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C D Bridges

Publications and source records attributed to C D Bridges.

At least 37 records · Page 2Linked to original sources
PubMed1987

Interstitial retinol-binding protein and cellular retinal-binding protein in the mammalian pineal.

Antibodies against bovine interstitial retinol-binding protein (IRBP) and cellular retinal-binding protein (CRA1BP) were used in immunochemical and immunocytochemical studies of the pineal glands of cattle, hamsters and rats (RCS and RCS-rdy+). On immunoblots, IRBP (Mr 144,000) was identified in cattle, hamster and rat pineal extracts. The abundance of IRBP in bovine pineals was 33 +/- 6 ng.mg-1 (mean +/- SD, n = 12) soluble protein. RCS (Royal College of Surgeons) rat pineals gave a strong IRBP reaction on immunoblots, even when virtually no IRBP could be found in the eye due to photoreceptor degeneration. In the hamster retina IRBP immunostaining was distributed throughout the entire interphotoreceptor matrix and the outer segment layer. The pineal also showed strong IRBP-like immunostaining scattered uniformly throughout the gland. Other hamster brain regions showed no specific immunostaining; however, an immunoreactive protein with the same Mr as IRBP was detected on Western blots of bovine cerebral cortex, spinal cord and brainstem soluble proteins. Immunoreactive proteins at lower Mr were also detected in these tissues. CRA1BP immunoreactivity (Mr about 32,000) was observed in immunoblots of bovine, hamster and rat pineal proteins. These findings suggest that some mammalian pinealocytes are related to the retinal cells that contain CRA1BP (i.e. pigment epithelium, Muller cells) while others are related to the photoreceptors, which synthesize IRBP.

Animals↗
PubMed1987

Levels of alpha- and gamma-tocopherol in human eyes: evaluation of the possible role of IRBP in intraocular alpha-tocopherol transport.

Alpha-tocopherol was distributed almost equally between the retina and its underlying pigmented layers (pigment epithelium and choroid). Only 8.4% of the total alpha-tocopherol occurred in the iris and ciliary body. Alpha-tocopherol content was expressed as amount per eye, per cm2, and per 100 g wet weight. The combined retina and pigment epithelium-choroid contained 2.9 +/- 1.0 mg/100 g wet weight (means +/- SD, n = 30 donors). Gamma-tocopherol represented 20.9 +/- 12.2 mol % of the alpha-tocopherol. The anterior tissues contained 0.4 +/- 0.2 mg/100 g (n = 19 donors). No significant correlation with age was found. Purified bovine interstitial retinol-binding protein (IRBP) bound exogenous 3H-alpha-tocopherol, which could be displaced by unlabeled all-trans retinol (KD = 10(-6) M). Much higher concentrations of unlabeled alpha-tocopherol were required to achieve a partial displacement of bound 3H-all-trans retinol. No endogenous alpha-tocopherol could be detected in bovine interphotoreceptor matrix.

Adult↗
PubMed1987

Rhodopsin, vitamin A, and interstitial retinol-binding protein in the rd chicken.

In order to determine whether blindness in the rd strain of Rhode Island Red chickens is due to a defect in the vitamin A (visual) cycle, spectroscopy, high performance liquid chromatography, and immunochemical techniques were used to compare the amounts of rhodopsin, interstitial retinol-binding protein, and vitamin A compounds in the dark-adapted eyes of homozygous rd and heterozygous carriers. In both groups of chickens, (up to 6 weeks post-hatching) the distribution of stored vitamin A differed from other vertebrates (mammals, amphibians, fish) in that more than half of the retinyl palmitate/stearate occurred in the neurosensory retina. The 11-cis isomer accounted for nearly 100% of the retinyl palmitate/stearate in the neurosensory retinas of both groups. In the pigmented layers (pigment epithelium and choroid) the 11-cis isomer amounted to 70.1 +/- 4.2% in the carrier, and 65.1 +/- 2.9% in the rd birds. With respect to their content of rhodopsin, IRBP, retinyl palmitate/stearate and unesterified retinol, (both 11-cis and all-trans isomers) no significant difference could be demonstrated between the eyes of rd and carrier chickens (3 days and 28 days post-hatching). These results therefore demonstrate that the ocular tissues of rd chickens do not lack IRBP, the putative extracellular transport protein for vitamin A, that these tissues synthesize and store the 11-cis isomer of vitamin A, and that the 11-cis isomer is used to form rhodopsin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

