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At least 145 records · Page 8Linked to original sources
PubMed2003

Time-dependent and tissue-specific accumulation of mtDNA and respiratory chain defects in chronic doxorubicin cardiomyopathy.

BACKGROUND: Doxorubicin causes a chronic cardiomyopathy of unknown pathogenesis. We investigated whether acquired defects in mitochondrial DNA (mtDNA) and interconnected respiratory chain dysfunction may represent a molecular mechanism for its late onset. METHODS AND RESULTS: Rats were treated weekly with intravenous doxorubicin (1 mg/kg) for 7 weeks, starting at 11 weeks of age (group B). Controls received saline. Group C received doxorubicin identically to group B, but the course was started at 41 weeks of age. All rats were killed at week 48. Doxorubicin was also injected once, either 6 days (group D) or 2 hours (group E) before euthanasia. Heart and skeletal muscle were examined. Only group B rats developed a significant clinical, macroscopic, histological, and ultrastructural cardiomyopathy. Group B hearts had the lowest cytochrome c oxidase (COX) activity (24% of controls; P=0.003), the highest citrate synthase activity (135% of controls; P=0.005), and the highest production of superoxide. In group B, the respiratory subunit COXI, which is encoded by mtDNA, was reduced (P<0.001), as was mtDNA (49% of controls, P<0.001). Group C hearts differed from group B in their lower cardiomyopathy score (P=0.006), higher COX activity (P=0.02), and higher mtDNA content (P=0.04). Group B and to a lesser extent group C hearts contained deleted mtDNA. There was no detectable mitochondrial toxicity in group D and E hearts or in skeletal muscle. CONCLUSIONS: In doxorubicin cardiomyopathy, mtDNA alterations, superoxide, and respiratory chain dysfunction accumulate long-term in the absence of the drug and are associated with a late onset.

Age Factors↗
PubMed1999

Mitochondrial DNA polymerase gamma deficiency and mtDNA depletion in a child with Alpers' syndrome.

Deficiency of mitochondrial DNA polymerase gamma activity was found in a patient with mtDNA depletion and Alpers' syndrome. Metabolic evaluation revealed fasting hypoglycemia, dicarboxylic aciduria, and reduced activity of the electron transport chain in skeletal muscle. The patient died in early childhood of fulminant hepatic failure, refractory epilepsy, lactic acidemia, and coma. mtDNA content was 30% of normal in skeletal muscle and 25% in the liver. The activity of mtDNA polymerase gamma was undetectable.

Ataxia↗
PubMed1997

Isolation of mitochondrial DNA-less mouse cell lines and their application for trapping mouse synaptosomal mitochondrial DNA with deletion mutations.

For isolation of mouse mtDNA-less (rho0) cell lines, we searched for various antimitochondrial drugs that were expected to decrease the mtDNA content and found that treatment with ditercalinium, an antitumor bis-intercalating agent, was extremely effective for completely excluding mtDNA in all the mouse cell lines we tested. The resulting rho0 mouse cells were successfully used for trapping the mtDNA of living nerve cells into dividing cultured cells by fusion of the rho0 cells with mouse brain synaptosomes, which represent synaptic endings isolated from nerve cells. With neuronal mtDNA obtained, all of the cybrid clones restored mitochondrial translation activity similarly regardless of whether the mtDNA was derived from young or aged mice, thus at least suggesting that defects in mitochondrial genomes are not involved in the age-associated mitochondrial dysfunction observed in the brain of aged mice. Furthermore, we could trap a very small amount of a common 5823-base pair deletion mutant mtDNA (DeltamtDNA5823) that was detectable by polymerase chain reaction in the cybrid clones. As the amount of mutant mtDNA with large scale deletions was expected to increase during prolonged cultivation of the cybrids, these cells should be available for establishment of mice containing the deletion mutant mtDNA.

3T3 Cells↗
PubMed2005

Analysis of proteome bound to D-loop region of mitochondrial DNA by DNA-linked affinity chromatography and reverse-phase liquid chromatography/tandem mass spectrometry.

