High chlorpyrifos resistance in Culex pipiens mosquitoes: strong synergy between resistance genes.

We investigated the genetic determinism of high chlorpyrifos resistance (HCR), a phenotype first described in 1999 in Culex pipiens mosquitoes surviving chlorpyrifos doses ⩾1 mg l(-1) and more recently found in field samples from Tunisia, Israel or Indian Ocean islands. Through chlorpyrifos selection, we selected several HCR strains that displayed over 10 000-fold resistance. All strains were homozygous for resistant alleles at two main loci: the ace-1 gene, with the resistant ace-1(R) allele expressing the insensitive G119S acetylcholinesterase, and a resistant allele of an unknown gene (named T) linked to the sex and ace-2 genes. We constructed a strain carrying only the T-resistant allele and studied its resistance characteristics. By crossing this strain with strains harboring different alleles at the ace-1 locus, we showed that the resistant ace-1(R) and the T alleles act in strong synergy, as they elicited a resistance 100 times higher than expected from a simple multiplicative effect. This effect was specific to chlorpyrifos and parathion and was not affected by synergists. We also examined how HCR was expressed in strains carrying other ace-1-resistant alleles, such as ace-1(V) or the duplicated ace-1(D) allele, currently spreading worldwide. We identified two major parameters that influenced the level of resistance: the number and the nature of the ace-1-resistant alleles and the number of T alleles. Our data fit a model that predicts that the T allele acts by decreasing chlorpyrifos concentration in the compartment targeted in insects.

Controlled surface density of RGD ligands for cell adhesion: evidence for ligand specificity by using QCM-D.

RGD peptides (Arg-Gly-Asp) are known to promote cell adhesion. As a consequence, numerous materials have been functionalized using these peptides for several medical applications. We report herein the controlled functionalization of surfaces to study the influence of RGD density on cell selectivity. For this purpose, we selected a quartz crystal microbalance QCM-D as this technique allows real-time monitoring of cell adhesion to RGD surfaces. We observed that a critical spacing of nearly 40 nm between RGD ligands is required to observe selective cell adhesion whereas a higher density is not specific.

Genetic variations in host IL28B links to the detection of peripheral blood mononuclear cells-associated hepatitis C virus RNA in chronically infected patients.

Hepatitis C virus (HCV) is mainly hepatotropic; however, several reports document the presence of genomic viral RNA in extrahepatic sites including peripheral blood mononuclear cells (PBMCs). In this study, the presence of HCV RNA was initially evaluated in the plasma and peripheral blood mononuclear cells (PBMCs) of 53 HCV-infected patients who were treated per protocol. PBMC-associated HCV RNA was detectable in 79% of patients. Early virological response to combined pegylated interferon-α (PegIFN) and ribavirin (RBV) therapy in patients with undetectable levels of PBMCs-associated HCV RNA was 100%, while it was 60% (P = 0.003) in those who had detectable levels of PBMC-associated HCV RNA. A sustained virological response was observed in 35% of patients with detectable PBMC-associated HCV RNA, but was 70% in patients with undetectable levels of PBMC-associated HCV RNA (P = 0.07). In a multivariate analysis incorporating parameters such as HCV genotype, viral load, presence of cirrhosis and absence of PBMC-associated HCV RNA, a significant relationship was observed between the detection of PBMC-associated HCV RNA and the sustained virological response (OR 19.4, 95% CI: 2.1-486.2, P = 0.0061). The association between single nucleotide polymorphism (SNP) in IL28B, known predictor of antiviral therapy outcome, and the occurrence of HCV RNA in PBMC in 84 chronically infected patients was then evaluated. Results suggest that the presence of a G allele in rs8099917, known to associate to a poor response to PegIFN/RBV therapy, also predicts an increased association of HCV RNA with PBMC (OR: 3.564; 95% CI: 1.114-11.40, P = 0.0437).

Unraveling the spatial distribution of immunoglobulins, enzymes, and polyelectrolytes within layer-by-layer self-assembled multilayers. Ellipsometric studies.

The ellipsometric characterization of a layer-by-layer electrostatically self-assembled multilayer of polyphenol oxidase and alkaline phosphatase with the polycation poly(dimethyldiallylammonium chloride) built on an immunologic layer formed by immunoglobulin G (IgG) and glucose oxidase-conjugated anti-IgG (IgG-GOD) on glassy carbon is reported. The step-by-step evolution of the psi-Delta ellipsometric angles was followed during film growth. Two optical models, named the three-layer film model and reorganization film model, were employed and found suitable for ellipsometric data interpretation. A comparative analysis of film optical properties, film thickness, and ellipsometric mass assessed from both models is also presented.

