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Blood Oct 1989Previously we showed that starvation of HL-60 promyelocytic leukemia cells for a single essential amino acid induced irreversible differentiation into more mature...
Previously we showed that starvation of HL-60 promyelocytic leukemia cells for a single essential amino acid induced irreversible differentiation into more mature monocyte-like cells. Although not an essential amino acid, glutamine is important in the growth of normal and neoplastic cells. The glutamine analogue, alpha S,5S-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin) inhibits several glutamine-utilizing enzymes and therefore depletes cells of certain metabolic end products. The current study was designed to examine in vitro the effects of acivicin on growth and differentiation of several established human myeloid leukemia cell lines, including the HL-60 cell line, and of freshly isolated cells from patients with acute nonlymphocytic leukemia (ANLL). Four-day culture of HL-60 cells with acivicin at concentrations of 0.1 to 10.0 micrograms/mL (0.56 to 56 nmol/L) decreased cell growth by 33% to 88% as compared with untreated control cells. Viability of cells was greater than 92% for untreated cells and 93% to 41% for acivicin-treated cells. Cells treated with acivicin differentiated along a monocytic pathway as shown by increased H2O2 production and alpha-naphthyl butyrate esterase (NSE) content. Differentiation was time and dose dependent, and was irreversible. Changes in H2O2 production and NSE content were partially abrogated by co-culture with 10 mmol/L exogenous cytidine and guanosine but not by co-culture with other nucleosides or glutamine. At these concentrations of acivicin, differentiation was associated with expression of the N-formyl-methyl-leucyl-phenylalanine-receptor (FMLP-R) on 8% to 29% of cells as compared with 8% for control cells. Acivicin potentiated the differentiating effects of interferon-gamma, tumor necrosis factor, dihydroxyvitamin D3, dimethylsulfoxide, and retinoic acid. Culture of cells from the U937 (monoblastic), K562 (erythroleukemia), and KG-1 (myeloblastic) cell lines resulted in decreased growth and viability, but not consistently in differentiation. Acivicin decreased survival of freshly isolated ANLL cells and increased their H2O2 production and NSE content. These results suggest that the glutamine analogue acivicin may be useful as a differentiating agent with antileukemia activity in patients with ANLL.
Topics: Antibiotics, Antineoplastic; Cell Differentiation; Cell Division; Cell Line; Cells, Cultured; Dose-Response Relationship, Drug; Humans; Isoxazoles; Kinetics; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Leukocytes; Oxazoles; Reference Values; Tumor Cells, Cultured
PubMed: 2790198
DOI: No ID Found -
PloS One 2012S-nitrosoglutathione (GSNO) involved in storage and transport of nitric oxide ((•)NO) plays an important role in vascular homeostasis. Breakdown of GSNO can be...
S-nitrosoglutathione (GSNO) involved in storage and transport of nitric oxide ((•)NO) plays an important role in vascular homeostasis. Breakdown of GSNO can be catalyzed by γ-glutamyltransferase (GGT). We investigated whether vascular GGT influences the vasorelaxant effect of GSNO in isolated rat aorta. Histochemical localization of GGT and measurement of its activity were performed by using chromogenic substrates in sections and in aorta homogenates, respectively. The role of GGT in GSNO metabolism was evaluated by measuring GSNO consumption rate (absorbance decay at 334 nm), (•)NO release was visualized and quantified with the fluorescent probe 4,5-diaminofluorescein diacetate. The vasorelaxant effect of GSNO was assayed using isolated rat aortic rings (in the presence or absence of endothelium). The role of GGT was assessed by stimulating enzyme activity with cosubstrate glycylglycine, as well as using two independent inhibitors, competitive serine borate complex and non-competitive acivicin. Specific GGT activity was histochemically localized in the endothelium. Consumption of GSNO and release of free (•)NO decreased and increased in presence of serine borate complex and glycylglycine, respectively. In vasorelaxation experiments with endothelium-intact aorta, the half maximal effective concentration of GSNO (EC50 = 3.2 ± 0.5.10(-7) M) increased in the presence of the two distinct GGT inhibitors, serine borate complex (1.6 ± 0.2.10(-6) M) and acivicin (8.3 ± 0.6.10(-7) M), while it decreased with glycylglycine (4.7 ± 0.9.10(-8) M). In endothelium-denuded aorta, EC(50) for GSNO alone increased to 2.3 ± 0.3.10(-6) M, with no change in the presence of serine borate complex. These data demonstrate the important role of endothelial GGT activity in mediating the vasorelaxant effect of GSNO in rat aorta under physiological conditions. Because therapeutic treatments based on GSNO are presently under development, this endothelium-dependent mechanism involved in the vascular effects of GSNO should be taken into account in a pharmacological perspective.
