Molecular structure of the Japanese hepatitis C viral genome

The amino acid sequence of the polyprotein deduced from the nucleotide sequence of the Japanese hepatitis C virus genome (N. Kato et. al. (1990) Proc. Natl. Acad. Sci. USA 87, 9524–9528)indicated that this virus is a member of a new class of positive-stranded RNA viruses. Several domains of this polyprotein also showed weak homology with those of flaviviruses and pestiviruses including the chymotrypsin-like serine proteinase. NTPase and RNA-dependent RNA polymerase     

Purification and Characterization of the Egg Jelly Macromolecules, Sialoglycoprotein and Fucose Sulfate Glycoconjugate, of the Sea Urchin Hemicentrotus Pulcherrimus

A sialoglycoprotein and a fucose sulfate glycoconjugate (FSG) were purified from egg jelly of the sea urchin Hemicentrotus pulcherrimus . Sialoglycoprotein which consisted of sialic acid (90%, w/w) and protein (10%, w/w) did not cause induction of the acrosome reaction and sperm isoagglutination. FSG which contained one mol sulfate/mol fucose possessed 2.0 times protein to fucose by weight. The proteins in intact FSG were separated to two major (258 kDa and 237 kDa) and one minor (120 kDa) proteins by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of 2-mercaptoethanol (2-ME) while the proteins could not be separated by HPLC in the presence of 0.1% SDS or SDS-PAGE without 2-ME. However, after carboxymethylation of FSG, two major (260 kDa and 240 kDa) proteins and two minor (140 kDa and 135 kDa) proteins were separated from the fucose sulfate moiety by HPLC in the presence of 0.1% SDS or SDS-PAGE without 2-ME. When FSG was first carboxymethylated with non-radioactive iodoacetic acid and then reduced with 2-ME and finally carboxymethylated with ¹⁴C-iodoacetic acid, the most of radioactivity was detected in 140 kDa and 135 kDa proteins. Carboxymethylted-FSG was less potent than intact FSG in induction of the acrosome reaction. Fucoidan, a fucose sulfate polymer, did not induce the acrosome reaction.     

Water-soluble glucans from the seed of Coix lacryma-jobi var. ma-yuen

The water-soluble major polysaccharides from the seed of Coix lacryma-jobi var. ma-yuen eluted as a broad peak by gel filtration on Sepharose CL-2B. The mixture (CS-Glucan) was resolved into 7 glucans by HPLC on the column of Asahi-Pak GS-510 + GS-320. Similarities were observed between M, shown in the gel filtration profile and the elution volume in HPLC. Methylation analysis indicated that the ethanol-fractionated CS-glucan contained 4-O- and 4,6-di-O-substituted glucosyl residues. ¹H and ¹³C NMR data accorded with the results of methylation analysis, and the glycosidic linkages were shown to have an α-configuration. Thus, CS-glucan contained (1 → 4) linked α-d-glucans to which are attached glucosyl side chains at O-6 of the main chain in a similar way to amylopectin. Each purified glucan was shown to have different absorption maxima ( > 550 nm or 530 nm) in the iodine reaction. The results of the methylation analysis and of the pullulanase digestion suggest that the 550 nm-glucan has a lower branching frequency and shorter side chains than the 530 nm-glucan. Although CS-glucan was found to have weak anti-complementary activity, HPLC-purified > 550 nm-glucan was found to be more potent than the 530 nm-glucan. Thus CS-glucan is highly heterogeneous, and the glucans which form a tight complex when tested with iodine, generally tend to have considerable anti-complementary activity.     

Testing the intermediate disturbance hypothesis on species diversity in herbaceous plant communities along a human trampling gradient using a 4-year experiment in an old-field

