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S J Fliesler

Publications and source records attributed to S J Fliesler.

At least 19 recordsLinked to original sources
PubMed1992

Primary structure of frog rhodopsin.

Amphibians have been employed extensively to study the anatomy, physiology, biochemistry, and cell biology of the visual system for decades, yet there have been no reports concerning the primary structure of amphibian visual transduction components. Thus, we have determined the entire nucleotide sequence of frog (Rana pipiens) rhodopsin cDNA, including a putative transcription start point and poly A tail, by sequence analysis of PCR products and mRNA. The open reading frame predicts an opsin of 354 residues, six residues longer than the mammalian rod opsins, containing 11 potential phosphorylation sites in the C-terminal domain. RNA blot analysis revealed two transcripts of ca. 1.7 and 3.1 kb. Frog rhodopsin exhibits approximately 85% identity to mammalian rhodopsin at the amino acid level. Sequence analysis of additional components will produce the framework from which a more detailed understanding of amphibian phototransduction can emerge.

Amino Acid Sequence↗
PubMed1992

Retinal and retinol promote membrane fusion.

Disk membranes from the bovine retinal rod outer segments (ROS) were found to fuse with vesicles made of lipids extracted from unbleached ROS disk membranes, using a lipid mixing assay for membrane fusion (relief of self-quenching of R18, octadecylrhodamine B chloride). If the retinal chromophore of rhodopsin was reductively linked to opsin before lipid extraction, the vesicles made of the extracted lipids were not suitable targets for fusion of the disk membranes. The addition of retinal and retinol to these vesicles restored their ability to fuse. Therefore, the presence of all-trans retinal was implicated in promoting membrane fusion in this system. To test this possibility, the ability of retinal and retinol to influence the phase behavior and the fusion capability of large unilamellar vesicles (LUV) of N-methyl dioleoylphosphatidylethanolamine (N-methyl-DOPE) was examined. Both retinal and retinol stimulated the fusion of vesicles of N-methyl-DOPE (contents mixing with ANTS, 1-aminonaphthalene-3,6,8-trisulfonic acid; DPX, p-xylylene bis(pyridinium bromide)). Both compounds reduced the onset temperature for isotropic resonances in the 31P-NMR spectra of N-methyl-DOPE dispersions and the onset temperature, TH, for formation of hexagonal II phase. These results were consistent with previous studies in which the onset temperature for the 31P-NMR isotropic resonances were correlated with stimulation of membrane fusion. These data suggested that both retinal and retinol may stimulate membrane fusion by destabilizing the bilayers of membranes.

Animals↗
PubMed1992

Ultrastructure of connecting cilia in different forms of retinitis pigmentosa.

The connecting (sensory) cilium of rods and cones is the stalk that separates the outer segment, which contains visual pigment in stacks of membrane discs, from the inner segment, which contains cytoplasmic organelles involved in protein synthesis. There are conflicting reports on the occurrence of abnormal motile cilia in patients with retinitis pigmentosa (RP) and very few ultrastructural studies of photoreceptor connecting cilia in retinas from patients with RP. Defective connecting cilia could lead to the outer segment atrophy and degeneration that are characteristic of RP. The present study addresses the hypothesis that photoreceptor connecting cilia, as observed in cross section by electron microscopy, are defective in RP. We examined retinas from five patients with RP and four controls and found morphologic defects in the connecting cilia of one RP patient with type 2 Usher syndrome (86% abnormal, P less than .0001) but not in our sample of patients with X-linked (n = 2), simplex (n = 1), or autosomal dominant (n = 1) RP.

Adolescent↗
PubMed1990

Relationship of cholesterol content to spatial distribution and age of disc membranes in retinal rod outer segments.

