1989 年度実績報告書

生体膜と電場との相互作用に関する総合的研究

研究課題

研究課題/領域番号	63302070
研究機関	九州大学
研究代表者	桐野豊九州大学, 薬学部, 教授 (10012668)
研究分担者	美宅成樹東京農工大学, 工学部, 助教授 (10107542) 木下一彦慶応大学, 理工学部, 教授 (30124366) 葛西道生大阪大学, 基礎工学部, 教授 (40022595) 岡田泰伸京都大学, 医学部, 講師 (10025661) 稲葉浩子放射線医学総合研究部, 遺伝研究所, 主任研究官 (70159953)
キーワード	電気穿孔法 / 電気的細胞融合法 / パルスレ-ザ-顕微鏡 / 遺伝子移入 / リン脂質平面膜 / リポソ-ム / 起電性イオンポンプ
研究概要	昨年度に続いて、細胞を電場によって操作する技術(電気穿孔法、誘電泳動法、電気融合法等)について、直接観察による検討をすすめると共に、これまで未開拓であった種々の細胞現象に対する電場効果を調べる手法を開発し、いくつかの興味深い知見を得た。主な研究成果を箇条書にする。(1)パルスレ-ザ-顕微鏡という新しい機器を開発し、これを用いて、細胞電気穿孔過程をマイクロ秒の時間分解能、マイクロメ-トルの空間分解能で解析した。その結果、穿孔は、誘起膜電位が閾値を越えると1マイクロ秒以内に起きることが分かった。脂質膜でも同様の現象が観察され、特に直径数マイクロメ-トルの大きな孔の形成も見られたことは特筆するに値する。(2)電気的細胞融合の機構を検討し、内因性チオ-ルプロテア-ゼが関与していることを見出した。(3)電気穿孔法による遺伝子移入の至適条件を検討し、効率のよい標準実験法を確立した。(4)電気穿孔により細胞膜に生じた孔の修復機構を検討し、これが二価金属イオンにより加速されることを見出した。(5)起電性イオンポンプであるH^+-ATPase及びCa^<2+>-ATPaseの能動輸送機構の電気生理学的解析を行うため、プロテオリポソ-ムからリン脂質平面膜に組み込む方法を開発し、起電性の直接的証明、外部電場の影響、速度論的解析を行った。(6)リポソ-ム中において、チュ-ブリンを重合させることにより、突起を持つリポソ-ムを形成できた。同様のリポソ-ムに高周波電場を印加することによりリポソ-ムの変形を起こすことができた。(7)直径100nm程度の膜小胞の融合過程を、電流、容量、画像の同時計測により実時間で測定するシステムを開発した。また、ヘルパ-T細胞が抗原提示細胞により活性化される際の細胞内カルシウム動態と膜電位変化を同時計測する手法を開発した。こうして、広範な細胞現象に対する電場効果を追究する方途が開かれた。

研究成果

(45件)

すべてその他

すべて文献書誌 (45件)

