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F S Walsh

Publications and source records attributed to F S Walsh.

At least 127 records · Page 7Linked to original sources
PubMed1991

Neurite outgrowth in response to transfected N-CAM and N-cadherin reveals fundamental differences in neuronal responsiveness to CAMs.

Different neuronal populations were used to compare the neurite outgrowth-promoting activities of N-CAM and N-cadherin expressed via gene transfer on the surface of nonneuronal cells. In contrast to a previously reported developmental loss of retinal ganglion cell responsiveness to N-CAM, these cells exhibited an increased and maintained responsiveness to N-cadherin over the same developmental period (E6-E11). N-CAM and N-cadherin responses could be specifically inhibited by their own antibodies, but not by antisera to the beta 1 integrin family or the L1/G4 glycoprotein. Cerebellar neurons showed qualitative differences in the nature of the dose-response curves for transfected N-CAM expression (highly cooperative) versus N-cadherin expression (linear). In addition "subthreshold" levels of N-CAM expression, which do not normally support neurite outgrowth, did so when coexpressed with functional levels of N-cadherin. These studies show fundamental differences in neuronal responsiveness to cell adhesion molecules and suggest a more dynamic regulation for N-CAM-dependent neurite outgrowth than for N-cadherin-dependent outgrowth.

Animals↗
PubMed1991

The contrasting roles of N-CAM and N-cadherin as neurite outgrowth-promoting molecules.

The neural cell adhesion molecule (N-CAM) is a prominent member of the immunoglobulin gene superfamily of recognition molecules. It operates in a calcium-independent manner to promote cell-cell adhesion. Alternative splicing of a single gene generates more than twenty N-CAM isoforms and these can be further modified by the differential addition of complex N- and O-linked carbohydrates. In contrast, N-cadherin is a major calcium-dependent adhesion molecule in the brain; it is not a member of the immunoglobulin gene superfamily and, as far as we know, exists as a single gene product with no evidence of differential post-translational modification. Both molecules are believed to operate through a homophilic binding mechanism and both are expressed at key developmental times in a number of tissues including the brain. Antibody perturbation experiments suggest that both of the above cell adhesion molecules (CAMs) can support neurite outgrowth over complex cellular substrata such as astrocytes and Schwann cells. In the present review we discuss the use of a molecular genetic approach to study the neurite outgrowth-promoting activity of these molecules. Using this approach we have found that both CAMs are potent inducers of neurite outgrowth from a variety of neurons. However, whereas a critical value of N-CAM expression is required for increased neurite outgrowth, with small increases above this value having substantial effects, N-cadherin promotes neurite outgrowth in a highly linear manner. In addition, whereas N-CAM promotes chick retinal ganglion cell (RGC) neurite outgrowth at E6 but not E11, N-cadherin does so throughout this developmental period.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Characterization of the human N-CAM promoter.

In contrast with the complex series of splicing choices that generate the various membrane-associated isoforms of the neural cell-adhesion molecule alternative splicing of 5' exons does not contribute to additional molecular diversity. A single regulatory unit in genomic DNA, mapping to a 5 kb restriction-endonuclease-HindIII fragment, controls the expression of all major RNA size classes. DNA sequence analysis of a 2 kb fragment spanning the two major identified transcriptional initiation sites (194 and 188 bp from the ATG codon) and translation start codon indicates that the regulatory unit does not possess classical TATA or CCAAT motifs. The region of the putative promoter exhibits a GC-rich content and a high frequency of the dinucleotide CpG, both characteristics of a HTF(HpaII tiny fragments)-island. Introduction of deletion-mutant chimaeric-gene constructs into human and rodent N-CAM-expressing cell lines defines an active promoter region of 467 bp (-144 to -611 bp from the ATG codon). This region of genomic DNA contains consensus sites for the interaction of known transcriptional factors.

Animals↗
PubMed1990

Role of myelin P0 protein as a homophilic adhesion molecule.

