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Biomedical subjects

R T Fletcher

Publications and source records attributed to R T Fletcher.

At least 19 recordsLinked to original sources
PubMed1992

Expression of guanylate cyclase-A mRNA in the rat retina: detection using polymerase chain reaction.

A technique based on RNA-PCR was successfully employed for the detection of guanylate cyclase-A (GC-A) mRNA in the rat retina. Three sets of primers designed from the published cDNA sequence of rat brain guanylate cyclase-A (GC-A) produced amplification products of expected sizes from the retina as well as brain. Analysis of retinal PCR products yielded a 970 bp sequence, which showed 100% homology to the cDNA sequence of GC-A (2343-3312 bp region). Northern blot analysis was not very sensitive for the detection of GC-A mRNA in the retina. The results indicate that the mRNA for GC-A (or a closely related form) is probably expressed in the retina, but at a lower level than that found in the brain.

Animals↗
PubMed1992

Strychnine-insensitive glycine receptors in embryonic chick retina: characteristics and modulation of NMDA neurotoxicity.

In the mammalian brain, the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor is coupled to a cation channel and a strychnine-insensitive glycine receptor. The present paper demonstrates the presence of NMDA receptor-coupled strychnine-insensitive glycine receptors in embryonic chick retina. Both glycine and 1-aminocyclopropanecarboxylic acid (ACPC) exhibited similar potencies (271 +/- 39 vs 247 +/- 39 nM, respectively) as inhibitors of strychnine-insensitive [3H]glycine binding to retinal membranes. Moreover, glycine and ACPC enhanced [3H]MK-801 binding to sites within the NMDA-coupled cation channel in retinal membranes with potencies comparable to those reported in rat brain. While the potency of ACPC was significantly higher than glycine (EC50 54 +/- 12 vs 256 +/- 57 nM, P < 0.02) in this measure, there were no significant differences in the maximum enhancement (efficacy) of [3H]MK-801 binding by these compounds. Since glycine appears to be required for the operation of NMDA-coupled cation channels, we examined the effects of glycine and ACPC on NMDA-induced acute excytotoxicity in the 14-day embryonic chick retina. Histological evaluation of retina revealed that either ACPC (10-100 microM) or glycine (200 microM) attenuated NMDA-induced (200 microM) retinal damage and a combination of these agents produced an enhanced protection against acute NMDA toxicity. ACPC (100 microM), but not MK-801 (1 microM) also afforded a modest protection against kainate-induced (25 microM) retinal damage. These findings demonstrate that while strychnine-insensitive glycine receptors are present in embryonic chick retina, occupation of these sites does not augment the cytotoxic actions of NMDA. Moreover, the ability of ACPC and glycine to attenuate NMDA-induced cytotoxicity does not appear to be mediated through occupation of these sites.

Amino Acids↗
PubMed1991

Insulin and IGF-I binding in developing chick neural retina and pigment epithelium: a characterization of binding and structural differences.

We have characterized insulin and insulin-like growth factor I (IGF-I) binding sites in developing chick retina and pigment epithelium (10- and 14-day embryonic, and 2-week post-hatched). For comparison, binding sites in brain and liver were also examined. Both the retina and pigment epithelium (PE) contain separate, specific, high affinity binding sites for insulin and IGF-I. In both tissues, IGF-I binding exceeds insulin binding by two to threefold. Insulin and IGF-I binding in the retina is four to six times greater than in PE. Insulin and IGF-I binding in the retina and PE exhibit independent developmental regulation. In the retina, the number of binding sites decreases by approximately 50% between embryonic day 10 and 2 weeks post-hatching. In the PE, binding decreases slightly between embryonic day 10 and 14 and then, in the 2-week post-hatched chick, increases threefold. Insulin receptor binding subunits in the retina and brain are similar in that both are neuraminidase insensitive and have apparent molecular weights of 116 kD. In contrast, binding subunits in the PE and liver have higher molecular weights (about 126 kD), and are sensitive to neuraminidase. At the embryonic stages examined, the levels of retinal insulin and IGF-I binding exceed those of the brain by five to 13-fold. Taken together, these data suggest that the retina is a major target of insulin and IGF-I and that the binding of these growth factors is developmentally regulated.

