Tissue-specific isoforms of dystrophin.
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Dystrophin↗
Biomedical subjects
G Dickson
Publications and source records attributed to G Dickson.
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↗
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↗
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↗
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↗
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↗
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↗
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↗
Cloning and expression of human nebulin cDNAs and assignment of the gene to chromosome 2q31-q32.
We have isolated two nonoverlapping cDNAs encoding human nebulin, a muscle-specific protein. Northern hybridization analysis shows that nebulin is encoded by a huge message at least 25 kb in length. By hybridizing two nonoverlapping cDNAs to DNA isolated from rodent X human cell hybrids, we assign this presumably single-copy gene to human chromosome 2; sublocalization studies indicate that the nebulin gene is on the long arm of the chromosome, in the region 2q31-q32.
Amino Acid Sequence↗
Expression of the messenger RNA encoding the glial fibrillary acidic protein in rat basal forebrain following fimbria-fornix lesion.
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Animals↗
Structure and expression of neural cell adhesion molecule complementary DNA clones in skeletal muscle.
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Amino Acid Sequence↗
Complete sequence and in vitro expression of a tissue-specific phosphatidylinositol-linked N-CAM isoform from skeletal muscle.
Neural cell adhesion molecules (N-CAMs) are a family of cell surface sialoglycoproteins encoded by a single copy gene. A full-length cDNA clone that encodes a nontransmembrane phosphatidylinositol (PI) linked N-CAM of Mr 125 x 10(3) has been isolated from a human skeletal muscle cDNA library. The deduced protein sequence encodes a polypeptide of 761 amino acids and is highly homologous to the N-CAM isoform in brain of Mr 120 x 10(3). The size difference between the 125 x 10(3). The size difference between the 125 x 10(3) Mr skeletal muscle form and the 120 x 10(3) Mr N-CAM form from brain is accounted for by the insertion of a block of 37 amino acids called MSD1, in the extracellular domain of the muscle form. Transient expression of the human cDNA in COS cells results in cell surface N-CAM expression via a putative covalent attachment to PI-containing phospholipid. Linked in vitro transcription and translation experiments followed by immunoprecipitation with anti-N-CAM antibodies demonstrate that the full-length clone of 761 amino acid coding potential produces a core polypeptide of Mr 110 x 10(3) which is processed by microsomal membranes to yield a 122 x 10(3) Mr species. Taken together, these results demonstrate that the cloned cDNA sequence encodes a lipid-linked, PI-specific phospholipase C releasable surface isoform of N-CAM with core glycopeptide molecular weight corresponding to the authentic muscle 125 x 10(3) Mr N-CAM isoform. This is the first direct correlation of cDNA and deduced protein sequence with a known PI-linked N-CAM isoform from skeletal muscle.
Amino Acid Sequence↗
Human muscle neural cell adhesion molecule (N-CAM): identification of a muscle-specific sequence in the extracellular domain.
cDNA clones encoding neural cell adhesion molecule (N-CAM) mRNAs of 6.7, 5.2, and 4.3 kb from human skeletal muscle cells were isolated. A 6.7 kb mRNA encodes a transmembrane N-CAM isoform, present predominantly in mononucleate myoblasts, that shows sequence homology with chick brain N-CAM-140 and is down-regulated during myotube formation. In contrast, the 5.2 and 4.3 kb mRNAs encode nontransmembrane N-CAM isoforms that greatly increase during myoblast fusion. Furthermore, a discrete muscle-specific sequence domain (MSD1) was detected in the extracellular coding regions of the 5.2 and 4.3 kb mRNAs. This encodes a unique run of 37 amino acids and is not expressed in 7.2 and 6.7 kb mRNAs from human or chick brain or in the corresponding 6.7 kb muscle transcript. The MSD1 is also absent from chick and mouse brain mRNAs of 4.0 and 2.9 kb. These results show that diversity in N-CAM primary structure can be found in the extracellular domain in a tissue-specific manner.
Amino Acid Sequence↗
Ketanserin and guanethidine in the treatment of causalgia.
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Administration, Oral↗
Nerve growth factor activates Thy-1 and neurofilament gene transcription in rat PC12 cells.
The effect of nerve growth factor (NGF) on the expression of neurofilament and Thy-1 genes in rat PC12 pheochromocytoma cells was examined at both the transcriptional and post-transcriptional levels. Addition of NGF to cultured PC12 cells produced increases in mRNAs corresponding to the 68 kd neurofilament protein (NF68) and the Thy-1 glycoprotein within 24 h, with maximal effects of some 90- and 45-fold stimulation (relative to beta-actin mRNA) being observed after 12 and 4 days of treatment, respectively. In addition, transcriptional run-off analyses using isolated nuclei showed that NGF treatment resulted directly in 8- and 4-fold increases in the rate of NF68 and Thy-1 gene transcription. These gene activation events were independent of overt morphological differentiation of PC12 cells occurring both under conditions permissive and non-permissive for neurite outgrowth, and once established the new molecular phenotype was dependent upon the continued presence of NGF. This is the first molecular evidence for the reversible activation of neuron-specific genes during NGF-induced differentiation in PC12 cells.
Journal Article↗
Breast self-examination: knowledge, attitudes and practice behaviors of working women.
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Adult↗
The prevention of acetaminophen-induced hepatotoxicity by the interferon inducer poly(rI . rC).
When mice were pretreated with the interferon inducer poly(rI . rC) for 24 hr, the mortality caused by acetaminophen was significantly reduced. Acetaminophen doses up to 600 mg/kg which killed 100% of the saline-treated control mice caused no deaths in poly(rI . rC)-treated animals. Even doses as high as 900 mg/kg acetaminophen killed only 38% of the animals. Histological examination of the livers demonstrated that acetaminophen-induced necrosis was decreased in poly(rI . rC)-treated animals compared to controls. Significant protection against necrosis was observed even with doses as high as 900 mg/kg acetaminophen. Following a dose of 300 mg acetaminophen, 0.67 +/- 0.5 nmol/mg protein were covalently bound to liver protein in control mice compared to 0.33 +/- 0.02 nmol/mg protein bound in poly(rI . rC)-treated mice. This protective effect of poly(rI . rC) did not result from an increase in hepatic glutathione content; in fact, the glutathione level was depressed in animals treated with poly(rI . rC). Since cytochrome P-450 levels were depressed in these experiments, it is concluded that poly(rI . rC) depresses the cytochrome P-450 species responsible for the formation of the toxic metabolite and less reactive species are available for binding to cell macromolecules. It is likely that the toxicity of acetaminophen will be decreased considerably during viral infections which promote the formation of interferon.
Acetaminophen↗