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F S Walsh

Publications and source records attributed to F S Walsh.

At least 145 records · Page 8Linked to original sources
PubMed1989

Neural cell adhesion molecule (N-CAM) expression during cardiac development in the rat.

Neural cell adhesion molecule (N-CAM) expression was examined in the rat heart using immunohistochemical and immunochemical techniques. N-CAM immunoreactivity was displayed by myocardial cells from embryonic day E12 and by cardiac nerves when first identified at day E18. Myocardial immunostaining increased up until about postnatal day 1 and then declined rapidly thereafter whereas neural immunoreactivity persisted in the adult. N-CAM cardiac isoforms also exhibited developmental changes from the main embryonic moieties (105 and 145 kDa) to the principal postnatal (125 and 155 kDa) and adult isoforms (125 kDa). Cardiac N-CAM expression is therefore subject to temporal regulation and may modulate cellular interactions in the developing heart.

Animals↗
PubMed1989

K-252a specifically inhibits the survival and morphological differentiation of NGF-dependent neurons in primary cultures of human dorsal root ganglia.

We have measured neurofilament protein expression in cultures of human dorsal root ganglia (DRG) grown in both the presence and absence of nerve growth factor (NGF). Cultures established from DRG of 8-13 weeks gestation contained populations of both NGF-dependent and -independent neurofilament-positive cells. A recently described kinase inhibitor named K-252a inhibited NGF-dependent increases in neurofilament protein expression with half-maximal and maximal inhibition apparent at concentrations of 6 and 25 nM. At up to 50 nM, K-252a had no effect on neurofilament protein expression from NGF-independent neurons.

Carbazoles↗
PubMed1989

Generation of multiple N-CAM polypeptides from a single gene.

The neural cell adhesion molecule (N-CAM) is believed to be a key regulator of adhesive events in the nervous system and skeletal muscle. The recent isolation of N-CAM cDNAs from different tissues has identified a high degree of diversity in primary amino acid sequence between different isoforms. In this article, we review these recent studies and discuss methods for unravelling the functional consequences of the generation of multiple N-CAM polypeptides using gene transfection approaches.

Amino Acids↗
PubMed1989

Monoclonal antibody 3F8 recognises the neural cell adhesion molecule (NCAM) in addition to the ganglioside GD2.

The monoclonal antibody 3F8 has been described as binding to the ganglioside GD2. This antibody, of the IgG3 isotype, has been used in immunotherapy, radioimmunolocalisation and targeted radiation therapy. 3F8 was originally observed to have a binding profile similar to two monoclonal antibodies, UJ13A and 5.1.H11, characterised as binding to the neural cell adhesion molecule (NCAM). This observation has also been confirmed using a hetero-antiserum prepared against purified NCAM. The cross-reactivity of 3F8 with NCAM has been confirmed by cross-blocking studies with an anti-NCAM antiserum, and by direct immunoprecipitation and gel electrophoresis. In addition, we show that 3F8 binds to human NCAM from 3T3 fibroblasts transfected with NCAM cDNA constructs. It is possible that the common epitope shared by GD2 ganglioside and NCAM involves sialic acid residues common to both the ganglioside and the glycoprotein.

Adult↗
PubMed1989

Neuronal process outgrowth of human sensory neurons on monolayers of cells transfected with cDNAs for five human N-CAM isoforms.

Full length cDNAs for a variety of human N-CAM isoforms have been transfected into mouse L-cells and/or 3T3 cells. Three independent clones of each cell line that were shown to express human N-CAM were tested for their ability to support the morphological differentiation of sensory neurons. The cell surface expression of N-CAM isoforms, linked to the membrane directly by an integral transmembrane spanning domain or indirectly via covalent attachment to a glycosyl-phosphatidylinositol moiety, were consistently found to be associated with a significant increase in the morphological differentiation of both human and rat dorsal root ganglion neurons. Modification of the extracellular structure of both classes of N-CAM, consequent to the expression of a glycosylated 37-amino acid sequence normally found expressed exclusively in muscle N-CAM isoforms did not obviously affect the ability of transfected cells to support increased neuronal differentiation. 3T3 cells that were transfected with a full length cDNA encoding a secreted N-CAM isoform, and that have previously been shown to secrete N-CAM into the growth media rather than link it to the membrane did not significantly differ from control cells in their ability to support neuronal differentiation. These data provide direct evidence for both transmembrane and lipid-linked N-CAM isoforms being components of the regulatory machinery that determines neuronal morphology and process outgrowth.

