Updated on 2024/11/24

写真a

 
FUTAMI Junichiro
 
Organization
Faculty of Interdisciplinary Science and Engineering in Health Systems Professor
Position
Professor
Profile
Medical protein engineering Lab is working to realize medical application of protein engineering. Our on-going efforts focus on innovative application of denatured protein solubilization techniques for immune-oncology.
External link

Degree

  • Ph.D. ( Okayama University )

  • 修士(工学)

Research Interests

  • Biochemistry

  • Immuno-Oncology

  • Biotechnology

  • Protein Engineering

Research Areas

  • Nanotechnology/Materials / Chemistry and chemical methodology of biomolecules

  • Life Science / Tumor diagnostics and therapeutics

  • Manufacturing Technology (Mechanical Engineering, Electrical and Electronic Engineering, Chemical Engineering) / Biofunction and bioprocess engineering

  • Life Science / Molecular biology

Education

  • Okayama University   自然科学研究科   生物資源科学専攻

    1996.4 - 1999.3

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    Country: Japan

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  • Okayama University   工学研究科   生体機能応用工学専攻

    1994.4 - 1996.3

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  • Okayama University   工学部   生体機能応用工学科

    1990.4 - 1994.3

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    Country: Japan

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Research History

  • 学術研究院ヘルスシステム統合科学学域 教授

    2022.4

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  • 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

    2019.12 - 2022.3

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  • Okayama University   Department of Interdisciplinary Science and Engineering in Health Systems   PI Associate Professor

    2018.4 - 2019.12

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  • Okayama University   Graduate School of Natural Science and Technology   PI Associate Professor

    2008.10 - 2018.3

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  • Okayama University   Graduate School of Natural Science and Technology   Senior Assistant Professor

    2005.10 - 2008.9

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  • NEDO-P/J PD fellow

    2003.2 - 2005.9

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  • Smimitomo Pharm. Genomics Research Group

    2002.4 - 2003.1

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  • NCI-Frederick, Guest PD Fellow

    2000.1 - 2000.12

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  • JSPS PD-Fellow

    1999.4 - 2002.3

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  • JSPS DC2 Fellow

    1997.4 - 1999.3

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Professional Memberships

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Committee Memberships

  • 日本生化学会   評議員  

    2016.9   

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    Committee type:Academic society

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  • 日本生物工学会   和文誌編集委員  

    2016.6 - 2022.6   

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    Committee type:Academic society

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  • 日本生物工学会   代議員  

    2013   

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    Committee type:Academic society

    日本生物工学会

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Papers

  • Enhanced design of pCMViR-TSC plasmid vector for sustainably high cargo gene expression in mammalian cells. International journal

    Masakiyo Sakaguchi, Rie Kinoshita, Nahoko Tomonobu, Yoshihiko Sakaguchi, Junichiro Futami, Akira Yamauchi, Hitoshi Murata, Ken-Ichi Yamamoto, Tetta Takahashi, Yuma Gohara, Toshiki Ochi, Fan Jiang, Ni Luh Gede Yoni Komalasari, Youyi Chen, I Made Winarsa Ruma, I Wayan Sumardika, Jin Zhou, Tomoko Honjo, Futoshi Kuribayashi, Kazumi Sagayama, Shinichi Toyooka, Eisaku Kondo, Yusuke Inoue

    In vitro cellular & developmental biology. Animal   2024.11

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    Language:English   Publishing type:Research paper (scientific journal)  

    The first-generation pCMViR-TSC, implemented through the promoter sandwich rule, yields 10- to 100-fold higher gene expression than the standard plasmid used with the CMV (cytomegalovirus) or CAG promoter. However, the vector's shortcomings limit its utility to transient expression only, as it is not suitable for establishing stable transformants in mammalian cells. To overcome this weakness, we here introduce the improved plasmid vector pSAKA-4B, derived from pCMViR-TSC as a second-generation chromosome-insertable vector. This vector facilitates the linear entry of the expression unit into the TTAA site of DNA universally with transposase assistance. The vector is helpful for the indefinite expression of our target gene. The new vector system is proven here to be efficient in establishing stable transformants with a high likelihood of positive clones that exhibit significantly elevated expression levels of the delivered foreign gene. This system, alongside the first-generation vector, is therefore instrumental for diverse basic research endeavors concerning genes, proteins, cells, and animals, and potentially for clinical applications such as gene therapy.

    DOI: 10.1007/s11626-024-00992-2

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  • Dissection of the signal transduction machinery responsible for the lysyl oxidase-like 4-mediated increase in invasive motility in triple-negative breast cancer cells: mechanistic insight into the integrin-β1-NF-κB-MMP9 axis Reviewed

    Fan Jiang, Youyi Chen, Nahoko Tomonobu, Rie Kinoshita, Ni Luh Gede, Yoni Komalasari, Carlos Ichiro, Kasano-Camones, Kazumi Ninomiya, Hitoshi Murata, Ken-ichi Yamamoto, Yuma Gohara, Toshiki Ochi, I Made Winarsa Ruma, I Wayan, Sumardika, Jin Zhou, Tomoko Honjo, Yoshihiko Sakaguchi, Akira Yamauchi, Futoshi Kuribayashi, Junichiro Futami, Eisaku Kondo, Yusuke Inoue, Shinichi Toyooka, Masakiyo Sakaguchi

    Front. Oncol. Sec. Molecular and Cellular Oncology.   14 ( 1371307 )   2024.5

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    DOI: 10.3389/fonc.2024.1371307

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  • Lysyl oxidase-like 4 promotes the invasiveness of triple-negative breast cancer cells by orchestrating the invasive machinery formed by annexin A2 and S100A11 on the cell surface. Reviewed International journal

    Tetta Takahashi, Nahoko Tomonobu, Rie Kinoshita, Ken-Ichi Yamamoto, Hitoshi Murata, Ni Luh Gede Yoni Komalasari, Youyi Chen, Fan Jiang, Yuma Gohara, Toshiki Ochi, I Made Winarsa Ruma, I Wayan Sumardika, Jin Zhou, Tomoko Honjo, Yoshihiko Sakaguchi, Akira Yamauchi, Futoshi Kuribayashi, Eisaku Kondo, Yusuke Inoue, Junichiro Futami, Shinichi Toyooka, Yoshito Zamami, Masakiyo Sakaguchi

    Frontiers in oncology   14   1371342 - 1371342   2024

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    BACKGROUND: Our earlier research revealed that the secreted lysyl oxidase-like 4 (LOXL4) that is highly elevated in triple-negative breast cancer (TNBC) acts as a catalyst to lock annexin A2 on the cell membrane surface, which accelerates invasive outgrowth of the cancer through the binding of integrin-β1 on the cell surface. However, whether this machinery is subject to the LOXL4-mediated intrusive regulation remains uncertain. METHODS: Cell invasion was assessed using a transwell-based assay, protein-protein interactions by an immunoprecipitation-Western blotting technique and immunocytochemistry, and plasmin activity in the cell membrane by gelatin zymography. RESULTS: We revealed that cell surface annexin A2 acts as a receptor of plasminogen via interaction with S100A10, a key cell surface annexin A2-binding factor, and S100A11. We found that the cell surface annexin A2/S100A11 complex leads to mature active plasmin from bound plasminogen, which actively stimulates gelatin digestion, followed by increased invasion. CONCLUSION: We have refined our understanding of the role of LOXL4 in TNBC cell invasion: namely, LOXL4 mediates the upregulation of annexin A2 at the cell surface, the upregulated annexin 2 binds S100A11 and S100A10, and the resulting annexin A2/S100A11 complex acts as a receptor of plasminogen, readily converting it into active-form plasmin and thereby enhancing invasion.

    DOI: 10.3389/fonc.2024.1371342

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  • Hydrophobicity and molecular mass‐based separation method for autoantibody discovery from mammalian total cellular proteins Reviewed

    Mirei Date, Ai Miyamoto, Tomoko Honjo, Tsugumi Shiokawa, Hiroko Tada, Nobuhiro Okada, Junichiro Futami

    Protein Science   32 ( 10 )   2023.9

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    Authorship:Last author, Corresponding author   Publishing type:Research paper (scientific journal)   Publisher:Wiley  

    Abstract

    Serum autoantibody profiles are unique to individuals and reflect the level and history of autoimmunity and tumor immunity. The identification of autoantibody biomarkers is critical for the development of immune monitoring systems for immune‐related disorders. Here, we present a practical method for large‐scale autoantibody discovery using total cellular proteins from cultured mammalian cells. We found that nucleic acid‐free and fully denatured water‐soluble total cellular proteins from mammalian cells were superior, allowing precise separation by reversed‐phase HPLC after preparing a large set of homogeneous total cellular proteins. After separating the proteins based on hydrophobicity, the fractionated samples were subjected to molecular mass analysis using conventional SDS‐PAGE. The resulting two‐dimensional gel electrophoresis was successfully employed for immune blotting and LC–MS/MS analysis. All procedures, including TRIzol‐based total cellular protein extraction, solubilization of denatured proteins, reversed‐phase HPLC separation, and SDS‐PAGE, were highly reproducible and easily scalable. We propose this novel two‐dimensional gel electrophoresis system as an alternative proteomics‐based methodology suitable for large‐scale autoantibody discovery.

    DOI: 10.1002/pro.4771

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  • Novel extracellular role of REIC/Dkk-3 protein in PD-L1 regulation in cancer cells. Reviewed International journal

    Yuma Gohara, Nahoko Tomonobu, Rie Kinoshita, Junichiro Futami, Léna Audebert, Youyi Chen, Ni Luh Gede Yoni Komalasari, Fan Jiang, Chikako Yoshizawa, Hitoshi Murata, Ken-Ichi Yamamoto, Masami Watanabe, Hiromi Kumon, Masakiyo Sakaguchi

    Journal of molecular medicine (Berlin, Germany)   101 ( 4 )   431 - 447   2023.3

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    The adenovirus-REIC/Dkk-3 expression vector (Ad-REIC) has been the focus of numerous clinical studies due to its potential for the quenching of cancers. The cancer-suppressing mechanisms of the REIC/DKK-3 gene depend on multiple pathways that exert both direct and indirect effects on cancers. The direct effect is triggered by REIC/Dkk-3-mediated ER stress that causes cancer-selective apoptosis, and the indirect effect can be classified in two ways: (i) induction, by Ad-REIC-mis-infected cancer-associated fibroblasts, of the production of IL-7, an important activator of T cells and NK cells, and (ii) promotion, by the secretory REIC/Dkk-3 protein, of dendritic cell polarization from monocytes. These unique features allow Ad-REIC to exert effective and selective cancer-preventative effects in the manner of an anticancer vaccine. However, the question of how the REIC/Dkk-3 protein leverages anticancer immunity has remained to be answered. We herein report a novel function of the extracellular REIC/Dkk-3-namely, regulation of an immune checkpoint via modulation of PD-L1 on the cancer-cell surface. First, we identified novel interactions of REIC/Dkk-3 with the membrane proteins C5aR, CXCR2, CXCR6, and CMTM6. These proteins all functioned to stabilize PD-L1 on the cell surface. Due to the dominant expression of CMTM6 among the proteins in cancer cells, we next focused on CMTM6 and observed that REIC/Dkk-3 competed with CMTM6 for PD-L1, thereby liberating PD-L1 from its complexation with CMTM6. The released PD-L1 immediately underwent endocytosis-mediated degradation. These results will enhance our understanding of not only the physiological nature of the extracellular REIC/Dkk-3 protein but also the Ad-REIC-mediated anticancer effects. KEY MESSAGES: • REIC/Dkk-3 protein effectively suppresses breast cancer progression through an acceleration of PD-L1 degradation. • PD-L1 stability on the cancer cell membrane is kept high by binding with mainly CMTM6. • Competitive binding of REIC/Dkk-3 protein with CMTM6 liberates PD-L1, leading to PD-L1 degradation.

