pyrophosphate and diphosphoric-acid

Conventionally, ATP is considered to be the principal energy source in cells. However, over the last few years, a novel role for ATP as a potent extracellular signaling molecule and the principal source of extracellular pyrophosphate, the main endogenous inhibitor of vascular calcification, has emerged. A large body of evidence suggests that two principal mechanisms are involved in the initiation and progression of ectopic calcification: high phosphate concentration and pyrophosphate deficiency. Pathologic calcification of cardiovascular structures, or vascular calcification, is a feature of several genetic diseases and a common complication of chronic kidney disease, diabetes, and aging. Previous studies have shown that the loss of function of several enzymes and transporters involved in extracellular ATP/pyrophosphate metabolism is associated with vascular calcification. Therefore, pyrophosphate homeostasis should be further studied to facilitate the design of novel therapeutic approaches for ectopic calcification of cardiovascular structures, including strategies to increase pyrophosphate concentrations by targeting the ATP/pyrophosphate metabolism cycle.

Topics: Adenosine Triphosphate; Diphosphates; Homeostasis; Humans; Vascular Calcification

Pathologic soft tissue calcification can occur in both genetic and acquired clinical conditions, causing significant morbidity and mortality. Although the pathomechanisms of pathologic calcification are poorly understood, major progress has been made in recent years in defining the underlying genetic defects in Mendelian disorders of ectopic calcification. This review presents an overview of the pathophysiology of five monogenic disorders of pathologic calcification: pseudoxanthoma elasticum, generalized arterial calcification of infancy, arterial calcification due to deficiency of CD73, ankylosis, and progeria. These hereditary disorders, caused by mutations in genes encoding ATP binding cassette subfamily C member 6, ectonucleotide pyrophosphatase/phosphodiesterase 1, CD73, progressive ankylosis protein, and lamin A/C proteins, respectively, are inorganic pyrophosphate (PPi) deficiency syndromes with reduced circulating levels of PPi, the principal physiologic inhibitor of calcium hydroxyapatite deposition in soft connective tissues. In addition to genetic diseases, PPi deficiency has been encountered in acquired clinical conditions accompanied by pathologic calcification. Because specific and effective treatments are lacking for pathologic calcification, the unifying finding of PPi deficiency suggests that PPi-targeted therapies may be beneficial to counteract pathologic soft tissue calcification in both genetic and acquired diseases.

Topics: Ankylosis; Calcinosis; Choristoma; Diphosphates; Humans; Pseudoxanthoma Elasticum; Syndrome; Vascular Calcification

Topics: Amyloidosis; Cardiomyopathies; Diphosphates; Diphosphonates; Humans; Prealbumin; Radiopharmaceuticals; Technetium Tc 99m Pyrophosphate

Cardiovascular complications due to accelerated arterial stiffening and atherosclerosis are the leading cause of morbimortality in Western society. Both pathologies are frequently associated with vascular calcification. Pathologic calcification of cardiovascular structures, or vascular calcification, is associated with several diseases (for example, genetic diseases, diabetes, and chronic kidney disease) and is a common consequence of aging. Calcium phosphate deposition, mainly in the form of hydroxyapatite, is the hallmark of vascular calcification and can occur in the medial layer of arteries (medial calcification), in the atheroma plaque (intimal calcification), and cardiac valves (heart valve calcification). Although various mechanisms have been proposed for the pathogenesis of vascular calcification, our understanding of the pathogenesis of calcification is far from complete. However, in recent years, some risk factors have been identified, including high serum phosphorus concentration (hyperphosphatemia) and defective synthesis of pyrophosphate (pyrophosphate deficiency). The balance between phosphate and pyrophosphate, strictly controlled by several genes, plays a key role in vascular calcification. This review summarizes the current knowledge concerning phosphate and pyrophosphate homeostasis, focusing on the role of extracellular pyrophosphate metabolism in aortic smooth muscle cells and macrophages.

Topics: Diphosphates; Humans; Phosphates; Vascular Calcification

In the past two decades, there has been great progress in identifying the molecular basis and pathomechanistic details in pseudoxanthoma elasticum (PXE), a heritable multisystem ectopic mineralization disorder. Although the identification of pathogenic variants in ABCC6 has been critical for understanding the disease process, genetic modifiers have been disclosed that explain the phenotypic heterogeneity of PXE. Adding to the genetic complexity of PXE are PXE-like phenotypes caused by pathogenic variants in other ectopic mineralization-associated genes. This review summarizes the current knowledge of the genetics and candidate modifier genes in PXE, a multifactorial disease at the genome-environment interface.

Topics: Animals; Diphosphates; Genetic Association Studies; Humans; Mice; Multidrug Resistance-Associated Proteins; Oxidative Stress; Phosphoric Diester Hydrolases; Pseudoxanthoma Elasticum; Pyrophosphatases; Vascular Calcification

In the conditions of [Mg2+] elevation that occur, in particular, under low oxygen stress and are the consequence of the decrease in [ATP] and increase in [ADP] and [AMP], pyrophosphate (PPi) can function as an alternative energy currency in plant cells. In addition to its production by various metabolic pathways, PPi can be synthesized in the combined reactions of pyruvate, phosphate dikinase (PPDK) and pyruvate kinase (PK) by so-called PK/PPDK substrate cycle, and in the reverse reaction of membrane-bound H+-pyrophosphatase, which uses the energy of electrochemical gradients generated on tonoplast and plasma membrane. The PPi can then be consumed in its active forms of MgPPi and Mg2PPi by PPi-utilizing enzymes, which require an elevated [Mg2+]. This ensures a continuous operation of glycolysis in the conditions of suppressed ATP synthesis, keeping metabolism energy efficient and less dependent on ATP.

Topics: Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Cell Membrane; Diphosphates; Energy Metabolism; Gene Expression Regulation, Plant; Intracellular Membranes; Magnesium; Plant Cells; Plant Proteins; Plants; Pyrophosphatases; Pyruvate Kinase; Pyruvate, Orthophosphate Dikinase

Topics: Diphosphates; Enzyme Assays; Humans; Hydrolysis; Inorganic Pyrophosphatase; Luciferases; Phosphates

Pathological (ectopic) mineralization of soft tissues occurs during aging, in several common conditions such as diabetes, hypercholesterolemia, and renal failure and in certain genetic disorders. Pseudoxanthoma elasticum (PXE), a multi-organ disease affecting dermal, ocular, and cardiovascular tissues, is a model for ectopic mineralization disorders. ABCC6 dysfunction is the primary cause of PXE, but also some cases of generalized arterial calcification of infancy (GACI). ABCC6 deficiency in mice underlies an inducible dystrophic cardiac calcification phenotype (DCC). These calcification diseases are part of a spectrum of mineralization disorders that also includes Calcification of Joints and Arteries (CALJA). Since the identification of ABCC6 as the "PXE gene" and the development of several animal models (mice, rat, and zebrafish), there has been significant progress in our understanding of the molecular genetics, the clinical phenotypes, and pathogenesis of these diseases, which share similarities with more common conditions with abnormal calcification. ABCC6 facilitates the cellular efflux of ATP, which is rapidly converted into inorganic pyrophosphate (PPi) and adenosine by the ectonucleotidases NPP1 and CD73 (NT5E). PPi is a potent endogenous inhibitor of calcification, whereas adenosine indirectly contributes to calcification inhibition by suppressing the synthesis of tissue non-specific alkaline phosphatase (TNAP). At present, therapies only exist to alleviate symptoms for both PXE and GACI; however, extensive studies have resulted in several novel approaches to treating PXE and GACI. This review seeks to summarize the role of ABCC6 in ectopic calcification in PXE and other calcification disorders, and discuss therapeutic strategies targeting various proteins in the pathway (ABCC6, NPP1, and TNAP) and direct inhibition of calcification via supplementation by various compounds.

Topics: 5'-Nucleotidase; Animals; ATP-Binding Cassette Transporters; Calcification, Physiologic; Calcinosis; Diphosphates; GPI-Linked Proteins; Humans; Joint Diseases; Mice; Multidrug Resistance-Associated Proteins; Phosphoric Diester Hydrolases; Pseudoxanthoma Elasticum; Pyrophosphatases; Rats; Vascular Calcification; Vascular Diseases

This review summarizes current understanding of generalized arterial calcification of infancy (GACI), emphasizing pathophysiology, clinical presentation, and approaches and controversies in management.. Identification of causative ENPP1 mutations revealed that GACI arises from deficiencies in inorganic pyrophosphate (leading to calcifications) and adenosine monophosphate (leading to intimal proliferation). Identification of genotypic and phenotypic overlap with pseudoxanthoma elasticum and autosomal recessive hypophosphatemic rickets further advanced understanding of GACI as a complex, multisystemic disease. Clinical data is limited to small, retrospective samples; it is therefore unknown whether commonly used medications, such as bisphosphonates and hypophosphatemia treatment, are therapeutic or potentially harmful. ENPP1-Fc replacement represents a promising approach warranting further study. Knowledge gaps in natural history place clinicians at high risk of assigning causality to interventions that are correlated with changes in clinical status. There is thus a critical need for improved natural history studies to develop and test targeted therapies.

Topics: Adenosine Monophosphate; Bone Density Conservation Agents; Calcinosis; Cardiovascular Agents; Chelating Agents; Diphosphates; Diphosphonates; Familial Hypophosphatemic Rickets; Genotype; Hearing Loss; Humans; Multidrug Resistance-Associated Proteins; Phenotype; Phosphoric Diester Hydrolases; Pseudoxanthoma Elasticum; Pyrophosphatases; Thiosulfates; Tooth Diseases; Vascular Calcification; Vitamin D

In the progression of osteoarthritis, pathological calcification in the affected joint is an important feature. The role of these crystallites in the pathogenesis and progression of osteoarthritis is controversial; it remains unclear whether they act as a disease initiator or are present as a result of joint damage. Recent studies reported that the molecular mechanisms regulating physiological calcification of skeletal tissues are similar to those regulating pathological or ectopic calcification of soft tissues. Pathological calcification takes place when the equilibrium is disrupted. Calcium phosphate crystallites are identified in most affected joints and the presence of these crystallites is closely correlated with the extent of joint destruction. These observations suggest that pathological calcification is most likely to be a disease initiator instead of an outcome of osteoarthritis progression. Inhibiting pathological crystallite deposition within joint tissues therefore represents a potential therapeutic target in the management of osteoarthritis.

Topics: Apoptosis; Bursa, Synovial; Calcinosis; Calcium; Cartilage; Chondrocytes; Collagen; Diphosphates; Extracellular Matrix; Extracellular Vesicles; Humans; Meniscus; Mitochondria; Osteoarthritis; Phosphates; Proteoglycans; Pyrophosphatases; Severity of Illness Index

Nudix proteins catalyse hydrolysis of pyrophosphate bonds in a variety of substrates and are ubiquitous in all domains of life. Their widespread presence and broad substrate specificity suggest that they have important cellular functions. In this review, we summarize the state of knowledge on microbial Nudix proteins involved in pathogenesis.

Topics: Amino Acid Sequence; Bacterial Proteins; Diphosphates; Nudix Hydrolases; Pyrophosphatases; Sequence Alignment; Viral Proteins; Virulence; Virulence Factors

Tissue-nonspecific alkaline phosphatase (TNAP) is a ubiquitously expressed enzyme that is best known for its role during mineralization processes in bones and skeleton. The enzyme metabolizes phosphate compounds like inorganic pyrophosphate and pyridoxal-5'-phosphate to provide, among others, inorganic phosphate for the mineralization and transportable vitamin B6 molecules. Patients with inherited loss of function mutations in the

Topics: Alkaline Phosphatase; Animals; Anxiety; Bone and Bones; Calcification, Physiologic; Depression; Diphosphates; Disease Models, Animal; Gene Expression; Humans; Hypophosphatasia; Mutation; Seizures; Severity of Illness Index; Tooth; Vitamin B 6

Pseudoxanthoma elasticum is a rare disease mainly due to

Topics: 5'-Nucleotidase; Diphosphates; GPI-Linked Proteins; Humans; Kidney Calculi; Multidrug Resistance-Associated Proteins; Mutation; Pseudoxanthoma Elasticum; Rare Diseases; Urolithiasis; Vascular Calcification

Ectopic mineralization is a global problem and leading cause of morbidity and mortality. The pathomechanisms of ectopic mineralization are poorly understood. Recent studies on heritable ectopic mineralization disorders with defined gene defects have been helpful in elucidation of the mechanisms of ectopic mineralization in general. The prototype of such disorders is pseudoxanthoma elasticum (PXE), a late-onset, slowly progressing disorder with multisystem clinical manifestations. Other conditions include generalized arterial calcification of infancy (GACI), characterized by severe, early-onset mineralization of the cardiovascular system, often with early postnatal demise. In addition, arterial calcification due to CD73 deficiency (ACDC) occurs late in life, mostly affecting arteries in the lower extremities in elderly individuals. These three conditions, PXE, GACI, and ACDC, caused by mutations in ABCC6, ENPP1, and NT5E, respectively, are characterized by reduced levels of inorganic pyrophosphate (PPi) in plasma. Because PPi is a powerful antimineralization factor, it has been postulated that reduced PPi is a major determinant for ectopic mineralization in these conditions. These and related observations on complementary mechanisms of ectopic mineralization have resulted in development of potential treatment modalities for PXE, including administration of bisphosphonates, stable PPi analogs with antimineralization activity. It is conceivable that efficient treatments may soon become available for heritable ectopic mineralization disorders with application to common calcification disorders.

Topics: 5'-Nucleotidase; Diphosphates; Diphosphonates; GPI-Linked Proteins; Humans; Multidrug Resistance-Associated Proteins; Phosphoric Diester Hydrolases; Pseudoxanthoma Elasticum; Pyrophosphatases; Vascular Calcification

Nonribosomal peptides are a prolific source of bioactive molecules biosynthesized on large, modular assembly line synthetases. Synthetic biologists seek to obtain tailored peptides with tuned or novel bioactivities by engineering modules and domains of these nonribosomal peptide synthetases. The activation step catalyzed by adenylation domains primarily selects which amino acids are incorporated into nonribosomal peptides. Here, we review experimental protocols for probing the adenylation reaction that are applicable in natural product discovery and engineering. Several alternatives to the established pyrophosphate exchange assay will be compared and potential pitfalls pointed out. Binding pocket mutagenesis of adenylation domains has been successfully conducted to adjust substrate preferences. Novel screening methods relying on yeast surface display, for instance, search a larger sequence space for improved mutants and thus allow more substantial changes in peptide structure.

Topics: Bioengineering; Cell Surface Display Techniques; Diphosphates; Kinetics; Peptide Biosynthesis, Nucleic Acid-Independent; Peptide Synthases; Peptides; Protein Domains; Substrate Specificity

Entamoeba histolytica has neither Krebs cycle nor oxidative phosphorylation activities; therefore, glycolysis is the main pathway for ATP supply and provision of carbon skeleton precursors for the synthesis of macromolecules. Glucose is metabolized through fermentative glycolysis, producing ethanol as its main end-product as well as some acetate. Amoebal glycolysis markedly differs from the typical Embden-Meyerhof-Parnas pathway present in human cells: (i) by the use of inorganic pyrophosphate, instead of ATP, as the high-energy phospho group donor; (ii) with one exception, the pathway enzymes can catalyze reversible reactions under physiological conditions; (iii) there is no allosteric regulation and sigmoidal kinetic behavior of key enzymes; and (iv) the presence of some glycolytic and fermentation enzymes similar to those of anaerobic bacteria. These peculiarities bring about alternative mechanisms of control and regulation of the PPi-dependent fermentative glycolysis in the parasite in comparison to the ATP-dependent and allosterically regulated glycolysis in many other eukaryotic cells. In this review, the current knowledge of the carbohydrate metabolism enzymes in E. histolytica is analyzed. Thermodynamics and stoichiometric analyses indicate 2 to 3.5 ATP yield per glucose metabolized, instead of the often presumed 5 ATP/glucose ratio. PPi derived from anabolism seems insufficient for PPi-glycolysis; hence, alternative ways of PPi supply are also discussed. Furthermore, the underlying mechanisms of control and regulation of the E. histolytica carbohydrate metabolism, analyzed by applying integral and systemic approaches such as Metabolic Control Analysis and kinetic modeling, contribute to unveiling alternative and promising drug targets.

Topics: Animals; Diphosphates; Entamoeba histolytica; Entamoebiasis; Glucose; Humans; Protozoan Proteins

In recent years, the mechanisms and clinical significance of vascular calcification have been increasingly investigated. For over a century, however, pathologists have recognized that vascular calcification is a form of heterotopic ossification. In this review, we aim to describe the pathology and molecular processes of vascular ossification, to characterize its clinical significance and treatment options, and to elucidate areas that require further investigation. The molecular mechanisms of vascular ossification involve the activation of regulators including bone morphogenic proteins and chondrogenic transcription factors and the loss of mineralization inhibitors like fetuin-A and pyrophosphate. Although few studies have examined the gross pathology of vascular ossification, the presence of these molecular regulators and evidence of microfractures and cartilage have been demonstrated on heart valves and atherosclerotic plaques. These changes are often triggered by common inflammatory and metabolic disorders like diabetes, hyperlipidemia, and chronic kidney disease. The increasing prevalence of these diseases warrants further research into the clinical significance of vascular ossification and future treatment options.

Topics: 5'-Nucleotidase; alpha-2-HS-Glycoprotein; Animals; Diphosphates; GPI-Linked Proteins; Humans; Ossification, Heterotopic; RANK Ligand; Vascular Calcification

Despite playing critical roles in the immune response and having significant potential in immunotherapy, γδ T cells have garnered little of the limelight. One major reason for this paradox is that their antigen recognition mechanisms are largely unknown, limiting our understanding of their biology and our potential to modulate their activity. One of the best-studied γδ subsets is the human Vγ9Vδ2T cell population, which predominates in peripheral blood and can combat both microbial infections and cancers. Although it has been known for decades that Vγ9Vδ2T cells respond to the presence of small pyrophosphate-based metabolites, collectively named phosphoantigens (pAgs), derived from microbial sources or malignant cells, the molecular basis for this response has been unclear. A major breakthrough in this area came with the identification of the Butyrophilin 3A (BTN3A) proteins, members of the Butyrophilin/Butyrophilin-like protein family, as mediators between pAgs and Vγ9Vδ2T cells. In this article, we review the most recent studies regarding pAg activation of human Vγ9Vδ2T cells, mainly focusing on the role of BTN3A as the pAg sensing molecule, as well as its potential impact on downstream events of the activation process.

Topics: Animals; Antigens, CD; Butyrophilins; Diphosphates; Humans; Lymphocyte Activation; Phosphorylation; T-Lymphocytes

Proton-translocating inorganic pyrophosphatase (H+-PPase) actively translocates protons across membranes coupled with the hydrolysis of inorganic pyrophosphate (PPi). H+-PPase, which is composed of a single protein and uses a simple compound as a substrate, has been recognized as a new type of ion pump in addition to the P-, F- and V-type ion-translocating ATPases. H+- and Na+-PPases are distributed in various organisms including plants, parasitic protozoa, Archaebacteria and bacteria, but are not present in animals or yeast. Vacuolar H+-PPase has dual functions in plant cells: hydrolysis of cytosolic PPi to maintain the levels of PPi, and translocation of protons into vacuoles to maintain the acidity of the vacuolar lumen. Acidification performed with the vacuolar-type H+-ATPase and H+-PPase is essential to maintain acidic conditions, which are necessary for vacuolar hydrolytic enzymes and for supplying energy to secondary active transporters. Recent studies using loss-of-function mutant lines of H+-PPase and complementation lines with soluble PPases have emphasized the physiological importance of the scavenging role of PPi. An overview of the main features of H+-PPases present in the vacuolar membrane is provided in terms of tissue distribution in plants, intracellular localization, structure-function relationship, biochemical potential as a proton pump and functional stability.

Topics: Cytosol; Diphosphates; Inorganic Pyrophosphatase; Plant Proteins; Vacuolar Proton-Translocating ATPases; Vacuoles

Pathologic cardiovascular calcification is associated with a number of conditions and is a common complication of chronic kidney disease. Because ambient calcium and phosphate levels together with properties of the vascular matrix favor calcification even under normal conditions, endogenous inhibitors such as pyrophosphate play a key role in prevention. Genetic diseases and animal models have elucidated the metabolism of extracellular pyrophosphate and demonstrated the importance of pyrophosphate deficiency in vascular calcification. Therapies based on pyrophosphate metabolism have been effective in animal models, including renal failure, and hold promise as future therapies to prevent vascular calcification.

Topics: Animals; Blood Vessels; Calcium; Diphosphates; Down-Regulation; Genetic Predisposition to Disease; Humans; Renal Insufficiency, Chronic; Risk Factors; Vascular Calcification

Topics: Adaptive Immunity; Antigens, Bacterial; Diphosphates; Hemiterpenes; Humans; Organophosphorus Compounds; Receptors, Antigen, T-Cell, gamma-delta; T-Lymphocyte Subsets

Pseudoxanthoma elasticum is a prototype of heritable ectopic mineralization disorders, with phenotypic overlap with generalized arterial calcification of infancy and arterial calcification due to CD73 deficiency. Recent observations have suggested that the reduced inorganic pyrophosphate/phosphate ratio is the cause of soft connective tissue mineralization in these disorders. PXE International, a patient advocacy organization, supports research in part by sponsoring biennial research symposia on these disorders; the latest meeting was held in September 2016 at Thomas Jefferson University, Philadelphia. This report summarizes the progress in pseudoxanthoma elasticum and other ectopic mineralization disorders, as presented in the symposium, with focus on translational aspects of precision medicine toward improved diagnostics and treatment development for these currently intractable disorders.

Topics: 5'-Nucleotidase; Alkaline Phosphatase; Animals; Biopsy, Needle; Clinical Trials as Topic; Congresses as Topic; Diphosphates; Disease Models, Animal; Etidronic Acid; Genetic Predisposition to Disease; GPI-Linked Proteins; Humans; Immunohistochemistry; Internationality; Mice; Mutation; Phosphoric Diester Hydrolases; Pseudoxanthoma Elasticum; Pyrophosphatases; Rare Diseases; Vascular Calcification

Pseudoxanthoma elasticum (PXE) is a genetic metabolic disease with autosomal recessive inheritance caused by mutations in the ABCC6 gene. The lack of functional ABCC6 protein leads to ectopic mineralization that is most apparent in the elastic tissues of the skin, eyes and blood vessels. The clinical prevalence of PXE has been estimated at between 1 per 100,000 and 1 per 25,000, with slight female predominance. The first clinical sign of PXE is almost always small yellow papules on the nape and sides of the neck and in flexural areas. The papules coalesce, and the skin becomes loose and wrinkled. The mid-dermal elastic fibers are short, fragmented, clumped and calcified. Dystrophic calcification of Bruch's membrane, revealed by angioid streaks, may trigger choroidal neovascularization and, ultimately, loss of central vision and blindness in late-stage disease. Lesions in small and medium-sized artery walls may result in intermittent claudication and peripheral artery disease. Cardiac complications (myocardial infarction, angina pectoris) are thought to be relatively rare but merit thorough investigation. Ischemic strokes have been reported. PXE is a metabolic disease in which circulating levels of an anti-mineralization factor are low. There is good evidence to suggest that the factor is inorganic pyrophosphate (PPi), and that the circulating low levels of PPi and decreased PPi/Pi ratio result from the lack of ATP release by hepatocytes harboring the mutant ABCC6 protein. However, the substrate(s) bound, transported or modulated by the ABCC6 protein remain unknown. More than 300 sequence variants of the ABCC6 gene have been identified. There is no cure for PXE; the main symptomatic treatments are vascular endothelial growth factor inhibitor therapy (for ophthalmic manifestations), lifestyle, lipid-lowering and dietary measures (for reducing vascular risk factors), and vascular surgery (for severe cardiovascular manifestations). Future treatment options may include gene therapy/editing and pharmacologic chaperone therapy.

Topics: Animals; Choroidal Neovascularization; Diphosphates; Humans; Metabolic Diseases; Peripheral Arterial Disease; Pseudoxanthoma Elasticum; Skin

Constitutive expression of the Arabidopsis vacuolar proton-pumping pyrophosphatase (H

Topics: Diphosphates; Gene Expression Regulation, Plant; Inorganic Pyrophosphatase; Plant Proteins; Sucrose; Vacuoles

Heritable ectopic mineralization disorders represent a phenotypically diverse group of conditions characterized by deposition of calcium phosphate complexes in soft connective tissues. The prototype of such conditions is pseudoxanthoma elasticum, and related conditions with overlapping clinical features include generalized arterial calcification of infancy and arterial calcification due to CD73 deficiency. Molecular genetic investigations have revealed mutations in the genes physiologically involved in generation of inorganic pyrophosphate and inorganic phosphate, and the findings suggest a unifying pathomechanism relating to reduced inorganic pyrophosphate/inorganic phosphate ratio. This hypothesis is based on the notion that inorganic pyrophosphate serves as a powerful inhibitor of mineralization, whereas inorganic phosphate is a promineralization factor, and an appropriate inorganic pyrophosphate/inorganic phosphate ratio is critical for prevention of ectopic mineralization under homeostatic conditions.

Topics: 5'-Nucleotidase; Animals; Biomedical Research; Diphosphates; Forecasting; Genetic Predisposition to Disease; GPI-Linked Proteins; Humans; Mice; Mice, Knockout; Molecular Biology; Mutation; Phenotype; Pseudoxanthoma Elasticum; Rare Diseases; Vascular Calcification

Inorganic pyrophosphate has long been known as a by-product of many intracellular biosynthetic reactions, and was first identified as a key endogenous inhibitor of biomineralisation in the 1960s. The major source of pyrophosphate appears to be extracellular ATP, which is released from cells in a controlled manner. Once released, ATP can be rapidly hydrolysed by ecto-nucleotide pyrophosphatase/phosphodiesterases to produce pyrophosphate. The main action of pyrophosphate is to directly inhibit hydroxyapatite formation thereby acting as a physiological 'water-softener'. Evidence suggests pyrophosphate may also act as a signalling molecule to influence gene expression and regulate its own production and breakdown. This review will summarise our current understanding of pyrophosphate metabolism and how it regulates bone mineralisation and prevents harmful soft tissue calcification.

Topics: Adenosine Triphosphate; Animals; Calcification, Physiologic; Calcinosis; Diphosphates; Gene Expression Regulation; Humans; Phosphoric Diester Hydrolases; Pyrophosphatases; Signal Transduction

Terpenoid cyclases catalyze the most complex reactions in biology, in that more than half of the substrate carbon atoms often undergo changes in bonding during the course of a multistep cyclization cascade that proceeds through multiple carbocation intermediates. Many cyclization mechanisms require stereospecific deprotonation and reprotonation steps, and most cyclization cascades are terminated by deprotonation to yield an olefin product. The first bacterial terpenoid cyclase to yield a crystal structure was pentalenene synthase from Streptomyces exfoliatus UC5319. This cyclase generates the hydrocarbon precursor of the pentalenolactone family of antibiotics. The structures of pentalenene synthase and other terpenoid cyclases reveal predominantly nonpolar active sites typically lacking amino acid side chains capable of serving general base-general acid functions. What chemical species, then, enables the Brønsted acid-base chemistry required in the catalytic mechanisms of these enzymes? The most likely candidate for such general base-general acid chemistry is the co-product inorganic pyrophosphate. Here, we briefly review biological and nonbiological systems in which phosphate and its derivatives serve general base and general acid functions in catalysis. These examples highlight the fact that the Brønsted acid-base activities of phosphate derivatives are comparable to the Brønsted acid-base activities of amino acid side chains.

Topics: Aspartate Carbamoyltransferase; Biocatalysis; Cyclization; Diphosphates; Geranyltranstransferase; Hydrolases; Intramolecular Lyases; Isomerases; L-Serine Dehydratase; Phosphates; Terpenes

Anoxia tolerance in plants is distinguished by direction of the sparse supply of energy to processes crucial to cell maintenance and sometimes to growth, as in rice seedlings. In anoxic rice coleoptiles energy is used to synthesise proteins, take up K(+) , synthesise cell walls and lipids, and in cell maintenance. Maintenance of electrochemical H(+) gradients across the tonoplast and plasma membrane is crucial for solute compartmentation and thus survival. These gradients sustain some H(+) -solute cotransport and regulate cytoplasmic pH. Pyrophosphate (PPi ), the alternative energy donor to ATP, allows direction of energy to the vacuolar H(+) -PPi ase, sustaining H(+) gradients across the tonoplast. When energy production is critically low, operation of a biochemical pHstat allows H(+) -solute cotransport across plasma membranes to continue for at least for 18 h. In active (e.g. growing) cells, PPi produced during substantial polymer synthesis allows conversion of PPi to ATP by PPi -phosphofructokinase (PFK). In quiescent cells with little polymer synthesis and associated PPi formation, the PPi required by the vacuolar H(+) -PPi ase and UDPG pyrophosphorylase involved in sucrose mobilisation via sucrose synthase might be produced by conversion of ATP to PPi through reversible glycolytic enzymes, presumably pyruvate orthophosphate dikinase. These hypotheses need testing with species characterised by contrasting anoxia tolerance.

Topics: Adenosine Triphosphate; Cotyledon; Diphosphates; Energy Metabolism; Germination; Glycolysis; Oryza; Oxygen; Pyruvate, Orthophosphate Dikinase; Seedlings

Carbonic anhydrases are a group of isoenzymes that catalyze the reversible conversion of carbon dioxide into bicarbonate. They participate in a constellation of physiological processes in humans, including respiration, bone metabolism, and the formation of body fluids, including urine, bile, pancreatic juice, gastric secretion, saliva, aqueous humor, cerebrospinal fluid, and sweat. In addition, carbonic anhydrase may provide carbon dioxide/bicarbonate to carboxylation reactions that incorporate carbon dioxide to substrates. Several isoforms of carbonic anhydrase have been identified in humans, but their precise physiological role and the consequences of their dysfunction are mostly unknown. Carbonic anhydrase isoenzymes are involved in calcification processes in a number of biological systems, including the formation of calcareous spicules from sponges, the formation of shell in some animals, and the precipitation of calcium salts induced by several microorganisms, particularly urease-producing bacteria. In human tissues, carbonic anhydrase is implicated in calcification processes either directly by facilitating calcium carbonate deposition which in turn serves to facilitate calcium phosphate mineralization, or indirectly via its action upon γ-glutamyl-carboxylase, a carboxylase that enables the biological activation of proteins involved in calcification, such as matrix Gla protein, bone Gla protein, and Gla-rich protein. Carbonic anhydrase is implicated in calcification of human tissues, including bone and soft-tissue calcification in rheumatological disorders such as ankylosing spondylitis and dermatomyositis. Carbonic anhydrase may be also involved in bile and kidney stone formation and carcinoma-associated microcalcifications. The aim of this review is to evaluate the possible association between carbonic anhydrase isoenzymes and vascular calcification in humans.

Topics: Animals; Blood Vessels; Calcium-Binding Proteins; Carbon-Carbon Ligases; Carbonic Anhydrases; Diphosphates; Extracellular Matrix Proteins; Humans; Isoenzymes; Matrix Gla Protein; Osteocalcin; Signal Transduction; Vascular Calcification

Currently, there is an ongoing interest in the synthesis of nucleoside diphosphate analogs as important regulators in catabolism/anabolism, and their potential applications as mechanistic probes and chemical tools for bioassays. However, the pyrophosphate bond formation step remains as the bottleneck. In this Digest, the chemical synthesis of the pyrophosphate bonds of representative bioactive nucleoside diphosphate analogs, i.e. phosphorus-modified analogs, nucleoside cyclic diphosphates, and nucleoside diphosphate conjugates, will be described.

Topics: Animals; Diphosphates; Humans; Nucleosides

In recent years, mechanisms of arterial calcifications are beginning to be elucidated. Arterial calcification is now considered as an actively regulated process resembling osteogenesis within the arterial wall orchestrated by a number of systemic or constitutively expressed mediators. Genetic studies of rare monogenic human disorders and studies of naturally occurring or mutant mouse models have identified specific inductors and inhibitors of arterial calcification, which can be classified according to the networks they participate in. These networks include ATP and pyrophosphate metabolism, phosphate homeostasis and vitamin D receptor signaling. Furthermore, intracellular signaling molecules, including SMAD6 and a number of systemic circulatory inhibitors of arterial calcification, including fetuin, tumor necrosis factor receptor superfamily member 11b, matrix GLA protein, adiponectin and family with sequence similarity 20 member A have been identified by human and mouse genetics. Based on the in vivo evidence of their functional relevance, these proteins will serve as excellent targets for the prevention and treatment of arterial calcification. In this review we discuss the functional role of the identified modulators of arterial calcification and describe the networks they belong to.

Topics: Adenosine Triphosphate; Animals; Diphosphates; Humans; Mice; Receptors, Calcitriol; Signal Transduction; Vascular Calcification

Soluble inorganic pyrophosphatases (PPases) catalyse an essential reaction, the hydrolysis of pyrophosphate to inorganic phosphate. In addition, an evolutionarily ancient family of membrane-integral pyrophosphatases couple this hydrolysis to Na(+) and/or H(+) pumping, and so recycle some of the free energy from the pyrophosphate. The structures of the H(+)-pumping mung bean PPase and the Na(+)-pumping Thermotoga maritima PPase solved last year revealed an entirely novel membrane protein containing 16 transmembrane helices. The hydrolytic centre, well above the membrane, is linked by a charged "coupling funnel" to the ionic gate about 20Å away. By comparing the active sites, fluoride inhibition data and the various models for ion transport, we conclude that membrane-integral PPases probably use binding of pyrophosphate to drive pumping.

Topics: Animals; Bacterial Proteins; Catalytic Domain; Diphosphates; Humans; Hydrogen Bonding; Hydrolysis; Models, Molecular; Protein Structure, Secondary; Pyrophosphatases

In its early history, life appeared to depend on pyrophosphate rather than ATP as the source of energy. Ancient membrane pyrophosphatases that couple pyrophosphate hydrolysis to active H(+) transport across biological membranes (H(+)-pyrophosphatases) have long been known in prokaryotes, plants, and protists. Recent studies have identified two evolutionarily related and widespread prokaryotic relics that can pump Na(+) (Na(+)-pyrophosphatase) or both Na(+) and H(+) (Na(+),H(+)-pyrophosphatase). Both these transporters require Na(+) for pyrophosphate hydrolysis and are further activated by K(+). The determination of the three-dimensional structures of H(+)- and Na(+)-pyrophosphatases has been another recent breakthrough in the studies of these cation pumps. Structural and functional studies have highlighted the major determinants of the cation specificities of membrane pyrophosphatases and their potential use in constructing transgenic stress-resistant organisms.

Topics: Animals; Biological Transport; Cation Transport Proteins; Cell Membrane; Diphosphates; Hydrolysis; Phylogeny; Prokaryotic Cells; Protons; Pyrophosphatases; Sodium

Loop-mediated isothermal amplification (LAMP) has several advantages: this technique involves gene amplification under isothermal conditions using only one high-specificity enzyme; the amplification efficiency is so high that large quantities of pyrophosphoric acid are formed as a by-product of DNA synthesis; furthermore, the results can be judged directly on the basis of turbidity. On the other hand, a PCR requires approximately 3.5-4.0 hours. The LAMP method is faster than the PCR method and is also relatively inexpensive. In the present study, we modified the composition of the reaction solutions to reduce the LAMP reaction time; more specifically, a thickener, either polyethylene glycol 8000 or 20000, was added. These results showed that the LAMP method was faster than the original method, and it is able to detect both turbidity and fluorescence. In conclusion, the LAMP reaction could be performed within 20 minutes when reaction mixture supplemented with a thickener was used. This method can be used for tests in various fields such as the diagnosis of hereditary disease and identification of viral infections as point-of-care testing.

Topics: Diphosphates; DNA; Genetic Diseases, Inborn; Humans; Nucleic Acid Amplification Techniques; Point-of-Care Systems; Polyethylene Glycols; Polymerase Chain Reaction; Solutions; Time Factors; Virus Diseases

Tooth sensitivity is a common, painful dental condition. Consumer dental products, mostly dentifrices, play an important role in sensitivity treatment. The objective of this review is to describe a new mouthwash-based desensitizing technology.. Background literature concerning desensitizing products is reviewed. Potassium salts are the most commonly used active ingredients in desensitizing dentifrices. Clinical studies show that while potassium salt dentifrices are generally effective; most formulations require several weeks to exert their desensitizing effect. Recently, a new desensitizing dentifrice containing the amino acid arginine was introduced. This dentifrice acts to occlude the dentinal tubules, and has been shown to be highly effective in multiple clinical studies. This arginine-containing dentifrice has also been shown to provide instant relief of sensitivity pain when applied directly to the sensitive tooth surface. In contrast to dentifrices, there are few desensitizing mouthwashes available. Building on the success of the arginine-based dentifrice, an arginine-based mouthwash formula was developed and tested.. Published studies in peer-reviewed publications.. Controlled and blinded clinical studies to provide evidence of efficacy. In vitro studies are included to indicate the mechanism of action. This review includes studies testing the new arginine-based desensitizing mouthwash.. Clinical findings indicate that this new desensitizing mouthwash, based on the Pro-Argin™ mouthwash technology effectively reduces sensitivity symptoms and can be used alone or as a adjunct to the use of the arginine-containing dentifrice in the home treatment of tooth sensitivity.

Topics: Arginine; Calcium Carbonate; Dentin Desensitizing Agents; Dentin Sensitivity; Dentinal Fluid; Diphosphates; Drug Combinations; Fluorides; Humans; Maleates; Mouthwashes; Phosphates; Polyethylenes; Sodium Fluoride

During the process of matrix vesicle (MV)-mediated initiation of mineralisation, chondrocytes and osteoblasts mineralise the extracellular matrix by promoting the seeding of basic calcium phosphate crystals of hydroxyapatite (HA) along the collagen fibrils. This orchestrated process is carefully regulated by the balanced action of propagators and inhibitors of calcification. The primary antagonistic regulators of extracellular matrix mineralisation are phosphate (Pi) and inorganic pyrophosphate (PPi). Studies in mouse models and in humans have established critical roles for Pi/PPi homeostasis in biomineralisation. In this review, we present the regulators of Pi/PPi, as derived from animal models, and discuss their clinical relevance to physiological and pathological mineralisation.

Topics: Animals; Bone Matrix; Calcification, Physiologic; Diphosphates; Extracellular Matrix; Humans; Mice; Phosphates

Pyrophosphate (PPi) is well known as a regulator of calcification, and the ANKH (ANK in mouse) protein has a role in the membrane transport of PPi. Earlier work concentrated on bones and joints, but ANKH is also likely to have important roles in the kidney, with newer studies focusing on vascular calcification in renal failure. Renal calcification can occur due to a naturally occurring ANK mouse mutation, yet other ANK mutations do not cause a renal phenotype. Despite evidence over 10 years of ANKH's involvement in PPi transport, efflux of PPi via ANKH has never been demonstrated. Rather than physically moving PPi, the ANKH protein may assist its membrane transport in other ways such as by hydrolysis and compartmentalisation. Protein complexes may account for effects of ANKH that are specific to particular tissues. In the kidney, recent localisation data may be helpful in suggesting physiological roles for ANKH, such as its co-localisation with aquaporin-2 and cilial proteins. Such diverse functions would reflect the ubiquitous nature of ANKH in tissues and its profound evolutionary conservation.

Topics: Animals; Aquaporin 2; Calcinosis; Cilia; Diphosphates; Humans; Kidney; Membrane Transport Proteins; Mice; Mutation; Nephrocalcinosis; Organ Specificity; Phosphate Transport Proteins; Renal Insufficiency

Topics: Bacteria; Biological Transport; Cytoplasm; Diphosphates; Hydrolysis; Inorganic Pyrophosphatase; Plant Cells; Plant Physiological Phenomena; Plants; Protons; Vacuoles

Topics: Cross-Over Studies; Diphosphates; Double-Blind Method; Humans; Phosphoric Diester Hydrolases; Pseudoxanthoma Elasticum

Calcium supplements have been associated with increased cardiovascular risk, but the mechanism is unknown. We investigated the effects of calcium supplements on the propensity of serum to calcify, based on the transition time of primary to secondary calciprotein particles (T50). Changes in serum calcium were related to changes in T50.. Calcium supplements have been associated with increased cardiovascular risk; however, it is unknown whether this is related to an increase in vascular calcification.. We investigated the acute and 3-month effects of calcium supplements on the propensity of serum to calcify, based on the transition time of primary to secondary calciprotein particles (T50), and on three possible regulators of calcification: fetuin-A, pyrophosphate and fibroblast growth factor-23 (FGF23). We randomized 41 postmenopausal women to 1 g/day of calcium as carbonate, or to a placebo containing no calcium. Measurements were performed at baseline and then 4 and 8 h after their first dose, and after 3 months of supplementation. Fetuin-A, pyrophosphate and FGF23 were measured in the first 10 participants allocated to calcium carbonate and placebo who completed the study.. T50 declined in both groups, the changes tending to be greater in the calcium group. Pyrophosphate declined from baseline in the placebo group at 4 h and was different from the calcium group at this time point (p = 0.04). There were no other significant between-groups differences. The changes in serum total calcium from baseline were significantly related to changes in T50 at 4 h (r = -0.32, p = 0.05) and 8 h (r = -0.39, p = 0.01), to fetuin-A at 3 months (r = 0.57, p = 0.01) and to pyrophosphate at 4 h (r = 0.61, p = 0.02).. These correlative findings suggest that serum calcium concentrations modulate the propensity of serum to calcify (T50), and possibly produce counter-regulatory changes in pyrophosphate and fetuin-A. This provides a possible mechanism by which calcium supplements might influence vascular calcification.

Topics: Aged; alpha-2-HS-Glycoprotein; Biomarkers; Bone Density Conservation Agents; Calcium; Calcium Carbonate; Calcium Citrate; Dietary Supplements; Diphosphates; Drug Administration Schedule; Female; Fibroblast Growth Factor-23; Fibroblast Growth Factors; Humans; Middle Aged; Vascular Calcification

The objective of this eight week, single-center, two-cell, double-blind, and randomized clinical study was to evaluate the dentin hypersensitivity reduction efficacy of a mouthwash using Pro-Argin™ Mouthwash Technology containing 0.8% arginine, PVM/MA copolymer, pyrophosphates, and 0.05% sodium fluoride in an alcohol-free base ("Arginine Mouthwash") compared to an ordinary mouthwash without any active ingredients ("Negative Control").. Qualifying subjects who presented two hypersensitive teeth with a tactile hypersensitivity score between 10 and 50 g of force, and an air blast hypersensitivity score of 2 or 3 participated in this study and were randomized into one of two treatment groups. Subjects brushed with the toothbrush and fluoride toothpaste provided and then rinsed with 20 mL of their assigned mouthwash for 30s twice daily. Subjects refrained from eating or drinking for 30 min after rinsing. Dentin hypersensitivity assessments, as well as examinations of oral hard and soft tissues, were conducted at the baseline visit and again after two weeks, four weeks and eight weeks of product use.. Ninety (90) subjects entered and completed the eight week study. After two weeks, four weeks and eight weeks of product use, subjects in the Arginine Mouthwash group exhibited statistically significant (p<0.05) improvements in mean tactile and air blast hypersensitivity scores as compared to the Negative Control Mouthwash.. The results of this study support the conclusion that the Arginine Mouthwash provides a significant reduction in dentin hypersensitivity after eight weeks of product use as compared to a Negative Control mouthwash.

Topics: Adult; Aged; Arginine; Calcium Carbonate; Chi-Square Distribution; Dentin Desensitizing Agents; Dentin Sensitivity; Diphosphates; Double-Blind Method; Drug Combinations; Female; Fluorides; Humans; Male; Maleates; Middle Aged; Mouthwashes; Phosphates; Polyethylenes; Prospective Studies; Sodium Fluoride; Touch; Young Adult

Evaluate the efficacy of 0.8% arginine, potassium nitrate and sodium fluoride mouthwashes on dentine hypersensitivity reduction.. Six week randomized, double blinded, two cell, parallel single centre clinical study in the Dominican Republic; subjects were randomized into three treatment groups: mouthwash containing 0.8% arginine, PVM/MA copolymer, pyrophosphates, and 0.05% sodium fluoride in an alcohol-free base (arginine); mouthwash containing 2.4% potassium nitrate and 0.022% sodium fluoride (potassium nitrate); a control mouthwash containing 0.05% sodium fluoride (negative control). Tactile and air-blast dentine hypersensitivity assessments were conducted at baseline, thirty minutes post rinsing and two, four, and six weeks of twice-daily product use. For treatment group comparisons, ANCOVA and post hoc Tukey's pair-wise comparisons (α=0.05) were done.. Seventy-five subjects were enrolled; 69 subjects completed the study. There were no differences after thirty minutes of a single use, among the three groups with respect to mean tactile and air blast hypersensitivity scores compared to potassium nitrate and negative control mouthwashes (p<0.05). The arginine group presented a statistically significant improvement in the mean tactile scores compared to potassium nitrate and negative control groups after two, four, and six weeks (p<0.001) of product use; the arginine group showed a statistically significant enhancement in air blast hypersensitivity mean scores compared to potassium nitrate and negative control groups after two (p=0.001), four (p<0.001), and six weeks (p<0.001) of product use.. A mouthwash containing arginine provides a significant and superior reduction in dentine hypersensitivity compared to potassium nitrate and a negative control mouthwash after two weeks.

Topics: Adult; Aged; Air; Analysis of Variance; Arginine; Calcium Carbonate; Chi-Square Distribution; Dentin; Dentin Desensitizing Agents; Dentin Sensitivity; Diphosphates; Double-Blind Method; Drug Combinations; Female; Fluorides; Humans; Male; Maleates; Middle Aged; Mouthwashes; Phosphates; Polyethylenes; Prospective Studies; Sodium Fluoride; Touch; Young Adult

Evaluate the efficacy of three regimens integrating toothpaste, toothbrush and mouthwash in reducing dentine hypersensitivity.. Eight-week single-centre, three-cell, double-blind, randomized study was conducted in the Dominican Republic. Subjects entered one of the three regimens: (1) toothpaste containing 8% arginine and 1450 ppm mono-fluorophosphate, in a calcium carbonate base, a soft-bristle toothbrush followed by a mouthwash containing 0.8% arginine, PVM/MA copolymer, pyrophosphates, and 0.05% sodium fluoride; (2) toothpaste containing 5% potassium nitrate and 1450 ppm sodium fluoride, a soft-bristle toothbrush, followed by a mouthwash containing 0.51% potassium chloride and 230 ppm sodium fluoride; and (3) toothpaste containing 1450 ppm mono-fluorophosphate, a soft-bristle toothbrush followed by a fluoride/arginine free mouthwash. Tactile and Air-Blast dentine hypersensitivity measurements were performed at baseline, two, four, and eight weeks. For treatment group comparisons, ANCOVA and post hoc Tukey's pair-wise (α=0.05) were used. Kaplan-Meier survival analysis was performed to evaluate Time to Treatment Improvement.. 120 subjects were enrolled, 118 completed the study. The Tactile hypersensitivity mean scores showed statistically significant improvement at two, four and eight (p ≤ 0.001) weeks in the arginine regime; the potassium regime did not show significant (p ≥ 0.05) improvement. Air-Blast Hypersensitivity scores had a statistically significant decrease at two (p=0.006), four (p=0.006) and eight (p=0.002) weeks in arginine and potassium regimes (p ≤ 0.05). The most effective treatment proved to be arginine (p ≤ 0.05) compared to the potassium regime.. Arginine regimen provided the greatest reduction in Tactile and Air-Blast dentine hypersensitivity compared to potassium and negative control regimens; and provides faster dentine hypersensitivity relief than potassium regimen.

Topics: Adolescent; Adult; Aged; Air; Analysis of Variance; Arginine; Calcium Carbonate; Dentin Desensitizing Agents; Dentin Sensitivity; Diphosphates; Double-Blind Method; Drug Combinations; Female; Fluorides; Humans; Kaplan-Meier Estimate; Male; Maleates; Middle Aged; Mouthwashes; Phosphates; Polyethylenes; Potassium; Prospective Studies; Sodium Fluoride; Statistics, Nonparametric; Time Factors; Toothbrushing; Toothpastes; Touch; Young Adult

The primary purpose of this randomized, controlled, six-week clinical trial was to determine the effectiveness and safety of a new whitening dentifrice in removing extrinsic tooth stain and whitening teeth. An additional two-week exploratory study was conducted to determine whether the whitening or stain-prevention activity of the dentifrice would persist following cessation of use.. In the first study (Phase I), one-hundred and forty-six qualifying subjects were randomly assigned to either a sodium bicarbonate whitening dentifrice group (Arm & Hammer Advance White Extreme Whitening Baking Soda and Peroxide Toothpaste) or a silica-based negative control dentifrice group, and brushed twice daily with their assigned dentifrice for six weeks. Tooth shade on the labial surfaces of the eight incisors was assessed using a Vita Classic shade guide, and extrinsic tooth stain was scored using a Modified Lobene Stain Index (MLSI) at baseline, week 4, and week 6. In Phase II (after the week 6 examination), volunteers from the Arm & Hammer whitening dentifrice group were randomly assigned to continue using the whitening dentifrice or to use the negative control dentifrice twice daily for two weeks. The six-week shade and stain index scores served as the baseline for this exploratory phase and were rescored after two weeks.. The whitening dentifrice group had statistically significant (p < 0.0001) mean shade score reductions of 1.82 and 2.57 from baseline to weeks 4 and 6, respectively. For the same periods, the negative control dentifrice group was virtually unchanged from baseline. For tooth stain, the MLSI total mean scores for the whitening dentifrice group showed statistically significant (p < 0.0001) decreases from baseline of 1.42 (41.6%) and 2.11 (61.6%) at weeks 4 and 6, respectively. In contrast, the negative control dentifrice group had a MLSI reduction of 0.07 at week 4 and a 0.06 increase at week 6. Between-group analyses using baseline-adjusted ANCOVA showed the whitening dentifrice to be statistically significantly more effective (p < 0.0001) than the negative control for shade and tooth stain reductions for all index comparisons. Compared to the six-week (baseline) scores, subjects who continued to use the whitening dentifrice for the additional two weeks experienced statistically significant (p < 0.0001) further mean reductions of 0.88 in shade score and 0.46 in MLSI score, while subjects who switched to the negative control dentifrice experienced smaller, statistically significant (p < 0.05) reductions of 0.34 in shade score and 0.13 in total MLSI score.. The Arm & Hammer whitening dentifrice tested in this study is effective for removing extrinsic tooth stain and whitening teeth. While the results also suggest that this dentifrice may have stain-prevention activity that persists following cessation of product use, such activity would need to be confirmed with further studies.

Topics: Adult; Aged; Analysis of Variance; Dentifrices; Diphosphates; Drug Combinations; Female; Follow-Up Studies; Humans; Hydrogen Peroxide; Male; Middle Aged; Prospective Studies; Silicon Dioxide; Sodium Bicarbonate; Tooth Bleaching; Tooth Bleaching Agents; Tooth Discoloration; Young Adult

Genetic testing has been increasingly used in several fields. In many applications, nucleic acid amplification technology is required. However, current methods to detect nucleic acid amplification require expensive reagents and special equipment or exhibit limited sensitivity, which hinders their use. To address this issue, this study reports an assay method for detecting occurrence of acid amplification in post-amplification samples using pyrophosphate, a highly sensitive byproduct of nucleic acid amplification. The method proposed requires two reagents and an automated analyzer. First, hydrogen peroxide is derived from pyrophosphate, an indicator of nucleic acid amplification, and the oxidizing power of hydrogen peroxide is used to produce Fe (III) from Fe (II). The specific metal chelator 5-Br-PAPS forms a complex with the trivalent iron produced, resulting in a highly sensitive coloration. The within-run reproducibility of our method (n = 20) was less than 3.67% at each concentration tested, and the detection limit was 0.075 μmol/L, sufficient for quantitative analysis. The technique described could detect pyrophosphate in a sample that was amplified using the loop-mediated isothermal amplification method after only 10 min. Therefore, the proposed method has the potential to be a new, rapid, and simple detection technique for amplified nucleic acids.

Topics: Diphosphates; Hydrogen Peroxide; Nucleic Acid Amplification Techniques; Nucleic Acids; Reproducibility of Results; Sensitivity and Specificity

Pyrophosphate (PYP) imaging has a high diagnostic accuracy for transthyretin cardiac amyloidosis (ATTR-CA). Indeterminate findings are often reported due to persistent blood pool activity, presumed to be from low cardiac output. We evaluated the relationship between blood pool activity on PYP imaging and echocardiographic indices of cardiac function.. Clinical and imaging data of 189 patients referred for PYP scintigraphy were evaluated. All patients underwent planar imaging and SPECT (diagnostic standard). Among those with a negative PYP SPECT, persistent left ventricular blood pool activity on planar images was inferred by a visual score ≥2 or a heart-to-contralateral (HCL) ratio ≥ 1.5. Absence of blood pool activity was inferred when both visual score was < 2 and HCL was < 1.5. Left ventricular ejection fraction (LVEF), global longitudinal strain (GLS), stroke volume index (SVi), and left atrial pressure (LAP) were calculated from standard transthoracic echocardiograms.. ATTR-CA was present in 43 (23%) patients. Among those with a negative PYP SPECT, 11 patients had significant blood pool activity. Patients with ATTR-CA had a lower LVEF, SVi, and GLS, with a higher LAP, compared to those without ATTR-CA. Among those without ATTR-CA, there were no significant differences in these parameters.. Approximately 8% of patients with a negative PYP SPECT have significant blood pool activity. Measures of cardiac function are not different among those with and without blood pool activity. PYP SPECT should be routinely performed in all patients to avoid false image interpretation.

Topics: Amyloid Neuropathies, Familial; Cardiomyopathies; Diphosphates; Echocardiography; Humans; Prealbumin; Radionuclide Imaging; Radiopharmaceuticals; Stroke Volume; Technetium Tc 99m Pyrophosphate; Ventricular Function, Left

A highly sensitive and selective sensor for the quantitative assay of inorganic pyrophosphatase (PPase) activity was developed based on a fluorescence "turn-off" strategy. Carbon quantum dots@Cu(II)-based metal-organic framework nanotubes (CQDs@Cu-MOF) with length less than 300 nm and width less than 20 nm were synthesized. CQDs in the nanotubes exhibited weak fluorescence owing to static quenching. The coordination reaction between pyrophosphate ion (PPi) and Cu(II) decomposed CQDs@Cu-MOF and led to the release of CQDs, of which the fluorescence recovered. In the presence of PPase, the hydrolysis of PPi generated phosphate ion (Pi). CQDs@Cu-MOF remained their structural stability and the fluorescence turned off. The fluorescence intensity difference of the mixture of CQDs@Cu-MOF and PPi in the absence and presence of PPase (-ΔF) was proportional to the PPase concentration from 0.1 to 5 mU mL

Topics: Carbon; Diphosphates; Fluorescence; Humans; Inorganic Pyrophosphatase; Metal-Organic Frameworks; Nanotubes, Carbon; Pyrophosphatases; Quantum Dots

Transthyretin amyloidosis (ATTR) proteins can infiltrate skeletal muscle and infrequently cause a myopathy.. Radioisotope uptake in the deltoid muscles of patients with ATTR was compared to uptake in control subjects without amyloidosis in a retrospective study.. Tracer uptake was significantly higher over the deltoids and heart but not the CC, in patients with ATTR than in control subjects. MC values were 120.1 ± 43.7 (mean ± SD) in ATTR patients and 78.9 ± 20.4 in control subjects over the heart (p = 0.005), 73.3± 21.0 and 63.5 ± 14.4 over CC (p = 0.09), and 37.0 ± 11.7 and 26.0 ± 7.1 averaged over both deltoid muscles (p = 0.014).

Topics: Amyloid Neuropathies, Familial; Biomarkers; Cardiomyopathies; Diphosphates; Humans; Muscle, Skeletal; Prealbumin; Retrospective Studies; Technetium; Technetium Tc 99m Pyrophosphate

Data were reviewed for 318 patients with suspected ATTR who underwent PYP SPECT/CT. Myocardial-to-blood pool count (MBP) ratios were computed and repeated independently > 1 month later. A physician independently scored LV myocardial-to-rib uptake on SPECT/CT as: 0 (negative), 1 < rib (equivocal), 2 = rib (positive) or 3 > rib (positive), and the image quality as: 1 (poor), 2 (adequate), and 3 (good). SPECT-only MBP ratios and visual scores were assessed separately for a subgroup of the first sequential 191 patients.. 25% of patients had positive myocardial uptake (myocardial-to-rib uptake score of ≥ 2). SPECT/CT MBP ratios were reproducible (1.35 ± .68 vs 1.33 ± .74, p = .09) and corresponded with visual scores ≥ 2 (ROC AUC = 99 ± 1%) more accurately than SPECT-only MBPs (93 ± 3%, p = .02). SPECT/CT image quality was better than that of SPECT-only (2.7 ± .5 vs 2.1 ± .5, p < .0001) with fewer equivocal results (2.6% vs 22.5%, p < .0001).. SPECT/CT produces MBP ratios that are reproducible and accurately identify a positive scan, with better image quality and fewer equivocal cases than SPECT-only.

Topics: Amyloid Neuropathies, Familial; Cardiomyopathies; Diphosphates; Humans; Reproducibility of Results; Single Photon Emission Computed Tomography Computed Tomography; Technetium Tc 99m Pyrophosphate; Tomography, Emission-Computed, Single-Photon

The purpose of this study was to examine the diagnostic value of planar. PYP images of 109 consecutive patients with suspected ATTR-CA were retrospectively reviewed. The myocardial PYP uptake was visually graded on a scale of 0 to 3 and quantified with the heart-to-contralateral (H/CL) ratio in accordance with the current expert consensus recommendations. The diagnostic value of planar images for identifying positive PYP SPECT was assessed by a receiver-operating characteristic curve analysis with the area under the curve (AUC). The uptake ratios of the ascending and descending aorta, left atrium, and trapezius muscle divided by the liver uptake were measured on SPECT images and compared to the renal function.. A total of 41 patients (38%) had myocardial PYP uptake on SPECT images. In comparison with the visual scores on 1-h anterior planar images, those on 3-h anterior planar images had lower sensitivity (80.5% vs. 97.6%) and higher specificity (86.8% vs. 55.9%) for identifying positive PYP SPECT. The ROC analysis showed that the combination of visual scores on both 1-h and 3-h anterior planar images had significantly higher AUC values in comparison with 1-h anterior planar images alone (0.90 [95% CI 0.83-0.94] vs. 0.83 [95% CI 0.75-0.88]; P < 0.001), which was comparable to the AUC values on 3-h anterior planar images alone (0.88 [95% CI 0.80-0.92]; P = 0.071). In comparison with visual scores on 1-h or 3-h anterior planar images alone, the combination of visual scores and H/CL ratio did not significantly improve the diagnostic value for identifying positive PYP SPECT (P = 0.73 and P = 0.50, respectively). The uptake ratios of ascending aorta/liver, descending aorta/liver, left atrium/liver, and trapezius muscle/liver were not significantly associated with the serum creatinine level or estimated glomerular filtration rate (P > 0.05 for all).. In the assessment of ATTR-CA using PYP imaging, visual scores on 3-h anterior planar images for identifying positive PYP SPECT had lower sensitivity and higher specificity in comparison with those on 1-h anterior planar images. The diagnostic value of the visual scores on 1-h and 3-h anterior planar images was not improved by adding the H/CL ratio. Blood pool activity of PYP was not significantly associated with renal dysfunction.

Topics: Amyloidosis; Cardiomyopathies; Diphosphates; Humans; Kidney Diseases; Prealbumin; Radionuclide Imaging; Radiopharmaceuticals; Retrospective Studies; Technetium Tc 99m Pyrophosphate; Tomography, Emission-Computed, Single-Photon

Isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) are the central five-carbon precursors to all terpenes. Despite their significance, exogenous, independent delivery of IPP and DMAPP to cells is impossible as the negatively charged pyrophosphate makes these molecules membrane impermeant. Herein, we demonstrate a facile method to circumvent this challenge through esterification of the β-phosphate with two self-immolative esters (SIEs) that neutralize the negatively charged pyrophosphate to yield membrane-permeant analogs of IPP and DMAPP. Following cellular incorporation, general esterase activity initiates cleavage of the SIEs, resulting in traceless release of IPP and DMAPP for metabolic utilization. Addition of the synthesized IPP and DMAPP precursor analogs rescued cell growth of glioblastoma (U-87MG) cancer cells concurrently treated with the HMG-CoA reductase inhibitor pitavastatin, which otherwise abrogates cell growth via blocking production of IPP and DMAPP. This work demonstrates a new application of a prodrug strategy to incorporate a metabolic intermediate and promises to enable future interrogation of the distinct biological roles of IPP and DMAPP.

Topics: Diphosphates; Hemiterpenes; Organophosphorus Compounds; Terpenes

Pseudomonas aeruginosa is a Gram-negative bacterium with an intrinsic resistance towards antibiotics due to the lack of a large diffusion pores. Exchange of substances with the environment is done mainly through a set of narrow and substrate-specific porins in its outer membrane that filter molecules according to their size and chemical composition. Among these proteins are OprP and OprO involved in the selective uptake of mono- and pyrophosphates, respectively. Both proteins are homotrimers and each monomer features an hourglass-shaped channel structure including a periplasmic cavity with a lysine cluster. In this study, we focus on the characterization of this lysine cluster in OprO. The importance of these lysine residues was shown with alanine substitutions in single channel conductance experiments, by titration of mono- and pyrophosphate in multi-channel analysis and by molecular dynamics simulations. All obtained data demonstrated that the closer the mutated lysine residues are to arginine 133, the lower gets the single channel conductance. It was found that the ion flow through each monomer can follow two different lysine paths indicating that phosphate ions have a larger freedom on the periplasmic side of the constriction region. Our results emphasize the important role of the lysine residue 121 in the binding site together with arginine 133 and aspartic acid 94. An improved understanding of the ion mobility across these channels can potentially lead to an optimized permeation of (phosphonic acid containing) antibiotics through the outer membrane of P. aeruginosa and the development of new drug molecules.

Topics: Anions; Anti-Bacterial Agents; Arginine; Bacterial Proteins; Diphosphates; Lysine; Pseudomonas aeruginosa

Topics: Diphosphates; Muscle, Skeletal; Radionuclide Imaging

Inorganic pyrophosphate (PPi) is a crucial extracellular mineralization regulator. Low plasma PPi concentrations underlie the soft tissue calcification present in several rare hereditary mineralization disorders as well as in more common conditions like chronic kidney disease and diabetes. Even though deregulated plasma PPi homeostasis is known to be linked to multiple human diseases, there is currently no reliable assay for its quantification. We here describe a PPi assay that employs the enzyme ATP sulfurylase to convert PPi into ATP. Generated ATP is subsequently quantified by firefly luciferase-based bioluminescence. An internal ATP standard was used to correct for sample-specific interference by matrix compounds on firefly luciferase activity. The assay was validated and shows excellent precision (< 3.5%) and accuracy (93-106%) of PPi spiked into human plasma samples. We found that of several anticoagulants tested only EDTA effectively blocked conversion of ATP into PPi in plasma after blood collection. Moreover, filtration over a 300,000-Da molecular weight cut-off membrane reduced variability of plasma PPi and removed ATP present in a membrane-enclosed compartment, possibly platelets. Applied to plasma samples of wild-type and Abcc6

Topics: Adenosine Triphosphate; Animals; Calcinosis; Diphosphates; Humans; Luciferases, Firefly; Rats

This case report describes an 80-year-old patient’s right-sided hemicranial headache, right-sided tongue hemiatrophy with fasciculations and deviation, right side of the tongue on protrusion, and mild dysarthria.

Topics: Calcium Pyrophosphate; Diphosphates; Humans; Hypoglossal Nerve Diseases; Tongue

Topics: Amyloid Neuropathies, Familial; Amyloidosis; Cardiomyopathies; Diphosphates; Heart Failure; Humans; Prealbumin; Radionuclide Imaging; Radiopharmaceuticals; Technetium

Phospholipids are the protective layer of modern cells, but it is challenging for the formation of phospholipids that require a simple abiotic synthesis before the advent of primitive cells. Here, we reported the abiotic synthesis for lysophosphatidic acids (LPAs) with prebiotically plausible reactants in aqueous microdroplets under ambient conditions. The LPAs formation is carried out by fusing two microdroplets streams: one contains glycerol and pyrophosphate in water and the other one contains fatty acids in acetonitrile. Compared with the bulk solution, LPAs were generated in microdroplets without the addition of catalyst and heating. Conditions of reactant concentrations and microdroplet size varied and suggested that LPAs formation occurred near or at the microdroplet surface. The LPAs formation also showed chemoselective toward on chain-length of fatty acids. Finally, the formation of LPAs underwent two-step reactions with glycerol phosphorylation eliminating one water molecule followed by esterification with fatty acids. These results also implicated that pyrophosphate functioned as both catalysts and precursors in prebiotic LPAs synthesis. The approach using fusion aqueous microdroplets has desirable features in studying the substance exchange and interaction in atmosphere.

Topics: Diphosphates; Fatty Acids; Glycerol; Phospholipids; Water

The flammability of polypropylene (PP) not only has negative effects on human health but also causes environmental pollution. Herein, from the molecular polarity point of view, rationally designed hyperbranched charring foaming agents (HCFA) modified black phosphorus nanosheets by in situ polymerization to solve the fire hazards of PP. Based on the UL-94 test V-0 rating, the conventional flame retardant of piperazine pyrophosphate (PAPP) is substituted partly by the BP@PPC. Surprisingly, compared with 27 wt% of PAPP/PP, composites consisting of only 2 wt% of BP@PPC and 20 wt% PAPP/PP also passes the V-0 rating. The results of the cone calorimeter test confirmed that adding BP@PPC decreases the total heat release (THR) and peak heat release (PHRR) by a large amount, which are decreased by 23.4%, 85.8% respectively compared with PP. Moreover, it is uncommon for the fire growth index of BP@PPC composites to be 66.7% lower than that of PAPP/PP composites. In addition, the incorporation of BP@PPC has almost no impact on the mechanical characteristics of PP composites. This study offers a reference for combining established flame retardants with novel compounds to modify the burning behaviors of PP.

