron donor than when they served as an electron acceptor. PCE was not reduced in controls without cells or when the current supply to cells was interrupted. These results demonstrate that G. lovleyi can use a poised electrode as a direct electron donor for reductive dechlorination of PCE. The ability to colocalize dechlorinating microorganisms with electrodes has several potential advantages for bioremediation of subsurface chlorinated contaminants, especially in source zones where electron donor delivery is challenging and often limits dechlorination.

Topics: Acetic Acid; Biofilms; Biomass; Electricity; Electrodes; Electrons; Ethylene Dichlorides; Fumarates; Geobacter; Graphite; Microscopy, Electron, Scanning; Succinic Acid; Tetrachloroethylene

Weakly basic drugs demonstrate higher solubility at lower pH, thus often leading to faster drug release at lower pH. The objective of this study was to achieve pH-independent release of weakly basic drugs from extended release formulations based on the naturally occurring polymer sodium alginate. Three approaches to overcome the pH-dependent solubility of the weakly basic model drug verapamil hydrochloride were investigated. First, matrix tablets were prepared by direct compression of drug substance with different types of sodium alginate only. Second, pH-modifiers were added to the drug/alginate matrix systems. Third, press-coated tablets consisting of an inner pH-modifier tablet core and an outer drug/sodium alginate coat were prepared. pH-Independent drug release was achieved from matrix tablets consisting of selected alginates and drug substance only. Alginates are better soluble at higher pH. Therefore, they are able to compensate the poor solubility of weakly basic drugs at higher pH as the matrix of the tablets dissolves faster. This approach was successful when using alginates that demonstrated fast hydration and erosion at higher pH. The approach failed for alginates with less-pronounced erosion at higher pH. The addition of fumaric acid to drug/alginate-based matrix systems decreased the microenvironmental pH within the tablets thus increasing the solubility of the weakly basic drug at higher pH. Therefore, pH-independent drug release was achieved irrespective of the type of alginate used. Drug release from press-coated tablets did not provide any further advantages as compound release remained pH-dependent.

Topics: Alginates; Drug Delivery Systems; Fumarates; Hardness; Hydrogen-Ion Concentration; Solubility; Tablets; Technology, Pharmaceutical; Verapamil

Membrane protein complexes can support both the generation and utilization of a transmembrane electrochemical proton potential (Deltap), either by supporting transmembrane electron transfer coupled to protolytic reactions on opposite sides of the membrane or by supporting transmembrane proton transfer. Regarding the first mechanism, this has been unequivocally demonstrated to be operational for Deltap-dependent catalysis of succinate oxidation by quinone in the case of the dihaem-containing SQR (succinate:menaquinone reductase) from the Gram-positive bacterium Bacillus licheniformis. This is physiologically relevant in that it allows the transmembrane Deltap to drive the endergonic oxidation of succinate by menaquinone by the dihaem-containing SQR of Gram-positive bacteria. In the case of a related but different respiratory membrane protein complex, the dihaem-containing QFR (quinol:fumarate reductase) of the epsilon-proteobacterium Wolinella succinogenes, evidence has been obtained indicating that both mechanisms are combined, so as to facilitate transmembrane electron transfer by proton transfer via a both novel and essential compensatory transmembrane proton transfer pathway ('E-pathway'). This is necessary because, although the reduction of fumarate by menaquinol is exergonic, it is obviously not exergonic enough to support the generation of a Deltap. This compensatory E-pathway appears to be required by all dihaem-containing QFR enzymes and the conservation of the essential acidic residue on transmembrane helix V (Glu-C180 in W. succinogenes QFR) is a useful key for the sequence-based discrimination of these QFR enzymes from the dihaem-containing SQR enzymes.

Topics: Bacillus; Bacterial Proteins; Catalysis; Electrochemistry; Electron Transport; Electron Transport Complex II; Fumarates; Models, Molecular; Molecular Structure; Multienzyme Complexes; Oxidation-Reduction; Oxidoreductases; Protein Conformation; Protons; Quinones; Wolinella

Dihydroorotate dehydrogenase (DHOD) from Trypanosoma cruzi (TcDHOD) is a member of family 1A DHOD that catalyzes the oxidation of dihydroorotate to orotate (first half-reaction) and then the reduction of fumarate to succinate (second half-reaction) in the de novo pyrimidine biosynthesis pathway. The oxidation of dihydroorotate is coupled with the reduction of FMN, and the reduced FMN converts fumarate to succinate in the second half-reaction. TcDHOD are known to be essential for survival and growth of T. cruzi and a validated drug target. The first-half reaction mechanism of the family 1A DHOD from Lactococcus lactis has been extensively investigated on the basis of kinetic isotope effects, mutagenesis and X-ray structures determined for ligand-free form and in complex with orotate, the product of the first half-reaction. In this report, we present crystal structures of TcDHOD in the ligand-free form and in complexes with an inhibitor, physiological substrates and products of the first and second half-reactions. These ligands bind to the same active site of TcDHOD, which is consistent with the one-site ping-pong Bi-Bi mechanism demonstrated by kinetic studies for family 1A DHODs. The binding of ligands to TcDHOD does not cause any significant structural changes to TcDHOD, and both reduced and oxidized FMN cofactors are in planar conformation, which indicates that the reduction of the FMN cofactor with dihydroorotate produces anionic reduced FMN. Therefore, they should be good models for the enzymatic reaction pathway of TcDHOD, although orotate and fumarate bind to TcDHOD with the oxidized FMN and dihydroorotate with the reduced FMN in the structures determined here. Cys130, which was identified as the active site base for family 1A DHOD (Fagan, R. L., Jensen, K. F., Bjornberg, O., and Palfey, B. A. (2007) Biochemistry 46, 4028-4036.), is well located for abstracting a proton from dihydroorotate C5 and transferring it to outside water molecules. The bound fumarate is in a twisted conformation, which induces partial charge separation represented as C 2 (delta-) and C 3 (delta+). Because of this partial charge separation, the thermodynamically favorable reduction of fumarate with reduced FMN seems to proceed in the way that C 2 (delta-) accepts a proton from Cys130 and C 3 (delta+) a hydride (or a hydride equivalent) from reduced FMN N 5 in TcDHOD.

Topics: Amino Acid Sequence; Animals; Cloning, Molecular; Crystallography, X-Ray; Dihydroorotate Dehydrogenase; Fumarates; Models, Molecular; Molecular Sequence Data; Orotic Acid; Oxidation-Reduction; Oxidoreductases Acting on CH-CH Group Donors; Protein Conformation; Sequence Homology, Amino Acid; Substrate Specificity; Trypanosoma cruzi

To study the content variation of furmarid acid and isofraxidin in Sarcandra glabra from 21 different provenances and provide the basis for resource utilization and quality optimization of S. glabra.. HPLC method was developed to determine the contents of furmarid acid and isofraxidin in 330 samples of S. glabra which were collected respectively from 21 different provenances.. There were significant differences in the contents of isofraxidin and furmarid acid in S. glabra from different provenances. The contents of isofraxidin and furmarid acid dropped off from low altitude to high altitude, which were also close with longitude and latitude. The content of isofraxidin in S. glabra at central area of natural distribution was the highest. The different parts of the plant had different results, the influence on the contents of the acitive components in stem were more obvious than the leaf.. This simple, accurate and reproducible method could be use to determine the contents of furmarid acid and isofraxidin in S. glabra. The results represented the status of medicines quality and difference of Chinese S. glabra. These agreed with the traditional views that the medicines quality of Sarcandra glabra in Jiangxi, Fujian, Zhejiang was better. These provenances were considered as important areas of medicines breeding and bases building on S. glabra in future.

Topics: Chromatography, High Pressure Liquid; Coumarins; Drugs, Chinese Herbal; Fumarates; Magnoliopsida; Plant Leaves; Plant Stems; Reproducibility of Results

To compare the different extraction methods of Sarcandra glabra (Thunb.) Nakai.. To compare the yield of extraction and determination content of extraction by 6 kinds of different extraction methods as cold-soaked extraction, percolation extraction, reflux extraction, continuous reflux extraction, ultrasonic extraction and microwave extraction, and the content of furmaric acid was determinated as index.. The yields of extraction and contents of availability component were very different through 6 kinds of different extraction methods. Among them, the yields of reflux extraction and continuous reflux extraction were higher, the contents of availability components of ultrasonic extraction and microwave extraction were higher.. Microwave extraction method is better than other methods. It is fast economical, solvent and energy, and also has higher yield of availability component.

Topics: Acetone; Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Ethanol; Fumarates; Magnoliopsida; Methanol; Plants, Medicinal; Solvents; Technology, Pharmaceutical

(1)H NMR spectrometry, FT-IR spectroscopy, as well as molecular modeling at the AM1 level and normal mode analysis were used to characterise the interactions and the formation of inclusion complexes between three organic acids: maleic, fumaric, L-tartaric acids and betaCD. In aqueous medium, the complexation was confirmed by (1)H NMR spectroscopy using two-dimensional technique. The stable geometries of the complexes were determined by molecular modeling. Experimental infrared frequencies were assigned on the base of the vibrational normal mode calculation at the fully optimized geometry for the inclusion complexes. All the results point out the presence of stable inclusion complexes between acids and betaCD at the solid state. These results show the double role of the acid. Correlated with the theoretical and experimental data previously obtained for the miconazole/CD/acids complexes, in function of both acids and CDs structures, the acids can either stabilize the complexes by formation of a multicomponent complex or form acid/CD inclusion complexes, hindering the guest inclusion.

Topics: beta-Cyclodextrins; Dicarboxylic Acids; Fumarates; Hydrogen Bonding; Magnetic Resonance Spectroscopy; Maleates; Miconazole; Models, Molecular; Pharmaceutical Vehicles; Spectroscopy, Fourier Transform Infrared; Tartrates; Thermodynamics

The aim of the experiment was to study the effect of dietary organic acids, fumaric and sorbic, on nitrogen corrected apparent metabolisable energy (AME(N)), metabolisability of nutrients, endogenous losses and performance on young broiler chickens. A total of 56 male Ross broilers were used in a growing experiment from 14 to 30d age. Seven experimental wheat-based (655g/kg) diets were formulated. The control diet did not contain organic acids. The other six diets were produced with the addition of fumaric or sorbic acids, replacing 0.5% , 1.0% or 1.5% of the wheat. The organic acid supplemented diets contained higher levels of AME(N) compared to the control diet. Overall, birds offered organic acids had lower feed intake. Dietary organic acids did not significantly affect weight gain or feed efficiency, however, birds offered supplemented diets had lower numbers of Lactic acid bacteria and Coliforms in the ileum and caeca. Birds offered organic acids had lower levels of endogenous losses compared to control fed birds. There was a negative relationship between AME(N) of the diets and excreted endogenous losses, measured as sialic acid. It can be concluded that the decrease in secretions from the gastrointestinal tract in the presence of fumaric and sorbic acids may be a mechanism involved in the mode of action of dietary organic acids.

Topics: Animal Feed; Animals; Chickens; Dietary Proteins; Digestion; Food Additives; Fumarates; Growth; Sorbic Acid

The geometry, frequency and intensity of the vibrational bands of miconazole were derived from the density functional theory (DFT) calculations with the hybrid functional B3LYP and the 6-31G(d) basis set. Starting from the fully AM1 optimized geometries of miconazole/betaCD/acids complexes, the miconazole/acid dimers were reoptimized at the B3LYP/6-31G(d) level. Three acids were studied: maleic, fumaric and l-tartaric acids. To begin with the vibrational spectral data obtained from solid phase in mid FT-IR spectrum of miconazole and its dimers are assigned based on the results of the normal modes calculations. All the observed spectra and the calculated ones are found to be in good agreement. In a second step, theoretical results allowed the assignment of FT-IR spectrum for the miconazole/HPgammaCD inclusion complex produced by supercritical carbon dioxide treatment and confirmed the inclusion of miconazole. The experimental spectra for the miconazole/HPgammaCD/acids complexes prepared by supercritical carbon dioxide processing were also assigned using theoretical results. The results confirmed the presence of a genuine inclusion complex and also the interaction between miconazole and the acid.

Topics: Cyclodextrins; Dimerization; Fumarates; Miconazole; Spectroscopy, Fourier Transform Infrared; Tartrates; Vibration

Fumaric acid is widely used as a food additive for flavor and preservation. Rhizopus oryzae ATCC 20344 is a fungus known for good fumaric acid production. It also has been reported that the fungal biomass has high chitin content. This study investigated the possibility of producing both fumaric acid and chitin via R. oryzae fermentation of dairy manure. Co-production of valuable bio-based chemicals such as fumaric acid and chitin could make the utilization of manure more efficient and more profitable. A three step fermentation process was developed which effectively utilized the nitrogen as well as the carbohydrate sources within the manure. These steps were: the culturing of pellet seed; biomass cultivation on liquid manure to produce both biomass and chitin; and fumaric acid production on the hydrolysate from the manure fiber. Under the identified optimal conditions, the fermentation system had a fumaric acid yield of 31%, and a biomass concentration of 11.5 g/L that contained 0.21 g chitin/g biomass.

Topics: Animals; Cattle; Chitin; Dairying; Fumarates; Lignin; Manure; Mycelium; Nitrogen; Rhizopus; Spores, Fungal

Tailor-made, pH-controlled matrix minitablets based on different HPMC types were developed comprising the weakly basic drug dipyridamole. The incorporation of pH modifiers, i.e., fumaric and succinic acid, enhanced the drug release at pH 6.8. Assessing the drug release, acid release, and the microenvironmental pH (pHM) provided detailed understanding of pH-controlled mini-matrices. The extent and duration of pHM alteration was more pronounced in presence of fumaric acid. Minitablets based on the fast dissolving Methocel K100LV (< or = 100 cps) showed simultaneous release rates of dipyridamole and fumaric acid with a constant low average pHM.

Topics: Chemistry, Pharmaceutical; Dipyridamole; Fumarates; Hydrogen-Ion Concentration; Hypromellose Derivatives; Methylcellulose; Molecular Weight; Solubility; Succinic Acid; Tablets

This study describes investigation of porous photocrosslinked oligo[(polyethylene glycol) fumarate] (OPF) hydrogels as potential matrix for osteoblastic differentiation of marrow stromal cells (MSCs). The porosity and interconnectivity of porous hydrogels were assessed using magnetic resonance microscopy (MRM) as a noninvasive investigative tool that could image the water construct inside the hydrogels at a high-spatial resolution. MSCs were cultured onto the porous hydrogels and cell number was assessed using PicoGreen DNA assay. Our results showed 10% of cells initially attached to the surface of scaffolds. However, cells did not show significant proliferation over a time period of 14 days. MSCs cultured on porous hydrogels had increased alkaline phosphatase activity as well as deposition of calcium, suggesting successful differentiation and maturation to the osteoblastic phenotype. Moreover, continued expression of type I collagen and osteonectin over 14 days confirmed osteoblastic differentiation of MSCs. MRM was also applied to monitor osteogenesis of MSCs on porous hydrogels. MRM images showed porous scaffolds became consolidated with osteogenic progression of cell differentiation. These findings indicate that porous OPF scaffolds enhanced MSC differentiation leading to development of bone-like mineralized tissue.

Topics: Alkaline Phosphatase; Animals; Biomechanical Phenomena; Bone Marrow Cells; Calcification, Physiologic; Cell Differentiation; Cell Survival; Cells, Cultured; Coated Materials, Biocompatible; Collagen Type I; Cross-Linking Reagents; Fumarates; Hydrogels; Male; Osteoblasts; Osteonectin; Phenotype; Polyethylene Glycols; Porosity; Rats; Rats, Sprague-Dawley; Spectroscopy, Fourier Transform Infrared; Stromal Cells; Time Factors; Tissue Engineering

The mechanism of function of the bacterial flagellar switch, which determines the direction of flagellar rotation and is essential for chemotaxis, has remained an enigma for many years. Here we show that the switch complex associates with the membrane-bound respiratory protein fumarate reductase (FRD). We provide evidence that FRD binds to preparations of isolated switch complexes, forms a 1:1 complex with the switch protein FliG, and that this interaction is required for both flagellar assembly and switching the direction of flagellar rotation. We further show that fumarate, known to be a clockwise/switch factor, affects the direction of flagellar rotation through FRD. These results not only uncover a new component important for switching and flagellar assembly, but they also reveal that FRD, an enzyme known to be primarily expressed and functional under anaerobic conditions in Escherichia coli, nonetheless, has important, unexpected functions under aerobic conditions.

Topics: Bacterial Proteins; Escherichia coli; Flagella; Fumarates; Gene Deletion; Genes, Switch; Protein Binding; Recombinant Fusion Proteins; Succinate Dehydrogenase

Supplementation of some organic acids to a P-deficient diet has been shown to improve phytate P utilization. Two experiments were conducted from 0 to 16 d in battery brooders to determine the effect of various organic acids supplementation on phytate P utilization. In both experiments, birds were fed P-deficient corn and soybean meal-based diets. In experiment 1, citric acid, malic acid, fumaric acid, and EDTA were supplemented. Experiment 2 had a 2 x 2 factorial design with 2 sources of Met, 2-hydroxy-4-(methylthio) butanoic acid (HMB) and dl-Met, with or without 500 U/kg of phytase. In experiment 1, the addition of citric, malic, and fumaric acids increased percentage of bone ash, but only the effect of citric acid was significant. The addition of citric and malic acids also significantly increased the retention of P and phytate P (P<0.05). In experiment 2, the addition of phytase to the diet significantly increased 16-d BW gain, feed intake, percentage of bone ash, milligrams of bone ash, phytate P disappearance, and decreased the incidence of P-deficiency rickets. Methionine source did not affect 16-d BW gain, feed intake, feed efficiency, milligrams of bone ash, or P rickets incidence. However, the birds fed HMB had a higher percentage of bone ash and phytate P disappearance compared with the groups fed dl-Met only when phytase was added to the diets. The additions of citric acid and HMB improved phytate P utilization. However, the reason why some organic acids are effective whereas others are not is not apparent.

Topics: 6-Phytase; Animal Nutritional Physiological Phenomena; Animals; Body Weight; Carboxylic Acids; Chelating Agents; Chickens; Citric Acid; Edetic Acid; Female; Fumarates; Malates; Male; Minerals; Phosphorus; Phytic Acid; Poultry Diseases; Random Allocation; Rickets

The purpose of this study was to obtain a nicorandil pulsatile release tablet that has a well-regulated release lag time. When nicorandil is used as an antiangina drug, administration time control is important. A pulsatile release tablet is one of the effective approaches to modified release to reduce daily administration frequency. In this study, a pulsatile release tablet of nicorandil was formulated by fumaric acid dry coating around the core tablet including nicorandil. The model tablets, which had different content ratios of excipients in the dry-coating layer, were characterized by a dissolution test. The results showed that the release lag time was generated with fast release profiles. Various lag time controls of tablets were achieved, from 60 to 310 min on average, by variation of outer layer composition. From an analysis of the relation between lag times and outer layer composition, the key ingredient for prolongation of lag time was found to be fumaric acid. To analyze the lag time generation mechanism, water penetration for tablet was measured. The results indicated that the penetration depth was proportionate to the square root of time and the lag time formation mechanism was simple water penetration through the matrix of fumaric acid to the tablet core. The results also showed that the Washburn equation could be used to design the lag time of the pulsatile release tablet in this study. In conclusion, novel release control technology using fumaric acid was appropriate to obtain a nicorandil pulsatile release tablet that has well regulated lag time.

Topics: Algorithms; Anti-Arrhythmia Agents; Chemistry, Pharmaceutical; Delayed-Action Preparations; Excipients; Fumarates; Indicators and Reagents; Nicorandil; Tablets; Water

Identification of the Diels-Alder adduct of Abietic Acid (AA) and maleic anhydride (MA) or Fumaric Acid (FA) using a Shimadzu QP 5050A Gas chromatograph Mass spectrometer revealed that AA and MA produced endo-maleopimaric acid (MPA) and endo-maleopimaric acid tricarboxylic acid. A reaction product of abietic acid and fumaric acid generated three peaks identified based on their mass spectra as fumaropimaric acid (FPA), FPA adducts and endo-MPA. To maximize the reaction between AA and MA or FA, molar ratio (AA and MA or FA), reaction time and reaction temperature were investigated. The best Diels-Alder reaction between abietic and maleic anhydride was at 125 degrees C for 1 h with a molar ratio of AA to MA of 1: 2. The best Diels-Alder reaction between AA and FA was at 200 degrees C for 1 h with a molar ratio of AA and FA of 1: 2.

Topics: Abietanes; Fumarates; Gas Chromatography-Mass Spectrometry; Kinetics; Maleic Anhydrides; Temperature

This study was directed to look at the chemical composition of maleo-pimaric and fumaro-pimaric rosins made of unmodified rosin with maleic anhydride (MA) or fumaric acid (FA). The results showed that one peak of a maleopimaric acid (MPA) adduct on the chromatogram was obtained when a sample of this reaction product was injected into a Gas liquid chromatography (GLC) or Gas chromatograph-Mass spectrometer (GC-MS) system. Identification of this adduct using a GC-MS showed that a reaction product of rosin and MA produced endo-maleopimaric acid methyl ester. This peak of the adduct had a similar profile as in a previous study with a base peak at m/z =146 and prominent ions at m/z = 386, m/z = 187 and at m/z = 121. In the making of MPA and fumaropimaric acid (FPA) adducts, the increase in molar ratio for both reaction temperatures of 125 and 2000C and all rosin samples used in the reaction process gave a higher yield of endo maleopimaric acid. The largest amount of this product was obtained at a molar ratio of 1:10 with the reaction at 200 degrees C. Among the origin of the rosin used in this experiment, rosin from Central Java produced the highest MPA, while the highest FPA was achieved by using rosin from North Sumatra. The relationship between the molar ratio and the concentration of MPA or FPA could be expressed as a quadratic equation. From the equation, one could predict the best molar ratio to produce the highest MPA or FPA adducts at a suitable molar ratio. Using the equation Y = -0.8475 X2 + 10.448X - 9.7125, at the reaction temperature 200 degrees C, the highest MPA (around 22.50%) could be achieved by using a molar ratio of rosin and MA of 1:6.2.

Topics: Anhydrides; Diterpenes; Fumarates; Gas Chromatography-Mass Spectrometry; Maleic Anhydrides; Models, Chemical; Pinus; Resins, Plant; Temperature; Time Factors; Triterpenes

We investigated the development of an injectable, biodegradable hydrogel composite of oligo(poly(ethylene glycol) fumarate) (OPF) with encapsulated rabbit marrow mesenchymal stem cells (MSCs) and gelatin microparticles (MPs) loaded with transforming growth factor-beta1 (TGF-beta1) for cartilage tissue engineering applications. Rabbit MSCs and TGF-beta1-loaded MPs were mixed with OPF, a poly(ethylene glycol)-diacrylate crosslinker and the radical initiators ammonium persulfate and N,N,N',N'-tetramethylethylenediamine, and then crosslinked at 37 degrees C for 8 min to form hydrogel composites. Three studies were conducted over 14 days in order to examine the effects of: (1) the composite formulation, (2) the MSC seeding density, and (3) the TGF-beta1 concentration on the chondrogenic differentiation of encapsulated rabbit MSCs. Bioassay results showed no significant difference in DNA amount between groups, however, groups with MPs had a significant increase in glycosaminoglycan content per DNA starting at day 7 as compared to controls at day 0. Chondrocyte-specific gene expression of type II collagen and aggrecan were only evident in groups containing TGF-beta1-loaded MPs and varied with TGF-beta1 concentration in a dose-dependent manner. Specifically, type II collagen gene expression exhibited a 161+/-49-fold increase and aggrecan gene expression a 221+/-151-fold increase after 14 days with the highest dose of TGF-beta1 (16 ng/ml). These results indicate that encapsulated rabbit MSCs remained viable over the culture period and differentiated into chondrocyte-like cells, thus suggesting the potential of OPF composite hydrogels as part of a novel strategy for localized delivery of stem cells and bioactive molecules.

Topics: Absorbable Implants; Aggrecans; Animals; Bone Marrow Cells; Cartilage; Cell Adhesion; Cell Differentiation; Cell Proliferation; Cell Survival; Cells, Cultured; Chondrocytes; Coated Materials, Biocompatible; Collagen Type II; Cross-Linking Reagents; Dose-Response Relationship, Drug; Fumarates; Gelatin; Gene Expression; Glycosaminoglycans; Hydrogel, Polyethylene Glycol Dimethacrylate; Mesenchymal Stem Cells; Microinjections; Nanoparticles; Particle Size; Peptides; Polyethylene Glycols; Polymers; Rabbits; Surface Properties; Time Factors; Tissue Engineering; Titanium; Transforming Growth Factor beta1

A general synthetic procedure leading to isotopomeric dihydro-2(3H)furanones (gamma-butyrolactones) containing two, four, or six deuterium atoms has been developed. The labeled dihydro-2(3H)furanones were synthesized in quantitative yield from the saturated diacid C4 (succinic) or unsaturated diacids C4 (fumaric, maleic, or acetylendicarboxylic) in the presence of Ru4H4(CO)8(PBu3)4 using a deuterium pressure of 180 bar at 180 degrees C. This methodology was applied to the total synthesis of a hexadeuterated matairesinol lignan: The 3,4-bis[[3-methoxy-4-(phenylmethoxy)phenyl]methyl]dihydro-2(3H)furanone-[7,7',8,8',9',9'-D6] (benzyl-protected matairesinol-D6) was fully characterized.

Topics: 4-Butyrolactone; Deuterium; Fumarates; Furans; Isotope Labeling; Lignans; Maleates; Ruthenium; Succinic Acid

Two incubation runs were conducted with Rusitec fermenters to investigate the effects of three additive treatments (mixed fibrolytic enzymes from Trichoderma longibrachiatum (FE), disodium fumarate (FUM) and both additives (MIX)) on rumen microbial growth and fermentation of a grass hay:concentrate (600 : 400 g/kg DM) substrate. Each fermenter received daily 20 g substrate DM. Application rate (per g substrate DM) was 34.3 endoglucanase, 0.57 exoglucanase, 24.7 xylanase and 5.51 amylase units for FE and 30 mg fumarate for FUM. MIX fermenters received both additives. Both FE and MIX increased (P 0.05). Supplementing with FUM increased (P 0.05) any other variable, thus suggesting that observed effects were due to fermentation of FUM itself. The lack of effects of FUM and the absence of differences between FE and MIX on most of the measured variables would indicate that beneficial effects found in MIX fermenters were mainly due to the action of FE. Combining FE and FUM as feed additives under the conditions of the present experiment did not further improve rumen fermentation, compared to FE alone.

Topics: Animal Feed; Animals; Fermentation; Fumarates; Methane; Rumen; Sheep

Due to specific adsorption to variable charge soils, low molecular weight organic acids (LMWOAs) have not been sufficiently extracted, even if common extractants, such as water and 0.1 M sodium hydroxide (NaOH), were employed. In this work, the method for extracting LMWOAs in soils with 0.1 M NaOH was improved for variable charge soils; e.g. 1.0 M potassium fluoride (KF) with pH 4.0 was applied as an extractant jointed with 0.1 M NaOH based on its stronger ability to change the electrochemical properties of variable charge soils by specific adsorption. With the proposed method, the recoveries of oxalic, tartaric, malic, citric and fumaric acids were increased from 83 +/- 4, 93 +/- 1, 22 +/- 2, 63 +/- 5 and 84 +/- 3% to 98 +/- 2, 100 +/- 2, 85 +/- 2, 90 +/- 2 and 89 +/- 2%, respectively, compared with NaOH alone. Simultaneously, the LMWOAs in Agri-Udic Ferrosol with field moisture were measured with a satisfactory result.

Topics: Acids; Citric Acid; Electrochemistry; Fluorides; Fumarates; Hydrogen-Ion Concentration; Lactic Acid; Malates; Molecular Weight; Organic Chemicals; Oxalic Acid; Potassium Compounds; Soil; Soil Pollutants; Tartrates; Time Factors

Rhizopus oryzae is subdivided into two groups based on genetic and phenotypic differences. Type-I isolates accumulate primarily lactic acid when grown in the presence of a fermentable carbon source and contain two lactate dehydrogenase genes, ldhA and ldhB. Type-II isolates synthesize predominantly fumaric acid and only have an ldhB gene. In this study, we determined that ldhB transcript is only minimally expressed in the Type-II isolate R. oryzae 99-880. LdhB enzyme purified from gene clones isolated from the Type-I isolate R. oryzae NRRL 395 and the Type-II isolate R. oryzae 99-880 each showed reductive LDH activity (pyruvate to lactate), while no oxidative LDH activity (lactate to pyruvate) was detected in either preparation. A transformation system was then developed for the first time with R. oryzae 99-880, using a uracil auxotrophic isolate that could be complemented with an orotate phosphoribosyltransferase gene, pyrF, isolated in this study. Transformation of this Type-II isolate with the ldhA gene from R. oryzae NRRL 395 resulted in reductive LDH activity between 1.0 and 1.8 U/mg total protein. Additionally, transformed isolates grown with glucose accumulated up to 27 g lactic acid/l with a concurrent decrease in fumaric acid, ethanol, and glycerol compared with the untransformed and vector-transformed control strains.

Topics: DNA, Fungal; Fumarates; Glucose; Isoenzymes; L-Lactate Dehydrogenase; Lactate Dehydrogenase 5; Lactic Acid; Orotate Phosphoribosyltransferase; Plasmids; Recombination, Genetic; Rhizopus; RNA, Messenger; Transcription, Genetic; Transformation, Genetic

The full-length cDNA encoding the maize (Zea mays) C(4) NADP-malic enzyme was expressed in Arabidopsis (Arabidopsis thaliana) under the control of the cauliflower mosaic virus 35S promoter. Homozygous transgenic plants (MEm) were isolated with activities ranging from 6- to 33-fold of those found in the wild type. The transformants did not show any differences in morphology and development when grown in long days; however, dark-induced senescence progressed more rapidly in MEm plants compared to the wild type. Interestingly, senescence could be retarded in the transgenic lines by exogenously supplying glucose, sucrose, or malate, suggesting that the lack of a readily mobilized carbon source is likely to be the initial factor leading to the premature induction of senescence in MEm plants. A comprehensive metabolic profiling on whole rosettes allowed determination of approximately 80 metabolites during a diurnal cycle as well as following dark-induced senescence and during metabolic complementation assays. MEm plants showed no differences in the accumulation and degradation of carbohydrates with respect to the wild type in all conditions tested, but accumulated lower levels of intermediates used as respiratory substrates, prominently malate and fumarate. The data indicated that extremely low levels of malate and fumarate are responsible for the accelerated dark-induced senescence encountered in MEm plants. Thus, in prolonged darkness these metabolites are consumed faster than in the wild type and, as a consequence, MEm plants enter irreversible senescence more rapidly. In addition, the data revealed that both malate and fumarate are important forms of fixed carbon that can be rapidly metabolized under stress conditions in Arabidopsis.

Topics: Arabidopsis; Carbon; Chloroplasts; Darkness; Energy Metabolism; Fumarates; Gene Expression; Gene Expression Profiling; Malate Dehydrogenase; Malates; Plants, Genetically Modified; Zea mays

The particle bridging behaviour of dicarboxylic acid bolaform compounds such as fumaric, oxalic,trans-beta-hydromuconic, trans,trans- and cis,cis-muconic acids were evaluated in terms of their effects on the yield stress of alpha-Al(2)O(3) dispersions. The adsorption behaviour of these additives and their effects on the particle zeta potential were also determined. This study aims to understand and identify molecular factors essential for particle bridging. Very rigid compounds like trans,trans- and cis,cis-muconic and fumaric acids were identified as excellent bridging compounds from the large increase in the maximum gel strength. This strength enhancement increases with chain length and is due to more bridging molecules located in the larger spherical cap bridging area and participating in bridging. Cis,cis-muconic acid with the same chain length as fumaric acid displayed a greater bridging capability as its bolaform carboxylate groups possessed a greater lateral reach. Trans-beta-hydromuconic acid with a more flexible backbone displayed a much diminished particle bridging capability. This study has revealed a number of new molecular structural factors essential for particle bridging attribute. These are (a) the degree of backbone rigidity, (b) chain length, (c) spatial position and (d) lateral displacement of the bolaform charged group. For fumaric, trans,trans- and cis,cis-muconic acids, the maximum gel strength was not located at the pH of zero zeta potential. A particle bridging model taking into account of electrostatic repulsive interactions between the interacting particles was proposed to explain the maximum gel strength enhancement by the bolaform compounds.

Topics: Aluminum Oxide; Cross-Linking Reagents; Dicarboxylic Acids; Fumarates; Hydrogen-Ion Concentration; Mechanics; Models, Molecular; Molecular Conformation; Molecular Structure; Oxalic Acid; Surface Properties

Four new colorimetric receptors (1-4) were synthesized and characterized. Upon addition of maleate to receptor in DMSO, the appearance of the solution of receptor 1 showed a color change from dark-blue to dark-red, which can be detected by the naked eye at parts per million. Similar experiments were repeated using receptors 2-4; the solution showed a distinct color change from blue to violet for receptor 2 and from blue-green to purple for both receptors 3 and 4, when they are formed as complexes with maleate. The striking color changes are thought to be due to the deprotonation of the thiourea moiety of the 4-nitronaphthyl chromophore. Whereas, in the addition of fumarate to receptors 1-4, the color of the solution changed from dark-blue to bright yellow for receptor and did not induce any color change for receptors 2-4. Thus, for a distinct color change, receptors 1-4 can act as optical chemosensors for recognition of maleate versus fumarate. Especially, only receptor 1 has a unique color change for the recognition of fumarate, accordingly it can be used for detection of the fumarate anion. In this research it was also found that the performance of the receptor is highly dependent on the substituent group on the phenyl ring; a stronger electron-withdrawing group resulted in a receptor with a higher binding constant with the maleate anion.

Topics: Anions; Colorimetry; Dicarboxylic Acids; Fumarates; Hydrogen Bonding; Isomerism; Kinetics; Magnetic Resonance Spectroscopy; Maleates; Protons; Spectrophotometry, Ultraviolet

PolyHIPEs show great promise as tissue engineering scaffolds due to the tremendous control of pore size and interconnectivity afforded by this technique. Highly porous, fully biodegradable scaffolds were prepared by polymerization of the continuous phase of high internal phase emulsions (HIPEs) containing the macromer poly(propylene fumarate) (PPF) and the cross-linker propylene fumarate diacrylate (PFDA). Toluene was used as a diluent to reduce the viscosity of the organic phase to enable HIPE formation. A range of polyHIPE scaffolds of different pore sizes and morphologies were generated by varying the diluent concentration (40-60 wt %), cross-linker concentration (25-75 wt %), and macromer molecular weight ( M n = 800-1000 g/mol). Although some formulations resulted in macroporous monoliths (pore diameter >500 microm), the majority of the polyHIPEs studied were rigid, microporous monoliths with average pore diameters in the range 10-300 microm. Gravimetric analysis confirmed the porosity of the microporous monoliths as 80-89% with most scaffolds above 84%. These studies demonstrate that emulsion templating can be used to generate rigid, biodegradable scaffolds with highly interconnected pores suitable for tissue engineering scaffolds.

Topics: Biocompatible Materials; Fumarates; Polymers; Styrenes; Tissue Engineering; Tissue Scaffolds

Dihydroorotate dehydrogenase (DHOD) is the fourth enzyme in the de novo pyrimidine biosynthetic pathway and is essential in Trypanosoma cruzi, the parasitic protist causing Chagas' disease. T. cruzi and human DHOD have different biochemical properties, including the electron acceptor capacities and cellular localization, suggesting that T. cruzi DHOD may be a potential chemotherapeutic target against Chagas' disease. Here, we report nucleotide sequence polymorphisms of T. cruzi DHOD genes and the kinetic properties of the recombinant enzymes. T. cruzi Tulahuen strain possesses three DHODgenes: DHOD1 and DHOD2, involved in the pyrimidine biosynthetic (pyr) gene cluster on an 800 and a 1000 kb chromosomal DNA, respectively, and DHOD3, located on an 800 kb DNA. The open reading frames of all three DHOD genes are comprised of 942 bp, and encode proteins of 314 amino acids. The three DHOD genes differ by 26 nucleotides, resulting in replacement of 8 amino acid residues. In contrast, all residues critical for constituting the active site are conserved among the three proteins. Recombinant T. cruzi DHOD1 and DHOD2 expressed in E. coli possess similar enzymatic properties, including optimal pH, optimal temperature, Vmax, and Km for dihydroorotate and fumarate. In contrast, DHOD3 had a higher Vmax and Km for both substrates. Orotate competitively inhibited all three DHOD enzymes to a comparable level. These results suggest that, despite their genetic variations, kinetic properties of the three T. cruziDHODs are conserved. Our findings facilitate further exploitation of T. cruzi DHOD inhibitors, as chemotherapeutic agents against Chagas' disease.

Topics: Animals; Base Sequence; Dihydroorotate Dehydrogenase; Fumarates; Genetic Variation; Hydrogen-Ion Concentration; Isoenzymes; Kinetics; Molecular Sequence Data; Orotic Acid; Oxidoreductases Acting on CH-CH Group Donors; Point Mutation; Polymorphism, Genetic; Recombinant Proteins; Sequence Alignment; Temperature; Trypanosoma cruzi

Geobacter sulfurreducens, generally considered to be a strict anaerobe, is a predominant microbe in subsurface environments, where it utilizes available metals as electron acceptors. To better understand the metabolic processes involved in the metal-reduction capability of this microbe, the proteins expressed by cells grown anaerobically with either fumarate or ferric citrate as electron acceptor were compared. Proteins were separated by 2-DE under denaturing or nondenaturing conditions, and proteins varying in abundance with a high level of statistical significance (p<0.0001) were identified by peptide mass analysis. Denaturing 2-DE revealed significant differences in the relative abundance of the membrane proteins OmpA and peptidoglycan-associated lipoprotein, several metabolic enzymes, and, in addition, superoxide dismutase and rubredoxin oxidoreductase. Nondenaturing 2-DE revealed elevated catalase in cells grown with ferric citrate. These results suggest that, in addition to adjustments in membrane transport and specific metabolic pathways in response to these two different electron acceptors, distinct differences exist in the oxidative environment within the cell when fumarate or soluble ferric citrate is provided as electron acceptor. Although an anaerobe, G. sulfurreducens appears to have alternate mechanisms for dealing with reactive oxygen species in response to increased intracellular soluble iron.

Topics: Anaerobiosis; Bacterial Proteins; Electrophoresis, Gel, Two-Dimensional; Ferric Compounds; Fumarates; Geobacter; Oxidation-Reduction; Proteomics; Reactive Oxygen Species; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

The mechanism of fumarate reduction in Geobacter sulfurreducens was investigated. The genome contained genes encoding a heterotrimeric fumarate reductase, FrdCAB, with homology to the fumarate reductase of Wolinella succinogenes and the succinate dehydrogenase of Bacillus subtilis. Mutation of the putative catalytic subunit of the enzyme resulted in a strain that lacked fumarate reductase activity and was unable to grow with fumarate as the terminal electron acceptor. The mutant strain also lacked succinate dehydrogenase activity and did not grow with acetate as the electron donor and Fe(III) as the electron acceptor. The mutant strain could grow with acetate as the electron donor and Fe(III) as the electron acceptor if fumarate was provided to alleviate the need for succinate dehydrogenase activity in the tricarboxylic acid cycle. The growth rate of the mutant strain under these conditions was faster and the cell yields were higher than for wild type grown under conditions requiring succinate dehydrogenase activity, suggesting that the succinate dehydrogenase reaction consumes energy. An orthologous frdCAB operon was present in Geobacter metallireducens, which cannot grow with fumarate as the terminal electron acceptor. When a putative dicarboxylic acid transporter from G. sulfurreducens was expressed in G. metallireducens, growth with fumarate as the sole electron acceptor was possible. These results demonstrate that, unlike previously described organisms, G. sulfurreducens and possibly G. metallireducens use the same enzyme for both fumarate reduction and succinate oxidation in vivo.

Topics: Culture Media; Dicarboxylic Acids; Fumarates; Geobacter; Molecular Sequence Data; Operon; Oxidation-Reduction; Recombinant Proteins; Substrate Specificity; Succinate Dehydrogenase; Succinic Acid

The recently reported CSA-amplified PASS experiment correlates the spinning sidebands at the true spinning frequency omega(r) with the spinning sidebands that would be obtained at the effective spinning frequency omega(r)/N, where N is termed the scaling factor. The experiment is useful for the measurement of small chemical shift anisotropies, for which slow magic-angle spinning frequencies, required to measure several spinning sidebands, can be unstable. We have experimentally evaluated the reliability of this experiment for this application. In particular we have demonstrated that large scaling factors of the order of N=27 may be used, whilst still obtaining accurate chemical shift sideband intensities at the effective spinning frequency from the F(1) projection. Moreover, the sideband intensities are accurately obtained even in the presence of significant pulse imperfections. A second application of the CSA-amplified PASS experiment is the measurement of the chemical shift anisotropy of sites that experience homonuclear dipolar coupling, as may be found in uniformly labelled biological molecules, or for nuclei with a high natural abundance. The effects of homonuclear dipolar coupling on CSA-amplified PASS spectra has been investigated by numerical simulations and are demonstrated using uniformly (13)C enriched l-histidine monohydrochloride monohydrate.

Topics: Anisotropy; Carbon Isotopes; Computer Simulation; Fumarates; Glycine; Histidine; Nuclear Magnetic Resonance, Biomolecular

Two obligate anaerobic bacterial strains (5-3-Z(T) and Y4-1) were isolated from river sediment and rice field mud, respectively. They degraded straight-chain fatty acids with 4-8 carbon atoms in syntrophic association with methanogens, however, neither tested branch-chain fatty acids nor could benzoate be degraded. The strains formed spores when cocultured with methanogens on butyrate, or when grew on butyrate plus dimethyl sulfoxide (DMSO) in pure culture. The cells were slightly curved rods with Gram-negative cell wall structure, and contained small amount of poly beta-hydroxyalkanoate. The strains could not degrade butyrate alone, nor could use fumarate, sulfate, thiosulfate, sulfur or nitrate as electron acceptors except DMSO for butyrate degradation. The generation time of strain 5-3-Z(T) was about 12h when growing on crotonate at 37 degrees C. The growth of the new strains occurred in the range of pH 5.5-8.4, and of temperature 20-48 degrees C, and at NaCl concentration of 0-700 mM. The G+C content of the genomic DNA of strain 5-3-Z(T) was 40.6mol%. Phylogenetic analysis based on 16S rRNA gene similarity showed the two strains to be a member of species Syntrophomonas erecta (98.4-98.9% sequence similarity), however they differed from the existing strains in both phenotypic and genetic characteristics. Therefore, a new subspecies of S. erecta, S. erecta subsp. sporosyntropha was proposed. The type strain was 5-3-Z(T) (=CGMCC1.5032(T)=JCM13344(T)).

Topics: Anaerobiosis; Base Composition; Benzoates; Butyrates; China; Crotonates; DNA, Bacterial; DNA, Ribosomal; Fatty Acids, Volatile; Fumarates; Genes, rRNA; Geologic Sediments; Gram-Positive Endospore-Forming Rods; Hydrogen-Ion Concentration; Methanobacterium; Microscopy, Electron, Transmission; Molecular Sequence Data; Nitrates; Phylogeny; Rivers; RNA, Bacterial; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Sequence Homology, Nucleic Acid; Spores, Bacterial; Sulfates; Temperature; Thiosulfates; Water Microbiology

Geobacter sulfurreducens is a well-studied representative of the Geobacteraceae, which play a critical role in organic matter oxidation coupled to Fe(III) reduction, bioremediation of groundwater contaminated with organics or metals, and electricity production from waste organic matter. In order to investigate G. sulfurreducens central metabolism and electron transport, a metabolic model which integrated genome-based predictions with available genetic and physiological data was developed via the constraint-based modeling approach. Evaluation of the rates of proton production and consumption in the extracellular and cytoplasmic compartments revealed that energy conservation with extracellular electron acceptors, such as Fe(III), was limited relative to that associated with intracellular acceptors. This limitation was attributed to lack of cytoplasmic proton consumption during reduction of extracellular electron acceptors. Model-based analysis of the metabolic cost of producing an extracellular electron shuttle to promote electron transfer to insoluble Fe(III) oxides demonstrated why Geobacter species, which do not produce shuttles, have an energetic advantage over shuttle-producing Fe(III) reducers in subsurface environments. In silico analysis also revealed that the metabolic network of G. sulfurreducens could synthesize amino acids more efficiently than that of Escherichia coli due to the presence of a pyruvate-ferredoxin oxidoreductase, which catalyzes synthesis of pyruvate from acetate and carbon dioxide in a single step. In silico phenotypic analysis of deletion mutants demonstrated the capability of the model to explore the flexibility of G. sulfurreducens central metabolism and correctly predict mutant phenotypes. These results demonstrate that iterative modeling coupled with experimentation can accelerate the understanding of the physiology of poorly studied but environmentally relevant organisms and may help optimize their practical applications.

Topics: Amino Acids; Electron Transport; Escherichia coli; Fumarates; Geobacter; Iron; Models, Biological; Mutation; Oxidation-Reduction; Phenotype; Protons; Quinones; Species Specificity

Incorporation of pH modifiers is a commonly used strategy to enhance the dissolution rate of weakly basic drugs from sustained release solid dosage forms. Electron paramagnetic resonance imaging (EPRI) was applied to spatially monitor pH(M) and the rotational correlation time (tau(R)), a parameter which is closely related to the surrounding microviscosity inside HPMC (hydroxypropylmethylcellulose) matrix tablets. Fumaric, citric, and succinic acid were employed as pH modifiers. 4-(methylamino)-2-ethyl-5,5-dimethyl-4-pyridine-2-yl-2,5-dihydro-1H-imidazole-1-oxyl (MEP) was used as spin label. Fumaric and citric acid reduced the pH(M) to equal extents in the initial phase. With the progress of hydration, the more soluble citric acid diffused out from the tablet resulting in an increase in pH(M), originating at the outer layers. In contrast, fumaric acid maintained a constantly reduced pH(M) inside the entire tablet. Due to its lower acidic strength, succinic acid did not reduce the pH(M) as effectively as the other pH modifiers used. The more water-soluble acids stimulated the water penetration into the matrix system, thereby rapidly decreasing tau(R). Once the matrix tablets were hydrated, the included pH modifiers influenced tau(R) insignificantly. EPRI, a novel approach for monitoring pH(M) and tau(R) non-invasively and spatially resolved, was used successfully for the optimization of an pH-controlled formulation.

Topics: Chemistry, Pharmaceutical; Citric Acid; Delayed-Action Preparations; Dipyridamole; Electron Spin Resonance Spectroscopy; Fumarates; Hydrogen-Ion Concentration; Hypromellose Derivatives; Methylcellulose; Polymers; Solubility; Succinic Acid; Tablets; Viscosity; Water

In a daily migration, the aquatic larvae of Chaoborus flavicans (a phantom midge) alternate oxygen-saturated and anoxic lake strata. To investigate this cycle, larvae were collected at a natural environment, and acetate, propionate, pyruvate, lactate, glycerol, phosphate, maleate, succinate, glucose and citrate were determined. Each larva was homogenized with 200 microL water and deproteinized with a spin-filter; 50 microL aliquots were mixed with 50 microL of a buffer containing 80 mM propylamine, 20 mM HCl and 0.06 mM 2,4-dihydroxybenzoic acid (internal standard) in methanol. The extracts were infused in an electrospray ionization ion-trap mass spectrometer. The limits of detection for the [M-H](-) peaks ranged from 2 microM for pyruvate and lactate to 200 microM for acetate and glycerol. The MS(2) ion-trap spectra obtained at pH 7 (ammonium acetate buffer) were used to distinguish maleate (cis-2-butenedioic), which gave [M-CO(2)-H](-) (m/z 71), from fumarate (trans-2-butenedioic), which showed first a loss of water yielding an instable peak at m/z 97. The compounds involved in the aerobic-anaerobic adjustment of the metabolism were revealed by linear discriminant analysis. Acetate, citrate, glucose, maleate (which decreased during the daytime), and particularly succinate (which increased), showed the maximal discrimination power between the day- and night-time samples.

Topics: Aerobiosis; Anaerobiosis; Animals; Ceratopogonidae; Discriminant Analysis; Fumarates; Larva; Models, Biological; Spectrometry, Mass, Electrospray Ionization

The Escherichia coli complex II homologues succinate:ubiquinone oxidoreductase (SQR, SdhCDAB) and menaquinol:fumarate oxidoreductase (QFR, FrdABCD) have remarkable structural homology at their dicarboxylate binding sites. Although both SQR and QFR can catalyze the interconversion of fumarate and succinate, QFR is a much better fumarate reductase, and SQR is a better succinate oxidase. An exception to the conservation of amino acids near the dicarboxylate binding sites of the two enzymes is that there is a Glu (FrdA Glu-49) near the covalently bound FAD cofactor in most QFRs, which is replaced with a Gln (SdhA Gln-50) in SQRs. The role of the amino acid side chain in enzymes with Glu/Gln/Ala substitutions at FrdA Glu-49 and SdhA Gln-50 has been investigated in this study. The data demonstrate that the mutant enzymes with Ala substitutions in either QFR or SQR remain functionally similar to their wild type counterparts. There were, however, dramatic changes in the catalytic properties when Glu and Gln were exchanged for each other in QFR and SQR. The data show that QFR and SQR enzymes are more efficient succinate oxidases when Gln is in the target position and a better fumarate reductase when Glu is present. Overall, structural and catalytic analyses of the FrdA E49Q and SdhA Q50E mutants suggest that coulombic effects and the electronic state of the FAD are critical in dictating the preferred directionality of the succinate/fumarate interconversions catalyzed by the complex II superfamily.

Topics: Alanine; Amino Acids; Binding Sites; Catalysis; Electrochemistry; Electron Spin Resonance Spectroscopy; Electron Transport Complex IV; Electrons; Enzyme Activation; Escherichia coli; Flavins; Fumarates; Hydrogen-Ion Concentration; Kinetics; Models, Molecular; Mutagenesis; Mutagenesis, Site-Directed; Mutation; Oxidoreductases; Oxygen; Plasmids; Potentiometry; Protein Binding; Protein Structure, Tertiary; Succinate Dehydrogenase; Synchrotrons; X-Ray Diffraction

As commonly recognized, the excretion of acetate by the aerobic growth of Escherichia coli on glucose is a manifestation of imbalanced flux between glycolysis and the tricarboxylic acid (TCA) cycle. Accordingly, this may restrict the production of recombinant proteins in E. coli, due to the limited amounts of precursor metabolites produced in TCA cycle. To approach this issue, an extra supply of intermediate metabolites in TCA cycle was made by conversion of aspartate to fumarate, a reaction mediated by the activity of L-aspartate ammonia-lyase (aspartase). As a result, in the glucose minimal medium containing aspartate, the production of two recombinant proteins, beta-galactosidase and green fluorescent protein, in the aspartase-producing strain was substantially increased by 5-fold in association with 30-40% more biomass production. This preliminary study illustrates the great promise of this approach used to enhance the production of these two recombinant proteins.

Topics: Aspartate Ammonia-Lyase; Aspartic Acid; beta-Galactosidase; Citric Acid Cycle; Escherichia coli; Fumarates; Glucose; Green Fluorescent Proteins; Recombinant Proteins

The effect of the disinfectant sodium hypochlorite (NaClO), with or without mild heat (50 degrees C) and fumaric acid, on native bacteria and the foodborne pathogens Staphylococcus aureus, Escherichia coli O157:H7, and Salmonella Typhimurium DT104 attached to iceberg lettuce leaves was examined. The retail lettuce examined consistently harbored 6 to 7 log CFU/g of native bacteria throughout the study period. Inner leaves supported 1 to 2 log CFU/g fewer bacteria than outer leaves. About 70% of the native bacterial flora was removed by washing five times with 0.85% NaCl. S. aureus, E. coli, and Salmonella allowed to attach to lettuce leaves for 5 min were more easily removed by washing than when allowed to attach for 1 h or 2 days, with more S. aureus being removed than E. coli or Salmonella Typhimurium. An increase of time for attachment of pathogens from 5 min to 2 days leads to decreased efficiency of the washing and sanitizing treatment. Treatment with fumaric acid (50 mM for 10 min at room temperature) was the most effective, although it caused browning of the lettuce, with up to a 2-log reduction observed. The combination of 200 ppm of sodium hypochlorite and mild heat treatment at 50 degrees C for 1 min reduced the pathogen populations by 94 to 98% (1.2- to 1.7-log reduction) without increasing browning.

Topics: Bacterial Adhesion; Colony Count, Microbial; Consumer Product Safety; Disinfectants; Dose-Response Relationship, Drug; Drug Synergism; Escherichia coli O157; Fumarates; Humans; Lactuca; Salmonella typhimurium; Sodium Hypochlorite; Staphylococcus aureus; Temperature; Time Factors

Fumarase (FH) deficiency is a rare autosomal recessive disease of the Krebs cycle causing severe neurological impairment in early childhood, characterized by encephalopathy with seizures and muscular hypotonia. Only a handful of patients with various recessive mutations in the FH gene have been described so far. Interestingly, autosomal dominant mutations in the same gene are associated with hereditary leiomyomatosis and renal cell cancer (HLRCC). We investigated a boy with developmental and growth delay, microcephaly, and muscular hypotonia recognized at the age of 3 months. No leiomyomatosis or renal cancer is known in the parents. Investigation of the patient's urine revealed massive fumarate excretion. FH activity was severely reduced in muscle and fibroblasts. Respirometric investigation of fibroblasts showed only modest changes indicating that fumarate mediated inhibition of enzymatic pathways other than oxidative phosphorylation might be more relevant in pathophysiology of FH deficiency. Molecular analysis revealed a known 435insK mutation on the paternal allele and a novel H275L mutation due to an A to T transversion of nucleotide 824 on the maternal allele. This mutation affects the same codon as a C to T transition of nucleotide 823, resulting in a H275Y mutation that was found in two families with HLRCC.

Topics: Amino Acid Substitution; Cell Respiration; Child, Preschool; Fatal Outcome; Fumarate Hydratase; Fumarates; Heterozygote; Humans; Infant; Infant, Newborn; Lysine; Male; Metabolism, Inborn Errors; Mitochondria; Models, Molecular; Muscle Hypotonia; Muscle, Skeletal; Mutation; Psychomotor Disorders

Tripeptidic structures were synthesized by using a combination of two independent consecutive catalytic procedures. Cross-metathesis of N-acroyl amino acid esters yields fumaric amide compounds with exclusive E double-bond geometry. This represents an unprecedented example of complete double-bond selectivity in this kind of reaction. A subsequent asymmetric aminohydroxylation of the chiral fumaric amides was carried out without the need of any further ligand and gave high yields and no side products. This reaction transforms the central fumaric amide unit into a hydroxy aspartic acid moiety and relies on the inherent stereochemistry of the starting fumaric diamides. An additional feature of our sequence is the ease of generating stereochemical diversification within the aminohydroxylation reaction. As a consequence, rapid conformational and configurational diversification can be achieved from the overall two-step catalytic sequence. The versatility of this approach is demonstrated by starting from two different N-acroyl amino esters, which led to the synthesis of eight structurally and stereochemically different tripeptides that could all be identified individually. As such, the present two-step catalytic approach should serve to efficiently synthesize large families of tripeptidic molecular probes.

Topics: Amides; Amines; Amino Acid Sequence; Amino Acids; Catalysis; Fumarates; Hydroxylation; Oligopeptides

The antinociceptive pharmacology of N-[[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]methyl]-2-[2-[[(4-methoxy-2,6-dimethylphenyl) sulfonyl]methylamino]ethoxy]-N-methylacetamide fumarate (LF22-0542), a novel nonpeptidic B1 antagonist, was characterized. LF22-0542 showed high affinity for human and mouse B1 receptors with virtually no affinity for the human B2 receptor; a selectivity index of at least 4000 times was obtained when LF22-0542 was profiled throughout binding or cell biology assays on 64 other G-protein-coupled receptor, 10 ion channels, and seven enzymes. LF22-0542 was a competitive B1 receptor antagonist and elicited significant antinociceptive actions in the mouse acetic acid-induced writhing assay, as well as in the second phases of formalin-induced nociception in mice and in both the first and second phases of the formalin response in rats. LF22-0542 was active after s.c. but not p.o. administration. In B1 receptor knockout (KO) mice, acetic acid and formalin responses were significantly reduced and LF22-0542 had no additional effects in these animals. LF22-0542 alleviated thermal hypersensitivity in both acute (carrageenan) and persistent inflammatory (complete Freund's adjuvant) pain models in rats. LF22-0542 produced a full reversal of experimental neuropathic thermal hypersensitivity but was inactive in reversing nerve injury-induced tactile hypersensitivity in rats. In agreement with this observation, B1 KO mice subjected to peripheral nerve injury did not show thermal hypersensitivity but developed nerve injury-induced tactile hypersensitivity normally. The data demonstrate the antihyperalgesic actions of a selective systemically administered B1 receptor antagonist and suggest the utility of this class of agents for the treatment of inflammatory pain states and for some aspects of neuropathic pain.

Topics: Acrylamides; Analgesics; Animals; Bradykinin B1 Receptor Antagonists; Cell Line; Dose-Response Relationship, Drug; Fumarates; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Pain Measurement; Rats; Rats, Sprague-Dawley; Rats, Wistar; Receptor, Bradykinin B1

A comparison of three different implementations of the chemical-shift recoupling experiment of Tycko et al. [R. Tycko, G. Dabbagh, P.A. Mirau, Determination of chemical-shift-anisotropy lineshapes in a two-dimensional magic-angle-spinning NMR experiment, J. Magn. Reson. 85 (1989) 265-274] is presented. The methods seek to reduce the effects of artefacts resulting from pulse imperfections and residual C-H dipolar coupling in organic solids. An optimised and constant time implementation are shown to give well-defined and artefact free powder pattern lineshapes in the indirectly observed dimension for both sp2 and sp3 carbon sites. Experimental setup is no more demanding than for the original experiment, and can be implemented using standard commercial hardware.

Topics: Anisotropy; Artifacts; Benzene; Carbon Isotopes; Fumarates; Glycine; Nuclear Magnetic Resonance, Biomolecular

A mutant gyrA allele resulting in an A271E substitution in the DNA gyrase protein generated a strain unable to grow on the C(4)-dicarboxylates succinate, malate, and fumarate. Bacteria harboring gyrA751 displayed decreased negative supercoiling in cells. Expression of the dctA gene, which encodes the C(4)-dicarboxylate transporter, was reduced in a gyrA751 mutant, providing the first evidence that dctA expression is supercoiling sensitive and uncovering a simple metabolic screen for lesions in gyrase that reduce negative supercoiling.

Topics: Alleles; Amino Acid Substitution; Carbon; DNA Gyrase; DNA, Superhelical; Fumarates; Gene Expression; Malates; Membrane Transport Proteins; Mutation, Missense; Salmonella typhimurium; Succinic Acid

S-(2-Succinyl)cysteine (2SC) has been identified as a chemical modification in plasma proteins, in the non-mercaptalbumin fraction of human plasma albumin, in human skin collagen, and in rat skeletal muscle proteins and urine. 2SC increases in human skin collagen with age and is increased in muscle protein of diabetic vs. control rats. The concentration of 2SC in skin collagen and muscle protein correlated strongly with that of the advanced glycation/lipoxidation end-product (AGE/ALE), N(epsilon)-(carboxymethyl)lysine (CML). 2SC is formed by a Michael addition reaction of cysteine sulfhydryl groups with fumarate at physiological pH. Fumarate, but not succinate, inactivates the sulfhydryl enzyme, glyceraldehyde-3-phosphate dehydrogenase in vitro, in concert with formation of 2SC. 2SC is the first example of spontaneous chemical modification of protein by a metabolic intermediate in the Krebs cycle. These observations identify fumarate as an endogenous electrophile and suggest a role for fumarate in regulation of metabolism.

Topics: Animals; Anticarcinogenic Agents; Citric Acid Cycle; Collagen; Cysteine; Diabetes Mellitus, Experimental; Female; Fumarates; Glycation End Products, Advanced; Glyceraldehyde-3-Phosphate Dehydrogenases; Humans; Insulin; Insulin, Long-Acting; Insulin, Regular, Human; Muscle Proteins; Protein Processing, Post-Translational; Radiation-Protective Agents; Rats; Rats, Sprague-Dawley; Serum Albumin; Serum Albumin, Human; Skin; Succinic Acid

Two psychrophilic, Gram-negative, rod-shaped, motile bacteria (strains 112T and 102T) that conserved energy from dissimilatory Fe(III) reduction concomitant with acetate oxidation were isolated from permanently cold Arctic marine sediments. Both strains grew at temperatures down to -2 degrees C, with respective temperature optima of 14 degrees C and 14-17 degrees C for strains 112T and 102T. The isolated strains reduced Fe(III) using common fermentation products such as acetate, lactate, propionate, formate or hydrogen as electron donors, and they also grew with fumarate as the sole substrate. As alternatives to Fe(III), they reduced fumarate, S0 and Mn(IV). Based on 16S rRNA gene sequence similarity, strain 112T was most closely related to Desulfuromonas acetoxidans (97.0 %) and Desulfuromonas thiophila NZ27T (95.5 %), and strain 102T to Malonomonas rubra Gra Mal 1T (96.3 %) and Desulfuromusa succinoxidans GylacT (95.9 %) within the Deltaproteobacteria. Strains 112T and 102T therefore represent novel species, for which the names Desulfuromonas svalbardensis sp. nov. (type strain 112T=DSM 16958T=JCM 12927T) and Desulfuromusa ferrireducens sp. nov. (type strain 102T=DSM 16956T=JCM 12926T) are proposed.

Topics: Acetic Acid; Arctic Regions; Bacterial Typing Techniques; Deltaproteobacteria; Fatty Acids; Ferric Compounds; Formates; Fumarates; Genes, rRNA; Geologic Sediments; Hydrogen; Lactic Acid; Manganese; Microscopy, Electron; Molecular Sequence Data; Movement; Oxidation-Reduction; Phylogeny; Propionates; RNA, Bacterial; RNA, Ribosomal, 16S; Soil Microbiology; Sulfur

In an attempt to understand the aromatic hydrocarbon metabolism by purple bacteria that do not grow at their expense, we earlier reported 2-aminobenzoate transformation by a purple non-sulfur bacterium, Rhodobacter sphaeroides OU5 (Sunayana et al., 2005, J Ind Microbiol Biotech 32:41-45), which is extended in the present study with aniline, a major environmental pollutant. Aniline did not support photo (light anaerobic) or chemo (dark aerobic) heterotrophic growth of Rhodobacter sphaeroides OU5 either as a sole source of carbon or nitrogen. However, light-dependent aniline transformation was observed in the culture supernatants and the products were identified as indole derivatives. The transformation was dependent on a tricarboxylate intermediate, fumarate. Five intermediates of the aniline biotransformation pathway were isolated and identified as indole esters having a mass of 443, 441, 279, 189, and 167 with unstoichiometric total indole yields of 0.16 mM: from 5 mM: of aniline consumed. The pathway proposed based on these intermediates suggest a novel xenobiotic detoxification process in bacteria.

