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D Bok

Publications and source records attributed to D Bok.

At least 73 records · Page 4Linked to original sources
PubMed1990

Uptake, processing and release of retinoids by cultured human retinal pigment epithelium.

Upon absorption of a photon, the 11-cis retinaldehyde chromophore of rhodopsin is isomerized and reduced to all-trans retinol (vitamin A) in the photoreceptor outer segments, whereupon it leaves the photoreceptors, and moves to the retinal pigment epithelium (RPE). To clarify the function of the RPE in the regeneration of 11-cis retinaldehyde, we delivered all-trans retinol to monolayer cultures of human RPE. During delivery the retinol was associated with its putative natural carrier, interphotoreceptor retinoid binding protein (IRBP). IRBP has been proposed as a carrier protein involved in the exchange of retinoids between the photoreceptors and the retinal pigment epithelium. The retinoid composition of RPE cells and culture medium was analyzed by HPLC following several incubation periods. The RPE monolayer was found to process all-trans retinol into two distinct end-products: all-trans retinyl palmitate, which remained within the RPE monolayer: and 11-cis retinaldehyde which was released into the culture medium. These results demonstrate retinoid isomerase, retinol oxidoreductase and retinyl ester synthetase activity in human RPE cells cultured under the appropriate conditions. They show that IRBP can serve as a carrier of retinol through an aqueous medium to the RPE, and they illustrate that the visual cycle can be studied in vitro.

Cells, Cultured↗
PubMed1990

Processing and transport of retinoids by the retinal pigment epithelium.

Recent developments regarding our understanding of retinoid processing and transport during the visual cycle and related events are reviewed. Retinoids are bound and protected by a cohort of retinoid binding proteins, each of which is unique. The production of retinol (Vitamin A) derivatives is accomplished by a group of membrane-bound enzymes, some of which appear to be coupled in their actions.

Animals↗
PubMed1990

Localization of cellular retinol-binding protein and retinol-binding protein in cells comprising the blood-brain barrier of rat and human.

Brain is not generally recognized as an organ that requires vitamin A, perhaps because no obvious histologic lesions have been observed in severely vitamin A-deficient animals. However, brain tissue does contain cellular vitamin A-binding proteins and a nuclear receptor protein for retinoic acid. In the present study, immunohistochemical techniques were used to determine the cell-specific location of cellular retinol-binding protein in human and rat brain tissue. Cellular retinol-binding protein was localized specifically within the endothelial cells of the brain microvasculature and within the cuboidal epithelial cells of the choroid plexus, two primary sites of the mammalian blood-brain barrier. In addition, autoradiographic procedures demonstrated binding sites for serum retinol-binding protein in the choroidal epithelium. These observations suggest that a significant movement of retinol across the blood-brain barrier may occur.

Animals↗
PubMed1990

Properties of channels reconstituted from the major intrinsic protein of lens fiber membranes.

Detergent-solubilized plasma membrane protein of either adult bovine or calf lens and high-performance liquid chromatography-purified major intrinsic protein (MIP) of the lens were reconstituted into unilamellar vesicles and planar lipid bilayers. Freeze-fracture studies showed that the density of intramembrane particles in the vesicles was proportional to the protein/lipid ratio. At high ratios, these particles crystallized into tetragonal arrays as does MIP in lens fibers. Channels induced by either purified MIP or detergent-solubilized protein had essentially identical properties. The conductance of multichannel membranes was maximal near 0 mV and decreased to 0.49 +/- 0.08 of the maximum value at voltages greater than 80 mV. The dependence of the conductance on voltage was well fit by a two-state Boltzmann distribution. Voltage steps greater than 30 mV elicited an ohmic current step followed by a slow (seconds) biexponential decrease. The amplitudes and time constants depended on the magnitude but not the sign of the voltage. Steps from 100 mV to voltages less than 30 mV caused the channels to open exponentially with a millisecond time constant. Analysis of latency to first closure after a voltage step gave nearly the same time constants as multichannel kinetics. Single-channel conductance is proportional to salt concentration from 0.1 to 1.0 M in KCl. In 0.1M KCl, the channel had two preferred conductance states with amplitudes of 380 and 160 pS, as well as three additional substates. Multi- and single-channel data suggest that the channel has two kinetically important open states. The channel is slightly anion selective. The properties of the channel do not vary appreciably from pH 7.4 to 5.8 or from pCa 7 to 2. We propose that a channel with these properties could contribute to maintenance of lens transparency and fluid balance.

