RESUMO
BACKGROUND: Glutathione (GSH) plays a role as a main antioxidant metabolite in all eukaryotes and many prokaryotes. Most of the organisms synthesize GSH by a pathway involving two enzymatic reactions, each one consuming one molecule of ATP. In a first step mediated by glutamate-cysteine ligase (GCL), the carboxylate of l-glutamic acid reacts with l-cysteine to form the dipeptide γ-glutamylcysteine (γ-GC). The second step involves the addition of glycine to the C-terminal of γ-GC catalyzed by glutathione synthetase (GS). In many bacteria, such as in the pathogen Leptospira interrogans, the main intracellular thiol has not yet been identified and the presence of GSH is not clear. METHODS: We performed the molecular cloning of the genes gshA and gshB from L. interrogans; which respectively code for GCL and GS. After heterologous expression of the cloned genes we recombinantly produced the respective proteins with high degree of purity. These enzymes were exhaustively characterized in their biochemical properties. In addition, we determined the contents of GSH and the activity of related enzymes (and proteins) in cell extracts of the bacterium. RESULTS: We functionally characterized GCL and GS, the two enzymes putatively involved in GSH synthesis in L. interrogans serovar Copenhageni. LinGCL showed higher substrate promiscuity (was active in presence of l-glutamic acid, l-cysteine and ATP, and also with GTP, l-aspartic acid and l-serine in lower proportion) unlike LinGS (which was only active with γ-GC, l-glycine and ATP). LinGCL is significantly inhibited by γ-GC and GSH, the respective intermediate and final product of the synthetic pathway. GSH showed inhibitory effect over LinGS but with a lower potency than LinGCL. Going further, we detected the presence of GSH in L. interrogans cells grown under basal conditions and also determined enzymatic activity of several GSH-dependent/related proteins in cell extracts. CONCLUSIONS: and General Significance. Our results contribute with novel insights concerning redox metabolism in L. interrogans, mainly supporting that GSH is part of the antioxidant defense in the bacterium.
Assuntos
Proteínas de Bactérias/metabolismo , Glutamato-Cisteína Ligase/metabolismo , Glutationa Sintase/metabolismo , Glutationa/metabolismo , Leptospira interrogans/metabolismo , Proteínas de Bactérias/genética , Clonagem Molecular , Glutamato-Cisteína Ligase/genética , Glutationa Sintase/genética , Leptospira interrogans/genética , Leptospira interrogans/crescimento & desenvolvimento , OxirreduçãoRESUMO
Glutathione synthetase deficiency (GSSD) is a rare inborn error of glutathione metabolism with autosomal recessive inheritance. The severe form of the disease is characterized by acute metabolic acidosis, usually present in the neonatal period with hemolytic anemia and progressive encephalopathy. A case of a male newborn infant who had severe metabolic acidosis with high anion gap, hemolytic anemia, and hyperbilirubinemia is reported. A high level of 5-oxoproline was detected in his urine and a diagnosis of generalized GSSD was made. DNA sequence analysis revealed the infant to be compound heterozygous with two mutations, c.738dupG in exon 8 of GSS gene resulting in p.S247fs and a repetitive sequence in exon 3 of GSS gene. Treatment after diagnosis of GSSD included supplementation with antioxidants and oral sodium hydrogen bicarbonate. However, he maintained a variable degree of metabolic acidosis and succumbed shortly after his parents requested discontinuation of therapy because of dismal prognosis and medical futility when he was 18 days old.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Glutationa Sintase/deficiência , Mutação , Acidose/etiologia , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Ácido Glutâmico/análise , Glutationa Sintase/genética , Glutationa Sintase/metabolismo , Humanos , Recém-Nascido , Masculino , Piroglutamato Hidrolase/deficiência , Piroglutamato Hidrolase/genética , Análise de Sequência de DNA/métodosRESUMO
Glutathione synthetase deficiency (GSSD) is a rare inborn error of glutathione metabolism with autosomal recessive inheritance. The severe form of the disease is characterized by acute metabolic acidosis, usually present in the neonatal period with hemolytic anemia and progressive encephalopathy. A case of a male newborn infant who had severe metabolic acidosis with high anion gap, hemolytic anemia, and hyperbilirubinemia is reported. A high level of 5-oxoproline was detected in his urine and a diagnosis of generalized GSSD was made. DNA sequence analysis revealed the infant to be compound heterozygous with two mutations, c.738dupG in exon 8 of GSS gene resulting in p.S247fs and a repetitive sequence in exon 3 of GSS gene. Treatment after diagnosis of GSSD included supplementation with antioxidants and oral sodium hydrogen bicarbonate. However, he maintained a variable degree of metabolic acidosis and succumbed shortly after his parents requested discontinuation of therapy because of dismal prognosis and medical futility when he was 18 days old.
