Inhibition of O-acetylserine sulfhydrylase by fluoroalanine derivatives

O-acetylserine sulfhydrylase (OASS) is the pyridoxal 5′-phosphate dependent enzyme that catalyses the formation of L-cysteine in bacteria and plants. Its inactivation is pursued as a strategy for the identification of novel antibiotics that, targeting dispensable proteins, holds a great promise for circumventing resistance development. In the present study, we have investigated the reactivity of Salmonella enterica serovar Typhimurium OASS-A and OASS-B isozymes with fluoroalanine derivatives. Monofluoroalanine reacts with OASS-A and OASS-B forming either a stable or a metastable α-aminoacrylate Schiff’s base, respectively, as proved by spectral changes. This finding indicates that monofluoroalanine is a substrate analogue, as previously found for other beta-halogenalanine derivatives. Trifluoroalanine caused different and time-dependent absorbance and fluorescence spectral changes for the two isozymes and is associated with irreversible inhibition. The time course of enzyme inactivation was found to be characterised by a biphasic behaviour. Partially distinct inactivation mechanisms for OASS-A and OASS-B are proposed.     

Proline synthesis in developing microspores is required for pollen development and fertility

Background

In many plants, the amino acid proline is strongly accumulated in pollen and disruption of proline synthesis caused abortion of microspore development in Arabidopsis. So far, it was unclear whether local biosynthesis or transport of proline determines the success of fertile pollen development.

Results

We analyzed the expression pattern of the proline biosynthetic genes PYRROLINE-5-CARBOXYLATE SYNTHETASE 1 & 2 (P5CS1 & 2) in Arabidopsis anthers and both isoforms were strongly expressed in developing microspores and pollen grains but only inconsistently in surrounding sporophytic tissues. We introduced in a p5cs1/p5cs1 p5cs2/P5CS2 mutant background an additional copy of P5CS2 under the control of the Cauliflower Mosaic Virus (CaMV) 35S promoter, the tapetum-specific LIPID TRANSFER PROTEIN 12 (Ltp12) promoter or the pollen-specific At5g17340 promoter to determine in which site proline biosynthesis can restore the fertility of proline-deficient microspores. The specificity of these promoters was confirmed by β-glucuronidase (GUS) analysis, and by direct proline measurement in pollen grains and stage-9/10 anthers. Expression of P5CS2 under control of the At5g17340 promoter fully rescued proline content and normal morphology and fertility of mutant pollen. In contrast, expression of P5CS2 driven by either the Ltp12 or CaMV35S promoter caused only partial restoration of pollen development with little effect on pollen fertility.

Conclusions

Overall, our results indicate that proline transport is not able to fulfill the demand of the cells of the male germ line. Pollen development and fertility depend on local proline biosynthesis during late stages of microspore development and in mature pollen grains.

     

Sex hormone-binding globulin selectively modulates estradiol-regulated genes in MCF-7 cells.

Human sex hormone-binding globulin inhibits the effects of estradiol on proliferation and apoptosis of breast cancer cells. We report here the effect of sex hormone-binding globulin on estradiol regulation of gene expression in MCF-7 breast cancer cells using a selected set of genes. Estradiol upregulates genes that are positive regulators of proliferation (e.g., bcl-2, c-fos, c-myc, cyclin D) or/and related to more aggressive form of breast cancer (e.g. BRCA-1, EGF-R) and downregulates two genes (c-jun and ERalpha). Sex hormone-binding globulin modulates only a selected group of estradiol-controlled genes (inhibiting upregulation of bcl-2, c-myc, EGF-R, PR, and downregulation of ERalpha), starting 48 hours after treatment. Our study demonstrates that in breast cancer cells, sex hormone-binding globulin is effective on few selected genes which are involved in cell growth and apoptosis or related to cell estrogen-dependence and that the protein regulation of estradiol effect is selected and specific. Sex hormone-binding globulin action in estrogen breast cancer cells is strongly associated to cell growth and estrogen-sensitivity.     

Functional Changes in Rat Nigral GABAA Receptors Induced by Degeneration of the Striatonigral GABAergic Pathway: An Electrophysiological Study of Receptors Incorporated into Xenopus Oocytes

Abstract: Expression of rat brain γ-aminobutyric acid type A (GABA_A) receptors in Xenopus laevis oocytes can be achieved by injection of the oocytes with synaptosomes. This approach has now been applied to evaluate changes in the function of nigral GABA_A receptors after degeneration of the striatonigral GABAergic pathway induced by the unilateral infusion of kainic acid into the rat striatum. Ten days after striatal injection, synaptosomal membranes were prepared from the substantia nigra and introduced into oocytes. Nigral GABA_A receptors incorporated into the oocyte cell membrane were then characterized electrophysiologically under voltage-clamp conditions. The maximal amplitude of GABA-induced Cl^? currents in oocytes injected with synaptosomes from denervated substantia nigra was twice that observed in oocytes injected with synaptosomes from control substantia nigra. The concentration of GABA required for the half-maximal response did not differ between the two groups of oocytes. In addition, the potentiation of GABA-induced currents by the benzodiazepine diazepam (1 µM) and the steroid derivative allopregnanolone (3 µM) was increased by ～65 and 60%, respectively, in oocytes injected with synaptosomes from denervated substantia nigra compared with those injected with control synaptosomes. The concentrations of diazepam and allopregnanolone giving half-maximal responses were not affected by denervation. In contrast, the inhibitory effects of the benzodiazepine receptor inverse agonists FG 7142 (10 µM) and 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylic acid ethyl ester (1 µM) were reduced by 48 and 38%, respectively, after denervation. These results indicate that the up-regulation of nigral GABA_A receptors induced by degeneration of the striatonigral GABAergic pathway is associated with an increased efficacy of positive allosteric modulators, such as benzodiazepines and steroids, and with a reduced efficacy of negative allosteric modulators such as β-carbolines.     

Corrugoside, a new immunostimulatory alpha-galactoglycosphingolipid from the marine sponge Axinella corrugata

Corrugoside (1a), a new immunostimulatory triglycosilated alpha-galactoglycosphingolipid, was isolated from the marine sponge Axinella corrugata, and its structure determined by spectral analysis and chemical degradation. Compound 1a activated murine NKT cells in vitro, with a potency of about 2 logs lower than that of alphaGalCer. Four stereoisomeric glycosphingolipids (2a-2d) were also obtained, beta-glucosylceramides bearing unusual endoperoxide and allylic hydroperoxide functionalities on the sphinganine chain. They were shown to be photooxidation artifacts of the known glycosphingolipids 3, also present in the sponge. A possible role of compound 3 as a singlet oxygen scavenger to protect the organism from oxidative damage is proposed.