flavin-adenine-dinucleotide and Disease-Models--Animal

Riboflavin (Rf), or vitamin B2, is the precursor of FMN and FAD, redox cofactors of several dehydrogenases involved in energy metabolism, redox balance and other cell regulatory processes. FAD synthase, coded by FLAD1 gene in humans, is the last enzyme in the pathway converting Rf into FAD. Mutations in FLAD1 gene are responsible for neuromuscular disorders, in some cases treatable with Rf. In order to mimic these disorders, the Caenorhabditis elegans (C. elegans) gene orthologue of FLAD1 (flad-1) was silenced in a model strain hypersensitive to RNA interference in nervous system. Silencing flad-1 resulted in a significant decrease in total flavin content, paralleled by a decrease in the level of the FAD-dependent ETFDH protein and by a secondary transcriptional down-regulation of the Rf transporter 1 (rft-1) possibly responsible for the total flavin content decrease. Conversely an increased ETFDH mRNA content was found. These biochemical changes were accompanied by significant phenotypical changes, including impairments of fertility and locomotion due to altered cholinergic transmission, as indicated by the increased sensitivity to aldicarb. A proposal is made that neuronal acetylcholine production/release is affected by alteration of Rf homeostasis. Rf supplementation restored flavin content, increased rft-1 transcript levels and eliminated locomotion defects. In this aspect, C. elegans could provide a low-cost animal model to elucidate the molecular rationale for Rf therapy in human Rf responsive neuromuscular disorders and to screen other molecules with therapeutic potential.

Topics: Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Disease Models, Animal; Flavin-Adenine Dinucleotide; Humans; Neuromuscular Diseases; Nucleotidyltransferases; Riboflavin; Vitamins

Short-term memory deficits have been associated with prefrontal cortex (PFC) dysfunction in Alzheimer's disease (AD) and AD mouse models. Extratelencephalic projection (ET) neurons in the PFC play a key role in short-term working memory, but the mechanism between ET neuronal dysfunction in the PFC and short-term memory impairment in AD is not well understood. Here, using fiber photometry and optogenetics, we found reduced neural activity in the ET neurons in the medial prefrontal cortex (mPFC) of the 5×FAD mouse model led to object recognition memory (ORM) deficits. Activation of ET neurons in the mPFC of 5×FAD mice rescued ORM impairment, and inhibition of ET neurons in the mPFC of wild type mice impaired ORM expression. ET neurons in the mPFC that project to supramammillary nucleus were necessary for ORM expression. Viral tracing and in vivo recording revealed that mPFC ET neurons received fewer cholinergic inputs from the basal forebrain in 5×FAD mice. Furthermore, activation of cholinergic fibers in the mPFC rescued ORM deficits in 5×FAD mice, while acetylcholine deficiency reduced the response of ET neurons in the mPFC to familiar objects. Taken together, our results revealed a neural mechanism behind ORM impairment in 5×FAD mice.

Topics: Acetylcholine; Alzheimer Disease; Animals; Disease Models, Animal; Flavin-Adenine Dinucleotide; Mice; Neurons; Prefrontal Cortex

Down syndrome (DS), or trisomy 21, is one of the critical risk factors for early-onset Alzheimer's disease (AD), implicating key roles for chromosome 21-encoded genes in the pathogenesis of AD. We previously identified a role for the deubiquitinase USP25, encoded on chromosome 21, in regulating microglial homeostasis in the AD brain; however, whether USP25 affects amyloid pathology remains unknown. Here, by crossing 5×FAD AD and Dp16 DS mice, we observed that trisomy 21 exacerbated amyloid pathology in the 5×FAD brain. Moreover, bacterial artificial chromosome (BAC) transgene-mediated USP25 overexpression increased amyloid deposition in the 5×FAD mouse brain, whereas genetic deletion of Usp25 reduced amyloid deposition. Furthermore, our results demonstrate that USP25 promoted β cleavage of APP and Aβ generation by reducing the ubiquitination and lysosomal degradation of both APP and BACE1. Importantly, pharmacological inhibition of USP25 ameliorated amyloid pathology in the 5×FAD mouse brain. In summary, we identified the DS-related gene USP25 as a critical regulator of AD pathology, and our data suggest that USP25 serves as a potential pharmacological target for AD drug development.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Amyloidosis; Animals; Aspartic Acid Endopeptidases; Brain; Disease Models, Animal; Down Syndrome; Flavin-Adenine Dinucleotide; Mice; Mice, Transgenic; Ubiquitin Thiolesterase

