論文 - 臼井 健二
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Effect of linearly polarized microwaves on nanomorphology of calcium carbonate mineralization using peptides 査読あり
Kenji Usui, Makoto Ozaki, Kan Hirao, Tsubasa Kosaka, Natsumi Endo, Shuhei Yoshida, Shin-ichiro Yokota, Yonejiro Arimoto, Ryuji Osawa, Nobuhiro Nakanishi, Kin-ya Tomizaki, Tomohiro Umetani, Fumihiro Kayamori
Scientific Reports 13 12027 2023年7月
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Construction of a Method to Design Fibril-Forming Peptides Applied to Biomaterials from 20 Beta-Sheet Peptides by Statistical Analysis 査読あり
Kazuya Iwata, Taisei Terao, Akira Takekawa, Tomohiro Umetani, Kenji Usui
Peptide Science 2022 141 - 142 2023年3月
担当区分：最終著者, 責任著者
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De novo design of a nanopore for single-molecule detection that incorporates a β-hairpin peptide. 査読あり 国際共著 国際誌
Keisuke Shimizu, Batsaikhan Mijiddorj, Masataka Usami, Ikuro Mizoguchi, Shuhei Yoshida, Shiori Akayama, Yoshio Hamada, Akifumi Ohyama, Kenji Usui, Izuru Kawamura, Ryuji Kawano
Nature nanotechnology 17 ( 1 ) 67 - 75 2022年1月
The amino-acid sequence of a protein encodes information on its three-dimensional structure and specific functionality. De novo design has emerged as a method to manipulate the primary structure for the development of artificial proteins and peptides with desired functionality. This paper describes the de novo design of a pore-forming peptide, named SV28, that has a β-hairpin structure and assembles to form a stable nanopore in a bilayer lipid membrane. This large synthetic nanopore is an entirely artificial device for practical applications. The peptide forms multidispersely sized nanopore structures ranging from 1.7 to 6.3 nm in diameter and can detect DNAs. To form a monodispersely sized nanopore, we redesigned the SV28 by introducing a glycine-kink mutation. The resulting redesigned peptide forms a monodisperse pore with a diameter of 1.7 nm leading to detection of a single polypeptide chain. Such de novo design of a β-hairpin peptide has the potential to create artificial nanopores, which can be size adjusted to a target molecule.
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ウォッシャーディスインフェクターのすすぎ排水中に含まれる洗浄剤残留量の測定によるすすぎ性能評価 査読あり
三軒隼人、藤田敏、川田原瑠勇、武川公、臼井健二、原田陽滋
医療機器学 91 315 - 332 2021年8月
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Ozaki, M., Yoshida, S., Tsuruoka, T., Usui, K.
Chemical Communications 57 ( 6 ) 2021年
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Ozaki, M., Imai, T., Tsuruoka, T., Sakashita, S., Tomizaki, K.-Y., Usui, K.
Communications Chemistry 4 ( 1 ) 2021年
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Miyazaki H , Hamada Y , Takaishi H , Minamino Y , Ikeda H , Mekata H , Takaishi M , Yamashita K , Usui K
Analyst 145 ( 9 ) 3211 - 3216 2020年5月
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Novel purification process for amyloid beta peptide(1-40) 招待あり 査読あり
Usui, K., Yokota, S.-I., Iwata, K., Hamada, Y.
Processes 8 ( 4 ) 2020年
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Miyazaki, H., Takaishi, H., Ikeda, H., Ariumi, H., Hamada, Y., Yamashita, K., Usui, K.
Processes 8 ( 10 ) 2020年
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Miyazaki, H., Samejima, Y., Iwata, K., Minamino, Y., Hikida, S., Ariumi, H., Ikeda, H., Hamada, Y., Yamashita, K., Usui, K.
International Journal of Molecular Sciences 21 ( 21 ) 2020年
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Effect of tryptophan residues on gold mineralization by a gold reducing peptide 査読あり
Ozaki, M., Yoshida, S., Oura, M., Tsuruoka, T., Usui, K.
RSC Advances 10 ( 66 ) 2020年
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Gold-Titania Nanocatalyst Generated by Mineralization Using Two Artificial Peptides with DNA 査読あり
Ozaki Makoto, Tomizaki Kin-Ya, Hamada Yoshio, Usui Kenji
JOURNAL OF PEPTIDE SCIENCE 24 S101 2018年9月
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Kenji Usui, Shin-ichiro Yokota, Makoto Ozaki, Shungo Sakashita, Takahito Imai, Kin-ya Tomizaki
Protein & Peptide Letters 25 ( 1 ) 42 - 47 2018年4月
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Yuki Tominaga, Kenji Usui, Akiyoshi Hirata, Hiro-O Ito, Kiyoshi Nokihara
Bioorganic & Medicinal Chemistry 26 ( 12 ) 3210 - 3216 2018年4月
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Editorial: Organic-Inorganic Hybrid Materials and Their Applications 査読あり
Kin-ya Tomizaki, Yoshio Hamada, Kenji Usui
Protein & Peptide Letters 25 2 - 3 2018年4月
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Peptides for Silica Precipitation: Amino Acid Sequences for Directing Mineralization 査読あり
Makoto Ozaki, Shungo Sakashita, Yoshio Hamada, Kenji Usui
Protein & Peptide Letters 25 ( 1 ) 15 - 24 2018年4月
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Design of Anti-Alzheimer's Disease Agents Focusing on a Specific Interaction with Target Biomolecules 査読あり
Yoshio Hamada, Kenji Usui
Neuromethods 132 207 - 228 2018年
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Channel current analysis estimates the pore-formation and the penetration of transmembrane peptides 査読あり
Sekiya, Y., Sakashita, S., Shimizu, K., Usui, K., Kawano, R.
