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Escherichia coli YggS is a highly conserved pyridoxal 5′-phosphate (PLP)-binding protein whose biochemical function is currently unknown. A previous study with a yggS-deficient E. coli strain (ΔyggS) demonstrated that YggS controls l-Ile- and l-Val-metabolism by modulating 2-ketobutyrate (2-KB), l-2-aminobutyrate (l-2-AB), and/or coenzyme A (CoA) availability in a PLP-dependent fashion. In this study, we found that ΔyggS accumulates an unknown metabolite as judged by amino acid analyses. LC/MS and MS/MS analyses of the compound with propyl chloroformate derivatization, and co-chromatography analysis identified this compound as γ-l-glutamyl-l-2-aminobutyryl-glycine (ophthalmic acid), a glutathione (GSH) analogue in which the l-Cys moiety is replaced by l-2-AB. We also determine the metabolic consequence of the yggS mutation. Absence of YggS initially increases l-2-AB availability, and then causes ophthalmic acid accumulation and CoA limitation in the cell. The expression of a γ-glutamylcysteine synthetase and a glutathione synthetase in a ΔyggS background causes high-level accumulation of ophthalmic acid in the cells (∼1.2 nmol/mg cells) in a minimal synthetic medium. This opens the possibility of a first fermentative production of ophthalmic acid.

Genome shuffling is an efficient and promising approach for the rapid improvement of microbial phenotypes. In this study, genome shuffling was applied to enhance the yield of glutathione produced by Saccharomyces cerevisiae YS86. Six isolates with subtle improvements in glutathione yield were obtained from populations generated by ultraviolet (UV) irradiation and nitrosoguanidine (NTG) mutagenesis. These yeast strains were then subjected to recursive pool-wise protoplast fusion. A strain library that was likely to yield positive colonies was created by fusing the lethal protoplasts obtained from both UV irradiation and heat treatments. After two rounds of genome shuffling, a high-yield recombinant YSF2-19 strain that exhibited 3.2- and 3.3-fold increases in glutathione production in shake flask and fermenter respectively was obtained. Comparative analysis of synthetase gene expression was conducted between the initial and shuffled strains using FQ (fluorescent quantitation) RT-PCR (reverse transcription polymerase chain reaction). Delta CT (threshold cycle) relative quantitation analysis revealed that glutathione synthetase gene (GSH-I) expression at the transcriptional level in the YSF2-19 strain was 9.9-fold greater than in the initial YS86. The shuffled yeast strain has a potential application in brewing, other food, and pharmaceutical industries. Simultaneously, the analysis of improved phenotypes will provide more valuable data for inverse metabolic engineering.

This work describes major features and specificities of glutathione (GSH) biosynthesis in the cold-adapted Pseudoalteromonas haloplanktis. Two steps of ATP hydrolysis, catalysed by γ-glutamyl-cysteine ligase (PhGshA) and glutathione synthetase (PhGshB), drive GSH formation; however, differently from other sources, this psychrophile contains two redundant PhGshAs. The biochemical properties of recombinant rPhGshB and rPhGshA II were previously reported; here we report the characterization of rPhGshA I, together with a structural comparison with rPhGshA II. The availability of the three enzymes involved in GSH biosynthesis allowed the reconstitution of two systems producing this thiol in P. haloplanktis. Both systems were active, although with different reaction rates; in particular, rPhGshB worked more efficiently with rPhGshA I compared to rPhGshA II, also because of the different expression of the corresponding genes. However, the lower KM of rPhGshA II for cysteine compared to rPhGshA I could permit an effective GSH production even under conditions of low cysteine content. Coupling of the kinetics of GSH production with that of ATP hydrolysis was realised for the first time and proved the rigorous stoichiometry of GSH biosynthesis, resulting in two moles of ATP hydrolysed per one mole of GSH formed. Furthermore, after dissecting the reaction rates for GSH biosynthesis, we demonstrated that synthesis of this thiol was rate-limited by the step catalysed by rPhGshB, at least in the cold-adapted source. Therefore, the GshA rate-limited step postulated in other sources could be revised or restricted to conditions of limiting substrate amounts.

Glutathione synthetase (GS) deficiency is a rare inborn error of glutathione (GSH) metabolism manifested by severe metabolic acidosis, hemolytic anemia, neurological problems and massive excretion of pyroglutamic acid (5-oxoproline) in the urine. The disorder has mild, moderate, and severe clinical variants. We aimed to report clinical and laboratory findings of four patients, effect of sodium hydrogen carbonate treatment and long-term follow up of three patients.Urine organic acid analysis was performed with gas chromatography-mass spectrometry. Molecular genetic analysis was performed in three patients, mutation was found in two of them. Enzyme analysis was performed in one patient. Clinical and laboratory findings of four patients were evaluated.One patient died at 4 months old, one patient’s growth and development are normal, two patients have developed intellectual disability and seizures in the long term follow up period. Three patients benefited from sodium hydrogen carbonate treatment.The clinical picture varies from patient to patient, so it is difficult to predict the prognosis and the effectiveness of treatment protocols. We reported long term follow up of four patients and demonstrated that sodium hydrogen carbonate is effective for treatment of chronic metabolic acidosis in GS deficieny.

Hemolytic anemia (HA) of the newborn should be considered in cases of rapidly developing, severe, or persistent hyperbilirubinemia. Several causes of corpuscular hemolysis have been described, among which red blood cell enzyme defects are of particular concern. We report a rare case of red blood cell enzyme defect in a male infant, who presented during his first months of life with recurrent and isolated neonatal hemolysis. All main causes were ruled out. At 6.5 months of age, the patient presented with gastroenteritis requiring hospitalization; fortuitously, urine organic acid chromatography revealed a large peak of 5-oxoproline. Before the association between HA and 5-oxoprolinuria was noted, glutathione synthetase deficiency was suspected and confirmed by a low glutathione synthetase concentration and a collapse of glutathione synthetase activity in erythrocytes. Moreover, molecular diagnosis revealed 2 mutations in the glutathione synthetase gene: a previously reported missense mutation (c.[656A>G]; p.[Asp219Gly]) and a mutation not yet described in the binding site of the enzyme (c.[902T>C]; p.[Leu301Pro]). However, 15 days later, a control sample revealed no signs of 5-oxoprolinuria and the clinical history discovered administration of acetaminophen in the 48 hours before hospitalization. Thus, in this patient, acetaminophen exposure allowed the diagnosis of a mild form of glutathione synthetase deficiency, characterized by isolated HA. Early diagnosis is important because treatment with bicarbonate, vitamins C and E, and elimination of trigger factors are recommended to improve long-term outcomes. Glutathione synthetase deficiency should be screened for in cases of unexplained newborn HA.

Conventionally, two consecutive enzymatic reactions catalyzed by γ-glutamylcysteine synthetase and glutathione synthetase are most commonly used for glutathione production. Here we demonstrate that bacterial bifunctional GshF can be used for glutathione production in a eukaryotic system without accumulation of the intermediate γ-glutamylcysteine.

