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C.C. McCormick, C.L. Balzli, A.R. Caballero, R.J. O'Callaghan; Degradation of Rabbit Tears by Specific Pseudomonas Proteases . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1911.
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To determine the ability of two Pseudomonas species and each of 4 P. aeruginosa proteases to degrade tear proteins in vitro.
Rabbit tears (25 µl) were incubated at 33oC for 24 hours with 5000 colony forming units of P. aeruginosa (PA01) or P. putida (non–ocular pathogen). Purified proteases, protease IV (PIV), alkaline protease (AP), elastase B (LasB), or Pseudomonas aeruginosa small protease (PASP) (3 µg of each) were incubated for 5 hours at 33oC with rabbit tears. After incubation, samples were analyzed for protein degradation by high pressure liquid chromatography (HPLC) and SDS–PAGE.
Incubation of tears with viable P. putida for 24 hours caused no detectable change in proteins by SDS–PAGE or HPLC. Incubation of rabbit tears with P. aeruginosa for 24 hours at 33oC resulted in 20% reduction of tear proteins in both SDS–PAGE and HPLC analyses. The proteolytic activity of purified PIV, after 5 hours incubation, reduced the by > 60% number of protein bands detectable by SDS–PAGE and HPLC, and revealed a major accumulation of degraded proteins at a molecular weight of 6–10 kDa. The AP activity accounted for a > 80% reduction in protein bands with no detectable accumulation of partially degraded proteins. The LasB activity accounted for approximately a 40% reduction in tear protein bands and an accumulation of partially degraded protein at 8 kDa. The PASP activity caused approximately 30% reduction in tear proteins. There was no accumulation of partially degraded proteins detected.
Pseudomonas aeruginosa proteases degrade rabbit tear proteins, a likely necessary reaction in the development of keratitis.
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