Aims: Glutamate cysteine ligase (GCL) enzyme is involved in the synthesis of glutathione, which functions as an antioxidant. Polymorphisms in the sequence of amino acids making up the gene GCLC will cause differences in enzyme expression and GCLC activity. Gene expression that is influenced by oxidative stress can be used to measure markers such as F2-isoprostanes. This study aims to examine the association between the polymorphism in the GCLC gene with glutathione plasma level and F2-isoprostanes in contacts of person with infectious tuberculosis (TB). Methodology and results: Samples are taken from the family members of pulmonary TB patients who seeks treatment at the Pulmonary Centre (Lung Health Center for Public = BBKPM) and Policlinic of Dr Wahidin Sudirohusodo Hospital, Makassar. Total of approximately 4 mL of venous blood are taken from each person with pulmonary TB contacts and furtherly analyzed using genomic PCR-RFLP method and ELISA. Our results described that contacts of person with infectious TB for approximately 6 months have polymorphism C/C genotype at 80.3%, C/T of 18.3% and T/T for 1.4% of the total 71 samples with high levels of glutathione from 0.167 to 0.548 mM/mL and F2-isoprostanes level 72.4 - 1343.9 pg/mL. Conclusion, significance and impact of study: There are no significant association between GCLC gene polymorphism with glutathione and F2-isoprostanes levels of individual who had contacted infection TB. In this study the elevation of F2-isoprostanes equal to the decrease levels of glutathione.
Glutamate-Cysteine Ligase

Previously, we demonstrated that galangin (3,5,7-trihydroxyflavone) protects human keratinocytes against ultraviolet B (UVB)-induced oxidative damage. In this study, we investigated the effect of galangin on induction of antioxidant enzymes involved in synthesis of reduced glutathione (GSH), and investigated the associated upstream signaling cascades. By activating nuclear factor-erythroid 2-related factor (Nrf2), galangin treatment significantly increased expression of glutamate-cysteine ligase catalytic subunit (GCLC) and glutathione synthetase (GSS). This activation of Nrf2 depended on extracellular signal-regulated kinases (ERKs) and protein kinase B (AKT) signaling. Inhibition of GSH in galangin-treated cells attenuated the protective effect of galangin against the deleterious effects of UVB. Our results reveal that galangin protects human keratinocytes by activating ERK/AKT-Nrf2, leading to elevated expression of GSH-synthesizing enzymes.
Catalytic Domain ; Extracellular Signal-Regulated MAP Kinases ; Glutamate-Cysteine Ligase ; Glutathione Synthase ; Glutathione* ; Humans* ; Keratinocytes* ; Proto-Oncogene Proteins c-akt

OBJECTIVETo investigate the possible association between the glutamate-cysteine ligase catalytic subunit gene (GCLC) C-129T and modifier subunit gene (GCLM) G-23T polymorphisms with coronary heart disease (CHD) in Chinese population.

METHODSGCLC C-129T and GCLM G-23T genotypes were determined in 212 CHD patients and 218 healthy individuals using a PCR-based restriction fragment length polymorphism (RFLP) method. Odds ratio (OR) for CHD and 95% confidence interval (CI) from unconditional logistic regression models were used to evaluate relative risks.

RESULTSThe T allele of the GCLC C-129T polymorphism was more frequently found in CHD cases than in controls (P < 0.01) and individuals with GCLC-129T allele had a significantly higher risk for CHD (OR = 2.38, 95% CI: 1.25 - 4.54) as compared to individuals with the -129C allele. When compared with CC homozygote, CT heterozygote had a 2.14-fold higher risk for CHD (95% CI: 1.08 - 4.24, P < 0.05) and carriers of the-129T allele (CT or TT genotype) also had a similarly 2.28-fold higher risk for CHD (95% CI: 1.16 - 4.49, P < 0.05). In contrast, the frequency of T allele of the GCLM G-23T polymorphism was lower in CHD patients than that of controls (0.174 vs. 0.264) and individuals with the GCLM-23T allele had a significantly lower risk for CHD (OR = 0.59, 95% CI: 0.42 - 0.82, P < 0.01) as compared to the -23G allele. When compared with GG homozygote, the OR of CHD for GT heterozygote was 0.71 (95% CI: 0.47 - 1.08, P > 0.05), for TT homozygote was 0.18 (95% CI: 0.06 - 0.55, P < 0.01), and for carriers of the -23T allele (GT or TT genotype) was 0.61 (95% CI: 0.42 - 0.92, P < 0.05).

