Assuntos
Doenças do Sistema Nervoso/prevenção & controle , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Metabolismo Energético , Humanos , Doenças do Sistema Nervoso/metabolismo , Doenças do Sistema Nervoso/patologia , Fármacos Neuroprotetores/uso terapêutico , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Vitamina D/uso terapêuticoRESUMO
The original version of this article unfortunately contained a mistake.
RESUMO
Oxidative stress plays an important role in neurodegenerative diseases and aging. The cellular defense mechanisms to deal with oxidative damage involve the activation of transcription factor related to NF-E2 (Nrf2), which enhances the transcription of antioxidant and phase II enzyme genes. S-allylcysteine (SAC) is an antioxidant with neuroprotective properties, and the main organosulfur compound in aged garlic extract. The ability of SAC to activate the Nrf2 factor has been previously reported in hepatic cells; however this effect has not been studied in normal brain. In order to determine if the chronic administration of SAC is able to activate Nrf2 factor and enhance antioxidant defense in the brain, male Wistar rats were administered with SAC (25, 50, 100 and 200 mg/kg-body weight each 24 h, i.g.) for 90 days. The activation of Nrf2, the levels of p65 and 8-hydroxy-2-deoxyguanosine (8-OHdG) as well as the activities of the enzymes glutathione peroxidase (GPx), glutathione reductase (GR), catalase (CAT), superoxide dismutase (SOD), and glutathione S-transferase (GST) were evaluated in the hippocampus, striatum and frontal cortex. Results showed that SAC activated Nrf2 factor in the hippocampus (25-200 mg/kg) and striatum (100 mg/kg) and significantly decreased p65 levels in the frontal cortex (25-200 mg/kg). On the other hand, SAC increased GPx, GR, CAT and SOD activities mainly in the hippocampus and striatum, but it did not change GST activity. Finally, no changes were observed in 8-OHdG levels mediated by SAC in any brain region, but the hippocampus showed a major level of 8-OHdG compared with the striatum and frontal cortex. All these results suggest that in the hippocampus, the observed increase in the activity of antioxidant enzymes could be associated with the ability of SAC to activate Nrf2 factor; however, a different mechanism could be involved in the striatum and frontal cortex, since no changes were found in Nrf2 activation and p65 levels.
Assuntos
Antioxidantes/metabolismo , Corpo Estriado/metabolismo , Cisteína/análogos & derivados , Lobo Frontal/metabolismo , Hipocampo/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Animais , Corpo Estriado/efeitos dos fármacos , Cisteína/administração & dosagem , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Lobo Frontal/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Masculino , Ratos , Ratos WistarRESUMO
BACKGROUND: Aged garlic extract (AGE) and its main constituent S-allylcysteine (SAC) are natural antioxidants with protective effects against cerebral ischemia or cancer, events that involve hypoxia stress. Cobalt chloride (CoCl2) has been used to mimic hypoxic conditions through the stabilization of the α subunit of hypoxia inducible factor (HIF-1α) and up-regulation of HIF-1α-dependent genes as well as activation of hypoxic conditions such as reactive oxygen species (ROS) generation, loss of mitochondrial membrane potential and apoptosis. The present study was designed to assess the effect of AGE and SAC on the CoCl2-chemical hypoxia model in PC12 cells. RESULTS: We found that CoCl2 induced the stabilization of HIF-1α and its nuclear localization. CoCl2 produced ROS and apoptotic cell death that depended on hypoxia extent. The treatment with AGE and SAC decreased ROS and protected against CoCl2-induced apoptotic cell death which depended on the CoCl2 concentration and incubation time. SAC or AGE decreased the number of cells in the early and late stages of apoptosis. Interestingly, this protective effect was associated with attenuation in HIF-1α stabilization, activity not previously reported for AGE and SAC. CONCLUSIONS: Obtained results show that AGE and SAC decreased apoptotic CoCl2-induced cell death. This protection occurs by affecting the activity of HIF-1α and supports the use of these natural compounds as a therapeutic alternative for hypoxic conditions.