The intermediate disturbance hypothesis predicts unimodal relationships between species diversity and disturbance frequency/intensity. To test this hypothesis, species diversity in herbaceous plant communities along a human trampling gradient was investigated by conducting a 4-year experiment in an old-field. In general, species richness (S), the Shannon–Weiner index (H) from plant cover data and species evenness (J) showed negative linear relationships with trampling frequency, in contrast to the prediction of the intermediate disturbance hypothesis. However, the significant relationships between trampling frequency and species diversity were not observed in the fourth year without J, which showed a unimodal relationship. In all experimental years, the number of new species that colonized the plots after 1year was small under frequent trampling, and the number of species lost from the plots was large under infrequent trampling. The relative number and the relative cover of perennial species increased as trampling frequency increased in the first and second years, but this pattern was not observed in the following years because the dominance of perennials further increased at decreasing frequencies of trampling. The similarity in the species composition and the yearly changes in species dominance indicated that trampling at higher frequencies eliminated more trampling-intolerant species only in the early years of the experiment. These results suggest that trampling mediated early changes in species diversity patterns, but competitive interactions were more important in the later experimental years. The time lag in the effects of trampling and competition appears to be attributable to the infrequent occurrence of unimodal patterns of species diversity.     

Ordered assembly of Sld3, GINS and Cdc45 is distinctly regulated by DDK and CDK for activation of replication origins

Initiation of chromosome DNA replication in eukaryotes is tightly regulated through assembly of replication factors at replication origins. Here, we investigated dependence of the assembly of the initiation complex on particular factors using temperature-sensitive fission yeast mutants. The psf3-1 mutant, a GINS component mutant, arrested with unreplicated DNA at the restrictive temperature and the DNA content gradually increased, suggesting a defect in DNA replication. The mutation impaired GINS complex formation, as shown by pull-down experiments. Chromatin immunoprecipitation assays indicated that GINS integrity was required for origin loading of Psf2, Cut5 and Cdc45, but not Sld3. In contrast, loading of Psf2 onto origins depended on Sld3 and Cut5 but not on Cdc45. These results suggest that Sld3 functions furthest upstream in initiation complex assembly, followed by GINS and Cut5, then Cdc45. Consistent with this conclusion, Cdc7-Dbf4 kinase (DDK) but not cyclin-dependent kinase (CDK) was required for Sld3 loading, whereas recruitment of the other factors depended on both kinases. These results suggest that DDK and CDK regulate distinct steps in activation of replication origins in fission yeast.     

High-efficiency CAG-FLPe deleter mice in C57BL/6J background.

The increasing popularity of conditional knockout (KO) technology has resulted in the demand for efficient FLP deleter mice. In addition, FLP deleters are needed in genetic backgrounds that are suited to behavioral studies. We generated CAG-FLPe transgenic (Tg) mice with the C57BL/6J genetic background, which is one of the most commonly-used strains in behavioral studies. We assessed the recombination efficiency of the CAG-FLPe-Tg lines by crossing them with a mouse line carrying a FRT-PGK-neo-FRT cassette. Four of five independent CAG-FLPe lines induced recombination in most (91%-100%) of their progenies, although a small fraction (0%-30%, depending on the line) showed mosaic recombination patterns. These animals are highly potent as deleters of FRT cassettes and are useful for behavioral studies involving conditional KO mice.     

N-terminal labeling of proteins by the Pictet-Spengler reaction

The Pictet-Spengler reaction was applied to the N-terminal labeling of horse heart myoglobin. This was performed in the following two steps: (1) conversion of the N-terminal glycine residue to an alpha-keto aldehyde by a transamination reaction and (2) condensation of the resulting activated myoglobin with tryptamine analogues by the Pictet-Spengler reaction. Ultraviolet (UV)/visible (vis) absorption and circular dichroism (CD) spectral data revealed that the tertiary structure of myoglobin was not altered by the Pictet-Spengler reaction.     

Two Alternative Conformations of a Voltage-Gated Sodium Channel

Activation and inactivation of voltage-gated sodium channels (Navs) are well studied, yet the molecular mechanisms governing channel gating in the membrane remain unknown. We present two conformations of a Nav from Caldalkalibacillus thermarum reconstituted into lipid bilayers in one crystal at 9 Å resolution based on electron crystallography. Despite a voltage sensor arrangement identical with that in the activated form, we observed two distinct pore domain structures: a prominent form with a relatively open inner gate and a closed inner-gate conformation similar to the first prokaryotic Nav structure. Structural differences, together with mutational and electrophysiological analyses, indicated that widening of the inner gate was dependent on interactions among the S4–S5 linker, the N-terminal part of S5 and its adjoining part in S6, and on interhelical repulsion by a negatively charged C-terminal region subsequent to S6. Our findings suggest that these specific interactions result in two conformational structures.