Aurora-A kinase Ser349 phosphorylation is required during Xenopus laevis oocyte maturation

Xenopus laevis Aurora-A is phosphorylated in vivo onto three amino acids: Ser53, Thr295 and Ser349. The activation of the kinase depends on its autophosphorylation on Thr295 within the T-loop. The phosphorylation of Ser53 by still unknown kinase(s) prevents its degradation. The present work focused on the regulation of Aurora-A function via Ser349 phosphorylation. Mutagenesis of Ser349 to alanine (S349A) had few impact in vitro on the capability of the kinase to autophosphorylate as well as on its activity. These data in addition to in gel kinase assays and site-specific proteolytic digestion experiments prove that Ser349 is clearly neither a primary autophosphorylation site, nor an autophosphorylation site depending on the priming phosphorylation of Thr295. Using specific antibodies, we also show that the phosphorylation of Aurora-A Ser349 is a physiological event during Xenopus oocyte maturation triggered by progesterone. A peak of phosphorylation paralleled the decrease of Aurora activity observed between meiosis I and II. In response to progesterone, X. laevis stage VI oocytes microinjected with the Aurora-A S349A mutant proceeded normally to germinal vesicle breakdown (GVBD), but degenerated rapidly soon after. Since phosphorylation of Ser349 is responsible for a decrease in kinase activity, our results suggest that a down-regulation of Aurora-A activity involving Ser349 phosphorylation is required in the process of maturation.     

Reduced fertility of female mice lacking CD81

In somatic cells, the tetraspanins CD81 and CD9 associate with each other, with additional tetraspanins and with non-tetraspanin molecules to form proteolipidic complexes. Here we show that CD81 is expressed on the surface of oocytes where it associates with tetraspanin-enriched membrane structures. A major CD9 and CD81 partner, CD9P-1, is also expressed by oocytes. Deletion of CD81 gene in mice results in a 40% reduction of female fertility. In vitro insemination indicated that this infertility is due to a deficiency of oocytes to fuse with sperm. While the fertility of CD9-/- mice is severely but not completely impaired, double knock-out CD9-/- CD81-/- mice were completely infertile indicating that CD9 and CD81 play complementary roles in sperm-egg fusion. Finally, a fraction of CD9 was transferred from CD81-/- oocytes to sperm present in the perivitelline space indicating that the defect of fusion of CD81-/- oocytes does not result from an impaired initial gamete interaction.     

Genetic variability in Puccinia striiformis f. sp. tritici populations sampled on a local scale during natural epidemics

This study investigated genetic polymorphism on a local scale in Puccinia striiformis f. sp. tritici populations during natural epidemics, in four fields located in northern France and sampled in 1998 or 1999. Two hundred and forty-seven isolates were analyzed for their amplified fragment length polymorphism (AFLP) pattern through four primer combinations, and 194 of them were also tested for their virulence factors. Only one or two pathotypes were found in each field, and all isolates had virulence v17, matching the recently introduced Yr17 resistance gene. Polymorphism on a field scale was low. Although 67 loci were polymorphic, 77% of the isolates had the same AFLP pattern, all other patterns being rare or unique. Analyses of the genetic distance between AFLP patterns based on the Jaccard index allowed us to define 12 groups, but a bootstrap analysis showed that all isolates could be assigned to a single clonal lineage. This leads us to conclude that P. striiformis f. sp. tritici populations are clonal on a field scale in northern France.     

Les Ostracodes et le Quaternaire d’Aigion (golfe de Corinthe, Grèce)

The Upper Pleistocene and the Holocene of Aigion have delivered an abundant microfauna with 35 ostracode species. The Pleistocene from Aigion borehole generally provides Ostracodes from oligohaline environment with Cyprideis torosa, Candona angulata and Tyrrhenocythere amnicola while, in the Holocene, marine infralittoral species dominate with Cytheridea neapolitana, Carinocythereis whitei, Loxoconcha ovulata and Cytherois frequens. The marine sedimentation occurred at depth from some meters to some tens of meters. In the Aghios Constantinos section, the lagoonal marls are characterised by Euxinocythere schuldtae and a dwarf species of Xestoleberis. Then, a drastic environmental change occurs around the Pleistocene-Holocene boundary and presumably affected the whole Corinth gulf.     

