Biomedical subjects

Melitta Schachner

Publications and source records attributed to Melitta Schachner.

140 records · Page 8Linked to original sources

PubMed2002

Localization and characterization of polysialic acid-containing N-linked glycans from bovine NCAM.

The neural cell adhesion molecule (NCAM) plays important roles during development, plasticity, and regeneration in the adult nervous system. Its function is strongly influenced by attachment of the unusual alpha 2-8-linked polysialic acid (PSA). Here we analyzed the N-glycosylation pattern of polysialylated NCAM from brains of newborn calves. Purified PSA-NCAM glycoprotein was digested with trypsin, and PSA-glycopeptides were separated by immunoaffinity chromatography. For determining the N-glycosylation sites, PNGase F-treated glycopeptides were analyzed by Edman degradation and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). They were found to be exclusively linked to the fifth (Asn 439) and sixth (Asn 468) N-glycosylation sites in the fifth immunoglobulin-like domain of NCAM. The chain length of PSA consisted of at least 30 sialic acid residues, as shown by anion exchange chromatography. For analysis of the core structures, endoneuraminidase N-treated PSA-NCAM was separated by SDS-PAGE and digested with PNGase F. The core structures of polysialylated glycans were characterized by MALDI-MS combined with exoglycosidase digestions and chromatographic fractionation. They include hybrid, di-, tri-, and small amounts of tetraantennary carbohydrates, which were all fucosylated at the innermost N-acetylglucosamine. For the triantennary glycans, the "2,6" arm was preferred in polysialylated structures. High levels of sulfated groups were found on polysialylated structures and to a lower extent also on nonpolysialylated glycans. In addition, high-mannose-type glycans could be detected on PSA-NCAM glycoforms ranging from (GlcNAc)(2)(Man)(5) up to (GlcNAc)(2)(Man)(9). In conclusion, we observed a structural variability and high regional selectivity for the PSA-glycans attached to the NCAM molecule that are most likely influencing its biological functions.

Animals↗

PubMed2002

Abnormal renal phenotype in L1 knockout mice: a novel cause of CAKUT.

L1, a member of the immunoglobulin superfamily, is a cell adhesion and signal transducing molecule. In the kidney, L1 is expressed in the mesonephric duct and the metanephros throughout collecting duct development. We show that mice with a targeted deletion of the L1 gene display diverse renal malformations including (i) a duplex kidney with two ureters partially or totally separated, accompanied by hydronephrosis; and (ii) an enlarged elongated kidney with a malformed or incorrectly positioned inner medulla. The type, penetrance and severity of these phenotypes are influenced by the genetic background. The development of a duplex kidney is initiated by double ureteral budding from the Wolffian duct or by an accessory budding from the main ureter, whereas medullary malformation is due to an improper growth and branching pattern of ureteral branches. Multiple developmental defects in formation of the collecting system promote subsequent renal damage and progression to renal insufficiency. Various features of mouse ureteral duplication resemble the human congenital anomalies of the kidney and urinary tract (CAKUT) although disturbances of medulla development have not yet been reported in men.

Animals↗

PubMed1992

Cell Type-specific Effects of the Neural Adhesion Molecules L1 and N-CAM on Diverse Second Messenger Systems.

We have previously shown that the neural adhesion molecules L1 and N-CAM influence second messenger systems when triggered with specific antibodies at the surface of the phaeochromocytoma PC12 cell line (Schuch et al., Neuron, 3, 13 - 20, 1989). To determine whether the two molecules are linked to the same intracellular signalling cascades, independent of the cell type expressing them, or whether different neural cell types respond with different signal transduction mechanisms, we have investigated the effects of antibodies to L1 and N-CAM, and the isolated molecules themselves, on second messenger systems in different neural cell types. We have investigated cultures of cerebellar and dorsal root ganglion neurons and transformed Schwann cells and related these results to those obtained with the PC12 cell line. Here we show that addition of L1 and N-CAM antibodies and the isolated molecules themselves elicit cell type-specific responses that can be modulated by the substrate on which the cells are maintained. Depending on the cell type, cells respond to the triggering of L1 and N-CAM with antibodies, or addition of the purified molecules, by either up-regulation or down-regulation of inositol phosphate turnover, by a rise in intracellular Ca2+ levels dependent on or independent of the opening of voltage-gated Ca2+ channels, or by an increase or decrease in intracellular pH. Moreover, cerebellar neurons expressing N-CAM respond to addition N-CAM, but not to N-CAM antibodies, in contrast to the other neural cell types studied, which respond to both triggers. Furthermore, cerebellar neurons were the only cells to show a rise in cAMP levels in response to any of the ligands tested. This stimulation of cAMP production by L1 antibodies depended on the cross-linking of L1 molecules at the cell surface, whereas the other responses did not depend on clustering of L1. Simultaneous addition of L1 and N-CAM antibodies either elicited an additive or more than additive effect on the intracellular responses which, for cerebellar neurons, depends on the substrate on which the cells are maintained. These observations indicate that L1 and N-CAM or their antibodies activate cell type-specific intracellular signalling systems and that the two molecules can act interdependently or independently of each other.

