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Biomedical subjects

Yukihiro Shirota

Publications and source records attributed to Yukihiro Shirota.

8 recordsLinked to original sources
PubMed2005

cDNA microarray analysis of autoimmune hepatitis, primary biliary cirrhosis and consecutive disease manifestation.

While autoimmune hepatitis (AIH) and primary biliary cirrhosis (PBC) usually have distinct clinical manifestations, some patients present with features of both conditions. Using cDNA microarrays, we analyzed and compared gene expression profiles in 8 patients with AIH, 9 with PBC, 8 with chronic hepatitis C (CHC), 8 with non-alcoholic steatohepatitis (NASH) and 9 with normal livers. We subsequently applied this method to a tissue sample from a 61-year-old woman with overlapping features of both AIH and PBC. A 61-year-old woman was admitted to our hospital for evaluation of elevated serum alkaline phosphatase. A liver biopsy showed accumulation of mononuclear cells around the bile duct cells, a feature characteristic of chronic non-suppurative destructive cholangitis (CNSDC). Three years later, her serum alanine aminotransferase (ALT) level had increased, and a liver biopsy demonstrated evidence of a severe form of hepatitis. A cDNA microarray analysis of both biopsies identified the molecular events associated with her altered histology. The expression profile of this patient, which was originally different from that of the other PBC patients, changed to an AIH pattern. Our results suggest that this patient has characteristics of both AIH and PBC.

Aged↗
PubMed2005

Differential gene alteration among hepatoma cell lines demonstrated by cDNA microarray-based comparative genomic hybridization.

We assayed chromosomal abnormalities in hepatoma cell lines using the microarray-based comparative genomic hybridization (array-CGH) method and investigated the relationship between genomic copy number alterations and expression profiles in these hepatoma cell lines. We modified a cDNA array-CGH assay to compare genomic DNAs from seven hepatoma cell lines, as well as DNA from two non-hepatoma cell lines and from normal cells. The mRNA expression of each sample was assayed in parallel by cDNA microarray. We identified small amplified or deleted chromosomal regions, as well as alterations in DNA copy number not previously described. We predominantly found alterations of apoptosis-related genes in Hep3B and HepG2, cell adhesion and receptor molecules in HLE, and cytokine-related genes in PLC/PRF/5. About 40% of the genes showing amplification or loss showed altered levels of mRNA (p < 0.05). Hierarchical clustering analysis showed that the expression of these genes allows differentiation between alpha-fetoprotein (AFP)-producing and AFP-negative cell lines. cDNA array-CGH is a sensitive method that can be used to detect alterations in genomic copy number in tumor cells. Differences in DNA copy alterations between AFP-producing and AFP-negative cells may lead to differential gene expression and may be related to the phenotype of these cells.

Apoptosis↗
PubMed2005

Differential gene expression profiles in stage I primary biliary cirrhosis.

OBJECTIVES: Primary biliary cirrhosis (PBC) is a progressive disease. However, little is understood about the molecular mechanisms underlying its features. METHODS: We analyzed gene expression profiles of liver biopsy samples from 16 patients with PBC, seven with autoimmune hepatitis, eight with chronic hepatitis C, and eight normal control livers. In addition to whole liver samples, we selectively analyzed chronic nonsuppurative destructive cholangitis (CNSDC) lesions by laser capture microdissection. RESULTS: Hierarchical clustering analysis using only early-stage liver disease demonstrated 85 genes were upregulated in stage I PBC specifically. Surprisingly, the expression of these genes was not maintained in advanced-stage PBC, while other gene clusters were upregulated. Expression analysis of CNSDC lesions in stage I PBC showed the presence of active inflammatory changes, characterized by the significant elevation of interferon-gamma and the development and maturation of lymphocytes. Expression of these genes was diminished in lymphoid cells aggregation in stage III PBC, and genes reflecting hepatocyte damage were upregulated with disease progression. CONCLUSION: Gene expression patterns in stage I PBC are different from others. There are distinct changes in molecular pathology from early- to late-stage PBC, which might be a clue to reveal the etiology and progression of PBC.

Aged↗
PubMed2003

Detection of hepatitis B virus DNA in sera from patients with chronic hepatitis B virus infection by DNA microarray method.

We have developed a sensitive and quantitative assay using a DNA microarray for the detection of hepatitis B virus (HBV) DNA in serum. Fluorescently labeled target cDNA prepared from cloned HBV DNA or serum HBV DNA was hybridized to capture DNA on a slide. A linear relationship was obtained between the intensities of the array spot and the amount of the cloned or serum HBV DNA, indicating the quantitative accuracy of this assay system. In addition, there was a significant correlation between the number of molecules of serum HBV DNA determined by the DNA microarray and that determined by a branched-DNA assay (n = 21, r = 0.89). Given these results, we conclude that the DNA microarray assay system may be useful as a diagnostic technique in the clinical laboratory.

