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Biomedical subjects

J C Saari

Publications and source records attributed to J C Saari.

At least 55 records · Page 3Linked to original sources
PubMed1987

Photochemistry and stereoselectivity of cellular retinaldehyde-binding protein from bovine retina.

11-cis-Retinaldehyde bound to cellular retinaldehyde-binding protein (CRALBP) is unaffected in bovine eyecup preparations by illumination that bleaches approximately 70% of the rhodopsin. Illumination of retinal homogenates to which CRALBP X [3H]11-cis-retinaldehyde had been added did not result in a reduction of the specific activity of recovered 11-cis-retinaldehyde, ruling out a bleaching regeneration cycle. The quantum efficiency of photoisomerization for CRALBP X 11-cis-retinaldehyde was determined by comparing the rate of photoisomerization of 11-cis-retinaldehyde bound to purified CRALBP and opsin. The low value obtained (0.07), coupled with a low molar extinction coefficient (15,400 M-1 cm-1), results in a photosensitivity only about 4% that of rhodopsin. CRALBP binds 9-cis- and 11-cis-retinaldehyde, producing complexes with absorption maxima at 405 and 425 nm, respectively. No complexes were detected with 13-cis- and all-trans-retinaldehyde. Following incubation of CRALBP X 11-cis-retinol with an equimolar mixture of 9-, 11-, 13-cis-, and all-trans-retinaldehydes, only 11-cis-retinaldehyde and residual 11-cis-retinol are present on the protein following separation from excess retinoids. A similar result is obtained following incubation of CRALBP X 11-cis-retinol with mixtures of 9- and 11-cis-retinaldehyde ranging in composition from 9:1 to 1:9 (9-cis-:11-cis-,mol/mol). The results indicate that CRALBP X 11-cis-retinol is sufficiently stereoselective in its binding properties to warrant consideration as a component of the mechanism for the generation of 11-cis-retinaldehyde in the dark.

Animals↗
PubMed1987

Uveoretinitis in rabbits following immunization with interphotoreceptor retinoid-binding protein.

Interphotoreceptor retinoid-binding protein (IRBP) is a glycoprotein found in the interphotoreceptor matrix between the neurosensory retina and the retinal pigment epithelium and is thought to shuttle retinol among cells that border the interphotoreceptor space. Immunization of rabbits with bovine IRBP caused subsequent photoreceptor degeneration, as documented by light- and electron microscopy. Beginning on post-injection day 18, scattered regions had photoreceptor outer segments that were disorganized and shortened or absent. Macrophages were found between the retinal pigment epithelium and neurosensory retina and within choroidal interstitium and blood vessels. Labeling of these cells with a marker specific for monocytic macrophages (RAM11) and absence of labeling with a marker for retinal pigment epithelium (rabbit anti-bovine cellular retinaldehyde-binding protein) suggest that these macrophages were hematogenous in origin. Staining of retinas with fluorescein isothiocyanate (FITC)-conjugated sheep anti-rabbit IgG revealed leakage of rabbit IgG into the interphotoreceptor matrix on and after day 18 in experimental animals but not in controls, suggesting breakdown of the outer blood-retinal barrier. Indirect immunofluorescence with anti-glial fibrillary acidic protein revealed labeling of Müller cells in experimental retinas on and after day 18, but not in control or shorter survival experimental retinas. There were foci of increased cellularity in the choroid on days 18, 26 and 39. From days 26 through 67, the retinal pathology became more widespread. Varying degrees of outer-segment degeneration were present in all parts of the retina and in many areas there was total loss of outer segments and loss of some photoreceptor-cell bodies. The inner retina appeared unaffected in all experimental and control retinas. These results demonstrate that injection of rabbits with bovine IRBP causes retinal photoreceptor degeneration as anti-IRBP titers increase and breakdown of the outer blood-retinal barrier ensues. Further studies will be required to elucidate factor(s) that control accessibility of the neurosensory retina to circulating antibodies against IRBP and other intrinsic retinal proteins.

