Enzymic, phylogenetic, and structural characterization of the unusual papain-like protease domain of Plasmodium falciparum SERA5.

Serine repeat antigen 5 (SERA5) is an abundant antigen of the human malaria parasite Plasmodium falciparum and is the most strongly expressed member of the nine-gene SERA family. It appears to be essential for the maintenance of the erythrocytic cycle, unlike a number of other members of this family, and has been implicated in parasite egress and/or erythrocyte invasion. All SERA proteins possess a central domain that has homology to papain except in the case of SERA5 (and some other SERAs), where the active site cysteine has been replaced with a serine. To investigate if this domain retains catalytic activity, we expressed, purified, and refolded a recombinant form of the SERA5 enzyme domain. This protein possessed chymotrypsin-like proteolytic activity as it processed substrates downstream of aromatic residues, and its activity was reversed by the serine protease inhibitor 3,4-diisocoumarin. Although all Plasmodium SERA enzyme domain sequences share considerable homology, phylogenetic studies revealed two distinct clusters across the genus, separated according to whether they possess an active site serine or cysteine. All Plasmodia appear to have at least one member of each group. Consistent with separate biological roles for members of these two clusters, molecular modeling studies revealed that SERA5 and SERA6 enzyme domains have dramatically different surface properties, although both have a characteristic papain-like fold, catalytic cleft, and an appropriately positioned catalytic triad. This study provides impetus for the examination of SERA5 as a target for antimalarial drug design.

First characterization of Candida albicans by random amplified polymorphic DNA method in Nicaragua and comparison of the diagnosis methods for vaginal candidiasis in Nicaraguan women

A total of 106 women with vaginitis in Nicaragua were studied. The positive rate for the identification of Candida species was 41% (44 positive cultures out of 106 women with vaginitis). The sensitivity of microscopic examination of wet mount with the potassium hydroxide (KOH) was 61% and 70% with Gram's stain when using the culture of vaginal fluid as gold standard for diagnosis of candidiasis. Among the 44 positives cultures, isolated species of yeast from vaginal swabs were C. albicans (59%), C. tropicalis (23%), C. glabrata (14%) and C. krusei (4%). This study reports the first characterization of 26 C. albicans stocks from Nicaragua by the random amplified polymorphic DNA method. The genetic analysis in this small C. albicans population showed the existence of linkage disequilibrium, which is consistent with the hypothesis that C. albicans undergoes a clonal propagation.

Structural characterization of Turtle Mountain anticline (Alberta, Canada) and impact on rock slope failure

his paper proposes a structural investigation of the Turtle Mountain anticline (Alberta, Canada) to better understand the role of the different tectonic features on the development of both local and large scale rock slope instabilities occurring in Turtle Mountain. The study area is investigated by combining remote methods with detailed field surveys. In particular, the benefit of Terrestrial Laser Scanning for ductile and brittle tectonic structure interpretations is illustrated. The proposed tectonic interpretation allows the characterization of the fracturing pattern, the fold geometry and the role of these tectonic features in rock slope instability development. Ten discontinuity sets are identified in the study area, their local variations permitting the differentiation of the study zone into 20 homogenous structural domains. The anticline is described as an eastern verging fold that displays considerable geometry differences along its axis and developed by both flexural slip and tangential longitudinal strain folding mechanisms. Moreover, the origins of the discontinuity sets are determined according to the tectonic phases affecting the region (pre-folding, folding, post-folding). The localization and interpretation of kinematics of the different instabilities revealed the importance of considering the discrete brittle planes of weakness, which largely control the kinematic release of the local instabilities, and also the rock mass damage induced by large tectonic structures (fold hinge, thrust).

Cloning and Molecular Characterization of the Schistosoma mansoni Genes RbAp48 and Histone H4

The human nuclear protein RbAp48 is a member of the tryptophan/aspartate (WD) repeat family, which binds to the retinoblastoma (Rb) protein. It also corresponds to the smallest subunit of the chromatin assembly factor and is able to bind to the helix 1 of histone H4, taking it to the DNA in replication. A cDNA homologous to the human gene RbAp48 was isolated from a Schistosoma mansoni adult worm library and named SmRbAp48. The full length sequence of SmRbAp48 cDNA is 1036 bp long, encoding a protein of 308 amino acids. The transcript of SmRbAp48 was detected in egg, cercariae and schistosomulum stages. The protein shows 84% similarity with the human RbAp48, possessing four WD repeats on its C-terminus. A hypothetical tridimensional structure for the SmRbAp48 C-terminal domain was constructed by computational molecular modeling using the b-subunit of the G protein as a model. To further verify a possible interaction between SmRbAp48 and S. mansoni histone H4, the histone H4 gene was amplified from adult worm genomic DNA using degenerated primers. The gene fragment of SmH4 is 294 bp long, encoding a protein of 98 amino acids which is 100% identical to histone H4 from Drosophila melanogaster.

