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M Hijikata

Publications and source records attributed to M Hijikata.

At least 19 recordsLinked to original sources
PubMed1998

Hepatitis C virus infection and mutations of mannose-binding lectin gene MBL.

We assessed the genetic polymorphism of mannose-binding lectin (MBL) in 93 patients with chronic hepatitis C (45 responders and 48 nonresponders to interferon) and 218 healthy controls. Mutant allele was identified only at codon 54 (Gly-->Asp), leading to three genotypes (54 m/m, 54 W/m, and 54 W/W). Frequency of 54 m/m was significantly lower in interferon-responders (2.2%), compared to those in nonresponders (14.6%) and controls (10.6%): p < 0.05. Our results suggest that homozygous carriage of the variant allele of codon 54 of MBL may predict poor response to interferon in chronic hepatitis C patients.

Adult↗
PubMed1998

Association of mannose-binding lectin gene haplotype LXPA and LYPB with interferon-resistant hepatitis C virus infection in Japanese patients.

BACKGROUND/AIMS: Mannose-binding lectin, a key factor of the innate immune system, has genetic polymorphism, and individuals who carry certain genotypes of mannose-binding lectin are known to be more prone to severe or prolonged infectious diseases. We aimed to find any relevance of mannose-binding lectin polymorphism to hepatitis C virus infection. METHODS: We determined the mannose-binding lectin genotypes by sequence specific priming-polymerase chain reaction in 159 hepatitis C virus-infected chronic hepatitis patients and 218 healthy controls in Japan by looking at 4 polymorphic loci: 2 (H/L and X/Y) within the promoter region and 2 (P/Q and A/B) within exon-1 of the mannose-binding lectin gene. RESULTS: A group of mannose-binding lectin genotypes designated "XB-type" (containing LXPA or LYPB haplotype at least heterozygously) was less frequently found in interferon-responsive patients (38.5%) than in interferon-resistant patients (60.7%, p=0.008) and controls (57.3%, p=0.014). Individuals with the "XB-type" had lower serum concentrations of mannose-binding lectin, compared to those with "YA-type", which is defined by homozygous carriage of both Y and A alleles: 0.63+/-0.61 vs 2.06+/-1.17 mg/l, p<0.001. CONCLUSIONS: Our results suggest that the mannose-binding lectin-related innate immune system plays an important role in elimination of hepatitis C virus during interferon therapy. Determining mannose-binding lectin genotypes may help in selecting the hepatitis C virus-infected patients to be treated with interferon.

Antiviral Agents↗
PubMed1997

Inhibition of protein tyrosine kinase by 5-S-GAD, a novel antibacterial substance from an insect.

The effect of N-beta-alanyl-5-S-glutathionyl-dopa (5-S-GAD), a compound originally isolated from Sarcophaga peregrina (a flesh fly) as an antibacterial substance, on protein phosphorylation was examined using v-src-transformed NIH3T3 cell lysates. 5-S-GAD was found to inhibit tyrosine phosphorylation of protein tyrosine kinase v-src, but not serine/threonine phosphorylation of protein kinase C. The potency of this compound was comparable to that of herbimycin A. Our results suggested that a substitution at position 5 of the catechol in 5-S-GAD with the sulfur of cysteine is essential for 5-S-GAD to inhibit protein tyrosine kinase v-src.

3T3 Cells↗
PubMed1997

Cleavage activity of hepatitis C virus serine proteinase.

To study the character of the hepatitis C virus (HCV) encoding serine proteinase and to search for inhibitors, a practical in vitro assay system using the purified enzyme and synthetic peptide substrates was established. The enzyme used was expressed in Escherichia coli as a fusion form with protein tags and purified to apparent homogeneity by single-step affinity chromatography. The purified enzyme exhibited proteolytic activity with pH optima of around eight, and the addition of NS4A fragments increased the activity as well as the thermal stability of the enzyme. The activity was inhibited by EDTA and some divalent ions, i.e., copper and zinc, though calcium, magnesium, and manganese were stimulative both in the presence and absence of the NS4A fragment. None of the common protease inhibitors, including serine protease inhibitors, effectively inhibited the activity. Based on the kinetic parameters of the cleavage reaction of the synthetic 20 mer peptides corresponding to the three cleavage sites, NS4A/4B, NS4B/5A, and NS5A/5B, the peptide with the NS5A/5B junction was found to be the most efficient substrate. Analysis of the minimal peptide substrate of NS5A/5B indicated that 5 to 7 amino acids on both sides of the junction were required for efficient cleavage. These findings are expected to contribute to the search for a proteinase inhibitor.

