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Despite the recent increase in use of mouse models in scientific research, researchers continue to use cognitive tasks that were originally designed and validated for rat use. The Radial Water Tread (RWT) maze test of spatial memory (designed specifically for mice and requiring no swimming) has been shown previously to successfully distinguish between controlled cortical impact-induced TBI mice and sham controls. Here, a detailed protocol for this task is presented. The RWT maze capitalizes on the natural tendency of mice to avoid open areas in favor of hugging the sides of an apparatus (thigmotaxis). The walls of the maze are lined with nine escape holes placed above the floor of the apparatus, and mice are trained to use visual cues to locate the escape hole that leads out of the maze. The maze is filled with an inch of cold water, sufficient to motivate escape but not deep enough to require that the mouse swim. The acquisition period takes only four training days, with a test of memory retention on day five and a long-term memory test on day 12. The results reported here suggest that the RWT maze is a feasible alternative to rat-validated, swimming-based cognitive tests in the assessment of spatial memory deficits in mouse models of TBI.

Introduction: The use of forced-swim, rat-validated cognition tests in mouse models of traumatic brain injury (TBI) raises methodological concerns; such models are vulnerable to a number of confounding factors including impaired motor function and stress-induced non-compliance (failure to swim). This study evaluated the ability of a Radial Water Tread (RWT) maze, designed specifically for mice, that requires no swimming to distinguish mice with controlled cortical impact (CCI) induced TBI and Sham controls., Methods: Ten-week-old, male C57BL6/J mice were randomly assigned to receive either Sham (n=14) or CCI surgeries (n=15). Mice were tested for sensorimotor deficits via Gridwalk test and Noldus CatWalk gait analysis at 1 and 32days post-injury. Mice received RWT testing at either 11days (early time point) or 35days (late time point) post-injury., Results: Compared to Sham-treated animals, CCI-induced TBI resulted in significant impairment in RWT maze performance. Additionally, CCI injured mice displayed significant deficits on the Gridwalk test at both 1day and 32days post-injury, and impairment in the CatWalk task at 1day, but not 32days, compared to Shams., Conclusions: The Radial Water Tread maze capitalizes on the natural tendency of mice to avoid open areas in favor of hugging the edges of an apparatus (thigmotaxis), and replaces a forced-swim model with water shallow enough that the animal is not required to swim, but aversive enough to motivate escape. Our findings indicate the RWT task is a sensitive species-appropriate behavioral test for evaluating spatial memory impairment in a mouse model of TBI., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Pharmacologic interventions for traumatic brain injury (TBI) hold promise to improve outcome. The purpose of this study was to determine if the microtubule stabilizing therapeutic paclitaxel used for more than 20 years in chemotherapy would improve outcome after TBI. We assessed neurological outcome in mice that received direct application of paclitaxel to brain injury from controlled cortical impact (CCI). Magnetic resonance imaging was used to assess injury-related morphological changes. Catwalk Gait analysis showed significant improvement in the paclitaxel group on a variety of parameters compared to the saline group. MRI analysis revealed that paclitaxel treatment resulted in significantly reduced edema volume at site-of-injury (11.92 ± 3.0 and 8.86 ± 2.2mm(3) for saline vs. paclitaxel respectively, as determined by T2-weighted analysis; p ≤ 0.05), and significantly increased myelin tissue preservation (9.45 ± 0.4 vs. 8.95 ± 0.3, p ≤ 0.05). Our findings indicate that paclitaxel treatment resulted in improvement of neurological outcome and MR imaging biomarkers of injury. These results could have a significant impact on therapeutic developments to treat traumatic brain injury., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Unlabelled: 3'-Fluoro-3'-deoxythymidine (FLT) has been proposed for positron emission tomography (PET)-based identification of tumor chemosensitivity that is mediated by the human equilibrative nucleoside transporter-1 (ENT1). ENT1 facilitates transport of FLT into cells and elevated levels of FLT are associated with both larger FLT-PET signals and increased response to nucleoside-based chemotherapies. FLT-PET is also used as a measure of tumor proliferation. The present study examined the extent to which ENT1 levels vary in a proliferation-dependent manner in tumor cells in vivo., Methods: The human adenocarcinoma cell line A549 was used to establish tumor xenografts in nude mice. FLT uptake was measured in vivo using PET, and further examined ex vivo using autoradiography. FLT uptake patterns were compared to immunohistochemical (IHC) analysis of ENT1 and the proliferation markers Ki67 and BrdU., Results: Regional differences in FLT uptake matched differences in IHC proliferation markers. All cells stained for ENT1, but the staining intensity was twice as high for Ki67(+) cells than for Ki67(-) cells., Conclusions: Under in vivo conditions, proliferating regions of tumors show increased FLT uptake and higher ENT1 levels than nonproliferating tumor regions., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Phosphorylation and regeneration of rhodopsin, the prototypical G-protein-coupled receptor, each can influence light and dark adaptation. To evaluate their relative contributions, we quantified rhodopsin, retinoids, phosphorylation, and photosensitivity in mice during a 90 min illumination followed by dark adaptation. During illumination, all-trans-retinyl esters and, to a lesser extent, all-trans-retinal accumulate and reach the steady state in <1 h. Each major phosphorylation site on rhodopsin reaches a steady state level of phosphorylation at a different time during illumination. The dominant factor that limits dark adaptation is isomerization of retinal. During dark adaptation, dephosphorylation of rhodopsin occurs in two phases. The faster phase corresponds to rapid dephosphorylation of regenerated rhodopsin present at the end of the illumination period. The slower phase corresponds to dephosphorylation of rhodopsin as it forms by regeneration. We conclude that rhodopsin phosphorylation has three physiological functions: it quenches phototransduction, reduces sensitivity during light adaptation, and suppresses bleached rhodopsin activity during dark adaptation.

