2021/10/21 更新

写真a

タダ ヒロコ
多田 宏子
TADA Hiroko
所属
自然生命科学研究支援センター 教授
職名
教授
外部リンク

学位

  • 博士(工学) ( 大阪大学 )

研究キーワード

  • Protein engineering

  • 蛋白質工学

  • Instrumental analysis of proteins

  • 蛋白質機器分析

研究分野

  • ライフサイエンス / 生体医工学

  • ライフサイエンス / 構造生物化学

  • ライフサイエンス / 生体材料学

  • ライフサイエンス / 機能生物化学

学歴

  • 大阪大学   School of Engineering  

    1978年4月 - 1982年3月

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  • 兵庫県立川西緑台高等学校    

    1975年4月 - 1978年3月

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    国名: 日本国

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経歴

  • - Professor,Advanced Science Research Center,Okayama University

    2014年

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  • - 岡山大学自然生命科学研究支援センター 教授

    2014年

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  • 岡山大学自然生命科学研究支援センター 准教授   Advanced Science Research Center

    2009年 - 2014年

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  • Associate Professor,Advanced Science Research Center,Okayama University

    2009年 - 2014年

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  • Assistant Professor,Chemical and Biological Technology,Graduate School of Natural Science and Technology,Okayama University

    2007年 - 2009年

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  • 岡山大学自然科学研究科物質生命工学専攻 助教

    2007年 - 2009年

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  • 岡山大学   工学部   助手

    1994年4月 - 2007年7月

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  • 九州大学   生体防御医学研究所   研究員(出向)

    1989年5月 - 1990年5月

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  • 武田薬品工業株式会社   中央研究所等   職員(研究系)

    1982年4月 - 1994年11月

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    国名:日本国

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▼全件表示

所属学協会

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論文

  • High-performance liquid chromatographic profile and 1H quantitative nuclear magnetic resonance analyses for quality control of a Xinjiang licorice extract. 査読 国際誌

    Atsumi Miyazaki, Eerdunbayaer, Tsugumi Shiokawa, Hiroko Tada, Yunhe Lian, Shoko Taniguchi, Tsutomu Hatano

    Bioscience, biotechnology, and biochemistry   84 ( 10 )   2128 - 2138   2020年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Various pharmacological properties of Xinjiang licorice flavonoids have been reported recently. We have investigated constituents corresponding to distinct peaks on the high-performance liquid chromatography (HPLC) profile of a flavonoid-rich extract from licorice, and identified 13 flavonoids, including licochalcone A (1), licochalcone B (3), glabrone (4), and echinatin (5), by isolating them and then performing high-resolution electrospray ionization mass spectrometry and 1H nuclear magnetic resonance (NMR) spectral analyses. We then applied the 1H quantitative NMR (qNMR) method for analysis of major flavonoids, 1 and 3-5 in the extract. The 1H qNMR results were supported by 13C NMR analysis. The results demonstrated the utility of the combination of HPLC profiling and qNMR analyses for quality control of Xinjiang licorice. Additionally, we observed a moderate inhibitory effect of the most abundant constituent, licochalcone A (1), on acetylcholine esterase activity, suggesting utility as a seed for drug development.

    DOI: 10.1080/09168451.2020.1785272

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  • Isolation and identification of the antimicrobial substance included in tempeh using Rhizopus stolonifer NBRC 30816 for fermentation. 査読 国際誌

    Masahiro Ito, Takashi Ito, Hideyuki Aoki, Koshi Nishioka, Tsugumi Shiokawa, Hiroko Tada, Yuki Takeuchi, Nobuyuki Takeyasu, Tadashi Yamamoto, Shogo Takashiba

    International journal of food microbiology   325   108645 - 108645   2020年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    In this study, we focus on the antimicrobial properties of tempeh, a soybean fermented food, against oral bacteria. Tempeh showed antimicrobial activity against dental caries pathogenic bacterium Streptococcus mutans at a final concentration of 1 mg/mL. An antimicrobial substance contained in tempeh was present in the 100 kDa or greater fraction generated by ultrafiltration, but it was found not to be proteinaceous by native-PAGE, SDS-PAGE and protein degradation tests. Next, when the fraction was purified with an ODS column, the 80% and 100% methanol eluates showed antimicrobial activity against S. mutans. The 100% methanol eluate was further subjected to a 2nd column purification, and isolation of the target was confirmed by HPLC. When the isolated material was analyzed by ESI-MS, the m/z was 279.234. Further analysis by Raman spectroscopy revealed a peak similar to linoleic acid. This substance also possessed antimicrobial properties equivalent to linoleic acid.

    DOI: 10.1016/j.ijfoodmicro.2020.108645

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  • Endogenous suppressor(s) in Arabidopsis thaliana 査読

    Thanh Luan Mai, Tatsuhiro Kawasaki, Aprilia Nur Fitrianti, Le Thi Phuong, Tsugumi Shiokawa, Hiroko Tada, Hidenori Matsui, Yoshiteru Noutoshi, Mikihiro Yamamoto, Yuki Ichinose, Tomonori Shiraishi, Kazuhiro Toyoda

    Journal of General Plant Pathology   86 ( 2 )   100 - 106   2020年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Springer Science and Business Media LLC  

    An ethyl acetate extract of Arabidopsis thaliana plants was tested for the presence of endogenous suppressor(s) (ES), and the active fraction, which partitioned into water phase contained a molecule(s) < 3000 Da based on a rough estimate using sized membrane filters. Foliar application of the ES enabled typically nonpathogenic fungi (non-adapted pathogens) to cause disease symptoms on A. thaliana. Consistently, the ES fraction severely suppressed the oxidative burst and the expression of defense-related genes such as FRK1, NHO1, WRKY22, WRKY29, PEN2, and PEN3 in plants challenged with non-adapted fungus Colletotrichum gloeosporioides or the fungal elicitor chitin.

    DOI: 10.1007/s10327-019-00897-z

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    その他リンク: http://link.springer.com/article/10.1007/s10327-019-00897-z/fulltext.html

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

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

    SCIENTIFIC REPORTS   7 ( 1 )   3268 - 3275   2017年9月

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

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

    DOI: 10.1038/s41598-017-12748-y

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  • Bortezomib combined with standard induction chemotherapy in Japanese children with refractory acute lymphoblastic leukemia 査読

    Akihiro Iguchi, Yuko Cho, Minako Sugiyama, Yukayo Terashita, Tadashi Ariga, Yosuke Hosoya, Shinsuke Hirabayashi, Atsushi Manabe, Keisuke Hara, Tetsuya Aiba, Tsugumi Shiokawa, Hiroko Tada, Norihiro Sato

    INTERNATIONAL JOURNAL OF HEMATOLOGY   106 ( 2 )   291 - 298   2017年8月

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

    Bortezomib has been shown to be effective and well-tolerated in patients with refractory acute lymphoblastic leukemia (ALL) in the Therapeutic Advances in Childhood Leukemia trial. However, the safety and efficacy of bortezomib have not been evaluated in Japanese pediatric patients. Here, we report the results of a clinical trial designed to evaluate the safety of bortezomib combined with induction chemotherapy in Japanese children with refractory ALL. A total of six patients with B-precursor ALL were enrolled in this study. Four-dose bortezomib (1.3 mg/m(2)/dose) combined with two standard induction chemotherapies was used. Prolonged pancytopenia (grade 4) was observed in all patients. Four of the six patients developed severe infectious complications. Peripheral neuropathy (grade 2) occurred in five patients. The individual plasma bortezomib concentration-time profiles were not related to toxicity and efficacy. Five patients were evaluable for response, and four patients achieved complete response (CR) or CR without platelet recovery (80%). In conclusion, four-dose bortezomib (1.3 mg/m(2)/dose) combined with standard re-induction chemotherapy was associated with a high risk of infectious complications induced by prolonged neutropenia, although high efficacy has been achieved for Japanese pediatric patients with refractory ALL. Attention must be given to severe infectious complications when performing re-induction chemotherapy including bortezomib.

    DOI: 10.1007/s12185-017-2235-z

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  • Synthesis and assembly of Hepatitis B virus envelope protein-derived particles in Escherichia coli 査読

    Hao Li, Keisuke Onbe, Qiushi Liu, Masumi Iijima, Kenji Tatematsu, Masaharu Seno, Hiroko Tada, Shun'ichi Kuroda

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   490 ( 2 )   155 - 160   2017年8月

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

    Hepatitis B virus (HBV) envelope particles have been synthesized in eukaryotic cells (e.g., mammalian cells, insect cells, and yeast cells) as an HB vaccine immunogen and drug delivery system (DDS) nano carrier. Many researchers had made attempts to synthesize the particles in Escherichia coli for minimize the cost and time for producing HBV envelope particles, but the protein was too deleterious to be synthesized in E. coli. In this study, we generated deletion mutants of HBV envelope L protein (389 amino acid residues (aa)) containing three transmembrane domains (TM1, TM2, TM3). The Delta NC mutant spanning from TM2 to N-terminal half of TM3 (from 237 aa to 335 aa) was found as a shortest form showing spontaneous particle formation. After the N-terminal end of ANC mutant was optimized by the N-end rule for E. coli expression, the modified ANC mutant (mANC) was efficiently expressed as particles in E. coli. The molecular mass of mANC particle was approx. 670 kDa, and the diameter was 28.5 +/- 6.2 nm (mean +/- SD, N = 61). The particle could react with anti-HBV envelope S protein antibody, indicating the particles exhibited S antigenic domain outside as well as HBV envelope particles. Taken together, the E. coli-derived mANC particles could be used as a substitute of eukaryotic cell-derived HBV envelope particles for versatile applications. (C) 2017 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.bbrc.2017.06.015

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  • Determination of CSF 5-methyltetrahydrofolate in children and its application for defects of folate transport and metabolism 査読

    Mari Akiyama, Tomoyuki Akiyama, Kaoruko Kanamaru, Mutsuko Kuribayashi, Hiroko Tada, Tsugumi Shiokawa, Soichiro Toda, Katsumi Imai, Yu Kobayashi, Jun Tohyama, Takafumi Sakakibara, Harumi Yoshinaga, Katsuhiro Kobayashi

    CLINICA CHIMICA ACTA   460   120 - 125   2016年9月

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

    Objective: To describe an assay of 5-methyltetrahydrofolate (5MTHF) in the cerebrospinal fluid (CSF) of children, to determine reference values, and to report the clinical significance of this assay in metabolic disorders affecting folate transport and metabolism.
    Methods: CSF 5MTHF was determined by high-performance liquid chromatography with fluorescent detection in pediatric patients including one with FOLR1 gene mutation and one with methylenetetrahydrofolate reductase (MTHFR) deficiency. CSF total folate was measured using an automated analyzer.
    Results: 5MTHF and total folate were determined in 188 and 93 CSF samples, respectively. CSF 5MTHF was high throughout the first six months of life and subsequently declined with age. Reference values of CSF 5MTHF and total folate were determined from 162 and 82 samples, respectively. The patient with FOLR1 gene mutation had extremely low CSF 5MTHF and total folate, though these values normalized after folinic acid supplementation. The patient with MTHFR deficiency had extremely low 5MTHF and moderately low total folate; these values were not associated and showed no significant change after folic acid supplementation.
    Conclusions: This 5MTHF assay is simple, rapid, sensitive, reliable, and cost-effective. It will aid in the diagnosis and therapeutic monitoring of metabolic disorders affecting folate transport and metabolism. (C) 2016 Elsevier B.V. All rights reserved.

    DOI: 10.1016/j.cca.2016.06.032

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  • Total folate and 5-methyltetrahydrofolate in the cerebrospinal fluid of children: correlation and reference values

    Tomoyuki Akiyama, Hiroko Tada, Tsugumi Shiokawa, Katsuhiro Kobayashi, Harumi Yoshinaga

    CLINICAL CHEMISTRY AND LABORATORY MEDICINE   53 ( 12 )   2009 - 2014   2015年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WALTER DE GRUYTER GMBH  

    Background: Cerebral folate deficiency (CFD) may be underdiagnosed, as it manifests with various non-specific neurological symptoms. The diagnosis of CFD requires a determination of 5-methyltetrahydrofolate (5MTHF) in the cerebrospinal fluid (CSF), which is available in a limited number of specialized laboratories. In clinical biochemistry laboratories, total folate (TF) determination in serum or plasma is routinely performed by automated analyzers. The aim of this study is to determine whether the automated assay of CSF TF is a helpful screening tool for CFD.
    Methods: We analyzed CSF samples collected from 73 pediatric patients. We measured CSF TF, serum TF, and CSF 5MTHF in 73, 70, and 48 patients, respectively. The assay of 5MTHF was conducted by a newly developed system utilizing liquid chromatography-tandem mass spectrometry (LC-MS/ MS). We investigated the correlation between TF and 5MTHF in the CSF.
    Results: There was a strong positive correlation between CSF TF and 5MTHF (rho=0.930, p&lt;0.0001, n=48). Age was negatively correlated with CSF TF (rho=-0.557, p&lt;0.0001, n=51), serum TF (rho=-0.457, p=0.0008, n=51), and CSF 5MTHF (rho=-0.387, p=0.0263, n=33), but not with the CSF/serum TF ratio.
    Conclusions: The automated assay of CSF TF is helpful to estimate CSF 5MTHF. The CSF TF assay may have a significant impact on the early diagnosis of CFD, because clinicians have better access to it than the 5MTHF assay.

