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| 1.
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Oishi, Naoki ; Shilagardi, Khurts ; Nakamoto, Yasunari ; Honda, Masao ; Kaneko, Shuichi ; Murakami, Seishi
概要:
医薬保健研究域医学系<br />Chronic infection with hepatitis B virus (HBV) is a major risk factor for hepatocellular carcinoma. The
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HBV X protein (HBx) is thought to have oncogenic potential, although the molecular mechanism remains obscure. Pathological roles of HBx in the carcinogenic process have been examined using rodent systems and no report is available on the oncogenic roles of HBx in human cells in vitro. We therefore examined the effect of HBx on immortalization and transformation in human primary cells. We found that HBx could overcome active RAS-induced senescence in human immortalized cells and that these cells could form colonies in soft agar and tumors in nude mice. HBx alone, however, could contribute to neither immortalization nor transformation of these cells. In a population doubling analysis, an N-terminal truncated mutant of HBx, HBx-D1 (amino acids 51-154), which harbors the coactivation domain, could overcome active RAS-induced cellular senescence, but these cells failed to exhibit colonigenic and tumorigenic abilities, probably due to the low expression level of the protein. By scanning a HBx expression library of the clustered-alanine substitution mutants, the N-terminal domain was found to be critical for overcoming active RAS-induced senescence by stabilizing full-length HBx. These results strongly suggest that HBx can contribute to carcinogenesis by overcoming active oncogene-induced senescence. © 2007 Japanese Cancer Association.
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| 2.
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Shimakami, Tetsuro ; Honda, Masao ; Shirasaki, Takayoshi ; Takabatake, Riuta ; Liu, Fanwei ; Murai, Kazuhisa ; Shiomoto, Takayuki ; Funaki, Masaya ; Yamane, Daisuke ; Murakami, Seishi ; Lemon, Stanley M. ; Kaneko, Shuichi
概要:
Clinical studies suggest that the oral acyclic retinoid Peretinoin may reduce the recurrence of hepatocellular carcinoma (HCC) following surgical ablation of primary tumours. Since hepatitis C virus (HCV) infection is a major cause of
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HCC, we assessed whether Peretinoin and other retinoids have any effect on HCV infection. For this purpose, we measured the effects of several retinoids on the replication of genotype 1a, 1b, and 2a HCV in vitro. Peretinoin inhibited RNA replication for all genotypes and showed the strongest antiviral effect among the retinoids tested. Furthermore, it reduced infectious virus release by 80-90% without affecting virus assembly. These effects could be due to reduced signalling from lipid droplets, triglyceride abundance, and the expression of mature sterol regulatory element-binding protein 1c and fatty acid synthase. These negative effects of Peretinoin on HCV infection may be beneficial in addition to its potential for HCC chemoprevention in HCV-infected patients.
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| 3.
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Hayashi, Naoyuki ; Kobayashi, Masahiko ; Shimizu, Hiroko ; Yamamoto, Ken-ichi ; Murakami, Seishi ; Nishimoto, Takeharu
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The Ran GTPase system regulates the direction and timing of several cellular events, such as nuclear-cytosolic transport
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, centrosome formation, and nuclear envelope assembly in telophase. To gain insight into the Ran system's involvement in chromatin formation, we investigated gene silencing at the telomere in several mutants of the budding yeast Saccharomyces cerevisiae, which had defects in genes involved in the Ran system. A mutation of the RanGAP gene, rna1-1, caused reduced silencing at the telomere, and partial disruption of the nuclear Ran binding factor, yrb2-Δ2, increased this silencing. The reduced telomere silencing in rna1-1 cells was suppressed by a high dosage of the SIR3 gene or the SIT4 gene. Furthermore, hyperphosphorylated Sir3 protein accumulated in the rna1-1 mutant. These results suggest that RanGAP is required for the heterochromatin structure at the telomere in budding yeast. © 2007 Elsevier Inc. All rights reserved.
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| 4.
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Mizuno, Hideki ; Khurts, Shilagardi ; Seki, Takahiko ; Hirota, Yasuhide ; Kaneko, Shuichi ; Murakami, Seishi
概要:
金沢大学がん研究所<br />Telomerase, a stable complex of telomerase reverse transcriptase (TERT) and template RNA (TERC), is respo
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nsible for telomere maintenance. During purification trials of recombinant human telomerase of the two components reconstituted in insect cells, we identified two complexes of human telomerase of molecular masses 680 and 380 kDa, both of which retain telomerase activity in vitro. We show here that the former complex does not include Hsp90 (heat shock protein 90) and its telomerase activity is resistant to Hsp90 inhibitors, whereas the latter contains Hsp90 and its telomerase activity is sensitive to Hsp90 inhibitors. N-terminal of FLAG-hTERT in the former is exposed, as this complex was efficiently purified with anti-FLAG M2 affinity resin. We also identified two different telomerase complexes in HeLa cells, in addition to ectopically expressed hTERT. Most of endogenous hTERT and FLAG-hTERT was detected around 680 kDa. These two complexes in HeLa cells have the same properties as their respective reconstituted telomerases. The unstable property of the telomerase complex with Hsp90, especially in the presence of Hsp90 inhibitors, was due to proteasome-mediated degradation of hTERT, since proteasome inhibitors prevented hTERT degradation in vivo. To our knowledge, this is the first demonstration of two distinct active complexes of human telomerase ectopically expressed in insect and mammalian cells. © 2007 The Japanese Biochemical Society.
