GSH Biosynthesis Research Articles

The glutathione synthesis may be regulated by cadmium-induced endogenous ethylene in Lycium chinense, and overexpression of an ethylene responsive transcription factor gene enhances tolerance to cadmium stress in tobacco

Glutathione (GSH) plays a pivotal role in heavy metal detoxification. Ethylene is one of the important plant hormones, which plays a critical role in triggering plant responses to different stresses such as cadmium (Cd) stress. Ethylene responsive transcription factor (ERF) belongs to one of the largest plant transcription factor families. ERF is known to be induced by ethylene and thus regulate multiple stress responses through the activation of stress-related genes. Until now, little has been done to explore the relationship among the accumulation of endogenous ethylene, ERF transcript levels and the GSH content in plants under Cd treatment and we will investigate these link. In this study, the gene transcript level of LchERF, LchGSH1 (gene responsible for the first-step GSH biosynthesis) and LchGSHS (gene responsible for the second-step GSH biosynthesis), endogenous ethylene accumulation, GSH content and Cd concentration in Lycium chinense with or without Cd stress treatment were studied. Furthermore, the transgenic tobacco expressing 35S::LchERF which grown under Cd stress condition was also investigated in this study. Our results showed that endogenous ethylene, LchERF, LchGSH1 and LchGSHS gene expression and GSH content can be induced by Cd treatment in L. chinense, however, reduced by co-treatment with 2-aminoethoxyvinlglycine (AVG), an inhibitor of ethylene biosynthesis. The transgenic tobacco expressing 35S::LchERF showed greater tolerance to Cd stress than non-transgenic plants. The expression of NtGSH1 and NtGSHS genes was increased in transgenic tobacco plants compared with non-transgenic plants, indicating that LchERF is associated with the expression level of GSH synthesis related genes in tobacco. Evidence was presented here that under Cd stress, GSH accumulation occurred at least partially via enhanced LchERF gene expression and the ethylene signal transduction pathways might be involved in this accumulation.

Vitamin D Inhibits ROS, ICAM‐1, and Monocyte Adhesion in High Glucose and Acetoacetate Treated Endothelial Cells by Upregulating GSH

The incidence of developing microvascular dysfunction is significantly higher in type 1 diabetic (T1D) patients. Monocyte adhesion to the endothelium plays a central role in the progression of endothelial dysfunction. This study examines whether vitamin D (VD) supplementation is beneficial in preventing endothelial dysfunction in diabetes. Human umbilical vein endothelial cells (HUVEC) were treated with 1, 25‐(OH)2‐D3 (0‐25 nM, 24 h) and later exposed to the ketone body acetoacetate (AA, 4 mM) or high glucose (HG, 25 mM) for 24 h. Treatment with AA or HG causes an increase in ROS, ICAM‐1 expression, and monocyte adhesion, along with a decrease in GSH levels in HUVEC. VD supplementation (25 nM) significantly lowered ROS (41%, p<0.05), ICAM‐1 (73%, p=0.05) and monocyte adhesion (37.5%, p=0.05) in AA treated HUVEC. VD had a similar beneficial effect on HG treated cells. Interestingly, we also saw an upregulation in GSH levels following treatment with VD. We hypothesize that the beneficial effect of VD is mediated by the upregulation of GSH. Glutamyl cysteine ligase (GCLC) catalyzes GSH biosynthesis. Knocking down GCLC using siRNA reduced GSH levels as well as abolished the beneficial effect of VD on ketone‐ or HG‐induced ROS, ICAM‐1, and monocyte adhesion. This suggests that VD mediates its beneficial effects via the upregulation of GCLC. This study provides the biochemical mechanism through which VD can lower ROS, ICAM‐1 expression, monocyte–endothelial adhesion, and thereby lower the risk of endothelial dysfunction in diabetes. Supported by the NIH, the Malcolm Feist chair in Diabetes, and a fellowship from the Malcolm Feist Cardiovascular Research Endowment, LSUHSC‐Shreveport.

