Assay of the enzymes of glutathione biosynthesis.

This article is dedicated to the late long-time Editor-in-Chief of Analytical Biochemistry, William Jakoby. As a graduate student, I remember reading many articles in Analytical Biochemistry and Methods in Enzymology, both volumes that Bill edited. I first met him as a graduate student presenting at the American Society of Biochemistry (and Molecular Biology) meetings. My Ph.D. advisor, Alton Meister, would bring over well-known biochemists and introduce me as Dr. Anderson, leaving me a bit tongue-tied being that I was still actually a humble graduate student! I next met Bill at my first Analytical Biochemistry Executive Editors meeting in San Diego when he was Editor-in-Chief Emeritus; I felt honored to be on the same board with him and serving the journal to which he had brought to prominence. His eyes were piercing and he was so sharp; his knowledge was both broad and deep. Since much of the large body of Bill's research was on glutathione S-transferases, my article focuses on the assay of the enzymes that synthesize glutathione, a substrate for glutathione S-transferases.

Effects of trypanocidal drugs on DNA synthesis: new insights into melarsoprol growth inhibition.

Trypanothione is the primary thiol redox carrier in Trypanosomatids whose biosynthesis and utilization pathways contain unique enzymes that include suitable drug targets against the human parasites in this family. Overexpression of the rate-limiting enzyme, γ-glutamylcysteine synthetase (GSH1), can increase the intracellular concentration of trypanothione. Melarsoprol directly inhibits trypanothione and has predicted the effects on downstream redox biology, including ROS management and dNTP synthesis that require further investigation. Thus, we hypothesized that melarsoprol treatment would inhibit DNA synthesis, which was tested using BrdU incorporation assays and cell cycle analyses. In addition, we analysed the effects of eflornithine, which interfaces with the trypanothione pathway, fexinidazole, because of the predicted effects on DNA synthesis, and pentamidine as an experimental control. We found that melarsoprol treatment resulted in a cell cycle stall and a complete inhibition of DNA synthesis within 24 h, which were alleviated by GSH1 overexpression. In contrast, the other drugs analysed had more subtle effects on DNA synthesis that were not significantly altered by GSH1 expression. Together these findings implicate DNA synthesis as a therapeutic target that warrants further investigation in the development of antitrypanosomal drugs.

γ-Glutamylcysteine synthetase and γ-glutamyl transferase as differential enzymatic sources of γ-glutamylpeptides in mice.

Some γ-glutamylpeptides in blood plasma are putative biomarkers for pathological conditions of the liver. γ-Glutamyltransferase (GGT) and γ-glutamylcysteine synthetase (γ-GCS) are two such potential enzymes that are responsible for the production of γ-glutamylpeptides. GGT produces γ-glutamylpeptides by transferring the γ-glutamyl moiety from glutathione to an amino acid or a peptide. γ-GCS normally catalyzes the production of γ-glutamylcysteine from glutamate and cysteine in the glutathione-synthesizing reaction, but other amino acids can also serve as an acceptor of a γ-glutamyl group, thus resulting in the formation of a variety of γ-glutamylpeptides. Based on liquid chromatography-mass spectrometry analyses, we observed differences in the distribution of γ-glutamylpeptides between the liver and kidney and were able to measure the activities of γ-GCS as well as the GGT reactions by quantifying the resulting γ-glutamylpeptides. The enzymatic characterization of γ-GCS in liver homogenates indicated that several γ-glutamylpeptides including γ-glutamyltaurine are actually produced. Cys showed the lowest Km value (0.06 mM) while other amino acids had much higher Km values (ranging from 21 to 1800 mM). The moderate Km values for these amino acids suggest that they were not the preferred amino acids in this conversion but were utilized as acceptor substrates for the production of the corresponding γ-glutamylpeptides by the γ-GCS reaction under Cys-deficient conditions. Thus, the production of these γ-glutamylpeptides by γ-GCS is directly correlated with a low Cys content, suggesting that their measurement in blood plasma could be useful for predicting the presymptomatic disease state of the liver with a defect in GSH redox balance.