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| 5.
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Le, Thi Thu Thuy ; Zhang, Shijun ; Hayashi, Naoyuki ; Yasukawa, Mami ; Delgermaa, Luvsanjav ; Murakami, Seishi
概要:
金沢大学がん研究所<br />RNA polymerase II (RNAPII) subunit 5 (RPB5) is positioned close to DNA downstream of the initiation site
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and is the site of interaction with several regulators. Hepatitis B virus X protein (HBx) binds the central part of RPB5 to modulate activated transcription, and TFIIF subunit RAP30 interacts with the same part of RPB5 that is critical for the association between TFIIF and RNAPII. However the residues necessary for these interactions remain unknown. Here we report systematic mutagenesis of the central part of RPB5 using two-step alanine scanning libraries to pinpoint critical residues for its binding to RAP30 in the TFIIF complex and/or to HBx, and identified these residues in both mammalian cells and in an in vitro binding assay. Four residues, F76, I104, T111 and S113, are critical for both TFIIF- and HBx-binding, indicating the overlapping nature of the sites of interaction. In addition, V74 and N98 are required for HBx-binding, and T56 and L58 are needed for RAP30-binding. Interestingly the residues exposed to solvent, T111 and S113, are very close to the DNA, implying that two factors may modulate the interaction between DNA and RPB5. © 2005 The Japanese Biochemical Society.
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| 6.
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Gantulga, Davaakhuu ; Tuvshintugs, Baljinnyam ; Endo, Yoshio ; Takino, Takahisa ; Sato, Hiroshi ; Murakami, Seishi ; Yoshioka, Katsuji
概要:
金沢大学がん研究所がん分子細胞制御<br />Scaffold proteins for MAP kinase (MAPK) signalling modules play an important role in the specific
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and efficient signal transduction of the relevant MAPK cascades. Here, we investigated the function of the scaffolding protein c-Jun NH2-terminal kinase (JNK)-associated leucine zipper protein (JLP) by depleting it in cultured cells using a short hairpin RNA (shRNA) against human JLP. HeLa and DLD-1 cells stably expressing the shRNA showed a defect in cell migration. The re-expression of full-length shRNA-resistant mouse JLP rescued the impaired cell migration of the JLP-depleted HeLa cells; whereas, a C-terminal deletion mutant of mouse JLP, which failed to bind the G protein Gα13, showed little or no effect on the cell migration defect. Furthermore, although a constitutively active Gα13 enhanced the migration of control HeLa cells, the Gα13-induced cell migration was significantly suppressed in the JLP-depleted HeLa cells. Taken together, these results suggest that JLP regulates cell migration through an interaction with Gα13. © The Authors 2008. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.
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| 7.
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Kusakawa, Takashi ; Shimakami, Tetsuro ; Kaneko, Shuichi ; Yoshioka, Katsuji ; Murakami, Seishi
概要:
金沢大学がん研究所<br />Hepatitis C Virus (HCV) non-structural proteins are major components of replication complex that is modul
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ated by several host factors. We previously reported that nucleolin, a representative nucleolar marker, interacts with the NS5B through two separated sequences, amino acids (aa) 208-214 and 500-506, and that W208 in the former stretch is essential for both nucleolin-binding and HCV replication. Here we evaluated the role of the latter stretch aa 500-506 of WRHRARS in nucleolin-binding and HCV replication scanned by alanine-substituted clustered mutant (cm) or point mutant (pm). One tryptophan and three arginine residues in the sequence were found to be essential both for nucleolin-binding in vivo and HCV replication detected with a HCV subgenomic replicon transfected into Huh7 cells. NS5B-binding of nucleolin was further delineated by truncation and clustered mutants of nucleolin. Arginine-glycine-glycine (RGG) repeat in the Glycine arginine rich (GAR) domain were defined to be indispensable for NS5B-binding immunologically detected in in vivo and in vitro although short internal-truncations of RGG repeat are tolerable for NS5B-binding. These results indicate that nucleolin is a critical host factor for HCV replication through the direct interaction between W208 and several residues at the sequence, aa 500-505, of NS5B, and the long-turn motif including RGG repeat at nucleolin C-terminal. © 2007 The Japanese Biochemical Society.
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| 8.
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Tang, H. ; Delgermaa, Luvsanjav ; Huang, F. ; Oishi, N. ; Liu, L. ; He, L. ; Zhao, L. ; Murakami, Seishi
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| 9.
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Oishi, T. ; Masutomi, Kenichi ; Khurts, S. ; Nakamoto, T. ; Kaneko, Shuichi ; Murakami, Seishi
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| 10.
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Le, TTT ; Zhang, S. ; Hayashi, Naoyuki ; Yasukawa, M. ; Delgermaa, Luvsanjav ; Murakami, Seishi
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