Faculty Profiles - SHIMANOUCHI Toshinori

Papers - SHIMANOUCHI Toshinori

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Amyloid-forming property of the N-terminal 1-70 residues of human apolipoprotein A-IV. International journal

Norihiro Namba, Tokiko Danjo, Yuichiro Kitagawa, Yoshito Naito, Takashi Ohgita, Toshinori Shimanouchi, Hiroyuki Saito

Scientific reports 15 ( 1 ) 13203 - 13203 2025.4

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

Apolipoprotein A-IV (apoA-IV), the largest member of the exchangeable apolipoprotein family, is a common constituent of amyloid deposits in renal and cardiac amyloidosis. In this study, we characterized the aggregation propensity of the apoA-IV N-terminal fragment to form amyloid fibrils using a variety of biophysical techniques. Thioflavin T fluorescence assay, circular dichroism measurement, and microscopic observations revealed that the N-terminal 1-70 amino acid fragment of apoA-IV readily forms amyloid fibrils by a transition from a random coil to a β-sheet-rich structure. Sequence-based analysis indicated that residues 7-16 and 38-42 are the major aggregation-prone segments within the N-terminal 1-70 residues of apoA-IV. Consistent with this, deletion of these residues strongly inhibited the β-transition and fibril formation of apoA-IV 1-70. Kinetic and thermodynamic analyses of fibril formation by the apoA-IV 1-70 fragment demonstrated that primary nucleation is the dominant step in fibril formation, for which the activation energy barrier is entirely entropic. In addition, we found that the presence of heparin, a representative glycosaminoglycan, accelerated fibril formation kinetics and enhanced the yield of apoA-IV 1-70 fibrils, and the positively charged residues K58-K59 play a critical role in heparin interaction. Overall, our results suggest that the strong amyloid-forming propensity of the N-terminal fragment of apoA-IV may play a key role in amyloid deposition associated with apoA-IV amyloidosis.

DOI： 10.1038/s41598-025-97992-3

Phase-separated cationic giant unilamellar vesicles as templates for the polymerization of tetraethyl orthosilicate (TEOS) Reviewed

Kohei Shiomi, Keita Hayashi, Haruyuki Ishii, Toshiyuki Kamei, Toshinori Shimanouchi, Hidemi Nakamura, Sosaku Ichikawa

Biochimica et Biophysica Acta (BBA) - Biomembranes 1867 ( 2 ) 184403 - 184403 2025.2

Preparation of Nano- and Microparticles Obtained from Polymerization Reaction and Their Application to Surface Coating of Woody Materials Reviewed

Toshinori Shimanouchi, Daichi Hirota, Masafumi Yoshida, Kazuma Yasuhara, Yukitaka Kimura

Applied Sciences 14 ( 23 ) 11326 - 11326 2024.12

Authorship：Lead author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：MDPI AG

A surface coating of polymer particles of different hydrophobicity and wide-ranged size is helpful for the surface modification of materials such as woody thin board (WTB) derived from biomass. A preparation method for polymer particles was, in this study, proposed using a capillary-type flow system. Under hydrothermal conditions, the refinement of dispersed oil droplets in water (O/W emulsions) and the polymerization reaction could be simultaneously advanced, and polymer particles of polystyrene (PS), polyvinyl alcohol (PVA), polymethyl methacrylate (PMMA), and poly-L-lactic acid (PLLA) with a particle size of about 100 nm could be synthesized. The coating of polymer particles gave an improved effect on the water repellency of WTBs due to the hydrophobicity of polymer particles and an alteration of surface roughness, and it also provided long-term stability (more than 6 years).

DOI： 10.3390/app142311326

Extraction of;ydroxymethylfurfural under hydrogen peroxide in liquid-liquid;slug flow of water;methylisobutylisoketone biphasic system;effect of hemin Reviewed

Toshinori Shimanouchi, Sota Sanagi, Satoko Fujioka, Koichi Terasaka, Yukitaka Kimura

Solvent Extr. Res. Dev. Japan 31 ( 2 ) 67 - 75 2024.4

Classification of binding property of amyloid β to lipid membranes: Membranomic research using quartz crystal microbalance combined with the immobilization of lipid planar membranes. Reviewed International journal

Toshinori Shimanouchi, Miki Iwamura, Yasuhiro Sano, Keita Hayashi, Minoru Noda, Yukitaka Kimura

Biochimica et biophysica acta. Proteins and proteomics 1872 ( 3 ) 140987 - 140987 2023.12

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

A biomembrane-related fibrillogenesis of Amyloid β from Alzheimer' disease (Aβ) is closely related to its accumulation behavior. A binding property of Aβ peptides from Alzheimer' disease to lipid membranes was then classified by a quartz crystal microbalance (QCM) method combined with an immobilization technique using thiol self-assembled membrane. The accumulated amounts of Aβ, Δfmax, was determined from the measurement of the maximal frequency reduction using QCM. The plots of Δfmax to Aβ concentration gave the slope and saturated value of Δfmax, (Δfmax)sat that are the parameters for binding property of Aβ to lipid membranes. Therefore, the Aβ-binding property on lipid membranes was classified by the slope and (Δfmax)sat. The plural lipid system was described as X + Y where X = L1, L1/L2, and L1/L2/L3. The slope and (Δfmax)sat values plotted as a function of mixing ratio of Y to X was classified on a basis of the lever principle (LP). The LP violation observed in both parameters resulted from the formation of the crevice or pothole, as Aβ-specific binding site, generated at the boundary between ld and lo phases. The LP violation observed only in the slope resulted from glycolipid-rich domain acting as Aβ-specific binding site. Furthermore, lipid planar membranes indicating strong LP violation favored strong fibrillogenesis. Especially, lipid planar membranes indicating the LP violation only in the slope induced lateral aggregated and spherulitic fibrillar aggregates. Thus, the classification of Aβ binding property on lipid membranes appeared to be related to the fibrillogenesis with a certain morphology.

DOI： 10.1016/j.bbapap.2023.140987

Amyloidogenic 60–71 deletion/ValThr insertion mutation of apolipoprotein A-I generates a new aggregation-prone segment that promotes nucleation through entropic effects

Norihiro Namba, Takashi Ohgita, Hiroko Tamagaki-Asahina, Kazuchika Nishitsuji, Toshinori Shimanouchi, Takeshi Sato, Hiroyuki Saito

Scientific Reports 13 ( 1 ) 2023.12

Publishing type：Research paper (scientific journal)

The N-terminal fragment of apolipoprotein A-I (apoA-I), comprising residues 1–83, contains three segments prone to aggregation: residues 14–22, 53–58, and 67–72. We previously demonstrated that residues 14–22 are critical in apoA-I fibril formation while residues 53–58 entropically drove the nucleation process. Here, we investigated the impact of amyloidogenic mutations (Δ60–71/VT, Δ70–72, and F71Y) located around residues 67–72 on fibril formation by the apoA-I 1–83 fragment. Thioflavin T fluorescence assay demonstrated that the Δ60–71/VT mutation significantly enhances both nucleation and fibril elongation rates, whereas the Δ70–72 and F71Y mutations had minimal effects. Circular dichroism measurements and microscopic observations revealed that all variant fragments formed straight fibrils, transitioning from random coils to β-sheet structures. Kinetic analysis demonstrated that primary nucleation is the dominant step in fibril formation, with fibril elongation reaching saturation at high protein concentrations. Thermodynamically, both nucleation and fibril elongation were enthalpically and entropically unfavorable in all apoA-I 1–83 variants, in which the entropic barrier of nucleation was almost eliminated for the Δ60–71/VT variant. Taken together, our results suggest the presence of new aggregation-prone segment in the Δ60–71/VT variant that promotes nucleation through entropic effects.

DOI： 10.1038/s41598-023-45803-y

Intramolecular interaction kinetically regulates fibril formation by human and mouse α-synuclein. International journal

Takashi Ohgita, Hiroki Kono, Izumi Morita, Hiroyuki Oyama, Toshinori Shimanouchi, Norihiro Kobayashi, Hiroyuki Saito

Scientific reports 13 ( 1 ) 10885 - 10885 2023.7

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

Regulation of α-synuclein (αS) fibril formation is a potent therapeutic strategy for αS-related neurodegenerative disorders. αS, an intrinsically disordered 140-residue intraneural protein, comprises positively charged N-terminal, hydrophobic non-amyloid β component (NAC), and negatively charged C-terminal regions. Although mouse and human αS share 95% sequence identity, mouse αS forms amyloid fibrils faster than human αS. To evaluate the kinetic regulation of αS fibrillation, we examined the effects of mismatched residues in human and mouse αS on fibril formation and intramolecular interactions. Thioflavin T fluorescence assay using domain-swapped or C-terminal-truncated αS variants revealed that mouse αS exhibited higher nucleation and fibril elongation than human αS. In mouse αS, S87N substitution in the NAC region rather than A53T substitution is dominant for enhanced fibril formation. Fӧrester resonance energy transfer analysis demonstrated that the intramolecular interaction of the C-terminal region with the N-terminal and NAC regions observed in human αS is perturbed in mouse αS. In mouse αS, S87N substitution is responsible for the perturbed interaction. These results indicate that the interaction of the C-terminal region with the N-terminal and NAC regions suppresses αS fibril formation and that the human-to-mouse S87N substitution in the NAC region accelerates αS fibril formation by perturbing intramolecular interaction.

DOI： 10.1038/s41598-023-38070-4

Hydrothermal Preparation of Faceted Vesicles Made of Span 40 and Tween 40 and Their Characterization

Toshinori Shimanouchi, Yui Komori, Kazuki Toramoto, Keita Hayashi, Kazuma Yasuhara, Ho Sup Jung, Yukitaka Kimura

Applied Sciences (Switzerland) 13 ( 12 ) 2023.6

Publishing type：Research paper (scientific journal)

The Span 40 (sorbitan monooleate)/Tween 40 (polyoxyethylene sorbitan monolaurate) system gives faceted vesicles with angular surfaces, rather than spherical vesicles. Herein, a continuous and facile preparation method, based on the subcritical water-assisted emulsification and solvent diffusion, was presented to yield faceted vesicles with two major and minor axes (Type A) and vesicles closer to a polyhedron (Type B). Type A, rather than Type B, vesicles were likely to be formed. From the measurements concerning ζ-potential, membrane fluidity, and the polarization environment of the membranes, faceted vesicles could be obtained at 0.25 wt% of the surfactant concentration. The phase-separated behavior of Span 40 and Tween 40 within vesicle membranes could explain the structural feature of faceted vesicles and calcein leakage behavior. The significant advantage is that Type A vesicles would be utilized as alternative drug carriers for others with low encapsulation efficiency, although the present technical limitations cause difficulty in the selective formation of Type A and B vesicles and the selection of adequate solvent to accelerate the solvent diffusion step.

DOI： 10.3390/app13126893

Cholesterol as a Subsidiary Component of Sorbitan Surfactant-Based Aggregates: A Study of Formation, Hydrophobicity, and Estimation of Localization of Embedded Molecules. International journal

Keita Hayashi, Hikaru Ota, Haruna Sugimura, Toshinori Shimanouchi, Tomoyuki Iwasaki, Sakiko Fujita, Hidemi Nakamura, Hiroshi Umakoshi

The journal of physical chemistry. B 127 ( 10 ) 2214 - 2223 2023.3

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

Aggregates of amphiphilic molecules can be used as drug carriers, for which the properties can be modified by mixing with other molecules such as cholesterol. It is important to understand the effects of such additives on the properties because they directly define the material functions. In this work, we investigated the effect of cholesterol on the formation and hydrophobicity of aggregates of sorbitan surfactants. As cholesterol changed its formation from micelles to vesicles, an increase in hydrophobicity was seen, particularly in the middle regions compared with the shallow and deep regions. We show that this gradual hydrophobicity is related to the localization of the embedded molecules. 4-Hydroxy-TEMPO and 4-carboxy-TEMPO were preferentially localized in the shallow region of the aggregates, whereas 4-PhCO2-TEMPO was preferentially localized in the deep region of the vesicle. The localization of molecules depends on their chemical structure. However, the localization of 4-PhCO2-TEMPO in micelles was not observed, despite the similar hydrophobicity in the hydrophobic region within the aggregates. The localization of embedded molecules was related to other properties, such as molecular mobility.

DOI： 10.1021/acs.jpcb.2c08153

Conversion of Glycerol to Lactic Acid by Using Platinum-supported Catalyst Combined with Phosphatidylcholine Vesicles

Toshinori Shimanouchi, Yuki Takahashi, Kazuma Yasuhara, Yukitaka Kimura

Chemistry Letters 52 ( 6 ) 426 - 429 2023

Possible Role of Vesicles on Metallocatalytic Reduction Reaction of 5-Hydroxymethylfurfural to 2,5-Dimethylfuran Reviewed

Toshinori Shimanouchi, Yuki Takahashi, Keita Hayashi, Kazuma Yasuhara, Yukitaka Kimura

Compounds 2 ( 4 ) 321 - 333 2022.11

Authorship：Lead author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：MDPI AG

A reduction reaction of 5-hydroxymethylfurfural to 2,5-dimethylfuran (2,5-DMF) has been previously performed in an organic solvent under high-temperature conditions. For the relaxation of such reaction conditions, conventional palladium on carbon (Pd/C) was combined with vesicles composed of phospholipids or surfactants. Pd/C combined with 1,2-dioleoyl-sn-glycero-3-phosphocholine indicated a yield (25%) at 60 °C compared with Pd/C (17%). Vesicles at the liquid crystalline phase were advantageous for the reduction reaction of HMF. The yield of 2,5-DMF catalyzed by Pd/C combined with the vesicles depended on the lipid composition of the vesicles. It was clarified that the yield of 2,5-DMF could be controlled by the hydration property of the vesicles. Compared with conventional 2,5-DMF synthesis in an organic solvent, the use of vesicles made it possible to reduce the burden of using organic solvents in high-temperature conditions, although limitedly.

DOI： 10.3390/compounds2040027

Possible Role of Bent Structure of Methylated Lithocholic Acid on Artificial and Plasma Membranes

Tomoyuki Iwasaki, Nobuyuki Endo, Yuta Nakayama, Toshiyuki Kamei, Toshinori Shimanouchi, Hidemi Nakamura, Keita Hayashi

Membranes 12 ( 10 ) 2022.10

Publishing type：Research paper (scientific journal)

Bile acids form micelles that are essential for the absorption of dietary lipids. However, excessive bile acid micelles can disrupt the plasma membrane by removing phospholipids, resulting in cell death. We hypothesized that the bent geometrical structure of the steroid scaffold of bile acids decreases the lipid order (similar to unsaturated phospholipids with cis double bonds), disrupting the plasma membrane. Here, lithocholic acid (LCA), a bile acid, was methylated to prevent micellization. Methylated lithocholic acid (Me-LCA) was mixed with a thin phase-separated lipid bilayer comprising 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and cholesterol (Chol). Me-LCA was not localized in the DPPC-rich rigid phase but localized in the DOPC-rich fluid phase, and excess Me-LCA did not affect the phase separation. Me-LCA is distributed in the plasma and organelle membranes. However, Me-LCA with bent structure did not affect the membrane properties, membrane fluidity, and hydrophobicity of liposomes composed of DOPC, DPPC, and Chol and also did not affect the proliferation of cells.

DOI： 10.3390/membranes12100997

Amyloid-β aggregates induced by β-cholesteryl glucose-embedded liposomes Reviewed

Toshinori Shimanouchi, Yasuhiro Sano, Kazuma Yasuhara, Yukitaka Kimura

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 1870 ( 8 ) 140816 - 140816 2022.8

Mechanisms of enhanced aggregation and fibril formation of Parkinson's disease-related variants of α-synuclein. Reviewed International journal

Takashi Ohgita, Norihiro Namba, Hiroki Kono, Toshinori Shimanouchi, Hiroyuki Saito

Scientific reports 12 ( 1 ) 6770 - 6770 2022.4

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

Aggregation of α-synuclein (α-syn) into amyloid fibrils is closely associated with Parkinson's disease (PD). Familial mutations or posttranslational truncations in α-syn are known as risk factor for PD. Here, we examined the effects of the PD-related A30P or A53T point mutation and C-terminal 123-140 or 104-140 truncation on the aggregating property of α-syn based on the kinetic and thermodynamic analyses. Thioflavin T fluorescence measurements indicated that A53T, Δ123‒140, and Δ104-140 variants aggregated faster than WT α-syn, in which the A53T mutation markedly increases nucleation rate whereas the Δ123‒140 or Δ104‒140 truncation significantly increases both nucleation and fibril elongation rates. Ultracentrifugation and western blotting analyses demonstrated that these mutations or truncations promote the conversion of monomer to aggregated forms of α-syn. Analysis of the dependence of aggregation reaction of α-syn variants on the monomer concentration suggested that the A53T mutation enhances conversion of monomers to amyloid nuclei whereas the C-terminal truncations, especially the Δ104-140, enhance autocatalytic aggregation on existing fibrils. In addition, thermodynamic analysis of the kinetics of nucleation and fibril elongation of α-syn variants indicated that both nucleation and fibril elongation of WT α-syn are enthalpically and entropically unfavorable. Interestingly, the unfavorable activation enthalpy of nucleation greatly decreases for the A53T and becomes reversed in sign for the C-terminally truncated variants. Taken together, our results indicate that the A53T mutation and the C-terminal truncation enhance α-syn aggregation by reducing unfavorable activation enthalpy of nucleation, and the C-terminal truncation further triggers the autocatalytic fibril elongation on the fibril surfaces.

DOI： 10.1038/s41598-022-10789-6

Sulfated glycosaminoglycans mediate prion-like behavior of p53 aggregates Reviewed International journal

Naoyuki Iwahashi, Midori Ikezaki, Taro Nishikawa, Norihiro Namba, Takashi Ohgita, Hiroyuki Saito, Yoshito Ihara, Toshinori Shimanouchi, Kazuhiko Ino, Kenji Uchimura, Kazuchika Nishitsuji

Proceedings of the National Academy of Sciences of the United States of America 117 ( 52 ) 33225 - 33234 2021.12

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

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) are heteropolysaccharides implicated in the pathology of protein aggregation diseases including localized and systemic forms of amyloidosis. Among subdomains of sulfated GAGs, highly sulfated domains of HS, called HS S-domains, have been highlighted as being critical for HS function in amyloidoses. Recent studies suggest that the tumor suppressor p53 aggregates to form amyloid fibrils and propagates in a prion-like manner; however, molecules and mechanisms that are involved in the prion-like behavior of p53 aggregates have not been addressed. Here, we identified sulfated GAGs as molecules that mediate prion-like behavior of p53 aggregates. Sulfated GAGs at the cell surface were required for cellular uptake of recombinant and cancer cell-derived p53 aggregates and extracellular release of p53 from cancer cells. We further showed that HS S-domains accumulated within p53 deposits in human ovarian cancer tissues, and enzymatic remodeling of HS S-domains by Sulf-2 extracellular sulfatase down-regulated cellular uptake of p53 aggregates. Finally, sulfated GAG-dependent cellular uptake of p53 aggregates was critical for subsequent extracellular release of the aggregates and gain of oncogenic function in recipient cells. Our work provides a mechanism of prion-like behavior of p53 aggregates and will shed light on sulfated GAGs as a common mediator of prions.

DOI： 10.1073/PNAS.2009931117

Fibril growth behavior of amyloid β on polymer-based planar membranes: Implications for the entanglement and hydration of polymers Reviewed

Toshinori Shimanouchi, Miki Iwamura, Shintaro Deguchi, Yukitaka Kimura

Applied Sciences (Switzerland) 11 ( 10 ) 4408 2021.7

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

The design of biosensors and artificial organs using biocompatible materials with a low affinity for amyloid β peptide (Aβ) would contribute to the inhibition of fibril growth causing Alzheimer’s disease. We systematically studied the amyloidogenicity of Aβ on various planar mem-branes. The planar membranes were prepared using biocompatible polymers, viz., poly(methyl methacrylate) (PMMA), polysulfone (PSf), poly(L-lactic acid) (PLLA), and polyvinylpyrrolidone (PVP). Phospholipids from biomembranes, viz., 1,2-dioleoyl-phosphatidylcholine (DOPC), 1,2-dipalmitoyl-phosphatidylcholine (DPPC), and polyethylene glycol-graft-phosphatidyl ethanolamine (PEG-PE) were used as controls. Phospholipid-and polymer-based membranes were prepared to determine the kinetics of Aβ fibril formation. Rates of Aβ nucleation on the PSf-and DPPC-based membranes were significantly higher than those on the other membranes. Aβ accumulation, cal-culated by the change in frequency of a quartz crystal microbalance (QCM), followed the order: PSf > PLLA > DOPC > PMMA, PVP, DPPC, and PEG-PE. Nucleation rates exhibited a positive correlation with the corresponding accumulation (except for the DPPC-based membrane) and a negative correlation with the molecular weight of the polymers. Strong hydration along the polymer backbone and polymer–Aβ entanglement might contribute to the accumulation of Aβ and subsequent fibrillation.

DOI： 10.3390/app11104408

Quantitative Determination of Relative Permittivity Based on the Fluorescence Property of Pyrene Derivatives: An Interpretation of Hydrophobicity in Self-Assembled Aggregates of Nonionic Amphiphiles. Reviewed International journal

Keita Hayashi, Haruna Sugimura, Toshiyuki Kamei, Toshinori Shimanouchi, Hidemi Nakamura, Hiroshi Umakoshi

The journal of physical chemistry. B 125 ( 23 ) 6192 - 6200 2021.6

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

Aggregates in aqueous solutions can embed hydrophobic molecules, and their interactions depend on the properties of the aggregates. The electric surface potential, molecular mobility, and gradual hydrophobicity are the properties that regulate the interactions, and it is essential to understand these to quantify the properties. Electric surface potential and molecular mobility are quantified using the zeta potential and NMR measurements. In this study, the quantification of gradual hydrophobicity within the aggregate based on the relative permittivity, also called the dielectric constant, has been estimated from fluorescence spectra of pyrene-dicarboxylic acid conjugates. The localization of the pyrene moiety was modified by conjugation with succinic acid, suberic acid, or dodecanedioic acid, and the conjugates were evaluated in the shallow, middle, and deep regions of the aggregates. Span and Tween surfactants have been employed to prepare these aggregates, because they form various kinds of aggregates such as micelles and vesicles. It was realized that the hydrophobicity gradually increased from the interface to the hydrophobic core. Alternatively, a comparison of hydrophobicity within the aggregates showed no remarkable difference. Moreover, the analyses suggested that there are a few water molecules in the deep region. These results support the idea of the localization of embedded molecules in aggregates.

DOI： 10.1021/acs.jpcb.1c00170

Microfluidic and hydrothermal preparation of vesicles using sorbitan monolaurate/polyoxyethylene (20) sorbitan monolaurate (Span 20/Tween 20). Reviewed International journal

Toshinori Shimanouchi, Tetsuya Hayashi, Kazuki Toramoto, Saki Fukuma, Keita Hayashi, Kazuma Yasuhara, Yukitaka Kimura

Colloids and surfaces. B, Biointerfaces 205 111836 - 111836 2021.5

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

Here, we present a method for preparing vesicles by combining hydrothermal emulsification with solvent diffusion (SD). The sorbitan monolaurate/polyoxyethylene (20) sorbitan monolaurate (Span 20/Tween 20) system was used as the target lipid because these lipids are cheap and advantageous for the production scale. The water-in-oil (W/O) emulsion stabilized with lipids was formed under hydrothermal conditions (240 °C under 10 MPa), followed by mixing with water that included lipids to obtain a W/O-in-water (W/O/W) emulsion. The SD for the W/O/W emulsion as a subsequent process yielded vesicles. The optimal preparation conditions were 50:50 wt% Span 20/Tween 20 as a mixing ratio (final lipid concentration 12 mM), octanoic acid as an organic solvent, 240 °C for 4 min during the hydrothermal treatment, and 4 °C for 24 h in the SD process. The diameter of the vesicles obtained was at most 100 nm, which was comparable to that of the W/O/W emulsion before SD. This suggested that the W/O/W emulsion acted as a template for vesicle formation. The number density, diameter, and membrane properties of vesicles depend on the mixing ratio of the water/oil/lipid system. Specifically, the number density of vesicles was low relative to that of vesicles prepared by the conventional method.

DOI： 10.1016/j.colsurfb.2021.111836

Characterization of water/organic biphasic system in liquid-liquid slug flow under hydrothermal conditions: Solvation, vorticity, and hydrophobicity Reviewed

Toshinori Shimanouchi, Satoko Fujioka, Tatsuya Tanifuji, Kenta Yamamoto, Koichi Terasaka, Yukitaka Kimura

Solvent Extraction Research and Development 28 ( 1 ) 21 - 35 2021.4

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

The microcapillary was combined with the liquid-liquid slug flow to build the separation field. The partition behavior of seventeen kinds of substrates was investigated by using six kinds of water/organic biphasic systems under hydrothermal conditions (25 - 190° C at 10 MPa). The scale of hydrophobicity (HF) of water/organic biphasic system was estimated. The HF value for the biphasic system used here was comparable to the conventional aqueous two-phase systems and depended on the (de)solvation of substrates by water and organic solvent. Besides, the vortex field in the slug contributed to the enhanced mass transfer of substrates. Those results are available for the selection of water/organic biphasic system as the reactive separation of target materials.

