-
Amino Acids Sep 2007We examined the effects of DON [glutamine-analogue and inhibitor of glutamine-requiring enzymes], alanyl-glutamine (regarding its role in neutrophil immunonutrition) and...
We examined the effects of DON [glutamine-analogue and inhibitor of glutamine-requiring enzymes], alanyl-glutamine (regarding its role in neutrophil immunonutrition) and alanyl-glutamine combined with L-NAME, SNAP, DON, beta-alanine and DFMO on neutrophil amino and alpha-keto acid concentrations or important neutrophil immune functions in order to establish whether an inhibitor of *NO-synthase [L-NAME], an *NO donor [SNAP], an analogue of taurine and a taurine transport antagonist [beta-alanine], an inhibitor of ornithine-decarboxylase [DFMO] as well as DON could influence any of the alanyl-glutamine-induced effects. In summary, irrespective of which pharmacological, metabolism-inhibiting or receptor-mediated mechanisms were involved, our results showed that impairment of granulocytic glutamine uptake, modulation of intracellular glutamine metabolisation and/or de novo synthesis as well as a blockade of important glutamine-dependent metabolic processes may led to significant modifications of physiological and immunological functions of the affected cells.
Topics: Adult; Amino Acids; Antibiotics, Antineoplastic; Diazooxonorleucine; Dipeptides; Eflornithine; Enzyme Inhibitors; Homeostasis; Humans; Hydrogen Peroxide; Immunocompetence; Keto Acids; Male; NG-Nitroarginine Methyl Ester; Neutrophils; Nitric Oxide Donors; Oxidants; Peroxidase; S-Nitroso-N-Acetylpenicillamine; Signal Transduction; Superoxides
PubMed: 17072790
DOI: 10.1007/s00726-006-0395-x -
Neurochemistry International 2006Brain edema and the subsequent increase in intracranial pressure are the major neurological complications in fulminant hepatic failure (FHF). Brain edema in FHF is...
Brain edema and the subsequent increase in intracranial pressure are the major neurological complications in fulminant hepatic failure (FHF). Brain edema in FHF is predominantly "cytotoxic" due principally to astrocyte swelling. It is generally believed that ammonia plays a key role in this process, although the mechanism by which ammonia brings about such swelling is yet to be defined. It has been postulated that glutamine accumulation in astrocytes subsequent to ammonia detoxification results in increased osmotic forces leading to cell swelling. While the hypothesis is plausible and has gained support, it has never been critically tested. In this study, we examined whether a correlation exists between cellular glutamine levels and the degree of cell swelling in cultured astrocytes exposed to ammonia. Cultured astrocytes derived from rat brain cortices were exposed to ammonia (5 mM) for different time periods and cell swelling was measured. Cultures treated with ammonia for 1-3 days showed a progressive increase in astrocyte cell volume (59-127%). Parallel treatment of astrocyte cultures with ammonia showed a significant increase in cellular glutamine content (60-80%) only at 1-4 h, a time when swelling was absent, while glutamine levels were normal at 1-3 days, a time when peak cell swelling was observed. Thus no direct correlation between cell swelling and glutamine levels was detected. Additionally, acute increase in intracellular levels of glutamine by treatment with the glutaminase inhibitor 6-diazo-5-oxo-L-norleucine (DON) after ammonia exposure also did not result in swelling. On the contrary, DON treatment significantly blocked (66%) ammonia-induced astrocyte swelling at a later time point (24 h), suggesting that some process resulting from glutamine metabolism is responsible for astrocyte swelling. Additionally, ammonia-induced free radical production and induction of the mitochondrial permeability transition (MPT) were significantly blocked by treatment with DON, suggesting a key role of glutamine in the ammonia-induced free radical generation and the MPT. In summary, our findings indicate a lack of direct correlation between the extent of cell swelling and cellular levels of glutamine. While glutamine may not be acting as an osmolyte, we propose that glutamine-mediated oxidative stress and/or the MPT may be responsible for the astrocyte swelling by ammonia.
