Effect of stage of follicular growth during superovulation on developmental competence of bovine oocytes

The final steps of oocyte capacitation and maturation are critical for embryonic development but detailed information is scarce on how the oocyte is affected during this period. In this study, 2033 oocytes were collected from 106 superovulated cattle at four different time points before ovulation. Follicular characteristics were measured and oocyte quality was assessed by morphology, mRNA expression of eight marker genes or developmental ability after in vitro/in vivo maturation and subsequent in vitro fertilization and culture. Approaching ovulation, expected increases in follicular size and cumulus expansion suggested progression of oocyte maturation. No differences were found in the expression patterns of analyzed genes, except for heat-shock-protein (Hsp) that was lower in in vivo matured oocytes collected shortly before ovulation. Oocytes collected at this time also had higher developmental ability measured as blastocyst rates (57.6%) after in vitro production while no differences were found between oocytes recovered earlier at the first three time points (39.3-41.5%). We conclude that oocytes recovered late in the preovulatory period are more developmentally competent than oocytes recovered at the pre-capacitation and the capacitation period, probably due to the former having matured in vivo. However, a precisely defined time for aspirating immature oocytes for subsequent in vitro development seems not to be crucial.     

Escape behaviour and ultimate causes of specific induced defences in an anuran tadpole

Induced defences, such as the predator avoidance morphologies in amphibians, result from spatial or temporal variability in predation risk. One important component of this variability should be the difference in hunting strategies between predators. However, little is known about how specific and effective induced defences are to different types of predators. We analysed the impact of both pursuing (fish, Gasterosteus aculeatus) and sit-and-wait (dragonfly, Aeshna cyanea) predators on tadpole (Rana dalmatina) morphology and performance (viz locomotive performance and growth rate). We also investigated the potential benefits of the predator-induced phenotype in the presence of fish predators. Both predators induced deeper tail fins in tadpoles exposed to threat of predation, and stickleback presence also induced longer tails and deeper tail muscles. Morphological and behavioural differences resulted in better escape ability of stickleback-induced tadpoles, leading to improved survival in the face of stickleback predation. These results clearly indicate that specific morphological responses to different types of predators have evolved in R. dalmatina. The specific morphologies suggest low correlations between the traits involved in the defence. Independence of traits allows prey species to fine-tune their response according to current predation risk, so that the benefit of the defence can be maximal.     

Crystal structure of a four-copper laccase complexed with an arylamine: insights into substrate recognition and correlation with kinetics

Laccases are multicopper oxidases that catalyze the oxidation of a wide range of phenols or arylamines, and their use in industrial oxidative processes is increasing. We purified from the white rot fungus Trametes versicolor a laccase that exists as five different isozymes, depending on glycosylation. The 2.4 A resolution structure of the most abundant isozyme of the glycosylated enzyme was solved. The four copper atoms are present, and it is the first crystal structure of a laccase in its active form. The crystallized enzyme binds 2,5-xylidine, which was used as a laccase inducer in the fungus culture. This arylamine is a very weak reducing substrate of the enzyme. The cavity enclosing 2,5-xylidine is rather wide, allowing the accommodation of substrates of various sizes. Several amino acid residues make hydrophobic interactions with the aromatic ring of the ligand. In addition, two charged or polar residues interact with its amino group. The first one is an histidine that also coordinates the copper that functions as the primary electron acceptor. The second is an aspartate conserved among fungal laccases. The purified enzyme can oxidize various hydroxylated compounds of the phenylurea family of herbicides that we synthesized. These phenolic substrates have better affinities at pH 5 than at pH 3, which could be related to the 2,5-xylidine binding by the aspartate. This is the first high-resolution structure of a multicopper oxidase complexed to a reducing substrate. It provides a model for engineering laccases that are either more efficient or with a wider substrate specificity.     

