Nucleotide sequence of Hungarian grapevine chrome mosaic nepovirus RNA1.

The nucleotide sequence of the RNA1 of hungarian grapevine chrome mosaic virus, a nepovirus very closely related to tomato black ring virus, has been determined from cDNA clones. It is 7212 nucleotides in length excluding the 3' terminal poly(A) tail and contains a large open reading frame extending from nucleotides 216 to 6971. The presumably encoded polyprotein is 2252 amino acids in length with a molecular weight of 250 kDa. The primary structure of the polyprotein was compared with that of other viral polyproteins, revealing the same general genetic organization as that of other picorna-like viruses (comoviruses, potyviruses and picornaviruses), except that an additional protein is suspected to occupy the N-terminus of the polyprotein.     

Heme acquisition by Shu1 requires Nbr1 and proteins of the ESCRT complex in Schizosaccharomyces pombe

Assimilation of heme is mediated by the cell surface protein Shu1 in Schizosaccharomyces pombe. Shu1 undergoes internalization from the cell surface to the vacuole in response to high concentrations of hemin. Here, we have identified cellular components that are involved in mediating vacuolar targeting of Shu1. Cells deficient in heme biosynthesis and lacking the polyubiquitin gene ubi4⁺ exhibit poor growth in the presence of exogenous hemin as a sole source of heme. Microscopic analyses of hem1Δ shu1Δ ubi4Δ cells expressing a functional HA₄‐tagged Shu1 show that Shu1 localizes to the cell surface. Ubiquitinated Nbr1 functions as a receptor for the endosomal sorting complexes required for transport (ESCRT) that delivers cargos to the vacuole. Inactivation of nbr1⁺, ESCRT‐0 hse1⁺ or ESCRT‐I sst6⁺ results in hem1Δ cells being unable to use exogenous hemin for the growth. Using lysate preparations from hemin‐treated cells, Shu1‐Nbr1 and Shu1‐Hse1 complexes are detected by coimmunoprecipitation experiments. Further analysis by immunofluorescence microscopy shows that Shu1 is unable to reach vacuoles of hemin‐treated cells harboring a deletion for one of the following genes: ubi4⁺, nbr1⁺, hse1⁺ and sst6⁺. Together, these results reveal that hemin‐mediated vacuolar targeting of Shu1 requires Ubi4‐dependent ubiquitination, the receptor Nbr1 and the ESCRT proteins Hse1 and Sst6.     

hierfstat,a package for r to compute and test hierarchical F‐statistics

The package hierfstat for the statistical software r , created by the R Development Core Team, allows the estimate of hierarchical F‐statistics from a hierarchy with any numbers of levels. In addition, it allows testing the statistical significance of population differentiation for these different levels, using a generalized likelihood‐ratio test. The package hierfstat is available at http://www.unil.ch/popgen/softwares/hierfstat.htm .     

Activity of novel Cdc25 inhibitors and preliminary evaluation of their potentiation of chemotherapeutic drugs in human breast cancer cells

Dual-specific phosphatases Cdc25 play a critical role in the cell cycle regulation by activating kinases of Cdk/cyclin complexes. Three Cdc25 isoforms (A, B and C) have been identified in mammalians. Cdc25A and B display oncogenic properties and are over-expressed in different tumors. Cdc25 phosphatases are therefore attractive targets for therapeutic strategies. Novel maleic anhydride derivatives bearing a fatty acid chain of variable size have been synthesized and tested for their Cdc25 inhibitory potential using an in vitro assay. We report biological activity of ineffective, moderate, and efficient inhibitors on breast cancer cells (MCF7) and its counterpart resistant to vincristine (Vcr-R). The most potent compounds induced Cdk2 inhibition and accumulation in G0/G1 phase of the cell cycle. Moreover, apoptosis was triggered within 48-h treatment, without oxidative burst and modulation of the Bax to Bcl-2 ratio. When used as pre-treatments, these derivatives were also able to potentiate adriamycin and cisplatin toxicity in both cell lines. Thus, maleic anhydride derivatives may mediate apoptosis through a cell cycle blockage via inhibition of Cdc25. This class of inhibitors may present potential interest in therapeutic strategies against cancer.     