Animals↗
PubMed1989

Intercellular adhesion mediated by human muscle neural cell adhesion molecule: effects of alternative exon use.

Mouse 3T3 fibroblasts were permanently transfected with cDNAs encoding isoforms of the neural cell adhesion molecule (N-CAM) present in human skeletal muscle and brain. Parental and transfected cells were then used in a range of adhesion assays. In the absence of external shear forces, transfection with cDNAs encoding either transmembrane or glycosylphosphatidylinositol (GPI)-linked N-CAM species significantly increased the intercellular adhesiveness of 3T3 cells in suspension. Transfection of a cDNA encoding a secreted N-CAM isoform was without effect on adhesion. Cells transfected with cDNAs containing or lacking the muscle-specific domain 1 sequence, a four-exon group spliced into the muscle but not the brain GPI-linked N-CAM species, were equally adhesive in the assays used. We also demonstrate that N-CAM-mediated intercellular adhesiveness is inhibited by 0.2 mg/ml heparin; but, at higher concentrations, reduced adhesion of parental cells was also seen. Coaggregation of fluorescently labeled and unlabeled cell populations was performed and measured by comparing their distribution within aggregates with distributions that assume nonspecific (random) aggregation. These studies demonstrate that random aggregation occurs between transfected cells expressing the transmembrane and GPI-linked N-CAM species and between parental cells and those expressing the secreted N-CAM isoform. Other combinations of these populations tested exhibited partial adhesive specificity, indicating homophilic binding between surface-bound N-CAM. Thus, the approach exploited here allows for a full analysis of the requirements, characteristics, and specificities of the adhesive behavior of individual N-CAM isoforms.

Animals↗
PubMed1989

Alternative splicing of the neural cell adhesion molecule gene generates variant extracellular domain structure in skeletal muscle and brain.

Myotube mRNA isoforms of the neural cell adhesion molecule (N-CAM) contain a novel sequence block termed muscle-specific domain 1 (MSD1), which is inserted within the extracellular coding region. Here, we report a characterization of the genomic organization of MSD1 and its pattern of expression within cellular N-CAM RNA and polypeptide species. S1 nuclease protection analyses and sequence analysis of an N-CAM human genomic clone containing MSD1 sequences indicated that MSD1 is comprised of three discrete exons of 15, 48, and 42 bp, designated MSD1a, MSD1b, and MSD1c, respectively. Although the MSD1a exon was present in a small proportion of mRNAs from both brain and muscle cells, the entire MSD1 sequence occurred predominantly in mRNAs from differentiated myotube cells. In addition, antiserum raised to a synthetic, MSD1b-encoded peptide sequence was found to stain the cell surface of human skeletal myotubes in culture, whereas myoblasts, fibroblasts, and neural cells were negative. MSD1a, MSD1b, and MSD1c sequences thus arise collectively in N-CAM mRNA and polypeptide isoforms as a result of muscle tissue-specific and developmentally regulated alternative mRNA splicing events. In addition, the occurrence of brain and muscle mRNAs containing only MSD1a indicate that alternative splicing may occur within the MSD region itself to generate further diversity.

Amino Acid Sequence↗
PubMed1989

Increased intracellular cyclic AMP differentially modulates nerve growth factor induction of three neuronal recognition molecules involved in neurite outgrowth.

