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L V Johnson

Publications and source records attributed to L V Johnson.

At least 37 records · Page 2Linked to original sources
PubMed1993

Effects of medium conditioned by retinal pigmented epithelial cells on neurotransmitter phenotype in retinoblastoma cells.

We previously reported that medium conditioned by retinal pigmented epithelial cells can induce cellular differentiation in human retinoblastoma cells. Extensive neurite outgrowth, increased expression of neuronal marker molecules and decreases expression of glial marker molecules are characteristic of the differentiated phenotype. In the studies described here, we examine whether modulations in the expression of potential neurotransmitter molecules, catecholamines and indolealkyl amines, might be associated with the differentiation of retinoblastoma cells. Concentrations of serotonin, 5-hydroxyindoleacetic acid, 3-methoxytyrosine, homovanillic acid, and 3-methoxy-4-hydroxyphenylacetic acid in extracts of differentiated and undifferentiated retinoblastoma cells were assessed by HPLC. The results show that serotonin and its metabolite, 5-hydroxyindoleacetic acid, are characteristically present in undifferentiated cells. Dopa metabolites, 3-methoxytyrosine, homovanillic acid and 3-methoxy-4-hydroxy-phenylacetic acid, are uniquely present in differentiated cells. It appears that differentiation of retinoblastoma cells induced by factors secreted by retinal pigmented epithelial cells involves a switch from a serotonergic phenotype to one dominated by metabolites of dopa. These findings may provide clues about the factors that control retinoblastoma cells and metastasis.

Cell Differentiation↗
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↗
PubMed1991

Structural and compositional analyses of isolated cone matrix sheaths.

The cone matrix sheath is a biochemically and structurally distinct domain of the retinal interphotoreceptor matrix that is specifically associated with cone photoreceptor cell inner and outer segments. An aqueous extraction technique for the isolation of cone matrix sheath-enriched retinal fractions was developed and used to examine the structure and composition of this extracellular matrix domain. A complex substructural organization of the cone matrix sheath was observed. Many longitudinally oriented, filamentous structures extend the entire length of the sheath and terminate in filamentous rings at both the apical and basal ends. These longitudinal filaments are interconnected by a finer, anastomosing filamentous network. The basal rings of cone matrix sheaths are interconnected with similar rings of matrix material associated with rod photoreceptor inner segments. Gel electrophoresis and lectin blot analyses of cone matrix sheath-enriched fractions reveal the presence of 17- and 32-kilodalton bands, labeled by peanut lectin, that are likely to be components of the structural elements of cone matrix sheaths. Thus, structural elements potentially capable of mediating adhesion between the neural retina and the retinal pigmented epithelium are present in the cone matrix sheath and may be at least partially responsible for the observation that cone matrix sheaths are firmly adherent to the pigmented epithelium and the neural retina.

Animals↗
PubMed1990

Transmembrane assemblage of the photoreceptor connecting cilium and motile cilium transition zone contain a common immunologic epitope.