Purpose: To determine molecular mechanisms for the release of 11-cis-retinal from the binding pocket of cellular retinaldehyde-binding protein (CRALBP)., Methods: Binding of CRALBP to lipid surfaces was assessed with a lipid-immunoblot assay. Lipids were presented to CRALBP as small unilamellar vesicles (SUVs) consisting of phosphatidylcholine (PC) plus other lipids. Release of 9-cis-retinal or 11-cis-retinal from CRALBP was measured with spectral and high performance liquid chromatography (HPLC) assays based on the protection of the protein-bound retinal carbonyl group from reaction with NH(2)OH. The electrostatic surface potential of CRALBP was calculated from a model of its structure using the program CCP4mg., Results: Incubation of CRALBP.11-cis-retinal with lipids absorbed on nitrocellulose revealed binding to the acidic lipids, phosphatidic acid (PA)>phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)]>phosphatidylserine (PS)> PI(4,5)P(2) and little or no binding to PC, phosphatidylethanolamine (PE), or PI(4)P. 11-cis-retinal was released during incubation of CRALBP with SUVs consisting of PC plus 50 mol% PA but not during incubation with those composed of 100 mol% PC. The efficacy of release of 9-cis-retinal or 11-cis-retinal from CRALBP by phospholipid-containing SUVs generally paralleled that of the binding of CRALBP to the lipids (PA>PS>PI>>PC). Examination of the electrostatic surface potential of the protein structure revealed a basic recess on one face of the protein, which may bind acidic lipids., Conclusions: Our results identify the first physiologic substances that release 11-cis-retinal from CRALBP. PA and PS are relatively minor membrane lipids that can be generated in the cytoplasmic leaflet of the plasma membrane in response to various signal transduction pathways, where they could interact with cytosolic CRALBP. The mechanism for release of retinal from CRALBP by acidic lipids remains to be determined but could involve binding of the acidic lipid in the 11-cis-retinal binding site or to the positive basic recess on the protein surface. These results open a new facet in our understanding of how CRALBP functions in the regeneration of visual pigments.

