Studies with cyanidium caldarium,an anomalously pigmented chlorophyte

Summary Cyanidium caldarium, an alga found in acid hot springs troughout the world, has a morphology and developmental history resembling those of Chlorella, but contains C-phycocyanin and no chlorophyll other than chlorophyll a. The reasons for considering it to be a member of the Chlorophyta are reviewed. Cyanidium is also remarkable for its thermal and acid tolerance. It grows readily in the dark on sugar media. However, light is required for the formation of chlorophyll and phycocyanin except in occasional variant cells which can form limited amounts of these pigments in the dark. Light-grown Cyanidium carries out normal green plant photosynthesis but resembles the red and some of the blue-green algae in that chlorophyll-absorbed light is used with lower efficiency than that absorbed by phycocyanin.The possible significance of the unusual pigmentation of Cyanidium is discussed.Contribution no.23 from the Laboratory of Comparative Physiology and Morphology of The Kaiser Foundation.     

Titration and Neutralization of Poliovirus in Micro Tissue Culture Under Increased Carbon Dioxide

Modification of the micro tissue culture technique, including incubation under increased CO(2), resulted in prolongation of the viability of the cells. As a consequence, satisfactory titrations of poliovirus and of poliovirus antiserum have been achieved by the micro method. The technique offers a number of advantages over conventional methods.     

Production of monoclonal antibodies against avian LHRH-I

Summary Production of antibodies against peptides or poorly antigenic proteins by conventional methods often requires either large quantities of the native immunogen or some chemical modification to increase their antigenicity. In this study an in vivo and in vitro immunization protocol has been used to generate monoclonal antibodies against the decapeptide luteinizing hormone-releasing hormone (LHRH). Two injections of 100 μg of avian LHRH-I into BALB/c mice were given 7 d apart. Dissociated splenocytes were collected under sterile conditions. They were incubated with 100 μg of the immunogen in 75-cm² tissue culture flasks in thymocyte-conditioned media. After 5 to 8 d exposure to the antigen, splenocytes were fused with SP2/O myeloma cells by polyethylene glycol. The cells were plated into 24 wells and then incubated in hypoxanthine aminopterin and thymidine selective media. After 14 d an initial screening was done by enzyme immunoassay. The positive wells (6/24) were expanded into 96-well plates and rescreened. Selected lines were cloned out 3 times by limiting dilution and the most positive expanded for ascites production. The antibody was affinity purified in a protein A column. The antibody cross-reacted with LHRH-I and II but preferentially to LHRH-I, as shown by competitive assay. A hypothalamic extract from a mature chick showed a higher response than preparations from whole brain explants of 1- to 3-d posthatched chicks, mature quail, and mature mouse. This work was funded by the Maryland Agricultural Experiment Station artical no. A4975, contribution no. 8019.     

Production and characterization of a compound inhibitory to Vibrio parahaemolyticus from Proteus vulgaris

The production and characteristics of a compound in Proteus vulgaris G cultures which was capable of inhibiting Vibrio parahaemolyticus and other food-borne pathogens was investigated. Production was influenced by medium composition, pH and temperature but not by the extent of aeration. The compound was most inhibitory at the optimum temperature for growth of V. parahaemolyticus. The inhibitor was most stable at pH 7·0 and inhibition occurred even after heating at 70°C for 30 min and after autoclaving. Ultrafiltration showed that the inhibitor had a molecular weight less than 1000. Thin layer chromatography of filtrates and subsequent peptidase digestion indicated that it was at least in part a peptide. The inhibitor purified by Sephadex G-15 gel filtration had a calculated molecular weight of 731 and contained only six amino acids.     

Studies on the Effect of Insulin-Like Growth Factor-I on Catecholamine Secretion from Chromaffin Cells

Chromaffin cells cultured in serum-free medium secreted a smaller percentage of their catecholamine stores in response to stimulation by high K+ (55 mM) than did cells cultured in serum-containing medium. Addition of insulin-like growth factor-I (IGF-I) to serum-free medium restored high K(+)-stimulated catecholamine secretion to the levels seen in serum-treated cultures. In contrast, addition of IGF-I to serum-containing medium had little effect on catecholamine secretion. These results suggest that serum contains IGF-I or another factor that maintains the secretory responsiveness of chromaffin cells. IGF-I not only enhanced high K(+)-stimulated catecholamine secretion, but also augmented secretion elicited by the nicotinic agonist dimethyl-phenylpiperazinium, the dihydropyridine agonist Bay K 8644, and Ba2+. IGF-I did not affect the dependence of catecholamine secretion on extracellular Ca2+ concentration nor did it affect the time course of secretion. Experiments using 45Ca2+ demonstrated that IGF-I treatment enhanced Ca2+ uptake into the cells. When cells were permeabilized by treatment with digitonin, Ca2(+)-dependent catecholamine secretion was slightly, but consistently, greater from IGF-I-treated cells than from untreated cells. Our results suggest that IGF-I may enhance catecholamine secretion partly by increasing Ca2+ entry into the cells and partly by affecting a step distal to Ca2+ entry.