Development of a highly efficient and specific L-theanine synthase.

γ-Glutamylcysteine synthetase (γ-GCS) from Escherichia coli, which catalyzes the formation of L-glutamylcysteine from L-glutamic acid and L-cysteine, was engineered into an L-theanine synthase using L-glutamic acid and ethylamine as substrates. A high-throughput screening method using a 96-well plate was developed to evaluate the L-theanine synthesis reaction. Both site-saturation mutagenesis and random mutagenesis were applied. After three rounds of directed evolution, 13B6, the best-performing mutant enzyme, exhibited 14.6- and 17.0-fold improvements in L-theanine production and catalytic efficiency for ethylamine, respectively, compared with the wild-type enzyme. In addition, the specific activity of 13B6 for the original substrate, L-cysteine, decreased to approximately 14.6% of that of the wild-type enzyme. Thus, the γ-GCS enzyme was successfully switched to a specific L-theanine synthase by directed evolution. Furthermore, an ATP-regeneration system was introduced based on polyphosphate kinases catalyzing the transfer of phosphates from polyphosphate to ADP, thus lowering the level of ATP consumption and the cost of L-theanine synthesis. The final L-theanine production by mutant 13B6 reached 30.4 ± 0.3 g/L in 2 h, with a conversion rate of 87.1%, which has great potential for industrial applications.

Inhibition of the glutathione biosynthetic pathway increases phytochemical toxicity to Spodoptera litura and Nilaparvata lugens.

Phytochemicals are toxic to insects, but their insecticidal efficiencies are usually low compared to synthetic insecticides. Understanding the mechanism of insect adaptation to phytochemicals will provide guidance for increasing their efficacy. Reduced glutathione (GSH) is a scavenger of reactive oxygen species (ROS) induced by phytochemicals. However, in insects, the pathway of GSH biosynthesis in response to phytochemicals is unclear. We found that exposure to 0.5% indole-3-methanol (I3C), xanthotoxin, and rotenone (ROT) significantly retarded the growth of Spodoptera litura larvae. The oxidative stress in S. litura larvae exposed to phytochemicals was increased. The up-regulation of glutamate cysteine ligase but not glutathione reductase revealed that the de novo synthesis pathway is responsible for GSH synthesis in phytochemical-treated larvae. Treatment with the inhibitor (BSO) of γ-glutamylcysteine synthetase (gclc), a subunit of glutamate cysteine ligase, resulted in decreases of GSH levels and GST activities, increases of ROS levels in I3C-treated larvae, which finally caused midgut necrosis and larval death. Treatment with BSO or I3C alone did not cause larval death. The addition of GSH could partly reduce the influence of I3C and BSO on S. litura growth. Nilaparvata lugens gclc RNAi confirmed the result of BSO treatment in S. litura. N. lugens gclc RNAi significantly increased the mortality of ROT-sprayed N. lugens, in which ROS levels were significantly increased. All data indicate that gclc is involved in insect response to phytochemical treatment. Treatment with dsgclc will increase the insecticidal efficacy of plant-derived compounds.

Cadmium induced glutathione bioaccumulation mediated by γ-glutamylcysteine synthetase in ectomycorrhizal fungus Hebeloma cylindrosporum.

Ectomycorrhizal fungi hold a potential role in bioremediation of heavy metal polluted areas because of its metal accumulation and detoxification property. We investigated the cadmium (Cd) induced bioaccumulation of glutathione (GSH) mediated by γ-glutamylcysteine synthetase (γ-GCS) in the ectomycorrhizal fungus Hebeloma cylindrosporum. In H. cylindrosporum, a demand driven synthesis of GSH has been observed in response to Cd. The expression and enzyme activity of H. cylindrosporum γ-GCS (Hcγ-GCS) increased as a function of external Cd stress resulting in increased GSH production. The function of Hcγ-GCS in providing heavy metal tolerance to H. cylindrosporum was justified by complementing the gene in gsh1Δ mutant of Saccharomyces cerevisiae. The metal sensitive mutant gsh1Δ successfully restored its metal tolerance ability when transformed with Hcγ-GCS gene. Sequence analysis of Hcγ-GCS showed homology with most of the reported γ-GCS proteins from basidiomycetes family. The active site of the Hcγ-GCS protein is composed of amino acids that were found to be conserved not only in fungi, but also in plants and mammals. From these results, it was concluded that Hcγ-GCS plays an important role in bioaccumulation of GSH, which is a core component in the mycorrhizal defense system under Cd stress for Cd homeostasis and detoxification.

