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G J Chader

Publications and source records attributed to G J Chader.

At least 127 records · Page 7Linked to original sources
PubMed1989

Rat T-cell lines specific to a nonimmunodominant determinant of a retinal protein (IRBP) produce uveoretinitis and pinealitis.

Rat lymphocyte lines were established, with specificity toward two synthetic peptides derived from the interphotoreceptor retinoid-binding protein (IRBP), which specifically localizes in the retina and pineal gland. One of the peptides, R4, is immunopathogenic, producing experimental autoimmune uveoretinitis (EAU) and pinealitis (EAP) in immunized rats, while the other peptide, R3, exhibits no detectable immunopathogenicity in rats. The cell lines carry surface markers specific for the helper/inducer subset of T-lymphocytes. When tested by the proliferation assay, the line cells demonstrated major histocompatibility-restricted vigorous responses against the immunizing (homologous) peptide, but failed to recognize the intact IRBP molecule. This finding is in line with other data indicating that peptides R3 and R4 are nonimmunodominant determinants of IRBP for the Lewis rat. Yet, the cell lines specific for R4 were highly immunopathogenic, producing EAU and EAP in naive rats at numbers as low as 0.25 x 10(6), with histopathological changes similar to those induced by active immunization with this peptide. The immunological capacity of the cell lines was further demonstrated by the finding that spleen cells from recipient rats of these lines responded well against the homologous peptides. The uniqueness of this system, in which lymphocytes specific toward a nondominant determinant are immunopathogenic, is underscored and the possible mechanisms of disease induction are discussed.

Animals↗
PubMed1989

Y-79 retinoblastoma cells: isolation and characterization of clonal lineages.

Y-79 cells are generally undifferentiated, but can be induced to express characteristics of various retinal cell types. Since specific changes occur in only low percentages of cells, it has not been clear whether different agents select different lineages, or if the same cells are differentially responsive. In order to answer this question, we isolated 16 clonal lineages and characterized their responses to various treatment regimes. Dibutyryl cyclic AMP (4 mM) had little effect on cell viability, but treatment with butyrate (1 mM), caused mortality ranging from 20 to 84%. Laminin (100 micrograms ml-1) did not decrease cell growth, and in some cases increased it. The frequency of attachment to a laminin-coated substrate also varied between clones. Comparison of the results for each clone, under all these conditions, indicated that the parental Y-79 line contains at least two different cell types. On the other hand, no obvious differences were found between clones in the morphological changes induced by different agents, evidencing the very plastic nature of Y-79 cells.

Bucladesine↗
PubMed1989

Abnormal expression of the RI subunit of cyclic AMP-dependent protein kinase in Y-79 retinoblastoma cells.

The cyclic AMP-dependent protein kinases of Y-79 retinoblastoma cells were analyzed by DEAE Sephacel column chromatography, SDS-polyacrylamide gel electrophoresis, and immunoblotting. Both type I and type II protein kinases (characteristic of fetal and mature retina respectively) were observed. In addition, free RI (type I regulatory subunit) was present in Y-79 cells; three other retinoblastoma cell lines also showed evidence of free RI in immunoblot analysis. Northern blot analysis of poly A+-enriched RNA from Y-79 cells showed a 4.5 kb RI transcript and a 6.0 kb RII transcript. The single 4.5 kb RI message is typical of normal retina, in contrast to other tissues and cell lines in which a 2.0 kb message predominates. Since RI expression appears to be post-transcriptionally inhibited in normal retina, it is suggested that free RI dimers in retinoblastoma is the result of loss of repression of translation of this transcript and that the free RI pool may contribute to the unregulated growth of these cells.

Cell Line↗
PubMed1989

Developmental expression of the RI subunit of cyclic AMP-dependent protein kinase in retina.

Type I and type II isoenzymes of cyclic AMP-dependent protein kinase were identified by anion exchange chromatography and immunoblotting in human retina at various stages of development. The type I form was typical of the early fetal stage during which proliferation and migration were the primary cellular processes. In later stages of differentiation and maturation, RI was no longer detected: the RII isoform, which appeared at the end of the proliferative phase, being the only isoform seen in mature retina, with a doubling of the specific activity of cyclic AMP-dependent protein kinase. Northern blot analyses showed that an RI transcript of 4.5 kb was present not only in fetal but also in mature human retina. The RI transcript was also found in fetal and adult bovine retina and in adult monkey retina. In vitro translation of mature retinal RNA produced a protein identical in size to authentic RI which was precipitated by anti-RI antiserum. Expression of RI in retina thus appears to be regulated during development. The presence of the mRNA for the RI subunit in the adult retina suggests that the regulation might be post-transcriptional.

Animals↗
PubMed1989

mRNA for interphotoreceptor retinoid-binding protein (IRBP): distribution and size diversity in vertebrate species.