    DOI: 10.1007/s00109-023-02292-w

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  • Toll-like receptor 4 promotes bladder cancer progression upon S100A8/A9 binding, which requires TIRAP-mediated TPL2 activation. Reviewed International journal

    Acosta Gonzalez Herik Rodrigo, Nahoko Tomonobu, Haruka Yoneda, Rie Kinoshita, Yosuke Mitsui, Takuya Sadahira, Shin-Ichi Terawaki, Yuma Gohara, Ni Luh Gede Yoni Komalasari, Fan Jiang, Hitoshi Murata, Ken-Ichi Yamamoto, Junichiro Futami, Akira Yamauchi, Futoshi Kuribayashi, Yusuke Inoue, Eisaku Kondo, Shinichi Toyooka, Masahiro Nishibori, Masami Watanabe, Yasutomo Nasu, Masakiyo Sakaguchi

    Biochemical and biophysical research communications   634   83 - 91   2022.12

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    Bladder cancer is an often widely disseminated and deadly cancer. To block the malignant outgrowth of bladder cancer, we must elucidate the molecular-level characteristics of not only bladder cancer cells but also their surrounding milieu. As part of this effort, we have long been studying extracellular S100A8/A9, which is elevated by the inflammation associated with certain cancers. Extracellularly enriched S100A8/A9 can hasten a shift to metastatic transition in multiple types of cancer cells. Intriguingly, high-level S100A8/A9 has been detected in the urine of bladder-cancer patients, and the level increases with the stage of malignancy. Nonetheless, S100A8/A9 has been investigated mainly as a potential biomarker of bladder cancers, and there have been no investigations of its role in bladder-cancer growth and metastasis. We herein report that extracellular S100A8/A9 induces upregulation of growth, migration and invasion in bladder cancer cells through its binding with cell-surface Toll-like receptor 4 (TLR4). Our molecular analysis revealed the TLR4 downstream signal that accelerates such cancer cell events. Tumor progression locus 2 (TPL2) was a key factor facilitating the aggressiveness of cancer cells. Upon binding of S100A8/A9 with TLR4, TPL2 activation was enhanced by an action with a TLR4 adaptor molecule, TIR domain-containing adaptor protein (TIRAP), which in turn led to activation of the mitogen-activated protein kinase (MAPK) cascade of TPL2. Finally, we showed that sustained inhibition of TLR4 in cancer cells effectively dampened cancer survival in vivo. Collectively, our results indicate that the S100A8/A9-TLR4-TPL2 axis influences the growth, survival, and invasive motility of bladder cancer cells.

    DOI: 10.1016/j.bbrc.2022.09.116

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  • Immune State Conversion of the Mesenteric Lymph Node in a Mouse Breast Cancer Model. Reviewed International journal

    Tsukasa Shigehiro, Maho Ueno, Mayumi Kijihira, Ryotaro Takahashi, Chiho Umemura, Eman A Taha, Chisaki Kurosaka, Megumi Asayama, Hiroshi Murakami, Ayano Satoh, Yoshimasa Nakamura, Junichiro Futami, Junko Masuda

    International journal of molecular sciences   23 ( 19 )   2022.9

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    Secondary lymphoid tissues, such as the spleen and lymph nodes (LNs), contribute to breast cancer development and metastasis in both anti- and pro-tumoral directions. Although secondary lymphoid tissues have been extensively studied, very little is known about the immune conversion in mesenteric LNs (mLNs) during breast cancer development. Here, we demonstrate inflammatory immune conversion of mLNs in a metastatic 4T1 breast cancer model. Splenic T cells were significantly decreased and continuously suppressed IFN-γ production during tumor development, while myeloid-derived suppressor cells (MDSCs) were dramatically enriched. However, T cell numbers in the mLN did not decrease, and the MDSCs only moderately increased. T cells in the mLN exhibited conversion from a pro-inflammatory state with high IFN-γ expression to an anti-inflammatory state with high expression of IL-4 and IL-10 in early- to late-stages of breast cancer development. Interestingly, increased migration of CD103+CD11b+ dendritic cells (DCs) into the mLN, along with increased (1→3)-β-D-glucan levels in serum, was observed even in late-stage breast cancer. This suggests that CD103+CD11b+ DCs could prime cancer-reactive T cells. Together, the data indicate that the mLN is an important lymphoid tissue contributing to breast cancer development.

    DOI: 10.3390/ijms231911035

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  • Histidine-Rich Glycoprotein Suppresses the S100A8/A9-Mediated Organotropic Metastasis of Melanoma Cells Reviewed

    Nahoko Tomonobu, Rie Kinoshita, Hidenori Wake, Yusuke Inoue, I Made Winarsa Ruma, Ken Suzawa, Yuma Gohara, Ni Luh Gede Yoni Komalasari, Fan Jiang, Hitoshi Murata, Ken-ichi Yamamoto, I Wayan Sumardika, Youyi Chen, Junichiro Futami, Akira Yamauchi, Futoshi Kuribayashi, Eisaku Kondo, Shinichi Toyooka, Masahiro Nishibori, Masakiyo Sakaguchi

    International Journal of Molecular Sciences   23 ( 18 )   10300 - 10300   2022.9

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    The dissection of the complex multistep process of metastasis exposes vulnerabilities that could be exploited to prevent metastasis. To search for possible factors that favor metastatic outgrowth, we have been focusing on secretory S100A8/A9. A heterodimer complex of the S100A8 and S100A9 proteins, S100A8/A9 functions as a strong chemoattractant, growth factor, and immune suppressor, both promoting the cancer milieu at the cancer-onset site and cultivating remote, premetastatic cancer sites. We previously reported that melanoma cells show lung-tropic metastasis owing to the abundant expression of S100A8/A9 in the lung. In the present study, we addressed the question of why melanoma cells are not metastasized into the brain at significant levels in mice despite the marked induction of S100A8/A9 in the brain. We discovered the presence of plasma histidine-rich glycoprotein (HRG), a brain-metastasis suppression factor against S100A8/A9. Using S100A8/A9 as an affinity ligand, we searched for and purified the binding plasma proteins of S100A8/A9 and identified HRG as the major protein on mass spectrometric analysis. HRG prevents the binding of S100A8/A9 to the B16-BL6 melanoma cell surface via the formation of the S100A8/A9 complex. HRG also inhibited the S100A8/A9-induced migration and invasion of A375 melanoma cells. When we knocked down HRG in mice bearing skin melanoma, metastasis to both the brain and lungs was significantly enhanced. The clinical examination of plasma S100A8/A9 and HRG levels showed that lung cancer patients with brain metastasis had higher S100A8/A9 and lower HRG levels than nonmetastatic patients. These results suggest that the plasma protein HRG strongly protects the brain and lungs from the threat of melanoma metastasis.

    DOI: 10.3390/ijms231810300

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  • The immunoreceptor SLAMF8 promotes the differentiation of follicular dendritic cell-dependent monocytic cells with B cell-activating ability. Reviewed International journal

    Masaki Magari, Miku Nishioka, Tomomi Hari, Sayaka Ogawa, Kaho Takahashi, Naoya Hatano, Naoki Kanayama, Junichiro Futami, Hiroshi Tokumitsu

    FEBS letters   2022.8

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    Follicular dendritic cells (FDCs) play a crucial role in generating high-affinity antibody-producing B cells during the germinal center (GC) reaction. Herein, we analysed the altered gene expression profile of a mouse FDC line, FL-Y, following lymphotoxin β receptor stimulation, and observed increased Slam-family member 8 (Slamf8) mRNA expression. Forced Slamf8 expression and SLAMF8-Fc addition enhanced the ability of FL-Y cells to induce FDC-induced monocytic cell (FDMC) differentiation. FDMCs accelerated GC-phenotype proliferation in cultured B cells, suggesting that they are capable of promoting GC responses. Furthermore, a pulldown assay showed that SLAMF8-Fc could bind to SLAMF8-His. Overall, the homophilic interaction of SLAMF8 promotes FDMC differentiation and SLAMF8 might act as a novel regulator of GC responses by regulating FDMC differentiation.

    DOI: 10.1002/1873-3468.14468

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  • Dkk3/REIC Deficiency Impairs Spermiation, Sperm Fibrous Sheath Integrity and the Sperm Motility of Mice. Reviewed International journal

    Ruizhi Xue, Wenfeng Lin, Hirofumi Fujita, Jingkai Sun, Rie Kinoshita, Kazuhiko Ochiai, Junichiro Futami, Masami Watanabe, Hideyo Ohuchi, Masakiyo Sakaguchi, Zhengyan Tang, Peng Huang, Yasutomo Nasu, Hiromi Kumon

    Genes   13 ( 2 )   2022.1

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    The role of Dickkopf-3 (Dkk3)/REIC (The Reduced Expression in Immortalized Cells), a Wnt-signaling inhibitor, in male reproductive physiology remains unknown thus far. To explore the functional details of Dkk3/REIC in the male reproductive process, we studied the Dkk3/REIC knock-out (KO) mouse model. By examining testicular sections and investigating the sperm characteristics (count, vitality and motility) and ultrastructure, we compared the reproductive features between Dkk3/REIC-KO and wild-type (WT) male mice. To further explore the underlying molecular mechanism, we performed RNA sequencing (RNA-seq) analysis of testicular tissues. Our results showed that spermiation failure existed in seminiferous tubules of Dkk3/REIC-KO mice, and sperm from Dkk3/REIC-KO mice exhibited inferior motility (44.09 ± 8.12% vs. 23.26 ± 10.02%, p < 0.01). The Ultrastructure examination revealed defects in the sperm fibrous sheath of KO mice. Although the average count of Dkk3/REIC-KO epididymal sperm was less than that of the wild-types (9.30 ± 0.69 vs. 8.27 ± 0.87, ×106), neither the gap (p > 0.05) nor the difference in the sperm vitality rate (72.83 ± 1.55% vs. 72.50 ± 0.71%, p > 0.05) were statistically significant. The RNA-seq and GO (Gene Oncology) enrichment results indicated that the differential genes were significantly enriched in the GO terms of cytoskeleton function, cAMP signaling and calcium ion binding. Collectively, our research demonstrates that Dkk3/REIC is involved in the process of spermiation, fibrous sheath integrity maintenance and sperm motility of mice.

