Applications of the Cartesian coordinate tensor transfer technique in the simulations of vibrational circular dichroism spectra of oligonucleotides

The application of the Cartesian coordinate tensor transfer (CCT) technique for simulations of the IR absorption and vibrational circular dichroism (VCD) spectra of relatively large nucleic acid fragments is demonstrated on several case studies. The approach is based on direct ab initio calculations of atomic tensors, determining molecular properties, for relatively small fragments, and subsequent transfer of these tensors to the larger systems in Cartesian coordinates. This procedure enables precise computations of vibrational spectra for large biomolecular systems, currently with up to several thousands of atoms. The versatile ability of the CCT methods is emphasized on the examples of VCD and IR absorption spectra calculations for B- and Z-forms of DNA, single-, double-, and triple-stranded RNA helices and DNA structures with different base content and sequences. The development and recent improvements of the methodology are followed, including utilization of the constrained normal mode optimization (NMO) strategy and combined quantum mechanics and molecular dynamics simulations. Advantages, drawbacks, and recommendations for future improvements of the CCT method as applied to nucleic acid spectra calculations are discussed.     

Subunit-specific coupling between gamma-aminobutyric acid type A and P2X2 receptor channels

ATP and gamma-aminobutyric acid (GABA) are two fast neurotransmitters co-released at central synapses, where they co-activate excitatory P2X and inhibitory GABAA (GABA type A) receptors. We report here that co-activation of P2X2 and various GABAA receptors, co-expressed in Xenopus oocytes, leads to a functional cross-inhibition dependent on GABAA subunit composition. Sequential applications of GABA and ATP revealed that alphabeta- or alphabetagamma-containing GABAA receptors inhibited P2X2 channels, whereas P2X2 channels failed to inhibit gamma-containing GABAA receptors. This functional cross-talk is independent of membrane potential, changes in current direction, and calcium. Non-additive responses observed between cation-selective GABAA and P2X2 receptors further indicate the chloride independence of this process. Overexpression of minigenes encoding either the C-terminal fragment of P2X2 or the intracellular loop of the beta3 subunit disrupted the functional cross-inhibition. We previously demonstrated functional and physical cross-talk between rho1 and P2X2 receptors, which induced a retargeting of rho1 channels to surface clusters when co-expressed in hippocampal neurons (Boue-Grabot, E., Emerit, M. B., Toulme, E., Seguela, P., and Garret, M. (2004) J. Biol. Chem. 279, 6967-6975). Co-expression of P2X2 and chimeric rho1 receptors with the C-terminal sequences of alpha2, beta3, or gamma2 subunits indicated that only rho1-beta3 and P2X2 channels exhibit both functional cross-inhibition in Xenopus oocytes and co-clustering/retargeting in hippocampal neurons. Therefore, the C-terminal domain of P2X2 and the intracellular loop of beta GABAA subunits are required for the functional interaction between ATP- and GABA-gated channels. This gamma subunit-dependent cross-talk may contribute to the regulation of synaptic activity.     

Transforming growth factor-β1 enhances the secretion of monocyte chemoattractant protein-1 by the IEC-18 intestinal epithelial cell line

Summary Intestinal epithelial cells (IEC) are known to produce monocyte chemoattractant protein-1 (MCP-1). However, MCP-1 production, as with many other cytokines, can be regulated by a network of cytokines present in the environment of the IEC. Both IEC and inflammatory cells have been shown to produce transforming growth factor-β (TGF-β), and the regulatory effect of this cytokine on MCP-1 secretion by IEC has not been determined. Using the IEC-18 cell line, we have found that TGF-β1 alone induced the secretion of high levels of MCP-1. Treatment with TGF-β1 also enhanced the levels of MCP-1 messenger ribonucleic acid. However, costimulation of the cells with TGF-β1 and interleukin-1β (IL-1β) resulted in significant, but less than additive, increases in MCP-1 secretion. Finally, the enhancing effect of TGF-β1 on MCP-1 secretion was not due to IL-6. These results suggest that TGF-β1 from IEC or inflammatory cells may significantly enhance the secretion of MCP-1 by IEC and play an important role in inflamed mucosal tissues.     

Phosphorylation of MAP kinase and p90rsk and its regulation during in vitro maturation of cumulus-enclosed rabbit oocytes

Numerous studies have demonstrated that activation of the mitogen-activated protein (MAP) kinase is involved in the maturation of oocytes. In this study, the expression and phosphorylation of MAP kinase and p90rsk, one of the substrates of MAP kinase, during rabbit oocyte maturation were studied. The results showed that MAP kinase phosphorylation began to occur after germinal vesicle breakdown (GVBD) and the active form was maintained until metaphase II. p90rsk was also activated after GVBD following MAP kinase activation. Immunofluorescent analysis showed that p90rsk was enriched in the nuclear area after GVBD and was gradually localised to the spindle. When GVBD was inhibited by increased cAMP or decreased protein kinase C activity, the phosphorylation of both MAP kinase and p9rsk was blocked. Our data suggest that (1) MAP kinase/p90rsk activation is not necessary for GVBD, but plays an important role in the post-GVBD events including spindle assembly in rabbit oocytes; and (2) MAP kinase/p9rsk activation is down-regulated by cAMP and up-regulated byprotein kinase C in cumulus-enclosed rabbit oocytes.     