Assuntos
Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/efeitos dos fármacos , Cisteína/análogos & derivados , Alho/química , Extratos Vegetais/farmacologia , Análise de Variância , Animais , Hipóxia Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cobalto , Cisteína/farmacologia , Citometria de Fluxo , Formazans , Células PC12 , Ratos , Espécies Reativas de Oxigênio/análise , Sais de TetrazólioRESUMO
BACKGROUND: Aged garlic extract (AGE) and its main constituent S-allylcysteine (SAC) are natural antioxidants with protective effects against cerebral ischemia or cancer, events that involve hypoxia stress. Cobalt chloride (CoCl2) has been used to mimic hypoxic conditions through the stabilization of the α subunit of hypoxia inducible factor (HIF-1α) and up-regulation of HIF-1α-dependent genes as well as activation of hypoxic conditions such as reactive oxygen species (ROS) generation, loss of mitochondrial membrane potential and apoptosis. The present study was designed to assess the effect of AGE and SAC on the CoCl2-chemical hypoxia model in PC12 cells. RESULTS: We found that CoCl2 induced the stabilization of HIF-1α and its nuclear localization. CoCl2 produced ROS and apoptotic cell death that depended on hypoxia extent. The treatment with AGE and SAC decreased ROS and protected against CoCl2-induced apoptotic cell death which depended on the CoCl2 concentration and incubation time. SAC or AGE decreased the number of cells in the early and late stages of apoptosis. Interestingly, this protective effect was associated with attenuation in HIF-1α stabilization, activity not previously reported for AGE and SAC. CONCLUSIONS: Obtained results show that AGE and SAC decreased apoptotic CoCl2-induced cell death. This protection occurs by affecting the activity of HIF-1α and supports the use of these natural compounds as a therapeutic alternative for hypoxic conditions
Assuntos
Animais , Ratos , Extratos Vegetais/farmacologia , Apoptose/efeitos dos fármacos , Cisteína/análogos & derivados , Fatores de Transcrição Hélice-Alça-Hélice Básicos/efeitos dos fármacos , Alho/química , Antioxidantes/farmacologia , Sais de Tetrazólio , Hipóxia Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Análise de Variância , Células PC12 , Espécies Reativas de Oxigênio/análise , Cobalto , Cisteína/farmacologia , Citometria de Fluxo , FormazansRESUMO
Hemeoxygenase (HO) system is responsible for cellular heme degradation to biliverdin, iron, and carbon monoxide. Two isoforms have been reported to date. Homologous HO-1 and HO-2 are microsomal proteins with more than 45% residue identity, share a similar fold and catalyze the same reaction. However, important differences between isoforms also exist. HO-1 isoform has been extensively studied mainly by its ability to respond to cellular stresses such as hemin, nitric oxide donors, oxidative damage, hypoxia, hyperthermia, and heavy metals, between others. On the contrary, due to its apparently constitutive nature, HO-2 has been less studied. Nevertheless, its abundance in tissues such as testis, endothelial cells, and particularly in brain, has pointed the relevance of HO-2 function. HO-2 presents particular characteristics that made it a unique protein in the HO system. Since attractive results on HO-2 have been arisen in later years, we focused this review in the second isoform. We summarize information on gene description, protein structure, and catalytic activity of HO-2 and particular facts such as its cellular impact and activity regulation. Finally, we call attention on the role of HO-2 in oxygen sensing, discussing proposed hypothesis on heme binding motifs and redox/thiol switches that participate in oxygen sensing as well as evidences of HO-2 response to hypoxia.
Assuntos
Heme Oxigenase (Desciclizante)/metabolismo , Oxigênio/metabolismo , Monóxido de Carbono/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Heme/metabolismo , Heme Oxigenase (Desciclizante)/química , Heme Oxigenase (Desciclizante)/genética , Humanos , Hipóxia , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Doadores de Óxido Nítrico/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismoRESUMO
Two hallmarks of Alzheimer diseases are the continuous inflammatory process, and the brain deposit of Amyloid b (Aß), a cytotoxic protein. The intracellular accumulation of Aß(25-35) fractions, in the absence of Heat Shock proteins (Hsps), could be responsible for its cytotoxic activity. As, pro-inflammatory mediators and nitric oxide control the expression of Hsps, our aim was to investigate the effect of Aß(25-35) on the concentration of IL-1ß, TNF-α and nitrite levels, and their relation to pHSF-1, Hsp-60, -70 and -90 expressions, in the rat C6 astrocyte cells. Interleukin-specific ELISA kits, immunohistochemistry with monoclonal anti-Hsp and anti pHSF-1 antibodies, and histochemistry techniques, were used. Our results showed that Aß25-35 treatment of C6 cells increased, significantly and consistently the concentration of IL-1ß, TNF-α and nitrite 3 days after initiating treatment. The immunoreactivity of C6 cells to Hsp-70 reached its peak after 3 days of treatment followed by an abrupt decrease, as opposed to Hsp-60 and -90 expressions that showed an initial and progressive increase after 3 days of Aß(25-35) treatment. pHSF-1 was identified throughout the experimental period. Nevertheless, progressive and sustained cell death was observed during all the treatment times and it was not caspase-3 dependent. Our results suggest that Hsp-70 temporary expression serves as a trigger to inhibit casapase-3 pathway and allow the expression of Hsp-60 and -90 in C6 astrocytoma cells stimulated with Aß(25-35).
