2003 Fiscal Year Annual Research Report

脳内胆汁酸の生理的意義解明に関する研究

Research Project

Project/Area Number	14370726
Research Institution	TOHOKU UNVERSITY
Principal Investigator	後藤順一東北大学, 医学部附属病院, 教授 (80006337)
Co-Investigator(Kenkyū-buntansha)	飯田隆日本大学, 文理学部, 教授 (60060125) 眞野成康東北大学, 大学院・薬学研究科, 助教授 (50323035) 小林典裕神戸薬科大学, 薬学部, 教授 (90205477)
Keywords	脳内胆汁酸 / ケノデオキシコール酸 / プロテオミクス / LC / ESI-MS / MALDI-TOFMS / アフィニティー抽出 / ディファレンシャルディスプレイ / アルツハイマー
Research Abstract	胆汁酸は、肝においてコレステロールより生合成される炭素数24個のカルボン酸であり、腸肝循環しているため、通常血中や末梢組織中にはごく僅かにしか存在しない。最近当研究グループにおいて、LC/ESI-MSを用いてラット脳組織中の胆汁酸の検索を行ったところ、3種の遊離型胆汁酸が存在し、しかもそれらのうち、ケノデオキシコール酸はグアニジンを用いる蛋白質変成操作を加えることにより初めて検出されることから、脳内においてある種の蛋白質と強くバウンドしていることが示唆された。そこで今回、ケノデオキシコール酸を固定化したアフィニティーゲルを調製し、これを用いて結合蛋白質の特異的抽出を試みるとともに、プロテオミクス手法を用いてその結合蛋白質の解析を行った。まず、ケノデオキシコール酸のβ-アラニン抱合体を調製し、これをカルボジイミド法を用いてアガロースゲルに固定化した。これに、ラツト脳細胞質画分を添加し、緩やかに混合した後、リン酸緩衝液、水/メタノール混液などを用いて順次洗浄して非特異的結合蛋白質を除去した。次に、先と同様に高濃度のグアニジン溶液を添加してケノデオキシコール酸と強く結合した蛋白質を抽出し、得られた蛋白質混合物を二次元ゲル電気泳動法を用いて分離したところ、数種の蛋白質が特異的に濃縮されることが判明した。トリプシンを用いるゲル内消化後、MALDI-TOFMSによるペプチドマスマッピングに付した。ペプチドマスフィンガープリンティング法によるデータベース検索を行ったところ、ピタミンD依存性Ca結合蛋白質の他、チューブリンや14-3-3蛋白質などのアルツハイマー病に関与する蛋白質が同定された。

Research Products
(21 results)

All Other

All Publications (21 results)

