Obtaining absorbance spectra from turbid retinal cell and tissue suspensions - Beating the light-scatter problem.

Light scattering and inability to uniformly expose the cuvette contents to an incident light beam are significant limitations of traditional spectrophotometers. The first of these drawbacks limits their usefulness in studies of turbid cellular and tissue suspensions; the second limits their use in photodecomposition studies. Our strategy circumvents both problems. Although we describe its potential usefulness in vision sciences, application of spherical integrating cuvettes has broad application. Absorbance spectra of turbid bovine rod outer segments and dispersed living frog retina were studied using a standard single-pass 1 cm cuvettes, or a spherical integrating cuvette (DeSa Presentation Chamber, DSPC). The DSPC was mounted on an OLIS Rapid Scanning Spectrophotometer configured to generate 100 spectral scans/sec. To follow rhodopsin bleaching kinetics in living photoreceptors, portions of dark-adapted frog retina were suspended in the DSPC. The incoming spectral beam at 2 scans/sec entered the chamber through a single port. Separate ports contained a 519 nm light emitting diode (LED), or window to the photomultiplier tube. The surface of the DSPC was coated with a highly reflective coating allowing the chamber to act as a multi-pass cuvette. The LED is triggered to flash and the PMT shutter temporarily closed during a "Dark-Interval" between each spectral scan. By interleafing scans with LED pulses, spectra changes can be followed in real time. Kinetic analysis of the 3-dimensional data was performed by Singular Value Decomposition. For crude bovine rod outer segment suspensions, the 1 cm single-pass traditional cuvette gave non-informative spectra dominated by high absorbances and Rayleigh scattering. In contrast, spectra generated using the DSPC showed low overall absorbance with peaks at 405 and 503 nm. The later peak disappeared with exposure to white light in presence of 100 mM hydroxylamine. For the dispersed living retinal, the sample was pulsed at 519 nm between the spectra. The 495 nm rhodopsin peak gradually reduced in size concomitant with the emergence of a 400 nm peak, probably representing Meta II. A conversion mechanism of two species, A → B with rate constant of 0.132 sec-1 was fit to the data. To our knowledge this is the first application of integrating sphere technology to retinal spectroscopy. Remarkably, the spherical cuvette designed for total internal reflectance to produce diffused light was efffectively immune to light scattering. Furthermore, the higher effective path length enhanced sensitivity and could be accounted for mathematically allowing determination of absorbance/cm. The approach, which complements the use of the CLARiTy RSM 1000 for photodecomposition studies (Gonzalez-Fernandez et al. Mol Vis 2016, 22:953), may facilitate studies of metabolically active photoreceptor suspensions or whole retinas in physiological assays.

Interphotoreceptor retinoid-binding protein removes all-trans-retinol and retinal from rod outer segments, preventing lipofuscin precursor formation.

Interphotoreceptor retinoid-binding protein (IRBP) is a specialized lipophilic carrier that binds the all-trans and 11-cis isomers of retinal and retinol, and this facilitates their transport between photoreceptors and cells in the retina. One of these retinoids, all-trans-retinal, is released in the rod outer segment by photoactivated rhodopsin after light excitation. Following its release, all-trans-retinal is reduced by the retinol dehydrogenase RDH8 to all-trans-retinol in an NADPH-dependent reaction. However, all-trans-retinal can also react with outer segment components, sometimes forming lipofuscin precursors, which after conversion to lipofuscin accumulate in the lysosomes of the retinal pigment epithelium and display cytotoxic effects. Here, we have imaged the fluorescence of all-trans-retinol, all-trans-retinal, and lipofuscin precursors in real time in single isolated mouse rod photoreceptors. We found that IRBP removes all-trans-retinol from individual rod photoreceptors in a concentration-dependent manner. The rate constant for retinol removal increased linearly with IRBP concentration with a slope of 0.012 min-1 µm-1 IRBP also removed all-trans-retinal, but with much less efficacy, indicating that the reduction of retinal to retinol promotes faster clearance of the photoisomerized rhodopsin chromophore. The presence of physiological IRBP concentrations in the extracellular medium resulted in lower levels of all-trans-retinal and retinol in rod outer segments following light exposure. It also prevented light-induced lipofuscin precursor formation, but it did not remove precursors that were already present. These findings reveal an important and previously unappreciated role of IRBP in protecting the photoreceptor cells against the cytotoxic effects of accumulated all-trans-retinal.

