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1.
Bioorg Med Chem Lett ; 28(8): 1330-1335, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29576509

RESUMO

Succinate-cytochrome c reductase (SCR) is composed of a mixture of mitochondrial complex II (succinate-ubiquinone oxidoreductase) and complex III (cytochrome bc1 complex). Meanwhile, complexes II and III are two promising targets of numerous antibiotics and fungicides. With an aim to identify new lead structures for SCR, complex II or III, a new series of 4-aryloxy-N-arylanilines were synthesized by introducing a 4-aryloxy phenyl group as one of the aryl groups in diaryl amines. With the economic Cu(OAc)2·H2O as the optimal copper promoter, a simple and facile protocol was utilized to afford 24 target products in 56-93% yields. Furthermore, extensive screening results suggested variable inhibitory activities of these compounds against SCR. Exceptionally, compounds 7k-7n showed excellent inhibition potency with their IC50 values in the nanomolar range, demonstrating higher potency than the commercial controls (penthiopyrad and azoxystrobin) by over one order of magnitude.


Assuntos
Compostos de Anilina/farmacologia , Inibidores Enzimáticos/farmacologia , Succinato Citocromo c Oxirredutase/antagonistas & inibidores , Compostos de Anilina/síntese química , Compostos de Anilina/química , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Estrutura Molecular , Pirazóis/farmacologia , Pirimidinas/farmacologia , Estrobilurinas/farmacologia , Tiofenos/farmacologia
2.
Toxicol Sci ; 134(2): 366-73, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23704229

RESUMO

Depletion of glutathione has been shown to occur in autopsied brains of patients with Parkinson's disease (PD) and in animal models of PD. The goal of this study was to determine whether chronic glutathione (GSH) deficiency per se resulted in complex I inhibition and/or dopamine depletion and whether these indices were further potentiated by aging or administration of paraquat, a redox-cycling herbicide that produces a PD-like neurodegeneration model in rodents (Brooks, A. I., Chadwick, C. A., Gelbard, H. A., Cory-Slechta, D. A., and Federoff, H. J. [1999]. Paraquat elicited neurobehavioral syndrome caused by dopaminergic neuron loss. Brain Res. 823, 1-10; McCormack, A. L., Thiruchelvam, M., Manning-Bog, A. B., Thiffault, C., Langston, J. W., Cory-Slechta, D. A., and Di Monte, D. A. [2002]. Environmental risk factors and Parkinson's disease: Selective degeneration of nigral dopaminergic neurons caused by the herbicide paraquat. Neurobiol. Dis. 10, 119-127.) Deletion of the rate-limiting GSH synthesis gene, glutamate-cysteine ligase modifier subunit (Gclm), leads to significantly lower GSH concentrations in all tissues including brain. Gclm null (Gclm (-/-)) mice provide a model of prolonged GSH depletion to explore the relationship between GSH, complex I inhibition, and dopamine loss in vivo. Despite ~60% depletion of brain GSH in Gclm (-/-) mice of ages 3-5 or 14-16 months, striatal complex I activity, dopamine levels, 3-nitrotyroine/tyrosine ratios, aconitase activity, and CoASH remained unchanged. Administration of paraquat (10mg/kg, twice/week, 3 weeks) to 3- to 5-month-old Gclm (-/-) mice resulted in significantly decreased aconitase activity, complex I activity, and dopamine levels but not in 3- to 5-month-old Gclm (+/+) mice. Furthermore, paraquat-induced inhibition of complex I and aconitase activities in Gclm (-/-) mice was observed in the striatum but not in the cortex. The results suggest that chronic deficiency of GSH in Gclm (-/-) mice was not sufficient to result in complex I inhibition or dopamine depletion perhaps due to homeostatic mechanisms but required an additional oxidative stress insult as shown with paraquat exposure.


Assuntos
Dopamina/metabolismo , Glutamato-Cisteína Ligase/genética , Glutationa/deficiência , Herbicidas/toxicidade , Paraquat/toxicidade , Succinato Citocromo c Oxirredutase/genética , Animais , Sequência de Bases , Primers do DNA , Camundongos , Camundongos Knockout
3.
Physiol Res ; 61(3): 259-65, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22480420

RESUMO

Digitonin solubilizes mitochondrial membrane, breaks the integrity of the respiratory chain and releases two mobile redox-active components: coenzyme Q (CoQ) and cytochrome c (cyt c). In the present study we report the inhibition of glycerol-3-phosphate- and succinate-dependent oxygen consumption rates by digitonin treatment. Our results show that the inhibition of oxygen consumption rates is recovered by the addition of exogenous synthetic analog of CoQ idebenone (hydroxydecyl-ubiquinone; IDB) and cyt c. Glycerol-3-phosphate oxidation rate is recovered to 148 % of control values, whereas succinate-dependent oxidation rate only to 68 %. We find a similar effect on the activities of glycerol-3-phosphate and succinate cytochrome c oxidoreductase. Our results also indicate that succinate-dependent oxidation is less sensitive to digitonin treatment and less activated by IDB in comparison with glycerol-3-phosphate-dependent oxidation. These findings might indicate the different mechanism of the electron transfer from two flavoprotein-dependent dehydrogenases (glycerol-3-phosphate dehydrogenase and succinate dehydrogenase) localized on the outer and inner face of the inner mitochondrial membrane, respectively.


