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D Bok

Publications and source records attributed to D Bok.

At least 37 records · Page 2Linked to original sources
PubMed1999

Molecular and biochemical characterization of lecithin retinol acyltransferase.

The enzyme responsible for conversion of all-trans-retinol into retinyl esters, the lecithin retinol acyltransferase (LRAT) has been characterized at the molecular level. The cDNA coding for this protein was cloned and its amino acid sequence deduced. LRAT is composed of a polypeptide of 230 amino acid residues with a calculated mass of 25.3 kDa. Tissue distribution analysis by Northern blot showed expression of a 5.0-kilobase transcript in the human retinal pigment epithelium as well as in other tissues that are known for their high LRAT activity and vitamin A processing. Affinity labeling experiments using specific compounds with high affinity for LRAT and monospecific polyclonal antibodies raised in rabbits against two peptide sequences for LRAT confirmed the molecular mass of LRAT as a 25-kDa protein. High performance liquid chromatography analysis of the reaction product formed by HEK-293 cells transfected with LRAT cDNA confirmed the ability of the transfected cells to convert [3H]all-trans-retinol into authentic [3H]all-trans-retinyl palmitate as chemically determined.

Acyltransferases↗
PubMed1999

Transgenic analysis of rds/peripherin N-glycosylation: effect on dimerization, interaction with rom1, and rescue of the rds null phenotype.

Rds/peripherin is an integral membrane glycoprotein that is present in the rims of photoreceptor outer segment disks. In mammals, it is thought to stabilize the disk rim through heterophilic interactions with the related nonglycosylated protein roml. Glycosylation of rds/peripherin at asparagine 229 is widely conserved in vertebrates. In this study, we investigated the role of rds/peripherin N-glycosylation. We generated transgenic mice that expressed only S231A-substituted rds/peripherin in their retinas. This protein was not glycosylated but formed covalent dimers with itself and with glycosylated rds/peripherin. Nonglycosylated rds/peripherin also interacted noncovalently with rom1 homodimers to form a heterooligomeric complex. The glycosylated rds/peripherin..rom1 complex bound to concanavalin A-Sepharose, suggesting that the glycan is not directly involved in the interaction between these proteins. In double transgenic mice expressing normal and S231A-substituted rds/peripherin, the mRNA-to-protein ratios were similar for both transgenes, indicating no effect of N-glycosylation on rds/peripherin stability. Finally, expression of nonglycosylated rds/peripherin in transgenic mice rescued the phenotype of outer segment nondevelopment in retinal degeneration slow (rds-/-) null mutants. These observations indicate that N-glycosylation of rds/peripherin is not required for its normal processing, stability, or in vivo function.

Animals↗
PubMed1999

Polarity of 11-cis retinal release from cultured retinal pigment epithelium.

PURPOSE: Fetal bovine retinal pigment epithelium (RPE) was grown on porous supports to investigate the polarity of 11-cis retinal (RAL) release from these cells and the influence that the interphotoreceptor retinoid-binding protein (IRBP) has on this process. METHODS: [3H]all-trans retinol (ROL) was delivered to the basal surface of the cultured RPE by serum retinol-binding protein (RBP). Apo IRBP was added to either the apical or basal medium, or was absent from the incubation entirely. RESULTS: The greatest level of [3H]11-cis RAL was detected in the apical medium but only when apo IRBP was present there. When apo IRBP was present only in the basal medium, or was absent from the incubation entirely, low levels of [3H]11-cis RAL were released apically and basally. CONCLUSIONS: If 11-cis RAL release were constitutive, one would expect to find elevated levels of this retinoid in the apical and basal media in the absence of apo IRBP. Instead, the enhancement of [3H]11-cis RAL release into the apical, but not the basal, medium in the presence of apo IRBP suggests that [3H]11-cis RAL release is polarized and dependent on the presence of apo IRBP. It is postulated, therefore, that a mechanism such as an IRBP membrane receptor in the apical plasma membrane may be responsible for this polarity.

Animals↗
PubMed1998

Non-cell-autonomous photoreceptor degeneration in rds mutant mice mosaic for expression of a rescue transgene.

