The role of s-nitrosothiols and rho-kinase 1 in glucose and lipid metabolism

RESUMO: Na sociedade contemporânea a diabetes tipo 2 e a obesidade estão a aumentar exponencialmente, representando um grave problema de saúde pública. De acordo com a IDF “A diabetes e a obesidade são o principal problema de saúde pública do século XXI’. Para além destas duas patologias, a prevalência de esteatose hepática não-alcoólica (NAFLD), entre a população obesa e diabética, é de cerca de 90%. O aumento da obesidade, diabetes e NAFLD tem uma forte correlação com o aumento do consumo de gorduras e açúcares, acompanhado de um decréscimo acentuado da actividade física. A obesidade, diabetes e NAFLD tem sido escrupolosamente investigada mas as terapêuticas disponíveis continuam a ser muito limitadas. Tendo em conta o número crescente e alarmante de obesos e diabéticos o conhecimento detalhado da patofisiologia da obesidade, diabetes e NAFLD, tendo em vista a necessidade extrema de desenvolvimento de novas estratégias terapêuticas, é da mais elevada urgência. O fígado é reconhecido como um orgão primordial no controlo da homeostase. No estado pós-prandial, o fígado converte a glucose em glicogénio e lípidos. Em contraste, no estado de jejum, o fígado promove a produção de glucose. Sistemas neuronais e hormonais, bem como o estado metabólico do fígado, controlam de forma muito precisa a alternância entre os diferentes substratos metabólicos, dependente do estado prandial. A insulina tem um papel central no controlo do metabolismo energético no fígado; se, por um lado, inibe a produção hepática de glucose e corpos cetónicos, por outro, promove a glicólise e a lipogénese. O metabolismo energético no fígado é também regulado por vários factores de transcrição e co-reguladores que, por sua vez, são regulados pela insulina, glucagina e outras hormonas metabólicas. Em conjunto, todos estes factores e reguladores vão controlar de forma muito estreita a gluconeogénese, a β-oxidação e a lipogénese, no fígado. Para além dos já conhecidos reguladores do metabolismo hepático, novas moléculas têm sido estudadas como tendo um papel fundamental na regulação do metabolismo energético no fígado. Qualquer desequilíbrio no metabolismo hepático vai contribuir para a insulino-resistência, NAFLD e diabetes tipo 2. O principal objectivo do trabalho de investigação aqui apresentado é o contributo para o estudo detalhado da patogénese da diabetes e obesidade, num contexto de dietas ricas em açúcares e gorduras, e com a perspectiva de explorar novas estratégias terapêuticas. Os objectivos específicos deste trabalho eram: primeiro, determinar se o tratamento com glutationo (GSH) e óxido nítrico (NO) era suficiente para melhorar a insulino-resistência associada ao elevado consumo de sacarose; segundo, determinar o papel da Rho-kinase 1 (ROCK1) na regulação do metabolismo hepático da glucose e dos lípidos; e terceiro, estudar o efeito do metilsulfonilmetano (MSM) em doenças metabólicas associadas à obesidade. Na primeira parte deste trabalho de investigação foram utilizados ratos Wistar machos sujeitos a uma dieta rica em sacarose (HS). Tal como esperado, estes animais apresentavam insulino-resistência e hiperinsulinémia. A dieta HS levou ao aumento dos níveis hepáticos de NO e ao decréscimo dos níveis de GSH no fígado. Em jejum, a administração intraportal de GSH e NO, a animais saudáveis promoveu um aumento significativo da sensibilidade à insulina. Também nestes animais, a administração intravenosa de S-nitrosotióis, compostos orgânicos que contém um grupo nitroso acoplado a um átomo de enxofre de um tiol, promoveu o aumento significativo da sensibilidade à insulina. Pelo contrário, em animais sujeitos à dieta HS, as doses padrão de GSH + NO e de S-nitrosotióis não conseguiram promover o aumento da sensibilidade à insulina. No entanto, ao aumentar a dose de S-nitrosotióis administrados por via intravenosa, foi possível observar o aumento da sensibilidade à insulina dependente da dose, indicando um possível papel dos S-nitrosotióis como sensibilizadores de insulina. O estudo detalhado do papel dos S-nitrosotióis na via de sinalização da insulina revelou que há um aumento da fosforilação do receptor da insulina (IR) e da proteína cinase B (Akt), sugerindo um efeito dos S-nitrosotióis nesta via de sinalização. Os resultados apresentados nesta primeira parte sugerem que os S-nitrosotióis promovem a correcta acção da insulina, podendo vir a ser importantes alvos terapêuticos. Na segunda parte deste trabalho de investigação utilizámos murganhos, com uma delecção específica da ROCK1 no fígado, e sujeitos a uma dieta rica em lípidos (HFD). Foi possível concluir que a ausência da ROCK1 no fígado previne a obesidade, melhora a sensibilidade à insulina e protege contra a esteatose hepática. A ausência de ROCK1 no fígado levou a um decréscimo significativo da expressão génica de genes associados à lipogénese, com uma diminuição acentuada do fluxo metabólico associado a esta via. Pelo contrário, a