[文献書誌] Kirino,Y.,Hirashima,N.& Saito,Y.: "Patch-Clamp Measurement of Ion Channels in Biomembrane Vesicles Reconstituted into Giant Proteoliposomes by Freeze-Thawing without Use of Detergent" Methods in Nurosciences. 4. (1990)
[文献書誌] Anzai,K.,et al.: "Formation of Ion Channels by Synthetic Basic Peptides in Planar Lipid Bilayer Membrane" Biochimica et Biophisica Acta.
[文献書誌] Wakamatsu,M.,et al.: "Effects of Anesthetic and Related Agents on Calciuminduced Calcium Release from Sarcoplasmic Reticulum Isolated from Rabbit Skeletal Muscle" Journal of Anesthetics. 3. 1-9 (1989)
[文献書誌] Anzai,K.,et al.: "Ion Channel Activity of Synthetic Basic Peptides in Planar Lipid Bilayer" Journal of Pharmacobio-Dynamics. 12. s-122 (1989)
[文献書誌] Anzai,K.,Yoshida,M.& Kirino,Y.: "Change in Intravesicular Volume of Liposomes by Freeze-Thawing Treatment as Studied by the ESR Stopped-Flow Technique" Biochemica et Biophysica Acta. 1021. 21-26 (1990)
[文献書誌] Nishie,I.,Anzai,K.,& Kirino,Y.: "Measurement of Steady-State Ca-Pump Current Caused by Purified Ca-ATPase of Sarcoplasmic Reticulum Incorporated into a Plannar Bilayer Lipid Membrane" Journal of Biological Chemistry. 265. 2488-2491 (1990)
[文献書誌] H.Hama-Inaba,T.Nishimoto M.Ohtsubo,K.Sato,M.Kasai: "Standard method of electroporation for introduction of plasmid and cosmid DNAs to mammalian cells" Studia Biophysica. 130. 193-196 (1989)
[文献書誌] H.Hama-Inaba,K.Sato,T.Nishimoto,M.Ohtsubo,M.Kasai: "Establishment and application of a standard method of electroporation for introduction of plasmid and cosmid DNAs to mammalian cells" Bioelectrochemistry and Bioenergetics. 21. 355-366 (1989)
[文献書誌] S.Oiki,T.Ohon-Shosaku,Y.Okada: "Electric currents associated with directed migration of fibroblasts." Biological Bullentin. 176(S). 123-125 (1989)
[文献書誌] S.Ueda,Y.Okada: "Acid secretagogues induce Ca^<2+> mobilization coupled to K^+ conductance activation in rat parietal cells in tissue culture." Biochemica et Biophysica Acta. 1012. 254-260 (1989)
[文献書誌] T.Yada,S.Oiki,S.Ueda,Y.Okada: "Intestinal secretagogues increase cytosolic Ca^<2+> concentration and K^+ conductance in a human intestinal epitheliac cell line." Journal of Membrane Biology. 112. 159-167 (1989)
[文献書誌] 岡田泰伸: "電気的細胞融合と電気的遺伝子移入" 実験医学. 7. 87-91 (1989)
[文献書誌] Kouyama,T.,et al.: "Correlation between Internal Motion and Emission Kinetics of Tryptophan Residues in Proteins" Eur.J.Biochem.182. 517-521 (1989)
[文献書誌] Itoh,H.,et al.: "Multi-Shot Pulsed Laser Fluorescence Microscope System" Proc.SPIE. (1990)
[文献書誌] Utsunomiya,N.Nakanishi,M.Arata,Y.: "Unidirectional Inhibition of Early Signal Transduction of Helper T-cells by Cloned Supprressor T-cells" International Immunol.1. 460-463 (1989)
[文献書誌] Mizuguchi,J.Utsunomiya,N.Nakanishi,M.: "Differential Sensitivity of Anti-IgM-induced and NaFinduced Inositol Phospholipid Metabolism to Serne Protease Inhibitors in BAL17 B Lymphoma Cells" Biochem.J.263. 641-646 (1989)
[文献書誌] Mizouchi,T.Asano,Y.Nakanishi,M.: "A Selective Signal Defection Helper T-cells Induced by Antigen Presenting Cells from Mice with Acquired Immunodeficiency Syndrome" J.Immunol.(1990)
[文献書誌] 中西守: "T細胞の抗原認識と情報処理" 薬学雑誌. 109. 707-717 (1989)
[文献書誌] 中西守: "T細胞の活性化と細胞内カルシウムイオンの動態" 細胞工学. 18. 271-274 (1989)
