Identifying new sex-linked genes through BAC sequencing in the dioecious plant Silene latifolia.

BACKGROUND: Silene latifolia represents one of the best-studied plant sex chromosome systems. A new approach using RNA-seq data has recently identified hundreds of new sex-linked genes in this species. However, this approach is expected to miss genes that are either not expressed or are expressed at low levels in the tissue(s) used for RNA-seq. Therefore other independent approaches are needed to discover such sex-linked genes. RESULTS: Here we used 10 well-characterized S. latifolia sex-linked genes and their homologs in Silene vulgaris, a species without sex chromosomes, to screen BAC libraries of both species. We isolated and sequenced 4 Mb of BAC clones of S. latifolia X and Y and S. vulgaris genomic regions, which yielded 59 new sex-linked genes (with S. vulgaris homologs for some of them). We assembled sequences that we believe represent the tip of the Xq arm. These sequences are clearly not pseudoautosomal, so we infer that the S. latifolia X has a single pseudoautosomal region (PAR) on the Xp arm. The estimated mean gene density in X BACs is 2.2 times lower than that in S. vulgaris BACs, agreeing with the genome size difference between these species. Gene density was estimated to be extremely low in the Y BAC clones. We compared our BAC-located genes with the sex-linked genes identified in previous RNA-seq studies, and found that about half of them (those with low expression in flower buds) were not identified as sex-linked in previous RNA-seq studies. We compiled a set of ~70 validated X/Y genes and X-hemizygous genes (without Y copies) from the literature, and used these genes to show that X-hemizygous genes have a higher probability of being undetected by the RNA-seq approach, compared with X/Y genes; we used this to estimate that about 30% of our BAC-located genes must be X-hemizygous. The estimate is similar when we use BAC-located genes that have S. vulgaris homologs, which excludes genes that were gained by the X chromosome. CONCLUSIONS: Our BAC sequencing identified 59 new sex-linked genes, and our analysis of these BAC-located genes, in combination with RNA-seq data suggests that gene losses from the S. latifolia Y chromosome could be as high as 30 %, higher than previous estimates of 10-20%.

Design and modeling of new platelet-activating factor antagonists. 2. Synthesis and biological activity of 1,4-bis-(3',4',5'-trimethoxybenzoyl)-2-alkyl and 2-alkyloxymethylpiperazines.

In the continuation of our investigations on the structure of platelet-activating factor (PAF)-receptor, 25 additional 2-substituted 1,4-bis-(poly- and mono methoxybenzoyl)-piperazines were synthesized and their in vitro biological activities measured. Substituent at position 2 is representative of the classical balance lipophilicity/hydrophilicity, i.e. alkyl, phenylalkyl, alkoxy and polyalkoxy groups. A potent PAF-induced platelet aggregation inhibitory activity measured in PRP medium is obtained with 5c, IC50 = 6 x 10(-8) M, which displaces the [3H]PAF from platelet membrane with an EC50 = 6 x 10(-8) M, and compound 4 presents an EC50 of 3 x 10(-8) M. Examination of structure-activity relationships shows that molecules bearing a hydrophilic or slightly hydrophobic appendix in position-2 are still potent; their IC50 being included between 10(-6) and 10(-7) M. After quantitative analysis, it seems that in PRP medium, the role of serum albumin must be taken into account instead of a pure hydrophobic interaction of the appendix Z into the receptor. The role of the methoxy groups in producing a potent antagonistic activity is demonstrated by syntheses of several 2-octylpiperazine analogs. These specific features will be quantitatively analysed in the following related publication (part 3).

Structure-activity relationships in platelet-activating factor (PAF). 7. Tetrahydrofuran derivatives as dual PAF antagonists and acetylcholinesterase inhibitors. Synthesis and PAF-antagonistic activity.