Topics: Diphosphates; Flame Retardants; Humans; Phosphorus; Piperazine; Polypropylenes

We previously showed that human hepatic intrasinusoidal (HI) natural killer (NK) T cells selectively eliminate hepatocellular carcinoma (HCC) cell lines. In this study, we investigated the underlying mechanisms on how HI γδ T cells, expanded with zoledronate, exhibit a superior cytotoxic effect on HI NK-resistant Huh7 HCC cells.. γδ T cells were obtained from living liver transplant donors or from peripheral blood mononuclear cells (PBMC) of healthy volunteers and were expanded in the presence of IL-2, IL-15, and zoledronate for 2 weeks. Cytotoxicity was measured using the lactate dehydrogenase (LDH) assay in vitro and by flow cytometry using carboxyfluorescein succinimidyl ester (CFSE) in vivo.. The cytotoxicity of expanded HI γδ T cells against Huh7 cells was associated with a higher pyrophosphate expression in Huh7 cells compared to SNU398 cells. In contrast, the cytotoxicity of HI γδ T cells against SNU398 cells depended on NKG2D. HI γδ T cells expressed less PD-1 than PB γδ T cells. The cytotoxicity of HI γδ T cells against Du145 and PC3 prostate cancer cells was also associated with pyrophosphate expression in these cells, as well as NKG2D and DNAM-1.. The expression levels of phospho-antigen in tumor cells determined the cytotoxicity of HI γδ T cells, although the NK activating receptors, death ligands, and immune checkpoint molecules also contribute to their cytotoxicity. γδ T cells are attractive candidates for cancer immune cell therapy.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cytotoxicity, Immunologic; Diphosphates; Humans; Leukocytes, Mononuclear; Liver Neoplasms; Male; NK Cell Lectin-Like Receptor Subfamily K; Receptors, Antigen, T-Cell, gamma-delta; Zoledronic Acid

Transthyretin (ATTR) amyloidosis is a debilitating systemic disease often associated with symptomatic cardiac involvement. Diagnosis has dramatically changed with the advent of Technetium-99 m pyrophosphate (Tc-PYP) single-photon emission computed tomography (SPECT). With the ability to diagnose ATTR amyloidosis noninvasively and offer newer therapies, it is increasingly important to identify which patients should be referred for this testing. Relative apical sparing of longitudinal strain on echocardiogram can be potentially used to screen such patients. We sought to describe electrocardiogram (ECG) and echocardiogram (TTE) findings, including relative apical sparing of longitudinal strain, in ATTR amyloidosis patients diagnosed non-invasively with

Topics: Amyloidosis; Cardiomyopathies; Diphosphates; Humans; Predictive Value of Tests; Radiopharmaceuticals; Retrospective Studies; Technetium; Technetium Tc 99m Pyrophosphate; Tomography, Emission-Computed, Single-Photon

The oxidative dissolution of As from arsenopyrite, one important arsenic mineral in reducing conditions, poses an environmental hazard to natural aquatic systems. The dissolution of arsenopyrite occurs slowly due to the surface precipitates of iron oxides in circumneutral oxic environments. However, the presence of natural ligands and coexisting metals may change the release of Fe species, which would be of critical importance to the dissolution of arsenopyrite. Here, we investigated the oxidative dissolution of arsenopyrite induced by pyrophosphate (PP) and dissolved Mn(III) species as a natural occurring Mn species with strong complexation affinity to PP. With the presence of PP, the formation of Fe(II)-PP complexes and its rapid oxidation to dissolved Fe(III)-PP species resulted in a substantial increase in the generation of hydroxyl radicals (

Topics: Arsenic; Diphosphates; Ferric Compounds; Minerals; Oxidation-Reduction; Solubility

Inositol pyrophosphates (PP-InsPs); are a functionally diverse family of eukaryotic molecules that deploy a highly-specialized array of phosphate groups as a combinatorial cell-signaling code. One reductive strategy to derive a molecular-level understanding of the many actions of PP-InsPs is to individually characterize the proteins that bind them. Here, we describe an alternate approach that seeks a single, collective rationalization for PP-InsP binding to an entire group of proteins, i.e., the multiple nucleolar proteins previously reported to bind 5-InsP

Topics: Diphosphates; Inositol; Phosphorylation; Signal Transduction

Polyphosphate kinase 1 (Ppk1) generates polyphosphates (polyPs) by catalyzing phosphate transfer from ATP. In the presence of ATP, Myxococcus xanthus Ppk1 showed the highest activity with polyP60-70 but also showed high activity with orthophosphate and pyrophosphate. Ppk1 synthesizes long-chain polyPs with >1 000 phosphate residues from orthophosphate or pyrophosphate present in high concentrations, suggesting that in M. xanthus, Ppk1 uses intracellular ortho/pyrophosphate as an initial primer for polyP production. During M. xanthus starvation-induced development, the specific activity of Ppk1 peaked at 12 h (300-800 nmol/min/mg) and then gradually decreased. The polyP concentration was highest during mound formation (45 nmol phosphate/mg protein); then, the level of long-chain polyPs decreased and that of short-chain polyPs increased during fruiting body and spore formation. Myxococcus xanthus expresses two exopolyphosphatases, Ppx1 and Ppx2, which mainly degrade short- and long-chain polyPs, respectively, both of which were highest in vegetative cells and were detected during starvation, which may account for the degradation of polyPs. Thus, polyPs synthesized by Ppk1 early in starvation-induced development could be degraded by exopolyphosphatases and may also be used as substrates by polyP:AMP phosphotransferases and polyphosphate/ATP-NAD kinases to generate ADP and NADP+, respectively.

Topics: Adenosine Triphosphate; Diphosphates; Myxococcus xanthus; Polyphosphates

Topics: Amyloidosis; Calcinosis; Cardiomyopathies; Diphosphates; Humans; Immunoglobulin Light-chain Amyloidosis; Prealbumin; Radiopharmaceuticals

Ectopic calcification is characterized by inappropriate deposition of calcium mineral in nonskeletal connective tissues and can cause significant morbidity and mortality, particularly when it affects the cardiovascular system. Identification of the metabolic and genetic determinants of ectopic calcification could help distinguish individuals at the greatest risk of developing these pathological calcifications and could guide development of medical interventions. Inorganic pyrophosphate (PP

Topics: Bone and Bones; Calcium, Dietary; Diphosphates; Humans; Minerals; Vascular Calcification

The optimal heart-to-contralateral chest (H/CL) ratio threshold for non-invasive diagnosis of transthyretin cardiac amyloidosis (ATTR-CA) using Tc. Using myocardial PYP retention by SPECT as the reference standard, we evaluated the diagnostic performance of different semi-quantitative and quantitative (H/CL chest ratio) planar parameters obtained from 3-hour PYP imaging in a prospectively recruited cohort of minority older adults with heart failure and increased LV wall thickness.. Of 229 patients, 14 were found to have ATTR-CA (6.1%). No PYP uptake (grade 0) was observed in 77% of scans, all grade 3 scans were ATTR-CA, and only 4 of 11 (36%) grade 2 scans were ATTR-CA. An H/CL threshold of ≥ 1.4 maximized specificity (99%) and positive predictive value (93%) but resulted in decreased sensitivity (93%), compared to the ≥ 1.3 threshold which had 100% sensitivity.. Among patients with a low pretest likelihood of ATTR-CA, planar interpretation, while useful to exclude disease, must be interpreted with caution. H/CL ratio threshold of ≥ 1.3 resulted in clinically important misclassifications. These data suggest that quantitative planar imaging thresholds may not be appropriate to apply in low pretest likelihood populations being evaluated for ATTR-CA.

Topics: Aged; Amyloidosis; Cardiomyopathies; Diphosphates; Humans; Prealbumin; Radiopharmaceuticals; Technetium; Technetium Tc 99m Pyrophosphate

Inorganic pyrophosphate anions (PPi) play a key role in various biological processes and act as an essential indicator for physiological function evaluation and disease diagnosis. However, there is still a lack of available approaches for straightforward, robust, and convenient PPi detection. Herein, we design an on-off-on fluorescent switching nanoprobe employing Fe

Topics: Coloring Agents; Diphosphates; Hydrogen Peroxide; Quantum Dots; Spectrometry, Fluorescence; Sulfur

A new concept to construct photoresponsive nanozyme through the in situ deposition of electron transporting material (ETM) on BiOBr nanoplates was proposed. That was, the spontaneous coordination of ferricyanide ions (i.e., [Fe(CN)

Topics: Biological Assay; Chloramphenicol; Diphosphates

Topics: Amyloidosis; Diphosphates; Humans; Prealbumin; Technetium

Topics: Amyloid Neuropathies, Familial; Amyloidosis; Cardiomyopathies; Diphosphates; Humans; Prealbumin; Predictive Value of Tests

The excessive use of pyrophosphate (PPi) anions as additives poses a serious threat to human health and the environment. Considering the current status of PPi probes, the development of metal-free auxiliary PPi probes has important applications. In this study, a novel near-infrared nitrogen and sulfur co-doped carbon dots (N,S-CDs) were prepared. The average particle size of N,S-CDs was 2.25 ± 0.32 nm with average height was 3.05 nm. The probe N,S-CDs showed a special response to PPi, and a good linear relationship was obtained with PPi concentrations ranging from 0 to 1 μM, with the limit of detection being 0.22 nM. Tap water and milk were used for practical inspection, and ideal experimental results were acquired. In addition, the probe N,S-CDs also showed good results in biological systems, such as cell and zebrafish experiments.

Topics: Animals; Carbon; Diphosphates; Fluorescent Dyes; Humans; Metals; Nitrogen; Quantum Dots; Zebrafish

Tafamidis has been used for treatment of transthyretin cardiac amyloidosis (ATTR-CA). However, Tc-99 m pyrophosphate (PYP) cardiac scan for follow-up after tafamidis therapy has not been reported.. From May 2017 to March 2022, five patients with or without tafamidis therapy had received two Tc-99 m PYP cardiac scans. Tc-99 m PYP cardiac scan was performed with planar image and single photon emission computed tomography/computed tomography (SPECT/CT) 3 h after administration of Tc-99 m PYP. Perugini grading system was applied to determine positive or negative result of the scan. Heart to contralateral lung (H/CL) ratio as well as the difference of H/CL ratio between first and second Tc-99 m PYP cardiac scans (ΔH/CL ratio) was calculated.. In the five patients participated in this study, three received tafamidis therapy and H/CL ratio was significantly decreased (p = 0.02) after tafamidis therapy. Besides, the ΔH/CL ratio was larger in patients with tafamidis therapy than that in those without tafamidis therapy, albeit not reaching statistical significance (p = 0.2).. A decrease in H/CL ratio was found after tafamidis therapy in patients with ATTR-CA, albeit the magnitude of changes in the H/CL ratio (ΔH/CL ratio) was not significantly different from that of patients without tafamidis therapy. Future study with larger population might be required to further clarify the effect of tafamidis therapy on myocardial uptake of Tc-99 m PYP.. No clinical trial was conducted in our retrospective study.

Topics: Amyloidosis; Cardiomyopathies; Diphosphates; Follow-Up Studies; Humans; Prealbumin; Retrospective Studies; Technetium Tc 99m Pyrophosphate

Selective and sensitive detection of inorganic pyrophosphate (PPi) are of great significance both for clinical applications and fundamental research. In this work, a ratiometric fluorescent probe was developed by decorating a porphyrin-based metal-organic framework (PCN-224) with sulfur nanodots (S-dots). The red-fluorescence of PCN-224 was significantly promoted (more than 6-fold) by S-dots through inhibiting molecular motion of porphyrin ligand, which also provided active sites for the detection of Cu

Topics: Biological Assay; Diphosphates; Fluorescence; Fluorescent Dyes; Humans; Ligands; Limit of Detection; Metal-Organic Frameworks; Quantum Dots; Spectrometry, Fluorescence

Topics: Adenosine Triphosphate; Clostridium thermocellum; Diphosphates; Fermentation; Glycolysis

Tissue non-specific alkaline phosphatase (TNSALP) is an enzyme that is tethered to the cell membrane by glycosylphosphatidylinositol (GPI) and converts inorganic pyrophosphate to inorganic phosphate. Inorganic phosphate combines with calcium to form hydroxyapatite, the main mineral in the skeleton. When TNSALP is defective, conversion of inorganic pyrophosphate to inorganic phosphate is impaired and the skeleton is at risk of under-mineralization. Phosphatidylinositol glycan anchor biosynthesis class N (PIGN) is one of more than 20 genes in the GPI-biosynthesis family. Pathogenic variants in PIGN have been identified in multiple congenital anomalies-hypotonia-seizures syndrome (OMIM 614080), although a metabolic bone disease or skeletal fragility phenotype has not been reported. We describe a female child with multiple congenital anomalies-hypotonia-seizures syndrome due to a compound heterozygous pathogenic variant in PIGN who sustained a low-trauma distal femur fracture at age 7.4 years. We hypothesized that the GPI synthesis defect may result in metabolic bone disease from inadequate anchoring of TNSALP in bone and initiated asfotase alfa, a human bone-targeted recombinant TNSALP-Fc-deca-aspartate peptide, as it could bypass the PIGN genetic defect that possibly caused her skeletal fragility. Asfotase alfa was begun at 8.5 years. Baseline X-rays revealed mild rachitic findings of wrists and knees, which resolved by 5 months of treatment. Bone mineral density (BMD) assessed by dual-energy X-ray absorptiometry (DXA) showed mild improvement in spine, hip and total body less head after 16 months of treatment, while radius declined. She sustained additional low trauma fractures at right tibia and left humeral neck at 11 and 15 months into treatment, which healed quickly. Calcium, phosphorus, and parathyroid hormone levels have remained within the normal range over the 18 months of treatment. For adverse effect, she experienced a rash and discomfort in the first week of treatment which resolved with ibuprofen and diphenhydramine. She also developed subcutaneous fat atrophy. Overall, in this child with a compound pathogenic variant in PIGN, off-label use of asfotase alfa has been generally well tolerated with minimal side effects and resolution of rickets, but she continues to remain skeletally fragile.

Topics: Alkaline Phosphatase; Bone and Bones; Bone Diseases, Metabolic; Calcinosis; Calcium; Calcium, Dietary; Child; Diphosphates; Female; Fracture Healing; Humans; Hypophosphatasia; Muscle Hypotonia; Osteoporotic Fractures; Seizures

Topics: Amyloidosis; Cardiomyopathies; Diphosphates; Humans; Magnetic Resonance Imaging; Positron-Emission Tomography; Prealbumin; Radionuclide Imaging; Radiopharmaceuticals; Technetium Tc 99m Pyrophosphate; Tomography, Emission-Computed, Single-Photon

The recent pyrophosphate shortages can limit the availability of

Topics: Amyloid Neuropathies, Familial; Diphosphates; Humans; Radiopharmaceuticals; Technetium Tc 99m Medronate

Templated assembly of small molecules into nano-structural architectures has been used extensively by nature throughout its evolution. These systems have also been studied in artificial systems to design a phosphate templated assembly. However, it is yet to be investigated how the molecules interact among themselves at the molecular level and whether the phosphate templated assembly has any role in the formation of prebiotic protocellular membranes. Here, we report the prebiotic synthesis of choline-based cationic amphiphiles (-N

Topics: Artificial Cells; Diphosphates; Polyphosphates

Topics: Diagnostic Imaging; Diphosphates; Heart; Humans; Technetium; Technetium Tc 99m Pyrophosphate

Sensing of pyrophosphate ion (PPi) has received much attention due to the strong demand for clinical diagnostics. Here, based on gold nanoclusters (Au NCs), a ratiometric optical detection method for PPi is developed by simultaneously detecting the dual signals of fluorescence (FL) and second-order scattering (SOS). The PPi is detected by inhibiting the formation of aggregates of Fe

Topics: Diphosphates; Fluorescent Dyes; Gold; Limit of Detection; Metal Nanoparticles; Spectrometry, Fluorescence

Topics: Animals; Diet; Diphosphates; Hyperglycemia; Inositol; Insulin; Insulin Resistance; Lipid Metabolism; Lipids; Liver; Mice; Mice, Inbred C57BL; Non-alcoholic Fatty Liver Disease; Obesity

Citrate is the only anticoagulant currently Food and Drug Administration (FDA)-approved for the long-term storage of blood for transfusion. Citrate inhibits phosphofructokinase and may play a pro-inflammatory role, suggesting that there may be an advantage to using alternative anticoagulants. Here, we examine the use of pyrophosphate as an anticoagulant.. Whole blood samples from healthy donors were anticoagulated either with citrate-phosphate-adenine-dextrose (CPDA-1) or our novel anticoagulant mixture pyrophosphate-phosphate-adenine-dextrose (PPDA-1). Samples were assessed for coagulation capacity by thromboelastography immediately after anticoagulation (T0) with and without recalcification, as well as 5 hours after anticoagulation (T1) with recalcification. Complete blood counts were taken at both timepoints. Flow cytometry to evaluate platelet activation as well as blood smears to evaluate cellular morphology were performed at T1.. No clotting was detected in samples anticoagulated with either solution without recalcification. After recalcification, clotting function was restored in both groups. R-Time in recalcified PPDA-1 samples was shorter than in CPDA-1 samples. A reduction in platelet count at T1 compared to T0 was observed in both groups. No significant platelet activation was observed in either group at T1. Blood smear indicated platelet clumping in PPDA-1.. We have shown initial proof of concept that pyrophosphate functions as an anticoagulant at the dose used in this study, though there is an associated loss of platelets over time that may limit its usefulness for blood storage. Further dose optimization of pyrophosphate may limit or reduce the loss of platelets.

Topics: Adenine; Anticoagulants; Blood Platelets; Blood Preservation; Citrates; Citric Acid; Diphosphates; Glucose; Humans; Phosphates

Topics: Amyloid Neuropathies, Familial; Diphosphates; Heart; Heart Failure; Humans

Cardiac amyloidosis (CA) is an infiltrative cardiomyopathy resulting from deposition of insoluble amyloid protein in the myocardial interstitium. The accumulation of amyloid protein causes the myocardium to thicken and stiffen, leading to diastolic dysfunction and, eventually, heart failure. Two primary types of amyloidosis-transthyretin and immunoglobulin light chain-account for nearly 95% of all CA diagnoses. Three case studies are presented. The first demonstrates a patient positive for transthyretin amyloidosis, the second demonstrates a patient positive for light-chain CA, and the third demonstrates a patient showing blood-pool uptake on the [

Topics: Amyloid Neuropathies, Familial; Amyloidogenic Proteins; Diphosphates; Humans; Radionuclide Imaging

Cardiac amyloidosis was thought to be rare, undiagnosable, and incurable. However, recently it has been discovered to be common, diagnosable, and treatable. This knowledge has led to a resurgence in nuclear imaging with

Topics: Amyloid Neuropathies, Familial; Cardiomyopathies; Diphosphates; Humans; Radionuclide Imaging; Radiopharmaceuticals

Technetium-labeled bone-avid radiotracers can be used to diagnose transthyretin cardiac amyloidosis (ATTR-CA). Extracardiac uptake of technetium pyrophosphate (Tc-99m PYP) in this context has not been extensively explored and its significance is not well characterized. We assessed extracardiac Tc-99m PYP uptake in individuals undergoing nuclear scintigraphy and the extent of clinically actionable findings.. The Screening for Cardiac Amyloidosis with Nuclear Imaging in Minority Populations (SCAN-MP) study utilizes Tc-99m PYP imaging to identify ATTR-CA in self-identified Black and Caribbean Hispanic participants ≥ 60 years old with heart failure. We characterized the distribution of extracardiac uptake, including stratification of findings by timing of scan (1 hour vs 3 hours after Tc-99m PYP administration) and noted any additional testing in these subjects.. Of 379 participants, 195 (51%) were male, 306 (81%) Black race, and 120 (32%) Hispanic ethnicity; mean age was 73 years. Extracardiac Tc-99m PYP uptake was found in 42 subjects (11.1%): 21 with renal uptake only, 14 with bone uptake only, 4 with both renal and bone uptake, 2 with breast uptake, and 1 with thyroid uptake. Extracardiac uptake was more common in subjects with Tc-99m PYP scans at 1 hour (23.8%) than at 3 hours (6.2%). Overall, four individuals (1.1%) had clinically actionable findings.. Extracardiac Tc-99m PYP uptake manifested in about 1 in 9 SCAN-MP subjects but was clinically actionable in only 1.1% of cases.

Topics: Aged; Amyloidosis; Cardiomyopathies; Diphosphates; Female; Humans; Male; Middle Aged; Prealbumin; Prevalence; Radiopharmaceuticals; Technetium; Technetium Tc 99m Pyrophosphate; Tomography, X-Ray Computed

Topics: Amyloid Neuropathies, Familial; Amyloidosis; Cardiomyopathies; Diphosphates; Humans; Prealbumin; Radionuclide Imaging

Technetium-99mm pyrophosphate (Tc-PYP) scintigraphy is a highly accurate non-invasive method for the diagnosis of transthyretin (ATTR) cardiac amyloidosis. Prognosis for this disease is improved following treatment with the transthyretin (TTR) stabilizer tafamidis. Although tafamidis slows disease progression, its effects on myocardial amyloid and Tc-PYP uptake remain unclear. We present a patient with ATTR cardiac amyloidosis who had a strongly positive initial Tc-PYP scan, with a dramatic decrease in Tc-PYP uptake on repeat scan after 3 years of tafamidis treatment. However, myocardial biopsy showed persistent diffuse amyloid deposits. This case highlights the need for further studies regarding the utility of serial Tc-PYP scans in monitoring the progress of ATTR cardiomyopathy.

Topics: Amyloidosis; Cardiomyopathies; Diphosphates; Humans; Prealbumin; Radionuclide Imaging; Radiopharmaceuticals; Technetium; Technetium Tc 99m Pyrophosphate

RNA polymerase I (Pol I) synthesizes ribosomal RNA (rRNA), which is the first and rate-limiting step in ribosome biosynthesis. A12.2 (A12) is a critical subunit of Pol I that is responsible for activating Pol I's exonuclease activity. We previously reported a kinetic mechanism for single-nucleotide incorporation catalyzed by Pol I lacking the A12 subunit (ΔA12 Pol I) purified from S. cerevisae and revealed that ΔA12 Pol I exhibited much slower incorporation compared to Pol I. However, it is unknown if A12 influences each nucleotide incorporation in the context of transcription elongation. Here, we show that A12 contributes to every repeating cycle of nucleotide addition and that deletion of A12 results in an entirely different kinetic mechanism compared to WT Pol I. We found that instead of one irreversible step between each nucleotide addition cycle, as reported for wild type (WT) Pol I, the ΔA12 variant requires one reversible step to describe each nucleotide addition. Reversibility fundamentally requires slow PPi release. Consistently, we show that Pol I is more pyrophosphate (PPi) concentration dependent than ΔA12 Pol I. This observation supports the model that PPi is retained in the active site of ΔA12 Pol I longer than WT Pol I. These results suggest that A12 promotes PPi release, revealing a larger role for the A12.2 subunit in the nucleotide addition cycle beyond merely activating exonuclease activity.

Topics: Diphosphates; Exonucleases; Nucleotides; RNA Polymerase I

The phosphofructokinase (Pfk) reaction represents one of the key regulatory points in glycolysis. While most organisms encode for Pfks that use ATP as phosphoryl donor, some organisms also encode for PP

Topics: Adenosine Triphosphate; Amino Acid Sequence; Bacteria; Clostridium thermocellum; Diphosphates; Guanosine Triphosphate; Kinetics; Phosphofructokinase-1; Phosphofructokinases

Topics: Alkyl and Aryl Transferases; Computational Biology; Diphosphates; Diterpenes

Topics: Antrodia; Diphosphates; Magnesium; Sesquiterpenes

Inositol pyrophosphates (PP-InsPs) are energetic signaling molecules with important functions in mammals. As their biosynthesis depends on ATP concentration, PP-InsPs are tightly connected to cellular energy homeostasis. Consequently, an increasing number of studies involve PP-InsPs in metabolic disorders, such as type 2 diabetes, aspects of tumorigenesis, and hyperphosphatemia. Research conducted in yeast suggests that the PP-InsP pathway is activated in response to reactive oxygen species (ROS). However, the precise modulation of PP-InsPs during cellular ROS signaling is unknown. Here, we report how mammalian PP-InsP levels are changing during exposure to exogenous (H

Topics: Adenosine Triphosphate; Cell Line, Tumor; Diabetes Mellitus, Type 2; Diphosphates; Humans; Hydrogen Peroxide; Inositol; NAD(P)H Dehydrogenase (Quinone); Naphthoquinones; Oxygen; Reactive Oxygen Species

Antimicrobial resistance is a leading mortality factor worldwide. Here, we report the discovery of clovibactin, an antibiotic isolated from uncultured soil bacteria. Clovibactin efficiently kills drug-resistant Gram-positive bacterial pathogens without detectable resistance. Using biochemical assays, solid-state nuclear magnetic resonance, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (C

Topics: Anti-Bacterial Agents; Bacteria; Biological Assay; Diphosphates; Soil Microbiology

(1R,2R-diaminocyclohexane)(dihydropyrophosphato) platinum(II), also abbreviated as RRD2, belongs to a class of potent antitumor platinum cytostatics called phosphaplatins. Curiously, several published studies have suggested significant mechanistic differences between phosphaplatins and conventional platinum antitumor drugs. Controversial findings have been published regarding the role of RRD2 binding to DNA in the mechanism of its antiproliferative activity in cancer cells. This prompted us to perform detailed studies to confirm or rule out the role of RRD2 binding to DNA in its antiproliferative effect in cancer cells. Here, we show that RRD2 exhibits excellent antiproliferative activity in various cancer cell lines, with IC

Topics: Antineoplastic Agents; Cisplatin; Diphosphates; DNA; Neoplasms; Organoplatinum Compounds; Oxaliplatin; Platinum

Eukaryotic cells control inorganic phosphate to balance its role as essential macronutrient with its negative bioenergetic impact on reactions liberating phosphate. Phosphate homeostasis depends on the conserved INPHORS signaling pathway that utilizes inositol pyrophosphates and SPX receptor domains. Since cells synthesize various inositol pyrophosphates and SPX domains bind them promiscuously, it is unclear whether a specific inositol pyrophosphate regulates SPX domains in vivo, or whether multiple inositol pyrophosphates act as a pool. In contrast to previous models, which postulated that phosphate starvation is signaled by increased production of the inositol pyrophosphate 1-IP

Topics: Animals; Cyclin-Dependent Kinases; Diphosphates; Mammals; Phosphates; Saccharomyces cerevisiae

With documented high specificity, 99m Tc-pyrophosphate (PYP) scan enables the diagnosis of transthyretin cardiomyopathy to be made reliably without endomyocardial biopsy in patients who do not have monoclonal gammopathy. We report a case with extensive myocardial uptake of Perugini 3 score in the 3-hour 99m Tc-PYP myocardial SPECT that suggested transthyretin cardiac amyloidosis. However, a followed endomyocardial biopsy revealed no amyloid deposition. In this case, hyperphosphatemia was the most likely and presumptive cause of the false-positive 99m Tc-PYP scan. With this case, our experiences of the potential causes of false-positive results of 99m Tc-PYP are further expanded.

Topics: Biopsy; Cardiomyopathies; Diphosphates; Humans; Hyperphosphatemia; Prealbumin; Technetium; Technetium Tc 99m Pyrophosphate

Recycling of undecaprenol pyrophosphate is critical to regenerate the pool of undecaprenol monophosphate required for cell wall biosynthesis. Undecaprenol pyrophosphate is dephosphorylated by membrane-associated undecaprenyl pyrophosphate phosphatases such as UppP or type 2 Phosphatidic Acid Phosphatases (PAP2) and then transferred across the cytoplasmic membrane by Und-P flippases such as PopT (DUF368-containing protein) or UptA (a DedA family protein). While the deletion of uppP in S. pneumoniae has been reported to increase susceptibility to bacitracin and reduce infectivity in a murine infection model, the presence of PAP2 family proteins or Und-P flippases and their potential interplay with UppP in S. pneumoniae remained unknown. In this report, we identified two PAP2 family proteins and a DUF368-containing protein and investigated their roles together with that of UppP in cell growth, cell morphology and susceptibility to bacitracin in S. pneumoniae. Our results suggest that the undecaprenol monophosphate recycling pathway in S. pneumoniae could result from a functional redundancy between UppP, the PAP2-family protein Spr0434 and the DUF368-containing protein Spr0889.

Topics: Animals; Bacitracin; Diphosphates; Mice; Streptococcus pneumoniae

Topics: Cardiomyopathies; Diphosphates; Heart; Humans; Prealbumin; Technetium Tc 99m Pyrophosphate; Tomography, Emission-Computed, Single-Photon

Vigna radiata H

Topics: Deuterium; Deuterium Exchange Measurement; Diphosphates; Hydrogen; Inorganic Pyrophosphatase; Mass Spectrometry; Protons; Vigna

Quantitation of myocardial. Consecutive patients who underwent. Seventy patients were identified, mean age 70.4 ± 11.4 years, with ATTR-CM and AL-CM diagnosed in 22 (31%) and 11 (16%) patients, respectively. In patients with ATTR-CM, there were significant correlations between CPA (r. CPA and VOI were correlated with CMR measures of myocardial fibrosis in patients with ATTR-CM.

Topics: Aged; Aged, 80 and over; Amyloidosis; Cardiomyopathies; Diphosphates; Humans; Magnetic Resonance Spectroscopy; Middle Aged; Prealbumin; Technetium; Technetium Tc 99m Pyrophosphate

Pseudoxanthoma elasticum (PXE; OMIM 264800) is a rare heritable multisystem disorder, characterized by ectopic mineralization affecting elastic fibres in the skin, eyes and the cardiovascular system. Skin findings often lead to early diagnosis of PXE, but currently, no specific treatment exists to counteract the progression of symptoms. PXE belongs to a group of Mendelian calcification disorders linked to pyrophosphate metabolism, which also includes generalized arterial calcification of infancy (GACI) and arterial calcification due to CD73 deficiency (ACDC). Inactivating mutations in ABCC6, ENPP1 and NT5E are the genetic cause of these diseases, respectively, and all of them result in reduced inorganic pyrophosphate (PP

Topics: Animals; Dietary Supplements; Diphosphates; Humans; Mice; Mutation; Phosphoric Diester Hydrolases; Pseudoxanthoma Elasticum; Pyrophosphatases; Vascular Calcification

Topics: Amyloidosis; Cardiomyopathies; Diphosphates; Humans; Prealbumin; Radiopharmaceuticals; Retrospective Studies; Single Photon Emission Computed Tomography Computed Tomography; Stroke Volume; Technetium Tc 99m Pyrophosphate; Ventricular Function, Left

Topics: Benzidines; Catalysis; Chromogenic Compounds; Colorimetry; Diphosphates; Drinking Water; Hydrogen Peroxide; Iron; Limit of Detection; Molybdenum; Nanospheres; Oxidation-Reduction; Water Pollutants, Chemical

Pyrophosphate-containing calcium phosphate implants promote osteoinduction and bone regeneration. The role of pyrophosphate for inflammatory cell-mesenchymal stem cell (MSC) cross-talk during osteogenesis is not known. In the present work, the effects of lipopolysaccharide (LPS) and pyrophosphate (PPi) on primary human monocytes and on osteogenic gene expression in human adipose-derived MSCs were evaluated in vitro, using conditioned media transfer as well as direct effect systems. Direct exposure to pyrophosphate increased nonadherent monocyte survival (by 120% without LPS and 235% with LPS) and MSC viability (LDH) (by 16-19% with and without LPS). Conditioned media from LPS-primed monocytes significantly upregulated osteogenic genes (ALP and RUNX2) and downregulated adipogenic (PPAR-γ) and chondrogenic (SOX9) genes in recipient MSCs. Moreover, the inclusion of PPi (250 μM) resulted in a 1.2- to 2-fold significant downregulation of SOX9 in the recipient MSCs, irrespective of LPS stimulation or culture media type. These results indicate that conditioned media from LPS-stimulated inflammatory monocytes potentiates the early MSCs commitment towards the osteogenic lineage and that direct pyrophosphate exposure to MSCs can promote their viability and reduce their chondrogenic gene expression. These results are the first to show that pyrophosphate can act as a survival factor for both human MSCs and primary monocytes and can influence the early MSC gene expression. Graphical abstract.

Topics: Bone Regeneration; Cell Communication; Cell Differentiation; Cell Survival; Cells, Cultured; Culture Media, Conditioned; Diphosphates; Down-Regulation; Humans; Lipopolysaccharides; Materials Testing; Mesenchymal Stem Cells; Monocytes; Osteogenesis; Up-Regulation

Many biosynthetic pathways produce pyrophosphate (PPi) as a by-product, which is cytotoxic if accumulated at high levels. Pyrophosphatases play pivotal roles in PPi detoxification by converting PPi to inorganic phosphate. A number of apicomplexan parasites, including Toxoplasma gondii and Cryptosporidium parvum, express a PPi-dependent phosphofructokinase (PPi-PFK) that consumes PPi to power the phosphorylation of fructose-6-phosphate. However, the physiological roles of PPi-PFKs in these organisms are not known. Here, we report that Toxoplasma expresses both ATP- and PPi-dependent phosphofructokinases in the cytoplasm. Nonetheless, only PPi-PFK was indispensable for parasite growth, whereas the deletion of ATP-PFK did not affect parasite proliferation or virulence. The conditional depletion of PPi-PFK completely arrested parasite growth, but it did not affect the ATP level and only modestly reduced the flux of central carbon metabolism. However, PPi-PFK depletion caused a significant increase in cellular PPi and decreased the rates of nascent protein synthesis. The expression of a cytosolic pyrophosphatase in the PPi-PFK depletion mutant reduced its PPi level and increased the protein synthesis rate, therefore partially rescuing its growth. These results suggest that PPi-PFK has a major role in maintaining pyrophosphate homeostasis in T. gondii. This role may allow PPi-PFK to fine-tune the balance of catabolism and anabolism and maximize the utilization efficiency for carbon nutrients derived from host cells, increasing the success of parasitism. Moreover, PPi-PFK is essential for parasite propagation and virulence in vivo but it is not present in human hosts, making it a potential drug target to combat toxoplasmosis.

Topics: Adenosine Triphosphate; Carbohydrate Metabolism; Diphosphates; Homeostasis; Mutation; Phosphorylation; Phosphotransferases; Toxoplasma; Toxoplasmosis

The plasma membrane protein ankylosis homologue (ANKH, mouse ortholog: Ank) prevents pathological mineralization of joints by controlling extracellular levels of the mineralization inhibitor pyrophosphate (PPi). It was long thought that ANKH acts by transporting PPi into the joints. We recently showed that when overproduced in HEK293 cells, ANKH mediates release of large amounts of nucleoside triphosphates (NTPs), predominantly ATP, into the culture medium. ATP is converted extracellularly into PPi and AMP by the ectoenzyme ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). We could not rule out, however, that cells also release PPi directly via ANKH. We now addressed the question of whether PPi leaves cells via ANKH using HEK293 cells that completely lack ENPP1. Introduction of ANKH in these ENPP1-deficient HEK293 cells resulted in robust cellular ATP release without the concomitant increase in extracellular PPi found in ENPP1-proficient cells. Ank activity was previously shown to be responsible for about 75% of the PPi found in mouse bones. However, bones of Enpp1

Topics: Adenosine Triphosphate; Animals; Bone and Bones; Calcification, Physiologic; Calcinosis; Diphosphates; HEK293 Cells; Humans; Mice; Phosphate Transport Proteins

Isoleucyl-tRNA synthetase (IleRS), leucyl-tRNA synthetase (LeuRS), and valyl-tRNA synthetase (ValRS) are enzymes that have potential for the determination of l-isoleucine, l-leucine, and l-valine in food products and plasma. However, the disadvantages of these enzymes are their specificity and sensitivity. Here, we examined the substrate specificity of IleRS, LeuRS, and ValRS under various conditions of pyrophosphate amplification to improve their specificity and sensitivity. The amount of pyrophosphate produced in IleRS, LeuRS, and ValRS reactions was amplified after the addition of excess adenosine-5'-triphosphate and magnesium ions, and was approximately 9-, 8-, and 7-fold higher, respectively, for each of the initial l-amino acid substrates (50 μM). However, in addition to their target amino acids, IleRS, LeuRS, and ValRS also reacted with l-valine, l-lysine, and l-threonine, respectively. This substrate misrecognition was overcome by making the reaction pH more acidic and by increasing the magnesium ion concentration. The pyrophosphate amplification in IleRS, LeuRS, and ValRS reactions resulted in the production of p

Topics: Adenosine; Amino Acids; Amino Acyl-tRNA Synthetases; Diphosphates; Escherichia coli; Isoleucine; Leucine; Leucine-tRNA Ligase; Magnesium; RNA, Transfer; Substrate Specificity; Valine; Valine-tRNA Ligase

The aim: To reveal the effect of pyrophosphates on the tooth germ structure in the mandible of embryos (17th day of pregnancy) gestated by females, kept on a pyrophosphate-rich diet since 30 days before fertilization to gestation.. Materials and methods: The effect of food supplements was studied in «Overload phosphates model». Experiments were carried out on white nonlinear outbred mice with mass 25-28g (n= 40). The females from the control group were fed with standard rodent food, whereas the experimental females were fed with pyrophosphate-enriched food. The material for the morphological study were the mandible of 17-day-old mouse embryos (E-17), which were examined under a microscope with subsequent photofixation.. Results: The examination of the mandible of 17-day-old mouse embryos, gestated by females on a pyrophosphate-rich diet, showed morphological changes in tooth germs at the dental follicle development stage.. Conclusions: The experimentation revealed that the pyrophosphate excessive intake during dental follicle development leads to early dentinogenesis and oppression of ectodermal structures of tooth germs.

Topics: Diet; Diphosphates; Female; Humans; Pregnancy; Tooth; Tooth Germ

Inositol pyrophosphates (IPPs) are signaling molecules that regulate cellular phosphate homeostasis in diverse eukaryal taxa. In fission yeast, mutations that increase 1,5-IP

Topics: Acid Phosphatase; Adenosine Triphosphate; Diphosphates; Gene Expression; Inositol Phosphates; Multifunctional Enzymes; Phosphotransferases (Phosphate Group Acceptor); Pyrophosphatases; Schizosaccharomyces; Schizosaccharomyces pombe Proteins

Inositol pyrophosphates are signaling molecules containing at least one phosphoanhydride bond that regulate a wide range of cellular processes in eukaryotes. With a cyclic array of phosphate esters and diphosphate groups around

Topics: Arabidopsis; Diphosphates; Dual-Specificity Phosphatases; Inositol Phosphates; Saccharomyces cerevisiae

Topics: Alkaline Phosphatase; Copper; Diphosphates; DNA, Single-Stranded; Fluorescent Dyes; Metal Nanoparticles; Spectrometry, Fluorescence

We included 318 patients in the analysis (n = 86 patients +ATTR-CM; n = 232 patients -ATTR-CM). The median follow-up time was 20.1 months. During the study period, 67% of +ATTR-CM patients received tafamidis (median treatment duration, 17 months). The median H/CL ratio was 1.58 (interquartile range, 1.40-1.75). An H/CL ratio of more than 1.6 or less than 1.6 did not seem to have an impact on survival probability in +ATTR-CM patients (P = .30; hazard ratio, 0.65; 95% confidence interval, 0.31-1.41). Cardiac biomarkers were poorly correlated with H/CL (troponin T, R

Topics: Amyloid Neuropathies, Familial; Cardiomyopathies; Diphosphates; Heart Failure; Humans; Natriuretic Peptide, Brain; Prealbumin; Retrospective Studies; Technetium Tc 99m Pyrophosphate; Troponin T

Topics: Diphosphates; NAD; Peptides; Phosphofructokinase-1; Phosphofructokinases

Inorganic pyrophosphatase (iPPase) is an enzyme that cleaves pyrophosphate into two phosphate molecules. This enzyme is an essential component of in vitro transcription (IVT) reactions for RNA preparation as it prevents pyrophosphate from precipitating with magnesium, ultimately increasing the rate of the IVT reaction. Large-scale RNA production is often required for biochemical and biophysical characterization studies of RNA, therefore requiring large amounts of IVT reagents. Commercially purchased iPPase is often the most expensive component of any IVT reaction. In this paper, we demonstrate that iPPase can be produced in large quantities and high quality using a reasonably generic laboratory facility and that laboratory-purified iPPase is as effective as commercially available iPPase. Furthermore, using size exclusion chromatography coupled with multi-angle light scattering and dynamic light scattering, analytical ultracentrifugation, and small-angle X-ray scattering, we demonstrate that yeast iPPase can form tetramers and hexamers in solution as well as the enzymatically active dimer. Our work provides a robust protocol for laboratories involved with RNA in vitro transcription to efficiently produce active iPPase, significantly reducing the financial strain of large-scale RNA production.

Topics: Diphosphates; Inorganic Pyrophosphatase; Magnesium; Pyrophosphatases; RNA

Topics: Alkaline Phosphatase; Copper; Diphosphates; Ligands; Phenylalanine

Topics: Amyloidosis; Cardiomyopathies; Diphosphates; Heart Failure; Humans; Prealbumin; Technetium

Nuclear spin relaxation mechanisms are often difficult to isolate and identify, especially in molecules with internal flexibility. Here we combine experimental work with computation in order to determine the major mechanisms responsible for

Topics: Anisotropy; Diphosphates; Magnetic Resonance Spectroscopy; Molecular Dynamics Simulation

Topics: alpha-Fetoproteins; Carcinoma, Hepatocellular; Diphosphates; Humans; Liver Neoplasms; Mutation Rate; Recurrence; Retrospective Studies; Tumor Suppressor Protein p53

Inorganic pyrophosphate (PPi) is an important biological functional anion and plays crucial roles in life science, environmental science, medicine, and chemical process. Quantification of PPi in water has far-reaching significance for life exploration, disease diagnosis, and water pollution control. The label-free quantitative detection of PPi anions with a nanofluidic sensing device based on a conical single nanochannel is demonstrated. The channel surface is functionalized with a synthetic PPi receptor, triazol-methanaminium-functionalized pillar[5]arene (TAMAP5), using carbodiimide coupling chemistry. Due to the specific binding between TAMAP5 and PPi, the functionalized nanochannel can discriminate PPi from other inorganic anions with high selectivity through ionic current recording, even in the presence of various interfering anions. The current response exhibits a linear correlation with PPi concentration in the range from 1 × 10

Topics: Anions; Diphosphates; Water

Patients with the renal phosphate-wasting disease X-linked hypophosphatemia (XLH) and Hyp mice, the murine homolog of XLH, are characterized by loss-of-function mutations in phosphate-regulating endopeptidase homolog X-linked (PHEX), leading to excessive secretion of the bone-derived phosphotropic hormone FGF23. The mineralization defect in patients with XLH and Hyp mice is caused by a combination of hypophosphatemia and local accumulation of mineralization-inhibiting molecules in bone. However, the mechanism by which PHEX deficiency regulates bone cell metabolism remains elusive. Here, we used spatial metabolomics by employing matrix-assisted laser desorption/ionization (MALDI) Fourier-transform ion cyclotron resonance mass spectrometry imaging (MSI) of undecalcified bone cryosections to characterize in situ metabolic changes in bones of Hyp mice in a holistic, unbiased manner. We found complex changes in Hyp bone metabolism, including perturbations in pentose phosphate, purine, pyrimidine, and phospholipid metabolism. Importantly, our study identified an upregulation of several biochemical pathways involved in intra- and extracellular production of the mineralization inhibitor pyrophosphate in the bone matrix of Hyp mice. Our data emphasize the utility of MSI-based spatial metabolomics in bone research and provide holistic in situ insights as to how Phex deficiency-induced changes in biochemical pathways in bone cells are linked to impaired bone mineralization.

Topics: Animals; Cortical Bone; Diphosphates; Familial Hypophosphatemic Rickets; Hormones; Metabolomics; Mice; Pentoses; PHEX Phosphate Regulating Neutral Endopeptidase; Phosphates; Phospholipids; Purines; Pyrimidines; Up-Regulation

The elementary steps of transcription as catalyzed by E. coli RNA polymerase during one and two rounds of the nucleotide addition cycle (NAC) were resolved in rapid kinetic studies. Modelling of stopped-flow kinetic data of pyrophosphate release in a coupled enzyme assay during one round of the NAC indicates that the rate of pyrophosphate release is significantly less than that for nucleotide incorporation. Upon modelling of the stopped-flow kinetic data for pyrophosphate release during two rounds of the NAC, it was observed that the presence of the next nucleotide for incorporation increases the rate of release of the first pyrophosphate equivalent; incorrect nucleotides for incorporation had no effect on the rate of pyrophosphate release. Although the next nucleotide for incorporation increases the rate of pyrophosphate release, it is still significantly less than the rate of incorporation of the first nucleotide. The results from the stopped-flow kinetic studies were confirmed by using quench-flow followed by thin-layer chromatography (QF-TLC) with only the first nucleotide for incorporation labeled on the gamma phosphate with 32P to monitor pyrophosphate release. Collectively, the results are consistent with an NTP-driven model for the NAC in which the binding of the next cognate nucleotide for incorporation causes a synergistic conformational change in the enzyme that triggers the more rapid release of pyrophosphate, translocation of the enzyme along the DNA template strand and nucleotide incorporation.

Topics: Diphosphates; DNA; DNA-Directed RNA Polymerases; Escherichia coli; Kinetics; Nucleotides; Transcription, Genetic

Glycolysis is an ancient, widespread, and highly conserved metabolic pathway that converts glucose into pyruvate. In the canonical pathway, the phosphofructokinase (PFK) reaction plays an important role in controlling flux through the pathway. Clostridium thermocellum has an atypical glycolysis and uses pyrophosphate (PP

Topics: Adenosine Triphosphate; Clostridium thermocellum; Diphosphates; Glycolysis; Phosphofructokinase-1; Phosphofructokinases; Thermodynamics

The article summarizes the current experience of the nuclear medicine department of the Chazov National Medical Research Center of Cardiology in the implementation of myocardium scintigraphy with 99mTc-pyrophosphate with the differential diagnosis of the types of cardiac amyloidosis. Causes of false-positive, equivocal and non-diagnostic results, are analyzed. Possible ways to eliminate mistakes, including by modifying protocols of planar and tomographic research and optimizing the whole diagnostic algorithm for amyloidosis of the heart, are discussed.. В статье суммируется текущий опыт отдела радионуклидной диагностики и позитронно-эмиссионной томографии ФГБУ НМИЦ кардиологии им. акад. Е.И. Чазова в выполнении сцинтиграфии миокарда с 99mTc-пирофосфатом при дифференциальной диагностике типов амилоидоза сердца. Проанализированы возможные причины ложноположительных, сомнительных и недиагностических результатов исследования, предложены способы их устранения, в том числе путем модификации протоколов планарного и томографического исследований и оптимизации диагностического алгоритма амилоидоза сердца в целом.

Topics: Algorithms; Amyloidosis; Cardiomyopathies; Diphosphates; Humans; Myocardial Perfusion Imaging; Radionuclide Imaging; Technetium Tc 99m Pyrophosphate

Comparison of the conventional method and our method showed no significant difference in sensitivity and AUC, while specificity was significantly improved to 97% (p=0.003) of ray-sum axial image and 90% (p=0.01) of ray-sum coronal image, and accuracy was significantly improved to 94% (p=0.02) of ray-sum axial image.. The H/CL calculation method using ray-sum images had higher diagnostic performance than the conventional method, with optimal cutoff of ray-sum axial images 3.07 and ray-sum coronal images 2.77.

Topics: Amyloidosis; Diphosphates; Heart; Humans; Radionuclide Imaging; Radiopharmaceuticals; Tomography, Emission-Computed, Single-Photon

Pyrophosphate (PP

Topics: Archaeal Proteins; Diphosphates; Inorganic Pyrophosphatase; Pyrophosphatases; Thermococcus; Uridine Diphosphate

Topics: Diphosphates; Hypoxanthines; Isotopes; Kinetics; Oxygen; Plasmodium falciparum

Expression of the fission yeast Schizosaccharomyces pombe phosphate regulon is sensitive to the intracellular level of the inositol pyrophosphate signaling molecule 1,5-IP

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Diphosphates; Humans; Inositol Phosphates; Multifunctional Enzymes; Phosphotransferases (Phosphate Group Acceptor); Pyrophosphatases; Schizosaccharomyces; Schizosaccharomyces pombe Proteins

Novel sulfur and selenium substituted 5',5'-linked dinucleoside pyrophate analogues were prepared in a vibration ball mill from the corresponding persilylated monophosphate. The chemical hydrolysis of pyrophosphorochalcogenolate-linked dimers was studied over a wide pH-range. The effect of the chalcogeno-substitution on the reactivity of dinucleoside pyrophosphates was surprisingly modest, and the chemical stability is promising considering the potential therapeutic or diagnostic applications. The chemical stability of the precursor phosphorochalcogenolate monoesters was also investigated. Hydrolytic desilylation of these materials was effected in aqueous buffer at pH 3, 7 or 11 and resulted in phosphorus-chalcogen bond scission which was monitored using

Topics: Chalcogens; Diphosphates; Hydrolysis; Nucleosides; Phosphates

Topics: Diphosphates; Heart; Humans; Technetium; Technetium Tc 99m Pyrophosphate

The pathogenesis of calcinosis cutis, a disabling complication of SSc, is poorly understood and effective treatments are lacking. Inorganic pyrophosphate (PPi) is a key regulator of ectopic mineralization, and its deficiency has been implicated in ectopic mineralization disorders. We therefore sought to test the hypothesis that SSc may be associated with reduced circulating PPi, which might play a pathogenic role in calcinosis cutis.. Subjects with SSc and age-matched controls without SSc were recruited from the outpatient rheumatology clinics at Rutgers and Northwestern Universities (US cohort), and from the Universities of Szeged and Debrecen (Hungarian cohort). Calcinosis cutis was confirmed by direct palpation, by imaging or both. Plasma PPi levels were determined in platelet-free plasma using ATP sulfurylase to convert PPi into ATP in the presence of excess adenosine 5' phosphosulfate.. Eighty-one patients with SSc (40 diffuse cutaneous, and 41 limited cutaneous SSc) in the US cohort and 45 patients with SSc (19 diffuse cutaneous and 26 limited cutaneous SSc) in the Hungarian cohort were enrolled. Calcinosis was frequently detected (40% of US and 46% of the Hungarian cohort). Plasma PPi levels were significantly reduced in both SSc cohorts with and without calcinosis (US: P = 0.003; Hungarian: P < 0.001).. Circulating PPi are significantly reduced in SSc patients with or without calcinosis. Reduced PPi may be important in the pathophysiology of calcinosis and contribute to tissue damage with chronic SSc. Administering PPi may be a therapeutic strategy and larger clinical studies are planned to confirm our findings.

Topics: Adult; Aged; Calcinosis; Diphosphates; Female; Humans; Male; Middle Aged; Scleroderma, Systemic

Topics: Cardiology; Diphosphates; Humans; Radionuclide Imaging; Technetium; Technetium Tc 99m Pyrophosphate

Inositol pyrophosphates (PP-InsPs) are highly phosphorylated molecules that have emerged as central nutrient messengers in eukaryotic organisms. They can bind to structurally diverse target proteins to regulate biological functions, such as protein-protein interactions. PP-InsPs are strongly negatively charged and interact with highly basic surface patches in proteins, making their quantitative biochemical analysis challenging. Here, we present the synthesis of biotinylated

Topics: Biosensing Techniques; Biotin; Biotinylation; Diphosphates; Inositol Phosphates; Phosphorylation; Phosphotransferases (Phosphate Group Acceptor); Phytic Acid; Protein Interaction Mapping; Signal Transduction

Guanosine triphosphate (GTP) cyclohydrolase II (RibA) is one of three enzymes that hydrolytically cleave the C8-N9 bond of the GTP guanine. RibA also catalyzes a subsequent hydrolytic attack at the base liberating formate and in addition cleaves the α-β phosphodiester bond of the triphosphate to form pyrophosphate (PPi). These hydrolytic reactions are promoted by tandem active-site metal ions, zinc and magnesium, that respectively function at the GTP guanine and triphosphate moieties. The RibA reaction is part of riboflavin biosynthesis and forms 2,5-diamino-6-β-pyrimidinone 5'-phosphate, an exocyclic pyrimidine nucleotide that ultimately forms the pyrimidine ring of the isoalloxazine of riboflavin. The stoichiometry of the RibA reaction was defined in the study that first identified this activity in

Topics: Biocatalysis; Diphosphates; Escherichia coli; Escherichia coli Proteins; GTP Cyclohydrolase; Guanosine Triphosphate; Inosine Triphosphate; Kinetics; Protein Binding; Pyrophosphatases

Bisphosphonates (BPs) are major anti-bone-resorptive drugs. Among them, the nitrogen-containing BPs (NBPs) exhibit much stronger anti-bone-resorptive activities than non-nitrogen-containing BPs (non-NBPs). However, BP-related osteonecrosis of the jaw (BRONJ) has been increasing without effective strategies for its prevention or treatment. The release of NBPs (but not non-NBPs) from NBP-accumulated jawbones has been supposed to cause BRONJ, even though non-NBPs (such as etidronate (Eti) and clodronate (Clo)) are given at very high doses because of their low anti-bone-resorptive activities. Our murine experiments have demonstrated that NBPs cause inflammation/necrosis at the injection site, and that Eti and Clo can reduce or prevent the inflammatory/necrotic effects of NBPs by inhibiting their entry into soft-tissue cells. In addition, our preliminary clinical studies suggest that Eti may be useful for treating BRONJ. Notably, Eti, when administered together with an NBP, reduces the latter's anti-bone-resorptive effect. Here, on the basis of the above background, we examined and compared in vitro interactions of NBPs, non-NBPs, and related substances with hydroxyapatite (HA), and obtained the following results. (i) NBPs bind rapidly to HA under pH-neutral conditions. (ii) At high concentrations, Eti and Clo inhibit NBP-binding to HA and rapidly expel HA-bound NBPs (potency Eti>>Clo). (iii) Pyrophosphate also inhibits NBP-binding to HA and expels HA-bound NBPs. Based on these results and those reported previously, we discuss (i) possible anti-BRONJ strategies involving the use of Eti and/or Clo to reduce jawbone-accumulated NBPs, and (ii) a possible involvement of pyrophosphate-mediated release of NBPs as a cause of BRONJ.

Topics: Calcium; Diphosphates; Diphosphonates; Durapatite; Hydrogen-Ion Concentration; Magnesium; Nitrogen

X-ray powder diffraction (XRPD) and thermal analysis (differential scanning calorimetry/derivative of thermogravimetry (DSC/DTG)) are solid-state techniques that can be successfully used to identify and quantify various chemical compounds in polycrystalline mixtures, such as dietary supplements or drugs. In this work, 31 dietary supplements available on the Polish market that contain iron compounds, namely iron gluconate, fumarate, bisglycinate, citrate and pyrophosphate, were evaluated. The aim of the work was to identify iron compounds declared by the manufacturer as food supplements and to try to verify compliance with the manufacturer's claims. Studies performed by X-ray and thermal analysis confirmed that crystalline iron compounds (iron (II) gluconate, iron (II) fumarate), declared by the manufacturers, were present in the investigated dietary supplements. Iron (II) bisglycinate proved to be semi-crystalline. However, depending on the composition of the formulation, it was possible to identify this compound in the tested supplements. For amorphous iron compounds (iron (III) citrate and iron (III) pyrophosphate), the diffraction pattern does not have characteristic diffraction lines. Food supplements containing crystalline iron compounds have a melting point close to the melting point of pure iron compounds. The presence of excipients was found to affect the shapes and positions of the endothermic peaks significantly. Widening of endothermic peaks and changes in their position were observed, as well as exothermic peaks indicating crystallization of amorphous compounds. Weight loss was determined for all dietary supplements tested. Analysis of the DTG curves showed that the thermal decomposition of most food supplements takes place in several steps. The results obtained by a combination of both simple, relatively fast and reliable XRPD and DSC/DTG methods are helpful in determining phase composition, pharmaceutical abnormalities or by detecting the presence of the correct polymorphic form.

Topics: Calorimetry, Differential Scanning; Dietary Supplements; Diphosphates; Fumarates; Gluconates; Iron; Thermogravimetry; X-Ray Diffraction

Trypanosoma cruzi, the causative agent of Chagas' disease, belongs to the Trypanosomatidae family. The parasite undergoes multiple morphological and metabolic changes during its life cycle, in which it can use both glucose and amino acids as carbon and energy sources. The glycolytic pathway is peculiar in that its first six or seven steps are compartmentalized in glycosomes, and has a two-branched auxiliary glycosomal system functioning beyond the intermediate phosphoenolpyruvate (PEP) that is also used in the cytosol as substrate by pyruvate kinase. The pyruvate phosphate dikinase (PPDK) is the first enzyme of one branch, converting PEP, PPi, and AMP into pyruvate, Pi, and ATP. Here we present a kinetic study of PPDK from T. cruzi that reveals its hysteretic behavior. The length of the lag phase, and therefore the time for reaching higher specific activity values is affected by the concentration of the enzyme, the presence of hydrogen ions and the concentrations of the enzyme's substrates. Additionally, the formation of a more active PPDK with more complex structure is promoted by it substrates and the cation ammonium, indicating that this enzyme equilibrates between the monomeric (less active) and a more complex (more active) form depending on the medium. These results confirm the hysteretic behavior of PPDK and are suggestive for its functioning as a regulatory mechanism of this auxiliary pathway. Such a regulation could serve to distribute the glycolytic flux over the two auxiliary branches as a response to the different environments that the parasite encounters during its life cycle.

Topics: Adenosine Monophosphate; Chagas Disease; Diphosphates; Glucose; Glycolysis; Hydrogen-Ion Concentration; Kinetics; Microbodies; Phosphoenolpyruvate; Pyruvate, Orthophosphate Dikinase; Pyruvates; Recombinant Proteins; Trypanosoma cruzi

Inorganic pyrophosphatases (PPases) catalyze the hydrolysis of pyrophosphate to phosphates. PPases play essential roles in growth and development, and are found in all kingdoms of life. Human possess two PPases, PPA1 and PPA2. PPA1 is present in all tissues, acting largely as a housekeeping enzyme. Besides pyrophosphate hydrolysis, PPA1 can also directly dephosphorylate phosphorylated c-Jun N-terminal kinases 1 (JNK1). Upregulated expression of PPA1 has been linked to many human malignant tumors. PPA1 knockdown induces apoptosis and decreases proliferation. PPA1 is emerging as a potential prognostic biomarker and target for anti-cancer drug development. In spite of the biological and physiopathological importance of PPA1, there is no detailed study on the structure and catalytic mechanisms of mammalian origin PPases. Here we report the crystal structure of human PPA1 at a resolution of 2.4 Å. We also carried out modeling studies of PPA1 in complex with JNK1 derived phosphor-peptides. The monomeric protein fold of PPA1 is similar to those found in other family I PPases. PPA1 forms a dimeric structure that should be conserved in animal and fungal PPases. Analysis of the PPA1 structure and comparison with available structures of PPases from lower organisms suggest that PPA1 has a largely pre-organized and relatively rigid active site for pyrophosphate hydrolysis. Results from the modeling study indicate the active site of PPA1 has the potential to accommodate double-phosphorylated peptides from JNK1. In short, results from the study provides new insights into the mechanisms of human PPA1 and basis for structure-based anti-cancer drug developments using PPA1 as the target.

Topics: Amino Acid Sequence; Catalytic Domain; Cloning, Molecular; Crystallography, X-Ray; Diphosphates; Escherichia coli; Gene Expression; Genetic Vectors; Humans; Inorganic Pyrophosphatase; Isoenzymes; Mitochondrial Proteins; Mitogen-Activated Protein Kinase 8; Models, Molecular; Phosphates; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Folding; Protein Interaction Domains and Motifs; Protein Multimerization; Recombinant Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Substrate Specificity

Topics: Adenosine Triphosphate; Binding Sites; Diphosphates; Enzyme Inhibitors; Humans; Hydrolysis; Kinetics; Methionine Adenosyltransferase; Polyphosphates; Protein Conformation; S-Adenosylmethionine

Protein O-linked β-D-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation), an essential post-translational as well as cotranslational modification, is the attachment of β-D-N-acetylglucosamine to serine and threonine residues of nucleocytoplasmic proteins. An aberrant O-GlcNAc profile on certain proteins has been implicated in metabolic diseases such as diabetes and cancer. Inhibitors of O-GlcNAc transferase (OGT) are valuable tools to study the cell biology of protein O-GlcNAc modification. In this study we report novel uridine-peptide conjugate molecules composed of an acceptor peptide covalently linked to a catalytically inactive donor substrate analogue that bears a pyrophosphate bioisostere and explore their inhibitory activities against OGT by a radioactive hOGT assay. Further, we investigate the structural basis of their activities via molecular modelling, explaining their lack of potency towards OGT inhibition.

Topics: Diphosphates; Drug Design; Humans; Models, Molecular; Molecular Structure; N-Acetylglucosaminyltransferases; Peptides; Protein Conformation

Anthranilate phosphoribosyltransferase (AnPRT) catalyzes the transfer of the phosphoribosyl group of 5'-phosphoribosyl-1'-pyrophosphate (PRPP) to anthranilate to form phosphoribosyl-anthranilate. Crystal structures of AnPRTs from bacteria and archaea have previously been determined; however, the structure of Saccharomyces cerevisiae AnPRT (ScAnPRT) still remains unsolved. Here, crystal structures of ScAnPRT in the apo form as well as in complex with its substrate PRPP and the substrate analogue 4-fluoroanthranilate (4FA) are presented. These structures demonstrate that ScAnPRT exhibits the conserved structural fold of type III phosphoribosyltransferase enzymes and shares the similar mode of substrate binding found across the AnPRT protein family. In addition, crystal structures of ScAnPRT mutants (ScAnPRT

Topics: Amino Acid Motifs; Amino Acid Sequence; Anthranilate Phosphoribosyltransferase; Binding Sites; Conserved Sequence; Crystallography, X-Ray; Diphosphates; ortho-Aminobenzoates; Protein Multimerization; Protein Structure, Secondary; Saccharomyces cerevisiae

Quantitative assessment of myocardial radiotracer activity with CPA or VOI have high diagnostic accuracy for ATTR-CM. Both measures are potential non-invasive markers to follow progression of disease or response to therapy.. La evaluación cuantitativa de la actividad del radiotrazador en miocárdico con CPA o VOI tiene una alta certeza diagnóstica para ATTR-CM. Ambas medidas son potenciales marcadores no invasivos para seguir la progresión de la enfermedad o la respuesta a terapia.. 背景:. L’imagerie au 99mTc-pyrophosphate est devenue une méthode non invasive importante pour le diagnostic de l’amyloidose cardiaque à transthyrétine (ATTR-CM). La quantification de l’activité du 99mTc-pyrophosphate sur les images SPECT, pourrait être un marqueur de l’intensité de la maladie. Dans cette étude, nous avons évalué la précision diagnostique et la signification clinique de la quantification au 99mTc-pyrophosphate. MéTHODES ET RéSULTATS: Les patients ayant bénéficié d’une imagerie au 99mTc-pyrophosphate pour suspicion d’ATTR-CM ont été inclus. Nous avons calculé l’activité (CPA) et le volume (VOI) du 99mTc-pyrophosphate (CPA) au niveau du myocarde sur les images SPECT en rapport à l’activité sanguine au niveau de la cavité ventriculaire gauche. La précision du test a été évaluée en utilisant la surface (AUC) sous la courbe ROC. Au total, 124 patients ont été étudiés (âge moyen de 73,9 ± 11,4). Quarante trois de ces patients (34.7%) furent diagnostiqués positivement pour l’ ATTR-CM. La précision diagnostique de la CPA s’est révélée la plus élevée (AUC 0,996, IC à 95% 0,987 - 1,00), et s’est avérée significativement plus élevée que le score de Perugini (AUC 0,952, p = 0,016). Chez les patients avec ATTR-CM, la CPA est associée à une fraction d’éjection ventriculaire gauche réduite (odds ratio ajusté de 1,28, p = 0,035) et une augmentation d’ hospitalisation pour insuffisance cardiaque (hazard ratio ajusté de 1,29, p = 0,006).. L’évaluation quantitative de l’activité du 99mTc-pyrophosphate est d’une grande précision diagnostique pour l’ ATTR-CM. Les mesures CAP et VOI sont des marqueurs non invasifs potentiels pour le suivi de la progression de la maladie ou réponse au traitement.

Topics: Aged; Aged, 80 and over; Amyloid Neuropathies, Familial; Cardiomyopathies; Diphosphates; Female; Humans; Male; Middle Aged; Observer Variation; Predictive Value of Tests; Prognosis; Radiopharmaceuticals; Retrospective Studies; ROC Curve; Stroke Volume; Technetium Tc 99m Pyrophosphate; Tomography, Emission-Computed, Single-Photon; Ventricular Function, Left

(1) Background: Tissue non-specific alkaline phosphatase (TNAP) is suspected to induce atherosclerosis plaque calcification. TNAP, during physiological mineralization, hydrolyzes the mineralization inhibitor inorganic pyrophosphate (PP

Topics: Adenosine Triphosphate; Alkaline Phosphatase; Animals; Aorta; Ascorbic Acid; Atherosclerosis; Cell Transdifferentiation; Chondrocytes; Diphosphates; Glycerophosphates; Humans; Magnetic Resonance Spectroscopy; Mice; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Phosphates; Vascular Calcification

Most kinases utilize ATP as a phosphate donor and phosphorylate a wide range of phosphate acceptors. An alternative phosphate donor is inorganic pyrophosphate (PPi), which costs only 1/1000 of ATP. To develop a method to engineer PPi-dependent kinases, we herein aimed to alter the product of PPi-dependent

Topics: Catalytic Domain; Crystallization; Diphosphates; Inositol Phosphates; Kinetics; Magnetic Resonance Spectroscopy; Mutant Proteins; Mutation; Phosphoric Monoester Hydrolases; Phosphorylation; Protein Conformation; Tandem Mass Spectrometry; Thermotoga maritima

Topics: Alkaline Phosphatase; Benzidines; Catalysis; Colorimetry; Diphosphates; Gold; Humans; Hydrolysis; Metal Nanoparticles; Oxidation-Reduction; Oxidoreductases; Reproducibility of Results

Bacterial replication is a fast and accurate process, with the bulk of genome duplication being catalyzed by the α subunit of DNA polymerase III within the bacterial replisome. Structural and biochemical studies have elucidated the overall properties of these polymerases, including how they interact with other components of the replisome, but have only begun to define the enzymatic mechanism of nucleotide incorporation. Using transient-state methods, we have determined the kinetic mechanism of accurate replication by PolC, the replicative polymerase from the Gram-positive pathogen Staphylococcus aureus. Remarkably, PolC can recognize the presence of the next correct nucleotide prior to completing the addition of the current nucleotide. By modulating the rate of pyrophosphate byproduct release, PolC can tune the speed of DNA synthesis in response to the concentration of the next incoming nucleotide. The kinetic mechanism described here would allow PolC to perform high fidelity replication in response to diverse cellular environments.