The initial events of visual transduction occur on disc membranes which are sequestered within the photoreceptor outer segment. In rod cells, the discs are stacked in the outer segment. Discs are formed at the base of the rod outer segment (ROS) from evaginations of the plasma membrane. As new discs form, older discs move toward the apical tip of the rod, from which they are eventually shed and subsequently phagocytosed by the adjacent pigment epithelium. Thus, disc membranes within a given rod cell are not of uniform age. We have recently shown that disc membranes are not homogeneous with respect to cholesterol content (Boesze-Battaglia, K., Hennessey, T., and Albert, A. D. (1989) J. Biol. Chem. 264, 8151-8155). In the present study, freshly isolated bovine retinas were incubated with [3H]leucine for 4 h in order to allow sufficient time for the radiolabeled proteins to become incorporated into the basal-most (newest) discs. Osmotically intact discs were then isolated. After the addition of digitonin, the discs were fractionated based on cholesterol content, and radioactivity (indicative of newly synthesized protein) was measured. Discs which exhibited high cholesterol content also exhibited high radio-activity. These results demonstrate that the cholesterol heterogeneity of ROS disc membranes is related to the age, and thus the position, of the discs in the ROS.

Animals↗
PubMed1990

Incorporation of squalene into rod outer segments.

We have reported previously that squalene is the major radiolabeled nonsaponifiable lipid product derived from [3H]acetate in short term incubations of frog retinas (Keller, R. K., Fliesler, S. J., and Nellis, S. W. (1988) J. Biol. Chem. 263, 2250-2254). In the present study, we demonstrate that newly synthesized squalene is incorporated into rod outer segments under similar in vitro conditions. We show further that squalene is an endogenous constituent of frog rod outer segment membranes; its concentration is approximately 9.5 nmol/mumol of phospholipid or about 9% of the level of cholesterol. Pulse-chase experiments with radiolabeled precursors revealed no metabolism of outer segment squalene to sterols in up to 20 h of chase. Taken together with our previous absolute rate studies (Keller, R. K., Fliesler, S. J., and Nellis, S. W. (1988) J. Biol. Chem. 263, 2250-2254), these results suggest that most, if not all, of the squalene synthesized by the frog retina is transported to rod outer segments. Synthesis of protein is not required for squalene transport since puromycin had no effect on squalene incorporation into outer segments. Conversely, inhibition of isoprenoid synthesis with mevinolin had no effect on the incorporation of opsin into the outer segment. These latter results support the conclusion that the de novo synthesis and subsequent intracellular trafficking of opsin and isoprenoid lipids destined for the outer segment occur via independent mechanisms.

Acetates↗
PubMed1990

Neural cell adhesion molecule (NCAM) in adult vertebrate retinas: tissue localization and evidence against its role in retina-pigment epithelium adhesion.

The presence of neural cell adhesion molecule (NCAM) was examined in the neural retina, interphotoreceptor matrix (IPM), and retinal pigment epithelium (RPE) of adult bovine and frog eyes. Using polyclonal antibodies raised against adult isoforms of NCAM. Western blot analyses revealed the presence of NCAM in the neural retina, but not in the IPM or RPE of these species. As a control, Western blot analysis was used to demonstrate the presence of interphotoreceptor retinoid-binding protein (IRBP) in the IPM preparations. NCAM immunoreactivity was detected by light microscopic immunocytochemistry primarily in the plexiform layers and nerve fibre layer of the frog retina. Minor immunoreactivity was also detected in the inner and outer nuclear layers, but there was no detectable NCAM immunoreactivity in the IPM, outer segments, or RPE. These results indicate that NCAM is not a likely participant in the process of retina-RPE adhesion in the adult eye.

Animals↗
PubMed1990

Glycogenesis in the amphibian retina: in vitro conversion of [2-3H]mannose to [3H]glucose and subsequent incorporation into glycogen.

