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PubMed2004

Induction of phase 2 genes by sulforaphane protects retinal pigment epithelial cells against photooxidative damage.

The retinal pigment epithelial cell (RPE cell) layer protects the photoreceptors of the retina against oxidative stress. The decline of this capacity is believed to be a major factor in the impairment of vision in age-related macular degeneration. Exposure of human adult RPE cells to UV light at predominantly 320-400 nm (UVA light) in the presence of all-trans-retinaldehyde results in photooxidative cytotoxicity. Significant protection of RPE cells was obtained by prior treatment with phase 2 gene inducers, such as the isothiocyanate sulforaphane or a bis-2-hydroxybenzylideneacetone Michael reaction acceptor. The degree of protection was correlated with the potencies of these inducers in elevating cytoprotective glutathione levels and activities of NAD(P)H:quinone oxidoreductase. In embryonic fibroblasts derived from mice in which the genes for the transcription factor Nrf2, the repressor Keap1, or both Nrf2 and Keap1 were disrupted, the magnitude of resistance to photooxidative damage paralleled the basal levels of glutathione and NAD(P)H:quinone oxidoreductase in each cell type. Demonstration of protection of RPE cells against photooxidative damage by induction of phase 2 proteins may shed light on the role of oxidative injury in ocular disease. Moreover, the finding that dietary inducers provide indirect antioxidant protection suggests novel strategies for preventing chronic degenerative diseases, such as age-related macular degeneration.

Animals↗
PubMed2003

Retinoid activation of retinoic acid receptor but not retinoid X receptor is sufficient to rescue lethal defect in retinoic acid synthesis.

Two isomers of retinoic acid (RA) may be necessary as ligands for retinoid signaling: all-trans-RA for RA receptors (RARs) and 9-cis-RA for retinoid X receptors (RXRs). This was explored by using retinaldehyde dehydrogenase (Raldh)2-/- mouse embryos lacking mesodermal RA synthesis that display early growth arrest unless rescued by all-trans-RA administration. Because isomerization of all-trans-RA to 9-cis-RA can occur, it is unclear whether both ligands are needed for rescue. We show here that an RAR-specific ligand can rescue Raldh2-/- embryos as efficiently as all-trans-RA, whereas an RXR-specific ligand has no effect. Further, whereas all-trans-RA was detected in embryos, 9-cis-RA was undetectable unless a supraphysiological dose of all-trans-RA was administered, revealing that 9-cis-RA is of pharmacological but not physiological significance. Because 9-cis-RA is undetectable and unnecessary for Raldh2-/- rescue, and others have shown that 4-oxo-RA is unnecessary for mouse development, all-trans-RA emerges as the only ligand clearly necessary for retinoid receptor signaling.

Aldehyde Oxidoreductases↗
PubMed2000

Biosynthesis of a major lipofuscin fluorophore in mice and humans with ABCR-mediated retinal and macular degeneration.

Increased accumulation of lipofuscin in cells of the retinal pigment epithelium (RPE) is seen in several forms of macular degeneration, a common cause of blindness in humans. A major fluorophore of lipofuscin is the toxic bis-retinoid, N-retinylidene-N-retinylethanolamine (A2E). Previously, we generated mice with a knockout mutation in the abcr gene. This gene encodes rim protein (RmP), an ATP-binding cassette transporter in rod outer segments. Mice lacking RmP accumulate A2E in RPE cells at a greatly increased rate over controls. Here, we identify three precursors of A2E in ocular tissues from abcr-/- mice and humans with ABCR-mediated recessive macular degenerations. Our results corroborate the scheme proposed by C. A. Parish, M. Hashimoto, K. Nakanishi, J. Dillon & J. Sparrow [Proc. Natl. Acad. Sci. USA (1998) 95, 14609-14613], for the biosynthesis of A2E: (i) condensation of all-trans-retinaldehyde (all-trans-RAL) with phosphatidylethanolamine to form a Schiff base; (ii) condensation of the amine product with a second all-trans-RAL to form a bis-retinoid; (iii) oxidation to yield a pyridinium salt; and (iv) hydrolysis of the phosphate ester to yield A2E. The latter two reactions probably occur within RPE phagolysosomes. As predicted by this model, formation of A2E was completely inhibited when abcr-/- mice were raised in total darkness. Also, once formed, A2E was not eliminated by the RPE. These data suggest that humans with retinal or macular degeneration caused by loss of RmP function may slow progression of their disease by limiting exposure to light. The precursors of A2E identified in this study may represent pharmacological targets for the treatment of ABCR-mediated macular degeneration.

