2021/11/07 更新

写真a

フタミ ジュンイチロウ
二見 淳一郎
FUTAMI Junichiro
所属
ヘルスシステム統合科学学域 准教授
職名
准教授
プロフィール
タンパク質工学を医療現場で応用するための研究を進めています。特に不安定で凝集しやすい物性のタンパク質を変性状態のまま可溶化する独自開発技術を腫瘍免疫学分野に応用する研究を展開しています。
2012年より蛋白質医用工学研究室を主宰しています。
外部リンク

学位

  • 修士(工学)

  • 博士(工学) ( 岡山大学 )

研究キーワード

  • 生化学

  • 腫瘍免疫学

  • 生物工学

  • タンパク質工学

研究分野

  • ナノテク・材料 / 生物分子化学

  • ライフサイエンス / 腫瘍診断、治療学

  • ものづくり技術(機械・電気電子・化学工学) / バイオ機能応用、バイオプロセス工学

  • ライフサイエンス / 分子生物学

学歴

  • 岡山大学大学院   自然科学研究科   生物資源科学専攻

    1996年4月 - 1999年3月

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    国名: 日本国

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  • 岡山大学    

    1994年4月 - 1996年3月

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  • 岡山大学   工学部   生体機能応用工学科

    1990年4月 - 1994年3月

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    国名: 日本国

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経歴

  • 岡山大学大学院ヘルスシステム統合科学研究科 研究教授

    2019年12月 - 現在

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  • 岡山大学   ヘルスシステム統合科学研究科   准教授

    2018年4月 - 現在

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  • 岡山大学大学院   自然科学研究科   准教授

    2008年10月 - 2018年3月

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  • 岡山大学大学院   自然科学研究科   講師

    2005年10月 - 2008年9月

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  • 株式会社日本触媒NEDO-P/J専任研究員(岡山大学工学部共同研究員)

    2003年2月 - 2005年9月

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  • 住友製薬株式会社ゲノム科学研究所 職員(技術系)

    2002年4月 - 2003年1月

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  • 米国癌研究所(NCI-Frederick)訪問研究員

    2000年1月 - 2000年12月

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  • 日本学術振興会特別研究員(PD)

    1999年4月 - 2002年3月

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  • 日本学術振興会特別研究員(DC2)

    1997年4月 - 1999年3月

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▼全件表示

所属学協会

▼全件表示

委員歴

  • 日本生化学会   評議員  

    2016年9月 - 現在   

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    団体区分:学協会

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  • 日本生物工学会   和文誌編集委員  

    2016年6月 - 現在   

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    団体区分:学協会

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  • 日本生物工学会   代議員  

    2013年 - 現在   

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    団体区分:学協会

    日本生物工学会

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論文

  • Unusual aggregation property of recombinantly expressed cancer-testis antigens in mammalian cells. 査読 国際誌

    Hannaneh Ahmadi, Kohei Shogen, Kana Fujita, Tomoko Honjo, Kazuhiro Kakimi, Junichiro Futami

    Journal of biochemistry   170 ( 3 )   435 - 443   2021年10月

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    担当区分:責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Transient expression of human intracellular proteins in human embryonic kidney (HEK) 293 cells is a reliable system for obtaining soluble proteins with biologically active conformations. Contrary to conventional concepts, we found that recombinantly expressed intracellular cancer-testis antigens (CTAs) showed frequent aggregation in HEK293 cells. Although experimental subcellular localization of recombinant CTAs displayed proper cytosolic or nuclear localization, some proteins showed aggregated particles in the cell. This aggregative property was not observed in recombinant housekeeping proteins. No significant correlation was found between the aggregative and biophysical properties, such as hydrophobicity, contents of intrinsically disordered regions and expression levels, of CTAs. These results can be explained in terms of structural instability of CTAs, which are specifically expressed in the testis and aberrantly expressed in cancer cells and function as a hub in the protein-protein network using intrinsically disordered regions. Hence, we speculate that recombinantly expressed CTAs failed to form this protein complex. Thus, unfolded CTAs formed aggregated particles in the cell.

    DOI: 10.1093/jb/mvab081

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  • 創意化学的な生体高分子の生物工学 特集によせて

    二見 淳一郎, 山口 哲志

    生物工学会誌   99 ( 4 )   162   2021年

  • 創意化学的な生体高分子の生物工学 変性タンパク質工学と免疫プロファイリング技術開発

    二見淳一郎

    生物工学会誌   99 ( 4 )   2021年

  • A suitable and effective stepwise oxidative refolding procedure for highly‐cationic tetrameric avidin in nucleic acid free conditions 査読 国際誌

    Shuichiro Kimura, Koreyoshi Imamura, Junichiro Futami

    Biotechnology Progress   36 ( 5 )   e3031   2020年9月

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    担当区分:責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Wiley  

    Optimized conditions are needed to refold recombinant proteins from bacterial inclusion bodies into their biologically active conformations. In this study, we found two crucial requirements for efficient refolding of cationic tetrameric chicken avidin. The first step is to eliminate nucleic acid contaminants from the bacterial inclusion body. The electrostatic interactions between the remaining nucleic acids and proteins strongly enhanced protein aggregation during the refolding process. The cysteine specific reversible S-cationization procedure was successfully employed for large-scale preparation of nucleic acid free denatured protein without purification tag system. The second step is the intramolecular disulfide formation prior to refolding in dialysis removing denaturant. Disulfide intact monomeric avidin showed efficient formation of biologically active tetrameric conformation during the refolding process. Using this optimized refolding procedure, highly cationic avidin derivative designed as an intracellular delivery carrier of biotinylated protein was successfully prepared.

    DOI: 10.1002/btpr.3031

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    その他リンク: https://onlinelibrary.wiley.com/doi/full-xml/10.1002/btpr.3031

  • Serum Antibody Against NY-ESO-1 and XAGE1 Antigens Potentially Predicts Clinical Responses to Anti-Programmed Cell Death-1 Therapy in NSCLC. 査読 国際誌

    Yoshihiro Ohue, Koji Kurose, Takahiro Karasaki, Midori Isobe, Takaaki Yamaoka, Junichiro Futami, Isao Irei, Takeshi Masuda, Masaaki Fukuda, Akitoshi Kinoshita, Hirokazu Matsushita, Katsuhiko Shimizu, Masao Nakata, Noboru Hattori, Hiroyuki Yamaguchi, Minoru Fukuda, Ryohei Nozawa, Kazuhiro Kakimi, Mikio Oka

    Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer   14 ( 12 )   2071 - 2083   2019年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    INTRODUCTION: Programmed cell death-1 (PD-1) inhibitors effectively treat NSCLC and prolong survival. Robust biomarkers for predicting clinical benefits of good response and long survival with anti-PD-1 therapy have yet to be identified; therefore, predictive biomarkers are needed to select patients with benefits. METHODS: We conducted a prospective study to explore whether serum antibody against NY-ESO-1 and/or XAGE1 cancer-testis antigens predicted primarily good clinical response and secondarily long survival with anti-PD-1 therapy for NSCLC. The serum antibody was detected by enzyme-linked immunosorbent assay, and tumor immune microenvironment and mutation burden were analyzed by immunohistochemistry and next-generation sequencing. RESULTS: In the discovery cohort (n = 13), six antibody-positive NSCLC cases responded to anti-PD-1 therapy (two complete and four partial responses), whereas seven antibody-negative NSCLC cases did not. Antibody positivity was associated with good response and survival, regardless of tumor programmed death ligand 1 (PD-L1) expression, mutation burden, and CD8+ T-cell infiltration. In the validation cohort (n = 75), 17 antibody-positive NSCLC cases responded well to anti-PD-1 therapy as compared with 58 negative NSCLC cases (objective response rate 65% versus 19%, p = 0.0006) and showed significantly prolonged progression-free survival and overall survival. Antibody titers highly correlated with tumor reduction rates. In the multivariate analysis, response biomarkers were tumor programmed death ligand 1 expression and antibody positivity, and only antibody positivity was a significantly better predictive biomarker of progression-free survival (hazard ratio = 0.4, p = 0.01) and overall survival (hazard ratio = 0.2, p = 0.004). CONCLUSIONS: Our results suggest that NY-ESO-1 and/or XAGE1 serum antibodies are useful biomarkers for predicting clinical benefits in anti-PD-1 therapy for NSCLC and probably for other cancers.

    DOI: 10.1016/j.jtho.2019.08.008

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  • 研究室主宰者の自戒 招待

    二見 淳一郎

    生物工学会誌   97 ( 11 )   671 - 674   2019年11月

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    記述言語:日本語  

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  • Upregulation of Mobility in Pancreatic Cancer Cells by Secreted S100A11 Through Activation of Surrounding Fibroblasts. 査読 国際誌

    Yosuke Mitsui, Nahoko Tomonobu, Masami Watanabe, Rie Kinoshita, I Wayan Sumardika, Chen Youyi, Hitoshi Murata, Ken-Ichi Yamamoto, Takuya Sadahira, Acosta Gonzalez Herik Rodrigo, Hitoshi Takamatsu, Kota Araki, Akira Yamauchi, Masahiro Yamamura, Hideyo Fujiwara, Yusuke Inoue, Junichiro Futami, Ken Saito, Hidekazu Iioka, Eisaku Kondo, Masahiro Nishibori, Shinichi Toyooka, Yasuhiko Yamamoto, Yasutomo Nasu, Masakiyo Sakaguchi

    Oncology research   27 ( 8 )   945 - 956   2019年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    S100A11, a member of the S100 family of proteins, is actively secreted from pancreatic ductal adenocarcinoma (PDAC) cells. However, the role of the extracellular S100A11 in PDAC progression remains unclear. In the present study, we investigated the extracellular role of S100A11 in crosstalking between PDAC cells and surrounding fibroblasts in PDAC progression. An abundant S100A11 secreted from pancreatic cancer cells stimulated neighboring fibroblasts through receptor for advanced glycation end products (RAGE) upon S100A11 binding and was followed by not only an enhanced cancer cell motility in vitro but also an increased number of the PDAC-derived circulating tumor cells (CTCs) in vivo. Mechanistic investigation of RAGE downstream in fibroblasts revealed a novel contribution of a mitogen-activated protein kinase kinase kinase (MAPKKK), tumor progression locus 2 (TPL2), which is required for positive regulation of PDAC cell motility through induction of cyclooxygenase 2 (COX2) and its catalyzed production of prostaglandin E2 (PGE2), a strong chemoattractive fatty acid. The extracellularly released PGE2 from fibroblasts was required for the rise in cellular migration as well as infiltration of their adjacent PDAC cells in a coculture setting. Taken together, our data reveal a novel role of the secretory S100A11 in PDAC disseminative progression through activation of surrounding fibroblasts triggered by the S100A11-RAGE-TPL2-COX2 pathway. The findings of this study will contribute to the establishment of a novel therapeutic antidote to PDACs that are difficult to treat by regulating cancer-associated fibroblasts (CAFs) through targeting the identified pathway.

    DOI: 10.3727/096504019X15555408784978

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  • Newly developed anti-S100A8/A9 monoclonal antibody efficiently prevents lung tropic cancer metastasis. 査読 国際誌

    Rie Kinoshita, Hiroki Sato, Akira Yamauchi, Yuta Takahashi, Yusuke Inoue, I Wayan Sumardika, Youyi Chen, Nahoko Tomonobu, Kota Araki, Kazuhiko Shien, Shuta Tomida, Hidejiro Torigoe, Kei Namba, Eisuke Kurihara, Yusuke Ogoshi, Hitoshi Murata, Ken-Ichi Yamamoto, Junichiro Futami, Endy Widya Putranto, I Made Winarsa Ruma, Hiromasa Yamamoto, Junichi Soh, Toshihiko Hibino, Masahiro Nishibori, Eisaku Kondo, Shinichi Toyooka, Masakiyo Sakaguchi

    International journal of cancer   145 ( 2 )   569 - 575   2019年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    The metastatic dissemination of cancer cells to remote areas of the body is the most problematic aspect in cancer patients. Among cancers, melanomas are notoriously difficult to treat due to their significantly high metastatic potential even during early stages. Hence, the establishment of advanced therapeutic approaches to regulate metastasis is required to overcome the melanoma disease. An accumulating mass of evidence has indicated a critical role of extracellular S100A8/A9 in melanoma distant metastasis. Lung S100A8/A9 is induced by melanoma cells from distant organs and it attracts these cells to its enriched lung environment since melanoma cells possess several receptors that sense the S100A8/A9 ligand. We hence aimed to develop a neutralizing antibody against S100A8/A9 that would efficiently block melanoma lung metastasis. Our protocol provided us with one prominent antibody, Ab45 that efficiently suppressed not only S100A8/A9-mediated melanoma mobility but also lung tropic melanoma metastasis in a mouse model. This prompted us to make chimeric Ab45, a chimera antibody consisting of mouse Ab45-Fab and human IgG2-Fc. Chimeric Ab45 also showed significant inhibition of the lung metastasis of melanoma. From these results, we have high hopes that the newly produced antibody will become a potential biological tool to block melanoma metastasis in future clinical settings.

    DOI: 10.1002/ijc.31982

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  • Critical role of the MCAM-ETV4 axis triggered by extracellular S100A8/A9 in breast cancer aggressiveness. 査読 国際誌

    Youyi Chen, I Wayan Sumardika, Nahoko Tomonobu, Rie Kinoshita, Yusuke Inoue, Hidekazu Iioka, Yosuke Mitsui, Ken Saito, I Made Winarsa Ruma, Hiroki Sato, Akira Yamauchi, Hitoshi Murata, Ken-Ichi Yamamoto, Shuta Tomida, Kazuhiko Shien, Hiromasa Yamamoto, Junichi Soh, Junichiro Futami, Miyoko Kubo, Endy Widya Putranto, Takashi Murakami, Ming Liu, Toshihiko Hibino, Masahiro Nishibori, Eisaku Kondo, Shinichi Toyooka, Masakiyo Sakaguchi

    Neoplasia (New York, N.Y.)   21 ( 7 )   627 - 640   2019年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Metastatic breast cancer is the leading cause of cancer-associated death in women. The progression of this fatal disease is associated with inflammatory responses that promote cancer cell growth and dissemination, eventually leading to a reduction of overall survival. However, the mechanism(s) of the inflammation-boosted cancer progression remains unclear. In this study, we found for the first time that an extracellular cytokine, S100A8/A9, accelerates breast cancer growth and metastasis upon binding to a cell surface receptor, melanoma cell adhesion molecule (MCAM). Our molecular analyses revealed an important role of ETS translocation variant 4 (ETV4), which is significantly activated in the region downstream of MCAM upon S100A8/A9 stimulation, in breast cancer progression in vitro as well as in vivo. The MCAM-mediated activation of ETV4 induced a mobile phenotype called epithelial-mesenchymal transition (EMT) in cells, since we found that ETV4 transcriptionally upregulates ZEB1, a strong EMT inducer, at a very high level. In contrast, downregulation of either MCAM or ETV4 repressed EMT, resulting in greatly weakened tumor growth and lung metastasis. Overall, our results revealed that ETV4 is a novel transcription factor regulated by the S100A8/A9-MCAM axis, which leads to EMT through ZEB1 and thereby to metastasis in breast cancer cells. Thus, therapeutic strategies based on our findings might improve patient outcomes.

