Structural changes of phenylalanine 338 and histidine 447 revealed by the crystal structures of tabun-inhibited murine acetylcholinesterase

Organophosphorus compounds (OPs) interfere with the catalytic mechanism of acetylcholinesterase (AChE) by rapidly phosphorylating the catalytic serine residue. The inhibited enzyme can at least partly be reactivated with nucleophilic reactivators such as oximes. The covalently attached OP conjugate may undergo further intramolecular dealkylation or deamidation reactions, a process termed "aging" that results in an enzyme considered completely resistant to reactivation. Of particular interest is the inhibition and aging reaction of the OP compound tabun since tabun conjugates display an extraordinary resistance toward most reactivators of today. To investigate the structural basis for this resistance, we determined the crystal structures of Mus musculus AChE (mAChE) inhibited by tabun prior to and after the aging reaction. The nonaged tabun conjugate induces a structural change of the side chain of His447 that uncouples the catalytic triad and positions the imidazole ring of His447 in a conformation where it may form a hydrogen bond to a water molecule. Moreover, an unexpected displacement of the side chain of Phe338 narrows the active site gorge. In the crystal structure of the aged tabun conjugate, the side chains of His447 and Phe338 are reversed to the conformation found in the apo structure of mAChE. A hydrogen bond between the imidazole ring of His447 and the ethoxy oxygen of the aged tabun conjugate stabilizes the side chain of His447. The displacement of the side chain of Phe338 into the active site gorge of the nonaged tabun conjugate may interfere with the accessibility of reactivators and thereby contribute to the high resistance of tabun conjugates toward reactivation.     

Palliacom : système multimodal d’aide à la communication

The Palliacom project, which is intended to be a communication support for persons with severe difficulties to talk and/or to write, focuses mainly on two objectives: the implementation of a writing assistance device based on either an alphabet or a set of icons, using personal or tablet computers; and the compilation of functional knowledge about how users exploit this writing assistance device, obtained through long term observation of utilization strategies. Depending on the user profile, different dynamic writing options are possible, for instance clicking or touching specific images displayed on the first screen of the writing assistance device enables to directly select the most appropriate set of configuration parameters. Otherwise, the written text can be printed, inserted within another text, or read automatically using a speech synthesizer. The system is currently being studied from a usability perspective, analyzing how impaired children express themselves with icons and handicapped adults communicate with alphabets.     

Primordial Enemies: Fungal Pathogens in Thrips Societies

Microbial pathogens are ancient selective agents that have driven many aspects of multicellular evolution, including genetic, behavioural, chemical and immune defence systems. It appears that fungi specialised to attack insects were already present in the environments in which social insects first evolved and we hypothesise that if the early stages of social evolution required antifungal defences, then covariance between levels of sociality and antifungal defences might be evident in extant lineages, the defences becoming stronger with group size and increasing social organisation. Thus, we compared the activity of cuticular antifungal compounds in thrips species (Insecta: Thysanoptera) representing a gradient of increasing group size and sociality: solitary, communal, social and eusocial, against the entomopathogen Cordyceps bassiana. Solitary and communal species showed little or no activity. In contrast, the social and eusocial species killed this fungus, suggesting that the evolution of sociality has been accompanied by sharp increases in the effectiveness of antifungal compounds. The antiquity of fungal entomopathogens, demonstrated by fossil finds, coupled with the unequivocal response of thrips colonies to them shown here, suggests two new insights into the evolution of thrips sociality: First, traits that enabled nascent colonies to defend themselves against microbial pathogens should be added to those considered essential for social evolution. Second, limits to the strength of antimicrobials, through resource constraints or self-antibiosis, may have been overcome by increase in the numbers of individuals secreting them, thus driving increases in colony size. If this is the case for social thrips, then we may ask: did antimicrobial traits and microbes such as fungal entomopathogens play an integral part in the evolution of insect sociality in general?     

Receptor protein tyrosine phosphatase mu expression as a marker for endothelial cell heterogeneity; analysis of RPTPmu gene expression using LacZ knock-in mice

The receptor-like protein tyrosine phosphatase mu (RPTPmu) belongs to the subfamily of meprin, A5, RPTPmu (MAM) domain-containing RPTPs, which are thought to play an important role in cell-cell adhesion mediated processes. The current study was designed to examine the expression pattern of RPTPmu in mice. We have generated RPTPmu-LacZ knock-in mice that express the beta-galactosidase (LacZ) reporter gene under the control of the RPTPmu promoter. LacZ expression patterns were analysed in embryos and adult mice by whole mount LacZ staining. Analysis of beta-galactosidase activity of heterozygous embryos and adult tissues revealed RPTPmu expression in endothelial cells of arteries and capillaries. In contrast, expression was virtually absent in endothelial cells of veins and in fenestrated endothelial cells in the adult liver and spleen. Moreover, RPTPmu expression was found in endothelial cells from the endocardium and the aorta in embryos, but not in adult mice. In addition to heterogeneous expression in endothelial cells, RPTPmu expression was found in cardiac muscle cells but not in skeletal muscle cells or smooth muscle cells. Expression was also found in Type II pneumonocytes in the lung alveoli and in Purkinje cells and other neurons in the brain. The specific expression of RPTPmu in arterial endothelial cells and in cardiac myocytes suggests that RPTPmu may play a role in the regulation of cardiovascular functions.     

