Gestational diabetes in women living with HIV in Botswana: lower rates with dolutegravir- than with efavirenz-based antiretroviral therapy.

BACKGROUND: There are few data on the prevalence of gestational diabetes (GDM) in pregnant women living with HIV (WLHIV) in sub-Saharan Africa, particularly those using integrase strand transfer inhibitors such as dolutegravir (DTG). METHODS: We prospectively enrolled pregnant WLHIV and pregnant women without HIV ≥18 years old in Gaborone, Botswana, excluding those with pre-existing diabetes. We screened for GDM using a 75 g oral glucose tolerance test (OGTT) performed at 24-28 weeks' gestation or at the earliest prenatal visit for those presenting after 28 weeks. Logistic regression models were fitted to assess the association between maternal HIV infection and GDM. Subgroup analyses were performed among WLHIV to assess the association between maternal antiretroviral therapy (ART) in pregnancy [DTG vs. efavirenz (EFV) with tenofovir/emtricitabine] and GDM. RESULTS: Of 486 pregnant women, 66.5% were WLHIV, and they were older than women without HIV (median age 30 vs. 25 years, P < 0.01). Among WLHIV, 97.8% had an HIV-1 RNA level < 400 copies/mL at enrolment. Overall, 8.4% had GDM with similar rates between WLHIV and those without HIV (9.0% vs. 7.4%). The WLHIV receiving DTG-based ART had a 60% lower risk for GDM compared with those on EFV-based ART (adjusted odds ratio = 0.40, 95% CI: 0.18-0.92) after adjusting for confounders. CONCLUSIONS: Pregnant WLHIV on ART in Botswana were not at increased risk of GDM compared with women without HIV. Among WLHIV, the risk of GDM was lower with DTG- than with EFV-based ART. Further studies with larger cohorts are warranted to confirm these findings.

Determinants of epidermal nerve fibre density in antiretroviral-naïve HIV-infected individuals.

OBJECTIVES: Distal leg epidermal nerve fibre density (ENFD) is a validated predictor of small unmyelinated nerve fibre damage and neuropathy risk in HIV infection. As pre-existing damage may increase the risk of neuropathy following antiretroviral (ARV) therapy, particularly when the regimen contains stavudine (d4T), we assessed the relationship between ENFD and various parameters including mitochondrial factors in HIV-infected Thai individuals naïve to ARV therapy. METHODS: Distal leg and proximal thigh ENFDs were quantified in HIV-infected Thai individuals without neuropathy prior to randomization to a HIV clinical trial that focused on mitochondrial toxicity issues. We assessed their association with various clinical and immunovirological parameters as well as with peripheral blood mononuclear cell (PBMC) mitochondrial (mt) DNA copies/cell, oxidative phosphorylation (OXPHOS) complex I (CI) and complex IV (CIV) enzyme activities, and mt 8-oxo-deoxyguanine (8-oxo-dG) break frequencies. RESULTS: In 132 subjects, the median (interquartile range) ENFD (fibres/mm) values were 21.0 (16.2-26.6) for the distal leg and 31.7 (26.2-40.0) for the proximal thigh. By linear regression, lower CD4 count (P < 0.01), older age (P < 0.01), increased body mass index (BMI) (P = 0.04), increased height (P = 0.02), and higher PBMC OXPHOS activity as measured by CIV activity (P = 0.02) were associated with lower distal leg ENFD. CONCLUSIONS: Older age, increased height, higher BMI, poorer immunological status and higher PBMC OXPHOS activity are associated with lower distal leg ENFD in HIV-infected subjects free of neuropathy prior to initiation of first-time ARV therapy.

Acetyl-l-carnitine and nucleoside reverse transcriptase inhibitor-associated neuropathy in HIV infection.