    DOI: 10.3390/genes13020285

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  • Engineering Cancer/Testis Antigens With Reversible S-Cationization to Evaluate Antigen Spreading. Reviewed International journal

    Ai Miyamoto, Tomoko Honjo, Mirei Masui, Rie Kinoshita, Hiromi Kumon, Kazuhiro Kakimi, Junichiro Futami

    Frontiers in oncology   12   869393 - 869393   2022

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    Authorship:Last author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)  

    Serum autoantibody to cancer/testis antigens (CTAs) is a critical biomarker that reflects the antitumor immune response. Quantitative and multiplexed anti-CTA detection arrays can assess the immune status in tumors and monitor therapy-induced antitumor immune reactions. Most full-length recombinant CTA proteins tend to aggregate. Cysteine residue-specific S-cationization techniques facilitate the preparation of water-soluble and full-length CTAs. Combined with Luminex technology, we designed a multiple S-cationized antigen-immobilized bead array (MUSCAT) assay system to evaluate multiple serum antibodies to CTAs. Reducible S-alkyl-disulfide-cationized antigens in cytosolic conditions were employed to develop rabbit polyclonal antibodies as positive controls. These control antibodies sensitively detected immobilized antigens on beads and endogenous antigens in human lung cancer-derived cell lines. Rabbit polyclonal antibodies successfully confirmed the dynamic ranges and quantitative MUSCAT assay results. An immune monitoring study was conducted using the serum samples on an adenovirus-mediated REIC/Dkk-3 gene therapy clinical trial that showed a successful clinical response in metastatic castration-resistant prostate cancer. Autoantibody responses were closely related to clinical outcomes. Notably, upregulation of anti-CTA responses was monitored before tumor regression. Thus, quantitative monitoring of anti-CTA antibody biomarkers can be used to evaluate the cancer-immunity cycle. A quality-certified serum autoantibody monitoring system is a powerful tool for developing and evaluating cancer immunotherapy.

    DOI: 10.3389/fonc.2022.869393

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  • Identification of Neoantigens in Two Murine Gastric Cancer Cell Lines Leading to the Neoantigen-Based Immunotherapy. Reviewed International journal

    Koji Nagaoka, Changbo Sun, Yukari Kobayashi, Takayuki Kanaseki, Serina Tokita, Toshihiro Komatsu, Kazuhiro Maejima, Junichiro Futami, Sachiyo Nomura, Keiko Udaka, Hidewaki Nakagawa, Toshihiko Torigoe, Kazuhiro Kakimi

    Cancers   14 ( 1 )   2021.12

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    To develop combination immunotherapies for gastric cancers, immunologically well-characterized preclinical models are crucial. Here, we leveraged two transplantable murine gastric cancer cell lines, YTN2 and YTN16, derived from the same parental line but differing in their susceptibility to immune rejection. We established their differential sensitivity to immune checkpoint inhibitors (ICI) and identified neoantigens. Although anti-CTLA-4 mAbs eradicated YTN16 tumors in 4 of 5 mice, anti-PD-1 and anti-PD-L1 mAbs failed to eradicate YTN16 tumors. Using whole-exome and RNA sequencing, we identified two and three neoantigens in YTN2 and YTN16, respectively. MHC class I ligandome analysis detected the expression of only one of these neoantigens, mutated Cdt1, but the exact length of MHC binding peptide was determined. Dendritic cell vaccine loaded with neoepitope peptides and adoptive transfer of neoantigen-specific CD8+ T cells successfully inhibited the YTN16 tumor growth. Targeting mutated Cdt1 had better efficacy for controlling the tumor. Therefore, mutated Cdt1 was the dominant neoantigen in these tumor cells. More mCdt1 peptides were bound to MHC class I and presented on YTN2 surface than YTN16. This might be one of the reasons why YTN2 was rejected while YTN16 grew in immune-competent mice.

    DOI: 10.3390/cancers14010106

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  • Unusual aggregation property of recombinantly expressed cancer-testis antigens in mammalian cells. Reviewed International journal

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

    Journal of biochemistry   170 ( 3 )   435 - 443   2021.10

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    Authorship:Corresponding author   Language:English   Publishing type:Research paper (scientific journal)  

    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

  • 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

  • A suitable and effective stepwise oxidative refolding procedure for highly‐cationic tetrameric avidin in nucleic acid free conditions Reviewed International journal

    Shuichiro Kimura, Koreyoshi Imamura, Junichiro Futami

    Biotechnology Progress   36 ( 5 )   e3031   2020.9

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    Authorship:Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher: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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    Other Link: 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. Reviewed International journal

    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   2019.12

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    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.

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  • 研究室主宰者の自戒 Invited

    二見 淳一郎

    生物工学会誌   97 ( 11 )   671 - 674   2019.11

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  • Upregulation of Mobility in Pancreatic Cancer Cells by Secreted S100A11 Through Activation of Surrounding Fibroblasts. Reviewed International journal

    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   2019.8

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    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.

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  • Newly developed anti-S100A8/A9 monoclonal antibody efficiently prevents lung tropic cancer metastasis. Reviewed International journal

    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   2019.7

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    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.

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  • Critical role of the MCAM-ETV4 axis triggered by extracellular S100A8/A9 in breast cancer aggressiveness. Reviewed International journal

    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   2019.7

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    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.

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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. Reviewed International journal

    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   2019.6

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    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.

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  • Extracellular S100A11 Plays a Critical Role in Spread of the Fibroblast Population in Pancreatic Cancers. Reviewed International journal

    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   2019.6

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    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.

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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. Reviewed International journal

    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   2019.6

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    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".

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  • Neuroplastin-β mediates S100A8/A9-induced lung cancer disseminative progression. Reviewed International journal

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

    Molecular carcinogenesis   58 ( 6 )   980 - 995   2019.6

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    Compiling evidence indicates an unusual role of extracellular S100A8/A9 in cancer metastasis. S100A8/A9 secreted from either cancer cells or normal cells including epithelial and inflammatory cells stimulates cancer cells through S100A8/A9 sensor receptors in an autocrine or paracrine manner, leading to cancer cell metastatic progression. We previously reported a novel S100A8/A9 receptor, neuroplastin-β (NPTNβ), which plays a critical role in atopic dermatitis when it is highly activated in keratinocytes by an excess amount of extracellular S100A8/A9 in the inflammatory skin lesion. Interestingly, our expression profiling of NPTNβ showed significantly high expression levels in lung cancer cell lines in a consistent manner. We hence aimed to determine the significance of NPTNβ as an S100A8/A9 receptor in lung cancer. Our results showed that NPTNβ has strong ability to induce cancer-related cellular events, including anchorage-independent growth, motility and invasiveness, in lung cancer cells in response to extracellular S100A8/A9, eventually leading to the expression of a cancer disseminative phenotype in lung tissue in vivo. Mechanistic investigation revealed that binding of S100A8/A9 to NPTNβ mediates activation of NFIA and NFIB and following SPDEF transcription factors through orchestrated upstream signals from TRAF2 and RAS, which is linked to anchorage-independent growth, motility and invasiveness. Overall, our results indicate the importance of the S100A8/A9-NPTNβ axis in lung cancer disseminative progression and reveal a pivotal role of its newly identified downstream signaling, TRAF2/RAS-NFIA/NFIB-SPDEF, in linking to the aggressive development of lung cancers.

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  • β-1,3-Galactosyl-O-Glycosyl-Glycoprotein β-1,6-N-Acetylglucosaminyltransferase 3 Increases MCAM Stability, Which Enhances S100A8/A9-Mediated Cancer Motility. Reviewed International journal

    I Wayan Sumardika, Chen Youyi, Eisaku Kondo, Yusuke Inoue, I Made Winarsa Ruma, Hitoshi Murata, Rie Kinoshita, Ken-Ichi Yamamoto, Shuta Tomida, Kazuhiko Shien, Hiroki Sato, Akira Yamauchi, Junichiro Futami, Endy Widya Putranto, Toshihiko Hibino, Shinichi Toyooka, Masahiro Nishibori, Masakiyo Sakaguchi

    Oncology research   26 ( 3 )   431 - 444   2018.4

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    We previously identified novel S100A8/A9 receptors, extracellular matrix metalloproteinase inducer (EMMPRIN), melanoma cell adhesion molecule (MCAM), activated leukocyte cell adhesion molecule (ALCAM), and neuroplastin (NPTN) β, that are critically involved in S100A8/A9-mediated cancer metastasis and inflammation when expressed at high levels. However, little is known about the presence of any cancer-specific mechanism(s) that modifies these receptors, further inducing upregulation at protein levels without any transcriptional regulation. Expression levels of glycosyltransferase-encoding genes were examined by a PCR-based profiling array followed by confirmation with quantitative real-time PCR. Cell migration and invasion were assessed using a Boyden chamber. Western blotting was used to examine the protein level, and the RNA level was examined by Northern blotting. Immunohistochemistry was used to examine the expression pattern of β-1,3-galactosyl-O-glycosyl-glycoprotein β-1,6-N-acetylglucosaminyltransferase 3 (GCNT3) and MCAM in melanoma tissue. We found that GCNT3 is overexpressed in highly metastatic melanomas. Silencing and functional inhibition of GCNT3 greatly suppressed migration and invasion of melanoma cells, resulting in the loss of S100A8/A9 responsiveness. Among the novel S100A8/A9 receptors, GCNT3 favorably glycosylates the MCAM receptor, extending its half-life and leading to further elevation of S100A8/A9-mediated cellular motility in melanoma cells. GCNT3 expression is positively correlated to MCAM expression in patients with high-grade melanomas. Collectively, our results showed that GCNT3 is an upstream regulator of MCAM protein and indicate the possibility of a potential molecular target in melanoma therapeutics through abrogation of the S100A8/A9-MCAM axis.

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  • Mitochondrial determinants of mammalian longevity Reviewed

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

    OPEN BIOLOGY   7 ( 10 )   2017.10

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    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.