Aging alters circadian regulation of redox in Drosophila

Circadian coordination of metabolism, physiology, and neural functions contributes to healthy aging and disease prevention. Clock genes govern the daily rhythmic expression of target genes whose activities underlie such broad physiological parameters as maintenance of redox homeostasis. Previously, we reported that glutathione (GSH) biosynthesis is controlled by the circadian system via effects of the clock genes on expression of the catalytic (Gclc) and modulatory (Gclm) subunits comprising the glutamate cysteine ligase (GCL) holoenzyme. The objective of this study was to determine whether and how aging, which leads to weakened circadian oscillations, affects the daily profiles of redox-active biomolecules. We found that fly aging is associated with altered profiles of Gclc and Gclm expression at both the mRNA and protein levels. Analysis of free aminothiols and GCL activity revealed that aging abolishes daily oscillations in GSH levels and alters the activity of glutathione biosynthetic pathways. Unlike GSH, its precursors and products of catabolism, methionine, cysteine and cysteinyl-glycine, were not rhythmic in young or old flies, while rhythms of the glutathione oxidation product, GSSG, were detectable. We conclude that the temporal regulation of GSH biosynthesis is altered in the aging organism and that age-related loss of circadian modulation of pathways involved in glutathione production is likely to impair temporal redox homeostasis.

Functional interplay between glutathione and hydrogen sulfide in regulation of thiol cascade during arsenate tolerance of common bean (Phaseolus vulgaris L.) genotypes.

Changes in expressions of up- and downstream thiol cascade were studied in leaves of Phaseolus vulgaris L. cv. VL-63 and its mutant, pvsod1 (deficient in superoxide dismutase activity) under 50 μM sodium arsenate (As), As + l-buthionine-sulfoximine (BSO) and As + BSO + Sodium hydrosulfide (NaHS)-treatments for 10 days. Main objective was to investigate the functional relationship between hydrogen sulfide (H2S) and glutathione (GSH) in regulation of sulfate transporters and cysteine metabolisms as up-stream thiol components and GSH, phytochelatins (PCs) and antioxidant defense response as downstream cascade under As-exposure. As treatment alone initiated coordinated inductions of sulfate transport, biosynthesis of cysteine, GSH, and PCs, and GSH-mediated antioxidant defense in the pvsod1 mutant. At As + BSO, GSH synthesis was blocked, resulting in significantly low GSH redox pool and steep decline in GSH-dependent antioxidant capacity of both the genotypes. However, unlike VL-63, cysteine-degradation pathway was induced in pvsod1 mutant, resulting in significant accumulation of endogenous H2S. The H2S-surge in the pvsod1 mutant stimulated ascorbate-dependent antioxidant defense and catalases and regulated O-acetylserine (thiol)lyase activity, preventing overaccumulation of H2O2 and free cysteine, respectively. No As-induced oxidative stress symptom was observed in the mutant. This trend was maintained at As + BSO + NaHS treatment, also. In contrast, failure to induce entire cascade from sulfate transport to downstream antioxidant defense led to onset of As-induced oxidative damage in VL-63 plant. Results revealed dual roles of H2S as (a) stimulator of GSH-independent antioxidant defense and (b) regulator of cysteine homeostasis through its metabolic diversion during As-exposure and blockage of GSH biosynthesis.

Succinobucol, a Lipid-Lowering Drug, Protects Against 3-Nitropropionic Acid-Induced Mitochondrial Dysfunction and Oxidative Stress in SH-SY5Y Cells via Upregulation of Glutathione Levels and Glutamate Cysteine Ligase Activity.

Succinobucol (succinyl ester of probucol) is a lipid-lowering compound with anti-inflammatory and antioxidant properties. Recent experimental evidence has highlighted the potential neuroprotective effects of succinobucol. In the present study, cultured neuroblastoma (SH-SY5Y) cells were used to investigate mechanisms mediating the potential protective effect of succinobucol against mitochondrial metabolic impairment and oxidative stress induced by 3-nitropropionic acid (3-NP), a succinate dehydrogenase inhibitor that has been used in experimental models of the Huntington disease (HD). 3-NP decreased cellular viability after 24 h of incubation. This decline in cellular viability was preceded by (i) reduced mitochondrial complex II activity, (ii) increased reactive species generation, (iii) decreased mitochondrial membrane potential (ΔΨm), and (iv) diminished glutathione (GSH) levels. Succinobucol pretreatment (6 days) significantly prevented 3-NP-induced loss of cellular viability, generation of reactive oxygen species, and decrease of ΔΨm. However, succinobucol pretreatment did not protect against 3-NP-induced inhibition of mitochondrial complex II activity, pointing to the mitigation of secondary events resultant from mitochondrial complex II inhibition. Succinobucol pretreatment (6 days) significantly increased (50 %) the levels of GSH in SH-SY5Y cells, and this event was paralleled by significant increases in glutamate cysteine ligase messenger RNA (mRNA) expression and activity (GCL; the first enzyme in the GSH biosynthesis). The present findings are the first to show that succinobucol increases GSH levels via upregulation of GCL activity (possibly through the activation of the nuclear (erythroid-derived 2)-related factor (Nrf2)/antioxidant response element (ARE) pathway), displaying protective effects against mitochondrial dysfunction-derived oxidative stress.

