Biomedical subjects

C Decker

Publications and source records attributed to C Decker.

34 records · Page 2Linked to original sources

PubMed1992

Analysis of chimeric mRNAs derived from the STE3 mRNA identifies multiple regions within yeast mRNAs that modulate mRNA decay.

In the yeast Saccharomyces cerevisiae unstable mRNAs decay 10-20 fold more rapidly than stable mRNAs. In order to examine the basis for the differences in decay rate of the unstable STE3 mRNA and the stable PGK1 and ACT1 mRNAs we have constructed and measured the decay rates of numerous chimeric mRNAs. These experiments indicate that multiple regions within yeast mRNAs are involved in modulating mRNA decay rates. Our results suggest that at least two regions within the STE3 mRNA are involved in stimulating rapid decay. One region is located within the coding region and requires sequences between codons 13 and 179. In addition, the STE3 3' UT can also function to stimulate decay. Surprisingly, the STE3 3' UT is not sufficient to accelerate the turnover of the stable PGK1 transcript unless portions of the PGK1 coding region are first deleted. These results not only identify sequences that function within yeast to stimulate mRNA turnover but also have important implications for an understanding of the basis of differences in eukaryotic mRNA decay rates.

Base Sequence↗

PubMed1992

Specific cleavage of pre-edited mRNAs in trypanosome mitochondrial extracts.

RNA editing in Trypanosoma brucei is a posttranscriptional processing event that results in the addition and deletion of uridine residues within several mitochondrial mRNAs. We have examined reactions involving pre-edited precursor RNAs in vitro. In this study, we report specific cleavage of pre-edited cytochrome b (CYb), cytochrome oxidase subunit II (COII), and cytochrome oxidase subunit III (COIII) mRNAs when incubated with T. brucei mitochondrial extracts. The pre-edited CYb RNA was cleaved near the 3'-most uridine addition sites, within the region where editing would be expected to commence. Pre-edited COII mRNA was similarly cleaved adjacent to its small editing domain, while pre-edited COIII RNA was cleaved at multiple sites in the region where uridine addition and deletion occurs in vivo. In contrast, edited versions of CYb, COII, and COIII RNAs were not cleaved within the editing domains. Such differential cleavage of the edited and pre-edited forms of these mRNAs suggests either a direct involvement in RNA editing or involvement in another aspect of mitochondrial gene expression requiring cleavage of pre-edited RNAs.

Animals↗

PubMed1989

Integrin on developing and adult skeletal muscle.

Avian integrin is a complex of integral membrane glycoproteins that appears to function as a dual receptors for both intracellular cytoskeletal and extracellular matrix components. Antibodies were raised against this complex and used to (1) immunolocalize integrin on cryosections of developing and adult muscle tissue and on developing myotube cultures in vitro and (2) immunoaffinity purify integrin from various fiber-type specific muscles. Integrin localization was compared with that of its putative cytoskeletal-associated and extracellular matrix ligands, talin and vinculin and fibronectin and laminin, respectively. The goal was to identify putative sites of interaction between the muscle sarcolemma and the cytoskeleton and the extracellular matrix and to reveal any differences in the molecular composition at these sites. Integrin's distribution on the sarcolemma of early (Day 12) embryonic limb muscle was random and punctate. On late embryonic (Days 17-19) limb muscle tissue its distribution was generally uniform but with occasional increased densities at specific sites along the sarcolemma. Posthatch (greater than 3 weeks) fast twitch muscle showed a highly regionalized distribution. These regions of integrin concentration coincided with densities of acetylcholine receptors, revealed by TRITC alpha-bungarotoxin labeling, and regions of muscle-tendon interaction, identified by morphological criteria. Tissue culture studies also demonstrated integrin densities at analogous sites in vitro, e.g., acetylcholine receptor clusters and sites at which myofibrils terminate at the sarcolemma. These integrin-rich sites were also shown to be Triton X-100 insoluble and therefore presumably are linked to the cytoskeleton or extracellular matrix. The localization of integrin on developing and adult muscle tissue was compared with that of fibronectin, laminin, vinculin, and talin using double, immunofluorescently labeled cryosections. In general, integrin did not colocalize exclusively with any one of its putative ligands. In the embryo, discrete densities of both talin and vinculin were observed at the myotendinous junction, whereas integrin immunoreactivity was widely distributed on muscle, vasculature, nerve, and connective tissue with no discernible sites of increased density. Laminin was primarily associated with muscle and nerve whereas fibronectin was prominent on connective tissue. On posthatch tissue, the distributions of talin, vinculin, laminin, and fibronectin were similar to those in the embryo, whereas the distribution of integrin was restricted to specific sites. The distribution of integrin was also examined for fiber-type specific differences on adu

