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ABSTRACT: The close packing of vertebrate photoreceptors presents a challenge to the exchange of molecules between the outer segments, retinal pigmented epithelium (RPE), and Müller glia. An extracellular hyaluronan scaffold separates these cells while soluble interphotoreceptor matrix (IPM) proteins traffic visual cycle retinoids, fatty acids, and other molecules between them. In the IPM, retinoids and fatty acids are carried by interphotoreceptor retinoid-binding protein (IRBP). The fact that much of the retina's IRBP can be extracted by saline wash has led to the notion that IRBP does not bind to the retina but freely distributes itself within the subretinal space. In this study, we challenge this idea by asking if there are specialized IPM domains that could bind IRBP perhaps facilitating its ability to target delivery/uptake of its ligands. Xenopus is an ideal animal model to study the role of the IPM in RPE photoreceptor interactions. Here, we took advantage of the large size of its photoreceptors, ability to detach the retina in light, sustainability of the retina in short term organ culture, and the availability of recombinant full-length Xenopus IRBP and antisera directed against Xenopus IRBP. We compared the distribution of wash resistant native IRBP, and that of IRBP-Alexa 647 binding in Xenopus retina. IRBP and cone opsin were localized using anti-Xenopus IRBP serum, and monoclonal COS-1 respectively. Cone matrix sheath proteoglycans were localized with wheat germ agglutinin (WGA), and diffuse IPM proteoglycans with peanut agglutinin (PNA). Wholemounts and frozen sections were compared by immunofluorescence from retinas detached under Ringer's followed by additional washes, or detached directly under 4% paraformaldehyde without Ringer's wash. Undetached Lowicryl embedded retinas were subjected to IRBP immunogold electron microscopy (EM). Immunogold labeled a diffuse network of filamentous structures, and a separate distinct flocculant material directly coating the outer segments, filling the rod periciliary ridge, and associated with Müller microvilli. By immunofluorescence, Ringer's wash removed most of the diffuse IRBP, but not that coating the outer segments. IRBP-Alexa 647 bound to the cone outer segments and Müller villi region, and to a much lesser degree that of the rods. Co-incubation with unlabeled IRBP markedly reduced this binding; ovalbumin-Alexa 647 and dye alone showed no binding. Our data suggest that the pericellular matrix of the cone outer segments and Müller microvilli provide specialized domains that facilitate IRBP's functions.
Experimental Eye Research 03/2013; · 3.26 Impact Factor
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ABSTRACT: Cones are critically dependent on interphotoreceptor retinoid-binding protein (IRBP) for retinoid delivery in the visual cycle. Cone-dominant vertebrates offer an opportunity to uncover the molecular basis of IRBP's role in this process. Here, we explore the association of IRBP with the interphotoreceptor matrix (IPM) of cones vs. rods in cone dominant retinas from chicken (Gallus domesticus), turkey (Meleagris gallopavo), and pig (Sus scrofa). Retinas were detached and fixed directly or washed in saline prior to fixation. Disassociated photoreceptors with adherent matrix were also prepared. Under 2 mM CaCl(2) , insoluble matrix was delaminated from saline washed retinas. The distribution of IRBP, as well as glycans binding peanut agglutinin (cone matrix) and wheat germ agglutinin (rod/cone matrix), was defined by confocal microscopy. Retina flat mounts showed IRBP diffusely distributed in an interconnecting, lattice-like pattern throughout the entire matrix. Saline wash replaced this pattern with fluorescent annuli surrounding individual cone outer segments. In isolated cones and matrix sheets, IRBP colocalized with the peanut agglutinin binding matrix glycans. Our results reveal a wash-resistant association of IRBP with a matrix domain immediately surrounding cone outer segments. The cone matrix sheath may be responsible for IRBP-mediated cone targeting of 11-cis retinoids.
The Journal of Comparative Neurology 09/2011; 520(4):756-69. · 3.81 Impact Factor
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ABSTRACT: Ocular sebaceous carcinoma can masquerade as benign lesions resulting in delay of diagnosis. Early recognition is even more difficult in young patients where the disease rarely occurs. Here, we provide a clinicopathological correlation of ocular sebaceous carcinoma in a young individual lacking history of hereditary cancer or immunosuppression.