Journal Article↗

PubMed1992

Immunohistological Localization of Tenascin in the Developing and Lesioned Adult Mouse Optic Nerve.

To gain insight into the morphogenetic functions of the recognition molecule tenascin in the central nervous system, we have studied its localization in the developing and lesioned adult mouse optic nerve using light and electron microscopic immunocytochemistry. Since tenascin is a secreted molecule, we have analysed the tenascin-synthesizing cells in tissue sections of retinae and optic nerves by in situ hybridization. A weak and homogeneous tenascin immunoreactivity was detectable in the developing retinal nerve fibre layer and optic nerve of 14-day-old mouse embryos, the earliest developmental age investigated. In the optic nerve of neonatal and 1-week-old animals, a high number of tenascin messenger RNA (mRNA)-containing cells were present, and antibodies to tenascin labelled the surfaces of astrocytes and unmyelinated retinal ganglion cell axons. With increasing age, expression of tenascin in the optic nerve was down-regulated at the mRNA and protein levels. At the fourth postnatal week, blood vessels in the optic nerve and collagen fibrils in the vicinity of meningeal fibroblast-like cells still showed significant immunoreactivity, but the optic nerve tissue proper no longer did so. In adult animals, tenascin was no longer detectable in association with blood vessels located in the myelinated part of the optic nerve, and meninges were only weakly immunoreactive. Also, tenascin mRNA-containing cells were no longer detectable in the myelinated part of the adult mouse optic nerve and few labelled cells were found in the meninges. In the retina, ganglion cells contained no detectable levels of tenascin mRNA at any of the developmental ages analysed. No significant up-regulation of tenascin expression was seen in the nerve tissue proper of transected proximal (i.e. retinal) and distal (i.e. cranial) optic nerve stumps of adult mice during the first 4 weeks after lesioning, the time period studied. However, collagen fibrils associated with meningeal fibroblast-like cells and located near the lesion site became strongly tenascin-immunoreactive 2 days after lesioning. Also, some blood vessels at the lesion site became immunoreactive. We conclude that tenascin in the optic nerve is synthesized by glial cells and not by retinal ganglion cells. The detectability of tenascin at embryonic ages suggests that it may mediate neurite growth in vivo. The absence of a strong, lesion-induced up-regulation of tenascin expression in the regeneration-prohibitive mouse optic nerve contrasts with the lesion-induced pronounced up-regulation in the regeneration-permissive peripheral nervous system, and may indicate a functional involvement of tenascin in regenerative processes. The high tenascin positivity of collagen fibrils at early postnatal ages and after lesioning suggests that tenascin expression may be correlated with mitotic activity of the associated meningeal fibroblast-like cells. Finally, tenascin may be involved in the process of vascularization, since the molecule is associated with blood vessels in developing and adult lesioned, but not intact adult, optic nerves.

Journal Article↗

PubMed1992

Expression of the Neural Recognition Molecule L1 by Cultured Neural Cells is influenced by K+ and the Glutamate Receptor Agonist NMDA.

