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Biomedical subjects

E H Ball

Publications and source records attributed to E H Ball.

At least 37 records · Page 2Linked to original sources
PubMed1988

Mapping of caldesmon: relationship between the high and low molecular weight forms.

Caldesmon is a widely distributed contractile protein that occurs in both a high molecular weight [120-150-kilodalton (kDa)] and a low molecular weight (71-80-kDa) form, depending on the tissue. The structural relationship between these two forms was examined by mapping techniques. Partial cyanogen bromide cleavage in conjunction with sodium dodecyl sulfate gel electrophoresis was used to construct a map of the cleavage points and determine the relative position of the fragments in a high molecular weight caldesmon from chicken gizzard (caldesmon125). By use of this map, markers for different regions of the protein were obtained: Antibodies directed toward certain areas were prepared by affinity purification, and specific 125I-labeled tryptic peptides were found to originate from terminal cyanogen bromide fragments. Mapping of a lower molecular weight form of caldesmon (caldesmon72 from chicken liver) revealed the presence of sequences located in both ends of caldesmon125. A terminal 38-kDa fragment of both proteins was apparently identical on the basis of arrangement of cleavage sites, antibody reactivity, and iodopeptide mapping. Fragments from the other end of both proteins exhibited an identical pattern of peptides. These results show that it is sequences located in the central area of caldesmon125 which are missing in caldesmon72, indicating that the smaller molecule is not simply a proteolytic product of the larger. The two forms of caldesmon may be derived from separate genes or by alternative splicing from a single gene.

Animals↗
PubMed1988

The relationship between the bilayer to hexagonal phase transition temperature in membranes and protein kinase C activity.

A number of substances affect the activity of protein kinase C. Among uncharged and zwitterionic compounds, those which activate protein kinase C also lower the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine while substances which inhibit protein kinase C raise this transition temperature. Using this criteria, we have identified 3 beta-chloro-5-cholestene, 5 beta-cholan-24-ol and eicosane as new protein kinase C activators and have shown that Z-Ser-Leu-NH2, Z-Gly-Leu-NH2, Z-Tyr-Leu-NH2, cyclosporin A and cholestan-3 beta, 5 alpha, 6 beta-triol are protein kinase C inhibitors.

Cholesterol↗
PubMed1988

A collagen-binding protein involved in the differentiation of myoblasts recognizes the Arg-Gly-Asp sequence.

We had earlier demonstrated that a 46-kDa glycoprotein is involved in the differentiation of rat skeletal myoblasts. We now show that the binding of this glycoprotein to collagen and gelatin is disrupted by Arg-Gly-Asp (RGD) containing peptide but not by Arg-Gly-Glu (RGE). The former peptide also selectively elutes the 46-kDa glycoprotein bound to gelatin-Sepharose. Since all other proteins which bind RGD sequences have been found at the cell surface, we attempted to localize the 46-kDa glycoprotein by means of immuno fluorescent staining and radioiodine labeling. Surprisingly, the majority of the protein was found to be localized in the endoplasmic reticulum. Protease treatment of a microsomal fraction revealed that the protein is in the interior of the reticulum. Immunoprecipitation experiments, using a polyclonal antibody against the 46-kDa protein, demonstrated that no closely related proteins exist in myoblasts and also confirmed that the protein was not a fragment of a cell-surface localized protein. These findings suggest that the RGD sequence is also used in protein recognition within the cell.

Amino Acid Sequence↗
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

Phosphorylation of caldesmon77 by protein kinase C in vitro and in intact human platelets.

Caldesmon is a widely distributed calmodulin- and actin-binding protein which occurs in different forms depending on the tissue or cell type under examination. On the basis of molecular weight, caldesmon species can be divided into two classes: caldesmon77 (Mr 70,000-80,000) and caldesmon150 (Mr 140,000-150,000). We have examined the phosphorylation of caldesmon77 by protein kinase C (the Ca2+/phospholipid-dependent enzyme) in vitro and in intact platelets. Caldesmon77, purified from bovine liver, could be phosphorylated by purified rat brain protein kinase C to a level of approximately 1.0 mol of phosphate per mol of caldesmon77 monomer. Two-dimensional tryptic peptide mapping and phosphoamino acid analysis reveals that caldesmon77 is phosphorylated at two major sites exclusively on serine residues. Following treatment of platelets with tumor-promoting phorbol ester, caldesmon77 phosphorylation was elevated 4-fold. Tryptic peptide mapping of phosphorylated platelet caldesmon77 demonstrates that phosphorylation is most significantly enhanced on two peptides which had migration patterns identical with those of the two major phosphopeptides of bovine liver caldesmon77 phosphorylated in vitro. The results of this study indicate that protein kinase C can phosphorylate caldesmon77 in vitro and in intact platelets, suggesting a role for protein kinase C in the regulation of caldesmon77 function or localization.