Topics: Ammonia; Ammonium Chloride; Animals; Animals, Newborn; Astrocytes; Cell Size; Cells, Cultured; Cerebral Cortex; Diazooxonorleucine; Free Radicals; Glutaminase; Glutamine; Hydrolysis; Membrane Potentials; Mitochondria; Permeability; Rats
PubMed: 16517020
DOI: 10.1016/j.neuint.2005.11.017 -
Journal of Experimental & Clinical... Dec 2004In this study the enzyme glutaminase, purified from the ascites fluid of ovarian cancer patients, was analysed for its antiangiogenic activity. Intraperitoneal...
In this study the enzyme glutaminase, purified from the ascites fluid of ovarian cancer patients, was analysed for its antiangiogenic activity. Intraperitoneal administration of this enzyme reduces the number of tumor directed capillaries in solid and ascites tumor bearing Swiss mice induced by transplantation of Ehrlich ascites cells. The enzyme has a critical role in regulating the secretion of vascular endothelial growth factor (VEGF) from tumor cell and in turn tumor growth. Glutamine analogue like 6-diazo, 5- oxo L-norleucine (DON) is also found to be effective in regulating vascular endothelial growth factor (VEGF) secretion from tumor cells in vitro. Treatment with enzyme reduced serum VEGF levels of the tumor induced animals. In vitro VEGF production by EAC cells was reduced in a concentration dependent manner in presence of glutamine analogue.
Topics: Animals; Antineoplastic Agents; Body Weight; Carcinoma, Ehrlich Tumor; Cell Line, Tumor; Cell Proliferation; Diazooxonorleucine; Dose-Response Relationship, Drug; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Glutaminase; Glutamine; Mice; Neoplasm Transplantation; Neoplasms; Neovascularization, Pathologic; Ovarian Neoplasms; Time Factors; Vascular Endothelial Growth Factor A
PubMed: 15743040
DOI: No ID Found -
Journal of Neuroscience ResearchGlutamine, a byproduct of ammonia detoxification, is found elevated in brain in hepatic encephalopathy (HE) and other hyperammonemic disorders. Such elevation has been... (Comparative Study)
Comparative Study
Glutamine, a byproduct of ammonia detoxification, is found elevated in brain in hepatic encephalopathy (HE) and other hyperammonemic disorders. Such elevation has been implicated in some of the deleterious effects of ammonia on the central nervous system (CNS). Recent studies have shown that glutamine results in the induction of the mitochondrial permeability transition (MPT) in cultured astrocytes. We examined whether glutamine shows similar effects in cultured neurons. Both cultured astrocytes and neurons were exposed to glutamine (6.5 mM) for 24 hr and the MPT was assessed by changes in cyclosporin A (CsA)-sensitive inner mitochondrial membrane potential (DeltaPsi(m)) using the potentiometric dye tetramethylrhodamine ethyl ester (TMRE). Glutamine significantly dissipated the DeltaPsi(m) in astrocytes as demonstrated by a decrease in mitochondrial TMRE fluorescence, a process that was blocked by CsA. On the other hand, treatment of cultured neurons with glutamine had no effect on the DeltaPsi(m). Dissipation of the DeltaPsi(m) in astrocytes by glutamine was blocked by treatment with 6-diazo-5-oxo-L-norleucine (DON; 100 microM), suggesting that glutamine hydrolysis and the subsequent generation of ammonia, which has been shown previously to induce the MPT, might be involved in MPT induction by glutamine. These data indicate that astrocytes but not neurons are vulnerable to the toxic effects of glutamine. The selective induction of oxidative stress and the MPT by glutamine in astrocytes may partially explain the deleterious affects of glutamine on the CNS in the setting of hyperammonemia, as well as account for the predominant involvement of astrocytes in the pathogenesis of HE and other hyperammonemic conditions.
Topics: Animals; Animals, Newborn; Antibiotics, Antineoplastic; Astrocytes; Cells, Cultured; Cerebral Cortex; Diazooxonorleucine; Drug Interactions; Glutamine; Membrane Potentials; Mitochondria; Neurons; Organometallic Compounds; Permeability; Rats
PubMed: 15573403
DOI: 10.1002/jnr.20295 -
European Journal of Biochemistry Feb 2004Genes SNO1 and SNZ1 are Saccharomyces cerevisiae homologues of PDX2 and PDX1 which participate in pyridoxine synthesis in the fungus Cercospora nicotianae. In order to...
