Repelling class discrimination: ephrin-A5 binds to and activates EphB2 receptor signaling

The interactions between Eph receptor tyrosine kinases and their ephrin ligands regulate cell migration and axon pathfinding. The EphA receptors are generally thought to become activated by ephrin-A ligands, whereas the EphB receptors interact with ephrin-B ligands. Here we show that two of the most widely studied of these molecules, EphB2 and ephrin-A5, which have never been described to interact with each other, do in fact bind one another with high affinity. Exposure of EphB2-expressing cells to ephrin-A5 leads to receptor clustering, autophosphorylation and initiation of downstream signaling. Ephrin-A5 induces EphB2-mediated growth cone collapse and neurite retraction in a model system. We further show, using X-ray crystallography, that the ephrin-A5-EphB2 complex is a heterodimer and is architecturally distinct from the tetrameric EphB2-ephrin-B2 structure. The structural data reveal the molecular basis for EphB2-ephrin-A5 signaling and provide a framework for understanding the complexities of functional interactions and crosstalk between A- and B-subclass Eph receptors and ephrins.

A genetic linkage map in autotetraploid lucerne adapted to northern Australia, and use of the map to identify DNA markers linked to resistance to Phytophthora medicaginis

Phytophthora root rot, caused by Phytophthora medicaginis, is a major limitation to lucerne ( Medicago sativa L.) production in Australia and North America. Quantitative trait loci (QTLs) involved in resistance to P. medicaginis were identified in a lucerne backcross population of 120 individuals. A genetic linkage map was constructed for tetraploid lucerne using 50 RAPD ( randomly amplified polymorphic DNA), 104 AFLP (amplified fragment length polymorphism) markers, and one SSR ( simple sequence repeat or microsatellite) marker, which originated from the resistant parent (W116); 13 markers remain unlinked. The linkage map contains 18 linkage groups covering 2136.5 cM, with an average distance of 15.0 cM between markers. Four of the linkage groups contained only either 2 or 3 markers. Using duplex markers and repulsion phase linkages the map condensed to 7 homology groups and 2 unassigned linkage groups. Three regions located on linkage groups 2, 14, and 18, were identified as associated with root reaction and the QTLs explained 6 - 15% of the phenotypic variation. The research also indicates that different resistance QTLs are involved in conferring resistance in different organs. Two QTLs were identified as associated with disease resistance expressed after inoculation of detached leaves. The marker, W11-2 on group 18, identified as associated with root reaction, contributed 7% of the phenotypic variation in leaf response in our population. This marker appears to be linked to a QTL encoding a resistance factor contributing to both root and leaf reaction. One other QTL, not identified as associated with root reaction, was positioned on group 1 and contributed to 6% of the variation. This genetic linkage map provides an entry point for future molecular-based improvement of lucerne in Australia, and markers linked to the QTLs we have reported should be useful for marker-assisted selection for partial resistance to P. medicaginis in lucerne.

Bounding film drainage in common thin films

A review of thin film drainage models is presented in which the predictions of thinning velocities and drainage times are compared to reported values on foam and emulsion films found in the literature. Free standing films with tangentially immobile interfaces and suppressed electrostatic repulsion are considered, such as those studied in capillary cells. The experimental thinning velocities and drainage times of foams and emulsions are shown to be bounded by predictions from the Reynolds and the theoretical MTsR equations. The semi-empirical MTsR and the surface wave equations were the most consistently accurate with all of the films considered. These results are used in an accompanying paper to develop scaling laws that bound the critical film thickness of foam and emulsion films. (c) 2005 Elsevier B.V. All rights reserved.

Response normalization and blur adaptation:data and multi-scale model

Adapting to blurred or sharpened images alters perceived blur of a focused image (M. A. Webster, M. A. Georgeson, & S. M. Webster, 2002). We asked whether blur adaptation results in (a) renormalization of perceived focus or (b) a repulsion aftereffect. Images were checkerboards or 2-D Gaussian noise, whose amplitude spectra had (log-log) slopes from -2 (strongly blurred) to 0 (strongly sharpened). Observers adjusted the spectral slope of a comparison image to match different test slopes after adaptation to blurred or sharpened images. Results did not show repulsion effects but were consistent with some renormalization. Test blur levels at and near a blurred or sharpened adaptation level were matched by more focused slopes (closer to 1/f) but with little or no change in appearance after adaptation to focused (1/f) images. A model of contrast adaptation and blur coding by multiple-scale spatial filters predicts these blur aftereffects and those of Webster et al. (2002). A key proposal is that observers are pre-adapted to natural spectra, and blurred or sharpened spectra induce changes in the state of adaptation. The model illustrates how norms might be encoded and recalibrated in the visual system even when they are represented only implicitly by the distribution of responses across multiple channels.

