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Biomedical subjects

G J Chader

Publications and source records attributed to G J Chader.

At least 163 records · Page 9Linked to original sources
PubMed1986

Interspecies conservation of structure of interphotoreceptor retinoid-binding protein. Similarities and differences as adjudged by peptide mapping and N-terminal sequencing.

Structural properties of the retinal extracellular-matrix glycolipoprotein interphotoreceptor retinoid-binding protein (IRBP) from human, monkey and bovine retinas have been compared. SDS/polyacrylamide-gel-electrophoretic analysis of limited tryptic and Staphylococcus aureus-V8-proteinase digests show virtually identical patterns for the monkey and human proteins, whereas both sets differ considerably from the bovine protein pattern. Time-course digestion shows monkey IRBP to be more readily cleaved than bovine IRBP and also cleaved to smaller fragments. Also, reversed-phase h.p.l.c. of complete tryptic digests of the IRBPs indicate that, although they have in common a similar preponderance of hydrophobic peptides, all three proteins differ extensively in their fine structure. The N-terminal sequences of monkey and bovine IRBPs have been extended beyond those presented in our previous report [Redmond, Wiggert, Robey, Nguyen, Lewis, Lee & Chader (1985) Biochemistry 24, 787-793] to over 30 residues each. The sequences yet show extensive homology, differing at only two positions, although the major monkey sequence has an additional five amino acid residues at its N-terminus ('n + 5' sequence) not observed with bovine IRBP ('n' sequence). The newly determined N-terminal sequence of human IRBP demonstrates the presence of equal amounts of the 'n' and 'n+5' sequences that are qualitatively identical with those of the monkey. The presence of the five-amino-acid-residue extension in primate, but not bovine, IRBP may indicate variation in post-translational processing.

Amino Acid Sequence↗
PubMed1986

Retinal photoreceptor neurons and pinealocytes accumulate mRNA for interphotoreceptor retinoid-binding protein (IRBP).

We have utilized cDNA probes and in situ hybridization techniques to define the subcellular localization of interphotoreceptor retinoid-binding protein (IRBP) mRNA in bovine and monkey retinas. Results suggest that the mRNA is mainly localized in rod photoreceptor neurons within the outer nuclear layer of the retina. IRBP mRNA is also abundant in cells of the pineal gland, strengthening the analogy between rod photoreceptor cells and pinealocytes.

Animals↗
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

In vivo cGMP levels in frog photoreceptor cells as a function of light exposure.

By employing a combination of highly sensitive radioimmunoassays and histochemical techniques, an in vivo time course of cGMP levels has been determined in the outer segment, photoreceptor cell and outer plexiform layers of frog retina. Frogs (Rana pipiens) were dark-adapted overnight and either frozen rapidly (approximately 3 sec) in liquid nitrogen or exposed to periods of light varying between 0.1 sec and 2 hr before freezing. Frozen retinal sections were cut, freeze-dried, and samples of individual layers dissected out and analysed for cGMP. In the outer plexiform layer, there was a 42% drop in cGMP concentration after 2 sec of light (250 ft candles) followed by a 34% rise after 2 min; a steep concentration gradient formed around the layer after the 2 min exposure. In both the outer-segment layer and photoreceptor-cell layer (which includes outer segments, inner segments and outer nuclear layers), cGMP levels declined from a dark value of 56 mumol kg-1 (dry) to 9 mumol kg-1 (dry) as a result of increasing exposure to several types of light source: levels appear to be primarily a function of total ft candle min. Cyclic GMP concentrations at the longest exposures (2 min with a fiber optic light source or 2 hr with fluorescent room light) reached identical minimum levels. In the outer segments, a 15% decrease in cGMP was observed after 0.1 sec of light exposure. Although the freezing time is too long to be able to say whether the 15% decrease in cGMP at the 0.1 sec exposure is involved in transduction, the low identical levels reached gradually after longer exposures appear to indicate that a light-induced biochemical adjustment in cGMP metabolism occurs over a relatively long time period separate from the msec time course of the transduction process.

Animals↗
PubMed1986

Retinoblastoma. A clinical, immunohistochemical, and electron microscopic case report.