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

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

    SCIENTIFIC REPORTS   7 ( 1 )   12471 - 3275   2017.9

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    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 Reviewed

    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   2017.8

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    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 Reviewed

    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   2017.7

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    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 Reviewed

    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   2017.4

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    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

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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. Reviewed

    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

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  • Active Secretion of Dimerized S100A11 Induced by the Peroxisome in Mesothelioma Cells. Reviewed International journal

    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   2016.12

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    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-kappa B and ROS formation upon ligand binding Reviewed

    I. Made Winarsa Ruma, Endy Widya Putranto, Eisaku Kondo, Hitoshi Murata, Masami Watanabe, Peng Huang, Rie Kinoshita, Junichiro Futami, Yusuke Inoue, Akira Yamauchi, I. Wayan Sumardika, Chen Youyi, Ken-Ichi Yamamoto, Yasutomo Nasu, Masahiro Nishibori, Toshihiko Hibino, Masakiyo Sakaguchi

    CLINICAL & EXPERIMENTAL METASTASIS   33 ( 6 )   609 - 627   2016.8

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    The dynamic interaction between tumor cells and their microenvironment induces a proinflammatory milieu that drives cancer development and progression. The S100A8/A9 complex has been implicated in chronic inflammation, tumor development, and progression. The cancer microenvironment contributes to the up-regulation of this protein complex in many invasive tumors, which is associated with the formation of pre-metastatic niches and poor prognosis. Changing adhesive preference of cancer cells is at the core of the metastatic process that governs the reciprocal interactions of cancer cells with the extracellular matrices and neighboring stromal cells. Cell adhesion molecules (CAMs) have been confirmed to have high-level expression in various highly invasive tumors. The expression and function of CAMs are profoundly influenced by the extracellular milieu. S100A8/A9 mediates its effects by binding to cell surface receptors, such as heparan sulfate, TLR4 and RAGE on immune and tumor cells. RAGE has recently been identified as an adhesion molecule and has considerably high identity and similarity to ALCAM and MCAM, which are frequently over-expressed on metastatic malignant melanoma cells. In this study, we demonstrated that ALCAM and MCAM also function as S100A8/A9 receptors as does RAGE and induce malignant melanoma progression by NF-kappa B activation and ROS formation. Notably, MCAM not only activated NF-kappa B more prominently than ALCAM and RAGE did but also mediated intracellular signaling for the formation of lung metastasis. MCAM is known to be involved in malignant melanoma development and progression through several mechanisms. Therefore, MCAM is a potential effective target in malignant melanoma treatment.

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

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

    Biochemistry and Biophysics Reports   6   94 - 100   2016.7

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    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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  • Development of Antibody Detection System to Evaluate Activation of Anti-Tumor Immune Response Invited

    FUTAMI Junichiro

    33 ( 5 )   19 - 24   2016.5

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

    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   2016.5

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    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.

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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

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

    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   2015.10

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    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 Reviewed

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

    ONCOLOGY REPORTS   33 ( 6 )   2908 - 2914   2015.6

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    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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  • Advanced Utilization of Denatured Protein by Cationization Techniques Invited Reviewed

    FUTAMI Junichiro

    KAGAKU TO SEIBUTSU   53 ( 4 )   245 - 251   2015.4

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    DOI: 10.1271/kagakutoseibutsu.53.245

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

    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   2015.4

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    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 Reviewed

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

    PLOS ONE   9 ( 11 )   e113295   2014.11

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    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 Reviewed

    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   2014.8

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    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 Reviewed

    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   2014.7

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    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 Reviewed

    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   2014.3

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    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 Reviewed

    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   2013.10

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    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.

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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 Invited

    二見 淳一郎

    バイオインダストリー   15 - 21   2013.7

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  • Protein cationization techniques for artificial control of physical property of protein and their medical applications Reviewed

    Junichiro Futami

    Seikagaku   85 ( 1 )   21 - 25   2013

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

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

    BIOCONJUGATE CHEMISTRY   23 ( 10 )   2025 - 2031   2012.10

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    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 Reviewed

    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   2011.3

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    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 Reviewed

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

    BIOTECHNOLOGY JOURNAL   5 ( 4 )   385 - 392   2010.4

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    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 Invited

    二見 淳一郎

    ケミカルエンジニアリング   55 ( 3 )   2010.3

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

    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   2010.2

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    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 Reviewed

    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   2009.3

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    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 Reviewed

    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   2008.10

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    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 Reviewed

    Junichiro Futami, Hidenori Yamada

    CURRENT PHARMACEUTICAL BIOTECHNOLOGY   9 ( 3 )   180 - 184   2008.6

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

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

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   105 ( 1 )   34 - 38   2008.1

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

    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   2008.1

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    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.

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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 Reviewed

    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   2007.12

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    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 Reviewed

    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   2007.7

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    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 Reviewed

    Junichiro Futami, Midori Kitazoe, Hiroshi Murata, Hidenori Yamada

    EXPERT OPINION ON DRUG DISCOVERY   2 ( 2 )   261 - 269   2007.2

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    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 Reviewed

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

    CHEMBIOCHEM   7 ( 11 )   1676 - +   2006.11

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    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 Reviewed

    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   2006.5

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    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 Reviewed

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

    ANTICANCER RESEARCH   26 ( 2A )   857 - 863   2006.3

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    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 Reviewed

    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

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher: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.

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  • 1P136 Crystal Lattice Engineering : Crystallization and Structure Determination of Human RNase 1 Mutants with a Hydrophobic Interface of Leucines(4. Protein engineering,Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

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

    Seibutsu Butsuri   46 ( 2 )   S180   2006

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    DOI: 10.2142/biophys.46.S180_4

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

    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   2005.7

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    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 Reviewed

    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   2005.6

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    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 Reviewed

    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   2005.2

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    Authorship:Lead author   Language:English   Publishing type:Research paper (scientific journal)   Publisher: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 Reviewed

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

    NUCLEIC ACIDS RESEARCH   33 ( 9 )   e88   2005

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    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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  • Protein Transduction using Cationic Polymers Invited

    FUTAMI Junichiro, YAMADA Hdenori

    21 ( 6 )   20 - 27   2004.6

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

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

    FEBS LETTERS   568 ( 1-3 )   39 - 43   2004.6

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    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 Reviewed

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

    Methods Mol Biol   207   283 - 304   2003

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

    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   2002.11

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    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 Reviewed

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

    JOURNAL OF BIOCHEMISTRY   132 ( 2 )   223 - 228   2002.8

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    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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  • EGFRを分子標的としたRNase-EGF recombinantタンパクの殺細胞効果

    星本 相淳, 上田 政和, 神野 浩光, 相浦 浩一, 渡辺 靖夫, 北島 政樹, 二見 淳一郎, 妹尾 昌治

    日本外科学会雑誌   103 ( 臨増 )   532 - 532   2002.3

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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 Reviewed

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

    BIOCHEMISTRY   40 ( 25 )   7518 - 7524   2001.6

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    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 Reviewed

    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   2001.4

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    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.

    DOI: 10.1107/S0907444901001147

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

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

    JOURNAL OF BIOCHEMISTRY   128 ( 2 )   245 - 250   2000.8

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    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 Reviewed

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

    JOURNAL OF BIOCHEMISTRY   127 ( 3 )   435 - 441   2000.3

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    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 Reviewed

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

    PROTEIN ENGINEERING   12 ( 11 )   1013 - 1019   1999.11

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    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 Reviewed

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

    Breast Cancer   4 ( 4 )   253 - 255   1997.12

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    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 Reviewed

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

    DNA AND CELL BIOLOGY   16 ( 4 )   413 - 419   1997.4

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    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 Reviewed

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

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   216 ( 1 )   406 - 413   1995.11

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    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   1995.4

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    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 Reviewed

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

    BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION   1218 ( 3 )   466 - 468   1994.8

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    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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Books

  • 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 

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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 

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    友信奈保子, 木下理恵, 合原勇馬, KOMALASARI Yoni, 二見淳一郎, 山内明, 近藤英作, 豊岡伸一, 阪口政清

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    宮本 愛, 本莊 知子, 益井 実鈴, 木下 理恵, 公文 裕巳, 垣見 和宏, 二見 淳一郎

    日本がん免疫学会総会プログラム・抄録集   26回   102 - 102   2022.6

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    峯苫 智晴, 垣見 和宏, 二見 淳一郎

    日本生物工学会大会講演要旨集   2019年   230 - 230   2019.8

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  • Melanoma cell adhesion molecule (MCAM) induces dissemination of melanoma upon S100A8/A9 binding

    友信奈保子, 木下理恵, 近藤英作, 山内明, 二見淳一郎, 豊岡伸一, 阪口政清

    日本癌学会学術総会抄録集(Web)   78th   2019

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    小坂恵, 山田秀徳, 二見淳一郎, 多田宏子, 今村維克, 玉田太郎

    日本結晶学会年会講演要旨集   2019   2019

  • パッキングのコントロールにより,難結晶性蛋白質の結晶化を促進する結晶化法

    小坂恵, 山田秀徳, 二見淳一郎, 多田宏子, 今村維克, 玉田太郎

    日本細胞生物学会大会(Web)   71st   2019

  • Novel therapeutic approach based on S100A8/A9-mediated organ tropic cancer metastasis

    木下理恵, 友信奈保子, 山内明, 枝園和彦, 冨田秀太, 村田等, 二見淳一郎, 近藤英作, 豊岡伸一, 阪口政清

    日本癌学会学術総会抄録集(Web)   78th   2019

  • 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

    峯苫 智晴, 垣見 和宏, 二見 淳一郎

    日本生物工学会大会講演要旨集   平成30年度   105 - 105   2018.8

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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, 本莊 知子, 吉岡 実咲, 勝河 祐希, Hannaneh Ahmadi, 木下 理恵, 藤枝 奈緒, 垣見 和宏

    日本がん免疫学会総会プログラム・抄録集   22回   122 - 122   2018.7

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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

    藤枝 奈緒, 大平 公亮, 小林 由香利, 神原 佳織, 泉 謙道, 高橋 卓也, 木村 真之介, 小林 史弥, 松下 博和, 二見 淳一郎, 垣見 和宏

    日本成人病(生活習慣病)学会会誌   44   77 - 77   2018.1

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    小坂恵, 山田秀徳, 二見淳一郎, 多田宏子, 今村維克, 玉田太郎

    日本結晶学会年会講演要旨集   2018   2018

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    日本生化学会大会(Web)   90th   2017

  • タンパク質のパッキングをコントロールする疎水性残基導入

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    日本結晶学会年会講演要旨集   2017   2017

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    二見 淳一郎, 細井 亮宏, 松下 博和, 垣見 和宏

    日本癌学会総会記事   75回   P - 2336   2016.10

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    小坂恵, 山田秀徳, 二見淳一郎, 多田宏子, 玉田太郎

    日本結晶学会年会講演要旨集   2016   2016

  • 疎水性残基の導入が蛋白質の結晶化に及ぼす影響

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  • 疎水性残基を変異導入する蛋白質結晶化促進法

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    日本結晶学会年会講演要旨集   2015   2015

  • 疎水性残基導入が難結晶性蛋白質の結晶化に及ぼす影響

    小坂恵, 山田秀徳, 二見淳一郎, 多田宏子, 安達基泰, 玉田太郎, 黒木良太

    日本蛋白質科学会年会プログラム・要旨集   15th   2015

  • 蛋白質のパッキングをコントロールする結晶化タグの開発と評価

    小坂恵, 山田秀徳, 二見淳一郎, 多田宏子, 安達基泰, 玉田太郎, 黒木良太

    日本結晶学会年会講演要旨集   2014   2014

  • 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

    小坂恵, 松田京子, 山田秀徳, 二見淳一郎, 多田宏子, 岡崎伸生, 玉田太郎, 黒木良太

    日本蛋白質科学会年会プログラム・要旨集   12th   2012

  • 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

    田路太地, 小坂恵, 山田秀徳, 二見淳一郎, 多田宏子, 岡崎伸生, 玉田太郎, 黒木良太

    日本蛋白質科学会年会プログラム・要旨集   12th   2012

  • タンパク質のパッキングをコントロールする結晶化法の開発

    小坂恵, 直井里美, 山田秀徳, 二見淳一郎, 多田宏子, 岡崎伸生, 玉田太郎, 黒木良太

    日本結晶学会年会講演要旨集   2011   2011

  • 結晶格子工学によるウシRNase Aの結晶空間群の変更

    土井慎一, 小坂恵, 二見淳一郎, 多田宏子, 玉田太郎, 岡崎伸生, 黒木良太, 山田秀徳

    生化学   2008

  • 内臓脂肪組織に由来するセリンプロテアーゼ阻害剤 : Vaspin の同定肥満状態でインスリン感受性を高める新規アディポサイトカイン

    肥田 和之, 和田 淳, 江口 潤, HONG Zhang, 馬場 雅子, 清田 綾, 橋本 泉, 岡田 達夫, 安原 章浩, 中司 敦子, 赤木 滋, 四方 賢一, 宝来 真志, 二見 淳一郎, 渡辺 英二郎, 松木 泰, 平松 隆司, 槇野 博史, KANWAR Yashpal S.

