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H Nomoto, T Yonezawa, A Ohtsuka, F Shiraga, H Ohtsuki, Y Ninomiya; Cloning and Immunohistochemical Studies of Limitrin in the Mouse Brain and Retina . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1436.
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Purpose: We first cloned cDNAs encoding a new transmembrane protein, limitrin, and designated it as a new member of the immunoglobulin superfamily. To examine the localization of limitrin in the mouse brain and eye, we herein performed immunohistochemical studies. Methods: We employed the PCR select cDNA subtraction method (Clontech) using poly (A+) RNAs isolated from cultured cells enriched in human brain capillary endothelial cells (HBCE), human adult skin endothelial cells and human umbilical vein endothelial cells. Overlapping cDNAs of limitrin derived from those of HBCE were isolated and the entire coding sequence was determined. Rabbit polyclonal antibody was raised against a human peptide sequence. For immunohistochemistry, cryostat sections were prepared from adult Balb/C murine brain and eye. The sections were examined with a BX50 microscope (Olympus) equipped with an Axio Vision system (Zeiss). Result: We determined the sequence of human and mouse limitrin. Their deduced amino acid sequence contained a N-terminal hydrophobic signal peptide, two v-type Ig domains, and a transmembrane region close to the carboxyl terminal end. M-limitrin mRNA was abundantly expressed in cultured mouse astrocytes rather than that of whole brain. We confirmed by Western-blotting analysis that rabbit anti h-limitrin antibody reacted to h- and m- limitrin. Using this antibody, immunohistochemical studies revealed that m-limitrin was localized around blood vessels in the parenchyma, optic nerve and retina; furthermore it was always present at the parenchymal side of basement membranes around blood vessels. Interestingly, however, m-limitrin was not expressed in the subarachnoid space, choroid plexus and choroidal vessels, suggesting that expression of limitrin is regulated in a tissue specific manner. Conclusions: These results indicated that astrocytes were the primary source of limitrin and suggested that limitrin was localized at the glia limitans of astrocytes and, according to its distribution, was related to the function of blood brain and retinal barriers.
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