Asymmetric lipid bilayer formation stabilized by DNA at the air/water interface

The lipid bis(guanidinium)-tren-cholesterol (BGTC) is a cationic cholesterol derivative bearing guanidinium polar headgroups used for gene transfection either alone or formulated as liposomes with the zwitterionic lipid 1,2-di-[cis-9-octadecenoyl]-sn-glycero-3-phosphoethanolamine (DOPE). Previous investigations have shown its ability to strongly interact with DNA and form asymmetric lipid bilayers at the air/water interface when mixed with DOPE. Here, with a view to further investigate its physicochemical behavior, we studied the interactions of mixtures of BGTC with another zwitterionic lipid, 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine, (DMPC), with DNA at the air/water interface by using the Langmuir monolayer technique coupled with Brewster Angle Microscopy (BAM) and Polarization Modulation Infra Red Reflexion Absorption (PMIRRAS) spectroscopy and we investigate DNA–BGTC/DMPC interactions. We demonstrate that when DNA is injected into the subphase in excess compared to the positive charges of BGTC, it adsorbs to BGTC/DMPC monolayers at 20 mN/m whatever the lipid monolayer composition (1/5, 2/3 or 3/2 BGTC/DMPC molar ratio) and forms an incomplete monolayer of either isotropic or anisotropic double strands depending on the BGTC content in the monolayer. Compression beyond the collapse of some mixed DNA–BGTC/DMPC (2/3 and 3/2 molar ratio) systems leads to the formation of DNA monolayers underneath asymmetric lipid bilayers characterized by a bottom layer of BGTC in contact with DNA and a top layer mainly constituted of DMPC.     

Study of pyoverdine type and production by Pseudomonas aeruginosa isolated from cystic fibrosis patients: prevalence of type II pyoverdine isolates and accumulation of pyoverdine-negative mutations

The lungs of cystic fibrosis patients are frequently colonized by Pseudomonas aeruginosa, which produces high-affinity fluorescent peptidic siderophores, pyoverdines. Three pyoverdines which differ in their peptide chain and are easily differentiated by isoelectric focusing exist, only one being produced by a given strain. P. aeruginosa isolates from cystic fibrosis patients of a German hospital were analyzed by sequential, pulse-field gel electrophoresis (PFGE) and for pyoverdine production and type. Only producers of type I and type II pyoverdine were found. There was a perfect correlation between the type of pyoverdine produced and the clonality determined by PFGE. PFGE clone C, the most prevalent among cystic fibrosis patients, and found in an aquatic environment, produced type II pyoverdine. Pyoverdine-negative mutants seemed to increase as a function of the lung colonization time, but retained the capacity to take up pyoverdines. Most isolates that took up type II pyoverdine were also able to utilize type I pyoverdine as judged by growth stimulation experiments. No correlation was observed between the loss of pyoverdine production and mucoidy.     

Expression of protein phosphatase 2A mutants and silencing of the regulatory B alpha subunit induce a selective loss of acetylated and detyrosinated microtubules

Carboxymethylation and phosphorylation of protein phosphatase 2A (PP2A) catalytic C subunit are evolutionary conserved mechanisms that critically control PP2A holoenzyme assembly and substrate specificity. Down-regulation of PP2A methylation and PP2A enzymes containing the B alpha regulatory subunit occur in Alzheimer's disease. In this study, we show that expressed wild-type and methylation- (L309 Delta) and phosphorylation- (T304D, T304A, Y307F, and Y307E) site mutants of PP2A C subunit differentially bind to B, B', and B'-type regulatory subunits in NIH 3T3 fibroblasts and neuro-2a (N2a) neuroblastoma cells. They also display distinct binding affinity for microtubules (MTs). Relative to controls, expression of the wild-type, T304A and Y307F C subunits in N2a cells promotes the accumulation of acetylated and detyrosinated MTs. However, expression of the Y307E, L309 Delta, and T304D mutants, which are impaired in their ability to associate with the B alpha subunit, induces their loss. Silencing of B alpha subunit in N2a and NIH 3T3 cells is sufficient to induce a similar breakdown of acetylated and detyrosinated MTs. It also confers increased sensitivity to nocodazole-induced MT depolymerization. Our findings suggest that changes in intracellular PP2A subunit composition can modulate MT dynamics. They support the hypothesis that reduced amounts of neuronal B alpha-containing PP2A heterotrimers contribute to MT destabilization in Alzheimer's disease.     

