No abstract

No abstract

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 ofoptic 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. Reagents. The calcium ionophore A23187 was purchased from Calbiochem-Behring and was dissolved in dimethyl sulfoxide. Theophylline, papaverine, and caffeine were purchased from Sigma. Verapamil was supplied by Knoll Pharmaceutical (Whippany, NJ). Trifluoperazine (TFP), [3H]TFP (67 ,uCi/mg; 1 Ci = 3.7 X 10O' becquerels), chlorpromazine (CPZ), and chlorpromazine sulfoxide (CPZ-S02) were the gifts of Smith Kline & French.Antisera. Sheep anticalmodulin (rat testis) was the gift of John Dedman (University of Texas Medical School). Fluorescein-conjugated rabbit anti-sheep IgG was purchased from GIBCO.Culture Experiments. All cultures were incubated at 37°C in medium 199 (GIBCO) in humid 5% C02/95% air. The inhibitory effects of various drugs were studied in an overnight culture system. The heads of stage 12 to 13 chicken embryos were excised, placed in medium containing a variety of concentrations of calcium antagonists or phenothiazines, and then incubated for 18 hr. The phenothiazines and papaverine were dissolved in water and diluted to the appropriate concentrations with medium. Verapamil was dissolved in 95% ethanol. Control heads were placed in medium containing no drug and incubated in parallel with each group of experimental heads. After 18 hr, experimental heads were examined by light microscopy and compared with controls for optic cup formation. Photographs were taken through a Wild M5 (Wild-Heerbrugg, Farmingdale, NY) dissecting microscope. Precocious optic cup formation in response to calcium agonists was assessed in a short-term (30-min) culture system.[3H]TFP Binding Studies. Heads of stage 13 chicken embryos were excised with extreme care to ensure equal size. They were incubated at 37°C in the presence of various concentrations of [3H]TFP (67 ,Ci/mg) for various periods of time as stated in the figures. After incubation, each head was individually transferred through three successive wash vials containing 10 ml of ice-cold medium. The medium used for washing was always the same as that used in the initial incubation except that it contained no drug. Each head was kept in each wash vi...

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.

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.