The photoreceptor connecting cilium bears a unique transmembrane assemblage which stably links cell surface glycoconjugates with the underlying axonemal cytoskeleton. Structural similarities between the photoreceptor connecting cilium and the transition zone of motile cilia suggests that this assemblage may also be present in motile cilia. Using a subcellular fraction enriched in detergent-extracted photoreceptor axonemes, three high molecular mass glycoconjugates (425, 600, and 700 kD) were previously identified as potential components of the assemblage. Through oligosaccharide characterization and binding of a specific monoclonal antibody, we have verified the localization of the 425 kD glycoconjugate to the transmembrane assemblage. Binding of the lectin peanut agglutinin (PNA) to the 425 kD glycoconjugate on nitrocellulose blots, and to isolated detergent-extracted axonemes, was assessed following treatment with the enzymes neuraminidase and O-glycanase. Changes in binding to the 425 kD glycoconjugate precisely paralleled changes in binding to intact axonemes, supporting the hypothesis that the 425 kD glycoconjugate is a component of the transmembrane assemblage. Furthermore, the results suggest that the 425 kD glycoconjugate contains sialated galactose-N-acetylgalactosamine oligosaccharides which are O-linked to the protein backbone. To directly assess the distribution of the 425 kD glycoconjugate, we produced a monoclonal antibody directed against this glycoconjugate. The antibody, K26, recognizes only the 425 kD on transblots of the axoneme fraction. K26 immunoreactivity of intact axonemes is identical to that seen by PNA staining. K26 staining of isolated photoreceptors and whole retina is uniquely localized to the region of the connecting cilium. Thus, in the photoreceptor, the 425 kD is not only a component of the transmembrane assemblage but is also completely restricted to the connecting cilium. Based on morphological similarities, the photoreceptor connecting cilium is thought to be homologous to the transition zone of the motile cilium. As such, we have stained oviduct epithelium with the K26 monoclonal antibody. Immunoreactivity is restricted to the region of the transition zone at the base of motile cilia.

Animals↗
PubMed1989

Carbohydrate components recognized by the cone-specific monoclonal antibody CSA-1 and by peanut agglutinin are associated with red and green-sensitive cone photoreceptors.

Previous investigations have demonstrated that the monoclonal antibody CSA-1 and peanut agglutinin label specifically cone photoreceptor cells. In the present study, we compared the binding of CSA-1 and peanut agglutinin to that of the monoclonal antibodies COS-1 and OS-2, which have been shown to recognize the red/green- and blue-sensitive cone visual pigments, respectively. Using lectin and immunocytochemistry on serial semithin sections of the pig retina, we have demonstrated in the present study that both CSA-1 and peanut agglutinin label specifically the red-, and green-sensitive, but not the blue-sensitive cone cell outer segments. Peanut agglutinin does bind, however, to the cone matrix sheaths associated with all three types of cones. These observations support the idea that red-, and green-sensitive cone cells share some common molecular epitopes and may represent a differentiation line of cones, considerably different from that of blue-sensitive cones.

Animals↗
PubMed1989

Collagen-induced alterations in intercellular adhesion and antigen expression in retinoblastoma cells.

Y79 human retinoblastoma cells, which typically grow as suspension cultures in vitro, show increased intercellular and cell-substratum adhesion, and form compact cellular aggregates when cultured on a collagen substratum. Concomitant with collagen-induced formation of compact cellular aggregates, is an increase in the binding of peanut lectin, especially at points of intercellular apposition. In addition, increases in the binding of antibodies against neuron-specific enolase and the cone-specific monoclonal antibody CSA-1 are noted following attachment and growth on collagen. In contrast, a decrease in the binding of antibodies against the glial marker, glial fibrillary acidic protein, is observed in collagen-attached cells. Thus, both the adhesive properties and the biochemical composition of Y79 retinoblastoma cells are altered by their attachment to and growth upon a collagenous substratum.

Antigens, Neoplasm↗
PubMed1989

Neuronal differentiation of retinoblastoma cells induced by medium conditioned by human RPE cells.

Tumor cells can be induced to differentiate in vitro by biochemical manipulation of their culture environment. In the studies described here, the effects of medium conditioned by human retinal pigmented epithelial (RPE) cells on Y79 human retinoblastoma cells have been examined. RPE-conditioned medium in conjunction with laminin and a poly-D-lysine substratum is observed to induce neuronal differentiation of Y79 cells. The cells extend long cellular processes and exhibit immunologically detectable neurotypic properties. In contrast, control Y79 cells not exposed to medium conditioned by RPE cells exhibit only infrequent neuronal phenotypes. This response of Y79 cells to RPE-conditioned medium indicates that factors secreted by RPE cells can act as inducers of neuronal differentiation in retinoblastoma cells and suggest that such factors may be of importance in the development and differentiation of the neural retina.