Nitrate efflux is an essential component of the cryptogein signaling pathway leading to defense responses and hypersensitive cell death in tobacco

There is much interest in the transduction pathways by which avirulent pathogens or derived elicitors activate plant defense responses. However, little is known about anion channel functions in this process. The aim of this study was to reveal the contribution of anion channels in the defense response triggered in tobacco by the elicitor cryptogein. Cryptogein induced a fast nitrate (NO(3)(-)) efflux that was sensitive to anion channel blockers and regulated by phosphorylation events and Ca(2+) influx. Using a pharmacological approach, we provide evidence that NO(3)(-) efflux acts upstream of the cryptogein-induced oxidative burst and a 40-kD protein kinase whose activation seems to be controlled by the duration and intensity of anion efflux. Moreover, NO(3)(-) efflux inhibitors reduced and delayed the hypersensitive cell death triggered by cryptogein in tobacco plants. This was accompanied by a delay or a complete suppression of the induction of several defense-related genes, including hsr203J, a gene whose expression is correlated strongly with programmed cell death in plants. Our results indicate that anion channels are involved intimately in mediating defense responses and hypersensitive cell death.     

Crystal structure of the measles virus phosphoprotein domain responsible for the induced folding of the C-terminal domain of the nucleoprotein

Measles virus is a negative-sense, single-stranded RNA virus belonging to the Mononegavirales order which comprises several human pathogens such as Ebola, Nipah, and Hendra viruses. The phosphoprotein of measles virus is a modular protein consisting of an intrinsically disordered N-terminal domain (Karlin, D., Longhi, S., Receveur, V., and Canard, B. (2002) Virology 296, 251-262) and of a C-terminal moiety (PCT) composed of alternating disordered and globular regions. We report the crystal structure of the extreme C-terminal domain (XD) of measles virus phosphoprotein (aa 459-507) at 1.8 A resolution. We have previously reported that the C-terminal domain of measles virus nucleoprotein, NTAIL, is intrinsically unstructured and undergoes induced folding in the presence of PCT (Longhi, S., Receveur-Brechot, V., Karlin, D., Johansson, K., Darbon, H., Bhella, D., Yeo, R., Finet, S., and Canard, B. (2003) J. Biol. Chem. 278, 18638-18648). Using far-UV circular dichroism, we show that within PCT, XD is the region responsible for the induced folding of NTAIL. The crystal structure of XD consists of three helices, arranged in an anti-parallel triple-helix bundle. The surface of XD formed between helices alpha2 and alpha3 displays a long hydrophobic cleft that might provide a complementary hydrophobic surface to embed and promote folding of the predicted alpha-helix of NTAIL. We present a tentative model of the interaction between XD and NTAIL. These results, beyond presenting the first measles virus protein structure, shed light both on the function of the phosphoprotein at the molecular level and on the process of induced folding.     

Interference of activated factor VII in apoptosis of erytholeukemic K562 cells

Coagulation factor VIIa (FVIIa) is a key protease initiating the coagulation cascade in the presence of its receptor, tissue factor (TF). FVIIa elicits several cellular responses, probably involving other receptors(s) than TF. This study investigates the implication of recombinant FVIIa on the apoptosis of K562 erythroleukemia cells. These cells undergo apoptosis when induced to differentiate towards the erythroid lineage by hemin. They do not express TF, but can be transfected to do so. FVIIa treatment significantly reduced the degree of hemin-induced apoptosis in K562 cells, but not in TF+ derived transfectants. Induction of apoptosis by hemin also elicited decrease in intracellular Ca2+ concentration ([Ca2+]i), but FVIIa restored this [Ca2+]i close to that of non-treated cells. These results suggest that FVIIa acts via a TF-independent pathway to counteract apoptosis by a mechanism involving its Gla domain and linked to the maintenance of Ca2+ homeostasis in K562 cells.     

