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Biomedical subjects

F S Walsh

Publications and source records attributed to F S Walsh.

At least 91 records · Page 5Linked to original sources
PubMed1993

Transmembrane neural cell-adhesion molecule (NCAM), but not glycosyl-phosphatidylinositol-anchored NCAM, down-regulates secretion of matrix metalloproteinases.

During embryogenesis interactions between cells and extracellular matrix play a central role in the modulation of cell motility, growth, and differentiation. Modulation of matrix structure is therefore crucial during development; extracellular matrix ligands, their receptors, extracellular proteinases, and proteinase inhibitors all participate in the construction, maintenance, and remodeling of extracellular matrix by cells. The neural cell-adhesion molecule (NCAM)-negative rat glioma cell line BT4Cn secretes substantial amounts of metalloproteinases, as compared with its NCAM-positive mother cell line BT4C. We have transfected the BT4Cn cell line with cDNAs encoding the human NCAM-B and -C isoforms. We report here that the expression of transmembrane NCAM-B, but not of glycosyl-phosphatidylinositol-linked NCAM-C, induces a down-regulation of 92-kDa gelatinase (matrix metalloproteinase 9) and interstitial collagenase (matrix metalloproteinase 1), indicating that cellular expression of the recognition molecule NCAM regulates the metabolism of the surrounding matrix.

Animals↗
PubMed1993

Neurite outgrowth of spinal neurons on tissue sections of embryonic muscle is largely integrin dependent.

Embryonic chick spinal neurons have been cultured over sections of human foetal muscle to determine which cell adhesion molecules present in embryonic muscle are important in promoting neurite outgrowth. Using blocking antibodies against the major cell adhesion molecules, neural cell adhesion molecule (NCAM), N-cadherin and the beta 1 subunit of the integrins, neurite outgrowth was significantly blocked only by anti-integrin antibodies. In addition other agents that block neurite outgrowth stimulated by NCAM, N-cadherin and L1, such as the calcium channel antagonists verapamil and omega-conotoxin and pertussis toxin which inactivates G-proteins also had no effect. This suggests that in this culture system integrins are able to promote neurite outgrowth whereas NCAM and N-cadherin are not.

Animals↗
PubMed1993

Trans-activation of the murine dystrophin gene in human-mouse hybrid myotubes.

Myotube cultures of the myogenic cell line, C2, produce significantly lower levels of dystrophin than primary mouse cultures. We demonstrate that expression of the C2 dystrophin gene increases 10-fold in hybrid myotubes formed by fusion of C2 and dystrophin-deficient human myoblasts from a Duchenne muscular dystrophy patient. These results indicate that C2 cells are deficient in endogenous gene regulatory factors which enhance dystrophin expression, and that the C2 cell line may therefore be used to identify putative trans-acting factors involved in the regulation of dystrophin gene expression.

Animals↗
PubMed1993

Epithelial (E-) and placental (P-) cadherin cell adhesion molecule expression in breast carcinoma.

Two members of the cadherin family of intercellular adhesion molecules are found in normal breast tissue: E-(epithelial) cadherin is present in both luminal and myoepithelial cells, whereas P- (placental) cadherin is confined to myoepithelium. There is experimental evidence that loss of E-cadherin is associated with increased invasiveness of malignant cells in vitro, which stimulated us to examine the presence and distribution of E- and P-cadherin in breast carcinomas by means of immunohistochemical staining. E-Cadherin was present in all in situ and invasive ductal carcinomas examined, although it had a patchy distribution and the staining was of variable intensity. However, in 83 per cent of invasive lobular carcinomas and all lobular carcinomas in situ there was complete loss of E-cadherin expression. In the remaining 17 per cent of invasive lobular tumours, E-cadherin appeared to have an abnormal distribution within the cytoplasm with variable expression on the cell membrane. P-Cadherin, by contrast, was absent from all benign breast luminal epithelium and 25 carcinomas of ductal and lobular type. It was found in only one carcinoma of lobular type. We suggest that loss of cell-cell adhesion mediated by E-cadherin plays a part in the characteristic morphology of lobular carcinomas.

Breast Neoplasms↗
PubMed1993

Migratory, invasive and metastatic capacity of NCAM transfected rat glioma cells.

