Hiroshi Kanamori

A case of biliary cystadenocarcinoma with recurrent jaundice. Diagnostic evaluation of computed tomography

A 73‐year‐old male patient with biliary cystadenocarcinoma and episodes of recurrent jaundice is reported. This very rare tumor was suggested as a possible diagnosis by the computed tomographic findings showing intrahepatic cystic masses with septations and papillary projections. The diagnosis of the mucin‐producing tumor was supported by aspiration of mucinous bile with a cannule inserted endoscopically via the major duodenal papilla. The computed tomographic findings and the diagnosis were verified by pathologic studies made on the material obtained surgically. The mucinous bile is assumed to have been responsible for the recurrent jaundice.

HTLV-1 p27rex stabilizes human interleukin-2 receptor alpha chain mRNA.

Expression of the pX gene products (p40tax, p27rex and p21X‐III) of human T cell leukemia virus type 1 (HTLV‐1), which is known to be a causative agent of adult T cell lymphoma/leukemia, induces expression of the interleukin‐2 receptor alpha chain (IL‐2R alpha) on infected T cells. Comparison of IL‐2R alpha promoter activities has revealed that the transcriptional activation of the promoter alone cannot explain the large numbers of IL‐2R alpha expressed on HTLV‐1 infected cells. We found that the rates of the IL‐2R alpha mRNA degradation were greatly reduced in pX‐positive cells as compared with pX‐negative cells. Simultaneous transfection of the expression vector plasmid containing IL‐2R alpha cDNA and similar plasmids containing various pX sequences showed that p27rex elongated the half life of IL‐2R alpha mRNA. As p27rex did not affect the transport of the IL‐2R alpha mRNA from nucleus to cytoplasm, prolongation of the IL‐2R alpha mRNA half life by p27rex is ascribed to stabilization of the mRNA. Experiments using deletion mutants and chimeric constructs of the IL‐2R alpha cDNA demonstrated that the coding sequence but not the 5′ or 3′ untranslated region of the IL‐2R alpha mRNA sequence is responsible for its protection by p27rex.

Measurement of HCV RdRp activity with C-terminal 21 aa truncated NS5b protein : optimization of assay conditions

The non-structural protein 5b (NS5b) of hepatitis C virus (HCV), bearing an RNA-dependent RNA polymerase (RdRp) activity, is considered as a new target of antiviral therapy. We expressed and purified the C-terminal 21 amino acid truncated NS5b protein fused with glutathione S-transferase (GST-5bC21) using Escherichia coli. With the highly purified GST-5bC21 protein, we established an in vitro assay system for RdRp activity by using poly(C) as the template and a 12 mer oligo(rG) as the primer. The optimal conditions for testing various concentrations of template, primer and proteins were determined to 22 degrees C and a pH of 7.5. The addition of 2.5 mM Mn(2+) increased the activity profoundly, to a level fivefold higher than that in the presence of 10 mM Mg(2+). At higher concentrations of Mn(2+), GST-5bC21 is stable as compared with previously reported full-length NS5b expressed using insect cells or NS5b protein with the C-terminal 18 amino acids deleted. This sensitive and easy to use quantitative assay system will provide a stable system for the screening of inhibitors for HCV RdRp.

RNA-dependent RNA polymerase of hepatitis C virus binds to its coding region RNA stem-loop structure, 5BSL3.2, and its negative strand

The hepatitis C virus NS5B RNA-dependent RNA polymerase (RdRp) is a key enzyme involved in viral replication. Interaction between NS5B RdRp and the viral RNA sequence is likely to be an important step in viral RNA replication. The C-terminal half of the NS5B-coding sequence, which contains the important cis-acting replication element, has been identified as an NS5B-binding sequence. In the present study, we confirm the specific binding of NS5B to one of the RNA stem-loop structures in the region, 5BSL3.2. In addition, we show that NS5B binds to the complementary strand of 5BSL3.2 (5BSL3.2N). The bulge structure of 5BSL3.2N was shown to be indispensable for tight binding to NS5B. In vitro RdRp activity was inhibited by 5BSL3.2N, indicating the importance of the RNA element in the polymerization by RdRp. These results suggest the involvement of the RNA stem-loop structure of the negative strand in the replication process.

