IFITM3 inhibits influenza A virus infection by preventing cytosolic entry

To replicate, viruses must gain access to the host cell's resources. Interferon (IFN) regulates the actions of a large complement of interferon effector genes (IEGs) that prevent viral replication. The interferon inducible transmembrane protein family members, IFITM1, 2 and 3, are IEGs required for inhibition of influenza A virus, dengue virus, and West Nile virus replication in vitro. Here we report that IFN prevents emergence of viral genomes from the endosomal pathway, and that IFITM3 is both necessary and sufficient for this function. Notably, viral pseudoparticles were inhibited from transferring their contents into the host cell cytosol by IFN, and IFITM3 was required and sufficient for this action. We further demonstrate that IFN expands Rab7 and LAMP1-containing structures, and that IFITM3 overexpression is sufficient for this phenotype. Moreover, IFITM3 partially resides in late endosomal and lysosomal structures, placing it in the path of invading viruses. Collectively our data are consistent with the prediction that viruses that fuse in the late endosomes or lysosomes are vulnerable to IFITM3's actions, while viruses that enter at the cell surface or in the early endosomes may avoid inhibition. Multiple viruses enter host cells through the late endocytic pathway, and many of these invaders are attenuated by IFN. Therefore these findings are likely to have significance for the intrinsic immune system's neutralization of a diverse array of threats.     

Levels of connectivity between longnose skate (Dipturus oxyrinchus) in the Mediterranean Sea and the north-eastern Atlantic Ocean

Sequencing of a partial region of the mitochondrial control region has revealed no shared haplotypes between longnose skate (Dipturus oxyrinchus L.) sampled in the north-eastern Atlantic (Norway and Rockall) and those sampled in the Mediterranean (Mallorca). Bayesian estimation of the migration rate suggests little, if any, gene flow occurs between the regions and that the populations separated 20,000?years ago. These conclusions provide a genetic basis for long-standing observations, based on egg capsule and adult size, that longnose skate in the Mediterranean may be genetically isolated from other stocks. This result has important conservation implications for the threatened longnose skate.     

Mi-2/NuRD complex function is required for normal S phase progression and assembly of pericentric heterochromatin

During chromosome duplication, it is essential to replicate not only the DNA sequence, but also the complex nucleoprotein structures of chromatin. Pericentric heterochromatin is critical for silencing repetitive elements and plays an essential structural role during mitosis. However, relatively little is understood about its assembly and maintenance during replication. The Mi2/NuRD chromatin remodeling complex tightly associates with actively replicating pericentric heterochromatin, suggesting a role in its assembly. Here we demonstrate that depletion of the catalytic ATPase subunit CHD4/Mi-2β in cells with a dampened DNA damage response results in a slow-growth phenotype characterized by delayed progression through S phase. Furthermore, we observe defects in pericentric heterochromatin maintenance and assembly. Our data suggest that chromatin assembly defects are sensed by an ATM-dependent intra-S phase chromatin quality checkpoint, resulting in a temporal block to the transition from early to late S phase. These findings implicate Mi-2β in the maintenance of chromatin structure and proper cell cycle progression.     

Regulation of intracellular Ca2+ release in corpus cavernosum smooth muscle: synergism between nitric oxide and cGMP

Tonic contraction of corpus cavernosum smooth muscle cells (SMCs) maintains the flaccid state of the penis, and relaxation is initiated by nitric oxide (NO), leading to erection. Our aim was to investigate the effect of NO on the smooth muscle cellular response to adrenergic stimulation in corpus cavernosum. Fura-2 fluorescence was used to record intracellular Ca²⁺ concentration ([Ca²⁺]_i) from freshly isolated SMCs from rat and human. Phenylephrine (PE) transiently elevated [Ca²⁺]_i in the presence and absence of extracellular Ca²⁺, indicating release from intracellular stores. Whereas the NO donor S-nitroso-N-acetylpenicillamine (SNAP) with sildenafil citrate (SIL) caused no change in basal [Ca²⁺]_i, the PE-induced rise of [Ca²⁺]_i was reversibly inhibited by 27 ± 7% (n = 21, P < 0.005) in rat and by 55 ± 15% (n = 9, P < 0.01) in human SMCs. SNAP and SIL also reduced the contractile response to PE. To investigate the mechanism, we applied mediators alone or in combination. The soluble guanylyl cyclase inhibitor ODQ reduced the effect of SNAP and SIL. SIL, cGMP analogs, and NO donors without SIL did not reduce the PE-induced rise of [Ca²⁺]_i. However, the combination of 8-bromo-cGMP with SNAP reduced the Ca²⁺ peak by 42 ± 9% (n = 22, P < 0.01). Our results demonstrate that NO and cGMP act synergistically to reduce Ca²⁺ release from intracellular stores. Reduction of intracellular Ca²⁺ release may contribute to relaxation of the corpus cavernosum, leading to erection. calcium stores; nitric oxide; sildenafil citrate; inositol 1,4,5-trisphosphate receptor     

Transcriptome analysis of recombinant protein secretion by Aspergillus nidulans and the unfolded-protein response in vivo

Filamentous fungi have a high capacity for producing large amounts of secreted proteins, a property that has been exploited for commercial production of recombinant proteins. However, the secretory pathway, which is key to the production of extracellular proteins, is rather poorly characterized in filamentous fungi compared to yeast. We report the effects of recombinant protein secretion on gene expression levels in Aspergillus nidulans by directly comparing a bovine chymosin-producing strain with its parental wild-type strain in continuous culture by using expressed sequence tag microarrays. This approach demonstrated more subtle and specific changes in gene expression than those observed when mimicking the effects of protein overproduction by using a secretion blocker. The impact of overexpressing a secreted recombinant protein more closely resembles the unfolded-protein response in vivo.     