A detailed histopathological study including p53 DNA sequencing was performed on an aggressive sebaceous carcinoma presenting in a healthy 32 year-old Caucasian woman. She had no history of retinoblastoma, evidence for a hereditary cancer syndrome, or radiation therapy. However, she potentially was at risk for excessive UV light exposure. A detailed review of the literature is also provided.A moderately well differentiated sebaceous carcinoma was established histopathologically arising from the meibomian gland of the upper eyelid. In most areas, the cytoplasm contained small but distinct Oil-red-O positive vacuoles. Direct sequencing of p53 identified a G:C→A:T mutation at a dipyrimidine site. The mutation results in substitution of arginine for the highly conserved glycine at residue 199 located at the p53 dimer-dimer interface. Energy minimization structural modeling predicts that G199R will neutralize negative charges contributed by nearby inter- and intramonomeric glutamate residues.
This study points to the importance of recognizing that sebaceous carcinoma can occur in young patients with no evidence for hereditary cancer risk or radiation therapy. The G199R substitution is anticipated to alter the stability of the p53 tetrameric complex. The role of UV light in the etiology of sebaceous carcinoma deserves further study. Our findings, taken together with those of others, suggest that different environmental factors could lead to the development of sebaceous carcinoma in different patients.
Diagnostic Pathology 09/2011; 6:81. · 1.64 Impact Factor
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ABSTRACT: The subretinal space, which borders the retinal pigment epithelium (RPE), photoreceptors, and Müller cells, is an ideal location to deliver genetic vectors, morpholino oligos, and nanopharmaceuticals. Unfortunately, materials injected into the space tend to stay localized, and degenerative changes secondary to retinal detachment limit its usefulness. Furthermore, such injection requires penetration of the sclera, RPE/choroid, or the retina itself. Here, we developed a strategy in Xenopus to utilize the continuity of the brain ventricle and optic vesicle lumen during embryogenesis as a means to access the subretinal space.
Wild-type and transgenic embryos expressing green fluorescent protein under the rod-opsin promoter were used for optic vesicle and brain ventricle injections. For injection directly into the optic vesicle, embryos were laid on one side in clay troughs. For brain ventricle injections, embryos were placed standing in foxholes cored from agarose dishes. Linear arrays with each embryo positioned dorsal side toward the micromanipulator facilitated high throughput injections. Twenty-five micrometer micropipettes, which were positioned with a micromanipulator or by hand, were used to pressure inject ~1.0 nl of test solution (brilliant blue, India ink, fluorescein isothiocyanate dextran, or 0.04 µm of latex polystyrene microspheres [FluoSpheres®]). FluroSpheres® were particularly useful in confirming successful injections in living embryos. Anesthetized embryos and tadpoles were fixed in 4% paraformaldehyde and cryoprotected for frozen sections, or dehydrated in ethanol and embedded in methacrylate resin compatible with the microspheres.
Direct optic vesicle injections resulted in filling of the brain ventricle, contralateral optic vesicle, and central canal. Stages 24 and 25 gave the most consistent results. However, even with experience, the success rate was only ~25%. Targeting the vesicle was even more difficult beyond stage 26 due to the flattening of the lumen. In contrast, brain ventricle injections were easier to perform and had a ~90% success rate. The most consistent results were obtained in targeting the diencephalic ventricle, which is located along the midline, and protrudes anteriorly just under the frontal ectoderm and prosencephalon. An anterior midline approach conveniently accessed the ventricle without disturbing the optic vesicles. Beyond stage 30, optic vesicle filling did not occur, presumably due to closure of the connection between the ventricular system and the optic vesicles. Securing the embryos in an upright position in the agarose foxholes allowed convenient access to the frontal cephalic region. On methacrylate sections, the RPE-neural retina interphase was intact and labeled with the microspheres. As development continued, no distortion or malformation of the orbital structures was detected. In green fluorescent protein (GFP), transgenic embryos allowed to develop to stage 41, retinal FluoSpheres® labeling and photoreceptor GFP expression could be observed through the pupil. On cryosections, it was found that the FluoSpheres® extended from the diencephalon along the embryonic optic nerve to the ventral subretinal area. GFP expression was restricted to rod photoreceptors. The microspheres were restricted to the subretinal region, except focally at the lip of the optic cup, where they were present within the retina; this was presumably due to incomplete formation of the peripheral zonulae adherens. Embryos showed normal anatomic relationships, and formation of eye and lens appeared to take place normally with lamination of the retina into its ganglion cell and the inner and outer nuclear layers.
