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C Goridis

Publications and source records attributed to C Goridis.

At least 91 records · Page 5Linked to original sources
PubMed1985

Identification of a previously unrecognized polypeptide associated with lymphocyte function associated antigen one (LFA-1).

In lymphocyte function associated antigen one (LFA-1) preparations from metabolically labeled lymphocytes we have observed a new polypeptide component of 86-kilodalton additional to the already described alpha- and beta-chains. This chain is cosynthesized with the alpha- and beta-chains and can be covalently cross-linked with them, resulting in a three-chain complex. This complex is recognized by the H35-89.9 anti-LFA-1 monoclonal antibody. Cleveland peptide mapping analysis indicates that the new chain is structurally different from the alpha- and beta-chains of the LFA-1 complex. The chain has been observed in B-cells as well as in T-cells. Labeling properties of the 86-kilodalton chain suggest that this molecule is not exposed on the membrane.

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PubMed1985

Individual neural cell types express immunologically distinct N-CAM forms.

The neural cell adhesion molecules, or N-CAMs, are a group of structurally and immunologically related glycoproteins found in vertebrate neural tissues. Adult brain N-CAMs have apparent molecular weights of 180,000, 140,000, and 120,000. In this article we identify, using monoclonal antibody (Mab) 3G6.41, an immunologically distinct adult rat N-CAM form and show that this form is selectively expressed by some clonal neural cell lines. Consecutive immunoprecipitation experiments indicate that rabbit anti-N-CAM can remove from solubilized cerebellar neuron primary cultures all 180,000- and 140,000-mol-wt N-CAM molecules that react with Mab 3G6.41. However Mab 3G6.41 cannot remove all N-CAM molecules that react with rabbit anti-N-CAM. Rabbit anti-N-CAM binds to and immunoprecipitates N-CAM forms from the rat neuronal cell lines B35, B65, and B104, the glial lines B12 and C6, and L6 myoblasts. Mab 3G6.41 does not bind to or immunoprecipitate N-CAM from the B12 and B65 lines but does react with the other four lines by both criteria. Many cells in primary cultures of postnatal rat that express glial fibrillary acidic protein also bind Mab 3G6.41. Thus a unique form of rat N-CAM recognized by Mab 3G6.41 is found on some but not all neuronal, glial, and muscle cells.

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PubMed1985

Selective expression of the 180-kD component of the neural cell adhesion molecule N-CAM during development.

The rodent neural cell adhesion molecule (N-CAM) consists of three glycoprotein chains of 180, 140, and 120 kD in their adult forms. Although the proportions of the three components are known to change during development and differ between brain regions, their individual distribution and function are unknown. Here we report studies carried out with a monoclonal antibody that specifically recognizes the 180-kD component of mouse N-CAM (N-CAM180) in its highly sialylated embryonic and less glycosylated adult forms. In primary cerebellar cell cultures, N-CAM180 antibody reacts intracellularly with all types of neural cells including astrocytes, oligodendrocytes, and neurons. During cerebellar, telencephalic, and retinal development N-CAM180 is detectable by indirect immunohistology in differentiated neural cells, but, in contrast to total N-CAM, not in their proliferating precursors in the ventricular zone and primordial and early postnatal external granular layer. In monolayer cultures of C1300 neuroblastoma cells, N-CAM180 appears by immunofluorescence more concentrated at contact points between adjacent cells, while N-CAM comprising the 180- and 140-kD component shows a more uniform distribution at the plasma membrane. Treatment of neuroblastoma cells with dimethylsulfoxide, which promotes differentiation, induces a shift toward the predominant expression of N-CAM180. These observations support the notion that N-CAM180 is expressed selectively in more differentiated neural cells and suggest a differential role of N-CAM180 in the stabilization of cell contacts.

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PubMed1985

Structural and functional studies on N-CAM neural cell adhesion molecules.