Topics: Bacterial Proteins; Diphosphates; DNA Replication; DNA-Directed DNA Polymerase; Humans; Kinetics; Staphylococcal Infections; Staphylococcus aureus

The catalytic subunit of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) contains two active sites that catalyze nucleotidyl-monophosphate transfer (NMPylation). Mechanistic studies and drug discovery have focused on RNA synthesis by the highly conserved RdRp. The second active site, which resides in a Nidovirus RdRp-Associated Nucleotidyl transferase (NiRAN) domain, is poorly characterized, but both catalytic reactions are essential for viral replication. One study showed that NiRAN transfers NMP to the first residue of RNA-binding protein nsp9; another reported a structure of nsp9 containing two additional N-terminal residues bound to the NiRAN active site but observed NMP transfer to RNA instead. We show that SARS-CoV-2 RdRp NMPylates the native but not the extended nsp9. Substitutions of the invariant NiRAN residues abolish NMPylation, whereas substitution of a catalytic RdRp Asp residue does not. NMPylation can utilize diverse nucleotide triphosphates, including remdesivir triphosphate, is reversible in the presence of pyrophosphate, and is inhibited by nucleotide analogs and bisphosphonates, suggesting a path for rational design of NiRAN inhibitors. We reconcile these and existing findings using a new model in which nsp9 remodels both active sites to alternately support initiation of RNA synthesis by RdRp or subsequent capping of the product RNA by the NiRAN domain.

Topics: Amino Acid Sequence; Catalytic Domain; Coenzymes; Coronavirus RNA-Dependent RNA Polymerase; Diphosphates; Diphosphonates; Guanosine Triphosphate; Manganese; Models, Molecular; Nidovirales; Nucleotides; Protein Domains; RNA-Binding Proteins; RNA-Dependent RNA Polymerase; SARS-CoV-2; Uridine Triphosphate; Viral Nonstructural Proteins

Tissue-nonspecific alkaline phosphatase (TNAP) is known to be involved in the degradation of extracellular ATP via the hydrolysis of pyrophosphate (PPi). We investigated, using three different computational methods, namely molecular docking, thermodynamic integration (TI) and conventional molecular dynamics (MD), whether TNAP may also be involved in the utilization of β,γ-modified ATP analogues. For that, we analyzed the interaction of bisphosphonates with this enzyme and evaluated the obtained structures using in silico studies. Complexes formed between pyrophosphate, hypophosphate, imidodiphosphate, methylenediphosphonic acid monothiopyrophosphate, alendronate, pamidronate and zoledronate with TNAP were generated and analyzed based on ligand docking, molecular dynamics and thermodynamic integration. The obtained results indicate that all selected ligands show high affinity toward this enzyme. The forming complexes are stabilized through hydrogen bonds, electrostatic interactions and van der Waals forces. Short- and middle-term molecular dynamics simulations yielded very similar affinity results and confirmed the stability of the protein and its complexes. The results suggest that certain effectors may have a significant impact on the enzyme, changing its properties.

Topics: Adenosine Triphosphate; Alendronate; Alkaline Phosphatase; Computational Biology; Diphosphates; Diphosphonates; Enzymes; Humans; Hydrogen Bonding; Ligands; Molecular Conformation; Molecular Docking Simulation; Molecular Dynamics Simulation; Pamidronate; Phosphates; Protein Conformation; Thermodynamics; Zoledronic Acid

Topics: Cell Cycle; Cell Survival; Diphosphates; DNA-Binding Proteins; Gene Expression Regulation, Fungal; Inositol; Osmotic Pressure; Oxidation-Reduction; Oxidative Stress; Peptides, Cyclic; Protein Tyrosine Phosphatases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Signal Transduction; Stress, Physiological; Transcription Factors

Traditional methods of identifying food-borne pathogens are time consuming and laborious, so innovative methods for their rapid identification must be developed. Testing for bioluminescence pyrophosphate is a convenient and fast method of detecting pathogens without complex equipment. However, the sensitivity of the method is not as high as that of other methods, and it has a very high detection limit. In this study, the method was optimized to improve its sensitivity. The shortcomings of the method were first identified and corrected using dATPαS instead of dATP for the polymerase chain reaction (PCR), therefore reducing the background signal. Also, when the DNA template extracted from the food-borne pathogens was purified, the new bioluminescence pyrophosphate assay had a limit of detection of <10 copy/μl or 10 colony-forming units/ml, and its sensitivity was higher than that of fluorescent real-time quantitative PCR. Moreover, a single copy of a food-borne pathogen could be detected when a single DNA template was included in the PCR. Salmonella was detected in and isolated from 60 samples of broiler chicken, and the accuracy of the results was verified using a culture method (GB 4789.4-2010). These results showed that the new bioluminescence pyrophosphate assay has the advantages of an intuitive detection process, convenient operation, and rapid measurements. Therefore, it can be used for the rapid detection of pathogenic bacteria and probiotics in various fields.

Topics: Diphosphates; DNA, Bacterial; Food Analysis; Food Microbiology; Polymerase Chain Reaction; Salmonella typhimurium

Supramolecular chemistry has provided versatile and affordable solutions for the design of tough, flexible polymers. However, application of supramolecular chemistry has been limited to the production of rigid, high-performance polymers due to weak segment mobility. This paper describes a new method of toughening rigid high-performance polymers using the synergistic effect between dual Cu

Topics: Copper; Diphosphates; Hydrogen Bonding; Materials Testing; Polymers; Recycling; Temperature

Calcifications can disrupt organ function in the cardiovascular system and the kidney, and are particularly common in patients with chronic kidney disease (CKD). Fetuin-A deficient mice maintained against the genetic background DBA/2 exhibit particularly severe soft tissue calcifications, while fetuin-A deficient C57BL/6 mice remain healthy. We employed molecular genetic analysis to identify risk factors of calcification in fetuin-A deficient mice. We sought to identify pharmaceutical therapeutic targets that could be influenced by dietary of parenteral supplementation. We studied the progeny of an intercross of fetuin-A deficient DBA/2 and C57BL/6 mice to identify candidate risk genes involved in calcification. We determined that a hypomorphic mutation of the Abcc6 gene, a liver ATP transporter supplying systemic pyrophosphate, and failure to regulate the Trpm6 magnesium transporter in kidney were associated with severity of calcification. Calcification prone fetuin-A deficient mice were alternatively treated with parenteral administration of fetuin-A dietary magnesium supplementation, phosphate restriction, or by or parenteral pyrophosphate. All treatments markedly reduced soft tissue calcification, demonstrated by computed tomography, histology and tissue calcium measurement. We show that pathological ectopic calcification in fetuin-A deficient DBA/2 mice is caused by a compound deficiency of three major extracellular and systemic inhibitors of calcification, namely fetuin-A, magnesium, and pyrophosphate. All three of these are individually known to contribute to stabilize protein-mineral complexes and thus inhibit mineral precipitation from extracellular fluid. We show for the first time a compound triple deficiency that can be treated by simple dietary or parenteral supplementation. This is of special importance in patients with advanced CKD, who commonly exhibit reduced serum fetuin-A, magnesium and pyrophosphate levels.

Topics: alpha-2-HS-Glycoprotein; alpha-Fetoproteins; Animals; Calcinosis; Diphosphates; Disease Models, Animal; Female; Kidney; Liver; Magnesium; Male; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Microvessels; Minerals; Multidrug Resistance-Associated Proteins; Renal Insufficiency, Chronic; TRPM Cation Channels

Aminoglycoside antibiotics have lost much of their effectiveness due to widespread resistance, primarily via covalent modification. One of the most ubiquitous enzymes responsible for aminoglycoside resistance is aminoglycoside

Topics: Adenosine Monophosphate; Amino Acid Sequence; Aminoglycosides; Anti-Bacterial Agents; Catalysis; Diphosphates; Drug Resistance, Microbial; Kinetics; Manganese; Models, Molecular; Molecular Conformation; Nucleotidyltransferases; Protein Binding; Signal Transduction; Structure-Activity Relationship; Substrate Specificity; Tobramycin

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel activated by protein kinase A (PKA) phosphorylation on the regulatory (R) domain. Phosphorylation at several R domain residues stimulates ATP-dependent channel openings and closings, termed channel gating. To explore the protein segment responsible for channel potentiation and PKA-dependent activation, deletion mutations were constructed by removing one to three protein segments of the R domain including residues 708-759 (ΔR

Topics: Adenosine Triphosphate; Cyclic AMP-Dependent Protein Kinases; Cystic Fibrosis Transmembrane Conductance Regulator; Deoxyadenosines; Diphosphates; Humans; Membrane Potentials; Mutagenesis, Site-Directed; Phosphorylation; Protein Domains

The control of pyrimidine nucleotide formation in the bacterium Pseudomonas aurantiaca ATCC 33663 by pyrimidines was studied. The activities of the pyrimidine biosynthetic pathway enzymes were investigated in P. aurantiaca ATCC 33663 cells and from cells of an auxotroph lacking orotate phosphoribosyltransferase activity under selected culture conditions. All activities of the pyrimidine biosynthetic pathway enzymes in ATCC 33663 cells were depressed by uracil addition to the minimal medium when succinate served as the carbon source. In contrast, all pyrimidine biosynthetic pathway enzyme activities in ATCC 33663 cells were depressed by orotic acid supplementation to the minimal medium when glucose served as the carbon source. The orotidine 5'-monophosphate decarboxylase activity in the phosphoribosyltransferase mutant strain increased by more than sixfold in succinate-grown cells and by more than 16-fold in glucose-grown cells after pyrimidine limitation showing possible repression of the decarboxylase by a pyrimidine-related compound. Inhibition by ATP, GTP, UTP and pyrophosphate of the in vitro activity of aspartate transcarbamoylase in ATCC 33663 was observed. The findings demonstrated control at the level of pyrimidine biosynthetic enzyme synthesis and activity for the P. aurantiaca transcarbamoylase. The control of pyrimidine synthesis in P. aurantiaca seemed to differ from what has been observed previously for the regulation of pyrimidine biosynthesis in related Pseudomonas species. This investigation could prove helpful to future work studying pseudomonad taxonomic analysis as well as to those exploring antifungal and antimicrobial agents produced by P. aurantiaca.

Topics: Aspartate Carbamoyltransferase; Biosynthetic Pathways; Diphosphates; Gene Expression Regulation, Bacterial; Orotate Phosphoribosyltransferase; Orotidine-5'-Phosphate Decarboxylase; Pseudomonas; Pyrimidine Nucleotides; Pyrimidines; Succinic Acid; Uracil

We have developed a simple and convenient route to prepare fluorescent carbon dots with dual emission peaks respectively at 470 and 570 nm. The prepared dual-emission carbon dots can be used for ratiometric detection of Fe3+ ions in the range from 0 to 50 μmol·L-1 with 0.8 μmol·L-1 detection limit based on the fluorescence quenching at 570 nm. The quenched fluorescence induced by Fe3+ ions could be recovered by pyrophosphate. We further used the carbon dots-Fe3+ ions-pyrophosphate mixed system for ratiometric detection of acid phosphatase in the range from 0.08 to 6.75 μg·mL-1 with 0.01 μg·mL-1 detection limit.

Topics: Acid Phosphatase; Carbon; Diphosphates; Ferric Compounds; Fluorescent Dyes; Ions; Molecular Structure; Particle Size; Quantum Dots; Spectrometry, Fluorescence; Surface Properties

It was the aim of this study to synthesize a phosphorylated emulsifier possessing a PEG-linker for establishment of a potent zeta potential changing system in self-emulsifying drug delivery systems (SEDDS).. N,N'-Bis(polyoxyethylene)oleylamine (POA) was phosphorylated utilizing pyrophosphoric acid. Successful synthesis of POA bisphosphate (POAP) was confirmed by NMR and HR CS MAS. After incorporation of 1% POAP into SEDDS (Kolliphor RH 40, Capmul PG-8, Labrafac Lipophile WL 1349, Labrafac PG; 30/20/20/30, v/v), according emulsions were incubated with intestinal alkaline phosphatase (IAP) and the zeta potential was measured. Additionally, the amount of released phosphate upon incubation with IAP or on Caco-2 cells was quantified by malachite green assay. Finally, cell viability studies on Caco-2 cells were performed and mucus permeation properties with and without IAP preincubation were assessed.. POAP was synthesized as brown viscous liquid with a yield of 36% and could be incorporated into SEDDS. By incubation with IAP a zeta potential shift from -15.1 to 6.5 mV was observed. A corresponding phosphate release in presence of isolated IAP as well as on Caco-2 cells was found. Assessment of the cytotoxic potential revealed no significant alteration in the safety profile of SEDDS by incorporation of POAP. Mucus permeation studies exposed a 2-fold higher permeation of fluorescein diacetate (FDA) having been embedded in SEDDS loaded with POAP in comparison to blank formulation and 3-fold higher permeability than for emulsions having been preincubated with phosphatase.. The novel phosphorylated surfactant exhibiting a PEG-linker facilitated a potent zeta potential change of SEDDS.

Topics: Alkaline Phosphatase; Caco-2 Cells; Diphosphates; Drug Carriers; Drug Compounding; Emulsifying Agents; Emulsions; GPI-Linked Proteins; Humans; Intestinal Absorption; Intestinal Mucosa; Permeability; Phosphates; Phosphorylation; Polyethylene Glycols; Surface Properties

Acidocalcisomes are membrane-bounded, electron-dense, acidic organelles, rich in calcium and polyphosphate. These organelles were first described in trypanosomatids and later found from bacteria to human cells. Some of the functions of the acidocalcisome are the storage of cations and phosphorus, participation in pyrophosphate (PP

Topics: Calcium Signaling; Cell Fractionation; Centrifugation, Density Gradient; Diphosphates; Enzyme Assays; Hydrogen-Ion Concentration; Microscopy, Electron; Organelles; Polyphosphates; Protozoan Proteins; Triiodobenzoic Acids; Trypanosoma brucei brucei

ANKH mutations are associated with calcium pyrophosphate deposition disease and craniometaphyseal dysplasia. This study investigated the effects of these ANKH mutants on cellular localisation and associated biochemistry. We generated four ANKH overexpression-plasmids containing either calcium pyrophosphate deposition disease or craniometaphyseal dysplasia linked mutations: P5L, E490del and S375del, G389R. They were transfected into CH-8 articular chondrocytes and HEK293 cells. The ANKH mutants dynamic differential localisations were imaged and we investigated the interactions with the autophagy marker LC3. Extracellular inorganic pyrophosphate, mineralization, ENPP1 activity expression of ENPP1, TNAP and PIT-1 were measured. P5L delayed cell membrane localisation but once recruited into the membrane it increased extracellular inorganic pyrophosphate, mineralization, and ENPP1 activity. E490del remained mostly cytoplasmic, forming punctate co-localisations with LC3, increased mineralization, ENPP1 and ENPP1 activity with an initial but unsustained increase in TNAP and PIT-1. S375del trended to decrease extracellular inorganic pyrophosphate, increase mineralization. G389R delayed cell membrane localisation, trended to decrease extracellular inorganic pyrophosphate, increased mineralization and co-localised with LC3. Our results demonstrate a link between pathological localisation of ANKH mutants with different degrees in mineralization. Furthermore, mutant ANKH functions are related to synthesis of defective proteins, inorganic pyrophosphate transport, ENPP1 activity and expression of ENPP1, TNAP and PIT-1.

Topics: Alkaline Phosphatase; Autophagy; Bone Diseases, Developmental; Carrier Proteins; Chondrocalcinosis; Chondrocytes; Craniofacial Abnormalities; Diphosphates; HEK293 Cells; Humans; Hyperostosis; Hypertelorism; Microscopy, Confocal; Mutation; Phosphate Transport Proteins; Phosphoric Diester Hydrolases; Protein Domains; Pyrophosphatases; Transcription Factor Pit-1

Laccase, a unique class of multicopper oxidase, presents promising potential as a biocatalyst in many industrial and biotechnological applications. Recently, it has been significantly applied in many metal-polluted sites due to its Manganese (Mn)-oxidation ability. Here, we demonstrate the Mn(II)-oxidase activity of laccase obtained from Bacillus sp. GZB. The CotA gene of GZB was transformed in E. coli BL21 and overexpressed. The purified laccase (LACREC3-laccase) displayed the absence of a peak at 610 nm that is usually found in blue-laccase. Further, the LACREC3-laccase exhibited high activity and stability at different pH and temperatures with substrates 2, 2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonate) and syringaldazine, respectively. It also functioned in the presence of various metals and enzyme inhibitors. Most notably, LACREC3-laccase formed insoluble brown Mn(III)/Mn(IV)-oxide particles from Mn(II) mineral, exhibiting its Mn(II)-oxidase activity. In addition to native polyacrylamide gel electrophoresis and buffer test, we developed an 'agarose gel plate' assay to evaluate Mn(II) oxidation activity of laccase. Furthermore, using the leucoberbelin blue assay, a total of 44.45 ± 0.45% Mn(IV)-oxides were quantified, in which 5.87 ± 0.61% autoxidized after 24 h. The Mn(II) oxidation mechanisms were further predicted by trapping Mn(III) using pyrophosphate during Mn(II) to Mn(IV) conversion by LACREC3-laccase. Overall, the laccase of GZB has excellent activity and stability plus an ability to oxidize Mn(II). This study is the first report on a non-blue laccase, exhibiting Mn(II)-oxidase activity. Thus, it offers a novel finding of the Mn(II) oxidation processes that can be a valuable way of Mn(II)-mineralization in various metal-polluted environments.

Topics: Bacillus; Diphosphates; Escherichia coli; Laccase; Manganese; Manganese Compounds; Oxidation-Reduction; Oxides

Antibiotics (AB) resistance is a major threat to global health, thus the development of novel AB classes is urgently needed. Lantibiotics (i.e. nisin) are natural compounds that effectively control bacterial populations, yet their clinical potential is very limited. Nisin targets membrane-embedded cell wall precursor - lipid II - via capturing its pyrophosphate group (PPi), which is unlikely to evolve, and thus represents a promising pharmaceutical target. Understanding of exact molecular mechanism of initial stages of membrane-bound lipid II recognition by water-soluble nisin is indispensable. Here, using molecular simulations, we demonstrate that the structure of lipid II is determined to a large extent by the surrounding water-lipid milieu. In contrast to the bulk solvent, in the bilayer only two conformational states remain capable of nisin binding. In these states PPi manifests a unique arrangement of hydrogen bond acceptors on the bilayer surface. Such a "pyrophosphate pharmacophore" cannot be formed by phospholipids, which explains high selectivity of nisin/lipid II recognition. Similarly, the "recognition module" of nisin, being rather flexible in water, adopts the only stable conformation in the presence of PPi analogue (which mimics the lipid II molecule). We establish the "energy of the pyrophosphate pharmacophore" approach, which effectively distinguishes nisin conformations that can form a complex with PPi. Finally, we propose a molecular model of nisin recognition module/lipid II complex in the bacterial membrane. These results will be employed for further study of lipid II targeting by antimicrobial (poly)cyclic peptides and for design of novel AB prototypes.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Computational Chemistry; Dimethyl Sulfoxide; Diphosphates; Hydrogen Bonding; Lipid Bilayers; Membrane Lipids; Models, Chemical; Models, Molecular; Molecular Conformation; Nisin; Nuclear Magnetic Resonance, Biomolecular; Phosphatidylethanolamines; Phosphatidylglycerols; Protein Binding; Protein Conformation; Solubility; Uridine Diphosphate N-Acetylmuramic Acid; Water

Farnesol is a sesquiterpene from propolis and citrus fruit that shows promising anti-bacterial activity for caries treatment and prevention, but its hydrophobicity limits the clinical application. We aimed to develop the novel polymeric micelles (PMs) containing a kind of derivative of farnesol and a ligand of pyrophosphate (PPi) that mediated PMs to adhere tightly with the tooth enamel.. Farnesal (Far) was derived from farnesol and successfully linked to PEG via an acid-labile hydrazone bond to form PEG-hyd-Far, which was then conjugated to PPi and loaded into PMs to form the aimed novel drug delivery system, PPi-Far-PMs. The in vitro test about the binding of PPi-Far-PMs to hydroxyapatite showed that PPi-Far-PMs could bind rapidly to hydroxyapatite and quickly release Far under the acidic conditions. Results from the mechanical testing and the micro-computed tomography indicated that PPi-Far-PMs could restore the microarchitecture of teeth with caries. Moreover, PPi-Far-PMs diminished the incidence and severity of smooth and sulcal surface caries in rats that were infected with Streptococcus mutans while being fed with a high-sucrose diet. The anti-caries efficacy of free Far can be improved significantly by PPi-Far-PMs through the effective binding of it with tooth enamel via PPi.. This novel drug-delivery system may be useful for the treatment and prevention of dental caries as well as the targeting therapy of anti-bacterial drugs in the oral disease.

Topics: Animals; Cariostatic Agents; Dental Caries; Diphosphates; Drug Carriers; Durapatite; Farnesol; Hydrophobic and Hydrophilic Interactions; Micelles; Molar; Polyethylene Glycols; Rats; Streptococcus mutans

Methanotrophs is a promising biocatalyst in biotechnological applications with their ability to utilize single carbon (C1) feedstock to produce high-value compounds. Understanding the behavior of biological networks of methanotrophic bacteria in different parameters is vital to systems biology and metabolic engineering. Interestingly, methanotrophic bacteria possess the pyrophosphate-dependent 6-phosphofructokinase (PPi-PFK) instead of the ATP-dependent 6-phosphofructokinase, indicating their potentials to serve as promising model for investigation the role of inorganic pyrophosphate (PPi) and PPi-dependent glycolysis in bacteria. Gene knockout experiments along with global-omics approaches can be used for studying gene functions as well as unraveling regulatory networks that rely on the gene product.. In this study, we performed gene knockout and RNA-seq experiments in Methylotuvimicrobium alcaliphilum 20Z to investigate the functional roles of PPi-PFK in C1 metabolism when cells were grown on methane and methanol, highlighting its metabolic importance in C1 assimilation in M. alcaliphilum 20Z. We further conducted adaptive laboratory evolution (ALE) to investigate regulatory architecture in pfk knockout strain. Whole-genome resequencing and RNA-seq approaches were performed to characterize the genetic and metabolic responses of adaptation to pfk knockout. A number of mutations, as well as gene expression profiles, were identified in pfk ALE strain to overcome insufficient C1 assimilation pathway which limits the growth in the unevolved strain.. This study first revealed the regulatory roles of PPi-PFK on C1 metabolism and then provided novel insights into mechanism of adaptation to the loss of this major metabolic enzyme as well as an improved basis for future strain design in type I methanotrophs.

Topics: Bacterial Proteins; Diphosphates; Gene Knockout Techniques; Glycolysis; Methane; Methanol; Methylococcaceae; Phosphotransferases; RNA-Seq

Hexameric inorganic pyrophosphatase from Mycobacterium tuberculosis (Mt-PPase) has a number of structural and functional features that distinguish it from homologous enzymes widely occurring in living organisms. In particular, it has unusual zones of inter-subunit contacts and lacks the N-terminal region common for other PPases. In this work, we constructed two mutant forms of the enzyme, Ec-Mt-PPase and R14Q-Mt-PPase. In Ec-Mt-PPase, the missing part of the polypeptide chain was compensated with a fragment of PPase from Escherichia coli (Ec-PPase). In R14Q-Mt-PPase, a point mutation was introduced to the contact interface between the two trimers of the hexamer. Both modifications significantly improved the catalytic activity of the enzyme and abolished its inhibition by the cofactor (Mg2+ ion) excess. Activation of Mt-PPase by low (~10 μM) concentrations of ATP, fructose-1-phosphate, L-malate, and non-hydrolyzable substrate analogue methylene bisphosphonate (PCP) was observed. At concentrations of 100 μM and higher, the first three compounds acted as inhibitors. The activating effect of PCP was absent in both mutant forms, and the inhibitory effect of fructose-1-phosphate was absent in Ec-Mt-PPase. The effects of other modulators varied only quantitatively among the mutants. The obtained data indicate the presence of allosteric sites in Mt-PPase, which are located in the zones of inter-subunit contact or associated with them.

Topics: Adenosine Triphosphate; Allosteric Site; Catalysis; Diphosphates; Escherichia coli; Fructosephosphates; Hydrogen-Ion Concentration; Hydrolysis; Inorganic Pyrophosphatase; Ions; Magnesium; Malates; Mutagenesis, Site-Directed; Mutation; Mycobacterium tuberculosis; Peptides; Point Mutation; Protein Conformation; Protein Domains; Temperature; Ultracentrifugation

The membrane protein ANKH was known to prevent pathological mineralization of joints and was thought to export pyrophosphate (PPi) from cells. This did not explain, however, the presence of ANKH in tissues, such as brain, blood vessels and muscle. We now report that in cultured cells ANKH exports ATP, rather than PPi, and, unexpectedly, also citrate as a prominent metabolite. The extracellular ATP is rapidly converted into PPi, explaining the role of ANKH in preventing ankylosis. Mice lacking functional Ank (Ankank/ank mice) had plasma citrate concentrations that were 65% lower than those detected in wild type control animals. Consequently, citrate excretion via the urine was substantially reduced in Ankank/ank mice. Citrate was even undetectable in the urine of a human patient lacking functional ANKH. The hydroxyapatite of Ankank/ank mice contained dramatically reduced levels of both, citrate and PPi and displayed diminished strength. Our results show that ANKH is a critical contributor to extracellular citrate and PPi homeostasis and profoundly affects bone matrix composition and, consequently, bone quality.

Topics: Adenosine Triphosphate; Animals; Bone and Bones; Bone Development; Calcinosis; Cell Differentiation; Cells, Cultured; Citric Acid; Diphosphates; Humans; Mechanical Phenomena; Mice; Mutation; Phosphate Transport Proteins

Discovering new functional genes, designing perfect crystal structures, and developing high-performance materials are the goals being pursued by scientists. Herein, the first antimony pyrophosphate, K

Topics: Antimony; Crystallography, X-Ray; Diphosphates; Fluorine; Models, Molecular; Optical Phenomena; Potassium; Spectrophotometry, Infrared

Trauma-induced calcification is the pathological consequence of complex injuries which often affect the central nervous system and other parts of the body simultaneously. We demonstrated by an animal model recapitulating the calcification of the above condition that adrenaline transmits the stress signal of brain injury to the calcifying tissues. We have also found that although the level of plasma pyrophosphate, the endogenous inhibitor of calcification, was normal in calcifying animals, it could not counteract the acute calcification. However, externally added pyrophosphate inhibited calcification even when it was administered after the complex injuries. Our finding suggests a potentially powerful clinical intervention of calcification triggered by polytrauma injuries which has no effective treatment.

Topics: Adrenergic Antagonists; Animals; Brain Injuries, Traumatic; Cardiotoxins; Diphosphates; Disease Models, Animal; Epinephrine; Female; Gene Expression Regulation; Mice, Inbred C57BL; Muscle, Skeletal; Ossification, Heterotopic; Receptors, Adrenergic; Vascular Calcification; X-Ray Microtomography

The hepatitis C virus RNA-dependent RNA polymerase NS5B is responsible for the replication of the viral genome. Previous studies have uncovered NTP-mediated excision mechanisms that may be responsible for aiding in maintaining fidelity (the frequency of incorrect incorporation events relative to correct), but little is known about the fidelity of NS5B. In this study, we used transient-state kinetics to examine the mechanistic basis for polymerase fidelity. We observe a wide range of efficiency for incorporation of various mismatched base pairs and have uncovered a mechanism in which the rate constant for pyrophosphate release is slowed for certain misincorporation events. This results in an increase in fidelity against these specific misincorporations. Furthermore, we discover that some mismatches are highly unfavorable and cannot be observed under the conditions used here. The calculated fidelity of NS5B ranges between 10

Topics: Diphosphates; Hepacivirus; RNA-Dependent RNA Polymerase; RNA, Viral; Viral Nonstructural Proteins

Coenzyme A (CoA) is a highly selective inhibitor of the mitotic regulatory enzyme Aurora A kinase, with a novel mode of action. Herein we report the design and synthesis of analogues of CoA as inhibitors of Aurora A kinase. We have designed and synthesised modified CoA structures as potential inhibitors, combining dicarbonyl mimics of the pyrophosphate group with a conserved adenosine headgroup and different length pantetheine-based tail groups. An analogue with a -SH group at the end of the pantotheinate tail showed the best IC50, probably due to the formation of a covalent bond with Aurora A kinase Cys290.

Topics: Aurora Kinase A; Coenzyme A; Diphosphates; Dose-Response Relationship, Drug; Drug Design; Humans; Models, Molecular; Molecular Structure; Pantetheine; Protein Kinase Inhibitors; Structure-Activity Relationship

Alkaline phosphatase (ALP) is extensively used as a clinical biomarker because of its close relevance with a variety of diseases. Thus, exploring reliable and practical methods for ALP analysis is of great significance. In the present work, we explored MIL-53(Fe) as a bifunctional platform with pyrophosphate (PPi)-mediated peroxidase-like activity and oxidation-stimulated fluorescence switching for ALP sensing. The proposed MIL-53(Fe) could exhibit favorable peroxidase-mimicking activity to catalytically decompose H2O2 to hydroxyl radicals, which had strong oxidizing ability to oxidize the terephthalic acid bridging ligand, resulting in the oxidation-stimulated turn-on fluorescence of MIL-53(Fe) itself. Due to the strong coordination interaction between PPi and Fe3+, the former with a relatively large molecular structure was able to inhibit the catalytic activity of MIL-53(Fe) via capping active Fe3+ sites, leading to the suppression of its self-fluorescence response. When ALP was present, it could hydrolyze the PPi inhibitor and restore the dual functions of MIL-53(Fe) to provide fluorescence again. With the above principle, highly sensitive and selective determination of ALP with a linear scope of 2-80 U L-1 and a detection limit down to 0.7 U L-1 was achieved. The MIL-53(Fe) was also demonstrated to be very reliable in measuring the target in human serum, indicating its great promise as an integrated tool for ALP detection in clinical practice.

Topics: Alkaline Phosphatase; Biomarkers; Catalysis; Colorimetry; Diphosphates; Fluorescence; Humans; Hydrogen Peroxide; Limit of Detection; Metal-Organic Frameworks; Oxidation-Reduction; Peroxidase; Phthalic Acids; Spectrometry, Fluorescence

Herein, an easy and effective ratiometric fluorescent nanoprobe for the selective detection of Pb2+ and pyrophosphate (PPi) was developed based on label-free carbon dots (CDs). The CDs showed dual emission bands at 477 and 651 nm under a single-wavelength excitation (391 nm). Pb2+ and PPi were sequentially detected by the sensor with high reliability and accuracy via the exploitation of the fluorescence intensity ratio (F477/F651). The dual emission sensor exhibited high selectivity and excellent sensitivity for Pb2+ and PPi than for other analytes with detection limits as low as 0.055 μM and 0.089 μM, respectively. Moreover, the fluorescent CDs changed from pink to cyan and from cyan to pink and could be used as a fluorescent paper sensor for the visual detection of Pb2+ and PPi. To the best of our knowledge, this is the first report on the ratiometric fluorescence detection of Pb2+ and PPi by CDs; therefore, the as-synthesized CDs show great potential for application in the determination of Pb2+ and PPi in real samples.

Topics: Carbon; Diphosphates; Fluorescent Dyes; Lead; Paper; Particle Size; Quantum Dots; Surface Properties

This work illustrates the highly selective fluorescence detection of ATP in the presence of other competing anions, such as AMP, ADP, PPi and other phosphates by using a set of hydroxide-bridged dizinc(ii) complexes offering a cavity lined with hydrogen bonds and other interactive forces. ATP, as a whole, was recognized by the synergic combination of Zn-phosphate bonding, ππ stacking between the adenine ring of ATP and the pyridine ring of the dizinc complex and hydrogen bonding interactions that modulate the cavity structure of the dizinc complexes.

Topics: Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Coordination Complexes; Crystallography, X-Ray; Diphosphates; Fluorescence; Hydrogen Bonding; Hydroxides; Models, Molecular; Molecular Structure; Zinc

The addition of organic amendment in soils affected by residual pollution of potentially harmful elements (PHEs) is evaluated. The area was polluted twenty years ago and remediation actions were intensively applied, but evidence of pollution are still detected in some sectors. The amendment application produces significant changes in the main soil properties and modifies the mobility and availability of the pollutants. In general, Cu, Zn, Cd and Pb, showed a significant reduction in soluble and exchangeable forms after the vermicompost addition (percentage of reduction ranging from 59% for soluble Pb to 95% for exchangeable Zn), both in highly (UVS) as in moderately (VS1) polluted soils. This reduction is strongly related to the rise in OC content and pH. Arsenic presented no significant reduction or even an increase in soluble forms in moderately polluted soils (VS1), where the competing effects of OC and phosphorous could be responsible for this increase. Pb also showed an increase in availability after vermicompost application, probably related to the competing effect of Mg

Topics: Animals; Carbon; Chemical Fractionation; Composting; Diphosphates; Edetic Acid; Environmental Restoration and Remediation; Horses; Hydrogen-Ion Concentration; Manure; Metals, Heavy; Soil; Soil Pollutants; Spain

Inorganic pyrophosphate (PP

Topics: Diphosphates; Humans; Models, Molecular; Phosphoenolpyruvate Carboxylase; Protein Conformation

Pyrophosphate deficiency may explain the excessive vascular calcification found in children with Hutchinson-Gilford progeria syndrome (HGPS) and in a mouse model of this disease. The present study found that hydrolysis products of ATP resulted in a <9% yield of pyrophosphate in wild-type blood and aortas, showing that eNTPD activity (ATP → phosphate) was greater than eNPP activity (ATP → pyrophosphate). Moreover, pyrophosphate synthesis from ATP was reduced and pyrophosphate hydrolysis (via TNAP; pyrophosphate → phosphate) was increased in both aortas and blood obtained from mice with HGPS. The reduced production of pyrophosphate, together with the reduction in plasma ATP, resulted in marked reduction of plasma pyrophosphate. The combination of TNAP inhibitor levamisole and eNTPD inhibitor ARL67156 increased the synthesis and reduced the degradation of pyrophosphate in aortas and blood ex vivo, suggesting that these combined inhibitors could represent a therapeutic approach for this devastating progeroid syndrome. Treatment with ATP prevented vascular calcification in HGPS mice but did not extend longevity. By contrast, combined treatment with ATP, levamisole, and ARL67156 prevented vascular calcification and extended longevity by 12% in HGPS mice. These findings suggest a therapeutic approach for children with HGPS.

Topics: Adenosine Triphosphate; Alkaline Phosphatase; Animals; Antigens, CD; Aortic Diseases; Apyrase; Calcinosis; Diphosphates; Disease Models, Animal; Gene Knock-In Techniques; Humans; Lamin Type A; Levamisole; Longevity; Male; Mice; Mice, Transgenic; Myocytes, Smooth Muscle; Phosphoric Diester Hydrolases; Progeria; Pyrophosphatases; Real-Time Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering

Topics: Air Pollutants, Occupational; China; Diphosphates; Dust; Silicon Dioxide; Spectrophotometry, Infrared

RNA degradation is one of several ways for organisms to regulate gene expression. In bacteria, the removal of two terminal phosphate moieties as orthophosphate (

Topics: Bacillus subtilis; Bacterial Proteins; Biocatalysis; Diphosphates; Hydrogen-Ion Concentration; Manganese; Mutagenesis, Site-Directed; Nucleic Acid Conformation; Pyrophosphatases; Recombinant Proteins; RNA, Bacterial; Substrate Specificity

Technetium-based bone scintigraphy is rapidly becoming the most common non-invasive imaging tool in the diagnosis of Transthyretin cardiac amyloidosis (ATTR). Skeletal muscle uptake has been described with technetium-99m-3,3-diphosphono-1,2-propanodicarboxylic acid (TcDPD), and may account for masking of bony uptake. We sought to investigate skeletal muscle uptake of technetium-99m-pyrophosphate (TcPYP) in patients with ATTR.. This was a retrospective analysis of 57 patients diagnosed with ATTR who underwent TcPYP scintigraphy. Cardiac uptake was assessed on whole-body planar imaging using a semiquantitative scale (grades 0 to 3) and on single-photon emission computed tomography (SPECT) with CT attenuation correction using total myocardial counts per voxel after a 3-hour incubation. Skeletal muscle (psoas and biceps), vertebral body, LV myocardium, and blood pool mean counts were calculated. In the cohort (age 78 ± 9 years, 77% male, and 30% hereditary ATTR), there was no visualized tracer uptake in skeletal muscle or soft tissue on qualitative SPECT assessment. Total and blood pool-corrected uptake in the muscle groups were significantly less than myocardium and bone (P < 0.001). Blood pool-corrected muscle uptake was not associated with semiquantitative grade 3 vs 2 uptake (psoas P = 0.66, biceps P = 0.13) or presence of hereditary ATTR (psoas P = 0.43, biceps P = 0.69). As bony uptake decreased, there was no corresponding increase in skeletal muscle uptake.. In patients with ATTR cardiac amyloidosis, skeletal muscle uptake of TcPYP is minimal when assessed by qualitative and quantitative metrics, and is not significantly different in patients with grade 2 vs 3 semiquantitative uptake. The properties of this tracer may be different than TcDPD with respect to non-cardiac uptake.

Topics: Aged; Aged, 80 and over; Amyloid Neuropathies, Familial; Biopsy; Diphosphates; Female; Humans; Male; Middle Aged; Muscle, Skeletal; Retrospective Studies; Technetium; Tomography, Emission-Computed, Single-Photon; Tomography, X-Ray Computed; Whole Body Imaging

A variety of cellular metabolic reactions generate inorganic pyrophosphate (PPi) as an ATP hydrolysis byproduct. The vacuolar H+-translocating pyrophosphatase (H+-PPase) loss-of-function fugu5 mutant is susceptible to drought and displays pleotropic postgerminative growth defects due to excess PPi. It was recently reported that stomatal closure after abscisic acid (ABA) treatment is delayed in vhp1-1, a fugu5 allele. In contrast, we found that specific removal of PPi rescued all of the above fugu5 developmental and growth defects. Hence, we speculated that excess PPi itself, rather than vacuolar acidification, might delay stomatal closure. To test this hypothesis, we constructed transgenic plants expressing the yeast IPP1 gene (encoding a cytosolic pyrophosphatase) driven by a guard cell-specific promoter (pGC1::IPP1) in the fugu5 background. Our measurements confirmed stomatal closure defects in fugu5, further supporting a role for H+-PPase in stomatal functioning. Importantly, while pGC1::IPP1 transgenics morphologically mimicked fugu5, stomatal closure was restored in response to ABA and darkness. Quantification of water loss revealed that fugu5 stomata were almost completely insensitive to ABA. In addition, growth of pGC1::IPP1 plants was promoted compared to fugu5 throughout their life; however, it did not reach the wild type level. fugu5 also displayed an increased stomatal index, in violation of the one-cell-spacing rule, and phenotypes recovered upon removal of PPi by pAVP1::IPP1 (FUGU5, VHP1 and AVP1 are the same gene encoding H+-PPase), but not in the pGC1::IPP1 line. Taken together, these results clearly support our hypothesis that dysfunction in stomata is triggered by excess PPi within guard cells, probably via perturbed guard cell metabolism.

Topics: Abscisic Acid; Arabidopsis; Diphosphates; Inorganic Pyrophosphatase; Mutation; Plant Stomata

An approach for the assembly of polyprenol pyrophosphate-based probes of O-polysaccharide biosynthesis in Klebsiella pneumoniae serotype O2a is described. This convergent route features high-yielding, diastereoselective glycosylations and the late-stage installation of the polyprenol pyrophosphate moiety. Although applied to the synthesis of a nonasaccharide bearing a farnesyl group (1), the modular nature of the route makes it amenable to the synthesis of additional derivatives containing either larger glycans or different lipid domains.

Topics: Diphosphates; Hemiterpenes; Klebsiella pneumoniae; Molecular Conformation; Molecular Probes; Pentanols; Polysaccharides

Topics: Biomimetic Materials; Blood Chemical Analysis; Catalysis; Cobalt; Diphosphates; Enzyme Assays; Ferric Compounds; Fluorescent Dyes; Humans; Hydroxides; Indoles; Peroxidase; Polymers; Pyrophosphatases

Pyrophosphate (PPi), a byproduct of macromolecule biosynthesis is maintained at low levels by soluble inorganic pyrophosphatases (sPPase) found in all eukaryotes. In plants, H

Topics: Acclimatization; Arabidopsis; Arabidopsis Proteins; Cold Temperature; Diphosphates; Hot Temperature; Inorganic Pyrophosphatase; Isoenzymes; Stress, Physiological; Sumoylation

This work reports the molecular cloning and heterologous expression of the genes coding for α and β subunits of pyrophosphate-dependent phosphofructokinase (PPi-PFK) from orange. When expressed individually, both recombinant subunits were produced as highly purified monomeric proteins able to phosphorylate fructose-6-phosphate at the expenses of PPi (specific activity of 0.075 and 0.017 units. mg

Topics: Citrus sinensis; Cloning, Molecular; Diphosphates; Fructosediphosphates; Fructosephosphates; Gene Expression; Kinetics; Multiprotein Complexes; Phosphofructokinases; Phosphorylation; Phosphotransferases; Plant Proteins; Recombinant Proteins

Sensing of pyrophosphate anion (PPi) in the presence of nucleotide triphosphates allows the real time monitoring of the polymerase chain reaction. To get a deeper understanding of the factors involved in PPi/nucleotide triphosphate discrimination, a detailed study on the performance of a dimethyltin (IV)-catecholate complex capable of both separate fluorimetric or electrochemical detection of PPi in the presence of adenosine triphosphate (ATP) has been undertaken. Dimethyltin (IV) tightly binds PPi or ATP, and forms a stable 1:1 complex with tiron (4,5-dihydroxy-1,3-benzenedisulfonic acid) in water. The complexation equilibria with all components are characterized quantitatively by potentiometric and spectroscopic titrations. Pyrophosphate anion can be detected owing to its ability to release free tiron from the complex by measuring either a fluorimetric or an electrochemical signal. On the contrary, ATP does not displace tiron but causes an interference with PPi in the fluorimetric detection method due to the formation of a ternary Me

Topics: Adenosine Diphosphate; Diphosphates; Electrochemistry; Fluorometry; Limit of Detection; Organotin Compounds; Spectrophotometry

Cyanobactin heterocyclases share the same catalytic domain (YcaO) as heterocyclases/cyclodehydratases from other ribosomal peptide (RiPPs) biosynthetic pathways. These enzymes process multiple residues (Cys/Thr/Ser) within the same substrate. The processing of cysteine residues proceeds with a known order. We show the order of reaction for threonines is different and depends in part on a leader peptide within the substrate. In contrast to other YcaO domains, which have been reported to exclusively break down ATP into ADP and inorganic phosphate, cyanobactin heterocyclases have been observed to produce AMP and inorganic pyrophosphate during catalysis. We dissect the nucleotide profiles associated with heterocyclization and propose a unifying mechanism, where the γ-phosphate of ATP is transferred in a kinase mechanism to the substrate to yield a phosphorylated intermediate common to all YcaO domains. In cyanobactin heterocyclases, this phosphorylated intermediate, in a proportion of turnovers, reacts with ADP to yield AMP and pyrophosphate.

Topics: Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Adenylyl Cyclases; Amino Acid Sequence; Animals; Bacterial Proteins; Cyclization; Cysteine; Diphosphates; Models, Chemical; Molecular Structure; Peptides, Cyclic; Prochloron; Threonine; Urochordata

During cementum formation, the key roles of osterix (Osx) and inorganic pyrophosphate (PPi), mainly controlled by nucleotide pyrophosphatase 1 (Npp1; encoded by the Enpp1 gene) and progressive ankylosis protein (Ank), have been demonstrated by animal models displaying altered cementum formation. In this study, we analyzed the relationship of Osx and local PPi during cementum formation using compound mutant mice with their wildtype and corresponding single gene mutants. Importantly, functional defects in PPi regulation led to the induction of Osx expression at the cervical cementum as demonstrated by Enpp1 mutant mice and cementoblasts with the retroviral transduction of small hairpin RNA for Enpp1 or Ank. Conversely, cementoblasts exposed to inorganic PPi or with the enforced expression of Enpp1 or Ank reduced Osx expression in a concentration-dependent manner. Furthermore, the loss of Osx induced the higher expression of Npp1 and Ank at the apical region of the developing tooth root as observed in Osx-deficient mice. The activity of PPi-generating ectoenzymes (nucleoside triphosphate pyrophosphohydrolase, NTPPPHase) and the level of extracellular PPi were significantly increased in Osx-knockdown cementoblasts. However, the formation of ectopic cervical cementum was not completely diminished by inactivation of Osx in Enpp1 mutant mice. In addition, fibroblast growth factor (FGF) receptor 1 (Fgfr1) was strongly localized in cementoblasts lining the acellular cementum and involved in the inhibitory regulation of matrix accumulation and further mineralization by supporting PPi production. Taken together, these results suggest that local PPi suppresses matrix accumulation and further mineralization through an antagonistic interaction with Osx under the synergistic influence of FGF signaling during cementum formation.

Topics: Animals; beta Catenin; Cell Line; Dental Cementum; Diphosphates; Immunohistochemistry; Mice; Mice, Mutant Strains; Phosphate Transport Proteins; Phosphoric Diester Hydrolases; Pyrophosphatases; Receptor, Fibroblast Growth Factor, Type 1; RNA, Small Interfering; Sp7 Transcription Factor

Currently,

Topics: Acid Anhydride Hydrolases; Diphosphates; Enzyme Assays; Fluorometry; Inorganic Pyrophosphatase; Magnetic Resonance Spectroscopy; NADP; Polyphosphates; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins

The UbiX-UbiD enzymes are widespread in microbes, acting in concert to decarboxylate alpha-beta unsaturated carboxylic acids using a highly modified flavin cofactor, prenylated FMN (prFMN). UbiX serves as the flavin prenyltransferase, extending the isoalloxazine ring system with a fourth non-aromatic ring, derived from sequential linkage between a dimethylallyl moiety and the FMN N5 and C6. Using structure determination and solution studies of both dimethylallyl monophosphate (DMAP) and dimethyallyl pyrophosphate (DMAPP) dependent UbiX enzymes, we reveal the first step, N5-C1' bond formation, is contingent on the presence of a dimethylallyl substrate moiety. Hence, an S

Topics: Aspergillus niger; Binding Sites; Carboxy-Lyases; Decarboxylation; Dimethylallyltranstransferase; Dinitrocresols; Diphosphates; Fungal Proteins; Prenylation; Terpenes

The terminal oxygen atoms of the pyrophosphate groups in the uranyl peroxide nanocluster U24Pp12 ([(UO2)24(O2)24(P2O7)12]48-) are not fully satisfied by bond valence considerations and can become protonated. This functionality could allow for specific interactions with mineral surfaces, as opposed to the electrostatically-driven interactions observed between non-functionalized uranyl peroxide nanoclusters and mineral surfaces. The sorption of U24Pp12 to goethite and hematite was studied using batch sorption experiments as a function of U24Pp12 concentration, mineral concentration, and pH. A suite of spectroscopic techniques, scanning electron microscopy, and electrophoretic mobility measurements were used to examine the minerals before and after reaction with U24Pp12, leading to a proposed conceptual model for U24Pp12 interactions with goethite. The governing rate laws were determined and compared to those previously determined for a non-functionalized uranyl peroxide nanocluster. The rate of uranyl peroxide nanocluster sorption depends on the charge density and functionalized component of the uranyl peroxide cage. Electrophoretic mobility and attenuated total reflectance Fourier transform infrared spectroscopy analyses show that an inner-sphere complex forms between the U24Pp12 cluster and the goethite surface through the terminal pyrophosphate groups, leading to a proposed conceptual model in which U24Pp12 interacts with the triply-coordinated reactive sites on the (110) plane of goethite. These results demonstrate that the behavior of U24Pp12 at the iron (hydr)oxide-water interface is unique relative to interactions of the uranyl ion and non-functionalized uranyl peroxide nanoclusters.

Topics: Adsorption; Diphosphates; Ferric Compounds; Iron Compounds; Minerals; Models, Theoretical; Nanostructures; Static Electricity; Uranium Compounds

Bordetella bronchiseptica, known to infect animals and rarely humans, expresses a lipopolysaccharide that plays an essential role in host interactions, being critical for early clearance of the bacteria. On a B. bronchiseptica 9.73 isolate, mutants defective in the expression of genes involved in the biosynthesis of the core region were previously constructed. Herein, a comparative detailed structural analysis of the expressed lipids A by MALDI-TOF mass spectrometry was performed. The Bb3394 LPS defective in a 2-amino-2-deoxy-D-galacturonic acid lateral residue of the core presented a penta-acylated diglucosamine backbone modified with two glucosamine phosphates, similar to the wild-type lipid A. In contrast, BbLP39, resulting in the interruption of the LPS core oligosaccharide synthesis, presented lipid A species consisting in a diglucosamine backbone N-substituted with C14:0(3-O-C12:0) in C-2 and C14:0(3-O-C14:0) in C-2', O-acylated with C14:0(3-O-C10:0(3-OH) in C-3' and with a pyrophosphate in C-1. Regarding Bb3398 also presenting a rough LPS, the lipid A is formed by a hexa-acylated diglucosamine backbone carrying one pyrophosphate group in C-1 and one phosphate in C-4', both substituted with ethanolamine groups. As far as we know, this is the first description of a phosphoethanolamine modification in B. bronchiseptica lipid A. Our results demonstrate that although gene deletions were not directed to the lipid A moiety, each mutant presented different modifications. MALDI-TOF mass spectrometry was an excellent tool to highlight the structural diversity of the lipid A structures biosynthesized during its transit through the periplasm to the final localization in the outer surface of the outer membrane. Graphical Abstract.

Topics: Bacterial Proteins; Bordetella bronchiseptica; Diphosphates; Glucosamine; Glycosyltransferases; Lipid A; Mutation; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tandem Mass Spectrometry

Polyphosphate kinase 2 (PPK2) transfer phosphate from inorganic polyphosphate to nucleotides. According to their activity, PPK2 enzymes are classified into three groups. Among them, class III enzymes catalyze both the phosphorylation of nucleotide mono- to diphosphates and di- to triphosphates by using polyphosphate, which is a very inexpensive substrate. Therefore, class III enzymes are very attractive for use in biotechnological applications. Despite several studies on class III enzymes, a detailed mechanism of how phosphate is transferred from the polyphosphate to the nucleotide remains to be elucidated. Herein, it is reported that PPK2 class III enzymes from two different bacterial species catalyze the phosphorylation of adenosine mono- (AMP) into triphosphate (ATP) not only through step-by-step phosphorylation, but also by pyrophosphorylation. These are the first PPK2 enzymes that have been shown to possess polyphosphate-dependent pyrophosphorylation activity.

Topics: Adenosine Diphosphate; Adenosine Monophosphate; Amino Acid Sequence; Biocatalysis; Deinococcus; Delftia; Diphosphates; Kinetics; Phosphates; Phosphorylation; Phosphotransferases (Phosphate Group Acceptor); Substrate Specificity

Wilson's disease (WD), which might lead to acute liver failure, is an inherited disorder characterized by accumulation of copper (Cu

Topics: Alkaline Phosphatase; Copper; Diphosphates; Fluorescent Dyes; HeLa Cells; Humans

Herein, a zinc ion (Zn

Topics: Diphosphates; Enzyme Assays; Fluorescence; Gold; Humans; Inorganic Pyrophosphatase; Limit of Detection; Metal Nanoparticles; Spectrometry, Fluorescence; Zinc

A simple ratiometric terpyridine-Cd(ll) complex was synthesized by the treatment of CdCl

Topics: Anions; Binding, Competitive; Cadmium; Diphosphates; HeLa Cells; Humans; Microscopy, Fluorescence; Pyridines; Sodium; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet

Topics: Animals; Antirheumatic Agents; Benzoates; Bone Remodeling; Diphosphates; Drug Evaluation, Preclinical; Drug Therapy, Combination; Levamisole; Male; Mice; Molecular Targeted Therapy; Naphthols; Ossification of Posterior Longitudinal Ligament; Random Allocation

To sense biologically important entities with different size and dimensions, a fluorenone based fluorescent receptor was designed and synthesized. Probe 1 displayed a distinct fluorescence enhancement emission at 565nm for pyrophosphate and 530nm for alanine in polar solvent. The fluorescence titration experiments confirm 1:1 stoichiometric ratio with high-binding constant and very low limit of detection (LoD) values. Receptor 1 showed a highly selective and sensitive recognition to HP

Topics: Alanine; Diphosphates; Fluorescence; Fluorescent Dyes; Limit of Detection; Spectrometry, Fluorescence

Topics: Amyloid Neuropathies, Familial; Cardiomyopathy, Dilated; Diagnosis, Differential; Diphosphates; Female; Heart Failure; Humans; Middle Aged; Radionuclide Imaging; Technetium; Tomography, Emission-Computed, Single-Photon

Traditionally, genomic DNA detection is relay on a rigorous DNA amplification process, which always accompanied with complicated gel electrophoresis or expensive fluorescence detection methods. In this work, we have translated genomic DNA detection into adenosine triphosphate (ATP) test based on a split aptamer-based electrochemical sandwich assay. The key characteristic of our method are list as follows: first, nucleic acid amplification of the target gene was performed by the use of a loop mediated isothermal amplification (LAMP) process. The pyrophosphate (PPi), which released as the byproduct during the LAMP reaction, were further converted into ATP in the presence of adenosine 5'-phosphosulfate (APS) and ATP sulfurylase. Thereafter, the converted ATP was detected by constructing an electrochemical sandwich aptasensor. With such design, the conversion from the difficult detecting target (genomic DNA) into a convenient measured object (ATP) has been achieved. This proposed strategy was highly sensitive for Nosema bombycis genomic DNA PTP1 detection with a detection limit as low as 0.47 fg/μL and a linear range from 0.001pg/μL to 50ng/μL. And we supposed that this novel target conversion electroanalytical strategy established a universal approach for quantitative analysis of any other kinds of nucleic acid in assistance of nucleic acid polymerization reaction.

Topics: Adenosine Triphosphate; Aptamers, Nucleotide; Biosensing Techniques; Diphosphates; DNA; Electrochemical Techniques; Fluorescence; Genomics; Limit of Detection; Nosema; Sulfate Adenylyltransferase

Crosslinked high-performance polymers have many industrial applications, but are difficult to recycle or rework. A novel class of recyclable crosslinking Cu(II)-metallo-supramolecular coordination polymers are successfully prepared, which possess outstanding thermal stability and mechanical property. More importantly, the Cu

Topics: Copper; Cross-Linking Reagents; Diphosphates; Magnetic Resonance Spectroscopy; Polymers; Pyridines; Spectroscopy, Fourier Transform Infrared

Positively charged polyhexamethylene guanidine hydrochloride-stabilized silver nanoparticles (PHMG-AgNPs) were prepared and applied as a colorimetric probe for single-step determination of pyrophosphate and sulfate. The approach is based on the nanoparticles aggregation leading to change in their absorption spectra and color of the solution. Due to both electrostatic and steric stabilization these nanoparticles show decreased sensitivity relatively to many common anions, which allows for simple and rapid direct single-step determination of pyrophosphate and sulfate. Effects of different factors (time of interaction, pH, concentrations of anions and the nanoparticles) on aggregation of PHMG-AgNPs and analytical performance of the procedure were investigated. The method allows for the determination of pyrophosphate and sulfate in the range of 0.16-2μgmL

Topics: Diphosphates; Food Analysis; Guanidine; Limit of Detection; Metal Nanoparticles; Silver; Spectrophotometry; Sulfates

In recent years, highly stable optical tweezers systems have enabled the characterization of the dynamics of molecular motors at very high resolution. However, the motion of many motors with angstrom-scale dynamics cannot be consistently resolved due to poor signal-to-noise ratio. Using an acousto-optic deflector to generate a "time-shared" dual-optical trap, we decreased low-frequency noise by more than one order of magnitude compared with conventional dual-trap optical tweezers. Using this instrument, we implemented a protocol that synthesizes single base-pair trajectories, which are used to test a Large State Space Hidden Markov Model algorithm to recover their individual steps. We then used this algorithm on real transcription data obtained in the same instrument to fully uncover the molecular trajectories of

Topics: Algorithms; Base Pairing; Diphosphates; DNA-Directed RNA Polymerases; Escherichia coli Proteins; Markov Chains; Optical Tweezers

We explored the co-localization of multiple enzymes on a DNA backbone via a DNA-binding protein, Gene-A* (A*-tag) to increase the efficiency of cascade enzymatic reactions.. Firefly luciferase (FLuc) and pyruvate orthophosphate dikinase (PPDK) were genetically fused with A*-tag and modified with single-stranded (ss) DNA via A*-tag. The components were assembled on ssDNA by hybridization, thereby enhancing the efficiency of the cascading bioluminescent reaction producing light emission from pyrophosphate. The activity of A*-tag in each enzyme was investigated with dye-labeled DNA. Co-localization of the enzymes via hybridization was examined using a gel shift assay. The multi-enzyme complex showed significant improvement in the overall efficiency of the cascading reaction in comparison to a mixture of free enzymes.. A*-tag is highly convenient for ssDNA modification of versatile enzymes, and it can be used for construction of functional DNA-enzyme complexes.

Topics: Diphosphates; DNA-Binding Proteins; DNA, Single-Stranded; Luciferases, Firefly; Luminescent Measurements; Pyruvate, Orthophosphate Dikinase

The cyclization mechanisms involved in the biosynthesis of sesterterpenes are not fully understood. For example, there are two plausible reaction pathways for sesterfisherol biosynthesis, which differ in the order of ring cyclization: A-D-B/C (Path a) and A-B-C/D (Path b). It is difficult to capture intermediates of terpene cyclization, which is a complex, domino-type reaction, and so here we employed a combination of experimental and computational methods. Density functional theory calculations revealed unexpected intermediates and transition states, and implied that C-H···π interaction between a carbocation intermediate and an aromatic residue of sesterfisherol synthase (NfSS) plays a critical role, serving to accelerate the 1,2-H shift (thereby preventing triquinane carbocation formation) and to protect reactive carbocation intermediates from bases such as pyrophosphate or water in the active site. Site-directed mutagenesis of NfSS guided by docking simulations confirmed that phenylalanine F191 is a critical amino acid residue for sesterfisherol synthase, as the F191A mutant of NfSS produces novel sesterterpenes, but not sesterfisherol. Although both pathways are energetically viable, on the basis of our computational and experimental results, NfSS-mediated sesterfisherol biosynthesis appears to proceed via Path a. These findings may also provide new insight into the cyclization mechanisms in related sesterterpene synthases.

Topics: Alanine; Alkyl and Aryl Transferases; Ascomycota; Catalytic Domain; Cloning, Molecular; Cyclization; Diphosphates; Escherichia coli; Fungal Proteins; Gene Expression; Genetic Vectors; Kinetics; Molecular Docking Simulation; Mutagenesis, Site-Directed; Phenylalanine; Quantum Theory; Recombinant Proteins; Sesterterpenes; Thermodynamics; Water

We report a series of Cu(II) and Zn(II) complexes with different ligands containing a dipicolyl unit functionalized with urea groups that may contain or not a phenylboronic acid function. These complexes were designed for the recognition of phosphorylated anions through coordination to the metal ion reinforced by hydrogen bonds involving the anion and NH groups of urea. The complexes were isolated and several adducts with pyrophosphate were characterized using Xray diffraction measurements. Coordination of one of the urea nitrogen atoms to the metal ion promoted the hydrolysis of the ligands containing 1,3-diphenylurea units, while ligands bearing 1-ethyl-3-phenylurea groups did not hydrolyze significantly at room temperature. Spectrophotometric titrations, combined with ¹H and

Topics: Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Anions; Boronic Acids; Coordination Complexes; Copper; Diphosphates; Ligands; Magnetic Resonance Spectroscopy; Molecular Structure; Phosphates; Picolinic Acids; Urea; Water; Zinc

Reverse transcriptase (RT) is responsible for replicating the HIV-1 genome and is a validated therapeutic target for the treatment of HIV infections. During each cycle of the RT-catalyzed DNA polymerization process, inorganic pyrophosphate is released as the by-product of nucleotide incorporation. Small molecules were identified that act as bioisosteres of pyrophosphate and can selectively freeze the catalytic cycle of HIV-1 RT at the pre-translocated stage of the DNA- or RNA-template-primer-enzyme complex.

Topics: Biocatalysis; Diphosphates; DNA, Viral; Dose-Response Relationship, Drug; HIV Reverse Transcriptase; Molecular Structure; Polymerization; Reverse Transcriptase Inhibitors; Small Molecule Libraries; Structure-Activity Relationship

The nucleotidyl transfer reaction, catalyzed by sugar nucleotidyltransferases (SNTs), is assisted by two active site Mg

Topics: Arginine; Bacterial Proteins; Biocatalysis; Catalytic Domain; Crystallography, X-Ray; Diphosphates; Magnesium; Models, Molecular; Molecular Dynamics Simulation; Multienzyme Complexes; Mycobacterium tuberculosis; Protein Conformation

A colorimetric detection scheme is introduced for the determination of alkaline phosphatase (ALP) activity based on Cu(II)-modulated G-quadruplex-based DNAzymes. It is exploiting the strong affinity of Cu(II) for pyrophosphate (PPi) upon which the cofactor PPi is trapped by Cu(II). Hence, the activity of the DNAzyme is inhibited. ALP catalyzes the hydrolysis of PPi, causing the release of Cu(II). DNAzyme, in turn, is activated and catalyzes the cleavage of the DNA probe substrate. The released G-rich sequence folds into the G-quadruplex, which can bind hemin and catalyze the oxidation of 2,2'-azinobis (3-ethylbenzothiozoline)-6-sulfonate (ABTS), and this leads to an increase in absorbance at 420 nm. Absorbance increases linearly with increasing ALP activity in 0.07 to 300 U

Topics: Alkaline Phosphatase; Colorimetry; Copper; Diphosphates; DNA, Catalytic; G-Quadruplexes; Hemin; Humans

A compartmental chemosensor probe HL has been designed and synthesized for the selective recognition of zinc ions over other transition metal ions via fluorescence "ON" strategy. The chemosensing behaviour of HL was demonstrated through fluorescence, absorption and NMR spectroscopic techniques. The molecular structure of the zinc complex derived from HL was determined by X-ray crystallography. A probable mechanism of this selective sensing behavior was described on the basis of spectroscopic results and theoretical studies by density functional theory (DFT). The biological applicability of the chemosensor HL was examined via cell imaging on HeLa cells. The HL-zinc complex served as a secondary fluorescent probe responding to the pyrophosphate anion specifically over other anions. The fluorescence enhancement of HL in association with Zn2+ ions was quenched in the presence of pyrophosphate (PPi). Thus, a dual response was established based on "OFF-ON-OFF" strategy for detection of both cation and anion. This phenomenon was utilized in the construction of a "INHIBIT" logic gate.

Topics: Crystallography, X-Ray; Diphosphates; Fluorescent Dyes; HeLa Cells; Humans; Hydrogen-Ion Concentration; Ions; Models, Molecular; Molecular Structure; Optical Imaging; Quantum Theory; Spectrophotometry, Ultraviolet; Tumor Cells, Cultured; Zinc

The terminal alkyne is a readily derivatized functionality valued for its diverse applications in material synthesis, pharmaceutical science, and chemical biology. The synthetic biology routes to terminal alkynes are highly desired and yet underexplored. Some marine natural products contain a terminal alkyne functionality, and the discovery of the biosynthetic gene clusters for jamaicamide B and carmabin A marked the beginning of a new era in the understanding and engineering of terminal alkyne biosynthesis. In this chapter, we will overview recent advances in understanding the biosynthetic machinery for terminal alkyne synthesis. We will first describe how to elucidate terminal alkyne biosynthetic mechanism through heterologous expression, purification, and in vitro biochemical assays of individual pathway proteins. This will be followed by the description of an in vivo reporting system for the characterization of a membrane-bound bifunctional desaturase/acetylenase involved in terminal alkyne formation. The chapter will also cover the strategies for discovering additional protein homologs for terminal alkyne synthesis from microbes as well as the applications of click chemistry to identify and quantify terminal alkyne-bearing metabolites from microbial cultures. We will conclude this chapter with current challenges and future directions in this field.

Topics: Alkynes; Aquatic Organisms; Biochemistry; Click Chemistry; Computational Biology; Culture Media; Detergents; Diphosphates; Enzymes; Membrane Proteins; Plasmids; Polyketide Synthases; Protein Engineering; Recombinant Proteins

The current study aimed to investigate the association of calcium pyrophosphate (CPP) and basic calcium phosphate (BCP) crystals in synovial fluid (SF) of patients with osteoarthritis (OA) with disease severity, clinical symptoms, and synovial inflammation. One-hundred-and-ten patients with knee OA completed the Western Ontario and McMaster Universities Arthritis Index (WOMAC) self-assessment questionnaire, the Lequesne algofunctional index survey, and the visual analogic scale forms; they also underwent power Doppler ultrasonography (PDUS) to assess synovial inflammation. Scanning electron microscopy (SEM) was used to detect SF crystals. SEM analyses uncovered CPP crystals in 26 patients (23.6%), BCP crystals in 24 patients (21.8%), and both types of crystals in 7 patients (6.3%). Categorizing patients according to SF crystal type, a strong association between BCP crystal presence, and higher WOMAC and Lequesne index scores has been uncovered. Classifying our patients according the severity Kellgre-Lawrence score, we found that the prevalence of CPP alone (27.8%) or in combination with BCP (11.1%) was higher in the late stage group with respect to the early one (CPP 21.6% and CPP + BCP 4.1%, respectively). The prevalence of BCP crystals alone was, instead, higher in the early (23%) with respect to the late group (19.4%). No association between the presence of crystals and the radiographic scores has been observed. Considering the growing evidence supporting a role of low-grade inflammation in OA pathogenesis, the results of this study suggest a role for calcium crystals in the development of the disease.