    岡山醫學會雜誌   118 ( 3 )   215 - 220   2007.1

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    Other Link: http://ousar.lib.okayama-u.ac.jp/13354

  • 2B11-4 Intracellular delivery of GST-fused proteins by polyethlenimineglutathione conjugates

    MURATA Hitoshi, FUTAMI Junichiro, KITAZOE Midori, KOSAKA Megumi, TADA Hiroko, KAI Takashi, SENO Masaharu, YAMADA Hidenori

    19   63 - 63   2007

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  • 1C16-5 Development of protein transduction technology by cationization and analysis of their mechanisms

    FUTAMI Junichiro, KITAZOE Midori, MURATA Hitoshi, WATANABE Yasuyoshi, YAGI Yasuyuki, TADA Hiroko, SENO Masaharu, Kai Hiroshi, YAMADA Hidenori

    17   103 - 103   2005

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  • 523 Design of fusion proteins by domain insertion

    Tada Hiroko, Onizuka Masayuki, Masuzawa Wataru, Futami Junichiro, Kosaka Megumi, Seno Masaharu, Yamada Hidenori

    14   78 - 78   2002

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  • Cytotoxic proteins generated by insertion of a carrier protein into ribonuclease.

    Tada Hiroko, Onizuka Masayuki, Futami Junichiro, Maeda takashi, Seno Masaharu, Yamada Hidenori

    13   266 - 266   2001

  • PP-815 上皮増殖因子受容体(EGFR)過剰発現癌に対する生理活性物質を用いた低免疫原性ターゲッテイング療法の開発

    神野 浩光, 上田 政和, 菊地 潔, 池田 正, 北島 政樹, 二見 淳一郎, 妹尾 昌治

    日本外科学会雑誌   101   411 - 411   2000.3

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  • Effects of Eosinophil Cationic Protein (ECP) on Mammalian Cell Lines.

    Maeda Takashi, Kitazoe Midori, Futami Junichiro, Tada Hiroko, Seno Masaharu, Yamada Hidenori

    12   198 - 198   2000

  • Effect of Eosinophil Cationic Protein (ECP) on Carcinoma Cells.

    Maeda Takashi, Kitazoe Midori, Futami Junichiro, Tada Hiroko, Seno Masaharu, Yamada Hidenori

    11   152 - 152   1999

  • 5種類のヒト膵臓由来型リボヌクレアーゼの比較

    村藤 裕, 二見 淳一郎, 津島 義明, 松本 佳世子, 堀井 史子, 多田 宏子, 妹尾 昌治, 山田 秀徳

    日本分子生物学会年会プログラム・講演要旨集   19   247 - 247   1996.8

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  • ヒトリボヌクレアーゼを用いた細胞増殖阻害剤の開発

    二見 淳一郎, 多田 慎太郎, 妹尾 昌治, 山田 秀徳

    日本分子生物学会年会プログラム・講演要旨集   19   722 - 722   1996.8

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  • SH基の保護試験alkyl methanethiosulfonateの蛋白質工学への応用

    竹内 倫太郎, 山口 博之, 谷川 真三郎, 津島 義明, 眞砂 明典, 二見 淳一郎, 小坂 恵, 多田 宏子, 妹尾 昌治, 山田 秀徳

    日本分子生物学会年会プログラム・講演要旨集   19   381 - 381   1996.8

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Presentations

  • 自己抗体バイオマーカーの網羅的定量評価系のバリデーション

    宮本 愛, 本莊 知子, 伊達 実鈴, 森 壮流, 大橋 圭明, 木浦 勝行, 垣見 和宏, 二見 淳一郎

    日本がん免疫学会総会プログラム・抄録集  2023.6  日本がん免疫学会

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    Event date: 2023.6

    Language:Japanese  

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  • 自己抗体バイオマーカーを用いたがん免疫サイクルを評価する精密測定法の開発

    宮本愛, 本莊知子, 益井実鈴, 木下理恵, 公文裕巳, 垣見和宏, 二見淳一郎

    第44回蛋白質の構造と機能に関する九州シンポジウム  2022.8.20 

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    Event date: 2022.8.21

    Presentation type:Oral presentation (general)  

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  • 代替2次元分離法を用いた自己抗体バイオマーカータンパク質の効率的な探索

    益井実鈴, 塩川つぐみ, 多田宏子, 二見淳一郎

    第44回蛋白質の構造と機能に関する九州シンポジウム  2022.8.20 

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    Event date: 2022.8.20 - 2022.8.21

    Presentation type:Poster presentation  

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  • がん免疫サイクルを評価する自己抗体バイオマーカー群の精密測定法の開発

    宮本愛, 本莊知子, 益井実鈴, 木下理恵, 公文裕巳, 垣見和宏, 二見淳一郎

    第26回日本がん免疫学会総会  2022.7.20 

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    Event date: 2022.7.22

    Presentation type:Oral presentation (general)  

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  • がん免疫サイクルを評価する自己抗体バイオマーカー群の精密測定法の開発

    宮本 愛, 本莊 知子, 益井 実鈴, 木下 理恵, 公文 裕巳, 垣見 和宏, 二見 淳一郎

    日本がん免疫学会総会プログラム・抄録集  2022.6  日本がん免疫学会

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    Event date: 2022.6

    Language:Japanese  

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  • 配列エピトープを認識する自己抗体バイオマーカーの効率的な探索法と評価系の改良

    伊達 実鈴, 塩川 つぐみ, 多田 宏子, 森 壮流, 本莊 知子, 宮本 愛, 二見 淳一郎

    第76回日本生物工学会大会  2024.9.9 

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  • 自己抗体バイオマーカーの網羅的定量評価システム開発と機械学習を用いたがん識別能の向上

    宮本 愛, 森 壮流, 伊達 実鈴, 本莊 知子, 大橋 圭明, 木浦 勝之, 垣見 和宏, 二見 淳一郎

    第76回日本生物工学会大会  2024.9.9 

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  • 自己抗体プロファイルの機械学習によるがん識別性能の評価

    森 壮流, 宮本 愛, 本莊 知子, 伊達 実鈴, 渡邉 洋美, 大橋 圭明, 木浦 勝之, 垣見 和宏, 二見 淳一郎

    第28回日本がん免疫学会総会・第37回日本バイオセラピィ学会学術集会総会 合同大会  2024.7.11 

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  • 変性状態の細胞内総タンパク質が示す溶解性を利用した新規プロテオミクス

    伊達実鈴, 塩川つぐみ, 多田宏子, 本莊知子, 宮本愛, 岡田宣宏, 二見 淳一郎

    第 75 回日本生物工学会大会  2023.9.5 

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  • 代替 2 次元分離法の開発による自己抗体バイオマーカー探索の効率化

    益井実鈴、塩川つぐみ、多田宏子、本莊知子、宮本愛、二見淳一郎

    第 23 回日本蛋白質科学会年会  2023.7.6 

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  • 自己抗体バイオマーカーの網羅的定量評価系による 免疫プロファイリング・モニタリング技術 Invited

    二見淳一郎

    第15回 日本血液疾患免疫療法学会学術集会シンポジウム:抗体マーカー  2023.6.23 

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  • 自己抗体免疫プロファイリング/モニタリング技術 と個別化医療サポートへの可能性 Invited

    二見淳一郎

    第96回日本内分泌学会学術総会:中堅若手の会 YECセミナー 『自己免疫とがん免疫研究のフロンティア』  2023.6.1 

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  • 自己抗体バイオマーカーの網羅的定量評価データの 統計解析による免疫プロファイリング

    森壮流, 宮本愛, 本莊知子, 益井実鈴, 二見淳一郎

    第64回日本生化学会中四国支部例会  2023.5.26 

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  • 自己抗体バイオマーカーのプロファイリング・ モニタリング技術による個別化医療のサポート Invited

    二見淳一郎

    第129回 岡山県医用工学研究会 オンラインセミナー 蛋白質機能の解明・創発に基づく医用工学・創薬への展開  2023.2.28 

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  • ヒト自己抗体が認識する構造/配列エピトープ解析

    山本莉加, 本莊知子, 宮本愛, 二見淳一郎

    日本生物工学会 西日本支部 第6回講演会  2022.11.26 

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  • がん免疫応答を末梢血で評価する自己抗体バイオマーカー群の 網羅的精密測定法開発

    宮本愛, 本莊知子, 益井実鈴, 木下理恵, 公文裕巳, 二見淳一郎

    第1回日本抗体学会設立記念学術大会  2022.11.26 

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  • タグなしタンパク質の高効率なrefoldingと精製法の開発

    山本航, 木村修一郎, 二見淳一郎

    日本生物工学会 西日本支部 第6回講演会  2022.11.26 

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  • 新規2次元分離法を用いた自己抗体バイオマーカータンパク質の効率的な探索法の開発 Invited

    益井実鈴

    日本生物工学会西日本支部大会2022(第6回講演会)第11回生物工学学生優秀賞(飛翔賞) 受賞講演  2022.11.26 

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  • 一人ひとりにあった医療をどう実現するか?-工学研究者と病院の現場の連携ー「個人に最適化された医療を生物工学で切り拓く」

    二見淳一郎

    岡山大学大学院ヘルスシステム統合科学研究科サイエンス・カフェ2022  2022.11.19 

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  • 代替 2 次元分離法の開発による自己抗体バイオマーカー探索の効率化

