Force Generation via β-Cardiac Myosin,Titin, and α-Actinin Drives Cardiac Sarcomere Assembly from Cell-Matrix Adhesions

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Total sleep deprivation does not significantly degrade semantic encoding

ABSTRACT

Sleep deprivation impairs performance on cognitive tasks, but it is unclear which cognitive processes it degrades. We administered a semantic matching task with variable stimulus onset asynchrony (SOA) and both speeded and self-paced trial blocks. The task was administered at the baseline and 24 hours later after 30.8 hours of total sleep deprivation (TSD) or matching well-rested control. After sleep deprivation, the 20% slowest response times (RTs) were significantly increased. However, the semantic encoding time component of the RTs remained at baseline level. Thus, the performance impairment induced by sleep deprivation on this task occurred in cognitive processes downstream of semantic encoding.     

The relationship of p53 and stress proteins in response to bleomycin and retinoic acid in the p53 heterozygous mouse.

A single, i.p. dose of bleomycin was administered simultaneously with [35S]methionine to 4-month-old p53 wild type (+/+) and p53 heterozygous (+/-) C57BL/6 mice. Following a period of 3.5 h from dosing, the bone marrow nuclei were examined by two-dimensional PAGE and fluorography for induction of stress proteins (sps). Eight sps ranging from 22000 to 100000 Mr were synthesized in p53+/- and p53+/+ mice following elicitation by bleomycin. No quantitative or qualitative differences were observed in sp expression in these two groups of animals. In a second experiment, three doses of retinoic acid were given i.p. to p53+/- and p53+/+ mice over a 36 h period. The p53 isoforms in bone marrow nuclei from these mice were analyzed by PAGE for incorporation of [35S]methionine following retinoic acid injections. Quantitative and qualitative alterations in p53 isotypes were substantially increased in p53+/+ as compared with p53+/- mice. The increased complexity in the synthesis patterns in both groups of dosed mice consisted of additional isoforms possessing more acidic isoelectric values. In an in vitro binding assay, individual p53 isoforms demonstrated varying degrees of association with sps 25a, 70i, 72c and 90 which was consistently greater in p53+/+ mice. Both the synthesis and binding of isoforms were greater in G1 than in S+G2 phase, in both groups of animals, reflecting a cell cycle regulated mechanism for these events. Collectively, these data implied that the synthesis and the binding characteristics of p53 isoforms with sps were enhanced in the p53+/+ mice relative to the p53+/- mouse; however, sp labeling was not affected by p53 genotype.     

DNA barcoding of South Africa’s ornamental freshwater fish – are the names reliable?

Trade in freshwater ornamental fish in South Africa is currently regulated by a ‘blacklist’ to prevent potentially invasive taxa from establishing in the country. Because its effective implementation requires accurate identification, the aim of the present study was to test whether DNA barcoding is a useful tool to identify freshwater fishes in the South African pet trade. A total of 351 aquarium fish specimens, representing 185 traded taxa, were sequenced for the mitochondrial COI barcoding marker in 2011 and 2012. Lake Malawi cichlids were treated as a single group due to a lack of resolution in their COI marker, resulting in a data set of 137 successfully sequenced taxa. The Barcode Of Life Database (BOLD) and GenBank were used for taxonomic assignment comparisons. The genetic identification matched the scientific name inferred from the trade name for 60 taxa (43.8%) using BOLD, and for 67 taxa (48.9%) using GenBank. A genetic ID could not be assigned in 47 (34.3%) cases using BOLD and in 37 cases (27%) using GenBank. Whereas DNA barcoding can be a useful tool to help identify imported freshwater fishes, it requires further development of publicly available databases to become a reliable means of identification.     