sobreexpressão de ROCK1, exclusivamente no fígado, promove a insulino-resistência e a esteatose hepática no contexto de obesidade induzida pela dieta. Para além disto, a delecção da ROCK1 no fígado de animais obesos e diabéticos, os murganhos deficientes em leptina, corroborou os dados obtidos no primeiro modelo animal, com a franca melhoria da hiperglicémia, hiperinsulinémia e esteatose hepática. Os dados que compõem esta parte do trabalho de investigação sugerem que a ROCK1 tem um papel crucial na regulação do metabolismo lipídico. Na terceira e última parte deste trabalho de investigação foi investigado o efeito do composto metilsulfunilmetano (MSM), um composto organosulfúrico naturalmente presente em plantas e utilizado também como suplemento dietético, em murganhos obesos e insulino-resistentes, por exposição a uma dieta rica em lípidos (DIO). O tratamento com MSM melhorou a insulino-resistência e protegeu contra a esteatose hepática. O conteúdo hepático em triglicéridos e colesterol também diminuíu de forma significativa nos animais DIO sujeitos ao tratamento com MSM, bem como a expressão génica associada à lipogénese. Para além disto, o tratamento com MSM levou a uma diminuição da expressão génica associada à inflamação. De realçar que o tratamento com MSM levou a uma melhoria do perfil hematopoiético destes animais, tanto na medula óssea como no sangue. Para comprovar o efeito benéfico do MSM na obesidade e insulino-resistência utilizámos murganhos deficientes no receptor da leptina, e por isso obesos e diabéticos, tendo observado um perfil semelhante ao obtido para murganhos sujeitos a uma dieta rica em lípidos e tratados com MSM. Concluímos, através dos dados recolhidos, que o MSM como suplemento pode ter efeitos benéficos na hiperinsulinémia, insulino-resistência e inflamação que caracterizam a diabetes tipo 2. Em resumo, os dados obtidos neste trabalho de investigação mostram que os S-nitrosotióis podem ter um papel importante como sensibilizadores da insulina, promovendo um aumento da sensibilidade à insulina num contexto de dietas ricas em sacarose. Para além disto, estudos in vitro, sugerem que os S-nitrosotióis regulam, especificamente, a via de sinalização da insulina. Este trabalho teve também como objectivo o estudo da ROCK1 como regulador do metabolismo da glucose e dos lípidos no fígado. Através do estudo de animais com uma delecção ou uma sobreexpressão da ROCK1 no fígado mostrou-se que esta tem um papel crucial na patogénese da obesidade e diabetes tipo 2, especificamente através do controlo da lipogénese de novo. Finalmente, foi também objectivo deste trabalho, explorar o efeito do MSM em animais DIO e deficientes em leptina. O tratamento com MSM protege de forma evidente contra a obesidade e insulino-resistência, com especial enfâse para a capacidade que esta molécula demonstrou ter na protecção contra a inflamação. Em conjunto os vários estudos aqui apresentados mostram que tanto os S-nitrosotióis como a ROCK1 têm um papel na patogénese da obesidade e diabetes tipo 2 e que a utilização de MSM como suplemento às terapêuticas convencionais pode ter um papel no tratamentos de doenças metabólicas.-------------------------------ABSTRACT: In modern western societies type 2 diabetes and obesity are increasing exponentially, representing a somber public concern. According to the International Diabetes Federation (IDF) ‘Diabetes and Obesity are the biggest public health challenges of the 21st century’. Aside from these the prevalence of nonalcoholic fatty liver disease (NAFLD), among the diabetic and obese population, is as high as 90%. It is now well established that the increase in obesity, diabetes and NAFLD strongly correlates with an increase in fat and sugar intake in our diet, alongside physical inactivity. The pathogenesis of obesity, diabetes and NAFLD has been thoroughly studied but the treatment options available are still narrow. Considering the alarming number in the obese and diabetic population the complete understanding of the pathogenesis, keeping in mind that new therapeutic strategies need to be attained, is of the highest urgency. The liver has been well established as a fundamental organ in regulating whole-body homeostasis. In the fed state the liver converts the glucose into glycogen and lipids. Conversely, in the fasted state, glucose will be produced in the liver. Neuronal and hormonal systems, as well as the hepatic metabolic states, tightly control the fast to fed switch in metabolic fuels. Insulin has a central role in controlling hepatic energy metabolism, by suppressing glucose production and ketogenesis, while stimulating glycolysis and lipogenesis. Liver energy metabolism is also regulated by various transcription factors and coregulators that are, in turn, regulated by insulin, glucagon and other metabolic hormones. Together, these regulators will act to control gluconeogenesis, β-oxidation and lipogenesis in the liver. Aside from the well-established regulators of liver energy