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Biomedical subjects

G J Chader

Publications and source records attributed to G J Chader.

At least 73 records · Page 4Linked to original sources
PubMed1993

Interphotoreceptor retinoid-binding protein (IRBP). Molecular biology and physiological role in the visual cycle of rhodopsin.

The regeneration of visual pigment in rod photoreceptors of the vertebrate retina requires an exchange of retinoids between the neural retina and the retina pigment epithelium (RPE). It has been hypothesized that interphotoreceptor retinoid-binding protein (IRBP) functions as a two-way carrier of retinoid through the aqueous compartment (interphotoreceptor matrix) that separates the RPE and the photoreceptors. The first part of this review summarizes the cellular and molecular biology of IRBP. Work on the IRBP gene indicates that the protein contains a four-fold repeat structure that may be involved in binding multiple retinoid and fatty acid ligands. These repeats and other aspects of the gene structure indicate that the gene has had an active and complex evolutionary history. IRBP mRNA is detected only in retinal photoreceptors and in the pineal gland; expression is thus restricted to the two photosensitive tissues of vertebrate organisms. In the second part of this review, we consider the results obtained in experiments that have examined the activity of IRBP in the process of visual pigment regeneration. We also consider the results obtained on the bleaching and regeneration of rhodopsin in the acutely detached retina, as well as in experiments testing the ability of IRBP to protect its retinoid ligand from isomerization and oxidation. Taken together, the findings provide evidence that, in vivo, IRBP facilitates both the delivery of all-trans retinol to the RPE and the transfer of 11-cis retinal from the RPE to bleached rod photoreceptors, and thereby directly supports the regeneration of rhodopsin in the visual cycle.

Animals↗
PubMed1993

Differential regulation of arylamine and arylalkylamine N-acetyltransferases in human retinoblastoma (Y-79) cells.

For the first time, arylamine and arylalkylamine N-acetyltransferase (NAT) activities are shown to be differentially regulated. In a human retinoblastoma (Y-79) cell line, arylalkylamine NAT activity, but not arylamine NAT activity increased (3-5-fold) rapidly (1-3 h) in response to treatment with dibutyryl cAMP. In contrast, treatment with butyrate showed a delayed (3-5 days) increase (3-5-fold) in arylamine NAT activity but not in arylalkylamine NAT activity. The differential response to these agents in Y-79 cells provides a model system for future studies on the regulatory relationship between the two enzyme activities.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1993

Modulation of retinoblastoma cell characteristics by hexamethylene bis-acetamide and other differentiating agents in culture.

The undifferentiated Y-79 retinoblastoma cell line can be induced by specific agents to express characteristics of mature retinal cells. In the present study, attached Y-79 cell cultures were treated with hexamethylene bis-acetamide (HMBA) and other differentiating agents and examined for "neuronal" and other properties. Immunocytochemical staining was performed with antibodies against neuron- and retina-specific antigens, [synaptophysin, interphotoreceptor retinoid-binding protein (IRBP), neural cell adhesion molecule (N-CAM), and rod- and cone-specific transducin (TR alpha and TC alpha)] and microtubule-associated protein (MAP-1) and tubulin. Enhanced expression of tubulin was observed with cAMP treatment in FBS media. Expression of N-CAM was observed in all groups. Morphological differentiation was pronounced with HMBA and butyrate treatment, with HMBA inducing increased tubulin expression after 2 weeks of treatment. Expression of TR alpha was minimal under all culture conditions, whereas TC alpha was ubiquitously expressed. This supports the concept that Y-79 retinoblastoma is predominantly of cone neuronal origin and that, surprisingly, immunocytochemical differentiation is not correlated with the marked morphological changes induced by the major differentiating agents used.

