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PubMed2001

Action spectrum for melatonin regulation in humans: evidence for a novel circadian photoreceptor.

The photopigment in the human eye that transduces light for circadian and neuroendocrine regulation, is unknown. The aim of this study was to establish an action spectrum for light-induced melatonin suppression that could help elucidate the ocular photoreceptor system for regulating the human pineal gland. Subjects (37 females, 35 males, mean age of 24.5 +/- 0.3 years) were healthy and had normal color vision. Full-field, monochromatic light exposures took place between 2:00 and 3:30 A.M. while subjects' pupils were dilated. Blood samples collected before and after light exposures were quantified for melatonin. Each subject was tested with at least seven different irradiances of one wavelength with a minimum of 1 week between each nighttime exposure. Nighttime melatonin suppression tests (n = 627) were completed with wavelengths from 420 to 600 nm. The data were fit to eight univariant, sigmoidal fluence-response curves (R(2) = 0.81-0.95). The action spectrum constructed from these data fit an opsin template (R(2) = 0.91), which identifies 446-477 nm as the most potent wavelength region providing circadian input for regulating melatonin secretion. The results suggest that, in humans, a single photopigment may be primarily responsible for melatonin suppression, and its peak absorbance appears to be distinct from that of rod and cone cell photopigments for vision. The data also suggest that this new photopigment is retinaldehyde based. These findings suggest that there is a novel opsin photopigment in the human eye that mediates circadian photoreception.

Adolescent↗
PubMed2001

Downregulation of differentiation specific gene expression by oxidative stress in ARPE-19 cells.

PURPOSE: To investigate how the differentiation of ARPE-19 cells affects the relative expression of the FGFR genes in response to oxidative stress. METHODS: After differentiation in vitro, APRE-19 cells were treated with t-butyl hydroperoxide (tBH) or hydrogen peroxide (H2O2) to induce oxidative stress. Viability and reactive oxygen intermediate (ROI) production were measured using standard assays. The mRNA expression of FGFR1, FGFR2, cellular retinaldehyde-binding protein (CRALBP), RPE65, and heme oxygenase-1 (HO-1) were measured by Northern blot analysis as a function of treatment with tBH and H2O2. RESULTS: ARPE-19 cells were viable at all tBH concentrations tested but showed progressive loss of viability at concentrations greater than 300 microM H2O2. Differentiated ARPE-19 cells treated with tBH or H2O2 resulted in upregulation of the HO-1 and FGFR1 transcripts. The expression of RPE-differentiated specific genes, including FGFR2, CRALBP, and RPE65 mRNAs, was downregulated with tBH or H2O2 treatment. CONCLUSIONS: Oxidative stress in differentiated ARPE-19 cells alters the expression of FGFR1, FGFR2, CRALBP, and RPE65 toward levels characteristic of the undifferentiated state. If similar changes take place in vivo, these events could alter the proliferative potential, viability, and even the function of the RPE.

Blotting, Northern↗
PubMed2001

Short-term study of allogeneic retinal pigment epithelium transplants onto debrided Bruch's membrane.

PURPOSE: To investigate the survival and behavior of retinal pigment epithelium (RPE) microaggregates transplanted onto hydraulically debrided Bruch's membrane and to compare results of using three different vehicles for cell delivery. METHODS: RPE microaggregates obtained from male cats were transplanted onto the tapetal area of female cats after native RPE was debrided. For the control, one of three vehicles was introduced into the debridements. Each transplant or control specimen was analyzed histologically and immunohistochemically. Transplanted male RPE cells were identified by in situ labeling of the cat Y chromosome. RESULTS: Histologically, significant numbers of condensed, darkly stained RPE nuclei were observed in all transplants compared with few TUNEL-positive RPE cells. Cellular retinaldehyde-binding protein was present up to day 7 in all RPE cells in transplants. In both transplant and control specimens, the antibody against the Ki-67 nuclear antigen labeled some RPE cells at day 3. TUNEL-positive outer nuclear layer nuclei were most frequently observed at day 1, but were much less frequent at 7 days in both transplant and control specimens. CONCLUSIONS: Transplanted RPE appeared to retain at least some markers of differentiation up to 7 days after surgery. Some proliferation of transplanted RPE cells was also seen. Apoptotic cell death of transplanted RPE, as judged by TUNEL staining was observed rarely. RPE transplants imposed no adverse effect on the overlying retina. RPE survival appeared to be similar with each of the three vehicles for cell delivery.

