Cyclophilin B is a functional regulator of hepatitis C virus RNA polymerase

Viruses depend on host-derived factors for their efficient genome replication. Here, we demonstrate that a cellular peptidyl-prolyl cis-trans isomerase (PPIase), cyclophilin B (CyPB), is critical for the efficient replication of the hepatitis C virus (HCV) genome. CyPB interacted with the HCV RNA polymerase NS5B to directly stimulate its RNA binding activity. Both the RNA interference (RNAi)-mediated reduction of endogenous CyPB expression and the induced loss of NS5B binding to CyPB decreased the levels of HCV replication. Thus, CyPB functions as a stimulatory regulator of NS5B in HCV replication machinery. This regulation mechanism for viral replication identifies CyPB as a target for antiviral therapeutic strategies.     

Targeting protein homodimerization: a novel drug discovery system

To identify a novel class of antibiotics, we have developed a high-throughput genetic system for targeting the homodimerization (HD system) of histidine kinase (HK), which is essential for a bacterial signal transduction system (two-component system, TCS). By using the HD system, we screened a chemical library and identified a compound, I-8-15 (1-dodecyl-2-isopropylimidazole), that specifically inhibited the dimerization of HK encoded by the YycG gene of Staphylococcus aureus and induced concomitant bacterial cell death. I-8-15 also showed antibacterial activity against MRSA (methicillin-resistant S. aureus) and VRE (vancomycin-resistant Enterococcus faecalis) with MICs at 25 and 50 microg/ml, respectively.     

Catalytic Pictet-Spengler reactions using Yb(OTf)3

The catalytic Pictet-Spengler reactions proceeded in high yields with high regioselectivity in the presence of a catalytic amount of Yb(OTf)3 and a dehydrating agent at room temperature. High regioselectivities were obtained in these reactions, and it is suggested that the reactions proceeded under kinetic control.     

Isolation, identification, and structure of a potent alkyl-peroxyl radical scavenger in crude canola oil, canolol

Alkylhydroperoxides in oxidized oil are undesirable components because they become alkylperoxyl radicals (ROO*) in the presence of heme, hemoglobin, or myoglobin in red meat. ROO* are biochemically reactive and damage nucleic acids and proteins, thereby harming living cells. We isolated a component, a highly potent ROO* scavenger, from crude canola oil (rapeseed), which we designated canolol, and identified its chemical structure, 4-vinyl-2,6-dimethoxyphenol. The canolol content of crude canola oil greatly increased after the seed was roasted as compared with that from unroasted seed, but it decreased in highly purified oil. This anti-ROO* activity was highest in crude oil, deceased after each refining step, and was lowest in highly purified refined oil. Canolol was thus generated during roasting. As shown previously, canolol is one of the most potent anti-ROO* components in crude canola oil and its potency is much greater than that of well-known antioxidants, including alpha-tocopherol, vitamin C, beta-carotene, rutin, and quercetin.     

Characterization of arginine as a solvent additive: a halophilic enzyme as a model protein

Arginine suppresses the aggregation of proteins. However, little is known about its mechanism. Here we have used HsNDK (Halobacterium salinarum nucleoside diphosphate kinase) to examine the solvent property of arginine. After exposure to 2 M arginine, HsNDK was diluted to a low salt buffer, resulting in fully active protein. Since unfolded HsNDK cannot refold in such low salt buffer, the observed activity indicates that HsNDK was in the native state in 2 M arginine. Enzyme activity was also examined directly in the presence of arginine, showing that it was active in the presence of 1 M arginine and, to less extent, 2 M arginine. Arginine, however, could not support refolding of heat-denatured HsNDK. HsNDK was stable at 40 degrees C for 19 h incubation in the presence of 1M arginine.     

Rapid categorization of pathogenic Escherichia coli by multiplex PCR

A one-shot multiplex polymerase chain reaction (PCR) was developed for detecting 12 virulence genes of diarrheagenic Escherichia coli. In order to differentiate between the five categories of diarrheagenic E. coli, we selected the target genes: stx1, stx2, and eaeA for enterohemorrhagic E. coli(EHEC); eaeA, bfpA, and EAF for enteropathogenic E. coli(EPEC); invE for enteroinvasive E. coli(EIEC); elt, estp, and esth for enterotoxigenic E. coli(ETEC); CVD432 and aggR for enteroaggregative E. coli(EAggEC); and astA distributed over the categories of diarrheagenic E. coli. In our multiplex PCR system, all 12 targeted genes (stx1, stx2, eaeA, invE, elt, estp, astA, esth, bfpA, aggR, EAF, and CVD432) were amplified in a single PCR reaction in one tube and detected by electrophoresis. Using our multiplex PCR, the 208 clinically isolated strains of diarrheagenic E. coli in our laboratory were successfully categorized and easily analyzed for the presence of virulence plasmids.     

