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Biomedical subjects

T Shikata

Publications and source records attributed to T Shikata.

At least 37 records · Page 2Linked to original sources
PubMed1995

Complete nucleotide sequences and the characteristics of two hepatitis B virus mutants causing serologically negative acute or chronic hepatitis B.

Hepatitis B virus (HBV) DNA was amplified by the polymerase chain reaction from the sera of a patient with acute hepatitis and a patient with chronic hepatitis. Both patients were negative for serum hepatitis B surface antigen and hepatitis B core antibodies and had been previously diagnosed as non-A, non-B, non-C, non-D, non-E hepatitis. The nucleotide sequence revealed an 8-nucleotide deletion in the X-gene coding region creating a C-terminally truncated X protein, and probable mutation of the enhancer II/core promoter element. In addition, DR2 showed a T-to-C mutation at the extreme 5'-terminus. These mutations within the X-gene coding region must suppress replication and expression of HBV DNA, and this seems to be responsible for absence of serological markers despite the presence of HBV infection.

Acute Disease↗
PubMed1995

Nucleotide sequence of hepatitis B virus isolated from subjects without serum anti-hepatitis B core antibody.

The nucleotide sequences of the precore/core and X open reading frames (ORFs) of hepatitis B virus (HBV) were studied in four subjects who were serologically negative for anti-hepatitis B core antibody. These subjects were positive for serum hepatitis B surface antigen and were considered to be asymptomatic HBV carriers. Sequencing of the precore/core ORF revealed precore wild type and 3 to 8 nucleotide substitutions (replacing 0 to 2 amino acids) in the core region compared with the sequence of subtype adr. These substitutions were not considered to have changed the epitope of the core antigen, resulting in the absence of anti-HBc as determined by a conventional diagnostic kit. The X ORF showed 1 to 5 nucleotide substitutions (replacing 1 to 3 amino acids) and the structure of the X protein and the core promoter/enhancer II complex appeared to be conserved. These findings strongly suggest that the absence of serum anti-HBc is not due to mutation of the HBV DNA but to an aberrant immune reaction of the host to HBV.

Adult↗
PubMed1995

Probable implication of mutations of the X open reading frame in the onset of fulminant hepatitis B.

A pathogenic role of precore-defective mutation in the onset of fulminant hepatitis B has been suggested. However, precore-defective mutants do not always cause fulminant hepatitis B and are not always isolated from affected patients. These findings strongly suggest the presence of some additional important mutations outside the precore region in fulminant hepatitis. In the present investigation an attempt was made to sequence the X open reading frame of hepatitis B virus DNA isolated from seven patients with fulminant hepatitis B and five patients with acute hepatitis B. The latter were used as controls. Since the X open reading frame encodes the X protein and contains the core promoter/enhancer II complex, some critical mutations may enhance or disrupt the replication and expression of hepatitis B virus DNA leading to fulminant hepatitis. A C-to-T substitution was found at nucleotide (nt) 1655, an A-to-T substitution at nt 1764 and a G-to-A substitution at nt 1766 in 4, 5 and 5 patients, respectively, out of the seven with fulminant hepatitis. These substitutions were not recognized in the patients with acute hepatitis. These mutations might change the function of the X protein and core promoter/enhancer II complex. It is suggested, therefore, that these mutations, as well as the precore-defective mutation, may play an important role in the pathogenesis of fulminant hepatitis.

Acute Disease↗
PubMed1995

Defective copper binding to apo-ceruloplasmin in a rat model and patients with Wilson's disease.

To examine the mechanism of decrease in serum ceruloplasmin (Cp) in Long-Evans Cinnamon (LEC) rats, a proposed model of Wilson's disease, we analyzed Cp products at the stages of transcription and translation. Northern blot analysis and immunoblot analysis showed that the level and the molecular size of Cp mRNA and protein in LEC rats were similar to those in control Long-Evans-Agouti (LEA) rats. However, the ferroxidase activity of Cp was significantly decreased in LEC rats. We separated serum Cp into two forms by native polyacrylamide gel electrophoresis with pH modification: one was a holo-Cp with copper and ferroxidase activity, and the other was an inactive apo-Cp without copper. Holo-Cp was the predominant form in LEA rats and normal humans, whereas apo-Cp was the major form in LEC rats and patients with Wilson's disease. The cosegregation of apo-Cp predominance with the disease in LEC rats was analyzed using backcross rats. Apo-Cp was dominant in 8 of 11 offspring with disease but in none of 19 normal offspring. These results indicate that a genetic disturbance of copper binding to apo-Cp may be closely associated with the pathogenesis in LEC rats, and probably in Wilson's disease.

