Assuntos
Crassostrea/microbiologia , Crassostrea/virologia , Herpesviridae/isolamento & purificação , Vibrio/isolamento & purificação , Animais , Brasil/epidemiologia , DNA Bacteriano , DNA Viral , Infecções por Herpesviridae/veterinária , Reação em Cadeia da Polimerase/veterinária , Vibrioses/veterináriaRESUMO
Methylglyoxal (MGO) is a major glycating agent that reacts with basic residues of proteins and promotes the formation of advanced glycation end products (AGEs) which are believed to play key roles in a number of pathologies, such as diabetes, Alzheimer's disease, and inflammation. Here, we examined the effects of MGO on immortalized mouse hippocampal HT22 nerve cells. The endpoints analyzed were MGO and thiol status, the glyoxalase system, comprising glyoxalase 1 and 2 (GLO1/2), and the cytosolic and mitochondrial Trx/TrxR systems, as well as nuclear Nrf2 and its target genes. We found that nuclear Nrf2 is induced by MGO treatment in HT22 cells, as corroborated by induction of the Nrf2-controlled target genes and proteins glutamate cysteine ligase and heme oxygenase 1. Nrf2 knockdown prevented MGO-dependent induction of glutamate cysteine ligase and heme oxygenase 1. The cystine/glutamate antiporter, system xc(-), which is also controlled by Nrf2, was also induced. The increased cystine import (system xc(-)) activity and GCL expression promoted GSH synthesis, leading to increased levels of GSH. The data indicate that MGO can act as both a foe and a friend of the glyoxalase and the Trx/TrxR systems. At low concentrations of MGO (0.3mM), GLO2 is strongly induced, but at high MGO (0.75 mM) concentrations, GLO1 is inhibited and GLO2 is downregulated. The cytosolic Trx/TrxR system is impaired by MGO, where Trx is downregulated yet TrxR is induced, but strong MGO-dependent glycation may explain the loss in TrxR activity. We propose that Nrf2 can be the unifying element to explain the observed upregulation of GSH, GCL, HO1, TrxR1, Trx2, TrxR2, and system xc(-) system activity.
Assuntos
Oxirredutases do Álcool/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hipocampo/metabolismo , Neurônios/metabolismo , Aldeído Pirúvico/farmacologia , Tiorredoxina Redutase 1/metabolismo , Tiorredoxinas/metabolismo , Oxirredutases do Álcool/genética , Animais , Apoptose , Western Blotting , Proliferação de Células , Células Cultivadas , Glutationa/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Imunoprecipitação , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tiorredoxina Redutase 1/genética , Tiorredoxinas/genéticaRESUMO
Membrane-bound GST transferase (GSTm) occurs in hepatic microsomal and plasma membranes as well as in the outer mitochondrial membrane, and it is known to be activated by N-ethylmaleimide. We recently analysed the activation by GSSG in some detail. The approximately 5-fold stimulation is reversed upon reduction of GSSG by GSSG reductase. In steady-state experiments, the Kox value was determined to be 0.05, i.e. 20 times more GSSG than GSH produces half-maximal activation. Kox is independent of the total glutathione concentration, indicating that S-thiolation by mixed disulfide formation, rather than interchain or intrachain disulfide bridge formation, is responsible for activation. In Western blots, a 17.7 kDa band, in addition to the 17.3 kDa band, was detected upon treatment with GSSG or with GSH plus t-butyl hydroperoxide. We suggest that under oxidative stress, GSTm is activated through direct S-thiolation of the enzyme. Dethiolation occurs via thiol disulfide exchange governed by the cellular glutathione redox state.