Assuntos
Digitonina/farmacologia , Glicerofosfatos/metabolismo , Mitocôndrias Hepáticas/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Ácido Succínico/metabolismo , Ubiquinona/análogos & derivados , Animais , Citocromos c/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Glicerolfosfato Desidrogenase/metabolismo , Hipertireoidismo/metabolismo , Cinética , Masculino , Mitocôndrias Hepáticas/metabolismo , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Oxirredução , Ratos , Ratos Wistar , Recuperação de Função Fisiológica , Succinato Citocromo c Oxirredutase/metabolismo , Ubiquinona/farmacologia
4.
Am J Emerg Med ; 30(8): 1540-8, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22386359

RESUMO

INTRODUCTION: Ventricular fibrillation (VF) and asphyxia account for most cardiac arrests but differ in cardiac arrest course, neurologic deficit, and myocardial damage. In VF resuscitation, cardiac mitochondria were known to be damaged via excess generation of reactive oxygen species. This study evaluated the difference of cardiac mitochondrial damages between VF and asphyxial cardiac arrests. METHODS: In the VF + electrical shock (ES) group, VF was induced and untreated for 5 minutes, followed by 1 minute of cardiopulmonary resuscitation (CPR) and 1 ES of 5 J. Animals were killed immediately after ES. In the asphyxia group, cardiac arrest was induced by airway obstruction, and then pulselessness was maintained for 5 minutes, followed by 1 minute of CPR. The animals were killed immediately after CPR. The histology and ultrastructural changes of myocardium and complex activities and respiration of mitochondria were evaluated. The mitochondrial permeability transition pore opening was measured based on mitochondrial swelling rate. RESULTS: The histopathologic examinations showed myocardial necrosis and mitochondrial damage in both cardiac arrests. Instead of regional damages of myocardium in the VF + ES group, the myocardial injury in the asphyxia group distributed diffusely. The asphyxia group demonstrated more severe mitochondrial damage than the VF + ES group, which had a faster mitochondrial swelling rate, more decreased cytochrome c oxidase activity, and more impaired respiration. CONCLUSIONS: Both VF and asphyxial cardiac arrests caused myocardial injuries and mitochondrial damages. Asphyxial cardiac arrest presented more diffuse myocardial injuries and more severe mitochondrial damages than VF cardiac arrest.


Assuntos
Asfixia/patologia , Parada Cardíaca/patologia , Mitocôndrias Cardíacas/patologia , Miocárdio/patologia , Fibrilação Ventricular/patologia , Animais , Asfixia/complicações , Parada Cardíaca/etiologia , Masculino , Malondialdeído/metabolismo , Microscopia Eletrônica de Transmissão , Mitocôndrias Cardíacas/metabolismo , Membranas Mitocondriais/patologia , NADH Desidrogenase/metabolismo , Estresse Oxidativo , Ratos , Ratos Wistar , Succinato Citocromo c Oxirredutase/metabolismo , Fibrilação Ventricular/complicações
5.
Metabolism ; 61(8): 1073-86, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22365040

RESUMO

Diabetic nephropathy is the most common cause of chronic renal failure in industrialized countries. Depletion of podocytes plays an important role in the progression of diabetic glomerulopathy. Various factors in the diabetic milieu lead to serious podocyte stress driving the cells toward cell cycle arrest (p27(Kip1)), hypertrophy, detachment, and apoptosis. Mitochondria are responsible for oxidative phosphorylation and energy supply in podocytes. Recent studies indicated that mitochondrial dysfunction is a key factor in diabetic nephropathy. In the present study, we investigated metabolic profiles of podocytes under diabetic conditions. We examined oxygen consumption rates (OCRs) and oxidative phosphorylation complex activities in murine podocytes. Cells were exposed to high glucose for 48 hours, cultured for 10 passages under high-glucose conditions (30 mmol/L), or incubated with transforming growth factor-ß (5 ng/mL) for 24 hours. After prolonged exposure to high glucose, podocytes showed a significantly increased OCR at baseline and also a higher OCR after addition of oligomycin, indicating significant changes in mitochondrial energy metabolism. Higher OCRs after inhibition of respiration by rotenone also indicated changes in nonmitochondrial respiration. Podocytes stimulated with a proapoptotic concentration of transforming growth factor-ß displayed similar bioenergetic profiles, even with decreased citrate synthase activity. In all tested conditions, we found a higher cellular nicotinamide adenine dinucleotide content and changes in activities of respiratory chain complexes. In summary, we provide for the first time evidence that key factors of the diabetic milieu induce changes in glucose metabolism and mitochondrial function in podocytes.


