日本農芸化学会関西支部 第 478 回 講演会

᪥ᮏ㎰ⱁ໬Ꮫ఍㛵すᨭ㒊

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᪥᫬㸸ᖹᡂ ᖺ ᭶ ᪥㸦ᅵ㸧 ᫬ ศ㛤఍

఍ሙ㸸ி㒔኱Ꮫᴦ཭఍㤋 㝵఍㆟࣭ㅮ₇ᐊ㸦ி㒔ᕷᕥி༊ྜྷ⏣஧ᮏᯇ⏫7(/㸧

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ி㜰㟁㌴ࠕ⚄ᐑ୸ኴ⏫㥐ࠖୗ㌴ᚐṌ ศ@

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⌮໬Ꮫ㸧

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ۑᓥ୪♸Ꮚࠊዟබ⚽ࠊᑉ㛵῟ࠊ㜰஭ᗣ⬟㸦^㸯ி኱㝔㎰࣭ᛂ⏝⏕࿨ࠊ^㸰ி኱Ꮫ㝿⼥ྜ࣭⏕⌮໬Ꮫ㸧

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ۑୡཱྀྂṌ⳹ࠊᐑୖἋ㈗ࠊᰘ⏣᭷ຍࠊ๓⏣⣧ኵ㸦ዉⰋዪ኱㝔ே㛫ᩥ໬࣭㣗≀ᰤ㣴ࠊዉⰋዪ኱⏕ά⎔ቃ࣭㣗≀ᰤ㣴㸧

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ࠐ୸㔝᫭Ⴙࠊᆏᮏᬛᘺࠊᚋ⸨๛ࠊ㧗ᶫಙஅࠊἙ⏣↷㞝㸦ி኱㝔㎰࣭㣗ရ⏕≀ࠊி኱Ꮫ㝿⼥ྜ࣭⏕⌮໬Ꮫ㸧

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ۑᒣ㷂㝧ኴࠊ㧗ᶫ᫓ᘺࠊᚋ⸨๛ࠊ㧗ᶫಙஅࠊᰘ⏣኱㍜ࠊἙ⏣↷㞝㸦ி኱㝔㎰࣭㣗ရ⏕≀ࠊி኱Ꮫ㝿⼥ྜ࣭⏕⌮

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ⱝᡭඃ⚽Ⓨ⾲㈹⾲ᙲᘧ㸦㸧

᠓ぶ఍㸦㹼㸧ி㒔኱Ꮫᴦ཭఍㤋㣗ᇽ ୍⯡ ෇ Ꮫ⏕↓ᩱ

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᪥᫬㸸ᖹᡂ ᖺ ᭶ ᪥㸦ᅵ㸧 ሙᡤ㸸ி㒔ᗓ❧኱Ꮫ

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mRNA ᰾እ㍺㏦ᅉᏊ UAP56 䛺䜙䜃䛻 URH49 䛾」ྜ

యᙧᡂ⬟䜢ᨭ㓄䛩䜛㡿ᇦ䛾ྠᐃ䛸ᶵ⬟ゎᯒ

ۑ⸨⏣㈼୍ࠊᖹᒣ⍞Ꮨࠊᐑ๓཭⟇ࠊ⚄ᡞ኱᭸ࠊỌᑿ㞞ဢࠊ ቑ⏣ㄔྖ㸦ி኱㝔⏕࿨࣭⤫ྜ⏕࿨⛉Ꮫ㸧

㻌

䛆┠ⓗ䛇┿᰾⏕≀䛻䛚䛔䛶᰾ෆ䛷㌿෗䛥䜜䛯๓㥑య 㼙㻾㻺㻭 䛿䚸ᵝ䚻䛺 㼙㻾㻺㻭 䝥䝻䝉䝅 䞁䜾䜢ཷ䛡䛶ᡂ⇍ 㼙㻾㻺㻭 䛸䛺䜚䚸⣽⬊㉁䛻㍺㏦䛥䜜䛶⺮ⓑ㉁䜈䛸⩻ヂ䛥䜜䜛䚹ᡂ⇍

㼙㻾㻺㻭 䛾᰾ෆ䛛䜙⣽⬊㉁䜈䛾㍺㏦䛻䛿 㼀㻾㻱㼄 」ྜయ䛜ᶵ⬟䛩䜛䚹㼁㻭㻼㻡㻢 䛿 㼀㻾㻱㼄 」

ྜయ䛾ᶵ⬟୰ᚰ䛸䛺䛳䛶ാ䛟ᅉᏊ䛷䛒䜚䚸㓝ẕ䛛䜙䝠䝖䜎䛷㐍໬ⓗ䛻ಖᏑ䛥䜜䛶䛔䜛䚹

୍᪉䚸㓝ẕ䜔䝝䜶䛺䛹䛷䚸㼁㻭㻼㻡㻢 䛿༢୍䛷Ꮡᅾ䛩䜛䛾䛻ᑐ䛧䛶䚸့ங㢮䛷䛿 㻥㻜㻑௨ୖ

䜒䛾┦ྠᛶ䜢ᣢ䛴 㼁㻾㻴㻠㻥 䛜Ꮡᅾ䛩䜛䚹ᙜ◊✲ᐊ䛷䛾ඛ⾜◊✲䛛䜙䚸䝠䝖䛻䛚䛔䛶 㼁㻾㻴㻠㻥 䛿䚸㼁㻭㻼㻡㻢 䛾ᙧᡂ䛩䜛 㼀㻾㻱㼄 」ྜయ䛸䛿඲䛟␗䛺䜛᪂つ䛾 㻭㻾㻱㼄 」ྜయ䜢ᙧ ᡂ䛩䜛䛣䛸䛜᫂䜙䛛䛻䛥䜜䛯䚹䜎䛯୧⪅䛜㍺㏦䛩䜛 㼙㻾㻺㻭 䛻䛿┦㐪䛜䜏䜙䜜䚸㑅ᢥⓗ

㼙㻾㻺㻭 ㍺㏦䛻ᶵ⬟䛩䜛䛣䛸䜒᫂䜙䛛䛻䛥䜜䛯䚹㼙㻾㻺㻭 䛾㑅ᢥⓗ䛺㍺㏦䛻䛿䚸」ྜయᙧ ᡂ䛾㐪䛔䛜 㼙㻾㻺㻭 䛾㑅ᢥᛶ䜢Ỵᐃ䛧䛶䛔䜛䛸ண᝿䛥䜜䜛䛜䚸㠀ᖖ䛻┦ྠᛶ䛾㧗䛔୧

⪅䛜䚸␗䛺䜛」ྜయ䜢ᙧᡂ䛩䜛ศᏊᶵᵓ䛿୙᫂䛷䛒䜛䚹䛭䛣䛷」ྜయᙧᡂ䛾㐪䛔䜢 ไᚚ䛩䜛䚸㼁㻭㻼㻡㻢 䛸 㼁㻾㻴㻠㻥 䛾⺮ⓑ㉁㡿ᇦ䛾ྠᐃ䜢ヨ䜏䛯䚹㻌

㻌

䛆᪉ἲ䞉⤖ᯝ䛇ゎᯒ᪉ἲ䛸䛧䛶䚸୧⪅䛷ẚ㍑ⓗ┦ྠᛶ䛾ప䛔୧ᮎ➃㡿ᇦ䜢䛿䛨䜑䚸 㼁㻭㻼㻡㻢 䛸 㼁㻾㻴㻠㻥 䛷␗䛺䜛ᵝ䚻䛺㡿ᇦ䜢ධ䜜᭰䛘䛯 㼁㻭㻼㻡㻢 䛸 㼁㻾㻴㻠㻥 䜻䝯䝷ኚ␗య䜢 సᡂ䛧䚸」ྜయᙧᡂ⬟䚸䛺䜙䜃䛻⣽⬊ෆ 㼙㻾㻺㻭 ㍺㏦⬟䜢ゎᯒ䛧䛯䚹」ྜయᙧᡂ⬟䛿 ච␿ỿ㝆ἲ䛻䜘䜚䚸ኚ␗య䛜 㼀㻾㻱㼄 」ྜయ䜢ᙧᡂ䛩䜛䛛䚸㻭㻾㻱㼄 」ྜయ䜢ᙧᡂ䛩䜛䛛 ゎᯒ䛧䛯䚹㼙㻾㻺㻭 ㍺㏦⬟䛿 㻾㻺㻭㻙㻲㻵㻿㻴 ἲ䛻䜘䜚䚸㼁㻭㻼㻡㻢 䜎䛯䛿 㼁㻾㻴㻠㻥 䛾 㻷㻰 䛻䜘䜚⏕

䛨䜛᰾ෆ䛾 㻼㼛㼘㼥㻌 㻔㻭㻕

^㻗

㻌 㻾㻺㻭 䛾⵳✚䛜䚸ኚ␗యⓎ⌧᫬䛻ᅇ᚟䛩䜛䛛ゎᯒ䛧䛯䚹౛䛘䜀 㼁㻭㻼㻡㻢 䛾䜻䝯䝷ኚ␗య䛜䚸ᮏ᮶ 㼁㻾㻴㻠㻥 䛾ᙧᡂ䛩䜛 㻭㻾㻱㼄 」ྜయ䜢ᙧᡂ䛧䚸㼁㻾㻴㻠㻥 䛸

ྠ䛨⣽⬊ෆ 㼙㻾㻺㻭 ㍺㏦⬟䜢䜒䛴ሙྜ䚸ኚ␗䜢ධ䜜䛯㡿ᇦ䛜」ྜయᙧᡂ䜢ไᚚ䛩䜛㡿 ᇦ䛸ุ᩿䛷䛝䜛䚹䛣䜜䜙 㻞 ✀㢮䛾ゎᯒἲ䛻䜘䜚ヲ⣽䛺」ྜయᙧᡂ䛾ศᏊᇶ┙ゎ᫂䜢⾜

䛳䛯䚹㻌

䜎䛪୧ᮎ➃㓄ิ䜢Ḟᦆ䛥䛫䛯Ḟᦆኚ␗య䛸䚸ᮎ➃㓄ิ䜢ධ䜜᭰䛘䛯 㼁㻭㻼㻡㻢 䛸 㼁㻾㻴㻠㻥 䛾䜻䝯䝷ኚ␗య䜢ゎᯒ䛧䛯䚹Ḟᦆኚ␗య䛾ゎᯒ䛛䜙䚸୧ᮎ➃㓄ิ䛿」ྜయᙧᡂ䛻ᚲ せ䛷䛒䜛䛣䛸䛜ุ᫂䛧䛯䚹䛧䛛䛧୧ᮎ➃䛾䜻䝯䝷ኚ␗య䛾ゎᯒ䛛䜙୧ᮎ➃䛿」ྜయᙧ ᡂ䛾㐪䛔䜢ไᚚ䛧䛺䛔䛣䛸䛜ุ᫂䛧䛯䚹⥆䛔䛶୧ᮎ➃௨እ䛾䝁䜰㡿ᇦ䛷␗䛺䜛䜰䝭䝜㓟 䜢 㻝 ṧᇶ䛪䛴ධ䜜᭰䛘䛯䜻䝯䝷ኚ␗య䜢ゎᯒ䛧䛯䚹䛭䛾⤖ᯝ䛒䜛 㻝 䜰䝭䝜㓟䜢ධ䜜᭰䛘 䛯 㼁㻾㻴㻠㻥 ኚ␗య䛜 㼁㻭㻼㻡㻢 䛸ྠᵝ䛾」ྜయᙧᡂ⬟䛸 㼙㻾㻺㻭 ㍺㏦⬟䜢䜒䛴䛣䛸䛜ุ᫂

䛧䛯䚹⌧ᅾ䚸䛣䛾䠍䜰䝭䝜㓟䜢ධ䜜᭰䛘䛯 㼁㻭㻼㻡㻢 ኚ␗య䛜 㼁㻾㻴㻠㻥 䛸ྠᵝ䛾」ྜయᙧᡂ

⬟䛸 㼙㻾㻺㻭 ㍺㏦⬟䜢䜒䛴䛾䛛䚸䛥䜙䛻䛺䛬䛣䛾䜰䝭䝜㓟䛜」ྜయᙧᡂ䜢ไᚚ䛩䜛䛾䛛䚸

ゎᯒ䜢㐍䜑䛶䛔䜛䚹

㸱

పள㖄ẕங䜢䜒䛯䜙䛩ள㖄䝖䝷䞁䝇䝫䞊䝍䞊ZnT2 䛾」ྜ䝦䝔䝻᥋ྜయኚ␗

ۑ㐓ᮧ┤ஓࠊ✄ẟᗣྖࠊᑎすᩥᜨࠊᒸᓮᩥᏊࠊᡂ⏣ᏹྐࠊඣᓥᾈᏊࠊ

⚄ᡞ኱᭸㸦ி኱㝔⏕࿨࣭⤫ྜ⏕࿨ࠊ᪥኱࣭⦎㤿ගࡀୣ⑓㝔࣭ᑠඣࠊிዪ

኱࣭㣗≀ᰤ㣴ࠊᖇிᖹᡂ኱࣭೺ᗣ࣓ࢹ࢕࣭࢝ࣝ೺ᗣᰤ㣴㸧

㻌

࠙┠ⓗࠚ

ள㖄ࡣࠊࣄࢺ࡟࡜ࡗ࡚Ḟ࠿ࡏ࡞࠸ᚲ㡲ᚤ㔞ඖ⣲࡛࠶ࡿࠋ࡜ࡃ࡟ࠊయ㔜ᙜࡓࡾ

࡛ᡂேࡢ⣙ 3 ಸࡶࡢள㖄ࢆᚲせ࡜ࡍࡿஙඣࡣࠊள㖄Ḟஈ࡟࡞ࡿ࡜㔜⠜࡞⓶⭵⅖

ࡸᡂ㛗ࡢ㐜ᘏ࡜࠸ࡗࡓ⑕≧ࢆ♧ࡍࠋஙඣࡢள㖄Ḟஈࡢせᅉ࡜ࡋ࡚ࠊẕங୰ࡢள 㖄ࡢῶᑡࡀ࠶ࡆࡽࢀࡿࠋᡃࠎࡣࠊள㖄Ḟஈ࡟ࡼࡾ㔜⠜࡞⓶⭵⅖ࢆⓎ⑕ࡋࡓஙඣ

࡜ࠊࡑࡢẕぶ࡛ࠊẕங୰ள㖄㔞ࡀ90%௨ୖῶᑡࡋࡓపள㖄ẕஙᝈ⪅ࢆぢฟࡋࡓࠋ పள㖄ẕஙࡢཎᅉ࡜ࡋ࡚ࠊẕங୰࡬ࡢள㖄㍺㏦ࢆᢸ࠺ள㖄ࢺࣛࣥࢫ࣏࣮ࢱ࣮

ZnT2ࡢ㑇ఏᏊኚ␗ࡀ▱ࡽࢀ࡚࠸ࡿࡇ࡜࠿ࡽࠊᡃࠎࡣẕぶࡢZnT2㑇ఏᏊ࡟╔┠

ࡋ࡚ゎᯒࢆ⾜ࡗࡓࠋ

࠙᪉ἲ࣭⤖ᯝࠚ

1) ẕぶࡢࢤࣀ࣒ DNA ࠿ࡽࠊZnT2 㑇ఏᏊ࡟ 2 ࡘࡢ᪂つ࣑ࢫࢭࣥࢫኚ␗

(W152R࣭S296L)ࢆࡑࢀࡒࢀูࡢ࢔ࣜࣝ࡟ぢฟࡋࡓࠋࡲࡓࠊZnT2㑇ఏᏊࡢࣉࣟ

࣮ࣔࢱ࣮㡿ᇦ࡟ኚ␗ࡣࡳࡽࢀ࡞࠿ࡗࡓࠋ

2) ྛ ZnT2 ࡢள㖄㍺㏦άᛶࢆࠊள㖄ឤཷᛶࡢ⣽⬊ᰴࢆ⏝࠸࡚ホ౯ࡋࡓ⤖ᯝࠊ ZnT2 S296LࡣZnT2㔝⏕ᆺ࡜࡯ࡰྠ➼ࡢள㖄㍺㏦άᛶࢆࡶࡘࡀࠊZnT2 W152R ࡣள㖄㍺㏦άᛶࢆࡶࡓ࡞࠸ࡇ࡜ࡀ♧ࡉࢀࡓࠋ