Topics: Aniline Compounds; Biodegradation, Environmental; Fumarates; Indoles; Light; Rhodobacter sphaeroides; Xenobiotics

The human mitochondrial NAD(P)+-dependent malic enzyme (m-NAD-ME) is a malic enzyme isoform with dual cofactor specificity and substrate binding cooperativity. Previous kinetic studies have suggested that Lys362 in the pigeon cytosolic NADP+-dependent malic enzyme has remarkable effects on the binding of NADP+ to the enzyme and on the catalytic power of the enzyme (Kuo, C. C., Tsai, L. C., Chin, T. Y., Chang, G.-G., and Chou, W. Y. (2000) Biochem. Biophys. Res. Commun. 270, 821-825). In this study, we investigate the important role of Gln362 in the transformation of cofactor specificity from NAD+ to NADP+ in human m-NAD-ME. Our kinetic data clearly indicate that the Q362K mutant shifted its cofactor preference from NAD+ to NADP+. The Km(NADP) and kcat(NADP) values for this mutant were reduced by 4-6-fold and increased by 5-10-fold, respectively, compared with those for the wild-type enzyme. Furthermore, up to a 2-fold reduction in Km(NADP)/Km(NAD) and elevation of kcat(NADP)/kcat(NAD) were observed for the Q362K enzyme. Mutation of Gln362 to Ala or Asn did not shift its cofactor preference. The Km(NADP)/Km(NAD) and kcat(NADP)/kcat(NAD) values for Q362A and Q362N were comparable with those for the wild-type enzyme. The DeltaG values for Q362A and Q362N with either NAD+ or NADP+ were positive, indicating that substitution of Gln with Ala or Asn at position 362 brings about unfavorable cofactor binding at the active site and thus significantly reduces the catalytic efficiency. Our data also indicate that the cooperative binding of malate became insignificant in human m-NAD-ME upon mutation of Gln362 to Lys because the sigmoidal phenomenon appearing in the wild-type enzyme was much less obvious that that in Q362K. Therefore, mutation of Gln362 to Lys in human m-NAD-ME alters its kinetic properties of cofactor preference, malate binding cooperativity, and allosteric regulation by fumarate. However, the other Gln362 mutants, Q362A and Q362N, have conserved malate binding cooperativity and NAD+ specificity. In this study, we provide clear evidence that the single mutation of Gln362 to Lys in human m-NAD-ME changes it to an NADP+-dependent enzyme, which is characteristic because it is non-allosteric, non-cooperative, and NADP+-specific.

Topics: Adenosine Triphosphate; Allosteric Site; Catalysis; Fumarates; Glutamine; Humans; Kinetics; Lysine; Malate Dehydrogenase; Mitochondria; NADP; Protein Binding; Substrate Specificity; Thermodynamics

The proteome of Geobacter sulfurreducens, a model for the Geobacter species that predominate in many Fe(III)-reducing subsurface environments, was characterized with ultra high-pressure liquid chromatography and mass spectrometry using accurate mass and time (AMT) tags as well as with more traditional two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Cells were grown under six different growth conditions in order to enhance the potential that a wide range of genes would be expressed. The AMT tag approach was able to identify a much greater number of proteins than could be detected with the 2-D PAGE approach. With the AMT approach over 3,000 gene products were identified, representing about 90% of the total predicted gene products in the genome. A high proportion of predicted proteins in most protein role categories were detected; the highest number of proteins was identified in the hypothetical protein role category. Furthermore, 91 c-type cytochromes of 111 predicted genes in the G. sulfurreducens genome were identified. Differences in the abundance of cytochromes and other proteins under different growth conditions provided information for future functional analysis of these proteins. These results demonstrate that a high percentage of the predicted proteins in the G. sulfurreducens genome are produced and that the AMT tag approach provides a rapid method for comparing differential expression of proteins under different growth conditions in this organism.

Topics: Bacterial Proteins; Bacteriological Techniques; Chromatography, High Pressure Liquid; Cytochrome c Group; Electrophoresis, Gel, Two-Dimensional; Ferric Compounds; Fumarates; Geobacter; Peptide Fragments; Proteome; Spectrometry, Mass, Electrospray Ionization

The objective of this study was to develop a rupturable, capsule-based pulsatile drug delivery system with pH-independent properties prepared using aqueous coating. The drug release is induced by rupturing of the top-coating, resulting by expanding of swellable layer upon water penetration through the top-coating. Croscarmellose sodium (AcDiSol) is a preferable superdisintegrant compared to low substituted hydroxypropylcellulose (L-HPC) and sodium starch glycolate (Explotab), because of controlled lag time, followed by a quick and complete drug release. However, due to its anionic character, AcDiSol showed pH-dependent swelling characteristics (pH 7.4 > 0.1N HCl) resulting in a pH-dependent lag time. The pH dependency could be eliminated by the addition of fumaric acid to the swelling layer, which allowed to keep an acidic micro-environment. Formation of the rupturable top-coating was successfully performed using an aqueous dispersion of ethylcellulose (Aquacoat) ECD), whereby sufficient drying during the coating was needed to avoid swelling of the AcDiSol layer. A higher coating level was required, when aqueous dispersion was used, compared to organic coatings. However, an advantageous aspect of the aqueous coating was the lower sensitivity of the lag time to a deviation in the coating level.

Topics: Capsules; Cellulose; Desiccation; Drug Compounding; Drug Delivery Systems; Drug Stability; Fumarates; Gelatin; Humidity; Hydrogen-Ion Concentration; Hydrophobic and Hydrophilic Interactions; Pulsatile Flow; Solubility; Surface Properties; Water

Sulphate- or sulphur-reducing bacteria with known or draft genome sequences (Desulfovibrio vulgaris, Desulfovibrio desulfuricans G20, Desulfobacterium autotrophicum [draft], Desulfotalea psychrophila and Geobacter sulfurreducens) all contain sdhCAB or frdCAB gene clusters encoding succinate : quinone oxidoreductases. frdD or sdhD genes are missing. The presence and function of succinate dehydrogenase versus fumarate reductase was studied. Desulfovibrio desulfuricans (strain Essex 6) grew by fumarate respiration or by fumarate disproportionation, and contained fumarate reductase activity. Desulfovibrio vulgaris lacked fumarate respiration and contained succinate dehydrogenase activity. Succinate oxidation by the menaquinone analogue 2,3-dimethyl-1,4-naphthoquinone depended on a proton potential, and the activity was lost after degradation of the proton potential. The membrane anchor SdhC contains four conserved His residues which are known as the ligands for two haem B residues. The properties are very similar to succinate dehydrogenase of the Gram-positive (menaquinone-containing) Bacillus subtilis, which uses a reverse redox loop mechanism in succinate : menaquinone reduction. It is concluded that succinate dehydrogenases from menaquinone-containing bacteria generally require a proton potential to drive the endergonic succinate oxidation. Sequence comparison shows that the SdhC subunit of this type lacks a Glu residue in transmembrane helix IV, which is part of the uncoupling E-pathway in most non-electrogenic FrdABC enzymes.

Topics: Amino Acid Sequence; Desulfovibrio; Fumarates; Molecular Sequence Data; Multigene Family; Oxidation-Reduction; Succinate Dehydrogenase; Succinic Acid; Sulfates

The procyclic stage of Trypanosoma brucei, a parasitic protist responsible for sleeping sickness in humans, converts most of the consumed glucose into excreted succinate, by succinic fermentation. Succinate is produced by the glycosomal and mitochondrial NADH-dependent fumarate reductases, which are not essential for parasite viability. To further explore the role of the succinic fermentation pathways, we studied the trypanosome fumarases, the enzymes providing fumarate to fumarate reductases. The T. brucei genome contains two class I fumarase genes encoding cytosolic (FHc) and mitochondrial (FHm) enzymes, which account for total cellular fumarase activity as shown by RNA interference. The growth arrest of a double RNA interference mutant cell line showing no fumarase activity indicates that fumarases are essential for the parasite. Interestingly, addition of fumarate to the medium rescues the growth phenotype, indicating that fumarate is an essential intermediary metabolite of the insect stage trypanosomes. We propose that trypanosomes use fumarate as an essential electron acceptor, as exemplified by the fumarate dependence previously reported for an enzyme of the essential de novo pyrimidine synthesis (Takashima, E., Inaoka, D. K., Osanai, A., Nara, T., Odaka, M., Aoki, T., Inaka, K., Harada, S., and Kita, K. (2002) Mol. Biochem. Parasitol. 122, 189-200).

Topics: Animals; Cytosol; Electrons; Fumarates; Glucose; Green Fluorescent Proteins; Microbodies; Mitochondria; Mutation; Phylogeny; Protein Isoforms; RNA Interference; Time Factors; Trypanosoma brucei brucei

Succinate dehydrogenase is an indispensable enzyme involved in the Krebs cycle as well as energy coupling in the mitochondria and certain prokaryotes. During catalysis, succinate oxidation is coupled to ubiquinone reduction by an electron transfer relay comprising a flavin adenine dinucleotide cofactor, three iron-sulfur clusters, and possibly a heme b556. At the heart of the electron transport chain is a [4Fe-4S] cluster with a low midpoint potential that acts as an energy barrier against electron transfer. Hydrophobic residues around the [4Fe-4S] cluster were mutated to determine their effects on the midpoint potential of the cluster as well as electron transfer rates. SdhB-I150E and SdhB-I150H mutants lowered the midpoint potential of this cluster; surprisingly, the His variant had a lower midpoint potential than the Glu mutant. Mutation of SdhB-Leu-220 to Ser did not alter the redox behavior of the cluster but instead lowered the midpoint potential of the [3Fe-4S] cluster. To correlate the midpoint potential changes in these mutants to enzyme function, we monitored aerobic growth in succinate minimal medium, anaerobic growth in glycerol-fumarate minimal medium, non-physiological and physiological enzyme activities, and heme reduction. It was discovered that a decrease in midpoint potential of either the [4Fe-4S] cluster or the [3Fe-4S] cluster is accompanied by a decrease in the rate of enzyme turnover. We hypothesize that this occurs because the midpoint potentials of the [Fe-S] clusters in the native enzyme are poised such that direction of electron transfer from succinate to ubiquinone is favored.

Topics: Electron Spin Resonance Spectroscopy; Electron Transport; Escherichia coli; Fumarates; Iron-Sulfur Proteins; Models, Molecular; Mutagenesis, Site-Directed; Mutation; Oxidation-Reduction; Plasmids; Protein Structure, Quaternary; Reactive Oxygen Species; Succinate Dehydrogenase; Ubiquinone

During L-lactic acid fermentation by Rhizopus oryzae, increasing the phosphate level in the fermentation medium from 0.1 g l(-1) to 0.6 g l(-1) KH2PO4 reduced the maximal concentration of L-lactic acid and fumaric acid from 85 g l(-1) to 71 g l(-1) and from 1.36 g l(-1) to 0.18 g l(-1), respectively; and it decreased the fermentation time from 72 h to 52 h. Phosphate at 0.40 g l(-1) KH2PO4 was suitable for both minimizing fumaric acid accumulation and benefiting L-lactic acid production.

Topics: Fermentation; Fumarates; Industrial Microbiology; Lactic Acid; Phosphates; Rhizopus

Culture supernatants of Rhodobacter sphaeroides OU5 grown in the presence of 2-aminobenzoate gave an orange-red color-reaction with Salper's reagent, suggesting the presence of an indole derivative. This production was light-dependent and inducible only with 2-aminobenzoate. Replacement of 2-aminobenzoate with other 2-substituted benzoates did not result in the formation of indole. Fumarate appeared to be the conjugating molecule with 2-aminobenzoate, resulting in the formation of an indole derivative. The purified indole derivative was orange-brown in color, with a yields 0.34 mM from 1 mM 2-aminobenzoate. Infrared analysis suggested an indole ester and (1)H NMR analysis indicated an indole carboxylate, esterified with a terpenoid alcohol. The indole ester has a mass of 441 with the molecular formula C(27)H(39)NO(4). The IUPAC name of the compound is (3 E,5 E)-14-hydroxy-3,7,11-trimethyl-3,5-tetradecadienyl 2-(hydroxymethyl)-1 H-indole-3-carboxylate; and the common name given to this compound is sphestrin.

Topics: Biotransformation; Fumarates; Indoles; Light; Magnetic Resonance Spectroscopy; ortho-Aminobenzoates; Rhodobacter sphaeroides; Spectrophotometry, Infrared

Arabidopsis (Arabidopsis thaliana) mutants lacking the tonoplastic malate transporter AttDT (A. thaliana tonoplast dicarboxylate transporter) and wild-type plants showed no phenotypic differences when grown under standard conditions. To identify putative metabolic changes in AttDT knock-out plants, we provoked a metabolic scenario connected to an increased consumption of dicarboxylates. Acidification of leaf discs stimulated dicarboxylate consumption and led to extremely low levels of dicarboxylates in mutants. To investigate whether reduced dicarboxylate concentrations in mutant leaf cells and, hence, reduced capacity to produce OH(-) to overcome acidification might affect metabolism, we measured photosynthetic oxygen evolution under conditions where the cytosol is acidified. AttDT::tDNA protoplasts showed a much stronger inhibition of oxygen evolution at low pH values when compared to wild-type protoplasts. Apparently citrate, which is present in higher amounts in knock-out plants, is not able to replace dicarboxylates to overcome acidification. To raise more information on the cellular level, we performed localization studies of carboxylates. Although the total pool of carboxylates in mutant vacuoles was nearly unaltered, these organelles contained a lower proportion of malate and fumarate and a higher proportion of citrate when compared to wild-type vacuoles. These alterations concur with the observation that radioactively labeled malate and citrate are transported into Arabidopsis vacuoles by different carriers. In addition, wild-type vacuoles and corresponding organelles from AttDT::tDNA mutants exhibited similar malate channel activities. In conclusion, these results show that Arabidopsis vacuoles contain at least two transporters and a channel for dicarboxylates and citrate and that the activity of AttDT is critical for regulation of pH homeostasis.

Topics: Arabidopsis; Arabidopsis Proteins; Biological Transport, Active; Citric Acid; Fumarates; Gene Expression Regulation, Plant; Homeostasis; Hydrogen-Ion Concentration; Malates; Membrane Potentials; Molecular Sequence Data; Mutation; Organic Anion Transporters; Oxygen Consumption; Photosynthesis; Plant Leaves; Vacuoles

The purpose of this investigation is to understand the influence of gastrointestinal (GI) pH on the gel layer formation and its dynamics for various hydrophilic/swellable matrices, in the process of developing a pH-independent controlled release system for a basic drug, oxybutynin hydrochloride (OXB). Cylindrical matrices (8-mm diameter) without and with fumaric acid, were readily prepared by direct compression. Formulations were evaluated for in vitro drug release, and gel layer dynamics was studied by viscosity measurements and texture profiling analysis. In the in vitro drug release study, OXB, which shows pH-dependent solubility, showed faster release from all the matrices in pH 1.2 medium. Release rates enhanced to a lesser extent with change of medium from pH 6.8 to pH 1.2, for HPMC polymer matrices. Anionic polymer matrices showed drastic differences in the release rates when medium was changed from pH 6.8 to pH 1.2. Addition of fumaric acid to matrices demonstrated pH-independent drug release, which was attributed to the micro-environmental pH manipulation within the hydrated gel layer. Viscosity and texture profiling studies revealed that saturation solubility of drug at swelling front play a major role in the pH-dependent drug release from HPMC matrices, while both saturation solubility and the altered gel consistency as a function of pH are involved with anionic polymer matrices. Presence of fumaric acid in HPMC matrices showed efficient retardation and pH-independent drug release. In conclusion, understanding the influence of GI physiological pH on the gel layer dynamics and manipulating the micro-environmental pH provides efficient and predictable in vivo performance from these swellable cylindrical matrices.

Topics: Drug Carriers; Fumarates; Gels; Hydrogen-Ion Concentration; Kinetics; Mandelic Acids; Models, Chemical; Polymers; Solubility; Tablets, Enteric-Coated; Viscosity

Tris(2,4,6-trimethoxyphenyl)phosphonium propylamine bromide (TMPP) has been used for the derivatisation of maleic, fumaric, sorbic and salicylic acids to facilitate determination using liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) in positive ion mode. Detection limits, achieved using multiple reaction monitoring mode, were 2, 4, 0.4 and 540 fmol (5 muL injection) for derivatised fumaric, sorbic, maleic and salicylic acids, respectively. In comparison, detection limits achieved in negative ion mode for the underivatised acids were 24, 51, 2, and 117 fmol, respectively. The method was successfully used for the determination of sorbic acid in a sample of Panadol. The derivatisation of salicylic acid was not as successful, probably due to poor reaction efficiency.

Topics: Acetaminophen; Analgesics, Non-Narcotic; Bridged Bicyclo Compounds, Heterocyclic; Carboxylic Acids; Chromatography, High Pressure Liquid; Fumarates; Maleates; Salicylic Acid; Sorbic Acid; Spectrometry, Mass, Electrospray Ionization

DcuS is a membrane-associated sensory histidine kinase of Escherichia coli specific for C(4) -dicarboxylates. The nature of the stimulus and its structural prerequisites were determined by measuring the induction of DcuS-dependent dcuB'-'lacZ gene expression. C(4)-dicarboxylates without or with substitutions at C2/C3 by hydrophilic (hydroxy, amino, or thiolate) groups stimulated gene expression in a similar way. When one carboxylate was replaced by sulfonate, methoxy, or nitro groups, only the latter (3-nitropropionate) was active. Thus, the ligand of DcuS has to carry two carboxylate or carboxylate/nitro groups 3.1-3.8 A apart from each other. The effector concentrations for half-maximal induction of dcuB'-'lacZ expression were 2-3 mm for the C(4)-dicarboxylates and 0.5 mm for 3-nitropropionate or d-tartrate. The periplasmic domain of DcuS contains a conserved cluster of positively charged or polar amino acid residues (Arg(107)-X(2)-His(110)-X(9)-Phe(120)-X(26)-Arg(147)-X-Phe(149)) that were essential for fumarate-dependent transcriptional regulation. The presence of fumarate or d-tartrate caused sharpening of peaks or chemical shift changes in HSQC NMR spectra of the isolated C(4)-dicarboylate binding domain. The amino acid residues responding to fumarate or d-tartrate were in the region comprising residues 89-150 and including the supposed binding site. DcuS(R147A) mutant with an inactivated binding site was isolated and reconstituted in liposomes. The protein showed the same (activation-independent) kinase activity as DcuS, but autophosphorylation of DcuS was no longer stimulated by C(4)-dicarboxylates. Therefore, the R147A mutation affected signal perception and transfer to the kinase but not the kinase activity per se.

Topics: Amino Acid Sequence; Binding Sites; Citric Acid; Dicarboxylic Acid Transporters; Dicarboxylic Acids; Enzyme Activation; Escherichia coli; Escherichia coli Proteins; Fumarates; Gene Expression Regulation, Bacterial; Histidine Kinase; Lac Operon; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Nitro Compounds; Peptide Fragments; Phosphorylation; Propionates; Protein Kinases; Recombinant Fusion Proteins; Sequence Alignment; Structure-Activity Relationship; Tartrates

The optimal concentrations of nutrient medium components, aeration conditions, and pH providing for maximum biomass yields, as well as fumarase and L-aspartase activities, during submerged cultivation of Erwinia sp. were determined. The data showed that different concentrations of carbon source (molasses) and pH of the nutrient medium were required to reach the maximum fumarase and L-aspartase activities. Calculations performed by application of the additive lattice model suggested that the combination of these optimized factors would result in 3.2-, 3.4-, and 3.8-fold increases as compared to the experimental means in Erwinia sp. biomass, and L-aspartase and fumarase activities, respectively. The conditions of the fumaric acid biotransformations into L-malic and L-aspartic acids were optimized on the basis of intact Erwinia sp. cells, a fumarase and L-aspartase producer. In the cases of fumarate transformation into L-malic acid and of fumarate transformation into L-aspartic acids, fumarase and L-aspartase activities increased 1.5- and 1.7-fold, respectively. The experimental data were consistent with these estimates to 80% accuracy. In comparison with the additive lattice model, the application of polynomial nonlinear model allowed the between-factor relations to be considered and analyzed, which resulted in 1.1-, 1.27-, and 1.1-fold increases in Erwinia sp. biomass and fumarase and L-aspartase activities for the case of cultivation. In the case of fumarate transformation into L-malic acid, this model demonstrated a 1.7-fold increase in fumarase activity, whereas during fumarate transformation into L-aspartic acid no significant change in aspartase activity was observed.

Topics: Algorithms; Aspartate Ammonia-Lyase; Aspartic Acid; Biomass; Culture Media; Erwinia; Fumarate Hydratase; Fumarates; Hydrogen-Ion Concentration; Industrial Microbiology; Malates; Models, Statistical; Polyvinyl Chloride

A DNA microarray representing the genome of Geobacter sulfurreducens was constructed for use in global gene expression profiling of cells under steady-state conditions with acetate as the electron donor and Fe(III) or fumarate as the electron acceptor. Reproducible differences in transcript levels were also observed in comparisons between cells grown with ammonia and those fixing atmospheric nitrogen. There was a high correlation between changes in transcript levels determined with microarray analyses and an evaluation of a subset of the genome with quantitative PCR. As expected, cells required to fix nitrogen had higher levels of transcripts of genes associated with nitrogen fixation, further demonstrating that the microarray approach could reliably detect important physiological changes. Cells grown with Fe(III) as the electron acceptor had higher levels of transcripts for omcB, a gene coding for an outer membrane c-type cytochrome that is essential for Fe(III) reduction. Several other c-type cytochrome genes also appeared to be up-regulated. An unexpected result was significantly higher levels of transcripts for genes which have a role in metal efflux, potentially suggesting the importance of maintaining metal homeostasis during release of soluble metals when reducing Fe(III). A substantial proportion (30%) of significantly expressed genes during Fe(III) reduction were genes of unknown function or hypothetical proteins, suggesting differences in Fe(III) reduction physiology among microorganisms which perform this metabolic process.

Topics: Ferric Compounds; Fumarates; Geobacter; Microarray Analysis; Nitrogen Fixation; Oxidation-Reduction; Polymerase Chain Reaction

Two obligate anaerobes, TB8106(T) and WZH410, which degraded propionate in syntrophic association with methanogens, were isolated from two upflow anaerobic sludge blanket reactors, one treating brewery wastewater and the other bean curd wastewater. The strains were Gram-negative, non-spore-forming and non-motile. Cells were egg-shaped, with a size of 1.0-1.3 x 1.8-2.2 microm. Growth was observed at 20-48 degrees C and pH 6.2-8.8. Both strains converted propionate to acetate and methane in co-culture with methanogens, and grew on propionate plus sulfate in pure culture, with a doubling time of 52-55 h at 37 degrees C. Sulfate and thiosulfate both served as electron acceptors for propionate degradation. The DNA G + C contents of the two strains were 58.5 and 58.7 mol%, respectively. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the strains were closely related to a propionate-oxidizing syntrophic bacterium, Syntrophobacter fumaroxidans DSM 10017(T) (94.7 % similarity). However, the novel strains could not ferment fumarate, and grew at a more alkaline pH range than Syntrophobacter fumaroxidans. Moreover, the novel isolates had obviously higher growth rates on propionate plus sulfate (0.12 day(-1)) than Syntrophobacter fumaroxidans DSM 10017(T) (0.024 day(-1)). Therefore, a novel species, Syntrophobacter sulfatireducens sp. nov., is proposed, with strain TB8106(T) (=AS 1.5016(T) = DSM 16706(T)) as the type strain.

Topics: Acetic Acid; Anaerobiosis; Base Composition; Deltaproteobacteria; DNA, Bacterial; DNA, Ribosomal; Fumarates; Genes, rRNA; Gentian Violet; Hydrogen-Ion Concentration; Methane; Molecular Sequence Data; Oxidation-Reduction; Phenazines; Phylogeny; Propionates; RNA, Bacterial; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Sewage; Sulfates; Temperature; Thiosulfates; Water Microbiology

Novel procedures and instrumentation are described for nuclear magnetic resonance (NMR) spectroscopy and imaging studies of live, in situ microbial films. A perfused NMR/optical microscope sample chamber containing a planar biofilm support was integrated into a recirculation/dilution flow loop growth reactor system and used to grow in situ Shewanella oneidensis strain MR-1 biofilms. Localized NMR techniques were developed and used to non-invasively monitor time-resolved metabolite concentrations and to image the biomass volume and distribution. As a first illustration of the feasibility of the methodology an initial 13C-labeled lactate metabolic pathway study was performed, yielding results consistent with existing genomic data for MR-1. These results represent progress toward our ultimate goal of correlating time- and depth-resolved metabolism and mass transport with gene expression in live in situ biofilms using combined NMR/optical microscopy techniques.

Topics: Biofilms; Bioreactors; Carbon Isotopes; Culture Media; Fumarates; Lactic Acid; Magnetic Resonance Spectroscopy; Microbiological Techniques; Shewanella

Benzylsuccinate synthase catalyzes a highly unusual reaction: the addition of toluene to fumarate to form (R)-benzylsuccinic acid. The stereochemistry of this reaction has been examined using [d3-methyl]toluene and either fumarate or its cis stereoisomer, maleate, as the substrates. We demonstrate that when fumarate is the cosubstrate, deuterium is transferred from toluene to the C-3 pro-(R) position of benzylsuccinate, implying a syn addition of toluene to the double bond of fumarate. However, when maleate is the cosubstrate, the addition of toluene occurs in an anti fashion, so that deuterium transfer to the C-3 pro-(R) position of benzylsuccinate is also observed. This is consistent with the formation of the C-3 radical of benzylsuccinate as an intermediate, in which rotation about the C-2-C-3 bond can occur to relieve the sterically unfavorable cis conformation of the carboxylate groups when maleate is the cosubstrate.

Topics: Carbon-Carbon Lyases; Deuterium; Fumarates; Hydrogen; Maleates; Nuclear Magnetic Resonance, Biomolecular; Stereoisomerism; Succinates; Toluene

ZK 811 752, a potent candidate for the treatment of autoimmune diseases, demonstrated pH-dependent solubility. The resulting release from conventional mini matrix tablets decreased with increasing pH-values of the dissolution medium. The aim of this study was to overcome this problem and to achieve pH-independent drug release. Mini matrix tablets were prepared by direct compression of drug, matrix former (polyvinylacetate/polyvinylpyrrolidone; Kollidon SR) and excipients (lactose, calcium phosphate or maize starch). To solve the problem of pH-dependent solubility fumaric acid was added to the drug-polymer excipient system. The addition of fumaric acid was found to maintain low pH-values within the mini tablets during release of ZK 811 752 in phosphate buffer pH 6.8. Thus, micro environmental conditions for the dissolution of the weakly basic drug were kept constant and drug release was demonstrated to be pH-independent. Incorporation of water-soluble (lactose) or highly swellable (maize starch) excipients accelerated drug release in a more pronounced manner compared to the water-insoluble excipient calcium phosphate. Stability studies demonstrated no degradation of the drug substance and reproducible drug release patterns for mini matrix tablets stored at 25 degrees C/60% RH and 30 degrees C/70% RH for up to 6 months.

Topics: Calcium Phosphates; Chemistry, Pharmaceutical; Delayed-Action Preparations; Drug Stability; Fumarates; Hydrogen-Ion Concentration; Lactose; Phenylurea Compounds; Piperidines; Polyvinyls; Povidone; Receptors, CCR1; Receptors, Chemokine; Solubility; Starch; Tablets

The study was designed to identify the key metabolic signals of glucose-stimulated proinsulin gene transcription and translation, focusing on the mechanism of succinate stimulation of insulin production.. Wistar rat islets were incubated in 3.3 mmol/l glucose with and without esters of different mitochondrial metabolites or with 16.7 mmol/l glucose. Proinsulin biosynthesis was analysed by tritiated leucine incorporation into newly synthesised proinsulin. Preproinsulin gene transcription was evaluated following transduction with adenoviral vectors expressing the luciferase reporter gene under the control of the rat I preproinsulin promoter. Steady-state preproinsulin mRNA was determined using relative quantitative PCR. The mitochondrial membrane potential was measured by microspectrofluorimetry using rhodamine-123.. Succinic acid monomethyl ester, but not other mitochondrial metabolites, stimulated preproinsulin gene transcription and translation. Similarly to glucose, succinate increased specific preproinsulin gene transcription and biosynthesis. The inhibitor of succinate dehydrogenase (SDH), 3-nitropropionate, abolished glucose- and succinate-stimulated mitochondrial membrane hyperpolarisation and proinsulin biosynthesis, indicating that stimulation of proinsulin translation depends on SDH activity. Partial inhibition of SDH activity by exposure to fumaric acid monomethyl ester abolished the stimulation of preproinsulin gene transcription, but only partially inhibited the stimulation of proinsulin biosynthesis by glucose and succinate, suggesting that SDH activity is particularly important for the transcriptional response to glucose.. Succinate is a key metabolic mediator of glucose-stimulated preproinsulin gene transcription and translation. Moreover, succinate stimulation of insulin production depends on its metabolism via SDH. The differential effect of fumarate on preproinsulin gene transcription and translation suggests that these processes have different sensitivities to metabolic signals.

Topics: Animals; Dose-Response Relationship, Drug; Fumarates; Gene Expression Regulation; Glutamine; In Vitro Techniques; Insulin; Islets of Langerhans; Male; Membrane Potentials; Mitochondria; Proinsulin; Protein Precursors; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Stimulation, Chemical; Succinate Dehydrogenase; Succinic Acid

Human mitochondrial NAD(P)+-dependent malate dehydrogenase (decarboxylating) (malic enzyme) can be specifically and allosterically activated by fumarate. X-ray crystal structures have revealed conformational changes in the enzyme in the absence and in the presence of fumarate. Previous studies have indicated that fumarate is bound to the allosteric pocket via Arg67 and Arg91. Mutation of these residues almost abolishes the activating effect of fumarate. However, these amino acid residues are conserved in some enzymes that are not activated by fumarate, suggesting that there may be additional factors controlling the activation mechanism. In the present study, we tried to delineate the detailed molecular mechanism of activation of the enzyme by fumarate. Site-directed mutagenesis was used to replace Asp102, which is one of the charged amino acids in the fumarate binding pocket and is not conserved in other decarboxylating malate dehydrogenases. In order to explore the charge effect of this residue, Asp102 was replaced by alanine, glutamate or lysine. Our experimental data clearly indicate the importance of Asp102 for activation by fumarate. Mutation of Asp102 to Ala or Lys significantly attenuated the activating effect of fumarate on the enzyme. Kinetic parameters indicate that the effect of fumarate was mainly to decrease the K(m) values for malate, Mg2+ and NAD+, but it did not notably elevate kcat. The apparent substrate K(m) values were reduced by increasing concentrations of fumarate. Furthermore, the greatest effect of fumarate activation was apparent at low malate, Mg2+ or NAD+ concentrations. The K(act) values were reduced with increasing concentrations of malate, Mg2+ and NAD+. The Asp102 mutants, however, are much less sensitive to regulation by fumarate. Mutation of Asp102 leads to the desensitization of the co-operative effect between fumarate and substrates of the enzyme.

Topics: Allosteric Site; Amino Acid Sequence; Aspartic Acid; Enzyme Activation; Fumarates; Gene Expression; Humans; Kinetics; Malate Dehydrogenase; Mitochondria; Models, Molecular; Molecular Sequence Data; Mutation; Recombinant Proteins; Substrate Specificity

We have cloned and functionally characterized a Na(+)-coupled dicarboxylate transporter, SdcS, from Staphylococcus aureus. This carrier protein is a member of the divalent anion/Na(+) symporter (DASS) family and shares significant sequence homology with the mammalian Na(+)/dicarboxylate cotransporters NaDC-1 and NaDC-3. Analysis of SdcS function indicates transport properties consistent with those of its eukaryotic counterparts. Thus, SdcS facilitates the transport of the dicarboxylates fumarate, malate, and succinate across the cytoplasmic membrane in a Na(+)-dependent manner. Furthermore, kinetic work predicts an ordered reaction sequence with Na(+) (K(0.5) of 2.7 mM) binding before dicarboxylate (K(m) of 4.5 microM). Because this transporter and its mammalian homologs are functionally similar, we suggest that SdcS may serve as a useful model for DASS family structural analysis.

Topics: Cations, Monovalent; Cloning, Molecular; Dicarboxylic Acid Transporters; Fumarates; Malates; Sodium; Staphylococcus aureus; Succinic Acid

Individuals with hemizygous germline fumarate hydratase (FH) mutations are predisposed to renal cancer. These tumors predominantly exhibit functional inactivation of the remaining wild-type allele, implicating FH inactivation as a tumor-promoting event. Hypoxia-inducible factors are expressed in many cancers and are increased in clear cell renal carcinomas. Under normoxia, the HIFs are labile due to VHL-dependent proteasomal degradation, but stabilization occurs under hypoxia due to inactivation of HIF prolyl hydroxylase (HPH), which prevents HIF hydroxylation and VHL recognition. We demonstrate that FH inhibition, together with elevated intracellular fumarate, coincides with HIF upregulation. Further, we show that fumarate acts as a competitive inhibitor of HPH. These data delineate a novel fumarate-dependent pathway for regulating HPH activity and HIF protein levels.

Topics: Adult; Alleles; Basic Helix-Loop-Helix Transcription Factors; Carcinoma, Renal Cell; DNA-Binding Proteins; Female; Fumarate Hydratase; Fumarates; Gene Expression Regulation, Neoplastic; Humans; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Ketoglutaric Acids; Kidney Neoplasms; Leiomyomatosis; Male; Middle Aged; Nuclear Proteins; Procollagen-Proline Dioxygenase; Syndrome; Transcription Factors; Up-Regulation

Fifteen potential precursors of propionate were tested for their ability to decrease CH4 production by ruminal fluid in vitro. Sodium acrylate and sodium fumarate produced the most consistent effects in batch cultures, with 50 % of the added precursors being fermented to propionate and CH4 production decreasing by between 8 and 17 %, respectively. Additives were more effective when added as free acids, but this also decreased the pH and may have inhibited fibre digestion. Changing the dietary substrate from predominantly grass hay to predominantly concentrate had no influence on the effectiveness of acrylate and fumarate. In an in vitro fermentor (the rumen simulating technique, Rusitec) with a grass hay-concentrate (50:50, w/w) diet as substrate, both compounds were again fermented to propionate (33 and 44 % conversion to propionate, respectively). However, fumarate appeared more effective as a H2 sink compound. It was calculated to capture 44 % of the H2 previously used for CH4 formation compared with a 22 % capture of H2 with acrylate. Fumarate also caused a stimulation in fibre digestion. Thus, sodium fumarate was the preferred propionate precursor for use as a feed ingredient to decrease CH4 emissions from ruminants.

Topics: Acids; Acrylates; Animals; Diet; Dietary Fiber; Digestion; Electrons; Fermentation; Food Additives; Fumarates; Hydrogen-Ion Concentration; Methane; Propionates; Rumen; Ruminants; Salts; Sheep

The effects of disodium fumarate on microbial growth, CH4 production and fermentation of three diets differing in their forage content (800, 500 and 200 g/kg DM) by rumen micro-organisms in vitro were studied using batch cultures. Rumen contents were collected from four Merino sheep. Disodium fumarate was added to the incubation bottles to achieve final concentrations of 0, 4 and 8 mm-fumarate, and (15)N was used as a microbial marker. Gas production was measured at regular intervals from 0 to 120 h of incubation. Fumarate did not affect (P>0.05) any of the measured gas production parameters. In 17 h incubations, the final pH and the production of acetate and propionate were increased linearly (P<0.001) by the addition of fumarate. Fumarate tended to increase (P=0.076) the organic matter disappearance of the diets and to decrease (P=0.079) the amount of NH3-N in the cultures. Adding fumarate to batch cultures tended (P=0.099) to decrease CH4 production, the mean values of the decrease being 5.4 %, 2.9 % and 3.8 % for the high-, medium- and low-forage diet, respectively. Fumarate tended to increase (P=0.082) rumen microbial growth for the high-forage diet, but no differences (P>0.05) were observed for the other two diets. These results indicate that the effects of fumarate on rumen fermentation depend on the nature of the incubated substrate, the high-forage diet showing the greatest response.

Topics: Acetates; Ammonia; Animal Feed; Animals; Dietary Supplements; Digestion; Fermentation; Fumarates; Hydrogen-Ion Concentration; Lactates; Methane; Propionates; Rumen; Sheep

A fumarase-deficient patient expressed a novel phenotype of congenital cerebral ventricular dilatation and periventricular cysts. The patient was a compound heterozygote for two mutations that are the only ones among the 12 published mutations that have been found in multiple, unrelated, fumarase-deficient patients.

Topics: Brain; Cysts; DNA Mutational Analysis; DNA, Complementary; Electroencephalography; Fatal Outcome; Female; Fumarate Hydratase; Fumarates; Heterozygote; Histidine; Humans; Infant, Newborn; Ketoglutaric Acids; Leukomalacia, Periventricular; Lysine; Metabolism, Inborn Errors; Methylmalonic Acid; Mutation; Phenotype; Pyruvic Acid; Ultrasonography

We examine the influence of continuous-wave heteronuclear decoupling on symmetry-based double-quantum homonuclear dipolar recoupling, using experimental measurements, numerical simulations, and average Hamiltonian theory. There are two distinct regimes in which the heteronuclear interference effects are minimized. The first regime utilizes a moderate homonuclear recoupling field and a strong heteronuclear decoupling field; the second regime utilizes a strong homonuclear recoupling field and a weak or absent heteronuclear decoupling field. The second regime is experimentally accessible at moderate or high magic-angle-spinning frequencies and is particularly relevant for many realistic applications of solid-state NMR recoupling experiments to organic or biological materials.

Topics: Alanine; Carbon Isotopes; Fumarates; Glycine; Molecular Structure; Nitrogen Isotopes; Nuclear Magnetic Resonance, Biomolecular

Human mitochondrial NAD(P)+-dependent malic enzyme is inhibited by ATP. The X-ray crystal structures have revealed that two ATP molecules occupy both the active and exo site of the enzyme, suggesting that ATP might act as an allosteric inhibitor of the enzyme. However, mutagenesis studies and kinetic evidences indicated that the catalytic activity of the enzyme is inhibited by ATP through a competitive inhibition mechanism in the active site and not in the exo site. Three amino acid residues, Arg165, Asn259, and Glu314, which are hydrogen-bonded with NAD+ or ATP, are chosen to characterize their possible roles on the inhibitory effect of ATP for the enzyme. Our kinetic data clearly demonstrate that Arg165 is essential for catalysis. The R165A enzyme had very low enzyme activity, and it was only slightly inhibited by ATP and not activated by fumarate. The values of K(m,NAD) and K(i,ATP) to both NAD+ and malate were elevated. Elimination of the guanidino side chain of R165 made the enzyme defective on the binding of NAD+ and ATP, and it caused the charge imbalance in the active site. These effects possibly caused the enzyme to malfunction on its catalytic power. The N259A enzyme was less inhibited by ATP but could be fully activated by fumarate at a similar extent compared with the wild-type enzyme. For the N259A enzyme, the value of K(i,ATP) to NAD+ but not to malate was elevated, indicating that the hydrogen bonding between ATP and the amide side chain of this residue is important for the binding stability of ATP. Removal of this side chain did not cause any harmful effect on the fumarate-induced activation of the enzyme. The E314A enzyme, however, was severely inhibited by ATP and only slightly activated by fumarate. The values of K(m,malate), K(m,NAD), and K(i,ATP) to both NAD+ and malate for E314A were reduced to about 2-7-folds compared with those of the wild-type enzyme. It can be concluded that mutation of Glu314 to Ala eliminated the repulsive effects between Glu314 and malate, NAD+, or ATP, and thus the binding affinities of malate, NAD+, and ATP in the active site of the enzyme were enhanced.

Topics: Adenosine Triphosphate; Amino Acids; Binding Sites; Binding, Competitive; Catalytic Domain; Fumarates; Humans; Kinetics; Malate Dehydrogenase; Mitochondria; Mitochondrial Proteins; Models, Molecular; Mutagenesis, Site-Directed; NAD; Recombinant Proteins

Previous research from our laboratory has shown that citric acid improves phytate P utilization in chicks fed a P-deficient corn-soybean meal diet. The current study was conducted to determine if other organic acids also are effective, with an emphasis on gluconic acid. Four experiments were conducted in which 4 replicate groups of 5 crossbred chicks (New Hampshire x Columbian) were fed a P-deficient diet (0.16% nonphyate P) from 8 to 22 d of age. In Experiment 1, chick weight gain and tibia ash were significantly increased (P < 0.05) by 1.5 and 3% sodium gluconate (NaGlu), 1.5% calcium gluconate (CaGlu), 1.5 and 3% glucono-delta-lactone, and 1% 2-hydroxy-4-methylthio butanoic acid (Alimet). In experiment 2, tibia ash was significantly increased (P < 0.05) by 2% NaGlu, CaGlu, and citric acid in chicks fed the P-deficient diet but not in chicks fed a 0.45% nonphytate P diet, indicating that the organic acid responses were due to increased P utilization. In experiments 3 and 4, tibia ash was significantly increased by 3% NaGlu and 3% citric acid, but not by 3% fumaric acid or 0.025, 0.05 and 0.1% EDTA. The results of this study showed that NaGlu, CaGlu, glucono-delta-lactone, Alimet, and citric acid, but not fumaric acid or EDTA, improved phytate P utilization in chicks fed a corn-soybean meal diet.

Topics: Animals; Biological Availability; Bone and Bones; Calcium Gluconate; Carboxylic Acids; Chickens; Citric Acid; Crosses, Genetic; Diet; Eating; Edetic Acid; Female; Fumarates; Gluconates; Glycine max; Male; Methionine; Phosphorus; Phytic Acid; Weight Gain; Zea mays

{[1-(3-Chloro-4-fluorobenzoyl)-4-fluoropiperidin-4yl]methyl}[(5-methylpyridin-2-yl)methyl]amine, fumaric acid salt (C(20)H(22)ClF(2)N(3)O, C(4)H(4)O(4)) (1) was synthesized and characterized by the complete (1)H, (13)C and (19)F NMR analyses. The conformation of the piperidin ring, in the solution state, was particularly studied from the coupling constants determined by recording a double-quantum filtered COSY experiment in phase-sensitive mode. (1)H NMR line-shape analysis was used, at temperatures varying between -5 and +60 degrees C, to determine the enthalpy of activation of the rotational barrier around the CN bond. Compound 1 crystallizes in the triclinic space group P1 with a=8.517(3) Angstrom, b=12.384(2) Angstrom, c=12.472(3) Angstrom, alpha=70.88(2) degrees, beta=82.04(2) degrees, gamma=83.58(2) degrees. The results strongly indicate that the solid and solution conformations are similar. Thermal stability and phases transitions were investigated by thermal gravimetric analysis (TGA) and differential scanning calorimetry (DSC). Furthermore polymorphism screening was studied from recrystallization of 1 performed in seven solvents and by slurry conversion in water. The X-ray powder diffraction (XRPD) and differential scanning calorimetry results suggested that 1 crystallizes into one crystalline form which melts at 157 degrees C (DeltaH=132 J g(-1)).

Topics: Calorimetry, Differential Scanning; Deuterium Oxide; Fumarates; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Conformation; Piperidines; Pyridines; Thermodynamics; X-Ray Diffraction

The chemometric resolution and quantification of overlapped peaks from comprehensive two-dimensional (2D) liquid chromatography (LCxLC) data are demonstrated. The LCxLC data is produced from an in-house LCxLC analyzer that couples an anion-exchange column via a multi-port valve with a reversed-phase column connected to a UV absorbance detector. Three test mixtures, each containing a target analyte, are subjected to partial LCxLC separations to simulate likely cases of signal overlap. The resulting unresolved target-analyte signals are then analyzed by the standard-addition method and two chemometric methods. The LCxLC analyses of a test mixture and its corresponding standard-addition mixture results in two data matrices, one for each mixture. The stacking of these two data matrices produces a data structure that can then be analyzed by trilinear chemometric methods. One method, the generalized rank annihilation method (GRAM), uses a non-iterative eigenvalue-based approach to mathematically resolve overlapped trilinear signals. The other method, parallel factor analysis (PARAFAC), uses an iterative approach to resolve trilinear signals by the optimization of initial estimates using alternating least squares and signal constraints. In this paper, GRAM followed by PARAFAC analysis is shown to produce better qualitative and quantitative results than using each method separately. For instance, for all three test mixtures, the GRAM-PARAFAC approach improved quantitative accuracy by at least a factor of 4 and quantitative precision by more than 2 when compared to GRAM alone. This paper also introduces a new means of correcting run-to-run retention time shifts in comprehensive 2D chromatographic data.

Topics: Benzoic Acid; Chemical Fractionation; Chlorobenzoates; Chromatography, Ion Exchange; Chromatography, Liquid; Factor Analysis, Statistical; Fumarates; Maleates; Organophosphorus Compounds; Pyruvic Acid; Uracil

In this study a single-well, "push- pull" test method is adapted for determination of in situ denitrification rates in groundwater aquifers. The rates of stepwise reduction of nitrate to nitrite, nitrous oxide, and molecular nitrogen were determined by performing a series of push-pull tests. The method consists of the controlled injection of a prepared test solution ("push") into an aquifer followed by the extraction of the test solution/ground water mixture ("pull") from the same location. The injected test solution consists of ground water containing a nonreactive tracer and one or more biologically reactive solutes. Reaction rate coefficients are computed from the mass of reactant consumed and/or product formed. A single Transport Test, one Biostimulation Test, and four Activity Tests were conducted for this study. Transport tests are conducted to evaluate the mobility of solutes used in subsequent tests. These included bromide (a conservative tracer), fumarate (a carbon and/or source), and nitrate (an electron acceptor). Extraction phase breakthrough curves for all solutes were similar, indicating apparent conservative transport of the solutes prior to biostimulation. Biostimulation tests were conducted to stimulate the activity of indigenous heterotrophic denitrifying microorganisms and consisted of injection of site ground water containing fumarate and nitrate. Biostimulation was detected by the simultaneous production of carbon dioxide and nitrite after each injection. Activity tests were conducted to quantify rates of nitrate, nitrite, and nitrous oxide reduction. Estimated zero-order degradation rates decreased in the order nitrate > nitrite > nitrous oxide. The series of push-pull tests developed and field tested in this study should prove useful for conducting rapid, low-cost feasibility assessments for in situ denitrification in nitrate-contaminated aquifers.

Topics: Biodegradation, Environmental; Bromides; Carbon Dioxide; Environmental Monitoring; Fumarates; Korea; Nitrates; Water; Water Microbiology; Water Pollutants, Chemical

Addition of 5 mM: fumarate to cultures of Klebsiella pneumoniae enhanced the rate of glycerol consumption and the production of 1,3-propanediol (PDO). Compared to the control, the activity of glycerol dehydrogenase increased by 35, 33 and 46%, the activity of glycerol dehydratase increased by 160, 210 and 115%, and the activity of 1,3-propanediol oxidoreductase increased by 25, 39 and 85% when, respectively, 5, 15 and 25 mM: fumarate were provided. At the same time, the ratio of NAD+ to NADH decreased by 20, 23 and 29%. Using a 5 l bioreactor with 5 mM: fumarate addition, the specific rate of glycerol consumption and the productivity of PDO was 30 mmol/l h and 17 mmol/l h, respectively, both increased by 35% over the control.

Topics: Cell Culture Techniques; Cell Proliferation; Dose-Response Relationship, Drug; Fumarates; Gene Expression Regulation, Bacterial; Glycerol; Hydro-Lyases; Klebsiella pneumoniae; Propylene Glycols

Of various yeasts tested in the conversion of fumaric to L-malic acid, Saccharomyces bayanus had the highest activity of fumarase. Cells permeabilized with 0.2% (w/v) CTAB for 5 min gave maximum enzyme activity. Under non-growth conditions, fumarase activity in the permeabilized cells was four times higher (271 U/g) than that of the intact cells (67 U/g). The proposed mathematical model for the batch production of L-malic acid was validated at different initial fumaric acid concentrations. The average conversion of fumaric acid was up to 82% and gave 21, 40, 83 and 175 mM L-malic acid from respectively, 25, 50, 100 and 210 mM: fumaric acid.

Topics: Cell Membrane Permeability; Cetrimonium; Cetrimonium Compounds; Fumarate Hydratase; Fumarates; Kinetics; Malates; Saccharomyces; Saccharomyces cerevisiae; Species Specificity; Surface-Active Agents; Time Factors

We report the fourth case of combined D-and L-2-hydroxyglutaric aciduria presenting with neonatal encephalopathy and subependymal cysts.

Topics: Brain; Brain Diseases, Metabolic, Inborn; Cysts; Electroencephalography; Fatal Outcome; Female; Fumarates; Glutarates; Humans; Infant, Newborn; Magnetic Resonance Imaging; Magnetic Resonance Spectroscopy; Male; Malonates; Muscle Hypotonia; Seizures; Stereoisomerism

The strength of the low-barrier hydrogen bond in hydrogen maleate in the gas phase was investigated by low-temperature photoelectron spectroscopy and ab initio calculations. Photoelectron spectra of maleic and fumaric acid monoanions (cis-/trans-HO(2)CCH=CHCO(2)(-)) were obtained at low temperatures and at 193 nm photon energy. Vibrational structure was observed for trans-HO(2)CCH=CHCO(2)(-) due to the OCO bending modes; however, cis-HO(2)CCH=CHCO(2)(-) yielded a broad and featureless spectrum. The electron binding energy of cis-HO(2)CCH=CHCO(2)(-) is about 1 eV blue-shifted relative to trans-HO(2)CCH=CHCO(2)(-) due to the formation of intramolecular hydrogen bond in the cis-isomer. Theoretical calculations (CCSD(T)/ aug-cc-pVTZ and B3LYP/aug-cc-pVTZ) were carried out to estimate the strength of the intramolecular hydrogen bond in cis-HO(2)CCH=CHCO(2)(-). Combining experimental and theoretical calculations yields an estimate of 21.5 +/- 2.0 kcal/mol for the intramolecular hydrogen bond strength in hydrogen maleate.

Topics: Chemical Phenomena; Chemistry, Physical; Fumarates; Gases; Hydrogen Bonding; Maleates; Molecular Conformation; Molecular Structure; Spectrum Analysis

Rhizopus oryzae is capable of producing high levels of lactic acid by the fermentation of glucose. Yields typically vary over 60-80%, with the remaining glucose diverted primarily into ethanol fermentation. The goal of this work was to increase lactate dehydrogenase (LDH) activity, so lactic acid fermentation could more effectively compete for available pyruvate. Three different constructs, pLdhA71X, pLdhA48XI, and pLdhA89VII, containing various lengths of the ldhA gene fragment, were transformed into R. oryzae. This fungus rarely integrates DNA used for transformation, but instead relies on extra-chromosomal replication in a high-copy number. Plasmid pLdhA48XI was linearized prior to transformation in order to facilitate integration into the pyrG gene used for selection. Isolates transformed with ldhA containing plasmid were compared with both the wild-type parent strain and the auxotrophic recipient strain containing vector only. All isolates transformed with pLdhA71X or pLdhA48XI had multiple copies of the ldhA gene that resulted in ldhA transcript accumulation, LDH specific activity, and lactic acid production higher than the controls. Integration of plasmid pLdhA48XI increased the stability of the strain, but did not seem to offer any benefit for increasing lactic acid production. Since lactic acid fermentation competes with ethanol and fumaric acid production, it was not unexpected that increased lactic acid production was always concomitant with decreased ethanol and fumaric acid. Plasmid pLdhA71X, containing a large ldhA fragment (6.1 kb), routinely yielded higher levels of lactic acid than the smaller region (3.3 kb) used to construct plasmid pLdhA48XI. The greatest levels of ldhA transcript and enzyme production occurred with isolates transformed with plasmid pLdhA89VII. However, these transformants always produced less lactic acid and higher amounts of ethanol, fumaric, and glycerol compared with the control.

Topics: Cloning, Molecular; DNA, Fungal; Ethanol; Fermentation; Fumarates; Gene Dosage; Genes, Fungal; Glucose; Glycerol; L-Lactate Dehydrogenase; Lactic Acid; Plasmids; Recombination, Genetic; Rhizopus; RNA, Messenger; Transcription, Genetic; Transformation, Genetic

Two main entry points for electrons into the mitochondrial respiratory chain are NADH:ubiquinone oxidoreductase (complex I) and succinate:ubiquinone oxidoreductase (complex II). Metabolic regulation of these two respiratory complexes is not understood in detail. It has been suggested that the Krebs cycle metabolic intermediate oxaloacetate (OAA) inhibits complex II in vivo, whereas complex I undergoes a reversible active/de-active transition. In normoxic and anoxic hearts it has been shown that the proportion of complex I in the active and de-active states is different suggesting a possible mode of regulation of the enzyme by oxygen concentration. In the current studies rapid isolation of mitochondrial membranes in a state that preserves the activity of both complex I and complex II has been achieved using Langendorff perfused rat hearts. The findings indicate that the state of activation of complex I is controlled by the oxygen saturation in the perfusate. In addition, these studies show that complex II is fully active in the mitochondrion and not inhibited by OAA regardless of the oxygen concentration.

Topics: Animals; Electron Transport Complex I; Electron Transport Complex II; Fumarates; Heart; Hypoxia; In Vitro Techniques; Intracellular Membranes; Male; Malonates; Mitochondria, Heart; Multienzyme Complexes; Myocardium; NADH, NADPH Oxidoreductases; Oxaloacetic Acid; Oxygen; Perfusion; Potassium Cyanide; Rats; Rats, Sprague-Dawley; Succinate Dehydrogenase

Desulfitobacterium hafniense and Desulfitobacterium sp. strain PCE-S grew under anoxic conditions with a variety of phenyl methyl ethers as electron donors in combination with fumarate as electron acceptor. The phenyl methyl ethers were O-demethylated to the corresponding phenol compounds. O-demethylation was strictly dependent on the presence of fumarate; no O-demethylation occurred with CO2 as electron acceptor. One mol phenyl methyl ether R-O-CH3 was O-demethylated to R-OH per 3 mol fumarate reduced to succinate. The growth yields with vanillate or syringate plus fumarate were approximately 15 g cells (dry weight) per mol methyl moiety converted. D. hafniense utilized vanillate or syringate as an electron donor for reductive dehalogenation of 3-Cl-4-hydroxyphenylacetate, whereas strain PCE-S was not able to dechlorinate tetrachloroethene with phenyl methyl ethers. Crude extracts of both organisms showed O-demethylase activity in the O-demethylase assay with vanillate or syringate as substrates when the organism was grown on syringate plus fumarate. Besides the homoacetogenic bacteria, only growing cells of Desulfitobacterium frappieri PCP-1 have thus far been reported to be capable of phenyl methyl ether O-demethylation. This present study is the first report of Desulfitobacteria utilizing phenyl methyl ethers as electron donors for fumarate reduction and for growth.

Topics: Anisoles; Desulfitobacterium; Electron Transport; Fumarates; Gallic Acid; Hydrocarbons, Chlorinated; Oxidation-Reduction; Oxidoreductases, O-Demethylating; Phenylacetates; Succinic Acid; Vanillic Acid

A simple HPLC method was developed for the determination of desloratadine in dog plasma and was used for evaluating the bioequivalence of desloratadine fumarate tablets and desloratadine tablets in dogs. Chromatographic separation was performed on a Hypersil CN column (150 mm x 5.0 mm, 5 microm) using a mixture of methanol, acetonitrile and phosphate buffer (pH 5.5; 0.01 mol/l) (35:35:30, v/v/v) as mobile phase delivered at a flow rate of 0.8 ml/min. The detection was set at 241 nm. The limit of quantitation was 5.0 ng/ml. The calibration range was from 5.0 to 800.0 ng/ml. Inter- and intra-day precision ranged from 1.8 to 3.8% and from 2.2 to 9.0%, respectively. The recovery of desloratadine from dog plasma ranged from 78.8 to 82.0%. The developed method was applied to the bioequivalence studies of desloratadine fumarate tablets (test preparation) and desloratadine tablets (reference preparation) in five dogs. Pharmacokinetic parameters t(max), C(max), AUC(0-t), AUC(0- infinity ), t(1/2) were determined from plasma concentration-time profiles of both preparations. The analysis of variance (ANOVA) did not show any significant difference between the two preparations and 90% confidence intervals fell within the acceptable range for bioequivalence. Based on these statistical inferences it was concluded that the two preparations exhibited comparable pharmacokinetic profiles and that desloratadine fumarate tablets was bioequivalent to desloratadine tablets.

Topics: Animals; Chromatography, High Pressure Liquid; Cross-Over Studies; Dogs; Drug Evaluation, Preclinical; Fumarates; Loratadine; Tablets; Therapeutic Equivalency

The fungal production of fumaric acid using rice bran and subsequent bacterial conversion of succinic acid using fungal culture broth were investigated. Since the rice bran contains abundant proteins, amino acids, vitamins, and minerals, it is suitable material that fungi use as a nitrogen source. The effective concentration of rice bran to produce fumaric acid was 5 g/L. A large amount of rice bran caused excessive fungal growth rather than enhance fumaric acid production. In addition, we could produce fumaric acid without the addition of zinc and iron. Fungal culture broth containing approx 25 g/L of fumaric acid was directly employed for succinic acid conversion. The amount of glycerol and yeast extract required for succinic acid conversion was reduced to 70 and 30%, respectively, compared with the amounts cited in previous studies.

Topics: Biochemistry; Biotechnology; Carbon; Cell Division; Dose-Response Relationship, Drug; Enterococcus faecalis; Fumarates; Glycerol; Oryza; Rhizopus; Succinic Acid; Time Factors

The function of the response regulator DcuR of the DcuSR fumarate two-component sensory system of Escherichia coli was analysed in vitro. Isolated DcuR protein was phosphorylated by the sensory histidine kinase, DcuS, and ATP, or by acetyl phosphate. In gel retardation assays with target promoters (frdA, dcuB, dctA), phosphoryl DcuR (DcuR-P) formed a high-affinity complex, with an apparent K(D) (app. K(D)) of 0.2-0.3 microM DcuR-P, and a low-affinity (app. K(D) 0.8-2 microM) complex. The high-affinity complex was formed only with promoters transcriptionally-regulated by DcuSR, whereas low-affinity binding was seen also with some DcuSR-independent promoters. The binding site of DcuR-P at the dcuB promoter was determined by DNase I footprinting. One binding site of 42-52 nt (position -359 to -400/-410 nt upstream of the transcriptional start) was identified in the presence of low and high concentrations of DcuR-P. Non-phosphorylated DcuR, or DcuR-D56N mutated in the phosphoryl-accepting Asp56 residue, showed low-affinity binding to target promoters. DcuR-D56N was still able to interact with DcuS. DcuR-D56N increased the phosphorylation of DcuS and competitively inhibited phosphoryl transfer to wild-type DcuR.

Topics: Base Sequence; Binding Sites; DNA-Binding Proteins; DNA, Bacterial; Escherichia coli; Escherichia coli Proteins; Fumarates; Gene Expression Regulation, Bacterial; Molecular Sequence Data; Phosphorylation; Promoter Regions, Genetic; Protein Kinases; Signal Transduction; Transcription Factors; Transcription, Genetic

Various situations observed when oxidizing organic compounds via ozone in a semi-batch reactor are illustrated. The resistance to the transfer of ozone from gas to liquid is accounted for using the film model. The mass balances are numerically solved simultaneously within the reactor and within the film to produce time dependent profiles of concentrations, Hatta, enhancement and depletion factors. Firstly, theoretical profiles are exemplified for various kinetic regimes from slow to fast; reaction occurs either in the bulk, in the film or in both. This shows the drastic importance of the shapes of the gas concentration profiles both at the exit of the reactor and in the liquid phase - in determining the regime. Then, a typical example dealing with fumaric acid ozonation is shown. Firstly, the acid itself oxidizes rapidly producing an intermediate regime: part of the reaction occurs within the film, part within the bulk and the rate constant can be determined. Then, the by-products oxidize more slowly producing a typical regime: reaction occurs within the bulk, the concentration of dissolved ozone is almost 0 and the mass transfer coefficient can be determined. Finally, when all organics have oxidized, the self-decomposition of ozone governs a slow kinetic regime: the concentration of dissolved ozone is close to equilibrium.

Topics: Anticarcinogenic Agents; Fumarates; Kinetics; Models, Theoretical; Organic Chemicals; Oxidants, Photochemical; Ozone; Waste Disposal, Fluid; Water Pollutants; Water Purification

The presence of Fe(III), but not that of Fe(II), resulted in ca. 20-fold-lower levels of mRNA for fumarate reductase, inhibiting fumarate reduction and favoring utilization of fumarate as an electron donor in chemostat cultures of Geobacter sulfurreducens, despite the fact that growth yield with fumarate was 3-fold higher than with Fe(III).

Topics: Electrons; Fumarates; Geobacter; Iron; Oxidation-Reduction; Succinate Dehydrogenase

Fumaric acid esters (FAE) are used as a systemic treatment for severe psoriasis in Germany but there has been only very little published experience from the U.K. The potential for use in combination with other systemic drugs has not been explored.. To present data relating to the efficacy of FAE in severe psoriasis and to examine the potential dose-sparing effect and safety issues when FAE are combined with other systemic agents.. We retrospectively analysed the records of patients who had received FAE for severe psoriasis either alone (in two cases) or along with other systemic medications (in 10 cases). We reviewed the efficacy of FAE and assessed whether dose reductions were achieved for other systemic drugs. Patients were monitored carefully for possible adverse effects.. Of 12 patients treated with FAE one discontinued the drug very early, due to flushing, while on a very low dose. The other 11 patients all demonstrated an improvement in psoriasis after starting FAE. Nine patients received FAE in combination with other systemic therapies including ciclosporin, acitretin, hydroxyurea and methotrexate. Seven achieved useful overall reductions in the dose of the other drugs. In two patients severe psoriasis was controlled using FAE alone. The side-effect profile of FAE was similar to that previously reported. There was no evidence of drug interactions.. FAE appear effective and less toxic than other systemic treatments for psoriasis. FAE were used successfully in combination with other systemic agents and generally enabled the doses of the more hazardous drugs to be reduced. Extremely careful monitoring is required when using FAE in such combined regimens as experience is currently very limited.

Topics: Adult; Aged; Cyclosporine; Dermatologic Agents; Drug Interactions; Drug Therapy, Combination; Esters; Female; Flushing; Fumarates; Humans; Male; Middle Aged; Psoriasis; Retrospective Studies; Time Factors; Treatment Outcome

Through pattern matching of the cytochrome c heme-binding site (CXXCH) against the genome sequence of Shewanella oneidensis MR-1, we identified 42 possible cytochrome c genes (27 of which should be soluble) out of a total of 4758. However, we found only six soluble cytochromes c in extracts of S. oneidensis grown under several different conditions: (1) a small tetraheme cytochrome c, (2) a tetraheme flavocytochrome c-fumarate reductase, (3) a diheme cytochrome c4, (4) a monoheme cytochrome c5, (5) a monoheme cytochrome c', and (6) a diheme bacterial cytochrome c peroxidase. These cytochromes were identified either through N-terminal or complete amino acid sequence determination combined with mass spectroscopy. All six cytochromes were about 10-fold more abundant when cells were grown at low than at high aeration, whereas the flavocytochrome c-fumarate reductase was specifically induced by anaerobic growth on fumarate. When adjusted for the different heme content, the monoheme cytochrome c5 is as abundant as are the small tetraheme cytochrome and the tetraheme fumarate reductase. Published results on regulation of cytochromes from DNA microarrays and 2D-PAGE differ somewhat from our results, emphasizing the importance of multifaceted analyses in proteomics.

Topics: Amino Acid Sequence; Cell Division; Cytochrome c Group; Cytochromes; Cytochromes c; Electrophoresis, Gel, Two-Dimensional; Fumarates; Genome, Bacterial; Heme; Molecular Sequence Data; Oligonucleotide Array Sequence Analysis; Proteome; Sequence Homology, Amino Acid; Shewanella; Spectrophotometry; Ultraviolet Rays

Geobacter sulfurreducens, a representative of the family Geobacteraceae that predominates in Fe(III)-reducing subsurface environments, can grow by coupling the oxidation of hydrogen to the reduction of a variety of electron acceptors, including Fe(III), fumarate, and quinones. An examination of the G. sulfurreducens genome revealed two operons, hya and hyb, which appeared to encode periplasmically oriented respiratory uptake hydrogenases. In order to assess the roles of these two enzymes in hydrogen-dependent growth, Hya- and Hyb-deficient mutants were generated by gene replacement. Hyb was found to be required for hydrogen-dependent reduction of Fe(III), anthraquinone-2,6-disulfonate, and fumarate by resting cell suspensions and to be essential for growth with hydrogen and these three electron acceptors. Hya, in contrast, was not. These findings suggest that Hyb is an essential respiratory hydrogenase in G. sulfurreducens.