Objective To explore the effects of interleukin-6 (IL-6) gene knockout on the cognitive function and pathological changes in 5×FAD transgenic mice of Alzheimer's disease.Methods IL-6

Topics: Alzheimer Disease; Animals; Cognition; Disease Models, Animal; Flavin-Adenine Dinucleotide; Gene Knockout Techniques; Interleukin-6; Mice; Mice, Knockout

Working memory capacity (WMC) is the ability to maintain information over a few seconds. Although it has been extensively studied in healthy subjects and neuropsychiatric patients, few tasks have been developed to measure such changes in rodents. Many procedures have been used to measure WM in rodents, including the radial arm maze, the WM version of the Morris swimming task, and various delayed matching and nonmatching-to-sample tasks. It should be noted, however, that the memory components assessed in these procedures do not include memory capacity.. We developed an olfactory working memory capacity (OWMC) paradigm to assess the WMC of 3-month-old 5×FAD mice, a mouse model of Alzheimer's disease. The task is divided into five phases: context adaptation, digging training, rule learning for nonmatching to a single sample odor (NMSS), rule learning for nonmatching to multiple sample odors (NMMS), and capacity testing.. In the NMSS rule-learning phase, there was no difference between wild-type (WT) mice and 5×FAD mice in the performance correct rate, correct option rate, and correct rejection rate. The WT mice and 5×FAD mice showed similar memory capacity in the NMMS rule-learning phase. After capacity test, we found that the WMC was significantly diminished in 5×FAD mice. As the memory load increased, 5×FAD mice also made significantly more errors than WT mice.. The OWMC task, based on a nonmatch-to-sample rule, is a sensitive and robust behavioral assay that we validated as a reliable method for measuring WMC and exploring different components of memory in mice.

Topics: Alzheimer Disease; Animals; Disease Models, Animal; Flavin-Adenine Dinucleotide; Humans; Maze Learning; Memory, Short-Term; Mice; Mice, Transgenic; Smell

Abundant recent evidence has shown that 3-phosphoinositide-dependent protein kinase 1 (PDK1) is activated in Alzheimer's disease (AD). However, it remains unknown whether inhibition of PDK1 in neurons may affect AD-like pathology in animal models of AD. Here, we aim to examine the effects of specific inactivation of neuronal PDK1 on pathology and behaviour in 5×FAD mice and to identify the underlying molecular mechanisms.. The Cre-loxP system was employed to generate Pdk1 cKO/5×FAD mice, in which PDK1 is inactivated in excitatory neurons in the adult forebrain. Cellular and behavioural techniques were used to examine plaque burden, inflammatory responses and spatial working memory in mice. Biochemical and molecular analyses were conducted to investigate relevant mechanisms.. First, Aβ deposition was massively decreased and gliosis was highly attenuated in Pdk1 cKO/5×FAD mice compared with 5×FAD mice. Second, memory deficits were significantly improved in Pdk1 cKO/5×FAD mice. Third, APP levels were notably decreased in Pdk1 cKO/5×FAD mice. Fourth, mammalian target of rapamycin (mTOR) signalling and ribosome biogenesis were reduced in Pdk1 cKO/5×FAD mice.. Neuron-specific deletion of PDK1 robustly ameliorates AD-like pathology and improves spatial working memory in 5×FAD mice. We propose that genetic approach to inhibit PDK1 may be an effective strategy to slow AD.

Topics: Alzheimer Disease; Animals; Disease Models, Animal; Flavin-Adenine Dinucleotide; Gliosis; Mice; Mice, Transgenic; Plaque, Amyloid; Pyruvate Dehydrogenase Acetyl-Transferring Kinase

