Molecular genetic study of autosomal dominant retinitis pigmentosa in Lithuanian patients. (1/191)

Lithuanian patients with visual problems were clinically examined for retinitis pigmentosa (RP). A total of 33 unrelated families with autosomal dominant RP (adRP) were identified. Screening for mutations in the rhodopsin (RHO) and peripherin/RDS (RDS) genes was performed using DNA heteroduplex analysis. Direct DNA sequencing in the cases of heteroduplex formation showed the presence of the following mutations and polymorphisms in 14 adRP patients: RHO gene - Lys248Arg (1 case), and Pro347Leu (2 cases); RDS gene - Glu304Gln (12 cases), Lys310Arg (5 cases), and Gly338Asp (12 cases). The presence of these mutations (except Lys248Arg in the RHO gene) was confirmed by relevant restriction enzyme digestion. The frequency of the RDS gene mutations Glu304Gln and Gly338Asp was estimated to be 36.4%, while mutation Lys310Arg was less frequent (15.2%). These 3 RDS gene mutations appear to be polypeptide polymorphisms not related to adRP.  (+info)

Detection of alterations in all three exons of the peripherin/RDS gene in Swedish patients with retinitis pigmentosa using an efficient DGGE system. (2/191)

AIMS: To develop a sensitive mutation screening procedure suitable for routine analysis of the peripherin/RDS gene, and to estimate the nature and prevalence of peripherin/RDS gene mutations in Swedish patients with autosomal dominant retinitis pigmentosa. METHODS: To make the method as sensitive as possible, as many as eight segments, covering the three exons and the flanking intron sequences of the peripherin/RDS gene, were analysed by denaturing gradient gel electrophoresis. A group of 38 Swedish patients with a clinical diagnosis of autosomal dominant retinitis pigmentosa were screened for mutations in the peripherin/RDS gene. RESULTS: Three point mutations were found in four of the patients and five polymorphisms were defined. One mutation in exon 1, R172W, has been described previously in other ethnic groups as causing a macular degeneration. Another mutation, in exon 2 and causing the substitution F211L, was found in two unrelated patients. A third mutation, resulting in the likely non-pathogenic substitution S289L, as well as a polymorphism not reported previously, was found in exon 3. CONCLUSIONS: The screening procedure described allows detection of mutations in all of the exons, including the polymorphic 5' and 3' ends of the gene, and is therefore suitable for routine screening of peripherin/RDS gene defects in patients with autosomal dominant retinitis pigmentosa. The frequency of mutations found in the Swedish patient group indicates that defects in the peripherin/RDS gene might be a more common cause of autosomal dominant retinitis pigmentosa than was thought previously.  (+info)

Neurons derived in vitro from ES cells express homeoproteins characteristic of motoneurons and interneurons. (3/191)

We have characterized different neuronal subpopulations derived from in vitro differentiation of embryonic stem (ES) cells using as markers the expression of several homeodomain transcription factors. Following treatment of embryo-like aggregates with retinoic acid (RA), Pax-6, a protein expressed by ventral central nervous system (CNS) progenitors is induced. In contrast, Pax-7 expressed in vivo by dorsal CNS progenitors, and erbB3, a gene expressed by neural crest cells and its derivatives, are almost undetectable. CNS neuronal subpopulations generated expressed combinations of markers characteristic of somatic motoneurons (Islet-1/2, Lim-3, and HB-9), cranial motoneurons (Islet-1/2 and Phox2b) and interneurons (Lim-1/2 or EN1). Molecular characterization of neuron subtypes generated from ES cells should considerably facilitate the identification of new genes expressed by restricted neuronal cell lineages.  (+info)

Identification of dividing, determined sensory neuron precursors in the mammalian neural crest. (4/191)

Sensory and autonomic neurons of the vertebrate peripheral nervous system are derived from the neural crest. Here we use the expression of lineage-specific transcription factors as a means to identify neuronal subtypes that develop in rat neural crest cultures grown in a defined medium. Sensory neurons, identified by expression of the POU-domain transcription factor Brn-3.0, develop from dividing precursors that differentiate within 2 days following emigration from the neural tube. Most of these precursors generate sensory neurons even when challenged with BMP2, a factor that induces autonomic neurogenesis in many other cells in the explants. Moreover, BMP2 fails to prevent expression of the sensory-specific basic helix-loop-helix (bHLH) transcription factors neurogenin1, neurogenin2 and neuroD, although it induces expression of the autonomic-specific bHLH factor MASH1 and the paired homeodomain factor Phox2a in other cells. These data suggest that there are mitotically active precursors in the mammalian neural crest that can generate sensory neurons even in the presence of a strong autonomic-inducing cue. Further characterization of the neurons generated from such precursors indicates that, under these culture conditions, they exhibit a proprioceptive and/or mechanosensory, but not nociceptive, phenotype. Such precursors may therefore correspond to a lineally (Frank, E. and Sanes, J. (1991) Development 111, 895-908) and genetically (Ma, Q., Fode, C., Guillemot, F. and Anderson, D. J. (1999) Genes Dev. 13, in press) distinct subset of early-differentiating precursors of large-diameter sensory neurons identified in vivo.  (+info)

Peripherin immunoreactivity labels small diameter vestibular 'bouton' afferents in rodents. (5/191)