Assuntos
Glicemia/metabolismo , Nefropatias Diabéticas/metabolismo , Hiperglicemia/metabolismo , Glomérulos Renais , Mitocôndrias/metabolismo , Consumo de Oxigênio , Podócitos/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Células Cultivadas , Diabetes Mellitus Experimental , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/fisiopatologia , Fluorometria/métodos , Membrana Basal Glomerular/efeitos dos fármacos , Membrana Basal Glomerular/metabolismo , Barreira de Filtração Glomerular/efeitos dos fármacos , Transportador de Glucose Tipo 1/metabolismo , Hiperglicemia/etiologia , Imuno-Histoquímica , Indicadores e Reagentes , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Glomérulos Renais/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos , Mitocôndrias/enzimologia , Oxazinas , Oxirredução , Fosforilação , Podócitos/efeitos dos fármacos , Podócitos/enzimologia , Podócitos/patologia , Espécies Reativas de Oxigênio/metabolismo , Succinato Citocromo c Oxirredutase/metabolismo , Succinato Desidrogenase/metabolismo , Xantenos
6.
Cell Res ; 22(1): 127-41, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21577235

RESUMO

Mitochondrial catastrophe can be the cause or consequence of apoptosis and is associated with a number of pathophysiological conditions. The exact relationship between mitochondrial catastrophe and caspase activation is not completely understood. Here we addressed the underlying mechanism, explaining how activated caspase could feedback to attack mitochondria to amplify further cytochrome c (cyto.c) release. We discovered that cytochrome c1 (cyto.c1) in the bc1 complex of the mitochondrial respiration chain was a novel substrate of caspase 3 (casp.3). We found that cyto.c1 was cleaved at the site of D106, which is critical for binding with cyto.c, following apoptotic stresses or targeted expression of casp.3 into the mitochondrial intermembrane space. We demonstrated that this cleavage was closely linked with further cyto.c release and mitochondrial catastrophe. These mitochondrial events could be effectively blocked by expressing non-cleavable cyto.c1 (D106A) or by caspase inhibitor z-VAD-fmk. Our results demonstrate that the cleavage of cyto.c1 represents a critical step for the feedback amplification of cyto.c release by caspases and subsequent mitochondrial catastrophe.


Assuntos
Caspase 3/metabolismo , Citocromos c1/metabolismo , Citocromos c/metabolismo , Mitocôndrias/enzimologia , Proteólise , Clorometilcetonas de Aminoácidos/farmacologia , Sequência de Aminoácidos , Animais , Inibidores de Caspase , Domínio Catalítico , Transporte de Elétrons , Ativação Enzimática , Retroalimentação Fisiológica , Células HeLa , Humanos , Potencial da Membrana Mitocondrial , Camundongos , Mitocôndrias/fisiologia , Dados de Sequência Molecular , Oxirredução , Fosforilação Oxidativa , RNA Interferente Pequeno , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/farmacologia , Especificidade por Substrato , Succinato Citocromo c Oxirredutase/metabolismo , Transfecção
7.
Bioorg Med Chem ; 19(15): 4608-15, 2011 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-21719298

RESUMO

The cytochrome bc1 complex (EC 1.10.2.2, bc1) is one of the most promising targets for new drugs and agricultural fungicides. Among the existing bc1 complex inhibitors specifically binding to the Q(o) site, oxazolidinedione derivatives have attracted great attention. With the aim to understand the substituent effects of oxazolidinedione derivatives on the inhibition activity against the bc1 complex, a series of new oxazolidinedione derivatives were designed, synthesized, and biologically evaluated. The further inhibitory kinetics studies against porcine succinate-cytochrome c reductase (SCR) revealed that the representative compound 8d and famoxadone are both non-competitive inhibitors with respect to the substrate cytochrome c, but competitive inhibitors with respect to substrate decylubiquinol (DBH2). In addition, compound 8d and famoxadone showed, respectively, 35-fold and 15-fold greater inhibitory activity against the porcine SCR than the porcine bc1 complex, indicating that these two inhibitors not only inhibited the activity of the bc1 complex, but possibly affect the interaction between the complex II and the bc1 complex. To our knowledge, this is the first report that famoxadone and its analogs have effects on the interaction between the complex II and the bc1 complex.


Assuntos
Desenho de Fármacos , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Oxazóis/química , Oxazóis/farmacologia , Animais , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Succinato Citocromo c Oxirredutase/antagonistas & inibidores , Succinato Citocromo c Oxirredutase/metabolismo , Suínos
8.
Anal Biochem ; 415(2): 209-11, 2011 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-21545784

RESUMO

This article describes a microplate-based kinetic assay for mitochondrial NADH-- and succinate--cytochrome c reductase activities in rat brain mitochondria. The assay reported here is based on the conventional spectrophotometric method and involves substrate-driven reduction of exogenous cytochrome c. Conditions regarding linearity with respect to time and protein concentration have been standardized. Furthermore, the methods were tested for inhibition of respective activities by specific inhibitors. The microplate format described here can be employed for rapid and simultaneous measurements of mitochondrial NADH-- and succinate--cytochrome c reductase activities in a large number of samples.