    益井実鈴, 塩川つぐみ, 多田宏子, 二見淳一郎

    第74回日本生物工学会大会  2022.10.20 

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  • がん免疫サイクルのレベルを末梢血で評価する自己抗体バイオマーカー群網羅的測定法の確立

    宮本愛, 本莊知子, 益井実鈴, 木下理恵, 公文裕巳, 垣見和宏, 二見淳一郎

    第74回日本生物工学会大会  2022.10.20 

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  • 変性タンパク質工学から見えてきた天然変性タンパク質の姿

    二見淳一郎

    第74回日本生物工学会大会 シンポジウム「生体分子の相互作用 における曖昧さの意義」  2022.10.18 

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  • 自己抗体免疫プロファイリング/モニタリング技術による個別化医療のサポート Invited

    二見淳一郎

    Basic Biology Seminar in Okayama  2022.10.14 

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  • 免疫プロファイリングプラットフォームの完成にはバイオバンクの検体がぜひとも必要である Invited

    二見淳一郎

    第7回クリニカルバイオバンク学会  2022.7.8 

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  • 新規2次元分離プロテオミクスによる自己抗体バイオマーカー探索法の開発

    益井 実鈴, 塩川 つぐみ, 多田 宏子, 二見 淳一郎

    第73回日本生物工学会大会  2021.10.29 

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  • 自己抗体バイオマーカーの網羅的迅定量評価による腫瘍免疫応答モニタリング

    宮本愛, 田中健介, 本莊知子, 二見 淳一郎

    第62回 日本生化学会 中国・四国支部例会  2021.9.10 

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  • Expi293での安価な分泌タンパク質生産

    森彩良, 宮本愛, 本荘知子, 二見淳一郎

    第62回 日本生化学会 中国・四国支部例会  2021.9.10 

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  • 新規2次元分離法を用いた自己抗体バイオマーカータンパク質の効率的な探索法の開発

    益井 実鈴, 塩川 つぐみ, 多田 宏子, 二見 淳一郎

    第62回 日本生化学会 中国・四国支部例会  2021.9.10 

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  • 自己抗体バイオマーカーの迅速定量評価技術によるがん免疫サイクルのモニタリング

    田中健介, 宮本愛, 本莊知子, 二見淳一郎

    日本生物工学会西日本支部大会2020(第5回講演会)  2020.11.14 

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  • 自己免疫疾患関連の自己抗原リソース強化から考察された抗原性との物性相関

    橋口万澄, 宮本愛, 本莊知子, 二見淳一郎

    日本生物工学会西日本支部大会2020(第5回講演会)  2020.11.14 

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  • 細胞内総タンパク質可溶化技術を活用した新規プロテオミクス解析技術の開発

    益井実鈴, 馬場龍之介, 塩川つぐみ, 多田宏子, 二見淳一郎

    日本生物工学会西日本支部大会2020(第5回講演会)  2020.11.14 

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  • Unusual aggregation properties of forced expressed cancer/testis antigens in mammalian cell

    Hannaneh Ahmadi, Kouhei Shogeni, Yuki Katsukawa, Kana Fujita, Junichiro Futami

    2019.9.19 

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  • 可逆的カチオン化転写因子タンパク質の生細胞内導入における効率と経路の検討

    中野智貴, 周伯揚, 二見翠, 二見淳一郎

    2019.9.17 

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  • エピゲノム薬を用いた複合免疫療法における診断薬開発の基礎検討

    勝河祐希, Hannaneh Ahmadi, 本莊 知子, 二見淳一郎

    第71回日本生物工学会大会  2019.9.17 

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  • 部位特異的ビオチン化とrefolding assayを組み合せたneo-antigenスクリーニング法の開発

    峯苫智晴, 垣見和宏, 二見淳一郎

    第71回日本生物工学会大会  2019.9.17 

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  • 抗がん抗原抗体が認識するエピトープ解析とがん免疫サイクル活性化の評価

    吉岡実咲, 本莊知子, 木下理恵, 二見淳一郎

    2019.9.17 

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  • MUSCAT-Assay法での自己抗体モニタリングによる腫瘍免疫応答評価

    二見淳一郎, 本莊知子, 吉岡実咲, 勝河祐希, Hannaneh Ahmadi, 尾崎龍之介, 木下理恵, 鵜殿平一郎, 垣見和宏

    第23回日本がん免疫学会  2019.8.23 

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  • 腫瘍免疫応答を迅速にモニタリングする MUSCAT-Assay

    二見淳一郎

    第1回ファーマラボEXPO  2019.7.5 

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  • 腫瘍免疫応答の活性化をモニタリングするマウス自己抗体のプロテオーム解析

    尾﨑龍之介, 勝河祐希, 二見淳一郎

    日本生物工学会 西日本支部 第4回講演会  2018.12.1 

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  • 分子内SS結合保護試薬による タンパク質の耐熱性向上

    萩本 惇史, 宮本 愛, 二見 淳一郎

    日本生物工学会 西日本支部 第4回講演会  2018.12.1 

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  • ペプチド転移酵素を利用した細胞質内送達リガンドの調製法の開発

    田中綾乃, 宮田尚也, 宮本愛, 二見淳一郎

    日本生物工学会 西日本支部 第4回講演会  2018.12.1 

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  • 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  2018.9.5 

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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  2018.9.5 

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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  2018.9.5 

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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  2018.9.5 

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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  2018.8.3 

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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  2018.5.26 

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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  2018.5.26 

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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  2018.5.26 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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

    2016 

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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 

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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 

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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 

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  • Quantitative analysis of activation of anti-tumor immunoresponce

    BioJAPAN2016  2016 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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

    Annual meeting of glycobiology 2013  2013 

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  • 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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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  • Protein Cationization Techniques for Biomedical Engineering

    Young Asian Biochemical Engineers' Community (YABEC)  2012 

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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 

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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 

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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 

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  • 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 

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  • 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 

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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

    日本組織培養学会  2010 

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  • 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 

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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 

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  • 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 

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  • 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 

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  • Exploiting protein cationization techniques in future cell therapy

    Joint Conference of 10th CTS and 36th JSOPMB  2009 

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  • 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 

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  • 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 

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  • 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 

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  • 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 

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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 

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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 

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  • 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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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  • Quantitative analysis of intracellular delivery of cationized proteins

    日本生化学会大会  2005 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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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 

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  • がん免疫サイクルのレベルを末梢血で評価する自己抗体バイオマーカー群網羅的測定法の確立

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  • 抗S100A8/A9抗体とその用途

    阪口 政清, 豊岡 伸一, 冨田 秀太, 枝園 和彦, 佐藤 博紀, 木下 理恵, 二見 淳一郎, 荒木 恒太, 岡▲崎▼ 幹生, 近藤 英作, 井上 裕介, 山内 明

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    Applicant:国立大学法人 岡山大学

    Application no:JP2019016100  Date applied:2019.4.15

    Publication no:WO2019-208290  Date published:20191031

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  • REIC/Dkk-3タンパク質を有効成分として含むTGFβ阻害剤

    二見淳一郎, 木下理恵, 公文裕巳

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    Applicant:桃太郎源株式会社

    Application no:特願2017-119919  Date applied:2017.6.19

    Announcement no:特開PCT/JP2017/022588  Date announced:2017.12.28

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  • 活性構造のREIC/Dkk-3タンパク質を特異的に認識して結合する抗体、及び該抗REIC/Dkk-3抗体を用いた癌治療のモニタリング

    公文 裕巳, 木下 理恵, 二見 淳一郎

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    Applicant:国立大学法人 岡山大学

    Application no:特願2017-543279  Date applied:2016.9.27

    Publication no:WO2017-057308  Date published:201746

    Patent/Registration no:特許第6975424号  Date registered:2021.11.10 

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  • 抗体検出用試薬の製造方法、及びその用途

    二見 淳一郎, 垣見 和宏, 前川 隆司, 白木 正人

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    Applicant:国立大学法人 岡山大学

    Application no:特願2013-531595  Date applied:2013.3.29

    Publication no:WO2013-147233  Date published:2013103

    Patent/Registration no:特許6168497  Date registered:2017.7.7  Date issued:2017.7.7

    6168497

    J-GLOBAL

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  • REIC/Dkk-3タンパク質の部分領域ポリペプチド

    公文 裕巳, 渡部 昌実, 二見 淳一郎, 藤井 康之, 植木 英雄, 落合 和彦

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    Applicant:国立大学法人 岡山大学

    Application no:特願2012-522733  Date applied:2011.7.1

    Publication no:WO2012-002582  Date published:201215

    Patent/Registration no:特許5936129  Date registered:2016.5.20  Date issued:2016.5.20

    5936129

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  • チオスルホナート化合物、タンパク質及び/又はペプチドの可逆的カチオン化剤並びに可溶化方法

    二見 淳一郎, 山田 秀徳, 久良木 豪, 矢木 恵一郎

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    Applicant:国立大学法人 岡山大学

    Application no:特願2012-507073  Date applied:2011.3.24

    Publication no:WO2011-118731  Date published:2011929

    Patent/Registration no:特許5713006  Date registered:2015.3.20 

    特許第5713006号

    J-GLOBAL

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  • タンパク質の細胞内導入剤

    山田 秀徳, 村田 等, 二見 淳一郎, 阪口 政清, 許 南浩, 八木 康行, 甲斐 敬

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    Applicant:株式会社日本触媒

    Application no:特願2007-046025  Date applied:2007.2.26

    Announcement no:特開2008-115150  Date announced:2008.5.22

    Patent/Registration no:特許第5097412号  Date registered:2012.9.28 

    特許第5097412号

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  • 細胞増殖剤及び細胞の増殖方法

    山田 秀徳, 二見 淳一郎, 村田 等, 阪口 政清, 許 南浩, 八木 康行, 甲斐 敬

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    Applicant:株式会社日本触媒

    Application no:特願2005-356631  Date applied:2005.12.9

    Announcement no:特開2007-159429  Date announced:2007.6.28

    J-GLOBAL

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  • タンパク質及び/又はペプチドの細胞内導入方法

    二見 淳一郎, 中西 秀高, 山田 秀徳, 甲斐 敬

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    Applicant:株式会社日本触媒

    Application no:特願2005-147250  Date applied:2005.5.19

    Announcement no:特開2006-006316  Date announced:2006.1.12

    Patent/Registration no:特許4885476  Date registered:2011.12.16  Date issued:2011.12.16

    特許第4885476号

    J-GLOBAL

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  • 可逆的カチオン化による変性タンパク質の細胞内導入と活性化の方法

    山田 秀徳, 二見 淳一郎, 村田 等, 前田 貴志, 阪口 政清

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    Applicant:株式会社日本触媒

    Application no:特願2003-356571  Date applied:2003.10.16

    Announcement no:特開2005-120017  Date announced:2005.5.12

    J-GLOBAL

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  • タンパク質及び/又はペプチドの細胞内導入用試験キット