Recent morphophysiological studies have described three different subpopulations of vestibular afferents. The purpose of this study was to determine whether peripherin, a 56-kDa type III intermediate filament protein present in small sensory neurons in dorsal root ganglion and spiral ganglion cells, would also label thin vestibular afferents. Peripherin immunohistochemistry was done on vestibular sensory organs (cristae ampullares, utriculi and sacculi) of chinchillas, rats, and mice. In these sensory organs, immunoreactivity was confined to the extrastriolar region of the utriculus and the peripheral region of the crista. The labelled terminals were all boutons, except for an occasional calyx. In vestibular ganglia, immunoreactivity was restricted to small vestibular ganglion cells with thin axons. The immunoreactive central axons of vestibular ganglion cells form narrow bundles as they pass through the caudal spinal trigeminal tract. As they exit this tract, several bundles coalesce to form a single, narrow bundle passing caudally through the ventral part of the lateral vestibular nucleus. Finally, we conclude that all labelled axons and terminals were vestibular afferents rather than efferents, as no immunoreactivity in the vestibular efferent nucleus of the brainstem was observed.  (+info)

Lactose promotes organized photoreceptor outer segment assembly and preserves expression of photoreceptor proteins in retinal degeneration. (6/191)

PURPOSE: We have previously shown that lactose promotes the proper assembly of photoreceptor outer segments in the absence of the retinal pigment epithelium (RPE). The purpose of this study was to determine if the difference between organized and disorganized membranes was a variation in the amounts of two structural proteins, opsin and rds/peripherin. METHODS: Eye rudiments were dissected from Xenopus laevis embryos and the RPE was removed prior to culturing in the following media: Niu-Twitty medium; Niu-Twitty with mannose; Niu-Twitty with lactose. Controls included retinas that matured in vitro with an adherent RPE. Photoreceptor ultrastructure was evaluated with emphasis on outer segment membrane organization. The relative amounts of opsin and rds/ peripherin, two outer segment-specific proteins, were determined, as were their immunolabeling patterns. RESULTS: In control retinas, outer segments were composed of stacked, flattened membranous saccules. Opsin labeling of rod outer segments was very dense, indicative of normally organized disc membranes, and rds/peripherin labeling was heavy at the outer segment disc periphery and incisures. In the absence of the RPE, a whorl-like profile of outer segments is present in what would be the sub-retinal space. Opsin immunolabeling was patchy and disorganized. Immunolabeling of rds/peripherin was present, but in a disorderly array. Mannose showed no protective effect. In contrast, lactose promoted the formation of organized outer segments and allowed for near normal expression of both photoreceptor markers. In retinas with disorganized outer segments, the expression of opsin is downregulated while the expression of rds/peripherin is maintained or upregulated. CONCLUSIONS: Lactose protects against the retinal degeneration induced by RPE removal by preserving the outer segment structure and the photoreceptor immunolabeling patterns. It also maintains constant the relative amounts of opsin and rds/peripherin. It is possible that in degenerating retinas, photoreceptors upregulate rds/peripherin expression in attempt to provide additional support for the proper folding of nascent membranes, however this is insufficient to permit organization of the photoreceptor outer segments. Our results suggest that rescue-effect of lactose is mediated by a non-rds/peripherin related mechanism.  (+info)

Analysis of the rds/peripherin.rom1 complex in transgenic photoreceptors that express a chimeric protein. (7/191)

Mice homozygous for the retinal degeneration slow (rds) mutation completely lack photoreceptor outer segments. The rds gene encodes rds/peripherin (rds), a membrane glycoprotein in the rims of rod and cone outer segment discs. rds is present as a complex with the related protein, rom1. Here, we generated transgenic mice that express a chimeric protein (rom/D2) containing the intradiscal D2 loop of rds in the context of rom1. rom/D2 was N-glycosylated, formed covalent homodimers, and interacted non-covalently with itself, rds, and rom1. The rds.rom/D2 interaction was significantly more stable than the non-covalent interaction between rds and rom1 by detergent/urea titration. Analysis of mice expressing rom/D2 revealed that rds is 2.5-fold more abundant than rom1, interacts non-covalently with itself and rom1 via the D2 loop, and forms a high order complex that may extend the entire circumference of the disc. Expression of rom/D2 fully rescued the ultrastructural phenotype in rds+/- mutant mice, but it had no effect on the phenotype in rds-/- mutants. Together, these observations explain the striking differences in null phenotypes and frequencies of disease-causing mutations between the RDS and ROM1 genes.  (+info)

A peptide analogue to a fusion domain within photoreceptor peripherin/rds promotes membrane adhesion and depolarization. (8/191)

Photoreceptor peripherin/rds promotes membrane fusion, through a putative fusion domain located within the C-terminus (Boesze-Battaglia et al., Biochemistry 37 (1998) 9477-9487). A peptide analogue to this region, PP-5, competitively inhibits peripherin/rds mediated fusion in a cell free assay system. To characterize how this region is involved in the fusion process we investigated two of the individual steps in membrane fusion, membrane adhesion and membrane destabilization inferred from depolarization studies. Membrane depolarization was measured as the collapse of a valinomycin induced K(+) diffusion potential in model membranes, using a potential sensitive fluorescent probe, diS-C(2)-5. PP-5 induced membrane depolarization in a concentration dependent manner. PP-5 has been shown by Fourier transform infrared spectroscopy to be an amphiphilic alpha-helix. Therefore, the requirement for an amphiphilic alpha-helix to promote depolarization was tested using two mutant peptides designed to disrupt either the amphiphilic nature of PP-5 (PP-5AB) or the alpha-helical structure (PP-5HB). PP-5AB inhibited PP-5 induced depolarization when added in an equimolar ratio to PP-5. Neither mutant peptide alone or in combination with PP-5 had any effect on calcium dependent vesicle aggregation. Using non-denaturing gel electrophoresis and size exclusion chromatography techniques PP-5 was shown to form a tetrameric complex. Equimolar mixtures of PP-5 and PP-5AB formed a heterotetramer which was unable to promote membrane depolarization. The hypothesis that PP-5 tetramers promote membrane depolarization is consistent with the calculated Hill coefficient of 3.725, determined from a Hill analysis of the depolarization data.  (+info)

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