2,5-Disubstituted tetrahydrofuran derivatives present a dual activity: they are effective PAF antagonists and acetylcholinesterase inhibitors. In this paper their synthesis and in vitro PAF-antagonistic effect are described. Introduction in position 2 of a long aliphatic chain bearing a carbamate group and a pyridinium moiety appears to be required for potent platelet aggregation inhibition. Substitution in position 5, or cis-trans isomerism do not induce any increase in activity. No correlation can be established between global lipophilicity and the anti-aggregant activity. Structural requirements for a potent activity are discussed and are consistent with the hypothesis we have proposed for the PAF receptor considered as a multipolarized structure with alternants of electropositive, electronegative and hydrophobic areas.

Structure-activity relationships in platelet-activating factor (PAF). 8. Tetrahydrofuran derivatives as dual PAF antagonists and acetylcholinesterase inhibitors: anti-acetylcholinesterase activity and comparative SAR.

2,5-disubstituted tetrahydrofuran derivatives display a dual functionality: they are PAF antagonists and acetylcholinesterase (AChE) inhibitors. In vitro anti-AChE activity and in vivo trials are presented herein. These compounds are competitive and potent AChE inhibitors. Structure-activity relationships are described and compared with PAF-antagonist results. The presence of an onium group, a suitable distance supplied by a chain of 7 or 10 carbon atoms separating the function from the polar head and an appreciable chain hydrophobicity (4 < sigma f < 7) are the main features required for a dual activity. The derivatives are evaluated in a mouse passive avoidance model. Only compounds with both activities are able to reverse scopolamine-induced amnesia. In addition, they display a very weak toxicity.

Design and modeling of new platelet-activating factor antagonists. 1. Synthesis and biological activity of 1,4-bis(3',4',5'-trimethoxybenzoyl)-2-[[(substituted carbonyl and carbamoyl)oxy]methyl]piperazines.

To further investigate our hypothesis on the structure of the platelet-activating factor (PAF) receptor, 35 compounds derived from 1,4-bis(3',4',5'-trimethoxybenzoyl)piperazine were synthesized and their in vitro antagonistic effect was measured. Substitution of the compounds in position 2, by ester or carbamate groups, giving increased steric hindrance and hydrophobicity, increased the platelet aggregation inhibitory activity from 2 microM (without substitution, compound 2) to 0.07 microM (compound 1h) and gave a maximum displacement of [3H]PAF from platelet membrane of 0.05 microM (compound 1k). It appears that the PAF antagonistic effect is only weakly enantiospecific, as observed in many cases including antagonists structurally related or not to PAF. 3D electrostatic potential maps (calculated at -10 kcal/mol) of such compounds revealed a double "Cache-oreilles" (ear-muffs) system. One of these systems has been previously described (distance between atoms generating negative wells, 11-14 A). The second shorter "Cache-oreilles" (6-7 A) system appears to be required for increased PAF antagonistic activity. This short distance between groups generating the negative wells is present in the gingkolides, a series of naturally occurring PAF antagonists. The present study indicates that the structure of the PAF receptor may be more complicated than our initial hypothesis and may be a tetrapolarized structure, with alternants of electropositive and hydrophobic areas. This modified hypothesis is in agreement with recent publications concerning PAF antagonists bearing a cationic moiety.

Structure-activity relationships in platelet-activating factor (PAF antagonists). 6. Synthesis and in vitro antagonistic activities of 2-substituted 5-oxotetrahydrofurans.

The synthesis of 2,5-disubstituted tetrahedrofuran compounds as potential in vitro PAF antagonists is described. Results demonstrate that the structural requirements for potent PAF antagonist activity are: a moderate lipophilic group or a trimethoxyphenyl group in position-5, and a long aliphatic chain terminated by a cationic polar head in position-2. The cis-trans configuration does not induce any difference in biological activity. Some conformational features of the putative PAF receptor are proposed in light of the present findings.

Models of hypoxia and cerebral ischemia.