The neural cell adhesion molecules N-CAM are to date the best characterized adhesion molecules of the nervous system. They have a high content of sialic acid residues which are present in the form of unusual sialic acid polymers. During development, a 3 fold decrease in the sialic acid content is observed. These changes in the degree of sialylation profoundly affect the binding properties of the molecules. A subpopulation of mouse brain N-CAM bears a carbohydrate determinant shared with other brain cell surface proteins and with the HNK-1 antigen of natural killer cells. Not only the carbohydrate side chains but also the protein moieties of the N-CAMs are heterogeneous. Three polypeptides of 180 K, 140 K and 120 K have been characterized in mouse brain. The 180 K and 140 K chains span the membrane. They differ mainly by the length of their cytoplasmic extensions. These intracellular domains are unusually long and contain phosphorylated serine residues. The 120 K chain exists in two forms, one membrane-bound and one soluble. Earlier studies had shown the presence of N-CAM on neurones and astrocytes of the mouse central nervous system, whereas cultured astrocytes had been reported to be N-CAM-negative. Recent results show that N-CAM is also expressed on astrocytes in culture. To study expression and heterogeneity of N-CAM polypeptides at the mRNA and gene level, cDNA clones for mouse N-CAM have been isolated. They reveal multiple mRNA species in mouse brain. By contrast, the corresponding sequences seem to be present only a few times, perhaps only once, in the mouse genome.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1984

Studies on the transmembrane disposition of the neural cell adhesion molecule N-CAM. A monoclonal antibody recognizing a cytoplasmic domain and evidence for the presence of phosphoserine residues.

The N-CAMs are a group of surface glycoproteins involved in adhesive interactions of neurones. Related molecules of the mouse nervous system, identified in our laboratory, have been called BSP-2 and shown to act as ligands in adhesion of neuroblastoma cells. Results presented in this report show that they are immunochemically identical with N-CAM. A monoclonal anti-(N-CAM) antibody, that recognized a determinant accessible only after permeabilization of intact cells, was used to define the mode of association of the N-CAMs with the plasma membrane. This antibody bound a 35 000-Mr fragment in lysates of trypsin-treated neuroblastoma cells. It is concluded that the antibody reacts with a transmembrane or cytoplasmic domain of the molecules. The same antibody recognized the Mr-180 000 and Mr-140 000 proteins but not the Mr-120 000 chain, which co-purify from adult mouse brain. The latter polypeptide was detected in the cytosol and could be partially released from brain membranes by osmotic shock. Part or all of the Mr-120 000 protein may thus lack a transmembrane segment. Our conclusion that the N-CAM forms of higher Mr are transmembrane proteins was further corroborated by our finding that they contain phosphoserine residues, which can be labeled with (32P)phosphate in intact neuroblastoma cells.

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PubMed1984

Studies on the transmembrane disposition of the neural cell adhesion molecule N-CAM. The use of liposome-inserted radioiodinated N-CAM to study its transbilayer orientation.

The transmembrane orientation of the polypeptide chains present in preparations of adult and neonatal mouse N-CAM was studied using, as a model system, liposome-inserted purified N-CAM preparations. N-CAM purified from adult or neonatal mouse brain was 125I-labeled and reconstituted into artificial lipid vesicles. After trypsin digestion, the peptides that remained associated with the liposomes were isolated by floatation of the vesicles on sucrose gradients. In control experiments the liposomes were lysed before trypsin treatment. Large, overlapping peptides were obtained after this treatment, several of which were protected by the liposome membrane. Sialic-acid-bearing peptides were revealed by their sensitivity to neuraminidase. To distinguish between peptides corresponding to intracellular or extracellular domains use was made of the P61 and H28.123 monoclonal antibodies, which recognize determinants located on the cytoplasmic and the extracellular part of the molecules respectively. There was no indication that the N-CAM chains were inserted in an inside-out configuration. Peptides protected from trypsin attack by the liposomes and recognized only by P61 had Mr values of 92 000, 42 000 and 35 000. The H28.123 determinant could be mapped to a 32 000-Mr peptide located close to the membrane at the vesicle's exterior. The bulk of the sialic acid seemed to be carried by a rather short sequence distal to the H28.123-reactive peptide but at some distance from the N terminus. Fragments of very similar Mr were generated from young and adult material. However, a 45 000-Mr peptide from neonatal N-CAM appeared to migrate in the higher-Mr region of sodium dodecyl sulfate/polyacrylamide gels in its fully sialylated form. It is concluded that (a) identical polypeptide chains are present in young and adult preparation, (b) the 180 000-Mr, 140 000-Mr and 120 000-Mr chains differ by the length of their cytoplasmic extensions and (c) the largest cytoplasmic sequences have a Mr close to 90 000. A tentative linear model of the transmembrane topography of the N-CAM polypeptides is presented.

