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Biomedical subjects

M Kohara

Publications and source records attributed to M Kohara.

At least 19 recordsLinked to original sources
PubMed2001

Assessment of markers of hepatitis C virus infection in a Japanese adult population.

Latent-class analysis was used to evaluate the usefulness of markers of hepatitis C virus (HCV) infection in characterizing the true, underlying infection in a community-based Japanese population. Antibodies to HCV were detected in 24%, HCV RNA in 22%, and HCV core protein in 19% of stored serum samples from 372 adults. A 2-class model suggested that positive results for any 2 virus markers defined the current HCV infection class, with an estimated prevalence of 22% (95% confidence interval, 18%-26%). The sensitivity for detection of current HCV infection was highest for anti-HCV (97%) and was more moderate for HCV RNA (91%) and HCV core protein (85%). The specificity for each marker was > or =96%. In general, the association between demographic factors and current HCV infection status was strengthened by use of latent-class analysis that combined data for markers of HCV infection, when compared with results of logistic regression analysis for each marker separately.

Agglutination Tests↗
PubMed2001

The hepatitis C virus nonstructural protein 4B is an integral endoplasmic reticulum membrane protein.

The hepatitis C virus (HCV) nonstructural protein 4B (NS4B) is a relatively hydrophobic 27-kDa protein of unknown function. A tetracycline-regulated gene expression system, a novel monoclonal antibody, and in vitro transcription-translation were employed to investigate the subcellular localization and to characterize the membrane association of this viral protein. When expressed individually or in the context of the entire HCV polyprotein, NS4B was localized in the endoplasmic reticulum (ER), as shown by subcellular fractionation, immunofluorescence analyses, and double-label confocal laser scanning microscopy. In this compartment NS4B colocalized with the other HCV nonstructural proteins. Association of NS4B with the ER membrane occurred cotranslationally, presumably via engagement of the signal recognition particle by an internal signal sequence. In membrane extraction and proteinase protection assays NS4B displayed properties of a cytoplasmically oriented integral membrane protein. Taken together, our findings suggest that NS4B is a component of a membrane-associated cytoplasmic HCV replication complex. An efficient replication system will be essential to further define the role of NS4B in the viral life cycle.

Animals↗
PubMed2001

Simple quantitative assay of alpha-fetoprotein mRNA in liver tissue using the real-time detection polymerase chain reaction assay - its application for clinical use.

Alpha-feto protein (AFP) mRNA levels increase in hepatocellular carcinoma (HCC) cells as compared with non-neoplastic tissue. Therefore, detection of AFP mRNA in blood nuclear cells is useful for the evaluation of treatment efficacy and prognosis of HCC. In this study, simple and reproducible methods were developed to quantify AFP mRNA using the real-time RT-PCR assay (Taq Man assay). By using in vitro synthesized AFP and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA, the sensitivity and dynamic range of the RT-PCR assay were established. AFP mRNA in both HCC and non-neoplastic tissue, as well as in cell lines, were measured using this assay system. The expression of the AFP mRNA level was normalized using the GAPDH house keeping gene product as an endogenous reference. AFP and GAPDH mRNA can be quantified in the range of 10-10(8) copies when using this quantitative assay. Among HCC cell lines, Huh 7 and HepG2 cells, respectively, represented 1.5x10(6) and 6.0x10(5) AFP mRNA/10(6) GAPDH mRNA, in contrast to 6, 23 and 230 AFP mRNA/10(6) GAPDH mRNA for HLE, HLF and PLC/PRF/5 cells, respectively. Other cell lines derived from stomach, pancreas, and colon cancers have 10 AFP mRNA copies/10(6) GAPDH mRNA. In liver tissue from patients with chronic hepatitis, and the non-neoplastic portion of the liver from HCC patients, AFP mRNA distributes from 2.5x10(3) to 5.8x10(4)/10(6) GAPDH transcripts. In contrast, AFP mRNA in tumor cells were more than 100-fold higher than that found in corresponding non-neoplastic portions in two patients who had a high level of AFP in serum. The establishment of the TaqMan quantifying system for AFP mRNA may have important clinical implications.

Journal Article↗
PubMed2001

Inhibition of cytochrome c release in Fas-mediated signaling pathway in transgenic mice induced to express hepatitis C viral proteins.