The inherited retinal dystrophies represent a large and heterogenous group of hereditary neurodegenerations, for many of which, the molecular defect has been defined. However, the mechanism of cell death has not been determined for any form of retinal degeneration. The retinal degeneration slow (rds-/-) mutation of mice is associated with nondevelopment of photoreceptor outer segments and gradual death of photoreceptor cell bodies, attributed to the absence of the outer segment protein rds/peripherin. Here, we examined the effects of a transgene encoding normal rds/peripherin that had integrated into the X-chromosome in male and female rds-/- mutant retinas. In 2-month-old transgenic males and homozygous-transgenic females on rds-/-, we observed virtually complete rescue of both the outer segment nondevelopment and photoreceptor degeneration. In contrast, hemizygous-transgenic rds-/- female littermates showed patchy distributions of the transgene mRNA, by in situ hybridization analysis, and of photoreceptor cells that contain outer segments. This pattern is consistent with random inactivation of the X-chromosome and mosaic expression of the transgene. Surprisingly, we observed significant photoreceptor cell loss in both transgene-expressing and nonexpressing patches in hemizygous female retinas. These observations were supported by nuclease protection analysis, which showed notably lower than predicted levels of transgene mRNA in retinas from hemizygous females compared with male and homozygous female littermates. This phenotype suggests an important component of non-cell-autonomous photoreceptor death in rds-/- mutant mice. These results have significance to both the etiology and potential treatment of human inherited retinal degenerations.

Animals↗
PubMed1998

Bovine epidermal fatty acid-binding protein: determination of ligand specificity and cellular localization in retina and testis.

The fatty acid-binding protein (FABP) family consists of small, cytosolic proteins believed to be involved in the uptake, transport, and solubilization of their hydrophobic ligands. Members of this family have highly conserved sequences and tertiary structures. Using an antibody against testis lipid-binding protein, a member of the FABP family, a protein was identified from bovine retina and testis that coeluted with exogenously added docosahexaenoic acid during purification. Amino acid sequencing and subsequent isolation of its cDNA revealed it to be nearly identical to a bovine protein expressed in the differentiating lens and to be the likely bovine homologue of the human epidermal fatty acid-binding protein (E-FABP). From quantitative Western blot analysis, it was estimated that bovine E-FABP comprised 0.9%, 0.1%, and 2.4% of retina, testis, and lens cytosolic proteins, respectively. Binding studies using the fluorescent probe ADIFAB indicated that this protein bound fatty acids of differing levels of saturation with relatively high affinities. Kd values ranged from 27 to 97 nM. In addition, the protein was immunolocalized to the Müller cells in the retina as well as to Sertoli cells in the testis. The location of bovine E-FABP in cells known to be supportive to other cell types in their tissues and the ability of E-FABP to bind a variety of fatty acids with similar affinities indicate that it may be involved in the uptake and transport of fatty acids essential for the nourishment of the surrounding cell types.

Amino Acid Sequence↗
PubMed1998

Structural and functional characterization of recombinant human cellular retinaldehyde-binding protein.

Cellular retinaldehyde-binding protein (CRALBP) is abundant in the retinal pigment epithelium (RPE) and Müller cells of the retina where it is thought to function in retinoid metabolism and visual pigment regeneration. The protein carries 11-cis-retinal and/or 11-cis-retinol as endogenous ligands in the RPE and retina and mutations in human CRALBP that destroy retinoid binding functionality have been linked to autosomal recessive retinitis pigmentosa. CRALBP is also present in brain without endogenous retinoids, suggesting other ligands and physiological roles exist for the protein. Human recombinant cellular retinaldehyde-binding protein (rCRALBP) has been over expressed as non-fusion and fusion proteins in Escherichia coli from pET3a and pET19b vectors, respectively. The recombinant proteins typically constitute 15-20% of the soluble bacterial lysate protein and after purification, yield about 3-8 mg per liter of bacterial culture. Liquid chromatography electrospray mass spectrometry, amino acid analysis, and Edman degradation were used to demonstrate that rCRALBP exhibits the correct primary structure and mass. Circular dichroism, retinoid HPLC, UV-visible absorption spectroscopy, and solution state 19F-NMR were used to characterize the secondary structure and retinoid binding properties of rCRALBP. Human rCRALBP appears virtually identical to bovine retinal CRALBP in terms of secondary structure, thermal stability, and stereoselective retinoid-binding properties. Ligand-dependent conformational changes appear to influence a newly detected difference in the bathochromic shift exhibited by bovine and human CRALBP when complexed with 9-cis-retinal. These recombinant preparations provide valid models for human CRALBP structure-function studies.

Amino Acid Sequence↗
PubMed1998

Rpe65 is necessary for production of 11-cis-vitamin A in the retinal visual cycle.