To investigate the influence of neuronal activity on the expression of neural recognition molecules, cultures of neural cell lines and dissociated cells of early postnatal mouse cerebellum were maintained in the presence of elevated concentrations of K+ and the glutamate agonist N-methyl-d-aspartate (NMDA). Levels of expression of the neural adhesion molecules L1 and N-CAM at the cell surface were measured by an enzyme-linked immunosorbent assay. Expression of L1 was up-regulated in neuroblastoma N2A cells after 1 day of maintenance in 40 and 60 mM K+, but not in phaeochromocytoma PC12 cells. Expression levels of N-CAM and antigens recognized by the monoclonal antibody A2B5 or by polyclonal antibodies to crude membrane fractions of liver were not significantly altered by elevated K+ concentrations in these two cell lines. In monolayer cultures of early postnatal mouse cerebellum, an increase of 60% in expression of L1, but not N-CAM or A2B5, was seen at 20 and 40 mM K+. This increase in L1 expression was specifically inhibitable by the Ca2+ channel blocker nicardipine. NMDA at a concentration of 100 microM increased levels of L1, but not of N-CAM. This increase was inhibitable by the NMDA antagonists 2-amino-5-phosphonovalerate and MK-801, but not significantly by the kainate/quisqualate antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. The increase in L1 expression at higher K+ concentrations was not inhibitable by the NMDA antagonists, indicating that the K+-mediated increase in L1 expression is not due to release of glutamate by cerebellar neurons. These observations indicate that compounds influencing neuronal membrane properties, and thus neuronal excitability, are capable of regulating the expression of L1. In a more general context, these findings suggest that previously observed changes in synaptic connectivity in situ, resulting from activity-dependent fine tuning of neuronal morphology, may be mediated by alterations in the expression of recognition molecules.

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PubMed1991

The Neural Cell Adhesion Molecule (N-CAM) Modulates K+ Channels in Cultured Glial Precursor Cells.

Application of antibodies against the neural cell adhesion molecule (N-CAM) to O4-positive murine glial precursor cells in vitro results in a reduction of two distinct K+ currents measured using the whole cell patch clamp technique. Both the A-type and delayed rectifier K+ currents are reduced in amplitude within a few minutes of the application of poly- or monoclonal antibodies against N-CAM. This effect is not due to the binding of any antibody to the surface of the glial precursor cells because monoclonal antibody directed against the O4 surface antigen, or polyclonal antibodies directed against liver cell membranes (which also bind to the surface of glial precursor cells), do not affect membrane currents. Activators of protein kinase C, such as phorbol esters or diacylglycerol, also induce changes in potassium currents that appear, both in magnitude and kinetics, to be similar to those induced by antibodies against N-CAM. In contrast, activation of G proteins upregulates K+ currents. Glial precursor cells thus respond to triggering of N-CAM by altering channel properties. These observations suggest that adhesive events between neural cells can influence the intracellular ionic milieu.

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PubMed1991

Divalent Cations Modulate the Inhibitory Substrate Properties of Murine Glia-derived J1-160 and J1-180 Extracellular Matrix Glycoproteins for Neuronal Adhesion.

J1-160 and J1-180 are developmentally late appearing J1 extracellular matrix glycoproteins derived from oligodendrocytes. They prevent adhesion of neurons (but not of astrocytes or fibroblasts) when offered as a substrate in mixture with laminin (Pesheva et al., J. Cell Biol., 109, 1765 - 1778, 1989). In the present study we have examined the influence of divalent cations on the inhibitory substrate properties of J1-160/180 glycoproteins towards adhesion of neurons. By metal chelate affinity chromatography, we show that J1-180, but not J1-160, binds Ca2+, while both J1 components are capable of binding Zn2+ and other divalent metal ions. Divalent cation binding was observed by gel filtration, aggregation assays with coated latex beads and electron microscopic examination to elicit aggregation of the molecules. Divalent cation binding also affects their non-permissive substrate properties towards neurons from early postnatal mouse cerebellum. Without divalent cations, J1-160 and J1-180 are inhibitory for substrate adhesion of neurons independently of the adhesive substrate present (laminin or poly-l-lysine). This effect is neutralized when J1-180 is preincubated with Ca2+ or Zn2+ prior to coating as substrate. In contrast, preincubation with Ca2+ ions does not affect the inhibitory substrate properties of J1-160 under these conditions. These observations show that J1-160/180 molecules may undergo self-aggregation in a divalent cation-dependent mechanism, which correlates with the neutralization of their inhibitory effect on neuronal adhesion. The aggregation state of the molecules may thus influence the process of myelination by a homophilic binding mechanism and determine the effectiveness of neurite extension during central nervous system development and under traumatic conditions in the adult.