Persistent hepatitis C virus (HCV) infection often progresses to chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Numerous viruses have been reported to escape from apoptotic mechanism to maintain persistent infection. In the present study, we characterized the effect of HCV proteins on the Fas signal using HCV transgenic mice, which expressed core, E1, E2, and NS2 proteins, regulated by the Cre/loxP switching system. The transgene expression of HCV transgenic mice caused resistance to Fas antibody stimulated lethality. Apoptotic cell death in the liver of HCV protein expressing mice was significantly reduced compared with nonexpressing mice. Histopathological analysis and DNA fragmentation analysis revealed that the HCV proteins suppressed Fas-mediated apoptotic cell death. To identify the target pathway of HCV proteins, we characterized caspase activity. The activation of caspase-9 and -3/7 but not caspase-8 was inhibited by HCV proteins. Cytochrome c release from mitochondria was inhibited in HCV protein expressing mice. These results indicated that the expression of HCV proteins may directly or indirectly inhibit Fas-mediated apoptosis and death in mice by repressing the release of cytochrome c from mitochondria, thereby suppressing caspase-9 and -3/7 activation. These results suggest that HCV may cause persistent infection, as a result of suppression of Fas-mediated cell death.

Animals↗
PubMed2001

Evaluation of the relative contribution of p53-mediated pathway in X-ray-induced apoptosis in human leukemic MOLT-4 cells by transfection with a mutant p53 gene at different expression levels.

There are several pathways leading to apoptosis. It is not clear whether cells choose one of them or use multiple processes when they commit to apoptosis. MOLT-4 cells undergo apoptosis after X-irradiation through the p53-dependent pathway and/or ceramide signal. To evaluate the relative contribution of these pathways, we studied effects of the expression of various levels of transfected murine mutant p53 cDNA (TGC-->CGC of codon 173, corresponding to codonl76 in human p53) on the induction of apoptosis in X-irradiated or heated MOLT-4 cells. When survival was determined by the dye-exclusion test at 24 h after irradiation, the percentage of X-ray- or heat-induced dead cells was markedly decreased, depending on the expression level of mutant p53 protein in transfected clones. The appearance of apoptotic cells as determined by morphological changes was also decreased. These inhibitions were almost complete at 24 h after irradiation with X-rays in the case of the highest-expressing clone. p21 WAF1 protein was increased in MOLT-4 cells after X-irradiation, but not in the transfectant. These results suggest that murine mutant p53 protein has a dominant-negative effect against normal p53 in MOLT-4, and that the X-ray-induced apoptosis in MOLT-4 is fully p53-dependent.

Apoptosis↗
PubMed2001

Hepatitis C virus core particle detected by immunoelectron microscopy and optical rotation technique.

Hepatitis C Virus (HCV) are 55-65 nm spherical particles, but the internal structure of the virion remains to be clarified. To clarify the morphology of HCV core particles, we performed an immune electron microscopy (IEM) using plasma samples from two blood donors with high HCV RNA titers and a detergent-treated anti-HCV core antibody-free plasma sample with high HCV RNA titer (1.5x10(8) copies/ml). Spherical particles, with 33-40 nm in diameter (an average diameter of 37 nm) were found in 1.22-1.25 g/ml fractions after sucrose density gradient centrifugation by conventional electron microscopy (EM). IEM using rabbit polyclonal antibody (RR8) specific to the putative HCV core protein and goat anti-rabbit IgG colloidal gold particles revealed that these spherical particles specifically reacted with RR8. This finding indicates that the spherical particles are naked HCV core particles. Some of the HCV core particles had an icosahedron-like structure. Optical rotation technique showed that the HCV core particle exhibits six-fold symmetry and that the length of regular hexagon side is approximately 20 nm. These findings showed that HCV core particles are spherical particles of 33-40 nm in diameter and that HCV core particles may possess an icosahedron-like structure and a buoyant density of 1.22-1.25 g/ml.

Journal Article↗
PubMed2001

Adipose tissue reduction in mice lacking the translational inhibitor 4E-BP1.