Animals↗
PubMed1990

Isolation and provisional identification of plasma membrane populations from cultured human retinal pigment epithelium.

We have attempted to isolate samples of apical and basal-lateral plasma membranes from cultured fetal human RPE. Cells from confluent, dome-forming cultures were disrupted with a Dounce apparatus. Nuclei and melanin granules were sedimented by centrifugation at 2600 g for 10 min. The supernates were layered over gradients of 17.5-65% sorbitol and centrifuged at 122,000 g for 5 hr. Fractions were grouped into "density windows" on the basis of their biochemical marker contents. Na,K-ATPase and alkaline phosphatase overlapped but did not precisely parallel one another, suggesting associations with two partially separated membrane populations; in density window I, alkaline phosphatase was enriched 4.3-fold, and Na,K-ATPase was enriched 1.7-fold, whereas in window II the corresponding enrichment factors were 7.7 and 6.7. These markers were well resolved from a mitochondrial marker, but they were overlapped by endoplasmic reticulum and Golgi markers. Additional density gradient centrifugations, performed after samples had been suspended in 55% sorbitol, further separated alkaline phosphatase- and Na,K-ATPase-containing membranes from endoplasmic reticulum and Golgi membranes, yielding alkaline phosphatase and Na,K-ATPase cumulative enrichment factors of 6.8 and 2.5 for the sample from window I and 9.3 and 10.9 for the sample from window II. Subsequent phase partitioning analysis of the sample from window I further enriched an alkaline-phosphatase-rich membrane population, which is believed to represent the RPE basal-lateral membranes. The sample from density window II contained two membrane populations, both enriched in Na,K-ATPase, alkaline phosphatase, and galactosyltransferase, and both of which appear to be derived from the apical plasma membrane. SDS-PAGE and Western blotting confirmed a correlation between Na,K-ATPase catalytic activity and Na,K-ATPase alpha subunit immunoreactivity.

Alkaline Phosphatase↗
PubMed1990

Beta adrenergic receptors on cultured human retinal pigment epithelium.

Cultured fetal human retinal pigment epithelium (RPE) was grown on a permeable substrate and sealed in an Ussing chamber. The average electrical resistance (R) was 330 ohm-cm2, the average transepithelial voltage (Ve) was 3.0 mV (apical side positive), and the average short circuit current (Isc) was 9.1 microA/cm2. When these RPE preparations were exposed to isoproterenol (a beta-adrenergic agonist), the Isc increased by 88%, R was reduced by 6%, and Ve increased by 85%. The effect of isoproterenol was blocked by propranolol (a beta-adrenergic antagonist). When cultured human RPE was exposed to isoproterenol, intracellular cyclic adenosine monophosphate (AMP) levels rose more than threefold. The effect of isoproterenol on cyclic AMP levels was blocked by propranolol. When the cultured RPE was exposed to dibutyryl cyclic AMP, both Ve and Isc rose by 47% with a time course similar to that which occurred when the cells were exposed to isoproterenol. Preparations treated with dibutyryl cyclic AMP did not respond to subsequently applied isoproterenol. These results indicate that cultured human RPE possesses a beta-adrenergic receptor and that stimulation of this receptor produces a change in cyclic AMP concentration which affects RPE electrical activity.

Bucladesine↗
PubMed1990

A histopathologic study of a choroideremia carrier.

We have examined eyes from a heterozygote (carrier) of choroideremia, an X-linked disease. Gross examination revealed irregular pigmentation at the level of the retinal pigment epithelium (RPE) except at the posterior pole, and islands of well defined depigmentation of 1-4 mm in diameter in the midperiphery. The optic nerve and retinal blood vessels appeared normal, and there was minimal pigment migration into the retina. Histopathologic examination showed normal photoreceptors in the posterior and anterior fundus, but the outer segments were short or absent in much of the equatorial region. Little gliosis was noted in areas of retinal atrophy. The RPE was abnormal, with irregular thickness and pigmentation associated with variable lipofuscin content from one RPE cell to another, as shown by fluorescence microscopy. There were areas of profound atrophy in the equatorial region, with abrupt transitions between relatively normal RPE and photoreceptors, and retina devoid of RPE and photoreceptors. Bruch's membrane was thickened to a greater extent than is common in age-related change. The choriocapillaris was normal in areas with normal photoreceptors, except for widening of the intercapillary pillars. In those regions with abnormal photoreceptors, choroidal capillaries were fewer in number, had reduced luminal diameter, and fenestrae were sparse. In some areas of intense atrophy, there were no choroidal capillaries. The findings are compatible with the primary defect residing in the RPE. The Lyon hypothesis of X-chromosome inactivation and mosaicism could explain the irregularity of change and areas of intense atrophy, but abrupt demarcation between grossly abnormal, and relatively well preserved retina also occurs in hemizygotes (affected males).

