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D Bok

Publications and source records attributed to D Bok.

At least 55 records · Page 3Linked to original sources
PubMed1995

Characterization and quantification of full-length and truncated Na,K-ATPase alpha 1 and beta 1 RNA transcripts expressed in human retinal pigment epithelium.

We have characterized cDNA clones encoding the alpha 1 and beta 1 subunits of Na,K-ATPase produced in the human retinal pigment epithelium (hRPE). In addition to isolating clones corresponding to known sequences of Na,K-ATPase subunits, we report hitherto unknown forms of Na,K-ATPase with unique deduced amino acid (aa) sequences in their C-termini. Truncated cDNA sequences were found for both the beta 1 and alpha 1 subunits. While the beta 1 sequence is truncated by two aa residues at the C terminus, in the alpha 1 sequence 342 aa have been replaced by a unique sequence containing only 44 aa. Interestingly, this new C-terminal polypeptide shows sequence similarities to the Ca(2+)-ATPase and contains consensus sequence elements for phosphorylation and cell adhesion, suggesting expression of Na,K-ATPase subunits with unique functions. Using reverse transcription-polymerase chain reaction, RNA sequences for alpha 1, beta 1 and their corresponding truncated isoforms were quantified. 4.0 x 10(5) alpha 1 and 2.3 x 10(5) beta 1 molecules were found per ng of mRNA from hRPE. Much lower levels were detected for truncated alpha 1 and beta 1 (3.6 x 10(3) and 2.7 x 10(3) molecules/ng, respectively). These data corroborate the expression of truncated transcripts coding for unique aa sequences in hRPE, and suggest that factors other than alpha 1 and beta 1 mRNA levels regulate the equimolar accumulation of alpha and beta subunits in the plasma membrane.

Amino Acid Sequence↗
PubMed1995

Expression, secretion, and age-related downregulation of pigment epithelium-derived factor, a serpin with neurotrophic activity.

Retinal pigment epithelial (RPE) cells form a functional complex with photoreceptor neurons of the retina, interacting through the interphotoreceptor matrix (IPM). We now provide evidence that the gene for pigment epithelium-derived factor (PEDF), a protein possessing neurotrophic and neuronal-survival activities, is highly expressed by both fetal and young adult RPE cells. PEDF mRNA is present in RPE cells of the human eye at 17 weeks of gestation, demonstrating its potential for action in vivo during early retinal development. The PEDF protein is secreted in vivo where it constitutes a part of the fetal and adult IPM surrounding photoreceptor outer segments. A polyclonal PEDF antibody recognizes at least four isoforms of secreted human and bovine PEDF by two dimensional gel analysis, and detects a similar 50 kDa protein in the IPM of several other vertebrate species. Within soluble extracts of RPE cells, however, where little, if any, of the 50 kDa species can be detected, an immunoreactive 36 kDa protein is observed by Western blot analysis. By immunofluorescence, PEDF is localized intracellularly in association with the nucleus, presumptive secretory granules, and cytoskeletal elements of cultured RPE cells with PEDF and actin antibodies colocalizing to the same cytoskeletal structures. During initial stages of attachment, PEDF and actin also concentrate at the tips of pseudopods extended by the cultured RPE cells. However, with successive passages, synthesis, and secretion of the PEDF protein as well as transcription of its mRNA decrease and are lost by about 10 passages. In parallel, cultured RPE cells lose their proliferative potential and change from an epithelial-like morphology in early passages to a more fibroblast-like appearance by about the 10th passage. PEDF is thus apparently present intracellularly and extracellularly in both fetal and early adult periods where it could be involved in cellular differentiation and survival and with its loss, in the onset of senescence.

Aging↗
PubMed1995

A human retinal pigment epithelial cell line that retains epithelial characteristics after prolonged culture.