Animals↗
PubMed2001

The ability of hyperoxia to limit the effects of experimental detachment in cone-dominated retina.

PURPOSE: To determine the ability of oxygen supplementation to ameliorate the effects of retinal detachment in a cone-dominated retina. METHODS: Retinal detachments were created in the right eyes of ground squirrels and the animals immediately placed in normoxic (room air) or hyperoxic (70% oxygen) conditions for 3 days. The retinas were sampled from different regions and investigated morphologically or immunocytochemically by light or confocal microscopy. Agarose embedded sections were immunostained with antibody probes to cytochrome oxidase, synaptophysin, medium-to-long wavelength-sensitive (M/L) cone opsin, rod opsin, excitatory amino acid transporter 1 (EAAT1), glutamate synthetase (GS), cellular retinaldehyde-binding protein (CRALBP), and peanut agglutinin (PNA) lectin. Retinal wholemounts were labeled with PNA and antibodies to short (S)-wavelength-sensitive cone opsin and rod opsin. Cell death was examined using a TUNEL assay on agarose sections or using toluidine blue staining on semithin sections. RESULTS: The percentage of dying cells relative to the total nuclei in the photoreceptor layer was significantly reduced, and the total number of nuclei was greater in hyperoxic animals. Triple labeling using TUNEL, anti-M/L cone opsin and anti-rod opsin showed that hyperoxia had a remarkable effect both on the reduction of cone cell death and the maintenance of the overall structure of cone photoreceptors. Analysis of the retinal wholemounts demonstrated the preservation of PNA, S-cone, and rod opsin antibody labeling in the detachments maintained in hyperoxic conditions. Although the disruption of cytochrome oxidase and synaptophysin was seen in normoxic animals, there was minimal disruption in hyperoxic animals. Labeling with anti-EAAT1, anti-GS, and anti-CRALBP was increased in the Müller cells of normoxic animals with detachments, but was decreased in the hyperoxic animals. CONCLUSIONS: Hyperoxia prevents the degeneration of both rods and cones in retinas heavily dominated by cones and mitigates the effect of detachment on Müller cell reactivity. The current results suggest that the rescue of cones is not secondary to that of rods.

Animals↗
PubMed2002

Characterization of genetically modified human retinal pigment epithelial cells developed for in vitro and transplantation studies.

PURPOSE: To develop, by specific genetic modification, a differentiated human retinal pigment epithelial (RPE) cell line with an extended life span that can be used for investigating their function in vitro and for in vivo transplantation studies. METHODS: Primary human RPE cells were genetically modified by transfecting with a plasmid encoding the simian virus (SV)40 large T antigen. After characterization, two cell lines, designated h1RPE-7 and h1RPE-116, were chosen for further investigation, along with the spontaneously derived RPE cell line ARPE-19. Factors reported to be important in RPE and photoreceptor cell function and survival in vivo were examined. RESULTS: Both h1RPE-7 and h1RPE-116 cells exhibited epithelial morphology, expressed cytokeratins, and displayed junctional distribution of ZO-1, p100-p120 and beta-catenin. The cells expressed mRNA for RPE65 and cellular retinaldehyde-binding protein (CRALBP) and the trophic and growth factors brain-derived neurotropic factor (BDNF), ciliary neurotrophic factor (CNTF), basic fibroblast growth factor (bFGF), pigment epithelium-derived factor (PEDF), nerve growth factor (NGF), platelet-derived growth factor (PDGF)-alpha, insulin-like growth factor (IGF)-1, and vascular endothelial growth factor (VEGF). Secreted BDNF, bFGF, and VEGF, but not CNTF, were identified in cell supernatants. The cell lines constitutively expressed HLA-ABC, CD54, CD58, and CD59. After activation with IFN-gamma both HLA-ABC and CD54 were upregulated, and the expression of HLA-DR was induced. Both cell lines failed to express CD80, CD86, CD40, or CD48 in vitro and in a mixed lymphocyte reaction were unable to induce T-cell proliferation. Fas ligand (CD95L) was not detected in vitro by RT-PCR. Similar results were obtained with the ARPE-19 cell line. CONCLUSIONS: RPE lines h1RPE-7 and h1RPE-116 retain many of the morphologic and biochemical characteristics of RPE cells in vivo and may serve as a source of cells for in vitro analysis of RPE cell function, as well as for orthotopic transplantation studies.