Mitochondrial dysfunction has been suggested as a causal factor for insulin resistance and diabetes. Previously we have shown a decrease of mitochondrial DNA (mtDNA) content in tissues of diabetic patients. The mitochondrial proteins, which regulate the mitochondrial biogensis, including transcription and replication of mtDNA, are encoded by nuclear DNA. Despite the potential function of the proteins bound to the D-loop region of mtDNA in regulating mtDNA transcription/replication, only a few proteins are known to bind the D-loop region of mtDNA. The functional association of these known proteins with insulin resistance is weak. In this study, we applied proteomic analysis to identify a group of proteins (proteome) that physically bind to D-loop DNA of mtDNA. We amplified D-loop DNA (1.1 kb) by PCR and conjugated the PCR fragments to CNBr-activated sepharose. Mitochondria fractions were isolated by both differential centrifugation and Optiprep-gradient ultracentrifugation. The D-loop DNA binding proteome fractions were enriched via this affinity chromatography and analyzed by SDS-PAGE. The proteins on the gel were transferred onto PVDF membrane and the peptide sequences of each band were subsequently analyzed by capillary reverse-phase liquid chromatography/tandem mass spectrometry (RPLC/MS/MS). We identified many D-loop DNA binding proteins, including mitochondrial transcription factor A (mtTFA, Tfam) and mitochondrial single-stranded DNA binding protein (mtSSBP) which were known to bind to mtDNA. We also report the possibility of novel D-loop binding proteins such as histone family proteins and high-mobility group proteins.

Amino Acid Sequence↗
PubMed1994

Quantitation of mitochondrial DNA in human lymphoblasts by a competitive polymerase chain reaction method: application to the study of inhibitors of mitochondrial DNA content.

With increasing awareness of the mitochondrial toxicity associated with certain 2',3'-dideoxynucleosides used in anti-human immunodeficiency virus therapy, procedures for quantitative analyses of drug effects on mitochondrial DNA (mtDNA) have assumed enhanced importance. For this reason we have developed a method to measure the copy numbers of mtDNA in cultured MOLT-4 cells. First a hybrid competitive DNA template was synthesized by conventional polymerase chain reaction (PCR), using two custom-synthesized 40-mer composite primers incorporating mitochondrial displacement loop sequences linked by a non-mitochondrial cDNA template (a 76-base pair sequence from the tat/rev region of human immunodeficiency virus cDNA). For the competitive assay, increasing known copy numbers of the hybrid competitive template were added as an internal control to samples containing total cellular DNA. With this approach, two competitive PCR products were generated, 1) a mitochondrial displacement loop-derived fragment (182 base pairs) and 2) a competitive DNA template-derived fragment (156 base pairs). Absolute quantitation was achieved by radiometric comparison of the relative amounts of the two products. To test the versatility of this method, varying amounts of competitive template (6.6 x 10(4) to 6.6 x 10(9) copies) were used with a fixed quantity of total cellular DNA taken from cells cultured for 9 days in the presence or absence of selected pyrimidine and purine dideoxynucleosides. The results showed that the copy number of cellular mtDNA is 823 +/- 71 copies/cell in MOLT-4 cells. Little selective depletion of mtDNA, compared with total cellular DNA, was seen with the purine dideoxynucleosides examined; however, when the cells were exposed to the pyrimidine dideoxynucleoside 2',3'-dideoxycytidine (50 nM) for 9 days, mtDNA content was specifically depleted, although total cellular DNA decreased by only 10%. Thus, in addition to the presently used methods of assessing mitochondrial impairment, i.e., Southern blot analysis and electron microscopy, the competitive PCR method provides a third and convenient assay, with particular applicability to determination of mtDNA in very small numbers of cells.

Base Sequence↗
PubMed1994

An unusual type of mitochondrial DNA inheritance in the blue mussel Mytilus.

