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Effect of Triton X-100. (A) The effect of Triton X-100 on platelet Polγ activity is shown expressed as pmol dTTP incorporated/min/mg platelets. The optimal concentration of Triton X-100 in the platelets was 3% (v/v). This represents a final Triton X-100 concentration of 0.6% during assay. Absence of Triton X-100 from the platelet suspensions resulted in minimal recovery of Polγ activity, even with up to 8 min sonication (5s/30s × 48 min; data not shown). (B) The effect of Triton X-100 was determined on CS activity. At the 3% concentration optima for Polγ, no adverse effect was observed. The data shown are expressed as relative units.
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Progressive age-related oxidative phosphorylation (OxPhos) decline is well known in human tissues. Depletion of mitochondrial DNA (mtDNA) causes OxPhos defects in patients with myopathic syndromes and deficient mtDNA replication has been observed in cells cultured from patients with mitochondrial disease. Patients undergoing treatment for AIDS deve...
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... of the assay described in this study was the suspension of platelets in Triton X-100. This factor significantly increased the activity recovered from the 50 mg/ml platelet preparations. Sonication of platelets without Triton X-100 for up to 8 min (10 s/min × 48 min) failed to liberate any significant level of Polγ activity (data not shown; Fig. 1A). Studies of platelet cytoskeletal components by others utilised Triton X-100 at a final concentration of 2% to expose the cytoskeleton (37). By titration (Fig. 1A), it was determined that a final concentration of 3% (v/v) of Triton X-100 was optimal for recovery of Polγ activity from 50 mg/ml platelet suspensions. It should be noted, ...
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... mg/ml platelet preparations. Sonication of platelets without Triton X-100 for up to 8 min (10 s/min × 48 min) failed to liberate any significant level of Polγ activity (data not shown; Fig. 1A). Studies of platelet cytoskeletal components by others utilised Triton X-100 at a final concentration of 2% to expose the cytoskeleton (37). By titration (Fig. 1A), it was determined that a final concentration of 3% (v/v) of Triton X-100 was optimal for recovery of Polγ activity from 50 mg/ml platelet suspensions. It should be noted, however, that the final assay concentration of Triton X-100 during Polγ assay was 0.6% due to a 1/5 dilution of the platelets in the reaction mixture. Since it was ...
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... suspensions. It should be noted, however, that the final assay concentration of Triton X-100 during Polγ assay was 0.6% due to a 1/5 dilution of the platelets in the reaction mixture. Since it was desirable to express Polγ activity relative to CS activity as an estimate of mt-eq, the effect of the Triton X-100 on CS activity was also determined (Fig. 1B), showing that no loss of CS activity occurred at the 3% Triton X-100 in which the platelets were suspended. This enabled expression of Polγ activity in terms of mt-eq utilising the same platelet suspension for both assays. This was not the case for the CytOx assays, which required use of 50 mg/ml platelets in the absence of the Triton ...
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... properties associated with the Polγ activity assayed in platelets are shown with appropriate units (with standard deviation between replicates). The assays were performed as outlined in Materials and Methods and mean counts incorporated over a 1 h reaction time are shown. Platelet homogenate Polγ function was inhibited by EtBr (Fig. 1A) and storage at -80_C over a period of 6 months did not affect the level of activity recovered in the homogenate (Fig. 1B). No activity was observed in platelets boiled prior to assay (Fig. 1C) and the minor variation between separate platelet preparations from the same individual (n = 6; variation between means of separate ...
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... standard deviation between replicates). The assays were performed as outlined in Materials and Methods and mean counts incorporated over a 1 h reaction time are shown. Platelet homogenate Polγ function was inhibited by EtBr (Fig. 1A) and storage at -80_C over a period of 6 months did not affect the level of activity recovered in the homogenate (Fig. 1B). No activity was observed in platelets boiled prior to assay (Fig. 1C) and the minor variation between separate platelet preparations from the same individual (n = 6; variation between means of separate experiments, not replicate assays) indicates good assay repeatability. The activity observed in intact WBC preparations ( Fig. 2A) was ...