N-terminal sequence homologies in interstitial retinol-binding proteins from 10 vertebrate species.

We report here the first comprehensive comparative NH2-terminal sequence studies of interstitial retinol-binding protein (IRBPs) from nine mammals (including cattle) and one amphibian. This study has revealed that in many species the N-terminus of IRBP includes a 3-6 amino acid extension. IRBP possessing this leader sequence is sometimes mixed with IRBP from which this sequence has been excised.

Amino Acid Sequence↗
PubMed1986

Distribution of interstitial retinol-binding protein (IRBP) in the vertebrates.

Immunoblots of interphotoreceptor matrix preparations from 20 species belonging to six vertebrate classes were probed with antibodies against bovine interstitial retinol-binding protein (b-IRBP). Each preparation displayed an immunoreactive protein band. In the Osteichthyes, the apparent Mr of this band was 67,600 +/- 2,700 (mean +/- SD, n = 8). In two of the Osteichthyes, the band was resolved into a closely spaced doublet. Including previously published data for five mammals and one amphibian, species from the other classes (Chondrichthyes, one species; Amphibia, four species; Reptilia, one species; Aves, one species; Mammalia, nine species) had IRBPs with Mr that averaged 2.0 times that of the Osteichthyes, namely 134,200 +/- 8,600 (mean +/- SD, n = 17). Frog IRBP was very similar to mammalian IRBP in terms of its immunohistochemical distribution (determined with rabbit anti-frog IRBP antibodies), its molecular weight (sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel-filtration chromatography), retinol- and concanavalin A-binding ability, and because it was synthesized and secreted in vitro by the isolated retina but not by the pigmented layers of eye. Goldfish IRBP apparently binds exogenous (3H)-retinol but does not bind concanavalin A and has about half the Mr of frog IRBP. The occurrence of IRBP-like proteins cross-reacting with anti b-IRBP antibodies in the interphotoreceptor matrix of all six major vertebrate classes is consistent with the hypothesis that IRBP is an important element in the vertebrate visual cycle.

Animals↗
PubMed1986

Rhodopsin, 11-cis vitamin A, and interstitial retinol-binding protein (IRBP) during retinal development in normal and rd mutant mice.

Biochemical and immunological techniques were used to determine the emergence of interstitial retinol binding protein (IRBP), rhodopsin, and stored retinyl esters (all-trans and 11-cis) during retinal development in normal and rd mice. IRBP could be demonstrated at embryonic Day 17 (E17), corresponding to an early stage of inner segment development. Although all-trans retinyl esters were present earlier, 11-cis retinyl esters did not appear until postnatal Days 6-7 (P6-P7), corresponding to rod outer segment (ROS) disc formation. Rhodopsin was detected at the same developmental stage. The proportion of 11-cis retinyl esters reached a maximum of 40-50% at P15-P20. Thereafter, the proportion dropped, due to more rapid accumulation of the all-trans isomer. Rhodopsin and IRBP increased in parallel with ROS elongation up to P25, when the ROS had reached their mature lengths. The increases then continued up to P40-P50. In rd (retinal degeneration) mice, IRBP and rhodopsin were identical with the controls until P12, but then dropped as the photoreceptors degenerated. Synthesis and secretion of IRBP in vitro was less than 10% of the controls in rd retinas at P26, when only 4-5% of the photoreceptors survived. The quantities of retinyl esters (mainly stearate and palmitate in the ratio of 6:1, respectively) stored in dark-adapted mouse eyes progressively increased as the animals aged, representing 0.5 mole eq. of the rhodopsin at 8 months. Although retinyl esters (11-cis and all-trans) also accumulated in rd mouse eyes up to P12, little further increase occurred. At P93, the retinyl esters (0.01 nmole X eye-1) were only 4% of the controls at P91. A peak in the proportion of 11-cis isomer occurred at P10-P20, but it averaged only 15% of the total ester and declined to 5% at P93. These findings support the hypothesis that IRBP is synthesized by the rods and cones, and suggest that its synthesis and secretion are initiated when the photoreceptor inner segments start to differentiate. 11-cis Retinoids and rhodopsin do not appear until the outer segments start to form. It is suggested that in the rd mouse the absence of photoreceptors, perhaps coupled with lack of normal interphotoreceptor matrix, leads to a loss in the ability of the pigment epithelium to store retinyl esters.