In animals, mitochondrial DNA (mtDNA) inheritance is predominantly maternal. In a few cases incidental transmission of paternal mtDNA was observed and estimated to account for only 10(-4)-10(-3) of an individual's mtDNA content. In contrast, biparental inheritance is common in mussels of the genus Mytilus. Here we present direct evidence that sex and mtDNA inheritance are coupled in Mytilus. Females inherit mtDNA only from their mother, but they transmit it to both daughters and sons. Males inherit mtDNA from both parents, but they transmit to sons only the mtDNA they inherited from their father. In pair matings, this mtDNA inheritance pattern is associated with a strong sex-ratio bias. These findings establish a newly discovered type of cytoplasmic DNA transmission. We also present evidence that the phenomenon breaks down in interspecific hybrids.

Aging↗
PubMed1991

Mitochondrial genome distribution in histochemically cytochrome c oxidase-negative muscle fibres in patients with a mixture of deleted and wild type mitochondrial DNA.

In situ hybridization studies were performed on a series of chronic progressive external ophthalmoplegia patients harbouring large mitochondrial DNA deletions, using intra- and extra-deletional probes. Clear differences in the distribution of wild type and deleted mitochondrial genomes were seen in both ragged-red and non-ragged red, cytochrome c oxidase-negative fibres, with an accumulation of deleted genomes in the subsarcolemmal zone. Wild type genome content was normal or decreased in the cytochrome c oxidase-negative regions of one case, but in two patients, wild type mtDNA content in cytochrome c oxidase-negative regions was either normal (most fibres) or increased (occasional fibres). The latter observation suggests there may be a stage in the natural history of ragged-red fibre evolution where wild type genomes are transiently increased. The significance of this finding is discussed.

Base Sequence↗
PubMed2003

Mitochondrial deoxyribonucleoside triphosphate pools in thymidine kinase 2 deficiency.

Deficiency of mitochondrial thymidine kinase (TK2) is associated with mitochondrial DNA (mtDNA) depletion and manifests by severe skeletal myopathy in infancy. In order to elucidate the pathophysiology of this condition, mitochondrial deoxyribonucleoside triphosphate (dNTP) pools were determined in patients' fibroblasts. Despite normal mtDNA content and cytochrome c oxidase (COX) activity, mitochondrial dNTP pools were imbalanced. Specifically, deoxythymidine triphosphate (dTTP) content was markedly decreased, resulting in reduced dTTP:deoxycytidine triphosphate ratio. These findings underline the importance of balanced mitochondrial dNTP pools for mtDNA synthesis and may serve as the basis for future therapeutic interventions.

Cell Nucleus↗
PubMed2005

Mitochondrial studies in HAART-related lipodystrophy: from experimental hypothesis to clinical findings.

Chronic use of antiretrovirals (ARVs) to treat HIV infection, along with more prolonged patient survival, has been associated with an increase in adverse drug effects in HIV-infected patients on treatment. It has been proposed that some of these adverse effects (including myopathy, cardiomyopathy, anaemia, hyperlactataemia/ lactic acidosis, pancreatitis, polyneuritis and lipodystrophy) could be mediated by mitochondrial (mt) toxicity. From the experimental data, it has been proposed that nucleoside analogue reverse transcriptase inhibitors (NRTIs) also inhibit gamma-polymerase, the enzyme devoted to replicate (and, to a lesser extent, repair) mtDNA. It is now widely accepted that the use of most NRTIs in HIV-infected patients is associated with mtDNA depletion. Although cross-sectional studies suggest that certain ARVs, especially stavudine, are more toxic to mitochondria, the differences among different highly active ARV therapy (HAART) schedules detected in the analysis of longitudinal studies are not so clear. These types of study in previously untreated individuals suggest that the greatest mtDNA loss appears at the beginning of the treatment. Conversely, in ARV-experienced patients, the potential beneficial effects of HAART changes in terms of mtDNA content remain controversial and must be further investigated. Functional studies accompanying genetic investigations are needed for the correct pathogenic interpretation of the mtDNA abnormalities.

Antiretroviral Therapy, Highly Active↗
PubMed2002

Stable heteroplasmy but differential inheritance of a large mitochondrial DNA deletion in nematodes.