Peripheral nervous system myelin is an extension of the Schwann cell's plasma membrane that tightly enwraps axons in many layers and permits nerve impulses to be rapidly conducted. It is not known how these multiple membrane layers are held together in this compact form. Here we present evidence supporting the hypothesis that the extracellular leaflets of myelin are held together by the most abundant protein of myelin of the peripheral nervous system, P0, by homophilic interaction of its extracellular domains. Transfected Chinese hamster ovary cells expressing P0 protein adhere to each other in suspension, to form large aggregates, whereas cells that are identical but which do not express P0 do not. We also show that this aggregation is mediated by homophilic binding between P0-expressing cells and that the apposing plasma membranes of these cells specifically form desmosomes, whereas control transfected cells do not. As the only difference between the two cell populations is the expression of P0, this protein is apparently responsible for the changes in morphology and adhesion in the cells that express it. The idea that P0 is a homophilic adhesion molecule is supported by its inclusion in the immunoglobulin supergene family, all members of which are involved in recognition and/or adhesion.

Animals↗
PubMed1990

Enhanced myogenesis in NCAM-transfected mouse myoblasts.

The fusion of mononucleate precursor myoblasts to form the multinucleated skeletal muscle fibre is proceeded by a series of complex cell-cell interactions but the cell-surface molecules involved in these events have not been characterized. During myogenesis in vivo and in vitro, expression of the neural cell adhesion molecule (NCAM) undergoes an isoform transition that precisely correlates with terminal myoblast differentiation and myotube formation. Altered processing of RNA results in the replacement of the transmembrane NCAM (relative molecular mass, 145,000 (145K) in proliferating myoblasts by a predominant 125K NCAM form linked to glycosyl phosphatidylinositol in myotubes. We now report that mouse myoblasts transfected to constitutively express the human muscle-specific 125K glycosylphosphatidylinositol-linked NCAM isoform more readily fuse to form myotubes. This suggests that NCAM plays a part in myoblast fusion and that the isoform switch may promote this function.

Animals↗
PubMed1990

A threshold effect of the major isoforms of NCAM on neurite outgrowth.

Interactions between recognition molecules on the surface of neuronal growth cones and guidance cues present in the local cellular environment are thought to account for the growth of neurites in the highly stereospecific manner that contributes to correct target cell innervation. In vitro assays have been used to identify candidate molecular components of this system, either directly by demonstrating their ability to promote neurite outgrowth, or indirectly by the ability of specific antibodies to inhibit neurite outgrowth. The role of the neural cell adhesion molecule (NCAM) in pathway finding is not fully understood. Some immunological studies support a positive role; others do not, and it has been reported that purified NCAM does not support neurite outgrowth. We have previously shown that an arbitrary biochemical index of neurite outgrowth, the relative level of immunoreactive neurofilament protein, is increased when human and rat dorsal root ganglion neurons are cultured on monolayers of cells expressing transfected human NCAM. But, the complexity of growth precluded a simple morphological analysis and we did not determine the 'dose-response' relationship between NCAM expression and neuronal response. Here, we report on the morphology of rat cerebellar neurons cultured on monolayers of 3T3 cells transfected with complementary DNAs encoding all of the main NCAM isoforms found in cells such as astrocytes, Schwann cells and skeletal muscle. The data indicate that both transmembrane and glycosyl-phosphatidylinositol linked NCAM isoforms are potent substrates for neurite extension. A critical threshold value of NCAM expression is required for increased neurite outgrowth. Above this threshold, small increases in NCAM induce substantial increases in neurite outgrowth.

Animals↗
PubMed1990

Expression of neural cell adhesion molecule immunoreactivity in hypertrophic myocardium.