The European freshwater landscape and hotspot areas of mass effects and regional connectivity

Aim

The maintenance of broad-scale connectivity patterns is suggested as a sustainable strategy for biodiversity preservation. However, explicit approaches for quantifying the functional role of different areas in biogeographic connectivity have been elusive. Freshwaters are spatially structured ecosystems critically endangered because of human activities and global change, demanding connectivity-based approaches for their conservation. Mass effects—the increase in local diversity by immigration—and corridor effects—the connections with distant communities—are basic and relevant mechanisms connecting diversity with landscape configuration. Here, we identified freshwater hotspots areas for mass and corridor effects across Europe.

Location

Europe.

Methods

Using satellite images, we quantified the areas of ephemeral, temporal and permanent freshwaters. The landscape structure of the freshwater ecoregions was represented as a directed-graph, and the link weights were determined by the distance between cells and the water cover. Three centrality metrics were used to rank freshwater areas with respect to their potential role in dispersal-mediated mechanisms. Out-degree represents the potential of an area to operate as a diversity source to other regions. In-degree reflects the importance that incoming dispersal may have in local diversity. Betweenness refers to the importance of local areas for connecting other distant areas.

Results

We detected great concentrations of source hotspots on the northern regions associated to lentic ecosystems, main European rivers acting as ecological corridors for all freshwaters, and a mixed distribution of connectivity hotspots in southern and Mediterranean ecoregions, associated with lentic and/or lotic systems.

Main Conclusions

We showed an explicit connection between landscape structure and dispersal process at large geographic scales, highlighting hotspots of connectivity for the European waterscape. The spatial distribution of hotspots points to differences in landscape configurations potentially accounting for biogeographic diversity patterns and for mechanisms that have to be considered in conservation planning.     

Oocyte meiosis in a trisomy 18 fetus. Behavior of the supernumerary chromosome and identification of the 18 bivalent]

Associations of the three n degrees 18 chromosomes were studied in a trisomy 18 fetus (the chromosomal constitution of which had been identified by amniocentesis). The three classes of associations observed were those observed in other trisomic organisms:trivalent, trivalent presenting an important asynaptic region, and bivalent accompanied by a univalent. In addition, the sequence was established of chromomeres, the number of which varied from 18 to 23 depending on the degree of chromosome contraction. In elongated pachytene oocyte bivalents each G-band of mitotic metaphase chromosomes could be subdivided into several sub-bands.     

Production and biochemical characterization of the recombinant Boophilus microplus Bm95 antigen from Pichia pastoris

The new antigen Bm95 from the cattle tick Boophilus microplus was recently isolated, cloned and expressed in the methylotrophic yeast Pichia pastoris. The recombinant protein has shown to induce protection in cattle against infestations of B. microplus under controlled and production conditions. In this paper we report the production and large-scale purification of the Bm95 protein, following a simple and cost-effective process. The antigen was obtained highly aggregated, forming particles ranging from 26 to 30 nm and with purity higher than 80%. The process yield was 0.55 g of pure Bm95 protein per liter of culture. The 98% of the primary structure of the recombinant protein was verified by mass spectrometry. Three amino acid changes in comparison with the sequence deduced from cDNA were detected by LC-MS/MS. The antigen was also obtained N-glycosylated, as previously reported for heterologous protein expression in P. pastoris.     

Percoll gradient-centrifuged capacitated mouse sperm have increased fertilizing ability and higher contents of sulfogalactosylglycerolipid and docosahexaenoic acid-containing phosphatidylcholine compared to washed capacitated mouse sperm

Although Percoll gradient centrifugation has been used routinely to prepare motile human sperm, its use in preparing motile mouse sperm has been limited. Here, we showed that Percoll gradient-centrifuged (PGC) capacitated mouse sperm had markedly higher fertilizing ability (sperm-zona pellucida [ZP] binding and in vitro fertilization) than washed capacitated mouse sperm. We also showed that the lipid profiles of PGC capacitated sperm and washed capacitated sperm differed significantly. The PGC sperm had much lower contents of cholesterol and phospholipids. This resulted in relative enrichment of male germ cell-specific sulfogalactosylglycerolipid (SGG), a ZP-binding ligand, in PGC capacitated sperm, and this would explain, in part, their increased ZP-binding ability compared with that of washed capacitated sperm. Analyses of phospholipid fatty acyl chains revealed that PGC capacitated sperm were enriched in phosphatidylcholine (PC) molecular species containing highly unsaturated fatty acids (HUFAs), with docosahexaenoic acid (DHA; C22: 6n-3) being the predominant HUFA (42% of total hydrocarbon chains of PC). In contrast, the level of PC-HUFAs comprising arachidonic acid (20:4n-6), docosapentaenoic acid (C22:5n-6), and DHA in washed capacitated sperm was only 27%. Having the highest unsaturation degree among all HUFAs in PC, DHA would enhance membrane fluidity to the uppermost. Therefore, membranes of PGC capacitated sperm would undergo fertilization-related fusion events at higher rates than washed capacitated sperm. These results suggested that PGC mouse sperm should be used in fertilization experiments and that SGG and DHA should be considered to be important biomarkers for sperm fertilizing ability.