Assuntos
Glutationa Transferase/metabolismo , Animais , Membrana Celular/enzimologia , Ativação Enzimática/efeitos dos fármacos , Dissulfeto de Glutationa/farmacologia , Membranas Intracelulares/enzimologia , Peroxidação de Lipídeos , Fígado/enzimologia , Microssomos Hepáticos/enzimologia , Mitocôndrias Hepáticas/enzimologia , Oxirredução , Estresse Oxidativo , Ratos , Compostos de Sulfidrila/metabolismoRESUMO
Starch gel electrophoresis pH 8.6, or PAGE pH 8.9, of the scalloped hammerhead shark hemolysates showed three hemoglobins (Hb). An additional Hb between the two most mobile electrophoretic components was seen in starch gel electrophoresis, pH 8.1, and also in highly loaded PAGE gels. The relative concentration of these Hbs was variable among individuals, when accessed at pH 8.1. Dilution of hemolysates led to a redistribution of the Hb tetramer subunits. Under denaturing conditions, the unfractionated hemolysate was resolved in 3 Hb subunits. Isolated Hbs, named SL I-SL IV, showed unusual subunit compositions: SL I, the least mobile, is "b3c"; SL II is "a2bc"; SL III and SL IV are composed only by "a" subunits. Hemoglobins in the whole hemolysate have an average of two reactive cysteines per tetramer, which were not easily S-thiolated by glutathione, as is the case for related species. After hemoglobin denaturation, six additional -SH groups were titrated by Ellman's reagent. Methemoglobin content was low in the erythrocytes of nine examined specimens, 1.13 +/- 1.90%. High values for total erythrocyte glutathione (GSH) were found: 4.5 +/- 0.7 mM; n = 7. The ratio of 1.4 +/- 0.4 GSH/Hb is higher than usually reported for mammalians.
Assuntos
Glutationa/sangue , Hemoglobinas/química , Tubarões/sangue , Compostos de Sulfidrila/análise , Animais , Eletroforese/métodos , Eritrócitos/química , Glutationa/análogos & derivados , Glutationa/química , Dissulfeto de Glutationa , Hematócrito , Concentração de Íons de Hidrogênio , Metemoglobina/análise , Metemoglobina/química , Amido , Compostos de Sulfidrila/químicaRESUMO
We evaluated the potential neuroprotective effect of 1-100 µM of four organoselenium compounds: diphenyl diselenide, 3'3-ditri-fluoromethyldiphenyl diselenide, p-methoxy-diphenyl diselenide, and p-chloro-diphenyl diselenide, against methylmercury-induced mitochondrial dysfunction and oxidative stress in mitochondrial-enriched fractions from adult Swiss mouse brain. Methylmercury (10-100 µM) significantly decreased mitochondrial activity, assessed by MTT reduction assay, in a dose-dependent manner, which occurred in parallel with increased glutathione oxidation, hydroperoxide formation (xylenol orange assay) and lipid peroxidation end-products (thiobarbituric acid reactive substances, TBARS). The co-incubation with diphenyl diselenide (100 µM) completely prevented the disruption of mitochondrial activity as well as the increase in TBARS levels caused by methylmercury. The compound 3'3-ditrifluoromethyldiphenyl diselenide provided a partial but significant protection against methylmercury-induced mitochondrial dysfunction (45.4 ± 5.8% inhibition of the methylmercury effect). Diphenyl diselenide showed a higher thiol peroxidase activity compared to the other three compounds. Catalase blocked methylmercury-induced TBARS, pointing to hydrogen peroxide as a vector during methylmercury toxicity in this model. This result also suggests that thiol peroxidase activity of organoselenium compounds accounts for their protective actions against methylmercury-induced oxidative stress. Our results show that diphenyl diselenide and potentially other organoselenium compounds may represent important molecules in the search for an improved therapy against the deleterious effects of methylmercury as well as other mercury compounds.
Assuntos
Encéfalo/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Intoxicação do Sistema Nervoso por Mercúrio/prevenção & controle , Compostos de Metilmercúrio/toxicidade , Mitocôndrias/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Compostos Organosselênicos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Análise de Variância , Animais , Derivados de Benzeno/farmacologia , Fracionamento Celular , Masculino , Camundongos , Modelos Animais , Fármacos Neuroprotetores/classificação , Compostos Organosselênicos/químicaRESUMO
The cellular prion protein (PrP(C)) is a neuronal-anchored glycoprotein that has been associated with various functions in the CNS such as synaptic plasticity, cognitive processes and neuroprotection. Here we investigated age-related behavioral and neurochemical alterations in wild-type (Prnp(+/+)), PrP(C) knockout (Prnp(0/0)) and the PrP(C) overexpressing Tg-20 mice. Three- or 11 month-old animals were submitted to a battery of behavioral tasks including open field, activity cages, elevated plus-maze, social recognition and inhibitory avoidance tasks. The 11 month-old Prnp(+/+) and Prnp(0/0) mice exhibited significant impairments in their locomotor activity and social recognition memory and increased anxiety-related responses. Remarkably, Tg-20 mice did not present these age-related impairments. The i.c.v. infusion of STI1 peptide 230-245, which includes the PrP(C) binding site, improved the age-related social recognition deficits in Prnp(+/+). In comparison with the two other age-matched genotypes, the 11 month-old Tg-20 mice also exhibited reduced activity of seric acetylcholinesterase, increased expression of the protein synaptophysin and decreased caspase-3 positive-cells in the hippocampus. The present findings obtained with genetic and pharmacological approaches provide convincing evidence that PrP(C) exerts a critical role in the age-related behavioral deficits in mice probably through adaptive mechanisms including apoptotic pathways and synaptic plasticity.