Many human mitochondrial diseases are associated with defects in the mitochondrial DNA (mtDNA). Mutated and wild-type forms of mtDNA often coexist in the same cell in a state called heteroplasmy. Here, we report the isolation of a Caenorhabditis elegans strain bearing the 3.1-kb uaDf5 deletion that removes 11 genes from the mtDNA. The uaDf5 deletion is maternally transmitted and has been maintained for at least 100 generations in a stable heteroplasmic state in which it accounts for approximately 60% of the mtDNA content of each developmental stage. Heteroplasmy levels vary between individual animals (from approximately 20 to 80%), but no observable phenotype is detected. The total mtDNA copy number in the uaDf5 mutant is approximately twice that of the wild type. The maternal transmission of the uaDf5 mtDNA is controlled by at least two competing processes: one process promotes the increase in the average proportion of uaDf5 mtDNA in the offspring, while the second promotes a decrease. These two forces prevent the segregation of the mtDNAs to homoplasmy.

Animals↗
PubMed2005

Extensive investigations of mitochondrial DNA genome in treated HIV-infected subjects: beyond mitochondrial DNA depletion.

BACKGROUND: Therapy with nucleoside reverse transcriptase inhibitor (NRTI) agents has been associated with lipoatrophy and lactic acidosis, presumably through inhibition of DNA polymerase-gamma and resultant mitochondrial DNA (mtDNA) depletion. In past investigations, studies have looked at mtDNA depletion and a few specific mutations but not at the entire mtDNA genome to correlate with clinical toxicity. METHODS: This is the largest prospective longitudinal study to date that has performed a complete analysis of the entire mtDNA genome in addition to mtDNA depletion. The study population included 54 HIV-infected NRTI-treated patients with or without clinical mitochondrial toxicities, 33 HIV-infected NRTI-naive patients, and 48 age-matched healthy volunteers. Data on demographics, treatment, and clinical characteristics were collected, and blood was drawn for mtDNA analysis, serum fasting lipids, and lactate. RESULTS: No depletion was found in blood mtDNA levels of subjects with clinical mitochondrial toxicities; duration of NRTI therapy was the only predictor of mtDNA levels. After complete analysis of the mtDNA genome, only 2 subjects showed development of mutations during the study period, after 14 and 52 months of antiretroviral therapy. CONCLUSION: Blood mtDNA content is not associated with the use of specific NRTIs, nor does it predict clinical symptoms such as lipoatrophy. The only factor associated with mtDNA depletion was duration of NRTI use. Complete mutational analysis of the mitochondrial genome revealed mtDNA mutations in 2 patients. More extensive studies of mtDNA mutation at the single molecule level are required to correlate mitochondrial dysfunction with NRTI-caused molecular defects in mtDNA.

Adult↗
PubMed2005

Mitochondrial DNA content is decreased in autosomal dominant optic atrophy.

BACKGROUND: Autosomal dominant optic atrophy (ADOA) is the commonest form of inherited optic neuropathy. Mutations in the OPA1 gene encoding a dynamin-related mitochondrial protein underlie ADOA and may perturb the biogenesis and maintenance of mitochondria. OBJECTIVE: To investigate the mutation spectrum of the OPA1 gene and assess alterations in mitochondrial content caused by OPA1 mutations. METHODS: Sixteen Korean patients with clinically suspected ADOA were studied. The mutation spectrum of the OPA1 gene was analyzed by PCR single-strand conformation polymorphism and sequencing, and mitochondrial DNA (mtDNA) content was quantified by real-time PCR. RESULTS: Eight different mutations were found, including five novel mutations. Quantitative real-time PCR analysis showed excellent linearity and precision for the determination of mtDNA copy numbers. The number of mtDNA copies per cell in patients with OPA1 gene mutations (ages 7 to 40) was significantly lower than those in all normal control subjects (p = 0.037), particularly lower than in normal control subjects ages 10 to 39 (p = 0.022). CONCLUSION: The mutation spectrum of the OPA1 gene disclosed marked genetic heterogeneity and the mitochondrial DNA content was found to be lower in autosomal dominant optic neuropathy, which provides direct evidence for a pathogenetic role of mutations of the OPA1 gene.