Diencephalic ventricular injection before stage 31 provides an efficient strategy to introduce molecules into the embryonic Xenopus subretinal space with minimal to the developing eye or retina.
Molecular vision 01/2011; 17:2956-69. · 2.20 Impact Factor
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ABSTRACT: Interphotoreceptor retinoid-binding protein (IRBP) appears to target and protect retinoids during the visual cycle. X-ray crystallographic studies had noted a betabetaalpha-spiral fold shared with crotonases and C-terminal protein transferases. The shallow cleft formed by the fold was assumed to represent the retinol-binding site. However, a second hydrophobic site consisting of a highly restricted cavity was more recently appreciated during in silico ligand-docking studies. In this study, the ligand-binding environment within the second module of Xenopus IRBP (X2IRBP) is defined.
Pristine recombinant polypeptide corresponding to X2IRBP was expressed in a soluble form and purified to homogeneity without its fusion tag. Phenylalanine was substituted for tryptophan at each of the putative retinol-binding domains (W450F, hydrophobic cavity; W587F, shallow cleft). Binding of 11-cis and all-trans retinol were observed in titrations monitoring retinol fluorescence enhancement, quenching of tryptophan fluorescence, and energy transfer. The effect of oleic acid on retinol binding was also examined.
A ligand-binding stoichiometry of approximately 1:1 was observed for 11-cis and all-trans with K(d) in the tens of nanomolar range. The substitution mutants showed little effect on retinol binding in titrations after fluorescence enhancement. However, the W450F and not the W587F mutant showed a markedly reduced capacity for fluorescence quenching for both 11-cis and all-trans retinol. Oleic acid inhibited the binding of 11-cis and all-trans retinol in an apparent noncompetitive manner.
The binding site for 11-cis and all-trans retinol is a novel hydrophobic cavity that is highly restrictive and probably distinct from the long chain fatty acid-binding site.
Investigative ophthalmology & visual science 08/2009; 50(12):5577-86. · 3.43 Impact Factor
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ABSTRACT: Interphotoreceptor retinoid-binding protein (IRBP) has been considered essential for normal rod and cone function, as it mediates the transport of retinoids between the photoreceptors and the retinal pigment epithelium. This study was performed to determine whether mutations in the IRBP gene (RBP3) are associated with photoreceptor degeneration.
A consanguineous family was ascertained in which four children had autosomal recessive retinitis pigmentosa (RP). Homozygosity mapping performed with SNP microarrays revealed only one homozygous region shared by all four affected siblings. Sequencing of RBP3, contained in this region, was performed in this family and others with recessive RP. Screening was also performed on patients with various other forms of retinal degeneration or malfunction.
Sequence analysis of RBP3 revealed a homozygous missense mutation (p.Asp1080Asn) in the four affected siblings. The mutation affects a residue that is completely conserved in all four homologous modules of the IRBP protein of vertebrate species and in C-terminal-processing proteases, photosynthesis enzymes found in bacteria, algae, and plants. Based on the previously reported crystal structure of Xenopus IRBP, the authors predict that the Asp1080-mediated conserved salt bridge that appears to participate in scaffolding of the retinol-binding domain is abolished by the mutation. No RBP3 mutations were detected in 395 unrelated patients with recessive or isolate RP or in 680 patients with other forms of hereditary retinal degeneration.
Mutations in RBP3 are an infrequent cause of autosomal recessive RP. The mutation Asp1080Asn may alter the conformation of the IRBP protein by disrupting a conserved salt bridge.