    DOI: 10.1515/cclm-2015-0208

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  • Influence of sugar surfactant structure on the encapsulation of oil droplets in an amorphous sugar matrix during freeze-drying 査読

    Nakayama Shota, Kimura Yoshifumi, Miki Sayuri, Oshitani Jun, Kobayashi Takashi, Adachi Shuji, Matsuura Tsutashi, Imanaka Hiroyuki, Ishida Naoyuki, Tada Hiroko, Nakanishi Kazuhiro, Imamura Koreyoshi

    FOOD RESEARCH INTERNATIONAL   70   143 - 149   2015年4月

  • Denatured mammalian protein mixtures exhibit unusually high solubility in nucleic acid-free pure water. (共著) 査読

    Futami J, Fujiyama H, Kinoshita R, Nonomura H, Honjo T, Tada H, Matsushita H, Abe Y, Kakimi K

    PLoS One.   9 ( 11 )   e113295   2014年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1371/journal.pone.0113295

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  • Characteristics of sugar surfactants in stabilizing proteins during freeze-thawing and freeze-drying. (共著) 査読

    Imamura K, Murai K, Korehisa T, Shimizu N, Yamahira R, Matsuura T, Tada H, Imanaka H, Ishida N, Nakanishi K

    Journal of Pharmaceutical Science   103 ( 6 )   1628 - 1637   2014年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1002/jps.23988

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

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

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

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

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

    DOI: 10.1021/bc300030d

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

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

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

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

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

    DOI: 10.1093/jb/mvn087

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  • Cell type dependent endocytic internalization of ErbB2 with an artificial peptide ligand that binds to ErbB2 査読

    Toshihiro Hashizume, Takayuki Fukuda, Tadahiro Nagaoka, Hiroko Tada, Hidenori Yamada, Kazuhide Watanabe, David S. Salomon, Masaharu Seno

    CELL BIOLOGY INTERNATIONAL   32 ( 7 )   814 - 826   2008年7月

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

    ErbB2, which is a member of the epidermal growth factor (erbB) receptor family, is frequently overexpressed in breast and ovarian cancers. Antibody and small molecule anti-tyrosine kinase inhibitors have been developed for targeted therapies for cancers overexpressing erbB2. Internalization and downregulation of erbB2, which is induced by a ligand, may be important for efficacious therapeutic effects. However, ligand-dependent erbB2 internalization has not been well characterized. Here we investigated the internalization of erbB2 in SKBr3 and SKOv3 cells, both overexpressing erbB2, using an EC-1 peptide fused to eGFP (EC-eGFP), which specifically binds to erbB2. ErbB2 was internalized in SKOv3 cells when the cells were treated with EC-eGFP. The accumulation of endosomal erbB2 was EC-eGFP dependent, which colocalized with transferrin implying endocytosis via clathrin-coated pits. In contrast, internalization of erbB2 was not observed in SKBr3 cells. As a result, two different mechanisms, which are cell type dependent for the internalization of erbB2, are proposed. (C) 2008 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

    DOI: 10.1016/j.cellbi.2008.03.012

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  • A betacellulin mutant promotes differentiation of pancreatic acinar AR42J cells into insulin-producing cells with low affinity of binding to ErbB1 査読

    Tadahiro Nagaoka, Takayuki Fukuda, Toshihiro Hlashizume, Tomoko Nishiyama, Hiroko Tada, Hidenori Yamada, David S. Salomon, Satoko Yamada, Itaru Kojima, Masaharu Seno

    JOURNAL OF MOLECULAR BIOLOGY   380 ( 1 )   83 - 94   2008年6月

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

    Betacellulin (BTC) is one of the members of the epidermal growth factor (EGF) ligand family of ErbB receptor tyrosine kinases. It is a differentiation factor as well as a potent mitogen. BTC promotes the differentiation of pancreatic acinar-derived AR42J cells into insulin-producing cells. It independently and preferentially binds to two type I tyrosine kinase receptors, the EGF receptor (ErbB1) and ErbB4. However, the physiochemical characteristics of BTC that are responsible for its preferential binding to these two receptors have not been fully defined. In this study, to investigate the essential amino acid residues of BTC for binding to the two receptors, we introduced point mutations into the EGF domain of BTC employing error-prone PCR. The receptor binding abilities of 190 mutants expressed in Escherichia coli were assessed by enzyme immunoassay. Replacement of the glutamic acid residue at position 88 with a lysine residue in BTC was found to produce a significant loss of affinity for binding to ErbB1, while the affinity of binding to ErbB4 was unchanged. In addition, the mutant of BTC-E/88/K showed less growth-promoting activity on BALB/c 3T3 cells compared with that of the wild-type BTC protein. Interestingly, the BTC mutant protein promoted differentiation of pancreatic acinar AR42J cells at a high frequency into insulin-producing cells compared with AR42J cells that were treated with wild-type BTC protein. These results indicate the possibility of designing BTC mutants, which have an activity of inducing differentiation only, without facilitating growth promotion. (C) 2008 Elsevier Ltd. All rights reserved.

    DOI: 10.1016/j.jmb.2008.03.054

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

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

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

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

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

    DOI: 10.1263/jbb.105.34

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

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

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

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

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

    DOI: 10.1110/ps.072851407

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  • Characterization of bio-nanocapsule as a transfer vector targeting human hepatocyte carcinoma by disulfide linkage modification 査読

    Tadahiro Nagaoka, Takayuki Fukuda, Shinnosuke Yoshida, Hirohito Nishimura, Dongwei Yu, Shun'ichi Kuroda, Katsuyuki Tanizawa, Akhko Kondo, Masakazu Ueda, Hidenori Yamada, Hiroko Tada, Masaharu Seno

    JOURNAL OF CONTROLLED RELEASE   118 ( 3 )   348 - 356   2007年4月

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

    The bio-nanocapsules (BNCs) composed of the recombinant envelope L-protein of hepatitis B virus constitute efficient delivery vectors specifically targeting human hepatocytes. Here, we have tried to enhance the stability of the BNCs because the L-proteins in the BNCs were aggregated due to random disulfide bridging when stored for a long period at 4 degrees C. The envelope protein contains fourteen cysteine residues in the S domain. Aggregation of the envelope proteins might be avoided if unessential cysteine residues are replaced or removed because the irreversible alkylation of the free sulfhydryl group protects against the aggregation and enhances the efficiency of encapsulation. In this study, the possibility of reducing the number of cysteine residues in the S domain to enhance the stability of the BNCs was assessed. The replacement of each cysteine residue by site-directed mutation showed that nine of fourteen cysteine residues were not essential to obtaining BNCs secreted into the culture media. Furthermore, upon evaluating the combination of these mutations, it was found that eight residues of replacement were acceptable. The mutant BNCs with replaced eight cysteine residues were not only more resistant against trypsin, but also more effective in transducing genes into human hepatoma-derived HepG2 cells than the original type BNC. Thus, we demonstrated that the minimized number of cysteine residues in the S domain could enhance the stability of the BNCs. (c) 2006 Elsevier B.V. All rights reserved.

    DOI: 10.1016/j.jconrel.2006.12.020

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  • Anti-tumor effect in an in vivo model by human-derived pancreatic RNase with basic fibroblast growth factor insertional fusion protein through antiangiogenic properties 査読

    Hiroshi Yagi, Masakazu Ueda, Hiromitsu Jinno, Koichi Aiura, Shuji Mikami, Hiroko Tada, Masaharu Seno, Hidenori Yamada, Masaki Kitajima

    CANCER SCIENCE   97 ( 12 )   1315 - 1320   2006年12月

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

    It is thought that the export of angiogenic fibroblast growth factors (FGF) from tumors may be involved in the onset of tumor angiogenesis. To create a new active targeting drug that inhibits the tumor angiogenic process without toxicities to normal cells, human basic FGF (h-bFGF) was inserted genetically into the Gly89 position of cross-linked RNase1 (the ribonuclease inhibitor protein [RI] binding site of cross-linked human pancreatic RNase) to prevent stereospecific binding to RI. The resultant insertional-fusion protein (CL-RFN89) was active both as h-bFGF and as RNase1. Furthermore, it acquired an additional ability of evading RI through steric blockade of RI binding caused by the fused h-bFGF domain. In the present study, the effect of the resultant protein, CL-RFN89, on the antitumor response though its antiangiogenic properties was investigated in an in vivo model. Continuous systemic treatment with CL-RFN89 significantly inhibited the growth of human A431 squamous cell carcinomas in vivo. Seven days of treatment with CL-RFN89 resulted in a 58.2% inhibition of tumor growth compared with control mice (P &lt; 0.0001). Furthermore, immunohistochemistry using a rat antimouse CD31 antibody showed that treatment with CL-RFN89 reduced tumor vascularization. These findings identify CL-RFN89 as a potent systemic inhibitor of tumor growth as a result of its antiangiogenic properties. This protein appears to be a new systemic antitumor agent.

    DOI: 10.1111/j.1349-7006.2006.00336.x

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  • Hydrogen peroxide and endothelin-1 are novel activators of betacellulin ectodomain shedding 査読

    Michael P. Sanderson, Catherine A. Abbott, Hiroko Tada, Masaharu Seno, Peter J. Dempsey, Andrew J. Dunbar

    JOURNAL OF CELLULAR BIOCHEMISTRY   99 ( 2 )   609 - 623   2006年10月

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

    The betacellulin precursor (pro-BTC) is a novel substrate for ADAM10-mediated ectodomain shedding. In this report, we investigated the ability of novel physiologically relevant stimuli, including G-protein coupled receptor (GPCR) agonists and reactive oxygen species (ROS), to stimulate pro-BTC shedding. We found that in breast adenocarcinoma MCF7 cells overexpressing pro-BTC, hydrogen peroxide (H2O2) was a powerful stimulator of ectodomain shedding. The stimulation of pro-BTC shedding by H2O2 was blocked by the broad-spectrum metalloprotease inhibitor TAPI-0 but was still functional in ADAM17 (TACE)-deficient stomach epithelial cells indicating the involvement of a distinct metalloprotease. H2O2-induced pro-BTC shedding was blocked by co-culturing cells in the anti-oxidant N-acetyl-L-cysteine but was unaffected by culture in calcium-deficient media. By contrast, calcium ionophore, which is a previously characterized activator of pro-BTC shedding, was sensitive to calcium depletion but was unaffected by co-culture with the anti-oxidant, identifying a clear distinction between these stimuli. We found that in vascular smooth muscle cells overexpressing pro-BTC, the GPCR agonist endothelin-1 (ET-1) was a strong inducer of ectodomain shedding. This was blocked by a metalloprotease inhibitor and by overexpression of catalytically inactive E385AADAM10. However, overexpression of wild-type ADAM10 or ADAM17 led to an increase in ET-1-induced pro-BTC shedding providing evidence for an involvement of both enzymes in this process. This study identifies ROS and ET-1 as two novel inducers of pro-BTC shedding and lends support to the notion of activated shedding occurring under the control of physiologically relevant stimuli. J. Cell. Biochem. 99: 609-623, 2006. (c) 2006 Wiley-Liss, Inc.

    DOI: 10.1002/jcb.20968

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

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

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

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

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

    DOI: 10.1021/bi052642a

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  • Engineered bio-nanocapsules, the selective vector for drug delivery system

    DW Yu, T Fukuda, Tuoya, S Kuroda, K Tanizawa, A Kondo, M Ueda, T Yamada, H Tada, M Seno

    IUBMB LIFE   58 ( 1 )   1 - 6   2006年1月

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    記述言語:英語   出版者・発行元:TAYLOR & FRANCIS INC  

    The bio-nanocapsule (BNC) is our concept of artificial hollow nanoparticles that have been designed and produced through biotechnological procedures. We proposed an empty virus-like particle, which consists of a recombinant L envelope protein of hepatitis B virus (HBV) and a lipid derived from the host cell, as an engineered BNC. Although this BNC was first developed as an immunogen of hepatitis B vaccine, the pre-S1 region in N-terminus of L envelope protein confers hepatocyte specific infectivity of HBV on the BNC. This recombinant BNC is now being developed as a novel platform of drug delivery system (DDS) vector for selective delivery.