Adolescent↗
PubMed2006

A low dose of ethidium bromide leads to an increase of total mitochondrial DNA while higher concentrations induce the mtDNA 4997 deletion in a human neuronal cell line.

Ethidium bromide (EtBr) is widely used to deplete mitochondrial DNA (mtDNA) and produce mitochondrial DNA-less cell lines. However, it frequently fails to deplete mtDNA in mouse cells. In this study we show by using a highly sensitive real-time PCR, that low doses of EtBr (10 microM) did lead to a three-fold increase of the total amount of mitochondrial DNA in a human neuronal cell line (Ntera 2). A higher dose of EtBr (25 microM) led to the expected decrease of mtDNA until day 22 when the cells almost died. Cell growth and mtDNA content could be restored after additional 22 days of non-EtBr treatment. The highest concentration of 50 microM also led to a significant increase of mtDNA. The cells died when they had only about 10% of mtDNA left, indicating a mtDNA threshold for cell survival. Additionally, the so-called common 4977 bp deletion could be induced by prolonged exposure to ethidium bromide. Whereas the higher doses led to significant higher amounts of deleted mtDNA.

Animals↗
PubMed1997

Reduced steady-state levels of mitochondrial RNA and increased mitochondrial DNA amount in human brain with aging.

The contribution of the mitochondrial genetic system in the degenerative processes of senescence remains unclear. This study deals with age-related changes in brain mtDNA expression in humans. Brain tissue from the frontal lobe cortex was obtained from autopsy of 13 humans aged between 21 and 84 years. No structural changes were detected in mtDNA, increased mtDNA content and reduced steady-state level of mitochondrial transcripts and transcription ratio (mtRNA/mtDNA) were associated with aging. These findings suggest that the increase of the mtDNA levels could be considered as an inefficient compensatory mechanism to maintain the normal levels of mtRNA transcripts. This unbalanced mitochondrial condition could play a role in the process of senescence in human brain.

Adult↗
PubMed2003

Placenta and cord blood mitochondrial DNA toxicity in HIV-infected women receiving nucleoside reverse transcriptase inhibitors during pregnancy.

Recent studies of pregnant women and animal models have raised concerns regarding potentially serious mitochondrial toxicity-related side effects in infants born to mothers who received nucleoside reverse transcriptase inhibitors (NRTIs) during their pregnancy to prevent HIV-1 perinatal transmission. The aim of this study was to assess mitochondrial DNA (mtDNA) content of cord blood and placenta in HIV-infected pregnant women receiving NRTI compared with HIV-negative women, hypothesizing that placenta and cord blood mtDNA copies per cell would be decreased in women on NRTI therapy. Immediately following delivery, placenta and cord blood were obtained from eight HIV-infected pregnant women on NRTIs and five HIV-negative women. Assessment of mtDNA copies per cell was accomplished by quantitative real-time PCR. The mean mtDNA copies per cell from the placenta of the HIV-infected women compared with HIV-negative women was 152 +/- 119 and 880 +/- 136 ( =.0016), respectively. Similarly, the mean mtDNA copies per cell from the cord blood of the HIV-positive women compared with HIV-negative women was 144 +/- 101 and 865 +/- 331 ( =.0026), respectively. There was a statistically significant decrease in mtDNA copies per cell in placenta and cord blood between the HIV-infected women on NRTIs compared with HIV-negative women. Further studies are needed to better understand the morbidity to infants and mothers treated with NRTI to prevent vertical transmission of HIV.

Base Sequence↗
PubMed2004

Mitochondrial DNA and human thyroid diseases.

