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Biomedical subjects

T Miyamura

Publications and source records attributed to T Miyamura.

At least 73 records · Page 4Linked to original sources
PubMed1999

Processing and functions of Hepatitis C virus proteins.

Hepatitis C virus (HCV) has a positive-stranded RNA genome of about 9.5 kb and a large open reading frame encoding a precursor polyprotein of ca. 3,000 amino acids (aa). This polyprotein is cleaved by host cellular signalase(s) and viral proteases into 10 viral proteins in the order of NH(2)-Core-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS 5B-COOH. Core and E1/E2 are considered to be a capsid protein and envelope glycoproteins, respectively. NS2-NS5B are putative nonstructural proteins involved in the replication of HCV. NS2/3 is a metalloprotease which cleaves in cis at the NS2/3 junction. NS3 possesses serine protease and RNA helicase activities and is responsible for the cleavage of the remaining nonstructural proteins. NS4A is suggested to be a cofactor for NS3 protease. Although the function of p7, NS4B and NS5A are still unknown, an association of a mutation in NS5A with a susceptibility to interferon (IFN) has been reported. NS5B possesses an RNA-dependent RNA polymerase activity. Most of the current findings in HCV proteins depend on expression studies of HCV cDNA clones because of the lack of an efficient replication system in cell cultures. Therefore, a final assignment of cleavages and functions of HCV proteins has to await the propagation of HCV in cell cultures.

Genome, Viral↗
PubMed1999

[Sex differences in subjective well-being and related factors in elderly people in the community aged 75 and over].

Sex differences in factors related to subjective well-being were evaluated in people in their late old age by interviewing individuals aged 75 years and over living in 2 regions of Enzan City, Yamanashi Prefecture. The 17-item revised "Philadelphia Geriatric Center (PGC) Morale Scale" was used for evaluation of subjective well-being. Factors related to family status, employment, health related factors, activities of daily living, and results of physical examinations were each classified into two or more categories, and PGC Morale Scale points were compared among the categories for all subjects and separately for males and females using the Mann-Whitney test and the Kruskal-Wallis test. 1) Although the mean age of males (80.0 +/- 4.4 years) and females (80.4 +/- 4.3 years) was no significantly difference, PGC Morale Scale points were significantly higher in males than in females (p < 0.05), indicating a sex difference in subjective well-being. 2) In elderly females, subjective well-being was related to many factors, and there was a particularly strong relationship between subjective well-being and both health related factors and activities of daily living. 3) In elderly males, the factors related to subjective well-being were fewer than in females, consisting only of "social opportunities", "hobbies", and "grip strength". Since factors related to subjective well-being differ between the sexes, these differences must be taken into account when evaluating subjective QOL of the elderly.

Aged↗
PubMed1999

Genetic analysis of wild polioviruses towards the eradication of poliomyelitis from the Western Pacific Region.

Since a laboratory network to eradicate poliomyelitis in the Western Pacific Region started in 1991, the Department of Virology II, National Institute of Infectious Diseases, has been functioning as a Regional Reference Laboratory. From 1992 to 1998, we examined 5453 stool samples collected from 3501 patients with acute flaccid paralysis in Cambodia, Vietnam, and Laos, and we isolated 392-type 1 and 10-type 3 wild polioviruses. As a result of the extensive immunization during this time in this area, the numbers of poliomyelitis cases by wild polioviruses have drastically decreased. In 1997, only nine type 1 wild polioviruses were isolated. Eight out of the nine cases were found in Cambodia, and one was in mid-Vietnam. Since then no wild polioviruses have been isolated in the Western Pacific Region for more than two years. A nucleotide sequence analysis of these 1997 isolates indicated that they all belonged to the same strain that has been prevailing in the Indochina area, suggesting the complete interruption of wild poliovirus transmission in the region.

Acute Disease↗
PubMed1999

Phylogenic analysis of echovirus type 30 isolated from a large epidemic of aseptic meningitis in Japan during 1997-1998.

