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Query: UNIPROT:P06889 (Mol)
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The present study was carried out to study de novo glutathione (GSH) synthesis and to evaluate the effect of stimulating GSH synthesis during in vitro maturation (IVM) of adult and prepubertal mouse oocytes on the embryo developmental rate. Adult (8 weeks old) and prepubertal mice (24-26 days old) were primed with 5 IU of PMSG and oocytes were retrieved from the ovary 48 hr later for IVM. After IVM (18 hr) Cumulus oocyte complexes (COC) were in vitro fertilized (IVF) and in vitro culture (IVC) in order to observe embryo development. The IVM medium was supplemented with: 0, 25, 50, 100, or 200 microM of cysteamine. To study the novo GSH synthesis, 5 mM BSO was added during IVM of adult or prepubertal oocyte. Developmental rates up to blastocyst were recorded for each group. Experiments also included a group of ovulated oocytes (in vivo matured) after priming with PMSG and HCG. After IVM of adult mice oocytes, an improvement was observed on embryo development in all the supplemented groups when compared with the untreated group (P < 0.05). No differences were observed in blastocyst rate among IVM oocytes with cysteamine and ovulated oocytes. Prepubertal IVM mouse oocytes had a lower cleavage rate compared with ovulated oocytes (P < 0.05). Cysteamine failed to improve prepubertal oocytes developmental rates (P > 0,05). 2-cell embryos, coming from IVM prepubertal oocytes and ovulated oocytes had the same preimplantation developmental rate up to the blastocyst stage. In prepubertal, and adult oocytes an inhibition of embryo development was observed when buthionine sulfoximide (BSO), a specific inhibitor of the gamma-glutamylcysteine synthetase, was added during oocyte maturation (P < 0.01). In conclusion, an improvement in mouse embryo development was observed when cysteamine was added to the IVM medium of adult mice oocytes. In prepubertal oocytes cysteamine addition during oocyte maturation failed to improve embryo developmental rates. The presence of BSO lowered or completely blocked blastocyst development. This proves that, de novo GSH synthesis during oocyte maturation of adult and prepubertal oocytes undoubtedly plays an important role in embryo development. The improvement on oocyte competence observed in adult mice oocytes is probably related to intracellular GSH synthesis stimulated by cysteamine. Nevertheless the reason why cysteamine failed to improve prepubertal oocytes competence remains as an open question.
Mol Reprod Dev 2003 Feb
PMID:Capacity of adult and prepubertal mouse oocytes to undergo embryo development in the presence of cysteamine. 1250 54

The fission yeast cells that contained the cloned glutathione synthetase (GS) gene showed 1.4-fold higher glutathione (GSH) content and 1.9-fold higher GS activity than the cells without the cloned GS gene. Interestingly, gamma-glutamylcysteine synthetase activity increased 2.1-fold in the S. pombe cells that contained the cloned GS gene. The S. pombe cells that harbored the multicopy-number plasmid pRGS49 (containing the cloned GS gene) showed a higher level of survival on solid media with cadmium chloride (1 mM) or mercuric chloride (10 microM) than the cells that harbored the YEp357R vector. The 506 bp upstream sequence from the translational initiation point and N-terminal 8 amino acid-coding region were fused into the promoterless beta-galactosidase gene of the shuttle vector YEp367R to generate the fusion plasmid pUGS39. Synthesis of beta-galactosidase from the fusion plasmid pUGS39 was significantly enhanced by cadmium chloride and NO-generating S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SN). It was also induced by L-buthionine-(S,R)-sulfoximine, a specific inhibitor of gamma-glutamylcysteine synthetase (GCS). We also found that the expression of the S. pombe GS gene is regulated by the Atf1-Spc1-Wis1 signal pathway.
J Biochem Mol Biol 2003 May 31
PMID:Regulation of the gene encoding glutathione synthetase from the fission yeast. 1278 90