Animals↗
PubMed1986

Bovine interstitial retinol-binding protein (IRBP)--isolation and sequence analysis of cDNA clones, characterization and in vitro translation of mRNA.

Three clones for b-IRBP were isolated by anti b-IRBP screening of two bovine retina libraries in the expression vector lambda gt11. The cDNA inserts were then used as hybridization probes to screen and isolate three more clones in a bovine retina library in the non-expression vector lambda gt10. The six overlapping clones generated a b-IRBP cDNA sequence of 3400 nucleotides. An open reading frame encoded the complete amino acid sequences of 8 of the 35 b-IRBP tryptic peptides purified in the present study. One tentative glycosylation site was identified. The coding region was followed by TAG translation terminating codon and an untranslated stretch of about 1700 nucleotides that ended in a sequence containing a presumptive AATAAA polyadenylation signal that was 18 nucleotides upstream from a 10 nucleotide oligo(A) tract. The coding region for b-IRBP would be expected to be 3300 bp long, but Northern blot hybridization experiments performed with bovine retina polyadenylated RNA and probes containing part of the coding region established that the mRNA for b-IRBP consisted of a major species of about 6300 bp, and a minor species of 5200 bp. In vitro translation of bovine retina polyadenylated RNA in a rabbit reticulocyte lysate system yielded an immunoreactive protein that was comparable in size with nonglycosylated, mature IRBP, showing that it is not synthesized from a large precursor, and supporting our finding that the mRNA contains an extensive non-coding region.

Amino Acid Sequence↗
PubMed1986

Electron microscopic immunocytochemistry of interstitial retinol-binding protein in vertebrate retinas.

Interstitial retinol binding protein (IRBP) is a soluble glycoprotein found in the interphotoreceptor matrix (IPM) and implicated in shuttling retinol between retina and pigment epithelium (PE) cells. The authors have studied the distribution of IRBP by EM immunocytochemistry. Thin sections of Lowicryl K4M embedded R. pipiens, X. laevis, bovine and human retinas were labeled sequentially with affinity purified rabbit antibovine IRBP, biotinyl-sheep antirabbit F(Ab')2, and avidin-ferritin, or with avidin and biotinyl-ferritin. Antigen was in the interphotoreceptor space and intercalated into the narrow spaces between PE cell microvilli. IRBP penetration between PE cells was delimited abruptly by the PE junctional complexes. IRBP was also observed in small vacuoles in the apical cytoplasm of PE cells and in PE cell phagosomes that contained IRBP surrounding ingested rod tips. IPM was heavily but inhomogeneously labeled. Antigen was usually deposited along the ROS and COS plasma membrane in a confluent layer, but sometimes it was distributed in large (ca. 0.2-micron thick) clumps. In bovine and human retinas, the connecting cilium was ensheathed by antigen at high density but an unlabeled halo surrounded its plasma membrane. The apical plasma membrane of the inner segment aligned along the connecting cilium was also densely coated by antigen. In both frog retinas, the ridges of the periciliary ridge complex (PRC) were coated with antigen. In none of the four species examined was Golgi labeling present. In bovine retinas, labeled vacuoles (granules) in the myoid region were found in very low numbers (15 vacuoles in 358 rod cells). Amphibian retinas also contained only small numbers of myoid vacuoles labeled by anti-IRBP. Absence of antibody binding to intracellular sites of synthesis in any of the cells that abut the interphotoreceptor matrix suggests that the antigen may be masked prior to its release from the synthetic cell(s) or that its level is below limits of detection.