Infection with avian influenza A H5N1 virus results in acute lung injury (ALI) and has a high mortality rate (52.79%) because there are limited therapies available for treatment. Drug repositioning is an economical approach to drug discovery. We developed a method for drug repositioning based on high-throughput RNA sequencing and identified several drugs as potential treatments for avian influenza A H5N1 virus. Using high-throughput RNA sequencing, we identified a total of 1,233 genes differentially expressed in A549 cells upon H5N1 virus infection. Among these candidate genes, 79 drug targets (corresponding to 59 approved drugs) overlapped with the DrugBank target database. Twenty-two of the 41 commercially available small-molecule drugs reduced H5N1-mediated cell death in cultured A549 cells, and fifteen drugs that protected A549 cells when administered both pre- and post-infection were tested in an H5N1-infection mouse model. The results showed significant alleviation of acute lung injury by amitriptyline HCl (an antidepressant drug), flavin adenine dinucleotide (FAD; an ophthalmic agent for vitamin B2 deficiency), azacitidine (an anti-neoplastic drug) and calcitriol (an active form of vitamin D). All four agents significantly reduced the infiltrating cell count and decreased the lung injury score in H5N1 virus-infected mice based on lung histopathology, significantly improved mouse lung edema by reducing the wet-to-dry weight ratio of lung tissue and significantly improved the survival of H5N1 virus-infected mice. This study not only identifies novel potential therapies for influenza H5N1 virus-induced lung injury but also provides a highly effective and economical screening method for repurposing drugs that may be generalizable for the prevention and therapy of other diseases.

Topics: Acute Lung Injury; Amitriptyline; Animals; Azacitidine; Calcitriol; Disease Models, Animal; Drug Repositioning; Female; Flavin-Adenine Dinucleotide; Humans; Influenza A Virus, H5N1 Subtype; Influenza, Human; Lung; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL

Macrophages play an important role in regulating the tumor microenvironment (TME). Here we show that classical (M1) macrophage polarization reduced expression of LSD1, nuclear REST corepressor 1 (CoREST), and the zinc finger protein SNAIL. The LSD1 inhibitor phenelzine targeted both the flavin adenine dinucleotide (FAD) and CoREST binding domains of LSD1, unlike the LSD1 inhibitor GSK2879552, which only targeted the FAD domain. Phenelzine treatment reduced nuclear demethylase activity and increased transcription and expression of M1-like signatures both

Topics: Animals; Cell Differentiation; Co-Repressor Proteins; Cytokines; Disease Models, Animal; Flavin-Adenine Dinucleotide; Histone Demethylases; Humans; Macrophage Activation; Macrophages; Mice; Nerve Tissue Proteins; Phenelzine; RAW 264.7 Cells; RNA, Small Interfering; Snail Family Transcription Factors; Th1 Cells; Triple Negative Breast Neoplasms; Tumor Microenvironment

Multiphoton FLIM microscopy offers many opportunities to investigate processes in live cells, tissue and animal model systems. For redox measurements, FLIM data is mostly published by cell mean values and intensity-based redox ratios. Our method is based entirely on FLIM parameters generated by 3-detector time domain microscopy capturing autofluorescent signals of NAD(P)H, FAD and novel FLIM-FRET application of Tryptophan and NAD(P)H-a2%/FAD-a1% redox ratio. Furthermore, image data is analyzed in segmented cells thresholded by 2 × 2 pixel Regions of Interest (ROIs) to separate mitochondrial oxidative phosphorylation from cytosolic glycolysis in a prostate cancer cell line. Hundreds of data points allow demonstration of heterogeneity in response to intervention, identity of cell responders to treatment, creating thereby different sub-populations. Histograms and bar charts visualize differences between cells, analyzing whole cell versus mitochondrial morphology data, all based on discrete ROIs. This assay method allows to detect subtle differences in cellular and tissue responses, suggesting an advancement over means-based analyses.

Topics: Animals; Cytosol; Disease Models, Animal; Doxorubicin; Flavin-Adenine Dinucleotide; Glucose; Heterografts; Humans; Microscopy, Fluorescence; Mitochondria; Molecular Imaging; NAD; NADP; Neoplasms; Oxidation-Reduction; Oxidative Phosphorylation

In this study, label-free fluorescence spectroscopy was used for the first time to determine spectral profiles of tryptophan, reduced nicotinamide adenine dinucleotide (NADH), and flavin denine dinucleotide (FAD) in fresh brain samples of a mouse model of Alzheimer's disease (AD). Our results showed that the emission spectral profile levels of tryptophan and NADH were higher in AD samples than normal samples. The intensity ratio of tryptophan to NADH and the change rate of fluorescence intensity with respect to wavelength also increased in AD brain. These results yield an optical method for detecting early stage of AD by comparing spectral profiles of biomolecules.