Assuntos
Envelhecimento/metabolismo , Encéfalo/metabolismo , Demência/metabolismo , Proteínas PrPC/metabolismo , Acetilcolinesterase/metabolismo , Envelhecimento/genética , Animais , Transtornos de Ansiedade/genética , Transtornos de Ansiedade/metabolismo , Transtornos de Ansiedade/fisiopatologia , Apoptose/genética , Comportamento Animal/fisiologia , Encéfalo/fisiopatologia , Caspase 3/metabolismo , Demência/genética , Demência/fisiopatologia , Hipocampo/metabolismo , Masculino , Aprendizagem em Labirinto/fisiologia , Transtornos da Memória/genética , Transtornos da Memória/metabolismo , Transtornos da Memória/fisiopatologia , Camundongos , Camundongos Knockout , Plasticidade Neuronal/genética , Testes Neuropsicológicos , Fragmentos de Peptídeos/farmacologia , Proteínas PrPC/genética , Estrutura Terciária de Proteína/genética , Sinaptofisina/metabolismoRESUMO
We evaluated the potential neuroprotective effect of 1-100 µM of four organoselenium compounds: diphenyl diselenide, 3’3-ditri-fluoromethyldiphenyl diselenide, p-methoxy-diphenyl diselenide, and p-chloro-diphenyl diselenide, against methylmercury-induced mitochondrial dysfunction and oxidative stress in mitochondrial-enriched fractions from adult Swiss mouse brain. Methylmercury (10-100 µM) significantly decreased mitochondrial activity, assessed by MTT reduction assay, in a dose-dependent manner, which occurred in parallel with increased glutathione oxidation, hydroperoxide formation (xylenol orange assay) and lipid peroxidation end-products (thiobarbituric acid reactive substances, TBARS). The co-incubation with diphenyl diselenide (100 µM) completely prevented the disruption of mitochondrial activity as well as the increase in TBARS levels caused by methylmercury. The compound 3’3-ditrifluoromethyldiphenyl diselenide provided a partial but significant protection against methylmercury-induced mitochondrial dysfunction (45.4 ± 5.8 percent inhibition of the methylmercury effect). Diphenyl diselenide showed a higher thiol peroxidase activity compared to the other three compounds. Catalase blocked methylmercury-induced TBARS, pointing to hydrogen peroxide as a vector during methylmercury toxicity in this model. This result also suggests that thiol peroxidase activity of organoselenium compounds accounts for their protective actions against methylmercury-induced oxidative stress. Our results show that diphenyl diselenide and potentially other organoselenium compounds may represent important molecules in the search for an improved therapy against the deleterious effects of methylmercury as well as other mercury compounds.
Assuntos
Animais , Masculino , Camundongos , Encéfalo/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Intoxicação do Sistema Nervoso por Mercúrio/prevenção & controle , Compostos de Metilmercúrio/toxicidade , Mitocôndrias/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Compostos Organosselênicos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Análise de Variância , Derivados de Benzeno/farmacologia , Fracionamento Celular , Modelos Animais , Fármacos Neuroprotetores/classificação , Compostos Organosselênicos/químicaRESUMO
1. Hemolysate from heavily stressed smooth hammerhead shark, Sphyrna zygaena, shows three electrophoretic components, SZ I, SZ II and SZ III, whose relative concentrations are 36.4 +/- 6.8, 36.4 +/- 5.0 and 20.8 +/- 5.7%, respectively. After reduction with DTE only SZ I remained. 2. SZ I reacted with glutathione disulfide reconstitute SZ II and SZ III. 3. Non-reduced, DTE-reduced, and denatured hemoglobin were found to have 2.0 +/- 0.4, 3.7 +/- 0.6, and 9.4 +/- 0.7-SH groups, respectively. 4. Erythrocyte non-protein--SH (NPSH), including glutathione present as mixed disulfide with SZ II and SZ III, is 1.7 NPSH/Hb.