3) ZnT2ࡣ஧㔞యࢆᙧᡂࡋ࡚ࡣࡌࡵ࡚ள㖄㍺㏦άᛶࢆⓎ᥹ࡍࡿࠋZnT2ྛኚ␗ᆺ ࡢ஧㔞యᙧᡂ⬟ࢆホ౯ࡍࡿࡓࡵࠊ㔝⏕ᆺࡸኚ␗ᆺࡢ ZnT2 ࢆ஧㔜Ⓨ⌧ࡉࡏࡓ⣽

⬊ᰴ࡟ࡼࡾඹච␿ỿ㝆ᐇ㦂ࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊZnT2 S296L ࡣ㔝⏕ᆺ࡜஧㔞 యࢆᙧᡂࡍࡿࡀࠊZnT2 W152Rࡣ㔝⏕ᆺ࡜஧㔞యࢆᙧᡂࡋ࡞࠸ࡇ࡜ࠊࡲࡓࠊZnT2 W152R࡜ZnT2 S296Lࡢ⤌ࡳྜࢃࡏࡶࠊ஧㔞యࢆᙧᡂࡋ࡞࠸ࡇ࡜ࡀ♧ࡉࢀࡓࠋ 4) ྛ ZnT2 ࡢࢱࣥࣃࢡ㉁ᏳᐃᛶࢆゎᯒࡍࡿࡓࡵࠊZnT2Ⓨ⌧⣽⬊ᰴࢆࢱࣥࣃࢡ

㉁ྜᡂ㜼ᐖ⸆࡛ฎ⌮ࡋࠊࢱࣥࣃࢡ㉁㔞ࡢ⥅᫬ⓗኚ໬ࢆồࡵࡓࠋ⤖ᯝࠊZnT2 S296L ࡢࢱࣥࣃࢡ㉁ࡣࠊ㔝⏕ᆺ࡟ẚ࡭࡚ⴭࡋࡃ୙Ᏻᐃ࡟࡞ࡗ࡚࠸ࡿࡇ࡜ࡀ♧ࡉࢀࡓࠋ

࠙⪃ᐹࠚ

ZnT2 W152Rࡣள㖄㍺㏦άᛶࢆࡶࡓࡎࠊࡲࡓ」ྜయᙧᡂ⬟ࡀ࡞࠸ࡇ࡜࠿ࡽࠊ

ඃᛶ㜼ᐖάᛶࡣ࡞࠸ࡇ࡜ࡀ♧၀ࡉࢀࡓࠋ୍᪉ࠊZnT2 S296L ࡣࠊள㖄㍺㏦άᛶ

࡜」ྜయᙧᡂ⬟ࢆࡶࡘࡀࠊࢱࣥࣃࢡ㉁ࡀ㠀ᖖ࡟୙Ᏻᐃ࡛࠶ࡿࡇ࡜ࡀุ᫂ࡋࡓࠋ ௨ୖ࠿ࡽࠊẕぶࡢZnT2㑇ఏᏊࡀࠊW152R࡜ S296Lࡢ」ྜ࣊ࢸࣟ᥋ྜయ࡛࠶

ࡿࡇ࡜ࡀࠊẕぶࡢపள㖄ẕஙࡢཎᅉ࡜⪃࠼ࡽࢀࡓࠋ

㸲

஝⇱⫙䝰䝕䝹䝬䜴䝇ヨ㦂䛻䛚䛡䜛䝟䜲䝘䝑䝥䝹ᯝᐇ⏤

᮶䜾䝹䝁䝅䝹䝉䝷䝭䝗⤒ཱྀᦤྲྀ䛾⓶⭵ᶵ⬟ᨵၿຠᯝཬ 䜃 in vitro ⭠⟶䝰䝕䝹䜈䛾ᙳ㡪㻌

㻌

ۑ‮ὸᘯᶞࠊす㇂ὒ㍜ࠊⓑ஭ᗣோࠊ㤿ሙ೺ྐࠊ㔝ᔱ ₶ࠊ኱ᡞಙ᫂ࠊ ᱔ཎᾈㄔࠊỈ㔝㞞ྐ㸦⚄኱㝔㎰࣭ᛂ⏝⏕࿨ࠊ⚄኱࣭⮬↛⛉Ꮫࠊ㜰኱㝔 ᕤ࣭⏕࿨ඛ➃ࠊ୸ၿ〇⸆㸧

࠙┠ⓗࠚࢫࣇ࢕ࣥࢦ⬡㉁ࡢ୍✀࡛࠶ࡿࢭ࣑ࣛࢻࡣゅ㉁ᒙࡢ⣽⬊㛫㝽ࢆᇙࡵ࡚࠸ࡿ

୺せᡂศ࡛࠶ࡿࠋ୺࡟ಖ‵࡞࡝⓶⭵ࡢࣂࣜ࢔ᶵ⬟࡟㔜せ࡞ᙺ๭ࢆᯝࡓࡋ࡚࠾ࡾ໬

⢝ရ࡞࡝࡟ࡶከࡃྵࡲࢀࡿࠋᮏ◊✲࡛ࡣࣃ࢖ࢼࢵࣉࣝᯝᐇࡼࡾᚓࡽࢀࡓࢢࣝࢥࢩ

ࣝࢭ࣑ࣛࢻྵ᭷⏬ศࢆ஝⇱⫙ࣔࢹ࣐ࣝ࢘ࢫ࡟⤒ཱྀᢞ୚ࡋࠊ⓶⭵ᶵ⬟࡟୚࠼ࡿᙳ㡪 ཬࡧLQYLWUR⭠⟶ࣔࢹࣝࢆ⏝࠸࡚ࡑࡢస⏝ᶵᵓࢆ᳨ウࡋࡓࠋ

࠙᪉ἲࠚ+RV+5 ࡬࢔ࣞࢫ࣐࢘ࢫ㸦㞝ᛶ 㐌㱋㸧ࢆ⏝࠸ࡓ஝⇱⫙ࣔࢹ࣐ࣝ࢘ࢫ ヨ㦂࡛ࡣࠊ≉Ṧ㣫ᩱ㸦+5$'㸧࡛ㄏᑟࡋࡓ஝⇱⫙࡟ᑐࡍࡿ⢒ࣃ࢖ࢼࢵࣉࣝᯝᐇ⏤

᮶ࢢࣝࢥࢩࣝࢭ࣑ࣛࢻ 㓄ྜ㣫ᩱ㸦3*㸧ࡢᙳ㡪ࢆ᳨ウࡋࡓࠋࡉࡽ࡟⤒ཱྀᦤྲྀ

࡟ࡼࡿ⭠⟶ච␿࡬ࡢᙳ㡪ࢆ᳨ウࡍࡿࡓࡵࠊࣃ࢖ࢼࢵࣉࣝᯝᐇ⏤᮶ࢢࣝࢥࢩࣝࢭࣛ

࣑ࢻ PJPO ࢆࣄࢺᑠ⭠ୖ⓶ᵝ⣽⬊ &DFR ⣽⬊࡜࣐࢘ࢫ࣐ࢡࣟࣇ࢓࣮ࢪᵝ⣽⬊

5$: ⣽⬊ࡢඹᇵ㣴⣔࡟౪ࡋࡓࠋ⟶⭍ഃ࠿ࡽࢧࣥࣉࣝฎ⌮ ᫬㛫ᚋ࡟ /36 ࡛ 5$: ⣽⬊ࢆ่⃭ࡋࠊࡉࡽ࡟ ᫬㛫ᇵ㣴ᚋ 5$: ⣽⬊ࡢ඲ 51$ ࢆᢳฟࡋࠊ ᐃ㔞 573&5 ἲ࡛ ,/P51$ Ⓨ⌧㔞ࢆ ᐃࡋࡓࠋࡲࡓࠊ⭠⟶ୖ⓶ࢆ௓ࡋࡓ௦ㅰ≀

ࡢᙳ㡪ࢆ᳨ウࡍࡿࡓࡵࠊࢺࣛࣥࢬ࢙࢘ࣝ⭷ୖ࡟ศ໬ࡉࡏࡓ &DFR ⣽⬊࡟ᑐࡋ࡚ࠊ

⟶⭍ഃ࠿ࡽࣃ࢖ࢼࢵࣉࣝᯝᐇ⏤᮶ࢢࣝࢥࢩࣝࢭ࣑ࣛࢻࢆῧຍࡋࠊ ᫬㛫ᚋ࡟ᇶᗏ

⭷ഃ࡟㏱㐣ࡋࡓᇵᆅࢆ 5$: ⣽⬊࡬࡜ῧຍࡋࡓࠋ ᫬㛫ᚋࠊ/36 ่⃭ࢆ⾜࠸ࠊ ࡉࡽ࡟ ᫬㛫ᇵ㣴ࡋࡓᚋ࡟ ,/P51$ Ⓨ⌧㔞ࢆ ᐃࡋࡓࠋ

࠙⤖ᯝࠚ㏻ᖖ㣫ᩱ⩌࡟ẚ࡭ࠊ≉Ṧ㣫ᩱ⩌࡛ 7(:/ ್ࡢୖ᪼ࡀㄆࡵࡽࢀࡓࠋ୍᪉ࠊ 3* ⩌࡛ࡣ 7(:/ ್ࡢୖ᪼ࢆ᭷ព࡟ᢚไࡋࡓࠋࡲࡓ 㐌㛫㣫⫱ᚋࠊ࣐࢘ࢫࡢ⫼㒊⾲

⓶ࢆほᐹࡋࡓ࡜ࡇࢁࠊ≉Ṧ㣫ᩱ⩌࡛ࡣ㢧ⴭ࡞ࢩ࣡ࡢᙧᡂཬࡧ⓶⭵ࡢ⫧ཌࡀ☜ㄆ࡛

ࡁࡓࡢ࡟ᑐࡋࠊ3* ⩌࡛ࡣ㏻ᖖ㣫ᩱ⩌࡜ྠ➼ࡢእほࢆ♧ࡋࡓࠋࡇࡢࡇ࡜࠿ࡽࣃ࢖

ࢼࢵࣉࣝᯝᐇ⏤᮶ࢢࣝࢥࢩࣝࢭ࣑ࣛࢻ࡟ࡣ⓶⭵ᶵ⬟ᨵၿຠᯝࡀ࠶ࡿࡇ࡜ࡀ♧၀ ࡉࢀࡓࠋࡇࢀࡲ࡛ࡢ◊✲࠿ࡽ⢭〇ࣃ࢖ࢼࢵࣉࣝᯝᐇ⏤᮶ࢢࣝࢥࢩࣝࢭ࣑ࣛࢻ 㸦3XUH3*㸧ࢆLQYLWUR⭠⟶ࣔࢹࣝ࡟౪ࡍࡿࡇ࡜࡛ 5$: ⣽⬊୰ࡢ ,/P51$

Ⓨ⌧㔞ࡀቑຍࡍࡿࡇ࡜ࡀศ࠿ࡗ࡚࠸ࡿࠋࢺࣛࣥࢬ࢙࢘ࣝ⭷ୖࡢ &DFR ⣽⬊༢ᒙ

⭷࡟ 3XUH3* ࢆῧຍࡋᇶᗏ⭷ഃᇵᆅࢆ 5$: ⣽⬊࡟┤᥋ῧຍࡋࡓ࡜ࡇࢁࠊඹ ᇵ㣴᫬࡜ྠᵝ࡟ 5$: ⣽⬊୰ࡢ ,/P51$ Ⓨ⌧㔞ࢆஹ㐍ࡋࡓࠋࡇࢀࡲ࡛ࡢᐇ 㦂࠿ࡽࠊ3XUH3* ࢆῧຍࡋࠊ ᫬㛫ᇵ㣴ᚋࡢṧᏑ㔞ࡣ⣙ ࡛࠶ࡗࡓࡇ࡜ࢆ⪃៖

ࡍࡿ࡜ࠊࣃ࢖ࢼࢵࣉࣝᯝᐇ⏤᮶ࢢࣝࢥࢩࣝࢭ࣑ࣛࢻࡣ⭠⟶ୖ⓶ࢆ௓ࡋࡓ௦ㅰࢆཷ

ࡅ࡚࠾ࡾࠊࡑࡢ௦ㅰ≀ࡀ࣐ࢡࣟࣇ࢓࣮ࢪ࡬࡜ᙳ㡪ࢆཬࡰࡋ࡚࠸ࡿྍ⬟ᛶࡀ♧၀ࡉ

ࢀࡓࠋ

㸳

ᾘ໬⟶䝩䝹䝰䞁䛻䜘䜛⫢⬡㉁௦ㅰไᚚ䛾ᶵᵓゎᯒ

㻌

ۑ㣤ሯࡳ࠶ࡁࠊ㧗ᶫಙஅࠊ᪂㇂⣪⧊ࠊ᐀ീᶞᏊࠊᚋ⸨๛ࠊ Ἑ⏣↷㞝㸦ி኱㝔࣭㎰࣭㣗ရ⏕≀ࠊி኱Ꮫ㝿⼥ྜ࣭⏕⌮໬Ꮫ㸧

㻌

࠙┠ⓗࠚ⏕యෆࡢ⬡㉁௦ㅰࡣ⚄⤒ࡸᾮᛶᅉᏊࢆ௓ࡋ࡚ከ⮚ჾ㛫࡛ไᚚࡉࢀࡿࡇ࡜

ࡀ㏆ᖺ᫂ࡽ࠿࡟ࡉࢀ࡚࠸ࡿࠋᙜ◊✲ᐊࡢඛ⾜◊✲࡟ࡼࡾࠊᑠ⭠⏤᮶ࡢᾘ໬⟶࣍ࣝ

࡛ࣔࣥ࠶ࡿcholecystokinin (CCK)ࡣ⫢⣽⬊࡛ࡢ⬡⫫⵳✚ࢆᢚไࡍࡿࡇ࡜ࡀ♧

ࡉࢀࠊCCKࡀᑠ⭠ࠊ⫢⮚㛫ࡢ⬡㉁௦ㅰ᝟ሗࢆఏ㐩ࡍࡿ㔜せ࡞ᅉᏊ࡛࠶ࡿྍ⬟ᛶ ࡀ⪃࠼ࡽࢀࡓࠋ࡜ࡇࢁࡀ᭱㏆ࠊCCKḞᦆ࣐࢘ࢫࡣ⬡⫫⫢ࢆⓎ⑕ࡋ࡞࠸ࡇ࡜ࡀሗ

࿌ࡉࢀࠊඛ⾜◊✲࠿ࡽࡢண ࡜▩┪ࡍࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋࡑࡇ࡛ᮏ◊✲࡛

ࡣCCK࡜ྠ୍ࡢཷᐜయ࡛࠶ࡿCCKB Receptor (CCKBR) ࢆㄆ㆑ࡍࡿᾘ໬⟶࣍

ࣝࣔࣥࠊgastrinࡀCCK࡟ࡼࡿస⏝ࢆ௦ൾࡋ࡚࠸ࡿࡶࡢ࡜௬ᐃࡋࠊgastrinࡀ⫢

⬡㉁௦ㅰ࡟ཬࡰࡍᙳ㡪ࠊ୪ࡧ࡟CCKBRࢆ௓ࡋࡓ⫢⬡⫫⵳✚ᢚไᶵᵓࡢヲ⣽ࢆ

ゎ᫂ࡍࡿࡇ࡜ࢆ┠ⓗ࡜ࡋࡓࠋ

࠙᪉ἲ࣭⤖ᯝࠚࣄࢺ⫢⣽⬊ࣔࢹ࡛ࣝ࠶ࡿHepG2⣽⬊࡟gastrinࢆฎ⌮ࡋࡓ⤖ᯝࠊ

⬡⫫⵳✚ࡣ᭷ព࡟ᢚไࡉࢀࠊࡑࡢస⏝ࡣ CCKBR ࡟ᑐࡍࡿ㜼ᐖ๣࡟ࡼࡾ᭷ព࡟

ᾘኻࡋࡓࠋඛ⾜◊✲ࡼࡾࠊCCKࡣ⫢⣽⬊࡟࠾࠸࡚⬡⫫㓟ǃ㓟໬ࢆஹ㐍ࡉࡏࡿࡇ

࡜࡛⬡⫫⵳✚ࢆᢚไࡍࡿࡇ࡜ࡀ♧၀ࡉࢀ࡚࠸ࡿࠋࡑࡇ࡛ࠊCCKBR࠿ࡽ⬡⫫㓟ǃ 㓟໬ࡢஹ㐍࡟⮳ࡿࢩࢢࢼࣝఏ㐩⤒㊰࡟㛵ࡍࡿ᳨ウࢆ⾜ࡗࡓࠋCCKBR ࡣ G protein coupled receptor࡛࠶ࡾࠊࡑࡢୗὶࡣCa²⁺ ࡀ఩⨨ࡍࡿࡇ࡜ࡀ▱ࡽࢀ࡚

