| Project/Area Number |
13356006
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
Applied animal science
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| Research Institution | The University of Tokyo |
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Principal Investigator |
SAKAI Senkiti The University of Tokyo, Graduate School of Agricultural and Life Sciences, Professor, 大学院・農学生命科学研究科, 教授 (80114487)
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| Co-Investigator(Kenkyū-buntansha) |
IMAKAWA Kazuhiko The University of Tokyo, Graduate School of Agricultural and Life Sciences, Associate Professor, 大学院・農学生命科学研究科, 助教授 (00291956)
HODATE Kouichi Kitazato University, School of Veterinary Medicine and Animal Science, Professor, 獣医畜産学部, 教授 (30348594)
AOKI Fugaku University of Tokyo, Graduate School of Frontier Sciences, Associate Professor, 大学院・新領域創成科学研究科, 助教授 (20175160)
伊藤 喜久治 東京大学, 大学院・農学生命科学研究科, 助教授 (50100045)
沢崎 徹 (澤崎 徹) 東京大学, 大学院・農学生命科学研究科, 教授 (00012047)
甫立 孝一 (独)畜産草地研究所, 生理部, 室長(研究職)
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| Project Period (FY) |
2001 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥49,790,000 (Direct Cost: ¥38,300,000、Indirect Cost: ¥11,490,000)
Fiscal Year 2004: ¥8,450,000 (Direct Cost: ¥6,500,000、Indirect Cost: ¥1,950,000)
Fiscal Year 2003: ¥9,100,000 (Direct Cost: ¥7,000,000、Indirect Cost: ¥2,100,000)
Fiscal Year 2002: ¥14,040,000 (Direct Cost: ¥10,800,000、Indirect Cost: ¥3,240,000)
Fiscal Year 2001: ¥18,200,000 (Direct Cost: ¥14,000,000、Indirect Cost: ¥4,200,000)
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| Keywords | L-amino acid oxidase / lactoperoxidase / antibacterial system / mastistis / mouse milk / cow milk / selection index / 過酸化水素産生能 / 化学発光 / 牛乳酵素 / 過酸化水素発生能 / 過酸化水素 / 化学発光法 / オキシダーゼ / L-アミノ酸オキシダーゼ / 乳蛋白質 / 基質特異性 / ミルク中の遊離アミノ酸 / 細菌毒性 |
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| Research Abstract |
Characterization of L-amino acid oxidase (LAO) in mouse milk
LAO was purified from mouse milk. LAO reacted with L-amino acids in an apparent order of Phe> Met, Tyr> Cys, Leu> His>> other 11 amino acids and produced hydrogen peroxide (H_2O_2) in a dose-and time-dependent manner. An apparent Km was 1.0μM for Phe, 8.1μM for Leu and 20.2μM for His. LAO in milk had a molecular weight of 113 kDa and was converted to a 60-kDa protein by SDS-PAGE. By ion-exchange chromatography, the LAO activity was detected in three different fractions. LAO consisted of two subunits but is electrically heterogeneous. The amino acid sequences of N- and C-terminals, and lysylendopeptidase-digested fragments were the same to those of mouse milk LAO deduced from cDNA. This is the first demonstration showing that LAO is present in milk.
Mechanism of Bacterial Killing by Mouse Milk
After gel-filtration, we detected lactoperoxidase (LPO) activity. Using LAO, LPO and amino acids, H_2O_2 was maintained between 190 and 23
… More
0 nmol/ml from 1 to 4 h. We cultured Escherichia (E) coli, Klebsiella (K) pneumoniae, Staphylococcus (S) aureus, and Streptococcus (S) agalactiae strains. Bacteria increased with a doubling time of 30-40 min. However, S.agalactiae and S.aureus did not grow ; the number of viable bacteria remained the same at 4 h as at 0 h. Escherichia coli and K.pneumoniae grew with doubling times of 98 and 84 min, respectively, and growth was suppressed. Bacterial growth was suppressed in the LAO/LPO/amino acid system.
Hydrogen peroxide generating substance in cow milk
Cow milk contained a H_2O_2 generating substance. After gel-filtration, the fraction with a molecular weight of 20 kDa had H_2O_2 generating activity, and generated it in a linear manner against the time. A H_2O_2 generating activity increased from early to mid-lactation and decreases at the end of lactation. The activity in mastitis milk was lower than that in normal milk. These results suggest that the H_2O_2 generating substance plays an important role for the activation of the LPO-system which is a potent antibacterial system formed by LPO, H_2O_2, and thiocyanate. Less
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