Topics: Aged; Aged, 80 and over; Calcium Phosphates; Diphosphates; Disease Progression; Female; Humans; Inflammation; Male; Microscopy, Electron, Scanning; Middle Aged; Osteoarthritis, Knee; Severity of Illness Index; Surveys and Questionnaires; Synovial Fluid; Ultrasonography, Doppler

Amorpha-4,11-diene synthase (ADS) cyclizes the substrate farnesyl pyrophosphate to produce amorpha-4,11-diene as a major product. This is considered the first committed and rate-limiting step in the biosynthesis of the antimalarial artemisinin. Here, we utilize a reported 3D model of ADS to perform mutability landscape guided enzyme engineering. A mutant library of 258 variants along sixteen active site residues was created then screened for catalytic activity and product profile. This allowed for identification of the role of some of these residues in the mechanism. R262 constrains the released pyrophosphate group along with magnesium ions. The aromatic residues (W271, Y519 and F525) stabilize the intermediate carbocations while T296, G400, G439 and L515 help with the 1,6- and 1,10-ring closures. Finally, W271 is suggested to act as active site base along with T399, which ensures regioselective deprotonation. The mutability landscape also helped determine variants with improved catalytic activity. H448A showed ~4 fold increase in catalytic efficiency and the double mutation T399S/H448A improved k

Topics: Alkyl and Aryl Transferases; Catalysis; Catalytic Domain; Diphosphates; Escherichia coli; Gas Chromatography-Mass Spectrometry; Mutagenesis, Site-Directed; Peptide Library; Polycyclic Sesquiterpenes; Protein Engineering; Recombinant Proteins; Sesquiterpenes

Objective- Hydroxyapatite deposition on the medial layer of the aortic walls is the hallmark of vascular calcification and the most common complication in aging individuals and in patients with diabetes mellitus and those undergoing hemodialysis. Extracellular pyrophosphate is a potent physicochemical inhibitor of hydroxyapatite crystal formation. This study analyzed changes in extracellular pyrophosphate metabolism during the phosphate-induced calcification process. Approach and Results- Phosphate-induced calcification of ex vivo-cultured aortic rings resulted in calcium accumulation after 7 days. This accumulation was enhanced when aortic walls were devitalized. BMP2 (bone morphogenic protein 2) expression was associated with calcium accumulation in cultured aortic rings, as well as in cultured vascular smooth muscle cells (VSMCs) and in calcitriol-induced calcification in rats. Hydroxyapatite dose dependently induced BMP2 overexpression in VSMCs. Moreover, TNAP (tissue nonspecific alkaline phosphatase) mRNA levels and activity were found to be downregulated in early phases and upregulated in later phases of calcification in all 3 models studied. eNPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1) increased from early to later phases of calcification, whereas eNTPD1 (ectonucleoside triphosphate diphosphohydrolase 1) was downregulated during later phases. Synthesis of pyrophosphate in VSMCs increased significantly over time, in all 3 models studied. Because the rate of pyrophosphate hydrolysis was 10× slower than the rate of pyrophosphate synthesis, pyrophosphate synthesis is determined mainly by the ratio of eNPP1 to eNTPD1 activity. Hydroxyapatite also induces increments both in TNAP and eNPP1/eNTPD1 ratio in VSMCs. Conclusions- Pyrophosphate synthesis increases in VSMCs during phosphate-induced calcification because of compensatory regulation of extracellular pyrophosphate metabolism.

Topics: Alkaline Phosphatase; Animals; Antigens, CD; Aorta; Apyrase; Bone Morphogenetic Protein 2; Cell Proliferation; Cells, Cultured; Diphosphates; Down-Regulation; Durapatite; Extracellular Space; Gene Expression; Hydrolysis; Male; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Phosphates; Phosphoric Diester Hydrolases; Pyrophosphatases; Rats, Sprague-Dawley; RNA, Messenger; Up-Regulation; Vascular Calcification

Topics: Adult; Aged; Aged, 80 and over; Alkaline Phosphatase; Animals; Aorta; Diphosphates; Extracellular Space; Female; Humans; Hydrolysis; Kinetics; Male; Middle Aged; Phosphates; Rats, Sprague-Dawley; Renal Dialysis

The detection of phosphates and their enzymatic hydrolysis is of great importance because of their essential roles in various biological processes and numerous diseases. Compared with individual sensors for detecting one given phosphate at a time, sensor arrays are able to discriminate multiple phosphates simultaneously. Although nanomaterial-based sensor arrays have shown great promise for the discrimination of phosphates, very few of them have been explored for probing phosphates involved enzymatic hydrolysis. To fill this gap, herein we fabricated two-dimensional-metal-organic-framework (2D-MOF)-nanozyme-based sensor arrays by modulating their peroxidase-mimicking activity with various phosphates, including AMP, ADP, ATP, pyrophosphate (PPi), and phosphate (Pi). The sensor arrays were used to successfully discriminate the five phosphates not only in aqueous solutions but also in biological samples. The practical application of the sensor arrays was then validated with blind samples, where 30 unknown samples containing phosphates were accurately identified. Moreover, the sensor arrays were successfully applied to probing hydrolytic processes involving ATP and PPi that are catalyzed by apyrase and PPase, respectively. This work demonstrates a nanozyme-based sensor array as a convenient and reliable analytical platform for probing phosphates and their related enzymatic processes, which could be applied to other analytes and enzymatic reactions.

Topics: Adenine Nucleotides; Biomimetic Materials; Diphosphates; Hydrolysis; Metal-Organic Frameworks; Nanostructures; Peroxidase; Phosphates

Here we report a novel bioluminescence (BL) method for exonuclease I (Exo I) detection based on terminal deoxynucleotidyl transferase (TdT)-mediated pyrophosphate release. An inert hairpin probe with a blocked protruding 3'-terminal biotinylated nucleotide was designed, and a blocked 3'-terminal nucleotide of the probe would be removed only in the presence of Exo I, thus rendering the probe with a free 3'-hydroxyl group. Subsequently, with nucleotide incorporation by TdT, huge amounts of pyrophosphates were generated. After the conversion of pyrophosphate to adenosine triphosphate (ATP) through ATP sulfurylase, BL was emitted by the reaction of d-luciferin and ATP with firefly luciferase. Therefore, Exo I activity was indirectly quantified in the range 1-500 mU with a detection limit of 0.05 mU/μl. Moreover, the developed approach was successfully applied to investigate the inhibitory effect of streptavidin on cleavage of Exo I and also determine Exo I activity in spiked serum samples. Overall, the proposed method has high sensitivity and selectivity, and can be universally extended to the detection of other nucleases using terminal extension as a signal amplification method and BL as a detection signal, having potential application in the diagnosis of nuclease-related diseases or evaluation of nuclease functions in biological systems.

Topics: Adenosine Triphosphate; Diphosphates; DNA Nucleotidylexotransferase; DNA Repair Enzymes; Enzyme Assays; Exodeoxyribonucleases; Firefly Luciferin; Humans; Limit of Detection; Luminescence; Luminescent Measurements

Detection of EGFR mutations in circulating cell-free DNA (cfDNA) is beneficial to monitor the therapeutic effect, tumor progression, and drug resistance in real time. However, it requires that the mutation detection method has the ability to quantify the mutation abundance accurately. Although the next-generation sequencing (NGS) and digital PCR showed high sensitivity for quantifying mutations in cfDNA, the use of expensive equipment and the high-cost hampered their applications in the clinic. Herein, we propose a highly sensitive and specific real-time PCR by employing serial invasive reaction as a sequence identifier for quantifying EGFR mutation abundance in cfDNA (termed as qPCR-Invader). The mutation abundance can be quantified by using the difference of Ct values between mutant and wild-type targets without the need of making a standard curve. The method can quantify a mutation level as lower as 0.1% (10 copies/tube). Thirty-six tissue samples from non-small-cell lung cancer (NSCLC) patients were detected by our method and 14/36 tissues gave EGFR L858R mutation-positive results, whereas ARMS-PCR just identified 12 of L858R mutant samples. The two inconsistent samples were confirmed as L858R mutant by pyrophosphorolysis-activated polymerization method, indicating that qPCR-Invader is more sensitive than ARMS-PCR for mutation detection. The L858R mutation abundances of 19 cfDNA samples detected by qPCR-Invader were close to that from NGS, indicating our method can precisely quantify mutation abundance in cfDNA. The qPCR-Invader just needs a common real-time PCR device to accomplish quantification of EGFR mutations, and the fluorescence probes are universal for any target detection. Therefore, it could be used in most laboratories to analyze mutations in cfDNA. Graphical abstract ᅟ.

Topics: Base Sequence; Carcinoma, Non-Small-Cell Lung; Cell-Free Nucleic Acids; Diphosphates; ErbB Receptors; Fluorescence; High-Throughput Nucleotide Sequencing; Humans; Limit of Detection; Lung Neoplasms; Mutation; Polymerization; Real-Time Polymerase Chain Reaction; Reproducibility of Results

Generalized arterial calcification of infancy (GACI) is a rare, life-threatening disorder caused by loss-of-function mutations in the gene encoding ectonucleotide pyrophosphatase phosphodiesterase 1 (

Topics: Animals; Blood Pressure; Cardiovascular System; Diphosphates; Disease Models, Animal; Enzyme Replacement Therapy; Humans; Mice; Mice, Inbred BALB C; Organ Specificity; Phosphoric Diester Hydrolases; Pyrophosphatases; Vascular Calcification

Sensing of pyrophosphate (PPi) is helpful to better understand many life processes and diagnose various early-stage diseases. However, many traditional reported methods based on artificial receptors for sensing of PPi exhibit some disadvantages including difficulties in designing appropriate binding sites and complicated multi-step assembly/functionalization. Thus, it is significantly important and a big challenge to know how to use a simple molecular self-assembly or an interaction system to solve the above-mentioned limits to achieve the quantitative analysis of specific substances in the system. Based on the natural connection and similarity (such as stimulus responsiveness) between sensing and logic computing, in this study, the Boolean logic tree of molecular self-assembly system based on the cobalt oxyhydroxide (CoOOH) nanoplatform is constructed and applied to organize and connect "plug and play" molecular events (fluorescent dye, acridine orange and anion, PPi). By using molecules as inputs and the corresponding fluorescence signal as the output, the CoOOH-based molecular self-assembly system can be programmed for three-input fluorescent Boolean logic computation, fluorescent three-state logic computation, detection of PPi (linear range from 50 to 6400 nM with a detection limit of 20 nM) and even for imaging in living cancer cells and in vivo (in systems such as Zebrafish and Carassius auratus). Our approach adds a new dimension for expanding molecular logic computing and sensing systems, which will not only provide more opportunities for developing novel logic computing paradigms, but also be helpful in promoting the development and applications of intelligent molecular computing and sensing systems.

Topics: Acridine Orange; Animals; Cobalt; Diphosphates; Fluorescent Dyes; Goldfish; Humans; Limit of Detection; Logic; Microscopy, Fluorescence; Nanostructures; Oxides; Zebrafish

DNA nanotechnology has been developed to construct a variety of functional two- and three-dimensional structures for versatile applications. Rolling circle amplification (RCA) has become prominent in the assembly of DNA-inorganic composites with hierarchical structures and attractive properties. Here, we demonstrate a one-pot method to directly encapsulate horseradish peroxidase (HRP) in DNA flowers (DFs) during RCA. The growing DNA strands and Mg

Topics: Aptamers, Nucleotide; Biosensing Techniques; Colorimetry; Diphosphates; DNA; DNA, Catalytic; Enzymes; Gold; Humans; Hydrogen Peroxide; Magnesium; Metal Nanoparticles; Microscopy, Electron, Transmission; Nanotechnology; Nanotubes; Nucleic Acid Amplification Techniques; Point-of-Care Systems; Serum; Spectrophotometry, Ultraviolet; Surface Plasmon Resonance; Thrombin

Peptidoglycan (PG) biosynthesis and assembly are needed for bacterial cell wall formation. Lipid II is the precursor in the PG biosynthetic pathway and carries a nascent PG unit that is processed by glycosyltransferases. Despite its immense therapeutic value as a target of several classes of antibiotics, the conformational ensemble of lipid II in bacterial membranes and its interactions with membrane-anchored enzymes remain elusive. In this work, lipid II and its elongated forms (lipid VI and lipid XII) were modeled and simulated in bilayers of POPE (palmitoyl-oleoyl-phosphatidyl-ethanolamine) and POPG (palmitoyl-oleoyl-phosphatidyl-glycerol) that mimic the prototypical composition of Gram-negative cytoplasmic membranes. In addition, penicillin-binding protein 1b (PBP1b) from Escherichia coli was modeled and simulated in the presence of a nascent PG to investigate their interactions. Trajectory analysis reveals that as the glycan chain grows, the non-reducing end of the nascent PG displays much greater fluctuation along the membrane normal and minimally interacts with the membrane surface. In addition, dihedral angles within the pyrophosphate moiety are determined by the length of the PG moiety and its surrounding environment. When a nascent PG is bound to PBP1b, the stem peptide remains in close contact with PBP1b by structural rearrangement of the glycan chain. Most importantly, the number of nascent PG units required to reach the transpeptidase domain are determined to be 7 or 8. Our findings complement experimental results to further understand how the structure of nascent PG can dictate the assembly of the PG scaffold.

Topics: Cell Membrane; Cell Wall; Diphosphates; Escherichia coli; Penicillin-Binding Proteins; Peptidoglycan; Peptidyl Transferases; Phosphatidylethanolamines; Phosphatidylglycerols; Polysaccharides; Uridine Diphosphate N-Acetylmuramic Acid

Phosphorus is an essential nutrient involved in many pathobiological processes. Less than 1% of phosphorus is found in extracellular fluids as inorganic phosphate ion (Pi) in solution. High serum Pi level promotes ectopic calcification in many tissues, including blood vessels. Here, we studied the effect of elevated Pi concentration on macrophage polarization and calcification. Macrophages, present in virtually all tissues, play key roles in health and disease and display remarkable plasticity, being able to change their physiology in response to environmental cues.. High-throughput transcriptomic analysis and functional studies demonstrated that Pi induces unpolarized macrophages to adopt a phenotype closely resembling that of alternatively-activated M2 macrophages, as revealed by arginine hydrolysis and energetic and antioxidant profiles. Pi-induced macrophages showed an anti-calcifying action mediated by increased availability of extracellular ATP and pyrophosphate.. We conclude that the ability of Pi-activated macrophages to prevent calcium-phosphate deposition is a compensatory mechanism protecting tissues from hyperphosphatemia-induced pathologic calcification.

Topics: Adenosine Triphosphate; Animals; Antioxidants; Calcium; Diphosphates; Flow Cytometry; Macrophages; Male; Mice; Mice, Inbred C57BL; Phosphates; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Vascular Calcification

Unique aggregation-induced emission (AIE) property has been found and widely applied in chemo/biosensors for thiolated gold nanoclusters and copper nanoclusters; however, little is known about this property of thiolate-protected silver nanoclusters. In this work, specific aggregation-induced emission enhancement (AIEE) of glutathione-capped silver nanoclusters (AgNCs) was verified via its solid-state luminescence and enhanced emission in poor solvent, three stimuli responsive nanoswitches were constructed based on its AIEE property, and a reliable and sensitive PPase assay was developed via ion-triggered luminescence switch. Glutathione-capped AgNCs from a facile one-pot synthesis were found to possess bright red luminescence and aggregation-induced emission enhancement property. This AIEE feature enables AgNCs in sensitive response to pH and temperature in a reversible way, allowing the two nanoswitches to precisely monitor the change of environmental pH and temperature. Complexation reactions among AgNCs, aluminum cation and PPi were also designed for an ion-triggered luminescence nanoswitch, which allows selective response to aluminum cation or PPi in luminescence. This ion-driven luminescence switch is further utilized to design a novel detection strategy for PPase activity through competitive coordination reactions. Our method illustrates a novel detection strategy mediated by complexation reaction between Al

Topics: Aluminum; Animals; Diphosphates; Enzyme Assays; Glutathione; Hydrogen-Ion Concentration; Inorganic Pyrophosphatase; Luminescent Measurements; Nanostructures; Rats; Silver; Temperature

Tooth hypersensitivity is a growing problem affecting both the young and ageing population worldwide. Since an effective and permanent solution is not yet available, we propose a new methodology for the restoration of dental enamel using femtosecond lasers and novel calcium phosphate biomaterials. During this procedure the irradiated mineral transforms into a densified layer of acid resistant iron doped β-pyrophosphate, bonded with the surface of eroded enamel. Our aim therefore is to evaluate this densified mineral as a potential replacement material for dental hard tissue. To this end, we have tested the hardness of β-pyrophosphate pellets (sintered at 1000°C) and its mineral precursor (brushite), the wear rate during simulated tooth-brushing trials and the cytocompatibility of these minerals in powder form. It was found that the hardness of the β-pyrophosphate pellets is comparable with that of dental enamel and significantly higher than dentine while, the brushing trials prove that the wear rate of β-pyrophosphate is much slower than that of natural enamel. Finally, cytotoxicity and genotoxicity tests suggest that iron doped β-pyrophosphate is cytocompatible and therefore could be used in dental applications. Taken together and with the previously reported results on laser irradiation of these materials we conclude that iron doped β-pyrophosphate may be a promising material for restoring acid eroded and worn enamel.

Topics: Animals; Biocompatible Materials; Calcium Phosphates; Cattle; Dental Enamel; Diphosphates; Microscopy, Electron, Scanning; X-Ray Diffraction

Soft tissue calcification occurs in several common acquired pathologies, such as diabetes and hypercholesterolemia, or can result from genetic disorders. ABCC6, a transmembrane transporter primarily expressed in liver and kidneys, initiates a molecular pathway inhibiting ectopic calcification. ABCC6 facilitates the cellular efflux of ATP, which is rapidly converted into pyrophosphate (PPi), a major calcification inhibitor. Heritable mutations in ABCC6 underlie the incurable calcification disorder pseudoxanthoma elasticum and some cases of generalized arterial calcification of infancy. Herein, we determined that the administration of PPi and the bisphosphonate etidronate to Abcc6

Topics: Acute Disease; Animals; ATP-Binding Cassette Transporters; Calcinosis; Chronic Disease; Diphosphates; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Etidronic Acid; Female; Liver; Male; Mice, Inbred C57BL; Mice, Knockout; Multidrug Resistance-Associated Proteins; Phenotype; Pseudoxanthoma Elasticum; Transgenes

We studied activation of macrophages with humic acids extracted from peat of large deposits in the Tomsk region by two extraction methods: by hydroxide or sodium pyrophosphate. Humic acid of lowland peat types containing large amounts of aromatic carbon, phenolic and alcohol groups, carbohydrate residues and ethers, irrespectively of the extraction methods contained LPS admixture that probably determines their activating properties. Humic acid of upland peat types characterized by high content of carbonyl, carboxyl, and ester groups enhance NO production and reduce arginase expression, but these effects were minimized when sodium hydroxide was used as an extraction solvent. Pyrophosphate samples of the upland peat types were characterized by aromaticity and diversity of functional groups and have a significant advantage because of they induce specific endotoxin-independent stimulating action on antigen presenting cells.

Topics: Animals; Arginase; Carbon; Complex Mixtures; Diphosphates; Female; Humic Substances; Lipopolysaccharides; Liquid-Liquid Extraction; Macrophages, Peritoneal; Male; Mice; Mice, Inbred C57BL; Nitric Oxide; Phenols; Polymyxin B; Primary Cell Culture; Sodium Hydroxide; Soil

Topics: Alkaline Phosphatase; Copper; Diphosphates; Enzymes; Humans; Hydrogen Peroxide; Limit of Detection; Luminescent Measurements; Luminol; Substrate Specificity

The Fenton process, one of several advanced oxidation processes, describes the reaction of Fe(II) with hydrogen peroxide. Fe(II) is oxidized to Fe(III) that reacts with hydrogen peroxide to Fe(II) and again initiates the Fenton reaction. In the course of the reactions reactive species, e.g. hydroxyl radicals, are formed. Conditions such as pH, ligand concentrations and the hydrogen peroxide/Fe(II) ratio may influence the OH radical yield. It could be shown that at pH < 2.7 and >3.5 the OH radical yield decreases significantly. Two ligands were investigated, pyrophosphate and sulfate. It was found that pyrophosphate forms a complex with Fe(III) that does not react with hydrogen peroxide and thus, the Fenton reaction is terminated and the OH radical yields do not further increase. The influence of sulfate is not as strong as that of pyrophosphate. The OH radical yield is decreased when sulfate is added but even at higher concentrations the Fenton reaction is not terminated.

Topics: Diphosphates; Ferrous Compounds; Hydrogen Peroxide; Hydrogen-Ion Concentration; Hydroxyl Radical; Iron; Ligands; Oxidation-Reduction; Sulfates

Two new Pt(II) derivatives of kiteplatin ([PtCl

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Cell Survival; Diphosphates; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Humans; Molecular Structure; Organoplatinum Compounds; Structure-Activity Relationship

A reaction of N

Topics: Antineoplastic Agents; Diphosphates; HCT116 Cells; Humans; Models, Molecular; Molecular Conformation; Organometallic Compounds; Schiff Bases; Water; Zinc

Mutations in the proximal tubular sodium-dependent phosphate co-transporters NPT2a and NPT2c have been reported in patients with renal stone disease and nephrocalcinosis, however the relative contribution of genotype, dietary calcium and phosphate, and modifiers of mineralization such as pyrophosphate (PPi) to the formation of renal mineral deposits is unclear. In the present study, we used Npt2a-/- mice to model the renal calcifications observed in these disorders. We observed elevated urinary excretion of PPi in Npt2a-/- mice when compared to WT mice. Presence of two hypomorphic Extracellular nucleotide pyrophosphatase phosphodiesterase 1 (Enpp1asj/asj) alleles decreased urine PPi and worsened renal calcifications in Npt2a-/- mice. These studies suggest that PPi is a thus far unrecognized factor protecting Npt2a-/- mice from the development of renal mineral deposits. Consistent with this conclusion, we next showed that renal calcifications in these mice can be reduced by intraperitoneal administration of sodium pyrophosphate. If confirmed in humans, urine PPi could therefore be of interest for developing new strategies to prevent the nephrocalcinosis and nephrolithiasis seen in phosphaturic disorders.

Topics: Animals; Diphosphates; Disease Models, Animal; Female; Humans; Injections, Intraperitoneal; Kidney Calculi; Male; Mice; Mice, Knockout; Mutation; Phosphoric Diester Hydrolases; Pyrophosphatases; Sodium-Phosphate Cotransporter Proteins, Type IIa; Treatment Outcome

The bacterial manganese oxidase MnxG of the Mnx protein complex is unique among multicopper oxidases (MCOs) in carrying out a two-electron metal oxidation, converting Mn(II) to MnO

Topics: Bacillus; Copper; Diphosphates; Manganese; Manganese Compounds; Models, Molecular; Nanoparticles; Oxidation-Reduction; Oxides; Oxidoreductases

Topics: Alkaline Phosphatase; Biological Assay; Carbon; Diphosphates; Humans; Nitrogen; Optical Phenomena; Quantum Dots; Spectrometry, Fluorescence; Time Factors

Inositol pyrophosphates are novel signaling molecules possessing high-energy pyrophosphate bonds and involved in a number of biological functions. Here, we report the correct identification and characterization of the kinases involved in the inositol pyrophosphate biosynthetic pathway in Trypanosoma brucei: inositol polyphosphate multikinase (TbIPMK), inositol pentakisphosphate 2-kinase (TbIP5K) and inositol hexakisphosphate kinase (TbIP6K). TbIP5K and TbIP6K were not identifiable by sequence alone and their activities were validated by enzymatic assays with the recombinant proteins or by their complementation of yeast mutants. We also analyzed T. brucei extracts for the presence of inositol phosphates using polyacrylamide gel electrophoresis and high-performance liquid chromatography. Interestingly, we could detect inositol phosphate (IP), inositol 4,5-bisphosphate (IP

Topics: Animals; Biosynthetic Pathways; Carbohydrate Metabolism; Diphosphates; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Phosphotransferases (Alcohol Group Acceptor); Phosphotransferases (Phosphate Group Acceptor); Phytic Acid; Polyphosphates; Recombinant Proteins; Signal Transduction; Trypanosoma brucei brucei

Adenosine diphosphate-ribose (ADP-ribose) and its derivatives play important roles in a series of complex physiological procedures. The design and synthesis of artificial ADP-ribosylated compounds is an efficient way to develop valuable chemical biology tools and discover new drug candidates. However, the synthesis of ADP-ribosylated compounds is currently difficult due to structural complexity, easily broken pyrophosphate bond and high hydrophilicity. In this paper, ADP-ribosyl-N₃ was designed and synthesized for the first time. With ADP-ribosyl-N₃ as the key precursor, a divergent post-modification strategy was developed to prepare structurally diverse ADP-ribosylated compounds including novel nucleotides and peptides bearing ADP-ribosyl moieties.

Topics: Adenosine Diphosphate Ribose; ADP-Ribosylation; Chemistry Techniques, Synthetic; Diphosphates; Hydrophobic and Hydrophilic Interactions; Magnetic Resonance Spectroscopy; Mass Spectrometry; Molecular Structure; Nucleotides; Peptides

YhdE is a Maf (multicopy associated filamentation) proteins from Escherichia coli which exhibits pyrophosphatase activity towards selected nucleotides, although its catalytic mechanism remains unclear. Herein we used a novel fluorescence probe (4-isoACBA-Zn(II) complex) to characterize the enzymatic properties of YhdE and its mutant, establishing a new method for assaying pyrophosphatase catalytic function. Our results reveal for the first time that the new fluorescence sensor confers high sensitivity and specificity and pyrophosphate (PPi) is the direct catalytic product of YhdE. Crystal structures of a mutant in the active-site loop (YhdE_E33A) show conformational flexibility implicated in the catalytic mechanism of YhdE. ITC experiments and computational docking further reveal that Asp70 and substrate dTTP coordinate Mn

Topics: Calorimetry; Catalysis; Catalytic Domain; Diphosphates; Enzyme Assays; Escherichia coli Proteins; Fluorescent Dyes; Hydrogen Bonding; Hydrolysis; Kinetics; Molecular Conformation; Molecular Docking Simulation; Molecular Dynamics Simulation; Protein Binding; Pyrophosphatases; Recombinant Proteins; Structure-Activity Relationship; Substrate Specificity

Enzymatic reactions usually occur in several steps: a step of substrate binding to the surface of the protein, a step of protein reorganization around the substrate and conduction of a chemical reaction, and a step of product release. The release of inorganic phosphate-PPi-from the matrix of the protein HIV reverse transcriptase is investigated computationally. Atomically detailed simulations with explicit solvent are analyzed to obtain the free energy profile, mean first passage time, and detailed molecular mechanisms of PPi escape. A challenge for the computations is of time scales. The experimental time scale of the process of interest is in milliseconds, and straightforward molecular dynamics simulations are in sub-microseconds. To overcome the time scale gap, we use the algorithm of Milestoning along a reaction coordinate to compute the overall free energy profile and rate. The methods of locally enhanced sampling and steered molecular dynamics determine plausible reaction coordinates. The observed molecular mechanism couples the transfer of the PPi to positively charged lysine side chains that are found on the exit pathway and to an exiting magnesium ion. In accord with experimental findings, the release rate is comparable to the chemical step, allowing for variations in substrate (DNA or RNA template) in which the release becomes rate determining.

Topics: Diphosphates; HIV Infections; HIV Reverse Transcriptase; HIV-1; Humans; Lysine; Magnesium; Molecular Dynamics Simulation; Substrate Specificity; Thermodynamics

The exosporium layer of Bacillus anthracis spores is rich in L-rhamnose, a common bacterial cell-wall component, which often contributes to the virulence of pathogens by increasing their adherence and immune evasion. The biosynthetic pathway used to form the activated L-rhamnose donor dTDP-L-rhamnose consists of four enzymes (RfbA, RfbB, RfbC and RfbD) and is an attractive drug target because there are no homologs in mammals. It was found that co-purifying and screening RfbC (dTDP-6-deoxy-D-xylo-4-hexulose 3,5-epimerase) from B. anthracis in the presence of the other three B. anthracis enzymes of the biosynthetic pathway yielded crystals that were suitable for data collection. RfbC crystallized as a dimer and its structure was determined at 1.63 Å resolution. Two different ligands were bound in the protein structure: pyrophosphate in the active site of one monomer and dTDP in the other monomer. A structural comparison with RfbC homologs showed that the key active-site residues are conserved across kingdoms.

Topics: Bacillus anthracis; Bacterial Proteins; Carbohydrate Epimerases; Catalytic Domain; Crystallography, X-Ray; Diphosphates; Models, Molecular; Protein Conformation; Structural Homology, Protein

Topics: Computed Tomography Angiography; Coronary Angiography; Diphosphates; Humans; Phosphates; Vascular Calcification

A strategy to develop water soluble, biocompatible nanocomposite probe for the detection of pyrophosphate (PPi) in physiological conditions and in in vitro live melanoma cancer cells (B16F10) is reported. The self-assembled nanocomposite probe comprised of amino acid (histidine) functionalized perylenediimide (PDI-HIS), copper ion and graphene oxide (GO) and that could be utilized as a highly effective sensing platform in biological conditions and cellular environment via fluorescence "turn-on" for PPi detection. This controlled fabrication of metal organic self-assembled spheres along with GO proved very valuable for the detection of PPi in unprecedented sensitivity over other competing ions. The PDI-HIS-Cu-GO (PCG) nanocomposite sensor provides a unique platform for the fluorogenic detection of PPi having a very low limit of detection (LOD) of 0.60×10

Topics: Animals; Cell Line, Tumor; Copper; Diphosphates; Graphite; Histidine; Imides; Melanoma; Mice; Nanostructures; Optical Imaging; Oxides; Perylene; Solubility; Spectrometry, Fluorescence; Water

The first two-photon fluorescent probe (PC) for selectively detecting biological Cu (Ⅱ) and pyrophosphate (PPi) has been developed based on 7-substituted coumarin in this study. The probe presented excellent selective two-photon "on-off-on" detection signal for Cu(II) /PPi in aqueous solution. The two-photon detection ensemble (PCCu) can detect PPi released from DNA amplification after the polymerase chain reactions (PCR). The probe showed low cytotoxicity and good biocompatibility, and therefore can be applied for imaging Cu(II)/PPi in living cells under two-photon excitation. Furthermore, the ensemble probe (PCCu) was also used to image PPi in deep living rat tissues (~100µm) and in 5-days old zebrafish.

Topics: Animals; Biosensing Techniques; Copper; Diphosphates; DNA; Fluorescent Dyes; HeLa Cells; Humans; Liver; Mice; Microscopy, Fluorescence, Multiphoton; Optical Imaging; Zebrafish

Bioactive compounds phytic acid (IP6) and pyrophosphate (PPi) are minor components of walnuts with the ability of being inhibitors of urolithiasis, among others. Since simultaneous analysis of IP6 and PPi have known drawbacks, a new method to determine their content in walnuts has been developed with emphasis on their extraction from walnuts by microwave-assisted extraction (MAE). Acid content of extracting solvent, extraction time and temperature were optimized. After extraction, compounds were purified by selective adsorption/desorption on an anion exchange solid phase extraction and analyzed by inductive coupled plasma/mass spectrometry. A mixture of H

Topics: Diphosphates; Juglans; Microwaves; Phytic Acid; Solid Phase Extraction

Adenosine 5'-triphosphate phosphoribosyltransferase (ATPPRT) catalyzes the first step in histidine biosynthesis, the condensation of ATP and 5-phospho-α-d-ribosyl-1-pyrophosphate to generate N

Topics: Acclimatization; Adenosine Triphosphate; Allosteric Regulation; Amino Acyl-tRNA Synthetases; ATP Phosphoribosyltransferase; Bacterial Proteins; Cold Temperature; Crystallography, X-Ray; Diphosphates; Enzyme Stability; Escherichia coli; Gene Expression; Histidine; Isoenzymes; Kinetics; Models, Molecular; Monosaccharide Transport Proteins; Phosphoribosyl Pyrophosphate; Protein Domains; Protein Multimerization; Protein Structure, Secondary; Psychrobacter; Recombinant Proteins; Thermodynamics

Two magnesium ions play important roles in nucleotide addition cycle (NAC) of gene transcription. However, at the end of each NAC, why does one ion stay in the active site while the other ion leaves with product pyrophosphate (PP

Topics: Bacteriophage T7; Catalytic Domain; Cations, Divalent; Diphosphates; DNA-Directed RNA Polymerases; Kinetics; Magnesium; Molecular Dynamics Simulation; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Thermodynamics; Transcription, Genetic; Viral Proteins

The soluble protein fraction of the extremely halophilic archaeon Haloarcula japonica exhibits substantial inorganic pyrophosphate (PPi) hydrolysis activity in the presence of 2-4 M NaCl (Wakai et al, J Biol Chem 288:29247-29251, 2013), which provides high ionic strength (2-4). In this study, much higher PPi hydrolysis activity was unexpectedly detected, even with 0 M NaCl in the presence of 100-200 mM MgSO

Topics: Archaeal Proteins; Diphosphates; Extreme Environments; Haloarcula; Osmolar Concentration; Pyrophosphatases; Salinity

Controlling the electromagnetic hot-spot generation is essential for surface-enhanced Raman scattering (SERS) assays. Current hot-spot-based SERS assays have been extensively studied in solutions or on substrates. However, probing biospecies by controlling the hot-spot assembly in living systems has not been demonstrated thus far. Herein, we report a background-free SERS probe for imaging pyrophosphate (PPi), a biochemically significant anion, in living cells. Intracellular PPi is able to induce the nanoparticle dimerization, thus creating an intense electromagnetic hot spot and dramatically enhancing the signal of the Raman reporters residing in the hot spot. More impressively, the reporter we used in this study provides a strong and sharp single peak in the cellular Raman-silent region (1800-2800 cm

Topics: Biomarkers; Diphosphates; Gold; HeLa Cells; Humans; Metal Nanoparticles; Molecular Conformation; Molecular Probes; Nitriles; Spectrum Analysis, Raman; Surface Properties; Tumor Cells, Cultured

Unguisin A (1) is a marine-derived, GABA-containing cyclic heptapeptide. The biological function of this flexible macrocycle is obscure. Here we show that compound 1 lacks any detectable activity in antimicrobial growth inhibition assays, a result that runs contrary to a previous report. However, we find that 1 functions as a promiscuous host molecule in a variety of anion-binding interactions, with high affinity particularly for phosphate and pyrophosphate. We also show that a series of rigidified, backbone-fluorinated analogues of 1 displays altered affinity for chloride ions.

Topics: Diphosphates; Models, Molecular; Peptides, Cyclic; Phosphates; Protein Conformation

Phosphorylated osteopontin (OPN) inhibits hydroxyapatite crystal formation and growth, and bone alkaline phosphatase (BALP) promotes extracellular mineralization via the release of inorganic phosphate from the mineralization inhibitor inorganic pyrophosphate (PPi). Tartrate-resistant acid phosphatase (TRAP), produced by osteoclasts, osteoblasts, and osteocytes, exhibits potent phosphatase activity towards OPN; however, its potential capacity as a regulator of mineralization has not previously been addressed. We compared the efficiency of BALP and TRAP towards the endogenous substrates for BALP, i.e., PPi and pyridoxal 5'-phosphate (PLP), and their impact on mineralization in vitro via dephosphorylation of bovine milk OPN. TRAP showed higher phosphatase activity towards phosphorylated OPN and PPi compared to BALP, whereas the activity of TRAP and BALP towards PLP was comparable. Bovine milk OPN could be completely dephosphorylated by TRAP, liberating all its 28 phosphates, whereas BALP dephosphorylated at most 10 phosphates. OPN, dephosphorylated by either BALP or TRAP, showed a partially or completely attenuated phosphorylation-dependent inhibitory capacity, respectively, compared to native OPN on the formation of mineralized nodules. Thus, there are phosphorylations in OPN important for inhibition of mineralization that are removed by TRAP but not by BALP. In conclusion, our data indicate that both BALP and TRAP can alleviate the inhibitory effect of OPN on mineralization, suggesting a potential role for TRAP in skeletal mineralization. Further studies are warranted to explore the possible physiological relevance of TRAP in bone mineralization.

Topics: Alkaline Phosphatase; Animals; Calcification, Physiologic; Cattle; Cell Line; Diphosphates; Humans; Osteoblasts; Osteopontin; Tartrate-Resistant Acid Phosphatase

A novel flow-through microfluidic device based on a magneto-controlled graphene sensing platform was designed for homogeneous electronic monitoring of pyrophosphatase (PPase) activity; enzymatic hydrolysate-induced release of inorganic copper ion (Cu(2+)) from the Cu(2+)-coordinated pyrophosphate ions (Cu(2+)-PPi) complex was assessed to determine enzyme activity. Magnetic graphene nanosheets (MGNS) functionalized with negatively charged Nafion were synthesized by using the wet-chemistry method. The Cu(2+)-PPi complexes were prepared on the basis of the coordination reaction between copper ion and inorganic pyrophosphate ions. Upon target PPase introduction into the detection system, the analyte initially hydrolyzed pyrophosphate ions into phosphate ions and released the electroactive copper ions from Cu(2+)-PPi complexes. The released copper ions could be readily captured through the negatively charged Nafion on the magnetic graphene nanosheets, which could be quantitatively monitored by using the stripping voltammetry on the flow-through detection cell with an external magnet. Under optimal conditions, the obtained electrochemical signal exhibited a high dependence on PPase activity within a dynamic range from 0.1 to 20 mU mL(-1) and allowed the detection at a concentration as low as 0.05 mU mL(-1). Coefficients of variation for reproducibility of the intra-assay and interassay were below 7.6 and 9.8%, respectively. The inhibition efficiency of sodium fluoride (NaF) also received good results in pyrophosphatase inhibitor screening research. In addition, the methodology afforded good specificity and selectivity, simplification, and low cost without the need of sample separations and multiple washing steps, thus representing a user-friendly protocol for practical utilization in a quantitative PPase activity.

Topics: Copper; Diphosphates; Dose-Response Relationship, Drug; Electrons; Enzyme Activation; Graphite; Hydrolysis; Ions; Magnetic Phenomena; Microfluidic Analytical Techniques; Nanostructures; Particle Size; Pyrophosphatases; Sodium Fluoride; Structure-Activity Relationship; Surface Properties; Time Factors

The presence of small phospho-anions, such as PPi and ATP in protein samples often complicates the robust detection of phosphoproteins by metal-based chemosensors and receptors. We herein report the development of a bis(Zn(2+)-cyclen)-triethylbenzene scaffold which can selectively sequester PPi and ATP without affecting the detection of a di-phosphorylated peptide by a ProxyPhos chemosensor.

Topics: Adenosine Triphosphate; Benzene Derivatives; Coordination Complexes; Diphosphates; Macrocyclic Compounds; Phosphopeptides; Zinc

A novel pyrene-functionalized polynorbornene (P1) bearing sulfonamide NH and triazolium donors has been synthesized for ratiometric fluorescence sensing of PPi in aqueous solution. In addition, P1 is also used to monitor intracellular PPi and to detect PPi released during polymerase chain reaction.

Topics: Diphosphates; Fluorescent Dyes; HeLa Cells; Humans; Microscopy, Confocal; Optical Imaging; Plastics; Pyrenes; Spectrometry, Fluorescence

We demonstrate a rationally designed fluorescent and colorimetric dual-readout strategy for the highly sensitive, quantitative determination of inorganic pyrophosphatase (PPase) activity, a key hydrolytic enzyme involved in a variety of metabolic processes. Inspired by the selective oxidative and chromogenic reaction of o-phenylenediamine (OPD) with Cu(2+), the special inhibitory effects of pyrophosphate (PPi) on the oxidative ability of Cu(2+), and the specific hydrolysis of PPi into orthophosphate by PPase, a convenient small molecule OPD-based analytical system was developed for Cu(2+)/PPi recognition and PPase activity assay. We have confirmed that Cu(2+) acts as the oxidant in the reaction and the main chromogenic product of OPD is 2,3-diaminophenazine (usually called OPDox) in the assay by combining the ESI-MS, (1)H NMR, and XPS spectra analysis. Direct electrochemical insights into the Cu(2+)-triggered and PPi-inhibited mechanism were performed by cyclic voltammetry characterizations of the Cu(2+) in the absence and presence of PPi for the first time. Furthermore, the proposed analytical system with clear response mechanism exhibits a promising outlook for the PPase activity assay in real biological samples and inhibitor screening.

Topics: Colorimetry; Copper; Diphosphates; Fluorescence; Inorganic Pyrophosphatase; Molecular Structure; Oxidation-Reduction; Phenylenediamines; Phosphates

Nonribosomal peptide synthetases (NRPSs) produce many important and structurally complex natural products. Because of their architectures, reprogramming NRPSs has long been attempted to access new bioactive compounds. However, detailed characterization of NRPS catalysis and substrate selectivity by adenylation (A) domains is needed to support such efforts. We present a simple coupled NADH/pyrophosphate (PPi  ) detection assay for analyzing A domain catalysis in vitro. PPi formation is coupled to the consumption of NADH by four enzymatic steps and is detected spectroscopically (λ=340 nm) for simple analysis. We demonstrate the effectiveness of this assay with several adenylation domains, including a stand-alone A domain (DltA, cell wall biosynthesis) and an embedded A domain (Tcp10, teicoplanin biosynthesis). Substrate acceptance of the Tcp10 A domain was explored for the first time, thus demonstrating the applicability of the assay for complex, multi-domain NRPSs.

Topics: Adenosine Monophosphate; Catalytic Domain; Diphosphates; Enzyme Assays; Kinetics; Peptide Synthases; Proteins; Teicoplanin

Three fluorescent asymmetric bis-urea receptors (L1-L3) have been synthesised. The binding properties of L1-L3 towards different anions (fluoride, acetate, hydrogencarbonate, dihydrogen phosphate, and hydrogen pyrophosphate HPpi(3-)) have been studied by means of (1)H-NMR, UV-Vis and fluorescence spectroscopy, single crystal X-ray diffraction, and theoretical calculations. In particular, a remarkable affinity for HPpi(3-) has been observed in the case L1 (DMSO-d6/0.5% H2O) which also acts as a fluorimetric chemosensor for this anion. Interestingly, when L1 is included in cetyltrimethylammonium (CTAB) micelles, hydrogen pyrophosphate recognition can also be achieved in pure water.

Topics: Diphosphates; Fluorescent Dyes; Magnetic Resonance Spectroscopy; Molecular Structure; Spectrometry, Fluorescence; Urea; Water; X-Ray Diffraction

Long-term (multiple-week or -month) release of small, water-soluble molecules from hydrogels remains a significant pharmaceutical challenge, which is typically overcome at the expense of more-complicated drug carrier designs. Such approaches are payload-specific and include covalent conjugation of drugs to base materials or incorporation of micro- and nanoparticles. As a simpler alternative, here we report a mild and simple method for achieving multiple-month release of small molecules from gel-like polymer networks. Densely cross-linked matrices were prepared through ionotropic gelation of poly(allylamine hydrochloride) (PAH) with either pyrophosphate (PPi) or tripolyphosphate (TPP), all of which are commonly available commercial molecules. The loading of model small molecules (Fast Green FCF and Rhodamine B dyes) within these polymer networks increases with the payload/network binding strength and with the PAH and payload concentrations used during encapsulation. Once loaded into the PAH/PPi and PAH/TPP ionic networks, only a few percent of the payload is released over multiple months. This extended release is achieved regardless of the payload/network binding strength and likely reflects the small hydrodynamic mesh size within the gel-like matrices. Furthermore, the PAH/TPP networks show promising in vitro cytocompatibility with model cells (human dermal fibroblasts), though slight cytotoxic effects were exhibited by the PAH/PPi networks. Taken together, the above findings suggest that PAH/PPi and (especially) PAH/TPP networks might be attractive materials for the multiple-month delivery of drugs and other active molecules (e.g., fragrances or disinfectants).

Topics: Biocompatible Materials; Chitosan; Diphosphates; Drug Delivery Systems; Drug Liberation; Fibroblasts; Humans; Hydrogels; Polyamines

Nonribosomal peptide synthetases (NRPSs) are multifunctional enzymes consisting of catalytic domains. The substrate specificities of adenylation (A) domains determine the amino-acid building blocks to be incorporated during nonribosomal peptide biosynthesis. The A-domains mediate ATP-dependent activation of amino-acid substrates as aminoacyl-O-AMP with pyrophosphate (PPi) release. Traditionally, the enzymatic activity of the A-domains has been measured by radioactive ATP-[(32)P]-PPi exchange assays with the detection of (32)P-labeled ATP. Recently, we developed a colorimetric assay for the direct detection of PPi as a yellow 18-molybdopyrophosphate anion ([(P2O7)Mo18O54](4-)). [(P2O7)Mo18O54](4-) was further reduced by ascorbic acid to give a more readily distinguishable blue coloration. Here we demonstrate the lab protocols for the colorimetric assay of PPi released in A-domain reactions.

Topics: Adenosine Monophosphate; Adenosine Triphosphate; Amino Acids; Bacteria; Catalytic Domain; Colorimetry; Diphosphates; Enzyme Assays; Peptide Synthases; Substrate Specificity

The object of the present study was to reveal the action of inosine-5'-monophosphate (IMP) toward myofibrils in postmortem muscles. IMP solubilized isolated actomyosin within a narrow range of KCl concentration, 0.19-0.20 mol/L, because of the dissociation of actomyosin into actin and myosin, but it did not solubilize the proteins in myofibrils with 0.2 mol/L KCl. However, IMP could solubilize both proteins in myofibrils with 0.2 mol/L KCl in the presence of 1 m mol/L pyrophosphate or 1.0-3.3 m mol/L adenosine-5'-diphosphate (ADP). Thus, we presumed that pyrophosphate and ADP released thin filaments composed of actin, and thick filaments composed of myosin from restraints of myofibrils, and then both filaments were solubilized through the IMP-induced dissociation of actomyosin. Thus, we concluded that IMP is a candidate agent to resolve rigor mortis because of its ability to break the association between thick and thin filaments.

Topics: Actins; Actomyosin; Adenosine Diphosphate; Animals; Chickens; Diphosphates; Dose-Response Relationship, Drug; Inosine Monophosphate; Meat; Muscle Proteins; Muscle, Skeletal; Myofibrils; Myosins; Postmortem Changes; Potassium Chloride; Proteolysis; Solubility; Swine

Acetate is an end-product of the PPi-dependent fermentative glycolysis in Entamoeba histolytica; it is synthesized from acetyl-CoA by ADP-forming acetyl-CoA synthetase (ACS) with net ATP synthesis or from acetyl-phosphate by a unique PPi-forming acetate kinase (AcK). The relevance of these enzymes to the parasite ATP and PPi supply, respectively, are analyzed here.. The recombinant enzymes were kinetically characterized and their physiological roles were analyzed by transcriptional gene silencing and further metabolic analyses in amoebae.. Recombinant ACS showed higher catalytic efficiencies (Vmax/Km) for acetate formation than for acetyl-CoA formation and high acetyl-CoA levels were found in trophozoites. Gradual ACS gene silencing (49-93%) significantly decreased the acetate flux without affecting the levels of glycolytic metabolites and ATP in trophozoites. However, amoebae lacking ACS activity were unable to reestablish the acetyl-CoA/CoA ratio after an oxidative stress challenge. Recombinant AcK showed activity only in the acetate formation direction; however, its substrate acetyl-phosphate was undetected in axenic parasites. AcK gene silencing did not affect acetate production in the parasites but promoted a slight decrease (10-20%) in the hexose phosphates and PPi levels.. These results indicated that the main role of ACS in the parasite energy metabolism is not ATP production but to recycle CoA for glycolysis to proceed under aerobic conditions. AcK does not contribute to acetate production but might be marginally involved in PPi and hexosephosphate homeostasis.. The previous, long-standing hypothesis that these enzymes importantly contribute to ATP and PPi supply in amoebae can now be ruled out.

Topics: Acetate Kinase; Acetate-CoA Ligase; Acetates; Adenosine Triphosphate; Diphosphates; Energy Metabolism; Entamoeba histolytica; Ethanol; Glycolysis

Trans-lesion synthesis polymerases, like DNA Polymerase-η (Pol-η), are essential for cell survival. Pol-η bypasses ultraviolet-induced DNA damages via a two-metal-ion mechanism that assures DNA strand elongation, with formation of the leaving group pyrophosphate (PPi). Recent structural and kinetics studies have shown that Pol-η function depends on the highly flexible and conserved Arg61 and, intriguingly, on a transient third ion resolved at the catalytic site, as lately observed in other nucleic acid-processing metalloenzymes. How these conserved structural features facilitate DNA replication, however, is still poorly understood. Through extended molecular dynamics and free energy simulations, we unravel a highly cooperative and dynamic mechanism for DNA elongation and repair, which is here described by an equilibrium ensemble of structures that connect the reactants to the products in Pol-η catalysis. We reveal that specific conformations of Arg61 help facilitate the recruitment of the incoming base and favor the proper formation of a pre-reactive complex in Pol-η for efficient DNA editing. Also, we show that a third transient metal ion, which acts concertedly with Arg61, serves as an exit shuttle for the leaving PPi. Finally, we discuss how this effective and cooperative mechanism for DNA repair may be shared by other DNA-repairing polymerases.

Topics: Adenosine Triphosphate; Amino Acid Motifs; Arginine; Biocatalysis; Cations, Divalent; Diphosphates; DNA; DNA Replication; DNA-Directed DNA Polymerase; Humans; Magnesium; Molecular Dynamics Simulation; Molecular Sequence Data; Protein Structure, Secondary; Protein Structure, Tertiary; Static Electricity; Thermodynamics

Low-cost activated carbons (ACs) were prepared from four kinds of solid wastes: petroleum coke, Enteromorpha prolifera, lignin from papermaking black liquid and hair, by pyrophosphoric acid (H4P2O7) activation. Thermo-gravimetric analysis of the pyrolysis of H4P2O7-precursor mixtures implied that H4P2O7 had different influences on the pyrolysis behavior of the four raw materials. N2 adsorption/desorption isotherms, scanning electron microscopy, Fourier transform infrared spectroscopy and adsorption capacities for dyes were used to characterize the prepared activated carbons. AC derived from E. prolifera exhibited the highest surface area (1094m(2)/g) and maximum monolayer adsorption capacity for malachite green (1250mg/g). Kinetic studies showed that the experimental data were in agreement with the pseudo-second-order model. The adsorption isotherms were well described by the Langmuir isotherm model, indicating the adsorption of dye onto the ACs proceeded by monolayers.

Topics: Adsorption; Charcoal; Coke; Diphosphates; Hair; Lignin; Microscopy, Electron, Scanning; Spectroscopy, Fourier Transform Infrared; Ulva

To analyze the influence of experimental conditions: Distribution of particulate and the filter condition on the pyrophosphoric acid method for quantitative analysis of free silica in dust.. According to Method for determination of dust in the air of workplace Part 3: Distribution of particulate (GBZ/T 192.3-2007) , Part 4: Content of free silica in dust (GBZ/T 192.4-2007) , the distribution of particulate of 5kinds of dust samples were observed. Different filter conditions were used to determinate the Content of free silica in the 4kinds ofdust samples: 1 filter paper, 2 filter papers, 3 filter papers, 2 filter papers with paper pulp in them.. The distribution of particulate of 4 kinds of dust sampleswere different. The order from high to low is defined with "I, II, III, IV, V" successively. For dust sample I, II, III, the results with different conditions increase successively (P<0.05) . The result in 2 filter papers with paper pulp were not significantly different compared with the reference value (P>0.05) . For dust sample IV, the resultin 1 filter paper were significantlylower thanthe reference value (P<0.05) . For dust sampleV, The results with different kinds of filter type were not significantly different (P>0.05) .. different filter conditions should be considered according to thecontent of free silica and the distribution of particulate in dust sample. For the dust sample which has the higher content of free silica and the distribution of particulate, 2 filter papers with paper pulp in themis the better filter condition compared with the traditional way.

Topics: Air Pollutants, Occupational; Diphosphates; Dust; Environmental Monitoring; Filtration; Particle Size; Silicon Dioxide

This paper reports a new and facile method for the synthesis of water-soluble thiolate-protected AuNCs via protein-ligand interaction. Using 3-mercaptopropionic acid (MPA) as a model ligand and bovine serum albumin (BSA) as a model protein, water-soluble AuNCs (BSA/MPA-AuNCs) with intense orange-yellow fluorescent emission (quantum yield=16%) are obtained. Results show that AuNCs produced with this method have hydrophobic interactions with BSA. The synthetic strategy is then successfully extended to produce water-soluble AuNCs protected by other thiolates. Moreover, a sensitive and eco-friendly sensing system is established for detection of the activity of inorganic pyrophosphatase (PPase), which relies on the selective coordination of Fe(3+)with BSA/MPA-AuNCs, the higher affinity between pyrophosphate (PPi) and Fe(3+), and the hydrolysis of PPi by PPase. A good linearity between the fluorescence intensity and PPase activity within the range from 0.1 to 3U/L is found, with a detection limit down to 0.07U/L. Additionally, the fluorescent assay developed here is utilized to assay the PPase activity in real biological samples and as well as to evaluate PPase inhibitor, illustrating the great potential for biological analysis.

Topics: 3-Mercaptopropionic Acid; Animals; Biosensing Techniques; Cattle; Diphosphates; Enzyme Assays; Fluorescent Dyes; Gold; Inorganic Pyrophosphatase; Nanostructures; Serum Albumin, Bovine; Solubility; Spectrometry, Fluorescence; Water

A detailed reaction mechanism is proposed for the hydrolysis of the phosphoanhydride bonds in adenosine triphosphate (ATP) in the presence of the binuclear Zr(IV)-substituted Keggin type polyoxometalate (Et2NH2)8[{α-PW11O39Zr(μ-OH)(H2O)}2]·7H2O (ZrK 2:2). The full reaction mechanism of ATP hydrolysis in the presence of ZrK 2:2 at pD 6.4 was elucidated by a combination of (31)P, (31)P DOSY, and (31)P EXSY NMR spectroscopy, demonstrating the potential of these techniques for the analysis of complex reaction mixtures involving polyoxometalates (POMs). Two possible parallel reaction pathways were proposed on the basis of the observed reaction intermediates and final products. The 1D (31)P and (31)P DOSY spectra of a mixture of 20.0 mM ATP and 3.0 mM ZrK 2:2 at pD 6.4, measured immediately after sample preparation, evidenced the formation of two types of complexes, I1A and I1B, representing different binding modes between ATP and the Zr(IV)-substituted Keggin type polyoxometalate (ZrK). Analysis of the NMR data shows that at pD 6.4 and 50 °C ATP hydrolysis in the presence of ZrK proceeds in a stepwise fashion. During the course of the hydrolytic reaction various products, including adenosine diphosphate (ADP), adenosine monophosphate (AMP), pyrophosphate (PP), and phosphate (P), were detected. In addition, intermediate species representing the complexes ADP/ZrK (I2) and PP/ZrK (I5) were identified and the potential formation of two other intermediates, AMP/ZrK (I3) and P/ZrK (I4), was demonstrated. (31)P EXSY NMR spectra evidenced slow exchange between ATP and I1A, ADP and I2, and PP and I5, thus confirming the proposed reaction pathways.

Topics: Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Diphosphates; Hydrolysis; Kinetics; Magnetic Resonance Spectroscopy; Models, Chemical; Oxides; Phosphorus Radioisotopes; Zirconium

Inorganic pyrophosphate (PPi) is produced from nucleoside triphosphates in important biosynthetic reactions and is considered a diagnostic marker for various diseases, such as cancer, crystal deposition disease, and arthritis. Traditional methods for biological PPi detection rely on off-line analytics after sample destruction. Molecular probes for imaging this biologically important analyte with temporal and spatial control in living cells are currently in demand. Herein, we report an Fe(III) -salen complex as the first small reaction-based probe for endogenous mitochondrial PPi following a disassembly approach. Significantly, we successfully applied this complex for the detection of increased cellular PPi levels, and its performance was not affected by the presence of mitochondrial ATP in living cells.

Topics: Adenosine Triphosphate; Aldehydes; Cell Membrane Permeability; Coordination Complexes; Diphosphates; Ferric Compounds; Fluorescent Dyes; HeLa Cells; Humans; Hydrogen-Ion Concentration; Mitochondria; Molecular Structure; Phosphate Transport Proteins; Probenecid

Ferric pyrophosphate (FePP) is a widely used iron source in food fortification and in nutritional supplements, due to its white colour, that is very uncommon for insoluble Fe salts. Although its dissolution is an important determinant of Fe adsorption in human body, the solubility characteristics of FePP are complex and not well understood. This report is a study on the solubility of FePP as a function of pH and excess of pyrophosphate ions. FePP powder is sparingly soluble in the pH range of 3-6 but slightly soluble at pH<2 and pH>8. In the presence of pyrophosphate ions the solubility of FePP strongly increases at pH 5-8.5 due to formation a soluble complex between Fe(III) and pyrophosphate ions, which leads to an 8-10-fold increase in the total ionic iron concentration. This finding is beneficial for enhancing iron bioavailability, which important for the design of fortified food, beverages, and nutraceutical products.

Topics: Beverages; Biological Availability; Dietary Supplements; Diphosphates; Food, Fortified; Humans; Iron; Solubility

Great efforts have been made to develop fluorescent probes for pyrophosphate (PPi) detection. Nucleus staining with fluorescence microscopy has been also widely investigated. But fluorescent probes for PPi detection with high sensitivity in water medium and nucleus staining with low-cost non-precious metal complexes in living cells are still challenging. Herein, we report simple terpyridine-Zn(II) complexes for selective nanomolar PPi detection over ATP and ADP in water based on aggregation induced emission (AIE) and intramolecular charge transfer (ICT). In addition, these terpyridine-Zn(II) complexes were successfully employed for nucleus staining in living cells. These results demonstrated simply obtained terpyridine-Zn(II) complexes are powerful tool for PPi detection and the development of PPi-related studies.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Cell Nucleus; Coordination Complexes; Diphosphates; Fluorescent Dyes; HeLa Cells; Humans; Organometallic Compounds; Spectrometry, Fluorescence; Water; Zinc

Self-assembled colorimetric sensors have been prepared from Zn(II)-DPA-attached phenylboronic acid (·Zn) and catechol-type dyes. The ·Zn-dye sensors display selectivity towards oligophosphate over monophosphates. The colorimetric sensor assay (·Zn-dye) is utilized to monitor a model of a metabolic reaction where ATP is hydrolyzed to pyrophosphate (PPi) and AMP.

Topics: Adenosine Monophosphate; Adenosine Triphosphate; Colorimetry; Diphosphates; Hydrolysis; Models, Biological; Water

Key nuclear processes in eukaryotes, including DNA replication, repair, and gene regulation, require extensive chromatin remodeling catalyzed by energy-consuming enzymes. It remains unclear how the ATP demands of such processes are met in response to rapid stimuli. We analyzed this question in the context of the massive gene regulation changes induced by progestins in breast cancer cells and found that ATP is generated in the cell nucleus via the hydrolysis of poly(ADP-ribose) to ADP-ribose. In the presence of pyrophosphate, ADP-ribose is used by the pyrophosphatase NUDIX5 to generate nuclear ATP. The nuclear source of ATP is essential for hormone-induced chromatin remodeling, transcriptional regulation, and cell proliferation.

Topics: Adenosine Diphosphate Ribose; Adenosine Triphosphate; Breast Neoplasms; Cell Nucleus; Cell Proliferation; Chromatin Assembly and Disassembly; Crystallography, X-Ray; Diphosphates; Energy Metabolism; Female; Gene Expression Regulation; Humans; Hydrolysis; MCF-7 Cells; Poly (ADP-Ribose) Polymerase-1; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerases; Progestins; Protein Multimerization; Pyrophosphatases

Sesterterpenoids are a group of terpenoid natural products that are primarily biosynthesized via cyclization of the C25 linear substrate geranylfarnesyl pyrophosphate (GFPP). Although the long carbon chain of GFPP in theory allows for many different cyclization patterns, sesterterpenoids are relatively rare species among terpenoids, suggesting that many intriguing sesterterpenoid scaffolds have been overlooked. Meanwhile, the recent identification of the first sesterterpene synthase has allowed the discovery of new sesterterpenoids by the genome mining approach. In this study, we characterized the unusual fungal sesterterpene synthase EvQS and successfully obtained the sesterterpene quiannulatene (1) with a novel and unique highly congested carbon skeleton, which is further oxidized to quiannulatic acid (2) by the cytochrome P450 Qnn-P450. A mechanistic study of its cyclization from GFPP indicated that the biosynthesis employs an unprecedented cyclization mode, which involves three rounds of hydride shifts and two successive C-C bond migrations to construct the 5-6-5-5-5 fused ring system of 1.

Topics: Alkyl and Aryl Transferases; Aspergillus oryzae; Biological Products; Carbon; Catalysis; Cyclization; Diphosphates; Emericella; Genome, Fungal; Hydrogen; Phylogeny; Recombinant Proteins; Sesterterpenes; Terpenes

Vascular calcification significantly contributes to mortality in chronic kidney disease (CKD) patients. Sevelamer and pyrophosphate (PPi) have proven to be effective in preventing vascular calcification, the former by controlling intestinal phosphate absorption, the latter by directly interfering with the hydroxyapatite crystal formation. Since most patients present with established vascular calcification, it is important to evaluate whether these compounds may also halt or reverse the progression of preexisting vascular calcification. CKD and vascular calcification were induced in male Wistar rats by a 0.75 % adenine low protein diet for 4 weeks. Treatment with PPi (30 or 120 µmol/kg/day), sevelamer carbonate (1500 mg/kg/day) or vehicle was started at the time point at which vascular calcification was present and continued for 3 weeks. Hyperphosphatemia and vascular calcification developed prior to treatment. A significant progression of aortic calcification in vehicle-treated rats with CKD was observed over the final 3-week period. Sevelamer treatment significantly reduced further progression of aortic calcification as compared to the vehicle control. No such an effect was seen for either PPi dose. Sevelamer but not PPi treatment resulted in an increase in both osteoblast and osteoid perimeter. Our study shows that sevelamer was able to reduce the progression of moderate to severe preexisting aortic calcification in a CKD rat model. Higher doses of PPi may be required to induce a similar reduction of severe established arterial calcification in this CKD model.

Topics: Animals; Aorta; Chelating Agents; Diphosphates; Durapatite; Male; Rats; Rats, Wistar; Renal Insufficiency, Chronic; Sevelamer; Vascular Calcification

Sudden unexpected death in infancy occurs in apparently healthy infants and remains largely unexplained despite thorough investigation. The vast majority of cases are sporadic. Here we report seven individuals from three families affected by sudden and unexpected cardiac arrest between 4 and 20 months of age. Whole-exome sequencing revealed compound heterozygous missense mutations in PPA2 in affected infants of each family. PPA2 encodes the mitochondrial pyrophosphatase, which hydrolyzes inorganic pyrophosphate into two phosphates. This is an essential activity for many biosynthetic reactions and for energy metabolism of the cell. We show that deletion of the orthologous gene in yeast (ppa2Δ) compromises cell viability due to the loss of mitochondria. Expression of wild-type human PPA2, but not PPA2 containing the mutations identified in affected individuals, preserves mitochondrial function in ppa2Δ yeast. Using a regulatable (doxycycline-repressible) gene expression system, we found that the pathogenic PPA2 mutations rapidly inactivate the mitochondrial energy transducing system and prevent the maintenance of a sufficient electrical potential across the inner membrane, which explains the subsequent disappearance of mitochondria from the mutant yeast cells. Altogether these data demonstrate that PPA2 is an essential gene in yeast and that biallelic mutations in PPA2 cause a mitochondrial disease leading to sudden cardiac arrest in infants.

Topics: Alleles; Death, Sudden, Cardiac; Diphosphates; Exome; Female; Gene Deletion; Genes, Essential; Genetic Complementation Test; Heterozygote; Humans; Infant; Inorganic Pyrophosphatase; Male; Membrane Potential, Mitochondrial; Microbial Viability; Mitochondria; Mitochondrial Proteins; Mutation; Mutation, Missense; Proton Pumps; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins

Syntrophus aciditrophicus is a model syntrophic bacterium that degrades key intermediates in anaerobic decomposition, such as benzoate, cyclohexane-1-carboxylate, and certain fatty acids, to acetate when grown with hydrogen-/formate-consuming microorganisms. ATP formation coupled to acetate production is the main source for energy conservation by S. aciditrophicus However, the absence of homologs for phosphate acetyltransferase and acetate kinase in the genome of S. aciditrophicus leaves it unclear as to how ATP is formed, as most fermentative bacteria rely on these two enzymes to synthesize ATP from acetyl coenzyme A (CoA) and phosphate. Here, we combine transcriptomic, proteomic, metabolite, and enzymatic approaches to show that S. aciditrophicus uses AMP-forming, acetyl-CoA synthetase (Acs1) for ATP synthesis from acetyl-CoA. acs1 mRNA and Acs1 were abundant in transcriptomes and proteomes, respectively, of S. aciditrophicus grown in pure culture and coculture. Cell extracts of S. aciditrophicus had low or undetectable acetate kinase and phosphate acetyltransferase activities but had high acetyl-CoA synthetase activity under all growth conditions tested. Both Acs1 purified from S. aciditrophicus and recombinantly produced Acs1 catalyzed ATP and acetate formation from acetyl-CoA, AMP, and pyrophosphate. High pyrophosphate levels and a high AMP-to-ATP ratio (5.9 ± 1.4) in S. aciditrophicus cells support the operation of Acs1 in the acetate-forming direction. Thus, S. aciditrophicus has a unique approach to conserve energy involving pyrophosphate, AMP, acetyl-CoA, and an AMP-forming, acetyl-CoA synthetase.. Bacteria use two enzymes, phosphate acetyltransferase and acetate kinase, to make ATP from acetyl-CoA, while acetate-forming archaea use a single enzyme, an ADP-forming, acetyl-CoA synthetase, to synthesize ATP and acetate from acetyl-CoA. Syntrophus aciditrophicus apparently relies on a different approach to conserve energy during acetyl-CoA metabolism, as its genome does not have homologs to the genes for phosphate acetyltransferase and acetate kinase. Here, we show that S. aciditrophicus uses an alternative approach, an AMP-forming, acetyl-CoA synthetase, to make ATP from acetyl-CoA. AMP-forming, acetyl-CoA synthetases were previously thought to function only in the activation of acetate to acetyl-CoA.