    DOI: 10.1016/j.neo.2019.04.006

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  • Melanoma cell adhesion molecule is the driving force behind the dissemination of melanoma upon S100A8/A9 binding in the original skin lesion. 査読 国際誌

    Youyi Chen, I Wayan Sumardika, Nahoko Tomonobu, I Made Winarsa Ruma, Rie Kinoshita, Eisaku Kondo, Yusuke Inoue, Hiroki Sato, Akira Yamauchi, Hitoshi Murata, Ken-Ichi Yamamoto, Shuta Tomida, Kazuhiko Shien, Hiromasa Yamamoto, Junichi Soh, Ming Liu, Junichiro Futami, Kaori Sasai, Hiroshi Katayama, Miyoko Kubo, Endy Widya Putranto, Toshihiko Hibino, Bei Sun, Masahiro Nishibori, Shinichi Toyooka, Masakiyo Sakaguchi

    Cancer letters   452   178 - 190   2019年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Since metastasis accounts for the majority of cancer-associated deaths, studies on the mechanisms of metastasis are needed to establish innovative strategies for cancer treatment. We previously reported that melanoma cell adhesion molecule (MCAM) functions as a critical receptor for S100A8/A9, and binding of S100A8/A9 to MCAM results in the migration of melanoma cells to lung tissue. However, the critical role of MCAM in the original melanoma skin lesion is still not clear. In this study, we aimed to determine the importance of the S100A8/A9-MCAM axis in melanoma dissemination in a skin lesion as a critical early step for metastasis. Mechanistic studies revealed the downstream signaling of MCAM that signaled the induction of metastasis. S100A8/A9-MCAM binding activates mitogen-activated protein kinase kinase kinase 8 (MAP3K8), also termed TPL2, leading to strong activation of the transcription factor ETV4 and subsequent induction of matrix metalloproteinase-25 (MMP25), and finally to induction of melanoma lung tropic metastasis. Collectively, our results demonstrate a crucial role of the S100A8/A9-MCAM signaling axis in metastatic onset of melanoma cells and indicate that strategies targeting the identified pathway may be useful for the establishment of innovative anti-cancer therapies.

    DOI: 10.1016/j.canlet.2019.03.023

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  • Extracellular S100A11 Plays a Critical Role in Spread of the Fibroblast Population in Pancreatic Cancers. 査読 国際誌

    Hitoshi Takamatsu, Ken-Ichi Yamamoto, Nahoko Tomonobu, Hitoshi Murata, Yusuke Inoue, Akira Yamauchi, I Wayan Sumardika, Youyi Chen, Rie Kinoshita, Masahiro Yamamura, Hideyo Fujiwara, Yosuke Mitsui, Kota Araki, Junichiro Futami, Ken Saito, Hidekazu Iioka, I Made Winarsa Ruma, Endy Widya Putranto, Masahiro Nishibori, Eisaku Kondo, Yasuhiko Yamamoto, Shinichi Toyooka, Masakiyo Sakaguchi

    Oncology research   27 ( 6 )   713 - 727   2019年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    The fertile stroma in pancreatic ductal adenocarcinomas (PDACs) has been suspected to greatly contribute to PDAC progression. Since the main cell constituents of the stroma are fibroblasts, there is crosstalking(s) between PDAC cells and surrounding fibroblasts in the stroma, which induces a fibroblast proliferation burst. We have reported that several malignant cancer cells including PDAC cells secrete a pronounced level of S100A11, which in turn stimulates proliferation of cancer cells via the receptor for advanced glycation end products (RAGE) in an autocrine manner. Owing to the RAGE+ expression in fibroblasts, the extracellular abundant S100A11 will affect adjacent fibroblasts. In this study, we investigated the significance of the paracrine axis of S100A11-RAGE in fibroblasts for their proliferation activity. In in vitro settings, extracellular S100A11 induced upregulation of fibroblast proliferation. Our mechanistic studies revealed that the induction is through RAGE-MyD88-mTOR-p70 S6 kinase upon S100A11 stimulation. The paracrine effect on fibroblasts is linked mainly to triggering growth but not cellular motility. Thus, the identified pathway might become a potential therapeutic target to suppress PDAC progression through preventing PDAC-associated fibroblast proliferation.

    DOI: 10.3727/096504018X15433161908259

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  • exSSSRs (extracellular S100 soil sensor receptors)-Fc fusion proteins work as prominent decoys to S100A8/A9-induced lung tropic cancer metastasis. 査読 国際誌

    Rie Kinoshita, Hiroki Sato, Akira Yamauchi, Yuta Takahashi, Yusuke Inoue, I Wayan Sumardika, Youyi Chen, Nahoko Tomonobu, Kota Araki, Kazuhiko Shien, Shuta Tomida, Hidejiro Torigoe, Kei Namba, Eisuke Kurihara, Yusuke Ogoshi, Hitoshi Murata, Ken-Ichi Yamamoto, Junichiro Futami, Endy Widya Putranto, I Made Winarsa Ruma, Hiromasa Yamamoto, Junichi Soh, Toshihiko Hibino, Masahiro Nishibori, Eisaku Kondo, Shinichi Toyooka, Masakiyo Sakaguchi

    International journal of cancer   144 ( 12 )   3138 - 3145   2019年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Within the "seed and soil" theory of organ tropic cancer metastasis is a growing compilation of evidence that S100A8/A9 functions as a soil signal that attracts cancer cells to certain organs, which prove beneficial to their growth. S100A8/A9-sensing receptors including Toll-like receptor 4 (TLR4), advanced glycation end products (RAGE), and also important receptors we recently succeeded in identifying (EMMPRIN, NPTNβ, MCAM, and ALCAM) have the potential to become promising therapeutic targets. In our study, we prepared extracellular regions of these novel molecules and fused them to human IgG2-Fc to extend half-life expectancy, and we evaluated the anti-metastatic effects of the purified decoy proteins on metastatic cancer cells. The purified proteins markedly suppressed S100A8/A9-mediated lung tropic cancer metastasis. We hence expect that our novel biologics may become a prominent medicine to prevent cancer metastasis in clinical settings through cutting the linkage between "seed and soil".

    DOI: 10.1002/ijc.31945

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  • Neuroplastin-β mediates S100A8/A9-induced lung cancer disseminative progression. 査読 国際誌

    Sumardika IW, Chen Y, Tomonobu N, Kinoshita R, Ruma IMW, Sato H, Kondo E, Inoue Y, Yamauchi A, Murata H, Yamamoto KI, Tomida S, Shien K, Yamamoto H, Soh J, Futami J, Putranto EW, Hibino T, Nishibori M, Toyooka S, Sakaguchi M

    Molecular carcinogenesis   58 ( 6 )   980 - 995   2019年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1002/mc.22987

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  • β-1,3-Galactosyl-<i>O</i>-Glycosyl-Glycoprotein β-1,6-<i>N</i>-Acetylglucosaminyltransferase 3 Increases MCAM Stability, Which Enhances S100A8/A9-Mediated Cancer Motility. 査読 国際誌

    Sumardika IW, Youyi C, Kondo E, Inoue Y, Ruma IMW, Murata H, Kinoshita R, Yamamoto KI, Tomida S, Shien K, Sato H, Yamauchi A, Futami J, Putranto EW, Hibino T, Toyooka S, Nishibori M, Sakaguchi M

    Oncology research   26 ( 3 )   431 - 444   2018年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.3727/096504017X15031557924123

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  • Mitochondrial determinants of mammalian longevity 査読

    Yasuhiro Kitazoe, Masami Hasegawa, Masashi Tanaka, Midori Futami, Junichiro Futami

    OPEN BIOLOGY   7 ( 10 )   2017年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ROYAL SOC  

    Current ageing theories are far from satisfactory because of the many determinants involved in ageing. The well-known rate-of-living theory assumes that the product (lifetime energy expenditure, LEE) of maximum lifespan (MLS) and mass-specific basal metabolic rate (msBMR) is approximately constant. Although this theory provides a significant inverse correlation between msBMR and MLS as a whole for mammals, it remains problematic for two reasons. First, several interspecies studies within respective orders (typically within rodents) have shown no inverse relationships between msBMR and MLS. Second, LEE values widely vary in mammals and birds. Here, to solve these two problems, we introduced a new quantity designated as mitochondrial (mt) lifetime energy output, mtLEO = MLS x mtMR, in place of LEE, by using the mt metabolic rate (mtMR) per mitochondrion. Thereby, we found that mtLEO values were distributed more narrowly than LEE ones, and strongly correlated with the four amino-acid variables (AAVs) of Ser, Thr and Cys contents and hydrophobicity of mtDNA-encoded membrane proteins (these variables were related to the stability of these proteins). Consequently, only these two mt items, mtMR and the AAVs, solved the above-mentioned problems in the rate-of-living theory, and thus extensively improved the correlation with MLS compared with that given by LEE.

    DOI: 10.1098/rsob.170083

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  • Evaluation of irreversible protein thermal inactivation caused by breakage of disulphide bonds using methanethiosulphonate 査読

    Junichiro Futami, Ai Miyamoto, Atsushi Hagimoto, Shigeyuki Suzuki, Midori Futami, Hiroko Tada

    SCIENTIFIC REPORTS   7 ( 1 )   12471 - 3275   2017年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NATURE PUBLISHING GROUP  

    Many extracellular globular proteins have evolved to possess disulphide bonds in their native conformations, which aids in thermodynamic stabilisation. However, disulphide bond breakage by heating leads to irreversible protein denaturation through disulphide-thiol exchange reactions. In this study, we demonstrate that methanethiosulphonate (MTS) specifically suppresses the heat-induced disulphide-thiol exchange reaction, thus improving the heat-resistance of proteins. In the presence of MTS, small globular proteins that contain disulphides can spontaneously refold from heat-denatured states, maintaining wild-type disulphide pairing. Because the disulphide-thiol exchange reaction is triggered by the generation of catalytic amounts of perthiol or thiol, rapid and specific perthiol/thiol protection by MTS reagents prevents irreversible denaturation. Combining MTS reagents with another additive that suppresses chemical modifications, glycinamide, further enhanced protein stabilisation. In the presence of these additives, reliable remnant activities were observed even after autoclaving. However, immunoglobulin G and biotin-binding protein, which are both composed of tetrameric quaternary structures, failed to refold from heat-denatured states, presumably due to chaperon requirements. Elucidation of the chemical modifications involved in irreversible thermoinactivation is useful for the development of preservation buffers with optimum constitutions for specific proteins. In addition, the impact of disulphide bond breakage on the thermoinactivation of proteins can be evaluated using MTS reagents.

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  • Robust cancer-specific gene expression by a novel cassette with hTERT and CMV promoter elements 査読

    Masakiyo Sakaguchi, Takuya Sadahira, Hideo Ueki, Rie Kinoshita, Hitoshi Murata, Ken-Ichi Yamamoto, Junichiro Futami, Yasutomo Nasu, Kazuhiko Ochiai, Hiromi Kumon, Nam-Ho Huh, Masami Watanabe

    ONCOLOGY REPORTS   38 ( 2 )   1108 - 1114   2017年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SPANDIDOS PUBL LTD  

    We developed and validated a novel hTERT/CMV promoter element-driven gene expression cassette that can robustly enhance cancer-specific gene expression. The following gene expressional elements were located in tandem within the plasmid construct: [hTERT core promoter, cytomegalovirus (CMV) minimized promoter, RU5' sequence, an inserted gene, BGH polyA, hTERT enhancer]; this is hereafter referred to as the hT/Cm-R-hT construct. Using various human cancer cell lines and normal cells, the cancer-specific transcription of the green fluorescent protein (GFP) gene was examined by western blotting and fluorescence microscopy. Cancer-specific gene expression was robustly achieved in the hT/Cm-R-hT plasmid in comparison to the other control hT/Cm-driven construct. Notably, the expression level of GFP observed in the hT/Cm-RhT-driven construct was superior to that of the control plasmid with the conventional CMV promoter in HEK293 cells, which are known to possess higher hTERT activity than normal cells. We next examined the availability of hT/Cm-R-hT in detecting the target GFP expressing cancer cells from human peripheral blood mononuclear cells (PBMCs). The hT/Cm-R-hT plasmid successfully induced cancer-specific gene expression; the robust expression of GFP was observed in target HeLa cancer cells, whereas GFP was not visibly expressed in normal PBMCs. The plasmid allowed for the selective visualization of viable HeLa cancer cells in mixed cell cultures containing up to 10000-fold more PBMCs. These findings indicate that the hT/Cm-R-hT expressional system is a valuable tool for detecting viable cancer cells mixed with normal cells. The current system can therefore be applied to the in vitro detection of cancer cells that are disseminated in the blood and other types of body fluid in vivo. Since the current system can also be applied to other types of vectors, including virus vectors, this approach using the hTERT promoter-based construct is expected to become a valuable tool for enhancing cancer specific gene expression.

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  • Expression of tumor suppressor REIC/Dkk-3 by a newly improved adenovirus vector with insertion of a hTERT promoter at the 3'-side of the transgene 査読

    Endy Widya Putranto, Rie Kinoshita, Masami Watanabe, Takuya Sadahira, Hitoshi Murata, Ken-Ichi Yamamoto, Junichiro Futami, Ken Kataoka, Yusuke Inoue, I. Made Winarsa Ruma, I. Wayan Sumardika, Chen Youyi, Miyoko Kubo, Yoshihiko Sakaguchi, Kenji Saito, Yasutomo Nasu, Hiromi Kumon, Nam-Ho Huh, Masakiyo Sakaguchi

    ONCOLOGY LETTERS   14 ( 1 )   1041 - 1048   2017年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SPANDIDOS PUBL LTD  

    Reduced expression in immortalized cells (REIC)/Dickkopf-3 (Dkk-3) overexpression, induced using an adenovirus (Ad)-REIC, has been revealed to have a dramatic therapeutic effect on multiple types of cancer. To achieve an improved therapeutic effect from Ad-REIC on cancer, our group previously developed an enhanced gene expression system, the C-TSC cassette [cytomegalovirus (CMV)-RU5' located upstream (C); another promoter unit composed of triple tandem promoters, human telomerase reverse transcriptase (hTERT), simian virus 40 and CMV, located downstream of the cDNA (TSC); plus a polyadenylation (polyA) signal]. When applied to the conventional Ad-REIC, this novel system induced the development of an enhanced product, Ad-C-TSC-REIC, which exhibited a noticeable anticancer effect. However, there were difficulties in terms of Ad-C-TSC-REIC productivity in HEK293 cells, which are a widely used donor cell line for viral production. Productivity of Ad-C-TSC-REIC was significantly reduced compared with the conventional Ad-REIC, as the Ad-C-TSC-REIC had a significantly higher ability to induce apoptotic cell death of not only various types of cancer cell, but also HEK293 cells. The present study aimed to overcome this problem by modifying the C-TSC structure, resulting in an improved candidate: A C-T cassette (C: CMV-RU5' located upstream; T: another promoter unit composed of a single hTERT promoter, located downstream of the cDNA plus a polyA signal), which demonstrated gene expression comparable to that of the C-TSC system. The improved adenovirus REIC/Dkk-3 product with the C-T cassette, named Ad-C-T-REIC, exhibited a higher expression level of REIC/Dkk3, similar to that of Ad-C-TSC-REIC. Notably, the vector mitigated the cell death of donor HEK293 cells, resulting in a higher rate of production of its adenovirus. These results indicated that Ad-C-T-REIC has the potential to be a useful tool for application in cancer gene therapy.

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  • Enhanced in-cell folding of reversibly cationized transcription factor using amphipathic peptide 査読

    Midori Futami, Tomoki Nakano, Mayu Yasunaga, Masahiro Makihara, Takashi Asama, Yoshihisa Hagihara, Yoshihiro Nakajima, Junichiro Futami

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   123 ( 4 )   419 - 424   2017年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SOC BIOSCIENCE BIOENGINEERING JAPAN  

    The intracellular delivery of functionally active transcription factor proteins is emerging as a promising technique for artificial regulation of cellular functions. However, in addition to the cell membrane, which acts as a barrier to macromolecules, the aggregation-favored properties of structurally flexible transcription factor proteins limit the application of this method. In-cell folding technique can be used to overcome these issues. This technique solubilizes denatured protein by reversible alkyl-disulfide cationization (S-cationization), and simultaneously endows efficient intracellular delivery and folding to the biologically active conformation in the reducing environment of the cytosol. Because cationized protein is internalized into cells by adsorption-mediated endocytosis, endosomal escape is crucial for this technique. In this study, we utilized a sensitive luciferase reporter gene assay to quantitatively evaluate in-cell folding of the artificial transcription factor GAIA-VP16. Although the cationic moiety of S-cationized protein was slightly affected, co-transduction of amphipathic peptide Endo-PORTER dramatically improved in-cell folding efficiency. Live cell imaging of fluorescent-labeled GAL4-VP16 revealed that some of the proteins diffused into the cytosol and nucleus through co-transduction with Endo-PORTER. Real-time monitoring of light output of luciferase revealed the kinetics of in-cell folding, supporting that endosomal-release assisted by Endo-PORTER was stimulated by endosome acidification. Because this method can transduce proteins uniformly and repeatedly into living cells, S-cationized transcription factor proteins are widely applicable for the artificial regulation of cellular functions. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.