Animals↗
PubMed1994

Completely or nearly identical hepatitis B virus strains replicate between patients with acute or fulminant hepatitis B and their respective infectious sources.

Five patients with acute hepatitis B and four with fulminant hepatitis B were selected for sequencing of the precore/core gene of the virus strains. Furthermore, identical sequencing was done with the HBV of the infectious sources, i.e., the sexual partner in eight cases and a natural child (chronic carrier) infecting the mother in one case. Of the subjects responsible for the infection, four were healthy HBV carriers, three suffered from chronic hepatitis B, and one from acute and one from fulminant hepatitis B. The nucleotide sequences of HBV from both the patients and the implicated sources of infection exhibited perfect identity of the precore region and perfect or high identity of the core region. The completely or nearly identical strain of virus seemed to proliferate successively in the patients following the transmission from the infecting individuals regardless of sequence variations and infectious status. In two cases a peculiar pattern of infection and disease was found: In one married couple the husband, during the incubation period of acute hepatitis B, infected his wife, who developed fulminant hepatitis. In another married couple, both partners ultimately developed fulminant hepatitis (the wife being the source of the infection).

Acute Disease↗
PubMed1994

Evolution of the hepatitis B virus gene during chronic infection in seven patients.

We analyzed the evolution of the precore/core gene of hepatitis B virus (HBV) over a period of 6 to 11 years in seven patients with chronic HBV infection, who exhibited a variety of clinical features. Sequence analysis revealed the following results: (1) HBeAg to anti-HBe seroconversion was correlated roughly with the occurrence of precore-defective mutants, and several years appeared to be required for complete replacement of wild types by mutants; (2) core gene mutations preceded precore-defective mutations and tended to increase with time, although not cumulatively. They occurred not only during serum alanine aminotransferase (ALT) elevations but also after ALT returned to normal; (3) ALT fluctuations appeared to subside with complete replacement of the wild type by the mutant type and/or substantial accumulation of core gene mutations; (4) unexpectedly, the anti-HBe-positive "healthy" carrier was found to harbor the wild type precore gene, as did the HBeAg-positive "healthy" carrier; however, the core gene of the former evolved at a rapid rate; and (5) a partial deletion was recognized in the core gene at the onset of fatal hepatic failure in one patient. Thus, the precore/core mutation was closely related with the clinical features in the patients.

Adolescent↗
PubMed1994

Analysis of upstream region of hepatitis B virus core gene using in vitro transcription system.

Transcription of the core (C) gene of hepatitis B virus DNA (HBV-DNA) was studied by an in vitro transcription system using nuclear extracts of human liver cell (HepG2) and non-liver cell (HeLa) origins. RNA polymerase II-dependent run-off transcription of 3.5-kb (C) mRNA was observed in both nuclear extracts; but the efficiency was much higher in the HepG2 nuclear extract. Analysis of run-off transcripts using upstream deletion mutants of HBV-DNA demonstrated that there are two transcription start sites located at approximately nucleotide numbers (nt) 1,797 +/- 5 and 1,815 +/- 5. This analysis also suggested that the minimum core promoter sequence and a cis-acting and liver-specific element for C mRNA transcription are located in the downstream region from -80 and -110 (HincII site) of transcription start sites, respectively. DNA-binding protein assays using synthetic double-stranded oligonucleotide probes corresponding to three regions in the upstream region (nt from 1,401 to 1,760) of transcription start sites revealed that there are some liver cell-specific and non-specific DNA-binding proteins in both nuclear extracts. The amount of those proteins was generally higher in the HepG2 nuclear extract. However, no obvious correlation was observed in the present study between the presence of DNA-binding proteins and transcription activity of nuclear extracts in our system. The possible causes of this discrepancy are discussed.

Base Sequence↗
PubMed1994

Immunoperoxidase staining of hepatitis C virus in formalin-fixed, paraffin-embedded needle liver biopsies.