All nuclear-encoded mRNAs contain a 5' cap structure (m7GpppN, where N is any nucleotide), which is recognized by the eukaryotic translation initiation factor 4E (eIF4E) subunit of the eIF4F complex. The eIF4E-binding proteins constitute a family of three polypeptides that reversibly repress cap-dependent translation by binding to eIF4E, thus preventing the formation of the eIF4F complex. We investigated the biological function of 4E-BP1 by disrupting its gene (Eif4ebp1) in the mouse. Eif4ebp1-/- mice manifest markedly smaller white fat pads than wild-type animals, and knockout males display an increase in metabolic rate. The males' white adipose tissue contains cells that exhibit the distinctive multilocular appearance of brown adipocytes, and expresses the uncoupling protein 1 (UCP1), a specific marker of brown fat. Consistent with these observations, translation of the peroxisome proliferator-activated receptor-gamma co-activator 1 (PGC1), a transcriptional co-activator implicated in mitochondrial biogenesis and adaptive thermogenesis, is increased in white adipose tissue of Eif4ebp1-/- mice. These findings demonstrate that 4E-BP1 is a novel regulator of adipogenesis and metabolism in mammals.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Acquisition of susceptibility to hepatitis C virus replication in HepG2 cells by fusion with primary human hepatocytes: establishment of a quantitative assay for hepatitis C virus infectivity in a cell culture system.

Hepatitis C virus (HCV) replicates in human and chimpanzee hepatocytes. To characterize the nature of HCV and evaluate antiviral agents, the development of an HCV replication system in a cell culture is essential. We developed a cell line derived from human hepatocytes by fusing them with a hepatoblastoma cell line, HepG2, and obtained several clones. When we tested the clones for their ability to support HCV replication by nested RT-PCR, we found 1 clone (IMY-N9) that was more susceptible to HCV replication than HepG2. The negative-strand HCV RNA was detected in IMY-N9 by strand-specific RT-PCR, and viral RNA was identified in culture supernatant during the culture. Then we monitored HCV RNA titers in IMY-N9 and HepG2, respectively, by real-time detection PCR throughout the culture. A significant increase in the HCV RNA titer was observed only in IMY-N9. Serial passages of HCV culture supernatant were shown in the culture system. Furthermore, we tested several infectious materials for viral infectivity by monitoring HCV RNA titers and/or 50% tissue culture infectious dose (TCID50) of HCV on IMY-N9. In each material, HCV showed various growth patterns and a different TCID50 even though the PCR titer in each material was identical. The results showed that HCV in each material served various growth patterns and different TCID50 even though PCR titer in each material was identical. This cell line is useful for estimating viral activity and for studying cellular factors that may be necessary to HCV replication in human hepatocytes.

Cell Fusion↗
PubMed2000

Complete genome structure of the nitrogen-fixing symbiotic bacterium Mesorhizobium loti.

The complete nucleotide sequence of the genome of a symbiotic bacterium Mesorhizobium loti strain MAFF303099 was determined. The genome of M. loti consisted of a single chromosome (7,036,071 bp) and two plasmids, designated as pMLa (351,911 bp) and pMLb (208, 315 bp). The chromosome comprises 6752 potential protein-coding genes, two sets of rRNA genes and 50 tRNA genes representing 47 tRNA species. Fifty-four percent of the potential protein genes showed sequence similarity to genes of known function, 21% to hypothetical genes, and the remaining 25% had no apparent similarity to reported genes. A 611-kb DNA segment, a highly probable candidate of a symbiotic island, was identified, and 30 genes for nitrogen fixation and 24 genes for nodulation were assigned in this region. Codon usage analysis suggested that the symbiotic island as well as the plasmids originated and were transmitted from other genetic systems. The genomes of two plasmids, pMLa and pMLb, contained 320 and 209 potential protein-coding genes, respectively, for a variety of biological functions. These include genes for the ABC-transporter system, phosphate assimilation, two-component system, DNA replication and conjugation, but only one gene for nodulation was identified.

ATP-Binding Cassette Transporters↗
PubMed2000

Sequence and analysis of chromosome 5 of the plant Arabidopsis thaliana.

