Automated Analysis of Mitochondrial Enzymes in Cultured Skin Fibroblasts

Williams, Andrew; Coakley, John; Christodoulou, John

doi:10.1006/abio.1998.2624

Cited by 24 publications

(15 citation statements)

References 8 publications

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“…This assay was carried out as described by Williams et al with minor modifications [15]. The citrate synthase activity was calculated using ε = 13.6 mM −1 cm −1 [16].…”

Section: Methodsmentioning

confidence: 99%

Mitochondrial dysfunction may explain the cardiomyopathy of chronic iron overload

Gao

Qian

Campian

et al. 2010

Free Radical Biology and Medicine

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In patients with hemochromatosis, cardiac dysfunction may appear years after they have reached a state of iron overload. We hypothesized that cumulative iron-catalyzed oxidant damage to mitochondrial DNA (mtDNA) might explain the cardiomyopathy of chronic iron overload. Mice were given repetitive injections of iron dextran for a total of 4 weeks after which the iron loaded mice had elevated cardiac iron, modest cardiac hypertrophy and cardiac dysfunction. qPCR amplification of near-full-length (~16 kb) mtDNA revealed >50% loss of full length product, whereas amounts of a qPCR product of a nuclear gene (13 kb region of beta globin) were unaffected. Quantitative rtPCR analyses revealed 60-70% loss of mRNA for proteins encoded by mtDNA with no change in mRNA abundance for nuclear encoded respiratory subunits. These changes coincided with proportionate reductions in complex I and IV activities and decreased respiration of isolated cardiac mitochondria. We conclude that chronic iron overload leads to cumulative iron-mediated damage to mtDNA and impaired synthesis of mitochondrial respiratory chain subunits. The resulting respiratory dysfunction may explain the slow development of cardiomyopathy in chronic iron overload and similar accumulation of damage to mtDNA may also explain the mitochondrial dysfunction observed in slowly progressing diseases such as neurodegenerative disorders.

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“…This assay was carried out as described by Williams et al with minor modifications [15]. The citrate synthase activity was calculated using ε = 13.6 mM −1 cm −1 [16].…”

Section: Methodsmentioning

confidence: 99%

Mitochondrial dysfunction may explain the cardiomyopathy of chronic iron overload

Gao

Qian

Campian

et al. 2010

Free Radical Biology and Medicine

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“…Citrate Synthase Assay-This assay was performed as described by Williams et al (33) with minor modifications. It is based on the reaction of oxaloacetate and acetyl-CoA to produce coenzyme A (CoASH), the latter being detected by dithiobis(nitrobenzoic acid), which reacts with sulfhydryls in CoASH producing free thionitrobenzoate.…”