Novel imprinted polymers as artificial enzymes

Derivatives of L-histidine were investigated as suitable models for the Asp-His couple found in the catalytic triad of serine proteases. A combination of molecular dynamics and IH NMR spectroscopy suggested that the most populous conformations of N-acetyl-L-histidine and the N-acetyl-L-histidine anion were predominated by those in which the carboxylate group was gauche to the imidazole ring overcoming steric and electrostatic repulsion, suggesting there is an interaction between the carboxylate group and the imidazole ring. Kinetic studies, using imidazole, N-acetyl-L-histidine and the N-acetyl-L-histidine anion showed that in a DMSO/H20 9: 1 v/v solution, the N-acetyl-L-histidine anion catalysed the hydrolysis of p-nitrophenyl acetate at a greater rate than using either imidazole or N-acetyl-L-histidine as catalyst. This indicates that the carboxylate group affects the nucleophilicity of the unprotonated imidazole ring. 31P MAS NMR spectroscopy was investigated as a new technique for the study of the template molecule environment within the polymer networks. It was found that it was possible to distinguish between template associated with the polymer and that which was precipitated onto the surface, though it was not possible to distinguish between polymer within imprinted cavities and that which was not. Attempts to study the effect of the carboxylate group/imidazole ring interaction in the imprinted cavity of a molecularly imprinted polymer network were hindered by the method used to follow the reaction. It was found though that in a pH 8.0 buffered solution the presence of imprinted cavities increased the rate of reaction for those polymers derived from L-histidine. Some preliminary investigations into the design and synthesis of an MIP which would catalyse the oxy-Cope rearrangement were carried out but the results were inconclusive.

The activity of pH membrane disruptive pseudopeptides and their subcellular fate in mammalian cells cultured in-vitro

This thesis describes investigations upon pseudopeptides which were conducted to improve our understanding of the fate of synthetic macromolecules in cells and to develop approaches to influence that fate. The low uptake of molecules across the external cellular membrane is the principal barrier against effective delivery of therapeutic products to within the cell structure. In nature, disruption of this membrane by amphiphilic peptides plays a central role in the pathogenesis by bacterial and toxin infections. These amphiphilic peptides contain both hydrophobic and weakly charged hydrophilic amino acid residues and upon activation they become integrated into the lipid bilayers of the extracellular or endosomal membranes. The architectures of the pseudopeptides described here were designed to display similar pH dependent membrane rupturing activity to that of peptides derived from the influenza virus hemagglutinin HA-2. This HA protein promotes fusion of the influenza virus envelope with the cell endosome membrane due to a change in conformation in response to the acidic pH of the endosome lumen (pH 5.0-6.0). The pseudopeptides were obtained by the copolymerisation of L-lysine and L-lysine ethyl-ester with various dicarboxylic acid moieties. In this way a linear polyamide comprising of alternating pendant carboxylic acids and pendant hydrophobic moieties was made. At physiological pH (pH 7.4), electrostatic repulsion of pendant anionic carboxyl groups along the polymer backbone is sufficient to overcome the intramolecular association of the hydrophobic groups resulting in an extended conformation. At low pH (typically pH 4.8) loss of charge results in increased intramolecular hydrophobic association and the polymer chain collapses to a compact conformation, leading to precipitation of the polymer. Consequently, a conformation dependent functional property could be made to respond to small changes in the environmental pH. Pseudopepides were investigated for their cytoxicity towards a well known cell line, namely C26 (colorectal adenocarcinoma) and were shown through the use of a cell viability assay, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) to be well tolerated by C26 cells over a range of concentrations (2-500,μg/ml) at physiological pH (pH 7.4). A modified version of a shorter 30-minute coupled enzymatic assay, the LDH (lactate dehydrogenase) assay was used to evaluate the ability of the pseudopeptides to disrupt the membrane of two different cell lines (COS-1; African green monkey, kidney and A2780; human ovarian carcinoma) at low pH (pH 5.5). The cell membrane disruption property of the pseudopeptides was successfully demonstrated for COS-I and A2780 cell lines at this pH (pH 5.5). A variety of cell lines were chosen owing to limited availability and to compare the cytotoxic action of these pH responsive psudopeptides towards normal and tumorogenic cell lines. To investigate the intracellular delivery of one of the pseudopeptides, poly (L-lysine iso-phthalamide) and its subcellular location, a Cy3 bisamine fluorophore was conjugated into its backbone, at ratios of dye:lysine of 1:20, 1:30, 1:40, 1:60 and 1:80. Native polyacrylacrylamide gel electrophoresis (PAGE) and high voltage paper electrophoresis (HVPE) studies of the polydyes were conducted and provided evidence that that the Cy3 bisamine fluorophore was conjugated into the backbone of the polymer, poly (L-lysine iso-phthalamide). The subcellular fate of the fluorescentlylabelled "polydye" (hereafter PD20) was monitored by laser scanning confocal microscopy (LSCM) in CHO (Chinese hamster ovary) cells cultured in-vitro at various pH values (pH 7.4 and 5.0). LSCM images depicting time-dependent internalisation of PD20 indicated that PD20 traversed the extracellular membrane of CHO cells cultured in-vitro within ten minutes and migrated towards the endosomal regions where the pH is in the region of 5.0 to 6.0. Nuclear localisation of PD20 was demonstrated in a subpopulation of CHO cells. A further study was completed in CHO and HepG2 (hepatocellular carcinoma) cells cultured in-vitro using a lower molecular weight polymer to demonstrate that the molecular weight of "polydye" could be tailored to attain nuclear trafficking in cells. Prospective use of this technology encompasses a method of delivering a payload into a living cell based upon the hypercoiling nature of the pseudopeptides studied in this thesis and has led to a patent application (GB0228525.2; 20(2).