We previously demonstrated by light and electron microscopic autoradiography that Xenopus retinas incubated with [3H]mannose exhibit tunicamycin-insensitive radiolabeling of glycogen storage compartments, especially cone parabaloids. In the present study, we utilized biochemical methods to evaluate the identity of the material presumed to be [3H]glycogen in Xenopus retinas obtained from eyecups incubated under similar conditions. A crude glycogen-containing fraction was isolated, solubilized with 8 M urea, and purified by Sepharose CL-4B column chromatography. The retinal glycogen was hydrolyzed either chemically or with specific amylolytic enzymes, followed by Sephacryl S-200 column chromatography and HPLC of the hydrolysis products. Under the conditions employed, [3H]glycogen represented at least 10% of the total radiolabeled macromolecules. Hydrolysis of the [3H]glycogen released all of the radiolabel in the form of [3H]glucose, not [3H]mannose, which indicated that direct incorporation of [3H]mannose into glycogen had not occurred. [3H]Glucose was distributed throughout the glycogen molecule, not just in the outer tiers, which indicated that de novo glycogenesis had occurred. Furthermore, enzymatic isomerization of the glycogen-derived [3H]glucose with glucose isomerase yielded fructose with retention of tritium. This demonstrated that positions other than the C-2 carbon of glucose were radiolabeled. Analysis of the medium after several hours of incubation revealed the presence of 3H2O as the major radiolabeled compound. These results support the conclusion that the in vitro incorporation of [2-3H]mannose into retinal glycogen involves initial catabolism of the radiolabeled substrate and subsequent reincorporation of the label via gluconeogenesis into precursors utilized for de novo glycogenesis.

Animals↗
PubMed1990

The effect of inhibitors of glycoprotein synthesis and processing on the phagocytosis of rod outer segments by cultured retinal pigment epithelial cells.

Retinal pigment epithelial cells selectively phagocytize rod outer segments by a process that may be mediated by specific cell surface receptors. Since many receptors are glycoproteins, we have studied the effect of tunicamycin, an inhibitor of N-linked oligosaccharide synthesis, and of castanospermine and swainsonine, which are inhibitors of oligosaccharide processing, on the ability of cultured retinal pigment epithelial cells to phagocytize rod outer segment. Tunicamycin inhibits the glycosylation of newly synthesized glycoproteins by 85-90%; concomitantly, the phagocytosis of rod outer segments is inhibited by 70-80%. The effect of tunicamycin is to initially reduce rod outer segments binding, and therefore the subsequent ingestion of rod outer segments. SDS-PAGE analysis and autoradiography of [35S]methionine labelled extracts of tunicamycin-treated cells, demonstrates the disappearance of a number of glycoprotein bands, and the appearance of a number of protein bands of lower Mr. Kinetic analysis of the disappearance and reappearance of specific glycoproteins suggests that the lower Mr bands are the non-glycosylated forms of the higher Mr bands. By contrast, castanospermine and swainsonine have no effect on the ability of retinal pigment epithelial cells to phagocytize rod outer segments, or on the SDS-PAGE pattern of treated cells, although they were shown to inhibit oligosaccharide processing as expected. These results support the hypothesis that rod outer segment phagocytosis by retinal pigment epithelial cells is mediated by specific glycoprotein receptors. N-Glycosylation of these receptors is required for their function, or for their insertion into the plasma membrane, whereas processing of the N-linked oligosaccharide chains of these receptors is not crucial for rod outer segment phagocytosis by retinal pigment epithelial cells.

Animals↗
PubMed1989

Biodegradable mechanical retinal fixation. A pilot study.

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 long-range 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 ensectomized/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.

Animals↗
PubMed1988

N-linked oligosaccharides are not required for neuron-neuron interactions mediated by neural cell adhesion molecule.

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.

Amidohydrolases↗
PubMed1988

Isoprenoid biosynthesis in the retina. Quantitation of the sterol and dolichol biosynthetic pathways.

The isoprenoid pathway provides several important products for retina function. In this study the sterol and dolichol pathways were investigated in retinas from Rana pipiens in order to assess the contribution of de novo synthesis. Levels of 5.9 +/- 2.0 (n = 13) nmol/retina for squalene, 134 +/- 27 (n = 16) nmol/retina for cholesterol, and 0.14 +/- 0.04 (n = 11) nmol/retina for dolichyl phosphate (Dol-P) were determined by high performance liquid chromatography analysis. When whole retinas were incubated with 3H2O, radioactivity was incorporated into compounds which chromatographed on reversed-phase and silica high performance liquid chromatography at the elution positions of squalene, cholesterol, lathosterol, and methyl sterols. From these results, the upper limit for the absolute rate of the sterol pathway was estimated to be 3.4 pmol/h. When retinas were incubated with [3H]acetate, the major labeled product was squalene. The relatively low level of incorporation into cholesterol was apparently due to a substantial pool of squalene which accumulated de novo incorporated [3H]acetate. Dol-P was also labeled with [3H]acetate, and by comparing the ratio of 3H incorporation into Dol-P/squalene with the absolute rate of the sterol pathway, the absolute rate of Dol-P synthesis was determined to be 0.022 pmol/h. Our calculations indicate that the retina does not synthesize sufficient quantities of cholesterol de novo to account for that which is utilized in the biogenesis of rod outer segment membranes.

