Differential actions of corticosterone on luteinizing hormone and follicle-stimulating hormone biosynthesis and release in cultured rat anterior pituitary cells: interactions with estradiol

Previous in vivo studies from our laboratory suggested that glucocorticoids antagonize estrogen-dependent actions on LH secretion. This study investigated whether corticosterone (B) may have similar actions on gonadotropin biosynthesis and secretion in vitro. Enzymatically dispersed anterior pituitary cells from adult female rats were cultured for 48 h in alpha-modified Eagle's medium containing 10% steroid-free horse serum with or without 0.5 nM estradiol (E2). The cells were then cultured for 24 h with or without B in the presence or absence of E2. To evaluate hormone release, 5 x 10(5) cells were incubated with varying doses of GnRH (0, 10(-11)-10(-7) M) or pulsatile GnRH (10(-9) M; 20 min/h) for 4 h. Cell and medium LH and FSH were measured by RIA. To evaluate LH biosynthesis, 5 x 10(6) cells were incubated for an additional 24 h with 10(-10) M GnRH, 60 microCi 3H-glucosamine (3H-Gln), 20 microCi 35S-methionine (35S-Met), and the appropriate steroid hormones. Radiolabeled precursor incorporation into LH subunits was determined by immunoprecipitation, followed by SDS-PAGE. Continuous exposure to GnRH stimulated LH release in a dose-dependent manner, and this response was enhanced by E2. B by itself had no effect on LH release, but inhibited LH secretion in E2-primed cells at low concentrations of GnRH (10(-10) M or less). Total LH content was not altered by GnRH or steroid treatment. Similar effects of B were observed in cells that were given a pulsatile GnRH stimulus. In contrast to LH, E2 or B enhanced GnRH-stimulated FSH release at the higher doses of GnRH, while the combination of E2 and B increased basal and further augmented GnRH-stimulated release. Total FSH content was also increased in the presence of B, but not E2 alone, and was further augmented in cells treated with both steroids. There were no effects of the steroids on the magnitude of FSH release in response to GnRH pulses, but the cumulative release of FSH was greater in the E2 + B group compared to controls, indicating an increased basal release. Independent of E2, B suppressed the incorporation of 3H-Gln into LH by more than 50% of control, with only subtle effects on the incorporation of 35S-Met.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of glycoinositol phospholipid linked and truncated forms of the scrapie prion protein

Analysis of carboxy-terminal peptides derived from endoproteinase Lys-C digests of the scrapie isoform of the hamster prion protein revealed that the majority of the molecules are glycoinositol phospholipid linked through ethanolamine attached at serin-231. However, approximately 15% of PrPSc had a carboxy-terminal peptide that ends at glycine-228. It is intriguing that this glycine is part of the PrP sequence Gly-Arg-Arg, which is an established target sequence for the proteolysis and release of bioactive peptides from larger precursors. The mechanism of formation, as well as the role of the truncated carboxy terminus in the dissemination and neuropathology of scrapie, remains to be determined.     

Purification and Characterization of an Autolysin from Clostridium acetobutylicum

A proteinaceous substance with antibiotic-like activity, resembling that of a bacteriocin, was isolated from an industrial-scale acetone-butanol fermentation of Clostridium acetobutylicum. The substance, purified by acetone precipitation, diethylaminoethyl cellulose chromatography, and polyacrylamide gel electrophoresis, was characterized as a glycoprotein with a molecular weight of 28,000. The glycoprotein was partially inactivated by certain protease enzymes. It had no effect on deoxyribonucleic acid, ribonucleic acid, or protein synthesis, and it did not result in the loss of intracellular adenosine triphosphate. The glycoprotein lysed sodium dodecyl sulfate-treated cells and cell wall preparations, and therefore it is referred to as an autolysin. The autolysin gene appeared to be chromosomal since plasmid deoxyribonucleic acid was not detected in the C. acetobutylicum strain.     

Cell-free synthesis of Chironomus thummi (Diptera) globins

RNA isolated from Chironomus thummi (Diptera) larvae directs the incorporation of amino acid into newly synthesized products in a cell-free translation system prepared from wheat germ. A fraction of the total cell-free product was specifically immunoprecipitable with antibody against total C. thummi hemoglobin. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis of the immunoreactive material revealed the cell-free product to have an apparent molecular mass approximately 3000 daltons greater than secreted C. thummi globin purified from hemolymph. In contrast, analysis of the immunoreactive material by polyacrylamide gel electrophoresis under nondenaturing conditions indicated several chemically distinct globins to be present in the cell-free immunoreactive products. These results provide evidence suggesting the possible existence of a preglobin and the data further provide the initial foundation required for elucidating the regulatory mechanisms that control the developmental stage-specific expression of the globin genes in C. thummi.     

Nature of the fast and slow refolding reactions of iron(III) cytochrome c

The fast and slow refolding reactions of iron(III) cytochrome c (Fe(III) cyt c), previously studied by Ikai et al. (Ikai, A., Fish, W. W., & Tanford, C. (1973) J. Mol. Biol. 73, 165--184), have been reinvestigated. The fast reaction has the major amplitude (78%) and is 100-fold faster than the slow reaction in these conditions (pH 7.2, 25 degrees C, 1.75 M guanidine hydrochloride). We show here that native cyt c is the product formed in the fast reaction as well as in the slow reaction. Two probes have been used to test for formation of native cyt c. absorbance in the 695-nm band and rate of reduction of by L-ascorbate. Different unfolded species (UF, US) give rise to the fast and slow refolding reactions, as shown both by refolding assays at different times after unfolding ("double-jump" experiments) and by the formation of native cyt c in each of the fast and slow refolding reactions. Thus the fast refolding reaction is UF leads to N and the slow refolding reaction is Us leads to N, where N is native cyt c, and there is a US in equilibrium UF equilibrium in unfolded cyt c. The results are consistent with the UF in equilibrium US reaction being proline isomerization, but this has not yet been tested in detail. Folding intermediates have been detected in both reactions. In the UF leads to N reaction, the Soret absorbance change precedes the recovery of the native 695-nm band spectrum, showing that Soret absorbance monitors the formation of a folding intermediate. In the US leads to N reaction an ascorbate-reducible intermediate has been found at an early stage in folding and the Soret absorbance change occurs together with the change at 695 nm as N is formed in the final stage of folding.     

Immunological identification of the human erythrocyte monosaccharide transporter

Antibodies to the purified cytochalasin B binding component of the human erythrocyte glucose transporter were prepared in rabbits. They precipitated detergent-solubilized transporter, and partially inhibited its binding of cytochalasin B. The antibodies were used to locate the transporter polypeptide in SDS-polyacrylamide gels of erythrocyte membranes prepared from freshly drawn blood in the presence of protease inhibitors. They labelled only the region of the gel corresponding to that occupied by the purified transporter, with an apparent molecular weight range of 45,000–75,000. These findings indicate that the isolated transporter does not arise by proteolytic degradation of a larger polypeptide, either during the storage of blood or during purification of the transporter.     

Diffusion of phosphate to plant roots in soil

Summary A method is described for computing the theoretical distribution of solutes around a root with root hairs. The P concentration profile around the primary root of a rape seedling with root hairs, growing in a low-P soil, showed considerably greater depletions than predicted by this method, using the relationship between P in soil and in solution obtained from a desorption isotherm experiment. This suggests an enhanced release of soil P into solution in the rhizosphere.Soil Science Laboratory, Department of Agricultural Science, University of Oxford