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.

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).

Kinetic studies of ferrochelatase in yeast. Zinc or iron as competing substrates.

Ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1) has been studied in yeast mitochondrial membranes with special reference to zinc-chelatase and iron-chelatase activities. Using physiological substrates (protoporphyrin IX, Fe(II) and Zn(II), anaerobic conditions of incubation and direct spectrophotometric assay, apparent Km values smaller than those previously described were found for the membrane-bound enzyme. Fe(II) but not Fe(III) was a strong competitive inhibitor of zinc-chelatase activity, while Zn(II) was a slight competitive inhibitor of iron-chelatase activity. These results could point to modes of control of ferrochelatase activity in yeast. We suggest that reduced supply of Fe(II) may explain the in vivo accumulation of zinc-protoporphyrin in yeast cells incubated under 'resting' conditions.

Iron-reductases in the yeast Saccharomyces cerevisiae.

Several NAD(P)H-dependent ferri-reductase activities were detected in sub-cellular extracts of the yeast Saccharomyces cerevisiae. Some were induced in cells grown under iron-deficient conditions. At least two cytosolic iron-reducing enzymes having different substrate specificities could contribute to iron assimilation in vivo. One enzyme was purified to homogeneity: it is a flavoprotein (FAD) of 40 kDa that uses NADPH as electron donor and Fe(III)-EDTA as artificial electron acceptor. Isolated mitochondria reduced a variety of ferric chelates, probably via an 'external' NADH dehydrogenase, but not the siderophore ferrioxamine B. A plasma membrane-bound ferri-reductase system functioning with NADPH as electron donor and FMN as prosthetic group was purified 100-fold from isolated plasma membranes. This system may be involved in the reductive uptake of iron in vivo.

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.

Protoporphyrinogen oxidase inhibition by three peroxidizing herbicides: oxadiazon, LS 82-556 and M&B 39279.

Three chemically unrelated peroxidizing molecules, namely oxadiazon [5-(t-butyl)-3-(2,4-dichloro-5-isopropoxyphenyl)-1,3,4-oxadiazol-2 -one], LS 82-556 [(S)3-N-(methylbenzyl)carbamoyl-5-propionyl-2,6-lutidine] and M&B 39279 [5-amino-4-cyano-1-(2,6-dichloro-4-trifluoromethylphenyl)pyrazol], are potent inhibitors of plant, yeast and mouse protoporphyrinogen oxidase.

Some properties of yeast mitochondria prepared by a rapid mechanical procedure.

A method is described for preparing yeast mitochondria rapidly (within one hour) by using the MSK Bronwill Cell homogenizer. Yeast mitochondria obtained by the method exhibit relatively good respiratory controls and ADP/O ratios. The method is convenient for small or large amounts of yeast cells (from 5 to a hundred grams, wet weight) and gave a yield of 3 to 5 mg protein/g wet weight of yeast mitochondria.

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.

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.

Ferric iron reduction and iron assimilation in Saccharomyces cerevisiae.

We have used the yeast Saccharomyces cerevisiae as a model organism to study the role of ferric iron reduction in eucaryotic iron uptake. S. cerevisiae is able to utilize ferric chelates as an iron source by reducing the ferric iron to the ferrous form, which is subsequently internalized by the cells. A gene (FRE1) was identified which encodes a protein required for both ferric iron reduction and efficient ferric iron assimilation, thus linking these two activities. The predicted FRE1 protein appears to be a membrane protein and shows homology to the beta-subunit of the human respiratory burst oxidase. These data suggest that FRE1 is a structural component of the ferric reductase. Subcellular fractionation studies showed that the ferric reductase activity of isolated plasma membranes did not reflect the activity of the intact cells, implying that cellular integrity was necessary for function of the major S. cerevisiae ferric reductase. An NADPH-dependent plasma membrane ferric reductase was partially purified from plasma membranes. Preliminary evidence suggests that the cell surface ferric reductase may, in addition to mediating cellular iron uptake, help modulate the intracellular redox potential of the yeast cell.

Improvement of poly(amphiphilic pyrrole) enzyme electrodes via the incorporation of synthetic laponite-clay-nanoparticles.

The electropolymerization of an enzyme-amphiphilic pyrrole ammonium-laponite nanoparticles mixture preadsorbed on the electrode surface provides the simultaneous immobilization of the enzyme and the hydrophilic laponite-clay-nanoparticles in a functionalized polypyrrole film. The presence of incorporated laponite particles within the electrogenerated polymer induces a strong improvement of the analytical performances (I(max) and sensitivity) of amperometric biosensors based on polyphenol oxidase. These beneficial effects have been attributed to a marked enhancement of the apparent specific activity of the immobilized enzyme (from 0.21 to 0.85% of the specific activity of the free enzyme), the permeability of the host polymer being unchanged. This strategy of biosensor performance improvement was tested with cholesterol oxidase as an enzyme model. The presence of laponite additive in the poly(amphiphilic pyrrole) host matrix induces a similar enhancement of sensitivity and I(max) for cholesterol biosensing as well as a large improvement of the storage stability of the polypyrrole-cholesterol oxidase electrode.

