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Biochemical and Biophysical Research... Jun 2011Glutamate, the principal excitatory neurotransmitter of the brain, participates in a multitude of physiologic and pathologic processes, including learning and memory....
Glutamate, the principal excitatory neurotransmitter of the brain, participates in a multitude of physiologic and pathologic processes, including learning and memory. Glutathione, a tripeptide composed of the amino acids glutamate, cysteine, and glycine, serves important cofactor roles in antioxidant defense and drug detoxification, but glutathione deficits occur in multiple neuropsychiatric disorders. Glutathione synthesis and metabolism are governed by a cycle of enzymes, the γ-glutamyl cycle, which can achieve intracellular glutathione concentrations of 1-10mM. Because of the considerable quantity of brain glutathione and its rapid turnover, we hypothesized that glutathione may serve as a reservoir of neural glutamate. We quantified glutamate in HT22 hippocampal neurons, PC12 cells and primary cortical neurons after treatment with molecular inhibitors targeting three different enzymes of the glutathione metabolic cycle. Inhibiting 5-oxoprolinase and γ-glutamyl transferase, enzymes that liberate glutamate from glutathione, leads to decreases in glutamate. In contrast, inhibition of γ-glutamyl cysteine ligase, which uses glutamate to synthesize glutathione, results in substantial glutamate accumulation. Increased glutamate levels following inhibition of glutathione synthesis temporally precede later effects upon oxidative stress.
Topics: Animals; Buthionine Sulfoximine; Cell Line, Tumor; Enzyme Inhibitors; Glutamic Acid; Glutathione; Hippocampus; Imidazolines; Isoxazoles; Mice; Neurons; Pyroglutamate Hydrolase; Rats; gamma-Glutamyltransferase
PubMed: 21539809
DOI: 10.1016/j.bbrc.2011.04.087 -
Liver Transplantation : Official... Nov 2003Hepatic ischemia-reperfusion (I-R) injury frequently is associated with cholestasis. However, the underlying mechanisms are not fully understood. The aim of the study is...
Hepatic ischemia-reperfusion (I-R) injury frequently is associated with cholestasis. However, the underlying mechanisms are not fully understood. The aim of the study is to assess bile secretory function in vivo in rats subjected to warm lobar hepatic ischemia at different times during reperfusion. A model of lobar 70% warm hepatic ischemia for 30 minutes was used with studies conducted at 1 and 6 hours and 1, 3, and 7 days after reperfusion. Bile secretory function was assessed after selective cannulation of bile ducts of ischemic (ILs) and nonischemic lobes (NILs). Serum activity of hepatic alanine and aspartate aminotransferase was slightly increased in rats subjected to I-R, whereas serum bile salt levels increased early during reperfusion, returning to control values after 7 days. ILs showed mild reversible leukocyte infiltration and no significant necrosis. Bile flow and bile salt excretion were significantly decreased in ILs during the first 24-hour reperfusion period compared with sham-operated rats and NILs. A marked reduction in glutathione (GSH) excretion occurred at 1 and 6 hours and 1 and 3 days, which returned to control values after 7 days. Total GSH and both reduced and oxidized GSH levels in liver homogenate and arterial blood GSH levels were unchanged at all times. Protein mass of multidrug resistance protein 2 and its function, assessed by the hepatic maximum secretory rate of ceftriaxone, did not show significant changes in ILs or NILs compared with sham-operated rats. Liver tissue gamma-glutamyl transpeptidase (GGT) and gamma-glutamylcysteine synthetase activities remained unchanged, whereas biliary GGT and cysteine secretory rates were significantly increased in ILs and NILs. Administration of acivicin, a GGT inhibitor, resulted in decreased secretion of this enzyme into bile and a parallel marked increase in biliary GSH secretion compared with untreated ischemic rats. In conclusion, warm hepatic I-R induces reversible cholestatic changes in ILs. GSH secretory rates from both ILs and NILs were markedly decreased during reperfusion. The reversibility of this effect after GGT inhibition, as well as increased release of active GGT into bile and cysteine biliary secretory rates, suggest increased GSH degradation in bile. These findings might be relevant for the I-R-induced clinical cholestasis, as well as cholangiocyte injury, seen after hepatic ischemia.
