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Biomedical subjects

M Hirn

Publications and source records attributed to M Hirn.

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

Animals↗
PubMed1984

Identification of a plasma membrane protein involved in Pi transport in the yeast Candida tropicalis.

A plasma membrane protein was found to contain antigenic determinants in common with a Pi-binding protein released by osmotic shock from Candida tropicalis. This plasma membrane protein (M.P. 30) has a molecular mass of 30 kDa as measured by SDS-PAGE and anti-M.P. 30 antibodies inhibit Pi uptake in protoplasts while only the corresponding Fab fragments inhibit Pi transport in whole cells. This plasma membrane protein may be the Pi plasma membrane carrier.

Antibodies↗
PubMed1983

Antigenic relationships between plasma membrane ATPases of two different yeasts, Candida tropicalis and Schizosaccharomyces pombe.

Antibodies raised against purified plasma membrane ATPase from the yeast S. pombe inhibit the plasma membrane-bound ATPase of another yeast, C. tropicalis. The kinetic constants, Vm and Km, of the ATPases are both modified by the antibodies. These results show antigenic relationships between the plasma membrane ATPases belonging to the two yeast genera.

Adenosine Triphosphatases↗
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.

Animals↗
PubMed1983

Anti-cell surface monoclonal antibodies which antagonize the action of VIP in a human adenocarcinoma cell line (HT 29 cells).

Hybridoma cells have been obtained by fusing RCY 3 Ag 1-2-3 rat myeloma cells with spleen cells from a rat hyperimmunized with human adenocarcinoma cells (HT 29 cell line) grown in serum-free medium. Immunoglobulins secreted by hybridomas were screened for: (i) specific binding to HT 29 cells; (ii) their ability to inhibit the binding of [125I]-vasoactive intestinal peptide (VIP) to HT 29 cells; (iii) their capacity to modulate the cAMP production induced by VIP. The monoclonal antibodies we have obtained from clones 109-10-16 and 109-10-19 compete for the binding of radiolabelled VIP to HT 29 cells and partially inhibit the production of cAMP induced by VIP while they are ineffective in reducing the intracellular level of cAMP attained after stimulation of HT 29 cells by isoproterenol. We never found antibodies which increase the cAMP level in HT 29 cells. The binding of the purified monoclonal antibody 109-10-16 Ig gamma 2c to HT 29 cells was visualized by indirect immunofluorescence and was not present at the surface of all cells. These observations strongly support the hypothesis that the monoclonal antibodies we have characterized interact with an antigenic determinant which belongs to the VIP receptor or at least to a cell surface component closely associated with the receptor.

Adenocarcinoma↗
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.

Animals↗
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.

Animals↗
PubMed1982

Tissue- and developmental stage-specific forms of a neural cell surface antigen linked to differences in glycosylation of a common polypeptide.

We have previously identified a cell surface glycoprotein of the mouse nervous system named brain cell surface protein-2 (BSP-2). Here we report that this antigen is not a single, discrete entity, but a family of antigenically and structurally related molecules. Three components of 180, 140, and 120 K were characteristic for more mature nervous tissues. Adult cerebral cortex contained the 140-K and 120-K antigens, adult spinal cord only the 120-K, and dorsal root ganglia from young mice mainly the 180-K component. Very different forms of the antigen that migrated as a diffuse zone from 180-250-K in SDS-polyacrylamide gels were found in immature nervous tissues. A molecule different from the previous ones was found in a neuroblastoma line. Evidence is presented that the structural diversity of BSP-2 is due to differences in glycosylation. This result indicates that cell type- and developmental stage-specific glycoprotein patterns previously found in the nervous system may in part be due to different glycosylation of identical polypeptides. The finding that a neural cell surface protein may be glycosylated in different ways has important implications for the generation of cell surface specificity.

Aging↗
PubMed1982

Monoclonal antibodies as neural cell surface markers.

A comparison is made of the immunohistochemistry at the ultrastructural level of three monoclonal antibodies directed against surface components of DNS cells. Hybridomas secreting these antibodies were obtained from two cell fusions of a rat myeloma cell line and immune splenocytes derived from rats immunized either with primary mouse brain cultured cells or membrane components. In cultures one antibody, anti-BSP2 (Brain Surface Protein-2), was preferentially directed against neurons while another, anti-BSP-3 (Brain Surface Protein-3), preferentially labeled astrocytes. In mouse cerebellar sections, both labeled the surface of Purkinje cells, granule cells and astrocytes. In addition a cytoplasm localization was apparent in granule cells and astrocytes. Another antibody anti-MESA-1 (Mouse Endothelial Surface Antigen-1) reacted exclusively with the surface of endothelial cells lining blood vessels. These data are discussed with reference to the biochemical nature of the corresponding antigens and to known glycoproteins of neural cell membranes.

Animals↗
PubMed1982

A new brain cell surface glycoprotein identified by monoclonal antibody.