Mutation of RPE65 can cause severe blindness from birth or early childhood, and RPE65 protein is associated with retinal pigment epithelium (RPE) vitamin A metabolism. Here, we show that Rpe65-deficient mice exhibit changes in retinal physiology and biochemistry. Outer segment discs of rod photoreceptors in Rpe65-/- mice are disorganized compared with those of Rpe65+/+ and Rpe65+/- mice. Rod function, as measured by electroretinography, is abolished in Rpe65-/- mice, although cone function remains. Rpe65-/- mice lack rhodopsin, but not opsin apoprotein. Furthermore, all-trans-retinyl esters over-accumulate in the RPE of Rpe65-/- mice, whereas 11-cis-retinyl esters are absent. Disruption of the RPE-based metabolism of all-trans-retinyl esters to 11-cis-retinal thus appears to underlie the Rpe65-/- phenotype, although cone pigment regeneration may be dependent on a separate pathway.

Animals↗
PubMed1998

Transplantation of retinal pigment epithelial cells and immune response in the subretinal space.

PURPOSE: To evaluate the fate of retinal pigment epithelial cell (RPE) allografts in the subretinal space. METHODS: Transplantation was performed in Royal College of Surgeons (RCS) rats. Two rat strains, BD IX and LEJ, which have incompatible major histocompatibility complex (MHC) haplotypes, were selected as healthy RPE donors. Transplantation was performed when recipients were 19 to 21 days old. Host systemic immunity was enhanced by challenge with donor spleen cells 2 weeks after RPE transplantation. Control subjects were administered injections of saline or host spleen cells. The animals were killed at 3 or 5 months of age. The extent of photoreceptor rescue was determined by counting the maximum layers of surviving photoreceptor nuclei in histologic sections. Reverse transcription-polymerase chain reaction analysis of the grafts was performed with host- and donor-specific primers. RESULTS: Despite the absence of acute immune rejection, chronic rejection occurred and was evidenced by an increased loss of photoreceptor cells in immunologically challenged RCS rats. Grafts with disparity at MHC class I and class II lost their ability to rescue photoreceptor cells more readily than did grafts with disparity at MHC class II alone. Furthermore, the donor RPE cells that were normally MHC class II-negative expressed MHC class II mRNA in the subretinal space after transplantation. CONCLUSIONS: Systemic immunity appeared to exert a slow but significant influence in the subretinal space. Therefore, in planning future trials involving human subjects, the immunologically privileged status of the subretinal space should be regarded as imperfect.

Animals↗
PubMed1997

Generation and analysis of transgenic mice expressing P216L-substituted rds/peripherin in rod photoreceptors.

PURPOSE: In this study, the authors present the biochemical, morphologic, and physiological analyses of a transgenic mouse model for retinal degeneration slow (RDS)-mediated retinitis pigmentosa caused by a proline 216 to leucine (P216L) amino acid substitution in rds/peripherin. METHODS: The authors assembled a mutant rds transgene that encodes rds/peripherin with a P216L substitution. Transgenic mice were generated on wild-type (+/+), heterozygous (rds-/+), and homozygous (rds-/rds-) null genetic backgrounds. These mice were analyzed biochemically, by light and electron microscopy, and by electroretinography. RESULTS: In P216L-transgenic mice on a +/- background, the authors observed expression-level-dependent photoreceptor degeneration and outer-segment shortening. Expression of the P216L transgene on an rds-/+ background resulted in more severe photoreceptor degeneration and outer-segment dysplasia than seen in nontransgenic rds-/+ mutants. Severely dysplastic outer segments were detectable in P216L transgenics on an rds-/rds-null background. The reduction in b-wave amplitudes by electroretinography were well correlated with the degree of photoreceptor degeneration, but not outer-segment dysplasia in these different rds mutants. CONCLUSIONS: The phenotype in P216L-transgenic mice on an rds-/+ genetic background probably is caused by a combination of two genetic mechanisms: a direct dominant effect of the P216L substituted protein, and a reduction in the level of normal rds/peripherin. The expression pattern of the normal and mutant genes in these animals is similar to that predicted for humans with RDS-mediated autosomal-dominant retinitis pigmentosa. These mice may thus be considered an animal model for this disease.

Animals↗
PubMed1996

Expression and synthesis of the Na,K-ATPase beta 2 subunit in human retinal pigment epithelium.