Analyst 143 ( 15 ) 2018年
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生体分子の挙動解析研究を目標としたマイクロ波照射システムの開発 ～ペプチドのバイオミネラリゼーションにおけるマイクロ波影響解析をモデルとして～ 査読あり
臼井健二, 富樫浩行, 圓東那津実, 尾崎誠, 有本米次郎, 裏鍛武史, 大沢隆二, 皆木幸一, 中西伸浩, 梅谷智弘
日本電磁波エネルギー応用学会論文誌 17 - 24 2017年12月
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Non-Covalent Loading of Anti-Cancer Doxorubicin by Modularizable Peptide Self-Assemblies for a Nanoscale Drug Carrier 査読あり
Kin-ya Tomizaki, Kohei Kishioka, Shunsuke Kataoka, Makoto Miyatani, Takuya Ikeda, Mami Komada, Takahito Imai, Kenji Usui
Molecules 22 ( 11 ) 1916 2017年11月
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DNA G-Wire Formation Using an Artificial Peptide is Controlled by Protease Activity 査読あり
Kenji Usui, Arisa Okada, Shungo Sakashita, Masayuki Shimooka, Takaaki Tsuruoka, Shu-ichi Nakano, Daisuke Miyoshi, Tsukasa Mashima, Masato Katahira, Yoshio Hamada
Molecules 22 ( 11 ) 1991 2017年11月
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Fluorescent and Luminescent Fusion Proteins for Analyses of Amyloid Beta Peptide Aggregation 査読あり
Usui K, Mie M, Andou T, Mihara H, Kobatake E.
J Pept Sci. 23 ( 7-8 ) 659 - 665 2017年7月
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Development of Peptide Microarray to Detect the Protein in Saliva for Periodontal Disease Test 査読あり
Yuki Tominaga, Kenji Usui, Akiyoshi Hirata, Atsushi Kitagawa, Hiro-O Ito and Kiyoshi Nokihara
Peptide Science 2016 131 - 132 2017年3月
共著
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A Systematic Study on Molecular Mechanism of Pore-Forming Peptides for Discovering Antimicrobial Medicine
Y. Sekiya, H. Watanabe, K. Usui, R. Kawano
Proceedings of MicroTAS 2016 595 - 596 2016年10月
共著
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Site-Specific Control of Silica Mineralization on DNA Using a Designed Peptide 査読あり
M. Ozaki, K. Nagai, H. Nishiyama, T. Tsuruoka, S. Fujii, T. Endoh, T. Imai, K.-y. Tomizaki, K. Usui
Chem. Commun. 52 4010 - 4013 2016年3月
共著
担当区分：筆頭著者
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Development of Designed Peptides with Secondary Structure for Use in Nanobiotechnology 招待あり 査読あり
K.Usui
Peptide Science 2015 5 - 8 2016年3月
単著
担当区分：筆頭著者
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A Cell Microarray Format: A Peptide Release System Using a Photo-Cleavable Linker for Cell Toxicity and Cell Uptake Analysis. 査読あり
Usui K, Tomizaki KY, Mihara H
Methods in molecular biology (Clifton, N.J.) 1352 199 - 210 2016年
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Site-specific control of multiple mineralizations using a designed peptide and DNA 査読あり
Kenji Usui, Makoto Ozaki, Aoi Yamada, Yoshio Hamada, Takaaki Tsuruoka, Takahito Imai, Kin-ya Tomizaki
NANOSCALE 8 ( 39 ) 17081 - 17084 2016年
出版者・発行元：ROYAL SOC CHEMISTRY
We have developed a site-specific method for precipitating multiple inorganic compounds using target DNA and a designed peptide consisting of a peptide nucleic acid (PNA) sequence and an inorganic compound-precipitating sequence. This system for controlled site-specific precipitation represents a powerful tool for use in nanobiotechnology and materials science.
DOI： 10.1039/c6nr03468c
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Site-specific control of silica mineralization on DNA using a designed peptide 査読あり
Makoto Ozaki, Kazuma Nagai, Hiroto Nishiyama, Takaaki Tsuruoka, Satoshi Fujii, Tamaki Endoh, Takahito Imai, Kin-ya Tomizaki, Kenji Usui
CHEMICAL COMMUNICATIONS 52 ( 21 ) 4010 - 4013 2016年
出版者・発行元：ROYAL SOC CHEMISTRY
We developed a site-specific method for precipitating inorganic compounds using organic compounds, DNA, and designed peptides with peptide nucleic acids (PNAs). Such a system for site-specific precipitation represents a powerful tool for use in nanobiochemistry and materials chemistry.
