Expression of a functional single chain antibody on the surface of extracellular enveloped vaccinia virus as a step towards selective tumour cell targeting.

Recombinant vaccinia virus with tumour cell specificity may provide a versatile tool either for direct lysis of cancer cells or for the targeted transfer of genes encoding immunomodulatory molecules. We report the expression of a single chain antibody on the surface of extracellular enveloped vaccinia virus. The wild-type haemagglutinin, an envelope glycoprotein which is not required for viral infection and replication, was replaced by haemagglutinin fusion molecules carrying a single chain antibody directed against the tumour-associated antigen ErbB2. ErbB2 is an epidermal growth factor receptor-related tyrosine kinase overexpressed in a high percentage of human adenocarcinomas. Two fusion proteins carrying the single chain antibody at different NH2-terminal positions were expressed and exposed at the envelope of the corresponding recombinant viruses. The construct containing the antibody at the site of the immunoglobulin-like loop of the haemagglutinin was able to bind solubilized ErbB2. This is the first report of replacement of a vaccinia virus envelope protein by a specific recognition structure and represents a first step towards modifying the host cell tropism of the virus.

Cell surface expression of the epithelial Na channel and a mutant causing Liddle syndrome: a quantitative approach.

The epithelial amiloride-sensitive sodium channel (ENaC) controls transepithelial Na+ movement in Na(+)-transporting epithelia and is associated with Liddle syndrome, an autosomal dominant form of salt-sensitive hypertension. Detailed analysis of ENaC channel properties and the functional consequences of mutations causing Liddle syndrome has been, so far, limited by lack of a method allowing specific and quantitative detection of cell-surface-expressed ENaC. We have developed a quantitative assay based on the binding of 125I-labeled M2 anti-FLAG monoclonal antibody (M2Ab*) directed against a FLAG reporter epitope introduced in the extracellular loop of each of the alpha, beta, and gamma ENaC subunits. Insertion of the FLAG epitope into ENaC sequences did not change its functional and pharmacological properties. The binding specificity and affinity (Kd = 3 nM) allowed us to correlate in individual Xenopus oocytes the macroscopic amiloride-sensitive sodium current (INa) with the number of ENaC wild-type and mutant subunits expressed at the cell surface. These experiments demonstrate that: (i) only heteromultimeric channels made of alpha, beta, and gamma ENaC subunits are maximally and efficiently expressed at the cell surface; (ii) the overall ENaC open probability is one order of magnitude lower than previously observed in single-channel recordings; (iii) the mutation causing Liddle syndrome (beta R564stop) enhances channel activity by two mechanisms, i.e., by increasing ENaC cell surface expression and by changing channel open probability. This quantitative approach provides new insights on the molecular mechanisms underlying one form of salt-sensitive hypertension.

Mutational analysis of cysteine-rich domains of the epithelium sodium channel (ENaC). Identification of cysteines essential for channel expression at the cell surface.

One of the characteristic features of the structure of the epithelial sodium channel family (ENaC) is the presence of two highly conserved cysteine-rich domains (CRD1 and CRD2) in the large extracellular loops of the proteins. We have studied the role of CRDs in the functional expression of rat alphabetagamma ENaC subunits by systematically mutating cysteine residues (singly or in combinations) into either serine or alanine. In the Xenopus oocyte expression system, mutations of two cysteines in CRD1 of alpha, beta, or gamma ENaC subunits led to a temperature-dependent inactivation of the channel. In CRD1, one of the cysteines of the rat alphaENaC subunit (Cys158) is homologous to Cys133 of the corresponding human subunit causing, when mutated to tyrosine (C133Y), pseudohypoaldosteronism type 1, a severe salt-loosing syndrome in neonates. In CRD2, mutation of two cysteines in alpha and beta but not in the gamma subunit also produced a temperature-dependent inactivation of the channel. The main features of the mutant cysteine channels are: (i) a decrease in cell surface expression of channel molecules that parallels the decrease in channel activity and (ii) a normal assembly or rate of degradation as assessed by nondenaturing co-immunoprecipitation of [35S]methionine-labeled channel protein. These data indicate that the two cysteines in CRD1 and CRD2 are not a prerequisite for subunit assembly and/or intrinsic channel activity. We propose that they play an essential role in the efficient transport of assembled channels to the plasma membrane.