Cations, Divalent↗
PubMed1997

Specific interaction of polypyrimidine tract-binding protein with the extreme 3'-terminal structure of the hepatitis C virus genome, the 3'X.

We previously identified a highly conserved 98-nucleotide (nt) sequence, the 3'X, as the extreme 3'-terminal structure of the hepatitis C virus (HCV) genome (T. Tanaka, N. Kato, M.-J. Cho, and K. Shimotohno, Biochem. Biophys. Res. Commun. 215:744-749, 1995). Since the 3' end of positive-strand viral RNA is the initiation site of RNA replication, the 3'X should contribute to HCV negative-strand RNA synthesis. Cellular factors may also be involved in this replication mechanism, since several cellular proteins have been shown to interact with the 3'-end regions of other viral genomes. In this study, we found that both 38- and 57-kDa proteins in the human hepatocyte line PH5CH bound specifically to the 3'-end structure of HCV positive-strand RNA by a UV-induced cross-linking assay. The 57-kDa protein (p57), which had higher affinities to RNA probes, recognized a 26-nt sequence including the 5'-terminal 19 nt of the 3'X and 7 flanking nt, designated the transitional region. This sequence contains pyrimidine-rich motifs and shows similarity to the consensus binding sequence of the polypyrimidine tract-binding protein (PTB), which has been implicated in alternative pre-mRNA splicing and cap-independent translation. We found that this 3'X-binding p57 is identical to PTB. The 3'X-binding p57 was immunoprecipitated by anti-PTB antibody, and recombinant PTB bound to the 3'X RNA. In addition, p57 bound solely to the 3'-end region of positive-strand RNA, not to this region of negative-strand RNA. We suggest that 3'X-PTB interaction is involved in the specific initiation of HCV genome replication.

Base Sequence↗
PubMed1996

Comparison of hypervariable regions (HVR1 and HVR2) in positive- and negative-stranded hepatitis C virus RNA in cancerous and non-cancerous liver tissue, peripheral blood mononuclear cells and serum from a patient with hepatocellular carcinoma.

Hepatitis C virus (HCV) infection is associated with a wide spectrum of liver diseases including cirrhosis and hepatocellular carcinoma (HCC). Although the biological relation between the virus and cirrhosis or HCC is unclear, such variable pathogenicity may be related to the genetic heterogeneity of HCV. Genetic variability of HCV was assessed by determining the nucleotide sequence corresponding to the hypervariable regions (HVR1 and HVR2) of the putative envelope protein (E2/NS1) in positive- and negative-stranded HCV RNA from the cancerous and surrounding non-cancerous liver tissue, peripheral blood mononuclear cells and serum of a patient with HCC. Nineteen distinct HVR1 amino acid sequences (deduced from the nucleotide sequences) were obtained from the patient and could be classified into 5 groups on the basis of the site and time of detection. Some viral isolates with the same HVR1 sequence were shown to replicate in both cancerous and non-cancerous liver tissue, whereas others replicated in HCC tissue only.

Amino Acid Sequence↗
PubMed1996

Antigenicity of hepatitis C virus envelope proteins expressed in Chinese hamster ovary cells.

A putative second envelope glycoprotein (E2) of hepatitis C virus (HCV) was constitutively produced in a Chinese hamster ovary cell line stably transformed with a plasmid expressing E2 protein under the control of an exogenous promoter and a signal sequence. E2 protein that lacked part of the C-terminal hydrophobic region was glycosylated with high-mannose type oligosaccharides and retained in the cells. On the other hand, E2 protein lacking the entire C-terminal hydrophobic region was glycosylated with complex type oligosaccharides (complex form) and excreted into the culture medium. Immunoreactivity of the high-mannose and complex forms of E2 proteins against sera from HCV infected patients were analyzed. We found that the antigenicity of the complex form of E2 protein was greater than that of the high-mannose form of E2 protein. This result indicated that the complex form of the E2 protein is superior for use in diagnosing HCV infection.

Animals↗
PubMed1996

Replication of hepatitis C virus.

The mode of replication of the hepatitis C virus (HCV) remains poorly understood. Attempts to produce a tissue culture model containing replicating HCV have been largely unsuccessful. Recent studies on sera from patients chronically infected with HCV have shown that viral particles may be found in high- or low-density fractions. High-density fractions contain non-infectious virions whilst infectious particles can be derived from low-density material. Using appropriate infectious fractions we have successfully infected a number of human cell lines allowing studies of HCV replication to be initiated.