PURPOSE: A spontaneously arising, apparently transformed, cell line has been cloned from a primary culture of human retinal pigment epithelial (RPE) cells and has been subcultured more than 200 times. The similarities of these cells to human RPE cells in vivo have been determined. METHODS: The structure of the transformed cells has been determined by light and electron microscopy and by immunocytochemistry using antibodies that detect cytoskeletal and other proteins. The ability of the cell line to bind and phagocytose photoreceptor material has also been assessed by fluorescence and electron microscopy. The metabolism of all-trans-retinol has been investigated by incubation of the cells with 3H-all-trans-retinol and analysis of the metabolic products by high-performance liquid chromatography. RESULTS: The transformed cells possess an epithelial cobblestone morphology with intercellular junctional complexes containing N-cadherin. The cytoskeleton of these cells comprises cytokeratins that are characteristic of epithelial cells, together with actin, spectrin, and vimentin. The keratins expressed are those typical of RPE cells. The cells also express cellular retinaldehyde binding protein and retinol dehydrogenase activity but do not express retinoid isomerase or lecithin retinol acyl transferase activities. These cells also exhibit phagocytic activity. CONCLUSIONS: This cell line retains many of the metabolic and morphologic characteristics of RPE cells in vivo although there are some differences, particularly the loss of some enzymatic activities and cytoskeletal polarization. These cells should be useful in further studies of RPE cell metabolism and other functions.

Cell Line, Transformed↗
PubMed1994

Synthesis and secretion of retinol-binding protein and transthyretin by cultured retinal pigment epithelium.

Recent studies indicate that the retinal pigment epithelium (RPE) may serve as an extrahepatic source of retinol-binding protein (RBP) and transthyretin (TTR) for the retina by virtue of the fact that this cell layer is the exclusive retinal location for mRNA coding for these proteins [Herbert, J., et al. (1991) Invest. Ophthalmol. Vis. Sci. 32, 302-309; Cavallaro, T., et al. (1990) Invest. Ophthalmol. Vis. Sci. 31, 497-501], although the proteins themselves are present in a variety of retinal neurons. It is therefore necessary to determine whether these mRNAs are translated and whether their translated products are secreted like hepatic RBP and TTR. Metabolic labeling of cultured bovine RPE with [35S]cysteine and [35S]methionine and subsequent analysis of newly synthesized proteins in the conditioned medium by affinity chromatography, gel filtration, partial amino acid sequence analysis, and autoradiography of electrophoretograms indicate that both RBP and TTR are synthesized and secreted by the RPE. Moreover, for cells grown in chambers with permeable supports, the predominant direction for secretion was into the apical medium. The mean apical:basal ratio after 72 h of incubation was 9.2 for TTR and 4.5 for RBP. A function for these proteins in the neurosensory retina remains speculative. They could be involved in the delivery of all-trans-retinol to amacrine and Müller cells as a precursor for retinoic acid, since these cells are known to contain cellular retinoic acid binding protein [Gaur, V.P., et al. (1990) Exp. Eye Res. 50, 505-511; Milam et al. (1990) J. Comp. Neurol. 296, 123-129].(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Lipoprotein (a) displays increased accumulation compared with low-density lipoprotein in the murine arterial wall.

Lipoprotein (a) (Lp(a)) is known to be an independent risk factor for cardiovascular disease, but the mechanisms by which it contributes to this disease remain unclear. Current evidence indicates that the closely related plasma particle, low-density lipoprotein (LDL), may initiate atherosclerosis through deposition in the arterial wall. This study has compared the ability of both lipoproteins to enter and accumulate within the arterial wall. Experiments were conducted in vivo with animals from two strains of mice: C57BL/6 mice, which develop fatty streak lesions upon challenge by a high-fat diet, and C3H/HeJ mice, which are resistant to lesion formation. Animals from both strains were maintained up to 16 weeks either on chow or high-fat diet. The mice were intravenously injected with 125I-labeled human Lp(a) or 125I-labeled human LDL in equimolar amounts and the lipoprotein allowed to circulate in vivo for 2 or 24 h. Transverse sections of the aortic root including sites of predilection for lesion formation at the commissures of the valve were prepared and examined after autoradiography. The autoradiographic grains over lesions and histologically uninvolved areas were enumerated and compared after normalization. Both Lp(a) and LDL demonstrated nearly ten times greater accumulation in lesions compared with histologically uninvolved areas from C57BL/6 mice. Analyses of histologically uninvolved areas from both strains of mice showed a significantly higher accumulation of Lp(a) than LDL. Finally, significantly higher accumulations of both Lp(a) and LDL occurred in the histologically uninvolved intima and subintima of lesion-prone C57BL/6 mice as compared with lesion-resistant C3H/HeJ mice after 5 weeks on the diets. We propose that enhanced accumulation of Lp(a) in the arterial wall accounts, in part, for the increased risk of cardiovascular disease.

Animals↗
PubMed1994

Localization of NaK ATPase on cultured human retinal pigment epithelium.

