Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.5.1.47 (cysteine synthase)
625 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have tested the tumoricidal potency of enzyme immunotoxins constructed of antibodies conjugated to glucose oxidase and to lactoperoxidase. Murine plasmacytoma cells were targeted in vitro with the use of affinity-purified rabbit anti-plasmacytoma membrane antibodies (conjugated to glucose oxidase or lactoperoxidase) or rabbit serum raised against plasmacytoma microsome membranes followed by goat anti-rabbit immunoglobulin conjugates (to glucose oxidase or lactoperoxidase). Cytotoxicity was generated subsequently by incubation of the washed cells in a medium supplemented with glucose and sodium iodide, which were the substrates of these enzymes. This resulted in the presumed metabolic release of highly toxic reduced oxygen species and iodinated derivatives. Targeting of tumor cells with both conjugates, as opposed to one of them alone, produced a synergistic killing effect. The gain of specific versus unspecific cytotoxicity was upwards of 10,000-fold. The killing rates were elevated (t10 values less than 30 min) and linear over time. The resultant reduction in tumor cell viability was in the order of 5 to 6 logs after only 20 to 90 min of incubation in the glucose/NaI medium. Cytotoxicity was enhanced by the gamma-glutamyl cysteine synthetase inhibitor buthionine-S,R-sulfoximine and by the glutathione reductase inhibitor 1,3-bis(2-chloroethyl)-1-nitrosourea, while catalase was inhibitory. The results suggest that these enzyme immunotoxins may be suitable for the ex vivo purging of autologous bone marrow grafts.
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PMID:Immunotoxins containing glucose oxidase and lactoperoxidase with tumoricidal properties: in vitro killing effectiveness in a mouse plasmacytoma cell model. 279 Jul 77

The ability of various concentrations of the differentiation-inducing agent sodium butyrate (NAB, 0-2 mM) to produce radiosensitization in human colon tumor cells when combined with varying concentrations of the irreversible inhibitor of gamma-glutamyl cysteine synthetase, buthionine sulfoximine (BSO, 0-0.75 mM) was studied. We have previously shown that high concentrations of each agent in combination (2 mM NAB + 0.5 mM BSO) produced a supra-additive effect in terms of radiosensitization as indicated by a decrease in the quasi-threshold value (Dq) of the single dose survival curve; we wished to define responses at other concentrations. Cells were adapted in vitro to growth in medium containing NAB for 3 passages prior to x-irradiation and BSO was given acutely 24 hrs before the x-irradiations. The most effective combination was 0.3 mM NAB + 0.75 mM BSO. These data suggest that adaptation of tumor cells to chronic low levels of a differentiation-inducing agent such as NAB followed by administration of BSO just prior to irradiation might be an effective combination in producing increased response of solid tumors.
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PMID:Potentiation of X ray sensitivity by combinations of sodium butyrate and buthionine sulfoximine. 318 35

The role of glutathione (GSH) in the preservation of renal function and the pathogenesis of renal injury has been investigated using the isolated perfused rat kidney as a model. In kidneys perfused for 80 min with 5 mM glucose as the only exogenous substrate, tissue GSH becomes depleted, renal function deteriorates, and a degenerative change appears, restricted to the medullary thick ascending limb. These abnormalities can be ameliorated by providing amino acid supplements or by adding GSH itself to the perfusion. To distinguish between the effects of amino acid supplementation and GSH depletion per se, selective depletion of GSH was accomplished in several different ways. Synthesis of GSH was inhibited by the addition of dl-buthionine-SR-sulfoximine, a specific inhibitor of gamma-glutamyl cysteine synthetase. GSH depletion was also produced by 2-cyclohexene-1-one and diethylmaleate, both known to diminish the concentration of GSH selectively without affecting protein thiols. Perfused kidneys selectively depleted of GSH showed significant impairment of concentrating ability, and less marked decreases in tubular reabsorption of sodium. The degenerative changes in the medullary thick ascending limb, on the other hand, were unaltered.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Selective glutathione depletion on function and structure of the isolated perfused rat kidney. 663 20

Cell-free extracts of Salmonella typhimurium synthesize a mutagenic azide metabolite from sodium azide and O-acetylserine. S. typhimurium mutant DW379 (O-acetylserine sulfhydrylase-deficient) extracts were neither able to carry out this reaction not produce the mutagenic azide metabolite in vivo. The in vitro reaction was inhibited by sulfide but not by L-cysteine. The catalytic activity responsible for the mutagenic metabolite synthesis was stable to brief heating up to 55 degrees C and had a pH optimum between 7-7.4. These results suggest that the enzyme O-acetylserine sulfhydrylase catalyzes the reaction of azide with O-acetylserine to form a mutagenic azide metabolite.
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PMID:In vitro synthesis of a mutagenic azide metabolite by cell-free bacterial extracts. 680 99