Assuntos
Ensaios Enzimáticos/métodos , Mitocôndrias/enzimologia , NADH Desidrogenase/metabolismo , Succinato Citocromo c Oxirredutase/metabolismo , Animais , Cinética , Masculino , NADH Desidrogenase/antagonistas & inibidores , Oxirredução , Ratos , Ratos Wistar , Rotenona/química , Espectrofotometria/métodos , Especificidade por Substrato , Succinato Citocromo c Oxirredutase/antagonistas & inibidores
9.
Biochim Biophys Acta ; 1807(5): 491-502, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21406178

RESUMO

Mitochondria-derived oxygen-free radical(s) are important mediators of oxidative cellular injury. It is widely hypothesized that excess NO enhances O(2)(•-) generated by mitochondria under certain pathological conditions. In the mitochondrial electron transport chain, succinate-cytochrome c reductase (SCR) catalyzes the electron transfer reaction from succinate to cytochrome c. To gain the insights into the molecular mechanism of how NO overproduction may mediate the oxygen-free radical generation by SCR, we employed isolated SCR, cardiac myoblast H9c2, and endothelial cells to study the interaction of NO with SCR in vitro and ex vivo. Under the conditions of enzyme turnover in the presence of NO donor (DEANO), SCR gained pro-oxidant function for generating hydroxyl radical as detected by EPR spin trapping using DEPMPO. The EPR signal associated with DEPMPO/(•)OH adduct was nearly completely abolished in the presence of catalase or an iron chelator and partially inhibited by SOD, suggesting the involvement of the iron-H(2)O(2)-dependent Fenton reaction or O(2)(•-)-dependent Haber-Weiss mechanism. Direct EPR measurement of SCR at 77K indicated the formation of a nonheme iron-NO complex, implying that electron leakage to molecular oxygen was enhanced at the FAD cofactor, and that excess NO predisposed SCR to produce (•)OH. In H9c2 cells, SCR-dependent oxygen-free radical generation was stimulated by NO released from DEANO or produced by the cells following exposure to hypoxia/reoxygenation. With shear exposure that led to overproduction of NO by the endothelium, SCR-mediated oxygen-free radical production was also detected in cultured vascular endothelial cells.


Assuntos
Radical Hidroxila/metabolismo , Óxido Nítrico/fisiologia , Succinato Citocromo c Oxirredutase/fisiologia , Animais , Bovinos , Células Cultivadas , Dietilaminas/farmacologia , Eletroquímica , Espectroscopia de Ressonância de Spin Eletrônica , Células Endoteliais/metabolismo , Mioblastos/metabolismo , Ácido Peroxinitroso/metabolismo , Ratos , Superóxidos/metabolismo
10.
Life Sci ; 88(1-2): 57-64, 2011 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-21050865

RESUMO

AIMS: Late phase ischemic preconditioning (LPC) protects the heart against ischemia-reperfusion (I/R) injury. However, its effect on myocardial tissue oxygenation and related mechanism(s) is unknown. The aim of the current study is to determine whether LPC attenuates post-ischemic myocardial tissue hyperoxygenation through preserving mitochondrial oxygen metabolism. MAIN METHODS: C57BL/6 mice were subjected to 30 min coronary ligation followed by 60 min or 24 h reperfusion with or without LPC (3 cycles of 5 min I/5 min R): Sham, LPC, I/R, and LPC+I/R group. Myocardial tissue Po(2) and redox status were measured with electron paramagnetic resonance (EPR) spectroscopy. KEY FINDINGS: Upon reperfusion, tissue Po(2) rose significantly above the pre-ischemic level in the I/R mice (23.1 ± 2.2 vs. 12.6 ± 1.3 mmHg, p<0.01). This hyperoxygenation was attenuated by LPC in the LPC+I/R mice (11.9 ± 2.0 mmHg, p<0.01). Activities of NADH dehydrogenase (NADH-DH), succinate-cytochrome c reductase (SCR) and cytochrome c oxidase (CcO) were preserved or increased in the LPC group, significantly reduced in the I/R group, and conserved in the LPC+I/R group. Manganese superoxide dismutase (Mn-SOD) protein expression was increased by LPC in the LPC and LPC+I/R mice compared to that in the Sham control (1.24 ± 0.01 and 1.23 ± 0.01, p<0.05). Tissue redox status was shifted to the oxidizing state with I/R (0.0268 ± 0.0016/min) and was corrected by LPC in the LPC+I/R mice (0.0379 ± 0.0023/min). Finally, LPC reduced the infarct size in the LPC+I/R mice (10.5 ± 0.4% vs. 33.3 ± 0.6%, p<0.05). SIGNIFICANCE: Thus, LPC preserved mitochondrial oxygen metabolism, attenuated post-ischemic myocardial tissue hyperoxygenation, and reduced I/R injury.