    山田 秀徳, 二見 淳一郎, 中西 秀高

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    Applicant:株式会社日本触媒

    Application no:特願2003-149359  Date applied:2003.5.27

    Announcement no:特開2004-350526  Date announced:2004.12.16

    J-GLOBAL

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  • 蛋白質またはペプチドの細胞内導入方法

    山田 秀徳, 二見 淳一郎, 中西 秀高

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    Applicant:株式会社日本触媒

    Application no:特願2002-342486  Date applied:2002.11.26

    Announcement no:特開2004-049214  Date announced:2004.2.19

    J-GLOBAL

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  • 蛋白質又はペプチドの細胞内導入方法

    山田 秀徳, 二見 淳一郎, 前田 貴志, 北添 翠

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    Applicant:株式会社日本触媒

    Application no:特願2002-287280  Date applied:2002.9.30

    Announcement no:特開2004-121041  Date announced:2004.4.22

    Patent/Registration no:特許第3996028号  Date registered:2007.8.10 

    J-GLOBAL

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Awards

  • ベストティーチャー賞(物理化学1)

    2023.3   岡山大学工学部  

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  • ベストティーチャー賞(工学基礎実験実習)

    2022.3   岡山大学工学部  

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  • 研究功績賞

    2021.3   岡山大学工学部  

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  • 新産業創出研究奨励大賞

    2019.10   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  

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    Country:Japan

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  • 内山勇三科学技術賞

    2015  

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Research Projects

  • Immunological analysis and POC establishment of comprehensive neoantigen-specific immunotherapy for cold tumor

    Grant number:23H03780  2023.04 - 2026.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (B)

    神垣 隆, 二見 淳一郎, 瀧本 理修, 近藤 聡英

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    Grant amount:\18850000 ( Direct expense: \14500000 、 Indirect expense:\4350000 )

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  • Study on immunogenicity, physicochemical property, and immune monitoring of cancer/testis antigens

    Grant number:22H01881  2022.04 - 2025.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (B)

    二見 淳一郎

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    Grant amount:\17420000 ( Direct expense: \13400000 、 Indirect expense:\4020000 )

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  • 免疫モニタリング情報を個別化医療に活用するための自己抗体reference database整備

    2022.04 - 2024.09

    バイオバンク・ネットワークの試料・情報を利用する第1回研究課題公募  https://biobank-search.megabank.tohoku.ac.jp/v2/news?id=6ftSYV0jy8bJEODBMUJMTD

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  • 簡易モニタリングのための好酸球を用いた頭頸部がん免疫治療バイオマーカーの開発

    Grant number:21K09666  2021.04 - 2025.03

    日本学術振興会  科学研究費助成事業 基盤研究(C)  基盤研究(C)

    西川 大輔, 花井 信広, 松下 博和, 二見 淳一郎, 澤部 倫

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    Grant amount:\2470000 ( Direct expense: \1900000 、 Indirect expense:\570000 )

    我々の研究グループでは、頭頸部がんで初めて、免疫チェックポイント阻害薬(ICI)治療前の末梢血の好酸球や、治療後の変化が、再発転移頭頸部がんに対するNivolumabの治療バイオマーカーとなることを報告してきた。本研究では、これらの研究成果を発展させ、頭頸部がんにおいて末梢血の好酸球と、抗腫瘍免疫を担うTリンパ球や、抗原提示の要因となる腫瘍抗原、それに関連するサイトカインの関係が、治療によってどう変化するかを評価する。本研究の目的は、どのような施設においても簡易に利用可能なバイオマーカーを開発することである。
    本研究の対象は、愛知県がんセンター頭頸部外科、薬物療法科(腫瘍内科)にて、再発・転移頭頸部がんでICI治療を行った患者である。研究開始以前にICI治療を受けた頭頸部扁平上皮がん患者(すでに保管されている凍結検体を使用)、研究開始後にICI治療を受ける頭頸部扁平上皮がん患者を対象とする。
    本研究では、研究協力者との連携体制の整備が重要であり、現在整備をすすめている状況である。末梢血をICI治療前後、増悪時に採取し、研究所の腫瘍免疫制御トランスレーショナルリサーチ分野(TR分野)にて、抹消単核細胞(PBMC)と血清に分離、凍結保存する具体的な連携手順書を作成中である。また、抗原パネルアッセイについて岡山大学工学部化学生命系学科と協議を行っている。また、実際の診療において、ICI投与の前後の前後の採血が必要となるため、患者の不利益が生じないような、計画を準備中である。
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  • 免疫プロファイリングプラットフォームによる疾患の早期診断・迅速モニタリングシステムの開発

    2019.11 - 2021.09

    JST  JST-STARTプログラム 

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  • がん免疫サイクルの観点からみたAd-REICと分泌REICタンパク質の分子機構の解明と新規創薬

    2019.06 - 2022.02

    特別電源所在県科学技術振興事業 

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  • REICタンパク質の機能解析による新たな創薬基盤プラットフォームの構築

    2016.06 - 2018.02

    特別電源所在県科学技術振興事業 

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  • Development of companion diagnostics for personalized cancer immunotherapy using denatured protein solubilization technology

    Grant number:16H04580  2016.04 - 2019.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)  Grant-in-Aid for Scientific Research (B)

    Futami Junichiro

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    Grant amount:\17810000 ( Direct expense: \13700000 、 Indirect expense:\4110000 )

    We have developed the technology for analysis of antitumor immune response by using anti-cancer antigen autoantibody biomarkers. This technology employed luminex technology combined with S-cationization techniques to expose linear epitopes. This antibody detection system was successfully demonstrated that sensitive autoantibody detection relating to the activation of cancer immunity cycles.

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  • 変性タンパク質の可溶化技術を活 用したがん抗原抗体検査試薬の開発

    2015.06 - 2016.03

    (公財)岡山工学振興会  岡山工学振興会学術研究助成 

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  • Development of membrane protein reconstitution technique in vivo.

    Grant number:24656506  2012.04 - 2014.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research  Grant-in-Aid for Challenging Exploratory Research

    FUTAMI JUNICHIRO

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    Grant amount:\4160000 ( Direct expense: \3200000 、 Indirect expense:\960000 )

    Combining protein transduction and in cell folding techniques, we tried to develop a novel in cell folding techniques for membrane protein in living cells. In order to achieve this technology, in cell folding technique was drastically improved in this study. Using denatured and reversibly cationized precursor protein, protein maturation events were observed in human cells without translation machinery.

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  • 高純度・水溶性がん抗原タンパク質の調製技術の確立とリソースの整備

    2011.07 - 2012.03

    JST  知財活用促進ハイウェイ 

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  • Application of protein cationization techniques for cancer immunotherapy

    Grant number:23360370  2011.04 - 2014.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)  Grant-in-Aid for Scientific Research (B)

    JUNICHIRO FUTAMI, KAKIMI Kazuhiro

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    Grant amount:\18330000 ( Direct expense: \14100000 、 Indirect expense:\4230000 )

    This study aimed development of chemical protein cationization techniques for cancer antigens. Since most of cancer antigen proteins are flexible and unstable, solubilization with protein cationization techniques were useful methodology to prepare water-soluble and full-length cancer antigens. Using these cationized antigens, antigen antibodies for cancer antigens raised in cancer patients were successfully detected with a highly sensitive assay. Combining these developed techniques and enhanced cancer antigen production resource, we have successfully developed tools for cancer immunotherapy.

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  • がん治療遺伝子REICによるナノバイオ標的医療の創成

    2010.06 - 2013.03

    特別 

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  • タンパク質カチオン化技術を活用した細胞再生医療への応用研究

    2009.04 - 2010.03

    財団法人山陽放送学術文化財団 

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  • タンパク質カチオン化技術を活用した医用工学の基盤技術開発

    2008.04 - 2010.05

    NEDO  産業技術研究助成 

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  • 変性タンパク質の可溶化技術を活用した細胞内イメージング技術の開発

    Grant number:20650070  2008 - 2009

    日本学術振興会  科学研究費助成事業 挑戦的萌芽研究  挑戦的萌芽研究

    山田 秀徳, 二見 淳一郎

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    Grant amount:\3300000 ( Direct expense: \3300000 )

    (1) N末端特異的な蛍光標識による高精細な細胞内イメージング
    平成20年度に細胞内イメージングのモデルタンパク質としてN末端特異的に蛍光基を標識したβアクチンタンパク質を可逆的変性カチオン化法により高い水溶性を保持した蛍光標識プローブを調製することに成功していた。本年度はマイクロインジェクション法で細胞内への注入を試みたが、培地中で蛍光標識プローブが重合しやすく極めて困難であった。そこで蛍光標識プローブの溶解度向上に向けた諸条件を検討した結果、新規カチオン化試薬:TAP3S-Sulfonateの利用で高い溶解性が得られた。また、逆相HPLCで高純度に精製することで、純水および生理食塩水中で高い溶解度を維持できることを見出し、本手法に適した蛍光標識プローブの調製方法を確立した。
    (2) 細胞内分泌経路のイメージングに関する技術開発
    可逆的変性カチオン化により可溶化した分泌シグナル付きの前駆体の酵素タンパク質を生細胞内に導入すると、一部が培養上清中に活性構造の成熟型の酵素タンパク質が再分泌されることを酵素活性レベルで確認していたが、その収量はごく微量であった。この効率を改善するため、エンドソームから細胞質内への放出を促進する両親媒性ペプチドの併用条件を検討し、再分泌量が大幅に向上する条件を得た。さらにこの手法で回収された成熟型タンパク質を逆相HPLCを用いて高度に精製し、N末端配列を確認したところ、正しくプロセッシングを受けていることが確認され、翻訳系を伴わないpost-translational経路によるタンパク質の成熟化が可能なことを立証できた。

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  • 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可溶化試薬の開発

    2007.06 - 2008.02

    特別電源所在県科学技術振興事業 

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  • Development of analytical and production method for problematic protein by artificial control of physical property of protein

    Grant number:19206085  2007 - 2010

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)  Grant-in-Aid for Scientific Research (A)

    YAMADA Hidenori, FUTAMI Jyunichirou, SENOO Masaharu, TADA Hiroko, KOSAKA Megumi

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    Grant amount:\40820000 ( Direct expense: \31400000 、 Indirect expense:\9420000 )

    This study aimed development of handling technology especially for problematic protein using chemical conjugation techniques and protein crystallization methodology. Novel chemical modification reagent and optimization of protein preparation techniques provided improved transduction of transcription factor protein into cells, and solubilization of total cellular protein from cancer cells. Together with a newly developed protein crystallization tag sequence, these technologies will give a useful methodology for structure and functional analysis of proteins and their medical applications.

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  • Development and application of in cell folding technology based on reversible cationization of denatured proteins.