OBJECTIVES: Antiretroviral toxic neuropathy (ATN) is associated with dideoxynucleoside reverse transcriptase inhibitor use in patients infected with HIV, possibly as a result of mitochondrial toxicity. Acetyl-l-carnitine (ALC) has been linked to symptomatic improvement in ATN. We present an open-label single-arm pilot study to evaluate changes in intra-epidermal nerve fibre (IENF) density and mitochondrial DNA (mtDNA) copies/cell among subjects treated with 3000 mg ALC daily. METHODS: Punch skin biopsies were examined at baseline and after 24 weeks of therapy. Participants reported neuropathic symptoms using the Gracely Pain Intensity Score. Neurological examinations were completed. RESULTS: Twenty-one subjects completed the study. ALC was generally well tolerated. The IENF density did not change in cases completing 24 weeks of ALC therapy, with median (90% confidence interval) IENF changes of -1.70 (-3.50, infinity) (P=0.98) and 2.15 (-0.10, infinity) (P=0.11) for the distal leg and proximal thigh, respectively. Fat mtDNA copies/cell did not change with therapy. Improvements in neuropathic pain (P<0.01), paresthesias (P=0.01), and symptoms of numbness (P<0.01) were noted. Similarly, improvement was noted on the Gracely Pain Intensity Score. CONCLUSIONS: ALC therapy coincided with improvements in subjective measures of pain in this open-label single-arm study. However, changes were not observed in objective measures of IENF density or mtDNA levels, providing little objective support for use of ALC in this setting.

Correlates of epidermal nerve fiber densities in HIV-associated distal sensory polyneuropathy.

OBJECTIVE: To demonstrate the relationship between epidermal nerve fiber density (ENFD) in the leg and the phenotype of HIV-associated distal sensory polyneuropathy (HIV-DSP) in a multicenter prospective study (ACTG A5117). METHODS: A total of 101 HIV-infected adults, with CD4 cell count <300 cells/mm(3) and who had received antiretroviral therapy (ART) for at least 15 consecutive weeks, underwent standardized clinical and electrophysiologic assessment. All 101 subjects were biopsied at the distal leg (DL) and 99 at the proximal thigh (PT) at baseline. ENFD was assessed by skin biopsy using PGP9.5 immunostaining. Associations of ENFD with demographics, ART treatment, Total Neuropathy Score (TNS), sural sensory nerve action potential (SNAP) amplitude and conduction velocity, quantitative sensory testing (QST) measures, and neuropathic pain were explored. RESULTS: ENFD at the DL site correlated with neuropathy severity as gauged by TNS (p < 0.01), the level of neuropathic pain quantified by the Gracely Pain Scale (GPS) (p = 0.01) and Visual Analogue Scale (VAS) (p = 0.01), sural SNAP amplitude (p < 0.01), and toe cooling (p < 0.01) and vibration (p = 0.02) detection thresholds. ENFD did not correlate with neurotoxic ART exposure, CD4 cell count, or plasma HIV-1 viral load. CONCLUSIONS: In subjects with advanced HIV-1 infection, epidermal nerve fiber density (ENFD) assessment correlates with the clinical and electrophysiologic severity of distal sensory polyneuropathy (DSP). ENFD did not correlate with previously established risk factors for HIV-DSP, including CD4 cell count, plasma HIV-1 viral load, and neurotoxic antiretroviral therapy exposure.

HIV neuropathy natural history cohort study: assessment measures and risk factors.