Animals↗
PubMed1986

Interstitial retinol-binding protein (IRBP) in subretinal fluid.

Antibodies against bovine interstitial retinol-binding protein (b-IRBP) were used to detect human IRBP (h-IRBP) on immunoblots of eight samples of subretinal fluid (SRF) from patients with retinal detachments of between 2 days' and more than 2 years' duration. Using this sensitive technique, it was found that seven of the samples contained h-IRBP in concentrations estimated to range from below 5% up to 19% of normal human IPM. One of these samples displayed two immunoreactive bands of roughly equal intensity, one at a molecular weight of 135,000 (h-IRBP), the other at 115,000. The latter may have been generated by proteolytic cleavage. No h-IRBP could be detected in an eighth sample from a patient with retrolental fibroplasia. It is concluded that the reduced concentration of h-IRBP in SRF may be due to a number of factors that include dilution, proteolytic degradation, and metabolic inactivation of photoreceptors at the detachment site.

Adolescent↗
PubMed1985

Development of the subretinal space in the preterm human eye: ultrastructural and immunocytochemical studies.

To investigate the development of the subretinal space in the human infant, eyes were obtained from 12 live-born, anomaly-free, preterm infants from 20 to 32 weeks gestation and from one 3-month postterm infant. The retinas were studied by light microscopy, electron microscopy, and immunocytochemistry. The immunocytochemical studies utilized rabbit antiserum against purified bovine interstitial retinol binding protein (IRBP). A subretinal space containing IRBP was present in the central retina at 20 weeks and extended further into the periphery (expressed as a percentage of the distance from the optic disc to the ora serrata in the temporal hemisphere) as the retina developed. At 28 weeks, IRBP was absent only from the most peripheral 25% of the retina and reached the temporal ora serrata at 32 weeks. At 3 months postterm, IRBP immunofluorescence outlined fully developed photoreceptors, which were present from the optic disc to the ora serrata. The appearance of IRBP in the subretinal space correlated with the development of the first photoreceptor outer segment discs.

Fluorescent Antibody Technique↗
PubMed1985

Varieties of rhodopsin in frog rod outer segment membranes: analysis by isoelectric focusing.

Purified ROS membranes from cattle, R. pipiens and R. catesbeiana adults and tadpoles were investigated by analytical and preparative isoelectric focusing and SDS-polyacrylamide gel electrophoresis. The rhodopsin from single specimens of R. pipiens displayed two closely-spaced bands with Mr at 34.7 and 37.0 k on SDS polyacrylamide gels. Both were found to be phosphorylated when prepared from retinas incubated with 32Pi and then exposed to light. When purified ROS membranes were solubilized in octyl glucoside and examined by isoelectric focusing followed by SDS-polyacrylamide gel electrophoresis, the low-Mr component focused in two bands (I, IIa) at pH 8.8 and 8.1. Band I and a trace amount of band IIa were observed if band I was eluted and refocused. The high-Mr component focused in one band (IIb) at pH 8.0. Identical isoelectric focusing patterns were observed with ROS membranes from R. catesbeiana tadpoles and the dorsal and ventral retina areas from R. catesbeiana adults. Bovine rhodopsin, on the other hand, had a single Mr component and focused in a major band at pH 6.2.

Adaptation, Ocular↗
PubMed1985

Conversion of retinol to 3,4-didehydroretinol in the tadpole.