During 1997 to 1998, a nationwide epidemic of aseptic meningitis occurred in Japan. More than 4,500 isolates from patients with aseptic meningitis were identified as echovirus type 30. To investigate the character of these isolates, we examined the nucleotide sequences of thirty-seven geographical representatives and compared them with 50 strains isolated during the past 20 years. The phylogenic analysis used partial sequences from either the VP1 or VP4-VP2 region of the viral capsid. This analysis revealed that the isolates were divided into six genomic groups. All isolates identified during 1997-1998 belonged to only two genomic groups; these two groups are thought to be the causative viral agents involved in the recent epidemic.

Enterovirus B, Human↗
PubMed1999

Enterovirus 71 from fatal and nonfatal cases of hand, foot and mouth disease epidemics in Malaysia, Japan and Taiwan in 1997-1998.

Enterovirus 71 (EV71), one of the major causative agents for hand, foot and mouth disease (HFMD), is sometimes associated with severe central nervous system diseases. In 1997, in Malaysia and Japan, and in 1998 in Taiwan, there were HFMD epidemics involving sudden deaths among young children, and EV71 was isolated from the HFMD patients, including the fatal cases. The nucleotide sequences of each EV71 isolate were determined and compared by phylogenetical analysis. EV71 strains from previously reported epidemics belonged to genotype A-1, while those from recent epidemics could be divided into two genotypes, A-2 and B. In Malaysia, genotype A-2 was more prevalent, while in Japan and Taiwan, B genotype was more prevalent. Two isolates from fatal cases in Malaysia and one isolate from a fatal case in Japan were genotype A-2. However, all isolates from three fatal cases in Taiwan belonged to genotype B. The severity of the HFMD did not link directly to certain genotypes of EV71.

DNA Primers↗
PubMed1998

A human liver cell line exhibits efficient translation of HCV RNAs produced by a recombinant adenovirus expressing T7 RNA polymerase.

An in vitro system that supports the efficient growth of hepatitis C virus (HCV) and reflects its complete in vitro replication cycle has not yet been established. The establishment of a minigene RNA of HCV in mammalian cells could facilitate the study of virus-cell interactions and the molecular pathogenesis of this virus. We constructed a replication-deficient recombinant adenovirus expressing bacteriophage T7 RNA polymerase under the control of CAG promoter (AdexCAT7). A high level of T7 RNA polymerase was detectable for at least 11 days after inoculation. Cells infected with AdexCAT7 were then transfected with plasmids carrying the authentic T7 promoter, the 5' untranslated region (UTR) of encephalomyocarditis virus, a luciferase gene, and a T7 terminator (pT7EMCVLuc) or carrying the modified T7 promoter, the 5'UTR of HCV, a luciferase gene, the coding region of C-terminal of NS5B and the 3'UTR of HCV, a ribozyme of hepatitis D virus and a T7 terminator (pT7HCVLuc). Most of the cell lines examined supported a higher expression of luciferase by transfection with pT7EMCVLuc than with pT7HCVLuc. However, one cell line, FLC4, derived from a human hepatocellular carcinoma, exhibited very high reporter gene expression with pT7HCVLuc. In this cell line, transfection with RNA synthesized in vitro from pT7HCVLuc induced a higher level of reporter gene expression than RNA from pT7EMCVLuc. The T7-adenovirus system for the synthesis of HCV minigenes in vivo provides useful information on the molecular mechanisms of HCV translation in human liver cells.

3' Untranslated Regions↗
PubMed1998

The structures of asparagine-linked oligosaccharides of rat liver cathepsin L reflect the substrate specificity of lysosomal alpha-mannosidase.

We have studied the structures of asparagine-linked oligosaccharides of cathepsin L purified from rat liver in detail. The oligosaccharides released from rat liver cathepsin L on glycopeptidase-F treatment were tagged with 2-aminopyridine at their reducing ends. The pyridylamino (PA) derivatives were separated into seven fractions according to molecular size by normal-phase HPLC. The structure of each oligosaccharide thus isolated was analyzed by reversed-phase HPLC and characterized by ion-spray mass spectrometry and high-resolution proton nuclear magnetic resonance (1H-NMR) spectroscopy. Our results indicate that the asparagine-linked oligosaccharides of rat liver cathepsin L are of the oligomannose type, having two to six mannose residues. Among them, the five major ones are Manalpha1-6Manbeta1-4-GlcNAcbeta1-4GlcNAc, Manalpha1 -6Manalpha1-6Manbeta1-4GIcNAcbeta1-4GlcNAc, Manalpha1-6(Manalpha1-3)-Manalpha1-6Manbeta1- 4GlcNAcbeta1-4GlcNAc, Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3) Manbeta1-4Glc-NAcbeta1-4GlcNAc, and Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-++ +2Manalpha1-3)Manbeta1-4GlcNAcbeta1-4Glc-NAc. Their structures are shown to be products of Man6GlcNAc2 hydrolysis with lysosomal alpha-mannosidase.

