Markers for trans-Golgi Membranes and the Intermediate Compartment Localize to Induced Membranes with Distinct Replication Functions in Flavivirus-Infected Cells

Replication of the flavivirus Kunjin virus is associated with virus-induced membrane structures within the cytoplasm of infected cells; these membranes appear as packets of vesicles associated with the sites of viral RNA synthesis and as convoluted membranes (CM) and paracrystalline arrays (PC) containing the components of the virus-specified protease (E. G. Westaway, J. M. Mackenzie, M. T. Kenney, M. K. Jones, and A. A. Khromykh, J. Virol. 71:6650-6661, 1997). To determine the cellular origins of these membrane structures, we compared the immunolabelling patterns of several cell markers in relation to these sites by immunofluorescence and immunoelectron microscopy. A marker for the trans-Golgi membranes and the trans-Golgi network, 1,4-galactosyltransferase (GalT), was redistributed to large foci in the cytoplasm of Kunjin virus-infected cells, partially coincident with immunofluorescent foci associated with the putative sites of viral RNA synthesis. As determined by immunoelectron microscopy, the induced vesicle packets contained GalT, whereas the CM and PC contained a specific protein marker for the intermediate compartment (ERGIC53). A further indicator of the role of cellular organelles in their biogenesis was the observation that the Golgi apparatus-disrupting agent brefeldin A prevented further development of immunofluorescent foci of induced membranes if added before the end of the latent period but that once formed, these membrane foci were resistant to brefeldin A dispersion. Reticulum membranes emanating from the induced CM and PC were also labelled with the rough endoplasmic reticulum marker anti-protein disulfide isomerase and were obviously redistributed during infection. This is the first report identifying trans-Golgi membranes and the intermediate compartment as the apparent sources of the flavivirus-induced membranes involved in events of replication.     

Engineering lysine‐rich caleosins as carrier proteins to render biotin as a hapten on artificial oil bodies for antibody production

It has been demonstrated that caleosin alone is sufficient to stabilize artificial oil bodies. A series of recombinant caleosins, mutated with 3, 5, 8, 11, 13, 15, and 17 extra Lys residues and over‐expressed in Escherichia coli, were used as carrier proteins to render biotin as a hapten on the surface of artificial oil bodies for antibody production. Biotinylation levels of the recombinant caleosins were step‐wisely elevated as the number of extra Lys residues increased, and the biotinylated Lys residues were identified by mass spectrometric analysis. Polyclonal antibodies against biotin were successfully generated in rats injected with artificial oil bodies constituted with each of the biotinylated caleosins. Moreover, those generated via the biotinylated caleosins with eight or more extra Lys residues no longer recognized caleosin. It appears that engineered Lys‐rich caleosins are suitable carrier proteins for the production of antibodies against small molecules. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Suppression of Viral RNA Binding and the Assembly of Infectious Hepatitis C Virus Particles In Vitro by Cyclophilin Inhibitors

Nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) is an indispensable component of the HCV replication and assembly machineries. Although its precise mechanism of action is not yet clear, current evidence indicates that its structure and function are regulated by the cellular peptidylprolyl isomerase cyclophilin A (CyPA). CyPA binds to proline residues in the C-terminal half of NS5A, in a distributed fashion, and modulates the structure of the disordered domains II and III. Cyclophilin inhibitors (CPIs), including cyclosporine (CsA) and its nonimmunosuppressive derivatives, inhibit HCV infection of diverse genotypes, both in vitro and in vivo. Here we report a mechanism by which CPIs inhibit HCV infection and demonstrate that CPIs can suppress HCV assembly in addition to their well-documented inhibitory effect on RNA replication. Although the interaction between NS5A and other viral proteins is not affected by CPIs, RNA binding by NS5A in cell culture-based HCV (HCVcc)-infected cells is significantly inhibited by CPI treatment, and sensitivity of RNA binding is correlated with previously characterized CyPA dependence or CsA sensitivity of HCV mutants. Furthermore, the difference in CyPA dependence between a subgenomic and a full-length replicon of JFH-1 was due, at least in part, to an additional role that CyPA plays in HCV assembly, a conclusion that is supported by experiments with the clinical CPI alisporivir. The host-directed nature and the ability to interfere with more than one step in the HCV life cycle may result in a higher genetic barrier to resistance for this class of HCV inhibitors.     

Nitroimidazole conjugates of bis(thiosemicarbazonato)Cu(II) - Potential combination agents for the PET imaging of hypoxia