Quantifying network properties in multi-electrode recordings: spatiotemporal characterization and inter-trial variation of evoked gamma oscillations in mouse somatosensory cortex in vitro.

Linking the structural connectivity of brain circuits to their cooperative dynamics and emergent functions is a central aim of neuroscience research. Graph theory has recently been applied to study the structure-function relationship of networks, where dynamical similarity of different nodes has been turned into a "static" functional connection. However, the capability of the brain to adapt, learn and process external stimuli requires a constant dynamical functional rewiring between circuitries and cell assemblies. Hence, we must capture the changes of network functional connectivity over time. Multi-electrode array data present a unique challenge within this framework. We study the dynamics of gamma oscillations in acute slices of the somatosensory cortex from juvenile mice recorded by planar multi-electrode arrays. Bursts of gamma oscillatory activity lasting a few hundred milliseconds could be initiated only by brief trains of electrical stimulations applied at the deepest cortical layers and simultaneously delivered at multiple locations. Local field potentials were used to study the spatio-temporal properties and the instantaneous synchronization profile of the gamma oscillatory activity, combined with current source density (CSD) analysis. Pair-wise differences in the oscillation phase were used to determine the presence of instantaneous synchronization between the different sites of the circuitry during the oscillatory period. Despite variation in the duration of the oscillatory response over successive trials, they showed a constant average power, suggesting that the rate of expenditure of energy during the gamma bursts is consistent across repeated stimulations. Within each gamma burst, the functional connectivity map reflected the columnar organization of the neocortex. Over successive trials, an apparently random rearrangement of the functional connectivity was observed, with a more stable columnar than horizontal organization. This work reveals new features of evoked gamma oscillations in developing cortex.