Journal Article↗

PubMed1991

Molecular Association of the Neural Adhesion Molecules L1 and N-CAM in the Surface Membrane of Neuroblastoma Cells is Shown by Chemical Cross-linking.

The two neural cell adhesion molecules L1 and N-CAM could be shown to be associated in the surface membrane of cultured neuroblastoma cells by chemical cross-linking with 3,3'-dithiobis(sulphosuccinimidyl-propionate) and subsequent immunopurification and precipitation using antibodies to L1 and N-CAM. Glycoproteins recognized in neuroblastoma cells by antibodies to mouse liver membranes were not chemically cross-linked to L1 or N-CAM. These observations suggest that a molecular association between the two molecules may be the basis for their functional cooperativity (Kadmon et al., 1990a,b, J. Cell Biol., 110, 193-208; 209-218).

Journal Article↗

PubMed1990

Accumulation of N-CAM 180 at Contact Sites Between Neuroblastoma Cells and Latex Beads Coated with Extracellular Matrix Molecules.

Neuronal cells expressing neural cell adhesion molecule (N-CAM) accumulate the largest N-CAM component (N-CAM 180) at cell - cell contact sites. To test whether this accumulation is induced by interactions at the surface membrane, latex beads coated with several purified adhesion molecules or extracellular matrix (ECM) components were co-cultured with neuroblastoma cells. Beads coated with L1, N-CAM, the L2/HNK-1 carbohydrate epitope-carrying molecules from adult mouse brain or laminin from Engelbreth-Holm-Swarm (EHS) sarcoma did not induce an accumulation of N-CAM 180 or L1 at sites of contact suggesting that these molecules are not directly involved in N-CAM 180 accumulation or that their mobility is required for this process. Beads coated with ECM components of the PF-HR9 cell line induced accumulation of N-CAM 180 at sites of contact with neuroblastoma cells. Accumulation was seen at cell bodies of undifferentiated and differentiated neuroblastoma cells, as well as on neurites and growth cones of differentiated neuroblastoma cells. Accumulation of the neural adhesion molecule L1 was also seen, but less prominently and reproducibly. These observations suggest that molecules of the ECM can directly or indirectly, e.g. via molecules linked to N-CAM 180 on the cell surface, induce accumulation of N-CAM 180.

Journal Article↗

PubMed1990

Two Monoclonal Antibodies Recognizing Carbohydrate Epitopes on Neural Adhesion Molecules Interfere with Cell Interactions.

We have studied two monoclonal antibodies raised against crude fractions of membrane glycoproteins from adult mouse brain and found them to react with two carbohydrate epitopes expressed on several neural cell adhesion molecules. Other identified and unidentified glycoproteins from different cell types, organs and species were also recognized by these antibodies. Both epitopes are N-glycosidically linked mannosidic or hybrid type oligosaccharides and co-expressed on all the glycoproteins so far tested. In spite of their remarkable similarities, the glycan epitopes are different as shown by ELISA competition assays. In microexplant outgrowth and cell adhesion assays, both antibodies interfere with neural cell adhesion, migration, and neurite outgrowth. These observations, together with previous studies on the L2/HNK-1 glycan (Künemund et al., 1988), indicate that adhesion molecules carry various carbohydrate epitopes mediating different cell interactions in in vitro assays.

Journal Article↗

PubMed1990

Lateral Mobility of the Cell Adhesion Molecule L1 Within the Surface Membrane of Morphologically Undifferentiated and Differentiated Neuroblastoma Cells.

Lateral mobility and localization in the surface membrane of the adhesion molecule L1 was studied in morphologically undifferentiated and differentiated neuroblastoma cells to gain insight into its possible association with the different molecular forms of N-CAM. In undifferentiated cells, the fraction of mobile L1 molecules is high and similar to that of N-CAM 140. Upon long-term morphological differentiation, the fraction of mobile L1 molecules is reduced by a factor of three and is similar to that of N-CAM 180, the predominant molecular form of N-CAM in differentiated neuroblastoma cells. Comparable to N-CAM 180, L1 is also preferentially accumulated at contact sites between these cells as seen by indirect immunofluorescence. These observations raise the question of whether at least part of the L1 molecules may be directly or indirectly (e.g. via N-CAM 180) linked to the cytoskeleton, thus stabilizing cell contacts between differentiated cells.

