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T Miyamura

Publications and source records attributed to T Miyamura.

At least 91 records · Page 5Linked to original sources
PubMed1997

A hybrid baculovirus-T7 RNA polymerase system for recovery of an infectious virus from cDNA.

We established a hybrid baculovirus-T7 RNA polymerase system for transient expression in mammalian cells. Two recombinant baculoviruses carrying cDNA of bacteriophage T7 RNA polymerase, with or without a nuclear localization signal, under the control of a mammalian promoter were constructed. High level expression of T7 RNA polymerase was observed in various mammalian cell lines after infection with the recombinant baculoviruses. After transfection of plasmids containing the luciferase gene under the control of the T7 promoter, high luciferase activity was detected in cells infected with the recombinant baculoviruses. We also constructed a plasmid containing an entire cDNA clone of type 1 poliovirus under the T7 promoter. Two days after transfection of the plasmid into the cells infected with the recombinant baculoviruses, a high titer of poliovirus was recovered. The use of the recombinant baculoviruses did not cause any cytopathic effects even at a high multiplicity of infection. The lack of replication ability and low toxicity are the advantageous features of the hybrid baculovirus-T7 polymerase system in comparison with the widely used vaccinia-T7 polymerase system for gene expression and recovery of infectious viruses from its cDNA.

Animals↗
PubMed1997

Sensitization to Fas-mediated apoptosis by hepatitis C virus core protein.

We have characterized viral-cell interactions of hepatitis C virus (HCV) and liver cells to study the pathogenesis of HCV infection. HepG2 cells constitutively expressing HCV core protein showed apoptotic changes in response to stimulation with anti-Fas monoclonal antibody. Cells treated with the antibody showed extensive cell rounding, shrinkage, and cytoplasmic blebbing and finally detached from plates. Fragmentation of the chromatin was observed in the nucleus and DNA ladders were detected. In contrast, cells expressing HCV envelope, nonstructural proteins or normal HepG2 cells did not exhibit such Fas-mediated apoptosis. However, expression of Fas receptor was not upregulated on the surface of the cells expressing HCV core protein. Apoptotic cell death was prevented by pretreatment with a specific inhibitor of the cysteine protease CPP32, while the specific inhibitor of interleukin-1 beta-converting enzyme did not show the preventive effect. The results suggest (i) that intracellular expression of HCV core protein makes cells prone to apoptotic death without upregulation of surface Fas expression and (ii) that the CPP32 protease plays a part in the apoptosis effector pathway of HCV core-expressing cells. HCV core protein may have a role in immune-mediated liver cell injury.

Apoptosis↗
PubMed1997

The N-terminal region of NS3 serine proteinase of hepatitis C virus is important to maintain its enzymatic integrity.

Hepatitis C virus codes a serine proteinase in nonstructural protein 3 (NS3) to produce viral replicative machinery. Recently, we reported that the activity of NS3 proteinase (region 1050-1214) was efficiently inhibited by some chelators. Kinetic analysis revealed that its K(m) value was 3.9 mM. In contrast, an enzyme covering region 1027-1214 (including N-terminal region of NS3) was found to show an improved K(m) of 0.3 mM and a remarkably reduced susceptibility to EDTA. These results suggest that the N-terminal region of NS3 is not essential for the proteinase activity but indispensable to maintain its structural integrity.

Amino Acid Sequence↗
PubMed1997

Hepatitis C virus core protein shows a cytoplasmic localization and associates to cellular lipid storage droplets.

There is now abundant evidence to substantiate an important role of hepatitis C virus (HCV) core protein in cellular gene expression as well as in the viral cycle. Thus the subcellular localization of this protein has important implications. However, several studies have shown controversial results: the HCV core has been, indeed, described as cytoplasmic or nuclear depending on the size of the protein or on the genotype analyzed. We have studied the localization of the HCV core protein in two different cell lines, one nonhepatic (CHO) and the other hepatic (HepG2). Double immunofluorescence staining using a nuclear membrane marker and confocal analysis showed the core protein pattern to be cytoplasmic and globular. This pattern is not cell cycle-regulated. Electron microscopy analysis revealed the nature of the globular staining observed in immunofluorescence. The HCV core protein accumulated at the surface of lipid droplets that were also the unique morphological feature of nonhepatic core transfected cells. The lipid droplets were isolated by sequential ultracentrifugation on the basis of their density; biochemical analysis revealed a prevalence of triglycerides. In addition the core protein colocalized with apolipoprotein AII at the surface of the lipid droplets as revealed by confocal microscopy. Moreover analysis of liver biopsies from chronically HCV-infected chimpanzees revealed that HCV core is cytoplasmic and localized on the endoplasmic reticulum and on lipid droplets. These results clearly define the subcellular localization of the HCV core protein and suggest a relationship between the expression of the HCV core protein and cellular lipid metabolism.