The relative expression of the immunoglobulin superfamily members Thy-1 and L1 and the neural cell adhesion molecule (N-CAM) in PC12 cells grown in the presence of nerve growth factor (NGF), cholera toxin, or both has been quantified. Whereas NGF treatment induced increases in the cell surface expression of all three glycoproteins, treatment with cholera toxin resulted in the specific induction of L1. During the first few days of culture, cholera toxin acted synergistically with NGF to promote increases in neuritic outgrowth and the synthesis and cell surface accumulation of the 140- and 180-kilodalton subunits of N-CAM. In contrast, over the same period of culture, cholera toxin inhibited the NGF induction of Thy-1 and L1. Over longer periods of culture (3-5 days), cholera toxin inhibited the NGF induction of N-CAM and neurite outgrowth. A similar pattern of synergistic and inhibitory responses was observed when differentiation was induced by fibroblast growth factor (FGF) rather than NGF or when cholera toxin was replaced with forskolin. These data suggest that intracellular cyclic AMP can differentially modulate cell surface glycoprotein expression induced by either NGF or FGF. Of the three cell surface glycoproteins we have studied, temporal changes in N-CAM expression correlate best with the morphological differentiation status of PC12 cells.

Antigens, Surface↗
PubMed1989

Cloning and characterization of a myoblast cell surface antigen defined by 24.1D5 monoclonal antibody.

Monoclonal antibody 24.1D5 reacts specifically with an epitope expressed on the cell surface of mononucleate myoblasts in primary cultures of human skeletal muscle cells, but not with either multinucleate myotubes or fibroblasts. Polypeptides of 60 and 100 X 10(3) Mr were identified by immunoblotting with the McAb. Human muscle cDNAs encoding the 24.1D5 epitope were used to study further the structure and expression of 24.1D5 during skeletal muscle development. Two mRNA species of 3.0 and 2.5 kb were identified in primary cultures of human skeletal muscle and in mouse muscle cell lines. The levels of both transcripts decreased during myotube formation in vitro and were similarly decreased during myogenesis in the mouse embryo. 24.1D5 mRNAs were expressed by multipotential cells and myoblast derivatives of the mouse embryonic cell line C3H10T1/2, suggesting that 24.1D5 is expressed at an early stage during skeletal muscle development.

Amino Acid Sequence↗
PubMed1989

Tissue specific O-linked glycosylation of the neural cell adhesion molecule (N-CAM).

We have shown previously that the predominant N-CAM isoform in skeletal muscle myotubes contains as a result of alternative splicing a novel domain (MSD1) in its extracellular region. Here we show that this region represents a site for O-linked carbohydrate attachment. The lipid tailed N-CAM in myotubes was found to bind peanut lectin while the transmembrane isoform from myoblasts lacking MSD1 did not. In addition, N-CAM from a variety of neural sources failed to bind the lectin. Analysis of 3T3 fibroblasts transfected with various N-CAM cDNAs, showed that peanut lectin binding was correlated specifically with the expression of the MSD1 region. The oligosaccharides isolated from a purified preparation of myotube N-CAM were shown to contain an O-linked oligosaccharide whose core structure was a sialylated version of Gal beta 1----3GalNac which is the structure recognized specifically by peanut lectin. These data provide the first evidence for the expression of O-linked carbohydrate on any N-CAM isoform and more specifically target this oligosaccharide to the MSD1 region of myotube N-CAM.

Autoradiography↗
PubMed1988

Alternative splicing generates a secreted form of N-CAM in muscle and brain.

A number of different membrane associated isoforms of the neural cell adhesion molecule (N-CAM) have previously been identified. Here the structure of a novel secreted isoform of N-CAM is established by analysis of a cDNA corresponding to an N-CAM mRNA from human skeletal muscle. The mRNA incorporates a novel sequence block into the extracellular domain, which introduces an in-frame stop codon and thus prematurely terminates the coding sequence, generating a truncated N-CAM polypeptide. Analysis of genomic clones indicates that the inserted sequence is present as a discrete exon within the human N-CAM gene, and Northern analysis shows it to be associated specifically with a 5.2 kb mRNA species from skeletal muscle and brain. Stable transfectants expressing the secreted isoform accumulate it in the cytoplasm and release it to the culture medium. In contrast, cells transfected with cDNA encoding lipid-tailed N-CAM express it predominantly at the cell surface. The existence of a secreted isoform may further expand the spectrum of N-CAM function beyond its known involvement in intercellular adhesion to extracellular matrix interactions.