To investigate a possible involvement of nitric oxide in gene regulation of glutathione and flavonoid synthesis pathways, maize seedlings were treated with sodium nitroprusside (SNP), a NO donor, and apocynin (APO), an inducer of NO production. After 12-h treatment, the transcripts of γ-glutamylcysteine synthetase (γ-ecs), glutathione synthetase (gsh-s), chalcone synthase (chs), phenylalanine ammonia lyase (pal.1), myb-related protein P (P1) and actin 1 (act) genes were quantified in maize leaves by real time PCR, using α-tubulin as standard transcript. The level of γ-ecs and gsh-s transcripts in maize leaves were increased 9-fold and 12-fold, respectively, following SNP treatment, while after APO treatment, those transcripts were not significantly different from control plants. SNP-treated maize leaves did not show significant changes in pal.1 and chs expression. NO content in maize leaves was increased in SNP and APO treated plants in comparison to control plants. In conclusion, our experiments suggested that genes involved in glutathione synthesis could be modulated by SNP in maize leaves. On the other hand, APO had no effect on γ-ecs and gshs gene expression.

Hepatic iron overload has been associated classically with the genetic disorder hereditary hemochromatosis. More recently, it has become apparent that mild-to-moderate degrees of elevated hepatic iron stores observed in other liver diseases also have clinical relevance. The goal was to use a mouse model of dietary hepatic iron overload and isobaric tag for relative and absolute quantitation proteomics to identify, at a global level, differentially expressed proteins in livers from mice fed a control or 3,5,5-trimethyl-hexanoyl-ferrocene (TMHF) supplemented diet for 4 weeks. The expression of 74 proteins was altered by ≥ ±1.5-fold, showing that the effects of iron on the liver proteome were extensive. The top canonical pathway altered by TMHF treatment was the NF-E2-related factor 2 (NRF2-)-mediated oxidative stress response. Because of the long-standing association of elevated hepatic iron with oxidative stress, the remainder of the study was focused on NRF2. TMHF treatment upregulated 25 phase I/II and antioxidant proteins previously categorized as NRF2 target gene products. Immunoblot analyses showed that TMHF treatment increased the levels of glutathione S-transferase (GST) M1, GSTM4, glutamate-cysteine ligase (GCL) catalytic subunit, GCL modifier subunit, glutathione synthetase, glutathione reductase, heme oxygenase 1, epoxide hydrolase 1, and NAD(P)H dehydrogenase quinone 1. Immunofluorescence, carried out to determine the cellular localization of NRF2, showed that NRF2 was detected in the nucleus of hepatocytes from TMHF-treated mice and not from control mice. We conclude that elevated hepatic iron in a mouse model activates NRF2, a key regulator of the cellular response to oxidative stress.

A kinetic model of trypanothione [T(SH)2] metabolism in Trypanosoma cruzi was constructed based on enzyme kinetic parameters determined under near-physiological conditions (including glutathione synthetase), and the enzyme activities, metabolite concentrations and fluxes determined in the parasite under control and oxidizing conditions. The pathway structure is characterized by a T(SH)2 synthetic module of low flux and low catalytic capacity, and another more catalytically efficient T(SH)2-dependent antioxidant/regenerating module. The model allowed quantification of the contribution of each enzyme to the control of T(SH)2 synthesis and concentration (flux control and concentration control coefficients, respectively). The main control of flux was exerted by γ-glutamylcysteine synthetase (γECS) and trypanothione synthetase (TryS) (control coefficients of 0.58–0.7 and 0.49–0.58, respectively), followed by spermidine transport (0.24); negligible flux controls by trypantothione reductase (TryR) and the T(SH)2-dependent antioxidant machinery were determined. The concentration of reduced T(SH)2 was controlled by TryR (0.98) and oxidative stress (−0.99); however, γECS and TryS also exerted control on the cellular level of T(SH2) when they were inhibited by more than 70%. The model predicted that in order to diminish the T(SH)2 synthesis flux by 50%, it is necessary to inhibit γECS or TryS by 58 or 63%, respectively, or both by 50%, whereas more than 98% inhibition was required for TryR. Hence, simultaneous and moderate inhibition of γECS and TryS appears to be a promising multi-target therapeutic strategy. In contrast, use of highly potent and specific inhibitors for TryR and the antioxidant machinery is necessary to affect the antioxidant capabilities of the parasites. Database The glutathione synthetase gene sequences from the Ninoa and Queretaro strains have been submitted to the GenBank database under accession numbers HQ398240 and HQ398239, respectively

Plants exposed to heavy metals rapidly induce changes in gene expression that activate and enhance detoxification mechanisms, including toxic-metal chelation and the scavenging of reactive oxygen species. However, the mechanisms mediating toxic heavy metal-induced gene expression remain largely unknown. To genetically elucidate cadmium-specific transcriptional responses in Arabidopsis, we designed a genetic screen based on the activation of a cadmium-inducible reporter gene. Microarray studies identified a high-affinity sulfate transporter (SULTR1;2) among the most robust and rapid cadmium-inducible transcripts. The SULTR1;2 promoter (2.2 kb) was fused with the firefly luciferase reporter gene to quantitatively report the transcriptional response of plants exposed to cadmium. Stably transformed luciferase reporter lines were ethyl methanesulfonate (EMS) mutagenized, and stable M(2) seedlings were screened for an abnormal luciferase response during exposure to cadmium. The screen identified non-allelic mutant lines that fell into one of three categories: (i) super response to cadmium (SRC) mutants; (ii) constitutive response to cadmium (CRC) mutants; or (iii) non-response and reduced response to cadmium (NRC) mutants. Two nrc mutants, nrc1 and nrc2, were mapped, cloned and further characterized. The nrc1 mutation was mapped to the γ-glutamylcysteine synthetase gene and the nrc2 mutation was identified as the first viable recessive mutant allele in the glutathione synthetase gene. Moreover, genetic, HPLC mass spectrometry, and gene expression analysis of the nrc1 and nrc2 mutants, revealed that intracellular glutathione depletion alone would be insufficient to induce gene expression of sulfate uptake and assimilation mechanisms. Our results modify the glutathione-depletion driven model for sulfate assimilation gene induction during cadmium stress, and suggest that an enhanced oxidative state and depletion of upstream thiols, in addition to glutathione depletion, are necessary to induce the transcription of sulfate assimilation genes during early cadmium stress.

32 Pags., 6 Figs. The definitive version is available at: http://jxb.oxfordjournals.org/, In plants and other organisms, glutathione (GSH) biosynthesis is catalysed sequentially by γ-glutamylcysteine synthetase (γECS) and glutathione synthetase (GSHS). In legumes, homoglutathione (hGSH) can replace GSH and is synthesized by γECS and a specific homoglutathione synthetase (hGSHS). The subcellular localization of the enzymes was examined by electron microscopy in several legumes and gene expression was analysed in Lotus japonicus plants treated for 1–48 h with 50 μM of hormones. Immunogold localization studies revealed that γECS is confined to chloroplasts and plastids, whereas hGSHS is also in the cytosol. Addition of hormones caused differential expression of thiol synthetases in roots. After 24–48 h, abscisic and salicylic acids downregulated GSHS whereas jasmonic acid upregulated it. Cytokinins and polyamines activated GSHS but not γECS or hGSHS. Jasmonic acid elicited a coordinated response of the three genes and auxin induced both hGSHS expression and activity. Results show that the thiol biosynthetic pathway is compartmentalized in legumes. Moreover, the similar response profiles of the GSH and hGSH contents in roots of non-nodulated and nodulated plants to the various hormonal treatments indicate that thiol homeostasis is independent of the nitrogen source of the plants. The differential regulation of the three mRNA levels, hGSHS activity, and thiol contents by hormones indicates a fine control of thiol biosynthesis at multiple levels and strongly suggests that GSH and hGSH play distinct roles in plant development and stress responses., Marina Gay and Joaquín Navascués were recipients of postdoctoral contracts of the Junta para la Ampliación de Estudios (JAE-Doc) programme, funded by Consejo Superior de Investigaciones Científicas-Fondo Social Europeo. This work was funded by Ministerio de Ciencia e Innovación-Fondo Europeo de Desarrollo Regional (AGL2008-01298 and AGL2011-24524) and Gobierno de Aragón (group A53).