DOI： 10.1039/c5cc07870a
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ATP-Binding Peptide-Hydrogel Composite Synthesized by Molecular Imprinting on Beads 査読あり
Ayana Takata, Kenji Usui, Jun Matsui
Molecular Imprinting 3 65 - 70 2015年12月
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A Cell Microarray Format: A Peptide Release System Using a Photo-Cleavable Linker for Cell Toxicity and Cell Uptake Analysis 査読あり
K. Usui, K.-y. Tomizaki, H. Mihara
Methods. Mol. Biol. 1352 199 - 210 2015年11月
共著
担当区分：筆頭著者
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Label and Label-Free Detection Techniques for Protein Microarrays 査読あり 国際共著
Amir Syahir, Kenji Usui, Kin-ya Tomizaki, Kotaro Kajikawa, Hisakazu Mihara
Microarrays 4 ( 4 ) 228 - 244 2015年4月
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Site-Specific Mineralization of Silica and Calcium on DNAs Using a Designed Peptide 査読あり
K. Usui, H. Nishiyama, A. Yamada, M. Ozaki, T. Tsuruoka, K.-y. Tomizaki
Peptide science 2014 325 - 326 2015年3月
共著
担当区分：筆頭著者
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Control of guanine-rich DNA secondary structures depending on the protease activity using a designed PNA peptide 査読あり
Kenji Usui, Arisa Okada, Keita Kobayashi, Naoki Sugimoto
ORGANIC & BIOMOLECULAR CHEMISTRY 13 ( 7 ) 2022 - 2025 2015年
出版者・発行元：ROYAL SOC CHEMISTRY
We constructed a regulation system for DNA secondary structure formation of G-rich sequences using a designed PNA peptide exhibiting an on-to-off switching functionality, depending on the protease activity. This study introduces the new concept of a simple and powerful system for regulating quadruplex-related important biological events.
DOI： 10.1039/c4ob02535k
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Cellular differentiation assessments by measuring the degree of cellular internalization and membrane adsorption using designed peptides 査読あり
Kenji Usui, Takuya Kikuchi, Kunio Kikuchi, Masayasu Mie, Eiry Kobatake, Hisakazu Mihara
BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 24 ( 17 ) 4129 - 4131 2014年9月
出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD
We demonstrate examples of cellular differentiation assessments, including cellular neurite outgrowth and fat cell maturation, by measuring the degree of membrane adsorption or cellular internalization using designed peptides. Because changes in the cellular membrane and cytosol during differentiation were shown to influence membrane adsorption and cellular internalization, we could successfully evaluate the extent of differentiation simply like stain indicators. (C) 2014 Elsevier Ltd. All rights reserved.
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A Designed Small Protein For Controlling Site-Specific Mineralization Of Silica And Calcium On Dnas 査読あり
Kenji Usui, Kazuma Nagai, Hiroto Nishiyama, Aoi Yamada, Makoto Ozaki, Takaaki Tsuruoka, Kin-ya Tomizaki
PROTEIN SCIENCE 23 143 - 143 2014年7月
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Site-Specific Control of Silica Mineralization on DNA Using Designed Peptides 査読あり
K. Usui, K. Nagai, H. Nishiyama, A. yamada, T. Tsuruoka, S. Fujii, K.-y. Tomizaki
Peptide Science 2013 2415 - 2416 2014年3月
共著
担当区分：筆頭著者
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Peptides targeting G-quadruplex structures 査読あり
Kenji Usui, Arisa Okada
Chemical Biology of Nucleic Acids: Fundamentals and Clinical Applications 459 - 475 2014年1月
出版者・発行元：Springer Berlin Heidelberg
Research in recent decades has revealed that some DNA and RNA secondary structures modulate a variety of cellular events. One secondary structure, the Guanine(G)-quadruplex, also regulates various cellular events that are mostly related to serious diseases. Systems capable of controlling DNA and RNA G-quadruplex structures would therefore be useful for the modulation of various cellular events to produce biological effects. Because of their biological importance, many G-quadruplex-targeting compounds have been described. However, the next generation of targeting molecules should exhibit increased G-quadruplex sequence specificity, a higher structure-inducing or -collapsing ability, and a greater degree of functionality, including on–off switches of binding ability and cellular penetration. Peptides might be good candidates for these next-generation Gquadruplex- targeting molecules due to the following advantages: (1) their easy design and synthesis, (2) their ability to mimic protein–G–quadruplex interactions, (3) the possibility of employing artificial amino acids in addition to naturally occurring amino acids, and (4) the ability to combine G-quadruplex-binding sequences with other functional sequences. Accordingly, several peptide-based compounds, such as furan-based cyclic peptides, PNA-conjugated peptides, and small molecule-peptide conjugates, have been developed. In this chapter, we introduce all these peptide ligands and describe most of the approaches for targeting G-quadruplex structures. We then conclude that peptides are among the most promising functional ligands for G-quadruplexes to control various biological events in next-generation approaches.
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Control of Site-Specific Silica Precipitation Using PNA Peptides and DNAs 査読あり
K. Usui, H. Nishiyama, K. Nagai, T. Tsuruoka, S. Fujii, K.-y. Tomizaki
Peptides 2013 162 - 163 2013年12月
共著
担当区分：筆頭著者
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Designed Peptide Libraries for Cell Analyzing Microarrays 査読あり
H. Mihara, K. Usui, H. Tsutsumi, K. Nokihara
Peptides 2013 22 - 23 2013年12月
共著
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Peptide-DNA Hybrid G-Quadruplex Structures for Regulation of Protein Expression Depending on Protease Activity 査読あり
A. Okada, M. Taniguchi, K. Usui, N. Sugimoto
Peptides 2013 180 - 181 2013年12月
共著
担当区分：筆頭著者
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Systematic screening of the cellular uptake of designed alpha-helix peptides 査読あり
Kenji Usui, Takuya Kikuchi, Masayasu Mie, Eiry Kobatake, Hisakazu Mihara
BIOORGANIC & MEDICINAL CHEMISTRY 21 ( 9 ) 2560 - 2567 2013年5月
出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD
The cellular penetration (CP) activity of functional molecules has attracted significant attention as one of the most promising new approaches for drug delivery. In particular, cell-penetrating peptides (CPPs) have been studied extensively in cellular engineering. Because there have been few large-scale systematic studies to identify peptide sequences with optimal CP activity or that are suitable for further applications in cell engineering, such as cell-specific penetration and cell-selective culture, we screened and compared the cellular uptake (CU) activity of 54 systematically designed a-helical peptides in HeLa cells. Furthermore, the CU activity of 24 designed peptides was examined in four cell lines using a cell fingerprinting technique and statistical approaches. The CU activities in various cells depended on amino acid residues of peptide sequences as well as charge, a-helical content and hydrophobicity of the peptides. Notably, the mutation of a single residue significantly altered the CU ability of a peptide, highlighting the variability of cell uptake mechanisms. Moreover, these results demonstrated the feasibility of cell-selective culture by conducting cell-selective permeation and death in cultures containing two cell types. These studies may lead to further peptide library design and screening for new classes of CPPs with useful functions. (C) 2013 Elsevier Ltd. All rights reserved.