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PubMed1984

Structural characteristics of the mouse transferrin receptor.

Rat monoclonal antibodies against mouse transferrin receptor have been used to isolate and characterize the mouse receptor molecule. The molecule is a dimeric glycoprotein of Mr 200 000 resembling its human homolog of Mr 190 000. Receptor molecules prepared from different lymphoid cell populations show structural differences which can be explained by variations in the carbohydrate moiety of the molecule. Both the antibody-binding site and the transferrin-binding site are located on tryptic fragments of Mr 80 000 on the extracellular part of the molecule. After trypsin treatment, these fragments are partially retained at the cell surface, probably non-covalently bound to one intact receptor subunit, but they are released at higher trypsin concentrations. The soluble fragments retain their ability to bind transferrin and appear to exist as dimers. In this fragment, there are no disulfide bonds present. Disulfide bonds are located near the plasma membrane. Studies using a cleavable cross-linker indicated the presence of cross-linking sites at the intramembranous or the cytoplasmic part of the molecule.

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PubMed1984

The neural cell adhesion molecule L1 is distinct from the N-CAM related group of surface antigens BSP-2 and D2.

The neural cell adhesion molecule L1 and the group of N-CAM related molecules, BSP-2 and D2 antigen, are immunochemically distinct molecular species. The two groups of surface molecules are also functionally distinct entities, since inhibition of Ca2+-independent adhesion among early post-natal mouse cerebellar cells by Fab fragments of both antibodies are at least additive, when compared with equal concentrations of the individual antibodies.

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PubMed1984

Recognition of sodium- and potassium-dependent adenosine triphosphatase on mouse lymphoid cells by means of a monoclonal antibody.

Previous evidence has established the similarity between (Na+ + K+)-ATPase (ATP phosphohydrolase, EC.3.6.1.3) and the antigen recognized by the rat antimouse monoclonal antibody anti-BSP-3. This antibody has been used for investigation of the surface expression and biochemical analysis of the enzyme in different mouse lymphoid populations. The BSP-3 determinant is found on almost all thymocytes and concanavalin A-induced thymocytes, to a lesser extent on bone marrow cells and also on a minor population of spleen cells. Spleen cells from athymic mice are negative. The (Na+ + K+)-ATPase purified from mouse thymus by affinity chromatography migrates in SDS-polyacrylamide gels in the form of two polypeptide chains of 105 000 and 51 000 daltons. Chains of the same molecular weight, fractionated on SDS-PAGE from microsomes of mouse thymuses, are shown to react with subunit-specific polyclonal antisera against ATPase in immunoblotting experiments. Immunoprecipitation with anti-BSP-3 from surface iodinated thymocytes yields only the small subunit. Comparison of the chains isolated from thymus and brain shows molecular weight differences in both subunits. These results, and variations in the reactivity pattern of the anti-BSP-3 antibody on several cell types, may indicate a possible heterogeneity of the (Na+ + K+)-ATPase expressed by various tissues and cells.

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PubMed1983

Expression of Ia antigens by cultured astrocytes treated with gamma-interferon.

The expression of major histocompatibility complex class II or Ia antigens by neural cells has been investigated by indirect immunofluorescence on dissociated cultures from mouse cerebellum, cerebral cortex and dorsal root ganglia. Ia antigen expression was not detectable under standard culture conditions. However, treatment of mixed cultures from the cerebellum and of astrocyte cultures from the cerebral cortex with gamma-interferon preparations induced expression of Ia antigens on a fraction of the astrocytes. Under the same conditions, Ia+ cells could be observed in dorsal root ganglion cultures.

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PubMed1983

Occurrence of alpha 2-8 linked polysialosyl units in a neural cell adhesion molecule.

A brain cell surface protein (BSP-2) was isolated from mice of different ages by affinity chromatography using a monoclonal antibody. Analysis of glycopeptides obtained after pronase digestion revealed that the embryonal and neonatal forms of the antigen contained an unusually high proportion of sialic acid, which decreased during development. Methylation analysis of native and neuraminidase treated glycopeptides indicated that the sialic acid occurred as alpha 2-8 bound polysialosyl units, similar to those of the recently described developmentally regulated polysialosyl glycopeptides of rat brain. Furthermore, the carbohydrate and amino acid composition, and electrophoretic mobility of BSP-2 antigen correspond to those reported for a neural cell adhesion molecule (N-CAM).

