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B D Sanwal

Publications and source records attributed to B D Sanwal.

At least 37 records · Page 2Linked to original sources
PubMed1987

Regulation of protein kinase C by cyclic adenosine 3':5'-monophosphate and a tumor promoter in skeletal myoblasts.

The specific activity of protein kinase C in rat skeletal myoblasts decreased when they were exposed for very short periods to isoproterenol, forskolin, dibutyryl cyclic AMP (Bt2cAMP), or the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). In the presence of Bt2cAMP or forskolin only the cytosolic but not the membrane-bound kinase activity was found to decrease. Treatment with TPA, however, led to a decrease in the activity of the enzyme both in the cytosolic as well as the membrane fractions. The effects observed in vivo could be duplicated in crude extracts of myoblasts incubated with cAMP analogues or TPA. In the presence of ATP, protein kinase C activity decreased considerably in crude cytosolic fractions treated with the cAMP analogues, but a requirement for ATP was not evident for the decrease in activity brought about by TPA. For the cAMP analogues the decrease in protein kinase C was also prevented by incubation of the extracts with an inhibitor of cAMP-dependent protein kinase. The regulation of protein kinase C by Bt2cAMP (but not by TPA) was altered in Rous sarcoma virus-transformed myoblasts. It is considered likely that a component affected by cAMP (probably a substrate for cAMP-dependent protein kinase) participates in the regulation of protein kinase C activity, and it is altered in unknown ways in transformed myoblasts.

Animals↗
PubMed1987

Phosphorylation of a gelatin-binding protein from L6 myoblasts by protein kinase C.

A gelatin-binding glycoprotein from L6 rat myoblasts, designated gp46, was shown to be phosphorylated in vivo. This phosphorylation was increased slightly (18%) by phorbol ester treatment of L6 suggesting protein kinase C involvement. Purified gp46 could be phosphorylated in vitro with protein kinase C, but not by the catalytic subunit of cAMP-dependent protein kinase. Comparison of the phosphotryptic peptide maps of in vitro and in vivo labeled gp46 suggested that in vivo phosphorylation of gp46 may be mediated by protein kinase C.

Animals↗
PubMed1987

In vivo and in vitro models of demyelinating disease: activation of the adenylate cyclase system influences JHM virus expression in explanted rat oligodendrocytes.

The specificity of JHM virus (JHMV) tropism for rat oligodendrocytes, as one of the primary host cells in the central nervous system, is maintained after explanation (S. Beushausen and S. Dales, Virology 141:89-101, 1985). The temporal correlation between onset of resistance to JHMV infection in vivo, completion of myelination, and maturation of the central nervous system can be simulated in vitro by inducers of oligodendrocyte differentiation (Beushausen and Dales, Virology, 1985). Stimulation of differentiation through the elevation of intracellular cyclic AMP (cAMP) levels suggests a possible connection between activation of the adenylate cyclase system and coronavirus expression. Chromatographic analysis of cAMP-dependent protein kinase activity in cytosol extracts prepared from astrocytes or oligodendrocytes revealed that both glial cell types were deficient in protein kinase I, indicating that expression of coronavirus in differentiated cells was not contingent upon the presence of protein kinase I. However, treatment with N6,2'-O-dibutyryladenosine-3',5'-cyclic monophosphate (dbcAMP) resulted in a severalfold enhancement of the free regulatory subunit (RI) in oligodendrocytes but not in astrocytes. The RII subunit in both neural cell types was relatively unaffected. Rapid increase in RI due to dbcAMP treatment was correlated with inhibition of JHMV expression. Other differentiation inducers, including 8-Br cAMP and forskolin which, by contrast, caused a decrease in detectable RI, also blocked JHMV expression. This apparent anomaly can be attributed to an increased turnover of RI due to destabilization of the molecule which occurs upon site-specific binding of the cyclic nucleotides. On the basis of these observations, we conclude that the state of oligodendrocyte differentiation manifested with the modulation of RI regulates JHMV expression. The differentiation process did not affect either virus adsorption or sequestration but appeared to inhibit the expression of viral RNA and proteins, implying that replication was inhibited at some step between penetration and initiation of genomic functions, perhaps at the stage of uncoating. We therefore examined the possibility that protein kinases and phosphatases, which influence cellular regulation during cAMP-induced differentiation, may be responsible for the phenomenon of coronavirus suppression in oligodendrocytes. Evidence was obtained indicating that normal processing of the phosphorylated nucleocapsid protein is inhibited in differentiated oligodendrocytes, consistent with the notion that JHMV replication might be arrested during uncoating.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1987

Differentiation defective mutants of skeletal myoblasts altered in a gelatin-binding glycoprotein.