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... outlined in Materials and Methods and mean counts incorporated over a 1 h reaction time are shown. Platelet homogenate Polγ function was inhibited by EtBr (Fig. 1A) and storage at -80_C over a period of 6 months did not affect the level of activity recovered in the homogenate (Fig. 1B). No activity was observed in platelets boiled prior to assay (Fig. 1C) and the minor variation between separate platelet preparations from the same individual (n = 6; variation between means of separate experiments, not replicate assays) indicates good assay repeatability. The activity observed in intact WBC preparations ( Fig. 2A) was not observed in intact nuclear preparations from WBC (Fig. 2B) or ...
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Multiple deletions of mitochondrial DNA (mtDNA) have recently been reported in familial progressive external ophthalmoplegia (PEO), in a case of progressive encephalomyopathy, and in inherited recurrent myoglobinuria. The inheritance of familial PEO has been autosomal dominant, which indicates that a mutation in an unknown nuclear gene results in s...
Mutations in the mitochondrial DNA (mtDNA) are responsible for several metabolic disorders, commonly involving muscle and the central nervous system1. Because of the critical role of mtDNA in oxidative phosphorylation, the majority of pathogenic mtDNA mutations are heteroplasmic, co-existing with wild-type molecules1. Using a mouse model with a het...
We have previously reported that neem limonoids (neem) induce multiple cancer cell death pathways. Here we dissect the underlying mechanisms of neem-induced apoptotic cell death in cancer. We observed that neem-induced caspase activation does not require Bax/Bak channel-mediated mitochondrial outer membrane permeabilization, permeability transition...
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... DNA polymerase g has a potent RNA-directed DNA polymerase activity. Assays based on this activity are routinely used to evaluate the polymerase activity of the enzyme [Kapsa et al., 1998;Longley et al., 1998;Naviaux et al., 1999]. We measured the incorporation of radioactive dTTP on a poly(rA):oligo(dT) primed template and used Mn 21 as cofactor to optimize polymerase activity [Gray and Wong, 1992]. ...
We studied six unrelated children with depletion of mitochondrial DNA (mtDNA). They presented with Leigh syndrome, infantile hepatocerebral mtDNA depletion syndrome, or Alpers-Huttenlocher syndrome. Several genes have been implicated in mtDNA depletion. Screening of candidate genes indicated that all six patients were compound heterozygous for missense mutations in the gene for the catalytic subunit of DNA polymerase (POLG). Three of the identified mutations, c.3328C>T (p.H1110Y), c.3401A>G (p.H1134R), and c.3406G>A (p.E1136K), have not been reported earlier. To investigate the functional consequences of the mutations, we carried out a series of biochemical assays in cultured fibroblasts. These studies revealed that fibroblast cultures from the patients with infantile hepatocerebral mtDNA depletion syndrome progressively lost their mtDNA during culturing, whereas fibroblast cultures from patients presenting with Leigh syndrome or Alpers-Huttenlocher syndrome had reduced but stable levels of mtDNA. DNA polymerase activity was below the normal range in all patient cultures, except for one; however, this culture showed low levels of the heterodimeric enzyme and poor DNA polymerase processivity. Parental fibroblast cultures had normal catalytic efficiency of DNA polymerase , consistent with the observation that all carriers are asymptomatic. Thus, we report the first patient with Leigh syndrome caused by POLG mutations. The cell culture experiments established the pathogenicity of the identified POLG mutations and helped to define the molecular mechanisms responsible for mtDNA depletion in the patients' tissues. The assays may facilitate the identification of those patients in whom screening for POLG mutations would be most appropriate. Hum Mutat 0, 1–8, 2008. © 2008 Wiley-Liss, Inc.
... Several recent reports indicate that disease-or aging-related depletion of mtDNA may cause bioenergetic defects in patients with mitochondrial diseases (6). It has also been shown that the age factor involved in mtDNA depletion syndrome is an age-related decline in the activity of mtDNA replication (28). It was demonstrated that age-dependent decline in mitochondrial energy production is involved in immunity change and often leads to aging process with concurrent activation of apoptotic pathways and lower ATP production in leukocytes (29). ...