ATP-Binding Cassette Transporters↗
PubMed1994

Hot spots of retinoic acid synthesis in the developing spinal cord.

The embryonic spinal cord is known to be rich in retinoic acid, and several indirect lines of evidence point to a dorsoventral concentration difference of this compound. Previous measurements of dorsoventral retinoic acid levels, however, showed only minor differences. By a combination of microdissection and bioassay techniques, we compared retinoic acid levels with retinaldehyde dehydrogenase levels along spinal cords from early embryonic to postnatal mice. Both parameters vary in parallel, indicating that the principal reason for regional retinoic acid differences in the developing spinal cord is different levels of retinoic acid-generating enzyme. Consistent with previous reports, we observed overall quite high synthesis, decreasing with age, and no dorsoventral difference throughout much of the spinal cord length. In two locations, however, ventral synthesis exceeds dorsal synthesis by several orders of magnitude. These hot spots colocalize with the origins of the limb innervations. They are highest during early stages of limb innervation and disappear slowly postnatally. The synthesis hot spots are likely to create local retinoic acid diffusion halos, which may influence the survival of neurons in the limb regions of the spinal cord and which probably promote innervation of the developing limbs.

Animals↗
PubMed1994

Retinoic acid is necessary for development of the ventral retina in zebrafish.

In the embryonic zebrafish retina, as in other vertebrates, retinoic acid is synthesized from retinaldehyde by two different dehydrogenases, one localized dorsally, the other primarily ventrally. Early in eye development only the ventral enzyme is present. Citral competitively inhibits the ventral enzyme in vitro and decreases the production of retinoic acid in the ventral retina in vivo. Treatment of neurula-stage zebrafish embryos with citral during the formation of the eye primordia results in eyes lacking a ventral retina. This defect can be partially rescued by retinoic acid. The results demonstrate that synthesis of retinoic acid can be selectively inhibited in vivo and suggest that retinoic acid is necessary for the proper development of the ventral retina.

Animals↗
PubMed1994

High levels of a retinoic acid-generating dehydrogenase in the meso-telencephalic dopamine system.

Retinoic acid is synthesized from retinaldehyde by several different dehydrogenases, which are arranged in conserved spatial and developmentally regulated patterns. Here we show for the mouse that a class-1 aldehyde dehydrogenase, characterized by oxidation and disulfiram sensitivity, is found in the brain at high levels only in the basal forebrain. It is present in axons and terminals of a subpopulation of dopaminergic neurons of the mesostriatal and mesolimbic system, forming a retinoic acid-generating projection from the ventral tegmentum to the corpus striatum and the shell of the nucleus accumbens. In the striatum the projection is heaviest to dorsal and rostral regions, declining gradually toward ventral. The enzyme is expressed early in development, shortly after appearance of tyrosine hydroxylase. Other dopaminergic neurons in the brain, as well as the chromaffin cells of the adrenal medulla, do not contain this dehydrogenase. The presence of this enzyme may be a factor in the long-term success of transplants of dopaminergic cells to the corpus striatum in Parkinson disease, and it may play a role in parkinsonism and catatonia due to disulfiram (Antabuse) neurotoxicity.