A cDNA encoding a transmembrane 140 kDa isoform of the neural cell adhesion molecule, NCAM, was transfected into the rat glioma cell line BT4Cn. Transfectants with a homogeneously high expression of NCAM-B showed a decreased capacity for penetration of an artificial basement membrane when compared to cells transfected with expression-vector alone or untransfected cells. However, when injected subcutaneously into nude mice, both NCAM expressing cells and control cells produced invasive tumors. Nude mice injected with NCAM positive cells developed tumors with slower growth rates as compared to those induced by NCAM negative cells. This implies that NCAM may not only be involved in adhesive and motile behaviour of glioma cells, but also in their growth regulation.

Animals↗
PubMed1993

Thy-1 antibody-triggered neurite outgrowth requires an influx of calcium into neurons via N- and L-type calcium channels.

We present evidence that the neurite out-growth stimulated by the binding of Thy-1 antibodies to PC12 cells is mediated by calcium influx through both N- and L-type calcium channels. PC12 cells cultured on a noncellular substratum in the presence of NGF, or on a cellular substratum in the absence of NGF, responded to soluble Thy-1 antibody by extending longer neurites. The response required bivalent antibody and could be blocked by removing Thy-1 from the surface of PC12 cells with phosphatidylinositol specific phospholipase C. The response could also be blocked by reducing extracellular calcium to 0.25 mM, or by antagonists of L- and N-type calcium channels. Additionally, the response could be fully inhibited by preloading PC12 cells with BAPTA/AM which buffers changes in intracellular calcium. A heterotrimeric G-protein is also implicated in the pathway as the response could be fully inhibited by pertussis toxin. These data suggest that antibody-induced clustering of Thy-1 stimulates neurite outgrowth by activating a second messenger pathway that has previously been shown to underlie cell adhesion molecule (NCAM, N-cadherin, and L1), but not integrin or NGF-dependent neurite outgrowth.

3T3 Cells↗
PubMed1993

Differential effects of over-expressed neural cell adhesion molecule isoforms on myoblast fusion.

We have used a transfection based approach to analyze the role of neural cell adhesion molecule (NCAM) in myogenesis at the stage of myoblast fusion to form multinucleate myotubes. Stable cell lines of myogenic C2 cells were isolated that express the transmembrane 140- or 180-kD NCAM isoforms or the glycosylphosphatidylinositol (GPI) linked isoforms of 120 or 125 kD. We found that expression of the 140-kD transmembrane isoform led to a potent enhancement of myoblast fusion. The 125-kD GPI-linked NCAM also enhanced the rate of fusion but less so when a direct comparison of cell surface levels of the 140-kD transmembrane form was carried out. While the 180-kD transmembrane NCAM isoform was effective in promoting C2 cell fusion similar to the 140-kD isoform, the 120-kD isoform did not have an effect on fusion parameters. It is possible that these alterations in cell fusion are associated with cis NCAM interactions in the plane of the membrane. While all of the transfected human NCAMs (the transmembrane 140- and 180-kD isoforms and the 125- and 120-kD GPI isoforms) could be clustered in the plane of the plasma membrane by species-specific antibodies there was a concomitant clustering of the endogenous mouse NCAM protein in all cases except with the 120-kD human isoform. These studies show that different isoforms of NCAM can undergo specific interactions in the plasma membrane which are likely to be important in fusion. While the transmembrane and the 125-kD GPI-anchored NCAMs are capable of enhancing fusion the 120-kD GPI NCAM is not. Thus it is likely that interactions associated with NCAM intracellular domains and also the muscle specific domain (MSD) region in the extracellular domain of the GPI-linked 125-kD NCAM are important. In particular this is the first role ascribed to the O-linked carbohydrate containing MSD region which is specifically expressed in skeletal muscle.

Animals↗
PubMed1993

Direct retroviral-mediated transfer of a dystrophin minigene into mdx mouse muscle in vivo.

At the cellular level, the primary pathology in Duchenne muscular dystrophy (DMD) is caused by deficiency of the sarcolemmal-associated protein, dystrophin, in the striated musculature. Here we describe the somatic transfer and long-term expression of a human dystrophin minigene corresponding to a mild Becker muscular dystrophy (BMD) phenotype in skeletal muscle tissues of the dystrophin-deficient mdx mouse by direct retroviral transduction. Following a single intramuscular injection of recombinant retrovirus, sarcolemmal expression of dystrophin was observed in an average of approximately 6% of myofibres in treated tibialis anterior muscles and was associated with activated reappearance of at least one component (43kD) of the dystrophin-glycoprotein membrane complex (DGC). Furthermore, expression of recombinant dystrophin was observed in muscle tissues up to 9 months after treatment and a significant enhancement of retrovirus-mediated myofibre transduction was obtained in mdx muscle undergoing experimentally-induced regeneration. The results clearly demonstrate that retroviral transduction of activated satellite cells in regenerating skeletal muscle is a feasible route for direct and stable dystrophin gene transfer into muscle tissues in vivo.