    DOI: 10.1080/15216540500484368

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  • Identification of cell surface marker candidates on SV-T2 cells using DNA microarray on DLC-coated glass

    Tuoya, K Hirayama, T Nagaoka, DW Yu, T Fukuda, H Tada, H Yamada, M Seno

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   334 ( 1 )   263 - 268   2005年8月

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

    We analyzed gene expression profiles of normal mouse fibroblast BALB/c 3T3 cells and its SV40 transformant SV-T2 cells using our originally developed cell surface marker DNA microarray, which is prepared on a diamond-like carbon-coated glass. As a result, CD62L and IL-6 receptor alpha gene expressions were upregulated in SV-T2 and were thought to be candidates for cell surface markers of the cells. The result of microarray analysis was validated by real-time quantitative PCR, immunohistochemistry and biological assays. These data show that our cell surface marker DNA microarray should be useful in finding the candidates of cell type-specific surface markers. (c) 2005 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.bbrc.2005.06.083

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  • Anti-angiogenic effect of an insertional fusion protein of human basic fibroblast growth factor and ribonuclease-1

    T Hayashida, M Ueda, K Aiura, H Tada, M Onizuka, M Seno, H Yamada, M Kitajima

    PROTEIN ENGINEERING DESIGN & SELECTION   18 ( 7 )   321 - 327   2005年7月

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

    Human pancreatic ribonuclease-1 (RNase1) does not exhibit its cytotoxicity unless it is artificially internalized into the cytosol. Furthermore, once it encounters the cytosolic RNase inhibitor (RI), the activity of RNase1 is seriously reduced. To achieve the cellular targeting of RNase1 and the blocking of RI binding simultaneously, the basic fibroblast growth factor (bFGF) sequence was inserted into RNase1 at the RI binding site using a gene fusion technique. The effect of this fusion protein, CL-RFN89, on the angiogenesis, which was accelerated by FGF-FGF receptor interaction, was investigated. It was shown by using fluorescein-labeled CL-RFN89, that the binding to human umbilical vein endothelial cells (HUVECs) was dependent on the existence of the FGF receptors. In addition, CL-RFN89 inhibited the cellular growth of HUVECs in vitro and also inhibited the tube formation, using a three-dimensional tube formation assay. Furthermore, this fusion protein was shown to prevent in vivo tumor cell-induced angiogenesis, using the mouse dorsal air sac assay. These results demonstrated that CL-RFN89 inhibits angiogenesis in vitro and in vivo and that it can be expected to be a potent antiangiogenic agent.

    DOI: 10.1093/protein/gzi040

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  • The specific delivery of proteins to human liver cells by engineered bio-nanocapsules

    DW Yu, C Amano, T Fukuda, T Yamada, S Kuroda, K Tanizawa, A Kondo, M Ueda, H Yamada, H Tada, M Seno

    FEBS JOURNAL   272 ( 14 )   3651 - 3660   2005年7月

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

    A bio-nanocapsule (BNC), composed of the surface antigen (sAg) of the hepatitis B virus, is an efficient nanomachine with which to accomplish the liver-specific delivery of genes and drugs. Approximately 110 molecules of sAg are associated to form a BNC particle with an average diameter of 130 nm. The L protein is an sAg peptide composed mainly of preS and S regions. The preS region, with specific affinity for human hepatocytes, is localized in the N-terminus. The S region following the preS has two transmembrane regions responsible for the formation of particles. In this study, the fusion of emerald green fluorescent protein (EGFP) at the C-terminus of the S region was designed to deliver proteins to human hepatocytes. Truncation of the C-terminus of the S region was required to obtain sufficient expression levels in Cos7 cells. The nanoparticles that were produced delivered EGFP to human hepatoma cells, displaying the EGFP moiety outside, or enclosing it inside. However, only a single orientation characterizes the particle, so that either type of L fusion particle could be effectively and independently separated by an antibody affinity column. The dual C-terminal topologies of the L fusion particles designed in this study could be applied to various proteins for the C-terminal moiety of the L fusion proteins, depending on the character of the proteins, such as cytoplasmic proteins, as well as cytokines or ligands to cell surface receptors. We suggest that this fusion design is the most efficient way to prepare a BNC that delivers proteins to specific cells or tissues.

    DOI: 10.1111/j.1742-4658.2005.04790.x

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  • Protein transduction assisted by polyethylenimine-cationized carrier proteins

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

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

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

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

    DOI: 10.1093/jb/mvi081

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

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

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

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

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

    DOI: 10.1263/jbb.99.095

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

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

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

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

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

    DOI: 10.1016/j.febslet.2004.05.007

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  • Nanoparticles for the delivery of genes and drugs to human hepatocytes

    T Yamada, Y Iwasaki, H Tada, H Iwabuki, MKL Chuah, T VandenDriessche, H Fukuda, A Kondo, M Ueda, M Seno, K Tanizawa, S Kuroda

    NATURE BIOTECHNOLOGY   21 ( 8 )   885 - 890   2003年8月

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

    Hepatitis B virus envelope L particles form hollow nanoparticles displaying a peptide that is indispensable for liver-specific infection by hepatitis B virus in humans. Here we demonstrate the use of L particles for the efficient and specific transfer of a gene or drug into human hepatocytes both in culture and in a mouse xenograft model. In this model, intravenous injection of L particles carrying the gene for green fluorescent protein (GFP) or a fluorescent dye resulted in observable fluorescence only in human hepatocellular carcinomas but not in other human carcinomas or in mouse tissues. When the gene encoding human clotting factor IX was transferred into the xenograft model using L particles, factor IX was produced at levels relevant to the treatment of hemophilia B. The yeast-derived L particle is free of viral genomes, highly specific to human liver cells and able to accommodate drugs as well as genes. These advantages should facilitate targeted delivery of genes and drugs to the human liver.

    DOI: 10.1038/nbt843

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

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

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

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

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

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  • Optimum modification for the highest cytotoxicity of cationized ribonuclease

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

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

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

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

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  • Solution structure of betacellulin, a new member of EGF-family ligands

    K Miura, H Doura, T Aizawa, H Tada, M Seno, H Yamada, K Kawano

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   294 ( 5 )   1040 - 1046   2002年6月

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

    The solution structure of the EGF-like domain of betacellulin (BTCe), a newly discovered member of the epidermal growth factor (EGF) family, has been determined using two-dimensional nuclear magnetic resonance spectroscopy. This is the first report to identify the solution structure of the EGF-family ligand monomers that interact with both ErbB-1 and ErbB-4. The solution structure of BTCe was calculated using 538 NMR-derived restraints. The overall structure of BTCe was stabilized by three disulfide bonds, a hydrophobic core, and 23 hydrogen bonds. It appears that BTCe is comprised of five beta-strands and one short 3(10) helical turn. The secondary structural elements of BTCe are basically similar to those of the other EGF-family proteins, except that several significant variations of the structural properties were found. It is suggested that the structural variations between BTCe and the other EGF-family ligands may affect the specific receptor-recognition properties of EGF-family ligands. (C) 2002 Elsevier Science (USA). All rights reserved.

    DOI: 10.1016/S0006-291X(02)00585-5

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  • Growth inhibition of mammalian cells by eosinophil cationic protein

    T Maeda, M Kitazoe, H Tada, R de Llorens, DS Salomon, M Ueda, H Yamada, M Seno

    EUROPEAN JOURNAL OF BIOCHEMISTRY   269 ( 1 )   307 - 316   2002年1月

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

    Eosinophil cationic protein (ECP), one of the major components of basic granules of eosinophils, is cytotoxic to tracheal epithelium. However, the extent of this effect on other cell types has not been evaluated in vitro. In this study, we evaluated the effect of ECP on 13 mammalian cell lines. ECP inhibited the growth of several cell lines including those derived from carcinoma and leukemia in a dose-dependent manner. The IC50 values on A431 cells, MDA-MB-453 cells, HL-60 cells and K562 cells were estimated to be approximate to1-5 muM. ECP significantly suppressed the size of colonies of A431 cells, and decreased K562 cells in G(1)/G(0) phase. However, there was little evidence that ECP killed cells in either cell line. These effects of ECP were not enhanced by extending its N-terminus. Rhodamine B isothiocyanate-labeled ECP started to bind to A431 cells after 0.5 h and accumulated for up to 24 h, indicating that specific affinity for the cell surface may be important. ne affinity of ECP for heparin was assessed and found to be reduced when tryptophan residues, one of which is located at a position in the catalytic subsite of ribonuclease in ECP, were modified. The growth-inhibitory effect was also attenuated by this modification. These results suggest that growth inhibition by ECP is dependent on cell type and is cytostatic.

    DOI: 10.1046/j.0014-2956.2001.02653.x

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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

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

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

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

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

    DOI: 10.1021/bi010248g

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

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

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

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

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

    DOI: 10.1107/S0907444901001147

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

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

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

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

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

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  • Three-dimensional Crystal Structure of Human Eosinophil Cationic Protein (RNase3) at 1.75 Å Resolution.

    Mallorqui-Fernandez, G, Pous J, Peracaula, R, Aymami, J, Maeda, T, Tada, H, Yamada, H, Seno, M, de Llorens, R, Gomis-Ruth, FX, Coll, M

    J. Mol.Biol.   300 ( 5 )   1297 - 1307   2000年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1006/jmbi.2000.3939

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  • Molecular cloning and expression of rat betacellulin cDNA

    H Tada, M Seno, H Yamada, R Sasada, K Igarashi

    BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION   1492 ( 1 )   285 - 288   2000年6月

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

    The cDNA encoding an entire open reading frame of rat betacellulin has been cloned from rat kidney. Expression of this cDNA in COS7 cells showed a significant amount of mitogenic activity in the culture media. Western blotting of the cell lysates suggested that the membrane-anchored precursor was cleaved to release its ectodomain very efficiently. (C) 2000 Published by Elsevier Science B.V.

    DOI: 10.1016/S0167-4781(00)00106-8

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

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

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

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

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

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  • Immunohistochemical localization of betacellulin, a new member of the EGF family, in normal human pancreas and islet tumor cells

    J Miyagawa, T Hanafusa, R Sasada, K Yamamoto, K Igarashi, K Yamamori, M Seno, H Tada, T Nammo, M Li, K Yamagata, H Nakajima, M Namba, M Kuwajima, Y Matsuzawa

    ENDOCRINE JOURNAL   46 ( 6 )   755 - 764   1999年12月

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

    Betacellulin (BTC) purified from mouse beta cell tumor (beta TC-3) is a new member of the epidermal growth factor (EGF) family which can bind receptor tyrosine kinase, EGF receptor (erbB1) and erbB4. It has been demonstrated that proBTC mRNA was abundantly expressed in human pancreas tissue, and that ETC converted amylase-secreting rat acinar cell line (AR42J) into insulin-secreting cells, suggesting that ETC might be important for the growth and/or differentiation of islet cells. However, the cell type producing ETC in the pancreas has not been clarified. In this study, we examined the localization of ETC in human pancreas and islet cell tumors. Immunohistochemistry using specific antibodies to human ETC revealed that this protein was produced in alpha cells and duct cells, and probably in beta cells in normal adult pancreas. Furthermore, strong immunoreactivity to ETC was detected in primitive duct cells of the fetal pancreas, and both insulinoma and glucagonoma cells also showed positive immunoreactivity to ETC. EGF receptor (erbB1) and erbB4 were expressed mainly in islet and duct cells, and duct cells, respectively. These results demonstrate the localization of ETC and its receptors, and suggest that ETC may be one of the factors that have physiologically important roles such as growth and differentiation of islet cells in the human pancreas.