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  • タンパク質を巻き戻すコツと原理

    二見淳一郎

    生物工学会誌   95 ( 6 )   328 - 332   2017年

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  • ß-1,3-galactosyl-O-glycosyl-glycoprotein ß-1,6-N-acetylglucosaminyltransferase 3 Increases MCAM Stability, Which Enhances S100A8/A9-Mediated Cancer Motility. 査読

    Sumardika IW, Youyi C, Kondo E, Inoue Y, Ruma IMW, Murata H, Kinoshita R, Yamamoto KI, Tomida S, Shien K, Satoh H, Yamauchi A, Futami J, Putranto EW, Hibino T, Toyooka S, Nishibori M, Sakaguchi M

    Oncol Res   2017年

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  • Active Secretion of Dimerized S100A11 Induced by the Peroxisome in Mesothelioma Cells. 査読 国際誌

    Satomi Saho, Hiroki Satoh, Eisaku Kondo, Yusuke Inoue, Akira Yamauchi, Hitoshi Murata, Rie Kinoshita, Ken-Ichi Yamamoto, Junichiro Futami, Endy Widya Putranto, I Made Winarsa Ruma, I Wayan Sumardika, Chen Youyi, Ken Suzawa, Hiromasa Yamamoto, Junichi Soh, Shuta Tomida, Yoshihiko Sakaguchi, Ken Saito, Hidekazu Iioka, Nam-Ho Huh, Shinichi Toyooka, Masakiyo Sakaguchi

    Cancer microenvironment : official journal of the International Cancer Microenvironment Society   9 ( 2-3 )   93 - 105   2016年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    S100A11, a small Ca2+ binding protein, acts extracellularly as a mediator of cancer progression. That raises the question of how a protein that lacks the classical secretory signal is able to be secreted outside cells without being damaged. Some insights into this question have been obtained, and there has been accumulating evidence indicating a pivotal role of a non-classical vesicle-mediated pathway using lysosomes or peroxisomes for the protein secretion. To obtain a more precise insight into the secretory mechanism of S100A11, we first screened representative cancer cells exhibiting significantly active secretion of S100A11. From the results of profiling, we turned our attention to aggressive cancer mesothelioma cells. In mesothelioma cells, we found that abundant dimeric S100A11 was produced selectively in the peroxisome after transportation of monomeric S100A11 through an interaction with PEX14, a peroxisome membrane protein, resulting in peroxisomal secretion of dimerized S100A11. In an extracellular environment in vitro, dimerized S100A11 promoted mesothelial cell invasion indirectly with the help of fibroblast cells. Overall, the results indicate that the peroxisome functions as an essential vesicle for the production of dimerized S100A11 and the subsequent secretion of the protein from mesothelioma cells and that peroxisome-mediated secretion of dimerized S100A11 might play a critical role in mesothelioma progression in a tumor microenvironment.

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  • MCAM, as a novel receptor for S100A8/A9, mediates progression of malignant melanoma through prominent activation of NF-κB and ROS formation upon ligand binding. 査読

    Ruma IM, Putranto EW, Kondo E, Murata H, Watanabe M, Huang P, Kinoshita R, Futami J, Inoue Y, Yamauchi A, Sumardika IW, Youyi C, Yamamoto KI, Nasu Y, Nishibori M, Hibino T, Sakaguchi M

    Clinical & experimental metastasis   33 ( 6 )   609 - 627   2016年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • An efficient method for the preparation of preferentially heterodimerized recombinant S100A8/A9 coexpressed in Escherichia coli 査読

    Junichiro Futami, Yuki Atago, Akari Azuma, Endy Widya Putranto, Rie Kinoshita, Hitoshi Murata, Masakiyo Sakaguchi

    Biochemistry and Biophysics Reports   6   94 - 100   2016年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Elsevier B.V.  

    It is now known that multicomponent protein assemblies strictly regulate many protein functions. The S100 protein family is known to play various physiological roles, which are associated with alternative complex formations. To prepare sufficient amounts of heterodimeric S100A8 and S100A9 proteins, we developed a method for bicistronic coexpression from a single-vector system using Escherichia coli cells as a host. The complex formation between S100A8 and S100A9 appears to be dependent on the thermodynamic stability of the protein during expression. The stable S100A8/A9 heterodimer complex spontaneously formed during coexpression, and biologically active samples were purified by cation-exchange chromatography. Semi-stable homodimers of S100A8 and S100A9 were also formed when expressed individually. These results suggest that the assembly of S100 protein complexes might be regulated by expression levels of partner proteins in vivo. Because protein assembly occurs rapidly after protein synthesis, coexpression of relevant proteins is crucial for the design of multicomponent recombinant protein expression systems.

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  • 【特集】がん医療の新たな展開:腫瘍免疫応答の活性化を測定する抗体検査技術の開発 招待

    二見 淳一郎

    バイオインダストリー   2016年5月

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    記述言語:日本語   掲載種別:研究論文(学術雑誌)  

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  • Identification of novel extracellular protein for PCB/biphenyl metabolism in Rhodococcus jostii RHA1 査読

    Yuki Atago, Jun Shimodaira, Naoto Araki, Nor'azizi Bin Othman, Zuriati Zakaria, Masao Fukuda, Junichiro Futami, Hirofumi Hara

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   80 ( 5 )   1012 - 1019   2016年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:TAYLOR & FRANCIS LTD  

    Rhodococcus jostii RHA1 (RHA1) degrades polychlorinated biphenyl (PCB) via co-metabolism with biphenyl. To identify the novel open reading frames (ORFs) that contribute to PCB/biphenyl metabolism in RHA1, we compared chromatin immunoprecipitation chip and transcriptomic data. Six novel ORFs involved in PCB/biphenyl metabolism were identified. Gene deletion mutants of these 6 ORFs were made and were tested for their ability to grow on biphenyl. Interestingly, only the ro10225 deletion mutant showed deficient growth on biphenyl. Analysis of Ro10225 protein function showed that growth of the ro10225 deletion mutant on biphenyl was recovered when exogenous recombinant Ro10225 protein was added to the culture medium. Although Ro10225 protein has no putative secretion signal sequence, partially degraded Ro10225 protein was detected in conditioned medium from wild-type RHA1 grown on biphenyl. This Ro10225 fragment appeared to form a complex with another PCB/biphenyl oxidation enzyme. These results indicated that Ro10225 protein is essential for the formation of the PCB/biphenyl dioxygenase complex in RHA1.

    DOI: 10.1080/09168451.2015.1127134

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  • 硬い肉をやわらかくする

    二見淳一郎

    Fuji Sankei Business i. よくわかるバイオ(第26回)   2016年

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  • Sensitive Multiplexed Quantitative Analysis of Autoantibodies to Cancer Antigens with Chemically S-Cationized Full-Length and Water-Soluble Denatured Proteins 査読

    Junichiro Futami, Hidenori Nonomura, Momoko Kido, Naomi Niidoi, Nao Fujieda, Akihiro Hosoi, Kana Fujita, Komako Mandai, Yuki Atago, Rie Kinoshita, Tomoko Honjo, Hirokazu Matsushita, Akiko Uenaka, Eiichi Nakayama, Kazuhiro Kakimi

    BIOCONJUGATE CHEMISTRY   26 ( 10 )   2076 - 2084   2015年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER CHEMICAL SOC  

    Humoral immune responses against tumor-associated antigens (TAAs) or cancer/testis antigens (CTAs) aberrantly expressed in tumor cells are frequently observed in cancer patients. Recent clinical studies have elucidated that anticancer immune responses with increased levels of anti-TAA/CTA antibodies improve cancer survival rates. Thus, these antibody levels are promising biomarkers for diagnosing the efficiency of cancer immunotherapy. Full-length antigens are favored for detecting anti-TAA/CTA antibodies because candidate antigen proteins contain multiple epitopes throughout their structures. In this study, we developed a methodology to prepare purified water-soluble and full-length antigens by using cysteine sulfhydryl group cationization (S-cationization) chemistry. S-Cationized antigens can be prepared from bacterial inclusion bodies, and they exhibit improved protein solubility but preserved antigenicity. Anti-TAA/CTA antibodies detected in cancer patients appeared to recognize linear epitopes, as well as conformational epitopes, and because the frequency of cysteine side-residues on the epitope-paratope interface was low, any adverse effects of S-cationization were virtually negligible for antibody binding. Furthermore, S-cationized antigen-immobilized Luminex beads could be successfully used in highly sensitive quantitative-multiplexed assays. Indeed, patients with a more broadly induced serum anti-TAA/CTA antibody level showed improved progression-free survival after immunotherapy. The comprehensive anti-TAA/CTA assay system, which uses S-cationized full-length and water-soluble recombinant antigens, may be a useful diagnostic tool for assessing the efficiency of cancer immunotherapy.

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  • The cysteine-rich core domain of REIC/Dkk-3 is critical for its effect on monocyte differentiation and tumor regression 査読

    Rie Kinoshita, Masami Watanabe, Peng Huang, Shun-Ai Li, Masakiyo Sakaguchi, Hiromi Kumon, Junichiro Futami

    ONCOLOGY REPORTS   33 ( 6 )   2908 - 2914   2015年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SPANDIDOS PUBL LTD  

    Reduced expression in immortalized cells (REIC)/Dickkopf (Dkk)-3 is a tumor-suppressor gene and has been studied as a promising therapeutic gene for cancer gene therapy. Intratumoral injection of an adenovirus vector carrying the human REIC/Dkk-3 gene (Ad-REIC) elicits cancer cell-specific apoptosis and anticancer immune responses. The cytokine-like effect of secretory REIC/Dkk-3 on the induction of dendritic cell (DC)-like cell differentiation from monocytes plays a role in systemic anticancer immunity. In the present study, we generated recombinant full-length and N-terminally truncated REIC/Dkk-3 to characterize the biological activity of the protein. During the purification procedure, we identified a 17 kDa cysteine-rich stable product (C17-REIC) showing limited degradation. Further analysis showed that the C17-REIC domain was sufficient for the induction of DC-like cell differentiation from monocytes. Concomitant with the differentiation of DCs, the REIC/Dkk-3 protein induced the phosphorylation of glycogen synthase kinase 3 beta (GSK-3 beta) and signal transducers and activators of transcription (STAT) at a level comparable to that of granulocyte/macrophage colony-stimulating factor. In a mouse model of subcutaneous renal adenocarcinoma, intraperitoneal injection of full-length and C17-REIC proteins exerted anticancer effects in parallel with the activation of immunocompetent cells such as DCs and cytotoxic T lymphocytes in peripheral blood. Taken together, our results indicate that the stable cysteine-rich core region of REIC/Dkk-3 is responsible for the induction of anticancer immune responses. Because REIC/Dkk-3 is a naturally circulating serum protein, the upregulation REIC/Dkk-3 protein expression could be a promising option for cancer therapy.

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  • 変性状態のタンパク質を高度に利用するカチオン化技術 招待 査読

    二見 淳一郎

    化学と生物   53 ( 4 )   245 - 251   2015年4月

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    記述言語:日本語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1271/kagakutoseibutsu.53.245

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  • A vaccine strategy with multiple prostatic acid phosphatase-fused cytokines for prostate cancer treatment 査読

    Kei Fujio, Masami Watanabe, Hideo Ueki, Shun-Ai Li, Rie Kinoshita, Kazuhiko Ochiai, Junichiro Futami, Toyohiko Watanabe, Yasutomo Nasu, Hiromi Kumon

    ONCOLOGY REPORTS   33 ( 4 )   1585 - 1592   2015年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SPANDIDOS PUBL LTD  

    Immunotherapy is one of the attractive treatment strategies for advanced prostate cancer. The US Food and Drug Administration (FDA) previously approved the therapeutic vaccine, sipuleucel-T, which is composed of autologous antigen-presenting cells cultured with a fusion protein [prostatic acid phosphatase (PAP) and granulocyte-macrophage colony-stimulating factor (GMCSF)]. Although sipuleucel-T has been shown to prolong the median survival of patients for 4.1 months, more robust therapeutic effects may be expected by modifying the vaccination protocol. In the present study, we aimed to develop and validate a novel vaccination strategy using multiple PAP-fused cytokines for prostate cancer treatment. Using a super gene expression (SGE) system that we previously established to amplify the production of a recombinant protein, significant amounts of PAP-fused cytokines [human GMCSF, interleukin-2 (IL2), IL4, IL7 and mouse GMCSF and IL4] were obtained. We examined the activity of the fusion proteins in vitro to validate their cytokine functions. A significant upregulation of dendritic cell differentiation from monocytes was achieved by PAP-GMCSF when used with the other PAP-fused cytokines. The PAP-fused human IL2 significantly increased the proliferation of lymphocytes, as determined by flow cytometry. We also investigated the in vivo therapeutic effects of multiple PAP-fused cytokines in a mouse prostate cancer model bearing prostate-specific antigen (PSA)- and PAP-expressing tumors. The simultaneous intraperitoneal administration of PAP-GMCSF, -IL2, -IL4 and -IL7 significantly prevented tumor induction and inhibited the tumor growth in the PAP-expressing tumors, yet not in the PSA-expressing tumors. The in vivo therapeutic effects with the multiple PAP-fused cytokines were superior to the effects of PAP-GMCSF alone. We thus demonstrated the advantages of the combined use of multiple PAP-fused cytokines including PAP-GMCSF, and propose a promising prostatic antigen-vaccination strategy to enhance the therapeutic effects.

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  • Denatured Mammalian Protein Mixtures Exhibit Unusually High Solubility in Nucleic Acid-Free Pure Water 査読

    Junichiro Futami, Haruna Fujiyama, Rie Kinoshita, Hidenori Nonomura, Tomoko Honjo, Hiroko Tada, Hirokazu Matsushita, Yoshito Abe, Kazuhiro Kakimi

    PLOS ONE   9 ( 11 )   e113295   2014年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PUBLIC LIBRARY SCIENCE  

    Preventing protein aggregation is a major goal of biotechnology. Since protein aggregates are mainly comprised of unfolded proteins, protecting against denaturation is likely to assist solubility in an aqueous medium. Contrary to this concept, we found denatured total cellular protein mixture from mammalian cell kept high solubility in pure water when the mixture was nucleic acids free. The lysates were prepared from total cellular protein pellet extracted by using guanidinium thiocyanate-phenol-chloroform mixture of TRIzol, denatured and reduced total protein mixtures remained soluble after extensive dialysis against pure water. The total cell protein lysates contained fully disordered proteins that readily formed large aggregates upon contact with nucleic acids or salts. These findings suggested that the highly flexible mixtures of disordered proteins, which have fully ionized side chains, are protected against aggregation. Interestingly, this unusual solubility is characteristic of protein mixtures from higher eukaryotes, whereas most prokaryotic protein mixtures were aggregated under identical conditions. This unusual solubility of unfolded protein mixtures could have implications for the study of intrinsically disordered proteins in a variety of cells.

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  • DNAX-activating Protein 10 (DAP10) Membrane Adaptor Associates with Receptor for Advanced Glycation End Products (RAGE) and Modulates the RAGE-triggered Signaling Pathway in Human Keratinocytes 査読

    Masakiyo Sakaguchi, Hitoshi Murata, Yumi Aoyama, Toshihiko Hibino, Endy Widya Putranto, I. Made Winarsa Ruma, Yusuke Inoue, Yoshihiko Sakaguchi, Ken-ichi Yamamoto, Rie Kinoshita, Junichiro Futami, Ken Kataoka, Keiji Iwatsuki, Nam-ho Huh

    JOURNAL OF BIOLOGICAL CHEMISTRY   289 ( 34 )   23389 - 23402   2014年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Background: RAGE receptor plays a critical role in many inflammatory disorders. Results: Functional interaction between RAGE and DAP10 coordinately regulates S100A8/A9-mediated cell survival. Conclusion: DAP10 membrane adaptor is critically involved in RAGE-mediated survival signaling upon S100A8/A9 binding. Significance: This is the first report demonstrating that RAGE-mediated survival signaling is critically regulated by DAP10 interaction.
    The receptor for advanced glycation end products (RAGE) is involved in the pathogenesis of many inflammatory, degenerative, and hyperproliferative diseases, including cancer. Previously, we revealed mechanisms of downstream signaling from ligand-activated RAGE, which recruits TIRAP/MyD88. Here, we showed that DNAX-activating protein 10 (DAP10), a transmembrane adaptor protein, also binds to RAGE. By artificial oligomerization of RAGE alone or RAGE-DAP10, we found that RAGE-DAP10 heterodimer formation resulted in a marked enhancement of Akt activation, whereas homomultimeric interaction of RAGE led to activation of caspase 8. Normal human epidermal keratinocytes exposed to S100A8/A9, a ligand for RAGE, at a nanomolar concentration mimicked the pro-survival response of RAGE-DAP10 interaction, although at a micromolar concentration, the cells mimicked the pro-apoptotic response of RAGE-RAGE. In transformed epithelial cell lines, A431 and HaCaT, in which endogenous DAP10 was overexpressed, and S100A8/A9, even at a micromolar concentration, led to cell growth and survival due to RAGE-DAP10 interaction. Functional blocking of DAP10 in the cell lines abrogated the Akt phosphorylation from S100A8/A9-activated RAGE, eventually leading to an increase in apoptosis. Finally, S100A8/A9, RAGE, and DAP10 were overexpressed in the psoriatic epidermis. Our findings indicate that the functional interaction between RAGE and DAP10 coordinately regulates S100A8/A9-mediated survival and/or apoptotic response of keratinocytes.