Topics: Alzheimer Disease; Animals; Brain; Disease Models, Animal; Early Diagnosis; Flavin-Adenine Dinucleotide; Humans; Mice; Mice, Transgenic; NAD; Spectrometry, Fluorescence; Tryptophan

The goal of the present study was to quantify and correlate the contribution of the cytosolic p67(phox) subunit of NADPH oxidase 2 to mitochondrial oxidative stress in the kidneys of the Dahl salt-sensitive (SS) hypertensive rat. Whole kidney redox states were uniquely assessed using a custom-designed optical fluorescence three-dimensional cryoimager to acquire multichannel signals of the intrinsic fluorophores NADH and FAD. SS rats were compared with SS rats in which the cytosolic subunit p67(phox) was rendered functionally inactive by zinc finger nuclease mutation of the gene (SS(p67phox)-null rats). Kidneys of SS rats fed a 0.4% NaCl diet exhibited significantly (P = 0.023) lower tissue redox ratio (NADH/FAD; 1.42 ± 0.06, n = 5) than SS(p67phox)-null rats (1.64 ± 0.07, n = 5), indicating reduced levels of mitochondrial electron transport chain metabolic activity and enhanced oxidative stress in SS rats. When fed a 4.0% salt diet for 21 days, both strains exhibited significantly lower tissue redox ratios (P < 0.001; SS rats: 1.03 ± 0.05, n = 9, vs. SS(p67phox)-null rats: 1.46 ± 0.04, n = 7) than when fed a 0.4% salt, but the ratio was still significantly higher in SS(p67phox) rats at the same salt level as SS rats. These results are consistent with results from previous studies that found elevated medullary interstitial fluid concentrations of superoxide and H2O2 in the medulla of SS rats. We conclude that the p67(phox) subunit of NADPH oxidase 2 plays an important role in the excess production of ROS from mitochondria in the renal medulla of the SS rat.

Topics: Animals; Disease Models, Animal; Flavin-Adenine Dinucleotide; Frozen Sections; Genotype; Hypertension; Image Processing, Computer-Assisted; Imaging, Three-Dimensional; Kidney; Male; Microscopy, Fluorescence; Mitochondria; NAD; Oxidation-Reduction; Oxidative Stress; Phenotype; Phosphoproteins; Rats, Inbred Dahl; Rats, Transgenic; Sodium Chloride, Dietary; Time Factors

Hereditary Hemorrhagic Telangiectasia-1 (HHT-1) is a vascular disease caused by mutations in the endoglin (Eng)/CD105 gene. The objective of this study was to quantify the oxidative state of a rodent model of HHT-1 using an optical imaging technique. We used a cryofluorescence imaging instrument to quantitatively assess tissue metabolism in this model. Mitochondrial redox ratio (FAD/NADH), FAD RR, was used as a quantitative marker of the metabolic status and was examined in the kidneys, and eyes of wild-type and Eng +/- mice. Kidneys and eyes from wild-type P21, 6W, and 10M old mice showed, respectively, a 9% (±2), 24% (±0.4), 15% (±1), and 23% (±4), 33% (±0.6), and 30% (±2) change in the mean FAD RR compared to Eng +/- mice at the same age. Thus, endoglin haploinsufficiency is associated with less oxidative stress in various organs and mitigation of angiogenesis.

Topics: Animals; Cells, Cultured; Disease Models, Animal; Endoglin; Eye; Flavin-Adenine Dinucleotide; Freezing; Imaging, Three-Dimensional; Intracellular Signaling Peptides and Proteins; Kidney; Lung; Mice, Transgenic; Mitochondria; NAD; Optical Imaging; Oxidative Stress; Reactive Oxygen Species; Telangiectasia, Hereditary Hemorrhagic

