Studies on the molecular basis of H+ translocation by cytochromec oxidase

We report here studies which characterize further the interaction ofN,N-dicyclohexylcarbodiimide with cytochromec oxidase leading to inhibition of H⁺ translocation by the enzyme. Further evidence is presented to show that the inhibition results from a real interaction of DCCD with the enzyme and cannot be accounted for by uncoupling and, contrary to recent criticisms, this interaction occurs specifically with subunit III of the enzyme even at relatively high inhibitor-to-enzyme stoichiometries. Use of a spin-label analogue of DCCD has enabled us to demonstrate that the carbodiimide-binding site is highly apolar and may not lie on the pathway of electron transfer.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - NCCD N-(2, 2, 6, 6-tetramethylpiperidyl-1-oxyl)-N-(cyclohexyl)carbodiimide - Hepes 2-(N-2-hydroxyethylpiperazin-N-yl) ethane sulfonate - TMPD N,N,N,N-tetramethylphenylenediamine     

Cytochrome c oxidases: polypeptide composition, role of subunits, and location of active metal centers

The general structure of the enzyme, its polypeptide composition, and a proposal for a rational nomenclature are discussed. The mitochondrially coded and bacterial cytochrome c oxidase subunits have been analyzed with more attention focused on elucidating the number of metals present in the enzyme and the ligands available for their coordination. The picture of a 2 Cu/2 Fe enzyme has been compared with that of a 3 Cu/2 Fe enzyme and a new model is proposed for the location of the metal centers in the enzyme.     

Interleukin-15 is a major regulator of the cell-microenvironment interactions in human renal homeostasis

Experiments in IL-15^?/? and IL-15Rα^?/? mice show that intra-renal IL-15, through IL-15Rα behaves as an epithelial survival factor. Recent data highlight new functions of IL-15 in renal homeostasis mediated by IL-15Rγ (CD132). Indeed, in CD132+ renal epithelial tubular cells IL-15 preserves E-cadherin expression inhibiting epithelial-mesenchymal transition (EMT). By contrast, during allograft rejection, the increased intra-graft IL-15 expression favors tubular destruction facilitating the intraepithelial recruitment of CD8 T cells expressing the E-cadherin ligand CD103. In renal cancer, loss of CD132 by epithelial cells defines a tumoral microenvironment where IL-15 triggers E-cadherin down-regulation and EMT. Finally, in CD132+ renal cancer stem cells IL-15 induces the generation of non-tumorigenic epithelial cells sensitive to cytotoxic drugs. These findings are discussed in the light of IL-15-based immunotherapy for renal cancer.     

Six Homeoproteins Directly Activate Myod Expression in the Gene Regulatory Networks That Control Early Myogenesis

In mammals, several genetic pathways have been characterized that govern engagement of multipotent embryonic progenitors into the myogenic program through the control of the key myogenic regulatory gene Myod. Here we demonstrate the involvement of Six homeoproteins. We first targeted into a Pax3 allele a sequence encoding a negative form of Six4 that binds DNA but cannot interact with essential Eya co-factors. The resulting embryos present hypoplasic skeletal muscles and impaired Myod activation in the trunk in the absence of Myf5/Mrf4. At the axial level, we further show that Myod is still expressed in compound Six1/Six4:Pax3 but not in Six1/Six4:Myf5 triple mutant embryos, demonstrating that Six1/4 participates in the Pax3-Myod genetic pathway. Myod expression and head myogenesis is preserved in Six1/Six4:Myf5 triple mutant embryos, illustrating that upstream regulators of Myod in different embryonic territories are distinct. We show that Myod regulatory regions are directly controlled by Six proteins and that, in the absence of Six1 and Six4, Six2 can compensate.     

Presence of TT virus DNA in bone marrow cells from hematologic patients

Recent observations suggest that TT virus (TTV), in addition to liver, may also infect bone marrow. In this study, bone marrow samples and sera from 33 patients with haematological disorders and sera from 16 healthy controls were investigated for TTV DNA presence. Altogether TTV DNA sequences were demonstrated in bone marrow cells of 84.84% of patients. Moreover TTV DNA was detected in sera from 72.72% of patients and from 93.75% of controls. N22 sequences amplified from bone marrow cells and serum of 3 patients were analysed, after cloning: all these isolates were of type 2c and 2 or 3 variants were present in each isolate. After single strand DNA degradation, replicative forms were detectable in BM cells. This finding, in addition to the detection of variants similar in the BM and in the serum of the same patient could suggest that BM is a site of TTV replication (or one of the sites) from which the virus is spread in blood.     

Drp-1-dependent division of the mitochondrial network blocks intraorganellar Ca2+ waves and protects against Ca2+-mediated apoptosis

By transiently or stably overexpressing the mitochondrial fission factor dynamin-related protein-1 (Drp-1), we evaluated the role of mitochondrial division in organelle Ca2+ homeostasis and apoptotic signaling. Quantitative 3D digital microscopy revealed a split mitochondrial network in Drp-1-overexpressing cells without changes in cell viability. High-speed mitochondrial [Ca2+] ([Ca2+]m) imaging revealed propagating intramitochondrial Ca2+ waves in intact cells, which were blocked in the Drp-1-fragmented network, leaving a fraction of individual mitochondria without substantial [Ca2+]m elevation. Consequently, in Drp-1-expressing cells the apoptotic efficacy of ceramide, which causes a Ca2+-dependent perturbation of mitochondrial structure and function, was drastically reduced. Conversely, the sensitivity to staurosporine-induced apoptosis, previously shown to be directly triggered by Drp-1-dependent recruitment of proapoptotic proteins to mitochondria, was enhanced. These results demonstrate that the regulated process of mitochondrial fusion and fission controls the spatiotemporal properties of mitochondrial Ca2+ responses and, thus, physiological and pathological consequences of cellular Ca2+ signals.     

Anti‐atherosclerotic effects of vitamin E – myth or reality?

Atherosclerosis and its complications such as coronary heart disease, myocardial infarction and stroke are the leading causes of death in the developed world. High blood pressure, diabetes, smoking and a diet high in cholesterol and lipids clearly increase the likelihood of premature atherosclerosis, albeit other factors, such as the individual genetic makeup, may play an additional role. Several epidemiological studies and intervention trials have been performed with vitamin E, and some of them showed that it prevents atherosclerosis. For a long time, vitamin E was assumed to act by decreasing the oxidation of LDL, a key step in atherosclerosis initiation. However, at the cellular level, vitamin E acts by inhibition of smooth muscle cell proliferation, platelet aggregation, monocyte adhesion, oxLDL uptake and cytokine production, all reactions implied in the progression of atherosclerosis. Recent research revealed that these effects are not the result of the antioxidant activity of vitamin E, but rather of precise molecular actions of this compound. It is assumed that specific interactions of vitamin E with enzymes and proteins are at the basis of its non-antioxidant effects. Vitamin E influences the activity of several enzymes (e.g. PKC, PP2A, COX-2, 5-lipooxygenase, nitric oxide synthase, NADPH-oxidase, superoxide dismutase, phopholipase A2) and modulates the expression of genes that are involved in atherosclerosis (e.g. scavenger receptors, integrins, selectins, cytokines, cyclins). These interactions promise to reveal the biological properties of vitamin E and allow designing better strategies for the protection against atherosclerosis progression.