    Grant number:17360399  2005 - 2006

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)  Grant-in-Aid for Scientific Research (B)

    YAMADA Hidenori, FUTAMI Junichiro, SENO Masaharu, TADA Hiroko, KOSAKA Megumi

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    Grant amount:\15100000 ( Direct expense: \15100000 )

    This project aimed for the development of two methodologies; first is efficient solubilization of denatured proteins with reversible protein chemical cationization techniques, and second is intracellular delivery of reversibly cationized proteins followed by simultaneous folding to the active conformations, called in cell folding techniques. As a model protein, we investigated human tumor-suppressor p53. Bacterially expressed p53 protein as insoluble inclusion body was successfully solubilized by introduction of polyethylenimine 600 (PEI600, molecular weight: 600) via disulfide bond. The analysis of resulted PEI600-SS-p53 revealed that six cysteins were conjugated by PEI600, but remaining four cysteins failed in conjugation of PEI600, probably because of steric hindrance. This result indicated that reversible blocking of remaining free sulfidryl groups by a small molecule of aminopropyl methanethiosulfonate is important for preparation of chemically stable samples. The 'in cell folding' of p53 was successfully demonstrated by treatment of p53-null Saos-2 cells with reversibly cationized p53. PEI600-SS-p53 treated cells 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. Cationic charge dependent protein transduction efficiency was seen on the comparison between APS-SS-p53 (net charge: +6) and PEI600-SS-p53 (net charge: +81.6) because more cationic PEI600-SS-p53 showed more efficient induction of p53 targeted gene expression. This project also revealed that most of denatured proteins possessing cysteine residues can be solubilized by introduction of excess cationic charges. Furthermore, protein transduction and induction of their biological functions in living cells by using in cell folding techniques were confirmed by using at least 6 kinds of proteins.

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  • 細胞内ネットワークのダイナミズム解析技術開発

    2002.10 - 2007.03

    NEDO 

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  • ヒト由来タンパク質をベースとした細胞特異的増殖阻害剤のデザイン

    Grant number:99J07764  1999 - 2001

    日本学術振興会  科学研究費助成事業 特別研究員奨励費  特別研究員奨励費

    二見 淳一郎

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    Grant amount:\4100000 ( Direct expense: \4100000 )

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  • ヒト膵由来型リボヌクレアーゼの生理学的意義の解明と細胞特異的増殖阻害剤への応用

    Grant number:97J08759  1998

    日本学術振興会  科学研究費助成事業 特別研究員奨励費  特別研究員奨励費

    二見 淳一郎

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    Grant amount:\900000 ( Direct expense: \900000 )

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  • English Engineering (2023academic year) 3rd and 4th semester  - 木3~4

  • English Engineering 1 (2023academic year) Third semester  - 木3~4

  • English Engineering 2 (2023academic year) Fourth semester  - 木3~4

  • Technical Writing and Presentation (2023academic year) Third semester  - 月5~6

  • Technical Writing and Presentation (2023academic year) Third semester  - 月5~6

  • Technical Writing and Presentation (2023academic year) Late  - その他

  • Thermodynamics II (2023academic year) Second semester  - 水3~4

  • Physical Chemistry 1 (2023academic year) 1st and 2nd semester  - [第1学期]水1~2, [第2学期]水3~4

  • Physical Chemistry 1 (2023academic year) 1st and 2nd semester  - [第1学期]水1~2, [第2学期]水3~4

  • Analytical Chemistry in Environmental Fields Ⅱ (2023academic year) Second semester  - 水1~2

  • Analytical Chemistry in Environmental Fields I (2023academic year) 1st semester  - 水1~2

  • Biotechnology Experiment 1 (2023academic year) 1st semester  - 月5~8,木5~8

  • Biophysics (2023academic year) Third semester  - 月1~2

  • Design of protein molecules (2023academic year) Late  - その他

  • General Biotechnology and Drug Discovery Technology (2022academic year) Prophase  - その他

  • General Biotechnology and Drug Discovery Technology (2022academic year) Prophase  - 木1~2

  • Advanced Internship for Interdisciplinary Medical Sciences and Engineering (2022academic year) Year-round  - その他

  • Technical English for Interdisciplinary Medical Sciences and Engineering (2022academic year) Late  - その他

  • Research Works for Interdisciplinary Medical Sciences and Engineering (2022academic year) Year-round  - その他

  • Advanced Interdisciplinary Medical Sciences and Engineering (2022academic year) Prophase  - その他

  • General Exercises for Interdisciplinary Medical Sciences and Engineering (2022academic year) Late  - その他

  • Analytical Chemistry (2022academic year) 1st and 2nd semester  - 水1~2

  • Analytical Chemistry (2022academic year) 1st and 2nd semester  - 水1~2

  • Analytical Chemistry 1 (2022academic year) 1st semester  - 水1~2

  • Analytical Chemistry 2 (2022academic year) Second semester  - 水1~2

  • Introduction to Chemistry and Bioengineering (2022academic year) 1st semester  - その他

  • English for Chemistry and Biotechnology (2022academic year) 1st and 2nd semester  - 水3~4

  • English for Chemistry and Biotechnology 1 (2022academic year) 1st semester  - 水3~4

  • English for Chemistry and Biotechnology 2 (2022academic year) Second semester  - 水3~4

  • English Engineering (2022academic year) 3rd and 4th semester  - 木3~4

  • English Engineering 1 (2022academic year) Third semester  - 木3~4

  • English Engineering 2 (2022academic year) Fourth semester  - 木3~4

  • Technical Writing and Presentation (2022academic year) 3rd and 4th semester  - 月5~6

  • Technical Writing and Presentation (2022academic year) 3rd and 4th semester  - 月5~6

  • Technical Writing and Presentation (2022academic year) Late  - その他

  • Technical Writing and Presentation (2022academic year) Late  - その他

  • Thermodynamics II (2022academic year) Second semester  - 水3~4

  • Thermodynamics I (2022academic year) 1st semester  - 水3~4

  • Physical Chemistry 1 (2022academic year) 1st and 2nd semester  - 水3~4

  • Physical Chemistry 1 (2022academic year) 1st and 2nd semester  - 水3~4

  • Analytical Chemistry in Environmental Fields Ⅱ (2022academic year) Second semester  - 水1~2

  • Analytical Chemistry in Environmental Fields I (2022academic year) 1st semester  - 水1~2

  • Biophysics (2022academic year) Second semester  - 月1~2,木3~4

  • Protein molecular engineering (2022academic year) Prophase  - 月1~2

  • Design of protein molecules (2022academic year) Late  - その他

  • SDGs:Life Science (2021academic year) Fourth semester  - 木7~8

  • General Biotechnology and Drug Discovery Technology (2021academic year) Prophase  - その他

  • General Biotechnology and Drug Discovery Technology (2021academic year) Prophase  - 木1~2

  • Advanced Internship for Interdisciplinary Medical Sciences and Engineering (2021academic year) Year-round  - その他

  • Technical English for Interdisciplinary Medical Sciences and Engineering (2021academic year) Late  - その他

  • Research Works for Interdisciplinary Medical Sciences and Engineering (2021academic year) Year-round  - その他

  • General Exercises for Interdisciplinary Medical Sciences and Engineering (2021academic year) Late  - その他

  • Analytical Chemistry (2021academic year) 1st and 2nd semester  - 木3,木4

  • Analytical Chemistry 1 (2021academic year) 1st semester  - 木3,木4

  • Analytical Chemistry 2 (2021academic year) Second semester  - 木3,木4

  • English Engineering (2021academic year) 3rd and 4th semester  - 木3,木4

  • English Engineering 1 (2021academic year) Third semester  - 木3,木4

  • English Engineering 2 (2021academic year) Fourth semester  - 木3,木4

  • Laboratory Work and Practice on Basic Engineering (2021academic year) 1st and 2nd semester  - 火5,火6,火7,火8

  • Laboratory Work and Practice on Basic Engineering (2021academic year) 1st and 2nd semester  - 火5,火6,火7,火8

  • Laboratory Work and Practice on Basic Engineering (2021academic year) 1st and 2nd semester  - 火5,火6,火7,火8

  • Technical Writing and Presentation (2021academic year) 3rd and 4th semester  - 月5,月6,月7,月8

  • Technical Writing and Presentation (2021academic year) 3rd and 4th semester  - 月5~6

  • Technical Writing and Presentation (2021academic year) Late  - その他

  • Technical Writing and Presentation (2021academic year) Late  - その他

  • Thermodynamics I (2021academic year) 1st semester  - 水3~4

  • Physical Chemistry and Exercises 1 (2021academic year) 1st semester  - 月3,月4,水1,水2

  • Physical Chemistry 1 (2021academic year) 1st semester  - 月3,月4,水1,水2

  • Physical Chemistry 1 (2021academic year) 1st and 2nd semester  - 水3,水4

  • Physiacal Chemistry A (2021academic year) 1-3 semesters  - [第1学期]水3~4, [第2学期]その他, [第3学期]金3~4

  • Physiacal Chemistry A (2021academic year) 1-3 semesters  - [第1学期]水3~4, [第2学期]その他, [第3学期]金3~4

  • Design of protein molecules (2021academic year) Late  - その他

  • General Biotechnology and Drug Discovery Technology (2020academic year) Prophase  - 木1,木2

  • Advanced Internship for Interdisciplinary Medical Sciences and Engineering (2020academic year) Year-round  - その他

  • Internship for Interdisciplinary Medical Sciences and Engineering (2020academic year) Year-round  - その他

  • Technical English for Interdisciplinary Medical Sciences and Engineering (2020academic year) Late  - その他

  • Research Works for Interdisciplinary Medical Sciences and Engineering (2020academic year) Year-round  - その他

  • General Exercises for Interdisciplinary Medical Sciences and Engineering (2020academic year) Late  - その他

  • Analytical Chemistry (2020academic year) 1st and 2nd semester  - 水3,水4

  • Analytical Chemistry 1 (2020academic year) 1st semester  - 水3,水4

  • Analytical Chemistry 2 (2020academic year) Second semester  - 水3,水4

  • Analytical Chemistry 2 (2020academic year) Fourth semester  - 水3~4

  • English for Chemistry and Biotechnology (2020academic year) 1st and 2nd semester  - 水3,水4

  • English for Chemistry and Biotechnology 1 (2020academic year) 1st semester  - 水3,水4

  • English for Chemistry and Biotechnology 2 (2020academic year) Second semester  - 水3,水4

  • Technical Writing and Presentation (2020academic year) 3rd and 4th semester  - 月5,月6

  • Technical Writing and Presentation (2020academic year) 3rd and 4th semester  - 月5,月6

  • Technical Writing and Presentation (2020academic year) Late  - その他

  • Biology (Introduction to Biotechnology) (2020academic year) special  - その他

  • Biology (Introduction to Biotechnology) (2020academic year) Fourth semester  - 木3,木4

  • Physical Chemistry and Exercises 1 (2020academic year) 1st semester  - 月3,月4,水1,水2

  • Physical Chemistry 1 (2020academic year) 1st semester  - 月3,月4,水1,水2

  • Protein molecular engineering (2020academic year) Prophase  - 月1,月2

  • Design of protein molecules (2020academic year) Late  - その他

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