Pericellular interphotoreceptor matrix dictates outer retina critical surface tension.

Retinal detachments create two pathological surfaces, the surface of the outer neural retinal, and an apical retinal-pigmented epithelium (RPE) surface. The physicochemical properties of these two new surfaces are poorly understood. At a molecular level little is known how detachments form, how to optimize reattachment, or prevent extension of the detachment. A major limitation is lack of information about the biophysical consequences of the retina-RPE separation. The primary challenge is determining the molecular properties of the pathological interface surfaces. Here, using detached bovine retina, we show that this hurdle can be overcome through a combination of biophysical and ultrastructural approaches. The outer surface of freshly detached bovine neural retina, and isolated molecular components of the outer retina were subjected to: 1) Contact angle goniometry to determine the critical surface tension of the outer retinal surface, isolated insoluble interphotoreceptor matrix (IPM) and purified interphotoreceptor retinoid binding protein (IRBP); 2) Multiple attenuated internal reflectance infrared (MAIR-IR) spectroscopy was used to characterize the molecular composition of the retinal surface. MAIR-IR depth penetration was established through ellipsometric measurement of barium-stearate films. Light microscopy, immunohistochemistry and electron microscopy defined the structures probed spectroscopically. Furthermore, the data were correlated to IR spectra of docosahexaenoic acid, hyaluronan, chondroitin-6-sulfate and IRBP, and imaging by IR-microscopy. We found that the retinal critical surface tension is 24 mN/m, similar to isolated insoluble IPM and lower than IRBP. Barium-stearate calibration studies established that the MAIR-IR spectroscopy penetration depth was 0.2 µm. Ultrastructural observations and MAIR-IR studies of isolated outer retina components determined that the pericellular IPM coating the outer retinal surface is primarily responsible for these surface properties. The critical surface tension of detached bovine retina is dictated not by the outer segments, but by a pericellular IPM covering the outer segment tips.

Technical brief: Pump-probe paradigm in an integrating cavity to study photodecomposition processes.

PURPOSE: Assaying photodecomposition is challenging because light must be used to initiate the photodamage and light must be used to monitor the photodecomposition. The experimental requirements are as follows: 1) During exposure of the actinic beam, continuously monitor the spectral characteristics of the sample, 2) uniformly expose the reactants to the actinic source, 3) obtain informative spectra in the presence of light scatter, and 4) achieve sufficient sensitivity for dilute reactants. Traditional spectrophotometers cannot address these issues due to sample turbidity, the inability to uniformly expose the cuvette contents to the incident beam, the inability to simultaneously perform spectral scans, and inherent low sensitivity. Here, we describe a system that meets these challenges in a practical way. METHODS: Light access to a 8.6 ml quartz integrating sphere containing 10 µM all-trans retinol in PBS was provided by three ports at right angles allowing for the following: 1) actinic light delivery from light-emitting diodes (LEDs) firing at 100 pulses/sec, 2) entry of a separate scanning beam at 100 scans/sec (10,000 µsec scan time) via an OLIS RSM 1000 ultraviolet/visual (UV/Vis) rapid-scanning spectrophotometer (RSM), and 3) light exit to the detector photomultiplier. The RSM spectral intermediate slit was partially covered to allow for a "dark" period of 2,000 µsec when no scanning light was admitted to the cuvette. During that interval, the LED was flashed, and the photomultiplier was temporarily blocked by a perforated spinning shutter disk. The absorbance per centimeter, which is increased due to the internal reflectance of the integrating sphere compared to a standard 1 cm rectangular cuvette, was calculated according to Fry et al. (2010) Applied Optics 49:575. Retinoid photodecomposition was confirmed with high-performance liquid chromatography (HPLC). RESULTS: Using the RSM to trigger the LED flash and photomultiplier shutter closure during the "dark" period allowed actinic flashes to be placed between scans. Exposure of the all-trans retinol to 366 nm flashes resulted in marked reduction in absorbance and a blue shift of the λmax. A white LED, despite its higher photon output, did not support all-trans retinol photolysis. Singular value decomposition (SVD) analysis revealed three spectral intermediates with mechanism, I -> II -> III. HPLC analysis of the reactants at the beginning and the conclusion of the light exposure confirmed the retinol photodecomposition. CONCLUSIONS: The highly reflecting cavity acts as a multipass cuvette that markedly increased the light path length and, thus, sensitivity. Triggering the LED during a dark period within the scan time allowed the actinic flashes to be interleafed between scans in a pump-probe paradigm. Furthermore, the entire sample was exposed to scan beam and actinic flashes, which is not possible in traditional spectrophotometers. Finally, the integrating cavity cuvette allowed use of turbid samples. SVD was useful for resolving spectral intermediates. Although the identity of the intermediates was not determined here, the ability to define molecular intermediates during photodecomposition reactions will allow future studies to isolate and identify the degradation products and determine the mechanism of light-induced retinoid degradation and that of retinoid-binding protein-mediated photoprotection.