Genes SNO1 and SNZ1 are Saccharomyces cerevisiae homologues of PDX2 and PDX1 which participate in pyridoxine synthesis in the fungus Cercospora nicotianae. In order to clarify their function, the two genes SNO1 and SNZ1 were expressed in Escherichia coli either individually or simultaneously and with or without a His-tag. When expressed simultaneously, the two protein products formed a complex and showed glutaminase activity. When purified to homogeneity, the complex exhibited a specific activity of 480 nmol.mg(-1).min(-1) as glutaminase, with a Km of 3.4 mm for glutamine. These values are comparable to those for other glutamine amidotransferases. In addition, the glutaminase activity was impaired by 6-diazo-5-oxo-L-norleucine in a time- and dose-dependent manner and the enzyme was protected from deactivation by glutamine. These data suggest strongly that the complex of Sno1p and Snz1p is a glutamine amidotransferase with the former serving as the glutaminase, although the activity was barely detectable with Sno1p alone. The function of Snz1p and the amido acceptor for ammonia remain to be identified.
Topics: Chromosome Mapping; Chromosomes, Fungal; DNA Primers; Diazooxonorleucine; Enzyme Inhibitors; Escherichia coli; Glutamic Acid; Glutaminase; Glutamine; Hydrolysis; Kinetics; Mutation; Plasmids; Pyridoxine; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 14764090
DOI: 10.1111/j.1432-1033.2003.03973.x -
The Journal of Biological Chemistry Dec 2003Glutamine stimulates the expression of the argininosuccinate synthetase (ASS) gene at both the level of enzyme activity and mRNA in Caco-2 cells. Searching to identify...
Glutamine stimulates the expression of the argininosuccinate synthetase (ASS) gene at both the level of enzyme activity and mRNA in Caco-2 cells. Searching to identify the pathway involved, we observed that (i) the stimulating effect of glutamine was totally mimicked by glucosamine addition, and (ii) its effect but not that of glucosamine was totally blocked by 6-diazo-5-oxo-l-norleucine (DON), an inhibitor of amidotransferases, suggesting that the metabolism of glutamine to glucosamine 6-phosphate was required. Moreover, run-on assays revealed that glucosamine was acting at a transcriptional level. Because three functional GC boxes were identified on the ASS gene promoter (Anderson, G. M., and Freytag, S. O. (1991) Mol. Cell Biol. 11, 1935-1943), the potential involvement of Sp1 family members was studied. Electrophoretic mobility shift assays using either the Sp1 consensus sequence or an appropriate fragment of the ASS promoter sequence as a probe demonstrated that both glutamine and glucosamine increased Sp1 DNA binding. Immunoprecipitation-Western blot experiments demonstrated that both compounds increased O-glycosylation of Sp1 leading to its translocation into nucleus. Again, the effect of glutamine on Sp1 was inhibited by the addition of DON but not of glucosamine. Taken together, the results clearly demonstrate that the metabolism of glutamine through the hexosamine pathway leads to the cytosolic O-glycosylation of Sp1, which, in turn, translocates into nucleus and stimulates the ASS gene transcription. Collectively, the results constitute the first demonstration of a functional relationship between a regulating signal (glutamine), a transcription factor (Sp1), and the transcription of the ASS gene.
Topics: Argininosuccinate Synthase; Blotting, Northern; Blotting, Western; Caco-2 Cells; Cell Line; Cell Line, Tumor; Cell Nucleus; Cytosol; DNA, Complementary; Diazooxonorleucine; Dose-Response Relationship, Drug; Fructosephosphates; Glucosamine; Glutamine; Glycosylation; Humans; Models, Biological; Precipitin Tests; Promoter Regions, Genetic; Protein Transport; RNA, Messenger; Sp1 Transcription Factor; Time Factors; Transcription, Genetic
PubMed: 14570901
DOI: 10.1074/jbc.M306752200 -
Endocrinology Oct 2003The present study investigated whether activation of the hexosamine biosynthesis pathway might mediate at least in part the high glucose effect on angiotensinogen (ANG)...