Assuntos
Precondicionamento Isquêmico Miocárdico , Mitocôndrias Cardíacas/metabolismo , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Animais , Western Blotting , Espectroscopia de Ressonância de Spin Eletrônica , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Cardíacas/fisiologia , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/fisiopatologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio/enzimologia , Miocárdio/metabolismo , NADH Desidrogenase/metabolismo , Oxirredução , Oxigênio/metabolismo , Fluxo Sanguíneo Regional/fisiologia , Succinato Citocromo c Oxirredutase/metabolismo
11.
J. physiol. biochem ; 66(4): 291-299, dic. 2010.
Artigo em Inglês | IBECS | ID: ibc-122816

RESUMO

No disponible


Enzymes in mitochondria play an important role in biological oxidation and energy production. To understand the effect of schistosomiasis on these important processes, succinate cytochrome creductase (SCR) from control and Schistosoma-infected mice was subjected for investigation. In this article, we report that SCR from Schistosoma-infected mouse showed a significant decrease in its Vmax and K m compared to control using both cytochrome c and 2,6-dichlorophenolindophenol as substrates. Furthermore, the kinetic studies of the purified SCR in the absence and presence of the schistosomicidal drugs praziquantel and Commiphora extract reveal that both drugs have an inhibitory action on the enzyme from the control and Schistosoma-infected mice and praziquantel changes the type of inhibition of SCR towards cytochrome c from mixed type in control to a competitive one in the case of the infection (AU)


Assuntos
Animais , Camundongos , Succinato Citocromo c Oxirredutase/farmacocinética , Esquistossomose/tratamento farmacológico , Schistosoma , Esquistossomicidas/farmacocinética , Modelos Animais de Doenças
12.
J Physiol Biochem ; 66(4): 291-9, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20680542

RESUMO

Enzymes in mitochondria play an important role in biological oxidation and energy production. To understand the effect of schistosomiasis on these important processes, succinate cytochrome c reductase (SCR) from control and Schistosoma-infected mice was subjected for investigation. In this article, we report that SCR from Schistosoma-infected mouse showed a significant decrease in its Vmax and Km compared to control using both cytochrome c and 2,6-dichlorophenolindophenol as substrates. Furthermore, the kinetic studies of the purified SCR in the absence and presence of the schistosomicidal drugs praziquantel and Commiphora extract reveal that both drugs have an inhibitory action on the enzyme from the control and Schistosoma-infected mice and praziquantel changes the type of inhibition of SCR towards cytochrome c from mixed type in control to a competitive one in the case of the infection.


Assuntos
Esquistossomose/enzimologia , Esquistossomicidas/uso terapêutico , Succinato Citocromo c Oxirredutase/antagonistas & inibidores , Succinato Citocromo c Oxirredutase/química , 2,6-Dicloroindofenol/química , Animais , Temperatura Alta , Técnicas In Vitro , Cinética , Masculino , Camundongos , Mitocôndrias/enzimologia , Praziquantel/farmacologia , Reprodutibilidade dos Testes , Temperatura
13.
Cell Biochem Funct ; 28(4): 283-92, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20517892

RESUMO

The specific activities of Complexes I-III, II-III, and IV of the respiratory chain, and citrate synthase, were determined in mitochondrial sonicates of six control passage 5 fibroblast cultures, cultivated in growth medium containing fetal calf serum as the only source of ascorbate. The enzymes were also assayed in serially subcultured fibroblasts which were characterized as aged at passage 20 and beyond. Results indicated a significant loss of all enzyme activities in aged cells at passage 20, 25, and 30. Further studies involved maintenance of serially subcultured cells in serum free media to which increasing ascorbate concentrations (100, 200, and 300 micromol 1(-1)) were added. Results indicated that ascorbate at 100 micromol 1(-1) was not sufficient to restore any of the enzyme activities in aged cells. An ascorbate concentration of 200 micromol 1(-1) however, could totally restore Complex IV and citrate synthase activities, but had no effect on complexes I-III and II-III activities which required 300 micromol 1(-1) ascorbate to be partially or totally restored respectively. In conclusion, this study demonstrates an age related drop in mitochondrial respiratory chain activity in cultured human fibroblasts. Enzyme activities could be completely or partially restored in the presence of double or triple normal human plasma ascorbate concentrations.


