2018年05月 第9回日本光合成学会年会, 優秀研究賞, パターン化人工生体膜を用いた光合成機構の再構成

米田 卓郎, 谷本 泰士, 高木 大輔, 森垣 憲一

国内学会・会議・シンポジウム等の賞

2009年10月 第82回日本生化学会大会, 優秀プレゼンテーション賞, ケージド化合物を用いたP450酵素活性の光制御

水谷 和幸, 森垣 憲一, 達 吉郎, 一色 邦夫, 今石 浩正

国内学会・会議・シンポジウム等の賞

Sophie A. Meredith, Takuro Yoneda, Ashley M. Hancock, Simon D. Connell, Stephen D. Evans, Kenichi Morigaki, Peter G. Adams

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研究論文（学術雑誌）

Sin-Ichi Odagaki, Shohei Maekawa, Fumio Hayashi, Toshinobu Suzaki, Kenichi Morigaki

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研究論文（学術雑誌）

Takuro Yoneda, Yasushi Tanimoto, Daisuke Takagi, Kenichi Morigaki

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研究論文（学術雑誌）

Yukito Kaneshige, Fumio Hayashi, Kenichi Morigaki, Yasushi Tanimoto, Hayato Yamashita, Masashi Fujii, Akinori Awazu

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研究論文（学術雑誌）

Kazuma Yasuhara, Kenichi Morigaki

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研究論文（学術雑誌）

Fuyuko Tamura, Yasushi Tanimoto, Rurika Nagai, Fumio Hayashi, Kenichi Morigaki

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研究論文（学術雑誌）

Hayashi F, Saito N, Tanimoto Y, Okada K, Morigaki K, Seno K, Maekawa S

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研究論文（学術雑誌）

Nomura K, Yamaguchi T, Mori S, Fujikawa K, Nishiyama K, Shimanouchi T, Tanimoto Y, Morigaki K, Shimamoto K

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研究論文（学術雑誌）

Tanabe M, Ando K, Komatsu R, Morigaki K

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研究論文（学術雑誌）

Kinoshita M, Suzuki K, Matsumori N, Takada M, Ano H, Morigaki K, Abe M, Makino A, Kobayashi T, Hirosawa K, Fujiwara T, Kusumi A, Murata M

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研究論文（学術雑誌）

Morigaki, Kenichi, Nishimura, Toshiki, Tamura, Fuyuko, Tanimoto, Yasushi, Ando, Koji, Sudo, Yuki, Hayashi, Fumio, Iwasaki, Yasuhiko

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研究論文（学術雑誌）

Nishimura, Toshiki, Tamura, Fuyuko, Kobayashi, Sawako, Tanimoto, Yasushi, Hayashi, Fumio, Sudo, Yuki, Iwasaki, Yasuhiko, Morigaki, Kenichi

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研究論文（学術雑誌）

Kaoru Nomura, Yasushi Tanimoto, Fumio Hayashi, Erisa Harada, Xiao-Yuan Shan, Masafumi Shionyu, Atsushi Hijikata, Tsuyoshi Shirai, Kenichi Morigaki, Keiko Shimamoto

Prod1 is a protein that regulates limb regeneration in salamanders by determining the direction of limb growth. Prod1 is attached to the membrane by a glycosylphosphatidylinositol (GPI) anchor, but the role of membrane anchoring in the limb regeneration process is poorly understood. In this study, we investigated the functional role of the anchoring of Prod1 to the membrane by using its synthetic mimics in combination with solid-state NMR spectroscopy and fluorescent microscopy techniques. Anchoring did not affect the three-dimensional structure of Prod1 but did induce aggregation by aligning the molecules and drastically reducing the molecular motion on the two-dimensional membrane surface. Interestingly, aggregated Prod1 interacted with Prod1 molecules tethered on the surface of opposing membranes, inducing membrane adhesion. Our results strongly suggest that anchoring of the salamander-specific protein Prod1 assists cell adhesion in the limb regeneration process.

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研究論文（学術雑誌）

Koji Ando, Masashi Tanabe, Kenichi Morigaki

The biological membrane is a natural biosensing platform that can detect specific molecules with extremely high sensitivity. We developed a biosensing methodology by combining a model biological membrane and a nanometer-sized gap structure on a glass substrate. The model membrane comprised lithographically patterned polymeric and fluid lipid bilayers. The polymeric bilayer was bonded to a poly(dimethylsiloxane) (PDMS) sheet by using an adhesion layer with a defined thickness (lipid vesicles). Extruded lipid vesicles having a biotin moiety on the surface were used as the adhesion layer in conjunction with the biotin streptavidin linkage. A gap structure was formed between the fluid bilayer and PDMS (nanogap junction). The thickness of the gap structure was several tens of nanometers, as determined by the thickness of the adhesion layer. The nanogap junction acted as a sensitive biosensing platform. From a mixture of proteins (cholera toxin and albumin), the target protein (cholera toxin) was selectively transported into the gap by the specific binding to a glycolipid (GMI) in the fluid bilayer and lateral diffusion. The target protein molecules were then detected with an elevated signal-to-noise ratio due to the reduced background noise in the nanometric gap. The combination of selective transport and reduced background noise drastically enhanced the sensitivity toward the target protein. The nanogap junction should have broad biomedical applications by realizing highly selective and sensitive biosensing in samples having diverse coexisting molecules.

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研究論文（学術雑誌）

Yasushi Tanimoto, Keisuke Okada, Fumio Hayashi, Kenichi Morigaki

Lipid rafts in the cell membrane are believed to affect various membrane functions, including the signaling by G-protein coupled receptors (GPCRs). However, the regulatory roles of lipid rafts on GPCRs' functions are still poorly understood, partially owing to the lack of the methods to quantitatively evaluate the affinity of membrane proteins to lipid raft (raftophilicity). Here, we describe a methodology to gauge the raftophilicity of a representative GPCR in vertebrate photoreceptor, i.e., rhodopsin (Rh), and its cognate G protein transducin (G(t)) by using a patterned model membrane. We generated a substrate-supported planar lipid bilayer that has patterned regions of liquid-ordered (L-0) and liquid-disordered (L-d) membrane domains. We reconstituted Rh and Gt into the patterned membrane and observed their lateral distribution and diffusion. Mobile and functional Rh molecules could be reconstituted through the rapid dilution of solubilized Rh, by optimizing the reconstitution conditions including the chamber design, protein/detergent concentrations, and solution mixing. We determined the partition and diffusion coefficients of Rh and Gt in the L-0-rich and L-d-rich regions. Both Rh and G(t) were predominantly localized in the L-d phase, suggesting their low affinity to lipid rafts. Patterned model membrane offers a robust and scalable platform for systematically and quantitatively studying the functional roles of lipid rafts in biological membranes including retinal disk membranes.

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研究論文（学術雑誌）

Kenji Sasahara, Kenichi Morigaki, Yasuko Mori

Amyloid aggregation and deposition of amyloid beta-peptide (A beta) are pathologic characteristics of Alzheimer's disease (AD). Recent reports have shown that the association of A beta with membranes containing ganglioside GM1 (GM 1) plays a pivotal role in amyloid deposition and the pathogenesis of AD. However, the molecular interactions responsible for membrane damage associated with A beta deposition are not fully understood. In this study, we microscopically observed amyloid aggregation of A beta in the presence of lipid vesicles and on a substrate-supported planar membrane containing raft components and GM1. The experimental system enabled us to observe lipid-associated aggregation of A beta, uptake of the raft components into A beta aggregates, and relevant membrane damage. The results indicate that uptake of raft components from the membrane into A beta deposits induces macroscopic heterogeneity of the membrane structure. (C) 2015 Elsevier Inc. All rights reserved.

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研究論文（学術雑誌）

Kuroda, Keita, Miyoshi, Hiromi, Fujii, Shota, Hirai, Tomoyasu, Takahara, Atsushi, Nakao, Aiko, Iwasaki, Yasuhiko, Morigaki, Kenichi, Ishihara, Kazuhiko, Yusa, Shin-ichi

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研究論文（学術雑誌）

Fumiko Okada, Kenichi Morigaki

The localization of lipids and proteins in microdomains (lipid rafts) is believed to play important functional roles in the biological membrane. Herein, we report on a micropatterned model membrane that mimics lipid rafts by quantitatively controlling the spatial distribution of lipid phases. We generated a composite membrane of polymeric and fluid lipid bilayers by lithographic polymerization of diacetylene phospholipid(1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine: DiynePC). The composite membrane comprised polymer free-region (R-0), partially polymerized region (R-1), and fully polymerized region (R-2). As a ternary mixture of saturated lipid, unsaturated lipid, and cholesterol was introduced into the voids between polymeric bilayers, liquid-ordered (L-o) and liquid-disordered (L-d) lipid phases were accumulated in R-0 and R-1, respectively. Local enrichment of L-d phase in R-1 (and L-o phase in R-0) was enhanced with a heightened coverage of polymeric bilayer in R-1, supporting the premise that polymeric bilayer domains are inducing the phase separation. The pattern geometry (the area fractions of R-0 and R-1) also affected the enrichment due to the balance of gross L-o/L-d area fractions. Therefore, we could control the local L-o/L-d ratios by modulating the pattern geometry and polymer coverage in R-1. Micropatterned model membrane with quantitatively controlled distribution of L-o/L-d phases offers a new tool to study the functional roles of lipid rafts by enabling to separate membrane-bound molecules according to their affinities to L-o and L-d phases.