Animals↗
PubMed1996

Characterization of long-term cultures of hepatitis C virus.

The human T- and B-cell lines HPBMa10-2 and Daudi produced infectious hepatitis C virus (HCV) for more than 1 year after infection. The infectivity titer of the cell culture-grown HCV and its genome titer were comparable. The virion density in sucrose was around 1.12 g/ml. Among the 13 variants detected in the inoculum, 7 were adsorbed by the cells and one particular HCV sequence which was present in minor quantities in the inoculum persisted.

Animals↗
PubMed1995

Mammalian karyopherin alpha 1 beta and alpha 2 beta heterodimers: alpha 1 or alpha 2 subunit binds nuclear localization signal and beta subunit interacts with peptide repeat-containing nucleoporins.

Although only 44% identical to human karyopherin alpha 1, human karyopherin alpha 2 (Rch1 protein) substituted for human karyopherin alpha 1 (hSRP-1/NPI-1) in recognizing a standard nuclear localization sequence and karyopherin beta-dependent targeting to the nuclear envelope of digitonin-permeabilized cells. By immunofluorescence microscopy of methanol-fixed cells, karyopherin beta was localized to the cytoplasm and the nuclear envelope and was absent from the nuclear interior. Digitonin permeabilization of buffalo rat liver cells depleted their endogenous karyopherin beta. Recombinant karyopherin beta can bind directly to the nuclear envelope of digitonin-permeabilized cells at 0 degree C (docking reaction). In contrast, recombinant karyopherin alpha 1 or alpha 2 did not bind unless karyopherin beta was present. Likewise, in an import reaction (at 20 degrees C) with all recombinant transport factors (karyopherin alpha 1 or alpha 2, karyopherin beta, Ran, and p10) import depended on karyopherin beta. Localization of the exogenously added transport factors after a 30-min import reaction showed karyopherin beta at the nuclear envelope and karyopherin alpha 1 or alpha 2, Ran, and p10 in the nuclear interior. In an overlay assay with SDS/PAGE-resolved and nitrocellulose-transferred proteins of the nuclear envelope, 35S-labeled karyopherin beta bound to at least four peptide repeat-containing nucleoporins--Nup358, Nup214, Nup153, and Nup98.

Animals↗
PubMed1995

Bacterial expression and analysis of cleavage activity of HCV serine proteinase using recombinant and synthetic substrate.

HCV encoding serine proteinase was expressed in E. coli as a fused form with maltose binding protein (MBP) and a six histidine tag. The enzyme was partially purified by using affinity chromatography for these fused peptides. Proteolytic cleavage activity of the partially purified enzyme was detected by means of an assay using both a recombinant protein and a synthetic peptide substrate which had an amino acid sequence corresponding to the most efficient cleavage site in vivo, the NS5A-NS5B junction. The cleavage occurred at the same site that was reported before.

ATP-Binding Cassette Transporters↗
PubMed1995

A novel method for analysis of viral proteinase activity encoded by hepatitis C virus in cultured cells.

We developed a novel method for analysis of hepatitis C viral proteinase activity in cultured cells, in which the proteinase activity was measured as the enhancement of reporter gene expression. In this system, plasmids encoding a reporter gene, the enzyme gene, and the substrate gene were simultaneously transfected into COS-1 cells. The reporter plasmid contains chloramphenicol acetyltransferase (CAT) gene downstream of an enhancer/promoter sequence derived from the human T-cell leukemia virus type-1 (HTLV-I) long-terminal repeat (LTR). The substrate expression plasmid was a triple chimera; HCV nonstructural protein 2 (NS2) and the Tax1 protein of HTLV-I sandwiched the substrate polypeptide, which was inserted upstream of Tax1. This method assumes that since the HCV NS2 appears to be located in the lipid bilayer of endoplasmic reticulum (ER) membranes, the Tax1 of the chimeric substrate was trapped on the surface of the ER in the absence of HCV proteinase activity. After release from the chimera by HCV proteinase-dependent cleavage, Tax1 could transactivate the expression of the CAT gene through the enhancer sequence of HTLV-I LTR. This system should enable us to simply and safely screen the potential antiviral activity of proteinase inhibitors in vivo, although this system may be limited to proteinase inhibitors that are permeable to the plasma membrane.