Riboflavin, also known as vitamin B2 , is converted by riboflavin kinase (RFK) into flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are essential cofactors of dehydrogenases, reductases, and oxidases including the phagocytic NADPH oxidase 2 (Nox2). Riboflavin deficiency is common in young adults and elderly individuals, who are at the coincidental risk for listeriosis. To address the impact of acute riboflavin deficiency on host defense against Listeria monocytogenes (L.m.), we generated conditional RFK knockout (KO) strains of mice. Phagocyte-specific RFK KO impaired the capability of phagocytes to control intracellular L.m., which corresponded to a greater susceptibility of mice to in vivo challenge with L.m. The oxidative burst of RFK-deficient phagocytes in response to L.m. infection was significantly reduced. Mechanistically, TNF-induced priming of Nox2, which is needed for oxidative burst, was defective in RFK-deficient phagocytes. Lack of riboflavin in wild-type macrophages for only 6 h shut down TNF-induced, RFK-mediated de novo FMN/FAD generation, which was accompanied by diminished ROS production and impaired anti-listerial activity. Vice versa, ROS production by riboflavin-deprived macrophages was rapidly restored by riboflavin supplementation. Our results suggest that acute riboflavin deficiency immediately impairs priming of Nox2, which is of crucial relevance for an effective phagocytic immune response in vivo.

Topics: Animals; Disease Models, Animal; Disease Resistance; Flavin-Adenine Dinucleotide; Immunity, Innate; Listeria monocytogenes; Listeriosis; Macrophages; Membrane Glycoproteins; Mice; Mice, Transgenic; NADPH Oxidase 2; NADPH Oxidases; Phagocytes; Riboflavin Deficiency; Tumor Necrosis Factor-alpha

Chronic quinolinic acid (QA) lesions in rats closely resemble Huntington's disease like conditions. Oxidative stress and mitochondrial dysfunction have long been implicated in the neurotoxic effects of QA acting through N-methyl-d-aspartate (NMDA) receptors. Reports suggest that inhibition of adenosine A2A receptor function elicits neuroprotective effect in QA induced neurotoxicity in rats. Caffeine, a preferential A2A receptor antagonist imitates antioxidant like actions and exerts neuroprotective effects in various neurodegenerative conditions. Thus, the present study was designed to evaluate the neuroprotective effects of caffeine against QA induced neurotoxicity in rats.. In the present study, QA (200nmol/2μl saline) has been administered bilaterally to the striatum of rats followed by chronic caffeine (10, 20 and 40mg/kg) administration for 21 days. Motor performance of the animals was evaluated in weekly intervals and subsequently after 21 days, the animals were sacrificed and measurement of mitochondrial complexes activity, respiration rate and endogenous antioxidant levels were carried out in the striatal region.. Single intrastriatal QA administration resulted in drastic reduction in body weight, marked motor impairment (decreased total locomotor activity in actophotometer and impaired grip strength in rotarod), increased oxidative stress, impaired mitochondrial complexes activities and decreased state 3 respiration (NAD(+)/FAD(+)-linked) in rats. However, chronic treatment of caffeine for 21 days significantly attenuated the QA induced behavioural, biochemical and mitochondrial alterations displaying neuroprotective efficacy.. The study highlights the possible involvement of A2A receptor antagonism in the neuroprotective effect of caffeine against QA induced mitochondrial dysfunction and oxidative stress in rats.

Topics: Animals; Antioxidants; Caffeine; Cell Respiration; Corpus Striatum; Disease Models, Animal; Dose-Response Relationship, Drug; Flavin-Adenine Dinucleotide; Huntington Disease; Mitochondria; NAD; Neuroprotective Agents; Neurotoxicity Syndromes; Oxidative Stress; Quinolinic Acid; Rats

Abnormal cellular metabolism is a hallmark of cancer, yet there is an absence of quantitative methods to dynamically image this powerful cellular function. Optical metabolic imaging (OMI) is a noninvasive, high-resolution, quantitative tool for monitoring cellular metabolism. OMI probes the fluorescence intensities and lifetimes of the autofluorescent metabolic coenzymes reduced NADH and flavin adenine dinucleotide. We confirm that OMI correlates with cellular glycolytic levels across a panel of human breast cell lines using standard assays of cellular rates of glucose uptake and lactate secretion (P < 0.05, r = 0.89). In addition, OMI resolves differences in the basal metabolic activity of untransformed from malignant breast cells (P < 0.05) and between breast cancer subtypes (P < 0.05), defined by estrogen receptor and/or HER2 expression or absence. In vivo OMI is sensitive to metabolic changes induced by inhibition of HER2 with the antibody trastuzumab (herceptin) in HER2-overexpressing human breast cancer xenografts in mice. This response was confirmed with tumor growth curves and stains for Ki67 and cleaved caspase-3. OMI resolved trastuzumab-induced changes in cellular metabolism in vivo as early as 48 hours posttreatment (P < 0.05), whereas fluorodeoxyglucose-positron emission tomography did not resolve any changes with trastuzumab up to 12 days posttreatment (P > 0.05). In addition, OMI resolved cellular subpopulations of differing response in vivo that are critical for investigating drug resistance mechanisms. Importantly, OMI endpoints remained unchanged with trastuzumab treatment in trastuzumab-resistant xenografts (P > 0.05). OMI has significant implications for rapid cellular-level assessment of metabolic response to molecular expression and drug action, which would greatly accelerate drug development studies.

