AN IDENTIFICATION AND CHARACTERIZATION OF MENBRANE PROTEIN ASSOCIATED WITH CELL ACTIVATIONS LY BACTERIAL LIPOPOLYSACCHARIDES

与细胞活化相关的细菌脂多糖膜蛋白的鉴定和表征

• 批准号: 11670270
• 负责人: KIRIKAE Teruo
• 金额: $ 2.24万
• 依托单位: INTERNATIONAL MEDICAL CENTER OF JAPAN
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670270/
• 关键词: LPS; LIPOPOLYSACCHARIDE; ENDOTOXIN; LIPIDA; MACROPHAGE; PE-406; PE-506; STOROMA-DERIVED CELL; Lipoplysaccharide; LPS結合タンパク質; タキソール

A bacterial endotoxin lipopolysaccharide (LPS) is known to induce the expression of various cytokines via activation of host cells, including macrophages. To identify and characterize the novel molecules which play integral roles in the LPS-induced cell activation, we utilized antibodies against membrane proteins and isotopically labeled lipid A, and detected some candidates. We have previously shown that murine stroma-derived ST2 cells do not express CD14, and have approximately the same LPS responsiveness as macrophages. We here raised polyclonal antibodies against purified cell membrane from ST2 cells, as well as LPS binding protein partially purified from the ST2 cell membrane fraction by HPLC.Expression of particular proteins in the macrophage membrane was observed by concentration, separation and visualization of macrophage membrane proteins by immunoprecipitation and subsequent Western blot with antibodies raised against the membrane fraction. We also detected 57 kDa and 53kDa proteins as lipid A binding proteins by ligand blot, respectively with [^3H]-lipid A precursor (PE-406) and [^3H]-lipid A (PE-506).

细菌内毒素脂多糖(LPS)通过激活宿主细胞，包括巨噬细胞，诱导多种细胞因子的表达。为了鉴定和鉴定在内毒素诱导的细胞激活中发挥重要作用的新分子，我们利用抗膜蛋白抗体和同位素标记的脂质A，并检测了一些候选分子。我们先前已经证明，小鼠基质来源的ST2细胞不表达CD14，并且具有与巨噬细胞大致相同的内毒素反应性。我们制备了针对ST2细胞纯化的细胞膜的多克隆抗体，以及用HPLC从ST2细胞膜部分纯化的内毒素结合蛋白。通过免疫沉淀浓缩、分离巨噬细胞膜蛋白和随后用针对膜部分的抗体进行免疫印迹，观察巨噬细胞膜上特定蛋白的表达。我们还通过配基印迹检测到57 kDa和53 kDa的蛋白质分别作为脂蛋白A的结合蛋白，分别与[~3H]-脂蛋白A前体(PE-406)和[~3H]-脂蛋白A(PE-506)结合。

项目成果

Hashimoto,M.,Kirikae,F.Toyooka,K.,Kaneko,A., et al.: "Protective Effect of OK-432 on Mice against Endotoxemia and Infection with Pseudomonas aeruginosa and Salmonella enteritidis."Microbiol.Immunol.. (in press).

Hashimoto,M.、Kirikae,F.Toyooka,K.、Kaneko,A. 等人：“OK-432 对小鼠内毒素血症和铜绿假单胞菌和肠炎沙门氏菌感染的保护作用。”Microbiol.Immunol..（

Swierzko, A.S., Kirikae, T., kirikae, F., Hirata, M., et al.: "Biological activities of Proteus lipopolysaccharides and their interaction with polymyxin B and a 18-kilodalton cationic antimicrobial protein (CAP18) -derived peptide."J.Med. Microbiol.. 49.

Swierzko, A.S.、Kirikae, T.、kirikae, F.、Hirata, M. 等人：“变形杆菌脂多糖的生物活性及其与多粘菌素 B 和 18 千道尔顿阳离子抗菌蛋白 (CAP18) 衍生肽的相互作用。

Kawano, C., Muroi, K., Yoshizaka, M., Kirikae, T., Ozawa, K., et al.: "Filamented Pseudomonas aeruginosa and a markedly high level of endotoxin in cerebrospinal fluid"J.Infect.. 41. 114-115 (2000)

Kawano, C.、Muroi, K.、Yoshizaka, M.、Kirikae, T.、Ozawa, K.等人：“丝状铜绿假单胞菌和脑脊液中内毒素水平显着升高”J.Infect.. 41

Swierzko,,A.S.,Kirikae,T.,Kirikae,F.,Hirata,M., et al.: "Biological activities of Proteus lipopolysaccharides and their interaction with polymyxin B and a 18-kilodalton cationic antimicrobial protein (CAP18)-derived peptide."J.Med.Microbiol.. 49. 127-138

Swierzko,,A.S.、Kirikae,T.、Kirikae,F.、Hirata,M. 等人：“变形杆菌脂多糖的生物活性及其与多粘菌素 B 和 18 千道尔顿阳离子抗菌蛋白 (CAP18) 衍生肽的相互作用

Kakinuma,N.,Iwai,H.,Takahashi,S.,Hamano,K., et al.: "Quinolactacins A, B and C: novel quinolone compounds from Penicillium sp.EPF-6."J.Antibiot. 53. 1252-1256 (2000)

Kakinuma, N.、Iwai, H.、Takahashi, S.、Hamano, K. 等人：“Quinolactacins A、B 和 C：来自青霉属 sp.EPF-6 的新型喹诺酮化合物。”J.Antibiot。