CRALBP, cellular retinaldehyde-binding protein, is a retinoid-binding protein necessary for efficient regeneration of rod and cone visual pigments. The C terminus of CRALBP binds to the PDZ domains of EBP50/NHERF-1, which in turn bind to ezrin and actin, proteins localized to the apical processes of the retinal pigment epithelium. In this study, we examined structural features associated with the interaction of the two proteins. The C-terminal amino-acid sequence of 11 orthologous CRALBPs is either ENTAL, ENTAF or EDTAL. Peptides ending in each of these sequences inhibited the interaction of CRALBP and EBP50/NHERF-1 with the use of an overlay assay. Molecular modeling showed that both NTAL and NTAF formed similar networks of H bonds with PDZ1 of EBP50/ NHERF-1, and the side chains of both C-terminal Leu and Phe fit into the peptide-binding groove of PDZ1x CRALBP.11-cis-retinal and EBP50/NHERF-1 migrated as single components when analyzed individually by gel filtration and as a complex when mixed together before gel filtration. Complex formation was abolished by preincubation of EBP50/NHERF-1 with peptide EVENTAL. The ligand absorption spectrum of the complex was identical with that of CRALBP x 11-cis-retinal, demonstrating that complex formation did not perturb the ligand-binding domain of CRALBP.

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.

Purpose: The underlying cause of involutional blepharoptosis is unknown. The carotenoid content of preaponeurotic and nasal orbital fat among patients with and without involutional ptosis was evaluated to investigate the hypothesis that development of ptosis may be related to low carotenoid content of preaponeurotic orbital fat., Methods: Through a case-control design, the carotenoid content of preaponeurotic and nasal fat of 10 patients with ptosis and 11 patients without ptosis was measured by spectrophotometry analysis. Differences in carotenoid content between patients with and without ptosis were evaluated in unadjusted analyses and in multivariate models adjusted for age, sex, race, and presence of thyroid eye disease as potential confounders., Results: The total carotenoid content of the preaponeurotic fat of patients with ptosis was 59% lower than patients without ptosis (2.98 versus 7.26 absorbance/mg, p = 0.005). When adjustments were made for age, sex, race, and presence of thyroid eye disease, this difference was attenuated, but there was still a trend toward lower preaponeurotic fat carotenoid content among patients with ptosis (p = 0.09). The carotenoid content of the nasal fat was not significantly different among patients with and without ptosis (2.69 versus 3.40 absorbance/mg, p = 0.33). A lower ratio of preaponeurotic to nasal carotenoid content was demonstrated among patients with ptosis compared with patients without ptosis (1.4 versus 2.1; p = 0.06 unadjusted, p = 0.10 adjusted)., Conclusions: Patients with involutional ptosis show trends toward having lower carotenoid content in preaponeurotic fat. Further investigation of the potential role of orbital fat carotenoids in the development of involutional ptosis is warranted.

Purpose: To determine whether the visual cycle is affected in mice without a functional gene for cellular retinol-binding protein type I (CRBPI(-/-) mice)., Methods: Visual-cycle retinoids and rhodopsin levels were analyzed in eyes of dark adapted (DA) CRBPI(-/-) and wild-type (wt) mice before and during recovery from a flash. The rate of dark adaptation was analyzed using electroretinography (ERG)., Results: all-trans-retinyl esters were reduced to approximately 33% of wt levels in DA CRBPI(-/-) mice. Recovery from a flash in wt mice produced transient accumulations of all-trans-retinal and all-trans-retinyl ester, as the pulse of retinoid produced by the flash traversed the visual cycle. In CRBPI(-/-) mice, all-trans-retinal accumulated transiently, as in wt mice. However, all-trans-retinol also accumulated transiently in the neural retina, and the transient increase in all-trans-retinyl ester of the wt was reduced. Rates of 11-cis-retinal and rhodopsin formation were comparable in wt and CRBPI(-/-) mice. Dark adaptation was delayed by a factor of approximately two., Conclusions: The accumulation of all-trans-retinol in neural retina, in the absence of CRBPI and the reduced amount of retinyl esters in the RPE suggest that the binding protein participates in a process that drives diffusion of all-trans-retinol from photoreceptor cells to RPE, perhaps by delivering vitamin A to lecithin-retinol acyltransferase (LRAT) for esterification. Because the perturbation occurred upstream of a slow step of the visual cycle, there was no major impairment of the rate of visual pigment regeneration.