Protein-protein interactions in ovalbumin solutions studied by small angle scattering: effect of ionic strength, and the chemical nature of cations

The influence of ionic strength and of the chemical nature of cations on the protein-protein interactions in ovalbumin solution was studied using small-angle X-ray and neutron scattering (SAXS/SANS). The globular protein ovalbumin is found in dimeric form in solutions as suggested by SANS/SAXS experiments. Due to the negative charge of the proteins at neutral pH, the protein-protein interactions without any salt addition are dominated by electrostatic repulsion. A structure factor related to screened Coulombic interactions together with an ellipsoid form factor was used to fit the scattering intensity. A monovalent salt (NaCl) and a trivalent salt (YCl3) were used to study the effect of the chemical nature of cations on the interaction in protein solutions. Upon addition of NaCl, with ionic strength below that of physiological conditions (150 mM), the effective interactions are still dominated by the surface charge of the proteins and the scattering data can be understood using the same model. When yttrium chloride was used, a reentrant condensation behavior, i.e., aggregation and subsequent redissolution of proteins with increasing salt concentration, was observed. SAXS measurements reveal a transition from effective repulsion to attraction with increasing salt concentration. The solutions in the reentrant regime become unstable after long times (several days). The results are discussed and compared with those from bovine serum albumin (BSA) in solutions.

Granular superconductors:from the nonlinear σ model to the Bose-Hubbard description

We modify a nonlinear σ model (NLσM) for the description of a granular disordered system in the presence of both the Coulomb repulsion and the Cooper pairing. We show that under certain controlled approximations the action of this model is reduced to the Ambegaokar-Eckern-Schön (AES) action, which is further reduced to the Bose-Hubbard (or “dirty-boson”) model with renormalized coupling constants. We obtain an effective action which is more general than the AES one but still simpler than the full NLσM action. This action can be applied in the region of parameters where the reduction to the AES or the Bose-Hubbard model is not justified. This action may lead to a different picture of the superconductor-insulator transition in two-dimensional systems.

Responsive hydrophobically associating polymers:a review of structure and properties

Responsive hydrophobically associating polymers can in many ways be considered to be analogous to proteins in their ability to form compact molecules with a defined secondary structure, and hence, functionality. These molecules are characterized by the presence of alternating charged and hydrophobic groups. The balance between charge repulsion and hydrophobic interactions is sensitive to environmental pH and therefore changes in pH produce controllable conformational changes. The change from a charged extended chain to a collapsed uncharged coil structure is sometimes referred to as hypercoiling behaviour and enables the polymer to act as a simple switch between an 'on' and 'off' state. The purpose of this review is to illustrate the structure and behaviour of polymers that exhibit hypercoiling behaviour and to highlight their potential pharmaceutical applications, which in terms of drug delivery is likely to be related to their surface behaviour and solubilizing activity. © 2001 Elsevier Science B.V. All rights reserved.