Dehalogenation of the herbicides bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) and ioxynil (3,5-diiodino-4-hydroxybenzonitrile) by Desulfitobacterium chlororespirans

Desulfitobacterium chlororespirans has been shown to grow by coupling the oxidation of lactate to the metabolic reductive dehalogenation of ortho chlorines on polysubstituted phenols. Here, we examine the ability of D. chlororespirans to debrominate and deiodinate the polysubstituted herbicides bromoxynil (3,5-dibromo-4-hydroxybenzonitrile), ioxynil (3,5-diiodo-4-hydroxybenzonitrile), and the bromoxynil metabolite 3,5-dibromo-4-hydroxybenzoate (DBHB). Stoichiometric debromination of bromoxynil to 4-cyanophenol and DBHB to 4-hydroxybenzoate occurred. Further, bromoxynil (35 to 75 microM) and DBHB (250 to 260 microM) were used as electron acceptors for growth. Doubling times for growth (means +/- standard deviations for triplicate cultures) on bromoxynil (18.4 +/- 5.2 h) and DBHB (11.9 +/- 1.4 h), determined by rate of [14C]lactate uptake into biomass, were similar to those previously reported for this microorganism during growth on pyruvate (15.4 h). In contrast, ioxynil was not deiodinated when added alone or when added with bromoxynil; however, ioxynil dehalogenation, with stoichiometric conversion to 4-cyanophenol, was observed when the culture was amended with 3-chloro-4-hydroxybenzoate (a previously reported electron acceptor). To our knowledge, this is the first direct report of deiodination by a bacterium in the Desulfitobacterium genus and the first report of an anaerobic pure culture with the ability to transform bromoxynil or ioxynil. This research provides valuable insights into the substrate range of D. chlororespirans.     

Comparative folate metabolism in humans and malaria parasites (part II): activities as yet untargeted or specific to Plasmodium

The folate pathway represents a powerful target for combating rapidly dividing systems such as cancer cells, bacteria and malaria parasites. Whereas folate metabolism in mammalian cells and bacteria has been studied extensively, it is understood less well in malaria parasites. In two articles, we attempt to reconstitute the malaria folate pathway based on available information from mammalian and microbial systems, in addition to Plasmodium-genome-sequencing projects. In part I, we focused on folate enzymes that are already used clinically as anticancer drug targets or that are under development in drug-discovery programs. In this article, we discuss mammalian folate enzymes that have not yet been exploited as potential drug targets, and enzymes that function in the de novo folate-synthesis pathway of the parasite--a particularly attractive area of attack because of its absence from the mammalian host.     

Application of molecular modeling to analysis of inhibition of kinesin motor proteins of the BimC subfamily by monastrol and related compounds

Application of molecular modeling approaches has potential to contribute to rational drug design. These approaches may be especially useful when attempting to elucidate the structural features associated with novel drug targets. In this study, molecular docking and molecular dynamics were applied to studies of inhibition of the human motor protein denoted HsEg5 and other homologues in the BimC subfamily. These proteins are essential for mitosis, so compounds that inhibit their activity may have potential as anticancer therapeutics. The discovery of a small-molecule cell-permeable inhibitor, monastrol, has stimulated research in this area. Interestingly, monastrol is reported to inhibit the human and Xenopus forms of Eg5, but not those from Drosophila and Aspergillus. In this study, homology modeling was used to generate models of the Xenopus, Drosophila, and Aspergillus homologues, using the crystal structure of the human protein in complex with monastrol as a template. A series of known inhibitors was docked into each of the homologues, and the differences in binding energies were consistent with reported experimental data. Molecular dynamics revealed significant changes in the structure of the Aspergillus homologue that may contribute to its relative insensitivity to monastrol and related compounds.     

Evidence for diversifying selection at the pyoverdine locus of Pseudomonas aeruginosa

Pyoverdine is the primary siderophore of the gram-negative bacterium Pseudomonas aeruginosa. The pyoverdine region was recently identified as the most divergent locus alignable between strains in the P. aeruginosa genome. Here we report the nucleotide sequence and analysis of more than 50 kb in the pyoverdine region from nine strains of P. aeruginosa. There are three divergent sequence types in the pyoverdine region, which correspond to the three structural types of pyoverdine. The pyoverdine outer membrane receptor fpvA may be driving diversity at the locus: it is the most divergent alignable gene in the region, is the only gene that showed substantial intratype variation that did not appear to be generated by recombination, and shows evidence of positive selection. The hypothetical membrane protein PA2403 also shows evidence of positive selection; residues on one side of the membrane after protein folding are under positive selection. R', previously identified as a type IV strain, is clearly derived from a type III strain via a 3.4-kb deletion which removes one amino acid from the pyoverdine side chain peptide. This deletion represents a natural modification of the product of a nonribosomal peptide synthetase enzyme, whose consequences are predictive from the DNA sequence. There is also linkage disequilibrium between the pyoverdine region and pvdY, a pyoverdine gene separated by 30 kb from the pyoverdine region. The pyoverdine region shows evidence of horizontal transfer; we propose that some alleles in the region were introduced from other soil bacteria and have been subsequently maintained by diversifying selection.