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.

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.

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.

Photoisomerization of 11-cis-retinal to all-trans-retinal and reduction to all-trans-retinol occur in photoreceptor outer segments whereas enzymatic esterification of all-trans-retinol, isomerization to 11-cis-retinol, and oxidation to 11-cis-retinal occur in adjacent cells. The processes are linked into a visual cycle by intercellular diffusion of retinoids. Knowledge of the mechanistic aspects of the visual cycle is very limited. In this study, we utilize chemical analysis of visual cycle retinoids to assess physiological roles for components inferred from in vitro experiments and to understand why excised mouse eyes fail to regenerate their bleached visual pigment. Flash illumination of excised mouse eyes or eyecups, in which regeneration of rhodopsin does not occur, produced a block in the visual cycle after all-trans-retinal formation; constant illumination of eyecups produced a block in the cycle after all-trans-retinol formation; and constant illumination of whole excised eyes resulted in a block of the cycle after formation of all-trans-retinyl ester. These blocks emphasize the role of cellular metabolism in the visual cycle. Interphotoreceptor retinoid-binding protein (IRBP) has been postulated to play a role in intercellular retinoid transfer in the retina; however, the rates of recovery of 11-cis-retinal and of regeneration of rhodopsin in the dark in IRBP-/- mice were very similar to those found with wild-type (wt) mice. Thus, IRBP is necessary for photoreceptor survival but is not essential for a normal rate of visual pigment turnover. Arrestin forms a complex with activated rhodopsin, quenches its activity, and affects the release of all-trans-retinal in vitro. The rate of recovery of 11-cis-retinal in arrestin-/- mice was modestly delayed relative to wt, and the rate of rhodopsin recovery was approximately 80% of that observed with wt mice. Thus, the absence of arrestin appeared to have a minor effect on the kinetics of the visual cycle.

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.

Cellular retinaldehyde-binding protein (CRALBP) is abundant in the retinal pigment epithelium and Müller glial cells of the retina, where it forms complexes with endogenous 11-cis-retinoids. We examined the distribution of CRALBP in extraretinal tissues using polyclonal antibodies (pAb) and monoclonal antibodies (mAb). A protein was detected by immunoblot analysis in extracts of bovine and rat brain and optic nerve but not in several other tissues. This protein had electrophoretic, chromatographic, and retinoid-binding properties identical to those of CRALBP from bovine retina. Comparison of the masses of tryptic peptides and of partial amino acid sequences derived from brain and retinal CRALBP indicated that the two proteins are probably identical. Immunoperoxidase cytochemistry and double labeling immunofluorescence revealed CRALBP(+) cells in brain that resembled oligondendrocytes and not astrocytes, microglial cells, or pinealocytes. In 11-day-old rat brain, approximately 11% of the CRALBP(+) cells were labeled with the Rip antibody, a marker for oligodendroglia. In developing rat optic nerve, the temporal appearance of CRALBP(+) cells corresponded to that of oligodendrocytes and not that of astrocytes. In adult rat and mouse optic nerves, the CRALBP(+) somata showed the same distribution as oligodendrocytes. No endogenous retinoids were associated with CRALBP isolated from dark-dissected adult bovine brain. The results suggest that CRALBP has functions in addition to retinoid metabolism and visual pigment regeneration.