Topics: Animals; Bile; Bile Acids and Salts; Cholestasis; Cysteine; Disease Models, Animal; Enzyme Inhibitors; Glutamate-Cysteine Ligase; Glutathione; Isoxazoles; Liver; Male; Mitochondrial Proteins; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Ribosomal Proteins; Saccharomyces cerevisiae Proteins
PubMed: 14586882
DOI: 10.1053/jlts.2003.50232 -
Molecules (Basel, Switzerland) Apr 2020is the aetiologic agent of Chagas disease, which affects people in the Americas and worldwide. The parasite has a complex life cycle that alternates among mammalian...
is the aetiologic agent of Chagas disease, which affects people in the Americas and worldwide. The parasite has a complex life cycle that alternates among mammalian hosts and insect vectors. During its life cycle, passes through different environments and faces nutrient shortages. It has been established that amino acids, such as proline, histidine, alanine, and glutamate, are crucial to survival. Recently, we described that can biosynthesize glutamine from glutamate and/or obtain it from the extracellular environment, and the role of glutamine in energetic metabolism and metacyclogenesis was demonstrated. In this study, we analysed the effect of glutamine analogues on the parasite life cycle. Here, we show that glutamine analogues impair cell proliferation, the developmental cycle during the infection of mammalian host cells and metacyclogenesis. Taken together, these results show that glutamine is an important metabolite for survival and suggest that glutamine analogues can be used as scaffolds for the development of new trypanocidal drugs. These data also reinforce the supposition that glutamine metabolism is an unexplored possible therapeutic target.
Topics: Animals; Azaserine; CHO Cells; Cell Cycle; Cell Proliferation; Cricetulus; Energy Metabolism; Glutamic Acid; Glutamine; Isoxazoles; Life Cycle Stages; Molecular Structure; Trypanocidal Agents; Trypanosoma cruzi
PubMed: 32252252
DOI: 10.3390/molecules25071628 -
ChemMedChem Apr 2017The natural product acivicin inhibits the glutaminase activity of cytidine triphosphate (CTP) synthetase and is a potent lead compound for drug discovery in the area of...
The natural product acivicin inhibits the glutaminase activity of cytidine triphosphate (CTP) synthetase and is a potent lead compound for drug discovery in the area of neglected tropical diseases, specifically trypanosomaisis. A 2.1-Å-resolution crystal structure of the acivicin adduct with the glutaminase domain from Trypanosoma brucei CTP synthetase has been deposited in the RCSB Protein Data Bank (PDB) and provides a template for structure-based approaches to design new inhibitors. However, our assessment of that data identified deficiencies in the model. We now report an improved and corrected inhibitor structure with changes to the chirality at one position, the orientation and covalent structure of the isoxazoline moiety, and the location of a chloride ion in an oxyanion binding site that is exploited during catalysis. The model is now in agreement with established chemical principles and allows an accurate description of molecular recognition of the ligand and the mode of binding in a potentially valuable drug target.
Topics: Bacillus subtilis; Carbon-Nitrogen Ligases; Catalytic Domain; Glutaminase; Helicobacter pylori; Hydrogen Bonding; Isoxazoles; Ligands; Trypanocidal Agents; Trypanosoma brucei brucei; gamma-Glutamyltransferase
PubMed: 28333400
DOI: 10.1002/cmdc.201700118 -
European Journal of Biochemistry Mar 1989Rat livers were perfused at constant pressure via the portal vein with media containing 5 mM glucose, 2 mM lactate and 0.2 mM pyruvate. 1. Leukotrienes C4 and D4...
Mechanism of action of cysteinyl leukotrienes on glucose and lactate balance and on flow in perfused rat liver. Comparison with the effects of sympathetic nerve stimulation and noradrenaline.