Although Yarrowia lipolytica is an important host strain, there have so far been few studies on the production of glutathione by the strain. We therefore performed a study to obtain an improved strain of Y. lipolytica ATCC20688, which could produce a high yield of glutathione. First, the capability of glutathione production in the ATCC20688 strain was estimated. In comparison with other yeasts, the yield of this strain was higher than those in Pichia strains. Furthermore, this strain could produce glutathione by assimilating sodium oleate. We next performed mutation and gene cloning to improve the yield. After the yield of glutathione was improved in the isolated methylglyoxal-resistant mutant (MGR3), the glutathione synthetase gene was cloned into the MGR3 strain. By using this recombinant strain, we could reach the maximum yield and intracellular content of glutathione of 54 mg/L-medium and 30 mg/g-dry cell weight, respectively.

Glutathione (γ-glutamylcysteinyl-glycine, GSH) has vital functions as thiol redox buffer and cofactor of antioxidant and detoxification enzymes. Plasmodium falciparum possesses a functional GSH biosynthesis pathway and contains mM concentrations of the tripeptide. It was impossible to delete in P. falciparum the genes encoding γ-glutamylcysteine synthetase (γGCS) or glutathione synthetase (GS), the two enzymes synthesizing GSH, although both gene loci were not refractory to recombination. Our data show that the parasites cannot compensate for the loss of GSH biosynthesis via GSH uptake. This suggests an important if not essential function of GSH biosynthesis pathway for the parasites. Treatment with the irreversible inhibitor of γGCS L-buthionine sulfoximine (BSO) reduced intracellular GSH levels in P. falciparum and was lethal for their intra-erythrocytic development, corroborating the suggestion that GSH biosynthesis is important for parasite survival. Episomal expression of γgcs in P. falciparum increased tolerance to BSO attributable to increased levels of γGCS. Concomitantly expression of glutathione reductase was reduced leading to an increased GSH efflux. Together these data indicate that GSH levels are tightly regulated by a functional GSH biosynthesis and the reduction of GSSG.

International audience; Human glutathione synthetase (hGS) catalyzes the second ATP-dependent step in the biosynthesis of glutathione (GSH) and is negatively cooperative to the γ-glutamyl substrate. The hGS active site is composed of three highly conserved catalytic loops, notably the alanine rich A-loop. Experimental and computational investigations of the impact of mutation of Asp458 are reported, and thus the role of this A-loop residue on hGS structure, activity, negativity cooperativity and stability is defined. Several Asp458 hGS mutants (D458A, D458N and D458R) were constructed using site-directed mutagenesis and their activities determined (10%, 15% and 7% of wild-type hGS, respectively). The Michaelis-Menten constant (K(m)) was determined for all three substrates (glycine, GAB and ATP): glycine K(m) increased by 30-115-fold, GAB K(m) decreased by 8-17-fold, and the ATP K(m) was unchanged. All Asp458 mutants display a change in cooperativity from negative cooperativity to non-cooperative. All mutants show similar stability as compared to wild-type hGS, as determined by differential scanning calorimetry. The findings indicate that Asp458 is essential for hGS catalysis and that it impacts the allostery of hGS.

Unlike neurons and various other non-neuronal cells, astrocytes have been reported to be resistant to morphine induced cytotoxicity. The present work demonstrates that primary cultures of astrocytes are also sensitive to morphine toxicity depending upon the thyroidal status of the culture medium. Chronic morphine treatment of astrocytes, cultured under thyroid hormone (TH)-deficient conditions, induced apoptotic cell death which was characterized by nuclear condensation, DNA fragmentation and activation of caspase-3 like enzymes. Cell death was accompanied with increase in nNOS level, nitration of cellular proteins and down regulation of pAKT level. Phosphorylation of ERK1/2 showed a biphasic response, an initial induction followed by sustained decline during chronic morphine treatment and the initial induction of pERK1/2 level appeared to be critical for apoptosis in the cells. Interestingly, supplementation with normal levels of TH to cells attenuated morphine-induced apoptosis as well as the biphasic response of pERK1/2 in the astrocytes. However, in the presence of glutathione synthetase inhibitor L-buthionine-S,R-sulfoximine, TH failed to protect astrocytes. Overall, the study demonstrates a possible signaling mechanism of morphine induced toxicity to cells and suggests that alteration of glutathione homeostasis by TH protect astrocytes from morphine by regulating NO and pERK1/2 pathways in the cells.

AtATM3, a member of the ATP-binding cassette transporter family, is localized at the mitochondrial membrane of Arabidopsis thaliana and is involved in the biogenesis of Fe–S clusters and iron homeostasis in plants. Through Agrobacterium-mediated genetic transformation, the AtATM3 gene driven by the cauliflower mosaic virus 35S promoter (CaMV35S) was introduced into Brassica juncea (Indian mustard), a plant species suitable for phytoremediation, with the aim of improving heavy metal tolerance and accumulation in plants. The presence of the AtATM3 gene in transgenic plants was confirmed by polymerase chain reaction (PCR). Reverse transcriptase-PCR analysis confirmed AtATM3 expression in transgenic plants, but the level of AtATM3 expression varied between lines. AtATM3 overexpression in B. juncea conferred enhanced tolerance to cadmium [Cd(II)] and lead [Pb(II)] stresses. Importantly, the shoot tissues of transgenic seedlings contained about 1.5- to 2.5-fold higher Cd(II) and Pb(II) levels than wild type (WT) seedlings, demonstrating significantly-increased accumulation of both Cd(II) and Pb(II) in transgenic plants. The enhanced capacity of heavy metal tolerance and accumulation by AtATM3 transgenic plants was attributed to higher BjGSHII (B. juncea glutathione synthetase II) and BjPCS1 (phytochelatin synthase 1) expression levels induced by AtATM3 overexpression. In addition, AtATM3 overexpression regulated the expression of several metal transporters in the transgenic B. juncea plants under heavy metal stress conditions. Therefore, AtATM3 transgenic plants are more tolerant of and can accumulate more heavy metals to enhance phytoremediation of contaminated soils.

