Pyochelin enantiomers and their outer-membrane siderophore transporters in fluorescent pseudomonads: structural bases for unique enantiospecific recognition.

Pyochelin (Pch) and enantiopyochelin (EPch) are enantiomeric siderophores, with three chiral centers, produced under iron limitation conditions by Pseudomonas aeruginosa and Pseudomonas fluorescens , respectively. After iron chelation in the extracellular medium, Pch-Fe and EPch-Fe are recognized and transported by their specific outer-membrane transporters: FptA in P. aeruginosa and FetA in P. fluorescens . Structural analysis of FetA-EPch-Fe and FptA-Pch-Fe, combined with mutagenesis and docking studies revealed the structural basis of the stereospecific recognition of these enantiomers by their respective transporters. Whereas FetA and FptA have a low sequence identity but high structural homology, the Pch and EPch binding pockets do not share any structural homology, but display similar physicochemical properties. The stereospecific recognition of both enantiomers by their corresponding transporters is imposed by the configuration of the siderophore's C4'' and C2'' chiral centers. This recognition involves specific hydrogen bonds between the Arg91 guanidinium group and EPch-Fe for FetA and between the Leu117-Leu116 main chain and Pch-Fe for FptA. FetA and FptA are the first membrane receptors to be structurally described with opposite binding enantioselectivities for their ligands, giving insights into the structural basis of their enantiospecificity.

Stabilized Petrov-Galerkin methods for the convection-diffusion-reaction and the Helmholtz equations

We present two new stabilized high-resolution numerical methods for the convection–diffusion–reaction (CDR) and the Helmholtz equations respectively. The work embarks upon a priori analysis of some consistency recovery procedures for some stabilization methods belonging to the Petrov–Galerkin framework. It was found that the use of some standard practices (e.g. M-Matrices theory) for the design of essentially non-oscillatory numerical methods is not feasible when consistency recovery methods are employed. Hence, with respect to convective stabilization, such recovery methods are not preferred. Next, we present the design of a high-resolution Petrov–Galerkin (HRPG) method for the 1D CDR problem. The problem is studied from a fresh point of view, including practical implications on the formulation of the maximum principle, M-Matrices theory, monotonicity and total variation diminishing (TVD) finite volume schemes. The current method is next in line to earlier methods that may be viewed as an upwinding plus a discontinuity-capturing operator. Finally, some remarks are made on the extension of the HRPG method to multidimensions. Next, we present a new numerical scheme for the Helmholtz equation resulting in quasi-exact solutions. The focus is on the approximation of the solution to the Helmholtz equation in the interior of the domain using compact stencils. Piecewise linear/bilinear polynomial interpolation are considered on a structured mesh/grid. The only a priori requirement is to provide a mesh/grid resolution of at least eight elements per wavelength. No stabilization parameters are involved in the definition of the scheme. The scheme consists of taking the average of the equation stencils obtained by the standard Galerkin finite element method and the classical finite difference method. Dispersion analysis in 1D and 2D illustrate the quasi-exact properties of this scheme. Finally, some remarks are made on the extension of the scheme to unstructured meshes by designing a method within the Petrov–Galerkin framework.

Capsule permeability via polymer and protein ingress/egress.

Static incubation tests, where microcapsules and beads are contacted with polymer and protein solutions, have been developed for the characterization of permselective materials applied for bioartificial organs and drug delivery. A combination of polymer ingress, detected by size-exclusion chromatography, and protein ingress/ egress, assessed by gel electrophoresis, provides information regarding the diffusion kinetics, molar mass cutoff(MMCO) and permeability. This represents an improvement over existing permeability measurements that are based on the diffusion of a single type of solute. Specifically, the permeability of capsules based on alginate, cellulose sulfate, polymethylene-co-guanidine were characterized as a function of membrane thickness. Solid alginate beads were also evaluated. The MMCO of these capsules was estimated to be between 80 and 90 kDa using polymers, and between 116-150 kDa with proteins. Apparently, the globular shape of the proteins (radius of gyration (Rg) of 4.2-4.6 nm) facilitates their passage through the membrane, comparatively to the polysaccharide coil conformation (Rg of 6.5-8.3 nm). An increase of the capsule membrane thickness reduced these values. The MMCO of the beads, which do not have a membrane limiting their permselective properties, was higher, between 110 and 200 kDa with dextrans, and between 150 and 220 kDa with proteins. Therefore, although the permeability estimated with biologically relevant molecules is generally higher due to their lower radius of gyration, both the MMCO of synthetic and natural watersoluble polymers correlate well, and can be used as in vitro metrics for the immune protection ability of microcapsules and microbeads. This article shows, to the authors' knowledge, the first reported concordance between permeability measures based on model natural and biological macromolecules.

N-terminal amino acid sequences of the major outer membrane proteins from a Neisseria meningitidis group B strain isolated in Brazil

The four dominant outer membrane proteins (46, 38, 33 and 28 kDa) were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in a semi-purified preparation of vesicle membranes of a Neisseria meningitidis (N44/89, B:4:P1.15:P5.5,7) strain isolated in Brazil. The N-terminal amino acid sequence for the 46 kDa and 28 kDa proteins matched that reported by others for class 1 and 5 proteins respectively, whereas the sequence (25 amino acids) for the 38 kDa (class 3) protein was similar to class 1 meningococcal proteins. The sequence for the 33 kDa (class 4) was unique and not homologous to any known protein.