Assuntos
Peptídeos beta-Amiloides/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Fragmentos de Peptídeos/metabolismo , Fatores de Transcrição/metabolismo , Peptídeos beta-Amiloides/farmacologia , Animais , Astrocitoma , Morte Celular , Citocinas/análise , Citocinas/metabolismo , Fatores de Transcrição de Choque Térmico , Inflamação/metabolismo , Óxido Nítrico/análise , Óxido Nítrico/metabolismo , Fragmentos de Peptídeos/farmacologia , Fosforilação , Ratos , Células Tumorais CultivadasRESUMO
BACKGROUND: In a previous report, we have characterized the antiperoxidative properties of alpha-mangostin in different toxic models tested in nerve tissue preparations. OBJECTIVES: Here, the modulatory effects of this xanthone on the glutathione system (reduced glutathione (GSH) levels, glutathione peroxidase (GPx), and glutathione S-transferase (GST) activities) were tested in synaptosomal P2 fractions isolated from rat brains in order to provide further information on key mechanisms exerted by this antioxidant in the nervous system. METHODS: Synaptosomes were exposed to increasing concentrations of the xanthone, and also challenged to the toxic actions of a free radical generator, ferrous sulfate (FeSO(4)). For comparative purposes, the mitochondrial toxin 3-nitropropionic acid (3-NP) was also explored. RESULTS: Alpha-mangostin significantly decreased the levels of GSH, and increased GPx activity. DISCUSSION: This finding was interpreted as a modulatory action of the GSH system in preparation to exert antioxidant responses. Although FeSO(4) exhibited similar effects, these were interpreted as a compensatory response to the toxic actions of the pro-oxidant. We came to this conclusion based on our previous report where alpha-mangostin produced antiperoxidative effects and FeSO(4) produced oxidative damage to lipids. GST activity remained unaffected by both the antioxidant and the pro-oxidant. Our results suggest that alpha-mangostin is able to modulate GPx activity as a potential antioxidant strategy, thereby transiently consuming GSH levels.
Assuntos
Encéfalo/metabolismo , Garcinia mangostana/química , Glutationa Peroxidase/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Sinaptossomos/efeitos dos fármacos , Xantonas/farmacologia , Animais , Anti-Hipertensivos/farmacologia , Antioxidantes/farmacologia , Compostos Ferrosos/farmacologia , Glutationa/metabolismo , Glutationa Transferase/metabolismo , Masculino , Nitrocompostos/farmacologia , Estresse Oxidativo/fisiologia , Propionatos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Espécies Reativas de Oxigênio/farmacologia , Sinaptossomos/metabolismoRESUMO
Huntington's and Parkinson's diseases are neurodegenerative disorders associated with unusual protein interactions. Although the origin and evolution of these diseases are completely different, characteristic deposits of protein aggregates (huntingtin and α-synuclein resp.), are a common feature in both diseases. After these observations, many studies are performed with both proteins. Some of them try to understand the nature and driving forces of the aggregation process; others try to find a correlation between the genetic and failure in protein function. Finally with the combination of both approaches, it was proposed that possible strategies deal with pathologic aggregation. Unfortunately, if protein aggregation is a cause or a consequence of the neurodegeneration observed in these pathologies, it is still debatable. This paper describes the process of aggregation of two proteins: huntingtin and α synuclein. The characteristics of the aggregation reaction of these proteins have been followed with novel methods both in vivo and in vitro; these studies include both the combination with other proteins and the presence of various chemical compounds. The ultimate goal of this study was to summarize recent findings on protein aggregation and its possible role as a therapeutic target in neurodegenerative diseases and their role in biomaterial science.