Topics: Animals; Endothelium, Vascular; Glutathione; In Vitro Techniques; Male; Nitric Oxide; Protein Transport; Rats; Rats, Wistar; S-Nitrosoglutathione; Vasodilator Agents; gamma-Glutamyltransferase
PubMed: 22984412
DOI: 10.1371/journal.pone.0043190 -
Acta Crystallographica. Section D,... Feb 2014γ-Glutamyltranspeptidase (GGT) is an enzyme that plays a central role in glutathione metabolism, and acivicin is a classical inhibitor of GGT. Here, the structure of...
Structure of Bacillus subtilis γ-glutamyltranspeptidase in complex with acivicin: diversity of the binding mode of a classical and electrophilic active-site-directed glutamate analogue.
γ-Glutamyltranspeptidase (GGT) is an enzyme that plays a central role in glutathione metabolism, and acivicin is a classical inhibitor of GGT. Here, the structure of acivicin bound to Bacillus subtilis GGT determined by X-ray crystallography to 1.8 Å resolution is presented, in which it binds to the active site in a similar manner to that in Helicobacter pylori GGT, but in a different binding mode to that in Escherichia coli GGT. In B. subtilis GGT, acivicin is bound covalently through its C3 atom with sp2 hybridization to Thr403 Oγ, the catalytic nucleophile of the enzyme. The results show that acivicin-binding sites are common, but the binding manners and orientations of its five-membered dihydroisoxazole ring are diverse in the binding pockets of GGTs.
Topics: Bacillus subtilis; Bacterial Proteins; Catalytic Domain; Crystallography, X-Ray; Enzyme Inhibitors; Escherichia coli; Glutamic Acid; Helicobacter pylori; Isoxazoles; Ligands; Models, Molecular; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Recombinant Proteins; Substrate Specificity; gamma-Glutamyltransferase
PubMed: 24531494
DOI: 10.1107/S1399004713031222 -
World Journal of Gastroenterology Mar 2011To assess whether glutamate plays a similar role to glutamine in preserving gut wall integrity.
AIM
To assess whether glutamate plays a similar role to glutamine in preserving gut wall integrity.
METHODS
The effects of glutamine and glutamate on induced hyperpermeability in intestinal cell lines were studied. Paracellular hyperpermeability was induced in Caco2.BBE and HT-29CL.19A cell lines by adding phorbol-12,13-dibutyrate (PDB) apically, after which the effects of glutamine and glutamate on horseradish peroxidase (HRP) diffusion were studied. An inhibitor of glutamate transport (L-trans-pyrrolidine-2,4-dicarboxylic acid: trans-PDC) and an irreversible blocker (acivicin) of the extracellular glutamine to glutamate converting enzyme, γ-glutamyltransferase, were used.
RESULTS
Apical to basolateral HRP flux increased significantly compared to controls not exposed to PDB (n = 30, P < 0.001). Glutamine application reduced hyperpermeability by 19% and 39% in the respective cell lines. Glutamate application reduced hyperpermeability by 30% and 20%, respectively. Incubation of HT29CL.19A cells with acivicin and subsequent PDB and glutamine addition increased permeability levels. Incubation of Caco2.BBE cells with trans-PDC followed by PDB and glutamate addition also resulted in high permeability levels.
CONCLUSION
Apical glutamate -similar to glutamine- can decrease induced paracellular hyperpermeability. Extracellular conversion of glutamine to glutamate and subsequent uptake of glutamate could be a pivotal step in the mechanism underlying the protective effect of glutamine.
Topics: Analysis of Variance; Caco-2 Cells; Dicarboxylic Acids; Enzyme Inhibitors; Epithelial Cells; Glutamate Plasma Membrane Transport Proteins; Glutamic Acid; Glutamine; Humans; Intestinal Absorption; Intestinal Mucosa; Isoxazoles; Permeability; Phorbol 12,13-Dibutyrate; Pyrrolidines; gamma-Glutamyltransferase
PubMed: 21472123
DOI: 10.3748/wjg.v17.i12.1569 -
Pharmaceuticals (Basel, Switzerland) Aug 2021Glaucoma is a leading cause of permanent vision loss and current drugs do not halt disease progression. Thus, new therapies targeting different drug targets with novel...