The localization of hepatitis C virus (HCV) in the liver has not been well clarified. We report successful indirect immunoperoxidase staining of the HCV core antigen using polyclonal antibodies raised in rabbits and conventional formalin-fixed, paraffin-embedded needle biopsy sections of liver. The core antigen was distributed in a fine granular pattern diffusely, perisinusoidally, or focally within the hepatocellular cytoplasm of livers from patients with HCV infection. The staining tended to show a more heterogeneous pattern in terms of intensity and distribution in cases of more advanced disease. Hepatocellular carcinoma cells were also frequently stained. HCV immunostaining will provide important information on the pathogenesis and treatment of HCV-related liver diseases.

Adult↗
PubMed1994

Variations of hepatitis B virus precore/core gene sequence in acute and fulminant hepatitis B.

Variations of the hepatitis B virus (HBV) precore/core sequence has been shown to play a role in the development of active liver disease in chronic hepatitis B. Whether this is also an important viral factor in the pathogenesis of acute and fulminant hepatitis B is unknown. To determine the precore/core gene sequence in patients with acute and fulminant hepatitis B, 11 patients with fulminant hepatitis B and seven patients with acute hepatitis B were studied. The sequences of precore/core gene were determined by direct sequencing of the polymerase chain reaction amplicons generated from the HBV isolated from patients' serum. For the 11 patients with fulminant hepatitis B, the precore/core regions were successfully amplified in 10 patients. Eight patients exhibited precore stop codon mutations. In addition, nine of the 10 fulminant hepatitis B patients had frequent nucleotide substitutions with corresponding changes in the predicted amino acid sequences in the mid-core and the 5' terminus region of the core gene. In contrast, precore stop codon mutants were not detected, and variations of the HBV core gene were minimal in patients with acute hepatitis B. The association of HBV precore mutants and HBV core gene variations with fulminant hepatitis B and not acute hepatitis B suggested that these variations may be important in modulating the clinical course of HBV infection.

Acute Disease↗
PubMed1994

"Silent" hepatitis B virus mutants are responsible for non-A, non-B, non-C, non-D, non-E hepatitis.

Since the recent introduction of diagnostic kits for hepatitis C and E, some cases of non-A, non-B, non-C, non-D, non-E hepatitis (so-called hepatitis F) have been revealed. We attempted to demonstrate that so-called hepatitis F is caused by hepatitis B virus (HBV) variants. Polymerase chain reaction (PCR) was used to amplify serum HBV DNAs from 20 patients with acute hepatitis and 20 patients with chronic hepatitis who had been diagnosed as having so-called hepatitis F on the basis of conventional serological markers. The PCR technique successfully amplified HBV DNAs in 18 (90%) cases of acute hepatitis and 17 (85%) cases of chronic hepatitis. Sequencing of HBV DNAs of six patients (acute 3, chronic 3) revealed equally a T-to-C mutation of DR2 and an 8-nucleotide deletion of the 3'-terminus of the X gene coding region, giving rise to the generation of a C-terminally truncated X protein and probable damage to the enhancer II/core promoter elements. These mutations of the X gene coding region may lead to suppression of replication and expression of HBV DNAs. Thus virtually all cases of so-called hepatitis F appear to be caused by "silent" HBV mutants, at least in Japan.

Acute Disease↗
PubMed1994

Increased expression of trk proto-oncogene by gamma-interferon in human neuroblastoma cell lines.

Three human neuroblastoma cell lines were examined to determine the effect of recombinant gamma-interferon (IFN-gamma) treatment on the expression of trk proto-oncogene. Increased levels of trk proto-oncogene mRNA were observed in two neuroblastoma cell lines (KP-N-RT and KP-N-SI(FA)) after IFN-gamma treatment. The levels of trk mRNA increased with growth inhibition and morphological change in a time- and dose-dependent manner. The decreased level of N-myc mRNA after IFN-gamma treatment in KP-N-RT was inversely correlated with trk mRNA. Our results suggest that IFN-gamma can modulate the signal transduction of nerve growth factor in human neuroblastoma cells.

Blotting, Northern↗
PubMed1994

Hepatitis C virus appears to replicate not only in hepatocytes but also in hepatocellular carcinoma cells as demonstrated by immunostaining.