DOI： 10.15261/SERDJ.28.21

バイセルのベシクルへの遷移法のための流体合流を用いたバイセルの連続調製 Reviewed

SungHak Choi, BongSu Kang, Toshinori Shimanouchi, Keesung Kim, HoSup Jung

Micro and Nano Systems Letters 9 ( 7 ) 2021

Language：English

Bicelle is one of the most stable phospholipid assemblies, which has tremendous applications in the research areas for drug delivery or structural studies of membrane proteins owing to its bio-membrane mimicking characteristics and high thermal stability. However, the conventional preparation method for bicelle demands complicated manufacturing processes and a long time so that the continuous synthesis method of bicelle using microfluidic chip has been playing an important role to expand its feasibility. We verified the general availability of hydrodynamic focusing method with microfluidic chip for bicelle synthesis using various kinds of lipids which have a phase transition temperature ranged from − 2 to 41 °C. Bicelle can be formed only when the inside temperature of microfluidic chip was over the phase transition temperature. Moreover, the concentration condition for bicelle formation varied depending on the lipids. Furthermore, the transition process characteristics from bicelle to vesicle were analyzed by effective q-value, mixing time and dilution condition. We verified that the size of transition vesicles was controlled according to the effective q-value, mixing time, and temperature.

DOI： 10.1186/s40486-021-00133-4

Sulfated glycosaminoglycans mediate prion-like behavior of p53 aggregates Reviewed

Naoyuki Iwahashi, Midori Ikezaki, Taro Nishikawa, Norihiro Namba, Takashi Ohgita, Hiroyuki Saito, Yoshito Ihara, Toshinori Shimanouchi, Kazuhiko Ino, Kenji Uchimura, Kazuchika Nishitsuji

Proceedings of the National Academy of Sciences of the United States of America 117 ( 52 ) 33225 - 33234 2020.12

Publishing type：Research paper (scientific journal)

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) are heteropolysaccharides implicated in the pathology of protein aggregation diseases including localized and systemic forms of amyloidosis. Among subdomains of sulfated GAGs, highly sulfated domains of HS, called HS S-domains, have been highlighted as being critical for HS function in amyloidoses. Recent studies suggest that the tumor suppressor p53 aggregates to form amyloid fibrils and propagates in a prion-like manner; however, molecules and mechanisms that are involved in the prion-like behavior of p53 aggregates have not been addressed. Here, we identified sulfated GAGs as molecules that mediate prion-like behavior of p53 aggregates. Sulfated GAGs at the cell surface were required for cellular uptake of recombinant and cancer cell-derived p53 aggregates and extracellular release of p53 from cancer cells. We further showed that HS S-domains accumulated within p53 deposits in human ovarian cancer tissues, and enzymatic remodeling of HS S-domains by Sulf-2 extracellular sulfatase down-regulated cellular uptake of p53 aggregates. Finally, sulfated GAG-dependent cellular uptake of p53 aggregates was critical for subsequent extracellular release of the aggregates and gain of oncogenic function in recipient cells. Our work provides a mechanism of prion-like behavior of p53 aggregates and will shed light on sulfated GAGs as a common mediator of prions.

DOI： 10.1073/PNAS.2009931117

Label-free, chronological and selective detection of aggregation and fibrillization of amyloid β protein in serum by microcantilever sensor immobilizing cholesterol-incorporated liposome Reviewed International journal

Tomoya Taniguchi, Toshinori Shimanouchi, Masayuki Sohgawa, Minoru Noda

Biotechnology and Bioengineering 117 ( 8 ) 2469 - 2478 2020.8

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

To facilitate the early diagnosis of Alzheimer's disease and mild cognitive impairment patients, we developed a cantilever-based microsensor that immobilized liposomes of various phospholipids to detect a trace amount of amyloid β (Aβ) protein, and investigated its aggregation and fibrillization on model cell membranes in human serum. Three species of liposomes composed of different phospholipids of 1,2-dipalmtoyl-sn-glycero-3-phosphocholine (DPPC), DPPC/phosphatidyl ethanolamine and 1,2-dipalmitoyl-sn-glycero-3-phosphorylglycerol having varied hydrophilic groups were applied, which showed different chronological interactions with Aβ(1–40) protein and varied sensitivities of the cantilever sensor, depending on their specific electrostatic charged conditions, hydrophilicity, and membrane fluidity. 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) having short hydrophobic carbon chains confirmed to show a large interaction with Aβ(1–40) and a high sensitivity. Furthermore, the incorporation of cholesterol into DMPC was effective to selectively detect Aβ(1–40) in human serum, which effect was also checked by quartz crystal microbalance. Finally, Aβ detection of 100-pM order was expected selectively in the serum by using the developed biosensor.

DOI： 10.1002/bit.27380

Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/bit.27380

Phosphatidylethanolamine accelerates aggregation of the amyloidogenic N-terminal fragment of apoA-I Reviewed International journal

Naoko Kurimitsu, Chiharu Mizuguchi, Kaho Fujita, Suzuno Taguchi, Takashi Ohgita, Kazuchika Nishitsuji, Toshinori Shimanouchi, Hiroyuki Saito

FEBS Letters 594 ( 9 ) 1443 - 1452 2020.5

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

Membrane lipid composition is known to influence aggregation and fibril formation of many amyloidogenic proteins. Here, we found that phosphatidylethanolamine (PE) accelerates aggregation of the N-terminal 1‒83 fragment of an amyloidogenic G26R variant of apoA-I on lipid membranes. Circular dichroism and isothermal titration calorimetry measurements demonstrated that PE does not affect the α-helical structure and lipid binding property of apoA-I 1-83/G26R. Rather, fluorescence measurements indicated that PE induces more ordered lipid packing at the interfacial and acyl chain regions, providing more hydrophobic environments especially around the highly amyloidogenic regions in apoA-I on the membrane surface. These results suggest that PE promotes aggregation of the amyloidogenic N-terminal fragment of apoA-I on lipid membranes by inducing hydrophobic membrane environments.

DOI： 10.1002/1873-3468.13737

Analysis of the Partitioning Behavior of Horseradish Peroxidase to Phospholipid and Surfactant Membranes Reviewed

Saki Fukuma, Toshinori Shimanouchi, Kazuma Yasuhara, Yukitaka Kimura

Solvent Extraction Research and Development 27 ( 2 ) 113 - 213 2020

Publishing type：Research paper (scientific journal)

The partitioning behavior of horseradish peroxidase (HRP) to phospholipid- and surfactant vesicles has been analyzed. Peripheral binding of the zwitterionic phospholipid vesicle membranes is proposed with insertion into the membrane interior of negatively charged surfactant vesicles, resulting from non-electrostatic interaction. The insertion of HRP is interpreted by considering the interaction between headgroups of phospholipid and between headgroups of surfactant.

DOI： 10.15261/serdj.27.113

Application of liposome membrane as the reaction field: A case study using the Horner–Wadsworth–Emmons reaction Reviewed

Toshinori Shimanouchi, Yuki Kitagawa, Yukitaka Kimura

Journal of Bioscience and Bioengineering 128 ( 2 ) 198 - 202 2019.8

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

The properties of the liposome membrane as a reaction field were investigated by focusing on the Horner–Wadsworth–Emmons reaction as a case study. Use of the liposomes existing in the gel phase resulted in the enhanced activity of the substrates and furnished the products with same E/Z stereoselectivity as in the liposome-free system. The membrane environment in the gel phase most likely assisted the formation of adducts that induced selective generation of the E-isomer. The possible role of liposomes is to assist the proton removal from the reactant, rather than providing the basic interfacial environment.

DOI： 10.1016/j.jbiosc.2019.01.015

Alteration of Membrane Physicochemical Properties by Two Factors for Membrane Protein Integration Reviewed International journal

K. Nomura, Toshiyuki Yamaguchi, S. Mori, K. Fujikawa, Ken ichi Nishiyama, Toshinori Shimanouchi, Yasushi Tanimoto, Kenichi Morigaki, K. Shimamoto

Biophysical Journal 117 ( 1 ) 99 - 110 2019.7

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

After a nascent chain of a membrane protein emerges from the ribosomal tunnel, the protein is integrated into the cell membrane. This process is controlled by a series of proteinaceous molecular devices, such as signal recognition particles and Sec translocons. In addition to these proteins, we discovered two endogenous components regulating membrane protein integration in the inner membrane of Escherichia coli. The integration is blocked by diacylglycerol (DAG), whereas the blocking is relieved by a glycolipid named membrane protein integrase (MPIase). Here, we investigated the influence of these integration-blocking and integration-promoting factors on the physicochemical properties of membrane lipids via solid-state NMR and fluorescence measurements. These factors did not have destructive effects on membrane morphology because the membrane maintained its lamellar structure and did not fuse in the presence of DAG and/or MPIase at their effective concentrations. We next focused on membrane flexibility. DAG did not affect the mobility of the membrane surface, whereas the sugar chain in MPIase was highly mobile and enhanced the flexibility of membrane lipid headgroups. Comparison with a synthetic MPIase analog revealed the effects of the long sugar chain on membrane properties. The acyl chain order inside the membrane was increased by DAG, whereas the increase was cancelled by the addition of MPIase. MPIase also loosened the membrane lipid packing. Focusing on the transbilayer movement, MPIase reduced the rapid flip-flop motion of DAG. On the other hand, MPIase could not compensate for the diminished lateral diffusion by DAG. These results suggest that by manipulating the membrane lipids dynamics, DAG inhibits the protein from contacting the inner membrane, whereas the flexible long sugar chain of MPIase increases the opportunity for interaction between the membrane and the protein, leading to membrane integration of the newly formed protein.

DOI： 10.1016/j.bpj.2019.05.014

Temperature Measurement by Sublimation Rate as a Process Analytical Technology Tool in Lyophilization Reviewed International journal

Hidenori Kawasaki, Toshinori Shimanouchi, Hiroyuki Sawada, Hiroshi Hosomi, Yuta Hamabe, Yukitaka Kimura

Journal of Pharmaceutical Sciences 108 ( 7 ) 2305 - 2314 2019.7

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

Product temperature (T ) and drying time constitute critical material attributes and process parameters in the lyophilization process and especially during the primary drying stage. In the study, we performed a temperature measurement by the sublimation rate (TMbySR) to monitor the T value and determine the end point of primary drying. First, the water vapor transfer resistance coefficient through the main pipe from the chamber to the condenser (C ) was estimated via the water sublimation test. The use of C value made it possible to obtain the time course of T from the measurement of pressure at the drying chamber and at the condenser. Second, a Flomoxef sodium bulk solution was lyophilized by using the TMbySR system. The outcome was satisfactory when compared with that obtained via conventional sensors. The same was applicable for the determination of the end point of primary drying. A laboratory-scale application of the TMbySR system was evidenced via the experiment using 220-, 440-, and 660-vial scales of lyophilization. The outcome was not dependent on the loading amount. Thus, the results confirmed that the TMbySR system is a promising tool in laboratory scale. b b r r b

DOI： 10.1016/j.xphs.2019.02.015

Kinetic pH Titration to Predict the Acid and Hydrothermal Conditions for the Hydrolysis of Disaccharides: Use of a Microcapillary System Reviewed

Toshinori Shimanouchi, Ryota Mano, Yu Yoshioka, Ayaka Fukuda, Kyung Min Park, Yukitaka Kimura

Journal of Chemistry 2019 3985915 2019

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

The hydrolysis of disaccharides was conducted using a microcapillary system under hydrothermal conditions (up to 190°C at 10 MPa and pH 4-11). The hydrolysis reaction showed a sigmoidal progression with time, especially under alkaline conditions. Analysis using a kinetic model yielded the reaction induction period. The specific pH value (pH ) at the induction time, which is the pH value corresponding to the progression of disaccharide hydrolysis, was peculiar to each disaccharide. Finally, the calculation of the electron density around the oxygen atom of the glycosidic bond between saccharides was found to roughly predict the pH value required for the progression of hydrolysis. amb amb

DOI： 10.1155/2019/3985915

Structural and physicochemical characteristics of granular malic acid-treated sweet potato starch containing heat-stable resistant starch Reviewed

Chinwoo Kwon, Ha Ram Kim, Tae Wha Moon, Seung Hyun Lee, Chang Joo Lee, Toshinori Shimanouchi

Journal of Chemistry 2019 2019

Publishing type：Research paper (scientific journal)

This study investigated the structural and physicochemical characteristics of malic acid-treated sweet potato starch. Sweet potato starch mixed with various concentrations of malic acid solution underwent either thermal or nonthermal treatment. Observation of samples under a light microscope ensured the maintenance of granular shape and the Maltese cross. FT-IR spectra displayed a distinct carbonyl peak at 1722 cm , and analysis of the degree of substitution (DS) indicated an increase in the extent of ester bonds with increasing concentrations of malic acid. The DS of 2.0M-130 (0.214) was the highest and that of 0.5M-130 was the lowest (0.088) among the reacted starches. In vitro digestion test revealed an increased amount of resistant starch when a high concentration of malic acid was used. In addition, thermally treated samples maintained a higher content of resistant starch (RS) after 30 min of cooking at 100°C. After cooking, 2.0M-130 had an RS fraction of 53.4% which was reduced to 49.9% after cooking, revealing greater heat stability compared with nonthermally treated samples. The structure of malic acid-treated starch was investigated using a differential scanning calorimeter (DSC), an X-ray diffractometer, a rapid visco analyzer (RVA), and analysis of apparent amylose content. The results showed that thermal and malic acid treatment of starch caused not only partial hydrolysis but also rearrangement of the crystalline area and helix structure of starch by esterification. Analysis of malic acid-treated starch, using a rapid visco analyzer showed no pasting properties, due to lack of its swelling caused by the malic acid cross link. -1

DOI： 10.1155/2019/2903252

Recent Development of Optimization of Lyophilization Process Reviewed

Hidenori Kawasaki, Toshinori Shimanouchi, Yukitaka Kimura

Journal of Chemistry 2019 2019

Publishing type：Research paper (scientific journal)

The objective of this review is to survey the development of the optimization of lyophilization. The optimization study of the lyophilizer has been roughly developing by the order of (i) trial-and-error approach, (ii) process modeling using mathematical models, (iii) scalability, and (iv) quality-by-design. From the conventional lyophilization studies based on the trial-and-error, the key parameters to optimize the operation of lyophilization were found out, i.e., critical material attributes (CMAs), critical process parameters (CPPs), and critical quality attributes (CQAs). The mathematical models using the key parameters mentioned above have been constructed from the viewpoints of the heat and mass transfer natures. In many cases, it is revealed that the control of the primary drying stage determines the outcome of the lyophilization of products, as compared with the freezing stage and the secondary drying stage. Thus, the understanding of the lyophilization process has proceeded. For the further improvement of the time and economical cost, the design space is a promising method to give the possible operation range for optimizing the lyophilization operation. This method is to search the optimized condition by reducing the number of key parameters of CMAs, CPPs, and CQAs. Alternatively, the transfer of lyophilization recipe among the lab-, pilot-, and production-scale lyophilizers (scale-up) has been examined. Notably, the scale-up of lyophilization requires the preservation of lyophilization dynamics between the two scales, i.e., the operation of lab- or pilot-scale lyophilizer under HEPA-filtrated airflow condition. The design space determined by focusing on the primary drying stage is large and involves the undesired variations in the quality of final products due to the heterogeneous size distribution of ice crystals. Accordingly, the control of the formation of the ice crystal with large size gave impact on the product quality and the productivity although the large water content in the final product should be improved. Therefore, the lyophilization should take into account the quality by design (QbD). The monitoring method of the quality of the product in lyophilization process is termed the "process analytical technology (PAT)." Recent PAT tools can reveal the lyophilization dynamics to some extent. A combination of PAT tools with a model/scale-up theory is expected to result in the QbD, i.e., a quality/risk management and an in situ optimization of lyophilization operation.

DOI： 10.1155/2019/9502856

Innovative Strategies and Emerging Technologies for Food Safety Reviewed

Kyung Min Park, Sang Hyun Park, Toshinori Shimanouchi

Journal of Chemistry 2019 2019

Effect of phosphatidylserine and cholesterol on membrane-mediated fibril formation by the N-terminal amyloidogenic fragment of apolipoprotein A-I Reviewed International journal

Chiharu Mizuguchi, Mitsuki Nakamura, Naoko Kurimitsu, Takashi Ohgita, Kazuchika Nishitsuji, Teruhiko Baba, Akira Shigenaga, Toshinori Shimanouchi, Keiichiro Okuhira, Akira Otaka, Hiroyuki Saito

Scientific Reports 8 ( 1 ) 5497 - 5497 2018.12

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

Here, we examined the effects of phosphatidylserine (PS) and cholesterol on the fibril-forming properties of the N-terminal 1-83 fragment of an amyloidogenic G26R variant of apoA-I bound to small unilamellar vesicles. A thioflavin T fluorescence assay together with microscopic observations showed that PS significantly retards the nucleation step in fibril formation by apoA-I 1-83/G26R, whereas cholesterol slightly enhances fibril formation. Circular dichroism analyses demonstrated that PS facilitates a structural transition from random coil to α-helix in apoA-I 1-83/G26R with great stabilization of the α-helical structure upon lipid binding. Isothermal titration calorimetry measurements revealed that PS induces a marked increase in capacity for binding of apoA-I 1-83/G26R to the membrane surface, perhaps due to electrostatic interactions of positively charged amino acids in apoA-I with PS. Such effects of PS to enhance lipid interactions and inhibit fibril formation of apoA-I were also observed for the amyloidogenic region-containing apoA-I 8-33/G26R peptide. Fluorescence measurements using environment-sensitive probes indicated that PS induces a more solvent-exposed, membrane-bound conformation in the amyloidogenic region of apoA-I without affecting membrane fluidity. Since cell membranes have highly heterogeneous lipid compositions, our findings may provide a molecular basis for the preferential deposition of apoA-I amyloid fibrils in tissues and organs.

DOI： 10.1038/s41598-018-23920-3

Design of Pyrene-Fatty Acid Conjugates for Real-Time Monitoring of Drug Delivery and Controllability of Drug Release Reviewed International journal

Keita Hayashi, Yuma Mitsuyoshi, Toshiyuki Kamei, Toshinori Shimanouchi, Keishi Suga, Yukihiro Okamoto, Hidemi Nakamura, Hiroshi Umakoshi

ACS Omega 3 ( 3 ) 3572 - 3580 2018.3

Language：English Publishing type：Research paper (scientific journal) Publisher：American Chemical Society

Fluorescence probes are usually employed to analyze pharmacokinetics of drug carriers; however, this method using usual probes is not suitable to monitor drug carriers in detail because fluorescence spectra do not change by the disruption of drug carriers. In this study, pyrene-fatty acid conjugates were investigated as probes to monitor the state of drug carriers in real time. 1-Pyrenemethanol was conjugated with fatty acids, such as lauric acid, stearic acid, and behenic acid, and the conjugates were stirred in ethanol, resulting in the formation of submicron particles; these particles exhibited excimer emission. When J774.1 and Colon 26 cells were treated with these particles, the associated fluorescence spectra shifted from excimer emission to monomer emission. Moreover, the degree of change was controlled by the type of fatty acid. These results support the design of drug carriers that can be used to monitor pharmacokinetics in real time and to control the disruption time.

DOI： 10.1021/acsomega.7b02061

Scale-up procedure for primary drying process in lyophilizer by using the vial heat transfer and the drying resistance Reviewed

Hidenori Kawasaki, Toshinori Shimanouchi, Masaharu Yamamoto, Kanako Takahashi, Yukitaka Kimura

Chemical and Pharmaceutical Bulletin 66 ( 11 ) 1048 - 1056 2018

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

The objective of this study is to design primary drying conditions in a production lyophilizer based on a pilot lyophilizer. Although the shelf temperature and the chamber pressure need to be designed to maintain the sublimation interface temperature of the formulation below the collapse temperature, it is difficult to utilize a production lyophilizer to optimize cycle parameters for manufacturing. In this report, we assumed that the water vapor transfer resistance (Rp) in the pilot lyophilizer can be used in the commercial lyophilizer without any correction, under the condition where both lyophilizers were operated in the high efficiency particulate air (HEPA)-filtrated airflow condition. The shelf temperature and the drying time for the commercial manufacturing were designed based on the maximum Rp value calculated from the pilot lyophilizer (1008 vials) under HEPA-filtrated airflow condition and from the vial heat transfer coefficient of the production lyophilizer (6000 vials). And, the cycle parameters were verified using the production lyophilizer of 60000 vials. It was therefore concluded that the operation of lab- or pilot-scale lyophilizer under HEPA-filtrated airflow condition was one of important factors for the scale-up.

DOI： 10.1248/cpb.c18-00516

Effect of controlled nucleation of ice crystals on the primary drying stage during lyophilization Reviewed

Hidenori Kawasaki, Toshinori Shimanouchi, Kanako Takahashi, Yukitaka Kimura

Chemical and Pharmaceutical Bulletin 66 ( 12 ) 1122 - 1130 2018

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

The freezing stage cannot be directly controlled, which leads to variation in product quality and low productivity during the lyophilization process. Our objective was to establish a robust design space for the primary drying stage using ice nucleation control based on the pressurization and depressurization technique. We evaluated the specific surface area (SSA), water content, scanning electron microscopy (SEM) images, and water vapor transfer resistance of the dried layer (R ) of the products. The ice nucleation control resulted in a reduction of the SSA value and in an increase in water content. SEM observation suggested that the ice nucleation control enabled formation of large ice crystals, which was consistent with the reduction in the R value. As a result, the generation of collapsed cakes was inhibited, whereas 18% of the collapsed cakes were observed without ice nucleation control. Finally, this technique succeeded in determining a robust design space for the primary drying stage to produce uniform products of higher productivity. It was considered, from the present findings, that controlling the formation of large ice crystals impacted the product quality and productivity. p p

DOI： 10.1248/cpb.c18-00494

A label-free fluorescent array sensor utilizing liposome encapsulating calcein for discriminating target proteins by principal component analysis Reviewed International journal

Ryota Imamura, Naoki Murata, Toshinori Shimanouchi, Kaoru Yamashita, Masayuki Fukuzawa, Minoru Noda

Sensors (Switzerland) 17 ( 7 ) 1630 2017.7

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

A new fluorescent arrayed biosensor has been developed to discriminate species and concentrations of target proteins by using plural different phospholipid liposome species encapsulating fluorescent molecules, utilizing differences in permeation of the fluorescent molecules through the membrane to modulate liposome-target protein interactions. This approach proposes a basically new label-free fluorescent sensor, compared with the common technique of developed fluorescent array sensors with labeling. We have confirmed a high output intensity of fluorescence emission related to characteristics of the fluorescent molecules dependent on their concentrations when they leak from inside the liposomes through the perturbed lipid membrane. After taking an array image of the fluorescence emission from the sensor using a CMOS imager, the output intensities of the fluorescence were analyzed by a principal component analysis (PCA) statistical method. It is found from PCA plots that different protein species with several concentrations were successfully discriminated by using the different lipid membranes with high cumulative contribution ratio. We also confirmed that the accuracy of the discrimination by the array sensor with a single shot is higher than that of a single sensor with multiple shots.

DOI： 10.3390/s17071630

Tailor-made drug carrier: Comparison of formation-dependent physicochemical properties within self-assembled aggregates for an optimal drug carrier Reviewed International journal

Keita Hayashi, Hideka Iwai, Toshiyuki Kamei, Kaede Iwamoto, Toshinori Shimanouchi, Sakiko Fujita, Hidemi Nakamura, Hiroshi Umakoshi

Colloids and Surfaces B: Biointerfaces 152 269 - 276 2017.4

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

Self-assembled surfactant aggregates, such as micelles and vesicles, have been investigated for their application as drug carriers in the treatment of various diseases. However, the characteristics that decide which aggregate is the best drug carrier for each disease have not yet been clarified. In order to design an optimal drug carrier for each disease, various kinds of self-assembled aggregates, such as spherical micelles, lens-like vesicles, and tube-like vesicles, were evaluated by “multiple techniques” including dynamic light scattering, differential scanning calorimetry, nuclear magnetic resonance spectroscopy, and fluorescence measurement using the Laurdan probe. These studies led to the compilation of a database on the formation-dependent properties of self-assembled aggregates. As the relationship between physicochemical properties of self-assembled aggregates and their functions as drug carriers have been extensively reported, this database can be utilized for designing an optimal drug carrier, i.e., a tailor-made drug carrier.

DOI： 10.1016/j.colsurfb.2017.01.012

Effect of heparin on amyloid fibril formation of apoA-I fragment peptides Reviewed

Shiho Mikawa, Chiharu Mizuguchi, Izumi Morita, Hiroyuki Oyama, Teruhiko Baba, Akira Shigenaga, Toshinori Shimanouchi, Norihiro Kobayashi, Akira Otaka, Kenichi Akaji, Hiroyuki Saito

Peptide Science 2016 149 - 151 2017.2

Calcein leakage behavior from vesicles induced by proteinvesicle interaction: A study by surface pressurearea isotherms Reviewed

Saki Fukuma, Toshinori Shimanouchi, Keita Hayashi, Yukitaka Kimura

Chemistry Letters 46 ( 7 ) 1036 - 1039 2017

Language：English Publishing type：Research paper (scientific journal) Publisher：CHEMICAL SOC JAPAN

The release behavior of substrates encapsulated in the vesicle can be controlled by the interaction between the vesicular membrane and the protein. However, its control mechanism is still now unknown. Therefore, the proteinvesicle interaction was investigated by a A isotherm. It was clarified that the released amount of encapsulated substrates increased with increasing proteinvesicle interaction. This result leads to the development of drug delivery systems (DDS) and of biosensor technology.