An enzyme of bovine retina that catalyzes oxidation of retinaldehyde to retinoic acid was purified to homogeneity and a monoclonal antibody (mAb H-4) was generated. MAb H-4 recognized a single component (Mr = 55,000) in extracts of bovine retina and other bovine tissues. The antibody showed no cross-reactivity with extracts of rat, monkey, or human retinas. A 2067 bp cDNA was selected from a retina cDNA expression library using mAb H-4. The cDNA hybridized with a similarly sized, moderately abundant mRNA prepared from bovine retina. Nucleotide sequence analysis indicated that the cDNA contained a single open reading frame encoding 501 amino acids that have 88% sequence identity with the amino-acid sequence of human hepatic Class 1 aldehyde dehydrogenase. Amino-acid sequence analysis of purified enzyme demonstrated that the cDNA encodes the isolated enzyme. MAb H-4 specifically labeled the somata and processes of a subset of amacrine cells in bovine retinal sections. Labeled amacrine somata were located on both sides of the inner plexiform layer, and their processes ramified into two laminae within the inner plexiform layer. The inner radial processes of Müller (glial) cells were weakly reactive with mAb H-4. Weak immunostaining of amacrine cells was found in monkey retina with mAb H-4, but no signal was detected in rat or human retina. The results provide further evidence for metabolism and function of retinoids within cells of the inner retina and define a novel class of retinal amacrine cells.

Two modifications of an extraction assay for retinoid dehydrogenases are described. The first method involves the transfer of tritium from carbon-15 of [15-3H]-retinol or [15-3H]retinaldehyde to NAD, whereas in the second method, tritium from [3H]NADPH is transferred to all-trans-retinaldehyde. Since both versions of the assay involve the interconversion of water-soluble and -insoluble tritium-labeled compounds, a simple phase partition is sufficient to separate labeled products from labeled reactants. The assays are shown to provide reliable estimations of the reaction progress for three retinoid dehydrogenases of the visual system when compared to HPLC analysis of retinoid products or gel filtration analysis of pyridine nucleotide. The assays will be useful in studying retinoid dehydrogenases from other tissues and in principle can be modified for other dehydrogenase reactions with water-insoluble substrates.

Cellular retinaldehyde-binding protein (CRALBP) carries 11-cis-retinol or 11-cis-retinaldehyde as endogenous ligands and may function as a substrate carrier protein that modulates interaction of these retinoids with visual cycle enzymes. As a first approach to identifying functional domains and protein recognition sites in CRALBP, a low resolution topological and epitope map has been developed using monoclonal and polyclonal antibodies and limited proteolysis. Fifteen peptides of 8-31 residues spanning 99% of the 316-residue bovine CRALBP were synthesized and used to prepare 13 anti-peptide polyclonal antibodies. Using a competitive ELISA procedure, peptide epitopes were classified as either accessible or inaccessible in the native protein based on the extent of their recognition by these site-specific antibodies. Use of the synthetic peptides to map the epitopes of a polyclonal antibody to intact CRALBP confirmed that the amino terminus and carboxyl terminus are immunodominate regions and hence likely to be exposed, at least in part. Limited tryptic proteolysis of native CRALBP produced three major fragments which were shown by microsequence and Western analysis to be derived from sequential loss of short peptides from the amino terminus. None of these major fragments reacted with four monoclonal antibodies (mAbs) to intact CRALBP although each mAb immunoprecipitated native CRALBP. These results and the lack of mAb recognition of any of the synthetic peptides indicates that the amino terminus of the protein is exposed and contains part of an assembly epitope recognized by the mAbs. Overall this study indicates that residues 1-30, 100-124, and 257-285 contain highly exposed segments in the native protein and therefore constitute potential interaction domains for CRALBP and visual cycle enzymes. Residues 30-99 and 176-229 are inaccessible in the native structure and may be involved with retinoid binding. These results provide a basis for a systematic higher resolution mutagenesis study directed toward correlating CRALBP structural domains with function.

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)

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)

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.