Search

Recent studies have described the role of various regions of the neural cell adhesion molecule (NCAM) in cell-cell interactions. Monoclonal antibodies (L2/HNK-1) directed against a sulfated, glucuronic acid-containing, N-linked carbohydrate epitope have also been shown to inhibit NCAM-mediated neural cell adhesion. In the present study we show that dissociated retinal neurons in an in vitro model system can bind as well to normal NCAM as to NCAM lacking the L2/HNK-1 epitope or to glycopeptidase F-treated NCAM. These data suggest that N-linked oligosaccharide chains do not confer upon NCAM the adhesional properties associated with its role in neuron-neuron interactions.

Isolated frog retinas were incubated with radiolabeled glycoprotein precursors in the presence or absence of tunicamycin (TM), a selective inhibitor of protein N-glycosylation. In dual-label incubations, TM inhibited the incorporation of [3H]mannose into total retina Cl3CCOOH-precipitable material by 85% relative to controls, whereas incorporation of [14C]leucine was not significantly affected. In a companion single-label incubation, TM blocked the incorporation of [3H]leucine into rod outer segment (ROS) membrane Cl3CCOOH-precipitable material by 95% relative to controls. When retinas were labeled with [35S]methionine, fluorograms of NaDodSO4/polyacrylamide gels from control retinas and ROS membranes exhibited a heavily labeled component (apparent Mr approximately 37,000) which had the electrophoretic and antigenic properties of opsin, the rod visual pigment apoglycoprotein. TM-treated retinas exhibited a substantially reduced labeling of the Mr 37,000 component and incorporation of label into a component (apparent Mr approximately 32,000) not found in control retinas, which exhibited the electrophoretic and antigenic behavior of nonglycosylated opsin. ROS membranes isolated from TM-treated retinas contained neither the Mr 37,000 nor the Mr 32,000 radiolabeled species. Light-microscope autoradiograms of retinas incubated with [3H]leucine in the absence of TM exhibited bands of silver grains at the base of ROS, indicative of new membrane assembly. However, no such bands were observed in autoradiograms of TM-treated retinas. These results suggest that glycosylation of opsin is required for its incorporation into ROS membranes.

Retinal tissue from human eye donors was incubated with [ 3 H]-labeled mannose, glucosamine, fucose, and leucine in the presence and absence of tunicamycin, a selective inhibitor of dolichol-dependent glycoprotein biosynthesis. The incorporation of labeled substrates was examined by quantitative light microscopic autoradiography and biochemical methods. [ 3 H]-Mannose and [ 3 H]-fucose were predominantly incorporated into the photoreceptor layer, while [ 3 H]-gluosamine and [ 3 H]-leucine labeled the entire retinal expanse. Tunicamycin caused a marked and selective reduction in the incorporation of [ 3 H]-mannose and [ 3 H]-glucosamine in the photoreceptor layer (especially the inner segments) without affecting the relative distribution of labeled products derived from [ 3 H]-fucose or [ 3 H]-leucine. All three [ 3 H]-labeled sugars were incorporated preferentially into rod inner segments, relative to cones. Dual-label experiments with [ 3 H]-sugars and [ 14 C]-leucine revealed that tunicamycin selectively inhibited the incorporation of [ 3 H]-mannose, but not the other substrates, into total retinal TCA-precipitable material. This inhibition was not due to decreased mannose uptake by retinal cells. Detergent-solubilized retinas were analyzed by polyacrylamide gel electrophoresis and fluorography. The results indicated that each labeled substrate was incorporated into a variety of glyoproteins, including a component having the electrophoretic mobility of opsin. These results suggest that the lipid intermediate pathway of glycoprotein synthesis is localized preferentially to the photoreceptor layer of the retina, and may subserve the biosynthesis of rod cell glycoproteins (e.g. opsin).

Isolation and biochemical characterization of goldfish retinal photoreceptor outer segment membranes are described. The lipid fraction is composed primarily of phospholipids (68 mol%) with substantial amounts of neutral lipids (32 mol%). Sterols account for only about 2 wt% of the membranes (about 9 mol% of the total lipids). The phospholipid class composition and fatty acid composition are similar to those of other vertebrate photoreceptor membranes. Two novel findings were the high levels of free fatty acids (21 mol% of the total lipids, primarily palmitic and docosahexaenoic acids) and the presence of relatively significant amounts of a C-32 diacylglycerol molecular species.