Acetates↗
PubMed1988

A 42,000-Da protein in rabbit tissues and in a glycogen synthase preparation cross-reacts with antibodies to glycogenin.

Rabbit skeletal muscle glycogen previously has been shown to be covalently bound to a 40,000-Da protein ("glycogenin") via a novel glucosyl-tyrosine linkage [I.R. Rodriguez and W.J. Whelan (1985) Biochem. Biophys. Res. Commun. 132, 829-836]. Antibodies raised against rabbit skeletal muscle glycogenin cross-react with a similar protein present in rabbit heart and liver glycogens, as well as with a 42,000-Da "acceptor protein" present in high-speed supernatants of rabbit muscle, heart, retina, and liver. This 42,000-Da protein incorporates [U-14C]Glc when an ammonium sulfate fraction prepared from the tissue supernatants is incubated with UDP-[U-14C]Glc. The [U-14C]Glc incorporated can be removed quantitatively by treatment with amylolytic enzymes, indicating that the [U-14C]Glc incorporation represents elongation of a preexisting glucan attached to the acceptor protein. Furthermore, a commercial preparation of rabbit skeletal muscle glycogen synthase contains this 42,000-Da protein. We propose that the 42,000-Da protein represents the free form of glycogenin in tissues, with its covalently attached glucan chain(s) providing a "primed" elongation site for glycogen synthesis.

Animals↗
PubMed1988

Tunicamycin-induced degeneration in cone photoreceptors.

Tunicamycin (TM), an inhibitor of dolichylphosphate-mediated protein glycosylation, was injected intravitreally into the eyes of diurnal rodents with cone-dominated retinas. Injection of 1 microgram of the B2 isomer led to a progressive degeneration of the photoreceptor outer segments and disruption of the RPE-photoreceptor interface that took place over a 10-day period. Cone outer segments were shortened by postinjection day 6 and virtually absent by day 9. The microvilli that normally protrude from the apical surface of the retinal pigment epithelium were replaced by a fringe of shortened processes. The other retinal layers showed no morphological evidence of disruption. Retinal sensitivity, as measured by electroretinographic b-wave threshold, showed a significant and progressive decline over the 10-day course of the experiment that paralleled the disruption of retinal morphology. These results suggest that TM leads to similar morphological and electrophysiological effects on rod and cone photoreceptors.

Animals↗
PubMed1987

Monensin stimulates glycerolipid incorporation into rod outer segment membranes.

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.

Animals↗
PubMed1987

Intravitreal vancomycin. Retinal toxicity, clearance, and interaction with gentamicin.

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.

Animals↗
PubMed1986

In vivo incorporation of [2-3H]-myo-inositol into frog opsin.

The in vivo incorporation of [2-3H]-myo-inositol into frog retinal rod outer segment membranes was examined. About 25% of the recovered radioactivity was found to be protein-associated. Following acid hydrolysis of this material and extraction with hexane, all the radioactivity remained in the aqueous phase, indicating that the label was not in fatty acids. Following ion exchange column chromatography of the hydrolysate, the major radioactive compound comigrated on TLC with an internal standard of [U-14C]-myo-inositol. SDS polyacrylamide gel electrophoresis of unextracted membranes indicated that the majority of the label was associated with opsin. These results indicate that [2-3H]-myo-inositol was incorporated in vivo into opsin, presumably with retention of its chemical identity.

Animals↗