metabolism new molecules are being studied has having a role in regulating hepatic metabolism. Any imbalance in the liver energy metabolism is a major contributor to insulin resistance, NAFLD and type 2 diabetes. The overall goal of this research work was to contribute to the understanding of the pathogenesis of diabetes and obesity, on a setting of high-sucrose and high-fat diets, and to explore potential therapeutic options. The specific aims were: first, to determine if treatment with glutathione (GSH) and nitric oxide (NO) was sufficient to ameliorate insulin resistance induced by high-sucrose feeding; second, to determine the physiological role of rho-kinase 1 (ROCK1) in regulating hepatic and lipid metabolism; and third, to study the effect of methylsulfonylmethane (MSM) on obesity-linked metabolic disorders. In the first part of this research work we used male Wistar rats fed a high-sucrose (HS) diet. As expected, rats fed a HS diet were insulin resistant and hyperinsulinemic. HS feeding increased hepatic levels of NO, while decreasing GSH. In fasted healthy animals administration of both GSH and NO, to the liver, was able to increase insulin sensitivity. Intravenous administration of S-nitrosothiols, organic compounds containing a nitroso group attached to the sulfur atom of a thiol, in fasted control animals also increased insulin sensitivity. Under HS feeding the standard doses of GSH + NO and S-nitrosothiols were unable to promote an increase in insulin sensitivity. However, the intravenous administration of increasing concentrations of S-nitrosothiols was able to restore insulin sensitivity, suggesting that S-nitrosothiols have an insulin sensitizing effect. Investigation of the effect of S-nitrosothiols on the insulin signaling pathway showed increased phosphorylation of the insulin receptor (IR) and protein kinase B (Akt), suggesting that S-nitrosothiols may have an effect on the insulin signaling pathway. Together, these data showed that S-nitrosothiols promote normal insulin action, suggesting that they may act as potential pharmacological tools. In the second part of this research work we used liver-specific ROCK1 knockout mice fed a high-fat (HF) diet. Liver-specific deletion of ROCK1 prevented obesity, improved insulin sensitivity and protected against hepatic steatosis. Deficiency of ROCK1 in the liver caused a significant decrease in the gene expression of lipogenesis associated gene, ultimately leading to decreased lipogenesis. Contrariwise, ROCK1 overexpression in the liver promoted insulin resistance and hepatic steatosis in diet-induced obesity. Furthermore, liver-specific deletion of ROCK1 in obese and diabetic mice, the leptin-deficient mice, improved the typical hyperglycemia, hyperinsulinemia and liver steatosis. Together, these data identify ROCK1 as a crucial regulator of lipid metabolism. In the third and final part of this research work we investigated the effect of MSM, an organosulfur compound naturally found in plants and used as a dietary supplement, on diet-induced obese (DIO) and insulin resistant mice. MSM treatment ameliorated insulin resistance and protected against hepatosteatosis. Hepatic content in triglycerides and cholesterol was significantly decreased by MSM treatment, as well as lipogenesis associated gene expression. Furthermore, MSM treated mice had decreased inflammation associated gene expression in the liver. Importantly, FACS analysis showed that MSM treatment rescued the inflammatory hematopoietic phenotype of DIO mice in the bone marrow and the peripheral blood. Moreover, MSM treatment of the obese and diabetic mice, the leptin-deficient mice, resulted in similar effects as the ones observed for DIO mice. Collectively, these data suggest that MSM supplementation has a beneficial effect on hyperinsulinemia, insulin resistance and inflammation, which are often found in type 2 diabetes. In conclusion, this research work showed that S-nitrosothiols may play a role as insulin sensitizers, restoring insulin sensitivity in a setting of high-sucrose induced insulin resistance. Furthermore, in vitro studies suggest that S-nitrosothiols specifically regulate the insulin signaling pathway. This research work also investigated the role of hepatic ROCK1 in regulation of glucose and lipid metabolism. Using liver-specific ROCK 1 knockout and ROCK1 overexpressing mice it was shown that ROCK1 plays a role in the pathogenesis of obesity and type 2 diabetes, specifically through regulation of the de novo lipogenesis pathway. Finally, this research work aimed to explore the effect of MSM in DIO and leptin receptor-deficient mice. MSM strongly protects against obesity and insulin resistance, moreover showed a robust ability to decrease inflammation. Together, the individual studies that compose this dissertation showed that S-nitrosothiols and ROCK1 play a role in the pathogenesis of obesity and type 2 diabetes and that MSM supplementation may have a role in the treatment of metabolic disorders.

