The sights along route 65.

Regeneration of rod visual pigments after photobleaching requires a protein called Rpe65. Several studies clarify its role in visual physiology and pathology, including a new one that shows it is required pigment regeneration in cone cells.

The accessibility of bovine rhodopsin in photoreceptor membranes.

Bovine photoreceptor membranes have been treated with proteases to determine the accessibility of rhodopsin to these large, water soluble molecules. The polypeptides that remain associated with the membranous structure after proteolysis were detected by sodium dodecyl sulfate gel electrophoresis. Thermolysin and chymotrypsin degraded rhodopsin (apparent mol wt 35,000-36,000) to fragments of 29,000 and 23,000 apparent mol wt, respectively, without affecting the chromophoric absorption of the molecule or removing the region of the polypeptide carrying carbohydrate. The two fragments were isolated and their amino acid compositions were determined. They do not appear to be more hydrophobic than rhodopsin. Subtilisin, at low concentration and temperature, produced a fragment with the same molecular weight as that produced by thermolysin. At higher concentrations, subtilisin yields major fragments of mol wt 23,000 and 20,000 without affecting the chromophoric absorption. Two intermediate fragments of apparent mol wt 29,000 and 26,000 were detected during the course of this degradation. Carbohydrate is retained by all but the smallest fragment. Bleaching of the photoreceptor pigment did not appreciably alter any of the fragmentation patterns. Trypsin did not alter the molecular weight of rhodopsin under the conditions of this study. Approximately 35-45% of rhodopsin appears to be accessible to the aqueous environment and can be removed without affecting the chromophoric properties of the retinaldehyde-carrying region which remains bound to the membrane.

Immunocytochemical localization of two retinoid-binding proteins in vertebrate retina.

The recent discovery and characterization of several proteins that purify with endogenous, bound retinoid have given rise to the suggestion that these proteins, which are abundant in retina, perform a role in transport and function of vitamin A. Immunocytochemical techniques were used to localize two retinoid-binding proteins in the retina of four species. Antisera to cellular retinal-binding protein (CRALBP) and an interphotoreceptor retinoid-binding protein (IRBP) were obtained from rabbits immunized with antigens purified from bovine retina. Antibodies from each antiserum reacted with a single component in retinal homogenates and supernatants which corresponded to the molecular weight and charge of the respective antigen (non-SDS and SDS PAGE, electrophoretic transfer to nitrocellulose, immunochemical staining). Immunocytochemistry controls were antibodies from nonimmune serum and antibodies absorbed with purified antigen. Antigens were localized on frozen-sectioned bovine, rat, monkey, and human retina using immunofluorescence and the peroxidase-antiperoxidase technique. Specific staining with anti-IRBP was found in the space that surrounds photoreceptor outer segments, with heaviest labeling in a line corresponding to the retinal pigment epithelium (RPE) apical surface. Cone outer segments were positive. Staining with anti-CRALBP was found in two cell types in all species: the RPE and the Müller glial cell. Within the RPE, labeling filled the cytoplasm and was heaviest apically, with negative nuclei. Labeling of Müller cells produced Golgi-like silhouettes with intense staining of all cytoplasmic compartments. Staining of the external limiting membrane was heavy, with labeled microvilli projecting into the interphotoreceptor space. Localization of IRBP to this space bordered by three cell types (RPE, photoreceptor, and Müller) is consistent with its proposed role in transport of retinoids among cells. Localization of CRALBP in RPE corroborates previous biochemical studies; its presence in the Müller cell suggests that this glial cell may play a hitherto unsuspected role in vitamin A metabolism in retina.

Fucosylated protein of retinal cone photoreceptor outer segments: morphological and biochemical analyses.