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  • Dramatic Increase in Expression of a Transgene by Insertion of Promoters Downstream of the Cargo Gene 査読

    Masakiyo Sakaguchi, Masami Watanabe, Rie Kinoshita, Haruki Kaku, Hideo Ueki, Junichiro Futami, Hitoshi Murata, Yusuke Inoue, Shun-Ai Li, Peng Huang, Endy Widya Putranto, I. Made Winarsa Ruma, Yasutomo Nasu, Hiromi Kumon, Nam-ho Huh

    MOLECULAR BIOTECHNOLOGY   56 ( 7 )   621 - 630   2014年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:HUMANA PRESS INC  

    For expression of genes in mammalian cells, various vectors have been developed using promoters including CMV, EF-1 alpha, and CAG promoters and have been widely used. However, such expression vectors sometimes fail to attain sufficient expression levels depending on the nature of cargo genes and/or on host cell types. In the present study, we aimed to develop a potent promoter system that enables high expression levels of cargo genes ubiquitously in many different cell types. We found that insertion of an additional promoter downstream of a cargo gene greatly enhanced the expression levels. Among the constructs we tested, C-TSC cassette (C: CMV-RU5' located upstream; TSC: another promoter unit composed of triple tandem promoters, hTERT, SV40, and CMV, located downstream of the cDNA plus a polyadenylation signal) had the most potent capability, showing far higher efficiency than that of potent conventional vector systems. The results indicate that the new expression system is useful for production of recombinant proteins in mammalian cells and for application as a gene therapeutic measure.

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  • A novel gene expression system strongly enhances the anticancer effects of a REIC/Dkk-3-encoding adenoviral vector 査読

    Masami Watanabe, Masakiyo Sakaguchi, Rie Kinoshita, Haruki Kaku, Yuichi Ariyoshi, Hideo Ueki, Ryuta Tanimoto, Shin Ebara, Kazuhiko Ochiai, Junichiro Futami, Shun-Ai Li, Peng Huang, Yasutomo Nasu, Nam-Ho Huh, Hiromi Kumon

    ONCOLOGY REPORTS   31 ( 3 )   1089 - 1095   2014年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SPANDIDOS PUBL LTD  

    Gene expression systems with various promoters, including the cytomegalovirus (CMV) promoter, have been developed to increase the gene expression in a variety of normal and cancer cells. In particular, in the clinical trials of cancer gene therapy, a more efficient and robust gene expression system is required to achieve sufficient therapeutic outcomes. By inserting the triple translational enhancer sequences of human telomerase reverse transcriptase (hTERT), Simian virus 40 (SV40) and CMV downstream of the sequence of the BGH polyA, we were able to develop a novel gene expression system that significantly enhances the expression of the genes of interest. We termed this novel gene expression cassette the super gene expression (SGE) system, and herein verify the utility of the SGE cassette for a replication-deficient adenoviral vector. We newly developed an adenoviral vector expressing the tumor suppressor, reduced expression in immortalized cells (REIC)/Dickkopf-3 (Dkk-3), based on the CMV promoter-driven SGE system (Ad-SGE-REIC) and compared the therapeutic utility of Ad-SGE-REIC with that of the conventional adenoviral vectors (Ad-CMV-REIC or Ad-CAG-REIC). The results demonstrated that the CMV promoter-SGE system allows for more potent gene expression, and that the Ad-SGE-REIC is superior to conventional adenoviral systems in terms of the REIC protein expression and therapeutic effects. Since the SGE cassette can be applied for the expression of various therapeutic genes using various vector systems, we believe that this novel system will become an innovative tool in the field of gene expression and gene therapy.

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  • Inhibition of RAGE signaling through the intracellular delivery of inhibitor peptides by PEI cationization 査読

    Endy Widya Putranto, Hitoshi Murata, Ken-Ichi Yamamoto, Ken Kataoka, Hidenori Yamada, Jun-Ichiro Futami, Masakiyo Sakaguchi, Nam-Ho Huh

    INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE   32 ( 4 )   938 - 944   2013年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SPANDIDOS PUBL LTD  

    The receptor for advanced glycation end products (RAGE) is a multi-ligand cell surface receptor and a member of the immunoglobulin superfamily. RAGE is involved in a wide range of inflammatory, degenerative and hyper-proliferative disorders which span over different organs by engaging diverse ligands, including advanced glycation end products, S100 family proteins, high-mobility group protein B1 (HMGB1) and amyloid beta. We previously demonstrated that the cytoplasmic domain of RAGE is phosphorylated upon the binding of ligands, enabling the recruitment of two distinct pairs of adaptor proteins, Toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP) and myeloid differentiation protein 88 (MyD88). This engagement allows the activation of downstream effector molecules, and thereby mediates a wide variety of cellular processes, such as inflammatory responses, apoptotic cell death, migration and cell growth. Therefore, inhibition of the binding of TIRAP to RAGE may abrogate intracellular signaling from ligand-activated RAGE. In the present study, we developed inhibitor peptides for RAGE signaling (RAGE-I) by mimicking the phosphorylatable cytosolic domain of RAGE. RAGE-I was efficiently delivered into the cells by polyethylenimine (PEI) cationization. We demonstrated that RAGE-I specifically bound to TIRAP and abrogated the activation of Cdc42 induced by ligand-activated RAGE. Furthermore, we were able to reduce neuronal cell death induced by an excess amount of S100B and to inhibit the migration and invasion of glioma cells in vitro. Our results indicate that RAGE-I provides a powerful tool for therapeutics to block RAGE-mediated multiple signaling.

    DOI: 10.3892/ijmm.2013.1467

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  • 【特集】タンパク質生産と溶解性制御:タンパク質の溶解性を操るタンパク質カチオン化技術 招待

    二見 淳一郎

    バイオインダストリー   15 - 21   2013年7月

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    記述言語:日本語   掲載種別:研究論文(学術雑誌)  

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  • タンパク質の物性を制御するカチオン化技術と工学的応用 査読

    Futami J

    生化学   85 ( 1 )   21 - 25   2013年

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    記述言語:日本語   掲載種別:研究論文(学術雑誌)  

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  • Uniformly Cationized Protein Efficiently Reaches the Cytosol of Mammalian Cells 査読

    Midori Futami, Yasuyoshi Watanabe, Takashi Asama, Hitoshi Murata, Hiroko Tada, Megumi Kosaka, Hidenori Yamada, Junichiro Futami

    BIOCONJUGATE CHEMISTRY   23 ( 10 )   2025 - 2031   2012年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER CHEMICAL SOC  

    Protein cationization techniques are powerful protein transduction methods for mammalian cells. As we demonstrated previously, cationized proteins with limited conjugation to polyethylenimine have excellent ability to enter into cells by adsorption-mediated endocytosis [Futami, J., et al. (2005) J. Biosci. Bioeng. 99, 95-103]. In this study, we show that proteins with extensive and uniform cationization covering the protein surface reach the cytoplasm and nucleus more effectively than proteins with limited cationic polymers or proteins that are fused to cationic peptides. Although extensive modification of carboxylates results in loss of protein function, chicken avidin retains biotin-binding ability even after extensive amidation of carboxylates. Using this cationized avidin carrier system, the protein transduction ability of variously cationized avidins was investigated using biotinylated protein as a probe. The results revealed that cationized avidins bind rapidly to the cell surface followed by endocytotic uptake. Small amounts of uniformly cationized avidin showed direct penetration into the cytoplasm within a 15 min incubation. This penetration route seemed to be energy dependent and functioned under cellular physiological conditions. A biotinylated exogenous transcription factor protein that penetrated cells was demonstrated to induce target gene expression in living cells.

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  • A New Cytosolic Pathway from a Parkinson Disease-associated Kinase, BRPK/PINK1 ACTIVATION OF AKT VIA MTORC2 査読

    Hitoshi Murata, Masakiyo Sakaguchi, Yu Jin, Yoshihiko Sakaguchi, Jun-ichiro Futami, Hidenori Yamada, Ken Kataoka, Nam-ho Huh

    JOURNAL OF BIOLOGICAL CHEMISTRY   286 ( 9 )   7182 - 7189   2011年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Accumulating evidence indicates that dysfunction of mitochondria is a common feature of Parkinson disease. Functional loss of a familial Parkinson disease-linked gene, BRPK/PINK1 (PINK1), results in deterioration of mitochondrial functions and eventual neuronal cell death. A mitochondrial chaperone protein has been shown to be a substrate of PINK1 kinase activity. In this study, we demonstrated that PINK1 has another action point in the cytoplasm. Phosphorylation of Akt at Ser-473 was enhanced by overexpression of PINK1, and the Akt activation was crucial for protection of SH-SY5Y cells from various cytotoxic agents, including oxidative stress. Enhanced Akt phosphorylation was not due to activation of phosphatidylinositol 3-kinase but due to activation of mammalian target of rapamycin complex 2 (mTORC2) by PINK1. Rictor, a specific component of mTORC2, was phosphorylated by overexpression of PINK1. Furthermore, overexpression of PINK1 enhanced cell motility. These results indicate that PINK1 exerts its cytoprotective function not only in mitochondria but also in the cytoplasm through activation of mTORC2.

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  • Polyethylenimine-cationized beta-catenin protein transduction activates the Wnt canonical signaling pathway more effectively than cationic lipid-based transduction 査読

    Midori Kitazoe, Junichiro Futami, Mitsuo Nishikawa, Hidenori Yamada, Yoshitake Maeda

    BIOTECHNOLOGY JOURNAL   5 ( 4 )   385 - 392   2010年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-V C H VERLAG GMBH  

    The Wnt canonical signaling pathway is essential for the early development of eukaryotic organisms and plays a key role in cell proliferation, differentiation, and oncogenesis. Moreover, the Wnt canonical signaling pathway contributes to the self-renewal of mouse hematopoietic stem cells (HSCs). Here, we demonstrate artificial activation of the Wnt canonical signaling pathway by beta-catenin protein transduction. Constitutively active beta-catenin protein was introduced into human embryonic kidney HEK-293 cells using a polyethylenimine (PEI) cationization method, or with the BioPORTER protein transduction reagent. We have previously shown that modification with PEI effectively causes proteins to be internalized by living mammalian cells. PEI-cationized, constitutively active beta-catenin protein was added to HEK-293 cells, and induction of several Wnt/beta-catenin target genes was detected by real-time PCR. However, using BioPORTER to introduce active beta-catenin did not activate the Wnt canonical signaling pathway. Introduction of eGFPNuc (enhanced green fluorescent protein variant containing a nuclear localization signal) into HEK-293 cells using the BioPORTER reagent caused significant cell death, as determined by propidium iodide staining. In contrast, the PEI-modified eGFPNuc did not impair survival of HEK-293 cells. These results indicate that the Wnt canonical signaling pathway could be successfully activated by transduction of PEI-cationized active beta-catenin, and the PEI-cationization method is an effective and safe technology for protein transduction into mammalian cells.

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  • タンパク質カチオン化技術の工学的応用 招待

    二見 淳一郎

    ケミカルエンジニアリング   55 ( 3 )   2010年3月

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  • Efficient cross-presentation of soluble exogenous antigens introduced into dendritic cells using a weak-based amphiphilic peptide 査読

    Nobuhito Ikeuchi, Junichiro Futami, Akihiro Hosoi, Shuichi Noji, Makoto Kurachi, Satoshi Ueha, Shin-ichiro Fujii, Hidenori Yamada, Koji Matsushima, Fuminori Moriyasu, Kazuhiro Kakimi

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   392 ( 2 )   217 - 222   2010年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS INC ELSEVIER SCIENCE  

    To develop a novel dendritic cell (DC)-based vaccine for inducing antigen-specific CD8(+) T cell responses by cross-presentation, we tested a novel antigen delivery system that introduces Soluble antigens into the cytosol of cells by an endocytosis-mediated mechanism which avoids damaging the plasma membrane ("Endo-Porter"(TM)). Proteins released from endosomes into the cytoplasm are degraded by the proteasome, and fragmented antigenic peptides are presented to the classical cytosolic MHC class I pathway. DCs pulsed with OVA protein in the presence of Endo-Porter efficiently stimulate OVA peptide-specific CD8(+) T (OT-I) cells. Although this agent diverts some of the endocytosed antigens away from the classical MHC class II-restricted presentation pathway to the class I pathway, the activation of CD4(+) T cells was found not to be hampered by Endo-Porter-mediated antigen delivery. On the contrary, it was rather augmented, probably due to the increased uptake of antigen. Because specific CD4(+) T cell help is required to license DCs for cross-priming, Endo-Porter-mediated antigen delivery is a promising approach for developing more efficient cancer vaccines targeting both CD4(+) and CD8(+) T cells. (C) 2010 Elsevier Inc. All rights reserved.

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  • Immunological aspects of REIC/Dkk-3 in monocyte differentiation and tumor regression 査読

    Masami Watanabe, Yuji Kashiwakura, Peng Huang, Kazuhiko Ochiai, Junichiro Futami, Shun-Ai Li, Munenori Takaoka, Yasutomo Nasu, Masakiyo Sakaguchi, Nam-Ho Huh, Hiromi Kumon

    INTERNATIONAL JOURNAL OF ONCOLOGY   34 ( 3 )   657 - 663   2009年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SPANDIDOS PUBL LTD  

    The REIC/Dkk-3 gene has been reported to be a tumor suppressor and the expression is significantly down-regulated in a broad range of cancer cell types. The protein is secretory, but the physiological function remains unclear. This study demonstrated that recombinant REIC/Dkk-3 protein induced the differentiation of human CD14(+) monocytes into a novel cell type ((REIC/Dkx-3)Mo). (REIC/Dkk-3)Mo resembles immature dendritic cells generated with IL-4 and GM-CSF. Both these cell populations exhibit similar proportions of CD11c(+), CD40(+), CD86(+) and HLA-DR(+) cells and endocytic capacity, but (REIC/Dkk-3)Mo is negative for CD1a antigen. An analysis of the signal transducers and activators of transcription (STAT) pathways revealed that REIC/Dkk-3 induces phosphorylation of STAT 1 and STAT 3. Furthermore, intratumoral administration of REIC/Dkk-3 protein significantly suppressed tumor growth with CD11c(+) and CD8(+) (dendritic and killer T cell marker, respectively) cell accumulation and enhanced anticancer cytolytic activity of splenocytes. These data indicated a cytokine-like role of REIC/Dkk-3 protein in monocyte differentiation that might be exploited therapeutically.

