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Cellular specificity in rat cerebellum monitored by monoclonal antibodies
420
Round
Table
IMMUNOLOGICAL APPROACHES TO NERVOUS SYSTEM DEVELOPMENT
15
CELLULAR SPECIFICITY IN RAT CEREBELLUMMONITOREDBY MONOCLONAL ANTIBODIES. G. Gombos, M.S. Ghandour, B. Foucaud, O.K. Langley, Y. Schladenhaufen, J. de Barry. (Centre de Neurochimie du CNRS, Strasbourg, France). Monoclonal antibodies (MAbs) against molecules of cerebellar structures were obtained by injecting, as immunogens, into mice, cerebellar “membrane” fractions from 12 day old rats; the mice immunocytes were fused with cells from a mouse myeloma line and the hybridomae were cloned. Immunocytology with the MAbs produced by each hybridoma cell line and differential binding tests of these !4Abs to 12 day old and to adult membrane were used for screening and for selecting developmentally regulated antigens. Several of the obtained MAbs showed specificity for a single cell type (either different neuronal types) or structures neurones or astrocytes) or subtype (i.e. Few MAbs the pinceau of basket cells, the parallel fibres, the glomeruli). (i.e. According to GJestern blots of reacted with developmentally regulated antigens. SDS-PAGE electrophoresis of proteins derived from cerebellar membranes, from cytosol and from cytosqueleton fractions none of the epitopes revealed by our MAbs are localized in known CNS antigens.
16
A NEW CELL SURFACE GLYCOPROTEIN INVOLVED 11 NEURITE Sigrid HENKE-FAHLE and Reiner BABIEL, Max-Planck-Institut fUr Entwicklungsbiologie, Spemannstr. 35, D 7400 TUbingen, FRG
OUTGROWTH
FROM THE CNS.
Outgrowth of neurites from central nervous system neurones is dependent on a variety of substances like trophic factors and appropriate substrates. We have recently described a monoclonal antibody (T61) which inhibits axonal outgrowth from chick retinal explants but does not affect axon growth from the peripheral nervous system. Here we report some investigations on the molecular nature of the antigen, its distribution within the chick embryo and its developmental expression. Immunoblot analysis of retinal proteins from the g-day old chick embryo indicated that it is a glycoprotein of molecular weight 300 kD and 180/170 kD. A significant increase in specific binding to solubilized brain proteins c.an be shown with T61 antibody between 7 and 11 days of incubation. These quantitative changes are accompanied by structural changes in the carbohydrate part of the molecule between days ES and E12, a period characterized by of T61 extensive neurite growth in the chick brain. No immunological crossreactivity antibody could be found with two other cell-surface glycoproteins, N-CAM and Ll, known to be involved in neuronal cell adhesion. By using a protein A-gold marker we localized the antigen on the surface of ganglion cell axons in culture and also bound to the substrate in the vicinity of fiber bundles, suggesting that it might be released by the axons themselves. Preliminary in vivo studies indicate that the T61 antibody inhibits the formation of an optic nemer being injected into the eye at early stages.
Round
Table
IMMUNOLOGICAL APPROACHES TO NERVOUS SYSTEM DEVELOPMENT
15
CELLULAR SPECIFICITY IN RAT CEREBELLUMMONITOREDBY MONOCLONAL ANTIBODIES. G. Gombos, M.S. Ghandour, B. Foucaud, O.K. Langley, Y. Schladenhaufen, J. de Barry. (Centre de Neurochimie du CNRS, Strasbourg, France). Monoclonal antibodies (MAbs) against molecules of cerebellar structures were obtained by injecting, as immunogens, into mice, cerebellar “membrane” fractions from 12 day old rats; the mice immunocytes were fused with cells from a mouse myeloma line and the hybridomae were cloned. Immunocytology with the MAbs produced by each hybridoma cell line and differential binding tests of these !4Abs to 12 day old and to adult membrane were used for screening and for selecting developmentally regulated antigens. Several of the obtained MAbs showed specificity for a single cell type (either different neuronal types) or structures neurones or astrocytes) or subtype (i.e. Few MAbs the pinceau of basket cells, the parallel fibres, the glomeruli). (i.e. According to GJestern blots of reacted with developmentally regulated antigens. SDS-PAGE electrophoresis of proteins derived from cerebellar membranes, from cytosol and from cytosqueleton fractions none of the epitopes revealed by our MAbs are localized in known CNS antigens.
16
A NEW CELL SURFACE GLYCOPROTEIN INVOLVED 11 NEURITE Sigrid HENKE-FAHLE and Reiner BABIEL, Max-Planck-Institut fUr Entwicklungsbiologie, Spemannstr. 35, D 7400 TUbingen, FRG
OUTGROWTH
FROM THE CNS.
Outgrowth of neurites from central nervous system neurones is dependent on a variety of substances like trophic factors and appropriate substrates. We have recently described a monoclonal antibody (T61) which inhibits axonal outgrowth from chick retinal explants but does not affect axon growth from the peripheral nervous system. Here we report some investigations on the molecular nature of the antigen, its distribution within the chick embryo and its developmental expression. Immunoblot analysis of retinal proteins from the g-day old chick embryo indicated that it is a glycoprotein of molecular weight 300 kD and 180/170 kD. A significant increase in specific binding to solubilized brain proteins c.an be shown with T61 antibody between 7 and 11 days of incubation. These quantitative changes are accompanied by structural changes in the carbohydrate part of the molecule between days ES and E12, a period characterized by of T61 extensive neurite growth in the chick brain. No immunological crossreactivity antibody could be found with two other cell-surface glycoproteins, N-CAM and Ll, known to be involved in neuronal cell adhesion. By using a protein A-gold marker we localized the antigen on the surface of ganglion cell axons in culture and also bound to the substrate in the vicinity of fiber bundles, suggesting that it might be released by the axons themselves. Preliminary in vivo studies indicate that the T61 antibody inhibits the formation of an optic nemer being injected into the eye at early stages.