Alcohol Dehydrogenase↗
PubMed1998

The identification of a 9-cis retinol dehydrogenase in the mouse embryo reveals a pathway for synthesis of 9-cis retinoic acid.

The ligand-controlled retinoic acid (RA) receptors and retinoid X receptors are important for several physiological processes, including normal embryonic development, but little is known about how their ligands, all-trans and 9-cis RA, are generated. Here we report the identification of a stereo-specific 9-cis retinol dehydrogenase, which is abundantly expressed in embryonic tissues known to be targets in the retinoid signaling pathway. The membrane-bound enzyme is a member of the short-chain alcohol dehydrogenase/reductase superfamily, able to oxidize 9-cis retinol into 9-cis retinaldehyde, an intermediate in 9-cis RA biosynthesis. Analysis by nonradioactive in situ hybridization in mouse embryos shows that expression of the enzyme is temporally and spatially well controlled during embryogenesis with prominent expression in parts of the developing central nervous system, sensory organs, somites and myotomes, and several tissues of endodermal origin. The identification of this enzyme reveals a pathway in RA biosynthesis, where 9-cis retinol is generated for subsequent oxidation to 9-cis RA.

Alcohol Oxidoreductases↗
PubMed1997

Identification and characterization of a stereospecific human enzyme that catalyzes 9-cis-retinol oxidation. A possible role in 9-cis-retinoic acid formation.

All-trans- and 9-cis-retinoic acid are active retinoids for regulating expression of retinoid responsive genes, serving as ligands for two classes of ligand-dependent transcription factors, the retinoic acid receptors and retinoid X receptors. Little is known, however, regarding 9-cis-retinoic acid formation. We have obtained a 1.4-kilobase cDNA clone from a normalized human breast tissue library, which when expressed in CHO cells encodes a protein that avidly catalyzes oxidation of 9-cis-retinol to 9-cis-retinaldehyde. This protein also catalyzes oxidation of 13-cis-retinol at a rate approximately 10% of that of the 9-cis isomer but does not catalyze all-trans-retinol oxidation. NAD+ was the preferred electron acceptor for oxidation of 9-cis-retinol, although NADP+ supported low rates of 9-cis-retinol oxidation. The rate of 9-cis-retinol oxidation was optimal at pHs between 7.5 and 8. Sequence analysis indicates that the cDNA encodes a protein of 319 amino acids that resembles members of the short chain alcohol dehydrogenase protein family. mRNA for the protein is most abundant in human mammary tissue followed by kidney and testis, with lower levels of expression in liver, adrenals, lung, pancreas, and skeletal muscle. We propose that this cDNA encodes a previously unknown stereospecific enzyme, 9-cis-retinol dehydrogenase, which probably plays a role in 9-cis-retinoic acid formation.

Alcohol Oxidoreductases↗
PubMed2000

Stereoisomeric specificity of the retinoid cycle in the vertebrate retina.

Understanding of the stereospecificity of enzymatic reactions that regenerate the universal chromophore required to sustain vision in vertebrates, 11-cis-retinal, is needed for an accurate molecular model of retinoid transformations. In rod outer segments (ROS), the redox reaction involves all-trans-retinal and pro-S-NADPH that results in the production of pro-R-all-trans-retinol. A recently identified all-trans-retinol dehydrogenase (photoreceptor retinol dehydrogenase) displays identical stereospecificity to that of the ROS enzyme(s). This result is unusual, because photoreceptor retinol dehydrogenase is a member of a short chain alcohol dehydrogenase family, which is often pro-S-specific toward their hydrophobic alcohol substrates. The second redox reaction occurring in retinal pigment epithelium, oxidation of 11-cis-retinol, which is largely catalyzed by abundantly expressed 11-cis-retinol dehydrogenase, is pro-S-specific to both 11-cis-retinol and NADH. However, there is notable presence of pro-R-specific activities. Therefore, multiple retinol dehydrogenases are involved in regeneration of 11-cis-retinal. Finally, the cellular retinaldehyde-binding protein-induced isomerization of all-trans-retinol to 11-cis-retinol proceeds with inversion of configuration at the C(15) carbon of retinol. Together, these results provide important additions to our understanding of retinoid transformations in the eye and a prelude for in vivo studies that ultimately may result in efficient pharmacological intervention to restore and prevent deterioration of vision in several inherited eye diseases.