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  • Intracellular delivery of glutathione S-transferase-fused proteins into mammalian cells by polyethylenimine-glutathione conjugates 査読

    Hitoshi Murata, Junichiro Futami, Midori Kitazoe, Takayuki Yonehara, Hidetaka Nakanishi, Megumi Kosaka, Hiroko Tada, Masakiyo Sakaguchi, Yasuyuki Yagi, Masaharu Seno, Nam-ho Huh, Hidenori Yamada

    JOURNAL OF BIOCHEMISTRY   144 ( 4 )   447 - 455   2008年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:OXFORD UNIV PRESS  

    The glutathione S-transferase (GST)-fused protein expression system has been extensively used to generate a large quantity of proteins and has served for functional analysis in vitro. In this study, we developed a novel approach for the efficient intracellular delivery of GST-fused proteins into living cells to expand their usefulness up to in vivo use. Since protein cationization techniques are powerful strategies for efficient intracellular uptake by adsorptive-mediated endocytosis, GST-fused proteins were cationized by forming a complex with a polycationic polyethylenimine (PEI)-glutathione conjugate. On screening of protein transduction, optimized PEI-glutathione conjugate for protein transduction was characterized by a partly oligomerized mixture of PEI with average molecular masses of 600 (PEI600) modified with multiple glutathiones, which could have sufficient avidity for GST. Furthermore, enhanced endosomal escape of transduced GST-fused proteins was observed when they were delivered with a glutathione-conjugated PEI600 derivative possessing a hydroxybutenyl moiety. These results were confirmed by both intracellular confocal imaging of GST-fused green fluorescent protein and activation of an endogenous growth signal transduction pathway by a GST-fused constitutively active mutant of a kinase protein. These PEI-glutathione conjugates seem to be convenient molecular tools for protein transduction of widely used GST-fused proteins.

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  • Design of cytotoxic ribonucleases by cationization to enhance intracellular protein delivery 査読

    Junichiro Futami, Hidenori Yamada

    CURRENT PHARMACEUTICAL BIOTECHNOLOGY   9 ( 3 )   180 - 184   2008年6月

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    記述言語:英語   出版者・発行元:BENTHAM SCIENCE PUBL LTD  

    The cytotoxic properties of naturally occurring or engineered RNases correlate well with their efficiency of cellular internalization and digestion level of cellular RNA. Cationized RNases are considered to adsorb to the anionic cellular surface by Coulombic interactions, and then become efficiently internalized into cells by an endocytosis-like pathway. The design of cytotoxic RNases by chemical modification of surface carboxylic residues is one of the powerful strategies for enhancing cellular internalization and is accompanied with a decreased sensitivity for the cytoplasmic RNase inhibitor. Although chemically modified cationized RNases showed decreased ribonucleolytic activity, improved endocytosis and decreased affinity to the endogenous RNase inhibitor conclusively contribute to their ability to digest cellular RNA. Furthermore, the cytotoxicity of cationized RNases can be drastically enhanced by co-endocytosis with an endosome-destabilizing peptide. Since efficient cellular internalization of proteins into living cells is an important technology for biotechnology, studies concerning the design of cytotoxic RNases provided general perceptions for protein-based drug design.

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  • S100A11, an dual mediator for growth regulation of human keratinocytes 査読

    Masakiyo Sakaguchi, Hiroyuki Sonegawa, Hitoshi Murata, Midori Kitazoe, Jun-ichiro Futami, Ken Kataoka, Hidenori Yamada, Nam-ho Huh

    MOLECULAR BIOLOGY OF THE CELL   19 ( 1 )   78 - 85   2008年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC CELL BIOLOGY  

    We previously revealed a novel signal pathway involving S100A11 for inhibition of the growth of normal human keratinocytes (NHK) caused by high Ca++ or transforming growth factor beta. Exposure to either agent resulted in transfer of S100A11 to nuclei, where it induced p21(WAF1). In contrast, S100A11 has been shown to be overexpressed in many human cancers. To address this apparent discrepancy, we analyzed possible new functions of S100A11, and we provide herein evidence that 1) S100A11 is actively secreted by NHK; 2) extracellular S100A11 acts on NHK to enhance the production of epidermal growth factor family proteins, resulting in growth stimulation; 3) receptor for advanced glycation end products, nuclear factor-kappa B, Akt, and cAMP response element-binding protein are involved in the S100A11-triggered signal transduction; and 4) production and secretion of S100A11 are markedly enhanced in human squamous cancer cells. These findings indicate that S100A11 plays a dual role in growth regulation of epithelial cells.

    DOI: 10.1091/mbc.E07-07-0682

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  • Transient cell proliferation with polyethylenimine-cationized N-terminal domain of simian virus 40 large T-antigen 査読

    Hitoshi Murata, Junichiro Futami, Midori Kitazoe, Megumi Kosaka, Hiroko Tada, Masaharu Seno, Hidenori Yamada

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   105 ( 1 )   34 - 38   2008年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SOC BIOSCIENCE BIOENGINEERING JAPAN  

    Polyethylenimine (PEI) cationization is a powerful strategy for protein transduction into cells. In this study, we attempted the artificial regulation of cell proliferation by protein transduction of the N-terminal domain (1-132 amino acids) of the simian virus 40 large T-antigen (SVLT-N), which inactivates retinoblastoma family proteins but not p53. To deliver SVLT-N into cells, we employed an indirect cationization method by forming a complex of biotynylated SVLT-N through disulfide bonds (biotin-SS-SVLT-N) and PEI-cationized avidin (PEI600-avidin). Using this complex, SVLT-N was transduced into the nucleus of confluent and quiescent Balb/c 3T3 cells and was found to be complexed with a cellular target protein, pRb. Furthermore, SVLT-N transduction induced cell proliferation in spite of confluent conditions. Because SVLT-N thus transduced into cells gradually degraded and was not detectable after a 4-d incubation, transiently transformed cells were obtained by this method. These results suggest that oncogene protein transduction technology has great potential for in vitro regulation of cell proliferation.

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  • Truncation of annexin A1 is a regulatory lever for linking epidermal growth factor signaling with cytosolic phospholipase A2 in normal and malignant squamous epithelial cells 査読

    Masakiyo Sakaguchi, Hitoshi Murata, Hiroyuki Sonegawa, Yoshihiko Sakaguchi, Jun-ichiro Futami, Midori Kitazoe, Hidenori Yamada, Nam-ho Huh

    JOURNAL OF BIOLOGICAL CHEMISTRY   282 ( 49 )   35679 - 35686   2007年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Regulation of cell growth and apoptosis is one of the pleiotropic functions of annexin A1 (ANXA1). Although previous reports on the overexpression of ANXA1 in many human cancers and on growth suppression and/or induction of apoptosis by ANXA1 may indicate the tumor-suppressive nature of ANXA1, molecular mechanisms of the function of ANXA1 remain largely unknown. Here we provide evidence that ANXA1 mechanistically links the epidermal growth factor-triggered growth signal pathway with cytosolic phospholipase A(2) (cPLA(2)), an initiator enzyme of the arachidonic acid cascade, through interaction with S100A11 in normal human keratinocytes (NHK). Ca2+ -dependent binding of S100A11 to ANXA1 facilitated the binding of the latter to cPLA2, resulting in inhibition of cPLA2 activity, which is essential for the growth of NHK. On exposure of NHK to epidermal growth factor, ANXA1 was cleaved solely at Trp(12), and this cleavage was executed by cathepsin D. In squamous cancer cells, this pathway was shown to be constitutively activated. The newly found mechanistic intersection may be a promising target for establishing new measures against human cancer and other cell growth disorders.

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  • 'Crystal lattice engineering,' an approach to engineer protein crystal contacts by creating intermolecular symmetry: Crystallization and structure determination of a mutant human RNase 1 with a hydrophobic interface of leucines 査読

    Hidenori Yamada, Taro Tamada, Megumi Kosaka, Kohei Miyata, Shinya Fujiki, Masaru Tano, Masayuki Moriya, Mamoru Yamanishi, Eijiro Honjo, Hiroko Tada, Takeshi Ino, Hiroshi Yamaguchi, Junichiro Futami, Masaharu Seno, Takashi Nomoto, Tomoko Hirata, Motonobu Yoshimura, Ryota Kuroki

    PROTEIN SCIENCE   16 ( 7 )   1389 - 1397   2007年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT  

    A protein crystal lattice consists of surface contact regions, where the interactions of specific groups play a key role in stabilizing the regular arrangement of the protein molecules. In an attempt to control protein incorporation in a crystal lattice, a leucine zipper-like hydrophobic interface (comprising four leucine residues) was introduced into a helical region (helix 2) of the human pancreatic ribonuclease 1 (RNase 1) that was predicted to form a suitable crystallization interface. Although crystallization of wild-type RNase 1 has not yet been reported, the RNase 1 mutant having four leucines (4L-RNase 1) was successfully crystallized under several different conditions. The crystal structures were subsequently determined by Xray crystallography by molecular replacement using the structure of bovine RNase A. The overall structure of 4L-RNase 1 is quite similar to that of the bovine RNase A, and the introduced leucine residues formed the designed crystal interface. To characterize the role of the introduced leucine residues in crystallization of RNase 1 further, the number of leucines was reduced to three or two (3L-and 2L-RNase 1, respectively). Both mutants crystallized and a similar hydrophobic interface as in 4L-RNase 1 was observed. A related approach to engineer crystal contacts at helix 3 of RNase 1 (N4L- RNase 1) was also evaluated. N4L- RNase 1 also successfully crystallized and formed the expected hydrophobic packing interface. These results suggest that appropriate introduction of a leucine zipper-like hydrophobic interface can promote intermolecular symmetry for more efficient protein crystallization in crystal lattice engineering efforts.

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  • Exploiting protein cationization techniques in future drug development 査読

    Junichiro Futami, Midori Kitazoe, Hiroshi Murata, Hidenori Yamada

    EXPERT OPINION ON DRUG DISCOVERY   2 ( 2 )   261 - 269   2007年2月

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    記述言語:英語   出版者・発行元:INFORMA HEALTHCARE  

    The development of a method for the efficient intracellular delivery of inherently non-permeable proteins is needed for manipulation of cellular phenotypes or the discovery of protein-based drugs. It has been demonstrated that proteins artificially cationized by chemical conjugation show efficient intracellular delivery via adsorptive-mediated endocytosis and then can exert their biological activity in cells. Studies have also revealed that cationic peptides known as cell-penetrating peptides (CPPs) provide a means to deliver molecules into mammalian cells. Although the internalization mechanisms remain controversial, it is now becoming clear that the main port of entry into cells by CPPs also involves adsorptive-mediated endocytosis rather than the direct penetration of the plasma membrane. As the mammalian cell membrane possesses an abundance of negatively charged glycoproteins and glycosphingolipids, cationization of proteins is a reasonable choice to endow them with the ability for intracellular delivery. Cationization of proteins is usually accompanied by drastic changes in protein properties, structure and biological activities. Recently developed sophisticated protein chemistry can minimize these side effects. Therefore, protein cationization techniques will hopefully prove to be powerful tools for innovative research and drug discovery. In this review, techniques for cationization of proteins and their intracellular delivery, as well as some of their potential therapeutic applications, are discussed.

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  • Leucyl/phenylalanyl-tRNA-protein transferase-mediated chemoenzymatic coupling of N-terminal arg/lys units in posttranslationally processed proteins with non-natural amino acids 査読

    Masumi Taki, Atsushi Kuno, Shinsuke Matoba, Yuki Kobayashi, Junichiro Futami, Hiroshi Murakami, Hiroaki Suga, Kazunari Taira, Tsunemi Hasegawa, Masahiko Sisido

    CHEMBIOCHEM   7 ( 11 )   1676 - +   2006年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-V C H VERLAG GMBH  

    The protein no longer terminates here. Non-natural amino acids were attached onto a tRNA and were then transferred with the aid of L/F-tRNA-protein transferase to the N termini of proteins possessing N-terminal Lys or Arg components. By this new technique, 1- and 2-naphthylalanine or 3-nitrotyrosine were coupled to the N termini of several proteins. A novel and convenient method for adding single Lys units at the N termini of various proteins was also developed.

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  • Denatured and reversibly cationized p53 readily enters cells and simultaneously folds to the functional protein in the cells 査読

    H Murata, M Sakaguchi, J Futami, M Kitazoe, T Maeda, H Doura, M Kosaka, H Tada, M Seno, N Huh, H Yamada

    BIOCHEMISTRY   45 ( 19 )   6124 - 6132   2006年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER CHEMICAL SOC  

    Cationization is a powerful strategy for internalizing a protein into living cells. On the other hand, a reversibly cationized denatured protein through disulfide bonds is not only soluble in water but also able to fold to the native conformation in vitro. When these advantages in cationization were combined, we developed a novel method to deliver a denatured protein into cells and simultaneously let it fold to express its function within cells. This "in-cell folding" method enhances the utility of recombinant proteins expressed in Escherichia coli as inclusion bodies; that is, the recombinant proteins in inclusion bodies are solubilized by reversible cationization through cysteine residues by disulfide bonds with aminopropyl methanethiosulfonate or pyridyldithiopropionylpolyethylenimine and then incubated with cells without an in vitro folding procedure. As a model protein, we investigated human tumor-suppressor p53. Treatment of p53-null Saos-2 cells with reversibly cationized p53 revealed that all events examined as indications of the activation of p53 in cells, such as reduction of disulfide bonds followed by tetramer formation, localization into the nucleus, induction of p53 target genes, and induction of apoptosis of cells, occurred. These results suggest that reversible cationization of a denatured protein through cysteine residues is an alternative method for delivery of a functional protein into cells. This method would be very useful when a native folded protein is not readily available.

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  • Mechanisms of the growth-inhibitory effect of the RNase-EGF fused protein against EGFR-overexpressing cells 査読

    S Hoshimoto, M Ueda, H Jinno, M Kitajima, J Futami, M Seno

    ANTICANCER RESEARCH   26 ( 2A )   857 - 863   2006年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:INT INST ANTICANCER RESEARCH  

    Background: We previously showed the usefulness of a fused protein of human pancreatic ribonuclease1 (hRNase1) with human epidermal growth factor (hEGF) for molecular targeting of EGF receptor (EGFR)-overexpressing cells. In this study, the mechanisms underlying the inhibition of cell growth by RNase-EGF fused proteins was confirmed. Materials and Methods: Des. 1-7 hRNase1 was genetically fused to hEGF. The fused proteins were expressed and isolated from Escherichia coli. The internalization of hRNase1-hEGF was confirmed by confocal fluorescence microscopy. The growth-inhibitory effect of the fused proteins was evaluated by MTT assay. Results: FITC-labelled hRNase1-hEGF was internalized into EGFR-overexpressing A431 cells. The internalization was not observed in A 431 cells pre-treated with hEGF and EGFR-deficient H69 cells. The growth-inhibitory effect of des.1-7 hRNase1-hEGF against A431 cells was statistically significantly more pronounced than that of hRNase1-hEGF. Conclusion: RNase-EGF fused proteins are internalized through EGFR and inhibit cell growth by exerting their ribonucleolytic activity in the cytosol.

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  • In vivo protein transduction to the CNS 査読

    LT Loftus, HF Li, AJ Gray, C Hirata-Fukae, BA Stoica, J Futami, H Yamada, PS Aisen, Y Matsuoka

    NEUROSCIENCE   139 ( 3 )   1061 - 1067   2006年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PERGAMON-ELSEVIER SCIENCE LTD  

    Proteins and peptides are useful research and therapeutic tools, however applications are limited because delivery to the desired location is not easily achievable. There are two hurdles in protein/peptide delivery to the brain: the blood-brain barrier and intracellular penetration. Penetration to both brain and the intracellular space can be achieved by adjusting hydrophilicity, and small molecule pharmacological agents have been successfully developed using this approach. But with proteins and peptides, it is difficult to modify the hydrophilicity without influencing biological functions. Trans-acting factor protein from the human immunodeficiency virus contains a highly conserved cationic peptide sequence necessary for transduction across the cell membrane. While trans-acting factor peptide has been used for in vitro protein transduction, its in vivo application is very limited because it is rapidly degraded by proteolysis. Polyethylenimine is a chemically synthesized small molecule cationization agent; the charge density is greater than a peptide-based cationic cluster such as trans-acting factor, and it is resistant to proteolysis in vivo. We first tested intracellular protein transduction following direct brain injection in mice using polyethylenimine-conjugated green fluorescence protein and beta-galactosidase (molecular weights 29 and 540 kDa, respectively). Polyethylenimine-conjugates penetrated to the intracellular space immediately surrounding the injection site within one hour. We further tested polyethylenimine-mediated protein transduction following intranasal administration, which bypasses the blood-brain barrier. Polyethylenimine-conjugates in pH 7.5 solution did not reach the brain, probably because the polyethylenimine-conjugates penetrated into the intracellular space where first exposed to the tissue, i.e. at the nasal mucosae. We temporarily reduced the electrostatic interaction between cationized polyethylenimine-conjugates and cellular surfaces by adjusting the pH to 4.5; solution rapidly reached the brain and penetrated to the intracellular space. This study suggests that polyethylenimine is a useful protein transduction agent in the brain in vivo, and adjusting cationic charge interaction can determine the extent of brain penetration. (c) 2006 IBRO. Published by Elsevier Ltd. All rights reserved.