Assuntos
Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Senescência Celular , Fibroblastos/enzimologia , Células Cultivadas , Citrato (si)-Sintase/metabolismo , Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Fibroblastos/metabolismo , Humanos , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , NADH Desidrogenase/metabolismo , Succinato Citocromo c Oxirredutase/metabolismo
14.
Ecotoxicol Environ Saf ; 73(6): 1246-54, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20570353

RESUMO

Pea seeds (Pisum sativum L.) were germinated by soaking in H2O or 5 mM CdCl2 during a 5-day period. Enzyme activities involved in respiratory metabolism were studied in cotyledons. Mitochondrial cytochrome c oxidase and NADH- and succinate-cytochrome c reductase activities were inhibited by cadmium treatment. The effects of Cd were performed in vivo and in vitro allowing to distinguish between the direct inhibition of the enzyme activities and the influence on the same enzymes into the cell environment. However, Cd exposure stimulated an enzyme activity of fermentation and inhibited the capacity of the enzyme inactivator (alcohol dehydrogenase inactivator). Moreover, the enzyme activities of NAD(P)H-recycling dehydrogenases via secondary pentose phosphate pathway, glucose-6-phosphate- and 6-phosphogluconate-dehydrogenases, were enhanced in Cd-stressed seeds. These disturbances suggest that cadmium may inflict a serious injury on renewal of respiration. The findings will help clarify the overall mechanisms that underlie cadmium-mediated toxicity in germinating seeds.


Assuntos
Cádmio/toxicidade , Cotilédone/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Ervilhas/efeitos dos fármacos , Respiração Celular/efeitos dos fármacos , Cotilédone/citologia , Cotilédone/enzimologia , Cotilédone/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Germinação/efeitos dos fármacos , Glucosefosfato Desidrogenase/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , NAD/metabolismo , Ervilhas/citologia , Ervilhas/enzimologia , Ervilhas/metabolismo , Fosfogluconato Desidrogenase/metabolismo , Sementes/citologia , Sementes/efeitos dos fármacos , Sementes/enzimologia , Sementes/metabolismo , Succinato Citocromo c Oxirredutase/metabolismo , Fatores de Tempo
15.
Transplant Proc ; 42(3): 721-4, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20430156

RESUMO

BACKGROUND: Ischemia-reperfusion (I-R) injury plays an important role in the immediate graft function in living-donor liver transplantation (LDLT). There is growing evidence that mitochondria play a pivotal role in I-R injury. Our aim was to evaluate changes in mitochondrial respiratory enzyme activities after I-R injury in LDLT. METHODS: Specimens from 8 donor recipient pairs enrolled in this study were obtained from the donor livers before harvest (before I-R injury) and after vascular anastomosis in the recipient (after I-R injury). Histidine-tryptophan-ketoglutarate solution was used to perfuse the organ during the cold ischemic period between harvesting and transplantation. We correlated changes in mitochondrial respiratory enzyme complex activity (succinate cytochrome c reductase [SCCR]; NADH cytochrome c reductase [NCCR]) after I-R injury with clinical data and graft status. RESULTS: NCCR and SCCR activities did not uniformly decrease after I-R injury. Two of 8 recipients experienced graft dysfunction after transplantation. The decrease in neither NCCR nor SCCR activity correlated with graft dysfunction in these 2 patients. Among the clinical factors, grafts from older donors tended to show decreased NCCR activity after I-R injury. CONCLUSIONS: In this study, changes in mitochondrial respiratory enzyme activity failed to predict the severity of I-R injury in LDLT. The organ preservation solution may play a protective role on mitochondrial respiratory enzymes during I-R injury.


Assuntos
Transplante de Fígado/efeitos adversos , Doadores Vivos , Mitocôndrias Hepáticas/enzimologia , NADH Desidrogenase/metabolismo , Traumatismo por Reperfusão/enzimologia , Succinato Citocromo c Oxirredutase/metabolismo , Adulto , Fatores Etários , Idoso , Biomarcadores , Feminino , Humanos , Cinética , Masculino , Pessoa de Meia-Idade , Falha de Tratamento , Resultado do Tratamento
16.
Am J Physiol Endocrinol Metab ; 298(1): E89-98, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19843872

RESUMO

Atherosclerotic cardiovascular disease is the leading cause of mortality in the Western world. Dysfunction of the mitochondrial respiratory chain and overproduction of reactive oxygen species (ROS) are associated with atherosclerosis and cardiovascular disease. Oxidation increases the atherogenecity of LDL. Oxidized LDL may be apoptotic or nonapoptotic for vascular endothelial cells (EC), depending on the intensity of oxidation. A previous study demonstrated that nonapoptotic oxidized LDL increased activity of mitochondrial complex I in human umbilical vein EC. The present study examined the impact of extensively oxidized LDL (eoLDL) on oxygen consumption and the activities of key enzymes in the mitochondrial respiratory chain of cultured porcine aortic EC. Oxygraphy detected that eoLDL significantly reduced oxygen consumption in various mitochondrial complexes. Treatment with eoLDL significantly decreased NADH-ubiquinone dehydrogenase (complex I), succinate cytochrome c reductase (complex II/III), ubiquinone cytochrome c reductase (complex III), and cytochrome c oxidase (complex IV) activities and the NAD+-to-NADH ratio in EC compared with mildly oxidized LDL, LDL, or vehicle. Butylated hydroxytoluene, a potent antioxidant, normalized eoLDL-induced reductions in complex I and III enzyme activity in EC. Mitochondria-associated intracellular ROS and release of ROS from EC were significantly increased after eoLDL treatment. These findings suggest that eoLDL impairs enzyme activity in mitochondrial respiratory chain complexes and increases ROS generation from mitochondria of arterial EC. Collectively, these effects could contribute to vascular injury and atherogenesis under conditions of hypercholesterolemia and oxidative stress.