A profound cytotoxic action of the antimalarial, artesunate (ART), was identified against 55 cancer cell lines of the U.S. National Cancer Institute (NCI). The 50% inhibition concentrations (IC50 values) for ART correlated significantly to the cell doubling times (P = 0.00132) and the portion of cells in the G0/G1 (P = 0.02244) or S cell cycle phases (P = 0.03567). We selected mRNA expression data of 465 genes obtained by microarray hybridization from the NCI data base. These genes belong to different biological categories (drug resistance genes, DNA damage response and repair genes, oncogenes and tumor suppressor genes, apoptosis-regulating genes, proliferation-associated genes, and cytokines and cytokine-associated genes). The constitutive expression of 54 of 465 (=12%) genes correlated significantly to the IC50 values for ART. Hierarchical cluster analysis of these 12 genes allowed the differentiation of clusters with ART-sensitive or ART-resistant cell lines (P = 0.00017). For exemplary validation, cell lines transduced with 3 of the 12 genes were used to prove a causative relationship. The cDNAs for a deletion-mutated epidermal growth factor receptor (EGFR) and for gamma-glutamylcysteine synthetase increased resistance to ART. The conditional expression of the CDC25A gene using a tetracycline repressor expression vector increased sensitivity toward ART. Multidrug-resistant cells differentially expressing the MDR1, MRP1, or BCRP genes were not cross-resistant to ART. ART acts via p53-dependent and- independent pathways in isogenic p53+/+ p21WAF1/CIP1+/+, p53-/- p21WAF1/CIP1+/+, and p53+/+ p21WAF1/CIP1-/- colon carcinoma cells.
Mol Pharmacol 2003 Aug
PMID:Molecular modes of action of artesunate in tumor cell lines. 1286 43

Reduced glutathione (GSH) is an essential, multifunctional tripepetide that controls redox-sensitive cellular processes, but its regulation in the heart is poorly understood. The present study used a pharmocological model of GSH depletion to examine cellular mechanisms controlling cardiac GSH. Inhibition of GSH metabolism was elicited in normal rats by daily injections of buthionine sulfoximine (BSO), a blocker of gamma-glutamylcysteine synthetase, plus 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase. After 3 d of BSO/BCNU treatment, intracellular [GSH] was measured in isolated-ventricular myocytes by fluorescence microscopy using the probe monochlorobimane. Basal [GSH] in left-ventricular myocytes from BSO/BCNU-treated rats (2.0 +/- 0.05 amol/microm(3), n = 146) was 50% less than control (4.0 +/- 0.13 amol/microm(3), n = 116; P < 0.05). Incubation of myocytes from BSO/BCNU rats with 0.1 microM insulin normalized [GSH] after a delay of 3-4 h (3.6 +/- 0.29 amol/microm(3), n = 66). This effect of insulin was blocked by pre-treating myocytes with cycloheximide. A protein tyrosine phosphatase inhibitor, bis-peroxovanadium-1,10-phenanthroline (bpV(phen), 1 microM), elicited a similar effect as insulin, while neither agent altered [GSH] in myocytes from control rats. Moreover, the effect of insulin and bpV(phen) to up-regulate GSH was blocked by inhibitors of PI 3-kinase (wortmannin, LY294002), MEK (PD98059) and p38 MAP kinases (SB203580). These data suggest that the insulin-signaling cascade regulates [GSH] in ventricular myocytes by a coordinated activation of PI 3-kinase and MAP kinase pathways. These signaling mechanisms may play essential roles in controlling intracellular redox state and normal function of cardiac myocytes.
J Mol Cell Cardiol 2003 Sep
PMID:Regulation of glutathione in cardiac myocytes. 1296 37

The thiol tripeptides, glutathione (GSH) and homoglutathione (hGSH), perform multiple roles in legumes, including protection against toxicity of free radicals and heavy metals. The three genes involved in the synthesis of GSH and hGSH in the model legume, Lotus japonicus, have been fully characterized and appear to be present as single copies in the genome. The gamma-glutamylcysteine synthetase (gamma(ecs)) gene was mapped on the long arm of chromosome 4 (70.0 centimorgans [cM]) and consists of 15 exons, whereas the glutathione synthetase (gshs) and homoglutathione synthetase (hgshs) genes were mapped on the long arm of chromosome 1 (81.3 cM) and found to be arranged in tandem with a separation of approximately 8 kb. Both genes consist of 12 exons of exactly the same size (except exon 1, which is similar). Two types of transcripts were detected for the gshs gene, which putatively encode proteins localized in the plastids and cytosol. Promoter regions contain cis-acting regulatory elements that may be involved in the plant's response to light, hormones, and stress. Determination of transcript levels, enzyme activities, and thiol contents in nodules, roots, and leaves revealed that gamma(ecs) and hgshs are expressed in all three plant organs, whereas gshs is significantly functional only in nodules. This strongly suggests an important role of GSH in the rhizobia-legume symbiosis.
Mol Plant Microbe Interact 2003 Nov
PMID:Molecular analysis of the pathway for the synthesis of thiol tripeptides in the model legume Lotus japonicus. 1460 72