Topics: Animals; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Disease Models, Animal; Female; Flavin-Adenine Dinucleotide; Glycolysis; Humans; Mice; Mice, Nude; NAD; Optical Imaging; Receptor, ErbB-2; Trastuzumab; Xenograft Model Antitumor Assays

D-Amino acid oxidase (DAAO; EC1.4.3.3) has been proposed to play a main role in the degradation of D-serine, an allosteric activator of the N-methyl-D-aspartate-type glutamate receptor in the human brain, and to be associated with the onset of schizophrenia. To prevent excessive D-serine degradation, novel drugs for schizophrenia treatment based on DAAO inhibition were designed and tested on rats. However, the properties of rat DAAO are unknown and various in vivo trials have demonstrated the effects of DAAO inhibitors on d-serine concentration in rats. In the present study, rat DAAO was efficiently expressed in Escherichia coli. The recombinant enzyme was purified as an active, 40 kDa monomeric flavoenzyme showing the basic properties of the dehydrogenase-oxidase class of flavoproteins. Rat DAAO differs significantly from the human counterpart because: (a) it possesses a different substrate specificity; (b) it shows a lower kinetic efficiency, mainly as a result of a low substrate affinity; (c) it differs in affinity for the binding of classical inhibitors; (d) it is a stable monomer in the absence of an active site ligand; and (e) it interacts with the mammalian protein modulator pLG72 yielding a ~100 kDa complex in addition to the ~200 kDa one, as formed by the human DAAO. Furthermore, the concentration of endogenous D-serine in U87 glioblastoma cells was not affected by transfection with rat DAAO, whereas it was significantly decreased when expressing the human homologue. These results raise doubt on the use of the rat as a model system for testing new drugs against schizophrenia and indicate a different physiological function of DAAO in rodents and humans.

Topics: Animals; Carrier Proteins; D-Amino-Acid Oxidase; Disease Models, Animal; Enzyme Inhibitors; Flavin-Adenine Dinucleotide; Glioblastoma; Humans; Intracellular Signaling Peptides and Proteins; Protein Binding; Protein Conformation; Rats; Recombinant Proteins; Schizophrenia; Serine; Substrate Specificity; Tumor Cells, Cultured

Diets deficient in an indispensable amino acid are known to suppress food intake in rats. Few studies were focused at understanding how amino acid-deficient diets may elicit biochemical changes at the mitochondrial level. The goal of this study was to evaluate mitochondrial function in rats fed diets with 0.00, 0.18, 0.36, and 0.88% threonine (Thr) (set at 0, 30, 60, and 140% of Thr requirement for growth). Here, it is described for the first time that Thr-deficient diets induce a specific uncoupling of mitochondria in liver, especially with NADH-linked substrates, not observed in heart (except for Thr-devoid diet). The advantage of this situation would be to provide ATP to support growth and maintenance when high-quality protein food (or wealth of high-quality food in general) is available, whereas Thr-deficient diets (or deficient-quality protein food) promote the opposite, increasing mitochondrial uncoupling in liver. The uncoupling with NADH substrates would favor the use of nutrients as energy sources with higher FADH-to-NADH ratios, such as fat, minimizing the first irreversible NADH-dependent catabolism of many amino acids, including Thr, thus enhancing the use of the limiting amino acid for protein synthesis when a low quality protein source is available.