PURPOSE: To localize NaK ATPase sites on cultured human retinal pigment epithelium (RPE). METHODS: Cultured human RPE from fetal, 2-year-old, and 21-year-old donors was grown to confluence in microporous culture wells for 4 months to 2 years, mounted in a small-volume Ussing chamber, and perfused with growth medium. Ouabain (10(-5)-M) was applied to the basal and apical sides of the RPE. Changes in transepithelial resistance (Rt), transepithelial potential (TEP), and apical and basal membrane potentials were measured. RESULTS: Application of ouabain to the basal side of RPE produced a small sustained increase in TEP after 6 minutes and, simultaneously, small depolarizations of both apical and basal membranes. During the continued presence of ouabain on the basal side, application of ouabain to the apical side produced a significantly larger TEP decrease and greater depolarization of both membranes. Significant changes in Rt were not observed. CONCLUSIONS: These results indicate that NaK ATPase sites are present on both the apical and basolateral membranes of cultured human RPE. The greater effect of ouabain when applied to the apical side suggests that functional NaK ATPase sites are more abundant on the apical membrane.

Adult↗
PubMed1993

Phagocytosis of outer segments by retinal pigment epithelium: phagosome-lysosome interaction.

We studied phagocytosis of rod outer segments (ROS) by the retinal pigmented epithelium (RPE) using rapid freezing, freeze-drying, and electron microscopic immunocytochemistry. Phagocytosis of photoreceptor outer segment tips by the RPE occurs daily, and in rats the shedding of these tips is light-entrained to a circadian rhythm. We studied the phagocytic process 5, 30, 90, and 150 min after light onset or after subjective light onset in rats entrained to a 12-hr dark-12-hr light cycle. Lysosomes were labeled with antibodies to cathepsin D, a major lysosomal enzyme responsible for opsin degradation. Phagosomes and phagolysosomes were recognized because of the lamellar structure of their photoreceptor-derived contents. We found a population of lysosomes that fuse with one another before they interact with phagosomes. This fusion can be triggered either by light or by endogenous circadian mechanisms. We also found that lysosome-phagosome interaction occurs after the ingestion stage is completed and that this interaction occurs in two steps. First, smaller lysosomes fuse with phagosomes. Subsequently, larger lysosomes appear to interact with phagosomes via pore-like or bridge-like structures. It is proposed that interchange of contents takes place through these structures.

Animals↗
PubMed1993

The retinal pigment epithelium: a versatile partner in vision.

The retinal pigment epithelium (RPE) is a monolayer of cuboidal cells that lies in close association with the rod and cone photoreceptors. This epithelium has diverse features, three of which are discussed in some detail in this review, namely the daily phagocytosis of rod and cone outer segment fragments that are shed from their distal ends; the uptake, processing, transport and release of vitamin A (retinol) and some of its visual cycle intermediates (retinoids); and some of the aspects of its apical and basolateral membrane polarity that are the reverse of most other epithelia. Phagocytosis takes place at the apical surface via membrane receptor-mediated processes that are not yet well defined. Retinol uptake occurs at both the basolateral and apical surfaces by what appear to be separate receptor-mediated processes. The release of a crucial retinoid, 11-cis retinaldehyde (11-cis retinal), occurs solely across the apical membrane. Delivery of retinol across the basolateral membrane is mediated by a retinol binding protein (RBP) that is secreted by the liver as a complex with retinol (vitamin A). Within the cell, retinol and its derivatives are solubilized by intracellular retinoid binding proteins that are selective for retinol (cellular retinol binding protein, CRBP) and 11-cis retinoids (cellular retinal binding protein, CRALBP). Release of 11-cis retinal across the apical membrane and re-uptake of retinol from the photoreceptors during the visual cycle is promoted by an intercellular retinoid binding protein (IRBP). Na,K-ATPase, the membrane-integrated enzyme required to set up the ion gradients that drive other ion transporters, is largely localized to the apical membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Direct RT-PCR amplification of mRNA supported on membranes.

We describe a simple and efficient technique that facilitates the amplification of specific mRNA for cloning and sequencing purposes. An mRNA bound to a small piece of membrane filter is used as a template to synthesize complementary DNA. The product of this reaction is then transferred to a new tube and amplified using a standard PCR protocol. By simple enzymatic treatment, this RNA membrane can be reused as many times as needed with no problems of low yield, mispriming or background. Multiple advantages and different applications can be gained with this procedure. We have been using this technique to characterize a 4.5-kb mRNA from human retinal pigment epithelial cells following identification by Northern blot. According to the size of the PCR amplification products, this mRNA band contains portions of the coding sequence for the Na+K(+)-ATPase beta 1 subunit.