BACKGROUND: Distal sensory polyneuropathy (DSP) is the most common neurologic complication of human immunodeficiency virus (HIV) infection. Risk factors for DSP have not been adequately defined in the era of highly active antiretroviral therapy. METHODS: The authors evaluated 101 subjects with advanced HIV infection over 48 weeks. Assessments included a brief peripheral neuropathy (PN) screen (BPNS), neurologic examination, nerve conduction studies, quantitative sensory testing (QST), and skin biopsies with quantitation of epidermal nerve fiber density. Data were summed into a Total Neuropathy Score (TNS). The presence, severity, and progression of DSP were related to clinical and laboratory results. RESULTS: The mean TNS (range 0 to 36) was 8.9, with 38% of subjects classified as PN-free, 10% classified as having asymptomatic DSP, and 52% classified as having symptomatic DSP. Progression in TNS from baseline to week 48 occurred only in the PN-free group at baseline (mean TNS change = 1.16 +/- 2.76, p = 0.03). Factors associated with progression in TNS were lower current TNS, distal epidermal denervation, and white race. As compared with the TNS diagnosis of PN at baseline, the BPNS had a sensitivity of 34.9% and a specificity of 89.5%. CONCLUSIONS: In this cohort of advanced human immunodeficiency virus (HIV)-infected subjects, distal sensory polyneuropathy was common and relatively stable over 48 weeks. Previously established risk factors, including CD4 cell count, plasma HIV RNA, and use of dideoxynucleoside antiretrovirals were not predictive of the progression of distal sensory polyneuropathy (DSP). Distal epidermal denervation was associated with worsening of DSP. As compared with the Total Neuropathy Score, the brief peripheral neuropathy screen had relatively low sensitivity and high specificity for the diagnosis of DSP.

Insulin resistance in the HIV-infected population: the potential role of mitochondrial dysfunction.

Insulin resistance is accepted as the underlying fundamental defect that predates and ultimately leads to the development of type 2 (adult onset) diabetes mellitus in the general non-human immunodeficiency virus (HIV)-infected population. Insulin resistance is also a major component of the metabolic syndrome that, in association with other factors such as hypertension, hypercholesterolemia, and central obesity, defines a pre-diabetic atherogenic state that leads to adverse cardiovascular events. Growing evidence now suggests that mitochondrial dysfunction in skeletal muscle may be the mechanism whereby insulin resistance is induced. The prevalence of insulin resistance, glucose intolerance, and diabetes in the HIV-infected population has dramatically increased following the common use of highly active antiretroviral therapy (HAART). The development of insulin resistance in the HIV-infected population is likely to be multifactorial reflecting genetic predisposition, direct and indirect effects of both the protease inhibitor (PI) and nucleoside reverse transcriptase inhibitor (NRTI) class of antiretroviral therapy, and a possible contribution from chronic inflammatory changes induced by HIV. Indirect effects of antiretroviral therapy on insulin resistance may be mediated through both the visceral adiposity and peripheral fat depletion components of lipodystrophy as well as through fatty infiltration in liver and muscle. Based on current knowledge, mitochondrial dysfunction can be hypothesized to play a key role in each of these components.

HIV disease progression and limited antiretroviral treatment options for a HIV-1 infected individual with myoclonic epilepsy associated with ragged red fibers.

We describe a 50-year-old Caucasian man with a family history of myoclonic epilepsy associated with ragged red fibers (MERRF) and a diagnosis of Human Immunodeficiency Virus (HIV). The patient had multiple risk factors for contracting HIV and was being followed in our clinic at the time of his diagnosis. Initial testing following seroconversion revealed a baseline CD4+ T-lymphocyte count of 652 x 10(6)cells/l and a HIV-1 RNA of 14,781 copies/ml. He reported exercise intolerance and had mild neurologic deficits, which worsened around the time of HIV seroconversion. These symptoms led to his subsequent diagnosis of MERRF by the detection of the A8344G point mutation in the tRNA(Lys) gene of mitochondrial DNA (mtDNA). The baseline estimated proportion of mutant genome was 39%. He showed a rapid course of HIV disease progression with a CD4+ T-lymphocyte nadir of 174 x 10(6) cells/l associated with a HIV-1 RNA of 238,178 copies/ml, within 17 months following HIV seroconversion. To avoid further mitochondrial insult, which could result from the use of a standard nucleoside reverse transcriptase inhibitor-containing regimen, a protease inhibitor regimen consisting of hard-gel saquinavir (Invirase), and lopinavir/ritonavir (Kaletra) was chosen for this patient. The patient's CD4+ T-lymphocyte count increased to 282 x 10(6)cells/l and his viral load became undetectable 7 months following the initiation of antiretroviral therapy. His neurologic symptoms did not worsen on this antiretroviral regimen. When initiating HIV therapy in individuals with metabolic myopathies related to mitochondrial dysfunction, it may be important to design an antiviral regimen that minimizes mitochondrial damage, yet effectively maintains durable viral suppression.