Topics: Anthraquinones; Ferric Compounds; Fumarates; Geobacter; Hydrogen; Operon; Oxidation-Reduction; Oxidoreductases; Phenotype

A strictly anaerobic bacterium, strain OX39, was isolated with o-xylene as organic substrate and sulfate as electron acceptor from an aquifer at a former gasworks plant contaminated with aromatic hydrocarbons. Apart from o-xylene, strain OX39 grew on m-xylene and toluene and all three substrates were oxidized completely to CO(2). Induction experiments indicated that o-xylene, m-xylene, and toluene degradation were initiated by different specific enzymes. Methylbenzylsuccinate was identified in supernatants of cultures grown on o-xylene and m-xylene, and benzylsuccinate was detected in supernatants of toluene-grown cells, thus indicating that degradation was initiated in all three cases by fumarate addition to the methyl group. Strain OX39 was sensitive towards sulfide and depended on Fe(II) in the medium as a scavenger of the produced sulfide. Analysis of the PCR-amplified 16S rRNA gene revealed that strain OX39 affiliates with the gram-positive endospore-forming sulfate reducers of the genus Desulfotomaculum and is the first hydrocarbon-oxidizing bacterium in this genus.

Topics: Anaerobiosis; Biodegradation, Environmental; Carbon Dioxide; Carbon-Carbon Lyases; Culture Media; Desulfotomaculum; DNA, Bacterial; DNA, Ribosomal; Ferrous Compounds; Fumarates; Genes, rRNA; Molecular Sequence Data; Oxidation-Reduction; Phylogeny; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Soil Microbiology; Succinates; Sulfides; Toluene; Xylenes

It has been demonstrated previously that Geobacter species can transfer electrons directly to electrodes. In order to determine whether electrodes could serve as electron donors for microbial respiration, enrichment cultures were established from a sediment inoculum with a potentiostat-poised graphite electrode as the sole electron donor and nitrate as the electron acceptor. Nitrate was reduced to nitrite with the consumption of electrical current. The stoichiometry of electron and nitrate consumption and nitrite accumulation were consistent with the electrode serving as the sole electron donor for nitrate reduction. Analysis of 16 rRNA gene sequences demonstrated that the electrodes supplied with current were specifically enriched in microorganisms with sequences most closely related to the sequences of known Geobacter species. A pure culture of Geobacter metallireducens was shown to reduce nitrate to nitrite with the electrode as the sole electron donor with the expected stoichiometry of electron consumption. Cells attached to the electrode appeared to be responsible for the nitrate reduction. Attached cells of Geobacter sulfurreducens reduced fumarate to succinate with the electrode as an electron donor. These results demonstrate for the first time that electrodes may serve as a direct electron donor for anaerobic respiration. This finding has implications for the harvesting of electricity from anaerobic sediments and the bioremediation of oxidized contaminants.

Topics: Anaerobiosis; Electrodes; Electrons; Fumarates; Geobacter; Geologic Sediments; Graphite; Kinetics; Microscopy, Electron, Scanning; Nitrates; Phylogeny; RNA, Ribosomal, 16S; Sequence Analysis, DNA

Shewanella oneidensis MR-1 uses several electron acceptors to support anaerobic respiration including insoluble species such as iron(III) and manganese(IV) oxides, and soluble species such as nitrate, fumarate, dimethylsulfoxide and many others. MR-1 has complex branched electron transport chains that include components in the cytoplasmic membrane, periplasm, and outer membrane (OM). Previous studies have implicated a role for anaerobically upregulated OM electron transport components in the use of insoluble electron acceptors, and have suggested that other OM components may also contribute to insoluble electron acceptor use. In this study, the role for an anaerobically upregulated 35-kDa OM protein (Omp35) in the use of anaerobic electron acceptors was explored.. Omp35 was purified from the OM of anaerobically grown cells, the gene encoding Omp35 was identified, and an omp35 null mutant (OMP35-1) was isolated and characterized. Although OMP35-1 grew on all electron acceptors tested, a significant lag was seen when grown on fumarate, nitrate, and Fe(III). Complementation studies confirmed that the phenotype of OMP35-1 was due to the loss of Omp35. Despite its requirement for wild-type rates of electron acceptor use, analysis of Omp35 protein and predicted sequence did not identify any electron transport moieties or predicted motifs. OMP35-1 had normal levels and distribution of known electron transport components including quinones, cytochromes, and fumarate reductase. Omp35 is related to putative porins from MR-1 and S. frigidimarina as well as to the PorA porin from Neisseria meningitidis. Subcellular fraction analysis confirmed that Omp35 is an OM protein. The seven-fold anaerobic upregulation of Omp35 is mediated post-transcriptionally.. Omp35 is a putative porin in the OM of MR-1 that is markedly upregulated anaerobically by a post-transcriptional mechanism. Omp35 is required for normal rates of growth on Fe(III), fumarate, and nitrate, but its absence has no effect on the use of other electron acceptors. Omp35 does not contain obvious electron transport moieties, and its absence does not alter the amounts or distribution of other known electron transport components including quinones and cytochromes. The effects of Omp35 on anaerobic electron acceptor use are therefore likely indirect. The results demonstrate the ability of non-electron transport proteins to influence anaerobic respiratory phenotypes.

Topics: Amino Acid Sequence; Anaerobiosis; Bacterial Proteins; Electron Transport; Ferric Compounds; Fumarates; Membrane Proteins; Molecular Sequence Data; Nitrates; Oxidation-Reduction; Sequence Alignment; Shewanella

To compare fermentation pattern in cultures of Bacteroides caccae supplied with pectin and glucose, and identify enzymes involved in metabolism of pectin.. A strain KWN isolated from the rabbit caecum was used. Fermentation pattern, changes of viscosity and enzyme reactions products were determined. Cultures grown on pectin produced significantly more acetate and less formate, lactate, fumarate and succinate than cultures grown on glucose. Production of cell dry matter and protein per gram of substrate used was the same in pectin- and glucose-grown cultures. The principal enzymes that participated in the metabolism of pectin were extracellular exopectate hydrolase (EC 3.2.1.67), extracellular endopectate lyase (EC 4.2.2.2) and cell-associated 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (EC 4.1.2.14). The latter enzyme is unique to the Entner-Doudoroff pathway. Activities of pectinolytic enzymes in cultures grown on glucose were low. Activity of KDPG aldolase was similar in pectin- and glucose-grown cells.. Metabolites and activities of pectin-degrading enzymes differed in cultures of B. caccae KWN grown on pectin and glucose. Yields of dry matter and protein were the same on both substrates.. Information on metabolism of pectin in animal strains of Bacteroides is incomplete. This study extends the knowledge on metabolism in bacteria from the rabbit caecum.

Topics: Acetates; Aldehyde-Lyases; Animals; Bacterial Proteins; Bacteroides; Biomass; Cecum; Fermentation; Formates; Fumarates; Glucose; Lactic Acid; Pectins; Polygalacturonase; Polysaccharide-Lyases; Rabbits; Succinic Acid

The mechanisms underlying the use of insoluble electron acceptors by metal-reducing bacteria, such as Shewanella oneidensis MR-1, are currently under intensive study. Current models for shuttling electrons across the outer membrane (OM) of MR-1 include roles for OM cytochromes and the possible excretion of a redox shuttle. While MR-1 is able to release a substance that restores the ability of a menaquinone (MK)-negative mutant, CMA-1, to reduce the humic acid analog anthraquinone-2,6-disulfonate (AQDS), cross-feeding experiments conducted here showed that the substance released by MR-1 restores the growth of CMA-1 on several soluble electron acceptors. Various strains derived from MR-1 also release this substance; these include mutants lacking the OM cytochromes OmcA and OmcB and the OM protein MtrB. Even though strains lacking OmcB and MtrB cannot reduce Fe(III) or AQDS, they still release a substance that restores the ability of CMA-1 to use MK-dependent electron acceptors, including AQDS and Fe(III). Quinone analysis showed that this released substance restores MK synthesis in CMA-1. This ability to restore MK synthesis in CMA-1 explains the cross-feeding results and challenges the previous hypothesis that this substance represents a redox shuttle that facilitates metal respiration.

Topics: Electron Transport; Ferric Compounds; Fumarates; Iron; Kinetics; Nitrates; Oxidation-Reduction; Shewanella; Vitamin K 2

This paper for the first time reports the isolation of 5 compounds from Phellinus linteus. A sphingolipid (1) and two tyrosinase inhibitory compounds (2, 3) along with two carboxylic acids (4, 5), were isolated from the fruiting body of Phellinus linteus (Berk & Curt) Aoshima. The structure of compound 1 was identified as 1-omicron-beta-D-glucopyranosyl-(2S, 3R, 4E, 8E)-2-[(2R)-2-hydroxyhexadecanoylamino]-9-methyl-4,8-octadecadiene-1,3-diol, known as cerebroside B, based on spectroscopic methods such as 1 D and 2D NMR as well as by acid hydrolysis. Compounds 2 -5 were identified as protocatechualdehyde (2), 5-hydroxymethyl-2-furaldehyde (HMF) (3), succinic acid (4), and fumaric acid (5) based on the spectroscopic evidence. Compounds 2 and 3 inhibited the oxidation of L-tyrosine catalyzed by mushroom tyrosinase with an IC50 of 0.40 and 90.8 microg/mL, respectively. The inhibitory kinetics, which were analyzed by the Lineweaver-Burk plots, were found to be competitive and noncompetitive inhibitors with a Ki of 1.1 microM and 1.4 mM, respectively.

Topics: Acetylation; Basidiomycota; Benzaldehydes; Catechols; Enzyme Inhibitors; Fumarates; Gas Chromatography-Mass Spectrometry; Kinetics; Monophenol Monooxygenase; Spectrometry, Mass, Fast Atom Bombardment; Sphingolipids; Succinic Acid

The effects of heat treatment (evaporation and excessive heating), and microbial contamination (Rhizopus stolonifer, Penicillium expansum and Lactobacillus plantarum) as sources of fumaric acid formation in apple juice concentrates were investigated. Fumaric acid formed in apple juice did not exceed 1.0 mg l(-1) during both commercial-scale evaporation and laboratory-scale excessive heat treatment, indicating that malic dehydration is not the primary source of fumaric acid formation. However, R. stolonifer and L. plantarum produced 18.23 +/- 0.82 and 5.39 +/- 0.31 mg l(-1) fumaric acid as determined in the raw juice obtained from contaminated apples after 5 days of incubation. The mean fumaric acid content of apple juice concentrates manufactured in 2000, 2001 and 2002 were determined as 4.9 +/- 1.9, 5.7 +/- 2.8 and 4.1 +/- 2.6 mg l(-1), respectively. The overall results suggest that the primary source of fumaric acid in apple juice concentrate is the use of apples decayed by certain microorganisms capable of producing fumaric acid.

Topics: Beverages; Chromatography, Liquid; Dehydration; Food Contamination; Food Handling; Food Industry; Fumarates; Hot Temperature; Lactobacillus plantarum; Malus; Penicillium; Reproducibility of Results; Rhizopus; Turkey

A crystal engineering strategy for designing cocrystals of pharmaceuticals is presented. The strategy increases the probability of discovering useful cocrystals and decreases the number of experiments that are needed by selecting API:guest combinations that have the greatest potential of forming energetically and structurally robust interactions. Our approach involves multicomponent cocrystallization of hydrochloride salts, wherein strong hydrogen bond donors are introduced to interact with chloride ions that are underutilized as hydrogen bond acceptors. The strategy is particularly effective in producing cocrystals of amine hydrochlorides with neutral organic acid guests. As an example of the approach, we report the discovery of three cocrystals containing fluoxetine hydrochloride (1), which is the active ingredient in the popular antidepressant Prozac. A 1:1 cocrystal was prepared with 1 and benzoic acid (2), while succinic acid and fumaric acid were each cocrystallized with 1 to provide 2:1 cocrystals of fluoxetine hydrochloride:succinic acid (3) and fluoxetine hydrochloride:fumaric acid (4). The presence of a guest molecule along with fluoxetine hydrochloride in the same crystal structure results in a solid phase with altered physical properties when compared to the known crystalline form of fluoxetine hydrochloride. On the basis of intrinsic dissolution rate experiments, cocrystals 2 and 4 dissolve more slowly than 1, and 3 dissolves more quickly than 1. Powder dissolution experiments demonstrated that the solid present at equilibrium corresponds to the cocrystal for 2 and 4, while 3 completely converted to 1 upon prolonged slurry in water.

Topics: Benzoic Acid; Carboxylic Acids; Chemistry, Pharmaceutical; Crystallization; Fluoxetine; Fumarates; Hydrogen Bonding; Models, Molecular; Succinic Acid; X-Ray Diffraction

Biodegradable hydrogel microspheres were synthesized by free radical suspension copolymerization of poly(ethylene glycol fumarate) macromer with bisacrylamide (PEGF/PAM). The acidic initiator ammonium persulphate in combination with the basic accelerator, N,N,N',N'-tetramethyethylenediamine, were used to form the PEGF/PAM hydrogel at a neutral pH. The equilibrium water content of the microspheres was greater than 90% w/w. A model double stranded plasmid DNA (dsDNA) coding for the enhanced green fluorescence protein (pEGFP) gene was encapsulated in the hydrogel and the effect of loading and water content before swelling on release kinetics was investigated. Fluorescent confocal microscopy demonstrated that the encapsulated dsDNA was in the biologically active double stranded configuration. The highest loading of 0.81 mg ml(-1) resulted in the best encapsulation efficiency of 95%. For that loading, 6% of the dsDNA was released in 25 days at a rate of 16 ng ml(-1). The highest water content of 70% resulted in the highest burst release of 27% and 14% of the dsDNA was released in 25 days at a rate of 30 ng ml(-1). For elucidating the release mechanism, the network mesh size was compared with the radius of gyration (Rg) of the dsDNA plasmid. The mesh size was 7 nm, which was less than Rg of the dsDNA (31 nm) but greater than the chain diameter of 1.1 nm. Since the mesh size was less than Rg, the release mechanism was by reptation of the segments of dsDNA within the tube formed by the network chains between crosslinks. These results indicate that the hydrogel mesh size and the size of the plasmid control the release mechanism.

Topics: Acrylamide; Biodegradation, Environmental; DNA; Drug Compounding; Fumarates; Green Fluorescent Proteins; Humans; Hydrogels; Microscopy, Fluorescence; Microspheres; Polyethylene Glycols

A novel piezotolerant, mesophilic, facultatively anaerobic, organotrophic, polarly flagellated bacterium (strain LT13a(T)) was isolated from a deep sediment layer in the Nankai Trough (Leg 190, Ocean Drilling Program) off the coast of Japan. This organism used a wide range of organic substrates as sole carbon and energy sources: pyruvate, glutamate, succinate, fumarate, lactate, citrate, peptone and tryptone. Oxygen, nitrate, fumarate, ferric iron and cystine were used as electron acceptors. Maximal growth rates were observed at a hydrostatic pressure of 10 MPa. Hydrostatic pressure for growth was in the range 0.1-50 MPa. Predominant cellular fatty acids were 16 : 1omega7c, 15 : 0 iso, 16 : 0 and 13 : 0 iso. The G+C content of the DNA was 44.9 mol%. On the basis of 16S rRNA gene sequences, strain LT13a(T) was shown to belong to the gamma-Proteobacteria, being closely related to Shewanella putrefaciens (98 %), Shewanella oneidensis (97 %) and Shewanella baltica (96 %). Levels of DNA homology between strain LT13a(T) and S. putrefaciens, S. oneidensis and S. baltica were <20 %, indicating that strain LT13a(T) represents a novel species. Genetic evidence and phenotypic characteristics showed that isolate LT13a(T) constitutes a novel species of the genus Shewanella. Because of the deep origin of the strain, the name Shewanella profunda sp. nov. is proposed, with LT13a(T) (=DSM 15900(T)=JCM 12080(T)) as the type strain.

Topics: Anaerobiosis; Base Composition; Cystine; DNA, Bacterial; DNA, Ribosomal; Fatty Acids; Ferric Compounds; Flagella; Fumarates; Genes, rRNA; Geologic Sediments; Hydrostatic Pressure; Japan; Molecular Sequence Data; Nitrates; Nucleic Acid Hybridization; Organic Chemicals; Oxygen; Pacific Ocean; Phylogeny; RNA, Bacterial; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Shewanella; Temperature; Water Microbiology

In situ methods are needed to evaluate the effectiveness of chemical amendments at enhancing reductive dechlorination rates in groundwater that is contaminated with the priority pollutant, trichloroethene (TCE). In this communication, a method that utilizes single-well, "push-pull" tests to quantify the effects of chemical amendments on in situ reductive dechlorination rates is presented and demonstrated. Five push-pull tests were conducted in each of five monitoring wells located in a TCE-contaminated aquifer at the site of a former chemical manufacturing facility. Rates for the reductive dechlorination of the fluorinated TCE-surrogate, trichlorofluoroethene (TCFE), were measured before (test 1) and after (test 5) three successive additions (tests 2-4) of fumarate. Fumarate was selected to stimulate the growth and activity of indigenous microorganisms with the metabolic capability to reduce TCFE and TCE. In three wells, first-order rate constants for the reductive dechlorination of TCFE increased by 8.2-92 times following fumarate additions. In two wells, reductive dechlorination of TCFE was observed after fumarate additions but not before. The transformation behavior of fumarate was also monitored following each fumarate addition. Correlations between the reductive dechlorination of TCFE and the reduction of fumarate to succinate were observed, indicating that these reactions were supported by similar biogeochemical conditions at this site.

Topics: Biodegradation, Environmental; Fresh Water; Fumarates; Hydrocarbons, Chlorinated; Oxidation-Reduction; Trichloroethylene; Water Pollutants, Chemical

Linear unsaturated oligo-anhydrides containing terminal acylchloride groups have been synthesized by polycondensation of fumaric or sebacic acid with either fumaryl chloride or sebacoyl chloride. Reaction of these oligo-anhydrides with poly(ethylene glycol) produce di- and tri-block copolymers. The oligo-anhydrides and block copolymers have been characterized by gel permeation chromatography (GPC), NMR and FT-IR spectroscopies. The tri-block copolymers composed of two PEG end blocks and an oligo-anhydride center block have been used in encapsulation of calcein (encapsulation efficiency up to 40%). Encapsulation and release profile of calcein (as a model hydrophilic drug) from the di-block copolymers (micelles) and tri-block copolymer vesicles (polymersomes) as well as their in-vitro hydrolytic degradation (pH=7.4 degrees C, 37 degrees C) are reported.

Topics: Biocompatible Materials; Biodegradation, Environmental; Chromatography, Gel; Decanoic Acids; Dicarboxylic Acids; Drug Carriers; Drug Delivery Systems; Fumarates; Magnetic Resonance Spectroscopy; Micelles; Polyethylene Glycols; Polymers; Spectroscopy, Fourier Transform Infrared

Complementation of a yeast acr1 mutant carrying a deletion of the succinate/fumarate carrier gene enabled functional identification of a mitochondrial succinate translocator from Arabidopsis thaliana (AtmSFC1). Thus complementation of yeast mutants is applicable also for identification and characterization of organellar transporters. Reverse transcription polymerase chain reaction and promoter-GUS fusion showed expression of AtmSFC1 in 2 day old dark grown seedlings, which declined in cotyledons during further development, consistent with a role in export of fumarate for gluconeogenesis during lipid mobilization at early germination of Arabidopsis seeds. In mature plants, expression was found in developing and germinating pollen, suggesting a role in ethanolic fermentation.

Topics: Amino Acid Sequence; Arabidopsis; Arabidopsis Proteins; Basic-Leucine Zipper Transcription Factors; Carrier Proteins; Cloning, Molecular; DNA-Binding Proteins; Fumarates; Gene Expression Regulation, Plant; Genetic Complementation Test; Mitochondria; Mitochondrial Proteins; Molecular Sequence Data; Mutation; Phylogeny; Plant Leaves; Promoter Regions, Genetic; Recombinant Fusion Proteins; Repressor Proteins; Saccharomyces cerevisiae Proteins; Seeds; Succinic Acid; Yeasts

The component bacteria of a three-membered mixed culture able to ferment glycolate to acetate, propionate and CO(2) were isolated in pure culture. All three strains were strict anaerobes that, on the basis of comparative 16S rRNA gene sequence analysis, belonged to the order Clostridiales in the phylum Firmicutes (low G+C gram-positive bacteria). Two of the strains were not involved in glycolate metabolism. The third, the glycolate-fermenting strain 19gly4 (DSM 11261), was related to members of the family Lachnospiraceae. The cells of strain 19gly4 were oval- to lemon-shaped, 0.85 microm long and 0.65 microm in diameter, occurring singly, in pairs, or in chains of up to 30 cells. Strain 19gly4 fermented glycolate or fumarate to acetate, succinate, and CO(2). Hydrogen was not formed, and strain 19gly4 was able to grow on glycolate in pure culture without any syntrophic hydrogen transfer and without the use of an external electron acceptor. There was no evidence for homoacetogenic metabolism. This bacterium therefore differs in metabolism from previously reported glycolate-utilising anaerobes.

Topics: Anaerobiosis; Biomass; Cells, Cultured; Evolution, Molecular; Fermentation; Fumarates; Glycolates; Gram-Positive Bacteria; Phylogeny

This study was designed to assess in vivo bone and soft tissue behavior of novel oligo(poly(ethylene glycol) fumarate) (OPF) hydrogels using a rabbit model. In vitro degradation of the OPF hydrogels was also investigated in order to compare with in vivo characteristics. Four groups of OPF hydrogel implants were synthesized by alternation of crosslinking density, poly(ethylene glycol) (PEG) block length of OPF, and cell-binding peptide content. The in vitro degradation rate of OPF hydrogels increased with decreasing crosslinking density of hydrogels, which was characterized by measuring weight loss and swelling ratio of hydrogels and medium pH change. Examination of histological sections of the subcutaneous and cranial implants showed that an uniform thin circumferential fibrous capsule was formed around the OPF hydrogel implants. Quantitative evaluation of the tissue response revealed that no statistical difference existed in capsule quality or thickness between implant groups, implantation sites or implantation times. At 4 weeks, there was a very limited number of inflammatory and multinuclear cells at the implant-fibrous capsule interface for all implants. However, at 12 weeks, OPF hydrogels with PEG block length of number average molecular weight 6090+/-90 showed extensive surface erosion and superficial fragmentation that was surrounded by a number of inflammatory cells, while OPF hydrogels with PEG block length of number average molecular weight 930+/-10 elicited minimal degradation. Constant fibrous capsule layers and number of inflammatory cells were observed regardless of the incorporation of cell-binding peptide and crosslinking density of OPF hydrogels with PEG block length of number average molecular weight 930+/-90. These results confirm that the degradation of implants can be controlled by tailoring the macromolecular structure of OPF hydrogels. Additionally, histological evaluation of implants proved that the OPF hydrogel is a promising material for biodegradable scaffolds in tissue engineering.

Topics: Animals; Biocompatible Materials; Biodegradation, Environmental; Bone and Bones; Cell Adhesion; Cross-Linking Reagents; Drug Delivery Systems; Drug Implants; Female; Fumarates; Hydrogel, Polyethylene Glycol Dimethacrylate; Hydrogels; Hydrogen-Ion Concentration; Materials Testing; Microscopy; Peptides; Polyesters; Polyethylene Glycols; Rabbits; Time Factors; Tissue Engineering

Shewanella oneidensis is a metal reducer that can use several terminal electron acceptors for anaerobic respiration, including fumarate, nitrate, dimethyl sulfoxide (DMSO), trimethylamine N-oxide (TMAO), nitrite, and insoluble iron and manganese oxides. Two S. oneidensis mutants, SR-558 and SR-559, with Tn5 insertions in crp, were isolated and analyzed. Both mutants were deficient in Fe(III) and Mn(IV) reduction. They were also deficient in anaerobic growth with, and reduction of, nitrate, fumarate, and DMSO. Although nitrite reductase activity was not affected by the crp mutation, the mutants failed to grow with nitrite as a terminal electron acceptor. This growth deficiency may be due to the observed loss of cytochromes c in the mutants. In contrast, TMAO reduction and growth were not affected by loss of cyclic AMP (cAMP) receptor protein (CRP). Fumarate and Fe(III) reductase activities were induced in rich medium by the addition of cAMP to aerobically growing wild-type S. oneidensis. These results indicate that CRP and cAMP play a role in the regulation of anaerobic respiration, in addition to their known roles in catabolite repression and carbon source utilization in other bacteria.

Topics: Anaerobiosis; Bacterial Proteins; Carrier Proteins; Culture Media; Cyclic AMP; Cyclic AMP Receptor Protein; Dimethyl Sulfoxide; Ferric Compounds; Fumarates; Manganese; Mutagenesis, Insertional; Nitrates; Oxidation-Reduction; Receptors, Cyclic AMP; Shewanella

Weakly basic drugs, such as verapamil hydrochloride, that are poorly soluble in neutral/alkaline medium may have poor oral bioavailability due to reduced solubility in the small intestine and colon. Film coated pellets were prepared using two strategies to enhance drug release at high pH values. Firstly, pellets were coated with Eudragit RS/hydroxypropyl methylcellulose acetate succinate (HMAS) mixtures in proportions of 10:1 and 10:3, respectively. The enteric polymer, HMAS, would dissolve in medium at pH > 6 creating pores through the insoluble Eudragit RS membrane to increase drug release. Secondly, an acidic environment was created within the core by the inclusion of fumaric acid at concentrations of 5 and 10% in order to increase drug solubility. Both strategies enhanced drug release into neutral medium in dissolution studies using the pH change method to simulate GIT transit. Dissolution profiles of samples tested in pH 1.2 for 12 hr were compared with those using the pH change method (pH 1.2 for first 1.5 hr, pH raised to 6.8 for remaining 10.5 hr) using the area under the dissolution curve (AUC), the dissolution half-life (t50%), and the amount of drug released in 3 hr (A3hr) values. Both strategies enhanced drug release into neutral medium although the strategy using HMAS in the film was more effective. The formulation least affected by pH change was a combination of the two strategies, i.e., pellets containing 5% fumaric acid coated with Eudragit RS 12% w/w and HMAS 1.2% w/w.

Topics: Acrylic Resins; Drug Compounding; Drug Implants; Excipients; Fumarates; Hydrogen-Ion Concentration; Kinetics; Methylcellulose; Microscopy, Electron, Scanning; Solubility; Surface Properties; Time Factors; Verapamil

The effect of the addition of small molecular weight anhydride oligomers to polymer microspheres was evaluated and increased bioadhesion of the composite was demonstrated. Blends of low molecular weight anhydride oligomers with thermoplastic poly(fumaric-co-sebacic anhydride) [p(FASA)] and polycaprolactone were examined. The effects of anhydride oligomers on polymer microsphere degradation, crystallinity, and surface morphology were also explored. The results demonstrated that fumaric anhydride oligomer remained within polymer microspheres for several hours after exposure to phosphate buffer, formed a homogenous crystalline blend, increased bioadhesion as measured on rat intestine, and enhanced drug delivery in vitro as measured by the everted sac technique.

Topics: Anhydrides; Animals; Biocompatible Materials; Drug Delivery Systems; Fumarates; Hydrogen-Ion Concentration; In Vitro Techniques; Jejunum; Microscopy, Electron, Scanning; Microspheres; Molecular Weight; Mucus; Polymers; Rats; Sodium Salicylate; Surface Properties; Temperature; Tissue Adhesions; Water

The effect of feeding diets containing either spray-dried porcine plasma (SDPP) or pea protein-isolate (PPI) supplemented with either egg yolk antibodies (EYA) from hens immunized with enterotoxigenic Escherichia coli (ETEC) (K88 and F18) antigens, ZnO, fumaric acid (FA), or carbadox (AB) on pig performance, incidence of scours, and gut morphology was studied in a 14-d experiment. Ninety 10-d-old weaned pigs were assigned to six dietary treatments in a completely randomized design to give five pens per treatment with three pigs per pen. The diets were SDPP without EYA (SDPP - EYA), PPI without EYA (PPI - EYA), PPI with EYA (PPI + EYA), PPI with ZnO (PPI + ZnO), PPI with FA (PPI + FA), or PPI with AB (PPI + AB). Diets were formulated to similar nutrient levels, with AB, EYA, FA, and ZnO at 0.25, 0.5, 2.0, and 0.4% of the diet, respectively. Pigs were weighed and bled on d 0, 7, and 14 to determine plasma urea N (PUN). Pigs were orally challenged with a 6-mL dose of 10(10) cfu/mL ETEC (K88) on d 7. On d 14, three pigs per treatment were killed to obtain sections of the small intestine for histological measurements. Weekly feed intake, BW changes, and gain:feed were determined. Incidence of scours and scour scores were monitored and fecal swabs were taken before and after ETEC challenge for PCR test to detect ETEC (K88). Feeding SDPP or supplementing PPI-based diets with EYA, ZnO, FA, or AB did not affect (P > 0.05) ADG, ADFI (as-fed basis), or gain:feed throughout the study. However, pigs fed PPI - EYA tended to have lower (P = 0.08) ADFI during wk 2 (137.9 g/d) and lower (P < 0.10) ADG from d 0 to 14 (100.1 g/d) than those fed the SDPP - EYA (156.6 g/d), PPI + EYA (151.2 g/d), PPI + ZnO (158.9 g/ d), PPI + FA (155.4 g/d), and PPI + AB (152.6 g/d) diets. Although scours was evident in all pigs 8 h after the ETEC challenge, it lasted only 3 to 5 d in pigs fed SDPP or PPI supplemented with EYA, ZnO, FA, or AB. Pigs fed PPI - EYA continued to have severe diarrhea, resulting in 40% mortality vs. 13% or less in the other groups. The PCR results showed that 81% of PPI-fed pigs continued to shed ETEC K88 7 d after ETEC challenge. Pigs fed PPI-EYA had shorter villi (P < 0.05), reduced villi:crypt ratio (P < 0.003), and higher intestinal pH (P < 0.001) and PUN (P < 0.001) than those fed SDPP or PPI supplemented with EYA, ZnO, FA, and AB. In conclusion, SDPP, EYA, ZnO, FA, and AB may have provided passive control to ETEC (K88) infection and potentially enabled young

Topics: Animal Feed; Animal Nutritional Physiological Phenomena; Animals; Animals, Newborn; Anti-Bacterial Agents; Antibodies, Bacterial; Diarrhea; Egg Yolk; Escherichia coli; Escherichia coli Infections; Fumarates; Pisum sativum; Plant Proteins; Plasma; Random Allocation; Swine; Swine Diseases; Weaning; Weight Gain; Zinc Oxide

Shewanella oneidensis MR-1 is a Gram-negative, nonfermentative rod with a complex electron transport system which facilitates its ability to use a variety of terminal electron acceptors, including fumarate, for anaerobic respiration. CMTn-3, a mutant isolated by transposon (TnphoA) mutagenesis, can no longer use fumarate as an electron acceptor; it lacks fumarate reductase activity as well as a 65-kDa soluble tetraheme flavocytochrome c. The sequence of the TnphoA-flanking genomic DNA of CMTn-3 did not align to those for fumarate reductase or related electron transport genes from other bacteria. Sequence analysis of the MR-1 genomic database demonstrated that an open reading frame encoding a 65-kDa tetraheme cytochrome c with sequence similarity to the fumarate reductase from S. frigidimarina NCIMB400 was found 8 kb away from the TnphoA-flanking genomic DNA of CMTn-3. PCR analysis demonstrated that a large deletion (>or=9.2 kb and

Topics: DNA Transposable Elements; Fumarates; Shewanella; Succinate Dehydrogenase

Chromate (Cr(VI)) reduction tests were performed with nitrate- and fumarate-grown stationary phase cultures of Shewanella oneidensis MR-1 (henceforth referred to as MR-1) and disappearance of Cr(VI) was monitored over time. A rapid initial decrease in Cr(VI) concentration was observed, which was followed by a slower, steady decrease. These observations appear to be consistent with our previous results indicating that Cr(VI) reduction in MR-1 involves at least two mechanisms (Viamajala et al., 2002b). Modeling of metal reduction kinetics is often based on single-enzyme Michaelis-Menten equations. However, these models are often developed using initial rates and do not always match actual reduction profiles. Based on the hypothesis that multiple Cr(VI) reduction mechanisms exist in MR-1, a model was developed to describe the kinetics of Cr(VI) reduction by two parallel mechanisms: (1) a rapid Cr(VI) reduction mechanism that was deactivated (or depleted) quickly, and (2) a slower mechanism that had a constant activity and was sustainable for a longer duration. Kinetic parameters were estimated by fitting experimental data, and model fits were found to correspond very closely to quantitative observations of Cr(VI) reduction by MR-1.

Topics: Cell Culture Techniques; Chromates; Enzymes; Fumarates; Kinetics; Mathematics; Models, Biological; Nitrates; Oxidation-Reduction; Reproducibility of Results; Shewanella

The structure of the water-soluble, periplasmic domain of the fumarate sensor DcuS (DcuS-pd) has been determined by NMR spectroscopy in solution. DcuS is a prototype for a sensory histidine kinase with transmembrane signal transfer. DcuS belongs to the CitA family of sensors that are specific for sensing di- and tricarboxylates. The periplasmic domain is folded autonomously and shows helices at the N and the C terminus, suggesting direct linking or connection to helices in the two transmembrane regions. The structure constitutes a novel fold. The nearest structural neighbor is the Per-Arnt-Sim domain of the photoactive yellow protein that binds small molecules covalently. Residues Arg107, His110, and Arg147 are essential for fumarate sensing and are found clustered together. The structure constitutes the first periplasmic domain of a two component sensory system and is distinctly different from the aspartate sensory domain of the Tar chemotaxis sensor.

Topics: Amino Acid Sequence; Arginine; Bacterial Proteins; Binding Sites; Cell Membrane; Chemoreceptor Cells; Escherichia coli; Escherichia coli Proteins; Fumarates; Histidine; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Sequence Data; Mutation; Periplasm; Protein Conformation; Protein Folding; Protein Kinases; Protein Structure, Secondary; Protein Structure, Tertiary; Receptors, Cell Surface; Signal Transduction

Dinoflagellate algae of the genus Symbiodinium in symbiosis with marine animals release much of their photosynthetic carbon to the animal host. The compounds translocated to the host ('mobile compounds') were investigated by metabolite comparison as follows: a substrate was identified as a candidate mobile compound when comparable profiles of metabolites were generated from host metabolism of this substrate (supplied exogenously) and the endogenous mobile compounds. When the sea anemone Anemonia viridis was incubated with NaH14CO2 under photosynthesizing conditions, most of the radioactivity in the animal tissue was recovered from the low-molecular-mass fraction and distributed in the ratio 1:2:1 between the neutral, acidic and basic sub-fractions. Prominent 14C-labelled compounds included glucose, malate and glucose-6-phosphate. When the symbiosis was incubated with 14C-labelled glucose plus succinate or fumarate (but none of eight other substrate combinations tested), the 14C-labelled metabolites closely matched those obtained with NaH14CO2. These data suggest that glucose and succinate/fumarate (or metabolically allied compounds) may be important photosynthetic compounds transferred from the Symbiodinium cells to the tissues of A. viridis. Metabolite comparisons can be applied to study nutritional interactions in symbioses involving photosynthetic algae and, with appropriate modification, other associations between microorganisms and plants or animals.

Topics: Animals; Carbon Radioisotopes; Dinoflagellida; Formates; Fumarates; Glucose; Photosynthesis; Sea Anemones; Succinic Acid; Symbiosis; Wales

This research investigates the in vitro release of transforming growth factor-beta1 (TGF-beta1) from novel, injectable hydrogels based on the polymer oligo(poly(ethylene glycol) fumarate) (OPF). These hydrogels can be used to encapsulate TGF-beta1-loaded-gelatin microparticles and can be crosslinked at physiological conditions within a clinically relevant time period. Experiments revealed that OPF formulation and crosslinking time may be adjusted to influence the equilibrium swelling ratio, elastic modulus, strain at fracture, and mesh size of these hydrogels. Studies with OPF-gelatin microparticle composites revealed that OPF formulation and crosslinking time, as well as microparticle loading and crosslinking extent, influence composite swelling. In vitro TGF-beta1 release studies demonstrated that burst release from OPF hydrogels with a mesh size of 136 A was approximately 53%, while burst release from hydrogels with a mesh size of 93 A was only 34%. For hydrogels with a large mesh size (136 A), encapsulation of loaded gelatin microparticles allowed burst release to be reduced to 29-32%, depending on microparticle loading. Likewise, final cumulative release after 28 days was reduced from 71% to 48-66% by encapsulation of loaded microparticles. However, inclusion of gelatin microparticles within OPF hydrogels of smaller mesh size (93 A) was seen to increase TGF-beta1 release rates. The equilibrium swelling ratio of the microparticle component of these composites was shown to be greater than the equilibrium swelling ratio of the OPF component. Therefore, increased release rates are the result of disruption of the polymer network during swelling. These combined results indicate that the kinetics of TGF-beta1 release can be controlled by adjusting OPF formulation and microparticle loading, factors affecting the swelling behavior these composites. By systematically altering these parameters, in vitro release rates from hydrogels and composites loaded with TGF-beta1 at concentrations of 200 ng/ml can be varied from 13 to 170 pg TGF-beta1/day for days 1-3 and from 7 to 47 pg TGF-beta1/day for days 6-21. Therefore, these studies demonstrate the potential of these novel hydrogels and composites in the sustained delivery of low dosages of TGF-beta1 to articular cartilage defects.

Topics: Capsules; Fumarates; Gelatin; Hydrogels; Polyethylene Glycols; Rheology; Tensile Strength; Transforming Growth Factor beta; Transforming Growth Factor beta1

This work presents a new molding process for photo-crosslinked, degradable polymeric networks of poly(propylene fumarate) (PPF) and the crosslinking agent poly(propylene fumarate)-diacrylate (PPF-DA). Transparent room temperature vulcanizing silicone molds were fabricated for parts ranging from simple test coupons to orthopaedic implants. The PPF/PPF-DA resin blend was injected into the cavity and photo-crosslinked as light was transmitted through the mold wall. The volumetric shrinkage, mechanical properties, and the effects of gamma sterilization were reported for molded PPF/PPF-DA networks prepared with varying compositions of the two polymer components. The shrinkage decreased while the mechanical properties displayed a general increasing trend when more of the crosslinking agent was incorporated into the network. Gamma irradiation resulted in an improvement of the mechanical properties. In addition, PPF/PPF-DA replicates of a 70:30 poly(L/DL-lactide) biodegradable fixation plate and a bone allograft interbody fusion spacer were produced to evaluate the performance of PPF/PPF-DA as an orthopaedic implant and allow for a comparison to be made with materials that have been established for clinical use.

Topics: Absorbable Implants; Biocompatible Materials; Drug Implants; Elasticity; Fumarates; Gamma Rays; Light; Materials Testing; Polymers; Polypropylenes; Silicones; Stress, Mechanical

Batch cultures of mixed rumen micro-organisms were used to study the effects of different concentrations of disodium fumarate on the fermentation of five concentrate feeds (maize, barley, wheat, sorghum and cassava meal). Rumen contents were collected from four Merino sheep fed lucerne hay ad libitum and supplemented with 300 g concentrate/d. Disodium fumarate was added to the incubation bottles to achieve final concentrations of 0, 4, 7 and 10 mM-fumarate. In 17 h incubations, the final pH and total volatile fatty acid production increased (P<0.001) linearly for all substrates as fumarate concentration increased from 0 to 10 mm. Propionate and acetate production increased (P<0.05), while the value of the acetate:propionate ratio decreased (P<0.05) linearly with increasing doses of fumarate. In contrast, l-lactate and NH3-N concentrations in the cultures were not affected (P>0.05) by the addition of fumarate. For all substrates, fumarate treatment decreased (P<0.05) CH4 production, the mean values of the decrease being 2.3, 3.8 and 4.8 % for concentrations of 4, 7 and 10 mM-fumarate respectively. Addition of fumarate did not affect (P>0.05) the total gas production. If the results of the present experiment are confirmed in vivo, fumarate could be used as a feed additive for ruminant animals fed high proportions of cereal grains, because it increased pH, acetate and propionate production and it decreased CH4 production.

Topics: Acetates; Animal Feed; Animals; Bacteriological Techniques; Dietary Supplements; Edible Grain; Fermentation; Fumarates; Hordeum; Hydrogen-Ion Concentration; Manihot; Methane; Propionates; Rumen; Sheep; Triticum; Zea mays

From a collection of over 2800 Salmonella enterica subspecies Enterica serotype Typhimurium F98 Tn5-TC1 insertion mutants 14 were identified as expressing growth-non-suppressive phenotype under strict anaerobic conditions. Sequence analysis of regions flanking the Tn insertions revealed that most of the selected mutants were defective in genes contributing to the anaerobic fumarate uptake and generation (insertions in dcuA, dcuB and aspA), or to the anaerobic L-arginine utilisation pathway (insertions in STM4467 encoding a putative arginine deiminase, and in between speF encoding ornithine decarboxylase and kdpE coding a response regulator protein). Mutants defective in flagellum synthesis (flhA) were also identified. In contrast to the in vitro results, all the mutants colonised 1-day-old chicks efficiently and suppressed the super-infection of chicks by the parent strain. This clearly indicates that neither of the metabolic pathways mentioned above nor motility play essential roles in lower intestinal tract colonisation.

Topics: Anaerobiosis; Animals; Arginine; Chickens; Colony Count, Microbial; Flagella; Fumarates; Gene Expression Regulation, Bacterial; Genes, Bacterial; Genotype; Intestines; Male; Mutagenesis, Insertional; Poultry Diseases; Salmonella Infections, Animal; Salmonella typhimurium; Specific Pathogen-Free Organisms

The aim of the experiment was to determine the effect of potential antioxidants (adenosine, L-cysteine hydrochloride, ascorbic acid, magnesium fumarate and prolactin) supplementing the Biosolwens extender on semen survival time and sperm chromatin structure. The semen motility was examined every day and the susceptibility of sperm chromatin to denaturation was evaluated on collection day and day 15 of storage. The addition of magnesium fumarate to Biosolwens extender increased sperm survival but resulted in the highest increment in the proportion of sperm with damaged chromatin. Biosolwens supplemented with 200 mg of L-cysteine hydrochloride brought the best results. It is possible that lower concentrations of this component would act in a more protective manner. The examination of the chromatin structure appears to be an useful tool for investigation of semen preservation.

Topics: Adenosine; Animals; Antioxidants; Ascorbic Acid; Chromatin; Cysteine; Fumarates; Male; Prolactin; Semen Preservation; Sperm Motility; Spermatozoa; Sus scrofa

The regulation of human mitochondrial NAD(P)+-dependent malic enzyme (m-NAD-ME) by ATP and fumarate may be crucial for the metabolism of glutamine for energy production in rapidly proliferating tissues and tumors. Here we report the crystal structure at 2.2 A resolution of m-NAD-ME in complex with ATP, Mn2+, tartronate, and fumarate. Our structural, kinetic, and mutagenesis studies reveal unexpectedly that ATP is an active-site inhibitor of the enzyme, despite the presence of an exo binding site. The structure also reveals the allosteric binding site for fumarate in the dimer interface. Mutations in this binding site abolished the activating effects of fumarate. Comparison to the structure in the absence of fumarate indicates a possible molecular mechanism for the allosteric function of this compound.

Topics: Adenosine Triphosphate; Allosteric Site; Amino Acid Sequence; Binding Sites; Cations, Divalent; Crystallography, X-Ray; Fumarates; Humans; Kinetics; Malate Dehydrogenase; Manganese; Mitochondria; Models, Molecular; Molecular Sequence Data; Mutation; Tartronates

The two-component regulatory system DcuSR of Escherichia coli controls the expression of genes of C(4)-dicarboxylate metabolism in response to extracellular C(4)- dicarboxylates such as fumarate or succinate. DcuS is a membrane-integral sensor kinase, and the sensory and kinase domains are located on opposite sides of the cytoplasmic membrane. The intact DcuS protein (His(6)-DcuS) was overproduced and isolated in detergent containing buffer. His(6)-DcuS was reconstituted into liposomes made from E. coli phospholipids. Reconstituted His(6)-DcuS catalyzed, in contrast to the detergent-solubilized sensor, autophosphorylation by [gamma-(33)P]ATP with an approximate K(D) of 0.16 mm for ATP. Up to 7% of the reconstituted DcuS was phosphorylated. Phosphorylation was stimulated up to 5.9-fold by C(4)-dicarboxylates, but not by other carboxylates. The phosphoryl group of DcuS was rapidly transferred to the response regulator DcuR. Upon phosphorylation, DcuR bound specifically to dcuB promoter DNA. The reconstituted DcuSR system therefore represents a defined in vitro system, which is capable of the complete transmembrane signal transduction by the DcuSR two-component system from the stimulus (fumarate) to the DNA, including signal transfer across the phospholipid membrane.

Topics: Adenosine Triphosphate; Cell Membrane; Detergents; DNA; DNA-Binding Proteins; Dose-Response Relationship, Drug; Escherichia coli; Escherichia coli Proteins; Fumarates; Histidine Kinase; Kinetics; Liposomes; Models, Biological; Phosphorylation; Promoter Regions, Genetic; Protein Binding; Protein Kinases; Proteolipids; Signal Transduction; Time Factors; Transcription Factors

The photoinduced gelation system based on 1 (non-gelling) to 2 (gelling) molecular photoisomerization in water results by microspheres (1) to gel fibers (2) transformation at the supramolecular level.

Topics: Amides; Delayed-Action Preparations; Fumarates; Gels; Maleates; Microspheres; Molecular Conformation; Phenylalanine; Photochemistry; Water

Torula corallina, a strain presently being used for the industrial production of erythritol, has the highest erythritol yield ever reported for an erythritol-producing microorganism. The increased production of erythritol by Torula corallina with trace elements such as Cu(2+) has been thoroughly reported, but the mechanism by which Cu(2+) increases the production of erythritol has not been studied. This study demonstrated that supplemental Cu(2+) enhanced the production of erythritol, while it significantly decreased the production of a major by-product that accumulates during erythritol fermentation, which was identified as fumarate by instrumental analyses. Erythrose reductase, a key enzyme that converts erythrose to erythritol in T. corallina, was purified to homogeneity by chromatographic methods, including ion-exchange and affinity chromatography. In vitro, purified erythrose reductase was significantly inhibited noncompetitively by increasing the fumarate concentration. In contrast, the enzyme activity remained almost constant regardless of Cu(2+) concentration. This suggests that supplemental Cu(2+) reduced the production of fumarate, a strong inhibitor of erythrose reductase, which led to less inhibition of erythrose reductase and a high yield of erythritol. This is the first report that suggests catabolite repression by a tricarboxylic acid cycle intermediate in T. corallina.

Topics: Aldehyde Reductase; Copper; Cryptococcus; Erythritol; Fumarates

The metabolite patterns obtained by ex vivo proton MR spectroscopy of fluid from different locations of hydatid cysts of sheep and humans (n = 16) and cysticercus cysts of swine and humans (n = 25) were compared with an objective of differentiating the two parasites on the basis of their metabolite pattern. The spectra from hydatid fluid differed from cysticercus cyst by the absence of creatine in the former. When the hydatid cyst was fertile, malate and/or fumarate was also observed, which was absent in cysticercus cyst. The most likely explanation for the presence of creatine only in the cysticercus fluid is its active diffusion from the surrounding host tissue along with a contribution from the musculature present in the bladder wall of the cyst.

Topics: Animals; Creatine; Cysticercosis; Diagnosis, Differential; Echinococcosis; Fumarates; Humans; Magnetic Resonance Spectroscopy; Malates; Protons; Sheep

A rotating fibrous-bed bioreactor (RFB) was developed for fermentation to produce L(+)-lactic acid from glucose and cornstarch by Rhizopus oryzae. Fungal mycelia were immobilized on cotton cloth in the RFB for a prolonged period to study the fermentation kinetics and process stability. The pH and dissolved oxygen concentration (DO) were found to have significant effects on lactic acid productivity and yield, with pH 6 and 90% DO being the optimal conditions. A high lactic acid yield of 90% (w/w) and productivity of 2.5 g/L.h (467 g/h.m(2)) was obtained from glucose in fed-batch fermentation. When cornstarch was used as the substrate, the lactic acid yield was close to 100% (w/w) and the productivity was 1.65 g/L.h (300 g/h.m(2)). The highest concentration of lactic acid achieved in these fed-batch fermentations was 127 g/L. The immobilized-cells fermentation in the RFB gave a virtually cell-free fermentation broth and provided many advantages over conventional fermentation processes, especially those with freely suspended fungal cells. Without immobilization with the cotton cloth, mycelia grew everywhere in the fermentor and caused serious problems in reactor control and operation and consequently the fermentation was poor in lactic acid production. Oxygen transfer in the RFB was also studied and the volumetric oxygen transfer coefficients under various aeration and agitation conditions were determined and then used to estimate the oxygen transfer rate and uptake rate during the fermentation. The results showed that the oxygen uptake rate increased with increasing DO, indicating that oxygen transfer was limited by the diffusion inside the mycelial layer.

Topics: Bioreactors; Cells, Cultured; Cells, Immobilized; Cotton Fiber; Equipment Design; Ethanol; Fermentation; Fumarates; Glucose; Hydrogen-Ion Concentration; Lactic Acid; Mycelium; Oxygen; Quality Control; Rhizopus; Rotation; Starch

Polymer microspheres (0.5-5.0 microm) are difficult to characterize in vivo because they degrade, migrate, and are endocytosed. A novel polyethylene mesh pouch containing microspheres allowed for retrieval of degraded polymeric products from rats without affecting the rate of degradation. Pouches containing poly(lactic-co-glycolic acid) (PLGA) or poly(fumaric-co-sebacic acid) (P(FASA)) microspheres were implanted intramuscularly, subcutaneously, and intraperitoneally and analyzed after 3, 7, 14, and 28 days. In vivo, subcutaneous or intraperitoneal implants experienced an immediate mass loss and a delayed decrease in molecular weight (Mw). Intramuscular implants behaved similarly to in vitro samples, decreasing in Mw immediately and lagging in mass loss. These results suggest that mass loss, which is usually dependent on Mw loss in vitro, may be directly due to enzymatic, rather than hydrolytic, degradation subcutaneously and intraperitoneally, while intramuscular implants appear to be mostly dependent on hydrolytic cleavage. This observation is further supported by histology. Additional experiments on pouches loaded with PLGA microspheres encapsulating osteoprotegerin, a protein drug used to prevent bone resorption, revealed that use of the device prevented the artifactual polymer compression inherent to microsphere centrifugation during release studies and allowed for the extraction of active protein from microspheres implanted for 3 days in vivo.

Topics: Absorbable Implants; Animals; Biocompatible Materials; Chromatography; Chromatography, High Pressure Liquid; Decanoic Acids; Dicarboxylic Acids; Fumarates; Glycoproteins; Lactic Acid; Microscopy, Electron, Scanning; Microspheres; Molecular Weight; Muscles; Organ Size; Osteoprotegerin; Polyethylenes; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Polymers; Rats; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Time Factors

The rotenone-insensitive NADH dehydrogenase isolated from mitochondria of the procyclic form of Trypanosoma brucei has the ability to produce superoxide anions (Biochemistry 41 (2002) 3065). Superoxide production by the purified enzyme was 60% inhibited by diphenyl iodonium (DPI), stimulated significantly by ubiquinone analogues, and unaffected by metal ions. Production of reactive oxygen species (ROS) in intact cells was not affected by addition of rotenone with proline and malate as substrates; however, addition of rotenone inhibited 41% ROS production with succinate as substrate. These results suggest that complex I is not involved in production of ROS and that succinate-linked reversed electron transport occurs in trypanosome mitochondria. Superoxide formation in mitochondria with NADH as substrate was stimulated by antimycin A but was unaffected by myxothiazol plus stigmatellin, indicating that bc(1) complex is not a source of superoxide. DPI and fumarate inhibited by 68 and 36%, respectively, the rate of superoxide production with NADH as substrate. Addition of both fumarate and DPI blocked 70% superoxide production in mitochondria, a total inhibition similar to that observed with DPI addition alone. These results suggest that the rotenone-insensitive NADH dehydrogenase in addition to NADH fumarate reductase is a potential source of superoxide production in procyclic trypanosome mitochondria.

Topics: Animals; Anti-Bacterial Agents; Antimycin A; Biphenyl Compounds; Fumarates; Malates; Methacrylates; Mitochondria; NAD; NADH Dehydrogenase; Onium Compounds; Polyenes; Proline; Rotenone; Substrate Specificity; Succinic Acid; Superoxides; Thiazoles; Trypanosoma brucei brucei; Ubiquinone; Uncoupling Agents

It has previously been suggested that organic acids enhance iron absorption. We have studied the effect of nine organic acids on the absorption of Fe(II) and Fe(III) in the human epithelial cell line Caco-2. The effect obtained was dose-dependent, and the greatest increase (43-fold) was observed for tartaric acid (4 mmol/L) on Fe(III) (10 micromol/L). Tartaric, malic, succinic, and fumaric acids enhanced Fe(II) and Fe(III) uptake. Citric and oxalic acid, on the other hand, inhibited Fe(II) uptake but enhanced Fe(III) uptake. Propionic and acetic acid increased the Fe(II) uptake, but had no effect on Fe(III) uptake. Our results show a correlation between absorption pattern and chemical structure; e.g. hydroxyl groups, in addition to carboxyls, were connected with a positive influence. The results may be important for elucidating factors affecting iron bioavailability in the small intestine and for the development of foods with improved iron bioavailability.

Topics: Absorption; Acetic Acid; Caco-2 Cells; Carboxylic Acids; Citric Acid; Ferric Compounds; Ferrous Compounds; Fumarates; Humans; Iron; Malates; Oxalic Acid; Propionates; Succinic Acid; Tartrates

Synthesis of ethylene in static cultures as well as the effect of endogenous and exogenous ethylene on the synthesis of polygalacturonase by Aspergillus niger were determined. This strain produced maximum ethylene amounts when cultured at 30 degrees C for 3 d. The effect of adding ethylene precursors (citrate-cycle intermediates) on ethylene production was investigated. Best intracellular and extracellular polygalacturonase production was obtained with 2-oxoglutaric, pyruvic and fumaric acids, and with glutamic acid too. Addition of ethylene to the culture medium also increased the synthesis of polygalacturonase, although to a lower degree than when glutamic acid was added.

Topics: Aspergillus niger; Biomass; Citric Acid Cycle; Culture Media; Ethylenes; Fumarates; Glutamic Acid; Ketoglutaric Acids; Kinetics; Polygalacturonase; Pyruvic Acid

Topics: Aged; Biopsy, Needle; Dose-Response Relationship, Drug; Drug Administration Schedule; Elbow; Esters; Female; Follow-Up Studies; Forearm; Fumarates; Granuloma Annulare; Granuloma, Giant Cell; Humans; Immunohistochemistry; Male; Severity of Illness Index; Skin Diseases; Treatment Outcome

Wolinella succinogenes performs oxidative phosphorylation with fumarate instead of O2 as terminal electron acceptor and H2 or formate as electron donors. Fumarate reduction by these donors ('fumarate respiration') is catalyzed by an electron transport chain in the bacterial membrane, and is coupled to the generation of an electrochemical proton potential (Deltap) across the bacterial membrane. The experimental evidence concerning the electron transport and its coupling to Deltap generation is reviewed in this article. The electron transport chain consists of fumarate reductase, menaquinone (MK) and either hydrogenase or formate dehydrogenase. Measurements indicate that the Deltap is generated exclusively by MK reduction with H2 or formate; MKH2 oxidation by fumarate appears to be an electroneutral process. However, evidence derived from the crystal structure of fumarate reductase suggests an electrogenic mechanism for the latter process.

Topics: Bacillus subtilis; Binding Sites; Catalysis; Cell Membrane; Electron Transport; Energy Metabolism; Formate Dehydrogenases; Fumarates; Hydrogenase; Models, Chemical; Oxidation-Reduction; Oxidative Phosphorylation; Succinate Dehydrogenase; Vitamin K 2; Wolinella

A membrane protein complex, succinate dehydrogenase (SQR) from Escherichia coli has been purified and crystallised. This enzyme is composed of four subunits containing FAD, three iron-sulphur clusters and one haem b as prosthetic groups. The obtained crystals belong to the hexagonal space group P6(3) with the unit-cell dimensions of a=b=123.8 A and c=214.6 A. An asymmetric unit of the crystals contains one SQR monomer (M(r) 120 kDa). A data set is now available at 4.0 A resolution with 88.1% completeness and 0.106 R(merge). We have obtained a molecular replacement solution that shows sensible molecular packing, using the soluble domain of E. coli QFR (fumarate reductase) as a search model. The packing suggests that E. coli SQR is a crystallographic trimer rather than a dimer as observed for the E. coli QFR.

Topics: Crystallography; Electron Transport Complex II; Escherichia coli; Flavin-Adenine Dinucleotide; Fumarates; Heme; Intracellular Membranes; Iron-Sulfur Proteins; Membrane Proteins; Models, Molecular; Multienzyme Complexes; Oxidoreductases; Quinone Reductases; Succinate Dehydrogenase; Succinic Acid

A simple unstructured model, which includes carbon source as the limiting and essential substrate and oxygen as an enhancing substrate for cell growth, has been implemented to depict cell population evolution of two Escherichia coli strains and the expression of their trimethylammonium metabolism in batch and continuous reactors. Although the model is applied to represent the trans-crotonobetaine to L-(-)-carnitine biotransformation, it is also useful for understanding the complete metabolic flow of trimethylammonium compounds in E. coli. Cell growth and biotransformation were studied in both anaerobic and aerobic conditions. For this reason we derived equations to modify the specific growth rate, mu, and the cell yield on the carbon source (glycerol), Y(xg), as oxygen increased the rate of growth. Inhibition functions representing an excess of the glycerol and oxygen were included to depict cell evolution during extreme conditions. As a result, the model fitted experimental data for various growth conditions, including different carbon source concentrations, initial oxygen levels, and the existence of a certain degree of cell death. Moreover, the production of enzymes involved within the E. coli trimethylammonium metabolism and related to trans-crotonobetaine biotransformation was also modeled as a function of both the cell and oxygen concentrations within the system. The model describes all the activities of the different enzymes within the transformed and wild strains, able to produce L-(-)-carnitine from trans-crotonobetaine under both anaerobic and aerobic conditions. Crotonobetaine reductase inhibition by either oxygen or the addition of fumarate as well as its non-reversible catalytic action was taken into consideration. The proposed model was useful for describing the whole set of variables under both growing and resting conditions. Both E. coli strains within membrane high-density reactors were well represented by the model as results matched the experimental data.

Topics: Algorithms; Betaine; Bioreactors; Carnitine; Computer Simulation; Escherichia coli; Fumarates; Membranes; Models, Biological; Multienzyme Complexes; Oxidoreductases; Time Factors

In this study, 0, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 g/l amounts of activated charcoal (AC) were added into apple juice with a patulin content of 62.3 ppb obtained from a well-established manufacturing company. Apple juice samples were then mixed for 0, 5, 10, 20, and 30 min, respectively. Considerable reduction in the patulin and HMF values was found while there is a dramatic improvement in the colour and clearness of apple juice. However, AC did not cause a significant decrease in the fumaric acid level of apple juice. The best result was obtained at 3.0 g/l AC mixed for 5 min. In addition, a negligible reduction in brix and pH values of samples was observed.

Topics: Anticarcinogenic Agents; Beverages; Charcoal; Fumarates; Hydrogen-Ion Concentration; Malus; Mutagens; Patulin; Time Factors

A study was conducted to develop a preservative treatment capable of the Food and Drug Administration-mandated 5-log reduction of Escherichia coli O157:H7 populations in apple cider. Unpreserved apple cider was treated with generally recognized as safe acidulants and preservatives before inoculation with E. coli O157:H7 in test tubes and subjected to mild heat treatments (25, 35, and 45 degrees C) followed by refrigerated storage (4 degrees C). Fumaric acid had significant (P < 0.05) bactericidal effect when added to cider at 0.10% (wt/vol) and adjusted to pH 3.3, but citric and malic acid had no effect. Strong linear correlation (R2 = 0.96) between increasing undissociated fumaric acid concentrations and increasing log reductions of E. coli O157:H7 in apple cider indicated the undissociated acid to be the bactericidal form. The treatment that achieved the 5-log reduction in three commercial ciders was the addition of fumaric acid (0.15%, wt/vol) and sodium benzoate (0.05%, wt/vol) followed by holding at 25 degrees C for 6 h before 24 h of refrigeration at 4 degrees C. Subsequent experiments revealed that the same preservatives added to cider in flasks resulted in a more than 5-log reduction in less than 5 and 2 h when held at 25 and 35 degrees C, respectively. The treatment also significantly (P < 0.05) reduced total aerobic counts in commercial ciders to populations less than those of pasteurized and raw ciders from the same source (after 5 and 21 days of refrigerated storage at 4 degrees C, respectively). Sensory evaluation of the same ciders revealed that consumers found the preservative-treated cider to be acceptable.

Topics: Anticarcinogenic Agents; Beverages; Colony Count, Microbial; Escherichia coli O157; Food Handling; Food Preservation; Food Preservatives; Fumarates; Hydrogen-Ion Concentration; Malus; Sodium Benzoate; Taste; Temperature; Time Factors

The purpose of this study was to examine if substantial bone loss occurs in weaned pigs by feeding a phosphorus-deficient diet with or without fumaric acid. Eighteen weaned pigs were used. The animals were assigned to three groups: group C (control; 0.65% P on DM basis), group LP (low phosphorus; 0.37% P on DM basis) and group LPF (low phosphorus plus fumaric acid; 0.35% P on DM basis plus 2% fumaric acid). These three diets were fed to the groups for a period of four weeks after a two-week adaptation period. Blood samples were collected once a week. Carboxyterminal telopeptide of type I collagen (ICTP) in serum was used as a bone resorption marker. Osteocalcin (OC) and bone-specific alkaline phosphatase (bAP) were used as bone formation markers. Bone mineral density (BMD) and content (BMC) were determined by peripheral quantitative computer tomography. BAP activities significantly increased (24%) in group LPF, and at the last sampling day group LPF had significantly increased activities in comparison to group C. In contrast, ICTP concentrations significantly increased with time in group LP and LPF, and at the last sampling day group LPF had significantly increased activities in comparison to group C. BMD and BMC in femur and tibia significantly decreased in group LP and LPF. The results show that P-deficient diets induce a bone loss. Fumaric acid did not influence the degree of bone loss. With a better understanding of its effect on bone, dietary phosphorus requirements in pigs could be more precisely defined.

