Hirofumi Okamura

Full-length GBV-C/HGV genomes from nine Japanese isolates: characterization by comparative analyses

SummaryThe genomes of nine GBV-C/HGV isolates from Japanese chronic hepatitis patients were fully sequenced and characterized. They shared 85% nucleotide sequence homology with previously characterized isolates from the US and West Africa. Homology studies and phylogenetic analyses showed that the Japanese isolates formed a third group distinct from the established groups 1 and 2. The genetic distances between the three groups of GBV-C/HGV were very similar to the distances between the two classical swine fever virus (CSFV) serotypes, which suggested that they might belong to a separate GBV-C/HGV serotype. Plot similarity analysis comparing the three groups exposed relatively conserved terminal non-coding regions. Hairpin structures predicted in the Japanese isolates are probably involved in viral replication. The region coding E1-E2-NS-2 showed the least similarity (80%); in HCV the similarity here is only 50% due to its hypervariablity. NS-3 and NS-5b that respectivity encode the helicase/protease and RNA-dependent RNA polymerase, had a high degree of amino acid homo- logy, suggesting a high degree of functional constraint in this region. The NS-5b nucleotide sequence was highly conserved perhaps because of constraints from RNA secondary structure and/or an open reading frame in the negative strand.

Slow evolutionary rate of GB virus C/hepatitis G virus.

Abstract. With the aim of elucidating evolutionary features of GB virus C/hepatitis G virus (GBV-C/HGV), molecular evolutionary analyses were conducted using the entire coding region of this virus. In particular, the rate of nucleotide substitution for this virus was estimated to be less than 9.0 × 10−6 per site per year, which was much slower than those for other RNA viruses. The phylogenetic tree reconstructed for GBV-C/HGV, by using GB virus A (GBV-A) as outgroup, indicated that there were three major clusters (the HG, GB, and Asian types) in GBV-C/HGV, and the divergence between the ancestor of GB- and Asian-type strains and that of HG-type strains first took place more than 7000–10,000 years ago. The slow evolutionary rate for GBV-C/HGV suggested that this virus cannot escape from the immune response of the host by means of producing escape mutants, implying that it may have evolved other systems for persistent infection.

Purification to apparent homogeneity of inactive kallikrein from rat urine

Inactive kallikrein was purified from rat urine by a procedure including ammonium sulfate fractionation, DEAE cellulose chromatography, phenyl-Sepharose CL-4B chromatography, and gel filtration on Sephadex G-100 and Sephadex G-75 columns. The resulting preparation was essentially homogeneous, as assessed by polyacrylamide gel electrophoresis. This preparation migrated as a single protein band on a SDS-polyacrylamide gel and the molecular weight was 41000. The purified material underwent marked activation by trypsin, but not by deoxycholate, Triton X-100, SDS or acidification. These results indicate that the purified inactive kallikrein is the precursor rather than a complex with a substance binding to the active form of kallikrein.

Isolation of inactive kallikrein from rat urine

Abstract An inactive kallikrein isolated from rat urine by DEAE cellulose chromatography could be activated with trypsin, but not with dithiothreitol and detergents such as SDS, Triton X-100 or deoxycholate. The molecular weight of this enzyme was estimated to be 44,000 by gel filtration, and the enzyme was converted to rat urinary kallikrein (RUK, 38,000) by trypsin treatment. There were no significant differences in the Km values for Pro-Phe-Arg-MCA hydrolysis and responses to several trypsin inhibitors between the RUK and the trypsin-activated form of inactive kallikrein. This inactive form of kallikrein in rat urine is probably a precursor proteolytically converted to its active form.

Activation of urinary inactive kallikrein by an extract from the rat kidney cortex

Activation of purified urinary inactive kallikrein by an extract from the rat kidney cortex was investigated. The extract produced a dose-dependent activation of the inactive kallikrein and the optimum pH for this activation was 5.0. Marked depression of the activation was observed when the extract was pre-incubated with E-64, p-CMB and iodoacetate, but not with DFP, PMSF or pepstatin A. The molecular weight of the inactive kallikrein (Mr 44,000) was reduced to 38,000 by treatment with the extract, this molecular weight value being identical with that of urinary active kallikrein. These results indicate that the rat kidney cortex contains a protease catalyzing conversion of urinary inactive kallikrein into its active form, and that the protease has properties compatible with those of a thiol protease, but not of trypsin which has been used as a tool for the activation of urinary inactive kallikrein. The thiol protease is probably one of regulators of the kallikrein-kinin system in the kidney.

Purification of Inactive Kallikrein from Rat Urine

An inactive kallikrein was purified from rat urine, and some of the properties of this enzyme were examined, in comparison with those of rat urinary kallikrein (RUK). The purified inactive kallikrein reacted with the antiserum against RUK and migrated slightly more slowly than RUK, on the immunoelectrophoresis. The molecular weights of the inactive kallikrein and RUK were estimated to be 44,000 and 38,000 by gel filtration, respectively. These results indicate that the rat urinary inactive kallikrein is immunologically identical with RUK, but this inactive enzyme has biochemical properties different from those of RUK, with respect to molecular weight and electrophoretical mobility.