Glutathione is involved in physiological response of Candida utilis to acid stress.

Candida utilis often encounters an acid stress environment when hexose and pentose are metabolized to produce acidic bio-based materials. In order to reveal the physiological role of glutathione (GSH) in the response of cells of this industrial yeast to acid stress, an efficient GSH-producing strain of C. utilis CCTCC M 209298 and its mutants deficient in GSH biosynthesis, C. utilis Δgsh1 and Δgsh2, were used in this study. A long-term mild acid challenge (pH 3.5 for 6 h) and a short-term severe acid challenge (pH 1.5 for 2 h) were conducted at 18 h during batch culture of the yeast to generate acid stress conditions. Differences in the physiological performances among the three strains under acid stress were analyzed in terms of GSH biosynthesis and distribution; intracellular pH; activities of γ-glutamylcysteine synthetase, catalase, and superoxide dismutase; intracellular ATP level; and ATP/ADP ratio. The intracellular GSH content of the yeast was found to be correlated with changes in physiological data, and a higher intracellular GSH content led to greater relief of cells to the acid stress, suggesting that GSH may be involved in protecting C. utilis against acid stress. Results presented in this manuscript not only increase our understanding of the impact of GSH on the physiology of C. utilis but also help us to comprehend the mechanism underlying the response to acid stress of eukaryotic microorganisms.

Isorhamnetin protects against oxidative stress by activating Nrf2 and inducing the expression of its target genes.

Isorhamentin is a 3'-O-methylated metabolite of quercetin, and has been reported to have anti-inflammatory and anti-proliferative effects. However, the effects of isorhamnetin on Nrf2 activation and on the expressions of its downstream genes in hepatocytes have not been elucidated. Here, we investigated whether isorhamnetin has the ability to activate Nrf2 and induce phase II antioxidant enzyme expression, and to determine the protective role of isorhamnetin on oxidative injury in hepatocytes. In HepG2 cells, isorhamnetin increased the nuclear translocation of Nrf2 in a dose- and time-dependent manner, and consistently, increased antioxidant response element (ARE) reporter gene activity and the protein levels of hemeoxygenase (HO-1) and of glutamate cysteine ligase (GCL), which resulted in intracellular GSH level increases. The specific role of Nrf2 in isorhamnetin-induced Nrf2 target gene expression was verified using an ARE-deletion mutant plasmid and Nrf2-knockout MEF cells. Deletion of the ARE in the promoter region of the sestrin2 gene, which is recently identified as the Nrf2 target gene by us, abolished the ability of isorhamnetin to increase luciferase activity. In addition, Nrf2 deficiency completely blocked the ability of isorhamnetin to induce HO-1 and GCL. Furthermore, isorhamnetin pretreatment blocked t-BHP-induced ROS production and reversed GSH depletion by t-BHP and consequently, due to reduced ROS levels, decreased t-BHP-induced cell death. In addition isorhamnetin increased ERK1/2, PKCδ and AMPK phosphorylation. Finally, we showed that Nrf2 deficiency blocked the ability of isorhamnetin to protect cells from injury induced by t-BHP. Taken together, our results demonstrate that isorhamnetin is efficacious in protecting hepatocytes against oxidative stress by Nrf2 activation and in inducing the expressions of its downstream genes.

Sulforaphane and phenylethyl isothiocyanate protect human skin against UVR-induced oxidative stress and apoptosis: role of Nrf2-dependent gene expression and antioxidant enzymes.