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T. Kakiyama, K. Usui, K.-y. Tomizaki, M. Mie, E. Kobatake, H. Mihara
Polym. J. 45 ( 5 ) 525 - 539 2013年5月
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Construction of Artificial Peptides for Control of DNA G-quadruplex Structures Depending on Protease Activity toward Regulation of Protein Expression 査読あり
Arisa Okada, Kenji Usui, Mai Taniguchi, Keita Kobayashi, Naoki Sugimoto
Peptide Science 2012 285 - 286 2013年3月
共著
担当区分：筆頭著者
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A PNA Peptide for the Control of Site-Specific Silica Precipitation on DNA 査読あり
Kenji Usui, Kazuma Nagai, Hiroto Nishiyama, Takaaki Tsuruoka, Satoshi Fujii
Peptide Science 2012 375 - 376 2013年3月
共著
担当区分：筆頭著者
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A novel array format for monitoring cellular uptake using a photo-cleavable linker for peptide release 査読あり
Kenji Usui, Takuya Kikuchi, Kin-ya Tomizaki, Takashi Kakiyama, Hisakazu Mihara
CHEMICAL COMMUNICATIONS 49 ( 57 ) 6394 - 6396 2013年
出版者・発行元：ROYAL SOC CHEMISTRY
We developed a novel peptide array format incorporating a photo-cleavable linker for monitoring cellular uptake. Model peptides were successfully immobilised via the photo-cleavable linker onto conventional plates and could be released spatiotemporally using UV irradiation. Incorporation of confocal microscopy allowed for detailed real-time monitoring of cellular internalisation of peptides.
DOI： 10.1039/c3cc41632a
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Screening of Designed Peptides for Binders to a DNA G-Quadruplex Structure 査読あり
N. Matsui, K. Kobayashi, K. Usui
Peptide Science 2011 2011 315 - 316 2012年3月
共著
担当区分：筆頭著者
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De Novo Designed Nitric Oxide Sensor Protein consisted of Iron Complex-Peptide Conjugate 査読あり
H. Miyazaki, K. Usui, S. Fujii
Peptide Science 2011 2011 303 - 304 2012年3月
共著
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Development of PNA-Peptides Controlling DNA G-Quadruplex Structures Depending on Protease Activity 査読あり
K. Usui, K. Kobayashi, N. Sugimoto
Peptide Science 2011 2012 315 - 316 2012年3月
共著
担当区分：筆頭著者
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Cell fingerprint patterns using designed α-helical peptides to screen for cell-specific toxicity. 査読あり
Usui K, Kakiyama T, Tomizaki KY, Mie M, Kobatake E, Mihara H
Bioorganic & medicinal chemistry letters 21 ( 21 ) 6281 - 6284 2011年11月
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Thermodynamic stability of Hoogsteen and Watson-Crick base pairs in the presence of histone H3-mimicking peptide 査読あり 国際共著
Smritimoy Pramanik, Kaori Nakamura, Kenji Usui, Shu-ichi Nakano, Sarika Saxena, Jun Matsui, Daisuke Miyoshi, Naoki Sugimoto
CHEMICAL COMMUNICATIONS 47 ( 10 ) 2790 - 2792 2011年
出版者・発行元：ROYAL SOC CHEMISTRY
We found that Hoogsteen base pairs were stabilized by molecular crowding and a histone H3-mimicking peptide, which was not observed for Watson-Crick base pairs. Our findings demonstrate that the type of DNA base pair is critical for the interaction between DNA and histones.
DOI： 10.1039/c0cc05776b
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Use of a designed peptide library to screen for binders to a particular DNA G-quadruplex sequence 査読あり
Keita Kobayashi, Noriko Matsui, Kenji Usui
Journal of Nucleic Acids 2011 572873 2011年
We demonstrated a method to screen for binders to a particular G-quadruplex sequence using easily designed short peptides consisting of naturally occurring amino acids and mining of binding data using statistical methods such as hierarchical clustering analysis (HCA). Despite the small size of the library used in this study, candidates of specific binders were identified. In addition, a selected peptide stabilized the G-quadruplex structure of a DNA oligonucleotide derived from the promoter region of the protooncogene c-MYC. This study illustrates how a peptide library can be designed and presents a screening guideline for construction of G-quadruplex binders. Such G-quadruplex peptide binders could be functionally modified to enable switching, cellular penetration, and organelle-targeting for cell and tissue engineering. © 2011 Keita Kobayashi et al.
