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Development of an analytical HPLC methodology to study the effects of thymosin β4 on actin in sputum of cystic fibrosis patients

机译:开发分析性HPLC方法以研究胸腺素β4对囊性纤维化患者痰中肌动蛋白的影响

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摘要

A high-performance liquid chromatography (HPLC) methodology is presented. Using bovine and rabbit F- and G-actin, this methodology results in both fractions as being well-resolved peaks, which were confirmed by dot blot immunoassay and fluorescence microscopy. F- and G-actin were incubated with thymosin β4 (Tβ4) and DNase and then analyzed by HPLC, which indicated that Tβ4 and DNase inhibit G-actin polymerization and that Tβ4 depolymerizes F-actin in a dose- and time-dependent manner. The F- and G-actin content in sputum from healthy controls and cystic fibrosis (CF) patients were measured by HPLC before and after incubation with Tβ4, DNase, and gelsolin. These data demonstrate higher quantities of F-actin in the sputum of CF patients compared to healthy individuals, and also demonstrate a significantly increased F/G-actin ratio in CF sputum. Further, Tβ4, DNase, and gelsolin each increase the depolymerization of F-actin in CF sputum in a dose-dependent fashion that is additive when these agents are combined.
机译:提出了一种高效液相色谱(HPLC)方法。使用牛和兔的F-和G-肌动蛋白,该方法将两个部分均分离为峰,并通过斑点印迹免疫分析法和荧光显微镜确认。将F-和G-肌动蛋白与胸腺素β4(Tβ4)和DNase孵育,然后通过HPLC分析,表明Tβ4和DNase抑制G-肌动蛋白聚合,并且Tβ4以剂量和时间依赖性解聚F-肌动蛋白。在与Tβ4,DNase和凝溶胶蛋白孵育之前和之后,通过HPLC测定健康对照组和囊性纤维化(CF)患者痰液中F-和G-肌动蛋白的含量。这些数据表明,与健康个体相比,CF患者的痰中F-肌动蛋白的含量更高,并且还表明CF痰中F / G-肌动蛋白的比率显着增加。此外,Tβ4,DNase和凝溶胶蛋白各自以剂量依赖的方式增加CF痰中F-肌动蛋白的解聚,当这些试剂合并时,这种作用是加和的。

著录项

  • 来源
    《Annals of the New York Academy of Sciences》 |2012年第2012期|p.86-92|共7页
  • 作者单位

    Department of Biochemistry and Molecular Biology, The George Washington University School of Medicine and Health Sciences, Washington DC, 10405 Chelsea Manors Court, Great Falls, VA 22066;

    Department of Internal Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts;

    Department of Biochemistry and Molecular Biology, The George Washington University School of Medicine and Health Sciences, Washington DC;

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  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

    thymosin β4; actin; cystic fibrosis; sputum; HPLC;

    机译:胸腺素β4;肌动蛋白囊性纤维化;痰;高效液相色谱;

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