Antigens, Polyomavirus Transforming↗
PubMed2002

In vitro characterization of a spontaneously immortalized human Müller cell line (MIO-M1).

PURPOSE: To characterize a spontaneously immortalized human Müller cell line and to determine whether it retains the characteristics of primary isolated cells without undergoing differentiation in vitro. METHODS: An immortalized cell line obtained from human retina was investigated for the expression of known markers of Müller cells, including cellular retinaldehyde binding protein (CRALBP), glutamine synthetase, epidermal growth factor receptor (EGF-R), alpha-smooth muscle actin (alpha-SMA), and glial fibrillary acidic protein (GFAP). Also examined were the morphologic features of these cells, by scanning and transmission electron microscopy, and their functional characteristics, by electrogenic responses to glutamate. In addition, comparative studies were made of these cells with primary cultures of freshly isolated human Müller cells. RESULTS: The cells expressed CRALBP, EGF-R, glutamine synthetase, and alpha-SMA, as judged by confocal microscopy and Western blot analysis of cell lysates. Western blot analysis did not detect GFAP in cell lysates, but confocal microscopy showed that occasional cells expressed GFAP after detachment from the monolayer. The morphologic features of the cells examined, as judged by scanning and transmission electron microscopy, resemble those of cells derived from primary cell cultures. They possess villous projections on their apical surfaces and contain loose bundles of microtubules aligned parallel to one another and the long axis of the cell process. Characteristically, they contain abundant deposits of glycogen particles that do not differ from those seen in primary isolated cells. Preliminary recordings with intracellular electrodes revealed that these cells have properties similar to those described for mammalian Müller cells and depolarize in response to L-glutamate without significant change in membrane resistance, consistent with the well-established electrogenic uptake of this amino acid. CONCLUSIONS: A spontaneously immortalized Müller cell line was characterized that retains the characteristics of primary isolated cells in culture. To the authors' knowledge, it constitutes the first human Müller cell line reported in the literature. It has been named MIO-M1 (Moorfields/Institute of Ophthalmology-Müller 1) after the authors' institution. Availability of this human cell line will facilitate studies designed to obtain a better understanding of the role of Müller cells in normal and pathologic conditions.

Actins↗
PubMed2002

In vitro differentiation capacity of telomerase immortalized human RPE cells.