࠸ࡿࠋࡑࡇ࡛ࠊHepG2⣽⬊ࢆ⏝࠸࡚[Ca²⁺]i࡟㛵ࡍࡿ᳨ウࢆ⾜ࡗࡓ⤖ᯝࠊgastrin ࡣCa²⁺ ࡢ⤒㊰ࢆ௓ࡍࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋḟ࡟ࠊCa²⁺ ࡢୗὶࡢࢩࢢࢼࣝఏ㐩⤒

㊰ࢆ᫂ࡽ࠿࡟ࡍࡿࡓࡵࠊྛ✀㜼ᐖ๣ࢆ⏝࠸ࡓ᳨ウࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊgastrin

࡟ࡼࡿ⬡⫫⵳✚ᢚไస⏝ࡣCaMKK࠾ࡼࡧAMPK࡟ᑐࡍࡿ㜼ᐖ๣࡟ࡼࡾ᭷ព࡟

ᾘኻࡋࠊCCK ࢆฎ⌮ࡋࡓሙྜ࡜ྠᵝࡢ⤖ᯝ࡜࡞ࡗࡓࠋࡉࡽ࡟ࠊCCK ࠾ࡼࡧ

gastrin࡟ࡼࡿస⏝ࢆ⏕యෆ᳨࡛ウࡍࡿࡓࡵࠊ⫢⮚≉␗ⓗ࡞㑇ఏᏊᑟධヨ⸆ࢆ⏝

࠸࡚CCKBR࡟ᑐࡍࡿsiRNAࢆ࣐࢘ࢫ࡟ᑟධࡋࡓࠋࡑࡢ⤖ᯝࠊCCKBR-siRNA

ࢆᑟධࡋࡓ࣐࢘ࢫࡢ⫢⬡⫫⵳✚ࡀ᭷ព࡟ቑຍࡋࡓࠋ௨ୖࡢ⤖ᯝࡼࡾࠊgastrinࡣ CCK࡜ྠᵝ࡟⫢⣽⬊࡟࠾࠸࡚CCKBRń[Ca²⁺]ińCaMKKńAMPKࢆ௓ࡋ࡚⬡

⫫⵳✚ᢚไస⏝ࢆ♧ࡍࡇ࡜ࠊ࠾ࡼࡧ⫢⬡⫫⵳✚࡟ᑐࡍࡿ CCKBR ࡢ⏕యෆ࡛ࡢ 㛵୚ࡀ♧၀ࡉࢀࡓࠋ

㸴

ABCG1 䛻䜘䜛 HMG-CoA 㑏ඖ㓝⣲䛾᪂つάᛶไᚚ

ᶵᵓ䛾ゎᯒ

㻌

ۑΏ㑔ኴ㑻{ࠊᖹ஭⤢Ꮚ{ࠊ᳜⏣࿴ග{tࠊᯇᑿ㐨᠇{㸦ி኱㝔㎰࣭ᛂ⏝⏕࿨ࠊ

ி኱࣭L&H06㸧

࠙┠ⓗࠚࢥࣞࢫࢸ࣮ࣟࣝࡣ⏕య⭷ࡢ㔜せ࡞ᵓᡂᡂศࡔࡀࠊ㐣๫࡞⵳✚ࡣ⣽⬊ẘᛶ

ࢆ♧ࡍࠋࡑࡢࡓࡵࠊ⣽⬊ෆࡢࢥࣞࢫࢸ࣮ࣟࣝ⃰ᗘࡣཝᐦ࡞ㄪ⠇ࢆཷࡅࡿࠋ ATP-binding cassette G1㸦ABCG1㸧ࡣࠊ⣽⬊ෆࡢవ๫ࢥࣞࢫࢸ࣮ࣟࣝࢆATP ຍỈศゎ౫Ꮡⓗ࡟⣽⬊እ࡬࡜᤼ฟࡍࡿࡇ࡜࡛ࠊࢥࣞࢫࢸ࣮ࣟࣝᜏᖖᛶ࡟㔜せ࡞ᙺ

๭ࢆᯝࡓࡍࠋ᰾ෆ㌿෗ᅉᏊLiver X receptor㸦LXR㸧ࡣ࢜࢟ࢩࢫࢸ࣮ࣟࣝࢆࣜ࢞

ࣥࢻ࡜ࡍࡿࡇ࡜࠿ࡽࠊLXR άᛶࡣ⣽⬊ෆࢥࣞࢫࢸ࣮ࣟࣝ㔞࡜ṇࡢ┦㛵㛵ಀࡀ࠶

ࡿࠋࡑࡢࡓࡵࠊLXR άᛶࢆᣦᶆ࡜ࡍࡿࡇ࡜࡛⣽⬊ෆࢥࣞࢫࢸ࣮ࣟࣝ㔞ࢆ㛫᥋ⓗ

࡟ホ౯࡛ࡁࡿࠋABCG1 ࢆ HEK293⣽⬊࡟Ⓨ⌧ࡉࡏࡿ࡜⣽⬊ෆࢥࣞࢫࢸ࣮ࣟࣝ

ࡀ᤼ฟࡉࢀࡿࡓࡵࠊLXRάᛶࡣῶᑡࡋࡓࠋ⯆࿡῝࠸ࡇ࡜࡟ࠊATPຍỈศゎάᛶ

ࢆᣢࡓࡎࢥࣞࢫࢸ࣮ࣟࣝࢆ᤼ฟ࡛ࡁ࡞࠸ABCG1ኚ␗యࢆⓎ⌧ࡉࡏࡓሙྜࡶࠊྠ

ᵝ࡟LXRάᛶࡀ኱ࡁࡃῶᑡࡋࡓࠋࡇࢀࡼࡾࠊABCG1ࡀ⬡㉁᤼ฟ࡟౫Ꮡࡏࡎࠊ ࢥࣞࢫࢸ࣮ࣟࣝ⏕ྜᡂࢆᢚไࡍࡿࡇ࡜࡛⣽⬊ෆࢥࣞࢫࢸ࣮ࣟࣝ㔞ࢆㄪ⠇ࡋ࡚࠸

ࡿ࡜௬ㄝࢆ❧࡚ࠊࡇࡢ௬ㄝࢆ᳨ウࡍࡿࡇ࡜ࢆᮏ◊✲ࡢ┠ⓗ࡜ࡋࡓࠋ

࠙᪉ἲ࣭⤖ᯝࠚື≀⣽⬊࡟࠾ࡅࡿࢥࣞࢫࢸ࣮ࣟࣝ⏕ྜᡂࡣࠊHMG-CoA㑏ඖ㓝⣲

㸦HMGCR㸧࡟ࡼࡾ HMG-CoA ࡀ࣓ࣂࣟࣥ㓟࡬ኚ᥮ࡉࢀࡿ཯ᛂࡀᚊ㏿ẁ㝵࡜࡞

ࡿࠋࡑࡇ࡛ࠊABCG1ࡀࢥࣞࢫࢸ࣮ࣟࣝ⏕ྜᡂ࡟୚࠼ࡿᙳ㡪ࢆࠊHMGCRⓎ⌧㔞

࡜ࢥࣞࢫࢸ࣮ࣟࣝྜᡂ㔞ࢆ ᐃࡋHMGCRάᛶࢆ⟬ฟࡍࡿࡇ࡜࡛ホ౯ࡋࡓࠋࡑ ࡢ⤖ᯝࠊHEK293 ⣽⬊ࡢ ABCG1 ཬࡧ ABCG1 ኚ␗యᏳᐃⓎ⌧ᰴ࡟࠾࠸࡚ࠊ

HMGCRࡢάᛶࡀపୗࡋ࡚࠸ࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋ㏫࡟ࠊTHP-1࣐ࢡࣟࣇ

࢓࣮ࢪ⣽⬊࡟࠾࠸࡚ABCG1ࢆࣀࢵࢡࢲ࢘ࣥࡍࡿ࡜ࠊHMGCRάᛶࡀୖ᪼ࡋࡓࠋ ࡇࢀࡽࡢ⤖ᯝࡣࠊABCG1ࡀ⬡㉁᤼ฟ࡟౫Ꮡࡏࡎ࡟ HMGCR ࡢάᛶࢆไᚚࡍࡿ

ࡇ࡜ࢆ♧၀ࡋࡓࠋࡋ࠿ࡋࠊHMGCRࡢάᛶไᚚᶵᵓ࡜ࡋ࡚ሗ࿌ࡉࢀ࡚࠸ࡿAMPK

࡟ࡼࡿHMGCRࣜࣥ㓟໬ࣞ࣋ࣝࡣࠊABCG1Ⓨ⌧࡟ࡼࡾኚ໬ࡋ࡞࠿ࡗࡓࠋḟ࡟ࠊ

ABCG1ࡀHMGCR࡜┤᥋┦஫స⏝ࡍࡿࡇ࡜࡟ࡼࡾHMGCRάᛶࢆไᚚࡍࡿ࡜

࠸࠺ྍ⬟ᛶࢆ᳨ウࡍࡿࡓࡵࠊච␿ඹỿ㝆ᐇ㦂ࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊABCG1 ࡜ HMGCRࡀ┤᥋┦஫స⏝ࡍࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋ௨ୖࡼࡾࠊABCG1ࡀHMGCR

࡜┦஫స⏝ࡍࡿࡇ࡜࡛HMGCRࡢάᛶࢆᢚไࡍࡿ࡜࠸࠺ࠊ᪂つࡢHMGCRάᛶ ไᚚᶵᵓࡢᏑᅾࡀ♧၀ࡉࢀࡓࠋ

㸵

Procyanidin-B1-3,3"-di- O -gallate 䛿 EGCG 䛸␗䛺䜛

స⏝䛷 HeLa S3 ⣽⬊䛾ቑṪ䜢㜼ᐖ䛩䜛䠛

㻌

ۑ▼ཎἋஓຍࠊ⥤㔝⩏༤ࠊᒸᮏಟᖹࠊᅵ஭⩧㤿ࠊᒸᮏὈ㍜ࠊ㡲⸨㱟ᙪࠊ 㛗⏣⿱அࠊ୰ᓥ⠊⾜ࠊ㰺⸨Ᏻ㈗Ꮚ኱㜰㟁㏻኱㝔࣭ᕤࠊ኱㜰㟁㏻኱࣭

ᕤࠊ⌮◊ᇶᖿ◊࣭ࢣ࣑࢝ࣝࣂ࢖࢜ࣟࢪ࣮ࠊᐩᒣ┴኱࣭ᕤ

࠙┠ⓗࠚProanthocyanidin(PAs)ࡣࠊከࡃࡢ᳜≀࡟ྵ

ࡲࢀࡿ㧗ᶵ⬟ᛶ࣏ࣜࣇ࢙ࣀ࣮ࣝ໬ྜ≀࡛࠶ࡾࠊ࠾Ⲕࡸ

࢝࢝࢜ࡢႴዲရࠊࣈࢻ࢘➼ࡢᯝ≀㢮ࠊࢫࢠ➼ࡢᶞ⓶࡞

࡝࡟ྵࡲࢀ࡚࠸ࡿࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿࠋࡇࢀࡽࡣᴟࡵ

࡚ᙉ࠸ᢠ㓟໬άᛶ࣭ᢠ⅖⑕స⏝➼ࠊᵝࠎ࡞⏕≀άᛶࢆ

♧ࡍࡀࠊኳ↛࠿ࡽ㧗⣧ᗘࡢࢧࣥࣉࣝࢆᚓࡿࡇ࡜ࡣ㠀ᖖ

࡟ᅔ㞴࡛࠶ࡾࠊ⣔⤫ⓗ࡞ᵓ㐀ïάᛶ┦㛵ࡣ࡯࡜ࢇ࡝ゎ

᫂ࡉࢀ࡚࠸࡞࠸ࠋࡑࡢ୍᪉࡛ࠊ࠾Ⲕ࢝ࢸ࢟ࣥ࡜ࡋ࡚▱

ࡽ ࢀ ࡿ(-)-Epigallocatechin-3-O-gallate (EGCG) 11 ࡣࠊ⣽⬊⭷ୖࡢཷᐜయ࡜ࡋ࡚ྠᐃࡉࢀࡓ67 kDa࣑ࣛ

ࢽࣥࣞࢭࣉࢱ࣮ (67LR)ࢆ௓ࡋ࡚ᵝࠎ࡞⏕⌮άᛶࢆ♧

ࡍࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ¹ࠋࡉࡽ࡟ࠊEGCGࡣ67LR

ࢆ௓ࡍࡿ᝟ሗఏ㐩ࡢ㐣⛬࡛p38MAPKࢆྵࡴMAPK ࡢࣜࣥ㓟໬ࢆ㜼ᐖࡍࡿࡇ࡜ࡀሗ࿌ࡉࢀࡓ²ࠋᡃࠎࡣࠊ PAsࡢᢠ⅖⑕స⏝࡞࡝ࡢάᛶⓎ⌧ࡀEGCG࡜ྠᵝࡢ

⤒㊰࡟ࡼࡿࡶࡢ࠿ࠊ໬Ꮫⓗ࣭⏕≀Ꮫⓗ࡟ド᫂ࡍࡿࡓࡵ

◊✲ࢆ㐍ࡵ࡚࠸ࡿࠋ

࠙᪉ἲ࡜⤖ᯝࠚ ❧య㑅ᢥⓗྜᡂ࡟ࡼࡗ࡚⣧⢋࡟ᚓࡽ

ࢀࡓ✀ࠎࡢPAsࢆ⏝࠸࡚HeLa S3⣽⬊࡟ᑐࡍࡿቑṪᢚไάᛶヨ㦂ࢆ⾜ࡗࡓࠋ ࡑࡢ⤖ᯝࠊB1-3,3”-di-O-gallate (22)࡜B4-3,3”-di-O-gallate (33)ࡀࠊEGCGࡼࡾࡶ

㧗࠸ቑṪᢚไάᛶࢆ♧ࡋࡓࠋࡉࡽ࡟ࠊࡇࢀࡽࡢ໬ྜ≀ࡀ⣽⬊ෆ p38MAPK 㔞࡟

ᙳ㡪ࢆ୚࠼ࡿࡇ࡜ࡀ☜ㄆ࡛ࡁࡓࠋEGCG ࡛ࡣྠᵝ࡞⌧㇟ࡀぢࡽࢀ࡞࠸஦࠿ࡽࠊ EGCG ࡜ࡣ␗࡞ࡿᶵᵓ࡛άᛶࢆⓎ᥹ࡋ࡚࠸ࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋຍ࠼࡚ࠊ௚

ࡢᐁ⬟ᇶ࡛ಟ㣭ࡋࡓ໬ྜ≀࡟ࡘ࠸࡚ࡶ᳨ウࢆ⾜ࡗ࡚࠾ࡾࠊࡑࢀࡽࢆࡲ࡜ࡵ࡚ሗ࿌

ࡍࡿࠋ

ཧ⪃ᩥ⊩) 1. Tachibana H, et al., Nat Struct Mol Biol. 2004, 111, 380-381 2. Eui-Baek Byun, et al., Biochem Biophys Res Commun. 2012, 426, 480-485