Evidence for Co-evolution of West Nile Virus and House Sparrows in North America

West Nile virus (WNV) has been maintained in North America in enzootic cycles between mosquitoes and birds since it was first described in North America in 1999. House sparrows (HOSPs; Passer domesticus) are a highly competent host for WNV that have contributed to the rapid spread of WNV across the U.S.; however, their competence has been evaluated primarily using an early WNV strain (NY99) that is no longer circulating. Herein, we report that the competence of wild HOSPs for the NY99 strain has decreased significantly over time, suggesting that HOSPs may have developed resistance to this early WNV strain. Moreover, recently isolated WNV strains generate higher peak viremias and mortality in contemporary HOSPs compared to NY99. These data indicate that opposing selective pressures in both the virus and avian host have resulted in a net increase in the level of host competence of North American HOSPs for currently circulating WNV strains.     

Isolation and characterization of an aggresome determinant in the NF2 tumor suppressor

Schwannomin (Sch) is the product of the NF2 tumor suppressor gene. The NF2 gene is mutated in patients affected by neurofibromatosis type 2, a syndrome associated with multiple tumors of the nervous system. Here we found that Sch, when its N-terminal FERM domain was misfolded by the pathogenetic mutation Delta F118, formed aggresomes, i.e. aggregates that cluster at the centrosome as a result of microtubule-dependent transport. Strikingly the related protein ezrin affected by the same mutation did not form aggresomes even though its FERM domain was similarly misfolded. By studying ezrin/Sch chimeras, we delineated a sequence of 61 amino acids in the C terminus of Sch that determined the formation of aggresomes. Aggresome formation by these chimeras was independent from their rate of degradation. Sch(535-595) was sufficient to induce aggresomes of a green fluorescent fusion protein in vivo and aggregates of a glutathione S-transferase fusion protein in vitro. Taken together, these results suggest that aggresome formation is controlled primarily by aggresome determinants, which are distinct from degradation determinants, or from misfolding, through which aggresome determinants might be exposed.     

Creatine uptake and creatine transporter expression among rat skeletal muscle fiber types

Total creatine (Cr(total) = phosphocreatine + creatine) concentrations differ substantially among mammalian skeletal muscle. Because the primary means to add Cr(total) to muscle is uptake of creatine through the sodium-dependent creatine transporter (CrT), differences in creatine uptake and CrT expression could account for the variations in [Cr(total)] among muscle fiber types. To test this hypothesis, hindlimbs of adult rats were perfused with 0.05-1 mM [(14)C]creatine for up to 90 min. Creatine uptake rates at 1 mM creatine were greatest in the soleus (140 +/- 8.8 nmol x h(-1) x g(-1)), less in the red gastrocnemius (117 +/- 8.3), and least in the white gastrocnemius (97 +/- 10.7). These rates were unaltered by time, insulin concentration, or increased perfusate sodium concentration. Conversely, creatine uptake rates were correspondingly decreased among fiber types by lower creatine and sodium concentrations. The CrT protein content by Western blot analysis was similarly greatest in the soleus, less in the red gastrocnemius, and least in the white gastrocnemius, whereas CrT mRNA was not different. Creatine uptake rates differ among skeletal muscle fiber sections in a manner reasonably assigned to the 58-kDa band of the CrT. Furthermore, creatine uptake rates scale inversely with creatine content, with the lowest uptake rate in the fiber type with the highest Cr(total) and vice versa. This suggests that the creatine pool fractional turnover rate is not common across muscle phenotypes and, therefore, is differentially regulated.     

Ilheus Virus Isolation in the Pantanal,West-Central Brazil

The wetlands of the Brazilian Pantanal host large concentrations of diverse wildlife species and hematophagous arthropods, conditions that favor the circulation of zoonotic arboviruses. A recent study from the Nhecolândia sub-region of Pantanal reported serological evidence of various flaviviruses, including West Nile virus and Ilheus virus (ILHV). According to the age of seropositive horses, at least three flaviviruses, including ILHV, circulated in the Brazilian Pantanal between 2005 and 2009. To extend this study, we collected 3,234 adult mosquitoes of 16 species during 2009 and 2010 in the same sub-region. Mosquito pool homogenates were assayed for infectious virus on C6/36 and Vero cell monolayers and also tested for flaviviral RNA by a group-specific real-time RT-PCR. One pool containing 50 non-engorged female specimens of Aedes scapularis tested positive for ILHV by culture and for ILHV RNA by real-time RT-PCR, indicating a minimum infection rate of 2.5 per 1000. Full-length genomic sequence exhibited 95% identity to the only full genome sequence available for ILHV. The present data confirm the circulation of ILHV in the Brazilian Pantanal.