Topics: Acetates; Acetyl Coenzyme A; Adenosine Triphosphate; Coenzyme A Ligases; Deltaproteobacteria; Diphosphates; Gene Expression Profiling; Metabolome; Proteome

We present a new copper-mediated on-off switch for detecting either pyrophosphate (PPi) or alkaline phosphatase (ALP) based on DNA-scaffolded silver nanoclusters (DNA/AgNCs) templated by a single-stranded sequence containing a 15-nt polythymine spacer between two different emitters. The switch is based on three favorable properties: the quenching ability of Cu(2+) for DNA/AgNCs with excitation at 550 nm; the strong binding capacity of Cu(2+) and PPi; and the ability of ALP to transform PPi into orthophosphate (Pi). The change in fluorescence of DNA/AgNCs depends on the concentrations of Cu(2+), PPi, and ALP. Copper(II) acts as a mediator to interact specifically with the Probe, while PPi and ALP convert the signal of the Probe by removing and recovering Cu(2+), operating as an on-off switch. In the presence of Cu(2+) only, DNA/AgNCs exhibit low fluorescence because the combination of Cu(2+) and DNA template disturbs the precise formation of DNA/AgNCs. When PPi is added to the system containing Cu(2+), free DNA template is obtained due to the stronger interaction of PPi and Cu(2+), leading to a significant fluorescence increase (ON state) which depends on the concentration of PPi. Further addition of ALP results in the release of free Cu(2+) via ALP-catalysis of hydrolysis of PPi into Pi, thereby returning the system to the low fluorescence OFF state. The switch allows the analysis of either PPi or ALP by observation of the fluorescence status, with the detection limit of 112.69 nM and 0.005 U/mL for PPi and ALP, respectively. The AgNCs on-off switch provides the advantages of simple design, convenient operation, and low experimental cost without need of chemical modification, organic dyes, or separation procedures.

Topics: Alkaline Phosphatase; Animals; Biosensing Techniques; Cattle; Copper; Diphosphates; DNA; Enzyme Assays; Humans; Limit of Detection; Nanostructures; Silver; Synovial Fluid

An effective, nontoxic, tumor-specific immunotherapy is the ultimate goal in the battle against cancer, especially the metastatic disease. Checkpoint blockade-based immunotherapies have been shown to be extraordinarily effective but benefit only the minority of patients whose tumors have been pre-infiltrated by T cells. Here, we show that Zn-pyrophosphate (ZnP) nanoparticles loaded with the photosensitizer pyrolipid (ZnP@pyro) can kill tumor cells upon irradiation with light directly by inducing apoptosis and/or necrosis and indirectly by disrupting tumor vasculature and increasing tumor immunogenicity. Furthermore, immunogenic ZnP@pyro photodynamic therapy (PDT) treatment sensitizes tumors to checkpoint inhibition mediated by a PD-L1 antibody, not only eradicating the primary 4T1 breast tumor but also significantly preventing metastasis to the lung. The abscopal effects on both 4T1 and TUBO bilateral syngeneic mouse models further demonstrate that ZnP@pyro PDT treatment combined with anti-PD-L1 results in the eradication of light-irradiated primary tumors and the complete inhibition of untreated distant tumors by generating a systemic tumor-specific cytotoxic T cell response. These findings indicate that nanoparticle-mediated PDT can potentiate the systemic efficacy of checkpoint blockade immunotherapies by activating the innate and adaptive immune systems in tumor microenvironment.

Topics: Animals; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Chlorophyll; Combined Modality Therapy; Diphosphates; Humans; Immunotherapy; Light; Lipids; Lung Neoplasms; Mice; Nanoparticles; Necrosis; Neoplasm Metastasis; Photochemotherapy; Photosensitizing Agents; Zinc

The proton-translocating inorganic pyrophosphatases (H(+)-PPases) are primary electrogenic H(+) pumps that derive energy from the hydrolysis of inorganic pyrophosphate (PPi). They are widely distributed among most land plants and have also been found in several species of protozoan parasites. Here we describe, for the first time, the molecular cloning and functional characterization of a gene encoding an H(+)-pyrophosphatase in the protozoan scuticociliate parasite Philasterides dicentrarchi, which infects turbot. The predicted P. dicentrarchi PPase (PdPPase) consists of 587 amino acids of molecular mass 61.7 kDa and an isoelectric point of 5.0. Several motifs characteristic of plant vacuolar H(+)-PPases (V-H(+)-PPases) were also found in the PdPPase, which contains all the sequence motifs of the prototypical type I V-H(+)-PPase from Arabidopsis thaliana vacuolar pyrophosphatase type I (AVP1) plant. The PdPPase has a characteristic residue that determines strict K(+)-dependence, but unlike AVP1, PdPPase contains an N-terminal signal peptide (SP) sequence. Antibodies generated by vaccination of mice with a genetic or recombinant protein containing a partial sequence of the PdPPase and a common motif with the polyclonal antibody PABHK specific to AVP1 recognized a single band of about 62 kDa in western blots. These antibodies specifically stained both vacuole and the alveolar membranes of trophozoites of P. dicentrarchi. H+ transport was partially inhibited by the bisphosphonate pamidronate (PAM) and completely inhibited by NaF. The bisphosphonate PAM inhibited both H+-translocation and gene expression. PdPPase and PAM also inhibited in vitro growth of the ciliates. The apparent lack of V-H(+)-PPases in vertebrates and the parasite sensitivity to PPI analogues may provide a molecular target for developing new drugs to control scuticociliatosis.

Topics: Amino Acid Sequence; Animals; Antibodies, Protozoan; Arabidopsis; Base Sequence; Ciliophora Infections; Diphosphates; DNA, Complementary; Fish Diseases; Flatfishes; Inorganic Pyrophosphatase; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Oligohymenophorea; Phylogeny; Proton Pump Inhibitors; Recombinant Proteins; Sequence Alignment

S1 nuclease has an important function in DNA transcription, replication, recombination, and repair. A label-free fluorescent method for the detection of S1 nuclease activity has been developed using polycytosine oligonucleotide-templated silver nanoclusters (dC12-Ag NCs). In this assay, dC12 can function as both the template for the stabilization of Ag NCs and the substrate of the S1 nuclease. Fluorescent Ag NCs could be effectively formed using dC12 as the template without S1 nuclease. In the presence of S1 nuclease, dC12 is degraded to mono- or oligonucleotide fragments, thereby resulting in a reduction in fluorescence. S1 nuclease with an activity as low as 5×10(-8)Uμl(-1) (signal/noise=3) can be determined with a linear range of 5×10(-7) to 1×10(-3)Uμl(-1). The promising application of the proposed method in S1 nuclease inhibitor screening has been demonstrated using pyrophosphate as the model inhibitor. Furthermore, the S1 nuclease concentrations in RPMI 1640 cell medium were validated. The developed method for S1 nuclease is sensitive and facile because its operation does not require any complicated DNA labeling or laborious fluorescent dye synthesis.

Topics: Diphosphates; DNA, Single-Stranded; Enzyme Inhibitors; Feasibility Studies; Fluorescent Dyes; Fungal Proteins; Metal Nanoparticles; Oligonucleotides; Poly C; Silver; Single-Strand Specific DNA and RNA Endonucleases; Spectrometry, Fluorescence

A reaction of DBU promoted ring opening in nucleoside-3'-O- and nucleoside-5'-O-(2-thio-4,4-pentamethylene-1,3,2-oxathiaphospholane) monomers with a pyrophosphate or a methylenediphosphonate anion proceeds with substantial loss of stereoselectivity. Depending on the absolute configuration of the phosphorus atom, so far widely accepted the stereoretentive mechanism of condensation is accompanied by a stereoinvertive one, most likely employing an intramolecular ligand-ligand exchange in an uncharged intermediate.

Topics: Anions; Bridged Bicyclo Compounds, Heterocyclic; Diphosphates; Diphosphonates; Magnetic Resonance Spectroscopy; Nucleotides; Phosphites; Ribose; Stereoisomerism

Cps2L, a thymidylytransferase, is the first enzyme in Streptococcus pneumoniae L-rhamnose biosynthesis and an antibacterial target. We herein report the evaluation of six sugar phosphate analogues selected to further probe Cps2L substrate tolerance. A modified continuous spectrophotometric assay was employed for facile detection of pyrophosphate (PPi) released from nucleotidylyltransfase-catalysed condensation of sugar 1-phosphates and nucleoside triphosphates to produce sugar nucleotides. Additionally, experiments using waterLOGSY NMR spectroscopy were investigated as a complimentary method to evaluate binding affinity to Cps2L.

Topics: Anti-Bacterial Agents; Bacterial Proteins; Diphosphates; Enzyme Assays; Enzyme Inhibitors; Glucosephosphates; Kinetics; Nucleotidyltransferases; Recombinant Proteins; Spectrophotometry; Streptococcus pneumoniae

RNA polymerase II catalyzes the nucleotidyl transfer reaction for messenger RNA synthesis in eukaryotes. Two crystal structures of this system have been resolved, each with its own defects in the coordination sphere of Mg(2+) (A) resulting from chemical modifications. We have used both structures and also a novel hybrid of the two that allows a better exploration of the parts of configuration space that reflect substrate-enzyme interactions. MD and QM/MM calculations have been performed, the latter with the semiempirical AM1/d-PhoT method, calibrated against density functional theory. Reaction path scans in 1-D provided insights about the role of Mg(2+) (A) which turns out to be more structural than catalytic. In contrast, 1-D scans of the incorporation of the nucleotidyl group yielded barriers that were much too high, necessitating the use of 2-D reaction coordinates. Three different proton acceptors for the initial reaction step were examined. For those models based on the two PDB structures the 2-D scans continued to yield very high barriers, indicating that the reaction is unlikely to proceed from these configurations. On the other hand, two hybrid models, chosen from the early and late parts of a 12 ns molecular dynamics simulation yielded greatly reduced barriers in the range of ∼ 17 to ∼ 27 kcal/mol for the three proton acceptors, as compared to the experimental estimate of 18 kcal/mol. The final step, release of pyrophosphate, was found to be facile. Our overall mechanism involves only active site residues or water without the need for external reactive agents such as the hydroxide ion previously proposed.

Topics: Catalytic Domain; Coordination Complexes; Diphosphates; Magnesium; Molecular Dynamics Simulation; Protein Binding; Protons; RNA Polymerase II; Transcription, Genetic

The terpyridine anthracene ligand was synthesized and characterized. is a ratiometric fluorescent probe for Cd(2+) with a recognition mechanism based on intramolecular charge transfer (ICT). An complex was isolated, and its structure was established using single-crystal XRD. The complex was able to serve as a novel reversible chemosensing ensemble to allow ratiometric response to pyrophosphate (PPi) in aqueous media. Moreover, the fluorescence imaging in living cells from these two emission channels suggested that was a ratiometric probe for Cd(2+), and the in situ generated complex was also a ratiometric ensemble for PPi detection in living cells.

Topics: Animals; Cadmium; Cell Line; Coordination Complexes; Crystallography, X-Ray; Diphosphates; Fluorescent Dyes; Mice; Microscopy, Confocal; Molecular Conformation; Pyridines; Solutions; Spectrometry, Fluorescence

Apple pomace is one of the major agricultural residues in Aomori prefecture, Japan, and it would be useful to develop effective applications for it. As apple pomace contains easily fermentable sugars such as glucose, fructose and sucrose, it can be used as a feedstock for the fermentation of fuels and chemicals. We previously isolated a new hydrogen-producing bacterium, Clostridium beijerinckii HU-1, which could produce H2 at a production rate of 14.5 mmol of H2/L/h in a fed-batch culture at 37 °C, pH 6.0. In this work we found that the HU-1 strain produces H2 at an approximately 20% greater rate when the fermentation medium contains the water-insoluble material from apple pomace. The water-insoluble material from apple pomace caused a metabolic shift that stimulated H2 production. HU-1 showed a decrease of lactate production, which consumes NADH, accompanied by an increase of the intracellular pyrophosphate content, which is an inhibitor of lactate dehydrogenase. The intracellular NAD(+)/NADH ratios of HU-1 during H2 fermentation were maintained in a more reductive state than those observed without the addition of the water insoluble material. To correct the abnormal intracellular redox balance, caused by the repression of lactate production, H2 production with NADH oxidation must be stimulated.

Topics: Batch Cell Culture Techniques; Bioreactors; Carbohydrate Metabolism; Clostridium; Diphosphates; Fermentation; Hydrogen; Hydrogen-Ion Concentration; Japan; L-Lactate Dehydrogenase; Lactic Acid; Malus; NAD; Oxidation-Reduction; Solubility; Temperature; Water

A highly selective and sensitive phenanthroimidazole tagged Mannich base type dizinc(II) fluorescent probe (R-Zn²⁺) has been developed for the pyrophosphate ion (PPi) with a very low limit of detection (LOD) of 0.25 ppm; this also assesses PPi from DNA polymerization chain reaction (PCR).

Topics: Diphosphates; DNA, Fungal; Fluorescent Dyes; Imidazoles; Polymerase Chain Reaction; Sequence Analysis, DNA; Xylariales; Zinc

Membrane-bound pyrophosphatase (mPPases) of various types consume pyrophosphate (PPi) to drive active H+ or Na+ transport across membranes. H+-transporting PPases are divided into phylogenetically distinct K+-independent and K+-dependent subfamilies. In the present study, we describe a group of 46 bacterial proteins and one archaeal protein that are only distantly related to known mPPases (23%-34% sequence identity). Despite this evolutionary divergence, these proteins contain the full set of 12 polar residues that interact with PPi, the nucleophilic water and five cofactor Mg2+ ions found in 'canonical' mPPases. They also contain a specific lysine residue that confers K+ independence on canonical mPPases. Two of the proteins (from Chlorobium limicola and Cellulomonas fimi) were expressed in Escherichia coli and shown to catalyse Mg2+-dependent PPi hydrolysis coupled with electrogenic H+, but not Na+ transport, in inverted membrane vesicles. Unique features of the new H+-PPases include their inhibition by Na+ and inhibition or activation, depending on PPi concentration, by K+ ions. Kinetic analyses of PPi hydrolysis over wide ranges of cofactor (Mg2+) and substrate (Mg2-PPi) concentrations indicated that the alkali cations displace Mg2+ from the enzyme, thereby arresting substrate conversion. These data define the new proteins as a novel subfamily of H+-transporting mPPases that partly retained the Na+ and K+ regulation patterns of their precursor Na+-transporting mPPases.

Topics: Bacterial Proteins; Cell Membrane; Cellulomonas; Chlorobium; Diphosphates; Escherichia coli; Ion Transport; Magnesium; Membrane Proteins; Potassium; Protons; Pyrophosphatases; Recombinant Proteins; Sodium

Glucose-1-phosphate uridylyltransferase in conjunction with UDP-glucose pyrophosphorylase was found to catalyse the conversion of a range of 5-substituted UTP derivatives into the corresponding UDP-galactose derivatives in poor yield. Notably the 5-iodo derivative was not converted to UDP-sugar. In contrast, UDP-glucose pyrophosphorylase in conjunction with inorganic pyrophosphatase was particularly effective at converting 5-substituted UTP derivatives, including the iodo compound, into a range of gluco-configured 5-substituted UDP-sugar derivatives in good yields. Attempts to effect 4"-epimerization of these 5-substituted UDP-glucose with UDP-glucose 4"-epimerase from yeast were unsuccessful, while use of the corresponding enzyme from Erwinia amylovora resulted in efficient epimerization of only 5-iodo-UDP-Glc, but not the corresponding 5-aryl derivatives, to give 5-iodo-UDP-Gal. Given the established potential for Pd-mediated cross-coupling of 5-iodo-UDP-sugars, this provides convenient access to the galacto-configured 5-substituted-UDP-sugars from gluco-configured substrates and 5-iodo-UTP.

Topics: Carbohydrate Conformation; Diphosphates; Erwinia amylovora; Phosphotransferases; UDPglucose 4-Epimerase; Uridine Diphosphate Sugars

A binuclear Cu(II) compound [Cu2(bpa)2(P2O7)(H2O)2]·2.5H2O, 1, (bpa = 2,2'-bipyridylamine), with pairs of Cu(II) ions bridged by one pyrophosphate tetra-anion, was synthesized and crystallized. Its triclinic structure was determined by single-crystal X-ray diffraction. Electron paramagnetic resonance (EPR) spectra of single crystal samples of 1 were recorded for a fixed orientation of the magnetic field (B0) as a function of temperature (T) between 4.7 and 293 K, and at T = 4.7, 50 and 293 K, as a function of the orientation of B0. Below ∼8 K, the spectra are assigned to two types of mononuclear crystal defects hyperfine-coupled to one copper and two nitrogen nuclei. The g-matrices and hyperfine couplings at these T provide information about the structures of these defects. Above 10 K, the spectrum is dominated by the response of the bulk binuclear Cu(II) material, showing hyperfine interactions with two copper nuclei, collapsing to a single peak above 18 K when the units are magnetically connected, and the magnetic behaviour becomes 3D. We attribute the results above 10 K to the interplay of an AFM intrabinuclear exchange interaction J0 = -28(3) cm(-1) (defined as Hex = -J0S1·S2), and three orders of magnitude weaker exchange coupling with average magnitude |J1| ≥ 0.022 cm(-1) between Cu(II) ions in neighbouring binuclear units. The interplays between structure, exchange couplings, magnetic dimension and spin dynamics in the binuclear compound are discussed. A previously unreported situation, where the structure of the spectra arising from the anisotropic spin-spin interaction term (D) within the binuclear unit is averaged out, but the forbidden half field transition is not, is observed and explained.

Topics: Copper; Crystallography, X-Ray; Diphosphates; Electron Spin Resonance Spectroscopy; Magnetic Phenomena; Models, Molecular

Herein, we report a new pyridine-biquinoline-derivative fluorophore L for effectively sensing pyrophosphate (PPi) and monohydrogen sulfide (HS(-)) in aqueous buffer and in living cells. L could selectively coordinate with metal ions (M(n+)) in Groups IB and IIB to form L-M(n+) complexes with 1:1 stoichiometry, resulting in fluorescence quenching via photoinduced electron transfer (PET) mechanism. L-Zn(2+) complex was applied to competitively coordinate with PPi to form a new "ate"-type complex, turning on the fluorescence by a 21-fold-increase. The limit of detection (LOD) of this assay for PPi detection in aqueous buffer is 0.85 μM. L-Cu(2+) complex was applied for highly selective detection of HS(-) with an excellent sensitivity by 25-fold decomplexation-induced fluorescence increase. LOD of L-Cu(2+) complex for HS(-) detection in aqueous buffer is 2.24 μM. With the in vitro data obtained, we successfully applied these two complexes for sequential imaging Zn(2+) and PPi, Cu(2+) and HS(-) in living cells, respectively. Since PPi and HS(-) occur in vascular calcification in positive correlation, our multifunctional probe L might help doctors to more precisely diagnose this disease in vivo in the future. For example, we could use radioactive tracer L-(64)Cu for qualitative and quantitative positron emission tomography/computed tomography (PET/CT) imaging of HS(-) in vivo.

Topics: Buffers; Coordination Complexes; Copper; Diphosphates; Fluorescent Dyes; Hep G2 Cells; Humans; Hydrogen Sulfide; Limit of Detection; Molecular Structure; Pyridines; Quinolines; Spectrometry, Fluorescence; Water; Zinc

Phloem loading is a critical process in plant physiology. The potential of regulating the translocation of photoassimilates from source to sink tissues represents an opportunity to increase crop yield. Pyrophosphate homeostasis is crucial for normal phloem function in apoplasmic loaders. The involvement of Arabidopsis (Arabidopsis thaliana) type I proton-pumping pyrophosphatase (AVP1) in phloem loading was analyzed at genetic, histochemical, and physiological levels. A transcriptional AVP1 promoter::GUS fusion revealed phloem activity in source leaves. Ubiquitous AVP1 overexpression (35S::AVP1 cassette) enhanced shoot biomass, photoassimilate production and transport, rhizosphere acidification, and expression of sugar-induced root ion transporter genes (POTASSIUM TRANSPORTER2 [KUP2], NITRATE TRANSPORTER2.1 [NRT2.1], NRT2.4, and PHOSPHATE TRANSPORTER1.4 [PHT1.4]). Phloem-specific AVP1 overexpression (Commelina Yellow Mottle Virus promoter [pCOYMV]::AVP1) elicited similar phenotypes. By contrast, phloem-specific AVP1 knockdown (pCoYMV::RNAiAVP1) resulted in stunted seedlings in sucrose-deprived medium. We also present a promoter mutant avp1-2 (SALK046492) with a 70% reduction of expression that did not show severe growth impairment. Interestingly, AVP1 protein in this mutant is prominent in the phloem. Moreover, expression of an Escherichia coli-soluble pyrophosphatase in the phloem (pCoYMV::pyrophosphatase) of avp1-2 plants resulted in severe dwarf phenotype and abnormal leaf morphology. We conclude that the Proton-Pumping Pyrophosphatase AVP1 localized at the plasma membrane of the sieve element-companion cell complexes functions as a synthase, and that this activity is critical for the maintenance of pyrophosphate homeostasis required for phloem function.

Topics: Arabidopsis; Arabidopsis Proteins; Diphosphates; Gene Expression; Gene Expression Regulation, Plant; Genes, Reporter; Homeostasis; Inorganic Pyrophosphatase; Mutation; Organ Specificity; Phenotype; Phloem; Plant Leaves; Plant Roots; Plant Shoots; Plants, Genetically Modified; Promoter Regions, Genetic; Seedlings; Sucrose

XAD-8 adsorption technique coupled with stepwise elution using pyrophosphate buffers with initial pH values of 3, 5, 7, 9, and 13 was developed to isolate Chinese standard fulvic acid (FA) and then separated the FA into five sub-fractions: FApH3, FApH5, FApH7, FApH9 and FApH13, respectively. Mass percentages of FApH3-FApH13 decreased from 42% to 2.5%, and the recovery ratios ranged from 99.0% to 99.5%. Earlier eluting sub-fractions contained greater proportions of carboxylic groups with greater polarity and molecular mass, and later eluting sub-fractions had greater phenolic and aliphatic content. Protein-like components, as well as amorphous and crystalline poly(methylene)-containing components were enriched using neutral and basic buffers. Three main mechanisms likely affect stepwise elution of humic components from XAD-8 resin with pyrophosphate buffers including: 1) the carboxylic-rich sub-fractions are deprotonated at lower pH values and eluted earlier, while phenolic-rich sub-fractions are deprotonated at greater pH values and eluted later. 2) protein or protein-like components can be desorbed and eluted by use of stepwise elution as progressively greater pH values exceed their isoelectric points. 3) size exclusion affects elution of FA sub-fractions. Successful isolation of FA sub-fractions will benefit exploration of the origin, structure, evolution and the investigation of interactions with environmental contaminants.

Topics: Acrylic Resins; Adsorption; Benzopyrans; Buffers; Chemical Fractionation; China; Diphosphates; Forests; Hydrogen-Ion Concentration; Ion Exchange Resins; Magnetic Resonance Spectroscopy; Molecular Weight; Polystyrenes; Soil; Spectrometry, Fluorescence; Spectroscopy, Fourier Transform Infrared

The importance of DNA polymerases in biology and biotechnology, and their recognition as potential therapeutic targets, drives development of methods for deriving kinetic characteristics of polymerases and their propensity to perform polynucleotide synthesis over modified DNA templates. Among various polymerases, translesion synthesis (TLS) polymerases enable cells to avoid the cytotoxic stalling of replicative DNA polymerases at chemotherapy-induced DNA lesions, thereby leading to drug resistance. Identification of TLS inhibitors to overcome drug-resistance necessitates the development of appropriate high-throughput assays. Since polymerase-mediated DNA synthesis involves the release of inorganic pyrophosphate (PPi), we established a universal and fast method for monitoring the progress of DNA polymerases based on the quantification of PPi with a fluorescence-based assay that we coupled to in vitro primer extension reactions. The established assay has a nanomolar detection limit in PPi and enables the evaluation of single nucleotide incorporation and DNA synthesis progression kinetics. The results demonstrated that the developed assay is a reliable method for monitoring TLS and identifying nucleoside and nucleotide-based TLS inhibitors.

Topics: Diphosphates; DNA; DNA-Directed DNA Polymerase; Enzyme Assays; Fluorescence; Nucleotides; Sulfolobus solfataricus

A hydroxyquinoline functionlized polynorbornene (P1) was designed and synthesized. In an aqueous solution, P1 shows a "turn-on" fluorescence response for Zn(2+) and Cd(2+) with a 50 nm blue shift. Furthermore, both P1-Zn(2+) and P1-Cd(2+) complexes were found to respond to pyrophosphate (PPi) over other important biological anions via a fluorescence quenching effect. P1 is also capable of monitoring intracellular Zn(2+) and PPi in real time.

Topics: Anions; Cadmium; Coordination Complexes; Diphosphates; Hep G2 Cells; Humans; Microscopy, Fluorescence; Plastics; Spectrometry, Fluorescence; Water; Zinc

Amino-functionalized fluorescent carbon dots have been prepared by hydrothermal treatment of glucosamine with excess pyrophosphate. The produced carbon dots showed stabilized green emission fluorescence at various excitation wavelengths and pH environments. Herein, we demonstrate the surface energy transfer between the amino-functionalized carbon dots and negatively charged hyaluronate stabilized gold nanoparticles. Hyaluronidase can degrade hyaluronate and break down the hyaluronate stabilized gold nanoparticles to inhibit the surface energy transfer. The developed fluorescent carbon dot/gold nanoparticle system can be utilized as a biosensor for sensitive and selective detection of hyaluronidase by two modes which include fluorescence measurements and colorimetric analysis.

Topics: Animals; Biosensing Techniques; Carbon; Cattle; Colorimetry; Diphosphates; Gold; Green Fluorescent Proteins; Humans; Hyaluronic Acid; Hyaluronoglucosaminidase; Hydrogen-Ion Concentration; Metal Nanoparticles; Nanotubes, Carbon; Quantum Dots; Quantum Theory; Spectrometry, Fluorescence

Aggressive cancers exhibit an efficient conversion of high amounts of glucose to lactate accompanied by acid secretion, a phenomenon popularly known as the Warburg effect. The acidic microenvironment and the alkaline cytosol create a proton-gradient (acid gradient) across the plasma membrane that represents proton-motive energy. Increasing experimental data from physiological relevant models suggest that acid gradient stimulates tumor proliferation, and can also support its energy needs. However, direct biochemical evidence linking extracellular acid gradient to generation of intracellular ATP are missing. In this work, we demonstrate that cancer cells can synthesize significant amounts of phosphate-bonds from phosphate in response to acid gradient across plasma membrane. The noted phenomenon exists in absence of glycolysis and mitochondrial ATP synthesis, and is unique to cancer. Biochemical assays using viable cancer cells, and purified plasma membrane vesicles utilizing radioactive phosphate, confirmed phosphate-bond synthesis from free phosphate (Pi), and also localization of this activity to the plasma membrane. In addition to ATP, predominant formation of pyrophosphate (PPi) from Pi was also observed when plasma membrane vesicles from cancer cells were subjected to trans-membrane acid gradient. Cancer cytosols were found capable of converting PPi to ATP, and also stimulate ATP synthesis from Pi from the vesicles. Acid gradient created through glucose metabolism by cancer cells, as observed in tumors, also proved critical for phosphate-bond synthesis. In brief, these observations reveal a role of acidic tumor milieu as a potential energy source and may offer a novel therapeutic target.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Cell Line, Tumor; Cell Membrane; Cell Proliferation; Cytosol; Diphosphates; Glucose; Glycolysis; Humans; Hydrogen-Ion Concentration; Ion Transport; Kinetics; Lactic Acid; Phosphates; Phosphorus Radioisotopes; Protons

We presented a novel approach for pyrophosphate (PPi) sensing. Two tetraphenylethene (TPE)-functionalised pyridine salts (TPM and TPH) were designed and synthesized. Both of them exhibited weak emission in the solution state that originates from intramolecular charge transfer (ICT) from TPE to the pyridine; the addition of PPi into the TPM aqueous solution would enhance the fluorescence intensity, which eliminates the emission quenching effect of the iodide ion by the formation of PPi-sensor nanoparticles. The detection limit of TPM was determined to be as low as 133 nM. Meanwhile, a thin solid film of TPM that could detect PPi rapidly was conveniently prepared.

Topics: Chemistry Techniques, Analytical; Diphosphates; Fluorescent Dyes; HeLa Cells; Humans; Limit of Detection; Pyridines; Salts; Spectrometry, Fluorescence; Stilbenes

Human tuberculosis is a chronic infectious disease affecting millions of lives. Because of emerging resistance to current medications, new therapeutic drugs are needed. One potential new target is hypoxanthine-guanine phosphoribosyltransferase (MtHGPRT), a key enzyme of the purine salvage pathway. Here, newly synthesized acyclic nucleoside phosphonates (ANPs) have been shown to be competitive inhibitors of MtHGPRT with Ki values as low as 0.69 μM. Prodrugs of these compounds arrest the growth of a virulent strain of M. tuberculosis with MIC50 values as low as 4.5 μM and possess low cytotoxicity in mammalian cells (CC50 values as high as >300 μM). In addition, the first crystal structures of MtHGPRT (2.03-2.76 Å resolution) have been determined, three of these in complex with novel ANPs and one with GMP and pyrophosphate. These data provide a solid foundation for the further development of ANPs as selective inhibitors of MtHGPRT and as antituberculosis agents.

Topics: Amino Acid Sequence; Antineoplastic Agents; Antitubercular Agents; Catalytic Domain; Cell Proliferation; Crystallography, X-Ray; Diphosphates; Enzyme Inhibitors; Guanosine Monophosphate; Humans; Hypoxanthine Phosphoribosyltransferase; Lung Neoplasms; Models, Molecular; Molecular Sequence Data; Molecular Structure; Mycobacterium tuberculosis; Organophosphonates; Prodrugs; Protein Conformation; Sequence Homology, Amino Acid; Structure-Activity Relationship; Tuberculosis; Tumor Cells, Cultured

In this work, a new fluorescent method for sensitive detection of pyrophosphate anion (P2O7(4-), PPi) in the synovial fluid was developed using fluorophore labeled single-stranded DNA-attached Fe3O4 NPs. The sensing approach is based on the strong affinity of PPi to Fe3O4 NPs and highly efficient fluorescent quenching ability of Fe3O4 NPs for fluorophore labeled single-stranded DNA. In the presence of PPi, the fluorescence would enhance dramatically due to desorption of fluorophore labeled single-stranded DNA from the surface of Fe3O4 NPs, which allowed the analysis of PPi in a very simple manner. The proposed sensing system allows for the sensitive determination of PPi in the range of 2.0 × 10(-7)-4 × 10(-6)M with a detection limit of 76 nM. Importantly, the protocol exhibits excellent selectivity for the determination of PPi over other phosphate-containing compounds. The method was successfully applied to the determination of PPi in the synovial fluid, which suggests our proposed method has great potential for diagnostic purposes.

Topics: Biosensing Techniques; Diphosphates; DNA, Single-Stranded; Fluorescent Dyes; Humans; Immobilized Nucleic Acids; Limit of Detection; Magnetite Nanoparticles; Spectrometry, Fluorescence; Synovial Fluid

A new colorimetric method for monitoring of rolling circle amplification was developed. At first H5N1 target hybrids with padlock probe (PLP) and then PLP is circularized upon the action of T4 ligase enzyme. Subsequently, the circular probe is served as a template for hyperbranched rolling circle amplification (HRCA) by utilizing Bst DNA polymerase enzyme. By improving the reaction, pyrophosphate is produced via DNA polymerization and chelates the Mg(2+) in the buffer solution. This causes change in solution color in the presence of hydroxy naphthol blue (HNB) as a metal indicator. By using pH shock instead of heat shock and isothermal RCA reaction not only the procedure becomes easier, but also application of HNB for colorimetric detection of RCA reaction further simplifies the assay. The responses of the biosensor toward H5N1 were linear in the concentration range from 0.16 to 1.20 pM with a detection limit of 28 fM.

Topics: Animals; Biosensing Techniques; Birds; Colorimetry; Diphosphates; DNA, Complementary; Humans; Influenza A Virus, H5N1 Subtype; Influenza in Birds; Influenza, Human; Limit of Detection; Magnesium; Naphthalenesulfonates; Nucleic Acid Amplification Techniques; RNA, Viral

In muscle food processing, where oxidation is inevitable, phosphates are usually added to improve water binding. This present study attempted to investigate the interactive roles of protein oxidation and pyrophosphate (PP) during thermal gelation of myosin. Myosin isolated from pork muscle was solubilized in 0.5 M NaCl at pH 6.2 then oxidatively stressed with an iron-redox cycling system that produces hydroxyl radicals with or without 1 mM PP and 2 mM MgCl2 at 4 °C for 12 or 24 h then heated to 50 °C at 1.3 °C/min. Protein conformational stability was measured by differential scanning calorimetry, and covalent cross-linking was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis following chymotrypsin digestion. The binding of PP to myosin suppressed disulfide bond formation in myosin subfragments 1 and 2 and partially inhibited oxidation-initiated cross-linking of heavy meromyosin during myosin gelation with a lesser effect on light meromyosin. In the presence of PP, myosin exhibited less loss of conformational integrity upon oxidation than myosin without PP. Rheological analysis from 20 to 75 °C indicated up to 32% decreases (P < 0.05) in elastic modulus (G') of myosin gels due to oxidation. However, the presence of 1 mM PP, which also lowered the gelling capacity of myosin, inhibited the oxidation-induced G' by nearly half (P < 0.05). These results suggest that the protection of myosin head from oxidative modification by PP can be a significant factor for the minimization of gelling property losses during cooking of comminuted meats.. The association of myosin S1 subfragment possibly S2 as well with pyrophosphate prevents extensive myosin head–head cross-linking. This alleviates the negative impact of oxidation on the gel formation of myosin, an expected main form of myofibrillar protein due to pyrophosphate-induced actomyosin dissociation. Likewise, oxidative S1–S1 association inhibits the binding of pyrophosphate thereby reducing its gel-weakening effect. The mutual constraining roles of oxidation and pyrophosphate can be a significant factor for the minimization of gelling property losses during the manufacture of comminuted meat products.

Topics: Animals; Calorimetry, Differential Scanning; Chymotrypsin; Diphosphates; Electrophoresis, Polyacrylamide Gel; Food Handling; Gels; Hydroxyl Radical; Magnesium Chloride; Meat; Muscle, Skeletal; Myosin Subfragments; Myosins; Oxidation-Reduction; Oxygen; Phosphates; Rheology; Swine; Temperature

An easy-to-make salicylimine (L) bearing an "O-N-O"-coordination site was used as a highly selective fluorescent sensor for Al(3+) and PPi in aqueous solution. Sensor L showed a significant fluorescence enhancement in the presence of Al(3+) over other competitive metal ions. It works based on the Al(3+)-induced formation of a 1 : 1 L-Al(3+) complex, producing a chelation-enhanced fluorescence effect, the fluorescence quantum yield reached 0.59. This L-Al(3+) ensemble is a subsequent fluorescent sensor for PPi due to the strong attraction between Al(3+) and PPi, it can selectively discriminate PPi overcoming the interference of the biological competitors including PO4(3-), ADP and ATP at physiological pH. L and L-Al(3+) exhibit high sensitivity and selectivity for Al(3+) and PPi, the detection limits were found to be as low as 2.94 × 10(-8) M and 2.74 × 10(-7) M, respectively. It was further confirmed that sensor L had potential practical applications through mapping of Al(3+) in live cells.

Topics: Aluminum; Coordination Complexes; Diphosphates; Fluorescent Dyes; HeLa Cells; Humans; Imines; Microscopy, Fluorescence

We have identified a phosphate transporter (TcPho91) localized to the bladder of the contractile vacuole complex (CVC) of Trypanosoma cruzi, the etiologic agent of Chagas disease. TcPho91 has 12 transmembrane domains, an N-terminal regulatory SPX (named after SYG1, Pho81 and XPR1) domain and an anion permease domain. Functional expression in Xenopus laevis oocytes followed by two-electrode voltage clamp showed that TcPho91 is a low-affinity transporter with a Km for Pi in the millimolar range, and sodium-dependency. Epimastigotes overexpressing TcPho91-green fluorescent protein have significantly higher levels of pyrophosphate (PPi ) and short-chain polyphosphate (polyP), suggesting accumulation of Pi in these cells. Moreover, when overexpressing parasites were maintained in a medium with low Pi , they grew at higher rates than control parasites. Only one allele of TcPho91 in the CL strain encodes for the complete open reading frame, while the other one is truncated encoding for only the N-terminal domain. Taking advantage of this characteristic, knockdown experiments were performed resulting in cells with reduced growth rate as well as a reduction in PPi and short-chain polyP levels. Our results indicate that TcPho91 is a phosphate sodium symporter involved in Pi homeostasis in T. cruzi.

Topics: Animals; Diphosphates; Gene Knockdown Techniques; Green Fluorescent Proteins; Homeostasis; Phosphates; Polyphosphates; Protozoan Proteins; Sodium; Symporters; Trypanosoma cruzi; Vacuoles; Xenopus laevis

We previously reported that oxidized low density lipoprotein (oxLDL) accelerated the calcification in aorta of rats and rat vascular smooth muscle cells (RVSMCs). However, the molecular mechanism underlying the acceleration remains poorly understood. The present study aimed to investigate the role of calpain-1, Ca2+-sensitive intracellular cysteine proteases, in the vascular calcification of rats treated with both high dose of vitamin D2 and high cholesterol diet. The results showed that calpain activity significantly increased in calcified aortic tissue of rats and RVSMCs treated with oxLDL. Specific calpain inhibitor I (CAI, 0.5mg/kg, intraperitoneal) inhibited the vascular calcification in rats with hypercholesterolemia accompanied by the increase in the level of extracellular inorganic pyrophosphate (PPi), the endogenous inhibitor of vascular calcification. In addition, CAI increased the content of adenosine triphosphate (ATP), decreased the activity, mRNA and protein expression of alkaline phosphatase (ALP) and reduced the production of superoxide anion in calcified aortic tissue. CAI also increased the activity of ATP synthase as well as protein expression of ATP5D, δ subunit of ATP synthase. In the in vitro study, suppression of calpain-1 using siRNA assay inhibited the calcium deposition, increased the levels of PPi and ATP, improved the activity of ATP synthase as well as protein expression of ATP5D in RVSMCs treated with oxLDL. Calpain-1 suppression also decreased the activity, mRNA and protein expression of ALP and reduced the mitochondrial ROS (Mito-ROS) production in RVSMCs. However, mito-TEMPO, the mitochondria-targeted ROS scavenger, reduced the calcium deposition, increased the PPi in culture medium, decreased the activity, mRNA and protein expression of ALP in RVSMCs treated with oxLDL. Taken together, the results suggested that calpain-1 activation plays critical role in vascular calcification caused by oxLDL, which might be mediated by PPi metabolism disorder. The results also implied that Mito-ROS might contribute to the PPi metabolism disorder through regulation of the activity and expression of ALP.

Topics: Animals; Aorta; Calcium; Calpain; Cell Line; Diphosphates; Glycoproteins; Hypercholesterolemia; Lipoproteins, LDL; Male; Mitochondria; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Proton-Translocating ATPases; Rats; Rats, Sprague-Dawley; Vascular Calcification

The three dimetallic compounds [Ga2(bpbp)(OH)2(H2O)2](ClO4)3, [In2(bpbp)(CH3CO2)2](ClO4)3 and [Zn2(bpbp)(HCO2)2](ClO4) (bpbp(-) = 2,6-bis((N,N'-bis(2-picolyl)amino)methyl)-4-tertbutylphenolate) were evaluated as stable solid state precursors for reactive solution state receptors to use for the recognition of the biologically important anion pyrophosphate in water at neutral pH. Indicator displacement assays using in situ generated complex-pyrocatechol violet adducts, {M2(bpbp)(HxPV)}(n+) M = Ga(3+), In(3+), Zn(2+), were tested for selectivity in their reactions with a series of common anions: pyrophosphate, phosphate, ATP, arsenate, nitrate, perchlorate, chloride, sulfate, formate, carbonate and acetate. The receptor employing Ga(3+) showed a slow but visually detectable response (blue to yellow) in the presence of one equivalent of pyrophosphate but no response to any other anion, even when they were present in much higher concentrations. The systems based on In(3+) or Zn(2+) show less selectivity in accord with visibly discernible responses to several of the anions. These results demonstrate a facile method for increasing anion selectivity without modification of an organic dinucleating ligand scaffold. The comfortable supramolecular recognition of pyrophosphate by the dimetallic complexes is demonstrated by the single crystal X-ray structure of [Ga2(bpbp)(HP2O7)](ClO4)2 in which the pyrophosphate is coordinated to the two gallium ions via four of its oxygen atoms.

Topics: Colorimetry; Crystallography, X-Ray; Diphosphates; Gallium; Indium; Ligands; Models, Molecular; Organometallic Compounds; Spectrophotometry, Ultraviolet; Water; Zinc

A series of linear peptide based anion receptors, in which the distance between the bis[zinc(II)dipicolylamine] binding sites and the peptide backbone was varied systematically, was prepared and their anion binding ability was investigated using indicator displacement assays. Shortening the distance between the binding site and the peptide backbone was found to enhance both the receptor affinity and selectivity for pyrophosphate over other organic polyphosphate anions in Krebs buffer with the maximum selectivity and affinity observed with a spacer length of two methylene units. The suitability of these receptors for the determination of pyrophosphate concentrations in Krebs buffer and in artificial urine was examined.

Topics: Diphosphates; Molecular Structure; Organometallic Compounds; Peptides; Picolinic Acids; Quantum Theory; Spectrophotometry, Ultraviolet

An anthracene-bridged dinuclear zinc(ii)-dipicolylamine complex was found to show high selectivity for ADP with a significant fluorescence enhancement over ATP, PPi and other common analytes in 100% aqueous solution. This complex can be used for fluorescence detection of ADP in living cells and for monitoring the activity of kinases.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Anthracenes; Coordination Complexes; Creatine Kinase; Crystallography, X-Ray; Diphosphates; HeLa Cells; Humans; Microscopy, Fluorescence; Molecular Conformation; Spectrometry, Fluorescence; Water; Zinc

A chemiluminescent method is proposed for quantitation of NO generation in cell cultures. The method is based on activation of soluble guanylyl cyclase by NO. The product of the guanylyl cyclase reaction, pyrophosphate, is converted to ATP by ATP sulfurylase and ATP is detected in a luciferin-luciferase system. The method has been applied to the measurement of NO generated by activated murine macrophages (RAW 264.7) and bovine aortic endothelial cells. For macrophages activated by lipopolysaccharide and γ-interferon, the rate of NO production is about 100 amol/(cell·min). The rate was confirmed by the measurements of nitrite, the product of NO oxidation. For endothelial cells, the basal rate of NO generation is 5 amol/(cell·min); the rate approximately doubles upon activation by bradykinin, Ca(2+) ionophore A23187 or mechanical stress. For both types of cells the measured rate of NO generation is strongly affected by inhibitors of NO synthase. The sensitivity of the method is about 50 pM/min, allowing the registration of NO generated by 10(2)-10(4) cells. The enzyme-linked chemiluminescent method is two orders of magnitude more sensitive than fluorescent detection using 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM).

Topics: Animals; Aorta; Biological Assay; Bradykinin; Cattle; Cell Line; Cyclic GMP; Diphosphates; Endothelial Cells; Firefly Luciferin; Guanosine Triphosphate; Guanylate Cyclase; Lipopolysaccharides; Luciferases; Luminescence; Luminescent Measurements; Macrophages; Mice; Nitric Oxide; Nitric Oxide Donors; Nitrites; Nitroso Compounds; Receptors, Cytoplasmic and Nuclear; Sensitivity and Specificity; Soluble Guanylyl Cyclase; Sulfate Adenylyltransferase

Family I inorganic pyrophosphatases (PPiases) are ubiquitous enzymes that are critical for phosphate metabolism in all domains of life. The detailed catalytic mechanism of these enzymes, including the identity of the general base, is not fully understood. We determined a series of crystal structures of the PPiase from Mycobacterium tuberculosis (Mtb PPiase) bound to catalytic metals, inorganic pyrophosphate (PPi; the reaction substrate) and to one or two inorganic phosphate ions (Pi; the reaction product), ranging in resolution from 1.85 to 3.30Å. These structures represent a set of major kinetic intermediates in the catalytic turnover pathway for this enzyme and suggest an order of association and dissociation of the divalent metals, the substrate and the two products during the catalytic turnover. The active site of Mtb PPiase exhibits significant structural differences from the well characterized Escherichia coli PPiase in the vicinity of the bound PPi substrate. Prompted by these differences, quantum mechanics/molecular mechanics (QM/MM) analysis yielded an atomic description of the hydrolysis step for Mtb PPiase and, unexpectedly, indicated that Asp89, rather than Asp54 that was proposed for E. coli PPiase, can abstract a proton from a water molecule to activate it for a nucleophilic attack on the PPi substrate. Mutagenesis studies of the key Asp residues of Mtb PPiase supported this mechanism. This combination of structural and computational analyses clarifies our understanding of the mechanism of family I PPiases and has potential utility for rational development of drugs targeting this enzyme.

Topics: Bacterial Proteins; Biocatalysis; Calcium; Catalytic Domain; Diphosphates; Escherichia coli; Hydrogen Bonding; Hydrolysis; Inorganic Pyrophosphatase; Kinetics; Molecular Dynamics Simulation; Mycobacterium tuberculosis; Protein Binding; Protein Structure, Secondary; Structural Homology, Protein

Highly fluorescent nitrogen-doped graphene quantum dots (N-GQDs) with greenish-yellow emission and quantum yield of 13.2% have been synthesized via a one-pot hydrothermal method. The obtained N-GQDs displayed excellent optical properties, high photostability and resistance to strong ion strength. Based on the higher affinity of pyrophosphate (PPi) than carboxyl and amido groups on the surface of the N-GQDs to Eu(3+), a Eu(3+)-modulated N-GQD off-on fluorescent probe for PPi detection was constructed with a detection limit of 0.074 μM. The detection process was simple in design, easy to operate, and showed a highly selective response to PPi in the presence of co-existing anions. This work widens the applications of N-GQDs with versatile functionality and reactivity in clinical diagnostics and as biosensors.

Topics: Biosensing Techniques; Cations; Diphosphates; Europium; Fluorescent Dyes; Graphite; Humans; Limit of Detection; Nitrogen; Quantum Dots

Lignin is a complex polymer that is a potential feedstock for aromatic compounds and carboxylic acids by cleaving the β-O-4 and 5-5' linkages. In this work, a syringe pump atomizes an alkaline solution of lignin into a catalytic fluidized bed operating above 600 K. The vanadium heterogeneous catalysts convert all the lignin into carboxylic acids (up to 25 % selectivity), coke, carbon oxides, and hydrogen. Aluminum-vanadium-molybdenum mostly produced lactic acid (together with formic acid, acrylic acid, and maleic anhydride), whereas the vanadium pyrophosphate catalyst produced more maleic anhydride.

Topics: Aluminum; Carboxylic Acids; Diphosphates; Gases; Lignin; Molybdenum; Oxidation-Reduction; Vanadium

The dynamic hydrolysis of tetrasodium pyrophosphate (TSPP), sodium tripolyphosphate (STPP) and polyphosphate compound, which was catalyzed by purified pyrophosphatase (PPase) and myosin- tripolyphosphatase (TPPase) from the silver carp dorsal muscle, was studied using (31) P NMR spectroscopy. In the PPase + TSPP system, the pyrophosphate (PP) was hydrolyzed quickly and completely within 8 h and the hydrolysis rate of PP was 12.51%/h. In the TPPase + STPP system, the first-order hydrolysis of tripolyphosphate (TPP) was not yet complete after 48 h, and the derived PP accumulated progressively. Given the coexistence of PPase and TPPase, only 1.20% of TPP in STPP alone remained after 48 h. However, the generation rate of Pi in the polyphosphate compound (TSPP: STPP: sodium hexametaphosphate = 1: 8: 1) was 0.76%/h, which was less than 0.88%/h in STPP alone. In the presence of polyphosphatases, the decrease of PP or TPP content in the polyphosphate compound was not as rapid as that in TSPP or STPP alone due to the inhibitory effect of PP on TPPase and the effect of low system pH on PPase. The understanding of polyphosphates hydrolysis mechanism was capable of developing the advanced polyphosphate mixture in order to reduce the phosphate residue in fish products.. Processors appreciate the proven value of phosphates to increase the yield and functionality of the fish meat products. Our studies showed that the hydrolysis rate of PP or TPP in the blend was slower than that of polyphosphate alone. Thus, it is likely that the addition of PP and TPP in a polyphosphate blend had a prolonged interaction with proteins in fish meat processing and the effectiveness of polyphosphates was enhanced.

Topics: Acid Anhydride Hydrolases; Animals; Carps; Diphosphates; Fish Products; Food Additives; Hydrogen-Ion Concentration; Hydrolysis; Magnetic Resonance Imaging; Magnetic Resonance Spectroscopy; Meat; Muscle, Skeletal; Myosins; Phosphates; Polyphosphates

Aberrant mineralization of soft connective tissues (ectopic calcification) may occur as a frequent age-related complication. Still, it remains unclear the role of mesenchymal cell donor's age and of replicative senescence on ectopic calcification. Therefore, the ability of cells to deposit in-vitro hydroxyapatite crystals and the expression of progressive ankylosis protein homolog (ANKH), ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), tissue non specific alkaline phosphatase (TNAP) and osteopontin (OPN) have been evaluated in human dermal fibroblasts derived from neonatal (nHDF) and adult (aHDF) donors (ex-vivo ageing model) or at low and high cumulative population doublings (CPD) up to replicative senescence (in-vitro ageing model). This study demonstrates that: 1) replicative senescence favours hydroxyapatite formation in cultured fibroblasts; 2) donor's age acts as a major modulator of the mineralizing potential of HDF, since nHDF are less prone than aHDF to induce calcification; 3) donor's age and replicative senescence play in concert synergistically increasing the calcification process; 4) the ANKH+ENPP1/TNAP ratio, being crucial for pyrophosphate/inorganic phosphate balance, is greatly influenced by donor's age, as well as by replicative senescence, and regulates mineral deposition; 5) OPN is only modulated by replicative senescence.

Topics: Adult; Age Factors; Alkaline Phosphatase; Calcification, Physiologic; Cells, Cultured; Cellular Senescence; Diphosphates; Fibroblasts; Humans; Infant, Newborn; Osteopontin; Phosphates; Phosphoric Diester Hydrolases; Pyrophosphatases

Pyrophosphate ion (PPi) release during transcription elongation is a signature step in each nucleotide addition cycle. The kinetics and energetics of the process as well as how it proceeds with substantial conformational changes of the polymerase complex determine the mechano-chemical coupling mechanism of the transcription elongation. Here we investigated detailed dynamics of the PPi release process in a single-subunit RNA polymerase (RNAP) from bacteriophage T7, implementing all-atom molecular dynamics (MD) simulations. We obtained a jump-from-cavity kinetic model of the PPi release utilizing extensive nanosecond MD simulations. We found that the PPi release in T7 RNAP is initiated by the PPi dissociation from two catalytic aspartic acids, followed by a comparatively slow jump-from-cavity activation process. Combining with a number of microsecond long MD simulations, we also found that the activation process is hindered by charged residue associations as well as by local steric and hydrogen bond interactions. On the other hand, the activation is greatly assisted by a highly flexible lysine residue Lys472 that swings its side chain to pull PPi out. The mechanism can apply in general to single subunit RNA and DNA polymerases with similar molecular structures and conserved key residues. Remarkably, the flexible lysine or arginine residue appears to be a universal module that assists the PPi release even in multi-subunit RNAPs with charge facilitated hopping mechanisms. We also noticed that the PPi release is not tightly coupled to opening motions of an O-helix on the fingers domain of T7 RNAP according to the microsecond MD simulations. Our study thus supports the Brownian ratchet scenario of the mechano-chemical coupling in the transcription elongation of the single-subunit polymerase.

Topics: Amino Acid Sequence; Computational Biology; Diphosphates; DNA-Directed RNA Polymerases; Lysine; Molecular Dynamics Simulation; Molecular Sequence Data; Promoter Regions, Genetic; Sequence Alignment; Viral Proteins

Most main oilfields in China have already entered a "double high" development stage (high water cut, high recovery degree). To further enhance oil recovery in reservoirs after polymer flooding (RAPFs), an efficient activator formulation for promoting metabolism of endogenous microorganism was studied by aerogenic experiments, physical simulation experiments, electron microscopy scanning and pyrophosphate sequencing. Results show that the activator could activate the endogenous microorganisms in the injected water and make the pressurized gas reach 2 MPa after 60 d static culture of the activator in a high pressure vessel. The oil recovery efficiency of natural core physical simulation flooding can be improved by more than 3.0% (OOIP) in RAPFs when injected 0.35 PV activator with 1.8% mass concentration, and a lot of growth and reproduction of activated endogenous microorganism in the core was observed by electron microscopy scanning. Field trial with 1 injector and 4 producers was carried out in the east of south II block of Sa Nan in December 2011. By monitoring four effective production wells, changes of carbon isotope δ13C (PDB) content of methane and carbon dioxide were -45 per thousand to -54 per thousand and 7 per thousand to 12 per thousand. Compared with east II of Sa Nan block, the oil amount increased by 35.9%, water cut stabled at 94%. The incremental oil was 5 957 t during the three and a half years, which provides an alternative approach for further improving oil recovery in similar reservoirs.

Topics: Carbon Dioxide; Carbon Isotopes; China; Diphosphates; Methane; Oil and Gas Fields; Polymers; Water; Water Microbiology

Examination of InhA mutants I16T, I21V, I47T, S94A, and I95P showed that direct and water mediated H-bond interactions between NADH and binding site residues reduced drastically. It allowed conformational flexibility to NADH, particularly at the pyrophosphate region, leading to weakening of its binding at dinucleotide binding site. The highly scattered distribution of pyrophosphate dihedral angles and chi1 side chain dihedral angles of corresponding active site residues therein confirmed weak bonding between InhA and NADH. The average direct and water mediated bridged H-bond interactions between NADH and mutants were observed weaker as compared to wild type. Further, estimated NADH binding free energy in mutants supported the observed weakening of InhA-NADH interactions. Similarly, per residue contribution to NADH binding was also found little less as compared to corresponding residues in wild type. This investigation clearly depicted and supported the effect of mutations on NADH binding and can be accounted for isoniazid resistance as suggested by previous biochemical and mutagenic studies. Further, structural analysis of InhA provided the crucial points to enhance the NADH binding affinity towards InhA mutants in the presence of direct InhA inhibitors to combat isoniazid drug resistance. This combination could be a potential alternative for treatment of drug resistant tuberculosis.

Topics: Amino Acid Substitution; Antitubercular Agents; Bacterial Proteins; Binding Sites; Crystallography, X-Ray; Diphosphates; Drug Resistance, Bacterial; Gene Expression; Hydrogen Bonding; Isoniazid; Kinetics; Ligands; Molecular Dynamics Simulation; Mutation; Mycobacterium tuberculosis; NAD; Oxidoreductases; Protein Binding; Thermodynamics; Water

Due to a fast setting reaction, good biological properties, and easily available starting materials, there has been extensive research within the field of brushite cements as bone replacing material. However, the fast setting of brushite cement gives them intrinsically low mechanical properties due to the poor crystal compaction during setting. To improve this, many additives such as citric acid, pyrophosphates, and glycolic acid have been added to the cement paste to retard the crystal growth. Furthermore, the incorporation of a filler material could improve the mechanical properties when used in the correct amounts. In this study, the effect of the addition of the two retardants, disodium dihydrogen pyrophosphate and citric acid, together with the addition of β-TCP filler particles, on the mechanical properties of a brushite cement was investigated. The results showed that the addition of low amounts of a filler (up to 10%) can have large effects on the mechanical properties. Furthermore, the addition of citric acid to the liquid phase makes it possible to use lower liquid-to-powder ratios (L/P), which strongly affects the strength of the cements. The maximal compressive strength (41.8MPa) was found for a composition with a molar ratio of 45:55 between monocalcium phosphate monohydrate and beta-tricalcium phosphate, an L/P of 0.25ml/g and a citric acid concentration of 0.5M in the liquid phase.

Topics: Bone Cements; Calcium Phosphates; Citric Acid; Diphosphates; Materials Testing; Mechanical Phenomena; Particle Size; Porosity; Structure-Activity Relationship; X-Ray Diffraction

Medial arterial calcification is accelerated in patients with CKD and strongly associated with increased arterial rigidity and cardiovascular mortality. Recently, a novel in vitro blood test that provides an overall measure of calcification propensity by monitoring the maturation time (T50) of calciprotein particles in serum was described. We used this test to measure serum T50 in a prospective cohort of 184 patients with stages 3 and 4 CKD, with a median of 5.3 years of follow-up. At baseline, the major determinants of serum calcification propensity included higher serum phosphate, ionized calcium, increased bone osteoclastic activity, and lower free fetuin-A, plasma pyrophosphate, and albumin concentrations, which accounted for 49% of the variation in this parameter. Increased serum calcification propensity at baseline independently associated with aortic pulse wave velocity in the complete cohort and progressive aortic stiffening over 30 months in a subgroup of 93 patients. After adjustment for demographic, renal, cardiovascular, and biochemical covariates, including serum phosphate, risk of death among patients in the lowest T50 tertile was more than two times the risk among patients in the highest T50 tertile (adjusted hazard ratio, 2.2; 95% confidence interval, 1.1 to 5.4; P=0.04). This effect was lost, however, after additional adjustment for aortic stiffness, suggesting a shared causal pathway. Longitudinally, serum calcification propensity measurements remained temporally stable (intraclass correlation=0.81). These results suggest that serum T50 may be helpful as a biomarker in designing methods to improve defenses against vascular calcification.

Topics: Aged; Aged, 80 and over; alpha-2-HS-Glycoprotein; Arteriosclerosis; Biomarkers; Calcinosis; Calcium Phosphates; Cardiovascular Diseases; Causality; Comorbidity; Diabetes Mellitus; Diphosphates; Disease Susceptibility; Female; Follow-Up Studies; Humans; Hypertension; Male; Middle Aged; Mortality; Osteoclasts; Phosphates; Prospective Studies; Pulse Wave Analysis; Renal Dialysis; Renal Insufficiency, Chronic; Risk; Serum Albumin; Smoking; Vascular Resistance

The evaluation of enzyme activities, especially their capacities, represents an important step towards the modelling of biochemical pathways in living organisms. The implementation of microplate technology enables the determination of up to >50 enzymes in relatively large numbers of samples and in various biological materials. Most of these enzymes are involved in central metabolism and several pathways are entirely covered. Direct or indirect assays can be used, as well as highly sensitive assays, depending on the abundance of the enzymes under study. To exemplify such methods, protocols for UDP-glucose pyrophosphorylase (E.C. 2.7.7.9) operating in real time and for pyrophosphate:fructose-6-phosphate 1-phosphotransferase (E.C. 2.7.1.90) are presented.

Topics: Diphosphates; Enzyme Assays; Kinetics; Phosphofructokinase-1; Plant Proteins; Plants; Reference Standards; Solutions; UTP-Glucose-1-Phosphate Uridylyltransferase

An enzymatic assay for L-methionine was developed by coupling adenosylmethionine synthetase (AdoMetS) to a pyrophosphate (PP(i)) detection system, which was constructed using pyruvate, phosphate dikinase. To expand the use of this assay, the PP(i) detection system was embodied as three different forms, which allowed PP(i) to be measured by UV, visible, and fluorescent light detectors. The assay system was robust and could tolerate the addition of inorganic phosphate and ATP to the assay mixtures. L-Methionine could be accurately determined by coupling the PP(i) detection system and AdoMetS. This AdoMetS coupling assay was highly selective to L-methionine and exhibited no significant activity to other proteinaceous amino acids, ammonia, or urea, unlike conventional enzymatic assays for L-methionine. Spike and recovery tests showed that the AdoMetS assay could accurately and reproducibly determine increases in L-methionine in human plasma samples without any pretreatment to remove proteins and potentially interfering low-molecular-weight molecules. The high selectivity and robustness of the AdoMetS assay provide rapid and high-throughput analysis of L-methionine in various kinds of analytes.

Topics: Adenosine Triphosphate; Biosensing Techniques; Diphosphates; Humans; Methionine; Methionine Adenosyltransferase; Propionibacterium; Pyruvate, Orthophosphate Dikinase; Time Factors

The stability of UO2 is critical to the success of reductive bioremediation of uranium. When reducing conditions are no longer maintained, Mn redox cycling may catalytically mediate the oxidation of UO2 and remobilization of uranium. Ligand-stabilized soluble Mn(III) was recently recognized as an important redox-active intermediate in Mn biogeochemical cycling. This study evaluated the kinetics of oxidative UO2 dissolution by soluble Mn(III) stabilized by pyrophosphate (PP) and desferrioxamine B (DFOB). The Mn(III)-PP complex was a potent oxidant that induced rapid UO2 dissolution at a rate higher than that by a comparable concentration of dissolved O2. However, the Mn(III)-DFOB complex was not able to induce oxidative dissolution of UO2. The ability of Mn(III) complexes to oxidize UO2 was probably determined by whether the coordination of Mn(III) with ligands allowed the attachment of the complexes to the UO2 surface to facilitate electron transfer. Systematic investigation into the kinetics of UO2 oxidative dissolution by the Mn(III)-PP complex suggested that Mn(III) could directly oxidize UO2 without involving particulate Mn species (e.g., MnO2). The expected 2:1 reaction stoichiometry between Mn(III) and UO2 was observed. The reactivity of soluble Mn(III) in oxidizing UO2 was higher at lower ratios of pyrophosphate to Mn(III) and lower pH, which is probably related to differences in the ligand-to-metal ratio and/or protonation states of the Mn(III)-pyrophosphate complexes. Disproportionation of Mn(III)-PP occurred at pH 9.0, and the oxidation of UO2 was then driven by both MnO2 and soluble Mn(III). Kinetic models were derived that provided excellent fits of the experimental results.

Topics: Deferoxamine; Diphosphates; Hydrogen-Ion Concentration; Kinetics; Ligands; Manganese; Oxidants; Oxidation-Reduction; Radioactive Pollutants; Solubility; Uranium Compounds

Several ecto-enzymatic activities have been described in the plasma membrane of the protozoan Leishmania amazonensis, which is the major etiological agent of diffuse cutaneous leishmaniasis in South America. These enzymes, including ecto-phosphatases, contribute to the survival of the parasite by participating in phosphate metabolism. This work identifies and characterizes the extracellular hydrolysis of inorganic pyrophosphate related to an ecto-pyrophosphatase activity of the promastigote form of L. amazonensis. This ecto-pyrophosphatase activity is insensitive to MnCl2 but is strongly stimulated by MgCl2. This stimulation was not observed during the hydrolysis of p-nitrophenyl phosphate (p-NPP) or β-glycerophosphate, two substrates for different ecto-phosphatases present in the L. amazonensis plasma membrane. Furthermore, extracellular PPi hydrolysis is more efficient at alkaline pHs, while p-NPP hydrolysis occurs mainly at acidic pHs. These results led us to conclude that extracellular PPi is hydrolyzed not by non-specific ecto-phosphatases but rather by a genuine ecto-pyrophosphatase. In the presence of 5mM MgCl2, the ecto-pyrophosphatase activity from L. amazonensis is sensitive to micromolar concentrations of NaF and millimolar concentrations of CaCl2. Moreover, this activity is significantly higher during the first days of L. amazonensis culture, which suggests a possible role for this enzyme in parasite growth.

Topics: Animals; Calcium Chloride; Cell Membrane; Chlorides; Cricetinae; Diphosphates; Dose-Response Relationship, Drug; Humans; Hydrogen-Ion Concentration; Hydrolysis; Leishmania mexicana; Leishmaniasis, Diffuse Cutaneous; Levamisole; Magnesium Chloride; Manganese Compounds; Pyrophosphatases; Sodium Fluoride; Tartrates; Vanadates

Ionically crosslinked chitosan particles with submicron dimensions attract widespread interest as materials for controlled release. To this end, we have examined the formation and dissolution of nanoparticles prepared by crosslinking chitosan with pyrophosphate (PPi). The formation of these particles required a critical PPi concentration (which increased with the chitosan concentration), and their z-average hydrodynamic diameters could be predictably tuned from roughly 60 to 220 nm by varying the concentration of the parent chitosan solutions. Unlike the nanoparticles crosslinked with the commonly used tripolyphosphate (TPP), which coagulated and precipitated when TPP was in excess, the chitosan/PPi nanoparticles remained colloidally stable even at high PPi concentrations. Moreover, the analysis of their dissolution revealed hysteresis in the particle formation/dissolution cycle, where portions of the crosslinked chitosan/PPi complexes remained stably intact at PPi concentrations below those required for their formation. This irreversible behavior was surmised to reflect the cooperativity of chitosan/PPi binding and was qualitatively modeled using the Bragg-Williams theory.

Topics: Chitosan; Colloids; Cross-Linking Reagents; Diphosphates; Hydrodynamics; Ions; Kinetics; Light; Models, Theoretical; Nanoparticles; Particle Size; Polyphosphates; Scattering, Radiation; Solubility; Solutions

A fluorescence sensing system for inorganic pyrophosphate based on ligand exchange of the Ce(III) complex of a xanthene-type probe is developed. This sensing system is successfully applied to the fluorescence detection of polymerase-catalyzed DNA amplification using loop-mediated isothermal amplification.