Topics: Adenosine Triphosphate; Animal Nutritional Physiological Phenomena; Animals; Body Weight; Dietary Proteins; Disease Models, Animal; Eating; Energy Metabolism; Flavin-Adenine Dinucleotide; Liver; Male; Mitochondria, Heart; Mitochondria, Liver; Myocardium; NAD; Oxidative Phosphorylation; Protein Deficiency; Rats; Rats, Sprague-Dawley; Threonine; Time Factors

Mutations in leukocyte NADPH oxidase genes lead to defective respiratory burst in leukocytes and cause chronic granulomatous diseases (CGD) in humans. The most common form of CGD is caused by mutations in the membrane-bound oxidase component gp91phox, which is encoded by the CYBB gene on the X chromosome. We previously reported on a patient with a CYBB mutation (H338Y) that prevents the intracellular trafficking and expression of gp91phox on leukocytes. The capacity of the leukocytes to produce reactive oxygen species (ROS) was rescued by treatment with thapsigargin and flavin adenine dinucleotide (FAD). The increase in ROS production was not due to the increase in cytoplasmic calcium induced by thapsigargin because the treatment of calcium ionophore did not have the same effect. Protein and cellular analyses on leukocytes and cells transfected with GFP-tagged gp91phox mutant showed that treated cells expressed more Endo H-resistant gp91phox protein on the cell surface and are more effective in killing bacteria. Thapsigargin- and FAD-treated CGD leukocytes had enhanced activity in protecting mice from Staphylococcus-induced peritoneal abscess formation in a mouse model of CGD. These results indicate that thapsigargin-FAD ex vivo treatment is effective in rescuing the ROS-producing activity of leukocytes in selected CGD patients.

Topics: Animals; Cytochrome b Group; Disease Models, Animal; Female; Flavin-Adenine Dinucleotide; Granulomatous Disease, Chronic; Humans; Leukocytes; Membrane Glycoproteins; Mice; Mice, Knockout; NADPH Oxidase 2; NADPH Oxidases; Reactive Oxygen Species; Staphylococcus aureus; Thapsigargin

The activity and expression of nitric oxide synthase (NOS) isoforms and protein nitrotyrosine (NT) residues were investigated in whole encephalic mass (WEM) homogenates during the development of experimental allergic encephalomyelitis (EAE) in Lewis rats. EAE stages (0-III) were daily defined by clinical evaluation, and in the end of each stage, WEMs were removed for analysis of NOS activity, protein NT residues and mRNA for the different NOS isoforms. In the presence of NADPH, WEMs from EAE-III rats showed lower Ca2+-dependent NOS activity than those from control group. These differences disappeared in the presence of exogenous calmodulin, flavin adenine dinucleotide (FAD), tetrahydrobiopterin (BH4) and NADPH. Of all the cofactors, just the omission of FAD caused comparable decrease of Ca2+-dependent NOS activity from both groups. Ca2+-independent NOS activity from EAE-III animals was insensitive to the omission of any of the cofactors, while in control animals this activity was significantly inhibited by the omission of either FAD or BH4. Increased levels of both iNOS mRNA and protein NT expression were observed in animals with EAE, which also showed lower levels of a thermolabile NOS inhibitor in WEM homogenates and sera than controls. In conclusion, during late EAE stages, constitutive Ca2+-dependent NOS activity decreases concomitantly with iNOS upregulation, which could be responsible for the high protein NT levels. The differential dependence of iNOS activity on cofactors and the absence of an endogenous thermolabile NOS inhibitor in animals with EAE could reflect additional control mechanisms of NOS activity in this model of multiple sclerosis.

Topics: Animals; Biopterins; Brain; Calcium; Calmodulin; Disease Models, Animal; Disease Progression; Encephalomyelitis, Autoimmune, Experimental; Female; Flavin-Adenine Dinucleotide; Male; NADP; Neurons; Nitric Oxide; Nitric Oxide Synthase; Protein Isoforms; Rats; Rats, Inbred Lew; RNA, Messenger; Subcellular Fractions; Tyrosine