We have previously described a myoblast cell surface glycoprotein of the molecular mass 46,000 (gp46), which is associated with myoblast differentiation. In this report we demonstrate that gp46 binds specifically to gelatin-Sepharose and in this respect is similar to a glycoprotein of the molecular mass 47,000, which has earlier been described as a cell surface localized protein in mouse parietal endoderm cells and in chick embryo fibroblasts. To ascertain the relationship of gp46 to myoblast differentiation, wild-type L6 myoblasts, as well as two concanavalin A (ConA) resistant, differentiation-negative, myoblast mutants (D-1 and C-8), were examined for gp46 expression. In the mutant designated D-1, which has a defect in dolichol mannosyl transferase, both mannose incorporation into gp46 and ConA binding to gp46 was reduced compared with L6, without markedly affecting the gelatin adhesion properties of gp46. Western blotting with a monoclonal antibody against gp46 was used to show that the expression of gp46 was normal in D-1 but was reduced in mutant C-8 compared with L6. Reduction occurred both in the plasma membrane and endoplasmic reticulum fractions of C-8 compared with wild-type L6. In L6 myoblasts, the expression of gp46 remained constant during myoblast replication and fusion but decreased markedly postfusion. In the nonfusing myoblast mutants D-1 and C-8 and in wild-type L6 cells that were prevented from fusing by treatment with 5-bromo-2'-deoxyuridine, the expression of gp46 remained invariant. We suggest that collagen interactions, mediated by gp46, are important for normal rat skeletal muscle differentiation.

Animals↗
PubMed1986

Purification and polyamine activation of a high-affinity 3',5'-cAMP phosphodiesterase from rabbit muscle.

A high-affinity phosphodiesterase, termed PDE II, has been purified about 1400-fold from rabbit skeletal muscle. This enzyme is activated by treatment with proteases. It is also activated specifically by polyarginine and arginine-rich histones, but not by other polyanions. The activation is counteracted nonspecifically by polycations, such as heparin and chondroitin sulphate. When the enzyme is fully activated by polyarginine it is no longer susceptible to activation by proteases. A conformational or structural change must thus occur in the enzyme by the binding of the polyanions.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1985

Regulation of protein kinase and its regulatory subunits during skeletal myogenesis.

Rat skeletal myoblasts contain two cytosolic cAMP-dependent protein kinases, types I and II. Photoaffinity labeling with 8-azido-cAMP reveals the presence of regulatory subunits of Mr = 52,000, 47,000, and 36,000. The Mr = 52,000 and 47,000 subunits are very likely RII and RI, respectively, while the Mr = 36,000 subunit appears to be a proteolytic product of RI, as judged by its cross-reactivity to anti-RI antiserum. The total protein kinase activity increases about 3-fold during the fusion of myoblasts. In parallel with this increase, the concentration of RI subunit also increases, while the levels of RII remain unchanged. Myoblast mutants which lack the capability to differentiate both biochemically and morphologically also lack the ability to increase the concentration of RI subunit. This ability is restored in complementing somatic hybrids which regain the capability to differentiate.

Affinity Labels↗
PubMed1985

Regulation of cyclic 3',5'-adenosine monophosphate phosphodiesterase in Friend erythroleukemia cells.

Friend erythroleukemia cells contain only a single form of cAMP phosphodiesterase with high affinity for substrate. It is activated by treatment with various proteases including those present in snake venom. The activity of this enzyme is increased about 2-fold when the cells are either cultivated in the presence of cAMP or in the presence of compounds which are capable of generating cAMP in vivo, such as isoproterenol, epinephrine and prostaglandins. The enzyme produced in the presence of cAMP is modified in such a way that it is no longer susceptible to activation by treatment with proteases. The activation and modification obtained in vivo can be duplicated in cell-free extracts of Friend cells, if they are incubated in the presence of cAMP and ATP. A possibility thus exists that the phosphodiesterase is activated by its substrate by phosphorylation.

1-Methyl-3-isobutylxanthine↗
PubMed1984

Possible involvement of a cell surface glycoprotein in the differentiation of skeletal myoblasts.