O O HO

OH

OH OH OH

O OH

OH 1 OH

O

O HO

OH

OH OH

O OH

OH OH O

O HO

OH

OH OH

O OH

OH 2: R1= H, R2= G OH

3: R1= G, R2= H R2

R1

G =

㸶

䜲䝛䛻䛚䛡䜛䜶䝸䝅䝍䞊ㄏᑟᛶ௦ㅰ≀䛾ရ✀㛫ẚ㍑

ۑ⥙ᖸ㈗Ꮚࠊᯇᮏࡩ࠺㤶ࠊᑎ▼ᨻ⩏ࠊዟᮏ⿱ࠊす⏣ᚊኵࠊ᳃┤ᶞ 㸦ி኱㝔㎰࣭ᛂ⏝⏕࿨ࠊி኱㝔㎰࣭㎰Ꮫ㸧

࠙┠ⓗࠚ

⑓ཎ⳦ࡢឤᰁࡸ᳜㣗ᛶ᪻⹸ࡢ㣗ᐖ࠿ࡽ㌟ࢆᏲࡿࡓࡵ࡟ࠊ᳜≀ࡣᢠ⳦άᛶࡸẘ ᛶࠊᚷ㑊άᛶࡢ࠶ࡿ໬ྜ≀࡞࡝ࢆ⏕ྜᡂࡋࠊ⵳✚ࡋ࡚࠸ࡿࠋ㏆ᖺࠊ᳜㣗ᛶ᪻⹸ࡢ 㣗ᐖࡸ⏘༸࡟ࡼࡾㄏᑟࡉࢀࡿ᳜≀ࡢ௦ㅰ≀ࡀ᢬ᢠᛶࢆ♧ࡍ⌧㇟ࡀሗ࿌ࡉࢀ࡚࠸

ࡿࠋ⯆࿡῝࠸ࡇ࡜࡟ࠊᩘ✀ࡢ᪻⹸ࡢྤࡁฟࡋᾮ㸦⭠⟶ෆᐜ≀㸧୰࠿ࡽࠊࢺ࢘ࣔࣟ

ࢥࢩࠊࢱࣂࢥࡸࢲ࢖ࢬ࡟᥹Ⓨᡂศࢆᨺฟࡉࡏࡿ࢚ࣜࢩࢱ࣮ࡀྠᐃࡉࢀ࡚࠸ࡿࠋᮏ

◊✲࡛ࡣࠊ㫣⩟┠ᗂ⹸ࡢ⭠⟶ෆᐜ≀ࡸയᐖᛂ⟅࡟㛵ࢃࡿࢪࣕࢫࣔࣥ㓟ฎ⌮࡟ࡼࡾ

ㄏᑟࡉࢀࡿ࢖ࢿ୙᥹Ⓨᛶ௦ㅰ≀࡟ὀ┠ࡋࡓࠋࡲࡓࠊࡑࡢရ✀㛫࡟࠾ࡅࡿᛂ⟅ࡢẚ

㍑࠿ࡽࠊᐖ⹸ࡢ㣗ᐖࢆ㜵ࡄㄏᑟᛶ௦ㅰ≀ࡢ⏕ྜᡂ⤒㊰ࡢゎ᫂ࡸࡑࡢ⏕ྜᡂ࡟㛵ࢃ

ࡿ㑇ఏᏊࡢྠᐃࢆ┠ᣦࡋ࡚࠸ࡿࠋ

࠙᪉ἲ࣭⤖ᯝࠚ

᳜≀࠿ࡽࡣࢱࣥࣃࢡ㉁ࢆᵓᡂࡍࡿ 20 ✀ࡢ࢔࣑ࣀ㓟ࡢ௚ࠊከࡃࡢ㠀ࢱࣥࣃࢡᛶ

࢔࣑ࣀ㓟ࡀྠᐃࡉࢀ࡚࠾ࡾࠊ୍㒊ࡣ᳜㣗⪅࡟ᑐࡋ࡚ẘᛶࡸᡂ㛗㜼ᐖάᛶࡀሗ࿌ࡉ

ࢀ࡚࠸ࡿࠋࡑࡇ࡛ࡲࡎࠊ࢚ࣜࢩࢱ࣮ฎ⌮᫬ࡢ࢖ࢿᗂⱑ࡟࠾ࡅࡿ࢔࣑ࣀ㓟ࡢኚືࢆ

ㄪ࡭ࡓࠋ

࢖ࢿᗂⱑ (4 ⴥ᏶඲ᒎ㛤ᮇ) ࡢⴥ࡟✰ࢆ㛤ࡅࠊ࢔࣡ࣚࢺ࢘ᗂ⹸⭠⟶ෆᐜ≀ࡢ⦆

⾪ ᾮ ᢳ ฟ ᾮ ࢆ ሬ ᕸ ࡋ ࠊ2ࠊ4ࠊ24ࠊ48 ᫬ 㛫 ᚋ ࡟ ࢔ ࣑ ࣀ 㓟 ࢆ ᢳ ฟ ࡋ ࠊ 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate ࡛ㄏᑟయᚋࠊLC/MS ࡟౪ヨࡋ ࡓࠋ࢖ࢿࡢရ✀ࡣ᪥ᮏᬕࠊ㖟ᆓ୺ࠊkasalath ࢆ⏝࠸ࡓࠋ᪥ᮏᬕ࡛ࡣ⭠⟶ෆᐜ≀ᢳ ฟᾮሬᕸ 2 ᪥ᚋࠊJ-aminobutylic acid (GABA) ࡀቑຍࡋࡓࡀࠊ㖟ᆓ୺ࠊkasalath ࡛ ࡣቑຍࡏࡎࠊရ✀㛫࡟ࡼࡿᛂ⟅࡟ᕪࡀ࠶ࡗࡓࠋ᪥ᮏᬕ࡛ࡣ GABA ࡢ๓㥑య࡛࠶

ࡿ ࢢ ࣝ ࢱ ࣑ ࣥ 㓟 ࡀ ῶ ᑡ ࡋ ࡚ ࠾ ࡾ ࠊ ⭠ ⟶ ෆ ᐜ ≀ ᢳ ฟ ᾮ ሬ ᕸ ࡟ ࡼ ࡾ glutamine decarboxylase ࡢάᛶ໬ࡀண᝿ࡉࢀࡓࠋ

୍᪉ࠊᗂⱑ࡟ࢪࣕࢫࣔࣥ㓟ࢆሬᕸࡍࡿ࡜ࠊkasalath ࡟࠾࠸࡚ GABA ࡢቑຍࡀ

☜ㄆࡉࢀࠊฎ⌮᪉ἲ࡟ࡼࡗ࡚ࡶရ✀㛫ࡢᛂ⟅࡟ᕪࡀ⏕ࡌࡿࡇ࡜ࡀࢃ࠿ࡗࡓࠋ

㸷

㓝ẕ Saccharomyces cerevisiae 䛻䛚䛡䜛䝨䝹䜸䜻䝅 䝋䞊䝮ෆ䝺䝗䝑䜽䝇ไᚚᅉᏊ䛾ゎᯒ㻌

㻌

ۑᓥ୪♸Ꮚࠊዟබ⚽ࠊᑉ㛵῟ࠊ㜰஭ᗣ⬟㸦ி኱㝔㎰࣭ᛂ⏝⏕࿨ࠊி

኱Ꮫ㝿⼥ྜ࣭⏕⌮໬Ꮫ㸧

࠙┠ⓗࠚ࣌ࣝ࢜࢟ࢩࢯ࣮࣒ࡣ࢜ࣞ࢖ࣥ㓟࡞࡝ࡢ⬡⫫㓟ࡢș㓟໬ࢆ⾜࠺⣽⬊ᑠჾᐁ

࡛࠶ࡿࠋ࢜ࣞ࢖ࣥ㓟ࢆ༢୍ࡢⅣ⣲※࡜ࡋࡓᇵᆅ࡛㓝ẕࡢᇵ㣴ࢆ⾜࠺࡜ࠊ࣌ࣝ࢜࢟

ࢩࢯ࣮࣒ෆ࡛⬡⫫㓟ࡢ௦ㅰࡀ⾜ࢃࢀ๪⏘≀࡜ࡋ࡚άᛶ㓟⣲ࡢ୍✀࡛࠶ࡿH2O2ࡀ

Ⓨ⏕ࡍࡿࠋάᛶ㓟⣲ࡣ㠀ᖖ࡟཯ᛂᛶࡀ㧗ࡃࠊࢱࣥࣃࢡ㉁ࡢኚᛶࡸDNAࡢᦆയࢆ

ᘬࡁ㉳ࡇࡍ࡞࡝⣽⬊࡟ᐖࢆ୚࠼ࡿࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿࠋࡑࡢࡓࡵ⏕≀࡟ࡣάᛶ㓟

⣲ࢆ↓ẘ໬ࡍࡿᢠ㓟໬㓝⣲ࢆ⏘⏕ࡋࠊ⣽⬊ෆࡢࣞࢻࢵࢡࢫ≧ែࢆ୍ᐃ࡟ಖࡘᶵ⬟

ࡀഛࢃࡗ࡚࠸ࡿࠋࡋ࠿ࡋࠊ⏕໬Ꮫⓗゎᯒࡢ㞴ࡋࡉ࠿ࡽ⣽⬊ᑠჾᐁෆ࡟࠾ࡅࡿࣞࢻ

ࢵࢡࢫไᚚࢩࢫࢸ࣒࡟ࡘ࠸࡚ヲࡋ࠸ࡇ࡜ࡣᮍࡔゎ᫂ࡉࢀ࡚࠸࡞࠸ࠋ

ࡑࡇ࡛ᮏ◊✲࡛ࡣࠊ⏕ࡁࡓ⣽⬊ෆࡢ㓟໬㑏ඖ≧ែࢆྍど໬࡛ࡁࡿ⺯ගࢱࣥࣃࢡ㉁

ࣉ࣮ࣟࣈ࡛࠶ࡿRedoxfluorࢆ㓝ẕ࣌ࣝ࢜࢟ࢩࢯ࣮࣒ෆ࡟Ⓨ⌧ࡉࡏࠊ࣌ࣝ࢜࢟ࢩ ࢯ࣮࣒ෆࡢࣞࢻࢵࢡࢫไᚚ࡟㛵ࢃࡿᅉᏊࡢゎᯒࢆ⾜ࡗ࡚࠸ࡿࠋ

࠙᪉ἲ࣭⤖ᯝࠚ࣌ࣝ࢜࢟ࢩࢯ࣮࣒ෆ࡛ാࡃ࢝ࢱ࣮ࣛࢮ࡛࠶ࡿ CTA1 㑇ఏᏊ◚ቯ

ᰴࠊࣞࢻࢵࢡࢫไᚚ࡟㔜せ࡞ാࡁࢆ⾜ࡗ࡚࠸ࡿศᏊࡢ1ࡘ࡛࠶ࡿTRX2㑇ఏᏊ

◚ቯᰴࠊCTA1TRX2ࡢ஧㔜◚ቯᰴࢆసᡂࡋࡓࠋࡇࢀࡽࡢ㑇ఏᏊ◚ቯᰴ࡟ࡘ࠸࡚

࢜ࣞ࢖ࣥ㓟ࢆ༢୍ࡢⅣ⣲※࡜ࡍࡿᇵᆅ࡛⏕⫱ࢆ⾜ࡗࡓ࡜ࡇࢁࠊCTA1㑇ఏᏊ◚ቯ

ᰴ࡛ࡣ⏕⫱ᗘࡢపୗࡀぢࡽࢀCTA1TRX2஧㔜◚ቯᰴ࡛ࡣࡉࡽ࡟⏕⫱ᗘࡀపୗࡍ

ࡿ࡜࠸࠺⤖ᯝࡀᚓࡽࢀࡓࠋࡇࢀࡽࡢᰴࡢ࣌ࣝ࢜࢟ࢩࢯ࣮࣒ෆࣞࢻࢵࢡࢫ≧ែࢆㄪ

࡭ࡿࡓࡵࠊ࣌ࣝ࢜࢟ࢩࢯ࣮࣒࡬ࡢ⛣⾜ࢩࢢࢼࣝ㓄ิࢆ௜ࡅࡓRedoxfluorࢆࡑࢀ

ࡒࢀࡢᰴ࡟Ⓨ⌧ࡉࡏ⺯ග㢧ᚤ㙾ほᐹࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝ CTA1 ^{◚ቯᰴ࡟࠾࠸࡚}

⺯ගᙉᗘࡢపୗࡀぢࡽࢀࠊCTA1TRX2஧㔜◚ቯᰴ࡛ࡣ᭦࡟ࡑࡢపୗࡀ㢧ⴭ࡟ぢ

ࡽࢀࡓࠋࡇࡢ᫬ࡢ࣌ࣝ࢜࢟ࢩࢯ࣮࣒ෆ࡟࠾ࡅࡿRedoxfluorࡢⓎ⌧㔞ࢆㄪ࡭ࡿࡓ

ࡵ⣽⬊ᢳฟᾮࢆ㐲ᚰศ㞳࡟ࡼࡗ࡚⣽⬊㉁⏬ศ࡜࢜ࣝ࢞ࢿࣛ⏬ศ࡟ศ⏬ࡋࠊ࢜ࣝ࢞

ࢿࣛ⏬ศ࡟ྵࡲࢀࡿRedoxfluor㔞ࡢẚ㍑ࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊCTA1TRX2^஧㔜

◚ቯᰴ࡟㛵ࡋ࡚ࡣ㔝⏕ᰴ࡜ẚ㍑ࡋ࡚࣌ࣝ࢜࢟ࢩࢯ࣮࣒ෆࡢRedoxfluor㔞ࡢῶᑡ ࡀ࠶ࡲࡾぢࡽࢀ࡞࠿ࡗࡓࠋࡇࡢࡇ࡜࠿ࡽCTA1TRX2㑇ఏᏊࡢ◚ቯࡀ࣌ࣝ࢜࢟ࢩ

ࢯ࣮࣒ෆ Redoxfluor ࡢ⺯ගࢱࣥࣃࢡ㉁ࡢᶵ⬟పୗࢆᘬࡁ㉳ࡇࡍࡇ࡜ࡀศ࠿ࡗ

ࡓࠋ

㸯㸮 㻌

Improvement in mitochondrial function by

antioxidants protects cellular oxidation caused by proteasome inhibition

Sunita Maharjan¹, Jun Hoseki^1,2, Masahide Oku¹, Yasuyoshi Sakai^1,2 (¹Division of Applied Life Sciences, Graduate School of Agriculture and

2Research Unit for Physiological Chemistry, the Center for the Promotion of Interdisciplinary Education and Research, Kyoto University)

࠙ObjectiveࠚUbiquitin proteasome system is essential for multiple physiological processes via selective degradation of target proteins. Proteasome impairment is linked to aging and many pathological conditions from cancer to neurodegenerative disorders like Alzheimer’s and Parkinson’s disease. We found that proteasome inhibition resulted in cellular oxidation. In this study, we aimed to investigate molecular mechanisms underlying protective effects of several antioxidant compounds from food against cellular oxidation caused by proteasome inhibition.

࠙Methods and ResultsࠚIntracellular redox state was observed with Redoxfluor following treatment with a proteasome inhibitor in mammalian cells, stably transfected with the redox probe. Redoxfluor detects intracellular redox state by its fluorescence resonance energy transfer (FRET) efficiency with a concomitant redox-dependent structural change of the protein. The proteasome inhibition induced cellular oxidation.

Simultaneous treatment with antioxidants in food prevented the cellular oxidation caused by proteasome inhibition and ultimately improved survival rate of cells under the proteasomal inhibition. Furthermore, treatment with the antioxidants maintained mitochondrial membrane potential and significantly reduced radioactive oxygen species (ROS) in mitochondria by using mitochondrial targeted molecular probes. Moreover, pretreatment with the antioxidants was found to significantly increase protein level of a mitochondrial antioxidative enzyme suggesting that the pretreatment of the antioxidants might stimulate a relevant signaling pathway(s) which subsequently increases antioxidant capacity in cells. The results suggest that improvement in mitochondrial function by the antioxidants prevents cellular oxidation caused by proteasome inhibition.