Cell Differentiation↗
PubMed1988

Ultrastructural visualization of primate cone photoreceptor matrix sheaths.

Glycoconjugates, including glycolipids, glycoproteins, and proteoglycans, are present in the plasma membrane of photoreceptor cells and in the interphotoreceptor matrix surrounding photoreceptor cell ellipsoids and outer segments. Although the precise function of these molecules is unknown, they may be important in mediating photoreceptor-pigment epithelial cell interactions, outer segment membrane assembly, and/or disc shedding. Lectins, affinity ligands for defined carbohydrate sequences, have proven particularly useful in studying the glycoconjugate composition of the interphotoreceptor matrix. The peanut lectin selectively binds to domains of the interphotoreceptor matrix surrounding cone ("cone matrix sheaths"), but not rod inner and outer segments. This is evidence for the existence of chemical and structural heterogeneity within the interphotoreceptor matrix. The studies described herein utilized ultrastructural pre-embedding histochemical labeling to assess whether, in addition to the surrounding interphotoreceptor matrix, peanut lectin binding is associated directly with that plasma membrane of cone inner and outer segments. This study confirms that ferritin-conjugated peanut agglutinin binds to cone matrix sheaths, and, in addition, provides ultrastructural evidence for the presence of binding to the plasma membrane surrounding cone inner and outer segments. The data suggest that cone membrane-associated peanut agglutinin-binding molecules may differ from those located within cone matrix sheaths.

Animals↗
PubMed1988

Ultrastructural evidence that specialized regions of the murine oviduct contribute a glycoprotein to the extracellular matrix of mouse oocytes.

Previous studies have identified a glycoprotein (GP215) that is secreted by the murine oviductal epithelium and subsequently becomes sequestered within the perivitelline space of oocytes and developing embryos (Kapur and Johnson, Dev. Biol. 112:89-93, 1985; J. Exp. Zool. 238:249-260, 1986). The ultrastructural localizations of GP215 in the perivitelline space of ovulated oocytes and in the oviductal epithelium are described here. The glycoprotein is shown to be associated with a morphologically discrete extracellular matrix that provides a unique microenvironment for fertilization and early developmental events. In addition, putative secretory granules that contain this glycoprotein are observed in specific segments of the murine oviductal epthelium, suggesting regional differences in the composition of oviductal secretions.

Animals↗
PubMed1988

Characterization of molecules bound by the cone photoreceptor-specific monoclonal antibody CSA-1.

The cone-specific monoclonal antibody, CSA-1, selectively labels cone photoreceptors in the neural retina. Immunofluorescence assays reveal that CSA-1 binds cone outer and inner segments, cell bodies, axons and synaptic pedicles. The binding of the antibody is membrane-associated, not cytoplasmic. Several lines of evidence, including sensitivity to mild alkaline hydrolysis and galactosidic enzymes, suggest that the CSA-1 detected determinant is a galactose-containing carbohydrate moiety. Immunoblotting analyses show CSA-1 immunoreactivity with at least two molecules of 54,000 daltons that differ slightly in isoelectric point, and an additional, higher molecular weight species. CSA-1 should prove to be of value in studies of cone photoreceptor biology, in analyses of the development and differentiation of these cells, and in assessing their fate in various retinal pathologies.

Animals↗
PubMed1987

Enzymatic characterization of peanut agglutinin-binding components in the retinal interphotoreceptor matrix.

