Homologous recombination partly restores the secretion defect of underglycosylated acid phosphatase in yeast.

The majority of secreted acid phosphatase in Saccharomyces cerevisiae is encoded by the PH05 gene. The secretion level of this acid phosphatase is directly determined by its level of glycosylation. Consequently, PHO5-11-encoded acid phosphatase which lacks 11 of 12 glycosylation sites is only poorly secreted. We have isolated and characterized both UV- and EMS-induced variants, which are partly able to restore the secretion of acid phosphatase. Our data indicate that the improved secretion is caused by mitotic intrachromosomal recombination between the PHO5-11 allele and the homologous tandemly repeated PHO3 sequences, resulting in the restoration of glycosylation sites in PHO5-11. Two different recombination mechanisms, unequal sister-chromatid exchange and sister-chromatid gene conversion, are responsible for these alterations of the PHO5-11 locus. Thus, recombination between mutant and wild-type sequences are able to restore the ability of mutant yeast cells to secrete acid phosphatase.

The Tn5 bleomycin resistance gene confers improved survival and growth advantage on Escherichia coli.

The bleomycin resistance gene (ble) of transposon Tn5 is known to decrease the death rate of Escherichia coli during stationary phase. Bleomycin is a DNA-damaging agent and bleomycin resistance is produced by improved DNA repair which also requires the host genes aidC and polA coding, respectively, for an alkylation-inducible gene product and DNA polymerase I. In the absence of the drug, this DNA repair system is believed to cause the slower death rate of bleomycin-resistant bacteria. In this study, the effect of ble and aidC genes on the viability of bacteria and their growth rate in chemostat competitions was studied. The results indicate, that bleomycin-resistant bacteria display greater fitness under these conditions. Another beneficial effect of transposon Tn5 had been previously attributed to the insertion sequence IS 50 R. We were not able to reproduce this result with IS 50 R, however, the complete transposon was beneficial under similar conditions. Moreover, we showed the Tn5 fitness effect to be aidC-dependent. The ble gene was discovered after the fitness effect of IS 50 R had been established; it has not previously been considered to mediate the beneficial effect of Tn5. This possibility is discussed based on the molecular mechanism of bleomycin resistance.

Involvement of GABAB receptors in the regulation of the hypothalamo-pituitary-adrenocortical (HPA) axis in rats.

Previous experiments have shown that the GABAB receptor agonist L-baclofen given subcutaneously to male rats significantly enhanced plasma concentrations of adrenocorticotropic hormone (ACTH) and the adrenocortical hormones corticosterone and aldosterone. The goal of the present study was to investigate whether the stimulatory effects on adrenocortical steroids elicited by L-baclofen in vivo could be reversed by the selective GABAB antagonist CGP 35 348. One hour before subcutaneous administration of 3 mg/kg L-baclofen, a dose of 600 mg/kg CGP 35 348 or saline was administered intraperitoneally. The stimulatory effect of L-baclofen on ACTH, corticosterone and aldosterone was significantly reduced by 60% after pretreatment with CGP 35 348. The GABAB antagonist CGP 35 348 by itself had no effect on ACTH or the adrenocortical hormones. These results indicate that GABAB receptors are involved in the L-baclofen-induced activation of the HPA axis in rats. In vitro, however, neither L-baclofen nor CGP 35 348 had any effects on corticosterone and aldosterone release from perifused adrenal cells. These results suggest that the participation of GABAB receptors in the activation of the HPA axis induced by L-baclofen in vivo does not occur at the level of the adrenal gland, and therefore must occur at the level of the pituitary or the brain.

Open dose-finding study of a new potent and selective nonsteroidal aromatase inhibitor, CGS 20 267, in healthy male subjects.