Animals↗
PubMed1997

Sialadenitis histologically resembling Sjogren syndrome in mice transgenic for hepatitis C virus envelope genes.

Hepatitis C virus (HCV), a major causative agent of non-A, non-B chronic hepatitis, is also suggested to be associated with extrahepatic manifestations such as mixed cryoglobulinemia and glomerulonephritis. Two independent lines of transgenic mice carrying the HCV envelope genes have been shown previously to express the HCV envelope proteins in organs, including the liver and salivary glands, which results in no pathological changes in the liver. Further analysis of these animals now has revealed that they develop an exocrinopathy involving the salivary and lachrymal glands. This pathology resembles Sjogren syndrome, which also is suggested to have a possible association with chronic hepatitis C. These observations suggest that HCV might be involved in the pathogenesis of sialadenitis in humans and that this transgenic mouse system would be a good animal model for the study of HCV infection.

Age Factors↗
PubMed1997

Interaction of BCR-ABL with the retinoblastoma protein in Philadelphia chromosome-positive cell lines.

The tyrosine kinase activity of BCR-ABL fusion proteins plays an important role in the pathogenesis of leukemia that is for the Philadelphia chromosome (Ph1). Because nuclear c-ABL is regulated during the cell cycle through a specific interaction with the retinoblastoma protein (pRB), the possible interaction of BCR-ABL with pRB in Ph1-positive cell lines was investigated. P145 c-ABL as well as P190 and P210 BCR-ABL proteins interacted with pRB. Furthermore, c-ABL and BCR-ABL associated with both phosphorylated and nonphosphorylated forms of pRB. These findings suggest that BCR-ABL interferes with pRB function and thereby regulates cell growth.

Animals↗
PubMed1997

Parvovirus B19-associated haemophagocytic syndrome with lymphadenopathy resembling histiocytic necrotizing lymphadenitis (Kikuchi's disease).

A 15-year-old girl developed a haemophagocytic syndrome caused by human parvovirus B19 (PVB19). The cervical lymph node histology, resembling that of histiocytic necrotizing lymphadenitis (HNL, Kikuchi's disease), included several transformed lymphocytes, numerous histocytes, and massive necrosis. We detected PVB19-positive cells in the lymph node by immunohistochemistry. Possible autoimmune mechanisms in HNL-like diseases triggered by PVB19 are discussed.

Adolescent↗
PubMed1997

Influence of pepsinogen gene polymorphisms on serum pepsinogen.

We identified pepsinogen C (PGC) gene polymorphisms by means of PCR, which amplified DNA in the region within the intron between exons 7 and 8, and by 6% polyacrylamide gel electrophoresis. Six alleles were found in a Japanese population. The frequencies of these alleles in 408 unrelated Japanese individuals were 0.074, 0.026, 0.335, 0.237, 0.016 and 0.314, respectively. The serum pepsinogen II level significantly decreased in the order of the allele 6 homozygote, the allele 6 heterozygote and the other genotypes (chi 2 = 7.850, D.F. = 2, p = 0.020). These findings indicated that the genetic background of serum pepsinogen should be considered when screening for stomach cancer by this procedure.

Adolescent↗
PubMed1997

Effects of interferon treatment on the antiviral T-cell response in hepatitis C virus genotype 1b- and genotype 2c-infected patients.

The viral genotype may influence the response to interferon (IFN) treatment in chronic hepatitis C virus (HCV) infection. To characterize potential mechanisms responsible for this effect, we assessed whether IFN modulation of HCV-specific T-cell responses differs in patients infected by different genotypes. The T-cell response to HCV core protein was sequentially analyzed before and during IFN treatment in two groups of patients chronically infected with HCV genotype 1b (eight patients) or 2c (eight patients). Overlapping 20 mer peptides corresponding to the amino acid sequence of the prevalent viral population identified in the serum of each patient were used for the analysis of the T-cell proliferative response to avoid possible problems caused by amino acid differences between infecting virus and HCV proteins used in vitro. Recombinant HCV core antigen was used in parallel. The level of viremia was monitored by competitive polymerase chain reaction (PCR). The T-cell response to HCV peptides and recombinant core protein detected throughout the follow-up was significantly more vigorous in genotype 2c- than in genotype 1b-infected patients. This difference was the result of a greater enhancement of the T-cell response caused by IFN treatment in genotype 2c- compared with genotype 1b-infected patients. The different IFN modulatory effect on T cells from genotype 1b- and genotype 2c-infected patients illustrates an aspect of the virus-host interaction, which may contribute toward the explanation of why different genotypes differ in responsiveness to IFN treatment.