PURPOSE: To investigate conditions promoting the differentiation of cultured human retinal pigment epithelial (RPE) cells and assess the differentiation potential of telomerase-immortalized RPE cells. METHODS: Serially passaged RPE 340 (parental) cells have limited replicative ability and senesce after 50 to 60 population doublings (PD)s. RPE 340 cells transfected with the catalytic component of human telomerase (hTERT) have an extended lifespan. RPE 340 and hTERT-transfected RPE (hTERT-RPE) cells were maintained at confluence without serum for up to 12 weeks. Morphologic, immunocytochemical, flow cytometric, and spectrophotometric analyses were performed to examine the extent of RPE differentiation. RESULTS: Parental RPE 340 and hTERT-RPE cells underwent growth arrest and differentiated in the absence of serum. In early-passage parental (PD11) and hTERT-RPE (PD115 and PD300) cells, serum deprivation for 4 weeks or more induced terminal differentiation as characterized by mature, growth-arrested, confluent sheets of polygonal and melanized cells that demonstrated diminished 5'-bromo-2'-deoxyuridine (BrdU) uptake and positive reactivity with antibodies to cellular retinaldehyde-binding protein (CRALBP), cytokeratin, and vimentin. Reintroduction of serum at 4 or 8 weeks allowed the cells to reenter the cell cycle and demelanize. Midpassage (PD 25) parental cells, however, were irreversibly arrested at G(1) after 8 weeks of serum deprivation. CONCLUSIONS: Cultured parental and hTERT-RPE cells express RPE-associated proteins and become stably melanized when density is arrested in the absence of serum. Moreover, hTERT-immortalized RPE cells retain the capacity to undergo terminal differentiation in vitro, even after long-term culture.

Carrier Proteins↗
PubMed2002

[Expression of mRNA for proteins involved in retinol metabolism in cultured bovine iris pigment epithelial cells in vitro].

OBJECTIVE: To assess the retinol metabolism of cultured bovine iris pigment epithelial (IPE) cells. METHODS: IPE cells were isolated from neonatal bovines with Hu's method, and were cultured. The total RNA of IPE cells was extracted by Trizol one step method. The specific primers for retinol-binding protein (RBP) and cellular retinaldehyde-binding protein (CRALBP) were designed according to their sequence from Genbank. The mRNA expression of these proteins in the IPE cells was analyzed by reverse transcription polymerase-chain reaction (RT-PCR). RESULTS: The extracted RNA from IPE cells of the second passage was ideal and had no degradation. RT-PCR analysis showed that RBP mRNA and CRALBP mRNA were expressed in cultured IPE cells in vitro. CONCLUSIONS: Similar to RPE cells, cultured IPE cells may have potential of transporting and metabolizing retinol.

Animals↗
PubMed2002

Synthesis and antioxidative properties of novel thiazolidinedione/imidazolidinedione compounds as retinoids.

The general term "retinoids" refers to both naturally occurring as well as synthetic compounds which exhibit biological activity similar to vitamin A (retinol). Vitamin A and its two metabolites, retinaldehyde and retinoic acid, are fat-soluble unsaturated isoprenoids necessary for the growth, differentiation and maintenance of epithelial tissues. In this study, we have synthesized thiazolidinedione/imidazolidinedione compounds as retinoids. Their in vitro effects on rat liver microsomal NADPH-dependent lipid peroxidation (LP) levels and superoxide anion formation were determined.

Animals↗
PubMed2003

The meninges is a source of retinoic acid for the late-developing hindbrain.

One general function for retinoic acid (RA) is pattern organization in the CNS. This regulatory factor has an essential role in spinal cord motor neuron and early posterior hindbrain development. In the anterior CNS, however, there is only a limited number of foci of RA synthesis, and less attention has been placed on regions such as the anterior hindbrain where RA synthesizing enzymes are absent. This study shows that a rich source of RA lies around the hindbrain from the RA synthetic enzyme retinaldehyde dehydrogenase-2 (RALDH2) present in the surrounding meninges and mesenchyme by embryonic day 13. RALDH2 is not distributed uniformly throughout the meninges but is restricted to territories over the developing hindbrain, suggesting that RA signaling may be localized to those regions. Further regulation of RA signaling is provided by the presence of a RA sink in the form of the CYP26B1 RA catabolic enzyme expressed in deeper regions of the brain. As a guide to the neural anatomy of hindbrain RA signaling, we used a mouse transgenic for a lacZ reporter gene driven by a RA response element (RAREhsplacZ) to identify regions of RA signaling. This reporter mouse provides evidence that RA signaling in the hindbrain after embryonic day 13 occurs in the regions of the cerebellum and precerebellar system adjacent to sources of RA, including the inferior olive and the pontine nuclei.