Combination agents comprising two different pharmacophores with the same biological target have the potential to show additive or synergistic activity. Bis(thiosemicarbazonato)copper(II) complexes (e.g. ⁶⁴Cu-ATSM) and nitroimidazoles (e.g. ¹⁸F-MISO) are classes of tracer used for the delineation of tumor hypoxia by positron emission tomography (PET). Three nitroimidazole-bis(thiosemicarbazonato)copper(II) conjugates were produced in order to investigate their potential as combination hypoxia imaging agents. Two were derived from the known bifunctional bis(thiosemicarbazone) H₂ATSM/A and the third from the new precursor diacetyl-2-(4-N-methyl-3-thiosemicarbazone)-3-(4-N-ethylamino-3-thiosemicarbazone) - H₂ATSM/en. Oxygen-dependent uptake studies were performed using the ⁶⁴Cu radiolabelled complexes in EMT6 carcinoma cells. All the complexes displayed appreciable hypoxia selectivity, with the nitroimidazole conjugates displaying greater selectivity than a simple propyl derivative used as a control. Participation of the nitroimidazole group in the trapping mechanism is indicated by the increased hypoxic uptake of the 2- vs. the 4-substituted ⁶⁴Cu-ATSM/A derivatives. The 2-nitroimidazole derivative of ⁶⁴Cu-ATSM/en demonstrated superior hypoxia selectivity to ⁶⁴Cu-ATSM over the range of oxygen concentrations tested. Biodistribution of the radiolabelled 2-nitroimidazole conjugates was carried out in EMT6 tumor-bearing mice. The complexes showed significantly different uptake trends in comparison to each other and previously studied Cu-ATSM derivatives. Uptake of the Cu-ATSM/en conjugate in non-target organs was considerably lower than for derivatives based on Cu-ATSM/A.     

Peripheral chemoreflex responsiveness is increased at elevated levels of carbon dioxide after episodic hypoxia in awake humans.

We hypothesized that the acute ventilatory response to hypoxia is enhanced after exposure to episodic hypoxia in awake humans. Eleven subjects completed a series of rebreathing trials before and after exposure to eight 4-min episodes of hypoxia. During the rebreathing trials, subjects initially hyperventilated to reduce the partial pressure of carbon dioxide (Pet(CO(2))) below 25 Torr. Subjects then breathed from a bag containing normocapnic (42 Torr), low (50 Torr), or high oxygen (140 Torr) gas mixtures. During the trials, Pet(CO(2)) increased while a constant oxygen level was maintained. The point at which ventilation began to rise in a linear fashion as Pet(CO(2)) increased was considered to be the ventilatory recruitment threshold. The ventilatory response below and above the recruitment threshold was determined. Ventilation did not persist above baseline values immediately after exposure to episodic hypoxia; however, Pet(CO(2)) levels were reduced compared with baseline. In contrast, compared with baseline, the ventilatory response to progressive increases in carbon dioxide during rebreathing trials in the presence of low but not high oxygen levels was increased after exposure to episodic hypoxia. This increase occurred when carbon dioxide levels were above but not below the ventilatory recruitment threshold. We conclude that long-term facilitation of ventilation (i.e., increases in ventilation that persist when normoxia is restored after episodic hypoxia) is not expressed in awake humans in the presence of hypocapnia. Nevertheless, despite this lack of expression, the acute ventilatory response to hypoxia in the presence of hypercapnia is increased after exposure to episodic hypoxia.     

Species co‐occurrence patterns and dietary resource competition in primates

Diamond (Assembly of species communities. In: Cody ML, Diamond JM, editors. Ecology and evolution of communities. Cambridge: Belknap. p 342–444 ( 1975 )) argued that interspecific competition between species occupying similar niches results in a nonrandom pattern of species distributions. In particular, some species pairs may never be found in the same community due to competitive exclusion. Rigorous analytical methods have been developed to investigate the possible role that interspecific competition has on the evolution of communities. Many studies that have implemented these methods have shown support for Diamond's assembly rules, yet there are numerous exceptions. We build on this previous research by examining the co‐occurrence patterns of primate species in 109 communities from across the world. We used EcoSim to calculate a checkerboard (C) score for each region. The C score provides a measure of the proportion of species pairs that do not co‐occur in a set of communities. High C scores indicate that species are nonrandomly distributed throughout a region, and interspecific competition may be driving patterns of competitive exclusion. We conducted two sets of analyses. One included all primate species per region, and the second analysis assigned each species to one of four dietary guilds: frugivores, folivores, insectivores, and frugivore‐insectivores. Using all species per region, we found significantly high C scores in 9 of 10 regions examined. For frugivores, we found significantly high‐C scores in more than 50% of regions. In contrast, only 23% of regions exhibited significantly high‐C scores for folivores. Our results suggest that communities are nonrandomly structured and may be the result of greater levels of interspecific competition between frugivores compared to folivores. Am J Phys Anthropol, 2010. © 2010 Wiley‐Liss, Inc.     

Precise identification of gene products in hearts after in vivo gene transfection, using Sendai virus-coated proteoliposomes.

Both efficient gene transfer and the exact identification of gene product are required for gene therapy. Gene transfection of green fluorescence protein (GFP) might be useful for the reporter. After in vivo cotransfection of GFP and beta-galactosidase (beta-Gal) genes in Sendai virus-coated proteoliposomes to rat hearts, we compared the sensitivity and specificity of three methods: GFP detection, histochemical staining (HC) of beta-Gal activity, and immunostaining (IS) of the beta-Gal protein. Fluorescence microscopy and double staining of HC and IS revealed that both GFP and IS were equally sensitive and fourfold superior to HC at the peak of gene expression. However, different from skeletal muscle, the GFP of transfected cardiomyocytes showed two demerits: the fluorescence quenching due to the intense staining of beta-Gal activity, and nonspecific autofluorescence from myocardium. Thus, specific IS would be so far the most reliable to identify the gene product in heart.