Characterization of root apoplastic barriers in Arabidopsis thaliana

In vascular plants, the best-known feature of a differentiated endodermal cell is the "Casparian Strip" (CS). This structure refers to a highly localized cell wall impregnation in the transversal and anticlinal walls of the cell, which surrounds the cell like a belt/ring and is tightly coordinated with respect to neighboring cells. Analogous to tight junctions in animal epithelia, CS in plants act as a diffusion barrier that controls the movement of water and ions from soil into the stele. Since its first description by Robert Caspary in 1865 there have been many attempts to identify the chemical nature of the cell wall deposition in CS. Suberin, lignin, or both have been claimed to be the important components of CS in a series of different species. However, the exact chemical composition of CS has remained enigmatic. This controversy was due to the confusion and lack of knowledge regarding the precise measurement of three developmental stages of the endodermis. The CS represent only the primary stage of endodermal differentiation, which is followed by the deposition of suberin lamellae all around the cellular surface of endodermal cells (secondary developmental stage). Therefore, chemical analysis of whole roots, or even of isolated endodermal tissues, will always find both of the polymers present. It was crucial to clarify this point because this will guide our efforts to understand which cell wall biosynthetic component becomes localized in order to form the CS. The main aim of my work was to find out the major components of (early) CS, as well as their spatial and temporal development, physiological roles and relationship to barrier formation. Employing the knowledge and tools that have been accumulated over the last few years in the model plant Arabidopsis thaliana, various histological and chemical assays were used in this study. A particular feature of my work was to completely degrade, or inhibit formation of lignin and suberin biopolymers by biochemical, classical genetic and molecular approaches and to investigate its effect on CS formation and the establishment of a functional diffusion barrier. Strikingly, interference with monolignol biosynthesis abrogates CS formation and delays the formation of function diffusion barrier. In contrast, transgenic plants devoid of any detectable suberin still develop a functional CS. The combination of all these assays clearly demonstrates that the early CS polymer is made from monolignol (lignin monomers) and is composed of lignin. By contrast, suberin is formed much later as a secondary wall during development of endodermis. These early CS are functionally sufficient to block extracellular diffusion and suberin does not play important role in the establishment of early endodermal diffusion barrier. Moreover, suberin biosynthetic machinery is not present at the time of CS formation. Our study finally concludes the long-standing debate about the chemical nature of CS and opens the door to a new approach in lignin research, specifically for the identification of the components of the CS biosynthetic pathway that mediates the localized deposition of cell walls. I also made some efforts to understand the patterning and differentiation of endodermal passage cells in young roots. In the literature, passage cells are defined as a non- suberized xylem pole associated endodermal cells. Since these cells only contain the CS but not the suberin lamellae, it has been assumed that these cells may offer a continued low-resistance pathway for water and minerals into the stele. Thus far, no genes have been found to be expressed specifically in passage cells. In order to understand the patterning, differentiation, and physiological role of passage it would be crucial to identify some genes that are exclusively expressed in these cells. In order to identify such genes, I first generated fluorescent marker lines of stele-expressed transporters that have been reported to be expressed in the passage cells. My aim was to first highlight the passage cells in a non-specific way. In order to find passage cell specific genes I then adapted a two-component system based on previously published methods for gene expression profiling of individual cell types. This approach will allow us to target only the passage cells and then to study gene expression specifically in this cell type. Taken together, this preparatory work will provide an entry point to understand the formation and role of endodermal passage cells. - Chez les plantes vasculaires, la caractéristique la plus commune des cellules différentiées de l'endoderme est la présence de cadres de Caspary. Cette structure correspond à une imprégnation localisée des parties transversales et anticlinales de la paroi cellulaire. Cela donne naissance, autour de la cellule, à un anneau/cadre qui est coordonné par rapport aux cellules voisines. De manière analogue aux jonctions serrées des épithéliums chez les animaux, les cadres de Caspary agissent chez les plantes comme barrière de diffusion, contrôlant le mouvement de l'eau et des ions à travers la racine entre le sol et la stèle. Depuis leur première description par Robert Caspary en 1865, beaucoup de tentatives ont eu pour but de définir la nature chimique de ces cadres de Caspary. Après l'étude de différentes espèces végétales, à la fois la subérine, la lignine ou les deux ont été revendiquées comme étant des composants importants de ces cadres. Malgré tout, leur nature chimique exacte est restée longtemps énigmatique. Cette controverse provient de la confusion et du manque de connaissance concernant la détermination précise des trois stades de développement de l'endoderme. Les cadres de Caspary représentent uniquement le stade primaire de différentiation de l'endoderme. Celui-ci est suivi par le second stade de différentiation, la déposition de lamelles de subérine tout autour de la cellule endodermal. De ce fait, l'analyse chimique de racines entières ou de cellules d'endoderme isolées ne permet pas de séparer les stades de différentiation primaire et secondaire et aboutit donc à la présence des deux polymères. Il est également crucial de clarifier ce point dans le but de connaître quelle machinerie cellulaire localisée à la paroi cellulaire permet l'élaboration des cadres de Caspary. En utilisant les connaissances et les outils accumulés récemment grâce à la plante modèle Arabidopsis thaliana, divers techniques histologiques et chimiques ont été utilisées dans cette étude. Un point particulier de mon travail a été de dégrader ou d'inhiber complètement la formation de lignine ou de subérine en utilisant des approches de génétique classique ou moléculaire. Le but étant d'observer l'effet de l'absence d'un de ces deux polymères sur la formation des cadres de Caspary et l'établissement d'une barrière de diffusion fonctionnelle. De manière frappante, le fait d'interférer avec la voie de biosynthèse de monolignol (monomères de lignine) abolit la formation des cadres de Caspary et retarde l'élaboration d'une barrière de diffusion fonctionnelle. Par contre, des plantes transgéniques dépourvues d'une quantité détectable de subérine sont quant à elles toujours capables de développer des cadres de Caspary fonctionnels. Mises en commun, ces expériences démontrent que le polymère formant les cadres de Caspary dans la partie jeune de la racine est fait de monolignol, et que de ce fait il s'agit de lignine. La subérine, quant à elle, est formée bien plus tard durant le développement de l'endoderme, de plus il s'agit d'une modification de la paroi secondaire. Ces cadres de Caspary précoces faits de lignine suffisent donc à bloquer la diffusion extracellulaire, contrairement à la subérine. De plus, la machinerie de biosynthèse de la subérine n'est pas encore présente au moment de la formation des cadres de Caspary. Notre étude permet donc de mettre un terme au long débat concernant la nature chimique des cadres de Caspary. De plus, elle ouvre la porte à de nouvelles approches dans la recherche sur la lignine, plus particulièrement pour identifier des composants permettant la déposition localisée de ce polymère dans la paroi cellulaire. J'ai aussi fais des efforts pour mettre en évidence la formation ainsi que le rôle des cellules de passage dans les jeunes racines. Dans la littérature, les cellules de passage sont définies comme de la cellule endodermal faisant face aux pôles xylèmes et dont la paroi n'est pas subérisée. Du fait que ces cellules contiennent uniquement des cadres de Caspary et pas de lamelle de subérine, il a été supposé qu'elles ne devraient offrir que peu de résistance au passage de l'eau et des nutriments entre le sol et la stèle. Le rôle de ces cellules de passage est toujours loin d'être clair, de plus aucun gène s'exprimant spécifiquement dans ces cellules n'a été découvert à ce jour. De manière à identifier de tels gènes, j'ai tout d'abord généré des marqueurs fluorescents pour des transporteurs exprimés dans la stèle mais dont l'expression avait également été signalée dans l'endoderme, uniquement dans les cellules de passage. J'ai ensuite développé un système à deux composants basé sur des méthodes déjà publiées, visant principalement à étudier le profil d'expression génique dans un type cellulaire donné. En recoupant les gènes exprimés spécifiquement dans l'endoderme à ceux exprimés dans la stèle et les cellules de passage, il nous sera possible d'identifier le transriptome spécifique de ces cellules. Pris dans leur ensemble, ces résultats devraient donner un bon point d'entrée dans la définition et la compréhension des cellules de passage.