Aldehyde Oxidoreductases↗
PubMed1992

Retinoic acid activation and thyroid hormone repression of the human alcohol dehydrogenase gene ADH3.

Mammalian alcohol dehydrogenase (ADH) catalyzes the oxidation of retinol to retinaldehyde, the rate-limiting step in the synthesis of retinoic acid. There exists a family of ADH isozymes encoded by unique genes, and it is unclear which isozymes are most important for regulation of retinoic acid synthesis during differentiation or development. A region in the human ADH3 promoter from -328 to -272 base pairs was shown previously to function as a retinoic acid response element (RARE), prompting an hypothesis for a positive feedback mechanism controlling retinoic acid synthesis (Duester, G., Shean, M. L., McBride, M. S., and Stewart, M. J. (1991) Mol. Cell. Biol. 11, 1638-1646). The ADH3 RARE contains three direct AGGTCA repeats which constitute the critical nucleotides of RAREs present in other genes. We dissected the ADH3 RARE and determined that receptor binding as well as transactivation are dependent upon only the two downstream AGGTCA motifs separated by 5 base pairs, a structure noticed previously for a RARE in the promoter for the retinoic acid receptor beta (RAR beta) gene. ADH3 and RAR beta RAREs functioned similarly in transfection assays, suggesting that the feedback mechanisms controlling ADH3 and RAR beta utilize a common RARE. We also found that the normal functioning of the ADH3 RARE was abrogated by thyroid hormone receptor in the presence of thyroid hormone. A negative thyroid hormone response element in the human ADH3 promoter was found to colocalize with the RARE. Since ADH production in rat liver is known to be repressed by thyroid hormone, these findings suggest that human ADH production may also be subject to thyroid hormone repression and that the mechanism involves an interference with retinoic acid induction.

Alcohol Dehydrogenase↗
PubMed1992

Expression of developmentally defined retinal phenotypes in the histogenesis of retinoblastoma.

Retinoblastoma, the most common intraocular tumor of childhood, is a malignant neoplasm that arises during retinal development. The embryonal cell target for neoplastic transformation is not yet clearly defined. To better understand the histogenetic potential of this tumor, the expression of photoreceptor and glial cell-associated proteins were examined in 22 primary retinoblastomas. Interphotoreceptor retinol-binding protein (IRBP), cone and rod opsins were selected as the photoreceptor specific proteins due to their different temporal patterns of expression during normal retinal development. Neoplastic Müller cell differentiation, and non-neoplastic reactive astrocytes were identified using cellular retinaldehyde binding-protein (CRAlBP), and glial fibrillary acidic protein (GFAP), respectively. Photoreceptor proteins were present in 16 cases and showed different cellular patterns of expression. IRBP and cone opsin were usually abundant. Although rod opsin was clearly identified in eight tumors, its expression was more restricted than either IRBP or cone opsin. This differential pattern of expression, opposite to the normal pattern of photoreceptor gene expression in the adult retina, corresponded to a marked decrease in mRNA for rod opsin. Cone opsin and IRBP colocalized in fleurettes demonstrating that neoplastic human cone cells are capable of IRBP synthesis. Müller cell differentiation was present in 12 of the 16 cases in which photoreceptor proteins were detected. In contrast, GFAP was only present in reactive, stromal astrocytes associated with blood vessels. Our data suggest that the retinoblastoma has the histogenetic potential of the immature neural retinal epithelium which can give rise to both photoreceptor and Müller cell lineages. The differential expression of cone and rod phenotypes in retinoblastoma is consistent with the "default" mechanism of cone cell differentiation.

Blotting, Northern↗
PubMed2005

Retinoid processing proteins in the ocular ciliary epithelium.

