The depletion of F1 subunit ε in yeast leads to an uncoupled respiratory phenotype that is rescued by mutations in the proton-translocating subunits of F0

The central stalk of the ATP synthase is an elongated hetero-oligomeric structure providing a physical connection between the catalytic sites in F₁ and the proton translocation channel in F₀ for energy transduction between the two subdomains. The shape of the central stalk and relevance to energy coupling are essentially the same in ATP synthases from all forms of life, yet the protein composition of this domain changed during evolution of the mitochondrial enzyme from a two- to a three-subunit structure (γ, δ, ε). Whereas the mitochondrial γ- and δ-subunits are homologues of the bacterial central stalk proteins, the deliberate addition of subunit ε is poorly understood. Here we report that down-regulation of the gene (ATP15) encoding the ε-subunit rapidly leads to lethal F₀-mediated proton leaks through the membrane because of the loss of stability of the ATP synthase. The ε-subunit is thus essential for oxidative phosphorylation. Moreover, mutations in F₀ subunits a and c, which slow the proton translocation rate, are identified that prevent ε-deficient ATP synthases from dissipating the electrochemical potential. Cumulatively our data lead us to propose that the ε-subunit evolved to permit operation of the central stalk under the torque imposed at the normal speed of proton movement through mitochondrial F₀.     

Mitochondrial DNA Mutations Provoke Dominant Inhibition of Mitochondrial Inner Membrane Fusion

Mitochondria are highly dynamic organelles that continuously move, fuse and divide. Mitochondrial dynamics modulate overall mitochondrial morphology and are essential for the proper function, maintenance and transmission of mitochondria and mitochondrial DNA (mtDNA). We have investigated mitochondrial fusion in yeast cells with severe defects in oxidative phosphorylation (OXPHOS) due to removal or various specific mutations of mtDNA. We find that, under fermentative conditions, OXPHOS deficient cells maintain normal levels of cellular ATP and ADP but display a reduced mitochondrial inner membrane potential. We demonstrate that, despite metabolic compensation by glycolysis, OXPHOS defects are associated to a selective inhibition of inner but not outer membrane fusion. Fusion inhibition was dominant and hampered the fusion of mutant mitochondria with wild-type mitochondria. Inhibition of inner membrane fusion was not systematically associated to changes of mitochondrial distribution and morphology, nor to changes in the isoform pattern of Mgm1, the major fusion factor of the inner membrane. However, inhibition of inner membrane fusion correlated with specific alterations of mitochondrial ultrastructure, notably with the presence of aligned and unfused inner membranes that are connected to two mitochondrial boundaries. The fusion inhibition observed upon deletion of OXPHOS related genes or upon removal of the entire mtDNA was similar to that observed upon introduction of point mutations in the mitochondrial ATP6 gene that are associated to neurogenic ataxia and retinitis pigmentosa (NARP) or to maternally inherited Leigh Syndrome (MILS) in humans. Our findings indicate that the consequences of mtDNA mutations may not be limited to OXPHOS defects but may also include alterations in mitochondrial fusion. Our results further imply that, in healthy cells, the dominant inhibition of fusion could mediate the exclusion of OXPHOS-deficient mitochondria from the network of functional, fusogenic mitochondria.     

Avian pox in Magellanic Penguins (Spheniscus magellanicus)

Avian pox is an enveloped double-stranded DNA virus that is mechanically transmitted via arthropod vectors or mucosal membrane contact with infectious particles or birds. Magellanic Penguins (Spheniscus magellanicus) from two colonies (Punta Tombo and Cabo Dos Bahías) in Argentina showed sporadic, nonepidemic signs of avian pox during five and two of 29 breeding seasons (1982-2010), respectively. In Magellanic Penguins, avian pox expresses externally as wart-like lesions around the beak, flippers, cloaca, feet, and eyes. Fleas (Parapsyllus longicornis) are the most likely arthropod vectors at these colonies. Three chicks with cutaneous pox-like lesions were positive for Avipoxvirus and revealed phylogenetic proximity with an Avipoxvirus found in Black-browed Albatross (Thalassarche melanophrys) from the Falkland Islands in 1987. This proximity suggests a long-term circulation of seabird Avipoxviruses in the southwest Atlantic. Avian pox outbreaks in these colonies primarily affected chicks, often resulted in death, and were not associated with handling, rainfall, or temperature.     