DOI： 10.4061/2011/572873
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PNA-Peptide Conjugates for Regulation of DNA and RNA G-Quadruplex Structures Depending on a Particular Protease Concentration 査読あり
Kenji Usui, Keita Kobayashi, Naoki Sugimoto
Peptides 2010 2010 608 - 609 2011年
共著
担当区分：筆頭著者
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Nanomolar aggregation of amyloid-beta peptides covalently and site-specifically modified by cholesterol oxidation products 査読あり 国際共著
Kenji Usui, Evan T. Powers, Johan F. Paulsson, Sarah J. Siegel & Jeffery W. Kelly
Peptides 2008 2008 588 - 589 2011年
共著
担当区分：筆頭著者
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Fluorescent and Luminescent Fusion Proteins for Detection of Amyloid Beta Peptide Localization and Aggregation 査読あり
Kenji Usui, Masayasu Mie, Takashi Andou, Naoki Sugimoto, Hisakazu Mihara, Eiry Kobatake
Peptides 2010 2010 488 - 489 2011年
共著
担当区分：筆頭著者
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Designed Peptide Libraries for Protein and Cell analyses 査読あり
K. Kikuchi, K. Usui, K.-Y. Tomizaki, T. Takahashi, H. Mihara
Peptide Science 2010 2010 21 - 21 2011年
共著
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Nanomolar Aggregation and Neurotoxicity of Amyloid beta Peptides with Site-Specific Modification of Oxidized Cholesterol 査読あり 国際共著
K. Usui, J. D. Hulleman, J. F. Paulsson, S. J. Siegel, E. T. Powers, J. W. Kelly
Peptide Science 2010 2010 169 - 169 2011年
共著
担当区分：筆頭著者
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A PNA Peptide Inducing DNAs to Form G-Quadruplex Structures Depending on a Protease Activity 査読あり
Kenji Usui, Keita Kobayashi, Naoki Sugimoto
Peptides 2011 2011 152 - 153 2011年
共著
担当区分：筆頭著者
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A Novel Nitric Oxide Sensor Using Fluorescent Peptides Attached to Iron Complexes 査読あり
Hiroshi Miyazaki, Kenji Usui, Satoshi Fujii
Peptides 2011 2011 224 - 255 2011年
共著
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Construction of Detection System for Amyloid beta Peptide Localization and Aggregation Using Fluorescent and Luminescent Fusion Proteins 査読あり
Kenji Usui, Masayasu Mie, Takashi Andou, Naoki Sugimoto, Hisakazu Mihara, Eiry Kobatake
Peptide Science 2009 2009 111 - 112 2010年
共著
担当区分：筆頭著者
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Site-specific modification of Alzheimer's peptides by cholesterol oxidation products enhances aggregation energetics and neurotoxicity 査読あり 国際共著
Kenji Usui, John D. Hulleman, Johan F. Paulsson, Sarah J. Siegel, Evan T. Powers, Jeffery W. Kelly
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 106 ( 44 ) 18563 - 18568 2009年11月
出版者・発行元：NATL ACAD SCIENCES
Accumulation of amyloid beta-peptide (A beta) and tau aggregates, possibly linked to age-associated deficiencies in protein homeostasis, appear to cause Alzheimer's disease. Schiff-base formation between A beta and the aldehyde-bearing cholesterol oxidation product 3-beta-hydroxy-5-oxo-5,6-secocholestan-6-al is known to increase A beta amyloidogenicity. Here, we synthesized A beta variants site-specifically modified with the cholesterol aldehyde at Asp-1, Lys-16, or Lys-28, rather than studying mixtures. These distinct modifications have a similar effect on the thermodynamic propensity for aggregation, enabling aggregation at low concentrations. In contrast, the modification site differentially influences the aggregation kinetics; Lys-16-modified A beta formed amorphous aggregates fastest and at the lowest concentration (within 2 h at a concentration of 20 nM), followed by the Lys-28 and Asp-1 conjugates. Also, the aggregates resulting from A beta Lys-16 cholesterol aldehyde conjugation were more toxic to primary rat cortical neurons than treatment with unmodified A beta under identical conditions and at the same concentration. Our results show that A beta modification by cholesterol derivatives, especially at Lys-16, renders it kinetically and thermodynamically competent to form neurotoxic aggregates at concentrations approaching the physiologic concentration of A beta.
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A designed peptide chip: Protein fingerprinting technology with a dry peptide array and statistical data mining 査読あり
Kenji Usui, Kin-Ya Tomizaki, Hisakazu Mihara
Methods in Molecular Biology 570 273 - 284 2009年
There has recently been increased interest in the potential for microarray technologies to study protein networks in a whole cell system within a single experiment. Protein-detecting microarrays are composed of numerous agents immobilized within a tiny area on solid surfaces to capture targeted proteins and to detect interactions in a high-throughput fashion. In this chapter, in order to extend the usability of peptide microarrays, we describe a novel dry peptide microarray format to obtain protein fingerprint (PFP) data sets and a statistical PFP data manipulation technique to quantitatively analyze targeted proteins. © 2009 Humana Press, a part of Springer Science+Business Media, LLC.