Engineering of fatty acid production and secretion in Saccharomyces cerevisiae

Tese de Doutoramento em Ciências - Especialidade em Biologia

Proteomic analysis of nitrate-dependent acetone degradation by Alicycliphilus denitrificans strain BC

Alicycliphilus denitrificans strain BC grows anaerobically on acetone with nitrate as electron acceptor. Comparative proteomics of cultures of A. denitrificans strain BC grown on either acetone or acetate with nitrate was performed to study the enzymes involved in the acetone degradation pathway. In the proposed acetone degradation pathway, an acetone carboxylase converts acetone to acetoacetate, an AMP-dependent synthetase/ligase converts acetoacetate to acetoacetyl-CoA, and an acetyl-CoA acetyltransferase cleaves acetoacetyl-CoA to two acetyl-CoA. We also found a putative aldehyde dehydrogenase associated with acetone degradation. This enzyme functioned as a -hydroxybutyrate dehydrogenase catalyzing the conversion of surplus acetoacetate to -hydroxybutyrate that may be converted to the energy and carbon storage compound, poly--hydroxybutyrate. Accordingly, we confirmed the formation of poly-?-hydroxybutyrate in acetone-grown cells of strain BC. Our findings provide insight in nitrate-dependent acetone degradation that is activated by carboxylation of acetone. This will aid studies of similar pathways found in other microorganisms degrading acetone with nitrate or sulfate as electron acceptor.

Evaluación del efecto del láser y magnetoterapia en miopatía experimental valorando biomarcadores de estrés oxidativo, histomorfometría y actividad enzimática mitocondrial. Evaluation of the effect of laser and magnetic therapy in experimental myopathy assessing oxidative stress biomarkers, histomorphometry and mitochondrial enzyme activity