Glaucoma is a leading cause of permanent vision loss and current drugs do not halt disease progression. Thus, new therapies targeting different drug targets with novel mechanisms of action are urgently needed. Previously, we identified CACNA2D1 as a novel modulator of intraocular pressure (IOP) and demonstrated that a topically applied CACNA2D1 antagonist-pregabalin (PRG)-lowered IOP in a dose-dependent manner. To further validate this novel IOP modulator as a drug target for IOP-lowering pharmaceutics, a homology model of CACNA2D1 was built and docked against the NCI library, which is one of the world's largest and most diverse compound libraries of natural products. Acivicin and zoledronic acid were identified using this method and together with PRG were tested for their plausible IOP-lowering effect on Dutch belted rabbits. Although they have inferior potency to PRG, both of the other compounds lower IOP, which in turn validates CACNA2D1 as a valuable drug target in treating glaucoma.
PubMed: 34577587
DOI: 10.3390/ph14090887 -
The Journal of Biological Chemistry Jul 1990The glutamine antagonists, acivicin (NSC 163501), azaserine (NSC 742), and 6-diazo-5-oxo-L-norleucine (DON) (NSC 7365), are potent inhibitors of many glutamine-dependent...
The glutamine antagonists, acivicin (NSC 163501), azaserine (NSC 742), and 6-diazo-5-oxo-L-norleucine (DON) (NSC 7365), are potent inhibitors of many glutamine-dependent amidotransferases in vitro. Experiments performed with mouse L1210 leukemia growing in culture show that each antagonist has different sites of inhibition in nucleotide biosynthesis. Acivicin is a potent inhibitor of CTP and GMP synthetases and partially inhibits N-formylglycineamidine ribotide (FGAM) synthetase of purine biosynthesis. DON inhibits FGAM synthetase, CTP synthetase, and glucosamine-6-phosphate isomerase. Azaserine inhibits FGAM synthetase and glucosamine-6-phosphate isomerase. Large accumulations of FGAR and its di- and triphosphate derivatives were observed for all three antagonists which could interfere with the biosynthesis of nucleic acids, providing another mechanism of cytotoxicity. Acivicin, azaserine, and DON are not potent inhibitors of carbamyl phosphate synthetase II (glutamine-hydrolyzing) and amidophosphoribosyltransferase in leukemia cells growing in culture although there are reports of such inhibitions in vitro. Blockade of de novo purine biosynthesis by these three antagonists results in a "complementary stimulation" of de novo pyrimidine biosynthesis.
Topics: Animals; Azaserine; Azo Compounds; Carbon-Nitrogen Ligases; Cell Line; Chromatography, High Pressure Liquid; Diazooxonorleucine; Glutamine; Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); Isoxazoles; Leukemia L1210; Ligases; Mice; Nucleotides; Oxazoles; Spectrophotometry, Ultraviolet
PubMed: 2358467
DOI: No ID Found -
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 -
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 -
The FEBS Journal Oct 2011GMP synthetase is a glutamine amidotransferase that incorporates ammonia derived from glutamine into the nucleotide xanthosine 5'-monophosphate (XMP) to form guanosine...
GMP synthetase is a glutamine amidotransferase that incorporates ammonia derived from glutamine into the nucleotide xanthosine 5'-monophosphate (XMP) to form guanosine 5'-monophosphate (GMP). Functional coordination of domains in glutamine amidotransferases leads to upregulation of glutamine hydrolysis in the presence of acceptor substrates and is a common feature in this class of enzymes. We have shown earlier that binding of substrates to the acceptor domain of Plasmodium falciparum GMP synthetase (PfGMPS) leads to enhancement in both glutaminase activity and rate of glutaminase inactivation, by the irreversible inhibitors acivicin and diazo-oxonorleucine [Bhat JY et al. (2008) Biochem J409, 263-273], a process that must be driven by conformational alterations. In this paper, through the combined use of biochemical assays, optical spectroscopy and mass spectrometry, we demonstrate that PfGMPS undergoes conformational transitions upon binding of substrates to the acceptor domain. Limited proteolysis and hydrogen-deuterium exchange in conjunction with mass spectrometry unveil region-specific conformational changes in the ATP + XMP bound state of PfGMPS. Decreased accessibility of R294 and K428 residues to trypsin in the ATP pyrophosphatase domain and reduced deuterium incorporation in the 143-155 region, pertaining to the glutaminase domain, suggest that in PfGMPS ligand-induced conformational changes are not only local but also transmitted over a long range across the domains. Overall, these results provide a detailed understanding of the substrate-induced changes in PfGMPS that could be essential for the overall catalytic process.
Topics: Carbon-Nitrogen Ligases; Circular Dichroism; Deuterium Exchange Measurement; Guanosine Monophosphate; Models, Molecular; Molecular Structure; Plasmodium falciparum; Protein Binding; Protein Conformation; Protozoan Proteins; Spectrometry, Fluorescence; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 21827625
DOI: 10.1111/j.1742-4658.2011.08296.x -
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