Cells of the thyroid tissue, either diseased or normal, can accumulate altered mitochondrial genomes in primary lesions and in surrounding parenchyma. Depending on the experimental approaches and the extent of the mutational process, it has been possible to demonstrate the occurrence of homoplasmic or heteroplasmic point mutations, presence of a common deletion and random large-scale mtDNA aberrations in various pathological states. Point somatic mutations documented in 5-60% of thyroid tumors do not concentrate in obvious hotspots but tend to cluster in certain regions of the mitochondrial genome and their distribution may differ between carcinomas and controls. Large-scale deletions in mtDNA are quite prevalent in healthy and diseased thyroid; however, the proportion of aberrant mtDNA molecules accounts for a very small part of total mtDNA and does not seem to correlate with pathological characteristics of thyroid tumors. Common deletion is most abundant in Hurthle cell tumors, yet it also occurs in other thyroid diseases as well as in normal tissue. The principal difference between the common deletion and other deletion-type mtDNA molecules is that the former does not depend on the relative mtDNA content in the tissue whereas in a subset of thyroid tumors, such as radiation-associated papillary carcinomas and follicular adenomas, there is a strong correlation between mtDNA levels and prevalence of large-scale deletions. Relative mtDNA levels by themselves are elevated in most thyroid tumors compared to normal tissue. Distinct differential distribution and prevalence of mutational mtDNA burden in normal tissue and thyroid lesions are suggestive of the implication of altered mtDNA in thyroid diseases, especially in cancer.

DNA, Mitochondrial↗
PubMed2004

Real-time PCR designs to estimate nuclear and mitochondrial DNA copy number in forensic and ancient DNA studies.

We explore different designs to estimate both nuclear and mitochondrial human DNA (mtDNA) content based on the detection of the 5' nuclease activity of the Taq DNA polymerase using fluorogenic probes and a real-time quantitative PCR detection system. Human mtDNA quantification was accomplished by monitoring the real-time progress of the PCR-amplification of two different fragment sizes (113 and 287 bp) within the hypervariable region I (HV1) of the mtDNA control region, using two fluorogenic probes to specifically determine the mtDNA copy of each fragment size category. This mtDNA real-time PCR design has been used to assess the mtDNA preservation (copy number and degradation state) of DNA samples retrieved from 500 to 1500 years old human remains that showed low copy number and highly degraded mtDNA. The quantification of nuclear DNA was achieved by real-time PCR of a segment of the X-Y homologous amelogenin (AMG) gene that allowed the simultaneous estimation of a Y-specific fragment (AMGY: 112 bp) and a X-specific fragment (AMGX: 106 bp) making possible not only haploid or diploid DNA quantitation but also sex determination. The AMG real-time PCR design has been used to quantify a set of 57 DNA samples from 4-5 years old forensic bone remains with improved sensitivity compared with the slot-blot hybridization method. The potential utility of this technology to improve the quality of some PCR-based forensic and ancient DNA studies (microsatellite typing and mtDNA sequencing) is discussed.

Amelogenin↗
PubMed2004

Regional differences in oxidative capacity of rat white adipose tissue are linked to the mitochondrial content of mature adipocytes.

Two metabolic pathways of the white adipocytes (i.e. de novo lipogenesis and lipolysis) require mitochondria functionality. In this report, the oxidative capacity of two white adipose tissues of rat and their respective isolated adipocytes were evaluated. Two major white fat pads, namely inguinal and epididymal tissues, were chosen as subcutaneous and visceral adipose tissues, respectively. The mitochondrial content of these tissues was estimated using cytological and biochemical analysis. Electron microscopy analysis showed higher mitochondrial density in epididymal than in inguinal adipocytes. The mitochondrial DNA content and mitochondrial enzymatic equipment were also higher in the former than in the latter tissue. A positive correlation between two mitochondrial enzymatic activities, namely cytochrome c oxidase and citrate synthase, and the mtDNA content of adipose tissue was reported. Moreover, NRF1 protein, which belongs to the transcriptional activator family and is thought to be involved in mitochondrial biogenesis regulation, was present in higher proportions in nuclei isolated from epididymal cells than in those from inguinal cells. Finally, greater abundance of mitochondria in epididymal tissue is in agreement with higher cytochrome c oxidase activity as well as increased respiration (i.e. basal and noradrenaline-stimulated) of adipocytes isolated from epididymal tissue as compared to adipocytes isolated from inguinal tissue. Therefore, white adipose tissue appears as a heterogeneous organ with marked variation in mitochondrial content depending on its anatomical location.

Adipocytes↗