Yukihiro Shirota

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.

Telomerase Activity Reconstituted in Vitro with Purified Human Telomerase Reverse Transcriptase and Human Telomerase RNA Component

Telomerase is a specialized reverse transcriptase that catalyzes elongation of the telomeric tandem repeat, TTAGGG, by addition of this sequence to the ends of existing telomeres. Human telomerase reverse transcriptase (hTERT) has been identified as a catalytic enzyme involved in telomere elongation that requires telomerase RNA, human telomerase RNA component (hTR), as an RNA template. We established a new method to express and purify soluble insect-expressed recombinant hTERT. The partially purified FLAG-hTERT retained the catalytic activity of telomerase in a complementation assay in vitro to exhibit telomerase activity in telomerase-negative TIG3 cell extract and in a reconstitution assay with FLAG-hTERT and purified hTR in vitro. FLAG-hTERT (D712A) with a mutation in the VDV motif exhibited no telomerase activity, confirming the authentic catalytic activity of FLAG-hTERT. The reconstituted complex of FLAG-hTERT and hTR in vitrowas detected by electrophoretic mobility shift assay, and its activity was stimulated by more than 30-fold by TIG3 cell extract. This suggested that some cellular component(s) in the extract facilitated the reconstituted telomerase activity in vitro. Geldanamycin had no effect on the reconstituted activity but partially reduced the stimulated activity of the reconstituted telomerase by the TIG3 cell extract, suggesting that Hsp90 may contribute to the stimulatory effect of the cellular components.

Ephrin-A1 expression contributes to the malignant characteristics of α-fetoprotein producing hepatocellular carcinoma

Background and aims: α-Fetoprotein (AFP), a tumour marker for hepatocellular carcinoma (HCC), is associated with poor prognosis. Using cDNA microarray analysis, we previously found that ephrin-A1, an angiogenic factor, is the most differentially overexpressed gene in AFP producing hepatoma cell lines. In the present study, we investigated the significance of ephrin-A1 expression in HCC. Methods: We examined ephrin-A1 expression and its effect on cell proliferation and gene expression in five AFP producing hepatoma cell lines, three AFP negative hepatoma cell lines, and 20 human HCC specimens. Results: Ephrin-A1 expression levels were lowest in normal liver tissue, elevated in cirrhotic tissue, and further elevated in HCC specimens. Ephrin-A1 expression was strongly correlated with AFP expression (r = 0.866). We showed that ephrin-A1 induced expression of AFP. This finding implicates ephrin-A1 in the mechanism of AFP induction in HCC. Ephrin-A1 promoted the proliferation of ephrin-A1 underexpressing HLE cells, and an ephrin-A1 antisense oligonucleotide inhibited the proliferation of ephrin-A1 overexpressing Huh7 cells. Thus ephrin-A1 affects hepatoma cell growth. cDNA microarray analysis showed that ephrin-A1 induced expression of genes related to the cell cycle (p21), angiogenesis (angiopoietin 1 and thrombospondin 1), and cell-cell interactions (Rho, integrin, and matrix metalloproteinases) in cultured hepatoma cells. These ephrin-A1 induced genes are also activated in HCC tissues that overexpress AFP. Conclusion: These findings suggest that the poor prognosis of patients with AFP producing HCC is partially caused by ephrin-A1 expression, which induces expression of genes related to tumour cell growth, angiogenesis, invasion, and metastasis.

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.

Electrical communication between horse heart cytochrome c and electrodes in the presence of DNA or RNA

Electrical communication between electrodes and enzymes has received active interest from the viewpoints of the clarification of electron transport mechanisms in biological systems, and of applications to biochemical sensors and biocatalyst electrodes. As has been reviewed by Armstrong et al. [l], direct electron exchange between electrodes and cytochrome c, one of the electron transport enzymes in mitochondria, has been successfully investigated by using metal oxide electrodes like In,O, [2], and promotors such as 4,4’-bipyridyl [3], dithiobis(ethanoic acid) [4], and bis(4-pyridyl)disulfide [5]. A feature common to the above promotors is that electron-rich nitrogen atoms or anions, which are able to interact with positively charged lysine residues in cytochrome c, are present at several tenths of a nm apart from electrode surfaces at the adsorbed state. We chose DNA or RNA and studied their suitability as a promotor, because they have negative charges and are actually present in mitochondria. As a result, we discovered that DNA or RNA works as an effective promotor by interacting electrostatically with the positively charged patch of cytochrome c.

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.

Detection of Hepatitis B Virus DNA in Sera from Patients with Chronic Hepatitis B Virus Infection by DNA Microarray Method

ABSTRACT 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.

