Domenico Marzulli

Cytochrome c-induced cytosolic nicotinamide adenine dinucleotide oxidation, mitochondrial permeability transition, and apoptosis

A catalytic amount of cytochrome c (cyto-c) added to the incubation medium of isolated mitochondria promotes the transfer of reducing equivalents from extramitochondrial nicotinamide adenine dinucleotide in its reduced state (NADH) to molecular oxygen inside the mitochondria, a process coupled to the generation of a membrane potential. This mimics in many aspects the early stages of those apoptotic pathways characterized by the persistence of mitochondrial membrane potential but with cyto-c already exported into the cytosol. In cyclosporin-sensitive and calcium-induced mitochondrial permeability transition (MPT) a release of cyto-c can also be observed. However, in MPT uncoupled respiration associated with mitochondrial swelling and preceded by the complete dissipation of the membrane potential which cannot be restored with ATP addition or any other source of energy is immediately activated. The results obtained and discussed with regard to intactness of mitochondrial preparations indicate that MPT could be an apoptotic event downstream but not upstream of cyto-c release linked to the energy-requiring processes. In the early stages of apoptosis cytosolic cyto-c participates in the activation of caspases and at the same time can promote the oxidation of cytosolic NADH, making more energy available for the correct execution of the cell death program. This hypothesis is not in contrast with available data in the literature showing that cyto-c is present in the cytosol of both control and apoptosis-induced cultured cell lines.

Effect of magnesium ions on the activity of the cytosolic NADH/cytochrome c electron transport system.

Cytochrome c (cyto‐c), added to isolated mitochondria, activates the oxidation of extramitochondrial NADH and the generation of a membrane potential, both linked to the activity of the cytosolic NADH/cyto‐c electron transport pathway. The data presented in this article show that the protective effect of magnesium ions on the permeability of the mitochondrial outer membrane, supported by previously published data, correlates with the finding that, in hypotonic but not isotonic medium, magnesium promotes a differential effect on both the additional release of endogenous cyto‐c and on the increased rate of NADH oxidation, depending on whether it is added before or after the mitochondria. At the same time, magnesium prevents or almost completely removes the binding of exogenously added cyto‐c. We suggest that, in physiological low‐amplitude swelling, magnesium ions may have the function, together with other factors, of modulating the amount of cyto‐c molecules transferred from the mitochondrial intermembrane space into the cytosol, required for the correct execution of the apoptotic programme and/or the activation of the NADH/cyto‐c electron transport pathway.

Ceramide-induced activation of cytosolic NADH/cytochrome c electron transport pathway: An additional source of energy for apoptosis.

We have investigated whether increase in the oxidation rate of exogenous cytochrome c (cyto-c), induced by long-chain ceramides, might be due to an increased rate of cytosolic NADH/cyto-c electron transport pathway. This process was identified in isolated liver mitochondria and has been studied in our laboratory for many years. Data from highly specific test of sulfite oxidase prove that exogenous cyto-c both in the absence and presence of ceramide cannot permeate through the mitochondrial outer membrane. However, the oxidation of added NADH, mediated by exogenous cyto-c and coupled to the generation of a membrane potential supporting the ATP synthesis, can also be stimulated by ceramide. The results obtained suggest that ceramide molecules, by increasing mitochondrial permeability, with the generation of either raft-like platforms or channels, may have a dual function. They can promote the release of endogenous cyto-c and activate, with an energy conserving process, the oxidation of cytosolic NADH either inducing the formation of new respiratory contact sites or increasing the frequency of the pre-existing porin contact sites. In agreement with the data in the literature, an increase of mitochondrial ceramide molecules level may represent an efficient strategy to activate and support the correct execution of apoptotic program.

Enzymatic activity in Mytilus galloprovincialis (Mollusca: Bivalvia) digestive gland

Mitochondrial dehydrogenases, antioxidant enzymes, transaminases and cytochrome P450 activity have been studied from the digestive gland of Mytilus galloprovincialis, reared in a non-polluted area of the Mar Grande of Taranto (Ionian Sea, Mediterranean).

Synthesis and Biological Evaluation of a Valinomycin Analog Bearing a Pentafluorophenyl Active Ester Moiety

A valuable analog of the K(+)-ionophore valinomycin (1), bearing a pentafluorophenyl ester moiety, has been obtained by selective reaction between the tertiary hydroxyl moiety of analog 2 (available from valinomycin hydroxylation) and the isocyanate group of pentafluorophenyl N-carbonyl glycinate (3) catalyzed by bis(N,N-dimethylformamide)dichlorodioxomolybdenum(VI). LC-HRMS studies show that analog 4 undergoes easy derivatization under mild conditions by reaction with OH- and NH2-containing compounds. Mitochondrial depolarization assays suggest that 4 acts as a K(+)-ionophore, provided that the glycine carboxyl group is appropriately masked.

Mitochondrial Oxygen Uptake as the Expression of both the One-Trans-Membrane and the Bi-Trans-Membrane Electron Transport Chains

Additional experimental data are presented showing that in rat liver mitochondria, together with the well known one-trans-membrane respiratory chain activity, a bi-trans-membrane electron channelling system, located at the contact sites, is present. This last electron transport pathway promotes the oxidation of substrates present either outside or inside the mitochondria.