Red blood cell structure and dynamics explored with digital holographic microspcopy

Digital holographic microscopy (DHM) is a technique that allows obtaining, from a single recorded hologram, quantitative phase image of living cell with interferometric accuracy. Specifically the optical phase shift induced by the specimen on the transmitted wave front can be regarded as a powerful endogenous contrast agent, depending on both the thickness and the refractive index of the sample. Thanks to a decoupling procedure cell thickness and intracellular refractive index can be measured separately. Consequently, Mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC), two highly relevant clinical parameters, have been measured non-invasively at a single cell level. The DHM nanometric axial and microsecond temporal sensitivities have permitted to measure the red blood cell membrane fluctuations (CMF) on the whole cell surface. ©2009 COPYRIGHT SPIE--The International Society for Optical Engineering.

GLUT2 surface expression and intracellular transport via the constitutive pathway in pancreatic beta cells and insulinoma: evidence for a block in trans-Golgi network exit by brefeldin A.

The biosynthesis, intracellular transport, and surface expression of the beta cell glucose transporter GLUT2 was investigated in isolated islets and insulinoma cells. Using a trypsin sensitivity assay to measure cell surface expression, we determined that: (a) greater than 95% of GLUT2 was expressed on the plasma membrane; (b) GLUT2 did not recycle in intracellular vesicles; and (c) after trypsin treatment, reexpression of the intact transporter occurred with a t1/2 of approximately 7 h. Kinetics of intracellular transport of GLUT2 was investigated in pulse-labeling experiments combined with glycosidase treatment and the trypsin sensitivity assay. We determined that transport from the endoplasmic reticulum to the trans-Golgi network (TGN) occurred with a t1/2 of 15 min and that transport from the TGN to the plasma membrane required a similar half-time. When added at the start of a pulse-labeling experiment, brefeldin A prevented exit of GLUT2 from the endoplasmic reticulum. When the transporter was first accumulated in the TGN during a 15-min period of chase, but not following a low temperature (22 degrees C) incubation, addition of brefeldin A (BFA) prevented subsequent surface expression of the transporter. This indicated that brefeldin A prevented GLUT2 exit from the TGN by acting at a site proximal to the 22 degrees C block. Together, these data demonstrate that GLUT2 surface expression in beta cells is via the constitutive pathway, that transport can be blocked by BFA at two distinct steps and that once on the surface, GLUT2 does not recycle in intracellular vesicles.

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.

The cover basement contact beneath the Rawil axial depression (Western Alps) - True amplitude seismic processing, petrophysical properties, and modeling

Since 1986, several near-vertical seismic reflection profiles have been recorded in Switzerland in order to map the deep geologic structure of the Alps. One objective of this endeavour has been to determine the geometries of the autochthonous basement and of the external crystalline massifs, important elements for understanding the geodynamics of the Alpine orogeny. The PNR-20 seismic line W1, located in the Rawil depression of the western Swiss Alps, provides important information on this subject. It extends northward from the `'Penninic front'' across the Helvetic nappes to the Prealps. The crystalline massifs do not outcrop along this profile. Thus, the interpretation of `'near-basement'' reflections has to be constrained by down-dip projections of surface geology, `'true amplitude'' processing, rock physical property studies and modelling. 3-D seismic modelling has been used to evaluate the seismic response of two alternative down-dip projection models. To constrain the interpretation in the southern part of the profile, `'true amplitude'' processing has provided information on the strength of the reflections. Density and velocity measurements on core samples collected up-dip from the region of the seismic line have been used to evaluate reflection coefficients of typical lithologic boundaries in the region. The cover-basement contact itself is not a source of strong reflections, but strong reflections arise from within the overlaying metasedimentary cover sequence, allowing the geometry of the top of the basement to be determined on the basis of `'near-basement'' reflections. The front of the external crystalline massifs is shown to extend beneath the Prealps, about 6 km north of the expected position. A 2-D model whose seismic response shows reflection patterns very similar to the observed is proposed.