The present study investigated whether activation of the hexosamine biosynthesis pathway might mediate at least in part the high glucose effect on angiotensinogen (ANG) gene expression and immortalized renal proximal tubular cell (IRPTC) hypertrophy. IRPTC were cultured in monolayer. ANG, renin, and beta-actin mRNA expression were determined by specific RT-PCR assays. Phosphorylation of p38 MAPK, activating transcription factor-2 (ATF-2), and cAMP-responsive element-binding protein (CREB) was determined by Western blot analysis. Cell hypertrophy was assessed by flow cytometry, intracellular p27kip1 protein levels, and [3H]leucine incorporation into proteins. Glucosamine stimulated ANG and renin mRNA expression and enhanced p38 MAPK, ATF-2, and CREB phosphorylation in normal glucose (5 mm) medium. Azaserine and 6-diazo-5-oxo-l-norleucine (inhibitors of glutamine: fructose-6-phosphate amino transferase enzyme) blocked the stimulatory effect of high glucose, but not that of glucosamine, on ANG gene expression in IRPTCs. SB 203580 (a specific p38 MAPK inhibitor) attenuated glucosamine action on ANG gene expression as well as p38 MAPK and ATF-2 phosphorylation, but not that of CREB. GF 109203X and calphostin C (inhibitors of protein kinase C) blocked the effect of glucosamine on ANG gene expression and CREB phosphorylation, but had no impact on p38 MAPK and ATF-2 phosphorylation. Finally, both glucosamine and high glucose induced IRPTC hypertrophy. The hypertrophic effect of glucosamine was blocked in the presence of GF 109203X, but not azaserine and SB 203580. In contrast, the hypertrophic effect of high glucose was blocked in the presence of azaserine and GF 109203X, but not SB203580. Our studies demonstrate that the stimulatory effect of high glucose on ANG gene expression and IRPTC hypertrophy may be mediated at least in part via activation of hexosamine biosynthesis pathway signaling.
Topics: Activating Transcription Factor 2; Angiotensinogen; Animals; Azaserine; Cell Line, Transformed; Cyclic AMP Response Element-Binding Protein; Diazooxonorleucine; Dose-Response Relationship, Drug; Gene Expression; Glucosamine; Glucose; Hexosamines; Hypertrophy; Kidney Tubules, Proximal; Mitogen-Activated Protein Kinases; Phosphorylation; RNA, Messenger; Rats; Transcription Factors; p38 Mitogen-Activated Protein Kinases
PubMed: 12960040
DOI: 10.1210/en.2003-0220 -
Biochemical and Biophysical Research... Jul 2003To elucidate the role of acetyl-L-carnitine in the brain, we used a novel method, 'Bioradiography,' in which the dynamic process could be followed in living slices by...
To elucidate the role of acetyl-L-carnitine in the brain, we used a novel method, 'Bioradiography,' in which the dynamic process could be followed in living slices by use of positron-emitter labeled compounds and imaging plates. We studied the incorporation of 2-[18F]fluoro-2-deoxy-D-glucose ([18F]FDG) into rat brain slices incubated in oxygenated Krebs-Ringer solution. Under the glucose-free condition, [18F]FDG uptake rate decreased with time and plateaued within 350 min in the cerebral cortex and cerebellum, and the addition of 1 or 5mM acetyl-L-carnitine did not alter the [18F]FDG uptake rate. When a glutaminase inhibitor, 0.5mM 6-diazo-5-oxo-L-norleucine (DON), was added under the normal glucose condition, [18F]FDG uptake rate decreased. Acetyl-L-carnitine (1mM), which decreased [18F]FDG uptake rate, reversed this DON-induced decrease in [18F]FDG uptake rate in the cerebral cortex. These results suggest that acetyl-L-carnitine can be used for the production of releasable glutamate rather than as an energy source in the brain.