Cabergoline protects dopaminergic neurons against rotenone-induced cell death in primary mesencephalic cell culture.

In the present study, primary mesencephalic cell cultures prepared from embryonic mouse mesencephala were used to investigate the neuroprotective effect of cabergoline, an ergoline D2 receptor agonist, against the pesticide and neurotoxin rotenone relevant to Parkinson disease (PD). Treatment of cultures with cabergoline alone significantly increased the number of tyrosine hydroxylase immunoreactive (THir) neurons and reduced the release of lactate dehydrogenase (LDH) into the culture medium compared to untreated controls. Against rotenone toxicity, cabergoline significantly rescued degenerating THir neurons, reduced the release of LDH into the culture medium and improved the morphology of surviving THir neurons. The neuroprotective effects afforded by cabergoline were independent of dopaminergic stimulation as blocking of dopamine receptors by the dopamine receptor antagonist sulpiride did not prevent them. Furthermore, rotenone-induced formation of reactive oxygen species (ROS) was significantly reduced by cabergoline. Although cabergoline increased the glutathione (GSH) content in the culture, the protective effect for dopaminergic neurons seemed not to be predominantly mediated by increasing GSH, as depletion of GSH by L-buthionine-(S,R)-sulfoximine (BSO), a GSH biosynthesis inhibitor, did not prevent cabergoline-mediated neuroprotection of THir neurons in rotenone-treated cultures. Moreover, cabergoline significantly increased the ATP/protein ratio in primary mesencephalic cell cultures when added alone or prior to rotenone treatment. These results indicate a neuroprotective effect of cabergoline for dopaminergic neurons against rotenone toxicity. This effect was independent of dopamine receptor stimulation and was at least partially mediated by reducing ROS production and increasing the ATP/protein ratio.

Protection of HepG2 cells against acrolein toxicity by 2-cyano-3,12-dioxooleana-1,9-dien-28-imidazolide via glutathione-mediated mechanism.

Acrolein is an environmental toxicant, mainly found in smoke released from incomplete combustion of organic matter. Several studies showed that exposure to acrolein can lead to liver damage. The mechanisms involved in acrolein-induced hepatocellular toxicity, however, are not completely understood. This study examined the cytotoxic mechanisms of acrolein on HepG2 cells. Acrolein at pathophysiological concentrations was shown to cause apoptotic cell death and an increase in levels of protein carbonyl and thiobarbituric acid reactive acid substances. Acrolein also rapidly depleted intracellular glutathione (GSH), GSH-linked glutathione-S-transferases, and aldose reductase, three critical cellular defenses that detoxify reactive aldehydes. Results further showed that depletion of cellular GSH by acrolein preceded the loss of cell viability. To further determine the role of cellular GSH in acrolein-mediated cytotoxicity, buthionine sulfoximine (BSO) was used to inhibit cellular GSH biosynthesis. It was observed that depletion of cellular GSH by BSO led to a marked potentiation of acrolein-mediated cytotoxicity in HepG2 cells. To further assess the contribution of these events to acrolein-induced cytotoxicity, triterpenoid compound 2-cyano-3,12-dioxooleana-1,9-dien-28-imidazolide (CDDO-Im) was used for induction of GSH. Induction of GSH by CDDO-Im afforded cytoprotection against acrolein toxicity in HepG2 cells. Furthermore, BSO significantly inhibited CDDO-Im-mediated induction in cellular GSH levels and also reversed cytoprotective effects of CDDO-Im in HepG2 cells. These results suggest that GSH is a predominant mechanism underlying acrolein-induced cytotoxicity as well as CDDO-Im-mediated cytoprotection. This study may provide understanding on the molecular action of acrolein which may be important to develop novel strategies for the prevention of acrolein-mediated toxicity.

