Revisiting Bond Breaking and Making in EuCo2 P2 : Where are the Electrons?

X-ray absorption spectroscopy (XAS) was used to elucidate changes in the electronic structure caused by the pressure-induced structural collapse in EuCo2 P2 . The spectral changes observed at the L3 -edge of Eu and K-edges of Co and P suggest electron density redistribution, which contradicts the formal charges calculated from the commonly used Zintl-Klemm concept. Quantum-chemical calculations show that, despite the increase in the oxidation state of Eu and the formation of a weak P-P bond in the high-pressure phase, the electron transfer from the Eu 4f orbitals to the hybridized 5d and 6s states causes strengthening of the Eu-P and P-P bonds. These changes explain the increased electron density on P atoms, deduced from the P K-edge XAS spectra. This work shows that the formal electron counting schemes do not provide an adequate description of changes associated with phase transitions in metallic systems with substantial mixing of the electronic states.

Extended Synaptotagmin Interaction with the Fibroblast Growth Factor Receptor Depends on Receptor Conformation, Not Catalytic Activity.

We previously demonstrated that ESyt2 interacts specifically with the activated FGF receptor and is required for a rapid phase of receptor internalization and for functional signaling via the ERK pathway in early Xenopus embryos. ESyt2 is one of the three-member family of Extended Synaptotagmins that were recently shown to be implicated in the formation of endoplasmic reticulum (ER)-plasma membrane (PM) junctions and in the Ca(2+) dependent regulation of these junctions. Here we show that ESyt2 is directed to the ER by its putative transmembrane domain, that the ESyts hetero- and homodimerize, and that ESyt2 homodimerization in vivo requires a TM adjacent sequence but not the SMP domain. ESyt2 and ESyt3, but not ESyt1, selectively interact in vivo with activated FGFR1. In the case of ESyt2, this interaction requires a short TM adjacent sequence and is independent of receptor autophosphorylation, but dependent on receptor conformation. The data show that ESyt2 recognizes a site in the upper kinase lobe of FGFR1 that is revealed by displacement of the kinase domain activation loop during receptor activation.

Taming the first-order transition in giant magnetocaloric materials.

Large magnetically driven temperature changes are observed in MnFe(P,Si,B) materials simultaneously with large entropy changes, limited (thermal or magnetic) hysteresis, and good mechanical stability. The partial substitution of B for P in MnFe(P,Si) compounds is found to be an ideal parameter to control the latent heat observed at the Curie point without deteriorating the magnetic properties, which results in promising magnetocaloric properties suitable for magnetic refrigeration.

The endocytic adapter E-Syt2 recruits the p21 GTPase activated kinase PAK1 to mediate actin dynamics and FGF signalling.

Fibroblast growth factor (FGF) signalling plays an essential role in early vertebrate development. However, the response to FGF requires endocytosis of the activated FGF receptor (FGFR) that is in part dependent on remodelling of the actin cytoskeleton. Recently we showed that the extended synaptotagmin family plasma membrane protein, E-Syt2, is an essential endocytic adapter for FGFR1. Here we show E-Syt2 is also an interaction partner for the p21-GTPase Activated Kinase PAK1. The phospholipid binding C2C domain of E-Syt2 specifically binds a site adjacent to the CRIB/GBD of PAK1. PAK1 and E-Syt2 selectively complex with FGFR1 and functionally cooperate in the FGF signalling. E-Syt2 binding suppresses actin polymerization and inhibits the activation of PAK1 by the GTPases Cdc42 and Rac. Interestingly, the E-Syt2 binding site on PAK1 extensively overlaps a site recently suggested to bind phospholipids. Our data suggest that PAK1 interacts with phospholipid membrane domains via E-Syt2, where it may cooperate in the E-Syt2-dependent endocytosis of activated FGFR1 by modulating cortical actin stability.

Extended-synaptotagmin-2 mediates FGF receptor endocytosis and ERK activation in vivo.

Targeting of activated plasma membrane receptors to endocytic pathways is important in determining the outcome of growth factor signaling. However, the molecular mechanisms are still poorly understood. Here, we show that the synaptotagmin-related membrane protein E-Syt2 is essential for rapid endocytosis of the activated FGF receptor and for functional signal transduction during Xenopus development. E-Syt2 depletion prevents an early phase of activated FGF receptor endocytosis that we show is required for ERK activation and the induction of the mesoderm. E-Syt2 interacts selectively with the activated FGF receptor and with Adaptin-2, and is required upstream of Ras activation and of receptor autophosphorylation for ERK activation and the induction of the mesodermal marker Xbra. The data identify E-Syt2 as an endocytic adaptor for the clathrin-mediated pathway whose function is conserved in human and suggest a broader role for the E-Syt subfamily in growth factor signaling.