Aged↗
PubMed1989

Degenerative changes in a retina affected with autosomal dominant retinitis pigmentosa.

The eyes of a 17-year-old male donor who was affected with autosomal dominant retinitis pigmentosa with variable expressivity have recently become available for study. Initial macroscopic examination of the fundus revealed bone spicules located in 180 degrees of the postequatorial fundus centered on the inferonasal quadrant. Light microscopic examination of the retina showed degeneration within each quadrant characterized by an absence of rods and cones in the equatorial areas, and the presence of photoreceptors in the more peripheral and central retina. Ultrastructural examination disclosed photoreceptors that were abnormal in all regions when compared to a control eye from a 26-year-old donor. Intact rods were restricted to the peripheral quadrants, and intact cones were identified in the fovea and far periphery. In areas of intermediate degeneration, many outer segments were either shortened and disorganized or absent. Regions of severe degeneration were characterized by the complete loss of the photoreceptors and apposition of the external limiting membrane to the retinal pigment epithelium. The density of rods and cones was found to be substantially lower than normal in all regions. In areas of relatively intact photoreceptor outer segments, we found ultrastructural evidence of recent phagocytic activity, and fluorescence microscopy revealed no unusual accumulation of lipofuscin within the pigment epithelium or subepithelial debris. The choroid and inner retina were normal throughout the eye. The normal condition of the choroid, retinal pigment epithelium, and inner retina implies that the primary disorder resides within the photoreceptor cell.

Adolescent↗
PubMed1988

Localization of retinal calmodulin kinase.

The localization of calmodulin kinase II (CaM kinase) was studied in the retina by light and electron microscopic immunocytochemistry, and by enzymatic and immunoblot assay of cellular and subcellular tissue fractions. By light microscopy, both mono- and polyclonal antibodies revealed CaM kinase-like immunoreactivity in the inner and outer plexiform regions (synaptic layers), retinal pigment epithelium (RPE), and ganglion cells. The inner nuclear layer and photoreceptor outer segments stained much less intensely, and the outer nuclear layer did not stain. Electron microscopy confirmed the high concentration of immunoreactive protein in RPE and minimal outer segment staining. In addition, photoreceptor inner segments also contained CaM kinase-like immunoreactivity. Calcium and calmodulin stimulated phosphate incorporation into proteins of retinal cytosol and of isolated and cultured RPE. Calcium- and calmodulin-dependent kinase activity was present to a lesser degree in crude nuclei and synaptic membranes and was absent in isolated rod outer segments. Immunoblot analyses were consistent with enzymatic assays and immunocytochemistry. These data suggest that retinal CaM kinase is ideally located to play an important role in synaptic transmission and modulation of visual processes. Furthermore, its presence in RPE implies that CaM kinase may have a more ubiquitous role in regulating cellular processes than was previously recognized.

Animals↗
PubMed1988

Lens-specific expression of recombinant ricin induces developmental defects in the eyes of transgenic mice.

An expression system for cell lineage ablation in transgenic mice was constructed in which a modified form of the A subunit of ricin, a toxic lectin produced by the castor bean Ricinus communis, can be expressed under the direction of tissue-specific regulatory signals. A chimeric gene was formed by fusing the promoter and 5'-flanking sequences of the lens-specific mouse alpha A-crystallin gene with a modified ricin A cDNA, and this construction was integrated into the germ line of transgenic mice. These animals develop profound microphthalmia with severe developmental defects of the eye, relating primarily to the disorganization and death of cells forming the lens. In addition, this defect is associated with several abnormalities, including eye size, folding of the retina, and ectopic lens material in other regions of the eye. The phenotype of this engineered developmental mutation suggests that the normal development of alpha A-crystallin-producing lens fiber cells is essential for the proper growth, organization, and orientation of optic structures.

