Extracellular annexins and dynamin are important for sequential steps in myoblast fusion

Myoblast fusion into multinucleated myotubes is a crucial step in skeletal muscle development and regeneration. Here, we accumulated murine myoblasts at the ready-to-fuse stage by blocking formation of early fusion intermediates with lysophosphatidylcholine. Lifting the block allowed us to explore a largely synchronized fusion. We found that initial merger of two cell membranes detected as lipid mixing involved extracellular annexins A1 and A5 acting in a functionally redundant manner. Subsequent stages of myoblast fusion depended on dynamin activity, phosphatidylinositol(4,5)bisphosphate content, and cell metabolism. Uncoupling fusion from preceding stages of myogenesis will help in the analysis of the interplay between protein machines that initiate and complete cell unification and in the identification of additional protein players controlling different fusion stages.     

Splicing Diversity of the Human OCLN Gene and Its Biological Significance for Hepatitis C Virus Entry

Persistent hepatitis C virus (HCV) infection is a primary etiological factor for the development of chronic liver disease, including cirrhosis and cancer. A recent study identified occludin (OCLN), an integral tight junction protein, as one of the key factors for HCV entry into cells. We explored the splicing diversity of OCLN in normal human liver and observed variable expression of alternative splice variants, including two known forms (WT-OCLN and OCLN-ex4del) and six novel forms (OCLN-ex7ext, OCLN-ex3pdel, OCLN-ex3del, OCLN-ex3-4del, OCLN-ex3p-9pdel, and OCLN-ex3p-7pdel). Recombinant protein isoforms WT-OCLN and OCLN-ex7ext, which retained the HCV-interacting MARVEL domain, were expressed on the cell membrane and were permissive for HCV infection in in vitro infectivity assays. All other forms lacked the MARVEL domain, were expressed in the cytoplasm, and were nonpermissive for HCV infection. Additionally, we observed variable expression of OCLN splicing forms across human tissues and cell lines. Our study suggests that the remarkable natural splicing diversity of OCLN might contribute to HCV tissue tropism and possibly modify the outcome of HCV infection in humans. Genetic factors crucial for regulation of OCLN expression and susceptibility to HCV infection remain to be elucidated.Hepatocellular carcinoma (HCC) is the most common primary cancer of the liver, the fifth most common malignancy worldwide, and the third leading cause of cancer-related death, after cancers of lung and stomach (WHO Mortality Database [http://www.who.int/healthinfo/morttables/en/index.html]). The estimated incidence of new HCC cases is about 500,000 to 1,000,000 annually, with mortality of 600,000 cases per year on a global scale (12, 16, 17, 20, 24). Various risk factors for HCC include infection with hepatitis C virus (HCV) or hepatitis B virus (HBV), toxic exposures (alcohol and aflatoxins), metabolic disease (diabetes, nonalcoholic fatty liver disease, and hereditary hemochromatosis), and immune-related conditions such as primary biliary cirrhosis and autoimmune hepatitis (15).The only established in vivo model for the study of HCV infection in an immunocompetent host is the chimpanzee (23). The inability of HCV to infect animals other than humans and chimpanzees has severely hampered efforts in developing a useful small animal model for the disease, specific antiviral therapies, and an effective vaccine against HCV-mediated liver cancer (18, 23).In the United States, chronic HCV infection is the major etiological agent of liver cancer. Among individuals infected with HCV, approximately 80% develop chronic HCV infection, of which 20% will progress to cirrhosis, and 1 to 5% will progress to liver cancer (14). Genetic factors might affect the risk of liver cancer by modifying the susceptibility to HCV infection and viral clearance. Recent studies identified occludin (OCLN), an integral tight junction (TJ) protein, as one of the key factors for HCV entry into cells (8, 18). HCV infectivity was exclusively mediated by the second extracellular loop (EC2) of the OCLN MARVEL membrane-associating domain (18). This domain is found in proteins involved in lipid-rich membrane apposition events, such as cell fencing contacts and formation of vesicular particles (19). OCLN also has a large intracellular protein (ELL) domain, found in C-terminal parts of OCLN and in the ELL family of RNA polymerase II elongation factors (7), but its role in HCV infection is unclear.We hypothesized that splicing diversity, generating multiple functionally distinct OCLN protein isoforms, might modulate susceptibility to HCV infection. Six splicing forms of OCLN and two distinct promoters, P1 and P2, have been described in cell lines (4, 5, 9, 10, 13). In the present study, we explored the splicing diversity of OCLN in normal human liver and observed variable expression of known and novel isoforms. Additionally, in vitro infectivity assays proved some of these forms to be nonpermissive for HCV infection. Our study suggests that naturally occurring splicing forms of OCLN might modify the outcome of HCV infection in humans.     