Northern blots of total retinal RNA from a number of different vertebrate species were probed with a cDNA fragment corresponding to translated portions of bovine IRBP mRNA. A hybridizing band was detected in normal human retina (4.6 kb), Y-79 human retinoblastoma cells (4.4 kb), and in retinas of monkey (4.6 kb), guinea-pig (4.9 kb), mouse (6.1 kb), rat (two bands at 5.4 kb and 6.6 kb), rabbit (6.3 kb), cow (6.5 kb), and hamster (7.6 kb). Thus, the IRBP gene is expressed in the retinas of a wide variety of mammalian species. The mRNAs could be readily detected in about 20 micrograms of total RNA, suggesting that, in these species, IRBP message is relatively abundant. In contrast, only very weak hybridization was detected on northern blots of the three bird species examined (chicken, duck, quail). IRBP mRNA is thus relatively well expressed in mammals, but not in birds.

Animals↗
PubMed1989

Immune responses to peptides derived from the retinal protein IRBP: immunopathogenic determinants are not necessarily immunodominant.

Interphotoreceptor retinoid-binding protein (IRBP), a glycoprotein specific to the retina and pineal gland, induces in immunized rats inflammatory changes in these organs (EAU and EAP, respectively). We report here on the immunological activities in Lewis rats of 10 IRBP-derived peptides. Only one of these peptides (R3) was found to induce high levels of antibodies in immunized rats, as detected by ELISA. On the other hand, the majority of the tested peptides stimulated substantial cellular immune responses, measured by the lymphocyte proliferation assay. None of the tested peptides were recognized, however, by antibodies or lymphocytes from rats immunized with the whole IRBP molecule, thus indicating that these synthetic peptides are nonimmunodominant in the Lewis rat. Two of these peptides, R4 and R9 (which contains R4), were previously found to be immunopathogenic, producing EAU and EAP in immunized Lewis rats. The immune responses to peptide R4 were further examined and the data show that it induces measurable lymphocyte responses only when injected at remarkably high doses (greater than or equal to 67 microgram/rat). Yet, peptide R4 was highly antigenic when tested for stimulation of specifically sensitized lymphocytes in culture. Furthermore, lymphocytes sensitized against R4 exhibited high capacity to adoptively transfer EAU and EAP to naive recipients. The finding of immunopathogenic but nonimmunodominant peptides is discussed.

Amino Acid Sequence↗
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↗
PubMed1989

Interphotoreceptor retinoid-binding protein: role in delivery of retinol to the pigment epithelium.

The ability of interphotoreceptor retinoid-binding protein (IRBP) to facilitate the incorporation of retinol into retinyl esters by the retinal pigment epithelium (RPE) was examined in toad (Bufo marinus) eyecup preparations devoid of neural retina (RPE-eyecup). Solutions containing purified bovine IRBP and all-trans[3H]retinol were introduced into the vitreal cavity of the RPE-eyecup. After incubation at 22 degrees C, [3H]retinyl ester was extracted from the RPE cells and isolated by high performance liquid chromatography. All-trans[3H]retinyl ester formed in the RPE increased with time of incubation (up to 2 hr) and with concentration of IRBP (up to 10 microM). The increase with IRBP concentration accompanied, and presumably resulted from, an increased transfer of [3H]retinol to the RPE-eyecup. With higher concentration of IRBP (20-30 microM), both the amount of [3H]retinyl ester formed (relative to the peak value at 10 microM IRBP) and the overall molar content of endogenous retinyl ester were reduced. On the other hand, bovine serum albumin at relatively high concentration (90 microM) was less effective than 3 microM IRBP in supporting the formation of [3H]retinyl ester, and it did not reduce the level of native retinyl ester in the RPE. Using 3 microM IRBP, levels of [3H]retinyl ester formed were comparable to or exceeded those obtained with phosphatidyl choline (0.9 mg ml-1) or serum retinol-binding protein (3 microM). The data are consistent with the hypothesized role of IRBP as a carrier of retinol between the retina and RPE in the operation of the visual cycle.

Animals↗
PubMed1989

Retinoid requirements for recovery of sensitivity after visual-pigment bleaching in isolated photoreceptors.

After visual-pigment bleaching, single isolated rod photoreceptors of Ambystoma tigrinum recover their sensitivity to light when supplied with 11-cis-retinal from liposomes or with 11-cis-retinal bound to interphotoreceptor retinoid-binding protein. Bleached rods do not recover sensitivity, or do so only very slowly, after exposure to 11-cis-retinol. The latter retinoid is "toxic" in that rods actually lose sensitivity in its presence. In contrast, bleached isolated cone cells recover sensitivity when either retinoid is supplied. It is suggested that the major pathway for rhodopsin regeneration during dark adaptation in the intact eye is transport of 11-cis-retinal from the pigment epithelium to the retina. The results also suggest that there may be separate pathways for visual-pigment regeneration in rods and cones during dark adaptation.