ATP synthesis is coupled to rat liver mitochondrial RNA synthesis.

Rat liver mitochondria respond to changes in energy demand by modulating the amount of RNA synthesized. Coupled rat liver mitochondria were used to determine the relationship between mitochondrial respiration, ATP levels, and mitochondrial transcription. This system included oxidizable substrates (malate and glutamate) and constituents that could support both mitochondrial respiration and transcription. The respiratory inhibitor rotenone, phosphorylation inhibitor oligomycin, and the uncoupler of oxidative phosphorylation carbonyl-cyanide p-triflouromethoxyphehylhydrazone inhibited RNA synthesis. Addition of ADP stimulated mitochondrial transcription and peak RNA synthesis was observed at 1-2 mM ADP. At ADP concentrations above 2 mM, RNA synthesis decreased. These results demonstrate that mitochondrial transcription is tightly coupled to ATP levels.

Chronic stavudine exposure induces hepatic mitochondrial toxicity in adult Erythrocebus patas monkeys.

OBJECTIVES: To understand the mitochondrial mechanisms underlying the lactic acidosis and hepatic steatosis seen in some HIV-1-infected individuals after long-term stavudine (d4T) exposure, we have explored mitochondrial integrity in adult monkeys (Erythrocebus patas) given a daily human equivalent dose of d4T for 78 days. STUDY DESIGN/METHODS: Three Erythrocebus patas (patas) monkeys were given 3 mg d4T orally twice daily (total 6 mg d4T), or approximately 1.2 mg d4T/kg body weight per day, for 78 days and compared with 3 unexposed animals. Blood taken from controls and from treated monkeys before and after drug exposure was subjected to a complete clinical chemistry profile. Liver and skeletal muscles were examined for oxidative phosphorylation enzyme specific activities, mitochondrial deoxyribonucleic acid (mtDNA) quantity by slot blot, and mtDNA integrity by Southern blot. RESULTS: Clinical chemistry assays demonstrated few significant differences; however, one d4T-exposed monkey had a serum lactate of 8.1 mmol/L after 78 days of oral d4T ingestion. Specific activities of oxidative phosphorylation Complexes I, II, and IV were significantly altered in both livers and skeletal muscles from the d4T-exposed animals, compared with the controls (p < or = 0.05). Significant depletion of mitochondrial DNA was observed in livers of drug-exposed monkeys, but not in skeletal muscle (p < or = 0.05). Further examination of liver DNA by Southern blot confirmed hepatic mtDNA depletion in drug exposed animals. CONCLUSIONS: The data suggest that direct examination of the liver may be required to elucidate clinical d4T-induced hepatotoxicity related to mitochondrial damage.

Cisplatin exposure induces mitochondrial toxicity in pregnant rats and their fetuses.

High levels of cis-diamminedicholorplatinum II (cisplatin)-DNA adducts have previously been observed at term in mitochondrial DNA (mtDNA) from organs of pregnant rats, and from their offspring, after administration of a single injection of cisplatin 15 mg/kg body weight (bw) to the pregnant rat on day 18 of gestation. The consequences of such DNA damage may be clinically relevant as cisplatin is given to pregnant women discovered to have ovarian cancer during pregnancy. In this study, kidneys, livers, and brains of exposed pregnant rats and their offspring were examined for mitochondrial functional integrity. Consistent with previous literature, the most severe toxicity occurred in maternal kidney, where oxidative phosphorylation (OXPHOS) enzyme activities were significantly (approximately 50%) impaired for Complexes II, III, and IV, mtDNA levels in drug-exposed animals were higher than in the unexposed controls, and abnormal mitochondrial morphology was observed by transmission electron microscopy (TEM). In fetal kidneys and livers, cisplatin exposure did not alter mitochondrial morphology or mtDNA quantity, but specific activities of OXPHOS Complexes II and IV were significantly decreased. Fetal brain sustained no discernible mitochondrial toxicity. Therefore, cisplatin-induced mitochondrial toxicity in maternal rat kidney is severe, while damage to mitochondria in fetal kidney and liver, occurring as a result of the transplacental drug exposure, appears to be mild.