Base Sequence↗
PubMed1992

A third form of the G protein beta subunit. 1. Immunochemical identification and localization to cone photoreceptors.

Vertebrate retinal cones play a major role in both photopic vision and color perception. Although the molecular mechanism of visual excitation in the cone is not as well understood as in the rod, it is generally thought to involve a cone-specific G protein (cone transducin) that couples the cone visual pigment to a cGMP phosphodiesterase. Like all other G proteins, cone transducin is most likely a heterotrimer consisting of G alpha, G beta, and G gamma subunits. A G alpha subunit of cone transducin has been localized to the outer segment of bovine cones, but its associated G beta and G gamma subunits are unknown. To identify the G beta subunit involved in the phototransduction process of cones, we have developed a panel of antipeptide antisera against the most diverse region of the amino acid sequences encoded by G beta 1, G beta 2, and G beta 3 cDNAs and used them to determine the distribution of the G beta isoforms in different retinal preparations. We found that the G beta 3 subunit is present in bovine retinal transducin and phosducin-T beta gamma complex preparations which were previously thought to contain only G beta 1. Analysis of its subcellular distribution indicated that G beta 3 is predominantly cytoplasmic. Immunocytochemical staining of bovine retinal sections with the anti-G beta 3 antiserum further revealed a specific localization of G beta 3 in cones but not in rods. In contrast, anti-G beta 1 antiserum stained only the rods. These results suggest that G beta 3 is the G beta subunit of cone transducin and confirms the proposition that rods and cones utilize distinct signaling proteins for phototransduction.

Amino Acid Sequence↗
PubMed1992

Promotion of the release of 11-cis-retinal from cultured retinal pigment epithelium by interphotoreceptor retinoid-binding protein.

This study investigates whether the interphotoreceptor retinoid-binding protein (IRBP) is necessary for the release of 11-cis-retinaldehyde (RAL) or if the retinoid is constitutively released from the retinal pigment epithelium (RPE) following synthesis. The strategic location of IRBP in the interphotoreceptor matrix (IPM) and its retinoid-binding ability make it a candidate for a role in 11-cis-RAL release. Fetal bovine RPE cells were grown in permeable chambers, and their apical surfaces were incubated with medium containing either apo-IRBP, the apo form of cellular retinaldehyde-binding protein (CRALBP), the apo form of serum retinol-binding protein (RBP), or bovine serum albumin (BSA) or with medium devoid of binding proteins. [3H]-all-trans-Retinol (ROL) was delivered to the basal surface of the cells by RBP. High-performance liquid chromatography demonstrated that [3H]-11-cis-RAL was optimally released into the apical medium when apo-IRBP was present. The most surprising result was the diminished level of [3H]-11-cis-RAL when apo-CRALBP was in the apical medium. Circular dichroism demonstrated that CRALBP had not been denatured by the photobleaching required for endogenous ligand removal. Therefore, apo-CRALBP should have been able to bind [3H]-11-cis-RAL if it was constitutively released into the apical medium. In addition, when proteins other than apo-IRBP were present, or if the cells were incubated with medium alone, the observed decrease in apical [3H]-11-cis-RAL was concomitant with a buildup of intracellular [3H]-all-trans-retinyl palmitate and [3H]-all-trans-ROL in the basal culture medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Polarized budding of vesicular stomatitis and influenza virus from cultured human and bovine retinal pigment epithelium.

The retinal pigment epithelium (RPE) is able to perform a variety of functions because of its high degree of plasma membrane polarity. Some aspects of this polarity such as the localization of the majority of Na-K ATPase to the apical membrane distinguish the RPE from kidney cells and most other transporting epithelia. The polarized budding of enveloped viruses such as vesicular stomatitis and influenza from the basolateral and apical membrane, respectively, has been used to study mechanisms underlying the domain-specific sorting of membrane proteins in cultured epithelial cell lines. These processes also serve as a useful index of the degree of polarization in epithelial cell cultures. Viral budding from apical and basolateral RPE membranes was used in this study to determine whether the sorting of viral envelope membrane proteins by the RPE is reversed in polarity from that of kidney cells and, if so, whether this might predict a fundamental difference in membrane protein sorting for RPE. The results clearly indicate that the polarity of viral membrane sorting and subsequent viral budding is the same in RPE as in other polarized epithelial cell lines examined to date.