Cone outer segments (OS) of the goldfish retina are diffusely labeled after intravitreal injection of [(3)H]fucose while rod OS remain unlabeled. By electron microscopic radioautography, the OS of red- and blue-sensitive cones are heavily labeled while green- sensitive cone OS are lightly labeled. The time-course and pattern of OS labeling in all cone types from 30 min to 24 h resemble that of incorporation of other sugars into rhodopsin in rod OS. The nature of the cone OS-specific fucosylated component(s) was examined using biochemical techniques. Cone OS were prelabeled by intravitreal injection of [(3)H]fucose 24 h before sacrifice. Photoreceptor OS were isolated using a discontinuous sucrose density gradient and it was verified by electron microscopic radioautography that the only source of radioactivity in the preparations was cone OS. The different cone types could be recognized by the heaviness of labeling, characteristic membrane spacing, and 'staining' of green cone OS in vitro with horseradish peroxidase. After acid hydrolysis of prelabeled photoreceptor membranes, 90 percent of the counts were in the neutral sugar fraction which was analyzed by thin-layer chromatography. Approximately 70 percent of the radioactivity co-chromatographed with authentic fucose. SDS-PAGE/fluorography of prelabeled photoreceptor membranes revealed a single radioactive component that was lightly stained with coomassie blue and showed an apparent molecular weight of 33,000. This cone-derived band was separated from unlabeled rod opsin which was well stained and showed an apparent mol wt of 38,000. Isoelectric focusing under denaturing conditions produced two major and one minor band of radioactivity with isoelectric points of 8.2, 8.6, and 8.8 respectively. No radioactivity was found in association with a stained band corresponding in isoelectric point to that of bovine opsin (pl, 6.2). The fucosylated component was readily digested by pronase, indicating its protein nature. Washing of the isolated OS with isotonic and hypotonic buffers failed to extract major amounts of the radioactivity, suggesting that the fucosylated component is an integral membrane protein. The presence of a fucosylated protein thus represents a major difference between cone and rod OS in the goldfish and has enabled us to identify cone OS in preparations of isolated photoreceptor membranes and to demonstrate the separation of a cone-derived glycoprotein from rod opsin.

Multiple phosphorylation of rhodopsin and the in vivo chemistry underlying rod photoreceptor dark adaptation.

Dark adaptation requires timely deactivation of phototransduction and efficient regeneration of visual pigment. No previous study has directly compared the kinetics of dark adaptation with rates of the various chemical reactions that influence it. To accomplish this, we developed a novel rapid-quench/mass spectrometry-based method to establish the initial kinetics and site specificity of light-stimulated rhodopsin phosphorylation in mouse retinas. We also measured phosphorylation and dephosphorylation, regeneration of rhodopsin, and reduction of all-trans retinal all under identical in vivo conditions. Dark adaptation was monitored by electroretinography. We found that rhodopsin is multiply phosphorylated and then dephosphorylated in an ordered fashion following exposure to light. Initially during dark adaptation, transduction activity wanes as multiple phosphates accumulate. Thereafter, full recovery of photosensitivity coincides with regeneration and dephosphorylation of rhodopsin.

Visual cycle impairment in cellular retinaldehyde binding protein (CRALBP) knockout mice results in delayed dark adaptation.

Mutations in the human CRALBP gene cause retinal pathology and delayed dark adaptation. Biochemical studies have not identified the primary physiological function of CRALBP. To resolve this, we generated and characterized mice with a non-functional CRALBP gene (Rlbp1(-/-) mice). The photosensitivity of Rlbp1(-/-) mice is normal but rhodopsin regeneration, 11-cis-retinal production, and dark adaptation after illumination are delayed by >10-fold. All-trans-retinyl esters accumulate during the delay indicating that isomerization of all-trans- to 11-cis-retinol is impaired. No evidence of photoreceptor degeneration was observed in animals raised in cyclic light/dark conditions for up to 1 year. Albino Rlbp(-/-) mice are protected from light damage relative to the wild type. These findings support a role for CRALBP as an acceptor of 11-cis-retinol in the isomerization reaction of the visual cycle.

Activation and inactivation steps in the visual transduction pathway.

Recent genetic, biochemical and electrophysiological evidence has provided insights into the molecular identity of the substance responsible for bleaching desensitization in vision. Studies examining the molecular defects that cause delayed dark adaptation suggest that the desensitizing substance is recognized by rhodopsin kinase and/or arrestin and, therefore, is probably a complex comprising all-trans-retinal and opsin.

Separable binding proteins for retinoic acid and retinol in bovine retina.