Topics: Adaptation, Physiological; Alkaline Phosphatase; Animals; Bone and Bones; Bone Density; Bone Development; Bone Resorption; Calcium; Collagen Type I; Digestion; Female; Fumarates; Magnesium; Nutritional Requirements; Osteocalcin; Peptide Fragments; Peptides; Phosphorus; Phosphorus, Dietary; Procollagen; Swine; Tomography, X-Ray Computed; Weaning

The anaerobic degradation pathway of the saturated hydrocarbon n-hexane in a denitrifying strain (HxN1) was examined by gas chromatography-mass spectrometry of derivatized extracts from cultures grown with unlabeled and deuterated substrate; several authentic standard compounds were included for comparison. The study was focused on possible reaction steps that follow the initial formation of (1-methylpentyl)succinate from n-hexane and fumarate. 4-Methyloctanoic, 4-methyloct-2-enoic, 2-methylhexanoic, 2-methylhex-2-enoic and 3-hydroxy-2-methylhexanoic acids (in addition to a few other methyl-branched acids) were detected in n-hexane-grown but not in n-hexanoate-grown cultures. Labeling indicated preservation of the original carbon chain of n-hexane in these acids. Tracing of the deuterium label of 3- d1-(1-methylpentyl)succinate in tentative subsequent products indicated a deuterium/carboxyl carbon exchange in the succinate moiety. This suggests that the metabolism of (1-methylpentyl)succinate employs reactions analogous to those in the established conversion of succinyl-CoA via methylmalonyl-CoA to propionyl-CoA. Accordingly, a pathway is proposed in which (1-methylpentyl)succinate is converted to the CoA-thioester, rearranged to (2-methylhexyl)malonyl-CoA and decarboxylated (perhaps by a transcarboxylase) to 4-methyloctanoyl-CoA. The other identified fatty acids match with a further degradation of 4-methyloctanoyl-CoA via rounds of conventional beta-oxidation. Such a pathway would also allow regeneration of fumarate (for n-hexane activation) from propionyl-CoA formed as intermediate and hence present a cyclic process.

Topics: Alkanes; Anaerobiosis; Bacteria; Caproates; Culture Media; Deuterium; Fumarates; Gas Chromatography-Mass Spectrometry; Hexanes; Nitrates; Oxidation-Reduction; Succinates

The purpose of this study was to evaluate under field conditions, the efficacy of mild organic acid solutions in the prevention of neonatal kid diarrhoea. At a goat farm, two experimental groups of approximately 120 kids each were formed. The kids of the first group were not submitted to any treatment and served as negative controls, whereas the kids of the second group received a solution of organic acids (Euroacid 50-L; Eurotec, Waterloo, Belgium) which was administered orally on the first and second day of life. Groups were compared with regard to the incidence of diarrhoea, its duration, and the mortality of the kids. The results showed that, in comparison with the control group, the morbidity, the mortality and the case fatality of the treated kids was significantly reduced (P < 0.05). Furthermore, the duration of diarrhoea per sick kid was markedly reduced in the acidifier-treated group in comparison with the control animals (P < 0.05). Cultures of the diarrhoeic faeces from kids indicated that enterotoxigenic strains of Escherichia coli, and more specifically strains that were positive for K88 and K99 antigens, were present in the particular farm. It was concluded that the administration of organic acids can be a helpful means in controlling scours in neonatal kids.

Topics: Administration, Oral; Animals; Animals, Newborn; Anti-Bacterial Agents; Dairying; Diarrhea; Escherichia coli Infections; Female; Fumarates; Goat Diseases; Goats; Solutions; Treatment Outcome

The organic acids present in several samples of quince fruit (pulp and peel) and quince jam (homemade and industrially manufactured) were analyzed by HPLC. The sample preparation was simple, involving only extraction with methanol (40 degrees C) and filtration through a Sep-pack C18 cartridge. The chromatographic separation was achieved using an ion exclusion column, Nucleogel Ion 300 OA (300 x 7.7 mm), in conjunction with a column heating device at 30 degrees C. An isocratic elution with H(2)SO(4) 0.01 N as the mobile phase, with a flow rate of 0.1 mL/min, and UV detection at 214 nm were used. These analyses showed that all samples presented a similar profile composed of at least six identified organic acids: citric, ascorbic, malic, quinic, shikimic, and fumaric acids. Several samples also contained oxalic acid. This study suggests that the organic acids levels and ratios may be useful for the determination of percent fruit content of quince jams. The citric acid value can also be used in the differentiation of the type of manufacture of the commercial quince jams (homemade or industrially manufactured).

Topics: Ascorbic Acid; Carboxylic Acids; Chromatography, Gel; Chromatography, High Pressure Liquid; Citric Acid; Food Handling; Fruit; Fumarates; Malates; Methanol; Oxalic Acid; Quinic Acid; Rosaceae; Shikimic Acid

Hydrogenase and fumarate reductase isolated from Wolinella succinogenes were incorporated into liposomes containing menaquinone. The two enzymes were found to be oriented solely to the outside of the resulting proteoliposomes. The proteoliposomes catalyzed fumarate reduction by H2 which generated an electrical proton potential (Delta(psi) = 0.19 V, negative inside) in the same direction as that generated by fumarate respiration in cells of W. succinogenes. The H+/e ratio brought about by fumarate reduction with H2 in proteoliposomes in the presence of valinomycin and external K+ was approximately 1. The same Delta(psi) and H+/e ratio was associated with the reduction of 2,3-dimethyl-1,4-naphthoquinone (DMN) by H2 in proteoliposomes containing menaquinone and hydrogenase with or without fumarate reductase. Proteoliposomes containing menaquinone and fumarate reductase with or without hydrogenase catalyzed fumarate reduction by DMNH2 which did not generate a Delta(psi). Incorporation of formate dehydrogenase together with fumarate reductase and menaquinone resulted in proteoliposomes catalyzing the reduction of fumarate or DMN by formate. Both reactions generated a Delta(psi) of 0.13 V (negative inside). The H+/e ratio of formate oxidation by menaquinone or DMN was close to 1. The results demonstrate for the first time that coupled fumarate respiration can be restored in liposomes using the well characterized electron transport enzymes isolated from W. succinogenes. The results support the view that Delta(psi) generation is coupled to menaquinone reduction by H2 or formate, but not to menaquinol oxidation by fumarate. Delta(psi) generation is probably caused by proton uptake from the cytoplasmic side of the membrane during menaquinone reduction, and by the coupled release of protons from H2 or formate oxidation on the periplasmic side. This mechanism is supported by the properties of two hydrogenase mutants of W. succinogenes which indicate that the site of quinone reduction is close to the cytoplasmic surface of the membrane.

Topics: Base Sequence; DNA Primers; Electron Transport; Fumarates; Liposomes; Oxidation-Reduction; Wolinella

Topics: Academies and Institutes; Ascorbic Acid; Biochemistry; Fumarates; History, 19th Century; History, 20th Century; Humans; Hungary; Nobel Prize; Sweden

A series of fumarate analogues has been used to explore the molecular mechanism of the activation of dopamine beta-mono-oxygenase by fumarate. Mesaconic acid (MA) and trans -glutaconic acid (TGA) both activate the enzyme at low concentrations, similar to fumarate. However, unlike fumarate, TGA and MA interact effectively with the oxidized enzyme to inhibit it at concentrations of 1-5 mM. Monoethylfumarate (EFum) does not activate the enzyme, but inhibits it. In contrast with TGA and MA, however, EFum inhibits the enzyme by interacting with the reduced form. The saturated dicarboxylic acid analogues, the geometric isomer and the diamide of fumaric acid do not either activate or inhibit the enzyme. The phenylethylamine-fumarate conjugate, N -(2-phenylethyl)fumaramide (PEA-Fum), is an approximately 600-fold more potent inhibitor than EFum and behaves as a bi-substrate inhibitor for the reduced enzyme. The amide of PEA-Fum behaves similarly, but with an inhibition potency approximately 20-fold less than that of PEA-Fum. The phenylethylamine conjugates of saturated or geometric isomers of fumarate do not inhibit the enzyme. Based on these findings and on steady-state kinetic analysis, an electrostatic model involving an interaction between the amine group of the enzyme-bound substrate and a carboxylate group of fumarate is proposed to account for enzyme activation by fumarate. Furthermore, in light of the recently proposed model for the similar copper enzyme, peptidylglycine alpha-hydroxylating mono-oxygenase, the above electrostatic model suggests that fumarate may also play a role in efficient electron transfer between the active-site copper centres of dopamine beta-mono-oxygenase.

Topics: Amides; Animals; Ascorbic Acid; Binding Sites; Cattle; Dopamine beta-Hydroxylase; Dose-Response Relationship, Drug; Electron Transport; Enzyme Activation; Fumarates; Glutarates; Kinetics; Maleates; Models, Chemical; Models, Molecular; Oxygen; Phenethylamines; Spectrophotometry; Tyramine

Trypanosoma cruzi, a protozoan causing Chagas' disease, excretes a considerable amount of succinate even though it uses the TCA cycle and the aerobic respiratory chain. For this reason, it was believed that unknown metabolic pathways participate in succinate production in this parasite. In the present study, we examined the molecular properties of dihydroorotate dehydrogenase (DHOD), the fourth enzyme of de novo pyrimidine biosynthetic pathway, as a soluble fumarate reductase (FRD) because our sequence analysis of pyr genes cluster showed that the amino acid sequence of T. cruzi DHOD is quite similar to that of type 1A DHOD of Saccharomyces cerevisiae, an enzyme that uses fumarate as an electron acceptor and produces succinate. Biochemical analyses of the cytosolic enzyme purified from the parasite and of the recombinant enzyme revealed that T. cruzi DHOD has methylviologen-fumarate reductase (MV-FRD) activity. In addition, T. cruzi DHOD was found to catalyze electron transfer from dihydroorotate to fumarate by a ping-pong Bi-Bi mechanism. The recombinant enzyme contained FMN as a prosthetic group. Dynamic light scattering analysis indicated that T. cruzi DHOD is a homodimer. These results clearly indicated that the cytosolic MV-FRD is attributable to T. cruzi DHOD. The DHOD may play an important role in succinate/fumarate metabolism as well as de novo pyrimidine biosynthesis in T. cruzi.

Topics: Animals; Binding Sites; Cytosol; Dihydroorotate Oxidase; Fumarates; Kinetics; Oxidation-Reduction; Paraquat; Recombinant Proteins; Solubility; Succinate Dehydrogenase; Succinates; Trypanosoma cruzi

The human gastrointestinal pathogen Campylobacter jejuni is a microaerophilic bacterium with a respiratory metabolism. The genome sequence of C. jejuni strain 11168 reveals the presence of genes that encode terminal reductases that are predicted to allow the use of a wide range of alternative electron acceptors to oxygen, including fumarate, nitrate, nitrite, and N- or S-oxides. All of these reductase activities were present in cells of strain 11168, and the molybdoenzyme encoded by Cj0264c was shown by mutagenesis to be responsible for both trimethylamine-N-oxide (TMAO) and dimethyl sulfoxide (DMSO) reduction. Nevertheless, growth of C. jejuni under strictly anaerobic conditions (with hydrogen or formate as electron donor) in the presence of any of the electron acceptors tested was insignificant. However, when fumarate, nitrate, nitrite, TMAO, or DMSO was added to microaerobic cultures in which the rate of oxygen transfer was severely restricted, clear increases in both the growth rate and final cell density compared to what was seen with the control were obtained, indicative of electron acceptor-dependent energy conservation. The C. jejuni genome encodes a single class I-type ribonucleotide reductase (RNR) which requires oxygen to generate a tyrosyl radical for catalysis. Electron microscopy of cells that had been incubated under strictly anaerobic conditions with an electron acceptor showed filamentation due to an inhibition of cell division similar to that induced by the RNR inhibitor hydroxyurea. An oxygen requirement for DNA synthesis can thus explain the lack of anaerobic growth of C. jejuni. The results indicate that strict anaerobiosis is a stress condition for C. jejuni but that alternative respiratory pathways can contribute significantly to energy conservation under oxygen-limited conditions, as might be found in vivo.

Topics: Anaerobiosis; Campylobacter jejuni; Dimethyl Sulfoxide; Fumarates; Methylamines; Nitrates; Nitrites; Oxygen Consumption

Twelve compounds containing two quadruply bonded Mo(2)(DAniF)(3) (DAniF = N,N'-di-p-anisylformamidinate) units linked by dicarboxylate anions have been prepared in high purity and good yields. All of these compounds have been characterized by crystallography and NMR. The dinuclear pairs display electrochemical behavior which is controlled by the nature of the bridging dicarboxylate group. As described by the linkers, the compounds are oxalate, 1; acetylene dicarboxylate, 2; fumarate, 3; tetrafluorophthalate, 4; carborane dicarboxylate, 5; ferrocene dicarboxylate, 6; malonate, 7; succinate, 8; propane-1,3-dicarboxylate, 9; tetrafluorosuccinate, 10; bicyclo[1.1.1]pentane-1,3-dicarboxylate, 11; and trans-1,4-cyclohexanedicarboxylate, 12.

Topics: Acetylene; Dicarboxylic Acids; Electrochemistry; Fumarates; Malonates; Models, Molecular; Molecular Structure; Molybdenum; Organometallic Compounds; Oxalates; Phthalic Acids; Propane; Spectrometry, X-Ray Emission; Succinates

Sulfate-reducing bacteria are considered as strict anaerobic microorganisms, in spite of the fact that some strains have been shown to tolerate the transient presence of dioxygen. This report shows that membranes from Desulfovibrio gigas grown in fumarate/sulfate contain a respiratory chain fully competent to reduce dioxygen to water. In particular, a membrane-bound terminal oxygen reductase, of the cytochrome bd family, was isolated, characterized, and shown to completely reduce oxygen to water. This oxidase has two subunits with apparent molecular masses of 40 and 29 kDa. Using NADH or succinate as electron donors, the oxygen respiratory rates of D. gigas membranes are comparable to those of aerobic organisms (3.2 and 29 nmol O(2) min(-1) mg protein(-1), respectively). This 'strict anaerobic' bacterium contains all the necessary enzymatic complexes to live aerobically, showing that the relationships between oxygen and anaerobes are much more complex than originally thought.

Topics: Anaerobiosis; Benzoquinones; Cell Membrane; Cytochrome b Group; Cytochromes; Desulfovibrio; Electron Transport; Electron Transport Chain Complex Proteins; Enzyme Inhibitors; Escherichia coli Proteins; Fumarates; NAD; Oxidation-Reduction; Oxidoreductases; Oxygen; Oxygen Consumption; Quinones; Succinic Acid; Sulfates

We report about a rare case of a pathological fracture of the shank following earlier pathological fractures at other locations in a comparatively young female patient with no history of trauma. There were no known diseases other than psoriasis. The shank fracture was treated surgically by osteosynthesis. Osteoporosis, myeloma, or malignancy as causative factors of this fracture could be excluded. Scintigraphy showed an enhancement, especially at the extremities. Other than reactive bone growth, histological examination revealed no further aspects. Laboratory analysis indicated a massive lack of vitamin D3. After transferring the patient to the internal department of our hospital, long-term medication with fumaric acid was determined to be the reason for the osteomalacia of a Fanconi's syndrome. Three months after cessation of these medicaments and treatment with active vitamin D3 metabolites, the patient was free of complaints. The radiographs showed an essential improvement of the demineralization.

Topics: Adult; Ankle Injuries; Diagnosis, Differential; Fanconi Syndrome; Female; Femoral Neck Fractures; Fracture Fixation, Internal; Fractures, Spontaneous; Fumarates; Humans; Psoriasis; Radiography; Reoperation; Tibial Fractures

The endogenous metabolites of the coelomic fluid of the earthworm Eisenia veneta were characterised using high-resolution one-dimensional and two-dimensional 1H nuclear magnetic resonance spectroscopy. Signals from common organic acids, such as acetate, fumarate, malonate, malate, formate, and succinate, were identified together with adenosine and nicotinamide mononucleotide. The potential use of this information as a baseline dataset for future toxicological or physiological studies was demonstrated by a metabonomic analysis: a series of earthworms were dosed with the model compound 3-fluoro-4-nitrophenol, and toxic effects followed by multivariate analysis of the spectral data of the coelomic fluid. Relative concentrations of acetate and malonate were decreased in the dosed worms compared to the controls.

Topics: Acetates; Animals; Fumarates; Magnetic Resonance Spectroscopy; Nitrophenols; Oligochaeta; Tritium

The flavoprotein dihydroorotate dehydrogenase (DHOD) catalyzes the oxidation of dihydroorotate to orotate. Dihydrooxonate is an analogue of dihydroorotate in which the C5 carbon is substituted by a nitrogen atom. We have investigated dihydrooxonate as a substrate of three DHODs, each representing a distinct evolutionary class of the enzyme, namely the two family 1 enzymes from Lactococcus lactis, DHODA and DHODB, and the enzyme from Escherichia coli, which, like the human enzyme, belongs to family 2. Dihydrooxonate was accepted as a substrate although much less efficiently than dihydroorotate. The first half-reaction was rate limiting according to pre-steady-state and steady-state kinetics with different electron acceptors. Cysteine and serine have been implicated as active site base residues, which promote substrate oxidation in family 1 and family 2 DHODs, respectively. Mutants of DHODA (C130A) and E. coli DHOD (S175A) have extremely low activity in standard assays with dihydroorotate as substrate, but with dihydrooxonate the mutants display considerable and increasing activity above pH 8.0. Thus, the absence of the active site base residue in the enzymes seems to be compensated for by a lower pK(a) of the 5-position in the substrate. Oxonate, the oxidation product of dihydrooxonate, was a competitive inhibitor versus dihydroorotate, and DHODA was the most sensitive of the three enzymes. DHODA was reinvestigated with respect to product inhibition by orotate. The results suggest a classical one-site ping-pong mechanism with fumarate as electron acceptor, while the kinetics with ferricyanide is highly dependent on the detailed reaction conditions.

Topics: Binding Sites; Catalysis; Cysteine; Dihydroorotate Dehydrogenase; Escherichia coli; Fumarates; Lactobacillus; Mutation; Orotic Acid; Oxidoreductases; Oxidoreductases Acting on CH-CH Group Donors; Recombinant Proteins; Serine; Substrate Specificity

High-level ab initio calculations (B3LYP/6-31+G and QCISD(T)/6-311+G**) were carried out to resolve the disagreement between recent experimental and computational estimates of the relative strength of the intramolecular hydrogen bond in Z-hydrogen maleate anion with respect to the normal hydrogen bond in maleic acid. The computational estimates for the strength of the intramolecular hydrogen bond in the gas-phase maleate anion are in a range of 14-28 kcal/mol depending on the choice of the reference structure. Computational data suggest that the electrostatic influence of a counterion such as a tetraalkylammonium cation can considerably weaken the hydrogen bonding interaction (by 1.5-2 times) in the complexed hydrogen maleate anion relative to that in the naked anion. The estimated internal H-bonding energies for a series of Z-maleate/R4N+ salts (R = CH3, C2H5, CH3CH2CH2CH2) range from 8 to 13 kcal/mol. The calculated energy differences between the E- and Z-hydrogen maleates complexed to Me4N+, Et4N+, and Bu4N+ cation are 4.9 (B3LYP/6-31+G(d,p)) and 5.7 and 5.8 kcal/mol (B3LYP/6-31G(d)). It is also demonstrated that the sodium cation exerts a similar electrostatic influence on the hydrogen bond strength in bifluoride anion (FHF-). The present study shows that while low-barrier short hydrogen bonds can exist in the gas phase (the barrier for the hydrogen transfer in maleate anion is only 0.2 kcal/mol at the QCISD(T)/6-311+G//QCISD/6-31+G level), whether they can also be strong in condensed media or not depends on how their interactions with their immediate environment affect their strength.

Topics: Enzyme Inhibitors; Fumarates; Hydrogen Bonding; Maleates; Models, Molecular; Molecular Conformation; Molecular Structure; Quaternary Ammonium Compounds; Thermodynamics

Quintuple mutants of Escherichia coli deficient in the C(4)-dicarboxylate carriers of aerobic and anaerobic metabolism (DctA, DcuA, DcuB, DcuC, and the DcuC homolog DcuD, or the citrate/succinate antiporter CitT) showed only poor growth on succinate (or other C(4)-dicarboxylates) under oxic conditions. At acidic pH (pH 6) the mutants regained aerobic growth on succinate, but not on fumarate. Succinate uptake by the mutants could not be saturated at physiological succinate concentrations (< or =5 mM), in contrast to the wild-type, which had a K(m) for succinate of 50 microM and a V(max) of 35 U/g dry weight at pH 6. At high substrate concentrations, the mutants showed transport activities (32 U/g dry weight) comparable to that of the wild-type. In the wild-type using DctA as the carrier, succinate uptake had a pH optimum of 6, whereas succinate uptake in the mutants was maximal at pH 5. In the mutants succinate uptake was inhibited competitively by monocarboxylic acids. Diffusion of succinate or fumarate across phospholipid membranes (liposomes) was orders of magnitude slower than the transport in the wild-type or the mutants. The data suggest that mutants deficient in DctA, DcuA, DcuB, DcuC, DcuD (or CitT) contain a carrier, possibly a monocarboxylate carrier, which is able to transport succinate, but not fumarate, at acidic pH, when succinate is present as a monoanion. Succinate uptake by this carrier was inhibited by addition of an uncoupler. Growth by fumarate respiration (requiring fumarate/succinate antiport) was also lost in the quintuple mutants, and growth was not restored at pH 6. In contrast, the efflux of succinate produced during glucose fermentation was not affected in the mutants, demonstrating that, for succinate efflux, a carrier different from, or in addition to, the known Dcu and CitT carriers is used.

Topics: Bacterial Proteins; Biological Transport; Carrier Proteins; Dicarboxylic Acid Transporters; Escherichia coli; Escherichia coli Proteins; Fumarates; Hydrogen-Ion Concentration; Mutation; Nitriles; Succinic Acid; Uncoupling Agents

The extent to and the mechanism by which fructose-1,6-bisphosphate (FDP) crosses cell membranes are unknown. We hypothesized that its transport is either via band 3 or a dicarboxylate transporter. The question was addressed in isolated Langendorff rat hearts perfused under normoxic conditions. Groups of hearts received the following metabolic substrates (in mM): 5 FDP; 5 FDP + either 5, 10, or 20 fumarate; 10 FDP and either 5, 10, or 20 fumarate; or 5 FDP + 2 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS), a band 3 inhibitor. FDP uptake and metabolism were measured as production of [(13)C]lactate from [(13)C]FDP or (14)CO(2) and [(14)C]lactate from uniformly labeled [(14)C]FDP in sample perfusates. During 30 min of perfusion, FDP metabolism was 12.4 +/- 2.6 and 31.2 +/- 3.0 micromol for 5 and 10 mM FDP, respectively. Addition of 20 mM fumarate reduced FDP metabolism over a 30-min perfusion period to 3.1 +/- 0.6 and 6.3 +/- 0.5 micromol for 5 and 10 mM FDP groups, respectively. DNDS did not affect FDP utilization. These data are consistent with transport of FDP by a dicarboxylate transport system.

Topics: Animals; Anion Exchange Protein 1, Erythrocyte; Biological Transport; Carbon Dioxide; Carbon Radioisotopes; Dicarboxylic Acid Transporters; Energy Metabolism; Fructosediphosphates; Fumarates; Glycolysis; Hydrogen-Ion Concentration; Lactic Acid; Magnetic Resonance Spectroscopy; Myocardial Ischemia; Myocardium; Rats; Sarcolemma

Using N2 cavitation, we established a protocol to prepare the active mitochondria from Plasmodium falciparum showing a higher succinate dehydrogenase activity than previously reported and a dihydroorotate-dependent respiration. The fact that fumarate partially inhibited the dihydroorotate dependent respiration suggests that complex II (succinate-ubiquinone reductase/quinol-fumarate reductase) in the erythrocytic stage cells of P. falciparum functions as a quinol-fumarate reductase.

Topics: Animals; Blotting, Western; Fumarates; Mitochondria; Orotic Acid; Oxygen; Oxygen Consumption; Plasmodium falciparum; Succinate Dehydrogenase

Topics: Antibodies, Monoclonal; Cholecalciferol; Clinical Trials as Topic; Combined Modality Therapy; Fumarates; Humans; Prognosis; Psoriasis; PUVA Therapy; Tumor Necrosis Factor-alpha

Three different surface-enhanced Raman scattering (SERS) spectra are recorded for aspartic acid on H(2)O silver sols under different concentrations and pH values. The analysis of the results shows that it interacts with the metal surface in its dianionic form in two different ways, depending on the pH and concentration. Moreover, in some cases the fumarate anion is detected, which results from the chemical surface transformation of the aspartate. The N-deuterated aspartic acid adsorbed on the D(2)O silver sols gives rise to only one SERS spectrum as a consequence of the interaction of amino and carboxylate functional groups of the dianion with the metal, independent of the concentration and pD.

Topics: Adsorption; Aspartic Acid; Colloids; Fumarates; Hydrogen-Ion Concentration; Models, Molecular; Silver; Spectrum Analysis, Raman

Based on the principle of coupling reaction and separation process, free cells containing fumarase were used for producing L-malic acid. The calcium fumarate was used as substrate to produce calcium malate directly. This new method was more advantageous than the traditional immobilized cells conversion system in aspects such as simple equipment and operation, high conversion efficiency and the yield of product. The results showed that at reaction temperature 40 degrees C, pH7.0-7.5, reaction time 20-28 h, the conversion efficiency was up to 99.9% and about 3.2 kg calcium fumarate was converted to calcium malate per liter enzyme suspension. Also, L-malic acid produced in free fumarase system satisfied USP criterion, the residual fumaric acid was less than 0.1% and the cost was approximately to that of DL-malic acid produced by chemical synthesis.

Topics: Biotransformation; Corynebacterium; Fumarate Hydratase; Fumarates; Hydrogen-Ion Concentration; Malates; Substrate Specificity; Temperature

Topics: Animals; Anticarcinogenic Agents; Fumarates; Gluconates; Ileum; In Vitro Techniques; Intestinal Absorption; Magnesium; Magnesium Chloride; Magnesium Compounds; Male; Rats; Rats, Wistar

The catalysts for many microbially mediated environmental processes such as the dechlorination of polychlorinated biphenyls (PCBs) have been difficult to identify by traditional isolation techniques. Numerous, as yet unsuccessful, attempts have been made to isolate and culture the dechlorinating species. To overcome this limitation, amplified rDNA restriction analysis (ARDRA) of a clone library, denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (TRFLP) were used concurrently to compare their effectiveness for characterizing an enriched microbial community. These methods were applied to enrichment cultures that selectively dechlorinated double-flanked chlorines in the PCB congener 2,3,4,5 chlorinated biphenyl. The methods have different biases, which were apparent from discrepancies in the relative clone frequencies (ARDRA), band intensities (DGGE) or peak heights (TRFLP) from the same enrichment culture. However, each method was effectively qualitative and identified the same organisms: a low G + C Gram-positive eubacterium, an organism most similar to the green non-sulphur bacteria, an Aminobacterium sp. and a Desulfovibrio sp. Overall, in community fingerprinting and preliminary identification, DGGE proved to be the most rapid and effective tool for the monitoring of microorganisms within a highly enriched culture. TRFLP results corroborated DGGE fingerprint analysis; however, identification required the additional step of creating a clone library. ARDRA provided an in-depth analysis of the community and this technique detected slight intraspecies sequence variation in 16S rDNA. These molecular methods are common in environmental microbiology, but rarely are they compared with the same sample site or culture. In general, all three methods detected similar community profiles, but inherent biases resulted in different detection limits for individual OTUs (operational taxonomic units).

Topics: Ampicillin; Anti-Bacterial Agents; Anticarcinogenic Agents; Bacteria; Chlorine; DNA, Bacterial; DNA, Ribosomal; Electrophoresis; Formates; Fumarates; Molecular Sequence Data; Penicillins; Phylogeny; Polychlorinated Biphenyls; Polymorphism, Restriction Fragment Length; Vancomycin

The metabolic consequences of supplying oxygen by two different modes were investigated. The effects of hypothermic liver preservation after cold hypoxic flush (Group I), oxygenated vascular persufflation (Group II), and continuous oxygenated perfusion (Group III) were compared. Adenine nucleotides were measured to assess energetics, and 1H nuclear magnetic resonance spectroscopy was employed to investigate other metabolic pathways. Energetics were maintained by both modes of oxygenation at 24 h. The mitochondrial redox state is indicated by the ratio of acetoacetate (Ace) and beta-hydroxybutyrate (betaHb). The detection of only betaHb or Ace in the hypoxic flush and perfused livers, respectively, suggested that the mitochondria of these livers were hyperreduced and hyperoxidized, respectively. In contrast, both components of the redox couple were detected in the persufflated livers, suggesting that persufflation may be a simple and effective method of maintaining hepatic energetics long-term while maintaining a more normal mitochondrial redox state.

Topics: Adenine Nucleotides; Amino Acids; Animals; Cryopreservation; Energy Metabolism; Fumarates; Glucose; In Vitro Techniques; Ketone Bodies; Kinetics; Lactic Acid; Liver; Magnetic Resonance Spectroscopy; Male; Mitochondria, Liver; Oxidation-Reduction; Oxygen; Perfusion; Rats; Rats, Sprague-Dawley; Spectrophotometry; Succinic Acid; Tissue Preservation

In this study, the immobilization characteristics of Enterococcus faecalis RKY1 for succinate production were examined. At first, three natural polymers--agar, kappa-carrageenan, and sodium alginate--were tried as immobilizing matrices. Among these, sodium alginate was selected as the best gel for immobilization of E. faecalis RKY1. Efficient conditions for immobilization were established to be with a 2% (w/v) sodium alginate solution and 2-mm-diameter bead. The bioconversion characteristics of the immobilized cells at various pH values and temperatures were examined and compared with those of free cells. The optimum pH and temperature of the immobilized cells were the same as for free cells, 7.0 and 38 degrees C respectively, but the conversion ratio was higher by immobilization for all the other pH and temperature conditions tested. When the seed volume of the immobilized cells was adjusted to 10% (v/v), 30 g/L of fumarate was completely converted to succinate (0.973 g/g conversion ratio) after 12 h. In addition, the immobilized cells maintained a conversion ratio of >0.95 g/g during 4 wk of storage at 4 degrees C in a 2% (w/v) CaCl2 solution. In repetitive bioconversion experiments, the activity of the immobilized cells decreased linearly according to the number of times of reuse.

Topics: Alginates; Bioreactors; Biotransformation; Cells, Immobilized; Enterococcus faecalis; Fumarates; Gels; Glucuronic Acid; Hexuronic Acids; Hydrogen-Ion Concentration; Particle Size; Succinic Acid; Temperature

Shewanella putrefaciens MR-1 possesses a complex electron transport system which facilitates its ability to use a diverse array of compounds as terminal electron acceptors for anaerobic respiration. A previous report described a mutant strain (CMTn-1) deficient in CymA, a tetraheme cytochrome c. However, the interpretation of the electron transport role of CymA was complicated by the fact that CMTn-1 was also markedly deficient in menaquinones. This report demonstrates that the depressed menaquinone levels were the result of the rifampin resistance phenotype of the parent of CMTn-1 and not the interruption of the cymA gene. This is the first report of rifampin resistance leading to decreased menaquinone levels, indicating that rifampin-resistant strains should be used with caution when analyzing electron transport processes. A site-directed gene replacement approach was used to isolate a cymA knockout strain (MR1-CYMA) directly from MR-1. While MR1-CYMA retained menaquinone levels comparable to those of MR-1, it lost the ability to reduce iron(III), manganese(IV), and nitrate and to grow by using fumarate as an electron acceptor. All of these functions were restored to wild-type efficacy, and the presence of the cymA transcript and CymA protein was also restored, by complementation of MR1-CYMA with the cymA gene. The requirement for CymA in anaerobic electron transport to iron(III), fumarate, nitrate, and manganese(IV) is therefore not dependent on the levels of menaquinone in these cells. This represents the first successful use of a suicide vector for directed gene replacement in MR-1.

Topics: Anaerobiosis; Cytochrome c Group; Drug Resistance, Microbial; Electron Transport; Enzyme Inhibitors; Fumarates; Genetic Complementation Test; Genetic Engineering; Iron; Manganese; Mutation; Nitrates; Quinones; Rifampin; Shewanella putrefaciens; Vitamin K

Following on from our previous discovery of Na+ pumping by the NADH:ubiquinone oxidoreductase (complex I) of Klebsiella pneumoniae, we show here that complex I from Escherichia coli is a Na+ pump as well. Our study object was the Escherichia coli mutant EP432, which lacks the Na+/H+ antiporter genes nhaA and nhaB and is therefore unable to grow on LB medium at elevated Na+ concentrations. During growth on mineral medium, the Na+ tolerance of E. coli EP432 was influenced by the organic substrate. NaCl up to 450 mM did not affect growth on glycerol and fumarate, but growth on glucose was inhibited. Correlated to the Na+ tolerance was an increased synthesis of complex I in the glycerol/fumarate medium. Inverted membrane vesicles catalysed respiratory Na+ uptake with NADH as electron donor. The sodium ion transport activity of vesicles from glycerol/fumarate-grown cells was 40 nmol mg-1 min-1 and was resistant to the uncoupler carbonyl-cyanide m-chlorophenylhydrazone (CCCP), but was inhibited by the complex I-specific inhibitor rotenone. With an E. coli mutant deficient in complex I, the Na+ transport activity was low (1-3 nmol mg-1 min-1), and rotenone was without effect.

Topics: Cell Membrane; Culture Media; Escherichia coli; Fumarates; Glucose; Glycerol; Ion Transport; Oxygen Consumption; Protons; Quinone Reductases; Sodium

Iron-induced flavocytochrome c(3), Ifc(3), from Shewanella frigidimarina NCIMB400, derivatized with a 2-pyridyl disulfide label, self-assembles on gold electrodes as a functional array whose fumarate reductase activity as viewed by direct electrochemistry is indistinguishable from that of Ifc(3) adsorbed on gold or graphite electrodes. The enhanced stability of the labeled protein's array permits analysis at a rotating electrode and limiting catalytic currents fit well to a Michaelis-Menten description of enzyme kinetics with K(M) = 56 +/- 20 microM, pH 7.5, comparable to that obtained in solution assays. At fumarate concentrations above 145 microM cyclic voltammetry shows the catalytic response to contain two features. The position and width of the lower potential component centered on -290 mV and corresponding to a one-electron wave implicates the oxidation state of the lowest potential heme of Ifc(3) as a defining feature in the mechanism of fumarate reduction at high turnover rates. We propose the operation of dual pathways for electron transfer to the active site of Ifc(3) with the lowest potential heme acting as an electron relay on one of these pathways.

Topics: Binding Sites; Cross-Linking Reagents; Cytochrome c Group; Disulfides; Electrochemistry; Electron Transport; Fumarates; Gold; Hemeproteins; Kinetics; Oxidation-Reduction; Shewanella; Succinate Dehydrogenase; Succinimides

A recent review suggested that the activity of NADH-fumarate reductase from trypanosomatids could be catalyzed by succinate dehydrogenase working in reverse (Tielens and van Hellemond, Parasitol. Today 14, 265-271, 1999). The results reported in this study demonstrate that the two activities can easily be separated without any loss in either activity, suggesting that fumarate reductase and succinate dehydrogenase are separate enzymes.

Topics: Animals; Fumarates; NAD; Succinate Dehydrogenase; Trypanosoma cruzi

The cerebral metabolism of lactate was investigated. Awake mice received [3-13C]lactate or [1-13C]glucose intravenously, and brain and blood extracts were analyzed by 13C nuclear magnetic resonance spectroscopy. The cerebral uptake and metabolism of [3-13C]lactate was 50% that of [1-13C]glucose. [3-13C]Lactate was almost exclusively metabolized by neurons and hardly at all by glia, as revealed by the 13C labeling of glutamate, gamma-aminobutyric acid and glutamine. Injection of [3-13C]lactate led to extensive formation of [2-13C]lactate, which was not seen with [1-13C]glucose, nor has it been seen in previous studies with [2-13C]acetate. This formation probably reflected reversible carboxylation of [3-13C]pyruvate to malate and equilibration with fumarate, because inhibition of succinate dehydrogenase with nitropropionic acid did not block it. Of the [3-13C]lactate that reached the brain, 20% underwent this reaction, which probably involved neuronal mitochondrial malic enzyme. The activities of mitochondrial malic enzyme, fumarase, and lactate dehydrogenase were high enough to account for the formation of [2-13C]lactate in neurons. Neuronal pyruvate carboxylation was confirmed by the higher specific activity of glutamate than of glutamine after intrastriatal injection of [1-14C]pyruvate into anesthetized mice. This procedure also demonstrated equilibration of malate, formed through pyruvate carboxylation, with fumarate. The demonstration of neuronal pyruvate carboxylation demands reconsideration of the metabolic interrelationship between neurons and glia.

Topics: Animals; Blood Glucose; Brain; Carbon; Carbon Isotopes; Carbon Radioisotopes; Cell Communication; Cytosol; Eating; Fasting; Female; Fumarates; Glucose; Lactic Acid; Magnetic Resonance Spectroscopy; Malate Dehydrogenase; Malates; Mice; Mice, Inbred Strains; Mitochondria; Neuroglia; Neurons; Pyruvic Acid; Synaptosomes

Cultured neocortical neurons were incubated in medium containing [U-13C]glucose (0.5 mM) and in some cases unlabeled glutamine (0.5 mM). Subsequently the cells were "superfused" for investigation of the effect of depolarization by 55 mM K+. Cell extracts were analyzed by 13C magnetic resonance spectroscopy and gas chromatography/mass spectrometry to determine incorporation of 13C in glutamate, GABA, aspartate and fumarate. The importance of the tricarboxylic acid (TCA) cycle for conversion of the carbon skeleton of glutamine to GABA was evident from the effect of glutamine on the labeling pattern of GABA and glutamate. Moreover, analysis of the labeling patterns of glutamate in particular indicated a depolarization induced increased oxidative metabolism. This effect was only observed in glutamate and not in neurotransmitter GABA. Based on this a hypothesis of mitochondrial compartmentation may be proposed in which mitochondria associated with neurotransmitter synthesis are distinct from those aimed at energy production and influenced by depolarization. The hypothesis of mitochondrial compartmentation was further supported by the finding that the total percent labeling of fumarate and aspartate differed significantly from each other. This can only be explained by the existence of multiple TCA cycles with different turnover rates.

Topics: Amino Acids; Animals; Carbon Isotopes; Cells, Cultured; Citric Acid Cycle; Electrophysiology; Fumarates; Gas Chromatography-Mass Spectrometry; Glucose; Glutamine; Magnetic Resonance Spectroscopy; Mice; Neocortex; Neurons

The Escherichia coli SixA protein is the first discovered prokaryotic phospho-histidine phosphatase, which was implicated in a His-to-Asp phosphorelay. The sixA gene was originally identified as the one that interferes with, at its multi-copy state, the cross-phosphorelay between the histidine-containing phosphotransmitter (HPt) domain of the ArcB anaerobic sensor and its non-cognate OmpR response regulator. Nevertheless, no evidence has been provided that the SixA phosphatase is indeed involved in a signaling circuitry of the authentic ArcB-to-ArcA phosphorelay in a physiologically meaningful manner. In this study, a SixA-deficient mutant was characterized with special reference to the ArcB signaling, which allows E. coli cells to respond to not only external oxygen, but also certain anaerobic respiratory conditions. Here evidence is provided for the first time that the SixA phosphatase is a crucial regulatory factor that is involved in the ArcB signaling, particularly, under certain anaerobic respiratory growth conditions. We propose a novel mechanism, involving an HPt domain and a phospho-histidine phosphatase, by which a given multi-step His-to-Asp signaling can be modulated.

Topics: Anaerobiosis; Aspartic Acid; Bacterial Outer Membrane Proteins; Bacterial Proteins; Dimethyl Sulfoxide; Electron Transport; Escherichia coli; Escherichia coli Proteins; Fumarates; Genes, Bacterial; Genes, Reporter; Histidine; Kinetics; Membrane Proteins; Methylamines; Mutation; Nitrates; Oxygen; Phosphorylation; Protein Kinases; Protein Structure, Tertiary; Recombinant Fusion Proteins; Repressor Proteins; Signal Transduction

We report the identification, expression, and characterization of a second Dihydroorotate dehydrogenase (DHODase A) from the human pathogen Enterococcus faecalis. The enzyme consists of a polypeptide chain of 322 amino acids that shares 68% identity with the cognate type A enzyme from the bacterium Lactococcus lactis. E. faecalis DHODase A catalyzed the oxidation of l-dihydroorotate while reducing a number of substrates, including fumarate, coenzyme Q(0), and menadione. The steady-state kinetic mechanism has been determined with menadione as an oxidizing substrate at pH 7.5. Initial velocity and product inhibition data suggest that the enzyme follows a two-site nonclassical ping-pong kinetic mechanism. The absorbance of the active site FMN cofactor is quenched in a concentration-dependent manner by titration with orotate and barbituric acid, two competitive inhibitors with respect to dihydroorotate. In contrast, titration of the enzyme with menadione had no effect on FMN absorbance, consistent with nonoverlapping binding sites for dihyroorotate and menadione, as suggested from the kinetic mechanism. The reductive half-reaction has been shown to be only partially rate limiting, and an attempt to evaluate the slow step in the overall reaction has been made by simulating orotate production under steady-state conditions. Our data indicate that the oxidative half-reaction is a rate-limiting segment, while orotate, most likely, retains significant affinity for the reduced enzyme, as suggested by the product inhibition pattern.

Topics: Amino Acid Sequence; Barbiturates; Binding Sites; Catalysis; Cloning, Molecular; Dihydroorotate Dehydrogenase; Enterococcus faecalis; Enzyme Stability; Escherichia coli; Fumarates; Humans; Kinetics; Models, Chemical; Molecular Sequence Data; Molecular Weight; Orotic Acid; Oxidation-Reduction; Oxidoreductases; Oxidoreductases Acting on CH-CH Group Donors; Oxygen; Recombinant Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Thermodynamics; Titrimetry; Vitamin K

Factors in a cultivation medium affecting fungal growth morphology and fumaric acid production by Rhizopus oryzae ATCC 20344 were investigated. These factors included the initial pH value and trace metals such as zinc, magnesium, iron, and manganese in the cultivation medium. It was found that a significant change in the growth morphology of R. oryzae ATCC 20344 occurs when the initial pH value is varied. A lower initial pH value in the cultivation medium was inhibitory to fungal growth, and fast growth in the cultivation medium at a higher initial pH value promoted the formation of large pellets or filamentous forms. Trace metals in the cultivation media also had significant effects on pellet formation and fumaric acid fermentation.

Topics: Cations, Divalent; Chromatography, High Pressure Liquid; Culture Media; Fumarates; Hydrogen-Ion Concentration; Rhizopus; Trace Elements

Fumaric acid is shown to be a competitive inhibitor of wheat germ lipoxygenase.

Topics: Binding, Competitive; Catalysis; Fumarates; Kinetics; Lipoxygenase; Lipoxygenase Inhibitors; Triticum

Topics: Adenosine; Aminoimidazole Carboxamide; Central Nervous System Diseases; Female; Fumarate Hydratase; Fumarates; Humans; Infant; Purines; Ribonucleosides

A novel dissimilatory, nitrate-reducing bacterium, designated strain N2460T, was isolated from an oil reservoir model column. Strain N2460T is a mesophilic, obligately anaerobic, marine, gram-negative bacterium. The cells are vibrio-shaped and motile by a bipolar flagellum. Strain N2460T reduces nitrate to ammonia in a mineral medium supplied by acetate. The presence of a 2-oxoglutarate dehydrogenase activity indicates that acetate is oxidized via the citric acid cycle. No growth is obtained on formate, higher fatty acids, malate, fumarate, benzoate, alcohols, sugar, yeast extract, crude oil, alkanes, proline, hydrogen, sulfur or thiosulfate with nitrate as electron acceptor. Oxygen, sulfate, thiosulfate and sulfur are not utilized as alternative electron acceptors. Strain N2460T grows fermentatively on fumarate, but not on pyruvate. The G+C content of the DNA is 42.6 mol%. 16S rRNA gene analysis shows that strain N2460T belongs to the Bacteria and that the closest relative is 'Geovibrio ferrireducens' (sequence similarity 86.9%). On the basis of phylogenetic as well as phenotypic data, it is proposed that strain N2460T represents the type strain of a new genus and species, Denitrovibrio acetiphilus gen. nov., sp. nov.

Topics: Acetates; Ammonia; Base Composition; DNA, Bacterial; DNA, Ribosomal; Fermentation; Fuel Oils; Fumarates; Gram-Negative Anaerobic Bacteria; Industrial Microbiology; Molecular Sequence Data; Nitrates; Phylogeny; RNA, Bacterial; RNA, Ribosomal, 16S; Sequence Homology, Nucleic Acid

Topics: Anticarcinogenic Agents; Clinical Trials as Topic; Dermatologic Agents; Fumarates; Humans; Kidney; Kidney Function Tests; Kidney Tubules; Prospective Studies; Psoriasis; Time Factors

An on-line coupled capillary isotachophoresis-capillary zone electrophoresis (cITP-CZE) method for the determination of the fumaric acid content in apple juice is presented. A clear separation of fumaric acid in real samples is achieved within 20 min. The leading, terminating and background electrolyte of the employed system consist of 10 mM HCl+beta-alanine+5 mM beta-cyclodextrin+0.05% hydroxypropylmethylcelullose (HPMC), pH 3, 10 mM citric acid and 20 mM citric acid+beta-alanine+5 mM beta-cyclodextrin+0.1% HPMC, pH 3.3, respectively. The linearity, recovery, repeatability and detection limit of the developed method are 25-1000 ng/ml, 1.07%, 95.4+/-3.5 (+/-s)% and 10 ng/ml, respectively. Low laboriousness (no sample pretreatment), sufficient sensitivity and low running cost are the important attributes of the cITP-CZE method which was successfully applied to analyses of real samples of apple juices.

Topics: Beverages; Electrolytes; Electrophoresis; Electrophoresis, Capillary; Fruit; Fumarates; Reproducibility of Results; Sensitivity and Specificity; Spectrophotometry, Ultraviolet

Eight barrows (Yorkshire x [Finnish Landrace x Dutch Landrace]), initially 30 kg BW, were fitted with ileal cannulas to evaluate the effects of supplementing Ca benzoate (2.4%) and organic acids (OA) in the amount of 300 mEq acid/kg feed on dietary buffering capacity (BC), apparent digestibility and retention of nutrients, and manure characteristics. Swine were allotted in a 2 x 4 factorial arrangement of treatments according to a cyclic (8 x 5) changeover design. Two tapioca-corn-soybean meal-based diets were formulated without and with acidogenic Ca benzoate. Each diet was fed in combination with OA (none, formic, fumaric, or n-butyric acid). Daily rations were equal to 2.8 x maintenance requirement (418 kJ ME/BW(.75)) and were given in two portions. Chromic oxide (.25 g/kg) was used as a marker. On average, Ca benzoate lowered BC by 54 mEq/kg feed. This salt enhanced (P < .05) the ileal digestibility (ID) of DM, OM, arginine, isoleucine, leucine, phenylalanine, alanine, aspartic acid, and tyrosine (by up to 2.4 percentage units). Also, the total tract digestibility (TD) of DM, ash, Ca and GE, and Ca retention (percentage of intake) was greater (P < .05) in swine fed Ca benzoate, whereas N retention remained unaffected. Addition of all OA (formic and n-butyric acid, in particular) exerted a positive effect (P < .05) on the ID of amino acids (except for arginine, methionine, and cysteine). A similar effect (P < .05) was found for the TD of DM, OM, CP, Ca and total P and for the retention of N and Ca. In swine fed Ca benzoate, urinary pH decreased by 1.6 units (P < .001). In conclusion, dietary OA have a beneficial effect on the apparent ileal/total tract nutrient digestibilities, and Ca benzoate increased urine acidity, which could be effective against a rapid ammonia emission from manure of swine.

Topics: Animal Feed; Animals; Buffers; Butyrates; Calcium; Dietary Supplements; Digestion; Feces; Formates; Fumarates; Hydrogen-Ion Concentration; Male; Random Allocation; Swine

We have further examined the mechanisms for a severe mitochondrial energetic deficit, deenergization, and impaired respiration in complex I that develop in kidney proximal tubules during hypoxia-reoxygenation, and their prevention and reversal by supplementation with alpha-ketoglutarate (alpha-KG) + aspartate. The abnormalities preceded the mitochondrial permeability transition and cytochrome c loss. Anaerobic metabolism of alpha-KG + aspartate generated ATP and maintained mitochondrial membrane potential. Other citric-acid cycle intermediates that can promote anaerobic metabolism (malate and fumarate) were also effective singly or in combination with alpha-KG. Succinate, the end product of these anaerobic pathways that can bypass complex I, was not protective when provided only during hypoxia. However, during reoxygenation, succinate also rescued the tubules, and its benefit, like that of alpha-KG + malate, persisted after the extra substrate was withdrawn. Thus proximal tubules can be salvaged from hypoxia-reoxygenation mitochondrial injury by both anaerobic metabolism of citric-acid cycle intermediates and aerobic metabolism of succinate. These results bear on the understanding of a fundamental mode of mitochondrial dysfunction during tubule injury and on strategies to prevent and reverse it.

Topics: Adenosine Triphosphate; Aerobiosis; Anaerobiosis; Animals; Aspartic Acid; Benzimidazoles; Carbocyanines; Cell Hypoxia; Citric Acid Cycle; Energy Metabolism; Female; Fluorescent Dyes; Fumarates; Ketoglutaric Acids; Kidney Tubules, Proximal; Malates; Membrane Potentials; Mitochondria; Oxygen; Rabbits; Rhodamines; Substrate Specificity

Photoassimilates are used by plants for production of energy, as carbon skeletons and in transport of fixed carbon between different plant organs. Many studies have been devoted to characterizing the factors that regulate photoassimilate concentrations in different plant species. Most studies examining photoassimilate concentrations in C3 plants have focused on analyzing starch and soluble sugars. However, work presented here demonstrates that a number of C3 plants, including the popular model organism Arabidopsis thaliana (L.) Heynh., and agriculturally important plants, such as soybean, Glycine max (L.) Merr., contain significant quantities of fumaric acid. In fact, fumaric acid can accumulate to levels of several milligrams per gram fresh weight in Arabidopsis leaves, often exceeding those of starch and soluble sugars. Fumaric acid is a component of the tricarboxylic acid cycle and, like starch and soluble sugars, can be metabolized to yield energy and carbon skeletons for production of other compounds. Fumaric acid concentrations increase with plant age and light intensity in Arabidopsis leaves. Moreover, Arabidopsis phloem exudates contain significant quantities of fumaric acid, raising the possibility that fumaric acid may function in carbon transport.

Topics: Arabidopsis; Carboxylic Acids; Crops, Agricultural; Fumarates; Glycine max; Photosynthesis; Plant Leaves; Species Specificity

Topics: Amniotic Fluid; Fatal Outcome; Female; Fumarate Hydratase; Fumarates; Humans; Infant, Newborn; Male; Pregnancy; Pregnancy Complications

The Saccharomyces cerevisiae succinate-ubiquinone reductase or succinate dehydrogenase (SDH) is a tetramer of non-equivalent subunits encoded by the SDH1, SDH2, SDH3, and SDH4 genes. In most organisms, SDH contains one or two endogenous b-type hemes. However, it is widely believed that the yeast SDH does not contain heme. In this report, we demonstrate the presence of a stoichiometric amount of cytochrome b562 in the yeast SDH. The cytochrome is detected as a peak present in fumarate-oxidized, dithionite-reduced mitochondria. The peak is centered at 562 nm and is present at a heme:covalent FAD molar ratio of 0.92+/-0.11. The cytochrome is not detectable in mitochondria isolated from SDH3 and SDH4 deletion strains. These observations strongly support our conclusion that cytochrome b562 is a component of the yeast SDH.

Topics: Animals; Cytochrome b Group; Dithionite; Electron Transport Complex II; Escherichia coli Proteins; Flavin-Adenine Dinucleotide; Fumarates; Fungal Proteins; Gene Deletion; Intracellular Membranes; Lactic Acid; Malonates; Mice; Mitochondria; Multienzyme Complexes; Oxidation-Reduction; Oxidoreductases; Saccharomyces cerevisiae; Spectrum Analysis; Succinate Dehydrogenase

2,3-Dimethoxy-5-methyl-1,4-benzoquinone (Q0), an analogue of ubiquinone, irreversibly paralyses the adult and microfilariae of the cattle filarial parasite Setaria digitata. The same concentration of Q0 that paralyses the microfilariae of S. digitata also paralyses the microfilariae of the human filarial parasite Wuchereria bancrofti within the same duration. Thus the experiments done in the model S. digitata system can well be extended to the human filarial system. A drug at the level of the quinone-centered energy generating system, perhaps an analogue of quinone like Q0, can inactivate the filarial parasites and may prove to be an effective drug to control filariasis.

Topics: Animals; Benzoquinones; Cattle; Disease Models, Animal; Dose-Response Relationship, Drug; Electron Transport; Filariasis; Fumarates; Glucose; Humans; Malates; Movement; NAD; Setaria Nematode; Setariasis; Sodium Lactate; Time Factors; Wuchereria bancrofti

Neutral red (NR) functioned as an electronophore or electron channel enabling either cells or membranes purified from Actinobacillus succinogenes to drive electron transfer and proton translocation by coupling fumarate reduction to succinate production. Electrically reduced NR, unlike methyl or benzyl viologen, bound to cell membranes, was not toxic, and chemically reduced NAD. The cell membrane of A. succinogenes contained high levels of benzyl viologen-linked hydrogenase (12.2 U), fumarate reductase (13.1 U), and diaphorase (109.7 U) activities. Fumarate reductase (24.5 U) displayed the highest activity with NR as the electron carrier, whereas hydrogenase (1.1 U) and diaphorase (0.8 U) did not. Proton translocation by whole cells was dependent on either electrically reduced NR or H2 as the electron donor and on the fumarate concentration. During the growth of Actinobacillus on glucose plus electrically reduced NR in an electrochemical bioreactor system versus on glucose alone, electrically reduced NR enhanced glucose consumption, growth, and succinate production by about 20% while it decreased acetate production by about 50%. The rate of fumarate reduction to succinate by purified membranes was twofold higher with electrically reduced NR than with hydrogen as the electron donor. The addition of 2-(n-heptyl)-4-hydroxyquinoline N-oxide to whole cells or purified membranes inhibited succinate production from H2 plus fumarate but not from electrically reduced NR plus fumarate. Thus, NR appears to replace the function of menaquinone in the fumarate reductase complex, and it enables A. succinogenes to utilize electricity as a significant source of metabolic reducing power.

Topics: Actinobacillus; Biological Transport; Cell Membrane; Energy Metabolism; Fermentation; Fumarates; Glucose; Models, Biological; Neutral Red; Oxidation-Reduction; Protons

The effect of fumarate used as a feed additive on the reduction of methanogenesis in the rumen was evaluated by in vitro experiments. The addition of fumarate to the culture of mixed ruminal microorganisms that were fermenting hay powder and concentrate reduced methane production. Most fumarate was metabolized to propionate, and a slight increase was noted in other volatile fatty acids. Fumarate was utilized by mixed bacteria but not by mixed protozoa. Fibrobacter succinogenes, Selenomonas ruminantium ssp. ruminantium, Selenomonas ruminantium ssp. lactilytica, Veillonella parvula, and Wollinella succinogenes oxidized H2 by using fumarate as a final electron acceptor, suggesting that these bacteria compete with methanogens for H2, which is the main substrate for methanogenesis in the rumen. However, the affinity of these bacteria to H2 was lower than their affinity to methanogens. These fumarate-utilizing bacteria metabolized malate to products that were similar to those from fumarate, suggesting the possession of fumarate dehydratase. Fibrobacter succinogenes, V. parvula, and W. succinogenes utilized formate, another substrate for methanogenesis, as an electron donor for fumarate reduction. The affinity of these bacteria to formate was higher than the affinity methanogenes have for formate. When methanogens were cocultured with an equal cellular amount of each of the fumarate-utilizing bacteria, methane production was markedly decreased, not only from formate, but also from H2. These results suggest that the addition of fumarate to ruminant feed reduces methanogenesis and enhances propionate production in the rumen.

Topics: Adenosine Triphosphate; Animals; Bacteria; Deuterium; Eukaryota; Euryarchaeota; Fumarates; Goats; Hydrogen-Ion Concentration; Methane; Oxidation-Reduction; Rumen

The influence of sodium fumarate on rumen fermentation was investigated in vitro using batch and semi-continuous cultures of mixed rumen micro-organisms taken from three sheep receiving a basal diet of hay, barley, molasses, fish meal and a mineral-vitamin supplement (500, 299.5, 100, 91 and 9.5 g/kg DM respectively). Batch cultures consisted of 10 ml strained rumen fluid in 40 ml anaerobic buffer containing 200 mg of the same feed given to the sheep. Sodium fumarate was added to achieve a final concentration of 0, 5 or 10 mmol/l, as a result of the addition of 0, 250 or 500 mumol, equivalent to 0, 200 and 400 g/kg feed. CH4 production at 24 h (360 mumol in the control cultures) fell (P < 0.05) by 18 and 22 mumol respectively (SED 7.5). Total gas production was increased by the addition of fumarate without significant accumulation of H2. Substantial increases in acetate production (92 and 194 mumol; SED 26.7, P < 0.01) were accompanied by increases in propionate formation (212 and 396 mumol; SED 13.0, P < 0.001). Longer-term effects of fumarate supplementation on ruminal fermentation and CH4 production were investigated using the rumen simulation technique (Rusitec). Eight vessels were given 20 g basal diet/d, and half of them received a supplement of fumarate (disodium salt) over a period of 19 d. The response to the daily addition of 6.25 mmol sodium fumarate was a decrease in CH4 production of 1.2 mmol (SED 0.39, P < 0.05), equivalent to the consumption of 4.8 mmol H2, and an increase in propionate production of 4.9 mmol (from 10.4 to 15.3 (SED 1.05) mmol/d, P < 0.01). The inhibition of CH4 production did not decline during the period of time that fumarate was added to the vessels. Thus, the decrease in CH4 corresponded well to the fraction of the fumarate that was converted to propionate. Fumarate had no significant (P > 0.05) effect on total bacterial numbers or on the number of methanogenic archaea, but numbers of cellulolytic bacteria were increased (8.8 v. 23.9 (SED 2.49) x 10(5) per ml, P < 0.01). Fumarate also increased DM digestibility of the basal diet after 48 h incubation (0.476 v. 0.508 (SED 0.0123), P < 0.05). Thus, it was concluded that sodium fumarate may be a useful dietary additive for ruminants, because it diverts some H2 from CH4 production and because it is able to stimulate proliferation of cellulolytic bacteria and digestion of fibre.

Topics: Acetates; Animals; Bacteria; Dietary Fiber; Fermentation; Fumarates; Methane; Microbiological Techniques; Propionates; Rumen; Sheep

The degradation of three poly(fumaric-co-sebacic anhydride) [P(FA:SA)] copolymers is examined in a composition of microspheres made by the hot melt encapsulation process. The emergence of low molecular weight oligomers occurs during degradation of the copolymer microspheres, as evidenced by a variety of characterization methods. Characterization was conducted to determine the extent of degradation of the polyanhydride microspheres using Fourier-transform infrared spectroscopy (FTIR), gel permeation chromatography (GPC), differential scanning calorimetry (DSC) and X-ray diffraction. It is demonstrated that degradation of P(FA:SA) is greatly accelerated at basic pH, yet there is little difference between degradation in neutral and acidic buffers. A good correlation exists between the results of each characterization method, which allows a better understanding of the degradation process and the resulting formation of low molecular weight oligomers in poly(fumaric-co-sebacic anhydride).

Topics: Anhydrides; Calorimetry, Differential Scanning; Chromatography, Gel; Decanoic Acids; Dicarboxylic Acids; Drug Delivery Systems; Fumarates; Microspheres; Polymers; Spectroscopy, Fourier Transform Infrared; X-Ray Diffraction

The integral membrane protein fumarate reductase catalyzes the final step of anaerobic respiration when fumarate is the terminal electron acceptor. The homologous enzyme succinate dehydrogenase also plays a prominent role in cellular energetics as a member of the Krebs cycle and as complex II of the aerobic respiratory chain. Fumarate reductase consists of four subunits that contain a covalently linked flavin adenine dinucleotide, three different iron-sulfur clusters, and at least two quinones. The crystal structure of intact fumarate reductase has been solved at 3.3 angstrom resolution and demonstrates that the cofactors are arranged in a nearly linear manner from the membrane-bound quinone to the active site flavin. Although fumarate reductase is not associated with any proton-pumping function, the two quinones are positioned on opposite sides of the membrane in an arrangement similar to that of the Q-cycle organization observed for cytochrome bc1.

Topics: Aerobiosis; Anaerobiosis; Binding Sites; Cell Membrane; Crystallization; Crystallography, X-Ray; Electron Transport; Energy Metabolism; Escherichia coli; Flavin-Adenine Dinucleotide; Fumarates; Iron-Sulfur Proteins; Models, Molecular; Oxidation-Reduction; Oxygen Consumption; Protein Conformation; Protein Folding; Quinones; Succinate Dehydrogenase

The respiratory chain of Helicobacter pylori has been investigated. The total insensitivity of activities of NADH dehydrogenase to rotenone and of NADH-cytochrome c reductase to antimycin is indicative of the absence of the classical complex I of the electron transfer chain in this bacterium. NADPH-dependent respiration was significantly stronger than NADH-dependent respiration, indicating that this is a major respiratory electron donor in H. pylori. Fumarate and malonate exhibited a concentration-dependent inhibitory effect on the activity of succinate dehydrogenase. The activity of succinate-cytochrome c reductase was inhibited by antimycin, implying the presence of a classical pathway from complex II to complex III in this bacterium. The presence of NADH-fumarate reductase (FRD) was demonstrated in H. pylori and fumarate could reduce H2O2 production from NADH, indicating fumarate to be an endogenous substrate for accepting electrons from NADH. The activity of NADH-FRD was inhibited by 2-thenoyltrifluoroacetone. A tentative scheme for the electron transfer pathway in H. pylori is proposed, which may be helpful in clarifying the pathogenesis of H. pylori and in opening new lines for chemotherapy against this bacterium.

Topics: Electron Transport; Fumarates; Helicobacter Infections; Helicobacter pylori; Humans; NADH Dehydrogenase; Oxidation-Reduction; Oxygen Consumption; Succinate Cytochrome c Oxidoreductase; Succinate Dehydrogenase; Succinic Acid; Thenoyltrifluoroacetone

Topics: 3-Hydroxybutyric Acid; Adsorption; Caprylates; Dicarboxylic Acids; Fumarates; Gas Chromatography-Mass Spectrometry; Glass; Glycolates; Humans; Hydroxy Acids; Lactic Acid; Malates; Malonates; Phenylbutyrates; Polytetrafluoroethylene; Valerates

The keto (linear) form of exogenous fructose 1,6-bisphosphate, a highly charged glycolytic intermediate, may utilize a dicarboxylate transporter to cross the cell membrane, support glycolysis, and produce ATP anaerobically. We tested the hypothesis that fumarate, a dicarboxylate, and 3-phosphoglycerate (3-PG), an intermediate structurally similar to a dicarboxylate, can support contraction in vascular smooth muscle during hypoxia. To assess ATP production during hypoxia we measured isometric force maintenance in hog carotid arteries during hypoxia in the presence or absence of 20 mM fumarate or 3-PG. 3-PG improved maintenance of force (p < 0.05) during the 30-80 min period of hypoxia. Fumarate decreased peak isometric force development by 9.5% (p = 0.008) but modestly improved maintenance of force (p < 0.05) throughout the first 80 min of hypoxia. 13C-NMR on tissue extracts and superfusates revealed 1,2,3,4-(13)C-fumarate (5 mM) metabolism to 1,2,3,4-(13)C-malate under oxygenated and hypoxic conditions suggesting uptake and metabolism of fumarate. In conclusion, exogenous fumarate and 3-PG readily enter vascular smooth muscle cells, presumably by a dicarboxylate transporter, and support energetically important pathways.