Monensin is an ionophore which disrupts the structure of the Golgi apparatus and inhibits vesicular transport in eukaryotic cells. In this study, we examined the effects of monensin on the incorporation of newly synthesized glycerolipids into retinal rod outer segment (ROS) membranes. Frog retinas were incubated in the presence or absence of monensin (50 nM) with either [1,2,3-3H]glycerol or [9,10-3H]palmitic acid as radiolabeled substrate. Total lipids were extracted from retinas and ROS membranes and resolved into individual phospholipid classes and neutral lipids by thin-layer chromatography. In the presence of monensin, the specific activity of ROS phospholipids was increased about 2-fold with [3H]glycerol and nearly 3-fold with [3H]palmitate as substrates relative to controls. In contrast, the specific activity of total retinal lipids, the relative incorporation of label into ROS and retinal phospholipids, and the total lipid phosphorous content of ROS membranes and retinas were not significantly different from control values. These data suggest that the enhanced labeling of ROS phospholipids in the presence of monensin was due to altered intracellular routing of lipids rather than increased glycerolipid synthesis. Under the same conditions, total retinal protein synthesis was about 90% of control, but light microscopic autoradiography indicated that newly synthesized proteins were not transported to the ROS for assembly into disc membranes. Thus, newly synthesized glycerolipids can be delivered to the ROS by a mechanism which is independent of protein transport to that cellular compartment.

Castanospermine (Cas), an inhibitor of alpha-glucosidase I, blocks "trimming" of the N-linked oligosaccharide Glc3Man9GlcNAc2, thus preventing normal glycoprotein maturation. With use of a dual-label protocol, Xenopus retinas incubated in the presence of Cas exhibited at least a 2.3-fold increase in the incorporation of [3H]mannose into total retina Cl3CCOOH-precipitable material, whereas incorporation of [14C]leucine was not significantly affected, relative to controls. Analysis of NaDodSO4/PAGE fluorograms of solubilized retinas and rod outer segment (ROS) membranes indicated a relatively selective effect of Cas on opsin (the rod visual pigment apoglycoprotein). The apparent molecular mass of opsin was increased by approximately 2500 in the presence of Cas; the incorporation of [3H]mannose into opsin was enhanced about 2.3-fold without a significant effect on [14C]leucine incorporation, relative to controls. Electron microscopic autoradiography of retinas incubated for 4 hr with [3H]mannose showed that the number of newly formed ROS discs in Cas-treated retinas was not significantly different from controls, but the silver grain density over those discs was about 2.6-fold greater than in controls. The morphology of the newly formed discs was comparable under both conditions. Thus, opsin bearing abnormally large oligosaccharides can be accommodated in the process of disc morphogenesis. These results suggest that the structural requirements for opsin's oligosaccharides, with regard to their potential role as determinants of disc morphogenesis, are not stringent. Furthermore, post-translational processing of N-linked oligosaccharides is not essential for the normal intracellular routing and cell surface expression of membrane glycoproteins.

The sterol composition of bovine retinal rod outer segment membranes and whole retinas was studied by detailed chromatographic analyses. Cholesterol represented at least 98% of the total 3 beta-monohydroxy sterols of rod outer segment membranes, accounting for 1.68 +/- 0.15% of the dry weight. Whole retinas contained 1.76 +/- 0.29% cholesterol by dry weight, representing at least 99% of the total 3 beta-monohydroxy sterols. Trace amounts of a component having the chromatographic properties of 5 alpha-cholestan-3 beta-ol were found in rod outer segment membranes and whole retinas. Very small amounts of a component having the chromatographic properties of 5 alpha-cholest-7-en-3 beta-ol were found in whole retinas, but not in rod outer segment membranes. The molar ratio of cholesterol to rhodopsin in bovine rod outer segment membranes was approximately 4.7. Cholesterol accounted for only 5-7 mol% of total rod outer segment membrane lipids.