DOI： 10.1246/cl.170119

Effect of wet and dry torrefaction process on fuel properties of solid fuels derived from bamboo and Japanese cedar Reviewed

Wei Yang, Shengji Wu, Hui Wang, Pengyu Ma, Toshiniri Shimanouchi, Yukitaka Kimura, Jie Zhou

BioResources 12 ( 4 ) 8629 - 8640 2017

Language：English Publishing type：Research paper (scientific journal) Publisher：North Carolina State University

Torrefaction is a promising pretreatment process to convert biomass into high energy density solid fuel for further thermal conversion systems. In this study, the effects of wet and dry torrefaction on the properties of solid fuels prepared from bamboo and Japanese cedar were investigated in a batch reactor. The yields of solid fuels decreased with increasing treatment temperature in both torrefaction processes, mainly due to the decomposition of cellulose and hemicellulose. Cellulose showed higher reactivity than hemicellulose in both biomasses. The higher heating values (HHV) of solid fuels prepared at the treatment temperatures higher than 240 °C in both torrefaction processes reached the same level as those of commercial coals. Wet torrefaction was better than dry torrefaction for decomposing bamboo and Japanese cedar. Dry torrefaction had more favorable impact than wet torrefaction on improving the fuel properties of bamboo and Japanese cedar because of its lower energy input, higher solid fuel yield, higher energy yield, and similar HHV under the same conditions. The crystalline structure of solid fuel had no great change below 260 °C in both torrefaction processes and was completely destroyed at 300 °C during dry torrefaction.

DOI： 10.15376/biores.12.4.8629-8640

The structural evaluation of amyloid beta on lipid membranes Reviewed

Satoka Aoyagi, Miki Iwamura, Toshinori Shimanouchi, Yuta Yokoyama, Hideo Iwai

Surface and Interface Analysis 48 ( 11 ) 1096 - 1099 2016.11

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL

Amyloid beta (Aβ) adsorption onto lipid membranes depending on the condition of a lipid was investigated by means of time-of-flight secondary ion mass spectrometry (ToF-SIMS). Aβ aggregation depending on the different hardness of lipid membrane has not been clarified yet although it is important to understand Alzheimer's disease. Aβ (1–40) on three different lipid membranes having different transition temperatures has been evaluated using ToF-SIMS in the previous study, and in this study, the differences between Aβ on liquid crystalline-phase lipid membranes and that on gel-phase lipid membranes were investigated in order to clarify the mechanisms of aggregation and peptide folding change. ToF-SIMS secondary ion images clearly showed Aβ distribution on lipid membranes. Because ToF-SIMS data is extremely complicated although it contains rich information, it was analysed by principal component analysis (PCA). Score images indicated by PCA are consistent with the images of secondary ions related to Aβ and are clearer than the secondary ion images. Moreover, PCA results suggest the difference between Aβ on different lipid membranes in terms of amino acid fragment ions, and the orientation of Aβ on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine was indicated. Copyright © 2016 John Wiley & Sons, Ltd.

DOI： 10.1002/sia.6086

Heparin promotes fibril formation by the N-terminal fragment of amyloidogenic apolipoprotein A-I. Reviewed International journal

Shiho Mikawa, Chiharu Mizuguchi, Kazuchika Nishitsuji, Teruhiko Baba, Akira Shigenaga, Toshinori Shimanouchi, Naomi Sakashita, Akira Otaka, Kenichi Akaji, Hiroyuki Saito

FEBS letters 590 ( 20 ) 3492 - 3500 2016.10

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL

Glycosaminoglycans are known to be associated with extracellular amyloid deposits of various amyloidogenic proteins. In this study, we found that the glycosaminoglycan heparin greatly accelerates the elongation step in fibril formation by the N-terminal 1-83 fragment of human apolipoprotein A-I (apoA-I), especially in the amyloidogenic W50R variant. Using fragment peptides, we demonstrate that heparin significantly promotes β-transition and fibril formation of the highly amyloidogenic region spanning residues 44-65 and colocalizes with fibrils formed by the W50R variant. These results suggest the possible role of glycosaminoglycans in fibril formation by amyloidogenic apoA-I variants.

DOI： 10.1002/1873-3468.12426

ToF-SIMS analysis of amyloid beta aggregation on different lipid membranes. International journal

Yuta Yokoyama, Satoka Aoyagi, Toshinori Shimanouchi, Miki Iwamura, Hideo Iwai

Biointerphases 11 ( 2 ) 02A314 - 6 2016.6

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER INST PHYSICS

Amyloid beta (Aβ) peptides are considered to be strongly related to Alzheimer's disease. Aβ peptides form a β-sheet structure on hard lipid membranes and it would aggregate to form amyloid fibrils, which are toxic to cells. However, the aggregation mechanism of Aβ is not fully understood. To evaluate the influence of the lipid membrane condition for Aβ aggregation, the adsorption forms of Aβ (1-40) on mixture membranes of lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and cholesterol β-d-glucoside (β-CG) were investigated by time-of-flight secondary ion mass spectrometry. As a result, Aβ adsorbed along the localized DMPC lipid on the mixture lipid membranes, whereas it was adsorbed homogeneously on the pure DMPC and β-CG membranes. Moreover, amino acid fragments that mainly existed in the n-terminal of Aβ (1-40) peptide were strongly detected on the localized DMPC region. These results suggested that the Aβ was adsorbed along the localized DMPC lipid with a characteristic orientation. These findings suggest that the hardness of the membrane is very sensitive to coexisting materials and that surface hardness is important for aggregation of Aβ.

DOI： 10.1116/1.4940706

Chemical Conversion and Liquid-Liquid Extraction of 5-Hydroxymethylfurfural from Fructose by Slug Flow Microreactor

Toshinori Shimanouchi, Yoshitaka Kataoka, Tatsuya Tanifuji, Yukitaka Kimura, Satoko Fujioka, Koichi Terasaka

AICHE JOURNAL 62 ( 6 ) 2135 - 2143 2016.6

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL

A dehydration of fructose in the water/methyl isobuthyl ketone (MIBK) biphasic system can yield 5hydroxymethylfulfural (IIMF) to be successfully extracted into the organic MIBK phase. The [IMF production and yield in MIRK phase was discussed by using a simplified model taking into consideration of the slug flow. The extraction resistance of IIMF across the interface between water and MIRK depended on the line velocity and the flow rate ratio. It was likely that the velocity field generated in the slug flow contributed to an increase in the mass transfer of IIMF. 2016 American Institute of Chemical Engineers AIChE J, 62: 2135-2143, 2016

DOI： 10.1002/aic.15201

Chemical Conversion and Liquid-Liquid Extraction of 5-Hydroxymethylfurfural from Fructose by Slug Flow Microreactor Reviewed

Toshinori Shimanouchi, Yoshitaka Kataoka, Tatsuya Tanifuji, Yukitaka Kimura, Satoko Fujioka, Koichi Terasaka

AICHE JOURNAL 62 ( 6 ) 2135 - 2143 2016.6

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL

A dehydration of fructose in the water/methyl isobuthyl ketone (MIBK) biphasic system can yield 5hydroxymethylfulfural (IIMF) to be successfully extracted into the organic MIBK phase. The [IMF production and yield in MIRK phase was discussed by using a simplified model taking into consideration of the slug flow. The extraction resistance of IIMF across the interface between water and MIRK depended on the line velocity and the flow rate ratio. It was likely that the velocity field generated in the slug flow contributed to an increase in the mass transfer of IIMF. 2016 American Institute of Chemical Engineers AIChE J, 62: 2135-2143, 2016

DOI： 10.1002/aic.15201

Detection of Fibrillization Process of Amyloid Beta Protein Using Arrayed Biosensor with Liposome Encapsulating Fluorescent Molecules

R. Imamura, T. Shimanouchi, N. Murata, K. Yamashita, M. Fukuzawa, M. Noda

PROCEEDINGS OF THE 30TH ANNIVERSARY EUROSENSORS CONFERENCE - EUROSENSORS 2016 168 1414 - 1417 2016

Language：English Publishing type：Research paper (international conference proceedings) Publisher：ELSEVIER SCIENCE BV

We have successfully detected fibrillization process of amyloid-beta(1-40) protein (A beta(1-40)) as causative compounds of Alzheimer's disease by our developed bio array sensing system utilizing different phospholipid liposomes encapsulating fluorescent molecules. The fluorescent intensity is dependent on the leakage of fluorescent molecules through perturbed lipid membrane of liposome. Therefore, the intensity considerably depends on the liposome-A beta interaction strength on fibrillization process of A beta(1-40). It is noted that the behavior of the fluorescent intensity corresponded to that of our cantilever sensor when measuring the interaction. Moreover, difference in A beta(1-40) concentration or liposome-A beta(1-40) interaction strength was successfully discriminated by principal component analysis. Consequently, we believe that the fluorescent liposome sensor is very effective for discriminating aggregation and fibrillization processes and concentrations of A beta(1-40). (C) 2016 Published by Elsevier Ltd.

DOI： 10.1016/j.proeng.2016.11.400

Formation of lens-like vesicles induced via microphase separations on a sorbitan monoester membrane with different headgroups. International journal

Keita Hayashi, Hideka Iwai, Toshinori Shimanouchi, Hiroshi Umakoshi, Tomoyuki Iwasaki, Ayako Kato, Hidemi Nakamura

Colloids and surfaces. B, Biointerfaces 135 235 - 242 2015.11

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

The microphase separation of lipid molecules on a vesicle membrane can be induced, depending on the difference in the geometric structures of their headgroups. Through cryo-transmission-electron-microscopy analysis, a lens-like vesicle was prepared by mixing 50 wt% Span 40 (sorbitan monopalmitate) and 50 wt% Tween 40 [polyoxyethylene (20) sorbitan monopalmitate]. Considering the molecular structures of Span 40 and Tween 40, the high-curvature region was mainly formed by Tween 40. As determined by Fourier-transform infrared spectroscopy, dielectric-dispersion analysis, and differential scanning calorimetry, a hydration layer was likely formed because polyoxyethylene conjugates with the headgroups of Tween 40. These investigations of the obtained self-assembled aggregates of nonionic surfactants with heterogeneous surfaces could contribute to the development of new types of biomaterials.

DOI： 10.1016/j.colsurfb.2015.07.071

Amyloidogenic Mutation Promotes Fibril Formation of the N-terminal Apolipoprotein A-I on Lipid Membranes. International journal

Chiharu Mizuguchi, Fuka Ogata, Shiho Mikawa, Kohei Tsuji, Teruhiko Baba, Akira Shigenaga, Toshinori Shimanouchi, Keiichiro Okuhira, Akira Otaka, Hiroyuki Saito

The Journal of biological chemistry 290 ( 34 ) 20947 - 20959 2015.8

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

The N-terminal amino acid 1-83 fragment of apolipoprotein A-I (apoA-I) has a strong propensity to form amyloid fibrils at physiological neutral pH. Because apoA-I has an ability to bind to lipid membranes, we examined the effects of the lipid environment on fibril-forming properties of the N-terminal fragment of apoA-I variants. Thioflavin T fluorescence assay as well as fluorescence and transmission microscopies revealed that upon lipid binding, fibril formation by apoA-I 1-83 is strongly inhibited, whereas the G26R mutant still retains the ability to form fibrils. Such distinct effects of lipid binding on fibril formation were also observed for the amyloidogenic prone region-containing peptides, apoA-I 8-33 and 8-33/G26R. This amyloidogenic region shifts from random coil to α-helical structure upon lipid binding. The G26R mutation appears to prevent this helix transition because lower helical propensity and more solvent-exposed conformation of the G26R variant upon lipid binding were observed in the apoA-I 1-83 fragment and 8-33 peptide. With a partially α-helical conformation induced by the presence of 2,2,2-trifluoroethanol, fibril formation by apoA-I 1-83 was strongly inhibited, whereas the G26R variant can form amyloid fibrils. These findings suggest a new possible pathway for amyloid fibril formation by the N-terminal fragment of apoA-I variants: the amyloidogenic mutations partially destabilize the α-helical structure formed upon association with lipid membranes, resulting in physiologically relevant conformations that allow fibril formation.

DOI： 10.1074/jbc.M115.664227

Surface properties of woody thin boards composed of commercially available lignin and cellulose: Relationship between the orientation of lignin and water repellency

Toshinori Shimanouchi, Tomoya Kamba, Wei Yang, Satoka Aoyagi, Yukitaka Kimura

APPLIED SURFACE SCIENCE 347 406 - 413 2015.8

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

Woody thin boards were prepared from lignin, cellulose, and water by compression molding at 180 degrees C and 25 MPa for 10 min. Boards with higher contact angles gave lower values of relative permittivity on their surface. Attenuated-total reflection Fourier transfer infrared spectroscopy suggested that more lignin existed on the surface of the boards with the high contact angle, which was also supported by scanning electron microscopy and atomic force microscopy. Our findings thus revealed that the orientation of lignin at the surface resulted in increased hydrophobicity of the surface and contributed to the enhancement of water repellency. (C) 2015 Published by Elsevier B.V.

DOI： 10.1016/j.apsusc.2015.03.139

Characterization of the Residue and Liquid Products Produced from Husks of Nuts from Carya cathayensis Sarg by Hydrothermal Carbonization

Wei Yang, Toshinori Shimanouchi, Yukitaka Kimura

ACS SUSTAINABLE CHEMISTRY & ENGINEERING 3 ( 4 ) 591 - 598 2015.4

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER CHEMICAL SOC

Hydrothermal carbonization is considered to be an environment-friendly method to pretreat biomass. Therefore, hydrothermal carbonization of the husks of nuts from Carya cathayensis Sarg,, at various temperatures and pHs were performed and the fuel properties of the obtained residue, water-soluble products (WSP) and acetone-soluble products (ASP) were characterized. It was found that the residue yield decreased with increasing hydrothermal carbonization temperature while the yield of total soluble products (WSP and ASP) was nearly the same. The residue and total soluble products yields had no large changes in the pH range of 4.0-10.0, but fewer residues and more total soluble products were obtained at pH 13.0. It was confirmed that hemicellulose was more reactive than cellulose and lignin during hydrothermal carbonization and that lignin could be effectively hydrolyzed/decomposed at pH 13.0. The higher heating values (HHVs) of residues were in the ranges of 22.0 to 28.2 MJ kg(-1) at temperatures from 180 to 260 inverted perpendicular C and pH from 4.0 to 10.0. The HHVs of these residues could be comparable with those of some commercial coals, such as lignite. However, the HI-IVs of residues decreased when the pH reached to 13.0. The WSP and ASP showed lower HHVs compared with heavy fuel oils, which meant the HHVs of WSP and ASP should be increased before being applied as bio-fuels.

DOI： 10.1021/acssuschemeng.5b00103

Elevating the fuel properties of Humulus lupulus, Plumeria alba and Calophyllum inophyllum L. through wet torrefaction

Wei Yang, Toshinori Shimanouchi, Miki Iwamura, Yuki Takahashi, Ryota Mano, Kohei Takashima, Tatsuya Tanifuji, Yukitaka Kimura

FUEL 146 88 - 94 2015.4

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCI LTD

Wet torrefaction is an effective process to improve the fuel properties of a biomass. However, different biomasses have different component weight ratios and it is possible that not all biomasses are suitable for wet torrefaction treatment. Here we conducted wet torrefaction using three types of biomass with different component weight ratios: Humulus lupulus (HL, the common hop), Plumeria alba (PA, an evergreen shrub) and Calophyllum inophyllum L. (CIL, an evergreen tree). The fuel properties of the obtained solid fuels were characterized. We found that lignin made the main contribution to the solid fuel yield. The reactivity of cellulose and hemicellulose in each biomass was affected by the biomass species and the component weight ratio of the biomass. The wet torrefaction was observed to efficiently elevate the fuel properties of carbon content, atomic H/C and O/C ratios, higher heating value (HHV) and hydrophobicity of all three types of biomass. The HHVs of the solid fuels prepared at 260 degrees C are comparable to those of commercial coals such as Northumerland No. 81/2 Sem. Anth. Coal, Jhanjra Bonbahal Seam Coal-R-VII, and German Braunkohole lignite. These solid fuels could be co-combusted with German Braunkohole lignite without a significant change in the combustion properties of German Braunkohole lignite because of their similar atomic H/C and O/C ratios as well as HHVs. At the wet torrefaction temperature of 260 degrees C, the solid fuel delivered from CIL had lower HHV compared to those from HL and PA although CIL contained the highest lignin content, which has a higher HHV than those of cellulose and hemicellulose. Scanning electron microscopy images of the solid fuels revealed that wet torrefaction was able to completely destroy the biomass surface and create numerous pores and cracks on the solid fuels surface, indicating that the solid fuels have the potential to be used as a source of carbon materials such as activated carbon in addition to their use as bio-fuels. (C) 2015 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.fuel.2015.01.005

ToF-SIMS observation for evaluating the interaction between amyloid β and lipid membranes. International journal

Satoka Aoyagi, Toshinori Shimanouchi, Tomoko Kawashima, Hideo Iwai

Analytical and bioanalytical chemistry 407 ( 10 ) 2859 - 63 2015.4

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

The adsorption behaviour of amyloid beta (Aβ), thought to be a key peptide for understanding Alzheimer's disease, was investigated by means of time-of-flight secondary ion mass spectrometry (ToF-SIMS). Aβ aggregates depending on the lipid membrane condition though it has not been fully understood yet. In this study, Aβ samples on different lipid membranes, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), were observed with ToF-SIMS and the complex ToF-SIMS data of the Aβ samples was interpreted using data analysis techniques such as principal component analysis (PCA), gentle-SIMS (G-SIMS) and g-ogram. DOPC and DMPC are liquid crystal at room temperature, while DPPC is gel at room temperature. As primary ion beams, Bi3(+) and Ar cluster ion beams were used and the effect of an Ar cluster ion for evaluating biomolecules was also studied. The secondary ion images of the peptide fragment ions indicated by G-SIMS and g-ogram were consistent with the PCA results. It is suggested that Aβ is adsorbed homogeneously on the liquid-crystalline-phase lipid membranes, while it aggregates along the lipid on the gel-phase lipid membrane. Moreover, in the results using the Ar cluster, the influence of contamination was reduced.

DOI： 10.1007/s00216-015-8527-3

Characterization of hydrochar prepared from hydrothermal carbonization of peels of Carya cathayensis sarg

Wei Yang, Toshinori Shimanouchi, Yukitaka Kimura

DESALINATION AND WATER TREATMENT 53 ( 10 ) 2831 - 2838 2015.3

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

Hydrothermal carbonization of peels of Carya cathayensis sarg (PCCS) by subcritical water or acetone- and ethanol-modified subcritical water was carried out at the temperature from 280 to 360 degrees C and coal-like hydrochar was obtained. The hydrochar yield decreased with increasing of treatment temperature, but the higher heating value (HHV) and carbon content of hydrochar were promoted at higher treatment temperature. The HHV of hydrochar was in range of 30-46 MJ/kg with an increase from 52.4 to 127.6%, compared to the original PCCS. The HHVs of hydrochars obtained at 360 degrees C could be comparable with those of heavy fuel oil (42.9 MJ/kg) and diesel oil (45.7 MJ/kg). The O/C and H/C values of hydrochar were similar to those of lignite and subbituminous, except the ash content. Especially, the O/C and H/C values of hydrochars prepared at 360 degrees C could be compatible with those of bituminous. When treated with acetone- or ethanol-modified subcritical water, a synergistic effect of acetone-water or ethanol-water for hydrothermal carbonization was observed; the liquefaction rate with acetone-water or ethanol-water mixture was larger than that of water or acetone or ethanol only. There was no significant change on the O/C and H/C values and HHV on the hydrochar, although more PCCS, was decomposed when using acetone-water or ethanol-water mixture.

DOI： 10.1080/19443994.2014.931537

Target Protein Discrimination Based on Array Sensor Using Calcein-Encapsulating Liposomes with Cholesterol by Principal Component Analysis

R. Imamura, Z. Zhang, T. Yoshikawa, T. Shimanouchi, N. Murata, K. Yamashita, M. Fukuzawa, M. Noda

EUROSENSORS 2015 120 699 - 702 2015

Language：English Publishing type：Research paper (international conference proceedings) Publisher：ELSEVIER SCIENCE BV

A bio array sensing system is newly composed utilizing different phospholipid liposomes encapsulating fluorescent molecules. We have confirmed a high output intensity of fluorescence emission due to the characteristics dependent on the concentration of fluorescent molecules when the fluorescent molecules leak from inside of the liposome through perturbed lipid membrane. After measuring a whole array image of fluorescence emission output from every element of liposome sensor by a new CMOS imager system, the outputs of fluorescence emission from all the elements were analyzed by a statistical method of principal component analysis (PCA). It was found from obtained PCA plots that different species of proteins with several concentrations were clearly discriminated with high cumulative contribution ratio. (C) 2015 Published by Elsevier Ltd.

DOI： 10.1016/j.proeng.2015.08.760

Discrimination of Target Proteins Using Arrayed Fluorescent Liposomes Incorporated with Cholesterol by Principal Component Analysis

R. Imamura, Z. Zhang, T. Yoshikawa, N. Murata, K. Yamashita, M. Fukuzawa, M. Noda, T. Shimanouchi

2015 IEEE SENSORS 1231 - 1234 2015

Language：English Publishing type：Research paper (international conference proceedings) Publisher：IEEE

A bio array sensing system is newly composed utilizing different phospholipid liposomes encapsulating fluorescent molecules. We have confirmed a high output intensity of fluorescence emission due to the characteristics dependent on the concentration of fluorescent molecules when the fluorescent molecules leak from inside of the liposome through perturbed lipid membrane. After measuring a whole array image of fluorescence emission output from every element of liposome sensor by a new CMOS imager system, the outputs of fluorescence emission from all the elements were analyzed by a statistical method of principal component analysis (PCA). It was found from obtained PCA plots that different species of proteins with several concentrations were discriminated with high cumulative contribution ratio.

DOI： 10.1109/ICSENS.2015.7370493

Systematical characterization of phase behaviors and membrane properties of fatty acid/didecyldimethylammonium bromide vesicles. International journal

Keishi Suga, Tomoya Yokoi, Dai Kondo, Keita Hayashi, Seiichi Morita, Yukihiro Okamoto, Toshinori Shimanouchi, Hiroshi Umakoshi

Langmuir : the ACS journal of surfaces and colloids 30 ( 43 ) 12721 - 8 2014.11

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER CHEMICAL SOC

Fatty acids (FAs) are known to form vesicle structures, depending on the surrounding pH conditions. In this study, we prepared vesicles by mixing FAs and a cationic surfactant, and then investigated their physicochemical properties using fluorescence spectroscopy and dielectric dispersion analysis (DDA). The assemblies formed from oleic acid (OA) and linoleic acid (LA) were modified by adding didecyldimethylammonium bromide (DDAB). The phase state of FA/DDAB mixtures was investigated with pH titration curves and turbidity measurements. The trigonal diagram of FA/ionized FA/DDAB was successfully drawn to understand the phase behaviors of FA/DDAB systems. The analysis of fluidities in the interior of the membrane with use of 1,6-diphenyl-1,3,5-hexatriene (DPH) indicated that the membrane fluidities of OA/DDAB and LA/DDAB at pH 8.5 slightly decreased in proportion to the molar ratio of DDAB in FA/DDAB systems. The fluorescent probe 6-lauroyl-2-dimethylamino naphthalene (Laurdan) indicated that the LA vesicle possessed a dehydrated surface, while the OA vesicle surface was hydrated. Modification of LA vesicles with DDAB induced the hydration of membrane surfaces, whereas modification of OA vesicles by DDAB had the opposite effect. DDA analysis indicated that the membrane surfaces were hydrated in the presence of DDAB, suggesting that the surface properties of FA vesicles are tunable by DDAB modification.

DOI： 10.1021/la503331r

Investigation of the Degradation Kinetic Parameters and Structure Changes of Microcrystalline Cellulose in Subcritical Water

Wei Yang, Toshinori Shimanouchi, Shengji Wu, Yukitaka Kimura

ENERGY & FUELS 28 ( 11 ) 6974 - 6980 2014.11

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER CHEMICAL SOC

Cellulose is the most abundant nature polymer and the main component in all biomass. In this study, the degradation kinetic parameters and structure changes of microcrystalline cellulose (MCC), which served as fundamental studies for degradation of biomass in subcritical water, were investigated at the temperature range from 100 to 300 degrees C. The yield of the MCC residue began to decrease at 205 degrees C and reached the lowest value at 275 degrees C. However, it howed an increase at the temperature higher than 275 degrees C. The degradation area of MCC (205275 degrees C) was separated into zones 1 and 2 with 245 degrees C as a boundary. The activation energy (E), pre-exponential factor (A), and reaction order (n) of MCC in each zone were 226.5 kJ mol(-1), 2.3 x 10(23) s(-1), and 0.6 (zone 1) and 423.1 kJ mol(-1), 9.0 x 10(40) s(-1), and 0.5 (zone 2), respectively. There showed a breaking point of 245 degrees C for the Arrhenius plot in the reaction area. The surface morphology of the MCC residue had no significant change below 260 degrees C, as indicated by scanning electron microscopy (SEM) images. However, it was completely destroyed at the temperature above 275 degrees C, and MCC residues with strong pore structures were obtained at higher temperatures. The structures of the crystalline region of the MCC residue below 275 degrees C had no significant change, although 89.2% MCC was degraded at 275 degrees C. However, they nearly disappeared at higher temperatures with the steeply reducing crystallinity index of MCC at the same time. As for the degradation mechanism of MCC, it was proposed that only the surface of MCC was degraded at 205-245 degrees C and the hydrogen and glycosidic bonds on the interior of MCC fibrils were destroyed at the temperature range from 245 to 275 degrees C. Finally, the remaining MCC as well as generated oligomer and monomer were further degraded at higher temperatures.