The genome of the model plant Arabidopsis thaliana has been sequenced by an international collaboration, The Arabidopsis Genome Initiative. Here we report the complete sequence of chromosome 5. This chromosome is 26 megabases long; it is the second largest Arabidopsis chromosome and represents 21% of the sequenced regions of the genome. The sequence of chromosomes 2 and 4 have been reported previously and that of chromosomes 1 and 3, together with an analysis of the complete genome sequence, are reported in this issue. Analysis of the sequence of chromosome 5 yields further insights into centromere structure and the sequence determinants of heterochromatin condensation. The 5,874 genes encoded on chromosome 5 reveal several new functions in plants, and the patterns of gene organization provide insights into the mechanisms and extent of genome evolution in plants.

Animals↗
PubMed2000

Possible role of cytotoxic T cells in acute liver injury in hepatitis C virus cDNA transgenic mice mediated by Cre/loxP system.

A line of hepatitis C virus (HCV) transgenic mice was established previously that was mediated by Cre/loxP system using HCV cDNA, including core, E1, E2 and NS2 genes. Intravenous infection of a recombinant adenovirus that expresses Cre DNA recombinase (AxCANCre) induced HCV structural protein expression in the liver of transgenic mice. HCV core protein production and transgene recombination in the mouse liver were serially evaluated after AxCANCre infusion. Core proteins were expressed efficiently and transgene was almost completely recombined in the liver of mice after 3 days and then the levels of both core protein production and transgene recombination decreased continuously for 28 days. However, 30.6% of the transgene recombination remained at 28 days and only 2.7% of core production remained at 28 days after infection. Compared with nontransgenic controls, the serum alanine aminotransferase levels in transgenic mice were significantly higher 10, 14, and 21 days after adenovirus infection. Histological scoring also indicated severe pathological changes in the liver of transgenic mice after adenovirus infection. AxCANCre infusion increased CD8+ lymphocyte infiltration into the liver of transgenic mice compared with that of non-transgenic controls. Furthermore, cytotoxic T lymphocytes (CTLs) isolated from transgenic mice during liver injury were specific for the HCV proteins. These results suggest that HCV structural proteins expressed in the liver of transgenic mice enhanced liver injury. HCV-specific CTLs may be to enhance hepatitis. Thus, the present HCV transgenic mouse model provides a useful model of liver injury due to HCV, and the host immune response may play a pivotal role(s) in the pathogenesis of HCV.

Adenoviridae↗
PubMed2000

HCV-core protein accelerates recovery from the insensitivity of liver cells to Fas-mediated apoptosis induced by an injection of anti-Fas antibody in mice.

BACKGROUND/AIMS: Hepatitis C virus (HCV) is a major etiologic agent of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The aim of this study was to elucidate pathological effects of HCV-core protein on liver cells. METHODS: We have generated transgenic mice carrying HCV-core cDNA (Px-core) and pathologically examined livers of Px-core mice. RESULTS: HCV-core protein was detectable in livers from lines 5 (C5) and 8 (C8) of Px-core transgenic mice. Since chronic hepatitis and cirrhosis precede hepatocellular carcinoma in patients with HCV infection, we tried to examine the effect of repetitive injection of a small dose of anti-Fas antibody in the transgenic mice. Surprisingly, an initial injection of anti-Fas antibody induced resistance of liver cells to the second injection of anti-Fas antibody in both Px-core and littermate control mice. The insensitivity of liver cells induced in the control mice continued for more than 24 weeks after the first injection but was broken within 1 week after partial hepatectomy. However, the sensitivity was restored in the Px-core mice within 12 weeks after the injection. CONCLUSION: HCV-core protein in liver cells may affect persistence of Fas-mediated liver cell injury.

Animals↗
PubMed2000

Hepatitis C virus replication and pathogenesis.

The mechanism involved in the development of persistent hepatitis C virus (HCV) infection and the pathogenesis remain unclear. The present review is an accumulation of evidence gathered to date. In addition, it discusses the system developed to characterize HCV.

Animals↗
PubMed1999

Phosphorylation of nonstructural 5A protein of hepatitis C virus: HCV group-specific hyperphosphorylation.

