The long term objective is to characterize, at a biochemical level, the process of corneal ulceration by identifying not only the proteases involved but other factors influencing the degradative process. Proteases are controlled by several means including inhibitory molecules. In order to understand the role of proteases in the degradative process, the identification of proteases in the cornea, understanding their inhibitory mechanism, factors which alter the levels of the inhibitors and the source of these inhibitors is important. Through a detailed analysis of the molecular mechanisms involved in protecting the normal cornea and those occurring during ulceration, a therapeutic means can be devised to prevent cornea ulceration, promote corneal matrix repair and restore transparency to the cornea.
The specific aims of this proposal are the following: 1) to determine what types of protease inhibitory activities are present in the normal corneal and to characterize their molecular properties. Inhibitors will be separated by electrophoresis and detected by enzymatic and immunological techniques. Corneal inhibitors will be isolated by chromatographic techniques and their inhibitory and molecular properties studied by biochemical techniques. 2) to determine whether corneal protease inhibitors are synthesized within the cornea or whether they are sequestered from the fluids which bathe the cornea. Corneas in short term organ culture will be tested for synthesis of the inhibitors using 35S-methionine followed by immunological isolation of the inhibitors and for sequestration of 125I-labeled inhibitors. 3) to determine the conditions which alter the synthesis and/or sequestration of proteases and protease inhibitors by the cornea. The effect of retinoid levels of synthesis and/or sequestration of corneal proteases and protease inhibitors and will be tested using the rat model system of vitamin A deficiency and retinoid excess.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Visual Sciences A Study Section (VISA)
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Medical College of Wisconsin
Schools of Medicine
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Twining, S S; Fukuchi, T; Yue, B Y et al. (1994) Corneal synthesis of alpha 1-proteinase inhibitor (alpha 1-antitrypsin). Invest Ophthalmol Vis Sci 35:458-62
Twining, S S; Fukuchi, T; Yue, B Y et al. (1994) Alpha 2-macroglobulin is present in and synthesized by the cornea. Invest Ophthalmol Vis Sci 35:3226-33
Twining, S S; Fukuchi, T; Yue, B Y et al. (1994) Alpha 1-antichymotrypsin is present in and synthesized by the cornea. Curr Eye Res 13:433-9
Twining, S S; Kirschner, S E; Mahnke, L A et al. (1993) Effect of Pseudomonas aeruginosa elastase, alkaline protease, and exotoxin A on corneal proteinases and proteins. Invest Ophthalmol Vis Sci 34:2699-712