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Screening of alpha-helical peptide ligands controlling a calcineurin-phosphatase activity 査読あり
Kenji Usui, Kin-ya Tomizaki, Hisakazu Mihara
BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 17 ( 1 ) 167 - 171 2007年1月
出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD
In this paper, we describe an application of 202-membered fluorescently labeled peptide library designed to take an alpha-helix secondary structure. As a proof-of-concept experiment, a calmodulin (CaM)/calcineurin (Cn) pair was chosen to screen alpha-helical peptide ligands that tightly bind to CaM and also control enzymatic functions of Cn. Three peptides were successfully selected from the library by assaying Cn-phosphatase activities and peptide-CaM interactions (dual check process). The strategy using a designed peptide. library shows real promise as a peptide-based high-throughput screening system. (c) 2006 Elsevier Ltd. All rights reserved.
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Interactions between Peptides Containing Nucleobase Amino Acids and T7 Phages Displaying S. Cerevisiae Proteins 査読あり
S. Watanabe, K.-Y. Tomizaki, T. Takahashi, K. Usui, K. Kajikawa and H. Mihara
Biopolymers 88 ( 2 ) 131 - 140 2007年
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Protein-fingerprint data mining of a designed alpha-helical peptide array 査読あり
Kenji Usui, Kin-ya Tomizaki, Hisakazu Mihara
MOLECULAR BIOSYSTEMS 2 ( 9 ) 417 - 420 2006年9月
出版者・発行元：ROYAL SOC CHEMISTRY
The data generated from protein fingerprints with an a-helical peptide array were analyzed using several statistical methods such as hierarchical clustering analysis and principal component analysis to discriminate target proteins. The data generated from protein fingerprints with an a-helical peptide array were analyzed using several statistical methods such as hierarchical clustering analysis and principal component analysis to discriminate target proteins.
DOI： 10.1039/b608875a
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A novel peptide microarray for protein detection and analysis utilizing a dry peptide array system 査読あり
K Usui, K Tomizaki, T Ohyama, K Nokihara, H Mihara
MOLECULAR BIOSYSTEMS 2 ( 2 ) 113 - 121 2006年2月
出版者・発行元：ROYAL SOC CHEMISTRY
A novel dry peptide microarray system has been constructed that affords a practical solution for protein detection and analysis. This system is an array preparation and assay procedure tinder dry conditions that uses designed peptides as non-immobilized capture agents for the detection of proteins. The system has several advantages that include its portability and ease-Of-Use, as well as the fact that vaporization of sample solutions need not be considered. In this study, various proteins have been characterized with an a-helical peptide mini-library. When proteins were added to the peptide library array, the fluorescent peptides showed different fluorescent intensities depending on their sequences. The patterns of these responses could be regarded as protein fingerprints' (PFPs), which are sufficient to establish the identities of the target proteins. Furthermore, statistical analysis of the resulting PFPs was performed using cluster analysis. The PFPs of the proteins were clustered successfully depending on their families and binding properties. Additionally, the target protein was characterized using a nanolitre system and could be detected down to 1.2 fmol. These Studies imply that the dry peptide array system is a promising too] for detecting and analyzing target proteins. The dry peptide array will play a role in development of high-throughput protein-detecting nano/micro arrays for proteomics and ligand screening studies.
DOI： 10.1039/b514263r
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A PNA-DNA hybridization chip approach for the detection of beta-secretase activity 査読あり
S Sano, KY Tomizaki, K Usui, H Mihara
BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 16 ( 3 ) 503 - 506 2006年2月
出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD
Developed was the addressable chip technology based on the PNA-DNA complementary hybridization equipped with short seven-mer PNA-encoded peptides that can be a versatile scaffold to monitor on-chip immunoassays. We also developed and validated a methodology to perform beta-secretase enzyme assay with a highly sensitive fashion, resulting that a peptide substrate tethering dual fluorescent probes allowed us to detect beta-secretase activity 10 times more sensitively than assays in solution. (c) 2005 Elsevier Ltd. All rights reserved.
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Designed peptide microarrays for protein detection and characterization 査読あり
Kenji Usui, Kin-ya Tomizaki, Kiyoshi Nokihara, Hisakazu Mihara
UNDERSTANDING BIOLOGY USING PEPTIDES 731 - + 2006年
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High throughput preparation of peptide arrays containing two fluorescent byes focusing on practical protein detection systems 査読あり
Kiyoshi Nokihara, Takafumi Ohyama, Koichi Yonemura, Yasuo Oka, Kenji Usui, Hisakazu Mihara
UNDERSTANDING BIOLOGY USING PEPTIDES 738 - + 2006年
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Anomalous reflection of gold applicable for a practical protein-detecting chip platform 査読あり
S Watanabe, K Usui, K Tomizaki, K Kajikawa, H Mihara
MOLECULAR BIOSYSTEMS 1 ( 5-6 ) 363 - 365 2005年12月
出版者・発行元：ROYAL SOC CHEMISTRY
A simple, convenient and label-free fiber optic detection system based on the characteristic property, 'anomalous reflection (AR)' of gold was developed and preliminary experiments showed that the AR signals were sensitive enough to monitor protein-peptide interactions on solid surfaces.