The use of human hepatocytes to investigate drug metabolism and CYP enzyme induction.

Over the past two decades, attrition of new drug candidates which entered into development increased strongly mainly due to sub-optimal ADME profiles. Major problems were linked to poor metabolic stability and drug-drug interactions linked to inhibition or induction of metabolism. Since most small molecule (MW below 1000) drugs are cleared from the body by the liver, primary cultures of human hepatocytes became the most predictive and widely used in vitro model for drug metabolism studies as well as enzyme induction. For this purpose, well-established and robust in vitro assays for the measurement of cell viability, metabolic activity, and cytochrome P450 (CYP) mRNA expression levels are needed to characterize the quality of the isolated and/or cryopreserved hepatocytes used to perform such studies.

Multiple promoters and alternative splicing: Hoxa5 transcriptional complexity in the mouse embryo.

BACKGROUND: The genomic organization of Hox clusters is fundamental for the precise spatio-temporal regulation and the function of each Hox gene, and hence for correct embryo patterning. Multiple overlapping transcriptional units exist at the Hoxa5 locus reflecting the complexity of Hox clustering: a major form of 1.8 kb corresponding to the two characterized exons of the gene and polyadenylated RNA species of 5.0, 9.5 and 11.0 kb. This transcriptional intricacy raises the question of the involvement of the larger transcripts in Hox function and regulation. METHODOLOGY/PRINCIPAL FINDINGS: We have undertaken the molecular characterization of the Hoxa5 larger transcripts. They initiate from two highly conserved distal promoters, one corresponding to the putative Hoxa6 promoter, and a second located nearby Hoxa7. Alternative splicing is also involved in the generation of the different transcripts. No functional polyadenylation sequence was found at the Hoxa6 locus and all larger transcripts use the polyadenylation site of the Hoxa5 gene. Some larger transcripts are potential Hoxa6/Hoxa5 bicistronic units. However, even though all transcripts could produce the genuine 270 a.a. HOXA5 protein, only the 1.8 kb form is translated into the protein, indicative of its essential role in Hoxa5 gene function. The Hoxa6 mutation disrupts the larger transcripts without major phenotypic impact on axial specification in their expression domain. However, Hoxa5-like skeletal anomalies are observed in Hoxa6 mutants and these defects can be explained by the loss of expression of the 1.8 kb transcript. Our data raise the possibility that the larger transcripts may be involved in Hoxa5 gene regulation. SIGNIFICANCE: Our observation that the Hoxa5 larger transcripts possess a developmentally-regulated expression combined to the increasing sum of data on the role of long noncoding RNAs in transcriptional regulation suggest that the Hoxa5 larger transcripts may participate in the control of Hox gene expression.

[Hoxa5: a master gene with multifaceted roles]. / Hoxa5: un maître d'oeuvre à multiples facettes.

The Hox gene family occupies a central position in the control of body patterning by regulating the transcription of downstream effectors that, in turn, direct the morphogenetic events leading to the complex body forms along the axes. Analysis of Hox mutant mouse lines has revealed a panoply of phenotypes indicative of the broad range of Hox genes action throughout embryonic and postnatal life. Although Hox genes have been the subject of extensive research in the last two decades, the comprehension of the mechanisms involved in their regulation and function still remains elusive. Here, we present an overview of our current knowledge about one Hox gene family member, Hoxa5. The phenotypic survey of Hoxa5 mutant mice has unveiled the crucial role of this gene in regulating morphogenesis and specifying regional identity along the embryo. A majority of Hoxa5 mutant pups die at birth from defective respiratory tract. Surviving mutants present deficient alveolar septation revealing the importance of Hoxa5 during formation and maturation of the lung. Hoxa5 also participates in the morphogenesis of the digestive tract as well as that of the thyroid and mammary glands. Hoxa5 expression is restricted to the mesenchyme, and its action appears to be mediated through the control of mesenchymal-epithelial interactions during organogenesis. The implication of Hoxa5 in tumorigenesis has also been documented. In breast cancer, Hoxa5 down-regulation may impact on p53 gene expression, contributing to the oncogenic process. In contrast, the loss of Hoxa5 function limits leukaemia associated with specific chromosomal translocations. Thus, inappropriate Hoxa5 gene expression may disrupt normal growth and differentiation programs causing neoplasia. Hox gene function is intimately linked to its correct expression. Regulation of Hoxa5 expression requires multiple cis-acting regions, some encompassing coding sequences from neighboring genes. Moreover, it is complicated by the presence of several transcription units. Together these data enlighten the importance of Hox cluster organization in Hoxa5 function.