DOI： 10.1021/ef501702q

Relationship between the mobility of phosphocholine headgroup and the protein-liposome interaction: a dielectric spectroscopic study. International journal

Toshinori Shimanouchi, Noriko Yoshimoto, Azusa Hiroiwa, Keiichi Nishiyama, Keita Hayashi, Hiroshi Umakoshi

Colloids and surfaces. B, Biointerfaces 116 343 - 50 2014.4

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

Proteins could affect the headgroup mobility of phospholipid within liposome membranes through the protein-liposome interaction. The variation of headgroup mobility of phospholipid was then investigated by using the dielectric dispersion analysis. The eight proteins (Mw = 4.2-28.7 kDa) were used to investigate the protein-liposome interaction. It has been revealed that the strength of the protein-liposome interaction at 25 °C was linearly correlated with the stability of intramolecular hydrogen bondings of proteins, better than with their hydrophobicity and the surface charge density. Overall, liposomes composed of binary lipid system, appeared to strongly interact with proteins, in contrast to liposomes composed of single, ternary, and quaternary lipid systems. This is probably because liposomes composed of binary lipid system favored to form the microscopic environment where proteins could interact. The present result suggested the heterogeneous phase state of lipid membranes was one of dominant factors for the interaction between proteins and lipid membranes.

DOI： 10.1016/j.colsurfb.2013.07.028

Rapid Conversion of Glycerol to Lactic Acid under Alkaline Hydrothermal Conditions, by Using a Continuous Flow Reaction System

Toshinori Shimanouchi, Shouhei Ueno, Kazuki Shidahara, Yukitaka Kimura

CHEMISTRY LETTERS 43 ( 4 ) 535 - 537 2014.4

Language：English Publishing type：Research paper (scientific journal) Publisher：CHEMICAL SOC JAPAN

A rapid conversion of glycerol to lactic acid (lactate) could be successfully achieved under alkaline hydrothermal conditions, by using a continuous flow reaction system. A rapid conversion by a continuous flow-type reaction system made it possible to achieve the reaction under high-temperature conditions without side reactions. The rapid conversion and high reaction yield (reaction: 2 min; yield: 90%) resulted from the rapid temperature-shift advantageous for the inhibition of side reactions.

DOI： 10.1246/cl.131160

Extraction of reducing sugar with anti-oxidative scavengers from peels of Carya cathayensis sarg.: Use of subcritical water

Toshinori Shimanouchi, Shohei Ueno, Wei Yang, Yukitaka Kimura

Environmental Engineering Research 19 ( 1 ) 41 - 45 2014.3

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

The peels of Carya cathayensis Sarg. (PCCS) were treated under subcritical water conditions (130°C to 280 °C for 0 to 120 min). The extract from PCCS included reducing sugar, proteins, and compounds with radical scavenging activity. Addressing the reducing sugar that is a resource of bioethanol, we could maximize the reducing sugar under the subcritical water (190°C for 60 min) and obtain 0.24 g/g-sample together with 9.7 units/mg-sample of radical scavenging activity. The obtained extract was estimated to correspond to 1 L of bioethanol/100 g-sample. It was therefore considered that the treatment by subcritical water could yield reducing sugar and natural compounds with radical scavenging activity. © 2014 Korean Society of Environmental Engineers.

DOI： 10.4491/eer.2014.19.1.041

Development of metal affinity-immobilized liposome chromatography and its basic characteristics

Hideto Nagami, Hiroshi Umakoshi, Takenori Kitaura, Gary Lee Thompson, Toshinori Shimanouchi, Ryoichi Kuboi

BIOCHEMICAL ENGINEERING JOURNAL 84 66 - 73 2014.3

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

Metal affinity-immobilized liposome chromatography (MA-ILC) was newly developed as a chromatographic technique to separate and analyze peptides. The MA-ILC matrix gel was first prepared by immobilizing liposomes modified with functional ligands. The functional ligand used to adsorb metal ions was N-hexadecyl iminodiacetic acid (HIDA), which is obtained by attaching a long alkyl chain to an iminodiacetic acid (IDA). Cu(II) ion was first adsorbed on the gel matrix through its complex formation with the HIDA on the surface of the immobilized liposome. Synthetic peptides of various types ranging in size from 5 to 40 residues were then used, and their retention properties on the MA-ILC were evaluated. The retention property of peptides on the MA-ILC by using a usual imidazole elution was compared with the retention property in the case of the immobilized metal affinity chromatography (IMAC) and an immobilized liposome chromatography (ILC). It was found that the retention property of peptides on the MA-ILC has the features of both the IMAC and the ILC; the retention ability of peptides depends on both the number of histidine residues in peptides and the liposome membrane affinity of the peptides. Histidine and tryptophan residues among amino acid residues in peptides indicated a high contribution coefficient for the peptide retention on the MA-ILC, probably due to their metal ion and membrane interaction properties, respectively. (C) 2014 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bej.2013.12.017

Water/Methyl isobutyl ketone (MIBK) Biphasic System in Slug Flow under High Temperature and Pressure Conditions

Toshinori Shimanouchi, Tatsuya Tanifuji, Satoko Fujioka, Koichi Terasaka, Yukitaka Kimurai

SOLVENT EXTRACTION RESEARCH AND DEVELOPMENT-JAPAN 21 201 - 209 2014

Language：English Publishing type：Research paper (scientific journal) Publisher：JAPAN ASSOC SOLVENT EXTRACTION

The clarification of the partition property of substrates in a water / organic biphasic system is useful in the development of a reaction / separation process for value-added materials, especially under high temperature and pressure conditions. In this study, the water / methyl isobuthyl ketone (MIBK) biphasic system was investigated under various temperatures (25 190 degrees C) at 10 MPa. The partitioning behavior of substrates such as furfural derivatives, amino acids, and saccharides, depended on the temperature and the flow rate ratio of MIBK to the water phase. The scale of hydrophobicity (HF) of the water / MIBK biphasic system to determine the partitioning behavior of substrates was estimated based on the partitioning behavior of amino acids. The HF value for the water / MIBK phase flow system of was greater than that for the batch system and comparable to that for conventional aqueous two-phase systems such as polyethylene glycol / dextran and liposome membrane systems. It was probably because an increase of the surface-to-volume ratio and the vortex field in the slug flow contributed to the mass transfer of substrates and their distribution.

DOI： 10.15261/serdj.21.201

Subcritical Water-Assisted Emulsification of Decane/Water: Influence of Surfactants

Toshinori Shimanouchi, Takashi Tange, Yukitaka Kimura

SOLVENT EXTRACTION RESEARCH AND DEVELOPMENT-JAPAN 21 103 - 110 2014

Language：English Publishing type：Research paper (scientific journal) Publisher：JAPAN ASSOC SOLVENT EXTRACTION

A finely monodispersed oil-in-water (O/W) emulsion was obtained under high temperature and pressure conditions (240 degrees C and 10 MPa). A coarse emulsion stabilized by Tween 20 was prepared in advance from the decane / water system to be treated under the subcritical conditions. The treated coarse emulsion was thereafter mixed with the aqueous solution including surfactants to improve the monodispersity of the oil droplets. The subcritical condition could supply sufficient energy to produce finely dispersed oil droplets (similar to 233 nm in diameter).

DOI： 10.15261/serdj.21.103

Comparison of the Interfacial Properties of Span 80 Vesicle, W/O Emulsions and Liposomes

Keita Hayash, Hiroshi Umakosile, Toshinori Shimanouchi

SOLVENT EXTRACTION RESEARCH AND DEVELOPMENT-JAPAN 21 191 - 199 2014

Language：English Publishing type：Research paper (scientific journal) Publisher：JAPAN ASSOC SOLVENT EXTRACTION

Vesicles composed of sorbitan monooleate (Span 80) were prepared by a two-step emulsification method. The water-in-oil (W/O) emulsions generated in the first step of emulsification exhibited a quite dynamic and large hydrophobic difference between water and hexane. A subsequent emulsification yielded the vesicles with a fluid and flexible interface, as compared with that of liposomes. It was furthermore demonstrated that the two-step emulsification method has the advantage of the introduction of a PEG layer into the outer surface of the vesicles, while retaining the membrane properties of the vesicles.

DOI： 10.15261/serdj.21.191

Subcritical Water-Assisted Emulsification of Oil/Water: The Effect of the Solubility of the Organic Solvent and Surfactants

Toshinori Shimanouchi, Takashi Tange, Yukitaka Kimura

SOLVENT EXTRACTION RESEARCH AND DEVELOPMENT-JAPAN 21 223 - 230 2014

Language：English Publishing type：Research paper (scientific journal) Publisher：JAPAN ASSOC SOLVENT EXTRACTION

The formation of finely monodispersed oil-in-water (O/W) emulsions under high temperature and pressure conditions (240 degrees C and 10 MPa) depended on the solubility of the oil in water. Fatty acids with large solubilities in water favored the formation of oil droplets in emulsions of less than 100 nm in diameter. The water dissolved in the oil droplets participated throughout the cooling process, followed by the break-down of the oil droplets. In contrast, organic solvents with lower solubilities in water formed oil droplets in emulsions that had at most a diameter of 300 nm.

DOI： 10.15261/serdj.21.223

Membrane interaction between Span 80 vesicle and phospholipid vesicle (liposome): Span 80 vesicle can perturb and hemifuse with liposomal membrane. International journal

Keita Hayashi, Tsuyoshi Tatsui, Toshinori Shimanouchi, Hiroshi Umakoshi

Colloids and surfaces. B, Biointerfaces 106 258 - 64 2013.6

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

We have focused on the interaction between the Span 80 vesicle and phospholipid vesicle (liposome). The Span 80 vesicle was mixed with a phospholipid vesicle (liposome), used as a simplified model of plasma membrane. From calcein leakage experiments, it was revealed that the interaction between the Span 80 vesicles and the liposomes could perturb the liposome membrane. In the experiments based on fluorescence resonance energy transfer, the lipid mixing between Span 80 vesicle and liposome was observed. The above phenomena were also supported by the fluorescence spectroscopic analysis using laurdan, resulting in the variation in the membrane properties of liposome after its mixing with Span 80 vesicle. These results suggest that the Span 80 vesicles can easily interact and hemifuse with the liposome membrane, which depend on the membrane properties of the Span 80 vesicle, "flexible" and "molecular structure" of the components.

DOI： 10.1016/j.colsurfb.2012.12.022

Membrane fusion mediated by phospholipase C under endosomal pH conditions

Toshinori Shimanouchi, Hidenori Kawasaki, Makoto Fuse, Hiroshi Umakoshi, Ryoichi Kuboi

Colloids and Surfaces B: Biointerfaces 103 75 - 83 2013.3

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

Phospholipase C (PLC) is considered to be one of key enzymes for the design of drug delivery system using the endocytosis route, because PLC can catalyze the membrane fusion between cell membranes and phospholipid vehicles (liposomes). Membrane fusion by PLC was then studied under various pHs to model the endosomal environment. The used liposomes were composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and cholesterol (Ch). The membrane fusion was dominated by the enzymatic reaction at pH 6-7.5. In contrast, the membrane perturbation effect due to the conformational change of PLC could induce the membrane fusion at around pH 4. The maximal value of membrane fusion was observed at around pH 5 for three liposomes in the order of DOPC &lt
DOPC/DPPC (1:1) &lt
DOPC/DPPC/Ch (1:1:1). From the experiments with the hydrophobic fluorescence probe and the circular dichroic method, it was revealed that PLC took a molten-globule state, with a large fluctuation and an enzymatic activity, at around pH 4-5. The DAG-rich domain enzymatically produced by PLC played role for the field for the membrane perturbation enough to lead to the membrane fusion. The present finding would be helpful to understand the behavior of membrane fusion under the late endosomal pH condition in cell system. © 2012 Elsevier B.V.

DOI： 10.1016/j.colsurfb.2012.10.011

Growth behavior of giant vesicles using the electroformation method: Effect of proteins on swelling and deformation

Toshinori Shimanouchi, Hiroshi Umakoshi, Ryoichi Kuboi

JOURNAL OF COLLOID AND INTERFACE SCIENCE 394 269 - 276 2013.3

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE

The growth of giant vesicles (GVs) can be considered as a consecutive process of swelling/detachment/deformation, which is a response of lipid membranes on solid surfaces to the solvent and environmental factors such as temperature and ionic strength. The electroformation method allows to visualize the responses to such factors. The additive effect of the protein on the growth of GVs, composed of zwitterionic phospholipids, was herein investigated using the electroformation method. Proteins denatured by a pH-shift (to be in the Molten Globule state) perturbed the lipid membranes, resulting in the acceleration of GV growth. The GVs detached from the electrode showed deformation close to a stomatocyte. It was revealed that common factor for the response of lipid membranes was the variation of the apparent area elastic modulus associated with the interaction between proteins and lipid membranes. The present finding affords better understanding about the response of lipid membranes on solid surfaces under a variety of environment factors. (C) 2012 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.jcis.2012.11.046

Growth behavior of A beta protofibrils on liposome membranes and their membrane perturbation effect

Toshinori Shimanouchi, Keiichi Nishiyama, Azusa Hiroiwa, Huong Thi Vu, Nachi Kitaura, Hiroshi Umakoshi, Ryoichi Kuboi

BIOCHEMICAL ENGINEERING JOURNAL 71 81 - 88 2013.2

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE SA

The protofibrils of amyloid fibrils have recently drawn an attention due to their cytotoxicity although the physicochemical properties of the protofibrils as an intermediate of fibrillation process have still remained unclear. We investigated the growth behavior of the protofibrils of amyloid beta protein with 40 amino acid residues in the presence of various liposornes and the membrane perturbation (calcein leakage). The growth behavior of protofibrils was apparently correlated with the protofibrils-induced calcein leakage. From the binding experiment using a hydrophobic fluorescence probe and the quartz crystal microbalance method combined with the planar lipid membrane, it is suggested that the apparent correlation between the growth behavior of protofibrils and their membrane perturbation resulted from the physicochemical properties of protofibrils such as hydrophobicity and the stability of hydrogen bonds. The protofibrils were found to show the highest hydrophobicity and the highest instability of hydrogen bonds of the proteins used in this study (monomer, protofibrils, matured fibrils, and other three typical proteins). It is anticipated that such properties of protofibrils were advantageous both for the binding of protofibrils with monomer (growth behavior) and for the binding of lipid bilayer membranes (membrane perturbation). (C) 2012 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bej.2012.11.006

Effect of liposome membranes on disaggregation of amyloid beta fibrils by dopamine

Huong Thi Vu, Toshinori Shimanouchi, Daisuke Ishikawa, Tadaharu Matsumoto, Hisashi Yagi, Yuji Goto, Hiroshi Umakoshi, Ryoichi Kuboi

BIOCHEMICAL ENGINEERING JOURNAL 71 118 - 126 2013.2

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE SA

The inhibition of fibril formation of amyloid beta (A beta) and the disaggregation of A beta fibrils are the promising approaches for a medical treatment of Alzheimer's disease (AD) therapy. In this study, we investigated the effects of liposomes on dopamine-induced disaggregation of A beta fibrils by using the variety of liposomes. The used liposomes were normal liposomes, raft-forming liposomes, charged liposomes and oxidized liposomes. Those liposome could accelerate the disaggregation rate of fibrils. From the comparison of normal and charged liposomes, a certain contribution of dopamine via an electrostatic interaction to the disaggregation was confirmed. From raft-forming and oxidized liposomes, we revealed a significant contribution of bound water to liposomes, which could assist the formation of the quinine-form of dopamine by a removal of its proton. It is, therefore, concluded that the membrane surface of liposomes is considered to be an adequate environment for the dopamine-induced disaggregation of fibrils. (c) 2012 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bej.2012.12.012

Simplified Model for Extraction of 5-Hydroxymethylfurfural from Fructose: Use of Water/Oil Biphasic System under High Temperature and Pressure Conditions

Toshinori Shimanouchi, Yoshitaka Kataoka, Masahiro Yasukawa, Tsutomu No, Yukitaka Kimura

SOLVENT EXTRACTION RESEARCH AND DEVELOPMENT-JAPAN 20 205 - 212 2013

Language：English Publishing type：Research paper (scientific journal) Publisher：JAPAN ASSOC SOLVENT EXTRACTION

5-Hydroxymethylfurfural (HMF) was obtained from the dehydration of fructose under high temperature and pressure conditions (180 degrees C and 10 MPa). To avoid the decomposition of HMF in the bulk aqueous phase, we dehydrated fructose in the water / metyl isobuthyl ketone (MIBK) biphasic system. The HMF could be successfully extracted into the organic MIBK phase without further decomposition of HMF. We present a simplified model taking into consideration of the slug flow with the wall film to maximize the HMF yield in the MIBK phase. It was likely that the slug flow contributed to an increase in the surface-to-volume ratio for the mass transfer of HMF.

DOI： 10.15261/serdj.20.205

Enhanced Cytotoxicity for Colon 26 Cells Using Doxorubicin-Loaded Sorbitan Monooleate (Span 80) Vesicles

Keita Hayashi, Tsuyoshi Tatsui, Toshinori Shimanouchi, Hiroshi Umakoshi

INTERNATIONAL JOURNAL OF BIOLOGICAL SCIENCES 9 ( 2 ) 142 - 148 2013

Language：English Publishing type：Research paper (scientific journal) Publisher：IVYSPRING INT PUBL

Span 80 (sorbitan monooleate) vesicles behaved differently from conventional phospholipid vesicles (liposomes) because the former had a more fluid interface. After doxorubicin hydrochloride (DOX) was encapsulated into the Span 80 vesicle (loading efficiency: 63 %), DOX-loaded Span 80 vesicles (DVs) were thereafter added to Colon 26 cells. It was suggested, from the flow cytometric analysis and confocal laser microscopic observation, that DVs directly deliver DOX into the cytoplasm of Colon 26 cells. DVs showed the different delivery manner from the DOX-loaded liposomes (DLs). It is considered that the difference of delivery manner between DVs and DLs resulted in the difference of cytotoxicity (IC50); i.e. IC50 values for DVs and DLs were 5 and > 30 mu M, respectively. The results obtained herein would give the fundamental findings which can contribute to the improvement of formulation of conventional liposome-based carrier and its cytotoxicity.

DOI： 10.7150/ijbs.5453

Secondary nucleation of A beta fibrils on liposome membrane

Toshinori Shimanouchi, Nachi Kitaura, Ryo Onishi, Hiroshi Umakoshi, Ryoichi Kuboi

AICHE JOURNAL 58 ( 12 ) 3625 - 3632 2012.12

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL

The amyloid fibrils of amyloid beta protein (A beta) from Alzheimer's disease are likely to show the cytotoxicity, depending on their morphology. The relationship between the nucleation kinetics of the A beta fibrils and their morphology has been investigated. From the perspective of a crystallization technique assuming primary/secondary nucleation steps and an elongation step, the secondary nucleation rate B [# m-3 s-1], was experimentally and coarsely determined by using total internal reflection fluorescence microscopy combined with thioflavin T. In an aqueous solution, linear and rigid fibrils were formed with a relatively smaller B value ((2.83 +/- 0.55) x 105 # m-3 s-1), whereas spherulitic amyloid assemblies were formed in the presence of negatively charged liposome including oxidized lipids, with a larger B value ((7.65 +/- 0.47) x 105 # m-3 s-1). Those findings should lead to a better understanding of the mechanism for the formation of fibrils and senile plaques in Alzheimer's disease. (C) 2012 American Institute of Chemical Engineers AIChE J, 2012

DOI： 10.1002/aic.13772

Formation of spherulitic amyloid beta aggregate by anionic liposomes

Toshinori Shimanouchi, Naoya Shimauchi, Ryo Ohnishi, Nachi Kitaura, Hisashi Yagi, Yuji Goto, Hiroshi Umakoshi, Ryoichi Kuboi

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 426 ( 2 ) 165 - 171 2012.9

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE

Alzheimer's disease is the most common form of senile dementia. This neurodegenerative disorder is characterized by an amyloid deposition in senile plaques, composed primarily of fibrils of an aggregated peptide, amyloid beta (A beta). The modeling of a senile plaque formation on a model neuronal membrane under the physiological condition is an attractive issue. In this study, we used anionic liposomes to model the senile plaque formation by A beta. The growth behavior of amyloid All fibrils was directly observed, revealing that the induction of the spherulitic A beta aggregates could result from the growth of seeds in the presence of anionic liposomes. The seeds of A beta fibrils strongly interacted with negatively charged liposome and the subsequent association of the seeds were induced to form the seed cluster with many growth ends, which is advantageous for the formation of spherulitic A beta aggregates. Therefore, anionic liposomes mediated not only fibril growth but also the aggregation process. These results imply that anionic liposome membranes would affect the aggregate form of All fibrils. The modeling of senile plaque reported here is considered to have great potential for study on the amyloidosis. (c) 2012 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2012.07.107

Effect of copper (II) ion against elongation behavior of amyloid ss fibrils on liposome membranes

T. Shimanouchi, R. Onishi, N. Kitaura, H. Umakoshi, R. Kuboi

CRYSTAL RESEARCH AND TECHNOLOGY 47 ( 1 ) 101 - 108 2012.1

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL

The fibril growth behavior of amyloid beta protein (A beta) on cell membranes is relating to the progression of Alzheimer's disease. This growth behavior of A beta fibrils is sensitively affected by the metal ions, neurotransmitters, or bioreactive substrate. The inhibitory effect of those materials was quantitatively estimated from the viewpoints of crystal growth. In a bulk aqueous solution, copper (II) ion showed the strong inhibitory effect on the growth of A beta fibrils. Meanwhile, the addition of a closed-phospholipid bilayer membrane (liposome) could reduce the above inhibitory effect of copper (II) ion. (c) 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

DOI： 10.1002/crat.201100344

A New Intact Immobilization of Liposome as Sensing Bio Nano-Particle on Oxidized Metal Electrode Surface

Minoru Noda, Keisuke Takada, Mariko Nakai, Kaoru Yamashita, Toshinori Shimanouchi, Hiroshi Umakoshi

2012 IEEE SENSORS PROCEEDINGS 25 - 28 2012

Language：English Publishing type：Research paper (international conference proceedings) Publisher：IEEE

We newly report that the intactness of liposome on the electrode surfaces of Pt and Au metals could be significantly improved on thermally oxidized metal surfaces, compared to those with non-oxidization or even with a SAM treatment. It is especially noted that the sub-gel phase of lipid membrane of the liposome on the oxidized Pt surface is much more suppressed than the case of non-oxidized, indicating that the fluidity of intact liposome greatly improves, as the sub-gel phase then crystalline phase will increase when the intact liposome loses its closed spherical shape. Finally, the retention time of the intact liposome on the oxidized surface becomes improved to about one day. These results indicate that the oxidation treatment made the metal surface hydrophilic enough to keep the stable and intact liposome. We think that the retention time up to one day is good enough for practical biosensor applications using intact liposome to improve device performance and its reliabilities.

DOI： 10.1109/ICSENS.2012.6411074

A New Biosensing by Dielectric Dispersion Analysis of Interaction between Lipid Membrane of Liposome and Target Biomolecules up to 20 GHz Range

Keisuke Takada, Kaoru Yamashita, Minoru Noda, Toshinori Shimanouchi, Hiroshi Umakoshi

2012 IEEE SENSORS PROCEEDINGS 935 - 938 2012

Language：English Publishing type：Research paper (international conference proceedings) Publisher：IEEE

This paper reports, firstly, that it was newly found by our specific Dielectric Dispersion Analysis (DDA) that plural type of bound water with different relaxation frequencies exist in a suspension of liposome of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Secondly, we observe that the interaction between the liposome and a specific target biomolecule depends on the type of target molecules (we used a protein of carbonic anhydrase from bovine (CAB) and/or cholesterol, respectively), leading to the change in dielectric relaxation at different relaxation frequency of bound water. It is finally concluded that the developed DDA method allows us to acquire the quantitative information with respect to bound water to liposomes, which gives novel physical insight on interaction between liposomes and target biomolecules (especially, protein).

DOI： 10.1109/ICSENS.2012.6411071

A planar-type leakage current and impedance microsensor for detection of interaction between electrolyte-entrapping liposome and protein

Y. Ohara, P. Lorchirachoonkul, K. Yamashita, M. Noda, O. A. Alberto, T. Shimanouchi

IMFEDK 2012 - 2012 International Meeting for Future of Electron Devices, Kansai 146 - 147 2012

Language：English Publishing type：Research paper (international conference proceedings)

We have developed a new leakage current microsensor by using simpler planar processes than Si-surface-bulk micromachining processes used in the previous microwell structure. This sensor fabrication and structure can easily make a target solution volume smaller than μL with excellent immobilization of the droplet and intact biomolecules as sensing elements, as a result, reduce effectively the background noise current in the microsensor and improve reproducibility of the results. The leakage current due to the biochemical interaction was successfully evaluated. Cole-Cole plots from the impedance analysis also show quantitative difference between with and without the interaction, depending on the charge-transfer impedance. © 2012 IEEE.

