Abstract
Publisher Summary This chapter describes the methods to manipulate the activity of the Rac1-regulated reduced Nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase in vivo using adenoviral vectors. The redox status of cells, and ultimately the whole tissue, is dependent on a balance between the reducing and oxidizing agents within cells. The reducing properties of the cell are affected by the expression of antioxidant enzymes, such as glutathione, catalase, thioredoxin, and superoxide dismutase among others. In addition, methods detailing the fluorescence and luminescence-based assays used for measuring the reactive O 2 species (ROS) generation regulated by the Rac1 GTPase are discussed, along with The use of recombinant adenoviruses as tools for manipulating Rac1-regulated signaling in vitro and in vivo . Procedure for the measurement of ischemia/reperfusion-induced, Rac1-regulated ROS production in mouse liver in vivo as well as as the measurement of hydrogen peroxide using amplex red reagent is also described.Concepts related to the introduction and detection of Rac1N17 in the liver tissue and adenovirus results in efficient expression of Rac1N17 in vivo , ischemia/reperfusion protocol and measurement of superoxide in liver tissue by lucigenin-enhanced chemiluminescence arediscussed.
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