Selection of a trioxaquine as an antimalarial drug candidate.

Trioxaquines are antimalarial agents based on hybrid structures with a dual mode of action. One of these molecules, PA1103/SAR116242, is highly active in vitro on several sensitive and resistant strains of Plasmodium falciparum at nanomolar concentrations (e.g., IC(50) value = 10 nM with FcM29, a chloroquine-resistant strain) and also on multidrug-resistant strains obtained from fresh patient isolates in Gabon. This molecule is very efficient by oral route with a complete cure of mice infected with chloroquine-sensitive or chloroquine-resistant strains of Plasmodia at 26-32 mg/kg. This compound is also highly effective in humanized mice infected with P. falciparum. Combined with a good drug profile (preliminary absorption, metabolism, and safety parameters), these data were favorable for the selection of this particular trioxaquine for development as drug candidate among 120 other active hybrid molecules.

Excretory-secretory products of larval Fasciola hepatica investigated using a two-dimensional proteomic approach.

So far, very few secreted proteins from trematodes have been characterized, although their role in the mechanisms that allow the parasite to escape host's immune response have been largely documented. Here we performed a proteomic analysis of excretory-secretory proteins from the intra-molluscan larval stages of Fasciola hepatica. We identified two antioxidative enzymes: a Cu/Zn-superoxide dismutase (Cu/Zn SOD) and a thioredoxin (TRX) previously characterized in ES products from adult stages. These results support the importance of parasite detoxication of reactive oxygen species in invertebrate hosts, and raise the question of the possible conservation of major immune evasion effectors across trematode developmental life-stages.

Contribution of the N-glucuronidation pathway to the overall in vitro metabolic clearance of midazolam in humans.

Midazolam (MDZ) is one of the most commonly used in vivo and in vitro CYP3A4 probe substrates for drug-drug interactions (DDI) studies. The major metabolic pathway of MDZ in humans consists of the CYP3A4-mediated 1'-hydroxylation followed by urinary excretion as 1'-O-glucuronide derivative. In the present study, following incubation of MDZ with human liver microsomes supplemented with UDP-glucuronic acid, two major high-performance liquid chromatography (HPLC) peaks were isolated. HPLC and liquid chromatography/tandem mass spectrometry analyses identified these two metabolites as quaternary direct N-glucuronides of MDZ, thus revealing an additional metabolic pathway for MDZ. (1)H NMR spectrometry studies were performed showing that these two glucuronides were beta-N-glucuronides, which could be considered as two different conformers of the same molecule. According to molecular modeling experiments, the two glucuronide derivatives could be involved in atropoisomerism equilibrium. The formation of MDZ N-glucuronide exhibited moderate intersubject variability (at most 4.5-fold difference, n = 10). Among the recombinant human UDP glucuronosyltransferase (UGT) isoforms tested, only isoform UGT1A4 catalyzed the N-glucuronidation of MDZ fitting a Michaelis-Menten model. K(m) and V(max) values were 29.9 +/- 2.4 microM and 659.6 +/- 19.0 pmol/min/mg protein, respectively. The N-glucuronide derivative was found in human hepatocytes incubated under control conditions but also in the presence of the well known CYP3A4 inhibitor, ketoconazole. In the context of the in vitro study of CYP3A4-mediated DDI using MDZ and ketoconazole, direct MDZ N-glucuronidation may partly compensate the decrease in MDZ metabolic clearance caused by the addition of the inhibitor, thus potentially leading to underestimation, at least in vitro, of the extent of DDI.

Excretory-secretory proteome of larval Schistosoma mansoni and Echinostoma caproni, two parasites of Biomphalaria glabrata.