Section: Methodsmentioning

confidence: 99%

Oxygen Tolerance and Coupling of Mitochondrial Electron Transport

Campian¹,

Qian

Gao

et al. 2004

Journal of Biological Chemistry

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Oxygen is critical to aerobic metabolism, but excessive oxygen (hyperoxia) causes cell injury and death. An oxygen-tolerant strain of HeLa cells, which proliferates even under 80% O 2 , termed "HeLa-80," was derived from wild-type HeLa cells ("HeLa-20") by selection for resistance to stepwise increases of oxygen partial pressure. Surprisingly, antioxidant defenses and susceptibility to oxidant-mediated killing do not differ between these two strains of HeLa cells. However, under both 20 and 80% O 2 , intracellular reactive oxygen species (ROS) production is significantly (ϳ2-fold) less in HeLa-80 cells. In both cell lines the source of ROS is evidently mitochondrial. Although HeLa-80 cells consume oxygen at the same rate as HeLa-20 cells, they consume less glucose and produce less lactic acid. Most importantly, the oxygen-tolerant HeLa-80 cells have significantly higher cytochrome c oxidase activity (ϳ2-fold), which may act to deplete upstream electron-rich intermediates responsible for ROS generation. Indeed, preferential inhibition of cytochrome c oxidase by treatment with n-methyl protoporphyrin (which selectively diminishes synthesis of heme a in cytochrome c oxidase) enhances ROS production and abrogates the oxygen tolerance of the HeLa-80 cells. Thus, it appears that the remarkable oxygen tolerance of these cells derives from tighter coupling of the electron transport chain.Oxygen is crucial to aerobic metabolism, but excess oxygen or, more likely, reactive oxygen species (ROS) 1 generated under hyperoxic conditions, will cause cell injury and death. Damage to cells and tissues caused by hyperoxia is clinically important. For example, both lung damage and retrolental fibroplasia occur in premature infants given oxygen as therapy for pulmonary insufficiency. The risk of these complications is further amplified by the immaturity of cellular antioxidant defenses in premature infants (1). Similar pulmonary damage is also observed in adults who, on prolonged exposure to high partial pressures of inhaled O 2 , exhibit cough, shortness of breath, decreased vital capacity, and increased alveolar-capillary permeability (2-4).Despite decades of work, it is still not known precisely how hyperoxia causes damage to cells and tissues. Nonetheless, it is commonly believed that free radicals play a key role in the pathophysiology of oxygen toxicity and cellular damage is probably mediated by increased production of ROS (5). This excessive production of ROS likely derives from the mitochondria that, under conditions of high oxygen, exhibit increased electron leak from the electron transport chain (5-7). We have approached the question of the nature of hyperoxic cell damage using a special line of HeLa cells selected for resistance to stepwise increases in the partial pressure of O 2 . These oxygentolerant HeLa cells are able to survive and grow under 80% O 2 , a partial pressure of oxygen under which normal HeLa cells and most other mammalian cells not only stop growing but die (8).If enhanced ROS production is, in fact, a...

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“…Catalase activity was measured by direct decomposition of H 2 O 2 (240 nm) in 50 mM potassium phosphate buffer, pH 7.0 [15]. Citrate synthase was measured by a method based on Williams et al [16]. The reduction of dithionitrobenzoic acid is measured at 412 nm in the presence of 0.3 mM acetyl-coenzyme A and 0.5 mM oxaloacetic acid in 100 mM Tris-HCl, pH 8.0.…”

Section: Enzyme Activities and Protein Measurementsmentioning

confidence: 99%

Decreased mitochondrial superoxide levels and enhanced protection against paraquat inDrosophilamelanogastersupplemented withRhodiola rosea

Schriner¹,

Abrahamyan

Avanessian³

et al. 2009

Free Radical Research

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The root extract from Rhodiola rosea has been reported to have numerous health benefits in human and animal studies. Its molecular mechanism is currently unknown; however, it has been suggested to act as an antioxidant. This study found that a formulation of R. rosea extract, SHR-5, from the Swedish Herbal Institute (SHI) could extend both mean (24% in both sexes) and maximum (16% in males and 31% in females) life span in Drosophila melanogaster when compared to controls. It also found that it lowered mitochondrial superoxide levels and afforded elevated protection against the superoxide generator paraquat in both sexes. The extract SHR-5 did not alter the activities of the major antioxidant enzymes, the superoxide dismutases or catalase, nor did it afford protection against H(2)O(2) or soluble iron. These results present a decrease in endogenous superoxide levels as a possible mode of action for the root extract of R. rosea.

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Automated Analysis of Mitochondrial Enzymes in Cultured Skin Fibroblasts

Cited by 24 publications

References 8 publications

Mitochondrial dysfunction may explain the cardiomyopathy of chronic iron overload

Mitochondrial dysfunction may explain the cardiomyopathy of chronic iron overload

Oxygen Tolerance and Coupling of Mitochondrial Electron Transport

Decreased mitochondrial superoxide levels and enhanced protection against paraquat inDrosophilamelanogastersupplemented withRhodiola rosea

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