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K.G. Shadrach, M.E. Rayborn, P. Senanayake, J.G. Hollyfield; Hyaluronan In The IPM: Localization, Synthesis And Turnover . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3651.
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Purpose:Hyaluronan (HA) is found in the interphotoreceptor matrix (IPM) and is proposed to be involved in matrix organization because of its ability to interact (bind) with other IPM molecules. Recent studies also indicate that HA may be more abundant around cones than rods. In this study we determine the rate of synthesis and turnover of HA in the IPM of the mouse Methods:Intraocular injections of 3H–glucosamine and 35S–sulfate (40 µCi/eye in a volume of 2–3 µl/eye) were administered to adult C57Bl/6J mice. Retinas and retinal pigment epithelium/choroid were recovered 1, 2, 4, and 8 days post–injection using 10 mice per recovery time. Immediately after sacrificing, the retinas were isolated and the vitreous removed under microscopic dissection from the inner retinal surface. The retinas were then rinsed with 1X PBS and extracted with TBS, pH 8.0. For FACE analysis, complex carbohydrates of retina , RPE/choroid and IPM were isolated and frozen. The tissues and retinal rinse/extracts were digested with proteinase K followed by ethanol precipitation of the complex carbohydrates. The precipitate fractions were digested with hyaluronidase SD and chondroitinase ABC to liberate disaccharides from the contained chondroitin and hyaluronan glycosaminoglycans (GAGs). The reducing end of the resulting saccharides were fluorotagged with AMAC and separated by electrophoresis. The fluorescent bands were digitized and their intensities quantified. Specific bands were cut from the gel for scintillation counting. Results:: Radiolabeled unsulfated disaccharides, 4–sulfated disaccharides and 6–sulfated disaccharides of chondroitin were present at 10–12 fold higher levels than were labeled HA disaccharides. HA specific activity was highest at day 1 and decreased exponentially thereafter, suggesting a half–life between 1 to 2 days. Conclusions:The mouse retina synthesizes hyaluronan, which can be extracted with procedures that remove the IPM. The short half–life of HA in this compartment suggests a dynamic turnover and assembly of this matrix molecule.
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