Determination of the physiological dimer interface of the PhoQ sensor domain

PhoQ is the transmembrane sensor kinase of the phoPQ two-component system, which detects and responds to divalent cations and antimicrobial peptides and can trigger bacterial virulence. Despite their ubiquity and importance in bacterial signaling, the structure and molecular mechanism of the sensor kinases is not fully understood. Frequently, signals are transmitted from a periplasmic domain in these proteins to the cytoplasmic kinase domains via an extended dimeric interface, and the PhoQ protein would appear to follow this paradigm. However, the isolated truncated periplasmic domain of PhoQ dimerizes poorly, so it has been difficult to distinguish the relevant interface in crystal structures of the PhoQ periplasmic domain. Thus, to determine the arrangement of the periplasmic domains of Escherichia coli PhoQ in the physiological homodimer, disulfide-scanning mutagenesis was used. Single cysteine substitutions were introduced along the N-terminal helix of the periplasmic region, and the degree of cross-linking in each protein variant was determined by Western blotting and immunodetection. The results were subjected to periodicity analysis to generate a profile that provides information concerning the C^β distances between corresponding residues at the interface. This profile, together with a rigid-body search procedure, side-chain placement, and energy minimization, was used to build a model of the dimer arrangement. The final model proved to be highly compatible with one of the PhoQ crystal structures, 3BQ8, indicating that 3BQ8 is representative of the physiological arrangement. The model of the periplasmic region is also compatible with a full-length PhoQ protein in which a four-helix bundle forms in the membrane. The membrane four-helix bundle has been proposed for other sensor kinases and is thought to have a role in the mechanism of signal transduction; our model supports the idea that signaling through a membrane four-helix bundle is a widespread mechanism in the transmembrane sensor kinases.     

Vascular endothelial growth factor modulates neutrophil transendothelial migration via up-regulation of interleukin-8 in human brain microvascular endothelial cells.

Hypoxia, a strong inducer for vascular endothelial growth factor (VEGF)/vascular permeable factor (VPF) expression, regulates leukocyte infiltration through the up-regulation of adhesion molecules and chemokine release. To determine whether VEGF/VPF is directly involved in chemokine secretion, we analyzed its effects on chemokine expression in human brain microvascular endothelial cells (HBMECs) by using a human cytokine cDNA array kit. Cytokine array analysis revealed a significant increase in expression of monocyte chemoattractant protein-1 and the chemokine receptor CXCR4 in HBMECs, a result similar to that described previously in other endothelial cells. Interestingly, we also observed that VEGF/VPF induced interleukin-8 (IL-8) expression in HBMECs and that IL-8 mRNA was maximal after 1 h of VEGF/VPF treatment of the cells. Enzyme-linked immunosorbent assay data and immunoprecipitation analysis revealed that although VEGF/VPF induced IL-8 expression at the translational level in HBMECs, basic fibroblast growth factor failed to induce this protein expression within 12 h. VEGF/VPF increased IL-8 production in HBMECs through activation of nuclear factor-KB via calcium and phosphatidylinositol 3-kinase pathways, whereas the ERK pathway was not involved in this process. Supernatants of the VEGF/VPF-treated HBMECs significantly increased neutrophil migration across the HBMEC monolayer compared with those of the untreated control. Furthermore, addition of anti-IL-8 antibody blocked this increased migration, indicating that VEGF/VPF induced the functional expression of IL-8 protein in HBMECs. Taken together, these data demonstrate for the first time that VEGF/VPF induces IL-8 expression in HBMECs and contributes to leukocyte infiltration through the expression of chemokines, such as IL-8, in endothelial cells.     

Optical Waveguide Lightmode Spectroscopic Techniques for Investigating Membrane-Bound Ion Channel Activities

Optical waveguide lightmode spectroscopic (OWLS) techniques were probed for monitoring ion permeation through channels incorporated into artificial lipid environment. A novel sensor set-up was developed by depositing liposomes or cell-derived membrane fragments onto hydrophilic polytetrafluoroethylene (PTFE) membrane. The fibrous material of PTFE membrane could entrap lipoid vesicles and the water-filled pores provided environment for the hydrophilic domains of lipid-embedded proteins. The sensor surface was kept clean from the lipid holder PTFE membrane by a water- and ion-permeable polyethylene terephthalate (PET) mesh. The sensor set-up was tested with egg yolk lecithin liposomes containing gramicidin ion channels and with cell-derived membrane fragments enriched in GABA-gated anion channels. The method allowed monitoring the move of Na⁺ and organic cations through gramicidin channels and detecting the Cl^–-channel functions of the (α₅β₂γ₂) GABA_A receptor in the presence or absence of GABA and the competitive GABA-blocker bicuculline.     

The effect of starch and of a heteropolysaccharide fraction from Phaseolus vulgaris on development and fecundity of Callosobruchus chinensis (Coleoptera: Bruchidae)

A heteropolysaccharide fraction was isolated from Phaseolus vulgaris beans in which it comprises at least 1% of the dry weight of the beans. This heteropolysaccharide increases larval mortality and decreases the rate of larval development and the number of eggs deposited by females of Callosobruchus chinensis, when incorporated in artificial beans in which the larvae feed. It is composed of galactose, glucose, xylose, arabinose and traces of rhamnose, as determined after acid hydrolysis. Of these individual sugars, arabinose and xylose affect adult fecundity as well. However, partial enzymic hydrolysis of the heteropolysaccharide fraction by C. chinensis larval midgut contents releases only glucose, galactose and trace amounts of arabinose, and the integral structure of heteropolysaccharide may be necessary for biological activity. the incorporation of the starch granules of Phaseolus vulgaris beans into artificial beans increases larval mortality and decreases the rate of larval development of C. chinensis. It is suggested that the heteropolysaccharide fraction as well as the starch are part of a complex of natural components of Phaseolus vulgaris beans that make these beans resistant to C. chinensis.