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研究論文（学術雑誌）

Kenji Sasahara, Kenichi Morigaki, Kyoko Shinya

Amyloid deposition of human islet amyloid polypeptide (hIAPP) within the islets of Langerhans is a pathological feature of type 2 diabetes mellitus. Substantial evidence indicates that the membrane-mediated aggregation and subsequent deposition of hIAPP are linked to dysfunction and death of pancreatic -cells, but the molecular processes of hIAPP deposition are poorly understood. In this study, we examined the membrane-mediated aggregation and deposition of hIAPP at supported planar lipid bilayers with and without raft components (i.e. cholesterol and sphingomyelin) to provide insight into hIAPP-induced membrane dysfunction. The adsorption of hIAPP onto the bilayers was studied using a quartz crystal microbalance with dissipation monitoring, which showed enhanced accumulation of the peptide onto the bilayer containing raft components. Microscope observations demonstrated the growth of the aggregates formed from the membrane-adsorbed hIAPP. The examination of the membrane interfaces revealed that hIAPP aggregates retained the ability to associate with the membranes during the aggregation process, resulting in insertion of the aggregates into the bilayers. We also report the inhibitory effect of insulin on the hIAPP deposition. These findings demonstrate the aggregation of hIAPP at the membrane interfaces leading to amyloid deposits associated with the membrane and suggest a role for insulin in hIAPP deposition. A presumed mechanism regulating hIAPP deposition at the membrane interfaces is discussed.

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研究論文（学術雑誌）

Masahiro Fujiwara, Kumi Shiokawa, Takayuki Kubota, Kenichi Morigaki

The encapsulation of fluorescent organic molecules into crystalline calcium carbonate was examined using calcium carbonate microcapsule, whose crystalline phase is vaterite as a metastable phase of calcium carbonate. A calcium carbonate microcapsule with impregnated pyrene that is a water insoluble fluorescent molecule was soaked into suitable aqueous solutions to promote the phase transition of vaterite toward calcite as the stable phase of calcium carbonate. When 0.2 M calcium chloride solution was used, the largest amount of pyrene (approximately 0.06 wt%) was encapsulated into the calcite particle. Pyrene thus included was not eliminated even after thorough washing with THF. The calcite particle thus prepared produced the excimer emission of pyrene by UV irradiation. Rhodamine B was also introduced into calcium carbonate by the immersion of the microcapsule into the aqueous solutions of Rhodamine B. The fluorescence of rhodamine B was observed from the calcium carbonate particles by visible light irradiation. Acetaminophen, a common drug poorly soluble in water, was also included in the calcium carbonate particle by the same procedures as the pyrene encapsulation. As acetaminophen thus encapsulated was released by the dissolution of the calcium carbonate particle in acidic solution, the particle is expected to be applied for a dissolution-triggered drug delivery. (C) 2014 The Society of Powder Technology Japan. Published by Elsevier B.V. and The Society of Powder Technology Japan. All rights reserved.

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研究論文（学術雑誌）

Emi Kanemura, Tatsushi Goto, Yoshiro Tatsu, Hiromasa Imaishi, Kenichi Morigaki

Assaying the activities of cytochrome P450 (P450) is important for evaluating the toxicity of chemicals in drugs, food, and the environment. We developed a methodology to immobilize multiple P450 isoforms with a heightened density and assay their activities in parallel. Inkjet printing technology was applied for the deposition of P450 onto a plastic substrate (cycloolefin polymer: COP) having arrayed microwells (diameter: 50 mu m, depth: 60 mu m). P450 was printed into each microwell and immobilized by gelation of co-printed agarose. Activities of printed P450 isoforms were measured by introducing a substrate solution into the microwells and sealing it with an elastomer sheet, isolating individual wells. In order to avoid uncontrolled initiation of the reaction, we photo-regulated the P450 catalysis by using caged glucose-6-phosphate. The combination of inkjet printing, immobilization in microwells, and photo-regulated initiation of the enzymatic reaction enabled us to assay multiple P450 isoforms on a chip with an unprecedented density. We demonstrate the parallel assay of single nucleotide polymorphs of P450, which play important roles in different drug efficacies in individuals.

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研究論文（学術雑誌）

Yamada,M, 森垣 憲一, 今石 浩正

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研究論文（学術雑誌）

Kenichi Morigaki, Kazuyuki Mizutani, Makoto Saito, Takashi Okazaki, Yoshihiro Nakajima, Yoshiro Tatsu, Hiromasa Imaishi

We describe a stable and functional model biological membrane based on a polymerized lipid bilayer with a chemically modified surface. A polymerized lipid bilayer was formed from a mixture of two diacetylene-containing phospholipids, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DiynePC) and 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphoethanolamine (DiynePE). DiynePC formed a stable bilayer structure, whereas the ethanolamine headgroup of DiynePE enabled functional molecules to be grafted onto the membrane surface. Copolymerization of DiynePC and DiynePE resulted in a robust bilayer. Functionalization of the polymeric bilayer provided a route to a robust and biomimetic surface that can be linked with biomolecules, cells, and three-dimensional (3D) microstructures. Biotin and peptides were grafted onto the polymeric bilayer for attaching streptavidin and cultured mammalian cells by molecular recognition, respectively. Nonspecific adsorption of proteins and cells on polymeric bilayers was minimum. DiynePE was also used to attach a microstructure made of an elastomer (polydimethylsiloxan: PDMS) onto the membrane, forming a confined aqueous solution between the two surfaces. The microcompartment enabled us to assay the activity of a membrane-bound enzyme (cyochrome P450). Natural (fluid) lipid bilayers were incorporated together with membrane-bound proteins by lithographically polymerizing DiynePC/DiynePE bilayers. The hybrid membrane of functionalized polymeric bilayers and fluid bilayers offers a novel platform for a wide range of biomedical applications including biosensor, bioassay, cell culture, and cell-based assay.

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研究論文（学術雑誌）

Kenji Sasahara, Kenichi Morigaki, Kyoko Shinya

Alzheimer's disease (AD) is the most prevalent age-dependent form of dementia, characterized by extracellular amyloid deposits comprising amyloid beta-peptide (A beta) in the cerebral cortex. Increasing evidence has indicated that ganglioside GM1 (GM1) in lipid rafts plays a pivotal role in amyloid deposition of A beta and the related cytotoxicity in AD. Despite recent efforts to characterize A beta-lipid interactions, the effect of A beta aggregation on dynamic properties and organization of lipid membranes is poorly understood. In this study, we examined the aggregation of A beta on supported lipid bilayers containing raft components (i.e., cholesterol, sphingomyelin, and GM1) and its effects on the membrane properties. We showed that the lateral fluidity of membranes was significantly affected by membrane binding and subsequent aggregation of A beta. Microscopic observations of the membrane surfaces demonstrated an enhancement in phase separation of lipids as a result of interactions between A beta and GM1 during induced aggregation of A beta. The uptake of GM1 into A beta aggregates and the attendant membrane damage were also observed under a microscope when the membrane-anchored aggregates were formed. On the basis of these observations, we propose that A beta aggregates formed in the presence of lipid membranes have a latent ability to trigger the uptake of raft components accompanied by phase separation of lipids.

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研究論文（学術雑誌）

Koyo Watanabe, Ryosuke Miyazaki, Goro Terakado, Takashi Okazaki, Kenichi Morigaki, Hiroshi Kano

We propose scanning localized surface plasmon microscopy of mixed lipid bilayers with submicron domain structures. Our observation technique, which employs localized surface plasmons excited on a flat metal surface as a sensing probe, provides non-label and non-contact imaging with the spatial resolution of similar to 170 nm. We experimentally show that submicron domain structures of mixed lipid bilayers can be observed. A detailed analysis finds that the domains are classified into two groups. (c) 2012 Optical Society of America

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研究論文（学術雑誌）

Kenji Sasahara, Kenichi Morigaki, Takashi Okazaki, Daizo Hamada

Amyloid deposition of human islet amyloid polypeptide (hIAPP) in the islets of Langerhans is closely associated with the pathogenesis of type II diabetes mellitus. Despite substantial evidence linking amyloidogenic hIAPP to loss of beta-cell mass and decreased pancreatic function, the molecular mechanism of hIAPP cytotoxicity is poorly understood. We here investigated the binding of hIAPP and nonamyloidogenic rat LAPP to substrate-supported planar bilayers and examined the membrane-mediated amyloid aggregation. The membrane binding of IAPP in soluble and fibrillar states was characterized using quartz crystal microbalance with dissipation monitoring, revealing significant differences in the binding abilities among different species and conformational states of IAPP. Patterned model membranes composed of polymerized and fluid lipid bilayer domains were used to microscopically observe the amyloid aggregation of hIAPP in its membrane-bound state. The results have important implications for lipid-mediated aggregation following the penetration of hIAPP into fluid membranes. Using the fluorescence recovery after photobleaching method, we show that the processes of membrane binding and subsequent amyloid aggregation are accompanied by substantial changes in membrane fluidity and morphology. Additionally, we show that the fibrillar hIAPP has a potential ability to perturb the membrane structure in experiments of the fibril-mediated aggregation of lipid vesicles. The results obtained in this study using model membranes reveal that membrane-bound hIAPP species display a pronounced membrane perturbation ability and suggest the potential involvement of the oligomeic forms of hAPP in membrane dysfunction.

