Although cellular oxidation reactions are normal and necessary for organisms growing aerobically, their by-products, in many cases, have been shown to be deleterious to the cell producing them, as well as to the surrounding tissues. This damage is most notable in diseases such as rheumatoid arthritis and immune damage to the kidney and lung. There is evidence to suggest that the oxidation of methionine residues (to methionine sulfoxide) in proteins plays a key role in this damage. The quantitation of methionine oxidation in these proteins as well as their identification is lacking because there is no easy and rapid assay. We propose to develop a quantitative ELISA based on raising antibodies that will specifically recognize methionine sulfoxide residues in proteins. These antibodies will also be used in Western analysis of the oxidized proteins as well as using the antibodies to affinity purify the methionine sulfoxide containing proteins. Synovial fluid obtained from patients with arthritis will initially be used for these experiments. This assay could potentially be used to assess the degree of inflammation as well as the effectiveness of an anti-inflammatory drug. It would be of major importance to study the regulation of an enzyme, called methionine sulfoxide reductase (MsrA), that is present in practically all tissues and specifically reduces methionine sulfoxide to methionine. This enzyme could play a major role in ensuring the recovery of a cell that is subjected to oxidants. Since nothing is known about the regulation of this enzyme, we propose to initially study the levels of this enzyme in neutrophils before and after activation with phorbol esters and to see whether there is any correlation with the amount of methionine sulfoxide as assayed by the ELISA described above. To fully understand the importance of MsrA to the organism, we propose to create a mouse deficient in this enzyme. If the animal develops normally, its response to inflammation in autoimmunity will be determined by backcrosses to mice with autoimmune or arthritis backgrounds.
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