Amino Acid Sequence↗
PubMed1988

Distinct dystrophin mRNA species are expressed in embryonic and adult mouse skeletal muscle.

We have examined dystrophin mRNA in embryonic, newborn and adult mouse skeletal muscle. A discrete nerve-independent increase in mRNA size was observed between embryonic and adult stages, indicating that a developmentally regulated mRNA isoform switch occurs in the expression of the Duchenne muscular dystrophy (DMD) gene in skeletal muscle. These distinct mRNAs are most likely generated via selection of alternative transcriptional start sites or RNA processing pathways. In addition, denervation of adult muscle was without effect on the expression pattern.

Animals↗
PubMed1988

Factors controlling the expression of the NGF receptor in PC12 cells.

An enzyme-linked immunoadsorbent assay (ELISA) has been used to study the relative expression of the nerve growth factor receptor (NGF-R) in PC12 cells. Both nerve growth factor (NGF) and fibroblast growth factor (FGF) were found to induce time-dependent increases in receptor expression. The former response was half-maximal at approximately 2 ng/ml and maximal at 25-50 ng/ml. The induction of NGF-R expression by NGF was associated with an increase in the relative level of a 3.8 kb mRNA species encoding this protein. Whereas the induction of the NGF-R by NGF and FGF were both fully inhibited by cholera toxin and cordycepin, the responses were differentially inhibited by the kinase inhibitor K-252a.

Animals↗
PubMed1988

Isolation of human myoblasts with the fluorescence-activated cell sorter.

We have established procedures for the rapid and efficient purification of human myoblasts using the fluorescence-activated cell sorter. Our approach capitalizes on the specific reaction of monoclonal antibody 5.1H11 with a human muscle cell surface antigen. For each of the five samples analyzed, an enrichment of myoblasts to greater than 99% of the cell population was immediately achieved. Following 3 to 4 weeks of additional growth in vitro, sorted myoblast cultures remained 97% pure. Differentiation of the sorted myoblast cultures, assessed by creatine kinase activity and isozyme content, was comparable to that of pure myoblast cultures obtained by cloning, and was significantly greater than that of mixed fibroblast and myoblast cultures. An average of 10(4) viable myoblasts can be obtained per 0.1 g tissue, each with the potential to undergo approximately 40 cell divisions. Accordingly, if only two-thirds of this proliferative capacity is utilized, the potential yield approximates 10(12) myoblasts, equivalent to 1 kg of cells. Human myogenesis in vitro is no longer limited by cell number and is now amenable to molecular and biochemical analysis on a large scale.+

Abortion, Therapeutic↗
PubMed1988

Analysis of PC12 cell adhesion to muscle and non-muscle cells and components of the extracellular matrix.

The relative strength of PC12 cell adhesion to monolayers of myoblasts and fibroblasts as well as to purified components of the extracellular matrix has been determined. PC12 cell adhesion to myoblasts and fibroblasts was dependent on temperature, and the relative strength of adhesion to both increased over a 4-h period. The strength of adhesion to myoblasts was consistently found to be greater than that to fibroblasts. Whereas coating tissue culture plastic with purified collagen and laminin increased its ability to support PC12 cell adhesion, coating with plasma fibronectin or gelatin reduced cell adhesion. Trypsin treatment of PC12 cells was without effect on their adhesion to collagen and laminin and only partially reduced the strength of adhesion to myoblasts. In contrast trypsinized cells showed a greatly reduced ability to adhere to fibroblast monolayers. Pretreatment of PC12 cells with NGF had no obvious effect on the ability of the cells to adhere to any of the above substrata.

Adrenal Gland Neoplasms↗