Topics: Acetylcarnitine; Animals; Antimetabolites, Antineoplastic; Brain; Cerebral Cortex; Diazooxonorleucine; Fluorodeoxyglucose F18; Glucose; Glutamic Acid; Male; Models, Biological; Radiography; Rats; Rats, Sprague-Dawley; Time Factors; Tomography, Emission-Computed
PubMed: 12821151
DOI: 10.1016/s0006-291x(03)01103-3 -
Journal of Molecular Biology Jun 2003Glutamate synthases (GltS) are crucial enzymes in ammonia assimilation in plants and bacteria, where they catalyze the formation of two molecules of L-glutamate from...
Glutamate synthases (GltS) are crucial enzymes in ammonia assimilation in plants and bacteria, where they catalyze the formation of two molecules of L-glutamate from L-glutamine and 2-oxoglutarate. The plant-type ferredoxin-dependent GltS and the functionally homologous alpha subunit of the bacterial NADPH-dependent GltS are complex four-domain monomeric enzymes of 140-165 kDa belonging to the NH(2)-terminal nucleophile family of amidotransferases. The enzymes function through the channeling of ammonia from the N-terminal amidotransferase domain to the FMN-binding domain. Here, we report the X-ray structure of the Synechocystis ferredoxin-dependent GltS with the substrate 2-oxoglutarate and the covalent inhibitor 5-oxo-L-norleucine bound in their physically distinct active sites solved using a new crystal form. The covalent Cys1-5-oxo-L-norleucine adduct mimics the glutamyl-thioester intermediate formed during L-glutamine hydrolysis. Moreover, we determined a high resolution structure of the GltS:2-oxoglutarate complex. These structures represent the enzyme in the active conformation. By comparing these structures with that of GltS alpha subunit and of related enzymes we propose a mechanism for enzyme self-regulation and ammonia channeling between the active sites. X-ray small-angle scattering experiments were performed on solutions containing GltS and its physiological electron donor ferredoxin (Fd). Using the structure of GltS and the newly determined crystal structure of Synechocystis Fd, the scattering experiments clearly showed that GltS forms an equimolar (1:1) complex with Fd. A fundamental consequence of this result is that two Fd molecules bind consecutively to Fd-GltS to yield the reduced FMN cofactor during catalysis.
Topics: Crystallography, X-Ray; Cyanobacteria; Diazooxonorleucine; Enzyme Activation; Enzyme Inhibitors; Ferredoxins; Glutamate Synthase; Ketoglutaric Acids; Models, Molecular; Protein Conformation; Quaternary Ammonium Compounds; Scattering, Radiation
PubMed: 12818206
DOI: 10.1016/s0022-2836(03)00522-9 -
Chemistry & Biology Mar 2003Recent studies have implicated a crucial role for tissue transglutaminase (TG2) in the pathogenesis of Celiac Sprue, a disorder of the small intestine triggered in... (Comparative Study)
Comparative Study
Recent studies have implicated a crucial role for tissue transglutaminase (TG2) in the pathogenesis of Celiac Sprue, a disorder of the small intestine triggered in genetically susceptible individuals by dietary exposure to gluten. Proteolytically stable peptide inhibitors of human TG2 were designed containing acivicin or alternatively 6-diazo-5-oxo-norleucine (DON) as warheads. In biochemical and cell-based assays, the best of these inhibitors, Ac-PQP-(DON)-LPF-NH(2), was considerably more potent and selective than other TG2 inhibitors reported to date. Selective pharmacological inhibition of extracellular TG2 should be useful in exploring the mechanistic implications of TG2-catalyzed modification of dietary gluten, a phenomenon of considerable relevance in Celiac Sprue.
Topics: Animals; Cells, Cultured; Diazooxonorleucine; Drug Design; Enzyme Inhibitors; GTP-Binding Proteins; Glutamic Acid; Glutens; Humans; Isoxazoles; Peptides; Protein Glutamine gamma Glutamyltransferase 2; Structure-Activity Relationship; Transglutaminases
PubMed: 12670536
DOI: 10.1016/s1074-5521(03)00045-0