Animals↗

PubMed1988

The role of protein kinase C in the inactivation of hepatic glycogen synthase by calcium-mobilizing agonists.

The regulation of glycogen synthase by Ca2+-mobilizing hormones was studied by using rat liver parenchymal cells in primary culture. Long-term exposure of hepatocytes to 4 beta-phorbol 12-myristate 13-acetate (TPA) resulted in a decrease in vasopressin or ATP inhibition of glycogen synthesis and glycogen synthase activity, without any change in the activation of glycogen phosphorylase. In contrast, treatment with TPA did not diminish the effects of glucagon, isoprenaline or A23187 on glycogen synthase or phosphorylase. TPA treatment for 18 h did not change specific [3H]vasopressin binding, but abolished protein kinase C activity in a concentration-dependent manner. The effects of TPA to decrease protein kinase C activity and to reverse the inactivation of glycogen synthase by vasopressin were well correlated and were mimicked by mezerein, but not by 4 alpha-phorbol. However, 1 microM-TPA totally inhibited protein kinase C activity, but reversed only 60% of the vasopressin effect on glycogen synthase. It is therefore concluded that Ca2+-mobilizing hormones inhibit glycogen synthase partly, but not wholly, through a mechanism involving protein kinase C.

Adenosine Triphosphate↗

PubMed1988

Platelet membrane fluidity and treatment response in cognitively-impaired, depressed elderly: initial results.

A prospective study of response to treatment with nortriptyline was carried out in a group of 14 inpatients with mixed symptoms of depression and dementia. Platelet membrane fluidity, as reflected by the fluorescence anisotropy of DPH labeled membranes, was determined for all 14 drug-free subjects prior to treatment by staff who were blind to clinical and demographic data. Patients who exhibited worsening of their cognitive impairment exhibited "increased" platelet membrane fluidity, an abnormally found in approximately 50% of patients with Alzheimer's disease.

Aged↗

PubMed1988

Combinations of monoclonal antibodies distinguish mesenchymal, myogenic, and chondrogenic precursors of the developing chick embryo.

Monoclonal antibodies (MAbs) were used as probes for molecular differences in the surfaces of nonterminally differentiated cells of the developing chick limb. The specificity of the MAbs was determined by immunofluorescent localization performed on cultured breast muscle and limb bud cells and cryosections of a variety of embryonic (stages 15-37) and neonatal tissues. Subpopulations of MAb-positive and -negative cells were isolated by fluorescence-activated cell sorting and their developmental potential was assessed in vitro. Cells of the compacted somite, lateral plate mesoderm, and early limb bud were labeled with the CSAT MAb. Myogenic precursors of the dermatome and limb bud were labeled with the CSAT and L4 MAbs. Chondrogenic precursors of the sclerotome and limb bud were labeled with the CSAT, L4, and C5 MAbs. These precursors were distinguished from fibroblasts which were labeled with the CSAT and C1 MAbs. The differentiation and maturation of muscle and cartilage were accompanied by alterations in the labeling patterns of the MAbs. These results indicate that combinations of these MAbs can be used to distinguish mesenchymal, myogenic, and chondrogenic precursors, identify their site of origin during development, and isolate subpopulations of embryonic cells.

Animals↗

PubMed1987

Degradation of rat hepatic cytochrome P-450 heme by 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine to irreversibly bound protein adducts.