Acetamides↗
PubMed1993

Human retinoblastoma cells express alpha B-crystallin in vivo and in vitro.

alpha B-crystallin is a major lens protein that is a member of the heat-shock family of proteins. Using immunohistochemical and northern blot techniques, we now demonstrate its presence in freshly-fixed retinoblastoma tissue. The protein is also abundantly expressed in cultured human retinoblastoma cells (Y-79 NEI, WERI Rb-1) as well as two subcultured Y-79 lines (ATCC and GM01232C). High expression of alpha B-crystallin may be involved in tumor growth and/or be a marker for general oncogenic "stress" in the tumor tissue.

Animals↗
PubMed1993

Identification and characterization of arylamine N-acetyltransferase activity from the bovine retinal pigment epithelium.

Arylamine N-acetyltransferase (NAT) activity was identified and characterized in bovine retinal pigment epithelium (RPE) cells. Upon examining the RPE supernatant for multiple ionic species, one major NAT activity peak was detected. Based upon its substrate specificity, it is best described as an arylamine NAT. However, there was detectable arylalkylamine NAT activity within this peak. Further purification via size-exclusion HPLC revealed multiple peaks of NAT activity, although the major peak (around 30 kDa) again predominantly exhibits arylamine NAT activity. However, substrate specificity studies indicate that this arylamine NAT activity is able to acetylate specific arylalkylamine substrates. This arylamine NAT demonstrates a monomorphic pattern of acetylation since it acetylates rho-aminobenzoic acid rather than sulfamethazine. It also demonstrates a low sensitivity to methotrexate inhibition indicated by the high IC50 value (570 microM). The mode of inhibition by methotrexate is uncompetitive as demonstrated by kinetic analysis.

Acetylation↗
PubMed1992

Matrigel promotes retinoblastoma cell growth in vitro and in vivo.

Cells derived from retinoblastomas grow slowly in vitro and only very rarely form tumors in nude mice. Matrigel, a mixture of components normally found in basement membranes, promotes the growth of Y-79 and WERI-Rb1 retinoblastoma (Rb) cells when added to suspension cultures of the 2 Rb cell lines. It also substantially increases cell adhesion in vitro. Y-79 cells, seeded into a Matrigel matrix, form round colonies over a 3-week period similar to those of control, weakly metastatic murine melanoma cells. In vivo, s.c. co-injection of Matrigel with either Y-79 or WERI-Rb 1 cells into nude mice promotes retinoblastoma tumor formation. Transplantation of as few as 1,000 cells allows for xenografting under these conditions, while no tumors were observed in the absence of Matrigel, even at 10 x 10(6) cells/inoculum. The tumors produced have the expected morphology and express an mRNA for a highly specific retina/retinoblastoma marker protein, the interphotoreceptor retinoid-binding protein. Thus, the xenografts obtained maintain the original morphological and molecular characteristics of the injected cells and represent a useful model for in vivo studies of retinoblastoma growth and treatment.

Animals↗
PubMed1992

Genetic control of susceptibility to experimental autoimmune uveoretinitis in the mouse model. Concomitant regulation by MHC and non-MHC genes.

Experimental autoimmune uveoretinitis (EAU) in animals is a T cell-mediated autoimmune response directed against cells of the neural retina, in particular the photoreceptors. EAU can be induced in susceptible strains of mice by immunization with purified retinal Ag, and serves as a model for human uveitis. Because strong HLA associations have been noted in a number of human uveitic diseases, we investigated the role of MHC vs non-MHC genes in the control of susceptibility to ocular autoimmunity using the mouse EAU model. Selected strains representing most of the known independent H-2 haplotypes, as well as several H-2-recombinant and congenic strains, were immunized with interphotoreceptor retinoid-binding protein. Ocular pathology was induced in strains of the H-2k haplotype and their I-A-matched congenics, as well as in strains of the H-2r, H-2b, and H-2d haplotypes. In a series of experiments utilizing intra-H-2 recombinant strains, MHC control of susceptibility was tentatively mapped to the I-A subregion of the H-2k. Expression of the I-Ek gene product was not required for susceptibility to EAU, and in fact appeared to have an ameliorating effect on disease. Incidence and severity of disease obtained in strains sharing the same H-2 on a different background, or sharing the same background in the context of a different H-2, indicated that non-MHC genes contribute significantly to the regulation of EAU. Disease expression of susceptible H-2 haplotypes was highest in strains with B10 background (permissive) and ranged from intermediate to absent in strains with other (nonpermissive) backgrounds. The data suggest that although the ability to develop ocular pathology is dependent on the I-A subregion of the H-2, the final expression of disease in susceptible haplotypes is largely determined by background, non-MHC genes.