Investigative ophthalmology & visual science 01/2009; 50(4):1864-72. · 3.43 Impact Factor
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Experimental Eye Research 03/2008; 86(2):169-70. · 3.26 Impact Factor
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ABSTRACT: Interphotoreceptor retinol-binding protein (IRBP), composed of two or four homologous modules in tandem, plays an important role in retinoid trafficking between the retinal pigmented epithelium, photoreceptors, and Müller cells. The exact nature of this role is not yet clear. Attempts to purify the full-length retinal IRBP to homogeneity for crystallization purposes have largely been unsuccessful, owing primarily to instability and denaturation of the protein at high concentrations in aqueous media.
A bacterial expression system was used for the production of the recombinant full-length four modules-containing Xenopus IRBP (xIRBP; 1197 amino acids; 131 kDa). An optimized purification strategy and the presence of molar excesses of a thiol-based reducing agent yielded highly pure xIRBP in a soluble, stable and active form, free of its fusion partner. Binding of all-trans retinol was characterized by fluorescence spectroscopy monitoring ligand-fluorescence enhancement, quenching of endogenous protein fluorescence, and energy transfer.
We grew the first diffraction-quality crystal of xIRBP. We have gathered diffraction data from these crystals to 2.46 A resolution, sufficient to yield an atomic model of the tertiary structure of IRBP. Retinol-binding results determined by fluorescence spectroscopy show roughly one retinol-binding site per polypeptide chain.
The binding stoichiometry taken together with modeling data suggest that not all modules are functionally equivalent. Determination of the full-length IRBP structure will be a significant breakthrough in understanding the functional roles of IRBP in the visual cycle. The advances presented here will not only lead to the structure of the full-length IRBP, but will allow us to understand how the modules interact in the function of IRBP. Furthermore, these studies will allow characterization of the ligand-binding site(s) with bound ligand(s).
Molecular vision 02/2007; 13:2275-81. · 2.20 Impact Factor
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ABSTRACT: Interphotoreceptor retinoid-binding protein's (IRBP) remarkable module structure may be critical to its role in mediating the transport of all-trans and 11-cis retinol, and 11-cis retinal between rods, cones, RPE and Müller cells during the visual cycle. We isolated cDNAs for Xenopus IRBP, and expressed and purified its individual modules, module combinations, and the full-length polypeptide. Binding of all-trans retinol, 11-cis retinal and 9-(9-anthroyloxy) stearic acid were characterized by fluorescence spectroscopy monitoring ligand-fluorescence enhancement, quenching of endogenous protein fluorescence, and energy transfer. Finally, the X-ray crystal structure of module-2 was used to predict the location of the ligand-binding sites, and compare their structures among modules using homology modeling.
The full-length Xenopus IRBP cDNA codes for a polypeptide of 1,197 amino acid residues beginning with a signal peptide followed by four homologous modules each approximately 300 amino acid residues in length. Modules 1 and 3 are more closely related to each other than either is to modules 2 and 4. Modules 1 and 4 are most similar to the N- and C-terminal modules of the two module IRBP of teleosts. Our data are consistent with the model that vertebrate IRBPs arose through two genetic duplication events, but that the middle two modules were lost during the evolution of the ray finned fish. The sequence of the expressed full-length IRBP was confirmed by liquid chromatography-tandem mass spectrometry. The recombinant full-length Xenopus IRBP bound all-trans retinol and 11-cis retinaldehyde at 3 to 4 sites with Kd's of 0.2 to 0.3 microM, and was active in protecting all-trans retinol from degradation. Module 2 showed selectivity for all-trans retinol over 11-cis retinaldehyde. The binding data are correlated to the results of docking of all-trans-retinol to the crystal structure of Xenopus module 2 suggesting two ligand-binding sites. However, homology modeling of modules 1, 3 and 4 indicate that both sites may not be available for binding of ligands in all four modules.
Although its four modules are homologous and each capable of supporting ligand-binding activity, structural differences between their ligand-binding domains, and interactions between the modules themselves will be critical to understanding IRBP's complex role in the visual cycle.