Fetal patas monkeys sustain mitochondrial toxicity as a result of in utero zidovudine exposure.

Mitochondrial toxicity was examined in near-term fetuses of pregnant Erythrocebus patas monkeys given human equivalent doses of 3'-azido-3'deoxythymidine (AZT) during the second half of gestation. Pregnant monkeys were dosed daily with 10 or 40 mg AZT, equivalent to about 21% and 86% of the daily AZT dose (500 mg) given to HIV-1-positive pregnant women to prevent maternal-fetal virus transmission. The fetal tissues examined include heart and skeletal muscle, which have high energy requirements, and placenta, which is less dependent on mitochondrial integrity. Slot blot quantitation of mitochondrial DNA (mtDNA) levels showed dose-dependent depletion in heart, skeletal muscle, and placenta from AZT-exposed fetuses compared to unexposed controls. Furthermore, mtDNA degradation, observed by Southern blot analysis, appeared more extensive in AZT-exposed tissues compared to unexposed controls. Mitochondrial functional integrity, as determined by oxidative phosphorylation (OXPHOS) enzyme assays, was also examined in heart, skeletal muscle, and placenta. All three tissues showed strong dose-related decreases in Complex I. In placenta, dose-related increases for Complexes II and IV and a decrease for Complex III were observed. Dose-related increases for Complexes II and IV observed in heart and skeletal muscle have been reported. The increase in Complex IV (cytochrome c oxidase) activity in heart and skeletal muscle tissue from patas fetuses exposed to 40 mg AZT/day has been confirmed here by histochemical staining. Overall, data demonstrate that mitochondrial toxicity, evidenced by depletion in mtDNA and OXPHOS enzyme abnormalities, is manifested similarly in heart, skeletal muscle, and placenta of AZT-exposed monkey fetuses. It is therefore possible that the placenta, which is a readily accessible tissue, might be an indicator of potential mitochondrial toxicity in human pregnancies involving nucleoside analog drug exposure.

Genotoxic and functional consequences of transplacental zidovudine exposure in fetal monkey brain mitochondria.

Mitochondrial toxicity was assessed in the brains of developing Erythrocebus patas monkey fetuses exposed in utero to the nucleoside analogue drug zidovudine (3'-azido-3'deoxythymidine or AZT). Pregnant E. patas monkeys were given 0 (n = 5), 10 (n = 3), and 40 (n = 3) mg of AZT/day, equivalent to 21 and 86% of the human daily dose, for the last half (about 10 weeks) of gestation. Mitochondria were isolated from fetal cerebrum and cerebellum at birth and mitochondrial morphology was examined in these tissues by transmission electron microscopy (TEM). Oxidative phosphorylation (OXPHOS) enzyme specific activities were measured spectrophotometrically. Mitochondrial DNA (mtDNA) integrity and quantity were determined by Southern blot and slot blot analysis. In the cerebral mitochondria, reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase (complex I) specific activity decreased by 25% in monkeys treated with 40 mg of AZT/day compared with unexposed monkeys (p > or = .05). At the same AZT dose in the cerebral mitochondria, succinate dehydrogenase (complex II) and cytochrome c reductase (complex IV)-specific activities showed dose-dependent increases (p > or = .05), compared with those in controls. In the cerebellum, no difference was seen in mitochondrial OXPHOS enzyme activities between unexposed and exposed fetuses. Furthermore, TEM demonstrated no difference in mitochondrial morphology in frontal cerebrum or cerebellum from unexposed and exposed fetuses, and all fetuses had similar amounts of mtDNA in both tissues. Cerebral mtDNA degradation was noted in the highest AZT dosage group, whereas mtDNA from cerebellum was uneffected. Thus, in fetal patas monkeys given a human equivalent daily dose of AZT during the last half of pregnancy, mitochondria in the fetal cerebrum appear to sustain moderate damage, while the fetal cerebellum mitochondria were not effected.