El láser de baja y media energía y la magnetoterapia son utilizados en desórdenes osteomioarticulares por sus efectos analgésico, antiinflamatorio y trófico, entre los más destacados. Sin embargo, son insuficientes las investigaciones sobre su mecanismo de acción y antecedentes científicos que avalen sus efectos. Es por ello, que la determinación de acontecimientos celulares y moleculares que ocurren durante la interacción de estos tipos de energía con el sistema muscular, sería relevante para el conocimiento y optimización de tales terapias en las ciencias biomédicas. En las miopatías inflamatorias idiopáticas, se encuentra afectada la estructura, morfología y bioquímica del tejido muscular. La energía que éste requiere para el normal funcionamiento es generada en la mitocondria. Esta organela también es la responsable de la generación de especies oxidantes provocando estrés oxidativo y el inicio de los procesos de apoptosis. Por lo antes dicho, consideramos que la determinación de los biomarcadores inflamatorios asociados a estrés oxidativo, realizando el análisis histomorfométrico ultraestructural y valorando la actividad de los complejos enzimáticos mitocondriales, permitiría una evaluación de la acción terapéutica del láser y la magnetoterapia en un modelo experimental de miopatía. Para ello se propone evaluar el efecto de la magnetoterapia y del láser de baja energía (He-Ne y As.Ga) en miopatía experimental determinando indicadores inflamatorios asociados a estrés oxidativo, análisis histomorfométrico y valoración de la actividad enzimática mitocondrial. Específicamente: -Determinar indicadores inflamatorios y de estrés oxidativo: Oxido Nítrico, Grupos carbonilos, L-citrulina, Fibrinógeno, Superóxido dismutasa, Glutation peroxidasa y Catalasa por espectrofotometría. -Identificar los cambios anatomopatológicos del músculo esquelético por microscopía óptica (MO): cuantificación del infiltrado inflamatorio; MO de alta resolución (MOAR) y por microscopía electrónica: histomorfometría de la ultraestructura miofibrilar y mitocondrial. -Valorar las actividades enzimáticas de la citrato sintasa y de los complejos: I (NADH-ubiquinona reductasa), II (succinato-ubiquinona-reductasa) III (ubiquinona-citocromo c-reductasa) y IV (citocromo c-oxidasa); en mitocondrias de tejido muscular por espectrofotometría. -Evaluar la actividad apoptótica en las fibras musculares de los diferentes grupos por ténica de T.U.N.E.L. Las mediciones mitocondriales (por ME) y de infiltrado inflamatorio (por MO) se realizarán en un total de 5 fotos de aumentos similares en forma aleatoria por grupo estudiado (n=10). Los cambios estructurales observados se analizarán en el programa Axiovision 4.8, para cuantificar el área total ocupada, número total y grado de alteración de las mitocondrias y el porcentaje de infiltrado inflamatorio determinando el grado de inflamación. Los resultados de los datos cuantitativos se analizarán aplicando ANAVA (test de Fisher para comparaciones múltiples); y para los datos categóricos se utilizará Chi cuadrado (test de Pearson), estableciéndose un nivel de significación de p < 0.05 para todos los casos. Importancia del Proyecto: La salud y el bienestar del hombre son los logros perseguidos por las ciencias de la salud. La obtención de terapias curativas o paliativas con un mínimo de efectos colaterales para el enfermo se incluye en estos logros. Por esto y todo lo anteriormente expuesto es que consideramos de gran importancia poder esclarecer desde las ciencias básicas los efectos celulares y moleculares en modelos experimentales la acción de la terapia con láser y magnetoterapia para una aplicación clínica con base científica en todas las áreas de las Ciencias Médicas. In the idiopathic inflammatory myopathies, is affected the structure, morphology and biochemistry of muscle tissue. The mitochondria is responsible for the generation of oxidizing species leading to oxidative stress and the beginning of the process of apoptosis. As said before, we consider the determination of inflammatory biomarkers related to oxidative stress, by ultrastructural morphometric analysis and assessing the activity of mitochondrial enzyme complexes, permit an evaluation of the therapeutic action of laser and magnetic therapy in an experimental model myopathy. We propose to evaluate the effect of the treatment identifying indicators in experimental inflammatory myopathy associated with oxidative stress, histomorphometric analysis and assessment of mitochondrial enzyme activity. Specifically -determining: Nitric oxide, carbonyl groups, L-citrulline, fibrinogen, superoxide dismutase, glutathione peroxidase and catalase by spectrophotometry. -Identify the pathological changes in skeletal muscle by optical microscopy (OM): quantification of the inflammatory infiltrate, OM high resolution (MOAR) and electron microscopy, histomorphometry of myofibrillar and mitochondrial ultrastructure. -Evaluate the enzymatic activity of citrate synthase and complexes: I, II, III and IV in mitochondria muscle tissue by spectrophotometry. -Evaluate apoptotic activity in muscle fibers by TUNEL technique of Mitochondrial measurements and inflammatory infiltration (by OM) was performed in a total of 5 photos of similar increases in random by the study group (n = 10). The structural changes observed are discussed in the program Axiovision 4.8, to quantify number, degree of alteration of mitochondria and the percentage of inflammatory infiltrate determining the degree of inflammation. The results of the quantitative data were analyzed using ANOVA (Fisher test), and categorical data with Chi-square (Pearson test), establishing a significance level of p <0.05.

Development of sensing systems for environmental monitoring

This project focused on the investigation and the development of a chemical sensing system for the determination of chromium Cr6+ and a bio-reactor followed by electrochemical detection at a glassy carbon electrode, for the determination of organochlorine compounds. The conjugation of Cr6+ with 1,5-diphenylcarbazide was studied at various types of electrodes such as glassy carbon, ultra-trace epoxy-graphite, chemically or un-modified carbon-paste and dropping-mercury. The cyclic voltammetric behaviour of the complex was also investigated. In addition, the possibility of developing a chemical sensor, Le. an electrochemical probe capable of sensing Cr6+ through its complexation with 1,5-diphenylacarbazide was studied. The conjugations of l-chloro-2,4-dinitrobenzene, 2,4-dichloronitrobenzene and ethacrynic, which are electrophilic organochlorine compounds, with reduced glutathione, were studied in order to test the bioreactor developed, based on the immobilisation of glutathione s-transferase. This was carried out at different types of electrodes such as glassy-carbon, gold, silver, platinum, epoxy-graphite, hangingmercury, and ferrocene-modified rotating-disc electrodes.