Animals↗
PubMed1998

Low stimulatory capacity of lymphoid dendritic cells expressing hepatitis C virus genes.

To determine the role of antigen presenting cells (APCs) during hepatitis C virus (HCV) infection, murine lymphoid dendritic cells (LDCs), the most potent APCs, were transfected with HCV genes using adenovirus vector-mediated gene transfer. At a multiplicity of infection (MOI) of 100, more than 90% of the transfected LDCs expressed the HCV genes (encoding core-E2 region). The stimulatory capacity of these LDCs (LDC-AxCA327) in the allogeneic mixed leukocyte reaction (Allo MLR) was significantly lower (P < 0.01) than that of LDCs infected with control adenovirus vector lacking the HCV genes (LDC-Axw1). LDC-AxCA327 also produced significantly lower levels of IL-12 than LDC-Axw1 (P < 0.05). These findings suggest that the inadequate APC capability of LDCs that express HCV genes is related to immunopathology during HCV infection and that IL-12 appears to be involved in this process.

Animals↗
PubMed1998

Critical point mutations for hepatitis C virus NS3 proteinase.

The hepatitis C virus NS3 proteinase plays an essential role in processing of HCV nonstructural precursor polyprotein. To detect its processing activity, we developed a simple trans-cleavage assay. Two recombinant plasmids expressing the NS3 proteinase region and a chimeric substrate polyprotein containing the NS5A/5B cleavage site between maltose binding protein and protein A were co-introduced into Escherichia coli cells. The proteinase processed the substrate at the single site during their polyprotein expression. Deletion analysis indicated that the functionally minimal domain of the NS3 proteinase was composed of 146 amino acids, 1059 to 1204. We isolated several cDNA clones encoding the functional domain of the NS3 proteinase from the sera of patients chronically infected with HCV and determined their proteinase activity by this trans-cleavage assay. Both active and inactive clones existed in the same patients. Comparative sequence analyses of these clones suggested that certain point mutations seemed to be related to the loss of proteolytic activity. This was confirmed by back mutation experiments. Among the critical mutations, Pro-1168 to Thr and Arg-1135 to Gly were intriguing. These amino acids, which are situated near the oxyanion hole, seem to be essential for maintaining the conformation of the active center of the NS3 proteinase.

Amino Acid Sequence↗
PubMed1998

Metabolic labeling of a subset of glial cells by UDP-galactose: implication for astrocyte lineage diversity.

Astrocytes are implicated in many aspects of brain function; however, it remains unclear whether astrocytes arise from a single cell lineage. It is therefore important to obtain new markers for the astrocyte cell lineage. We show that exogenously added UDP-galactose (UDP-Gal) can be used to metabolically label a subset of glial fibrillary acidic protein-positive (GFAP+) cells. UDP-Gal was incorporated into the cultured embryonic mouse brain slices in a time-dependent manner. Surprisingly, the transferred sugar moiety was no longer Gal but was mainly glucose. Most of the radioactivity was transferred to a polymer of glucose, most likely to be glycogen, and also to glucosyl ceramide. In the slice culture, the reaction products were distributed densely in the ventricular zone and also on process-like structures extending to the pial surface. In dissociation culture, UDP-Gal labeled some of the GFAP+ cells and some of the vimentin+ cells. Because radial glial cells (RGCs) contain glycogen and change from vimentin+ to GFAP+, it is strongly suggested that UDP-Gal labeled RGCs and their descendants. Only 27% of the GFAP+ cells were labeled with UDP-Gal, which suggests that only a subset of astrocytes are derived from RGCs and that there is a discrete group of GFAP+ cells that is not generated from RGCs.