Chronic UVR-exposure may impair the stress response and antioxidant defense mechanisms of human skin. The transcription factor nuclear factor erythroid-2 related factor 2 (Nrf2) orchestrates the expression of genes coding for the stress response and antioxidant proteins. Here, we tested sulforaphane (SFN) and phenylethyl isothiocyanate (PEITC) for their ability to counteract UVR-induced oxidative stress and apoptosis in ex vivo human full-thickness skin combined with in vitro HaCaT keratinocytes. Investigation of Nrf2 transactivation and induction of genes coding for Nrf2-dependent phase II antioxidative enzymes (γ-glutamylcysteine-synthetase (γGCS), heme oxygenase 1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO1)) was performed in HaCaT keratinocytes. Comparative investigations in human ex vivo skin were conducted for analysis of gene expression of above mentioned phase II enzymes and catalase (CAT) as well as hematoxylin/eosin (H&E) and immunofluorescence (catalase, cleaved Casp-3). UVR exposure of human skin (300mJ/cm(2)) resulted in a significant time-dependent increase of the number of sunburn cells and caspase-3 activation as biomarkers of apoptosis for up to 48h (p<0.001) and induced a significant decrease of the antioxidant enzyme catalase (p<0.001). This was significantly counteracted by the pre-treatment of human skin with SFN and PEITC (5µM and 10µM). Mechanistic cell culture studies revealed SFN and PEITC to increase Nrf2 activity and Nrf2-dependent gene expression (γGCS, HO-1, NQO1); this was paralleled in human full skin mRNA. In conclusion, the induction of Nrf2-dependent antioxidant pathways seems to be a potential mechanism by which SFN and PEITC protect against UVR-induced oxidative stress and apoptosis in human skin.

[Electroacupuncture promotes expression of glutathione related regulatory enzymes in ovary tissue of rats with diminished ovarian reserve].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on ovarian function and expression of glutathione (GSH) related regulatory enzymes γ-glutamylcysteine synthetase (γ-GCS), glutathione reductase (GR) protein and gene in rats with diminished ovarian reserve (DOR), so as to explore its mechanisms underlying up-regulation of antioxidant stress ability. METHODS: A total of 30 female SD rats with normal estrous cycle were randomly divided into blank control, model and EA groups, with 10 rats in each group. The DOR model was established by gavage of tripterygium wilfordii polyglycoside suspension (50 mg·kg-1·d-1) for 14 consecutive days, while the rats in the blank group were given equal volume of 0.9% sodium chloride solution. One hour after daily gavage, EA (1.0 mA, 100 Hz) was applied alternately to bilateral "Shenshu"(BL23), and "Zhongwan"(CV12)+"Guanyuan"(CV4) for 10 min, for 14 consecutive days. Estrous cycles of rats in each group were observed and recorded daily during intervention.After the intervention, H.E.staining was used to observe histopathological changes of the ovarian tissue. The contents of serum sex hormones ï¼»follicle stimulating hormone (FSH), anti-mullerian hormone (AMH), estradiol (E2)ï¼½ and oxidative damage markers ï¼»8-hydroxydeoxyguanosine (8-OHDG) and nitrotyrosine (NTY)ï¼½ were determined by ELISA. The contents of GSH and oxidized glutathione (GSSG) in the liver tissue were determined by colorimetry, and their ratios were calculated. Immunohistochemistry and real-time fluorescence quantitative PCR were used to detect the immunoactivity and gene expression levels of γ-GCS and GR in the ovarian tissues, respectively. RESULTS: Compared with the blank group, the model group had a marked increase in the disorder rate of estrous cycle, serum FSH, 8-OHDG and NTY contents (P<0.01) and a considerable decrease in the levels of serum AMH and E2, liver GSH and GSSG contents and GSH/GSSG ratio, ovarian optical density and cell number as well as the expression of γ-GCS and GR mRNAs (P<0.05, P<0.01). After EA intervention, the increase of the disorder rate of estrous cycle, serum FSH, 8-OHDG and NTY contents and the decrease of serum AMH and E2, liver GSH and GSSG contents and GSH/GSSG ratio, ovarian optical density and cell number of γ-GCS and GR as well as the expression of γ-GCS genes were all reversed (P<0.01, P<0.05). H.E. staining showed degenerative changes of the ovarian tissue, fewer follicles at every level and increase of atretic follicles, disarrangement and layer number decrease of granulosa cells, and atrophy of corpus luteum in the model group, which were relatively milder in the EA group. CONCLUSION: EA can improve ovarian function, and reduce oxidative stress damage in DOR rats, which may be associated with its functions in up-regulating the expression of γ-GCS and GR protein and gene in the ovarian tissue.