Fold conservation and proteolysis in zebrafish IRBP structure: Clues to possible enzymatic function?

Multiple functions for Interphotoreceptor Retinoid-Binding Protein (IRBP) may explain its localization in the retina, vitreous and pineal gland and association with retinitis pigmentosa and myopia. We have been engaged in uncovering the structure-function relationships of this interesting protein long thought to bind visual-cycle retinoids and fatty acids in the subretinal space. Although hydrophobic domains capable of binding such ligands have now been found, we ask what other structural domains might be present that could predict new functions? Interestingly, IRBP possesses a fold similar to C-terminal processing proteases (CTPases) but is missing the PDZ domain. Here we present structural evidence that this fold may have a role in a recently observed autoproteolytic activity of the two-module zebrafish (z) IRBP (Ghosh et al. Exp. Eye Res., 2015). When the structure of Scenedesmus obliquus D1 CTPase (D1P) is superimposed with the first module of zIRBP (z1), the PDZ domain of D1P occupies roughly the same position in the amino acid sequence as the inter-domain tether in z1, between residues P71 and P85. The catalytic triad K397, S372 and E375 of D1P is located at the inter-domain interfacial cleft, similarly as the tetrad K241, S243, D177 and T179 of z1 residues, presumed to have proteolytic function. Packing of two adjacent symmetry-related molecules within the z1 crystal show that the helix α8 penetrates the interfacial cleft underneath the inter-domain tether, forming a simple intermolecular "knot". The full-length zIRBP is cleaved at or immediately after T309, which is located at the end of α8 and is the ninth residue of the second module z2. We propose that the helix α8 within intact zIRBP bends at P301, away from the improbable knotted fold, and positions the cleavage site T309 near the putative catalytic tetrad of the neighboring zIRBP to be proteolytically cleaved. The conservation of this functional catalytic domain suggests that possible physiological roles of IRBP as a hydrolase needs to be considered.

Structure of zebrafish IRBP reveals fatty acid binding.

Interphotoreceptor retinoid-binding protein (IRBP) has a remarkable role in targeting and protecting all-trans and 11-cis retinol, and 11-cis retinal during the rod and cone visual cycles. Little is known about how the correct retinoid is efficiently delivered and removed from the correct cell at the required time. It has been proposed that different fatty composition at that the outer-segments and retinal-pigmented epithelium have an important role is regulating the delivery and uptake of the visual cycle retinoids at the cell-interphotoreceptor-matrix interface. Although this suggests intriguing mechanisms for the role of local fatty acids in visual-cycle retinoid trafficking, nothing is known about the structural basis of IRBP-fatty acid interactions. Such regulation may be mediated through IRBP's unusual repeating homologous modules, each containing about 300 amino acids. We have been investigating structure-function relationships of Zebrafish IRBP (zIRBP), which has only two tandem modules (z1 and z2), as a model for the more complex four-module mammalian IRBP's. Here we report the first X-ray crystal structure of a teleost IRBP, and the only structure with a bound ligand. The X-ray structure of z1, determined at 1.90 Å resolution, reveals a two-domain organization of the module (domains A and B). A deep hydrophobic pocket with a single bound molecule of oleic acid was identified within the N-terminal domain A. In fluorescence titrations assays, oleic acid displaced all-trans retinol from zIRBP. Our study, which provides the first structure of an IRBP with bound ligand, supports a potential role for fatty acids in regulating retinoid binding.

Thiol-dependent antioxidant activity of interphotoreceptor retinoid-binding protein.