PURPOSE: To identify retinoids and retinoid processing proteins in the ocular ciliary epithelium (CE), and to compare in cultured ciliary epithelial cell lines promoter activities of the cellular retinaldehyde binding protein (CRALBP) and interphotoreceptor retinoid binding protein (IRBP). METHODS: Retinoid processing proteins were detected by RT-PCR, western analysis and immunocytochemistry. PCR products were verified by DNA sequence analysis. Retinoids were measured by normal phase HPLC and UV visible spectroscopy. Reporter product from CRALBP and IRBP promoter fragments was measured following transient transfection in bovine pigmented and nonpigmented CE cells. RESULTS: CRALBP, IRBP, cellular retinol binding protein (CRBP), 11-cis-retinol dehydrogenase (11-cis-RDH), lecithin:retinol acyltransferase (LRAT), and ATP binding cassette receptor (ABCR) were detected in human CE tissue by RT-PCR. Retinal pigment epithelium specific protein 65 kDa (RPE65) mRNA and protein were also detected. CRALBP and IRBP were detected by western analysis in tissue extracts from bovine CE and were localized to the PE and NPE cell layers, respectively, by immunocytochemistry. IRBP immunoreactivity was also detected in aqueous humor. Retinoids identified in the bovine CE include retinyl esters (7.4+/-3.5 pMol/mg of protein) and all-trans-retinol (14.9+/-1.1 pMol/mg of protein). Betacarotene was also tentatively identified. 11-cis-Retinoids were not detected. In CE cell cultures, the CRALBP p2.1-kb promoter construct exhibited reporter activity 15-30 fold above basal level, with 2 fold more activity in pigmented than nonpigmented CE cells. IRBP promoter constructs exhibited low level reporter activities in vitro in both CE cell layers. CONCLUSIONS: The ocular CE expresses genes encoding components of the rod visual cycle. The differential localization of CRALBP and IRBP along the bilayer of the CE suggests a potential role in retinoid transport and/or retinoid metabolism. However, the absence of 11-cis-retinoids suggests that the function of retinoid processing proteins in the CE differs from that of the retina.

ATP-Binding Cassette Transporters↗
PubMed1991

Alteration of the binding specificity of cellular retinol-binding protein II by site-directed mutagenesis.

Rat cellular retinol-binding protein II (CRBP II) is an abundant 134-residue intestinal protein that binds all-trans-retinol and all-trans-retinal. It belongs to a family of homologous, 15-kDa cytoplasmic proteins that bind hydrophobic ligands in a noncovalent fashion. These binding proteins include a number of proteins that bind long chain fatty acids. X-ray analyses of the structure of two family members, rat intestinal fatty acid-binding protein and bovine myelin P2 protein, indicate that they have a high degree of conformational similarity and that the carboxylate group of their bound fatty acid interacts with a delta-guanidium group of at least 1 of 2 "buried" arginine residues. These 2 Arg residues are conserved in other family members that bind long chain fatty acids and in cellular retinoic acid-binding protein, but are replaced by Gln109 and Gln129 in CRBP II. We have genetically engineered two amino acid substitutions in CRBP II: 1) Gln109 to Arg and 2) Gln129 to Arg. The purified Escherichia coli-derived CRBP II mutant proteins were analyzed by fluorescence and nuclear magnetic resonance spectroscopy. Both mutants exhibit markedly decreased binding of all-trans-retinol and all-trans-retinaldehyde, but no increased binding of all-trans-retinoic acid. Arg substitution for Gln109 but not for Gln129 produces a dramatic increase in palmitate binding activity. Analysis of the endogenous fatty acids associated with the purified E. coli-derived proteins revealed that E. coli-derived intestinal fatty acid binding protein and the Arg109 CRBP II mutant are complexed with endogenous fatty acids in a qualitatively and quantitatively similar manner. These results provide evidence that this internal Arg may play an important role in the binding of long chain fatty acids by members of this protein family.

Amino Acid Sequence↗
PubMed1990

Immunocytochemical identification of Müller's glia as a component of human epiretinal membranes.