Most models of hypoxia and ischemia are used for evaluating the metabolic consequences of cerebral insult. They have also been used for inducing cognitive disturbance. The pathological cascade after severe hypoxia or ischemia includes decreased ATP, influx of Ca2+ and Na+ with decrease in intracellular K+ leading to depolarization, release of glutamate, noradrenaline and acetylcholine, changes in neuronal plasticity, cell death, and cognitive impairment. Possible pharmacological mechanisms for protecting brain function include blockade of Ca2+ influx, inhibition of cell swelling, regulation of membrane potential, inhibition of neurotransmitter release and inhibition of excitatory amino-acid receptors. Among the existing models, many suffer from poor reproducibility and standardization. Two models which are more satisfactory in this respect are global transient ischemia in gerbils induced by bilateral carotid occlusion and focal ischemia in rats induced by occlusion of the middle cerebral artery. Although clear protective effects have been observed in both kinds of model (e.g., with NMDA antagonists, Ca2+ antagonists, PAF antagonists) it is frequently difficult to extrapolate these effects to disorders associated with memory impairment.

Involvement of platelet-activating factor (PAF) in cerebral post-ischemic phase in Mongolian gerbils.

Platelet-activating factor (PAF) is a potent mediator of anaphylaxis and shock. In addition, evidence for PAF participation in gastric, intestinal and heart post-ischemic phase has been recently demonstrated. Ginkgo biloba extracts improve cerebral metabolism and protect brain against hypoxic damage in various models of cerebral ischemia. Potent and specific antagonists of PAF have been found in Ginkgo biloba and termed Ginkgolides: BN 52020, BN 52021, BN 52022, BN 52024. We therefore undertook the investigation of the role of Ginkgolides in cerebral ischemia obtained by bilateral ligature of the common carotid for 10 min and 6 h of recirculation in male Mongolian adult gerbils. Given preventively (one week treatment 10 mg/kg/day orally) or at the time of clamping, BN 52021 and related Ginkgolides dose-dependently antagonize morbidity assessed by the stroke-index. Similarly the mitochondrial respiration evaluated by the respiratory control ratio is significantly improved. In both determinations, the range of activity: BN 52021 greater than, BN 52020 greater than BN 52022 greater than BN 52024 shows that the effect of Ginkgolides in cerebral ischemia are correlated with their PAF antagonistic properties. Given curatively, 1 h after declamping, BN 52021 is able to reverse the cerebral impairment trend. Kadsurenone and brotizolam, two other chemically unrelated PAF antagonists led to similar recovery. Therefore PAF appears to play an important role in the post-ischemic phase after bilateral carotid ligation in Mongolian gerbils.

[Effects of Ginkgo biloba extract on a cerebral ischemia model in gerbils]. / Effets de l'extrait de Ginkgo biloba sur un modèle d'ischémie cérébrale chez la gerbille.

Certain anatomical characteristics peculiar to the gerbil make it the animal model best adapted to experimental pathology studies of acute ischaemia. In this animal species, devoid of any substitute vertebro-basilar vascular tissue, unilateral ligature of the carotid artery produces a cerebral ischaemia with neurological signs (well quantifiable), metabolic perturbations (especially mitochondrial) and cerebral oedema development closely resembling the symptoms revealed by physiopathology in human clinical studies. Using this model and under the experimental conditions described, clear-cut, highly significant results were obtained with Ginkgo biloba, whether by oral or intravenous administration. These results were normalization of mitochondrial respiration, diminution of cerebral oedema, correction of the accompanying ionic perturbations, and practically total functional restoration revealed by a normal neurological index in the gerbils treated with Ginkgo biloba extract.

Further characterization of the anorexic action of orally-administered THIP, a GABA-analogue, in the rat.

Sprague-Dawley rats were used to further characterize the anorexic action of orally-administered THIP, a GABA-analogue. The anorexic action of THIP (5 or 10 mg/kg) was antagonized by prior subcutaneous injection of bicuculline (1 mg/kg), but not by prior subcutaneous injection of bicuculline-methobromide (1.5 mg/kg), strychnine-SO4 (0.75 mg/kg), pentylenetetrazol (25 mg/kg), or picrotoxin (1 mg/kg). Orally-administered GABA (50-300 mg/kg), bicuculline (1-10 mg/kg) or picrotoxin (1-10 mg/kg) generally did not inhibit food intake. These results indicate that the anorexic action of THIP is mediated by central GABA-receptors.