Recent studies of transgenic poplars over-expressing the genes gsh1 and gsh2 encoding γ-glutamylcysteine synthetase (γ-ECS) and glutathione synthetase, respectively, provided detailed information on regulation of GSH synthesis, enzymes activities and mRNA expression. In this experiment, we studied quantitative parameters of leaves, assimilating tissues, cells and chloroplasts, mesophyll resistance for CO(2) diffusion, chlorophyll and carbohydrate content in wild-type poplar and transgenic plants over-expressing gsh1 in the cytosol after 3 years of growth in relatively clean (control) or heavy metal-contaminated soil in the field. Over-expression of gsh1 in the cytosol led to a twofold increase of intrafoliar GSH concentration and influenced the photosynthetic apparatus at different levels of organisation, i.e., leaves, photosynthetic cells and chloroplasts. At the control site, transgenic poplars had a twofold smaller total leaf area per plant and a 1.6-fold leaf area per leaf compared to wild-type controls. Annual aboveground biomass gain was reduced by 50% in the transgenic plants. The reduction of leaf area of the transformants was accompanied by a significant decline in total cell number per leaf, indicating suppression of cell division. Over-expression of γ-ECS in the cytosol also caused changes in mesophyll structure, i.e., a 20% decrease in cell and chloroplast number per leaf area, but also an enhanced volume share of chloroplasts and intercellular airspaces in the leaves. Transgenic and wild poplars did not exhibit differences in chlorophyll and carotenoid content of leaves, but transformants had 1.3-fold fewer soluble carbohydrates. Cultivation on contaminated soil caused a reduction of palisade cell volume and chloroplast number, both per cell and leaf area, in wild-type plants but not in transformants. Biomass accumulation of wild-type poplars decreased in contaminated soil by more than 30-fold, whereas transformants showed a twofold decrease compared to the control site. Thus, poplars over-expressing γ-ECS in the cytosol were more tolerant to heavy metal stress under field conditions than wild-type plants according to the parameters analysed. Correlation analysis revealed strong dependence of cell number per leaf area unit, chloroplast parameters and mesophyll resistance with the GSH level in poplar leaves.

Objective: To determine the profile, relative quantitation, and correlation of gene expression of the antioxidant enzymes copper-zinc superoxide dismutase (CuZn-SOD), Mn-superoxide dismutase (Mn-SOD), extracellular superoxide dismutase (EC-SOD), catalase (CAT), glutathione synthetase (GSS), and glutathione reductase (GSR) in conjunctival samples from healthy subjects. Design: Descriptive study. Participants: Sixteen healthy donors (32 eyes) were included in this study. Methods: Conventional and real-time polymerase chain reactions (PCR) were performed using total RNA isolated from conjunctival impression cytology taken from bulbar superior conjunctiva from both eyes. Results: Products amplified by conventional PCR had only 1 band with the expected size for each target gene. Melt-curve analysis of realtime PCR products also identified a single amplified product for each gene. Log-transformed antioxidant enzyme mRNA expression levels, relative to glyceraldehyde-3-phosphate dehydrogenase expression (mean ± standard error of the mean [SEM]), were CuZn-SOD, 1.52 ± 0.13; Mn-SOD, 1.72 ± 0.08; EC-SOD, 0.35 ± 0.08; GSS, 2.43 ± 0.20; GSR, 2.52 ± 0.16; and CAT, 0.90 ± 0.08. The mRNA levels for CuZn-SOD were strongly correlated with GSS, GSR, Mn-SOD, and EC-SOD. Similarly, the levels of mRNA of GSS and GSR were strongly correlated with each other and with Mn-SOD and EC-SOD. Conclusions: Normal human conjunctiva expresses the antioxidant enzymes genes CuZn-SOD, Mn-SOD, EC-SOD, CAT, GSS, and GSR. The relative quantitation of these genes expressed in conjunctivas of normal eyes will allow further comparisons in pathological circumstances. Knowledge of correlated gene expression will provide a better understanding of the antioxidant balance in the ocular surface.

Redox status affects various cellular activities, such as proliferation, differentiation, and death. Recent studies suggest pivotal roles of reactive oxygen species not only in pathogenesis under oxidative insult but also in intracellular signal transduction. Glutathione is present in several millimolar concentrations in the cytoplasm and has multiple roles in the regulation of cellular homeostasis. Two enzymes, γ-glutamylcysteine synthetase and glutathione synthetase, constitute the de novo synthesis machinery, while glutathione reductase is involved in the recycling of oxidized glutathione. Multidrug resistant proteins and some other transporters are responsible for exporting oxidized glutathione, glutathione conjugates, and S-nitrosoglutathione. In addition to antioxidation, glutathione is more positively involved in cellular activity via its sulfhydryl moiety of a molecule. Animals in which genes responsible for glutathione metabolism are genetically modified can be used as beneficial and reliable models to elucidate roles of glutathione in vivo. This review article overviews recent progress in works related to genetically modified rodents and advances in the elucidation of glutathione-mediated reactions.

Previous research demonstrated that soft wheat cultivars have better post-harvest storage tolerance than harder cultivars during accelerated ageing. To better understand this phenomenon, a tandem mass tag-based quantitative proteomic analysis of soft wheat seeds was performed at different storage times during accelerated ageing (germination ratios of 97%, 45%, 28%, and 6%). A total of 1,010 proteins were differentially regulated, of which 519 and 491 were up- and downregulated, respectively. Most of the differentially expressed proteins were predicted to be involved in nutrient reservoir, enzyme activity and regulation, energy and metabolism, and response to stimulus functions, consistent with processes occurring in hard wheat during artificial ageing. Notably, defense-associated proteins including wheatwin-2, pathogenesis-related proteins protecting against fungal invasion, and glutathione S-transferase and glutathione synthetase participating in reactive oxygen species (ROS) detoxification, were upregulated compared to levels in hard wheat during accelerated ageing. These upregulated proteins might be responsible for the superior post-harvest storage-tolerance of soft wheat cultivars during accelerated ageing compared with hard wheat. Although accelerated ageing could not fully mimic natural ageing, our findings provided novel dynamic proteomic insight into soft wheat seeds during seed deterioration.

Aims: Schizophrenia is a major psychiatric disorder with complex genetic, environmental, and psychological causes, and oxidative stress may be involved in the pathogenesis of the disease. Glutathione (GSH), one of the main cellular non-protein antioxidants and redox regulators, and altered GSH levels have been reported in various regions in patients with schizophrenia. Three enzymes are responsible for GSH synthesis: glutamate cysteine ligase modifier (GCLM), glutamate cysteine ligase catalytic subunit (GCLC), and glutathione synthetase (GSS). Previously, positive associations between GCLM and schizophrenia were reported in Europeans, but not in the Japanese population. Thus, in this study, we investigated the association between the GSH synthesis genes (GCLM, GCLC, and GSS) and schizophrenia in Japanese individuals. Methods: Seventeen single-nucleotide polymorphisms (SNP) in GCLM, GCLC, and GSS were genotyped in 358 patients with schizophrenia and in 359 controls. Results: No SNP showed a significant association between their allelic or genotypic frequencies and schizophrenia. Case–control haplotype association analysis using windows of two or three SNP showed no significant associations with schizophrenia. The case–control haplotype analyses based on the ascertained linkage disequilibrium blocks also showed no significant associations in any genes with schizophrenia. Conclusions: The three primary GSH synthesis genes do not have an apparent degree of association with schizophrenia in the Japanese population.