The therapeutic efficiency of conventional cancer chemotherapies is often hampered by their poor tumor cell-selectivity and the drug resistance developed in most tumor cells. To improve the clinical outcome of cancer chemotherapy, it is a practical strategy to use the differences in metabolic charcteristics of mitochondria between tumor and normal cells as drug targets. Mitochondria play key roles in the oxidative metabolism of tricarboxylic acids and fatty acids for energy production to support normal function and act as an arbitrator in the initiation and execution of apoptosis of mammalian cells. In the early 1930s, Otto Warburg put forward a hypothesis that defective mitochondrial function is involved in tumorigenesis, and that tumor cells would thus reply more on glycolysis for energy supply. This proposal first raised the possibility that mitochondria play a role in tumor progression. In the past decade, it has been established that tumor cells usually have higher rate of mitochondrial DNA (mtDNA) mutation and that some cancers have lower mtDNA content. Moreover, some of the nuclear DNA-encoded mitochondrial proteins such as succinate dehydrogenase (SDH), Bcl-2 superfamility and mitochondrial permeability transition pore (MPTP) proteins have been found to be mutated in several kinds of human cancers. The alterations in the structure and function of these proteins might prevent tumor cells from apoptosis under normal conditions and lead to the development of drug resistance to anticancer agents that induce apoptosis. Therefore, it holds great promise to develop anticancer drugs targeting at mitochondria. In recent years, many mitochondria-targeting compounds with anticancer activities have been developed, and some of them have entered clinical trials. In this review, we discuss various alterations in mitochondria and mtDNA occurring in tumors and the potential in the development of mitochondria-targeting agents for cancer chemotherapy.
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Objectives – We investigated whether mutation of mitochondrial DNA (mtDNA) affects the copy number of the mitochondrial genome in patients with mitochondrial myopathy encephalopathy with lactic acidosis and stroke-like episodes (MELAS) and those with myoclonic epilepsy with ragged-red fiber (MERRF) syndromes.
Materials and methods – Forty-eight Taiwanese patients with MELAS syndrome and 20 patients with MERRF syndrome were recruited in this study.
Results – In relation to controls, the copy numbers of mtDNA in leukocytes of patients with MELAS or MERRF syndrome were significantly higher at a young age but lower at an advanced age. In addition, MELAS patients harboring higher proportions of mtDNA with A3243G transition had lower mtDNA copy numbers. The MELAS or MERRF patients with multi-system disorders had lower mtDNA copy numbers in leukocytes. Furthermore, higher proportions of mtDNA with 4977 bp deletion were found in leukocytes of MERRF patients with multi-system involvement.
Conclusion – In leukocytes, alteration in the copy number of mtDNA is related to the proportion of mtDNA with a point mutation or large-scale deletion, which may serve as a biomarker in the pathogenesis and disease progression of MELAS and MERRF syndromes.
A decline in respiratory function and increase in oxidative stressin mitochondria have been proposed as important contributors to human aging. A wide spectrum of alterations in mitochondria and mitochondrial DNA (mtDNA) has been observed in aged individuals and senescent cells. These include: • Decline in mitochondrial respiratory function • Accumulation of mtDNA mutations • Alteration in the expression of the mitochondrial genes • Increase in the rate of production of reactive oxygen species (ROS) • Increase in the extent of oxidative damage to DNA, proteins, and lipids. Responses to oxidative stress and their subsequent consequences in affected tissues play an important role in the deleterious effects of ROS on cellular function and on the apoptotic process, which culminate in aging and degenerative diseases. In this review, we focus on the roles that ROS play in aging-associated oxidative damage to mtDNA and proteins and on the oxidative stress responses of human cells at the molecular and cellular levels. The alterations of gene expression profiles elicited by oxidative stress in aging animals are discussed. Taking recent findings from this and other laboratories together, we suggest that the decline in respiratory function and increase in mitochondrial production of ROS and subsequent accumulation of mtDNA mutations and alteration in the expression of a few clusters of genes play an important role in the aging process.