Analysis of cadmium translocation, partitioning and tolerance in six barley (Hordeum vulgare L.) cultivars as a function of thiol metabolism

Six barley cultivars widely differing for cadmium (Cd) tolerance, partitioning, and translocation were analyzed in relation to their thiol metabolism. Results indicated that Cd tolerance was not clearly related to the total amount of Cd absorbed by plants, resulting instead closely dependent on the capacity of the cultivars to trap the metal into the roots. Such behaviors suggested the existence of root mechanisms preserving shoots from Cd-induced oxidative damages, as indicated by the analysis of thiobarbituric acid-reactive substances—diagnostic indicators of oxidative stress—whose increases in the shoots were negatively related to Cd root retention and tolerance. Cd exposure differentially affected glutathione (GSH) and phytochelatin (PC) levels in the tissues of each barley cultivar. The capacity to produce PCs appeared as a specific characteristic of each barley cultivar, since it did not depend on Cd concentration in the roots and resulted negatively related to the concentration of the metal in the shoots, indicating the existence of a cultivar-specific interference of Cd on GSH biosynthesis, as confirmed by the existence of close positive linear relationships between the effect of Cd on GSH levels and PC accumulation in both roots and shoots. The six barley cultivars also differed for their capacity to load Cd ions into the xylem, which was negatively related to PC content in the roots. Taken as a whole, these data indicated that the different capacity of each cultivar to maintain GSH homeostasis under Cd stress may strongly affect PC accumulation and, thus, Cd tolerance and translocation.

Skin resistance to oxidative stress induced by resveratrol: From Nrf2 activation to GSH biosynthesis

Skin is particularly exposed to oxidative stress, either from environmental insults such as sunlight or pollution or as a consequence of specific impairments in antioxidant status resulting from pathologies or aging. Traditionally, antioxidant products are exogenously provided to neutralize pro-oxidant species. However, another approach based on stimulation of endogenous antioxidant defense pathways is more original. Resveratrol (RSV) was reported to display such a behavior in various tissues, but data about the mechanisms of action in skin are scarce. We show here that, in primary culture of normal human keratinocytes (NHKs) or in full-thickness reconstructed human skin, RSV activated the Nrf2 pathway at nontoxic doses, from 20 µM up to 100µM. Among the Nrf2 downstream genes, glutamylcysteinyl ligase and glutathione peroxidase-2 were induced at the mRNA and protein levels. In parallel, a significant increase in glutathione content, assessed by LC/MS analysis, was observed in both models. Nrf2 gene silencing experiments performed in NHKs confirmed that Nrf2 was involved in RSV-induced modulation of cellular antioxidant status, in part by increasing cellular glutathione content. Finally, improvement of endogenous defenses induced in RSV-pretreated reconstructed skin ensured protection against the toxic oxidative effects of cumene hydroperoxide (CHP). In fact after RSV pretreatment, in response to CHP stress, glutathione content did not decrease as in unprotected samples. Cellular alterations at the dermal–epidermal junction were clearly prevented. Together, these complementary experiments demonstrated the beneficial effects of RSV on skin, beyond its direct antioxidant properties, by upregulation of a cutaneous endogenous antioxidant pathway.

Selenium inhibits root elongation by repressing the generation of endogenous hydrogen sulfide in Brassica rapa.

Selenium (Se) has been becoming an emerging pollutant causing severe phytotoxicity, which the biochemical mechanism is rarely known. Although hydrogen sulfide (H2S) has been suggested as an important exogenous regulator modulating plant physiological adaptions in response to heavy metal stress, whether and how the endogenous H2S regulates Se-induce phytotoxicity remains unclear. In this work, a self-developed specific fluorescent probe (WSP-1) was applied to track endogenous H2S in situ in the roots of Brassica rapa under Se(IV) stress. Se(IV)-induced root growth stunt was closely correlated with the inhibition of endogenous H2S generation in root tips. Se(IV) stress dampened the expression of most LCD and DCD homologues in the roots of B. rapa. By using various specific fluorescent probes for bio-imaging root tips in situ, we found that the increase in endogenous H2S by the application of H2S donor NaHS could significantly alleviate Se(IV)-induced reactive oxygen species (ROS) over-accumulation, oxidative impairment, and cell death in root tips, which further resulted in the recovery of root growth under Se(IV) stress. However, dampening the endogenous H2S could block the alleviated effect of NaHS on Se(IV)-induced phytotoxicity. Finally, the increase in endogenous H2S resulted in the enhancement of glutathione (GSH) in Se(IV)-treated roots, which may share the similar molecular mechanism for the dominant role of H2S in removing ROS by activating GSH biosynthesis in mammals. Altogether, these data provide the first direct evidences confirming the pivotal role of endogenous H2S in modulating Se(IV)-induced phytotoxicity in roots.