[文献書誌] Kagawa,Y.,Ohta,S.,Yohda,M.,Hamamoto,T.and Hirata,H.: "Biotechnological Applications of Thermophilic ATP Synthase:Membrane Electronics and Genetics." J.Membr.Sci.41. 237-247 (1989)
[文献書誌] Muneyuki,E.,Kagawa,Y.and Hirata,H.: "Steady State Kinetics of Protein Translocation Catalyzed by Thermophilic F_0F_1 ATPase Reconstituted in Planar Bilayer Membranes." J.Biol.Chem.264. 6092-6096 (1989)
[文献書誌] Tsuda,M.,Tsuda,T.and Hirata,H.: "Cyclic Nucleotides and GTP Analogues stimulate Light-induced Phosphorylation of Octopus Rhodopsin." FEBS Lett.257. 38-40 (1989)
[文献書誌] Matsushita,T.,Hirata,H. and Kusaka,I.: "Calcium Channels in Bacteria…Purification and Characterization." Ann.N.Y.Acad.Sci.560. 426-429 (1989)
[文献書誌] N.Yanagihara,M.Suwa and S.Mitaku: "A Theoretical Method of Distingishing Between Soluble and Membrane Proteins" Biophys.Chem.
[文献書誌] Norimitsu Hirose,Kazunori Yamamoto and Shigeki Mitaku: "Ultrasonic Measurement Of Lipid Bilayer Transition In The Presence Of Melittin" Japan.J.Appl.Phys.
[文献書誌] 加藤知、宮本宏: "生体試料の走査トンネル顕微鏡観測" 生物物理. 30. (1990)
[文献書誌] Sokabe,M.: "The structure and dynamics of patch clamped membranes" J.Cell Biol.
[文献書誌] Sokabe,M.: "Quantitative videomicroscopy of patch clamped membranes:stress strain,capacitance,and stretch channel activation" Science.
[文献書誌] Sokabe,M.: "Effects of ions drugs on the responses of sensory axon terminals of decapsulated frog muscle spindle" Neurosci.Res.
[文献書誌] Nomura,K.: "Aminoglycoside blockade of Ca activated K channel from rat brain synaptosomal membranes incorporated into planar bilayers" J.Membr.Biol.
[文献書誌] 曽我部正博: "機械受容器変換・符号化機序におけるCAMPの関与" 生物物理. 29. 111-117 (1989)
[文献書誌] 桐野豊: "“電子スピン共鳴"(新基礎生化学実験法第5巻、「高次構造・状態分析」松橋通生、中島暉躬編)" 丸善, 107-126 (1989)
[文献書誌] 桐野豊、小沢俊彦: "ESR実験法" 廣川書店,
[文献書誌] Shigemori,M.,et al.: "Electroporation of Cell Membrane under a Pulsed Laser Fluorescence Microscope,in “Dynamic Behabior of macromolecules,Colloids,Liquid Crystals and Biological Systems by Optical and Electro-optical Methods"(Watanabe,H.,ed.)" Hirokawa Publishing Co.(Tokyo), 311-316 (1989)
[文献書誌] 岡田泰伸: "臨床生理学シリ-ズ(3)胃" 南山,
[文献書誌] T.Ohno-Shosaku,Y.Okada: "Role of proteases in electrofusion of mammalian cells.In “Electroporation and Electrofusion in Cell Biology" Pienum Publishing Corporation(Eas.,E.Neumann,A.E.Sowers,C.A.iordan), 9 (1989)
[文献書誌] Kinosita,K.,Jr.,et al.: "Submicrosecond Imaging under a Pulsed-Laser Fluorescence Microscope.Electroporation of Cell Membrane Time-and Space-Resolved.in “Science on Form"(ed.,Ishizaka,S.)" KTK Scientific Publishers(Tokyo), (1990)
[文献書誌] 木下一彦、神山勉、池上明: "蛍光、蛍光偏光、新基礎生化学実験法第5巻(中島暉躬編)" 丸善, 69-84 (1989)
[文献書誌] 木下一彦: "蛍光新生化学実験講座第1巻(日本生化学会編)" 東京化学同人, (1990)
[文献書誌] 木下一彦: "-瞬を見る(パルスレ-ザ-顕微鏡)限界を越える生物顕微鏡-見えないものを見る(宝谷紘一、木下一彦編)" 学会出版センタ-, (1990)
[文献書誌] 美宅成樹、幾田一哉、柳原憲那、諏訪牧子: "蛋白質核酸酵素造刊号「視覚の分子メカニズム」「膜タンパク質における立体構造の予測法」" 共立出版、東京,
[文献書誌] Hiroshi Miyamoto and Hirokazu Hotani: "Dynamics of Microtubules,Proceeding of 14th International Symposium Division of Biophysics" Taniguchi Foundation, 415 (1989)
[文献書誌] 宮本宏、石渡信一他: "生体物理工学" 丸善株式会社, (1990)
[文献書誌] Sokabe,M.: "Towards a quantitative study of stretch activated channel.In “Comparative Aspects of Mechanoreceptor Systems"" Springer Verlag(Heidelberg),
[文献書誌] Ito,F.: "Effects of intracellular calcium on thr frog muscle spindle in relation to cyclic AMP action.In“Mechanoreceptor"" Plenum Press, 195-199 (1988)

