The NS5B RNA-dependent RNA polymerase encoded by hepatitis C virus (HCV) plays a key role in viral replication. Reported here is evidence that HCV NS5B polymerase acts as a functional oligomer. Oligomerization of HCV NS5B protein was demonstrated by gel filtration, chemical cross-linking, temperature sensitivity, and yeast cell two-hybrid analysis. Mutagenesis studies showed that the C-terminal hydrophobic region of the protein was not essential for its oligomerization. Importantly, HCV NS5B polymerase exhibited cooperative RNA synthesis activity with a dissociation constant, K d , of Ϸ22 nM, suggesting a role for the polymerase-polymerase interaction in the regulation of HCV replicase activity. Further functional evidence includes the inhibition of the wild-type NS5B polymerase activity by a catalytically inactive form of NS5B. Finally, the X-ray crystal structure of HCV NS5B polymerase was solved at 2.9 Å. Two extensive interfaces have been identified from the packing of the NS5B molecules in the crystal lattice, suggesting a higher-order structure that is consistent with the biochemical data.Hepatitis C virus (HCV) belongs to the Flaviviridae family and is responsible for a significant proportion of acute and chronic hepatitis in humans worldwide (7,8). Similar to other flaviviruses, HCV is a small positive-stranded RNA virus with a genome size of Ϸ9.6 kb encoding a single polyprotein (30). This viral polyprotein is processed by both host and virally encoded proteases to generate mature structural and nonstructural proteins essential for virus replication (see references 9, 34, and 39 for review). One of the nonstructural proteins, designated NS5B, is the virally encoded RNA-dependent RNA polymerase (RdRp), which contains the GDD signature motif of RNA polymerases (3).It has been shown that NS5B is a membrane-associated protein, which contains a C-terminal domain comprising Ϸ21 hydrophobic amino acids that is responsible for membrane anchorage (41). NS5B may form a complex with cellular proteins (37) or other HCV nonstructural proteins, including NS3, the viral protease and helicase; NS4A, a cofactor of NS3 protease activity; and NS5A, a phosphoprotein containing a putative interferon sensitivity region (15). Although the HCV replication mechanism is not clearly understood, the essential role of NS5B polymerase in the HCV replication and infection process has been demonstrated in chimpanzees (22). Accordingly, it has been viewed as an attractive target for antiviral intervention.Recombinant HCV NS5B polymerase has been produced and purified from both bacterial and insect cells by several groups (3,11,16,19,25,27,31,41). The availability of highly purified protein has facilitated the biochemical characterization of HCV NS5B polymerase. Similar to other viral RdRps, purified HCV NS5B is able to synthesize RNA using various RNAs as templates in vitro (3,11,16,19,25,27,31,41). In this regard, two RNA synthesis reaction modes have been described for this enzyme: RNA elongation using a preannealed primer and RNA in...

Intracoronary infusion of VEGF165 significantly improves blood flow to the ischemic myocardium. Concomitant administration of L-NAME inhibits VEGF-induced hypotension while most likely preserving VEGF-induced angiogenesis. Intravenous infusion of VEGF165 was not effective in augmenting either myocardial flow or function in this model.

Our goal was to investigate effects of long-term exposure to pulsed microwave radiation. The major emphasis was to expose a large sample of experimental animals throughout their lifetimes and to monitor them for effects on general health and longevity. An exposure facility was developed that enabled 200 rats to be maintained under specific-pathogen-free (SPF) conditions while housed individually in circularly-polarized waveguides. The exposure facility consisted of two rooms, each containing 50 active waveguides and 50 waveguides for sham (control) exposures. The experimental rats were exposed to 2,450-MHz pulsed microwaves at 800 pps with a 10-microseconds pulse width. The pulsed microwaves were square-wave modulated at 8-Hz. Whole body calorimetry, thermographic analysis, and power-meter analysis indicated that microwaves delivered at 0.144 W to each exposure waveguide resulted in an average specific absorption rate (SAR) that ranged from 0.4 W/kg for a 200-g rat to 0.15 W/kg for an 800-g rat. Two hundred male, Sprague-Dawley rats were assigned in equal numbers to radiation-exposure and sham-exposure conditions. Exposure began at 8 weeks of age and continued daily, 21.5 h/day, for 25 months. Animals were bled at regular intervals and blood samples were analyzed for serum chemistries, hematological values, protein electrophoretic patterns, thyroxine, and plasma corticosterone levels. In addition to daily measures of body mass, food and water consumption by all animals, O2 consumption and CO2 production were periodically measured in a sub-sample (N = 18) of each group. Activity was assessed in an open-field apparatus at regular intervals throughout the study. After 13 months, 10 rats from each group were euthanatized to test for immunological competence and to permit whole-body analysis, as well as gross and histopathological examinations. At the end of 25 months, the survivors (11 sham-exposed and 12 radiation-exposed rats) were euthanatized for similar analyses. The other 157 animals were examined histopathologically when they died spontaneously or were terminated in extremis.

