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C D Bridges

Publications and source records attributed to C D Bridges.

At least 55 records · Page 3Linked to original sources
PubMed1985

Participation of photoreceptor cells in retrieval and degradation of components in the interphotoreceptor matrix.

Evidence for endocytosis of components of the interphotoreceptor matrix by photoreceptor inner segments is presented. Photoreceptors cultured in the presence of horseradish peroxidase (HRP) rapidly interiorized this tracer as evidenced by the presence of HRP-reaction product within primary endocytotic vesicles in the inner segment cytoplasm. The tracer was also observed in multivesicular bodies. Retinas were incubated in a suspension of interstitial retinol-binding protein (IRBP) coated colloidal gold (CG). IRBP-CG was interiorized by photoreceptors where it was found in endocytotic vesicles and in multivesicular bodies in both rod and cone inner segments. Acid phosphatase reaction product co-localized with IRBP-CG in multivesicular bodies suggesting that IRBP is degraded following endocytosis and is not recycled.

Culture Techniques↗
PubMed1985

The carbohydrate of bovine interstitial retinol-binding protein.

Pronase digestion of IRBP yielded one major glycopeptide (IRBP-GP1) of approximate Mr 2,500 IRBP-GP1 was heterogeneous by anion exchange chromatography, an observation that was attributed to the presence of varying numbers of sialic acid residues. Asialo-IRBP-GP1 was also heterogeneous by gel-filtration chromatography, possibly because of varying degrees of fucosylation. A minimum of four (possibly five) concanavalin A-binding glycopeptides was generated by cyanogen bromide cleavage of IRBP. These findings, in conjunction with earlier observations, suggest that bovine IRBP contains 4-5 N-linked oligosaccharide chains. These chains appear to have the same basic complex biantennary structure. They contain galactose, mannose and N-acetylglucosamine, in addition to sialic acid and fucose. Nearly all of the IRBP secreted by bovine retinas incubated with (3H)-leucine in the presence of tunicamycin was nonglycosylated and did not bind to concanavalin A. On SDS polyacrylamide gels non-glycosylated IRBP had an Mr that was 5,000-6,000 below that of the glycosylated protein. Non-glycosylated IRBP had an Mr of 250,000 on gel-filtration columns, a value that was identical with that of glycosylated IRBP. It is concluded that the oligosaccharide has little influence on the molecular conformation of IRBP, which is believed to be responsible for the anomalously high Mr seen on these columns. When mixed, the glycosylated and nonglycosylated forms of IRBP appeared to associate as stable aggregates. Bovine retinas incubated with (14C)-leucine and (3H)-fucose in the presence of castanospermine, an inhibitor of glucosidase I, secreted IRBP that appeared to have a reduced number of fucose residues. Swainsonine, an inhibitor of mannosidase II, effected only a marginal reduction in the degree of fucosylation of secreted IRBP.

Alkaloids↗
PubMed1985

Synthesis and secretion of interstitial retinol-binding protein by the human retina.

Interstitial retinol-binding protein (IRBP) is a soluble glycoprotein present between the retina and pigmented epithelium, which may function to shuttle vitamin A derivatives between these tissues. While previous studies have shown that the retina is solely responsible for IRBP synthesis, the specific retinal cell(s) in which this occurs has not been established. Since the carbohydrate moiety of IRBP contains fucose, the authors have analyzed the sites of incorporation of 3H-fucose in the human retina in vitro, using autoradiography. Following a 30-min pulse incubation, all retinal layers exhibited incorporation of label; however, the rod photoreceptor inner segments contained one- to two-fold more radioactivity than was present in any other retinal compartment. In autoradiographs of retinas recovered following a 4 hr chase incubation, all retinal layers retained similar levels of radioactivity with the exception of the rod photoreceptors, cone photoreceptors and cells in the inner nuclear layer, which lost 75, 11, and 14 percent, respectively of the radioactivity present immediately following the 30-min pulse. Proteins present in the chase incubation medium were analyzed by polyacrylamide gel electrophoresis and fluorography. The principal labeled component in the chase medium was identified as IRBP by immunoprecipitation with antibovine-IRBP immunoglobulins. Thus, the major loss of 3H-fucose radioactivity from rod photoreceptors coupled with the appearance of 3H-labeled IRBP in the incubation media suggests that the rod photoreceptors are primarily responsible for the synthesis and secretion of IRBP.