HepG2 cells expressing CYP2E1 (E47 cells) are more susceptible to toxicity by arachidonic acid (AA) or after glutathione depletion with an inhibitor of glutamate-cysteine ligase, l-buthionine-(S,R)-sulfoximine (BSO), compared with control HepG2 cells (C34 cells). The ability of nitric oxide (NO) to protect against CYP2E1-dependent toxicity has not been evaluated. We therefore studied the ability of O2-vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO), a liver-selective NO donor, to protect against CYP2E1-dependent toxicity and compared this with protection by chemical NO donors. E47 cells incubated with V-PYRRO/NO produced NO, whereas C34 cells did not. Incubation of E47 cells with 50 microM AA or 100 microM BSO for 2 days resulted in a 50% loss of cell viability. VPYRRO/NO (1 mM) blocked this toxicity of AA and BSO by a mechanism involving NO release via CYP2E1 metabolism of VPYRRO/NO. NO scavengers hemoglobin and 2-(4-carboxophenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide blocked the protective effects of V-PYRRO/NO. V-PYRRO/NO inhibited CYP2E1 activity and production of reactive oxygen species, whereas hemoglobin prevented these events. AA and BSO induced lipid peroxidation and decreased mitochondrial membrane potential; both of these effects were blocked by V-PYRRO/NO. Unlike V-PYRRO/NO, the chemical donors spermine/NO and (S)-nitroso-N-acetylpenicillamine release NO directly when added to the medium; however, they could partially protect against the CYP2E1-dependent toxicity. These results suggest that VPYRRO/NO protects HepG2 cells against CYP2E1-dependent toxicity through inhibition of CYP2E1-derived reactive oxygen species production and lipid peroxidation by the generated NO and that this compound may be valuable in protecting against CYP2E1-dependent toxicity via liver P450-specific generation of NO.
Mol Pharmacol 2004 Jan
PMID:The liver-selective nitric oxide donor O2-vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO) protects HepG2 cells against cytochrome P450 2E1-dependent toxicity. 1472 44

Retinoid X receptor alpha (RXRalpha) plays a pivotal role in regulating liver metabolism. RXRalpha-mediated gene expression involved in amino acid metabolism was examined using the NIA Mouse 15K cDNA microarray containing 15,000 different expressed sequence tags. Seven amino acid metabolic genes, three of which encode enzymes involved in phase II detoxification process, were identified as RXRalpha target genes in mouse liver. Glutamate-cysteine ligase catalytic subunit (GCLC), glutathione S-transferasemu, and glutathione peroxidase 1 were down-regulated in the liver of hepatocyte RXRalpha-deficient mice. The down-regulation of GCLC in RXRalpha-deficient mice led to 40% and 45% reductions in the rate of glutathione (GSH) synthesis and level of hepatic GSH, respectively. Primary hepatocytes from RXRalpha-deficient mice were more sensitive to t-butylhydroperoxide-induced oxidative stress. However, GSH diminished RXRalpha-deficient mice were resistant to acetaminophen (APAP)-induced hepatotoxicity. Analysis of phase I detoxification genes revealed that CYP1A2 and CYP3A11 were up-regulated in wild-type mice but down-regulated in RXRalpha-deficient mice after APAP administration. Taken together, the data indicate that RXRalpha centrally regulates both phase I and phase II drug metabolism and detoxification. Regulation of hepatic GSH levels by RXRalpha is essential to protect hepatocytes from oxidative stress, whereas up-regulation of phase I drug metabolism genes by RXRalpha may render the liver more sensitive to APAP-induced toxicity.
Mol Pharmacol 2004 Mar
PMID:Retinoid X receptor alpha regulates glutathione homeostasis and xenobiotic detoxification processes in mouse liver. 1497 33