Animals↗
PubMed1992

Laminin-induced retinoblastoma cell differentiation: possible involvement of a 100-kDa cell-surface laminin-binding protein.

Gene and protein expression of Y-79 retinoblastoma cells growing on poly(D-lysine) is switched from a photoreceptor-like to a conventional neuron-like pathway by the basement membrane glycoprotein laminin. Unlike other cell systems where laminin influences differentiation, Y-79 cells can neither attach to nor chemotactically respond to laminin. However, laminin increases attachment to poly(D-lysine). The laminin effects therefore seem to occur via an adhesion- and chemotaxis-independent mechanism. Moreover, these tumor cells do not exhibit high-affinity laminin binding, having only a single binding site of intermediate affinity. Laminin-Sepharose affinity chromatography of Y-79 cell surface proteins labeled with 125I revealed a single major radiolabeled 100-kDa protein eluted by 20 mM EDTA, with an electrophoretic behavior different from that of integrins. No other proteins were eluted under more stringent conditions. This material, which we call LBM-100 (100-kDa laminin-binding molecule), may be a "differentiative" laminin-binding protein through which laminin influences gene expression and development independently of attachment.

Amino Acid Sequence↗
PubMed1992

Neurotrophic activity of interphotoreceptor matrix on human Y79 retinoblastoma cells.

A neurotrophic activity of adult monkey and bovine interphotoreceptor matrix (IPM) was examined by using cultured human Y79 retinoblastoma cells as a model system. The cells were stimulated for 7 days in suspension culture with soluble IPM components and then attached to poly-D-lysine substratum. IPMs from both species induced greater than 80% neuronal differentiation of Y79 cell aggregates after 11 days of attachment as adjudged morphologically by the extension of lengthy, neurite-like processes. Immunocytochemical studies indicate that differentiated Y79 cells had an increased level of expression of neuron-specific enolase and a concomitant decreased expression of glial fibrillary acidic protein. This neurotrophic activity cannot be ascribed to nerve growth factor, platelet-derived growth factor, fibroblast growth factor, epidermal growth factor, or transforming growth factor beta. Although the nature of the factor and its cellular source have yet to be characterized, it may be related to a recently described neurotrophic protein secreted by human fetal retinal pigment epithelial cells in culture. Our findings provide evidence supporting the neuroblastic potential of the Y79 cell line and indicate that the IPM contains a potent neurotrophic activity. Such factors may be important to normal differentiation and maintenance of function of the neural retina.

Animals↗
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

Monkey retinal pigment epithelial cells in vitro synthesize, secrete, and degrade insulin-like growth factor binding proteins.