Effects of exogenous glutathione and cysteine on growth, lead accumulation, and tolerance of Iris lactea var. chinensis

Effects of exogenous reduced glutathione (GSH) and cysteine (Cys) on growth, lead (Pb) accumulation, and nonprotein thiol (NPT) contents of Iris lactea var. chinensis under 100 and 500 mg L(-1) Pb stress were studied. Our results showed that 500 mg L(-1) Pb stress caused a dramatical decline in fresh weights, while the reduction of aboveground biomass was alleviated by exogenous GSH and Cys even though keeping higher Pb contents in roots and shoots. Exogenous GSH and Cys could enhance Pb accumulation in the shoots and roots compared with single Pb treatment. The promoting effect of GSH to Pb accumulation was larger than the effect of Cys, and the Pb contents in the shoots and roots treated with 500 mg L(-1) Pb + GSH reached 1,712 and 14,603 mg kg(-1), about 4.19 and 2.78 times of single 500 mg L(-1) Pb treatment, respectively. Microscopic imaging of Pb in roots and leaves showed that higher intensive fluorescence was observed in cell wall of root epidermis, stele, vascular tissues of the roots, and sclerenchyma cells of leaves treated with 500 mg L(-1) Pb + GSH and treated with 500 mg L(-1) Pb + Cys. Exogenous GSH had an apparent promoting effect on root and shoot GSH synthesis, while exogenous Cys reduced the synthesis of cellular GSH in shoot and increased Cys contents. Pb only induced the synthesis of phytochelatin (PC)2 in roots, and the PC2 content declined in GSH- and Cys-treated plant roots. These results suggested that GSH synthesis was a more effective approach to improve Pb accumulation and translocation of I. lactea var. chinensis. Further analysis of protein expression in plants by exogenous GSH and buthionine sulfoximine (BSO) application showed that the proteins regulated by GSH and BSO may constitute various enzymes involved in GSH biosynthesis and play certain roles in Pb accumulation and tolerance of I. lactea var. chinensis.

Abstract 215: The Redox Activation of DUSP4 by N-Acetyl Cysteine Protects Endothelial Cells and the Myocardium against Oxidative Damage

Background: Redox imbalance is the primary cause for endothelial dysfunction (ED), obstructed blood flow, and subsequent heart attack and failure. Under oxidant stress, many critical proteins regulating endothelial function undergo oxidative modifications that lead to ED. Cellular levels of glutathione (GSH), the primary reducing source, can significantly regulate cell function via reversible protein thiol modification. N-Acetyl cysteine (NAC), a precursor for GSH biosynthesis, is beneficial for many vascular diseases; however, the detailed mechanism of these benefits is still not clear. Methods: We employed EPR spin-trapping, HPLC, fluorescent microscopy, immunoblotting, and qPCR of both in vitro and ex vivo experiments using either cultured cells or the Langendorff heart preparation. Results: NAC treatment increases NO generation from endothelial cells, as well as the enzyme and cofactor responsible for its production, ie eNOS and BH4. Interestingly, NAC treatment also increased the expression of DUSP4, an inducible nuclear dual-specificity phosphatase implicated in cardiovascular function. We hereby establish that DUSP4 redox modulates two important kinases (p38 and ERK1/2) of MAPK signaling pathways and provides protection against Cd2+-induced ROS damage as well as hypoxia-reoxygenation insult to endothelial cells. Furthermore, a four week oral NAC pre-treatment promotes DUSP4 both protein and mRNA expression in the rat myocardium and renders the heart less susceptible to ex vivo ischemia-reperfusion injury. Protein expression profiles in the myocardium closely mimic those observed with cultured endothelial cells. The infarct size of NAC-treated hearts is significantly reduced. The myocardial rate pressure product is much improved above vehicle treated rats. Conclusion: NAC serves as a direct antioxidant and as a regulator of transcription and translation of DUSP4, modulating the activity of its downstream effectors: ERK1/2 and p38, and thus protects against ROS-induced damage both in vitro and ex vivo. As such, NAC-derived drugs can provide a novel therapy to oxidant-induced diseases via the specific up-regulation of protective proteins such as DUSP4 and eNOS.