On the worldwide spread of an insecticide resistance gene: a role for local selection.

Adaptation occurs by gene replacement (or transient balanced polymorphism). Replacement may be caused by selection (local or global) and/or genetic drift among alleles. In addition, historical events may blur the respective effects of selection and drift during the course of replacement. We address the relative importance of these processes in the evolution of insecticide resistance genes in the mosquito Culex pipiens. The resistance allele, Ester2, has a broad geographic distribution compared to the other resistance alleles. To distinguish between the different processes explaining this distribution, we reviewed the literature and analysed updated data from the Montpellier area of southern France. Overall, our data indicate that Ester2 prevails over other Ester resistance alleles in moderately treated areas. Such conditions are common and favour the hypothesis of selection acting at a local level. This places an emphasis on the importance of ecological conditions during the evolution of resistance. Finally, we highlight that historical events have contributed to its spread in some areas.

Multivalent ion/polyelectrolyte exchange processes in exponentially growing multilayers.

We show, in this paper that multivalent ferrocyanide anions can penetrate into exponentially growing (PGA/PAH)n multilayer films whatever the nature of the last deposited layer. These ions are not able to diffuse out of the film when it is brought in contact with a pure buffer solution. However, the contact of this film with a poly(allylamine) (PAH) or a poly(L-glutamic acid) (PGA) solution leads to the release of ferrocyanide ions from the multilayer. It is shown that the release of ferrocyanide anions, when the film is in contact with a PGA solution, is due to the diffusion of the PGA chains into the film so that an exchange between ferrocyanide ions and PGA chains takes place inside the film. On the other hand, PAH chains do not diffuse into PGA/PAH multilayers. When the film is then brought in contact with a PAH solution, the PAH chains from the solution are expected to strongly interact with the ferrocyanide ions and thus induce a diffusion mechanism of the multivalent anions out of the film, the film/solution interface playing the role of a sink for these ions. This work thus shows that interactions between multivalent ions and exponentially growing films are much more complex than expected at first sight and that polyelectrolyte multilayers must be seen as dynamic entities in which diffusion and exchange processes can take place.

Phosphate tungsten bronze series: crystallographic and structural properties of low-dimensional conductors.

Phosphate tungsten bronzes have been shown to be conductors of low dimensionality. A review of the crystallographic and structural properties of this huge series of compounds is given here, corresponding to the present knowledge of the different X-ray studies and electron microscopy investigations. Three main families are described, monophosphate tungsten bronzes, Ax(PO2)4(WO3)2m, either with pentagonal tunnels (MPTBp) or with hexagonal tunnels (MPTBh), and diphosphate tungsten bronzes, Ax(P2O4)2(WO3)2m, mainly with hexagonal tunnels (DPTBh). The general aspect of these crystal structures may be described as a building of polyhedra sharing oxygen corners made of regular stacking of WO3-type slabs with a thickness function of m, joined by slices of tetrahedral PO4 phosphate or P2O7 diphosphate groups. The relations of the different slabs with respect to the basic perovskite structure are mentioned. The structural description is focused on the tilt phenomenon of the WO6 octahedra inside a slab of WO3-type. In this respect, a comparison with the different phases of the WO3 crystal structures is established. The various modes of tilting and the different possible connections between two adjacent WO3-type slabs involve a great variety of structures with different symmetries, as well as the existence of numerous twins in MPTBp's. Several phase transitions, with the appearance of diffuse scattering and modulation phenomena, were analysed by X-ray scattering measurements and through the temperature dependence of various physical properties for the MPTBp's. The role of the W displacements within the WO3-type slabs, in two modulated structures (m = 4 and m = 10), already solved, is discussed. Finally, the complexity of the structural aspects of DPTBh's is explained on the basis of the average structures which are the only ones solved.

Amplification of amperometric biosensor responses by electrochemical substrate recycling. 3. Theoretical and experimental study of the phenol-polyphenol oxidase system immobilized in Laponite hydrogels and layer-by-layer self-assembled structures.

The amperometric response toward phenol of PPO-based rotating disk bioelectrodes is analyzed on the basis of a kinetic model taking into account internal and external mass transport effects and a CEC' electroenzymatic mechanism. Monophenolase activity of PPO catalyses the oxidation of phenol to o-quinone (step C). o-Quinone can then enter an amplification recycling process involving electrochemical reduction (step E) and enzymatic reoxidation (step C': catecholase activity). The rate-limiting steps such as monophenolase activity, catecholase recycling, permeability of the membrane, and activity and accessibility of the catalytic enzyme sites are theoretically considered and experimentally demonstrated for different electrode configurations including PPO immobilized in Laponite hydrogels and layer-by-layer self-assembled multilayers of PPO and poly(diallyldimethylammonium).