Photoperiodic regulation of a 24-kD dehydrin-like protein in red-osier dogwood (Cornus sericea L.) in relation to freeze-tolerance

A predominant 24-kD dehydrin-like protein was previously found to fluctuate seasonally within red-osier dogwood (Cornus sericea L.) stems. The current study attempted to determine what environmental cues triggered the accumulation of the 24-kD protein and to assess its potential role in winter survival. Controlled photoperiod and field studies confirmed that photoperiod regulates a reduction of stem water content (SWC), freeze-tolerance enhancement and accumulation of the 24-kD protein. Diverse climatic ecotypes, which are known to respond to different critical photoperiods, displayed differential reduction of SWC and accumulation of the 24-kD protein. A time-course study confirmed that prolonged exposure to short days is essential for SWC reduction, 24-kD protein accumulation, and freeze-tolerance enhancement. Water deficit induced 24-kD protein accumulation and enhanced freeze-tolerance under long-day conditions. In all instances, freeze-tolerance enhancement and 24-kD protein accumulation was preceded by a reduction of SWC. These results are consistent with the hypothesis that C. sericea responds to decreasing photoperiod, which triggers a reduction in SWC. Reduced SWC in turn may trigger the accumulation of the 24-kD protein and a parallel increase in freeze-tolerance.     

Phosphorylation-dependent paxillin-ERK association mediates hepatocyte growth factor-stimulated epithelial morphogenesis

Activation of the hepatocyte growth factor (HGF) receptor c-met results in the regulation of cell-matrix interactions, including the MAPK-dependent stimulation of epithelial cell morphogenesis. In the present study we demonstrate that HGF stimulates the localization of ERK to sites of cell-matrix interactions and that this is mediated by the tyrosine phosphorylation-dependent association of inactive ERK and the focal adhesion complex protein paxillin. In addition, paxillin was found to associate with the upstream MAP kinases Raf and MEK, resulting in a complex that can mediate localized ERK activation. Mutation of the ERK binding site in paxillin prevented HGF-stimulated ERK-paxillin association and eliminated HGF-induced cell spreading and branching process formation. These experiments reveal that paxillin-dependent ERK activation at sites of cell-matrix interaction is critical for HGF-stimulated epithelial morphogenesis.     

Field evidence for density-dependent effects in the trematode Microphallus papillorobustus in its manipulated host, Gammarus insensibilis

Numerous studies have demonstrated that parasites with complex life cycles frequently manipulate the phenotype of their hosts to increase their transmission rate. Little is known, however, concerning density-dependent processes within infrapopulations of manipulative parasites--whether parasites cooperate to manipulate the host, whether competition counteracts with these potential cooperative benefits, or both. Here we explored these ideas, focusing on the association between the manipulative trematode Microphallus papillorobustus and its second intermediate host, the gammarid Gammarus insensibilis. From the data collected in the field, we found no evidence that co-occurring M. papillorobustus individuals benefit from the presence of conspecifics; instead, individuals in larger infrapopulations suffered reduced size and fecundity. Thus, the net effect of increasing density suggests that competition rather than cooperation is the dominant force in infrapopulations of M. papillorobustus.     

Tracking a genetic signal of extinction-recolonization events in a neotropical tree species: Vouacapoua americana Aublet in French Guiana

Drier periods from the late Pleistocene and early Holocene have been hypothesized to have caused the disappearance of various rainforest species over large geographical areas in South America and restricted the extant populations to mesic sites. Subsequent improvement in climatic conditions has been associated with recolonization. Changes in population size associated with these extinction-recolonization events should have affected genetic diversity within species. However, these historical hypotheses and their genetic consequences have rarely been tested in South America. Here, we examine the diversity of the chloroplast and nuclear genomes in a Neotropical rainforest tree species, Vouacapoua americana (Leguminosae, Caesalpinioideae) in French Guiana. The chloroplast diversity was analyzed using a polymerase chain reaction-restriction fragment length polymorphism method (six pairs of primers) in 29 populations distributed over most of French Guiana, and a subset of 17 populations was also analyzed at nine polymorphic microsatellite loci. To determine whether this species has experienced extinction-recolonization, we sampled populations in areas supposedly not or only slightly affected by climatic changes, where the populations would not have experienced frequent extinction, and in areas that appear to have been recently recolonized. In the putatively recolonized areas, we found patches of several thousands of hectares homogeneous for chloroplast variation that can be interpreted as the effect of recolonization processes from several geographical origins. In addition, we observed that, for both chloroplast and nuclear genomes, the populations in newly recolonized areas exhibited a significantly smaller allelic richness than others. Controlling for geographic distance, we also detected a significant correlation between chloroplast and nuclear population differentiation. This result indicates a cytonuclear disequilibrium that can be interpreted as a historical signal of a genetic divergence between fragmented populations. In conclusion, the spatial genetic structure of contemporary V. americana populations shows evidence that this species has experienced large extinction-recolonization events, which were possibly caused by past climatic change.     

