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A Goldwich, CR Ethier, ER Tamm; Perfusion With the Olfactomedin Domain of Myocilin Does Not Show Major Effects on Outflow Facility in Anterior Eye Segment Organ Cultures . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1036.
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Purpose: To examine the effects of the olfactomedin domain of myocilin, which harbors most of the mutations in GLC1A-linked glaucoma, on aqueous humor outflow facility. Methods: Eucaryotic 293-EBNA cells were transfected with the cDNA of human myocilin linked to a polyhistidine tag at the C-terminus. Recombinant myocilin was proteolyzed by 293-EBNA cells in two major fragments. The C-terminal fragment containing the entire olfactomedin domain (OD-His) was isolated by Ni2+ chelate chromatography. The identity of the fragment was confirmed by N-terminal sequencing, and one- and two-dimensional gel electrophoresis with western blots. Eight pairs of porcine eyes and three pairs of human eyes were perfused in anterior segment organ cultures with 14µg/ml of OD-His for at least 20 h. The morphology of the anterior eye segments was investigated by light and electron microscopy. Results: The N-terminal sequence of OD-His was LKSELTEV, the molecular weight was 29-32 kDa (under both reducing and non-reducing conditions) and the pI was 5.2 to 5.4. Perfusion of porcine anterior eye segments (n=8) resulted in a facility increase of 5 % which was not statistically significant. In human eyes (n=3), there was a modest increase in facility of 39% which was again not statistically significant. Conclusion: The isolated olfactomedin domain of myocilin does not significantly affect outflow facility in perfused anterior eye segment organ cultures. Any direct effects of myocilin on outflow facility appear to require N-terminal sequences containing the leucine zipper region.
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