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T Shikata

Publications and source records attributed to T Shikata.

At least 19 recordsLinked to original sources
PubMed1992

Heterogeneity within the nonstructural protein 5-encoding region of hepatitis C viruses from a single patient.

Nucleotide (nt) sequence heterogeneity of the hepatitis C virus (HCV) genome derived from a single carrier was investigated. A polymerase chain reaction (PCR) product of 311 bp in the putative nonstructural protein 5-encoding region was directly sequenced, while part of a PCR product was cloned, and sequence analyses were carried out for 27 independent clones. Although 14 of the 27 clones were conserved, ten other types of nt sequences were found. The difference was at most 3 nt (1.1%). A directly determined sequence showed the major sequence of the cloned products. Since most of the nt changes occurred in the third letter of a codon, these nt changes might not have originated from random misincorporation during the PCR. These results of natural divergence of genome population in a single carrier suggest that HCV is a typical RNA virus with a quasi-species nature due to high mutation rates.

Base Sequence↗
PubMed1992

Three different patterns of hepatitis C virus infection in chimpanzees.

The relationship between hepatitis C virus RNA and hepatitis C virus-associated antibodies (antibody against the putative capsid protein and C-100 antibody) was determined by nested polymerase chain reaction and enzyme-linked immunosorbent assay in serial serum samples obtained from eight chimpanzees experimentally infected with hepatitis C virus. Three different patterns emerged from the polymerase chain reaction data: the first (group 1) was acute resolving hepatitis with transient appearance of HCV RNA (two cases). The second (group 2) had chronic hepatitis with persistent hepatitis C virus RNA positivity (four cases) and the third (group 3) had chronic hepatitis with intermittent appearance of hepatitis C virus RNA (two cases). In four of eight animals, hepatitis C virus RNA was first detectable in serum 1 wk after inoculation. Although serum HCV RNA was detected in all infected chimpanzees, two were positive only for antibody against the putative capsid protein, whereas two were positive only for antibody to C-100 antigen. In four of eight cases, antibody against the putative capsid protein appeared earlier than did antibody to C-100 antigen, was detected just before or coincident with rising glutamate pyruvate transaminase values and remained positive for a long time even after recovery. Six of eight animals (75%) were still hepatitis C virus RNA positive 1 yr after inoculation, suggesting that the risk of development of the chronic carrier state is high in hepatitis C virus infection. Furthermore, there did not appear to be a good correlation between antibody titer in serum and hepatitis C virus infectivity titer.

Alanine Transaminase↗
PubMed1992

Evaluation of three hepatitis C virus-related antibodies C100, KCL-163, JCC. Tests for screening blood donors.

To evaluate the most effective method for detecting hepatitis C virus (HCV) carriers in a large population of blood donors, HCV-related antibodies were measured in 919 donor serum samples using three different enzyme-linked immunosorbent assays. The antibodies were C100 and KCL-163, nonstructural proteins of HCV, as well as JCC, a translation product of the presumptive HCV core gene. Fourteen (1.5%), 12 (1.3%), and 13 (1.4%) specimens were positive for anti-C100, anti-KCL-163, and anti-JCC, respectively. HCV RNA was detected by the polymerase chain reaction in seven (25.0%) of the 28 specimens that were anti-HCV-positive by at least one of the three assays. Four of the seven specimens were detected by anti-C100 screening, while the remaining three were not. All seven specimens were positive for KCL-163 and/or JCC antibodies. These findings suggest that screening for both KCL-163 and JCC antibodies may be of particular use in accurately identifying HCV-positive blood.

Adult↗
PubMed1992

Interferon-inducible gene expression in chimpanzee liver infected with hepatitis C virus.

The molecular host response to hepatitis C virus (HCV) infection was examined by isolation of HCV-induced genes from a cDNA library constructed from chimpanzee liver during the acute phase of hepatitis C. Two cDNA clones, 130-7 and 130-51, were obtained by differential hybridization with cDNA probes prepared from poly(A)+ RNAs of infected and uninfected livers. Northern blot analysis revealed that the 130-7 and 130-51 cDNAs were expressed as 1.5- and 1.0-kb products, respectively, in chimpanzee liver and that their induction rates of the two were 20 and 4, respectively. Nucleotide sequence analyses of these cDNA inserts showed that the sequence of cDNA 130-7 was that of a class I major histocompatibility antigen and that the sequence of cDNA 130-51 was 98% homologous with a human interferon-inducible mRNA. These results suggest that HCV infection may actively induce interferon, which in turn induces the expressions of these interferon-inducible genes. Furthermore, the high expression of HLA class I antigen in the acute phase of hepatitis C suggests that liver cell injury in HCV infection may be mediated by cytotoxic T cells that recognize viral antigen in association with HLA class I antigen.

Animals↗
PubMed1992

Hepatitis E virus: cDNA cloning and expression.

Viral hepatitis E is endemic, frequently provoking epidemic outbreaks in many developing countries. We have attempted to clone the viral genome and to develop an antibody assay system. A lambda gt11 cDNA library was constructed from the bile juice containing putative causative viruses and was immunoscreened by the antisera obtained from patients and monkeys infected with hepatitis E. Three virus-specific clones were isolated and were revealed to overlap one another in sequence, with 1,459 nucleotides in total length. These clones direct the synthesis of polypeptides probably having common immunological epitope(s). Immunoplaque assay revealed the occurrence of antibodies against this epitope in the sera from experimental monkeys with the convalescent phase and from patients of Myanmar, Nepal and India. The data indicate that the cDNA fragments are useful for immunodiagnosis of hepatitis E.

Amino Acid Sequence↗
PubMed1992

Sequence comparison of the capsid region of hepatitis E viruses isolated from Myanmar and China.