We have explored the use of the Na+-H+ ionophore monensin as a potential tool for the investigation of membrane assembly and transport in retinal photoreceptors. Autoradiographic analysis of frog retinas incubated with [3H]leucine in the presence of monensin revealed a lack of concentrated silver grains (“bands”) at the base of the rod outer segments, in contrast to controls. This is indicative of a pronounced monensin-induced decrease in disc membrane assembly. Biochemical analyses of whole retinas and isolated rod outer segment membranes showed that protein synthesis (including opsin synthesis) was not significantly inhibited under these conditions, whereas passage of membrane protein to the rod outer segment was blocked. Glycerolipid synthesis was not significantly affected by monensin. The results suggest that membrane proteins (e.g., opsin) destined for incorporation into the rod outer segment must pass through the Golgi apparatus and demonstrate the potential utility of monensin for inhibiting aspects of marcomolecule transport in photoreceptors.

The antibiotic tunicamycin inhibits the biosynthesis of N-acetyl-glucosaminylpyrophosphoryl polyisoprenol1–3, a key intermediate in the formation of the asparagine-linked oligosaccharides of glycoproteins4. The effects of tunicamycin have been studied in various biological systems5, primarily with the aim of elucidating the role of the carbohydrate moieties in the cellular function of glycoproteins6. Rhodopsin, the visual pigment of retinal rod photoreceptor cells, is a membrane glycoprotein which consists of a single polypeptide chain (opsin) to which a chromophoric prosthetic group (II-cis-retinaldehyde) and two asparagine-linked oligosaccharide chains are covalently attached7. The glycosylation of opsin can be blocked with tunicamycin in vitro in conditions where polypeptide synthesis is only slightly decreased8,9. We have reported9 that tunicamycin can disrupt the normal assembly of rod outer segment membranes in vitro without significantly inhibiting the biosynthesis or intracellular transport of opsin. Here we report that intraocular injection of tunicamycin produces a photoreceptor-specific degeneration characterized by (1) progressive shortening of rod outer segment, (2) decreased electroretinogram amplitudes, (3) cessation of rod outer segment membrane assembly, and (4) eventual photoreceptor cell death.

When human retinas are cultured in the presence of various phosphodiesterase (PDE) inhibitors or cyclic nucleotide analogues, rod photoreceptors undergo degenerative changes followed by cell death within 8 hours of incubation, whereas cone photoreceptors and other retinal cells are affected minimally. In the present study we found that the PDE inhibitor, isobutylmethylxanthine (IBMX), as well as the dibutyryl analogues of cGMP and cAMP, inhibited protein synthesis in short-term cultures of human retinas under conditions where uptake of exogenously supplied 3H-leucine was not diminished (relative to controls). The results of an autoradiographic analysis suggested that inhibition of protein synthesis by these drugs occurred to a greater extent in rod photoreceptors than in cones or other retinal cells, and that this phenomenon happened prior to the onset of any morphological changes. When retinas incubated in IBMX for 4 hours were returned to control medium for an additional incubation, rods recovered their ability to synthesize proteins and cell viability was maintained. The results of polyacrylamide gel electrophoresis indicated that IBMX caused a general inhibition of retinal protein synthesis rather than affecting the synthesis of specific retinal proteins. These observations are discussed with regard to possible mechanisms underlying rod-specific photoreceptor degeneration.

• Some of the gram-positive isolates from exogenous bacterial endophthalmitis cases treated at our institution have been found to be resistant to either cefazolin sodium, gentamicin sulfate, or both. However, all of these isolates have been sensitive to vancomycin. These findings prompted us to reevaluate the retinal toxicity and clearance of intravitreal vancomycin in pigmented rabbits. Doses up to 2 mg were found to be nontoxic in phakic and aphakic-vitrectomized eyes. Clearance was determined in phakic and aphakic-vitrectomized rabbit eyes with or without intact lens capsules. The antibiotic was cleared most slowly in phakic eyes. Aphakic-vitrectomized eyes without an intact lens capsule cleared antibiotic most rapidly, while aphakic-vitrectomized eyes with intact capsules exhibited an intermediate clearance rate. In addition, the interaction between vancomycin and gentamicin on gram-positive endophthalmitis isolates was found to be additive or synergistic depending on the bacterial species. Based on these data, we recommend the combination of vancomycin and an aminoglycoside as the initial antibiotic therapy for exogenous bacterial endophthalmitis.