Adult↗
PubMed1997

Two major strains of type 1 wild poliovirus circulating in Indochina.

Two hundred ninety-four isolates from 329 patients with acute flaccid paralysis in Cambodia and Vietnam during 1992-1995 were identified as type 1 wild polioviruses. Among these isolates, 85 were selected as geographic representatives and were examined by determining the nucleotide sequences of their genome in the VP1 region. The phylogenic analysis revealed that all of the isolates examined were classified into groups A and B. Isolates belonging to group A had been found only in northern Vietnam until 1993 but not in 1994 and 1995. Group B isolates were located in both northern and southern Vietnam and Cambodia. In 1994 and 1995, however, only group B isolates were found in the Mekong Delta area in southern Vietnam and Cambodia. Isolates of groups A and B were genetically different from strains previously isolated in other Asian countries. One of the two indigenous wild polioviruses still remains to be eliminated in this area.

Antigens, Viral↗
PubMed1997

Efficient gene transfer into various mammalian cells, including non-hepatic cells, by baculovirus vectors.

A baculovirus (Autographa californica nucleopolyhedrovirus) vector containing a strong promoter, the CAG promoter, was developed to introduce foreign genes into mammalian cells. Recombinant baculoviruses carrying a reporter gene under the control of the CAG promoter were inoculated into various mammalian cell lines. High-level expression was observed not only in hepatocytes but also in other non-hepatic cell lines tested. Expression of the reporter gene was detected even 14 days after infection. The infectious titre of the recovered baculoviruses decreased significantly after infection, indicating that the baculoviruses did not replicate in mammalian cells. We then compared the efficiencies of gene expression by the baculovirus vector with that of a replication-defective adenovirus vector by using the same expression unit. The same level of expression was observed in HepG2, HeLa and COS7 cells by both vectors. Efficient expression and proper processing were observed in mammalian cells infected with baculoviruses carrying genes coding for structural regions of hepatitis C virus. These results suggest that the baculovirus vector is a good tool for gene delivery into various mammalian cells in order to study the function of foreign genes.

Adenoviridae↗
PubMed1997

Hepatitis C virus core protein induces hepatic steatosis in transgenic mice.

Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide, which finally leads to development of hepatocellular carcinoma. Chronic hepatitis C is characterized by several histological features in the liver which discriminate it from other forms of hepatitis: bile duct damage, lymphoid follicles and steatosis (fatty change). Little is known, however, about the role of HCV or its viral proteins in the pathogenesis of hepatitis. Recently, the core protein of HCV has been suggested to have a transcriptional regulatory function, and thereby to be involved in inducing phenotypic changes in hepatocytes. To clarify whether or not the HCV core protein has an effect on pathological phenotypes in the liver, two independent transgenic mouse lines carrying the HCV core gene were established. These mice developed progressive hepatic steatosis, indicating that the HCV core protein plays a direct role in the development of hepatic steatosis, which characterizes hepatitis C. This transgenic mouse system would be a good animal model for the study of pathogenesis in human HCV infection.

Animals↗
PubMed1997

Expression and self-assembly of empty virus-like particles of hepatitis E virus.

Hepatitis E virus (HEV) is a pathogenic agent that causes fecally-orally transmitted acute hepatitis. The genome, a single-stranded positive-sense RNA, encodes three forward open reading frames (ORFs), in which an approximately 2-kb structural protein is located in the 3' end. To produce HEV-like particles the structural protein, with its N terminus truncated (amino acid residues 112 to 660 of ORF2), was expressed in insect Tn5 cells by a recombinant baculovirus. In addition to the primary translation product with a molecular mass of 58 kDa, a large amount of a further-processed molecule with a molecular mass of 50 kDa was generated and efficiently released into the culture medium. Electron microscopic observation of the culture medium revealed that the 50-kDa protein self-assembled to form empty virus-like particles (VLPs). The buoyant density of the VLPs in CsCl was 1.285 g/cm3 and their diameter was 23.7 nm, a little smaller than the 27 nm of native HEV particles secreted into the bile or stools of experimentally infected monkeys. The yield of the VLPs was 1 mg per 10(7) cells as a purified form. The particles possess antigenicity similar to that of authentic HEV particles and, consequently, they appear to be a good antigen for the sensitive detection of HEV-specific immunoglobulin G (IgG) and IgM antibodies. Furthermore, the VLP may be the most promising candidate yet for an HEV vaccine, owing to its potent immunogenicity.