Glutathione, a nonribosomal thiol tripeptide, has been shown to be critical for many processes in plants. Much less is known about the roles of glutathione in cyanobacteria, oxygenic photosynthetic prokaryotes that are the evolutionary precursor of the chloroplast. An understanding of glutathione metabolism in cyanobacteria is expected to provide novel insight into the evolution of the elaborate and extensive pathways that utilize glutathione in photosynthetic organisms. To investigate the function of glutathione in cyanobacteria, we generated deletion mutants of glutamate-cysteine ligase (gshA) and glutathione synthetase (gshB) in Synechocystis sp. PCC 6803. Complete segregation of the ƊgshA mutation was not achieved, suggesting that GshA activity is essential for growth. In contrast, fully segregated ƊgshB mutants were isolated and characterized. The ƊgshB strain lacks reduced glutathione (GSH) but instead accumulates the precursor compound γ-glutamylcysteine (γ-EC). The ƊgshB strain grows slower than the wild-type strain under favorable conditions and exhibits extremely reduced growth or death when subjected to conditions promoting oxidative stress. Furthermore, we analyzed thiol contents in the wild type and the ƊgshB mutant after subjecting the strains to multiple environmental and redox perturbations. We found that conditions promoting growth stimulate glutathione biosynthesis. We also determined that cellular GSH and γ-EC content decline following exposure to dark and blue light and during photoheterotrophic growth. Moreover, a rapid depletion of GSH and γ-EC is observed in the wild type and the ƊgshB strain, respectively, when cells are starved for nitrate or sulfate.

Overexpression of thioredoxin (TRX) confers oxidative stress resistance and extends lifespan in mammals and insects. However, less is known about phenotypes associated with loss of TRX. We investigated loss-of-function phenotypes of Trx-2 in Drosophila, and found that the mutant flies are hyper-susceptible to paraquat, a free radical generator, but not to hydrogen peroxide. They contain a high amount of protein carbonyl, which dramatically increases with age. Trx-2 mutants express high levels of anti-oxidant genes, such as superoxide dismutase, catalase, and glutathione synthetase. This is the first demonstration of biochemical and physiological consequences caused by loss of Trx-2 in Drosophila.

The ratio of reduced glutathione to its oxidized form (GSH/GSSG) is essential for cell viability including its role in redox-dependent regulation of gene expression. The aim of our study was to estimate GSH/GSSG ratio and expression of redox-dependent genes (glutaredoxin, thioredoxin, peroxiredoxin) under development of cancer cell resistance to cisplatin (CDDP) possessed pro-oxidant action. Under development of resistance of human erythroleukemia K562 and ovarian carcinoma SKOV-3 cells to CDDP co-ordinative enhanced expression of genes encoding glutathione synthetase (GS), subunits of γ-glutamylcysteine synthetase (γ-GCSH, γ-GCSL) was found. In addition, the growth of GSH/GSSG, enhanced expression of glutaredoxin (GLRX1, GLRX2), thioredoxin (TRX2), peroxiredoxin (PRDX6) genes as well as elevated level of transcription factor Nrf2 were observed in both types of resistant cells. We suggest that mechanism of development of cancer cell resistance to CDDP can include activation of GSH synthesis de novo, redox-dependent increase in expression of GLRX1, GLRX2, TRX2, PRDX6 genes which are co-ordinative regulated by redox-dependent transcription factor Nrf2 and elevated level of GSH/GSSG. The publication was prepared with the support of the “RUDN University Program 5–100”.

Chlorothalonil (CHT) and carbendazim (CAR) are two widely used fungicides in agriculture. Despite their agronomic importance in pest control, little is known about their detoxification in the plant. In this study, we investigated the effects of these fungicides on glutathione (GSH) content, GSH-dependent enzyme activities and gene expression in tomato leaves. Results showed that exposure to CHT resulted in increases in GSH content, activities of glutathione S-transferases (GSTs) and glutathione reductase (GR), as well as the transcriptional levels of glutathione S-transferase genes (GST1, GST2 and GST3), glutathione synthetase gene (GSH), glutathione reductase gene (GR) and glutathione peroxidase gene (GPX) in tomato leaves, but such increases were not observed in leaves exposed to CAR. In addition, GSTs, GR, peroxidase (POD) activities and most of GSH-dependent gene expression were induced by CHT in a concentration- and time-dependent manner. These results suggest that GSH-dependent pathway plays an important role in the CHT detoxification but not in the CAR detoxification in tomato leaves.

Glycine protects mammalian intestine against oxidative damage caused by ischaemia-reperfusion (IR) injury and prevents or reverses experimentally-induced colitis. However the mechanism of protection remains largely unknown. The objectives of the current study were to demonstrate directly glycine-mediated protection of human intestinal epithelial cells and to determine the requirement for glycine uptake by the specific transporter GLYT1. Exogenous glycine protected human intestinal Caco-2 and HCT-8 cells against the oxidative agent tert-butylhydroperoxide and reduced the intracellular concentration of reactive oxygen species, when applied prior to but not concomitant with the oxidative challenge. Glycine given prior to oxidative challenge preserved intracellular glutathione concentration but had no effect on the rate of glycine uptake. Protection was dependent on GLYT1 activity, being blocked by a specific GLYT1 inhibitor, supporting a requirement for intracellular glycine accumulation. Maintained intracellular glutathione content is indicated as a mechanism through which the protective effect may in part be mediated. However expression of the genes encoding GLYT1 and the glutathione synthesising enzymes glutamate-cysteine ligase, both catalytic and modifier subunits, and glutathione synthetase was not altered by glycine or tert-butylhydroperoxide, suggesting transcriptional regulation is not involved. This work has demonstrated a novel role of GLYT1 in intestine and shown that intestinal epithelial cells respond directly to oxidative challenge without reliance on extra-epithelial tissues or functions such as neurone, blood-flow or immune responses for antioxidant defence. The protective actions of glycine and maintenance of epithelial antioxidant defences suggest it may be beneficial in treatment of inflammatory bowel disease.

Graphical abstract The close similarity of Trypanosoma brucei glutathione synthetase to the human orthologue indicates that the enzyme would be a difficult target for drug discovery., Glutathione synthetase catalyses the synthesis of the low molecular mass thiol glutathione from l-γ-glutamyl-l-cysteine and glycine. We report the crystal structure of the dimeric enzyme from Trypanosoma brucei in complex with the product glutathione. The enzyme belongs to the ATP-grasp family, a group of enzymes known to undergo conformational changes upon ligand binding. The T. brucei enzyme crystal structure presents two dimers in the asymmetric unit. The structure reveals variability in the order and position of a small domain, which forms a lid for the active site and serves to capture conformations likely to exist during the catalytic cycle. Comparisons with orthologous enzymes, in particular from Homo sapiens and Saccharomyces cerevisae, indicate a high degree of sequence and structure conservation in part of the active site. Structural differences that are observed between the orthologous enzymes are assigned to different ligand binding states since key residues are conserved. This suggests that the molecular determinants of ligand recognition and reactivity are highly conserved across species. We conclude that it would be difficult to target the parasite enzyme in preference to the host enzyme and therefore glutathione synthetase may not be a suitable target for antiparasitic drug discovery.