Fetal mitochondrial heart and skeletal muscle damage in Erythrocebus patas monkeys exposed in utero to 3'-azido-3'-deoxythymidine.

3'-azido-3'-deoxythymidine (AZT) is given to pregnant women positive for the human immunodeficiency virus type 1 (HIV-1) to reduce maternal-fetal viral transmission. To explore fetal mitochondrial consequences of this exposure, pregnant Erythrocebus patas monkeys were given daily doses of 1.5 mg (21% of the human daily dose) and 6.0 mg (86% of the human daily dose) of AZT/kg body weight (bw), for the second half of gestation. At term, electron microscopy of fetal cardiac and skeletal muscle showed abnormal and disrupted sarcomeres with myofibrillar loss. Some abnormally shaped mitochondria with disrupted cristae were observed in skeletal muscle myocytes. Oxidative phosphorylation (OXPHOS) enzyme assays showed dose-dependent alterations. At the human-equivalent dose of AZT (6 mg of AZT/kg bw), there was an approximately 85% decrease in the specific activity of NADH dehydrogenase (complex I) and three- to sixfold increases in specific activities of succinate dehydrogenase (complex II) and cytochrome-c oxidase (complex IV). Furthermore, a dose-dependent depletion of mitochondrial DNA levels was observed in both tissues. The data demonstrate that transplacental AZT exposure causes cardiac and skeletal muscle mitochondrial myopathy in the patas monkey fetus.

Oxidative DNA damage in fetal tissues after transplacental exposure to 3'-azido-3'-deoxythymidine (AZT).

The nucleoside analogue 3'-azido-3'-deoxythymidine (AZT) has been used successfully to reduce the incidence of transplacental and perinatal transmission of the HIV virus. However, prolonged treatment with high doses of AZT is utilized in this therapy, and AZT has been found to be a perinatal carcinogen in mice. Any possible perinatal carcinogenic side effects in the human can best be managed if the mechanism is understood. AZT targets mitochondria and might cause increased intracellular production of reactive oxygen species (ROS). We tested whether transplacental AZT may cause oxidative damage in nuclear DNA of fetal tissues. CD-1 Swiss pregnant mice were treated with the transplacental carcinogenesis regimen (25 mg/day AZT, for gestation days 12-18) and tissues collected on the day of birth. Significant increases in 8-oxo-2'-deoxyguano- sine (8-oxo-dG) were found in the livers, a target tissue for transplacental carcinogenesis, and in the kidneys. A non-significant increase occurred in brain, with no change in lung. Tissues were also obtained from fetal patas monkeys (Erythrocebus patas), whose mothers had received 10 mg AZT/day during the last half of gestation. Although limited numbers of samples were available, possible increases in 8-oxo-dG were noted, relative to controls, for placenta and for fetal lung and brain (P = 0.055 for treatment-related increases in these tissues). These results suggest that an increase in reactive oxygen species could contribute to the mechanism of transplacental carcinogenesis by AZT in mice, and that this may also occur in primates.

Endonuclease G from mammalian nuclei is identical to the major endonuclease of mitochondria.

Two Mg(2+)-dependent DNA endonucleases have been isolated from mammalian cells which have a strong preference to nick within long tracts of guanine residues in vitro. One endonuclease activity is mitochondrial (mt). The other endonuclease, called Endonuclease G, is associated with isolated nuclei, and is released when the nuclear chromatin is exposed to moderate ionic strength. Our laboratory has previously purified the mt endonuclease to near homogeneity from mitochondria of bovine heart and reported the enzyme to be a homodimer of a approximately 29 kDa polypeptide [Cummings, O. W. et al. (1987) J. Biol. Chem., 262, 2005-2015]. Although the purified mt endonuclease will extensively fragment M13 viral ssDNA and plasmid dsDNAs in vitro, the enzyme displays an unusually strong preference to nick within a (dG)12:(dC)12 sequence tract which resides just upstream from the origin of DNA replication in the mitochondrial genome. The nuclear Endonuclease G first identified from its selective targeting of several (dG)n:(dC)n tracts in vitro (where N = 3-29), was subsequently purified from calf thymus nuclei and shown to be a homodimer of a approximately 26-kDa polypeptide [Côté, J. et al. (1989) J. Biol. Chem., 264, 3301-3310]. In the present study, we find that Endonuclease G partially purified from calf thymus nuclei will extensively degrade both viral ss- and dsDNAs in vitro, and that the enzyme possesses biochemical properties and specificity for nucleotide sequences in DNA that are strongly related or identical to those of the mt endonuclease. These findings and the discovery of sequence identity between the proteins strengthen the conclusion that the nuclear Endonuclease G is the same enzyme as the mt endonuclease.