A long term exposure to some nucleoside reverse transcriptase inhibitors induces a severe depletion of mitochondrial DNA (mtDNA) throughout the inhibition of polymerase γ. Even though the mtDNA depletion has been correlated to the incidence of side effects in several studies, the mechanisms underlying this phenomenon are still unclear. The mtDNA depletion may not be responsible for the rise of mitochondrial toxicity but would provide a good toxicity marker because of the ease of its exploration. In order to evaluate this marker, there is a need determine in which tissue, at what time and with which tool quantify mtDNA.
The cytopathic hypoxia theory proposes that there is an impaired cellular oxygen utilization during sepsis. Respiratory complex IV, or cytochrome c oxidase, was only previously studied in muscle biopsies of 16 surviving and 12 nonsurviving septic patients. We hypothesized that higher activities and quantities of this enzyme complex could be associated with septic patient survival. The objective was to evaluate the relationship between cytochrome c oxidase activities and quantities and 6-month survival in a larger series of septic patients using a less invasive method (circulating platelets).
Prospective, multicenter, observational study.
The study was carried out in six Spanish intensive care units.
We included 96 septic patients.
We determined the cytochrome c oxidase activity per citrate synthase activity ratio and cytochrome c oxidase quantity per citrate synthase activity ratio in circulating platelets at the time of diagnosis and related them to 6-month survival. The written informed consent from the family members was obtained.
Survivor patients (n = 54) showed higher cytochrome c oxidase activity per citrate synthase activity ratio (p = .04) and cytochrome c oxidase quantity per citrate synthase activity ratio (p = .006) than nonsurvivors (n = 42). Logistic regression analyses confirmed that the cytochrome c oxidase activity per citrate synthase activity ratio (p = .04) and cytochrome c oxidase quantity per citrate synthase activity ratio (p = .02) were independent predictors of 6-month survival. The area under the curve to predict 6-month survival was 0.62 (95% confidence interval 0.51-0.74; p = .04) for the cytochrome c oxidase activity per citrate synthase activity ratio and 0.67 (95% confidence interval 0.56-0.76; p = .003) for the cytochrome c oxidase quantity per citrate synthase activity ratio. A negative correlation was found between the cytochrome c oxidase quantity per citrate synthase activity ratio and Sepsis-Related Organ Failure Assessment score (p = .04).
Platelet cytochrome c oxidase activity and quantity were independent predictors of 6-month survival and could be used as biomarkers of sepsis mortality. This is a rapid, easy, and less invasive protocol to assess mitochondrial function. Patients with lower cytochrome c oxidase activity and quantity could benefit from drugs that improve mitochondrial function.
Synthesis of mitochondrial DNA is performed by DNA polymerase gamma. Mutations in POLG, the gene encoding the catalytic subunit of DNA polymerase gamma, are a major cause of neurological disease. A large proportion of patients carry rare nucleotide substitutions leading to single amino acid changes. Confirming that these replacements are pathogenic can be problematic without biochemical evidence. Here, we provide a hands-on protocol for an in vitro kinetic assay of DNA polymerase gamma which allows assessment of the K(m) and V(max) for the incoming nucleotide of the polymerization reaction. To avoid measurement of contaminating nuclear DNA polymerases, platelet extracts are used since platelets do not contain a nucleus. Moreover, platelets have the advantage of being obtainable relatively non-invasively. Polymerization activity is determined by measurement of the incorporation of radioactive thymidine 5'-triphosphate (dTTP) on the homopolymeric RNA substrate poly(rA).oligo(dT)(12-18). To further minimize nuclear DNA polymerase activity, aphidicolin, an inhibitor of most nuclear DNA polymerases, is included in the reaction. In addition, reactions are carried out in the absence and presence of the competitive inhibitor of DNA polymerase gamma, 2',3'-dideoxythymidine 5'-triphosphate (ddTTP), to allow calculation of the ddTTP-sensitive incorporation. With this method, platelets from healthy control subjects extracted with 3% Triton X-100 showed a K(m) for dTTP of 1.42 microM and a V(max) of 0.83 pmol min(-1)mg(-1).