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研究論文（学術雑誌）

Gang Chang, Yoshinao Mori, Saori Mori, Takashi Irie, Hidenori Nagai, Tatsushi Goto, Yoshiro Tatsu, Hiromasa Imaishi, Kenichi Morigaki

A microarray chip containing human P450 isoforms was constructed for the parallel assay of their metabolic activities. The chip had microwells that contained vertically integrated P450 and oxygen sensing layers. The oxygen sensing film was made of an organically modified silica film (ORMOSIL) doped with tris(4,7-diphenyl-1,10-phenanthroline) ruthenium dichloride (Ru(dpp)(3)Cl-2). Human P450s (23 types) expressed in E. coli and purified as membrane fractions were immobilized in agarose matrixes on the oxygen sensing layer. The activities of P450s were determined by evaluating the fluorescence intensity enhancement of the oxygen sensor due to the oxygen consumption by the metabolic reaction. By normalizing the responses with the amounts of oxygen sensor and P450 enzymes in microwells, we could obtain fluorescence enhancement patterns that were characteristic to the combination of P450 isoforms and substrate material. The patterns obtained from two psoralen derivatives resembled each other, whereas a structurally different substrate (capsaicin) resulted in a distinct pattern. These results suggest the potential of the microarray to analyze the activities of diverse P450 isoforms in a high-throughput fashion. Furthermore, mechanism-based inactivation (MBI) of P450 could be detected by successively incubating a chip with different substrate solutions and measuring the residual activities.

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研究論文（学術雑誌）

Kenichi Morigaki, Shigeki Kimura, Keisuke Okada, Takashi Kawasaki, Kazunori Kawasaki

We studied the formation of substrate-supported planar phospholipid bilayers (SPBs) on glass and silica from mixtures of long- and short-chain phospholipids to assess the effects of detergent additives on SPB formation. 1,2-Hexyanoyl-sn-glycero-3-phosphocholine (DHPC-C6) and 1,2-heptanoyl-sn-glycero-3-phosphocholine (DHPC-C7) were chosen as short-chain phospholipids. 1-Palmitoyl-2-oleol-sn-glycero-3-phosphocholine (POPC) was used as a model long-chain phospholipid. Kinetic studies by quartz crystal microbalance with dissipation monitoring (QCM-D) showed that the presence of short-chain phospholipids significantly accelerated the formation of SPBs. Rapid rinsing with a buffer solution did not change the adsorbed mass on the surface if POPC/DHPC-C6 mixtures were used below the critical micelle concentration (cmc) of DHPC-C6, indicating that an SPB composed of POPC molecules remained on the surface. Fluorescence microscopy observation showed homogeneous SPBs, and the fluorescence recovery after photobleaching (FRAP) measurements gave a diffusion coefficient comparable to that for SPBs formed from POPC vesicles. However, mixtures of POPC/DHPC-C7 resulted in a smaller mass of lipid adsorption on the substrate. FRAP measurements also yielded significantly smaller diffusion coefficients, suggesting the presence of defects. The different behaviors for DHPC-C6 and DHPC-C7 point to the dual roles of detergents to enhance the formation of SPBs and to destabilize them, depending on their structures and aggregation properties.

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研究論文（学術雑誌）

Kenichi Morigaki, Makoto Saito, Kazuyuki Mizutani, Takashi Okazaki, Yoshihiro Nakajima, Yoshiro Tatsu, Hiromasa Imaishi

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Kenichi Morigaki, Kazuyuki Mizutani, Emi Kanemura, Yoshiro Tatsu, Noboru Yumoto, Hiromasa Imaishi

Cytochrome P450 (P450) species play an important role in the metabolism of xenobiotics, and assaying the activities of P450 is important for evaluating the toxicity of chemicals in drugs and food. However, the lag time caused by the introduction and mixing of sample solutions can become sources of error as the throughput is heightened by increasing the sample number and decreasing the sample volume. To amend this technological obstacle, we developed a methodology to photoregulate the activity of P450 by using photoprotected (caged) compounds. We synthesized caged molecules of nicotinamide adenine dinucleotide phosphate (NADP(+)) and glucose 6-phosphate (G6P), which are involved in the generation of NADPH (cofactor of P450). The use of caged-G6P completely blocked the P450 catalysis before the UV illumination, whereas caged-NADP+ resulted in a little background reaction. Upon UV illumination, more than 90% of the enzymatic activity could be restored. The use of caged-G6P enabled assays in isolated microchambers (width, 50 mu m; height, 50 mu m) by encapsulating necessary ingredients in advance and initiating the reaction by UV illumination. The initiation of enzymatic reaction could be observed in a single microchamber. Minimizing uncertainties caused by the introduction and mixing of solutions led to significantly reduced errors of obtained kinetic constants.

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研究論文（学術雑誌）

Kaoru Nomura, Masami Lintuluoto, Kenichi Morigaki

Inhomogeneous line broadening due to conformational distributions of molecules is one of the troublesome problems in solid-state NMR spectroscopy. The best possible way to avoid it is to crystallize the sample. Here, we present a highly resolved C-13 cross-polarization (CP) magic angle spinning (MAS) NMR spectrum of the highly ordered crystalline 1,2-dimyrystoyl-sn-glycero-3-phosphocholine (DMPC) and completely assigned it using two-dimensional (2D) solid-state NMR spectra, dipolar heteronuclear correlation (HETCOR) spectra, scalar heteronuclear J coupling based chemical shift correlation (MAS-J-HMQC) spectra, and Dipolar Assisted Rotational Resonance (DARR) spectra. A comparison between assigned chemical shift values by solid-state NMR in this study and the calculated chemical shift values for X-ray crystal DMPC structures shows good agreement, indicating that the two isomers in the crystalline DMPC take the same conformation as the X-ray crystal structure. The phase diagram of the low hydration level of DMPC (3 <= n(w) <= 12) determined by H-1 and C-13 NMR spectra indicates that DMPC takes a crystalline state only in a very narrow region around n(w) = 4 and T < 313 K. These findings provide us with conformational information on crystalline DMPC and the physical properties of DMPC at a low hydration level and can possibly help us obtain a highly resolved solid-state NMR spectrum of microcrystalline membrane-associated protein samples.

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研究論文（学術雑誌）

Takehiko Inaba, Yoshiro Tatsu, Kenichi Morigaki

We studied the peptide-induced membrane fusion process between small unilamellar vesicles (SUVs) and supported planar bilayers (SPBs) with the aim of developing a method for incorporating membrane components into SPBs. As fusogenic peptides, two analogues of the N-terminal region of an influenza membrane fusion protein hemaggulutinin, anionic E5 and cationic K5, were synthesized, and the membrane fusion was investigated using SPB and SUVs composed of phosphatidylcholine from egg yolk (EggPC). We directly visualized the process of lipid transfer from SUVs to SPB by total internal reflection fluorescence (TIRE) microscopy. The transfer of fluorescent lipids was effectively induced only by the combination of two peptides. The TIRE microscopy observations of single SUV fusion events also revealed that lipid membranes from SUV could completely fuse into the SPB. However, the presence of single peptide (either E5 or K5) rather inhibited the lipid transfer, presumably due to the electrostatic repulsion between SUVs and SPB. The opposite effects induced by the peptides indicate the possibility for a designed application of two peptides as a means to control the membrane fusion spatially and temporally.

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研究論文（学術雑誌）

Keiko Tawa, Xiaoqiang Cui, Kenji Kintaka, Junji Nishii, Kenichi Morigaki

Grating-coupled surface plasmon-field enhanced fluorescence (GC-SPF) was applied to biosensing. Although the greatest enhancement of GC-SPF on our plasmonic chips compared with that on a glass slide was found to be 40 times, this was due to the enhanced excitation field. Therefore, grating-coupled fluorescence using a reverse coupling mode is here explored to achieve further 4-5 times enhancement. As a result of using both the excitation field enhanced by grating-coupled surface plasmon resonance and the directional emission enhanced by reverse coupling mode, an increase in fluorescence of more than 110 times compared with that on the glass slide was recorded. The reverse coupling mode was also applied to the sensitive fluorescence microscopic imaging of Cy5-streptavidin (Cy5-SA) in microfluidic channels on a two dimensional nanohole array substrate. We performed a Cy5-SA concentration series analysis in which the plasmonic substrate demonstrated 26.3x enhancement of sensitivity and a limit of detection (LOD) of ca. 100 pM, which is at least one order of magnitude lower than in glass slides with identical surface chemistry. This plasmonic nanostructure will be invaluable for colorimetric detection in applications such as microfluidic enzyme-linked immune-sorbent assay (ELISA) device, and portable microarray biosensing, because the optical setup can be simplified. (C) 2011 Elsevier B.V. All rights reserved.