    DOI: 10.1016/j.neuroscience.2006.01.041

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  • Visceral adipose tissue-derived serine protease inhibitor: A unique insulin-sensitizing adipocytokine in obesity 査読

    K Hida, J Wada, J Eguchi, H Zhang, M Baba, A Seida, L Hashimoto, T Okada, A Yasuhara, A Nakatsuka, K Shikata, S Hourai, J Futami, E Watanabe, Y Matsuki, R Hiramatsu, S Akagi, H Makino, YS Kanwar

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   102 ( 30 )   10610 - 10615   2005年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NATL ACAD SCIENCES  

    There is a rapid global rise in obesity, and the link between obesity and diabetes remains somewhat obscure. We identified an adipocytokine, designated as visceral adipose tissue-derived serpin (vaspin), which is a member of serine protease inhibitor family. Vaspin cDNA was isolated by from visceral white adipose tissues (WATs) of Otsuka Long-Evans Tokushima fatty (OLETIF) rat, an animal model of abdominal obesity with type 2 diabetes. Rat, mouse, and human vaspins are made up of 392,394, and 395 amino acids, respectively; exhibit approximate to 40% homology with alpha(1)-antitrypsin; and are related to serine protease inhibitor family. Vaspin was barely detectable in rats at 6 wk and was highly expressed in adipocytes of visceral WATs at 30 wk, the age when obesity, body weight, and insulin levels peak in OLETF rats. The tissue expression of vaspin and its serum levels decrease with worsening of diabetes and body weight loss at 50 wk. The expression and serum levels were normalized with the treatment of insulin or insulin-sensitizing agent, pioglitazone, in OLETF rats. Administration of vaspin to obese CRL:CD-1 (ICR) (ICR) mice fed with high-fat high-sucrose chow improved glucose tolerance and insulin sensitivity reflected by normalized serum glucose levels. It also led to the reversal of altered expression of genes relevant to insulin resistance, e.g., leptin, resistin, TNF alpha, glucose transporter-4, and adiponectin. In DNA chip analyses, vaspin treatment resulted in the reversal of expression in approximate to 50% of the high-fat high-sucrose-induced genes in WATs. These findings indicate that vaspin exerts an insulin-sensitizing effect targeted toward WATs in states of obesity.

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  • Protein transduction assisted by polyethylenimine-cationized carrier proteins 査読

    M Kitazoe, H Murata, J Futami, T Maeda, M Sakaguchi, M Miyazaki, M Kosaka, H Tada, M Seno, N Huh, M Namba, M Nishikawa, Y Maeda, H Yamada

    JOURNAL OF BIOCHEMISTRY   137 ( 6 )   693 - 701   2005年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:JAPANESE BIOCHEMICAL SOC  

    Previously, we have reported that cationized-proteins covalently modified with polyethylenimine (PEI) (direct PEI-cationization) efficiently enter cells and function in the cytosol [Futami et al. (2005) J. Biosci. Bioeng. 99,95-1031. However, it may be more convenient if a protein could be delivered into cells just by mixing the protein with a PEI-cationized carrier protein having a specific affinity (indirect PEI-cationization). Thus, we prepared PEI-cationized avidin (PEI-avidin), streptavidin (PEI-streptavidin), and protein G (PEI-protein G), and examined whether they could deliver biotinylated proteins and antibodies into living cells. PEI-avidin (and/or PEI-streptavidin) carried biotinylated GFPs into various mammalian cells very efficiently. A GFP variant containing a nuclear localization signal was found to arrive even in the nucleus. The addition of a biotinylated RNase A derivative mixed with PEI-streptavidin to a culture medium of 3T3-SV-40 cells resulted in remarkable cell growth inhibition, suggesting that the biotinylated RNase A derivative entered cells and digested intracellular RNA molecules. Furthermore, the addition of a fluorescein-labeled antiS100C (beta-actin binding protein) antibody mixed with PEI-protein G to human fibroblasts resulted in the appearance of a fluorescence image of actin-like filamentous structures in the cells. These results indicate that indirect PEI-cationization using non-covalent interaction is as effective as the direct PEI-cationization for the transduction of proteins into living cells and for expression of their functions in the cytosol. Thus, PEI-cationized proteins having a specific affinity for certain molecules such as PEI-streptavidin, PEI-avidin and PEI-protein G are concluded to be widely applicable protein transduction carrier molecules.

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  • Intracellular delivery of proteins into mammalian living cells by polyethylenimine-cationization 査読

    J Futami, M Kitazoe, T Maeda, E Nukui, M Sakaguchi, J Kosaka, M Miyazaki, M Kosaka, H Tada, M Seno, Y Sasaki, NH Huh, M Namba, H Yamada

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   99 ( 2 )   95 - 103   2005年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SOC BIOSCIENCE BIOENGINEERING JAPAN  

    In the post-genomic era, there is pressing need for development of protein manipulation methodology to analyze functions of proteins in living cells. For this purpose, techniques to deliver functional proteins into living cells are currently being evaluated as alternative approaches to the introduction of transcriptionally active DNA. Here, we describe a novel method for efficient protein transduction into living cells in which a protein is simply cationized with polyethylenimine (PEI) by limited chemical conjugation. PEI-cationized proteins appear to adhere to the cell surface by ionic charge interaction and then internalize into cells in a receptor- and transporter-independent fashion. Since PEI is an organic macromolecule with a high cationic-charge density, limited coupling with PEI results in endowment of sufficient cationic charge to proteins without causing serious decline in their fundamental functions. A number of PEI-cationized proteins, such as ribonuclease (RNase), green fluorescent protein (GFP) and immunoglobulin (IgG), efficiently entered cells and functioned in the cytosol. Our results suggest that protein cationization techniques using PEI will be useful for the development of protein transduction technology.

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  • Intracellular delivery of proteins into mammalian living cells by polyethylenimine-cationization 査読

    J Futami, M Kitazoe, T Maeda, E Nukui, M Sakaguchi, J Kosaka, M Miyazaki, M Kosaka, H Tada, M Seno, Y Sasaki, NH Huh, M Namba, H Yamada

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   99 ( 2 )   95 - 103   2005年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SOC BIOSCIENCE BIOENGINEERING JAPAN  

    In the post-genomic era, there is pressing need for development of protein manipulation methodology to analyze functions of proteins in living cells. For this purpose, techniques to deliver functional proteins into living cells are currently being evaluated as alternative approaches to the introduction of transcriptionally active DNA. Here, we describe a novel method for efficient protein transduction into living cells in which a protein is simply cationized with polyethylenimine (PEI) by limited chemical conjugation. PEI-cationized proteins appear to adhere to the cell surface by ionic charge interaction and then internalize into cells in a receptor- and transporter-independent fashion. Since PEI is an organic macromolecule with a high cationic-charge density, limited coupling with PEI results in endowment of sufficient cationic charge to proteins without causing serious decline in their fundamental functions. A number of PEI-cationized proteins, such as ribonuclease (RNase), green fluorescent protein (GFP) and immunoglobulin (IgG), efficiently entered cells and functioned in the cytosol. Our results suggest that protein cationization techniques using PEI will be useful for the development of protein transduction technology.

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  • Targeted disruption of transcriptional regulatory function of p53 by a novel efficient method for introducing a decoy oligonucleotide into nuclei 査読

    M Sakaguchi, T Nukui, H Sonegawa, H Murata, J Futami, H Yamada, N Huh

    NUCLEIC ACIDS RESEARCH   33 ( 9 )   e88   2005年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:OXFORD UNIV PRESS  

    Decoy oligonucleotides have been used for functional sequestering of transcription factors. Efficient introduction into cells is a prerequisite for the oligonucleotides to exert their blocking function. Lipofection is the most widely used technique for that purpose because of its convenience and relatively high efficiency. However, the transduction efficiency of lipofection largely depends on cell types and experimental conditions and the introduced nucleotides are not specifically directed to nuclei where they exert their major function. In the present study, we designed a new system for transporting oligonucleotides into cell nuclei. The vehicle is composed of glutathione-S-transferase, 7 arginine residues, the DNA- binding domain of GAL4 and a nuclear localization signal, which are linked with flexible glycine stretches. The p53-responsive element linked to the GAL4 upstream activating sequence was efficiently transferred by the vehicle protein into nuclei of primary cultures of neuronal cells, embryonic stem cells and various human normal cells. Transcriptional activation of p21(WAF1/CIP1) and Bax by p53 on exposure to cisplatin was completely blocked by introducing the p53 decoy oligonucleotide. Thus, the system developed in the present study can be a convenient and powerful tool for specifically disrupting the function of DNA- binding proteins in culture.

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  • 【特集】一細胞・一分子生物学の夜明け:カチオン性ポリマーによるタンパク質の細胞内導入 招待

    二見淳一郎, 山田秀徳

    バイオインダストリー   20 - 27   2004年6月

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  • Insertional-fusion of basic fibroblast growth factor endowed ribonuclease 1 with enhanced cytotoxicity by steric blockade of inhibitor interaction 査読

    H Tada, M Onizuka, K Muraki, W Masuzawa, J Futami, M Kosaka, M Seno, H Yamada

    FEBS LETTERS   568 ( 1-3 )   39 - 43   2004年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE BV  

    Basic fibroblast growth factor (bFGF) was inserted in the middle of human ribonuclease 1 (RNase1) sequence at an RNase inhibitor (RI)-binding site (Gly89) by a new gene fusion technique, insertional-fusion. The resultant insertional-fusion protein (CL-RFN89) was active both as bFGF and as RNase. Furthermore, it acquired an additional ability of evading RI through steric blockade of RI-binding caused by fused bFGF domain. As a result, CL-RFN89 showed stronger growth inhibition on B16/BL6 melanoma cells than an RI-sensitive tandem fusion protein. Thus, the insertional-fusion technique increases accessible positions for gene fusion on RNase, resulting in construction of a potent cytotoxic RNase. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

    DOI: 10.1016/j.febslet.2004.05.007

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  • Construction and characterization of RNase-based targeted therapeutics 査読

    Newton D. L, Futami J, Ruby D, Rybak S. M

    Methods Mol Biol   207   283 - 304   2003年

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  • RNase 3 (ECP) is an extraordinarily stable protein among human pancreatic-type RNases 査読

    T Maeda, K Mahara, M Kitazoe, J Futami, A Takidani, M Kosaka, H Tada, M Seno, H Yamada

    JOURNAL OF BIOCHEMISTRY   132 ( 5 )   737 - 742   2002年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:JAPANESE BIOCHEMICAL SOC  

    There have been some attempts to develop immunotoxins utilizing human RNase as a cytotoxic domain of antitumor agents. We have recently shown that only human RNase 3 (eosinophil cationic protein, ECP) among five human pancreatic-type RNases excels in binding to the cell surface and has a growth inhibition effect on several cancer cell lines, even though the RNase activity of RNase 3 is completely inhibited by the ubiquitously expressed cytosolic RNase inhibitor. This phenomenon may be explained by that RNase 3 is very stable against proteolytic degradation because RNase 3 internalized through endocytosis could have a longer life time in the cytosol, resulting in the accumulation of enough of it to exceed the concentration of RNase inhibitor, which allows the degradation of cytosolic RNA molecules. Thus, we compared the stabilities of human pancreatic-type RNases (RNases 1-5) and bovine RNase A by means of guanidium chloride-induced denaturation experiments based on the assumption of a two-state transition for unfolding. It was demonstrated that RNase 3 is extraordinarily stabler than either RNase A or the other human RNases (by more than 25 kJ/mol). Thus, our data suggest that in addition to its specific affinity for certain cancer cell lines, the stability of RNase 3 contributes to its unique cytotoxic effect and that it is important to stabilize a human RNase moiety through protein engineering for the design of human RNase-based immunotoxins.

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  • Optimum modification for the highest cytotoxicity of cationized ribonuclease 査読

    J Futami, E Nukui, T Maeda, M Kosaka, H Tada, M Seno, H Yamada

    JOURNAL OF BIOCHEMISTRY   132 ( 2 )   223 - 228   2002年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:JAPANESE BIOCHEMICAL SOC  

    Cationization of a protein is considered to be a powerful strategy for internalizing a functional protein into cells. Cationized proteins appear to adsorb to the cell surface by electrostatic interactions, then enter the cell in a receptor- and transporter-independent fashion. Thus, in principle, all cell types appear to take up cationized proteins. Since ribonucleases (RNases) have a latent cytotoxic potential, cationized RNases could be useful cancer chemotherapeutics. In this study, we investigated the effect of the degree of cationization on the cytotoxicity of RNase A by modifying carboxyl groups with ethylenediamine. We found that there is an optimum degree of modification for cytotoxicity, in which 5 to 7 out of 11 carboxyl groups in RNase A are modified, toward MCF-7 and 3T3-SV-40 cells. More interestingly, the cytotoxicity of cationized RNase As correlates well with the value of [RNase activity] x [estimated concentration of RNase free from RNase inhibitor], mimicking the practical enzymatic activity of cationized RNase As in cytosol. The results indicate that cationization of a protein to an optimum level is important for maintaining protein function in the cytosol. Sophisticated protein cationization techniques will help to advance protein transduction technology.

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  • Preparation of potent cytotoxic ribonucleases by cationization: Enhanced cellular uptake and decreased interaction with ribonuclease inhibitor by chemical modification of carboxyl groups 査読

    J Futami, T Maeda, M Kitazoe, E Nukui, H Tada, M Seno, M Kosaka, H Yamada

    BIOCHEMISTRY   40 ( 25 )   7518 - 7524   2001年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER CHEMICAL SOC  

    Carboxyl groups of bovine RNase A were amidated with ethylenediamine (to convert negative charges of carboxylate anions to positive ones), 2-aminoethanol (to eliminate negative charges), and taurine (to keep negative charges), respectively, by a carbodiimide reaction. Human RNase 1 was also modified with ethylenediamine. Surprisingly, the modified RNases were all cytotoxic toward 3T3-SV-40 cells despite their decreased ribonucleolytic activity. However, their enzymatic activity was not completely eliminated by the presence of excess cytosolic RNase inhibitor (RI). As for native RNase A and RNase 1 which were not cytotoxic, they were completely inactivated by RI. More interestingly, within the cytotoxic RNase derivatives. cytotoxicity correlated well with the net positive charge. RNase 1 and RNase A modified with ethylenediamine were more cytotoxic than naturally occurring cytotoxic bovine seminal RNase. An experiment using the fluorescence-labeled RNase derivatives indicated that the more cationic RNases were more efficiently adsorbed to the cells. Thus, it is suggested that the modification of carboxyl groups could change complementarity of RNase to RI and as a result endow RNase cytotoxicity and that cationization enhances the efficiency of cellular uptake of RNase so as to strengthen its cytotoxicity. The finding that an extracellular human enzyme such as RNase 1 could be effectively internalized into the cell by cationization suggests that cationization is a simple strategy for efficient delivery of a protein into cells and may open the way of the development of new therapeutics.

    DOI: 10.1021/bi010248g

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  • Three-dimensional structure of human RNase 1 Delta N7 at 1.9 angstrom resolution 査読

    J Pous, G Mallorqui-Fernandez, R Peracaula, SS Terzyan, J Futami, H Tada, H Yamada, M Seno, R de Llorens, FX Gomis-Ruth, M Coll

    ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY   57   498 - 505   2001年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:MUNKSGAARD INT PUBL LTD  

    Human pancreatic ribonuclease 1 (RNase 1) is considered to be the human counterpart of bovine pancreatic RNase A. Truncation of seven amino-acid residues from the aminoterminal sequence resulted in RNase 1 Delta N7, which has a reduced ribonucleolytic activity and a lower affinity for the human placental RNase inhibitor (PRI). This RNase 1 variant has been cloned, heterologously overexpressed, purified and crystallized. Its crystal structure has been determined and refined using data to 1.9 Angstrom resolution. The molecule displays the alpha + beta folding topology typical of members of the RNase A superfamily. The main distinct features found in RNase 1 Delta N7 are basically located in three loops affecting the fitting of the enzyme to the active site of subtilisin and the shape of the B2 subsite. These changes, taken with the lack of the catalytically active residue Lys7, may explain the reduced affinity of RNase 1 Delta N7 for PRI and the low ribonucleolytic activity of the protein when compared with the native enzyme.