Administration of 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP) (a structural analog of the dihydropyridine Ca2+ antagonists) to untreated, phenobarbital-, or dexamethasone-pretreated rats results in time-dependent losses of hepatic cytochrome P-450 content. Functional markers for various cytochrome P-450 isozymes have permitted the identification of P-450h, P-450 PB-1/k, and P-450p as the isozymes inactivated preferentially by the drug. DDEP-mediated cytochrome P-450 destruction may be reproduced in vitro, is most prominent after pretreatment of rats with dexamethasone, pregnenolone 16 alpha-carbonitrile or phenobarbital, and is blocked by triacetyloleandomycin. These findings together with the observation that DDEP markedly inactivates hepatic 2 beta- and 6 beta-testosterone hydroxylase and erythromycin N-demethylase tend to indict the steroid-inducible P-450p isozyme as a key protagonist in this event. The precise mechanism of such DDEP-mediated P-450p heme destruction is unclear, but involves prosthetic heme alkylation of the apocytochrome at its active site in what appears to be a novel mechanism-based "suicide" inactivation. Such inactivation appears to involve fragmentation of the heme to reactive metabolites that irreversibly bind to the protein, but the chemical structure of the heme-protein adducts is yet to be established. Intriguingly, such DDEP-mediated P-450p destruction in vivo also results in accelerated loss of immunochemically detectable apocytochrome P-450p. It remains to be determined whether or not this loss is due to enhanced proteolysis triggered by the structural modification of the apocytochrome.

Animals↗

PubMed1986

Inactivation of rat hepatic cytochrome P-450 by spironolactone.

Administration of antimineralocorticoid spironolactone (SPL) to rats results in modest destruction of hepatic cytochrome P-450 with parallel loss of heme. This process is accentuated by pretreatment with dexamethasone (DEX), an inducer of cytochrome P-450p and is associated with marked functional loss of cytochrome P-450p-dependent hydroxylases. Cytochrome P-450 destruction may be replicated in vitro when microsomes from DEX-pretreated rats are incubated with SPL and NADPH and is impaired when these rats are given triacetyloleandomycin, an inhibitor of cytochrome P-450p. In vitro SPL-mediated cytochrome P-450 destruction is accompanied by a loss of heme, which appears to be converted to reactive intermediates which covalently bind to microsomes or are converted to polar metabolites.

Animals↗

PubMed1985

The cell substrate attachment (CSAT) antigen has properties of a receptor for laminin and fibronectin.

The cell substrate attachment (CSAT) antigen is an integral membrane glycoprotein complex that participates in the adhesion of cells to extracellular molecules. The CSAT monoclonal antibody, directed against this complex, inhibited adhesion of cardiac and tendon fibroblasts and skeletal myoblasts to both laminin and fibronectin, thus implicating the CSAT antigen in adhesion to these extracellular molecules. Equilibrium gel filtration was used to explore the hypothesis that the CSAT antigen functions as a cell surface receptor for both laminin and fibronectin. In this technique, designed for rapidly exchanging equilibria, the gel filtration column is pre-equilibrated with extracellular ligand to ensure receptor occupancy during its journey through the column. Both laminin and fibronectin formed complexes with the CSAT antigen. The association with laminin was inhibited by the CSAT monoclonal antibody; the associations with both fibronectin and laminin were inhibited by synthetic peptides containing the fibronectin cell-binding sequence. Estimates of the dissociation constants by equilibrium gel filtration agree well with those available from other measurements. This suggests that these associations are biologically significant. SDS PAGE showed that all three glycoproteins comprising the CSAT antigen were present in the antigen-ligand complexes. Gel filtration and velocity sedimentation were used to show that the three bands comprise and oligomeric complex, which provides an explanation for their functional association. The inhibition of adhesion by the CSAT monoclonal antibody and the association of the purified antigen with extracellular ligands are interpreted as strongly implicating the CSAT antigen as a receptor for both fibronectin and laminin and perhaps for other extracellular molecules as well.

Animals↗

PubMed1984

The kinetics of myoblast fusion.