The aim of this open, dose-finding study was to evaluate the effects of single dose CGS 20 267, a new oral nonsteroidal aromatase inhibitor, on the inhibition of estrogen production and also on the production of adrenal and testicular steroids in healthy male subjects. Nine dose levels ranging from 0.02-30 mg and placebo were tested, each dose being given to 3 subjects only. A total of 18 subjects were included; 12 of them received 2 single administration, the remaining 6 were exposed only once to one of the 2 highest dose levels. A reduction in serum estrogen levels when compared to baseline was already observed after 2 h, reaching maximum suppression between 10 and 48 h after administration. After 24 h, a suppression of estrone levels by 60-85% from baseline was achieved with all tested doses. A reduction in estradiol levels by about 30% from baseline was observed at the lowest dose (0.02 mg). This reduction was further enhanced dose dependently to a maximum of about 90% from baseline at 24 h after administration of the highest dose (30 mg). With the higher doses (10 and 30 mg), estrogen suppression was maintained up to 3 days. A dose-dependent increase of testosterone, LH, and FSH was observed and was most pronounced in the 10- and 30-mg dose groups, which can be considered as a consequence of the long-lasting aromatase inhibition achieved with these high doses. No effect on serum cortisol and aldosterone levels was observed up to the highest dose. No clinically relevant changes were observed in blood chemistry and hematology tests. The systemic and subjective tolerability of CGS 20 267 was good at all doses. This study has shown that CGS 20 267 is a well tolerated, potent, selective, and long-acting inhibitor of the aromatase enzyme after single administration.

An in vitro method to determine the selective inhibition of estrogen biosynthesis by aromatase inhibitors.

Potency and selectivity of aromatase inhibition are parameters which ultimately influence the therapeutic efficacy of aromatase inhibitors. This report describes an in vitro model which allows an assessment of the selectivity with which aromatase inhibitors inhibit estrogen biosynthesis. Estrogen production was stimulated by incubating adult female hamster ovarian tissue with ovine LH. The production rates of estrogens (E), testosterone (T) and progesterone (P) were determined using radioimmunoassays to measure the amount of these steroids released into the incubation medium over a 4-hour incubation period. The selectivity of aromatase inhibition was assessed by determining the IC50S with which each inhibitor inhibited the production of E (end product), T (immediate precursor of E) and P (early precursor of E). Selectivity was studied for each of the 4 aromatase inhibitors, CGS 16949A (a new non-steroidal compound), 4-OH-androstenedione, aminoglutethimide and testolactone. CGS 16949A was the most potent of the four, followed by 4-OH-androstenedione, aminoglutethimide and testolactone. As far as selectivity was concerned, both CGS 16949A and 4-OH-androstenedione selectively inhibited aromatase judging from the IC50s for E and P production (CGS 16949A: IC50 for E & P = 0.03 & 160 microM, resp.; 4-OH-androstenedione: IC50 for E & P = 0.88 & greater than or equal to 330 microM, resp.). Aminoglutethimide was the least selective inhibitor of aromatase (IC50 for E & P = 13 & 60 microM, resp.). For testolactone, the least potent of the four (IC50 for E = 130 microM), no conclusive data were obtained concerning the selectivity of aromatase inhibition. Thus a simple, effective and reproducible method is described for assessing the selectivity with which aromatase inhibitors inhibit aromatase.

Evidence that corticosterone is not an obligatory intermediate in aldosterone biosynthesis in the rat adrenal.

CGS 16949A is a potent inhibitor of aromatase in vitro with an IC50 of 0.03 microM for the inhibition of LH-stimulated estrogen biosynthesis in hamster ovaries. In vivo, CGS 16949A leads to sequelae of estrogen deprivation (e.g. regression of DMBA-induced mammary tumors) without causing adrenal hypertrophy in adult rats. To complement these in vitro and in vivo findings, the effect of CGS 16949A on adrenal steroid biosynthesis in rats was investigated in vitro and in vivo. The surprising finding in vitro was that CGS 16949A inhibited aldosterone biosynthesis (IC50 = 1 microM) at concentrations 100 times lower than those for inhibition of corticosterone biosynthesis (IC50 = 100 microM). Moreover, deoxycorticosterone (DOC) concentrations were elevated at all concentrations of CGS 16949A which inhibited aldosterone synthesis. The classical biosynthetic pathway for aldosterone is DOC----corticosterone----18-OH-corticosterone----aldosterone. Thus inhibition of aldosterone biosynthesis, reflected in DOC accumulation, without affecting corticosterone concentrations, indicates that corticosterone is not an obligatory intermediate in the conversion of DOC to aldosterone in the rat. In vivo, CGS 16949A showed a suppression of plasma aldosterone in ACTH-stimulated male rats at doses which did not significantly affect plasma corticosterone. In conclusion, aldosterone measured both in vitro and in vivo must be derived primarily from a biosynthetic pathway in which corticosterone is not obligatory intermediate.