Trypanosoma cruzi ribosomal protein S4: characterization of its coding locus, analysis of transcripts, and antigenicity of the protein

Two allelic genomic fragments containing ribosomal protein S4 encoding genes (rpS4) from Trypanosoma cruzi (CL-Brener strain) were isolated and characterized. One allele comprises two complete tandem repeats of a sequence encoding an rpS4 gene. In the other, only one rpS4 gene is found. Sequence comparison to the accessed data in the genome project database reveals that our two-copy allele corresponds to a variant haplotype. However, the deduced aminoacid sequence of all the gene copies is identical. The rpS4 transcripts processing sites were determined by comparison of genomic sequences with published cDNA data. The obtained sequence data demonstrates that rpS4 genes are expressed in epimastigotes, amastigotes, and trypomastigotes. A recombinant version of rpS4 was found to be an antigenic: it was recognized by 62.5% of the individuals with positive serology for T. cruzi and by 93.3% of patients with proven chronic chagasic disease.

Characterization and monitoring of the Åknes landslide using terrestrial laser scanning

Terrestrial laser scanning (TLS) provides high-resolution point clouds of the topography and new TLS instruments with ranges exceeding 300 m or even 1000 m are powerful tools for characterizing and monitoring slope movements. This study focuses on the 35 million m3 Åknes rockslide in Western Norway, which is one of the most investigated and monitored rockslides in the world. The TLS point clouds are used for the structural analysis of the steep, inaccessible main scarp of the rockslide, including an assessment of the discontinuity sets and fold axes. TLS acquisitions in 2006, 2007 and 2008 provide information on 3-D displacements for the entire scanned area and are not restricted like conventional survey instruments to single measurement points. The affine transformation matrix between two TLS acquisitions precisely describes the rockslide displacements and enables their separation into translational components, such as the displacement velocity and direction, and rotational components, like toppling. This study shows the ability of TLS to obtain reliable 3-D displacement information over a large, unstable area. Finally, a possible instability model for the upper part of Åknes rockslide explains the measured translational and rotational displacements by a combination of southward planar sliding along the gneiss foliation, gravitational vertical settlement along the complex, stepped basal sliding surface and northward toppling toward the opened graben structure.

Characterization of the 3-HKT gene in important malaria vectors in India, viz: Anopheles culicifacies and Anopheles stephensi (Diptera: Culicidae)

The 3-hydroxykynurenine transaminase (3-HKT) gene plays a vital role in the development of malaria parasites by participating in the synthesis of xanthurenic acid, which is involved in the exflagellation of microgametocytes in the midgut of malaria vector species. The 3-HKT enzyme is involved in the tryptophan metabolism of Anophelines. The gene had been studied in the important global malaria vector, Anopheles gambiae. In this report, we have conducted a preliminary investigation to characterize this gene in the two important vector species of malaria in India, Anopheles culicifacies and Anopheles stephensi. The analysis of the genetic structure of this gene in these species revealed high homology with the An. gambiae gene. However, four non-synonymous mutations in An. stephensi and seven in An. culicifacies sequences were noted in the exons 1 and 2 of the gene; the implication of these mutations on enzyme structure remains to be explored.

Molecular dissection of Arabidopsis thaliana endodermis development, structure and function