Aging↗
PubMed1983

Molecular heterogeneity and structural evolution during cerebellar ontogeny detected by monoclonal antibody of the mouse cell surface antigen BSP-2.

The monoclonal antibody anti-BSP-2 defines a set of glycoproteins present on the neuronal cell surface in dissociated mouse cerebellar cultures and on neurons and astrocytes in sections of the mouse cerebellum. This antibody was used in the present study to characterize the antigens recognized in cerebellar cultures and in the developing and adult mouse cerebellum in vivo. In extracts from cerebellar cultures and from late postnatal or adult cerebellum, the anti-BSP-2 antibody reacted with a triplet of glycosylated polypeptide chains of 180,000, 140,000 and 120,000 mol. wt. Early postnatal cerebellum contained a different form of BSP-2 antigen which migrated as one broad or several closely spaced diffuse bands in the 190,000-250,000 mol. wt. region of SDS polyacrylamide gels. During cerebellar ontogeny, the adult pattern emerged gradually between postnatal days 5 and 13. The cellular expression of the BSP-2 antigen was studied by immunohistochemistry on sections of the developing cerebellum. At postnatal day 3, the antigen was found mainly on cell bodies and fibers of the Bergmann glia and on astrocytes of the granular layer. Immature granule cells of the outer zone of the external granular layer lacked the antigen, but they appeared to acquire the antigen during their migration to the internal granular layer. At postnatal day 13, the immunofluorescence pattern was not different from the one seen in the adult. These results suggest that the neonatal 190,000-250,000 mol. wt. form of BSP-2 may at least in part be expressed by astroglial cells and they show a close correlation between the emergence of the adult forms of the antigen and the appearance of labeled granule cells in the internal granular layer. In vitro degradation implying cleavage of sialic acid residues, but probably also proteolysis and/or cleavage of different glycans converted the neonatal form of BSP-2 into the triplet pattern and ultimately into a p120 component. Neuraminidase digestion of the adult antigens produced small molecular weight shifts without converting one band into the other, but endogenous enzyme activities were capable of degrading the p180 and p140 bands by converting them into the p120 protein. Our findings support the idea that distinct, but structurally similar surface glycoproteins created by post-translational modifications from a common precursor molecule may be expressed by different cell types or during different developmental stages. As shown by sequential immunoprecipitation experiments, BSP-2 and the rat neuronal membrane protein D2 may belong to the same family of surface glycoproteins.

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PubMed1983

Recognition of sodium- and potassium-dependent adenosine triphosphatase in organs of the mouse by means of a monoclonal antibody.

The antigen detected by the rat anti-mouse monoclonal antibody (m Ab), anti-BSP-3, has been initially described as a brain cell-surface protein. Evidence is presented that this m Ab recognizes mouse (Na+ + K+)-ATPase (ATP phosphohydrolase, E.C.3.6.1.3). The antigen, purified from mouse brain by means of affinity chromatography, migrated in SDS-polyacrylamide gels in the form of two polypeptide chains of 100 000 and 48 000 molecular weight, which could be shown to react with subunit-specific polyclonal antisera against ATPase in immunoblotting experiments. Purified BSP-3 antigen was bound to the specific (Na+ + K+)-ATPase inhibitor ouabain. Finally, the anti-BSP-3 m Ab was capable of immunoprecipitating the ATPase activity of a microsomal fraction from mouse kidney. The m Ab was used to study the localization of (Na+ + K+)-ATPase in different organs of the mouse. It stained the basolateral plasma membranes of polarized cells in immunofluorescence experiments, while the entire cell surface of unpolarized cells was labeled. Interestingly, several cell types did not react with the m Ab, indicating a possible heterogeneity of ATPases. Such a m Ab could prove to be a useful tool for studying localization, structure and function of (Na+ + K+)-ATPase.

Animals↗
PubMed1983

Mouse monoclonal anti-Ia antibodies recognize cross-reacting determinants expressed on distinct subsets of human Ia-like cell-surface molecules.