Detailed 3D seismic imaging of a fault zone beneath Lake Geneva, Switzerland

An efficient high-resolution (HR) three-dimensional (3D) seismic reflection system for small-scale targets in lacustrine settings was developed. In Lake Geneva, near the city of Lausanne, Switzerland, the offshore extension of a complex fault zone well mapped on land was chosen for testing our system. A preliminary two-dimensional seismic survey indicated structures that include a thin (<40 m) layer of subhorizontal Quaternary sediments that unconformably overlie south-east-dipping Tertiary Molasse beds and a major fault zone (Paudeze Fault Zone) that separates Plateau and Subalpine Molasse (SM) units. A 3D survey was conducted over this test site using a newly developed three-streamer system. It provided high-quality data with a penetration to depths of 300 m below the water bottom of non-aliased signal for dips up to 30degrees and with a maximum vertical resolution of 1.1 m. The data were subjected to a conventional 3D processing sequence that included post-stack time migration. Tests with 3D pre-stack depth migration showed that such techniques can be applied to HR seismic surveys. Delineation of several horizons and fault surfaces reveals the potential for small-scale geologic and tectonic interpretation in three dimensions. Five major seismic facies and their detailed 3D geometries can be distinguished. Three fault surfaces and the top of a molasse surface were mapped in 3D. Analysis of the geometry of these surfaces and their relative orientation suggests that pre-existing structures within the Plateau Molasse (PM) unit influenced later faulting between the Plateau and SM. In particular, a change in strike of the PM bed dip may indicate a fold formed by a regional stress regime, the orientation of which was different from the one responsible for the creation of the Paudeze Fault Zone. This structure might have later influenced the local stress regime and caused the curved shape of the Paudeze Fault in our surveyed area.

Use of an in-house approach to study the three-dimensional structures of various outer membrane proteins: structure of the alcaligin outer membrane transporter FauA from Bordetella pertussis.

Bordetella pertussis is the bacterial agent of whooping cough in humans. Under iron-limiting conditions, it produces the siderophore alcaligin. Released to the extracellular environment, alcaligin chelates iron, which is then taken up as a ferric alcaligin complex via the FauA outer membrane transporter. FauA belongs to a family of TonB-dependent outer membrane transporters that function using energy derived from the proton motive force. Using an in-house protocol for membrane-protein expression, purification and crystallization, FauA was crystallized in its apo form together with three other TonB-dependent transporters from different organisms. Here, the protocol used to study FauA is described and its three-dimensional structure determined at 2.3 A resolution is discussed.

Direct visualization of the trimeric structure of the ASIC1a channel, using AFM imaging.

There has been confusion about the subunit stoichiometry of the degenerin family of ion channels. Recently, a crystal structure of acid-sensing ion channel (ASIC) 1a revealed that it assembles as a trimer. Here, we used atomic force microscopy (AFM) to image unprocessed ASIC1a bound to mica. We detected a mixture of subunit monomers, dimers and trimers. In some cases, triple-subunit clusters were clearly visible, confirming the trimeric structure of the channel, and indicating that the trimer sometimes disaggregated after adhesion to the mica surface. This AFM-based technique will now enable us to determine the subunit arrangement within heteromeric ASICs.