CONCLUSIONThe GCLC C-129T polymorphism may be one of the genetic risk factor while the GCLM G-23T polymorphism may be one of the genetic protective factors for CHD in this Chinese population.

Aged ; Alleles ; Coronary Disease ; genetics ; Female ; Genetic Predisposition to Disease ; Genotype ; Glutamate-Cysteine Ligase ; genetics ; Humans ; Male ; Polymorphism, Single Nucleotide

Bronchi ; cytology ; metabolism ; Cell Line ; Epithelial Cells ; cytology ; metabolism ; Erythromycin ; pharmacology ; Glutamate-Cysteine Ligase ; genetics ; metabolism ; Glutathione ; genetics ; metabolism ; Humans ; RNA, Messenger ; genetics ; metabolism ; Up-Regulation ; drug effects

AIMTo investigate the expression and relationship of gamma-glutamylcysteine synthetase (gamma-GCS) and NF-E2-related factor2 (NRR2) in lung of rat with chronic obstructive pulmonary disease (COPD)in order to elucidate the possible important role of gamma-GCS and NRF2 in pathogenesis of COPD.

METHODSThe rat COPD model was established by intratracheal instillation of lipopolysaccharide twice and exposed to cigarette smoke daily. The gamma-GCS activity was measured, the expression of gamma-GCS mRNA in lung was examined by in situ hybridization (ISH) and reverse transcription-polymerase chain reaction (RT-PCR), the protein expressions of NRF2, gamma-GCS in lung were detected by immunohistochemical (IH) and Western blot respectively.

RESULTSThe gamma-GCS activity was higher in COPD group than that in control group. The expressions of gamma-GCS mRNA in COPD group was stronger than those in control group. ISH showed that the gamma-GCS mRNA was expressed in alveolar epithelium and bronchial smooth muscle cell in COPD. The protein expressions of NRF2, gamma-GCS were significantly higher than the control group. IH showed that NRF2, gamma-GCS proteins were expressed in alveolar and bronchial epithelium in the COPD group. There was a positive correlation between NRF2 and gamma-GCS and gamma-GCS mRNA.

CONCLUSIONNRF2 may play an important role in the mechanism of COPD oxidative stress vis up-regulation of gamma-GCS.

Animals ; Glutamate-Cysteine Ligase ; metabolism ; Lung ; metabolism ; Male ; NF-E2-Related Factor 2 ; metabolism ; Oxidative Stress ; Pulmonary Disease, Chronic Obstructive ; metabolism ; physiopathology ; Random Allocation ; Rats ; Rats, Wistar

AIMTo investigate the effects of Nrf2 (Nuclear-E2 related factor) on gamma-glutamylcysteine synthase (gamma-GCS) in lung of guinea pigs with bronchial asthma.

METHODS20 adult male guinea pigs were randomly divided into two groups (n = 10): control group (C group) and asthmatic group (A group), asthmatic model was established by ovalbumin intraperitoneal and ovalbumin inhalation. The reactive oxygen piece (ROS), reduced glutathione (GSH), oxidant glutathione (GSSG) and total GSH in lung tissue were examined respectively. Inflammatory cell infiltration and index of remodeling of bronchiole were detected. In situ hybridization detected the gamma-GCS heavy subunit (gamma-GCS h) mRNA in lung tissue. Immunohistochemistry detected the expression of Nrf2 protein and gamma-GCS protein in lung tissue. RT-PCR measured the expression of Nrf2 mRNA in lung tissue. The activity of gamma-GCS was measured by coupled enzyme assay.

RESULTS(1) The number of eosinophils and lymphocytes in bronchiole of A group were significantly higher than that of C group (P < 0.05), the remodeling of bronchiole in A group was definite. (2) ROS (U/mg pro), GSSG (micromol/g pro) and total GSH in lung tissue of A group were significantly higher than that of C group (P < 0.01). The GSH/GSSG in lung tissue of A group was much lower than that of C group (P < 0.01), GSH in lung tissue showed no difference between A group and C group. (3) Immunohistochemistry indicated that Nrf2 protein and gamma-GCS protein were more positively expressed in A group than that in C group (P < 0.01). In situ hybridization discovered that the expression of gamma-GCS-h mRNA in lung tissue of A group was more positive than that of C group. (4) RT-PCR showed that the expression of Nrf2 mRNA was no difference between A group and C group (P > 0.05). (5) The activity of gamma-GCS of A group was (28 +/- 8)U which was significantly higher than that of C group (9 +/- 2)U (P < 0.01). (6) Linear correlation analysis indicated that in lung tissue of guinea pig with asthma there existed strongly positive relationship among ROS, GSSG and the expression of Nrf2, gamma-GCS mRNA, gamma-GCS protein, the activity of gamma-GCS, there existed strongly negative relationship among GSH/GSSG and the expression of Nrf2, gamma-GCS mRNA, gamma-GCS protein, the activity of gamma-GCS.