From a highly myogenic permanent line of rat skel-myoblasts (L6), we have isolated two classes of single step concanavalin A-resistant mutants. The RI class is about 2-fold and RII about 5-fold more resistant than the parental cells to the lethal action of concanavalin A. In all of the mutants, both the morphological differentiation (i.e. fusion to form myotubes) and biochemical differentiation, measured by the appearance of creatine kinase and acetylcholine receptors, are absent. The biochemical lesion in the RI type of mutants is not known, but RII type of mutants is unable to catalyze transfer of mannose from GDP-mannose into a lipid-linked form. Concanavalin A binding to separated membrane proteins from RII type of mutants on polyacrylamide gels is reduced 80% compared to wild type cells. In the RI type of mutants, however, only one major band, approximately 46,000 daltons, does not bind concanavalin A to the same extent as the wild type cells. In somatic cell hybridizations, RI type of mutants complements the RII type. In the hybrids, fusion as well as creatine kinase and acetylcholine receptors reappear, although not to the same extent as in the wild type cells. The 46,000-dalton band also reappears in the complementing hybrids. Thus, this protein may play some crucial role in myogenesis.

Animals↗
PubMed1984

Inhibition of fusion of skeletal myoblasts by tunicamycin and its reversal by N-acetylglucosamine.

The differentiation of a permanent line of rat skeletal myoblasts is inhibited by a low concentration of tunicamycin in the growth medium. At a level of 0.9 micrograms/mL, mannose incorporation in trichloroacetic-acid-insoluble material is inhibited to the extent of about 50% by the antibiotic. Blotting of glycoproteins of the cytoplasmic membrane of myoblasts separated by gel electrophoresis by radioiodinated concanavalin A revealed that four major glycoproteins of 230,0000, 145,000, 119,000, and 46,000 daltons were present in lower relative amounts in the plasma membrane following tunicamycin treatment. The 119,000 dalton glycoprotein was a major radioiodinated protein in intact cells and was presumably localized on the periphery of the membrane. The effect of tunicamycin on both fusion and glycosylation of membrane proteins could be reversed by N-acetylglucosamine, but not by the protease inhibitors leupeptin and pepstatin.

Acetylglucosamine↗
PubMed1984

Relationship between cell surface asparagine-linked glycoproteins and myoblast differentiation. Analysis of wheat germ agglutinin-resistant mutants.

Alterations in the oligosaccharides of complex surface glycoproteins have been studied in wild-type rat skeletal myoblasts (L6-9/1) and two wheat germ agglutinin resistant mutants, selected for either high level of resistance (WGARI) or low level of resistance (WGARII). All three lines possessed high mannose oligosaccharides of the structure Man(n)GlcNAc, where n = 6, 7, or 8. L6-9/1 had complex oligosaccharides of the type (+/- NeuNAc alpha 2-3Gal beta GlcNAc beta)n-Man3GlcNAc(+/- Fuc)GlcNAc where n = 3 or 4, in addition to small amounts of biantennary and higher order structures. WGARII, the mutant with low resistance to wheat germ agglutinin, possessed similar complex oligosaccharides except that they had undergone some base-sensitive modification which rendered them resistant to Clostridium perfringens sialidase. WGARI, mutant of high resistance to wheat germ agglutinin, possessed complex oligosaccharides of the type (GlcNAc beta)nMan3GlcNAc(+/- Fuc)GlcNAc, with n = 2, 3, or 4. Since the WGARI mutants were unaltered in differentiation, it is concluded that the terminal sialic acid and galactose residues on the protein-bound oligosaccharides are not required for differentiation.

Animals↗
PubMed1984

Regulation of phosphatidylcholine synthesis and the activity of CTP-cholinephosphate cytidylytransferase in myoblasts by 12-O-tetradecanoylphorbol-13-acetate.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) increases rate of incorporation of 32P into phosphatidylcholine and phosphatidylethanolamine about twofold in a line of rat skeletal myoblasts (L6). This effect is specific for TPA and is not caused by the nontumor-promoting analogue 4 alpha-phorbol didecanoate (alpha-PDD). Cycloheximide and actinomycin D have no effect on the TPA-dependent increase in labelling. There is no significant difference in the rate of disappearance of [14C]glycerol from phospholipids in cells exposed to TPA. Several enzymes of phospholipid synthesis in myoblasts exposed to TPA are unaltered in activity, except CTP:cholinephosphate cytidylyltransferase (EC 2.7.7.15) whose activity is enhanced 1.5- to 2-fold compared with cells exposed to alpha-PDD. Exposure of cells for only 15 min to a concentration of 1 X 10(-6)M TPA is sufficient to cause maximum activation of the enzyme. The cytidylyltransferase activity is associated largely with the particulate fraction in the cell-free extracts of myoblasts. Following gel filtration on Bio-Gel A-1.5m, the enzyme activity appears in the void volume and smaller molecular weight forms are not discernible. The kinetic parameters of cytidylyltransferase in preparations from treated cells are unaltered except for an increased Vmax. The apparent Km value for CTP for the enzyme from treated and untreated cells is about 0.9mM. Both enzyme preparations are weakly inhibited by lysophosphatidylcholine, but are unaffected by several other phospholipids.