Animals↗
PubMed1995

Selective transmission of hepatitis C virus in vivo and in vitro.

A human plasma containing quasi-species of hepatitis C virus (HCV) was inoculated to a chimpanzee and to human lymphocytic cell lines, HPB-Ma clone 10-2, AD HPB, and Daudi, which support replication of HCV. Among six different hypervariable region (HVR) sequences detected in the inoculum, the same two were recovered both in vivo and in vitro.

Amino Acid Sequence↗
PubMed1995

Expression and processing of putative nonstructural proteins of hepatitis C virus in insect cells using baculovirus vector.

Processing of the putative nonstructural (NS) proteins, p70(NS3), p4(NS4A), p27(NS4B), p58/56(NS5A), and p66(NS5B), of Japanese type hepatitis C virus (HCV) in insect cells was analyzed by using a baculovirus expression system. Products processed by the HCV serine proteinase (Cpro-2) were essentially identical to those found in mammalian cultured cells transiently producing the NS region of the HCV precursor polyprotein. A series of internal and carboxy (C)-terminal deletion experiments coupled with epitope scanning analysis showed that efficient cleavage at the Cpro-2-dependent processing sites, except at the p4(NS4A)/p27(NS4B) site, is not significantly influenced by those mutations. Efficient cleavage at p4(NS4A)/p27(NS4B) required about 40% of the NS5A N-terminal region. Estimation of the processing sites by determination of the N-terminal amino acid sequences of the processed products revealed that all the Cpro-2-dependent cleavages occurred at essentially identical sites to those reported for another HCV genotype, suggesting that Cpro-2 is a possible target for the development of a strain-independent anti-HCV agent.

Amino Acid Sequence↗
PubMed1995

Hepatitis C virus-encoded nonstructural protein NS4A has versatile functions in viral protein processing.

A transient protein expression system in COS-1 cells was used to study the role of hepatitis C virus (HCV)-encoded NS4A protein on HCV nonstructural polyprotein processing. By analyzing the protein expression and processing of a deletion mutant polypeptide, NS delta 4A, which encodes the entire putative HCV nonstructural polyprotein except the region encoding NS4A, the versatile functions of NS4A were revealed. Most of the NS3 processed from NS delta 4A was localized in the cytosol fraction and was degraded promptly. Coproduction of NS4A stabilizes NS3 and assists in its localization in the membrane. NS4A was found to be indispensable for cleavage at the 4B/5A site but not essential for cleavage at the 5A/5B site in NS delta 4A. The functioning of NS4A as a cofactor for cleavage at the 4B/5A site was also observed when 30 amino acids around this site was used as a substrate and a serine proteinase domain of 167 amino acids, from Gly-1049 to Ser-1215, was used as an enzyme protein, suggesting that possible domains for the interaction of NS4A were in those regions of the enzyme protein (NS3) and/or the substrate protein. Two proteins, p58 and p56, were produced from NS5A. For the production of p58, equal or excess molar amounts of NS4A relative to NS delta 4A were required. Deletion analysis of NS4A revealed a minimum functional domain of NS4A of 10 amino acids, from Gly-1678 to Ile-1687.

Amino Acid Sequence↗
PubMed1995

The N-terminal region of hepatitis C virus nonstructural protein 3 (NS3) is essential for stable complex formation with NS4A.

Hepatitis C virus proteins are produced by proteolytic processing of the viral precursor polyprotein that is encoded in the largest open reading frame of the viral genome. Processing of the nonstructural viral polyprotein requires the viral serine-type proteinase present in nonstructural protein 3 (NS3). The cleavage of the junction between NS4B and NS5A is mediated by NS3 only when NS4A is present. NS4A is thought to be a cofactor that enhances the cleavage efficiency of NS3 in hepatitis C virus protein-producing cells. Stable NS3-NS4A complex formation required the N-terminal 22 amino acid residues of NS3. This interaction contributed to stabilization of the NS3 product as well as increased the efficiency of cleavage at the NS4B/5A site. The N-terminal 22 amino acid residues fused to Escherichia coli dihydrofolate reductase also formed a stable complex with NS4A. NS3 derivatives which lacked the N-terminal 22 amino acid residues showed drastically reduced cleavage activity at the NS4B/5A site even in the presence of NS4A. These data suggested that the interaction with NS4A through the 22 amino acid residues of NS3 is primarily important for the NS4A-dependent processing of the NS4B/5A site by NS3.

Base Sequence↗