A 4-year, 9-month-old boy had a history of leukocoria of the right eye for approximately six months prior to admission. The other eye was normal. There was no family history of retinoblastoma. Funduscopy disclosed a large white mass extending from the nasal pars plana to the mid-pupillary zone and the posterior pole with a near total retinal detachment in the superior temporal quadrant. A B-scan ultrasound showed an echo dense area of the anterior portion of the mass. A CT scan showed intraocular tissue densities with no evidence of optic nerve involvement or extraocular extension. Immunohistochemistry of a fresh frozen portion of tumor revealed reactivity with antibodies directed against interphotoreceptor retinoid-binding-protein (IRBP), neuron-specific enolase, glial fibrillary acidic protein, S-antigen, focal reactivity with opsin, and scattered cytoplasmic staining for cyclic GMP and cyclic GMP phosphodiesterase. Biochemical analysis of fresh frozen tissue samples confirmed the presence of IRBP. Transmission electron microscopy disclosed occasional Flexner-Wintersteiner rosettes connected by zonula adherens-like junctions. These showed inner segment-like structures containing prominent mitochondria, portions of cilia and fragments of outer segment material. These data, along with the immunocytochemistry indicates a predominant neuronal nature of the tumor cells with significant photoreceptor-like differentiation.

Child, Preschool↗
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↗
PubMed1986

Human Y-79 retinoblastoma cells exhibit specific insulin receptors.

The presence of insulin receptors was investigated in human Y-79 retinoblastoma cells grown in suspension culture. The binding of [125I] insulin to these cells was time, temperature, and pH dependent, was competed for by insulin and proinsulin but not other peptides, and was inhibited by antibodies against the insulin receptor. The Scatchard plot of insulin competition data was curvilinear and was resolved into a high-affinity (KD approximately 0.5 X 10(-9) M)/low-capacity (approximately 3,000 sites/cell) and a low-affinity (KD approximately 1 X 10(-7) M)/high-capacity (approximately 155,000 sites/cell) component. Negative cooperativity was not found, in agreement with other studies in rodent neural cells. However, in contrast to studies with rodent cells, insulin specifically down-regulated its receptor on human Y-79 cells after prolonged exposure. In conclusion, these data show for the first time the presence of specific insulin receptors in human Y-79 retinoblastoma cells. Because these cells were previously shown to have several characteristics typical of neural cells, we propose their use as a model to study the effects of insulin on neural and retinal tissues of human origin.

Cell Line↗
PubMed1986

Retinoblastoma: a primitive tumor with multipotential characteristics.

The histogenesis of retinoblastoma, the most common intraocular tumor of childhood, has been recently attributed to a primitive retinal cell which is capable of differentiation into both neuronal and glial elements. By manipulating the culture medium constituents and using differentiating agents, such as dibutyryl-cyclic AMP and butyrate, we have now been able to differentiate a population of Y-79 human retinoblastoma cells into cells of pigment epithelial character, as well as into cells of neuronal or glial nature. These studies additionally implicate the outer layer of the optic vesicle in retinoblastoma etiology, and introduce a new system for studying the biologic properties of primitive neural tumors as well as the embryonic development of the retina and central nervous system.

Bucladesine↗
PubMed1986

Differentiation of human retinoblastoma in vitro into cell types with characteristics observed in embryonal or mature retina.

The capacity of a primitive human retinoblastoma cell line (Y-79) to differentiate into several cell types of normal human retina was investigated. Cells were studied in suspension and monolayer cultures, in serum-free or serum-supplemented medium, and in the presence or absence of differentiating agents such as N6O12-dibutyryl adenosine 3',5'-cyclic monophosphate (dbc-AMP) and sodium butyrate (Nabut). Electron microscopy, immunohistochemistry for detection of myelin basic protein (MBP), and formaldehyde-induced fluorescence (FIF) for catecholamines were performed. Treated cells exhibited morphologic characteristics supportive of differentiation toward photoreceptors, conventional neurons and glial cells, increased FIF reactivity, and MBP expression. Growth in serum-free medium without differentiating agents led to a similar but less enhanced morphologic differentiation. These results confirm the concept that human retinoblastoma originates from a primitive neuroectodermal multipotential cell.