BMC Biochemistry 02/2007; 8:15. · 1.99 Impact Factor
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ABSTRACT: Although often associated with holoprosencephaly, little detail of the histopathology of cyclopia is available. Here, we describe the ocular findings in a case of trisomy 13 to better understand the histogenesis of the rosettes, or tubules, characteristic of the retinal dysplasia associated with this condition.
A full pediatric autopsy was performed of a near term infant who died shortly after birth from multiple congenital anomalies including fused facial-midline structures. A detailed histopathological study of the ocular structures was performed. The expression of interphotoreceptor retinoid-binding protein (IRBP), cellular retinal-binding protein (CRALBP), rod opsin, and Sonic Hedgehog (Shh) were studied by immunohistochemistry.
Holoprosencephaly, and a spectrum of anatomical findings characteristic of Patau's syndrome, were found. Cytogenetic studies demonstrated trisomy 13 [47, XY, +13]. The eyes were fused but contained two developed separate lenses. In contrast, the cornea, and angle structures were hypoplastic, and the anterior chamber had failed to form. The retina showed areas of normally laminated neural retina, whereas in other areas it was replaced by numerous neuronal rosettes. Histological and immunohistochemical studies revealed that the rosettes were composed of differentiated retinal neurons and Müller cell glia. In normally laminated retina, Shh expression was restricted to retinal-ganglion cells, and to a population of neurons in the inner zone of the outer nuclear layer. In contrast, Shh could not be detected in the dysplastic rosettes.
The histopathology of cyclopia appears to be more complex than what may have been previously appreciated. In fact, the terms "cyclopia" and "synophthalmia" are misnomers as the underlying mechanism is a failure of the eyes to form separately during development. The rosettes found in the dysplastic retina are fundamentally different than those of retinoblastoma, being composed of a variety of differentiated cell types. The dysplastic rosettes are essentially laminated retina failing to establish a polarized orientation, resulting in the formation of tubules. Finally, our findings suggest that defective ganglion cell Shh expression may contribute to the ocular pathology of cyclopia.
Diagnostic Pathology 02/2007; 2:48. · 1.64 Impact Factor
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ABSTRACT: Retinoblastoma (RB) is the most common intraocular tumor of early childhood. The early onset of RB, coupled with our previous findings of cancer stem cell characteristics in RB, led us to hypothesize that subpopulations of RB tumors harbor markers and behaviors characteristic of embryonic and neuronal origin.
Our RB sources included: human pathological tissues, and the human RB cell lines Y79 and WERI-RB27. Microarray screening, single and dual-label immunocytochemistry and RT-PCR were performed to detect embryonic and neuronal stem cell markers, such as Oct3/4, Nanog, CD133, and Musashi-1. To test for functional evidence of stem cell behavior, we examined RB cells for their ability to form neurospheres and retain BrdU label as indicators of self-renewal and slow cell cycling, respectively.
Microarray comparisons of human RB tumors with normal retinal tissue detected upregulation of a number of genes involved in embryonic development that were also present in Y79 cells, including Oct3/4, Nanog, Musashi-1 and Musashi-2, prominin-1 (CD133), Jagged-2, Reelin, Thy-1, nestin, Meis-1,NCAM, Patched, and Notch4. Expression of Musashi-1, Oct3/4 and Nanog was confirmed by immunostaining and RT-PCR analyses of RB tumors and RB cell lines. CD133 expression was confirmed by PCR analysis. Y79 and WERI-RB27 contained populations of Hoechst-dim/ABCG2-positive cells that co-localized with embryonic stem cell markers Oct3/4-ABCG2 and Nanog-ABCG2. Subpopulations of Y79 and WERI-RB27 cells were label-retaining (as seen by BrdU incorporation) and were able to generate neurospheres, both hallmarks of a stem cell phenotype.
Small subpopulation(s) of RB cells express human embryonic and neuronal stem cell markers. There are also subpopulations that demonstrate functional behavior (label retention and self-renewal) consistent with cancer stem cells. These findings support the hypothesis that RB is a heterogeneous tumor comprised of subpopulation(s) with stem cell-like properties.