Symmetry and twins in the monophosphate tungsten bronze series (P02)4(W03)2m (2 < or = m < or = 14)

Monophosphate tungsten bronze with pentagonal tunnels (PO2)4(WO3)2m are low-dimensional materials with charge density wave (CDW)-type electron instabilities. Two forms of the structure can thus be expected for all the members of the series: a low-temperature form (LT) corresponding to the CDW state and a high-temperature form (HT) corresponding to a normal metallic state. The HT form is described here for m = 9 and compared with that of the m = 5 and m = 7 counterparts. It is shown that a systematic twin phenomenon must be taken into account for HT members because of two possible configurations of the tilting mode of WO6 octahedra, The structure is also compared with that of m = 10, which exhibits the modulated CDW-LT form at room temperature. Owing to two possible polarization directions of the segments built of m WO6 octahedra, a twin phenomenon is also encountered in the LT forms. A review of all the structures known at present (m = 2, 4, 5, 6, 7, 8, 9, 10, 12) leads us to propose a structural law based on the building mode of W06 octahedra in W03-type slabs to explain the symmetry changes observed between even and odd members of the series.

Cluster configurations in modulated EuVxMo8+/-yO14 crystals.

Three orthorhombic crystals of chemical formula Eu(x)V(y)Mo(8+/-z)O(14) were investigated by X-ray diffraction (Mo Kalpha radiation, lambda = 0.71073 Å). They have nearly the same lattice parameters (a approximately 11.3, b approximately 10.0, c approximately 9.2 Å), display one-dimensional incommensurate modulations of wavevector q* = gammac* and are characterized by the same superspace group Cmca(00gamma)s00. The crystals differ both in their compositions (namely Eu(0.976(6))V(1.13(5))Mo(7.10(5))O(14), Eu(0.986(4))V(1.10(3))Mo(7.30(1))O(14) and EuMo(7.96(1))O(14)) and in their gamma components [0.195 (2), 0.245 (2) and 0.286 (3), respectively]. The average structures of these crystals appear closely related to the structures of LaMo(7.7)O(14) (not modulated) and LaMo(8)O(14) (modulated); however, two main differences are outlined: first, the modulation direction is c in the Eu-containing crystals but b in the modulated La-containing crystal [q* = (1/3)b*], second, the Eu-containing crystals have centrosymmetric structures while the La-containing crystals have polar structures (space group C2ca). The Mo (or Mo and V) atoms are stacked to form (001) layers of metallic clusters. The density modulation of these structures implies the existence of the new types of clusters Mo(9), Mo(10), Mo(6)V(4), Mo(7)V(3) and Mo(8)V(2) besides the clusters M(8) (Mo(8), Mo(6)V(2) and Mo(7)V) and M(7) (Mo(7) and Mo(6)V) which are already known. Mo(8) units with cis and trans configurations and Mo(6)V(2) units with a trans configuration appear as the main cluster types in these crystals. The nature of the metallic clusters changes along c, but inside one (001) layer it is likely that only one cluster type with a given configuration is present. The main structural result is the formation, in some unit cells, of strong intercluster Mo-Mo, Mo-V or V-V bonds with distances close to 2.6 Å within a layer as well as between two neighbouring layers.

Functional responses of human neutrophils to sodium sulfite (Na2SO3) in vitro.

An influx of neutrophils into the airways is a common feature observed during pulmonary inflammation induced by air pollutants, including sulfur dioxide and sulfates. In the present study focusing on the in vitro interactions of sodium sulfite (Na2SO3) with human neutrophils, we confirm results indicating that this sulfite induces superoxide production (O2-) by itself. We demonstrated that this response can occur more rapidly than previously reported (within 5 min), and that Na2SO3 can act as a priming agent, in a concentration-dependent fashion, to the bacterial tripeptide N-formyl-methionine-leucine-phenylalanine (fMLP) by increasing O2-production. In addition, our results show that Na2SO3 induces gene expression in human neutrophils in a concentration-dependent manner as assessed by incorporation of 5-[3H] uridine into total RNA. However, it does not induce cell shape changes. We also demonstrated that Na2SO3 does not modulate neutrophil apoptosis nor reverse the well-known delaying effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on apoptosis. We conclude that Na2SO3 acts rapidly on neutrophil physiology, within a few minutes with respect to superoxide production, and a few hours (4 h) with respect to gene expression without altering a biological process such as the rate of apoptosis evaluated after a long period of incubation (20 h). We further conclude that Na2SO3-induced production of O2does not drive neutrophils to undergo apoptosis, a mechanism known to occur in other conditions. Therefore, the potential toxicity of Na2SO3 during pulmonary inflammation or lung-associated diseases may be related to its ability to induce superoxide production without altering neutrophil apoptosis rate.