Assuntos
Humanos , Masculino , Recém-Nascido , Erros Inatos do Metabolismo dos Aminoácidos/genética , Glutationa Sintase/deficiência , Mutação , Acidose/etiologia , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Ácido Glutâmico/análise , Glutationa Sintase/genética , Glutationa Sintase/metabolismo , Piroglutamato Hidrolase/deficiência , Piroglutamato Hidrolase/genética , Análise de Sequência de DNA/métodosRESUMO
Leishmaniasis is a neglected tropical disease that affects millions of people worldwide and represents a major public health problem. Information on protein expression patterns and functional roles within the context of Leishmania-infected human monocyte-derived macrophages (MDMs) under drug treatment conditions is essential for understanding the role of these cells in leishmaniasis treatment. We analyzed functional changes in the expression of human MDM genes and proteins during in vitro infection by Leishmania braziliensis and treatment with Glucantime (SbV), using quantitative PCR (qPCR) arrays, Western blotting, confocal microscopy, and small interfering RNA (siRNA) human gene inhibition assays. Comparison of the results from gene transcription and protein expression analyses revealed that glutathione S-transferase π1 (GSTP1), glutamate-cysteine ligase modifier subunit (GCLM), glutathione reductase (GSR), glutathione synthetase (GSS), thioredoxin (TRX), and ATP-binding cassette, subfamily B, member 5 (ABCB5), were strongly upregulated at both the mRNA and protein levels in human MDMs that were infected and treated, compared to the control group. Subcellular localization studies showed a primarily phagolysosomal location for the ABCB5 transporter, indicating that this protein may be involved in the transport of SbV By inducing a decrease in L. braziliensis intracellular survival in THP-1 macrophages, siRNA silencing of GSTP1, GSS, and ABCB5 resulted in an increased leishmanicidal effect of SbV exposure in vitro Our results suggest that human MDMs infected with L. braziliensis and treated with SbV express increased levels of genes participating in antioxidant defense, whereas our functional analyses provide evidence for the involvement of human MDMs in drug detoxification. Therefore, we conclude that GSS, GSTP1, and ABCB5 proteins represent potential targets for enhancing the leishmanicidal activity of Glucantime.
Assuntos
Leishmania braziliensis/efeitos dos fármacos , Leishmania braziliensis/patogenicidade , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Meglumina/farmacologia , Compostos Organometálicos/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antioxidantes/metabolismo , Glutamato-Cisteína Ligase/metabolismo , Glutationa Redutase/metabolismo , Glutationa S-Transferase pi/metabolismo , Glutationa Sintase/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Antimoniato de Meglumina , Reação em Cadeia da PolimeraseRESUMO
Females are born with a finite number of oocyte-containing follicles and ovary damage results in reduced fertility. Cadmium accumulates in the reproductive system, damaging it, and the cigarette smoke is a potential exposure route. Natural therapies are relevant to health benefits and disease prevention. This study verified the effect of cadmium exposure on the ovaries of mice and the blueberry extract as a potential therapy. Blueberry therapy was effective in restoring reactive species levels and δ-aminolevulinate dehydratase activity, and partially improved the viability of cadmium-disrupted follicles. This therapy was not able to restore the 17 ß-hydroxysteroid dehydrogenase activity. Extract HPLC evaluation indicated the presence of quercetin, quercitrin, isoquercetin, and ascorbic acid. Ascorbic acid was the major substance and its concentration was 620.24 µg/mL. Thus, cadmium accumulates in the ovaries of mice after subchronic exposure, inducing cellular damage, and the blueberry extract possesses antioxidant properties that could protect, at least in part, the ovarian tissue from cadmium toxicity. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 188-196, 2017.
