Nuclear DNA Ratio Research Articles

Prospective evaluation of blood concentration of mitochondrial DNA as a marker of toxicity in 157 consecutively recruited untreated or HAART-treated HIV-positive patients

Highly active antiretroviral therapy (HAART) can cause mitochondrial toxicity. The concentration of mitochondrial DNA (mtDNA) in peripheral blood cells has been reported to be a marker of this toxicity. However, these observations are controversial and were drawn from small series. Thus, we analysed the value of blood mtDNA as a marker of mitochondrial toxicity in a large cohort of human immunodeficiency virus (HIV)-infected out-patients during routine clinical evaluations. Real-time quantitative PCR was used to determine the mtDNA to nuclear DNA (nDNA) ratio in peripheral blood mononuclear cells from 157 consecutive HIV-1-infected patients (13 naive, 144 receiving HAART) and 30 HIV-1-uninfected patients. The mtDNA to nDNA ratio was significantly lower in both groups of HIV-infected patients than in the control group. No significant difference was observed between treated and naive HIV-infected patients. Lactataemia was significantly lower in controls than in the group of HIV-treated patients. None of the treated patients had lactataemia >5 mmol/l or bicarbonates <20 mmol/l. Triglyceride levels were significantly higher in the HAART-treated patients than in the nontreated patients. Clinical symptoms of lipodystrophy were observed in 62 HAART-treated patients. These symptoms were not associated with an abnormal mtDNA to nDNA ratio or plasma triglyceride concentration. The mtDNA to nDNA ratio was lower in DDI/D4T-treated patients than in AZT/3TC-treated patients. In conclusion, there are no obvious links between the mtDNA to nDNA ratio in peripheral mononuclear cells and any clinical symptoms or lactate level. Thus, the mtDNA to nDNA ratio in leukocytes does not seem to be an accurate marker of mild and/or long-term mitochondrial toxicity.

Mitochondrial Effects of Antiretroviral Therapies in Asymptomatic Patients

A decrease in the mitochondrial (mt) DNA to nuclear DNA ratio has gained acceptance as a marker of mitochondrial toxicity in treated HIV-infected patients, but the functional meaning of this alteration is unclear. We assessed mtDNA content, mitochondrial content and function in peripheral blood mononuclear cells (PBMCs) of consecutive asymptomatic HIV-infected patients. Patients selected had been receiving a first-line highly active antiretroviral therapy (HAART) regimen for at least 6 months, consisting of zidovudine plus lamivudine or stavudine plus didanosine plus either nelfinavir or nevirapine, or were antiretroviral-naive. The mtDNA content was assessed by quantitative real-time PCR, mitochondrial content by citrate synthase activity, enzyme activity of complexes III and IV (both partially encoded by mtDNA) of the electron transport chain by spectrophotometry, oxygen consumption by polarography, and oxidative damage in cell membranes by monitoring cis-parinaric acid fluorescence. Mitochondrial content was significantly lower in all treated groups. Patients receiving stavudine plus didanosine had mtDNA depletion and a decrease in complex IV activity. However, oxygen consumption capacity and lipid peroxidation were unaffected in all groups. Long-term HAART may induce mitochondrial abnormalities in PBMC mitochondria, which do not necessarily translate into functional abnormalities, at least in asymptomatic patients.

Mitochondrial:nuclear DNA ratios in peripheral blood cells from human immunodeficiency virus (HIV)-infected patients who received selected HIV antiretroviral drug regimens.

Mitochondrial:nuclear DNA (mtDNA:nDNA) ratios in blood cells were investigated in relation to selected human immunodeficiency virus antiretroviral drug regimens. Patients (n = 214) continually received a regimen consisting of either (1) saquinavir (SAQ) + ritonavir (RTV) + either nevirapine (NVP) or lamivudine (3TC) (n=32) or (2) SAQ+RTV (+/- either NVP or 3TC) + either stavudine (d4T) (n=127), didanosine (ddI) (n=19), d4T+ddI (n=21), or zidovudine (ZDV) (n=15), for >/=4 months. NVP- or 3TC-only regimens were associated with median mtDNA:nDNA ratios that were significantly higher than those for ddI- and d4T+ddI-containing regimens (P<.01) but that were not significantly higher than those for d4T- or ZDV-containing regimens. Patients received thymidine analogue- and/or ddI-containing regimens for a shorter time (median, 14 vs. 24 months; P<.01). Because of survivor-bias effect, these results may represent a conservative estimate of these nucleosides' effect on mtDNA.

Mitochondrial DNA content of human spermatozoa.

Sperm mitochondria play an important role in spermatozoa because of the high ATP demand of these cells. Different mitochondrial DNA (mtDNA) mutations and haplogroups influence sperm function. The mtDNA dose also contributes to genetic variability and pathology in different tissues and organs, but nothing is known about its relevance in the performance of spermatozoa. We estimated the variability in mtDNA content within a population of men. Different mtDNA:nuclear DNA ratios were characteristic of progressive and nonprogressive spermatozoa, confirming the influence of mtDNA content on sperm functionality. We also estimated that the absolute content of mtDNA was 700 and 1200 mtDNA copies per cell in progressive and nonprogressive human spermatozoa, respectively. These results suggest that a marked increase of mtDNA copy number per cell volume takes place during spermatogenesis.