Dihydrofolate reductase activity and resistance to aminopterin in various species of Drosophila

Summary The aim of our work was to compare the mechanisms of resistance to aminopterin, inhibitor of the dihydrofolate reductase enzyme, between different Drosophila species and those described for cultured cells. Moreover we compared the systematic species divisions based on morphological traits and those based on a molecular approach. For this purpose, the effect of aminopterin on viability and wing phenotype was studied in different Drosophila species. Dihydrofolate reductase was measured in adult flies. We found an important dihydrofolate reductase activity in the melanogaster sub-group compared to the other species studies. Wing effect was observed only in this sub-group. The effects of aminopterin on the wing phenotype were very similar to the phenotype of rudimentary mutants. Both deplete the pyrimidine pool and it has been shown by the studies of the structural genes of the nucleotide pyrimidine pathway that the wing tissue is very sensitive to every pertubation of this metabolism.The D. ananassae species was found to be fully resistant at the concentrations of the inhibitor tested. No or very little dihydrofolate reductase activity was detected. The binding of the enzyme to the inhibitor was comparable to that found in the Oregon strain of D. melanogaster. The purine and pyrimidine salvage pathways were investigated and the D. ananassae species displayed an important thymidine kinase activity. The D. ananassae flies were sensitive on Sang medium compared to the Oregon flies but were able to use exogenous bases or nucleosides more efficiently. Therefore the mechanism of resistance to aminopterin in Drosophila may be different from those described for methotrexate in mammalian cultured cells, as indicated by the results obtained for D. ananassae.     

Synthesis and structural study of a fully protected α-C-galactosyl model dipeptide

Summary The synthesis of three N-alkyl-6,7-dideoxy-1,2:3,4-di-O-isopropylidene-7-[(alkyl-carbonyl)amino]-L-glycero-α-D-galacto-octopyranuronamides6a-c, analogous model dipeptides containing two amide groups connected to the α-carbon bearing the fully protected galactose as a side chain, has been realized with the aim of determining the conformational influence of the galactosyl moiety on the peptide backbone. Molecular modeing of6a, X-ray crystallography of6c and IR and NMR experiments on6a-c in organic solvents show that the carbohydrate ring assumes a twist boat conformation. In non-polar organic solvents, the NH of the left amide group interacts with one ketal oxygen of the galactosyl group.     

Disturbed nitrogen metabolism associated with the hyperhydric status of fully habituated callus of sugarbeet

The content of polyamines and proline was much lower in a normal (N) callus of Beta vulgaris L. than in a fully habituated hyperhydric (H) callus. The H callus also contained more glutamate and had a higher glutamate dehydrogenase activity. The excess of glutamate, in this chlorophyll-deficient callus, was linked to accumulation of proline and polyamines. Experiments with α-difluoromethylornithine (DFMO) and α-difluoromethylarginine (DFMA) showed that both ornithine decarboxylase and arginine decarboxylase participated in the synthesis of polyamines (especially spermidine and putrescine) and removal of ammonia. It is hypothesized that the H callus was subjected to ammonia stress from the start of the culture. Experiments with gabaculine, an inhibitor of ornithine aminotransferase, showed that this enzyme linked proline degradation to polyamine synthesis through the production of ornithine. This disturbed nitrogen metabolism appeared to be characteristic of the fully habituated callus and might explain the low growth of this hyperhydric tissue.     

Soil seed banks in a heavy-metal polluted grassland at Prayon (Belgium)

Seed banks were investigated in a grassland dominated byAgrostis capillaris and Thlaspicaerulescens, established on soils polluted by atmospheric depositsof SO₂ and heavy-metal rich dust (Zn, Pb, Cd) from an ore-smelter inPrayon (E Belgium). A total of 155 soil cores (0–7 depth)distributed among 10 plots were sampled in November and March. Each samplingplot was characterised for the botanical composition and standing biomass of theestablished vegetation as well as for four environmental parameters (soil pH andextractable zinc, volume percentage of stones, relative altitude within thesite). Very high densities of germinable seeds were found (grand mean ±SE, pooled over dates and plots: 28,950 ± 6830 ). Agrostis capillaris represented 95% of theseeds, followed by Thlaspi caerulescens (3%); the remaining2% were shared among 24 other species. Two thirds of the seeds were concentratedin the upper 3 of the soil. The high seed density might be dueto a low rate of microbial decay of seeds due to heavy metal toxicity. The seedbank is almost entirely derived from those few heavy-metal tolerant species thathave re-colonised the polluted area in the last three decades. Only two species(Anagallis arvensis and Urtica dioica)not represented in the established vegetation and with seeds concentrated in thelower soil layer are probably derived from seed buried prior to the onset ofheavy-metal pollution. This study provides the first data suggesting that theheavy-metal hyperaccumulator Thlaspi caerulescens possessespersistent seed banks. Seed densities of Agrostiscapillaris, Thlaspi caerulescens andRumex acetosa were positively correlated with the standingbiomass of grasses and negatively correlated with the volume percentage occupiedby stones in the soil. It is argued that small-scale variation in microclimaticfactors that cue germination (fluctuations in soil temperature and moisture) canaccount for the observed pattern of spatial variation in seed density. From thestandpoint of restoration management, the results indicate that little change inthe floristic composition of the heavy-metal tolerant grasslands of Prayon canbe expected from the recruitment of buried seeds.