㸯㸯

㓝ẕ Saccharomyces cerevisiae 䛻䛚䛡䜛⬡⫫⁲ศゎ 䛾䛯䜑䛾䜸䞊䝖䝣䜯䝆䞊⤒㊰䛾ᶵ⬟ゎᯒ㻌

㻌

ۑᒣ⏣㯞⾰ࠊዟබ⚽ࠊ㜰஭ᗣ⬟㸦ி኱㝔㎰࣭ᛂ⏝⏕࿨㸧

࠙┠ⓗࠚ⬡⫫⁲ࡣ┿᰾⣽⬊࡟ᗈࡃಖᏑࡉࢀࡓ࢚ࢿࣝࢠ࣮㈓ⶶჾᐁ࡛࠶ࡾࠊࢺࣜ࢔

ࢩࣝࢢࣜࢭ࣮ࣟࣝࡸࢫࢸ࣮࢚ࣟࣝࢫࢸࣝ࡞࡝ࡢ୰ᛶ⬡㉁ࡀࣜࣥ⬡㉁୍㔜⭷࡟そ

ࢃࢀࡓᵓ㐀ࢆࡶࡘࠋ⌧ᅾࠊ⬡⫫⁲ࡢศゎᶵᵓࡢ୍ࡘ࡜ࡋ࡚ࠊ࣮࢜ࢺࣇ࢓ࢪ࣮ࡢᏑ ᅾࡀ♧၀ࡉࢀ࡚࠸ࡿࠋ࣮࢜ࢺࣇ࢓ࢪ࣮࡜ࡣࠊ⣽⬊㉁ᡂศࢆศゎࢥࣥࣃ࣮ࢺ࣓ࣥࢺ 㸦ᾮ⬊㸭ࣜࢯࢯ࣮࣒㸧࡬㍺㏦ࡍࡿศゎ⣔࡛࠶ࡾࠊ࣐ࢡ࣮ࣟ࢜ࢺࣇ࢓ࢪ࣮࡜࣑ࢡࣟ

࣮࢜ࢺࣇ࢓ࢪ࣮ࡢ㸰✀㢮ࡢᶵᵓࡀᏑᅾࡍࡿࠋ๓⪅࡛ࡣࠊศゎᑐ㇟ࢆ᪂⏕⭷ࡀໟࡳ

㎸ࡳࠊᙧᡂࡉࢀࡓ࣮࢜ࢺࣇ࢓ࢦࢯ࣮࣒࡜࿧ࡤࢀࡿᵓ㐀యࡀࠊᾮ⬊⭷࡜⼥ྜࡍࡿࡇ

࡜࡛ᾮ⬊ෆ࡬ᨺฟࡍࡿࠋ୍᪉ࠊᚋ⪅࡛ࡣᾮ⬊⭷ࡢ㝗ධ࡟ࡼࡾᑐ㇟ࢆ┤᥋ໟࡳ㎸ࡴࠋ

࣮࢜ࢺࣇ࢓ࢪ࣮ࡣ୍⯡ⓗ࡟㠀㑅ᢥⓗ࡞ࢱࣥࣃࢡ㉁ศゎ⤒㊰࡜ࡋ࡚▱ࡽࢀࡿࡀࠊ࣌

ࣝ࢜࢟ࢩࢯ࣮࣒➼ࡢ࢜ࣝ࢞ࢿࣛ≉␗ⓗ࡞ࡶࡢࡶ࠶ࡾࠊ့ங㢮࡟࠾࠸࡚⬡⫫⁲㑅ᢥ

ⓗ࡞ࠕ࣏ࣜࣇ࢓ࢪ࣮ࠖࡀᥦၐࡉࢀ࡚࠸ࡿࠋࡋ࠿ࡋ㓝ẕ࡟࠾࠸࡚ࡣࡑࡢᏑᅾࡸ࢔ࢵ

ࢭ࢖⣔ࡣ☜❧ࡉࢀ࡚࠸࡞࠸ࠋࡑࡇ࡛ᮏ◊✲࡛ࡣࠊฟⱆ㓝ẕࢆ⏝࠸࡚࣏ࣜࣇ࢓ࢪ࣮

ࡢᐃ㔞ⓗ࡞࢔ࢵࢭ࢖⣔ࢆᵓ⠏ࡋࠊ࣏ࣜࣇ࢓ࢪ࣮࡟ᶵ⬟ࡍࡿ㑇ఏᏊࡢ᥈⣴࠾ࡼࡧゎ ᯒࢆ⾜࠺ࡇ࡜ࢆ┠ⓗ࡜ࡋࡓࠋ

࠙᪉ἲ࣭⤖ᯝࠚ⬡⫫⁲ࡢᾮ⬊ෆ㍺㏦࠾ࡼࡧศゎࢆ᳨ฟࡍࡿࡓࡵࠊ⥳Ⰽ⺯ගࢱࣥࣃ

ࢡ㉁ GFP ࡢᾮ⬊ෆ࡛ࡢᏳᐃᛶࢆ฼⏝ࡋࡓࣉࣟࢭࢵࢩࣥࢢ࢔ࢵࢭ࢖⣔ࢆᵓ⠏ࡋ ࡓࠋࡲࡎࠊ⬡⫫⁲ᒁᅾࢱࣥࣃࢡ㉁࡜GFPࡢ⼥ྜࢱࣥࣃࢡ㉁ࢆⓎ⌧ࡍࡿ㓝ẕᰴࢆ

స〇ࡋࡓࠋ⼥ྜࢱࣥࣃࢡ㉁Ⓨ⌧ᰴ࠿ࡽᢳฟࡋࡓࢱࣥࣃࢡ㉁ࢆศ㞳ࡋGFPᢠయࢆ

⏝࠸ࡓ࢙࢘ࢫࢱࣥࣈࣟࢵࢸ࢕ࣥࢢࢆ⾜ࡗࡓ࡜ࡇࢁࠊ㔝⏕ᰴ࡛ࡣ⼥ྜࢱࣥࣃࢡ㉁ࡢ ศゎ⏘≀࡛࠶ࡿ㐟㞳ᆺGFPࡀ᳨ฟࡉࢀࡓࠋ୍᪉ࠊᾮ⬊ෆศゎࡢḞᦆࢆ♧ࡍ㑇ఏ Ꮚ◚ቯᰴ࡛ࡣ㐟㞳ᆺGFPࡣ᳨ฟࡉࢀ࡞࠿ࡗࡓࠋࡲࡓ⺯ග㢧ᚤ㙾࡟ࡼࡾࠊᾮ⬊ෆ ศゎḞᦆᰴ࡟࠾࠸࡚GFPࡢᾮ⬊ෆᣑᩓࡀほᐹࡉࢀࡓࡇ࡜࠿ࡽࠊ⬡⫫⁲ࡀ࣮࢜ࢺ

ࣇ࢓ࢪ࣮࡟ࡼࡾᾮ⬊ෆ࡟㍺㏦ࡉࢀࡓᚋࠊศゎࢆཷࡅࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋ ࡉࡽ࡟ࠊࣉࣟࢭࢵࢩࣥࢢ࢔ࢵࢭ࢖࡟ࡼࡾࠊࣜࣥ⬡㉁PI3P㸦phosphatidylinositol 3-phosphate㸧ࢆ⏘⏕ࡍࡿࢱࣥࣃࢡ㉁ࡸPI3P࡟⤖ྜࡍࡿࢱࣥࣃࢡ㉁ࡀ࣏ࣜࣇ࢓

ࢪ࣮࡟㛵୚ࡋ࡚࠸ࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋ

㸯㸰

ᅛ┦㻙Ẽ┦䝞䜲䜸䝣䜱䝹䝮ᇵ㣴䛜኱⭠⳦䛾 persister cell ᙧᡂ䛻ཬ䜌䛩ᙳ㡪

㻌

ۑᐑୖἋ㈗ࠊᮡᾆ༓᫂ࠊୡཱྀྂṌ⳹ࠊ๓⏣⣧ኵ㸦ዉⰋዪᏊ኱Ꮫ኱Ꮫ 㝔 㣗ᰤᑓᨷࠊዉⰋዪᏊ኱Ꮫ 㣗≀ᰤ㣴Ꮫ⛉㸧

࠙┠ⓗࠚ㏆ᖺࠊ⣽⳦࡟࠾࠸࡚persister cell࡜࿧ࡤࢀࡿᢠ⏕≀㉁⪏ᛶ⣽⬊ࡢᏑᅾ ࡀὀ┠ࡉࢀ࡚࠸ࡿࠋpersister cell ࡜ࡣ⣽⳦㞟ᅋ୰ࡢ୍㒊ࡢ⣽⬊ࡀ୍᫬ⓗ࡟ఇ╀

≧ែ࡜࡞ࡾᢠ⏕≀㉁⪏ᛶࢆ♧ࡍࡼ࠺࡟࡞ࡗࡓࡶࡢ࡛࠶ࡿࠋpersister cell ࡣኚ␗

ࡸ㑇ఏᏊࡢ⋓ᚓ࡞࡝ࡢ㑇ఏⓗኚ໬ࢆకࢃ࡞࠸➨୕ࡢᢠ⏕≀㉁⪏ᛶ⣽⬊࡜ࡋ࡚ࠊ⣽

⳦ ⏕ ⌮ ࠾ ࡼ ࡧ ⾨ ⏕ ⟶ ⌮ ࡢ ほ Ⅼ ࠿ ࡽ ࡶ ࡑ ࡢ 㔜 せ ᛶ ࡀ ㄆ ㆑ ࡉ ࢀ ࡘ ࡘ ࠶ ࡿ^>@ࠋ persister cell ࡣẕ㞟ᅋ࡟ᑐࡍࡿᏑᅾ๭ྜࡢᑠࡉࡉ࠿ࡽࠊࡇࢀࡲ࡛༑ศ◊✲ࡀ㐍

ࢇ࡛࠸࡞࠿ࡗࡓࠋࡋ࠿ࡋ㏆ᖺࠊpersister cell ᙧᡂࡀ㑇ఏⓗ࡟ࣉࣟࢢ࣒ࣛࡉࢀࡓ

⌧㇟࡛࠶ࡿࡇ࡜ࢆ♧၀ࡍࡿሗ࿌ࡀ┦ḟ࠸࡛࡞ࡉࢀࠊࡑࡢ⌧㇟ࡢヲ⣽࠾ࡼࡧᶵᵓࡢ ゎᯒࡀ◊✲ࡢ↔Ⅼ࡜࡞ࡗ࡚ࡁ࡚࠸ࡿࠋࡑࡇ࡛ᮏ◊✲࡛ࡣࠊ᪂ࡓ࡞ほⅬ࠿ࡽ

persister cell ᙧᡂࢆゎᯒࡍࡿࡓࡵࠊ୍⯡ⓗ࡞◊✲࡛⏝࠸ࡽࢀ࡚࠸ࡿᾮయᇵ㣴࡛

ࡣ࡞ࡃࠊᅛ┦Ẽ┦ࣂ࢖࢜ࣇ࢕࣒ࣝᇵ㣴࡟╔┠ࡋࠊࡑࡢ᮲௳ୗ࡛ࡢ኱⭠⳦ࡢ persister cellᙧᡂࡢヲ⣽ゎ᫂ࢆヨࡳࡓࠋ

࠙᪉ἲ࣭⤖ᯝࠚ኱⭠⳦ࢆᢠ⏕≀㉁↓ῧຍࡢLBᇵᆅ࡛ Υࠊ ᫬㛫ࡢᾮయᇵ㣴

࠶ࡿ࠸ࡣᅛ┦Ẽ┦ࣂ࢖࢜ࣇ࢕࣒ࣝᇵ㣴ᚋࠊࡑࢀࡒࢀࡢ⣽⬊ࢆᢠ⏕≀㉁ῧຍ LB ᇵᆅ࡟ᠱ⃮ࡋࠊΥࠊѸ ᫬㛫ࡢฎ⌮ࢆ⾜ࡗࡓࠋฎ⌮ᚋࡢ⣽⬊ࢆᢠ⏕≀㉁↓ῧ ຍࡢLBᐮኳୖ࡟ࣉ࣮ࣞࢸ࢕ࣥࢢࡋࠊ⏕ṧ⣽⬊㸦 persister cell㸧ᩘࢆ ᐃࡋࡓࠋ

⏕ṧ⣽⬊࠿ࡽ⏕ࡌࡓࢥࣟࢽ࣮ࡣᢠ⏕≀㉁ࣉ࣮ࣞࢺ࡟෌ࢫࢺ࣮ࣜࢡࡋࠊᜏஂⓗ࡞⪏

ᛶ⋓ᚓኚ␗ࡀ㉳ࡇࡗ࡚࠸࡞࠸ࡇ࡜ࢆ☜ㄆࡋࡓࠋྛ✀᮲௳᳨ウࡢ⤖ᯝࠊᅛ┦Ẽ┦

ࣂ࢖࢜ࣇ࢕࣒ࣝᇵ㣴ᚋࡢ⣽⬊࡛ࡣࠊᢠ⏕≀㉁ฎ⌮ࡢ≉ᐃ᮲௳ୗ᳨࡛ฟࡉࢀࡿ

persister cellࡀࠊᾮయᇵ㣴ᚋࡢ⣽⬊࡜ẚ࡭᭱኱ ಸࡶࡢ㧗࠸㢖ᗘ࡛ฟ⌧ࡍࡿࡇ

࡜ࢆ᫂ࡽ࠿࡟ࡋࡓࠋࡇࡢഴྥࡣస⏝ᵝᘧࡢ␗࡞ࡿ࡯࡜ࢇ࡝ࡢᢠ⏕≀㉁࡟࠾࠸࡚ࡶ

ྠᵝ࡛࠶ࡾࠊᢠ⏕≀㉁ࡢ✀㢮࡟≉␗ⓗ࡞⪏ᛶᶵᵓࡢⓎ⌧࡛ࡣ࡞࠸ࡇ࡜ࡀ♧၀ࡉࢀ

ࡓࠋࡲࡓୖグ࡜ྠᵝࡢ≉ᐃ᮲௳ୗ࡛ࡢᢠ⏕≀㉁ฎ⌮᫬㛫ࡢ᳨ウࢆ⾜ࡗࡓ࡜ࡇࢁࠊ ᾮయᇵ㣴ᚋࡢ⣽⬊࡛ࡣ ᫬㛫௨ෆ࡟⏕⣽⬊ࡀᾘኻࡍࡿࡢ࡟ᑐࡋࠊᅛ┦Ẽ┦ࣂ

࢖࢜ࣇ࢕࣒ࣝᚋࡢ⣽⬊࡛ࡣ ᫬㛫ᚋࡶ㧗࠸๭ྜ࡛persister cellࡀᏑᅾࡋ⥆ࡅ

ࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋࡇࡢ⤖ᯝ࠿ࡽࠊ ᫬㛫ࡢᅛ┦Ẽ┦ࣂ࢖࢜ࣇ࢕࣒ࣝᇵ 㣴࡟ࡼࡗ࡚ࠊࡑࡢᚋࠊ⣽⬊ࡀᾮయᾋ㐟≧ែ࡟࡞ࡗ࡚ࡶ༑᪥㛫௨ୖ࡟ࢃࡓࡗ࡚ಖᣢ ࡉࢀࡿ㠀㑇ఏⓗ࡞⏕⌮ኚ໬ࡀ⏕ࡎࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋ

[1] Keren, I. et al. FEMS Microbiol lett. 230, 13-18. 2004 [2] Kim, L. Annual Review of Microbiology. 64, 357-372. 2010