In vascular plants, the endodermis establishes a protective diffusion barrier surrounding the vasculature preventing the passive, uncontrolled entry of nutrients absorbed by the plant. It does so by means of a differentiation feature, the "Casparian Strip" (CS), a highly localized cell wall impregnation made of lignin, which seals the extracellular space. Although the existence of this differentiation feature has been intensively described, the mechanisms establishing this hallmark remain obscure. In this work I report, the developmental sequence of events that leads to a differentiated endodermis, in the plant model Arabidopsis thaliana. In addition, my descriptive approach gave important insights as to how these cells define membrane domains involved in the directional transport of nutrients. I also participated in characterizing a new transmembrane protein family, the CASPs, localized to the membrane domain underlying the CS, which we accordingly named the Casparian Strip membrane Domain (CSD). Our molecular analysis indicates that these proteins drive CS establishment. To identify more molecular factors of CS establishment, I performed a forward genetic screen. This screen led to the identification of 11 endodermis permissive mutants, which we named schengen (sgn) mutants. The causative mutations have been mapped to 5 independent loci: SGN1 to SGN5. SGN1 and SGN3 encode Receptor Like Kinases involved in the correct establishment of the CSD. A lack of those kinases leads to an incomplete CSD, which gives rise to interrupted CS barriers. Interestingly, SGN1 seems to also regulate CSD positioning to the middle of endodermal transversal walls. SGN4 encodes an NADPH oxidase involved in lignin polymerization essential for CS formation. The sgn5 mutant induces extra divisions of cortical cells strongly affecting the cell identity, but also leading to incorrect differentiation. A thorough characterization of the sgn2 mutant will follow elsewhere, yet preliminary results indicate that SGN2 encodes an Acyl-CoA N-acyltransferase. . In summary, with my work I have contributed a first set of molecular players of Casparian strip formation and initiated their characterization. Eventually, this might lead to an understanding of the molecular mechanisms of CS establishment in A.thaliana . This in turn will hopefully help to better understand nutrient uptake in higher plants and their response to environmental stresses. - Au sein des plantes vasculaires, l'endoderme représente un tissu protecteur mettant en place une barrière imperméable, empêchant n'importe quel élément de rejoindre les tissus conducteurs par simple diffusion. Cette barrière, appelée « Cadre de Caspary », correspond à une lignification de la paroi de l'endoderme et donne lieu à un cloisonnement de l'espace intercellulaire. Bien que cet élément de différenciation soit décrit en détail, sa mise en place reste incomprise. Cette étude indique la suite d'événements aboutissant à l'établissement du cadre de Caspary chez la plante modèle Arabidopsis thaliana. De plus, ce travail apporte de nouvelles connaissances expliquant comment ces cellules définissent des domaines membranaires importants pour le transport des nutriments. Nous décrivons une nouvelle famille de protéines membranaires, les CASPs (« CAparian Strip membrane domain Proteins »), localisées dans un domaine membranaire longeant le cadre de Caspary : le domaine de Caspary (CSD). L'analyse moléculaire des CASPs indique qu'elles dirigent la formation du cadre de Caspary. Par ailleurs, une approche génétique directe nous a permis d'identifier 11 mutants ayant un endoderme perméable. Nous avons nommé ces mutants Schengen, en référence à la zone de libre échange européenne. Les mutations impliquées dans ces mutants affectent 5 gènes désignés de SGN1 à SGN5. SGN1 et SGN3 produisent des protéines de type kinases (« Receptor-like Kinases », RLK) qui participent à la délimitation du CSD. L'absence de ces kinases aboutit à un domaine CSD incomplet, se traduisant par un cadre de Caspary discontinu. De plus, SGN1 semble réguler le positionnement du CSD au milieu de la paroi transversale de l'endoderme. SGN4 produit une enzyme de type NADPH oxydase impliquée dans la polymérisation du cadre de Caspary. Dans le mutant sgn5, on observe une division anormale des cellules du cortex créant ainsi une nouvelle couche cellulaire incapable d'achever sa différenciation en endoderme. Quant à la mutation sgn2, bien que nous pensons qu'elle affecte une Acyl-CoA N-acyltransferase, sa caractérisation ne sera réalisée que prochainement. Au final, ce travail procure de nouveaux éléments sur l'établissement du cadre de Caspary qui pourraient être importants afin de comprendre comment les plantes sélectionnent leurs nutriments et résistent à des conditions environnementales parfois hostiles. - De par leur immobilité, les plantes terrestres n'ont pas d'autre choix que de puiser leurs ressources dans leur environnement direct. La plante extrait du sol les nutriments qui lui sont nécessaires et les redistribue grâce à des tissus conducteurs. Afin de ne pas s'intoxiquer, il est donc essentiel de pouvoir sélectionner les éléments entrant dans la racine. Etonnement, ce n'est pas la surface des racines qui permet ce contrôle mais un tissu interne appelé endoderme. Ce dernier forme une barrière imperméable qui entoure chaque cellule et crée une jointure permettant de bloquer le passage des éléments entre les cellules. Cette structure, appelée « cadre de Caspary », oblige les éléments à entrer dans les cellules de l'endoderme et à être ainsi sélectionnés. Bien que cette structure soit décrite en détail, sa mise en place reste incomprise. Cette étude indique la suite d'événements qui aboutit à la formation du cadre de Caspary chez la plante modèle Arabidopsis thaliana. Ce travail apporte également de nouvelles connaissances expliquant comment ces cellules définissent, organisent et dirigent le transport des nutriments. Nous décrivons comment certains éléments de la cellule, les protéines CASPs (CAsparian Strip membrane domain Proteins), sont organisées un domaine particulier des membranes afin de créer une plateforme de construction longeant le cadre de Caspary : le domaine de Caspary (CSD). Afin de déterminer ce qu'il se passerait si une plante ne possédait pas de cadre de Caspary, nous avons réalisé une mutagénèse, ou approche génétique directe, et identifié 11 mutants (individu ayant un gène défectueux conduisant à la perte d'une fonction) ayant un endoderme perméable. Nous avons nommé ces mutants schengen, en référence à la zone de libre échange européenne. Les mutations impliquées dans ces mutants affectent 5 gènes désignés de SGN1 à SGN5. Les gènes SGN1 et SGN3 produisent des protéines de type kinases (« Receptor-like Kinases », RLK) servant à l'établissement de la plateforme de construction. L'absence de ces kinases aboutit à une base incomplète, se traduisant par un cadre de Caspary discontinu. Qui plus est, la kinase SGN1 semble réguler le positionnement de la plateforme au milieu de l'endoderme. Le gène SGN4 est par contre, impliqué dans la construction à proprement dite du cadre de Caspary. Dans le mutant sgn5, on observe une nouvelle couche de cellules ressemblant à de l'endoderme mais incapable de former correctement une barrière identique au cadre de Caspary. Quant au dernier mutant, sgn2, bien que cette étude fournisse des indices permettant de comprendre pourquoi le mutant sgn2 est défectueux, nous n'expliquerons ce cas que prochainement. En résumé, ce travail procure de nouvelles connaissances sur l'établissement du cadre de Caspary qui pourraient être importantes afin de comprendre comment les plantes sélectionnent leurs nutriments et résistent à des conditions environnementales parfois hostiles.