Assuntos
Aterosclerose/metabolismo , Células Endoteliais/metabolismo , Lipoproteínas LDL/metabolismo , Mitocôndrias/metabolismo , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Aorta/citologia , Aterosclerose/patologia , Hidroxitolueno Butilado/metabolismo , Hidroxitolueno Butilado/farmacologia , Células Cultivadas , Transporte de Elétrons/fisiologia , Complexo I de Transporte de Elétrons/metabolismo , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Hipercolesterolemia/metabolismo , Hipercolesterolemia/patologia , Lipoproteínas LDL/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Consumo de Oxigênio/fisiologia , Succinato Citocromo c Oxirredutase/metabolismo , Suínos
17.
J Membr Biol ; 232(1-3): 47-57, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19921325

RESUMO

Effects of treatment with a single intraperitoneal injection of cadmium (Cd) on oxidative energy metabolism and lipid/phospholipid profiles of rat liver mitochondria were examined at the end of 1 week and 1 month. Following Cd treatment the body weight increased only in the 1 month group, whereas the liver weight increased in both groups. State 3 and 4 respiration rates in general decreased significantly, with the maximum effect being seen with succinate. The 1 week Cd group showed decreased respiratory activity with glutamate, pyruvate + malate, and succinate as the substrates. In the 1 month Cd-treated group respiration rates recovered with glutamate and pyruvate + malate but not with succinate. All cytochrome contents decreased in the 1 week Cd-treated group but recovered in the 1 month group. ATPase activity registered an increase in both Cd-treated groups. Dehydrogenase activities increased in the 1 week group but decreased in the 1 month Cd-treated group. The mitochondrial cholesterol content increased in the 1 week Cd-treated group. In the 1 week Cd-treated group the lysophospholipid (Lyso), sphingomyelin (SPM), and diphosphatidylglycerol (DPG) components increased. By contrast, the phosphatidylethanolamine (PE) component decreased. In the 1 month Cd-treated group the phosphatidylinositol, phosphatidylserine, and DPG components increased, whereas the Lyso, SPM, and phosphatidylcholine components decreased. The results demonstrate that single-dose Cd treatment can have adverse effects on liver mitochondrial oxidative energy metabolism and lipid/phosphopholipid profiles, which in turn can affect membrane structure-function relationships.


Assuntos
Cádmio/farmacologia , Metabolismo Energético/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Glutamato Desidrogenase/metabolismo , Malato Desidrogenase/metabolismo , Masculino , Fluidez de Membrana/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Fosfolipídeos/metabolismo , Ratos , Succinato Citocromo c Oxirredutase/metabolismo
18.
Eur Respir J ; 33(5): 1045-52, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19129279

RESUMO

Several cellular and molecular alterations have been described in skeletal and respiratory muscles of patients with chronic obstructive pulmonary disease (COPD), but information on potential abnormalities of mitochondrial function is scarce. The aim of the present study was to investigate mitochondrial function in the vastus lateralis (VL) and external intercostalis (EI) of COPD patients. Biopsies from VL and EI were obtained during surgery for lung cancer in 13 patients with mild to moderate COPD (age 68+/-6 yrs, forced expiratory volume in one second (FEV(1)) 66+/-15% predicted) and 19 control subjects (age 67+/-9 yrs, FEV(1) 95+/-18% pred). State 3 and 4 mitochondrial oxygen consumption (V'(O(2),m)), ATP synthesis, citrate synthase, cytochrome oxidase (COX) and complex I-III activities, as well as reactive oxygen species (ROS) production, were determined. In COPD patients, in both muscles, COX activity (VL: COPD 3.0+/-0.8 versus control 2.0+/-0.8; EI: 3.7+/-1.6 versus 2.4+/-0.9 micromol min(-1) mg(-1)) and ROS production (VL: 1,643+/-290 versus 1,285+/-468; EI: 1,033+/-210 versus 848+/-288 arbitrary units) were increased, whereas state 3 V'(O(2),m) was reduced (VL: 2.9+/-0.3 versus 3.6+/-0.4; EI: 3.6+/-0.3 versus 4.1+/-0.4 mmol min(-1) kg(-1)). Skeletal muscle mitochondria of patients with chronic obstructive pulmonary disease show electron transport chain blockade and excessive production of reactive oxygen species. The concurrent involvement of both vastus lateralis and external intercostalis suggests a systemic (rather than a local) mechanism(s) already occurring in relatively early stages (Global Initiative for Chronic Obstructive Lung Disease stage II) of the disease.


