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:1.2.1.13 (glyceraldehyde-3-phosphate dehydrogenase)
6,511 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glutathione (GSH) and glutathione-related enzyme systems in astrocytes play an important role in cellular defense against oxidative stress in the nervous system. The present study was designed to characterize the cellular responses of cultured astrocytes to chemically-induced perturbations of mitochondrial and cytosolic GSH homeostasis. Treatment of astrocytes in culture with ethacrynic acid (EA), a mitochondrion-penetrating thiol reagent, induced rapid and extensive depletion of both cytosolic and mitochondrial pools of GSH. Concomitant with the effects of EA on cellular GSH were significant and concentration-dependent increases in intracellular generation of reactive oxygen species (ROS) as indicated by the oxidation of preloaded 2',7'-dichlorofluorescein diacetate. Significant elevation of intracellular ROS occurred by 15 min following exposure to 100 microM EA and reached peak levels by 30 min which were approximately 7-fold higher than corresponding control levels. Ethacrynic acid-induced GSH depletion and intracellular ROS elevation was followed by marked decreases in glutathione reductase (GR) activity in mitochondria, and to a lesser extent, in cytosolic fractions of cultured astrocytes. This inhibitory effect was time- and concentration-dependent, and other GSH-related enzymes, glutathione peroxidase and glutathione S-transferase, were not or only slightly affected. Kinetic studies showed that EA markedly diminished V(max) values of both mitochondrial and cytosolic GR without affecting K(m), suggesting noncompetitive inhibition of this thiol-dependent enzyme. Another thiol-dependent enzyme glyceraldehyde-3-phosphate dehydrogenase was also markedly inhibited by EA in a time-dependent fashion. Subsequent decline of mitochondrial transmembrane potential (rhodamine 123 uptake) and cellular ATP production following EA treatment occurred prior to the onset of loss of cell viability as indicated by lactate dehydrogenase leakage. These results suggest that the loss of mitochondrial GSH may render the astrocytes unable to combat the pathological sequelae of endogenous oxidative stress, leading to perturbations of thiol-dependent enzyme activities, mitochondrial function and energy metabolism.
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PMID:Cellular responses of cultured cerebellar astrocytes to ethacrynic acid-induced perturbation of subcellular glutathione homeostasis. 868 Aug 62

4-Hydroxy-2-nonenal (HNE), one of the major products of membrane lipid peroxidation, has been shown recently to be present in a form covalently attached to proteins in the renal proximal tubules of rats treated with a renal carcinogen, ferric nitrilotriacetate (Toyokuni, S., et al. (1994) Proc. Natl. Acad. Sci. USA 91, 2616-2620; Uchida, K., et al. (1995) Arch. Biochem. Biophys. 317, 405-411). In the present study, the mechanism of HNE cytotoxicity was studied using the renal tubular epithelial cells (LLC-PK1), focusing on the protein modification and alteration of cellular redox status induced by HNE. Upon treatment with HNE for 2 h, the LLC-PK1 cells were found to be resistant to the low concentration (10 microM) of HNE, while HNE at higher concentrations (> or = 50 microM) mediated cell death. The cytotoxicity of HNE appeared to be correlated with the HNE modification of cellular proteins. Among a number of proteins modified by HNE, a glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase was detected as one of the major targets of HNE in the cells. On the other hand, exposure of LLC-PK1 cells to HNE resulted in rapid reduction of cellular glutathione (GSH) levels, suggesting that HNE influenced primarily the redox status of the cells. Depletion of GSH with buthionine sulfoximine, a potent suppressor of GSH biosynthesis, before HNE treatment caused the cells to be sensitive to HNE cytotoxicity and to HNE modification of cellular proteins, whereas the increase in intracellular GSH levels by treatment with N-acetylcysteine before HNE treatment resulted in a dose-dependent inhibition of HNE-mediated protein modification. These results suggest that intracellular GSH is a determinant on cellular resistance against the HNE-mediated cytotoxicity.
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PMID:4-Hydroxy-2-nonenal cytotoxicity in renal proximal tubular cells: protein modification and redox alteration. 880 82