Alcohol Oxidoreductases↗
PubMed2000

Omega -crystallin of the scallop lens. A dimeric aldehyde dehydrogenase class 1/2 enzyme-crystallin.

While many of the diverse crystallins of the transparent lens of vertebrates are related or identical to metabolic enzymes, much less is known about the lens crystallins of invertebrates. Here we investigate the complex eye of scallops. Electron microscopic inspection revealed that the anterior, single layered corneal epithelium overlying the cellular lens contains a regular array of microvilli that we propose might contribute to its optical properties. The sole crystallin of the scallop eye lens was found to be homologous to Omega-crystallin, a minor crystallin in cephalopods related to aldehyde dehydrogenase (ALDH) class 1/2. Scallop Omega-crystallin (officially designated ALDH1A9) is 55-56% identical to its cephalopod homologues, while it is 67 and 64% identical to human ALDH 2 and 1, respectively, and 61% identical to retinaldehyde dehydrogenase/eta-crystallin of elephant shrews. Like other enzyme-crystallins, scallop Omega-crystallin appears to be present in low amounts in non-ocular tissues. Within the scallop eye, immunofluorescence tests indicated that Omega-crystallin expression is confined to the lens and cornea. Although it has conserved the critical residues required for activity in other ALDHs and appears by homology modeling to have a structure very similar to human ALDH2, scallop Omega-crystallin was enzymatically inactive with diverse substrates and did not bind NAD or NADP. In contrast to mammalian ALDH1 and -2 and other cephalopod Omega-crystallins, which are tetrameric proteins, scallop Omega-crystallin is a dimeric protein. Thus, ALDH is the most diverse lens enzyme-crystallin identified so far, having been used as a lens crystallin in at least two classes of molluscs as well as elephant shrews.

Aldehyde Dehydrogenase↗
PubMed2001

Isomerization of 11-cis-retinoids to all-trans-retinoids in vitro and in vivo.

The regeneration of 11-cis-retinal, the universal chromophore of the vertebrate retina, is a complex process involving photoreceptors and adjacent retinal pigment epithelial cells (RPE). 11-cis-Retinal is coupled to opsins in both rod and cone photoreceptor cells and is photoisomerized to all-trans-retinal by light. Here, we show that RPE microsomes can catalyze the reverse isomerization of 11-cis-retinol to all-trans-retinol (and 13-cis-retinol), and membrane exposure to UV light further enhances the rate of this reaction. This conversion is inhibited when 11-cis-retinol is in a complex with cellular retinaldehyde-binding protein (CRALBP), providing a clear demonstration of the protective effect of retinoid-binding proteins in retinoid processes in the eye, a function that has been long suspected but never proven. The reverse isomerization is nonenzymatic and specific to alcohol forms of retinoids, and it displays stereospecific preference for 11-cis-retinol and 13-cis-retinol but is much less efficient for 9-cis-retinol. The mechanism of reverse isomerization was investigated using stable isotope-labeled retinoids and radioactive tracers to show that this reaction occurs with the retention of configuration of the C-15 carbon of retinol through a mechanism that does not eliminate the hydroxyl group, in contrast to the enzymatic all-trans-retinol to 11-cis-retinol reaction. The activation energy for the conversion of 11-cis-retinol to all-trans-retinol is 19.5 kcal/mol, and 20.1 kcal/mol for isomerization of 13-cis-retinol to all-trans-retinol. We also demonstrate that the reverse isomerization occurs in vivo using exogenous 11-cis-retinol injected into the intravitreal space of wild type and Rpe65-/- mice, which have defective forward isomerization. This study demonstrates an uncharacterized activity of RPE microsomes that could be important in the normal flow of retinoids in the eye in vivo during dark adaptation.