Revisiting the heteromultimeric structure of ENaC in Xenopus leavis oocytes

The epithelial sodium channel (ENaC) regulates the sodium reabsorption in the principal cells of collecting duct of the nephron, and is essential for the maintenance of Na+ balance and blood pressure. ENaC is regulated by hormones such as aldosterone and vasopressin, by serine proteases. The functional ENaC channel expressed at the cell surface is a hetemultimeric complex composed by the homologous a, ß and y subunits. Several functional and biochemical studies have provided evidence that the ENaC is a heterotetramer formed by 2a lß and ly subunits. Recently, a channel homologue of ENaC, the acid-sensing ion channel ASIC1 has been crystallized as a homotrimer. This discrepancy in the subunit composition of these two channels of the same family, motivated us to revisit the subunit oligomerization of the purified functional abg EnaC channel complex. His(6)ENaC a ß y subunits were expressed in Xenopus leavis oocytes. The three ENaC subunits copurify on Ni+2-NTA agarose beads in a aßy ENaC complex. On Western blot, the ENaC subunits show typical post-translation modifications associated with a functional channel. Using differentially tagged ENaC subunits, we could demonstrate that 2 different a ENaC co- purify with ß and y subunits, whereas only one single ß and y are detected in the ENaC complex. Comparison of the mass of the aßy ENaC complex on Western blot under non reducing conditions with different ENaC dimeric, trimmeric and tetratemeric concatamers indicate that the ENaC channel complex is a heterotetramer made of 2a-, lß-, and ly ENaC subunits. Our result will certainly not provide the last words on the subunit stoichiometry of the ENaC/ASIC channels, but hopefully will promote the réévaluation of the cASICl crystal structure for its functional relevance. -- Le canal épithélial sodique ENaC est responsable de la réabsorption du sodium dans les cellules principales du tubule collecteur rénal et joue un rôle important dans le maintien de l'homéostasie sodique et le maintien de la pression artérielle. Ce canal est régulé par des hormones telles que l'aldostérone ou la Vasopressine mais également par des sérines protéases. ENaC est un canal multimerique constitué des trois sous-unités homologues a, ß and y. De nombreuses études fonctionnelles et biochimiques ont montré que le canal ENaC fonctionnel exprimé à la surface cellulaire est un canal formé de 4 sous unités avec une stoichiometric préférentielle de 2 sous-unités a, 1 sous-unité ß et 1 sous-unité y. Récemment, la cristallisation du canal sodique sensible au pH acide, ASIC, un autre membre de la famille ENaC/Deg, a mis en évidence un canal homotrimérique. Cette divergence dans la composition en sous-unités formant les complexes ENaC et ASIC, deux canaux de la même famille de gènes, nous a motivé à réinvestiguer le problème de l'oligomérisation du complexe fonctionnel ENaC après purification. Dans ce but le complexe ENaC fait des sous-unités aßy marquées par un épitope His 6 ont été exprimées dans l'ovocyte de Xenopus leavis. Les trois sous-unités aßy du complexe ENaC peuvent être co-purifiées sur des billes d'agarose Ni+2-NTA et montrent les modifications post-traductionnelles attendues pour le complexe fonctionnel ENaC exprimé en surface. Nous avons pu démontrer que ce complexe ENaC fonctionnel, est formé de deux sous-unités a différentes, mais de une seule sous-unité ß et une seule sous-unité y, suggérant un complexe ENaC formé de plus de trois sous-unités. L'estimation de la masse du complexe fonctionnel ENaC par Western blot, en comparaison avec des constructions concatemériques de ENaC faites de 2, 3, ou 4 sous-unités indique que le complexe aßy ENaC fonctionnel est une hétérotétramère composé de 2 sous-unités a, une ß et une y. Ces expériences ne représentent pas le fin d'une controverse quant à la structure des canaux ENaC et ASIC, mais soulèvent la question de la relevance fonctionnelle de la structure tridimentionelle du canal ASIC révélée par crystallographie.

Apoplastic polyesters in Arabidopsis surface tissues--a typical suberin and a particular cutin.

Cutinized and suberized cell walls form physiological important plant-environment interfaces as they act as barriers limiting water and nutrient loss and protect from radiation and invasion by pathogens. Due to the lack of protocols for the isolation and analysis of cutin and suberin in Arabidopsis, the model plant for molecular biology, mutants and transgenic plants with a defined altered cutin or suberin composition are unavailable, causing that structure and function of these apoplastic barriers are still poorly understood. Transmission electron microscopy (TEM) revealed that Arabidopsis leaf cuticle thickness ranges from only 22 nm in leaf blades to 45 nm on petioles, causing the difficulty in cuticular membrane isolation. We report the use of polysaccharide hydrolases to isolate Arabidopsis cuticular membranes, suitable for depolymerization and subsequent compositional analysis. Although cutin characteristic omega-hydroxy acids (7%) and mid-chain hydroxylated fatty acids (8%) were detected, the discovery of alpha,omega-diacids (40%) and 2-hydroxy acids (14%) as major depolymerization products reveals a so far novel monomer composition in Arabidopsis cutin, but with chemical analogy to root suberin. Histochemical and TEM analysis revealed that suberin depositions were localized to the cell walls in the endodermis of primary roots and the periderm of mature roots of Arabidopsis. Enzyme digested and solvent extracted root cell walls when subjected to suberin depolymerization conditions released omega-hydroxy acids (43%) and alpha,omega-diacids (24%) as major components together with carboxylic acids (9%), alcohols (6%) and 2-hydroxyacids (0.1%). This similarity to suberin of other species indicates that Arabidopsis roots can serve as a model for suberized tissue in general.