Sections of seven human epiretinal membranes of diverse pathologic origin were labeled with antibodies to cellular retinaldehyde binding protein (CRALBP) and two intermediate filament proteins: glial fibrillary acidic protein (GFAP) and vimentin. All of the membranes studied contained heterogeneous cell populations that exhibited diverse morphologic characteristics. Double labeling with both anti-GFAP and anti-CRALBP positively identified one of the cellular components in these membranes as Müller's glia. In addition, other epiretinal cells exhibited immunolabeling patterns consistent with those found in fibrous astrocytes and retinal pigment epithelial (RPE) cells normally. The results demonstrate that a double-labeling method using CRALBP antibodies, in combination with antibodies to other appropriate antigens, can be used to distinguish between the different epiretinal cell types.

Adult↗
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↗
PubMed1982

Retinol dehydrogenase from bovine retinal rod outer segments. Kinetic mechanism of the solubilized enzyme.

Retinol dehydrogenase solubilized by Lubrol 12A9 from bovine retinal rod outer segments forms mixed micelles of Stokes radius 8.5 nm. The kinetic properties of the solubilized retinol dehydrogenase were examined and retinaldehyde reduction and retinol oxidation were seen to proceed at pH 8.3 by a sequential Ordered Bi Bi mechanism. This conclusion was supported by bisubstrate initial velocity studies, dead-end and product inhibition. The kinetic mechanism of retinol dehydrogenase is not altered by the effect of Lubrol until a concentration of 2 mM is reached, at which the detergent lowers the values of the Michaelis and dissociation constants. The catalytic rate of the retinol dehydrogenase is significantly lowered by detergent in the range of pH 3 to 9.

Alcohol Oxidoreductases↗
PubMed1994

Growth factor localization in choroidal neovascular membranes of age-related macular degeneration.

PURPOSE: Because several polypeptide growth factors are known to influence capillary endothelial cell mitogenesis, the authors investigated the presence of some of these molecules in choroidal neovascular membranes (CNVMs) removed surgically from human subjects with age-related macular degeneration (ARMD). METHODS: The authors performed immunoelectron microscopic studies on surgically removed submacular CNVMs from nine subjects with ARMD and from one subject with ARMD whose eye was studied after death. These were compared with retinal pigment epithelial (RPE) and choroidal tissue from eight normal subjects whose eyes were received after death and one received after massive trauma. RESULTS: RPE cells from the CNVMs were strongly immunoreactive for acidic and basic fibroblast growth factor (aFGF and bFGF) and for transforming growth factor beta (TGF beta). Some of the immunoreactivity was intracytoplasmic, but most was intralysosomal. In addition, some choriocapillary endothelial cells located close to the RPE layer in these CNVMs were immunopositive for bFGF and for FGF receptor. Reaction product for these two substances was located at regular intervals along the endothelial plasma membrane on both the anteluminal and the luminal side of the cells, suggesting a physiological reaction between the growth factor and its receptor. Choriocapillary endothelial cells deeper within the stroma were unreactive to bFGF and FGF receptor antibodies. There was little immunoreactivity for the growth factors in RPE or choriocapillary endothelial cells from normal eyes. The aFGF and bFGF immunoreactivity was highly specific because aFGF positivity was abolished when the antibody was incubated with 10(-6) M aFGF but not a with the same concentration of bFGF, whereas bFGF immunoreactivity was abolished by incubation of the antibody with bFGF but not with aFGF. RPE cells from normal eyes and from eyes affected by ARMD showed strong cytoplasmic immunoreactivity to antibodies for cytoplasmic retinaldehyde-binding protein and superoxide dismutase and weak reactivity to antibodies for vimentin. CONCLUSIONS: These results are consistent with the hypothesis that one or both FGFs are causally related to the development of choroidal neovascularization. The authors have reported similar observations in experimental choroidal neovascularization in pigmented rats after red krypton laser photocoagulation. TGF beta may serve to modulate the effects of these mitogens. The authors suggest that growth factor production is induced in RPE cells after physical or chemical damage. Because of the damage to these cells, FGF molecules can be released from the cells despite the absence of a "signal sequence" in the DNA coding for FGF production.

Aged↗