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

Annals of the New York Academy of Sciences2012PMID: 23050822

View on PubMed DOI: 10.1111/j.1749-6632.2012.06671.x

Animal StudyTB-500

Plain Language Summary

Develops an HPLC methodology for quantifying F-actin and G-actin separately and studies the effects of thymosin β4 on actin in cystic fibrosis sputum. TB4 depolymerized F-actin dose- and time-dependently in vitro; CF sputum showed altered F/G-actin ratios compared to healthy controls. Establishes a clinically relevant application of TB4's actin-depolymerizing activity: dissolving the viscous actin-containing mucus in CF airways—the extracellular actin released from dying neutrophils that greatly increases CF sputum viscosity and contributes to airways obstruction.

Abstract

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.

Authors

Badamchian, Mahnaz; Damavandy, Ali A; Goldstein, Allan L