Estudo in silico das interações da protease NS3-NS2B de DENV-2 com o inibidor peptídico Bz-nKRR-H com finalidades terapêuticas

Dengue virus is an important patogen that causes Dengue desease in all world, and belongs to Flavivirus gender. The virus consists of enveloped RNA with a single strand positive sense, 11Kb genome. The RNA is translated into a polyprotein precursor, wich is cleaved into 3 structural proteins (C, prM e E) and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B e NS5). The NS3 is a multifunctional protein, that besides to promote the polyprotein precursor cleavage, also have NTPase, helicase and RTPase activity. The NS3 needs a hydrophilic segment of 40 residues from the transmembrane NS2B protein (who acts like cofator) to realize this functions. Actually, there's no vacines available on the market, and the treatment are just symptomatic. The tetrapeptide inhibitor Bz-Nle-Lys-Arg-Arg-H (Ki de 5,8-7,0 M) was showed as a potent inhibitor μ for NS3prot in Dengue virus. That is a inteligent alternative to treat the dengue desease. The present work aimed analyse the interactions of the ligand bounded to the activity site to provid a clear and depth vision of that interaction. For this purpouse, it was conducted an in silico study, by using quantum mechanical calculations based on Density Functional Theory (DFT), with Generalized Gradient approximation (GGA) to describe the effects of exchange and correlation. The interaction energy of each amino acid belonging to the binding site to the ligand was calculated the using the method of molecular fragmentation with conjugated caps (MFCC). Besides energy, we calculated the distances, types of molecular interactions and atomic groups involved. The theoretical models used were satisfactory and show a more accurate description when the dielectric constant = 20 ε and 80 was used. The results demonstrate that the interaction energy of the system reached convergence at 13.5 A. Within a radius of 13,5A the most important residues were identified. Met49, Met84 and Asp81 perform interactions of hydrogen with the ligant. The Asp79 and Asp75 residues present high energy of attraction. Arg54, Arg85 and Lys 131 perform hydrogen interactions with the ligand, however, appear in BIRD graph having high repulsion energy with the inhibitor. The data also emphasizes the importance of residue Tyr161 and the involvement of the catalytic triad composed by Asp75, His51 and Ser135

Rôle des récepteurs aux protéines G (GPR55, GPR91 et GPR99) dans la croissance et le guidage axonal au cours du développement du système visuel

Lors de l’attribution du prix Nobel de chimie aux docteurs Robert Leftkowitz et Brian Kobika pour leurs travaux essentiels sur les récepteurs couplés à des protéines G (RCPGs), Sven Lindin, membre du comité Nobel, a affirmé que « jusqu'à la moitié » des médicaments « reposent sur une action ciblant les RCPG ». En raison de leurs rôles importants, leurs mécanismes d'activation et l’action de leurs ligands, les RCPG demeurent les cibles potentielles de la majorité des recherches pour le développement de nouveaux médicaments et de leurs applications cliniques. Dans cette optique, nous avons concentré nos recherches à travers cette thèse pour élucider les rôles, les mécanismes d’action et les effets des ligands de trois RCPG : GPR55; GPR91 et GPR99 au cours du développement des axones des cellules ganglionnaires de la rétine (CGRs). Les résultats de nos études confirment l’expression des récepteurs lors du développement embryonnaire, postnatal et adulte des CGRs ainsi qu’au cours de l’établissement de la voie rétinothalamique. In vitro, la modulation pharmacologique et génétique de l’activité de ces RCPGs réorganise la morphologie du cône de croissance des CGRs, celle des neurones corticaux et elle modifie la croissance axonale globale. De plus, les effets de la stimulation avec des ligands des ces trois RCPGs sur le guidage axonal varient d’aucun effet (GPR91 et GPR99) à la répulsion ou l’attraction (GPR55). La voie de signalisation MAPK-ERK1/2 joue un rôle essentiel dans la médiation des effets des ligands de ces récepteurs avec une implication de la voie de RhoA à hautes concentrations pour l’agoniste endogène de GPR55. In vivo, cette recherche démontre également l’implication de GPR55 dans les processus de sélection des cibles thalamiques et de raffinement au cours du développement du système nerveux visuel.