[Publications] Nariyasu Mano: "Inhibition of the rat hepatic microsomal flurbiprofen acyl glucuronidation by bile acids"Journal of Pharmaceutical Sciences. 92. 2098-2108 (2003)
[Publications] Takashi Iida: "^1H and ^<13>C NMR signal assignments of carboxyl-linked glucuronides of bile acids"Magnetic Resonance Chemistry. 41. 260-264 (2003)
[Publications] Takashi Iida: "Functionalization of unactivated carbons in 3α,6- and 3α,24-dihydroxy-5β-cholane derivatives by dimethyldioxirane"Lipids. 38. 281-287 (2003)
[Publications] Nariyasu Mano: "Synthesis and characterization of deoxycholyl 2-deoxyglucuronide : A water-soluble affinity labeling reagent"Lipids. 38. 873-879 (2003)
[Publications] Norihiro Kobayashi: "Idiotype-anti-idiotype based noncompetitive enzyme-liked immunosorbent assay of ursodeoxycholic acid 7-N-acetylglucosaminides in human urine with subfemtomole range sensitivity"Journal of Immunological Methods. 272. 1-10 (2003)
[Publications] Naoto Suzuki: "Determination of urinary 12(S)-hydroxyeicosatetraenoic acid by liquid chromatography/tandem mass spectrometry with column-switching technique : Sex difference in healthy voluntees and patients with diabetes mellitus"Journal of Chromatography B. 783. 383-389 (2003)
[Publications] Lilian Rumi Tsuruta: "Characterization of 11-dehydro-thromboxane B_2 recombinant antibbody obtained by phage display technology"Prostaglandins, Leukotrienes & Essential Fatty Acids. 68. 273-284 (2003)
[Publications] Naoto Nakagawa: "Brain and heart specific alteration of methamphetamine (MAP) distribution in MAP-senstized rat"Biological Pharmaceutical Bulletin. 26. 506-509 (2003)
[Publications] Takehiko Suzuki: "Identification and characterization of novel rat and human gonad specific organic anion transporters"Molecular Endocrinology. 17. 1203-1215 (2003)
[Publications] Takashi Iida: "An improved method for the capillary gas chromatographic derivatization of polyhydroxylated steroids having tert.-hydroxyl groups"Analytical Sciences. 19. 1317-1321 (2003)
[Publications] Takashi Iida: "A Comparative study of remote-oxyfunctionalization of unactivated carbons in 5β-steroids by dimethyldioxirane and 2,6-dichloropyridine N-oxide/Ruthenium"Arkivoc. 2003. 170-179 (2003)
[Publications] Sookie La: "Simultaneous chiral discrimination of multiple profens by cyclodextrin-modifeid capillary electrophoesis in normal and reversed polarity modes"Electrophoresis. 24. 2642-2649 (2003)
[Publications] Nariyasu Mano: "Exopeptidase degradation for the analysis of phosphorylation site in a mono-phosphorylated peptide with matrix-assisted laser desorption mass spectrometry"Analytical Sciences. 19. 1469-1472 (2003)
[Publications] Mami Kida: "Vectorial transport of bile acids in immortalized mouse bile duct cells"Hepatology Research. 27. 151-157 (2003)
[Publications] Nariyasu Mano: "Presence of protein-bound unconjugated bile acids in the cytoplasmic fraction of rat brain"Journal of Lipid Research. 45. 295-300 (2004)
[Publications] Nariyasu Mano: "Analysis of the antigen binding site of anti-deoxycholate monoclonal antibody using novel affinity labeling reagent, acyl adenylate"Biochemistry. 43. 2041-2048 (2004)
[Publications] Genta Kakiyama: "Chemical synthesis of (22E)-3α,6β,7β-trihydroxy-5β-chol-22-en-24-oic acid and its taurine and glycine conjugates : a major bile acid in the rat"Journal of Lipid Research. (in press). (2004)
[Publications] Tsuyoshi Mikkaichi: "Isolation and characterization of a novel human kidney-specific digoxin transporter and its rat homologue"Proceedings of National Academy of Sciences in USA. (in press). (2004)
[Publications] Shoujiro Ogawa: "Remote-oxyfunction of unactivated carbons in (5β)-3-oxobile acids by 2,6-dichloropyridine N-oxide catalyzed by ruthenium-porphyrin and HBr : direct"Organic Biomolecular Chemistry. (in press). (2004)
[Publications] Naoto Suzuki: "Quantitative liquid chromatography-tandem mass spectrometric analysis of 11-dehydroTXB_2 in the urine"Prostagrandins & Other Lipid Metabolites. (in press). (2004)
[Publications] Hiroki Tsukamoto: "Thiazolidinediones increase arachidonic acid release and subsequent prostanoid production in peroxisome proliferator-activated receptor γ-independent manner"Prostagrandins & Other Lipid Metabolites. (in press). (2004)

2003 Fiscal Year Annual Research Report

脳内胆汁酸の生理的意義解明に関する研究

Principal Investigator

後藤 順一 東北大学, 医学部附属病院, 教授 (80006337)

Research Products

[Publications] Nariyasu Mano: "Inhibition of the rat hepatic microsomal flurbiprofen acyl glucuronidation by bile acids"Journal of Pharmaceutical Sciences. 92. 2098-2108 (2003)

[Publications] Takashi Iida: "^1H and ^<13>C NMR signal assignments of carboxyl-linked glucuronides of bile acids"Magnetic Resonance Chemistry. 41. 260-264 (2003)

[Publications] Takashi Iida: "Functionalization of unactivated carbons in 3α,6- and 3α,24-dihydroxy-5β-cholane derivatives by dimethyldioxirane"Lipids. 38. 281-287 (2003)