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  • Stabilization of human RNase 1 by introduction of a disulfide bond between residues 4 and 118 査読

    J Futami, H Tada, M Seno, S Ishikami, H Yamada

    JOURNAL OF BIOCHEMISTRY   128 ( 2 )   245 - 250   2000年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:JAPANESE BIOCHEMICAL SOC  

    In order to stabilize human RNase 1 by introduction of an intramolecular cross-link, a mutant protein (4-118CL RNase 1), in which Arg4 and Val118 are replaced with cysteine residues and linked by a disulfide bond, was designed and expressed in Escherichia coli as inclusion bodies. The 4-118CL RNase 1 that refolded under redox conditions was a monomer without free SH groups and retained 11% of the activity of the wild-type recombinant RNase 1, indicating that the mutant enzyme was correctly folded with the formation of an additional disulfide bond between Cys4 and Cys118, From guanidium chloride denaturation experiments based on the assumption of a two-state transition for unfolding, it was demonstrated that the introduction of the present cross-link increased the thermodynamic stability of RNase 1 by 2.0 kcal/mol. This value was lower than that, 5.4 kcal/mol, theoretically calculated from the reduction of chain entropy of the unfolded state due to the introduction of the cross-link. These results suggest that the present cross-link also destabilized the folded state of RNase 1 by 3.4 kcal/mol. Along with the increase in the thermodynamic stability, the stability of RNase 1 against trypsin digestion was also significantly increased by the introduction of this cross-link. It is likely, although not proven, that stabilized human RNases are favorable for clinical use, because human RNase-based immunotoxins should have long half-lives as to proteolytic degradation after endocytosis.

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  • Convenient and efficient in vitro folding of disulfide-containing globular protein from crude bacterial inclusion bodies 査読

    J Futami, Y Tsushima, H Tada, M Seno, H Yamada

    JOURNAL OF BIOCHEMISTRY   127 ( 3 )   435 - 441   2000年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:JAPANESE BIOCHEMICAL SOC  

    We investigated how the folding yield of disulfide-containing globular proteins having positive net charges from crude bacterial inclusion bodies was affected by additives in the folding buffer. In screening folding conditions for human ribonucleases and its derivative, we found that addition of salt (about 0.4 M) to a folding buffer increased the folding yield. This suggested that electrostatic interaction between polyanionic impurities such as nucleic acids and cationic unfolded protein led to the formation of aggregates under the low-salt conditions. Since inclusion bodies were found to contain nucleic acids regardless of the electrostatic nature of the expressed protein, the electrostatic interaction between phosphate moieties of nucleic acids and basic amino acid residues of a denatured protein may be large enough to cause aggregation, and therefore the addition of salt in a folding buffer may generally be useful for promotion of protein folding from crude inclusion bodies. We further systematically investigated additives such as glycerol, guanidium chloride, and urea that are known to act as chemical chaperons, and found that these additives, together with salt, synergistically improved folding yield. This study, suggesting that the addition of salt into the folding buffer is one of the crucial points to be considered, may pave the way for a systematic investigation of the folding conditions of disulfide-containing foreign proteins from crude bacterial inclusion bodies.

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  • Inhibition of cell growth by a fused protein of human ribonuclease 1 and human basic fibroblast growth factor 査読

    J Futami, M Seno, M Ueda, H Tada, H Yamada

    PROTEIN ENGINEERING   12 ( 11 )   1013 - 1019   1999年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:OXFORD UNIV PRESS  

    Pancreatic-type RNases are considered to have cytotoxic potential due to their ability to degrade RNA molecules when they enter the cytosol. However, most of these RNases show little cytotoxicity because cells have no active uptake mechanism for these RNases and because the ubiquitous cytoplasmic RNase inhibitor is considered to play a protective role against the endocytotic leak of RNases from the outside of cells. To study the cytotoxic potential of RNase toward malignant cells targeting growth factor receptors, the C-terminus of human RNase 1 was fused to the N-terminus of human basic fibroblast growth factor (bFGF). This RNase-FGF fused protein effectively inhibited the growth of mouse melanoma cell line B16/BL6 with high levels of cell surface FGF receptor. This effect appeared to result from prolongation of the overall cell cycle rather than the killing of cells or specific arrest in a particular phase of the cell cycle. Thus, human RNase 1 fused to a ligand of cell surface molecules, such as the FGF receptor, is shown to be an effective candidate for a selective cell targeting agent with low toxic effects on normal cell types.

    DOI: 10.1093/protein/12.11.1013

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  • Molecular targeting for epidermal growth factor receptor expressed on breast cancer cells by human fusion protein 査読

    Masakazu Ueda, Kyriakos Psarras, Hiromitsu Jinno, Tadashi Ikeda, Kohji Enomoto, Masaki Kitajima, Junichiro Futami, Hidenori Yamada, Masaharu Seno

    Breast Cancer   4 ( 4 )   253 - 255   1997年12月

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    記述言語:英語   掲載種別:研究論文(国際会議プロシーディングス)  

    Recombinant human ribonuclease 1 (RNasel) was chemically linked to recombinant human epidermal growth factor (EGF). The cytotoxicity of this conjugate was assayed using MTT assay. The EGF-RNase conjugate showed dose-dependent cytotoxicity against breast and squamous cell carcinomas overexpressing the EGF receptor (EGFR). The cytotoxicity of the conjugate correlated positively with the level of EGFR expression by each cell line. These results suggest that the EGF-RNase conjugate is a more effective anticancer agent with less immunogenicity and toxicity than conventional chimeric breast cancer toxins.

    DOI: 10.1007/BF02966516

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  • Tissue-specific expression of pancreatic-type RNases and RNase inhibitor in humans 査読

    J Futami, Y Tsushima, Y Murato, H Tada, J Sasaki, M Seno, H Yamada

    DNA AND CELL BIOLOGY   16 ( 4 )   413 - 419   1997年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:MARY ANN LIEBERT INC PUBL  

    The tissue-specific expression of five human pancreatic-type RNases and RNase inhibitor was analyzed by Northern hybridization against poly(A)(+)RNA prepared from 16 normal tissues. The widespread expression of RNase 1 was observed in almost all of the tissues. RNase 4 and angiogenin showed a similar distribution of expression abundantly present in the liver. This suggested the identity of the cell types producing these two molecules. However, no relativity appeared to be present between the vascularization of the tissues and the angiogenin expression. A narrow range of expression of the eosinophil-derived neurotoxin gene was observed. This localization seems related to the phagocytic cells in the tissues. The undetectable level of the eosinophil cationic protein mRNA in normal tissues suggests that the differentiation of eosinophils, triggered by inflammation and/or atopy, is required. The expression of RNase inhibitor was found to be ubiquitous. The regulatory function of inhibitor against RNases in the cell should be considered in studying the physiological significance of the pancreatic-type RNase family.

    DOI: 10.1089/dna.1997.16.413

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  • RECOMBINANT HUMAN PANCREATIC RIBONUCLEASE PRODUCED IN ESCHERICHIA-COLI - IMPORTANCE OF THE AMINO-TERMINAL SEQUENCE 査読

    J FUTAMI, M SENO, M KOSAKA, H TADA, S SENO, H YAMADA

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   216 ( 1 )   406 - 413   1995年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

    Human pancreatic ribonuclease I (hRNase 1) in the mature form has been produced in E.coli using T7 expression system. The recombinant hRNase 1 protein was solubilized from the inclusion bodies, refolded in glutathione redox system, and purified through chromatographic procedures by utilizing cation-exchange and reversed-phase columns. The ribonucleolytic activity of recombinant hRNase 1 was examined on yeast RNA and cytidylyl-3',5'-adenosine revealing the distinctive ribonucleolytic activity. The activity was perfectly inhibited by human placental RNase inhibitor. Truncation of 7 amino acid residues in the amino-terminal sequence resulted in much reduction in ribonucleolytic activity and in affinity to human placental RNase inhibitor with the disintegration of secondary structures of the protein observed by circular dichroism spectra. The present study has revealed the important contribution of the amino-terminal sequence of hRNase I to the characteristics of the protein. (C) 1995 Academic Press, Inc.

    DOI: 10.1006/bbrc.1995.2638

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  • MOLECULAR-CLONING AND EXPRESSION OF HUMAN RIBONUCLEASE-4 CDNA

    M SENO, J FUTAMI, Y TSUSHIMA, K AKUTAGAWA, M KOSAKA, H TADA, H YAMADA

    BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION   1261 ( 3 )   424 - 426   1995年4月

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    記述言語:英語   出版者・発行元:ELSEVIER SCIENCE BV  

    A cDNA coding for human ribonuclease 4 was isolated from a pancreas cDNA library and sequenced. This cDNA (996 bp) includes an entire open reading frame encoding mature protein (119 aa) following signal peptide (28 aa). Expression of mature protein in Escherichia coli showed an apparent molecular mass of about 16 kDa, which was slightly lower than the mature form of human RNase 1, in SDS-PAGE.

    DOI: 10.1016/0167-4781(95)00040-N

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  • MOLECULAR-CLONING AND EXPRESSION OF HUMAN RIBONUCLEASE-4 CDNA 査読

    M SENO, J FUTAMI, Y TSUSHIMA, K AKUTAGAWA, M KOSAKA, H TADA, H YAMADA

    BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION   1261 ( 3 )   424 - 426   1995年4月

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    記述言語:英語   出版者・発行元:ELSEVIER SCIENCE BV  

    A cDNA coding for human ribonuclease 4 was isolated from a pancreas cDNA library and sequenced. This cDNA (996 bp) includes an entire open reading frame encoding mature protein (119 aa) following signal peptide (28 aa). Expression of mature protein in Escherichia coli showed an apparent molecular mass of about 16 kDa, which was slightly lower than the mature form of human RNase 1, in SDS-PAGE.

    DOI: 10.1016/0167-4781(95)00040-n

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  • NUCLEOTIDE-SEQUENCE ENCODING HUMAN PANCREATIC RIBONUCLEASE 査読

    M SENO, J FUTAMI, M KOSAKA, S SENO, H YAMADA

    BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION   1218 ( 3 )   466 - 468   1994年8月

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    記述言語:英語   出版者・発行元:ELSEVIER SCIENCE BV  

    A cDNA coding for human pancreatic ribonuclease was isolated from a pancreas cDNA library and sequenced. This cDNA (1620 bp) includes an entire open reading frame encoding mature protein (128 aa) following a signal peptide (28 aa) as well as 5'- and 3'-untranslated regions.

    DOI: 10.1016/0167-4781(94)90208-9

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▼全件表示

書籍等出版物

  • 酵素利用技術体系 3-1表面修飾による酵素機能の向上

    NTS  2010年 

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  • ゲノミクス・プロテオミクスの新展開:生物情報の解析と応用

    2004年 

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講演・口頭発表等

  • 複合免疫療法に向けたがんの抗原性向上に関する基礎検討

    第70回日本生物工学会大会  2018年9月5日 

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  • REIC/Dkk-3タンパク質の相互作用分子解析と抗がん免疫活性化の分子機構解明

    第70回日本生物工学会大会  2018年9月5日 

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  • 変性タンパク質への部位特異的biotin化条件の最適化とneoantigenスクリーニングへの応用

    第70回日本生物工学会大会  2018年9月5日 

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    会議種別:口頭発表(一般)  

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  • 抗腫瘍関連抗原抗体をバイオマーカーとした高感度測定系の開発

    第70回日本生物工学会大会  2018年9月5日 

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  • 抗がん抗原抗体で腫瘍免疫応答をモニタリングするMUSCAT-Assay

    二見 淳一郎

    第22回日本がん免疫学会総会  2018年8月3日 

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  • 変性タンパク質への部位特異的biotin化技術を活用したNeo-antigenスクリーニング法の開発

    第59回 日本生化学会 中国・四国支部例会  2018年5月26日 

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  • 腫瘍関連抗原の生産系の構築とAntigen-Spreading測定系の開発

    第59回 日本生化学会 中国・四国支部例会  2018年5月26日 

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    会議種別:口頭発表(一般)  

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  • エピゲノム薬を用いたがんの抗原性向上に関する基礎検討

    第59回 日本生化学会 中国・四国支部例会  2018年5月26日 

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  • 不溶化する組換えタンパク質の巻き戻しと高度利用

    ライフサイエンス技術部会/反応分科会 技術セミナー  2018年 

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  • がん免疫細胞治療(γδT 細胞治療)におけるバイオマーカーの検索

    第52回日本成人病学会学術集会  2018年 

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  • SH保護試薬によるジスルフィド結合含有タンパク質の加熱不可逆失活の抑制

    第69回日本生物工学会大会  2017年 

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  • 変性タンパク質の可溶化技術を利用した腫瘍免疫応答の活性化をモニタリングするコンパニオン診断薬の開発

    第41回蛋白質と酵素の構造と機能に関する九州シンポジウム  2017年 

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  • 腫瘍免疫を活性化するREIC/Dkk-3タンパク質の相互作用分子の解析

    第69回日本生物工学会大会  2017年 

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  • 腫瘍関連抗原に対するAntigen-Spreading測定系の開発

    生物工学若手研究者の集い(若手会)夏のセミナー2017  2017年 

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  • SH保護試薬を用いたジスルフィド結合含有タンパク質の加熱不可逆失活機構の評価

    2017年度生命科学系学会合同年次大会  2017年 

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  • 腫瘍免疫応答の活性化をモニタリングするMUSCAT-assay

    第112回 岡山県医用工学研究会 平成29年度 第2回セミナー・交流会  2017年 

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  • 腫瘍免疫応答の活性化をモニタリングする抗体検査診断薬の標準化

    第69回日本生物工学会大会  2017年 

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  • REIC/Dkk-3タンパク質の相互作用分子解析による抗がん免疫活性機構の解明

    2017年度生命科学系学会合同年次大会  2017年 

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  • Analysis of structural propensity of epitopes for antibody against cancer specific antigen observed in cancer patients

    2017年度生命科学系学会合同年次大会  2017年 

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  • 複合がん免疫療法でのコンパニオン診断薬開発に向けた自家製陽性コントロールの評価

    生物工学若手研究者の集い(若手会)夏のセミナー2017  2017年 

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  • 自己抗原・がん抗原タンパク質の固定化法の開発

    生物工学若手研究者の集い(若手会)夏のセミナー2017  2017年 

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  • ジスルフィド結合含有タンパク質の加熱不可逆失活抑制法の開発

    第3回 日本生物工学会 西日本支部 講演会  2016年 

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  • HEK293細胞内で不溶性として発現する全長がん抗原タンパク質の可溶化法

    第39回日本分子生物学会年会  2016年 

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  • 腫瘍免疫応答の活性化測定の標準化

    第3回 日本生物工学会 西日本支部 講演会  2016年 

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  • REIC/DKK3タンパク質の免疫賦活化機構の解明

    第3回 日本生物工学会 西日本支部 講演会  2016年 

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  • 受容体を介したタンパク質細胞内導入技術の開発

    第3回 日本生物工学会 西日本支部 講演会  2016年 

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  • Hek293細胞を用いた全長がん抗原タンパク質の網羅的調製条件の最適化

    第68回日本生物工学会大会  2016年 

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  • Optimization of preparation methodology for multiple full-length cancer antigens using Hek293 cells

    2016年 

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  • Quantitative analysis of antigen-spreading using water-soluble and full-length cancer antigens

    第75回日本癌学会学術総会  2016年 

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  • 動物細胞内で強制発現させたがん抗原タンパク質の細胞内凝集

    第68回日本生物工学会大会  2016年 

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  • 受容体を介したヒト細胞内タンパク質の標的細胞質内への送達技術の開発

    第39回日本分子生物学会年会  2016年 

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  • Quantitative analysis of activation of anti-tumor immunoresponce

    BioJAPAN2016  2016年 

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  • 疎水性残基の導入が蛋白質の結晶化に及ぼす影響

    第39回日本分子生物学会年会  2016年 

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  • フレキシブルな構造のヒト細胞内タンパク質を標的細胞内へ送達する技術開発