Cultured monkey retinal pigment epithelial (RPE) cells rapidly secrete large amounts of insulin-like growth factor binding proteins (IGF-BPs). IGF-II tracer binding activity in conditioned media is two to three times greater than that of IGF-I. Under reducing SDS-PAGE conditions, 125I-IGF affinity-crosslinked binding protein (BP) is visualized as a broad band between 36 +/- 2.9 and 49 +/- 3.3 kDa. Because the electrophoretic mobility of the crosslinked BP is increased under non-reducing conditions (33-45 kDa), intramolecular sulfhydryl bonding may be present. Frequently, the radiographic band representing affinity-crosslinked binding protein exhibits a complex pattern of non-uniform densities that suggests structural or functional IGF-BP micro-heterogeneity. IGF-BPs synthesized by RPE also exhibit heterogeneity with respect to the absence or presence of oligosaccharide side chains. In particular, the larger, but not the mid-sized or smaller IGF-BPs exhibit side chains linked to the core protein with N-glycosidic linkage. None of the crosslinked IGF-BPs exhibit O-linked side chains. Long-term (12, 24, 48 hr) conditioning studies revealed that IGF-BP fails to accumulate in culture media beyond 12 hr, but that replacement of conditioned media with fresh media allows a second period of binding protein accumulation. Other short-term (12 hr) experiments indicate that, in fresh medium, the levels of IGF-BP increase during the first 6-8 hr and then remain stable. To examine the processes contributing to these steady state levels of IGF-BP, aliquots of 8-hr conditioned medium were removed from the cells and either frozen on dry ice or incubated at 37 degrees C for 16 hr. Importantly, it was found that incubation at 37 degrees C resulted in a near total loss of binding activity. This is the first report of IGF-BP degrading activity in a cell culture system. These findings indicate that 1) primate RPE cells rapidly secrete a complex mixture of N-glycosylated and non-glycosylated IGF-BPs, and 2) the steady state levels of secreted IGF-BP are tightly regulated at least in part through a concomitant IGF-BP inactivating activity. Cultured RPE cells may be of utility in examining the mechanisms of IGF-BP synthesis, secretion, and degradation at the cellular level.

Animals↗
PubMed1992

Cellular immune responses to retinal antigens in retinitis pigmentosa.

Patients with retinitis pigmentosa and a group of controls were tested for their cellular immune response toward two retinal proteins, S-antigen and interphotoreceptor retinoid-binding protein (IRBP), as well as their reaction against two synthetic peptides ("M" and "N") derived from the sequence of S-antigen and peptide "R14", derived from IRBP. Positive responses to the retinal antigens were found in larger proportions and with higher levels in the patient group than in the controls. The difference between the two groups was statistically significant in their response to S-antigen, but the patients reacted better than the controls against the other antigens as well. Of particular interest was the finding that several patients responded to both retinal proteins and/or to their peptides. These patients suffered from severe retinal changes and the data are thus interpreted as suggesting that the responses to the retinal antigens are secondary to these changes and to nonphysiological release of retinal antigens.

Adult↗
PubMed1992

Immunocytochemical demonstration of interphotoreceptor retinoid-binding protein in cerebellar medulloblastoma.

Previously, immunoreactive rod-opsin and S-antigen (arrestin), two highly characteristic markers of retinal photoreceptors and pinealocytes, were shown to be present in certain medulloblastoma cells. It, thus, has been suggested that such cells differentiate along the photoreceptor lineage. This is corroborated in the present immunocytochemical investigation using antibodies against another photoreceptor-cell marker, the interphotoreceptor retinoid-binding protein (IRBP). As shown in preparations of human retina and pineal organ, IRBP can be successfully demonstrated in formalin-fixed and paraffin-embedded tissue: the IRBP immunoreaction is located to the outer and inner segments of retinal photoreceptor cells and to perikarya of certain pinealocytes. Examination of formalin-fixed, paraffin-embedded biopsy specimens of 66 cerebellar medullo-blastomas revealed varying numbers of IRBP-immuno-reactive tumor cells in 19 cases that were formerly shown to contain rod-opsin and S-antigen immunoreaction. IRBP-immunoreactive tumor cells were also found in a retinoblastoma and a pineocytoma, but not in neuroblastoma, ganglioneuroblastoma, glioblastoma, oligodendroglioma and astrocytoma. The results indicate: (1) cerebellar medulloblastomas are heterogeneous in their differentiation potential; (2) one type of medulloblastoma displays photoreceptor characteristics; (3) this type appears to be closely related to retinoblastoma and pineal cell tumors; and (4) all three types of tumors may display additional common features to be explored in future studies.