Tramadol Alleviates Myocardial Injury Induced by Acute Hindlimb Ischemia Reperfusion in Rats

AbstractBackground:Organ injury occurs not only during periods of ischemia but also during reperfusion. It is known that ischemia reperfusion (IR) causes both remote organ and local injuries.Objective:This study evaluated the effects of tramadol on the heart as a remote organ after acute hindlimb IR.Methods:Thirty healthy mature male Wistar rats were allocated randomly into three groups: Group I (sham), Group II (IR), and Group III (IR + tramadol). Ischemia was induced in anesthetized rats by left femoral artery clamping for 3 h, followed by 3 h of reperfusion. Tramadol (20 mg/kg, intravenous) was administered immediately prior to reperfusion. At the end of the reperfusion, animals were euthanized, and hearts were harvested for histological and biochemical examination.Results:The levels of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were higher in Groups I and III than those in Group II (p < 0.05). In comparison with other groups, tissue malondialdehyde (MDA) levels in Group II were significantly increased (p < 0.05), and this increase was prevented by tramadol. Histopathological changes, including microscopic bleeding, edema, neutrophil infiltration, and necrosis, were scored. The total injuryscore in Group III was significantly decreased (p < 0.05) compared with Group II.Conclusion:From the histological and biochemical perspectives, treatment with tramadol alleviated the myocardial injuries induced by skeletal muscle IR in this experimental model.

Charakterisierung von Teratomcybriden mit der primären LHON-Mutation G11778A

LHON, cybrids, ROS, ATP, glutathione, oxidative stress, SOD, GPx1, glutathione peroxidase

Pentoxifylline Attenuates Cardiac Remodeling Induced by Tobacco Smoke Exposure

Abstract Background: Tobacco smoke exposure is an important risk factor for cardiac remodeling. Under this condition, inflammation, oxidative stress, energy metabolism abnormalities, apoptosis, and hypertrophy are present. Pentoxifylline has anti‑inflammatory, anti-apoptotic, anti-thrombotic and anti-proliferative properties. Objective: The present study tested the hypothesis that pentoxifylline would attenuate cardiac remodeling induced by smoking. Methods: Wistar rats were distributed in four groups: Control (C), Pentoxifylline (PX), Tobacco Smoke (TS), and PX-TS. After two months, echocardiography, invasive blood pressure measurement, biochemical, and histological studies were performed. The groups were compared by two-way ANOVA with a significance level of 5%. Results: TS increased left atrium diameter and area, which was attenuated by PX. In the isolated heart study, TS lowered the positive derivate (+dp/dt), and this was attenuated by PX. The antioxidants enzyme superoxide dismutase and glutathione peroxidase were decreased in the TS group; PX recovered these activities. TS increased lactate dehydrogenase (LDH) and decreased 3-hydroxyacyl Coenzyme A dehydrogenases (OH-DHA) and citrate synthase (CS). PX attenuated LDH, 3-OH-DHA and CS alterations in TS-PX group. TS increased IL-10, ICAM-1, and caspase-3. PX did not influence these variables. Conclusion: TS induced cardiac remodeling, associated with increased inflammation, oxidative stress, apoptosis, and changed energy metabolism. PX attenuated cardiac remodeling by reducing oxidative stress and improving cardiac bioenergetics, but did not act upon cardiac cytokines and apoptosis.

Glutathion no sangue humano normal

1.) - Woodward-Fry's and Okuda-Hess technics were employed in the determination of blood glutathione in normal healthy adults of both sexes. 2.) - It was found more accurately results with the technic of Woodward and Fry than of any others for the dosage of G. S. H. of blood. 3.) - When the process of Okuda-Hess is modified by the use of an intern indicator (starch) the readings of the end-point are much more easy and therefore the results more exacts. 4.) - The averages of the data obtained for normal blood by the technic of Woodward and Fry were for men per hundred cubic cent. 27 mgrs (G.S.H); 6.6 mgrs. (G.S.S) and 33.6 mges. (G. T) and for women: 28.4 mgrs. (G.S.H), 7.8 mgrs. (G.S.S) and 36,2 mgrs. (G.T). 5.) - Autoxidation in the blood filtrate is only apreciated after 24 hs. In the first eight hours autoxidation is never observed. 6.) - The increase of glutathione in hyperglobulia is a function of the amount of red corpuscles. In acrocyanosis arterial blood is richest in these component than venous blood and this fact is in accordance with the observation of Blanchetière, Mélon and Binet for the experimental asphyxia of dogs.

Endothelial mineralocorticoid receptor activation mediates endothelial dysfunction in diet-induced obesity.