Animals↗
PubMed2003

Characterization of mouse short-chain aldehyde reductase (SCALD), an enzyme regulated by sterol regulatory element-binding proteins.

Sterol regulatory element-binding proteins (SREBPs) enhance transcription of genes encoding all of the proteins required for the cellular synthesis and uptake of cholesterol and unsaturated fatty acids. Here, we use suppression subtractive hybridization to identify a previously unrecognized SREBP-enhanced gene in mice. The gene encodes a membrane-bound enzyme that we designate SCALD, for short-chain aldehyde reductase. We expressed SCALD in bacteria, purified it extensively, and studied its catalytic properties in detergent solution. The enzyme specifically uses NADPH to reduce a variety of short-chain aldehydes, including nonanal and 4-hydroxy-2-nonenal. The enzyme also reduces retinaldehydes, showing equal activity for all-trans-retinal and 9-cis-retinal. Northern blot analysis indicates that SCALD is expressed most abundantly in mouse liver and testis. In the liver of mice, SCALD is suppressed by fasting and induced by refeeding, consistent with regulation by SREBPs. In testis, SCALD expression is restricted to pachytene spermatocytes, as revealed by visualization of mRNA and protein. SCALD is also expressed in four layers of the retina, including the outer segment of rods and cones, as revealed by immunohistochemistry. SCALD appears to be the mouse ortholog of the human protein that has been designated variously as prostate short-chain dehydrogenase/reductase 1, retinal reductase 1, and retinol dehydrogenase 11. In view of its ability to reduce short-chain aldehydes in addition to retinals, we propose that SCALD may be induced by SREBP in liver and other tissues to prevent toxicity from fatty aldehydes that are generated from oxidation of unsaturated fatty acids that are synthesized as a result of SREBP activity.

Aldehyde Reductase↗
PubMed2003

A novel human cytochrome P450, CYP26C1, involved in metabolism of 9-cis and all-trans isomers of retinoic acid.

Retinoids are potent regulators of cell proliferation, cell differentiation, and morphogenesis and are important therapeutic agents in oncology and dermatology. The gene regulatory activity of endogenous retinoids is effected primarily by retinoic acid isomers (all-trans and 9-cis) that are synthesized from retinaldehyde precursors in a broad range of tissues and act as ligands for nuclear retinoic acid receptors. The catabolism of all-trans-retinoic acid (atRA) is an important mechanism of controlling RA levels in cell and tissues. We have previously identified two cytochrome P450s, P450RAI-1 and P450RAI-2 (herein named CYP26A1 and CYP26B1), which were shown to be responsible for catabolism of atRA both in the embryo and the adult. In this report, we describe the identification, molecular cloning, and substrate characterization of a third member of the CYP26 family, named CYP26C1. Transiently transfected cells expressing CYP26C1 convert atRA to polar water-soluble metabolites similar to those generated by CYP26A1 and -B1. Competition studies with all-trans, 13-cis, and 9-cis isomers of retinoic acid demonstrated that atRA was the preferred substrate for CYP26C1. Although CYP26C1 shares extensive sequence similarity with CYP26A1 and CYP26B1, its catalytic activity appears distinct from those of other CYP26 family members. Specifically, CYP26C1 can also recognize and metabolize 9-cis-RA and is much less sensitive than the other CYP26 family members to the inhibitory effects of ketoconazole. CYP26C1 is not widely expressed in the adult but is inducible by RA in HPK1a, transformed human keratinocyte cell lines. This third CYP26 member may play a specific role in catabolizing both all-trans and 9-cis isomers of RA.

Adult↗
PubMed2004

Retinoic acid synthesis controlled by Raldh2 is required early for limb bud initiation and then later as a proximodistal signal during apical ectodermal ridge formation.