    DOI: 10.1507/endocrj.46.755

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

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

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

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

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

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  • Processing and juxtacrine activity of membrane-anchored betacellulin

    H Tada, R Sasada, Y Kawaguchi, Kojima, I, WJ Gullick, DS Salomon, K Igarashi, M Seno, H Yamada

    JOURNAL OF CELLULAR BIOCHEMISTRY   72 ( 3 )   423 - 434   1999年3月

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

    Betacellulin (BTC) was originally isolated as a secreted growth factor from a mouse pancreatic beta-tumor cell line, whereas the cDNA sequence predicts that ETC is synthesized as a larger transmembrane protein. In the present study, we have characterized the membrane-anchored forms of BTC, using Chinese hamster ovary (CHO) cells, mouse fibroblast A9 cells, and a human breast cancer cell line MCF-7, all of which were stably transfected with human ETC cDNA. A9 and MCF-7 transfectants produced membrane-anchored ETC isoforms of 21, 25, 29, and 40 kDa on the cell surface, as well as a secreted BTC isoform. CHO transfectants secreted little ETC but accumulated the membrane-anchored isoforms. The cleavage of the membrane-anchored forms to release a secreted from of ETC was not enhanced by biological mediators such as a phorbol ester, which stimulates the cleavage of other membrane-anchored growth factors. The membrane-anchored forms of ETC expressed on the transfected cells induced the insulin production and/or promoted the growth in subclones of AR42J rat pancreatic cells. These results suggest that the membrane-anchored ETC can function as a juxtacrine factor in regulating the growth and differentiation of pancreatic endocrine cells. J. Cell. Biochem. 72.423-134, 1999. (C) 1999 Wiley-Liss, Inc.

    DOI: 10.1002/(SICI)1097-4644(19990301)72:3<423::AID-JCB11>3.0.CO;2-P

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  • BALB/c 3T3 cells co-expressing FGF-2 and soluble FGF receptor acquire tumorigenicity

    Masaharu Seno, Akinori Masago, Atsushi Nishimura, Hiroko Tada, Megumi Kosaka, Reiko Sasada, Koichi Igarashi, Satimaru Seno, Hidenori Yamada

    Cytokine   10 ( 4 )   290 - 294   1998年

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

    The physiological significance of the soluble fibroblast growth factor (FGF) receptors is not clear yet although they are present in blood, vitreous fluid and in the extracellular matrix of vascular endothelial cells. A hypothesis that they might help FGF-2 release from cells is very interesting because FGF-2 does not have clear secretion signal and the mechanism of the secretion of FGF-2 is still unclear. Single overexpression of FGF-2 is related neither to the secretion potential of the molecule nor to the tumorigenicity of the cells. In this report, BALB/c 3T3 cells transformed with the full length of human FGF-2 cDNA are further transformed with the cDNA coding the extracellular domain of human FGF receptor 1. The obtained transformants co-expressing FGF-2 and soluble FGF receptor are highly tumorigenic in nude mice, while the parental cells do not show any tumorigenicity. In the conditioned medium of the double-transformants, FGF-2 is immunologically detected. These results suggest that naturally produced soluble form of FGF receptor supports the release of FGF-2 from the cells and that over-expression of these two molecules leads to induce the malignant tumours in vivo.

    DOI: 10.1006/cyto.1997.0286

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  • Efficient production of a functional mouse/human chimeric Fab' against human urokinase-type plasminogen activator by Bacillus brevis

    Y. Inoue, T. Ohta, H. Tada, S. Iwasa, S. Udaka, H. Yamagata

    Applied Microbiology and Biotechnology   48 ( 4 )   487 - 492   1997年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Expression/secretion vectors for the production of Fab' and single-chain (Sc) Fab' by Bacillus brevis have been constructed. For the production of Fab', the cDNAs encoding the L chain and Fd' fragment (Fd with the hinge region) of a mouse-human chimeric Fab' against human urokinase-type plasminogen activator were fused directly with the translation-start and signal-peptide-encoding regions of the mwp gene, the gene for one of the major cell-wall proteins of Bacillus brevis. The two fused genes were placed tandemly downstream from the promoter of the cell-wall protein gene operon (cwp) of B. brevis. For the production of scFab', the two cDNAs were linked with a synthetic oligonucleotide encoding a flexible peptide linker of 17 or 24 amino acids, and fused with the translation start and signal-peptide-encoding regions of the mwp gene. Fab' was efficiently produced by B. brevis, being accumulated at a level of 100 mg/l in the culture medium in a simple shake-flask culture, which is the highest level obtained so far for a gram-positive bacterium. On the other hand, the scFab' remained at a level of a few milligrams per liter in the culture medium. The Fab' produced by B. brevis showed comparable antigen-binding activity to that of the parental antibody. The L chain and Fd' fragment, constituting the Fab', had the correct N-terminal amino acid sequences. These results indicate that B. brevis is a very promising host for the production of native Ig fragments.

    DOI: 10.1007/s002530051084

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  • Human betacellulin, a member of the EGF family dominantly expressed in pancreas and small intestine, is fully active in a monomeric form

    M Seno, H Tada, M Kosaka, R Sasada, K Igarashi, Y Shing, J Folkman, M Ueda, H Yamada

    GROWTH FACTORS   13 ( 3-4 )   181 - 191   1996年

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

    Betacellulin (BTC) was found to be expressed mainly in human pancreas and small intestine. This finding suggests that ETC possesses some specific function distinguished from the other members of epidermal growth factor (EGF) family. To clarify this function, the released form of human ETC has been expressed in E.coli, purified, and characterized. The recombinant human ETC was produced as an inclusion body. This material was dissolved in guanidine-HCl under reducing conditions, refolded, and purified through sequential liquid chromatography. Purified ETC was electrophoresed under reducing conditions and a molecular size of 18 kDa was determined, which is the supposed size of a dimer of the peptide. However, chemical analysis failed to show a covalently linked dimer. The molecular mass of ETC analyzed by mass spectrometry revealed it to be 9 kDa, which is consistent with theoretical value for a monomer. Recombinant ETC showed growth promoting activity for mouse fibroblasts and rat aortic smooth muscle cells which was equivalent to EGF. On the other hand, ETC was found to exhibit a growth inhibitory effect on the cells overexpressing EGF receptor.

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  • Molecular Cloning and Expression of Human Ribonuclease 4 cDNA.(jointly worked) 査読

    Biochimica et Biophysica Acta   ( 1261 )   424 - 426   1995年

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  • Recombinant human pancreatic ribonuclease produced in e. coli : Importance of the amino-terminal sequence

    Junichiro Futami, Masaharu Seno, Megumi Kosaka, Hiroko Tada, Satimaru Seno, Hidenori Yamada

    Biochemical and Biophysical Research Communications   216 ( 1 )   406 - 413   1995年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    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. © 1995 by Academic Press, Inc.

    DOI: 10.1006/bbrc.1995.2638

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  • Expression and Characterization of a Chimeric Bispecific Antibody against Fibrin and against Urokinase-type Plasminogen Activator.(jointly worked) 査読

    Journal of Biotechnology   ( 33 )   137 - 174   1994年

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    記述言語:英語  

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  • An Enzyme Immunoassay of Human Lymphotoxin(jointly worked) 査読

    Journal of Immunoassay   ( 10 )   93 - 105   1989年

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    担当区分:筆頭著者   記述言語:英語  

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  • Bispecific antibody-producing hybrid hybridoma and its use in one-step immunoassays for human lymphotoxin

    H. Tada, H. Toyoda, S. Iwasa

    Hybridoma   8 ( 1 )   73 - 83   1989年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1089/hyb.1989.8.73

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  • AN IMPROVED COLORIMETRIC ASSAY FOR INTERLEUKIN-2 査読

    H TADA, O SHIHO, K KUROSHIMA, M KOYAMA, K TSUKAMOTO

    JOURNAL OF IMMUNOLOGICAL METHODS   93 ( 2 )   157 - 165   1986年

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

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  • PURIFICATION AND PARTIAL SEQUENCE-ANALYSIS OF HUMAN INTERLEUKIN-2 DERIVED FROM PERIPHERAL-BLOOD LEUKOCYTES 査読

    K KATO, K NARUO, M KOYAMA, K KAWAHARA, S HINUMA, H TADA, H SUGINO, K TSUKAMOTO

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   127 ( 1 )   182 - 190   1985年

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

    DOI: 10.1016/S0006-291X(85)80142-X

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  • COMPARISON OF THE BIOLOGICAL PROPERTIES OF PURIFIED NATURAL AND RECOMBINANT HUMAN INTERLEUKIN-2 査読

    K NARUO, S HINUMA, K KATO, M KOYAMA, H TADA, O SHIHO, K TSUKAMOTO

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   128 ( 1 )   257 - 264   1985年

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

    DOI: 10.1016/0006-291X(85)91672-9

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▼全件表示

書籍等出版物

  • Bioinformatics. A Practical Approach.

    Chspman & Hall/CRC  2007年 

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  • Monoclonal Antibody Technology(jointly worked)

    Encyclopedia of Human Biology, Second Edition, Academic Press 

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  • Monoclonal Antibody Technology(jointly worked)

    Encyclopedia of Human Biology, Second Edition, Academic Press 

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MISC

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

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

    SCIENTIFIC REPORTS   7 ( 1 )   3268 - 3275   2017年9月

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    記述言語:英語   出版者・発行元:NATURE PUBLISHING GROUP  

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

    DOI: 10.1038/s41598-017-12748-y

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  • Synthesis and assembly of Hepatitis B virus envelope protein-derived particles in Escherichia coli

    Hao Li, Keisuke Onbe, Qiushi Liu, Masumi Iijima, Kenji Tatematsu, Masaharu Seno, Hiroko Tada, Shun'ichi Kuroda

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   490 ( 2 )   155 - 160   2017年8月

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    記述言語:英語   出版者・発行元:ACADEMIC PRESS INC ELSEVIER SCIENCE  

    Hepatitis B virus (HBV) envelope particles have been synthesized in eukaryotic cells (e.g., mammalian cells, insect cells, and yeast cells) as an HB vaccine immunogen and drug delivery system (DDS) nano carrier. Many researchers had made attempts to synthesize the particles in Escherichia coli for minimize the cost and time for producing HBV envelope particles, but the protein was too deleterious to be synthesized in E. coli. In this study, we generated deletion mutants of HBV envelope L protein (389 amino acid residues (aa)) containing three transmembrane domains (TM1, TM2, TM3). The Delta NC mutant spanning from TM2 to N-terminal half of TM3 (from 237 aa to 335 aa) was found as a shortest form showing spontaneous particle formation. After the N-terminal end of ANC mutant was optimized by the N-end rule for E. coli expression, the modified ANC mutant (mANC) was efficiently expressed as particles in E. coli. The molecular mass of mANC particle was approx. 670 kDa, and the diameter was 28.5 +/- 6.2 nm (mean +/- SD, N = 61). The particle could react with anti-HBV envelope S protein antibody, indicating the particles exhibited S antigenic domain outside as well as HBV envelope particles. Taken together, the E. coli-derived mANC particles could be used as a substitute of eukaryotic cell-derived HBV envelope particles for versatile applications. (C) 2017 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.bbrc.2017.06.015

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  • Bortezomib combined with standard induction chemotherapy in Japanese children with refractory acute lymphoblastic leukemia

    Akihiro Iguchi, Yuko Cho, Minako Sugiyama, Yukayo Terashita, Tadashi Ariga, Yosuke Hosoya, Shinsuke Hirabayashi, Atsushi Manabe, Keisuke Hara, Tetsuya Aiba, Tsugumi Shiokawa, Hiroko Tada, Norihiro Sato

    International Journal of Hematology, Nippon Ketsueki Gakkai zasshi : journal of Japan Haematological Society, Acta Haematologica Japonica   1 - 8   2017年

  • Determination of CSF 5-methyltetrahydrofolate in children and its application for defects of folate transport and metabolism

    Mari Akiyama, Tomoyuki Akiyama, Kaoruko Kanamaru, Mutsuko Kuribayashi, Hiroko Tada, Tsugumi Shiokawa, Soichiro Toda, Katsumi Imai, Yu Kobayashi, Jun Tohyama, Takafumi Sakakibara, Harumi Yoshinaga, Katsuhiro Kobayashi

    CLINICA CHIMICA ACTA   460   120 - 125   2016年9月

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

    Objective: To describe an assay of 5-methyltetrahydrofolate (5MTHF) in the cerebrospinal fluid (CSF) of children, to determine reference values, and to report the clinical significance of this assay in metabolic disorders affecting folate transport and metabolism.
    Methods: CSF 5MTHF was determined by high-performance liquid chromatography with fluorescent detection in pediatric patients including one with FOLR1 gene mutation and one with methylenetetrahydrofolate reductase (MTHFR) deficiency. CSF total folate was measured using an automated analyzer.
    Results: 5MTHF and total folate were determined in 188 and 93 CSF samples, respectively. CSF 5MTHF was high throughout the first six months of life and subsequently declined with age. Reference values of CSF 5MTHF and total folate were determined from 162 and 82 samples, respectively. The patient with FOLR1 gene mutation had extremely low CSF 5MTHF and total folate, though these values normalized after folinic acid supplementation. The patient with MTHFR deficiency had extremely low 5MTHF and moderately low total folate; these values were not associated and showed no significant change after folic acid supplementation.
    Conclusions: This 5MTHF assay is simple, rapid, sensitive, reliable, and cost-effective. It will aid in the diagnosis and therapeutic monitoring of metabolic disorders affecting folate transport and metabolism. (C) 2016 Elsevier B.V. All rights reserved.

