Stimulation of rat luteinizing hormone-beta messenger RNA levels by gonadotropin releasing hormone. Apparent role for protein kinase C

The ability of gonadotropin releasing hormone (GnRH) to elevate cellular levels of mRNA for beta-subunit of luteinizing hormone (LH) has been examined in monolayer cultures from rat pituitary. Low concentrations of GnRH (100 pM) induced a 6.8-fold increase in LH-beta mRNA, while higher concentrations of GnRH were less effective. The low concentrations of GnRH (100 pM) did not result in altered GnRH receptor levels (92 +/- 12% compared to controls) after 24 h treatment but did increase protein kinase C activity to 249 +/- 16%. The protein kinase C activator, phorbol 12-myristate 13-acetate, at concentrations (2-20 nM) which did not deplete protein kinase C, stimulated LH-beta mRNA levels 2-5-fold after 24 h. Higher concentrations of phorbol 12-myristate 13-acetate, which depleted protein kinase C activity, substantially reduced the ability of 100 pM GnRH to stimulate increases in LH-beta mRNA levels. As previously observed, protein kinase C-depleted cells exhibited normal LH release in response to GnRH stimulation. These studies demonstrate that low concentrations of GnRH may have an important role in regulation of gonadotropin biosynthesis. Furthermore, the results suggest that activation of protein kinase C is sufficient to stimulate increases in LH-beta mRNA levels and that protein kinase C is necessary for normal GnRH stimulation of LH-beta mRNA levels. Accordingly, we postulate that protein kinase C may mediate the action of GnRH on LH-beta mRNA levels.     

D-Ala6-des-Gly10-gonadotropin-releasing hormone ethylamide: absence of binding sites and lack of a direct effect in rabbit corpora lutea

Rat ovarian tissue has been shown to contain high-affinity gonadotropin-releasing hormone (GnRH) receptors, and synthetic GnRH analogues have been shown to inhibit steroid production by rat corpora lutea in vivo and in vitro. These results raise the possibility that an ovarian GnRH-like peptide may be involved in normal luteal regression. We have examined binding of D-Ala6-des-Gly10-GnRH ethylamide (D-Ala) to rabbit corpora lutea, and have investigated the luteolytic activity of this analogue in hypophysectomized, pseudopregnant rabbits. Three hypophysectomized estrogen-treated rabbits were injected with 0.25 mg D-Ala s.c. every 6 h for 48 h during mid-pseudopregnancy, and three were injected with vehicle only. Treatment with D-Ala produced no acute changes in serum progesterone, nor was the time of luteal regression altered. Rabbit anterior pituitary tissue was found to contain high-affinity GnRH receptors (Ka = 7.0 X 10(9) M-1; 188.2 +/- 35.6 fmol/mg protein). However, no similar high-affinity GnRH receptors were detected in rabbit luteal tissue from any stage of pseudopregnancy. Some apparent low-affinity binding was observed, but this displaceable binding was subsequently observed in all control tissues tested. Thus, a potent GnRH analogue does not have any detectable direct effect on steroidogenesis in the rabbit corpus luteum, nor are high-affinity GnRH binding sites present in rabbit luteal tissue.     

Two Enzymes, Both of Which Process Recombination Intermediates, Have opposite Effects on Adaptive Mutation in Escherichia Coli

Reversion of a lac(-) frameshift allele carried on an F' episome in Escherichia coli occurs at a high rate when the cells are placed under lactose selection. Unlike Lac(+) mutations that arise during nonselective growth, the production of these adaptive mutations requires the RecA-RecBCD pathway for recombination. In this report, we show that enzymes that process recombination intermediates are involved in the mutagenic process. RuvAB and RecG, E. coli's two enzymes for translocating Holliday junctions, have opposite effects: RuvAB is required for RecA-dependent adaptive mutations, whereas RecG inhibits them.     

Occurrence of 28- and 27-Carbon ecdysteroids and sterols in developing worker pupae of the leaf-cutting ant acromyrmex octospinosus (reich) (hymenoptera,formicidae: Attini)

The major ecdysteroids in large worker pupae of the leaf-cutting ant Acromyrmex octospinosus were characterized at the peak ecdysteroid concentration by using high-performance liquid chromatography, enzyme immunoassay, and mass spectrometry. In decreasing amounts, they were determined to be makisterone A, an unidentified C₂₈ ecdysteroid bearing a molecular weight of 494, 20-hydroxyecdysone (ratio of 1 to 6 as compared to makisterone A), and putative but negligible ecdysone. The presence of both C₂₈ and C₂₇ ecdysteroids is discussed in relation to the content of 4-desmethylsterols determined by gas chromatography and mass spectrometry to be ergosta-5,7,24 (28)-trien-3β-ol, ergosterol, ergosta-5,7-dien-3β-ol and ergosta-7,24(28)-dien-3β-ol for the main sterols, and with a small amount of cholesterol.     

Isolation of a specific granulopoiesis inhibitor from calf spleen and identification as glutathione

A small peptide was isolated from calf spleen, specifically inhibiting murine granulopoiesis in vitro. Purification involved ultrafiltration, anion-exchange chromatography with a FPLC system and reversed-phase chromatography on HPLC. Determination of the amino acid composition, following acid hydrolysis and phenyl isothiocyanate and dabsyl chloride derivatization, revealed the amino acids glutamic acid, cysteine and glycine. Although the N-terminal amino group was not blocked, peptide sequencing with common techniques was not possible. Comparison of the isolated peptide with the well-known tripeptide glutathione by HPLC and fast atom bombardment (FAB)/tandem mass spectrometry showed the identity of both substances. Moreover, glutathione was found to be a specific granulopoiesis inhibitor in vitro at 10-100 nM, a so far unknown property of this well-known peptide.