The kinetics of myoblast fusion were estimated using two complementary assays. Both utilized suspensions of fusion-competent cells, i.e. 48-52-h cultures of chick pectoral muscle grown in a low-calcium medium, thus minimizing contributions arising from cellular migration. One assay, designed to measure the onset of membrane contiguity, relies on the transfer of a lipid dye, diI-C18-[3], from labelled to unlabelled cells. The other assay, designed to estimate the kinetics of appearance of morphologically distinct multinucleate cells in suspension (myoballs), relies on enzymic dissociation of cellular aggregates followed by nuclear staining. The assays demonstrate significant membrane contiguity within 20-30 min after initiating the fusion process; however, the multinucleate myoball morphology does not appear for at least one additional hour.

Animals↗

PubMed1984

Adhesive multiplicity in the interaction of embryonic fibroblasts and myoblasts with extracellular matrices.

Neff et al. (1982, J. Cell Biol., 95:654-666) have described a monoclonal antibody, CSAT, directed against a cell surface antigen that participates in the adhesion of skeletal muscle to extracellular matrices. We used the same antibody to compare and parse the determinants of adhesion and morphology on myogenic and fibrogenic cells. We report here that the antigen is present on skeletal and cardiac muscle and on tendon, skeletal, dermal, and cardiac fibroblasts; however, its contribution to their morphology and adhesion is different. The antibody produces large alterations in the morphology and adhesion of skeletal myoblasts and tendon fibroblasts; in contrast, its effects on the cardiac fibroblasts are not readily detected. The effects of CSAT on the other cell types, i.e., dermal and skeletal fibroblasts, cardiac muscle, 5-bromodeoxyuridine-treated skeletal muscle, lie between these extremes. The effects of CSAT on the skeletal myoblasts depends on the calcium concentration in the growth medium and on the culture age. We interpret these differential responses to CSAT as revealing differences in the adhesion of the various cells to extracellular matrices. This interpretation is supported by parallel studies using quantitative assays of cell-matrix adhesion. The likely origin of these adhesive differences is the progressive display of different kinds of adhesion-related molecules and their organizational complexes on increasingly adhesive cells. The antigen to which CSAT is directed is present on all of the above cells and thus appears to be a lowest common denominator of their adhesion to extracellular matrices.

Animals↗

PubMed1982

A monoclonal antibody detaches embryonic skeletal muscle from extracellular matrices.

We have described a monoclonal antibody that rounds and detaches chick skeletal myoblasts and myotubes from extracellular substrata. The antibody also inhibits the attachment of myogenic cells to a gelatin-coated substratum but has no detectable effect on myoblast fusion. The cellular response to antibody treatment varies with differentiation and cell type. Young myoblasts and myotubes are rapidly rounded and detached by the antibody. Older myotubes require longer incubation times or higher antibody titers for rounding and detachment. Chick embryo fibroblasts, cardiac cells, and neurons are not similarly rounded and remain attached. Since the antibody also detaches cells from embryonic muscle tissue explants, the cell-substratum interaction perturbed by the antibody appears relevant to the in vivo interaction of myogenic cells with their extracellular matrices. Binding studies using iodinated antibody revealed 2-4 x 10(5) sites per myoblast with an apparent Kd in the range of 2-5 x 10(-9) molar. Embryo fibroblasts bind antibody as well and display approximately twice the number of binding sites per cell. The fluorescence distribution of antigen on myoblasts and myotubes is somewhat punctate and particularly bright along the edge of the myotube. The distribution on fibroblasts was also punctate and was particularly bright along the cell periphery and portions of stress fibers. For both cell types the binding was distinctly different than that reported for collagen, fibronectin, and other extracellular molecules. The antigen, as isolated by antibody affinity chromatography, inhibits antibody-induced rounding. SDS PAGE reveals two unique polypeptides migrating in the region of approximately 120 and 160 kilodaltons (kd). The most straightforward mechanism for the antibody-induced rounding and detachment is the perturbation of a membrane molecule involved in adhesion. The hypothesized transmembrane link between extracellular macromolecules and the cytoskeleton provides an obvious candidate.

Animals↗

PubMed1970