Myocardial neural cell adhesion molecule (N-CAM) is temporally regulated, being expressed during cardiac morphogenesis and innervation and suppressed in the adult heart. We have investigated the plasticity of N-CAM expression in hypertrophic muscle using the rat model of chronic hypoxia to selectively induce right ventricular hypertrophy over a 14 day time course. Sarcolemmal and intercalated disc N-CAM immunostaining was more extensive in the ventricular myocardium of hypoxic rats compared to normoxic controls. Quantitative assessment of the immunoreactivity in tissue extracts demonstrated a selective increase in the amount of N-CAM immunoreactivity in the hypertrophic myocardium of the right ventricle of rats exposed to hypoxia and this was associated with an increase of the 125 kDa isoform. We conclude that myocardial hypertrophy may be a factor influencing N-CAM expression in the heart and adhesion molecules may have a role in cardiac remodelling.

Animals↗
PubMed1990

Neurite outgrowth in response to transfected N-CAM changes during development and is modulated by polysialic acid.

We have used monolayers of control 3T3 cells and 3T3 cells transfected with a cDNA encoding human N-CAM as a culture substrate for embryonic chick retinal ganglion cells (RGCs). At embryonic day 6 (E6), but not at E11, RGCs extended longer neurites on monolayers of N-CAM-transfected cells. This loss of RGC responsiveness was not associated with substantial changes in the level of N-CAM expression on RGC growth cones. The neurite outgrowth response from E6 RGCs could be inhibited by removal of N-CAM from the monolayer, by removal of alpha 2-8-linked polysialic acid from neuronal N-CAM, or by antibodies that bind exclusively to chick (neuronal) N-CAM. In contrast, the response was not dependent on neuronal beta 1 integrin function. These data provide substantive evidence for a homophilic binding mechanism directly mediating N-CAM-dependent neurite outgrowth, and suggest that changes in polysialic acid expression on neuronal N-CAM may modulate N-CAM-dependent axonal growth during development.

Animals↗
PubMed1990

N-cadherin gene maps to human chromosome 18 and is not linked to the E-cadherin gene.

cDNA clones encoding the human N-cadherin cell adhesion molecule have been isolated from an embryonic muscle library by screening with an oligonucleotide probe complementary to the chick brain sequence and chick brain cDNA probe lambda N2. Comparison of the predicted protein sequences revealed greater than 91% homology between chick brain, mouse brain, and human muscle N-cadherin cDNAs over the 748 amino acids of the mature, processed protein. A single polyadenylation site in the chick clone was also present and duplicated in the human muscle sequence. Immediately 3' of the recognition site in chick a poly(A) tail ensued; however, in human an additional 800 bp of 3' untranslated sequence followed. Northern analysis identified a number of major N-cadherin mRNAs. These were of 5.2, 4.3, and 4.0 kb in C6 glioma, 4.3 and 4.0 kb in human foetal muscle cultures, and 4.3 kb in human embryonic brain and mouse brain with minor bands of 5.2 kb in human muscle and embryonic brain. Southern analysis of a panel of somatic cell hybrids allowed the human N-cadherin gene to be mapped to chromosome 18. This is distinct from the E-cadherin locus on chromosome 16. Therefore, it is likely that the cadherins have evolved from a common precursor gene that has undergone duplication and migration to other chromosomal locations.

Amino Acid Sequence↗
PubMed1990

Myocardial localization and isoforms of neural cell adhesion molecule (N-CAM) in the developing and transplanted human heart.

Neural cell adhesion molecule (N-CAM) has been implicated in cellular interactions involved in cardiac morphogenesis and innervation. Immunohistochemical techniques and Western blot analysis were used to determine the localization and isoforms of N-CAM in the developing and extrinsically denervated human heart. Myocardial and conducting cells in the fetal heart (7-24 wk gestation) exhibited sarcolemmal immunoreactivity, the major desialo N-CAM isoforms being 150, 145, 120, 115, and 110 kD. N-CAM expression appeared to be downregulated in the myocardium during adult life, with relatively little sarcolemmal immunoreactivity being detected in normal donor tissues. In contrast to the temporal changes observed in the myocardium, both the developing and mature cardiac innervation displayed N-CAM immunofluorescence staining, localized to neuronal cell bodies, nerve fascicles and fibres. Extrinsically denervated cardiac allografts, obtained 2 d to 91 mo after transplantation, showed extensive sarcolemmal and intercalated disc immunostaining and expression of 125-, 120-, and 115-kD isoforms. Tissues from explanted recipient hearts and atrial appendage samples obtained during coronary bypass graft operations were also examined and displayed varying amounts of N-CAM immunoreactivity. We conclude that the expression of N-CAM immunoreactivity and isoforms in the human heart is developmentally regulated and may be modulated by factors such as cardiac innervation and myocardial hypertrophy.

