Spectrum of Mutations in the RPGR Gene That Are Identified in 20% of Families with X-Linked Retinitis Pigmentosa

The RPGR (retinitis pigmentosa GTPase regulator) gene for RP3, the most frequent genetic subtype of X-linked retinitis pigmentosa (XLRP), has been shown to be mutated in 10%-15% of European XLRP patients. We have examined the RPGR gene for mutations in a cohort of 80 affected males from apparently unrelated XLRP families, by direct sequencing of the PCR-amplified products from the genomic DNA. Fifteen different putative disease-causing mutations were identified in 17 of the 80 families; these include four nonsense mutations, one missense mutation, six microdeletions, and four intronic-sequence substitutions resulting in splice defects. Most of the mutations were detected in the conserved N-terminal region of the RPGR protein, containing tandem repeats homologous to those present in the RCC-1 protein (a guanine nucleotide-exchange factor for Ran-GTPase). Our results indicate that mutations either in as yet uncharacterized sequences of the RPGR gene or in another gene located in its vicinity may be a more frequent cause of XLRP. The reported studies will be beneficial in establishing genotype-phenotype correlations and should lead to further investigations seeking to understand the mechanism of disease pathogenesis.     

RGNTF单抗与多抗的特性和抑制作用(简报)

继本实验室周明华等新近发现顶盖提取液(Te)中,有视网膜神经节细胞诱向因子(RGNTF)之后,为了进一步验证它的生物     

Dopamine Induces Light-Adaptive Retinomotor Movements in Bullfrog Cones via D2 Receptors and in Retinal Pigment Epithelium via D1 Receptors

In the eyes of lower vertebrates, retinal photoreceptors and melanin pigment granules of the retinal pigment epithelium (RPE) exhibit characteristic retinomotor movements in response to changes in ambient illumination and to signals from an endogenous circadian clock. We previously reported that 3,4-dihydroxyphenylethylamine (dopamine) mimicked the effect of light on these movements in photo-receptors and RPE cells of green sunfish, Lepomis cyanellus, by interacting with D2 dopaminergic receptors. Here, we report that dopamine also mimics the effect of light on cone and RPE retinomotor movements in bullfrogs, Rana catesbeiana, i.e., dopamine induces cone contraction and RPE pigment dispersion. Dopamine induced cone contraction in isolated dark-adapted bullfrog retinas incubated in constant darkness in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). This effect of dopamine was inhibited by a D2 but not a D1 antagonist and mimicked by a D2 but not a D1 agonist. These results suggest that induction of cone contraction by dopamine is mediated by D2 dopaminergic receptors and that cone adenylate cyclase activity is inhibited. Thus, dopamine acts via the same type of receptor in both bullfrog and green sunfish retinas to induce cone contraction. In contrast, dopamine influences RPE retinomotor movement via different receptors in fish and bullfrog. Dopamine induced light-adaptive pigment dispersion in isolated dark-adapted bullfrog RPE-eyecups incubated in constant darkness in normal Ringer's solution. Because the retina was not present, these experiments demonstrate a direct effect of dopamine on bullfrog RPE. This effect of dopamine on bullfrog RPE was inhibited by a D1 but not a D2 antagonist and mimicked by a D1 but not a D2 agonist. Furthermore, agents that increase the concentration of intracellular cyclic AMP also induced pigment dispersion in dark-adapted bullfrog RPE-eyecups incubated in the dark. These results suggest that dopamine induces pigment dispersion in bullfrog RPE via D1 dopaminergic receptors. Thus, dopamine acts via different receptors on bullfrog (D1) versus green sunfish (D2) RPE to induce pigment dispersion. In addition, inhibitor studies indicate that pigment dispersion is actin dependent in teleost but not in bullfrog RPE. Dopamine-induced pigment dispersion was inhibited by cytochalasin D in isolated RPE sheets of green sunfish but not in RPE-eyecups of bullfrogs. Together, these observations indicate that dopamine mimics the effect of light on cone and RPE retinomotor movements in both fish and bullfrogs. However, in the RPE, different receptors mediate the effect of dopamine, and different cytoskeletal mechanisms are used to affect pigment transport.(ABSTRACT TRUNCATED AT 400 WORDS)     

2-Amino-4-Phosphonobutyric Acid Exerts a Light-Dependent Effect on Post-Gabaculine Levels of Retinal γ-Aminobutyric Acid (GABA): Evidence that ON Synaptic Pathways Regulate Retinal GABAergic Transmission