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研究論文（学術雑誌）

Gang Chang, Kenichi Morigaki, Yoshiro Tatsu, Takashi Hikawa, Tatsushi Goto, Hiromasa Imaishi

An assaying method of cytochrome P450 (P450 or CYP) monooxygenase activities for toxicological evaluation of drugs and environmental pollutants was developed by immobilizing P450 on an oxygen sensoring layer. Membrane fractions from E. coli expressing human P450 were entrapped in agarose or silica-based gels and immobilized on 96-well microarrays having an oxygen sensing film at the bottom. The oxygen sensing film was made of an organically modified silica film (ORMOSIL) doped with Tris(4,7-diphenyl-1,10-phenanthroline) ruthenium dichloride (Ru(dpp)(3)Cl(2)). P450 activity toward the substrates was monitored through the fluorescence intensity enhancement due to the oxygen consumption by the metabolic reactions. For the metabolism of chlortoluron, a selective herbicide used to control grass weeds, CYP1A1 immobilized in agarose gel showed a higher activity and stability compared with those in silica gels and free suspensions. The luminescence changing rate evaluated by the dynamic transient method (DTM) could be correlated with the substrate concentration. We also compared the metabolic responses of human P450s (CYP1A1, CYP2C8, CYP2E1, CYP3A4) toward various substances. The use of immobilized P450 on an oxygen sensing layer provides a versatile assaying platform owing to the following features. First, the oxygen sensor can detect metabolic reactions of any P450 species, in contrast with assays using fluorogenic substrates. Second, vertical integration of the oxygen sensor and immobilized P450 enhanced the sensitivity because of the effective depletion of oxygen in the vicinity of the oxygen sensing layer. Third, immobilization enables repeated use of P450 by replacing the substrate solutions using a flow cell. Furthermore, the activity of immobilized P450 was retained at least for 3 weeks at 4 degrees C, suggesting its long-term stability, which is an additional attractive feature.

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研究論文（学術雑誌）

Tatsushi Goto, Hiroshi Moriuchi, Xuejun Fu, Tomoyo Ikegawa, Toshiyuki Matsubara, Gang Chang, Tomohide Uno, Kenichi Morigaki, Kunio Isshiki, Hiromasa Imaishi

A number of studies have demonstrated that cytochrome P450 (P450) converts furanocoumarin derivatives into reactive molecules, which form covalent bonds to biomolecules. 5-Methoxypsoralen (5-MOP) is a natural furanocoumarin from apiaceous plants. In this study, we examined the effect on 5-MOP metabolism of single nucleotide polymorphisms (SNPs) in CYP2A13. We used Escherichia coli-generated recombinant enzymes of wild-type CYP2A13(star)1 and five variants, CYP2A13(star)4 (R101Q), CYP2A13(star)5 (F453Y), CYP2A13(star)6 (R494C), CYP2A13(star)8 (D158E), and CYP2A13(star)9 (V323L). In high-performance liquid chromatography analyses of 5-MOP metabolic products, CYP2A13(star)1 converted 5-MOP into 5-MOP dihydrodiol; K(m) and V(max) values of the reaction were 1.44 +/- 0.17 mu M and 4.23 +/- 0.36 nmol/(min . nmol P450), respectively. The generation of a dihydrodiol from 5-MOP implies that conversion by CYP2A13 causes toxicity due to the formation of covalent bonds with DNA or proteins. Most of the CYP2A13 variants could metabolize 5-MOP; K(m) values for CYP2A13(star)5, (star)6, (star)8, and (star)9 were 1.63 +/- 0.12, 1.36 +/- 0.10, 0.85 +/- 0.09, and 0.58 +/- 0.06 mu M, respectively, and V(max) values were 3.20 +/- 0.13, 4.69 +/- 0.13, 2.34 +/- 0.07, and 1.84 +/- 0.09 nmol/(min . nmol P450), respectively. However, the processing of 5-MOP by CYP2A13(star)4 was not detectable. Based on this data, we hypothesize that SNPs within the CYP2A13 gene affect metabolism of 5-MOP in humans.

[査読有り]

研究論文（学術雑誌）

Keiko Tawa, Kenichi Morigaki

A parallel microscopic imaging technique, surface plasmon microscopy (SPM)-surface plasmon fluorescence microscopy (SPFM), is introduced as a versatile analytical tool to monitor biochips. In spite of the fact that the fluorescence excited by surface plasmon is 1-2 order stronger compared with the total internal reflection fluorescence microscopy, SPFM has not fully utilized its advantages because fluorescence from fluorophores near the gold surface is almost entirely quenched due to the Forster energy transfer. In this study, SiO(2) layer sputtered on the gold substrate suppressed the quenching of fluorescence and enabled a parallel measurement of SPM and SPFM. As a model system, micropatterned membranes composed of polymeric and fluid phospholipid bilayers were employed. The difference of film thickness could be detected by SPM, and SPFM provided information on the composition and structure of membranes, enabling the distinction between polymeric and fluid phospholipid bilayers. These results suggest the general applicability of SPM-SPFM for various formats of biochips. (C) 2010 Elsevier B.V. All rights reserved.

[査読有り]

研究論文（学術雑誌）

Gang Chang, Yoshiro Tatsu, Tatsushi Goto, Hiromasa Imaishi, Kenichi Morigaki

Optical biosensor arrays for rapidly determining the glucose concentrations in a large number of beverage and blood samples were developed by immobilizing glucose oxidase (GOD) on oxygen sensor layer Glucose oxidase was first encapsulated in silica based gels through sol-gel approach and then immobilized on 96-well microarrays integrated with oxygen sensing film at the bottom The oxygen sensing film was made of an organically modified silica film (ORMOSIL) doped with tris(4 7-diphenyl-1 10-phenanthroline) ruthenium dichloride (Ru(dpp)(3)Cl(2)) The oxidation reaction of glucose by glucose oxidase could be monitored through fluorescence intensity enhancement due to the oxygen consumption in the reaction The luminescence changing rate evaluated by the dynamic transient method (DIM) was correlated with the glucose concentration with the wide linear range from 0 1 to 50mM (Y = 13 28X-0 128 R = 0 9968) and low detection limit (0 06 mM) The effects of pH and coexisting ions were systemically studied The results showed that the optical biosensor arrays worked under a wide range of pH value and normal interfering species such as Na(+) K(+) Cl(-) PO(4)(3-) and ascorbic acid did not cause apparent interference on the measurement The activity of glucose oxidase was mostly retained even after 2-month storage indicating their long-term stability (C) 2010 Elsevier B V All rights reserved

[査読有り]

研究論文（学術雑誌）

Masahiro Fujiwara, Kumi Shiokawa, Miyuki Araki, Nobuyuki Ashitaka, Kenichi Morigaki, Takayuki Kubota, Yoshiko Nakahara

Phase transition from vaterite to calcite is a general behavior of CaCO3 materials. The interfacial reaction method using water-in-oil-in-water (W/O/W) emulsion we reported before is an effective method to produce hollow CaCO3 particles (microcapsules) with vaterite crystalline structure. These vaterite CaCO3 microcapsules underwent the phase transition to calcite in various aqueous solutions. When some proteins were mixed in the solution used for the phase transition, their encapsulations were achieved satisfactorily at room temperature regardless of their molecular weight. Insulin, lysozyme, bovine serum albumin, and chicken IgY were successfully encapsulated into the phase transition CaCO3 particles, while the encapsulation of lysozyme was unsuccessful by the interfacial reaction method. Protein included in the phase transition CaCO3 particle was not discharged by simple water washing but by the dissolution of the CaCO3 matrix in an acid solution, being advantageous to a responsive protein delivery technology. The recycle uses of the insulin solution used for the encapsulation improved the utilization efficiency of insulin. It was ascertained that the phase transition of vaterite CaCO3 microcapsule to calcite is a simple, general, and convenient method to encapsulate proteins into CaCO3 small particles under very mild conditions.

