reported that mt pools of rat tissues instead are highly asymmetric, with the dGTP pool in some cases being several-hundred-fold larger than the dTTP pool, and suggested that the asymmetry contributes to increased mutagenesis during mt DNA replication. We have now investigated this discrepancy and determined the size of each dNTP pool in mouse liver mitochondria. We found large variations in pool sizes that closely followed variations in the ATP pool and depended on the length of time spent in the preparation of mitochondria. The proportion between dNTPs was in all cases without major asymmetries and similar to those found earlier in cultured resting cells. We also investigated the import and export of thymidine phosphates in mouse liver mitochondria and provide evidence for a rapid, highly selective, and saturable import of dTMP, not depending on a functional respiratory chain. At nM external dTMP the nucleotide is concentrated 100-fold inside the mt matrix. Export of thymidine phosphates was much slower and possibly occurred at the level of dTDP.mitochondrial diseases ͉ mitochondrial DNA ͉ regulation of mitochondrial dNTP pools M itochondria contain pools of the four canonical dNTPs for the synthesis of mitochondrial (mt) DNA, separated by the mt double membrane from the corresponding cytosolic dNTP pools for nuclear DNA synthesis (1). mt DNA synthesis occurs throughout the whole life of a cell, independent of nuclear DNA synthesis in S-phase, and mt dNTPs must therefore be available also in cells outside S-phase, when dNTP pools are not required for nuclear DNA synthesis. In cultured cells we found much larger dNTP pools in the cytosol than in mitochondria, but we found roughly equal proportions among the four dNTPs in these two compartments. This applied to both cycling (2) and resting (3) cells even though pool sizes were much smaller in the latter. Pulse-chase experiments with radioactively labeled thymidine demonstrated separate metabolic compartments for mt and cytosolic dTTP pools, in rapid communication with each other. mt dTTP was formed by two separate pathways: (i) de novo synthesis from ribonucleotides in the cytosol, followed by import into mitochondria; or (ii) phosphorylation of imported thymidine by a mt thymidine kinase. Cycling cells largely depended on the first pathway whereas resting cells used the second. These results may explain why genetic defects (4-6) involving enzymes of the second pathway mostly affect tissues with terminally differentiated cells.A recent article in PNAS by Song et al. (7) reported a large asymmetry among the four mt dNTP pools in various rat organs, with dGTP in some cases being several-hundred-fold more abundant than dTTP. In model experiments the asymmetries greatly affected the fidelity of DNA synthesis in vitro. They were suggested to lead to an increased mutability of the mt genome in vivo. The pool data appear to be at variance with the scenario for mt dNTP synthesis in cultured cells suggested by us.Earlier analyses of the size and composition of adenosine ...

-/-mice, a model of muscular dystrophies due to defects of collagen VI. Experimental approach: We studied calcineurin activity based on NFAT translocation; T cell activation based on expression of CD69 and CD25; propensity to open the permeability transition pore in mitochondria and skeletal muscle fibres based on the ability to retain Ca 2+ and on membrane potential, respectively; muscle ultrastructure by electronmicroscopy; and apoptotic rates by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assays in Col6a1 -/-mice before after treatment with Debio 025. Key results: Debio 025 did not inhibit calcineurin activity, yet it desensitizes the mitochondrial permeability transition pore in vivo. Treatment with Debio 025 prevented the mitochondrial dysfunction and normalized the apoptotic rates and ultrastructural lesions of myopathic Col6a1 -/-mice. Conclusions and implications:Desensitization of the mitochondrial permeability transition pore can be achieved by selective inhibition of matrix cyclophilin D without inhibition of calcineurin, resulting in an effective therapy of Col6a1 -/-myopathic mice. These findings provide an important proof of principle that collagen VI muscular dystrophies can be treated with Debio 025. They represent an essential step towards an effective therapy for Ullrich Congenital Muscular Dystrophy and Bethlem Myopathy, because Debio 025 does not expose patients to the potentially harmful effects of immunosuppression.

We studied the effects of cyclosporin A (CsA) administration 1) on the properties of the permeability transition pore (PTP) in mitochondria isolated from the liver and 2) on the susceptibility to hepatotoxicity induced by lipopolysaccharide of Escherichia coli (

These results demonstrate that the MSO target glutamine synthetase is required for the early steps of the cytokine response to endotoxins, and that its pharmacological inhibition may be exploited to treat inflammation.

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