Stimulation of sugar transport in rat diaphragm by 8-bromoguanosine 3', 5'-monophosphate.

The cyclic GMP derivative, 8-bromo cyclic GMP, increases the uptake of D-xylose and of 2-deoxy D-glucose into intact rat diaphragm incubated in vitro. 8-Bromo cyclic GMP does not stimulate the incorporation of [14C]glucose into glycogen in the diaphragm, or the uptake of alphalpha-amino isobutyric acid into this tissue. The effect of 8-bromo cyclic GMP on the diaphragm is consistent with the hypothesis that cyclic GMP plays a role in the regulation of sugar transport in muscle.     

Stimulation of sugar transport in rat diaphragm by 8-bromoguanosine 3′,5′-monophosphate

The cyclic GMP derivative, 8-bromo cyclic GMP, increases the uptake of D-xylose and of 2-deoxy D-glucose into intact rat diaphragm incubated in vitro. 8-Bromo cyclic GMP does not stimulate the incorporation of [¹⁴C] glucose into glycogen in the diaphragm, or the uptake of α-amino isobutyric acid into this tissue. The effect of 8-bromo cyclic GMP on the diaphragm is consistent with the hypothesis that cyclic GMP plays a role in the regulation of sugar transport in muscle.     

Porcine reproductive and respiratory syndrome virus (PRRSV) infection spreads by cell-to-cell transfer in cultured MARC-145 cells, is dependent on an intact cytoskeleton, and is suppressed by drug-targeting of cell permissiveness to virus infection

Background

Porcine reproductive and respiratory syndrome virus (PRRSV) is the etiologic agent of PRRS, causing widespread chronic infections which are largely uncontrolled by currently available vaccines or other antiviral measures. Cultured monkey kidney (MARC-145) cells provide an important tool for the study of PRRSV replication. For the present study, flow cytometric and fluorescence antibody (FA) analyses of PRRSV infection of cultured MARC-145 cells were carried out in experiments designed to clarify viral dynamics and the mechanism of viral spread. The roles of viral permissiveness and the cytoskeleton in PRRSV infection and transmission were examined in conjunction with antiviral and cytotoxic drugs.

Results

Flow cytometric and FA analyses of PRRSV antigen expression revealed distinct primary and secondary phases of MARC-145 cell infection. PRRSV antigen was randomly expressed in a few percent of cells during the primary phase of infection (up to about 20–22 h p.i.), but the logarithmic infection phase (days 2–3 p.i.), was characterized by secondary spread to clusters of infected cells. The formation of secondary clusters of PRRSV-infected cells preceded the development of CPE in MARC-145 cells, and both primary and secondary PRRSV infection were inhibited by colchicine and cytochalasin D, demonstrating a critical role of the cytoskeleton in viral permissiveness as well as cell-to-cell transmission from a subpopulation of cells permissive for free virus to secondary targets. Cellular expression of actin also appeared to correlate with PRRSV resistance, suggesting a second role of the actin cytoskeleton as a potential barrier to cell-to-cell transmission. PRRSV infection and cell-to-cell transmission were efficiently suppressed by interferon-γ (IFN-γ), as well as the more-potent experimental antiviral agent AK-2.

Conclusion

The results demonstrate two distinct mechanisms of PRRSV infection: primary infection of a relatively small subpopulation of innately PRRSV-permissive cells, and secondary cell-to-cell transmission to contiguous cells which appear non-permissive to free virus. The results also indicate that an intact cytoskeleton is critical for PRRSV infection, and that viral permissiveness is a highly efficient drug target to control PRRSV infection. The data from this experimental system have important implications for the mechanisms of PRRSV persistence and pathology, as well as for a better understanding of arterivirus regulation.     

Cloning, mapping, and expression of two novel actin genes, actin-like-7A (ACTL7A) and actin-like-7B (ACTL7B), from the familial dysautonomia candidate region on 9q31.

Two novel human actin-like genes, ACTL7A and ACTL7B, were identified by cDNA selection and direct genomic sequencing from the familial dysautonomia candidate region on 9q31. ACTL7A encodes a 435-amino-acid protein (predicted molecular mass 48.6 kDa) and ACTL7B encodes a 415-amino-acid protein (predicted molecular mass 45. 2 kDa) that show greater than 65% amino acid identity to each other. Genomic analysis revealed ACTL7A and ACTL7B to be intronless genes contained on a common 8-kb HindIII fragment in a "head-to-head" orientation. The murine homologues were cloned and mapped by linkage analysis to mouse chromosome 4 in a region of gene order conserved with human chromosome 9q31. No recombinants were observed between the two genes, indicating a close physical proximity in mouse. ACTL7A is expressed in a wide variety of adult tissues, while the ACTL7B message was detected only in the testis and, to a lesser extent, in the prostate. No coding sequence mutations, genomic rearrangements, or differences in expression were detected for either gene in familial dysautonomia patients.     