Lymphocyte mitochondrial biomarkers in asymptomatic HIV-1-infected individuals treated with nucleoside reverse transcriptase inhibitors

Mitochondrial toxicity was evaluated in peripheral lymphocytes obtained from a cohort of 10 subjects with extensive exposure to nucleoside reverse transcriptase inhibitors (NRTI) and matched controls. Mitochondrial copy number was expressed as the ratio of mitochondrial (cytochrome b)-to-nuclear (pyruvate kinase) genes as measured by real-time polymerase chain reaction. There was no significant difference between the mtDNA : nuclear DNA ratios for the NRTI-treated subjects (mean ratio 187.2) versus the matched controls (mean ratio 199).

Quantification of OXPHOS Gene Transcripts during Muscle Cell Differentiation in Patients with Mitochondrial Myopathies

The transcript levels of nuclear and mitochondrial genes involved in oxidative phosphorylation were quantified in human myoblasts and myotubes cultured from biopsies of patients harboring either heteroplasmic point mutation or deletion of mitochondrial DNA. The transcript patterns were determined by two different methodologies, competitive reverse-transcription polymerase chain reaction and classical Northern blot analysis, both referred to the mitochondrial to nuclear DNA ratio. In myoblasts from the patients with MELAS (myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) and KSS (Kearns–Sayre) syndromes, both methodologies revealed an increase of mtDNA transcript levels. A higher level of the nuclear ATP synthase β transcript was observed in the MELAS patient cells and could be the consequence of a feedback effect of the mitochondrial DNA mutation. Moreover, the nuclear and mitochondrial transcript accumulation is more pronounced after myoblast differentiation. Thus, the OXPHOS expression is specifically altered in patients with mitochondrial diseases. The competitive RT-PCR, a rapid and sensitive technique, could be applied to investigation of mitochondrial myopathies.

The effects of age and physical activity on cardiac mitochondrial DNA template availability

The purpose of this study was to examine the effects of aging and physical activityon heart mtDNA template availability. Young, mature and aged C57BL6 mice were used in this study with half of the animals having access to exercise wheels. Cardiac DNA was isolated and the mitochondrial DNA content was assessed by slot blots with a specific mtDNA probe. Mitochondrial DNA template availability increased in mouse cardiac tissue with senescence. This was reflected in increases in mitochondrial DNA content per milligram of cardiac tissue and mitochondrial DNA relative to nuclear DNA ratios. Oxidative capacity, as determined by citrate synthase activity, was not significantly decreased with aging. Compromises to the erLergy transducing pathway due to aging responses in cardiac tissue may have led to higher replicative rates of mtDNA to induce more mitochondrial specific proteins. The mature mice responded positively to the voluntary chronic physical activity displaying increased mitochondrial DNA to nuclear DNA ratios and elevated citrate synthase activities. Thus the increasing metabolic demand put on the mitochondrial system was associated with elevated mtDNA template levels in these animals. In contrast, the senescent animals’ mtDNA template content was unaffected by the physical activity. This lack of a relationship in the senescent animals is most likely due to low running velocities and therefore relative workloads in this age group, but this suggestion remains to be confirmed.

Post-transcriptional control of plastid mRNA accumulation during adaptation of chloroplasts to different light quality environments.

The adaptation of germinating spinach seedlings to yellow and red light was studied and compared with plants grown in white light. Spinach chloroplasts isolated from cotyledons and leaves of yellow and white light-grown plants showed similar membrane structures and compositions, while chloroplasts from plants grown in red light have significant adaptive changes. Based on an equal amount of chlorophyll, these changes include a reduction in the number of photosystem I complexes, an increase of photosystem II antenna size, and an increased ratio of stacked to unstacked membranes in red light-adapted chloroplasts. The decrease in the number of photosystem I complexes per unit of chlorophyll in these chloroplasts was qualitatively correlated with an approximately 10-fold decrease in the level of the psaA mRNA encoding the photosystem I 65-kilodalton to 70-kilodalton chlorophyll apoprotein, as well as with a differential decrease in mRNA levels of other photosynthetic proteins. Light quality adaptations do not significantly affect the plastid to nuclear DNA ratio or the overall chloroplast transcription activity. The relative transcriptional activities of 10 plastid genes, as determined by run-on transcription assays, are similar in chloroplasts from cotyledons and leaves of plants grown under the three light qualities. Only the psaA gene shows a 30% to 40% decrease in transcription activity in chloroplasts of plants adapted to red light. This decrease in psaA transcription activity, however, cannot fully account for the decrease of its mRNA level. We conclude, therefore, that post-transcriptional mechanisms are primarily responsible for the control of differential chloroplast mRNA accumulation in light quality adaptations.

Quantitative Cytochemical Differences Between Young and Old Patients with Lung Cancer

In order to study differences in the tumor growth and frequency of metastases between younger and older patients with lung cancer, we investigated the nuclear DNA and nuclear protein according to age by means of cytophotometry after combined staining with Feulgen and Naphthol Yellow S. On the basis of Feulgen-Naphthol Yellow S staining method, 13 patients less than 40 years old and 16 patients older than 70 years were investigated. Our results showed no significant difference in nuclear DNA contents between young and old patients, but there were significantly higher nuclear protein contents (p less than 0.05) and nuclear protein to nuclear DNA (NP/DNA) ratios (p less than 0.001) in young patients than in old patients. This suggests that young patients may have higher tumor proliferation (high nuclear protein contents and NP/DNA ratios). The lower nuclear protein content and NP/DNA ratio of older cases is in keeping with the general phenomenon of slower tumor growth and less frequent metastases in such cases.