Liver hyperplasia and paradoxical regulation of glycogen metabolism and glucose-sensitive gene expression in GLUT2-null hepatocytes. Further evidence for the existence of a membrane-based glucose release pathway.

We investigated the impact of GLUT2 gene inactivation on the regulation of hepatic glucose metabolism during the fed to fast transition. In control and GLUT2-null mice, fasting was accompanied by a approximately 10-fold increase in plasma glucagon to insulin ratio, a similar activation of liver glycogen phosphorylase and inhibition of glycogen synthase and the same elevation in phosphoenolpyruvate carboxykinase and glucose-6-phosphatase mRNAs. In GLUT2-null mice, mobilization of glycogen stores was, however, strongly impaired. This was correlated with glucose-6-phosphate (G6P) levels, which remained at the fed values, indicating an important allosteric stimulation of glycogen synthase by G6P. These G6P levels were also accompanied by a paradoxical elevation of the mRNAs for L-pyruvate kinase. Re-expression of GLUT2 in liver corrected the abnormal regulation of glycogen and L-pyruvate kinase gene expression. Interestingly, GLUT2-null livers were hyperplasic, as revealed by a 40% increase in liver mass and 30% increase in liver DNA content. Together, these data indicate that in the absence of GLUT2, the G6P levels cannot decrease during a fasting period. This may be due to neosynthesized glucose entering the cytosol, being unable to diffuse into the extracellular space, and being phosphorylated back to G6P. Because hepatic glucose production is nevertheless quantitatively normal, glucose produced in the endoplasmic reticulum may also be exported out of the cell through an alternative, membrane traffic-based pathway, as previously reported (Guillam, M.-T., Burcelin, R., and Thorens, B. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 12317-12321). Therefore, in fasting, GLUT2 is not required for quantitative normal glucose output but is necessary to equilibrate cytosolic glucose with the extracellular space. In the absence of this equilibration, the control of hepatic glucose metabolism by G6P is dominant over that by plasma hormone concentrations.

A geometric mechanism of diffusion: Rigorous verification in a priori unstable Hamiltonian systems

In this paper we consider a representative a priori unstable Hamiltonian system with 2+1/2 degrees of freedom, to which we apply the geometric mechanism for diffusion introduced in the paper Delshams et al., Mem.Amer.Math. Soc. 2006, and generalized in Delshams and Huguet, Nonlinearity 2009, and provide explicit, concrete and easily verifiable conditions for the existence of diffusing orbits. The simplification of the hypotheses allows us to perform explicitly the computations along the proof, which contribute to present in an easily understandable way the geometric mechanism of diffusion. In particular, we fully describe the construction of the scattering map and the combination of two types of dynamics on a normally hyperbolic invariant manifold.

Conversion of membrane-bound Fas(CD95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity.

Human Fas ligand (L) (CD95L) and tumor necrosis factor (TNF)-alpha undergo metalloproteinase-mediated proteolytic processing in their extracellular domains resulting in the release of soluble trimeric ligands (soluble [s]FasL, sTNF-alpha) which, in the case of sFasL, is thought to be implicated in diseases such as hepatitis and AIDS. Here we show that the processing of sFasL occurs between Ser126 and Leu127. The apoptotic-inducing capacity of naturally processed sFasL was reduced by >1,000-fold compared with membrane-bound FasL, and injection of high doses of recombinant sFasL in mice did not induce liver failure. However, soluble FasL retained its capacity to interact with Fas, and restoration of its cytotoxic activity was achieved both in vitro and in vivo with the addition of cross-linking antibodies. Similarly, the marginal apoptotic activity of recombinant soluble TNF-related apoptosis-inducing ligand (sTRAIL), another member of the TNF ligand family, was greatly increased upon cross-linking. These results indicate that the mere trimerization of the Fas and TRAIL receptors may not be sufficient to trigger death signals. Thus, the observation that sFasL is less cytotoxic than membrane-bound FasL may explain why in certain types of cancer, systemic tissue damage is not detected, even though the levels of circulating sFasL are high.