Topics: Animals; Biological Transport; Carbon Isotopes; Carotid Arteries; Cell Membrane; Fumarates; Glyceric Acids; Hypoxia; Muscle, Smooth, Vascular; Nuclear Magnetic Resonance, Biomolecular; Swine; Time Factors

In this study, a facultative bacterium that converts fumarate to succinate at a high yield was isolated. The yield of bioconversion was enhanced about 1.2 times by addition of glucose into culture medium at an initial concentration of 6 g/L. When the initial cell density was high (2 g/L), the succinate produced at pH 7.0 for initial fumarate concentrations of 30, 50, 80, and 100 g/L were 29.3, 40.9, 63.6, and 82.5 g/L, respectively, showing an increase with the initial fumarate concentration. The high yield of 96.8%/mole of fumarate in just 4 h was obtained at the initial fumarate concentration of 30 g/L. Comparing these values to those obtained with low cell culture (0.2 g/L), we found that the amount of succinate produced was similar, but the production rate in the high cell culture was about three times higher than was the case in the low cell culture. This strain converted fumarate to succinate at a rate of 3.5 g/L.h under the sparge of CO2.

Topics: Biodegradation, Environmental; Carbon; Cell Division; Dose-Response Relationship, Drug; Enterococcus; Fumarates; Glycerol; Hydrogen-Ion Concentration; Succinic Acid; Time Factors

Topics: Anaerobiosis; Bacillus subtilis; Binding Sites; Cell Membrane; Crystallography, X-Ray; Dimerization; Electron Transport; Energy Metabolism; Escherichia coli; Evolution, Molecular; Fumarates; Mitochondria; Oxidation-Reduction; Oxygen Consumption; Protein Conformation; Protein Structure, Secondary; Succinate Dehydrogenase; Succinic Acid

A gas chromatography tandem mass spectrometry method using an ion trap GC/MS system was developed to quickly screen urine samples for 14 organic acids associated with multiple organic acidemias. The following organic acids are used as diagnostic markers: methylmalonic acid, glutaric acid, 2-ketoisocaproic acid, succinylacetone, 3-methylcrotonylglycine, tiglylglycine, isovalerylglycine, fumaric acid, butyrylglycine, propionylglycine, hexanoylglycine, adipic acid, suberic acid, and sebacic acid. 2-ketocaproic acid is used as an internal standard. The samples are prepared using a solid-phase extraction and converted to trimethylsilyl derivatives. The extraction efficiency for the 14 compounds is between 57 and 106%. A derivatized standard mixture of the 14 markers is run prior to the patient samples to determine the accurate absolute and relative retention times. The samples are then injected and the product ion spectra monitored. For data analysis, one characteristic product ion plot is extracted for each of the 14 marker compounds, and the presence of a peak with the expected retention time is determined. The areas of the product ion peaks are compared with the reference range determined from 30 normal controls. Ten samples of patients with known organic acidemias were measured. For all patients, diagnostic peaks at the expected retention times of at least five times the upper limit of the reference range were detected. The method, with its relatively fast sample preparation, short 10.0 min run time and simple data analysis, is suitable for use as a quick metabolic screen of very sick patients in whom there is concern regarding the possibility of a treatable inborn error.

Topics: Acids; Adipates; Automation; Biomarkers; Caprylates; Decanoic Acids; Dicarboxylic Acids; Fumarates; Gas Chromatography-Mass Spectrometry; Glutarates; Humans; Keto Acids; Mass Screening; Metabolism, Inborn Errors; Methylmalonic Acid; Reference Values; Sensitivity and Specificity; Urinalysis

The Gram-negative bacterium Rhizobium meliloti contains two distinct malic enzymes. We report the purification of the two isozymes to homogeneity, and their in vitro characterization. Both enzymes exhibit unusually high subunit molecular weights of about 82 kDa. The NAD(P)(+) specific malic enzyme [EC 1.1.1.39] exhibits positive co-operativity with respect to malate, but Michaelis-Menten type behavior with respect to the co-factors NAD(+) or NADP(+). The enzyme is subject to substrate inhibition, and shows allosteric regulation by acetyl-CoA, an effect that has so far only been described for some NADP(+) dependent malic enzymes. Its activity is positively regulated by succinate and fumarate. In contrast to the NAD(P)(+) specific malic enzyme, the NADP(+) dependent malic enzyme [EC 1.1.1.40] shows Michaelis-Menten type behavior with respect to malate and NADP(+). Apart from product inhibition, the enzyme is not subjected to any regulatory mechanism. Neither reductive carboxylation of pyruvate, nor decarboxylation of oxaloacetate, could be detected for either malic enzyme. Our characterization of the two R. meliloti malic enzymes therefore suggests a number of features uncharacteristic for malic enzymes described so far.

Topics: Acetyl Coenzyme A; Cloning, Molecular; Decarboxylation; Enzyme Activation; Enzyme Inhibitors; Escherichia coli; Fumarates; Isoenzymes; Kinetics; Malate Dehydrogenase; Sinorhizobium meliloti; Succinic Acid

In contrast to the classic concept of direct conversion of glutamine to gamma-aminobutyric acid (GABA; via glutamate), this process may involve alpha-ketoglutarate as an intermediary metabolite and tricarboxylic acid (TCA) cycle activity. To obtain information about a possible differential role of these pathways for the synthesis of cytosolic and vesicular GABA, cultured neocortical neurons were incubated in medium containing [U-(13)C]glucose (0.5 mM) and in some cases unlabeled glutamine (0.5 mM). Subsequently, the cells were "superfused" for investigation of the effect of depolarization by 55 mM K+. To make sure that depolarization by 55 mM K+ released only vesicular GABA, tiagabin, a nontransportable inhibitor of the plasma membrane GABA carriers, was included in the medium to prevent GABA release from the cytoplasmic pool by reversal of the carriers. The importance of the TCA cycle for conversion of the carbon skeleton of glutamine to GABA was evident from the effect of glutamine on the labeling pattern of GABA. Percentage of labeling by GABA released into the depolarizing medium was the same as that in the corresponding cell extracts and was unaffected by the presence of glutamine during incubation. Despite the existence of multiple forms of glutamate decarboxylase, compartmentation of glutamate pools, and functionally different compartments within neurons, there appears to be full equilibration between the vesicular and cytosolic pools of GABA. However, during depolarization, the newly synthesized pool of GABA from glutamine does not rapidly equilibrate with the vesicular pool.

Topics: Amino Acids; Animals; Cells, Cultured; Citric Acid Cycle; Fumarates; gamma-Aminobutyric Acid; Gas Chromatography-Mass Spectrometry; Glutamine; Magnetic Resonance Spectroscopy; Membrane Potentials; Mice; Mitochondria; Neocortex; Neurons; Synaptic Vesicles

The gene (dctA) encoding the aerobic C(4)-dicarboxylate transporter (DctA) of Escherichia coli was previously mapped to the 79-min region of the linkage map. The nucleotide sequence of this region reveals two candidates for the dctA gene: f428 at 79.3 min and the o157a-o424-o328 (or orfQMP) operon at 79.9 min. The f428 gene encodes a homologue of the Sinorhizobium meliloti and Rhizobium leguminosarum H(+)/C(4)-dicarboxylate symporter, DctA, whereas the orfQMP operon encodes homologues of the aerobic periplasmic-binding protein- dependent C(4)-dicarboxylate transport system (DctQ, DctM, and DctP) of Rhodobacter capsulatus. To determine which, if either, of these loci specify the E. coli DctA system, the chromosomal f428 and orfM genes were inactivated by inserting Sp(r) or Ap(r) cassettes, respectively. The resulting f428 mutant was unable to grow aerobically with fumarate or malate as the sole carbon source and grew poorly with succinate. Furthermore, fumarate uptake was abolished in the f428 mutant and succinate transport was approximately 10-fold lower than that of the wild type. The growth and fumarate transport deficiencies of the f428 mutant were complemented by transformation with an f428-containing plasmid. No growth defect was found for the orfM mutant. In combination, the above findings confirm that f428 corresponds to the dctA gene and indicate that the orfQMP products play no role in C(4)-dicarboxylate transport. Regulation studies with a dctA-lacZ (f428-lacZ) transcriptional fusion showed that dctA is subject to cyclic AMP receptor protein (CRP)-dependent catabolite repression and ArcA-mediated anaerobic repression and is weakly induced by the DcuS-DcuR system in response to C(4)-dicarboxylates and citrate. Interestingly, in a dctA mutant, expression of dctA is constitutive with respect to C(4)-dicarboxylate induction, suggesting that DctA regulates its own synthesis. Northern blot analysis revealed a single, monocistronic dctA transcript and confirmed that dctA is subject to regulation by catabolite repression and CRP. Reverse transcriptase-mediated primer extension indicated a single transcriptional start site centered 81 bp downstream of a strongly predicted CRP-binding site.

Topics: Aerobiosis; Bacterial Proteins; Base Sequence; Biological Transport; Carrier Proteins; Chromosome Mapping; Chromosomes, Bacterial; Cloning, Molecular; Dicarboxylic Acid Transporters; Dicarboxylic Acids; Escherichia coli; Fumarates; Gene Expression Regulation, Bacterial; Molecular Sequence Data; Mutagenesis, Insertional; Plasmids; Recombinant Proteins; Restriction Mapping; Rhizobiaceae; Rhizobium leguminosarum; Succinates; Transcription, Genetic

Benzylsuccinate synthase, which catalyzes the anaerobic addition of the methyl carbon of toluene to fumarate, has recently been reported in several denitrifying and sulfate-reducing, toluene-degrading bacteria. In substrate range studies with partially purified benzylsuccinate synthase from denitrifying Azoarcus sp. strain T, benzylsuccinate analogs were observed as a result of fumarate addition to the following toluene surrogates: xylenes, monofluorotoluenes, benzaldehyde, and 1-methyl-1-cyclohexene (but not 4-methyl-l-cyclohexene or methylcyclohexane). Benzylsuccinate was also observed as a result of toluene addition to maleate, but no products were observed from assays with toluene and either crotonate or trans-glutaconate. Toluene-maleate addition, like toluene-fumarate addition, resulted in highly stereospecific formation of the (+)-benzylsuccinic acid enantiomer [(R)-2-benzyl-3-carboxypropionic acid]. The previously reported finding that the methyl H atom abstracted from toluene is retained in the succinyl moiety of benzylsuccinate was found to apply to several toluene surrogates. The implications of these observations for the mechanism of benzylsuccinate synthase will be discussed.

Topics: Carbon-Carbon Lyases; Fumarates; Gram-Negative Facultatively Anaerobic Rods; Toluene

Escherichia coli succinate-ubiquinone oxidoreductase (SQR) and menaquinol-fumarate reductase (QFR) are excellent model systems to understand the function of eukaryotic Complex II. They have structural and catalytic properties similar to their eukaryotic counterpart. An exception is that potent inhibitors of mammalian Complex II, such as thenoyltrifluoroacetone and carboxanilides, only weakly inhibit their bacterial counterparts. This lack of good inhibitors of quinone reactions and the higher level of side reactions in the prokaryotic enzymes has hampered the elucidation of the mechanism of quinone oxidation/reduction in E. coli Complex II. In this communication DT-diaphorase and an appropriate quinone are used to measure quinol-fumarate reductase activity and E. coli bo-oxidase and quinones are used to determine succinate-quinone reductase activity. Simple Michaelis kinetics are observed for both enzymes with ubiquinones and menaquinones in the succinate oxidase (forward) and fumarate reductase (reverse) reactions. The comparison of E. coli SQR and QFR demonstrates that 2-n-heptyl 4-hydroxyquinoline-N-oxide (HQNO) is a potent inhibitor of QFR in both assays; however, SQR is not sensitive to HQNO. A series of 2-alkyl-4,6-dinitrophenols and pentachlorophenol were found to be potent competitive inhibitors of both SQR and QFR. In addition, the isolated E. coli SQR complex demonstrates a mixed-type inhibition with carboxanilides, whereas the QFR complex is resistant to this inhibitor. The kinetic properties of SQR and QFR suggest that either ubiquinone or menaquinone operates at a single exchangeable site working in forward or reverse reactions. The pH activity profiles for E. coli QFR and SQR are similar showing maximal activity between pH 7.4 and 7.8, suggesting the importance of similar catalytic groups in quinol deprotonation and oxidation.

Topics: Anilides; Dinitrophenols; Electron Transport Complex II; Enzyme Inhibitors; Escherichia coli; Eukaryotic Cells; Fumarates; Hydrogen-Ion Concentration; Hydroxyquinolines; Kinetics; Multienzyme Complexes; Naphthols; Oxidoreductases; Pentachlorophenol; Prokaryotic Cells; Succinate Dehydrogenase; Succinic Acid; Terpenes; Ubiquinone

The prevalence of 3-methylglutaconic aciduria was evaluated among children with developmental language disorders. A urine specimen was obtained from 40 children referred for developmental language delay to the Tel-Aviv Child Development Center during 12/96-6/97 and from 50 age-matched controls. Urine organic acids were analysed by gas chromatography-mass spectrometry. Urinary 3-methylglutaconic acid was quantified. A mildly increased excretion of 3-methylglutaconic acid was found in 8 children with developmental language delay. The combined excretion of 3-methylglutaconic and 3-methylglutaric acid was increased in 9 patients. There were no differences in the excretion of other organic acids. The patients with elevated 3-methylglutaconic acid did not differ from the other patients with developmental language disorders in any of the parameters evaluated. Mildly elevated urinary levels of 3-methylglutaconic acid may be a marker of a still undefined metabolic disorder presenting with developmental language delay. A further study in large groups of children with different developmental disorders is mandatory.

Topics: Child; Child, Preschool; Female; Fumarates; Glutarates; Humans; Language Development Disorders; Male; Malonates; Mass Screening; Meglutol

beta-Poly(L-malate) is supposed to function in the storage and transport of histones, DNA polymerases and other nuclear proteins in the giant syncytical cells (plasmodia) of myxomycetes. Here we report on the biosynthesis of [14C]beta-poly(L-malate) from injected L-[14C]malate in the plasmodium of Physarum polycephalum. The effects of KCN, arsenate, adenosine 5'-(alpha, beta-methylene)triphosphate, adenosine 5'-(beta, gamma-methylene)triphosphate, guanosine 5'-(beta, gamma-methylene)triphosphate, desulfo coenzyme A and phenylarsinoxid on beta-poly(L-malate) synthesis were studied after their coinjection with L-[14C]malate. The synthesis was not affected by KCN or desulfo coenzyme A, but was blocked by arsenate and adenosine 5'-(alpha,beta-methylene)triphosphate. The plasmodium lysate catalysed an L-malate-dependent ATP-[32P]pyrophosphate exchange, but was devoid of beta-poly(L-malate) synthetic activity under all experimental conditions tested. The results suggested an extramitochondrial synthesis of beta-poly(L-malate), involving the polymerization of beta-L-malyl-AMP. It is assumed that the lack of synthesis in the lysate is caused by the inactivation of beta-poly(L-malate) polymerase involving a cell injury kinase pathway. Because injected guanosine 5'-(beta, gamma-methylene)triphosphate blocks the synthesis, the injury signal is likely to be GTP dependent.

Topics: Acyltransferases; Adenosine Triphosphate; Animals; Aspartic Acid; Citric Acid; Energy Metabolism; Fumarates; Ligases; Malates; Microinjections; Physarum polycephalum; Polymers

We have cloned a polyspecific organic anion transporter from Caenorhabditis elegans and elucidated its functional characteristics. The C. elegans anion transporter (CeOAT1) codes for a protein of 526 amino acids containing 12 putative transmembrane domains. It exhibits significant homology at the level of amino acid sequence to the C. elegans organic cation transporter and to the mammalian organic cation and anion transporters. The function of CeOAT1 was investigated by expressing the transporter heterologously in mammalian cells. CeOAT1 transports p-aminohippurate (PAH) in a Na(+)-independent manner. The transport mechanism appears to involve anion exchange because CeOAT1-mediated PAH transport is stimulated by a cell-to-medium concentration gradient of alpha-ketoglutarate or fumarate generated by coexpression in the cells of a mammalian Na(+)-coupled dicarboxylate transporter. CeOAT1 exhibits broad specificity, accepting anions such as folate, indomethacin, furosemide, probenecid, and benzylpenicillin as substrates. The Michaelis-Menten constant for the prototypical organic anion PAH is 0.43+/-0.07 mM. This constitutes the first report of the molecular and functional identification of a polyspecific organic anion transporter in C. elegans.

Topics: Animals; Anions; Anticarcinogenic Agents; Base Sequence; Biological Transport, Active; Caenorhabditis elegans; Cloning, Molecular; Dicarboxylic Acids; Dose-Response Relationship, Drug; Fumarates; In Vitro Techniques; Ion Exchange; Ketoglutaric Acids; Molecular Sequence Data; p-Aminohippuric Acid; Radiation-Protective Agents; Sodium

The seven nap genes at minute 47 on the Escherichia coli K-12 chromosome encode a functional nitrate reductase located in the periplasm. The molybdoprotein, NapA, is known to be essential for nitrate reduction. We now demonstrate that the two c-type cytochromes, the periplasmic NapB and the membrane-associated NapC, as well as a fourth polypeptide, NapD, are also essential for nitrate reduction in the periplasm by physiological substrates such as glycerol, formate and glucose. None of the three iron-sulphur proteins, NapF, NapG or NapH, are essential, irrespective of whether the bacteria are grown anaerobically in the presence of nitrate or fumarate as a terminal electron acceptor, or by glucose fermentation. Mutation of napD resulted in the total loss of Methyl Viologen-dependent nitrate reductase activity of the molybdoprotein, NapA, consistent with an earlier suggestion by others that NapD might be required for post-translational modification of NapA.

Topics: Bacterial Proteins; Base Sequence; Cytochrome c Group; DNA Primers; Electron Transport; Escherichia coli; Escherichia coli Proteins; Fermentation; Fumarates; Gene Deletion; Genes, Bacterial; Glucose; Mutation; Nitrate Reductase; Nitrate Reductases; Nitric Acid; Oxidation-Reduction; Periplasm; Plasmids

A new xylanolytic bacterium designated strain HESP1T (T = type strain) was isolated from a methanogenic digester. Strain HESP1T was a motile, rod shaped, spore-forming bacterium, which possessed a Gram-positive type cell wall. Glucose, fructose, lactose, trehalose, maltose, raffinose, sucrose, xylan, mannitol, cellobiose, galactose, mannose, melibiose, ribose were fermented to produce, acetate, butyrate, H2, CO2, formate, isobutyrate, and ethanol. Fumarate was fermented to acetate. Glycerol and methanol were also utilized. Sulfate, thiosulfate, nitrate, sulfur and fumarate were not used as electron acceptors. Strain HESP1T had a G + C content of 40 mol% and grew optimally at 37 degrees C and pH 7 on a fructose containing medium. Phylogenetically, strain HESP1T was most related to Clostridium aminovalericum (similarity of 94%) than to C. populeti, C. herbivorans and Eubacterium xylanophilum (average similarity of 92%), all members of subcluster XIVa of the low G + C containing Gram-positive branch. However, strain HESP1T shared little phenotypic and genotypic traits with C. aminovalericum and on the basis of this and phylogenetic evidence, we propose to tentatively designate strain HESP1T as a new species of the genus Clostridium, Clostridium xylanovorans sp. nov. The type strain is HESP1T (= DSM 12503).

Topics: Base Sequence; Biodegradation, Environmental; Carbohydrate Metabolism; Chromatography, High Pressure Liquid; Clostridium; Fermentation; Fumarates; Methanol; Molecular Sequence Data; Phylogeny; Polymerase Chain Reaction; RNA, Bacterial; RNA, Ribosomal, 16S; Xylans

Newest studies have shown that psoriasis is not primarily a skin disorder but an immunological disturbance under the skin. The skin manifestations are a result of overstimulation of superficial skin cells (Langerhans cells) due to increased production of interleukin 2, 6 and 8 as well as transforming growth-factor-alpha. Interleucin-10 production is diminished. In a recent study (11 German University Skin Clinics) fumaric acid was shown to improve the skin leasons in 80% of treated patients.. Our own studies on 54 patients which where treated in addition with intravenous thymus extract (Thymoject) and selenium (Selenase) showed a faster healing rate with fumaric acid alone. From these results one can postulate that the above treatments (fumaric acid, thymus and selenium) have a synergystic effect.. Internal immunmodulating treatments should therefore have preference over external symptomatic treatments like UV-light, ointments, salt water bath, etc.

Topics: Administration, Oral; Adult; Anticarcinogenic Agents; Drug Synergism; Drug Therapy, Combination; Female; Fumarates; Humans; Infusions, Intravenous; Male; Middle Aged; Psoriasis; Selenium; Thymus Extracts

Experiments were conducted to determine the effect of fumaric acid supplementation and buffering capacity of the diet on ileal and fecal digestibilities of CP, GE, and amino acids in early-weaned pigs. In two experiments, 12 pigs each were weaned at 14 d of age and fitted with a simple T-cannula at the distal ileum between 15 and 17 d of age. In both experiments, the pigs were fed four diets, based on wheat and soybean meal without (control) or with 1, 2, or 3% fumaric acid according to a balanced two-period change-over design. In Exp. 2, the buffering capacity of the diets was increased by supplementation of 3% sodium bicarbonate. The pigs were fed three times daily: equal amounts at 8-h intervals. The diets were supplied at a rate of 5% (wt/wt) of body weight. The inclusion of fumaric acid to the diet with a low buffering capacity increased (P < .05) the ileal digestibilities of CP, GE, and the majority of amino acids. The highest responses were found at an inclusion level of 2% fumaric acid. The improvements in apparent ileal amino acid digestibilities ranged from 4.9 to 12.8 percentage units. Supplementation of fumaric acid to a diet with a high buffering capacity led only to numerical increases in ileal digestibilities of CP, GE, and amino acids. In both studies, fumaric acid supplementation had no effect (P > .05) on the fecal digestibilities of CP, GE, and amino acids, except histidine. A high buffering capacity of the diet decreased the ileal amino acid digestibilities by 1 to 10 percentage units compared with diets with the low buffering capacities. In both experiments, ileal and fecal digestibilities were higher (P < .05) in Period 2 (on d 24 after weaning) than in Period 1 (on d 11 after weaning). A positive correlation (P < .05) between ileal digestibilities and fumaric acid supplementation to the diets with the low buffering capacities was observed in Period 1 (on d 11 after weaning), but not in Period 2 (on d 24 after weaning). No relationship (P > .05) between ileal digestibilities and fumaric acid supplementation to the diets with a high buffering capacity could be established. In conclusion, supplementation of fumaric acid to starter diets during the first 3 to 4 wk after weaning increases the ileal digestibilities of GE, CP, and amino acids.

Topics: Amino Acids; Animal Feed; Animals; Buffers; Dietary Proteins; Digestion; Feces; Female; Fumarates; Ileum; Male; Swine; Weaning

Fumarate reductases and succinate dehydrogenases play central roles in the metabolism of eukaryotic and prokaryotic cells. A recent medium resolution structure of the Escherichia coli fumarate reductase (Frd) has revealed the overall organization of the membrane-bound complex. Here we present the first high resolution X-ray crystal structure of a water-soluble bacterial fumarate reductase in an open conformation. This structure reveals a mobile domain that modulates substrate access to the active site and provides new insights into the mechanism of this widespread and important family of FAD-containing respiratory proteins.

Topics: Amino Acid Sequence; Catalytic Domain; Crystallization; Crystallography, X-Ray; Cytochrome c Group; Dimerization; Flavins; Fumarates; Heme; Isoenzymes; Models, Molecular; Molecular Sequence Data; Molecular Weight; Motion; Oxidation-Reduction; Protein Conformation; Shewanella; Solubility; Static Electricity; Structure-Activity Relationship; Succinate Dehydrogenase

The 1.8 A resolution crystal structure of the tetraheme flavocytochrome c3, Fcc3, provides the first mechanistic insight into respiratory fumarate reductases or succinate dehydrogenases. The multi-redox center, three-domain protein shows a 40 A long 'molecular wire' allowing rapid conduction of electrons through a new type of cytochrome domain onto the active site flavin, driving the reduction of fumarate to succinate. In this structure a malate-like molecule is trapped in the enzyme active site. The interactions between this molecule and the enzyme suggest a clear mechanism for fumarate reduction in which the substrate is polarized and twisted, facilitating hydride transfer from the reduced flavin and subsequent proton transfer. The enzyme active site in the oxidized form is completely buried at the interface between the flavin-binding and the clamp domains. Movement of the cytochrome and clamp domains is postulated to allow release of the product.

Topics: Amino Acid Sequence; Binding Sites; Catalysis; Crystallization; Crystallography, X-Ray; Cytochrome c Group; Electrons; Flavins; Fumarates; Heme; Models, Molecular; Molecular Sequence Data; Oxidation-Reduction; Protein Conformation; Protons; Shewanella; Structure-Activity Relationship; Succinate Dehydrogenase

Fumarate respiration is one of the most widespread types of anaerobic respiration. The soluble fumarate reductase of Shewanella putrefaciens MR-1 is a periplasmic tetraheme flavocytochrome c. The crystal structures of the enzyme were solved to 2.9 A for the uncomplexed form and to 2.8 A and 2.5 A for the fumarate and the succinate-bound protein, respectively. The structures reveal a flexible capping domain linked to the FAD-binding domain. A catalytic mechanism for fumarate reduction based on the structure of the complexed protein is proposed. The mechanism for the reverse reaction is a model for the homologous succinate dehydrogenase (complex II) of the respiratory chain. In flavocytochrome c fumarate reductase, all redox centers are in van der Waals contact with one another, thus providing an efficient conduit of electrons from the hemes via the FAD to fumarate.

Topics: Amino Acid Oxidoreductases; Amino Acid Sequence; Binding Sites; Catalytic Domain; Crystallization; Crystallography, X-Ray; Cytochrome c Group; Electrons; Escherichia coli; Escherichia coli Proteins; Flavin-Adenine Dinucleotide; Fumarates; Heme; Models, Molecular; Molecular Sequence Data; Oxidation-Reduction; Oxidoreductases; Protein Folding; Protein Structure, Secondary; Shewanella putrefaciens; Succinate Dehydrogenase; Succinic Acid

In this study, we investigated the metabolite permeability of isolated coupled Saccharomyces cerevisiae mitochondria. The occurrence of a fumarate/malate antiporter activity was shown. The activity differs from that of the dicarboxylate carrier (which catalyses the succinate/malate antiport) in (a) kinetics (Km and Vmax values are about 27 microM and 22 nmol min(-1) mg protein(-1) and 70 microM and 4 nmol min(-1) mg protein(-1), respectively), (b) sensitivity to inhibitors, (c) Ki for the competitive inhibitor phenylsuccinate and (d) pH profiles.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Antiporters; Biological Transport, Active; Fumarates; Hydrogen-Ion Concentration; Kinetics; Malates; Mitochondria; Saccharomyces cerevisiae; Succinates

Fumaric acid preparations are used as longterm and effective treatment of psoriasis. Apart from gastrointestinal, dermatological and hematological side-effects, transient renal damage was observed during treatment with fumaric acid. The case of a 38 year old woman who was treated with fumaric acid (420 mg bid) for 5 years before she complained of fatigue and weakness. According to clinical laboratory she had developed severe proximal tubular damage. Hypophosphatemia, glycosuria and proteinuria persisted although medication was stopped immediately.

Topics: Adult; Fanconi Syndrome; Female; Fumarates; Glycosuria; Humans; Hypophosphatemia; Proteinuria; Psoriasis; Time

A reliable method for the simultaneous determination of oxalic, fumaric, maleic, and succinic acids in tartaric and malic acids for pharmaceutical use by reversed-phase ion-suppression high performance liquid chromatography is presented. HPLC was achieved on a Nova-Pak C18 column by isocratic elution using water adjusted to pH 2.10-2.15 with perchloric acid, and detection was by UV adsorption at a wavelength of 210 nm. This method was found to be superior to previous liquid chromatography as well as other classical assay, and to be an attractive choice for the analysis of these compounds.

Topics: Chromatography, High Pressure Liquid; Dicarboxylic Acids; Drug Contamination; Evaluation Studies as Topic; Fumarates; Hydrogen-Ion Concentration; Malates; Maleates; Oxalic Acid; Perchlorates; Reproducibility of Results; Succinic Acid; Tartrates

The possibility that microorganisms might use reduced humic substances (humics) as an electron donor for the reduction of electron acceptors with a more positive redox potential was investigated. All of the Fe(III)- and humics-reducing microorganisms evaluated were capable of oxidizing reduced humics and/or the reduced humics analogue anthrahydroquinone-2,6,-disulphonate (AHODS), with nitrate and/or fumarate as the electron acceptor. These included Geobacter metallireducens, Geobacter sulphurreducens, Geothrix fermentans, Shewanella alga, Wolinella succinogenes and 'S. barnesii'. Several of the humics-oxidizing microorganisms grew in medium with AHQDS as the sole electron donor and fumarate as the electron acceptor. Even though it does not reduce Fe(III) or humics, Paracoccus denitrificans could use AHQDS and reduced humics as electron donors for denitrification. However, another denitrifier, Pseudomonas denitrificans, could not. AHODS could also serve as an electron donor for selenate and arsenate reduction by W. succinogenes. Electron spin resonance studies demonstrated that humics oxidation was associated with the oxidation of hydroquinone moieties in the humics. Studies with G. metallireducens and W. succinogenes demonstrated that the anthraquinone-2,6-disulphonate (AQDS)/AHQDS redox couple mediated an interspecies electron transfer between the two organisms. These results suggest that, as microbially reduced humics enter less reduced zones of soils and sediments, the reduced humics may serve as electron donors for microbial reduction of several environmentally significant electron acceptors.

Topics: Aerobiosis; Anaerobiosis; Anthraquinones; Arsenates; Bacteria; Electron Transport; Fumarates; Humic Substances; Selenic Acid; Selenium Compounds; Tumor Cells, Cultured

An Escherichia coli knockout ubiCA mutant has been constructed using a gene replacement method and verified using both Southern hybridization and PCR. The mutant, which was unable to synthesize ubiquinone (Q), showed severely diminished growth yields aerobically but not anaerobically with either nitrate or fumarate as terminal electron acceptors. Low oxygen uptake rates were demonstrated in membrane preparations using either NADH or lactate as substrates. However, these rates were greatly stimulated by the addition of ubiquinone-1 (Q-1). The rate of electron transfer to those oxidase components observable by photodissociation of their CO complexes was studied at sub-zero temperatures. In the ubiCA mutant, the reduced form of haemoproteins--predominantly cytochrome b595--was reoxidized significantly faster in the presence of oxygen than in a Ubi+ strain, indicating the absence of Q as electron donor. Continuous multiple-wavelength recordings of the oxidoreduction state of cytochrome(s) b during steady-state respiration showed greater reduction in membranes from the ubiCA mutant than in wildtype membranes. A scheme for the respiratory electron-transfer chain in E. coli is proposed, in which Q functions downstream of cytochrome(s) b.

Topics: Aerobiosis; Alkyl and Aryl Transferases; Alleles; Anaerobiosis; Cell Membrane; Cloning, Molecular; Cytochrome b Group; DNA, Bacterial; Electron Transport; Escherichia coli; Fumarates; Lactic Acid; NAD; Nitrates; Oxidation-Reduction; Oxo-Acid-Lyases; Oxygen; Plasmids; Polymerase Chain Reaction; Ubiquinone

Four agents, fumaric acid (FA), N-acetylcysteine (NAC), N-(4-hydroxyphenyl) retinamide (4-HPR) and beta-carotene (beta-CT), were evaluated for potential chemopreventive activity using the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumor model in female A/J mice. The agents were evaluated in both 16-week and 52-week bioassays at two dose levels corresponding to 0.8 maximum tolerated dose (MTD) and 0.4 MTD administered throughout the bioassay either in the diet (FA, 160 and 80 mmol/kg diet; NAC, 160 and 80 mmol/kg diet; 4-HPR, 4 and 2 mmol/kg diet) or by subcutaneous injection twice a week (beta-CT, 32 and 16 mg/kg b.w.). Mice were treated with a single i.p. dose of 10 micromol NNK in saline 1 week after administration of test agent. Lung adenomas were evaluated in the 16-week bioassay, whereas both adenomas and adenocarcinomas of the lung were determined in the 52-week bioassay. Both bioassays showed that all four agents did not significantly inhibit the total tumor incidence and multiplicity of the lung. However, the incidence of adenocarcinomas was reduced (P < 0.01) at 52 weeks in NNK groups given either 0.8 MTD NAC or 0.8 MTD beta-CT compared with the NNK control group. The decreases in adenocarcinomas were accompanied by corresponding increases in adenomas in these treatment groups. Thus, this study showed that FA, NAC, 4-HPR and beta-CT did not inhibit the total tumor formation, however, at the higher doses both NAC and beta-CT significantly retarded the malignant progression in the lung of NNK-treated A/J mice.

Topics: Acetylcysteine; Animals; Anticarcinogenic Agents; beta Carotene; Carcinogens; Dose-Response Relationship, Drug; Female; Fenretinide; Fumarates; Lung Neoplasms; Mice; Mice, Inbred A; Nicotiana; Nitrosamines; Plants, Toxic

In order to gain some insight into the fate of fumarate synthesised in the cytosol in the purine nucleotide cycle and in amino acid catabolism, the capability of both rat kidney mitochondria and acidotic rat kidney mitochondria to take up either externally synthesised, via adenylsuccinate lyase, or added fumarate in exchange with intramitochondrial malate or aspartate was tested by means of both spectrophotometric and isotopic techniques. The appearance of either malate or aspartate caused by the presence of fumarate was revealed outside normal and acidotic mitochondria by using specific substrate detecting systems. Consistently, externally added fumarate was found to cause efflux of either [14C]-malate or [14C]-aspartate from loaded mitochondria. The occurrence in rat kidney mitochondria of two separate translocators, i.e., fumarate/malate and fumarate/aspartate carriers, is shown in the light of saturation kinetics and the different inhibitor sensitivity. The fumarate/aspartate antiporters found in normal and acidotic mitochondria appear to differ from each other.

Topics: Acidosis; Adenylosuccinate Lyase; Animals; Aspartic Acid; Biological Transport; Carrier Proteins; Fumarates; Kidney; Malates; Male; Mitochondria; NAD; NADP; Oxaloacetates; Rats; Rats, Wistar

During growth with fumarate as the terminal electron transport acceptor and either formate or sulfide as the electron donor, Wolinella succinogenes induced a peri-plasmic protein (54 kDa) that reacted with an antiserum raised against the periplasmic fumarate reductase (Fcc) of Shewanella putrefaciens. However, the periplasmic cell fraction of W. succinogenes did not catalyze fumarate reduction with viologen radicals. W. succinogenes grown with polysulfide instead of fumarate contained much less (< 10%) of the 54-kDa antigen, and the antigen was not detectable in nitrate-grown bacteria. The antigen was most likely encoded by the fccA gene of W. succinogenes. The antigen was absent from a DeltafccABC mutant, and its size is close to that of the protein predicted by fccA. The fccA gene probably encodes a pre-protein carrying an N-terminal signal peptide. The sequence of the mature FccA (481 residues, 52.4 kDa) is similar (31% identity) to that of the C-terminal part (450 residues) of S. putrefaciens fumarate reductase. As indicated by Northern blot analysis, fccA is cotranscribed with fccB and fccC. The proteins predicted from the fccB and fccC gene sequences represent tetraheme cytochromes c. FccB is similar to the N-terminal part (150 residues) of S. putrefaciens fumarate reductase, while FccC resembles the tetraheme cytochromes c of the NirT/NapC family. The DeltafccABC mutant of W. succinogenes grew with fumarate and formate or sulfide, suggesting that the deleted proteins were not required for fumarate respiration with either electron donor.

Topics: Amino Acid Sequence; Cell Membrane; Cytochrome c Group; Formates; Fumarates; Genes, Bacterial; Gram-Negative Facultatively Anaerobic Rods; Molecular Sequence Data; Molecular Weight; Periplasm; Restriction Mapping; RNA, Bacterial; RNA, Messenger; Sequence Deletion; Sequence Homology, Amino Acid; Succinate Dehydrogenase; Sulfides; Wolinella

The modified nucleoside 2-methylthio-N-6-isopentenyl adenosine (ms2i6A) is present in position 37 (adjacent to and 3' of the anticodon) of tRNAs that read codons beginning with U except tRNA(i.v. Ser) in Escherichia coli. In Salmonella typhimurium, 2-methylthio-N-6-(cis-hydroxy)isopentenyl adenosine (ms2io6A; also referred to as 2-methylthio cis-ribozeatin) is found in tRNA, most likely in the species that have ms2i6A in E. coli. Mutants (miaE) of S. typhimurium in which ms2i6A hydroxylation is blocked are unable to grow aerobically on the dicarboxylic acids of the citric acid cycle. Such mutants have normal uptake of dicarboxylic acids and functional enzymes of the citric acid cycle and the aerobic respiratory chain. The ability of S. typhimurium to grow on succinate, fumarate, and malate is dependent on the state of modification in position 37 of those tRNAs normally having ms2io6A37 and is not due to a second cellular function of tRNA (ms2io6A37)hydroxylase, the miaE gene product. We suggest that S. typhimurium senses the hydroxylation status of the isopentenyl group of the tRNA and will grow on succinate, fumarate, or malate only if the isopentenyl group is hydroxylated.

Topics: Chromosome Mapping; Citric Acid Cycle; Fumarates; Genes, Bacterial; Genetic Complementation Test; Isopentenyladenosine; Malates; Mutation; Phenotype; RNA, Transfer; Salmonella typhimurium; Succinic Acid

The yeast Saccharomyces cerevisiae has been used to efficiently produce L-malic acid from fumaric acid. Fumarase is responsible for the reversible conversion of fumaric and L-malic acids in the TCA cycle. To investigate the function of mitochondrial and cytoplasmic fumarase isoenzymes in L-malic acid bioconversion, a wild-type strain and a cytoplasmic respiratory-deficient mutant devoid of functional mitochondria were employed. The mutant strain, which only contained the cytoplasmic fumarase, was still functional in fumaric acid to L-malic acid bioconversion However, its specific conversion rate was much lower (0.20 g/g.h) than that of the wild-type strain (0.55 g/g.h).

Topics: Catalysis; Coloring Agents; Cytoplasm; Fermentation; Fumarate Hydratase; Fumarates; Malates; Mitochondria; Oxygen Consumption; Saccharomyces cerevisiae; Stereoisomerism; Tetrazolium Salts

Succinic acid was produced efficiently from fumaric acid by a recombinant E. coli strain DH5 alpha/pGC1002 containing multicopy fumarate reductase genes. The effects of initial fumaric acid and glucose concentration on the production of succinic acid were investigated. Succinic acid reached 41 to over 60 g/L in 48.5 h starting with 50 to 64 g/L fumaric acid. Significant substrate inhibition was observed at initial fumaric acid concentration of 90 g/L. L-Malic acid became the major fermentation product under these conditions. Provision of glucose (5-30 g/L) to the fermentation medium stimulated the initial succinic acid production rate over two folds.

Topics: Acetic Acid; Escherichia coli; Fermentation; Fumarates; Glucose; Malates; Succinate Dehydrogenase; Succinic Acid

The effect of CheY and fumarate on switching frequency and rotational bias of the bacterial flagellar motor was analyzed by computer-aided tracking of tethered Escherichia coli. Plots of cells overexpressing CheY in a gutted background showed a bell-shaped correlation curve of Switching frequency and bias centering at about 50% clockwise rotation. Gutted cells (i.e., with cheA to cheZ deleted) with a low CheY level but a high cytoplasmic fumarate concentration displayed the same correlation of switching frequency and bias as cells overexpressing CheY at the wild-type fumarate level. Hence, a high fumarate level can phenotypically mimic CheY overexpression by simultaneously changing the switching frequency and the bias. A linear correlation of cytoplasmic fumarate concentration and clockwise rotation bias was found and predicts exclusively counter-clockwise rotation without switching when fumarate is absent. This suggests that (i) fumarate is essential for clockwise rotation in vivo and (ii) any metabolically induced fluctuation of its cytoplasmic concentration will result in a transient change in bias and switching probability. A high fumarate level resulted in a dose-response curve linking bias and cytoplasmic CheY concentration that was offset but with a slope similar to that for a low fumarate level. It is concluded that fumarate and CheY act additively presumably at different reaction steps in the conformational transition of the switch complex from counterclockwise to clockwise motor rotation.

Topics: Arabinose; Bacterial Proteins; Chemotaxis; Escherichia coli; Escherichia coli Proteins; Flagella; Fumarates; Gene Deletion; Genotype; Histidine Kinase; Kinetics; Membrane Proteins; Methyl-Accepting Chemotaxis Proteins; Time Factors

Switching flagellar rotation from one direction to another is an essential part of bacterial chemotaxis. Fumarate has been shown to possess the capacity to restore to flagella of cytoplasm-free, CheY-containing bacterial envelopes the ability to switch directions and to increase the probability of reversal in intact cells. Neither the target of fumarate action nor the mechanism of function is known. To distinguish between the two potential targets of fumarate, the response regulator CheY and the flagellar switch-motor complex, we compared flagellar rotation between isogenic strains that lacked CheY and had either low or high levels of fumarate. The difference in the fumarate levels was due to a deletion of the genes encoding the enzymes that synthesize and metabolize fumarate; succinate dehydrogenase and fumarase, respectively. The strains were in a gutted background (i.e. a background deleted for the cytoplasmic chemotaxis proteins and some of the receptors), and switching was achieved by carrying out the measurements at 2.5 degreesC, where it has been demonstrated that gutted cells switch spontaneously. The flagellar rotation of the strain with the highest level of fumarate was the most clockwise-biased and had the highest reversal frequency, indicating that fumarate is effective even in the absence of CheY. Fumarate reduced the free energy difference of the counterclockwise-to-clockwise transition and had no appreciable effect on the activation energy of this transition. Similar observations were made at room temperature, provided that intracellular CheY was present. In a wild-type background, both mutants made rings on semi-solid agar typical of normal chemotaxis. Taken together, the results suggest that the target of fumarate is the switch-motor complex, that fumarate acts by increasing the probability of the clockwise state, and that a fumarate level as low as that found in succinate dehydrogenase mutants is sufficient for normal chemotaxis.

Topics: Bacterial Proteins; Chemotaxis; Energy Metabolism; Escherichia coli; Escherichia coli Proteins; Flagella; Fumarates; Membrane Proteins; Methyl-Accepting Chemotaxis Proteins; Rotation; Temperature; Thermodynamics

Chemical antioxidants and catalase have been shown to be ineffective in improving the motility of ram spermatozoa in a chemically-defined diluent (RSD-1). In an attempt to identify the biochemical basis of this observation, the activity of aromatic amino acid oxidase (AAAO), the enzyme responsible for generation of hydrogen peroxide in ram and bull spermatozoa, has been investigated. Ram spermatozoa contained higher levels of AAAO activity than bull spermatozoa, although the physico-chemical properties of the enzyme were generally similar in both species. Components of the medium had a marked effect upon AAAO activity. In the presence of glutamate and 3-(N-morpholino)propanesulfonic acid (MOPS), AAAO activity was decreased. Pyruvate appeared to increase AAAO activity. This was due to the ability of this substance to destroy hydrogen peroxide. Pyruvate in RSD-1 works as an effective antioxidant and therefore eliminates the need for other antioxidants in the semen diluent.

Topics: Amino Acid Oxidoreductases; Animals; Antioxidants; Buffers; Catalase; Cattle; Enzyme Inhibitors; Ethylmaleimide; Fumarates; Glucose; Glutamic Acid; Hot Temperature; Hydrogen-Ion Concentration; Male; Morpholines; Oximetry; Pyruvic Acid; Reactive Oxygen Species; Semen; Sheep; Sperm Motility; Spermatozoa

Impaired oxidative and energy metabolism are important features in Alzheimer's disease. These metabolic abnormalities may induce functional disturbances and are associated with significant cognitive impairment.. To determine whether mitochondrial function is altered by Alzheimer's disease, a quantitative analysis of substrates that enter the tricarboxylic acid cycle was carried out in cerebrospinal fluid (CSF) from Alzheimer patients.. Organic acid levels related to carbohydrate oxidation were measured in CSF from patients affected by dementia of Alzheimer type (n = 17) and from nondemented elderly controls (n = 17) using a gas chromatography/mass spectrometry system. CSF glucose and glutamine concentrations were determined by a quantitative enzymatic method and by ion exchange chromatography, respectively.. Compared to age-matched controls, patients had a higher CSF level of lactate (p = 0.002) and a lower mean level of succinate (p = 0.002), fumarate (p = 0.003) and glutamine (p = 0.0005). The CSF glucose level was not modified.. Our results suggest an impairment of mitochondrial oxidative metabolism in brain cells of patients with Alzheimer's disease.

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Cerebrospinal Fluid; Citric Acid Cycle; Female; Fumarates; Glutamine; Humans; Lactic Acid; Male; Mitochondria; Reference Values; Substrate Specificity; Succinic Acid

Topics: Fumarate Hydratase; Fumarates; Humans; Infant

In Escherichia coli the genes encoding the anaerobic fumarate respiratory system are transcriptionally regulated by C4-dicarboxylates. The regulation is effected by a two-component regulatory system, DcuSR, consisting of a sensory histidine kinase (DcuS) and a response regulator (DcuR). DcuS and DcuR are encoded by the dcuSR genes (previously yjdHG) at 93.7 min on the calculated E. coli map. Inactivation of the dcuR and dcuS genes caused the loss of C4-dicarboxylate-stimulated synthesis of fumarate reductase (frdABCD genes) and of the anaerobic fumarate-succinate antiporter DcuB (dcuB gene). DcuS is predicted to contain a large periplasmic domain as the supposed site for C4-dicarboxylate sensing. Regulation by DcuR and DcuS responded to the presence of the C4-dicarboxylates fumarate, succinate, malate, aspartate, tartrate, and maleate. Since maleate is not taken up by the bacteria under these conditions, the carboxylates presumably act from without. Genes of the aerobic C4-dicarboxylate pathway encoding succinate dehydrogenase (sdhCDAB) and the aerobic succinate carrier (dctA) are only marginally or negatively regulated by the DcuSR system. The CitAB two-component regulatory system, which is highly similar to DcuSR, had no effect on C4-dicarboxylate regulation of any of the genes.

Topics: Antiporters; Bacterial Proteins; Carrier Proteins; Dicarboxylic Acid Transporters; Dicarboxylic Acids; DNA-Binding Proteins; Escherichia coli; Escherichia coli Proteins; Fumarates; Gene Expression Regulation, Bacterial; Genes, Bacterial; Histidine Kinase; Membrane Proteins; Mutagenesis, Insertional; Protein Kinases; Signal Transduction; Succinate Dehydrogenase; Transcription Factors

Recent studies of anaerobic toluene catabolism have demonstrated a novel reaction for anaerobic hydrocarbon activation: the addition of the methyl carbon of toluene to fumarate to form benzylsuccinate. In vitro studies of the anaerobic benzylsuccinate synthase reaction indicate that the H atom abstracted from the toluene methyl group during addition to fumarate is retained in the succinyl moiety of benzylsuccinate. Based on structural studies of benzylsuccinate formed during anaerobic, in vitro assays with denitrifying, toluene-mineralizing strain T, we now report the following characteristics of the benzylsuccinate synthase reaction: (i) it is highly stereospecific, resulting in >95% formation of the (+)-benzylsuccinic acid enantiomer [(R)-2-benzyl-3-carboxypropionic acid], and (ii) active benzylsuccinate synthase does not contain an abstracted methyl H atom from toluene at the beginning or at the end of a catalytic cycle.

Topics: Anaerobiosis; Biodegradation, Environmental; Carbon-Carbon Lyases; Fumarates; Stereoisomerism; Substrate Specificity; Succinates; Toluene

A eukaryotic fumarase is for the first time unequivocally shown to contain two distinct substrate-binding sites. Pig heart fumarase is a tetrameric enzyme consisting of four identical subunits of 50 kDa each. Besides the true substrates L-malate and fumarate, the active sites (sites A) also bind their analogs D-malate and oxaloacetate, as well as the competitive inhibitor glycine. The additional binding sites (sites B) on the other hand also bind the substrates and their analogs D-malate and oxaloacetate, as well as L-aspartate which is not an inhibitor. Depending on the pH, the affinity of sites B for ligands (Kd being in the millimolar range) is 1-2 orders of magnitude lower than the affinity of sites A (of which Kd is in the micromolar range). However, saturating sites B results in an increase in the overall activity of the enzyme. The benzenetetracarboxyl compound pyromellitic acid displays very special properties. One molecule of this ligand is indeed able to bind into a site A and a site B at the same time. Four molecules of pyromellitic acid were found to bind per molecule fumarase, and the affinity of the enzyme for this ligand is very high (Kd = 0.6 to 2.2 microM, depending on the pH). Experiments with this ligand turned out to be crucial in order to explain the results obtained. An essential tyrosine residue is found to be located in site A, whereas an essential methionine residue resides in or near site B. Upon limited proteolysis, a peptide of about 4 kDa is initially removed, probably at the C-terminal side; this degradation results in inactivation of the enzyme. Small local conformational changes in the enzyme are picked up by circular dichroism measurements in the near-UV region. This spectrum is built up of two tryptophanyl triplets, the first one of which is modified upon saturating the active sites (A), and the second one upon saturating the low affinity binding sites (B).

Topics: Animals; Catalytic Domain; Circular Dichroism; Fumarate Hydratase; Fumarates; Kinetics; Ligands; Macromolecular Substances; Malates; Models, Molecular; Molecular Weight; Myocardium; Protein Denaturation; Spectrophotometry; Swine; Urea

The rates of p-aminohippurate (PAH) and of uric acid uptake by basolateral membrane vesicles isolated from proximal tubules of rat kidney have been investigated. Accumulation of both substrates against the concentration gradient within the vesicles was shown to occur in the presence of alpha-ketoglutarate (alpha-KG) and Na+ gradient in the incubation medium. The mechanism of the coupling between Na(+)-dicarboxylate symport and organic anion transport is discussed based on the differences between the rates of PAH and of uric acid uptake. It is proposed that the limiting step of coupling between two transporters is the step of alpha-KG-organic anion exchange.

Topics: Animals; Anion Transport Proteins; Anions; Biological Transport; Carrier Proteins; Cell Membrane; Cell Polarity; Citric Acid; Dicarboxylic Acids; Fumarates; Glutarates; Ion Transport; Isocitrates; Ketoglutaric Acids; Kidney Tubules, Proximal; Oxaloacetic Acid; p-Aminohippuric Acid; Rats; Sodium; Sodium Channels; Succinic Acid; Uric Acid

Topics: Acetyl-CoA C-Acyltransferase; Amino Acid Metabolism, Inborn Errors; Cells, Cultured; Dicarboxylic Acids; Energy Metabolism; Fibroblasts; Fumarates; Humans; Infant; Isoleucine; Malates; Male; Neurodegenerative Diseases; Phenotype; Succinic Acid

Hepatocytes prepared from overnight fasted rats were incubated for 120 min in the presence of the dimethyl ester of [2,3-(13)C]succinic acid (10 mM). The identification and quantification of 13C-enriched metabolites in the incubation medium were performed by a novel computational strategy for the deconvolution of NMR spectra with multiplet structures and constraints. The generation of 13C-labelled metabolites, including succinate, fumarate, malate, lactate, alanine, aspartate and glucose accounted for about half of the initial amount of the ester present in the incubation medium. A fair correlation was observed between the experimental abundance of each 13C-labelled glucose isotopomer and the corresponding values derived from a model for the metabolism of [2,3-(13)C]succinate. Newly formed glucose was more efficiently labelled in the carbon C5 than C2, as well as the carbon C6 than C1, supporting the concept that D-glyceraldehyde-3-phosphate may undergo enzyme-to-enzyme channelling between glyceraldehyde-3-phosphate dehydrogenase and phosphofructoaldolase.

Topics: Alanine; Animals; Esters; Female; Fumarates; In Vitro Techniques; Lactic Acid; Liver; Magnetic Resonance Spectroscopy; Malates; Models, Chemical; Rats; Rats, Wistar; Succinates; Succinic Acid

Recycling of yeast fumarase to permit repetition of its reaction chemistry requires two proton transfers and two conformational changes, in pathways that are different in detail but thematically similar in the two directions. In the malate --> fumarate direction, simple anions such as acetate accelerate the fumarate-off step producing E(H(f)), a fumarate-specific isoform that retains the C3R-proton of malate. Fumarate specificity is shown with S-2,3-dicarboxyaziridine, which is competitive vs fumarate and noncompetitive with malate as substrate. The steady-state level of E(H(f)), based on Kii (S-2,3-dicarboxyaziridine), is increased by D2O and decreased by imidazole acting as a general acid for conversion of E(H(f)) to E(H(f))H. E(H(f))H is fumarate-specific as shown by the inhibition pattern with ClO4-. The pKa of this step is approximately 7.25 based on the pH dependence of Kii (ClO4-). A conformational change occurs next as shown by high sensitivity of k(cat) but not k(cat)/Km, to the microviscosogen, glycerol, and change to a nonspecific isoform, E(H(mf))H, probably the same species formed in the fumarate --> malate direction from malate-specific intermediates by a different conformational change. Malate enters the cycle by reaction with E(H(mf))H and returns to E(m)H x malate after a second conformational change. When fumarate-off is slow, as in low anion medium, malate itself becomes an activator of malate --> fumarate. This effect occurs with changes in inhibition patterns suggestive of the bypass of the slow E(f) --> E(mf) conversion in favor of direct formation of E(mf) when free fumarate is formed. 3-Nitro-2-hydroxypropionate, a strong inhibitor of fumarase [Porter, D. J. T., and Bright, H. J. (1980) J. Biol. Chem. 255, 4772-4780] in its carbanion form, is competitive with both malate and fumarate. Therefore, 3-nitro-2-hydroxypropionic acid interacts with E(H(mf))H and not with E(m) or E(f) isoforms. Occurrence of two different conformational changes in the recycling process suggests that the reaction chemistry employs a two-step mechanism. The specificity of inhibition for E(H(mf))H is consistent with the expected intermediate of a carbanion mechanism, E(H)H x carbanion-. The proton transfers and conformational changes of recycling occur in the same sequence that is expected for this reaction chemistry. Several examples of ligand-activated conformational changes are reported.

Topics: Animals; Anions; Binding, Competitive; Citric Acid Cycle; Enzyme Activation; Fumarate Hydratase; Fumarates; Isoenzymes; Lactates; Malates; Protein Conformation; Saccharomyces cerevisiae; Substrate Specificity; Swine

Anaerobic assays conducted with strain T, a denitrifying bacterium capable of mineralizing toluene to carbon dioxide, demonstrated that toluene-grown, permeabilized cells catalyzed the addition of toluene to fumarate to form benzylsuccinate. This reaction was not dependent on the presence of coenzyme A (CoA) or ATP. In the presence of CoA, formation of E-phenylitaconate from benzylsuccinate was also observed. Kinetic studies demonstrated that the specific rate of benzylsuccinate formation from toluene and fumarate in assays with permeabilized cells was >30% of the specific rate of toluene consumption in whole-cell suspensions with nitrate; this observation suggests that benzylsuccinate formation may be the first reaction in anaerobic toluene degradation by strain T. Use of deuterium-labeled toluene and gas chromatography-mass spectrometry indicated that the H atom abstracted from the toluene methyl group during addition to fumarate was retained in the succinyl moiety of benzylsuccinate. In this study, no evidence was found to support previously proposed reactions of toluene with acetyl-CoA or succinyl-CoA. Toluene-grown, permeabilized cells of strain T also catalyzed the addition of o-xylene to fumarate to form (2-methylbenzyl)succinate. o-Xylene is not a growth substrate for strain T, and its transformation was probably cometabolic. With the exception of specific reaction rates, the observed characteristics of the toluene-fumarate addition reaction (i.e., retention of a methyl H atom and independence from CoA and ATP) also apply to the o-xylene-fumarate addition reaction. Thus, addition to fumarate may be a biochemical strategy to anaerobically activate a range of methylbenzenes.

Topics: Anaerobiosis; Benzylidene Compounds; Biodegradation, Environmental; Cell Membrane Permeability; Fumarates; Gram-Negative Aerobic Bacteria; Kinetics; Mass Spectrometry; Succinates; Toluene; Xylenes

The cymA gene, which encodes a tetraheme cytochrome c, was cloned from Shewanella putrefaciens MR-1. This gene complemented a mutant which had a TnphoA insertion in cymA and which was deficient in the respiratory reduction of iron(III), nitrate, fumarate, and manganese(IV). The 561-bp nucleotide sequence of cymA encodes a protein of 187 amino acids with a predicted molecular mass of 20.8 kDa. No N-terminal signal sequence was readily apparent; consistent with this, a cytochrome with a size of 21 kDa was detected in the wild type but was absent in the insertional mutant. The cymA gene is transcribed into an mRNA; the major transcript was approximately 790 bases, suggesting that it is not part of a multicistronic operon. This RNA transcript was not detected in the cymA mutant. The CymA protein was found in the cytoplasmic membrane and soluble fraction of MR-1, and it shares partial amino acid sequence homology with multiheme c-type cytochromes from other bacteria. These cytochromes are ostensibly involved in the transfer of electrons from the cytoplasmic membrane to acceptors in the periplasm. The localization of the fumarate and iron(III) reductases to the periplasm and outer membrane of MR-1, respectively, suggests the possibility of a similar electron transfer role for CymA.

Topics: Amino Acid Sequence; Base Sequence; Cloning, Molecular; Cytochrome c Group; DNA Transposable Elements; Electron Transport; Ferric Compounds; Fumarates; Genes, Bacterial; Genetic Complementation Test; Gram-Negative Facultatively Anaerobic Rods; Molecular Sequence Data; Molecular Weight; Mutagenesis, Insertional; Nitrates; Open Reading Frames; Oxidation-Reduction; Sequence Homology, Amino Acid

The cardioprotective effects of fumarate have been linked to its metabolism to succinate through both oxidative and reductive pathways. To date, the relative contribution of these pathways is a subject of controversy. To address this question, we designed a protocol with 13C substrates and took advantage of 13C isotopomer analysis by gas chromatography-mass spectrometry. Rat hearts were perfused with 11 mM glucose, 1 mM lactate, 0.2 mM pyruvate, 0.2 mM [1-13C]octanoate, and 0.04 or 0.4 mM [U-13C4]fumarate. On reoxygenation after 40 min of severe hypoxia, hearts perfused with 0.4 mM fumarate showed a better recovery of contractile function and released less lactate dehydrogenase (an index of cellular necrosis) than those perfused with 0.04 mM fumarate. The 13C data showed that, in hypoxic hearts, fumarate conversion to succinate occurred only through reduction, although it accounted for only 16% of total succinate release. Most of the succinate was formed through the oxidation of alpha-ketoglutarate or its precursors (50 +/- 5%) and by another yet-unidentified pathway (34 +/- 4%). These data show that, in a model of hypoxia-reoxygenation, the cardioprotective effects of fumarate were associated with its predominant metabolism to succinate through the reductive pathway.

Topics: Animals; Carbon Isotopes; Citric Acid Cycle; Fumarates; Gas Chromatography-Mass Spectrometry; Heart; In Vitro Techniques; Ketoglutaric Acids; Male; Myocardium; Perfusion; Rats; Rats, Sprague-Dawley; Succinates; Succinic Acid

The synthesis of pyruvate carboxylase (PC) was studied by using quantitative immunoblot analysis with an antibody raised against PC purified from Rhodobacter capsulatus and was found to vary 20-fold depending on the growth conditions. The PC content was high in cells grown on pyruvate or on carbon substrates metabolized via pyruvate (lactate, D-malate, glucose, or fructose) and low in cells grown on tricarboxylic acid (TCA) cycle intermediates or substrates metabolized without intermediate formation of pyruvate (acetate or glutamate). Under dark aerobic growth conditions with lactate as a carbon source, the PC content was approximately twofold higher than that found under light anaerobic growth conditions. The results of incubation experiments demonstrate that PC synthesis is induced by pyruvate and repressed by TCA cycle intermediates, with negative control dominating over positive control. The content of PC in R. capsulatus cells was also directly related to the growth rate in continuous cultures. The analysis of intracellular levels of pyruvate and TCA cycle intermediates in cells grown under different conditions demonstrated that the content of PC is directly proportional to the ratio between pyruvate and C4 dicarboxylates. These results suggest that the regulation of PC synthesis by oxygen and its direct correlation with growth rate may reflect effects on the balance of intracellular pyruvate and C4 dicarboxylates. Thus, this important enzyme is potentially regulated both allosterically and at the level of synthesis.

Topics: Aerobiosis; Amino Acid Sequence; Anaerobiosis; Chloramphenicol; Citric Acid Cycle; Culture Media; Darkness; Dicarboxylic Acids; Enzyme Induction; Enzyme Repression; Fumarates; Lactic Acid; Molecular Sequence Data; Protein Synthesis Inhibitors; Pyruvate Carboxylase; Pyruvic Acid; Rhodobacter capsulatus

Biologically adhesive delivery systems offer important advantages over conventional drug delivery systems. Here we show that engineered polymer microspheres made of biologically erodable polymers, which display strong adhesive interactions with gastrointestinal mucus and cellular linings, can traverse both the mucosal absorptive epithelium and the follicle-associated epithelium covering the lymphoid tissue of Peyer's patches. The polymers maintain contact with intestinal epithelium for extended periods of time and actually penetrate it, through and between cells. Thus, once loaded with compounds of pharmacological interest, the microspheres could be developed as delivery systems to transfer biologically active molecules to the circulation. We show that these microspheres increase the absorption of three model substances of widely different molecular size: dicumarol, insulin and plasmid DNA.

Topics: Adhesiveness; Administration, Oral; Area Under Curve; beta-Galactosidase; Biological Availability; Blood Glucose; Decanoic Acids; Dicarboxylic Acids; Dicumarol; Drug Delivery Systems; Fumarates; Gene Transfer Techniques; Insulin; Intestinal Mucosa; Microscopy, Electron; Microspheres; Mucous Membrane; Peyer's Patches; Plasmids; Polymers; Tissue Distribution

Actinobacillus sp. 130Z fermented glucose to the major products succinate, acetate, and formate. Ethanol was formed as a minor fermentation product. Under CO2-limiting conditions, less succinate and more ethanol were formed. The fermentation product ratio remained constant at pH values from 6.0 to 7.4. More succinate was produced when hydrogen was present in the gas phase. Actinobacillus sp. 130Z grew at the expense of fumarate and l-malate reduction, with hydrogen as an electron donor. Other substrates such as more-reduced carbohydrates (e.g., d-sorbitol) resulted in higher succinate and/or ethanol production. Actinobacillus sp. 130Z contained the key enzymes involved in the Embden-Meyerhof-Parnas and the pentose-phosphate pathways and contained high levels of phosphoenolpyruvate (PEP) carboxykinase, malate dehydrogenase, fumarase, fumarate reductase, pyruvate kinase, pyruvate formate-lyase, phosphotransacetylase, acetate kinase, malic enzyme, and oxaloacetate decarboxylase. The levels of PEP carboxykinase, malate dehydrogenase, and fumarase were significantly higher in Actinobacillus sp. 130Z than in Escherichia coli K-12 and accounted for the differences in succinate production. Key enzymes in end product formation in Actinobacillus sp. 130Z were regulated by the energy substrates.