Na,K-ATPase in the retinal pigment epithelium (RPE) is apically localized, whereas in most other tissues this pump is found predominantly in the basolateral membrane domain. As part of our investigations into the molecular aspects of this pump in the RPE, we have cloned the cDNA and characterized the expression of the gene encoding the beta 2 subunit isoform of Na,K-ATPase in human, rat and bovine RPE and in the bovine choroid plexus. We have also detected the beta 2 isoform polypeptide in the human RPE (hRPE). Comparison of complete coding sequences derived from cloned cDNAs revealed that all beta 2 sequences from RPE, and the choroid plexus, differed uniformly at positions: P51/L, M121/I, and L148/R from the published sequences for human retina and liver. However, analysis of 10 RT-PCR clones derived from 5 fetal and 2 adult human retinas sequenced in our laboratory, revealed that only the P51/L residue was different with the hRPE beta 2 subunit sequence. Northern blot analysis indicated a 3.4-kb RNA transcript for the beta 2 subunit, a 4.5-kb RNA for the alpha 1 subunit and a doublet of 2.3 and 2.6 kb for the beta 1 subunit, respectively. alpha 1 (100 kDa), beta 1 (45 kDa) and beta 2 (65 kDa) isoforms were detected in hRPE extracts by immunoblotting. No alpha 2 and alpha 3 RNA transcripts were found in the hRPE. Quantification of beta 2 mRNA by RT-PCR revealed 2.7 x 10(5) molecules per ng of poly A+ RNA. This is similar to the beta 1 isoform levels reported previously from our laboratory. These data demonstrate the coexistence of significant amounts of alpha 1, beta 1 and beta 2 Na,K-ATPase subunits in the RPE. It is therefore reasonable to suggest that both alpha 1 beta 1 and alpha 1 beta 2 heterodimers are present in these cells.

Adult↗
PubMed1996

TIMP-3 is expressed in the human retinal pigment epithelium.

TIMP-3 is the most recent member of the tissue inhibitor of metalloproteinases (TIMP) family. In the present study, we describe the expression of TIMP-3 messenger RNA (mRNA) by the retinal pigment epithelium of the normal human eye (hRPE). In addition to the three predominant transcripts of approximately 5.1, 2.8, and 2.4 Kbp found in several other human tissues at adult and fetal stages. The hRPE also expresses two RNA species of 1.2 and 1.0 Kbp. Based on the sequence analysis of cDNA clones isolated from a hRPE cDNA library, the use of alternate polyadenylation signals could account for the expression of these smaller transcripts. The possibility of an alternative mechanism of regulation of the expression of TIMP-3 by the RPE is not discarded. The number of RNA transcripts specific for TIMP-3 per nanogram of poly A+ RNA was quantified by RT-PCR. 9.6 x 10(5) transcripts per nanogram of polyA +RNA were found at the adult stage and 1.2 x 10(6) transcripts per nanogram of polyA +RNA were detected at the fetal stage. These findings were supported by the predominant labeling in the RPE layer of retinal tissue sections in in situ hybridization experiments. All of these data support the hypothesis that the production of TIMP-3 by the RPE may be crucial for the maintenance of Bruch's membrane, the complex layer of extracellular matrix that provides a structural substrate for the RPE in the healthy retina and is perturbed during the ageing process and in Sorby's Fundus Dystrophy a inherited disease.

Adult↗
PubMed1996

Characterization of the 3' UTR sequence encoded by the AQP-1 gene in human retinal pigment epithelium.

The complete 3' UTR sequence encoded by the human aquaporin-1 gene is reported. The sequence encompassed by two cDNA clones showed, 33 nucleotides of 5' UTR sequence, a coding sequence of 807 nucleotides and 1886 nucleotides corresponding to the complete 3' UTR sequence. High similarity with 3' UTR sequences from rat and mouse counterparts was found. Northern blot analysis of several human tissues revealed a 2.8 kbp transcript. These data confirm the existence of water channels in the human retinal pigment epithelium.

Animals↗
PubMed1996

Chloride transport in cultured fetal human retinal pigment epithelium.