Animals↗
PubMed1986

Quantitation of proteins by elution of Coomassie brilliant blue R from stained bands after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

A simple method for the extraction of Coomassie brilliant blue R from stained protein bands excised from polyacrylamide gels is described. Spectrophotometric measurement of the eluted dye forms the basis of a sensitive assay to quantitate proteins in gels in the range 0.5-10 micrograms. The method requires no unusual equipment and is suitable for measurement of multiple samples. The polypeptide is not extracted and remains available for further analysis. The technique has been applied to three proteins and gels of various acrylamide percentages.

Electrophoresis, Polyacrylamide Gel↗
PubMed1986

Phosphorylation of the cytoskeletal protein talin by protein kinase C.

Talin, a component of the focal contact of cultured cells, is an in vitro substrate for protein kinase C. Immunoprecipitation confirms that talin is the phosphorylated protein. Phosphorylation is dependent on both phosphatidylserine and calcium and reaches a level of incorporation of 0.8 mol phosphate/mol protein. Phosphoamino acid analysis demonstrates the presence of phosphoserine and phosphothreonine, but no phosphotyrosine. Two dimensional mapping of tryptic peptides, and V8 peptides reveals the existence of multiple phosphorylation sites. The identification of talin as a substrate for protein kinase C implicates talin as a potential regulator of focal contact organization and perhaps cell morphology.

Animals↗
PubMed1986

Vinculin interaction with permeabilized cells: disruption and reconstitution of a binding site.

Fluorescently labeled vinculin binds to focal contact areas in permeabilized cells independent of actin (Avnur, Z., J. V. Small, and B. Geiger, 1983, J. Cell Biol., 96:1622-1630), but the nature of the binding site is unknown. In this study we have examined the interaction of vinculin with these sites in permeabilized L6 myoblasts to define conditions that perturb the binding and subsequently to reconstitute it. Mild treatment with low concentrations of protease prevents vinculin incorporation without gross changes in the cytoskeleton or extensive protein breakdown. Exposure to buffers of moderate ionic strength also reduces subsequent vinculin binding without large morphological effects. These extraction conditions were used to obtain a fraction from gizzard which was able to restore the vinculin localization. Talin, actin, and vinculin itself were able to alter the binding of labeled vinculin to permeabilized cells and each also interacted with vinculin in gel overlays; however, they were unable to reconstitute the binding site in treated permeabilized cells. The results show a requirement for an as yet unidentified protein to capacitate vinculin binding to focal contact sites and suggest that this protein is peripheral and interacts directly with the binding site.

Animals↗
PubMed1982

Mitochondria are associated with microtubules and not with intermediate filaments in cultured fibroblasts.

Triple-immunofluorescence experiments with antibodies to cytochrome c oxidase, tubulin, and vimentin have been used to immunolabel the mitochondria, microtubules, and intermediate filaments inside the same cultured fibroblasts. In particular, fibroblasts were immunolabeled after they had either been transformed by infection with Rous sarcoma virus or given long-term treatment with cycloheximide. These treatments induced redistribution of the intermediate filaments into a perinuclear arrangement, segregated away from the microtubules, which remained extended to the cell periphery. In such cells, many labeled mitochondria were observed to be codistributed with the peripherally located microtubules. From these results, we infer that an association, probably involving some type of chemical linkage(s), between mitochondria and microtubules exists in these cells that is independent of the intermediate filaments.

Animals↗
PubMed1981

Vinculin: a cytoskeletal target of the transforming protein of Rous sarcoma virus.

Vinculin, a protein associated with the cytoplasmic face of the focal adhesion plaques which anchor actin-containing microfilaments to the plasma membrane and attach a cell to the substratum, contains 8-fold more phosphotyrosine in cells transformed by Rous sarcoma virus than in uninfected cells. Because the transforming protein of RSV, p60src, is a protein kinase that modifies cellular proteins through the phosphorylation of tyrosine and because phosphotyrosine is a very rare modified amino acid, this result is a very rare modified amino acid, this result suggests that vinculin is a primary substrate of p60src. Only trace amounts of phosphotyrosine were detected in myosin heavy chains, alpha-actinin, filamin, and the intermediate filament protein vimentin. The modification of vinculin by p60src may be responsible in part for the disruption of the microfilament organization and for the changes in cell shape and adhesiveness which accompany transformation by Rous sarcoma virus.

