RESUMO
Mitochondrial adrenodoxins (ADXs) are small iron-sulfur proteins with electron transfer properties. In animals, ADXs transfer electrons between an adrenodoxin reductase (ADXR) and mitochondrial P450s, which is crucial for steroidogenesis. Here we show that a plant mitochondrial steroidogenic pathway, dependent on an ADXR-ADX-P450 shuttle, is essential for female gametogenesis and early embryogenesis through a maternal effect. The steroid profile of maternal and gametophytic tissues of wild-type (WT) and adxr ovules revealed that homocastasterone is the main steroid present in WT gametophytes and that its levels are reduced in the mutant ovules. The application of exogenous homocastasterone partially rescued adxr and P450 mutant phenotypes, indicating that gametophytic homocastasterone biosynthesis is affected in the mutants and that a deficiency of this hormone causes the phenotypic alterations observed. These findings also suggest not only a remarkable similarity between steroid biosynthetic pathways in plants and animals but also a common function during sexual reproduction.
Assuntos
Adrenodoxina/metabolismo , Arabidopsis/embriologia , Ferredoxina-NADP Redutase/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Sistema Enzimático do Citocromo P-450/fisiologia , Transporte de Elétrons , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/fisiologia , Desenvolvimento Embrionário/genética , Gametogênese/fisiologia , Células Germinativas Vegetais/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Fitosteróis/biossíntese , Ligação ProteicaRESUMO
Amidines are chemically characterized by the presence of two nitrogen atoms that bind to the same carbon atom in its structure. Several biological activities have been ascribed to these compounds. Pentamidine, an aromatic diamidine, is effective in the treatment against Pneumocystis carinii and leishmaniasis, but it can also have severe side effects. New amidine derivatives have been synthesized, among them N,N'-diphenyl-4-methoxy-benzamidine (methoxyamidine), which is effective against Leishmania amazonensis (LD50 = 20 µM) and Trypanosoma cruzi (LD50 = 59 nM). In the present study, methoxyamidine toxicity was evaluated in isolated rat liver mitochondria at the same range of concentrations that exert antiprotozoal activity. In these organelles, actively oxidizing glutamate + malate inhibited state 3 respiration (25 nmol mg-1 of protein) by â¼15%. The sites of inhibition in the respiratory chain were complex I and the segment between ubiquinone and complex III. Methoxyamidine also stimulated state 4 respiration by â¼32% and â¼43% at 50 and 65 nmol mg-1 of protein, respectively. Its uncoupling effect was confirmed by a dose-dependent increase in oxygen consumption in state 4 respiration that was induced by oligomycin, reaching up to â¼69% (65 nmol mg-1 of protein) and an increase in ATPase activity in intact mitochondria by â¼27% and â¼83% at 50 and 65 nmol mg-1 protein, respectively. Swelling that was supported by the oxidation of glutamate + malate in the presence of sodium acetate was reduced by methoxyamidine by â¼16% and 32% at 50 and 65 nmol mg-1 protein, respectively. Mitochondrial swelling in the absence of substrate and in the presence of K+ and valinomycin was inhibited by â¼20% at the same concentrations, suggesting that methoxyamidine affects mitochondrial membrane permeability and fluidity. Our data show that methoxyamidine has slight effects on the energy-linked functions of isolated mitochondria at concentrations that correspond to the LD50 against Leishmania amazonensis and Trypanosoma cruzi. These findings may prompt further studies that evaluate methoxyamidine toxicity in vivo.
Assuntos
Antiprotozoários/farmacologia , Benzamidinas/farmacologia , Metabolismo Energético/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Animais , Antiprotozoários/química , Benzamidinas/química , Complexo de Proteínas da Cadeia de Transporte de Elétrons/fisiologia , Masculino , Consumo de Oxigênio/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
Reactive oxygen species are a by-product of mitochondrial oxidative phosphorylation, derived from a small quantity of superoxide radicals generated during electron transport. We conducted a comprehensive and quantitative study of oxygen consumption, inner membrane potentials, and H(2)O(2) release in mitochondria isolated from rat brain, heart, kidney, liver, and skeletal muscle, using various respiratory substrates (alpha-ketoglutarate, glutamate, succinate, glycerol phosphate, and palmitoyl carnitine). The locations and properties of reactive oxygen species formation were determined using oxidative phosphorylation and the respiratory chain modulators oligomycin, rotenone, myxothiazol, and antimycin A and the uncoupler CCCP. We found that in mitochondria isolated from most tissues incubated under physiologically relevant conditions, reactive oxygen release accounts for 0.1-0.2% of O(2) consumed. Our findings support an important participation of flavoenzymes and complex III and a substantial role for reverse electron transport to complex I as reactive oxygen species sources. Our results also indicate that succinate is an important substrate for isolated mitochondrial reactive oxygen production in brain, heart, kidney, and skeletal muscle, whereas fatty acids generate significant quantities of oxidants in kidney and liver. Finally, we found that increasing respiratory rates is an effective way to prevent mitochondrial oxidant release under many, but not all, conditions. Altogether, our data uncover and quantify many tissue-, substrate-, and site-specific characteristics of mitochondrial ROS release.
Assuntos
Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio , Animais , Encéfalo/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo de Proteínas da Cadeia de Transporte de Elétrons/fisiologia , Rim/metabolismo , Fígado/metabolismo , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Moduladores de Transporte de Membrana/farmacologia , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Especificidade de Órgãos , Fosforilação Oxidativa , Estresse Oxidativo , Consumo de Oxigênio , Ratos , Ratos Sprague-Dawley , Especificidade por SubstratoRESUMO
There is growing evidence to suggest that chagasic myocardia are exposed to sustained oxidative stress-induced injuries that may contribute to disease progression. Pathogen invasion- and replication-mediated cellular injuries and immune-mediated cytotoxic reactions are the common source of reactive oxygen species (ROS) in infectious etiologies. However, our understanding of the source and role of oxidative stress in chagasic cardiomyopathy (CCM) remains incomplete. In this review, we discuss the evidence for increased oxidative stress in chagasic disease, with emphasis on mitochondrial abnormalities, electron transport chain dysfunction and its role in sustaining oxidative stress in myocardium. We discuss the literature reporting the consequences of sustained oxidative stress in CCM pathogenesis.
Assuntos
Cardiomiopatia Chagásica/etiologia , Mitocôndrias Cardíacas/metabolismo , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio , Animais , Cardiomiopatia Chagásica/imunologia , Cardiomiopatia Chagásica/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/fisiologia , Humanos , Trypanosoma cruzi/imunologiaRESUMO
Há evidências que sugerem que as miocardites chagásicas são devidas aos danos induzidos pelo estresse oxidativo, podendo contribuir para a evolução da doença de Chagas. Em doenças infecciosas, a formação de espécies reativas do oxigênio (ROS) é, principalmente, derivada de danos celulares mediados pela invasão e replicação do patógeno e por reações citotóxicas mediadas pelo sistema imune. No entanto, como as ROS são formadas e sua função no estresse oxidativo na cardiomiopatia chagásica (CCM) não estão completamente elucidadas. Nesta revisão, nós discutimos as evidências para o aumento do estresse oxidativo na doença de Chagas, com ênfase nas anormalidades mitocondriais, na disfunção da cadeia de transporte de elétrons e seu papel na manutenção do estresse oxidativo no miocárdio. Discutimos ainda, os resultados da literatura que relatam as conseqüências da manutenção do estresse oxidativo na patogênese da CCM.