1989 年度 実績報告書

生体膜と電場との相互作用に関する総合的研究

研究代表者

桐野 豊 九州大学, 薬学部, 教授 (10012668)

研究成果

[文献書誌] Kirino,Y.,Hirashima,N.& Saito,Y.: "Patch-Clamp Measurement of Ion Channels in Biomembrane Vesicles Reconstituted into Giant Proteoliposomes by Freeze-Thawing without Use of Detergent" Methods in Nurosciences. 4. (1990)

[文献書誌] Anzai,K.,et al.: "Formation of Ion Channels by Synthetic Basic Peptides in Planar Lipid Bilayer Membrane" Biochimica et Biophisica Acta.

[文献書誌] Wakamatsu,M.,et al.: "Effects of Anesthetic and Related Agents on Calciuminduced Calcium Release from Sarcoplasmic Reticulum Isolated from Rabbit Skeletal Muscle" Journal of Anesthetics. 3. 1-9 (1989)

[文献書誌] Anzai,K.,et al.: "Ion Channel Activity of Synthetic Basic Peptides in Planar Lipid Bilayer" Journal of Pharmacobio-Dynamics. 12. s-122 (1989)

[文献書誌] Anzai,K.,Yoshida,M.& Kirino,Y.: "Change in Intravesicular Volume of Liposomes by Freeze-Thawing Treatment as Studied by the ESR Stopped-Flow Technique" Biochemica et Biophysica Acta. 1021. 21-26 (1990)

[文献書誌] Nishie,I.,Anzai,K.,& Kirino,Y.: "Measurement of Steady-State Ca-Pump Current Caused by Purified Ca-ATPase of Sarcoplasmic Reticulum Incorporated into a Plannar Bilayer Lipid Membrane" Journal of Biological Chemistry. 265. 2488-2491 (1990)

[文献書誌] H.Hama-Inaba,T.Nishimoto M.Ohtsubo,K.Sato,M.Kasai: "Standard method of electroporation for introduction of plasmid and cosmid DNAs to mammalian cells" Studia Biophysica. 130. 193-196 (1989)

[文献書誌] H.Hama-Inaba,K.Sato,T.Nishimoto,M.Ohtsubo,M.Kasai: "Establishment and application of a standard method of electroporation for introduction of plasmid and cosmid DNAs to mammalian cells" Bioelectrochemistry and Bioenergetics. 21. 355-366 (1989)

[文献書誌] S.Oiki,T.Ohon-Shosaku,Y.Okada: "Electric currents associated with directed migration of fibroblasts." Biological Bullentin. 176(S). 123-125 (1989)

[文献書誌] S.Ueda,Y.Okada: "Acid secretagogues induce Ca^<2+> mobilization coupled to K^+ conductance activation in rat parietal cells in tissue culture." Biochemica et Biophysica Acta. 1012. 254-260 (1989)

[文献書誌] T.Yada,S.Oiki,S.Ueda,Y.Okada: "Intestinal secretagogues increase cytosolic Ca^<2+> concentration and K^+ conductance in a human intestinal epitheliac cell line." Journal of Membrane Biology. 112. 159-167 (1989)

[文献書誌] 岡田泰伸: "電気的細胞融合と電気的遺伝子移入" 実験医学. 7. 87-91 (1989)

[文献書誌] Kouyama,T.,et al.: "Correlation between Internal Motion and Emission Kinetics of Tryptophan Residues in Proteins" Eur.J.Biochem.182. 517-521 (1989)

[文献書誌] Itoh,H.,et al.: "Multi-Shot Pulsed Laser Fluorescence Microscope System" Proc.SPIE. (1990)

[文献書誌] Utsunomiya,N.Nakanishi,M.Arata,Y.: "Unidirectional Inhibition of Early Signal Transduction of Helper T-cells by Cloned Supprressor T-cells" International Immunol.1. 460-463 (1989)

[文献書誌] Mizuguchi,J.Utsunomiya,N.Nakanishi,M.: "Differential Sensitivity of Anti-IgM-induced and NaFinduced Inositol Phospholipid Metabolism to Serne Protease Inhibitors in BAL17 B Lymphoma Cells" Biochem.J.263. 641-646 (1989)

[文献書誌] Mizouchi,T.Asano,Y.Nakanishi,M.: "A Selective Signal Defection Helper T-cells Induced by Antigen Presenting Cells from Mice with Acquired Immunodeficiency Syndrome" J.Immunol.(1990)

[文献書誌] 中西守: "T細胞の抗原認識と情報処理" 薬学雑誌. 109. 707-717 (1989)

[文献書誌] 中西守: "T細胞の活性化と細胞内カルシウムイオンの動態" 細胞工学. 18. 271-274 (1989)

[文献書誌] Kagawa,Y.,Ohta,S.,Yohda,M.,Hamamoto,T.and Hirata,H.: "Biotechnological Applications of Thermophilic ATP Synthase:Membrane Electronics and Genetics." J.Membr.Sci.41. 237-247 (1989)

[文献書誌] Muneyuki,E.,Kagawa,Y.and Hirata,H.: "Steady State Kinetics of Protein Translocation Catalyzed by Thermophilic F_0F_1 ATPase Reconstituted in Planar Bilayer Membranes." J.Biol.Chem.264. 6092-6096 (1989)

[文献書誌] Tsuda,M.,Tsuda,T.and Hirata,H.: "Cyclic Nucleotides and GTP Analogues stimulate Light-induced Phosphorylation of Octopus Rhodopsin." FEBS Lett.257. 38-40 (1989)

[文献書誌] Matsushita,T.,Hirata,H. and Kusaka,I.: "Calcium Channels in Bacteria…Purification and Characterization." Ann.N.Y.Acad.Sci.560. 426-429 (1989)

[文献書誌] N.Yanagihara,M.Suwa and S.Mitaku: "A Theoretical Method of Distingishing Between Soluble and Membrane Proteins" Biophys.Chem.