Animals↗
PubMed1997

Comparison between the single deepest pocket and amniotic fluid index in predicting fetal distress in small-for-gestational age fetuses.

BACKGROUND: The relationship between reduced amniotic fluid volume and increased risk of fetal distress in small-for-gestational age fetuses is not yet clear. Furthermore, the criteria used for the diagnosis of 'oligohydramnios' are different between the single deepest pocket method and amniotic fluid index. We investigated the relationship between the single deepest pocket and amniotic fluid index and their ability in predicting fetal distress in small-for-gestational age fetuses. METHOD: We examined 69 pregnant women with singleton pregnancy, intact membranes, birth weight below the 50% percentile - 1.5 s.d., in whom delivery occurred within seven days of the last estimation of amniotic fluid volume. Amniotic fluid volume was measured in each woman simultaneously by single deepest pocket and amniotic fluid index. Fetal distress was determined by intrapartum monitoring of fetal heart rate. RESULTS: Estimates of amniotic fluid volume measured by the two methods showed a significant correlation at 24 to 41 weeks of gestation. A cut-off value for single deepest pocket of 3.0 cm was associated with the highest accuracy in predicting fetal distress in small-for-gestational age fetuses. The rate of cesarean section among patients with single deepest pocket <3.0 cm was higher than that in amniotic fluid index <8.0 cm. CONCLUSION: Our results demonstrated that a single deepest pocket measurement of <3 cm is the most useful criterion for 'oligohydramnios' in the prediction of fetal distress.

Amniotic Fluid↗
PubMed1997

[Relationship between knee pain and X-ray findings of osteoarthritis with reference to bone mineral density measured by computed X-ray densitometry].

We studied the relation between performance of activities of daily living and X-ray findings of osteoarthritis of the knee in aged persons. We made Covariance Structure Analysis models of knee pain, X-ray findings and bone mineral density as measured by computed X-ray densitometry. The subjects were 257 women aged from 47 to 88 years who were outpatients at an orthopedic clinic. The best-fit model indicated that loss of bone mineral density in metacarpals was associated with X-ray findings of knee-joint degeneration, as well as with knee pain. No relationship was found between knee-joint degeneration and knee pain. These results suggest that bone mineral density should be taken into consideration when interpreting X-ray findings of knee pain. This model incorporates the effects of degeneration of the subchondral bone. In summary, we should use measurements of bone mineral density along with X-ray findings for the diagnosis and treatment of knee osteoarthritis. We may be able to precisely predict knee pain caused by osteoarthritis, by analysis of a model that includes a lesion in joint cartilage.

Absorptiometry, Photon↗
PubMed1996

Enzymatic characterization of purified NS3 serine proteinase of hepatitis C virus expressed in Escherichia coli.

Non-structural protein 3 (NS3) of the hepatitis C virus (HCV) has been shown to be a serine proteinase which cleaves the HCV polyprotein thus activating its replicative machinery. To characterize enzymatic activities of NS3 serine proteinase, the proteinase region was expressed in Escherichia coli and purified. The purified proteinase specifically cleaved a purified fusion protein sandwiching the NS5A/5B cleavage sequence. In addition to serine proteinase inhibitors, some chelators also inhibited the cleavage activity. Metal ions were not required for its activity, suggesting that the proteinase may be a novel serine proteinase having a unique binding site for chelators.

ATP-Binding Cassette Transporters↗
PubMed1996

Genetic basis of the neurovirulence of type 1 polioviruses isolated from vaccine-associated paralytic patients.

We examined four type 1 polioviruses isolated from the stools of patients with vaccine-associated paralytic poliomyelitis in China. All of these isolates were shown to be Sabin derived viruses by restriction fragment length polymorphism assay after polymerase chain reaction and by sequencing of the viral genome encoding the viral coat protein, VP1. However, the same analysis of the 3D coding region suggested that two of the four isolates had the sequence of wild type poliovirus in the tested region. Furthermore there were also point mutations in the 5' non-coding region. One was a single base change from U to C at nucleotide position 525, and the other three were from G to A at position 480. All the four strains were more neurovirulent that Sabin type 1 virus in transgenic mice with human poliovirus receptor gene. The data showed that the nucleotide positions of type 1 poliovirus which were identified to be in favor of the high neurovirulence were indeed changed during natural transmission, and suggested that the point mutation alone or a recombination of the vaccine type with wild type genome results in an acquisition of neurovirulence.

Animals↗