Characterization and binding affinities of SmLANP: a new Schistosoma mansoni member of the ANP32 family of regulatory proteins.

Members of the leucine-rich repeat protein family are involved in diverse functions including protein phosphatase 2-inhibition, cell cycle regulation, gene regulation and signalling pathways. A novel Schistosoma mansoni gene, called SmLANP, presenting homology to various genes coding for proteins that belong to the super family of leucine-rich repeat proteins, was characterized here. SmLANP was 1184bp in length as determined from cDNA and genomic sequences and encoded a 296 amino acid open reading frame that spanning from 6 to 894bp. The predicted amino acid sequence had a calculated molecular weight of 32kDa. Analysis of the predicted sequence indicated the presence of 3 leucine-rich domains (LRR) located in the N-terminal region and an aspartic acid rich region in the C-terminal end. SmLANP transcript is expressed in all stages of the S. mansoni life cycle analyzed, exhibiting the highest expression level in males. The SmLANP protein was expressed in a GST expression system and antibodies raised in mice against the recombinant protein. By immunolocalization assay, using adult worms, it was shown that the protein is mainly present in the cell nucleus through the whole body and strongly expressed along the tegument cell body nuclei of adult worms. As members of this family are usually involved in protein-protein interaction, a yeast two hybrid assay was conducted to identify putative binding partners for SmLANP. Thirty-six possible partners were identified, and a protein ATP synthase subunit alpha was confirmed by pull down assays, as a binding partner of the SmLANP protein.

Growth and characterization of CoO ultra thin films

In this report we present the growth process of the cobalt oxide system using reactive electron beam deposition. In that technique, a target of metallic cobalt is evaporated and its atoms are in-flight oxidized in an oxygen rich reactive atmosphere before reaching the surface of the substrate. With a trial and error procedure the deposition parameters have been optimized to obtain the correct stoichiometry and crystalline phase. The evaporation conditions to achieve the correct cobalt oxide salt rock structure, when evaporating over amorphous silicon nitride, are: 525 K of substrate temperature, 2.5·10-4 mbar of oxygen partial pressure and 1 Å/s of evaporation rate. Once the parameters were optimized a set of ultra thin film ranging from samples of 1 nm of nominal thickness to 20nm thick and bulk samples were grown. With the aim to characterize the samples and study their microstructure and morphology, X-ray diffraction, transmission electron microscopy, electron diffraction, energy dispersive X-ray spectroscopy and quasi-adiabatic nanocalorimetry techniques are utilised. The final results show a size dependent effect of the antiferromagnetic transition. Its Néel temperature becomes depressed as the size of the grains forming the layer decreases.