Reconstruction of 90Sr intake for breast-fed infants in the Techa riverside settlements

The Techa River (Southern Urals, Russia) was contaminated as a result of radioactive releases by the Mayak plutonium production facility during 1949-1956. The persons born after the onset of the contamination have been identified as the "Techa River Offspring Cohort" (TROC). The TROC has the potential to provide direct data on health effects in progeny that resulted from exposure of a general parent population to chronic radiation. The purpose of the present investigation is the estimation of (90)Sr intake from breast milk and river water in the period from birth to 6 months of life, necessary for an infant dose calculation. The investigation is based on all available data concerning radioactive contamination due to global fallouts and Mayak releases in the Southern Urals where extensive radiometric and radiochemical investigations of human tissues and environmental samples were conducted during the second half of the twentieth century. The strontium transfer factor from mother's daily diet to breast milk was estimated as 0.05 (0.01-0.13) d L(-1). Based on this transfer factor and data on (90)Sr water contamination, the average total (90)Sr intake for an infant born in the middle Techa River region was found to be equal to 60-80 kBq in 1950-1951. For the same period, calculations of (90)Sr intake using ICRP models gave values of 70-100 kBq. From 1952 onwards, the differences in intakes calculated using the two approaches increased, reaching a factor of 2-3 in 1953. The Techa River data provide the basis for improving and adapting the ICRP models for application to Techa River-specific population.     

Characterization of bacterial isolates from industrial wastewater according to probable modes of hexadecane uptake

Bacterial isolates from industrial wastewater were characterized according to probable modes of hexadecane uptake based on data for cell surface hydrophobicity, emulsifying activity, glycoside content and surface tension of cell-free culture medium. The results obtained suggested that both modes of biosurfactant-enhanced hexadecane uptake by bacterial strains take place, direct uptake and alkane transfer. The increase in cell surface hydrophobicity and glycoside production by the strains suggested the existence of biosurfactant-enhanced interfacial uptake of the alkane. Such mechanism is probably predominant for three isolates, Staphylococcus sp. HW-2, Streptococcus sp. HW-9 and Bacillus sp. HW-4. Secreted biosurfactants enhanced mainly alkane emulsification for most hydrophobic isolate Arthrobacter sp. HW-8, and micellar transfer for most hydrophilic isolate Streptococcus sp. HW-5. For other strains (67%) both mechanisms of biosurfactant-enhanced hexadecane uptake probably take place in similar degree, interfacial uptake and alkane emulsification. The results obtained could contribute to clarifying the natural relationships between the members of water ecosystem studied as well as will reveal potential producers of surface active compounds.     

Seasonal covariation in progesterone and odorant emissions among breeding crested auklets (Aethia cristatella)

Crested auklets emit a citrus-like odorant that is seasonally modulated, suggesting that it is a secondary sexual trait. We hypothesized that expression of the chemical odorant is facilitated by steroid hormones, similar other secondary sexual traits in birds. Therefore we examined variation in concentrations of hormones in blood plasma and odor production during incubation and early chick rearing. A novel method was used to obtain and measure chemical emissions of crested auklets. Blood plasma samples were analyzed by radioimmunoassay. Progesterone was detected in all birds, and it varied during the breeding season. Octanal emissions covaried with progesterone levels in males but not in females. No seasonal patterns were detected in testosterone, estrogen or DHT, and these hormones were not detected in all breeding adults. Covariance of progesterone and octanal emissions in males suggests there could be at least an indirect relationship between odor emissions and steroid hormones in this species. Thus expression of the citrus-like odorant of crested auklets, like other secondary sexual traits in birds, could be regulated by steroid hormones.     

Membrane interactions of designed cationic antimicrobial peptides: the two thresholds

Novel cationic antimicrobial peptides (CAPs) designed in our lab-typified by sequences such as KKKKKKAAX-AAXAAXAA-NH(2), where X = Phe/Trp-display high antibacterial activity but exhibit little or no hemolytic activity towards human red blood cells even at high doses. To clarify the mechanism of their selectivity for bacterial versus mammalian membranes and to increase our understanding of the relationships between primary sequence and bioactivity, a library of derivatives was prepared by increasing segmental hydrophobicity, in which systematic substitutions of Ala for two, three, or four Leu residues were made. Conformationally constrained dimeric and cyclic derivatives were also synthesized. The peptides were examined for activity against pathogenic bacteria (Pseudomonas aeruginosa), hemolytic activity on human red blood cells, and insertion into models of natural bacterial membranes (containing anionic lipids) and mammalian membranes (containing zwitterionic lipids + cholesterol). Results were compared with corresponding properties of the natural CAPs magainin and cecropin. Using circular dichroism and fluorescence spectroscopy, we found that peptide conformation and membrane insertion were sequence dependent, both upon the number of Leu residues, and upon their positions along the hydrophobic core. Membrane disruption was likely enhanced by the fact that the peptides contain potent dimerization-promoting sequence motifs, as assessed by SDS-PAGE gel analysis. The overall results led us to identify distinctions in the mechanism of actions of these CAPs for disruption of bacterial versus mammalian membranes, the latter dependent on surpassing a "second hydrophobicity threshold" for insertion into zwitterionic membranes.