[査読有り]

研究論文（学術雑誌）

Takashi Okazaki, Yoshiro Tatsu, Kenichi Morigaki

We developed a micropatterned model biological membrane on a solid substrate that can induce phase separation of lipid microdomains in a designed geometry. Micropatterned lipid bilayers were generated by the photolithographic polymerization of a diacetylene phospholipid, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine(DiynePC). By changing the UV dose for the photopolymerization, we could modulate the coverage of the surface by the polymeric bilayer domains. After removing nonpolymerized DiynePC, natural phoshoplipid membranes were incorporated into the micropatterned polymeric bilayer matrix by a self-assembly process (vesicle fusion). As we incorporated a ternary lipid mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), sphingomyelin (SM), and cholesterol (Chol) (1:1:1), liquid ordered domains (Lo: rich in SM and Chol) were accumulated in the polymer free regions, whereas liquid disordered domains (Ld: rich in DOPC) preferentially participated into the partially polymeric bilayer regions. It was postulated that Ld domains preferentially came in contact with the polymeric bilayer boundaries because of their lower elastic moduli and a smaller thickness mismatch at the boundary. The effect of polymeric bilayer matrix to hinder the size growth of Lo domains should also be playing an important role. The controlled phase separation should open new possibilities to locally concentrate membrane proteins and other nanometer-sized materials on the substrate by associating them with the lipid microdomains.

[査読有り]

研究論文（学術雑誌）

Koyo Watanabe, Miyazaki Ryosuke, Goro Terakado, Takashi Okazaki, Kenichi Morigaki, Hiroshi Kano

We report on microscopic imaging of phospholipid membranes. To achieve nonlabel, noncontact, and high spatial resolution imaging of the membranes, we use optically excited localized surface plasmons as a virtual measurement probe to obtain the local refractive index. This enables significantly higher lateral resolution of similar to 170 nm. We reveal that the developed microscope has the capability of observing lipid bilayers with thickness of 3.0 nm deposited into the gaps in a patterned lipid bilayer with thickness of 4.6 nm. We find that the thickness resolution against the deposited lipid bilayer is similar to 0.33 nm. (C) 2010 Optical Society of America

[査読有り]

研究論文（学術雑誌）

Iwasaki, Yasuhiko, Nakai, Kosuke, Morigaki, Kenichi

[査読有り]

研究論文（学術雑誌）

今石 浩正, 後藤 達志, 宇野 知秀, 森垣 憲一

[査読有り]

研究論文（学術雑誌）

Nakai, K, Morigaki, K, Iwasaki, Y

[査読有り]

研究論文（学術雑誌）

Takashi Okazaki, Takehiko Inaba, Yoshiro Tatsu, Ryugo Tero, Tsuneo Urisu, Kenichi Morigaki

Substrate Supported planar lipid bilayers (SPBs) are versatile models of the biological membrane in biophysical studies and biomedical applications. We previously developed a methodology for generating SPBs composed of polymeric and fluid phospholipid bilayers by using a photopolymerizable diacetylene phospholipid (DiynePC). Polymeric bilayers could be generated with micropatterns by conventional photolithography, and the degree of polymerization could be controlled by modulating UV irradiation doses. After removing nonreacted monomers, fluid lipid membranes could be integrated with polymeric bilayers. Herein, we report on a quantitative study of the morphology of polymeric bilayer domains and their obstruction toward lateral diffusion of membrane-associated Molecules. Atomic force microscopy (AFM) observations revealed that polymerized DiynePC bilayers were formed as nanometer-sized domains. The ratio of polymeric and fluid bilayers could be modulated quantitatively by changing the UV irradiation dose for photopolymerization. Lateral diffusion coefficients of lipid molecules in fluid bilayers were measured by fluorescence recovery after photobleaching (FRAP) and correlated with the amount of polymeric bilayer domains on the substrate. Controlled domain structures, lipid compositions, and lateral mobility in the model membranes should allow us to fabricate model membranes that mimic complex features of biological membranes with well-defined structures and physicochemical properties.

[査読有り]

研究論文（学術雑誌）

Kaoru Nomura, Takehiko Inaba, Kenichi Morigaki, Klaus Brandenburg, Ulrich Seydel, Shoichi Kusumoto

Lipopolysaccharide (LPS), which constitutes the outermost layer of Gram-negative bacterial cells as a typical component essential for their life, induces the first line defense system of innate immunity of higher animals. To understand the basic mode of interaction between bacterial LPS and phospholipid cell membranes, distribution patterns were studied by various physical methods of deep rough mutant LPS (ReLPS) of Escherichia coli incorporated in phospholipid bilayers as simple models of cell membranes. Solid-state P-31-NMR spectroscopic analysis suggested that a substantial part of ReLPS is incorporated into 1,1,2-dimyristoyl-sn-glycero-3-phosphocholine lipid bilayers when multilamellar vesicles were prepared from mixtures of these. In egg L-a-phosphatidylcholine (egg-PC)-rich membranes, ReLPS undergoes micellization. In phosphatidylethanolamine-rich membranes, however, micellization was not observed. We studied by microscopic techniques the location of ReLPS in membranes of ReLPS/egg-PC (1:10 M/M) and ReLPS/egg-PC/1 -palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) (1:9:1 M/M/M). The influence of ReLPS on the physicochemical properties of the membranes was studied as well. Microscopic images of both giant unilamellar vesicles and supported planar lipid bilayers showed that LPS was uniformly incorporated in the egg-PC lipid bilayers. In the egg-PC/POPG (9:1 M/M) lipid bilayers, however, ReLPS is only partially incorporated and becomes a part of the membrane in a form of aggregates (or as mixed aggregates with the lipids) on the bilayer surface. The lipid lateral diffusion coefficient measurements at various molar ratios of ReLPS/egg-PC/POPG indicated that the incorporated ReLPS reduces the diffusion coefficients of the phospholipids in the membrane. The retardation of diffusion became more significant with increasing POPG concentrations in the membrane at high ReLPS/phospholipid ratios. This work demonstrated that the phospholipid composition has critical influence on the distribution of added ReLPS in the respective lipid membranes and also on the morphology and physicochemical property of the resulting membranes. A putative major factor causing these phenomena is reasoned to be the miscibility between ReLPS and individual phospholipid compositions.

[査読有り]

研究論文（学術雑誌）

Yoshiko Ishizuka-Katsura, Tetsuichi Wazawa, Tadato Ban, Kenichi Morigaki, Shigeru Aoyama

We describe a technique to form a biotin-containing phospholipid vesicle layer on a self-assembled monolayer (SAM) deposited on a gold surface to immobilize biotinylated receptor proteins for a surface plasmon resonance (SPR) biosensor. The adsorption of vesicle of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) was examined by SPR on the SAMs of dithiobis(1-deoxy-glucitol-1-carbamoyl pentane) (DDGP), 11-mercaptoundecanoic acid, 11-mercaptoundecanol, 11-amino-1-undecanethiol, and 12-mercaptododecane, and it was found that the DOPC vesicle rapidly adsorbed on the DDGP SAM to achieve the highest coverage of the surface. By quartz crystal microbalance with dissipation monitoring (QCM-D), the DOPC layer formed on the DDGP SAM was shown to be a vesicle layer, in which intact DOPC vesicles physisorbed on the SAM surface. To immobilize a biotinylated receptor protein, one of three biotinylated phospholipids, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl) (biotin-DOPE), N-((6-(biotinoyl)amino)hexanoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (biotin-X-DHPE) and N-(biotinoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (biotin-DHPE), was mixed with DOPC to form a biotin-containing vesicle layer on the DDGP SAM. A comparative binding study of NeutrAvidin and the biotin-containing vesicle layers showed that the use of biotin-X-DHPE achieved the most rapid immobilization of NeutrAvidin on the vesicle layer at the highest surface density. Furthermore, biotinylated protein A, as a receptor protein, could be immobilized through NeutrAvidin on the vesicle layer containing DOPC and biotin-X-DHPE, and its reaction with immunoglobulin G, as an analyte, was successfully observed by SPR. The results demonstrate that the biotin-containing vesicle layer on the DDGP SAM must be a useful component for SPR biosensor surfaces.

[査読有り]

研究論文（学術雑誌）

Masahiro Fujiwara, Kurni Shiokawa, Kenichi Morigaki, Yoshiro Tatsu, Yoshiko Nakahara

We reported before that inorganic reaction occurring at the interface of W/O/W emulsion is advantageous to produce hollow spheres (microcapsules) of inorganic matrices such as silica. This process enables us to include various materials into inorganic matrices directly. Calcium phosphates were also produced from NH4H2PO4 and Ca(OH)(2) by this interfacial reaction method. Various biomaterials are directly incorporated into crystalline calcium phosphate matrices, when the biomaterials are added to the inner water phase of the W/O/W emulsion. ZrO2 and Al2O3 powders were effectively encapsulated in calcium phosphates such as hydroxyapatite (HAp). The images of backscattered electron of FE-SEM observations indicated that ZrO2 particles were included in HAp, while they adhered to the surface of HAp in the case of a simple precipitation method. Biomacromolecules such as BSA and duplex DNA were also included in HAp using the inner water phases dissolving them. Fluorescent microscopy observations revealed that biomacromolecules incorporated in HAp localized in some domains of the HAp matrices. Biomacromolecules thus included were scarcely liberated into deionized water, indicating their strong encapsulation in HAp. This general and simple methodology will provide various composite materials of calcium phosphates, which are applicable to regenerative medicine, DDS, GDS and more. (c) 2007 Elsevier B.V. All rights reserved.