Nitric oxide (NO) has been demonstrated to play a protective role in cell injury. In this study, we have explored the effect of NO and two NO donors (sodium nitroprusside [SNP] and isosorbide dinitrate [ISDN]) on cellular glutathione (GSH) levels in a rat lung fibroblast cell line (RFL6 cells). SNP and ISDN significantly increased cellular GSH in RFL6 cells (5 x 10(-4) M SNP: 21.9 +/- 3.6 nmol/10(6) cells and 5 x 10(-3) M ISDN: 27.6 +/- 1.7 nmol/10(6) cells versus control: 13.2 +/- 0.4 nmol/10(6) cells; P < 0.05). The stimulatory effect of SNP and ISDN on GSH was first seen at 6 h and peaked at 12 to 24 h. A similar increase in GSH was observed in RFL6 cells exposed to 400 ppm NO for 7.5 h (NO: 20.5 +/- 3.4 nmol/10(6) cells versus control: 11.9 +/- 2.4; P < 0.05). SNP and ISDN also increased cellular GSH in bovine pulmonary artery smooth muscle cells (BPSMC) and bovine pulmonary artery endothelial cells (BPAEC). Buthionine sulfoximine (BSO) (0.01 mM), an inhibitor of the GSH synthetic enzyme gamma-glutamyl cysteine synthetase, blocked the increase in GSH in RFL6 cells seen with both SNP and ISDN. In BPAEC, exposure to NO donors for 24 h stimulated glutamate uptake (SNP: 441 +/- 19 pmol/10 min/10(6) cells and ISDN: 677 +/- 48 pmol/10 min/10(6) min/10(6) cells versus control: 222 +/- 9 pmol/10 min/10(6); P < 0.05). This effect paralleled the increase in GSH. In RFL6 cells, only SNP increased glutamate uptake after 24 h of incubation. In summary, NO and NO donors increase cellular GSH in RFL6 cells, BPAEC, and BPSMC. The mechanism of this effect is unclear but may involve upregulation of the normal GSH synthetic pathways. This observation may explain in part the protective effect of NO seen in some cell culture systems and may contribute to a protective effect against oxidant injury in vivo.
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PMID:Nitric oxide increases cellular glutathione levels in rat lung fibroblasts. 754 74

Time-dependent alterations in glutathione (GSH) concentration and the activities of several key enzymes of GSH metabolism were studied in a rat model of experimental Fanconi syndrome induced by i.p. injection of sodium maleate (400 mg/kg BW). The changes in the parameters tested were monitored 0, 2, 4, and 12 h after sodium maleate administration. A significant decrease in renal GSH level was observed 2 and 4 h after sodium maleate treatment (27% and 38% of control values, respectively). The renal GSH depletion did not appear to be due to the decreased production rate or to an increased degradation of the tripeptide. This suggestion is based on the findings that the activities of the GSH synthesis (gamma-glutamyl cysteine synthetase and glutathione reductase) and those of the catabolic pathways (gamma-glutamyl transpeptidase) were unaltered at the same time points. The unchanged activity of gamma-glutamyl transpeptidase also suggests preserved luminal membrane integrity in experimental Fanconi syndrome. The decreased activity of glutathione peroxidase, which utilizes GSH as a cosubstrate in the course of inactivation of free radicals, in the first hours after treatment could facilitate lipid peroxidation reactions in this model of acute renal failure. The observed changes in all parameters tested were transient, with recovery to baseline levels in a period of 12 h after sodium maleate administration. At the same time a pronounced functional impairment still existed. The beneficial effect of fast recovery of renal GSH level on the functional and morphological restitution in experimental Fanconi syndrome is suggested.
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PMID:Time course of renal glutathione levels in experimental Fanconi syndrome: an enzyme-based approach. 915 54

The reactions of the pyridoxal 5'-phosphate-dependent enzyme O-acetylserine sulfhydrylase with the substrate O-acetyl-L-serine and substrate analogs have been investigated in the crystalline state by single-crystal polarized absorption microspectrophotometry. This approach has allowed us to examine the catalytic competence of the enzyme in different crystalline states, one of which was used to determine the three-dimensional structure; experimental conditions were defined for the accumulation of catalytic intermediates in the crystal suitable for crystallographic analyses.O-Acetyl-L-serine reacts with the enzyme in one of the crystal forms leading via a beta-elimination reaction to the accumulation of the alpha-aminoacrylate Schiff base, absorbing maximally at 320 and 470 nm, as in solution. The dissociation constant for the alpha-aminoacrylate Schiff base is in the millimolar range, 500-fold higher than in solution, suggesting that crystal lattice interactions may oppose functionally relevant conformational changes. The dissociation constant exhibits a bell-shaped dependence on pH centered at pH 7. At this pH the alpha-aminoacrylate species slowly decays with time (30% decrease in 24 hours). The alpha-aminoacrylate intermediate readily reacts with sodium azide, an analog of sulfide, the natural nucleophilic agent, to give a new amino acid and the native enzyme, indicating that the crystalline enzyme catalyzes the overall beta-replacement reaction as in solution. In other crystal forms, including that used for the X-ray investigation, O-acetyl-L-serine either has an even higher dissociation constant or causes crystal damage upon binding. When the crystalline enzyme reacts with either L-cysteine or L-serine, the external aldimine intermediate is formed. The dissociation constants for both substrate analogs are closer to those observed in solution and are modulated by pH as in solution. Findings demonstrate that O-acetylserine sulfhydrylase is catalytically competent in the crystal although some regions of the molecule, likely involved in an open-closed transition induced by O-acetyl-L-serine binding, may have a limited flexibility. The accumulation in the crystal of both the external aldimine and the alpha-aminoacrylate intermediate makes feasible their structural determination and, therefore, the elucidation of the catalytic pathway at the molecular level.
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PMID:Catalytic competence of O-acetylserine sulfhydrylase in the crystal probed by polarized absorption microspectrophotometry. 976 79