    DOI: 10.1016/j.cca.2016.06.032

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  • Total folate and 5-methyltetrahydrofolate in the cerebrospinal fluid of children: correlation and reference values

    Tomoyuki Akiyama, Hiroko Tada, Tsugumi Shiokawa, Katsuhiro Kobayashi, Harumi Yoshinaga

    CLINICAL CHEMISTRY AND LABORATORY MEDICINE   53 ( 12 )   2009 - 2014   2015年11月

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    記述言語:英語   出版者・発行元:WALTER DE GRUYTER GMBH  

    Background: Cerebral folate deficiency (CFD) may be underdiagnosed, as it manifests with various non-specific neurological symptoms. The diagnosis of CFD requires a determination of 5-methyltetrahydrofolate (5MTHF) in the cerebrospinal fluid (CSF), which is available in a limited number of specialized laboratories. In clinical biochemistry laboratories, total folate (TF) determination in serum or plasma is routinely performed by automated analyzers. The aim of this study is to determine whether the automated assay of CSF TF is a helpful screening tool for CFD.
    Methods: We analyzed CSF samples collected from 73 pediatric patients. We measured CSF TF, serum TF, and CSF 5MTHF in 73, 70, and 48 patients, respectively. The assay of 5MTHF was conducted by a newly developed system utilizing liquid chromatography-tandem mass spectrometry (LC-MS/ MS). We investigated the correlation between TF and 5MTHF in the CSF.
    Results: There was a strong positive correlation between CSF TF and 5MTHF (rho=0.930, p&lt;0.0001, n=48). Age was negatively correlated with CSF TF (rho=-0.557, p&lt;0.0001, n=51), serum TF (rho=-0.457, p=0.0008, n=51), and CSF 5MTHF (rho=-0.387, p=0.0263, n=33), but not with the CSF/serum TF ratio.
    Conclusions: The automated assay of CSF TF is helpful to estimate CSF 5MTHF. The CSF TF assay may have a significant impact on the early diagnosis of CFD, because clinicians have better access to it than the 5MTHF assay.

    DOI: 10.1515/cclm-2015-0208

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  • Denatured mammalian protein mixtures exhibit unusually high solubility in nucleic acid-free pure water. (共著)

    Futami J, Fujiyama H, Kinoshita R, Nonomura H, Honjo T, Tada H, Matsushita H, Abe Y, Kakimi K

    PLoS One.   9 ( 11 )   e113295   2014年11月

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  • Characteristics of sugar surfactants in stabilizing proteins during freeze-thawing and freeze-drying. (共著)

    Imamura K, Murai K, Korehisa T, Shimizu N, Yamahira R, Matsuura T, Tada H, Imanaka H, Ishida N, Nakanishi K

    Journal of Pharmaceutical Science   103 ( 6 )   1628 - 1637   2014年6月

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

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

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

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    記述言語:英語   出版者・発行元:OXFORD UNIV PRESS  

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

    DOI: 10.1093/jb/mvn087

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  • Cell type dependent endocytic internalization of ErbB2 with an artificial peptide ligand that binds to ErbB2

    Toshihiro Hashizume, Takayuki Fukuda, Tadahiro Nagaoka, Hiroko Tada, Hidenori Yamada, Kazuhide Watanabe, David S. Salomon, Masaharu Seno

    CELL BIOLOGY INTERNATIONAL   32 ( 7 )   814 - 826   2008年7月

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    記述言語:英語   出版者・発行元:ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD  

    ErbB2, which is a member of the epidermal growth factor (erbB) receptor family, is frequently overexpressed in breast and ovarian cancers. Antibody and small molecule anti-tyrosine kinase inhibitors have been developed for targeted therapies for cancers overexpressing erbB2. Internalization and downregulation of erbB2, which is induced by a ligand, may be important for efficacious therapeutic effects. However, ligand-dependent erbB2 internalization has not been well characterized. Here we investigated the internalization of erbB2 in SKBr3 and SKOv3 cells, both overexpressing erbB2, using an EC-1 peptide fused to eGFP (EC-eGFP), which specifically binds to erbB2. ErbB2 was internalized in SKOv3 cells when the cells were treated with EC-eGFP. The accumulation of endosomal erbB2 was EC-eGFP dependent, which colocalized with transferrin implying endocytosis via clathrin-coated pits. In contrast, internalization of erbB2 was not observed in SKBr3 cells. As a result, two different mechanisms, which are cell type dependent for the internalization of erbB2, are proposed. (C) 2008 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

    DOI: 10.1016/j.cellbi.2008.03.012

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  • A betacellulin mutant promotes differentiation of pancreatic acinar AR42J cells into insulin-producing cells with low affinity of binding to ErbB1

    Tadahiro Nagaoka, Takayuki Fukuda, Toshihiro Hlashizume, Tomoko Nishiyama, Hiroko Tada, Hidenori Yamada, David S. Salomon, Satoko Yamada, Itaru Kojima, Masaharu Seno

    JOURNAL OF MOLECULAR BIOLOGY   380 ( 1 )   83 - 94   2008年6月

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    記述言語:英語   出版者・発行元:ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD  

    Betacellulin (BTC) is one of the members of the epidermal growth factor (EGF) ligand family of ErbB receptor tyrosine kinases. It is a differentiation factor as well as a potent mitogen. BTC promotes the differentiation of pancreatic acinar-derived AR42J cells into insulin-producing cells. It independently and preferentially binds to two type I tyrosine kinase receptors, the EGF receptor (ErbB1) and ErbB4. However, the physiochemical characteristics of BTC that are responsible for its preferential binding to these two receptors have not been fully defined. In this study, to investigate the essential amino acid residues of BTC for binding to the two receptors, we introduced point mutations into the EGF domain of BTC employing error-prone PCR. The receptor binding abilities of 190 mutants expressed in Escherichia coli were assessed by enzyme immunoassay. Replacement of the glutamic acid residue at position 88 with a lysine residue in BTC was found to produce a significant loss of affinity for binding to ErbB1, while the affinity of binding to ErbB4 was unchanged. In addition, the mutant of BTC-E/88/K showed less growth-promoting activity on BALB/c 3T3 cells compared with that of the wild-type BTC protein. Interestingly, the BTC mutant protein promoted differentiation of pancreatic acinar AR42J cells at a high frequency into insulin-producing cells compared with AR42J cells that were treated with wild-type BTC protein. These results indicate the possibility of designing BTC mutants, which have an activity of inducing differentiation only, without facilitating growth promotion. (C) 2008 Elsevier Ltd. All rights reserved.

    DOI: 10.1016/j.jmb.2008.03.054

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

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

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

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    記述言語:英語   出版者・発行元:SOC BIOSCIENCE BIOENGINEERING JAPAN  

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

    DOI: 10.1263/jbb.105.34

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  • 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   4P-0116   2008年

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    記述言語:日本語  

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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

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

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

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    記述言語:英語   出版者・発行元:COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT  

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

    DOI: 10.1110/ps.072851407

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  • Characterization of bio-nanocapsule as a transfer vector targeting human hepatocyte carcinoma by disulfide linkage modification

    Tadahiro Nagaoka, Takayuki Fukuda, Shinnosuke Yoshida, Hirohito Nishimura, Dongwei Yu, Shun'ichi Kuroda, Katsuyuki Tanizawa, Akhko Kondo, Masakazu Ueda, Hidenori Yamada, Hiroko Tada, Masaharu Seno

    JOURNAL OF CONTROLLED RELEASE   118 ( 3 )   348 - 356   2007年4月

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

    The bio-nanocapsules (BNCs) composed of the recombinant envelope L-protein of hepatitis B virus constitute efficient delivery vectors specifically targeting human hepatocytes. Here, we have tried to enhance the stability of the BNCs because the L-proteins in the BNCs were aggregated due to random disulfide bridging when stored for a long period at 4 degrees C. The envelope protein contains fourteen cysteine residues in the S domain. Aggregation of the envelope proteins might be avoided if unessential cysteine residues are replaced or removed because the irreversible alkylation of the free sulfhydryl group protects against the aggregation and enhances the efficiency of encapsulation. In this study, the possibility of reducing the number of cysteine residues in the S domain to enhance the stability of the BNCs was assessed. The replacement of each cysteine residue by site-directed mutation showed that nine of fourteen cysteine residues were not essential to obtaining BNCs secreted into the culture media. Furthermore, upon evaluating the combination of these mutations, it was found that eight residues of replacement were acceptable. The mutant BNCs with replaced eight cysteine residues were not only more resistant against trypsin, but also more effective in transducing genes into human hepatoma-derived HepG2 cells than the original type BNC. Thus, we demonstrated that the minimized number of cysteine residues in the S domain could enhance the stability of the BNCs. (c) 2006 Elsevier B.V. All rights reserved.

    DOI: 10.1016/j.jconrel.2006.12.020

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  • Anti-tumor effect in an in vivo model by human-derived pancreatic RNase with basic fibroblast growth factor insertional fusion protein through antiangiogenic properties

    Hiroshi Yagi, Masakazu Ueda, Hiromitsu Jinno, Koichi Aiura, Shuji Mikami, Hiroko Tada, Masaharu Seno, Hidenori Yamada, Masaki Kitajima

    CANCER SCIENCE   97 ( 12 )   1315 - 1320   2006年12月

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    記述言語:英語   出版者・発行元:BLACKWELL PUBLISHING  

    It is thought that the export of angiogenic fibroblast growth factors (FGF) from tumors may be involved in the onset of tumor angiogenesis. To create a new active targeting drug that inhibits the tumor angiogenic process without toxicities to normal cells, human basic FGF (h-bFGF) was inserted genetically into the Gly89 position of cross-linked RNase1 (the ribonuclease inhibitor protein [RI] binding site of cross-linked human pancreatic RNase) to prevent stereospecific binding to RI. The resultant insertional-fusion protein (CL-RFN89) was active both as h-bFGF and as RNase1. Furthermore, it acquired an additional ability of evading RI through steric blockade of RI binding caused by the fused h-bFGF domain. In the present study, the effect of the resultant protein, CL-RFN89, on the antitumor response though its antiangiogenic properties was investigated in an in vivo model. Continuous systemic treatment with CL-RFN89 significantly inhibited the growth of human A431 squamous cell carcinomas in vivo. Seven days of treatment with CL-RFN89 resulted in a 58.2% inhibition of tumor growth compared with control mice (P &lt; 0.0001). Furthermore, immunohistochemistry using a rat antimouse CD31 antibody showed that treatment with CL-RFN89 reduced tumor vascularization. These findings identify CL-RFN89 as a potent systemic inhibitor of tumor growth as a result of its antiangiogenic properties. This protein appears to be a new systemic antitumor agent.

    DOI: 10.1111/j.1349-7006.2006.00336.x

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  • Hydrogen peroxide and endothelin-1 are novel activators of betacellulin ectodomain shedding

    Michael P. Sanderson, Catherine A. Abbott, Hiroko Tada, Masaharu Seno, Peter J. Dempsey, Andrew J. Dunbar

    JOURNAL OF CELLULAR BIOCHEMISTRY   99 ( 2 )   609 - 623   2006年10月

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    記述言語:英語   出版者・発行元:WILEY-LISS  

    The betacellulin precursor (pro-BTC) is a novel substrate for ADAM10-mediated ectodomain shedding. In this report, we investigated the ability of novel physiologically relevant stimuli, including G-protein coupled receptor (GPCR) agonists and reactive oxygen species (ROS), to stimulate pro-BTC shedding. We found that in breast adenocarcinoma MCF7 cells overexpressing pro-BTC, hydrogen peroxide (H2O2) was a powerful stimulator of ectodomain shedding. The stimulation of pro-BTC shedding by H2O2 was blocked by the broad-spectrum metalloprotease inhibitor TAPI-0 but was still functional in ADAM17 (TACE)-deficient stomach epithelial cells indicating the involvement of a distinct metalloprotease. H2O2-induced pro-BTC shedding was blocked by co-culturing cells in the anti-oxidant N-acetyl-L-cysteine but was unaffected by culture in calcium-deficient media. By contrast, calcium ionophore, which is a previously characterized activator of pro-BTC shedding, was sensitive to calcium depletion but was unaffected by co-culture with the anti-oxidant, identifying a clear distinction between these stimuli. We found that in vascular smooth muscle cells overexpressing pro-BTC, the GPCR agonist endothelin-1 (ET-1) was a strong inducer of ectodomain shedding. This was blocked by a metalloprotease inhibitor and by overexpression of catalytically inactive E385AADAM10. However, overexpression of wild-type ADAM10 or ADAM17 led to an increase in ET-1-induced pro-BTC shedding providing evidence for an involvement of both enzymes in this process. This study identifies ROS and ET-1 as two novel inducers of pro-BTC shedding and lends support to the notion of activated shedding occurring under the control of physiologically relevant stimuli. J. Cell. Biochem. 99: 609-623, 2006. (c) 2006 Wiley-Liss, Inc.