Topics: Actinobacillus; Carbohydrate Metabolism; Carbon Dioxide; Electron Transport; Energy Metabolism; Escherichia coli; Fermentation; Fumarates; Glucose; Hydrogen; Hydrogen-Ion Concentration; Phosphoenolpyruvate; Species Specificity; Succinates; Succinic Acid

Most studies on the antipsoriatic mode of action of dimethylfumarate focused on its antiproliferative effects in keratinocytes. Because inflammatory skin diseases are associated with an upregulation of endothelial cell adhesion molecules and because the presence of inflammatory cells in dermis and epidermis is considered an important feature in psoriasis, we tested the effect of DMF on cytokine-induced adhesion molecule expression in HUVEC, using in situ ELISA and Northern blotting. Dimethylfumarate inhibited ICAM-1, VCAM-1, and E-selectin expression and reduced adhesion of U937 cells to stimulated HUVEC. Monoethylfumarate and fumaric acid had no effect. Similar inhibitory effects for DMF on VCAM-1 expression were observed after stimulation of HUVEC with LPS, PMA, IL-4, and IL-1 alpha or in combinations with TNF alpha. These data are in agreement with previously reported effects of DMF on intracellular thiol levels and inhibition of NF-kappa B activation. The inhibitory effect on cytokine-induced endothelial adhesion molecule expression may represent another target of dimethylfumarate in psoriasis.

Topics: Blotting, Northern; Cell Adhesion; Cell Adhesion Molecules; Cytokines; Dimethyl Fumarate; E-Selectin; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Fumarates; Gene Expression Regulation; Humans; Intercellular Adhesion Molecule-1; Interleukins; Lipopolysaccharides; Psoriasis; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha; Umbilical Cord; Vascular Cell Adhesion Molecule-1

Inactivation of the enzyme L-aspartase from Escherichia coli by the substrate analog aspartate beta-semialdehyde has previously been shown to occur by the mechanism-based conversion to the corresponding product aldehyde, followed by covalent modification of cysteine-273 (F. Giorgianni et al. (1995) Biochemistry 34, 3529). Inactivation by the product analog, fumaric acid aldehyde (FAA), has now been examined directly by adding a reduction step to the modification protocol in order to stabilize the resulting enzyme-FAA derivative(s). HPLC and mass spectrometric analyses of proteolytic digests of inactivated L-aspartase have confirmed the modification at cysteine-273, and have also identified an additional modified peptide. The inactivation at this additional site involves a crosslink between cysteine-140 and an adjacent lysine. Site-directed mutagenesis studies have shown that cysteine-140 is a very reactive and accessible nucleophile that is not, however, directly involved in enzyme activity. The adjacent lysine-139 that is modified does appear to play a role in substrate binding. A double mutant in which both of the reactive cysteines have been replaced is almost completely insensitive to modification by these substrate and product analogs.

Topics: Aspartate Ammonia-Lyase; Aspartic Acid; Bacterial Proteins; Chromatography, High Pressure Liquid; Cysteine; Deamination; Enzyme Inhibitors; Escherichia coli; Fumarates; Lysine; Mutagenesis, Site-Directed; Peptide Fragments

Fruit juices and purees are defined as fermentable, but unfermented, products obtained by mechanical processing of fresh fruits. The presence of undesired metabolites derived from microbial growth can arise from the use of unsuitable fruit or from defects in the production line or subsequent contamination. This involves a loss in the overall quality that cannot be resolved by thermal treatment following the start of fermentation. With these considerations, together with microbiological control, the analysis of different metabolites, which can be considered as microbial growth markers, such as alcohols (i.e. ethanol, etc.), acids (i.e. acetic, fumaric, lactic, etc.) is fundamental in order to achieve a better evaluation of product quality. Enzymatic determination and other single-component analytical techniques are often used for the determination of these metabolites. When the microbial spoilage is not well known, this results in a long and cumbersome procedure. A versatile technique that is capable of determining many metabolites in one analysis could be helpful in improving routine quality control. For this purpose, an ion chromatographic technique, such as ion exclusion, for separation, and diode array spectrophotometry and conductivity, for detection, were evaluated. Both different industrial samples and inoculated samples were analyzed.

Topics: Acetic Acid; Beverages; Chromatography, Ion Exchange; Electric Conductivity; Food Microbiology; Food-Processing Industry; Fruit; Fumarates; Lactic Acid; Reproducibility of Results; Sensitivity and Specificity; Spectrophotometry, Ultraviolet

An anti-thrombin substance (M2) was isolated from a culture broth of Rhizopus javanicus. Accumulation of M2 reached a maximum peak after 14 to 15 days of incubation and then decreased. The yield of M2 was 500 mg from 1 L of culture broth. M2 inhibited thrombin activity, and its 50% inhibition concentration in a reaction mixture containing 50 microL of 12.5 NIH unit/mL thrombin and 200 microL of 0.33% bovine fibrinogen was 63 microM. M2 had a specific activity for thrombin, but it was less responsive to plasmin, tissue-type plasminogen activator, urokinase, plasma kallikrein and glandular kallikrein. The structure of M2 was identified as fumaric acid by elementary analysis, FAB/MS, 1H-NMR, 13C-NMR and IR spectra.

Topics: Animals; Antithrombins; Blood Coagulation Tests; Cattle; Culture Media; Fumarates; Magnetic Resonance Spectroscopy; Mass Spectrometry; Rhizopus; Spectrophotometry, Infrared

A previous report from our laboratory (Collier et al 1993) showed that the elongated tubules of mitochondria in the cytoplasm of cultured chicken embryo fibroblasts collapsed to irregularly shaped structures surrounding the nuclear membrane after a 1 h heat shock treatment. The normal mitochondrial morphology reappeared upon removal of the thermal stress. We have now determined that several changes occurred in mitochondrial-related metabolites under these same heat shock and recovery conditions. Among these were significant decreases in the levels of fumarate and malate and increases in the amounts of aspartate and glutamate. In contrast, other intermediates of the tri-carboxylic acid cycle were unaltered as were levels of ATP and phosphocreatine. The changes observed might result from heat shock-induced changes in enzyme activities of the mitochondria, from alterations in the membrane-embedded specialized carrier proteins that transport metabolites between cytosol and mitochondria or from a disorganization of the electron-transport system normally coupled to oxidative metabolism. The rapid recovery, however, suggested that these changes were transient and readily reversible.

Topics: Adenosine Triphosphate; Animals; Aspartic Acid; Cells, Cultured; Chick Embryo; Citric Acid; Energy Metabolism; Fibroblasts; Fumarates; Glutamic Acid; Glycerophosphates; Glycolysis; Hot Temperature; Ketoglutaric Acids; Malates; Mitochondria; Phosphocreatine; Pyruvates; Stress, Physiological

In a metabolic trial 4 groups of 8 piglets of 5 kg weight each were kept individually for 45 days (final weight 23 kg) and fed a practical diet. At the beginning of the experiment the body amino acid contents of an additional group of 8 piglets were determined by carcass analysis, and at the end of the experiment the body amino acid contents of the 4 test group piglets (A = control fed ad libitum, B and C = supplement of 1.5% fumaric acid fed ad libitum or restrictively, D = supplement of 1.5% citric acid fed ad libitum) were also analysed. The amino acid retention during the experimental period was determined by difference. The supplements of fumaric or citric acid did not influence the amount of the amino acid retention. The quotient of amino acid retention to amino acid consumed or the "productive amino acid value" was calculated and the maintenance requirements of essential amino acids for piglets were used to estimate the productive amino acid value for both retention and maintenance. The mean amino acid retention amounted to about 56 g/d, i.e. 3.49 g/kg W0.75.d of essential amino acids. The essential amino acid requirements for maintenance was 2.0 g, i.e. 0.29 g/kg W0.75.d, showing a variation of 4% (Leu) to 20% (Met+Cys) when related to the amount of the corresponding amino acid retention. With regard to the amino acid pattern for retention of the nutritionally most important amino acids, the following ratios were found: Lys, 100 (6.27 g/16 g N): Met+Cys, 48 (3.03 g): Thr, 56 (3.49 g): Trp, 13 (0.80 g). The productive amino acid values ranged from 40% (Trp), 55% (Thr), 66% (Met) to 80% (Lys). Under the conditions investigated, neither the supplements of organic acids nor the feed restriction influenced the amino acid utilization.

Topics: Amino Acids; Animals; Citric Acid; Diet; Eating; Food, Fortified; Fumarates; Male; Swine; Time Factors; Weight Gain

Saccharomyces cerevisiae accumulates L-malic acid through a cytosolic pathway starting from pyruvic acid and involving the enzymes pyruvate carboxylase and malate dehydrogenase. In the present study, the role of malate dehydrogenase in the cytosolic pathway was studied. Overexpression of cytosolic malate dehydrogenase (MDH2) under either the strong inducible GAL10 or the constitutive PGK promoter causes a 6- to 16-fold increase in cytosolic MDH activity in growth and production media and up to 3.7-fold increase in L-malic acid accumulation in the production medium. The high apparent Km of MDH2 for L-malic acid (11.8 mM) indicates a low affinity of the enzyme for this acid, which is consistent with the cytosolic function in the enzyme and differs from the previously published Km of the mitochondrial enzyme (MDH1, 0.28 mM). Under conditions of MDH2 overexpression, pyruvate carboxylase appears to be a limiting factor, thus providing a system for further metabolic engineering of L-malic acid production. The overexpression of MDH2 activity also causes an evaluation in the accumulation of fumaric acid and citric acid. Accumulation of fumaric acid is presumably caused by high intracellular L-malic acid concentrations and the activity of the cytosolic fumarase. The accumulation of citric acid may suggest the intriguing possibility that cytosolic L-malic acid is a direct precursor of citric acid in yeast.

Topics: Citric Acid; Cytosol; Fumarates; Kinetics; Malate Dehydrogenase; Malates; Saccharomyces cerevisiae

Changes in the active site of fumarase (yeast fumarase II) that occur when fumarate is converted to malate (E.F --> E.M) must be reversed for another cycle of reaction to take place. As shown here, recycling of the enzyme includes two proton transfers and one conformational change. These events, together with the M-off step, are variously rate-determining depending on the medium. In very low salt the release of M is limited by the conformational change. Thus, (V/Km)F decreases with increased viscosity, shown with glycerol. A variety of simple anions, such as Cl- at approximately 50 mM and F itself at low concentration, activate the dissociation of M. This nonspecific anion effect is the basis for the >4-fold apparent cooperative activation by substrate. The M-off step and the conformational change are independent and random-order events. Thus, even when M-off is made rapid the rate of recycling is inhibited by glycerol, which in 100 mM NaCl inhibits Vmax but not V/Km. The enzyme form that results when M is released is M-specific, Em. Thus mesotartarate, competitive toward M, is noncompetitive toward F. The slow conformational change required for recycling of Em is activated by Pi and chaotropic anions such as azide and thiocyanate, giving rise to a nonspecific intermediate, Emf (mesotartarate becomes competitive toward F and Britton's countertransport property disappears with these activators). Evidence is presented for the locations and rates of the two proton transfer steps required to complete the cycle.

Topics: Anions; Binding Sites; Buffers; Enzyme Activation; Fumarate Hydratase; Fumarates; Hydrogen-Ion Concentration; Kinetics; Malates; Organophosphonates; Phosphates; Sodium Chloride; Tartrates

A partially saturated linear polyester based on poly(propylene fumarate) (PPF) was synthesized for potential application in filling skeletal defects. The synthesis was carried out according to a two-step reaction scheme. Propylene glycol and fumaryl chloride were first combined to form an intermediate fumaric diester. The intermediate was then subjected to a transesterification to form the PPF-based polymer. This method allowed for production of a polymer with a number average molecular weight up to 1500 and a polydispersity index of 2.8 and below. The polymeric backbone structure was investigated through the use of FTIR and NMR. Kinetic studies of the transesterification allowed mapping of the molecular weight increase with reaction time. The final product was also characterized by thermal and solubility analysis.

Topics: Bone Cements; Calorimetry, Differential Scanning; Esterification; Fumarates; Magnetic Resonance Spectroscopy; Molecular Weight; Polypropylenes; Propylene Glycol; Solubility; Spectrometry, Mass, Fast Atom Bombardment; Spectroscopy, Fourier Transform Infrared

A transposon mutant, designated CMTn-3, of Shewanella putrefaciens MR-1 that was deficient in fumarate reduction was isolated and characterized. In contrast to the wild-type, CMTn-3 could not grow anaerobically with fumarate as the electron acceptor, and it lacked benzyl viologen-linked fumarate reductase activity. Consistent with this, CMTn-3 lacked a 65 kDa c-type cytochrome, which is the same size as the fumarate reductase enzyme. CMTn-3 retained the wild-type ability to use nitrate, iron(III), manganese(IV) and trimethylamine N-oxide (TMAO) as terminal electron acceptors. The results indicate that the loss of the fumarate reductase enzyme does not affect other anaerobic electron transport systems in this bacterium.

Topics: Base Sequence; DNA Primers; DNA Transposable Elements; Electron Transport; Fumarates; Genes, Bacterial; Gram-Negative Facultatively Anaerobic Rods; Mutagenesis, Insertional; Mutation; Subcellular Fractions; Succinate Dehydrogenase

The ortho-cleavage pathways of catechol and 3-chlorocatechol are central catabolic pathways of Pseudomonas putida that convert aromatic and chloroaromatic compounds to tricarboxylic acid (TCA) cycle intermediates. They are encoded by the evolutionarily related catBCA and clcABD operons, respectively. Expression of the cat and clc operons requires the LysR-type transcriptional activators CatR and ClcR, respectively, and the inducer molecules cis,cis-muconate and 2-chloro-cis,cis-muconate, respectively. The regulation of the cat and clc promoters has been well studied, but the extent to which these operons are repressed by growth in TCA cycle intermediates has not been explored. We demonstrate by transcriptional fusion studies that the expression from the clc promoter is repressed when the cells are grown on succinate, citrate, or fumarate and that this repression is ClcR dependent and occurs at the transcriptional level. The presence of these organic acids did not affect the expression from the cat promoter. In vitro transcription assays demonstrate that the TCA cycle intermediate fumarate directly and specifically inhibits the formation of the clcA transcript. No such inhibition was observed when CatR was used as the activator on either the cat or clc template. Titration studies of fumarate and 2-chloromuconate show that the fumarate effect is concentration dependent and reversible, indicating that fumarate and 2-chloromuconate most probably compete for the same binding site on ClcR. This is an interesting example of the transcriptional regulation of a biodegradative pathway by the intracellular sensing of the state of the TCA cycle.

Topics: Bacterial Proteins; Biodegradation, Environmental; Catechols; Chlorophenols; Citric Acid Cycle; Enzyme Repression; Fumarates; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Maleates; Operon; Pseudomonas putida; Sorbic Acid; Trans-Activators; Transcription, Genetic

Regulation of the katB catalase gene in the anaerobic bacterium Bacteroides fragilis was studied. Northern blot hybridization analyses revealed that katB was transcribed as an approximately 1.6-kb monocistronic mRNA. The levels of katB mRNA increased > 15-fold when anaerobic, mid-logarithmic-phase cultures were exposed to O2, O2 with paraquat, or hydrogen peroxide. Under anaerobic conditions, the low levels of katB mRNA increased in a growth-dependent manner, reaching maximum expression at late logarithmic or early stationary phase, followed by a decrease in stationary phase. Under anaerobic conditions, the expression of katB mRNA was strongly repressed by glucose and to a lesser extent by xylose. However, glucose repression was completely abolished upon exposure to oxygen. The nonfermentable carbon sources fumarate, succinate, acetate, and pyruvate did not significantly affect expression. Phosphate, nitrogen, and hemin limitation did not affect the expression of katB mRNA, suggesting that the nutritional control of katB expression is restricted to carbon and energy sources and not other forms of nutrient limitation. Primer extension analysis revealed that during both oxidative stress and carbon or energy limitation, katB utilized the same promoter region but transcription initiation occurred at two different nucleotides separated by 3 or 4 bases. Interestingly, a 6-bp inverted repeat sequence present in the katB regulatory region was also observed upstream of the B. fragilis superoxide dismutase gene sod. It is possible that this is a recognition site for a DNA binding protein involved in the regulation of oxidative stress genes in this organism.

Topics: Acetates; Anaerobiosis; Bacteroides fragilis; Base Sequence; Carbon; Catalase; Fumarates; Gene Expression Regulation, Bacterial; Glucose; Hemin; Herbicides; Hydrogen Peroxide; Kinesins; Molecular Sequence Data; Nitrogen; Oxidative Stress; Oxygen; Paraquat; Phosphates; Promoter Regions, Genetic; Pyruvic Acid; RNA, Messenger; S Phase; Succinic Acid; Superoxide Dismutase; Transcription, Genetic; Xylose

The protein encoded by the ACR1 gene in Saccharomyces cerevisiae belongs to a family of 35 related membrane proteins that are encoded in the fungal genome. Some of them are known to transport various substrates and products across the inner membranes of mitochondria, but the functions of 28 members of the family are unknown. The yeast ACR1 gene was introduced into Escherichia coli on an expression plasmid. The protein was over-produced as inclusion bodies, which were purified and solubilised in the presence of sarkosyl. The solubilised protein was reconstituted into liposomes and shown to transport fumarate and succinate. Its physiological role in S. cerevisiae is probably to transport cytoplasmic succinate, derived from isocitrate by the action of isocitrate lyase in the cytosol, into the mitochondrial matrix in exchange for fumarate. This exchange activity and the subsequent conversion of fumarate to oxaloacetate in the cytosol would be essential for the growth of S. cerevisiae on ethanol or acetate as the sole carbon source.

Topics: Acetates; Carrier Proteins; Escherichia coli; Ethanol; Fumarates; Fungal Proteins; Genetic Vectors; Kinetics; Recombinant Fusion Proteins; Saccharomyces cerevisiae; Substrate Specificity; Succinic Acid

A procedure in which anionic analytes, trapped on ion exchange resin, are simultaneously methylated and released using methyl iodide in either supercritical carbon dioxide or acetonitrile has been extended to polyfunctional organic acids. The combined SFE methylation of fruit juice acids trapped onto ion exchange resin proceeds in good yield producing the methyl esters of fumaric, succinic, malic, tartaric, isocitric and citric acids which are readily separated by GC. Using this procedure low concentrations of one acid can be detected and quantitated in the presence of very high concentrations of another. This new method detects tartaric acid at levels of 10 ppm in juices containing 10,000 ppm citric acid. Quantitation was performed either by using GC-FID with triethyl citrate or diethyl tartrate as internal standards or with the element specific calibration capability of the GC-AED. A simple new technique for the determination of citric/isocitric acid ratio is now available. Also, in contrast to HPLC methods, the identity of an analyte is readily confirmed by GC-MS.

Topics: Beverages; Citric Acid; Fruit; Fumarates; Gas Chromatography-Mass Spectrometry; Hydrogen-Ion Concentration; Isocitrates; Malates; Methylation; Succinic Acid; Tartrates

Although schistosomes were thought to be one of the few parasitic helminths that do not produce succinate via fumarate reduction, it was recently demonstrated that sporocysts of Schistosoma mansoni produce, under certain conditions, succinate in addition to lactate. This succinate production was only observed when the respiratory chain activity of the sporocysts was inhibited, which suggested that succinate is produced by fumarate reduction. In this report the presence of essential components for fumarate reduction was investigated in various stages of S. mansoni and it was shown that, in contrast to adults, sporocysts contained a substantial amount of rhodoquinone which is essential for efficient fumarate reduction in eukaryotes. This rhodoquinone was not made by modification of ubiquinone obtained from the host, but was synthesized de novo. Furthermore, it was shown that complex II of the electron-transport chain in schistosomes has the kinetic properties of a dedicated fumarate reductase instead of those of a succinate dehydrogenase. The presence of such an enzyme, together with the substantial amounts of rhodoquinone, shows that in S. mansoni sporocysts succinate is produced via fumarate reduction. Therefore, the energy metabolism of schistosomes does not differ in principle from most other parasitic helminths, which are known to rely heavily on fumarate reduction.

Topics: Animals; Fumarates; Oxidation-Reduction; Schistosoma mansoni; Succinate Dehydrogenase; Succinic Acid; Ubiquinone

A method for the determination of fumaric acid in Sarcandra glabra is reported. After the sample was pretreated with an anion exchange resin column, fumaric acid was determined on a Spherisorb C18 column, with methanol-0.02 mol/L monobasic potassium (20:80, pH2.5) as the mobile phase and detected at 210 nm. The method is simple and accurate.

Topics: Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Fumarates; Magnoliopsida; Plants, Medicinal

Fumarase from the syntrophic propionate-oxidizing bacterium strain MPOB was purified 130-fold under anoxic conditions. The native enzyme had an apparent molecular mass of 114 kDa and was composed of two subunits of 60 kDa. The enzyme exhibited maximum activity at pH 8.5 and approximately 54 degrees C. The Km values for fumarate and L-malate were 0.25 mM and 2.38 mM, respectively. Fumarase was inactivated by oxygen, but the activity could be restored by addition of Fe2+ and β-mercaptoethanol under anoxic conditions. EPR spectroscopy of the purified enzyme revealed the presence of a [3Fe-4S] cluster. Under reducing conditions, only a trace amount of a [4Fe-4S] cluster was detected. Addition of fumarate resulted in a significant increase of this [4Fe-4S] signal. The N-terminal amino acid sequence showed similarity to the sequences of fumarase A and B of Escherichia coli (56%) and fumarase A of Salmonella typhimurium (63%).

Topics: Amino Acid Sequence; Anaerobiosis; Bacteria, Anaerobic; Electron Spin Resonance Spectroscopy; Escherichia coli; Fumarate Hydratase; Fumarates; Hydrogen-Ion Concentration; Kinetics; Malates; Molecular Sequence Data; Molecular Weight; Oxidation-Reduction; Oxygen; Propionates; Salmonella typhimurium; Temperature

The objective of this study was to evaluate the effect of extracellular H2 on organic acid utilization by two lactate-utilizing strains of Selenomonas ruminantium (HD4, H18). Both strains were able to grow (optical density at 600 nm > or = after 9 h) on either aspartate, fumarate, or malate in the presence of 1 atmosphere (atm) of H2. Succinate was the major end product produced in these fermentations. When cells were incubated with lactate plus 1 atm H2, growth was minimal little lactate was fermented. The electron transport inhibitor, acriflavine, was strong inhibitor of growth when either strain was incubated in the presence of organic acid plus H2. Compared with glucose- or lactate-grown cells, cellular carbohydrate levels were lower for both strains in cells grown on either organic acid plus H2. These results suggest that electron transport plays a role in organic acid utilization by S. ruminantium.

Topics: Animals; Aspartic Acid; Culture Media; Fermentation; Fumarates; Gram-Negative Bacteria; Growth Inhibitors; Hydrogen; Lactates; Lactic Acid; Malates; Rumen

Topics: Administration, Oral; Dermatologic Agents; Follow-Up Studies; Fumarates; Humans; Psoriasis; Tablets

Toluene is degraded anoxically to CO2 by the denitrifying bacterium Thauera aromatica. Toluene first becomes oxidized to benzoyl-CoA by O2-independent reactions. Benzoyl-CoA is then reduced to non-aromatic products by benzoyl-CoA reductase. We set out to study the reactions employed for the initial activation of toluene and its oxidation to the level of benzoate. Evidence is provided for a novel way of toluene degradation based on experiments with cell-free extracts and with whole toluene-grown cells: Cell-free extracts oxidized [14C]toluene to [14C]benzoyl-CoA via several radioactive intermediates. This reaction was strictly dependent on the presence of fumarate, coenzyme A and nitrate as electron acceptor; acetyl-CoA and ATP were not necessary for the reaction. The first product formed in vitro was benzylsuccinate; (2H8)toluene was converted to (2H7)benzylsuccinate. Formation of benzylsuccinate from toluene was independent of coenzyme A and nitrate, but it required the presence of fumarate. Other tricarboxylic acid cycle intermediates were converted to fumarate in cell extracts and therefore could partially substitute for fumarate. [14C]Benzylsuccinate was oxidized further to [14C]benzoyl-CoA and [14C]benzoate in cell extracts if coenzyme A and nitrate were present. No benzyl alcohol and benzaldehyde and no phenylpropionate could be detected as intermediates. In isotope trapping experiments with cell suspensions, two intermediates from [14C]toluene were detected, benzoate and benzylsuccinate. This corroborates the sequence of reactions deduced from in vitro experiments. A hypothetical degradation pathway for the anaerobic oxidation of toluene to benzoyl-CoA via an initial addition of fumarate to the methyl group of toluene and following beta-oxidation of the benzylsuccinate formed is suggested.

Topics: Acyl Coenzyme A; Benzoates; Benzoic Acid; Carbon Radioisotopes; Cell Extracts; Fumarates; Gram-Negative Anaerobic Bacteria; Kinetics; Oxidation-Reduction; Succinates; Toluene

Fumaric acid, fumaric acid dimethylester, and the dithranol derivative C4-lactone were studied in the mouse tail test to evaluate their effects on epidermal cell differentiation compared with other topical antipsoriatic drugs, such as betamethasone, calcipotriol, and dithranol. Mouse tails were treated for 2 weeks and longitudinal histological sections prepared of the tail skin. The length of the orthokeratotic regions (stratum granulosum) was measured on 10 sequential scales per tail and expressed as percentage of the full length of the scale. In addition, epidermal thickness was measured and the efficacy of the various compounds evaluated. In comparison to 2% salicylic acid ointment, all tested compounds except fumaric acid significantly (p < or = 0.05) increased the proportion of the orthokeratotic region. C4-lactone and calcipotriol were less effective than dithranol, fumaric acid dimethylester only moderately influenced cell differentiation, and betamethasone showed the least potent effect. Dithranol was the most potent substance inducing orthokeratosis without increasing epidermal thickness.

Topics: Administration, Topical; Animals; Anti-Inflammatory Agents; Anticarcinogenic Agents; Cell Differentiation; Epidermal Cells; Epidermis; Fumarates; Keratosis; Male; Mice; Psoriasis; Tail

Topics: Animals; Deuterium; Fumarates; Hydrogen Peroxide; Liver; Oxidation-Reduction; Rats; Succinates; Succinic Acid

Fumarate restores to flagella of cytoplasm-free, Che Y-containing envelopes of Escherichia coli and Salmonella typhimurium the ability to switch from one direction of rotation to another. To examine the specificity of this effect, we studied flagellar rotation of envelopes which contained, instead of fumarate, one of its analogues. Malate, maleate and succinate promoted switching, but to a lesser extent than fumarate. These observations were made both with wild-type envelopes and with envelopes of a mutant which lacks the enzymes succinate dehydrogenase and fumarase, indicating that the switching-promoting activity of the analogues was not caused by their conversion to fumarate. Aspartate and lactate did not promote switching. Using strains defective in specific enzymes of the tricarboxylic acid cycle and lacking the cytoplasmic chemotaxis proteins as well as some of the chemotaxis receptors, we demonstrated that, in intact bacteria, unlike the situation in envelopes, fumarate promoted clockwise rotation via its metabolites acetyl phosphate and acetyladenylate, but did not promote switching (presumably because of the presence of cytoplasmic fumarate). All of the results are consistent with the notion that fumarate acts as a switching factor, presumably by lowering the activation energy of switching. Thus fumarate and some of its metabolites may serve as a connection point between the bacterial metabolic state and chemotactic behaviour.

Topics: Aspartic Acid; Bacterial Proteins; Chemotaxis; Escherichia coli; Flagella; Fumarates; Lactic Acid; Malates; Maleates; Membrane Proteins; Methyl-Accepting Chemotaxis Proteins; Rotation; Salmonella typhimurium; Succinates; Succinic Acid

The interactions of reductants with dopamine beta-monooxygenase (DbetaM) were examined using two novel classes of reductants. The steady-state kinetics of the previously characterized DbetaM reductant, N,N-dimethyl-1,4-p-phenylenediamine (DMPD), were parallel to the ascorbic acid-supported reaction with respect to pH dependence and fumarate activation. DMPD also displayed pH and fumarate-dependent apparent negative cooperativity demonstrating that the previously reported cooperative behavior of DbetaM toward the reductant is not unique to ascorbic acid. The 6-OH phenyl and alkylphenyl-substituted ascorbic acid derivatives were more efficient reductants for the enzyme than ascorbic acid. Kinetic studies suggested that these derivatives behave as pseudo bisubstrates with respect to ascorbic acid and the amine substrate. The lack of apparent cooperative behavior with these derivatives suggests that this behavior of DbetaM is not common for all the reductants. Based on these findings and additional kinetic evidence, the proposal that the apparent negative cooperativity in the interaction of ascorbic acid with DbetaM was due to the presence of a distinct allosteric regulatory site has been ruled out. In contrast to previous models, where fumarate was proposed to interact with a distinct anion binding site, the effect of fumarate on the steady-state kinetics of these novel reductants suggests that fumarate and the reductant may interact with the same site of the enzyme. In accordance with these observations and mathematical analysis of the experimental data, a unified model for the apparent negative cooperativity and fumarate activation of DbetaM in which both fumarate and the reductant interact with the same site of all forms of the enzyme with varying affinities under steady-state turnover conditions has been proposed.

Topics: Anticarcinogenic Agents; Chromatography, High Pressure Liquid; Dopamine beta-Hydroxylase; Enzyme Activation; Fumarates; Hydrogen-Ion Concentration; Kinetics; Models, Chemical; Phenylenediamines

Bacterial chemotaxis is based on modulation of the probability to switch the direction of flagellar rotation. Responses to many stimuli are transduced by a two-component system via reversible phosphorylation of CheY, a small cytoplasmic protein that directly interacts with the switch complex at the flagellar motor. We found that the chemorepellents indole and benzoate induce motor switching in Escherichia coli cells with a disabled phosphorylation cascade. This phosphorylation-independent chemoresponse is explained by reversible inhibition of fumarase by indole or benzoate which leads to an increased level of cellular fumarate, a compound involved in motor switching for bacteria and archaea. Genetic deletion of fumarase increased the intracellular concentration of fumarate and enhanced the switching frequency of the flagellar motors irrespective of the presence or absence of the phosphorylation cascade. These correlations provide evidence for fumarate-dependent metabolic signal transduction in bacterial chemosensing.

Topics: Bacterial Proteins; Benzoates; Benzoic Acid; Chemotaxis; Cytoplasm; Enzyme Inhibitors; Escherichia coli; Escherichia coli Proteins; Fumarate Hydratase; Fumarates; Gene Deletion; Indoles; Membrane Proteins; Methyl-Accepting Chemotaxis Proteins; Oxidation-Reduction; Phosphorylation; Signal Transduction

Topics: Anticarcinogenic Agents; Fumarates; Humans; Psoriasis; Treatment Outcome

The human brain contains a cytosolic and mitochondrial form of NADP(+)-dependent malic enzyme. To investigate their possible metabolic roles we compared the regulatory properties of these two iso-enzymes. The mitochondrial malic enzyme exhibited a sigmoid substrate saturation curve at low malate concentration which was shifted to the right at both higher pH values and in the presence of low concentration of Mn2+ or Mg2+. Succinate or fumarate increased the activity of the mitochondrial malic enzyme at low malate concentration. Both activators shifted the plot of reaction velocity versus malate concentration to the left, and removed sigmoidicity, but the maximum velocity was unaffected. The activation was associated with a decrease in Hill coefficient from 2.3 to 1.1. The human brain cytosolic malic enzyme displayed a hyperbolic substrate saturation kinetics and no sigmoidicity was detected even at high pH and low malate concentrations. Succinate or fumarate exerted no effect on the enzyme activity. Excess of malate inhibited the oxidative decarboxylation catalysed by cytosolic enzyme at pH 7.0 and below. In contrast, decarboxylation catalysed by mitochondrial malic enzyme, was unaffected by the substrate. These results suggest that under in vivo conditions, cytosolic malic enzyme catalyses both oxidative decarboxylation of malate and reductive carboxylation of pyruvate, whereas the role of mitochondrial enzyme is limited to decarboxylation of malate. One may speculate that in vivo the reaction catalysed by cytosolic malic enzyme supplies dicarboxylic acids (anaplerotic function) for the formation of neurotransmitters, while the mitochondrial enzyme regulates the flux rate via Krebs cycle by disposition of the tricarboxylic acid cycle intermediates (cataplerotic function).

Topics: Brain; Cytoplasm; Fumarates; Humans; Hydrogen-Ion Concentration; Magnesium Chloride; Malate Dehydrogenase; Malates; Mitochondria; NADP; Succinates; Succinic Acid

Topics: Campylobacter; Colloids; Energy Metabolism; Fumarates; Humans; Hydrogen; Mouth; Oxidation-Reduction; Sulfur

Malate dehydrogenase catalyzes the interconversion of malate and oxaloacetate. It participates as a member of the tricarboxylic acid cycle and the branched noncyclic pathways under aerobic and anaerobic cell growth conditions, respectively. To investigate how the mdh gene is expressed under these different conditions, an mdh-lacZ operon fusion was constructed and analyzed in vivo. The mdh-lacZ fusion was expressed about twofold higher under aerobic conditions than under anaerobic cell growth conditions on most media tested. This anaerobic response is modulated by the ArcA protein, which functions as a repressor of mdh gene expression under both aerobic and anaerobic conditions. In contrast, mutations in the fnr gene did not affect mdh gene expression. Interestingly, cells grown anaerobically with glycerol and trimethylamine N-oxide or fumarate showed higher levels of mdh expression than did cells that were grown aerobically. Depending on the type of carbon compound used for cell growth, mdh expression varied by 11-fold and 5-fold under aerobic and anaerobic conditions, respectively. While mdh transcription was shown to be inversely proportional to the cell growth rate, cellular heme limitation stimulated a fivefold increase in mdh gene expression. The mdh gene appears to be highly regulated to adapt to changing conditions of aerobic and anaerobic cell growth with various types of carbon substrates.

Topics: 2,2'-Dipyridyl; Aerobiosis; Anaerobiosis; Bacterial Outer Membrane Proteins; Bacterial Proteins; Carbon; Culture Media; Escherichia coli; Escherichia coli Proteins; Fumarates; Gene Expression Regulation, Bacterial; Glycerol; Heme; Iron Chelating Agents; Iron-Sulfur Proteins; Malate Dehydrogenase; Methylamines; Oxidants; Oxygen; Recombinant Fusion Proteins; Repressor Proteins

Topics: Administration, Cutaneous; Administration, Oral; Dimethyl Fumarate; Fumarates; Humans; Psoriasis

The fumarate reductase (FR) and succinate dehydrogenase (SDH) activities of isolated submitochondrial particles (SMPs) prepared from axenised L3 larvae of S. ratti were characterised with respect to their response to a selected range of inhibitors. Rotenone (a specific inhibitor of electron transport Complex I) inhibited the S. ratti FR (EC50 = 3.0 x 10(-7) M) but not SDH. This strongly suggests that the S. ratti FR is functionally linked with the S. ratti ET-Complex I. 2-Thenoyltrifluoroacetone (TTFA, an inhibitor of ET-Complex II) inhibited FR (EC50 = 2.6 x 10(-5) M) and SDH (EC50 = 2.8 x 10(-5) M) with similar effectiveness. Sodium malonate (substrate analogue of succinate) had a greater affinity for SDH (EC50 = 6.8 x 10(-4) M), than FR (EC50 = 1.9 x 10(-2) M). Sodium fumarate was ca. 8-fold more effective in inhibiting the S. ratti FR (EC50 = 6.0 x 10(-4) M) than SDH (EC50 = 4.8 x 10(-3) M). The S. ratti FR was more sensitive to inhibition by thiabendazole (TBZ; EC50 = 4.6 x 10(-4) M) than SDH (EC50 > 1.0 x 10(-3) M), suggesting that one of the sites-of-action of TBZ to be the FR of S. ratti mitochondria. More potent inhibitors of S. ratti FR, if developed, may prove to be effective chemotherapeutic agents in the management of human strongloidiasis.

Topics: Animals; Electron Transport; Female; Fumarates; Kinetics; Larva; Malonates; Rats; Rats, Sprague-Dawley; Rotenone; Sensitivity and Specificity; Strongyloides ratti; Submitochondrial Particles; Succinate Dehydrogenase; Thenoyltrifluoroacetone; Thiabendazole

There are two stereochemical classes of hydratase-dehydratase enzymes. Those that catalyze the addition of water to alpha, beta-unsaturated thioesters give syn addition-elimination stereochemistry, whereas those that catalyze the addition of water to conjugated carboxylate substrates give anti stereochemistry. This dichotomy could reflect different adaptive advantages or contingencies of separate evolutionary histories. Determination of the nonenzymatic stereochemistry of deuterium oxide addition to fumarate and to S-crotonyl N-acetylcysteamine has provided direct evidence for the importance of the contingencies of evolutionary history, rather than chemical efficiency, in the pathways of these hydratase-dehydratase enzymes.

Topics: Biological Evolution; Catalysis; Cysteamine; Deuterium Oxide; Enoyl-CoA Hydratase; Fumarate Hydratase; Fumarates; Hydro-Lyases; Hydrolysis; Molecular Conformation; Temperature

A novel poly(ether urethane amide) was synthesized by chain-extending the prepolymer with fumaric acid, resulting in a polymer exhibiting superior mechanical properties and containing chemically active derivatization sites, stemming from the fumaramide double bond. The derivatization of the active sites was performed via a Michael-like addition of amine-terminated poly(ethylene oxide) chains, which when performed in bulk, greatly affected the mechanical properties of the polymer, except in the case in which a relatively high molecular weight was grafted. Surface grafting of the polymer led to a significantly lower effect on its mechanical properties, which was minimal when a high molecular weight molecule was used.

Topics: Absorption; Amides; Cross-Linking Reagents; Fumarates; Molecular Weight; Polyethylene Glycols; Polyurethanes; Spectroscopy, Fourier Transform Infrared; Thermography

An acid-base chemical mechanism is proposed for Hafnia alvei aspartase in which a proton is abstracted from C-3 of the monoanionic form of L-aspartate by an enzyme general base with a pK of 6.3-6.6 in the absence and presence of Mg2+. The resulting carbanion is presumably stabilized by delocalization of electrons into the beta-carboxyl with the assistance of a protonated enzyme group in the vicinity of the beta-carboxyl. Ammonia is then expelled with the assistance of a general acid group that traps an initially expelled NH3 as the final NH4+ product. In agreement with the function of the general acid group, potassium, an analog of NH4+, binds optimally when the group is unprotonated. The pK for the general acid is about 7 in the absence of Mg2+, but is increased by about a pH unit in the presence of Mg2+. Since the same pK values are observed in the pKi(succinate) and V/K pH profile, both enzyme groups must be in their optimum protonation state for efficient binding of reactant in the presence of Mg2+. At the end of a catalytic cycle, both the general base and general acid groups are in a protonation state opposite that in which they started when aspartate was bound. The presence of Mg2+ causes a pH-dependent activation of aspartase exhibited as a partial change in the V and V/Kasp pH profiles. When the aspartase reaction is run in D2O to greater than 50% completion no deuterium is found in the remaining aspartate, indicating that the site is inaccessible to solvent during the catalytic cycle.

Topics: Acid-Base Equilibrium; Aspartate Ammonia-Lyase; Aspartic Acid; Enterobacteriaceae; Escherichia coli; Fumarates; Hydrogen-Ion Concentration; Kinetics; Magnesium; Models, Chemical; Succinates; Succinic Acid

Shewanella putrefaciens 200 is a nonfermentative bacterium that is capable of dehalogenating tetrachloromethane to chloroform and other, unidentified products under anaerobic conditions. Since S. putrefaciens 200 can respire anaerobically by using a variety of terminal electron acceptors, including NO3-, NO2-, and Fe(III), it provides a unique opportunity to study the competitive effects of different electron acceptors on dehalogenation in a single organism. The results of batch studies showed that dehalogenation of CT by S. putrefaciens 200 was inhibited by O2, 10 mM NO3-, and 3 mM NO2-, but not by 15 mM Fe(III), 15 mM fumarate, or 15 mM trimethylamine oxide. Using measured O2, Fe(III), NO2-, and NO3- reduction rates, we developed a speculative model of electron transport to explain inhibition patterns on the basis of (i) the kinetics of electron transfer at branch points in the electron transport chain, and (ii) possible direct inhibition by nitrogen oxides. In additional experiments in which we used 20 mM lactate, 20 mM glucose, 20 mM glycerol, 20 mM pyruvate, or 20 mM formate as the electron donor, dehalogenation rates were independent of the electron donor used. The results of other experiments suggested that sufficient quantities of endogenous substrates were present to support transformation of tetrachloromethane even in the absence of an exogenous electron donor. Our results should be significant for evaluating (i) the bioremediation potential at sites contaminated with both halogenated organic compounds and nitrogen oxides, and (ii) the bioremediation potential of iron-reducing bacteria at contaminated locations containing significant amounts of iron-bearing minerals.

Topics: Carbon Tetrachloride; Electron Transport; Fumarates; Gram-Negative Facultatively Anaerobic Rods; Halogens; Methylamines; Models, Biological; Oxidation-Reduction; Oxygen

Nine moderately alkalitolerant thermophilic bacteria with similar properties were isolated from water and soil samples obtained from Yellowstone National Park. These Gram-type-positive, rod-shaped bacteria produce cells with primary branches. The cells are peritrichous and exhibit only slight tumbling motility. At 60 degrees C the pH range for growth is 6.9 to 10.3, and the optimum pH is 8.5. At pH 8.5 the temperature range for growth is 34 to 66 degrees C, with an optimum temperature of 57 degrees C. The strains are mainly proteolytic. The fermentation products from yeast extract are acetate, CO2, and H2. Fumarate added to minimal medium containing yeast extract is stoichiometrically converted to succinate, indicating that it is used as an alternative electron acceptor. The DNA G + C content is 33 to 34 mol%. On the basis of its unique properties, such as branch formation, growth at alkaline pH values at elevated temperatures, and the relative distance of its 16S rRNA sequence from those of other known bacteria, we propose that strain JW/YL-138T (T = type strain) and eight similar strains represent a new genus and species, Anaerobranca horikoshii. Strain JW/YL-138 is designated the type strain of the type species, A. horikoshii, which was named in honor of Koki Horikoshi, a pioneer in the field of alkaliphilic bacteria.

Topics: Aminopeptidases; Anaerobiosis; Base Composition; Biological Evolution; Culture Media; DNA, Bacterial; DNA, Ribosomal; Fermentation; Fumarates; Glycoside Hydrolases; Gram-Positive Rods; Hydrogen-Ion Concentration; Molecular Sequence Data; Phylogeny; RNA, Ribosomal, 16S; Soil Microbiology; Temperature; Water Microbiology

Shewanella putrefaciens MR-1 can grow either aerobically or anaerobically at the expense of many different electron acceptors and is often found in abundance at redox interfaces in nature. Such redox interfaces are often characterized by very strong gradients of electron acceptors resulting from rapid microbial metabolism. The coincidence of S. putrefaciens abundance with environmental gradients prompted an examination of the ability of MR-1 to sense and respond to electron acceptor gradients in the laboratory. In these experiments, taxis to the majority of the electron acceptors that S. putrefaciens utilizes for anaerobic growth was seen. All anaerobic electron acceptor taxis was eliminated by the presence of oxygen, nitrate, nitrite, elemental sulfur, or dimethyl sulfoxide, even though taxis to the latter was very weak and nitrate and nitrite respiration was normal in the presence of dimethyl sulfoxide. Studies with respiratory mutants of MR-1 revealed that several electron acceptors that could not be used for anaerobic growth nevertheless elicited normal anaerobic taxis. Mutant M56, which was unable to respire nitrite, showed normal taxis to nitrite, as well as the inhibition of taxis to other electron acceptors by nitrite. These results indicate that electron acceptor taxis in S. putrefaciens does not conform to the paradigm established for Escherichia coli and several other bacteria. Carbon chemo-taxis was also unusual in this organism: of all carbon compounds tested, the only positive response observed was to formate under anaerobic conditions.

Topics: Bacteria, Anaerobic; Carbon; Chemotaxis; Dimethyl Sulfoxide; Electron Transport; Environmental Microbiology; Fumarates; Methylamines; Nitrates; Nitrites; Oxidants; Oxidation-Reduction; Thiosulfates

The sorbitol fermentation by Actinomyces viscosus and Actinomyces naeslundii was studied with washed sorbitol-grown cells. The fermentation was followed by titration of acids produced at pH 7.0 under anaerobic conditions. Metabolic end-products and intracellular levels of NAD, NADH and glycolytic intermediates during the fermentation were also analyzed. Cell extracts were examined for certain enzyme activities. Bicarbonate was required for acid production from sorbitol and from a mixture of glucose and sorbitol. Malate and fumarate could also support the acid production of A. viscosus. The main end-products were succinate and lactate but not ethanol. Cell extracts showed no activities of alcohol and aldehyde dehydrogenases, but they had activities of malate dehydrogenase and fumarate reductase. In the absence of bicarbonate, malate or fumarate, the intracellular NADH/NAD ratio increased and the levels of 3- and 2-phosphoglycerate and phosphoenolpyruvate decreased. The results indicate that oral sorbitol-fermenting actinomyces lack the ethanol pathway that can contribute to NADH oxidation. To maintain intracellular redox balance during anaerobic sorbitol fermentation, these bacteria can oxidize surplus NADH through a succinate pathway.

Topics: Actinomyces; Actinomyces viscosus; Dental Plaque; Fermentation; Fumarates; Glucose; Glycolysis; Humans; Lactates; Maleates; NAD; Sodium Bicarbonate; Sorbitol; Succinates

Systemic administration of fumaric acid (FA) derivatives was originally an empirical antipsoriatic treatment, which showed promising clinical results. In the present study, FURA-2-loaded suspensions of cultured normal keratinocytes and SV40-transformed keratinocytes (SVK-14 cells) were used to study the effects of FA derivatives on the intracellular free calcium concentration ([Ca2+]i). Monomethylfumarate (MMF), dimethylfumarate (DMF) and monoethylfumarate (MEF) induced a rapid, transient [Ca2+]i increase in both cell types. This immediate increase reached maximal values of 396 nmol/l 10s after addition of MMF, and fell to basal values within 90-120 s (173 nmol/l for normal keratinocytes and 68 nmol/l for transformed keratinocytes). This increase was not affected by the prior addition of EGTA, indicating that FA derivatives released Ca2+ mainly from intracellular stores into the cytoplasm. Subsequently, dose-dependent inhibitory effects of FA derivatives on keratinocyte proliferation were demonstrated. The results of these experiments revealed that DMF was the most potent, MMF and MEF intermediate, and FA and malonic acid the least potent growth inhibitors. These antiproliferative effects of FA derivatives might be linked to the observed, transient [Ca2+]i elevations.

Topics: Anticarcinogenic Agents; Calcium; Cell Division; Cell Line, Transformed; Cells, Cultured; Depression, Chemical; Dimethyl Fumarate; Dose-Response Relationship, Drug; Fumarates; Humans; Intracellular Fluid; Keratinocytes; L-Lactate Dehydrogenase; Male; Maleates

Metabolism of [2-13C]-, [3-13C]-, and [1,2,3-13C]propionate in perfused rat livers and [2-13C]-acetate in perfused rat hearts has been examined in tissue extracts by 13C NMR. Label from [2-13C]-propionate was preferentially incorporated into the C2 carbon of lactate, alanine, and aspartate in liver tissue while label from [3-13C]propionate appeared preferentially in the C3 carbon of those same molecules. These data suggest that 13C may not be completely randomized in the symmetric citric acid cycle intermediates succinate and fumarate as is normally assumed but that some fraction of those intermediates may be transferred between enzymes in this span of the cycle with conservation of spatial orientation, consistent with recent results obtained in yeast [Sumegi et al. (1990) Biochemistry 29, 9106-9110]. This was confirmed by performing similar experiments with [1,2,3-13C]propionate. Time-dependent asymmetry was also observed between the intensities of the glutamate C2 and C3 resonances and between the aspartate C2 and C3 resonances in 13C NMR spectra of intact hearts and heart extracts during early perfusion with [2-13C]-acetate. A model is presented which predicts that isotopic asymmetry is observed only during the first 2-3 turns of the cycle pools when isotope enters the cycle via acetyl-CoA even if all symmetric cycle intermediates retain a unique molecular orientation on each pass through the citric acid cycle.

Topics: Acetates; Animals; Biological Transport; Carbon Isotopes; Citrates; Citric Acid; Citric Acid Cycle; Fumarates; Glutamates; Glutamic Acid; Lactates; Lactic Acid; Liver; Magnetic Resonance Spectroscopy; Myocardium; Propionates; Pyruvates; Pyruvic Acid; Rats; Succinates; Succinic Acid

A histological-immunohistological study was conducted to investigate the effect of systemically administered fumaric acid esters (FAEs) on epidermal thickness and composition of the inflammatory infiltrate in psoriatic plaques. The very first effect of systemic therapy with FAEs is the disappearance of CD 15-positive cells in the beneath the epidermis, accompanied by a significant reduction in T-helper cells beneath the epidermis, pointing to an immunosuppressive effect. This is followed after some delay by a reduction in acanthosis and hyperkeratosis. The reduction in infiltrating T-lymphocytes corresponds to that seen after systemic or intralesional therapy with cyclosporin. However, the normalization of the psoriatic plaques takes longer under the influence of FAEs than under cyclosporin.

Topics: Adult; Aged; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Esters; Female; Fumarates; Humans; Lewis X Antigen; Lymphocyte Subsets; Male; Middle Aged; Psoriasis; Skin

The nucleotide sequences of two Escherichia coli genes, dcuA and dcuB (formerly designated genA and genF), have been shown to encode highly homologous products, M(r) 45,751 and 47,935 (434 and 446 amino acid residues) with 36% sequence identity (63% similarity). These proteins have a high proportion (approximately 61%) of hydrophobic residues and are probably members of a new group of integral inner membrane proteins. The locations of the dcu genes, one upstream of the aspartase gene (dcuA-aspA) and the other downstream of the anaerobic fumarase gene (fumB-dcuB), suggested that they may function in the anaerobic transport of C4-dicarboxylic acids. Growth tests and transport studies with mutants containing insertionally inactivated chromosomal dcuA and dcuB genes show that their products perform analogous and mutually complementary roles as anaerobic dicarboxylate carriers. The anaerobic dicarboxylate transport systems (Dcu) are genetically and functionally distinct from the aerobic system (Dct).

Topics: Aerobiosis; Amino Acid Sequence; Anaerobiosis; Aspartic Acid; Bacterial Proteins; Base Sequence; Biological Transport; Carrier Proteins; Dicarboxylic Acid Transporters; Escherichia coli; Escherichia coli Proteins; Fumarates; Genes, Bacterial; Membrane Proteins; Molecular Sequence Data; Protein Structure, Secondary; Repressor Proteins; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Substrate Specificity; Succinates; Succinic Acid; Transcription Factors

Amino acid enrichment of cardioplegic solutions has been shown to improve both the metabolic and functional recovery of ischemic myocardium. However, because of the marked systemic vasodilatation involved, use of amino acid enrichment is limited to the periods of induction and reperfusion. Fumarate is a Krebs' cycle intermediate whose conversion to succinate is responsible for the generation of adenosone triphosphate and the oxidation of the reduced form of nicotinamide-adenine nucleotide which is the pathway by which aspartate exerts its effect. Fumarate may also function as a free-radical scavenger and is involved in calcium transport. To determine if fumarate-enriched blood cardioplegia would improve the functional recovery of the neonatal heart, 14 neonatal piglet hearts were isolated and placed on a blood-perfused working heart circuit. After the baseline functional and metabolic assessment was done, cold ischemic arrest was initiated with either standard blood cardioplegic solution (group I; N = 7) or fumarate-enriched (13 mmol/L) blood cardioplegic solution (group II; N = 7). Cardioplegic solution was given at a pressure of 40 mm Hg every 20 minutes for 2 hours, and topical hypothermia was used. Sixty minutes after warm whole blood reperfusion, the functional recovery at left atrial pressures of 3, 6, 9, and 12 mm Hg was 70%, 66%, 66%, and 65%, respectively, in group I, versus 102%, 106%, 105%, and 109%, respectively, in group II (p < 0.05). The tissue creatinine phosphate levels after reperfusion were significantly higher in group II hearts (15.0 +/- 1.2 mumol/g dry heart tissue) than in group I hearts (9.2 +/- 1.9 mumol/g dry heart tissue), although the adenosine triphosphate levels were not significantly different.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine Triphosphate; Animals; Animals, Newborn; Atrial Function; Blood; Blood Pressure; Cardiac Output; Cardioplegic Solutions; Fumarates; Heart; Heart Arrest, Induced; Hypothermia, Induced; Lactates; Myocardial Reperfusion; Myocardium; Phosphocreatine; Stroke Volume; Swine; Time Factors; Ventricular Function, Left

Transport activities for uptake, efflux and exchange of C4-dicarboxylates were observed in anaerobically grown Escherichia coli. All three transport modes were only present in strains containing the transcriptional activator FNR of anaerobic respiration, and were repressed by nitrate and O2. The kinetic and energetic parameters of C4-dicarboxylate transport and the mechanism of the uptake, efflux and exchange reactions were analyzed in whole cells and in membrane vesicles. Fumarate/succinate exchange could be characterized as homologous or heterologous 1:1 counter-exchange. The external substrate was determined as divalent fumarate2- (or succinate2-) at pH 6-9, whereas monovalent H-fumarate dominated as the substrate at pH 3-4. The exchange was not inhibited by dissipation of delta p or constituents of it (delta psi or delta pH). We conclude that this transport mode functions as an electroneutral exchange of C4-dicarboxylates. The uptake of C4-dicarboxylates did not depend on internal counter-substrate and resulted in an accumulation of the substrate. Similar to antiport, fumarate was accepted in the divalent form at pH values greater than or equal to 6 and in the monovalent form at pH 3.5-6. The uptake was inhibited by dissipation of delta p or delta psi. Artificially imposed delta pH, delta psi or fumarate gradients were able to drive fumarate uptake. An involvement of Na+ could not be detected. Thus the uptake is likely to operate as an electrophoretic H+/fumarate symport. Independent of the presence of an external counter-substrate, the substrates were secreted from cells or membrane vesicles loaded with succinate or fumarate. The efflux was electrogenic. Energizing the cells or membrane vesicles inhibited efflux, maximal efflux rates were obtained only after dissipation of delta p or delta psi. An imposed K(+)-diffusion potential (outside positive) inhibited succinate excretion. The efflux of succinate from de-energized membrane vesicles generated a delta psi of -70 mV. It is thus suggested that succinate efflux functions as a H+/succinate symport.

Topics: Anaerobiosis; Binding, Competitive; Biological Transport; Cell Membrane; Dicarboxylic Acids; Edetic Acid; Escherichia coli; Fumarates; Glycerol; Hydrogen-Ion Concentration; Kinetics; Lactose; Substrate Specificity; Succinates; Succinic Acid

Succinate dehydrogenase of the bacterial or inner mitochondrial membrane catalyses the oxidation of succinate to fumarate and directs reducing equivalents into the electron-transport chain. The enzyme is also able to catalyse the reverse reaction, the reduction of fumarate to succinate. The enzyme is composed of four subunits. These subunits include a catalytic dimer composed of a flavoprotein subunit with a covalently bound FAD, and an iron-sulfur protein subunit with three different iron-sulfur centres, which is anchored to the membrane by two smaller integral membrane proteins. The FAD moiety is attached to the flavoprotein subunit by an 8 alpha-[N(3)-histidyl]FAD linkage at a conserved histidine residue, His90 of the Saccharomyces cerevisiae succinate dehydrogenase. By mutating His90 to a serine residue, we have constructed a flavoprotein subunit that is unable to covalently bind FAD. The mutant flavoprotein is targeted to mitochondria, translocated across the mitochondrial membranes, and is assembled with the other subunits where it binds FAD non-covalently. The resulting holoenzyme has no succinate-dehydrogenase activity but retains fumarate reductase activity. The covalent attachment of FAD is therefore necessary for succinate oxidation but is dispensable for both fumarate reduction and for the import and assembly of the flavoprotein subunit.

Topics: Binding Sites; Electron Spin Resonance Spectroscopy; Electron Transport; Electrophoresis, Polyacrylamide Gel; Flavin-Adenine Dinucleotide; Flavoproteins; Fumarates; Iron-Sulfur Proteins; Mitochondria; Mutagenesis, Site-Directed; Oxidation-Reduction; Saccharomyces cerevisiae; Succinate Dehydrogenase; Succinates; Succinic Acid

Isomerization of free enzyme can be detected in kinetic patterns of dead-end inhibition because competitive substrate analogs yield noncompetitive inhibition versus product in reverse reaction kinetics. The ratio of slope and intercept inhibition constants allows a quantitative estimation of the relative kinetic significance of the isomerization to a catalytic turnover. Applying this kinetic analysis theoretically to inhibition data for bovine carbonic anhydrase II by anions [Y. Pocker and T. L. Deits (1982) J. Am. Chem. Soc. 104, 2424] provides an estimate of 43 +/- 13% for how rate-limiting the isomerization segment is at pH 6.6. Applying the analysis experimentally to porcine heart fumarase provides a competitive pattern of inhibition by trans-aconitate versus fumarate with Ki(s) = 2.0 +/- 0.5 mM, together with a non-competitive pattern versus malate, with Ki(s) = 0.8 +/- 0.1 mM and Kii = 2.3 +/- 0.4 mM. Assuming that the isomerization segment of fumarase is the reprotonation of an active site carboxyl and imidazole with pK1 = 5.53 and pK2 = 7.78 [Blanchard and Cleland (1980) Biochemistry 19, 4506], an apparent rate constant for the isomerization segment of fumarate hydration is estimated as 95 +/- 22 s-1, compared to 42 +/- 13 s-1 for the chemical segment and 29 +/- 0.7 s-1 for a complete turnover. In contrast, the values are 17000 +/- 5200, 82 +/- 25, and 82 +/- 3 s-1, respectively, for malate dehydration. Hence, the isomerization segment is 30 +/- 7% rate-limiting during fumarate hydration but less than 1% during malate dehydration.

Topics: Aconitic Acid; Animals; Carbon Dioxide; Carbonic Anhydrase Inhibitors; Carbonic Anhydrases; Catalysis; Cattle; Fumarate Hydratase; Fumarates; Isomerism; Kinetics; Malates; Models, Chemical; Swine; Water

Four experiments were conducted to characterize the interactions between fumaric (FA), malic (MA), or citric acid (CA) and NaHCO3. In two experiments, seven diets were formulated containing 2.5% FA, MA, and CA, with or without 2.3, 1.9, or 1.4% NaHCO3, respectively, as well as a control diet (no addition of organic acids or NaHCO3) for 28-d-old pigs (Exp. 1, corn-soy protein concentrate-based diet) and 1-wk-old chicks (Exp. 4, corn-soy-based diet). In Exp. 1, at 2 and 4 wk, the FA+NaHCO3 treatment resulted in greater average daily gain (ADG) and feed intake (ADFI) compared with the control (P < .05). In Exp. 2, 28-d-old pigs were fed corn-soy diets with .9, 1.6, and 2.3% NaHCO3 in addition to 2.5% FA. After wk 2, there was a quadratic response in ADG (P < .08) and ADFI (P < .05) when increasing levels of NaHCO3 were added to the diet. This was true at wk 4 for both ADG and ADFI (P < .05). In Exp. 3, finishing pigs were fed corn-soy diets containing 2.5% FA or 2.5% FA + 2.3% NaHCO3 added to a control diet. No effect (P < .05) of FA or NaHCO3 was observed. In Exp. 4, the combination of CA+NaHCO3 or MA+NaHCO3 was superior to FA+NaHCO3 for ADG (P < .08) and ADFI (P < .05) when fed to young chicks.

Topics: Animal Feed; Animal Nutritional Physiological Phenomena; Animals; Carboxylic Acids; Chickens; Citrates; Citric Acid; Eating; Female; Fumarates; Hydrogen-Ion Concentration; Malates; Male; Random Allocation; Sodium Bicarbonate; Swine; Weight Gain

1. This study investigated the effects of fructose and 2-deoxyglucose on the uptake and release of phosphate from everted intestinal sacs of mice. 2. Both the sugars significantly decreased the release of phosphate without affecting the uptake. 3. Succinate and fumarate were able to partially reverse the inhibition of phosphate release exerted by fructose but not that exerted by 2-deoxyglucose. 4. Pre-loading with mannoheptulose, a known inhibitor of hexokinase, improved the release of phosphate in the presence of either of these sugars. 5. Adrenaline, known to inhibit phosphorylation of 2-deoxyglucose, reduced the inhibition exerted by this sugar on phosphate release. 6. These results indicate that the inhibition of phosphate release caused by these sugars may be due to the trapping of free phosphate during their metabolism in the gut wall.

Topics: Animals; Deoxyglucose; Epinephrine; Fructose; Fumarates; Intestinal Mucosa; Intestines; Male; Mannoheptulose; Mice; Phosphates; Phosphorylation; Succinates; Succinic Acid

Oral administration with complex mixtures of fumaric acid derivatives is known to have antipsoriatic efficacy. The present studies aimed to clarify the mode of action and toxicity of the individual compounds. Hyperproliferative HaCaT keratinocytes in monolayer cultures were exposed to fumaric acid, dimethylfumarate, zinc monoethylfumarate, calcium monoethylfumarate and magnesium monoethylfumarate at concentrations between 0.4 microM and 960 microM for 48 h. Cell proliferation was studied by [3H]thymidine incorporation. In addition 14C-labelled amino acid uptake and total protein content were measured. Direct cytotoxicity was determined by the release of cytoplasmic lactate dehydrogenase (LDH) into the culture medium. The corresponding 50% inhibition concentrations (IC50) were calculated for DNA/protein synthesis: 2.3/2.5 microM (dimethylfumarate), 133/145 microM (zinc monoethylfumarate), 215/230 microM (calcium monoethylfumarate), 275/270 microM (magnesium monoethylfumarate), > 960/> 960 microM (fumaric acid). The total protein content was less sensitive. Antiproliferative activity was found for dimethylfumarate and to a lesser degree for calcium monoethylfumarate already at the subtoxic concentrations of 1.3 and 4 microM, respectively. In the case of magnesium monoethylfumarate, zinc monoethylfumarate and fumaric acid there was no such dissociation between their cytotoxic and antiproliferative potential. These data indicate that most of the antipsoriatic potential of fumaric therapies is due to the dimethylfumarate compound.

Topics: Cell Death; Cell Division; Cells, Cultured; Dimethyl Fumarate; DNA; Fumarates; Humans; Keratinocytes; L-Lactate Dehydrogenase; Protein Biosynthesis; Psoriasis

The yeast Saccharomyces cerevisiae was entrapped within polyacrylamide gel beads by employing a procedure that uses sodium dodecylsulfate as a detergent to improve the spherical configuration of the beads. The resulting preparation showed a rate of fumarate bio-conversion to L-malic acid about 60 times higher than that found for the free cells. Almost all fumarate was converted in 30 min of incubation. The thermal stability of the immobilized cells did not significantly differ from the free cells. An optimal pH of 5.7 was found for the immobilized preparation and no succinic acid was detected as a byproduct in the incubation mixture.

Topics: Acrylic Resins; Fumarates; Hot Temperature; Hydrogen-Ion Concentration; Malates; Microspheres; Saccharomyces cerevisiae

The present study was undertaken to determine whether naftidrofuryl oxalate, a cerebral vasodilator, may improve or attenuate microsphere embolism-induced damage to the mitochondrial tricarboxylic acid cycle. For this purpose, the intermediates in the tricarboxylic acid cycle were determined using cerebral cortex isolated from microsphere-injected rats with and without naftidrofuryl oxalate treatment. Seven-hundred microspheres, with a diameter of 48 microns were injected into the right hemisphere through the right common carotid artery. The presence of cerebral infarction on the 3rd day after the operation was confirmed by the development of triphenyltetrazolium chloride-unstained areas in brain sections. Succinate, fumarate, malate, citrate and alpha-ketoglutarate, but not oxaloacetate, contents were significantly decreased in the right hemisphere of rats on the 3rd day following microsphere embolism. In the left hemisphere, a similar but smaller decrease in these intermediates was seen. The rats, which showed typical stroke-like symptoms, were treated with 15 mg/kg naftidrofuryl oxalate i.p., twice daily for 2.5 days, resulting in a significant reversal of the intermediate content of both hemispheres toward the control and an increased in the triphenyltetrazolium-stained area of a coronal section of the right hemisphere relative to the untreated animals. The results suggest that naftidrofuryl oxalate attenuates the development of microsphere embolism-induced cerebral infarction and improves microsphere-induced impairment of the mitochondrial tricarboxylic acid cycle. The observed effects provided evidence for a possible site of action of the agent on ischemic brain energy metabolism.