Animals↗
PubMed1988

[3H]ouabain localization of Na-K ATPase in the epithelium of rabbit ciliary body pars plicata.

The secretion of the aqueous humor has been proposed to occur as the result of active Na+ transport by a ouabain-sensitive Na-K ATPase. We have examined the localization of this enzyme in the epithelium of rabbit ciliary body pars plicata using [3H]ouabain autoradiography. Single ciliary processes were isolated and incubated in Ringer containing [3H]ouabain. Processes were then rapidly frozen, freezedried, sectioned and exposed for autoradiography. In the light microscope, silver grains were found predominantly over the nonpigmented epithelial cells. In the electron microscope, grains could be localized for the most part to the interdigitations of the nonpigmented cell basolateral membrane. Label could also be observed at a much lower density above other membranes and above the pigmented and nonpigmented cell cytoplasm. No label was found in sections of control tissue which had been incubated in [3H]ouabain with an excess of cold ouabain. To show that the [3H]ouabain had free access to all of the membrane surfaces within the epithelium, in parallel experiments we incubated isolated processes in horseradish peroxidase. Our experiments suggest that most of the active Na+ transport in ciliary body epithelium occurs across the basolateral membrane of nonpigmented cells into the posterior chamber. Furthermore, the placement of the Na-K ATPase within the narrow membrane infoldings of the interdigitations is consistent with a role for this enzyme in water transport and the production of the aqueous.

Animals↗
PubMed1988

A diurnal rhythm in opsin content of Rana pipiens rod inner segments.

Quantitative electron microscope immunocytochemistry, employing an antibody specific to opsin, was used to evaluate the amount and location of opsin in Rana pipiens rod photoreceptors throughout a 24 hr light/dark cycle. We found a distinct diurnal rhythm in the density of anti-opsin labeling of the rough endoplasmic reticulum (RER) and Golgi apparatus in the myoid region of the rod inner segment. Opsin labeling of these organelles was lowest at light onset, increasing thereafter by three- to four-fold, and remained high until 2 hr into the dark phase. A fall in labeling density occurred within the following 4 hr, and remained low for the remainder of the dark phase. Our finding of a diurnal rhythm regulating inner segment opsin transport in Rana pipiens contrasts with published observations on outer segment membrane turnover, since it has been shown that the rates of disc formation and disc shedding are governed by environmental lighting alone in this species. These results imply that there is opsin pooling in the inner segment during the first 14 hr of a 24 hr light/dark cycle; thereafter the loss of inner segment opsin due to mobilization of this protein from the Golgi exceeds the rate of formation of new opsin. There was no evidence of accumulation of opsin-containing vesicles near the cilium or in the ellipsoid just prior to light onset. At light onset, prominent opsin labeling was identified at the proximal portion of the outer segment in regions separate from the disc stack. In two separate experiments, additional groups of frogs were killed around the time of light onset and were examined by conventional transmission electron microscopy. Disordered disc membranes were seen at the base of the outer segment which were not in register with the disc stack. These disordered membranes were observed as early as 2 hr before light onset, and were no longer observed by 1 hr after light onset. We suggest that these disordered membranes reflect a step in the biogenesis of new discs, serving as a pool of membrane that forms during the later part of the dark cycle. It appears that light onset triggers the ordering of neatly registered discs from this new membrane, rather than assembly of new membrane from pooled transport vesicles in the inner segment.

Animals↗
PubMed1988

The potential usefulness to research of retina obtained by biopsy.

Retinal biopsy has been performed on normal rabbits and dogs. It was shown that retinal samples could be obtained by internal and external routes in rabbits, but in dogs inability to achieve adequate vitrectomy precluded useful retinal biopsy by the internal route. A single external biopsy specimen of 3 mm diameter was more than adequate to undertake standard histopathological examination, immunocytochemical experiments and determination of cyclic nucleotide levels. The quality of the micrographs, immunocytochemical labelling of rhodopsin and phosphodiesterase, and cyclic nucleotide analyses were similar to those obtained with retinas from freshly enucleated eyes. The surgical exercise was well tolerated by most eyes and does not preclude serial biopsies being undertaken. It is concluded that retinal biopsy provides material of sufficient quantity and quality to satisfy many laboratory needs in retinal research.