Animals↗
PubMed1998

Full-length complementary DNA of hepatitis C virus genome from an infectious blood sample.

We constructed a full-length complementary DNA (cDNA) clone of hepatitis C virus (HCV) from a blood sample of an HCV carrier. The blood from the carrier was eventually transfused to a patient who later developed typical posttransfusion hepatitis C. It was also shown to be infectious to chimpanzees. We obtained 12 overlapping cDNA fragments altogether, covering the entire HCV genome. By subcloning and sequencing, clones considered to constitute the major population were selected. We could also detect 98 base pairs of extra sequences at the 3' end of the genome. After confirming the overlapping sequences, we combined the fragments to make a full-length cDNA. The HCV population in the donor was heterogeneous, as determined by their nucleotide sequences of the hypervariable region in envelope protein, but a few virus clones were selected in the recipient after transmission. The similar convergence of the virus population was previously observed when the same blood sample was injected into a chimpanzee. Interestingly, virus clones isolated during the acute phase in the recipient and the chimpanzee had sequences in the hypervariable region identical to that of the full-length cDNA clone. The full-length cDNA clone of HCV constructed in this study may originate from infectious virus clones.

Amino Acid Sequence↗
PubMed1998

High titers of antibodies inhibiting the binding of envelope to human cells correlate with natural resolution of chronic hepatitis C.

Most cases of hepatitis C virus (HCV) infection result in chronic disease; however, a very small fraction of patients naturally clear the virus and resolve chronic hepatitis. In an attempt to correlate immune response with chronic disease resolution, we compared the antibody response in patients with different outcomes of the infection. Antibody responses to HCV structural proteins were assessed in 34 patients originally diagnosed with acute hepatitis. Five cases resolved acute infection, 22 developed chronic hepatitis, and 7 naturally resolved chronic hepatitis C. To estimate HCV neutralizing antibodies we used the neutralization of binding (NOB) assay, which evaluates inhibition of the envelope-2 protein binding to human cells. Enzyme-linked immunosorbent assay was used for the quantitative assessment of serum antibodies. The presence of HCV RNA was ascertained by reverse transcription-polymerase chain reaction. In 6 of 7 patients naturally recovered from chronic hepatitis C, the emergence and the persistence (for more than 3 months) of high serum titers (>1/600) of NOB antibodies coincided with virus clearance and clinical resolution of hepatitis. NOB antibody activity was observed in only 2 of 5 patients recovered from acute hepatitis C. Chronic patients who did not show any resolution during the course of the study developed low or no NOB antibodies. Because of the correlation between prolonged high NOB titers and natural resolution of chronic hepatitis C, vaccination or passive immunization aimed at high titers of NOB antibodies may be valuable new therapeutic approaches for chronic hepatitis C.

Adult↗
PubMed1998

The core protein of hepatitis C virus induces hepatocellular carcinoma in transgenic mice.

Hepatitis C virus (HCV) is the main cause of chronic hepatitis worldwide. Chronic hepatitis ultimately results in the development of hepatocellular carcinoma (HCC). However, the mechanism of hepatocarcinogenesis in chronic HCV infection is still unclear. The ability of the core protein of HCV to modulate gene transcription, cell proliferation and cell death may be involved in the pathogenesis of HCC. Here, we report the development of HCC in two independent lines of mice transgenic for the HCV core gene, which develop hepatic steatosis early in life as a histological feature characteristic of chronic hepatitis C. After the age of 16 months, mice of both lines developed hepatic tumors that first appeared as adenomas containing fat droplets in the cytoplasm. Then HCC, a more poorly-differentiated neoplasia, developed from within the adenomas, presenting in a 'nodule-in-nodule' manner without cytoplasmic fat droplets; this closely resembled the histopathological characteristics of the early stage of HCC in patients with chronic hepatitis C. These results indicate that the HCV core protein has a chief role in the development of HCC, and that these transgenic mice provide good animal models for determining the molecular events in hepatocarcinogenesis with HCV infection.