It is becoming more and more apparent that most genetic disorders are caused by biochemical abnormalities. Recent advances in human genome project and related research have showed us to detect and understand most of the inborn errors of metabolism. These are often caused by point mutations manifested as single-nucleotide-polymorphisms (SNPs). The GSS gene inquested in this work was analyzed for potential mutations with the help of computational tools like SIFT, PolyPhen and UTRscan. It was noted that 84.38% nsSNPs were found to be deleterious by the sequence homology based tool (SIFT), 78.13% by the structure homology based tool (PolyPhen) and 75% by both the SIFT and PolyPhen servers. Two major mutations occurred in the native protein (2HGS) coded by GSS gene at positions R125C and R236Q. Then a modeled structure for the mutant proteins (R125C and R236Q) was proposed and compared with that of the native protein. It was found that the total energy of the mutant (R125C and R236Q) proteins were -31893.846 and -31833.818 Kcal/mol respectively and that of the native protein was -31977.365 Kcal/mol. Also the RMSD values between the native and mutant (R125C and R236Q) type proteins were 1.80A and 1.54A. Hence, we conclude based on our study that the above mutations could be the major target mutations in causing the glutathione synthetase deficiency.

The response of Camellia sinensis (L.) O. Kuntze cultivars Chinary and Assamica to Cu and Al stresses was investigated. Exposure to 100 µM CuSO4 or 100 µM AlCl3 led to accumulation of reactive oxygen species (ROS) more in Assamica than in Chinary. Proline content was higher in Chinary compared to Assamica, while chlorophyll and protein contents decreased upon Cu and Al exposure in both the cultivars. Expression of glutathione biosynthetic enzymes γ-glutamylcysteinyl synthetase (γ-ECS) and glutathione synthetase (GSHS) was elevated. Phytochelatin synthase (PCS), an enzyme involved in phytochelatins synthesis by using glutathione as a substrate was up-regulated at its transcript level more in Chinary than in Assamica. These results suggest that Chinary could be more tolerant than Assamica.

While the toxicity of hexavalent chromium is well established, trivalent chromium is an essential nutrient involved in insulin and glucose homeostasis. To study the antioxidant effects of Cr(III)His, cDNA arrays were used to investigate the modulation of gene expression by trivalent chromium histidinate (Cr(III)His) in HaCaT human keratinocytes submitted to hydrogen peroxide (H2O2). Array was composed by a set of 81 expressed sequences tags (ESTs) essentially represented by antioxidant and DNA repair genes. HaCaT were preincubated for 24 h with 50 microM Cr(III)His and were treated with 50 muM H2O2. Total RNAs were isolated immediately or 6 h after the stress. In Cr(III)His preincubated cells, transcripts related to antioxidant family were upregulated (glutathione synthetase, heme oxygenase 2, peroxiredoxin 4). In Cr(III)His preincubated cells and exposed to H2O2, increased expressions of polymerase delta 2 and antioxidant transcripts were observed. Biochemical methods performed in parallel to measure oxidative stress in cells showed that Cr(III)His supplementation before H2O2 stress protected HaCaT from thiol groups decrease and thiobarbituric acid reactive substances increase. In summary, these results give evidence of antioxidant gene expression and antioxidant protection in HaCaT preincubated with Cr(III)His and help to explain the lack of toxicity reported for Cr(III)His.

Glutathione synthetase (GSS) deficiency is a rare disorder of glutathione metabolism with varying clinical severity. Patients may present with haemolytic anaemia alone or together with acidosis and central nervous system impairment. Diagnosis is made by clinical presentation and detection of elevated concentrations of 5-oxoproline in urine and low GSS activity in erythrocytes or cultured skin fibroblasts. Diagnosis can be confirmed by mutational analysis. Treatment consists of the correction of acidosis, blood transfusion, and supplementation with antioxidants. The most important determinants for outcome and survival in patients with GSS deficiency are early diagnosis and early initiation of treatment. The case of a newborn with GSS deficiency diagnosed by tandem mass spectrometry (MS/MS)-based newborn screening is described. After onset of clinical symptoms on the 2nd day of life, expanded newborn screening revealed normal results for all disorders included in the German screening programme; however, selective MS/MS screening revealed a >10-fold elevation of 5-oxoproline in dried blood, leading to the presumptive diagnosis of GSS deficiency by the 5th day of life. Diagnosis was later confirmed by detection of markedly reduced glutathione concentration in erythrocytes and mutational analysis of the GSS gene. Presently, GSS deficiency is not included in newborn screening programmes in Europe. As outcome depends significantly on early start of treatment, routine inclusion of this disorder in newborn screening panels should be considered.

We investigated whether N-acetylcysteine (NAC), a precursor of glutathione, could protect rabbit articular chondrocytes against nitric oxide (NO)-induced apoptosis and could prevent cartilage destruction in an experimental model of osteoarthritis (OA) in rats. Isolated chondrocytes were treated with various concentrations of NAC (0-2 mM). Apoptosis was induced by 0.75 mM sodium nitroprusside (SNP) dehydrate, which produces NO. Cell viability was assessed by MTT assay, while apoptosis was evaluated by Hoechst 33342 and TUNEL staining. Intracellular reactive oxygen species (ROS) and glutathione levels were measured, and expression of p53 and caspase-3 were determined by Western blotting. To determine whether intraarticular injection of NAC prevents cartilage destruction in vivo, cartilage samples of an OA model were subjected to H&E, Safranin O, and TUNEL staining. NAC prevented NO-induced apoptosis, ROS overproduction, p53 up-regulation, and caspase-3 activation. The protective effects of NAC were significantly blocked by buthionine sulfoximine, a glutathione synthetase inhibitor, indicating that the apoptosis-preventing activity of NAC was mediated by glutathione. Using a rat model of experimentally induced OA, we found that NAC also significantly prevented cartilage destruction and chondrocyte apoptosis in vivo. These results indicate that NAC inhibits NO-induced apoptosis of chondrocytes through glutathione in vitro, and inhibits chondrocyte apoptosis and articular cartilage degeneration in vivo.