Aging↗
PubMed1990

Cyclic AMP and butyrate modulate melatonin synthesis in Y79 human retinoblastoma cells.

Melatonin is synthesized by cultured Y79 human retinoblastoma cells and is secreted into the medium. Activity of the two key enzymes involved in the synthesis of melatonin, N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT), are present in retinoblastoma cells. The activity of these enzymes and the resulting synthesis and release of melatonin are modulated by the addition of a cyclic AMP analogue and butyrate to the culture medium. Melatonin levels increase dramatically over control levels after the addition of dibutyryl cyclic AMP (dbcAMP), whereas melatonin levels decrease after butyrate treatment. HIOMT activity is inhibited by both dbcAMP and butyrate, and NAT activity is stimulated by both of these differentiating agents, suggesting that the rise in melatonin levels in response to dbcAMP is the result of increased activity of NAT, whereas the decline in melatonin levels in response to butyrate may be due to a drop in HIOMT activity. Melatonin synthesis is dose- and time-dependent, and the effect of dbcAMP is readily reversible, whereas the effect of butyrate does not appear to be reversible. These effects probably reflect basic differences in the regulatory mechanisms of the inducing agents.

Acetylserotonin O-Methyltransferase↗
PubMed1990

Retinal cyclic GMP and AMP in rod-cone dysplasia: comparison of assays in fixed and unfixed retinal tissues.

The effects of 3 fixatives (formalin, Bouin's solution and glutaraldehyde-paraformaldehyde) on retinal cyclic AMP and GMP content were determined in canine eyes with a known abnormality of cyclic GMP metabolism. Cyclic nucleotides were measured by radioimmunoassay on perchloric acid (PCA)-precipitated retinal samples from fixed (left) and unfixed (right) eyes from nine 6-week-old rod-cone-dysplasia-affected Irish setter dogs. Cyclic AMP values were significantly lower for fixed retinas than for unfixed controls. Retinal cyclic GMP values were also lower from glutaraldehyde-paraformaldehyde-fixed samples, but were not different from control values for formalin- or Bouin's-fixed eyes, when the retina was PCA-precipitated promptly after fixation.

Animals↗
PubMed1990

Insulin and IGF-1 binding in chick sclera.

The sclera of embryonic (days 10 and 14) and young adult (2-week posthatching chicks) contains distinct binding sites for insulin and for insulin-like growth factor-1 (IGF-1). Since there is a nearly 50% decrease in insulin and IGF-1 binding between embryonic day 10 and the 2nd week posthatching, it is clear that these sites are developmentally regulated. The affinity of each binding site for its ligand is stable across development. This suggests that the developmental decrease in binding is the result of a decrease in the number of binding sites. The insulin binding site in the sclera is specific for insulin since it has a high affinity for insulin and a lower affinity for IGF-1 (IC50 for unlabeled insulin = 0.4 nM; unlabeled IGF-1 = 5.0 nM). The embryonic chick sclera also contains two high-affinity IGF-1 binding sites. One of these sites exhibits poor binding specificity since it has an equal affinity for insulin and IGF-1. However, the specificity of this site increases in the young adult. The second IGF-1 binding site exhibits a more conventional specificity in that it has a higher affinity for IGF-1 than for insulin. The specificity of this binding site also improves in the young adult. The presence of insulin and IGF-1 receptor binding site subtypes is not correlated with structurally different receptor binding subunits since only a single population of binding subunits is observed (apparent molecular weight of 125 +/- 2.7 kD) in embryonic and adult sclera.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1989

Non-allelism of three genes (rcd1, rcd2 and erd) for early-onset hereditary retinal degeneration.