Antiretroviral therapy, although successful in reducing HIV load and accordingly decreasing the incidence of HIV infection-related symptoms, has its drawbacks in the form of severe side effects. Recognized drug-related side effects are, for example, nausea, fatigue, lactic acidosis, neuropathy, lipodystrophy, and myopathy. Because not all patients experience these side effects, genetic factors could be involved. It is believed that the main toxicity of nucleoside analog drugs is due to a decrease in mitochondrial function, possibly by inhibition of mitochondrial DNA (mtDNA) replication. mtDNA is replicated by a multienzyme complex, the main component of which is the nuclear-encoded DNA polymerase gamma. Presently, the only known variation in the DNA polymerase gamma gene is variation in the number of CAG repeats in the second exon. To investigate whether CAG repeat expansion or mutations in the DNA polymerase gamma (POLG) gene could predispose to peripheral neuropathy or lactic acidosis, we have sequenced part of the second exon of the DNA polymerase gamma gene, containing the CAG repeat, of 59 drug-treated HIV-infected patients, 11 of whom experienced drug-induced neuropathy, and 3 of whom died from lactic acidosis. No correlation was found between numbers of CAG repeats and any of the symptoms. The coding regions of the POLG gene from the three lactic acidosis patients were then completely sequenced, but no mutations were found. In addition, no variation was detected in exons 3, 8, and 19 of seven neuropathy patients and three control subjects without symptoms. These exons were the only sites of amino acid changes between human and chimpanzee POLG genes, and were chosen as targets of tolerated variation.
To extend previous observations demonstrating differences in number, morphology, and activity of mitochondria in spontaneously immortalized vim(+) and vim(-) fibroblasts derived from wild-type and vimentin knockout mice, some structural and functional aspects of mitochondrial genome performance and integrity in both types of cells were investigated. Primary Vim(+/+) and Vim(-/-) fibroblasts, which escaped terminal differentiation by immortalization were characterized by an almost twofold lower mtDNA content in comparison to that of their primary precursor cells, whereby the average mtDNA copy number in two clones of vim(+) cells was lower by a factor of 0.6 than that in four clones of vim(-) cells. However, during serial subcultivation up to high passage numbers, the vim(+) and vim() fibroblasts increased their mtDNA copy number 1.5- and 2.5-fold, respectively. While early-passage cells of the vim(+) and vim(-) fibroblast clones differed only slightly in the ratio between mtDNA content and mitochondrial mass represented by mtHSP70 protein, after ca. 300 population doublings the average mtDNA/mtmass ratio in the vim(+) and vim() cells was increased by a factor of 2 and 4.5, respectively. During subcultivation, both types of cells acquired the fully transformed phenotype. These findings suggest that cytoskeletal vimentin filaments exert a strong influence on the mechanisms controlling mtDNA copy number during serial subcultivation of immortalized mouse embryo fibroblasts, and that vimentin deficiency causes a disproportionately enhanced mtDNA content in high-passage vim(-) fibroblasts. Such a role of vimentin filaments was supported by the stronger retention potential for mtDNA and mtDNA polymerase (gamma) detected in vim(+) fibroblasts by Triton X-100 extraction of mitochondria and agaroseembedded cells. Moreover, although the vim(+) and vim(-) fibroblasts were equally active in generating free radicals, the vim(-) cells exhibited higher levels of immunologically detectable 8-oxoG and mismatch repair proteins MSH2 and MLH1 in their mitochondria. Because in vim(-) fibroblasts only one point mutation was detected in the mtDNA D-loop control region, these cells are apparently able to efficiently remove oxidatively damaged nucleobases. On the other hand, a number of large-scale mtDNA deletions were found in high-passage vim(-) fibroblasts, but not in low-passage vim(-) cells and vim(+) cells of both low and high passage. Large mtDNA deletions were also induced in young vim(-) fibroblasts by treatment with the DNA intercalator ethidium bromide, whereas no such deletions were found after treatment of vim(+) cells. These results indicate that in immortalized vim(-) fibroblasts the mitochondrial genome is prone to large-scale rearrangements, probably due to insufficient control of mtDNA repair and recombination processes in the absence of vimentin.