We previously showed that two proteins with molecular weights of 56 and 58 kDa are produced from nonstructural protein 5A (NS5A) derived from hepatitis C virus (HCV)-1b genotype. The 56-kDa protein is phosphorylated at serine residues in NS5A, including those located in the C-terminal region of NS5A, while the 58-kDa protein, the hyperphosphorylated form of the 56-kDa protein, is phosphorylated at serine residues in the central region. This hyperphosphorylation is dependent on the presence of HCV NS4A protein. To clarify whether NS4A-dependent phosphorylation also occurs in other HCV genotypes, phosphorylation of NS5A was analyzed by two-dimensional gel electrophoresis. Here, we report that NS5A from the HCV-2a genotype was phosphorylated. However, hyperphosphorylation of NS5A occurs in the HCV-1b genotype but not in the -2a genotype. This result suggests that modification of NS5A phosphorylation reflects the virological features of HCV and that there are physiological differences in the roles of differently phosphorylated NS5A between HCV genotypes.

Alkaline Phosphatase↗
PubMed1999

Correlation of interferon treatment response with GBV-C/HGV genomic RNA and anti-envelope 2 protein antibody.

The clinical significance of GB virus C/hepatitis G virus (GBV-C/HGV) co-infection was studied retrospectively in 100 consecutive patients with hepatitis C virus (HCV) infection. All 100 patients had been treated with interferon-alpha (IFN-alpha). Co-infection with GBV-C/HGV and HCV was detected in 10 of the 100 patients (10%) and anti-envelope 2 region (anti-E2) antibody was detected in 25 patients. None of the patients with GBV-C/HGV RNA had anti-E2 antibody. Co-infected patients were younger (P < .005) and their serum transaminase levels were lower than HCV-only infected patients (P< .01). In 7 of the 10 co-infected patients, HCV RNA was eradicated from serum after IFN-alpha treatment and normal alanine transaminase (ALT) levels continued in 6 of these 7 patients. In one patient who was negative for HCV RNA but positive for GBV-C/HGV RNA, the ALT level relapsed transiently. The rate of clearance of HCV and normalization of the ALT level was significantly higher in co-infected patients than in HCV-only infected patients (P < .05). GBV-C/HGV RNA disappeared from 6 of the 10 co-infected patients (60%) upon cessation of IFN-alpha treatment. However, continuous clearance of GBV-C/HGV was observed in only two patients and anti-E2 antibody could not be detected in the serum of these patients. These results indicate that co-infected patients tend to be younger and more sensitive to IFN-alpha treatment. However, long-term clearance of GBV-C/HGV after IFN-alpha treatment may be difficult. Moreover, anti-E2 antibody may act to neutralize GBV-C/ HGV.

Adult↗
PubMed1999

Hepatitis C virus structural proteins induce liver cell injury in transgenic mice.

To develop an animal model of hepatitis C virus (HCV) infection, transgenic mice carrying part of the HCV cDNA (C980) encoding HCV-core and envelope proteins under control of the mouse class I major histocompatibility complex gene (H-2K) regulatory region were produced. HCV-C980 RNA and HCV-core protein were present in livers from line H36 as determined by RNase protection assay and immunostaining, respectively. More than 40 animals from line H36 were examined histologically. Most of these H36 mice after 10 months of age developed spontaneous focal infiltration of lymphocytes, hepatocyte necrosis, degeneration, and altered foci with mitotic hepatocytes. These pathological lesions were absent in livers from the age-matched control littermates. Liver cells from these H36 mice were sensitive to damage induced by intravenous administration of an anti-Fas antibody. It is suggested that HCV-C980 proteins by themselves may be one causative agent of liver cell injury in subjects with HCV infection.

Animals↗
PubMed1999

Hepatitis C virus core protein binds to apolipoprotein AII and its secretion is modulated by fibrates.

Several lines of evidence suggest that hepatitis C virus (HCV) core protein may modulate cellular transduction signals and alter lipid metabolism. We have investigated the binding of HCV core protein to cellular proteins by combining 2 yeast hybrid, confocal, and surface plasmon resonance assays. Our results show the direct binding of the viral protein to apolipoprotein AII (apoAII) and map the interaction domain to the C-terminal of HCV core protein. To investigate the biological relevance of the interaction between HCV core and lipid metabolism, we took advantage of the well-established increase in apoAII expression caused by fibrates in HepG2 cells. After fenofibric acid treatment, we show a parallel increase in apoAII and core protein secretion, this effect being abolished by brefeldin A. Our study identifies apoAII as one of the cellular targets for HCV core protein. We also show that the intervention of fenofibric acid in cellular lipid metabolism directly affects the expression pattern of HCV core protein.

Apolipoprotein A-II↗