The Regulation of Pea (Pisum sativum L.) Symbiotic Nodule Infection and Defense Responses by Glutathione, Homoglutathione, and Their Ratio.

In this study, the roles of glutathione (GSH), homoglutathione (hGSH), and their ratio in symbiotic nodule development and functioning, as well as in defense responses accompanying ineffective nodulation in pea (Pisum sativum) were investigated. The expression of genes involved in (h)GSH biosynthesis, thiol content, and localization of the reduced form of GSH were analyzed in nodules of wild-type pea plants and mutants sym33-3 (weak allele, "locked" infection threads, occasional bacterial release, and defense reactions) and sym33-2 (strong allele, "locked" infection threads, defense reactions), and sym40-1 (abnormal bacteroids, oxidative stress, early senescence, and defense reactions). The effects of (h)GSH depletion and GSH treatment on nodule number and development were also examined. The GSH:hGSH ratio was found to be higher in nodules than in uninoculated roots in all genotypes analyzed, with the highest value being detected in wild-type nodules. Moreover, it was demonstrated, that a hGSHS-to-GSHS switch in gene expression in nodule tissue occurs only after bacterial release and leads to an increase in the GSH:hGSH ratio. Ineffective nodules showed variable GSH:hGSH ratios that correlated with the stage of nodule development. Changes in the levels of both thiols led to the activation of defense responses in nodules. The application of a (h)GSH biosynthesis inhibitor disrupted the nitrogen fixation zone in wild-type nodules, affected symbiosome formation in sym40-1 mutant nodules, and meristem functioning and infection thread growth in sym33-3 mutant nodules. An increase in the levels of both thiols following GSH treatment promoted both infection and extension of defense responses in sym33-3 nodules, whereas a similar increase in sym40-1 nodules led to the formation of infected cells resembling wild-type nitrogen-fixing cells and the disappearance of an early senescence zone in the base of the nodule. Meanwhile, an increase in hGSH levels in sym40-1 nodules resulting from GSH treatment manifested as a restriction of infection similar to that seen in untreated sym33-3 nodules. These findings indicated that a certain level of thiols is required for proper symbiotic nitrogen fixation and that changes in thiol content or the GSH:hGSH ratio are associated with different abnormalities and defense responses.

Exogenous γ-aminobutyric acid strengthens phenylpropanoid and nitrogen metabolism to enhance the contents of flavonoids, amino acids, and the derivatives in edamame.

γ-aminobutyric acid (GABA) has been reported to improve stress resistance in plants. Nonetheless, little is known about the effects of GABA on the nutritional quality and regulatory mechanisms of edamame. Therefore, we analyzed the flavonoid and amino acid (AA) metabolism and the effects of GABA on the nutrient content of edamame seeds through physiological and metabolomic analyses. Exogenous GABA increased endogenous GABA metabolism and GABA transaminase activity and enhanced the oxoglutarate content, which entered into nitrogen metabolism and increased the activity and expression of nitrogen metabolism-related enzymes, to accumulate AAs and bioactive peptides. Meanwhile, exogenous GABA induced the metabolism of flavonoids, including total flavonoids, anthocyanins, 6''-o-malonyglycitin, glycitin, ononin, cyanin, and ginkgetin, by increasing the activity and expression of flavonoid biosynthetic enzymes. This is the first study to reveal that GABA effectively improves the nutritional quality of edamame through the accumulation of AAs, bioactive peptides, isoflavones, anthocyanins, sugars, and organic acids.