[査読有り]

研究論文（学術雑誌）

Hisashi Yagi, Tadato Ban, Kenichi Morigaki, Hironobu Naiki, Yuji Goto

Deposition of amyloid beta (A beta) fibrils has been suggested to play a central role in Alzheimer's disease. In clarifying the mechanism by which fibrils form and moreover in developing new treatments for amyloidosis, direct observation is important. Focusing on the interactions with surfaces at the early stages, we studied the spontaneous formation of A beta(1-40) fibrils on quartz slices, monitored by total internal reflection fluorescence microscopy combined with thioflavin T, an amyloid-specific fluorescence dye. Self-assembly of A beta(1-40), accelerated by a low concentration of sodium dodecyl sulfate, produced various remarkable amyloid assemblies. Densely packed spherulitic structures with radial fibril growth were typically observed. When the packing of fibrils was coarse, extremely long fibrils often protruded from the spherulitic cores. In other cases, a large number of wormlike fibrils were formed. Transmission electron microscopy and atomic force microscopy revealed relatively short and straight fibrillar blocks associated laterally without tight interaction, leading to random-walk-like fibril growth. These results suggest that, during spontaneous fibrillation, the nucleation occurring in contact with surfaces is easily affected by environmental factors, creating various types of nuclei, and hence variations in amyloid morphology. A taxonomy of amyloid supramolecular assemblies will be useful in clarifying the structure-function relationship of amyloid fibrils.

[査読有り]

研究論文（学術雑誌）

Kenichi Morigaki, Holger Schoenherr, Takashi Okazaki

Micropatterned phospholipid bilayers on solid substrates offer an attractive platform for various applications, such as high throughput drug screening. We have previously developed a photopolymerization-based methodology for generating micropatterned bilayers composed of polymerized and fluid lipid bilayers. Lithographic photopolymerization of a diacetylene-containing phospholipid (DiynePC) allowed facile fabrication of compartmentalized arrays of fluid lipid membranes. Herein, we report on a key experimental parameter that significantly influences the homogeneity and quality of the fabricated polymeric bilayers, namely the temperature at which monolayers of monomeric DiynePC were formed on the water surface and transferred onto solid substrates by the Langmuir-Blodgett/Langmuir-Schaefer (LB/LS) technique. Using fluorescence microscopy and atomic force microscopy, it was found that polymerized bilayers were homogeneous, if bilayers of DiynePC were prepared below the triple point temperature (ca. 20 degrees C) of the monolayer, where a direct transition from the gaseous state to the liquid condensed state occurred. Bilayers prepared above this temperature had a markedly increased number of crack-like line defects. The differences were attributed to the domain structures in the monolayer that were transferred from the water surface to the substrate. Domain size, rather than the molecular packing in each domain, was concluded to play a critical role in the formation of defects. The spontaneous curvature and area changes of bilayers were postulated to cause destabilization and detachment of the films from the substrate upon polymerization. Our present results highlight the importance of controlling the domain structures for the homogeneity of polymerized bilayers required in technological applications.

[査読有り]

研究論文（学術雑誌）

Yoshihiro Ueda, Kenichi Morigaki, Yoshiro Tatsu, Noboru Yumoto, Hiromasa Imaishi

We report on a methodology for immobilizing cytochrome P450 on the surface of micropatterned lipid bilayer membranes and measuring the enzymatic activity. The patterned bilayer comprised a matrix of polymeric lipid bilayers and embedded fluid lipid bilayers. The polymeric lipid bilayer domains act as a barrier to confine fluid lipid bilayers in defined areas and as a framework to stabilize embedded membranes. The fluid bilayer domains, on the other hand, can contain lipid compositions that facilitate the fusion between lipid membranes, and are intended to be used as the binding agent of microsomes containing rat CYP1A1 By optimizing the membrane compositions of the fluid bilayers, we could selectively immobilize microsomal membranes on these domains. The enzymatic activity was significantly higher on lipid bilayer substrates compared with direct adsorption on glass. Furthermore, competitive assay experiment between two fluorogenic substrates demonstrated the feasibility of bioassays based on immobilized P450s. (c) 2007 Elsevier Inc. All rights reserved.

[査読有り]

研究論文（学術雑誌）

Masahiro Fujiwara, Kumi Shiokawa, Kaoru Hayashi, Kenichi Morigaki, Yoshiko Nakahara

We reported before that silica hollow spheres (microcapsules) are prepared by interfacial reaction method that W/O emulsion with the aqueous solution of sodium silicate and n-hexane solution of Tween 80 and Span 80 is combined with another aqueous solution of silica precipitant such as NH4HCO3 and NH4CI. This procedure using W/O/W emulsion fabricates the hollow structures of silica particles directly, and additional steps such as the removal of core parts, that are often essential for the preparation of hollow particles via core-shell materials, are not required. When biomacromolecules such as protein and nucleic acid are mixed in the aqueous solution of sodium silicate, these macromolecules can be encapsulated into the microcapsules. We succeeded the direct encapsulation of bovine serum albumin (BSA) and duplex DNA. Most of encapsulated BSA and DNA cannot be released from the microcapsules without the destruction of microcapsule shell. These microcapsule materials encapsulating biomacromolecules will be applied to biotechnologies such as immobilized enzyme and so on. (c) 2006 Wiley Periodicals, Inc. J Biomed Mater Res 81A: 103-112, 2007.

[査読有り]

研究論文（学術雑誌）

Takehiko Inaba, Kenichi Morigaki, Yoshiro Tatsu

[査読有り]

Trishool Namani, Takashi Ishikawa, Kenichi Morigaki, Peter Walde

In dilute aqueous solution and at room temperature, cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) self-assembles into vesicles (self-closed bilayers), if the molar ratio of the neutral form of DHA to anionic DHA is kept between 1: 1 and 1:3 (corresponding to a bulk pH between 8.5 and 9.2 for a system with 10 mM DHA). By using polycarbonate membrane extrusion, stable unilamellar DHA vesicles with an average diameter of 80 nm can be prepared at pH 8.8. Cryo-transmission electron microscopy indicates that the width of the DHA bilayers in the vesicles is clearly below twice the length of an extended DHA molecule, indicating a high conformational flexibility of DHA within the vesicle bilayer. These DHA bilayers have a similar thickness like bilayers of vesicles prepared at pH 8.5 from oleic acid (cis-9-octadecenoic acid). Using calcein as fluorescent reference compound, it is shown that water-soluble molecules can be encapsulated inside DHA vesicles which may make them interesting for medical or food applications. (C) 2006 Elsevier B.V. All rights reserved.

[査読有り]

研究論文（学術雑誌）

Tadato Ban, Kenichi Morigaki, Hisashi Yagi, Takashi Kawasaki, Atsuko Kobayashi, Shunsuke Yuba, Hironobu Naiki, Yuji Goto

In Alzheimer disease, amyloid beta, a 39-43-residue peptide produced by cleavage from a large amyloid precursor protein, undergoes conformational change to form amyloid fibrils and deposits as senile amyloid plaques in the extracellular cerebral cortices of the brain. However, the mechanism of how the intrinsically linear amyloid fibrils form spherical senile plaques is unknown. With total internal reflection fluorescence microscopy combined with the use of thioflavin T, an amyloid-specific fluorescence dye, we succeeded in observing the formation of the senile plaque-like spherulitic structures with diameters of around 15 mu m on the chemically modified quartz surface. Real-time observation at a single fibrillar level revealed that, in the absence of tight contact with the surface, the cooperative and radial growth of amyloid fibrils from the core leads to a huge spherulitic structure. The results suggest the underlying physicochemical mechanism of senile plaque formation, essential for obtaining insight into prevention of Alzheimer disease.

[査読有り]

研究論文（学術雑誌）

森垣憲一

[査読有り][招待有り]

記事・総説・解説・論説等（学術雑誌）

Kenichi Morigaki

[査読有り][招待有り]

記事・総説・解説・論説等（学術雑誌）

森垣憲一

[査読有り][招待有り]

記事・総説・解説・論説等（学術雑誌）

Morigaki K, Tanimoto Y

[査読有り]

記事・総説・解説・論説等（学術雑誌）

森垣 憲一

[査読有り][招待有り]

記事・総説・解説・論説等（学術雑誌）

森垣 憲一

[招待有り]

記事・総説・解説・論説等（学術雑誌）

Morigaki K

[査読有り]

記事・総説・解説・論説等（学術雑誌）

古川 一暁, 並河 英紀, 村越 敬, 森垣 憲一, 手老 龍吾

Kenichi Morigaki

Micropatterned phospholipid bilayers on solid substrates offer an attractive platform for various applications, such as high-throughput drug screening. The authors have developed a photopolymerization-based methodology for generating micropatterned bilayers composed of polymerized and fluid lipid bilayers. Lithographic photopolymerization of a diacetylene-containing phospholipid (DiynePC) allowed facile fabrication of compartmentalized arrays of fluid lipid membranes. Herein, the authors report on the present state of research and discuss on the prospective application of the model membranes.

