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1From the Laboratory of Retinal Cell and Molecular Biology, 3Laboratory of Mechanisms of Ocular Diseases, National Eye Institute, National Institutes of Health, Bethesda, Maryland; and 4Department of Ophthalmology, University of Maryland School of Medicine, Baltimore, Maryland.
PURPOSE. ß-Carotene 15,15' monooxygenase (ß-CM) catalyzes the central cleavage of ß-carotene to all-trans-retinal, the first step in vitamin A synthesis. This study was conducted to determine the expression of ß-CM in the mammalian retina and RPE, to assess its relevance in carotenoid-retinoid metabolism in the retina and RPE.
METHODS. RT-PCR was used to detect expression of ß-CM mRNA in the retina and RPE-choroid of the mouse, cow, human, and monkey and in RPE cells and other cell lines. Immunofluorescence microscopy was used to localize ß-CM in mouse and monkey retina with an anti-peptide antibody specific for ß-CM.
RESULTS. By RT-PCR, ß-CM mRNA was detected at a low level in mouse and monkey retina and in the RPE-choroid of the monkey but not of the mouse. Conversely, ß-CM mRNA was expressed at a low level in both human and bovine RPE-choroid, but not in the retina of either. RPE primary cultured cells of the monkey also showed ß-CM mRNA expression, although the three human lines did not. In addition, of nine other cell lines tested, only COS-7 was positive for ß-CM. Immunofluorescence microscopy showed weak immunoreactivity in the inner retina in both the mouse and monkey. ß-CM immunoreactivity was not detectable in RPE of the mouse. Use of a long-wavelength exciting and emitting secondary probe to mitigate lipofuscin autofluorescence, facilitated the detection of a low level of ß-CM immunoreactivity in monkey RPE.
CONCLUSIONS. ß-CM mRNA and protein are expressed at low levels in the mammalian retina and RPE-choroid. Given the low and variable expression of ß-CM in the retina and RPE, it can be concluded that ß-CM is not necessary for a conserved retina or RPE-specific function, but may be necessary for a species-specific function.
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