Arsenic toxicity and its mitigation in ectomycorrhizal fungus Hebeloma cylindrosporum through glutathione biosynthesis.

Arsenic (As) contamination is one of the most daunting environmental problem bothering the whole world. Exploring a suitable bioremediation technique is an urgent need of the hour. The present study focusses on scrutinizing the ectomycorrhizal (ECM) fungus for its potential role in As detoxification and understanding the molecular mechanisms responsible for its tolerance. When exposed to increasing concentrations of external As, the ECM fungus H. cylindrosporum accumulated the metalloid intracellularly, inducing the glutathione biosynthesis pathway. The genes coding for GSH biosynthesis enzymes, γ-glutamylcysteine synthetase (Hcγ-GCS) and glutathione synthetase (HcGS) were highly regulated by As stress. Arsenic coordinately upregulated the expression of both Hcγ-GCS and HcGS genes, thus resulting in increased Hcγ-GCS and HcGS protein expressions and enzyme activities, with substantial increase in intracellular GSH. Functional complementation of the two genes (Hcγ-GCS and HcGS) in their respective yeast mutants (gsh1Δ and gsh2Δ) further validated the role of both enzymes in mitigating As toxicity. These findings clearly highlight the potential importance of GSH antioxidant defense system in regulating the As induced responses and its detoxification in ECM fungus H. cylindrosporum.

Metabolic Control Analysis for Drug Target Prioritization in Trypanosomatids.

To validate therapeutic targets in metabolic pathways of trypanosomatids, the criterion of enzyme essentiality determined by gene knockout or knockdown is usually being applied. Since, it is often found that most of the enzymes/proteins analyzed are essential, additional criteria have to be implemented for drug target prioritization. Metabolic control analysis (MCA), often in conjunction with kinetic pathway modeling, offers such possibility for prioritization. MCA is a theoretical and experimental approach to analyze how metabolic pathways are controlled. It involves strategies to perform quantitative analyses to determine the degree in which an enzyme controls a pathway flux, a value called flux control coefficient ([Formula: see text]). By determining the [Formula: see text] of individual steps in a metabolic pathway, the distribution of control of the pathway is established, that is, the identification of the main flux-controlling steps. Therefore, MCA can help in ranking pathway enzymes as drug targets from a metabolic perspective. In this chapter, three approaches to determine [Formula: see text] are reviewed: (1) In vitro pathway reconstitution, (2) manipulation of enzyme activities within parasites, and (3) in silico kinetic modeling of the metabolic pathway. To perform these methods, accurate experimental data of enzyme activities, metabolite concentrations and pathway fluxes are necessary. The methodology is illustrated with the example of trypanothione metabolism of Trypanosoma cruzi and protocols to determine such experimental data for this metabolic process are also described. However, the MCA strategy can be applied to any metabolic pathway in the parasite and general directions to perform it are provided in this chapter.

Effects of enhancing endogenous and exogenous glutathione in roots on cadmium movement in Arabidopsis thaliana.

Glutathione (GSH) is a thiol-containing compound involved in many aspects of plant metabolism. In the present study, we investigated how enhancing endogenous and exogenous GSH affects cadmium (Cd) movement and distribution in Arabidopsis plants cultured hydroponically. Transgenic Arabidopsis plants with a strong ability to synthesize GSH in roots were generated　by transforming the gene encoding the bifunctional γ-glutamylcysteine synthetase-glutathione synthetase enzyme from Streptococcus thermophiles (StGCS-GS). Enhancing endogenous and exogenous GSH decreased the Cd translocation ratio in different ways. Only exogenous GSH significantly inhibited Cd translocation from roots to shoots in wild-type and transgenic Arabidopsis plants. Our study demonstrated that GSH mainly functions outside root cells to inhibit Cd translocation from roots to shoots.

Glutathione ethyl ester supplementation prevents airway hyper-responsiveness in mice.

