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R C Brady

Publications and source records attributed to R C Brady.

28 records · Page 2Linked to original sources
PubMed1986

Identification of a 52-kD calmodulin-binding protein associated with the mitotic spindle apparatus in mammalian cells.

A pool of 10 calmodulin-binding proteins (CBPs) was isolated from Chinese hamster ovary (CHO) cells via calmodulin (CaM)-Sepharose affinity chromatography. One of these ten isolated CBPs with a molecular mass of 52 kD was also found to be present in isolated CHO cell mitotic spindles. Affinity-purified antibodies generated against this pool of isolated CBPs recognize a single 52-kD protein in isolated CHO cell mitotic spindles by immunoblot analysis. Immunofluorescence examination of CHO, 3T3, NRK, PTK-2, and HeLa cells resulted in a distinct pattern of mitotic spindle fluorescence. The localization pattern of this 52-kD CBP directly parallels that of CaM in the spindle apparatus throughout the various stages of mitosis. Interestingly, there was no association of this 52-kD CBP with cytoplasmic microtubules. As is the case with CaM, the localization pattern of the 52-kD CBP in interphase cells is diffuse within the cytoplasm and is not associated with any discrete, cellular structures. This 52-kD CBP appears to represent the first mitotic spindle-specific calmodulin-binding protein identified and represents an initial step toward the ultimate determination of CaM function in the mitotic spindle apparatus.

Animals↗
PubMed1986

Subcellular distribution of calmodulin and its binding proteins within the rat submandibular gland.

The involvement of calmodulin (CaM) in rat submandibular gland mucin secretion was investigated in vitro using a dispersed cell preparation. The CaM antagonist trifluoperazine (TFP) inhibited mucin secretion in response to both isoproterenol and dibutyryladenosine 3',5'-cyclic monophosphate. The inhibitory concentrations of TFP were between 10 and 100 microM. One millimolar TFP was toxic to submandibular cells and resulted in decreased levels of cellular ATP and a significant release of lactate dehydrogenase. Determination of CaM levels via radioimmunoassay within various subcellular fractions indicated that the majority of the CaM within the rat submandibular cell was located within the cytoplasm. CaM binding proteins were also identified within these subcellular fractions utilizing a gel overlay procedure. The two major, specific CaM binding proteins present within rat submandibular cells were a 59-kDa cytosolic protein and a 47-kDa membrane-associated protein.

Animals↗
PubMed1985

Colonic glycoprotein secretion and calmodulin-acceptor proteins in the reserpine-treated rat.

The rate of radioactive precursor-labeled colonic glycoprotein secretion in chronically reserpine-treated versus saline-injected control rats was examined. Everted colonic sacs prepared from reserpine-treated rats were found to be hypersecretory, exhibiting a basal rate of glycoprotein secretion that was threefold higher than control everted sacs. Furthermore, glycoprotein secretion in control tissue was stimulated by the secretagogue carbachol, and this stimulation was precluded by 10 microM trifluoperazine, the calmodulin antagonist. Reserpine-treated tissue, in contrast, was refractory to treatment with carbachol as well as trifluoperazine. While reserpine-treated and control colonic mucosae were demonstrated to contain equivalent levels of calmodulin via radioimmunoassay, reserpine-treated tissue was determined to lack two calmodulin-acceptor proteins with molecular weights of 29 and 47 kilodaltons. The data suggest that the mechanism by which reserpine elicits this cystic fibrosislike, hypersecretory state of glycoprotein secretion within the colonic mucosa entails the loss of calmodulin function in the regulation of this secretory process. We speculate that the biochemical defect present in cystic fibrosis could also entail such a loss of calmodulin function in the regulation of glycoprotein secretion.

Animals↗
PubMed1984

Reserpine-induced alterations in mucus production and calmodulin-binding proteins in a human epithelial cell line.

The characterization of a mucus-producing human cell line (HC-84) derived from a colon carcinoma and its response to in vitro reserpine treatment is reported. Mucous granules were demonstrated within these cells on the basis of electron microscopic examination and incorporation of [3H]glucosamine with subsequent autoradiographic analysis. Fluorographic analysis of total HC-84 cell protein after incubation with [3H]glucosamine indicated that the majority of tritium was incorporated into two proteins with molecular weights of 115 and 120 kD. When total HC-84 protein was subjected to immuno-blot analysis utilizing rabbit antibody against human intestinal mucus, only these two proteins (115K and 120K) reacted positively, indicating a direct correlation between [3H]glucosamine incorporation and mucus production. Immunofluorescence localization of mucus within HC-84 cells utilizing this same antibody resulted in a punctate pattern of fluorescence within the cytoplasm. Treatment of HC-84 cells with 30 microM reserpine for 7 days resulted in a three-fold increase in mucus production compared with controls. There was also a concomitant loss of a 30K calmodulin-binding protein in cells treated with reserpine. These cells represent a useful system for studying the effect of reserpine on the processes of mucus synthesis and secretion.

Calcium-Binding Proteins↗
PubMed1984

Effect of cAMP and calmodulin inhibitors on water absorption in rat proximal tubule.