Molecular vision 02/2007; 13:823-32. · 2.20 Impact Factor
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ABSTRACT: The clinicopathologic findings of this patient were typical for Vogt-Koyanagi-Harada syndrome. Clinically, the patient had sudden onset of blindness, pleocytosis, and temporary hearing loss, with no history of eye trauma or surgery. Histopathologically, the choroid and iris showed chronic inflammation, loss of choroidal melanocytes, and dense chorioretinal adhesions. Presence of HLA-DRB1*0405 was consistent with the chronic phase of this syndrome.
Archives of pathology & laboratory medicine 08/2006; 130(7):1070-2. · 2.58 Impact Factor
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ABSTRACT: A case of Nocardia asteroides keratitis occurring 3 weeks after laser in situ keratomileusis (LASIK) in a nontraumatized eye is reported. The patient presented with decreased vision, inflammation, and stromal melting of the LASIK flap, discrete infiltrates, and an anterior chamber cellular reaction. Cultures for acid-fast bacteria grew Nocardia asteroides after 5 days. Infection progressed despite treatment with topical antibiotics and eventually required penetrating keratoplasty (PKP). Postoperatively, the patient was placed on moxifloxacin, a fourth-generation flouroquinolone. The patient experienced a recurrence of Nocardia keratitis at the graft-host interface 2 months after the PKP. This eventually resolved with a combination of topical moxifloxacin and imipenem therapy.
Journal of Cataract [?] Refractive Surgery 11/2005; 31(10):2012-5. · 2.26 Impact Factor
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ABSTRACT: Despite decades of investigation, the function of interphotoreceptor retinoid binding protein (IRBP), the most abundant protein in the interphotoreceptor matrix of vertebrates, remains enigmatic. Roles for IRBP in the visual cycle of rod photoreceptors and in the independent visual cycle of cone photoreceptors have been suggested, yet very little is known of the biology of IRBP in cone-dominant retinas, such as those of diurnal birds. Our aim was to identify and characterize expression of the IRBP of the cone-dominant chicken (Gallus gallus domesticus).
Chicken IRBP mRNA was identified by PCR cloning. Primary protein structure, genomic organization, and phylogenies were determined through comparative sequence analyses. Expression of IRBP mRNA was characterized by northern analysis and by in situ hybridization on cryosectioned chicken retina. Expression of the IRBP protein was characterized by western blotting and by indirect immunofluorescence on cryosectioned retina and on retinal whole mounts.
The chicken IRBP gene encodes a secreted protein with a predicted 1,252 amino acid length. The gene structure for chicken IRBP resembles that of most other vertebrates, with four homologous, modular repeats and introns within only the fourth module. Each module is more homologous with the corresponding module in other species than it is with the remaining chicken modules. Chicken retinal tissue contains a single IRBP mRNA transcript of approximately 4.8 kb and western analysis of chicken retina shows a single major band of 140 kDa. Chicken IRBP mRNA is expressed exclusively by retinal photoreceptor cells and the intensity of the hybridization signal shows light/dark rhythmicity. The IRBP protein is localized to the interphotoreceptor matrix of the chicken retina and to intracellular regions of photoreceptors, with a spatial distribution indicating an association with cone outer segments.
The high degree of conservation of IRBP's primary structure, genomic organization, and cell-specific expression within the retinas of all vertebrates examined to date, including those with cone-dominant retinas, implies a conserved role for IRBP in photoreceptor function and/or health. Expression of chicken IRBP and its mRNA are functionally regulated. This report provides a necessary first step to explore a specific function for IRBP in the cone visual cycle.
Molecular vision 02/2005; 11:833-45. · 2.20 Impact Factor
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ABSTRACT: There is increasing evidence that cancer stem cells contribute to tumor progression and chemoresistance in a variety of malignancies, including brain tumors, leukemias, and breast carcinomas. In this study, we tested the hypothesis that retinoblastomas contain subpopulations of cells that exhibit cancer stem cell properties.