Levels of the mitochondrial endonuclease during rat cardiac development implicate a role for the enzyme in repair of oxidative damage in mitochondrial DNA.

Mammalian heart mitochondria (MT) contain a potent Mg(2+)-dependent DNA endonuclease that becomes soluble once isolated mitochondria are disrupted using detergent. The level of this endonuclease was previously found to be markedly elevated in adult rat heart compared to other adult rat tissues. Among tissues, the level of the MT endonuclease does not appear to be correlated with the rate of MT DNA replication but rather with the rate of oxidative metabolism [Houmiel, K.L., Gerschenson, M. and Low, R.L., 1991. Biochimica Biophysica Acta 1079: 197-202]. In the present study, the level of the endonuclease has been quantitated both during rat cardiac development, from gestational day 18 through adulthood, and in cultured rat heart myoblasts. Surprisingly, the specific activity of the MT endonuclease in fetal and newborn mitochondria is high. The values are greater than 50% of that seen in the adult even though the mitochondria at this period of heart development are few and structurally disorganized. Remarkably, there is a burst of endonuclease activity at day 2 which accompanies a similar, transient elevation of respiratory complex I and IV activities. At later times, the endonuclease activity gradually increases until adulthood and correlates with steady increases in MT DNA and DNA polymerase-gamma. In cultured myoblasts, the level of the endonuclease increases about seven-fold as the growing cells reach confluency and differentiate into myotubes. These variations in the specific activity of the endonuclease, when considered along with other properties of the enzyme suggest that the endonuclease may serve a role in the removal of oxidative damage in MT DNA incurred from respiration.

Mitochondrial endonuclease activity in the rat varies markedly among tissues in relation to the rate of tissue metabolism.

Rat heart mitochondria contain a potent endonuclease activity that closely resembles the endonuclease of bovine and human heart mitochondria, and shows a striking preference for an evolutionarily conserved sequence that resides just upstream from the heavy (H)-strand origin of DNA replication (Ori H), (Low, R.L. et al. (1988) Nucleic Acids Res. 16, 6427-6425). This study reports that while the site-directed endonuclease is evident in the mt fractions of several rat organs, the levels of activity among them varies in an unexpected and marked fashion. There is nearly 200-times more of this endonuclease activity per mg of mt protein in the heart than in the liver (or spleen). Levels intermediate to those in heart and liver are found in the kidney and brain. The large variations in endonuclease activity do not correlate with reported rates of mtDNA turnover among tissues and are in contrast to the much smaller variations in levels of mtDNA and DNA polymerase-gamma activity. However, there may be some relationship between the amount of the endonuclease and the rate of oxidative phosphorylation.

Regulation of melanoma by the embryonic skin.

This report focuses on the regulation of murine melanoma by the embryonic skin. A surgical technique was developed to allow injection of B16 melanoma cells into the embryo in utero. A significant decrease in incidence of tumors was noted, which correlated with the time of arrival of normally migrating premelanocytes into the skin. Media were conditioned from skin explanted at the time premelanocytes arrive in it; these media inhibited the growth of melanoma cells in vitro. Under optimal conditions the growth of melanoma cells ceased; the cells had altered morphology and failed to proliferate when placed in fresh growth media.