Assuntos
Hemoglobinas/isolamento & purificação , Estresse Fisiológico/sangue , Adolescente , Animais , Cromatografia em Gel , Dissulfetos/análise , Ditiotreitol/farmacologia , Eletroforese em Gel de Poliacrilamida , Eletroforese em Gel de Amido , Hemólise , Humanos , Substâncias Macromoleculares , TubarõesRESUMO
1. Adult chicken hemoglobins Hb A and Hb D interact with glutathione disulfide, GSSG. The major hemoglobin, Hb A, forms at least two new components, termed GHb AI and GHb AII, and Hb D forms at least one, GHb DI. 2. At pH 8.0 and 5 degrees C, glutathione disulfide (GSSG) in a molar excess of 50 x took 6 days to complete the reaction, although at pH 8.6 and 41 degrees C only 1 hr was needed, where the hemoglobins Hb A and Hb D were converted to their most mobile forms GHb AII and GHb DI. 3. Slight molar excess (2.7 GSSG/Hb, pH 7.4, 41 degrees C), reacting for 1 hr, showed extensive formation of GHb AI and some GHb AII. 4. Electrophoretic patterns, from the reaction products of 54 GSSG/Hb excess at different times, showed a marked pH dependence. 5. Titration with pCMB (p-chloromercuribezoic acid) of DTE (dithioerythrytol)-reduced samples showed 8.0 +/- 0.4 (N = 5) -SH (sulfhydryl) per tetramer. In hemolysates not reacted with DTE, 6.0 +/- 0.4 (N = 3) -SH were detected. 6. DTE-reduced and GSSG-reacted hemoglobins showed 4.6 +/- 0.5 (N = 7) -SH and 1.5 +/- 0.4 (N = 6) -SH, respectively, as titrated by DTNB, pH 8.0. DTE-reduced hemoglobins showed four fast-reacting -SH groups, no longer present in GSSG-reacted hemoglobins. 7. Our data indicate that chicken GHb AI and GHb DI probably have two glutathionyl residues per tetramer whereas GHb AII has four.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Galinhas/sangue , Glutationa/análogos & derivados , Hemoglobinas/química , Animais , Glutationa/biossíntese , Dissulfeto de Glutationa , Hemoglobinas/metabolismo , Ponto IsoelétricoRESUMO
Time courses of total (GSH-t), disulfide (GSSG), and mixed disulfide (PSSG) forms of glutathione were studied in chicken blood submitted to oxidative stress induced by diamide or by the reactive oxygen species (ROS)-producing system xanthine/xanthine oxidase (X/XO). Diamide-treated blood induced an immediate increase in GSSG and PSSG, while X/XO produced a slow and sustained stress with increased values of GSSG and PSSG only after 30 and/or 60 min of incubation. Both total protein S-thiolation (mixed disulfide with glutathione) and dethiolation and hemoglobin A S-thiolation and dethiolation were clearly observed. Hemoglobin A (Hb A) was the major S-thiolated protein. We further characterized chicken Hb S-thiolation through the reaction of Hb with GSSG or the GSH/GSSG redox couple. Methemoglobin levels did not change with diamide or with X/XO treatment. Present results suggest that the most reactive cysteine pair of Hb A, the major chicken Hb, might function as an antioxidant under in vivo oxidative stress conditions.