In vitro selection of RNA aptamers that bind the RNA-dependent RNA polymerase of hepatitis C virus: A possible role of GC-rich RNA motifs in NS5B binding

We employed SELEX (systematic evolution of ligands by exponential enrichment) and identified high affinity RNA aptamers to the hepatitis C virus NS5B RNA-dependent RNA polymerase (RdRp). GC-rich stretches were identified in many of the aptamers. Deletion of the 5-end single-stranded GC-stretch (CGGG) of the highest binding RNA impaired the binding and the inhibitory activity of the RNA to NS5B RdRp. The majority of the mutants with a single base substitution on the CGGG motif exhibited weaker binding to NS5B. Interestingly, the CGGG motif is present on the stem structure of the NS5B coding RNA (5BSL3.2), which is considered to be an important cis-acting replication element. The 5BSL3.2 RNA showed substantial binding to NS5B, while a point mutation on the CGGG motif reduced the binding of RNA to NS5B. These results suggest a GC-stretch to be the RNA element recognized by NS5B.

Treatment of unresectable hepatocellular carcinoma less than 2 centimeters by transcatheter arterial chemoembolization with autologous blood clot

Goals To assess the efficacy of transcatheter arterial chemoembolization using autologous blood clot as an embolizing agent (short-TAE [S-TAE]) for the treatment of unresectable hepatocellular carcinoma less than 2 cm. Study Twenty-eight consecutive patients with unresectable hepatocellular carcinoma less than 2 cm in diameter were treated by S-TAE alone. All patients had documented cirrhosis (Child class B:C = 20:8). S-TAE was performed by injecting a mixture of iodized oil and anticancer drugs followed by embolization of hepatic arteries with autologous blood clot. Results A total of 147 sessions of embolization with clots were performed. S-TAE maintained patency of hepatic arteries. The overall survival rates at 1, 3, 5, and 8 years were estimated to be 89%, 52%, 34%, and 17%, respectively, which were better compared with prior records for the gelfoam method. The survival rates for Child class B patients were significantly better than that for Child class C patients (P < 0.05). The Cox proportional hazard model also demonstrated that Child staging of cirrhosis was the sole factor significantly predicting the survival (P < 0.05). Conclusions The long-term outcomes of S-TAE for unresectable hepatocellular carcinoma less than 2 cm are satisfactory. Prognosis of these patients was significantly dependent on clinical stages of coexisting liver cirrhosis.

In vitro selection of the 3'-untranslated regions of the human liver mRNA that bind to the HCV nonstructural protein 5B.

Hepatitis C virus (HCV) nonstructural protein 5B (NS5B) has RNA-dependent RNA polymerase (RdRp) activity. Because NS5B recognizes various RNA motifs besides the HCV genome, NS5B has the potential of interacting with host RNA molecules. In this study, an RNA pool enriched with the 3-UTR sequences was generated and mRNA molecules with high affinity binding to NS5B were selected by iterative selection. Among the high binding mRNA 3-UTR segments, we analyzed the housekeeping ribosomal protein S4, X-linked [RPS4X] mRNA 3-UTR and the 3-UTR of galectin-1 (GAL-1) mRNA, which is known to be one of the genes upregulated in HCV-infected liver cells and to have a wide spectrum of biological properties. By means of IP-RT-PCR, it was demonstrated that both of the mRNA molecules bind to NS5B in the cytoplasm. Interestingly, GAL-1 and RPS4X mRNA can serve as templates for NS5B RdRp, suggesting these RNA molecules are regulated in vivo by NS5B.