Microorganisms can complex and sequester heavy metals, rendering them promising living factories for nanoparticle production. Glutathione (GSH) is pivotal in cadmium sulfide (CdS) nanoparticle formation in yeasts and its synthesis necessitates two enzymes: γ-glutamylcysteine synthetase (γ-GCS) and glutathione synthetase (GS). Hereby, we constructed two recombinant E. coli ABLE C strains to over-express either γ-GCS or GS and found that γ-GCS over-expression resulted in inclusion body formation and impaired cell physiology, whereas GS over-expression yielded abundant soluble proteins and barely impeded cell growth. Upon exposure of the recombinant cells to cadmium chloride and sodium sulfide, GS over-expression augmented GSH synthesis and ameliorated CdS nanoparticles formation. The resultant CdS nanoparticles resembled those from the wild-type cells in size (2–5 nm) and wurtzite structures, yet differed in dispersibility and elemental composition. The maximum particle yield attained in the recombinant E. coli was ≈2.5 times that attained in the wild-type cells and considerably exceeded that achieved in yeasts. These data implicated the potential of genetic engineering approach to enhancing CdS nanoparticle biosynthesis in bacteria. Additionally, E. coli-based biosynthesis offers a more energy-efficient and eco-friendly method as opposed to chemical processes requiring high temperature and toxic solvents. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009

We previously reported that under the stress condition caused by the addition of 2-hydroxyethyl disulfide, a thiol-specific oxidant, to growing cultures of Escherichia coli BL21(DE3), a population of stress-responsive proteins [peptidyl-prolyl cis–trans isomerase B (PpiB), bacterioferritin (Bfr), putative HTH-type transcriptional regulator yjdC (YjdC), dihydrofolate reductase (FolA), chemotaxis protein cheZ (CheZ), and glutathione synthetase (GshB)] were significantly upregulated when compared with the nonstress condition. When those stress-responsive proteins were used as fusion partners for the expression of human granulocyte colony-stimulating factor (hG-CSF), the solubility of hG-CSF was dramatically enhanced in E. coli cytoplasm, whereas almost all of the directly expressed hG-CSF were aggregated to inclusion bodies. In addition, the spectra of circular dichroism measured with the purified hG-CSF were identical to that of standard hG-CSF, implying that the synthesized hG-CSF has native conformation. These results indicate that the bacterial stress-responsive proteins could be potent fusion expression partners for aggregation-prone heterologous proteins in E. coli cytoplasm.

PURPOSE: To describe the retinal findings in 2 young adults with glutathione synthetase (GS) deficiency, an autosomal-recessive inborn error of glutathione (GSH) metabolism. DESIGN: Report of 2 cas ...

Morphologically identical transgenic mint (Mentha arvensis L.) with bacterial glutathione synthetase gene has been developed. Transformed plants were obtained by co-cultivation of leaf disks with Agrobacterium tumefaciens strain LBA 4404 harbouring a binary vector pCAMBIA-CpGS that carried E. coli glutathione synthetase (GS), β-glucuronidase as reporter gene and nptII as selective marker gene for kanamycin resistance. Using a constitutive double CaMV 35S promoter and an rbcS transit peptide, we successfully addressed CpGS to the chloroplasts through pJIT 117 vector. Preculture and the presence of AS in the co-cultivation medium played a significant role in enhancing transformation frequency. The highest transformation frequency was achieved with MS selection medium supplemented with 25% coconut water, 1.12 mg l−1 BAP, 0.2 mg l−1 NAA, 50 mg l−1 kanamycin and 125 mg l−1 cefotaxime. Robust rooting of regenerated shoots was obtained in half-strength liquid MS medium containing 0.2 mg l−1 NAA and 50 mg l−1 kanamycin. The presence and expression of transgenes in transgenics (T0) was evidenced by GUS histoenzymatic assay, PCR and RT-PCR analysis of nptII and the gene of interest, i.e., GS of putative transgenic leaves. Chromosomal integration of GS gene was confirmed by Southern blot analysis. Transgenic plants were successfully acclimatized in the greenhouse. An overall transformation frequency of 15% was achieved in approximately 3 months of time period. These results are discussed in relation to heavy metal trafficking pathways in higher plants and to the interest of using plastid expression of PCS for biotechnological applications.

To investigate the hepatotoxic potential of tienilic acid in vivo , we administered a single oral dose of tienilic acid to Sprague–Dawley rats and performed general clinicopathological examinations and hepatic gene expression analysis using Affymetrix microarrays. No change in the serum transaminases was noted at up to 1000 mg/kg, although slight elevation of the serum bile acid and bilirubin, and very mild hepatotoxic changes in morphology were observed. In contrast to the marginal clinicopathological changes, marked upregulation of the genes involved in glutathione biosynthesis [glutathione synthetase and glutamate-cysteine ligase (Gcl)], oxidative stress response [heme oxygenase-1 and NAD(P)H dehydrogenase quinone 1] and phase II drug metabolism (glutathione S -transferase and UDP glycosyltransferase 1A6) were noted after 3 or 6 h post-dosing. The hepatic reduced glutathione level decreased at 3–6 h, and then increased at 24 or 48 h, indicating that the upregulation of NF-E2-related factor 2 (Nrf2)-regulated gene and the late increase in hepatic glutathione are protective responses against the oxidative and/or electrophilic stresses caused by tienilic acid. In a subsequent experiment, tienilic acid in combination with l -buthionine-(S,R)-sulfoximine (BSO), an inhibitor of Gcl caused marked elevation of serum alanine aminotransferase (ALT) with extensive centrilobular hepatocyte necrosis, whereas BSO alone showed no hepatotoxicity. The elevation of ALT by this combination was observed at the same dose levels of tienilic acid as the upregulation of the Nrf2-regulated genes by tienilic acid alone. In conclusion, these results suggest that the impairment of glutathione biosynthesis may play a critical role in the development of tienilic acid hepatotoxicity through extensive oxidative and/or electrophilic stresses.

GSH, GSSG, vitamin E, and ascorbate were measured in 14-day cultures of chick astrocytes and neurons and compared with levels in the forebrains of chick embryos of comparable age. Activities of enzymes involved in GSH metabolism were also measured. These included -γ-glutamylcysteine synthetase, GSH synthetase, γ-glutamyl cyclotransferase, γ-glutamyltranspeptidase, glutathione transferase (GST), GSH peroxidase, and GSSG reductase. The concentration of lipid-soluble vitamin E in the cultured neurons was found to be comparable with that in the forebrain. On the other hand, the concentration of vitamin E in the astrocytes was significantly greater in the cultured astrocytes than in the neurons, suggesting that the astrocytes are able to accumulate exogenous vitamin E more extensively than neurons. The concentrations of major fatty acids were higher in the cell membranes of cultured neurons than those in the astrocytes. Ascorbate was not detected in cultured cells although the chick forebrains contained appreciable levels of this antioxidant. GSH, total glutathione (i.e., GSH and GSSG), and GST activity were much higher in cultured astrocytes than in neurons. γ-Glutamylcysteine synthetase activity was higher in the cultured astrocytes than in the cultured neurons. GSH reductase and GSH peroxidase activities were roughly comparable in cultured astrocytes and neurons. The high levels of GSH and GST in cultured astrocytes appears to reflect the situation in vivo. The data suggest that astrocytes are resistant to reactive oxygen species (and potentially toxic xenobiotics) and may play a protective role in the brain. Because enzymes of GSH metabolism are generally well represented in cultured astrocytes and neurons these cells may be ideally suited as probes for manipulating GSH levels in neural tissues in vitro. Cultured astrocytes and neurons should be amenable to the study of the effects of various metabolic insults on the GSH system. Such studies may provide insights into the design of therapeutic strategies to combat oxidative and xenobiotic stresses.

