The long-term objectives of this research are to identify the oxidative toxins generated by phagocytic cells (neutrophils, macrophages) and to determine their microbicidal mechanisms. This knowledge is expected to lead to improved/new methods of pharmacological intervention in combating infection, as well as provide insights into the causes and progression of human diseases associated with oxidative stress. Oxidative reactions within stimulated phagocytes will be probed by using unique fluorescein-conjugated polyacrylamide microspheres that, when opsonized, are avidly phagocytosed by these cells. Fluorescence changes of the engulfed particles will be used to monitor in real time the intracellular oxidation processes; recovery of the dye and subsequent chemical analyses will identify the oxidant(s) generated by the cells. These studies will resolve major issues concerning the function of the neutrophil enzyme, myeloperoxidase, and the microbicidal competence of the putative macrophage-generated toxin, peroxynitrous acid (ONOOH). In other studies, radiobiological methods will be used to examine the microbial toxicity of short-lived oxidants that can be formed from peroxynitrite decomposition in physiological environments, namely the radicals 0H, C03-, N02, and the nitrosating agent N203. For C03-, ONOOH, and other reactive nitrogen species (RNS) that are found to be toxic to selected microbes, the metabolic dysfunctions associated with cellular death will be identified by various biophysical and biochemical analyses. The membrane permeabilities of the RNS will be determined using model liposomal systems containing entrapped reductants; this information will be used along with the extensive kinetic data available on oxidation rates by the RNS to mathematically simulate the fate of the short-lived oxidants within the phagosome. Finally, the molecular sites leading to oxidative inactivation will be investigated using advanced mass spectral analyses for a P-type H+-ATPase whose functional loss is implicated in the fungicidal mechanisms of these oxidants.
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