[文献書誌] Norimitsu Hirose,Kazunori Yamamoto and Shigeki Mitaku: "Ultrasonic Measurement Of Lipid Bilayer Transition In The Presence Of Melittin" Japan.J.Appl.Phys.

[文献書誌] 加藤知、宮本宏: "生体試料の走査トンネル顕微鏡観測" 生物物理. 30. (1990)

[文献書誌] Sokabe,M.: "The structure and dynamics of patch clamped membranes" J.Cell Biol.

[文献書誌] Sokabe,M.: "Quantitative videomicroscopy of patch clamped membranes:stress strain,capacitance,and stretch channel activation" Science.

[文献書誌] Sokabe,M.: "Effects of ions drugs on the responses of sensory axon terminals of decapsulated frog muscle spindle" Neurosci.Res.

[文献書誌] Nomura,K.: "Aminoglycoside blockade of Ca activated K channel from rat brain synaptosomal membranes incorporated into planar bilayers" J.Membr.Biol.

[文献書誌] 曽我部正博: "機械受容器変換・符号化機序におけるCAMPの関与" 生物物理. 29. 111-117 (1989)

[文献書誌] 桐野豊: "“電子スピン共鳴"(新基礎生化学実験法第5巻、「高次構造・状態分析」松橋通生、中島暉躬編)" 丸善, 107-126 (1989)

[文献書誌] 桐野豊、小沢俊彦: "ESR実験法" 廣川書店,

[文献書誌] 岡田泰伸: "臨床生理学シリ-ズ(3)胃" 南山,

[文献書誌] T.Ohno-Shosaku,Y.Okada: "Role of proteases in electrofusion of mammalian cells.In “Electroporation and Electrofusion in Cell Biology" Pienum Publishing Corporation(Eas.,E.Neumann,A.E.Sowers,C.A.iordan), 9 (1989)

[文献書誌] Kinosita,K.,Jr.,et al.: "Submicrosecond Imaging under a Pulsed-Laser Fluorescence Microscope.Electroporation of Cell Membrane Time-and Space-Resolved.in “Science on Form"(ed.,Ishizaka,S.)" KTK Scientific Publishers(Tokyo), (1990)

[文献書誌] 木下一彦、神山勉、池上明: "蛍光、蛍光偏光、新基礎生化学実験法第5巻(中島暉躬編)" 丸善, 69-84 (1989)

[文献書誌] 木下一彦: "蛍光新生化学実験講座第1巻(日本生化学会編)" 東京化学同人, (1990)

[文献書誌] 木下一彦: "-瞬を見る(パルスレ-ザ-顕微鏡)限界を越える生物顕微鏡-見えないものを見る(宝谷紘一、木下一彦編)" 学会出版センタ-, (1990)

[文献書誌] 美宅成樹、幾田一哉、柳原憲那、諏訪牧子: "蛋白質核酸酵素造刊号「視覚の分子メカニズム」「膜タンパク質における立体構造の予測法」" 共立出版、東京,

[文献書誌] Hiroshi Miyamoto and Hirokazu Hotani: "Dynamics of Microtubules,Proceeding of 14th International Symposium Division of Biophysics" Taniguchi Foundation, 415 (1989)

[文献書誌] 宮本宏、石渡信一 他: "生体物理工学" 丸善株式会社, (1990)

[文献書誌] Sokabe,M.: "Towards a quantitative study of stretch activated channel.In “Comparative Aspects of Mechanoreceptor Systems"" Springer Verlag(Heidelberg),

[文献書誌] Ito,F.: "Effects of intracellular calcium on thr frog muscle spindle in relation to cyclic AMP action.In“Mechanoreceptor"" Plenum Press, 195-199 (1988)

1989 年度実績報告書

桐野豊九州大学, 薬学部, 教授 (10012668)

[文献書誌] 宮本宏、石渡信一他: "生体物理工学" 丸善株式会社, (1990)