The bifunctional trypanothione synthetase-amidase catalyzes biosynthesis and hydrolysis of the glutathione-spermidine adduct trypanothione, the principal intracellular thiol-redox metabolite in parasitic trypanosomatids. These parasites are unique with regard to their reliance on trypanothione to determine intracellular thiol-redox balance in defense against oxidative and chemical stress and to regulate polyamine levels. Enzymes involved in trypanothione biosynthesis provide essential biological activities, and those absent from humans or for which orthologues are sufficiently distinct are attractive targets to underpin anti-parasitic drug discovery. The structure of Leishmania major trypanothione synthetase-amidase, determined in three crystal forms, reveals two catalytic domains. The N-terminal domain, a cysteine, histidine-dependent amidohydrolase/peptidase amidase, is a papain-like cysteine protease, and the C-terminal synthetase domain displays an ATP-grasp family fold common to C:N ligases. Modeling of substrates into each active site provides insight into the specificity and reactivity of this unusual enzyme, which is able to catalyze four reactions. The domain orientation is distinct from that observed in a related bacterial glutathionylspermidine synthetase. In trypanothione synthetase-amidase, the interactions formed by the C terminus, binding in and restricting access to the amidase active site, suggest that the balance of ligation and hydrolytic activity is directly influenced by the alignment of the domains with respect to each other and implicate conformational changes with amidase activity. The potential inhibitory role of the C terminus provides a mechanism to control relative levels of the critical metabolites, trypanothione, glutathionylspermidine, and spermidine in Leishmania.

We report on gene silencing of glutathione synthetase (GSHS) that reduces reduced glutathione (GSH) content in somatic embryos of Camellia sinensis L. Using degenerate primers with cDNA of Camellia sinensis, a 457 bp GSHS gene fragment was cloned through polymerase chain reaction. This fragment was used in making ihpRNA. For this it was cloned in sense at AscI and SwaI and in anti-sense at Bam HI and XbaI restriction sites of pFGC5941 that has chalcone synthase (Chs) intron between SwaI and BamHI restriction sites. Resultant RNAi construct was used for C. sinensis somatic embryos transformation through Agrobacterium. After 11, 13 and 15 d of transformation, embryo GSHS transcript levels and GSH content decreased to a great extent which documented the feasibility of RNAi based gene silencing in C. sinensis.

Intracellular defence mechanisms against oxidative stress may play an important role in the progression of liver diseases, including cholangiopathies. The multifunctional anti-apoptotic hepatocyte growth factor (HGF) has been suggested to have antioxidant functions. The effect of HGF upon cell viability, the generation of ROS, the expression of genes that play a role in ROS defence, and the activation of caspase-3 were measured in bile duct epithelial (BDE) cells in the presence or absence of H(2)O(2). HGF reduced H(2)O(2)-induced loss of viability, diminished H(2)O(2)-mediated ROS generation and abrogated H(2)O(2)-triggered changes in GSH/GSSG ratio. Furthermore, HGF increased the gene-expression of gamma-glutamylcysteine synthetase (GCLC) and glutathione reductase (GSR), while no effect was seen upon the gene-expression of superoxide dismutase 1 (SOD1), catalase (CAT), glutathione peroxidase (GPX1), and glutathione synthetase (GSR). Finally, HGF diminished the proteolytical activation of the key mediator of apoptosis (caspase-3) after H(2)O(2) exposure. Together, HGF may improve viability in bile duct epithelia cells after H(2)O(2) induced toxicity by proliferation, strengthening the intrinsic antioxidant defences, and/or by an attenuation of apoptosis. These in vitro results support the evaluation of HGF as antioxidative factor in hepatobiliary pathologies.

In the last century mercury levels in the global environment have tripled as a result of increased pollution from industrial, occupational, medicinal and domestic uses \cite{BaMe03}. Glutathione is known to be the main agent responsible for the excretion of mercury (we refer to \cite{Thim05}, \cite{ZalBar99} and \cite{Lyn02}). It has also been shown that mercury inhibits glutathione synthetase (an enzyme acting in the synthesization of Glutathione), therefore leading to decreased glutathione levels (we refer to \cite{Thim05}, \cite{GeGe05}, \cite{GeGe06} and \cite{RDeth04}). Mercury also interferes with the production of heme in the porphyrin pathway \cite{WoMaEc93}. Heme is needed for biological energy production and ability to detox organic toxins via the P450 enzymes \cite{Boy06}. The purpose of this paper is to show that body's response to mercury exposure is hysteretic, i.e. when this feedback of mercury on its main detoxifying agents is strong enough then mercury body burden has two points of equilibrium: one with normal abilities to detoxify and low levels of mercury and one with inhibited abilities to detoxify and high levels of mercury. Furthermore, a small increase of body's mercury burden may not be sufficient to trigger observable neurotoxic effects but it may be sufficient to act as a switch leading to an accumulation of mercury in the body through environmental exposure until its toxicity becomes manifest., Comment: To appear in Journal of Biological Systems

Treatment of 10 days old maize seedlings with metribuzin and pretilachlor near the recommended field-dose resulted in differential reductions in shoot fresh and dry weights during the following 16 days. Metribuzin showed great and consistent reductions, however, the reduction induced by pretilachlor, mostly nullified by the end of the experiment. Moreover, there were differential accumulations of lipid peroxides, carbonyl groups and H2O2 in maize leaves; metribuzin caused the greatest accumulation. Meanwhile, levels of thiol forms and reduced glutathione (GSH) were much more induced by pretilachlor than metribuzin; the contrary was true regarding oxidized glutathione (GSSG). The ratio of GSH/GSSG was highest following pretilachlor treatment and least by metribuzin. On the other hand, activities of glutathione-S-transferases (GSTs, EC 2.5.1.18), γ-glutamyl-cysteine synthetase (γ-GCS, EC 6.3.2.2), glutathione synthetase (GS, EC 6.3.2.3), glutathione peroxidase (GPX, EC 1.15.1.1) and glutathione reductase (GR, EC 1.6.4.2) were more enhanced in maize leaves by pretilachlor than metribuzin. These findings suggest the occurrence of an oxidative stress differentially induced in maize by the herbicides, a state that was most pronounced with metribuzin. Pretilachlor was concluded to be the least phytotoxic to maize, while metribuzin was the most, this differential tolerance seemed to be related to the induction of GSH and GSH-associated enzymes.

Loss of intracellular neuronal glutathione (GSH) is an important feature of neurodegenerative disorders including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. The consequences of GSH depletion include increased oxidative damage to proteins, lipids, and DNA and subsequent cytotoxic effects. GSH is also an important modulator of cellular copper (Cu) homeostasis and altered Cu metabolism is central to the pathology of several neurodegenerative diseases. The cytotoxic effects of Cu in cells depleted of GSH are not well understood. We have previously reported that depletion of neuronal GSH levels results in cell death from trace levels of extracellular Cu due to elevated Cu(I)-mediated free radical production. In this study we further examined the molecular pathway of trace Cu toxicity in neurons and fibroblasts depleted of GSH. Treatment of primary cortical neurons or 3T3 fibroblasts with the glutathione synthetase inhibitor buthionine sulfoximine resulted in substantial loss of intracellular GSH and increased cytotoxicity. We found that both neurons and fibroblasts revealed increased expression and activation of p53 after depletion of GSH. The increased p53 activity was induced by extracellular trace Cu. Furthermore, we showed that in GSH-depleted cells, Cu induced an increase in oxidative stress resulting in DNA damage and activation of p53-dependent cell death. These findings may have important implications for neurodegenerative disorders that involve GSH depletion and aberrant Cu metabolism.