㸯㸱

኱⭠⳦ᶆ‽㔝⏕ᰴ䛻䛚䛡䜛䝣䜯䞊䝆⏘⏕ᰴ䛾⥙⨶ⓗ

᥈⣴

㻌

ۑᰘ⏣᭷ຍࠊ㧘ᶫᮥዉࠊ㬼ஂ᳃༓㔛ࠊୡཱྀྂṌ⳹ࠊ๓⏣⣧ኵ㸦ዉⰋ ዪᏊ኱㝔ே㛫ᩥ໬࣭㣗≀ᰤ㣴ࠊዉⰋዪᏊ኱⏕ά⎔ቃ࣭㣗≀ᰤ㣴㸧

㻌

࠙┠ⓗࠚ㏆ᖺࠊୡ⏺ྛᅜ࡛༢㞳ࡉࢀ࡚࠸ࡿ✀ࠎࡢ⑓ཎᛶ኱⭠⳦ᰴ࡛ࡣࠊ㑇ఏᏊ⩌

ࡢከ㔜⋓ᚓ࡟ࡼࡿ㧗⑓ཎᛶ໬ࡸከ๣⪏ᛶ໬ࡀၥ㢟࡜࡞ࡗ࡚࠸ࡿࠋࡇ࠺ࡋࡓ㑇ఏᏊ

⩌ࡢ⋓ᚓࡣࠊ⳦࠿ࡽ⳦࡬ࡢ㑇ఏᏊࡢỈᖹఏ᧛࡟ࡼࡗ࡚⾜ࢃࢀ࡚࠾ࡾࠊࡑࡢ୺せᶵ ᵓࡢ୍ࡘ࡟ࠊࣇ࢓࣮ࢪ࡟ࡼࡿᙧ㉁ᑟධࡀ࠶ࡿࠋࡋ࠿ࡋࣇ࢓࣮ࢪ࡟ࡼࡿ㑇ఏᏊỈᖹ ఏ᧛ࡀࠊᐇ㝿ࡢ⎔ቃ୰࡛࡝ࡢࡼ࠺࡟㉳ࡇࡿࡢ࠿࡜࠸࠺Ⅼ࡟ࡘ࠸࡚ࡣࠊ༑ศ࡞◊✲

ࡀ࡞ࡉࢀ࡚࠸࡞࠸ࠋࡑࡇ࡛ᡃࠎࡣ⎔ቃ୰࡛ࡢ࢖࣋ࣥࢺࢆࡼࡾᛅᐇ࡟཯࡛ᫎࡁࡿࣔ

ࢹࣝᐇ㦂⣔ࢆᵓ⠏ࡍࡿࡓࡵ࡟ࠊ኱⭠⳦ࡢᶆ‽㔝⏕ᰴࢥࣞࢡࢩ࡛ࣙࣥ࠶ࡿ ECOR (E. coli Collection of Reference) 㸵㸰ᰴ¹⁾ ࡢ฼⏝ࢆ⪃࠼ࡓࠋECORᰴࡢࣇ࢓࣮

ࢪಖ᭷࣭⏘⏕࡟㛵ࡋ࡚ࡣࠊ㐣ཤ஧ࠊ୕ࡢሗ࿌౛ ^{2), 3)} ࡣ࠶ࡿࡶࡢࡢᮍࡔ༑ศ࡞ゎ

᫂ࡣ࡞ࡉࢀ࡚࠸࡞࠸ࠋᮏ◊✲࡛ࡣࠊECOR ᰴࢆ⏝࠸ࡓᙧ㉁ᑟධࣔࢹࣝᐇ㦂⣔ࢆ

ᵓ⠏ࡍࡿࡓࡵࡢ➨୍ẁ㝵࡜ࡋ࡚ࠊECOR ᰴ୰ࡢࣇ࢓࣮ࢪಖ᭷࣭⏘⏕ᰴࡢ⥙⨶ⓗ

᥈⣴ࢆヨࡳࡓࠋ

࠙᪉ἲࠚྛECORᰴࢆMitomycin Cῧຍ࡟ࡼࡿSOSᛂ⟅ㄏᑟ࠾ࡼࡧ↓ฎ⌮ࡢ 㠀ㄏᑟࡢ2ࡘࡢ᮲௳ୗ࡛ᇵ㣴ᚋࠊ㐲ᚰศ㞳࡜ࣇ࢕ࣝࢱ࣮⁛⳦࠶ࡿ࠸ࡣࢡࣟࣟ࣍ࣝ

࣒ฎ⌮࡟ࡼࡾୖΎࢆㄪ〇ࡋࡓࠋࡇࢀࡽࡢୖΎࢆูࡢᐇ㦂ᐊᰴ࡟ῧຍࡋࠊࢫ࣏ࢵࢺ

࢔ࢵࢭ࢖࠾ࡼࡧࣉ࣮ࣛࢡ࢔ࢵࢭ࢖࡟ࡼࡾᨺฟࣇ࢓࣮ࢪࡢ᳨ฟࢆ⾜ࡗࡓࠋ

࠙⤖ᯝࠚୖグ᥈⣴ࡢ⤖ᯝ࠿ࡽࠊECOR㸵㸰ᰴ࠿ࡽィ㸱㸴ᰴࡢࣇ࢓࣮ࢪ⏘⏕ᰴࢆ≉

ᐃࡋࡓࠋࡇࡢෆࠊ㸯㸲ᰴࡣࡇࢀࡲ࡛࡟ࣇ࢓࣮ࢪಖ᭷࣭⏘⏕ࡢሗ࿌ࡢ࡞࠸ᰴ࡛࠶ࡗ ࡓࠋ⌧ᅾࠊࡇࢀࡽࣇ࢓࣮ࢪࡢ༢㞳࣭ྠᐃࢆ㐍ࡵ࡚࠸ࡿࠋ

1) Ochman, H., and Selander, R. K. 1984. J. Bacteriol. 157: 690-693.

2) Riley, M. A., and Gordon D. M. 1992. J. Gen. Microbiol. 138: 1345-1352 3) Nilsson, A. S., Karlsson, J. L., and Haggard-Ljungquist, E.

2004. Mol. Biol. Evol. 21 : 1-13

㸯㸲

㔝⏕኱⭠⳦ᰴ䜢⏝䛔䛯㑇ఏᏊỈᖹఏ᧛䛾ゎᯒ 䇲䝰䝕䝹⣔䛾☜❧䛸㣗ရᡂศ䛾ᙳ㡪ホ౯

㻌

ۑୡཱྀྂṌ⳹ࠊᐑୖἋ㈗ࠊᰘ⏣᭷ຍࠊ๓⏣⣧ኵ㸦ዉⰋዪ኱㝔ே㛫ᩥ

໬࣭㣗≀ᰤ㣴ࠊዉⰋዪ኱⏕ά⎔ቃ࣭㣗≀ᰤ㣴㸧

㻌

࠙┠ⓗࠚ㏆ᖺࠊከᩘࡢ⑓ཎᛶࡸᢠ⏕≀㉁⪏ᛶࢆ⋓ᚓࡋࡓ᪂ࡓ࡞⑓ཎᛶ኱⭠⳦ࡢฟ

⌧ࡀୡ⏺ⓗ࡞ၥ㢟࡜࡞ࡗ࡚࠸ࡿࠋ኱⭠⳦࡛ࡣ୍⯡࡟⮬↛⎔ቃୗ࡛ࡢᙧ㉁㌿᥮ࡣ࡯

࡜ࢇ࡝㉳ࡇࡽ࡞࠸࡜ࡉࢀ࡚ࡁࡓࡀࠊ㏆ᖺᡃࠎࡢ◊✲ᐊ࡛ࡣࠊΰྜᇵ㣴ࡢ኱⭠⳦ᰴ

㛫࡛ᙧ㉁㌿᥮࡟ࡼࡾ㠀᥋ྜᛶࣉࣛࢫ࣑ࢻࡀỈᖹఏ᧛ࡍࡿ⌧㇟ࢆⓎぢࡋࡓࠋࡇࡢⓎ

ぢࡣࠊ⎔ቃ୰࡛ࡢ኱⭠⳦ࡢ㑇ఏᏊỈᖹఏ᧛࡟㛵ࡍࡿᐃㄝࡢ෌᳨ウࢆಁࡍࡶࡢ࡛࠶

ࡿࠋ⎔ቃ୰ࡢ⌧㇟ࢆṇ☜࡟෌⌧ࡍࡿࡓࡵ࡟ࡣࠊỗ⏝ࡢᐇ㦂ᐊᰴ࡛ࡣ࡞ࡃ㔝⏕⳦ᰴ

ࢆ⏝࠸ࡓᐇ㦂⣔ࡀᚲ㡲࡜࡞ࡿࠋࡑࡇ࡛ᮏ◊✲࡛ࡣࠊ㔝⏕኱⭠⳦ᰴࡢ௦⾲࡜ࡋ࡚ࠊ ECOR㸦E. coli Collection of Reference㸧ࡢ฼⏝ࢆ⪃࠼ࡓࠋECOR࡜ࡣ1984ᖺ

࡟Ochmanࡽࡀ㑅ᢤࡋࡓࠊ㔝⏕኱⭠⳦ᰴࡢᩘ༓✀㢮ࢆ72ᰴ࡛௦⾲ࡍࡿᶆ‽ᰴࢥ

ࣞࢡࢩ࡛ࣙࣥ࠶ࡿࠋᮏ◊✲࡛ࡣࠊࡲࡎECORᰴࢆ⏝࠸ࡓ㑇ఏᏊỈᖹఏ᧛ࣔࢹࣝ

ᐇ㦂⣔ࡢ☜❧࡜ఏ᧛ᵝᘧࡢุูࢆ⾜࠸ࠊḟ࠸࡛ྠᐇ㦂⣔ࢆ⏝࠸ࠊ㣗ရࡀᰤ㣴※࡜

࡞ࡿ᮲௳ୗ࡛ࡢ㑇ఏᏊỈᖹఏ᧛ࡢⓎ⏕᳨ド࡜ࠊ࣮ࣚࢢࣝࢺ࡟ྵࡲࢀࡿங㓟ࡸ㓑㓟

࡟╔┠ࡋࡓᙳ㡪ホ౯ࢆヨࡳࡓࠋ

࠙᪉ἲࠚ⳦ᰴࡣࠊኳ↛ࣉࣛࢫ࣑ࢻಖ᭷㸭㠀ಖ᭷ࡢECORᰴࠊேᕤࣉࣛࢫ࣑ࢻᑟ ධ㸭ᮍᑟධࡢECORᰴࠊ࠾ࡼࡧᐇ㦂ᐊᰴࢆ⏝࠸ࡓࠋ㑇ఏᏊỈᖹఏ᧛ࡣࠊ2 ✀ࡢ

⳦ᰴࢆඹᇵ㣴ᚋࠊ᪂ࡓ࡞ᢠ⏕≀㉁⪏ᛶࢆ⋓ᚓࡋࡓࢥࣟࢽ࣮ࡢⓎ⏕ᩘ࠿ࡽᐃ㔞໬ࡋ ࡓࠋ㑇ఏᏊỈᖹఏ᧛ࡢᶵᵓࡣࠊDNase ࠾ࡼࡧ࣓ࣥࣈࣞࣥࢆ⏝࠸ࡓᐇ㦂࡟ࡼࡾุ

ูࡋࡓࠋ㣗ရᢳฟᾮࡣ㐲ᚰศ㞳ᚋࡢୖΎࢆࣇ࢕ࣝࢱ࣮⁛⳦ࡋ࡚⏝࠸ࡓࠋᅛᙧ㸭༙

ᅛᙧ㣗ရ࡛ࡣ㣗ရୖ࡟⨨࠸ࡓ࣓ࣥࣈࣞࣥୖ࡛⳦ࢆᇵ㣴ࡋࡓࠋ

࠙⤖ᯝࠚୖグ⳦ᰴ࠿ࡽࠊᢠ⏕≀㉁⪏ᛶࡢỈᖹఏ᧛ࡀ㉳ࡇࡿ⤌ࡳྜࢃࡏࢆ㑅ᢥᚋࠊ ᶵᵓุูᐇ㦂࠿ࡽᙧ㉁㌿᥮࠶ࡿ࠸ࡣ᥋ྜ㸦ྍື໬㸧ࡀ㉳ࡇࡿ௦⾲ⓗ⤌ࡳྜࢃࡏࢆ

ࡑࢀࡒࢀ㸰✀㢮ࡎࡘ㸦ィ㸲✀㢮㸧㑅ᐃࡋࡓࠋḟ࠸࡛ࡇࢀࡽ⳦ᰴࢆ⏝࠸࡚ྛ✀㣗ရ ᡂศ࡛ࡢᇵ㣴ࢆ⾜࠸ࠊ㑇ఏᏊỈᖹఏ᧛ࡢⓎ⏕ࢆ᳨ドࡋࡓࠋࡑࡢ⤖ᯝࠊྛ✀ᢳฟᾮ

୰ࡸᅛᙧ㣗ရୖ࡛ࡶ㏻ᖖࡢᐇ㦂ᇵᆅ࡜࡯ࡰྠ➼ࣞ࣋ࣝࡢ㑇ఏᏊఏ᧛ࡀ㉳ࡇࡿࡇ

࡜ࢆ᫂ࡽ࠿࡟ࡋࡓࠋࡲࡓࡑࡢ୰࡛࣮ࣚࢢࣝࢺࡢᾮయᡂศࡀ㑇ఏᏊỈᖹఏ᧛ࡢ㢧ⴭ

࡞ᢚไຠᯝࢆ♧ࡋࡓࡓࡵࠊࡑࡢ୺࡞ᡂศ࡛࠶ࡿங㓟ࡸ㓑㓟ࡢస⏝࡟㛵ࡋࠊࡉࡽ࡟

ヲ⣽࡞ゎᯒࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊங㓟ࡸ㓑㓟ࡣ㑇ఏᏊỈᖹఏ᧛࡟ᑐࡋࠊሷ㓟࡛ࡣ ぢࡽࢀ࡞࠸⊂⮬ࡢᢚไస⏝ࢆⓎ᥹ࡍࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋ

࠙ㅰ㎡ࠚᮏ◊✲ࡣࠊ㈈ᅋἲே᪥ᮏ⎔ቃ㈈ᅋࡢ◊✲ຓᡂ㔠࠾ࡼࡧH23ዉⰋዪᏊ኱

Ꮫ◊✲᥎㐍ࣉࣟࢪ࢙ࢡࢺ⤒㈝ࡢຓᡂࢆཷࡅ࡚ᐇ᪋ࡋࡓࠋ

㸯㸳

䜾䜰䝜䝅䞁䛾⏕యෆ㓟໬ᅉᏊ䛿⬡㉁䝠䝗䝻䝨䝹䜸䜻䝅 䝗䛷䛒䜛

㻌

ۑᆏᮏᮍ᮶ࠊ⸛ཎ⚈Ꮚࠊᶫᮏᇽྐࠊ㔠ἑ࿴ᶞ

㸦⚄ᡞ኱㝔㎰࣭⏕࿨ᶵ⬟㸧

࠙┠ⓗࠚ㑇ఏᏊኚ␗ࡢཎᅉࡢ୍ࡘ࡟ࠊ

2’-

ࢹ࢜࢟ࢩࢢ࢔ࣀࢩࣥ㸦

dG

㸧ࡢ

8-

ࣄࢻࣟ

࢟ࢩ

-2’-

ࢹ࢜࢟ࢩࢢ࢔ࣀࢩࣥ㸦

8-OHdG

㸧࡬ࡢ㓟໬ࡀ࠶ࡿࠋ⏕యෆ㓟໬ᅉᏊࡣࠊ୍

⯡ⓗ࡟ࠊ㐣㓟໬Ỉ⣲࠿ࡽ⏕ࡌࡿࣄࢻࣟ࢟ࢩࣛࢪ࢝ࣝ࡜⪃࠼ࡽࢀ࡚࠸ࡿࠋࡋ࠿ࡋࠊ

ࣄࢻࣟ࢟ࢩࣛࢪ࢝ࣝࡢ༙ῶᮇࡣ

1

ࢼࣀ⛊࡜▷ࡃࠊࡲࡓࠊࡑࡢ⏕ᡂ࡟ࡣ㑄⛣㔠ᒓࢆ

ᚲせ࡜ࡍࡿࠋࡋࡓࡀࡗ࡚ࠊ㐣㓟໬Ỉ⣲ࡣ⏕యෆ㓟໬ᅉᏊ࡜⪃࠼࡟ࡃ࠸ࠋ୍᪉ࠊ⬡

㉁ࣄࢻࣟ࣌ࣝ࢜࢟ࢩࢻࡢศゎ࡛⏕ࡌࡿ࣌ࣝ࢜࢟ࢩࣛࢪ࢝ࣝࡢ༙ῶᮇࡣ

7

⛊࡛࠶

ࡿࠋᮏ◊✲࡛ࡣࠊ㠀⣽⬊⣔࡜⣽⬊⣔࡛ࠊ㐣㓟໬Ỉ⣲࡜⬡⫫㓟ࣄࢻࣟ࣌ࣝ࢜࢟ࢩࢻ

ࡢ

8-OHdG

ࡢ⏕ᡂ㔞ࢆẚ㍑ࡋࡓࠋࡲࡓࠊdG ࡢ㓟໬ࢆᢚไࡍࡿ⏕యෆ࡛᭷ຠ࡜ᛮ

࠼ࡿᢠ㓟໬ᡂศࢆ᳨⣴ࡋࡓࠋ

࠙᪉ἲ࣭⤖ᯝࠚࣜࣀ࣮ࣝ㓟ࣄࢻࣟ࣌ࣝ࢜࢟ࢩࢻ㸦

LAHPO

㸧ࡣࠊࣜࣀ࣮ࣝ㓟ࢆ⮬

ື㓟໬ࡋ࡚ᚓࡓ㐣㓟໬≀࠿ࡽࠊ

HPLC

࡛

98%

௨ୖࡢ⣧ᗘ࡟⢭〇ࡋ࡚⏝࠸ࡓࠋ

dG

250 μMࠊdG 250 μM

ࢆྵࡴ࣏ࣜࢯ࣮࣒ࠊ࠶ࡿ࠸ࡣ

10 μg / ml

ࡢ௘࢘ࢩ⬚⭢

DNA

࡟ࠊ

50 μM

ࡢ

LAHPO

࠶ࡿ࠸ࡣ㐣㓟໬Ỉ⣲ࢆῧຍࡋࠊ⏕ᡂࡋࡓ

8-OHdG

ࢆ㟁Ẽ໬

Ꮫ᳨ฟჾ࡜

UV

᳨ฟჾࢆഛ࠼ࡓ

HPLC

࡛ᐃ㔞ࡋࡓࠋ

LAHPO

ࢆ dG ࡟ῧຍࡍࡿ࡜

10⁵ dG

࠶ࡓࡾ

3.35

ࡢ

8-OHdG

ࡀࠊ࣏ࣜࢯ࣮࣒࡛ࡣ

6.04 / 10⁵ dG

ࡀࠊ⬚⭢

DNA

࡛ࡣ

27.16 / 10⁵ dG

ࡢ⏕ᡂࡀㄆࡵࡽࢀࠊ㐣㓟໬Ỉ⣲ῧຍ࡛ࡣࡑࢀࡒࢀ

1.91 / 10⁵ dGࠊ2.17 / 10⁵ dGࠊ9.28 / 10⁵ dG

࡛࠶ࡗࡓࠋLAHPO ࡣ㐣㓟໬Ỉ⣲ࡼࡾࡶ᭷ព

࡟

8-OHdG

ࢆከࡃ⏕ᡂࡋࡓࠋࡲࡓࠊࡇࢀࡽࡢ⣔࡟

10 μM

ࡢ

FeSO4

ࢆຍ࠼ࡿ࡜ࠊ

LAHPO

࡜㐣㓟໬Ỉ⣲ࡢ㛫࡟

8-OHdG

ࡢ⏕ᡂ㔞ࡢᕪࡣ࡞ࡃ࡞ࡗࡓࠋࡑࡇ࡛ࠊࣛࢵ

ࢺ⫢࣑ࢺࢥࣥࢻࣜ࢔࡟

100 μM

ࡢ

LAHPO

ࢆῧຍࡍࡿ࡜

34.43 / 10⁵ dG

ࡢ

8-OHdG

ࡀࠊ㐣㓟໬Ỉ⣲ࡢῧຍ࡛ࡣ

23.96 / 10⁵ dG

ࡀ⏕ᡂࡋࡓࠋࡑࡋ࡚ࠊ࣓ࣝ࢝ࣉࢺࢥࣁ

ࢡ㓟࡛

24

᫬㛫ฎ⌮ࡋࡓ

HepG2

⣽⬊࡟

200 μM

ࡢ

LAHPO

ࢆ୚࠼ࡿ࡜

0.61 / 10⁵ dG

ࡀࠊ㐣㓟໬Ỉ⣲࡛ࡣ

0.34 / 10⁵ dG

ࡢ

8-OHdG

ࡀ⏕ᡂࡋࡓࠋࡇࡢࡼ࠺࡟

LAHPO

ࡀ

᭷ព࡟

8-OHdG

ࢆ⏕ᡂࡋࡓࠋࡑࡇ࡛ࠊ

10 μM

ࡢ

Į-

ࢺࢥࣇ࢙࣮ࣟࣝࠊࣝࢸ࢜ࣜࣥࡲ

ࡓࡣࢣࣝࢭࢳࣥࢆ

HepG2

⣽⬊࡟

1

᫬㛫୚࠼ࡓᚋࠊ ࡑࡢ᰾ࢆ༢㞳ࡋ࡚ࠊ ᰾࡟

100 μM

ࡢ

LAHPO

ࢆῧຍࡋ࡚

8-OHdG

⏕ᡂࢆᐃ㔞ࡍࡿࡇ࡜࡛ᢠ㓟໬ຠᯝࢆ ᐃࡋࡓࠋ࠸

ࡎࢀࡢᡂศࡶ

8-OHdG

⏕ᡂࢆ⣙

60

㸣ᢚ࠼ࡓࠋ௨ୖࡢ⤖ᯝ࠿ࡽࠊ㑄⛣㔠ᒓࡀ㧗⃰

ᗘ࡛Ꮡᅾࡋ࡞࠸⏕యෆ᮲௳࡛ࡣࠊ

dG

ࡢ㓟໬ᅉᏊࡣ㐣㓟໬Ỉ⣲࡛ࡣ࡞ࡃ⬡㉁ࣄࢻ

ࣟ࣌ࣝ࢜࢟ࢩࢻ࡛࠶ࡾࠊࡲࡓࠊࡑࡢ㓟໬ࢆ

Į-

ࢺࢥࣇ࢙࣮ࣟࣝࡸ࢝ࢸࢥ࣮ࣝᵓ㐀

ࡢࣇࣛ࣎ࣀ࢖ࢻࡀᢚ࠼ࡿࡇ࡜ࡀ࡛ࡁࡿ࡜⪃࠼ࡓࠋ

㸯㸴

ᢪྜᛶ䝣䝷䝪䝜䜲䝗䛾ᣕᢠⓗ⭠⟶྾཰䛻䛴䛔䛶

㻌

ۑᅵ஭ᙬ཭⨾

ࠊ⸛ཎ⚈Ꮚ

ࠊᶫᮏᇽྐ

ࠊ㔠ἑ࿴ᶞ

㸦

⚄ᡞ኱㝔㎰࣭⏕࿨ᶵ

⬟㸧 㻌 㻌

࠙┠ⓗࠚ᳜≀ᛶ㣗ရ࡟ྵࡲࢀ࡚࠸ࡿከᵝ࡞ࣇࣛ࣎ࣀ࢖ࢻࡣࠊᢠ㓟໬⬟ࡸࢱࣥࣃࢡ

㉁ᶵ⬟ㄪ⠇స⏝ࢆ௓ࡋࡓ⏕ά⩦័⑓ண㜵ຠᯝࡀᮇᚅࡉࢀ࡚࠸ࡿࠋண㜵ຠᯝࢆᮇᚅ ࡍࡿ࡞ࡽࡤࠊࡑࡢࣇࣛ࣎ࣀ࢖ࢻࡢయෆ྾཰⃰ᗘࡀ㧗࠸᪉ࡀᮃࡲࡋ࠸ࠋࡲࡓࠊࣇࣛ

࣎ࣀ࢖ࢻࡣయෆ྾཰᫬࡟ࢢࣝࢡࣟࣥ㓟࠶ࡿ࠸ࡣ◲㓟ᢪྜࢆཷࡅࡿࡀࠊᢪྜయࡼࡾ

ࡶ࢔ࢢࣜࢥࣥࡢ᪉ࡀ⏕⌮άᛶࡣ㧗࠸ࡢ࡛ࠊయෆ࢔ࢢࣜࢥࣥ⃰ᗘࡀ㧗࠸ࡇ࡜ࡀዲࡲ

ࡋ࠸ࠋᮏ◊✲࡛ࡣࠊࣇࣛ࣎ࣀ࢖ࢻࡢ୰࡛ࡶᢪྜࢆཷࡅࡸࡍ࠸࡜ሗ࿌ࡉࢀ࡚࠸ࡿࢣ

ࣝࢭࢳࣥ࡟╔┠ࡋࠊࢣࣝࢭࢳࣥࠊࡑࡢㄏᑟయ࠾ࡼࡧ㢮⦕໬ྜ≀ࢆ⤌ࡳྜࢃࡏ࡚ࣛ

ࢵࢺ࡟ᢞ୚ࡍࡿࡇ࡜࡛ࠊࢣࣝࢭࢳࣥࡢయෆᚠ⎔⃰ᗘࢆୖࡆࡿ⤌ࡳྜࢃࡏࢆ᥈ࡋฟ ࡍࡇ࡜ࢆヨࡳࡓࠋ

࠙᪉ἲ࣭⤖ᯝࠚ୍ᬌ⤯㣗ࡉࡏࡓ

Wistar ST

ࣛࢵࢺ㸦㞝ࠊ11 㐌㱋㸧࡟ࠊ50 mg/kg య㔜ࡎࡘࡢࢣࣝࢭࢳࣥ࡜࢖ࢯࢣࣝࢭࢳࣥࠊࢣࣝࢭࢳࣥ࡜ࣉࣟࢺ࢝ࢸࢡ㓟ࠊࢣࣝࢭ ࢳࣥ࡜࢖ࢯࢣࣝࢭࢳࣥ࠾ࡼࡧࣉࣟࢺ࢝ࢸࢡ㓟ࢆ⤌ࡳྜࢃࡏࡓヨᩱࢆࣉࣟࣆࣞࣥ

ࢢࣜࢥ࣮ࣝ࡟⁐ゎࡋ࡚⤒ཱྀᢞ୚ࡋࠊࢣࣝࢭࢳࣥࢆ༢⊂࡛୚࠼ࡓሙྜ࡜ẚ㍑ࡋࡓࠋ

ᢞ୚ᚋ

0.5ࠊ1ࠊ2ࠊ4ࠊ6

᫬㛫࡟ᑿ㟼⬦᥇⾑ࡋࠊ⾑₢୰ࡢࢣࣝࢭࢳࣥ࢔ࢢࣜࢥࣥ

࡜ࢣࣝࢭࢳࣥᢪྜయ㔞ࢆࠊࢡ࣮ࣟ࢔ࣞ࢖᳨ฟჾࢆഛ࠼ࡓ

HPLC

࡛ᐃ㔞ࡋࡓࠋࢣ

ࣝࢭࢳࣥ༢⊂ᢞ୚࡜ẚ㍑ࡋ࡚ࠊ࢖ࢯࢣࣝࢭࢳࣥࢆྠ᫬ᢞ୚ࡍࡿ࡜ࠊ⾑₢୰ࡢࢣࣝ

ࢭࢳࣥᢪྜయ㔞ࡣ᭷ព࡟ቑຍࡋࡓࠋࡲࡓࠊྠ᫬ᢞ୚ࡍࡿ࢖ࢯࢣࣝࢭࢳࣥ㔞ࢆࢣࣝ

ࢭࢳࣥ࡟ᑐࡋ࡚

1/1ࠊ1/2ࠊ1/4ࠊ1/10

࡜ࡋ࡚ࠊ0.5ࠊ1ࠊ2ࠊ5 ᫬㛫ᚋࡢࣛࢵࢺࡢ⾑

₢୰ࡢࢣࣝࢭࢳࣥࡢ࢔ࢢࣜࢥࣥ࡜ᢪྜయ㔞ࢆẚ㍑ࡋࡓࠋ࠸ࡎࢀࡢΰྜẚ⋡࡛ࡶࢣ

ࣝࢭࢳࣥᢪྜయ㔞ࡶ࢔ࢢࣜࢥࣥࡢ㔞ࡶኚࢃࡽ࡞࠿ࡗࡓࠋࡋ࠿ࡋࠊࢣࣝࢭࢳࣥ࡜࢖

ࢯࢣࣝࢭࢳࣥࢆ➼㔞ᢞ୚ࡋࡓ⩌ࡢࡳࠊࢣࣝࢭࢳࣥ༢⊂ᢞ୚࡜ẚ㍑ࡋ࡚ࠊ⾑₢୰ࡢ ࢣࣝࢭࢳࣥᢪྜయ㔞ࡀ᭷ព࡟ቑຍࡋࡓࠋࡑࡇ࡛ࠊࢣࣝࢭࢳࣥ࡟࢔ࣆࢤࢽࣥࠊ࢔ࣆ

ࢤࢽࣥ

O-

㓄⢾యࡢ࢔ࣆࢤࢺࣜࣥࠊ࢔ࣆࢤࢽࣥ

C-

㓄⢾యࡢࣅࢸ࢟ࢩࣥࢆΰྜࡋ࡚

ᢞ୚ࡋࡓࠋࢣࣝࢭࢳࣥ༢⊂ᢞ୚࡜ẚ㍑ࡋ࡚ࠊ࢔ࣆࢤࢺࣜࣥࡢྠ᫬ᢞ୚⩌࡛ࢣࣝࢭ ࢳࣥᢪྜయ㔞ࡢ᭷ព࡞ቑຍࡀほᐹࡉࢀࡓࠋࡑࡋ࡚ࠊࣅࢸ࢟ࢩࣥྠ᫬ᢞ୚࡛ࡣ᭷ព

࡞ᙳ㡪ࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࠋ௨ୖࡢࡼ࠺࡟ࠊࣇࣛ࣎ࣀ࢖ࢻ

O-

㓄⢾యࡢྠ᫬ᢞ୚

ࡣࢣࣝࢭࢳࣥࡢయෆ⃰ᗘࢆቑຍࡉࡏࠊࡑࡢᢪྜయ㔞ࢆቑຍࡉࡏࡿࡀࠊ࢔ࢢࣜࢥࣥ

㔞࡟ࡣᙳ㡪ࢆ୚࠼࡞࠿ࡗࡓࠋ

㸯㸵

Interaction of wheat E-amylase with maltose and glucose as examined by fluorescence

ۑTadessa Daba, Kenji Kojima, and Kuniyo Inouye

䠄Division of Food

Science and Biotechnology, Graduate School of Agriculture, Kyoto University䠅

࠙ObjectiveࠚE-Amylases [EC 3.2.1.2] are exo-acting enzymes, with successive removal of E-anomeric maltose from the non-reducing ends of polysaccharides. The fluorescence quenching effects of maltose and glucose on wheat E-amylase (WBA) were studied by fluorescence titration. The temperature and pH-dependences of the dissociation constants (K^d) of WBA-maltose and WBA-glucose complexes were evaluated.