Previous histochemical and biochemical studies have documented the presence of carbohydrate-containing molecules in the retinal interphotoreceptor matrix (IPM). The lectin peanut agglutinin (PNA), which preferentially binds galactose-containing carbohydrates, especially galactose-galactosamine linkages, selectively labels cone photoreceptor-associated domains of the IPM ('cone matrix sheaths') in a variety of vertebrate retinas. In the studies described here, the nature of these PNA-binding components was investigated by monitoring the effects of proteolytic and glycosidic enzymes on binding of the lectin in the retina and IPM. All proteolytic enzymes tested cause a marked reduction in PNA-binding to cone matrix sheaths, suggesting that proteinaceous components are important to their organization. Exposure to O-glycanase, but not N-glycanase, markedly reduces binding of PNA to cone matrix sheaths indicating that O-linked oligosaccharides are probably responsible for its binding. Galactose oxidase treatment reduces PNA-binding throughout the retina and IPM, confirming that galactose moieties are involved in its binding. beta-Galactosidase (either before or after neuraminidase treatment) does not alter the pattern of PNA binding, suggesting that neither terminal nor penultimate beta-linked galactose residues constitute a major proportion of the lectin's binding sites in the retina. Neuraminidase treatment markedly increases the density and distribution of PNA binding throughout the retina and IPM, however, this effect appears to be, at least in part, the result of the binding of the lectin to neuraminidase molecules that become associated with tissue sections in addition to binding to carbohydrate groups unmasked by desialation. Exposure to chondroitinases causes disruption of the morphological integrity of cone matrix sheaths and slight diminution of PNA binding. Other enzymes acting on common constituents of extracellular matrices do not have similar effects. Taken together, these observations suggest that PNA-binding to cone matrix sheaths is due to the presence of glycoconjugates with galactose-containing, O-linked oligosaccharide chains.

Animals↗
PubMed1987

Chondroitin 6-sulfate glycosaminoglycan is a major constituent of primate cone photoreceptor matrix sheaths.

Recent work suggests that chemically and structurally distinct domains of the interphotoreceptor matrix ("cone matrix sheaths") surround cone photoreceptor outer segments and ellipsoids. This specific regionalization of at least some molecular constituents of the interphotoreceptor matrix may establish a unique cone-associated microenvironment. Previous histochemical and biochemical investigations have shown that a variety of glycosaminoglycans are components of the interphotoreceptor matrix and that the structural integrity of cone sheaths is slightly disrupted by glycosaminoglycan-degrading enzymes. In order to pursue the possibility that specific glycosaminoglycan species establish cone matrix sheath domains, monoclonal antibodies directed against various unsaturated glycosaminoglycans have been screened on sections of primate retina. The results of these studies identify chondroitin 6-sulfate glycosaminoglycan as a specific component of primate cone matrix sheaths.

Animals↗
PubMed1987

Appearance of PNA-binding cells within the outer nuclear layer coinciding with photoreceptor degeneration in rd mice.

Peanut agglutinin (PNA), a lectin with affinity for galactose-galactosamine disaccharides, has been employed to monitor alterations in carbohydrate expression in retinal degenerative (rd) mice. Mice homozygous for the rd gene exhibit a rapid loss of rod photoreceptor cells in the first postnatal month; outer segment degeneration is first detected between postnatal days (P) 12-P14 and most rod photoreceptors degenerate by P30. In the rd mouse, PNA-binding cells are observed in the outer nuclear layer (ONL) between P10 and P18, a time corresponding to the intense phase of photoreceptor cell death (LaVail et al., 1982). At no time are PNA-positive cells identified within the ONL of control retinas. PNA-binding cells typically occur in groups and binding of the lectin appears to be restricted to granular elements of the cytoplasm. This report represents the first documentation of changes in the expression and/or accessibility of glycoconjugates in the degenerating ONL of the rd retina. Further analysis of the relationship between regional distribution of PNA-binding cells in the ONL and degenerating photoreceptors of the rd may lead to a better understanding of the mechanism of cell death induced by this mutation.

Animals↗
PubMed1986

Vascular atrophy in the retinal degenerative rd mouse.