High-resolution seismic images of potentially seismogenic structures beneath the northwest Canterbury Plains, New Zealand

The transpressional boundary between the Australian and Pacific plates in the central South Island of New Zealand comprises the Alpine Fault and a broad region of distributed strain concentrated in the Southern Alps but encompassing regions further to the east, including the northwest Canterbury Plains. Low to moderate levels of seismicity (e. g., 2 > M 5 events since 1974 and 2 > M 4.0 in 2009) and Holocene sediments offset or disrupted along rare exposed active fault segments are evidence for ongoing tectonism in the northwest plains, the surface topography of which is remarkably flat and even. Because the geology underlying the late Quaternary alluvial fan deposits that carpet most of the plains is not established, the detailed tectonic evolution of this region and the potential for larger earthquakes is only poorly understood. To address these issues, we have processed and interpreted high-resolution (2.5 m subsurface sampling interval) seismic data acquired along lines strategically located relative to extensive rock exposures to the north, west, and southwest and rare exposures to the east. Geological information provided by these rock exposures offer important constraints on the interpretation of the seismic data. The processed seismic reflection sections image a variably thick layer of generally undisturbed younger (i.e., < 24 ka) Quaternary alluvial sediments unconformably overlying an older (> 59 ka) Quaternary sedimentary sequence that shows evidence of moderate faulting and folding during and subsequent to deposition. These Quaternary units are in unconformable contact with Late Cretaceous-Tertiary interbedded sedimentary and volcanic rocks that are highly faulted, folded, and tilted. The lowest imaged unit is largely reflection-free Permian Triassic basement rocks. Quaternary-age deformation has affected all the rocks underlying the younger alluvial sediments, and there is evidence for ongoing deformation. Eight primary and numerous secondary faults as well as a major anticlinal fold are revealed on the seismic sections. Folded sedimentary and volcanic units are observed in the hanging walls and footwalls of most faults. Five of the primary faults represent plausible extensions of mapped faults, three of which are active. The major anticlinal fold is the probable continuation of known active structure. A magnitude 7.1 earthquake occurred on 4 September 2010 near the southeastern edge of our study area. This predominantly right-lateral strike-slip event and numerous aftershocks (ten with magnitudes >= 5 within one week of the main event) highlight the primary message of our paper: that the generally flat and topographically featureless Canterbury Plains is underlain by a network of active faults that have the potential to generate significant earthquakes.

Role of CD44H carbohydrate structure in neuroblastoma adhesive properties.

BACKGROUND: CD44 represents a heterogeneous group of surface glycoproteins involved in cell-cell and cell-matrix interactions. CD44H is the major receptor for hyaluronate, and most if not all CD44H known functions are attributed to its ability to recognize hyaluronate. We have previously demonstrated a lack of CD44 expression in high stages and NMYC-amplified tumors and further have shown that NMYC-amplified cell lines either did not express CD44 at all or expressed a nonfunctional receptor. On the other hand, nonamplified cells constitutively expressed an active receptor, suggesting that absence of CD44-mediated hy aluronate binding could be related to increased malignancy in human neuroblastoma. PROCEDURE: In the present study we have compared the glycosylated structure of CD44 expressed by NMYC amplified vs. nonamplified cell lines in relation to their adhesive properties for hyaluronate. These adhesive properties were measured after modifications of the carbohydrate structure with enzymes and inhibitors of N- or O-linked glycosylation. RESULTS AND CONCLUSIONS: Our results indicate that increased sialylation, defective N-linked glycosylation, and substitution of the CD44 glycoprotein with keratan sulfate glycosaminoglycan might include modifications observed on neuroblastoma cells that could account for the inability of the receptor to bind hyaluronate.

Model structure of human APOBEC3G.

BACKGROUND: APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G) has antiretroviral activity associated with the hypermutation of viral DNA through cytosine deamination. APOBEC3G has two cytosine deaminase (CDA) domains; the catalytically inactive amino-terminal domain of APOBEC3G (N-CDA) carries the Vif interaction domain. There is no 3-D structure of APOBEC3G solved by X-ray or nuclear magnetic resonance. METHODOLOGY/PRINCIPAL FINDINGS: We predicted the structure of human APOBEC3G based on the crystal structure of APOBEC2. To assess the model structure, we evaluated 48 mutants of APOBEC3G N-CDA that identify novel variants altering DeltaVif HIV-1 infectivity and packaging of APOBEC3G. Results indicated that the key residue D128 is exposed at the surface of the model, with a negative local electrostatic potential. Mutation D128K changes the sign of that local potential. In addition, two novel functionally relevant residues that result in defective APOBEC3G encapsidation, R122 and W127, cluster at the surface. CONCLUSIONS/SIGNIFICANCE: The structure model identifies a cluster of residues important for packaging of APOBEC3G into virions, and may serve to guide functional analysis of APOBEC3G.