Structural analysis of Canavalia maritima and Canavalia gladiata lectins complexed with different dimannosides: new insights into the understanding of the structure-biological activity relationship in legume lectins

Plant lectins, especially those purified from species of the Leguminosae family, represent the best studied group of carbohydrate-binding proteins. The legume lectins from Diocleinae subtribe are highly similar proteins that present significant differences in the potency/efficacy of their biological activities. The structural studies of the interactions between lectins and sugars may clarify the origin of the distinct biological activities observed in this high similar class of proteins. In this way, this work presents a crystallographic study of the ConM and CGL (agglutinins from Canavalia maritima and Canavalia gladiata, respectively) in the following complexes: ConM/CGL:Man(alpha1-2)Man(alpha1-O)Me, ConM/CGL:Man(alpha1-3)Man(alpha1-O)Me and ConM/CGL:Man(alpha1-4)Man(alpha1-O)Me, which crystallized in different conditions and space group from the native proteins. The structures were solved by molecular replacement, presenting satisfactory values for R(factor) and R(free). Comparisons between ConM, CGL and ConA (Canavalia ensiformis lectin) binding mode with the dimannosides in subject, presented different interactions patterns, which may account for a structural explanation of the distincts biological properties observed in the lectins of Diocleinae subtribe.     

Software package for automatic microarray image analysis (MAIA)

Although various software solutions are currently available for microarray image analysis, one would still expect to develop algorithms ensuring higher level of intelligence and robustness. We present a fully functional software package for automatic processing of the two-color microarray images including spot localization, quantification and quality control. The developed algorithms aim at making ratio estimates more resistant to array contamination and offer automatic tools to evaluate spot quality. Availability: A demo version of the software can be downloaded from http://bioinfo.curie.fr/projects/maia. A full version is freely available to non-commercial users upon request from the authors.     

Molecular and karyological aspects of Batoidea (Chondrichthyes, Elasmobranchi) phylogeny

Although considerable progress has been made in elucidating the relationships within the Chondrichthyes, there is no agreement as it concerns the systematics of Batoidea, the most derived superorder among cartilaginous fishes, and many different interpretations exist. Our investigation provides the first assessment of relationships among the described batoid species using sequences from both mtDNA and nuclear genes as well as karyological morphology. Our work consists primarily in reconstructing the phylogenetic relationships of Batoidea by examining the mtDNA (16S) and nuclear gene (18S) sequences from 11 batoid species. The three analytical methods (NJ, MP and Bayesian analysis) grouped Rajiformes, Myliobatiformes and Rhinobatiformes. In these trees the two torpedoes diverge from the other batoid fishes. We also compare the molecular data with the available karyological evidence, which consist of the diploid number and the karyotype morphology of eight species belonging to the four orders examined. The results show that the karyological structure in the different species is generally consistent with the various phylogenetical trees, and that Torpediniformes confirm their unique genome organization.     

Nitric oxide during ischemia attenuates oxidant stress and cell death during ischemia and reperfusion in cardiomyocytes

Nitric oxide (NO) has been implicated as a cardioprotective agent during ischemia/reperfusion (I/R), but the mechanism of protection is unknown. Oxidant stress contributes to cell death in I/R, so we tested whether NO protects by attenuating oxidant stress. Cardiomyocytes and murine embryonic fibroblasts were administered NO (10-1200 nM) during simulated ischemia, and cell death was assessed during reperfusion without NO. In each case, NO abrogated cell death during reperfusion. Cells overexpressing endothelial NO synthase (NOS) exhibited a similar protection, which was abolished by the NOS inhibitor N(omega)-nitro-l-arginine methyl ester. Protection was not mediated by guanylate cyclase or the mitochondrial K(ATP) channel, as inhibitors of these systems failed to abolish protection. NO did not prevent decreases in mitochondrial potential, but cells protected with NO demonstrated recovery of potential at reperfusion. Measurements using C11-BODIPY reveal that NO attenuates lipid peroxidation during ischemia and reperfusion. Measurements of oxidant stress using the ratiometric redox sensor HSP-FRET demonstrate that NO attenuates protein oxidation during ischemia. These findings reveal that physiological levels of NO during ischemia can attenuate oxidant stress both during ischemia and during reperfusion. This response is associated with a remarkable attenuation of cell death, suggesting that ischemic cell death may be a regulated event.     

The third dimension bridges the gap between cell culture and live tissue

Moving from cell monolayers to three-dimensional (3D) cultures is motivated by the need to work with cellular models that mimic the functions of living tissues. Essential cellular functions that are present in tissues are missed by 'petri dish'-based cell cultures. This limits their potential to predict the cellular responses of real organisms. However, establishing 3D cultures as a mainstream approach requires the development of standard protocols, new cell lines and quantitative analysis methods, which include well-suited three-dimensional imaging techniques. We believe that 3D cultures will have a strong impact on drug screening and will also decrease the use of laboratory animals, for example, in the context of toxicity assays.     

The use of ASB-14 in combination with CHAPS is the best for solubilization of human brain proteins for two-dimensional gel electrophoresis.

Protein extraction is the most important step to reveal a proteome by Two-Dimensional Gel Electrophoresis. Usually, the urea/thiourea based standard protein extraction buffer (SB) is combined with detergents with the aim of achieving better resolution and solubilization of different classes of proteins. In order to produce better gels and achieve the greatest spot resolution of Human Brain Proteins, comparisons using 2-DE of extracted proteins from Human Brain Frontal Cortex with SB constituents (7M Urea, 2M Thiourea and 100mM DTT) were made, using different detergent compositions in the buffer. SB preparations in combination with CHAPS and ASB-14 as well as with ASB-16 (reported for the first time in 2-DE experiments) have been tested. Our results confirm that the most efficient solubilizing solution for 2-DE analysis of cytosolic and membrane Human Brain Proteins is SB combined with 4% CHAPS and 2% ASB-14.     

Space exploration by dendritic cells requires maintenance of myosin II activity by IP3 receptor 1

Dendritic cells (DCs) patrol the interstitial space of peripheral tissues. The mechanisms that regulate their migration in such constrained environment remain unknown. We here investigated the role of calcium in immature DCs migrating in confinement. We found that they displayed calcium oscillations that were independent of extracellular calcium and more frequently observed in DCs undergoing strong speed fluctuations. In these cells, calcium spikes were associated with fast motility phases. IP₃ receptors (IP₃Rs) channels, which allow calcium release from the endoplasmic reticulum, were identified as required for immature DCs to migrate at fast speed. The IP₃R1 isoform was further shown to specifically regulate the locomotion persistence of immature DCs, that is, their capacity to maintain directional migration. This function of IP₃R1 results from its ability to control the phosphorylation levels of myosin II regulatory light chain (MLC) and the back/front polarization of the motor protein. We propose that by upholding myosin II activity, constitutive calcium release from the ER through IP₃R1 maintains DC polarity during migration in confinement, facilitating the exploration of their environment.