Topics: Animals; Brain Ischemia; Cerebral Cortex; Citrates; Citric Acid; Citric Acid Cycle; Fumarates; Intracranial Embolism and Thrombosis; Ketoglutaric Acids; Malates; Male; Microspheres; Nafronyl; Oxaloacetates; Rats; Rats, Wistar; Staining and Labeling; Succinates; Succinic Acid; Tetrazolium Salts

Halobacterium salinarium responds to blue light by reversing its swimming direction. Fumarate has been proposed as one of the molecular components of this sensory system and is involved in the switching process of the flagellar motor. In order to obtain chemical proof for this role of fumarate, cells were stimulated with a pulse of blue light and lysed by rapid mixing with distilled water. The lysate contained fumarate in free and bound form, which were separated by ultrafiltration. The fumarate concentration in the low-molecular-mass fraction (< 5 kDa) of the lysate was assayed enzymatically and a light-induced increase was observed. Additionally, the total cellular fumarate content decreased in response to light, indicating that fumarate was released from a cellular pool rather than being formed by de novo synthesis. The light-induced release was not detected in a mutant defective in sensory rhodopsin-I and -II. Therefore it is concluded that photoreceptor activation rather than a direct effect of light on the activity of metabolic enzymes causes fumarate release. For each photoactivated sensory rhodopsin-II molecule at least 350 molecules of fumarate were liberated demonstrating efficient amplification. The rate of light-induced fumarate release is at least 10-times faster than the fumarate turnover number of the citric acid cycle which was estimated as approximately 4300 per cell and second. Therefore this metabolic process is not expected to be part of the signal transduction chain in the halobacterial cell.

Topics: Cell Membrane; Cell Movement; Citric Acid Cycle; Cytoplasm; Flagella; Fumarate Hydratase; Fumarates; Halobacterium; Kinetics; Light; Mutation; Rhodopsin; Signal Transduction

The metabolism of fumarate by Helicobacter pylori was investigated employing one- and two-dimensional 1H and 13C nuclear magnetic resonance spectroscopy. Metabolically competent cells generated malate and succinate from fumarate as the sole substrate indicating the presence of fumarase and fumarate reductase activities in the bacterium. In incubations of fumarate with cell lysates accumulation of lactate, acetate, formate and alanine was observed after the initial production of malate and succinate. The results indicate the existence of active fumarate catabolism in H. pylori and suggest the possibility of an ATP generating mechanism which may play an important role in the bioenergetics of the bacterium.

Topics: Acetates; Alanine; Energy Metabolism; Formates; Fumarates; Helicobacter pylori; Kinetics; Lactates; Lactic Acid; Magnetic Resonance Spectroscopy; Malates; Succinates; Succinic Acid

Reduction of fumarate by soluble beef heart succinate dehydrogenase has been shown previously by voltammetry to become increasingly retarded as the potential is lowered below a threshold potential of -80 mV at pH 7.5. The behaviour resembles that of a tunnel diode, an electronic device exhibiting the property of negative resistance. The enzyme thus acts to oppose fumarate reduction under conditions of high thermodynamic driving force. We now provide independent evidence for this phenomenon from spectrophotometric kinetic assays. With reduced benzylviologen as electron donor, we have studied the reduction of fumarate catalysed by various enzymes classified either as succinate dehydrogenases or fumarate reductases. For succinate dehydrogenases, the rate increases as the concentration of reduced dye (driving force) decreases during the reaction. In contrast, authentic fumarate reductases of anaerobic cells (and 'succinate dehydrogenase' from Bacillus subtilis) neither exhibit the electrochemical effect nor deviate from simple kinetic behaviour in the cuvette assay. The 'tunnel-diode' effect may thus represent an evolutionary adaptation to aerobic metabolism.

Topics: Animals; Ascaris suum; Bacillus subtilis; Benzyl Viologen; Cattle; Electrochemistry; Escherichia coli; Female; Fumarates; Oxidation-Reduction; Placenta; Rats; Spectrophotometry; Succinate Dehydrogenase; Succinates; Succinic Acid

The argininosuccinate lyase activity of duck delta-crystallin was inactivated by diethyl pyrocarbonate at 0 degrees C and pH 7.5. The inactivation followed pseudo-first-order kinetics after appropriate correction for the decomposition of the reagent during the modification period. The plot of the observed pseudo-first-order rate constant versus diethyl pyrocarbonate concentration in the range of 0.17-1.7 mM was linear and went through the origin with a second-order rate constant of 1.45 +/- 0.1 M-1.s-1. The double-logarithmic plot was also linear, with slope of 1.13, which suggested a 1:1 stoichiometry for the reaction between diethyl pyrocarbonate and delta-crystallin. L-Arginine, L-norvaline or L-citrulline protected the argininosuccinate lyase activity of delta-crystallin from diethyl pyrocarbonate inactivation. The dissociation constants for the delta-crystallin-L-arginine and delta-crystallin-L-citrulline binary complexes, determined by the protection experiments, were 4.2 +/- 0.2 and 0.12 +/- 0.04 mM respectively. Fumarate alone had no protective effect. However, fumarate plus L-arginine gave synergistic protection with a ligand binding interacting factor of 0.12 +/- 0.02. The double-protection data conformed to a random Uni Bi kinetic mechanism. Fluorescence-quenching studies indicated that the modified delta-crystallin had minimum, if any, conformational changes as compared with the native delta-crystallin. Inactivation of the enzyme activity was accompanied by an increasing absorbance at 240 nm of the protein. The absorption near 280 nm did not change. Treatment of the modified protein with hydroxylamine regenerated the enzyme activity to the original level. These results strongly indicated the modification of an essential histidine residue. Calculation from the 240 nm absorption changes indicated that only one histidine residue per subunit was modified by the reagent. This super-active histidine residue has a pKa value of approximately 6.8 and acts as a general acid-base catalyst in the enzyme reaction mechanism. Our experimental data are compatible with an E1cB mechanism [Raushel (1984) Arch. Biochem. Biophys. 232, 520-525] for the argininosuccinate lyase with the essential histidine residue close to the arginine-binding domain of delta-crystallin. L-Citrulline, after binding to this domain, might form an extra hydrogen bond with the essential histidine residue.

Topics: Animals; Arginine; Argininosuccinate Lyase; Crystallins; Diethyl Pyrocarbonate; Ducks; Fumarates; Histidine; Hydrogen-Ion Concentration; Hydroxylamine; Hydroxylamines; Spectrometry, Fluorescence

DL-3-Arsonoalanine has been synthesized by the Strecker synthesis from the unstable compound arsonoacetaldehyde. It inactivates pig heart cytosolic aspartate aminotransferase and inhibits aspartate ammonia-lyase by competing with aspartate (Ki/Km 0.23). The fumarate analogue (E)-3-arsonoacrylic acid and the malate analogue (RS)-3-arsonolactate also inhibit fumarate hydratase, competing with fumarate (Ki/Km 1.8) and malate (Ki/Km 1.6) respectively. Attempted non-enzymic transamination of 3-arsonoalanine gave elimination of arsenite, in contrast with the transamination of 3-phosphonoalanine, which is either successful or leads to loss of phosphate.

Topics: Alanine; Arsenic; Arsenicals; Aspartate Aminotransferases; Aspartate Ammonia-Lyase; Enzyme Inhibitors; Fumarate Hydratase; Fumarates; Malates

Human hemoglobin was reacted with five dicarboxylic acid bis(methyl phosphate) reagents under different ligand conditions. The bis(methyl phosphate) reagents tested were derived from fumaric, isophthalic, terephthalic, trans-stilbene-3,3'-dicarboxylic, and trans-stilbene-4,4'-dicarboxylic acids. These acyl phosphate mixed anhydrides are anionic electrophiles and will react with N-terminal amino and lysyl epsilon-amino groups to form amides. The major and many of the minor reaction products that result have been isolated and structurally characterized by globin chain and peptide analysis. Products which are not cross-linked, intrachain linked, and interchain singly and doubly cross-linked occur in proportions which depend upon the reaction conditions and reagent. Modifications of the beta chains were limited to the amino groups of beta 1Val, beta 82Lys, and, to a minor extent, beta 144Lys. In the case of the smaller reagents, the amino groups of alpha 1Val, alpha 99Lys, and, to a minor extent, alpha 139Lys were modified. The oxygen binding affinities of most of the major modified hemoglobins have been measured and are characterized by P50 values from about 1/2 to over 5 times that of unmodified human hemoglobin. Most show strong cooperativity with Hill coefficients (n) of 2.0 or greater. Several of the products that are cross-linked between the beta 1Val of one chain and the beta 82Lys of the other chain have oxygen affinities in a physiologically useful range for oxygen transport and delivery. An inverse linear correlation has been found between the log of P50 and bridging distances for the hemoglobins cross-linked between beta 1Val of one chain and the beta 82Lys of the other chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acid Sequence; Chromatography, High Pressure Liquid; Cross-Linking Reagents; Dicarboxylic Acids; Electrophoresis, Polyacrylamide Gel; Fumarates; Globins; Hemoglobins; Humans; Molecular Sequence Data; Oxygen; Phosphates; Phthalic Acids

The Ascaris larval respiratory chain, particularly complex II (succinate-ubiquinone oxidoreductase), was characterized in isolated mitochondria. Low-temperature difference spectra showed the presence of substrate-reducible cytochromes aa3 of complex IV, c+c1 and b of complex III (ubiquinol-cytochrome c oxidoreductase) in mitochondria from second-stage larvae (L2 mitochondria). Quinone analysis by high-performance liquid chromatography showed that, unlike adult mitochondria, which contain only rhodoquinone-9, L2 mitochondria contain ubiquinone-9 as a major component. Complex II in L2 mitochondria was kinetically different from that in adult mitochondria. The individual oxidoreductase activities comprising succinate oxidase, and fumarate reductase were determined in mitochondria from L2 larvae, from larvae cultured to later stages, and from adult nematodes. The L2 mitochondria exhibited the highest specific activity of cytochrome c oxidase, indicating that L2 larvae have the most aerobic respiratory chain among the stages studied. The Cybs subunit of complex II in L2 and cultured-larvae mitochondria exhibited different reactivities against anti-adult Cybs antibodies. Taken together, these results indicate that the complex II of larvae is different from its adult counterpart. In parallel with this change in mitochondrial biogenesis, biosynthetic conversion of quinones occurs during development in Ascaris nematodes.

Topics: Animals; Ascaris suum; Cattle; Electron Transport Complex II; Fumarates; Larva; Mitochondria; Models, Biological; Multienzyme Complexes; Myocardium; NAD(P)H Dehydrogenase (Quinone); Oxidoreductases; Quinones; Succinate Dehydrogenase; Succinates; Succinic Acid; Ubiquinone

A two-component sensor-regulator system has been identified in the purple photosynthetic bacterium Rhodobacter capsulatus, which controls the expression of high-affinity C4-dicarboxylate transport activity in these cells. Nucleotide sequencing has revealed the existence of two genes, dctS and dctR, which together form an operon linked to, but divergently transcribed from, the previously identified dctP gene, which encodes the periplasmic binding protein of the transport system. The DctS protein is predicted to be a membrane-bound sensor-kinase with two potential membrane-spanning sequences in the N-terminal region. DctR was found to have sequence similarity throughout its entire length with proteins in the FixJ subfamily of response-regulators, especially to FixJ itself (42% identical residues). Insertional inactivation of the dctS and dctR genes resulted in the inability of the resulting mutants to grow on or transport malate, succinate or fumarate under aerobic conditions in the dark, and such mutants did not express the DctP protein. The mutants were complemented in trans by plasmids containing intact copies of the dctS and dctR genes.

Topics: Amino Acid Sequence; Bacterial Proteins; Base Sequence; Biological Transport; Carrier Proteins; Dicarboxylic Acids; Fumarates; Histidine Kinase; Malates; Membrane Proteins; Molecular Sequence Data; Mutagenesis, Insertional; Operon; Protein Structure, Secondary; Pyruvates; Restriction Mapping; Rhodobacter capsulatus; Sequence Alignment; Sequence Analysis; Sequence Homology, Amino Acid; Signal Transduction; Succinates; Succinic Acid; Transcription Factors

Fumarate reductase (Escherichia coli) can be immobilized in an extremely electroactive state at an electrode, with retention of native catalytic properties. The membrane-extrinsic FrdAB component adsorbs to monolayer coverage at edge-oriented pyrolytic graphite and catalyzes reduction of fumarate or oxidation of succinate, depending upon the electrode potential. In the absence of substrates, reversible redox transformations of centers in the enzyme are observed by cyclic voltammetry. The major component of the voltammograms is a pair of narrow reduction and oxidation signals corresponding to a pH-sensitive couple with formal reduction potential E degree' = -48 mV vs SHE at pH 7.0 (25 degrees C). This is assigned to two-electron reduction and oxidation of the active-site FAD. A redox couple with E degree' = -311 mV at pH 7 is assigned to center 2 ([4Fe-4S]2+/1+). Voltammograms for fumarate reduction at 25 degrees C, measured with a rotating-disk electrode, show high catalytic activity without the low-potential switch-off that is observed for the related enzyme succinate dehydrogenase. The catalytic electrochemistry is interpreted in terms of a basic model incorporating mass transport of substrate, interfacial electron transfer, and intrinsic kinetic properties of the enzyme, each of these becoming a rate-determining factor under certain conditions. Electrochemical reversibility is approached under conditions of low turnover rate, for example, as the supply of substrate to the active site is limited. In this situation, electrocatalytic half-wave potentials, E1/2, are similar for oxidation of bulk succinate and reduction of bulk fumarate and coincide closely with the E degree' value assigned to the FAD. At 25 degrees C and pH 7, the apparent KM for fumarate reduction is 0.16 mM, and kcat is 840 s-1. Accordingly the second-order rate constant, kcat/KM, is 5.3 x 10(6) M-1 s-1. Under the same conditions, oxidation of succinate is much slower. As the supply of fumarate to the enzyme is raised to increase turnover, the electrochemical reaction eventually becomes limited by the rate of electron transfer from the electrode. Under these conditions a second catalytic wave becomes evident, the E1/2 value of which corresponds to the reduction potential of the redox couple suggested to be center 2. This small boost to the catalytic current indicates that the low-potential [4Fe-4S] cluster can function as a second center for relaying electrons to the FAD.

Topics: Adsorption; Catalysis; Electrochemistry; Electrodes; Electron Transport; Enzymes, Immobilized; Flavin-Adenine Dinucleotide; Fumarates; Hydrogen-Ion Concentration; Kinetics; Oxidation-Reduction; Succinate Dehydrogenase; Succinates; Succinic Acid

Anticarcinogenic effects of the fumaric acid was studied in two rat models of carcinogenesis. Tumors of the esophagus, forestomach, tongue and throat were induced by peroral instillation of 35 mg/kg body weight N-methyl-N-benzylnitrosamine, and neurogenic and renal ones--by transplacental injection of 75 mg/kg body weight N-ethyl-N-nitrosourea. The fumaric acid given in drinking water in the dose of 1 g/l at the postinitiation stage of the carcinogenesis was shown to inhibit the development of esophageal papilloma, brain glioma and mesenchymal tumors of the kidney.

Topics: Animals; Anticarcinogenic Agents; Carcinogens; Chi-Square Distribution; Dimethylnitrosamine; Disease Models, Animal; Drug Screening Assays, Antitumor; Esophageal Neoplasms; Ethylnitrosourea; Female; Fumarates; Kidney Neoplasms; Male; Nervous System Neoplasms; Pregnancy; Prenatal Exposure Delayed Effects; Rats

Using 3T[14C]malate it was possible to show intermolecular T-transfer to unlabeled fumarate. The rate of dissociation of ET derived from the malate was not rapid, only about as fast as required for KMcat. Because of the slow dissociation of ET derived from T-malate, the awkward complex ET-malate is readily formed. As shown by the effect of added malate on the partition of ET, otherwise captured by fumarate, ET.malate must be functional. Its rate of dissociation to E.M determines the V/Km value of malate. Hydrogen dissociation of the complex ET.F was linearly related to the concentration and basicity of the buffer provided, varying from < 10% to > 60% of the overall rate with alkyl phosphonates. Partition of EH.F to free malate or fumarate occurs in a ratio approximately 2:1 at both low and high buffer. This agrees well with the comparison of the equilibrium exchange rates: malate with [18O]water to malate with [14C]-fumarate [Hansen, J.N., Dinovo, E.C., & Boyer, P.D. (1969) J. Biol. Chem. 244, 6270-6279]. Therefore, the abstracted hydroxyl group is fully exchanged from the enzyme when the bound hydrogen and fumarate return to malate and must be much more accessible to the medium than the abstracted proton. The fact that buffer increases the rate of proton transfer to the medium in the central complex makes it appear that a proton relay connects the active site donor with a remote site that interfaces with the ultimate proton source, water.

Topics: Binding, Competitive; Buffers; Catalysis; Enzyme Activation; Fumarate Hydratase; Fumarates; Hydrogen-Ion Concentration; Kinetics; Malates; Protons; Tritium; Water

1. Transport of citrate and fumarate across the pig intestinal brush-border membrane (BBM) was investigated using isolated BBM vesicles. 2. Citrate and fumarate uptake was stimulated by an inwardly directed Na+ gradient consistent with Na+/citrate (fumarate) co-transport. Cis-inhibition and trans-stimulation experiments strongly suggest the existence of a common transport site for tri- and dicarboxylates. 3. The protonated forms of citrate (citrate-1, citrate-2) seem to be much better transported than citrate-3, indicated by the strong stimulation of citrate uptake at an extravesicular pH of 5.6 compared to pH 7.8. 4. Uptake of tri- and dicarboxylates seems to be potential-sensitive since citrate and in particular fumarate transport was enhanced by an inside negative potential difference. 5. Kinetics of succinate transport revealed a single carrier-mediated component with apparent kinetic constants of 0.43 nmol/mg protein-3 s (Vmax) and 0.14 mmol/l (Km).

Topics: Animals; Biological Transport; Cations, Monovalent; Cattle; Citrates; Citric Acid; Fumarates; Hydrogen-Ion Concentration; Jejunum; Kinetics; Membrane Potentials; Microvilli; Sodium; Succinates; Succinic Acid; Swine

Purine nucleotide concentrations in skin- and blood-cells of psoriatic patients are abnormal: The increase in the steady state level of cGMP and the decrease in the cAMP concentrations are associated with an enhanced rate of cellular proliferation. Concomitantly we found in the present study decreased ADP and ATP concentrations in blood cells (p less than 0.0001). The change in nucleotide concentrations suggests a defective purine nucleotide synthesis pathway. Stimulation of the Krebs cycle with fumaric acid raises ATP (p less than 0.0001) and most probably cAMP levels and at the same time slows down the purine nucleotide synthesis through end-product inhibition. Both effects can inhibit DNA and protein synthesis activity, which results in inhibition of cellular proliferation. Fumaric acid seems therefore a useful treatment for psoriatic lesions if liver and kidney functions (purine nucleotide and urea cycle) are controlled during treatment.

Topics: Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Adult; Cell Division; Fumarates; Humans; Middle Aged; Psoriasis; Purine Nucleotides

Corynebacterium callunae (NCIB 10338) grows faster on glutamate than ammonia when used as sole nitrogen sources. The levels of glutamine synthetase (GS; EC 6.3.1.2) and glutamate synthase (GOGAT; EC 1.4.1.13) of C. callunae were found to be influenced by the nitrogen source. Accordingly, the levels of GS and GOGAT activities were decreased markedly under conditions of ammonia excess and increased under low nitrogen conditions. In contrast, glutamate dehydrogenase (GDH; EC 1.4.1.4) activities were not significantly affected by the type or the concentration of the nitrogen source supplied. The carbon source in the growth medium could also affect GDH, GS and GOGAT levels. Of the carbon sources tested in the presence of 2 mM or 10 mM ammonium chloride as the nitrogen source pyruvate, acetate, fumarate and malate caused a decrease in the levels of all three enzymes as compared with glucose. GDH, GS and GOGAT levels were slightly influenced by aeration. Also, the enzyme levels varied with the growth phase. Methionine sulfoximine, an analogue of glutamine, markedly inhibited both the growth of C. callunae cells and the transferase activity of GS. The apparent Km values of GDH for ammonia and glutamate were 17.2 mM and 69.1 mM, respectively. In the NADPH-dependent reaction of GOGAT, the apparent Km values were 0.1 mM for alpha-ketoglutarate and 0.22 mM for glutamine.

Topics: Ammonia; Corynebacterium; Culture Media; Fumarates; Glucose; Glutamate Dehydrogenase; Glutamate Synthase; Glutamate-Ammonia Ligase; Glutamates; Glutamic Acid; Kinetics; Malates; Methionine Sulfoximine

Fumaric acid production by Rhizopus arrhizus from commercial hydrolysates of corn starch (i.e. glucose molasses) was studied at different initial concentrations of glucose (S) and C:N ratios (R) by performing a 3(2) factorial experiment. By using the response surface methodology and statistical analysis, fumaric acid (YF) and mycelial biomass (YX) yields, as referred to the initial concentration of glucose and fumaric acid productivity (PF), were fitted to the only significant first-order effects of S and R with mean percentage errors ranging from 11 to 15%. The resulting empiric models were used to determine the optimal values of S (100-130 g dm-3) and R (150-210 g-atom C per g-atom N) associated with YF and PF varying in the ranges 40-49% and 7-8.5 g dm-3 day-1, respectively. After establishing the validity of these data at the 95% confidence level, an optimal operating condition (S = 120 g dm-3 and R = 150) was further tested using other substrates (i.e. glucose and acid or enzymatic hydrolysates of cassava, corn and potato flours). Statistically significant improvements in the fumaric acid yield and productivity were determined with respect to the predicted values. Since the highest values of YF and PF were obtained from the acid hydrolysates of the starch-based materials and such values were also found to be insensitive to the substrate used (at a probability level of 0.05), the above operating condition might be further employed to minimise fumaric acid production costs as a function of the feedstock used.

Topics: Culture Media; Fermentation; Flour; Fumarates; Glucose; Hydrolysis; Industrial Microbiology; Malates; Manihot; Models, Biological; Molasses; Rhizopus; Solanum tuberosum; Starch

A case of fully reversible tubular toxicity with consecutive metabolic osteopathy following systemic fumaric acid therapy is reported. This secondary effect of oral fumaric acid therapy obviously occurs very rarely, never having been described before. A 46-year-old female patient with a long history of recurrent palmoplantar psoriasis underwent oral treatment with fumaric acid and its esters in accordance with the Schäfer method, preceding attempts at curative treatment with conventional antipsoriatic agents having proved unsatisfactory. Two months later, the patient started to experience arthralgia, back pain in the early hours of the morning and myalgia with increasing frequency, progressing to disablement in moving and walking and, finally, to total immobility. Not until 9 months later was the reason for these severe disabilities found: they stemmed from hypophosphataemic osteomalacia as a result of a complex disturbance of the renal tubular system. The clinical symptoms and the results of laboratory chemistry tests returned to normal as soon as fumaric acid medication was discontinued. Two attempts at reexposure confirmed the causal relationship. Oral fumaric acid medication should never be administered without clinical and chemical controls.

Topics: Administration, Oral; Biopsy; Bone and Bones; Fanconi Syndrome; Female; Fumarates; Humans; Kidney Function Tests; Middle Aged; Osteomalacia; Psoriasis

A 5-year-old girl with a previous diagnosis of cerebral palsy, nonprogressive psychomotor retardation, and hypotonia was found to excrete excessive fumaric acid in urine. Fumarate hydratase activity in skin fibroblasts was 10% of the control value. This case underscores the clinical heterogeneity of neurometabolic disorders and the importance of organic acid analysis in the diagnosis of static encephalopathy.

Topics: Cerebral Palsy; Child, Preschool; Diagnostic Errors; Female; Fumarate Hydratase; Fumarates; Humans; Intellectual Disability; Metabolism, Inborn Errors; Muscle Hypotonia; Succinates; Succinic Acid

Two dead-end metabolites of anaerobic toluene transformation, benzylsuccinic acid and benzylfumaric acid, accumulated in sulfate-reducing enrichment cultures that were fed toluene as the sole carbon source. Stable isotope-labeled toluene and gas chromatography-mass spectrometry were used to confirm that the compounds resulted from toluene metabolism. The two metabolites constituted less than 10% of the toluene carbon (over 80% was mineralized to carbon dioxide, according to a previous study). This study demonstrates that the novel nonproductive pathway proposed by Evans and coworkers (P. J. Evans, W. Ling, B. Goldschmidt, E. R. Ritter, and L. Y. Young, Appl. Environ. Microbiol. 58:496-501, 1992) for a denitrifying pure culture applies to disparate anaerobic bacteria.

Topics: Bacteria, Anaerobic; Biodegradation, Environmental; Culture Media; Fumarates; Oxidation-Reduction; Succinates; Sulfates; Toluene

Trypanosoma cruzi epimastigotes permeabilized with digitonin (65 micrograms (mg protein)-1) to measure mitochondrial respiration were exposed to different substrates. Although none of the NADH-dependent substrates stimulated respiration, succinate supported not only oxygen consumption but also oxidative phosphorylation (respiratory control ratio of 1.9 +/- 0.3) indicating that the mitochondria were coupled. The rate of NADH-dependent oxygen consumption by membrane fractions (9.4 +/- 0.7 nmol min-1 (mg protein)-1) was reduced by 50% upon addition of catalase indicating that the electrons from NADH oxidation reduced oxygen to H2O2. NADH-dependent H2O2 production (16 +/- 1 nmol min-1 (mg protein)-1) was confirmed using cytochrome c peroxidase. This activity was inhibited by fumarate by 70%, suggesting a competition between fumarate and oxygen for the electrons from NADH, probably at the fumarate reductase level. The respiratory chain inhibitor antimycin blocked both respiration by intact cells and succinate-dependent cytochrome c by isolated membranes. No inhibition by antimycin was observed when NADH replaced succinate as an electron donor, indicating that the electrons from NADH oxidation reduced cytochrome c through a different route. Malonate blocked not only succinate-cytochrome c reductase and fumarate reductase, but also intact cell motility. These results suggest that succinate has a central role in the intermediate metabolism of i. cruzi, as it may be used for respiration or excreted to the extracellular space under anaerobic conditions. In addition, 2 potential sources of H2O2 were tentatively identified as: (a) the enzyme fumarate reductase; and (b) a succinate-dependent site, which may be the semiquinone form of Coenzyme Q9, as in mammalian mitochondria.

Topics: Animals; Fumarates; Glucose; Hydrogen Peroxide; NAD; Oxygen Consumption; Proline; Succinates; Succinic Acid; Trypanosoma cruzi

Pigs weaned at 21 d of age (n = 72) were fed a 20% CP corn-soybean meal-based diet (control) with 1.5% fumaric or 1.5% citric acid added to observe the effect of these acids on the pH, chloride ion concentration (Cl-), VFA profile, and microflora population in the stomach, jejunum, cecum, and lower colon contents at -2, 0, 3, 7, 14, and 21 d postweaning. Feeding organic acids had no appreciable effect (P greater than .10) on stomach jejunum, cecum, or lower colon pH, Cl-, VFA profile, or microflora populations, except for increasing the concentration of fumaric acid in the stomachs of pigs fed fumaric acid. The pH of the gastrointestinal tract generally decreased from -2 to 21 d postweaning with no corresponding change in Cl- over time. No age effects on total anaerobic culture counts were observed except in the stomach, where counts decreased from -2 to 3 d postweaning. Clostridia counts generally decreased after weaning in all intestinal sections. Lactobacillus counts were usually lower at d 0 and 3 and greatest at d 7 postweaning in the stomach, jejunum, and lower colon, but no age effect was observed for concentration of cecum lactobacilli. Escherichia coli counts generally increased after weaning to 3 and 7 d postweaning. Intestinal content measurements were affected by postweaning age but were not affected by feeding organic acids.

Topics: Age Factors; Animals; Bacteria, Anaerobic; Chlorides; Citrates; Citric Acid; Clostridium; Colony Count, Microbial; Escherichia coli; Fatty Acids; Fatty Acids, Volatile; Fumarates; Gastrointestinal Contents; Hydrogen-Ion Concentration; Lactobacillus; Random Allocation; Swine; Weaning

CGS 5649 B improves the learning performance of aged rats in a one-way active-avoidance situation. If, under reversed conditions, treated aged rats are also tested for passive avoidance, they show "place learning," which our findings have demonstrated to be typical of young rats. The effects of the substance are not confined to these experimental models nor are they species specific: it also facilitates passive avoidance in mice and social learning in rats. The compound is effective if administered before or immediately after the learning trial.

Topics: Aging; Animals; Avoidance Learning; Dose-Response Relationship, Drug; Ergoloid Mesylates; Fumarates; Male; Mental Recall; Mice; Mice, Inbred Strains; Piracetam; Premedication; Rats; Rats, Inbred Strains; Reversal Learning; Social Behavior

A nuclear mutant of Saccharomyces cerevisiae deficient in mitochondrial fumarase has been identified through the in vitro biochemical assay of enzyme activity after visual selection due to an increased acidification ability of its colonies. Cells of the fumarase-deficient mutant fermenting glucose accumulated extracellular fumaric acid. This accumulation was observed only in growing cultures and required functional mitochondrial electron transport from succinate dehydrogenase to oxygen.

Topics: Anaerobiosis; Antifungal Agents; Chromatography, High Pressure Liquid; Fatty Acids, Unsaturated; Fumarate Hydratase; Fumarates; Hydrogen-Ion Concentration; Kinetics; Methacrylates; Mutation; Oxygen Consumption; Saccharomyces cerevisiae; Strobilurins

Flagella of cytoplasm-free envelopes of Escherichia coli or Salmonella typhimurium can rotate in either the counterclockwise or clockwise direction, but they never switch from one direction of rotation to another. Exogenous fumarate, in the intracellular presence of the chemotaxis protein CheY, restored switching ability to envelopes, with a concomitant increase in clockwise rotation. An increase in clockwise rotation was also observed after fumarate was added to partially lysed cells of E. coli, but the proportion of switching cells remained unchanged.

Topics: Cell Membrane; Cell Wall; Chemotaxis; Escherichia coli; Flagella; Fumarates

Three growth experiments were conducted to assess the effect of fumaric acid (FA) supplementation on the performance of pigs fed different types of diets. These 4-wk experiments involved 550 crossbred pigs weaned at 30 +/- 3 d of age. Pigs in Exp. 1 were fed either a simple corn-soybean meal (SBM) diet or a 75% corn-SBM, 25% dried skim milk (DSM) diet. Zero, 2, or 3% FA were added to the diets to create a 2 x 3 factorial arrangement of treatments. Supplementing diets with DSM improved gain (P less than .05) and gain/feed (P less than .001). Fumaric acid addition improved efficiency of feed utilization (P less than .01). Experiment 2 was conducted to evaluate the effect of protein source (casein or soy protein concentrate [SPC]) on the response to 3% FA. Sodium bicarbonate (BCB; 2.75%) was added to one diet containing casein and FA to negate the effect of FA on growth performance. Addition of FA improved gain and feed efficiency (P less than .05) during the first 2 wk.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animal Feed; Animals; Bicarbonates; Diet; Dietary Proteins; Female; Fumarates; Hydrogen-Ion Concentration; Male; Sodium; Sodium Bicarbonate; Swine; Weight Gain

1. Phebrol (sodium 2,5-dichloro-4-bromophenol), a synthetic molluscicide against Oncomelania nosophora, showed a dual effect on rat liver submitochondria, acting as an uncoupler at low concentrations (approximately 10 microM) and an inhibitor of succinate-cytochrome c reductase at high concentrations. 2. Phebrol also inhibited the enzymes responsible for succinate-fumarate conversion, i.e. the succinate-cytochrome c reductase, fumarate reductase and NADH-cytochrome c reductase of the mitochondrial fraction from Biomphalaria glabrata. 3. Kinetic inhibition studies showed succinate-cytochrome c reductase of B. glabrata and O. nosophora to be more sensitive than that of rat liver toward phebrol. 4. Phebrol accumulated in whole tissues of B. glabrata and O. nosophora and had significant effects on the production of succinate, fumarate and malate by these snails. 5. On the basis of these results, the possible sites of inhibition by phebrol of snail respiratory chains are proposed.

Topics: Animals; Biomphalaria; Chlorophenols; Fumarates; Kinetics; Liver; Malates; Mitochondria; Molluscacides; NADH Dehydrogenase; Oxygen Consumption; Rats; Snails; Succinate Cytochrome c Oxidoreductase; Succinate Dehydrogenase; Succinates; Succinic Acid; Uncoupling Agents

An examination was made on the effect of fumaric acid on an experimental systemic candidiasis. Male ICR mice were innoculated into the tail veins with 10(6) yeast cells of Candida albicans and treated with daily intraperitoneal injections of fumaric acid at the dose of 40 mg/kg/d. The results indicated that the administration of fumaric acid was effective in prolonging the survival of animals after the fungal challenge and prevented one-fifth of the treated animals from dying of candidiasis.

Topics: Animals; Antifungal Agents; Candidiasis; Fumarates; Male; Mice; Mice, Inbred ICR

Three chemical compounds isolated from the leaves and stems of Ipomoea batatas were identified as fumaric acid, succinic acid and 7,3',4'-trimethylquercetin by chemical and spectroscopic analysis. In this paper, an analysis of the amino-acids of Ipomoea batatas is also reported.

Topics: Amino Acids; Drugs, Chinese Herbal; Fumarates; Quercetin; Succinates; Succinic Acid

A radiochemical assay for adenylosuccinase, an enzyme which intervenes twice in the biosynthesis of adenine nucleotides, has been developed. The two substrates of the enzyme, succinylaminoimidazole carboxamide ribotide (SAICAR) and adenylosuccinate (S-AMP), were synthesized in radioactive form by incubating [2,3-14C]fumarate and, respectively, AICAR and AMP with partially purified adenylosuccinase from yeast. Enzyme activities were determined by measuring the release of labeled fumarate after its separation from the substrate by chromatography on polyethyleneimine thin-layer plates. The ratio of the activity of adenylosuccinase measured with SAICAR compared to that with S-AMP was about 1 in crude extracts of rat liver and muscle and around 0.5 in human liver. In rat and human liver, but not in rat muscle, 20 to 40% of both activities of adenylosuccinase were lost after freezing at -80 degrees C followed by thawing. In the liver of patients with adenylosuccinase deficiency, in whom the deficiency had hitherto been measured only with S-AMP, the activity of the enzyme toward S-AMP and SAICAR was found to be lost in parallel. This is in accordance with the finding that both SAICA-riboside and succinyladenosine accumulate in adenylosuccinase-deficient patients.

Topics: Adenosine Monophosphate; Adenylosuccinate Lyase; Aminoimidazole Carboxamide; Animals; Carbon Radioisotopes; Chromatography, Thin Layer; Cold Temperature; Enzyme Stability; Fumarates; Humans; Liver; Male; Muscles; Rats; Rats, Inbred Strains; Ribonucleotides; Saccharomyces cerevisiae; Substrate Specificity

Two experiments were conducted to determine the apparent ileal digestibility of DM and N by young pigs fed diets supplemented with different protein sources or organic acids. Pigs were surgically fitted with silicone cannulas at 2 wk of age. Following surgery, pigs were allowed to recuperate with their dams while suckling normally. After weaning at 24 d, pigs were assigned to treatment diets at 28 d of age. A 3-d adjustment and 4-d collection sequence was followed for the duration of the 4-wk experiment. Four treatment diets were fed in each experiment in a weekly rotation until each diet had been fed to each pig. Diet samples and digesta collected through the ileal cannulas were analyzed for chromic oxide (used as an indigestible marker), DM, and N. Pigs in Exp. 1 were fed isolysinic (1.0%) corn-based diets supplemented with casein, soybean meal, soy protein concentrate, or isolated soy protein. Casein addition resulted in improved DM (P less than .001) and N (P less than .05) digestibility but reduced gain (P less than .05) compared with the average of the soy protein sources. Nitrogen from diets formulated with soybean meal was digested more completely (P less than .05) than N from diets based on soy protein concentrate and isolated soy protein. Experiment 2 was an evaluation of the effect of dried skim milk (25%) and fumaric acid (2%) addition on apparent ileal digestibility of N and DM in corn-soybean meal diets. Addition of dried skim milk improved DM (P less than .01) and N (P less than .05) digestibility and daily gain (P less than .001). Fumaric acid supplementation did not affect nutrient digestibility or gain (P greater than .10).

Topics: Animal Feed; Animals; Caseins; Dietary Proteins; Digestion; Fumarates; Glycine max; Ileum; Nitrogen; Plant Proteins, Dietary; Soybean Proteins; Swine; Weight Gain

Two studies were conducted to examine the effects of dietary inclusion of fumaric acid on performance and carcass composition of broiler chickens. Fumaric acid was added to nutritionally complete diets at levels of 0, .125, .25, and .5% and fed from 1 to 49 days under simulated commercial conditions. In one trial, samples of birds were processed to determine dressing percentage and abdominal fat content. In the first trial (mixed sex broilers), the addition of .125% fumaric acid significantly (P less than .05) improved 49-day body weight of females and average weight gain of both sexes with no effect on feed utilization. Feed consumption was significantly increased when diets contained .125 or .50% fumaric acid. In the second trial (male broilers), 49-day body weight was significantly (P less than .05) improved by the addition of .125 and .25% fumaric acid. There were no significant differences in feed consumption; feed utilization was improved by the addition of all levels of fumaric acid. Dietary fumaric acid had no adverse effects on dressing percentage, abdominal fat content, or mortality rate.

Topics: Animals; Body Weight; Chickens; Eating; Female; Fumarates; Male; Random Allocation; Sex Characteristics; Weight Gain

Bacteroides gracilis is a gram-negative anaerobic bacillus which requires formate and fumarate for growth; it has been implicated in periodontal disease and serious infections of the head and neck. In this study, Bacteroides ureolyticus (NTU) medium was tested for its ability to allow the growth of B. gracilis and other formate-fumarate requiring gram-negative anaerobes and to enable the recovery of these organisms from clinical specimens. All reference strains grew on NTU medium with the exception of Wolinella recta and formate-fumarate requiring organisms were isolated from 18 of 20 samples of subgingival dental plaque from patients with chronic periodontitis. B. gracilis was the commonest species isolated (14 of the 29 isolates); B. ureolyticus was not found.

Topics: Bacteroides; Culture Media; Formates; Fumarates; Humans; Periodontitis

L-Malate was produced from fumarate by using immobilized Saccharomyces cerevisiae cells entrapped in polyacrylamide. This preparation performed better when pretreated with malonate. Under the experimental conditions described here, succinate was not detected as a by-product of the reaction, as had been reported for other microorganisms.

Topics: Acrylic Resins; Biotechnology; Detergents; Fumarate Hydratase; Fumarates; Malates; Saccharomyces cerevisiae; Succinates; Succinic Acid

Setaria digitata, a cattle filarial parasite, is known to have peculiarities such as hydrogen peroxide (H2O2) production, cyanide insensitivity, absence of cytochromes and presence of quinones. Estimation of mitochondrial H2O2 with different substrates and inhibitors showed that salicylhydroxamic acid (SHAM), the alternative oxidase inhibitor, inhibited the H2O2 production maximally. Based on the inhibitory studies with rotenone, antimycin A, o-hydroxydiphenyl, SHAM and 2 thenoyltrifluoroacetone, a mechanism for the electron transport is proposed. Quinone Q8 seems to have a central role, hence inhibitors at the level of quinones might prove to be effective in designing drugs for filariasis.

Topics: Animals; Antimycin A; Biphenyl Compounds; Cyanides; Electron Transport; Filarioidea; Fumarates; Glycerophosphates; Hydrogen Peroxide; Malates; Mitochondria; Quinones; Rotenone; Salicylamides; Succinates; Succinic Acid; Thenoyltrifluoroacetone

Topics: Animals; Fumarates; Kidney; Rats

Succinate is effectively taken up by washed cells of Corynebacterium glutamicum. The apparent Km value of uptake is about 150 microM and the Vmax 4-7 nmol (mg dry weight)-1 min-1 and uptake can be competetively inhibited by fumarate and oxaloacetate. The activation energy was determined to be 50 kJ/mol. The transport activity is clearly dependent on the presence of Na+ ions in the incubation medium and on the membrane potential and has a pH optimum around 8.5. It is concluded that succinate is taken up in C. glutamicum via a specific carrier by a secondary active, Na+ coupled mechanism.

Topics: Binding, Competitive; Corynebacterium; Escherichia coli; Fumarates; Hydrogen-Ion Concentration; Kinetics; Membrane Potentials; Sodium; Succinates; Succinic Acid

Experimental data showed a significant improvement of growth rate and efficiency of feed utilization of young animals (piglets) by the dietary inclusion of organic acids. These ergotropic effects were mainly observed with citric acid, fumaric acid and formic acid as well as with Ca and Na formate. The merely dietary pH lowering with an inorganic acid (ortho-phosphoric acid) failed to show a nutritive efficacy. Studies on the mode of action of organic acids indicated a higher protein and energy digestibility, a lower stomach pH and reduced levels of NH3 and lactic acid in the stomach and duodenal digesta. Furthermore, the duodenum mainly contained a significant lower bacterial population for E. coli and enterococci. By this way the burden of metabolism of the host may be reduced which results in a higher overall performance.

Topics: Animal Feed; Animals; Carboxylic Acids; Citrates; Citric Acid; Dietary Proteins; Digestion; Duodenum; Energy Metabolism; Formates; Fumarates; Gastric Mucosa; Hydrogen-Ion Concentration; Phosphoric Acids; Swine

Topics: Bacteroides fragilis; Carboxylic Acids; Chromatography, Gas; Fumarates; Lactates; Lactic Acid; Malonates; Methylmalonic Acid; Oxalates; Oxalic Acid; Pyruvates; Pyruvic Acid; Succinates; Succinic Acid

The effects of pH and inhibitors on the spectra and redox properties of the haems b of the bc1 complex of beef heart submitochondrial particles were investigated. The major findings were: (1) both haems have a weakly redox-linked protonatable group with pKox and pKred of around 6 and 8; (2) at pH values above 7, haem bH becomes heterogeneous in its redox behaviour. This heterogeneity is removed by the Qi site inhibitors antimycin A, funiculosin and HQNO, but not by the Qo site inhibitors myxothiazol or stigmatellin; (3) of all inhibitors tested only funiculosin had a large effect on the Em/pH profile of either haem b. In all cases where definite effects were found, the haem most affected was that thought to be closest to the site of inhibitor binding; (4) spectral shifts of haem groups caused by inhibitor binding were usually, but not always, of the haem group closest to the binding site; (5) titrations with succinate/fumarate were in reasonable agreement with redox-mediated data provided that strict anaerobiosis was maintained. Apparent large shifts of haem midpoint potentials with antimycin A and myxothiazol could be produced in aerobic succinate/fumarate titrations in the presence of cyanide, as already reported in the literature, but these were artefactual; (6) the heterogeneous haem bH titration behaviour can be simulated with a model similar to that proposed by Salerno et al. (J. Biol. Chem. (1989) 264, 15398-15403) in which there is redox interaction between haem bH and ubiquinone species bound at the Qi site. Simulations closely fit both the haem bH data and known semiquinone data only if it is assumed that semiquinone bound to oxidised haem bH is EPR-silent.

Topics: Animals; Anthraquinones; Antimycin A; Cattle; Electron Spin Resonance Spectroscopy; Electron Transport Complex III; Fumarates; Heme; Hydrogen-Ion Concentration; Hydroxyquinolines; Mitochondria, Heart; Oxidation-Reduction; Protons; Submitochondrial Particles; Succinates; Succinic Acid

Two sisters, aged 25 and 29 years, with generalized psoriasis guttata since childhood, developed nausea, upper-abdominal pain, loss of appetite, palpitations and flushes in the course of local and oral administration of fumaric acid esters. Because of these side effects the treatment was discontinued after about two weeks, and the symptoms disappeared. But proteinuria and haematuria were subsequently noted, creatinine concentration rose to 2.2 and 2.5 mg/dl, respectively, while creatinine clearance fell to 44 and 27 ml/min, respectively. Examination of urinary sediments and analysis of urinary proteins gave results compatible with tubular-interstitial renal damage. The abnormal renal functions and urinary findings proved reversible within three weeks.

Topics: Administration, Oral; Adult; Baths; Combined Modality Therapy; Digestive System; Drug Therapy, Combination; Female; Fumarates; Humans; Kidney; Kidney Diseases; Ointments; Psoriasis; Time Factors

Fumarate reductase (FRD) of Escherichia coli is a four-subunit membrane-bound complex that is synthesized during anaerobic growth when fumarate is available as a terminal oxidant. The two subunits that comprise the catalytic domain, FrdA and FrdB, are anchored to the cytoplasmic membrane surface by two small hydrophobic polypeptides, FrdC and FrdD, which are also required for the enzyme to interact with quinone. To better define the individual roles of the FrdC and FrdD polypeptides in FRD complex formation and quinone binding, we selectively mutagenized the frdCD genes. Frd- strains were identified by their inability to grow on restrictive media, and the resulting mutant FRD complexes were isolated and biochemically characterized. The majority of the frdC and frdD mutations were identified as single base deletions that caused premature termination in either FrdC or FrdD and resulted in the loss of one or more of the predicted transmembrane helices. Two additional frdC mutants were characterized that contained single base changes resulting in single amino acid substitutions. All mutant enzyme complexes were incapable of oxidizing the physiological electron donor, menaquinol-6, in the presence of fumarate. Additionally, the ability of the mutant complexes to oxidize reduced benzyl viologen or reduce the ubiquinone analogue 2,3-dimethoxy-5-methyl-6-pentyl-1,4-benzoquinone and phenazine methosulfate with succinate as electron donor were also affected but to varying degrees. The separation of oxidative and reductive activities with quinones suggests there are two quinone binding sites in the fumarate reductase complex and that electron transfer occurs in two le- steps carried out at these separate sites.

Topics: Amino Acid Sequence; Catalysis; Cell Membrane; Cloning, Molecular; Electron Transport; Escherichia coli; Fumarates; Macromolecular Substances; Molecular Sequence Data; Mutagenesis, Site-Directed; Naphthols; Plasmids; Structure-Activity Relationship; Succinate Dehydrogenase; Terpenes; Transformation, Bacterial

Topics: Dermatologic Agents; Fumarates; Humans; Psoriasis

The inhibitory effect of fumaric acid (FA) on hepatocarcinogenesis was examined in mice fed thioacetamide (TAA). A group of male ICR mice was fed TAA at a level of 0.035% in the diet for 40 weeks and then fed a basal diet for 48 weeks. Hepatic tumors developed in 11 of the 24 animals of this group and they were diagnosed as hepatocellular carcinomas. However, cirrhotic lesions and the enlargement of hepatocyte nucleoli were not as marked in mice as in previous findings in rats fed TAA. The effect of FA on the carcinogenesis was examined in a group of mice fed this compound at a level of 1% in a basal diet after ingestion of TAA. The inhibitory effect of FA on TAA carcinogenesis was so marked that no hepatic carcinomas were found in any of the 15 animals fed FA in combination with TAA.

Topics: Animals; Fumarates; Liver; Liver Neoplasms, Experimental; Male; Mice; Mice, Inbred ICR; Thioacetamide

Cloning of the Saccharomyces cerevisiae FUM1 gene downstream of the strong GAL10 promoter resulted in inducible overexpression of fumarase in the yeast. The overproducing strain exhibited efficient bioconversion of fumaric acid to L-malic acid with an apparent conversion value of 88% and a conversion rate of 80.4 mmol of fumaric acid/h per g of cell wet weight, both of which are much higher than parameters known for industrial bacterial strains. The only product of the conversion reaction was L-malic acid, which was essentially free of the unwanted by-product succinic acid. The GAL10 promoter situated upstream of a promoterless FUM1 gene led to production and correct distribution of the two fumarase isoenzyme activities between cytosolic and mitochondrial subcellular fractions. The amino-terminal sequence of fumarase contains the mitochondrial signal sequence since (i) 92 of 463 amino acid residues from the amino terminus of fumarase are sufficient to localize fumarase-lacZ fusions to mitochondria and (ii) fumarase and fumarase-lacZ fusions lacking the amino-terminal sequence are localized exclusively in the cytosol. The possibility that both mitochondrial and cytosolic fumarases are derived from the same initial translation product is discussed.

Topics: Cloning, Molecular; Enzyme Induction; Fumarate Hydratase; Fumarates; Gene Expression; Genes, Fungal; Malates; Saccharomyces cerevisiae

The isolation of a straight-swimming mutant of Halobacterium halobium is reported which has a defect in switching the rotational sense of its flagellar motor. Cells of this mutant strain could be complemented with an extract from wild-type cells by mild sonication and resealing of the cells in fresh medium. The switch factor responsible for restoration of wild-type behaviour was isolated from membrane vesicle preparations. Its chemical nature is proposed to be that of fumarate on the basis of chemical, chromatographic and mass spectrometric analysis. Since the switch factor (fumarate) was released from a membrane-bound state by heat and was accumulated into mutant cells that lack this compound, it is proposed that a membrane-bound protein exists which specifically binds the switch factor. Both the switch factor and fumarate cause stimulus-induced responses in cells at the level of one or few molecules.

Topics: Cell Movement; Fumarates; Genetic Complementation Test; Halobacterium; Kinetics; Light; Models, Biological; Mutation; Signal Transduction; Software

24 days after starting treatment of psoriasis with fumaric acid derivatives (0.8-1.0 g orally, plus unknown quantities locally) a 21-year-old woman developed acute oliguric renal failure with a rise of serum creatinine levels to 1094 mumol/l (12.4 mg/dl). Deterioration of renal function had been preceded by severe abdominal symptoms with nausea, vomiting and colicky pain. On admission to hospital she was dehydrated with hyponatraemia and hypokalaemia. There was glomerular microhaematuria, increased excretion of renal epithelia, and tubular proteinuria. Renal biopsy demonstrated acute tubular damage with vacuolization of proximal epithelia, dilated tubules and scattered necroses. After intermittent haemodialysis (13 courses over two weeks) renal function gradually recovered, as demonstrated at a follow-up examination four months after discharge.

Topics: Acute Kidney Injury; Adult; Biopsy; Chronic Disease; Drug Combinations; Drug Therapy, Combination; Female; Fumarates; Humans; Kidney; Powders; Psoriasis

In the fully grown Bufo arenarum oocyte, carbohydrate breakdown during the autumn-winter season is accomplished mainly through the glycolytic pathway followed by the Krebs cycle. During the breeding season (spring-summer), carbohydrates are used mainly through the pentose phosphate cycle and through the variant of the Krebs cycle known as the glutamic aspartic cycle. The metabolism operating in the oocytes was determined using the following parameters: 1) the capacity of isolated mitochondria to oxidize citrate and fumarate; 2) the enzymatic activities of phosphofructokinase (PFK) and glucose-6-phosphate dehydrogenase (G-6-PDH); and 3) citrate and ATP compartmentalization. The present paper shows that follicle-stimulating hormone (FSH) would be one of the factors responsible for summer metabolism, since ovary fractions obtained from winter specimens treated with the hormone acquired the metabolic characteristics corresponding to oocytes obtained from breeding-season animals. From dose-response, and response in the function of time curves, it could be assumed that the optimum doses and times were 0.1 micrograms FSH/ml of incubation medium and 30 min treatment, respectively. The metabolic effect of FSH upon oocytes could be mediated by the adenylate cyclase-cAMP system, since treatment of ovaric fractions with cAMP 10(-3) M reproduced the effects obtained with the hormone. In addition, 0.02 mg/ml tetracycline proved to block the effect of FSH. A direct metabolic action of FSH on body cavity oocytes (without follicle cells) was observed when submitting these oocytes to the same hormonal treatment.

Topics: Animals; Bufo arenarum; Cell Division; Citrates; Citric Acid; Female; Follicle Stimulating Hormone; Fumarates; Luteinizing Hormone; Mitochondria; Oocytes; Progesterone; Seasons

An unusual fumarate reductase was purified from cell extracts of Methanobacterium thermoautotrophicum and partially characterized. Two coenzymes previously isolated from cell extracts, 2-mercaptoethane-sulfonic acid (HS-CoM) and N-(7-mercaptoheptanoyl)threonine-O3-phosphate (HS-HTP), were established as direct electron donors for fumarate reductase. By measuring the consumption of free thiol, we determined that fumarate reductase catalyzed the oxidation of HS-CoM and HS-HTP; by the direct measurement of succinate and the heterodisulfide of HS-CoM and HS-HTP (CoM-S-S-HTP), we established that these compounds were products of the fumarate reductase reaction. A number of thiol-containing compounds did not function as substrates for fumarate reductase, but this enzyme had high specific activity when HS-CoM and HS-HTP were used as electron donors. HS-CoM and HS-HTP were quantitatively oxidized by the fumarate reductase reaction, and results indicated that this reaction was irreversible. Additionally, by measuring formylmethanofuran, we demonstrated that the addition of fumarate to cell extracts activated CO2 fixation for the formation of formylmethanofuran. Results indicated that this activation resulted from the production of CoM-S-S-HTP (a compound known to be involved in the activation of formylmethanofuran synthesis) by the fumarate reductase reaction.

Topics: Disulfides; Electron Transport; Electrophoresis, Polyacrylamide Gel; Euryarchaeota; Fumarates; Furans; Mercaptoethanol; Mesna; Oxidation-Reduction; Phosphothreonine; Substrate Specificity; Succinate Dehydrogenase; Succinates; Succinic Acid; Sulfhydryl Compounds

Modification by covalent FAD attachment to a histidine residue via an 8 alpha-(N3-histidyl)-riboflavin linkage occurs in several flavoenzymes. Among them is 6-hydroxy-D-nicotine oxidase (6-HDNO) of Arthrobacter oxidans and the flavoprotein subunits of the fumarate reductase and succinate dehydrogenase complex of Escherichia coli and other bacterial and eukaryotic cells. We found that 6-HDNO holoenzyme formation from apo-6-HDNO, monitored by [14C]FAD incorporation and increase in enzyme activity, can be mediated not only by phosphoenolpyruvate [Nagursky, H., Bichler, V. and Brandsch, R. (1988) Eur. J. Biochem. 177, 319-325], but also by one of the glycolytic intermediates glyceraldehyde-3-P, glycerate-3-P, or the intermediate in glycerol utilization by bacteria, glycerol-3-P. Apoflavoprotein of fumarate reductase and succinate dehydrogenase was obtained in an E. coli riboflavin-requiring strain (E. coli RR28rf) overexpressing the frdABCD or the sdhCDAB operon from the recombinant plasmids pGS39 and pGS141, respectively. In extracts obtained from these cells, flavoprotein flavinylation, analyzed as covalent [14C]FAD incorporation into the apoflavoprotein polypeptide by polyacrylamide gel electrophoresis and fluorography, was stimulated severalfold by the citric acid cycle intermediates citrate, isocitrate, succinate and fumarate. Our results suggest that covalent modification and thus activation of these enzymes is dependent on specific metabolic intermediates which may act as allosteric effectors in the reaction.

Topics: Citrates; Citric Acid Cycle; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Escherichia coli; Flavin-Adenine Dinucleotide; Flavoproteins; Fumarates; Isocitrates; Plasmids; Pyruvates; Spectrometry, Fluorescence; Succinate Dehydrogenase; Succinates

It was found that the nonspecific effect of ionic strength of the external solution on the enzymatic activity of E. coli cells consists in rapid changes in the permeability of cell membranes interacting with the substrate. This effect depends on the initial substrate concentration, i.e., ionic strength of the external solution, and is maintained for some time as the substrate concentration decreases. Chloramphenicol, a protein synthesis inhibitor, and sodium azide, a respiration inhibitor (300 micrograms/ml and 200 microM, respectively) do not change the enzymatic activity of E. coli cells during the synthesis of L-aspartic and L-malic acids from fumaric acid. The kinetic equations of L-aspartate and L-malate synthesis are described by equations of zero and intermediate (between zero and first) order, respectively.

Topics: Ammonia-Lyases; Aspartate Ammonia-Lyase; Aspartic Acid; Azides; Catalysis; Cell Membrane Permeability; Chloramphenicol; Escherichia coli; Fumarate Hydratase; Fumarates; Kinetics; Malates; Osmolar Concentration; Sodium Azide; Substrate Specificity

Formation of adenine nucleotides, IMP, malate + fumarate, ammonia, adenosine, and inosine + hypoxanthine + uric acid were measured in cytosolic extracts from renal cortex and medulla. The order of substrate addition was IMP, then 2-deoxyglucose, then P-creatine. Compared with cortex, medulla showed greater rates of formation of adenosine triphosphate (ATP) from P-creatine, of adenosine monophosphate (AMP) from 2-deoxyglucose, and of total adenine nucleotides from IMP. These results suggest that the purine nucleotide cycle is more active in medulla than in cortex. This cycle may provide a mechanism in medulla for storing purine nucleotides which can be used to restore ATP pools in the relatively hypoxic conditions of this part of the kidney.

Topics: Adenosine Monophosphate; Adenosine Triphosphate; Animals; Aspartic Acid; Cytosol; Fumarates; In Vitro Techniques; Inosine Monophosphate; Kidney Cortex; Kidney Medulla; Malates; Purine Nucleotides; Rats; Rats, Inbred Strains

Comparison of hindlimb muscles of rats flown on Spacelab-3 or tail-traction-suspended showed that 11-17 h reloading post-flight might have altered the results. Soleus atrophied, plantaris, gastrocnemius and extensor digitorum longus grew slower, and tibialis anterior grew normally. In both flight and simulated soleus and plantaris, higher tyrosine and greater glutamine/glutamate ratio indicated negative protein balance and increased glutamine production, respectively, relative to controls. Aspartate was lower in these muscles. Reloading generally decreased tyrosine, but increased aspartate and glutamine/glutamate. These data showed that at 12 h of reloading after flight is characterized by reversal to varying extents of effects of unloading.

Topics: Adaptation, Physiological; Amino Acids, Dicarboxylic; Animals; Fumarates; Glycogen; Hindlimb Suspension; Male; Muscle Proteins; Muscle, Skeletal; Muscular Atrophy; Rats; Research Design; Space Flight; Tail; Tyrosine; Weightlessness; Weightlessness Simulation

O2- generation in mitochondrial electron transport systems, especially the NADPH-coenzyme Q10 oxidoreductase system, was examined using a model system, NADPH-coenzyme Q1-NADPH-dependent cytochrome P-450 reductase. One electron reduction of coenzyme Q1 produces coenzyme Q1-. and O2- during enzyme-catalyzed reduction and O2+ coenzyme Q1-. are in equilibrium with O2- + coenzyme Q1 in the presence of enough O2. The coenzyme Q1-. produced can be completely eliminated by superoxide dismutase, identical to bound coenzyme Q10 radical produced in a succinate/fumarate couple-KCN-submitochondrial system in the presence of O2. Superoxide dismutase promotes electron transfer from reduced enzyme to coenzyme Q1 by the rapid dismutation of O2- generated, thereby preventing the reduction of coenzyme Q1 by O2-. The enzymatic reduction of coenzyme Q1 to coenzyme Q1H2 via coenzyme Q1-. is smoothly achieved under anaerobic conditions. The rate of coenzyme Q1H2 autoxidation is extremely slow, i.e., second-order constant for [O2][coenzyme Q1H2] = 1.5 M-1.s-1 at 258 microM O2, pH 7.5 and 25 degrees C.

Topics: Anaerobiosis; Animals; Cattle; Electron Spin Resonance Spectroscopy; Electron Transport; Free Radicals; Fumarates; Hydrogen-Ion Concentration; Kinetics; Mitochondria; Mitochondria, Heart; Models, Biological; NADP; NADPH-Ferrihemoprotein Reductase; Oxidation-Reduction; Oxygen Consumption; Potassium Cyanide; Submitochondrial Particles; Succinates; Succinic Acid; Superoxide Dismutase; Superoxides; Ubiquinone; Vitamin K

Alternaria alternata produces the polyketides alternariol (AOH) and alternariol monomethyl ether (AME) during the stationary growth phase. Addition of 12 mM NaNO3 to the cultures before initiation of polyketide production reduced the AOH and AME content to 5 to 10% of that of controls. Glutamate and urea also reduced AOH and AME accumulation, whereas increasing the ionic strength did not affect the polyketide content. Adding NaNO3 after polyketide production had started did not inhibit further AOH accumulation, although over 90% of the added NO3- disappeared from the medium within 24 h. Activity of an AME-synthesizing enzyme, alternariol-O-methyltransferase (AOH-MT), appeared in control mycelia during the early stationary growth phase. No AOH-MT activity appeared in mycelia blocked in polyketide synthesis by addition of NaNO3. Later addition of NaNO3 reduced the AOH-MT specific activity to 50% of that of the control, whereas the total of activity per mycelium was the same. The AOH-MT activity in vitro was not affected by 100 mM NaNO3. The results suggest that nitrogen in some way inhibited the formation of active enzymes in the polyketide-synthesizing pathway in A. alternata when it was added before these enzymes were formed.

Topics: Alternaria; Fumarates; Glutamates; Glutamic Acid; Hydrogen-Ion Concentration; Lactones; Mitosporic Fungi; Mycotoxins; Nitrates; Nitrogen; Urea

Two mutant strains of Escherichia coli, AK11 and AK22, express normal levels of hydrogenase activity, assayed by deuterium exchange, when grown on glucose or complex medium but cannot reduce methyl viologen by H2 nor grow on fumarate plus H2. The mutant strains also lack formate hydrogenlyase and formate dehydrogenase activities. The mutation in these strains was located near minute 17 of the genome map and a single mutation was shown to be responsible for loss of both hydrogen uptake and formate-related activities. Membrane vesicles and solubilized membranes of strains AK11 and AK22 were capable of methyl viologen reduction by H2 and had the normal complement of hydrogenase isoenzymes 1 and 2. Intact cells of the mutant strains could reduce fumarate by H2 but could not grow under these conditions. A plasmid, pAK11, was isolated, as well as smaller plasmids derived from it, which restored the hydrogen uptake activities in the two mutant strains, the smallest active DNA fragment being 1.4 kb. The formate activities were partially restored by some of the plasmids. The plasmids which restored hydrogen uptake activities led to synthesis of a polypeptide of subunit molecular mass 30 kDa.