AIMS: Aldosterone plays a crucial role in cardiovascular disease. 'Systemic' inhibition of its mineralocorticoid receptor (MR) decreases atherosclerosis by reducing inflammation and oxidative stress. Obesity, an important cardiovascular risk factor, is an inflammatory disease associated with increased plasma aldosterone levels. We have investigated the role of the 'endothelial' MR in obesity-induced endothelial dysfunction, the earliest stage in atherogenesis. METHODS AND RESULTS: C57BL/6 mice were exposed to a normal chow diet (ND) or a high-fat diet (HFD) alone or in combination with the MR antagonist eplerenone (200 mg/kg/day) for 14 weeks. Diet-induced obesity impaired endothelium-dependent relaxation in response to acetylcholine, whereas eplerenone treatment of obese mice prevented this. Expression analyses in aortic endothelial cells isolated from these mice revealed that eplerenone attenuated expression of pro-oxidative NADPH oxidase (subunits p22phox, p40phox) and increased expression of antioxidative genes (glutathione peroxidase-1, superoxide dismutase-1 and -3) in obesity. Eplerenone did not affect obesity-induced upregulation of cyclooxygenase (COX)-1 or prostacyclin synthase. Endothelial-specific MR deletion prevented endothelial dysfunction in obese (exhibiting high 'endogenous' aldosterone) and in 'exogenous' aldosterone-infused lean mice. Pre-incubation of aortic rings from aldosterone-treated animals with the COX-inhibitor indomethacin restored endothelial function. Exogenous aldosterone administration induced endothelial expression of p22phox in the presence, but not in the absence of the endothelial MR. CONCLUSION: Obesity-induced endothelial dysfunction depends on the 'endothelial' MR and is mediated by an imbalance of oxidative stress-modulating mechanisms. Therefore, MR antagonists may represent an attractive therapeutic strategy in the increasing population of obese patients to decrease vascular dysfunction and subsequent atherosclerotic complications.

High-resolution spatial mapping of changes in the neurochemical profile after focal ischemia in mice.

After ischemic stroke, the ischemic damage to brain tissue evolves over time and with an uneven spatial distribution. Early irreversible changes occur in the ischemic core, whereas, in the penumbra, which receives more collateral blood flow, the damage is more mild and delayed. A better characterization of the penumbra, irreversibly damaged and healthy tissues is needed to understand the mechanisms involved in tissue death. MRSI is a powerful tool for this task if the scan time can be decreased whilst maintaining high sensitivity. Therefore, we made improvements to a (1) H MRSI protocol to study middle cerebral artery occlusion in mice. The spatial distribution of changes in the neurochemical profile was investigated, with an effective spatial resolution of 1.4 μL, applying the protocol on a 14.1-T magnet. The acquired maps included the difficult-to-separate glutamate and glutamine resonances and, to our knowledge, the first mapping of metabolites γ-aminobutyric acid and glutathione in vivo, within a metabolite measurement time of 45 min. The maps were in excellent agreement with findings from single-voxel spectroscopy and offer spatial information at a scan time acceptable for most animal models. The metabolites measured differed with respect to the temporal evolution of their concentrations and the localization of these changes. Specifically, lactate and N-acetylaspartate concentration changes largely overlapped with the T(2) -hyperintense region visualized with MRI, whereas changes in cholines and glutathione affected the entire middle cerebral artery territory. Glutamine maps showed elevated levels in the ischemic striatum until 8 h after reperfusion, and until 24 h in cortical tissue, indicating differences in excitotoxic effects and secondary energy failure in these tissue types. Copyright © 2011 John Wiley & Sons, Ltd.

GP38, P28-I and P28-II: candidates for a vaccine against Schistosomiasis

Three antigens protective against Schistosoma mansoni have been extensively characterized. The schistosomulum surface antigen GP38 possesses an immunodominant carbohydrate epitope of which the structure has been defined. Protection can be achieved via the transfer of monoclonal antibodies recognizing the epitope or by immunization with anti-idiotype monoclonal antibodies. The glycan epitope is shared with the intermediate host, Biomphalaria glabrata as well as being present on other molluscs, including the Keyhole Limpet. A group of molecules at 28 kDa were initially characterized in adult worms and shown to protect rats and mice against a challenge infection. One of these molecules, P28-I, was cloned and expressed in E. coli, yeast and vaccinia virus. The recombinant antigen significantly protected rats, hamsters and baboons against a challenge infection. P28-I is a glutathione-S-transferase and the recombinant antigen produced in yeast exhibits the enzyme activity and has been purified to homogeneity by affinity chromatography. A second P28 antigen, P28-II, has also been cloned, fully sequenced and expressed. This recombinant antigen also protects against S. mansoni infection.