[査読有り]

記事・総説・解説・論説等（学術雑誌）

稲葉 岳彦, 達 吉郎, 森垣 憲一

岡崎 敬, 稲葉 岳彦, 達 吉郎, 森垣 憲一

Kenichi Morigaki, Peter Walde

Results obtained from recent studies on the preparation and application of fatty acid vesicles are reviewed, focusing on some of the particular properties of fatty acid vesicles in comparison with conventional phospholipid vesicles (liposomes): (i) pH dependency which allows reversible transformations from non-vesicular to vesicular aggregates, and (ii) dynamic features that place fatty acid vesicles in between conventional vesicles formed from double-chain amphiphiles and micelles formed from single-chain surfactants. There are two main research areas in which fatty acid vesicles have been studied actively during the last years: (i) basic physico-chemical properties, and (ii) applications as protocell models. Applications of fatty acid vesicles in the fields of food additives and drug delivery are largely unexplored, which is at least partially due to concerns regarding the colloidal stability of fatty acid vesicles (pH- and divalent cation-sensitivity). Recently, fatty acid vesicles were prepared from highly unsaturated fatty acids (docosahexaenoic acid) and the pH range of vesicle formation could be extended to high or low pH values by preparing mixed vesicles through addition of a second type of single-chain amphiphile that stabilizes the vesicle bilayer but itself is not a fatty acid. (c) 2007 Elsevier Ltd. All rights reserved.

[査読有り]

書評論文,書評,文献紹介等

Morigaki K

学術書

Morigaki K

学術書

今石 浩正, 後藤 達志, 宇野 知秀, 森垣 憲一

学術書

Morigaki K

学術書

石原 悠人, 岸本 龍典, 杭田 芙子, 工藤 卓, 森垣 憲一, 細川 千絵

ポスター発表

金重 先人, 藤井 雅史, 林 文夫, 森垣 憲一, 山下 隼人, 粟津 暁紀

ポスター発表

干鰯谷 和彦, 山下 隼人, 林 文夫, 森垣 憲一, 藤井 雅史, 粟津 暁紀, 阿部 真之

ポスター発表

高山 宙輝, 曲師 香緒里, 森垣 憲一, 茶谷 絵理

ポスター発表

Kenichi Morigaki

口頭発表（招待・特別）

永井るりか, 谷本泰士, 笠井倫志, 鈴木健一, 林 文夫, 森垣憲一

口頭発表（一般）

Kenichi Morigaki

[招待有り]

口頭発表（招待・特別）

杭田 芙子

ポスター発表

TakuroYoneda, Yasushi Tanimoto, Daisuke Takagi, Kenichi Morigaki

ポスター発表

杭田 芙子, 谷本 泰士, 林 文夫, 森垣 憲一

ポスター発表

永井 るりか, 谷本 泰士, 笠井 倫志, 鈴木 健一, 林 文夫, 森垣 憲一

ポスター発表

林 文夫, 杭田 扶子, 森垣 憲一, 妹尾 圭司

ポスター発表

干鰯谷 和彦, 谷本 泰士, 山下 隼人, 森垣 憲一, 林 文夫, 阿部 真之

ポスター発表

谷本 泰士, 山田 美紗登, 林 文夫, 森垣 憲一

ポスター発表

金重 先人, 谷本 泰士, 西森 拓, 森垣 憲一, 林 文夫, 粟津 暁紀

ポスター発表

米田 卓郎, 谷本 泰士, 高木 大輔, 森垣 憲一

ポスター発表

Kenichi Morigaki

口頭発表（基調）

Kenichi Morigaki

公開講演，セミナー，チュートリアル，講習，講義等

米田 卓郎, 谷本 泰士, 高木 大輔, 森垣 憲一

ポスター発表

Kenichi Morigaki, Fuyuko Tamura, Yasushi Tanimoto, Rurika Nagai, Misato Yamada, Fumio Hayashi

[招待有り]

口頭発表（招待・特別）

Kenichi Morigaki

[招待有り]

口頭発表（招待・特別）

Kenichi Morigaki

[招待有り]

口頭発表（招待・特別）

Kenichi Morigaki

[招待有り]

口頭発表（招待・特別）

○Kaori Mageshi, Naoki Yamamoto, Ryota Komatsu, Kenichi Morigaki, Masatomo So, Yuji Goto, Eri Chatani

ポスター発表

小林 佐和子, 小松 亮太, 森垣 憲一

ポスター発表

小松 亮太, 谷本 泰士, 林 文夫, 森垣 憲一

口頭発表（一般）

米田 卓郎, 谷本 泰士, 高木 大輔, 森垣 憲一

口頭発表（一般）

森垣 憲一, 谷本 泰士, 山下 隼人, 粟津 暁紀, 林 文夫

[招待有り]

口頭発表（招待・特別）

谷本 泰士, 山下 隼人, 野村 健人, 阿部 真之, 林 文夫, 森垣 憲一

ポスター発表

永井 るりか, 谷本 泰士, 笠井 倫志, 鈴木 健一, 林 文夫, 森垣 憲一

ポスター発表

Kenichi Morigaki

[招待有り]

口頭発表（招待・特別）

金重 先人, 粟津 暁紀, 西森 拓, 林 文夫, 森垣 憲一, 谷本 泰士

ポスター発表

曲師 香緒里, 山本 直樹, 森垣 憲一, 茶谷 絵理

ポスター発表

Kenichi Morigaki

[招待有り]

口頭発表（招待・特別）

Kenichi Morigaki

[招待有り]

口頭発表（招待・特別）

Koji Ando, Fumio Hayashi, Kenichi Morigaki

口頭発表（一般）

Yasushi Tanimoto, Sakiko Kojima, Akinori Awazu, Fumio Hayashi, Kenichi Morigaki

口頭発表（一般）

Kenichi Morigaki

[招待有り]

口頭発表（招待・特別）

Kenichi Morigaki

[招待有り]

口頭発表（招待・特別）

森垣 憲一, 谷本 泰士, 小嶋 佐妃子, 粟津 暁紀, 林 文夫

[招待有り]

口頭発表（招待・特別）

小松 亮太, 小林 佐和子, 安藤 公二, 岩崎 泰彦, 遊佐 真一, 森垣 憲一

ポスター発表

安藤 公二, 林 文夫, 森垣 憲一

ポスター発表

Kenichi Morigaki

[招待有り]

口頭発表（招待・特別）

Sawako Kobayashi, Fuyuko Tamura, Kazuhiko Iwasaki, KenichiMorigaki

ポスター発表

小嶋 佐妃子, 谷本 泰士, 林 文夫, 前川 昌平, 森垣 憲一

ポスター発表

谷本 泰士, 小嶋 佐妃子, 粟津 暁紀, 林 文夫, 森垣 憲一

ポスター発表

Koji Ando, Kenichi Morigaki

ポスター発表

Yasushi Tanimoto, Kenichi Morigaki

ポスター発表

Yasushi Tanimoto, Kenichi Morigaki

口頭発表（一般）

Kenichi Morigaki

[招待有り]

口頭発表（招待・特別）

Kenichi Morigaki

[招待有り]

口頭発表（招待・特別）

Sakiko Kojima, Kenichi Morigaki

ポスター発表

Ryota Komatsu, Kenichi Morigaki

ポスター発表

Ryota Komatsu, Kenichi Morigaki

口頭発表（一般）

Fuyuko Tamura, Kenichi Morigaki

ポスター発表

Fuyuko Tamura, Kenichi Morigaki

口頭発表（一般）

Sawako Kobayashi, Kenichi Morigaki

ポスター発表

Sawako Kobayashi, Kenichi Morigaki

口頭発表（一般）

森垣 憲一

[招待有り]

口頭発表（招待・特別）

Koji Ando, Fumio Hayashi, Kenichi Morigaki

口頭発表（一般）

Kenichi Morigaki

[招待有り]

口頭発表（招待・特別）

Yasushi Tanimoto, Sakiko Kojima, Fumio Hayashi, Kenichi Morigaki

口頭発表（一般）

Kenichi Morigaki

[招待有り]

口頭発表（招待・特別）

Kenichi Morigaki

[招待有り]

口頭発表（招待・特別）

Koji Ando, Fumio Hayashi, Kenichi Morigaki

ポスター発表

Fuyuko Tamura, Yasushi Tanimoto, Yasuhiko Iwasaki, Fumio Hayashi, Yuki Sudo, Kenichi Morigaki

ポスター発表

林 文夫, 齋藤 夏美, 谷本 泰士, 森垣 憲一, 妹尾 圭司

[招待有り]

口頭発表（招待・特別）

Sakiko Kojima, Yasushi Tanimoto, Fumio Hayashi, Shohei Maekawa, Kenichi Morigaki

ポスター発表

Yasushi Tanimoto, Sakiko Kojima, Fumio Hayashi, Kenichi Morigaki

ポスター発表

Kenichi Morigaki, Yasushi Tanimoto, Fumio Hayashi

[招待有り]