CONCLUSIONThere existed oxidative stress in lung of guinea pigs with bronchial asthma, which possibly positively regulated gamma-GCS via up regulating transcription factor Nrf2.

Animals ; Asthma ; metabolism ; Glutamate-Cysteine Ligase ; metabolism ; Guinea Pigs ; Lung ; metabolism ; Male ; NF-E2-Related Factor 2 ; metabolism ; Oxidative Stress ; RNA, Messenger ; genetics

BACKGROUND/OBJECTIVES: This study investigated the antioxidant activities and hepatoprotective effects of Schisandra chinensis Baillon extract (SCE) against tert-butyl hydroperoxide (t-BHP)-induced oxidative hepatic damage in rats. MATERIALS/METHODS: Sprague-Dawley (SD) rats were pretreated with SCE (300, 600, and 1,200 mg/kg BW) or saline once daily for 14 consecutive days. On day 14, each animal, except those belonging to the normal control group, were injected with t-BHP (0.8 mmol/kg BW/i.p.), and all of the rats were sacrificed 16 h after t-BHP injection. RESULTS: Although no significant differences in AST and ALT levels were observed among the TC and SCE groups, the high-dose SCE group showed a decreasing tendency compared to the TC group. However, erythrocyte SOD activity showed a significant increase in the low-dose SCE group compared with the TC group. On the other hand, no significant differences in hepatic total glutathione (GSH) level, glutathione reductase (GR), and glutathione peroxidase (GSH-Px) activities were observed among the TC and SCE groups. Hepatic histopathological evaluation revealed that pretreatment with SCE resulted in reduced t-BHP-induced incidence of lesions, such as neutrophil infiltration, swelling of liver cells, and necrosis. In particular, treatment with a high dose of SCE resulted in induction of phase II antioxidant/detoxifying enzyme expression, such as glutathione S-transferase (GST) and glutamate-cysteine ligase catalytic subunit (GCLC). CONCLUSIONS: Based on these results, we conclude that SCE exerts protective effects against t-BHP induced oxidative hepatic damage through the reduction of neutrophil infiltration, swelling of liver cells, and necrosis. In addition, SCE regulates the gene expression of phase II antioxidant/detoxifying enzymes independent of hepatic antioxidant enzyme activity.
Animals ; Catalytic Domain ; Erythrocytes ; Gene Expression* ; Glutamate-Cysteine Ligase ; Glutathione ; Glutathione Peroxidase ; Glutathione Reductase ; Glutathione Transferase ; Hand ; Incidence ; Liver ; Necrosis ; Neutrophil Infiltration ; Rats* ; Rats, Sprague-Dawley ; Schisandra* ; tert-Butylhydroperoxide

BACKGROUND: Exposure to ethanol abuse and severe oxidative stress are risk factors for hepatocarcinoma. The aim of this study was to evaluate the effects of S-adenosylmethionine (SAMe) and its combinations with taurine and/or betaine on the level of glutathione (GSH), a powerful antioxidant in the liver, in acute hepatotoxicity induced by ethanol. METHODS: To examine the effects of SAMe and its combinations with taurine and/or betaine on ethanol-induced hepatotoxicity, AML12 cells and C57BL/6 mice were pretreated with SAMe, taurine, and/or betaine, followed by ethanol challenge. Cell viability was detected with an MTT assay. GSH concentration and mRNA levels of GSH synthetic enzymes were measured using GSH reductase and quantitative real-time reverse transcriptase-PCR. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured with commercially available kits. RESULTS: Pretreatment of SAMe, with or without taurine and/or betaine, attenuated decreases in GSH levels and mRNA expression of the catalytic subunit of glutamate-cysteine ligase (GCL), the rate-limiting enzyme for GSH synthesis, in ethanol-treated cells and mice. mRNA levels of the modifier subunit of GCL and glutathione synthetase were increased in mice treated with SAMe combinations. SAMe, taurine, and/or betaine pretreatment restored serum ALT and AST levels to control levels in the ethanol-treated group. CONCLUSIONS: Combinations of SAMe with taurine and/or betaine have a hepatoprotective effect against ethanol-induced liver injury by maintaining GSH homeostasis.
Alanine Transaminase ; Animals ; Aspartate Aminotransferases ; Betaine* ; Catalytic Domain ; Cell Survival ; Ethanol ; Glutamate-Cysteine Ligase ; Glutathione Synthase ; Glutathione* ; Homeostasis* ; Liver ; Mice ; Oxidative Stress ; Oxidoreductases ; Risk Factors ; RNA, Messenger ; S-Adenosylmethionine* ; Taurine*