DOI： 10.1109/IMFEDK.2012.6218624

Copper-mediated growth of amyloid beta fibrils in the presence of oxidized and negatively charged liposomes

Toshinori Shimanouchi, Ryo Onishi, Nachi Kitaura, Hiroshi Umakoshi, Ryoichi Kuboi

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 112 ( 6 ) 611 - 615 2011.12

Language：English Publishing type：Research paper (scientific journal) Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN

Amyloid beta protein (A beta) from Alzheimer's disease formed fibrillar aggregates and their morphology depended on oxidized and negatively charged liposomes. The morphology of fibrillar aggregates was affected by Cu2+, together with their growth kinetics. This is because Cu2+ inhibited the nucleation step in the formation of amyloid A beta fibrillar aggregates by forming A beta/Cu complex inactive to the growth of fibrillar aggregates. In addition, this is probably because Cu2+ affected the fibrillar aggregate formed on the surface of liposomes. These findings would give a better understanding of the formation mechanism of amyloid fibrils on biomembranes. (C) 2011, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2011.08.015

Relationship between the mobility of phosphocholine headgroups of liposomes and the hydrophobicity at the membrane interface: A characterization with spectrophotometric measurements

Toshinori Shimanouchi, Masashi Sasaki, Azusa Hiroiwa, Noriko Yoshimoto, Kazuya Miyagawa, Hiroshi Umakoshi, Ryoichi Kuboi

COLLOIDS AND SURFACES B-BIOINTERFACES 88 ( 1 ) 221 - 230 2011.11

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

In this study, we investigated the dynamics of a membrane interface of liposomes prepared by eight zwitterionic phosphatidylcholines in terms of their headgroup mobility, with spectroscopic methods such as dielectric dispersion analysis (DDA), fluorescence spectroscopy. The DDA measurement is based on the response of the permanent dipole moment to a driving electric field and could give the information on the axial rotational Brownian motion of a headgroup with the permanent dipole moment. This motion depended on kinds of phospholipids, the diameter of the liposomes, and the temperature. The activation energy required to overcome the intermolecular force between headgroups of phospholipids depended on the strength of the interaction between headgroups such as hydrogen bonds and/or dipole-dipole interaction. Hydration at the phosphorous group of phospholipid and the molecular order of lipid membrane impaired the interaction between headgroups. Furthermore, the hydrophobicity of membrane surface increased parallel to the increase in headgroup mobility. It is, therefore, concluded that hydration of headgroup promoted its mobility to make the membrane surface hydrophobic. The lipid membrane in liquid crystalline phase or the lipid membrane with the larger curvature was more hydrophobic. (C) 2011 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.colsurfb.2011.06.036

Conformational change of single-stranded RNAs induced by liposome binding

Keishi Suga, Tomoyuki Tanabe, Hibiki Tomita, Toshinori Shimanouchi, Hiroshi Umakoshi

NUCLEIC ACIDS RESEARCH 39 ( 20 ) 8891 - 8900 2011.11

Language：English Publishing type：Research paper (scientific journal) Publisher：OXFORD UNIV PRESS

The interaction between single-stranded RNAs and liposomes was studied using UV, Fourier Transform Infrared spectroscopy (FTIR) and Circular Dichroism spectroscopy (CD). The effect of the surface characteristics of liposomes, which were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and modified with cholesterol (Ch) or 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), on the liposome-RNA interaction was investigated. The fluorescence of 6-(p-toluidino)naphthalene-2-sulfonate (TNS) embedded in the liposome surface (epsilon = 30-40) was decreased in the presence of tRNA, suggesting that single-stranded tRNA could bind onto the liposome. The dehydration of -PO(2)(-) -, guanine (G) and cytosine (C) of tRNA molecules in the presence of liposomes suggested both an electrostatic interaction (phosphate backbone of tRNA and trimethylammonium group of POPC, DOTAP) and a hydrophobic interaction (guanine or cytosine of tRNA and aliphatic tail of lipid). The tRNA conformation on the liposome was determined by CD spectroscopy. POPC/Ch (70/30) maintained tRNA conformation without any denaturation, while POPC/DOTAP(70/30) drastically denatured it. The mRNA translation was evaluated in an Escherichia coli cell-free translation system. POPC/Ch(70/30) enhanced expression of green fluorescent protein (GFP) (116%) while POPC/DOTAP(70/30) inhibited (37%), suggesting that the conformation of RNAs was closely related to the translation efficiency. Therefore, single-stranded RNAs could bind to liposomal membranes through electrostatic and hydrophobic attraction, after which conformational changes were induced depending on the liposome characteristics.

DOI： 10.1093/nar/gkr568

Sensitivity Enhancement of Leakage Current Microsensor for Detection of Target Protein by Using Protein Denaturant

Minoru Noda, Prinya Lorchirachoonkul, Toshinori Shimanouchi, Kaoru Yamashita, Hiroshi Umakoshi, Ryouichi Kuboi

IEEE SENSORS JOURNAL 11 ( 11 ) 2749 - 2755 2011.11

Language：English Publishing type：Research paper (scientific journal) Publisher：IEEE-INST ELECTRICAL ELECTRONICS ENGINEERS INC

We have developed a highly-sensitive leakage current microsensor by using 1,2-dipalmitoyl-sn-glycero-3-phosphocholline (DPPC) liposome entrapping K-4[Fe(CN)(6)] solution to detect the existence of proteins. In this work, the addition of guanidinium hydrochloride (GuHCl) as protein denaturant successfully resulted in a prominent improvement in sensitivity by 129-folds of magnitude. This is because the modulation of conformation of the target proteins, carbonic anhydrase from bovine (CAB) and lysozyme could contribute to the promotion of the interaction between the liposome and the target protein.

DOI： 10.1109/JSEN.2011.2159109

Span 80 vesicles have a more fluid, flexible and "wet" surface than phospholipid liposomes

Keita Hayashi, Toshinori Shimanouchi, Keiichi Kato, Tatsuhiko Miyazaki, Atsushi Nakamura, Hiroshi Umakoshi

COLLOIDS AND SURFACES B-BIOINTERFACES 87 ( 1 ) 28 - 35 2011.10

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

The surface properties of Span 80 vesicles at various cholesterol contents, together with those of various liposomes, were characterized by using fluorescence probes. The membrane fluidity of the Span 80 vesicles was measured by 1,6-dipheny1-1.3.5-hexatriene (DPH) and trimethlyammonium-DPH (TMA-DPH), and the results suggested that the surface of the Span 80 vesicles was fluid due to the lateral diffusion of Span 80 molecules. The depolarization measured by TMA-DPH and the headgroup mobility measured by dielectric dispersion analysis indicated the high mobility of the head group of Span 80 vesicles. This suggested that the surface of Span 80 vesicles was flexible due to the head group structure of Span 80, sorbitol. In addition, spectrophotometric analysis with 6-dodecanoyl-N, N-dimethy1-2-naphthylamine and 8-anilino-1-naphthalenesulfonic acid indicated that the water molecules could easily invade into the interior of the Span 80 vesicle membrane, suggesting that the membrane surface was more wet than the liposome surface. These surface properties indicated that the protein could interact with the interior of vesicle membranes, which was similar to the case of cholesterol. Thus the present results confirmed that the Span 80 vesicle surfaces showed the unique characteristics of fluidity, flexibility, and "wetness", whereas the liposome surfaces did not. (C) 2011 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.colsurfb.2011.04.029

Span 80 vesicles have a morefluid, flexibleand"wet"surface than phospholipid liposomes. Reviewed

Hayashi Keita, Shimanouchi Toshinori, Kato Keiichi, Miyazaki Tatsuhiko, Nakamura Atsushi, Umakoshi Hiroshi

Colloids and surfaces. B, Biointerfaces 87 ( 1 ) 28 2011.10

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

The surface properties of Span 80 vesicles at various cholesterol contents, together with those of various liposomes, were characterized by using fluorescence probes. The membrane fluidity of the Span 80 vesicles was measured by 1,6-diphenyl-1.3.5-hexatriene (DPH) and trimethlyammonium-DPH (TMA-DPH), and theresults suggested that the surface of the Span 80 vesicles was fluid due to the lateral diffusion of Span 80 molecules. The depolarization measured by TMA-DPH and the headgroup mobility measured by dielectric dispersion analysis indicated the high mobility of the head group of Span 80 vesicles. This suggested that the surface of Span 80 vesicles was flexible due to the head group structure of Span 80, sorbitol. In addition, spectrophotometric analysis with 6-dodecanoyl-N, N-dimethyl-2-naphthylamine and 8-anilino-1-naphthalenesulfonic acid indicated that the water molecules could easily invade into the interior of the Span 80 vesicle membrane, suggesting that the membrane surface was more wet than the liposome surface. These surface propertiesindicated that the protein could interact with the interior of vesicle membranes, which was similar to the case of cholesterol. Thus the pr

DOI： 10.1016/j.colsurfb.2011.04.029

Oxidative Stress Can Affect the Gene Silencing Effect of DOTAP Liposome in an In Vitro Translation System

Hiroshi Umakoshi, Tomoyuki Tanabe, Keishi Suga, Huong Thi Bui, Toshinori Shimanouchi, Ryoichi Kuboi

INTERNATIONAL JOURNAL OF BIOLOGICAL SCIENCES 7 ( 3 ) 253 - 260 2011

Language：English Publishing type：Research paper (scientific journal) Publisher：IVYSPRING INT PUBL

Oxidative stress can affect in vitro GFP expression through its control of the gene silencing effect of the liposome prepared by 1,2- dioleoyl-3-trimethyl-ammonium propane ( DOTAP). The gene silencing effect of cationic DOTAP liposome in in vitro GFP expression, especially focusing on its translation process, and the effects of oxidative stress on its silencing effect were investigated. GFP expression, initiated by mRNA, was found to be thoroughly inhibited in the presence of DOTAP liposome at concentration of more than 2.5 mM, though its inhibitory effect was reduced in the presence of hydrogen peroxide. The analyses of (i) the interaction of mRNA with DOTAP, (ii) the chemical structure of DOTAP, and (iii) the membrane fluidity of DOTAP liposome imply the possible role of gene expression by the liposome membrane and stress conditions.

DOI： 10.7150/ijbs.7.253

Development of LIPOzymes Based on Biomembrane Process Chemistry

Hiroshi Umakoshi, Toshinori Shimanouchi, Ryoichi Kuboi

Pharmaceutical Process Chemistry 421 - 441 2010.10

Cationic liposome can interfere mRNA translation in an E. coli cell-free translation system

Huong Thi Bui, Hiroshi Umakoshi, Keishi Suga, Tomoyuki Tanabe, Kien Xuan Ngo, Toshinori Shimanouchi, Ryoichi Kuboi

BIOCHEMICAL ENGINEERING JOURNAL 52 ( 1 ) 38 - 43 2010.10

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

The effect of cationic liposome prepared from 1,2-dioleoyl-3-trimethyl- ammonium-propane (DOTAP) on the gene expression at the mRNA translation level was investigated using an E. coli cell-free translation system. DOTAP liposome at 3 mM inhibited the mRNA translation of green fluorescent protein (GFP), as indicated both by the fluorescence intensity of GFP and by SDS-PAGE analysis. The role of DOTAP liposome on the inhibition of mRNA translation was revealed that the cationic quaternary amine groups on the liposome surface can interact and neutralize the anionic phosphate groups on mRNA by an electrostatic interaction. mRNA molecules still existed without any degradation in the presence of DOTAP liposome although it could not be translated. These results clearly illustrated that the DOTAP liposome could knock down mRNA and silence its activity of translation in an E. coli cell-free system. (C) 2010 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bej.2010.07.001

Catechol derivatives inhibit the fibril formation of amyloid-beta peptides

Vu Thi Huong, Toshinori Shimanouchi, Naoya Shimauchi, Hisashi Yagi, Hiroshi Umakoshi, Yuji Goto, Ryoichi Kuboi

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 109 ( 6 ) 629 - 634 2010.6

Language：English Publishing type：Research paper (scientific journal) Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN

The inhibition of fibril formation of amyloid beta proteins (A beta) would be attractive therapeutic targets for the treatment of Alzheimer's disease (AD). Dopamine (DA) and other catechol derivatives were used as inhibitory factors for A beta fibril formation. The fibril formation of A beta was monitored by Thioflavin T fluorescence, a transmission electron microscopy (TEM) and a total internal reflection fluorescence microscopy (TIRFM). Catechol and its derivatives showed the dose-dependent inhibitory effects on the spontaneous A beta fibril formation. The inhibitory activity depended on the chemical structure of catechol derivatives both in the presence and absence of the liposome a model of biomembrane. Formation of catechol quinone-conjugated-A beta adduct by a Schiff-base is a key step for the inhibition effect of A beta fibril formation. (C) 2009, The Society for Biotechnology, japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2009.11.010

Laser-induced Propagation and Destruction of Amyloid beta Fibrils

Hisashi Yagi, Daisaku Ozawa, Kazumasa Sakurai, Toru Kawakami, Hiroki Kuyama, Osamu Nishimura, Toshinori Shimanouchi, Ryoichi Kuboi, Hironobu Naiki, Yuji Goto

JOURNAL OF BIOLOGICAL CHEMISTRY 285 ( 25 ) 19660 - 19667 2010.6

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

The amyloid deposition of amyloid beta (A beta) peptides is a critical pathological event in Alzheimer disease (AD). Preventing the formation of amyloid deposits and removing preformed fibrils in tissues are important therapeutic strategies against AD. Previously, we reported the destruction of amyloid fibrils of beta 2-microglobulin K3 fragments by laser irradiation coupled with the binding of amyloid-specific thioflavin T. Here, we studied the effects of a laser beam on A beta fibrils. As was the case for K3 fibrils, extensive irradiation destroyed the preformed A beta fibrils. However, irradiation during spontaneous fibril formation resulted in only the partial destruction of growing fibrils and a subsequent explosive propagation of fibrils. The explosive propagation was caused by an increase in the number of active ends due to breakage. The results not only reveal a case of fragmentation-induced propagation of fibrils but also provide insights into therapeutic strategies for AD.

DOI： 10.1074/jbc.M109.076505

Development of liposome-based mimics of superoxide dismutase and peroxidase based on the "LIPOzyme" concept

Hiroshi Umakoshi, Kengo Morimoto, Naoto Yasuda, Toshinori Shimanouchi, Ryoichi Kuboi

JOURNAL OF BIOTECHNOLOGY 147 ( 1 ) 59 - 63 2010.5

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

An antioxidative liposome catalyst. LIPOzyme, that mimics both superoxide dismutase (SOD) and peroxidase (POD)-like activities has been developed by using liposomes modified with simple ligands (dodecanoyl-histidine, Dodec-His) and metal ions (Mn) The SOD-like activity is dependent on the stability of the ligand-metal complex on the liposome membrane, with the value being higher for the DPPC liposome and at a higher pH The POD-like activity was found to be maximal in the case of DMPC liposome, in which the ligand-metal complex is inserted more deeply into the membrane. It was thus shown that liposome modified with simple ligands can exhibit different enzyme-like activities depending on the characteristics of the liposome membrane. (C) 2010 Elsevier B V All rights reserved.

DOI： 10.1016/j.jbiotec.2010.02.013

Analysis of the 22-NBD-cholesterol transfer between liposome membranes and its relation to the intermembrane exchange of 25-hydroxycholesterol

Haruyuki Ishii, Toshinori Shimanouchi, Hiroshi Umakoshi, Peter Walde, Ryoichi Kuboi

COLLOIDS AND SURFACES B-BIOINTERFACES 77 ( 1 ) 117 - 121 2010.5

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

The transfer of 22-NBD-cholesterol (22-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3-ol) between two liposome membranes was quantitatively analyzed by using the fluorescence resonance energy transfer (FRET) method. Liposomes labeled with both 22-NBD-cholesterol and a rhodamine-labeled phosphatidylethanolamine (Rh-DHPE) were used as donor liposomes, and the 22-NBD-cholesterol transfer from these donor liposomes to acceptor liposomes prepared from same type of phosphatidylcholine was monitored. The transfer kinetics was found to be composed of a fast and a slow phase, and all kinetic measurements could be fitted with a bi-exponential model. The results obtained indicate that the 22-NBD-cholesterol transfer kinetics between liposome membranes depends on the fluidity of the liposome used and that the curvature may affect the kinetics. Furthermore, the behavior of 22-NBD-cholesterol in lipid membrane is similar to that of the oxysterol 25-hydroxycholesterol rather than cholesterol. It is proposed that 22-NBD-cholesterol can be a useful fluorescent probe to mimic the intermembrane transfer of oxidized cholesterols like 25-hydroxycholesterol, rather than that of cholesterol itself. (C) 2010 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.colsurfb.2010.01.002

Preparation of superoxide dismutase LIPOzyme in hollow fiber membrane module

Hiroyuki Sugaya, Hiroshi Umakoshi, K. B. M. A. Fadzil, Le Quoc Tuan, Toshinori Shimanouchi, Ryoichi Kuboi

DESALINATION AND WATER TREATMENT 17 ( 1-3 ) 281 - 287 2010.5

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

Liposome-loaded membrane module (LLM) was prepared in hollow fiber module (HF-LLM), where the liposome was loaded into the hollow fiber membrane. The filtration property of the LLM was characterized. The oxidized and fragmented superoxide dismutase (SOD) was applied to the prepared LLM to separate the peptide to give a SOD-like activity on the liposome membrane, resulting in the recovery of the specific peptide. It was found that the SOD-like activity could be obtained in the SOD LIPOzyme prepared, resulting in the effective elimination of the superoxide in the HF-LLM.

DOI： 10.5004/dwt.2010.1730

Monitoring of membrane damages by dialysis treatment: Study with membrane chip analysis

Toshinori Shimanouchi, Ena Oyama, Huong Thi Vu, Haruyuki Ishii, Hiroshi Umakoshi, Ryoichi Kuboi

DESALINATION AND WATER TREATMENT 17 ( 1-3 ) 45 - 51 2010.5

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

The liposome immobilized on indium tin-oxide electrode was prepared as a "Membrane Chip'' to evaluate the liposome-liposome interaction. Three kinds of neutral liposomes entrapping the fluorescence probe, calcein, were utilized as a sensory liposome. Twenty four kinds of liposomes were used to construct a membrane library based on the calcein release behavior. The loading of liposome including the unsaturated phospholipid into the dialyzer induced the variation of surface state of liposome membrane. We analyzed the above liposome by a comparison of it with the membrane library constructed by Membrane Chip. The liposome after its dialysis treatment was found to show the membrane property of the liposome with domain-like structure prior to the interaction with amyloid beta protein. Also the above liposome showed the oxidized liposome-like membrane property. In conclusion, we demonstrated a membrane library-based method to evaluate the surface state of the model biomembranes with unclear surface property.

DOI： 10.5004/dwt.2010.1697

Liposomes destabilize tRNA during heat stress

Keishi Suga, Hiroshi Umakoshi, Hibiki Tomita, Tomoyuki Tanabe, Toshinori Shimanouchi, Ryoichi Kuboi

BIOTECHNOLOGY JOURNAL 5 ( 5 ) 526 - 529 2010.5

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-V C H VERLAG GMBH

Biomembranes play an important role in cellular response to heat stress. In this study, we focus on the interaction between liposomes and tRNA. Upon heat treatment we determined circular dichroism spectra of tRNA in presence of liposomes prepared from POPC (1-palmitoyl-2-oleoylsn-glycero-3-phosphocholine) and cholesterol (Ch). To compare thermal stability, midpoint temperature (T(m)) of tRNA was calculated from normalized theta(208). Addition of POPC/Ch liposomes decreased the T(m) value of tRNA from 48 degrees C to 38 degrees C. We conclude that POPC/Ch liposomes interact with tRNA and destabilize its conformation under heat stress.

DOI： 10.1002/biot.200900289

A sensitive thermochemical detector with a new target droplet supply and immobilization process

M. Noda, T. Shimanouchi, T. Asai, K. Yamashita, M. Okuyama, H. Umakoshi, R. Kuboi

SENSORS AND ACTUATORS B-CHEMICAL 147 ( 1 ) 337 - 342 2010.5

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE SA

A thermal detector structure of bio-thermochemical sensor with a new target droplet supply is considered, analyzed and developed. The basic structure of the bio-thermochemical sensor is a Pt film microbolometer thermally insulated from a silicon thermal conductor substrate to enhance the temperature sensitivity. In the detector structure, it is calculated from a finite element analysis that the variation in lateral size of immobilized droplet, larger than detector film size of 50 mu m, affects little on the temperature change, while the real vertical size of the droplet keeps nearly the same as around 70 mu m. These indicate the validity of formation of the target droplet onto the detector part as an accurate thermal measurement. Based on this, a new supplying and immobilizing process of the droplet is developed to realize a soft formation of the droplet on the detector surface with retained intact immobilization of liposome at least for 24 h, not giving the membrane a mechanical stress and destruction.
Thereafter, we have evaluated a bio-thermochemical reaction in a droplet of mixed solution of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposome and lysozyme by the newly developed thermochemical sensor. Some new peaks in thermal spectra were observed for the case of the mixture, which were not found so far in the case of each single solution of liposome or lysozyme, indicating that their bio-thermochemical interactions were newly detected in this method. It was also found that the thermal sensor with new forming process of droplet target detects the thermochemical reaction of respective DPPC liposome and lysozyme, consistent with the same as those reported by a differential scanning calorimetry. (C) 2010 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.snb.2010.03.012

Chitosanase displayed on liposome can increase its activity and stability

Kien Xuan Ngo, Hiroshi Umakoshi, Toshinori Shimanouchi, Hiroyuki Sugaya, Ryoichi Kuboi

JOURNAL OF BIOTECHNOLOGY 146 ( 3 ) 105 - 113 2010.4

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

The strategy to prepare a novel biocatalyst by the immobilization of chitosanase onto liposome (ICL) was carried out based on the direct interaction of liposomes with cell membrane of Streptomyces griseus cell. The ICL was characterized in relation to the molecular weight of protein, the chitosanase activity, the effect of the surface hydration of various liposomes on hydrolysis activity of immobilized chitosanase and the stability of ICL under various extreme conditions. The SOS-PAGE analysis of the purified ICL sample shows the existence of a protein with approximately 39 kDa that corresponded to the sum of weight of the mature chitosanase and its signal peptide (38.8 kDa). The above protein of ICL also expresses the chitosanase activity that is significantly higher than that of the conventional chitosanase. Furthermore, the surface hydration of liposomes used to prepare ICL that affected the activity of immobilized chitosanase verified the importance of liposome surfaces. Indeed, the stability of ICL assayed by measuring the chitosanase activity is significantly higher than that of conventional chitosanase under various temperatures and pH conditions. These characteristics of ICL show the possible preparation of the biocatalysts that can be prepared by immobilizing enzymes onto liposome vesicles properly. (C) 2010 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jbiotec.2010.01.014

A beta/Cu-catalyzed oxidation of cholesterol in 1,2-dipalmitoyl phosphatidylcholine liposome membrane

Toshinori Shimanouchi, Makoto Tasaki, Huong Thi Vu, Haruyuki Ishii, Noriko Yoshimoto, Hiroshi Umakoshi, Ryoichi Kuboi

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 109 ( 2 ) 145 - 148 2010.2

Language：English Publishing type：Research paper (scientific journal) Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN

The amyloid beta protein with 42 amino acid residues (A beta), which is a causative protein of Alzheimer's disease (AD), forms the complex with copper (II) to induce the cholesterol oxidase-like activity by the proton transfer from the cholesterol. In this study, the oxidation of cholesterol by A beta/Cu complex was investigated on the surface of the zwitterionic phospholipid liposome including the bound water advantageous for the enhancement of the proton transfer. The bound water was pooled by the formation of cholesterol-rich domain within liposomes. The resulting reactivity was enhanced by the proton transfer mediated by the bound water. (C) 2009, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2009.08.003

A Highly-Sensitive Leakage Current Microsensor by Using Denaturant: Detection of Target Protein by DPPC Liposome

P. Lorchirachoonkul, T. Shimanouchi, K. Yamashita, H. Umakoshi, R. Kuboi, M. Noda

EUROSENSORS XXIV CONFERENCE 5 812 - 815 2010

Language：English Publishing type：Research paper (international conference proceedings) Publisher：ELSEVIER SCIENCE BV

We have developed a highly-sensitive leakage current microsensor by using the DPPC liposome entrapping K-4[Fe(CN)(6)] solution to detect existence of biomolecules especially proteins and their dynamic conditions. In this work, the addition of guanidinium hydrochloride (GuHCl) as protein denaturant successfully obtain a prominent improvement in sensitivity by 129-fold of magnitude, although target protein, which is carbonic anhydrase from bovine ( CAB), weakly interacts with liposome under the normal condition. Moreover, we can use it to evaluate the conformation state of CAB protein such as 'Native', 'Molten-Globule' and 'Unfold' states. (C) 2009 Published by Elsevier Ltd.

DOI： 10.1016/j.proeng.2010.09.232

Characterization of Amyloid beta Fibrils with An Aqueous Two-Phase System: Implications of Fibril Formation

Toshinori Shimanouchi, Naoya Shimauchi, Keiichi Nishiyama, Huong Thi Vu, Hisashi Yagi, Yuji Goto, Hiroshi Umakoshi, Ryoichi Kuboi

SOLVENT EXTRACTION RESEARCH AND DEVELOPMENT-JAPAN 17 121 - 128 2010

Language：English Publishing type：Research paper (scientific journal) Publisher：JAPAN ASSOC SOLVENT EXTRACTION

The pathological process of Alzheimer's disease is closely related to amyloid fibril formation by the causative protein, amyloid beta (A beta). The growth behavior of A beta fibrils is predominated by the seeds-monomeric A beta interaction. In this study, the local hydrophobicity of seeds of A beta fibrils was investigated by the aqueous two-phase partitioning method to evaluate the hydrophobic interaction between the seed-monomeric A beta. The seeds showed a high local hydrophobicity relative to the monomer and fibrils. From the fibril growth experiment and the additive effect of Triton X-100, we could demonstrate the contribution of the hydrophobic seeds-monomer interaction to fibril formation.