Amino Acids↗
PubMed1981

Association of microtubules and intermediate filaments in normal fibroblasts and its disruption upon transformation by a temperature-sensitive mutant of Rous sarcoma virus.

By double indirect immunofluorescence, using primary rabbit antibodies to tubulin and guinea pig antibodies to vimentin, we have simultaneously labeled microtubules and intermediate filaments in several types of cultured normal fibroblasts. With well-spread interphase cells there was an extensive but not complete correspondence of the labeling patterns for the two filamentous structures out to the cell periphery. This correspondence existed both at a gross level, where parallel but not coincident arrays of thickly labeled strands of the two types of filaments were observed, and at a fine level, where thinly labeled strands of the two were superimposed. The results suggest that there may be some type(s) of molecular linkages between microtubules and vimentin intermediate filaments that is under metabolic control. With NRK fibroblasts infected with a temperature-sensitive mutant (LA23) of Rous sarcoma virus, cells grown at the nonpermissive temperature (39 degrees C) showed the correspondence of the distributions of the microtubules and intermediate filaments characteristic of the normal phenotype but within 1 hr after a shift to the permissive temperature (33 degrees C) there was an extensive retraction of the intermediate filaments around the cell nucleus whereas the microtubules remained dispersed into the cell periphery. These results suggest that one of the functions carried out by p60src, the protein kinase responsible for transformation by Rous sarcoma virus, may be to modify the component(s) involved in the putative linkages between microtubules and intermediate filaments in the normal cells.

Animals↗
PubMed1981

Unusual response of a yeast to imidazole antifungals.

Thiamin was shown to relieve preferentially the toxicity of econazole for a yeast which showed an unusual response to imidazole drugs. The possible role of this vitamin in the response of the yeast to imidazoles is discussed. An investigation of the effect of a second exposure of yeasts to imidazole drugs showed that there is an increase in sensitivity when the cells are transferred directly to fresh medium containing the same concentrations of the drug, and a decrease in sensitivity following transfer via drug-free medium.

Candida↗
PubMed1980

A synergistic effect of glucocorticoids and insulin on the differentiation of myoblasts.

Glucocorticoids and insulin were found to act synergistically to promote differentiation in some clones of the L6 rat myoblast cell line. Other hormone effects on these cells were investigated to determine the extent of the synergism. The insulin stimulation of sugar transport was unaffected by glucocorticoids although they did by themselves slightly enhance transport. Glucocorticoids were found to increase the adhesiveness of the cells--an effect not influenced by insulin. Cyclic AMP levels were found to peak just prior to the time of the onset of fusion and insulin broadened this peak, while the combination of both hormones further lengthened the time for which cyclic AMP levels remained elevated.

Animals↗
PubMed1979

Experimental infection of guinea-pigs with atypical and dysgonic strains of microsporum canis.

Pathogenicity tests with one dysgonic and six atypical strains of Microsporum canis were carried out on guinea-pigs. Five of the atypical strains were laboratory mutants from dysgonic strains isolated from living hosts. As sporulation and viability varied greatly between the strains, inocula consisted of suspensions of fungal fragments of known viable count. When a sufficiently active inoculum was used, lesions and fluorescent hairs were induced in the guinea-pigs by all but one of the strains tested. In each case the strain inoculated was reisolated from the lesions in pure culture. The significance of the results is discussed in the light of the unusual nature and origin of the strains.

Animals↗
PubMed1979

The cyclic adenosine monophosphate phosphodiesterases of myoblasts, fibroblasts, and their somatic cell hybrids.

Three forms of cAMP phosphodiesterases are found in mouse L cells (fibroblasts) and rat skeletal myoblasts. The myoblast enzymes can be resolved by chromatography on DEAE-cellulose and the fibroblast enzymes by chromatography on DEAE-Biogel. The myoblast enzymes are "high affinity" cAMP specific forms and have different molecular weights, while all L-cell enzymes have an apparent molecular weight of 450,000. Only one of the L-cell enzymes is able to hydrolyze both cyclic guanosine monophosphate (cGMP) and cAMP. Hydrolysis of the latter is stimulated by micromolar amounts of cGMP. The myoblast x L cell hybrids possess at least five phosphodiesterases, three of which can be identified as being of myoblast or fibroblast origin. One of the fibroblast enzymes appears to be modified in hybrids. The entire phosphodiesterase regulatory system of the myoblasts is active in the hybrids.

3',5'-Cyclic-AMP Phosphodiesterases↗