Animals↗
PubMed1998

Two major antigenic polypeptides of molluscum contagiosum virus.

A library of molluscum contagiosum virus (MCV) transferred into the cowpox vector expression system was screened with 12 sera from molluscum patients. Two recombinant proteins of 70 and 34 kDa were detected by immunoblotting and mapped to the open-reading frames MC133L and MC084L, respectively. Consensus sites were found between the C-terminus of the 70-kDa MCV protein and the 14-kDa fusion protein of vaccinia and variola virus, and between the 34-kDa MCV protein and the 37.5-kDa viral membrane-associated protein of vaccinia and variola virus. Rabbit antisera against these two proteins were prepared. An immunofluorescence study demonstrated that the 70- and 34-kDa proteins were predominantly expressed on the surface of recombinant virus-infected HeLa cells, indicating the potential to be inserted into the membrane. On immunoelectron microscopy, antiserum against 70-kDa protein showed significant labeling of the MCV membrane, while the antiserum against 34-kDa protein failed to do so.

Amino Acid Sequence↗
PubMed1998

Expression of target genes by coinfection with replication-deficient viral vectors.

An in vivo transcription system was developed by coinfection of cells with replication-deficient viral vectors. Recombinant baculovirus (AcT7HCVLuc) and fowlpox virus (FPVT7HCVLuc) carrying a cDNA of the hepatitis C virus (HCV) minigene encoding the HCV 5' untranslated region (UTR), a luciferase gene and the 3' UTR, including the 98 nt extra sequence, under the control of the T7 promoter were constructed. The HCV minigene was synthesized in various cells by coinfection with one of these two viruses and recombinant baculovirus (AcCAT7) or adenovirus (AdexCAT7) expressing T7 RNA polymerase under the control of a mammalian promoter. Only a low level of luciferase expression was obtained in cells coinfected with AcT7HCVLuc and either AcCAT7 or AdexCAT7. In contrast, high-level luciferase expression was detected when the same cells were coinfected with FPVT7HCVLuc and either AcCAT7 or AdexCAT7. We further constructed a recombinant fowlpox virus with its HCV minigene extended to contain the whole HCV core protein region. Significantly high levels of expression of HCV core protein were detected in MT-2, COS7 and Vero cells by coinfection with the recombinant fowlpox virus and AdexCAT7. A coinfection system consisting of recombinant fowlpox virus and AdexCAT7 was established for high level of expression of a target gene in various cells.

Adenoviridae↗
PubMed1998

Complete nucleotide sequence and genetic organization of Aichi virus, a distinct member of the Picornaviridae associated with acute gastroenteritis in humans.

The complete nucleotide sequence of a novel enteric virus, Aichi virus, associated with nonbacterial acute gastroenteritis in humans was determined. The Aichi virus genome proved to be a single-stranded positive-sense RNA molecule with 8,251 bases excluding a poly(A) tail; it contains a large open reading frame with 7,302 nucleotides that encodes a potential polyprotein precursor of 2,433 amino acids. The genome contains a 5' nontranslated region (NTR) with 712 bases and a 3' NTR with 240 bases followed by a poly(A) tail. The structure of the genome, VPg-5' NTR-leader protein-structural proteins-nonstructural proteins-3' NTR-poly(A), was found to be typical of a picornavirus. The VP0-VP3 and VP3-VP1 cleavage sites were determined to be Q-H and Q-T, respectively, by N-terminal amino acid sequence analyses using purified virion proteins. Possible cleavage sites, Q-G, Q-A, and Q-S, which cleave P2 and P3 polyproteins were found to be similar to those of picornaviruses. A dendrogram based on 3Dpol proteins indicated that Aichi virus is genetically distinct from the known six genera of picornaviruses including entero-, rhino-, cardio-, aphtho-, and hepatovirus and echovirus 22. Considering this together with other properties of the virus (T. Yamashita, S. Kobayashi, K. Sakae, S. Nakata, S. Chiba, Y. Ishihara, and S. Isomura, J. Infect. Dis. 164:954-957, 1991), we propose that Aichi virus be regarded as a new genus of the family Picornaviridae.

Acute Disease↗