Faculty Profiles - SHIMANOUCHI Toshinori

Papers - SHIMANOUCHI Toshinori

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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

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