Abstract: The effects of light, 2-amino-4-phosphonobutyric acid (APB), and kainic acid on rat retinal γ-aminobutyric acid (GABA)-ergic transmission were studied by measuring levels of retinal GABA following subcutaneous injection of gabaculine, an irreversible inhibitor of GABA-transaminase. Post-gabaculine levels of retinal GABA in light-exposed rats were significantly greater than those in rats held in darkness. The synaptic mechanism of this effect of light was examined by measuring post-gabaculine levels of retinal GABA in rats placed into either lighted or darkened conditions after receiving unilateral intravitreal injections of APB, a glutamate analogue that selectively decreases the activity of ON synaptic pathways in the retina. APB attenuated the post-gabaculine accumulation of GABA in rats held in the light, but not in those placed into darkness. Furthermore, the light-dependent increment in post-gabacu line accumulation of retinal GABA was entirely APB sensitive, and the effect of APB was entirely light dependent. In contrast to APB, kainic acid stimulated the post-gabaculine accumulation of retinal GABA in vivo. Our findings suggest that APB and kainic acid influence GABAergic transmission at different sites in the retina and that some retinal GABAergic neurons are either ON or ON-OFF amacrine cells.     

Guanine Nucleotides Regulate 2-[125I]Iodomelatonin Binding Sites in Chick Retinal Pigment Epithelium but Not in Neuronal Retina

The characteristics of the binding sites labeled by the radioligand 2-[125I]iodomelatonin were compared in chicken neuronal retina and retinal pigment epithelium (RPE). Specific binding of 2-[125I]iodomelatonin in both sites was stable, saturable, reversible, and of high affinity. Scatchard analysis revealed an affinity constant (KD) of 446 +/- 55 pM and a total number of binding sites (Bmax) of 25.4 +/- 2.2 fmol/mg of protein for neuronal retina. For RPE the KD was 34.1 +/- 2.2 pM and the Bmax 59.5 +/- 5.2 fmol/mg of protein. Competition experiments with various melatonin analogues gave the following order of affinities: 2-iodomelatonin greater than 2-chloromelatonin greater than melatonin greater than 6-chloromelatonin greater than 6-hydroxymelatonin greater than N-acetylserotonin greater than 6-methoxyharmalan greater than 5-hydroxytryptamine. Linear regression of log Ki values from neuronal retina and RPE gave a highly significant correlation (r = 0.994, n = 8; p less than 0.001). GTP inhibited specific binding to RPE membranes in a concentration-dependent manner, but not in neuronal retinal membranes. The present results strongly suggest that a single type of melatonin receptor is found in neuronal retina and RPE, and that the site in RPE is coupled to a guanine nucleotide-binding regulatory protein (G protein), but that in neuronal retina is not.     

Evidence for an Insulin-Like Growth Factor Autocrine-Paracrine System in the Retinal Photoreceptor-Pigment Epithelial Cell Complex

The interphotoreceptor matrix (IPM), lying between retinal photoreceptor and pigment epithelial (RPE) cells, contains insulin-like growth factor I (IGF-I) immunoreactivity that co-elutes with authentic human IGF-I in HPLC analyses. Cultured human RPE cells synthesize and release IGF-I, raising the possibility that the RPE serves as a source of IPM IGF-I in vivo. Photoreceptor rod outer segments and cultured monkey RPE cells express specific IGF-I receptors with alpha-subunits of 120 and 138 kDa, respectively. They thus appear to be of the "brain" (in photoreceptors) and "peripheral" (in RPE cells) receptor subtypes. Additionally, the IPM contains high levels of an IGF binding protein (IGF-BP) that specifically binds IGF-I and IGF-II. The IPM-BP is visualized as a single radiographic band by both ligand blot and affinity cross-linking procedures. With enzymes specific for removing N- and O-linked oligosaccharides, the IPM-BP was found to contain O- but not N-linked glycosylated side chains. The distinctive size and glycosylation pattern of the IPM-BP indicate that it is not derived from the vitreous or serum but instead is synthesized locally. The presence of IGF-I and IGF-BP in the IPM, together with the presence of IGF-I receptors on both photoreceptor and RPE cells, suggests the presence of an outer retina autocrine-paracrine system.     

Retinal ganglion cells of high cytochrome oxidase activity in the rat

Retinal ganglion cells in the rat were studied using the heavy metal intensified cytochrome oxidase and horseradish peroxidase histochemical methods.The results show that a population of large retinal ganglion cells was consistently observed with the cytochrome oxidase staining method in retinas of normal rats or rats which received unilateral thalamotomy at birth.These cytochrome oxidase rich ganglion cells appeared to have large somata,3-6 primary dendrites and extensive dendritic arbors,and are comparable to ganglion cells labeled by the wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP).However,the morphological details of some of the cells revealed by the cytochrome oxidase staining method are frequently better than those shown by the HRP histochemical method.These results suggest that the mitochondrial enzyme cytochrome oxidase can be used as a simple but reliable marker for identifying and studying a population of retinal genglion cells with high metabolic rate in the rat.     

