Evaluation of methods for the determination of mitochondrial respiratory chain enzyme activities in human skeletal muscle samples.
ABSTRACT The quantification of mitochondrial enzyme activities in skeletal muscle samples of patients suspected of having mitochondrial myopathies is problematic. Therefore, we have evaluated different methods for the determination of activities cytochrome c oxidase and NADH:CoQ oxidoreductase in human skeletal muscle samples. The measurement of cytochrome c oxidase activity in the presence of 200 microM ferrocytochrome c and the detection of NADH:CoQ oxidoreductase as rotenone-sensitive NADH:CoQ(1) reductase resulted in comparable citrate synthase-normalized respiratory chain enzyme activities of both isolated mitochondria and homogenates from control human skeletal muscle samples. These methods allowed the precise detection of deficiencies of respiratory chain enzymes in skeletal muscle of two patients harboring only 20 and 27% of deleted mitochondrial DNA, respectively. Therefore, citrate synthase-normalized respiratory chain activities can serve as stable reference values for the determination of a putative mitochondrial defect in human skeletal muscle.
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ABSTRACT: Although novel drugs have contributed immensely to improving outcomes of patients with multiple myeloma (MM), many patients develop drug resistance and ultimately succumb to MM. Here, we show that artesunate, an anti-malarial drug, reliably induces cell death in vitro in naïve as well as drug-resistant MM cells at concentrations shown to be safe in humans. Artesunate induced apoptosis predominantly through the non-caspase mediated pathway by primarily targeting mitochondria and causing outer mitochondrial membrane permeabilization that led to cytosolic and subsequent nuclear translocation of mitochondrial proteins apoptosis inducing factor (AIF) and endonuclease G (EndoG). Nuclear translocation of AIF and EndoG was accompanied by low levels of reactive oxygen species (ROS) and increased mitochondrial production of superoxide. These effects were present before apoptosis was evident and were related to intracellular levels of bivalent iron (Fe+2). Artesunate's unique mechanism probably was at least partially responsible for, its ability to act synergistically with multiple anti-myeloma agents. Our findings suggest that artesunate acts through iron to affect the mitochondria and induce low ROS and non-caspase-mediated apoptosis. Its potency, toxicity profile, and synergism with other drugs make it an intriguing new candidate for MM treatment.Oncotarget 03/2014; · 6.63 Impact Factor
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ABSTRACT: Oxidative stress has been implicated in the pathology of the lethal skeletal muscle disease Duchenne Muscular Dystrophy (DMD), and various antioxidants have been investigated as a potential therapy. Recently, treatment of the mdx mouse model for DMD with the antioxidant and cysteine and glutathione (GSH) precursor n-acetylcysteine (NAC) was shown to decrease protein thiol oxidation and improve muscle pathology and ex vivo muscle strength. This study further investigates the mechanism for the benefits of NAC on dystrophic muscle by administering L-2-Oxothiazolidine-4-Carboxylate (OTC) which also upregulates intracellular cysteine and GSH, but does not directly function as an antioxidant. We observed that OTC, like NAC, decreases protein thiol oxidation, decreases pathology and increases strength, suggesting that the both NAC and OTC function via increasing cysteine and GSH content of dystrophic muscle. We demonstrate that mdx muscle is not deficient in either cysteine or GSH and that these are not increased by OTC treatment. However, we show that dystrophic muscle of 12 week old mdx mice is deficient in taurine, a by-product of disposal of excess cysteine, a deficiency that is ameliorated by OTC treatment. These data suggest that in dystrophic muscles, apart from the strong association of increased oxidative stress and protein thiol oxidation with dystropathology, another major issue is an insufficiency in taurine that can be corrected by increasing the availability of cysteine. This study provides new insight into the molecular mechanism underlying the benefits of NAC in muscular dystrophy and supports the use of OTC as an alternative drug for potential clinical applications to DMD.The international journal of biochemistry & cell biology 07/2013; DOI:10.1016/j.biocel.2013.07.009 · 4.24 Impact Factor
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ABSTRACT: Background Chronic ethanol (EtOH) administration to experimental animals induces hepatic oxidative stress and up-regulates mitochondrial biogenesis. The mechanisms by which chronic EtOH up-regulates mitochondrial biogenesis have not been fully explored. In this work, we hypothesized that oxidative stress is a factor that triggers mitochondrial biogenesis after chronic EtOH feeding. If our hypothesis is correct, co-administration of antioxidants should prevent up-regulation of mitochondrial biogenesis genes.Methods Rats were fed an EtOH-containing diet intragastrically by total enteral nutrition for 150 days, in the absence or presence of the antioxidant N-acetylcysteine (NAC) at 1.7 g/kg/d; control rats were administered isocaloric diets where carbohydrates substituted for EtOH calories.ResultsEtOH administration significantly increased hepatic oxidative stress, evidenced as decreased liver total glutathione and reduced glutathione/glutathione disulfide ratio. These effects were inhibited by co-administration of EtOH and NAC. Chronic EtOH increased the expression of mitochondrial biogenesis genes including peroxisome proliferator-activated receptor gamma-coactivator-1 alpha and mitochondrial transcription factor A, and mitochondrial DNA; co-administration of EtOH and NAC prevented these effects. Chronic EtOH administration was associated with decreased mitochondrial mass, inactivation and depletion of mitochondrial complex I and complex IV, and increased hepatic mitochondrial oxidative damage, effects that were not prevented by NAC.Conclusions These results suggest that oxidative stress caused by chronic EtOH triggered the up-regulation of mitochondrial biogenesis genes in rat liver, because an antioxidant such as NAC prevented both effects. Because NAC did not prevent liver mitochondrial oxidative damage, extra-mitochondrial effects of reactive oxygen species may regulate mitochondrial biogenesis. In spite of the induction of hepatic mitochondrial biogenesis genes by chronic EtOH, mitochondrial mass and function decreased probably in association with mitochondrial oxidative damage. These results also predict that the effectiveness of NAC as an antioxidant therapy for chronic alcoholism will be limited by its limited antioxidant effects in mitochondria, and its inhibitory effect on mitochondrial biogenesis.Alcoholism Clinical and Experimental Research 12/2014; 38(12). DOI:10.1111/acer.12576 · 3.31 Impact Factor