DOI： 10.15261/serdj.17.121

Oxidative/heat stress enhanced production of chitosanase from Streptomyces griseus cells through its interaction with liposome

Kien Xuan Ngo, Hiroshi Umakoshi, Haruyuki Ishii, Huong Thi Bui, Toshinori Shimanouchi, Ryoichi Kuboi

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 108 ( 6 ) 471 - 476 2009.12

Language：English Publishing type：Research paper (scientific journal) Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN

The effective production and secretion of chitosanase from S. griseus cells, in the presence of hydrogen peroxide, were studied by the treatment of the cells with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) liposomes under heat condition. The variation of the conformation and activity of the chitosanase caused by the interaction of liposomes with target chitosanase under stress condition was systematically investigated by using circular dichroism (CD) spectra and dielectric dispersion analysis (DDA). The effect of the oxidative stress (hydrogen peroxide) on the lipid and protein peroxide of cell membrane of S. griseus pretreated with and without liposomes under heat stress at 41 degrees C was further carried out. The possible utilization of membrane-membrane interaction between liposomes and cell membrane induced by the heat treatment was further investigated to enhance the production of chitosanase from S. griseus under oxidative stress condition. (C) 2009, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2009.06.010

Membranomics Research on Interactions between Liposome Membranes with Membrane Chip Analysis

SHIMANOUCHI Toshinori, OYAMA Ena, ISHII Haruyuki, UMAKOSHI Hiroshi, KUBOI Ryoichi

MEMBRANE 34 ( 6 ) 342 - 350 2009.11

Language：English Publisher：THE MEMBRANE SOCIETY OF JAPAN

The liposome immobilized on indium tin–oxide (ITO) electrode was prepared to evaluate the liposome–liposome interaction to develop "Membrane Chip". Three kinds of standard liposomes entrapping the fluorescence probe, calcein, were used for their immobilization, where the immobilized liposomes show the different characteristics in their phase transition temperature and membrane fluidity. Membrane Chip could give the set of the quantitative information relating to the membrane–membrane interaction. The interaction between three immobilized liposomes and 22 kinds of liposomes as samples was then systematically investigated on a basis of the calcein release from the immobilized liposomes, which could give the information in relation to "Membranome". It was, therefore, found that the Membrane Chip as a means of Membranomics research was effective to evaluate the Membranome information of liposomes.

DOI： 10.5360/membrane.34.342

Other Link： https://jlc.jst.go.jp/DN/JALC/00341564957?from=CiNii

Charged liposome affects the translation and folding steps of in vitro expression of green fluorescent protein

Hiroshi Umakoshi, Keishi Suga, Huong Thi Bui, Masato Nishida, Toshinori Shimanouchi, Ryoichi Kuboi

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 108 ( 5 ) 450 - 454 2009.11

Language：English Publishing type：Research paper (scientific journal) Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN

The role of the charged liposome on the in vitro expression of green fluorescent protein (GFP) was investigated, focusing on its elemental steps such as transcription, translation and folding. The total GFP expression was enhanced to 145% when a neutral liposome (POPC: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocoline) was added externally to a cell-free translation system. On the contrary, the addition of the charged liposome composed of POPC with anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) or cationic stearyl amine (SA) inhibited the total GFP expression, depending on the surface charge density of liposome. In transcription, the RNA synthesis was enhanced regardless of the variation of the surface charge, indicating that transcription was enhanced due to the stabilization of RNA structure by its hydrophobic interaction with liposome. Translation was inhibited by cationic liposome although it was enhanced by anionic liposome and neutral liposome. On the other hand, the folding was not inhibited in the presence of neutral liposome, whereas anionic liposome and cationic liposome inhibited the folding in proportion to the their surface charges, suggesting that the total GFP expression was controlled by a charged liposome in the translation step and folding step. (C) 2009, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2009.05.012

Analysis of interaction between liposome membranes induced by stress condition: Utilization of liposomes immobilized on indium tin oxide electrode Reviewed

Haruyuki Ishii, Toshinori Shimanouchi, Hiroshi Umakoshi, Ryoichi Kuboi

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 108 ( 5 ) 425 - 428 2009.11

Language：English Publishing type：Research paper (scientific journal) Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN

NBD-cholesterol (NBD-Ch)-modified liposome was immobilized on indium tin oxide (ITO) electrode via the covalent binding method. The transfer of NBD-Ch between the immobilized liposomes and the target liposomes was observed by using a fluorescent microscope. The addition of liposome suspension co-incubated with alpha-chymotrypsin or stimuli-responsive polymer to the surface of the above ITO electrode, enhanced the liposome-liposome interaction, resulting in the promotion of NBD-Ch transfer. The apparent transfer rate constant of NBD-Ch was found to be correlated with the index for the liposome-liposome interaction evaluated by an immobilized liposome chromatography. This suggests that the present method using the liposome-immobilized ITO electrode was effective to evaluate the liposome-liposome interaction induced by the protein or the stimuli-responsive polymer under stress conditions. (C) 2009, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2009.05.009

Preparation of liposome immobilized membrane module and its application Reviewed

Hiroshi Umakoshi, Hiroyuki Sugaya, Yuji Tohtake, Toshinori Shimanouchi, Ryoichi Kuboi

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 108 S65 - S65 2009.11

Membrane interference: Role of liposome on in vitro gene expression of GFP Reviewed

Suga Keishi, Umakoshi Hiroshi, Bui Huong Thi, Tanabe Tomoyuki, Shimanouchi Toshinori, Kuboi Ryoichi

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 108 ( 1 ) S102 - S103 2009.11

Disaggregation of catecholamine derivative-induced amyloid fibrils on biomembranes Reviewed

Shimanouchi Toshinori, Vu Huong Thi, Umakoshi Hiroshi, Kuboi Ryoichi

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 108 ( 1 ) S31 - S32 2009.11

Development of membrane chip system for study on membrane-protein interaction Reviewed

Shimanouchi Toshinori, Ishii Haruyuki, Oyama Ena, Shimauchi Naoya, Umakoshi Hiroshi, Kuboi Ryoichi

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 108 S162 - S163 2009.11

Calcein permeation across phosphatidylcholine bilayer membrane: Effects of membrane fluidity, liposome size, and immobilization

Toshinori Shimanouchi, Haruyuki Ishii, Noriko Yoshimoto, Hiroshi Umakoshi, Ryoichi Kuboi

COLLOIDS AND SURFACES B-BIOINTERFACES 73 ( 1 ) 156 - 160 2009.10

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

The permeation of calcein across the phospholipid bilayer membrane is a key phenomenon in the detection system using liposomes as a sensor unit. The behavior of the calcein release from the liposome was analyzed by a first-order kinetic to obtain the permeability coefficient, P(s) [cm/s]. The P(s) value for the neutral liposome, prepared by zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), was found to depend on both the diameter of liposome and the temperature. The membrane fluidity of the POPC liposome, evaluated by the hydrophobic probe, 1-(4-trimethyl-aminophenyl)-6-diphenyl-1,3,5-hexatriene, was also dependent on the liposome diameter and the temperature. The P, values for various neutral liposomes under gel phase or liquid-crystalline phase were correlated with their membrane fluidity, although some data were a little scattered, possibly due to the lamellarity. It is therefore considered that the membrane fluidity dominates the permeability of calcein across the neutral phospholipid membrane. Based on the above results, the P, value for liposomes immobilized on the solid surface is discussed. (C) 2009 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.colsurfb.2009.05.014

Characterization of heat-induced interaction of neutral liposome with lipid membrane of Streptomyces griseus cell

Kien Xuan Ngo, Hiroshi Umakoshi, Toshinori Shimanouchi, Ryoichi Kuboi

COLLOIDS AND SURFACES B-BIOINTERFACES 73 ( 2 ) 399 - 407 2009.10

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

The interaction between the neutral 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) liposomes and cell membrane of Streptomyces griseus induced by the heat treatment at specific temperature was investigated, focusing on the internalization of the neutral POPC liposomes with S. griseus cells. In an attempt to clarify the modes of liposome internalization, various kinds of inhibitors of endocytotic pathways were used to treat S. griseus cells. The efficiency of the heat treatment on liposome-cell membrane interactions was finally characterized based on the hydrophobic, electrostatic interactions and hydration effect. In fact, the internalization of the neutral liposomes into these cells was found to show higher rate and greater amount at higher temperatures. The kinetic study showed that the maximum amount of the internalized liposomes was, respectively, 469 x 10(5) and 643 x 10(5) liposomes/cell at 37 and 41 degrees C. The internalization of the neutral liposomes induced by the heat treatment was characterized, implying that the endocytosis occurred. The interactions involving the internalization, adsorption, and fusion of these liposomes with S. griseus cells were mainly contributed by the hydrophobic interaction and the unstable hydrogen bonds caused by the loss of water of surface hydration of cell membrane rather than the electrostatic interaction under the specific heat condition. (c) 2009 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.colsurfb.2009.06.015

Negatively charged liposome as a potent inhibitor of post-translation during in vitro synthesis of green fluorescent protein

Huong Thi Bui, Hiroshi Umakoshi, Keishi Suga, Masato Nishida, Toshinori Shimanouchi, Ryoichi Kuboi

BIOCHEMICAL ENGINEERING JOURNAL 46 ( 2 ) 154 - 160 2009.10

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE SA

The effect of negatively charged liposome on in vitro synthesis of a reporter protein, green fluorescent protein (UP), was investigated using a cell-free translation system. GFP was expressed with and without negatively charged liposome prepared with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylchorine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), resulting in the GFP fluorescence being reduced to approximately 60% in the presence of POPC/POPG at more than 30% POPG, depending oil its concentration. However, the amount of synthesized GFP products, as analyzed by SDS-PAGE, did not change with and without the POPC/POPG liposome. The results of the ultrafiltration operation indicate that the liposome interacts not only with synthesized polypeptide GFP but also with folded synthesized GFP (mature GFP). Liposome also inhibited refolding of unfolded GFP to its native state. The above results show that the POPC/POPG could inhibit the folding of GFP in the post-translational process of the gene expression product. (C) 2009 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bej.2009.05.002

Polymethylthiophene/Nafion-modified glassy carbon electrode for selective detection of dopamine in the presence of ascorbic acid

Vu Thi Huong, Toshinori Shimanouchi, Do Phuc Quan, Hiroshi Umakoshi, Pham Hung Viet, Ryoichi Kuboi

JOURNAL OF APPLIED ELECTROCHEMISTRY 39 ( 10 ) 2035 - 2042 2009.10

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

The possible use of an electrode modified with electroactive conductive poly(3-methylthiophene) (PMeT)/Nafion as a chemical sensor was investigated for the voltammetric analysis of Dopamine (DA). The electrochemical behavior of dopamine was examined by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) techniques. By using a PMeT-modified glassy carbon (GC/PMeT) electrode, DA and Ascorbic Acid (AA) signals could be separated but the AA at high concentrations still caused significant interference by overlapping the DA peak. In comparison to the GC/PMeT electrode, the glassy carbon (GC/Nafion/PMeT) electrode modified with hybrid film Nafion/PMeT was found to permit a superior separation by shifting the oxidation of AA peak toward the less positive potential. The DPV curves for a mixture of DA and AA at an GC/Nafion/PMeT electrode in a 0.1 M H(2)SO(4) solution showed peaks of DA and AA, at 0.45 and 0.21 V, respectively, indicating that the difference in the oxidation potential was 240 mV. In the 0.1 M H(2)SO(4) solution, the oxidation peak current on the differential pulse voltammograms for the GC/PMeT electrode increased linearly with the concentration of DA in the range 1 x 10(-6) to 1 x 10(-3) M, and the oxidation peak current on the differential pulse voltammograms for the GC/Nafion/PMeT electrode in the range 5 x 10(-7) to 2 x 10(-4) M. The DA detection sensitivity of the GC/Nafion/PMeT electrode (26.7 mu A mu M(-1) cm(-2)) was 22 times higher than that of the GC/PMeT electrode (1.21 mu A mu M(-1) cm(-2)).

DOI： 10.1007/s10800-009-9860-z

Preparation of Hollow Fiber Immobilized Liposome Membrane

SUGAYA Hiroyuki, UMAKOSHI Hiroshi, TOHTAKE Yuji, OYAMA Ena, SHIMANOUCHI Toshinori, KUBOI Ryoichi

MEMBRANE 34 ( 5 ) 272 - 280 2009.9

Language：English Publisher：THE MEMBRANE SOCIETY OF JAPAN

Immobilized liposome membrane (ILM) was prepared in hollow fiber module (HF–ILM), where the liposome was loaded into the hollow fiber membrane module and was physically immobilized by forming the hydrogel in the cavity of the fiber membrane. Basic nature of the hydrogels prepared with xanthan gum (XG) and polyethyleneimine (PEI) via amino coupling method was first investigated in relation to the water contents of the gel matrix, so that the optimal condition for the liposome immobilization was obtained. The HF–ILM was prepared at the optimal condition, resulting in the high contents of immobilized liposome with high stability.

DOI： 10.5360/membrane.34.272

Other Link： https://jlc.jst.go.jp/DN/JALC/00338580205?from=CiNii

Immobilization of intact liposomes on solid surfaces: A quartz crystal microbalance study

Thi Huong Vu, Toshinori Shimanouchi, Haruyuki Ishii, Hiroshi Umakoshi, Ryoichi Kuboi

JOURNAL OF COLLOID AND INTERFACE SCIENCE 336 ( 2 ) 902 - 907 2009.8

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE

Immobilization of model cell membranes (liposomes) on sensing devices is useful for the detection, evaluation, and analysis of interaction between cell membranes and proteins or other biological materials. The method to immobilize the liposomes on the solid surface was investigated by using the quartz crystal microbalance (QCM) method. focusing on the density of immobilized liposomes and their stability. The different liposomes were applied onto the planar surface of the QCM electrode by varying the immobilization method. The results showed that the immobilized liposomes depended not only on the immobilization method but also on the properties of the liposomes. The liposome with low membrane fluidity likely showed intact immobilization on a solid surface. An electrostatic interaction also affected the amount of immobilized liposomes on the functionalized quartz crystal. By using the amino-coupling method, intact liposomes could be immobilized on a solid surface and immobilized liposomes could stabilize for 10 h with frequency changes less than 5%. (C) 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.jcis.2009.04.048

Inversion of the Configuration of a Single Stereocenter in a beta-Heptapeptide Leads to Drastic Changes in its Interaction with Phospholipid Bilayers

Toshinori Shimanouchi, Peter Walde, James Gardiner, Stefania Capone, Dieter Seebach, Ryoichi Kuboi

CHEMBIOCHEM 10 ( 12 ) 1978 - 1981 2009.8

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-V C H VERLAG GMBH

In summary, a change in the configuration of a single amino acid in a β3-heptapeptide can lead to remarkable changes in the physicochemical properties of the peptide with respect to the interaction with a (protein-free) biomimetic environment. Thus, the presence of an unnatural amino acid configuration in a peptide can have consequences not only with respect to specific interactions with proteins, but also, indirectly, with respect to the interaction with membrane lipids. © 2009 Wiley-VCH Verlag GmbH & Co. KGaA.

DOI： 10.1002/cbic.200900175

Kinetic Study on Giant Vesicle Formation with Electroformation Method

Toshinori Shimanouchi, Hiroshi Umakoshi, Ryoichi Kuboi

LANGMUIR 25 ( 9 ) 4835 - 4840 2009.5

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER CHEMICAL SOC

Giant vesicles (GVs) composed of zwitterionic phospholipids were prepared by the electroformation method. The growth behavior of GVs was quantitatively analyzed as a first-order kinetics of the radius of GVs to obtain the apparent growth rate constant k(Gr). On the basis of the dependence of the k(Gr) value on the preparation temperature and the lipid composition of GVs, the membrane fluidity of vesicle membranes was found to be dominant in the growth behavior of GVs. The comparison of the k(Gr) values with the membrane fluidity for GVs suggested the validity of the model that the swelling of the lipid membrane could induce the growth of GVs.

DOI： 10.1021/la8040488

Cationic Liposome Inhibits Gene Expression in an E. coli Cell-Free Translation System Reviewed

Bui, H. T, Umakoshi, H, Suga, K, Tanabe, T, Shimanouchi, T, Kuboi, R

Membrane 34 ( 3 ) 146 - 151 2009.3

Liposome membrane can act like molecular and metal chaperones for oxidized and fragmented superoxide dismutase

Le Quoc Tuan, Hiroshi Umakoshi, Toshinori Shimanouchi, Ryoichi Kuboi

ENZYME AND MICROBIAL TECHNOLOGY 44 ( 2 ) 101 - 106 2009.2

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE INC

A mechanism for liposome-recruited activity of oxidized and fragmented superoxide dismutase (Fr.-SOD) [Tuan LQ, Umakoshi H, Shimanouchi T, Kuboi R. Liposome-recruited activity of oxidized and fragmented superoxide dismutase. Langmuir 2008;24:350-4] was further investigated, focusing on the secondary structure of Fr.-SOD. Liposome membrane was found to assist the conformational change of Fr.-SOD and reactivate the enzymatic activity. like molecular and metal chaperones. The loss of SOD activity and its secondary structure was observed during 6 h oxidation in 2 mM hydrogen peroxide. The contents of the alpha-helix and beta-sheet structures in the oxidized and fragmented SOD (2 mu M) were increased only in the presence of 10 mu M Cu2+ and Zn2+ together, or in the presence of 2 mM POPC liposomes. The mixture of all of these elements (fragmented SOD and POPC liposomes with Cu2+ and Zn2+) gave not only the increase of the alpha-helix and beta-sheet contents but also the mediation of the high SOD-like activity. (c) 2008 Published by Elsevier Inc.

DOI： 10.1016/j.enzmictec.2008.10.012

A Microbolometer of Bio-Thermochemical Sensor Immobilized Lipo some for Detection of Causative Protein of Alzheimer's Disease, Amyloid Beta

M. Noda, T. Asai, T. Shimanouchi, K. Yamashita, H. Umakoshi, M. Okuyama, R. Kuboi

PROCEEDINGS OF THE EUROSENSORS XXIII CONFERENCE 1 ( 1 ) 1071 - + 2009

Language：English Publishing type：Research paper (international conference proceedings) Publisher：ELSEVIER SCIENCE BV

Forming process of liposome droplet and its stable immobilization on a Si-based microbolometer are investigated and applied for detection of causative protein of Alzheimer's disease, amyloid beta (A beta) protein, where the disease is closely related to the amyloid fibril formation. This work is a preliminary but innovative approach applying a bio-thermochemical microsensor to monitor the formation. It is observed that the bolometer surface keeps a long time intact behavior of the liposome up to 24 hours. It seems from time course results with A beta that the variation in the temperature profile corresponds to that observed in fluorescence analysis result of the formation.

DOI： 10.1016/j.proche.2009.07.267

A Leakage Current Microsensor for Detection of Interaction between an Electrolyte-Entrapping Liposome and Protein

Minoru Noda, Takeshi Asai, Kaoru Yamashita, Toshinori Shimanouchi, Hiroshi Umakoshi, Masanori Okuyama, Ryouichi Kuboi

2009 IEEE SENSORS, VOLS 1-3 1883 - + 2009

Language：English Publishing type：Research paper (international conference proceedings) Publisher：IEEE

We have newly made a specific biomolecule microsensor measuring leakage current and impedance with minute liposome solution that interacts with biomolecules such as protein. The microsensor was fabricated with a Si surface-bulk-micromachining process. K-4[Fe(CN)(6)] solution was entrapped inside of liposome of 1,2-dipalmitoyl-sn-glycero-3-phosphocholline (DPPC) and Carbonic Anhydrase from Bovine (CAB) was used as added protein for the interaction. A clear leakage in nA order was observed for the CAB, indicating the microsensor detects the CAB through the liposome-protein interaction. The detectivity increased by about 7 times and the volume of solution was decreased by about 10,000 from the previous result.

DOI： 10.1109/ICSENS.2009.5398390

Characterization of Hollow Fiber Immobilized Liposome Membrane by Using Aqueous Two-Phase Partitioning Systems

Hiroyuki Sugaya, Hiroshi Umakoshi, Yuji Tohtake, Toshinori Shimanouchi, Ryoichi Kuboi

SOLVENT EXTRACTION RESEARCH AND DEVELOPMENT-JAPAN 16 103 - 109 2009

Language：English Publishing type：Research paper (scientific journal) Publisher：JAPAN ASSOC SOLVENT EXTRACTION

The modification of the surface of a polysulfone (PSf) membrane in a hollow fiber module was performed based on the evaluated hydrophobicity of the polymers used in its modification to develop a new type of membrane module immobilizing liposome. The surface net hydrophobicity of the polyvinylpyrrolidone (PVP), to be modified, was first characterized by using the aqueous two-phase partitioning method. The hollow fiber module using the PVP-modified PSf membrane was characterized based on the adsorption behaviors of standard amino acids, showing that the membrane surface was found to have a hydrophilic surface nearly equal to the difference in aqueous two-phase systems. The immobilized liposome membrane (ILM) in the above module was found to have a hydrophobic potential, thus promoting the adsorption of the hydrophobic molecules on its surface.

Characterization of Green Fluorescent Protein Using Aqueous Two-Phase Systems

Hiroshi Umakoshi, Masato Nishida, Keishi Suga, Huong Thi Bui, Toshinori Shimanouchi, Ryoichi Kuboi

SOLVENT EXTRACTION RESEARCH AND DEVELOPMENT-JAPAN 16 145 - 150 2009

Language：English Publishing type：Research paper (scientific journal) Publisher：JAPAN ASSOC SOLVENT EXTRACTION

The surface net hydrophobicity and local hydrophobicity of green fluorescent protein was evaluated by using the aqueous two-phase partitioning method. The surface net hydrophobicity of GFP allowed it to be classified as a hydrophobic protein although the local hydrophobicity was low. The local hydrophobicity of the GFP was increased by a pH shift to acidic conditions and was maximal at pH 4. The surface net hydrophobicity increased at acidic conditions less than pH 4. The conformational change of GFP at acidic pH is discussed based on the above results.

A Protein Microsensor by Detection of Leakage Current from Interaction between an Electrolyte-Entrapping Liposome and Protein

M. Noda, T. Asai, T. Shimanouchi, K. Yamashita, H. Umakoshi, M. Okuyama, R. Kuboi

PROCEEDINGS OF THE EUROSENSORS XXIII CONFERENCE 1 ( 1 ) 1295 - + 2009

Language：English Publishing type：Research paper (international conference proceedings) Publisher：ELSEVIER SCIENCE BV

We have newly made a specific biomolecule microsensor measuring leakage current and impedance with minute liposome solution that interacts with biomolecules such as protein. The microsensor was fabricated with a Si surface-bulk-micromachining process. K-4[Fe(CN)(6)] solution was entrapped inside of liposome of 1,2-dipalmitoyl-sn-glycero-3-phosphocholline (DPPC) and Carbonic Anhydrase from Bovine (CAB) was used as added protein for the interaction. A clear leakage in nA order was observed for the CAB, indicating the microsensor detects the CAB through the liposome-protein interaction. The detectivity increased by about 7 times and the volume of solution was decreased by about 10,000 from the previous result.

DOI： 10.1016/j.proche.2009.07.323

A Microbolometer of Bio-Thermochemical Sensor Immobilized Lipo some for Detection of Causative Protein of Alzheimer's Disease, Amyloid Beta

M. Noda, T. Asai, T. Shimanouchi, K. Yamashita, H. Umakoshi, M. Okuyama, R. Kuboi

PROCEEDINGS OF THE EUROSENSORS XXIII CONFERENCE 1 ( 1 ) 1071 - + 2009

Language：English Publishing type：Research paper (international conference proceedings) Publisher：ELSEVIER SCIENCE BV

Forming process of liposome droplet and its stable immobilization on a Si-based microbolometer are investigated and applied for detection of causative protein of Alzheimer's disease, amyloid beta (A beta) protein, where the disease is closely related to the amyloid fibril formation. This work is a preliminary but innovative approach applying a bio-thermochemical microsensor to monitor the formation. It is observed that the bolometer surface keeps a long time intact behavior of the liposome up to 24 hours. It seems from time course results with A beta that the variation in the temperature profile corresponds to that observed in fluorescence analysis result of the formation.

DOI： 10.1016/j.proche.2009.07.267

A Bio-Thermochemical Sensor of Microbolometer Immobilized Liposome for Detection of Causative Protein of Alzheimer's Disease, Amyloid Beta

Minoru Noda, Takeshi Asai, Kaoru Yamashita, Toshinori Shimanouchi, Hiroshi Umakoshi, Masanori Okuyama, Ryouichi Kuboi

2009 IEEE SENSORS, VOLS 1-3 838 - + 2009

Language：English Publishing type：Research paper (international conference proceedings) Publisher：IEEE

Forming process of Liposome droplet and its stable immobilization on a Si-based microbolometer are investigated and applied for detection of causative protein of Alzheimer's disease, amyloid beta (A beta) protein, where the disease is closely related to the amyloid fibril formation. This work is a preliminary but innovative approach applying a bio-thermochemical microsensor to monitor the formation. It is observed that the bolometer surface keeps a long time intact behavior of the liposome up to 24 hours. It seems from time course results with A beta that the variation in the temperature profile corresponds to that observed in fluorescence analysis result of the formation.

A Leakage Current Microsensor for Detection of Interaction between an Electrolyte-Entrapping Liposome and Protein

Minoru Noda, Takeshi Asai, Kaoru Yamashita, Toshinori Shimanouchi, Hiroshi Umakoshi, Masanori Okuyama, Ryouichi Kuboi

2009 IEEE SENSORS, VOLS 1-3 1883 - + 2009

Language：English Publishing type：Research paper (international conference proceedings) Publisher：IEEE

We have newly made a specific biomolecule microsensor measuring leakage current and impedance with minute liposome solution that interacts with biomolecules such as protein. The microsensor was fabricated with a Si surface-bulk-micromachining process. K-4[Fe(CN)(6)] solution was entrapped inside of liposome of 1,2-dipalmitoyl-sn-glycero-3-phosphocholline (DPPC) and Carbonic Anhydrase from Bovine (CAB) was used as added protein for the interaction. A clear leakage in nA order was observed for the CAB, indicating the microsensor detects the CAB through the liposome-protein interaction. The detectivity increased by about 7 times and the volume of solution was decreased by about 10,000 from the previous result.