Adolescent↗
PubMed1985

Vitamin A and interstitial retinol-binding protein in an eye with recessive retinitis pigmentosa.

The composition and amount of vitamin A stored in the retinal pigment epithelium and choroid (RPE-Ch) was evaluated in postmortem donor eyes from a patient with retinitis pigmentosa that was probably inherited by an autosomal recessive mode. Additionally, the soluble proteins in the neural retina and RPE-Ch cytosols and interphotoreceptor matrix were examined collectively for the presence of interstitial retinol-binding protein (IRBP). Although there was depletion of the amount of vitamin A stored in the RPE, this was commensurate with the histopathologic findings on the RPE extent and thickness. No evidence was found for an accumulation of free retinol. Nearly all of the vitamin A stored in the RPE was esterified. As in normal eyes, the retinyl esters consisted mainly of palmitate mixed with a small proportion of stearate. Eleven-cis retinyl esters were present, although their proportion was lower than that reported for normals. IRBP could not be detected in stained gels of the soluble proteins, or by autoradiography of these gels after treatment with 125I-concanavalin A. These findings suggest that depletion of stored vitamin A, accumulation of free retinol, or deficiency of 11-cis isomer are unlikely to be causative factors in the retinal degeneration examined here. Although the depletion of IRBP seen at this advanced stage might be secondary to the advanced loss of photoreceptors, the authors cannot rule out the possibility that a relative deficiency or abnormality in this protein at earlier disease stages may contribute to the pathogenesis of retinitis pigmentosa.

Aged↗
PubMed1985

Interstitial retinol-binding protein (IRBP) in the RCS rat: effect of dark-rearing.

The effect of light- and dark-rearing on the amounts of rhodopsin and interstitial retinol-binding protein (IRBP) in RCS rats and their congenic controls (RCS-rdy+) was determined. Rhodopsin was measured spectroscopically and IRBP by dot-blot enzyme immunoassay utilizing rabbit antibovine IRBP IgG. After P15-20, dark-reared RCS and RCS-rdy+ rats always had more rhodopsin than their light-reared, age-matched counterparts. The rhodopsin in the light-reared RCS rats peaked at about 2 nmol/eye at P20-25. The rhodopsin in the dark-reared RCS rats peaked at about 4 nmol/eye at P60-70. Maintenance of RCS-rdy+ rats in darkness had no effect on their IRBP content, which continued to increase up to P80-110. In both groups of RCS rats, the amount of IRBP reached a peak at P22. In RCS rats maintained in darkness, the amount of IRBP attained at this peak was about twice that in the corresponding light-reared group and in RCS-rdy+ animals at this age. The decline of IRBP after P22 in RCS rats was slowed in darkness by approximately 10 days. This slowed decline of IRBP is associated with a decreased rate of photoreceptor degeneration, and the results are therefore consistent with the hypothesis that the photoreceptors synthesize and secrete IRBP. The layer of membranous debris would restrict the diffusion of IRBP in the subretinal space and could partially exclude this retinol transport protein from access to the zone adjacent to the apical surface of the retinal pigment epithelium (RPE).

Animals↗
PubMed1985

Rod photoreceptors in the human retina synthesize and secrete interstitial retinol-binding protein.

3H-fucose has been used as a probe to identify the retinal cells responsible for the synthesis of the fucose-containing glycoprotein, IRBP. While all retinal cells utilize 3H-fucose, rod photoreceptors show the highest levels of incorporation. Rods, cones and some cells in the inner nuclear layer lose 75%, 11% and 14%, respectively, of their radioactivity during the chase incubation period. Radioactivity lost during the chase incubation period can be recovered from the incubation medium and characterized immunochemically as radiolabeled IRBP. While cones and cells of the inner nuclear layer (Muller's cells?) cannot be excluded as minor contributors, the major share of radioactivity lost from rods indicates that rod photoreceptors are principally responsible for the synthesis and secretion of IRBP.