Bucladesine↗
PubMed1986

Retinitis pigmentosa: immunohistochemical and biochemical studies of the retina.

Several immunohistochemical and biochemical characteristics of retinas from two patients with retinitis pigmentosa (RP) were examined. Histochemically, interphotoreceptor retinoid-binding protein (IRBP) was found in the interphotoreceptor space in small areas of preserved peripheral retina, although enzyme-linked immunosorbent assay suggested that little IRBP was present. Antibodies to glial fibrillary acidic protein and factor VIII-related antigen corroborated retinal gliosis and retinal vessel attenuation, respectively, in the RP cases. Fibronectin showed similar staining in normal and affected tissue.

Aged↗
PubMed1986

Uveoretinitis and pinealitis induced by immunization with interphotoreceptor retinoid-binding protein.

Rats immunized with microgram amounts of interphotoreceptor retinoid-binding protein (IRBP), a glycoprotein which localizes specifically in the eye and pineal gland, developed uveoretinitis and pinealitis. The severity and onset of changes were found to be dose-related and to be enhanced by B. pertussis bacteria. In general, the inflammatory changes induced by IRBP resembled those provoked by S-antigen (S-Ag), but significant differences were noted between the two diseases. The possible usefulness of the new experimental autoimmune disease is discussed.

Animals↗
PubMed1986

Distribution of the regulatory subunit of type II cAMP-dependent protein kinase in Y-79 retinoblastoma cells. Effect of butyrate.

An indirect immunofluorescence technique was employed to determine the intracellular distribution of the regulatory subunit of type II cyclic adenosine 3':5'-monophosphate-dependent protein kinase (RII) in Y-79 human retinoblastoma cells growing in monolayer culture. During the initial phases of growth (6 hr-6 days after seeding), RII was confined to the cytoplasmic areas of the Y-79 cells, seemingly, the Golgi apparatus. Treatment of cells for 3-5 days with 4 mM butyrate resulted in translocation of RII from cytoplasm to nuclei (mainly nucleoli) of cells. In a later stage of growth (24-day-old cultures), RII immunofluorescence was significantly decreased in all compartments within the untreated cells. In contrast, about 70% of the butyrate-treated cells yet showed nucleoli and/or cytoplasmic localization of RII at this stage. The nucleolar appearance of RII was parallel to the growth arrest and differentiation induced by butyrate.

Biological Transport↗
PubMed1986

Immunochemical distribution of interphotoreceptor retinoid-binding protein in selected species.

An enzyme-linked immunosorbent assay (ELISA) was used to quantitate interphotoreceptor retinoid-binding protein (IRBP) in tissues of monkey and other species using rabbit antiserum against monkey IRBP. A 1:7500 antiserum dilution was found optimal with the linear range extending to 250 micrograms IRBP/ml. The highest IRBP concentration was found in cannulation fluid of the monkey interphotoreceptor space, although vitreous and aqueous humors also contained IRBP. The presence of IRBP in the vitreous was confirmed by Western blot and 3H-retinol binding studies. The pineal gland of monkey and rat also was found to contain IRBP, as assessed by ELISA and immunocytochemistry; IRBP was below the limits of detection in turtle and chicken retina. IRBP levels were uniformly low in retinas of human cases with hereditary retinal degeneration, including retinitis pigmentosa (three cases) and choroideremia (two cases). The presence of IRBP in pineal as well as in the vitreous and aqueous humors may indicate a broader role for this putative retinoid-transport protein than previously suspected.

Animals↗
PubMed1985

Endogenous fatty acids are covalently and noncovalently bound to interphotoreceptor retinoid-binding protein in the monkey retina.