The aim of this study was to estimate the anticataract action of vitamin E using an in vitro methylprednisolone (MP)-induced cataract model. The same severity of early cortical cataract was induced in lenses isolated from male Wistar rats aged 6 weeks by incubation with MP (1.5 mg/ml) in TC-199 medium. The cataractous lenses showed slight increases in lipid peroxide (LPO) content and Na+/K+ ratio and slight decreases in reduced glutathione (GSH) content and glyceraldehyde-3-phosphate dehydrogenase (GAP-DH), a sensitive index of oxidative stress, and Na+,K(+)-ATPase activities. When the cataractous lenses were further incubated in TC-199 medium with and without vitamin E (250 micrograms/ml) for 48 h, the progression of cataract was prevented in the vitamin E-treated lenses, but not in the vitamin E-untreated lenses. The vitamin E-untreated lenses showed a decrease in vitamin E content and an increase in water content in addition to further increases in LPO content and Na+/K+ ratio and further decreases in GSH content and GAP-DH and Na+,K(+)-ATPase activities. In contrast, the changes of these components and enzymes except for GSH were attenuated in the vitamin E-treated lenses. From these results, it can be estimated that vitamin E prevents in vitro cataractogenesis in rat lenses treated with MP by protecting the lenses against oxidative damage and loss of membrane function.
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PMID:Anticataract action of vitamin E: its estimation using an in vitro steroid cataract model. 888 85

4-Hydroxy-2-nonenal (HNE), one of the major products of lipid peroxidation, inactivated the rate-limiting enzymes (from animal sources) of the glycolytic pathway and the pentose phosphate pathway when incubated at 37 degrees C for 1 h in the absence of glutathione (GSH). The HNE concentration for half-maximal inactivation of 6-phosphofructokinase (PFK) and glyceraldehyde-3-phosphate dehydrogenase was 3-10 microM; and that value for pyruvate kinase, glucose-6-phosphate dehydrogenase, and hexokinases I and II was 0.15-0.6 mM. In the presence of 5 mM GSH, however, only PFK, irrespective of the source (muscle, liver, or erythrocyte), was inactivated by 40-50% when incubated with 0.1 mM HNE for 1 h. Even PFK was not inactivated in the presence of both GSH and its substrate, ATP (2 mM). Glycolysis in human erythrocytes was not affected by treatment of cells with 0.1 mM HNE at 37 degrees C for 30 min. The results suggest that HNE, at concentrations observable under physiological and pathological conditions, hardly affects glycolysis in cells.
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PMID:4-Hydroxy-2-nonenal hardly affects glycolysis. 921 6

Pretreatment of 5-fluorouracil (5-FU), but not posttreatment, has been shown to augment the cytotoxicity of cisplatin (CDDP) or even circumvent CDDP resistance by inhibiting repair of platinum-DNA interstrand crosslinks as well as by reducing the cellular glutathione (GSH) contents in CDDP-resistant HST-1/CP0.2 human squamous carcinoma cells. Because exogenous thymidine, which compensates for 5-FU-mediated inhibition of de novo DNA synthesis via salvage pathway, did not affect this schedule-dependent synergism, the modulatory effect of 5-FU on CDDP resistance would be attributed to the 5-FU-induced RNA damage. We therefore examined the effect of 5-FU on the steady-state levels of messenger RNA (mRNA) of a human excision repair gene ERCC1 and gamma-glutamylcysteine synthetase (gamma-GCS) gene coding for a rate-limiting enzyme for GSH synthesis. The HST-1/ CP0.2 cells were found to have significantly more mRNA expression of these respective genes than do parental HST-1 cells. In these cells, 5-FU pretreatment progressively inhibited mRNA expression of both ERCC1 and gamma-GCS after removal of 5-FU, without affecting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. A maximal mRNA suppression was observed at 48 h posttreatment. Such 5-FU-induced suppression of mRNA transcripts of these genes seems to be consistent with its inhibitory activity on DNA repair capacity and cellular GSH contents. In contrast, 5-FU did not reduce the level of glutathione-S-transferase-pi (GST-pi) or DNA topoisomerase 1 mRNA. Although not convinced, our data suggest that 5-FU, when incorporated into RNA, may inhibit both GSH synthesis and repair of platinum-DNA adducts by downregulating the ERCC1 and gamma-GCS genes, thereby enhancing antitumor activity of CDDP and reversing resistance to CDDP in HST-1/CP0.2 cells.
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PMID:Inhibition by 5-fluorouracil of ERCC1 and gamma-glutamylcysteine synthetase messenger RNA expression in a cisplatin-resistant HST-1 human squamous carcinoma cell line. 926 87