Journal Article↗

PubMed1990

[Alternatives to Animal Experimentation in University Education: Principles of Neurophysiology Exemplified by the Computer]

The basic physiology courses for students in biology and medicine include experiments on live animals or animal tissue. This article describes alternatives by replacing such experiments with simulation programs on personal computers. Programs for a neurophysiological experiment were developed at the Department of Neurobiology, University of Heidelberg, within the last years and have served to replace animal experiments in the basic training of biologists.

Journal Article↗

PubMedDate not supplied

Localization of defined carbohydrate epitopes in bovine polysialylated NCAM.

Polysialylated neural cell adhesion molecule (NCAM) was immunoaffinity-purified from the brains of newborn calves. A degree of polymerization of up to 40 was chromatographically determined for released polysialic acid (PSA) chains. For characterization of N-glycan structures and attachment sites, PSA-NCAM was digested with trypsin, and the generated glycopeptides were fractionated by serial immunoaffinity chromatography using immobilized monoclonal antibodies specific for PSA or the HNK1 epitope, i.e., HSO(3)-3GlcA(beta 1-3)Gal(beta 1-4)GlcNAc(beta 1-, yielding PSA-glycopeptides, HNK-glycopeptides and non-PSA/HNK1-(glyco) peptides. Using a combination of enzymatic deglycosylation, peptide fractionation, mass spectrometry and Edman degradation, HNK1-N-glycans could be assigned to glycosylation sites 2, 4, 5 and 6. Non-PSA/HNK1-glycans were assigned to glycosylation site 2, whereas PSA-N-glycans of bovine NCAM had been already previously shown to be restricted to glycosylation sites 5 and 6 (Glycobiology 12 (2002) 47). Respective oligosaccharides were enzymatically released, labeled with 2-aminopyridine and characterized by linkage analysis and mass spectrometry. Carbohydrate chains bearing PSA or the HNK1 epitope comprised mainly fucosylated, partially sulfated diantennary, triantennary or tetraantennary glycans without bisecting GlcNAc or fucosylated diantennary and triantennary species carrying, in part, bisecting GlcNAc residues, respectively. Some N-glycans simultaneously contained both the HNK1-epitope and PSA. Non-PSA/HNK1-glycans exhibited a heterogeneous pattern of partially truncated, mostly diantennary structures with one to three fucose residues, bisecting GlcNAc and/or sulfate residues. In addition, they were demonstrated to carry, to some extent, the Lewis X epitope. When compared with previous data on murine NCAM glycosylation, our results indicate a conservation of structural features and attachment sites for the different types of NCAM N-glycans.

Animals↗

PubMedDate not supplied

Absence of L1 in pancreatic masses distinguishes adenocarcinomas from poorly differentiated neuroendocrine carcinomas.

BACKGROUND: Pancreatic adenocarcinoma is a tumor with fatal outcome. Cell adhesion molecules, such as L1 (CD171), have an essential function in tumor progression. L1 has been shown to be specifically expressed in poorly differentiated neuroendocrine carcinomas of the pancreas. The aim of this study was to determine the expression of L1 in pancreatic adenocarcinomas to evaluate whether L1 might differentiate between pancreatic carcinomas of neuroendocrine and ductal origin. MATERIALS AND METHODS: L1 expression was retrospectively analyzed in 111 cases of pancreatic adenocarcinomas by immunohistochemistry on paraffin sections of primary tumors. Staining was performed by the peroxidase technique with monoclonal antibody against human L1. All tumors were classified according to the most recent TNM classification. RESULTS: The focal expression of L1 was detected in 2 (2%) out of 111 pancreatic carcinomas only, the remaining 109 (98%) being L1-negative. No expression was found in acinar or ductal cells of normal pancreatic tissue. CONCLUSION: Our data suggest that L1 is expressed in few cases of pancreatic ductal adenocarcinoma. Since L1 was previously found to be expressed specifically in neuroendocrine pancreatic carcinomas, its absence in unclear pancreatic masses might hint at a ductal origin for a malignant pancreatic tumor.

Adenocarcinoma↗