Epidemiology and optimal management of polymyalgia rheumatica.

Polymyalgia rheumatica (PMR) is a disease of unknown aetiology that occurs in elderly patients, predominantly affecting the Caucasian population. The disease has a slightly higher prevalence in women than in men. There is ongoing discussion regarding the relationship between PMR and giant cell arteritis; an increasing number of studies indicate that they are closely related. PMR has also been linked with rheumatoid arthritis, myopathy and malignant disease. Oral corticosteroids remain the mainstay of drug therapy for PMR. These drugs usually induce prompt relief of symptoms, and some authors consider this dramatic response to be diagnostic for PMR. However, the ideal initial dosage, the duration of treatment and the optimal tapering schedule are much debated. Other drugs, such as methotrexate and azathioprine, have been suggested as corticosteroid sparing agents. Nonsteroidal anti-inflammatory drugs are generally considered to be unsuitable for the long term treatment of PMR.

Cytochrome P-450 reductase is responsible for the ferrireductase activity associated with isolated plasma membranes of Saccharomyces cerevisiae.

Cytochrome P-450 reductase (encoded by the NCP1 gene) was found to catalyse all the NADPH-dependent ferrireductase activities associated with isolated plasma membranes of the yeast Saccharomyces cerevisiae. We therefore examined the contribution of this enzyme to the ferrireductase activity of cells in vivo. Cytochrome P-450 reductase was shown to be not essential for the cell ferrireductase activity, but it influenced this activity, with different effects on the Fre1- and the Fre2-dependent reductase systems. Overexpression of FRE1 did not lead to an increased ferrireductase activity of the cells when NCP1 was repressed. In contrast, cells that overexpressed FRE2 had maximal ferrireductase activity when NCP1 was repressed. The degree of NCP1 expression also affected the amount of iron and copper accumulated by the cells during growth. The biochemical implications and the physiological significance of these observations are discussed.

Human coproporphyrinogen oxidase. Biochemical characterization of recombinant normal and R231W mutated enzymes expressed in E. coli as soluble, catalytically active homodimers.

To obtain recombinant human coproporphyrinogen oxidase (CPX), a cDNA for the coding region of mature human CPX has been expressed in E. coli. CPX was produced as a fusion protein with glutathione S-transferase followed by the hexapeptide recognition site for thrombin cleavage just preceding first amino acid of the CPX protein. The human CPX was found to be in the soluble fraction. This previously unobtainable human heme synthetic enzyme was purified to electrophoretic homogeneity with a specific activity of 4200 nmol/hr./mg of protein using a Glutathione Sepharose 4B column and gel filtration. Recombinant human CPX exhibits homogeneous behavior during high performance liquid chromatography (HPLC) and the N-terminal sequence, confirmed by protein sequencing, revealed a single polypeptide chain. In its active form, human CPX is a homodimer. According to the hydrodynamic properties derived from analytical ultracentrifugation, dimeric CPX has a nearly globular shape. Additionally, naturally occurring Arg to Trp (R231W)-mutated CPX has been also expressed in E. coli and further characterized. The mutated enzyme has a Km value of 0.55 microM as compared to 0.30 microM for the wild type. The catalytic efficiency (specificity constant, kcat/Km) of the mutated CPX was four fold lower than wild-type enzyme. The activity measurement of the mutated enzyme showed higher thermal sensitivity as compared with wild type CPX. The measured pI for mutated CPX is 5.65, compared to 6.40 for wild type. The pH optima for the mutated and wild-type protein are 6.6 and 6.8, respectively. The R231W mutation of CPX does not affect dimer formation and both normal and mutated CPX exhibit identical sedimentation properties. The thermal denaturation of both wild type and mutant CPX was found to be irreversible. The mutated CPX contained a significant amount of tightly bound porphyrin coproporphyrin. No metal association was found either in wild type or in mutated CPX. The availability of the recombinant human CPX will aid in structural and mechanistic studies.