BACKGROUND: Oxidative stress plays an important role in the pathogenesis of asthma. Glutathione (GSH) is considered to be one of the most important antioxidants. Our study systematically investigated the effect of the GSH alternative, glutathione ethyl ester (GSH-EE), on airway hyper-responsiveness (AHR) in mice. METHODS: Sixty-three male specific pathogen-free mice were used. Asthma was induced using a single dose of ovalbumin (OVA). The normal group (n=15) received vehicle only [Al(OH)3 in saline]. Then, 48 mice were divided into two groups, including a control group who received sodium phosphate buffer (pH =7.4), and the GSH-EE group who received 0.1% GSH-EE. AHR was measured 2, 6, and 12 hours after exposure to nebulized OVA (0.01%). The animals were then sacrificed, and lung tissue and the bronchi-alveolar lavage fluid (BALF) were harvested. Factors involved in the antioxidant response to asthma were then measured in these tissues, including thiol content (from GSH and protein), γ-glutamylcysteine synthetase (γ-GCS) activity and expression, and nuclear factor-erythroid-2-related factor (Nrf2) expression. RESULTS: The GSH-EE group showed a significant attenuation of AHR (P<0.01) 2 hours after OVA challenge, and significantly enhanced thiol contents by approximately 45% (P<0.05) at 2 and 6 hours after the last OVA challenge, compared to the control group. γ-GCS activity was also higher in the GSH-EE group compared to the control group at different time points (P<0.01). γ-GCSh and Nrf2 protein expression increased in the GSH-EE group and the control group compared with the normal group, but there was no statistically significant difference (P>0.05) between the GSH-EE group and the control group. CONCLUSIONS: GSH-EE supplementation can prevent AHR in asthmatic mice during the early stages. It may function by serving as a precursor for GSH biosynthesis and by protecting sulfhydryl groups from oxidation.

Activating PPARγ Increases NQO1 and γ-GCS Expression via Nrf2 in Thrombin-activated Microglia.

The present study aimed to explore the molecular mechanisms underlying the increase of nicotinamide adenine dinucleotide phosphate:quinine oxidoreductase 1 (NQO1) and γ-glutamylcysteine synthetase (γ-GCS) in brain tissues after intracerebral hemorrhage (ICH). The microglial cells obtained from newborn rats were cultured and then randomly divided into the normal control group (NC group), model control group (MC group), rosiglitazone (RSG) intervention group (RSG group), retinoic-acid intervention group (RSG+RA group), and sulforaphane group (RSG+SF group). The expression levels of NQO1, γ-GCS, and nuclear factor E2-related factor 2 (Nrf2) were measured by real-time polymerase chain reaction (RT-PCR) and Western blotting, respectively. The results showed that the levels of NQO1, γ-GCS and Nrf2 were significantly increased in the MC group and the RSG group as compared with those in the NC group (P<0.01). They were found to be markedly decreased in the RSG+RA group and increased in the RSG+SF group when compared with those in the MC group or the RSG group (P<0.01). The RSG+SF group displayed the highest levels of NQO1, γ-GCS, and Nrf2 among the five groups. In conclusion, a medium dose of RSG increased the anti-oxidative ability of thrombin-activated microglia by increasing the expression of NQO1 and γ-GCS. The molecular mechanisms underlying the increase of NQO1 and γ-GCS in thrombin-activated microglia may be associated with the activation of Nrf2.

In-Silico Identification of the Best Compound Against Leishmania infantum: High Throughput Screening of All FDA Approved Drugs