RESUMO
Metabolic alterations in the nervous system can be produced at early stages of toxicity and are linked with oxidative stress, energy depletion and death signaling. Proteases activation is responsible for triggering deadly cascades during cell damage in toxic models. In this study we evaluated the early time-course of toxic events (oxidative damage to lipids, mitochondrial dysfunction and LDH leakage, all at 1, 3 and 6h) in rat striatal slices exposed to quinolinic acid (QUIN, 100 microM) as an excitotoxic/pro-oxidant model, 3-nitropropionic acid (3-NP, 1mM) as an inhibitor of mitochondrial succinate dehydrogenase, and a combined model produced by the co-administration of these two toxins at subtoxic concentrations (21 and 166 microM for QUIN and 3-NP, respectively). In order to further characterize a possible causality of caspases or calpains on the toxic mechanisms produced in these models, the broad calpain inhibitor IC1 (50 microM), and the pan-caspase inhibitor Z-VAD (100 microM) were tested. Lipid peroxidation (LP) was increased at all times and in all models evaluated. Both IC1 and Z-VAD exerted significant protection against LP in all models and at all times evaluated. Mitochondrial dysfunction (MD) was consistently affected by all toxic models at 3 and 6h, but was mostly affected by 3-NP and QUIN at 1h. IC1 differentially protected the slices against 3-NP and QUIN at 1h and against QUIN at 3h, while Z-VAD exhibited positive actions against QUIN and 3-NP at all times tested, and against their combination at 3 and 6h. LDH leakage was enhanced at 1 and 3h in all toxic models, but this effect was evident only for 3-NP + QUIN and 3-NP at 6h. IC1 protected against LDH leakage at 1h in 3-NP + QUIN and 3-NP models, at 3h in all toxic models, and at 6h in 3-NP + QUIN and 3-NP models. In turn, Z-VAD protected at 1 and 6h in all models tested, and at 3h in the combined and QUIN models. Our results suggest differential chronologic and mechanistic patterns, depending on the toxic insult. Although LP, MD and membrane cell rupture are shared by the three models, the occurrence of each event seems to obey to a selective recruitment of damaging signals, including a differential activation of proteases in time. Proteases activation is likely to be an up-stream event influencing oxidative stress and mitochondrial dysfunction in these toxic models.
Assuntos
Corpo Estriado/efeitos dos fármacos , Nitrocompostos/toxicidade , Oxidantes/toxicidade , Propionatos/toxicidade , Inibidores de Proteases/farmacologia , Ácido Quinolínico/toxicidade , Animais , Calpaína/antagonistas & inibidores , Calpaína/metabolismo , Inibidores de Caspase , Caspases/metabolismo , Morte Celular/efeitos dos fármacos , Corpo Estriado/ultraestrutura , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos , Técnicas In Vitro , Cinética , L-Lactato Desidrogenase/metabolismo , Leupeptinas/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Mitocôndrias/fisiologia , Modelos Animais , Oligopeptídeos/farmacologia , Ratos , Ratos Wistar , Succinato Desidrogenase/antagonistas & inibidoresRESUMO
It is assumed that amino acid sequence differences in highly homologous enzymes would be found at the peripheral level, subtle changes that would not necessarily affect catalysis. Here, we demonstrate that, using the same set of mutations at the level of the interface loop 3, the activity of a triosephosphate isomerase monomeric enzyme is ten times higher than that of a homologous enzyme with 74% identity and 86% similarity, whereas the activity of the native, dimeric enzymes is essentially the same. This is an example of how the dimeric biological unit evolved to compensate for the intrinsic differences found at the monomeric species level. Biophysical techniques of size exclusion chromatography, dynamic light scattering, X-ray crystallography, fluorescence and circular dichroism, as well as denaturation/renaturation assays with guanidinium hydrochloride and ANS binding, allowed us to fully characterize the properties of the new monomer.
Assuntos
Triose-Fosfato Isomerase/química , Sequência de Aminoácidos/genética , Animais , Catálise , Cristalografia por Raios X , Estabilidade Enzimática/genética , Guanidina/química , Luz , Mutação , Conformação Proteica , Desnaturação Proteica , Espalhamento de Radiação , Triose-Fosfato Isomerase/genética , Trypanosoma cruzi/enzimologiaRESUMO
Excitotoxicity and oxidative stress are mechanisms involved in the neuronal cell death induced by the intrastriatal injection of quinolinic acid (QUIN) as a model of Huntington's disease. Production of nitric oxide by nitric oxide synthase (NOS) has been proposed to participate in QUIN-induced neurotoxicity; however, the precise role of NOS in QUIN-induced toxicity still remains controversial. In order to provide further information on the role of NOS isoforms in QUIN toxicity, we performed real time RT-PCR and immunohistochemistry of inducible NOS (iNOS), endothelial NOS (eNOS) and neuronal NOS (nNOS) and determined Ca(2+)-dependent and Ca(2+)-independent NOS activity in a temporal course (3-48h), after an intrastriatal injection of QUIN to rats. NOS isoforms exhibited a transitory expression of mRNA and protein after QUIN infusion: eNOS increased between 3 and 24h, iNOS between 12 and 24h, while nNOS at 35 and 48h. Ca(2+)-independent activity (iNOS) did not show any change, while Ca(2+)-dependent activity (constitutive NOS: eNOS/nNOS) exhibited increased levels at 3h. Our results support the participation of Ca(2+)-dependent NOS isoforms during the toxic events produced at early times after QUIN injection.