Antigens↗
PubMed1992

The existence of two completely distinct antigenic sites within a decapeptide.

A decapeptide (1182-1191) derived from the bovine interphotoreceptor retinoid-binding protein (IRBP) was found to contain two completely distinct antigenic sites when tested in Lewis rats. One site, localized in sequence 1182-1191, is the core of the immunodominant and highly uveitogenic determinant of IRBP. The second site localizes within sequence 1183-1191 and becomes detectable only when tryptophan at position 1182 is deleted. Lymphocytes sensitized against the first, larger site recognized all longer peptides within sequence 1169-1191, as well as whole IRBP. In contrast, lymphocytes sensitized against the second, short epitope recognized only two peptides, 1184-1191 and (to a lesser degree) 1183-1191. The responses to both sites were restricted by the same major histocompatibility complex (MHC) product (I-A), as shown by monoclonal antibody blocking and by the finding that the lymphocyte response to 1184-1191 was competitively inhibited by peptide 1181-1191. The unique finding of two completely distinct antigenic sites within a decapeptide could be explained by the hypothesis that peptides of the two sites combine with the MHC molecule on antigen-presenting cells by different configurations, thus forming two distinct antigenicities.

Amino Acid Sequence↗
PubMed1992

Photoreceptor-specific mRNAs in mice carrying different allelic combinations at the rd and rds loci.

Several retinal mRNAs encoding photoreceptor-specific proteins have been examined in congenic lines of mice carrying different allelic combinations at the rd and rds loci, to determine how mRNA expression is affected by the presence of both the rd and rds genes together or by the presence of one or two rd and rds alleles in the visual cells. Slot blots with retinal RNA from 9 to 30 days old C3H +/+, +/+; rd/+, +/+; rd/rd, +/+; +/+, +/rds; +/+, rds/rds; and the rd/rd, rds/rds double homozygous mutant mice were hybridized successively to several [32P]cDNA probes encoding proteins involved in phototransduction. The increases and decreases in the levels of mRNA coding for opsin, the alpha-subunit of transducin, 48 kDa protein and the beta-subunit of cGMP-phosphodiesterase in the heterozygous and homozygous rds and rd mouse retinas reflected the growth and degeneration of the photoreceptor cells. The heterozygous rd mouse (rd/+, +/+) retina expressed cGMP-phosphodiesterase beta mRNA levels halfway between those in the control (+/+, +/+) and homozygous (rd/rd, +/+) mice, indicating a possible dosage effect. The double homozygous mutant (rd/rd, rds/rds) showed intermediate levels between those observed in the two homozygotes for all mRNAs studied, suggesting a possible interaction between the rd and rds genes.

Alleles↗
PubMed1992

Synthesis and secretion of interphotoreceptor retinoid-binding protein (IRBP) and developmental expression of IRBP mRNA in normal and rd mouse retinas.

The synthesis and secretion of interphotoreceptor retinoid-binding protein (IRBP) was quantitatively assessed in retinas of normal and rd mutant mice using short-term organ culture with [35S]methionine. Retinas were studied at ages P9-P12, time points prior to and immediately after the onset of the degeneration of the rd retina. Soluble proteins of the retinal pellet and the incubation medium were subjected to SDS-polyacrylamide gel electrophoresis. Analysis of labeled protein bands utilized a radioactivity scanning system to quantify [35S]methionine incorporation into newly synthesized IRBP. The synthesis and secretion into the incubation medium of IRBP by rd mouse retinas was comparable to normal retinas at P9-P10 but decreased by more than 50% by P12. IRBP mRNA levels were evaluated in retinas of normal and rd mice ages P7-P14. Although IRBP mRNA expression increased in the rd mouse through P10, it decreased markedly thereafter. Previously reported immunocytochemical studies suggested that IRBP was not secreted in the rd mouse retina. The results of this study indicate, however, that rd mouse retinas, when removed from the eye, have the capacity to synthesize and secrete IRBP.

Animals↗