Of 207 monoclonal antibodies produced against cultured mouse cerebellar cells, 16 reacted with cerebellar cell surfaces and 4 reacted with glycoproteins. One of them, called an anti-BSP-3 (Brain cell Surface Protein-3) defines a 48,000 molecular weight protein which can be iodinated at the surface of cultured cerebellar cells. Lectin-binding and sugar incorporation studies established the glycoprotein nature of the antigen. Astroglia (glial fibrillary acidic protein-positive cells) in primary cerebellar cultures were labelled intensely for this antigen by the indirect immunofluorescence method while neuronal cells and their processes were more weakly labelled. Fibronectin-positive cells were negative for BSP-3. In cerebellar sections using the immunoperoxidase method at both the optical and electron microscope levels, the difference in staining intensity between astrocytes and neuronal cells was not significant: in Purkinje cells and in the large neurones present in the deep cerebellar nuclei the immunoperoxidase percipitate was confined to the plasma, membrane while in both astrocytes and granule cells cytoplasmic labelling was also observed. Oligodendrocytes do not appear to react with the anti-BSP-3 monoclonal antibody; neither do endothelial or leptomeningeal cells. The availability of a monoclonal antibody produced by a stable hybridoma line will be a powerful tool in attempts to purify the BSP-3 antigen and to elucidate its function.

Animals↗
PubMed1982

Effect of maternal cigarette smoke exposure on foetuses of inbred strains of mice responsive and non-responsive to polycyclic aromatic hydrocarbons.

To evaluate the role of genetically controlled responsiveness to polycyclic aromatic hydrocarbons (PAH) in the effect of cigarette smoke on birth weight, pregnant mice were exposed to cigarette smoke from day 1 to day 17 of pregnancy and the offspring studied on day 18. An unequivocal phenotyping of foetuses with respect to responsiveness was achieved by measuring the activity of aryl hydrocarbon hydroxylase (AHH) in the foetal liver and corresponding placenta after pretreatment of the dam with beta-naphthoflavone. The foetuses of dams exposed to standard non-filter cigarettes were consistently smaller than those of the controls, whereas smoke from commercial filter cigarettes was only marginally effective. No statistically significant association with the responsiveness to PAH could be found with respect to this effect on weight. These results suggest that genetically determined responsiveness to PAH, although of importance for PAH foetotoxicity in mice, is not important in the effect of cigarette smoke on birth weight in the strains studied.

Animals↗
PubMed1982

Detection of ecdysteroids in the human trematode, Schistosoma mansoni.

Freeze fracture study of Schistosoma mansoni membrane differentiation enabled us to describe the characteristic features of the tegumental membrane complex at various stages of development of this parasite. The observation of a membrane exuviation during the 10-20 day period of infection of the definitive host led us to search for possible hormonal control. The development of an appropriate radioimmunoassay has allowed the detection of significant production of ecdysteroids in a human platyhelminth. This hormonal material undergoes level variation during the development of post-cercarial stages of S. mansoni and its precise chemical characterization is presently being investigated.

Animals↗
PubMed1982

A surface marker for murine vascular endothelial cells defined by monoclonal antibody.

Spleen cells from a rat immunized with mouse cerebellar cells were fused with mouse myeloma cells. One of the hybridomas secreted a monoclonal antibody that reacts with a surface antigen on vascular endothelial cells. The antibody stained endothelial cells lining blood vessels in brain, heart, lung, kidney, and liver. It did not, however, stain endothelial cells lining hepatic sinusoids. Parenchymal cells were always negative. So far, an antigen of similar tissue distribution has not been described in the mouse and we have called it mouse endothelial surface antigen-1 (MESA-1). The antibody could be used as a highly specific usefulness for identifying endothelium-derived cells in culture has been demonstrated on cultures of dissociated mouse cerebellum, where it stained a subclass of fibronectin-expressing cells.

Animals↗
PubMed1981

Monoclonal antibody against cell surface glycoprotein of neurons.

A monoclonal antibody, named BSP-2, has been produced against glycoproteins extracted from neonatal mouse brain. Its reactivity with live cells established the surface location of the antigen. In primary cultures of dissociated cerebellar cells, the antibody bound to neuronal cell types, but not to astrocytes nor to fibroblasts. Immunoprecipitates prepared with the BSP-2 antibody contained a triplet of high-molecular weight glycoproteins with apparent molecular weights of 180,000, 140,000 and 120,000.

Animals↗
PubMed1981

A solid-phase radioimmunoassay for detecting antibodies to brain cell surface antigens.

A trace radioimmuno-binding assay is described which utilizes microcultures of dissociated cerebellar cells from neonatal mice as targets for the binding of antibodies to surface antigens on normal brain cells. This microassay is easy to perform, highly sensitive and economical in terms of cells and antibodies. Results are presented showing its specificity for antigens accessible at the cell surface. For the assay, dissociated cells are cultured in polylysine-coated wells of 60-well Terasaki microtest plates. After a recovery period allowing extensive outgrowth of neurites, the cultures are incubated with antibody solution, and bound antibody is revealed by an iodinated second antibody. This monolayer binding assay is particularly suited to the screening of large numbers of samples, for instance when trying to raise monoclonal antibodies against brain cells by the hybridoma methodology. The test can also be used for the analysis of anti-surface specificities in conventional antisera.

Animals↗