Assuntos
Gânglios da Base/enzimologia , Regulação Enzimológica da Expressão Gênica , Doença de Huntington/enzimologia , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/metabolismo , Animais , Cálcio/metabolismo , Modelos Animais de Doenças , Doença de Huntington/induzido quimicamente , Imuno-Histoquímica , Masculino , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo I , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo III , Ácido Quinolínico , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Regulação para CimaRESUMO
Reactive oxygen and nitrogen species formation leads to DNA damage in animals treated with quinolinic acid. Poly(ADP-ribose) polymerase-1 (PARP-1) is a protein involved in the DNA base excision repair system. Its overactivation promotes cellular energy deficit and necrosis. Here, we evaluated the effect of PJ-34, a potent inhibitor of PARP-1, on the neuronal damage induced by quinolinic acid. Animals were administered with PJ-34 (10 mg/kg, i.p.), 1 h before and 1 h after a striatal infusion of 1 microl of quinolinic acid (240 nmol). PJ-34 clearly attenuated the circling behavior produced by quinolinic acid and completely prevented the histological damage induced by the toxin. The protective effect of PJ-34 suggests that PARP-1 activation is playing an active role in the neuronal death induced by quinolinic acid.
Assuntos
Neurônios/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/metabolismo , Ácido Quinolínico/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Encéfalo/citologia , Morte Celular/efeitos dos fármacos , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Masculino , Fenantrenos/farmacologia , Ratos , Ratos Wistar , Comportamento Estereotipado/efeitos dos fármacosRESUMO
The reversible guanidinium hydrochloride-induced unfolding of Trypanosoma cruzi triosephosphate isomerase (TcTIM) was characterized under equilibrium conditions. The catalytic activity was followed as a native homodimeric functional probe. Circular dichroism, intrinsic fluorescence, and size-exclusion chromatography were used as secondary, tertiary, and quaternary structural probes, respectively. The change in ANS fluorescence intensity with increasing denaturant concentrations was also determined. The results show that two stable intermediates exist in the transition from the homodimeric native enzyme to the unfolded monomers: one (N(2*)) is a slightly more expanded, non-native, and active dimer, and the other is a partially expanded monomer (M) that binds ANS. Spectroscopic and activity data were used to reach a thermodynamic characterization. The results indicate that the Gibbs free energies for the partial reactions are 4.5 (N(2) <==> N(2*)), 65.8 (N(2*) <==> 2M), and 17.8 kJ/mol (M <==> U). It appears that TcTIM monomers are more stable than those found for other TIM species (except yeast TIM), where monomer stability is only marginal. These results are compared with those for the guanidinium hydrochloride-induced denaturation of TIM from different species, where despite the functional and three-dimensional similarities, a remarkable heterogeneity exists in the unfolding pathways.
Assuntos
Renaturação Proteica , Triose-Fosfato Isomerase/química , Trypanosoma cruzi/enzimologia , Animais , Guanidina , Desnaturação Proteica , Espectrometria de Fluorescência , Termodinâmica , Triose-Fosfato Isomerase/metabolismoRESUMO
In triosephosphate isomerase, Cys126 is a conserved residue located close to the catalytic glutamate, Glu165. Although it has been mentioned that Cys126 and other nearby residues are required to maintain the active site geometry optimal for catalysis, no evidence supporting this idea has been reported to date. In this work, we studied the catalytic and stability properties of mutants C126A and C126S of Saccharomyces cerevisiae TIM (wtTIM). None of these amino acid replacements induced significant changes in the folding of wtTIM, as indicated by spectroscopic studies. C126S and C126A have K(M) and k(cat) values that are concomitantly reduced by only 4-fold and 1.5-fold, respectively, compared to those of wtTIM; in either case, however, the catalytic efficiency (k(cat)/K(M)) of the enzyme is barely affected. The affinity of mutated TIMs for the competitive inhibitor 2-phosphoglycolate augmented also slightly. In contrast, greater susceptibility to thermal denaturation resulted from mutation of Cys126, especially when it was changed to Ser. By using values of the rate constants for unfolding and refolding, we estimated that, at 25 degrees C, C126A and C126S are less stable than wtTIM by about 5.0 and 9.0 kcal mol(-)(1), respectively. Moreover, either of these mutations slows down the folding rate by a factor of 10 and decreases the recovery of the active enzyme after thermal unfolding. Thus, Cys126 is required for proper stability and efficient folding of TIM rather than for enzymatic catalysis.