    DOI: 10.1002/jcb.20968

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

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

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

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    記述言語:英語   出版者・発行元:AMER CHEMICAL SOC  

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

    DOI: 10.1021/bi052642a

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  • Engineered bio-nanocapsules, the selective vector for drug delivery system

    DW Yu, T Fukuda, Tuoya, S Kuroda, K Tanizawa, A Kondo, M Ueda, T Yamada, H Tada, M Seno

    IUBMB LIFE   58 ( 1 )   1 - 6   2006年1月

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    記述言語:英語   掲載種別:書評論文,書評,文献紹介等   出版者・発行元:TAYLOR & FRANCIS INC  

    The bio-nanocapsule (BNC) is our concept of artificial hollow nanoparticles that have been designed and produced through biotechnological procedures. We proposed an empty virus-like particle, which consists of a recombinant L envelope protein of hepatitis B virus (HBV) and a lipid derived from the host cell, as an engineered BNC. Although this BNC was first developed as an immunogen of hepatitis B vaccine, the pre-S1 region in N-terminus of L envelope protein confers hepatocyte specific infectivity of HBV on the BNC. This recombinant BNC is now being developed as a novel platform of drug delivery system (DDS) vector for selective delivery.

    DOI: 10.1080/15216540500484368

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  • Identification of cell surface marker candidates on SV-T2 cells using DNA microarray on DLC-coated glass

    Tuoya, K Hirayama, T Nagaoka, DW Yu, T Fukuda, H Tada, H Yamada, M Seno

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   334 ( 1 )   263 - 268   2005年8月

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    記述言語:英語   出版者・発行元:ACADEMIC PRESS INC ELSEVIER SCIENCE  

    We analyzed gene expression profiles of normal mouse fibroblast BALB/c 3T3 cells and its SV40 transformant SV-T2 cells using our originally developed cell surface marker DNA microarray, which is prepared on a diamond-like carbon-coated glass. As a result, CD62L and IL-6 receptor alpha gene expressions were upregulated in SV-T2 and were thought to be candidates for cell surface markers of the cells. The result of microarray analysis was validated by real-time quantitative PCR, immunohistochemistry and biological assays. These data show that our cell surface marker DNA microarray should be useful in finding the candidates of cell type-specific surface markers. (c) 2005 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.bbrc.2005.06.083

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  • The specific delivery of proteins to human liver cells by engineered bio-nanocapsules

    DW Yu, C Amano, T Fukuda, T Yamada, S Kuroda, K Tanizawa, A Kondo, M Ueda, H Yamada, H Tada, M Seno

    FEBS JOURNAL   272 ( 14 )   3651 - 3660   2005年7月

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    記述言語:英語   出版者・発行元:BLACKWELL PUBLISHING  

    A bio-nanocapsule (BNC), composed of the surface antigen (sAg) of the hepatitis B virus, is an efficient nanomachine with which to accomplish the liver-specific delivery of genes and drugs. Approximately 110 molecules of sAg are associated to form a BNC particle with an average diameter of 130 nm. The L protein is an sAg peptide composed mainly of preS and S regions. The preS region, with specific affinity for human hepatocytes, is localized in the N-terminus. The S region following the preS has two transmembrane regions responsible for the formation of particles. In this study, the fusion of emerald green fluorescent protein (EGFP) at the C-terminus of the S region was designed to deliver proteins to human hepatocytes. Truncation of the C-terminus of the S region was required to obtain sufficient expression levels in Cos7 cells. The nanoparticles that were produced delivered EGFP to human hepatoma cells, displaying the EGFP moiety outside, or enclosing it inside. However, only a single orientation characterizes the particle, so that either type of L fusion particle could be effectively and independently separated by an antibody affinity column. The dual C-terminal topologies of the L fusion particles designed in this study could be applied to various proteins for the C-terminal moiety of the L fusion proteins, depending on the character of the proteins, such as cytoplasmic proteins, as well as cytokines or ligands to cell surface receptors. We suggest that this fusion design is the most efficient way to prepare a BNC that delivers proteins to specific cells or tissues.

    DOI: 10.1111/j.1742-4658.2005.04790.x

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  • Anti-angiogenic effect of an insertional fusion protein of human basic fibroblast growth factor and ribonuclease-1

    T Hayashida, M Ueda, K Aiura, H Tada, M Onizuka, M Seno, H Yamada, M Kitajima

    PROTEIN ENGINEERING DESIGN & SELECTION   18 ( 7 )   321 - 327   2005年7月

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    記述言語:英語   出版者・発行元:OXFORD UNIV PRESS  

    Human pancreatic ribonuclease-1 (RNase1) does not exhibit its cytotoxicity unless it is artificially internalized into the cytosol. Furthermore, once it encounters the cytosolic RNase inhibitor (RI), the activity of RNase1 is seriously reduced. To achieve the cellular targeting of RNase1 and the blocking of RI binding simultaneously, the basic fibroblast growth factor (bFGF) sequence was inserted into RNase1 at the RI binding site using a gene fusion technique. The effect of this fusion protein, CL-RFN89, on the angiogenesis, which was accelerated by FGF-FGF receptor interaction, was investigated. It was shown by using fluorescein-labeled CL-RFN89, that the binding to human umbilical vein endothelial cells (HUVECs) was dependent on the existence of the FGF receptors. In addition, CL-RFN89 inhibited the cellular growth of HUVECs in vitro and also inhibited the tube formation, using a three-dimensional tube formation assay. Furthermore, this fusion protein was shown to prevent in vivo tumor cell-induced angiogenesis, using the mouse dorsal air sac assay. These results demonstrated that CL-RFN89 inhibits angiogenesis in vitro and in vivo and that it can be expected to be a potent antiangiogenic agent.

    DOI: 10.1093/protein/gzi040

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  • Protein transduction assisted by polyethylenimine-cationized carrier proteins

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

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

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    記述言語:英語   出版者・発行元:JAPANESE BIOCHEMICAL SOC  

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

    DOI: 10.1093/jb/mvi081

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

    Junichiro Futami, Midori Kitazoe, Takashi Maeda, Masakiyo Sakaguchi, Jun Kosaka, Megumi Kosaka, Hiroko Tada, Masaharu Seno, Junzo Sasaki, Nam-Ho Huh, Masayoshi Namba, Hidenori Yamada

    Journal of Bioscience and Bioengineering   2005年

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

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

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

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

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

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  • Nanoparticles for the delivery of genes and drugs to human hepatocytes

    T Yamada, Y Iwasaki, H Tada, H Iwabuki, MKL Chuah, T VandenDriessche, H Fukuda, A Kondo, M Ueda, M Seno, K Tanizawa, S Kuroda

    NATURE BIOTECHNOLOGY   21 ( 8 )   885 - 890   2003年8月

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    記述言語:英語   出版者・発行元:NATURE PUBLISHING GROUP  

    Hepatitis B virus envelope L particles form hollow nanoparticles displaying a peptide that is indispensable for liver-specific infection by hepatitis B virus in humans. Here we demonstrate the use of L particles for the efficient and specific transfer of a gene or drug into human hepatocytes both in culture and in a mouse xenograft model. In this model, intravenous injection of L particles carrying the gene for green fluorescent protein (GFP) or a fluorescent dye resulted in observable fluorescence only in human hepatocellular carcinomas but not in other human carcinomas or in mouse tissues. When the gene encoding human clotting factor IX was transferred into the xenograft model using L particles, factor IX was produced at levels relevant to the treatment of hemophilia B. The yeast-derived L particle is free of viral genomes, highly specific to human liver cells and able to accommodate drugs as well as genes. These advantages should facilitate targeted delivery of genes and drugs to the human liver.

    DOI: 10.1038/nbt843

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  • バイオナノ粒子を用いる遺伝子、薬剤のピンポイントドラッグデリバリーシステム

    妹尾昌治, 黒田俊一, 近藤昭彦, 多田宏子, 谷沢克行, 上田政和

    BIO INDUSTRY   29(4)   2003年

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  • Growth inhibition of mammalian cells by eosinophil cationic protein

    Takashi Maeda, Midori Kitazoe, Hiroko Tada, Rafael de Llorens, David S. Salomon, Masakazu Ueda, Hidenori Yamada, Masaharu Seno

    Eur. J. Biochem.   2002年

  • RNase 3 (ECP) Is an Extraordinarily Stable Protein among Human Pancreatic-Type RNases

    Takashi Maeda, Kazutoshi Mahara, Midori Kitazoe, Junichiro Futami, Aiko Takidani, Megumi Kosaka, Hiroko Tada, Masaharu Seno, Hidenori Yamada

    J. Biochem.   2002年

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  • Optimum Modification for the Highest Cytotoxicity of Cationized Ribonuclease 1

    Junichiro Futami, Emiko Nukui, Takashi Maeda, Megumi Kosaka, Hiroko Tada, Masaharu Seno, Hidenori Yamada

    J. Biochem.   2002年

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  • Solution structure of betacellulin, a new member of EGF-family ligands

    Kazunori Miura, Hideki Doura, Tomoyasu Aizawa, Hiroko Tada, Masaharu Seno, Hidenori Yamada, Keiichi Kawano

    Biochem. Biophys. Res. Commun.   2002年

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

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

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

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    記述言語:英語   出版者・発行元:AMER CHEMICAL SOC  

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

    DOI: 10.1021/bi010248g

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

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

    ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY   2001年

  • Stabilization of Human RNase 1 by Introduction of a Disulfide Bond between Residures 4 and 118.

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

    J. Biochem   2000年

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  • Three-dimensional Crystal Structure of Human Eosinophil Cationic Protein (RNase3) at 1.75 Å Resolution.

    Mallorqui-Fernandez, G, Pous J, Peracaula, R, Aymami, J, Maeda, T, Tada, H, Yamada, H, Seno, M, de Llorens, R, Gomis-Ruth, FX, Coll, M

    J. Mol.Biol.   2000年

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  • Molecular Cloning and Expression of Rat Betacellulin cDNA.

    Tada, H, Seno, M, Yamada, H, Sasada, R, Igarashi, K

    Biochim. Biophys. Acta   2000年

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

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

    J. Biochem   2000年

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  • Immunohistochemical localization of betacellulin, a new member of the EGF family, in normal human pancreas and islet tumor cells

    Miyagawa J, Hanafusa T, Sasada R, Yamamoto K, Igarashi K, Yamamori K, Seno M, Tada H, Nammo T, Li M, Yamagata K, Nakajima H, Namba M, Kuwajima M, Matsuzawa Y

    ENDOCRINE JOURNAL   1999年

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  • Inhibition of Cell Growth by a Fused Protein of Human Ribonuclease 1 and Human Basic fibroblast Growth Factor.

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

    Protein Eng   1999年

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  • Processing and Juxtacrine Activity of Membrane-Anchored Betacellulin

    Hiroko Tada, Reiko Sasada, Yasuko Kawaguchi, Itaru Kojima, Wiliam J. Gullick, David Salomon, Koichi Igarashi, Hidenori Yamada

    Journal of Cellular Biochemistry   1999年

  • BALB/c 3T3 cells co-expressing FGF-2 and soluble FGF receptor acquire tumorigenicity

    Masaharu Seno, Akinori Masago, Atsushi Nishimura, Hiroko Tada, Megumi Kosaka, Reiko Sasada, Koichi Igarashi, Satimaru Seno, Hidenori Yamada

    Cytokine   10 ( 4 )   290 - 294   1998年

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    記述言語:英語   出版者・発行元:Academic Press  

    The physiological significance of the soluble fibroblast growth factor (FGF) receptors is not clear yet although they are present in blood, vitreous fluid and in the extracellular matrix of vascular endothelial cells. A hypothesis that they might help FGF-2 release from cells is very interesting because FGF-2 does not have clear secretion signal and the mechanism of the secretion of FGF-2 is still unclear. Single overexpression of FGF-2 is related neither to the secretion potential of the molecule nor to the tumorigenicity of the cells. In this report, BALB/c 3T3 cells transformed with the full length of human FGF-2 cDNA are further transformed with the cDNA coding the extracellular domain of human FGF receptor 1. The obtained transformants co-expressing FGF-2 and soluble FGF receptor are highly tumorigenic in nude mice, while the parental cells do not show any tumorigenicity. In the conditioned medium of the double-transformants, FGF-2 is immunologically detected. These results suggest that naturally produced soluble form of FGF receptor supports the release of FGF-2 from the cells and that over-expression of these two molecules leads to induce the malignant tumours in vivo.