Ambystoma↗
PubMed1989

Postnatal development of photoreceptor proteins in mutant mice and Abyssinian cats with retinal degeneration.

Using immunocytochemical techniques, development of opsin, transducin alpha and S-antigen in photoreceptor cells of the mice homozygous or heterozygous for the rd or rds genes has been found to be similar to that of control animals during the first postnatal week. Even though the absolute amounts of these proteins are low (eg. opsin in the rds retina) or decrease in the postnatal period (as in the rd retina), we can demonstrate their persistence during the entire degeneration process. In fact, the content of the proteins in the photoreceptor perikarya actually appear to be higher after postnatal day 11 in all mutants studied. Thus, one of the major manifestations of the mutant retinae is a loss of polarity of the photoreceptor cells at the time of ROS degeneration without a loss in the ability to synthesize these important proteins of the visual cycle. This correlates well with the apparent defect in IRBP secretion and its intracellular accumulation in mutant photoreceptor cells as previously observed (van Veen et al. 1986). In the Abyssinian cat model for progressive retinal atrophy, the development an cellular distribution of all the proteins studied are similar in affected and control retinae until the beginning of stage 2 of the disease. At this time, outer segments begin to degenerate and immunoreactivity increases in the photoreceptor perikarya. The IRBP content of the retina declines markedly at stage 2, preceding extensive loss of photoreceptors.

Aging↗
PubMed1988

Anomalous insulin-binding activity in the bovine neural retina: a possible mechanism for regulation of receptor binding specificity.

Crude membrane from the bovine neural retina contains one IGF-I and two insulin binding sites. Although both insulin binding sites have a high affinity for insulin (IC50 = 0.1 and 7.0 nM), only one exhibits "classical" specificity and binds insulin with higher affinity than IGF-I. The second insulin binding site is "non-classical" in that it has an equal affinity for IGF-I and insulin. Retinal IGF-I binding exceeds insulin binding by a factor of 10-20. Despite this high level of IGF-I binding it is unlikely that non-classical insulin binding represents insulin binding to an IGF-I receptor because 1) anomalous binding is 30 times greater than that predicted from cross-specificity, 2) low concentrations of unlabeled IGF-I increase IGF-I binding to the IGF-I binding site but do not increase IGF-I binding to the non-classical insulin binding site and 3) the IGF-I receptor's affinity for insulin (and IGF-I) increases greatly during receptor purification. In contrast, the insulin affinity of the non-classical insulin binding site is largely unaffected by this process. Although receptor solubilization and purification had no effect on the insulin receptor's affinity for insulin, it did markedly increase this site's affinity for IGF-I. Thus, the major proportion of purified retinal "insulin receptors" have a higher affinity for IGF-I than insulin. The evidence presented here is consistent with the view that the bovine retina contains one IGF-I and two insulin binding sites and that a detergent-sensitive factor regulates IGF-I affinity of both classes of binding sites.

Animals↗
PubMed1988

Coexpression of neuronal, glial, and major histocompatibility complex class II antigens on retinoblastoma cells.

This study identifies the presence of major histocompatibility complex class II antigens on retinoblastoma cells. In addition, the modulation of HLA-DR by interferon-gamma as well as the preferential expression of this major histocompatibility complex molecule over HLA-DQ is described. Double labeling experiments revealed that HLA-DR antigen is shared concomitantly with cells of glial and neuronal character. Investigations such as these underscore the possibility that expression of major histocompatibility complex class II antigens may function as immunological components in the host or play a role in the cellular differentiation of these tumor cells.

Antigens↗
PubMed1988

Retinal insulin receptors: localization using a polyclonal anti-insulin receptor antibody.

Although retinal insulin receptors have recently been described biochemically, the location of these receptors within the retina is unknown. The study presented here used a polyclonal anti-insulin receptor antibody (B10), immunofluorescence and immunoelectron microscopy to determine the location of insulin receptors in bovine, monkey and human retina. It was found that antibody immunofluorescence formed discrete bands localized predominantly to photoreceptor and neuronal cell bodies. In addition to the strong association with neuronal perikarya, a lower level of antibody binding was observed in photoreceptor outer segments. In human retina, some of the antibody immunofluorescence also had a pattern that suggested B10 binding to glial-like cells.

Animals↗
PubMed1988

A new model of autoimmune disease. Experimental autoimmune uveoretinitis induced in mice with two different retinal antigens.