Binding proteins for retinoic acid and retinol were separated from a supernatant prepared from bovine retina. Fraction IV from DEAE-cellulose chromatography bound exogenous [3H] retinoic acid which could not be effectively displaced by retinol, retinal, retinyl acetate or palmitate, but which was readily displaced with excess retinoic acid. [3H] Retinol was bound by fraction V from DEAE-cellulose chromatography and was not displaced by retinal, retinoic acid, retinyl acetate or retinyl palmitate, but was readily displaced by excess retinol. Unlike bovine serum retinol-binding protein, neither intracellular binding protein formed a complex with purified human serum prealbumin. The supernatant from bovine retinas was estimated to contain five times more retinoic acid binding than retinol binder.

Immunolocalization of cellular retinoic acid binding protein to Müller cells and/or a subpopulation of GABA-positive amacrine cells in retinas of different species.

Retinoic acid (RA) and its specific binding protein, cellular RA binding protein (CRABP), are found in relative abundance in bovine and rat retinas. Since RA does not participate in the visual cycle, the presence of RA and its binding protein in retina suggests that they may be involved in other aspects of retinoid action. As an initial step in identifying the role of RA and its binding protein in retina, monoclonal antibodies were prepared against CRABP purified from bovine retina and used to localize this antigen by immunocytochemistry in retinas of different species. Human and monkey retinas showed specific cytoplasmic labeling of Müller cells. Cat, bovine, rabbit, rat, turtle, and chick retinas showed specific cytoplasmic labeling of some somata in the inner nuclear and ganglion cell layers and characteristic strata in the inner plexiform layer. Cat and bovine retinas also showed cytoplasmic labeling of Müller cells. Immunoreactivity in these species was absent with nonimmune serum or abolished when the antibodies were preabsorbed with purified antigen. Chameleon, goldfish, and frog retinas were nonreactive. We used double-labeling immunofluorescence experiments to determine if the CRABP-positive cells were also positive for known neurotransmitters or associated enzymes. CRABP-positive amacrine cells of cat, cow, rabbit, rat, and chick represented a subset of the more numerous gamma-aminobutyric acid (GABA)-positive amacrine cells. However, turtle CRABP-positive amacrine cells were negative for GABA despite the fact that turtle retina contains many GABA positive cells. CRABP-positive amacrine cells in rat retinas were not immunoreactive for glycine, choline acetyltransferase, somatostatin, or tyrosine hydroxylase.(ABSTRACT TRUNCATED AT 250 WORDS)

Occurrence of 11-cis-retinal-binding protein restricted to the retina.

A binding protein for retinal (vitamin A aldehyde), the 11-cis-retinal-binding protein, was present in the soluble protein fraction of rat retina but was absent from brain, lung, heart, skeletal muscle, liver, kidney, spleen, small intestine, and testes. The binding protein was also found in human retina but not human liver. The binding protein for retinal from human retina did not bind retinol and was larger than, and readily separable from, the cellular retinol-binding protein which did not bind retinal. It would appear that the occurrence of a soluble binding protein for retinal, unlike the more widely distributed binding proteins for retinol and retinoic acid, may be unique to the retina.

Immunocytochemical localization of interphotoreceptor retinoid-binding protein in developing normal and RCS rat retinas.

Interphotoreceptor retinoid-binding protein (IRBP) was localized immunocytochemically in developing normal and RCS rat retinas. IRBP was present in normal and RCS neural retinas on the day after birth (postnatal day 2, P2) to P8 in the space between the neuroblastic layer and the retinal pigment epithelium (RPE). The presence of IRBP prior to the development of outer segments (OS) suggests that OS formation is not linked temporally with IRBP secretion. On P10, staining was confined to the interphotoreceptor space with an intense band of label adjacent to the RPE. This staining pattern persisted in normal rats throughout development and until P18 in RCS rats. On P18, anti-IRBP staining in the RCS was spread evenly throughout the OS layer with no intense band of label adjacent to the RPE and after P18, there was decreased staining with anti-IRBP. On P45 and later, no staining of the RCS retina was found with anti-IRBP. Immunoblots of normal and RCS retinas corroborated the results from immunocytochemical staining. These findings suggest that IRBP may be synthesized in the photoreceptors, but is not abnormal in amount or distribution prior to onset of retinal degeneration in the RCS rat.