Topics: Cerium; Coordination Complexes; Diphosphates; DNA; DNA-Directed DNA Polymerase; Ligands; Molecular Probes; Nucleic Acid Amplification Techniques; Spectrometry, Fluorescence; Xanthenes

Vascular calcification (VC) is a risk factor for cardiovascular mortality in the setting of chronic kidney disease (CKD). Pyrophosphate (PPi), an endogenous molecule that inhibits hydroxyapatite crystal formation, has been shown to prevent the development of VC in animal models of CKD. However, the possibility of harmful effects of exogenous administration of PPi on bone requires further investigation. To this end, we examined by histomorphometry the bone of CKD mice after intraperitoneal PPi administration. After CKD creation or sham surgery, 10-week-old female apolipoprotein-E knockout (apoE(-/-)) mice were randomized to one non-CKD group or 4 CKD groups (n = 10-35/group) treated with placebo or three distinct doses of PPi, and fed with standard diet. Eight weeks later, the animals were killed. Serum and femurs were sampled. Femurs were processed for bone histomorphometry. Placebo-treated CKD mice had significantly higher values of osteoid volume, osteoid surface and bone formation rate than sham-placebo mice with normal renal function. Slightly higher osteoid values were observed in CKD mice in response to very low PPi dose (OV/BV, O.Th and ObS/BS) and, for one parameter measured, to high PPi dose (O.Th), compared to placebo-treated CKD mice. Treatment with PPi did not modify any other structural parameters. Mineral apposition rates, and other parameters of bone formation and resorption were not significantly different among the treated animal groups or control CKD placebo group. In conclusion, PPi does not appear to be deleterious to bone tissue in apoE(-/-) mice with CKD, although a possible stimulatory PPi effect on osteoid formation may be worth further investigation.

Topics: Animals; Apolipoproteins E; Bone Density; Dialysis Solutions; Diphosphates; Female; Femur; Mice; Mice, Knockout; Peritoneal Dialysis; Renal Insufficiency, Chronic; Vascular Calcification

Pseudoxanthoma elasticum (PXE) is a genetic disorder characterized by progressive calcification of soft connective tissues. The pathogenesis is still hard to pin down. In PXE dermal fibroblasts, in addition to impaired carboxylation of the vitamin K-dependent inhibitor matrix Gla protein (MGP), we have also demonstrated an up-regulation of alkaline phosphatase activity. In the light of these data we have suggested that both calcium and phosphate metabolism might be locally altered, both pathways acting in synergy on the occurrence of matrix calcification.. This study aims to better explore if cultured PXE fibroblasts, compared to control cells, exhibit a modified inorganic pyrophosphate (PPi) metabolism and are more responsive to pro-calcifying stimuli.. Primary human dermal fibroblasts isolated from healthy individuals and from PXE patients were cultured for different time points in standard and in pro-calcifying media. The expression of ANKH/ANKH, ENPP1/PC1, ALPL/TNAP, SPP1/OPN was evaluated by qRT-PCR and Western blot, respectively. TNAP activity was measured by spectrophotometric analyses, whereas calcification was investigated by light and electron microscopy as well as by micro-analytical techniques.. In the presence of pro-calcifying stimuli, dermal fibroblasts alter their phenotype favouring matrix mineralization. In particular, ENPP1/PC1 and SPP1/OPN expression, as well as TNAP activity, was differently expressed in control and in PXE fibroblasts. Moreover, in pathologic cells the ratio between factors favouring and reducing PPi availability exhibits a more pronounced shift towards a pro-calcifying balance.. PXE fibroblasts are more susceptible to pro-calcifying stimuli and in these cells an altered PPi metabolism contributes to matrix calcification.

Topics: Adult; Alkaline Phosphatase; Calcinosis; Calcium; Calcium-Binding Proteins; Diphosphates; DNA Primers; Extracellular Matrix Proteins; Female; Fibroblasts; Humans; Matrix Gla Protein; Microscopy, Electron, Scanning; Middle Aged; Pseudoxanthoma Elasticum; Skin; Spectrometry, X-Ray Emission; Up-Regulation

In eukaryotes, the tRNA(His) guanylyltransferase (Thg1) catalyzes 3'-5' addition of a single guanosine residue to the -1 position (G-1) of tRNA(His), across from a highly conserved adenosine at position 73 (A73). After addition of G-1, Thg1 removes pyrophosphate from the tRNA 5'-end, generating 5'-monophosphorylated G-1-containing tRNA. The presence of the 5'-monophosphorylated G-1 residue is important for recognition of tRNA(His) by its cognate histidyl-tRNA synthetase. In addition to the single-G-1 addition reaction, Thg1 polymerizes multiple G residues to the 5'-end of tRNA(His) variants. For 3'-5' polymerization, Thg1 uses the 3'-end of the tRNA(His) acceptor stem as a template. The mechanism of reverse polymerization is presumed to involve nucleophilic attack of the 3'-OH from each incoming NTP on the intact 5'-triphosphate created by the preceding nucleotide addition. The potential exists for competition between 5'-pyrophosphate removal and 3'-5' polymerase reactions that could define the outcome of Thg1-catalyzed addition, yet the interplay between these competing reactions has not been investigated for any Thg1 enzyme. Here we establish transient kinetic assays to characterize the pyrophosphate removal versus nucleotide addition activities of yeast Thg1 with a set of tRNA(His) substrates in which the identity of the N-1:N73 base pair was varied to mimic various products of the N-1 addition reaction catalyzed by Thg1. We demonstrate that retention of the 5'-triphosphate is correlated with efficient 3'-5' reverse polymerization. A kinetic partitioning mechanism that acts to prevent addition of nucleotides beyond the -1 position with wild-type tRNA(His) is proposed.

Topics: Base Pair Mismatch; Base Pairing; Biocatalysis; Diphosphates; Guanosine Triphosphate; Kinetics; Nucleotides; Nucleotidyltransferases; Reproducibility of Results; RNA, Transfer; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins

Studies on thiamin biosynthesis have so far been achieved in eubacteria, yeast and plants, in which the thiamin structure is formed as thiamin phosphate from a thiazole and a pyrimidine moiety. This condensation reaction is catalyzed by thiamin phosphate synthase, which is encoded by the thiE gene or its orthologs. On the other hand, most archaea do not seem to have the thiE gene, but instead their thiD gene, coding for a 2-methyl-4-amino-5-hydroxymethylpyrimidine (HMP) kinase/HMP phosphate kinase, possesses an additional C-terminal domain designated thiN. These two proteins, ThiE and ThiN, do not share sequence similarity. In this study, using recombinant protein from the hyperthermophile archaea Pyrobaculum calidifontis, we demonstrated that the ThiN protein is an analog of the ThiE protein, catalyzing the formation of thiamin phosphate with the release of inorganic pyrophosphate from HMP pyrophosphate and 4-methyl-5-β-hydroxyethylthiazole phosphate (HET-P). In addition, we found that the ThiN protein can liberate an inorganic pyrophosphate from HMP pyrophosphate in the absence of HET-P. A structure model of the enzyme-product complex of P. calidifontis ThiN domain was proposed on the basis of the known three-dimensional structure of the ortholog of Pyrococcus furiosus. The significance of Arg320 and His341 residues for thiN-coded thiamin phosphate synthase activity was confirmed by site-directed mutagenesis. This is the first report of the experimental analysis of an archaeal thiamin synthesis enzyme.

Topics: Alkyl and Aryl Transferases; Amino Acid Sequence; Archaeal Proteins; Catalytic Domain; Chitin; Diphosphates; Escherichia coli; Hydrolysis; Isoenzymes; Models, Molecular; Molecular Sequence Data; Protein Binding; Pyrimidines; Pyrobaculum; Pyrococcus furiosus; Recombinant Proteins; Structural Homology, Protein; Substrate Specificity; Thermodynamics; Thiamine Monophosphate

Rapid determination of L-citrulline and L-arginine, physiologically important amino acids, is a beneficial technique from the scientific and medical viewpoints. In this study, enzymatic assays for L-citrulline and L-arginine were established and evaluated. L-Citrulline assay was constructed by coupling argininosuccinate synthetase to a pyrophosphate detection system, in which pyruvate phosphate dikinase was employed, so that the citrulline-dependent production of pyrophosphate could be determined. Furthermore, the L-arginine assay was developed by coupling arginine deiminase to the L-citrulline assay. Both assays exhibited high selectivity to L-citrulline and L-arginine without any significant reactivity to other proteinaceous amino acids. These assays were also resistant to various contaminants that interfered with the conventional L-citrulline and L-arginine assays. The high accuracy of these assays was demonstrated by measurements in the presence of human plasma. Because these assays can be conducted under the neutral pH without terminating the reaction progress, they allow not only measurements in static analyte solutions, but also real-time monitoring of L-citrulline and L-arginine synthesis in the reaction mixture. The features of these assays also demonstrated that the pyrophosphate detection system served as a useful platform to develop selective and robust enzymatic assays by being coupled to a pyrophosphate-producing enzyme.

Topics: Arginine; Argininosuccinate Synthase; Bacterial Proteins; Blood Chemical Analysis; Citrulline; Diphosphates; Enzyme Assays; Escherichia coli; Escherichia coli Proteins; Genes, Bacterial; Humans; Hydrolases; Propionibacterium; Pseudomonas aeruginosa; Pyruvate, Orthophosphate Dikinase; Recombinant Proteins; Urea

Two dinuclear copper complexes with and without ammonium moieties were synthesized. The complexes exhibited selective binding affinity to pyrophosphate in aqueous solution. The dinuclear copper complex, with ammonium arms, showed a ca. 527-fold enhancement in pyrophosphate binding affinity compared with its analogue without ammonium units.

Topics: Ammonium Compounds; Colorimetry; Copper; Crystallography, X-Ray; Diphosphates; Hydrogen-Ion Concentration; Models, Molecular; Molecular Structure; Organometallic Compounds

Plasma levels of pyrophosphate, an endogenous inhibitor of vascular calcification, are reduced in end-stage renal disease and correlate inversely with arterial calcification. However, it is not known whether the low plasma levels are directly pathogenic or are merely a marker of reduced tissue levels. This was tested in an animal model in which aortas were transplanted between normal mice and Enpp1(-/-) mice lacking ectonucleotide pyrophosphatase phosphodiesterase, the enzyme that synthesizes extracellular pyrophosphate. Enpp1(-/-) mice had very low plasma pyrophosphate and developed aortic calcification by 2 months that was greatly accelerated with a high-phosphate diet. Aortas of Enpp1(-/-) mice showed no further calcification after transplantation into wild-type mice fed a high-phosphate diet. Aorta allografts of wild-type mice calcified in Enpp1(-/-) mice but less so than the adjacent recipient Enpp1(-/-) aorta. Donor and recipient aortic calcium contents did not differ in transplants between wild-type and Enpp1(-/-) mice, demonstrating that transplantation per se did not affect calcification. Histology revealed medial calcification with no signs of rejection. Thus, normal levels of extracellular pyrophosphate are sufficient to prevent vascular calcification, and systemic Enpp1 deficiency is sufficient to produce vascular calcification despite normal vascular extracellular pyrophosphate production. This establishes an important role for circulating extracellular pyrophosphate in preventing vascular calcification.

Topics: Animals; Aorta; Aortic Diseases; Calcium; Diphosphates; Disease Models, Animal; Disease Progression; Mice, Inbred C57BL; Mice, Knockout; Phosphoric Diester Hydrolases; Phosphorus, Dietary; Pyrophosphatases; Time Factors; Vascular Calcification

Combining metal-anion coordination and π-π stacking interactions, a zinc complex of a novel anthracene-based cyclophane was designed to recognise adenine nucleoside polyphosphates. This complex was found to show selective fluorescence enhancement for ATP, ADP, AMP and PPi in neutral aqueous solution. Among them, ADP induced the largest fluorescence change to the complex, while ATP showed the strongest binding affinity to the complex. This property was used to sense ATP in the presence of excess amounts of other phosphates such as ADP, AMP, PPi and Pi.

Topics: Adenine Nucleotides; Anthracenes; Coordination Complexes; Diphosphates; Ethers, Cyclic; Ions; Kinetics; Spectrometry, Fluorescence; Water; Zinc

The glutathione S-transferase from Plasmodium falciparum presents distinct features which are absent from mammalian GST isoenzyme counterparts. Most apparent among these are the ability to tetramerize and the presence of a flexible loop. The loop, situated between the 113-119 residues, has been reported necessary for the tetramerization process. In this article, we report that a residue outside of this loop, Asn112, is a key to the process - to the point where the single Asn112Leu mutation prevents tetramerization altogether. We propose that a structural pattern involving the interaction of the Asn112 and Lys117 residues from two neighboring subunits plays a role in keeping the tetramer structure stable. We also report that, for the tetramerization of the wild-type PfGST to occur, phosphate or pyrophosphate anions must be present. In other words, tetramerization is a phosphate- or pyrophosphate-induced process. Furthermore, the presence of magnesium reinforces this induction. We present experimental evidence for these claims as well as a preliminary calorimetric and kinetic study of the dimeric Asn112Leu PfGST mutant. We also propose a putative binding site for phosphate or pyrophosphate anions through a comparative structural analysis of PfGST and pyrophosphatases from several organisms. Our results highlight the differences between PfGST and the human isoenzymes, which make the parasite enzyme a suitable antimalarial target.

Topics: Asparagine; Cations, Divalent; Diphosphates; Escherichia coli; Gene Expression; Glutathione Transferase; Humans; Kinetics; Magnesium; Models, Molecular; Mutation; Phosphates; Plasmodium falciparum; Protein Multimerization; Protein Stability; Protein Structure, Secondary; Protein Structure, Tertiary; Protein Subunits; Protozoan Proteins; Recombinant Proteins; Species Specificity; Thermodynamics

The purpose of this study was to develop a novel, drug-free therapy that can reduce the over-accumulation of cariogenic bacteria on dental surfaces.. We designed and synthesized a polyethylene glycol (PEG)-based hydrophilic copolymer functionalized with a pyrophosphate (PPi) tooth-binding anchor using "click" chemistry. The polymer was then evaluated for hydroxyapatite (HA) binding kinetics and capability of reducing bacteria adhesion to artificial tooth surface.. The PPi-PEG copolymer can effectively inhibit salivary protein adsorption after rapid binding to an artificial tooth surface. As a result, the in vitro S. mutans adhesion study showed that the PPi-PEG copolymer can inhibit saliva protein-promoted S. mutans adhesion through the creation of a neutral, hydrophilic layer on the artificial tooth surface.. The results suggested the potential application of a PPi-PEG copolymer as a drug-free alternative to current antimicrobial therapy for caries prevention.

Topics: Bacterial Adhesion; Dental Caries; Diphosphates; Hydrophobic and Hydrophilic Interactions; Polyethylene Glycols; Polymers; Saliva; Streptococcus mutans; Tooth

Acetate kinase (AK) generally utilizes ATP as a phosphoryl donor, but AK from Entamoeba histolytica (PPi-ehiAK) uses pyrophosphate (PPi), not ATP, and is PPi-specific. The determinants of the phosphoryl donor specificity are unknown. Here, we inferred 5 candidate amino acid residues associated with this specificity, based on structural information. Each candidate residue in Escherichia coli ATP-specific AK (ATP-ecoAK), which is unable to use PPi, was substituted with the respective PPi-ehiAK amino acid residue. Each variant ATP-ecoAK had an increased Km for ATP, indicating that the 5 residues are the determinants for the specificity to ATP in ATP-ecoAK. Moreover, Asn-337 of ATP-ecoAK was shown to be particularly significant for the specificity to ATP. The 5 residues are highly conserved in 2625 PPi-ehiAK homologs, implying that almost all organisms have ATP-dependent, rather than PPi-dependent, AK.

Topics: Acetate Kinase; Adenosine Triphosphate; Amino Acid Sequence; Amino Acid Substitution; Diphosphates; Entamoeba histolytica; Escherichia coli; Kinetics; Models, Molecular; Molecular Sequence Data; Substrate Specificity

A novel and label-free fluorescence polarization (FP) method for the determination of pyrophosphate (PPi) is developed based on the change in FP signals during fast reversible transformation between CdTe zero-dimensional (0D) nanocrystals (NCs) and one-dimensional (1D) nanorods (NRs) induced by addition of PPi. Under optimum conditions, the FP ratio was linearly proportional to the logarithm of the concentration of PPi between 2.0 × 10(-5) and 1.0 × 10(-9) M with a detection limit of 8.0 × 10(-10) M. The developed method, with high signal selectivity and stability, was successfully applied to the detection of PPi in human urine samples.

Topics: Cadmium Compounds; Diphosphates; Fluorescence Polarization; Humans; Limit of Detection; Nanoparticles; Nanotubes; Tellurium

Mutations in ABCC6 underlie the ectopic mineralization disorder pseudoxanthoma elasticum (PXE) and some forms of generalized arterial calcification of infancy, both of which affect the cardiovascular system. Using cultured cells, we recently showed that ATP-binding cassette subfamily C member 6 (ABCC6) mediates the cellular release of ATP, which is extracellularly rapidly converted into AMP and the mineralization inhibitor inorganic pyrophosphate (PPi). The current study was performed to determine which tissues release ATP in an ABCC6-dependent manner in vivo, where released ATP is converted into AMP and PPi, and whether human PXE ptients have low plasma PPi concentrations.. Using cultured primary hepatocytes and in vivo liver perfusion experiments, we found that ABCC6 mediates the direct, sinusoidal, release of ATP from the liver. Outside hepatocytes, but still within the liver vasculature, released ATP is converted into AMP and PPi. The absence of functional ABCC6 in patients with PXE leads to strongly reduced plasma PPi concentrations.. Hepatic ABCC6-mediated ATP release is the main source of circulating PPi, revealing an unanticipated role of the liver in systemic PPi homeostasis. Patients with PXE have a strongly reduced plasma PPi level, explaining their mineralization disorder. Our results indicate that systemic PPi is relatively stable and that PXE, generalized arterial calcification of infancy, and other ectopic mineralization disorders could be treated with PPi supplementation therapy.

Topics: Adenosine Monophosphate; Adenosine Triphosphate; Aged; Animals; ATP-Binding Cassette Transporters; Cells, Cultured; Culture Media, Conditioned; Diphosphates; Female; HEK293 Cells; HeLa Cells; Hepatocytes; Homeostasis; Humans; Liver; Male; Mice; Middle Aged; Multidrug Resistance-Associated Proteins; Pseudoxanthoma Elasticum; Rats

The Japanese marine sponge Discodermia calyx contains a major cytotoxic compound, calyculin A, which exhibits selective inhibition of protein phosphatases 1 and 2A. It has long been used as a chemical tool to evaluate intracellular signal transduction regulated by reversible protein phosphorylation. We describe the identification of the biosynthetic gene cluster of calyculin A by a metagenome mining approach. Single-cell analysis revealed that the gene cluster originates in the symbiont bacterium 'Candidatus Entotheonella' sp. A phosphotransferase encoded in the gene cluster deactivated calyculin A to produce a newly discovered diphosphate, which was actually the biosynthetic end product. The diphosphate had been previously overlooked because of the enzymatic dephosphorylation that occurred in response to sponge tissue disruption. Our work presents what is to our knowledge the first evidence for the biosynthetic process of calyculin A along with a notable phosphorylation-dephosphorylation mechanism to regulate toxicity, suggesting activated chemical defense in the most primitive of all multicellular animals.

Topics: Animals; Antineoplastic Agents; Diphosphates; Drug Screening Assays, Antitumor; HeLa Cells; Humans; Leukemia; Marine Toxins; Metagenome; Mice; Molecular Sequence Data; Multigene Family; Organophosphates; Oxazoles; Phosphorylation; Phosphotransferases; Phylogeny; Polyketide Synthases; Porifera; RNA, Ribosomal, 16S; Symbiosis

Individuals with neurofibromatosis type-1 (NF1) can manifest focal skeletal dysplasias that remain extremely difficult to treat. NF1 is caused by mutations in the NF1 gene, which encodes the RAS GTPase-activating protein neurofibromin. We report here that ablation of Nf1 in bone-forming cells leads to supraphysiologic accumulation of pyrophosphate (PPi), a strong inhibitor of hydroxyapatite formation, and that a chronic extracellular signal-regulated kinase (ERK)-dependent increase in expression of genes promoting PPi synthesis and extracellular transport, namely Enpp1 and Ank, causes this phenotype. Nf1 ablation also prevents bone morphogenic protein-2-induced osteoprogenitor differentiation and, consequently, expression of alkaline phosphatase and PPi breakdown, further contributing to PPi accumulation. The short stature and impaired bone mineralization and strength in mice lacking Nf1 in osteochondroprogenitors or osteoblasts can be corrected by asfotase-α enzyme therapy aimed at reducing PPi concentration. These results establish neurofibromin as an essential regulator of bone mineralization. They also suggest that altered PPi homeostasis contributes to the skeletal dysplasias associated with NF1 and that some of the NF1 skeletal conditions could be prevented pharmacologically.

Topics: Adolescent; Alkaline Phosphatase; Animals; Bone Development; Bone Diseases, Developmental; Bone Morphogenetic Protein 2; Calcification, Physiologic; Cells, Cultured; Child; Child, Preschool; Collagen Type I; Collagen Type I, alpha 1 Chain; Collagen Type II; Diphosphates; Disease Models, Animal; Durapatite; Humans; Immunoglobulin G; Infant; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitogen-Activated Protein Kinases; Neurofibromatosis 1; Neurofibromin 1; Osteoblasts; Osteogenesis; Phosphate Transport Proteins; Phosphoric Diester Hydrolases; Pyrophosphatases; Recombinant Fusion Proteins; Sp7 Transcription Factor; Transcription Factors

A new zinc(ii) complex with a condensed hydroxynaphthyl pyridine (SPHN) as the coordinated ligand has been synthesized for the selective recognition of pyrophosphate (PPi) over other anions including phosphate in a mixed aqueous solution. The fluorescence enhancement of SPHN in association with Zn(2+) ions is quenched in the presence of intracellular pyrophosphate. This phenomenon is utilized in the construction of a logic gate. The binding of SPHN with Zn(2+) and its displacement by PPi have been established by photophysical investigation and supported by the DFT level of studies. The development of blue fluorescence in the {} complex upon binding of zinc with is shown to be useful as a nucleus marker in a cell similar to the commercially available staining compound, DAPI (diamino-2-phenylindole).

Topics: Diphosphates; Fluorescent Dyes; HeLa Cells; Humans; Indoles; Staining and Labeling

We have designed a series of triazolium-pyrene-based dyads to probe their potential as fluorescent chemosensors for anion recognition through combinations of hydrogen and halogen bonding. Cooperation between the two distinct noncovalent interactions leads to an unusual effect on receptor affinity, as a result of fundamental differences in the interactions of halogen and hydrogen bond donor groups with anions. Absorption, emission spectrophotometries and proton and phosphorus NMR spectroscopies indicate that the two interactions act in concert to achieve the selective binding of the hydrogen pyrophosphate anion, a conclusion supported by computational studies. Hence, as clearly demonstrated with respective halogen- and hydrogen-bonding triazolium receptors, the integration of a halogen atom into the anion receptor at the expense of one hydrogen-bonding receptor greatly influences the anion recognition affinity of the receptor. The association constant values of the halogen-bonding complexes are larger than the hydrogen-bonding counterpart. Thus, halogen bonding has been exploited for the selective fluorescent sensing of hydrogen pyrophosphate anion. Halogen bonding has been demonstrated to increase the strength of hydrogen pyrophosphate binding, as compared to the hydrogen-bonded analogue. Grimme's PBE-D functional, which adequately reproduces the pyrene stacking energies, has been successfully applied to model the affinity for anions, especially hydrogen pyrophosphate, of the new receptors.

Topics: Anions; Diphosphates; Halogens; Hydrogen; Hydrogen Bonding; Magnetic Resonance Spectroscopy; Models, Molecular; Pyrenes; Triazoles

Chronic airway infection in adults with cystic fibrosis (CF) is polymicrobial and the impact of intravenous antibiotics on the bacterial community composition is poorly understood. We employed culture-independent molecular techniques to explore the early effects of i.v. antibiotics on the CF airway microbiome. DNA was extracted from sputum samples collected from adult subjects with CF at three time-points (before starting treatment, and at day 3 and day 8-10 of i.v. antibiotics) during treatment of an infective pulmonary exacerbation. Microbial community profiles were derived through analysis of bacterial-derived 16S ribosomal RNA by pyrosequencing and changes over time were compared. 59 sputum samples were collected during 24 pulmonary exacerbations from 23 subjects. Between treatment onset and day 3 there was a significant reduction in the relative abundance of Pseudomonas and increased microbial diversity. By day 8-10, bacterial community composition was similar to pre-treatment. Changes in community composition did not predict improvements in lung function. The relative abundance of Pseudomonas falls rapidly in subjects with CF receiving i.v. antibiotic treatment for a pulmonary exacerbation and is accompanied by an increase in overall microbial diversity. However, this effect is not maintained beyond the first week of treatment.

Topics: Adolescent; Adult; Anti-Bacterial Agents; Cystic Fibrosis; Diphosphates; DNA, Bacterial; Female; Humans; Injections, Intravenous; Lung; Male; Microbiota; Middle Aged; Sequence Analysis, DNA; Young Adult

Topics: Animals; Diphosphates; Humans; Marine Toxins; Oxazoles; Porifera

One of the well-known strategies for anion sensing is an indicator (dye) displacement assay. However, the disadvantage of the dye displacement assays is the low sensitivity due to the excess of the dye used. To overcome this setback, we have developed an "Intramolecular Indicator Displacement Assay (IIDA)". The IIDAs comprise a receptor and a spacer with an attached anionic chromophore in a single-molecule assembly. In the resting state, the environment-sensitive anionic chromophore is bound by the receptor, while the anionic substrate competes for binding into the receptor. The photophysical properties of the dye exhibit change in fluorescence when displaced by anions, which results in cross-reactive response. To illustrate the concept, we have prepared IID sensors 1 and 2. Here, the characterization of sensors and microtiter arrays comprising the IIDA are reported. The microtiter array including IID sensors 1 and 2 is capable of recognizing biological phosphates in water. The utility of the IIDA approach is demonstrated on sensing of a phosphonate herbicide glyphosate and other biologically important anions such as pyrophosphate in the presence of interferent sodium chloride.

Topics: Anions; Chlorides; Diphosphates; Discriminant Analysis; Electrolytes; Fluorescent Dyes; Glycine; Glyphosate; Herbicides; Magnetic Resonance Spectroscopy; Multivariate Analysis; Phosphates; Polymers; Sodium Chloride; Water; X-Ray Diffraction

A novel and highly sensitive colorimetric and fluorescence assay for the accurate determination of alkaline phosphatase (ALP) activity has been developed. The assay takes advantage of the inhibition of the peroxidase activity of Cu(2+) ions caused by complexation with pyrophosphate (PPi), a natural substrate for ALP. This inhibition disappears when PPi undergoes ALP catalyzed hydrolysis to generate phosphate, which does not bind to Cu(2+) ions. Thus, ALP causes generation of uncomplexed Cu(2+) ions, which promote multiple oxidation reactions of Amplex UltraRed in the presence of hydrogen peroxide in conjunction with the production of intense fluorescence and colorimetric signals. By employing the fluorescence and colorimetric assay strategies, ALP can be detected at respective concentrations as low as 4.3 pM and 5.4 pM, detection limits that are much lower than those associated with previously described methods. The practical diagnostic capability of the assay system has been demonstrated by its use to detect ALP in human blood serum.

Topics: Alkaline Phosphatase; Cations, Divalent; Colorimetry; Copper; Diphosphates; Enzyme Assays; Humans; Hydrolysis; Spectrometry, Fluorescence

To investigate the conformational dynamics of human DNA polymerase κ (hpol κ), we generated two mutants, Y50W (N-clasp region) and Y408W (linker between the thumb and little finger domains), using a Trp-null mutant (W214Y/W392H) of the hpol κ catalytic core enzyme. These mutants retained catalytic activity and similar patterns of selectivity for bypassing the DNA adduct 7,8-dihydro-8-oxo-2'-deoxyguanosine, as indicated by the results of steady-state and pre-steady-state kinetic experiments. Stopped-flow kinetic assays with hpol κ Y50W and T408W revealed a decrease in Trp fluorescence with the template G:dCTP pair but not for any mispairs. This decrease in fluorescence was not rate-limiting and is considered to be related to a conformational change necessary for correct nucleotidyl transfer. When a free 3'-hydroxyl was present on the primer, the Trp fluorescence returned to the baseline level at a rate similar to the observed kcat , suggesting that this change occurs during or after nucleotidyl transfer. However, polymerization rates (kpol ) of extended-product formation were fast, indicating that the slow fluorescence step follows phosphodiester bond formation and is rate-limiting. Pyrophosphate formation and release were fast and are likely to precede the slower relaxation step. The available kinetic data were used to fit a simplified minimal model. The extracted rate constants confirmed that the conformational change after phosphodiester bond formation was rate-limiting for hpol κ catalysis with the template G:dCTP pair.

Topics: Amino Acid Substitution; Catalytic Domain; Deoxycytosine Nucleotides; Diphosphates; DNA Adducts; DNA-Directed DNA Polymerase; Humans; Kinetics; Mutagenesis, Site-Directed; Protein Binding

In this research, the dinuclear Zn(ii) complex of anthracene based tripodal tetramine Zn2L was synthesized, and its sensing abilities towards anions was investigated using the indicator displacement assay (IDA) approach with four complexometric indicators: pyrocatechol violet (PV), bromopyrogallol red (BPG), methylthymol blue (MTB) and xylenol orange (XO). UV-vis spectrophotometry results indicated that the Zn2L-MTB ensemble sensor could discriminate the pyrophosphate anion (PPi) from other phosphate containing anions. (1)H and (31)P NMR spectroscopy as well as DFT calculations confirmed that PPi bound to Zn2L in a 2 : 2 manner. Both NMR spectroscopy and UV-vis spectrophotometry suggested that the two bulky tripodal tetramine units in Zn2L played an important role to provide the ensemble cleft for MTB, giving rise to an ensemble that could be displaced exclusively by PPi. The detection limit of PPi for the reported IDA system was 0.3 μM in 20% (v/v) water-acetonitrile buffered at pH 7.4 with HEPES.

Topics: Amides; Colorimetry; Diphosphates; Indicators and Reagents; Magnetic Resonance Spectroscopy; Molecular Structure; Phosphates; Zinc

In this work, chemiluminescence (CL) reagent and catalyst metal ion complexes bifunctionalized gold nanoparticles (BF-AuNPs) with high CL efficiency were synthesized via an improved synthesis strategy. Biothiols, such as cysteine (Cys), cysteinyl-glycine (Cys-Gly), homocysteine (Hcy), and glutathione (GSH), instead of 2-[bis[2-[carboxymethyl-[2-oxo-2-(2-sulfanylethylamino)ethyl]amino]ethyl]amino]acetic acid (DTDTPA), were used as new chelators. N-(aminobutyl)-N-(ethylisoluminol) (ABEI) was used as a model of CL reagents and Cu(2+) as a model of metal ion. In this strategy, biothiols were first grafted on the surface of ABEI-AuNPs by Au-S bond. Then, Cu(2+) was captured onto the surface of ABEI-AuNPs by the coordination reaction to form BF-AuNPs. The CL intensity of Cu(2+)-Cys/ABEI-AuNPs was 1 order of magnitude higher than that of DTDTPA/Cu(2+)-ABEI-AuNPs synthesized by the previous work. Moreover, strong CL emission of Cu(2+)-Cys/ABEI-AuNPs was also observed in neutral pH conditions. In addition, the present BF-AuNPs synthesis method exhibited advantages over the previous method in CL efficiency, simplicity, and synthetic rate. Finally, by virtue of Cu(2+)-Cys/ABEI-AuNPs as a platform, a simple CL chemosensor for the sensitive and selective detection of pyrophosphate ion (PPi) was established based on the competitive coordination interactions of Cu(2+) between Cys and PPi. The method exhibited a wide detection range from 10 nM to 100 μM, with a low detection limit of 3.6 nM. The chemosensor was successfully applied to the detection of PPi in human plasma samples. It is of great application potential in clinical analysis. This work reveals that BF-AuNPs could be used as ideal nanointerface for the development of novel analytical methods.

Topics: Biosensing Techniques; Chelating Agents; Copper; Diphosphates; Gold; Humans; Hydrogen Peroxide; Hydrogen-Ion Concentration; Ions; Kinetics; Luminescence; Luminol; Metal Nanoparticles; Sulfhydryl Compounds

The ability to acquire iron from various sources has been demonstrated to be a major determinant in the pathogenesis of Neisseria meningitidis. Outside the cells, iron is bound to transferrin in serum, or to lactoferrin in mucosal secretions. Meningococci can extract iron from iron-loaded human transferrin by the TbpA/TbpB outer membrane complex. Moreover, N. meningitidis expresses the LbpA/LbpB outer membrane complex, which can extract iron from iron-loaded human lactoferrin. Iron transport through the outer membrane requires energy provided by the ExbB-ExbD-TonB complex. After transportation through the outer membrane, iron is bound by periplasmic protein FbpA and is addressed to the FbpBC inner membrane transporter. Iron-complexing compounds like citrate and pyrophosphate have been shown to support meningococcal growth ex vivo. The use of iron pyrophosphate as an iron source by N. meningitidis was previously described, but has not been investigated. Pyrophosphate was shown to participate in iron transfer from transferrin to ferritin. In this report, we investigated the use of ferric pyrophosphate as an iron source by N. meningitidis both ex vivo and in a mouse model. We showed that pyrophosphate was able to sustain N. meningitidis growth when desferal was used as an iron chelator. Addition of a pyrophosphate analogue to bacterial suspension at millimolar concentrations supported N. meningitidis survival in the mouse model. Finally, we show that pyrophosphate enabled TonB-independent ex vivo use of iron-loaded human or bovine transferrin as an iron source by N. meningitidis. Our data suggest that, in addition to acquiring iron through sophisticated systems, N. meningitidis is able to use simple strategies to acquire iron from a wide range of sources so as to sustain bacterial survival.

Topics: Animals; Bacterial Proteins; Biological Transport; Deferoxamine; Diphosphates; Disease Models, Animal; Humans; Iron; Membrane Proteins; Meningitis, Meningococcal; Mice; Microbial Viability; Neisseria meningitidis; Transferrin

Arterial medial calcification is a major complication in patients with chronic kidney disease and diabetes. It has been hypothesized that a high concentration of inorganic phosphate (Pi) induces calcification in vascular smooth muscle cells (vSMCs). However, the role of transforming growth factor-β (TGF-β)/Smad3 signaling in Pi-induced vascular calcification remains controversial. The aim of this study was to investigate the possible involvement of Smad3 in Pi-induced vascular calcification. We compared the degree of Pi-induced vSMC calcification between vSMCs isolated from wild-type (Smad3(+/+)) and Smad3-deficient (Smad3(-/-)) mice. We found that vSMCs from Smad3(+/+) mice had less calcium (Ca) than those from Smad3(-/-) mice when they were exposed to high concentrations of Pi and Ca (Pi+Ca). The phosphorylation of Smad3 was induced in Smad3(+/+) vSMCs by exposure to Pi+Ca. The concentration of extracellular pyrophosphate (ePPi) was lower in Smad3(-/-) vSMCs than in Smad3(+/+) vSMCs and was significantly increased in Smad3(+/+) vSMCs by treatment with TGF-β1. Also, the addition of a small amount of PPi to culture medium significantly decreased the deposition of Ca in both Smad3(+/+) and Smad3(-/-) vSMCs. Ectonucleotide phosphatase/phosphodiesterase1 (Enpp1) was decreased at the mRNA, protein, and enzymatic activity levels in Smad3(-/-) vSMCs compared with Smad3(+/+) vSMCs. A ChIP assay showed that phosphorylated Smad3 directly binds to the Enpp1 gene. Furthermore, the calcification of aortic segments was attenuated by treatment with TGF-β1 only in Smad3(+/+) mice. Taken together, we conclude that Pi-induced vSMC calcification is suppressed by Smad3 via an increase in ePPi.

Topics: Animals; Blotting, Western; Chromatin Immunoprecipitation; Diphosphates; Male; Mice; Mice, Knockout; Muscle, Smooth, Vascular; Phosphoric Diester Hydrolases; Pyrophosphatases; Real-Time Polymerase Chain Reaction; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta1; Vascular Calcification

Entamoeba histolytica, an amitochondriate protozoan parasite that relies on glycolysis as a key pathway for ATP generation, has developed a unique extended PPi-dependent glycolytic pathway in which ADP-forming acetyl-coenzyme A (CoA) synthetase (ACD; acetate:CoA ligase [ADP-forming]; EC 6.2.1.13) converts acetyl-CoA to acetate to produce additional ATP and recycle CoA. We characterized the recombinant E. histolytica ACD and found that the enzyme is bidirectional, allowing it to potentially play a role in ATP production or in utilization of acetate. In the acetate-forming direction, acetyl-CoA was the preferred substrate and propionyl-CoA was used with lower efficiency. In the acetyl-CoA-forming direction, acetate was the preferred substrate, with a lower efficiency observed with propionate. The enzyme can utilize both ADP/ATP and GDP/GTP in the respective directions of the reaction. ATP and PPi were found to inhibit the acetate-forming direction of the reaction, with 50% inhibitory concentrations of 0.81 ± 0.17 mM (mean ± standard deviation) and 0.75 ± 0.20 mM, respectively, which are both in the range of their physiological concentrations. ATP and PPi displayed mixed inhibition versus each of the three substrates, acetyl-CoA, ADP, and phosphate. This is the first example of regulation of ACD enzymatic activity, and possible roles for this regulation are discussed.

Topics: Acetate-CoA Ligase; Acetyl Coenzyme A; Adenosine Diphosphate; Adenosine Triphosphate; Diphosphates; Entamoeba histolytica; Kinetics; Magnesium; Protozoan Proteins; Recombinant Proteins; Substrate Specificity

Pyrophosphate (PPi) is a potent inhibitor of ectopic mineralization but its role during aortic valve calcification is not known.. Anti-calcific effect of PPi was investigated by using an in vitro model of serum-driven calcification of collagen sponges and decellularized porcine aortic valve leaflets. Bovine interstitial valve cells (VIC), seeded either within the collagen matrices or in transwell chambers, were used to test cellular ability to inhibit serum-induced calcification. PPi metabolism was investigated in clonal VIC harboring different calcifying potential.. In a cell-free system, high serum levels induced a dose-dependent calcification of type I collagen matrices which was prevented by PPi and ATP supplementation. Blockade of serum-driven calcification by PPi and ATP was also observed when using decellularized porcine aortic valve leaflets. A similar anti-calcific effect was also seen for bovine VIC, either statically seeded into the collagen matrices or co-cultured by using a transwell system. However, when we performed co-culture experiments by using clonal VIC harboring different calcifying potential, we observed that the subset of cells acquiring a pro-calcific profile lost the ability to protect the collagen from serum-driven calcification. Pro-calcific differentiation of the clonal VIC was accompanied by increase in ALP along with significant reduction in NPP activity and ATP/PPi extracellular accumulation. These changes were not observed in the clonal subtype with lower propensity towards calcification.. We showed that PPi and ATP are potent inhibitors of serum-driven calcification of collagen matrix and that their extracellular accumulation is reduced in calcifying VIC.

Topics: Adenosine Triphosphate; Alkaline Phosphatase; Animals; Aorta; Aortic Valve; Aortic Valve Stenosis; Calcinosis; Calcium; Cattle; Cell Differentiation; Cell-Free System; Cloning, Molecular; Collagen; Diphosphates; Microscopy, Electron, Scanning; Nucleotides; Swine; X-Ray Diffraction

Aortic valve (AV) calcification is a major cause of morbidity and mortality, yet the molecular mechanisms involved are poorly understood. Hence, an ex vivo model of calcification in intact AVs was developed in order to test the role of orthophosphate and pyrophosphate (PPi), both of which factors are known to influence vascular calcification.. Porcine AV leaflets were cultured in serum-free medium under static conditions for eight days, over which time leaflet architecture and viability were preserved. Calcification was measured as the incorporation of 45Ca, with confirmation by Alizarin Red staining.. Calcification required both a high phosphate concentration (3.8 mM) and removal of PPi with alkaline phosphatase or inorganic pyrophosphatase. Calcification occurred predominantly on the fibrosa and was arrested by the bisphosphonate etidronate, a non-hydrolyzable analog of PPi. Leaflets released PPi into the medium, and this was enhanced by MLS38949, a specific inhibitor of tissue non-specific alkaline phosphatase (TNAP). Furthermore, leaflets synthesized PPi from extracellular ATP, which was reduced by β,γ-methylene-ATP, an inhibitor of ectonucleotide pyrophosphorylase phosphodiesterase (NPP1).. The ex vivo AV calcification model developed in the present study showed that extracellular PPi, produced by valvular tissue, is a potent inhibitor of valvular calcification. In addition to synthesis, hydrolysis by TNAP also controls PPi levels and calcification. The results suggest that a decreased synthesis or increased hydrolysis of pyrophosphate may contribute to valvular calcification, and that bisphosphonates or inhibitors of TNAP are potential preventive strategies of the process. TNAP are potential preventive strategies.

Topics: Alkaline Phosphatase; Animals; Aortic Valve; Aortic Valve Stenosis; Calcinosis; Diphosphates; Etidronic Acid; Female; Phosphates; Pyrophosphatases; Swine; Tissue Culture Techniques

Cofactor specificities of glycolytic enzymes in Clostridium thermocellum were studied with cellobiose-grown cells from batch cultures. Intracellular glucose was phosphorylated by glucokinase using GTP rather than ATP. Although phosphofructokinase typically uses ATP as a phosphoryl donor, we found only pyrophosphate (PPi)-linked activity. Phosphoglycerate kinase used both GDP and ADP as phosphoryl acceptors. In agreement with the absence of a pyruvate kinase sequence in the C. thermocellum genome, no activity of this enzyme could be detected. Also, the annotated pyruvate phosphate dikinase (ppdk) is not crucial for the generation of pyruvate from phosphoenolpyruvate (PEP), as deletion of the ppdk gene did not substantially change cellobiose fermentation. Instead pyruvate formation is likely to proceed via a malate shunt with GDP-linked PEP carboxykinase, NADH-linked malate dehydrogenase, and NADP-linked malic enzyme. High activities of these enzymes were detected in extracts of cellobiose-grown cells. Our results thus show that GTP is consumed while both GTP and ATP are produced in glycolysis of C. thermocellum. The requirement for PPi in this pathway can be satisfied only to a small extent by biosynthetic reactions, in contrast to what is generally assumed for a PPi-dependent glycolysis in anaerobic heterotrophs. Metabolic network analysis showed that most of the required PPi must be generated via ATP or GTP hydrolysis exclusive of that which happens during biosynthesis. Experimental proof for the necessity of an alternative mechanism of PPi generation was obtained by studying the glycolysis in washed-cell suspensions in which biosynthesis was absent. Under these conditions, cells still fermented cellobiose to ethanol.

Topics: Adenosine Triphosphate; Bacterial Proteins; Cellobiose; Clostridium thermocellum; Diphosphates; Enzymes; Fermentation; Glucose; Glycogen; Glycolysis; Guanosine Triphosphate; Phosphorylation; Pyruvate, Orthophosphate Dikinase; Sequence Deletion

Inorganic pyrophosphatases are required for anabolism to take place in all living organisms. Defects in genes encoding these hydrolytic enzymes are considered inviable, although their exact nature has not been studied at the cellular and molecular physiology levels. Using a conditional mutant in IPP1, the Saccharomyces cerevisiae gene encoding the cytosolic soluble pyrophosphatase, we show that respiring cells arrest in S phase upon Ipp1p deficiency, but they remain viable and resume growth if accumulated pyrophosphate is removed. However, fermenting cells arrest in G1/G0 phase and suffer massive vacuolization and eventual cell death by autophagy. Impaired NAD(+) metabolism is a major determinant of cell death in this scenario because demise can be avoided under conditions favoring accumulation of the oxidized pyridine coenzyme. These results posit that the mechanisms related to excess pyrophosphate toxicity in eukaryotes are dependent on the energy metabolism of the cell.

Topics: Autophagy; Diphosphates; Energy Metabolism; Inorganic Pyrophosphatase; NAD; S Phase Cell Cycle Checkpoints; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins

Deterioration of ventricular fibrillation (VF) into asystole or severe bradycardia (electrical failure) heralds a fatal outcome of cardiac arrest. The role of metabolism in the timing of electrical failure remains unknown.. To determine metabolic factors of early electrical failure in an ex-vivo canine model of cardiac arrest (VF+global ischemia).. Metabolomic screening was performed in left ventricular biopsies collected before and after 0.3, 2, 5, 10 and 20 min of VF and global ischemia. Electrical activity was monitored via plunge needle electrodes and pseudo-ECG. Four out of nine hearts exhibited electrical failure at 10.1±0.9 min (early-asys), while 5/9 hearts maintained VF for at least 19.7 min (late-asys). As compared to late-asys, early-asys hearts had more ADP, less phosphocreatine, and higher levels of lactate at some time points during VF/ischemia (all comparisons p<0.05). Pre-ischemic samples from late-asys hearts contained ∼25 times more inorganic pyrophosphate (PPi) than early-asys hearts. A mechanistic role of PPi in cardioprotection was then tested by monitoring mitochondrial membrane potential (ΔΨ) during 20 min of simulated-demand ischemia using potentiometric probe TMRM in rabbit adult ventricular myocytes incubated with PPi versus control group. Untreated myocytes experienced significant loss of ΔΨ while in the PPi-treated myocytes ΔΨ was relatively maintained throughout 20 min of simulated-demand ischemia as compared to control (p<0.05).. High tissue level of PPi may prevent ΔΨm loss and electrical failure at the early phase of ischemic stress. The link between the two protective effects may involve decreased rates of mitochondrial ATP hydrolysis and lactate accumulation.

Topics: Animals; Cardiotonic Agents; Cells, Cultured; Diphosphates; Dogs; Female; Heart Arrest; Male; Membrane Potential, Mitochondrial; Mitochondria, Heart; Myocardial Ischemia; Myocytes, Cardiac; Rabbits

On the basis of the inhibition of double strand DNA (dsDNA)-templated fluorescent copper nanoparticles (CuNPs) by pyrophosphate (PPi), a novel label-free turn-on fluorescent strategy to detect alkaline phosphatase (ALP) under physiological conditions has been developed. This method relies on the strong interaction between PPi and Cu(2+), which would hamper the effective formation of fluorescent CuNPs, leading to low fluorescence intensity. The ALP-catalyzed PPi hydrolysis would disable the complexation between Cu(2+) and PPi, facilitating the formation of fluorescent CuNPs through the reduction by ascorbate in the presence of dsDNA templates. Thus, the fluorescence intensity was recovered, and the fluorescence enhancement was related to the concentration of ALP. This method is cost-effective and convenient without any labels or complicated operations. The present strategy exhibits a high sensitivity and the turn-on mode provides a high selectivity for the ALP assay. Additionally, the inhibition effect of phosphate on the ALP activity was also studied. The proposed method using a PPi substrate may hold a potential application in diagnosis of ALP-related diseases or evaluation of ALP functions in biological systems.

Topics: Alkaline Phosphatase; Ascorbic Acid; Copper; Diphosphates; DNA; Enzyme Assays; Fluorescent Dyes; Humans; Hydrolysis; Metal Nanoparticles; Sensitivity and Specificity; Spectrometry, Fluorescence

A library of bis[zinc(II)dipicolylamine]-functionalised linear peptides was prepared using an efficient solid phase peptide synthesis approach and their use as chemosensors for anions in water was investigated using indicator displacement assays. High affinity and selectivity for pyrophosphate (PPi) over adenosine triphosphate (ATP) and adenosine diphosphate (ADP) were observed and additional aromatic side chains provided enhanced discrimination between PPi and ATP.

Topics: Diphosphates; Organometallic Compounds; Peptides; Picolinic Acids; Water

Zymomonas mobilis is the only known microorganism that utilizes the Entner-Doudoroff (ED) pathway anaerobically. In this work, we investigated whether the overexpression of a phosphofructokinase (PFK), the only missing Embden-Meyerhof-Parnas (EMP) pathway enzyme, could establish the pathway in this organism. Introduction of a pyrophosphate-dependent PFK, along with co-expression of homologous fructose-1,6-bisphosphate aldolase and triosephosphate isomerase, did not result in an EMP flux to any appreciable level. However, the metabolism of glucose was impacted significantly. Eight percent of glucose was metabolized to form a new metabolite, dihydroxyacetone. Reducing flux through the ED pathway by as much as 40 % through antisense of a key enzyme, ED aldolase, did not result in a fully functional EMP pathway, suggesting that the ED pathway, especially the lower arm, downstream from glyceraldehyde-3-phosphate, is very rigid, possibly due to redox balance.

Topics: Bacterial Proteins; Cloning, Molecular; Dihydroxyacetone; Diphosphates; Escherichia coli; Fermentation; Fructose-Bisphosphate Aldolase; Genes, Bacterial; Glucose; Glycolysis; Phosphofructokinases; Triose-Phosphate Isomerase; Zymomonas

Progerin is a mutant form of lamin A responsible for Hutchinson-Gilford progeria syndrome (HGPS), a premature aging disorder characterized by excessive atherosclerosis and vascular calcification that leads to premature death, predominantly of myocardial infarction or stroke. The goal of this study was to investigate mechanisms that cause excessive vascular calcification in HGPS.. We performed expression and functional studies in wild-type mice and knock-in Lmna(G609G/+) mice expressing progerin, which mimic the main clinical manifestations of HGPS. Lmna(G609G/+) mice showed excessive aortic calcification, and primary aortic vascular smooth muscle cells from these progeroid animals had an impaired capacity to inhibit vascular calcification. This defect in progerin-expressing vascular smooth muscle cells is associated with increased expression and activity of tissue-nonspecific alkaline phosphatase and mitochondrial dysfunction, which leads to reduced ATP synthesis. Accordingly, Lmna(G609G/+) vascular smooth muscle cells are defective for the production and extracellular accumulation of pyrophosphate, a major inhibitor of vascular calcification. We also found increased alkaline phosphatase activity and reduced ATP and pyrophosphate levels in plasma of Lmna(G609G/+) mice without changes in phosphorus and calcium. Treatment with pyrophosphate inhibited vascular calcification in progeroid mice.. Excessive vascular calcification in Lmna(G609G) mice is caused by reduced extracellular accumulation of pyrophosphate that results from increased tissue-nonspecific alkaline phosphatase activity and diminished ATP availability caused by mitochondrial dysfunction in vascular smooth muscle cells. Excessive calcification is ameliorated on pyrophosphate treatment. These findings reveal a previously undefined pathogenic process in HGPS that may also contribute to vascular calcification in normal aging, because progerin progressively accumulates in the vascular tissue of individuals without HGPS.

Topics: Adenosine Triphosphate; Alkaline Phosphatase; Animals; Aorta; Cells, Cultured; Diphosphates; Disease Models, Animal; Lamin Type A; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Mitochondria, Muscle; Muscle, Smooth, Vascular; Progeria; Treatment Outcome; Vascular Calcification

Topics: Animals; Diphosphates; Male; Progeria; Vascular Calcification

Pyrophosphate ions are both inhibitors of HA formation and substrates for phosphatase enzymes. Unlike polyphosphates their hydrolysis results simultaneously in the complete loss of mineral formation inhibition and a localised elevation in orthophosphate ion concentration. Despite recent advances in our knowledge of the role of the pyrophosphate ion, very little is known about the effects of pyrophosphate on bone formation and even less is known about its local delivery. In this work we first developed a self setting pyrophosphate based calcium cement system with appropriate handling properties and then compared its in vivo degradation properties with those of a non-pyrophosphate containing control. Contrary to expectation, the presence of the pyrophosphate phase in the cement matrix did not inhibit mineralisation of the healing bone around the implant, but actually appeared to stimulate it. In vitro evidence suggested that enzymatic action accelerated dissolution of the inorganic pyrophosphate ions, causing a simultaneous loss of their mineralisation inhibition and a localised rise in supersaturation with respect to HA. This is thought to be a rare example of a biologically responsive inorganic material and these materials seem to be worthy of further investigation. Bioceramics to date have mainly been limited to orthophosphate, silicate and carbonate salts of calcium, here we report the successful application of a pyrophosphate material as a degradable osteoconductive bone repair cement.

Topics: Animals; Bone Cements; Calcium Phosphates; Diphosphates; Polyphosphates; Sheep; X-Ray Diffraction

We examined the mode of IMP and pyrophosphate enhancement of myosin and actin extraction from porcine meat. Extractabilities were determined after homogenates, prepared by adding 9 volumes of 0.3, 0.4, or 0.5 M NaCl solutions containing 0 to 36 mM IMP and 0 to 9 mM tetrapotassium pyrophosphate (KPP) to minced pork, were incubated at 4 °C for 0 or 12 h. Irrespective of the NaCl concentrations, IMP-induced extraction of both proteins increased with increasing extraction time. In contrast, that of KPP did not. When 0.3 M NaCl solutions containing both IMP and KPP were used, the solutions with 1.5 mM KPP showed marked enhancement of IMP-induced myosin and actin extraction. Incorporating these results with our previously published data (Nakamura et al., Biosci. Biotechnol. Biochem., 76, 1611-1615 (2012)), we hypothesized that IMP and KPP have the ability to release thick and thin filaments from restraints in myofibrils, in addition to the ability to dissociate actomyosin into myosin and actin, and that the restraint-releasing ability of IMP is dependent on reaction time and NaCl concentration while that of KPP is not.

Topics: Actins; Actomyosin; Animals; Diphosphates; Inosine Monophosphate; Meat; Myofibrils; Myosins; Potassium Compounds; Sodium Chloride; Swine

Development of dentotropic (tooth-binding) micelle formulations to improved efficacy and safety of antimicrobial therapy for dental plaque prevention and treatment.. Because of their excellent biocompatibility and biodegradability, diphosphoserine peptide and pyrophosphate were selected as the tooth-binding moieties to replace alendronate, which was used previously. Diphosphoserine peptide was conjugated to Pluronic P123 using "click" chemistry, whereas pyrophosphate was attached to P123 through an ester bond. The tooth-binding micelles (TBMs) were prepared by self-assembly of the modified P123 with the antimicrobial agent triclosan. The influence of human saliva and/or its components on TBMs' drug-releasing profile, tooth-binding potential and binding stability was evaluated in vitro. S. mutans UA159 biofilm formed on hydroxyapatite (HA) discs was used to evaluate the TBMs' therapeutic potential.. Saliva does not affect triclosan release from TBMs. More than 60% of TBMs' HA binding capacity was maintained in the presence of saliva. Less than 5% of TBMs bound to HA was released over 24 h in human saliva, protease or phosphatase, suggesting the retention properties of the TBMs will not be compromised due to the biodegradable nature of the binding moieties. In both in vitro biofilm prevention and treatment studies, the TBM treated group showed significantly lower CFU per HA disc compared to the controls (2-log reduction, p < 0.05).. The data from these studies suggest that the novel dentotropic micelle formulations bearing biodegradable tooth-binding moieties can be used as an effective and safe delivery tool for antimicrobials to improve dental plaque prevention and treatment.

Topics: Anti-Infective Agents, Local; Biofilms; Dental Plaque; Diphosphates; Drug Carriers; Drug Delivery Systems; Humans; Micelles; Phosphoserine; Poloxalene; Streptococcus mutans; Tooth; Triclosan

Generalized arterial calcification of infancy (GACI), an autosomal recessive disorder, is characterized by early mineralization of blood vessels, often diagnosed by prenatal ultrasound and usually resulting in demise during the first year of life. It is caused in most cases by mutations in the ENPP1 gene, encoding an enzyme that hydrolyzes ATP to AMP and inorganic pyrophosphate, the latter being a powerful anti-mineralization factor. Recently, a novel mouse phenotype was recognized as a result of ENU mutagenesis - those mice developed stiffening of the joints, hence the mutant mouse was named 'ages with stiffened joints' (asj). These mice harbor a missense mutation, p.V246D, in the Enpp1 gene. Here we demonstrate that the mutant ENPP1 protein is largely absent in the liver of asj mice, and the lack of enzymatic activity results in reduced inorganic pyrophosphate (PPi) levels in the plasma, accompanied by extensive mineralization of a number of tissues, including arterial blood vessels. The progress of mineralization is highly dependent on the mineral composition of the diet, with significant shortening of the lifespan on a diet enriched in phosphorus and low in magnesium. These results suggest that the asj mouse can serve as an animal model for GACI.

Topics: Animals; Base Sequence; Calcification, Physiologic; Calcium; Diet; Diphosphates; Disease Models, Animal; DNA Mutational Analysis; Genotyping Techniques; Kaplan-Meier Estimate; Kidney; Mice; Mice, Mutant Strains; Molecular Sequence Data; Phenotype; Phosphoric Diester Hydrolases; Phosphorus; Pyrophosphatases; RNA, Messenger; Spectrometry, X-Ray Emission; Tomography, X-Ray Computed; Vascular Calcification; Vibrissae

Reactive Mn(IV) oxide minerals are ubiquitous in the environment and control the bioavailability and distribution of many toxic and essential elements and organic compounds. Their formation is thought to be dependent on microbial enzymes, because spontaneous Mn(II) to Mn(IV) oxidation is slow. Several species of marine Bacillus spores oxidize Mn(II) on their exosporium, the outermost layer of the spore, encrusting them with Mn(IV) oxides. Molecular studies have identified the mnx (Mn oxidation) genes, including mnxG, encoding a putative multicopper oxidase (MCO), as responsible for this two-electron oxidation, a surprising finding because MCOs only catalyze single-electron transfer reactions. Characterization of the enzymatic mechanism has been hindered by the lack of purified protein. By purifying active protein from the mnxDEFG expression construct, we found that the resulting enzyme is a blue (absorption maximum 590 nm) complex containing MnxE, MnxF, and MnxG proteins. Further, by analyzing the Mn(II)- and (III)-oxidizing activity in the presence of a Mn(III) chelator, pyrophosphate, we found that the complex facilitates both electron transfers from Mn(II) to Mn(III) and from Mn(III) to Mn(IV). X-ray absorption spectroscopy of the Mn mineral product confirmed its similarity to Mn(IV) oxides generated by whole spores. Our results demonstrate that Mn oxidation from soluble Mn(II) to Mn(IV) oxides is a two-step reaction catalyzed by an MCO-containing complex. With the purification of active Mn oxidase, we will be able to uncover its mechanism, broadening our understanding of Mn mineral formation and the bioinorganic capabilities of MCOs.

Topics: Bacillus; Cloning, Molecular; Diphosphates; DNA Primers; Escherichia coli; Manganese; Manganese Compounds; Mass Spectrometry; Multiprotein Complexes; Oxidation-Reduction; Oxides; Oxidoreductases; X-Ray Absorption Spectroscopy

A new naphthalene carbohydrazone based dizinc(II) complex has been synthesized and investigated to act as a highly selective fluorescence and visual sensor for a pyrophosphate ion with a quite low detection limit of 155 ppb; this has also been used to detect the pyrophosphate ion released from polymerase-chain-reaction.

Topics: Chemistry Techniques, Analytical; Colorimetry; Diphosphates; DNA; Fluorescent Dyes; Limit of Detection; Naphthalenes; Organometallic Compounds; Polymerase Chain Reaction; Zinc

In this work, based on the fact that pyrophosphate (PPi) could regulate the activity of Zn(2+)-dependent DNAzyme, we for the first time report a fluorescence turn-on sensing system for alkaline phosphatase (ALP) with improved sensitivity via nonprotein-enzymatic signal amplification. A catalytic and molecular beacon (CAMB) design was employed to further improve its sensitivity. Taking advantage of the strong interactions between PPi and the Zn(2+), the cofactor Zn(2+) was caged, and the DNAzyme activity was effectively inhibited. The introduction of ALP, however, could catalyze the hydrolysis of PPi and release free Zn(2+), resulting in the activation of DNAzyme to catalyze the cleavage of the molecular beacon substrate with a remarkable increase of fluorescent signal. These optimized designs together allow a high sensitivity for ALP, with a detection limit of 20 pM observed, much lower than previously reported methods. It has also been used for detection of ALP in human serum with satisfactory results, demonstrating its potential applications in clinical diagnosis.

Topics: Alkaline Phosphatase; Biosensing Techniques; Diphosphates; DNA, Catalytic; Enzyme Assays; Humans; Hydrolysis; Limit of Detection; Spectrometry, Fluorescence; Zinc

A diformyl-quinoline based receptor (L1) exhibits selective colorimetric and fluorometric sensing of Zn(2+) in aqueous medium at pH 7.4 based on the intraligand charge transfer (ICT) process. The in situ formed phenoxo-bridged complex, L1·2Zn can selectively and specifically sense PPi among all the other biologically important anions including ATP through reversible binding. The detection limit for Zn(2+) and PPi were found to be approximately 56 and 2 ppb, respectively. The unique selectivity of the PPi by the L1-Zn ensemble could be used as an analytical tool to probe PPi generation in a prototype polymerase chain reaction (PCR) setup and track DNA amplification with higher sensitivity as compared to conventional agarose gel electrophoresis. Interestingly, the principle of PPi estimation in PCR rendered rapid estimation of bacterial cell numbers with a limit of detection of 10 CFU of Escherichia coli MTCC 433 in as early as 10 PCR cycles. The proposed method of PPi sensing offers interesting application potential in PCR-based rapid diagnostics for pathogenic agents and microbiological quality control.

Topics: Cell Count; Diphosphates; DNA, Bacterial; Escherichia coli; Polymerase Chain Reaction; Zinc

We report that magnetite nanoparticles (Fe3O4 NPs) act as an efficient quencher for boron dipyrromethene-conjugated adenosine 5'-triphosphate (BODIPY-ATP) that is highly fluorescent in bulk solution. BODIPY-ATP molecules attached to the surface of Fe3O4 NPs through the coordination between the triphosphate group of BODIPY-ATP and Fe(3+)/Fe(2+) on the NP surface. The formed complexes induced an apparent reduction in the BODIPY-ATP fluorescence resulting from an oxidative-photoinduced electron transfer (PET) from the BODIPY-ATP excited state to an unfilled d shell of Fe(3+)/Fe(2+) on the NP surface. A comparison of the Stern-Volmer quenching constant between Fe(3+) and Fe(2+) suggests that Fe(3+) on the NP surface dominantly controls this quenching process. The efficiency for Fe3O4 NP-induced fluorescence quenching of the BODIPY-ATP was enhanced by increasing the concentration of Fe3O4 NPs and lowering the pH of the solution to below 6.0. We found that pyrophosphate and ATP compete with BODIPY-ATP for binding to Fe3O4 NPs. Thus, we amplified BODIPY-ATP fluorescence in the presence of increasing the pyrophosphate and ATP concentration; the detection limits at a signal-to-noise ratio of 3 for pyrophosphate and ATP were determined to be 7 and 30 nM, respectively. The Fe3O4 NP-based competitive binding assay detected ATP and pyrophosphate in only 5 min. The selectivity of this assay for ATP over metal ions, amino acids, and adenosine analogues is particularly high. The practicality of using the developed method to determine ATP in a single drop of blood is also validated.

Topics: Adenosine Triphosphate; Biosensing Techniques; Boron Compounds; Diphosphates; Fluorescent Dyes; Humans; Magnetite Nanoparticles; Spectrometry, Fluorescence

Transcription elongation consists of repetition of the nucleotide addition cycle: phosphodiester bond formation, translocation and binding of the next nucleotide. Inhibitor of multi-subunit RNA polymerase tagetitoxin (TGT) enigmatically slows down addition of nucleotides in a sequence-dependent manner, only at certain positions of the template. Here, we show that TGT neither affects chemistry of RNA synthesis nor induces backward translocation, nor competes with the nucleoside triphosphate (NTP) in the active center. Instead, TGT increases the stability of the pre-translocated state of elongation complex, thus slowing down addition of the following nucleotide. We show that the extent of inhibition directly depends on the intrinsic stability of the pre-translocated state. The dependence of translocation equilibrium on the transcribed sequence results in a wide distribution (~1-10(3)-fold) of inhibitory effects of TGT at different positions of the template, thus explaining sequence-specificity of TGT action. We provide biochemical evidence that, in pre-translocated state, TGT stabilizes folded conformation of the Trigger Loop, which inhibits forward and backward translocation of the complex. The results suggest that Trigger Loop folding in the pre-translocated state may serve to reduce back-tracking of the elongation complex. Overall, we propose that translocation may be a limiting and highly regulated step of RNA synthesis.

Topics: Dicarboxylic Acids; Diphosphates; DNA-Directed RNA Polymerases; Nucleic Acid Synthesis Inhibitors; Organophosphorus Compounds; Transcription Elongation, Genetic; Transcriptional Elongation Factors

During endochondral bone formation, chondrocytes and osteoblasts synthesize and mineralize the extracellular matrix through a process that initiates within matrix vesicles (MVs) and ends with bone mineral propagation onto the collagenous scaffold. pH gradients have been identified in the growth plate of long bones, but how pH changes affect the initiation of skeletal mineralization is not known. Tissue-nonspecific alkaline phosphatase (TNAP) degrades extracellular inorganic pyrophosphate (PPi), a mineralization inhibitor produced by ectonucleotide pyrophosphatase/phosphodiesterase-1 (NPP1), while contributing Pi from ATP to initiate mineralization. TNAP and NPP1, alone or combined, were reconstituted in dipalmitoylphosphatidylcholine liposomes to mimic the microenvironment of MVs. The hydrolysis of ATP, ADP, AMP, and PPi was studied at pH 8 and 9 and compared to the data determined at pH 7.4. While catalytic efficiencies in general were higher at alkaline pH, PPi hydrolysis was maximal at pH 8 and indicated a preferential utilization of PPi over ATP at pH 8 versus 9. In addition, all proteoliposomes induced mineral formation when incubated in a synthetic cartilage lymph containing 1 mM ATP as substrate and amorphous calcium phosphate or calcium-phosphate-phosphatidylserine complexes as nucleators. Propagation of mineralization was significantly more efficient at pH 7.5 and 8 than at pH 9. Since a slight pH elevation from 7.4 to 8 promotes considerably more hydrolysis of ATP, ADP, and AMP primarily by TNAP, this small pH change facilitates mineralization, especially via upregulated PPi hydrolysis by both NPP1 and TNAP, further elevating the Pi/PPi ratio, thus enhancing bone mineralization.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Adenosine Triphosphate; Alkaline Phosphatase; Animals; Biomimetics; Calcium Phosphates; Chlorocebus aethiops; COS Cells; Diphosphates; Electrolytes; Hydrogen-Ion Concentration; Hydrolysis; Liposomes; Mice; Phosphates; Phosphatidylserines; Phosphoric Diester Hydrolases; Plasmids; Polidocanol; Polyethylene Glycols; Proteolipids; Pyrophosphatases; Rats

The analysis of free amino acids in urine and plasma is useful for estimating disease status in clinical diagnoses. Changes in the concentration of free amino acids in foods are also useful markers of freshness, nutrition, and taste. In this study, the specific interaction between aminoacyl-tRNA synthetase (aaRS) and its corresponding amino acid was used to measure amino acid concentrations. Pyrophosphate released by the amino acid-aaRS binding reaction was detected by luminol chemiluminescence; the method provided selective quantitation of 1.0-30 μM histidine and 1.0-60 μM lysine.

