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.     

Nitrogenase activity,hydrogen evolution and biomass production in different Casuarina symbioses

Nitrogenase activity, hydrogen evolution, biomass production and nodulation were studied in threeCasuarina species,C. equisetifolia Forst.,C. glauca Sieber ex Spreng andC. obesa Miq., either inoculated with a crushed nodule inoculum prepared fromC. glauca nodules or inoculated with the pure cultureHFP CcI3. Nodulation was also studied inC. cristata Miq. inoculated with the above mentionedFrankia sources. C. equisetifolia, C. glauca andC. obesa were nodulated when inoculated with both of theFrankia inoculum, whileC. cristata was very poorly nodulated. Nitrogenase activity per plant and on a nodule dry weight basis was significantly highest inC. glauca inoculated withC. glauca inoculum after 150 days from planting. This difference decreased and at 217 days from planting there was no significant difference between the symbioses, except forC. obesa inoculated withC. glauca inoculum which showed the significantly lowest nitrogenase activity. After 150 days from planting relative efficiency of nitrogenase was lowest inC. equisetifolia inoculated withHFP CcI3 and inC. equisetifolia inoculated withC. glauca inoculum. Biomass production was similar inC. glauca inoculated withC. glauca inoculum, inC. equisetifolia inoculated withHFP CcI3 and inC. obesa inoculated withHFP CcI3 at the final harvest. The data presented here show that there is a strong interrelationship between host plant and endobiont. This interrelationship is of considerable importance when introducing Casuarina symbioses for production of fuel wood.     

Angiotensin III and IV activation of the brain AT1 receptor subtype in cardiovascular function

The present investigation determined that native angiotensins II and III (ANG II and III) were equipotent as pressor agents when ICV infused in alert rats, whereas native angiotensin IV (ANG IV) was less potent. An analogue of each of these angiotensins was prepared with a hydroxyethylamine (HEA) amide bond replacement at the N-terminus, yielding additional resistance to degradation. These three angiotensin analogues, HEA-ANG II, HEA-ANG III, and HEA-ANG IV, were equivalent with respect to maximum elevation in pressor responses when ICV infused; and each evidenced significantly extended durations of effect compared with their respective native angiotensin. Comparing analogues, HEA-ANG II had a significantly longer effect compared with HEA-ANG III, and HEA-ANG IV, whereas the latter were equivalent. Pretreatment with the AT₁ receptor subtype antagonist, Losartan (DuP753), blocked subsequent pressor responses to each of these analogues, suggesting that these responses were mediated by the AT₁ receptor subtype. Pretreatment with the specific AT₄ receptor subtype antagonist, Divalinal (HED 1291), failed to influence pressor responses induced by the subsequent infusion of these analogues. These results suggest an important role for Ang III, and perhaps ANG IV, in brain angiotensin pressor responses mediated by the AT₁ receptor subtype.     

Heme binding and polymerization by Plasmodium falciparum histidine rich protein II: influence of pH on activity and conformation.

The histidine rich protein II (HRPII) from Plasmodium falciparum has been implicated as a heme polymerase which detoxifies free heme by its polymerization to inactive hemozoin. Histidine-iron center coordination is the dominant mechanism of interaction between the amino acid and heme. The protein also contains aspartate allowing for ionic/coordination interactions between the carboxylate side chain and the heme metal center. The pH profile of heme binding and polymerization shows the possibility of these two types of binding sites being differentiated by pH. Circular dichroism studies of the protein show that pH and heme binding cause a change in conformation above pH 6 implying the involvement of His-His+ transitions. Heme binding at pHs above 6 perturbs HRPII conformation, causing an increase in helicity.     

Mammalian Fbh1 is important to restore normal mitotic progression following decatenation stress

We have addressed the role of the F-box helicase 1 (Fbh1) protein during genome maintenance in mammalian cells. For this, we generated two mouse embryonic stem cell lines deficient for Fbh1: one with a homozygous deletion of the N-terminal F-box domain (Fbh1^f/f), and the other with a homozygous disruption (Fbh1^?/?). Consistent with previous reports of Fbh1-deficiency in vertebrate cells, we found that Fbh1^?/? cells show a moderate increase in Rad51 localization to DNA damage, but no clear defect in chromosome break repair. In contrast, we found that Fbh1^f/f cells show a decrease in Rad51 localization to DNA damage and increased cytoplasmic localization of Rad51. However, these Fbh1^f/f cells show no clear defects in chromosome break repair. Since some Rad51 partners and F-box-associated proteins (Skp1-Cul1) have been implicated in progression through mitosis, we considered whether Fbh1 might play a role in this process. To test this hypothesis, we disrupted mitosis using catalytic topoisomerase II inhibitors (bisdioxopiperazines), which inhibit chromosome decatenation. We found that both Fbh1^f/f and Fbh1^?/? cells show hypersensitivity to topoisomerase II catalytic inhibitors, even though the degree of decatenation stress was not affected. Furthermore, following topoisomerase II catalytic inhibition, both Fbh1-deficient cell lines show substantial defects in anaphase separation of chromosomes. These results indicate that Fbh1 is important for restoration of normal mitotic progression following decatenation stress.     

Chromosome Specificity of Polysomy Promotion by Disruptions of the Saccharomyces cerevisiae RNA1 Gene

Previously, we showed that a disruption of the yeast RNA1 gene with LEU2 sequences promotes polysomy for chromosome XIII. Here we demonstrate that this phenotype is due to sequences specific to the RNA1 gene and that the disruption allele does not affect nondisjunction of three other chromosomes or polysomy of a minichromosome. Hence polysomy appears to be restricted to chromosome XIII.     

Resolution of genetic and uterine environmental effects in a family study of new dermatoglyphic measure: sole pattern ridge counts

The heritability of sole pattern ridge counts was examined in two family studies of endogamous castes from peninsular India. The phenotypes included ridge counts for each of the eight configurational areas separately, all areas combined, and only distal areas combined. Differences in heritability estimates were found between populations as well as among the individual configurational areas. Although some ridge counts do not show familial resemblance, others appear to be moderately heritable. Estimates of h2 range from 0.36 to 0.63 in one family series and from 0.22 to 0.51 in the other. In addition, significant uterine environmental effects were detected in one family series but not in the other.     

Actinomycetes inducing phytotoxic or fungistatic activity in a Douglas-fir Forest and in an adjacent area of repeated regeneration failure in Southwestern Oregon

Actinomycetes were isolated from the upper 1 - 3 cm of the soil layer in a well-developed forest and in an adjacent clearcut area where Douglas-fir [Pseudotsuga menziesii (MIRB.) Franco] regeneration had been impaired for two decades. The population density in the clearcut area was two times as high as that in the forested area. The percentage of actinomycetes that inhibited seed germination of the test plants was significantly higher in isolates obtained from the clearcut area than in those obtained from the forested area, and isolates from the clearcut showed five times the phytotoxic effect of those from the forest. Some actinomycete isolates, 4 % from the clearcut and 2.6 % from the forest, significantly reduced in vitro growth of two common ectomycorrhizal fungi of Douglas-fir,Laccaria laccata andHebeloma ovstuliniforme. Two actinomycete isolates from the clearcut reduced fungal growth by 40 % and 73 %. Reduction of the nutrient in the growth medium did not affect the antifungal activity of the actinomycetes. The data support the idea that, along with other factors, phytotoxic and antifungal actinomycetes may suppress natural regeneration or establishment of planted seedlings - either directly or. indirectly - through inhibition of seed germination or of mycorrhizal fungi.