Interphotoreceptor retinoid-binding protein (IRBP) purified from monkey interphotoreceptor matrix contains relatively high concentrations of endogenous fatty acids, 6.51 mol/mol of protein. Sixty-five percent of the total fatty acid bound to IRBP was found to be noncovalently attached, with the remainder covalently bound. The fatty acids are not residual components of phospholipids or neutral lipids, as judged by microchemical methods. The major fatty acids bound to IRBP are: palmitic (35%), stearic (21%), palmitoleic (7%), oleic (29%), linoleic (6%) and docosahexaenoic acids (2%). These fatty acids account for about 90% of the total fatty acid bound to interphotoreceptor matrix proteins extracted with organic solvents. Thus, IRBP may function as an intercellular fatty acid carrier and may depend on the covalently bound fatty acids for anchoring in the outer leaflet of cell membranes.

Animals↗
PubMed1985

cDNA clones encoding bovine interphotoreceptor retinoid binding protein.

We have isolated a cDNA clone (lambda IRBP-1) for bovine interphotoreceptor retinoid-binding protein (IRBP) by immunological screening of a bovine retinal lambda gt11 cDNA expression library. This clone contained a cDNA insert 325 bp in length. A 250 bp fragment of this cDNA was used to screen a bovine retina lambda gt10 cDNA library, resulting in the isolation of two larger cDNA clones containing inserts of 2.5 kb (lambda IRBP-2) and 1.5 kb (lambda IRBP-3). Restriction endonuclease mapping revealed all three clones to have an EcoR I restriction site. The 250 bp fragment of lambda IRBP-1 and the 2000 bp fragment of lambda IRBP-2 both hybridized to a single bovine retinal mRNA species approximately 8 kb in length; there was no hybridization with either chicken lens or liver RNA. The amino acid sequence of a tryptic peptide from authentic IRBP has been obtained. The deduced amino acid sequence from the cDNA nucleotide sequence is the same as this authentic peptide. This definitively establishes the identity of the cDNA clones as encoding bovine IRBP.

Amino Acid Sequence↗
PubMed1985

Characteristics of protein tyrosine kinase activities of Y-79 retinoblastoma cells and retina.

Protein tyrosine kinase activities were determined in retina and Y-79 retinoblastoma cells. Kinase activity was associated with particulate subcellular fractions. Specific activities were similar in both retina and Y-79 cells; apparent Km values for ATP and casein were also similar. Retinoblastoma-derived growth factor stimulated endogenous protein tyrosine phosphorylation in Y-79 cells significantly more than in retina. Differences in protein tyrosine phosphorylation between retina and Y-79 retinoblastoma appear to be due to differences in regulation of the activity by the growth factor or differences in the endogenous protein substrates present in the Y-79 cells.

Animals↗
PubMed1985

Extracellular cGMP phosphodiesterase related to the rod outer segment phosphodiesterase isolated from bovine and monkey retinas.

A phosphodiesterase (PDE) has been characterized in the interphotoreceptor matrix (IPM) of light-adapted fresh bovine retinas. It is obtained through a gentle rinsing of the retinal surface under conditions where the light-activated rod outer segment (ROS) enzyme remains attached. The enzyme has an apparent native molecular weight of 350 000 by gel filtration and appears as a doublet at Mr 47 000 and 45 000 on sodium dodecyl sulfate-polyacrylamide gels. It has an apparent Km value for cGMP of 33 microM and an apparent Km value for cAMP of 2200 microM. It is activated 3-6-fold by protamine and over 40-fold by trypsin. Protamine has no effect on the Km for cGMP while trypsin decreases the Km for cGMP by a factor of 2. The enzyme occurs in at least two forms as evidenced by two distinct peaks of activity after gel electrophoresis under nondenaturing conditions. A heat-stable inhibitor is tightly bound to the enzyme. The inhibitor obtained from the IPM PDE inhibits 98% of the activity of the trypsin-activated ROS PDE: conversely, the inhibitor obtained by boiling the ROS PDE completely inhibits the trypsin-activated IPM enzyme. A high-affinity monoclonal antibody to the active site of the ROS PDE, ROS 1 [Hurwitz, R., Bunt-Milan, A.H., & Beavo, J. (1984) J. Biol. Chem. 259, 8612-8618], quantitatively absorbs the IPM PDE. These observations indicate a clear relationship between these two PDEs even though their location, sizes, and specific functions in the retina appear to be distinct.

3',5'-Cyclic-GMP Phosphodiesterases↗