Interphotoreceptor retinoid-binding protein (IRBP), which is critical to photoreceptor survival and function, is comprised of homologous tandem modules each ∼300 amino acids, and contains 10 cysteines, possibly 8 as free thiols. Purification of IRBP has historically been difficult due to aggregation, denaturation and precipitation. Our observation that reducing agent 1,4-dithiothreitol dramatically prevents aggregation prompted investigation of possible functions for IRBP's free thiols. Bovine IRBP (bIRBP) was purified from retina saline washes by a combination of concanavalin A, ion exchange and size exclusion chromatography. Antioxidant activity of the purified protein was measured by its ability to inhibit oxidation of 2,2'-azinobis [3-ethylbenzothiazoline-6-sulfonate] by metmyoglobin. Homology modeling predicted the relationship of the retinoid binding sites to cysteine residues. As a free radical scavenger, bIRBP was more active than ovalbumin, thioredoxin, and vitamin E analog Trolox. Alkylation of free cysteines by N-ethylmaleimide inhibited bIRBP's antioxidant activity, but not its ability to bind all-trans retinol. Structural modeling predicted that Cys 1051 is at the mouth of the module 4 hydrophobic ligand-binding site. Its free radical scavenging activity points to a new function for IRBP in defining the redox environment in the subretinal space.

The First Steps of the Visual Cycle in Human Rod and Cone Photoreceptors.

Purpose: Light detection destroys the visual pigment. Its regeneration, necessary for the recovery of light sensitivity, is accomplished through the visual cycle. Release of all-trans retinal by the light-activated visual pigment and its reduction to all-trans retinol comprise the first steps of the visual cycle. In this study, we determined the kinetics of all-trans retinol formation in human rod and cone photoreceptors. Methods: Single living rod and cone photoreceptors were isolated from the retinas of human cadaver eyes (ages 21 to 90 years). Formation of all-trans retinol was measured by imaging its outer segment fluorescence (excitation, 360 nm; emission, >420 nm). The extent of conversion of released all-trans retinal to all-trans retinol was determined by measuring the fluorescence excited by 340 and 380 nm. Measurements were repeated with photoreceptors isolated from Macaca fascicularis retinas. Experiments were carried out at 37°C. Results: We found that ∼80% to 90% of all-trans retinal released by the light-activated pigment is converted to all-trans retinol, with a rate constant of 0.24 to 0.55 min-1 in human rods and ∼1.8 min-1 in human cones. In M. fascicularis rods and cones, the rate constants were 0.38 ± 0.08 min-1 and 4.0 ± 1.1 min-1, respectively. These kinetics are several times faster than those measured in other vertebrates. Interphotoreceptor retinoid-binding protein facilitated the removal of all-trans retinol from human rods. Conclusions: The first steps of the visual cycle in human photoreceptors are several times faster than in other vertebrates and in line with the rapid recovery of light sensitivity exhibited by the human visual system.

Carotenoids as possible interphotoreceptor retinoid-binding protein (IRBP) ligands: a surface plasmon resonance (SPR) based study.

Uptake, transport and stabilization of xanthophylls in the human retina are important components of a complex multistep process that culminates in a non-uniform distribution of these important nutrients in the retina. The process is far from understood; here, we consider the potential role of interphotoreceptor retinoid-binding protein (IRBP) in this process. IRBP is thought to facilitate the exchange of 11-cis-retinal, 11-cis-retinol and all-trans-retinol between the retinal pigment epithelium (RPE), photoreceptors and Müller cells in the visual cycle. Structural and biochemical studies suggest that IRBP has a variety of nonequivalent ligand binding sites that function in this process. IRBP is multifunctional, being able to bind a variety of physiologically significant molecules including fatty acids in the subretinal space. This wide range of binding activities is of particular interest because it is unknown whether the lutein and zeaxanthin found in the macula originate from the choroidal or retinal circulations. If from the choroidal circulation, then IRBP is a likely mediator for their transport across the interphotoreceptor matrix. In this report, we explore the binding interactions of retinoids, fatty acids, and carotenoids with IRBP using surface plasmon resonance (SPR)-based biosensors. IRBP showed similar affinity toward retinoids and carotenoids (1-2 µM), while fatty acids had approximately 10 times less affinity. These results suggest that further studies should be carried out to evaluate whether IRBP has a physiologically relevant role in binding lutein and zeaxanthin in the interphotoreceptor matrix.