    DOI: 10.1006/cyto.1997.0286

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  • Efficient production of a functional mouse/human chimeric Fab' against human urokinase-type plasminogen activator by Bacillus brevis

    Y. Inoue, T. Ohta, H. Tada, S. Iwasa, S. Udaka, H. Yamagata

    Applied Microbiology and Biotechnology   48 ( 4 )   487 - 492   1997年

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    記述言語:英語  

    Expression/secretion vectors for the production of Fab' and single-chain (Sc) Fab' by Bacillus brevis have been constructed. For the production of Fab', the cDNAs encoding the L chain and Fd' fragment (Fd with the hinge region) of a mouse-human chimeric Fab' against human urokinase-type plasminogen activator were fused directly with the translation-start and signal-peptide-encoding regions of the mwp gene, the gene for one of the major cell-wall proteins of Bacillus brevis. The two fused genes were placed tandemly downstream from the promoter of the cell-wall protein gene operon (cwp) of B. brevis. For the production of scFab', the two cDNAs were linked with a synthetic oligonucleotide encoding a flexible peptide linker of 17 or 24 amino acids, and fused with the translation start and signal-peptide-encoding regions of the mwp gene. Fab' was efficiently produced by B. brevis, being accumulated at a level of 100 mg/l in the culture medium in a simple shake-flask culture, which is the highest level obtained so far for a gram-positive bacterium. On the other hand, the scFab' remained at a level of a few milligrams per liter in the culture medium. The Fab' produced by B. brevis showed comparable antigen-binding activity to that of the parental antibody. The L chain and Fd' fragment, constituting the Fab', had the correct N-terminal amino acid sequences. These results indicate that B. brevis is a very promising host for the production of native Ig fragments.

    DOI: 10.1007/s002530051084

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  • Recombinant human pancreatic ribonuclease produced in e. coli : Importance of the amino-terminal sequence

    Junichiro Futami, Masaharu Seno, Megumi Kosaka, Hiroko Tada, Satimaru Seno, Hidenori Yamada

    Biochemical and Biophysical Research Communications   216 ( 1 )   406 - 413   1995年

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    記述言語:英語  

    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. © 1995 by Academic Press, Inc.

    DOI: 10.1006/bbrc.1995.2638

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  • Bispecific antibody-producing hybrid hybridoma and its use in one-step immunoassays for human lymphotoxin

    H. Tada, H. Toyoda, S. Iwasa

    Hybridoma   8 ( 1 )   73 - 83   1989年

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▼全件表示

講演・口頭発表等

  • 難結晶性蛋白質のパッキングをコントロールする結晶化法の開発と評価

    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年11月1日 

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    開催年月日: 2018年11月1日

    記述言語:日本語  

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  • A CEP peptide acts as an endogenous suppressor in Arabidopsis

    平成30年度日本植物病理学会関西部会 

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    開催年月日: 2018年9月27日 - 2018年9月28日

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  • 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

    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  2017年11月23日 

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    開催年月日: 2017年11月23日

    記述言語:日本語  

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  • 疎水相互作用がカギを握る難結晶性タンパク質の結晶化法

    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年11月17日 

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    開催年月日: 2016年11月17日

    記述言語:日本語  

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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

    小坂恵, 山田秀徳, 二見淳一郎, 多田宏子, 玉田太郎

    日本蛋白質科学会年会プログラム・要旨集  2016年5月19日 

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    開催年月日: 2016年5月19日

    記述言語:日本語  

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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

    小坂恵, 山田秀徳, 二見淳一郎, 多田宏子, 玉田太郎

    日本分子生物学会年会プログラム・要旨集(Web)  2016年 

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    開催年月日: 2016年

    記述言語:日本語  

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  • 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  2015年5月26日 

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    開催年月日: 2015年5月26日

    記述言語:日本語  

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  • Total folate and 5-methyltetrahydrofolate in the cerebrospinal fluid of children

    第13回アジア・オセアニア小児神経学会議  2015年5月 

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    開催年月日: 2015年5月14日 - 2015年5月17日

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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

    小坂恵, 直井里美, 松下里美, 山田秀徳, 二見淳一郎, 多田宏子, 安達基泰, 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  2014年5月26日 

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    開催年月日: 2014年5月26日

    記述言語:日本語  

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  • 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年 

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    開催年月日: 2014年

    記述言語:日本語  

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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

    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  2012年5月31日 

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    開催年月日: 2012年5月31日

    記述言語:日本語  

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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

    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  2012年5月31日 

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    開催年月日: 2012年5月31日

    記述言語:日本語  

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  • 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年 

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    開催年月日: 2011年

    記述言語:日本語  

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  • Analysis on the Cleavage of Membrane-Bound Betacellulin.(jointly worked)

    The FASEB Journal  1996年4月 

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    開催年月日: 1996年4月

    記述言語:英語  

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  • Construction of Isogenic Polyploid Series of Yeast Strains.(jointly worked)

    Proceedings of IVth International Symposium on Genetics of Industrial Microorganisms  1982年 

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    開催年月日: 1982年

    記述言語:英語  

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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

    日本生化学会中国・四国支部例会 P16-C2  2021年9月10日 

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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  2020年11月14日 

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    会議種別:口頭発表(一般)  

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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

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

    第20回日本蛋白質科学会年会 WEB開催  2020年7月28日 

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  • パッキング相互作用に着目したタンパク質結晶化法

    小坂 恵, 山田 秀徳, 二見 淳一郎, 多田 宏子, 今村 維克, 玉田 太郎

    日本結晶学会令和元年年会  2019年11月20日 

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  • パッキングのコントロールにより、難結晶性タンパク質の結晶化を促進する結晶化

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

    第19回日本蛋白質科学会年会第71回日本細胞生物学会大会 合同年次大会 3WDp-03  2019年6月29日 

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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

    二見 淳一郎, Junichiro Futami, 勝瀬 奈津美, Natsumi Katsuse, 野々村 英典, Hidenori Nonomura, 木下 理恵, Rie Kinoshit, 本荘 知子, Tomoko Honjo, 多田 宏子, Hiroko Tada

    第15回日本蛋白質科学会年会 1P-113  2015年6月24日 

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  • Total folate and 5-methyltetrahydrofolate in the cerebrospinal fluid of children

    第13回アジア・オセアニア小児神経学会議  2015年 

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  • 難結晶性蛋白質の結晶化を促進する疎水性残基導入

    日本結晶学会年会  2015年 

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

    第15回日本蛋白質科学会年会  2015年 

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  • 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

    日本生物工学会  2012年 

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  • 蛋白質の質量分析におけるS-カチオン化試薬の有用性

    第11回日本蛋白質科学会年会  2011年 

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  • Utility of S-cationization regents for mass spectrometry of proteins

    The11th Annual Meeting of the Protein Scientific Society of Japan  2011年 

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  • ヒトD型肝炎ウィルス形成メカニズムを利用したHBsAg粒子内部への機能蛋白質封入法

    第9回日本蛋白質科学会年会  2009年 

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  • 人工転写因子蛋白質を用いた細胞内導入条件の最適化

    第31回日本分子生物学会年会・第81回日本生化学会大会合同年会(BMB2008)  2008年 

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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

    第31回日本分子生物学会年会・第81回日本生化学会大会合同年会(BMB2008)  2008年 

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  • 中空ナノ粒子形成に対するHBVエンベロープ蛋白質粒子内部領域の影響

    第31回日本分子生物学会年会・第81回日本生化学会大会合同年会(BMB2008)  2008年 

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  • 変性タンパク質の可溶化と細胞内導入を目的とした新規SH基多価カチオン化試薬の合成

    第31回日本分子生物学会年会・第81回日本生化学会大会合同年会(BMB2008)  2008年 

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  • B型肝炎ウィルスエンベロープ蛋白質によるウィルス様粒子形成にTM1ヘリックスは不必要である。

    第8回日本蛋白質科学会年会  2008年 

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  • Eosinophil derived cationinc protein enhances stress fiber formation and cardiomyogenesis

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

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  • 好酸球由来タンパク質によるP19細胞の心筋細胞への分化促進

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

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  • Cell surface marker candidates in brain tumors identified by an oligonucleotide microarray coupled with spherical SOMs.

    2007日本バイオインフォマティクス学会  2007年 

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  • Cell surface marker candidates in brain tumors.

    47th Annual Meeting, The American Society for Cell Bioligy,  2007年 

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  • カチオン化アビジンによるビオチン化タンパク質細胞導入における導入効率の解析と評価

    2007年日本化学会西日本大会  2007年 

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  • 人工転写因子を用いたタンパク質細胞内導入効率の定量化

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

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

    第59回日本生物工学会大会  2007年 

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  • 粒子形成に必要なヒトB型肝炎ウィルスエンベロープ蛋白質配列の特定

    第7回日本蛋白質科学会年会  2007年 

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  • Cell type dependent internalization of ErbB2.

    第66回日本癌学会学術総会  2007年 

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  • Identification of cell surface marker candidates in breast cancer using an oligonucleotide-based microarray system.

    第66回日本癌学会学術総会  2007年 

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  • Interpreting patterns of gene expression with spherical self-organizing maps (sSOM): application to search for cell surface markers

    The 17th International Conference on Genome Informatics  2006年12月18日 

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  • Separation of Dual Affinity of Betacellulin to ErbB Receptors

    The American Society for Cell Biology 46th Annual Meeting  2006年12月10日 

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  • β-cateninタンパク質導入による人為的Wntシグナル制御

    2006年分子生物学フォーラム  2006年12月6日 

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

    5th East Asian Biophysics Symposium & 44th Annual Meeting of the Biophysical Society of Japan  2006年11月 

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

    20th IUBMB international congress of biochemistry and molecular biology and 11th FAOBMB congress  2006年6月23日 

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

    20th IUBMB international congress of biochemistry and molecular biology and 11th FAOBMB congress  2006年6月20日 

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  • Pinpoint Drug and Gene Delivery System Using ZZ tag-displaying Bio-nanocapsule and Targeting Molecule-fused IgG Fc protein.

    20th IUBMB international congress of biochemistry and molecular biology and 11th FAOBMB congress  2006年6月20日 

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  • Development of a novel DDS vector ? the engineered Bio-nanocapsule for specific delivery of proteins to human liver cells.

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

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  • Specific delivery of protein to human liver cells by engineered Bio-nanocapsule.

    4th European-Japanese Bioorganic Conference & Chemical Biology COE Program Sponsored by Okayama University (Joint Symposium)  2005年 

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  • Comparison of cell surface proteins expressed in BALB/c 3T3 and SV-T2 cells by novel DNA microarray.

    4th European-Japanese Bioorganic Conference & Chemical Biology COE Program Sponsored by Okayama University (Joint Symposium)  2005年 

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  • Effect of cys-residue replacement from envelope protein of Bio-nanocapsule (BNC) for drug delively system.

    4th European-Japanese Bioorganic Conference & Chemical Biology COE Program Sponsored by Okayama University (Joint Symposium)  2005年 

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  • 新しいDDSのためのバイオナノパーティクルテクノロジー

    第一回医歯工学先端技術シンポジウムOKAYAMA  2005年 

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  • Design of soluble ErbBs and interacting with their ligands.

    4th European-Japanese Bioorganic Conference & Chemical Biology COE Program Sponsored by Okayama University (Joint Symposium)  2005年 

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  • Biological activity of the extracellular domain of tomoregulin.

    4th European-Japanese Bioorganic Conference & Chemical Biology COE Program Sponsored by Okayama University (Joint Symposium)  2005年 

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  • The application of cell surface marker DNA microarray in the search for molecular targets.

    The 16th International Conference on Genome Informatics  2005年 

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  • Artificial control of cell proliferation using an N-terminal domain of simian virus 40 large T antigen by means of PEI-cationization.