Gastrointestinal amyloidosis secondary to hypersensitivity vasculitis presenting with intestinal pseudoobstruction

Hypersensitivity vasculitis is characterized byinflammation and necrosis of small blood vesselssecondary to allergic or hypersensitivity mechanisms (1,2). Gastrointestinal involvement with edema and bleeding also has been reported (3-5).Long-standing inflammation, such as rheumatic disease,infectious disease, inflammatory bowel disease, familialMediterranean fever, and malignancy, may lead tosystemic amyloidosis (6). Gastrointestinal involvementmay induce anorexia, nausea and vomiting, diarrhea,constipation, bleeding, malabsorption, andpseudoobstruction (6, 10-12). In this report we discussa patient with hypersensitivity vasculitis with severeintestinal bleeding who developed systemic amyloidosiswith intestinal pseudoobstruction 29 months after onset.Secondary amyloidosis due to hypersensitivity vasculitis has not been previously reported,and the causal relationship is discussed in thisreport.

Assessment of still and moving images in the diagnosis of gastric lesions using magnifying narrow-band imaging in a prospective multicenter trial.

Objectives Magnifying narrow-band imaging (M-NBI) is more accurate than white-light imaging for diagnosing small gastric cancers. However, it is uncertain whether moving M-NBI images have additional effects in the diagnosis of gastric cancers compared with still images. Design A prospective multicenter cohort study. Methods To identify the additional benefits of moving M-NBI images by comparing the diagnostic accuracy of still images only with that of both still and moving images. Still and moving M-NBI images of 40 gastric lesions were obtained by an expert endoscopist prior to this prospective multicenter cohort study. Thirty-four endoscopists from ten different Japanese institutions participated in the prospective multicenter cohort study. Each study participant was first tested using only still M-NBI images (still image test), then tested 1 month later using both still and moving M-NBI images (moving image test). The main outcome was a difference in the diagnostic accuracy of cancerous versus noncancerous lesions between the still image test and the moving image test. Results Thirty-four endoscopists were analysed. There were no significant difference of cancerous versus noncancerous lesions between still and moving image tests in the diagnostic accuracy (59.9% versus 61.5%), sensitivity (53.4% versus 55.9%), and specificity (67.0% versus 67.6%). And there were no significant difference in the diagnostic accuracy between still and moving image tests of demarcation line (65.4% versus 65.5%), microvascular pattern (56.7% versus 56.9%), and microsurface pattern (48.1% versus 50.9%). Diagnostic accuracy showed no significant difference between the still and moving image tests in the subgroups of endoscopic findings of the lesions. Conclusions The addition of moving M-NBI images to still M-NBI images does not improve the diagnostic accuracy for gastric lesions. It is reasonable to concentrate on taking sharp still M-NBI images during endoscopic observation and use them for diagnosis. Trial registration Umin.ac.jp UMIN-CTR000008048

Transcatheter pancreatoscopy-guided electrohydraulic lithotripsy for large pancreatic duct stones

A 61-year-old man was admitted for treatment of alcohol-related chronic pancreatitis with recurrent acute exacerbations. Computed tomography (CT) scanning showed a large cluster of stones in the main pancreatic duct of the head of the pancreas and upstream duct dilatation (▶Fig. 1). We used endoscopic retrograde cholangiopancreatography (ERCP) and pancreatoscopy with electrohydraulic lithotripsy (EHL) to reduce the patient’s stone burden and ductal hypertension. Pancreatoscopy was attempted using the SpyGlass Direct Visualization System with a SpyScope catheter (Boston Scientific, Natick, Massachusetts, USA), but the catheter was too large to pass through the duct without dilation. Therefore, we used a double-lumen catheter (Introducer of a CytoMax II Double Lumen Biliary Cytology Brush; Cook Medical, Winston-Salem, North Carolina, USA), which is smaller than the SpyScope catheter. A SpyGlass optical probe and an EHL probe (Walz Elektronik GmbH, Rohrdorf, Germany) could then be passed together through the lumen of the catheter. After pancreatic sphincterotomy had been performed, the impacted stones were identified by transcatheter pancreatoscopy, with irrigation being provided through the lumen of the catheter containing the probe (▶Fig. 2). EHL successfully fragmented most of the impacted stones under direct vision, and a guidewire was successfully passed through the stones (▶Video1). The fragments were removed with an ERCP basket, and a pancreatogram showed improvement in the upstream duct dilatation (▶Fig. 3). There were no procedure-related complications. Pancreatoscopy-guided EHL is often difficult because of limited control of the field of view, especially when a tortuous and narrow pancreatic duct is involved. Transcatheter endoscopy with an ERCP catheter and Spyglass optical probe has been previously reported to be useful for approaching and observing a narrow portion of the pancreaticobiliary tract, although the technique was used for diagnosis only [1]. Transcatheter pancreatoscopy with EHL using a double-lumen catheter for fragmentation of a large E-Videos