Branched DNA Signal Amplification Assay↗
PubMed2002

Direct interaction between nucleolin and hepatitis C virus NS5B.

Hepatitis C virus (HCV) NS5B is an RNA-dependent RNA polymerase (RdRP), a central catalytic enzyme in HCV replication. While studying the subcellular localization of a NS5B mutant lacking the C-terminal membrane-anchoring domain, NS5Bt, we found that expression of the green fluorescent protein (GFP)-fused form was exclusively nucleolar. Interestingly, the distribution of endogenous nucleolin changed greatly in the cells expressing GFP-NS5B, with nucleolin colocalized with GFP-NS5B in perinuclear regions in addition to the nucleolus, suggesting that NS5B retains the ability to bind nucleolin. The interaction between nucleolin and NS5B was demonstrated by GST pull-down assay. GST pull-down assay results indicated that C-terminal region of nucleolin was important for its binding to NS5B. Scanning clustered alanine substitution mutants library of NS5B revealed two sites on NS5B that binds nucleolin. NS5B amino acids 208-214 and 500-506 were both found to be indispensable for the nucleolin binding. We reported that the latter sequence is essential for oligomerization of NS5B, which is a prerequisite for the RdRP activity. C-terminal nucleolin inhibited the NS5B RdRP activity in a dose-dependent manner. Taken together, this indicates the binding ability of nucleolin may be involved in NS5B functions.

Animals↗
PubMed2002

Hepatitis C virus (HCV) NS5A binds RNA-dependent RNA polymerase (RdRP) NS5B and modulates RNA-dependent RNA polymerase activity.

Hepatitis C virus (HCV) NS5B is RNA-dependent RNA polymerase (RdRP), the essential catalytic enzyme for HCV replication. Recently, NS5A has been reported to be important for the establishment of HCV replication in vitro by the adaptive mutations, although its role in viral replication remains uncertain. Here we report that purified bacterial recombinant NS5A and NS5B directly interact with each other in vitro, detected by glutathione S-transferase (GST) pull-down assay. Furthermore, complex formation of these proteins transiently coexpressed in mammalian cells was detected by coprecipitation. Using terminally and internally truncated NS5A, two discontinuous regions of NS5A (amino acids 105-162 and 277-334) outside of the adaptive mutations were identified to be independently essential for the binding both in vivo and in vitro (Yamashita, T., Kaneko, S., Shirota, Y., Qin, W., Nomura, T., Kobayashi, K., and Mkyrakami, S. (1998) J. Biol. Chem. 273, 15479-15486). We previously examined the effect of His-NS5A on RdRP activity of the soluble recombinant NS5Bt in vitro (see Yamashita et al. above). Wild NS5A weakly stimulated at first (when less than 0.1 molar ratio to NS5B) and then inhibited the NS5Bt RdRP activity in a dose-dependent manner. The internal deletion mutants defective in NS5B binding exhibited no inhibitory effect, indicating that the NS5B binding is necessary for the inhibition. Taken together, our results support the idea that NS5A modulates HCV replication as a component of replication complex.

Animals↗
PubMed2002

Detection of measles virus genomic RNA in tear samples from a patient with measles keratitis.

PURPOSE: To report a case of measles keratitis, in which measles virus genomic RNA was detected in tear samples. METHODS: Samples of tears and pharyngeal mucus were obtained from a 19-year-old man with active systemic measles on day 7 after illness onset. These specimens were then subjected to reverse-transcriptase polymerase chain reaction (RT-PCR), using measles-specific primers. RESULTS: One week after systemic measles outbreak, the patient presented with a chief complaint of irritation in both eyes. Slit-lamp examination revealed bilateral superficial punctate keratopathy in the central cornea with small diffuse subepithelial infiltrations, and RT-PCR detected measles virus genomic RNA in tears and pharyngeal mucus. The patient was treated with topical 0.1% fluorometholone and 0.5% levofloxacin three times a day for 1 week, and the keratopathy resolved without sequelae. CONCLUSION: This report demonstrates for the first time the presence of measles virus genomic RNA in the tears of a patient with measles keratitis. It also highlights the utility of RT-PCR for rapid detection of measles RNA in tear or mucus samples. Furthermore, the presence of virus in bodily fluids and secretions signifies the potential contagiousness of this type of keratopathy.

Adult↗