Molecular determinants for membrane association of the hepatitis C virus NS2 protease domain

Background: Hepatitis C virus (HCV) nonstructural protein 2 (NS2) plays essential roles in particle assembly and polyprotein processing. It harbors an N-terminal membrane domain comprising three putative transmembrane s egments ( amino acids [aa] 1-93) a nd a C-terminal cysteine protease domain (aa 94-217). Given that the latter has been predicted to be membrane-associated, we aimed to identify molecular determinants for membrane association of the NS2 protease domain. Methods: A comprehensive panel of NS2 deletion constructs was analyzed by fluorescence microscopy, selective membrane extraction, and m embrane flotation assays. Candidate aa r esidues involved in membrane association were substituted by site-directed mutagenesis. Results: The NS2 protease domain alone was found to associate with membranes. Two N-terminal α-helices comprising aa 102-114 and aa 123-136 were found to m ediate this a ssociation, w ith c onserved hydrophobic and positively charged aa residues representing the key determinants. I nterestingly, m utagenesis analyses r evealed that electrostatic interactions involving a positively charged aa residue in α-helix aa 123-136 are required for membrane association. Mono- and bicistronic (i.e. NS2 c leavage-independent) HCV constructs were prepared to i nvestigate the effect o f these substitutions on RNA replication and infectious viral particle formation. Conclusions: T he NS2 protease d omain itself harbors m olecular determinants for membrane association within α-helices aa 102-114 and aa 1 23-136 which may contribute to p roper p ositioning of t he active site. These results provide new insights i nto the membrane topology and t he p oorly understood f unction of t his essential viral protease.

Artificial Feeding of Amblyomma cajennense (Fabricius, 1787) (Acari: Ixodidae) through Silicone Membrane

An artificial feeding system was used where citrated bovine blood was offerred to male and female Amblyomma cajennense. Vestiges of blood, sweat, hair and exfoliated skin were used as phago-stimulants placed on the surface of the silicone membrane. The ticks were collected, as engorged nymphs, from naturally infested equines, with the ecdysis occurring in the laboratory. Four hundred ticks were used, 50% being female, at three to four weeks post-ecdysis. Vestiges of blood on the silicone membrane were the most efficient phago-stimulant and the association of vestiges of blood and sweat residue smears yielded better results compared to the other phago-stimulants used

Rapid, combinatorial analysis of membrane compartments in intact plants with a multicolor marker set.

Plant membrane compartments and trafficking pathways are highly complex, and are often distinct from those of animals and fungi. Progress has been made in defining trafficking in plants using transient expression systems. However, many processes require a precise understanding of plant membrane trafficking in a developmental context, and in diverse, specialized cell types. These include defense responses to pathogens, regulation of transporter accumulation in plant nutrition or polar auxin transport in development. In all of these cases a central role is played by the endosomal membrane system, which, however, is the most divergent and ill-defined aspect of plant cell compartmentation. We have designed a new vector series, and have generated a large number of stably transformed plants expressing membrane protein fusions to spectrally distinct, fluorescent tags. We selected lines with distinct subcellular localization patterns, and stable, non-toxic expression. We demonstrate the power of this multicolor 'Wave' marker set for rapid, combinatorial analysis of plant cell membrane compartments, both in live-imaging and immunoelectron microscopy. Among other findings, our systematic co-localization analysis revealed that a class of plant Rab1-homologs has a much more extended localization than was previously assumed, and also localizes to trans-Golgi/endosomal compartments. Constructs that can be transformed into any genetic background or species, as well as seeds from transgenic Arabidopsis plants, will be freely available, and will promote rapid progress in diverse areas of plant cell biology.

Wnt secretion and gradient formation.

Concentration gradients formed by the lipid-modified morphogens of the Wnt family are known for their pivotal roles during embryogenesis and adult tissue homeostasis. Wnt morphogens are also implicated in a variety of human diseases, especially cancer. Therefore, the signaling cascades triggered by Wnts have received considerable attention during recent decades. However, how Wnts are secreted and how concentration gradients are formed remains poorly understood. The use of model organisms such as Drosophila melanogaster has provided important advances in this area. For instance, we have previously shown that the lipid raft-associated reggie/flotillin proteins influence Wnt secretion and spreading in Drosophila. Our work supports the notion that producing cells secrete Wnt molecules in at least two pools: a poorly diffusible one and a reggie/flotillin-dependent highly diffusible pool which allows morphogen spreading over long distances away from its source of production. Here we revise the current views of Wnt secretion and spreading, and propose two models for the role of the reggie/flotillin proteins in these processes: (i) reggies/flotillins regulate the basolateral endocytosis of the poorly diffusible, membrane-bound Wnt pool, which is then sorted and secreted to apical compartments for long-range diffusion, and (ii) lipid rafts organized by reggies/flotillins serve as "dating points" where extracellular Wnt transiently interacts with lipoprotein receptors to allow its capture and further spreading via lipoprotein particles. We further discuss these processes in the context of human breast cancer. A better understanding of these phenomena may be relevant for identification of novel drug targets and therapeutic strategies.

Identification of Novel Membrane Structures in Plasmodium falciparum Infected Erythrocytes

Little is known about the molecular mechanisms underlying the release of merozoites from malaria infected erythrocytes. In this study membranous structures present in the culture medium at the time of merozoite release have been characterized. Biochemical and ultrastructural evidence indicate that membranous structures consist of the infected erythrocyte membrane, the parasitophorous vacuolar membrane and a residual body containing electron dense material. These are subcellular compartments expected in a structure that arises as a consequence of merozoite release from the infected cell. Ultrastructural studies show that a novel structure extends from the former parasite compartment to the surface membrane. Since these membrane modifications are detected only after merozoites have been released from the infected erythrocyte, it is proposed that they might play a role in the release of merozoites from the host cell