Enhanced Release of Chitosanase from Streptomyces griseus through Direct Interaction of Liposome with Cell Membrane under Heat Stress

Kien Xuan Ngo, Hiroshi Umakoshi, Toshinori Shimanouchi, Huong Thi Bui, Ryoichi Kuboi

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 106 ( 6 ) 602 - 605 2008.12

Language：English Publishing type：Research paper (scientific journal) Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN

A direct interaction of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes with membrane of Streptomyces griseus cell under the heat stress at 41 degrees C increased the chitosanase production and release to 2.2 times higher than that at 37 degrees C without the POPC liposomes. Amount of chitosanase released across the lipid mimicking cell membrane (LMCM) liposome under a heat at 41 degrees C in the presence of POPC liposomes was 17% of initially-entrapped chitosanase while it was only 1% in the absence of POPC liposomes, even under a heat stress at 41 degrees C, clearly showing the importance of the direct interaction between membrane and membrane.

DOI： 10.1263/jbb.106.602

A bio-thermochemical microbolometer with immobilized intact liposome on sensor solid surface Reviewed

M. Noda, T. Shimanouchi, M. Okuyama, R. Kuboi

SENSORS AND ACTUATORS B-CHEMICAL 135 ( 1 ) 40 - 45 2008.12

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE SA

We have developed a scheme of bio-thermochemical microbolometer with immobilized intact liposome on sensor solid surface. A liposome of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was confirmed to be deposited intact by X-ray diffraction (XRD) and AFM analysis on a Pt film that is used as resistive bolometer material. By using a fabricated surface micromachined thermal cavity structure beneath the Pt resistor film with the immobilized intact liposome, thermochemical reactions of DPPC on phase transition among gel, ripple and fluid phases have been successfully detected. It is also found from analysis of fluorescence polarization that endothermic reaction especially at around 40 degrees C (phase change from ripple to fluid) corresponds to the change in mobility of phospholipid molecule. As a result we believe that the developed scheme of the microbolometer is effective for detecting thermochemical reaction of biochemical molecules that interact with the liposome. (c) 2008 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.snb.2008.07.022

Liposome membrane itself can affect gene expression in the Escherichia coli cell-free translation system

Huong Thi Bui, Hiroshi Urnakoshi, Kien Xuan Ngo, Masato Nishida, Toshinori Shimanouchi, Ryoichi Kuboi

LANGMUIR 24 ( 19 ) 10537 - 10542 2008.10

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER CHEMICAL SOC

A possible role of a model biomembrane, liposome, in gene expression was investigated by using the cell-free translation system. A reporter protein, green fluorescence protein (GFP), was expressed in vitro with and without liposome prepared with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyl chorine (POPC) and cholesterol (Ch) (5.7 mM lipid concentration). In the presence of POPC/Ch liposome, the fluorescence intensity of produced GFP was found to be 1.67 times higher than that in the control after 18 h of expression. The results of the SDS-PAGE analysis also show the above promotion effect of the liposome on the net expression of the GFP gene (1.58 times more). The amounts of mRNA were found to be promoted to 1.29 times higher than those in the control. The differences among mRNA, net expression of the GFP gene, and GFP fluorescence indicate that the enhanced GFP expression in the presence of POPC/Ch liposome could primarily affect the transcription and translation of the GFP gene among the possible steps of gene expression. The variation of in vitro gene expression with various liposomes also shows that the biomembrane could act as a modulator to split the genotype and phenotype in a biological cell.

DOI： 10.1021/la801962j

Arsenic (V) induces a fluidization of algal cell and liposome membranes

Le Quoc Tuan, Tran Thi Thanh Huong, Pham Thi Anh Hong, Tomonori Kawakami, Toshinori Shimanouchi, Hiroshi Umakoshi, Ryoichi Kuboi

TOXICOLOGY IN VITRO 22 ( 6 ) 1632 - 1638 2008.9

Language：English Publishing type：Research paper (scientific journal) Publisher：PERGAMON-ELSEVIER SCIENCE LTD

Arsenate is one of the most poisonous elements for living cells. When cells are exposed to arsenate, their life activities are immediately affected by various biochemical reactions, such as the binding of arsenic to membranes and the substitution of arsenic for phosphate or the choline head of phospholipids in the biological membranes. The effects of arsenate on the life activities of algae Chlorella vulgaris were investigated at various concentrations and exposure times. The results demonstrated that the living activities of algal cells (10(10) cells/L) were seriously affected by arsenate at a concentration of more than 7.5 mg As/L within 24 h. Algal cells and the artificial membranes (liposomes) were exposed to arsenate to evaluate its effects on the membrane fluidization. In the presence of arsenate, the membranes were fluidized due to the binding and substitution of arsenate groups for phosphates or the choline head on the their membrane surface. This fluidization of the biological membranes was considered to enhance the transport of toxicants across the membrane of algal cells. (C) 2008 Published by Elsevier Ltd.

DOI： 10.1016/j.tiv.2008.05.012

Characterization of Oxidized and Fragmented Superoxide Dismutase Recruited on Liposome Surface

TUAN Le Quoc, UMAKOSHI Hiroshi, SHIMANOUCHI Toshinori, KUBOI Ryoichi

MEMBRANE 33 ( 4 ) 173 - 179 2008.7

Language：English Publisher：THE MEMBRANE SOCIETY OF JAPAN

The interaction of oxidized and fragmented SOD with liposomes has been reported to induce the original SOD-likeenzymatic activity (L.Q. Tuan et al., Langmuir, 24 (2008) 350-354).The effect of several kinds of liposomes on theirrecruited activity of oxidized and fragmented SOD was first investigated. The addition of zwitterionic liposomes withhigh membrane fluidity or that with positive charge was found to increase the SOD-like activity of fragmented SODalthough the negatively charged liposome has no effect on its activity. The SOD-like activity was found to be relatedto the adsorbed amount of the peptides on the liposome surface. The analyzed characteristics of the peptide, togeth-er with the above findings, imply that the liposome-recruited activity of the fragmented SOD was related with therecognition of the SOD fragment by the liposome caused by the combination of electrostatic and hydrophobic inter-action, and hydrogen bonding between the peptide and liposome membrane.

DOI： 10.5360/membrane.33.173

Other Link： https://jlc.jst.go.jp/DN/JALC/00316941158?from=CiNii

Superoxide Dismutase-like Activity of Liposomes Modified with Dodecanoyl His and Metal Ions

UMAKOSHI Hiroshi, TUAN Le Quoc, MORIMOTO Kengo, OHAMA Yuji, SHIMANOUCHI Toshinori, KUBOI Ryoichi

MEMBRANE 33 ( 4 ) 180 - 187 2008.7

Language：English Publisher：THE MEMBRANE SOCIETY OF JAPAN

The liposome modified with simple ligand and metal ions shows the superoxide dismutase-like activity. The mem-brane fluidity of various liposomes modified with the functional ligand (Dodecanoyl-His; Dodec-His) and the cluster-ing of the ligand on the liposome surface were first characterized, showing that the clustering of Dodec-His could beinduced on the liposome surface at gel-phase. The capacity of adsorption of Cu and Zn was found to be increaseddependently on the type of liposome, resulting in the maximal adsorption in liposome prepared by 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) at gel state and with higher ligand clustering state. As a result on the SOD-likeactivity of the metal/ligand-modified liposome, the SOD-like activity was found to be induced by using the above lipo-somes although its activity level is not so high.

DOI： 10.5360/membrane.33.180

Other Link： https://jlc.jst.go.jp/DN/JALC/00316941165?from=CiNii

Liposome modified with Mn-porphyrin complex can simultaneously induce antioxidative enzyme-like activity of both superoxide dismutase and peroxidase

Hiroshi Umakoshi, Kengo Morimoto, Yuji Ohama, Hideto Nagami, Toshinori Shimanouchi, Ryoichi Kuboi

LANGMUIR 24 ( 9 ) 4451 - 4455 2008.5

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER CHEMICAL SOC

An antioxidative liposome catalysis that mimics both superoxide dismutase (SOD) and peroxidase (POD) activities has been developed by using the liposomes modified with lipophilic Mn - (5,10,15,20-tetrakis[1-hexadecylpyridium-4-yl]-21H,23H-porphyrin) (Mn - HPyP). The SOD- and POD-like activities of the Mn - HPyP-modified liposome were first investigated by varying the type of phospholipid, such as 1,2-distearyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-dilauroyi-sn-glycero-3-phosphocholine (DLPC). Higher SOD-like activity was obtained in the case of DLPC and DMPC liposomes, in which the ligands were well-dispersed on the membrane in the liquid crystalline phase. The POD-like activity was maximal in the case of DMPC liposome, in which the Mn - HPyP complex was appropriately clustered on the membrane in the gel phase. On the basis of the above results, the co-induction of the SOD and POD activities to eliminate the superoxide and also hydrogen peroxide as a one-pot reaction was finally performed by using the Mn - HPyP-modified DMPC liposome, resulting in an increase in the efficiency of the elimination of both superoxide and hydrogen peroxide.

DOI： 10.1021/la800174n

Liposome-recruited activity of oxidized and fragmented superoxide dismutase

Le Quoc Tuan, Hiroshi Umakoshi, Toshinori Shimanouchi, Ryoichi Kuboi

LANGMUIR 24 ( 2 ) 350 - 354 2008.1

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER CHEMICAL SOC

The peptide fragment of H2O2-treated Cu,Zn-superoxidedismutase (SOD) was found to be reactivated with liposomes prepared by 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The fragmentation of SOD was observed by 2 mM H2O2 treatment as well as by SOD inactivation and the loss of an alpha-helix in the neighborhood of its activity center. The H2O2-treated SOD which lost its activity at different incubation times was dramatically reactivated only by adding POPC liposomes resulting in 1.3-2.8 times higher enzymatic activity. The ultrafiltration analysis of H2O2-treated SOD co-incubated with liposomes shows that some specific peptide fragments of the oxidized SOD can interact with POPC liposomes. A comparison of the fractions detected in reverse-phase chromatography shows that specific SOD fragments are able to contribute to the reactivation of oxidized and fragmented SOD in the presence of POPC liposomes. The liposomes can recruit the potentially active fragment of SOD among the lethally damaged SOD fragments to elucidate the antioxidative function.

DOI： 10.1021/la702690a

Variation of surface properties of Streptomyces griseus cells after heat treatment with liposome

Kien Xuan Ngo, Hiroshi Umakoshi, Toshinori Shimanouchi, Ryoichi Kuboi

SOLVENT EXTRACTION RESEARCH AND DEVELOPMENT-JAPAN 15 133 - 137 2008

Language：English Publishing type：Research paper (scientific journal) Publisher：JAPAN ASSOC SOLVENT EXTRACTION

Surface properties of Streptomyces griseus cells, such as surface net charge and surface net hydrophobicity, were characterized by using the aqueous two-phase system (ATPS) method. It was found that the surface net charge of S. griseus cells was reduced after the POPC liposome-treatment of the cells at 41 degrees C. The surface net hydrophobicities of S. griseus cells and liposome-treated cells cultivated at 41 degrees C were about - 122 and -510 kJ/mol, respectively, evaluated by the ATPS method. The above results show that the variation of the cell surface properties could be obtained by the cell-liposome interaction under heat stress conditions.

Bio-thermochemical sensor with liposome immobilized intact for protein detection using their interaction and membrane dynamics

Minoru Noda, Takeshi Asai, Kaoru Yamashita, Toshinori Shimanouchi, Masanori Okuyama, Ryouichi Kuboi

Proceedings of IEEE Sensors 882 - 885 2008

Language：English Publishing type：Research paper (international conference proceedings)

We have developed a new scheme of biothermochemical microbolometer with a small amount of droplet solution consisting from liposome and biochemical target molecules. And newly detected bio-thermochemical reaction between 2-dipalmitoyl-sn-glycero-3-phosphocholline) liposome immobilized intact and protein of lysozyme (enzyme) in a droplet on a Pt thin film microbolometer, where they are dissolved with concentration of 30 mM and 30 μ?, respectively, a droplet with volume of about 65 nL is immobilized on the surface of Pt film bolometer. This paper reports our first experimental results showing interaction between the DPPC liposome and lysozyme by the new scheme of biothermochemical sensor. © 2008 IEEE.

DOI： 10.1109/ICSENS.2008.4716582

Characterization of surface properties of microbial transglutaminase using aqueous two-phase partitioning method

Hiroshi Umakoshi, Noriko Yoshimoto, Makoto Yoshimoto, Toshinori Shimanouchi, Ryoichi Kuboi

SOLVENT EXTRACTION RESEARCH AND DEVELOPMENT-JAPAN 15 111 - 115 2008

Language：English Publishing type：Research paper (scientific journal) Publisher：JAPAN ASSOC SOLVENT EXTRACTION

The surface properties of recombinant microbial transglutaminase (MTG) were characterized by varying the pH through the aqueous two-phase system (ATPS) method in order to select the effective refolding pathways. It was found that the MTG activity was lost and intrinsic tryptophan fluorescence was varied at pH values less than 3.8. In this pH region, the surface net charge and surface net hydrophobicity of the MTG, determined by the ATPS method, gradually increased. At around pH 3.5, the local hydrophobicity of the MTG was found to be maximal.

Permeation of a beta-heptapeptide derivative across phospholipid bilayers

Toshinori Shimanouchi, Peter Walde, James Gardiner, Yogesh R. Mahajan, Dieter Seebach, Anita Thomae, Stefanie D. Kraemer, Matthias Voser, Ryoichi Kuboi

BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 1768 ( 11 ) 2726 - 2736 2007.11

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

Based on a number of experiments it is concluded that the fluorescein labeled beta-heptapeptide fluoresceinyl-NH-CS-(S)-beta(3)hAla-(S)-beta(3)hArg-(R)beta(3)hLeu-(S)- beta(3)hPhe-(S)-beta(3)hAla-(S)-beta(3)hAla-(S)-beta(3)hLys-OH translocates across lipid vesicle bilayers formed from DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine). The conclusion is based on the following observations: (i) addition of the peptide to the vicinity of micrometer-sized giant vesicles leads to an accumulation of the peptide inside the vesicles; (ii) if the peptide is injected inside individual giant vesicles, it is released from the vesicles in a time dependent manner; (iii) if the peptide is encapsulated within sub-micrometer-sized large unilamellar vesicles, it is released from the vesicles as a function of time; (iv) if the peptide is submitted to immobilized liposome chromatography, the peptide is retained by the immobilized DOPC vesicles. Furthermore, the addition of the peptide to calcein-containing DOPC vesicles does not lead to significant calcein leakage and vesicle fusion is not observed. The finding that derivatives of the beta-heptapeptide (S)-beta(3)hAla-(S)-beta(3)hArg-(R)-beta(3)hLeu-(S)-beta(3)hPhe-(S)-beta(3)hAla-(S)-beta(3)hAla-(S)-beta(3)hLys-OH can translocate across phospholipid bilayers is supported by independent measurements using Tb3+-containing large unilamellar vesicles prepared from egg phosphatidylcholine and wheat germ phosphatidylinositol (molar ratio of 9: 1) and a corresponding peptide that is labeled with dipicolinic acid instead of fluorescein. The experiments show that this dipicolinic acid labeled beta-heptapeptide derivative also permeates across phospholipid bilayers. The possible mechanism of the translocation of the particular beta-heptapeptide derivatives across the membrane of phospholipid vesicles is discussed within the frame of the current understanding of the permeation of certain oligopeptides across simple phospholipid bilayers. (c) 2007 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbamem.2007.07.011

Immobilized-Liposome Sensor System for Detection of Proteins under Stress Conditions

JUNG Ho-Sup, ISHII Haruyuki, SHIMANOUCHI Toshinori, UMAKOSHI Hiroshi, KUBOI Ryoichi

MEMBRANE 32 ( 5 ) 294 - 301 2007.9

Study on Stress-Mediated Behavior and Preparation of Giant Vesicles

Toshinori Shimanouchi, Hiroshi Umakoshi, Ryoichi Kuboi

Giant Vesicles: Perspectives in Supramolecular Chemistry 6 369 - 377 2007.3

Fluorescence study on the domain formation of N-dodecanoyl-L-tryptophan within a liposome membrane

Kazuma Yasuhara, Toshinori Shimanouchi, Hiroshi Umakoshi, Ryoichi Kuboi

COLLOID AND POLYMER SCIENCE 285 ( 2 ) 239 - 243 2006.11

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

The aggregation behavior of N-dodecanoyl-L-tryptophan within a liposome membrane was investigated by fluorescence spectroscopy. Liposomes with a mean diameter of 100 nm, formed from either 1-2-dilauroyl-sn-glycero3-phosphocholine, 1-2-myristoyl-sn-glycero-3-phosphocholine, or 1-2-palmitoyl-sn-glycero-3-phosphocholine, were used. Fluorescence measurements indicate that the sodium salt of N-dodecanoyl-L-tryptophan forms domains with the liposome membranes below the main phase transition temperature, T-m. Above T-m, the molecules are homogeneously dispersed in the liquid crystalline state of the membranes.

DOI： 10.1007/s00396-006-1547-y

Immobilization of liposomes onto quartz crystal microbalance to detect interaction between liposomes and proteins

S Morita, M Nukui, R Kuboi

JOURNAL OF COLLOID AND INTERFACE SCIENCE 298 ( 2 ) 672 - 678 2006.6

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE

To study the interaction between liposomes and proteins, intact liposomes were immobilized on a metal planar support by chemical binding and/or bioaffinity using a quartz crystal microbalance (QCM). A large decrease in the resonance frequency of quartz crystal was observed when the QCM, modified by a self-assembled monolayer (SAM) of carboxythiol, was added to liposome solutions. The stable chemical immobilization of intact liposomes onto SAM was judged according to the degree with which adsorbed mass depended on the prepared size of liposomes, as well as on the activation time of SAMs when amino-coupling was introduced, where the liposome coverage of electrodes was 69 +/- 8% in optimal conditions. When avidin-biotin binding was used on amino-coupling liposome layers, liposome immobilization finally reached 168% coverage of the electrode surface. Denatured protein was also successfully detected according to the change in the frequency of the liposome-immobilized QCM. The adsorbed mass of denatured carbonic anhydrase from bovine onto immobilized liposomes showed a characteristic peak at a concentration of guanidine hydrochloride that corresponded to a molten globule-like state of the protein, although the mass adsorbed onto deactivated SAM increased monotonously. (c) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.jcis.2005.12.043

Evaluation of temperature and guanidine hydrochloride-induced protein-liposome interactions by using immobilized liposome chromatography

N Yoshimoto, M Yoshimoto, K Yasuhara, T Shimanouchi, H Umakoshi, R Kuboi

BIOCHEMICAL ENGINEERING JOURNAL 29 ( 3 ) 174 - 181 2006.4

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE SA

Hydrophobic interactions between nine model proteins and net-neutral lipid bilayer membranes (liposomes) under stress conditions were quantitatively examined by using immobilized liposome chromatography (ILC). Small or large unilamellar liposomes were composed of 1-palinitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and immobilized in a gel matrix by utilizing covalent coupling between amino-containing lipids and activated gel beads or avidin-biotin biospecific binding. Retardation of bovine carbonic anhydrase (CAB) in ILC was pronounced at particular temperatures (50 and 60 degrees C) where the local hydrophobicity of theses protein molecules becomes sufficiently large. Protein-induced leakage of a hydrophilic dye (calcein) from immobilized liposomes interior was also drastically enhanced at particular temperatures where large retardation was observed. For other proteins examined, similar results were also observed. The specific capacity factor of the proteins characteristic for the ILC and the amount of calcein released from immobilized liposomes were successfully expressed as a function of the product of the local hydrophobicities of proteins and liposomes, regardless of protein species and the type of the stress conditions applied (denaturant and heating). These findings indicate that lipid membranes have an ability to non-specifically recognize local hydrophobicities of proteins to form stress-mediated supramolecular assemblies with proteins, which may have potential applications in bioprocesses such as protein refolding and separation. ILC was thus found to be a very useful method for the quantitative detection of dynarnic protein-liposome interactions triggered by stress conditions. (c) 2005 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bej.2005.08.030

Evaluation of carboxylic acid-induced conformational transitions of beta-lactoglobulin: Comparison of the alcohol effects on beta-lactoglobulin

BK Lee, T Shimanouchi, H Umakoshi, R Kuboi

BIOCHEMICAL ENGINEERING JOURNAL 28 ( 1 ) 79 - 86 2006.2

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE SA

Conformational transitions of bovine P-lactoglobulin A (beta-LG) induced by carboxylic acid were systematically studied by steady-state tryptophan (Trp) fluorescence. The behavior of beta-LG denaturation depends upon the species and concentration of carboxylic acid, as well as on the pH of solutions. The order of the effectiveness of the respective carboxylic acids was described as follows: MeCOOH < EtCOOH < PrCOOH < ClEtCOOH < TFA < iBuCOOH < nBuCOOH < PFPA. The conformational chan-e of beta-LG through the carboxylic acid-induced transitions of the beta-LG conformation were analyzed assuming a two-state mechanism between unfold and native states in order to obtain the m value, a measure of the dependence of the free energy change on the concentration of carboxylic acid. The m values of various carboxylic acids were compared with those of various alcohols based on the role of each group constituting the carboxylic acid and alcohol molecules, namely, the hydrocarbon group, hydroxyl group, halogen substituents, and the carboxyl group. Among these groups, the hydrophobic hydrocarbon groups and halogen substituents contributed positively to the in value, whereas the hydrophilic carboxyl and hydroxyl group contributed negatively. The present results can therefore be interpreted as a simple correlation based on the accessible surface area (ASA) of each groups of carboxylic acids and alcohols. These results suggest that the conformational transition of the protein due to the addition of carboxylic acids and alcohols can be explained both by hydrophobicity as well as clustering effects of each carboxylic acid and alcohol molecule. (c) 2005 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bej.2005.08.040

Heat-enhanced production of chitosanase from Streptomyces griseus in the presence of liposome

KX Ngo, H Umakoshi, T Shimanouchi, HS Jung, S Morita, R Kuboi

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 100 ( 5 ) 495 - 501 2005.11

Language：English Publishing type：Research paper (scientific journal) Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN

The effects of heat stress and liposome treatment on the growth of Streplomyces griseus cells and chitosanase production were investigated on the basis of using the designed strategy of a stress-mediated bioprocess. The effective conditions for increasing the interaction between chitosanase and the 1-palmitoyl-2-oleoyl-3-phosphocholine (POPC) liposome under heat stress condition were determined on the basis of the results of circular dichroism (CD) and dielectric dispersion analysis (DDA). Under these effective conditions, S. griseus cells were cultivated for the effective production of chitosanase. The results obtained from both CD spectra and DDA showed that heat stress enhances the interaction of the POPC liposomes and chitosanase. The strongest interaction between them could be obtained in the specific temperature range of 40-45 degrees C. The enhancement of the target chitosanase production was conducted under heat stress at WC in the presence and absence of the POPC liposomes. The growth rates of S. griseus cells in the cases of heat (41 degrees C) and heat (41 degrees C)/POPC treatments were respectively 1.2 and 1.4 times higher than that obtained under the control condition. In the heat (41 degrees C) and heat (41 degrees C)/POPC treatments, chitosanase activity increased to 1.8 and 2 times, respectively, higher than that obtained under the control condition. Heat stress and the addition of the POPC liposomes could therefore be utilized to induce the potential functions of bacterial cells for the enhancement of the final target production.

DOI： 10.1263/jbb.100.495

Oxidation of cholesterol catalyzed by amyloid beta-peptide (A beta)-Cu complex on lipid membrane

N Yoshimoto, M Tasaki, T Shimanouchi, H Umakoshi, R Kuboi

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 100 ( 4 ) 455 - 459 2005.10

Language：English Publishing type：Research paper (scientific journal) Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN

A catalytic reaction of H2O2 production by an amyloid beta-peptide (A beta)-Cu complex with cholesterol incorporated in a liposome was kinetically analyzed. The Michaelis-Menten model was applied to the H2O2 production reaction using cholesterol as the substrate catalyzed by the A beta-Cu complex. The K-m value for the A beta-Cu complex catalytic reaction with cholesterol-containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes (K-m=0.436 mu M for A beta(1-40); K-m=0.641 mu M for A beta(1-42)) was found to be smaller than that with cholesterol-containing 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes (K-m=0.585 mu M for A beta(1-40), K-m=0.890 mu M for A beta(1-42)). The results imply that membrane properties could play an important role in the interactions of the A beta-Cu complex with cholesterol in these liposomes. Considering the physical states of the cholesterol/POPC (liquid disordered phase) and cholesterol/DPPC (liquid ordered phase) liposomes in the present reaction conditions, the data obtained suggests that the H2O2-gencrating activity of the A beta-Cu complex, accompanied by oxidation of membrane-incorporated cholesterol, could be effected by the phase of the liposome membranes.