Animals↗
PubMed1992

Effect of phytanic acid on cultured retinal pigment epithelium: an in vitro model for Refsum's disease.

Refsum's disease (heredopathia atactica polyneuritiformis) is an autosomal recessive retinitis pigmentosa syndrome caused by the excessive deposition of phytanic acid in ocular tissues. It is thought that phytanic acid causes retinal degeneration either by interfering with vitamin A metabolism in the retinal pigment epithelium or by altering photoreceptor cell membrane structure. Efforts to elucidate the molecular mechanism of phytanic acid's retinal toxicity have been hampered by the rarity of human pathological specimens and by the inability to reproduce the disease in living animal models. In this study, an in vitro model for Refsum's disease was established by exposing cultured human and bovine retinal pigment epithelial cells to phytanic acid bound to bovine serum albumin at concentrations comparable to levels found in affected humans. Ultrastructural studies show that these cells exhibit morphological changes consistent with those observed in pathological specimens from patients with Refsum's disease. Biochemical assays of retinoid metabolism by cell membranes from control cells and from cells exposed to 200 microM phytanic acid demonstrate that the ability to esterify retinol and to isomerize all-trans retinoids to 11-cis retinoids remains intact despite the deposition of large amounts of phytanic acid. The work described here is strong evidence against the hypothesis that phytanic acid inhibits vitamin A metabolism in the retinal pigment epithelium, and it demonstrates the potential use of cultured retinal pigment epithelial cells in modeling this and other degenerative diseases of the retina.

Animals↗
PubMed1992

Complete rescue of photoreceptor dysplasia and degeneration in transgenic retinal degeneration slow (rds) mice.

retinal degeneration slow (rds) is a semidominant mutation of mice with the phenotype of abnormal development of rod and cone photoreceptors, followed by their slow degeneration. The rds gene has been putatively cloned and its novel protein product initially characterized biochemically. In the present study we undertook to correct in vivo the retinal phenotype of mice with the rds mutation. We assembled a transgene containing a regulatory segment of the opsin gene positioned upstream of the wild-type rds coding region. Mice from three transgenic lines, homozygous for the rds mutation, were analyzed for expression of the transgene and for their retinal phenotypes. In two high expressing lines, we observed complete reversion to wild-type retinal morphology. In a third, low expressing line, we observed a retinal phenotype intermediate between wild type and rds/rds, suggesting partial rescue of the mutation. These results constitute formal proof that we have cloned the rds gene.

Animals↗
PubMed1991

The retinal degeneration slow (rds) gene product is a photoreceptor disc membrane-associated glycoprotein.

Mice homozygous for the retinal degeneration slow (rds) mutation exhibit abnormal development of photoreceptor cells, followed by their slow degeneration. We have recently cloned the rds gene and determined the structure of the wild-type rds mRNA. Here we show that the gene is expressed exclusively in photoreceptor cells. We demonstrate that it encodes a 39 kd membrane-associated glycoprotein that is restricted to photoreceptor outer segments. By electron microscopy, we show that the rds protein is distributed uniformly within outer segment discs. The developmental appearance of the rds protein coincides with outer segment disc formation. We propose that the rds protein functions as an adhesion molecule for stabilization of the outer segment discs.

Amino Acid Sequence↗
PubMed1991

Detection of interphotoreceptor retinoid binding protein (IRBP) mRNA in human and cone-dominant squirrel retinas by in situ hybridization.

Interphotoreceptor retinoid binding protein (IRBP) is a soluble glycolipoprotein located between the neurosensory retina and pigment epithelium, which may serve to transport vitamin A derivatives between these tissues. The specific cell type responsible for IRBP synthesis has not been well established. To address this issue, we have examined the expression of IRBP mRNA in human and cone-dominant ground squirrel retinas by in situ hybridization. Optimal labeling and histological resolution were achieved with 35S- and 3H-labeled anti-sense riboprobes made from a human IRBP cDNA clone, and semi-thin wax-embedded retinal sections. In human retina, label was localized over the inner segments of both rod and cone photoreceptors. Quantitative analysis demonstrated a fourfold higher density of label over rod inner segments. In ground squirrel retina, labeling was found almost exclusively over the inner segments of cones. The results indicate that in human retina both rods and cones express IRBP mRNA, albeit at different levels. In cone-dominant species such as the ground squirrel, cones are the principal cell type responsible for IRBP mRNA synthesis.

Animals↗