Cation-distribution in developing follicles of the fruit fly Drosophila melanogaster

Abstract. Cations were precipitated with potassium antimonate in ovarian follicles of Drosophila and the distribution of the formed precipitates was studied. The precipitates were analyzed with a laser microprobe mass analyzer (LAMMA) and found to contain a high concentration of calcium; potassium and sodium were also detected. On counting the antimon precipitates in stage 10B follicles with the electron microscope, few precipitates per unit area were found in anterior nurse cells, but more in posterior nurse cells; the highest precipitate density occurred consistently in the oocyte. When follicles of different stages were compared, the precipitate density was found to increase in the ooplasm and in the posterior nurse cells during vitellogenesis, whereas it remained nearly constant in the anterior nurse cells. Thus, the ratio of precipitates between the posterior and anterior end of the follicle increases during vitellogenesis. It begins to decrease at the time when the nurse cells collapse. These results suggest that the electrical polarity observed in polytrophic ovarioles may be based on differences in the cation distribution along the antero-posterior axis of the follicle.     

Production of pro-opiomelanocortin (POMC) by a vaccinia virus transient expression system and in vitro processing of the expressed prohormone by POMC-converting enzyme

Pro-opiomelanocortin (POMC) was expressed in CV-1 (green monkey kidney) cells using a vaccinia virus transient expression system [(1986) Proc. Natl. Acad. Sci. USA 83, 8122]. The system involved infection of cells with a recombinant vaccinia virus carrying the T7 RNA polymerase gene and transfection with a plasmid containing the mouse POMC sequence flanked by the T7 RNA polymerase promoter at its 5'-end and the T7 RNA polymerase terminator at its 3'-end. Assay of the medium from transfected cells showed that 1-2 micrograms of immunoreactive ACTH was produced/10(6) cells. Analysis of the same medium by SDS-PAGE/Western blots revealed a band of 30-36 kDa, which was immunostained with both ACTH and beta-endorphin antisera. Labeling the transfected cells with [3H]Arg, followed by immunoprecipitation and SDS-PAGE showed the synthesis of a major peak of POMC, 33 kDa. Purified [3H]POMC expressed by CV-1 cells was cleaved in vitro by bovine intermediate lobe secretory vesicle pro-opiomelanocortin-converting enzyme to ACTH intermediates (19-25 kDa), beta-lipotropin and beta-endorphin. Thus, this work has demonstrated a technique for expressing microgram quantities of prohormones in mammalian cells, suitable for use as substrates for prohormone-converting enzymes in vitro.     

Inhibitors of reverse serotonin uptake and of specific imipramine binding in human blood plasma

Human blood plasma contains low-molecular substances that inhibit in a dose-dependent manner both high-affinity specific binding of imipramine and reverse serotonin uptake by platelets. Incubation of human blood plasma with alumina was made use of to extract and study these imipramine-like inhibitors. The extract obtained from human blood plasma inhibited imipramine binding and reverse uptake of serotonin with median inhibitory concentrations of 0.18 +/- 0.1 and 0.36 +/- 0.15 mg/ml, respectively. After gel chromatography on Biogel P-2 the elution profile of the extract showed 2 major peaks of reverse serotonin uptake and imipramine binding inhibition and 3 additional peaks of reverse serotonin uptake inhibition, which did not have any considerable effect on imipramine specific binding. It is assumed that endogenous inhibitors of imipramine binding and reverse serotonin uptake are involved in the development of affective disorders.