Objective: Current in-silico research was designed and administered for the screening of 20000 Food and Drug Administration-approved drug compounds with the goal of finding promising drugs against lipophosphoglycan (LPG) and γ-glutamylcysteine synthetase (γ-GCS) of Leishmania infantum. Methods: After the protein sequence of both targets was taken, the 3D structures of protein of interest were predicted and validated. Molecular docking was done among the two putative targets (LPG and γ-GCS) and approved compounds were selected using AutoDock 4.2 program to predict ligand-receptor interactions. Results: After docking experiment was done on 20000 drug compounds, a total number of seven ligands, two for γ-GCS receptor and five for LPG receptor, were assigned as novel, potent anti-leishmanial drugs based on their binding affinity and energy. Of those, five ligands possessed cytotoxic and anti-cancer characteristics and showed good binding capacity to LPG receptor with ΔGbinding up to 8.5 kcal/mol more negative; while two compounds showed good binding capacity to glutamyl receptor with ΔGbinding up to 7.8 kcal/mol more negative. Conclusion: The latest software-based methods are powerful tools for scanning and predicting new peptide templates specific to biological targets in organisms for new drug discovery. However, the use of in vitro and in vivo techniques is a requirement for better evaluation of the potential of projected ligands with the help of in-silico approaches, identifying molecular mechanism of action of the more active compounds is possible. This can help in defining the most likely molecular target, so that the subsequent optimization using in vitro and in vivo techniques can be undertaken.

Elevated glutathione synthesis in leaves contributes to zinc transport from roots to shoots in Arabidopsis.

Glutathione (GSH) is a vital compound involved in several plant metabolic pathways. Our previous study indicated that foliar GSH application can increase zinc (Zn) levels in leafy vegetables. The objective of this study was to determine the mode of action of GSH as it relates to Zn transport from roots to shoots. Two types of transgenic Arabidopsis plants with genes for GSH synthesis, including StGCS-GS or AtGSH1 driven by the leaf-specific promoter of chlorophyll a/b-binding protein (pCab3) gene were generated. Both types of transgenic Arabidopsis plants showed significant increases in shoot GSH concentrations compared to the wild type (WT). Monitoring 65Zn movement by positron-emitting tracer imaging system (PETIS) analysis indicated that the 65Zn amount in the shoots of both types of transgenic Arabidopsis plants were higher than that in the WT. GSH concentration in phloem sap was increased significantly in WT with foliar applications of 10 mM GSH (WT-GSH), but not in transgenic Arabidopsis with elevated foliar GSH synthesis. Both types of transgenic Arabidopsis with elevated foliar GSH synthesis and WT-GSH exhibited increased shoot Zn concentrations and Zn translocation ratios. These results suggest that enhancement of endogenous foliar GSH synthesis and exogenous foliar GSH application affect root-to-shoot transport of Zn.

γ-Glutamylcysteine synthetase (γ-GCS) as a target for overcoming chemo- and radio-resistance of human hepatocellular carcinoma cells.

AIMS: This study uncovered that the genetically endowed intracellular glutathione contents (iGSH) regulated by the catalytic subunit of γ­glutamylcysteine synthetase heavy chain (γ­GCSh) as a prime target for overcoming both the inherited and stimuli-activated chemo- and radio-resistance of hepatocellular carcinoma (HCC) cells. MAIN METHODS: Reactive oxygen species (ROS) production and mitochondrial membrane potential (Δψm) were determined by the probe-based flow cytometry. The TUNEL assay was used as an index of radio-sensitivity and the MTT assay was used as an index of chemo-sensitivity against various anti-cancer agents. iGSH and γ­GCSh activity were measured by HPLC methods. γ­GCSh-overexpressing GCS30 cell line was established by tetracycline-controlled Tet-OFF gene expression system in SK-Hep-1 cells. KEY FINDINGS: The relative radio-sensitivities of a panel of five HCC cells were found to be correlated negatively with both the contents of iGSH and their corresponding γ­GCSh activities with an order of abundance being Hep G2 > Hep 3B > J5 > Mahlavu > SK-Hep-1, respectively. Similarly, the cytotoxicity response patterns of these HCC cells against arsenic trioxide (ATO), a ROS-producing anti-cancer drug, were exactly identical to the order of ranking instigated by the radiotherapy (RT) treatment. Next, γ­GCSh-overexpressing GCS30 cells were found to possess excellent ability to profoundly mitigate both the drop of Δψm and apoptotic TUNEL-positive cell population engendered by ATO, cisplatin, doxorubicin, and RT treatments. SIGNIFICANCE: Our data unequivocally demonstrate that γ­GCSh may represent a prime target for overcoming anti-cancer drugs and RT resistance for HCC cells.