Animals↗
PubMed1986

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.

Animals↗
PubMed1985

Properties of an interphotoreceptor retinoid-binding protein from bovine retina.

Washes and extracts of frozen and fresh cattle retina contain a water-soluble high-molecular-weight, retinoid-binding protein that is distinct from three other retinoid-binding proteins previously isolated from this tissue. The protein can be purified to apparent homogeneity from retinal homogenates by a combination of gel filtration, lectin, and ion-exchange chromatography. Overestimation of the protein molecular weight was observed in several systems involving migration of the protein through a porous network. The approximate molecular weight obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 140,000, a value consistent with those reported by other laboratories. However, a more detailed analysis using the method of Ferguson revealed the protein to behave anomalously relative to several proteins used as sodium dodecyl sulfate-polyacrylamide gel electrophoresis standards. The apparent radius of the native protein, estimated from calibrated gel filtration, corresponded to a globular protein with a molecular weight of 240,000-280,000, suggesting that the protein was a dimer. However, when the molecular weight of native interphotoreceptor retinoid-binding protein (IRBP) was determined by a method with no shape dependence, sedimentation equilibrium, a value of 131,700 +/- 3,900 g/mol, was obtained. Sedimentation equilibrium in a dissociating solvent (6 M guanidine HCl) yielded a molecular weight of the smallest component of 120,100 +/- 2,300 g/mol. The similarity of values for the denatured and native molecular weight by sedimentation equilibrium demonstrates that the protein is a monomer. In further support of this, no evidence for a dimer was observed in cross-linking experiments with dimethyl suberimidate. The sedimentation coefficient (S0(20),w = 5.73 +/- 0.15 S) and molecular weight from sedimentation equilibrium were employed to calculate the frictional coefficient and Stokes radius of IRBP (f/f0 = 1.64, Rs = 55 A). The high value of f/f0 of the protein provides a reasonable explanation for the over-estimation of the molecular weight of native IRBP on gel filtration. Approximately 2 mol of exogenous all-trans- or 11-cis-retinol were bound per mol of protein (131,000). Approximately 7% of the binding sites were saturated with endogenous ligand (11-cis-retinol, 88%; all-trans-retinol, 12%) following isolation from partially bleached cattle eyes.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1985

Localization of retinoid-binding proteins in developing rat retina.

Antibodies to cellular retinol-binding protein (CRBP) and cellular retinal-binding protein (CRALBP) were obtained from rabbits immunized with antigens purified from bovine retina. Antigens were localized on frozen sections of rat retina using indirect FITC immunofluorescence. In the retinal pigment epithelium of rats from postnatal day 1 (the day of birth) to postnatal day 32, specific staining with anti-CRBP was restricted to the cytoplasm; the nuclei were unstained. In the neurosensory retina, Muller cell endfeet were stained with anti-CRBP at all ages examined. No CRBP reactivity was found in the pigment epithelium of the ciliary body at any age examined. On postnatal days 14 and 32, in addition to Muller cell endfeet and radial processes, two fine laminae in the inner plexiform layer were faintly positive for CRBP. Anti-CRALBP stained the cytoplasm of the RPE and Muller cells in the adult rat. In developing rat retina, in addition to Muller cells and the retinal pigment epithelium, the ciliary body pigment epithelium and the outer epithelium of the iris were stained with anti-CRALBP in the first postnatal week. The intensity of staining of the ciliary body pigment epithelium decreased gradually from postnatal day 8 until postnatal day 14, at which point a clear line of demarcation was found between the positively stained retinal pigment epithelium and the adjacent, lightly stained, pigment epithelial cells of the ciliary body.

Aging↗
PubMed1985

Immunocytochemical localization of retinoid-binding proteins in developing normal and RCS rats.