Autoradiography↗
PubMed1984

Purification and characterization of a retinol-binding glycoprotein synthesized and secreted by bovine neural retina.

A retinol-binding glycoprotein ( IRBP ) was purified in milligram quantities from the extracellular matrix ( interphotoreceptor matrix) that occupies the subretinal space in bovine eyes. IRBP binds 2.2 molecules of all-trans retinol with a KD of approximately 10(-6) M. The holoprotein has lambda max at 280 nm (E1%1 cm = 10.99) and at 330 nm (E1%1 cm = 7.88). When freshly isolated from light-exposed eyes, IRBP contains up to 0.6 molecule of all-trans retinol, together with small amounts of the 11-cis and 13-cis isomers. IRBP also binds exogenous cholesterol, alpha-tocopherol, and all-trans retinoic acid, all of which are completely displaced by all trans retinol. The affinity of alpha-tocopherol for IRBP was at least several orders of magnitude less than that of all-trans retinol. IRBP contains 8.4% by weight of carbohydrate, which consists of sialic acid, neutral hexoses, and glucosamine in the molar ratio of approximately 1:3:2. No galactosamine was detected. Observations on the binding of 125I-labeled lectins to IRBP in sodium dodecyl sulfate-polyacrylamide gels before and after desialosylation suggest that at least one oligosaccharide chain is of the sialated biantennary complex type and contains fucose. The Mr of IRBP on calibrated size-exclusion columns averaged 249,000; on sodium dodecyl sulfate-polyacrylamide gels (with or without dithiothreitol) the apparent Mr was 144,000. IRBP exists in at least four isoelectric forms that bind concanavalin A and have pI values ranging from 4.4 to 4.8. Rabbit anti-bovine IRBP antiserum gave a single precipitin line against purified bovine IRBP , which showed a line of complete identity with crude bovine interphotoreceptor matrix and a line of partial identity with human interphotoreceptor matrix. The human material contains a prominent protein with lectin-binding properties similar to bovine IRBP but with a somewhat faster electrophoretic mobility. When isolated bovine neural retinas were incubated with 3H-labeled fucose, glucosamine, or leucine, a solitary labeled protein identified as IRBP was secreted into the medium. Labeled IRBP could not be detected in the medium when retinal pigment epithelium was incubated with these precursors under the same conditions. Neural retinas incubated with 3H-labeled leucine in the presence of tunicamycin secreted a form of IRBP that did not bind concanavalin A and had an Mr reduced by approximately 5,000.

Amino Acids↗
PubMed1984

A serum-free defined medium for retinal pigment epithelial cells.

Human and bovine RPE cells underwent changes in morphology and culture doubling times when passaged in serum-supplemented medium (CM). Furthermore, late passage human RPE cells subcultured in CM medium increased synthesis of three acidic, 43 000-63 000 D proteins. In order to provide a controlled environment for the study of RPE cells in vitro, we have developed a method for growing human and bovine RPE in a serum-free defined medium (DM). RPE cells grown in DM required a 24 h pretreatment with CM to allow the cells to attach and spread on the substrate. Cells grown in DM retained an epithelioid morphology, a stable culture doubling time, and similar 2-D PAGE patterns through several subculturings.

Animals↗
PubMed1984

Use of high-performance liquid chromatography in the analysis of retinyl and 3,4-didehydroretinyl compounds in tissue extracts of bullfrog tadpoles and goldfish.

HPLC (high-performance liquid chromatography) was used to analyse retinyl and 3,4-didehydroretinyl compounds in tissue extracts from goldfish and bullfrog tadpoles. Using silica columns (packed with 10-micron mu Porasil or 5-micron Ultrasphere particles) eluted with n-hexane (containing a small amount of dioxane or diethyl ether), the authentic all-trans retinyl and 3,4-didehydroretinyl palmitates, retinal and 3,4-didehydroretinal, retinol and 3,4-didehydroretinol were completely separated. Liver and eye extracts of the goldfish and bullfrog tadpoles had mainly esterified all-trans retinol and all-trans 3,4-didehydroretinol. In the liver, these vitamin A were conjugated to a number of fatty acids whereas in the eye, principally one fatty acid was used. Moreover, the relative proportions of all-trans retinol and all-trans 3,4-didehydroretinol (obtained by analysing the saponified esters) were significantly different between some of these body compartments.