Assuntos
Mitocôndrias Musculares/metabolismo , Músculo Esquelético/fisiopatologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Músculos Respiratórios/fisiopatologia , Trifosfato de Adenosina/metabolismo , Idoso , Biópsia , Citrato (si)-Sintase/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Volume Expiratório Forçado/fisiologia , Humanos , Masculino , Músculo Esquelético/metabolismo , Consumo de Oxigênio/fisiologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Músculos Respiratórios/metabolismo , Espirometria , Succinato Citocromo c Oxirredutase/metabolismo
19.
Folia Microbiol (Praha) ; 54(6): 475-82, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20140712

RESUMO

Membrane fragments of two mutant strains of Paracoccus denitrificans genetically modified in the bc(1) complex have been studied for comparison of enzymic activities of succinate-cytochrome-c reductase and its components, viz. succinate dehydrogenase (Complex II) and ubiquinol-cytochrome-c reductase (Complex III) and their response to changes in concentration of succinate, cytochrome c, ionic strength, pH, temperature and sensitivity to antimycin A. The mutants synthesized and assembled the b and c hemes in the ratio characteristic for the wild type strain. The mutant strain M 71 expressing the truncated copy of cytochrome c(1) (devoid of a stretch of 150 mainly acidic amino acids) was less sensitive to increasing concentration of cytochrome c and changes in ionic strength of the medium, but maintained the original affinity to succinate and sensitivity to antimycin A. The mutant strain M 36 with an overexpressed bc(1) content showed the highest response to changes in ionic strength and physical parameters, exhibited the lowest turnover number values with succinate-cytochrome-c reductase, but positively affected the succinate dehydrogenase. In view of the interaction of the redox components in native membranes the functional analyses of separated Complexes II and III should be regarded with caution.


Assuntos
Elétrons , Óperon , Paracoccus denitrificans/metabolismo , Antibacterianos/farmacologia , Antimicina A/farmacologia , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Complexo III da Cadeia de Transporte de Elétrons , Concentração de Íons de Hidrogênio , Mutação , Pressão Osmótica , Oxirredução , Estresse Fisiológico , Succinato Citocromo c Oxirredutase , Temperatura
20.
Chemosphere ; 72(9): 1347-54, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18511104

RESUMO

Methoprene (isopropyl(2E,4E)-11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate) is an insect growth regulator generally used to control insect populations by preventing insect maturation. So far, the effects of the insecticide on mitochondrial bioenergetics were not investigated. In the present work, liver mitochondria from Wistar rats were isolated and features of mitochondrial physiology were characterized in the presence of methoprene. High concentrations of methoprene, in the range of 40-100 nmol/mg of protein could decrease the transmembrane electric potential (Delta Psi) developed by mitochondria and, at the highest concentration, methoprene prevented complete Delta Psi repolarization after ADP addition. The effect was more evident using succinate than with ascorbate+TMPD as substrate. State 3 respiration was approximately 60% inhibited by 80 nmol of methoprene/mg of protein, while state 4 respiration, within the same range of methoprene concentrations, showed a slight increase, when both glutamate-malate and succinate were used as substrates. Additionally, FCCP-stimulated respiration was inhibited to an extent comparable to the effect on state 3, which suggests an interaction of methoprene with the respiratory chain, more evident with glutamate/malate as substrate. The activity of complex I (NADH-ubiquinone oxidorreductase) and that of the segment comprehending complexes II and III (succinate-cytochrome c reductase) were decreased in the presence of methoprene (approximately 60% and 85% of inhibition, respectively, with 300 nmol of methoprene/mg of protein), while the activities of cytochrome c oxidase and ATPase do not seem to be affected. Furthermore, the action of methoprene on the mitochondrial permeability transition was also studied, showing that the insecticide (in the range of 30-80 nmol mg(-1) of protein) decreases the susceptibility of liver mitochondria to the opening of the transition pore, even in non-energized mitochondria. These results lead to the conclusion that methoprene interference with hepatic mitochondrial function occurs only for high concentrations, which implies that the noxious effects of the insecticide reported for a number of non-target organisms are not fully attributable to mitochondrial effects. Therefore, it seems that mitochondrial activity does not represent the primary target for methoprene toxic action.


Assuntos
Metabolismo Energético , Hormônios Juvenis/toxicidade , Metoprene/toxicidade , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Complexo I de Transporte de Elétrons/metabolismo , Eletrofisiologia , Técnicas In Vitro , Masculino , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias Hepáticas/enzimologia , Consumo de Oxigênio/efeitos dos fármacos , Permeabilidade/efeitos dos fármacos , Ratos , Ratos Wistar , Succinato Citocromo c Oxirredutase/metabolismo
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