Human Ia-like cell-surface molecules from a homozygous HLA-DR (6/6) B lymphoblastoid cell line have been analyzed using five mouse anti-Ia m.Ab cross-reacting with HLA-DR antigens. The surface-iodinated molecules immunoprecipitated by these m.Ab were analyzed by SDS-PAGE under reducing conditions and by SDS-PAGE followed by isoelectrofocusing. As read from the different migration patterns, three distinct combinations of human Ia-like molecules were identified by these m.Ab. Three anti-I-E-reactive m.Ab immunoprecipitated two-chain molecules whose apparent mol. wt (32K, 29K) corresponded to those of the classical HLA-DR antigens. One m.Ab which on mouse cells recognized a determinant shared by the I-A and I-E molecules precipitated not only the 32-29K bands, but also a 26K band from human cell extracts. Finally, an I-A reactive m.Ab precipitated a complex set of polypeptides including in addition to the 32-29K bands, three additional chains of 30, 28 and 26K. Sequential immunoprecipitation demonstrated that removal of the classical 29-32K HLA-DR chains by an anti-I-E m.Ab did not affect the subsequent immunoprecipitation of the additional chains by the anti-I-A or the anti-I-A + I-E m.Abs. These patterns and those obtained by 2D-gels analysis which demonstrated the complexity of the 26K band are compatible with the coexpression of at least three different subsets of molecules: (1) Ia-like molecules of 29-32K, recognized by all the m.Ab used; (b) molecules of 28-30K recognized by the anti-I-A m.Ab and (c) molecules apparently constituted by 26K chains, precipitated by the anti-I-A m.Ab and by the anti-I-A + I-E m.Ab.

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PubMed1983

A monoclonal antibody recognizing subpopulations of neurones in mouse brain.

A monoclonal antibody, designated C138, was raised against a particulate fraction from young postnatal mouse cerebellum. In sections from adult mouse brain and cerebellum, this antibody stained a sub-population of neurons. Cell bodies of large neurones and fibre tracts were negative in all brain regions examined, whereas neuropil was heavily labelled. Immune-electron microscopy confirmed the specificity of the antibody for certain types of neurones and indicated that the antigen may be associated with microtubular structures. Immunofluorescence studies on dissociated cerebellar cultures showed that the antigen was expressed inside all tetanus toxin-positive neurones but not by non-neuronal cells.

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PubMed1983

Monoclonal antibody to neural cell surface protein: identification of a glycoprotein family of restricted cellular localization.

A monoclonal antibody, designated anti-NSP-4 (anti-Neural cell Surface Protein-4), was obtained from a hybridoma generated by fusing rat myeloma cells with splenocytes of a rat immunized with membranes from the cerebella of weaver mutant mice. This antibody reacted with several high-molecular weight polypeptides in extracts prepared from the newborn and adult CNS of wild-type mice. The main NSP-4-reactive bands from neonatal cerebellum and spinal cord migrated with apparent molecular weights of 220,000 and 140,000. Major bands of 160,000 and of 175,000, 160,000 and 140,000 molecular weight were revealed in the adult cerebellum and spinal cord, respectively. Reaction of the antibodies with concanavalin A-binding proteins demonstrated the glycoprotein nature of the antigen. Cell types expressing NSP-4 antigen were determined using indirect immunofluorescence on monolayer cultures of early postnatal mouse cerebellar and dorsal root ganglion cells and on sections of developing and adult mouse cerebellum. In cerebellar cultures, the antibody reacted with the surface membrane of a subpopulation of astrocytes and of a small subset of neurones. In dorsal root ganglion cultures, anti-NSP-4 antibodies were highly specific for a subclass of small neurones. Staining for NSP-4 in sections of adult cerebellum was confined to the granular layer where the antibody seemed to label astroglia. In the developing cerebellum, NSP-4 staining outlined cell bodies of neuroblasts and migrating granule cells in the external granular layer. Post-migratory granule cells and Purkinje cells were negative. As in the adult, the labeled structures in the internal granular layer were probably astrocytes. Our results on the in vivo and in vitro localization of NSP-4 show its expression by subclasses of neurones and astrocytes in the cerebellum and by a subclass of neurones in cultures from the peripheral nervous system. The developmentally-regulated changes in the molecular weight forms of the NSP-4 antigen together with the shift in its cellular localization during cerebellar ontogeny suggest a functional significance for this antigen in developmental processes.

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