Anterograde labelling from the optic nerve reveals multiple central targets in the teleost,Lethrinus chrysostomus (Perciformes)

Summary Cobaltous-lysine is transported anterogradely from the optic nerve of the teleost, Lethrinus chrysostomus (Lethrinidae, Perciformes). The marginal optic tract is labelled in longtitudinal bands of light and dark staining fibres which persists caudally within the ventral division but not in the dorsal division. This species possesses multiple central targets in the contralateral preoptic, diencephalic, pretectal, periventricular and tectal regions of the brain. In addition, a greater subdivision of the marginal optic tract is found to project to various nuclei. Ipsilateral projections are found in the suprachiasmatic nucleus and in the region of the horizontal commissure. Projections are also found in the telencephalic region of the nucleus olfactoretinalis and the thalamic region of the nucleus thalamoretinalis. The retinotopicity of some of these nuclei, found in previous studies, is discussed in relation to the possibility of specific sub-populations of retinal ganglion cells having different central targets.Abbreviations used in the Text and Figures A nucleus anteriorthalami - AO accessory optic nucleus - AOT accessory optic tract - AxOT axial optic tract - BO nucleus of the basal optic root - C cerebellum - HCv ventral division of horizontal commissure - I nucleus intermedius thalami - IL inferior lobe - MdOT medial optic tract - MO medulla oblongata - MOTd dorsal division of the marginal optic tract - MOTi intermediate division of the marginal optic tract - MOtv ventral division of the marginal optic tract - O olfactory bulb - OT optic tract - PC nucleus pretectalis centralis - PCo posterior commissure - Pd nucleus pretectalis dorsalis - PG preglomerular complex - PPd nucleus pretectalis periventricularis, pars dorsalis - PPv nucleus pretectalis periventricularis, pars ventralis - PSm nucleus pretectalis superficial pars magnocellularis - PSp nucleus pretectalis superficialis, pars parvocellularis - Sn suprachiasmatic nucleus - TEL telencephalon - TeO optic tectum - TL torus longtitudinalis - TrOlfR tractus olfactoretinalis - VCg granular layer of the valvula cerebelli - VCm molecular layer of the valvula cerebelli - VM nucleus medialis thalami - VL nucleus ventrolateralis thalami - VMdOT ventro-medial optic tract     

The glucose transport in retinal pigment epithelium is via passive facilitated diffusion

The glucose transport across the bovine retinal pigment epithelium (RPE) was studied in a modified Ussing chamber. Unidirectional fluxes were recorded with radioactive tracers L-[14C]-glucose (LG) and 3-O-methyl-D-[3H]-glucose (MDG). There was no significant difference between the unidirectional MDG fluxes (retina to choroid, and choroid to retina directions) with or without ouabain. The effects of two glucose transporter inhibitors, phloretin and cytochalasin B, on the glucose fluxes from choroid to retina cells were also investigated. The MDG flux was found to be inhibited by 45.5% by phloretin (10(-4) M) and 87.4% by cytochalasin B (10(-4) M). These inhibitory characteristics resembled the facilitated diffusion mode of glucose transport. The glucose transporter protein in the plasma membrane of RPE was located by means of photolabeling [3H]-cytochalasin B. The labeled plasma membrane enriched fraction was analysed by SDS-PAGE. The glucose transporter of bovine RPE was found to have a molecular weight range of 46-53 kDa. The molecular weight range of this transporter protein agreed with those of facilitated glucose transporters in other tissues indicating a molecular similarity between them. The results indicated that the glucose transport across the RPE is via passive facilitated diffusion.     

The quantitative relationship between olfactory axons and mitral/tufted cells in developing Xenopus with partially deafferented olfactory bulbs

Partial deafferentation of the olfactory bulb in Xenopus embryos was performed to analyze the effects of afferent innervation on the development of the central olfactory structure. In an attempt to analyze a possible early inductive role of the olfactory axons, one olfactory placode was removed before differentiation of the neural tube began (stages 26–31). A morphological and quantitative analysis was performed on larvae at the onset of metamorphic climax (stage 58). When the single olfactory nerve innervated one side of the rostral telencephalon, a single olfactory bulb developed on that side and no olfactory bulb formed on the contralateral side. When the nerve innervated the midline of the rostral telencephalon, a smaller-than-normal, fused olfactory bulb developed. Partial deafferentation at these early stages resulted in a significant reduction in the number of olfactory axons (to approximately one-half of control values) and a corresponding decrease in the number of mitral/tufted cells (output neurons of the olfactory bulb). To control for possible damage to the neural tube during olfactory-placode removal, a portion of the neural tube directly beneath one of the olfactory placodes was removed in embryos. In these animals, the neural tube regenerated within 24 h and formed a normal olfactory bulb; olfactory axon and mitral/tufted-cell numbers were not significantly different from controls. In conclusion, olfactory-afferent innervation was critical for differentiation of the olfactory bulb, and decreasing the number of olfactory axons resulted in a reduction in the number of output neurons of the olfactory bulb. © 1993 John Wiley & Sons, Inc.