࠙ Methods and Results ࠚ The source of WBA, Himaltosin which is an industrially-applicable commercial preparation from wheat bran, was purchased from HBI Enzymes, Osaka¹⁾. The fluorescence emission of WBA was observed by excitation at 280 nm at 25^oC, pH 5.4. The fluorescence intensity (FI) of WBA was partly quenched by the addition of maltose and glucose. The K^d values of the enzyme-inhibitor complexes (EI) dissociations determined by titration of FI were 0.20s0.12 M for maltose and 0.36s0.11 M for glucose. The Kd values slightly increased while the Gibbs energy changes (¨G^o) decreased with increasing temperature. The respective Kd values were 0.22s0.09, 0.20s0.12, and 0.17s0.04 M for maltose and 0.29s0.04, 0.36s 0.11, 0.26s0.07 M for glucose at pH 3.0, 5.4, and 9.0, respectively at 25^oC. The inhibitor constants (Ki) of our previous inhibition kinetics data²⁾ are in reasonable agreement with the Kd values obtained in this study. The ¨H^o and ¨G^o values indicate that the EI dissociations are endothermic and enthalpy-driven. The temperature and pH-dependences of the K^d values suggest that the changes in conformation and the environment of tryptophan and/or tyrosine residues around the binding site of WBA are governed by pH and temperature. It is therefore, essential to consider the temperature and pH of starch hydrolysis to reduce the end-product inhibition.

1. Daba T, Kojima K, Inouye K, Enzyme. Microb. Technol.51, 245-251 (2012) 2. Daba T, Kojima K, Inouye K, submitted

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⫧‶䛻క䛖⅖⑕≧ែ䛜⬡⫫⤌⧊䛻䛚䛡䜛 UCP1 Ⓨ⌧

ㄏᑟ䛻୚䛘䜛ᙳ㡪㻌

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ۑ୸㔝᫭Ⴙࠊᆏᮏᬛᘺࠊᚋ⸨๛ࠊ㧗ᶫಙஅࠊἙ⏣↷㞝㸦ி኱

㝔࣭㎰࣭㣗ရ⏕≀ࠊி኱Ꮫ㝿⼥ྜ࣭⏕⌮໬Ꮫ㸧

࠙┠ⓗࠚ့ங㢮࡟Ꮡᅾࡍࡿ〓Ⰽ⬡⫫⣽⬊ࡣࠊࡑࡢ࣑ࢺࢥࣥࢻࣜ࢔ෆ⭷࡟Ꮡᅾࡍࡿ

uncoupling protein-1 (UCP1) ࡢാࡁ࡟ࡼࡾ࢚ࢿࣝࢠ࣮ࢆᾘ㈝ࡋࠊ⇕ࢆ⏘⏕ࡍ

ࡿࠋUCP1ࢆⓎ⌧ࡍࡿ⬡⫫⣽⬊ࡣࠊ〓Ⰽ⬡⫫⤌⧊ (BAT) ࡜ࡋ࡚⫪⏥㦵࿘㎶࡞࡝

࡟Ꮡᅾࡍࡿࠋࡉࡽ࡟ᐮ෭࡞࡝ࡢ่⃭࡟ࡼࡾࠊ㰡㋣㒊ⓑⰍ⬡⫫⤌⧊ (I-WAT) ࡞࡝

୍㒊ࡢⓑⰍ⬡⫫⤌⧊࡟࠾࠸࡚ࡶࠊUCP1ࡢⓎ⌧㔞ࡀቑຍࡋࠊ࢚ࢿࣝࢠ࣮ᾘ㈝㔞ࡀ ቑ኱ࡍࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋ୍᪉࡛ࠊ⫧‶࡜ UCP1 ࡢⓎ⌧㔞ࡢ㛫࡟ࡣ㧗࠸

┦㛵ࡀࡳࡽࢀࠊ⫧‶≧ែࡢ⬡⫫⤌⧊࡛ࡣ UCP1 ࡢⓎ⌧ࡀపୗࡋ࡚࠸ࡿࠋᮏ◊✲

࡛ࡣࠊࡑࡢ࣓࢝ࢽࢬ࣒ࢆ᫂ࡽ࠿࡟ࡍࡿࡇ࡜ࢆ┠ⓗ࡜ࡋ࡚ࠊ⫧‶≧ែࡢ⬡⫫⤌⧊࡛

⏕ࡌࡿ⅖⑕≧ែ࡟╔┠ࡋࠊ⅖⑕≧ែࡀBAT࠾ࡼࡧI-WATࡢ୧⬡⫫⤌⧊࡟࠾ࡅࡿ

UCP1ࡢⓎ⌧ㄏᑟ࡟୚࠼ࡿᙳ㡪ࢆື≀ಶయ᳨࡛ࣞ࣋ࣝウࡋࡓࠋ

࠙᪉ἲ࣭⤖ᯝࠚ⫧‶≧ែࡢBAT࠾ࡼࡧI-WATࡢ୧⬡⫫⤌⧊࡟࠾ࡅࡿUCP1Ⓨ

⌧ㄏᑟ࡟ࡘ࠸࡚ㄪ࡭ࡿࡓࡵࠊ㧗⬡⫫㣗 (HFD) ࢆ16㐌㛫ᦤ㣗ࡉࡏࡓC57BL/6J

࣐࢘ࢫࢆ⏝࠸ࡓ᳨ウࢆ⾜ࡗࡓࠋ࣐࢘ࢫ࡟ᐮ෭่⃭ (4Υࠊ24᫬㛫) ࢆ୚࠼ࡿ࡜ࠊ HFDᦤ㣗⩌ࡢBAT࠾ࡼࡧI-WAT࡛ㄏᑟࡉࢀࡿUCP1ࡢⓎ⌧㔞ࡀࠊᬑ㏻㣗 (ND) ᦤ㣗⩌࡜ẚ㍑ࡋ࡚᭷ព࡟ῶᑡࡋࡓࠋࡲࡓࠊHFDᦤ㣗⩌ࡢBAT࠾ࡼࡧI-WAT࡛ ࡣࠊF4/80 (࣐ࢡࣟࣇ࢓࣮ࢪࡢ࣐࣮࣮࢝㑇ఏᏊ) ࡜⅖⑕ᛶࢧ࢖ࢺ࢝࢖ࣥ tumor necrosis factor D (TNFD) mRNAࡢⓎ⌧㔞ࡀNDᦤ㣗⩌࡜ẚ㍑ࡋ࡚᭷ព࡟ቑຍ ࡋࡓࠋ௨ୖࡢ⤖ᯝࡼࡾࠊ࣐ࢡࣟࣇ࢓࣮ࢪࡀᾐ₶ࡋ⅖⑕≧ែࡀច㉳ࡉࢀࡓBAT࠾

ࡼࡧI-WAT࡛ࡣࠊᐮ෭่⃭࡟ࡼࡾ⏕ࡌࡿUCP1ࡢⓎ⌧ㄏᑟࡀᢚไࡉࢀࡿྍ⬟ᛶ ࡀ⪃࠼ࡽࢀࡓࠋ

ḟ࡟࣐ࢡࣟࣇ࢓࣮ࢪࡀᾐ₶ࡋ⅖⑕≧ែࡀច㉳ࡉࢀࡓ⬡⫫⤌⧊࡛Ⓨ⌧ࡀቑຍࡍ

ࡿTNFDࡀࠊBAT࠾ࡼࡧI-WAT࡟࠾ࡅࡿUCP㸯Ⓨ⌧ㄏᑟ࡟୚࠼ࡿᙳ㡪ࢆㄪ࡭

ࡓࠋ㠀⫧‶≧ែࡢ5㐌㱋C57BL/6J࣐࢘ࢫ࡟TNFDࢱࣥࣃࢡ㉁mg/kg body weightࢆ⭡⭍ෆ࡟ᢞ୚ࡋࡓࠋࡑࡢ⤖ᯝࠊBAT࠾ࡼࡧI-WAT࡟࠾࠸࡚ᐮ෭่⃭

࡟ࡼࡾㄏᑟࡉࢀࡿUCP1 ࡢⓎ⌧㔞ࡀࠊ⏕⌮㣗ሷỈࢆᢞ୚ࡋࡓ࣐࢘ࢫ࡜ẚ㍑ࡋ࡚

᭷ព࡟ῶᑡࡋࡓࠋ௨ୖࡢ⤖ᯝࡼࡾࠊ⫧‶࡟ࡼࡗ࡚⏕ࡌࡿ⅖⑕≧ែࡀBAT࠾ࡼࡧ

I-WATࡢ୧⬡⫫⤌⧊࡟࠾࠸࡚ࠊUCP1Ⓨ⌧ㄏᑟࢆᢚไࡍࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋ

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䝯䝍䝪䝻䞊䝮ゎᯒ䜢⏝䛔䛯 PPAR άᛶ໬䛻䜘䜚ኚື䛩 䜛⏕యෆ௦ㅰ≀䛾᥈⣴㻌

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ۑᒣ㷂㝧ኴࠊ㧗ᶫ᫓ᘺࠊᚋ⸨๛ࠊ㧗ᶫಙஅࠊᰘ⏣኱㍜ࠊἙ⏣↷㞝 㸦ி኱㝔㎰࣭㣗ရ⏕≀ࠊி኱Ꮫ㝿⼥ྜ࣭⏕⌮໬Ꮫࠊ࠿ࡎࡉ '1$ ◊㸧

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࠙┠ⓗࠚPeroxisome proliferator-activated receptor㸦PPAR㸧ࡣࠊ᰾ෆཷᐜయ ᆺ㌿෗ㄪ⠇ᅉᏊ࡛࠶ࡾࠊ⢾࣭⬡㉁௦ㅰࡢ୺せ࡞ไᚚᅉᏊ࡛࠶ࡿࠋPPAR࡟ࡣࠊĮࠊ Ȗࠊįࡢ 3ࡘࡢࢧࣈࢱ࢖ࣉࡀᏑᅾࡍࡿࠋࡑࡢ୰࡛ࠊPPARȖࡣ୺࡟⬡⫫⤌⧊࡟Ⓨ⌧

ࡋࠊ⬡⫫⣽⬊ศ໬ࡸ࢖ࣥࢫࣜࣥឤཷᛶࡢㄪ⠇࡟㛵୚ࡋ࡚࠸ࡿࠋࡑࡢࡓࡵࠊPPARȖ ࡢ⏕⌮ᶵ⬟ࢆ⌮ゎࡍࡿࡇ࡜ࡣࠊ⢾ᒀ⑓ࡸ⬡㉁␗ᖖ⑕࡜࠸ࡗࡓ⏕ά⩦័⑓ࡢண㜵࣭

ᨵၿ࡟ࡘ࡞ࡀࡿࡇ࡜ࡀᮇᚅ࡛ࡁࡿࠋPPARȖ άᛶ໬ࡣࠊ୍⯡ⓗ࡟ࠊࡑࡢᶆⓗ㑇ఏ ᏊࡢⓎ⌧ኚື࡛ホ౯ࡉࢀࡿࠋࡋ࠿ࡋࠊPPARȖ ࡢ⏕⌮ᶵ⬟ࢆໟᣓⓗ࡟⌮ゎࡍࡿࡓ

ࡵ࡟ࡣࠊ⏕࿨ࢩࢫࢸ࣒ࡢ᭱⤊⏘≀࡛࠶ࡿ௦ㅰ≀ࡢ࡛ࣞ࣋ࣝࠊPPARȖ άᛶ໬ࢆホ ౯ࡍࡿࡇ࡜ࡶ㔜せ࡛࠶ࡿࠋࡑࡇ࡛ᮏ◊✲࡛ࡣࠊ௦ㅰ඲యࢆಠ▔ⓗ࡟ᢕᥱࡍࡿࡇ࡜

ࡀ࡛ࡁࡿ࣓ࢱ࣮࣒࣎ࣟゎᯒࢆ⏝࠸࡚ࠊPPARȖ άᛶ໬࡟ࡼࡾኚືࡍࡿ⏕యෆ௦ㅰ

⏘≀ࡢ᥈⣴ࢆ⾜ࡗࡓࠋ

࠙᪉ἲ࣭⤖ᯝࠚPPARȖ࢔ࢦࢽࢫࢺ࡛࠶ࡿpioglitazone㸦௨ୗpio㸧ࢆ⫧‶ࣔࢹࣝ

࣐࢘ࢫ࡟ᢞ୚ࡋࠊ4㐌㛫㣫⫱ࡋࡓࠋࡑࡢᚋࠊゎ๗ࢆ⾜࠸ࠊ⬡⫫⤌⧊ᢳฟ≀ࡢศᯒ

࡟ࡼࡾࠊ⸆๣ࡀ⬡⫫⤌⧊࡬฿㐩ࡋ࡚࠸ࡿࡇ࡜ࢆ☜ㄆࡋࡓࠋࡉࡽ࡟ࠊ⬡⫫⤌⧊୰ࡢ ᶆⓗ㑇ఏᏊⓎ⌧ࡀ᭷ព࡟ቑຍࡋ࡚࠸ࡿࡇ࡜࠿ࡽࠊPPARȖ ࡀάᛶ໬ࡉࢀ࡚࠸ࡿࡇ

࡜ࢆ☜ㄆࡋࡓࠋࡑࡢᚋࠊࡇࡢ࣐࢘ࢫࡢ⾑₢࣭⬡⫫⤌⧊ࢆ 80% methanol ࡛ᢳฟ ࡋࠊLC-MSࢆ⏝࠸࡚௦ㅰ≀ࡢ⥙⨶ⓗ࡞ゎᯒ㸦࣓ࢱ࣮࣒࣎ࣟゎᯒ㸧ࢆ⾜ࡗࡓࠋゎ ᯒࡢ⤖ᯝࠊpioࢆᢞ୚ࡋࡓ࣐࢘ࢫࡢ⾑୰࣭⬡⫫⤌⧊୰࡟࠾࠸࡚ࠊ࢔࣑ࣀ㓟ࡀ᭷ព

࡟ቑຍࡍࡿࡇ࡜ࢆぢฟࡋࡓࠋḟ࡟ࠊቑຍࡋࡓ࢔࣑ࣀ㓟ࡀ⬡⫫⣽⬊⏤᮶࡛࠶ࡿ࠿ࢆ

࣐࢘ࢫ⏤᮶๓㥑⬡⫫⣽⬊3T3-L1ࢆ⏝࠸᳨࡚ウࡋࡓࠋศ໬ㄏᑟᚋࡢ3T3-L1⬡⫫

⣽⬊࡟pioࢆ୍㐌㛫ῧຍࡋࡓᚋࠊ⣽⬊ෆࡢ௦ㅰ≀ࢆ80% methanol ࡛ᢳฟࡋࠊ

LC-MS ࢆ⏝࠸࡚ゎᯒࡋࡓࠋࡑࡢ⤖ᯝࠊpio ῧຍ࡟ࡼࡾࠊື≀ᐇ㦂࡛ቑຍࡀぢࡽ

ࢀࡓ࢔࣑ࣀ㓟ࡀ᭷ព࡟ቑຍࡍࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋࡲࡓࠊࡇࢀࡽࡢ࢔࣑ࣀ㓟 ࡢኚືࡣࠊpioᢞ୚࣐࢘ࢫࡢ⫢⮚୰࡟࠾࠸࡚ㄆࡵࡽࢀ࡞࠿ࡗࡓࠋࡉࡽ࡟ࠊPPARĮ

࢔ࢦࢽࢫࢺᢞ୚࣐࢘ࢫࡢ⾑୰ࠊ⫢⮚୰ࠊ⬡⫫⤌⧊୰ࡢ࣓ࢱ࣮࣒࣎ࣟゎᯒࢆ⾜ࡗࡓ

⤖ᯝࠊPPARĮ ࢔ࢦࢽࢫࢺࡢᢞ୚࡛ࡣࠊpio ᢞ୚࡟ࡼࡾቑຍࡋࡓ࢔࣑ࣀ㓟ࡢኚື

ࡀㄆࡵࡽࢀ࡞࠸ࠊࡲࡓࡣኚື⋡ࡀᑠࡉ࠸ࡇ࡜ࡀ♧ࡉࢀࡓࠋࡇࢀࡽࡢ⤖ᯝ࠿ࡽࠊ

PPARȖ ࡢ᪂ࡓ࡞ᶵ⬟࡜ࡋ࡚㸪⬡⫫⣽⬊࡟࠾ࡅࡿ࢔࣑ࣀ㓟௦ㅰ࡬ࡢ㛵୚ࡀ♧၀ࡉ

ࢀࡓࠋ