The following sources of retinoblastoma cells and tissues were examined for the presence of stem cell markers by immunocytochemistry: retinoblastoma tumors from mice transgenic for the SV40 T-antigen (driven by the beta-luteinizing hormone promoter), cell pellets of human Y79 and WERI-Rb27 retinoblastoma cell lines, and archival human retinoblastoma pathological specimens. Hoechst dye exclusion, mediated by the stem cell surface marker ABCG2 (ATP-binding cassette transporter, G2 subfamily), was assessed by flow cytometry in mouse tumors and WERI-Rb27 cells.
Small numbers of retinoblastoma cells (less than 1%) exhibited immunoreactivity to stem cell markers ABCG2, aldehyde dehydrogenase 1 (ALDH1), MCM2 (minichromosome maintenance marker 2), SCA-1 (Stem cell antigen-1), and p63. Hoechst dye uptake in mouse tumors and WERI-Rb27 cells was enhanced by addition of 50 microM Verapamil, consistent with activity of the calcium-sensitive ABCG2 protein. Flow cytometric analysis confirmed the presence of small subpopulations of cells excluding Hoechst dye in mouse retinoblastoma tumors (0.3%) and WERI-Rb27 cells (0.1%) in a verapamil-sensitive manner. ABCG2 and ALDH1 positive cells were Hoechst-dim, as seen by dual labeling in vitro.
Mouse and human retinoblastoma tumor cells contain a small subpopulation of cells that exhibit a cancer stem cell-like phenotype. Especially significant is the expression of ABCG2 in mouse and human tumor cells, a calcium-sensitive cell surface protein that not only acts to exclude Hoechst dye, but also confers resistance to over 20 different chemotherapeutic agents. These findings point to a heterogeneity in retinoblastoma tumors that may have significant impact on future treatment strategies.
Molecular vision 02/2005; 11:729-37. · 2.20 Impact Factor
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ABSTRACT: The interphotoreceptor matrix mediates interactions between the retinal pigment epithelium, photoreceptors, and Muller cells. Each of these cells contributes to specific proteoglycans, proteins, and growth factors in the interphotoreceptor matrix. Some components, such as interphotoreceptor retinoid binding protein (IRBP), are virtually unique to the interphotoreceptor matrix. It has been proposed that serum albumin, thought to be present in the interphotoreceptor matrix, could act as a surrogate retinoid binding protein within the subretinal space of transgenic mice lacking interphotoreceptor retinoid binding protein. To address this question, we sought to determine whether albumin is present in the interphotoreceptor matrix of IRBP+/+ mice or IRBP-/- mice.
We examined the distribution of albumin in IRBP-/- mice and IRBP+/+ mice using immunofluorescence and immunoperoxidase histochemistry.
The distribution of albumin within the corneal stroma, sclera, and capillaries is consistent with previous work. Serum albumin could not be detected in the interphotoreceptor matrix. The distribution of albumin in IRBP-/- mice was similar to that of their wildtype counterparts.
Serum albumin is not a component of the interphotoreceptor matrix of IRBP+/+ mice or IRBP-/- mice.
Molecular vision 01/2005; 10:1038-46. · 2.20 Impact Factor
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ABSTRACT: Xenopus rods and cones secrete into the interphotoreceptor matrix (IPM) a 124-kDa glycoprotein termed interphotoreceptor retinoid-binding protein (IRBP; Hessler et al. [1996] J. Comp. Neurol. 367:329-341). IRBP is confined to the IPM, being too large to diffuse through the zonulae adherentes between adjacent photoreceptor and Müller cells. Despite this physical entrapment within the subretinal space, IRBP is rapidly cleared from the IPM by an unknown mechanism. Immunohistochemistry and immunoelectron microscopy were used to localize IRBP in intact and detached retina-retinal pigment epithelium (RPE) eyecups. The effects of light, dark, and time of day on the compartmentalization of IRBP were characterized by quantitative Western blot analysis and by immunoprecipitation of IRBP labeled in vivo by intraocular injection of [(35)S]methionine. Immunohistochemistry showed that the apparent intercellular IRBP in both the RPE and the photoreceptors is resistant to saline extraction, in contrast to that in the IPM. In the RPE, IRBP was associated with matrix material within phagosomes and endosomes. The IPM, RPE, and retina contained 75%, 18%, and 7% of the total IRBP in the eye, respectively. The IPM and RPE contain 130 +/- 14 pmoles and 34 +/- 4 pmoles of IRBP, respectively. The amounts of IRBP in the RPE at middark and midlight were the same. Furthermore, the in vivo uptake of [(35)S]methionine-labeled IRBP was light independent. Our studies suggest that IRBP is not strictly confined to the subretinal space but rather that significant amounts are present intracellularly, particularly within the RPE, which does not synthesize IRBP. Furthermore, IRBP secreted by the photoreceptors is taken up from the IPM mainly through a light-independent endocytic pathway separate from outer segment phagocytosis. The role of RPE endocytosis should be explored in relation to the function of IRBP.