    日本分子生物学会・日本生化学会合同年会BMB2015  2015年 

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  • ハイマンノース型遊離糖鎖のタンパク質リフォールディング促進機能

    日本農芸化学会中四国支部第41回講演会  2015年 

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  • 動物細胞由来の変性状態の総タンパク質混合物が核酸除去により示す高い水溶性

    第15回日本蛋白質科学会年会  2015年 

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  • 放線菌RHA1株のビフェニル代謝に関与するタンパク質の新規同定と解析

    日本農芸化学会2015年度岡山大会  2015年 

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  • カチオン化全長・水溶性がん抗原タンパク質の調製条件の最適化

    第15回日本蛋白質科学会年会  2015年 

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  • 受容体を介したタンパク質細胞内導入技術の開発

    第15回日本蛋白質科学会年会  2015年 

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  • 難結晶性蛋白質の結晶化を促進する疎水性残基導入

    日本結晶学会年会  2015年 

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  • 疎水性基導入が難結晶性蛋白質の結晶化に及ぼす影響

    第15回日本蛋白質科学会年会  2015年 

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  • S-カチオン化全長・水溶性抗原を用いた高感度抗体検出技術による腫瘍免疫応答の定量評価

    第67回日本生物工学会大会  2015年 

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  • カチオン化全長・水溶性がん抗原タンパク質を用いた高感度抗体検出試薬の調製条件の最適化

    日本分子生物学会・日本生化学会合同年会BMB2015  2015年 

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  • 高純度・水溶性ヒト全長Cancer/Testis 抗原リソースの整備

    第66回日本生物工学会  2014年 

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  • 遊離N―グリ力ンはタンパク質リフォールディングを促進する

    第 33 回日本糖質学会年会  2014年 

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  • 放線菌RHA1 株のビフェニル代謝に関与する新規同定タンパク質の機能解析

    第66回日本生物工学会  2014年 

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  • Yoshito Abe, Takatoshi Ohkuri, 木村吉伸, 前田 恵, 二見淳一郎, Tadashi Ueda

    日本薬学会  2014年 

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  • ヒト抗体 L 鎖変異体の安定性に対する添加剤の影響

    第14回日本蛋白質科学会年会  2014年 

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  • ヒト全長Cancer/Testis抗原の効率的な生産系の開発

    第14回日本蛋白質科学会年会  2014年 

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  • 遊離N-グリカンのタンパク質リフォールディング促進機能

    日本生化学会  2014年 

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  • タンパク質カチオン化技術を用いた高感度抗体検出技術の開発

    第66回日本生物工学会  2014年 

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  • 多種類ヒトCT抗原の効率的な生産系システムの開発

    日本生物工学会  2013年 

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  • 用途に応じたタンパク質の効率的生産

    中性子利用研究セミナー  2013年 

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  • ヒト全長CT 抗原の効率的生産システムの開発

    日本蛋白質科学会  2013年 

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  • Physiological Function of Free N-glycan involved in Protein Folding

    Annual meeting of glycobiology 2013  2013年 

     詳細を見る

  • 放線菌RHA1株のビフェニル代謝に関する新規遺伝子の同定と解析

    日本生物工学会  2013年 

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  • 変性状態のタンパク質の高度利用と診断薬への応用

    化学工学会 第45回秋季大会  2013年 

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  • 全長・水溶性がん抗原タンパク質を用いた抗体検査法の開発

    日本生物工学会  2013年 

     詳細を見る

  • Protein Cationization Techniques for Biomedical Engineering

    Young Asian Biochemical Engineers' Community (YABEC)  2012年 

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  • 細胞内導入型転写因子と両親媒性ペプチドの併用による機能発現の向上

    日本生物工学会西日本支部・日本農芸化学会中四国支部合同講演会  2012年 

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  • 難結晶性蛋白質の結晶化に向けた結晶化タグの開発と評価

    第12回日本蛋白質科学会年会  2012年 

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  • タンパク質カチオン化技術を用いた高純度・可溶性がん抗原タンパク質の調製技術の開発

    第34回東大病院22世紀医療センター:産学連携メディカルフロンティアセミナー  2012年 

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  • 細胞内導入型RAGE阻害ペプチドの開発

    日本生物工学会西日本支部第2回講演会  2012年 

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  • 疎水性結晶化タグの導入による蛋白質結晶化促進法の開発

    第12回日本蛋白質科学会年会  2012年 

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  • 転写因子タンパク質のin cell folding法 による機能発現技術の開発

    第64回日本生物工学会大会  2012年 

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  • 変性状態の細胞内総タンパク質が示す溶解性に関する研究

    第64回日本生物工学会大会  2012年 

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  • カチオン化アビジンを介したビオチン化タンパク質細胞導入法における導入効率の最適化

    第64回日本生物工学会大会  2012年 

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  • 大腸菌を用いた膜貫通蛋白質の高発現

    第64回日本生物工学会大会  2012年 

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  • 両親媒性ペプチドの併用による可逆的変性カチオン化タンパク質のin cell folding技術の改善

    日本生物工学会西日本支部第2回講演会  2012年 

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  • 可逆的変性カチオン化法を活用した高純度・水溶性がん抗原タンパク質の調製

    第64回日本生物工学会大会  2012年 

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  • TAPS-sulfonateを用いた高純度・水溶性がん抗原タンパク質の調製方法

    日本生物工学会西日本支部第2回講演会  2012年 

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  • タンパク質カチオン化技術を活用した細胞機能制御

    日本高分子学会・バイオ・高分子研究会  2011年 

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  • 可逆的変性カチオン化法を用いた変性タンパク質の高度精製とin cell foldin

    第63回日本生物工学会大会  2011年 

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  • 凍結乾燥保存が可能な細胞内導入型転写因子

    第11回日本蛋白質科学会年会  2011年 

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  • タンパク質カチオン化技術によるタンパク質の物性操作と医用応用

    第8回産総研健康工学研究部門講演会  2010年 

     詳細を見る

  • 変性タンパク質の可溶化技術:原理と応用

    第163回農芸化学特別セミナー  2010年 

     詳細を見る

  • 変性タンパク質の可溶化技術と工学的応用

    日本蛋白質科学会年会  2010年 

     詳細を見る

  • タンパク質カチオン化技術を活用した細胞機能制御

    日本組織培養学会  2010年 

     詳細を見る

  • タンパク質可逆的修飾試薬を活用したがん免疫への応用

    第33回日本分子生物学会年会会  2010年 

     詳細を見る

  • 可逆的タンパク質マンノース化試薬の合成と評価

    第32回日本分子生物学会年会  2009年 

     詳細を見る

  • タンパク質カチオン化技術を駆使した医用工学への応用研究

    第2回高度医療都市を創出する未来技術国際シンポジウム  2009年 

     詳細を見る

  • 可逆的変性カチオン化法を用いた細胞内導入型転写因子の開発

    日本蛋白科学会  2009年 

     詳細を見る

  • Exploiting protein cationization techniques in future cell therapy

    Joint Conference of 10th CTS and 36th JSOPMB  2009年 

     詳細を見る

  • 細胞内総タンパク質の可逆的変性カチオン化と溶解性に関する研究

    第32回日本分子生物学会年会  2009年 

     詳細を見る

  • ナノ構造体内部への高密度タンパク質封入に向けたタンパク質カチオン化技術の検討

    日本生化学・分子生物学会合同年会BMB2008  2008年 

     詳細を見る

  • Differential delivery of antigen to dendoritic cells potentiates the MHC class I and/or II-restricted T cell responses

    67回 日本癌学会総会  2008年 

     詳細を見る

  • タンパク質カチオン化技術の医用工学への応用

    大阪大学蛋白質研究所セミナー:蛋白質を創る、知る、使う 〜蛋白質科学と産業応用〜  2008年 

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  • タンパク質カチオン化技術を利用したがん治療技術の開発戦略

    第1回高度医療都市を創出する未来技術国際シンポジウム  2008年 

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  • 人工転写因子蛋白質を用いた細胞内導入条件の最適化

    日本生化学・分子生物学会合同年会BMB2008  2008年 

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  • タンパク質の化学修飾(カチオン化)を駆使した創薬支援研究

    第3回 新薬創生研究会  2007年 

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  • ポリエチレンイミン(PEI)-グルタチオンキャリアーを用いたGST-融合タンパク質の細胞導入

    第59回日本生物工学会大会  2007年 

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  • 細胞内導入型転写因子の開発に向けた基礎検討

    第31回蛋白質と酵素の構造と機能に関する九州シンポジウム  2007年 

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  • 人工転写因子を用いたタンパク質細胞内導入効率の定量化

    第30回日本分子生物学会年会・第80回日本生化学会大会 合同大会(BMB2007)  2007年 

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  • β-cateninタンパク質導入による人為的Wntシグナル制御

    2006年分子生物学フォーラム  2006年 

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  • Design and characterization of hybrid proteins constructed by domain insertion fusion.

    20th IUBMB international congress of biochemistry and molecular biology and 11th FAOBMB congress  2006年 

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  • Artificial regulation of cell proliferation by protein transduction of N-terminal domain of simian virus 40 large T antigen using PEI-cationization method.

    20th IUBMB international congress of biochemistry and molecular biology and 11th FAOBMB congress  2006年 

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  • タンパク質カチオン化技術 -タンパク質の細胞内導入と生産技術への応用-

    蛋白質と酵素の構造と機能に関する九州シンポジウム  2006年 

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  • Crystal Lattice Engineering: Crystallization and Structure Determination of Human RNase 1 Mutants with a Hydrophobic Interface of Leucines.

    5th East Asian Biophysics Symposium & 44th Annual Meeting of the Biophysical Society of Japan  2006年 

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  • Applications of intracellular delivery of a protein by cationiczation. From in vitro folding to in cell folding of denatured proteins.

    A satellite Meeting of International Conference of 43rd Japanese Symposium and 4th Peptide Engeneering Meeting. Membrane-permeable peptides: Chemistry, biology and therapeutic applications  2006年 

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  • 蛋白質カチオン化法とHIV-TATペプチドを介した蛋白質細胞内導入の比較

    日本分子生物学会年会  2005年 

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  • 分子間疎水相互作用の導入によるヒト RNase1 の結晶化と構造解析

    第5回日本蛋白質科学会年会  2005年 

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  • ポリエチレンイミン(PEI)カチオン化法によるタンパク質細胞内導入技術の開発

    第23回 バイオテクノロジーシンポジウム  2005年 

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  • カチオン性ポリマーを用いた変性蛋白質の可溶化技術と細胞内導入技術の開発

    産学連携を指向した九州バイオサイエンスシンポジウム・疾患プロテオミクス最前線  2005年 

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  • Quantitative analysis of intracellular delivery of cationized proteins

    日本生化学会大会  2005年 

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  • 蛋白質カチオン化による細胞内導入技術開発と機構解明

    日本生物工学会大会  2005年 

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  • Artificial Control of Cell Proliferation Using an N-terminal Domain of Simian Virus 40 Large T Antigen by Means of PEI-cationization.

    The American Society for Cell Biology 45th Annual Meeting  2005年 

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  • 大腸菌インクルージョンボディからのニワトリアビジン四量体の酸化的リフォールディング

    日本生化学大会  2004年 

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  • カチオン性キャリアーによるタンパク質導入技術の開発

    第22回バイオシンポジウム  2004年 

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  • 蛍光共鳴エネルギー移動原理に基づく免疫学的測定法を利用した細胞内シグナル伝達経路の解析技術とPEIを用いたタンパク質の細胞内導入技術の開発

    第22回バイオシンポジウム  2004年 

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  • カチオン化SV40T抗原N末端ドメインによる細胞増殖の人工制御

    第27回日本分子生物学会年会  2004年 

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  • タンパク質生細胞内導入法によるliving cell imagingにおけるバックグラウンド低減化技術

    第27回日本分子生物学会年会  2004年 

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  • T7 RNA Polymeraseの細胞内導入による外部制御可能な一過性遺伝子発現

    日本生化学大会  2004年 

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受賞

  • 新産業創出研究奨励大賞

    2019年10月   公益財団法人両備檉園記念財団  

    二見 淳一郎

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  • BBB Awards for Excellence to Authors (2016) Vol. 80

    2017年  

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    受賞国:日本国

    researchmap

  • 内山勇三科学技術賞

    2015年  

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    受賞国:日本国

    researchmap

 

担当授業科目

  • SDGs:生命科学 (2021年度) 第4学期  - 木7~8

  • バイオ・創薬科学概論 (2021年度) 前期  - その他

  • バイオ・創薬科学概論 (2021年度) 前期  - 木1~2

  • ヘルスシステム統合科学アドバンストインターンシップ (2021年度) 通年  - その他

  • ヘルスシステム統合科学専門英語 (2021年度) 後期  - その他

  • ヘルスシステム統合科学特別研究 (2021年度) 通年  - その他

  • ヘルスシステム統合科学総合演習 (2021年度) 後期  - その他

  • 分析化学 (2021年度) 1・2学期  - 木3,木4

  • 分析化学1 (2021年度) 第1学期  - 木3,木4

  • 分析化学2 (2021年度) 第2学期  - 木3,木4

  • 専門英語 (2021年度) 3・4学期  - 木3,木4

  • 専門英語1 (2021年度) 第3学期  - 木3,木4

  • 専門英語2 (2021年度) 第4学期  - 木3,木4

  • 工学基礎実験実習 (2021年度) 1・2学期  - 火5,火6,火7,火8

  • 工学基礎実験実習 (2021年度) 1・2学期  - 火5,火6,火7,火8

  • 工学基礎実験実習 (2021年度) 1・2学期  - 火5,火6,火7,火8

  • 技術表現法 (2021年度) 3・4学期  - 月5,月6,月7,月8

  • 技術表現法 (2021年度) 3・4学期  - 月5~6

  • 技術表現発表学 (2021年度) 後期  - その他

  • 技術表現発表学 (2021年度) 後期  - その他

  • 熱力学I (2021年度) 第1学期  - 水3~4

  • 物理化学及び演習1 (2021年度) 第1学期  - 月3,月4,水1,水2

  • 物理化学1 (2021年度) 第1学期  - 月3,月4,水1,水2

  • 物理化学1 (2021年度) 1・2学期  - 水3,水4

  • 物理化学A (2021年度) 1~3学期  - [第1学期]水3~4, [第2学期]その他, [第3学期]金3~4

  • 物理化学A (2021年度) 1~3学期  - [第1学期]水3~4, [第2学期]その他, [第3学期]金3~4

  • 蛋白質分子設計学 (2021年度) 後期  - その他

  • バイオ・創薬科学概論 (2020年度) 前期  - 木1,木2

  • ヘルスシステム統合科学アドバンストインターンシップ (2020年度) 通年  - その他

  • ヘルスシステム統合科学インターンシップ (2020年度) 通年  - その他

  • ヘルスシステム統合科学専門英語 (2020年度) 後期  - その他

  • ヘルスシステム統合科学特別研究 (2020年度) 通年  - その他

  • ヘルスシステム統合科学総合演習 (2020年度) 後期  - その他

  • 分析化学 (2020年度) 1・2学期  - 水3,水4

  • 分析化学1 (2020年度) 第1学期  - 水3,水4

  • 分析化学2 (2020年度) 第2学期  - 水3,水4

  • 分析化学2 (2020年度) 第4学期  - 水3~4

  • 化学生命系英語 (2020年度) 1・2学期  - 水3,水4

  • 化学生命系英語1 (2020年度) 第1学期  - 水3,水4

  • 化学生命系英語2 (2020年度) 第2学期  - 水3,水4

  • 技術表現法 (2020年度) 3・4学期  - 月5,月6

  • 技術表現法 (2020年度) 3・4学期  - 月5,月6

  • 技術表現発表学 (2020年度) 後期  - その他

  • 教養生物学(バイオテクノロジー) (2020年度) 特別  - その他

  • 教養生物学(バイオテクノロジー) (2020年度) 第4学期  - 木3,木4

  • 物理化学及び演習1 (2020年度) 第1学期  - 月3,月4,水1,水2

  • 物理化学1 (2020年度) 第1学期  - 月3,月4,水1,水2

  • 蛋白質分子工学 (2020年度) 前期  - 月1,月2

  • 蛋白質分子設計学 (2020年度) 後期  - その他

▼全件表示