Epigenetic modifications of Nrf2-mediated glutamate–cysteine ligase: implications for the development of diabetic retinopathy and the metabolic memory phenomenon associated with its continued progression

Diabetes increases oxidative stress in the retina and decreases the levels of the intracellular antioxidant glutathione (GSH). The transcriptional factor Nrf2 regulates the expression of Gclc, the enzyme important in the biosynthesis of GSH, and in diabetes the binding of Nrf2 at the antioxidant response element region 4 (ARE4) is decreased. Our aim was to investigate the role of epigenetic modifications in the decreased Nrf2 binding at Gclc-ARE4 in the development of diabetic retinopathy and in the metabolic memory associated with its continued progression. The effect of hyperglycemia on H3K4 methylation in Nrf2 binding at Gclc-ARE4 was investigated by chromatin immunoprecipitation in the rat retina and was confirmed in retinal endothelial cells in which histone demethylase (LSD1) was manipulated. The role of histone methylation at Gclc-ARE4 in the metabolic memory was examined in rats maintained under poor control for 3 months followed by good control (GC) for 3 months. Although H3K4me2 at Gclc-ARE4 was increased in diabetes, H3K4me3 and H3K4me1 were decreased. LSD1 siRNA abrogated the glucose-induced decrease in H3K4me1 at Gclc-ARE4 and ameliorated decreases in Nrf2 binding at Gclc-ARE4 and Gclc transcripts. Reestablishment of GC failed to provide any benefits to histone methylation, and Nrf2 binding activity remained compromised. Thus, in diabetic retinopathy, histone methylation at Gclc-ARE4 plays an important role in regulating the Nrf2–Gclc–GSH cascade. Targeting histone methylation could help inhibit/slow down this blinding disease.

Redox activation of DUSP4 by N-acetylcysteine protects endothelial cells from Cd2+-induced apoptosis

Redox imbalance is a primary cause of endothelial dysfunction (ED). Under oxidant stress, many critical proteins regulating endothelial function undergo oxidative modifications that lead to ED. Cellular levels of glutathione (GSH), the primary reducing source in cells, can significantly regulate cell function via reversible protein thiol modification. N-acetylcysteine (NAC), a precursor for GSH biosynthesis, is beneficial for many vascular diseases; however, the detailed mechanism of these benefits is still not clear. From HPLC analysis, NAC significantly increases both cellular GSH and tetrahydrobiopterin levels. Immunoblotting of endothelial NO synthase (eNOS) and DUSP4, a dual-specificity phosphatase with a cysteine as its active residue, revealed that both enzymes are upregulated by NAC. EPR spin trapping further demonstrated that NAC enhances NO generation from cells. Long-term exposure to Cd2+ contributes to DUSP4 degradation and the uncontrolled activation of p38 and ERK1/2, leading to apoptosis. Treatment with NAC prevents DUSP4 degradation and protects cells against Cd2+-induced apoptosis. Moreover, the increased DUSP4 expression can redox-regulate the p38 and ERK1/2 pathways from hyperactivation, providing a survival mechanism against the toxicity of Cd2+. DUSP4 gene knockdown further supports the hypothesis that DUSP4 is an antioxidant gene, critical in the modulation of eNOS expression, and thus protects against Cd2+-induced stress. Depletion of intracellular GSH by buthionine sulfoximine makes cells more susceptible to Cd2+-induced apoptosis. Pretreatment with NAC prevents p38 overactivation and thus protects the endothelium from this oxidative stress. Therefore, the identification of DUSP4 activation by NAC provides a novel target for future drug design.

An interaction between glutathione and the capsid is required for the morphogenesis of C-cluster enteroviruses.