Cross-breeding experiments were utilized to study the genetics of three autosomal recessive, early onset retinal degenerations in dogs. Irish setters affected with rod-cone dysplasia type 1 (rcd1) were bred to Norwegian elkhounds affected with early retinal degeneration (erd). All offspring (15 pups-two litters) surviving to diagnostic age were phenotypically normal, as assessed by electroretinography, retinal morphology and assay of retinal cyclic nucleotide content. One phenotypically normal female Irish setter-Norwegian elkhound crossbred dog (the progeny of the above breeding and thus heterozygous at both the rcd1 and the erd locus) was bred to a collie dog affected with rod-cone dysplasia type 2 (rcd2). All 11 pups from this breeding also proved phenotypically normal by the above methods. These results establish that the genes rcd1, rcd2 and erd are non-allelic. Biochemical data are also presented that establish that erd, unlike rcd1 and rcd2, is not associated with abnormal metabolism of retinal cyclic guanosine monophosphate (cyclic GMP).

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1988

IGF-I receptors in the bovine neural retina: structure, kinase activity and comparison with retinal insulin receptors.

The retina contains specific high-affinity receptors for insulin-like growth factor-I (IGF-I). Although IGF-I binding was observed in photoreceptor outer segments, the level of this binding was only 10% of that found in whole retina or mixed preparations of rod outer (ROS) and inner (RIS) segments. The higher IGF-I binding activity in RIS and non-photoreceptor regions of the retina suggests these sites as candidates for putative IGF-I action. Data from crosslinking experiments with and without neuraminidase treatment indicate that the binding subunits of the retinal IGF-I receptor exist in two subpopulations (Mr = 121- and 131 kDa), and that the larger of the two subunits has either a greater number or more exposed sialic acid residues. In these characteristics, the retinal IGF-I receptor is similar to the retinal insulin receptor. Retinal IGF-I and insulin receptors possess kinase activity towards their own beta-subunits, a tyrosine containing copolymer, and various molecular forms and subunits of transducin (T alpha-GDP, T alpha-GTP, T beta). The transducin forms are phosphorylated with different efficiencies (e.g. T alpha-GDP is 10-15 times more effective than T alpha-GTP as substrate). These differences are also observed in basal conditions and may reflect differences in transducin subunit affinity for the IGF-I and insulin receptor. In all retinal areas examined, tracer IGF-I binding is 10 to 20-fold higher than insulin binding; however, autophosphorylation levels are approximately equal.

Animals↗
PubMed1987

Retinal insulin receptors. 1. Structural heterogeneity and functional characterization.

Neural cells of the bovine retina contain specific, high-affinity receptors for insulin. When solubilized and wheat-germ purified, these receptors exhibit a kinase activity that is capable of phosphorylating the receptor's beta-subunit (autophosphorylation) and a tyrosine-containing exogenous substrate, poly (Glu, Tyr) 4:1. Studies of the structure of retinal insulin receptors revealed the existence of two insulin receptor subpopulations. For these populations, the apparent molecular weights of the alpha-subunit were 120- and 133 kDa. This structural heterogeneity does not appear to be related to the presence of vascular contamination and stands in contrast to the brain and liver where a single alpha-subunit type was found (120 kDa for brain and 133 kDa for liver). In addition to being distinguishable by their molecular weights, the two populations of retinal insulin receptors could be distinguished in terms of (a) their solubility in Triton X-100, (b) glycosylation, and (c) recognition by anti-insulin receptor antibody. Despite these structural differences, the two populations of retinal insulin receptors appear to have similar insulin binding affinities.

Animals↗
PubMed1987

Retinal insulin receptors. 2. Characterization and insulin-induced tyrosine kinase activity in bovine retinal rod outer segments.

Bovine retinal rod outer segments (ROS) possess specific, high-affinity receptors for insulin. These receptors exhibit an insulin-stimulatable tyrosine-specific activity that is capable of phosphorylating the receptor's own beta-subunit and exogenous substrate. ROS insulin receptors exhibit heterogeneity in the apparent molecular weight of the receptor's alpha-subunit. In this regard, insulin receptors from this single cell type resemble insulin receptors obtained from whole retina, but are unlike receptors from brain and liver.