Aggregating Brain Cell Cultures as a Model to Study Ischaemia-induced Neurodegeneration.

Rotation-mediated aggregating brain cell cultures at two different maturational stages (DIV 11 and DIV 20) were subjected for 1 or 2 hours to ischaemic conditions by transient immobilization (arrest of media circulation). During recovery, cell damage was evaluated by measuring changes in cell type-specific enzyme activities and total protein content. It was found that in immature cultures (DIV 11), immobilization for 1 or 2 hours did not affect the parameters measured. By contrast, at DIV 20, ischaemic conditions for 1 hour caused a pronounced decrease in the activities of glutamic acid decarboxylase and choline acetyltransferase. A significant decrease in these neuron-specific enzyme activities was found at post-ischaemic days 1-14, indicating immediate and irreversible neuronal damage. The activity of the astrocyte-specific enzyme, glutamine synthetase, was significantly increased at 4 days post-treatment; equal to control values at 6 days; and significantly decreased at 14 days after the ischaemic insult. Immobilization of DIV 20 cultures for 2 hours caused a drastic reduction in all the parameters measured at post-ischaemic day 6. Generally, the ischaemic conditions appeared to be more detrimental to neurons than to astrocytes, and GABAergic neurons were more affected than cholinergic neurons.

NEUROPATHOPHYSIOLOGICAL MECHANISMS IN GLUTARIC ACIDURIA TYPE I: 3-HYDROXYGLUTARIC ACID LEADS TO AMMONIA INCREASE AND NON-APOPTOTIC CELL DEATH

A 3D in vitro model of rat organotypic brain cell cultures in aggregates was used to investigate neurotoxicity mechanisms in glutaric aciduria type I (GA-I). 1 mM glutarate (GA) or 3-hydroxyglutarate (3OHGA) were repeatedly added to the culture media at two different time points. In cultures treated with 3OHGA, we observed an increase in lactate in the medium, pointing to a possible inhibition of Krebs cycle and respiratory chain. We further observed that 3OHGA and to a lesser extend GA induced an increase in ammonia production with concomitant decrease of glutamine concentrations, which may suggest an inhibition of the astrocytic enzyme glutamine synthetase. These previously unreported findings may uncover a pathogenic mechanism in this disease which has deleterious effects on early stages of brain development. By immunohistochemistry we showed that 3OHGA increased non-apoptotic cell death. On the cellular level, 3OHGA and to a lesser extend GA led to cell swelling and loss of astrocytic fibers whereas a loss of oligodendrocytes was only observed for 3OHGA. We conclude that 3OHGAwas the most toxic metabolite in our model for GA-I. 3OHGA induced deleterious effects on glial cells, an increase of ammonia production, and resulted in accentuated cell death of non-apoptotic origin.

Effects of the naturally occurring food mycotoxin ochratoxin A on brain cells in culture.

The potential of ochratoxin A (OTA) to damage brain cells was studied by using a three-dimensional cell culture system as model for the developing brain. Aggregating cell cultures of foetal rat telencephalon were tested either during an early developmental period, or during a phase of advanced maturation, over a wide range of OTA concentrations (0.4 nM to 50 microM). By monitoring changes in activities of cell type-specific enzymes (ChAt and GAD, for cholinergic and GABAergic neurones, respectively, GS for astrocytes and CNP for oligodendrocytes), the concentration-dependent toxicity and neurodevelopmental effects of OTA were determined. OTA proved to be highly toxic, since a 10-day treatment at 50 nM caused a general cytotoxicity in both mature and immature cultures. At 10 nM of OTA, cell type-specific effects were observed: in immature cultures, a loss in neuronal and oligodendroglial enzyme activities, and an increase in the activity of the astroglial marker glutamine synthetase were found, Furthermore, at 2 and 10 nM of OTA, a clustering of microglial cells was observed. In mature cultures, OTA was somewhat less potent, but caused a similar pattern of toxic effects. A 24 h-treatment with OTA resulted in a concentration-dependent decrease in protein synthesis, with IC50 values of 25 nM and 33 nM for immature and mature cultures respectively. Acute (24 h) treatment at high OTA concentrations (10 to 50 microM) caused a significant increase in reactive oxygen species formation, as measured by the intracellular oxidation of 2',7'-dichlorofluorescin. These results suggest that OTA has the potential to be a potent toxicant to brain cells, and that its effects at nanomolar concentrations are primarily due to the inhibition of protein synthesis, whereas ROS seem not to be involved in the toxicity mediated by a chronic exposure to OTA at such low concentrations.