This study identifies bumetanide-sensitive chloride transport in cultured fetal human retinal pigment epithelium (RPE). Ion flux and electrophysiologic studies were performed on fetal human RPE grown to confluence on microporous culture wells mounted in modified Ussing chambers. Unidirectional transepithelial Cl- fluxes were measured along with the transepithelial potential (TEP), resistance (Rt), and short circuit current (Isc). In separate experiments the apical and basal membrane voltages (Vap and Vba) and membrane resistance ratio (Rap/Rba) were measured using standard electrophysiological techniques. The average electrical parameters under control conditions were TEP = 3.6 +/- 2.1 mV, Rt = 385 +/- 98 ohm cm2, and Isc = 8.8 +/- 3.6 microA cm-2. Apical bumetanide (10(-5) M) reduced the TEP and Isc an average of 0.6 +/- 0.4 mV and 1.6 +/- 0.8 microA cm-2, respectively. Under open-circuit conditions, we measured a net apical to basal 36Cl flux of 0.30 microEq cm-2 hr-1. Bumetanide reduced this net Cl- flux by 83% by reducing the unidirectional apical-to-basal Cl- flux. Apical bumetanide hyperpolarized Vba from -58 +/- 14 to -66 +/- 17 mV and reduced Rap/Rba from 1.17 +/- 0.50 to 0.85 +/- 0.38, consistent with blockade of Cl- uptake and reduction of the Cl- equilibrium potential at a basal membrane Cl- channel. Basal bumetanide had no effect on electrical parameters. We identified a bumetanide sensitive Cl- uptake mechanism at the apical membrane which may represent the Na-K-2Cl cotransporter reported in explant and other cultured preparations of mammalian RPE.

Biological Transport, Active↗
PubMed1996

Three homologs of rds/peripherin in Xenopus laevis photoreceptors that exhibit covalent and non-covalent interactions.

We have isolated and characterized three homologs of mammalian rds/peripherin from Xenopus retinae. One (xrds38) is likely the Xenopus ortholog, while the other two (xrds36 and -35) are more distant relatives. By immunocytochemical analysis of retinal sections, xrds38 is distributed in both rod and cone photoreceptors, while xrds36 and xrds35 are present in rods only. At the EM level, xrds38 is present specifically in the rims and incisures of rod and cone outer segment discs. All are N-glycosylated and form covalent dimers. Immunoprecipitation analysis showed that in rods, these three proteins interact to form heterotetrameric or higher-order complexes. The pattern of sequence conservation among the xrds proteins, mammalian rds/peripherin, and mammalian rom-1 suggest that the central portion of the intradiscal D2 loop contains the interacting structural elements.

Amino Acid Sequence↗
PubMed1995

Arginine-427 in the Na+/glucose cotransporter (SGLT1) is involved in trafficking to the plasma membrane.

To investigate the role of charged intramembrane residues in the function of the rabbit Na+/glucose cotransporter (rbSGLT1) we substituted arginine-427 (R427) by alanine in the putative domain M9 SGLT1. This residue is conserved in all the members of the SGLT1 family. The mutant protein (R427A) was expressed in Xenopus oocytes and, although Western blot analysis revealed that it was produced in amounts comparable to wild-type, no function was measured. Freeze-fracture analysis showed that R427A SGLT1 was not in the plasma membrane while immunocytochemical experiments localized the transporter to just beneath it. These results indicate that arginine-427 plays a critical role in SGLT1 trafficking to the plasma membrane.

Animals↗
PubMed1995

Molecular cloning and characterization of the G protein gamma subunit of cone photoreceptors.

The phototransduction process in cones has been proposed to involve a G protein that couples the signal from light-activated visual pigment to the effector cyclic GMP phosphodiesterase. Previously, we have identified and purified a G beta gamma complex composed of a G beta 3 isoform and an immunochemically distinct G gamma subunit (G gamma 8) from bovine retinal cones (Fung, B. K.-K., Lieberman, B. S., and Lee, R. H. (1992) J. Biol. Chem. 267, 24782-24788; Lee, R. H., Lieberman, B.S., Yamane, H. K., Bok, D., and Fung, B. K.-K. (1992a) J. Biol. Chem. 267, 24776-24781). Based on the partial amino acid sequence of this cone G gamma 8, we screened a bovine retinal cDNA library and isolated a cDNA clone encoding G gamma 8. The cDNA insert of this clone includes an open reading frame of 207 bases encoding a 69-amino acid protein. The predicted protein sequence of G gamma 8 shares a high degree of sequence identity (68%) with the G gamma (G gamma 1) subunit of rod transducin. Similar to rod G gamma 1, it terminates in a CIIS motif that is the site for post-translational modification by farnesylation. Messenger RNA for G gamma 8 is present at a high level in the retina and at a very low level in the lung, but is undetectable in other tissues. Immunostaining of bovine retinal sections with an antipeptide antibody against the N-terminal region of G gamma 8 further shows a differential localization of G gamma 8 to cones with a pattern indistinguishable from that of G beta 3. This finding suggests that G beta 3 gamma 8 is a component of cone transducin involved in cone phototransduction and color vision.

Amino Acid Sequence↗