Basic Study for the Design and the Preparation of the Artificial Drug Binding Protein Modules and its Pharmaceutical Applications.

人工药物结合蛋白模块的设计、制备及其医药应用的基础研究。

• 批准号: 15590122
• 负责人: TOMIOKA Yoshihisa
• 金额: $ 1.98万
• 依托单位: Josai International University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15590122/
• 关键词: phage display; neocarzinostatin; antibody-like protein; amino acid substitution; ethidium bromide; genetic library; megaprimer PCR mutagenesis; supernatural product; エチジウムブロミド

In order to investigate the amino acid residues in apo-NCS involving the binding with the NCS-chr, the amino acid-substituted apo-NCS mutants (S98C, S98G, S98T and S98A) were prepared. The binding of the non-natural chromophore, EtdBr, with the each mutated apo-NCS was evaluated by the monitoring for the total fluorescence intensity and fluorescence polarization. The S98G mutant had the best binding property compared with another apo-NCS. The result suggested that the strategy of the substitution for the proper amino acid residues in apo-NCS could be expected to create supernatural apo-NCS. The randomized libraries were successfully prepared using the megaprimer PCR mutagenesis, the overlap extension PCR mutagenesis with dam methylase / DpnI digestion method. The mutated amino acids were total 14, and their sites were region A (49-52, PADF), region B (76-80, FLFDG), and region C (94-98, QVGLS), which are involving the binding with NCS-chr. The randomized library was successfully inserted into the vector DNA for the phage display, and performed a screening using bio-panning method against the glycyrrhetic acid conjugated bovine serum albumin (GA-BSA), and then isolated the clones which had a specific binding property to the GA-BSA. Hereafter, the detail structure analysis will be investigated for the isolated clones.

为了研究apo-NCS中涉及与NCS-CHR结合的氨基酸残基，制备了氨基酸取代的apo-NCS突变体(S98C、S98G、S98T和S98A)。通过监测总荧光强度和荧光偏振来评价非天然发色团EtdBR与每个突变的apo-NCs的结合情况。与另一种apo-NCS相比，S98G突变体具有最好的结合性能。这一结果表明，用适当的氨基酸残基替代脱氧核糖核酸的策略有望创造出超自然的脱氧核糖核酸。采用大分子聚合酶链式反应突变、重叠延伸聚合酶链式反应和DAM甲基酶/DPNI双酶切法，成功制备了随机文库。突变的氨基酸共有14个，分别位于A区(49-52，PADF)、B区(76-80，FLFDG)和C区(94-98，QVGLS)，涉及与NCS-Chr的结合。将随机文库成功插入载体DNA中进行噬菌体展示，利用生物淘洗法对甘草次酸结合的牛血清白蛋白(GA-BSA)进行筛选，筛选出与GA-BSA具有特异结合特性的克隆。此后，将对分离克隆进行详细的结构分析。

项目成果

The induction of prostaglandin E synthetase and upregulation of cyclooxygenase-2 by 9-cis retinoic acid.

9-顺式视黄酸诱导前列腺素 E 合成酶并上调环氧合酶 2。

• 发表时间: 2004
• 期刊: Prostaglandins and Other Lipid Mediators 74
• 作者: Yoko Nagumo;Hiroki Tsukamoto;Shunji Ishiwata;Hiroki Tsukamoto
• 通讯作者: Hiroki Tsukamoto

IL-17 and IL-17F modulate GM-CSF production by lung microvascular endothelial cells stimulated with IL-1β and/or TNF-α.

IL-17 和 IL-17F 调节用 IL-1β 和/或 TNF-α 刺激的肺微血管内皮细胞产生 GM-CSF。

• 发表时间: 2004
• 期刊: Immunology Letters 95
• 作者: Muneo Numasaki;Hidenori Takahashi;Yoshihisa Tomioka;Hidetada Sasaki;Yoko Nagumo;Muneo Numasaki;Muneo Numasaki
• 通讯作者: Muneo Numasaki

Thiazolidinediones increase arachidonic acid release and subsequent prostanoid production inn a PPARg-independent manner.

噻唑烷二酮以不依赖 PPARg 的方式增加花生四烯酸的释放和随后的前列腺素的产生。

• 发表时间: 2004
• 期刊: Prostaglandins and other Lipid Mediators 73
• 作者: Yoko Nagumo;Hiroki Tsukamoto
• 通讯作者: Hiroki Tsukamoto

Regulatory roles of IL-17 and IL-17F in G-CSF production by lung microvascular endothelial cells stimulated with IL-1β and/or TNF-α

• DOI: 10.1016/j.imlet.2004.06.010
• 发表时间: 2004-08-15
• 期刊: IMMUNOLOGY LETTERS
• 影响因子: 4.4
• 作者: Numasaki, M;Takahashi, H;Sasaki, H
• 通讯作者: Sasaki, H

IL-17 and IL-17F modulate GM-CSF production by lung microvascular endothelial cells stimulated with IL-1beta and/or TNF-alpha.

IL-17 和 IL-17F 调节用 IL-1β 和/或 TNF-α 刺激的肺微血管内皮细胞产生 GM-CSF。

• 发表时间: 2004
• 期刊: Immunol Lett 95(2)
• 作者: Numasaki M;et al.
• 通讯作者: et al.