Recombinant plasmid pICG was constructed by replacing the internal fragment of a-acetohydroxyacid synthase (AHAS) gene (ILV2) with a copy of gamma-glutamylcysteine synthetase gene (GSH1) and copper chelatin gene (CUP1) from the industrial brewing yeast strain YSF31. YSF31 was transformed with plasmid pICG linearized by Kpn I and Pst I. A recombinant strain with high-glutathione and low-diacetyl production was selected. The results of fermentation in 100-L bioreactor showed that the lagering time of beer produced for recombinant strain T2 was shortened by 3 days and the shelf life of the beer was prolonged about 50%. It may be more acceptable for the commercial application, as it does not contain foreign DNA.
Acetolactate Synthase ; genetics ; metabolism ; Beer ; microbiology ; Cloning, Molecular ; Diacetyl ; metabolism ; Fermentation ; Gene Expression Regulation, Fungal ; Glutamate-Cysteine Ligase ; genetics ; metabolism ; Glutathione ; biosynthesis ; Metallothionein ; genetics ; metabolism ; Organisms, Genetically Modified ; genetics ; Saccharomyces cerevisiae ; genetics ; metabolism ; Saccharomyces cerevisiae Proteins ; genetics ; metabolism

OBJECTIVETo investigate the expressions of PPAR-gamma/PGC-1alpha and Nrf2/gamma-GCS-h in lung of guinea pigs with bronchial asthma, and to explore the roles of them.

METHODSForty adult male guinea pigs were randomly divided into 4 groups: the control group (group A), asthmatic group ( group B), dexamethasone group (group C) and rogridone group (group D), 10 guinea pigs in each group. The asthmatic model was established by the ovalbumin challenge method. Expressions of PPAR-gamma/PGC-1alpha and Nrf2/gamma-GCS-h mRNA in lung tissue were assayed by in situ hybridization. Expressions of PPAR-gamma/PGC-1alpha and Nrf2/gamma-GCS-h protein were detected by immunohischemistry and by Western blot.

RESULTSIn situ hybridization showed that the expressions of PPAR-gamma/PGC-1alpha and Nrf2/gamma-GCS-h mRNA in lung tissue were the lowest in group B and the comparison among groups showed statistical significant (all P < 0.01). Immunohistochemistry and Western blot indicated that the value of PPAR-gamma/PGC-1alpha and Nrf2/gamma-GCS-h protein in lung tissue were the lowest in group B, and expressed primarily in nucleus, the differences being statistically significant (all P < 0.01). There was positive correlation between PPAR-gamma and PGC-1. gamma-GCS-h mRNA also positively correlated between PPAR-gamma/PGC-1alpha and Nrf2 in nucleus, and the expression of Nrf2 was also positively correlated with PPAR-gamma/ PGC-1alpha.

CONCLUSIONIn acute asthmatic models induced by ovalbumin, the expressions of PPAR-alpha/PGC-1alpha and Nrf2/gamma-GCS-h were decreased, and PPARgamma/PGC-1alpha could up-regulate the expressions of Nrf2/gamma-GCS-h to increase the antioxidant defense of tissues, thus being implicated that PPARgamma/PGC-1alpha might play important roles in the pathogenesis and prevention of asthma.

Animals ; Asthma ; chemically induced ; physiopathology ; Glutamate-Cysteine Ligase ; genetics ; metabolism ; Guinea Pigs ; Lung ; metabolism ; Male ; NF-E2-Related Factor 2 ; genetics ; metabolism ; Ovalbumin ; PPAR gamma ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Transcription Factors ; genetics ; metabolism