Localization of cellular retinal-binding protein in bovine retina and retinal pigment epithelium, with a consideration of the pigment epithelium isolation technique.

Bovine RPE was isolated by commonly used brushout procedures and analyzed by light and electron microscopy. The preparation was found to consist almost entirely of cells with retained organelles (mitochondria, pigment, and other granules) but with broken surface membranes and extracted cytoplasm. In keeping with this, the wash obtained by sedimenting these broken cells contained approximately 97 percent of the cellular retinol-binding protein present in the suspension. Cellular retinoic acid-binding protein, present in bovine retinal extracts, was found in low amounts in the wash from RPE. The cellular retinol-binding protein present in the RPE wash was of high specific activity and similar in properties to that obtained from bovine retina. Supernatant obtained from sonicated rod outer segments contained approximately 10 percent of the retinol-binding protein of the retina. No retinoic acid-binding protein was found. The relatively large amount of cellular retinol-binding protein present in the RPE (more than is found in the retina) is consistent with a functional role of this protein in uptake and transport of retinol by the RPE.

Zonulae adherentes pore size in the external limiting membrane of the rabbit retina.

The interphotoreceptor space (IPS) of the retina is bordered by the retinal pigment epithelium, photoreceptors, and Müller cells and surrounds the photoreceptor outer and inner segments. It contains a matrix composed of glycosaminoglycans and proteins, including interphotoreceptor retinol-binding protein (IRBP). The matrix does not diffuse sclerad through the tight junctions that link cells of the pigment epithelium or vitread beyond the point at which photoreceptors and Müller cells are linked by zonulae adherentes that comprise the external limiting membrane (ELM). Biotinylated protein probes of known Stokes' radius were used to determine the pore size of the ELM. Following exposure of the photoreceptor side of isolated rabbit retinas to each protein, the extent of diffusion of the probe through the retina was determined by avidin D-horseradish peroxidase histochemistry. Each protein with a Stokes' radius of 30 A or less diffused freely through the neurosensory retina while each protein with a Stokes' radius greater than 36 A was blocked abruptly at the ELM. Thus, the pore radius of the zonulae adherentes of the ELM lies between 30 and 36 A, which is sufficiently small to account for containment of IRBP (55 A) within the IPS. This study emphasizes that in addition to providing structural support, the zonulae adherentes of the ELM serve to define an important extracellular space of the retina. This has clinical relevance, since two serum proteins tested, albumin and gamma-globulin, are too large to diffuse through an intact ELM. This may explain why protein-rich fluid accumulates in the IPS when the outer blood retinal barrier is compromised by disease or injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoantibodies against retinal bipolar cells in cutaneous melanoma-associated retinopathy.

PURPOSE: This study's goal was to determine the pathophysiology of the retinopathy that occurs in patients with metastatic cutaneous melanoma and sudden onset of night blindness, the so-called melanoma-associated retinopathy (MAR) syndrome. We tested the hypothesis that sera from two MAR patients contained autoantibodies that reacted with "on" bipolar cells of the human retina. METHODS: Immunofluorescence was performed on cryostat sections of unfixed normal human retinas. Sera and IgG fractions were tested from the two MAR patients and 38 control subjects (28 patients with metastatic melanoma, but no visual symptoms; two patients with non-MAR retinopathy; and eight normal subjects). RESULTS: The sera and IgG fractions from both MAR patients but from none of the control subjects produced heavy immunostaining of bipolar cells, which were identified as rod bipolars by a double labeling procedure using anti-protein kinase C. CONCLUSIONS: We hypothesize that MAR patients generate autoantibodies against a melanoma antigen that cross react with bipolar cells of the retina. These antibodies, by an unknown mechanism, may cause abnormalities of the rod and cone systems that are characteristic of MAR.

Retinoid-binding proteins in cone-dominant retinas.