[Publications] Nariyasu Mano: "Synthesis and characterization of deoxycholyl 2-deoxyglucuronide : A water-soluble affinity labeling reagent"Lipids. 38. 873-879 (2003)

[Publications] Norihiro Kobayashi: "Idiotype-anti-idiotype based noncompetitive enzyme-liked immunosorbent assay of ursodeoxycholic acid 7-N-acetylglucosaminides in human urine with subfemtomole range sensitivity"Journal of Immunological Methods. 272. 1-10 (2003)

[Publications] Naoto Suzuki: "Determination of urinary 12(S)-hydroxyeicosatetraenoic acid by liquid chromatography/tandem mass spectrometry with column-switching technique : Sex difference in healthy voluntees and patients with diabetes mellitus"Journal of Chromatography B. 783. 383-389 (2003)

[Publications] Lilian Rumi Tsuruta: "Characterization of 11-dehydro-thromboxane B_2 recombinant antibbody obtained by phage display technology"Prostaglandins, Leukotrienes & Essential Fatty Acids. 68. 273-284 (2003)

[Publications] Naoto Nakagawa: "Brain and heart specific alteration of methamphetamine (MAP) distribution in MAP-senstized rat"Biological Pharmaceutical Bulletin. 26. 506-509 (2003)

[Publications] Takehiko Suzuki: "Identification and characterization of novel rat and human gonad specific organic anion transporters"Molecular Endocrinology. 17. 1203-1215 (2003)

[Publications] Takashi Iida: "An improved method for the capillary gas chromatographic derivatization of polyhydroxylated steroids having tert.-hydroxyl groups"Analytical Sciences. 19. 1317-1321 (2003)

[Publications] Takashi Iida: "A Comparative study of remote-oxyfunctionalization of unactivated carbons in 5β-steroids by dimethyldioxirane and 2,6-dichloropyridine N-oxide/Ruthenium"Arkivoc. 2003. 170-179 (2003)

[Publications] Sookie La: "Simultaneous chiral discrimination of multiple profens by cyclodextrin-modifeid capillary electrophoesis in normal and reversed polarity modes"Electrophoresis. 24. 2642-2649 (2003)

[Publications] Nariyasu Mano: "Exopeptidase degradation for the analysis of phosphorylation site in a mono-phosphorylated peptide with matrix-assisted laser desorption mass spectrometry"Analytical Sciences. 19. 1469-1472 (2003)

[Publications] Mami Kida: "Vectorial transport of bile acids in immortalized mouse bile duct cells"Hepatology Research. 27. 151-157 (2003)

[Publications] Nariyasu Mano: "Presence of protein-bound unconjugated bile acids in the cytoplasmic fraction of rat brain"Journal of Lipid Research. 45. 295-300 (2004)

[Publications] Nariyasu Mano: "Analysis of the antigen binding site of anti-deoxycholate monoclonal antibody using novel affinity labeling reagent, acyl adenylate"Biochemistry. 43. 2041-2048 (2004)

[Publications] Genta Kakiyama: "Chemical synthesis of (22E)-3α,6β,7β-trihydroxy-5β-chol-22-en-24-oic acid and its taurine and glycine conjugates : a major bile acid in the rat"Journal of Lipid Research. (in press). (2004)

[Publications] Tsuyoshi Mikkaichi: "Isolation and characterization of a novel human kidney-specific digoxin transporter and its rat homologue"Proceedings of National Academy of Sciences in USA. (in press). (2004)

[Publications] Shoujiro Ogawa: "Remote-oxyfunction of unactivated carbons in (5β)-3-oxobile acids by 2,6-dichloropyridine N-oxide catalyzed by ruthenium-porphyrin and HBr : direct"Organic Biomolecular Chemistry. (in press). (2004)

[Publications] Naoto Suzuki: "Quantitative liquid chromatography-tandem mass spectrometric analysis of 11-dehydroTXB_2 in the urine"Prostagrandins & Other Lipid Metabolites. (in press). (2004)

[Publications] Hiroki Tsukamoto: "Thiazolidinediones increase arachidonic acid release and subsequent prostanoid production in peroxisome proliferator-activated receptor γ-independent manner"Prostagrandins & Other Lipid Metabolites. (in press). (2004)

後藤順一東北大学, 医学部附属病院, 教授 (80006337)