DOI： 10.1039/b513075c
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Kenji Usui, Tetsunori Ojima, Kin-Ya Tomizaki, Hisakazu Mihara
Nanobiotechnology 1 ( 2 ) 191 - 199 2005年11月
For the realization of a practical high-throughput protein detection and analysis system, a novel peptide array has been constructed using a designed glycopeptide model library with an α-helical secondary structure. This study will contribute the increment of the diversity of such an array system and the application to focused proteomics and ligand screening by effective detection of sugar-binding proteins. Fluorescent glycopeptides with an α-helix, a β-strand, or a loop structure were designed initially to select a suitable scaffold for the detection of a model protein. After selection of the α-helical structure as the best scaffold, a small model library with various saccharides was constructed to have charge and hydrophobicity variations in the peptide sequences. When various sugar-binding proteins were added to the peptide library array, the fluorescent peptides showed different responses in fluorescence intensities depending on their sequences as well as saccharides. The patterns of these responses could be regarded as "protein fingerprints" (PFPs), which are able to establish the identities of the target proteins. The resulting PFPs reflected the recognition properties of the proteins. Furthermore, statistical data analysis from obtained PFPs was performed using a cluster analysis. The PFPs of sugar-binding proteins were clustered successfully depending on their families and binding properties. These studies demonstrate that arrays with glycopeptide libraries based on designed structures can be promising tools to detect and analyze the target proteins. Designed peptides with functional groups such as sugars will play roles as the capturing agents of high-throughput protein nano/micro arrays for focused proteomics and ligand screening studies. Copyright © 2005 Humana Press Inc. All rights of any nature whatsoever are reserved.
DOI： 10.1385/NBT:1:2:191
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Difference in self-assembling morphology of peptide nanorings 査読あり
H Okamoto, T Yamada, H Miyazaki, T Nakanishi, K Takeda, K Usui, Obataya, I, H Mihara, H Azehara, W Mizutani, K Hashimoto, H Yamaguchi, Y Hirayama
JAPANESE JOURNAL OF APPLIED PHYSICS PART 1-REGULAR PAPERS BRIEF COMMUNICATIONS & REVIEW PAPERS 44 ( 11 ) 8240 - 8248 2005年11月
出版者・発行元：JAPAN SOC APPLIED PHYSICS
We synthesized the peptide nanorings of cyclo[-(D-Ala-L-Gln)(3)], cyclo[-(D-CyS-L-Gln)(3)], CyC10[-D-Cys-L-HiS-D-Ala-L-Asn-Gly-L-Gln-1 and Cyc1o[-(L-Gln)(5)], and studied the way in which the difference in the type and/or number of component amino acid residues changes the self-assembling morphology of the nanorings on gold substrates by atomic force microscopy. The study revealed that CyClo[-(D-Ala-L-Gln)(3)] formed nanotube bundles through inter-ring hydrogen bonds, while the nanorings of CyC10[-(D-CyS-L-Gln)3] adhered to the gold surface directly due to the high affinity of thiol to gold. In contrast, a random amino acid sequence of cyclo[-D-CyS-L-HiS-D-Ala-L-Asn-GlY-L-Gln-] resulted in many isolated nanotubes, which were first observed in the present study. While the D,L-peptide nanotubes have very straight forms, the homo-L-peptide of cyclo[-(L-Gln)(5)] formed interesting randomly branching nanotubes that were entwined and grew on the substrate. Scanning tunneling microscopy was also performed and high-resolution images of both the peptide nanotubes and the nanotube bundles were obtained.
DOI： 10.1143/JJAP.44.8240
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IR study on stacking manner of peptide nanorings in peptide nanotubes 査読あり
Y Nagai, T Nakanishi, H Okamoto, K Takeda, Y Furukawa, K Usui, H Mihara
JAPANESE JOURNAL OF APPLIED PHYSICS PART 1-REGULAR PAPERS BRIEF COMMUNICATIONS & REVIEW PAPERS 44 ( 10 ) 7654 - 7661 2005年10月
出版者・発行元：JAPAN SOC APPLIED PHYSICS
We here report our theoretical as well as experimental studies on the stacking manner of peptide nanorings (PNRs) in peptide nanotubes (PNTs). We focus on the molecular vibrations of N-H and C=O stretching modes and discuss this subject via their infrared (IR) spectroscopy, because PNTs are formed by the inter-ring H bonds between the adjacent PNRs via -N-H(...)O=C-. Symmetry analysis based on group theory reveals that parallel stacking causes two IR active modes in these molecular vibrations while three modes are active in the antiparallel stacking. This difference in the number of IR active modes is determined only by the stacking manner and not by the number of amino acid residues composing the PNRs. By using two typical PNRs Of cyclo[-((L)-Gln-D-Ala)(3)] and cyclo[-((L)-Gln-(D)-Ala)(4)], we further studied the favorable stacking manners of PNRs via IR observation. Our IR experiments as well as the ab initio energetics show that the former PNRs create a PNT by stacking themselves in parallel while the latter PNRs do so by stacking themselves in an antiparallel manner.