Dynamics of vector rogue waves in a fiber laser with a ring cavity

A pulse–pulse interaction that leads to rogue wave (RW) generation in lasers was previously attributed either to soliton–soliton or soliton–dispersive-wave interaction. The beating between polarization modes in the absence of a saturable absorber causes similar effects. Accounting for these polarization modes in a laser resonator is the purpose of the distributed vector model of laser resonators. Furthermore, high pump power, high amplitude, and short pulse duration are not necessary conditions to observe pulse attraction, repulsion, and collisions and the resonance exchange of energy between among them. The regimes of interest can be tuned just by changing the birefringence in the cavity with the pump power slightly higher than the laser threshold. This allows the observation of a wide range of RW patterns in the same experiment, as well as to classify them. The dynamics of the interaction between pulses leads us to the conclusion that all of these effects occur due to nonlinearity induced by the inverse population in the active fiber as well as an intrinsic nonlinearity in the passive part of the cavity. Most of the mechanisms of pulse–pulse interaction were found to be mutually exclusive. This means that all the observed RW patterns, namely, the “lonely,” “twins,” “three sisters,” and “cross,” are probably different cases of the same process.

Dynamical Heterogeneity and Nonlinear Susceptibility in Short-Ranged Attractive Supercooled Liquids

Recent work has demonstrated the strong qualitative differences between the dynamics near a glass transition driven by short-ranged repulsion and one governed by short-ranged attraction. Here, we study in detail the behavior of non-linear, higher-order correlation functions that measure the growth of length scales associated with dynamical heterogeneity in both types of systems. We find that this measure is qualitatively different in the repulsive and attractive cases with regards to the wave vector dependence as well as the time dependence of the standard non-linear four-point dynamical susceptibility. We discuss the implications of these results for the general understanding of dynamical heterogeneity in glass-forming liquids.

Phase behavior and far-from-equilibrium gelation in charged attractive colloids

In this Rapid Communication we demonstrate the applicability of an augmented Gibbs ensemble Monte Carlo approach for the phase behavior determination of model colloidal systems with short-ranged depletion attraction and long-ranged repulsion. This technique allows for a quantitative determination of the phase boundaries and ground states in such systems. We demonstrate that gelation may occur in systems of this type as the result of arrested microphase separation, even when the equilibrium state of the system is characterized by compact microphase structures.

Rôle des récepteurs aux protéines G (GPR55, GPR91 et GPR99) dans la croissance et le guidage axonal au cours du développement du système visuel.

Lors de l’attribution du prix Nobel de chimie aux docteurs Robert Leftkowitz et Brian Kobika pour leurs travaux essentiels sur les récepteurs couplés à des protéines G (RCPGs), Sven Lindin, membre du comité Nobel, a affirmé que « jusqu'à la moitié » des médicaments « reposent sur une action ciblant les RCPG ». En raison de leurs rôles importants, leurs mécanismes d'activation et l’action de leurs ligands, les RCPG demeurent les cibles potentielles de la majorité des recherches pour le développement de nouveaux médicaments et de leurs applications cliniques. Dans cette optique, nous avons concentré nos recherches à travers cette thèse pour élucider les rôles, les mécanismes d’action et les effets des ligands de trois RCPG : GPR55; GPR91 et GPR99 au cours du développement des axones des cellules ganglionnaires de la rétine (CGRs). Les résultats de nos études confirment l’expression des récepteurs lors du développement embryonnaire, postnatal et adulte des CGRs ainsi qu’au cours de l’établissement de la voie rétinothalamique. In vitro, la modulation pharmacologique et génétique de l’activité de ces RCPGs réorganise la morphologie du cône de croissance des CGRs, celle des neurones corticaux et elle modifie la croissance axonale globale. De plus, les effets de la stimulation avec des ligands des ces trois RCPGs sur le guidage axonal varient d’aucun effet (GPR91 et GPR99) à la répulsion ou l’attraction (GPR55). La voie de signalisation MAPK-ERK1/2 joue un rôle essentiel dans la médiation des effets des ligands de ces récepteurs avec une implication de la voie de RhoA à hautes concentrations pour l’agoniste endogène de GPR55. In vivo, cette recherche démontre également l’implication de GPR55 dans les processus de sélection des cibles thalamiques et de raffinement au cours du développement du système nerveux visuel.