A new mtDNA mutation showing accumulation with time and restriction to skeletal muscle.

We have identified a new mutation in mtDNA, involving tRNALeu(CUN) in a patient manifesting an isolated skeletal myopathy. This heteroplasmic A-->G transition at position 12320 affects the T psi C loop at a conserved site and was not found in 120 controls. Analysis of cultured fibroblasts, white blood cells/platelets, and skeletal muscle showed that only skeletal muscle contained the mutation and that only this tissue demonstrated a biochemical defect of respiratory-chain activity. In a series of four muscle-biopsy specimens taken over a 12-year period, there was a gradual increase, from 70% to 90%, in the overall level of mutation, as well as a marked clinical deterioration. Single-fiber PCR confirmed that the proportion of mutant mtDNA was highest in cytochrome c oxidase-negative fibers. This study, which reports a mutation involving tRNALeu(CUN), demonstrates clearly that mtDNA point mutations can accumulate over time and may be restricted in their tissue distribution. Furthermore, clinical deterioration seemed to follow the increase in the level of mutation, although, interestingly, the appearance of fibers deficient in respiratory-chain activity showed a lag period.     

Cultured AIDS-related Kaposi's sarcoma cells retain a proliferative bioenergetic profile but demonstrate reduced cytoprotective capabilities

Features of AIDS-related Kaposi's sarcoma (AIDS-KS), such as the multifocal presentation at mucosal and epidermal sites subjected to trauma, suggest that AIDS-KS is initially a reactive hyperplasia that subsequently progresses to a neoplasia. It is recognized that there is an association between sustained states and the subsequent development of neoplasia (e.g., ulcerative colitis/colonic adenocarcinoma). Furthermore, patients who develop AIDS-KS experience both a constant immune stimulation due to sustained high levelsof virus-induced cytokines and, because of a sparing effect on their phagoctic cells, retention of the phagocytic inflammatory response. A component of phygocytic activation is the initiation of the oxidative brust, resulting in the generation of reactive oxygen species (ROS), which can be mutagenic to host cells if released beyond the phagolysosome and not inactivated. Our results demonstrate that cultured AIDS-KS cells possess drcreased cytoprotective capabilities. Relativeto either dermal fibroblasts, or human microvascular endothelial cells (HMECs), AIDS-KS cells contained significantly lower levels of glutathione, a tripeptide integral in both cytoprotection and maintenance of cellular thiol status. While HMECs increased catalase activity during culture in the cytokine-rich KS milieu (control medium supplement with conditioned medium from MOT, an TLV II-infected cell line), AIDS-KS cells demonstrated reduced catalase function under these conditions. Furthermore, HMEC cultures showed in inherent biochemical responsiveness, by increasing catalase activity following exposure to exaogenous H₂(O₂). In contrast, the catalase activity of AIDS-KS cells decreased following (H₂O₂) challenge. Our results show that an inherent deficiency in cellular cytoprotection is present in AIDS-KS cells and suggest that oxidant stress may function in the development and progression AIDS-KS.     

Degradation and resynthesis of adenine nucleotides in adult rat heart myocytes

The degradation and short-term resynthesis of adenine nucleotides have been examined in a preparation of isolated rat heart myocytes. These myocyte preparations are essentially free of vascular and endothelial cells, contain levels of adenine nucleotides quite comparable to those of intact heart tissue, and retain these components remarkably well for up to 2 h of aerobic incubation in the presence of 1 mM Ca2+. When the cells are rapidly and synchronously de-energized by addition of uncoupler, an inhibitor of respiration and iodoacetate, cellular ATP is degraded almost quantitatively to AMP. The AMP is then converted to either intracellular adenosine, which accumulates to high concentrations before release to the cell exterior, or to IMP. The relative contribution of these two pathways depends on the metabolic state of the cells just prior to de-energization, with IMP production favored when respiring cells are de-energized and adenosine formation predominant when glycolyzing myocytes are subjected to this treatment. Cells de-energized by anaerobiosis in the absence of glucose lose ATP and adenine nucleotides with the production of IMP and adenosine. Upon reoxygenation, these cells restore a high adenylate energy charge and about 60% of control levels of GTP. There is a net resynthesis of 5-7 nmol of adenine nucleotides.mg-1 protein with a corresponding decline in IMP. Added [14C]adenosine labels the adenine nucleotide pool, but little net resynthesis of adenine nucleotides via adenosine kinase can be detected. It therefore appears that a rapid regeneration of adenine nucleotides can occur via the enzymes of the purine nucleotide cycle in heart myocytes and is limited by the size of the IMP pool retained.     