Topics: Amino Acyl-tRNA Synthetases; Calibration; Diphosphates; Enzyme Assays; Histidine; Hydrogen Peroxide; Inorganic Pyrophosphatase; Limit of Detection; Luminescence; Luminescent Measurements; Luminol; Lysine; Protein Binding; Pyruvate Oxidase; Reproducibility of Results; Solutions; Temperature

Dinuclear zinc complex [Zn2(TQHPN)(AcO)](2+) exhibits characteristic fluorescence response (λex = 317 nm and λem = 455 nm) toward pyrophosphate (PPi) with maximum fluorescence upon 1:1 Zn2(TQHPN)-PPi complex formation. The crystallographic investigation utilizing P(1)P(2)-Ph2PPi revealed that the fluorescent response mechanism is due to intramolecular excimer formation of two quinoline rings.

Topics: Diphosphates; Fluorescence; Models, Molecular; Molecular Structure; Organometallic Compounds; Quinolines; Spectrometry, Fluorescence; Zinc

In this study we demonstrate a new colorimetric method for real-time pyrophosphatase (PPase) activity assay based on reversible tuning of the dispersion/aggregation states of gold nanoparticles (Au-NPs) by controlling the coordination of Cu(2+) between cysteine and pyrophosphate ion (PPi) with PPase. The addition of Cu(2+) to the cysteine-stabilized Au-NP dispersion results in the aggregation of Au-NPs, while the further addition of PPi to this aggregation turns the aggregated Au-NPs into their dispersed state because of the higher coordination reactivity between Cu(2+) and PPi than that between Cu(2+) and cysteine. The subsequent addition of PPase to the PPi-triggered dispersed Au-NPs restores the aggregation state of Au-NPs because PPase catalyzes the hydrolysis of PPi into orthophosphate and thus consumes PPi in the reaction system. In this study, we utilize this reversibility of the change between the aggregation/dispersion states of Au-NPs for real-time colorimetric monitoring of PPase activity by continuously measuring the ratio of absorbance at the wavelength of 650 nm (A650) to that at 522 nm (A522) in the time-dependent UV-vis spectra of Au-NP dispersions containing different activities of PPase. To calculate the kinetics of the PPase-catalyzed hydrolysis of PPi, the A650/A522 values are converted into PPi concentrations to obtain the time-dependent changes of PPi concentrations in the dispersions containing different activities of PPase. The initial reaction rates (v0) are thus achieved from the time-dependent logarithm of PPi concentrations with the presence of different PPase activities. Under the experimental conditions employed here, the v0 values are linear with the PPase activity within a range from 0.025 to 0.4 U with a detection limit down to 0.010 U (S/N = 3). Moreover, the colorimetric method developed here is also employed for PPase inhibitor evaluation. This study offers a simple yet effective method for real-time PPase activity assay.

Topics: Colorimetry; Copper; Cysteine; Diphosphates; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Gold; Ions; Metal Nanoparticles; Molecular Structure; Pyrophosphatases; Sodium Fluoride; Structure-Activity Relationship; Surface Properties; Time Factors

ATP sulfurylase (ATPS) catalyzes a key reaction in the global sulfur cycle by reversibly converting inorganic sulfate (SO4 (2-)) with ATP to adenosine 5'-phosphosulfate (APS) and pyrophosphate (PPi). In this work we report on the sat encoded dissimilatory ATP sulfurylase from the sulfur-oxidizing purple sulfur bacterium Allochromatium vinosum. In this organism, the sat gene is located in one operon and co-transcribed with the aprMBA genes for membrane-bound APS reductase. Like APS reductase, Sat is dispensible for growth on reduced sulfur compounds due to the presence of an alternate, so far unidentified sulfite-oxidizing pathway in A. vinosum. Sulfate assimilation also proceeds independently of Sat by a separate pathway involving a cysDN-encoded assimilatory ATP sulfurylase. We produced the purple bacterial sat-encoded ATP sulfurylase as a recombinant protein in E. coli, determined crucial kinetic parameters and obtained a crystal structure in an open state with a ligand-free active site. By comparison with several known structures of the ATPS-APS complex in the closed state a scenario about substrate-induced conformational changes was worked out. Despite different kinetic properties ATPS involved in sulfur-oxidizing and sulfate-reducing processes are not distinguishable on a structural level presumably due to the interference between functional and evolutionary processes.

Topics: Adenosine Phosphosulfate; Adenosine Triphosphate; Amino Acid Sequence; Bacterial Proteins; Chromatiaceae; Diphosphates; Escherichia coli; Molecular Sequence Data; Protein Conformation; Recombinant Proteins; Sequence Homology, Amino Acid; Sulfate Adenylyltransferase; Sulfates

To evaluate the influence of chronic stress (CS) on urine composition of calcium oxalate (CaOx) stone patients and controls.. This case-control study enrolled 128 patients during a period of 20 months. The cases were CaOx stone formers with a recent stone episode. Controls were matched by sex and age. Dimensions of CS were evaluated in cases and controls by validated self-report questionnaires measuring stressful life events, perceived stress, anxiety, depression, burnout, and satisfaction with life. Blood and urine samples were collected to determine cortisol levels and urinary composition.. More relations between CS dimensions and blood and urine parameters were observed in cases than in controls. In cases, the blood cortisol level was related positively with the number of stressful life events (P = .03), intensity of these events (P = .04), and anxiety (P = .04). In addition, urinary magnesium (P = .03) and pyrophosphate (P = .05) levels were positively related with satisfaction with life and burnout, respectively. In contrast, urinary magnesium levels were negatively related with perceived stress (P = .01), anxiety (P = .016), and depression (P = .03). In controls, the number of stressful life events and the intensity of stressful life events was related positively with magnesium (P = .06, P = .02) levels and negatively with blood cortisol levels (P = .03, P = .004).. Based on the variation between cases and controls in relations between CS dimensions and biochemical parameters, we hypothesize that CS may trigger a differential biological response in CaOx stone formers and controls, which in turn may promote or protect against CaOx stone formation.

Topics: Adult; Aged; Anxiety; Case-Control Studies; Depression; Diphosphates; Female; Humans; Hydrocortisone; Magnesium; Male; Middle Aged; Nephrolithiasis; Stress, Psychological; Urine; Young Adult

Tuberculosis (TB) causes up to 10 million incident cases worldwide per annum. Multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains are leading factors in the resurgence of TB cases and the need to produce new agents to combat such infection. Herein, we describe Co(II) and Cu(II) metal based complexes that feature the pyrophosphate ligand with notable selectivity and marked potency against Mycobacterium tuberculosis, including MDR strains. Such complexes are confirmed to be bacteriocidal and not affected by efflux inhibitors. Finally, while susceptibility to copper has recently been established for M. tuberculosis, the greater efficacy of cobalt observed herein is of considerable note and in line with the discovery of a copper metallothionein in M. tuberculosis.

Topics: Anti-Bacterial Agents; Cobalt; Copper; Diphosphates; Dose-Response Relationship, Drug; Microbial Sensitivity Tests; Models, Molecular; Mycobacterium; Organometallic Compounds; Structure-Activity Relationship

A colorimetric pyrophosphate assay based on the formation and reduction of the 18-molybdopyrophosphate ([(P2O7)Mo18O54](4-)) anion in an acetonitrile-water mixed solvent was modified and improved. The [(P2O7)Mo18O54](4-) anion is precipitated from the acetonitrile-water solution containing MoO4(2-) and HCl, and is re-dissolved in neat acetonitrile or propylene carbonate. This separation process decreases the interference by ATP, and prevents a yellow coloration of the reducing agent, ascorbic acid, due to excess Mo(VI) species. In the organic solvent, the [(P2O7)Mo18O54](4-) anion is reduced to a more intense blue molybdopyrophosphate species. The application of the colorimetry to the assay of adenylation enzymes is also described in this note.

Topics: Colorimetry; Diphosphates; Ions; Peptide Synthases

Pseudoxanthoma elasticum (PXE) is an autosomal recessive disease characterized by progressive ectopic mineralization of the skin, eyes, and arteries, for which no effective treatment exists. PXE is caused by inactivating mutations in the gene encoding ATP-binding cassette sub-family C member 6 (ABCC6), an ATP-dependent efflux transporter present mainly in the liver. Abcc6(-/-) mice have been instrumental in demonstrating that PXE is a metabolic disease caused by the absence of an unknown factor in the circulation, the presence of which depends on ABCC6 in the liver. Why absence of this factor results in PXE has remained a mystery. Here we report that medium from HEK293 cells overexpressing either human or rat ABCC6 potently inhibits mineralization in vitro, whereas medium from HEK293 control cells does not. Untargeted metabolomics revealed that cells expressing ABCC6 excrete large amounts of nucleoside triphosphates, even though ABCC6 itself does not transport nucleoside triphosphates. Extracellularly, ectonucleotidases hydrolyze the excreted nucleoside triphosphates to nucleoside monophosphates and inorganic pyrophosphate (PPi), a strong inhibitor of mineralization that plays a pivotal role in several mineralization disorders similar to PXE. The in vivo relevance of our data are demonstrated in Abcc6(-/-) mice, which had plasma PPi levels <40% of those found in WT mice. This study provides insight into how ABCC6 affects PXE. Our data indicate that the factor that normally prevents PXE is PPi, which is provided to the circulation in the form of nucleoside triphosphates via an as-yet unidentified but ABCC6-dependent mechanism.

Topics: Animals; Dinucleoside Phosphates; Diphosphates; DNA Primers; DNA, Complementary; HEK293 Cells; Humans; Metabolic Diseases; Metabolomics; Mice; Mice, Knockout; Multidrug Resistance-Associated Proteins; Mutagenesis, Site-Directed; Mutation; Pseudoxanthoma Elasticum; Rats

Diphosphate (pyrophosphate, PPi) is vital for organisms, and therefore its detection is of special importance. In this paper, one cadmium complex of terpyridine (tpy) derivative, 4'-(aminomethylphenyl)-2,2':6',2"-terpyridine (aptpy), has been reported for the ratiometric fluorescence recognition of PPi. When added with cadmium, the emission of aptpy at 358 nm was greatly enhanced and red shifted to 397 nm due to the complexation-induced ICT process, which then blue shifted to 349 nm upon the further addition of PPi. Based on the different response of dual fluorescence emissions at 349 and 397 nm, a ratiometric fluorescence method could be successfully established for the fluorescence recognition of PPi. With that, PPi could be successfully discriminated from other structurally similar anions, including nucleotide triphosphates.

Topics: Diphosphates; Pyridines; Spectrometry, Fluorescence

The simple proton-translocating inorganic pyrophosphatase (H(+)-PPase) found in plants and protists is an evolutionally conserved, essential enzyme that catalyzes the hydrolysis of pyrophosphate (PPi). Little is known about the functional contribution of H(+)-PPase to the cellular response to abiotic stresses, except its high salinity and drought stress. To investigate the role of H(+)-PPase during response to cellular stress, we isolated the cDNA of Arabidopsis thaliana H(+)-PPase (AVP1) and Oryza sativa H(+)-PPase (OVP1) and constructed transgenic Saccharomyces cerevisiae and Escherichia coli lines that express AVP1 and OVP1. In S. cerevisiae, the expression of a chimeric derivative of the AVP1 and OVP1 alleviated the phenotype associated with ipp2-deficient cells in the presence of high salinity (NaCl) and metal stressors (Cd, Mn, and Zn). In E. coli, AVP1 and OVP1 overexpression conferred enhanced tolerance to abiotic stresses, including heat shock and H(2)O(2), as well as NaCl, Cd, Mn, Zn, Ca, and Al. Interestingly, AVP1 and OVP1 overexpression resulted in hypersensitivity to menadione and cobalt. These results demonstrate the cellular capacity of AVP1- and OVP1-expressing transgenic yeast and E. coli in response to physiological, abiotic stresses. Moreover, our results suggest new ways of engineering stress-tolerant plants that are capable of responding to climate change. Here, we provide an outline of an experimental system to examine the alternative roles of plant H(+)-PPase.

Topics: Arabidopsis; Arabidopsis Proteins; Diphosphates; Escherichia coli; Gene Transfer Techniques; Inorganic Pyrophosphatase; Oryza; Saccharomyces cerevisiae; Stress, Physiological

Here, we describe a high-throughput, single-tube, allele-specific ribonucleotide analog pyrophosphorolysis-activated polymerization (ribo-PAP) PCR multiplex genotyping and resequencing method. An RNA/DNA chimeric PCR product is generated using genomic DNA as starting template, a panel of allele-selective 5'-tagged primers, a reverse primer, one nucleotide in the ribo-form (90-100%), the other nucleotides in the deoxy-form, a DNA polymerase capable of incorporating ribonucleotides, a suitable buffer and thermal cycling. The RNA/DNA chimeric PCR products are fragmented by treatment with alkali and analyzed by mass spectrometry. All allele-selective primers have a 5' repetitive motif where each repeat unit has a unique, distinct mass upon reverse copying and alkali fragmentation. The mass of the complement repeat fragment or flag identifies the primer or primers that were recruited in the ribo-PAP PCR. The method readily identifies homozygous and heterozygous positions in simplex or duplex ribo-PAP PCR. Many different tags can be analyzed simultaneously. The assay can genotype several SNPs in a single tube. It thus constitutes the simplest genotyping protocol with multiplex analysis. This novel genotyping and resequencing protocol was applied to different genomic loci: NOS1 and H19 in 30 individuals in simplex ribo-PAP PCR and at two SLCO1B1 loci in 95 individuals in duplex ribo-PAP PCR.

Topics: Alkalies; Alleles; Diphosphates; DNA; DNA Primers; Genotype; Genotyping Techniques; Humans; Hydrogen-Ion Concentration; Nitric Oxide Synthase Type I; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Reproducibility of Results; Ribonucleotides; RNA; RNA, Long Noncoding; Sequence Analysis, DNA; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

A kinetic method has been developed for highly sensitive and selective detection of pyrophosphate anions (PPi) by using a fluorophore labelled single-stranded-DNA-Al(III) complex system. The facile and spectrum-tunable fluorescent sensor enables rapid detection of PPi in urine and cell lysate solutions without the need for complex pre-cleanup procedures.

Topics: Aluminum; Anions; Cell Line; Coordination Complexes; Diphosphates; DNA, Single-Stranded; Fluorescent Dyes; Humans; Kinetics; Sensitivity and Specificity; Spectrometry, Fluorescence

Highly selective detection of pyrophosphate in the presence of inorganic phosphates, halides, acetate, ATP, other nucleotides in neutral aqueous solutions is achieved by a fluorescent Zn-ensemble based system. The implementation of the first molecule based JK-latch sequential logic function is also described.

Topics: Anthraquinones; Diphosphates; Fluorescent Dyes; Molecular Structure; Organometallic Compounds; Spectrometry, Fluorescence

To investigate the ability of a novel mouthwash comprised of 0.8% arginine, PVM/MA copolymer, pyrophosphates, and 0.05% sodium fluoride in an alcohol-free base (Pro-Argin™ Mouthwash Technology) to reduce dentine permeability.. Hydraulic conductance was used to assess the dentine permeability effects of the arginine mouthwash. Aqueous solutions containing arginine and PVM/MA copolymer were studied in the initial stage of the method development. The acid resistance was tested with a cola drink challenge. Finally, a blinded study was carried out to determine the occlusion of the arginine mouthwash in comparison to a negative control mouthwash.. Dentine discs treated with the arginine mouthwash showed an average fluid reduction of 42%, which was statistically, significantly better than the fluid reduction for the negative control mouthwash. In addition, experiments using simple solutions of arginine and PVM/MA copolymer, alone and in combination, demonstrated that the combination of the two was required to provide a relevant occlusion benefit. Finally, the occlusion provided by the arginine mouthwash was maintained after exposure to an acid challenge.. The exclusive combination of ingredients in the arginine mouthwash has been proven to be efficacious in decreasing dentine fluid flow as measured by hydraulic conductance. The new mouthwash works by occlusion, due to the unique combination of arginine, PVM/MA copolymer and pyrophosphates.

Topics: Arginine; Calcium Carbonate; Dentin Desensitizing Agents; Diphosphates; Drug Combinations; Drug Compounding; Fluorides; Humans; Hydrodynamics; Maleates; Mouthwashes; Phosphates; Polyethylenes; Sodium Fluoride

The mode of action of an arginine mouthwash using the Pro-Argin™ Mouthwash Technology, containing 0.8% arginine, PVM/MA copolymer, pyrophosphates and 0.05% sodium fluoride, has been proposed and confirmed as occlusion using a variety of in vitro techniques.. Quantitative and qualitative laboratory techniques were employed to investigate the mode of action of the new arginine mouthwash. Confocal laser scanning microscopy (CSLM) and atomic force microscopy (AFM) investigated a hydrated layer on dentine surface. Electron spectroscopy for chemical analysis (ESCA), secondary ion mass spectroscopy (SIMS) and near-infrared spectroscopy (NIR) provided information about its chemical nature.. CLSM was used to observe the formation of a hydrated layer on exposed dentine tubules upon application of the arginine mouthwash. Fluorescence studies confirmed penetration of the hydrated layer in the inner walls of the dentinal tubules. The AFM investigation confirmed the affinity of the arginine mouthwash for the dentine surface, supporting its adhesive nature. NIR showed the deposition of arginine after several mouthwash applications, and ESCA/SIMS detected the presence of phosphate groups and organic acid groups, indicating the deposition of copolymer and pyrophosphates along with arginine.. The studies presented in this paper support occlusion of the dentine surface upon the deposition of an arginine-rich layer together with copolymer and phosphate ions from an alcohol-free mouthwash containing 0.8% arginine, PVM/MA copolymer, pyrophosphates and 0.05% sodium fluoride.

Topics: Arginine; Calcium Carbonate; Dentin Desensitizing Agents; Diphosphates; Drug Combinations; Fluorides; Humans; Maleates; Mouthwashes; Phosphates; Polyethylenes; Sodium Fluoride

Acanthamoeba polyphaga mimivirus tyrosyl-tRNA synthetase (TyrRSapm) was the first reported aminoacyl-tRNA synthetase of viral origin. The previous crystal structure of TyrRSapm showed a non-canonical orientation of the dimer conformation and the CP1 domain, responsible for dimer formation, displays a major modification of a motif structurally conserved in other TyrRS structures. An earlier study reported that Bacillus stearothermophilus N-terminal TyrRS exists as a dimer under native conditions. N-terminal TyrRSapm (ΔTyrRSapm, 1-234 aa) was constructed to remove the C-terminal anticodon-binding domain. Here we show by Ferguson plot analysis and analytical ultracentrifugation that ΔTyrRSapm exists as a monomer and contains a disulfide-bridge. The ΔTyrRSapm loses the ability to bind tRNA(Tyr), however it remains active in pyrophosphate exchange with similar ligand dissociation constants as the full-length enzyme.

Topics: Amino Acid Motifs; Circular Dichroism; Diphosphates; Disulfides; Humans; Kinetics; Ligands; Mimiviridae; Mutation; Protein Multimerization; Protein Structure, Secondary; Protein Structure, Tertiary; Recombinant Proteins; Solutions; Thermodynamics; Tyrosine-tRNA Ligase; Ultracentrifugation; Viral Proteins

A new family of bis-ureidic receptors (L(1)-L(6)) has been synthesised. The binding properties of L(1)-L(6) towards different anions (acetate, benzoate, glutarate, malonate, dihydrogen phosphate, hydrogen pyrophosphate, triphosphate, AMP and ADP) have been studied by means of (1)H-NMR, UV-Vis and fluorescence spectroscopies and a remarkable affinity for HPpi(3-) has been observed in the case L(3) (in DMSO-d6 and DMSO-d6-5% H2O) which also acts as a fluorimetric chemosensor, even to the naked eye, for this anion. Theoretical calculations helped us explain the binding properties observed.

Topics: Chemistry Techniques, Analytical; Diphosphates; Models, Molecular; Molecular Conformation; Optical Phenomena; Urea

Mutations in the liver/bone/kidney alkaline phosphatase (ALPL) gene in hypophosphatasia (HPP) reduce the function of tissue non-specific alkaline phosphatase (ALP), resulting in increased pyrophosphate (PP(i)) and a severe deficiency in acellular cementum. We hypothesize that exogenous phosphate (P(i)) would rescue the in vitro mineralization capacity of periodontal ligament (PDL) cells harvested from HPP-diagnosed patients, by correcting the P(i)/PP(i) ratio and modulating expression of genes involved with P(i)/PP(i) metabolism.. Ex vivo and in vitro analyses were used to identify mechanisms involved in HPP-associated PDL/tooth root deficiencies. Constitutive expression of PP(i)-associated genes was contrasted in PDL versus pulp tissues obtained from healthy individuals. Primary PDL cell cultures from patients with HPP (monozygotic twin males) were established to assay ALP activity, in vitro mineralization, and gene expression. Exogenous P(i) was provided to correct the P(i)/PP(i) ratio.. PDL tissues obtained from healthy individuals featured higher basal expression of key PP(i) regulators, genes ALPL, progressive ankylosis protein (ANKH), and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), versus paired pulp tissues. A novel ALPL mutation was identified in the twin patients with HPP enrolled in this study. Compared to controls, HPP-PDL cells exhibited significantly reduced ALP and mineralizing capacity, which were rescued by addition of 1 mM P(i). Dysregulated expression of PP(i) regulatory genes ALPL, ANKH, and ENPP1 was also corrected by adding P(i), although other matrix markers evaluated in our study remained downregulated.. These findings underscore the importance of controlling the P(i)/PP(i) ratio toward development of a functional periodontal apparatus and support P(i)/PP(i) imbalance as the etiology of HPP-associated cementum defects.

Topics: Adolescent; Alkaline Phosphatase; Case-Control Studies; Dental Cementum; Dental Pulp; Diphosphates; Diseases in Twins; DNA Mutational Analysis; Female; Gene Expression Profiling; Gene Expression Regulation, Enzymologic; Humans; Hypophosphatasia; Male; Periodontal Ligament; Phosphate Transport Proteins; Phosphates; Phosphoric Diester Hydrolases; Primary Cell Culture; Pyrophosphatases; Tooth Calcification; Young Adult

The WaaL-mediated ligation of O-antigen onto the core region of the lipid A-core block is an important step in the lipopolysaccharide (LPS) biosynthetic pathway. Although the LPS biosynthesis has been largely characterized, only a limited amount of in vitro biochemical evidence has been established for the ligation reaction. Such limitations have primarily resulted from the barriers in purifying WaaL homologues and obtaining chemically defined substrates. Accordingly, we describe herein a chemical biology approach that enabled the reconstitution of this ligation reaction. The O-antigen repeating unit (O-unit) of Escherichia coli O86 was first enzymatically assembled via sequential enzymatic glycosylation of a chemically synthesized GalNAc-pyrophosphate-undecaprenyl precursor. Subsequent expression of WaaL through use of a chaperone co-expression system then enabled the demonstration of the in vitro ligation between the synthesized donor (O-unit-pyrophosphate-undecaprenyl) and the isolated lipid A-core acceptor. The previously reported ATP and divalent metal cation dependence were not observed using this system. Further analyses of other donor substrates revealed that WaaL possesses a highly relaxed specificity toward both the lipid moiety and the glycan moiety of the donor. Lastly, three conserved amino acid residues identified by sequence alignment were found essential for the WaaL activity. Taken together, the present work represents an in vitro systematic investigation of the WaaL function using a chemical biology approach, providing a system that could facilitate the elucidation of the mechanism of WaaL-catalyzed ligation reaction.

Topics: Carbon-Oxygen Ligases; Cell Membrane; Diphosphates; Escherichia coli; Escherichia coli Proteins; Mutation; O Antigens; Substrate Specificity

Pyrophosphate ion (PP(i)) release after nucleotide incorporation is a necessary step for RNA polymerase II (pol II) to enter the next nucleotide addition cycle during transcription elongation. However, the role of pol II residues in PP(i) release and the mechanistic relationship between PP(i) release and the conformational change of the trigger loop remain unclear. In this study, we constructed a Markov state model (MSM) from extensive all-atom molecular dynamics (MD) simulations in the explicit solvent to simulate the PP(i) release process along the pol II secondary channel. Our results show that the trigger loop has significantly larger intrinsic motion after catalysis and formation of PP(i), which in turn aids PP(i) release mainly through the hydrogen bonding between the trigger loop residue H1085 and the (Mg-PP(i))(2-) group. Once PP(i) leaves the active site, it adopts a hopping model through several highly conserved positively charged residues such as K752 and K619 to release from the pol II pore region of the secondary channel. These positive hopping sites form favorable interactions with PP(i) and generate four kinetically metastable states as identified by our MSM. Furthermore, our single-mutant simulations suggest that H1085 and K752 aid PP(i) exit from the active site after catalysis, whereas K619 facilitates its passage through the secondary channel. Finally, we suggest that PP(i) release could help the opening motion of the trigger loop, even though PP(i) release precedes full opening of the trigger loop due to faster PP(i) dynamics. Our simulations provide predictions to guide future experimental tests.

Topics: Diphosphates; Ions; Models, Molecular; Molecular Dynamics Simulation; RNA Polymerase II

The molecular basis of fructose-2,6-bisphosphatase (F-2,6-P(2)ase) of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB) was investigated using the crystal structures of the human inducible form (PFKFB3) in a phospho-enzyme intermediate state (PFKFB3-P•F-6-P), in a transition state-analogous complex (PFKFB3•AlF(4)), and in a complex with pyrophosphate (PFKFB3•PP(i)) at resolutions of 2.45, 2.2, and 2.3 Å, respectively. Trapping the PFKFB3-P•F-6-P intermediate was achieved by flash cooling the crystal during the reaction, and the PFKFB3•AlF(4) and PFKFB3•PP(i) complexes were obtained by soaking. The PFKFB3•AlF(4) and PFKFB3•PP(i) complexes resulted in removing F-6-P from the catalytic pocket. With these structures, the structures of the Michaelis complex and the transition state were extrapolated. For both the PFKFB3-P formation and break down, the phosphoryl donor and the acceptor are located within ~5.1 Å, and the pivotal point 2-P is on the same line, suggesting an "in-line" transfer with a direct inversion of phosphate configuration. The geometry suggests that NE2 of His253 undergoes a nucleophilic attack to form a covalent N-P bond, breaking the 2O-P bond in the substrate. The resulting high reactivity of the leaving group, 2O of F-6-P, is neutralized by a proton donated by Glu322. Negative charges on the equatorial oxygen of the transient bipyramidal phosphorane formed during the transfer are stabilized by Arg252, His387, and Asn259. The C-terminal domain (residues 440-446) was rearranged in PFKFB3•PP(i), implying that this domain plays a critical role in binding of substrate to and release of product from the F-2,6-P(2) ase catalytic pocket. These findings provide a new insight into the understanding of the phosphoryl transfer reaction.

Topics: Aluminum Compounds; Amino Acid Sequence; Catalytic Domain; Conserved Sequence; Diphosphates; Enzyme Activation; Enzyme Assays; Enzyme Stability; Escherichia coli; Fluorides; Humans; Isoenzymes; Ligands; Models, Molecular; Molecular Sequence Data; Multiprotein Complexes; Phosphofructokinase-2; Protein Binding; Protein Isoforms; Proteolysis; Sequence Alignment; Structure-Activity Relationship; Water

The synthesis of DNA, RNA, and de novo proteins is fundamental for early development of the seedling after germination, but such processes release pyrophosphate (PPi) as a byproduct of ATP hydrolysis. The over-accumulation of the inhibitory metabolite PPi in the cytosol hinders these biosynthetic reactions. All living organisms possess ubiquitous enzymes collectively called inorganic pyrophosphatases (PPases), which catalyze the hydrolysis of PPi into two orthophosphate (Pi) molecules. Defects in PPase activity cause severe developmental defects and/or growth arrest in several organisms. In higher plants, a proton-translocating vacuolar PPase (H+-PPase) uses the energy of PPi hydrolysis to acidify the vacuole. However, the biological implications of PPi hydrolysis are vague due to the widespread belief that the major role of H+-PPase in plants is vacuolar acidification. We have shown that the Arabidopsis fugu5 mutant phenotype, caused by a defect in H+-PPase activity, is rescued by complementation with the yeast cytosolic PPase IPP1. In addition, our analyses have revealed that increased cytosolic PPi levels impair postgerminative development in fugu5 by inhibiting gluconeogenesis. This led us to the conclusion that the role of H+-PPase as a proton-pump is negligible. Here, we present further evidence of the growth-boosting effects of removing PPi in later stages of plant vegetative development, and briefly discuss the biological role of PPases and their potential applications in different disciplines and in various organisms.

Topics: Adenosine Triphosphate; Animals; Cytosol; Diphosphates; Homeostasis; Hydrolysis; Inorganic Pyrophosphatase; Plant Development

Complexes of phi29 DNA polymerase and DNA fluctuate on the millisecond time scale between two ionic current amplitude states when captured atop the α-hemolysin nanopore in an applied field. The lower amplitude state is stabilized by complementary dNTP and thus corresponds to complexes in the post-translocation state. We have demonstrated that in the upper amplitude state, the DNA is displaced by a distance of one nucleotide from the post-translocation state. We propose that the upper amplitude state corresponds to complexes in the pre-translocation state. Force exerted on the template strand biases the complexes toward the pre-translocation state. Based on the results of voltage and dNTP titrations, we concluded through mathematical modeling that complementary dNTP binds only to the post-translocation state, and we estimated the binding affinity. The equilibrium between the two states is influenced by active site-proximal DNA sequences. Consistent with the assignment of the upper amplitude state as the pre-translocation state, a DNA substrate that favors the pre-translocation state in complexes on the nanopore is a superior substrate in bulk phase for pyrophosphorolysis. There is also a correlation between DNA sequences that bias complexes toward the pre-translocation state and the rate of exonucleolysis in bulk phase, suggesting that during DNA synthesis the pathway for transfer of the primer strand from the polymerase to exonuclease active site initiates in the pre-translocation state.

Topics: ATP-Binding Cassette Transporters; Bacillus Phages; Catalytic Domain; Diphosphates; DNA Replication; DNA-Directed DNA Polymerase; DNA, Viral; Enzyme Activation; Exonucleases; Hemolysin Proteins; Inverted Repeat Sequences; Molecular Motor Proteins; Nanopores; Nucleic Acid Conformation

The sulfonamide antibiotics inhibit dihydropteroate synthase (DHPS), a key enzyme in the folate pathway of bacteria and primitive eukaryotes. However, resistance mutations have severely compromised the usefulness of these drugs. We report structural, computational, and mutagenesis studies on the catalytic and resistance mechanisms of DHPS. By performing the enzyme-catalyzed reaction in crystalline DHPS, we have structurally characterized key intermediates along the reaction pathway. Results support an S(N)1 reaction mechanism via formation of a novel cationic pterin intermediate. We also show that two conserved loops generate a substructure during catalysis that creates a specific binding pocket for p-aminobenzoic acid, one of the two DHPS substrates. This substructure, together with the pterin-binding pocket, explains the roles of the conserved active-site residues and reveals how sulfonamide resistance arises.

Topics: 4-Aminobenzoic Acid; Amino Acid Sequence; Anti-Bacterial Agents; Bacillus anthracis; Biocatalysis; Catalytic Domain; Crystallization; Crystallography, X-Ray; Dihydropteroate Synthase; Diphosphates; Drug Resistance, Bacterial; Magnesium; Models, Chemical; Models, Molecular; Molecular Sequence Data; Mutagenesis; Parabens; Protein Conformation; Sulfamethoxazole; Sulfathiazole; Sulfathiazoles; Yersinia pestis

Phosphorus is an essential element for all living cells, but its availability is often limiting in the soil. Plants have adapted to such limitation and respond to phosphorus deficiency. The soluble inorganic pyrophosphatases (PPase; EC 3.6.1.1) recycle the pyrophosphate produced by many biosynthetic reactions, and may play a role in the plant adaptation to phosphorus deficiency. In this work, three PPase mRNAs were identified from the Phaseolus vulgaris EST international database and their sequences were corroborated and completed using 3'RACE. After design and validation of the appropriate oligonucleotide primers, the PPase mRNA expression was measured by qRT-PCR in leaves, stems, and roots of bean plants grown with 1mM phosphate or under phosphate starvation. The plant tissues were classified according to their position on the plant, and some physiological signs of stress were recorded. qRT-PCR revealed changes in mRNA expression, but not for all isozymes under analysis, and not for all tissues. In addition, changes in the activity of some PPases were observed in zymograms. Our data are consistent with an important role for pyrophosphate in the adaptation of the plant to phosphate starvation.

Topics: Adaptation, Physiological; Base Sequence; Diphosphates; DNA Primers; Gene Expression; Gene Expression Regulation, Plant; Inorganic Pyrophosphatase; Isoenzymes; Phaseolus; Phosphates; Phosphorus; Plant Structures; RNA, Messenger; Soil; Solubility; Stress, Physiological

Vascular calcification is a major risk factor of cardiovascular mortality, particularly for patients with end-stage renal disease and diabetes. Although chronic inflammation is one of the etiologic factors, the underlying mechanism is not fully understood. To clarify this, we studied how nuclear factor-kappa B (NF-κB) induction, a mediator of inflammation, might promote vascular calcification. Activation of NF-κB by tumor necrosis factor (TNF) promoted inorganic phosphate-induced calcification in human aortic smooth muscle cells. Pyrophosphate (an inhibitor of calcification) efflux to the extracellular matrix was suppressed along with the decreased expression of ankylosis protein homolog (ANKH), a transmembrane protein that controls pyrophosphate efflux of cells. The restoration of ANKH expression in these cells overcame the decreased pyrophosphate efflux and calcification. Tristetraprolin, a downstream product of NF-κB activation, may mediate destabilization of ANKH mRNA as its knockdown by shRNA increased ANKH expression and decreased calcification. Furthermore, a rat chronic renal failure model, with increased serum TNF levels, activated NF-κB and decreased ANKH levels. In contrast, the inhibition of NF-κB maintained ANKH expression and attenuated vascular calcification both in vivo and in vitro. Both human calcified atherosclerotic lesions and arteries from patients with chronic kidney disease had activated NF-κB and decreased ANKH expression. Thus, TNF-activated NF-κB promotes inflammation-accelerated vascular calcification by inhibiting ankylosis protein homolog expression and consequent pyrophosphate secretion.

Topics: Animals; Atherosclerosis; Diphosphates; Disease Models, Animal; Disease Progression; Down-Regulation; Genes, Reporter; HEK293 Cells; Humans; I-kappa B Proteins; Inflammation Mediators; Kidney Failure, Chronic; Male; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; NF-kappa B; NF-KappaB Inhibitor alpha; Osteogenesis; Phosphate Transport Proteins; Promoter Regions, Genetic; Rats; Rats, Wistar; RNA Interference; RNA Stability; Signal Transduction; Time Factors; Transfection; Tristetraprolin; Tumor Necrosis Factor-alpha; Vascular Calcification

Adenosine triphosphate (ATP)-binding cassette (ABC) transporters, ubiquitous proteins found in all kingdoms of life, catalyze substrates translocation across biological membranes using the free energy of ATP hydrolysis. Cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of this superfamily in that it functions as an ATP-gated chloride channel. Despite difference in function, recent studies suggest that the CFTR chloride channel and the exporter members of the ABC protein family may share an evolutionary origin. Although ABC exporters harness the free energy of ATP hydrolysis to fuel a transport cycle, for CFTR, ATP-induced dimerization of its nucleotide-binding domains (NBDs) and subsequent hydrolysis-triggered dimer separation are proposed to be coupled, respectively, to the opening and closing of the gate in its transmembrane domains. In this study, by using nonhydrolyzable ATP analogues, such as pyrophosphate or adenylyl-imidodiphosphate as baits, we captured a short-lived state (state X), which distinguishes itself from the previously identified long-lived C2 closed state by its fast response to these nonhydrolyzable ligands. As state X is caught during the decay phase of channel closing upon washout of the ligand ATP but before the channel sojourns to the C2 closed state, it likely emerges after the bound ATP in the catalysis-competent site has been hydrolyzed and the hydrolytic products have been released. Thus, this newly identified post-hydrolytic state may share a similar conformation of NBDs as the C2 closed state (i.e., a partially separated NBD and a vacated ATP-binding pocket). The significance of this novel state in understanding the structural basis of CFTR gating is discussed.

Topics: Adenosine Triphosphate; Adenylyl Imidodiphosphate; Animals; Binding Sites; Biocatalysis; CHO Cells; Cricetinae; Cystic Fibrosis Transmembrane Conductance Regulator; Diphosphates; Hydrolysis; Protein Multimerization

Pyridyl-based triazole-linked calix[4]arene conjugates, viz. L(1) and L(2), were synthesized and characterized. These two conjugates were shown to be selective and sensitive for Zn(2+) among the 12 metal ions studied in HEPES buffer medium by fluorescence, absorption, and visual color change with the detection limit of ~31 and ~112 ppb, respectively, by L(1) and L(2). Moreover, the utility of the conjugates L(1) and L(2) in showing the zinc recognition in live cells has also been demonstrated using HeLa cells as monitored by fluorescence imaging. The zinc complexes of L(1) and L(2) were isolated, and the structure of [ZnL(1)] has been established by single-crystal XRD and that of [ZnL(2)] by DFT calculations. TDDFT calculations were performed in order to demonstrate the electronic properties of receptors and their zinc complexes. The isolated zinc complexes, viz. [ZnL(1)] and [ZnL(2)], have been used as molecular tools for the recognition of anions on the basis of their binding affinities toward Zn(2+). [ZnL(2)] was found to be sensitive and selective toward phosphate-bearing ions and molecules and in particular to pyrophosphate (PPi) and ATP among the other 18 anions studied; however, [ZnL(1)] was not sensitive toward any of the anions studied. The selectivity has been shown on the basis of the changes observed in the emission and absorption spectral studies through the removal of Zn(2+) from [ZnL(2)] by PPi. Thus, [ZnL(2)] has been shown to detect PPi up to 278 ± 10 ppb at pH 7.4 in aqueous methanolic (1/2 v/v) HEPES buffer.

Topics: Absorption; Adenosine Triphosphate; Calixarenes; Chemistry Techniques, Analytical; Diphosphates; HeLa Cells; Humans; Imines; Intracellular Space; Models, Molecular; Molecular Conformation; Organometallic Compounds; Phenols; Pyridines; Quantum Theory; Solutions; Spectrometry, Fluorescence; Zinc

A one-dimensional (1D) motif usually comprises conserved essential residues involved in catalysis, ligand binding, or maintaining a specific structure. However, it cannot be easily detected in proteins with low sequence identity because it is difficult to (1) identify protein sequences suspected to contain the motif, and (2) align sequences with little sequence identity to spot the conserved residues. Here, we present a strategy for discovering phosphate-binding 1D motifs in NAD(P)-binding proteins sharing low sequence identity that overcomes these two hurdles by determining all distinct locally conserved pyrophosphate-binding structures and aligning the same-length sequences comprising each of these structures to identify the conserved residues. We show that the sequence motifs derived from the distinct pyrophosphate-binding structures yield different numbers/spacing of conserved Gly residues. We also show that they depend on the side chain orientations and cofactor type (NAD or NADP). Thus, sequence motifs derived from local similarity of backbone structures without consideration of the cofactor type and/or side chain orientations would reduce their reliability in annotating protein function from sequence alone. The three-dimensional (3D) and 1D motifs comprising conserved residues in nonredundant proteins reveal hidden relationships between the protein structure/function and sequence as well as protein-cofactor interactions.

Topics: Amino Acid Motifs; Amino Acid Sequence; Binding Sites; Conserved Sequence; Diphosphates; Glycine; Models, Molecular; NAD; NADP; Phosphate-Binding Proteins; Protein Binding; Protein Folding; Protein Structure, Tertiary; Proteins

Multisubunit RNA polymerase (RNAP) is the central information-processing enzyme in all cellular life forms, yet its mechanism of translocation along the DNA molecule remains conjectural. Here, we report direct monitoring of bacterial RNAP translocation following the addition of a single nucleotide. Time-resolved measurements demonstrated that translocation is delayed relative to nucleotide incorporation and occurs shortly after or concurrently with pyrophosphate release. An investigation of translocation equilibrium suggested that the strength of interactions between RNA 3' nucleotide and nucleophilic and substrate sites determines the translocation state of transcription elongation complexes, whereas active site opening and closure modulate the affinity of the substrate site, thereby favoring the post- and pre-translocated states, respectively. The RNAP translocation mechanism is exploited by the antibiotic tagetitoxin, which mimics pyrophosphate and induces backward translocation by closing the active site.

Topics: Bacteria; Catalytic Domain; Dicarboxylic Acids; Diphosphates; DNA; DNA-Directed RNA Polymerases; Enzyme Inhibitors; Fluorescent Dyes; Nucleotides; Organophosphorus Compounds; Protein Subunits; Protein Transport; RNA; Transcription, Genetic

A simple, straightforward, reliable, and efficient method for the chemical synthesis of sodium salt of 2'-deoxynucleoside-5'-O-triphosphates (dNTPs), starting from the corresponding nucleoside, is described. This improved "one-pot, three-step" synthetic strategy involves the monophosphorylation of nucleoside, followed by reaction with tributylammonium pyrophosphate and hydrolysis of the resulting cyclic intermediate to provide the corresponding dNTP in good yields (65% to 70%). It is noteworthy that the protocol holds good for both the purine deoxynucleotides, such as 2'-deoxyguanosine-5'-O-triphosphate (dGTP) and 2'-deoxyadenosine-5'-O-triphosphate (dATP), and pyrimidine deoxynucleotides, such as 2'-deoxycytidine-5'-O-triphosphate (dCTP), thymidine-5'-O-triphosphate (TTP), and 2'-deoxyuridine-5'-O-triphosphate (dUTP).

Topics: Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Diphosphates; Hydrolysis; Nucleosides; Purine Nucleotides; Pyrimidine Nucleotides; Thymine Nucleotides

Mutations in the gene ALPL in hypophosphatasia (HPP) reduce the function of tissue nonspecific alkaline phosphatase, and the resulting increase in pyrophosphate (PP(i)) contributes to bone and tooth mineralization defects by inhibiting physiologic calcium-phosphate (P(i)) precipitation. Although periodontal phenotypes are well documented, pulp/dentin abnormalities have been suggested in the clinical literature although reports are variable and underlying mechanisms remains unclear. In vitro analyses were used to identify mechanisms involved in HPP-associated pulp/dentin phenotypes.. Primary pulp cells cultured from HPP subjects were established to assay alkaline phosphatase (ALP) activity, mineralization, and gene expression compared with cells from healthy controls. Exogenous P(i) was provided to the correct P(i)/PP(i) ratio in cell culture.. HPP cells exhibited significantly reduced ALP activity (by 50%) and mineral nodule formation (by 60%) compared with the controls. The expression of PP(i) regulatory genes was altered in HPP pulp cells, including reduction in the progressive ankylosis gene (ANKH) and increased ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). Odontoblast marker gene expression was disrupted in HPP cells, including reduced osteopontin (OPN), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP), and matrix extracellular phosphoprotein (MEPE). The addition of P(i) provided a corrective measure for mineralization and partially rescued the expression of some genes although cells retained altered messenger RNA levels for PP(i)-associated genes.. These studies suggest that under HPP conditions pulp cells have the compromised ability to mineralize and feature a disrupted odontoblast profile, providing a first step toward understanding the molecular mechanisms for dentin phenotypes observed in HPP.

Topics: Adolescent; Alkaline Phosphatase; Amino Acid Substitution; Analysis of Variance; Calcium; Case-Control Studies; Dental Pulp; Dentin; Diphosphates; Diseases in Twins; Down-Regulation; Extracellular Matrix Proteins; Female; Gene Expression; Glycoproteins; Humans; Hypophosphatasia; Male; Mutation, Missense; Odontoblasts; Osteopontin; Phosphate Transport Proteins; Phosphoproteins; Phosphoric Diester Hydrolases; Primary Cell Culture; Pyrophosphatases; Sialoglycoproteins; Statistics, Nonparametric; Tooth Calcification; Young Adult

A novel NBD-phenoxo-bridged dinuclear Zn(II) complex is found to be an effective colorimetric sensor for pyrophosphate (PPi) in pure aqueous solution over a wide pH range. This sensor shows high binding affinity (K(a)≈ 3 × 10(8) M(-1)) and high selectivity for PPi, and can be also used to assay the activity of pyrophosphatase in real time.

Topics: Colorimetry; Coordination Complexes; Crystallography, X-Ray; Diphosphates; Hydrogen-Ion Concentration; Kinetics; Pyrophosphatases; Water; Zinc

Extracellular ATP is an important signaling molecule mediating quite divergent specific biological effects. Even though recent studies suggest a potential role of ATP in cancer progress, its real impact in chemotherapeutic efficacy remains unclear. In the present study, we investigated the effect of ATP on the cytotoxicity of doxorubicin in various cancer cell types and found that ATP had no effect on doxorubicin cytotoxicity in colon, prostate, breast, and cervical cancers or in osteosarcoma. In contrast, ATP has divergent effects on lung cancer cells: it can protect against doxorubicin-induced cell death in non-metastatic lung cancer CL1.0 cells, but not in highly metastatic CL1.5 cells. Both apoptotic (characterized by sub-G(1) peak, caspase 3 activation, poly(ADP-ribose) polymerase-1 cleavage) and necrotic (characterized by propidium iodide uptake and ROS production) features induced by doxorubicin in CL1.0 cells were reduced by ATP. In addition, ATP attenuated p53 accumulation, DNA damage (assessed by poly(ADP-ribose) formation and the comet assay) and topoisomerase II inhibition after doxorubicin treatment, and doxorubicin cytotoxicity was diminished by the p53 inhibitor pifithrin-α. Moreover, UTP, UDP, ADP, and pyrophosphate sodium pyrophosphate tetrabasic decahydrate diminished the antitumor effect of doxorubicin in CL1.0 cells, whereas purinergic P2 receptors antagonists did not abrogate the action of ATP. In summary, ATP fails to alter the antitumor efficacy of doxorubicin in most cancer cell types, except in CL1.0 cells, in which pyrophosphate mediates the cell protection afforded by ATP via attenuation of reactive oxygen species production, DNA damage, p53 accumulation, and caspase activation.

Topics: Adenosine Triphosphate; Antibiotics, Antineoplastic; Apoptosis; Blotting, Western; Cell Line, Tumor; Cell Survival; Comet Assay; Diphosphates; Doxorubicin; Humans; Necrosis; Real-Time Polymerase Chain Reaction

Treatment of HIV-1 with nucleoside reverse transcription inhibitors leads to the emergence of resistance mutations in the reverse transcriptase (RT) gene. Resistance to 3'-azido-3'-deoxythymidine (AZT) and to a lesser extent to 2'-3'-didehydro-2'-3'-dideoxythymidine is mediated by phosphorolytic excision of the chain terminator. Wild-type RT excises AZT by pyrophosphorolysis, while thymidine-associated resistance mutations in RT (TAMs) favour ATP as the donor substrate. However, in vitro, resistant RT still uses pyrophosphate more efficiently than ATP. We performed in vitro (-) strong-stop DNA synthesis experiments, with wild-type and AZT-resistant HIV-1 RTs, in the presence of physiologically relevant pyrophosphate and/or ATP concentrations and found that in the presence of pyrophosphate, ATP and AZTTP, TAMs do not enhance in vitro (-) strong-stop DNA synthesis. We hypothesized that utilisation of ATP in vivo is driven by intrinsic low pyrophosphate concentrations within the reverse transcription complex, which could be explained by the packaging of a cellular pyrophosphatase. We showed that over-expressed flagged-pyrophosphatase was associated with HIV-1 viral-like particles. In addition, we demonstrated that when HIV-1 particles were purified in order to avoid cellular microvesicle contamination, a pyrophosphatase activity was specifically associated to them. The presence of a pyrophosphatase activity in close proximity to the reverse transcription complex is most likely advantageous to the virus, even in the absence of any drug pressure.

Topics: Adenosine Triphosphate; Anti-HIV Agents; Dideoxynucleotides; Diphosphates; DNA, Viral; Drug Resistance, Viral; HIV Reverse Transcriptase; HIV-1; Kinetics; Mutation; Pyrophosphatases; Stavudine; Substrate Specificity; Thymine Nucleotides; Virion; Zidovudine

Rapid identification of the causative bacteria of sepsis in patients can contribute to the selection of appropriate antibiotics and improvement of patients' prognosis. Genotypic identification is an emerging technology that may provide an alternative method to, or complement, established phenotypic identification procedures. We evaluated a rapid protocol for bacterial identification based on PCR and pyrosequencing of the V1 and V3 regions of the 16S rRNA gene using DNA extracted directly from positive blood culture samples. One hundred and two positive blood culture bottles from 68 patients were randomly selected and the bacteria were identified by phenotyping and pyrosequencing. The results of pyrosequencing identification displayed 84.3 and 64.7 % concordance with the results of phenotypic identification at the genus and species levels, respectively. In the monomicrobial samples, the concordance between the results of pyrosequencing and phenotypic identification at the genus level was 87.0 %. Pyrosequencing identified one isolate in 60 % of polymicrobial samples, which were confirmed by culture analysis. Of the samples identified by pyrosequencing, 55.7 % showed consistent results in V1 and V3 targeted sequencing; other samples were identified based on the results of V1 (12.5 %) or V3 (31.8 %) sequencing alone. One isolate was erroneously identified by pyrosequencing due to high sequence similarity with another isolate. Pyrosequencing identified one isolate that was not detected by phenotypic identification. The process of pyrosequencing identification can be completed within ~4 h. The information provided by DNA-pyrosequencing for the identification of micro-organisms in positive blood culture bottles is accurate and could prove to be a rapid and useful tool in standard laboratory practice.

Topics: Bacteria; Bacterial Infections; Bacteriological Techniques; Base Sequence; Candida albicans; Candidiasis; Diphosphates; DNA, Bacterial; DNA, Fungal; DNA, Ribosomal; Gene Expression Regulation, Bacterial; Humans; Polymerase Chain Reaction; RNA, Bacterial; RNA, Ribosomal, 16S; Sepsis; Sequence Analysis, DNA; Species Specificity

Acetate kinase (ACK) catalyzes the reversible synthesis of acetyl phosphate by transfer of the γ-phosphate of ATP to acetate. Here we report the first biochemical and kinetic characterization of a eukaryotic ACK, that from the protist Entamoeba histolytica. Our characterization revealed that this protist ACK is the only known member of the ASKHA structural superfamily, which includes acetate kinase, hexokinase, and other sugar kinases, to utilize inorganic pyrophosphate (PP(i))/inorganic phosphate (P(i)) as the sole phosphoryl donor/acceptor. Detection of ACK activity in E. histolytica cell extracts in the direction of acetate/PP(i) formation but not in the direction of acetyl phosphate/P(i) formation suggests that the physiological direction of the reaction is toward acetate/PP(i) production. Kinetic parameters determined for each direction of the reaction are consistent with this observation. The E. histolytica PP(i)-forming ACK follows a sequential mechanism, supporting a direct in-line phosphoryl transfer mechanism as previously reported for the well-characterized Methanosarcina thermophila ATP-dependent ACK. Characterizations of enzyme variants altered in the putative acetate/acetyl phosphate binding pocket suggested that acetyl phosphate binding is not mediated solely through a hydrophobic interaction but also through the phosphoryl group, as for the M. thermophila ACK. However, there are key differences in the roles of certain active site residues between the two enzymes. The absence of known ACK partner enzymes raises the possibility that ACK is part of a novel pathway in Entamoeba.

Topics: Amino Acid Sequence; Binding Sites; Catalytic Domain; Diphosphates; Entamoeba histolytica; Molecular Sequence Data; Organophosphates; Phosphotransferases (Carboxyl Group Acceptor); Protozoan Proteins

The anion binding ability of a family of bis(Zn(II)-Dpa) functionalized cyclic peptides has been investigated using displacement assays with a fluorescent coumarin indicator in water, saline solution, and Krebs buffer. Non-binding side-chain steric bulk, the relative position of binding sites, and the scaffold size were all found to affect the ability of these receptors to discriminate between polyphosphate ions. Most receptors showed some selectivity for pyrophosphate over ATP and ADP in water and saline, and this selectivity was significantly enhanced in the biologically relevant Krebs buffer giving chemosensing ensembles capable of selective recognition of pyrophosphate in the presence of excess ATP.

Topics: Anions; Diphosphates; Peptides, Cyclic; Protein Binding; Sodium Chloride; Zinc

Medulloblastoma (MB) is the most common malignant brain tumor in children and occurs mainly in the cerebellum. Important intracellular signaling molecules, such those present in the Sonic Hedgehog and Wnt pathways, are involved in its development and can also be employed to determine tumor grade and prognosis. Ectonucleotidases, particularly ecto-5'NT/CD73, are important enzymes in the malignant process of different tumor types regulating extracellular ATP and adenosine levels. Here, we investigated the activity of ectonucleotidases in three malignant human cell lines: Daoy and ONS76, being representative of primary MB, and the D283 cell line, derived from a metastatic MB. All cell lines secreted ATP into the extracellular medium while hydrolyze poorly this nucleotide, which is in agreement with the low expression and activity of pyrophosphate/phosphodiesterase, NTPDases and alkaline phosphatase. The analysis of AMP hydrolysis showed that Daoy and ONS76 completely hydrolyzed AMP, with parallel adenosine production (Daoy) and inosine accumulation (ONS76). On the other hand, D283 cell line did not hydrolyze AMP. Moreover, primary MB tumor cells, Daoy and ONS76 express the ecto-5'NT/CD73 while D283 representative of a metastatic tumor, revealed poor expression of this enzyme, while the ecto-adenosine deaminase showed higher expression in D283 compared to Daoy and ONS76 cells. Nuclear beta-catenin has been suggested as a marker for MB prognosis. Further it can promotes expression of ecto-5'NT/CD73 and suppression of adenosine deaminase. It was observed that Daoy and ONS76 showed greater nuclear beta-catenin immunoreactivity than D283, which presented mainly cytoplasmic immunoreactivity. In summary, the absence of ecto-5'NT/CD73 in the D283 cell line, a metastatic MB phenotype, suggests that high expression levels of this ectonucleotidase could be correlated with a poor prognosis in patients with MB.

Topics: 5'-Nucleotidase; Adenosine; Adenosine Deaminase; Adenosine Monophosphate; Adenosine Triphosphate; Alkaline Phosphatase; beta Catenin; Biomarkers, Tumor; Brain Neoplasms; Cell Line, Tumor; Cerebellum; Child; Diphosphates; Gene Expression Regulation, Neoplastic; Humans; Medulloblastoma; Neoplasm Metastasis; Organ Specificity; Phosphoric Diester Hydrolases; Prognosis; Pyrophosphatases; Signal Transduction

Topics: Diphosphates; Humans; N-Acetylglucosaminyltransferases; Nucleotides

Protein O-GlcNAcylation is an essential post-translational modification on hundreds of intracellular proteins in metazoa, catalyzed by O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) using unknown mechanisms of transfer and substrate recognition. Through crystallographic snapshots and mechanism-inspired chemical probes, we define how human OGT recognizes the sugar donor and acceptor peptide and uses a new catalytic mechanism of glycosyl transfer, involving the sugar donor α-phosphate as the catalytic base as well as an essential lysine. This mechanism seems to be a unique evolutionary solution to the spatial constraints imposed by a bulky protein acceptor substrate and explains the unexpected specificity of a recently reported metabolic OGT inhibitor.

Topics: Adaptor Proteins, Signal Transducing; Catalysis; Catalytic Domain; Crystallography, X-Ray; Diphosphates; Humans; Kinetics; Lysine; Models, Molecular; Molecular Sequence Data; N-Acetylglucosaminyltransferases; Nucleotides; Phosphates; Protein Binding; Protein Conformation; Protein Processing, Post-Translational; Stereoisomerism; Substrate Specificity; Surface Plasmon Resonance; Uridine Diphosphate Galactose

Burbank and Norkotah potato slices were dipped into 3% sodium acid sulfate (SAS), citric acid (CA), sodium erythorbate (SE), malic acid (MA), sodium acid pyrophosphate (SAPP), or a combination of SAS-CA-SE. Browning by polyphenol oxidase (PPO) obtained from potato extract with 0.04 to 0.016 g/mL of antibrowning solutions at pH 2.0 to 6.9 were measured by UV-Vis spectroscopy. The color of slices dipped in antibrowning solutions at pHs 2 to 7 and stored at 4 °C for 15 d was measured every 5 d by colorimeter. Headspace analysis of volatiles in raw and cooked potato samples was performed by selected ion flow tube mass spectrometer (SIFT-MS) and soft independent modelling by class analogy (SIMCA) analysis of the calculated odor activity values (OAV) determined interclass distances. Microbial growth was measured at 15 d. At unadjusted pHs (1.1 to 7.1), the PPO browning of the control and samples with SAPP was not significantly different, SAS, CA, and MA produced some inhibition and SE and SAS-CA-SE prevented browning. At pH 5 to 7, only SE and SAS-CA-SE were effective browning inhibitors. Based on the color of potato slices, SE was the most effective at pH 2 to 7, but SAS was most effective at unadjusted pH. Cooking increased volatile levels in the treated potatoes and decreased differences between volatile profiles. Differences between cooked samples may not be noticeable by the consumer because volatiles with high discriminating powers have low OAVs. SAS, CA, and SAS-CA-SE treatments inhibited microbial growth but SAPP, control, and SE did not, most likely due to pH.. Antibrowning agents inhibit polyphenol oxidase, increasing shelf life and consumer acceptability of processed raw potato products by preserving the color. Their effectiveness was shown to be mainly due to a pH effect, except SE, which was not pH dependent. MA, CA, and SAS-CA-SE are better acidulants for inhibition of color change as well as growth of spoilage bacteria, yeast, and mold than SAPP, the industry standard.

Topics: Ascorbic Acid; Catechol Oxidase; Citric Acid; Colony Count, Microbial; Color; Consumer Behavior; Diphosphates; Food Contamination; Food Handling; Food Microbiology; Food Preservation; Hydrogen-Ion Concentration; Maillard Reaction; Malates; Odorants; Solanum tuberosum; Sulfates; Volatile Organic Compounds

We have investigated the applicability of different chemical methods for pyrophosphate bond formation to the synthesis of 5-substituted UDP-galactose and UDP-N-acetylglucosamine derivatives. The use of phosphoromorpholidate chemistry, in conjunction with N-methyl imidazolium chloride as the promoter, was identified as the most reliable synthetic protocol for the preparation of these non-natural sugar-nucleotides. Under these conditions, the primary synthetic targets 5-iodo UDP-galactose and 5-iodo UDP-N-acetylglucosamine were consistently obtained in isolated yields of 40-43%. Both 5-iodo UDP-sugars were used successfully as substrates in the Suzuki-Miyaura cross-coupling with 5-formylthien-2-ylboronic acid under aqueous conditions. Importantly, 5-iodo UDP-GlcNAc and 5-(5-formylthien-2-yl) UDP-GlcNAc showed moderate inhibitory activity against the GlcNAc transferase GnT-V, providing the first examples for the inhibition of a GlcNAc transferase by a base-modified donor analogue.

Topics: Animals; CHO Cells; Cricetinae; Diphosphates; Enzyme Activation; Enzyme Assays; Enzyme Inhibitors; Galactosephosphates; Magnetic Resonance Spectroscopy; N-Acetylglucosaminyltransferases; Recombinant Proteins; Solvents; Tetrazoles; Time Factors; Uridine Diphosphate; Uridine Diphosphate Galactose; Uridine Diphosphate N-Acetylglucosamine

The kinetics of the oxidative degradation of dipeptide glycyl-glycine (Gly-Gly) by water-soluble colloidal MnO(2) in acidic medium has been studied by employing visible spectrophotometer in the aqueous and micellar media at 35°C. To obtain the rate constants as functions of [Gly-Gly], [MnO(2)] and [HClO(4)], pseudo-first-order conditions were maintained in each kinetic run. The first-order-rate is observed with respect to [MnO(2)], whereas fractional-order-rates are determined in both [Gly-Gly] and [HClO(4)]. The addition of sodium pyrophosphate and sodium fluoride has composite effects (catalytic and inhibition). The reaction proceeds through the fast adsorption of Gly-Gly on the surface of the colloidal MnO(2). The observed results are discussed in terms of Michaelis-Menten/Langmuir-Hinshelwood model. The Arrhenius and Eyring equations are found valid for the reaction over a range of temperatures and different activation parameters have been evaluated. A probable reaction mechanism, in agreement with the observed kinetic results, has been proposed and discussed. The influence of changes in the surfactant concentrations on the observed rate constant is also investigated and the reaction followed the same type of kinetic behavior in micellar media. The pseudo-first-order rate constant (k(ψ)) is found to increase about two-fold with increase in [TX-100]. The catalytic effect of nonionic surfactant TX-100 is explained in terms of the mathematical model proposed by Tuncay et al.

Topics: Adsorption; Aldehydes; Ammonia; Colloids; Diphosphates; Glycylglycine; Kinetics; Manganese Compounds; Micelles; Octoxynol; Oxidation-Reduction; Oxides; Perchlorates; Sodium Fluoride; Solubility; Spectrophotometry, Ultraviolet; Temperature; Water

The developing periodontium is sensitive to local levels of inorganic phosphate (P(i)) and inorganic pyrophosphate (PP(i)) as demonstrated by cementum phenotypes resulting from the loss of function of protein regulators of P(i)/PP(i) homeostasis. The progressive ankylosis protein (ANK) regulates the transport of PP(i), and progressive ankylosis gene (Ank) and knock-out (KO) mice feature a rapidly forming and thick cementum. We hypothesized that, besides affecting cementum formation, decreased extracellular PP(i) levels in Ank KO mice would also impact cementum regeneration.. Periodontal fenestration defects (approximately 2 mm in length, 1 mm in width, and 0.5 mm in depth) were created on buccal aspects of mandibular molars in Ank KO and wild-type (WT) mice. Mandibles were harvested at 15 and 30 days post-surgery for histology, histomorphometry, evaluation of in vivo fluorochrome labeling, and immunohistochemistry (IHC) for proteins including bone sialoprotein (BSP), osteopontin (OPN), dentin matrix protein 1 (DMP1), and ectonucleotide pyrophosphatase/phosphodiesterase 1 (NPP1).. A greater amount of new cementum was observed in Ank KO mice at 15 and 30 days post-surgery (P <0.05), which was confirmed by fluorochrome labeling showing a higher new cementum appositional activity in defect areas in Ank KO mice versus controls. At days 15 and 30 during healing, regenerating cementum and associated cells in Ank KO samples recapitulated expression patterns mapped during development, including limited BSP and positive OPN and DMP1 in the cementum matrix as well as elevated NPP1 in cementoblasts.. Within the limits of the study, these findings suggest that reduced local levels of PP(i) could promote increased cementum regeneration. Therefore, the local modulation of P(i)/PP(i) may be a potential therapeutic approach for achieving improved cementum regeneration.

Topics: Animals; Dental Cementum; Diphosphates; Extracellular Matrix Proteins; Integrin-Binding Sialoprotein; Mice; Mice, Knockout; Osteopontin; Phosphate Transport Proteins; Phosphates; Phosphoric Diester Hydrolases; Pyrophosphatases; Regeneration

The solvothermal reactions of silver(I) salts with mono-organophosphonic acids, i.e. 3-thienylphosphonic acid (3-TPA), phenylphosphonic acid (PPA), α-naphthylphosphonic acid (α-NPA) and cyclohexylphosphonic acid (CPA), yield four new silver(I) pyrophosphonates, namely: [Ag(2)(ptp)] (1), [Ag(2)(ppp)] (2), [Ag(3)(CH(3)CN)(pnp)(pnpH)] (3), and [Ag(3)(pcp)(pcpH)] (4) [ptp(2-) = pyro-3-thienylphosphonate, ppp(2-) = pyrophenylphosphonate, pnp(2-) = pyro-α-naphthylphosphonate, pcp(2-) = pyrocyclohexylphosphoante]. In all cases, the pyrophosphonate ligands are generated in situ from their relative mono-organophosphonic acids, mediated by silver(I) ions. Single crystal structural determinations reveal that compounds 1 and 2 display two-dimensional layer architectures, while 3 and 4 show one-dimensional chain structures. Structure 1 can be best described as a layer made up of Ag(4)O(P)(6) clusters linked by O-P-O units and AgAg contacts, with the organic groups grafted on the two sides of the inorganic layer. A similar layer structure is found in 2 except that the AgAg interactions are absent. Compound 3 shows a chain structure where the silver ions are bridged by the phosphonate oxygen atoms forming an infinite Ag-O(P) chain which is decorated by the pyrophosphonate ligand and CH(3)CN. Compound 4 has another type of chain structure made up of Ag-O(P) with extensive Ag···Ag argentophilic interactions. Solid state photoluminescent properties and thermal expansion behaviors are also investigated.