Phospho-NHE3 forms membrane patches and interacts with beta-actin to sense and maintain constant direction during cell migration

The Na(+)/H(+) exchanger NHE3 colocalizes with beta-actin at the leading edge of directionally migrating cells. Using human osteosarcoma cells (SaOS-2), rat osteoblasts (calvaria), and human embryonic kidney (HEK) cells, we identified a novel role for NHE3 via beta-actin in anode and cathode directed motility, during electrotaxis. NHE3 knockdown by RNAi revealed that NHE3 expression is required to achieve constant directionality and polarity in migrating cells. Phosphorylated NHE3 (pNHE3) and beta-actin complex formation was impaired by the NHE3 inhibitor S3226 (IC50 0.02 µM). Fluorescence cross-correlation spectroscopy (FCCS) revealed that the molecular interactions between NHE3 and beta-actin in membrane protrusions increased 1.7-fold in the presence of a directional cue and decreased 3.3-fold in the presence of cytochalasin D. Data from flow cytometric analysis showed that membrane potential of cells (V_mem) decreases in directionally migrating, NHE3-deficient osteoblasts and osteosarcoma cells whereas only V_mem of wild type osteoblasts is affected during directional migration. These findings suggest that pNHE3 has a mechanical function via beta-actin that is dependent on its physiological activity and V_mem. Furthermore, phosphatidylinositol 3,4,5-trisphosphate (PIP3) levels increase while PIP2 remains stable when cells have persistent directionality. Both PI3 kinase (PI3K) and Akt expression levels change proportionally to NHE3 levels. Interestingly, however, the content of pNHE3 level does not change when PI3K/Akt is inhibited. Therefore, we conclude that NHE3 can act as a direction sensor for cells and that NHE3 phosphorylation in persistent directional cell migration does not involve PI3K/Akt during electrotaxis.     

B cells regulate murine gammaherpesvirus 68 latency

The dynamics of the establishment of, and reactivation from, gammaherpesviruses latency has not been quantitatively analyzed in the natural host. Gammaherpesvirus 68 (gammaHV68) is a murine gammaherpesvirus genetically related to primate gammaherpesviruses that establishes a latent infection in infected mice. We used limiting dilution reactivation (frequency of cells reactivating gammaHV68 in vitro) and limiting dilution PCR (frequency of cells carrying gammaHV68 genome) assays to compare gammaHV68 latency in normal (C57BL/6) and B-cell-deficient (MuMT) mice. After intraperitoneal (i.p.) inoculation, latent gammaHV68 was detected in the spleen, bone marrow, and peritoneal cells. Both B-cell-deficient and C57BL/6 mice established latent infection in peritoneal cells after either i.p. or intranasal (i.n.) inoculation. In contrast, establishment of splenic latency was less efficient in B-cell-deficient than in C57BL/6 mice after i.n. inoculation. Analysis of reactivation efficiency (reactivation frequency compared to frequency of cells carrying gammaHV68 genome) revealed that (i) regardless of route or mouse strain, splenic cells reactivated gammaHV68 less efficiently than peritoneal cells, (ii) the frequency of cells carrying gammaHV68 genome was generally comparable over the course of infection between C57BL/6 and B-cell-deficient mice, (iii) between 28 and 250 days after infection, cells from B-cell-deficient mice reactivated gammaHV68 10- to 100-fold more efficiently than cells from C57BL/6 mice, (iv) at 7 weeks postinfection, B-cell-deficient mice had more genome-positive peritoneal cells than C57BL/6 mice, and (v) mixing cells (ratio of 3 to 1) that reactivated inefficiently with cells that reactivated efficiently did not significantly decrease reactivation efficiency. Consistent with a failure to normally regulate chronic gammaHV68 infection, the majority of infected B-cell-deficient mice died between 100 and 200 days postinfection. We conclude that (i) B cells are not required for establishment of gammaHV68 latency, (ii) there are organ-specific differences in the efficiency of gammaHV68 reactivation, (iii) B cells play a crucial role in regulating reactivation of gammaHV68 from latency, and (iv) B cells are important for controlling chronic gammaHV68 infection.     

The structural basis of cooperative regulation at an alternate genetic switch

Bacteriophage lambda is a paradigm for understanding the role of cooperativity in gene regulation. Comparison of the regulatory regions of lambda and the unrelated temperate bacteriophage 186 provides insight into alternate ways to assemble functional genetic switches. The structure of the C-terminal domain of the 186 repressor, determined at 2.7 A resolution, reveals an unusual heptamer of dimers, consistent with presented genetic studies. In addition, the structure of a cooperativity mutant of the full-length 186 repressor, identified by genetic screens, was solved to 1.95 A resolution. These structures provide a molecular basis for understanding lysogenic regulation in 186. Whereas the overall fold of the 186 and lambda repressor monomers is remarkably similar, the way the two repressors cooperatively assemble is quite different and explains in part the differences in their regulatory activity.     

GTPase from rod outer segments: characterization by photoaffinity labeling and tryptic peptide mapping

The photoaffinity label [γ-³²P]8-N₃GTP has been used to identify GTP-binding components in highly purified preparations of GTPase from bovine rod outer segments. These preparations contain two major polypeptides of 37,000 and 39,000 daltons. In the presence of photolyzing radiation, [γ-³²P]8-N₃GTP is covalently attached to the 37,000 dalton polypeptide. Tryptic peptide mapping of this polypeptide indicates that it is highly related to the 39,000 dalton species that has been previously identified as a GTP-binding component.