Experimental autoimmune uveoretinitis (EAU) is an organ-specific, T lymphocyte-mediated autoimmune disease, which serves as a model for several human ocular inflammations of an apparently autoimmune nature. EAU pathology in some rodents and in monkeys can readily be induced by immunization with several different retinal proteins; however, advancing research into the cellular mechanisms of this disease has raised the need for an EAU model in an immunologically and genetically well defined species. We report here the induction of EAU in the mouse, which has hitherto been considered a species refractory to EAU, with two retinal Ag, the retinal soluble Ag and the interphotoreceptor retinoid-binding protein. Although all the mouse strains tested exhibited lymphocyte responses and antibody titers to both retinal Ag, EAU was inducible in only some of the strains, and the uveitogenic responses to retinal soluble Ag and interphotoreceptor retinoid-binding protein appeared to be mutually exclusive. The EAU model in mice was found to differ in several respects from the EAU model in other rodent species. Induction of the disease was achieved with a relatively high dose of Ag and an intensified immunization protocol, and the onset of disease was later, the duration was longer, and the course was less acute. Anterior segment involvement was slight or nonexistent, and damage to the retina and uvea was of a focal rather than of a diffuse nature. Murine EAU appeared to approximate some types of human uveitis more closely than the EAU models described in other rodent species with respect to its pathologic manifestations as well as its more chronic course. The relatively longer duration of the active stage of disease in murine EAU should facilitate therapeutic intervention in established disease, which was not feasible in the more acute models of EAU. The extensive knowledge of the immunologic parameters of the mouse and the availability of genetically defined strains should be of great value in the study of cellular mechanisms and immunogenetics of ocular autoimmune disease.

Animals↗
PubMed1988

Interphotoreceptor retinoid-binding protein levels in subretinal fluid from rhegmatogenous retinal detachment and retinopathy of prematurity.

Interphotoreceptor retinoid-binding protein (IRBP) has been detected qualitatively in subretinal fluid associated with rhegmatogenous retinal detachments. We have used a quantitative enzyme-linked immunosorbent assay to determine the IRBP concentrations in 24 fluid specimens collected at retinal reattachment surgery from 22 patients, and from 17 patients who underwent surgery for retinopathy of prematurity with traction detachment. We observed a wide range of concentrations (1 to 176 micrograms of IRBP per milligram of protein). Higher concentrations were found only in fluid from the more recent rhegmatogenous detachments. The lowest concentrations were detected among fluids drained from under rhegmatogenous detachments ranging from five days' to one year's duration. The IRBP concentration in subretinal fluid showed no correlation with visual outcome and may be influenced by IRBP turnover in the vitreous cavity. Most strikingly, none of the 17 samples from patients with retinopathy of prematurity contained detectable amounts of IRBP.

Adult↗
PubMed1988

A developmental study of interphotoreceptor retinoid-binding protein (IRBP) in single and double homozygous rd and rds mutant mouse retinae.

Interphotoreceptor retinoid-binding protein (IRBP) was studied using immunochemical and immunocytochemical techniques in retinae of mice with allelic combinations at the rd and rds loci at different stages of development and degeneration. Until postnatal day 7 (P7), IRBP is located intracellularly in developing retinae of the different genotypes. Thereafter, IRBP is present mainly in the interphotoreceptor matrix. As previously noted, cell death is slowest in the heterozygous +/+,rds/+ mutant with loss increasing in order in +/+,rds/rds, rd/rd, rds/rds and rd/rd,+/+ animals. The IRBP content of the total retina also approximates this pattern, with lowest amounts by far in rd/rd, rds/rds and rd/rd,+/+ mutants (after P14). Interestingly though, IRBP loss significantly precedes visual cell loss in the rd/rd,rds/rds retina. In all the mutants, the remaining rod cells in the outer nuclear layer exhibit synthesis of intracellularly located IRBP at late stages of degeneration. In the single homozygous rd/rd,+/+ and the double homozygous rd/rd,rds/rds mutants, IRBP is present intracellularly during the entire degenerative process with somewhat less intracellular IRBP in the rd/rd,rds/rds mutant. Retinae of homozygous +/+,rds/rds and heterozygous +/+,rds/+ animals exhibit a normal distribution pattern of IRBP immunoreactivity until loss of photoreceptor cells becomes pronounced at later stages of the disease. Many of the remaining cells at this time are probably cone elements although they are structurally changed. Double labeling with IRBP and S-antigen demonstrates, in many but not all, the presence of both proteins in the same cell body. Immunocytochemistry clearly demonstrated the presence of IRBP in remaining photoreceptor cells at late stages of the disease. Thus, the biochemically measured loss of IRBP appears to be a complex process neither directly dependent on the loss of photoreceptor outer segments and reduced interphotoreceptor matrix space (e.g. there is a sustained IRBP level in rodless rds mutants) nor simply due to cell death (e.g. in the rd/rd,rds/rds mutant, IRBP loss significantly precedes cell loss). That this IRBP is mainly intracellular, however, may indicate an abnormality in secretion which, combined with other factors, induces a degenerated and less differentiated phenotype.

Animals↗
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↗