Schistosoma mansoni and Echinostoma caproni are two trematode species that use different strategies (mimicry and immunosuppression, respectively) to interfere with the snail innate immune system. Parasites excretory-secretory (ES) products have been shown to play a key role in these host-parasite immune interactions. However, they remain largely uncharacterized in larval trematodes. We developed a global proteomic approach to characterize the ES proteome of S. mansoni and E. caproni primary sporocysts. In ES products of both parasites, we found proteins involved in reactive oxygen species scavenging, glycolysis, signalling or calcium binding (superoxide dismutase Cu/Zn; glutathione S-transferase; aldo-keto-reductase; triose-phosphate isomerase; glyceraldehyde-3-phosphate dehydrogenase; aldolase, enolase, MICAL-like, calreticulin). According to their predicted functions, we propose a model in which these proteins (i) are involved in antioxidant activity, (ii) prevent hemocyte encapsulation process or (iii) favor invasion and migration of sporocysts in host tissues. These results suggest that S. mansoni and E. caproni sporocysts develope a strong immune protection during the first hours of infection giving them enough time to build up a long lasting immune evasion strategy relying on molecular mimicry or immunosuppression, respectively.

Identification and expression of gene transcripts generated during an anti-parasitic response in Biomphalaria glabrata.

In order to gain further insights into the molecular basis of gastropod anti-parasite immune responses, we investigated transcripts of Biomphalaria glabrata regulated during hemocytic encapsulation. Using a snail strain that is resistant to the parasite Echinostoma caproni, we performed suppression subtractive hybridization (SSH) to construct cDNA libraries of transcripts more abundantly expressed in unexposed or parasite-exposed snails. After sequence analysis and quantitative PCR analysis of expression, we identified 10 candidates of particular interest. They belonged to various functional groups such as detoxification enzymes (GST, SOD), antimicrobial proteins (LBI/BPI), protease inhibitors (cystatins), calcium-binding proteins, or C-type lectins. In situ hybridization (ISH) analysis revealed that one overexpressed cystatin-like candidate is specifically expressed in hemocytes participating in parasite encapsulation or aggregating at the site of infection. Two other candidates (C-type lectin and a LBP/BPI) were expressed in the albumen gland, further supporting the role of this organ in immunity and/or host-parasite interaction.

In vitro metabolism of ferroquine (SSR97193) in animal and human hepatic models and antimalarial activity of major metabolites on Plasmodium falciparum.

Ferroquine (SSR97193) has been shown to be a promising antimalarial, both on laboratory clones and on field isolates. So far, no resistance was documented in Plasmodium falciparum. In the present work, the metabolic pathway of ferroquine, based on experiments using animal and human hepatic models, is proposed. Ferroquine is metabolized mainly via an oxidative pathway into the major metabolite mono-N-demethyl ferroquine and then into di-N,N-demethyl ferroquine. Some other minor metabolic pathways were also identified. Cytochrome P450 isoforms 2C9, 2C19, and 3A4 and, possibly in some patients, isoform 2D6, are mainly involved in ferroquine oxidation. The metabolites were synthesized and tested against the 3D7 (chloroquine-sensitive) and W2 (chloroquine-resistant) P. falciparum strains. According to the results, the activity of the two main metabolites decreased compared with that of ferroquine; however, the activity of the mono-N-demethyl derivative is significantly higher than that of chloroquine on both strains, and the di-N-demethyl derivative remains more active than chloroquine on the chloroquine-resistant strain. These results further support the potential use of ferroquine against human malaria.

An inter-laboratory study to evaluate the effects of medium composition on the differentiation and barrier function of Caco-2 cell lines.

Differentiated human intestinal Caco-2 cells are frequently used in toxicology and pharmacology as in vitro models for studies on intestinal barrier functions. Since several discrepancies exist among the different lines and clones of Caco-2 cells, comparison of the results obtained and optimisation of models for use for regulatory purposes are particularly difficult, especially with respect to culture conditions and morphological and biochemical parameters. An inter-laboratory study has been performed on the parental cell line and on three clonal Caco-2 cell lines, with the aim of standardising the culture conditions and identifying the best cell line with respect to parameters relevant to barrier integrity, namely, trans-epithelial electrical resistance (TEER) and mannitol passage, and of epithelial differentiation (alkaline phosphatase activity). Comparison of the cell lines maintained in traditional serum-supplemented culture medium or in defined medium, containing insulin, transferrin, selenium and lipids, showed that parameter performance was better and more reproducible with the traditional medium. The maintenance of the cell lines for 15 days in culture was found to be sufficient for the development of barrier properties, but not for full epithelial differentiation. Caco-2/TC7 cells performed better than the other three cell lines, both in terms of reproducibility and performance, exhibiting low TEER and mannitol passage, and high alkaline phosphatase activity.