Assuntos
Mirtilos Azuis (Planta)/química , Intoxicação por Cádmio/tratamento farmacológico , Doenças Ovarianas/induzido quimicamente , Doenças Ovarianas/tratamento farmacológico , Extratos Vegetais/farmacologia , Sintase do Porfobilinogênio/metabolismo , 11-beta-Hidroxiesteroide Desidrogenases/metabolismo , Animais , Intoxicação por Cádmio/patologia , Feminino , Glutationa Peroxidase/metabolismo , Glutationa Sintase/metabolismo , Camundongos , Doenças Ovarianas/patologia , Folículo Ovariano/efeitos dos fármacos , Sintase do Porfobilinogênio/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismoRESUMO
The aim of the present study was to evaluate the effects of heat stress (HS) and methionine supplementation on the markers of stress and on the gene expression levels of uncoupling proteins (UCP), betaine-homocysteine methyltransferase (BHMT), cystathionine ß-synthase (CBS), glutathione synthetase (GSS) and glutathione peroxidase 7 (GPx7). Broilers from 1 to 21 d and from 22 to 42 d of age were divided into three treatment groups related to methionine supplementation: without methionine supplementation (MD); recommended level of methionine supplementation (DL1); excess methionine supplementation (DL2). The broilers were either kept at a comfortable thermal temperature or exposed to HS (38°C for 24 h). During the starter period, we observed the effects of the interaction between diet and environment on the gene expression levels of UCP, BHMT and GSS. Higher gene expression levels of UCP and BHMT were observed in broilers that were maintained at thermal comfort conditions and received the MD diet. HS broilers fed the DL1 and DL2 diets had the highest expression level of GSS. The expression levels of the CBS and GPx7 genes were influenced by both the environment and methionine supplementation. During the grower period, the gene expression levels of BHMT, CBS, GSS and GPx7 were affected by the diet × environment interaction. A higher expression level of BHMT was observed in broilers maintained at thermal comfort conditions and on the MD diet. HS induced higher expression levels of CBS, GSS and GPx7 in broilers that received the DL1 and DL2 diets. The present results suggest that under HS conditions, methionine supplementation could mitigate the effects of stress, since methionine contributed to the increased expression levels of genes related to antioxidant activity.
Assuntos
Doenças das Aves/prevenção & controle , Dieta/veterinária , Regulação da Expressão Gênica no Desenvolvimento , Transtornos de Estresse por Calor/veterinária , Metionina/uso terapêutico , Estresse Oxidativo , Músculos Peitorais/enzimologia , Animais , Animais Endogâmicos , Antioxidantes/administração & dosagem , Antioxidantes/uso terapêutico , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Betaína-Homocisteína S-Metiltransferase/genética , Betaína-Homocisteína S-Metiltransferase/metabolismo , Biomarcadores/sangue , Biomarcadores/metabolismo , Doenças das Aves/dietoterapia , Doenças das Aves/metabolismo , Doenças das Aves/patologia , Galinhas , Ingestão de Energia , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Glutationa Sintase/genética , Glutationa Sintase/metabolismo , Transtornos de Estresse por Calor/dietoterapia , Transtornos de Estresse por Calor/metabolismo , Transtornos de Estresse por Calor/prevenção & controle , Homocisteína/sangue , Canais Iônicos/genética , Canais Iônicos/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Metionina/administração & dosagem , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Músculos Peitorais/metabolismo , Músculos Peitorais/patologia , Proteína Desacopladora 1 , Aumento de PesoRESUMO
The present study was conducted to assess the development, quality and gene expression profile of oxidative stress-related genes of bovine embryos cultured in different culture systems with low oxygen tension (5% CO2, 5% O2 and 90% N2). The systems assessed included: (1) an incubator chamber; (2) a plastic bag; and (3) a foil bag. The choice of culture system had no effect on cleavage rate at 72 h. However, significant differences (P < 0.01) were observed in the rate of blastocysts registered at day 7 (29.8, 20.2 and 12.7% for incubator chamber, plastic bag and foil bag, respectively). Total number of cells did not differ between systems, although the proportion of ICM:total cells was affected particularly in the plastic bag (19.5%), compared with the incubator chamber (31.4%). In addition, significant differences were found in the apoptotic:total cell ratio (3.3, 6.5 and 8.8% for the incubator chamber, plastic bag and foil bag, respectively), with apoptotic nuclei localised mainly in the ICM compartment of the embryo. The amount of reactive oxygen species was also different between culture systems and this effect was correlated with a higher expression of SOD2, GSS and GPX1 genes in embryos cultured in the gassed bags as compared with embryos cultured in the incubator chamber. In conclusion, these results give evidence that, under low oxygen tension, the incubator chamber is more efficient and generates higher number of, and better quality, embryos than gassed bag systems evaluated here and this effect was probably due to an increased level of reactive oxygen species in the gassed bags, which upregulates the expression of some antioxidant enzymes to compensate for hyperoxia conditions.