DOI： 10.1143/JJAP.44.7654
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Protein-Detecting Microarrays: Current Accomplishments and Requirements 査読あり
Kin-ya Tomizaki, Kenji Usui, Hisakazu Mihara
ChemBioChem 6 ( 6 ) 782 - 799 2005年5月
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Studies on Fluorescent Dyes and Surface Chemistry focusing on Practical Peptide Array Preparation 査読あり
NOKIHARA Kiyoshi, USUI Kenji, YONEMURA Koichi, OHYAMA Takafumi, OKA Yasuo, TOMIZAKI Kin‐ya, MIHARA Hisakazu
Pept Sci 2004 145 - 148 2005年3月
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Optimization of peptide arrays on chip as novel practical protein characterization system 査読あり
Kiyoshi Nokihara, Takafumi Ohyama, Koichi Yonemura, Kenji Usui, Kin-ya Tomizaki, Hisakazu Mihara
Peptides 2004, Proceedings 176 - 177 2005年
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Protein-detection microarrays using structure-based designed peptide libraries 査読あり
Kenji Usui, Tetsunori Ojima, Masato Suzuki, Sin-ya Watanabe, Kin-ya Tomizaki, Kiyoshi Nokihara, Hisakazu Mihara
Peptides 2004, Proceedings 401 - 402 2005年
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Peptide Arrays with Designed Secondary Structures for Protein Characterization Using Fluorescent Fingerprint Patterns. 査読あり
臼井健二
Biopolymers ( 76 ) 129 - 139 2004年11月
単著
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Peptide arrays with designed alpha-helical structures for characterization of proteins from FRET fingerprint patterns 査読あり
Kenji Usui, Mizuki Takahashi, Kiyoshi Nokihara, Hisakazu Mihara
MOLECULAR DIVERSITY 8 ( 3 ) 209 - 218 2004年9月
出版者・発行元：SPRINGER
A practical high-throughput protein detection system is described, based on synthetic peptide arrays consisting of designed alpha-helical peptides, detected by fluorescence resonance energy transfer (FRET). Initially a model alpha-helical peptide known to interact with a structured protein, calmodulin, was selected to establish the strategy for high-throughput detection. In comparison to peptides with a single probe, a much higher FRET response has been observed with two fluorescent probes (7-diethylaminocoumarin-3-carboxylic acid and 5(6)-carboxy-fluorescein) at both termini of the synthetic peptides. To establish a reproducible high-throughput detection system, peptides were also immobilized onto a solid surface for detection of the target proteins. A small library of 112 different peptides was constructed, based on a model of the alpha-helical peptide with systematic replacement of residues carrying specific charges and/or hydrophobicities. The library was used to effectively characterize various proteins, giving their own 'protein fingerprint' patterns. The resulting 'protein fingerprints' correlate with the recognition properties of the proteins. The present microarray with designed synthetic peptides as the capturing agents is promising for the development of protein detection chips.
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Development of a practical protein-chip using designed synthetic peptide-arrays 査読あり
K Nokihara, T Ohyama, K Usui, K Yonemura, K Tomizaki, H Mihara
KOBUNSHI RONBUNSHU 61 ( 10 ) 523 - 532 2004年
出版者・発行元：SOC POLYMER SCIENCE JAPAN
Novel high-throughput technologies, which can replace conventional electrophoresis and mass spectroscopic analyses, are in great demand for understanding the structure-function relationships of proteins. For a practical protein-detection system, arrays with immobilized designed synthetic peptides have been constructed. Peptides were labeled with fluorescent dyes in order to achieve high sensitivity. Two different types of prototype-arrayer have been constructed: one has a micro-dispensing system and the other a spot-printing system. Combinatorial peptide libraries, which consisted of beta-loop, beta-strand and alpha-helical peptides, were constructed by improved highly efficient solid-phase synthesis. Labeled peptides were covalently immobilized on solid supports such as precision glass plates. Various proteins were characterized with these peptide-arrays using a fluorescent scanner to give the "protein fingerprint" which is characteristic of the individual proteins. The results of the present prototype system demonstrate the practicality of protein-chips as a new generation of biochips.
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Peptide arrays with designed secondary structures for protein characterization using fluorescent fingerprint patterns 査読あり
K Usui, T Ojima, M Takahashi, K Nokihara, H Mihara
BIOPOLYMERS 76 ( 2 ) 129 - 139 2004年
出版者・発行元：JOHN WILEY & SONS INC
To realize a practical high-throughput protein-detection system, novel peptide arrays have been constructed using designed peptide libraries with loop, a-helix, or P-strand structures. Here, we describe the overview of the reported deigned peptide arrays with loop and a-helix structures and the new results of those with beta-strand structures. Initially, several model peptides known to interact with model structured proteins were selected to establish the present strategy for high-throughput detection of proteins. The fluorescent probes and suitable scaffolds of peptides were examined for the effective detection of proteins. The detection methods were established in solution and in an immobilized manner using the model systems. In the case of a-helix peptide, the response of a peptide with fluorescent resonance energy tran, fer between two probes at both termini was several times higher than that of a peptide with a single probe. In the cases of peptides with other structures, however, proteins were effectively detectable even by the fluorescent change of one probe. Furthermore, structurally focused libraries consisting of a total of ca. 250 different peptides based on the model pcptides with secondary and/or tertiary structures were constructed with systematic replacement of residues. Using these libraries, various proteins were characterized effectively to give their own fluorescent "protein fingerprint" patterns. The resulting protein fingerprints correlated with the recognition properties of the proteins. These studies demonstrate that arrays with peptide libraries based on designed structures can be promising tools for detecting the target proteins. Designed synthetic peptides play roles as the capturing agents to be developed for practical protein chips. (C) 2004 Wiley Periodicals, Inc.
DOI： 10.1002/bip.10568
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Development of Peptide-Chips Focusing on a High Throughput Protein-Detection System 査読あり
Kiyoshi Nokihara, Takafumi Ohyama, Kenji Usui, Koichi. Yoneyama, Mizuki Takahashi, Hisakazu Mihara
Innovation and Perspectives in Solid Phase Synthesis & Combinatorial Libraries 83 - 88 2004年
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Peptide microarrays using structure-based peptide libraries for protein chips 査読あり
Kenji Usui, Mizuki Takahashi, Tetsunori Ojima, Masato Suzuki, Kiyoshi Nokihara, Eiichi Tamiya, Hisakazu Mihara
Peptide Revolution: Genomics, Proteomics & Therapeutics 258 - 259 2004年