Inhibition of foot intake in the rat.

The effects of single oral administrations of tricyclic antidepressants (imipramine and desipramine), an atypical antidepressant (nomifensine), known anorexic agents, haloperidol, and diazepam on food intake were compared in Sprague-Dawley rats over a 4-day test period. The tricyclic antidepressants produced decreases in food intake during the total 4-day test period following their administration. In contrast, the anorexic agents (d-amphetamine, cocaine, mazindol, fenfluramine and quipazine), and nomifensine, and haloperidol produced decreases in food intake only on their administration. Diazepam produced an increase in food intake only on the day of its decrease food intake, this model appears to show some specificity for tricyclic antidepressants.

Inhibition of food intake in the rat by cyproheptadine.

In a model of conditioned feeding behavior, oral administration of cyproheptadine (1-100 mg/kg), 30 min before presentation of food, produced a dose-dependent reduction of food intake in the rat (ED50 congruent to 17 mg/kg during the 1st h of testing). This anorexic effect persisted for at least 24 h. These results provide further evidence that under certain conditions cyproheptadine, which is used as an orectic agent in man, can produce anorexia.

Studies on food intake in the fasted rat.

1. The effects of orally- and intravenously-administered d-amphetamine-SO4, fenfluramine-HCl and cocaine-HCl on food intake in the fasted rat were compared. 2. During the first hour after presentation of food to the animals, ED50 values for suppression of food intake after intravenous injection were: d-amphetamine-SO4, 0.1 mg/kg; fenfluramine-HCl, 0.6 mg/kg; cocaine-HCl, 1.0 mg/kg, ED50 values for suppression of food intake after oral administration were: d-amphetamine-SO4, 0.3 mg/kg; fenfluramine-HCl, 0.7 mg/kg; cocaine-HCl, 9 mg/kg. 3. Over a 4 hr test period, d-amphetamine, fenfluramine and cocaine produced dose-dependent anorexigenic effects only when orally administered. 4. Orally-administered quipazine-maleate and mazindol also decreased food intake, their respective ED50 values being 7 mg/kg and 1.2 mg/kg for the first hour of testing; the anorexigenic effect of mazindol appeared to be more sustained than those of the other anorexigenic agents tested in that it was evident on the day after drug administration. 5. Orally-administered diazepam increased food intake on the day of its administration, but decreased food intake on the day after its administration. 6. In addition to providing further evidence for the effects of several typical anorexigenic agents, the model described might be useful for further studies on anorexigenic agents and minor tranquillizers.

Lack of effect of the peptide pyro-Glu-His-Gly-OH on food consumption in mice and rats.

1. Single subcutaneous injections (0.5-10 mg/kg) or repeated oral administration (5 mg/kg, twice daily for 3 or 5 days) of the proposed anorexigenic peptide pyro-GLU-HIS-GLY-OH did not affect food intake in the mouse. 2. Single intravenous injections (0.05-1 mg/kg) or daily intravenous injections (0.3-10 mg/kg, for 5 days) of pyro-GLU-HIS-GLY-OH did not affect food intake in the rat. 3. These results, together with results obtained recently by other workers, indicate that pyro-GLU-HIS-GLY-OH does not have anorexigenic properties in rodents.

THIP inhibits feeding behavior in fasted rats.

The effects of 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol (THIP) were compared with those of d-amphetamine and GABA in fasted rats. Intravenously-administered THIP produced a dose-dependent decrease in food consumption (ED50 congruent to 1.5 mg/kg) by an action that was not reversed by prior subcutaneous or simultaneous intravenous (IV) injection of bicuculline. d-Amphetamine-SO4 also produced a decrease in food consumption in this model (ED50 congruent to 0.2 mg/kg, IV). Unlike THIP, GABA (in doses up to 100 mg/kg, IV) did not produce a marked anorexigenic effect. These results provide further evidence that THIP can penetrate the "blood-brain barrier", and that central GABA-ergic systems are involved in controlling food intake.