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.

[HLA DRB1 polymorphism in rhizomelic pseudo-polyarthritis and Horton disease]. / Etude du polymorphisme HLA DRB1 au cours de la pseudopolyarthrite rhizomélique et de la maladie de Horton.

Some studies have suggested that distribution of HLA DRB1 alleles in polymyalgia rheumatica (PMR) resembles that found in giant cell arteritis (GCA). However these data are controversial. OBJECTIVE--To evaluate in French native patients whether PMR immunogenetically resembles GCA in determining HLA DRB1 alleles. PATIENTS AND METHODS--Fourty-five patients were included in the study. Twenty-one patients with PMR alone (Bird's criteria) and 24 with GCA (ACR criteria). In 11 patients, GCA was associated with PMR. HLA DRB1 genotype was determined by PCR-RFLP analysis. Statistical analysis was performed by the chi 2 test and determination of the odds ratio (OR). Two hundred and thirty-three unselected normal healthy subjects served as controls. RESULTS--A significant increased prevalence of HLA DR1 was observed in patient with PMR alone and an absence of DR7 (0% vs 10.3%, p = 0.02, OR = 0.1). An increased incidence of DR4 and particularly *0401 allele was only found in patients with GCA (OR = 2.4). No patient with isolated PMR had DR7 genotype compared with 25% in GCA (p < 0.001, OR = 0.03). A comparative study between isolated PMR versus GCA showed a significant increased in DR1 and DR3 alleles in isolated PMR and a significant increased prevalence of DR4 and DRB1 *0701 allele in GCA. CONCLUSION--The present study emphasizes the absence of similarity in HLA DRB1 allele distribution between PMR and GCA. The association of DR7 in patient with GCA seems characteristic in French native patients.

[Osteoporotic vertebral crush fractures with severe neurologic manifestations. Apropos of 6 cases]. / Tassements vertébraux ostéoporotiques avec complications neurologiques sévères. A propos de six observations.

Osteoporotic vertebral crush fractures with neurologic complications are rarely reported in the literature. We report six new cases particularly severe in which death occurred in two cases. The study group included four women and two men with a mean age of 75 years (range: 72-79). Vertebral collapse causing neurological deficit was T5, T9, T11 in two cases, L1 and L3. The mean number of vertebral collapses was three per patient (range: 1-9). Back pain appeared without traumatism 6 weeks before admission (range: 1-24). Neurological complications appeared 2.5 weeks after back pain (range: 1-8). One patient suffered from a paraplegia, three from a paraparesia with bladder dysfunction (n = 1). In one case there was a severe weakness of the levator muscles of the foot and in another a L3 femoral neuralgia with severe bowel and bladder dysfunction. X-rays demonstrated backwards displacement of the posterior cortex in three cases, an intravertebral vacuum phenomenon in two cases and a heterogeneous appearance suggesting a malignancy in two cases. Computed tomography, performed in four patients and tomography in one patient, demonstrated fragmentation of the vertebral body in all the cases and vacuum phenomenon in four cases. Magnetic resonance imaging performed in four cases has confirmed the absence of epiduritis and a compression due to bony structures in two cases. A vertebral biopsy was performed in three cases. Osteoporosis was observed in all the cases and in two cases there was also an osteonecrosis. Surgical treatment was performed in three cases and conservative medical treatment in the other cases. After surgical treatment we have observed an absence of improvement of neurological complications in one case, an improvement in another and finally a full recovery in the last case. After conservative treatment we have noted in two cases an absence of improvement of neurological complications and in one case an improvement of neurological deficit. Two patients died (one after medical treatment and another after surgical treatment).

[Rheumatoid polyarthritis in the elderly--rhizomelic pseudopolyarthritis: what differences?]. / Polyarthrite rhumatoïde du sujet âgé--pseudopolyarthrite rhizomélique: quelles différences?

The authors have conducted a comparative retrospective study between polymyalgia rheumatic (N = 26) and rheumatoid arthritis in the elderly (N = 44), including HLA DRB1 genotype determination by PCR-RFLP analysis. No clinical nor biological differences were significant between the 2 groups of patients. However 70% of RA patients had one ore more susceptibility alleles (shared epitope hypothesis) and 50% in polymyalgia rheumatica.