Animals↗
PubMed1984

Visual cycle in the mammalian eye. Retinoid-binding proteins and the distribution of 11-cis retinoids.

This work was designed to provide an insight into the mammalian visual cycle by investigating the possible function of retinoid-binding proteins in this system, and the distribution and type of 11-cis retinoids present in the interphotoreceptor matrix and the cytosols of the retinal pigment epithelium and retina. The total retinol and retinal in the soluble fractions from these three compartments was 8% (3.31 nmol/eye) of the retinyl palmitate and stearate stored in the pigment epithelium membrane fractions (39 nmol/eye). Only small amounts of retinoids were detected in the rod outer segment cytosol. The insoluble fractions also contained retinol, nearly all of which was found in the retina. The retinoids in the soluble fractions appeared to be bound to cellular retinol-binding protein (CRBP), cellular retinal-binding protein (CRA1BP) and interstitial retinol-binding protein (IRBP, a high-Mr glycoprotein). Using immunospecific precipitation, immunoblot and immunocytochemical techniques it was demonstrated that IRBP was localized in the interphotoreceptor matrix and was synthesized and secreted by the retina, a process that did not require the protein to be glycosylated. The amount of retinol bound to IRBP increased if the eyes were exposed to light, when it was estimated that the protein carried up to 30% of its full capacity for all-trans retinol. In addition to all-trans retinol, IRBP carried smaller amounts of 11-cis retinol. The proportion of 11-cis retinol was frequently higher in eyes that had been protected from illumination, suggesting that IRBP plays a role in rhodopsin regeneration during dark-adaptation. Additionally, endogenous 11-cis retinoids in the retina and RPE cytosols were bound to an Mr 33,000 protein tentatively identified as CRA1BP. The 11-cis retinoid in the retina cytosol was mainly in the form of retinol, while in the RPE cytosol it was mainly in the form of retinal. Substantial amounts of 11-cis retinol were also found in the insoluble (membrane) fraction from the retina. It is suggested that in the mammalian retina 11-cis retinol is generated from all-trans retinol (possibly in the Muller cells). Lack of an 11-cis retinol oxidoreductase in the retina prevents it from being utilized for rhodopsin regeneration until it has been transported to the pigment epithelium, where it is converted to 11-cis retinal and returned to the rod outer segments. It is also suggested that IRBP may be implicated in the transport of retinoids between the rod outer segments, the Muller cells and the pigment epithelium.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1984

An extracellular retinol-binding glycoprotein in the eyes of mutant rats with retinal dystrophy: development, localization, and biosynthesis.

Interstitial retinol-binding protein (IRBP) is a soluble glycoprotein in the interphotoreceptor matrix of bovine, human, monkey, and rat eyes. It may transport retinol between the retinal pigment epithelium and the neural retina. In light-reared Royal College of Surgeons (RCS) and RCS retinal dystrophy gene (rdy)+ rats, the amount of IRBP in the interphotoreceptor matrix increased in corresponding proportion to the amount of total rhodopsin through postnatal day 22 (P22). In the RCS-rdy+ rats, the amount increased slightly after P23. However, in the RCS rats there was a rapid fall in the quantity of IRBP as the photoreceptors degenerated between P23 and P29. No IRBP was detected by immunocytochemistry in rats at P28. The amount of rhodopsin fell more slowly. Although retinas from young RCS and RCS-rdy+ rats were able to synthesize and secrete IRBP, this ability was lost in retinas from older RCS rats (P51, P88) but not their congenic controls. The photoreceptor cells have degenerated at these ages in the RCS animals, and may therefore be the retinal cells responsible for IRBP synthesis. The putative function of IRBP in the extracellular transport of retinoids during the visual cycle is consistent with a defect in retinol transport in the RCS rat reported by others.

Aging↗
PubMed1984

Characterization, localization, and biosynthesis of an interstitial retinol-binding glycoprotein in the human eye.