Biological and biochemical characteristics of prion strains conserved in persistently infected cell cultures

Abnormal prion protein (PrP(Sc)) plays a central role in the transmission of prion diseases, but the molecular basis of prion strains with distinct biological characteristics remains to be elucidated. We analyzed the characteristics of prion disease by using mice inoculated with the Chandler and Fukuoka-1 strains propagated in a cultured mouse neuronal cell line, GT1-7, which is highly permissive to replication of the infectious agents. Strain-specific biological characteristics, including clinical manifestations, incubation period as related to the infectious unit, and pathological profiles, remained unchanged after passages in the cell cultures. We noted some differences in the biochemical aspects of PrP(Sc) between brain tissues and GT1-7 cells which were unlikely to affect the strain phenotypes. On the other hand, the proteinase K-resistant PrP core fragments derived from Fukuoka-1-infected tissues and cells were slightly larger than those from Chandler-infected versions. Moreover, Fukuoka-1 infection, but not Chandler infection, gave an extra fragment with a low molecular weight, approximately 13 kDa, in both brain tissues and GT1-7 cells. This cell culture model persistently infected with different strains will provide a new insight into the understanding of the molecular basis of prion diversity.     

Hematopoietic tissue-specific expression of mouse Neil3 for endonuclease VIII-like protein

We cloned cDNA and genomic DNA containing exon 1 of mouse Neil3. Neil3 spans 52.4 kb and consists of 10 exons. Northern blot analysis revealed that Neil3 mRNA was selectively expressed in thymus, spleen and bone marrow. High levels of Neil3 mRNA were also detected in various mouse B cell lines by RT-PCR. Immunofluorescence microscopy using anti-NEIL3 revealed that recombinant mouse NEIL3 is localized in the nuclei. In mouse splenocytes, the level of Neil3 mRNA significantly increased after mitogen stimulation in vitro. We established NEIL3-null mice, which are viable and fertile. We found candidate sequences for NEIL3 orthologues in a DNA database from dog and zebrafish in addition to human and mouse, but not invertebrates. NEIL3 may function exclusively in vertebrates, such as mammals.     

Proteome analysis of human metaphase chromosomes

DNA is packaged as chromatin in the interphase nucleus. During mitosis, chromatin fibers are highly condensed to form metaphase chromosomes, which ensure equal segregation of replicated chromosomal DNA into the daughter cells. Despite >1 century of research on metaphase chromosomes, information regarding the higher order structure of metaphase chromosomes is limited, and it is still not clear which proteins are involved in further folding of the chromatin fiber into metaphase chromosomes. To obtain a global view of the chromosomal proteins, we performed proteome analyses on three types of isolated human metaphase chromosomes. We first show the results from comparative proteome analyses of two types of isolated human metaphase chromosomes that have been frequently used in biochemical and morphological analyses. 209 proteins were quantitatively identified and classified into six groups on the basis of their known interphase localization. Furthermore, a list of 107 proteins was obtained from the proteome analyses of highly purified metaphase chromosomes, the majority of which are essential for chromosome structure and function. Based on the information obtained on these proteins and on their localizations during mitosis as assessed by immunostaining, we present a four-layer model of metaphase chromosomes. According to this model, the chromosomal proteins have been newly classified into each of four groups: chromosome coating proteins, chromosome peripheral proteins, chromosome structural proteins, and chromosome fibrous proteins. This analysis represents the first compositional view of human metaphase chromosomes and provides a protein framework for future research on this topic.     

Bacillolysin MA, a novel bacterial metalloproteinase that produces angiostatin-like fragments from plasminogen and activates protease zymogens in the coagulation and fibrinolysis systems

We isolated a novel protease that converts plasminogen to angiostatin-like fragments (BL-angiostatins) from a culture of Bacillus megaterium A9542 through a single-step chromatography on CM-cellulose. The protease, designated bacillolysin MA (BL-MA), belongs to a family of neutral metalloproteinases based on the nucleotide sequence of its gene. At an enzyme:substrate ratio of 1:540, BL-MA cleaved human plasminogen mainly at Ser441-Val442 to form BL-angiostatin and miniplasminogen with a K(m) of 3.0 +/- 0.8 microM and a k(cat) of 0.70 +/- 0.09 s(-1). The resulting BL-angiostatins inhibited the proliferation, migration, and tube formation of vascular endothelial cells at concentrations of 1-10 microg/ml. Although BL-MA failed to activate plasminogen, it increased urokinase-catalyzed activation of plasminogen caused by production of miniplasminogen, which is highly susceptible to activation. In addition, BL-MA was active in converting prourokinase, prothrombin, coagulation factor X, and protein C to their active forms. BL-MA enhanced both the clotting of human plasma and clot dissolution in the presence of prourokinase. Thus, BL-MA affects blood coagulation and fibrinolysis systems and can be used to produce angiostatin-like plasminogen fragments and active serine proteases of human plasma.