Assuntos
Diamida/farmacologia , Hemoglobina A/metabolismo , Líquido Intracelular/metabolismo , Estresse Oxidativo , Compostos de Sulfidrila/sangue , Xantina Oxidase/farmacologia , Animais , Galinhas , Eritrócitos/metabolismo , Glutationa/sangue , Dissulfeto de Glutationa/sangue , Hemoglobina A/química , Oxirredução/efeitos dos fármacosRESUMO
Microsomal glutathione transferase (GSTm) is activated up to fivefold by incubation with glutathione disulfide (GSSG). The process is reversed by the addition of an NADPH-regenerating system consisting of glutathione reductase and glucose 6-phosphate/glucose-6-phosphate dehydrogenase. By treating the microsomes at different GSH/GSSG ratios a Kox value of 0.047 is found, i.e., 21 times more GSSG than GSH is necessary to produce half-maximal activation. The Kox is independent of the total glutathione concentration, indicating that S-thiolation by GSH rather than interchain or intrachain disulfide bridge formation is responsible for activation. Further evidence for S-thiolation of GSTm comes from SDS-PAGE under nonreducing conditions and Western blotting. Treating microsomes with GSSG or with GSH and t-butyl hydroperoxide or cumene hydroperoxide results in the appearance of a second GSTm band at approximately 17.7 kDa in addition to the native band at 17.3 kDa, the size difference approximately corresponding to the molecular mass of glutathione. The 17.7-kDa band is not seen in the presence of mercaptoethanol. Microsomal preparations from rat livers perfused with t-butyl hydroperoxide or cumene hydroperoxide also contain both GSTm forms. We suggest that under oxidative stress the microsomal GST in the cell can be activated through direct hydroperoxide-mediated S-thiolation of the enzyme with GSH, its reversal occurring via a thiol exchange-mediated dethiolation imposed by the intracellular glutathione redox state.
Assuntos
Glutationa Transferase/metabolismo , Glutationa/metabolismo , Microssomos Hepáticos/metabolismo , Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Ativação Enzimática/efeitos dos fármacos , Glutationa/análogos & derivados , Glutationa/farmacologia , Dissulfeto de Glutationa , Glutationa Transferase/genética , Técnicas In Vitro , Cinética , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Dados de Sequência Molecular , Oxirredução , Estresse Oxidativo , Perfusão , Peróxidos/farmacologia , Ratos , Ratos Wistar , Compostos de Sulfidrila/metabolismoRESUMO
1. A comparative study involving 80 species (14 ray, 14 shark and 52 teleost species) of marine fish found at the southeastern Brazilian coast is presented. 2. Active species displayed higher values for all hematological parameters studied when compared to the less active forms. 3. Mean values of hematocrit, hemoglobin concentration and red blood cell counts increased according to the sequence: rays, sharks, teleosts. 4. As a group, cartilaginous fish blood displayed larger and fewer erythrocytes containing more hemoglobin than teleosts; mean cell hemoglobin concentration was significantly higher in rays and sharks than in teleosts. 5. For all but the hemoglobin concentration, the hematological values studied revealed a marked contrast between bony and cartilaginous fishes which suggests distinct ways to accomplish their oxygen demands.
Assuntos
Peixes/sangue , Animais , Análise Química do Sangue , Análise de Regressão , Especificidade da EspécieRESUMO
The original pso3-1 mutant isolate of the yeast Saccharomyces cerevisiae exhibits a pleiotropic mutagen-sensitivity phenotype that includes sensitivity to UVA-activated 3-carbethoxypsoralen, to UVC-light, to mono- and bi-functional nitrogen mustard, to paraquat, and to cadmium; on the other hand, it shows hyper-resistance (HYR) to nitrosoguanidine when compared to established wild-type strains. Also, the original pso3-1 mutant exhibits a low UVC-induced mutability and mitotic gene conversion and a high rate of spontaneous and UVC-induced petite mutations. Since the HYR to the nitrosoguanidine (MNNG) phenotype resembles that of low glutathione-containing yeast cells, the original pso3-1 mutant was crossed to a gsh1 knock-out mutant that lacks the enzyme for the first step in glutathione biosynthesis and the resulting diploid was tested for complementation. While there was none for HYR to nitrosoguanidine, and other low glutathione-related phenotypes, some other phenotypic characteristics of pso3-1, e.g. UVC sensitivity and UVC-induced mutability were restored to a wild-type level. Tetrad analysis of a diploid derived from a cross of the original haploid pso3-1 isolate with a repair-proficient, normal glutathione-containing, PSO3 GSH1 wild-type led to the separation of a leaky gsh1 mutation phenotype from that of the repair-deficient pso3-1 phenotype. Linkage studies by tetrad and random spore analyses indicated no linkage of the two genes. This shows that the low glutathione content in the original pso3-1 isolate is due to a second, additional, mutation in the GSH1 locus and is unrelated to the pso3-1 mutation. Thus, the original pso3-1 isolate is a pso3-1 gsh1 double mutant with most of the particular characteristics of the pleiotropic sensitivity phenotype contributed by either the pso3-1 or the gsh1-leaky mutant allele. The expression of a few phenotypic characteristics of pso3, however, were most pronounced in pso3-1 mutants with a low glutathione pool.