We identified and localized interphotoreceptor (or interstitial) retinoid-binding protein (IRBP) and cellular retinaldehyde-binding protein (CRALBP) in the cone-dominant retinas of diurnal squirrels. Western blots were prepared from sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) from whole retina, and from retina proximal and distal to the photoreceptor nuclei. Blots were incubated with purified rabbit IgG's specific for the bovine retinal antigens, and the labeled components were visualized using immunoperoxidase techniques. Anti-bovine IRBP and anti-bovine CRALBP recognized single components on gels of retinal supernatants that corresponded to the electrophoretic migration of the bovine antigens. The component recognized by anti-bovine IRBP on blots of outer retinal proteins (Mr 146,000) was absent on blots of inner retinal proteins. Twelve and 24 hr after intravitreal injection of 3H-L-fucose, electropherograms showed one major peak of radioactivity that coincided with the component recognized by anti-bovine IRBP. By immunoelectron microscopy, anti-bovine CRALBP labeling was restricted to the cytoplasm of both RPE and Muller cells, with light labeling of nuclear euchromatin in both cell types. In contrast, anti-bovine IRBP recognized antigenic sites primarily in the interphotoreceptor space (IPS). Intracellular labeling was limited to occasional granules in the photoreceptor myoids and the apical RPE cytoplasm. Extracellular labeling with anti-bovine IRBP was strongly associated with patches or small clumps of amorphous, electron opaque material distributed throughout the IPS. This material was particularly prominent near the cone outer segment plasma membranes, and was tentatively identified as the residual interphotoreceptor matrix that remained after exposure to the solvents used during tissue processing. In general, the results are consistent with those obtained in rod-dominant species. In addition, they imply that cones as well as rods are responsible for IRBP synthesis in the ground squirrel.

Cellular retinol- and retinoic acid-binding proteins in transformed mammalian cells.

Extracts prepared from several lines of transformed cells were examined for the presence of cellular binding proteins specific for retinoids. Extracts of human retinoblastoma cell line WERI-Rb1 contained a cellular binding protein specific for retinoic acid, whereas extracts of human retinoblastoma cell line Y-79 contained cellular binding proteins for both retinol and retinoic acid. Upon purification, the latter two binding proteins proved to have properties similar to those of the corresponding proteins obtained from bovine retina. Smaller amounts of these binding proteins were detected in extracts of undifferentiated and differentiated neuroblastoma and McCoy cells. HeLa and rat glioma cells had no detectable amount of binding proteins. The 11-cis-retinal-binding protein, present in extracts of human, rat, and bovine retina, was not found in any of the cell lines examined.

Molecular characterization of the mouse gene encoding cellular retinaldehyde-binding protein.

PURPOSE: To clone and characterize the mouse gene encoding cellular retinaldehyde-binding protein (CRALBP). CRALBP appears to modulate enzymatic generation and processing of 11-cis-retinol and regeneration of visual pigment in the vertebrate visual cycle. Mutations in human CRALBP segregate with autosomal recessive retinitis pigmentosa. METHODS: A genomic clone encompassing the 5' end of the CRALBP gene through exon 6 was isolated from a mouse 129/Sv genomic DNA library. Exons 7 and 8 were PCR amplified from mouse eye cDNA and 129/SvJ genomic DNA. The gene structure was determined by automated DNA sequence analysis. RESULTS: The sequence of 6855 nucleotides was determined, including all 8 exons, 3 introns plus 3932 and 629 bases from the 5'- and 3'-flanking regions, respectively. The lengths of introns 3-6 were determined by PCR amplification. Northern analysis identifies a approximately 2.1 kb transcript in mouse eye; Southern analysis supports a single copy gene. CONCLUSIONS: The mouse CRALBP gene is similar to the human gene; the coding sequence is approximately 87% identical, the non-coding sequence approximately 65% identical. In contrast to the human gene, the mouse gene contains a consensus TATA box. One of two photoreceptor consensus elements important for CRALBP expression in human retinal pigment epithelium is also present in the mouse gene. Additional conserved and species-specific consensus sequences are identified. The mouse CRALBP genomic clones and structure provide valuable tools for developing an in vivo model to study protein function and gene regulation.

The color difference in orbital fat.