Biochemical characterization of recombinant human telomerase

Résumé Les télomères sont les structures ADN-protéines des extrémités des chromosomes des eucaryotes. L'ADN télomérique est constitué de courtes séquences répétitives. L'intégrité des télomères est essentielle pour protéger les extrémités des chromosomes contre les systèmes de dégradations et pour les distinguer des cassures de l'ADN double brin. Parce que la machinerie de la réplication de l'ADN n'est pas capable de répliquer l'extrémité des chromosomes, les télomères raccourcissent au fur et à mesure des cycles de réplication. Dès que les télomères atteignent une longueur critique, leur structure protectrice est perdue. Cela induit un signal de dommage de l'ADN et l'arrêt du cycle cellulaire. Pour contrebalancer le raccourcissement des télomères, les cellules qui s'auto régénèrent, dont les cellules de la moelle osseuse, les lymphocytes activés et 80-90% des cellules cancéreuses, expriment la télomérase. C'est une ribonucléoprotéine qui a la capacité de synthétiser des séquences télomériques par transcription inverse d'une courte séquence contenue dans sa propre sous-unité ARN avec laquelle elle est associée. La télomérase humaine est une enzyme processive au niveau de l'addition des nucléotides et aussi des répétitions télomériques. La télomérase de levure et la télomérase humaine sont toutes deux dimériques et il a été montré que la télomérase humaine recombinante contient deux ARN qui coopèrent pour fonctionner ainsi que deux sous-unités catalytiques. Cependant, il n'a pas encore été montré quel est le rôle de la dimérisation dans l'activité de la télomérase. Afin d'élucider ce rôle, nous avons exprimé, reconstitué et purifié la télomérase humaine dimérique recombinante. Et pour étudier l'effet d'ARN mutants sur l'activité de la télomérase, nous avons développé une méthode pour reconstituer et enrichir en hétérodimères de télomérase. Les hétérodimères contiennent une sous-unité ARN sauvage et une sous-unité ARN mutée au niveau de la séquence de la matrice. Sur l'ARN muté nous avons introduit une étiquette aptamer ARN-S1 puis nous avons purifié la télomérase via l'etiquette Si. Nous avons montré que la dimérisation est essentielle pour l'activité de la télomérase. Nos données indiquent que chaque télomérase du dimère allonge leur substrat, l'ADN télomérique, indépendamment l'une de l'autre à chaque cycle d'élongation mais que l'addition itérative de répétitions télomériques nécessite une coopération entre les deux télomérases du dimère. Nous proposons donc un modèle dans lequel les deux télomérases du dimères se lient et allongent deux substrats télomères et que pendant l'élongation processive les deux enzymes subissent un changement de conformation de manière coordonnée, ce changement va permettre le repositionnement des substrats pour d'autres cycles d'additions de répétitions télomériques. Dyskeratosis congenita est une maladie mortelle due majoritairement au disfonctionnement de la moelle osseuse. Dans la forme autosomale de la maladie, l'ARN de la télomérase contient des mutations. En utilisant notre système de reconstitution, nous avons montré que ces ARN mutés, qui ont perdu leur activité enzymatique dans le cas d'un homodimère de mutants, sont dominant négatifs quand ils sont présents dans les hétérodimères sauvage/mutant. Cet effet trans-dominant négatif pourrait contribuer à la progression de la maladie. Abstract Telomeres are protein-DNA structures at the ends of linear eukaryotic chromosomes. The telomeric DNA consists of tandemly repeated sequences. Telomeric integrity is essential to protect chromosomal ends from nucleolytic degradation and to prevent their recognition as DNA double strand breaks. Due to the inability of the conventional DNA replication machinery to replicate terminal DNA stretches, telomeres shorten with continuous rounds of DNA replication. As soon as telomeres reach a critical length, their protective structure is lost and the deprotected telomeres will induce a DNA damage response leading to cell cycle arrest. To counteract telomere shortening, self-renewing cells, including bone marrow cells, activated lymphocytes and 80-90% of cancer cells express the cellular reverse transcriptase telomerase, which has the capacity to synthesize telomeric repeats by reverse transcription of a short template sequence encoded by its stably associated RNA subunit. Human telomerase is a processive enzyme for nucleotide as well as repeat addition. Both yeast and human telomerase are dimeric enzymes and recombinant human telomerase has been shown to contain two functionally cooperating RNAs and most probably also two protein subunits. However, it has remained unclear how dimerization may contribute to telomerase activity. To study the role of dimerization, we expressed, reconstituted and purified recombinant human telomerase. We also developed a new method to reconstitute and enrich for telomerase heterodimers containing wild-type (wt) and mutant telomerase RNA subunits. To this end we introduced an S1-RNA-aptamer tag into telomerase RNA and purified telomerase reconstituted with a mixture of untagged and tagged RNA via the S1-tag. Using this experimental system, we introduced template mutations in the tagged RNA subunit and examined the effect of mutant RNAs on wt telomerase activity in wt/mutant heterodimers. We obtained evidence that dimerization is essential for telomerase activity. Our data indicate that the two subunits elongate telomere substrates independently of each other during single rounds of elongation, but that iterative addition of telomeric repeats requires cooperation between the two subunits. We suggest a model, in which dimeric telomerases bind and elongate two telomere substrates and that the two subunits undergo coordinated conformational changes during processive elongation that enable repositioning the substrates for subsequent rounds of repeat addition. Dyskeratosis congenita is a multisystemic disease with bone marrow failure as the major cause of death. The autosomal form of this disease was found to harbor mutations in the telomerase RNA. Using our reconstitution system, we tested whether mutant dyskeratosis telomerase RNAs behaved in a dominant negative manner. We observed that dyskeratosis telomerase RNA mutants, which lacked enzymatic activity were dominant negative, when present in wt/ mutant heterodimers. The transdominant negative effect of these mutants may contribute to disease progression.