Siderophore-mediated iron acquisition in the entomopathogenic bacterium Pseudomonas entomophila L48 and its close relative Pseudomonas putida KT2440

Pseudomonas entomophila L48 is a recently identified entomopathogenic bacterium which, upon ingestion, kills Drosophila melanogaster, and is closely related to P. putida. The complete genome of this species has been sequenced and therefore a genomic, genetic and structural analysis of the siderophore-mediated iron acquisition was undertaken. P. entomophila produces two siderophores, a structurally new and unique pyoverdine and the secondary siderophore pseudomonine, already described in P. fluorescens species. Structural analysis of the pyoverdine produced by the closely related P. putida KT2440 showed that this strain produces an already characterised pyoverdine, but different from P. entomophila, and no evidence was found for the production of a second siderophore. Growth stimulation assays with heterologous pyoverdines demonstrated that P. entomophila is able to utilize a large variety of structurally distinct pyoverdines produced by other Pseudomonas species. In contrast, P. putida KT2440 is able to utilize only its own pyoverdine and the pyoverdine produced by P. syringae LMG 1247. Our data suggest that although closely related, P. entomophila is a more efficient competitor for iron than P. putida.     

The Enterobacter aerogenes outer membrane efflux proteins TolC and EefC have different channel properties

The outer membrane proteins TolC and EefC from Enterobacter aerogenes are involved in multidrug resistance as part of two resistance-nodulation-division efflux systems. To gain more understanding in the molecular mechanism underlying drug efflux, we have undertaken an electrophysiological characterization of the channel properties of these two proteins. TolC and EefC were purified in their native trimeric form and then reconstituted in proteoliposomes for patch-clamp experiments and in planar lipid bilayers. Both proteins generated a small single channel conductance of about 80 pS in 0.5 M KCl, indicating a common gated structure. The resultant pores were stable, and no voltage-dependent openings or closures were observed. EefC has a low ionic selectivity (P(K)/P(Cl)= approximately 3), whereas TolC is more selective to cations (P(K)/P(Cl)= approximately 30). This may provide a possible explanation for the difference in drug selectivity between the AcrAB-TolC and EefABC efflux systems observed in vivo. The pore-forming activity of both TolC and EefC was severely inhibited by divalent cations entering from the extracellular side. Another characteristic of the TolC and EefC channels was the systematic closure induced by acidic pH. These results are discussed in respect to the physiological functions and structural models of TolC and EefC.     

Nuclear localization of two steroid receptor-associated proteins, hsp90 and p59

It has been proposed that the unliganded nontransformed form of steroid hormone receptor is a heterooligomer comprising, in addition to the hormone-binding subunit, two associated proteins: a heat shock protein of MW 90,000 (hsp90) and another protein of MW 59,000 (p59). Using monoclonal antibodies, we demonstrate immunocytochemically the presence of both hsp90 and p59 in cell nuclei of progesterone target cells of the rabbit uterus. While steroid receptors (e.g., progesterone receptors) appear to be exclusively nuclear, we find p59 predominantly in the cell nuclei and hsp90 in both the nucleus and the cytoplasm. In addition, Western blotting of high-salt extracts of nuclear proteins detects the presence of hsp90 and p59 in the nuclei of rabbit uterus. These observations are consistent with the presence of the untransformed heterooligomeric form of steroid hormone receptors in the nuclei of target cells.     

Modeling the three-dimensional structure of H+-ATPase of Neurospora crassa.

Homology modeling in combination with transmembrane topology predictions are used to build the atomic model of Neurospora crassa plasma membrane H+-ATPase, using as template the 2.6 A crystal structure of rabbit sarcoplasmic reticulum Ca2+-ATPase [Toyoshima, C., Nakasako, M., Nomura, H. & Ogawa, H. (2000) Nature 405, 647-655]. Comparison of the two calcium-binding sites in the crystal structure of Ca2+-ATPase with the equivalent region in the H+-ATPase model shows that the latter is devoid of most of the negatively charged groups required to bind the cations, suggesting a different role for this region. Using the built model, a pathway for proton transport is then proposed from computed locations of internal polar cavities, large enough to contain at least one water molecule. As a control, the same approach is applied to the high-resolution crystal structure of halorhodopsin and the proton pump bacteriorhodopsin. This revealed a striking correspondence between the positions of internal polar cavities, those of crystallographic water molecules and, in the case of bacteriorhodopsin, the residues mediating proton translocation. In our H+-ATPase model, most of these cavities are in contact with residues previously shown to affect coupling of proton translocation to ATP hydrolysis. A string of six polar cavities identified in the cytoplasmic domain, the most accurate part of the model, suggests a proton entry path starting close to the phosphorylation site. Strikingly, members of the haloacid dehalogenase superfamily, which are close structural homologs of this domain but do not share the same function, display only one polar cavity in the vicinity of the conserved catalytic Asp residue.