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

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  • ファージディスプレイを用いたErbB2に特異的に結合するペプチドの同定

    第28回日本分子生物学会年会  2005年 

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  • ヒトベータセルリンの受容体結合性分離のための変異体ライブラリー構築と解析

    第28回日本分子生物学会年会  2005年 

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

    第28回日本分子生物学会年会  2005年 

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  • Quantitative analysis of intracellular delivery of cationized proteins.

    第78回日本生化学会大会  2005年 

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  • Characterization of tumor cells using DNA microarray designed for cell surface markers.

    The European Life Scientist Organization 2005 Meeting  2005年 

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  • Engineered Bio-nanocapsule for specific delivery of proteins to human liver cells as a novel DDS vector.

    The 3rd Science and Research Symposium Hepatitis and Liver fibrosis from Basic Research to the Clinic  2005年 

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  • 蛋白質カチオン化による細胞内導入技術開発と機構解明

    日本生物工学会大会  2005年 

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

    第5回日本蛋白質科学会年会  2005年 

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  • Screening for receptor binding motif of betacellulin.

    4th European-Japanese Bioorganic Conference & Chemical Biology COE Program Sponsored by Okayama University (Joint Symposium)  2005年 

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  • Effect of cys-residue replacement on envelope protein of Bio-nanocapsule (BNC) for drug delively system.

    The European Life Scientist Organization 2005 Meeting  2005年 

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  • DDS用バイオナノカプセル(BNC)を構成する蛋白質へのCys残基欠損の効果

    第27回日本分子生物学会年会  2004年 

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  • 細胞表面マーカー・マイクロアレイを用いるBALB/c 3T3細胞とそのSV40形質転換細胞SV-T2の比較

    第27回日本分子生物学会年会  2004年 

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  • 免疫グロブリンのヒンジ領域を用いた可溶性ErbBのデザイン

    第27回日本分子生物学会年会  2004年 

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  • Diamond-like Carbonコーティングスライドグラスを用いたヒトベータセルリン変異体アレイによる受容体結合性の解析

    第27回日本分子生物学会年会  2004年 

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  • Imagine of human liver cells by specific delivery of Bio-nanocapsule fused to EGFP

    第27回日本分子生物学会年会,  2004年 

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  • Molecular design of soluble ErbBs with IgG-hinge region

    The American Society for Cell Biology 44th Annual Meeting,  2004年 

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  • カチオン化SV40T抗原N末端ドメインによる細胞増殖の人工制御

    第27回日本分子生物学会年会  2004年 

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

    第27回日本分子生物学会年会  2004年 

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  • 大腸菌性インクルージョンボディからのニワトリアビジン4量体の酸化的リフォールディング

    第77回日本生化学会大会  2004年 

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  • Characterization of cell surface marker proteins in SV-T2 cells

    The American Society for Cell Biology 44th Annual Meeting  2004年 

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  • T7 RNA polymerase の細胞ない導入による外部制御可能な一過性遺伝子発現

    第77回日本生化学会大会  2004年 

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  • 変異導入によるHBウイルスエンベロープ蛋白質のC末端領域構造の解析

    第26回日本分子生物学会年会  2003年 

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  • Detection of the interaction between Betacellulin and soluble ErbB receptors by peptide-microarray

    THE AMERICAN SOCIETY FOR CELL BIOLOGY, 43rd Annual Meeting,  2003年 

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  • Criptoを標的するバイオナノ粒子の分子デザイン

    第26回日本分子生物学会年会  2003年 

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  • Design of Bio-nanoparticle, in which proteins are enclosed

    第26回日本分子生物学会年会  2003年 

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  • Engineering of Pancreatic RNases for Conversion to Cytotoxin

    第76回日本生化学会大会  2003年 

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  • 変異導入による蛋白質の結晶化とそのX線結晶構造解析

    第3回日本蛋白質科学会  2003年 

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  • バイオナノ粒子を用いるドラッグデリバリーシステム

    第4回VBLシーズ講演会, 講演番号3  2003年 

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  • Reversible cationization, a new method for delivery of a denatured protein into living cells. Application to p53.

    第76回 日本生化学会大会  2003年 

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  • ドメイン挿入型融合タンパク質の設計

    日本生物工学会大会  2002年 

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  • カチオン性キャリアによる蛋白質細胞内導入法の改良

    第25回日本分子生物学会年会  2002年 

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  • B型肝炎ウィルス表面抗原におけるCys残基の粒子構築への寄与

    第25回日本分子生物学会  2002年 

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  • PEIカチオン化法による蛋白質細胞内導入機構の解明

    第75回日本生化学会年会  2002年 

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  • バイオナノ粒子を用いるヒト肝細胞癌特異的遺伝子導入

    遺伝子・デリバリー研究会第2回シンポジウム  2002年 

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  • 疎水性パッキング部位の構築によるヒトRNase 1の結晶化と構造解析

    第2回日本蛋白質科学会年会  2002年 

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  • ドメイン内部ループへのドメイン挿入融合による「新規融合ドメイン」の形成

    第2回日本蛋白質科学会年会  2002年 

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  • ヒト組換え膵臓型RNase及びウシRNase Aの速度論的安定性に関する研究

    第75回日本生化学大会  2002年 

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  • Optimum Modification of Cationization For the Potent Cytotoxic Ribonuclease

    6th International Conferencee on Ribonucleases  2002年 

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  • 免疫グロブリンのヒンジ領域のみを用いて二量体化した可溶性ErbB蛋白質による ベータセルリンの検出

    日本生物工学会大会  2002年 

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  • ECP のBALB/c 3T3 細胞に対する生理活性

    第24回 日本分子生物学会  2001年 

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  • 組み換え型ラット及びヒトベータセルリンの調製とドメイン解析

    第24回 日本分子生物学会  2001年 

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  • 化学修飾による蛋白質細胞内導入法の開発

    平成13年度日本分子生物学会年会  2001年 

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  • PEI カチオン化法による蛋白質細胞内導入とバイオイメージング

    第24回 日本分子生物学会  2001年 

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  • 膜透過性ペプチドの部位特異的導入によるヒトリボヌクレア-ゼ1 の細胞障害活性発揮へのアプロ-チ

    第24回 日本分子生物学会  2001年 

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  • ヒト好酸球由来RNase (ECP) 各種変異体における細胞増殖阻害活性の比較

    第24回日本分子生物学会年会  2001年 

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  • 蛋白質カチオン化による蛋白質細胞内導入

    平成13年度日本蛋白科学会年会  2001年 

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  • 組み換え型ヒトECPの糖質認識機構

    第1回日本蛋白質科学会年会  2001年 

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  • ヒト組換え型膵臓型RNase及びウシRNaseAの安定性の比較

    平成13年度日本蛋白質科学会  2001年 

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  • ヒトBetacellulinのNMR法による構造解析

    第1回日本蛋白質科学会年会  2001年 

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  • ヒトリボヌクレアーゼ1のドメイン間へbFGFを挿入した融合タンパク質の作製

    第1回日本蛋白質科学会年会  2001年 

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  • NMR法によるHuman BetacellulinのEGF-domain の構造解析

    第121年会日本薬学会  2001年 

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  • ベータセルリン結合蛋白のクローニングと解析

    第24回 日本分子生物学会  2001年 

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  • リボヌクレアーゼ分子内部へのキャリヤタンパク質挿入融合による細胞傷害性物質作製

    平成13年度日本生物工学会大会  2001年 

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  • サブユニット分子間SS架橋導入により安定化したヒトRNase1ダイマーの解析

    第23回日本分子生物学会大会  2000年 

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  • RNaseの細胞増殖阻害活性におけるRNA分解活性の関与

    第23回日本分子生物学会大会  2000年 

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  • 組み換え型ヒト好酸球由来ECPの哺乳動物株化細胞に対する効果

    平成12年度日本生物工学会大会  2000年 

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  • サブユニット間相互作用導入による安定なヒトリボヌクレアーゼ二量体の作製

    蛋白質合同年会東京2000  2000年 

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  • ヒト好酸球由来ECPの細胞に対する効果

    第73回日本生化学会大会  2000年 

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  • ポリエチレンイミンの導入によるRNaseAの細胞毒性機能の発現

    第73回日本生化学会大会  2000年 

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  • Eosinophil Cationic protein の Balb/c 3T3 細胞に対する生理活性の検討

    第23回日本分子生物学会大会  2000年 

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  • ECPの動物細胞に対する吸着および細胞内移行

    第53回日本細胞生物学会大会  2000年 

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  • 膵臓型リボヌクレアーゼファミリー蛋白質が形成するダイマーの安定性

    第23回日本分子生物学会大会  2000年 

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  • RNaseAのスワッピングダイマーの鎖間架橋による構造の固定化

    第72回日本生化学会大会  1999年 

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  • Cytotoxic Effects of Eosinophil Cationic Protein on Carcinoma Cells

    Fifth International Meeting on Ribonucleas  1999年 

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  • 疎水結合性接触面の導入によるヒトリボヌクレアーゼのダイマー四次構造の制御

    第11回蛋白質工学会年会  1999年 

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  • ヒト膵臓リボヌクレアーゼファミリー蛋白質のmetastableなダイマーの形成

    第11回蛋白質工学会年会  1999年 

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  • 正電荷の導入に伴うRNaseの細胞増殖機能の発現

    第22回日本分子生物学会年会  1999年 

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  • ヒトEosinophil Cationic Protein によry細胞増殖阻害活性の検討

    平成11年度生物工学会大会  1999年 

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  • ヒト好酸球由来リボヌクレアーゼECPの癌細胞に対する効果

    平成11年度生物工学会大会  1999年 

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  • ベータセルリンが誘導するマウス乳腺上皮細胞株HC-11の分化

    第22回日本分子生物学会年会  1999年 

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  • TAPS-sulfonate (SH基のカチオン性保護試薬)の蛋白質工学への応用-インクルージョンボディになりにくい小さな蛋白質の精製と活性化-

    第22回日本分子生物学会年会  1999年 

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  • ヒト膵臓型リボヌクレアーゼファミリー蛋白質のダイマー形成

    第22回日本分子生物学会年会  1999年 

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  • 分泌型FGF受容体の機能的役割の解析(共著)

    第18回日本分子生物学会年会要旨集 

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産業財産権

  • ウイルス粒子様ナノカプセル

    多田 宏子, 妹尾 昌治, 黒田 俊一

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    出願人:国立大学法人 岡山大学

    出願番号:特願2011-162596  出願日:2011年7月25日

    公開番号:特開2013-021999  公開日:2013年2月4日

    特許番号/登録番号:特許第5892741号  登録日:2016年3月4日 

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  • 低抗原性のHBsAg粒子及びその作製法

    山田 一朗, 妹尾 昌治, 多田 宏子

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    出願人:国立大学法人 岡山大学

    出願番号:JP2007065646  出願日:2007年8月9日

    公表番号:WO2008-018555  公表日:2008年2月14日

    特許番号/登録番号:特許第5147697号  登録日:2012年12月7日 

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  • 生体構造認識部位を提示する中空ナノ粒子およびその生産方法、並びにその利用

    妹尾 昌治, 多田 宏子, 黒田 俊一, 谷澤 克行, 近藤 昭彦, 上田 政和

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    出願人:独立行政法人科学技術振興機構

    出願番号:JP2004017282  出願日:2004年11月19日

    公表番号:WO2005-049824  公表日:2005年6月2日

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  • システイン残基を改変したタンパク質からなる中空ナノ粒子およびそれを用いた薬剤

    黒田 俊一, 谷澤 克行, 近藤 昭彦, 上田 政和, 妹尾 昌治, 多田 宏子

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    出願人:科学技術振興事業団

    出願番号:特願2003-183863  出願日:2003年6月27日

    公開番号:特開2004-081210  公開日:2004年3月18日

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  • ヒト生理活性物質とヒト蛋白質合成阻害物質との挿入融合体

    上田 政和, 多田 宏子, 妹尾 昌治

     詳細を見る

    出願人:上田 政和

    出願番号:特願2003-078355  出願日:2003年3月20日

    公開番号:特開2004-284977  公開日:2004年10月14日

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  • 中空ナノ粒子を形成するタンパク質に疾患治療用の細胞導入物質を融合させた薬剤

    黒田 俊一, 谷澤 克行, 近藤 昭彦, 上田 政和, 妹尾 昌治, 多田 宏子

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    出願人:科学技術振興事業団

    出願番号:特願2002-097280  出願日:2002年3月29日

    公開番号:特開2003-286189  公開日:2003年10月7日

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受賞

  • 岡山工学振興会科学技術賞

    1999年  

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    受賞国:日本国

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