The redox homeostasis is controlled by several enzyme systems. Sulfhydryl groups in lens proteins are very sensitive to oxidative stress and can easily conjugate with nonprotein thiols (S-thiolation) to form protein-thiol mixed disulfides. We have observed an elevation of protein S-S-glutathione (PSSG) and protein-S-S-cysteine (PSSC) in cataractous lenses from humans and from animal models subjected to oxidative stress. We also observed that these protein-thiol mixed disulfides could be spontaneously dissociated and lowered to basal levels if the lens which was pre-exposed to H2O2 was subsequently cultured in H2O2-free medium. This suggests that the lens has a system to repair oxidative damage through dethiolation thereby restoring its redox homeostasis. In other tissues, an enzyme, thioltransferase (TTase), has been shown to be responsible for thiol/disulfide regulation. We recently demonstrated the presence of this enzyme in the lens and in cultured lens epithelial cells. Here, we investigated the response of TTase to H2O2 stress and its possible repair function in cultured lens epithelial cells. Rabbit lens epithelial cell line N/N 1003A was raised to confluence, trypsinized and plated at 0.8 million cells per 60 mm culture dish. The cells were incubated overnight in Eagle's minimum essential medium (MEM) with 1% rabbit serum and then in serum-free MEM for 30 min before a bolus of 0.5 mm H2O2 was added. At intervals of 5, 15, 30 min and up to 3 hr, the cells were harvested and used for enzyme assays for TTase, glutathione reductase (GR), glutathione peroxidase (GPx) and glyceraldehyde-3-phosphate dehydrogenase (G-3PD). Free GSH, total SH and PSSG and PSSC were also determined. Hydrogen peroxide in the medium was measured at each time point. Cells incubated without H2O2 were used as controls. The results showed that the H2O2 concentration was reduced to 50% within 30 min and was undetectable at 2 hr. Cellular GSH dropped to 40% within 5 min and stayed at this level before it began to increase at 90 min and completely recovered by 2 hr. The total SH groups were similar to free GSH. PSSG and PSSC increased 6.5 and 2 times respectively before 30 min and then decreased when GSH started to recover. G-3PD was most sensitive to H2O2 and lost 95% activity within 5 min. The activity was regained quickly when H2O2 diminished in the medium. A similar but less severe pattern was observed in both GPx (60% loss at 60 min) and GR (30% loss at 90 min). In contrast, TTase activity remained constant during the entire 3 hr. Only when a higher dose of H2O2 (0.8-1.0 mM) was used, did TTase activity show a brief loss (<30% at 60 min) and a swift recovery. Cells exposed to H2O2 exhibited a normal morphology with no evidence of DNA fragmentation. The lens epithelial cells showed a remarkable ability to repair the early damages induced by H2O2. The unusual oxidative stress-resistant property displayed by TTase, coupled with its known function suggest that it plays an important role in the repair of oxidative damage.
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PMID:Thioltransferase is present in the lens epithelial cells as a highly oxidative stress-resistant enzyme. 959 40

This study was devoted to the continued search for an explanation of the neurodegeneration found in a severely TPI deficient Hungarian patient whose brother with genomically completely identical TPI defect was completely free of neurological disorders. The changes found in the molecular species composition of the major PL subclasses and the decrease in PE plasmalogens explain the earlier round increase in membrane fluidity interfering thereby with the physiological function of membrane enzymes, receptors, signal transduction, protein-protein interactions and vesicle fusion. Plasmalogens have also the capacity to protect against oxidative stress, that is deemed to contribute to neurodegenerative processes. The presence of chronic oxidative stress was well reflected in the decreased levels of GSH and alpha-tocopherol in the affected brothers. Decrease in plasmalogens have been described recently in Zellweger's syndrome, in other peroxisomal neurodegenerative disorders, in demyelinating processes and in Alzheimer's disease. The brain in normal individuals is highly enriched in plasmalogens. The pathological decrease found in TPI deficient lymphocytes will presumably be more pronounced in excitatory tissues. The recently described role of expanding nucleotide triplets in the development of neurodegeneration is suggested to result through the selective binding via their polyglutamine repeats to GAPDH. The role of GAPDH in TPI deficiency may be of crucial help in the elucidation of the development of neurodegeneration, since the enzymatic defect of TPI can be partially bypassed by means of the HMP shunt which generates GAP via GAPDH without the participation of TPI. Considering the results found in TPI deficiency in comparison to the new literary findings in different neurodegenerative diseases the following pathomechanism may be proposed. The protein products of the defective genes due to their abnormal steric structure bind GAPDH in a different manner or in differing quantity than their normal counterparts. The PL composition and the resulting differences in the biophysical properties of the cell membranes have crucial impact on these protein-protein interactions and on the activity of enzymes and membrane transport functions. The plasmalogen decrease impairs the protection against oxidative stress with consecutive worsening of the neurodegenerative process. The final common pathway to neuronal death leads through destabilization of intracellular Ca2+ homeostasis via elevation of intracellular Ca2+ to apoptosis. The most important conclusion is that lipids are not an inert environment of membrane proteins. Unravelling of the pathogenesis of neurodegeneration needs more concerted investigation of the interactions between genetic changes with biophysical and biochemical cell membrane lipid alterations.
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PMID:[Glycolytic enzyme defects and neurodegeneration]. 987 6