Animals↗
PubMed1983

Regulation of cyclic adenosine 3':5'-monophosphate phosphodiesterases: altered pattern in transformed myoblasts.

Rat skeletal myoblasts, L6 and L8, have two major forms of phosphodiesterases, PDE II and PDE III. Only the former is activated by treatment with proteases. When the myoblasts are exposed to cAMP for 10-16 h, the activity of PDE III increases considerably. This increase is accompanied by a loss of activatability of PDE II by proteases. Leupeptin prevents the increase in the levels of PDE III suggesting that a protease in vivo may be responsible for the formation of PDE III from PDE II. Spontaneously or Rous sarcoma virus-transformed myoblasts, however, show altered regulation of the two forms of PDE. In the presence of cAMP in the medium, unlike the nontransformed cells, the levels of PDE III do not increase but the activity of PDE II rises. Simultaneously, PDE II becomes refractory to activation by proteases. The altered mode of PDE regulation in transformed cells is dominant in hybrids between normal and transformed myoblasts, which suggests that altered regulation is due to an "acquisition" of some new property by transformed cells.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1983

Isolation and characterization of rat skeletal myoblast mutants resistant to lectins.

Mutants resistant to the lethal action of the lectins phytohemagglutinin A (PHA) and wheat germ agglutinin (WGA) have been made in a line of differentiating rat skeletal myoblasts. The WGA mutants are of two types, WGArII, resistant to low concentrations of the lectin, and WGArI, resistant to high concentrations of the lectin. WGArII and PHAr mutants are unable to differentiate, whereas WGArI mutants differentiate normally. WGArII mutants are not impaired in the binding of wheat germ agglutinin, but WGArI mutants bind the lectin only to the extent of about 50% of the wild-type values. All of the mutants are cross-resistant to lectins other than those used in their selection.

Animals↗
PubMed1983

Metabolic labelling of oligosaccharide chains of glycoproteins in a rat myoblast cell line and its lectin resistant mutants.

We have determined the experimental conditions under which optimum hydrolysis of the various oligosaccharides of membrane glycopeptides can be achieved in a permanent line of rat skeletal myoblasts and its wheat germ agglutinin resistant mutants. The labelled glycopeptides were isolated by extensive pronase digestion of whole cells. Labelling studies demonstrated that label from fucose and galactose remained largely in these two sugars, while glucosamine labelled sialic acid and N-acetylglucosamine. Mannose label was found in both mannose and fucose.

Acetylation↗
PubMed1981

Temperature-sensitive mutation affecting synthesis of phosphoenolpyruvate carboxykinase in Escherichia coli.

A mutation has been characterized in Escherichia coli which results in temperature-sensitive expression of phosphoenolpyruvate carboxykinase activity and antigen. The enzyme produced by the mutant strain at a permissive temperature or by cells treated with chloramphenicol at nonpermissive temperatures had normal activity and stability in extracts. Since phosphoenolpyruvate carboxykinase had a monomeric structure, the mutation probably affects the synthesis, rather than the structure or assembly, of the enzyme.

Escherichia coli↗
PubMed1981

Regulation of the activity of a calcium-activated neutral protease during differentiation of skeletal myoblasts.

A calcium-activated neutral protease activity appears concomitantly with myotube formation during the differentiation of a cell line of rat skeletal myoblasts. Other proteases such as cathepsin D and plasminogen activator, however, do not show any changes in their activities. The appearance of the protease is not fusion dependent, as judged by assays of fusion defective myoblast mutants. The formation of the protease is suppressed along with differentiation in the presence of 5-bromodeoxyuridine. Undifferentiated myoblasts contain a potent inhibitor of the protease. The inhibitor, which is probably proteinaceous in nature, is lost during the differentiation of the cells into myotubes. This mode of regulation of an enzyme during differentiation seems so far to be an unique example of its kind.

Animals↗