We have utilized cDNA probes and in situ hybridization techniques to define the subcellular localization of interphotoreceptor retinoid-binding protein (IRBP) mRNA in bovine and monkey retinas. Results suggest that the mRNA is mainly localized in rod photoreceptor neurons within the outer nuclear layer of the retina. IRBP mRNA is also abundant in cells of the pineal gland, strengthening the analogy between rod photoreceptor cells and pinealocytes.Interphotoreceptor retinoid-binding protein cDNA
S-Antigen is a major soluble protein of the retina and pineal. It is capable of inducing experimental autoimmune uveitis (EAU) in laboratory animals and also seems to play an important role in the visual cycle. The results of partial cDNA sequence analysis reveal interesting homologies with a-transducin, a GTP-binding protein of retina and other purine nucIeotide-binding proteins. In particular S-antigen shows over 50% identity to the proposed pertussis toxin ADP-~~sylation site of a-transducin. It also contains the Gly-X-X-X-X-Giy-Lys pattern common to phosphoryl binding sites. A possible relationship between S-antigen and purine nucleotide-binding proteins is discussed. There is also evidence for a repetitious /I-structure in the C-terminal half of S-antigen, with a monoclonal antibody epitope in a helical region at the C-terminus.{Bovine retina) S-Antigen Uveitb eDNA seqwnce a-Transducin ADP-ribosylation
Using in situ hybridization and platelet-derived growth factor (PDGF) cDNA probes labeled with horseradish peroxidase, PDGF-A and -B (c-cis proto-oncogene) mRNA transcripts were identified and localized in proliferating cultures. A human retinal pigment epithelial (RPE) cell line and a glial cell line were treated with either transforming growth factor beta-1 (TGFB1), phorbol-12-myristate-13-acetate (PMA), or thrombin from human plasma and compared for their ability to stimulate the production of PDGF-A and -B. Expression of both PDGF-A and -B transcripts were found to be localized predominantly in the cytoplasm of TGFB1-treated RPE cells, with a portion of these cells displaying a hybridization response in the nuclear region. When compared to PMA- and thrombin-treated cells, TGFB1 stimulated the RPE cell line to yield the greatest amount of detectable PDGF mRNA. In addition, the hybridization response observed in TGFB1-treated cells was shown to be RNA dependent.
The microsomal prqparation from the lactating bovine mammary tissue was solubilized by treatment with nonionic detergent, NP-40, at a protein/detergent ratio of 1.5: 1 and a detergent concentration of 0.5 %. Following centrifugation at 147000 x g for 120 min, the supernatant fraction was incubated with labeled sugar nucleotides, GDP-Man and UDP-GlcNAc. It was found to synthesize a series of lipid-linked saccharides up to (Man),-(GlcNAc)2. The solubilized glycosyltransferases retained up to about 60 % of the activity after two weeks of storage at 4°C. The biosynthesis of glycolipids was stimulated by a mixture of lipids obtained by extracting the mammary microsomes with CHCl,/CH,OH (2: 1).A labeled lipid-linked tetrasaccharide of the structure Manal + 3Man~-+GlcNAcj+GlcNAc was isolated by labeling baby hamster kidney cells with [2-,H]mannose under conditions of glucose starvation followed by extraction of the cells with CHCI,/CH,OH (2: 1) and separation of the lipids by high-performance liquid chromatography. When this lipid-linked tetrasaccharide was incubated with the solubilized bovine mammary microsomes and GDP-Man, it was elongated to a lipid-linked heptasaccharide having the structure M a d + 2Manctl+2Manal --t 3(Mancrl+ 6)Manj+ GlcNAcj+ GlcNAc. The kinetics of the elongation reaction also revealed the intermediary formation of smaller amounts of lipid-linked pentasaccharide and hexasaccharide. The elongation reaction did not require any divalent metal ion and had a broad pH optimum between 6.8 and 7.6. The lack of inhibition of the elongation reaction by EDTA or amphomycin support earlier studies that GDP-Man rather than mannosylphosphoryldolichol, is the direct donor of mannosyl residues for the biosynthesis of glycolipids up to (Man),(GlcNAc),. Mannosylphosphorylretinol was ineffective as mannosyl donor for the elongation reaction.It is now well established that for most of the eukaryotic systems, the lipid-linked tetradecasaccharide, (Glc),(Man),-(GlcNAc),, serves as the major precursor for an en bloc glycosylation of the nascent polypeptides of asparagine-linked glycoproteins [I]. Following transfer, the oligosaccharide is extensively processed and modified to givc rise to highmannose, complex and hybrid glycoproteins [I].Previous reports from our laboratory have shown that microsomal preparations from both the lactating bovine mammary tissue and Saccharomyces cerevisiae catalyze a stepwise assembly of the branched tetradecasaccharide [2 -41. Even though these incubations yield multiple, minor isomers of several intermediates during the lipid-linked assembly of the precursor [2, 5 -81, supplementation of incubation mixtures with dolichyl phosphate to optimize and drive the biosynthetic sequence, causes a dramatic shift in the relative proportion of the isomers in favor of an ordered sequence [5, 61, identical to that proposed for biosynthesis of (Glc),(Man),(GlcNAc)2 unit in vivo in Chinese hamster overy (CHO) cells [9].On the assumption that one glycosyltransferase is required per linkage assembly in ...
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