Topics: Bacterial Proteins; Cloning, Molecular; Culture Media; DNA, Bacterial; Escherichia coli; Fumarates; Genes, Bacterial; Hydrogen; Mutation; Phenotype; Plasmids; Restriction Mapping

The effect of dietary citric acid (CA) and fumaric acid (FA) on pig weight gain (ADG) and gain/feed (G/F) was studied in two trials using 192 crossbred, 4-wk-old weaning pigs. Three dietary levels (0, 1.5 or 3.0%) of either FA (Trial 1) or CA (Trial 2) with or without an antibiotic supplement (110 mg chlortetracycline, 110 mg sulfamethazine and 55 mg penicillin/kg diet) formed six treatment combinations in each trial. These six diets were fed to two replicate pens of eight pigs each for a 4-wk period. In Trial 1, ADG was improved (P less than .01) during wk 1, and G/F was improved during wk 1 (P less than .01) as well as during wk 1 to 2 (P less than .05) for pigs consuming FA-supplemented diets. In Trial 2, CA had no beneficial influence on ADG during the 4-wk trial. However, feed intake during wk 1 was depressed (P less than .05) by adding CA, as was G/F during wk 1 to 2 (P less than .05). Based on these results, FA was selected to be used in a nutrient balance study. Twelve 4-wk-old weanling pigs were fed one of three diets: control (C), C + 1.5% FA, or C + antibiotic supplement (A). Diet digestible energy (DE), ME and N-corrected ME (MEN) were not different among treatments. Nitrogen balance, percentage N retained and apparent N digestibility were not affected by dietary treatment. Calcium balance and percentage of Ca retained were unaffected by diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animal Feed; Animals; Anti-Bacterial Agents; Body Weight; Citrates; Citric Acid; Fumarates; Swine

Two feeding trials were carried out to determine the potential use of fumaric acid and calcium formate in diets for broiler chickens. In the first study using male broiler chicks fed to 21 days of age, fumaric acid was added at 0, .5, 1.0 and 1.5% and calcium formate was added at 0, .72, 1.48, 2.20, and 2.89%. All combinations of fumaric acid and calcium formate were evaluated in a 4 x 5 factorial arrangement of treatments. Addition of .5 or 1.0% fumaric acid significantly (P less than .05) improved body weights of broilers but did not influence feed utilization. Addition of calcium formate at levels greater than .72% significantly reduced both body weight and feed utilization. There was no significant interaction between levels of fumaric acid and calcium formate. In a second trial, male and female broiler chicks were grown to 49 days of age using diets containing 0, .5, 1.0, and 1.5% calcium formate. Addition of .5 or 1.0% calcium formate had no significant effect on weight gains or feed utilization. Addition of 1.5% calcium formate significantly reduced body weights of both males and females at 21 and 42 days. At 49 days of age, body weights of males fed 1.5% calcium formate did not differ from those of males fed the control diet, but were significantly less than those of males fed diets with .5 or 1.0% calcium formate. Female body weights at 49 days of age were significantly depressed by addition of 1.5% calcium formate.

Topics: Animals; Body Weight; Chickens; Diet; Formates; Fumarates; Male

The b haems of the bc1 complex of bovine heart mitochondria were poised with succinate and fumarate so that only the high-potential haem (b-562) was reduced, and then isolated from further redox exchange with the ubiquinone pool by adding antimycin and myxothiazol. A transmembrane electric potential difference was then developed, either by electron flow from [Ru(NH3)6]Cl2 to oxygen or by ATP hydrolysis. The small difference spectrum, caused by the electric field, indicated 32-55% oxidation of b-562 with concomitant reduction of b-566. No lag greater than 0.1 s was detectable between the initiation of respiration and the development of the difference spectrum, thus providing a direct demonstration of (fairly) rapid electron transfer between the b haems.

Topics: Animals; Antimycin A; Cattle; Cytochrome b Group; Electron Transport; Escherichia coli Proteins; Fumarates; Membrane Potentials; Methacrylates; Mitochondria, Heart; Oxidation-Reduction; Spectrophotometry; Succinates; Succinic Acid; Thiazoles

The effect of twelve detergents on aspartate ammonia-lyase activity of Escherichia alcalescens used for the production of L-aspartic acid was tested. Best permeabilization was found with Triton X-100, Slovafol 910 and Corona, a mixed commercial preparation. In contrast to Triton X-100 and Slovafol 910, a much narrower range of suitable concentrations was observed with Corona.

Topics: Ammonia-Lyases; Aspartate Ammonia-Lyase; Aspartic Acid; Biotransformation; Catalysis; Detergents; Enzyme Activation; Escherichia; Escherichia coli; Fumarates; Octoxynol; Polyethylene Glycols; Surface-Active Agents

N-Phenyl-1-naphthylamine (NPN), a reagent which has been used previously to probe the fluidity or microviscosity of the membrane lipids of intact cells of Escherichia coli, was found to respond to metabolic changes in everted inner membrane vesicles from this organism. NPN was bound to the vesicles to produce a steady-state level of fluorescence intensity. Addition of substrate or ATP did not alter the fluorescence. However, following complete removal of oxygen from the medium by oxidation of substrate through the respiratory chain, there was an increase in the fluorescence of NPN. Reoxidation of the components of the respiratory chain by the addition of oxygen, ferricyanide, fumarate or nitrate decreased fluorescence to the steady-state level until the oxidant had been completely reduced. The fluorescence changes were insensitive to the state of energization of the membrane. It is proposed that NPN responds to the state of reduction of components of the respiratory chain either directly by reacting with a component of the chain or indirectly through an effect transmitted to the membrane by a change in the conformation of respiratory chain components.

Topics: 1-Naphthylamine; Adenosine Triphosphate; Anaerobiosis; Electron Transport; Escherichia coli; Ferricyanides; Fluorescence; Fluorescent Dyes; Fumarates; Intracellular Membranes; Lactates; Lactic Acid; Naphthalenes; Nigericin; Nitrates; Oxidation-Reduction; Oxygen; Potassium Cyanide; Quinacrine

The fumarate reductase enzyme complex, encoded by the frdABCD operon, allows Escherichia coli to utilize fumarate as a terminal electron acceptor for anaerobic oxidative phosphorylation. To analyze the expression of fumarate reductase, protein and operon fusions were constructed between the frdA and the lacZ genes and introduced onto the E. coli chromosome at the lambda attachment site. Expression of beta-galactosidase from either fusion was increased 10-fold during anaerobic versus aerobic cell growth, increased an additional 1.5-fold by the presence of fumarate, the substrate, and decreased 23-fold by nitrate, a preferred electron acceptor. The addition of trimethylamine-N-oxide as an electron acceptor did not significantly alter frdA'-'lacZ expression. Control of frd operon expression is therefore exerted at the transcriptional level in response to the availability of the electron acceptors oxygen, fumarate, and nitrate. Anaerobic induction of frdA'-'lacZ expression was impaired in an fnr mutant and was restored when the fnr+ gene was provided in trans, thus establishing that the fnr gene product, Fnr, is responsible for the anaerobic activation of frd operon expression. Nitrate repression of frdA'-'lacZ expression was observed under either aerobic or anaerobic cell growth conditions in both wild-type and fnr mutant strains, demonstrating that the mechanism for nitrate repression is independent of nitrate respiration and oxygen control imparted by Fnr. Studies performed with a fnr'-'lacZ protein fusion confirmed that the fnr gene is expressed both aerobically and anaerobically. A model is proposed for the regulation of frdABCD operon expression in response to the availability of the alternate terminal electron acceptors oxygen, nitrate, and fumarate.

Topics: Aerobiosis; Anaerobiosis; Chromosomes, Bacterial; Electron Transport; Escherichia coli; Fumarates; Gene Expression Regulation; Genes, Bacterial; Methylamines; Nitrates; Operon; Oxygen; Recombinant Fusion Proteins; Repressor Proteins; Succinate Dehydrogenase

Topics: Bacteroides; Campylobacter; Culture Media; Eikenella corrodens; Formates; Fumarates; Vibrio

It has been demonstrated that perfusion of myocardium with glutamic acid or tricarboxylic acid cycle intermediates during hypoxia or ischemia, improves cardiac function, increases ATP levels, and stimulates succinate production. In this study isolated adult rat heart cells were used to investigate the mechanism of anaerobic succinate formation and examine beneficial effects attributed to ATP generated by this pathway. Myocytes incubated for 60 min under hypoxic conditions showed a slight loss of ATP from an initial value of 21 +/- 1 nmol/mg protein, a decline of CP from 42 to 17 nmol/mg protein and a fourfold increase in lactic acid production to 1.8 +/- 0.2 mumol/mg protein/h. These metabolite contents were not altered by the addition of malate and 2-oxoglutarate to the incubation medium nor were differences in cell viability observed; however, succinate release was substantially accelerated to 241 +/- 53 nmol/mg protein. Incubation of cells with [U-14C]malate or [2-U-14C]oxoglutarate indicates that succinate is formed directly from malate but not from 2-oxoglutarate. Moreover, anaerobic succinate formation was rotenone sensitive. We conclude that malate reduction to succinate occurs via the reverse action of succinate dehydrogenase in a coupled reaction where NADH is oxidized (and FAD reduced) and ADP is phosphorylated. Furthermore, by transaminating with aspartate to produce oxaloacetate, 2-oxoglutarate stimulates cytosolic malic dehydrogenase activity, whereby malate is formed and NADH is oxidized. In the form of malate, reducing equivalents and substrate are transported into the mitochondria where they are utilized for succinate synthesis.

Topics: Adenosine Triphosphate; Animals; Cell Survival; Chromatography, High Pressure Liquid; Deoxyglucose; Fumarates; Hypoxia; Ketoglutaric Acids; Malates; Myocardium; Phosphocreatine; Rats; Succinates; Succinic Acid

An inhibitory effect of fumaric acid (FA) on hepatocarcinogenesis was examined in rats fed thioacetamide (TAA). A group of male DONRYU rats were fed TAA at a level of 0.035% in the diet for 40 weeks and then fed a basal diet for 40 weeks. Hepatic carcinomas developed in 9 of 41 animals of this group fed TAA alone. The effect of FA on the carcinogenesis was examined in 2 groups fed both TAA and FA; one group of rats were fed FA at 1% in a basal diet after ingestion of TAA, and another group of rats were fed TAA plus a supplement of 1% FA in the diet. The inhibitory effect of FA on TAA carcinogenesis was so marked that no hepatic carcinomas were found in both groups fed FA in combination with TAA.

Topics: Acetamides; Animals; Fumarates; Liver; Liver Neoplasms, Experimental; Male; Methyldimethylaminoazobenzene; Rats; Thioacetamide

A male infant, whose parents were first cousins, presented at 6 mo of age with hypotonia, microcephaly, and delayed development. He was found to have large amounts of fumaric and succinic acids present in the urine. In lysed cultured skin-fibroblast preparations, the activity of fumarase was found to be 22.7% of that in controls. Cell fractionation by homogenization and by digitonin treatment indicated that the residual activity in the cells of the patient was primarily located in the mitochondrial fraction rather than in the cytosolic fraction. Isoelectric focusing of fibroblast extracts showed that six bands of fumarase activity were discernible in control cell lines, two of them cytosolic with pI's of 5.53 and 5.60 and four of them mitochondrial with a pI of 5.65-6.8. In contrast, isoelectric focusing of fibroblast extracts from the fumarase-deficient patient showed only a single band of activity with a pI corresponding to the mitochondrial type seen in the controls. Immunoprecipitation of proteins with rabbit antifumarase antibody in (35S)-methionine-labeled fibroblasts indicated that a protein of correct size (Mr = 44,000 daltons) corresponding to fumarase was synthesized in similar amounts in both the patients and controls. It is proposed that in the patient's cells a single active species of fumarase that is mitochondrial in location is synthesized. Since it is known that mitochondrial and cytosolic fumarases are encoded by the same gene but differ slightly in amino acid sequence, it is possible that a point mutation might explain these findings.

Topics: Abnormalities, Multiple; Cells, Cultured; Consanguinity; Cytosol; Fibroblasts; Fumarate Hydratase; Fumarates; Humans; Infant; Intellectual Disability; Isoelectric Focusing; Isoenzymes; Male; Microcephaly; Mitochondria; Muscle Hypotonia

Succinate dehydrogenase was purified from the particulate fraction of Desulfobulbus. The enzyme catalyzed both fumarate reduction and succinate oxidation but the rate of fumarate reduction was 8-times less than that of succinate oxidation. Quantitative analysis showed the presence of 1 mol of covalently bound flavin and 1 mol of cytochrome b per mol of succinate dehydrogenase. The enzyme contained three subunits with molecular mass 68.5, 27.5 and 22 kDa. EPR spectroscopy indicated the presence of at least two iron sulfur clusters. 2-Heptyl-4-hydroxy-quinoline-N-oxide inhibited the electron-transfer between succinate dehydrogenase and a high redox potential cytochrome c3 from Desulfobulbus elongatus.

Topics: Bacteria; Bacterial Proteins; Fumarates; Membrane Proteins; Substrate Specificity; Succinate Dehydrogenase; Succinates; Succinic Acid

Penetration of fumarate into rat brain mitochondria has been investigated, as required in brain ammoniogenesis. Mitochondria swell in ammonium fumarate and this swelling is increased by both Pi and malate. According to a carrier mediated process, fumarate translocation, which occurs in exchange with intramitochondrial malate or Pi shows saturation characteristics. By photometrically investigating the kinetics of fumarate/malate, fumarate/Pi and malate/Pi exchanges, different Km values were obtained (10, 22 and 250 microM, respectively), whereas no significant difference was found for Vmax values (40 nmol NAD(P)+ reduced/min X mg protein). This suggests that fumarate and malate share a single carrier to enter mitochondria, namely the dicarboxylate carrier. Both comparison made of the Vmax values and inhibition studies exclude a fumarate translocation via either the tricarboxylate carrier, whose occurrence in brain is here demonstrated, or oxodicarboxylate carrier. Kinetic investigation of the dicarboxylate translocator shows the existence of thiol group/s and metal ion/s at or near the substrate binding sites. The experimental findings are discussed in the light of fumarate uptake in vivo in brain ammoniogenesis.

Topics: Animals; Biological Transport; Brain; Carrier Proteins; Chemical Phenomena; Chemistry; Fumarates; In Vitro Techniques; Malates; Male; Mitochondria; Phenanthrolines; Phosphates; Rats; Rats, Inbred Strains; Zinc

Seventeen patients with recent cerebral infarction were included in the study. The general condition of the patients was evaluated within 3 days after the incidence (rating 1) and 7-9 days after the incidence (rating 2) using a clinical rating scale for internal, psychiatric and neurological findings. The statistical evaluation showed a clinical improvement of the score of 32%. The extent of the brain lesions was determined by cranial computed tomography. Routine analysis of the cerebrospinal fluid included determination of protein, cell count, cell type, glucose, lactic acid, and hemoglobin. In addition, spinal fluid and serum concentration of pyruvic acid, citric acid, fumaric acid, alpha-ketoglutaric acid and alpha-keto-beta-methylvaleric acid (3-MKV) were determined at the two rating times. CSF and serum values of fumaric acid, 3-MKV and pyruvic acid correlated at rating 1, while at rating 2 a correlation existed only for fumaric acid. A trend analysis infarction - postinfarction period showed the concentration of all metabolites to decrease significantly. This is interpreted as a reconstitution of the initially disturbed blood brain barrier function.

Topics: Aged; Aged, 80 and over; Blood-Brain Barrier; Cerebral Infarction; Citrates; Citric Acid; Female; Follow-Up Studies; Fumarates; Humans; Keto Acids; Ketoglutaric Acids; Lactates; Lactic Acid; Male; Middle Aged; Pyruvates; Pyruvic Acid

The reactions of cytochrome b561 with other redox-active components of the adrenal chromaffin granule were examined using optical difference spectroscopy. It was shown that there is no direct electron transfer between the cytochrome and dopamine beta-hydroxylase, but that in the presence of ascorbate, turnover of dopamine beta-hydroxylase causes an oxidation of the cytochrome, which is partially reversed by the action of the mitochondrial NADH:A-. oxidoreductase. Thus, these three proteins may be functionally coupled via ascorbate. A quantitative study of the relationship between the redox state of the cytochrome and the ascorbate radical concentration measured by EPR showed that ascorbate reduces the cytochrome in a one-electron transfer reaction. Generation of a proton electrochemical gradient across the granule membrane causes only a small (20 mV) increase in the cytochrome midpoint potential suggesting the cytochrome is not a proton pump. The data are consistent with a model in which cytochrome b561, by reacting with ascorbate or ascorbate free radical on either side of the granule membrane, could couple the ascorbate-consuming reaction of the dopamine beta-hydroxylase inside the chromaffin granule to the ascorbate-regenerating reaction of the NADH:A-. oxidoreductase on the outer mitochondrial membrane. The H+-ATPase of the granule membrane could both drive the flow of electrons in the direction from cytosol to granule and replenish protons consumed by the turnover of dopamine beta-hydroxylase inside the granule.

Topics: Animals; Ascorbic Acid; Cattle; Chromaffin Granules; Chromaffin System; Cytochrome b Group; Dopamine; Dopamine beta-Hydroxylase; Electron Spin Resonance Spectroscopy; Epinephrine; Ethylmaleimide; Free Radicals; Fumarates; Fusaric Acid; Intracellular Membranes; NAD; Norepinephrine; Oxidation-Reduction; Spectrophotometry; Tyramine

The Mu dl (ApR lac) bacteriophage was used to generate mutants of Escherichia coli which were defective in formate hydrogenlyase. Three mutants were chosen for further analysis: they lacked hydrogenase (hydrogen: benzyl viologen oxidoreductase) activity, but produced normal levels of fumarate reductase activity and two- to three-fold reduced levels of benzyl viologen (BV)-dependent formate dehydrogenase activity. Two of them (hydC) were shown to contain about 4-fold reduced amounts of formate hydrogenlyase and fumarate-dependent H2 uptake activities. The third one (hydD) was totally devoid of both activities. Their insertion sites were located at 77 min on the E. coli map. Subdivision of these mutants into two classes was subsequently based on the restoration capacity of hydrogenase activity with high concentration of nickel in the growth media. Addition of 500 microM NiCl2 led to a complete recovery of hydrogenase activity, and to the concomitant restoration of normal BV-linked formate dehydrogenase, formate hydrogenlyase and fumarate-dependent H2 uptake activities in the hydC mutants. The hydD mutant was insensitive to the effect of nickel. Expression of the lac operon in hydC and hydD mutants was induced by anaerobiosis. It was not increased by the addition of formate under anaerobic conditions. The presence of nitrate resulted in slightly reduced beta-galactosidase activities in the hydC mutants, whereas those found in the hydD mutant reached only one third of the level obtained in its absence. Fumarate had no effect on both classes. Moreover, in contrast to the hydD locus, the hydC::Mu dl fusions were found to be dependent upon the positive control exerted by the nirR gene product and were totally repressed by an excess of nickel. In addition, the low levels of overall hydrogenase-dependent activities found in a nirR strain were also relieved by the presence of nickel. Our results strongly suggest that the pleiotropic regulatory gene nirR is essential for the expression of a gene (hydC) involved in either transport or processing of nickel in the cell, whose alteration leads to a loss of hydrogenase activity.

Topics: Alleles; Benzyl Viologen; beta-Galactosidase; Conjugation, Genetic; Escherichia coli; Formate Dehydrogenases; Formates; Fumarates; Gene Expression Regulation; Genes, Bacterial; Hydrogen; Hydrogenase; Mutation; Nickel; Transcription, Genetic; Transduction, Genetic

The effect of fumaric acid was examined on DNA synthesis in hepatocytes or hepatoma cells from rats treated with toxic agents. Male Donryu rats were injected with mitomycin C or aflatoxin B1, singly or in combination with fumaric acid. After a specified period, hepatocytes were isolated from the liver by the collagenase perfusion method and placed in culture, and their activities for DNA synthesis were measured. The iv injection of rats with mitomycin C (0.5 mg/kg) reduced the semiconservative DNA synthesis of the hepatocytes, but simultaneous dosing of fumaric acid (40 mg/kg) enhanced the recovery of the DNA synthesis. The DNA synthesis of hepatoma cells, a 3'-methyl-4-(dimethylamino)azobenzene-induced transplantable cell line growing in the abdominal ascites of rats, was also reduced by the iv injection of mitomycin C but, in contrast to that of the hepatocytes, was little influenced by the simultaneous dosing of fumaric acid. The ip injection of fumaric acid also reduced the toxicity of aflatoxin B1 (0.25 mg/kg, ip), preventing the reduction of DNA synthesis as well as the occurrence of nuclear degenerative changes in the aflatoxin B1-exposed hepatocytes.

Topics: Aflatoxin B1; Aflatoxins; Animals; Cell Line; Cell Nucleus; DNA; Fumarates; Liver; Liver Neoplasms, Experimental; Male; Mitomycin; Mitomycins; Rats

A new method for the determination of dissociation rates of enzyme-substrate complexes has been developed. The rate of exchange of a labeled product back into the substrate is measured during catalysis of the forward reaction when the forward reaction is kept far from equilibrium by the enzymatic removal of the nonexchanging product. The ratio of the exchange rate and the net rate for product formation is then determined at various concentrations of the exchanging product. A plot of this ratio is a diagnostic indication of the kinetic mechanism and the relative rates of product dissociation from the binary and ternary enzyme complexes. This technique has been applied to the reaction catalyzed by bovine liver argininosuccinate lyase. The ratio for the rate of exchange of fumarate into argininosuccinate and the net rate for product formation was found to increase with the concentration of fumarate but to reach a limit of 3.3. The ratio of rates was half-maximal at 36 mM fumarate. The data have been interpreted to indicate the argininosuccinate lyase has a random kinetic mechanism. The calculated lower limit for the rate of release of arginine from the enzyme-fumarate-arginine complex is 0.35 times as fast as the Vmax in the reverse direction. The rate of release of arginine from the enzyme-arginine binary complex is 210 times faster than Vmax in the reverse direction.

Topics: Animals; Argininosuccinate Lyase; Argininosuccinic Acid; Cattle; Fumarates; Kinetics; Liver; Lyases; Magnetic Resonance Spectroscopy; Mathematics; Radioisotope Dilution Technique

The kinetics of the action of fumarase on L-malate and fumarate were investigated at constant ionic strength. This was done to evaluate reports that fumarase follows simple Michaelis-Menten kinetics. However, when pH, buffer concentration and ionic strength are all maintained at constant values, the Lineweaver-Burk plots exhibit pronounced downward curvature, characteristic of negative kinetic co-operativity.

Topics: Animals; Fumarate Hydratase; Fumarates; Kinetics; Malates; Myocardium; Osmolar Concentration; Swine

Rhizobium sp. strain ORS571 conducts synergistic, free-living N2 fixation and nicotinate oxidation. Explicitly, ORS571 is able to fix N2 aerobically because 6-OH-nicotinate acts as an intracellular O2 sink. Because 6-OH-nicotinate oxidation is mandatory for aerobic, free-living N2 fixation and because the synergistic processes yield ammonium from substrates (as the nitrogen source for growth), ORS571 is not a diazotroph.

Topics: Fumarates; Mutation; Niacin; Nicotinic Acids; Nitrogen; Nitrogen Fixation; Quaternary Ammonium Compounds; Rhizobium

Developing Nicotiana tabacum L. cv. Wisconsin-38 callus grown on modified Murashige-Skoog (MS) medium with Kao organic acids (pyruvic, citric, malic and fumaric acids) contains abnormally high levels of nornicotine and total alkaloids when compared with the leaves of the donor plant. Nornicotine/nicotine ratios observed during callus development suggest that nicotine is converted into nornicotine in the callus, with subsequent movement of alkaloids into roots formed on the callus and into the agar medium. Addition of Kao organic acids to the medium increases alkaloid levels, but cannot account for the abnormal increase in nicotine demethylation. This study thus reports two new findings: (a) that the total alkaloid content of tobacco callus can be greatly enhanced to 3.75% on a dry weight basis by exogenous organic acids, and (b) that endogenous nornicotine can accumulate in tobacco tissue cultures.

Topics: Alkaloids; Anabasine; Carboxylic Acids; Citric Acid; Culture Media; Culture Techniques; Fumarates; Malates; Nicotiana; Nicotine; Plant Roots; Plants, Toxic; Pyridines; Pyrrolidines; Pyruvic Acid

Topics: Animals; Carbon Radioisotopes; Fumarates; Glutamates; Glutamic Acid; Male; Mitochondria, Liver; Oxaloacetates; Pyruvates; Pyruvic Acid; Rats; Rats, Inbred Strains

A radioligand that selectively labels beta 2-adrenoceptors, 3H-ICI 118,551 (3H-ICI), is introduced. Experiments were performed on guinea-pig tissues. The binding characteristics of 3H-ICI on lung membrane particles are compared with the blocking characteristics of ICI 118,551 against the tracheo-relaxant effects of (-)-noradrenaline, (-)-adrenaline and (+/-)-fenoterol. Binding to both beta 1- and beta 2-adrenoceptors were also performed with 3H-(-)-bupranolol on lung and ventricular myocardium. The binding inhibition characteristics of unlabelled ICI 118,551 on ventricle were compared with its characteristics as antagonist of the positive chronotropic effects of (-)-noradrenaline, (-)-adrenaline and (+/-)-fenoterol in spontaneously beating right atria. 1. ICI 118,551 blocked more the relaxant effects of (+/-)-fenoterol and (-)-adrenaline than those of (-)-noradrenaline on trachea. The positive chronotropic effects of (+/-)-fenoterol on sinoatrial node were blocked more than those of both (-)-adrenaline and (-)-noradrenaline. A non-linear regression analysis of blocking data with ICI 118,551 according to the model of Lemoine and Kaumann (1983) revelas that both beta 1- and beta 2-adrenoceptors contribute to the tracheo-relaxant and positive chronotropic effects of agonists. The estimated equilibrium dissociation constants pKB (-log KB = pKB; mol/l) were 7.1 and 9.6 for beta 1- and beta 2-adrenoceptors, respectively. Tracheal beta 2-adrenoceptors contribute 99%, 97% and 7%, sinoatrial beta 2-adrenoceptors contribute 76%, 3% and 0% to the fractional stimuli induced by (+/-)-fenoterol, (-)-adrenaline and (-)-noradrenaline, respectively. 2. 3H-ICI associated to beta 2-adrenoceptors of lung membranes with a kon of 0.521 X nmol-1 X min-1 and dissociated with a koff of 0.19 min-1. 3H-ICI bound to lung beta 2-adrenoceptors with an equilibrium dissociation constant pKL* of 9.2. Unlabelled ICI 118,551, (-)-bupranolol, (+)-bupranolol, (-)-adrenaline, (-)-noradrenaline and (+/-)-fenoterol competed with 3H-ICI for lung beta 2-adrenoceptors with pKL-values of 9.0, 9.4, 8.1, 5.9, 4.9 and 6.4, respectively. 3. 3H-(-)-bupranolol associated to beta-adrenoceptors of lung membranes with a kon 1.21 X nmol-1 X min-1 and dissociated with a koff of 0.26 min-1. 3H-(-)-bupranolol bound to lung beta 2-adrenoceptors and to heart beta 1-adrenoceptors with a pKL of 9.6 and a pKL of 8.8, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Adrenergic beta-Antagonists; Animals; Bisoprolol; Bupranolol; Female; Fumarates; Glutamates; Glutamic Acid; Guinea Pigs; In Vitro Techniques; Kinetics; Lung; Male; Membranes; Myocardium; Propanolamines; Pyruvates; Pyruvic Acid; Receptors, Adrenergic, beta; Sinoatrial Node; Trachea

Topics: Aconitic Acid; Animals; Anti-Inflammatory Agents, Non-Steroidal; Female; Fumarates; Male; Medicine, Chinese Traditional; Medicine, East Asian Traditional; Mice; Plants, Medicinal; Rabbits; Rats; Succinates; Succinic Acid

The fumarate reductase enzyme complex allows Escherichia coli to grow anaerobically with fumarate as a terminal electron acceptor for oxidative phosphorylation when the preferred compounds oxygen and nitrate are not available. We used the pKO promoter test vectors to identify a single promoter for the frdABCD genes which encode fumarate reductase. Expression of galactokinase from the frd promoter-galK operon fusion plasmid was repressed by oxygen and by nitrate and was induced by fumarate, indicating that frd gene expression is regulated at the transcriptional level by these terminal electron acceptors. S1 nuclease analysis, using a single-stranded DNA probe from the frd promoter region and mRNA isolated from a fumarate reductase-induced culture, revealed that the frd mRNA transcript initiates with an adenine residue 93 bases prior to the start of frdA translation. No promoters internal to the frd genes were revealed with the plasmid promoter screening system. S1 nuclease analysis revealed that the frd mRNA terminates in a uridine-rich region centered at 46 bases after the last codon of frdD. A stem and loop structure previously described as the growth rate-dependent attenuator for the linked ampC gene precedes the frd mRNA terminus. This result confirms the proposal that the stem and loop structure serves the dual role of a frd terminator anaerobically and an ampC attenuator aerobically. The four frd genes encoding the subunits of the fumarate reductase complex thus comprise an operon which is regulated at the transcriptional level in response to the cellular availability of the alternate electron acceptors oxygen, nitrate, and fumarate.

Topics: Base Sequence; DNA Restriction Enzymes; DNA, Bacterial; DNA, Single-Stranded; Endonucleases; Escherichia coli; Fumarates; Galactokinase; Gene Expression Regulation; Nitrates; Nucleic Acid Conformation; Operon; Oxidative Phosphorylation; Oxygen; RNA, Messenger; Single-Strand Specific DNA and RNA Endonucleases; Succinate Dehydrogenase; Transcription, Genetic

Six experiments involving 706 newly weaned 28- to 32-d-old pigs were conducted to evaluate the efficacy of copper (Cu) sulfate (to provide 250 mg/kg Cu), antibiotic-sulfa combinations [chlortetracycline, 110 mg/kg + penicillin, 55 mg/kg + sulfamethazine, 110 mg/kg; i.e., Aureo-Sulfa-Penicillin (ASP) or tylosin, 110 mg/kg + sulfamethazine, 110 mg/kg; i.e., Tylosin-Sulfa (TS)] and anhydrous citric or fumaric acid (.75 to 1.5% of the diet). The basal experimental diet was a 19% crude protein (CP)-fortified corn-soybean meal diet (1.08% lysine) containing 7% dried whey and 3% fish meal. Marked and consistent gain and gain/feed responses occurred from the Cu supplement, particularly during the first week postweaning. The antibiotic-sulfa combinations were less efficacious than Cu during the 1-wk postweaning stress period. During either the 1- or 3-wk growth periods, ASP and TS showed additivity with Cu in promoting rate and efficiency of weight gain. Liver Cu was increased by Cu addition to the diet, but neither ASP nor TS affected Cu deposition in the liver. In a factorial experiment involving 17% (1.01% lysine) or 20% CP (1.23% lysine) corn-soybean meal diets containing either no dried whey or an addition of 25% whey, Cu supplementation elicited marked improvements in rate and efficiency of weight gain, particularly in diets without added whey. Likewise, whey addition improved pig performance, especially when added to the diets containing no supplemental Cu.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Anti-Bacterial Agents; Body Weight; Citrates; Citric Acid; Copper; Copper Sulfate; Drug Combinations; Food Additives; Fumarates; Liver; Sulfonamides; Swine

Topics: Adult; Aged; Drug Evaluation; Female; Fumarates; Humans; Male; Middle Aged; Psoriasis

Four growth experiments were conducted to assess the effects of organic acid supplementation on performance of starter and finisher pigs. Three 4-wk starter experiments utilized 392 pigs fed simple corn-soybean meal diets. A fourth experiment employed 135 finisher pigs in a 6-wk study. Each of the starter experiments was initiated immediately after weaning; piglets were 30 +/- 3 d of age. In Exp. 1, weanling pigs fed a 19% crude protein, simple corn-soybean meal diet were compared with pigs fed similar diets supplemented with 2% propionic, fumaric, or citric acid. Addition of each acid improved (P less than .07) efficiency of gain, while propionate depressed (P less than .05) feed intake. Additions of 1, 2, 3 or 4% fumarate were made in Exp. 2, resulting in linear daily gain and feed efficiency improvements (P less than .05). In Exp. 3, a possible protein-sparing effect of fumaric acid was investigated. Increasing protein levels from 16 to 20% improved daily gain (P less than .01) and feed efficiency (P less than .0001); fumarate supplementation (2%) increased (P less than .01) gain:feed. However, there was no protein X fumaric acid interaction. In Exp. 4, no treatment effects were noted with performance of finisher pigs fed a 14% crude protein, corn-soybean meal diet was compared with that of pigs fed similar diets supplemented with 1.5 or 3% fumaric acid.

Topics: Animals; Body Weight; Citrates; Citric Acid; Dietary Proteins; Energy Metabolism; Female; Food, Fortified; Fumarates; Glycine max; Hydrogen-Ion Concentration; Male; Propionates; Swine; Zea mays

The thermodynamics of the conversion of aqueous fumarate to L-(-)-malate has been investigated using both heat conduction microcalorimetry and a gas chromatographic method for determining equilibrium constants. The reaction was carried out in aqueous Tris-HCl buffer over the pH range 6.3-8.0, the temperature range 25-47 degrees C, and at ionic strengths varying from 0.0005 to 0.62 mol kg-1. Measured enthalpies and equilibrium ratios have been adjusted to zero ionic strength and corrected for ionization effects to obtain the following standard state values for the conversion of aqueous fumarate 2- to malate 2- at 25 degrees C: K = 4.20 +/- 0.05, delta G degrees = -3557 +/- 30 J mol-1, delta H degrees = -15670 +/- 150 J mol-1, and delta C degrees p = -36 +/- J mol-1 K-1. Equations are given which allow one to calculate the combined effects of pH and temperature on equilibrium constants and enthalpies of this reaction.

Topics: Animals; Chickens; Fumarate Hydratase; Fumarates; Hydrogen-Ion Concentration; Kinetics; Malates; Myocardium; Thermodynamics

Fumarate permeation in isolated rat liver mitochondria was demonstrated by measuring malate and phosphate efflux caused by fumarate added externally to the mitochondrial suspension. The existence of two specific fumarate translocators, fumarate/malate and fumarate/phosphate, is shown here. These carriers are distinguished in the light of different kinetic parameters (Km values are 50 microM and 150 microM, and Vmax values are 17 and 40 nmoles/min X mg mitochondrial protein, respectively) and of differing sensitivity to non-penetrant compounds. Fumarate was found to cause oxaloacetate efflux from mitochondria by means of an indirect process which involves the cooperation of both fumarate/malate and malate/oxaloacetate translocators. Results are discussed in the light of the physiological role played by fumarate translocation in both ureogenesis and aminoacid metabolism.

Topics: Animals; Biological Transport, Active; Citric Acid Cycle; Ethylmaleimide; Fumarates; Kinetics; Malates; Malonates; Mersalyl; Mitochondria, Liver; Models, Biological; Phosphates; Rats; Succinates

Glucose and other hexoses as well as amino acids have been shown to inhibit the renal transport of phosphate (Pi). Although studies with renal brush border membrane vesicles showed that such an inhibitory effect on Pi transport is due to the dissipation of the Na electrochemical gradient, the mechanism(s) responsible for such an action in the intact cell is not clear. The present study examined the effects of tricarboxylic acid (TCA) cycle intermediates (succinate and fumarate) and acetate on the uptake of Pi and alpha-methylglucoside (AMG) at 37 degrees C by intact rabbit renal cells. These TCA cycle compounds significantly (p less than 0.05) inhibited the uptake of both Pi and AMG. In the presence of 5-10 mM succinate the ATP content of the renal cells increased by 40% (p less than 0.02). Inhibition of succinate-induced gluconeogenesis by 3-mercaptopicolinic acid did not modify the inhibition of Pi uptake. Studies with renal brush border membrane vesicles showed that succinate inhibited Pi uptake at 15 and 60 s but not at 1 s and only under conditions of Na gradient (outside greater than inside). Succinate did not inhibit Pi uptake during Na equilibrium conditions. The data demonstrate that the succinate-induced inhibition of the Pi uptake by intact proximal renal cells is not due to competition for metabolic energy, is not related to stimulation of gluconeogenesis nor due to allosteric interaction between Pi carrier and succinate transporter. The results support the notion that the inhibition of Pi uptake by succinate in the intact renal cell is due to dissipation of the Na chemical gradient.

Topics: Animals; Citric Acid Cycle; Fumarates; Glucose; Kidney; Male; Methylglucosides; Microvilli; Phosphates; Picolinic Acids; Rabbits; Sodium; Succinates; Succinic Acid

The kinetic mechanism of dopamine beta-hydroxylase (dopamine beta-monooxygenase EC 1.14.17.1) was studied either in the absence or the presence of the nonessential activator fumarate. In the absence of fumarate, intersecting initial velocity patterns were obtained, consistent with a sequential mechanism. In the presence of saturating concentrations of fumarate, initial velocity patterns became parallel. Other activating anions, such as acetate and chloride, could replicate the effects of fumarate. Since previous initial rate studies of dopamine beta-hydroxylase have been performed in the presence of saturating concentrations of fumarate, the present results may explain why parallel initial velocity patterns, apparently consistent with a ping-pong mechanism, have been so far observed. As a plausible mechanism of the anion effect it is proposed that activating anions induce saturation of the enzyme with oxygen.

Topics: Adrenal Glands; Animals; Cattle; Dopamine beta-Hydroxylase; Fumarates; Kinetics; Tyramine

The maximum Gibbs free energies of reverse electron transfer from succinate to NAD+ and from cytochrome c to fumarate driven by ATP hydrolysis in submitochondrial particles from beef heart were measured as a function of the Gibbs free energy of ATP hydrolysis. The ratio of the energies delta G'redox/delta G'ATP was 1.40 from succinate to NAD+ and 0.89 from cytochrome c to succinate. The ratio, equivalent to a thermodynamic P/2e-ratio, was dependent on whether the electrochemical proton gradient was primarily a membrane potential or a pH gradient for the cytochrome c to fumarate reaction. The results are consistent with H+/ATP = 3 for F1 ATPase, H+/2e- = 4 for NADH-CoQ reductase, and H+(matrix)/2e- = 2 for succinate-cytochrome c reductase.

Topics: Adenosine Triphosphate; Animals; Cattle; Cytochrome c Group; Electron Transport; Fumarates; Mathematics; Mitochondria; Mitochondria, Heart; NAD; Oxidation-Reduction; Submitochondrial Particles; Succinates; Succinic Acid

Enzyme activities forming extracellular products from succinate, fumarate, and malate were examined using washed cell suspensions of Pseudomonas fluorescens from chemostat cultures. Membrane-associated enzyme activities (glucose, gluconate, and malate dehydrogenases), producing large accumulations of extracellular oxidation products in carbon-excess environments, have previously been found in P. fluorescens. Investigations carried out here have demonstrated the presence in this microorganism of a malic enzyme activity which produces extracellular pyruvate from malate in carbon-excess environments. Although the three membrane dehydrogenase enzymes decrease significantly in carbon-limited chemostat cultures, malic enzyme activity was found to increase fourfold under these conditions. The regulation of malate dehydrogenase and malic enzyme by malate or succinate was similar. Malate dehydrogenase increased and malic enzyme decreased in carbon-excess cultures. The opposite effect was observed in carbon-limited cultures. When pyruvate or glucose was used as the carbon source, malate dehydrogenase was regulated similarly by the available carbon concentration, but malic enzyme activity producing extracellular pyruvate was not detected. While large accumulations of extracellular oxalacetate and pyruvate were produced in malate-excess cultures, no extracellular oxidation products were detected in succinate-excess cultures. This may be explained by the lack of detectable activity for the conversion of added external succinate to extracellular fumarate and malate in cells from carbon-excess cultures. In cells from carbon-limited (malate or succinate) cultures, very active enzymes for the conversion of succinate to extracellular fumarate and malate were detected. Washed cell suspensions from these carbon-limited cultures rapidly oxidized added succinate to extracellular pyruvate through the sequential action of succinate dehydrogenase, fumarase, and malic enzyme. Succinate dehydrogenase and fumarase activities producing extracellular products were not detected in cells from chemostat cultures using pyruvate or glucose as the carbon source. Uptake activities for succinate, malate, and pyruvate also were found to increase in carbon-limited (malate or succinate) and decrease in carbon-excess cultures. The role of the membrane-associated enzymes forming different pathways for carbon dissimilation in both carbon-limited and carbon-excess environments is discussed.

Topics: Ammonia; Carbohydrate Dehydrogenases; Cell Membrane; Citric Acid Cycle; Fumarate Hydratase; Fumarates; Glucose; Glucose 1-Dehydrogenase; Glucose Dehydrogenases; Malate Dehydrogenase; Malates; Oxaloacetates; Oxidation-Reduction; Pseudomonas; Pseudomonas fluorescens; Pyruvates; Pyruvic Acid; Succinate Dehydrogenase; Succinates; Succinic Acid

We investigated the viability of isolated rabbit renal cortical cells and the effect of metabolic intermediates on phosphate uptake. Glucose production and its stimulation by hormones was similar to that previously reported for renal tubules. The uptake of alpha-methylglucoside and phosphate was reduced by 90% when sodium was removed from the media. ATP content of the cells was 9.4 +/- 0.7 nmol/mg protein. Succinate and fumarate stimulated phosphate uptake in a dose-dependent manner by 30-60%. Valeric acid (1 mM) and butyric acid (10 mM) stimulated phosphate uptake by 20-30%. Glucose and fructose stimulated phosphate uptake by 18% but only at low concentrations (0.1 mM). Exogenous nucleotides had no effect on phosphate uptake at 0.2 mM, but inhibited 2.4 mM phosphate uptake at 2 mM. We conclude: rabbit renal cortical cells in suspension are metabolically and functionally viable; tricarboxylic acid cycle and glycolytic intermediates as well as short chain fatty acids can stimulate phosphate uptake, and exogenous nucleotides are hydrolyzed to produce free phosphate causing inhibition of isotopic phosphate uptake.

Topics: Animals; Fumarates; Gluconeogenesis; Glycolysis; Isoproterenol; Kidney Cortex; Male; Methylglucosides; NAD; Nucleotides; Phosphates; Rabbits; Succinates; Succinic Acid; Time Factors

Coupled spectrophotometric assays that monitor the formation of fumarate and ammonia in the direction of aspartate deamination and aspartate in the direction of fumarate amination were used to collect initial velocity data for the aspartase reaction. Data are consistent with rapid equilibrium ordered addition of Mg2+ prior to aspartate but completely random release of Mg2+, NH4+, or fumarate. In addition to Mg2+, Mn2+ can also be used as a divalent metal with Vmax 80% and a Kaspartate 3.5-fold lower than when Mg2+ is used. Monovalent cations such as Li+, K+, Cs+, and Rb+ are competitive vs. either aspartate or NH4+ but noncompetitive vs. fumarate. A primary deuterium isotope effect of about 1 on both V and V/Kaspartate is obtained with (3R)-L-aspartate-3-d, while a primary 15N isotope effect on V/Kaspartate of 1.0239 +/- 0.0014 is obtained in the direction of aspartate deamination. A secondary isotope effect on V of 1.13 +/- 0.04 is obtained with L-aspartate-2-d. In addition, a secondary isotope effect of 0.81 +/- 0.05 on V is obtained with fumarate-d2, while a value of 1.18 +/- 0.05 on V is obtained by using (2S,3S)-L-aspartate-2,3-d2. These data are interpreted in terms of a two-step mechanism with an intermediate carbanion in which C-N bond cleavage limits the overall rate and the rate-limiting transition state is intermediate between the carbanion and fumarate.

Topics: Ammonia; Ammonia-Lyases; Aspartate Ammonia-Lyase; Aspartic Acid; Enterobacteriaceae; Fumarates; Kinetics; Magnesium; Manganese; Mathematics

The protective effect of dicarboxylates on the active-site-directed inhibition of the membrane-bound succinate dehydrogenase by N-ethylmaleimide, steady-state kinetics methods for Ki and Ks determinations, and equilibrium studies were employed to quantitate the relative affinities of succinate, fumarate, malonate and oxaloacetate to the reduced and oxidized species of the enzyme. A more than 10-fold difference in the relative affinities of the reduced and oxidized succinate dehydrogenase to succinate, fumarate and oxaloacetate is found, whereas the reactivity of the active-site sulphydryl group does not depend on the redox state of the enzyme. The redox-state-dependent changes in the affinity of the membrane-bound succinate dehydrogenase to oxaloacetate can be quantitatively accounted for by a 10-fold increase in the rate of dissociation of the enzyme-inhibitor complex which occurs upon reduction of the enzyme. The data obtained give no support for either the existence of a sulphydryl group other than the active-site one important for the catalysis or for the presence of a separate dicarboxylate-specific regulatory site in the succinate dehydrogenase molecule.

Topics: Animals; Binding Sites; Binding, Competitive; Enzyme Inhibitors; Ethylmaleimide; Fumarates; Kinetics; Malonates; Succinate Dehydrogenase; Succinates; Succinic Acid

Twenty subjects recorded perceived sourness of solutions of citric + fumaric and of citric + tartaric acids, at pH 3.5 and titratable acidity (TiA) of 4.0 g/l on a moving chart, while parotid saliva flow was recorded via a sialometer . Sourness intensity and flow were greater when citric was the minor acid than when it was dominant. Subjects varied widely in calculated volume of saliva reservoir, but not flow rate (time to 2/3 reservoir vol.). In tartaric-fumaric acid mixtures varying in pH (3.0-3.75) at a constant TiA of 4.0 g/l, and varying in TiA (3.7-4.6 g/l) at a constant pH of 3.5, sourness intensity and parotid flow increased with acidity and decreased with pH. However, eight subjects with a high flow (HF = 1.2 +/- 0.28 g/2 min) and nine subjects with a low flow (LF = 0.43 +/- 0.11 g/2 min) differed widely: (a) In response to variation in stimulus pH and TiA, HF demonstrated marked alteration in flow, but little change in sourness ; LF responded at a lower absolute level, but showed marked changes in sourness and little change in flow; (b) Salivary pH was higher and Na+ was three times greater for the HF than for the LF subjects; and (c) Salivary Ca++ showed a direct relationship with flow and pH among the HF, but an inverse relationship for the LF subjects.

Topics: Adult; Calcium; Citrates; Citric Acid; Female; Fumarates; Humans; Hydrogen-Ion Concentration; Male; Potassium; Saliva; Salivation; Sodium; Tartrates; Taste

Fumaric acid may not be regarded as an antipsoriatic drug. Beneficial effects on psoriatic lesions may be explained by secondary changes such as the acidification of gastric juice in cases of anacidity or hypacidity . Monoethylfumarate exerts true antipsoriatic activities but is by far too toxic for clinical use. Experimental investigations have confirmed an inhibitory action of the above-mentioned fumarate on nucleic acid synthesis and protein synthesis of PHA-stimulated human lymphocytes.

Topics: DNA; Fumarates; Gastric Acidity Determination; Humans; Proteins; Psoriasis

Incorporation of 32Pi into organic phosphate by mitochondria of Cotugnia digonopora was supported maximally by malate. Fumarate and succinate induced lower but significant production of ATP. Pyruvate, alpha-ketoglutarate and oxalacetate proved to be poor substrates and citrate and isocitrate had no effect. A net phosphorylation of approximately 2 mol of ADP was observed for each mol of CO2 liberated from malate or succinate. In contrast, with pyruvate, in spite of a high rate of decarboxylation, the production of ATP was extremely low. 2,4-Dinitrophenol inhibited phosphorylation. All anthelmintics examined interfered with the mitochondrial phosphorylation of ADP, with maximum inhibition by salicylanilide compounds. The anticestodal activity of the latter group of compounds, niclosamide for example, may, therefore, be attributed to their ability to inhibit mitochondrial phosphorylation.

Topics: Animals; Anthelmintics; Cestoda; Energy Metabolism; Fumarates; Malates; Mitochondria; Phosphates; Phosphorylation; Pyruvates; Pyruvic Acid

Two recombinant plasmid Escherichia coli strains containing amplified fumarate reductase activity converted fumarate to succinate at significantly higher rates and yields than a wild-type E. coli strain. Glucose was required for the conversion of fumarate to succinate, and in the absence of glucose or in cultures with a low cell density, malate accumulated. Two-dimensional gel electrophoretic analysis of proteins from the recombinant DNA and wild-type strains showed that increased quantities of both large and small fumarate reductase subunits were expressed in the recombinant DNA strains.

Topics: Ammonium Sulfate; DNA, Recombinant; Escherichia coli; Fumarates; Glucose; Malates; Oxidoreductases; Oxidoreductases Acting on CH-CH Group Donors; Plasmids; Succinates; Succinic Acid

We have demonstrated that the transport of succinate into the cells of Rhizobium japonicum strains USDA 110 and USDA 217 is severely inhibited by cyanide, azide, and 2,4-dinitrophenol, but not by arsenate. These results suggest an active mechanism of transport that is dependent on an energized membrane, but does not directly utilize ATP. The apparent Km for succinate was 3.8 microM for strain USDA 110 and 1.8 microM for strain USDA 217; maximal transport velocities were 1.5 and 3.3 nmol of succinate per min per mg of protein, respectively. The expression of the succinate uptake activity was inducible rather than constitutive, with succinate and structurally related compounds being the most effective inducers. The mechanism showed some specificity for succinate and similar organic acids; fumarate and L-malate were classical competitive inhibitors of the system. In general, the best competing compounds were also the best carbon substrates for induction of succinate uptake activity. EDTA inhibited the transport of succinate, implying a role for divalent cations in the system. When various divalent cations were used to reconstitute EDTA-inhibited activity, Ca2+ was most effective, followed by Mg2+, which restored activity at about half the efficiency of Ca2+. Growth media that were supplemented with increased Ca2+ concentration supported more rapid growth with succinate as the carbon substrate, and cells from such media showed higher specific activities of succinate transport.

Topics: Biological Transport; Cations, Divalent; Edetic Acid; Fumarates; Kinetics; Malates; Rhizobium; Succinates; Succinic Acid

The electron-transport chain catalyzing fumarate reduction by formate has recently been reconstituted from the formate dehydrogenase complex and the fumarate reductase complex from Vibrio succinogenes, in a liposomal preparation containing vitamin K-1 (Unden, G. and Kröger, A. (1982) Biochim. Biophys. Acta 682, 258-263). We have now investigated the structural properties of this preparation. The preparation was found to consist of a homogeneous population of unilamellar proteoliposomes with an average diameter of about 100 nm and an internal volume of 2-4 ml/g phospholipid. The buoyant density (1.07 g/ml) was consistent with the protein/phospholipid ratio (0.2 g/g) of the preparation. Leakage of glucose from the internal spaces of the proteoliposomes was negligibly slow. Proteoliposomes prepared with either of the enzyme complexes showed peripheral projections mainly on the outer surface, when examined by electron microscopy after negative staining. The size, orientation and surface density of the projections were consistent with those of the enzymes. Most of the substrate and dye-reactive sites (70-90%) of the enzymes in the proteoliposomes were accessible to external non-permeant substrates. The proteoliposomes catalyzing electron transport were formed by freeze-thawing a mixture of liposomes and protein-phospholipid complexes which did not perform electron transport from formate to fumarate. Nearly the entire amount of the enzymes supplied (0.2 g protein/g phospholipid) was incorporated into the liposomes by this procedure. The transformation of liposomes into proteoliposomes was accompanied by exchange of the internal solutes with the external medium.

Topics: Aldehyde Oxidoreductases; Electron Transport; Formate Dehydrogenases; Formates; Fumarates; Kinetics; Liposomes; Microscopy, Electron; Multienzyme Complexes; Proteolipids; Succinate Dehydrogenase; Vibrio

The inhibitory effect of fumaric acid (FA) on carcinogenesis by potassium 1-methyl-7-[2-(5-nitro-2-furyl)vinyl]-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylate (trans, NFN) was examined histologically with male ICR/JCL mice. NFN was fed to mice at a dose level of 0.012% in the diet for 14 weeks. These mice were then divided into 2 groups. One group was given a basal diet, and the other group was given a diet containing 1% FA in the subsequent 39 weeks. In the group of 30 mice fed NFN alone, squamous cell carcinomas were found in the stomachs of 7 mice, multiple papillomas in the stomachs of 13 mice, and multiple and large papillary adenocarcinomas in the lungs of 27 animals. The administration of FA suppressed the NFN-induced stomach and lung carcinogenesis. In the group of 32 mice fed NFN and FA, no stomach tumors developed except 1 early-stage of squamous cell carcinoma. In the lungs, only a small focus of mild atypical hyperplasia and a few early-stage adenocarcinomas were noted in 7 and 11 animals, respectively.

Topics: Adenocarcinoma; Animals; Carcinoma, Squamous Cell; Drug Interactions; Fumarates; Lung Neoplasms; Mice; Mice, Inbred ICR; Nalidixic Acid; Papilloma; Stomach Neoplasms

Topics: Animals; Body Composition; Body Weight; Diet; Dose-Response Relationship, Drug; Female; Fumarates; Growth; Rats; Rats, Inbred Strains

Substitution of half-time parameters in the integrated form of the Michaelis-Menten equation for any enzyme-catalysed reaction yields an equation that gives a linear relationship between the half-time of the reaction and the substrate concentration at that point of the reaction. The logarithmic term of the integrated equation becomes a constant as a result of the substitution, which means that the use of the half-time plot of the equation requires calculation only of half-time and substrate-concentration values at various stages of the reaction. The half-time method is both simple and exact, being analogous to an [S(0)]/v(i) against [S(0)] plot. A direct linear form of the half-time plot has been devised that allows very simple estimation of Michaelis parameters and/or initial velocities from progress-curve data. This method involves no approximation and is statistically valid. Simulation studies have shown that linear-regression analysis of half-time plots provides unbiased estimates of the Michaelis parameters. Simulation of the effect of error in estimation of the product concentration at infinite time [P(infinity)] reveals that this is always a cause for concern, such errors being magnified approximately an order of magnitude in the estimate of the Michaelis constant. Both the half-time plot and the direct linear form have been applied to the analysis of a variety of experimental data. The method has been shown to produce excellent results provided certain simple rules are followed regarding criteria of experimental design. A set of rules has been formulated that, if followed, allows progress-curve data to be acquired and analysed in a reliable fashion. It is apparent that the use of modern spectrophotometers in carefully designed experiments allows the collection of data characterized by low noise and accurate [P(infinity)] estimates. [P(infinity)] values have been found, in the present work, to be precise to within +/-0.2% and noise levels have always been below 0.1% (signal-to-noise ratio approximately 1000). As a result of the considerations above, it is concluded that there is little to be feared with regard to the analysis of enzyme kinetics using complete progress curves, despite the generally lukewarm recommendations to be found in the literature. The saving in time, materials and experimental effort amply justify analysis of enzyme kinetics by progress-curve methods. Half-time plots linear to >/=90% of reaction have been obtained for some alp

Topics: Chymotrypsin; Fumarate Hydratase; Fumarates; Glycine; Hydrolysis; Kinetics; Malates; Methods; Microcomputers; Models, Biological; Papain

Topics: Animals; Body Weight; Female; Food Additives; Fumarates; Minerals; Rats; Rats, Inbred Strains; Trace Elements

The NAD-dependent conversion of malate to lactate in human erythrocytes was studied by spin echo proton NMR. A pathway involving the decarboxylation of oxaloacetate catalysed by haemoglobin is proposed to account for the observed reaction. NADP-dependent reaction was negligible. The rate of the reaction was measured in intact erythrocytes under controlled conditions. This rate correlates with that obtained with lysates at 30 microM free NAD and that obtained with purified human erythrocyte enzymes at about 15 microM NAD. The total extractable NAD in the intact cells was 70-90 microM. Experiments with cells containing elevated NAD levels could be explained by a significant fraction of the NAD being weakly bound (Kd about 1 mM) to haemoglobin.

Topics: Biological Transport; Erythrocytes; Fumarate Hydratase; Fumarates; Humans; Kinetics; Lactates; Lactic Acid; Magnetic Resonance Spectroscopy; Malates; NAD

Total and high-density lipoprotein (HDL) cholesterol were estimated in 131 blood samples obtained from women who had been taking the oral contraceptive Noriday 1 + 50Fe (one packet contains 21 tablets of 1 mg of norethindrone + 0.05 mg of mestranol, and 7 of 75 mg of ferrous fumarate) for 1-60 months. Thirty five women who had never used oral contraception (OC) formed the control group. There was a significantly higher mean HDL cholesterol level, and HDL cholesterol/total cholesterol ratio but not total cholesterol level in the women who had been using OC for 19-60 months. The values in women who had been using OC for 1-18 months were not significantly different from those in the control group. The increase in the HDL cholesterol level may not depend on the oestrogen content of the oral contraceptive but on the duration of its intake.. Total and high-density lipoprotein (HDL) cholesterol were estimated in 131 blood samples obtained from women who had been taking the oral contraceptive (OC) Noriday 1+50Fe (1 packet contains 21 tablets of 1 mg of norethindrone + 0.05 mg of mestranol, and 7 of 75 mg of ferrous fumarate) for 1-60 months. 35 women who had never used OCs formed the control group. There was a significantly higher mean HDL cholesterol level and HDL cholesterol/total cholesterol ratio but not total cholesterol level in the women who had been using OCs for 19-60 months. Values in women who had been using OCs for 1-18 months were not significantly different from the control group. The increase in the HDL cholesterol level may not depend on the estrogen content of the OC but on the duration of its intake. This increase may be beneficial as HDL-cholesterol has been reported to have an anti-atherogenic effect.

Topics: Adult; Cholesterol; Cholesterol, HDL; Contraceptives, Oral, Combined; Contraceptives, Oral, Hormonal; Female; Fumarates; Humans; Lipoproteins, HDL; Mestranol; Norethindrone; Time Factors

Fumaric acid, isolated as the active component of Capsella bursa-pastoris herb for inhibiting the solid growth of Ehrlich tumor in mice, was found to reduce markedly the growth and viability of Ehrlich, MH134, and L1210 mouse tumor cells in culture at concentration of 0.3 approximately 1.2 mg/ml. In contrast, fumaric acid at these concentrations in the culture medium had no deleterious effect on the monolayer development of mouse and chick embryo cells but exhibited activity to enhance the recovery of the cells from the toxic effects of mitomycin C, aflatoxin B1, N-methyl-N'-nitro-N-nitrosoguanidine, and potassium 1-methyl-7-[2-(5-nitro-2-furyl) vinyl]-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylate.

Topics: Animals; Antineoplastic Agents; Antioxidants; Carcinoma, Ehrlich Tumor; Cell Division; Cell Survival; Cells, Cultured; Chick Embryo; Dicarboxylic Acids; DNA, Neoplasm; Embryo, Mammalian; Fumarates; Leukemia L1210; Liver; Mice; Mice, Inbred Strains; Neoplasms, Experimental

Strains of Pasteurella multocida use L-aspartate, L-malate and furmarate, respectively, as substrates for production of succinic acid which accumulates in the medium. As was established by studies with 14C and 3H labelled substrates, the degradation of these substances proceeds analogous via the citric acid cycle.

Topics: Aspartic Acid; Carbon Radioisotopes; Citric Acid Cycle; Fumarates; Kinetics; Malates; Pasteurella; Tritium

Topics: Carbon Dioxide; Cyanides; Erythrocytes; Fumarates; Hydrazines; Lactates; Malates; Manometry; Metabolism; NAD; Pharmacology; Research

Wegener, Warner S. (University of Cincinnati, Cincinnati, Ohio), and Antonio H. Romano. Control of isocitratase formation in Rhizopus nigricans. J. Bacteriol. 87:156-161. 1964.-A fumaric acid-producing strain of Rhizopus nigricans was found to produce a fair level of isocitratase in a casein hydrolysate medium. Glucose repressed enzyme formation. When glucose was utilized during growth, there was a relief of repression, and enzyme synthesis was resumed at a rate equivalent to that found in nonrepressed cells. Zinc stimulated isocitratase formation in glucose-repressed cultures by stimulating growth and glucose utilization, thereby decreasing accumulation of repressor metabolites derived from glucose. The effectiveness of acetate as an inducer was greater on glucose-repressed cells than on nonrepressed cells; cells grown in the presence of glucose formed higher levels of isocitratase when subsequently replaced with an acetate-containing inductive medium than did cells grown without glucose. Moreover, addition of 2 ppm of Zn(++) during the inductive replacement phase resulted in a twofold increase in isocitratase formation. The hypothesis is submitted that Zn(++) exerts its action by stimulating ribonucleic acid (RNA) synthesis, thereby facilitating the formation of a specific RNA during induction. Preliminary evidence implicating Zn(++) in the stimulation of RNA synthesis in this organism is presented.

Topics: Acetates; Caseins; Fumarates; Glucose; Isocitrate Lyase; Lyases; Proteins; Research; Rhizopus; RNA; RNA, Bacterial; Zinc

Topics: Acetates; Amino Acids; Antimetabolites; Aspartic Acid; Carbamates; Carbon Dioxide; Citric Acid Cycle; Citrulline; Fumarates; Glutamates; Glutamic Acid; In Vitro Techniques; Liver; Pharmacology; Rats; Research

Topics: Chemistry Techniques, Analytical; Fumarates; Iron; Microchemistry; Research

Topics: Chemical Phenomena; Chemistry; Fumarates; Plants; Senecio

Wiley, W. R. (Washington State University, Pullman), and J. L. Stokes. Effect of pH and ammonium ions on the permeability of Bacillus pasteurii. J. Bacteriol. 86:1152-1156. 1963.-Cell suspensions of Bacillus pasteurii require an alkaline pH (8.5 to 9.0) and NH(4) (+) for the oxidation of low concentrations (4 mum) of fumaric acid, glutamic acid, alanine, and other oxidizable substrates. In contrast, cells disrupted by a French press or by lysozyme oxidize these substrates at pH 7.2 and without NH(4) (+). Moreover, the alkaline pH and NH(4) (+) inhibit substrate oxidation by the broken cells. These striking differences between whole and disrupted cells suggest that pH and NH(4) (+) affect whole cells externally and not internally. It appears that the alkaline pH is needed to convert NH(4) (+) to free NH(3). The latter in turn is required by the cells for the transport of low concentrations of substrate across the cell membrane. At high concentrations (20 to 250 mum), substrates force entry into the cells by simple diffusion, thereby eliminating the need for a high pH and NH(4) (+) for oxidation.

Topics: Alanine; Ammonium Compounds; Bacillus; Biological Transport; Fumarates; Glutamic Acid; Hydrogen-Ion Concentration; Ions; Isoleucine; Metabolism; Muramidase; Permeability; Quaternary Ammonium Compounds; Research

Topics: Fumarates; Liver; Malates; Radionuclide Imaging; Rats; Research; Tritium; Water

Topics: Adrenal Cortex Hormones; Adrenal Insufficiency; Adrenalectomy; Citric Acid Cycle; Cold Temperature; Fumarates; Pharmacology; Rats; Research; Starvation; Stress, Physiological; Succinates; Succinic Acid; Vitamin A; Vitamin K; Vitamins

Topics: Biophysical Phenomena; Enzymes; Fumarates; Hydroxylamine; Hydroxylamines; Maleates

Topics: Anemia; Anemia, Hypochromic; Ferrous Compounds; Fumarates; Iron; Maleates

Topics: Fumarates; Maleates; Rhizopus

Topics: Fermentation; Fumarates; Malates

Topics: Fumarates; Maleates; Micrococcus

Topics: Acids; Arginine; Argininosuccinic Acid; Biophysical Phenomena; Cytokinesis; Fumarates; Succinates; Urea

Topics: Acids; Adrenochrome; Epinephrine; Fumarates; Maleates; Oxidation-Reduction

Topics: Acids; Animals; Fumarates; Sarcoma, Experimental; Sarcoma, Yoshida

Topics: Cyclohexanes; Enzymes; Fatty Acids, Unsaturated; Fumarates; Malates; Sesquiterpenes

Topics: Acids; Female; Fumarates; Fungi; Humans; Rhizopus; Vagina

Topics: Acids; Fermentation; Fumarates; Fungi; Rhizopus

Topics: Dicarboxylic Acids; Fumarates; Glycogen; Succinic Acid

Topics: Acids; Fumarates; Fungi; Rhizopus

Topics: Acids; Colorimetry; Fumarates

Topics: Deuterium; Fumarates; Humans

Topics: Animals; Chromatography; Chromatography, Liquid; Fumarates

Topics: Copper; Fumarates

Topics: Anthracenes; Citraconic Anhydrides; Fumarates; Maleates

Topics: Citraconic Anhydrides; Fumarates; Maleates

Topics: Fumarates; Fungi; Rhizopus

Topics: Fumarates; Maleates; Oxalates; Oxalic Acid; Tartrates

Topics: Bacillus; Deuterium; Escherichia coli; Fumarates; Hydrogen; Hydrogenation

Topics: Fumarates; Humans

Topics: Animals; Brain; Columbidae; Fumarates; Oxidation-Reduction; Pyruvic Acid

Topics: Cathartics; Fumarates; Humans; Salts