Animals↗
PubMed1993

The cell adhesion molecule M-cadherin is specifically expressed in developing and regenerating, but not denervated skeletal muscle.

The spatiotemporal distribution of M-cadherin mRNA has been determined by in situ hybridization in the mouse embryo and in adult skeletal muscle following experimental regeneration and denervation. M-cadherin mRNA is highly tissue specific and is found only in developing skeletal muscle. In contrast, N-cadherin mRNA has a broader tissue distribution in the embryo, being found on both neural elements and skeletal and cardiac muscle. M-cadherin is expressed in the myotomes shortly after they form, along with the myogenic regulatory factor myogenin. M-cadherin is expressed in muscles derived from the myotomes and is detected in forelimb bud precursor cells at embryonic day 11.5. In the latter case M-cadherin expression appears co-ordinately with that of myogenin and cardiac alpha-actin. Shortly before birth, M-cadherin expression is down regulated. M-cadherin can, however, be re-expressed following experimental regeneration of skeletal muscle. Here M-cadherin is transiently expressed on regenerating myoblasts but not myotubes. Following muscle denervation no evidence was found for re-expression of M-cadherin under conditions where there was strong expression of the nicotinic acetylcholine receptor on myofibres. The highly specific tissue distribution and unique developmental profile distinguishes M-cadherin from other cadherins and suggests a role in cell surface events during early myogenesis.

Actins↗
PubMed1993

Expression of beta 1 integrins in sensory neurons of the dorsal root ganglion and their functions in neurite outgrowth on two laminin isoforms.

Integrins are heterodimeric receptors that mediate responses of neurons and many other cell types to components of the extracellular matrix. In the present article, we examine the roles of individual integrin receptors expressed by spinal sensory neurons of the dorsal root ganglion (DRG) in mediating interactions with laminin, an extracellular matrix glycoprotein that promotes neurite outgrowth. DRG neurons were shown to express three beta 1 integrins that have been shown in other cell types to function as laminin receptors--high levels of alpha 1 beta 1 and alpha 3 beta 1 and low levels of alpha 6 beta 1. In addition, DRG neurons were shown to express a known fibronectin receptor, alpha 5 beta 1, and an integrin with undefined ligands, alpha 6 beta 4. Function-inhibitory monoclonal antibodies specific for the alpha 1, alpha 2, alpha 3, alpha 5, and alpha 6 integrin subunits were used to determine the roles of individual integrins in mediating neurite outgrowth by DRG neurons on laminin. The results demonstrate that alpha 1 beta 1 and alpha 3 beta 1 function as laminin receptors on these neurons. As many as 18 distinct isoforms of laminin may exist, assembled as heterotrimers containing one each of the different A, B1, or B2 subunit homologs. In the present study, we characterize neurite outgrowth in response to two of these isoforms, the AeB1eB2e isoform and the AmB1eB2e isoform. Results utilizing DRG neurons and a pheochromocytoma cell line (PC12) indicate that these two isoforms exhibit differential selectivities for the alpha 1 beta 1 and alpha 3 beta 1 integrins.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Alternative splicing of neural-cell-adhesion molecule mRNA in human small-cell lung-cancer cell line H69.

The neural-cell-adhesion molecule (NCAM) is expressed in all small-cell lung cancers (SCLC) and in approximately 20% of non-small-cell lung tumors (non-SCLC). These NCAM-positive lung tumors have a poor prognosis compared with NCAM-negative tumors. Multiple NCAM protein isoforms are expressed from a single-copy gene as a result of alternative splicing and/or post-translational modifications. Therefore, we studied the NCAM isoforms expressed in a human small-cell lung-cancer cell line, H69. NCAM mRNA transcripts of 7.2, 6.7, 4.3 and 4.0 were detected in these cells on Northern blots. Since the various NCAM isoforms may have different biological properties, we performed a more precise examination of NCAM mRNAs, using polymerase chain reactions (PCR) with primers flanking the various NCAM exon boundaries. The shortest alternatively spliced sequence that we found was the trinucleotide AAG located between exon 12 and 13 in the so-called hinge region of the NCAM protein. This AAG trinucleotide was present in the majority of the NCAM mRNAs. A second alternatively spliced 30 nt-exon VASE (immunoglobulin-variable domain-like alternatively spliced exon) was present in all NCAM transcript isoforms at the exon 7/exon 8 junction. VASE resulted in the insertion of 10 amino acids into the 4th immunoglobulin-like loop of the NCAM protein. Within the limits of the PCR methodology, no evidence for the presence of mRNA containing exon 15, encoding the glycosyl-phosphatidylinositol-linked (GPI-linked) NCAM isoform in H69 cells was obtained. Considering that H69 cells express 2 major NCAM protein classes (NCAM-180 and NCAM-140), and that the VASE and AAG alternative mRNA splice variants result in minor differences in protein sizes, at least 8 polypeptide isoforms of NCAM might be expressed in H69 cells that contribute to the binding interactions of NCAM.