Animals↗
PubMed1987

Conformational properties of the main intrinsic polypeptide (MIP26) isolated from lens plasma membranes.

The conformational properties of the main intrinsic polypeptide (MIP26) isolated from lens plasma membranes were studied by using near- and far-ultraviolet circular dichroism. The far-ultraviolet spectrum of MIP26 solubilized with octyl beta-D-glucopyranoside indicates an alpha-helical content of approximately 50% and a beta-structure content of approximately 20%. A detergent-free membrane suspension of MIP26 produced a typically distorted far-ultraviolet spectrum which was caused by differential light scattering and absorption flattening. However, decreasing the size of the membrane fragments by sonication produced a far-ultraviolet spectrum free of distortion, and with a rotatory strength profile similar to that obtained for MIP26 solubilized with octyl beta-D-glucopyranoside. This implies similar secondary structure properties for the protein in both the suspension and the sugar detergent. The cleavage of MIP26 with Staphylococcus aureus protease, which results in removal of a 5-kilodalton peptide and which mimics the age-dependent posttranslational changes that take place in the lens, did not significantly affect the conformation of the core protein as judged by the near-ultraviolet circular dichroism spectra.

Animals↗
PubMed1987

Immunocytochemical localization of photopigments in cephalopod retinae.

The photopigments, rhodopsin and retinochrome, have been localized in cephalopod retinae using light and electron microscopic immunocytochemical methods. Polyclonal antibodies prepared against squid opsin demonstrated the presence of this protein in the photoreceptor rhabdomes, Golgi zone, Golgi-associated vesicles, plasma membrane, large cytoplasmic vesicles, and axonal membranes of octopus retinae. Monoclonal anti-opsin immunostained the rhabdomes and multivesicular bodies in the photoreceptor inner segments of squid. We believe the multivesicular bodies are involved in rhabdome turnover. Polyclonal anti-retinochrome localized this photopigment to the myeloid bodies of the photoreceptor inner segments, the rhabdomes, and to the extracellular space between opposing rhabdomeres in octopus retina. The results suggest some interesting functional relationships between rhodopsin and retinochrome with regard to chromophore exchange between illuminated forms of these photopigments and chromophore addition to newly synthesized opsin.

Animals↗
PubMed1987

Changes in the localization and content of opsin during retinal development in the rds mutant mouse: immunocytochemistry and immunoassay.

Electron-microscope immunocytochemistry and antibody staining of nitrocellulose replicas of SDS gels (Western blots) were used in a developmental study to detect the presence and localization of opsin in the developing photoreceptors of rds (020/A) mutant mice and their BALB/c normal controls. Western blot analysis of isolated retinal membranes first detected opsin at 10 postnatal days in both strains. Opsin levels rose progressively with development in BALB/c normal retinas. In contrast, levels in the rds retina became undetectable by 30 days after peaking at 15 days. Specific binding of anti-opsin antibodies was first observed by immunocytochemistry at postnatal 5 days in the distal plasma membrane of the connecting cilium in both BALB/c and rds retinas. Thereafter, labeling intensity increased progressively with development in the BALB/c retina. Anti-opsin labeling remained localized primarily to the plasma membrane of the distal cilium and to the outer segment with the exception that light labeling of the inner-segment plasma membrane was observed from 5-15 postnatal days. Antibody binding to photoreceptors in the rds mouse retina predominated in the plasma membrane of the connecting cilium at 5 postnatal days, but opsin was present at higher density in the inner segment plasma membrane at 5-, 10-, 15- and 20 postnatal days, when compared with BALB/c photoreceptors. From 10-20 postnatal days opsin-rich vesicles were observed in the ventricular (subretinal) space of the rds retina. Maximum intensity of labeling was observed at 15 postnatal days. By 30 postnatal days, labeling of the ciliary and inner-segment plasma membrane decreased to near background levels.

Aging↗
PubMed1987

Hydroxyindole-O-methyltransferase in Y-79 human retinoblastoma cells: effect of cell attachment.

Hydroxyindole-O-methyltransferase (HIOMT), the enzyme in the final step of melatonin synthesis, is present in the Y-79 human retinoblastoma cell line. Using electroblot immunolabellings, a single band corresponding to HIOMT was observed. Immunofluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and quantification of enzyme activity all revealed dramatic increases in HIOMT in cells attached to substrate compared to cells in suspension culture.

Acetylserotonin O-Methyltransferase↗