The Journal of Comparative Neurology 12/2003; 466(3):331-42. · 3.81 Impact Factor
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Federico Gonzalez-Fernandez
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ABSTRACT: Evolving 40 times independently, eyes are striking examples of convergent evolution in that 11-cis retinaldehyde is always used for photon capture, yet the mechanism for its regeneration may be dramatically different in between systems. In particular, insects, cephalopods and vertebrates show varying physical separation of the cis-->trans photoisomerization and chromphore reisomerization. In the vertebrate retina, these two processes are actually distributed between different cells. This compartmentalization is made possible by the phylogenetic innovation of the two-layered optic cup of the vertebrate retina. This unprecedented design created the subretinal space as a novel anatomical compartment allowing photoreceptors access to the retinal pigment epithelium (RPE) and Müller cells, the two cell types which share the burden of 11-cis retinoid regeneration. To take advantage of this arrangement, early vertebrates appear to have recruited for retinoid binding, the betabetaalpha-spiral fold proven useful in enoyl-CoA isomerase/hydratases, and the carboxy-terminal proteases for stabilizing hydrophobic ligands. Quadruplication of this functional domain within a single polypeptide lead to the emergence of interphotoreceptor retinoid-binding protein (IRBP). IRBP is the main soluble component of the IPM, and is prevented from diffusing out of the subretinal space because its large size excludes it from the photoreceptor/Müller cell zonulae adheretes. Despite this physical entrapment, IRBP is rapidly turned over within the IPM through a process that coordinates secretion of the protein by the photoreceptors, and its removal from the matrix by RPE and photoreceptor endocytosis. The present review will summarize what is known about the structure and function of IRBP to anticipate future avenues of research.
Vision Research 12/2003; 43(28):3021-36. · 2.41 Impact Factor
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ABSTRACT: Interphotoreceptor retinoid binding protein (IRBP), the major soluble component of the interphotoreceptor matrix, is critical to the function, integrity, and development of the vertebrate retina. Although its role is poorly understood, IRBP has been thought to protect 11-cis retinal and all-trans retinol while facilitating their exchange between the photoreceptors and retinal-pigmented epithelium. We determined the X-ray structure of one of the functional units, or modules, of Xenopus laevis IRBP to 1.8 A resolution by multiwavelength anomalous dispersion. The monomeric protein consists of two domains separated by a hydrophobic ligand binding site. A structural homology to the recently solved photosystem II D1 C-terminal-processing protease and the enoyl-CoA isomerase/hydratase family suggests the utility of a common fold used in diverse settings, ranging from proteolysis to fatty acid isomerization to retinoid transport.
Structure 02/2002; 10(1):43-9. · 6.35 Impact Factor
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ABSTRACT: Interphotoreceptor retinoid-binding protein (IRBP) is unusual for a lipid-binding protein in that its gene is expressed uniquely by cells of photoreceptor origin and consists of four homologous repeats, each coding for a module of~300 amino acid residues. All-trans retinol binding domains, which appear to be present in each module, are composed of conserved hydrophobic regions [Baer et al, Exp Eye Res 1998; 66:249-262]. Here we investigate the role of highly conserved arginines contained in these regions.
Chemistry.