Amino Acid Sequence↗
PubMed1992

The VASE exon downregulates the neurite growth-promoting activity of NCAM 140.

Axonal growth, guidance and synapse formation are controlled by receptors on neuronal growth cones that can recognize positive and inhibitory cues in the local microenvironment. Four well characterized receptor systems are known that recognize the growth-promoting activities associated with the extracellular matrix and the membranes of cells such as astrocytes, muscle cells and Schwann cells; these are the integrins and the homophilically binding cell adhesion molecules neural-cell adhesion molecule (NCAM), N-cadherin and L1 (refs 5-12). Alternative splicing generates 20-30 isoforms of NCAM and these can also be differentially glycosylated. There are two sites where alternative splicing changes the extracellular structure of membrane-bound NCAM and one of these (the MSD1 region) does not obviously affect function. Here we report that the variable alternatively spliced exon (VASE) in immunoglobulin domain 4 downregulates the neurite outgrowth-promoting activity of NCAM. The high level of VASE expression in the adult central as compared with peripheral nervous system could contribute to the poor regenerative capacity of the former.

Age Factors↗
PubMed1992

Retroviral-mediated transfer of a dystrophin minigene into mdx mouse myoblasts in vitro.

We have demonstrated expression of a 6.3 kb Becker muscular dystrophy (BMD) human dystrophin cDNA following retroviral-mediated transduction of cultured myoblasts from the dystrophin-deficient mdx mouse. The truncated dystrophin protein was localised to the sarcolemma of differentiated myotubes by antibodies against the C-terminus of the molecule, and produced an identical immunostaining pattern to that observed in control myotubes expressing normal endogenous dystrophin. These results indicate that retroviral-mediated gene transfer may be useful for experimental in vivo studies on the complementation of dystrophin gene mutations.

Animals↗
PubMed1992

Expression of NCAM isoforms during skeletal myogenesis in the mouse embryo.

We have examined the developmental patterns of neural cell adhesion molecule (NCAM) gene expression in embryonic mouse skeletal muscle cells by in situ hybridization. Moreover, by utilising exon-specific cRNA probes, we have examined tissue specific splicing of the NCAM gene. We show that there is a distinct sequence of NCAM isoform expression during skeletal muscle development. Since NCAMs are also expressed in other cell types, particularly neurons, NCAM mRNAs have been colocalised with acetylcholine receptor alpha (AChR alpha) gene transcripts to identify muscle-specific expression. NCAM is first detected in somites as they first form, prior to their differentiation into muscle and nonmuscle compartments. Myotomes, the first skeletal muscle masses to form in the embryo, express mRNAs for the transmembrane 180 and 140 kDa isoforms of NCAM. Both of these transcripts are also detected in the neural tube, and their spatial pattern of expression changes with development. Transcripts containing the muscle-specific domain (MSD) of the NCAM gene are not detected prior to 11 days postcoitum (p.c.), at a time when rostral somites already contain well-developed myotomes. As the level of MSD mRNAs increases at 12 days p.c., the 140 and 180 kDa transcript levels decrease in skeletal muscle masses. The level of all NCAM isoform transcripts declines between 13 and 15 days p.c. in muscle. However, the 180 and 140 kDa NCAM isoforms are expressed at a high level in neural tissue and in other locations in the developing embryo such as in smooth muscle, around vibrissae follicles, and in the perichondrial zone of digits.

Animals↗
PubMed1992

Cell adhesion molecules, second messengers and axonal growth.

Recent studies on NCAM-related molecules suggest that individual cell adhesion molecules might function to both promote axonal growth during development and maintain synaptic structure in the adult. Evidence that differential alternative splicing contributes to this apparent bifunctionality of cell adhesion molecules is discussed.

Animals↗