OBJECTIVE: To identify and quantify carotenoids found in white and yellow orbital fat. METHODS: Specimens of nasal (white) and preaponeurotic (yellow) orbital fat were obtained from patients during upper eyelid blepharoplasty. Carotenoids and retinoids were extracted and subjected to spectral and high-performance liquid chromatography analyses. RESULTS: The chromophore content of extracts from unsaponified fat, as measured by absorbance at 425 nm per gram of fat, was 2- to 4-fold higher in preaponeurotic fat than in nasal fat. High-performance liquid chromatography analysis from enzymatically digested fat revealed large amounts of lutein, beta-carotene, and retinol and small amounts of other unidentified carotenoids. The amount of beta-carotene and lutein in preaponeurotic fat was approximately 4-fold higher than in nasal fat. CONCLUSIONS: The higher carotenoid content of preaponeurotic fat might cause it to be more yellow than other orbital fat, and lutein and beta-carotene might be selectively absorbed from plasma by preaponeurotic fat. CLINICAL RELEVANCE: The results provide baseline information for studies of the physiological features of orbital fat in normal and diseased conditions.

Reduction of all-trans-retinal limits regeneration of visual pigment in mice.

Absorption of photons by pigments in photoreceptor cells results in photoisomerization of the chromophore, 11-cis-retinal, to all-trans-retinal and activation of opsin. Photolysed chromophore is converted back to the 11-cis-configuration via several enzymatic steps in photoreceptor and retinal pigment epithelial cells. We investigated the levels of retinoids in mouse retina during constant illumination and regeneration in the dark as a means of obtaining more information about the rate-limiting step of the visual cycle and about cycle intermediates that could be responsible for desensitization of the visual system. All-trans-retinal accumulated in the retinas during constant illumination and following flash illumination. Decay of all-trans-retinal in the dark following constant illumination occurred without substantial accumulation of all-trans-retinal, generated by constant approximately equal to visual pigment regeneration (t1/2 approximately 5 and t1/2 approximately 7 min, respectively). All-trans-retinal, generated by constant illumination, decayed approximately 3 times more rapidly than that generated by a flash and, as shown previously, the rate of rhodopsin regeneration following a flash was approximately 4 times slower than after constant illumination. The retinyl ester pool (> 95% all-trans-retinyl ester) did not show a statistically significant change in size or composition during illumination. In addition, constant illumination increased the amount of photoreceptor membrane-associated arrestin. The results suggest that the rate-limiting step of the visual cycle is the reduction of all-trans-retinal to all-trans-retinol by all-trans-retinol dehydrogenase. The accumulation of all-trans-retinal during illumination may be responsible, in part, for the reduction in sensitivity of the visual system that accompanies photobleaching and may contribute to the development of retinal pathology associated with light damage and aging.

Properties and immunocytochemical localization of three retinoid-binding proteins from bovine retina.

Cellular retinal-binding protein (CRALBP) complexed with 11-cis-retinal has several properties characteristic of a visual pigment. Interaction of the protein and retinoid results in a bathochromic shift in the absorption spectrum of the chromophore from 380 to 425 nm, accompanied by a decrease in the extinction coefficient (25,000-15,000 M-1 cm-1). Illumination of the complex results in the progressive loss of absorbance at 425 nm and an increase at 375 nm, consistent with the production of a geometrical isomer of retinal that lacks affinity for the binding protein. Analysis by HPLC of the retinoids after illumination reveals that the basis of the spectral transition is a photoisomerization of 11-cis-retinal to all-trans-retinal. Only small amounts (less than 10%) of 13-cis-retinal are produced during the photoisomerization, showing the stereospecificity of the process. Although CRALBP has the spectral characteristics of a blue-sensitive visual pigment, there is no evidence that this is related to its function. This protein may serve as a model for the interactions of 11-cis-retinal and protein. Eleven-cis-retinol bound to CRALBP is a better substrate for esterification by microsomes from retinal pigment epithelium (RPE) than all-trans-retinol bound to cellular retinol-binding protein (CRBP). The product of the reaction, retinyl ester, does not remain bound to either binding protein but becomes associated with the microsomal fraction. Esterification is the first described process, occurring in the dark, by which retinoids can be removed from CRBP and CRALBP. Antibodies to bovine CRBP have been produced in rabbits following injection of the performic acid-oxidized protein.(ABSTRACT TRUNCATED AT 250 WORDS)