Glutathione (GSH) is the most abundant cellular thiol playing an essential role in preserving a reduced cellular environment. Cellular GSH levels can be efficiently reduced by the GSH biosynthesis inhibitor, L-buthionine sulfoximine (BSO). The aim of our study was to determine the role of GSH in the growth of two C-cluster enteroviruses, poliovirus type 1 (PV1) and coxsackievirus A20 (CAV20). Our results show that the growth of both PV1 and CAV20 is strongly inhibited by BSO and can be partially reversed by the addition of GSH. BSO has no effect on viral protein synthesis or RNA replication but it strikingly reduces the accumulation of 14S pentamers in infected cells. GSH-pull down assays show that GSH directly interacts with capsid precursors and mature virus made in the absence of BSO whereas capsid precursors produced under GSH-depletion do not bind to GSH. In particular, the loss of binding of GSH may debilitate the stability of 14S pentamers, resulting in their failure to assemble into mature virus. Immunofluorescence cell imaging demonstrated that GSH-depletion did not affect the localization of viral capsid proteins to the replication complex. PV1 BSO resistant (BSOr) mutants evolved readily during passaging of the virus in the presence of BSO. Structural analyses revealed that the BSOr mutations, mapping to VP1 and VP3 capsid proteins, are primarily located at protomer/protomer interfaces. BSOr mutations might, in place of GSH, aid the stability of 14S particles that is required for virion maturation. Our observation that BSOr mutants are more heat resistant and need less GSH than wt virus to be protected from heat inactivation suggests that they possess a more stable capsid. We propose that the role of GSH during enterovirus morphogenesis is to stabilize capsid structures by direct interaction with capsid proteins both during and after the formation of mature virus particles.

Reduced Expression of miRNA-27a Modulates Cisplatin Resistance in Bladder Cancer by Targeting the Cystine/Glutamate Exchanger SLC7A11

Resistance to cisplatin-based chemotherapy is a major obstacle to bladder cancer treatment. We aimed to identify microRNAs (miRNA) that are dysregulated in cisplatin-resistant disease, ascertain how these contribute to a drug-resistant phenotype, and how this resistance might be overcome. miRNA expression in paired cisplatin-resistant and -sensitive cell lines was measured. Dysregulated miRNAs were further studied for their ability to mediate resistance. The nature of the cisplatin-resistant phenotype was established by measurement of cisplatin/DNA adducts and intracellular glutathione (GSH). Candidate miRNAs were examined for their ability to (i) mediate resistance and (ii) alter the expression of a candidate target protein (SLC7A11); direct regulation of SLC7A11 was confirmed using a luciferase assay. SLC7A11 protein and mRNA, and miRNA-27a were quantified in patient tumor material. A panel of miRNAs were found to be dysregulated in cisplatin-resistant cells. miRNA-27a was found to target the cystine/glutamate exchanger SLC7A11 and to contribute to cisplatin resistance through modulation of GSH biosynthesis. In patients, SLC7A11 expression was inversely related to miRNA-27a expression, and those tumors with high mRNA expression or high membrane staining for SLC7A11 experienced poorer clinical outcomes. Resistant cell lines were resensitized by restoring miRNA-27a expression or reducing SLC7A11 activity with siRNA or with sulfasalazine. Our findings indicate that miRNA-27a negatively regulates SLC7A11 in cisplatin-resistant bladder cancer, and shows promise as a marker for patients likely to benefit from cisplatin-based chemotherapy. SLC7A11 inhibition with sulfasalazine may be a promising therapeutic approach to the treatment of cisplatin-resistant disease.

Oleanolic acid enhances the radiosensitivity of tumor cells under mimetic hypoxia through the reduction in intracellular GSH content and HIF-1α expression

We previously found that oleanolic acid (OA), a naturally pentacyclic triterpenoid, enhances the radiosensitizing effect on tumor cells. However, it is unclear whether or not OA enhances the radiosensitivity of hypoxic cells. Therefore, the aim of the present study was to further observe the influence of OA on hypoxic tumor cells, and the relative mechanism was also investigated. The radiosensitivity of rat glioma C6 cells and human lung cancer A549 cells with different treatments, under mimetic hypoxia, was evaluated by clonogenic assay. A micronucleus (MN) test, meanwhile, was utilized to observe the alteration in intracellular DNA damage. For determining the mechanism involved in the OA influence on the radiosensitivity of hypoxic cells, we determined the levels of intracellular reduced glutathione (GSH) using the glutathione reductase/5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) recycling assay. Simultaneously, the activities of γ-glutamylcysteine synthetase (γ-GCS) and GSH synthase (GSS), both enzymes for GSH synthesis, were tested using appropriate methods. Due to the involvement of hypoxia inducible factor-1α (HIF-1α) in the resistence of hypoxic cells to radiation damage, its levels were also observed by western blot method. The results from this study demonstrated that the clonogenic growth of irradiated cells was increased under mimetic hypoxia while the refractory effect of hypoxic cells to radiation was decreased following OA treatment. Moreover, the (MN) frequencies in the hypoxic cells treated with OA were augmented after irradiation compared with the cells without OA treatment. In the subsequent experiment, OA significantly reduced the biosynthesis of intracellular GSH via the attenuation of γ-GCS activity. Additionally, there was an obvious reduction in HIF-1α expression in irradiated cells treated with OA at different concentrations. In conclusion, OA significantly enhanced the radiosensitivity of tumor cells under mimetic hypoxia, through the reduction in intracellular GSH content and HIF-1α expression.