Animals↗
PubMed1987

Retinitis pigmentosa with segmental massive retinal gliosis. An immunohistochemical, biochemical, and ultrastructural study.

A morphologic, immunohistologic, and biochemical study was made on the eyes of a 79-year-old woman with clinically documented retinitis pigmentosa (RP). The methods included light and electron microscopy, immunohistologic staining, and biochemical analysis of interphotoreceptor retinoid-binding protein (IRBP) and cyclic nucleotides. Results from a histopathologic examination showed marked equatorial pigmentary retinal degeneration as well as peripheral chorioretinal atrophy corresponding to areas of paving stone chorioretinal changes. An unusual finding was a localized equatorial nodule in the right eye that stained with anti-glial fibrillary acidic protein (GFAP) antibodies, and showed lipid infiltrates in its margin and base. The equatorial retina showed marked gliosis of the outer layers. Photoreceptor cells were present only in the posterior retina, macula, and focally, in the far periphery. These areas corresponded to detectable IRBP assessed by immunohistochemical staining and biochemical analysis using the enzyme-linked immunosorbent assay (ELISA). Cyclic nucleotides were reduced in the peripheral retina, in areas of photoreceptor cell loss.

Aged↗
PubMed1986

Laminin: an initiating role in retinoblastoma cell attachment and differentiation.

The effect of laminin (LN) on the attachment and differentiation of a human retinoblastoma cell line (Y-79) was investigated. We found that 10 micrograms/ml LN in the serum-free culture medium for 3 to 4 d induces 20 to 30% of the cells to firmly attach to a plastic substratum. This effect seems to be complex because the short-term effect of LN is inhibition of cell attachment. The specificity of LN may be indicated because antilaminin antibody counteracted this effect. The attached cells form small processes immediately after attachment and continue to proliferate, forming small colonies. Treatment of these cells with 4 mM dibutyryl-cyclic AMP (db-cAMP) or 2 mM sodium butyrate starting on the 3rd or 4th d of culture results in extensive differentiation of all the attached cells. Db-cAMP provokes the formation of long ramifying neuritelike processes whereas butyrate induces the appearance of epithelial-like cells with a flat morphology. Thus, laminin seems to act in concert with other agents promoting attachment and potentiating differentiation.

Antibodies↗
PubMed1986

Genetic expression of cyclic GMP phosphodiesterase activity defines abnormal photoreceptor differentiation in neurological mutants of inherited retinal degeneration.

We have examined cyclic GMP concentrations, guanylate cyclase activities, and cyclic GMP phosphodiesterase (PDE) activities in developing retinas of congenic mice with different allelic combinations at the retinal degeneration (rd) and retinal degeneration slow (rds) loci. Although guanylate cyclase activities were found to be uniformly low in the mutant retinas, striking differences in PDE activity and cyclic GMP levels were observed in retinas of the various genotypes. Homozygous rds mice, which lack receptor outer segments, showed reduced retinal PDE activity and cyclic GMP concentration in comparison to normal animals. In heterozygous rds/+ mice with abnormal outer segments, the levels were intermediate. In retinas of homozygous rd mice, PDE activity was lower than in rds retinas and cyclic GMP levels were much higher. In mice homozygous for both rd and rds genes, retinal PDE activities were even lower than in single homozygous rd mice; the cyclic GMP level reached the same high value as in the rd animals, persisted for a longer time at this high level, and did not correlate with the rate of photoreceptor cell loss. Thus, a marked variation in PDE activity appears to be the major manifestation of abnormal outer segment differentiation and eventual degeneration of photoreceptor cells in these neurological mutants. An increased cyclic GMP level seems to be an essential corollary in the expression of the rd gene even in the absence of outer segments, but it appears unlikely that an abnormally high nucleotide level in itself causes photoreceptor cell death.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1985

Dominantly inherited retinitis pigmentosa. Ultrastructure and biochemical analysis.