The conversion of retinol to 3,4-didehydroretinol in bullfrog tadpoles was studied by injecting [3H] all-trans retinol into the peritoneal cavity. The specific activities of retinoids in the eye and the rest of the body at various time intervals after the injection were then determined by HPLC (high-performance liquid chromatography). Radioactivity was observed in ocular 3,4-didehydroretinyl esters after 2 days and their specific activity increased throughout the 2 weeks of experiment. This demonstrates that tadpoles can convert retinol to its 3,4-didehydro derivative. In vitro experiments performed on isolated eye cups also suggested that the ocular tissues could convert retinol to 3,4-didehydroretinol. In the eye, the specific activity of porphyropsin or all-trans 3,4-didehydroretinal (extracted by the denaturing solvent acetone) exceeded that of the all-trans 3,4-didehydroretinyl esters in storage. This suggests that the main ocular store of 3,4-didehydroretinyl esters does not constitute a precursor pool for porphyropsin synthesis.

Animals↗
PubMed1985

Adult human retinal cells in culture. Identification of cell types and expression of differentiated properties.

A method for culturing adult mammalian retinal neurons in serum-free N2 medium supplemented with nerve growth factor (NGF) is described. Identification of neurons in cultures of dispersed human retina was based upon morphology, immunocytochemical localization of bound tetanus toxin, and autoradiographic localization of 3H-neurotransmitter candidates (gamma-aminobutyric acid, glycine, dopamine) accumulated by high-affinity uptake mechanisms. Neurons would not attach to glass or plastic substrates, consequently the present studies were performed using neurons plated upon a feeder layer. Serum was required for the initial phase of attachment. The feeder layer was derived from retinal cells that had been plated on glass or plastic in the presence of serum and had later been passaged. Since these cells exhibited glial fibrillary acidic protein (GFAP) immunoreactivity, they were tentatively identified as being glial in origin. Under these conditions, neuron- and glia-specific properties were retained up to 28 days. The presence of interstitial retinol-binding protein (IRBP) in medium of cultures of neuronal cells on feeder layers was demonstrated by an immunoblot technique using rabbit antibovine IRBP antibodies. No IRBP was detected in medium in which the feeder layers alone had been cultured. IRBP biosynthesis was demonstrated by incubation of the cultures with [35S]methionine. Immunoprecipitable [35S]IRBP was detected only in medium from cultures containing neurons; cells of the feeder layer did not synthesize and secrete this glycoprotein. These findings are consistent with the hypothesis that IRBP, a 135K constituent of the interphotoreceptor matrix, is synthesized in vivo by a neuronal cell, specifically, the photoreceptors.

Adult↗
PubMed1985

Endocytosis and degradation of interstitial retinol-binding protein: differential capabilities of cells that border the interphotoreceptor matrix.

Between the pigment epithelium and the outer limiting membrane of the retina is an extracellular compartment filled with the interphotoreceptor matrix (IPM). A prominent component of the IPM is a glycoprotein known as interstitial retinol-binding protein (IRBP). Using in vitro techniques, we compared the ability of the cells that border this compartment to internalize colloidal gold (CG) coated with either IRBP or ovalbumin, a glycoprotein not found in the IPM. Neither IRBP-CG nor ovalbumin-CG was internalized by the Muller's cells. Both rod and cone photoreceptors take up IRBP-CG, which is observed in small vesicles and multivesicular bodies. Neither photoreceptor type takes up ovalbumin-CG. Acid phosphatase cytochemistry indicates that acid phosphatase reaction product in the multivesicular bodies co-localizes with IRBP-CG, which suggests that this molecule is degraded by rod and cone photoreceptors and is not recycled. The pigment epithelium internalizes IRBP-CG and ovalbumin-CG, both of which remain in small cytoplasmic vesicles near the apical plasma membrane. There is no indication that vesicles that contain either IRBP-CG or ovalbumin-CG fuse with the lysosomal system in the pigment epithelial cells during the incubation.

Animals↗