We have observed that the lectin Ricinus communis agglutinin I (RCA), which binds to terminal galactose moieties, serves as a marker for vasculature in the mouse retina. The binding of fluorescein-isothiocyanate-conjugated RCA was used to study the development of retinal vasculature in normal mice and in rd (retinal degeneration) mutant mice, which exhibit a massive loss of photoreceptor cells during the first month of life. In the normal mouse, retinal capillaries develop in an ordered manner and are concentrated in three major zones between the inner limiting membrane and the outer plexiform layer. In the rd mouse, the vessels appear to form normally but begin to degenerate by the end of the second postnatal week. By the end of the fourth postnatal week there is approximately a 35% reduction in the total number of vascular profiles in the rd retina compared to normal littermate controls. This reduction in vascularity is temporally associated with the photoreceptor degeneration.

Animals↗
PubMed1986

Biochemical characterization of the major peanut-agglutinin-binding glycoproteins in vertebrate retinae.

Peanut agglutinin (PNA), a lectin that binds D-galactose-beta (1----3) N-acetyl-D-galactosamine disaccharide linkages, selectively labels cone photoreceptors in the retinae of a variety of species. PNA binds consistently to domains of the interphotoreceptor matrix associated with cone, but not rod, inner and outer segments, to cone cell body and axonal membranes, to cone synaptic pedicles, and to portions of the inner plexiform layer. In order to begin the characterization of the molecular species responsible for cone-specific PNA binding, chick, turkey, rat, dog, pig, monkey, and human retinal extracts were separated by SDS-polyacrylamide gel electrophoresis and probed with peroxidase-conjugated PNA. The results reveal the presence of six major groups of PNA-binding glycoproteins ranging from 30 to 88 kilodaltons. Most of these are shared by the seven species examined; however, some interspecies variation is present. Three groups, designated GP39/40, GP42/45, and GP60, are the most intensely labeled by PNA and are common to all species analyzed, while groups GP29/31 and GP88 are less intensely labeled and are present in most but not all of the species investigated. Labeling of the GP54 group is variable but is most consistently associated with extracts of rat and pig retinae. Trypsin treatment, which results in the loss of cone-associated PNA binding in the interphotoreceptor matrix, causes a visually detectable reduction in three of the six groups of PNA-binding glycoproteins in porcine retinal extracts. Of these, GP54 is the most sensitive, being undetectable on PNA-stained blots after only 5 minutes of enzyme exposure; GP88 and GP45 are less sensitive but both are markedly reduced after 15 minutes of trypsinization. Trypsin-sensitive molecules thus may be involved in the establishment of the cone-specific domains of interphotoreceptor matrix identified by PNA binding. These, as well as the other groups of PNA-binding molecules, are being utilized to develop more specific immunologic probes with which to further study of their distribution and function.

Animals↗
PubMed1986

Selective sequestration of an oviductal fluid glycoprotein in the perivitelline space of mouse oocytes and embryos.

Previously, we identified a 215 kd glycoprotein, GP215, which is associated with postovulatory oocytes and embryos, but not with preovulatory oocytes (Kapur and Johnson, '85). In this paper a polyclonal antibody that specifically recognizes GP215 has been used to study the distribution of the molecule in association with ova and preimplantation embryos and in the female reproductive tract. GP215 is present in epithelial cells lining the cranial portions of the oviduct and in oviductal fluid, ovarian bursal fluid, and medium conditioned by oviductal tissue in vitro. Immunofluorescence assays of the ovum and early embryo show that GP215 is sequestered in the perivitelline space. Since preovulatory oocytes exposed to bursal fluid in vitro acquire GP215, we hypothesize that GP215 is synthesized and secreted by the oviductal epithelium and secondarily associates with the ovulated oocyte. Sequestration of GP215 within the perivitelline space is relatively specific since mouse serum albumin, a major constituent of oviductal fluid, and other high molecular weight proteins are not similarly retained. These observations indicate that the composition of the perivitelline space may be significantly different from the greater environment external to the zona pellucida such that fertilization and early development of mammalian ova potentially take place in a distinct perivitelline microenvironment.

Animals↗