The possible role of calmodulin in solute transport was examined in the kidney of the rat. Utilizing a radioimmunoassay, calmodulin was identified and quantitated in homogenates of the cortex of the kidney. The physiologic significance of these findings was examined utilizing in vivo microperfusion techniques applied to the proximal convoluted tubule of the thyroparathyroidectomized rat. The addition of dibutyryl cyclic adenosine monophosphate (cAMP) to the luminal perfusion solution resulted in a lower rate of water absorption of 1.67 +/- 0.09 nl min-1 mm-1 as compared to 2.46 +/- 0.11 in controls. The addition of either of two compounds with affinity for calmodulin, trifluoperazine (TFP) or W-13, reversed the cAMP-induced inhibition of water absorption. In the absence of cAMP, neither agent affected water absorption. Analogs of TFP and W-13 with lower binding affinities for calmodulin had no effect on water absorption and did not reverse the cAMP effect. None of the above experimental maneuvers affected the absorption of phosphate. These results demonstrate the presence of calmodulin in the kidney of the rat and suggest that calmodulin may be involved in cAMP-associated inhibition of water and electrolyte transport in the proximal tubule of the rat.

Absorption↗
PubMed1983

The role of calmodulin in cell transformation.

Two cell lines transformed with temperature sensitive retroviruses were examined for: their ability to grow in low Ca2+ medium, their calmodulin levels and changes in calmodulin acceptor proteins. Both cell lines grow in low Ca2+ medium at the permissive temperature 34 degrees C while both lines did not replicate at the non-permissive temperature 39 degrees C. The NRKLA23 cells have nearly twice as much calmodulin at the permissive temperature than they do at the non-permissive temperature while the 6M2 cells have an equal amount of calmodulin at both temperatures. Both cell lines exhibit changes in the calmodulin acceptor proteins going from the permissive to the non-permissive temperature. We suspect that the changes in the calmodulin acceptor proteins may be involved in the altered Ca2+-sensitivity of growth in the cells going from the permissive to non-permissive temperature.

Animals↗
PubMed1983

Production of monoclonal antibodies against calmodulin by in vitro immunization of spleen cells.

Monoclonal antibodies against the highly conserved ubiquitous calcium-binding protein, calmodulin (CaM), were produced by immunization of mouse primary spleen cell cultures. Dissociated spleen cells were cultured for 5 d in the presence of mixed thymocyte culture conditioned media (TCM) and purified bovine testes CaM (50 ng-1 mg). Following immunization, cells were fused with mouse myeloma cells (SP2/0, Ag 8.653) and cultured for 2-3 wk before initial screening for antibody. In five independent immunizations there was a range of 25-44% of the initial polyclonal cultures which produced antibodies reacting with purified CaM as determined by immunoassay. 80% of the cloned hybridoma produced IgM immunoglobulins while the remaining clones were IgG producers. This ratio was changed to 50% IgM and 50% IgG by subsequent extension of the in vitro immunization periods and reduced amounts of antigen and extended in vitro culturing. In vitro immunization introduces a new dimension to monoclonal antibody production where limited antigen or poorly antigenic proteins are of interest. The monoclonal antibodies produced in this study have enabled us to to selectively localize CaM in association with distinct subcellular structures, mitochondria, stress fibers, centrioles, and the mitotic spindle.

Animals↗
PubMed1982

Optic cup formation: a calcium-regulated process.

Invagination of the optic vesicle to form the optic cup is an important event in the formation of the eye in the early embryo. To obtain support for earlier conclusions that a contractile process is involved, calcium dependency of optic cup formation was tested. Heads were excised from chicken embryos at the optic vesicle stage of development (stage 13) and incubated in nutrient medium containing antagonists or agonists of calcium transport. Invagination was reversibly inhibited by the Ca2+ antagonists verapamil and papaverine. It was initiated in a precocious fashion by the Ca2+ ionophore A23187 but only in the presence of external Ca2+. Neither caffeine, theophylline, nor A23187 (in the absence of external Ca2+) were able to initiate precocious optic cup formation. Trifluoperazine and chlorpromazine reversibly inhibited optic cup formation while chlorpromazine sulfoxide had no effect at the concentrations used. The binding of [3H]trifluoperazine to isolated stage 13 heads revealed a class of Ca2+-dependent binding sites having a Kd similar to that of calmodulin. These results indicate a Ca2+-dependence for optic cup formation and that the source of the Ca2+ may be extracellular. This Ca2+ dependence probably is conferred to the system by calmodulin.

Animals↗
PubMed1981

Trifluoperazine inhibits spreading and migration of cells in culture.

Trifluoperazine (TFP) blocks spreading and migration of cultured mammalian cells. These are calcium-dependent and microfilament-mediated processes. Calmodulin, a regulator of many calcium-dependent processes in cells, is selectively inhibited by TFP. Cell spreading on a plastic- or collagen-coated substratum was reversibly inhibited by 10 micro M TFP. The drug blocks cell spreading even in the presence of 1 mM cAMP. TFP is as effective as cytochalasin B (CB), in inhibitor of microfilament function, in blocking cell spreading. All cell lines tested, whether "normal" or virally transformed, failed to spread to TFP. The drug, at a concentration sufficient to inhibit spreading, does not interfere with the initial attachment of a cell to a plastic surface. Cells plated in the presence of 10 micro M TFP attach at a rate and to an extent equal to untreated controls. TFP added to already spread cells results in a reversible cell rounding. Detection of fibronectin by indirect immunofluorescence suggests TFP-induced cell rounding is not due to shedding of fibronectin from the cell surface. TFP reversibly blocks cell migration into a would edge almost as effectively as CB. We suggest that TFP interferes with these microfilament-mediated functions by direct action on the microfilaments or indirect action by inactivating calmodulin.

Animals↗