Glutathione S-Transferase activities and glutathione levels in needles of drought stressed Pinus brutia Ten. trees

Aim: Glutathione S-transferases (GST) take roles under stress conditions by the conjugation of Glutathione (GSH) to electrophilic substrates to increase their solubility and facilitating further metabolic processing. This is one of the main pathways functioning in stress resistance and, for dought stress, there is not yet a study on Pinus brutia Ten. which is a very important forest tree of Turkey. Materials and Methods: The needles of 30 different individuals of Pinus brutia Ten. were collected three times in the season: at the begining, in the middle and at the end of summer (June-August). The total precipitation and the temperature of the region were surveyed during sampling period. The osmotic pressure values, total GST activities and the GSH pools of the needle samples were measured and statistically analysed. Results: The osmotic pressure values showed 16% increase throughout the season. Moreover, total precipitation and temperature values demonstrated that the highest drought stress was observed at the end of sampling period. Approximately 80% of increase in total GST activity between the beginning and the end of summer probably indicated the response against drought; although there was no significant change in the GSH pool. Conclusion: The presence of drought, elevated osmotic pressure and a satistically significant (p<0.005) increase in total GST activities in needle samples were detected. The lack of direct correlation between the GSH pool and total GST activities was explained by the rate of GSH biosynthesis and its redox cycling.

Glutathione initiates the development of Dictyostelium discoideum through the regulation of YakA

Reduced glutathione (GSH) is an essential metabolite that performs multiple indispensable roles during the development of Dictyostelium. We show here that disruption of the gene (gcsA¯) encoding γ-glutamylcysteine synthetase, an essential enzyme in GSH biosynthesis, inhibited aggregation, and that this developmental defect was rescued by exogenous GSH, but not by other thiols or antioxidants. In GSH-depleted gcsA¯ cells, the expression of a growth-stage-specific gene (cprD) was not inhibited, and we did not detect the expression of genes that encode proteins required for early development (cAMP receptor, carA/cAR1; adenylyl cyclase, acaA/ACA; and the catalytic subunit of protein kinase A, pkaC/PKA-C). The defects in gcsA¯ cells were not restored by cAMP stimulation or by cAR1 expression. Further, the expression of yakA, which initiates development and induces the expression of PKA-C, ACA, and cAR1, was regulated by the intracellular concentration of GSH. Constitutive expression of YakA in gcsA¯ cells (YakAOE/gcsA¯) rescued the defects in developmental initiation and the expression of early developmental genes in the absence of GSH. Taken together, these findings suggest that GSH plays an essential role in the transition from growth to development by modulating the expression of the genes encoding YakA as well as components that act downstream in the YakA signaling pathway.

Redox signaling mediates the expression of a sulfate‐deprivation‐inducible microRNA395 in Arabidopsis

MicroRNA395 (miR395) is a conserved miRNA that targets a low-affinity sulfate transporter (AST68) and three ATP sulfurylases (APS1, APS3 and APS4) in higher plants. In this study, At2g28780 was confirmed as another target of miR395 in Arabidopsis. Interestingly, several dicots contained genes homologous to At2g28780 and a cognate miR395 complementary site but possess a gradient of mismatches at the target site. It is well established that miR395 is induced during S deprivation in Arabidopsis; however, the signaling pathways that mediate this regulation are unknown. Several findings in the present study demonstrate that redox signaling plays an important role in induction of miR395 during S deprivation. These include the following results: (i) glutathione (GSH) supplementation suppressed miR395 induction in S-deprived plants (ii) miR395 is induced in Arabidopsis seedlings exposed to Arsenate or Cu(2+) , which induces oxidative stress (iii), S deprivation-induced oxidative stress, and (iv) compromised induction of miR395 during S deprivation in cad2 mutant (deficient in GSH biosynthesis) that is defective in glutaredoxin-dependent redox signaling and ntra/ntrb (defective in thioredoxin reductases a and b) double mutants that are defective in thioredoxin-dependent redox signaling. Collectively, these findings strongly support the involvement of redox signaling in inducing the expression of miR395 during S deprivation in Arabidopsis.