Topics: Coordination Complexes; Crystallography, X-Ray; Diphosphates; Luminescent Agents; Molecular Conformation; Silver; Temperature

A variety of chiral bisphosphoric acids derived from binaphthols have been evaluated for enantioselective 1,3-dipolar cycloaddition reactions, revealing that the feature of the linker in the catalysts exerted great impact on the stereoselectivity. Among them, the oxygen-linked bisphosphoric acid 1a provided the highest level of stereoselectivity for the 1,3-dipolar cycloaddition reaction tolerating a wide range of substrates including azomethine ylides, generated in situ from a broad scope of aldehydes and α-amino esters, and various electron-deficient dipolarophiles such as maleates, fumarates, vinyl ketones, and esters. This reaction actually represents one of the most enantioselective catalytic approaches to access structurally diverse pyrrolidines with excellent optical purity. Theoretical calculations with DFT method on the formation of azomethine ylides and on the transition states of the 1,3-dipolar cycloaddition step showed that the dipole and dipolarophile were simultaneously activated by the bifunctional chiral bisphosphoric acids through the formation of hydrogen bonds. The effect of the bisphosphoric acids on reactivity and stereochemistry of the three-component 1,3-dipolar cycloaddition reaction was also theoretically rationalized. The bisphosphoric acid catalyst 1a may take on a half-moon shape with the two phosphoric acid groups forming two intramolecular hydrogen bonds. In the case of maleates, one phosphate acts as a base to activate the 1,3-dipole, and simultaneously, the two hydroxyl groups in the catalyst 1a may respectively form two hydrogen bonds with the two ester groups of maleate to make it more electronically deficient as a much stronger dipolarophile to participate in a concerted 1,3-dipolar cycloaddition with azomethine ylide. However, in the cases involving acrylate and fumarate dipolarophiles, only one hydroxyl group forms a hydrogen bond with the ester functional group to lower the LUMO of the C-C double bond and another one is remained to adjust the acidity and basicity of two phosphoric acids to activate the dipole and dipolarophile more effectively.

Topics: Alkenes; Azo Compounds; Crystallography, X-Ray; Cyclization; Diphosphates; Models, Molecular; Stereoisomerism; Thiosemicarbazones

Calcium phosphate deposition (CPD) is the hallmark of vascular smooth muscle cell (VSMC) calcification. CPD is a thermodynamically-favored process under physiological conditions. Hydroxyapatite, the most common calcium phosphate in calcified arteries, is passively formed during VSMC calcification, independently on any direct cellular activity. Furthermore, in recent years it has been demonstrated there is an anti-calcifying effect by extracellular pyrophosphate, an endogenous inhibitor of CPD, both in vitro and in vivo, which directly blocks hydroxyapatite formation.. We have used the in vitro calcification model without cellular activity, by treating confluent rat aortic VSMC with paraformaldehyde. Fixed cells were incubated with the indicated media to obtain or inhibit calcification. The calcium content was determined colorimetrically. Calcification was observed after 3 weeks (21 days) using a physiological concentration of calcium (1.8 mmol/L) and phosphate (1 mmol/L). Calcium deposition was directly proportional to the amount of phosphate in the media, with a calcification rate of 3.5, 7.5, and 14.3 µg·cm⁻²·day⁻¹, using 1, 2, and 4 mmol/L of phosphate, respectively. Under physiological conditions, pyrophosphate inhibits CPD with an IC₅₀ of ≍200 nmol/L.. CPD occurs under a physiological concentration of calcium and phosphate, but this deposition is completely inhibited in the presence of a physiological concentration of pyrophosphate (3-5 µmol/L).

Topics: Animals; Calcium; Cells, Cultured; Diphosphates; Durapatite; Models, Biological; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Rats; Time Factors; Vascular Calcification

PP(i) is a critical element of cellular metabolism as both an energy donor and as an allosteric regulator of several metabolic pathways. The apicomplexan parasite Toxoplasma gondii uses PP(i) in place of ATP as an energy donor in at least two reactions: the glycolytic PP(i)-dependent PFK (phosphofructokinase) and V-H(+)-PPase [vacuolar H(+)-translocating PPase (pyrophosphatase)]. In the present study, we report the cloning, expression and characterization of cytosolic TgPPase (T. gondii soluble PPase). Amino acid sequence alignment and phylogenetic analysis indicates that the gene encodes a family I soluble PPase. Overexpression of the enzyme in extracellular tachyzoites led to a 6-fold decrease in the cytosolic concentration of PP(i) relative to wild-type strain RH tachyzoites. Unexpectedly, this subsequent reduction in PP(i) was associated with a higher glycolytic flux in the overexpressing mutants, as evidenced by higher rates of proton and lactate extrusion. In addition to elevated glycolytic flux, TgPPase-overexpressing tachyzoites also possessed higher ATP concentrations relative to wild-type RH parasites. These results implicate PP(i) as having a significant regulatory role in glycolysis and, potentially, other downstream processes that regulate growth and cell division.

Topics: Amino Acid Sequence; Base Sequence; Cytosol; Diphosphates; Glycolysis; Inorganic Pyrophosphatase; Molecular Sequence Data; Organelles; Phosphotransferases; Toxoplasma

Topics: Arabidopsis; Arabidopsis Proteins; Diphosphates; Inorganic Pyrophosphatase; Saccharomyces cerevisiae Proteins; Sucrose; Vacuoles

Postgerminative growth of seed plants requires specialized metabolism, such as gluconeogenesis, to support heterotrophic growth of seedlings until the functional photosynthetic apparatus is established. Here, we show that the Arabidopsis thaliana fugu5 mutant, which we show to be defective in AVP1 (vacuolar H(+)-pyrophosphatase), failed to support heterotrophic growth after germination. We found that exogenous supplementation of Suc or the specific removal of the cytosolic pyrophosphate (PPi) by the heterologous expression of the cytosolic inorganic pyrophosphatase1 (IPP1) gene from budding yeast (Saccharomyces cerevisiae) rescued fugu5 phenotypes. Furthermore, compared with the wild-type and AVP1(Pro):IPP1 transgenic lines, hypocotyl elongation in the fugu5 mutant was severely compromised in the dark but recovered upon exogenous supply of Suc to the growth media. Measurements revealed that the peroxisomal β-oxidation activity, dry seed contents of storage lipids, and their mobilization were unaffected in fugu5. By contrast, fugu5 mutants contained ~2.5-fold higher PPi and ~50% less Suc than the wild type. Together, these results provide clear evidence that gluconeogenesis is inhibited due to the elevated levels of cytosolic PPi. This study demonstrates that the hydrolysis of cytosolic PPi, rather than vacuolar acidification, is the major function of AVP1/FUGU5 in planta. Plant cells optimize their metabolic function by eliminating PPi in the cytosol for efficient postembryonic heterotrophic growth.

Topics: Arabidopsis; Arabidopsis Proteins; Cotyledon; Cytosol; Darkness; Diphosphates; Gene Expression Regulation, Plant; Germination; Gluconeogenesis; Heterotrophic Processes; Hydrogen-Ion Concentration; Hypocotyl; Inorganic Pyrophosphatase; Mutation; Oxidation-Reduction; Peroxisomes; Phenotype; Plants, Genetically Modified; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Seedlings; Sucrose; Vacuoles

Mononuclear Zn(II)-DPA and Cu(II)-DPA complexes crafted on 2-hydroxy-6-cyanonaphthalene fluorophore selectively recognize PPi over ATP and other anions including inorganic phosphates in aqueous medium, showing turn-on type fluorescence enhancements. Coordination of a hydroxyl group of the fluorophore, directly or in alkoxy form, to the central metal ion is crucial for the sensing processes. Both the complexes elicit a fluorescence increase in a time-dependent fashion.

Topics: Amines; Anions; Copper; Diphosphates; Fluorescence; Fluorescent Dyes; Molecular Probes; Naphthols; Organometallic Compounds; Phosphates; Picolinic Acids; Solutions; Spectrometry, Fluorescence; Water; Zinc

Carbon nanotubes (CNTs) can efficiently quench the fluorescence of the adsorbed fluorophores and nonconvalently interact with soft single-stranded DNA (ssDNA). Upon disruption of CNTs-fluorescent oligonucleotides hybrid by nuclease S1, fluorescence turn-on was observed. Using this strategy, a platform based on fluorescence signal for monitoring the activity of nuclease with advantages of high sensitivity and commonality was established, and a linear relationship between initial cleavage reaction rate and nuclease S1 concentration is found in the range of 0.6-8.0 U mL(-1) with a detection limit of 0.08 U mL(-1). Furthermore, by taking pyrophosphate as an example, we use the assay to evaluate the prohibition effect on nuclease, and the extent of fluorescence recovery decreased linearly with increasing the concentration of pyrophosphate in the range of 0.2-1.4 mM, implying that the cleavage reaction by nuclease S1 was prohibited, and therefore this fluorescence assay can also be conveniently utilized for inhibitor screening of nuclease.

Topics: Diphosphates; DNA Cleavage; DNA, Single-Stranded; Enzyme Inhibitors; Fluorescent Dyes; Kinetics; Nanotubes, Carbon; Oligonucleotides; Rhodamines; Single-Strand Specific DNA and RNA Endonucleases; Spectrometry, Fluorescence

Replicative DNA polymerases (DNA pols) increase their fidelity by removing misincorporated nucleotides with their 3' → 5' exonuclease activity. Exonuclease activity reduces translesion synthesis (TLS) efficiency and TLS DNA pols lack 3' → 5' exonuclease activity. Here we show that physiological concentrations of pyrophosphate (PP(i)) activate the pyrophosphorolytic activity by DNA pol-λ, allowing the preferential excision of the incorrectly incorporated A opposite a 7,8-dihydro-8-oxoguanine lesion, or T opposite a 6-methyl-guanine, with respect to the correct C. This is the first example of an alternative proofreading mechanism used during TLS.

Topics: Deoxyadenine Nucleotides; Diphosphates; DNA Breaks, Single-Stranded; DNA Polymerase beta; DNA Repair; DNA Replication; Enzyme Activation; Guanosine; Humans

A supramolecular platform based on self-assembled monolayers (SAMs) has been implemented in a microfluidic device. The system has been applied for the sensing of two different analyte types: biologically relevant phosphate anions and aromatic carboxylic acids, which are important for anthrax detection. A Eu(III)-EDTA complex was bound to β-cyclodextrin monolayers via orthogonal supramolecular host-guest interactions. The self-assembly of the Eu(III)-EDTA conjugate and naphthalene β-diketone as an antenna resulted in the formation of a highly luminescent lanthanide complex on the microchannel surface. Detection of different phosphate anions and aromatic carboxylic acids was demonstrated by monitoring the decrease in red emission following displacement of the antenna by the analyte. Among these analytes, adenosine triphosphate (ATP) and pyrophosphate, as well as dipicolinic acid (DPA) which is a biomarker for anthrax, showed a strong response. Parallel fabrication of five sensing SAMs in a single multichannel chip was performed, as a first demonstration of phosphate and carboxylic acid screening in a multiplexed format that allows a general detection platform for both analyte systems in a single test run with μM and nM detection sensitivity for ATP and DPA, respectively.

Topics: Adenosine Triphosphate; Anions; Bacillus anthracis; beta-Cyclodextrins; Biomarkers; Diphosphates; Luminescence; Microchip Analytical Procedures; Microfluidic Analytical Techniques; Phosphates; Picolinic Acids

A particulate formation-laser scattering detector (PFLSD) was developed and used for evaluating the crystallization efficiency of inorganic polyphosphates (PPs) that reacted with either magnesium or calcium cations. As the solutions for reactive crystallization, 0.5 M ammonium buffer (pH 9.6) containing either 0.15 M MgCl(2) or 0.15 M CaCl(2) (MAP: magnesium ammonium phosphate and HAP: hydroxyapatite solution) were used. In the case of mono- and diphosphate (P1 and P2), the significant dependences of the particulate formation efficiency on various types of both P1/P2 and MAP/HAP reaction solutions were observed with the direct sample injection mode. The PFLSD was hyphenated with the anion-exchange chromatography and the dependence of the particulate formation efficiency on the polymerization degree (n(p)) of PP oligomers, separated chromatographically, was evaluated sequentially. The significant suppression of the particulate formation for PP oligomers was clearly confirmed, i.e., the MAP and HAP reaction solutions did not produce the particulates of the PP oligomers having an n(p) value of more than 3 and 5, respectively. As the overall tendency, the particulate formation efficiency in the case of the HAP solution was superior to that in the case of the MAP solution.

Topics: Calcium Chloride; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Crystallization; Diphosphates; Lasers; Magnesium Chloride; Phosphates; Polyphosphates; Potassium Compounds; Scattering, Radiation

Sphagnum peat has been found to efficiently remove heavy metals, oil, detergents, dyes, pesticides and nutrients from contaminated waters since its major constituents, i.e., unesterified polyuronic acids, cellulose, and fulvic and humic acids (HA), show functional groups (e.g., alcohols, aldehydes, carboxylic acids, ketones and phenolic hydroxides) which may adsorb pollutant species. The influence of the extractant on the analytical characteristics of HA is an old but still open topic that should be studied in relation to the nature of the matrix from which they originate. While a number of works have been published on the effects of different reagents on the extraction yields and structural properties of HA from soils, relatively little attention has been devoted to peat HA. In this work, the contents of some major and trace elements (As, Ca, Cr, K, Mn, Sr, and Ti) in five Sphagnum-peat samples and in their corresponding HA fractions isolated using three common extractant solutions, i.e., 0.5M NaOH, 0.1M Na(4)P(2)O(7), and 0.5M NaOH+0.1M Na(4)P(2)O(7), where investigated by X-ray fluorescence spectroscopy. In general, Cr, Mn, and Ti concentrations of bulk peat samples were higher than those of the corresponding HA fractions regardless of the extractant used. Arsenic, Ca, K, and Sr concentrations in the HA fractions were affected by the extraction procedure, although at different extents depending on the extractant utilized. In particular, compared to both NaOH and NaOH+Na(4)P(2)O(7), the Na(4)P(2)O(7) extractant yielded HA generally richer in As, Ca, K, and Sr, and poorer in Ti. These results may be related to both the nature of each HA fraction and the physical and chemical form of each element supplied to the studied bog via atmospheric deposition.

Topics: Diphosphates; Humic Substances; Metals, Heavy; Sodium Hydroxide; Soil; Solutions

The present paper deals with the chemiluminescent determination of the herbicide Karbutilate on the basis of its previous photodegradation by using a low-pressure Hg lamp as UV source in a continuous-flow multicommutation assembly (a solenoid valves set). The pesticide solution was segmented by a solenoid valve and sequentially alternated with segments of the 0.001 mol l(-1) of NaOH solution, the suitable media for the formation of photo-fragments; then it passes through the photo-reactor and was lead to the flow-cell after being divided in small segments which were sequentially alternated with the oxidizing system; 2 x 10(-5) mol l(-1) of potassium permanganate in 0.2% pyrophosphoric acid. The studied calibration range, from 0.1 microg l(-1) to 65 mg l(-1), resulted in a linear behaviour over the range 20 microg l(-1)-20 mg l(-1) and fitting the linear equation: I=(1180+/-30)C+(15+/-5) with the correlation coefficient 0.9998. The limit of detection was 10 microg l(-1) and the sample throughput 17 h(-1). After testing the influence of a large series of potential interfering species, the method was applied to water and human urine samples.

Topics: Calibration; Carbamates; Diphosphates; Female; Flow Injection Analysis; Herbicides; Humans; Luminescent Measurements; Male; Molecular Structure; Oxidants; Oxidation-Reduction; Photolysis; Potassium Permanganate; Reproducibility of Results; Sodium Hydroxide; Temperature; Ultraviolet Rays; Water

Biodetection and biosensing have been developed based on the concept of sensitivity toward specific molecules. However, current demand may require more levelheaded or far-sighted methods, especially in the field of biological safety and security. In the fields of hygiene, public safety, and security including fighting bioterrorism, the detection of biological contaminants, e.g., microorganisms, spores, and viruses, is a constant challenge. However, there is as yet no sophisticated method of detecting such contaminants in situ without oversight. The authors focused their attention on diphosphoric acid anhydride, which is a structure common to all biological phosphoric substances. Interestingly, biological phosphoric substances are peculiar substances present in all living things and include many different substances, e.g., ATP, ADP, dNTP, pyrophosphate, and so forth, all of which have a diphosphoric acid anhydride structure. The authors took this common structure as the basis of their development of an artificial enzyme membrane with selectivity for the structure common to all biological phosphoric substances and studied the possibility of its application to in situ biosurveillance sensors. The artificial enzyme membrane-based amperometric biosensor developed by the authors can detect various biological phosphoric substances, because it has a comprehensive molecular selectivity for the structure of these biological phosphoric substances. This in situ detection method of the common diphosphoric acid anhydride structure brings a unique advantage to the fabrication of in situ biosurveillance sensors for monitoring biological contaminants, e.g., microorganism, spores, and viruses, without an oversight, even if they were transformed.

Topics: Anhydrides; Animals; Bacteria; Biological Warfare; Biosensing Techniques; Bioterrorism; Diphosphates; Electrochemistry; Environmental Exposure; Environmental Pollutants; Enzymes, Immobilized; Humans; Membranes, Artificial; Public Health; Sensitivity and Specificity; Spores, Bacterial; Viruses

Synthetic colloidal calcium hydroxyapatite (Ca(10)(PO(4))(6)(OH)(2): CaHap) was treated with pyrophosphoric acid (H(4)P(2)O(7): PP) in acetone and the materials were characterized by XRD, TEM, FTIR, and N(2) and H(2)O adsorption measurements. XRD patterns and morphology of CaHap particles were essentially not changed by the modification. The additional amount of PO(4) of CaHap was increased with an increase of PP concentration and the Ca/P molar ratio of the particles decreased from 1.62 to 0.81. IR results indicated that the isolated surface POH band developed with increasing the PP concentration up to 6.0 mmol dm(-3) by the reaction of isolated surface POH groups of CaHap and pyrophosphoric acids. Above 10.2 mmol dm(-3), a hydrogen-bonding surface POH band appeared at 2913 cm(-1) and enlarged with increasing the PP concentration, while the isolated surface POH band was weakened. The results of N(2) and H(2)O adsorption measurements revealed that the modified particles aggregated compared to the unmodified ones, which would be due to the formation of hydrogen-bonding surface POH groups among the particles.

Topics: Adsorption; Diphosphates; Durapatite; Nitrogen; Particle Size; Surface Properties; Water

The surface of synthetic colloidal calcium hydroxyapatite (Ca(10)(PO(4))(6)(OH)(2); CaHap) was treated with pyrophosphoric acid (H(4)P(2)O(7); PP) in acetone and the materials were characterized by XRD, thermal analysis, N(2) adsorption, TEM, and FTIR. No remarkable change in XRD patterns or in particle morphology by the modification was observed. The additional amount of PO(4) of CaHap was increased with an increase in PP concentration. The Ca/P molar ratio of CaHap was decreased from 1.62 to 1.35 by the modification. IR results indicated that the PP reacts with surface P-OH groups of CaHap to form additional surface P-OH groups as follows: surface P-OH+H(4)P(2)O(7)--> surface P-O-PO(OH)(2) + H(3)PO(4). When the modified CaHap with Ca/P molar ratio less than 1.50 was treated at 850 degrees C in air, the materials transformed into beta-Ca(3)(PO(4))(2).

Topics: Diphosphates; Durapatite; Surface Properties

The quantities of two forms of crystalline silica, tridymite and cristobalite, in heated rice husk ash (RHA) samples were determined by X-ray diffraction (XRD) and chemical methods. Two RHA samples, containing 93% SiO2 and 2-3% K2O, were prepared from charcoaled rice husk products and heated to above 900 degrees C. The crystalline silica made up over 60-80% of the total silica in the heated RHA samples based on the XRD analysis. The crystalline phases in the two samples were somewhat different: The sample heated in the temperature range of 900 to 1,200 degrees C contained 52-62% cristobalite and 10-17% tridymite, but the other sample heated at a comparable temperature, above 1,100 degrees C, contained 46-66% tridymite and 37-16% cristobalite. Based on a correlation of lower tridymite crystallization temperature with higher potassium content, it was concluded that higher potassium levels were responsible for this difference. The pyrophosphoric acid analysis did not give exact results in the evaluation of total crystalline silica content in these RHA samples. As the combustion of rice husk was considered to cover the demands for energy and silica resource in Asian countries, cristobalite and tridymite crystallized in RHA by burning of rice husk should be assessed precisely by XRD analysis and the airborne dust in relevant workplace be controlled.

Topics: Crystallization; Diphosphates; Environmental Monitoring; Heating; Oryza; Plant Epidermis; Silicon Dioxide; X-Ray Diffraction

The structure and reactivity of gaseous H5P2O8- ions obtained from the chemical ionization (CI) of an H4P2O7/H2O mixture and from electrospray ionization (ESI) of CH3CN/H2O/H4P2O7 solutions were investigated by Fourier transform ion cyclotron (FTICR) and triple quadrupole mass spectrometry. Theoretical calculations performed at the B3LYP/6-31+G* level of theory and collisionally activated dissociation (CAD) mass spectrometric results allowed the ionic population obtained in the CI conditions to be structurally characterized as a mixture of gaseous [H3P2O7...H2O]-, [H3PO4...H2PO4]-, and [PO3...H3PO4...H2O]- clusters. The energy profile emerging from theoretical calculations affords insight into the mechanism of diphosphate ion hydrolysis and synthesis.

Topics: Computers; Diphosphates; Hydrolysis; Ions; Isomerism; Molecular Structure; Spectrum Analysis; Temperature

The characterization of naturally occurring variations in the human genome has evoked an immense interest during recent years. Variations known as biallelic Single-Nucleotide Polymorphisms (SNPs) have become increasingly popular markers in molecular genetics because of their wide application both in evolutionary relationship studies and in the identification of susceptibility to common diseases. We have addressed the issue of SNP genotype determination by investigating variations within the Renin-Angiotensin-Aldosterone System (RAAS) using pyrosequencing, a real-time pyrophosphate detection technology. The method is based on indirect luminometric quantification of the pyrophosphate that is released as a result of nucleotide incorporation onto an amplified template. The technical platform employed comprises a highly automated sequencing instrument that allows the analysis of 96 samples within 10 to 20 minutes. In addition to each studied polymorphic position, 5-10 downstream bases were sequenced for acquisition of reference signals. Evaluation of pyrogram data was accomplished by comparison of peak heights, which are proportional to the number of incorporated nucleotides. Analysis of the pyrograms that resulted from alternate allelic configurations for each addressed SNP revealed a highly discriminating pattern. Homozygous samples produced clear-cut single base peaks in the expected position, whereas heterozygous counterparts were characterized by distinct half-height peaks representing both allelic positions. Whenever any of the allelic bases of an SNP formed a homopolymer with adjacent bases, the nonallelic signal was added to those of the SNP. This feature did not, however, influence SNP readability. Furthermore, the multibase reading capacity of the described system provides extensive flexibility in regard to the positioning of sequencing primers and allows the determination of several closely located SNPs in a single run.

Topics: Alleles; Diphosphates; DNA; DNA Primers; Genotype; Humans; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Reproducibility of Results; Sequence Analysis, DNA; Templates, Genetic

Equilibrium constants for the binding of anions to apotransferrin, to the recombinant N-lobe half transferrin molecule (Tf/2N), and to a series of mutants of Tf/2N have been determined by difference UV titrations of samples in 0.1 M Hepes buffer at pH 7.4 and 25 degrees C. The anions included in this study are phosphate, sulfate, bicarbonate, pyrophosphate, methylenediphosphonic acid, and ethylenediphosphonic acid. There are no significant differences between anion binding to Tf/2N and anion binding to the N-lobe of apotransferrin. The binding of simple anions like phosphate appears to be essentially equivalent for the two apotransferrin binding sites. The binding of pyrophosphate and the diphosphonates is inequivalent, and the studies on the recombinant Tf/2N show that the stronger binding is associated with the N-terminal site. Anion binding constants for phosphate, pyrophosphate, and the diphosphonates with the N-lobe mutants K206A, K296A, and R124A have been determined. Anion binding tends to be weakest for the K296A mutant, but the variation in log K values among the three mutants is surprisingly small. It appears that the side chains of K206, K296, and R124 all make comparable contributions to anion binding. There are significant variations in the intensities of the peaks in the difference UV spectra that are generated by the titrations of the mutant apoproteins with these anions. These differences appear to be related more to variations in the molar extinction coefficients of the anion-protein complexes rather than to differences in binding constants.

Topics: Anions; Apoproteins; Binding Sites; Diphosphates; Diphosphonates; Models, Molecular; Mutation; Recombinant Proteins; Spectrophotometry, Ultraviolet; Thermodynamics; Transferrin

The pharmacological treatment of calcium urinary stones, most of which are made of calcium oxalate (CaOx), is only prophylactic. The causes of nephrolithiasis are often unclear, and a number of patients were found to be deficient in physiological inhibitors, e.g. citrate, pyrophosphate, magnesium, and specific proteins. The identification and characterization of these inhibitors can be performed in vitro by a number of methods, most of which are complex and time-consuming. Thus, we developed a simple turbidimetric method based on the precipitation of CaOx from a supersaturated solution. Using this approach, we determined that ionic strengths > 0.2 and pH < 5 inhibited the precipitation of CaOx. The first observation is of interest if one considers that the osmolarity of urine varies in the range of 50-1,400 mmol/kg, while the effect of pH is not usually seen in vivo because of the influence of other phenomena, such as the precipitation of uric acid. The activity of sodium chloride, magnesium and citrate was displayed at concentrations not far from their normal urinary level. Among several mono-, di- and tricarboxylic acids, like acetic, ascorbic, citric, isocitric, formic, fumaric, gluconic, glutaric, alpha-ketoglutaric, maleic, malic, malonic, propionic, pyruvic, succinic, and tartaric acid, only isocitric acid was more potent than citric acid. Pyrophosphate was a potent inhibitor in vitro, but its urinary level may not be sufficient for a significant effect in vivo. Amino acids like Ala, Arg, Asp, Glu, Gly, and Ser which are known to bind calcium, showed little activity. Work is in progress to search for new compounds potentially useful in the treatment of nephrolithiasis.

Topics: Calcium Oxalate; Chemical Precipitation; Citric Acid; Diphosphates; Humans; Hydrogen-Ion Concentration; Kidney Calculi; Kinetics; Manganese; Nephelometry and Turbidimetry; Sensitivity and Specificity; Urine

A newly produced bioceramic, beta-Ca2P2O7 with addition of Na4P2O7.10H2O (SDCP), has been implanted into the femoral condyle of rabbits. Within 6 weeks after implantation, most of the bioceramic is replaced by new woven bone. On the contrary, block from hydroxyapatite (HA) and beta-tricalcium phosphate (beta-TCP), which are osteoconductible, do not resorb within a short period of time. We believe that the biodegradable behaviour of SDCP may occur in two steps. The first and most important step is the digestion of particles and migration of the particles by phagocytosis. The object of this study is to examine the change in morphologies, chemical compositions and crystal structure of SDCP after soaking in distilled water for a certain period of time. The SDCP ceramic was also co-cultured with leucocytes to observe how the SDCP particles were digested by the leucocytes, so that the mechanism of biodegradable behaviour of SDCP ceramic in vivo might be clarified. Four types of sintered calcium phosphate ceramics were tested in the experiment: SDCP, pure beta-Ca2P2O7 (DCP), HA and beta-TCP. They wee soaked in distilled water at 37 degrees C for up to 30 days. The microstructure and morphology of crystals deposited on the surface were observed using scanning electron microscopy. Sodium, calcium and phosphorus ion contents in the supernatant solution were detected by atomic absorption analysis and ion coupled plasma. In summary, HA and DCP showed no significant evidence of dissolution in distilled water. In static distilled water, calcium ions may be released from beta-TCP into solution during the initial 7 days and then converted into HA by reprecipitation. The results showed that the SDCP was firstly dissolved into small grains or fragments by the solution. The small fragments should be so small as to be digested by the phagocytes in a physiological environment.

Topics: Animals; Biocompatible Materials; Biodegradation, Environmental; Calcium; Cells, Cultured; Ceramics; Diphosphates; Kinetics; Leukocytes; Male; Microscopy, Electron, Scanning; Phagocytosis; Phosphates; Rabbits; Time Factors; X-Ray Diffraction

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Carbon Isotopes; Chromatography; Diphosphates; Escherichia coli; Hot Temperature; Ligases; Magnesium; Metabolism; Phosphorus Isotopes; Polyphosphates; Research; RNA

Topics: Biochemical Phenomena; Biochemistry; Chromatography; Diphosphates; Edetic Acid; Glucose; Guanine Nucleotides; Mannose; Nucleotides; Nucleotidyltransferases; Research; Saccharomyces; Uracil Nucleotides

Topics: Biochemical Phenomena; Biochemistry; Diphosphates; Edetic Acid; Guanine Nucleotides; Hexosephosphates; Kinetics; Mannose; Nucleotides; Nucleotidyltransferases; Research; Uracil Nucleotides

Topics: Animals; Cytosine Nucleotides; Diabetes Mellitus, Experimental; Diphosphates; Glucose; Glucose-6-Phosphatase; Inorganic Pyrophosphatase; Insulin; Kinetics; Liver; Microsomes; Microsomes, Liver; Phosphotransferases; Polyphosphates; Pyrophosphatases; Rats; Research; Surface-Active Agents; Toxicology

Topics: Alcohols; Diphosphates; Enzyme Inhibitors; Hexoses; Liver; Mannose; Metabolism; Microsomes; Pentoses; Phosphotransferases; Rats; Research

Topics: Carps; Detergents; Diphosphates; Glutamate Dehydrogenase; Liver; Research; Subcellular Fractions

Topics: Adenine; Adenine Nucleotides; Animals; Carbohydrate Metabolism; Carcinoma; Carcinoma, Ehrlich Tumor; Dinitrophenols; Diphosphates; Glucose; Humans; In Vitro Techniques; Iodoacetates; Lactates; Methylene Blue; Pharmacology; Phosphoribosyl Pyrophosphate; Phosphotransferases; Research

Topics: Adenine; Animals; Carcinoma; Carcinoma, Ehrlich Tumor; Dinitrophenols; Diphosphates; Guanine; In Vitro Techniques; Iodoacetates; Metabolism; Methylene Blue; Nucleotides; Pharmacology; Phosphoribosyl Pyrophosphate; Research; Xanthines

Topics: Animals; Carbohydrate Metabolism; Carcinoma; Carcinoma, Ehrlich Tumor; Dinitrophenols; Diphosphates; Glucose; In Vitro Techniques; Iodoacetates; Lactates; Metabolism; Nucleosides; Pharmacology; Phosphoribosyl Pyrophosphate; Research; Ribonucleosides; Uridine

Topics: Bone Neoplasms; Bone Resorption; Chromatography; Diphosphates; Fluids and Secretions; Humans; Hydroxyproline; Hyperparathyroidism; Hyperthyroidism; Osteitis Deformans; Phosphorus; Urine

Topics: Adrenal Glands; Animals; Cattle; Chromatography; Diphosphates; Farnesol; Ligases; Metabolism; Mevalonic Acid; Microsomes; NADP; Research; Squalene; Terpenes

Topics: Alkaline Phosphatase; Calcification, Physiologic; Calcinosis; Chromatography; Diphosphates; Fluids and Secretions; Hypophosphatasia; Metabolism, Inborn Errors; Phosphorus Isotopes; Urine

Topics: Aspartate Carbamoyltransferase; Aspartic Acid; Carbamates; Catalysis; Diphosphates; Enzyme Inhibitors; Escherichia coli; Kinetics; Research; Transferases

Topics: Adenine; Adenosine Triphosphate; Animals; Antimetabolites; Ascites; Carbon Isotopes; Diphosphates; Metabolism; Mice; Neoplasms; Neoplasms, Experimental; Nucleosides; Nucleotides; Phosphoribosyl Pyrophosphate; Research; Tissue Culture Techniques; Xylose

Oster, M. O. (A. & M. College of Texas, College Station), and N. P. Wood. Formate-pyruvate exchange reaction in Streptococcus faecalis. II. Reaction conditions for cell extracts. J. Bacteriol. 87:104-113. 1964.-In contrast to intact cells of Streptococcus faecalis, no stimulation of the formate-pyruvate exchange reaction was observed in cell extracts when yeast extract was added to the reaction mixture. A heated extract of Micrococcus lactilyticus, vitamin K(5), ferrous sulfate, and ferrous ammonium sulfate stimulated an active exchange by protecting the system from oxygen. Tetrahydrofolate, 2,3-dimercaptopropanol, and sodium sulfide provided partial protection, whereas ascorbate, glutathione, sodium hydrosulfite, ammonium sulfide, and sodium bisulfite gave insufficient protection or were inhibitory. Oxidation-reduction (O-R) indicators were not inhibitory and were used to estimate the O-R potentials of reaction mixtures. A potential at least as negative as -125 mv was estimated to be necessary to preserve or initiate formate-pyruvate exchange activity. The reaction operated over a narrow pH range when strict anaerobic conditions were not maintained but, when the system was suitably poised, the pH range was broader. The influence of high phosphate concentrations was less under strictly anaerobic conditions, and orthophosphate could be replaced by small amounts of pyrophosphate. Effect of temperature, time, and amount of extract is presented. Addition of reduced benzyl viologen and hydrogen-saturated palladium in the buffer during 8 hr of dialysis prevented inactivation of extracts. Recovery of activity could be obtained after ammonium sulfate treatment when a combination of palladium chloride, neutral red, and hydrogen bubbling were used.

Topics: Adenosine Triphosphate; Ammonium Compounds; Cell Extracts; Dialysis; Dimercaprol; Diphosphates; Enterococcus faecalis; Folic Acid; Formates; Glutathione; Iron; Micrococcus; Palladium; Phosphates; Pyruvates; Pyruvic Acid; Quaternary Ammonium Compounds; Renal Dialysis; Research; Sodium; Temperature; Vitamin K

Topics: Biomedical Research; Calcium; Diphosphates; Humans; Pharmacology; Phosphates; Preventive Medicine; Urinary Calculi; Urine; Urolithiasis

Topics: Diphosphates; Humans; Metabolism; Phosphates; Pyrophosphatases; Urinary Calculi; Urine; Urolithiasis

Topics: Adenine; Adenine Nucleotides; Animals; Antimetabolites; Carbon Isotopes; Carcinoma; Carcinoma, Ehrlich Tumor; Deoxyadenine Nucleotides; Diphosphates; Neoplasms, Experimental; Pharmacology; Phosphoribosyl Pyrophosphate; Phosphotransferases; Research

Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Antimetabolites; Benzoates; Chloromercuribenzoates; Chromatography; Dinitrophenols; Diphosphates; Edetic Acid; Luminescence; Muscle Proteins; Myosins; Pharmacology; Pyrroles; Research; Spectrophotometry

Topics: Adenosine Triphosphate; Amino Acids; Animals; Chromatography; Diphosphates; Diptera; DNA; Electron Transport Complex II; Glycerophosphates; Magnesium; Metamorphosis, Biological; Mitochondria; Oxidoreductases; Phosphorus Isotopes; Proteins; Research; RNA; Spectrophotometry; Succinate Dehydrogenase

Topics: Adenine Nucleotides; Adenosine Triphosphate; Animals; Cattle; Cytosine Nucleotides; Dinitrophenols; Diphosphates; Guanine Nucleotides; Mitochondria; Mitochondria, Heart; Myocardium; NAD; NADP; Nucleotides; Pharmacology; Phosphates; Phospholipids; Proteins; Research; Spectrophotometry; Uracil Nucleotides

Topics: Calcium Carbonate; Calcium Phosphates; Carbonates; Chromatography; Colorimetry; Diphosphates; Heating; Hot Temperature; Hydroxides; Infrared Rays; Magnesium; Research; Strontium

Topics: Calcium; Calcium Pyrophosphate; Chelating Agents; Diphosphates; Ions; Magnesium; Muscle Proteins; Research; Solutions; Viscosity

Topics: Bone and Bones; Diphosphates; Fluorescence; Minerals; Oxytetracycline; Research

Topics: 4-Aminobenzoic Acid; Diphosphates; Enterococcus faecalis; Enzyme Inhibitors; Folic Acid; Glutamates; Metabolism; Pterins; Research; Sulfathiazoles; Veillonella

Topics: Adenosine Triphosphate; Amino Acids; Diphosphates; Glutathione; Isoleucine; Leucine; Liver; Magnesium; Rats; Research; RNA; Species Specificity

Topics: Diphosphates; Enzyme Inhibitors; Glucose; Glucose-6-Phosphatase; Hexosephosphates; Inorganic Pyrophosphatase; Liver; Mannose; Microsomes; Phosphotransferases; Pyrophosphatases; Rats; Research; Spectrophotometry

Topics: Diphosphates; Enzyme Inhibitors; Galactose; Glucosamine; Glucose; Glucose-6-Phosphatase; Hexosephosphates; Inorganic Pyrophosphatase; Kinetics; Liver; Metabolism; Microsomes; Microsomes, Liver; Phosphotransferases; Pyrophosphatases; Research

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Amino Acids; Chloramphenicol; Demeclocycline; Diphosphates; DNA; DNA, Bacterial; Edetic Acid; Escherichia coli; Nucleotides; Partial Pressure; Pharmacology; Polymyxins; Research; Streptomycin; Sulfadiazine

Topics: Adenine Nucleotides; Adenosine; Adenosine Diphosphate; Blood Platelet Disorders; Blood Platelets; Collagen; Diphosphates; Epinephrine; Hemophilia A; Humans; Leukemia; Leukemia, Myeloid; Norepinephrine; Research; Serotonin

Mathemeier, Paul F. (Oregon State University, Corvallis), and Richard Y. Morita. Influence of substrate-cofactor ratios on partially purified inorganic pyrophosphatase activity at elevated temperatures. J. Bacteriol. 88:1661-1666. 1964.-Inorganic pyrophosphatase of Bacillus stearothermophilus was studied for optimal substrate-cofactor ratios at 60 to 100 C. Mg(++) was the primary cofactor, and Co(++) resulted in 50% enzyme activity at 60 C. The pH optima differed for the Mg(++) activated and Co(++) activated pyrophosphatase. At 80 C and above, Co(++) replaced Mg(++) as the optimal cofactor in the enzyme reaction. The optimal ratio of pyrophosphate to Mg(++) varied from 2 to 0.25, dependent on enzyme concentration. The optimal pyrophosphate-cobalt ratio was constant at 1.0. The enzyme catalyzed appreciable pyrophosphate hydrolysis at 95 C.

Topics: Bacillus; Calcium; Cobalt; Diphosphates; Geobacillus stearothermophilus; Hot Temperature; Hydrolysis; Inorganic Pyrophosphatase; Kinetics; Magnesium; Manganese; Metabolism; Oregon; Pharmacology; Pyrophosphatases; Research; Temperature

Benziman, Moshe (The Hebrew University of Jerusalem, Jerusalem, Israel), and N. Heller. Oxaloacetate decarboxylation and oxaloacetate-carbon dioxide exchange in Acetobacter xylinum. J. Bacteriol. 88:1678-1687. 1964.-Extracts of Acetobacter xylinum, prepared by sonic treatment, were shown to catalyze the decarboxylation of oxaloacetate (OAA) to pyruvate and CO(2), and the exchange of C(14)-carbon dioxide into the beta-carboxyl of OAA. Fractionation of the extracts with ammonium sulfate resulted in a 10-fold increase of the specific activity of the enzyme system catalyzing the CO(2) exchange and OAA decarboxylation reactions. The purified preparation catalyzed the exchange of pyruvate-3-C(14) into OAA. Similar pH curves with a pH optimum of 5.6 were obtained for the CO(2) exchange and OAA decarboxylation reactions. Both reactions require the presence of Mn(2+) or Mg(2+) ions. OAA decarboxylation was more strongly inhibited than the exchange of CO(2) by dialysis or metal-chelating agents. Avidin did not inhibit either reaction. Adenosine triphosphate (ATP), adenosine diphosphate (ADP), guanosine triphosphate (GTP), guanosine diphosphate (GDP), pyrophosphate, or inorganic phosphate did not promote OAA decarboxylation and the CO(2)-exchange reaction catalyzed by the purified preparation. The purified preparation failed to catalyze the carboxylation of phosphoenolpyruvate in the presence of GDP, ADP, or inorganic phosphate, and that of pyruvate in the presence of ATP or GTP, even when supplemented with an OAA-trapping system. A scheme for OAA decarboxylation which could account for the observed exchange reactions and for the failure to obtain net fixation of CO(2) is proposed. The relation between the exchange reaction and the synthesis of cellulose from pyruvate by A. xylinum is discussed.

Topics: Acetates; Acetobacter; Adenosine Triphosphate; Carbon Dioxide; Carbon Isotopes; Cellulose; Chelating Agents; Decarboxylation; Diphosphates; Gluconacetobacter xylinus; Guanine Nucleotides; Hydrogen-Ion Concentration; Israel; Magnesium; Manganese; Metabolism; Oxaloacetates; Oxaloacetic Acid; Pharmacology; Pyruvates; Pyruvic Acid; Renal Dialysis; Research

Topics: Animals; Diphosphates; Enzyme Inhibitors; Fetus; Hydrogen-Ion Concentration; Kidney; Kinetics; Liver; Magnesium; Microsomes; Phosphorus; Phosphotransferases; Pregnancy; Pregnancy, Animal; Rats; Research

Topics: Diphosphates; Glucose-6-Phosphatase; Hexosephosphates; Hydrogen-Ion Concentration; Inorganic Pyrophosphatase; Liver; Metabolism; Microsomes; Phosphotransferases; Pyrophosphatases; Rats; Research

Topics: Chemical Phenomena; Chemistry; Diphosphates; Nucleotides; Phenylalanine; Research

Topics: Adenine Nucleotides; Adenosine; Adenosine Diphosphate; Adenosine Monophosphate; Diphosphates; Erythrocytes; Female; Humans; Labor, Obstetric; Pregnancy; Work

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Amino Acids; Aminoacylation; Catalysis; Diphosphates; Pyrophosphatases; RNA; Triticum

Topics: Dactinomycin; Diphosphates; Humans; Nucleosides; Nucleotides; Polyphosphates

Topics: Diphosphates; Polyphosphates; Saccharomyces cerevisiae; Yeast, Dried; Yeasts

Topics: Adenosine Triphosphate; Diphosphates; Nonmuscle Myosin Type IIA; Phosphates

Topics: Adenine Nucleotides; Adenosine; Adenosine Triphosphate; Antimetabolites; Diphosphates; DNA; DNA, Bacterial; Escherichia coli; Phosphorylation; Radiation Effects

Topics: Biological Transport; Body Fluids; Diphosphates; Humans; Urine

Akagi, J. M. (University of Illinois, Urbana) and L. Leon Campbell. Inorganic pyrophosphatase of Desulfovibrio desulfuricans. J. Bacteriol. 86:563-568. 1963.-The inorganic pyrophosphatase of Desulfovibrio desulfuricans was purified 136-fold by (NH(4))(2)SO(4) and ethanol fractionation and diethylaminoethyl cellulose chromatography. Mg(++) or Mn(++) was required for optimal activity; Co(++) was only 65% as effective as Mg(++). The optimal ratio of Mg(++) to pyrophosphate was 1.0 at pH 8.0. The K(s) for the pyrophosphatase was found to be in the region of 1.9 x 10(-3)m. Sulfhydryl inhibitors and sodium fluoride had no effect on enzyme activity at a concentration of 10(-3)m. The purified enzyme did not hydrolyze adenosine triphosphate, glycerol phosphate, diphenyl phosphate, or p-nitrophenyl phosphate. Thermal stability studies showed that the enzyme is rapidly inactivated at temperatures above 40 C.

Topics: Adenosine Triphosphate; Desulfovibrio; Desulfovibrio desulfuricans; Diphosphates; Inorganic Pyrophosphatase; Nitrophenols; Organophosphorus Compounds; Phosphates; Pyrophosphatases; Research; Temperature

Topics: Adenine Nucleotides; Adenosine Triphosphate; Chemical Phenomena; Chemistry; Diphosphates; Edetic Acid; Guanine Nucleotides; Hydrogen-Ion Concentration; Hydrolysis; Liver; Magnesium; Microsomes; Microsomes, Liver; Mitochondria; Pyrophosphatases; Rats; Research

Topics: Chemistry, Pharmaceutical; Diphosphates; Morpholines; Nucleosides; Nucleotides; Polyphosphates; Research; Thymine Nucleotides

Topics: Adenosine Triphosphate; Animals; Ascites; Carbohydrate Metabolism; Carbohydrates; Carcinoma; Carcinoma, Ehrlich Tumor; Diphosphates; Erythrocytes; Glucose; Insulin; Metabolism; Neoplasms, Experimental; Pharmacology; Poultry; Research

Topics: Adenine Nucleotides; Adenosine Monophosphate; Diphosphates; Inosine; Liver; Nucleosides; Nucleotides; Phosphates

Topics: Adenine Nucleotides; Adenosine Diphosphate; Adenosine Triphosphate; Diphosphates; Muscle Proteins; Myosins

Topics: Calcification, Physiologic; Diphosphates; Humans

Topics: Adenine Nucleotides; Adenosine; Diphosphates; Female; Humans; Labor, Obstetric; Polyphosphates; Pre-Eclampsia; Pregnancy; Work

Van Vunakis, Helen (Brandeis University, Waltham, Mass.) and Roger M. Herriott. Inactivation of the T-even coliphages by pyrophosphate. J. Bacteriol. 83:590-596. 1962.-Pyrophosphate liberates the deoxyribonucleic acid from the T-even coliphages. The reaction is dependent on pH, temperature, time, and ionic environment. The reaction can be inhibited by magnesium, putrescine, spermidine, and neutral molecules above a certain molecular weight. Possible mechanisms of the reaction are discussed.

Topics: Bacteriophages; Coliphages; Diphosphates; Hydrogen-Ion Concentration; Temperature

Topics: Carotenoids; Diphosphates; Fruit; Lycopene; Solanum lycopersicum; Terpenes

Topics: Adenosine Triphosphate; Amino Acids; Diphosphates; Plants; Root Nodules, Plant

Topics: Calcification, Physiologic; Calcinosis; Diphosphates; Humans

Topics: Calcification, Physiologic; Calcinosis; Calcium; Calcium, Dietary; Diphosphates; Humans; Urine

Topics: Amides; Anesthetics, Local; Chromatography; Chromatography, Paper; Diphosphates; Phosphates

Topics: Adenosine Triphosphate; Diphosphates; Humans; Liver

Topics: Dialysis; Diphosphates; Renal Dialysis

Topics: Adenine Nucleotides; Adenosine; Blood Circulation; Coenzyme A; Diphosphates; Muscle, Smooth; NAD; NADP; Respiration

Schlesinger and Coon's report that crystalline yeast inorganic pyrophosphatase, in addition to its known ability to hydrolyze inorganic pyrophosphate in the presence of Mg ions, is also able to catalyze the hydrolysis of ATP and ADP in the presence of Zn ions was confirmed. A systematic study showed that the ratio of 370 of PPase-Mg over ATPase-Zn activities per milligram protein in various preparations of pyrophosphatase obtained in the course of isolation of crystalline pyrophosphatase from baker's yeast was nearly identical in all the preparations, independent of their purity. The course of hydrolysis of ATP by crystalline pyrophosphatase in the presence of Zn was carried out with the aid of ion exchange on Dowex 1. The finding of Schlesinger and Coon that the hydrolysis proceeds from ATP to ADP and then slowly to AMP was confirmed. The kinetics of the first phase of the reaction was found to depend on the molar ratio of Zn/ATP in the reaction mixture. Mg ions in the presence of Zn ions have an accelerating effect on the rate of hydrolysis of ATP. This suggests strongly that both activities-ATPase and PPase-are manifestations of the same active group in the protein molecule of crystalline pyrophosphatase.

Topics: Adenine Nucleotides; Adenosine Triphosphate; Catalysis; Diphosphates; Hydrolysis; Kinetics; Magnesium; Pyrophosphatases; Saccharomyces cerevisiae; Zinc

Topics: Adenine Nucleotides; Adenosine; Adenosine Triphosphate; Adenylyl Cyclases; Diphosphates; Enzymes; Phosphates

Topics: Biochemical Phenomena; Diphosphates; RNA

Topics: Adenine Nucleotides; Adenosine; Digitalis; Digitalis Glycosides; Digitoxin; Diphosphates; Myocardium; Phosphates; Phosphocreatine; Polyphosphates

Topics: Acetaldehyde; Decarboxylation; Diphosphates; Lyases; Oxythiamine; Pyruvate Decarboxylase; Pyruvates; Pyruvic Acid; Saccharomyces cerevisiae; Vitamin A

Topics: Chromatography; Diphosphates; Esters

Topics: Biochemical Phenomena; Diphosphates; Enzymes; Nucleosides; Nucleotides

Topics: Biochemical Phenomena; Diphosphates; Mycobacterium

Topics: Adenine Nucleotides; Adenosine Triphosphate; Amino Acids; Animals; Diphosphates; Embryo, Mammalian; Embryo, Nonmammalian; Leucine; Phosphates; Pyrophosphatases; Xenopus laevis

Topics: Amino Acids; Diphosphates; Ions; Magnesium; RNA; Sulfhydryl Compounds

Topics: Alcohols; Carotenoids; Diphosphates; Farnesol; Fungi; Phycomyces; Pyrophosphatases

Topics: Amino Acids; Biochemical Phenomena; Catalysis; Diphosphates; Magnesium; Pyrophosphatases; RNA

Topics: Animals; Diphosphates; Hawks; Insecta; Moths

Topics: Diphosphates; Mycobacterium; Pyrophosphatases

Topics: Diphosphates; Research; Thiamine Pyrophosphate

Topics: Adenine Nucleotides; Adenosine Triphosphate; Argininosuccinate Synthase; Diphosphates; Enzymes; Hydrolysis

Topics: Animals; Diphosphates; Magnesium; Muscle Proteins; Nonmuscle Myosin Type IIB; Rabbits; Solutions

Topics: Animals; Cattle; Diphosphates; DNA; Nucleotides; Phosphates; Pyrophosphatases; RNA; Semen

Topics: Body Fluids; Diphosphates; Enzymes; Nucleosides; Nucleotides; Phosphates; Semen

Topics: Chymotrypsin; Diphosphates; Ions; Sulfates

Topics: Cytokinesis; Diphosphates; Hydrolysis; Nucleosides; Nucleotides; Phosphates; Polyphosphates; Pyrophosphatases

Topics: Diphosphates

Topics: Adenine Nucleotides; Adenosine; Diphosphates; Eukaryota; Galactose; Guanosine Diphosphate; Mannose; Nucleosides; Nucleotides; Porphyra

Topics: Animals; Diphosphates; Heart; Heart Atria; Membrane Potentials; Organophosphorus Compounds; Pacemaker, Artificial; Rabbits

Topics: Diphosphates; Liver; Mitochondria, Liver

Topics: Adenosine Triphosphate; Amidohydrolases; Diphosphates; Glutaminase; Hydrolysis

Topics: Diphosphates; Humans

The soluble methanesulphonate of the oxime 2-hydroxyiminomethyl-N-methylpyridinium (P2S) has been used to treat animals poisoned with sarin or ethyl pyrophosphate. The effect of the size of the dose, and its time of administration in relation to poisoning, have been examined. This oxime is very efficient in conjunction with atropine when given either before or after poisoning. About 30 mg./kg. seems to be the optimum therapeutic dose of the methanesulphonate. The significance of this optimum is discussed in relation to the treatment of accidental poisoning by organophosphate insecticides in man.

Topics: Animals; Atropine; Diphosphates; Humans; Insecticides; Male; Mesylates; Organophosphate Poisoning; Oximes; Phosphates; Pralidoxime Compounds; Pyrans; Pyridinium Compounds; Sarin

Topics: Adenosine Triphosphate; Amino Acids; Animals; Diphosphates; Liver; Rats; Ribonucleases

Topics: Adenosine Triphosphate; Amino Acids; Diphosphates; Photosynthesis; Spinacia oleracea

Topics: Anti-Bacterial Agents; Antibiotics, Antitubercular; Bacillus; Diphosphates; Glutamates; Kanamycin; Mycobacterium tuberculosis; Phosphates; Potassium; Sodium Glutamate

Topics: Diphosphates; Fatty Acids; Hemiterpenes; Mevalonic Acid; Organophosphorus Compounds

Topics: Diphosphates

Topics: Coenzymes; Diphosphates; Muscles; Organic Chemicals

Topics: Biochemical Phenomena; Diphosphates; Escherichia coli

Topics: Adenosine Triphosphate; Diphosphates; Pentosephosphates; Ribose; Ribosemonophosphates

Anticholinesterases (eserine, ethyl pyrophosphate, paraoxon) were injected into the parotid ducts and found to cause a secretion of saliva. After previous preganglionic, parasympathetic denervation the effects were increased above normal; this was probably due to the supersensitivity to acetylcholine which develops after the operation. After previous postganglionic, parasympathetic denervation, on the other hand, the effects were much reduced, in spite of a pronounced supersensitivity towards, for example, acetylcholine. The cause of this "subsensitivity" towards cholinesterase inhibitors is discussed.

Topics: Acetylcholine; Cholinesterase Inhibitors; Denervation; Diphosphates; Humans; Parasympathectomy; Parasympathetic Nervous System; Parotid Gland; Phosphates; Physostigmine; Politics; Saliva

Topics: Bone and Bones; Cartilage; Diphosphates; Humans; Rickets

Topics: Amino Acids; Animals; Diphosphates; Liver; Microsomes; Microsomes, Liver; Nucleoproteins; Rats

Topics: Diphosphates; Glyceraldehyde 3-Phosphate; Glyceraldehyde-3-Phosphate Dehydrogenases; Oxidoreductases

Topics: Adenosine Triphosphate; Amino Acids; Animals; Diphosphates; Sea Urchins

Topics: Diphosphates; Thiamine Pyrophosphate

Topics: Adenosine Triphosphatases; Diphosphates; Hydrolysis; Ion Transport; Muscle Proteins; Myosins

Topics: Biochemical Phenomena; Diphosphates; Humans; Liver; Microsomes; Phosphates

Topics: Diphosphates; Humans; Male; Phosphoric Monoester Hydrolases; Prostate; Stilbenes

An association between the blood cholinesterase (ChE) levels and the toxicity of sarin and ethyl pyrophosphate has been established. This has been demonstrated in two types of experiment. In the first, guinea-pigs were given a non-lethal dose of sarin (isopropyl methylphosphonofluoridate) which reduced the blood ChE to 20% of normal, and at intervals, as the ChE level of the blood gradually recovered, separate batches were given a second dose of the same size. A comparison was then made between the blood ChE levels immediately prior to the injection of the second dose and the mortality rate. In the second, a relatively small dose of sarin or ethyl pyrophosphate was given daily to rabbits until the enzyme value fell to a steady level. LD50 values were then determined on such groups and compared with those found in saline treated ones. In both guinea-pigs and rabbits it has been shown that a depression of blood ChE to below 40% indicated an increased toxicity of sarin and ethyl pyrophosphate to these species. The approximate quantitative relation appears to be that susceptibility is increased in the ratio 1.5 when the blood ChE is reduced by half.

Topics: Animals; Cholinesterase Inhibitors; Cholinesterases; Diphosphates; Guinea Pigs; Phosphates; Rabbits; Sarin

Topics: Antineoplastic Agents; Diphosphates; Humans; Neoplasms

The mechanism responsible for the protection against lethal organophosphate poisoning by pyridine-2-aldoxime methiodide (P-2-AM) was studied in the mouse. Two types of organophosphates were used: ethyl pyrophosphate (TEPP), E 600, Ro 3-0340, and Ro 3-0422 which form with true cholinesterase a diethylphosphoryl enzyme (1) and DFP, D 600, and Ro 3-0351 which form with true cholinesterase a diisopropylphosphoryl enzyme (2).In vitro and under the experimental conditions used more than 50% reactivation of (1) was obtained within 1 hr. by concentrations of P-2-AM ranging from 0.5 to 1x10(-5) M; 30 times higher concentrations of the oxime were required to achieve the same effect with (2). In vivo reactivation of phosphorylated true cholinesterases in blood amounted to 10 to 24% within the first 30 min. if 25 mg./kg. P-2-AM was injected (i.p.) 5 min. before a sublethal dose of TEPP, E 600, Ro 3-0340, or Ro 3-0422 and reactivation reached a maximum within 1 to 2 hr. after the injection of the oxime. P-2-AM was more effective when given 30 min. after the organophosphate. The effect of 25 mg./kg. P-2-AM on the phosphorylated true cholinesterase in brain (experiments with TEPP and E 600) was negligible. A dose of 25 mg./kg. P-2-AM had no consistent effect on the phosphorylated true cholinesterases in blood and brain of mice injected with sublethal doses of DFP, D 600, or Ro 3-0351.The protection by 25 mg./kg. P-2-AM against lethal doses of TEPP, E 600, Ro 3-0422, and Ro 3-0340 was greater than that obtained with 50 mg./kg. atropine sulphate, but the degree of protection was determined by the organophosphate itself and not its dialkylphosphoryl group. Protection by 25 mg./kg. P-2-AM against lethal doses of DFP, D 600, and Ro 3-0351 was negligible. The antidotal effect of P-2-AM was potentiated by atropine. Mice which were injected with atropine and P-2-AM were protected to a greater extent against DFP than against Ro 3-0422, and protection against DFP was only slightly less than protection against TEPP. This is difficult to reconcile with a specific action of P-2-AM on phosphorylated cholinesterases.

Topics: Acetylcholinesterase; Animals; Atropine; Brain; Cholinesterases; Diphosphates; Mice; Organophosphates; Oximes; Paraoxon; Phosphates; Phosphorylation; Picolines; Pralidoxime Compounds

The effects of two potent inhibitors of cholinesterase, isopropylmethylphosphonofluoridate (sarin) and ethyl pyrophosphate (TEPP), were investigated on the bronchioles and pulmonary blood vessels in isolated dog lung preparations perfused with heparinized blood and ventilated artificially. These anticholinesterases caused gradual bronchoconstriction as indicated by a diminution in tidal air volume, a reduction in compliance and an increase in expiratory resistance. These effects were antagonized by atropine. An increase in pulmonary vascular resistance occurred which was enhanced by the addition of small quantities of acetylcholine to the perfusing blood. The pulmonary vascular effects of acetylcholine, sarin and TEPP were prevented and abolished by atropine. Evidence is presented which suggests that the site of action of these anticholinesterases is, at least in part, peripheral to the autonomic ganglia.

Topics: Acetylcholine; Animals; Bronchi; Bronchioles; Bronchoconstriction; Cholinesterase Inhibitors; Diphosphates; Dogs; Lung; Phosphoric Monoester Hydrolases; Tidal Volume; Vascular Resistance

Topics: Adenosine Triphosphate; Amino Acids; Biochemical Phenomena; Diphosphates

Topics: Adenosine Triphosphate; Amino Acids; Biochemical Phenomena; Diphosphates; Enzymes; Ribonucleases; Vitamin B 12

Topics: Antineoplastic Agents; Aziridines; Diphosphates; Humans

Topics: Cervix Uteri; Diphosphates; Female; Humans; Neoplasms; Thiamine; Thiamine Pyrophosphate

Topics: Blood; Diphosphates; Humans

Topics: Adenosine Triphosphate; Amino Acids; Bacteria; Diphosphates; Fungi

Using a technique for determining Ca and Mg based on Schwarzenberg's method of titration with ethylenediamine tetraacetic acid (EDTA), it was found that glycerol-extracted muscle fibers contain on the average 0.58 millimole Ca and 0.55 millimole Mg per kg. muscle. The fibers take up additional Ca or Mg from dilute solutions of these metals, but in KCl solutions, the excess is exchanged for K ions. Inorganic pyrophosphate (PP) removes part of the bound Mg, no Ca; EDTA removes predominantly Ca, but never more than about one-half the total amount. These results are discussed in relation to previous observations on the effects of PP and EDTA on mechanical properties and contractility of extracted muscle fibers. After the partial loss of bound divalent metals, muscle fibers swell in dilute salt solutions; they also contract slightly and become more translucent.

Topics: Calcium; Cations, Divalent; Diphosphates; Magnesium; Muscle Contraction; Muscles

Topics: Adenosine Triphosphate; Amino Acids; Bacteria; Biochemical Phenomena; Diphosphates

Topics: Adenosine; Adenosine Triphosphate; Diphosphates; Esters; Humans; Ions; Manganese; Nucleotides; Salts

Topics: Animals; Diphosphates; Liver; Rats; Thiamine Pyrophosphate

Topics: Diphosphates; Humans; Hydrolysis; Inorganic Pyrophosphatase; Magnesium; Phosphoric Monoester Hydrolases

Topics: Diphosphates; Humans; Myasthenia Gravis; Organophosphorus Compounds

Topics: Biological Assay; Buffers; Diphosphates; Oxidoreductases; Xanthine Oxidase

1. If glycerol-extracted muscle fibers are alternately immersed in solutions of inorganic pyrophosphate (PP) or ethylenediamme tetraacetate (EDTA) and KCl two to three times, PP no longer increases extensibility. The original condition is restored by solutions containing Mg. EDTA prevents completely the softening action of PP, but has no effect in the presence of an excess of Mg. These observations are explained by assuming that PP and EDTA remove bound Mg. Evidently PP has no softening action without Mg. 2. If PP is washed out from muscle fibers by solutions containing Mg, the softening action of PP persists for many minutes, but washing out with KCl solution promptly abolishes the effect of PP. Also, if fibers which have been made refractory to PP are immersed into a solution of PP and then into a KCl solution free of PP for several minutes, the addition of MgCl(2) alone increases extensibility. It is concluded that PP forms a complex with protein. The formation of this complex, however, has no influence on the mechanical properties, unless Mg also is present. 3. In the presence of PP and Mg the viscous resistance of muscle fibers drops with diminishing temperatures.

Topics: Diphosphates; Magnesium; Muscle Contraction; Muscles

Topics: Colorimetry; Diphosphates; Humans

Topics: Analgesics; Autonomic Nervous System; Diphosphates; Esters; Humans; Thiamine

Topics: Diphosphates; Phosphates

Topics: Adenosine Triphosphate; Carbohydrate Metabolism; Citrates; Citric Acid Cycle; Diphosphates; Fermentation; Glucose; Glycolysis; Hydrolysis

Topics: Diphosphates; Humans; Phosphates

Topics: Animals; Diphosphates; Fireflies; Luminescence

Topics: Adenosine Triphosphate; Diphosphates; Humans; Muscles; Stem Cells

Topics: Diphosphates; Organophosphate Poisoning; Parathion

Topics: Diphosphates; Phosphates

Topics: Bacteria; Bacteriolysis; Diphosphates; Fabaceae; Glycine; Glycine Agents

Topics: Actin Cytoskeleton; Actomyosin; Adenosine Triphosphate; Diphosphates; Magnesium; Magnesium Compounds; Muscle Proteins

Topics: Diphosphates; Muscle Weakness; Myasthenia Gravis

Crystalline inorganic pyrophosphatase has been isolated from baker's yeast. The crystalline enzyme is a protein of the albumin type with an isoelectric point near pH 4.8. Its molecular weight is of the order of 100,000. It contains about 5 per cent tyrosine and 3.5 per cent tryptophane. It is most stable at pH 6.8. The new crystalline protein acts as a specific catalyst for the hydrolysis of inorganic pyrophosphate into orthophosphate ions. It does not catalyze the hydrolysis of the pyrophosphate radical of such organic esters as adenosine di- and triphosphate, or thiamine pyrophosphate. Crystalline pyrophosphatase requires the presence of Mg, Co, or Mn ions as activators. These ions are antagonized by calcium ions. Mg is also antagonized by Co or Mn ions. The rate of the enzymatic hydrolysis of inorganic pyrophosphate is proportional to the concentration of enzyme and is a function of pH, temperature, concentration of substrate, and concentration of activating ion. The approximate conditions for optimum rate are: 40 degrees C. and pH 7.0 at a concentration of 3 to 4 x 10(-3)M Na(4)P(2)O(7) and an equivalent concentration of magnesium salt. The enzymatic hydrolysis of Na(4)P(2)O(7) or K(4)P(2)O(7) proceeds to completion and is irreversible under the conditions at which hydrolysis is occurring. Details are given of the method of isolation of the crystalline enzyme.

Topics: Diphosphates; Inorganic Pyrophosphatase; Kinetics; Magnesium; Phosphates; Phosphoric Monoester Hydrolases; Pyrophosphatases; Saccharomyces cerevisiae; Temperature; Yeast, Dried

Topics: Diphosphates; Myocardium; Myocytes, Cardiac; Phosphates; Phosphorus; Phosphorus Radioisotopes; Phosphorus, Dietary

Topics: Animals; Characidae; Cholinesterases; Diphosphates; Humans; Muscle Weakness; Myasthenia Gravis

Topics: Diphosphates; Fructose; Humans; Liver

Topics: Biophysical Phenomena; Coenzymes; Cytokinesis; Diphosphates; NAD

Topics: Adenosine Triphosphate; Diphosphates; Snake Venoms; Venoms

Topics: Diabetes Mellitus; Diphosphates; Hexosephosphates; Hexoses; Humans; Polysaccharides

Topics: Diphosphates; Enzymes; Phosphorus; Phosphorus Compounds; Phosphorus, Dietary; Polyphosphates

Topics: Diphosphates; Isotopes; Myocardium; Phosphates; Phosphorus; Phosphorus Isotopes; Radioactivity

Topics: Animals; Butterflies; Diphosphates; Insecta; Phosphorus; Phosphorus Compounds

Topics: Diphosphates; Fluorides; Insecticides; Neostigmine; Organophosphorus Compounds; Phosphates; Phosphorus; Phosphorus Compounds; Physostigmine

Topics: Animals; Diphosphates; Erythrocytes; Humans; Leukocytes; Male; Rats; Thiamine; Thiamine Pyrophosphate

Topics: Animals; Characidae; Diphosphates; Drug-Related Side Effects and Adverse Reactions; Hypokalemia; Myasthenia Gravis; Potassium; Potassium Compounds; Potassium Deficiency; Tetracycline

Topics: Adenine; Diphosphates; Fabaceae; Fruit; Pentoses; Phosphorus; Phosphorus Compounds; Phosphorus, Dietary; Vegetables

Topics: Diphosphates; NAD

Topics: Adenine; Diphosphates; Pentoses; Plants

Topics: Bacillus; Diphosphates; Glycerol; Gram-Positive Bacteria; Humans; Inositol; Mycobacterium tuberculosis; Phospholipids

Topics: Bacteria; Diphosphates; Phosphorus; Saccharomyces cerevisiae; Yeasts