Persistent infection with hepatitis C virus is a major risk factor for the development of hepatocellular carcinoma (HCC). It has been unclear whether hepatitis C virus replicates in HCC. A total of 39 hepatitis B surface-antigen-negative HCC were resected surgically at Nihon University Itabashi Hospital between January 1990 and December 1992. Of them, serum anti-hepatitis C was positive in 28 cases, negative in three and not examined in eight. The indirect immunoperoxidase technique was used for detection of the hepatitis C core antigen on formalin-fixed, paraffin-embedded sections. Positive staining was found within non-tumor hepatocytes in 22 (78.6%), one and four cases, respectively. Fourteen (58.3%) of these 24 cases, which were positively immunostained in non-tumor hepatocytes (three were excluded because of complete necrosis of HCC), were also stained for the core antigen in HCC cells. Core antigen was stained in both hepatocytes and HCC cells within the cytoplasm, diffusely or focally with varying intensity in a local or patchy distribution. Hepatitis C virus appears to replicate in HCC cells and the significance of such viral replication in hepatocarcinogenesis remains to be clarified.

Adolescent↗
PubMed1994

Hepatitis C virus and liver diseases.

Hepatitis C virus (HCV) was identified molecularly and a procedure for its diagnosis was developed. In Japan, 70-80% of all cases of chronic liver disease, including hepatocellular carcinoma, are associated with HCV infection. Hepatitis C virus is a typical RNA virus with a high mutation rate. At least six variants of HCV have been identified by their nucleotide sequences. These variants are still classified into three types each containing at least two subtypes; that is, 1a (type I) and 1b (type II), 2a (type III) and 2b (type IV), and 3a (type V) and 3b (type VI). Type 1b (type II) is the predominant HCV in Japan. Even HCV cDNA clones isolated from a single patient showed mutations of HCV, especially in envelope-coding regions. Thus HCV may change during the course of chronic hepatitis due to the high mutation rate of HCV itself and elimination of some clones by immune reactions or interferon therapy. These findings explain the higher rate of chronic HCV infection and indicate that production of an effective vaccine is difficult.

Amino Acid Sequence↗
PubMed1994

Pathology of livers infected with "silent" hepatitis B virus mutant.

We have discovered that non-A, non-B, non-C, non-D, non-E (so-called type F) acute and chronic hepatitis is caused by a hepatitis B virus (HBV) variant with mutations in the X open reading frame. This silent HBV mutant does not induce immunoserological markers. In the present investigation we attempted to elucidate the putative mechanism of hepatocellular necrosis and expression patterns of hepatitis B surface antigen (HBsAg) and core antigen (HBcAg) in biopsied liver tissue. The subjects consisted of 14 patients with acute hepatitis, 11 with chronic hepatitis and eight with liver cirrhosis, all of whom had been previously diagnosed as having so-called hepatitis F. Nine of the 14, 10 of the 11 and all eight, respectively, of the above patients exhibited significant positive immunostaining for HBsAg within their hepatocellular cytoplasm, diffusely or focally. HBcAg stained in a few hepatocellular nuclei in 24.2% of the patients. Histological features were characterized by necroinflammation, indicating immune-mediated hepatocellular necrosis. Despite the serological-marker negativity, the results of immunostaining for HBsAg and HBcAg support replication and expression of HBV DNA, though weak.

Acute Disease↗
PubMed1993

Secretion and purification of hepatitis C virus NS1 glycoprotein produced by recombinant baculovirus-infected insect cells.

Recombinant baculoviruses that produce a putative non-structural protein 1 (NS1) of hepatitis C virus (HCV), predicted to be the second envelope glycoprotein, were constructed. The recombinant NS1 protein (re-NS1) produced in infected insect cells was localized on the cell surface and was apparently glycosylated, because it was susceptible to treatment with both tunicamycin and N-glycanase. Furthermore, re-NS1 was effectively secreted into the culture supernatant when the putative NS1 signal peptide (SP) was replaced by the SP of rabies virus G protein, and the C-terminal hydrophobic region was eliminated. The secreted re-NS1 was tagged with six His residues at the C terminus and purified simply by native Ni(2+)-nitrilotriacetic acid (Ni(2+)-NTA) affinity column chromatography. An enzyme-linked immunosorbent assay (ELISA) was developed for the serological diagnosis of HC using purified re-NS1. Anti-NS1 antibody (Ab) was detected in 55 of 60 patients (92%) with chronic HC liver diseases. Thus, this ELISA for Ab directed against HCV re-NS1 produced in insect cells is useful for the detection of chronic HC patients.

Amino Acid Sequence↗