口頭発表（招待・特別）

森垣 憲一, 安藤 公二, 田邊 真志

[招待有り]

口頭発表（招待・特別）

Kaoru Nomura, Erisa Harada, Kenji Sugase, Keiko Shimamoto, Fumio Hayashi, Kenichi Morigaki

ポスター発表

Koji Ando, Masashi Tanabe, Kenichi Morigaki

ポスター発表

Yasushi Tanimoto, Fumio Hayashi, Kenichi Morigaki

ポスター発表

Kenichi Morigaki

[招待有り]

口頭発表（招待・特別）

Toshiki Nishimura, Kenichi Morigaki

ポスター発表

谷本 泰士, 小嶋 佐妃子, 森垣 憲一, 林 文夫

口頭発表（一般）

林 文夫, 齋藤 夏美, 谷本 泰士, 森垣 憲一, 妹尾 圭司

ポスター発表

鈴木 健一, 安藤 弘宗, 河村 奈緒子, 山崎 彩乃, 石田 秀治, 古川 鋼一, 森垣 憲一, 楠見 明弘, 木曽 真

ポスター発表

森垣 憲一

シンポジウム・ワークショップパネル（公募）

森垣 憲一

[招待有り]

口頭発表（招待・特別）

森垣 憲一

[招待有り]

口頭発表（招待・特別）

森垣 憲一

公開講演，セミナー，チュートリアル，講習，講義等

安藤 公二, 森垣 憲一

ポスター発表

谷本 泰士, 森垣 憲一, 林 文夫

ポスター発表

森垣 憲一, 谷本 泰士, 岡田 文子, 林 文夫

[招待有り]

口頭発表（招待・特別）

Morigaki K

[招待有り]

口頭発表（招待・特別）

岡田 文子, 森垣 憲一

ポスター発表

谷本 泰士, 森垣 憲一, 林 文夫

ポスター発表

森垣 憲一

ポスター発表

森垣 憲一

口頭発表（一般）

山田 美紗登, 森垣 憲一

口頭発表（一般）

森垣 憲一

口頭発表（一般）

Okada F, Morigaki K

口頭発表（一般）

Okada k, Morigaki K, F Hayashi

ポスター発表

Morigaki K

[招待有り]

口頭発表（招待・特別）

Morigaki K

口頭発表（一般）

Morigaki, K, Saito, M, Okazaki, T, Nakajima, Y, Tatsu, Y, Imaishi, H

口頭発表（一般）

今石 浩正, 森垣 憲一, 後藤 達志, 森 大気

[招待有り]

口頭発表（招待・特別）

Morigaki K, Saito M, Mizutani K, Okazaki T, Nakajima Y, Tatsu Y, Imaishi, H

[招待有り]

口頭発表（招待・特別）

Kanemura E, Yamada M, Goto T, Tatsu Y, Imaishi H, Morigaki K

口頭発表（一般）

清水 泰博, 松原 俊之, 後藤 達志, 森垣 憲一, 今石 浩正

口頭発表（一般）

Okada, K, Morigaki, K, Imaishi, H, Hyashi, H

口頭発表（一般）

森垣 憲一, 水谷 和幸, 齋藤 慎, 今石 浩正

[招待有り]

口頭発表（招待・特別）

森 芳直, 常 鋼, 後藤 達志, 今石 浩正, 森垣 憲一

ポスター発表

金村 絵美, 山田 美紗登, 後藤 達志, 今石 浩正, 森垣 憲一

ポスター発表

後藤 達志, 森 大気, 森垣 憲一, 宇野 知秀, 清水 泰博, 有澤 章, 今石 浩正

ポスター発表

Morigaki K

[招待有り]

口頭発表（招待・特別）

金村 絵美, 水谷 和幸, 森垣 憲, 達 吉郎, 今石 浩正

ポスター発表

後藤 達志, 森垣 憲一, 宇野 知秀, 清水 泰博, 有澤 章, 今石 浩正

ポスター発表

後藤 達志, 森垣 憲一, 今石 浩正

[招待有り]

シンポジウム・ワークショップパネル（指名）

森垣 憲一, 岡田 佳祐, 水谷 和幸, 今石 浩正

ポスター発表

Goto T, Morigaki K, Uno T, Arisawa A, Imaishi H

ポスター発表

水谷 和幸, 田和 圭子, 今石 浩正, 森垣 憲一

ポスター発表

岡田 佳祐, 今石 浩正, 森垣 憲一

ポスター発表

K Morigaki, T Okazaki, Y Tatsu, H Imaishi

[招待有り]

口頭発表（招待・特別）

森垣 憲一, 岡田 佳祐, 水谷 和幸, 今石 浩正

口頭発表（一般）

Morigaki K

ポスター発表

森垣 憲一

[招待有り]

口頭発表（招待・特別）

岡崎 敬, 森垣 憲一

ポスター発表

森垣 憲一, 村 成, 稲葉 岳, 岡崎 敬, 川崎 隆史, 今石 浩正

口頭発表（一般）

Gang Chang, 森垣 憲一, 達 吉郎, 宇野 知秀, 一色 邦夫, 今石 浩正

口頭発表（一般）

後藤 達志, 桧皮 貴史, 宇野 知秀, 一色 邦夫, 森垣 憲一, 今石 浩正

口頭発表（一般）

平井 洋輔, 後藤 達志, 宇野 知秀, 一色 邦夫, 森垣 憲一, 今石 浩正

口頭発表（一般）

水谷 和幸, 森垣 憲一, 達 吉郎, 一色 邦夫, 今石 浩正

ポスター発表

桧皮 貴史, 後藤 達志, 宇野 知秀, 一色 邦夫, 森垣 憲一, 今石 浩正

口頭発表（一般）

常 鋼, 森垣 憲一

ポスター発表

Morigaki K

[招待有り]

口頭発表（招待・特別）

森垣 憲一

ポスター発表

水谷 和幸, 森垣 憲一, 田和 圭子, 今石 浩正

口頭発表（一般）

Morigaki K

[招待有り]

口頭発表（招待・特別）

Morigaki K

[招待有り]

口頭発表（招待・特別）

池川 朋代, 森内 寛, 付 学軍, 森垣 憲一, 一色 邦夫, 今石 浩正

口頭発表（一般）

池川 朋代, 森内 寛, 付 学軍, 森垣 憲一, 一色 邦夫, 今石 浩正

ポスター発表

付 学軍, 平井 洋輔, 森内 寛, 池川 朋代, 森垣 憲一, 一色 邦夫, 今石 浩正

口頭発表（一般）

付 学軍, 森内 寛, 池川 朋代, 宇野 知秀, 森垣 憲一, 一色 邦夫, 今石 浩正

ポスター発表

森内 寛, 付 学軍, 池川 朋代, 平井 洋輔, 森垣 憲一, 一色 邦夫, 今石 浩正

ポスター発表

森垣 憲一, 達 吉郎

ポスター発表

Morigaki K

[招待有り]

口頭発表（招待・特別）

Morigaki K

[招待有り]

口頭発表（招待・特別）

上田 佳弘, 森垣 憲一, 達 吉郎, 湯元 昇, 今石 浩正

ポスター発表

上田 佳弘, 森垣 憲一, 達 吉郎, 湯元 昇, 今石 浩正

ポスター発表

森垣 憲一

競争的資金

森垣 憲一

競争的資金

森垣 憲一

競争的資金

森垣 憲一

競争的資金

笹原 健二

競争的資金

森垣 憲一

競争的資金

森垣 憲一

競争的資金

森垣 憲一

競争的資金

ナノギャップ構造型基板

森垣 憲一

特許権

シトクロムP450酵素代謝活性パターンによる化合物の毒性予測法

森垣 憲一, 堀本 勝久, 今石 浩正

特許権

多様なチトクロムＰ４５０分子種の酵素活性を網羅的かつ高効率で測定する方法及びキット

今石 浩正, 森垣 憲一, 常 鋼, 達 吉郎

特許権

ポリマー脂質二分子膜を用いた機能性基板

森垣 憲一, 岡崎 敬, 達 吉郎, 中島 芳浩

特許権

ベシクル融合法による光重合性脂質二分子膜の作製方法

森垣 憲一, 岡崎 敬

特許権

固定化チトクロムＰ４５０と酸素センサーを有する積層基板

森垣 憲一, 常 鋼, 今石 浩正

特許権

ＮＡＤＰＨ依存性酵素又は該依存性酵素により還元される酸化酵素の酵素活性を測定する方法及びキット

森垣 憲一, 達 吉郎, 今石 浩正

特許権

基板上に固定化された膜結合型チトクローム P450の活性測定

今石 浩正, 森垣 憲一, 達 吉郎, 湯元 昇

特許権

光重合性脂質膜による膜内分子の水平拡散制御

森垣 憲一

特許権