A 66-year-old white man had dominant retinitis pigmentosa. He developed progressive restriction of his visual field, night blindness, pallor of the optic discs, pigmentary retinopathy and posterior subcapsular cataracts. Postmortem examination of the eyes included electron microscopy and biochemical analysis of cyclic nucleotides and interphotoreceptor retinoid-binding protein (IRBP). Except for the fovea and periphery, the retina showed extensive gliosis and neuronal loss with loss of photoreceptor cells. The choriocapillaris was variably occluded in the regions of absent retinal pigment epithelium (RPE). In places, the pigment epithelium invaded the retina to the level of the internal limiting membrane. Biochemical analysis revealed that the interphotoreceptor retinoid-binding protein (IRBP), an important glycoprotein of the interphotoreceptor space, was virtually absent even in retinal areas where photoreceptor cells were still present. Cyclic nucleotide determinations indicated a decrease in the cyclic GMP concentration that reflected the general loss of photoreceptor elements. On the other hand the cyclic AMP levels in all retinal areas tested were abnormally elevated, indicating the possible involvement of this nucleotide in the pathogenesis of the disease.

Aged↗
PubMed1985

Qualitative and quantitative analysis of cytosol retinoid binding proteins in human skin.

The distribution of the cytosol retinol and retinoic acid binding proteins are known to vary greatly within the different layers of the eye, a retinoid target organ. We have analyzed the cytosol retinoid binding from adult human skin, another retinoid target organ, and examined the relative contribution of the epidermis and dermis to the total retinoid binding. The mean specific activity of [3H]retinol (0.52 +/- 0.06 pmol/mg protein) and [3H]retinoic acid (3.20 +/- 0.45 pmol/mg protein) binding to cytosol preparations from different specimens of adult human skin was determined. On the average these skins bound 7-fold more retinoic acid than retinol. When skin was treated with EDTA and separated into epidermal and dermal fractions, [3H]retinol and [3H]retinoic acid binding was found in the cytosol derived from epidermis (0.36 +/- 0.03 pmol/mg protein, 3.69 +/- 0.13 pmol/mg protein, respectively) but not from dermis. To confirm that the absence of dermal binding was not due to loss during the EDTA separation, we assayed skin keratomed at 0.1, 0.2, and 0.3 mm. The skin obtained at 0.1 mm was upper epidermis and exhibited binding for both retinol and retinoid acid. The 0.2 mm skin, which added lower epidermis but little dermal contamination, had higher specific activities for both retinol and retinoic acid binding. The 0.3 mm skin which added primarily dermis, had lower specific activities for binding both retinoids. This is consistent with the concept that the epidermis is responsible for the majority of retinoid binding in adult human skin obtained from the lower limb.

Adult↗
PubMed1984

Choroideremia: a clinical, electron microscopic, and biochemical report.

An asymptomatic 19-year-old male with choroideremia had diffuse loss of retinal pigment epithelium (RPE) and choroid except for the periphery and macula. Fluorescein angiography of the arteriovenous phase showed absence of retinal pigment epithelium and exaggerated visualization of choroidal vessels in involved areas. The mother was a typical carrier with pigment stippling of the midperipheral retina. Histopathologic examination of affected areas of one eye showed marked degeneration of the outer and midretina with loss of retinal pigment epithelium and Bruch's membrane, absence of choriocapillaris, chorioretinal adhesions and gliosis. Atrophy of inner and mid-choroid was also observed. Pigmented macrophage-like cells had migrated into the outer and midretinal layers. Electron microscopy disclosed macrophage-like cells with trilaminar structures and photoreceptor phagosomes in the RPE and outer retina. Remnants of photoreceptor outer segments were adherent to the plasma membranes of the macrophage-like cells. Biochemical analysis of retinal tissue samples for interphotoreceptor retinoid-binding protein (IRBP) showed marked reduction in the 146K bands in the equator and posterior pole in the patient compared to controls. Cyclic nucleotide content was altered in the retinal equator. Cyclic AMP was several-fold higher in the RPE-choroid complex of the affected eye than in the control.

Adult↗