Clinical resistance to pentavalent antimonials, in the form of pentostam (sodium stibogluconate) or glucantime (N-methylglucamine antimoniate), has long been recognized as a problem in Leishmaniasis. However, the mechanisms of resistance are unclear. We selected in vitro a Leishmania tropica line resistant to 1.2 mg/mL of Sb(V) of glucantime (GLU-R10). The cell line has a stable phenotype for at least 6 months and a resistance index of 1400-fold. The resistant line has no cross-resistance to pentostam or to SbCl3 and SbCl5. The resistance to glucantime was reverted by buthionine sulfoximine (BSO) and chlorambucil (CLB); however, thiol analyses by HPLC of wild-type and GLU-R10 cell lines, in the presence or absence of the drug, showed no differences between these two cell lines. The resistant line had a DNA amplification shown as a circular extrachromosomal element (G-circle) of approximately 22 kb. However, the specific probes for gamma-glutamyl cysteine synthetase, ornithine decarboxylase and trypanothione reductase did not recognize the G-circle amplified in the GLU-R10. The G-circle did not arise from the H region and was not related with P-glycoprotein Pgp-MDR- or Pgp-MRP-like genes. Northern blot analysis of the G-circle showed that a single transcript of approximately 6 kb was overexpressed in the resistant line. Molecular characterization of the G-circle would lead to the determination of the gene(s) involved in resistance to glucantime in Leishmania.
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PMID:Involvement of thiol metabolism in resistance to glucantime in Leishmania tropica. 980 32

The relationship between l-cystine transport and intracellular glutathione (GSH) levels was investigated in cultured pancreatic AR42J acinar and betaTC3 islet cells exposed to diethylmaleate, an electrophilic agent known to activate cellular antioxidant responses. Cystine transport was mediated predominantly by the Na+-independent anionic amino acid transport system x-c, with influx inhibited potently by glutamate and homocysteate but unaffected by cationic or neutral amino acids. Saturable cystine transport was 10-fold higher in AR42J (531 pmol (mg protein)-1 min-1) than in betaTC3 (49 pmol (mg protein)-1 min-1) cells, and GSH levels were higher in AR42J cells. Treatment with 2-mercaptoethanol increased GSH levels in betaTC3 cells from 7.5 to 36 nmol (mg protein)-1, whilst the GSH content in AR42J cells (64 nmol (mg protein)-1) was not altered significantly. Incubation of AR42J or betaTC3 cells with homocysteate (2.5 mM, 0-48 h), a competitive inhibitor of cystine transport via system x-c, reduced intracellular GSH levels and resulted in a time-dependent (6-24 h) induction of system x-c transport activity. Treatment of AR42J cells with diethylmaleate (100 microM, 0-48 h) resulted in a time- (5-10 h) and protein synthesis-dependent induction of cystine transport, with intracellular GSH levels initially decreasing and then increasing 2-fold above control levels after 24 h. Diethylmaleate also depressed GSH levels in betaTC3 cells, but cystine transport was not elevated significantly. In both AR42J and betaTC3 cells, inhibition of gamma-glutamyl cysteine synthetase by buthionine sulphoximine (100 microM, 24 h) reduced GSH levels but had no effect on cystine transport. The present findings establish that induction of system x-c leads to changes in GSH levels in pancreatic AR42J acinar and betaTC3 islet cells, with changes in the intracellular redox state stimulating transporter expression. Induction of activity of system x-c, together with adaptive increases in GSH synthesis in response to oxidative stress, may contribute to cellular antioxidant defences in pancreatic disease.
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PMID:Induction of cystine transport via system x-c and maintenance of intracellular glutathione levels in pancreatic acinar and islet cell lines. 980 3

Aerial parts (shoots) of maize seedlings fed hydroponically with 300 muM sodium arsenate [As(V)] or 250 muM sodium arsenite [As(III)] for 24 h were analyzed for differentially expressed proteins by 2-DE and digital image analysis. About 15% of total detected proteins (74 out of 500) were up- or, mainly, down-regulated by arsenic, among which 14 were selected as being those most affected by the metalloid. These proteins were analyzed by MALDI-TOF MS and 7 of them were identified: translation initiation factor eIF-5A, ATP synthase, cysteine synthase, malate dehydrogenase, protein kinase C inhibitor, Tn10 transposase-like protein, and guanine nucleotide binding protein. Each of these proteins was completely repressed by As(V) and/or As(III), except protein kinase C inhibitor, which was newly detected after exposure to As(V).
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PMID:Maize response to acute arsenic toxicity as revealed by proteome analysis of plant shoots. 1653 46


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