Systemic injection of [2-3H]myo-inositol into frogs resulted in the incorporation of more than half of the label into glycerolipid classes other than phosphoinositides in retinal rod outer segment membranes. Following methanolysis and differential extraction of isolated lipid classes, radioactivity was recovered primarily in the aqueous phase. After phospholipase C hydrolysis of the total membrane lipids, 97% of the radioactivity was extractable with organic solvents, and 70% of the label in lipids was in 1,2-diglycerides. These results indicate that the label was incorporated primarily into the glyceryl moiety of the membrane glycerolipids. Intraocular injection of frog eyes or in vitro incubation of frog retinas with [2-3H]myo-inositol resulted in the incorporation of radioactivity almost exclusively into phosphoinositides in rod outer segment membranes. Incubation of retinas with [U-14C]glucuronic acid did not result in the formation of labeled retinal lipids. These results suggest that myo-inositol can be catabolized systemically to precursors utilized for glycerolipid biosynthesis in the retina.

Oviduct tissue slices were incubated with [3H]-leucine or [3H]-mannose in the presence and absence of tunicamycin, a specific inhibitor of lipid-mediated protein glycosylation. Conditions were established where tunicamycin had maximal effect on [3H]-mannose incorporation (greater than 90% inhibition) but a minimal effect on [3H]-leucine incorporation (less than 10% inhibition) into total TCA-insoluble products. Analysis of incubated tissues by SDS-polyacrylamide gel electrophoresis revealed that in the absence of tunicamycin, [3H]-mannose was incorporated into only a few proteins, of which ovalbumin represented the major radiolabeled component. Tunicamycin markedly reduced the incorporation of [3H]-mannose into ovalbumin and other oviduct glycoproteins. In contrast, analysis by SDS-polyacrylamide gel electrophoresis showed that [3H]-leucine was incorporated into a variety of proteins in the absence of tunicamycin. The radioactivity profile of some of these proteins was shifted toward lower Mr when oviduct slices were incubated in the presence of tunicamycin, with only a minimal decrease in protein labeling. Light microscopic autoradiograms of tissue incubated with [3H]-leucine in either the presence or absence of tunicamycin exhibited extensive labeling of tubular gland and epithelial cells. In the absence of tunicamycin, these cell types also become markedly labeled with [3H]-mannose; however, incorporation of label in both cell types was substantially reduced in the presence of tunicamycin. Qualitatively, labeling of tubular gland cells appeared greater than that of epithelial cells, largely due to the concentration of silver grains over the dense population of secretory vesicles in the tubular gland cells.(ABSTRACT TRUNCATED AT 250 WORDS)

• Mechanical retinal fixation is a useful tool in the armamentarium of vitreoretinal surgeons. Unfortunately, the use of metallic retinal tacks on a permanent basic mandates a retained intraocular foreign body that poses some potential for longrange toxic effects. Herein, we report the results of our initial experience with a temporary, biodegradable mechanical retinal fixation device ("biopin") in rabbits. When compared with metallic tacks, the biopins appeared to be well tolerated intraocularly, with minimal damage detectable at the light microscopic level. Disinsertion of the biopins occurred in 46% of the eyes within two weeks and in all eyes by four weeks. Initial signs of degradation of the biopins were observed at six weeks (median) in the lensectomized/vitrectomized eyes and at 12 weeks (median) in the nonvitrectomized eyes. Differences were found in the occurrence of retinal detachment between animals that underwent lensectomy/vitrectomy with insertion of two metallic tacks and animals that underwent insertion of one biopin without vitrectomy. The biopin has a potential as a vehicle for sustained release of pharmacologic agents to inhibit directly the development of vitreoretinal proliferation, thereby retarding or preventing subsequent retinal traction and detachment.