Rat livers were perfused at constant pressure via the portal vein with media containing 5 mM glucose, 2 mM lactate and 0.2 mM pyruvate. 1. Leukotrienes C4 and D4 enhanced glucose and lactate output and reduced perfusion flow to the same extent and with essentially identical kinetics. They both caused half-maximal alterations (area under the curve) of carbohydrate metabolism at a concentration of about 1 nM and of flow at about 5 nM. The leukotriene-C4/D4 antagonist CGP 35949 B inhibited the metabolic and hemodynamic effects of 5 nM leukotrienes C4 and D4 with the same efficiency, causing 50% inhibition at about 0.1 microM. 2. Leukotriene C4 elicited the same metabolic and hemodynamic alterations with the same kinetics as leukotriene D4 in livers from rats pretreated with the gamma-glutamyltransferase inhibitor, acivicin. 3. The calcium antagonist, nifedipine, at a concentration of 50 microM did not affect the metabolic and hemodynamic changes caused by 5 nM leukotriene D4. The smooth-muscle relaxant, nitroprussiate, at a concentration of 10 microM reduced flow changes, without significantly affecting the metabolic alterations. 4. Leukotriene D4 not only reduced flow; it also caused an intrahepatic redistribution of flow, restricting some areas from perfusion. Thus, leukotrienes increased glucose and lactate output directly in the accessible parenchyma and, in addition, indirectly by washout from restricted areas during their reopening upon termination of application. 5. The phospholipase A2 inhibitor, bromophenacyl bromide, but not the cyclooxygenase inhibitor, indomethacin, at a concentration of 20 microM reduced the metabolic and hemodynamic effects of 5 mM leukotriene D4. 6. Stimulation of the sympathetic hepatic nerves with 2-ms rectangular pulses at 20 Hz and infusion of 1 microM noradrenaline increased glucose and lactate output and decreased flow, similar to 10 nM leukotrienes C4 and D4. The kinetics of the metabolic and hemodynamic changes caused by the leukotrienes differed, however, from those due to nerve stimulation and noradrenaline. 7. The leukotriene-C4/D4 antagonist, CGP 35949 B, even at very high concentrations (20 microM) inhibited the metabolic and hemodynamic alterations caused by nerve stimulation or noradrenaline infusion only slightly and unspecifically.(ABSTRACT TRUNCATED AT 400 WORDS)
Topics: Animals; Electric Stimulation; Glucose; Kinetics; Lactates; Liver; Liver Circulation; Male; Norepinephrine; Perfusion; Rats; Rats, Inbred Strains; SRS-A; Structure-Activity Relationship; Tetrazoles; gamma-Glutamyltransferase
PubMed: 2564341
DOI: 10.1111/j.1432-1033.1989.tb14644.x -
BMC Microbiology Jun 2021The global rise in the incidence of non-tuberculosis mycobacterial infections is of increasing concern due their high levels of intrinsic antibiotic resistance. Although...
BACKGROUND
The global rise in the incidence of non-tuberculosis mycobacterial infections is of increasing concern due their high levels of intrinsic antibiotic resistance. Although integrated viral genomes, called prophage, are linked to increased antibiotic resistance in some bacterial species, we know little of their role in mycobacterial drug resistance.
RESULTS
We present here for the first time, evidence of increased antibiotic resistance and expression of intrinsic antibiotic resistance genes in a strain of Mycobacterium chelonae carrying prophage. Strains carrying the prophage McProf demonstrated increased resistance to amikacin. Resistance in these strains was further enhanced by exposure to sub-inhibitory concentrations of the antibiotic, acivicin, or by the presence of a second prophage, BPs. Increased expression of the virulence gene, whiB7, was observed in strains carrying both prophages, BPs and McProf, relative to strains carrying a single prophage or no prophages.
CONCLUSIONS
This study provides evidence that prophage alter expression of important mycobacterial intrinsic antibiotic resistance genes and additionally offers insight into the role prophage may play in mycobacterial adaptation to stress.
Topics: Anti-Bacterial Agents; Bacterial Proteins; Drug Resistance, Bacterial; Mycobacterium chelonae; Prophages; Virulence Factors
PubMed: 34107872
DOI: 10.1186/s12866-021-02224-z -
Asian Pacific Journal of Cancer... 2007Treatment for metastatic melanoma has mostly been unsatisfactory despite advances in ongoing medical research. Here we investigated the role of acivicin, a glutamine...
Treatment for metastatic melanoma has mostly been unsatisfactory despite advances in ongoing medical research. Here we investigated the role of acivicin, a glutamine analogue, singly and in combination with either E. coli glutaminase or cisplatin, on the growth, angiogenic activity and invasiveness of B16F10 cells in vitro and after allografting in C57BL/6 mice. B16F10 melanoma colonization in the lungs of mice was measured by monitoring colony counts. Host toxicity was assessed with reference to tumor bearing host's weight and survivability. Acivicin promoted melanoma dormancy and reduced melanoma associated angiogenic factors like VEGF level and vessel diameter. Acivicin in combination with glutaminase significantly suppressed tumor growth by 66.7% and increased life-span by 43.5% without host toxicity. Tumor VEGF content was significantly lowered by combination therapy as assessed by ELISA. Accelerated cytotoxicity, reduced invasion and enhanced apoptosis of melanoma cells were exhibited in vitro by combined than by single agent treatment. Moreover, invasion of melanoma cells through matrigel chambers was reduced in presence of acivicin and glutaminase combination. These findings support future studies of acivicin in combination with other anticancer agents for prevention of melanoma metastasis.