Assuntos
Embrião de Mamíferos/metabolismo , Glutationa Peroxidase/genética , Glutationa Sintase/genética , Estresse Oxidativo , Oxigênio/metabolismo , Superóxido Dismutase/genética , Animais , Antioxidantes/metabolismo , Blastocisto/metabolismo , Bovinos , Desenvolvimento Embrionário , Fertilização in vitro , Glutationa Peroxidase/metabolismo , Glutationa Sintase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Glutationa Peroxidase GPX1RESUMO
In order to identify tobacco (Nicotiana megalosiphon) genes involved in broad-spectrum resistance to tobacco blue mold (Peronospora hyoscyami f. sp. tabacina), suppression subtractive hybridization was used to generate cDNA from transcripts that are differentially expressed during an incompatible interaction. After differential screening by membrane-based hybridization, clones corresponding to 182 differentially expressed genes were selected, sequenced, and analyzed. The cDNA collection comprised a broad repertoire of genes associated with various processes. Northern blot analysis of a subset of these genes confirmed the differential expression patterns between the compatible and incompatible interaction. Subsequent virus-induced gene silencing (VIGS) of four genes that were found to be differentially induced was pursued. While VIGS of a lipid transfer protein gene or a glutamate decarboxylase gene in Nicotiana megalosiphon did not affect blue mold resistance, silencing of an EIL2 transcription factor gene and a glutathione synthetase gene was found to compromise the resistance of Nicotiana megalosiphon to P. hyoscyami f. sp. tabacina. Potentially, these genes can be used to engineer resistance in blue mold-susceptible tobacco cultivars.
Assuntos
Glutationa Sintase/metabolismo , Nicotiana/metabolismo , Nicotiana/microbiologia , Peronospora/fisiologia , Doenças das Plantas/microbiologia , Fatores de Transcrição/metabolismo , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Glutationa Sintase/genética , Dados de Sequência Molecular , Mutação , Folhas de Planta/microbiologia , Nicotiana/enzimologia , Nicotiana/genética , Fatores de Transcrição/genéticaRESUMO
To study the effects of aluminium (Al) on glutathione (GSH) metabolism in the small intestine, adult male Wistar rats were orally treated with AlCl3.6H2O at doses of 30, 60, 120 and 200 mg/kg body weight (b.w.) per day, during seven days. Controls received deionized water. At doses above 120 mg/kg b.w., Al produced both a significant reduction of GSH content and an increase of oxidized/reduced glutathione ratio (P < 0.05). The index of oxidative stress of the intestine mucosa in terms of lipid peroxidation evaluated by thiobarbituric acid reactive substances was significantly increased (52%) at higher Al dose used. The duodenal expression of the multidrug resistance-associated protein 2 in brush border membranes, determined by Western blot technique, was increased 2.7-fold in rats treated with 200mg AlCl3/kg b.w (P < 0.01). Intestine activities of both GSH-synthase (from 60 mg/kg b.w.) and GSSG-reductase (from 120 mg/kg b.w.) were significantly reduced (26% and 31%, respectively) while glutathione-S-transferase showed to be slightly modified in the Al-treated groups. Conversely, gamma-glutamyltranspeptidase activity was significantly increased (P < 0.05) due to the Al treatment. Al reduced in vitro mucosa-to-lumen GSH efflux (P < 0.05). A positive linear correlation between the intestine GSH depletion and reduction of in situ 45Ca intestinal absorption, both produced by Al, was found (r = 0.923, P = 0.038). Taking as a whole, these results show that Al would alter GSH metabolism in small intestine by decreasing its turnover, leading to an unbalance of redox state in the epithelial cells, thus contributing to deteriorate GSH-dependent absorptive functions.