S-Nitrosylation of protein thiol groups by nitric oxide (NO) is a widely recognized protein modification. In this study we show that nitrosonium tetrafluoroborate (BF4NO), a NO+ donor, modified the thiol groups of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by S-nitrosylation and caused enzyme inhibition. The resultant protein-S-nitrosothiol was found to be unstable and to decompose spontaneously, thereby restoring enzyme activity. In contrast, the NO-releasing compound S-nitrosoglutathione (GSNO) promoted S-glutathionylation of a thiol group of GAPDH both in vitro and under cellular conditions. The GSH-mixed protein disulfide formed led to a permanent enzyme inhibition, but upon dithiothreitol addition a functional active GAPDH was recovered. This S-glutathionylation is specific for GSNO because GSH itself was unable to produce protein-mixed disulfides. During cellular nitrosative stress, the production of intracellular GSNO might channel signaling responses to form protein-mixed disulfide that can regulate intracellular function.
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PMID:Nitric oxide-induced S-glutathionylation and inactivation of glyceraldehyde-3-phosphate dehydrogenase. 1009 23

A 3-h exposure to NO donors (spermine-NO, DETA-NO, or SNAP), or to NOS II-expressing cells (activated macrophages or EMT6 cells) reversibly inhibited DNA synthesis in K562 tumor cells. In GSH-depleted K562 cells, cytostasis remained reversible when induced by DETA-NO or NOS II activity, but became irreversible after exposure to spermine-NO or SNAP. Only SNAP and spermine-NO efficiently inhibited GAPDH, an enzyme with a critical thiol, in GSH-depleted cells. Thus, the irreversible cytostasis induced in GSH-depleted cells by spermine-NO or SNAP can be tentatively attributed to S-nitrosating or oxidizing species derived from NO. However, these species did not contribute significantly to the early antiproliferative effects of macrophages. Ribonucleotide reductase, a key enzyme in DNA synthesis. has been shown to be inhibited by NO. Supplementation of the medium with deoxyribonucleosides to bypass RNR inhibition restored DNA synthesis in target cells exposed to DETA-NO and NO-producing cells, but was inefficient for GSH-depleted cells previously submitted to spermine-NO or SNAP. These cells also exhibited a persistent depletion of the dATP pool. In conclusion, GSH depletion reveals striking qualitative differences in the nature of the toxic effectors released by various NO sources, questioning the significance of S-nitrosating or oxidizing nitrogen oxides in NOS II-dependent cytostasis.
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PMID:Differential cytostatic effects of NO donors and NO producing cells. 1038 Dec

A phagocytic challenge with immunoglobulin G (IgG)-coated erythrocytes (EIgGs) has been shown to cause a subsequent depression of macrophage respiratory burst capacity and phagocytic function. The present study evaluated the hypothesis that this macrophage dysfunction is caused by an oxidative stress. An oxidative stress induced by ferric ammonium citrate (FAC) plus cumene hydroperoxide (CHP) caused a depression of macrophage function that was attenuated by antioxidants and iron chelators. In contrast, the same antioxidants and iron chelators did not alter changes caused by a challenge with EIgGs. EIgG challenge caused an increase in lipid peroxidation but failed to deplete glutathione (GSH) or decrease the activity of glyceraldehyde-3-phosphate dehydrogenase (GA-3-PD), suggesting that there was only a slight oxidative stress. Inhibition of the Fc gamma receptor (Fc gammaR) stimulated respiratory burst by removing calcium during the challenge did not attenuate the changes caused by an EIgG challenge. A phagocytic challenge with nonerythrocyte particles, IgG-coated beads (BIgGs), did not depress the respiratory burst capacity but did depress phagocytic function. Fc gammaR expression was depressed following a phagocytic challenge but not an oxidative stress. Thus, an oxidative stress can depress macrophage function, but the dysfunction caused by a phagocytic challenge with EIgGs involves Fc gammaR depletion and the erythrocyte contents rather than an oxidative stress.
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PMID:Role of an oxidative stress in the macrophage dysfunction caused by erythrophagocytosis. 1064 41


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