Cone outer segment and Müller microvilli pericellular matrices provide binding domains for interphotoreceptor retinoid-binding protein (IRBP).

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 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 comparably less to rod outer segments. Co-incubation with unlabeled IRBP markedly reduced this binding; ovalbumin-Alexa 647 and Alexa 647 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.

Novel strategy for subretinal delivery in Xenopus.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Liver Damage and Exposure to Toxic Concentrations of Endogenous Retinoids in the Pathogenesis of COVID-19 Disease: Hypothesis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a marked tropism for the biliary tract; it damages the bile ducts and hepatocytes and can lead to liver decompensation, cirrhosis, and sepsis. The pathogenesis of liver damage and its association with damage to the lung, heart, and brain and to the other protean manifestations of COVID-19 disease are not fully understood. In particular, tissue damage from thinning and leaky blood vessels appears to result from an inflammatory response to the virus rather than the virus itself. This article outlines a new hypothesis of the nature of the inflammatory factor responsible for tissue damage in COVID-19. Review of the literature reveals that COVID-19 disease closely resembles an endogenous form of hypervitaminosis A. We propose that SARS-CoV-2 virus-induced liver damage causes retinoic acid and stored retinyl esters to be released into the circulation in toxic concentrations, unbound to protein, with resulting damage to organs including the lungs, heart, blood vessels, and skin. Several lines of evidence support this model of disease causation. Subject to testing, strategies for the effective treatment and prevention of COVID-19 could include targeting the action and accumulation of retinoids.

Congenital Zika Virus Infection in Immunocompetent Mice Causes Postnatal Growth Impediment and Neurobehavioral Deficits.

A small percentage of babies born to Zika virus (ZIKV)-infected mothers manifest severe defects at birth, including microcephaly. Among those who appeared healthy at birth, there are increasing reports of postnatal growth or developmental defects. However, the impact of congenital ZIKV infection in postnatal development is poorly understood. Here, we report that a mild congenital ZIKV-infection in pups born to immunocompetent pregnant mice did not display apparent defects at birth, but manifested postnatal growth impediments and neurobehavioral deficits, which include reduced locomotor and cognitive deficits that persisted into adulthood. We found that the brains of these pups were smaller, had a thinner cortical layer 1, displayed increased astrogliosis, decreased expression of microcephaly- and neuron development- related genes, and increased pathology as compared to mock-infected controls. In summary, our results showed that even a mild congenital ZIKV infection in immunocompetent mice could lead to postnatal deficits, providing definitive experimental evidence for a necessity to closely monitor postnatal growth and development of presumably healthy human infants, whose mothers were exposed to ZIKV infection during pregnancy.

Purification of the full-length Xenopus interphotoreceptor retinoid binding protein and growth of diffraction-quality crystals.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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).

Human embryonic and neuronal stem cell markers in retinoblastoma.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Module structure of interphotoreceptor retinoid-binding protein (IRBP) may provide bases for its complex role in the visual cycle - structure/function study of Xenopus IRBP.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Crystal structure of the functional unit of interphotoreceptor retinoid binding protein.

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.

The Value of Cytology Smears for Acanthamoeba Keratitis.

Purpose. Acanthamoeba keratitis remains a difficult diagnosis despite advances in genetic and imaging technologies. The purpose of this paper is to highlight the utility of cytology smears for diagnosis of Acanthamoeba keratitis. Methods. This is a case study of the diagnostic course for a patient with suspected Acanthamoeba keratitis. Results. A 40-year-old male with poor contact lens hygiene presented with severe left eye pain. Slit lamp examination showed two peripheral ring infiltrates without an epithelial defect. The epithelium over both infiltrates was removed with a Kimura spatula. Half of the sample was smeared on a dry microscope slide and the other half was submitted for Acanthamoeba culture and PCR. Both culture and PCR were negative for Acanthamoeba, but hematoxylin and eosin stain of the smear revealed double-walled cysts. Conclusion. H&E staining of corneal cytology specimens is an efficient and readily available test for diagnosis of Acanthamoeba keratitis.

Cancer stem cell characteristics in retinoblastoma.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

The interphotoreceptor retinoid-binding protein (IRBP) of the chicken (Gallus gallus domesticus).

PURPOSE: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.