We present evidence for the existence of two phases of retinoic acid (RA) signaling required for vertebrate limb development. Limb RA synthesis is under the control of retinaldehyde dehydrogenase-2 (Raldh2) expressed in the lateral plate mesoderm, which generates a proximodistal RA signal during limb outgrowth. We report that Raldh2(-/-) embryos lack trunk mesodermal RA activity and fail to initiate forelimb development. This is associated with deficient expression of important limb determinants Tbx5, Meis2, and dHand needed to establish forelimb bud initiation, proximal identity, and the zone of polarizing activity (ZPA), respectively. Limb expression of these genes can be rescued by maternal RA treatment limited to embryonic day 8 (E8) during limb field establishment, but the mutant forelimbs obtained at E10 display a significant growth defect associated with a smaller apical ectodermal ridge (AER), referred to here as an apical ectodermal mound (AEM). In these RA-deficient forelimbs, a ZPA expressing Shh forms, but it is located distally adjacent to the Fgf8 expression domain in the AEM rather than posteriorly as is normal. AER formation in Raldh2(-/-) forelimbs is rescued by continuous RA treatment through E10, which restores RA to distal ectoderm fated to become the AER. Our findings indicate the existence of an early phase of RA signaling acting upstream of Tbx5, Meis2, and dHand, followed by a late phase of RA signaling needed to expand AER structure fully along the distal ectoderm. During ZPA formation, RA acts early to activate expression of dHand, but it is not required later for Shh activation.

Aldehyde Oxidoreductases↗
PubMed2006

Avian and canine aldehyde oxidases. Novel insights into the biology and evolution of molybdo-flavoenzymes.

Aldehyde oxidases are molybdo-flavoenzymes structurally related to xanthine oxidoreductase. They catalyze the oxidation of aldehydes or N-heterocycles of physiological, pharmacological, and toxicological relevance. Rodents are characterized by four aldehyde oxidases as follows: AOX1 and aldehyde oxidase homologs 1-3 (AOH1, AOH2, and AOH3). Humans synthesize a single functional aldehyde oxidase, AOX1. Here we define the structure and the characteristics of the aldehyde oxidase genes and proteins in chicken and dog. The avian genome contains two aldehyde oxidase genes, AOX1 and AOH, mapping to chromosome 7. AOX1 and AOH are structurally very similar and code for proteins whose sequence was deduced from the corresponding cDNAs. AOX1 is the ortholog of the same gene in mammals, whereas AOH represents the likely ancestor of rodent AOH1, AOH2, and AOH3. The dog genome is endowed with two structurally conserved and active aldehyde oxidases clustering on chromosome 37. Cloning of the corresponding cDNAs and tissue distribution studies demonstrate that they are the orthologs of rodent AOH2 and AOH3. The vestiges of dog AOX1 and AOH1 are recognizable upstream of AOH2 and AOH3 on the same chromosome. Comparison of the complement and the structure of the aldehyde oxidase and xanthine oxidoreductase genes in vertebrates and other animal species indicates that they evolved through a series of duplication and inactivation events. Purification of the chicken AOX1 protein to homogeneity from kidney demonstrates that the enzyme possesses retinaldehyde oxidase activity. Unlike humans and most other mammals, dog and chicken are devoid of liver aldehyde oxidase activity.

Aldehyde Oxidase↗
PubMed2006

Identification and functional validation of novel autoantigens in equine uveitis.

The development, progression, and recurrence of autoimmune diseases are frequently driven by a group of participatory autoantigens. We identified and characterized novel autoantigens by analyzing the autoantibody binding pattern from horses affected by spontaneous equine recurrent uveitis to the retinal proteome. Cellular retinaldehyde-binding protein (cRALBP) had not been described previously as autoantigen, but subsequent characterization in equine recurrent uveitis horses revealed B and T cell autoreactivity to this protein and established a link to epitope spreading. We further immunized healthy rats and horses with cRALBP and observed uveitis in both species with typical tissue lesions at cRALBP expression sites. The autoantibody profiling outlined here could be used in various autoimmune diseases to detect autoantigens involved in the dynamic spreading cascade or serve as predictive markers.