Discovery of cell-active phenyl-imidazole Pin1 inhibitors by structure-guided fragment evolution

Pin1 is an emerging oncology target strongly implicated in Ras and ErbB2-mediated tumourigenesis. Pin1 isomerizes bonds linking phospho-serine/threonine moieties to proline enabling it to play a key role in proline-directed kinase signalling. Here we report a novel series of Pin1 inhibitors based on a phenyl imidazole acid core that contains sub-μM inhibitors. Compounds have been identified that block prostate cancer cell growth under conditions where Pin1 is essential.     

A deletion mutant of vitronectin lacking the somatomedin B domain exhibits residual plasminogen activator inhibitor-1-binding activity

Vitronectin and plasminogen activator inhibitor-1 (PAI-1) are important physiological binding partners that work in concert to regulate cellular adhesion, migration, and fibrinolysis. The high affinity binding site for PAI-1 is located within the N-terminal somatomedin B domain of vitronectin; however, several studies have suggested a second PAI-1-binding site within vitronectin. To investigate this secondary site, a vitronectin mutant lacking the somatomedin B domain (rDeltasBVN) was engineered. The short deletion had no effect on heparin-binding, integrin-binding, or cellular adhesion. Binding to the urokinase receptor was completely abolished while PAI-1 binding was still observed, albeit with a lower affinity. Analytical ultracentrifugation on the PAI-1-vitronectin complex demonstrated that increasing NaCl concentration favors 1:1 versus 2:1 PAI-1-vitronectin complexes and hampers formation of higher order complexes, pointing to the contribution of charge-charge interactions for PAI-1 binding to the second site. Furthermore, fluorescence resonance energy transfer between differentially labeled PAI-1 molecules confirmed that two independent molecules of PAI-1 are capable of binding to vitronectin. These results support a model for the assembly of higher order PAI-1-vitronectin complexes via two distinct binding sites in both proteins.     

RAB-5- and RAB-11-dependent vesicle-trafficking pathways are required for plasma membrane repair after attack by bacterial pore-forming toxin

Sequence variation of antigenic proteins allows pathogens to evade antibody attack. The variable protein commonly includes a hypervariable region (HVR), which represents a key target for antibodies and is therefore predicted to be immunodominant. To understand the mechanism(s) of antibody evasion, we analyzed the clinically important HVR-containing M proteins of the human pathogen Streptococcus pyogenes. Antibodies elicited by M proteins were directed almost exclusively against the C-terminal part and not against the N-terminal HVR. Similar results were obtained for mice and humans with invasive S.?pyogenes infection. Nevertheless, only anti-HVR antibodies protected efficiently against infection, as shown by passive immunizations. The HVR fused to an unrelated protein elicited no antibodies, implying that it is inherently weakly immunogenic. These data indicate that the M protein HVR evades antibody attack not only through antigenic variation but also by weak immunogenicity, a paradoxical observation that may apply to other HVR-containing proteins.     

High Virulence of Wolbachia after Host Switching: When Autophagy Hurts

Wolbachia are widespread endosymbionts found in a large variety of arthropods. While these bacteria are generally transmitted vertically and exhibit weak virulence in their native hosts, a growing number of studies suggests that horizontal transfers of Wolbachia to new host species also occur frequently in nature. In transfer situations, virulence variations can be predicted since hosts and symbionts are not adapted to each other. Here, we describe a situation where a Wolbachia strain (wVulC) becomes a pathogen when transfected from its native terrestrial isopod host species (Armadillidium vulgare) to another species (Porcellio d. dilatatus). Such transfer of wVulC kills all recipient animals within 75 days. Before death, animals suffer symptoms such as growth slowdown and nervous system disorders. Neither those symptoms nor mortalities were observed after injection of wVulC into its native host A. vulgare. Analyses of wVulC''s densities in main organs including Central Nervous System (CNS) of both naturally infected A. vulgare and transfected P. d. dilatatus and A. vulgare individuals revealed a similar pattern of host colonization suggesting an overall similar resistance of both host species towards this bacterium. However, for only P. d. dilatatus, we observed drastic accumulations of autophagic vesicles and vacuoles in the nerve cells and adipocytes of the CNS from individuals infected by wVulC. The symptoms and mortalities could therefore be explained by this huge autophagic response against wVulC in P. d. dilatatus cells that is not triggered in A. vulgare. Our results show that Wolbachia (wVulC) can lead to a pathogenic interaction when transferred horizontally into species that are phylogenetically close to their native hosts. This change in virulence likely results from the autophagic response of the host, strongly altering its tolerance to the symbiont and turning it into a deadly pathogen.     

High light exposure leads to a sign change in gravitaxis of the flagellate Euglena gracilis

In the absence of other external stimuli the motile, unicellular freshwater flagellate Euglena gracilis normally swims upward in the water column (negative gravitaxis). This behavior is most likely triggered by active physiological orientation mechanisms. Recently it was found that negative gravitaxis often inverts to a positive one upon high light exposure. This response is not mediated by the photoreceptor (the paraxonemal body - PAB), because PAB-free mutants do also show this response after high radiation. It is very likely that the phenomenon is triggered by reactive oxygen species, because in the absence of oxygen no gravitaxis sign change was observed. Also increased salinity inverses the sign of gravitaxis, leading to the assumption that environmental stressors induce the formation of reactive oxygen species, serving as signal molecules.