Human eyes contain an Mr 135K retinol-binding protein that is analogous to interstitial retinol-binding protein ( IRBP ) in the subretinal space of bovine eyes. It is a glycoprotein, because it binds 125I-concanavalin A, 125I-wheat germ agglutinin and 125I-Lens culinaris hemagglutinin. It does not bind Ricinus communis agglutinin I. After desialation, it binds Ricinus communis agglutinin I, but loses its capacity to bind wheat germ agglutinin. These observations, coupled with the known specificities of these lectins, suggest that at least one of the oligosaccharide chains is a sialated , biantennary complex type containing fucose. Both by direct analysis of dissected ocular tissues and by immunocytochemistry it was shown that human interstitial retinol binding protein is an extracellular protein that is confined predominantly to the subretinal space. Monkey retinas incubated in vitro in medium containing [3H]leucine were shown to synthesize and secrete this protein into the medium, a conclusion that was confirmed by immunoprecipitation with an immunoglobulin fraction prepared from rabbit antibovine IRBP serum. Virtually no other labeled proteins were detectable in the medium. It is concluded that interstitial retinol-binding protein meets many of the requirements for a putative transport protein implicated in the transfer of retinol between the pigment epithelium and retina during the visual cycle, and that the neural retina may play an important role in regulating its amount in the subretinal space.

Amino Acids↗
PubMed1983

Utilization of exogenous retinol by frog pigment epithelium.

High-performance liquid chromatography was used to investigate the utilization of exogenous 11,12-3H2-retinol by frog pigment epithelium (RPE) in vitro or after intraocular injection into the dark-adapted, whole animal. Isolated frog RPE contains an adequate supply of acyl donors and can esterify all-trans, 11-cis and 13-cis isomers of retinol. The esterifying activity is restricted to the particulate fraction. Homogenates of choroid cannot esterify retinol. The ester formed by the RPE is primarily palmitate, and is therefore identical with the endogenous retinyl ester. Frog RPE also formed 13-cis retinyl palmitate from all-trans retinol, probably by esterification of 13-cis retinol formed non-enzymatically from the all-trans isomer. None of the in vitro experiments provided any evidence for the formation of 11-cis retinoid. There was slow appearance of label in 11-cis retinyl palmitate when 3H-all-trans retinol was injected intraocularly into the intact frog. After 15 hr its specific activity was only 20% of that of the all-trans retinyl palmitate. This rate of formation of 11-cis retinoid is inadequate for rhodopsin regeneration. However, it is more than an order of magnitude too fast to be accounted for by phagocytosis of rhodopsin. It is suggested that 11-cis retinoid is generated in the retina and is slowly transferred to the site of esterification in the RPE.

Animals↗
PubMed1983

Vitamin A utilization in human retinal pigment epithelial cells in vitro.

Vitamin A (vit A) metabolism was studied in freshly isolated and cultured human retinal pigment epithelial (RPE) cells obtained from postmortem donor eyes. Fluorometric determination of vit A in human RPE cells demonstrated that freshly isolated cells contained approximately 1.0 to 4.0 pg vit A/cell which decreased with increasing time in culture; after 48 hrs in culture cellular vit A was reduced 80%. High performance liquid chromatography (hplc) profiles of the retinyl esters in freshly isolated RPE cells showed the presence of 11-cis retinyl stearate and palmitate and all-trans retinyl stearate, palmitate and oleate; all-trans palmitate was the major ester. Hplc analyses of cell cultures supplemented with all-trans retinol, using fatty acid-free bovine serum albumin as a carrier, showed that the cells in primary and subcultures took up all-trans retinol and esterified it to form palmitate, stearate, and oleate. Palmitate was the major ester synthesized by the cells in primary cultures. In the subcultures the esters synthesized differed from that found in freshly isolated cells and in the cells in primary culture; in the subcultures, the overall synthesis of ester was reduced and oleate was more prominent. The esters that were synthesized in culture were all-trans; the formation of 11-cis isomers was not observed in human RPE cells in culture. Electron microscopy of retinol-supplemented cultures indicated that vit A doses up to 1.0 micrograms/ml had no obvious effects on the cells; at higher doses the cells no longer adhered to the culture surface.

Cells, Cultured↗