Molecular basis for resistance to myxothiazol, mucidin (strobilurin A), and stigmatellin. Cytochrome b inhibitors acting at the center o of the mitochondrial ubiquinol-cytochrome c reductase in Saccharomyces cerevisiae

The respiratory bc1 complex transfers the electrons from ubiquinol to cytochrome c oxidase. Myxothiazol, strobilurin A (mucidin), and stigmatellin are center o inhibitors preventing electron transfer at the ubiquinone redox site Qo, which is located closer to the outer side of the inner mitochondrial membrane. The cytochrome b gene is carried by the organelle DNA. Yeast mutants resistant to myxothiazol and mucidin have been previously isolated and mapped to specific loci of the cytochrome b gene. In the present work, stigmatellin-resistant mutants were isolated and genetically analyzed. The mutated amino acid residues from seven myxothiazol-, four mucidin-, and six stigmatellin-resistant mutants have been identified by sequencing the relevant segments of the resistant cytochrome b gene. A third myxothiazol-resistant locus and the first stigmatellin-resistant locus were identified. The mutated codons were found to be clustered in two regions of the cytochrome b protein which appeared to be responsible for the resistance to Qo site inhibitors. The first region is within the end of the first, the second, and the beginning of the third exon whereas the second region is within exon five and the beginning of the sixth exon.     

Cytochrome P450 aromatase expression in human seminoma

Background  

The enzyme cytochrome P450 aromatase, catalysing the conversion of androgens into estrogens, has been detected in normal human testicular cells suggesting a physiological role of local estrogen biosynthesis on spermatogenesis control. Estrogens, regulating cell growth and apoptosis, can also be involved in tumorigenesis process, but the possible link between estrogens and testicular neoplastic process is, up to now, scarcely known. This study examined aromatase expression in human seminoma, which is the most common germ cell tumour of the testis.     

Immunolocalization of estrogen receptor beta in the epididymis of mature and immature pigs

A growing body of evidence suggests a role of estrogens in the male reproduction via their specific estrogen receptors (ERalpha/ERbeta). Estrogen receptor distribution along the genital tract tissues has been described in different species, but it is unknown in the pig. Therefore, the aim of the present study was to localize ERbeta in the epididymis of mature and immature pigs (aged 2 and 18 months, respectively). Immunohistochemistry was carried out on paraffin-embedded tissues using a mouse anti-human monoclonal IgG against ERbeta as the primary antibody, and a goat anti-mouse biotinylated IgG as the secondary antibody. Avidin-biotin-peroxidase complex was then applied followed by diaminobenzidine. In immature pigs, the epithelial cells from the caput, corpus and cauda epididymis showed no or very weak immunoreactivity for ERbeta, whereas they were all strongly immmunoreactive in mature pigs. A various intensity of immunostaining from weak to strong in the smooth muscle cells as well as in the connective tissue cells were detected in the epididymis of both, young and adult pigs. This is the first report on the cellular localization of ERbeta protein in porcine epidydimis. The present study demonstrated that (1) irrespectively of the epididymal region, the epithelial cells of caput, corpus and cauda epididymis of mature pigs revealed a strong immunoreactivity for ERbeta, and (2) ERbeta expression in the epididymal epithelium is regulated by puberty. Finally, although the biological activity of ERbeta has not yet been established, the results of the present study suggest its involvement in estrogen modulation of pig epididymal function.     

Facile methods for generating human somatic cell gene knockouts using recombinant adeno-associated viruses

Emerging evidence suggests that recombinant adeno-associated viral (rAAV) vectors can be used for specific gene targeting in human somatic cells. We have developed an rAAV vector construction procedure employing fusion PCR and a single cloning step that considerably simplifies the knockout process. We demonstrate its utility by disrupting genes at specific positions within human colon cancer cells as well as within immortalized normal epithelial cells. This technology should be broadly applicable to in vitro studies that require the manipulation of the human genome.