Hepatitis E viruses (HEVs) were isolated during epidemics, one from Myanmar (formerly called Burma) and one from China and were partially sequenced. Another HEV Myanmar strain from sporadic hepatitis was previously sequenced by us. A cDNA sequence comparison was performed among them in the 3'-terminal region, approximately 750-base long. This region contained at least two immunological epitopes and was considered to correspond to the structural protein. The nucleotide sequence identity was 97.2% between the two Myanmar strains and 93.3 and 92.5% between the two Myanmar and the China strain. The deduced amino acid sequence identity ranged from 98.4 to 100.0% among the three strains. Thus this segment was well conserved on the amino acid level among the different strains isolated from these two Asian countries, although the China strain diverged more from the Myanmar strains on the nucleotide sequence level. This data may provide important information for the development of a vaccine and for identification of the virological link between different geographical locations.

Amino Acid Sequence↗
PubMed1991

Oncogene expression in the liver tissue of patients with nonneoplastic liver disease.

The expression of cellular oncogenes in nonneoplastic human liver tissue was examined to determine if there was a correlation between oncogene expression and physiologic regeneration in liver disease. Human liver tissue specimens from 70 patients with various histologic findings from almost normal to cirrhosis were examined (using northern blot analysis) for the expression of nine cellular oncogenes. With c-K-ras, four RNA bands (5.6-kilobase [kb], 2.1-kb, 1.5-kb, and 1.2-kb RNA species) were detected in all liver tissue examined. Expression of c-fos was also detected in a few samples examined when 50-micrograms samples of total RNA were applied. Other oncogenes such as H-ras, myc, erbB, raf, fms, fes, and myb were not detected. These results indicate that particular oncogene(s) may not be highly expressed during liver regeneration in human liver disease, or that populations of regenerating hepatocytes may be too small to show significant elevations of oncogene expression. The new finding of a constant expression of c-K-ras in human liver tissue suggests that it is linked to essential hepatocellular function rather than carcinogenesis.

Adult↗
PubMed1991

Animal model, virology and gene cloning of hepatitis E.

We have developed animal models of viral hepatitis E using cynomolgus and rhesus monkeys. They developed acute biochemical and histological hepatitis after the inoculation of virus particles with identical kinetics and magnitude for the sixth subpassage. Virus particles multiplied in hepatocytes and were excreted into feces via bile. Additionally, a transient viremia was recognized. Molecular cloning of virus gene cDNA was successfully accomplished from two separate libraries (HT3 and NE). These clones were expressed into polypeptides having immunological epitopes, which were used for antibody assay of sera of monkeys and patients with positive results.

Animals↗
PubMed1991

In vitro transcription of the hepatitis B virus gene by nuclear extracts of human hepatoma cells.

In vitro transcription of hepatitis B virus DNA (HBV DNA) was studied using nuclear extracts of human hepatoma cell lines. RNA polymerase II-dependent run-off transcription of pre-S mRNA under the control of pre-S1 promoter was observed in nuclear extracts obtained from HepG2 and PLC/PRF/5 cells, and the efficiencies in these extracts were significantly higher than those in nuclear extracts of non-liver cells such as HeLa, Molt-4, and Ehrlich. Analysis of run-off transcripts by the pre-S1 promoter, using deletion mutants of HBV DNA as templates and synthetic oligonucleotides as competitors, showed that hepatocyte nuclear factor 1 was necessary for initiation of in vitro transcription of pre-S mRNA. The run-off transcript of pregenome RNA was also detected and its initiation site was determined. Nuclear extracts of not only hepatoma cells but non-liver cells were active in transcription of pregenome RNA in vitro. However, run-off transcripts of S mRNA and X mRNA were not observed in this system. These results suggest that there were some differences between the mechanisms of HBV DNA transcription in vitro and in vivo. This in vitro transcription system will be useful for clarifying the mechanism regulating transcription of HBV DNA since the biochemical and functional characteristics of the nuclear factors can readily be analyzed.

Base Sequence↗
PubMed1991

Cloning and expression of cDNAs from enterically-transmitted non-A, non-B hepatitis virus.

The fragment gene of enterically-transmitted non-A, non-B hepatitis virus (ET-NANBHV) was cloned as a cDNA and inserted into an expression vector pUEX2. The recombinant protein was expressed in Escherichia coli HB101 as a fusion protein with beta-galactosidase (beta-Gal). The fusion protein reacted with the sera of infected cynomolgus monkeys and of patients from Myanmar. This reaction was highly related with ET-NANBHV infection, and obviously demonstrates in that the recombinant protein can be used for the detection of ET-NANBHV infection.

Amino Acid Sequence↗
PubMed1991

Transplantation studies on human and duck hepatocytes in athymic nude mice.

For determination of the most suitable tissue for heterotopic transplantation of exogenous hepatocytes, dissociated hepatocytes or small pieces of liver tissue were transplanted into the spleen, adipose tissue and inside the capsule of the kidney of BALB/c mice. Survival of syngeneic grafts of dissociated hepatocytes was highest in the spleen and that of pieces of liver tissue in the adipose tissue, but only the latter system was suitable for xenogeneic transplantation. Histological examination showed that a total of 50% of the human or duck liver tissue implants survived in the inguinal fat pad of athymic nude mice (BALB/c-nu). Histochemical analyses revealed that most hepatocytes transplanted into the fat pad gave positive reactions for glucose-6-phosphatase and with periodic acid-Schiff reagent at least 28 days after transplantation. Electron microscopic observation showed that these cells also maintained characteristic cellular organelles. This xenogeneic transplantation into adipose tissue should be useful in the studies on replication and infection of human hepatotropic viruses such as hepatitis B and C viruses.

Adipose Tissue↗