Animals↗
PubMed1997

A transformed neonatal rat retinal pigment epithelial cell line: secreted protein analysis and fibroblast growth factor and receptor expression.

PURPOSE: A newly-derived transformed neonatal rat retinal pigment epithelial (tnrRPE) cell line was investigated: for secreted proteins by electrophoresis, and for basic and acidic fibroblast growth factor (FGF) by immunocytochemistry, Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR). The FGFR-1 (flg) receptor, which is recognized by aFGF and bFGF, was studied by RT-PCR. METHODS: Retinal pigment epithelial (RPE) cells were isolated from 6-day-old pigmented normal Long Evans rats, and became spontaneously transformed after the second passage. RESULTS: RPE cells at the 5th through 28th passages expressed the epithelial cell marker cytokeratin and cellular retinaldehyde binding protein (CRALBP), an RPE cell marker, but were negative for glial fibrillary acidic protein (GFAP), as shown by immunofluorescence. Secreted proteins of late passage tnrRPE cells were in a narrow molecular weight range of 60-80kDa, while early passage cells exhibited multiple proteins from 20-200kDa. These tnrRPE cells increased by 17-30 fold over a 4-day culture period. At 5th and 28th passage, immunostaining for bFGF and aFGF was dense within nuclei, but light and diffuse within the cytoplasm of transformed RPE cells. As shown by Northern blot, similar levels of message for bFGF were detected in 5th and 30th passage RPE cells. As shown by Northern blot, similar levels of message for bFGF were detected in 5th and 30th passage RPE cells. Furthermore, as shown by RT-PCR, bFGF mRNA was found in freshly isolated and transformed neonatal rat RPE cells. However, the message for FGFR-1(flg) receptor was detected only in the transformed RPE cells. CONCLUSIONS: This study demonstrated a neonatal rat RPE cell line that proliferated rapidly in vitro, expressed high levels of message for hFGF and FGFR-1(flg) receptor, and continued to express RPE-cell characteristics. Importantly, mRNA levels of confluent cultures of these cells were sufficient for bFGF mRNA blot analysis, which eliminates the necessity for PCR and for using excessive numbers of animals for such studies.

Animals↗
PubMed2001

Evaluation of RLBP1 in 50 autosomal recessive retinitis pigmentosa and 4 retinitis punctata albescens Spanish families.

Defects in retinal vitamin A metabolism or in genes expressed in the retinal pigment epithelium (RPE) are related to nonsyndromic retinitis pigmentosa (RP). The RLBP1 gene encodes the cellular retinaldehyde-binding protein which, in the RPE and Müller cells of the retina, is thought to play a role in retinoid metabolism and visual pigment regeneration. We describe a study of the involvement of the RLBP1 gene in 50 autosomal recessive retinitis pigmentosa (ARRP) and four retinitis punctata albescens Spanish families. Cosegregation and homozygosity studies using an intragenic polymorphism and three close markers (D15S116, D15S127, and D15S130) ruled out RLBP1 as the cause of ARRP in 26 pedigrees. In the remaining families, SSCP analysis of the coding region and sequencing of the abnormal migrating bands did not detect any disease-causing mutation. These results indicate that mutations in the RLBP1 gene are not responsible for the ARRP or retinitis punctata albescens in this set of Spanish families. We did, however, identify two frequent polymorphisms (3'UTR + 167 G > T, T: 0.23 and G: 0.77; IVS6 + 20 T > C, T: 0.36 and C: 0.64), a silent substitution (S218S), and a rare variant (5'UTR-101 G > A).

Carrier Proteins↗