Enteroviruses, the most common cause of acute myocarditis, are also supposed aetiological agents of dilated cardiomyopathy. Autoantibodies (anti-M7; Klein & Berg, Clin Exp Immunol 1990; 58:283-92) directed against flavoproteins with covalently bound flavin (alphaFp-Ab; Otto et al., Clin Exp Immunol 1998; 111:541-2) are detected in up to 30% of sera of patients with myocarditis and idiopathic dilated cardiomyopathy (IDCM). Mice inoculated with a myocarditic variant of coxsackievirus B3 (CVB3) were employed to study the occurrence of serum alphaFp-Ab following viral infection. The presence of alphaFp-Ab was analysed by Western blotting with the flavoprotein antigens 6-hydroxy-D-nicotine oxidase (6HDNO) and sarcosine oxidase (SaO). Of 10 sera from CVB3-infected mice, five showed a strong reaction with both antigens. The sera were reactive also to the mitochondrial covalently flavinylated proteins dimethylglycine dehydrogenase and sarcosine dehydrogenase. Sera of non-infected mice did not react with these antigens. A 6HDNO mutant protein with non-covalently bound FAD no longer reacted on Western blots with sera of CVB3-infected mice. Preincubation with FAD abolished or reduced the reaction of the sera with the 6HDNO antigen. At 2 weeks p.i. the alphaFp-Ab were of the IgM and IgG isotypes, at 7 and 9 weeks p.i. of the IgG isotype. The sera of CVB3-infected mice reproduced closely the antigenic specificity of the anti-M7 sera of patients, lending further support to the role of coxsackieviruses in the pathogenesis of IDCM.

Topics: Animals; Autoantibodies; Cardiomyopathy, Dilated; Coxsackievirus Infections; Disease Models, Animal; Enterovirus B, Human; Flavin-Adenine Dinucleotide; Flavoproteins; Humans; Immunoglobulin G; Immunoglobulin M; Male; Metalloendopeptidases; Mice; Mitochondria, Liver; Myocarditis; Myocardium; Neutralization Tests; Oxidoreductases; Oxidoreductases, N-Demethylating; Peptides; Rats; Sarcosine Oxidase; Trypsin

Glucose-stimulated insulin secretion is impaired in GK (Goto-Kakizaki) rats, perhaps because of abnormalities in glucose metabolism in pancreatic islet beta cells. The glycerol phosphate shuttle plays a major role in glucose metabolism by reoxidizing cytosolic NADH generated by glycolysis. In the pancreatic islets of GK rats, the activity of mitochondrial FAD-linked glycerol-3-phosphate dehydrogenase (mGPDH), the key enzyme of the glycerol phosphate shuttle, is decreased and this abnormality may be responsible, at least in part, for impaired glucose-stimulated insulin secretion. To investigate this possibility, we overexpressed mGPDH in islets isolated from GK rats via recombinant adenovirus-mediated gene transduction, and examined glucose-stimulated insulin secretion. In islets isolated from diabetic GK rats at 8 to 10 weeks of age, glucose-stimulated insulin secretion was severely impaired, and mGPDH activity was decreased to 79 % of that in non-diabetic Wistar rats. When mGPDH was overexpressed in islets from GK rats, enzyme activity and protein content increased 2- and 6-fold, respectively. Basal (3 mmol/l glucose) and glucose-stimulated (20 mmol/l) insulin secretion from the Adex1CAlacZ-infected GK rat islets were, respectively, 4.4 +/- 0.7 and 8.1 +/- 0.7 ng. x islet(-1) x 30 min(-1), and those from mGPDH-overexpressed GK rat islets 4.7 +/- 0.3 and 9.1 +/- 0.8 ng x islet(-1) x 30 min(-1), in contrast to those from the AdexlCAlacZ-infected non-diabetic Wistar rat islets (4.7 +/- 1.6 and 47.6 +/- 11.9 ng x islet(-1) x 30 min(-1)). Thus, glucose-stimulated insulin secretion is severely impaired in GK rats even in the stage when mGPDH activity is modestly decreased, and at this stage, overexpression of mGPDH cannot restore glucose-stimulated insulin secretion. We conclude that decreased mGPDH activity in GK rat islets is not the defect primarily responsible for impaired glucose-stimulated insulin secretion.

Topics: Adenoviridae; Animals; Blotting, Western; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 2; Disease Models, Animal; Female; Flavin-Adenine Dinucleotide; Gene Expression; Gene Expression Regulation, Enzymologic; Glucose; Glycerolphosphate Dehydrogenase; Insulin; Insulin Secretion; Islets of Langerhans; Lac Operon; Male; Mitochondria; Rats; Rats, Inbred Strains; Rats, Wistar; Recombinant Fusion Proteins; Transcriptional Activation; Transfection