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Résumé Ce travail cherche à préciser pourquoi les graines de haricot (Phaseolus vulgaris) ne sont pas attaquées par la Buche chinoise (Callosobruchus chinensis), espèce cependant très polyphage.Les graines de haricot renferment 1% d'un hétéropolysaccharide, qui s'est révélé accroître la mortalité et ralentir le développement des larves de cette Bruche, mais qui aussi réduit la fécondité des femelles adultes. Ces observations ont été faites à partir d'élevages sur des milieux artificiels normalement favorables, présentés sous forme de haricots, par un moulage approprié.Une hydrolyse acide de cet hétéropolysaccharide libère du galactose, du glucose, du xylose, de l'arabinose et des traces de rhamnose. Ces sucres simples sont testés: l'arabinose et le rhamnose influencent le développement des larves, tandis que le galactose, le xylose et à nouveau l'arabinose, ont un effet sur la fécondité des femelles (réduction de la fécondité à 40% de la normale avec 1% d'arabinose).Toutefois l'hydrolyse enzymatique de cette fraction hétéropolysaccharidique, par le contenu stomacal de larves de C. chinensis reste partielle et libère seulement du glucose, du galactose et des traces d'arabinose. II est possible que la structure intégrale de l'hétéropolysaccharide soit nécessaire à son activité biologique.Un autre facteur défavorable à C. chinensis pourrait être la nature même des grains d'amidon de Phaseolus. Cet amidon incorporé à l'aliment artificiel accroît en effet la mortalité larvaire et ralentit la vitesse de développement. Cet effet défavorable pourrait être dû à la non digestibilité des grains d'amidon entiers.Il est suggéré que la fraction hétéropolysaccharide et l'amidon de Phaseolus sont deux des facteurs présents dans la graine de haricot qui lui confèrent sa résistance naturelle à la Bruche chinoise.

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Supported in part by the Research and Development Authority of the Hebrew University of Jerusalem.     

Experimental and Theoretical Assessment of Ni‐Based Binary Compounds for the Hydrogen Evolution Reaction

Metallic binary compounds have emerged in recent years as highly active and stable electrocatalysts toward the hydrogen evolution reaction. In this work, the origin of their high activity from a theoretical and experimental point of view is elucidated. Here, different metallic ceramics as Ni₃S₂, Ni₃N, or Ni₅P₄ are grown directly on Ni support in order to avoid any contaminations. The correlation of theoretical calculations with detailed material characterization and electrochemical testing paves the way to a deeper understanding of possible active adsorption sites for each material and the observed catalytic activity. It is shown that heteroatoms as P, S, and N actively take part in the reaction and do not act as simple spectator. Due to the anisotropic nature of the materials, a variety of adsorption sites with highly coverage‐dependent properties exists, leading to a general shift in hydrogen adsorption free energies ΔG _H close to zero. Extending the knowledge gained about the here described materials, a new catalyst is prepared by modifying a high surface Ni foam, for which current densities up to 100 mA cm^?2 at around 0.15 V (for Ni₃N) are obtained.     

Generation of diverse neuronal subtypes in cloned populations of stem-like cells

Background  

The central nervous tissue contains diverse subtypes of neurons with characteristic morphological and physiological features and different neurotransmitter phenotypes. The generation of neurons with defined neurotransmitter phenotypes seems to be governed by factors differently expressed along the anterior-posterior and dorsal-ventral body axes. The mechanisms of the cell-type determination, however, are poorly understood. Selected neuronal phenotypes had been generated from embryonic stem (ES) cells, but similar results were not obtained on more restricted neural stem cells, presumably due to the lack of homogeneous neural stem cell populations as a starting material.     

A leukotriene receptor antagonist modulates iNOS in the lung and in a leukotriene-free cell model.

Nitric oxide (NO), an important cell signaling molecule, is considered a marker of inflammatory response and is elevated in asthmatics. This study investigated the effects of montelukast (a leukotriene receptor antagonist) on iNOS expression and activity in a Brown Norway (BN) rat allergic inflammation model and in L2 lung epithelial cells. Allergic inflammation was induced by ovalbumin (OVA) injection in BN rats followed by treatment with either montelukast or dexamethasone (DX). Allergen inhalation was performed, and post-allergen Penh was measured 5 min after the challenge. Cysteinyl leukotriene levels were measured in bronchoalveolar lavage (BAL) fluid and lung iNOS expression and activity determined. These parameters were also measured in cytokine stimulated L2 lung epithelial cells. iNOS expression was significantly higher in OVA challenged rats compared to the naive, DX, and montelukast treated groups, as confirmed by immunohistochemistry and Western blot analysis. However, no significant differences in NOS activity were found. Cysteinyl leukotriene measured in BAL was significantly higher in all OVA challenged rats compared to naive controls. Incubation of L2 cells with a mixture of interferon gamma (IFNgamma), lipopolysaccharide (LPS), and tumor necrosis factor (TNFalpha) resulted in high levels of nitrite formation resulting from iNOS induction. Treatment of cytokine stimulated cells with DX or montelukast significantly decreased iNOS expression and activity. No detectable cysteinyl leukotrienes were found in the supernatant fluid of L2 cells. This study confirms the ability of montelukast to modulate iNOS function and raises the possibility that changes in iNOS expression and activity may occur via pathways independent of cysteinyl leukotrienes.