DOI： 10.1263/jbb.100.455

Liposome-assisted activity of superoxide dismutase under oxidative stress

H Nagami, N Yoshimoto, H Umakoshi, T Shimanouchi, R Kuboi

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 99 ( 4 ) 423 - 428 2005.4

Language：English Publishing type：Research paper (scientific journal) Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN

A biological membrane is the front line of defense for cells against various environmental stresses such as heat and reactive oxygen species (ROS) and is expected to play an important role in the antioxidant system with antioxidant enzymes, similarly to its chaperone-like function in cooperation with heat shock proteins. The oxidative stress response of superoxide dismutase (SOD), which is known to catalyze the dismutation of O-2(-) to H2O2 was investigated in the presence of artificial membranes, liposomes, in order to obtain fundamental information on the biological ROS scavenging system. SOD lost its activity in the presence of H2O, and was found to have two loops including one which contains an a-helix which presents the substrate O-2(-) to the activity center of SOD (Cu(II)). From circular dichroism analysis of SOD in the presence of H2O2, the contents of the a-helix in SOD were found to decrease in correspondence with the inactivation and conformational change of SOD, suggesting that the conformation of the a-helix loops affects SOD activity. In the presence of liposomes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), SOD was not inactivated in the presence of H2O2 although the contents of its a-helix structure were decreased. The oxidized SOD was found to interact with the liposome surface under oxidative stress using dielectric dispersion analysis. Based on these results, a possible mechanism of SOD protection against ROS on liposomes was presented.

DOI： 10.1263/jbb.99.423

PBL Education for the 2nd Year Undergraduate Students in Chemical Engineering and Continuous Outcomes Assessment

KUBOI Ryoichi, SHIMADA Wataru, EGASHIRA Yasuyuki, EBITANI Kohki, KINO-OKA Masahiro, TAKAHASHI Hideaki, SATO Hiroshi, UMAKOSHI Hiroshi, SHIRAISHI Yasuhiro, SHIBA Sadataka, SHIMAUCHI Hisanori

Journal of JSEE 53, 52-57/, ( 6 ) 52 - 57 2005

Language：Japanese Publisher：Japanese Society for Engineering Education

A Project-Based Learning (PBL) program has been examined for the 2nd year students who spontaneously set the project themes. The questionnaires to the same students who had taken the PBL course were carried out just after promotion to the 4th year as well as just before graduation. The results demonstrated the effectiveness of the PBL program and the usefulness of the PBL continuous assessment of the outcomes.

DOI： 10.4307/jsee.53.6_52

Variable SOD-like activity of liposome modified with Mn(II)-porphyrin derivative complex

H Nagami, H Umakoshi, T Shimanouchi, R Kuboi

BIOCHEMICAL ENGINEERING JOURNAL 21 ( 3 ) 221 - 227 2004.11

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE SA

A novel artificial superoxide dismutase (SOD), which can vary its activity in response to oxidative stress, was developed by using the liposome modified with a metal-functional ligand complex. A lipophilic chloro-(5,10,15,20-tetrakis[1-hexadecylpyridinium-4-yl]-21H,23H-porphyrin) (HPyP) was selected as the functional ligand to modify the liposome surface. The effects of the molar ratio of the HPyP and metal ion in the liposome membrane on two kinds of SOD-like functions, i.e. SOD activity and the antioxidative function against phospholipid composed of liposome was investigated by varying the membrane properties of liposomes. The modification of liposome with HPyP complex resulted in conflicting functions, such as an inhibitive and a promotional function during the induction of the antioxidation function against phospholipid, depending on the molar content of HPyP. The Mn(II)-HPyP complex can efficiently convert O-2(-) to H2O2 and O-2 at low HPyP content, while the efficiency of the SOD activity of Mn(II)-HPyP was significantly reduced due to the cluster formation of Mn(II)-HPyP complex in the liposome at a HPyP content exceeding 1 mol%. It was considered that the cluster formation of the Mn(II)-HPyP complex in the liposome may decrease the reactivity of the active center of the disproportional reaction of Mn(II)-HPyP since the complex was buried into the aggregate of Mn(II)-HPyP itself. (C) 2004 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bej.2004.05.006

Comparison of the extraction behavior of metal ions by metal affinity ligands in an organic-aqueous two-phase system and in a liposome system

H Nagami, H Umakoshi, T Shimanouchi, R Kuboi, Y Baba

SOLVENT EXTRACTION RESEARCH AND DEVELOPMENT-JAPAN 11 159 - 165 2004

Language：English Publishing type：Research paper (scientific journal) Publisher：JAPAN ASSOC SOLVENT EXTRACTION

The interaction of various metal ions with simple metal affinity ligands, 3(hexadecylthio)propionic acid (HTP) and 2-(hexadecylthio)benzoic acid (HTB), was investigated in an organic/aqueous two-phase system and in a liposome system. Both ligands exhibited a high silver(I) affinity at low pH in the organic/aqueous two phase system but low selectivity in the extraction of other transition metal ions such as Cu(II), Ni(II), Co(II), Zn(II), Cd(II), and Hg(II) above pH 6. On the other hand, remarkably different behavior for metal adsorption was observed in the liposome system with HTP and HTB. HTB lost the ability to interact with metal ions when modified on the liposome surface. This may be attributed to the formation of a hydrogen bond between the carboxyl group of the HTB and the phosphate group of the phospholipid. The HTP-modified liposome effectively adsorbed metal ions of the zinc family, such as Zn(II) and Cd(II), which could interact with the phosphate group of phospholipid, and also adsorbed those transition metal ions, which could be extracted in the organic/aqueous two-phase system. This may be caused by two functions of the liposome, i.e., a gate function for metal ions at the surface of the HTP-modified liposome and additional stabilization by interaction between Cu(II) and the phosphate group of the phospholipid.

Development of PPy films doped with thiol-SAM-Cu particles for NH3 gas sensing

T Shimanouchi, S Morita, HS Jung, Y Sakurai, Y Suzuki, R Kuboi

SENSORS AND MATERIALS 16 ( 5 ) 255 - 265 2004

Language：English Publishing type：Research paper (scientific journal) Publisher：MYU, SCIENTIFIC PUBLISHING DIVISION

We have developed a high-sensitivity polypyrrole (PPy) film with a porous structure using a simple and easy method of doping thiol self-assembled monolayer (SAM)-Cu particles into a PPy film. The thiol-Cu-SAM particles were doped by the adsorption of thioxylenol or decanethiol into the porous PPy film surface, resulting in an improvement in sensitivity of the film to NH3. Owing to the addition of thiol-Cu-SAM particles formed using various 4-tent-butylcalix-6-arene thiol derivatives, the sensitivity correspondingly decreased. In particular, for the case of poly(propylene oxide)-calix-6-arenethiol, a marked decrease in sensitivity was observed. Using this composite nanomaterial, we found that the detection limit for NH3 increased to concentration levels of several ppm. We present a novel preparation method for these sensor films aimed at controlling their porosity and sensitivity.

Detection of protein conformation under stress conditions using liposomes as sensor materials

R Kuboi, T Shimanouchi, M Yoshimoto, H Umakoshi

SENSORS AND MATERIALS 16 ( 5 ) 241 - 254 2004

Language：English Publishing type：Research paper (scientific journal) Publisher：MYU, SCIENTIFIC PUBLISHING DIVISION

Calcein release from liposomes was analyzed kinetically in the presence of various proteins under the stress condition. In the case of bovine carbonic anhydrase (CAB), a significant release of calcein was observed at pH 4. The conformational change of CAB from the native to the molten-globule (MG) state was confirmed using an aqueous two-phase partitioning method and immobilized liposome chromatography. The CAB-liposome interaction was maximum at a specific pH (4.0), where the CAB conformation was an MG-like state. The results may show that the hydrophobic interaction between CAB and liposomes enhances the perturbation of the liposome membrane, leading to a significant release of calcein from liposomes. These phenomena depended not only on the characteristics of proteins (local hydrophobicity, LHpr) but also on the dynamic properties of liposomes (membrane fluidity). These results obtained with CAB may be extended to other proteins. The manner of protein-induced calcein release was classified into at least two types. Calcein release due to the addition of proteins having either disulfide bonds or being rich in beta-sheet structure was not observed. On the contrary, a significant release of calcein was observed in the case of a reduced protein with a cleaved disulfide bond and in the case of a protein with relatively a low content of beta-sheet structure. These findings suggest that a liposome containing calcein can be used as an effective sensor element for the detection of proteins having large structural fluctuations.

Novel fabrication of conductive polymers contained micro-array gas sensitive films using a micromanipulation method

HS Jung, T Shimanouchi, S Morita, R Kuboi

ELECTROANALYSIS 15 ( 18 ) 1453 - 1459 2003.10

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-V C H VERLAG GMBH

A novel method for fabricating a micro gas sensor film on an indium tin oxide (ITO) electrode patterned using micromachining technology was developed. A micromanipulation system equipped with a counter electrode (Au; circle divide10 pm) and a microsyringe, which was connected to a microinjection system, was first constructed. With this system, micro gas sensor arrays could be successfully prepared on ITO electrodes. Two kinds of micro gas sensor films were prepared, based on polythiophene (PTh) and poly(3-n-dodecylthiophene) (PD). The response behavior of conventional PTh and micro-PTh films against NH3 at three different operating temperatures (25, 40 and 60degreesC) was investigated by measuring the resistance of the film. With the micro-PTh film, a reversible response was observed against NH3 when measured at 40 and 60degreesC. In addition, the responsive characteristics of the microsensor films against different testing gases were examined at the three operating temperatures. The resistance of the microsensor films of PTh and PD changed considerably, depending on the type of testing gas, allowing these sensor films to be used for the detection of various gases. Furthermore, the microsensor films had a high stability compared with conventional films prepared from the same polymer.

DOI： 10.1002/elan.200302660

Characterization of the surface properties of chitosanase under heat stress condition using aqueous two-phase systems and an immobilized-liposome sensor system

HS Jung, H Umakoshi, SY Son, T Shimanouchi, R Kuboi

SOLVENT EXTRACTION RESEARCH AND DEVELOPMENT-JAPAN 10 123 - 132 2003

Language：English Publishing type：Research paper (scientific journal) Publisher：JAPAN ASSOC SOLVENT EXTRACTION

The surface properties of chitosanase from Storeptomyces griseus were characterized under heat stress conditions by using aqueous two-phase systems and by using an immobilized-liposome sensor. The surface net (HFS) and local hydrophobicities (LH), were first characterized by using the poly (ethylene glycol) (PEG)/dextran 100-200k (Dex) aqueous two-phase system at various temperatures (25similar to50degreesC). The LH values of dithiothreitol (DTT)-treated chitosanase showed a maximum at a specific temperature (45degreesC) while the HFS values gradually increased with increasing temperature from 40 to 50degreesC. At the same temperature conditions as above, the surface properties of chitosanase were also analyzed by using a sensor tip immobilized-liposome electrode. Similarly in the case of the variation of LH but not HFS, the detected signal (maxima in electric current) was maximized at 45degreesC, and a linear correlation between the LH values and the signal values of the new immobilized-liposome sensor system was obtained. It was thus found that the new sensor system was effective for the characterization of the surface properties and LH values, of proteins, as well as the aqueous two-phase system method.

Detection of a heat stress-mediated interaction between protein and phospholipid membrane using dielectric measurement

Seiichi Morita, Toshinori Shimanouchi, Masashi Sasaki, Hiroshi Umakoshi, Ryoichi Kuboi

Journal of Bioscience and Bioengineering 95 ( 3 ) 252 - 256 2003

Language：English Publishing type：Research paper (scientific journal) Publisher：Society of Fermentation and Bioengineering

The dielectric response of lipid bilayer membrane vesicles (liposomes) prepared using either phosphatidylcholine from egg (EPC) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was analyzed at a frequency range of 0.1 to 100 MHz. A marked dielectric dispersion for EPC and POPC liposome suspensions was observed above 1 MHz. An appropriate analysis of the dielectric dispersion curve was performed using the Cole-Cole equation and the Debye equation and was found to provide a method for the determination of dielectric parameters. Among the dielectric parameters, the characteristic frequency of a second dispersion around 50 MHz varied corresponding with changes in the test conditions. Of particular note is that an anomalous change in the characteristic frequency in the presence of protein corresponded to the degree of hydrophobic interaction between proteins and liposomes. The value of the frequency around 50 MHz, as well as the decrease in permittivity over the frequency range tested, are indicators of the interaction between proteins and liposomes.

DOI： 10.1263/jbb.95.252

Characterization and control of stimuli-induced membrane fusion of liposomes in the presence of proteins and stimuli responsive polymers

MM Felix, H Umakoshi, T Shimanouchi, M Yoshimoto, R Kuboi

BIOCHEMICAL ENGINEERING JOURNAL 12 ( 1 ) 7 - 19 2002.10

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE SA

The process of fusion of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes in the presence of stimuli responsive copolymers (poly(N-isopropylacrylaniide-co-methacrylic acid) and poly(N-isopropylacrylamide-co-methacrylic acid-co-octadecylacrylamide)) or proteins ((x-chymotrypsin (alpha-CT), bovine carbonic anhydrase (CAB), and p-galactosidase (p-gal)) was quantitatively characterized under the stimuli by varying pH and temperature. After the liposomes were exposed to the specific pH and heat stimuli in the presence of the stimuli responsive polymers or proteins, the percentage of the fusion was determined by using two kinds of liposomes entrapping cobalt-calcein and EDTA (cobalt-calcein method). In the presence of stimuli responsive copolymers, the percentage of fusion was increased to 20% above the phase transition condition of the copolymer (above 37 degreesC and below pH 5.7). The percentage of fusion in the presence of proteins was varied under the stimuli, depending on the type of proteins. In the case of the alpha-CT, the maximal percentage of fusion was only 8%. The addition of the CAB and p-gal improved the percentage of fusion to 15 and 20%, respectively, by selecting the optimal stimuli conditions. Although some disagreements were observed in the region of strong acidic conditions, it was found that the increase of the percentage of fusion was well corresponding with the increase of the membrane fluidity of the POPC liposome, followed by the increase of the local hydrophobicity of proteins or copolymers under the stimuli. Based on the above results, a possible model for the stimuli-induced fusion of POPC liposome membranes was finally presented. (C) 2002 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S1369-703X(02)00014-1

Novel array-type gas sensors using conducting polymers, and their performance for gas identification

Y Sakurai, HS Jung, T Shimanouchi, T Inoguchi, S Morita, R Kuboi, K Natsukawa

SENSORS AND ACTUATORS B-CHEMICAL 83 ( 1-3 ) 270 - 275 2002.3

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE SA

Novel gas sensor devices have been developed using polythiophene (pTh) film and poly(3-n-dodecylthiophene) (pDpTh.) film coated over pTh film. These polymer films were electrochemically deposited and doped by cyclic voltammetry on thin-film electrodes where the isolation gap was formed by a micromachining process. We examined the response characteristics of the conducting polymer films against various sample gases over a range of temperatures of the sensitive layer. The resistance changes of both sensitive layers of pTh and pDpTh were highly dependent on the kind of layer. In particular, pTh film responded to ammonia gas and pDpTh films clearly responded to hydrophobic gases, such as chloroform, methane and ethanol. The response of these films to several gases was analyzed with a pattern recognition (PARC) algorithm. It was found that our simple gas sensor device could discriminate between the gases that were used here. (C) 2002 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0925-4005(01)01069-3

Evaluation of interaction between liposome membranes induced by stimuli responsive polymer and protein

Matundu Menayame Félix, Hirosh Umakoshi, Toshinor Shimanouchi, Makot Yoshimoto, Ryoich Kuboi

Journal of Bioscience and Bioengineering 93 ( 5 ) 498 - 501 2002

Language：English Publishing type：Research paper (scientific journal) Publisher：Society of Fermentation and Bioengineering

Immobilized liposome chromatography was utilized as a novel method for the quantitative evaluation of the interaction between liposome membranes. The capacity factors evaluated from the elution profile showed that interaction between 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposome membranes occurred in the presence of a stimuli responsive polymer and protein under specific stimulus conditions. The occurrence of such interaction was supported by experimental results for POPC liposome membrane fusion under corresponding stimuli conditions.

DOI： 10.1016/S1389-1723(02)80098-6

Characterization of stimuli-induced membrane fusion of liposomes

Matundu Menayame Félix, Toshinori Shimanouchi, Hiroshi Umakoshi, Makoto Yoshimoto, Ryoichi Kuboi

Kagaku Kogaku Ronbunshu 28 ( 4 ) 481 - 484 2002

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Society of Chemical Engineers

The phenomena of aggregation and fusion of liposome membranes under the stimuli conditions (heat and pH) were shown to be triggered by the conformational change of the stimuli-responsive polymers and proteins. It is found that the percentage of liposome fusion and the liposome size under the specific stimuli condition were well corresponding with the capacity factor obtained from the peak shift of the elution profile on the immobilized liposome chromatography.

DOI： 10.1252/kakoronbunshu.28.481

Highly sensitive and selective gas sensors using the polythiophen derivatives controlled the temperature Reviewed

Y Sakurai, HS Jung, T Inoguchi, T Shimanouchi, M Umakoshi, R Kuboi, K Natsukawa

TRANSDUCERS '01: EUROSENSORS XV, DIGEST OF TECHNICAL PAPERS, VOLS 1 AND 2 1696 - 1699 2001

Language：English Publishing type：Research paper (international conference proceedings) Publisher：SPRINGER-VERLAG BERLIN

The advanced odor gas sensor devices were developed using the polythiophene derivatives. These polymers have been found to sense gases and vapours by monitoring the change in resistance on exposure of the polymer to the gas sample. These polymer films were electrochemically deposited and doped by cyclic voltammetry on thin-film electrodes where the isolation gap was formed by the micro-machining process. The breakthrough resulted in sensor devices with unique benefits, such as the very high selectivity and the operation possibility at ambient temperatures (steady and nonsteady state).

Immobilized liposome chromatography for refolding and purification of protein

Makoto Yoshimoto, Toshinori Shimanouchi, Hiroshi Umakoshi, Ryoichi Kuboi

Journal of Chromatography B: Biomedical Sciences and Applications 743 ( 1-2 ) 93 - 99 2000.6

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

Small unilamellar liposomes were utilized as a kind of aqueous two-phase system and artificial chaperone which specifically recognize protein conformation with fluctuated structure. Liposomes showed highly selective binding ability to conformationally changed proteins treated with various concentrations of guanidinium hydrochloride, as evaluated by immobilized liposome chromatography (ILC). In refolding of proteins, liposomes bound to refolding intermediate of proteins and prevented them from forming intermolecular aggregates. Refolding of bovine carbonic anhydrase, lysozyme and ribonuclease A was significantly improved in the presence of liposomes. Furthermore, by utilizing ILC, refolding of proteins was also successfully and simply carried out with considerable high reactivation yield. © 2000 Elsevier Science B.V.

DOI： 10.1016/S0378-4347(00)00051-7

Stimuli-responsive separation of proteins using immobilized liposome chromatography

Toshinori Shimanouchi, Seiichi Morita, Hiroshi Umakoshi, Ryoichi Kuboi

Journal of Chromatography B: Biomedical Sciences and Applications 743 ( 1-2 ) 85 - 91 2000.6

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

The possibility of the stimuli-responsive separation of proteins was investigated using immobilized liposome chromatography (ILC) as novel aqueous two-phase systems. The specific capacity factor (k(s)) of β-galactosidase, obtained by analysis of ILC, was varied by changing the pH of the solution and was maximized at the specific pH of 5 (k(s, max) = 5.57). The k(s) values were found to correspond well with their local hydrophobicities, which can be determined by the aqueous two-phase partitioning method. The variation of k(s), therefore, indicates a change in the surface properties of a protein during conformational change under pH stimuli. A similar phenomenon is observed in the case of other proteins (α-glucosidase, k(s, max) = 11.3 at pH 4
carbonic anhydrase from bovine, k(s, max) = 6.53 at pH 4). The difference in the height and/or the position of the peaks of the k(s)-pH curves of each protein suggests a difference in their pH denaturation in the ILC column. Based on these results, the mutual separation of the above proteins at pH 4 could be successfully performed by selecting their specific capacity factor as a design parameter. © 2000 Elsevier Science B.V.

DOI： 10.1016/S0378-4347(00)00061-X

Stimuli-responsive separation of proteins using immobilized liposome chromatography Reviewed

T Shimanouchi, S Morita, H Umakoshi, R Kuboi

JOURNAL OF CHROMATOGRAPHY B 743 ( 1-2 ) 85 - 91 2000.6

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

The possibility of the stimuli-responsive separation of proteins was investigated using immobilized liposome chromatography (ILC) as novel aqueous two-phase systems. The specific capacity factor (k(s)) of beta-galactosidase, obtained by analysis of ILC, was varied by changing the pH of the solution and was maximized at the specific pH of 5 (k(s,max) = 5.57). The k(s) values were found to correspond well with their local hydrophobicities, which can be determined by the aqueous two-phase partitioning method. The variation of k(s), therefore, indicates a change in the surface properties of a protein during conformational change under pH stimuli. A similar phenomenon is observed in the case of other proteins (alpha-glucosidase, k(s,max) = 11.3 at pH 4; carbonic anhydrase from bovine, k(s,max) = 6.53 at pH 4). The difference in the height and/or the position of the peaks of the k(s)-pH curves of each protein suggests a difference in their pH denaturation in the ILC column. Based on these results, the mutual separation of the above proteins at pH 4 could be successfully performed by selecting their specific capacity factor as a design parameter. (C) 2000 Elsevier Science B.V. All rights reserved.

Immobilized liposome chromatography for refolding and purification of protein Reviewed

M Yoshimoto, T Shimanouchi, H Umakoshi, R Kuboi

JOURNAL OF CHROMATOGRAPHY B 743 ( 1-2 ) 93 - 99 2000.6

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

Small unilamellar liposomes were utilized as a kind of aqueous two-phase system and artificial chaperone which specifically recognize protein conformation with fluctuated structure. Liposomes showed highly selective binding ability to conformationally changed proteins treated with various concentrations of guanidinium hydrochloride, as evaluated by immobilized liposome chromatography (ILC). In refolding of proteins, liposomes bound to refolding intermediate of proteins and prevented them from forming intermolecular aggregates. Refolding of bovine carbonic anhydrase, lysozyme and ribonuclease A was significantly improved in the presence of liposomes. Furthermore, by utilizing ILC, refolding of proteins was also successfully and simply carried out with considerable high reactivation yield. (C) 2000 Elsevier Science B.V. All rights reserved.

Selective separation process of proteins based on the heat stress- induced translocation across phospholipid membranes

Hiroshi Umakoshi, Toshinori Shimanouchi, Ryoichi Kuboi

Journal of Chromatography B: Biomedical Applications 711 ( 1-2 ) 111 - 116 1998.6

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

Heating of several protein solutions at 40-47°C for 5-60 min in the presence of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes induced the translocation of β-galactosidase (β-gal), α-glucosidase (α- glu) and bovine carbonic anhydrase (CAB) from outer to inner aqueous phase across the liposome membrane. The translocated amounts of β-gal at various temperatures were maximized under suitable heating conditions (45°C, 30 min). Those of α-glu and CAB were maximized at 40-45 and 60°C, respectively. Each maximum value could be correlated with the corresponding local hydrophobicity of each protein evaluated by the aqueous two-phase partitioning method. The possibility to apply these heat-induced translocation phenomena to the bioseparation of proteins was successfully demonstrated for the model mixture solution of β-gal, α-glu and CAB.

DOI： 10.1016/S0378-4347(97)00659-2

Selective separation process of proteins based on the heat stress-induced translocation across phospholipid membranes Reviewed

H Umakoshi, T Shimanouchi, R Kuboi

JOURNAL OF CHROMATOGRAPHY B 711 ( 1-2 ) 111 - 116 1998.6

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

Heating of several protein solutions at 40-47 degrees C for 5-60 min in the presence of 1-palmitoyl-2-oleoyI-sn-glycero-3-phosphocholine (POPC) liposomes induced the translocation of beta-galactosidase (beta-gal), alpha-glucosidase (alpha-glu) and bovine carbonic anhydrase (CAB) from outer to inner aqueous phase across the liposome membrane. The translocated amounts of P-gal at various temperatures were maximized under suitable heating conditions (45 degrees C, 30 min). Those of alpha-glu and CAB were maximized at 40-45 and 60 degrees C, respectively. Each maximum value could be correlated with the corresponding local hydrophobicity of each protein evaluated by the aqueous two-phase partitioning method. The possibility to apply these heat-induced translocation phenomena to the bioseparation of proteins was successfully demonstrated for the model mixture solution of P-gal, a-glu and CAB. (C) 1998 Elsevier Science B.V. All rights reserved.

Model system for heat-induced translocation of cytoplasmic beta-galactosidase across phospholipid bilayer membrane

H Umakoshi, M Yoshimoto, T Shimanouchi, R Kuboi, Komasawa, I

BIOTECHNOLOGY PROGRESS 14 ( 2 ) 218 - 226 1998.3

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER CHEMICAL SOC

The possibility of the translocation of the enzyme across the phospholipid bilayer membrane was investigated by using the liposomes prepared by 1-palmitoyl-2-oleoylsn-glycero-3-phosphocholine (POPC) in which beta-galactosidase (beta-gal) was entrapped. Exposing the POPC liposomes entrapping beta-gal inside to heat treatment (40-50 degrees C, 1-60 min) was found to induce its translocation across the liposome membrane. The translocated activity of beta-gal from inner to outer aqueous phase of liposomes indicated the maximal value when the liposomes entrapping beta-gal were heated at 45 degrees C for 30 min. The gel permeation profiles of the liposomes before and after heat treatment (45 degrees C, 30 min) also supported the translocation of beta-gal across the liposome membrane. The membrane fluidity of liposomes was found to be increased with increasing temperature, so that the hydrophobicity of liposome membrane was also increased. The local hydrophobicity of beta-gal was maximized at the temperature of 40-50 degrees C. The mechanisms of beta-gal translocation have been suggested to be triggered by the enhancement of hydrophobic interaction between the liposome surface and beta-gal molecules. Finally, a minimal scheme of possible mechanism on the heat-induced translocation of beta-gal has been presented on the basis of the hydrophobic interaction between the liposome and the proteins. The experimental data on the heat-induced translocation of beta-gal were well corresponding to those from model calculation.

DOI： 10.1021/bp9800135

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