Characterization of a plant-derived peptide displaying water clarifying and antimicrobial activities

SUMMARY Drinking water is currently a scarce world resource, the preparation of which requires complex treatments that include clarification of suspended particles and disinfection. Seed extracts of Moringa oleifera Lam., a tropical tree, have been proposed as an environment- friendly alternative, due to their traditional use for the clarification of drinking water. However, the precise nature of the active components was unknown. Here, we show that recombinant or synthetic forms of a cationic seed polypeptide mediate efficient sedimentation of suspended mineral particles and bacteria. Unexpectedly, the polypeptide was also found to possesses a bactericidal activity capable of disinfecting heavily contaminated water. Furthermore, the polypeptide has been shown to efficiently kill several pathogenic bacteria, including antibiotic-resistant isolates of Pseudomona, Streptococcus and Legionella species. Structural modeling of the peptide coupled to the functional analysis of synthetic peptide derivatives delineated distinct structural determinants for the flocculation and antibacterial activities. Our results suggest that a glutamine-rich portion of the polypeptide is involved in the sedimentation process; alternatively, the antibacterial activity depends on a amphiphilic loop. Assembly of multiple copies of this loop into a branched peptide derivative strongly enhances antibacterial activity without displaying hemolytic effect. In conclusion, this polypeptide displays the unprecedented feature of combining efficient water purification and disinfectant properties indicating different molecular mechanisms involved in each case. This work not only identified the features responsible for these activities but also provides useful information that has implications for the further development of this cationic polypeptide as a potent antibacterial agent. RESUME L'eau potable est actuellement une ressource limitée dans le monde. La production d'eau propre à la consommation exige des traitements complexes, incluant la clarification des particules en suspension ainsi que sa désinfection par des additifs chimiques. Les extraits de la graine d'un arbre tropical, Moringa oleifera, sont utilisés traditionnellement en Afrique afin de clarifier l'eau. Quoique la nature exacte des composants actifs était inconnue, on a pu mettre en évidence un polypeptide cationique contenu dans ces graines, capable de sédimenter de manière efficace des particules minérales en suspension ainsi que des bactéries. Ce travail a aussi mis en évidence que ce polypeptide a une activité bactéricide, permettant une désinfection d'eau fortement contaminée. De plus, nous avons démontré que ce polypeptide est efficace contre de nombreuses souches bactériennes pathogènes, également celles résistantes aux antibiotiques comme Pseudomonas, Streptococcus et Legionella. L'analyse de la structure moléculaire de ce polypeptide, couplée à son analyse fonctionnelle a mis en évidence deux domaines structuraux distinct, un pour l'activité de floculation et l'autre pour l'activité antibactérienne. Nos résultats suggèrent que le domaine riche en glutamine est impliqué dans le processus de sédimentation et que l'activité antimicrobienne dépend d'un domaine formant une boucle amphiphilique. En ramifiant plusieurs copies de cette boucle on a pu augmenter de manière significative l'activité antibactérienne. En conclusion, nous avons pu démontrer que ce polypeptide à la capacité unique de combiner des propriétés de purification et de désinfection de l'eau, ce qui implique des mécanismes moléculaires distincts pour ces deux activités. Ce travail a permis d'identifier les domaines du polypeptide qui sont responsables de ses activités et offre une perspective pour le développement d'un nouvel agent antimicrobien.

Characterization of alkaline lignins for use in penol-formaldehyde and epoxy resins

Besides polyurethanes and polyesters, phenolic and epoxy resins are the most prominent applications for technical lignins in thermosetting materials. To evaluate the potential application of lignin raw materials in phenol formaldehyde and epoxy resins, three types of alkaline lignins were characterized in terms of their structures and thermal properties. The lignin samples analyzed were kraft lignin (LIG-1), soda–rice straw lignin (LIG-2), and soda-wheat straw lignin (LIG-3). FTIR and 1H-NMR methods were used to determine their structure. Gel permeation chromatography (GPC) was used to determine the molecular weight distribution (MWD). Differential scanning calorimetry (DSC) was used to measure the glass transition temperature (Tg), and thermogravimetric analysis (TGA) to determine the thermal stability of lignin samples. Results showed that kraft lignin (LIG-1) has moderate hydroxyl-group content, is rich in G-type units, and has good thermal stability. These properties make it more suitable for direct use in phenol formaldehyde resins, and it is therefore a good raw material for this purpose. The alkaline soda-rice straw lignin (LIG-2) with a high hydroxyl-group content and excellent thermal stability is most suited to preparing lignin-based epoxy resin