A gene (mgt) encoding a monofunctional glycosyltransferase (MGT) from Staphylococcus aureus has been identified. This first reported gram-positive MGT shared significant homology with several MGTs from gram-negative bacteria and the N-terminal glycosyltransferase domain of class A high-molecular-mass penicillin-binding proteins from different species. S. aureus MGT contained an N-terminal hydrophobic domain perhaps involved with membrane association. It was expressed in Escherichia coli cells as a truncated protein lacking the hydrophobic domain and purified to homogeneity. Analysis by circular dichroism revealed that secondary structural elements of purified truncated S. aureus MGT were consistent with predicted structural elements, indicating that the protein might exhibit the expected folding. In addition, purified S. aureus MGT catalyzed incorporation of UDP-N-acetylglucosamine into peptidoglycan, proving that it was enzymatically active. MGT activity was inhibited by moenomycin A, and the reaction product was sensitive to lysozyme treatment. Moreover, a protein matching the calculated molecular weight of S. aureus MGT was identified from an S. aureus cell lysate using antibodies developed against purified MGT. Taken together, our results suggest that this enzyme is natively present in S. aureus cells and that it may play a role in bacterial cell wall biosynthesis.

The yeast gene MCKI encodes a serine/threonine protein kinase that is thought to function in regulating kinetochore activity and entry into meiosis. Disruption of MCKI confers a cold-sensitive phenotype, a temperature-sensitive phenotype, and sensitivity to the microtubule-destabilizing drug benomyl and leads to loss of chromosomes during growth on benomyl. A dosage suppression selection was used to identify genes that, when present at high copy number, could suppress the cold-sensitive phenotype of mckl:::HIS3 mutant cells.Several unique classes of clones were identified, and one of these, designated MDSI, has been characterized in some detail. Nucleotide sequence data reveal that MDSI encodes a serine/threonine protein kinase that is highly homologous to the shaggy/zw3 kinase in Drosophila melanogaster and its functional homolog, glycogen synthase kinase 3, in rats. The presence of MDSI in high copy number rescues both the cold-sensitive and the temperature-sensitive phenotypes, but not the benomyl-sensitive phenotype, associated with the disruption of MCK1. Analysis of strains harboring an mdsl null mutation demonstrates that MDSI is not essential during normal vegetative growth but appears to be required for meiosis. Finally, in vitro experiments indicate that the proteins encoded by both MCKI and MDS1 possess protein kinase activity with substrate specificity similar to that of mammalian glycogen synthase kinase 3.The maintenance of correct chromosomal ploidy in eukaryotic cells during mitotic cell division requires the faithful execution of a number of complex processes. These processes are temporally controlled by a regulatory system that comprises the machinery of the eukaryotic cell cycle. An essential component of the mitotic apparatus is the kinetochore, a complex macromolecular structure which provides the site of chromosome attachment to the mitotic spindle. Although the kinetochore has been morphologically described in higher eukaryotes, relatively little is known about the molecular mechanisms responsible for kinetochore function. Elucidation of the roles of individual proteins that comprise and regulate the activity of this structure will require a combination of biochemical, cell biological, and genetic approaches.The centromere (CEN) DNA from Saccharomyces cerevisiae, on which the kinetochore assembles prior to mitosis, has been extensively characterized (for a review, see reference 4). This region consists of three conserved centromere DNA elements (CDEs). Mutational analyses of CEN DNA has demonstrated that one of these elements, CDEIII, is essential for centromere function. In previous studies, a large number of point mutations were introduced into CDEIII, and their effects on the mitotic segregation of a marker chromosome fragment were examined in a sensitive visual assay (17,23 fragments harboring a mutant CEN. One of the clones identified through the use of this screen, MCK1 (37), was independently identified as a regulator of meiosis in S. cerevisiae (29) and as a gene encoding a protein...

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