Intracellular free Ca2+ and the hypercontracture of adult rat heart myocytes

The Ca2+ sensitivity of a population of isolated adult rat heart myocytes has been related to the Na+ content of the cells prior to Ca2+ exposure, and the intracellular free Ca2+ as reported by quin2 fluorescence when the cells are challenged with millimolar external Ca2+. Myocytes exposed to Ca2+ during quin2 loading show a resting intracellular free Ca2+ of 150 +/- 30 nM and retain the rod cell morphology of heart cells in situ. The myocytes take up Na+ and lose K+ when incubated in the cold in the absence of Ca2+. Large numbers of these rod-shaped, Na+-loaded myocytes hypercontract into grossly distorted round cell forms when exposed to physiological levels of Ca2+. The number of cells that hypercontract is proportional to the Na+ content of the cells prior to Ca2+ addition and can be directly related to the intracellular free Ca2+ concentration attained following Ca2+ addition. Fifty percent of the cells in a myocyte population hypercontract when the internal free Ca2+ concentration reported by quin2 reaches 400 nM and virtually all of the cells hypercontract when this value reaches 1 microM. The entry of Ca2+ into Na+-loaded myocytes is biphasic with one phase inhibited by Ca2+ channel blockade. This suggests that Ca2+ enters Na+-loaded myocytes by the Ca2+ channel as well as by Na+/Ca2+ exchange.     

Characteristics of a moderately thermophilic and acidophilic iron-oxidizing Thiobacillus

Summary Thiobacillus TH1 is an acidophilic chemolithotrophic heterotroph growing at temperatures up to about 50°C on media containing ferrous iron or pyrite when supplemented with yeast extract or glutathione. Virtually no carbon dioxide fixation occurred during growth on iron with yeast extract. Its DNA contains 48 mol % guanine + cytosine. The organism effects the thermophilic leaching of metals from pyrite, chalcopyrite, CuS, and copper concentrates. Oxidation of soluble ferrous iron at pH 1.6 was competitively inhibited by ferric iron and had a K_m of 7.3 mM FeSO₄.     

Regulation of mitochondrial K+/H+ antiport activity by hydrogen ions

The effect of matrix pH (pHi) on the activity of the mitochondrial K+/H+ antiport has been studied using the fluorescence of 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) to monitor pHi and passive swelling in K+ acetate to follow antiport activity. Heart mitochondria suspended in hypotonic K+ acetate in the absence of respiration show an initial delta pH of -0.4 (interior acid) that decays slowly. Addition of A23187 to deplete matrix Mg2+ results in a further acid shift in pHi followed by equilibration of delta pH. This equilibration appears to depend on K+/H+ antiport and is slow at acid pHi but very rapid when the matrix is alkaline. Swelling of Mg(2+)-depleted mitochondria in K+ acetate is multiphasic with a slow initial rate, a period of maximum swelling, and a final period in which the rate declines. At constant external pH (pH0), the initial rate of swelling is faster with increasing pHi and the time to the onset of the maximum swelling rate decreases. The maximum swelling rate is initiated at pHi 7.4 when pH0 is 7.8 and at pHi 7.1 when pH0 is 7.4. The maximum rate of swelling increases linearly with increasing pH0 in the range from 7.0 to 8.2. This rate also shows a linear relationship to the value of pHi at the time the maximum rate is attained. Dixon plots of the reciprocal of the maximum swelling rate vs [H+]0 suggest that external [H+] is a noncompetitive inhibitor of K+ entry on the antiport. It is concluded that K+/H+ antiport in Mg(2+)-depleted heart mitochondria can be regulated by matrix [H+] (see Beavis, A. D., and Garlid, K. D. (1990) J. Biol. Chem. 265, 2538-2545), but that this antiport is also sensitive to external [H+] or to delta pH when it acts in the direction of K+ uptake.