Real-Time Imaging of NADPH Oxidase Activity in Living Cells Using a Novel Fluorescent Protein ReporterReportar como inadecuado




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Production of reactive oxygen species ROS has been implicated in the pathology of many conditions, including cardiovascular, inflammatory and degenerative diseases, aging, muscular dystrophy, and muscle fatigue. NADPH oxidases Nox have recently gained attention as an important source of ROS involved in redox signaling. However, our knowledge of the source of ROS has been limited by the relatively impoverished array of tools available to study them and the limitations of all imaging probes to provide meaningful spatial resolution. By linking redox-sensitive GFP roGFP to the Nox organizer protein, p47phox, we have developed a redox sensitive protein to specifically assess Nox activity p47-roGFP. Stimulation of murine macrophages with endotoxin resulted in rapid, reversible oxidation of p47-roGFP. In murine skeletal muscle, both passive stretch and repetitive electrical stimulation resulted in oxidation of p47-roGFP. The oxidation of p47-roGFP in both macrophages and skeletal muscle was blocked by a Nox specific peptide inhibitor. Furthermore, expression of p47-roGFP in p47phox deficient cells restored Nox activity. As Nox has been linked to pathological redox signaling, our newly developed Nox biosensor will allow for the direct assessment of Nox activity and the development of therapeutic Nox inhibitors.



Autor: Rituraj Pal, Poulami Basu Thakur, Shumin Li, Charles Minard, George G. Rodney

Fuente: http://plos.srce.hr/



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