Adult↗
PubMed1989

Monoclonal antibody UJ13A recognizes the neural cell adhesion molecule (NCAM).

Monoclonal antibody (MAb) UJ13A, raised against 16-week-old human foetal brain, recognizes an antigen present on the majority of tissues of neuro-ectodermal origin. The binding profile of this antibody is similar to the known distribution of the neural cell adhesion molecule (NCAM). Although detailed immunohistological and immuno-cytochemical studies with the MAb have been published previously, we demonstrate here, through investigations of selected tissues and cell lines, that the binding profile of an anti-NCAM antiserum is similar to UJ13A. Additional indirect evidence suggesting that UJ13A recognizes NCAM comes from Northern blot analysis of cell lines either binding or not binding UJ13A as determined by indirect immunofluorescence. Only those cell lines known to bind UJ13A express high levels of NCAM mRNA. Western blot analysis of extracts of human brain show that UJ13A recognizes proteins of 180 and 140 kDa in addition to a very weak band of 120 kDa. This data corroborates the suggestion that UJ13A recognizes NCAM as these 3 isoforms of the protein are identified in human brain. Final confirmation of the specificity of UJ13A comes from the study of 3T3 fibroblasts transfected with a cDNA coding for the 125 kDa isoform of human muscle NCAM. UJ13A selectively recognizes these transfectants and binds to a 125 kDa protein isolated from the cells by Western blot analysis. Thus we conclude from these immunological, biochemical and molecular studies that the antigen recognized by the UJ13A MAb is the neural cell adhesion molecule.

Antibodies, Monoclonal↗
PubMed1989

Neural cell adhesion molecule (NCAM) is the antigen recognized by monoclonal antibodies of similar specificity in small-cell lung carcinoma and neuroblastoma.

We describe reagents from 2 workshops which had been identified as recognizing the same or very similar antigens based on their tissue reactivity. Examination of their tissue specificity led us to the conclusion that this was similar to the expression of the neural cell adhesion molecule (NCAM). We also describe the use of a transfection-based assay to show that these reagents do recognize NCAM. 3T3 cells were transfected with a full-length clone of human NCAM. Indirect immunofluorescence studies showed binding of all related antibodies to the transfectants, but not to the control 3T3 cells. In addition, biochemical analysis using certain antibodies in the cluster confirm that they detect NCAM in the transfectants. Our study shows the benefits of using workshops to compare monoclonal antibodies and a molecular approach to define the antigens recognized by such reagents.

Antibodies, Monoclonal↗
PubMed1989

Unmasking N-CAM.

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Animals↗
PubMed1989

An autosomal transcript in skeletal muscle with homology to dystrophin.

The Duchenne muscular dystrophy (DMD) gene has been localized to chromosome Xp21 and codes for a 14-kilobase (kb) transcript and a protein called dystrophin, of relative molecular mass 427,000. Dystrophin is associated with the cytoplasmic face of muscle fibre membranes and its C-terminal domain is thought to mediate membrane attachment. Although N-terminal and central domain structures share common features with other cytoskeletal components, no significant sequence similarity between the C-terminal region of dystrophin and other previously characterized proteins has been described. Here we report that fragments from the C-terminal domain of the DMD complementary DNA detect a closely related sequence which exhibits nucleic-acid and predicted amino-acid identities with dystrophin of approximately 65 and 80%, respectively. The dystrophin-related sequence identifies a 13-kb transcript in human fetal muscle and maps to chromosome 6. Thus, dystrophin may be a member of a family of functionally related large structural proteins in muscle.

Base Sequence↗