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D.T. Organisciak, R.M. Darrow, L.S. Barsalou, B. McDonald, P. Wong; Circadian and Stress Induced Changes in Rod Outer Segment Crystallins . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4594.
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Crystallins may serve a protective, chaperone–like role in post mitotic tissues such as retina and reportedly migrate within cells during stress. In this study we sought to determine conditions under which crystallin migration might occur within rat photoreceptors.
60 day old male Sprague–Dawley rats were dark adapted for 16 hours and then sacrificed at 8 hour intervals during the day or night. Retinas were excised without vitreous and ROS purified by discontinuous sucrose gradient ultracentrifugation. ROS isolated from 16 retinas were used for 2D gel electrophoresis and proteins visualized by Coomassie staining with Western analysis of crystallins. Other rats were treated with intense visible light beginning at 9 am, for 8 hours and immunohistochemistry performed on the excised eyes. Some rats were given an antioxidant and then treated under hyperthermic conditions for 2 hours in darkness before 2D gel electrophoresis of ROS proteins.
Immunohistochemistry revealed a marked accumulation of αB–crystallin staining in ROS of light stressed rats and a substantially reduced level of γS–crystallin. 2D gel/Western analysis of ROS isolated from unexposed rats at 1am, 9am and 5pm exhibited a clear gradient of increased α– and ß–crystallin staining at 5pm along with an isoelectric pH charge train suggestive of α–crystallin phosphorylation. ROS γ–crystallin levels were elevated at 1am, but greatly reduced at 5pm. ROS from rats stressed only by heat at these same times had reduced levels of all crystallins, but 5pm ROS still retained higher levels than those from rats at 1am or 9am. Pretreatment of euthermic or hyperthermic rats with the antioxidant dimethylthiourea resulted in the loss of αB – and γ–crystallins from ROS with little effect on ß–crystallin levels.
The marked differences in αB– and γS– crystallin staining in ROS during light exposure suggests that movement occurs during stress. Differences in ROS crystallin levels also occur during the course of the day and night and following brief hyperthermic stress at various times. Antioxidant pretreatment of rats appears to promote αB– and γ–crystallin migration out of ROS. The protection afforded retinal photoreceptors in light exposed rats by either antioxidants or by light treatment beginning at 5pm may be related to the levels and types of crystallins within the ROS compartment and the photoreceptor cell body.
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