Topics: Animals; Antineoplastic Agents; Cell Division; Cell Survival; Cisplatin; Disease Models, Animal; Drug Synergism; Glutaminase; Male; Melanoma; Mice; Mice, Inbred C57BL; Neoplasm Invasiveness; Vascular Endothelial Growth Factor A
PubMed: 17696751
DOI: No ID Found -
British Journal of Cancer Jun 1987
Topics: Antimetabolites; Azo Compounds; Cell Cycle; Cell Line; Colonic Neoplasms; Diazooxonorleucine; Drug Evaluation, Preclinical; Glutaminase; Glutamine; Humans; Isoxazoles; Lung Neoplasms; Neoplasms, Experimental; Oxazoles
PubMed: 3620309
DOI: 10.1038/bjc.1987.133 -
The Journal of Biological Chemistry Aug 1994Inactivation of gamma-glutamyl transpeptidase by acivicin (L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazole acetic acid) is rapid, thought to be irreversible,...
Inactivation of gamma-glutamyl transpeptidase by acivicin (L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazole acetic acid) is rapid, thought to be irreversible, and associated with binding of close to 1 mol of inhibitor/mol of enzyme. Previous studies with [3-14C]acivicin indicated binding (prevented by substrate) to a specific hydroxyl group (threonine 523) of the rat kidney enzyme. In the present work, we found that such inactivation can be reversed by treating the inhibited enzyme with hydroxylamine. Reactivation (more than 85% complete) is associated with release from the inactivated enzyme of compounds that exhibit the properties of threo-beta-hydroxy-L-gamma-glutamyl hydroxamate and 3-hydroxypyrrolidone-2-carboxylate. We found that the enzyme acts very slowly on acivicin, at a rate that is about 10(-9) that of its normal catalytic rate with glutathione, to form threo-beta-hydroxy-L-glutamate and hydroxylamine. The findings indicate that inhibition by acivicin involves its transformation on the enzyme to an inhibitory species which is attached, apparently by ester linkage, to a specific hydroxyl group of the enzyme. The very slow rate of release of this intermediate appears to account for the observed inhibition.
Topics: Animals; Catalysis; Enzyme Reactivators; Glutamates; Glutamic Acid; Hydroxylamine; Hydroxylamines; Isoxazoles; Rats; gamma-Glutamyltransferase
PubMed: 7914892
DOI: No ID Found -
The Journal of Biological Chemistry Jun 1986High concentrations of glutathione (GSH) and two of its constituent amino acids, glutamate and glycine, are normally found in rat bile. To examine the role of...
High concentrations of glutathione (GSH) and two of its constituent amino acids, glutamate and glycine, are normally found in rat bile. To examine the role of intrabiliary GSH hydrolysis as a source of these amino acids, as well as of cystine in bile, the biliary excretion of GSH and free amino acids was measured in normal male Sprague-Dawley rats; in animals given either phenol 3,6-dibromphthalein disulfonate or diethyl maleate, inhibitors of GSH secretion into bile; and after a retrograde intrabiliary infusion of (alpha S, 5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125), an irreversible inhibitor of gamma-glutamyl transferase activity. Total concentration of amino acids in normal rat bile ranged from 4 to 7 mM and was more than double the concentration in plasma (2-3 mM). Although most amino acids were detected in bile, glutamate and glycine were the most prevalent (1.2 and 1.0 mM, respectively), followed by the branched chain amino acids valine and leucine. The administration of phenol 3,6-dibromphthalein disulfonate (180 mumol/kg, intravenous), or of diethyl maleate (1 mmol/kg, intraperitoneal), resulted in a marked decrease in the biliary excretion of GSH, as well as a decrease in the excretion of glutamate, cystine, and glycine; however, the effects of these agents were not specific for the amino acid constituents of GSH. Following retrograde intrabiliary infusion of AT-125 (10 mumol/kg), there was an immediate and sustained doubling in the rate of biliary excretion of both GSH and glutathione disulfide and a marked decrease in the rate of excretion of glutamate. Varying the dose of AT-125 (0-20 mumol/kg) resulted in an inverse linear relation between hepatic gamma-glutamyl transferase activity and the biliary excretion of intact GSH. These findings suggest that most, if not all, of the free glutamate in excreted bile is formed from the intrabiliary hydrolysis of GSH. Prior to hydrolysis within the biliary tree, substantial concentrations of GSH must be transported from liver cells into bile; minimal canalicular concentrations of this tripeptide are estimated at 5 mM.
Topics: Amino Acids; Animals; Antimetabolites; Bile; Gallbladder; Glutamates; Glutamic Acid; Glutathione; Glutathione Reductase; Hydrolysis; Isoxazoles; Kinetics; Liver; Male; Organ Specificity; Rats; Rats, Inbred Strains; gamma-Glutamyltransferase
PubMed: 2872220
DOI: No ID Found