The genes encoding enzymes involved in glutathione (GSH) metabolism may modulate responses to antimalarial drugs, but the role of most of them in antimalarial drug resistance has scarcely been investigated. Using an in silico/PCR combined approach, we have isolated from Plasmodium chabaudi, full sequences of five Plasmodium falciparum gene orthologues involved in GSH metabolism: the gamma-glutamylcysteine synthetase (Pc-gammagcs), glutathione-synthetase (Pc-gs), glutathione peroxidase (Pc-gpx), glutathione reductase (Pc-gr) and glutathione-S-transferase (Pc-gst). DNA sequencing of these genes from drug sensitive parasites, P. chabaudi AS (0CQ), and ones isolated from parasite lines that show genetically stable resistance to chloroquine (CQ) at low, intermediate and high levels, AS (3CQ), AS (15CQ) and AS (30CQ), respectively, revealed no point mutations in the resistant parasites. We used these sequences to design internal oligonucleotide primers to compare relative mRNA amounts of these genes between all P. chabaudi clones, in untreated mice or following CQ treatment with sub-curative doses, by real-time PCR. Analysis of three independent experiments revealed that transcription levels of the Pc-gammagcs, Pc-gs, Pc-gpx, Pc-gr and Pc-gst genes were not changed between chloroquine sensitive and resistant parasite clones, and that treatment with chloroquine did not induce an alteration in the expression of these genes in sensitive or resistant parasites. We concluded that chloroquine resistance in this species is determined by a mechanism that is independent of these genes, and most likely, of GSH metabolism.
Mol Biochem Parasitol 2004 Jul
PMID:Is the expression of genes encoding enzymes of glutathione (GSH) metabolism involved in chloroquine resistance in Plasmodium chabaudi parasites? 1513 66

Vanin-1 is an epithelial ectoenzyme with pantetheinase activity and generating the amino-thiol cysteamine through the metabolism of pantothenic acid (vitamin B(5)). Here we show that Vanin-1(-/-) mice, which lack cysteamine in tissues, exhibit resistance to oxidative injury induced by whole-body gamma-irradiation or paraquat. This protection is correlated with reduced apoptosis and inflammation and is reversed by treating mutant animals with cystamine. The better tolerance of the Vanin-1(-/-) mice is associated with an enhanced gamma-glutamylcysteine synthetase activity in liver, probably due to the absence of cysteamine and leading to elevated stores of glutathione (GSH), the most potent cellular antioxidant. Consequently, Vanin-1(-/-) mice maintain a more reducing environment in tissue after exposure to irradiation. In normal mice, we found a stress-induced biphasic expression of Vanin-1 regulated via antioxidant response elements in its promoter region. This process should finely tune the redox environment and thus change an early inflammatory process into a late tissue repair process. We propose Vanin-1 as a key molecule to regulate the GSH-dependent response to oxidative injury in tissue at the epithelial level. Therefore, Vanin/pantetheinase inhibitors could be useful for treatment of damage due to irradiation and pro-oxidant inducers.
Mol Cell Biol 2004 Aug
PMID:Vanin-1-/- mice exhibit a glutathione-mediated tissue resistance to oxidative stress. 1528 20

The antioxidant responsive element (ARE) is a cis-acting regulatory element of genes encoding phase II detoxification enzymes and antioxidant proteins, such as NAD(P)H: quinone oxidoreductase 1, glutathione S-transferases, and glutamate-cysteine ligase. Interestingly, it has been reported that Nrf2 (NF-E2-related factor 2) regulates a wide array of ARE-driven genes in various cell types. Nrf2 is a basic leucine zipper transcription factor, which was originally identified as a binding protein of locus control region of beta-globin gene. The DNA binding sequence of Nrf2 and ARE sequence are very similar, and many studies demonstrated that Nrf2 binds to the ARE sites leading to up-regulation of downstream genes. The function of Nrf2 and its downstream target genes suggests that the Nrf2-ARE pathway is important in the cellular antioxidant defense system. In support of this, many studies showed a critical role of Nrf2 in cellular protection and anti-carcinogenicity, implying that the Nrf2-ARE pathway may serve as a therapeutic target for neurodegenerative diseases and cancers, in which oxidative stress is closely implicated.
J Biochem Mol Biol 2004 Mar 31
PMID:An important role of Nrf2-ARE pathway in the cellular defense mechanism. 1546 87


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