Infection of the cockle Cerastoderma edule in the Baie des Veys (France) by the microsporidian parasite Steinhausia sp

We report the occurrence of the microsporidian parasite Steinhausia sp. in the oocytes of the common cockle Cerastoderma edule in a natural population in France, where high mortalities occurred. Steinhausia sp. appeared primarily as sporocysts containing many small spores, and putative earlier developmental stages were also observed. Both its prevalence and infection intensity were low, and no host defence reaction was recognized, suggesting that Steinhausia sp. had no detrimental effect on C. edule. Its prevalence was higher in cockles lying on the sediment surface, but the significance of this observation could not be explained given the poor knowledge of the Steinhausia life cycle. The present data did not allow specific identification of the parasite, and further studies are required to determine whether Steinhausia sp. in the cockle is a new species, or a microsporidian infecting multiple host species.     

N,N,N',N'-tetramethyl-p-phenylenediamine initiates the appearance of a well-resolved I peak in the kinetics of chlorophyll fluorescence rise in isolated thylakoids

Addition of N,N,N',N'-tetramethyl-p-phenylendiamine (TMPD) to thylakoid membranes isolated from pea leaves initiates the appearance of peak I in the polyphasic rise of chlorophyll (Chl) fluorescence observed during strong illumination, making it similar to that observed in leaves or intact chloroplasts. This effect depends on TMPD concentration and incubation period of isolated thylakoids with TMPD. The resolution of I-peak in the presence of weak concentrations of TMPD which reduced the overlap between I- and P-peaks, resulted from a decreased reduction of both fast and slow plastoquinone (PQ) pools of the granal and stromal thylakoids, respectively, as TMPD effectively accepts electrons from reduced PQ. High concentrations of TMPD markedly decreased the J-I-P phase of fluorescence rise and greatly retarded the I-P step rise. Accumulation of oxidized TMPD in the thylakoid lumen accelerated the re-oxidation of the acceptor side of Photosystem II (PSII) as illustrated by a two-fold increase in the magnitude of the fast component and complete suppression of the middle component of the variable Chl fluorescence (F(v)) decay in the dark. Evidently, exogenous addition of high concentrations of TMPD prevented the light-induced reduction of the slow PQ pool.     

The BRET2/arrestin assay in stable recombinant cells: a platform to screen for compounds that interact with G protein-coupled receptors (GPCRS)

In BRET2 (Bioluminescence Resonance Energy Transfer), a Renilla luciferase (RLuc) is used as the donor protein, while a Green Fluorescent Protein (GFP2) is used as the acceptor protein. In the presence of the cell permeable substrate DeepBlueC, RLuc emits blue light at 395 nm. If the GFP2 is brought into close proximity to RLuc via a specific biomolecular interaction, the GFP2 will absorb the blue light energy and reemit green light at 510nm. BRET2 signals are therefore easily determined by measuring the ratio of green over blue light (510/395nm) using appropriate dual channel luminometry instruments (e.g., Fusion Universal Microplate Analyzer, Packard BioScience). Since no light source is required for BRET2 assays, the technology does not suffer from high fluorescent background or photobleaching, the common problems associated with standard FRET-based assays. Using BRET2, we developed a generic G Protein-Coupled Receptor (GPCR) assay based on the observation that activation of the majority of GPCRs by agonists leads to the interaction of beta-arrestin (a protein that is involved in receptor desensitization and sequestration) with the receptor. We established a cell line stably expressing the GFP2:beta-arrestin 2 fusion protein, and showed that it can be used to monitor the activation of various transiently expressed GPCRs, in BRET2/arrestin assays. In addition, using the HEK 293/GFP2:beta-arrestin 2 cell line as a recipient, we generated a double-stable line co-expressing the vasopressin 2 receptor (V2R) fused to RLuc (V2R:RLuc) and used it for the pharmacological characterization of compounds in BRET2/arrestin assays. This approach yields genuine pharmacology and supports the BRET2/arrestin assay as a tool that can be used with recombinant cell lines to characterize ligand-GPCR interactions which can be applied to ligand identification for orphan receptors.