Interstitial retinol-binding protein (IRBP), cellular retinol-binding protein (CRBP), and cellular retinal-binding protein (CRALBP) were localized in developing normal and RCS rat retinas. IRBP is found in the presumptive interphotoreceptor space in normal and RCS rats on P1. It reaches adult levels on P18. In the RCS rat, the level of IRBP decreases after P18 until there is only a small amount of staining on P38. CRBP and CRALBP are localized in the retinal pigment epithelium from P1 to adult. CRALBP is also found in the ciliary body pigment epithelium and in the outer epithelium of the iris during the first week, but decreases during the second week to a very low level as found in the adult. The significance of the early presence of IRBP and the transient appearance of CRALBP in the ciliary body and iris epithelia is discussed.

Age Factors↗
PubMed1985

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.

Animals↗
PubMed1985

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)

Animals↗
PubMed1985

Characterization of the interstitial space: immunocytochemical and biochemical studies.

The interstitial space has been characterized using immunocytochemical and biochemical techniques. The pore size of the external limiting membrane that forms the vitread margin of this extracellular space has been probed using a series of biotinylated proteins of known Stokes' radius, followed by avidin-horseradish peroxidase histochemistry. The pore size of the zonulae adherentes that collectively comprise the external limiting membrane has been found to lie between 30 and 36A. This is sufficiently small to block passage of interstitial retinol-binding protein (IRBP, Stokes' radius = 55A) from the interstitial space vitread into the retina. IRBP has been localized to the interstitial space of bovine retina by use of indirect immunogold electron microscopy. IRBP has also been identified in cytoplasmic vesicles of rod and cone inner segments. The function of these vesicles may be for secretion and/or uptake of IRBP from the interstitial space. A postulated function of IRBP is transfer of vitamin A between the retinal pigment epithelium and photoreceptors during the visual cycle. Biochemical experiments have corroborated previous studies that IRBP becomes loaded with all-trans-retinol following bleaching illumination, while the amount of endogenous 11-cis-retinol remains unchanged. These data are consistent with the emerging concept that IRBP plays a role in transport of vitamin A among the cell types that border the interstitial space.

Animals↗
PubMed1984

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)

Animals↗
PubMed1983

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.

Animals↗
PubMed1982

Identification of the endogenous retinoids associated with three cellular retinoid-binding proteins from bovine retina and retinal pigment epithelium.

The endogenous retinoids associated with three cellular retinoid-binding proteins from bovine retina and retinal pigment epithelium have been identified by their spectral characteristics and their co-migration with authentic retinoids on high performance liquid chromatography. All-trans-retinol is the only retinoid associated with the cellular retinol-binding protein from retina and retinal pigment epithelium. The saturation of the binding site with native ligand is 0.95 +/- 0.05 mol of retinoid/mol of protein for cellular retinol-binding protein purified from frozen and fresh retina and retinal pigment epithelium. All-trans-retinoic acid has been identified as the endogenous ligand associated with the cellular retinoic acid-binding protein of cattle retina. The saturation of the binding site of cellular retinoic acid-binding protein from frozen and fresh retina (the protein is absent in extracts of retinal pigment epithelium) is about 0.2 mol of retinoid/mol of protein, probably a minimum value due to losses of the ligand. Retinoic acid has not been detected previously in retina. Cellular retinaldehyde-binding protein from retina purifies with two endogenous ligands which cochromatograph on high performance liquid chromatography with 11-cis-retinaldehyde and 11-cis-retinol and occur in a ratio of approximately 3:1, respectively. The binding site of cellular retinaldehyde-binding protein from frozen retina is nearly fully occupied with these two ligands (saturation greater than 0.9 mol of retinoids/mol of protein). In contrast, purified cellular retinaldehyde-binding protein from retinal pigment epithelium carries only 11-cis-retinaldehyde as an endogenous ligand. The saturation of the binding site with this ligand is greater than 0.95.

Animals↗
PubMed1982

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.

Animals↗