The N-terminal Amphipathic ��-Helix of Viperin Mediates Localization to the Cytosolic Face of the Endoplasmic Reticulum and Inhibits Protein Secretion

Viperin is an evolutionarily conserved interferon-inducible protein that localizes to the endoplasmic reticulum (ER) and inhibits a number of DNA and RNA viruses. In this study, we report that viperin specifically localizes to the cytoplasmic face of the ER and that an amphipathic α-helix at its N terminus is necessary for the ER localization of viperin and sufficient to promote ER localization of a reporter protein, dsRed. Overexpression of intact viperin but not the amphipathic α-helix fused to dsRed induced crystalloid ER. Consistent with other proteins that induce crystalloid ER, viperin self-associates, and it does so independently of the amphipathic α-helix. Viperin expression also affected the transport of soluble but not membrane-associated proteins. Expression of intact viperin or an N-terminal α-helix-dsRed fusion protein significantly reduced secretion of soluble alkaline phosphatase and reduced its rate of ER-to-Golgi trafficking. Similarly, viperin expression inhibited bulk protein secretion and secretion of endogenous α₁-antitrypsin and serum albumin from HepG2 cells. Converting hydrophobic residues in the N-terminal α-helix to acidic residues partially or completely restored normal transport of soluble alkaline phosphatase, suggesting that the extended amphipathic nature of the N-terminal α-helical domain is essential for inhibiting protein secretion.Type I interferons are the first line of defense against viral infections. The significance of the interferon pathway is illustrated by the susceptibility of interferon signaling mutants to infection and by viral mechanisms that counteract this pathway (1, 2). Although many genes are induced upon interferon stimulation, very few of these genes have been functionally characterized. Viperin is highly induced by both Type I and II interferons and has a broad range of antiviral activity, inhibiting DNA viruses, notably human cytomegalovirus (3); RNA viruses such as influenza, hepatitis C virus (HCV),² and alphaviruses (4-6); and retroviruses such as human immunodeficiency virus (7). Upon expression, viperin localizes to the endoplasmic reticulum (ER), where it interacts with farnesyl-diphosphate synthase, an enzyme involved in lipid biosynthesis. This interaction appears to result in the disruption of lipid raft microdomains and prevention of influenza virus from budding from the plasma membrane (4).Although recent studies have explored the antiviral functions of viperin, the general biochemical properties of this protein remain largely undefined. Viperin is highly conserved across both mammals and lower vertebrates and shares homology with the MoaA family of “radical S-adenosylmethionine” enzymes that bind Fe-S clusters (3, 8). In addition to a putative Fe-S cluster-binding domain, viperin has a 42-amino acid residue N-terminal amphipathic α-helix, and similar domains in other proteins have been shown to bind membranes and induce membrane curvature (9, 10).In this study, we examined the role of the viperin N-terminal α-helical domain in both cellular localization and ER membrane morphology and analyzed the biochemical properties of viperin. We discovered that viperin forms dimers and induces a tightly ordered, visually striking array of ER membranes, known as crystalloid ER(11-13), upon overexpression. In addition, viperin expression impedes the secretion of a variety of soluble proteins. Although the N-terminal amphipathic α-helix is not sufficient to induce crystalloid ER formation, it is both necessary and sufficient to mediate ER localization and to inhibit protein secretion.     

Retinoic acid induction of stress proteins in fetal mouse limb buds

Retinoic acid (RA) is teratogenic in rodent embryos. Several teratogens have been shown to induce the synthesis of a subset of heat shock proteins (stress proteins) in Drosophila. To determine if RA induces the synthesis of these proteins in rodent embryos, pregnant ICR mice were dosed with 100 mg/kg RA on Day 11 of gestation. Forelimb buds were removed from embryos 2.5 hr post-RA-treatment and nuclei were isolated, stained, and sorted from stages of the cell cycle. Nuclear proteins were extracted and analyzed by two-dimensional polyacrylamide gel electrophoresis. Nuclear proteins with molecular weights of approximately 84 and 25 kDa were synthesized in embryos in the G0 + G1 phase after pregnant dams were treated with RA. Isoelectric points, molecular weights, immunochemical blotting, and polypeptide mapping demonstrated that these proteins are indistinguishable from stress proteins isolated under a variety of conditions from rat submaxillary glands and mouse lymphoma cells. These results suggest that treatment with RA induces the synthesis of a subset of stress proteins; the role of these proteins in the teratogenic effects of RA is not known.