CYP6 P450 Enzymes and ACE-1 Duplication Produce Extreme and Multiple Insecticide Resistance in the Malaria Mosquito Anopheles gambiae

Malaria control relies heavily on pyrethroid insecticides, to which susceptibility is declining in Anopheles mosquitoes. To combat pyrethroid resistance, application of alternative insecticides is advocated for indoor residual spraying (IRS), and carbamates are increasingly important. Emergence of a very strong carbamate resistance phenotype in Anopheles gambiae from Tiassal??, C??te d'Ivoire, West Africa, is therefore a potentially major operational challenge, particularly because these malaria vectors now exhibit resistance to multiple insecticide classes. We investigated the genetic basis of resistance to the most commonly-applied carbamate, bendiocarb, in An. gambiae from Tiassal??. Geographically-replicated whole genome microarray experiments identified elevated P450 enzyme expression as associated with bendiocarb resistance, most notably genes from the CYP6 subfamily. P450s were further implicated in resistance phenotypes by induction of significantly elevated mortality to bendiocarb by the synergist piperonyl butoxide (PBO), which also enhanced the action of pyrethroids and an organophosphate. CYP6P3 and especially CYP6M2 produced bendiocarb resistance via transgenic expression in Drosophila in addition to pyrethroid resistance for both genes, and DDT resistance for CYP6M2 expression. CYP6M2 can thus cause resistance to three distinct classes of insecticide although the biochemical mechanism for carbamates is unclear because, in contrast to CYP6P3, recombinant CYP6M2 did not metabolise bendiocarb in vitro. Strongly bendiocarb resistant mosquitoes also displayed elevated expression of the acetylcholinesterase ACE-1 gene, arising at least in part from gene duplication, which confers a survival advantage to carriers of additional copies of resistant ACE-1 G119S alleles. Our results are alarming for vector-based malaria control. Extreme carbamate resistance in Tiassal?? An. gambiae results from coupling of over-expressed target site allelic variants with heightened CYP6 P450 expression, which also provides resistance across contrasting insecticides. Mosquito populations displaying such a diverse basis of extreme and cross-resistance are likely to be unresponsive to standard insecticide resistance management practices.

Effects of chronic sublethal exposure to waterborne Cu, Cd or Zn in rainbow trout 2: tissue specific metal accumulation

Tissue specific metal accumulations (gills, liver, kidney and whole body) in rainbow trout ( Oncorhynchus mykiss) were compared during chronic exposure (up to 100 days) to sublethal levels of waterborne Cd (3 μg·l 1), Cu (75 μg·l 1) or Zn (250 μg·l 1) in moderately hard water (hardness of 140 mg·l 1, pH 8.0). A general pattern of tissue metal increase and stabilization was evident for all three metals, although the degree and time course of accumulation varied. The exception to this general pattern was a lack of Zn accumulation in the liver and kidney although small amounts did accumulate in the gills and whole body. Accumulation of Cu occurred primarily in the liver while for Cd the kidney was the major organ of accumulation. Exponential modeling was employed to compare and contrast the saturation concentration and time to half saturation of various tissues. Accumulation of essential metals (Cu and Zn), if it occurred, was rapid and increases were relatively low. For example the time to half saturation during Cu exposures was always less than 2 weeks and the maximum level of accumulation was less than four times background levels. For non-essential Cd, time to half saturation for the liver and kidney was always longer than 5 weeks and modeled saturation concentrations were up to 80-fold higher than background. The response to Cu and Zn suggested an active regulation of tissue burdens while that of Cd appears to be more passive, resulting in continuous metal accumulation over an extended time course. While the initial patterns of accumulation for each metal were generally consistent with the damage, repair and acclimation pattern from concurrent physiological measurements it was clear that tissue metal accumulation was not a good indicator of either exposure of physiological impact.

Metodi per lo studio dei polifenoli dell'uva

A detailed description of a practical scheme to quantify polyphenolic components in grape skins, seeds and musts is proposed. Details of berry sampling and extraction as well as an introduction to spectroscopic and HPLC data acquisition are included.

Exhalation flow and pressure-controlled reservoir collection of exhaled nitric oxide for remote and delayed analysis

Expiratory flow rate, soft palate closure, and dead space air may influence exhaled levels of nitric oxide (NO). These factors have not been evaluated in the reservoir collection of NO.Exhaled NO was collected into a reservoir during a single flow and pressure controlled exhalation.NO collected in a reservoir containing silica gel was stable for 24 hours. Nasally delivered 4.8% argon measured by mass spectrometry did not contaminate exhaled argon levels (0.1 (0.02)%) in five volunteers during exhalation against a resistance (10 (0.5) cmH2O), hence proving an effective soft palate closure. Exhaled NO in the reservoir was 11 (0.2) ppb, 8.6 (0.1) ppb, 7.1 (0.6) ppb, and 6.6 (0.4) ppb in five normal subjects and 48.3 (18) ppb, 20.3 (12) ppb, 16.9 (0.3) ppb and 10.1 (0.4) ppb in 10 asthmatic subjects at four studied expiratory flows (5-6, 7-8, 10-11, and 12-13 l/min, respectively), with NO levels equal to direct measurement (7.3 (0.5) ppb and 17.4 (0.5) ppb for normal and asthmatic subjects respectively, p < 0.05) at the flow rate 10-11 l/min. Elimination of dead space proved necessary to provide NO levels comparable to the direct measurement. Exhaled NO collected into the reservoir without dead space during flow controlled exhalation against mild resistance provided close agreement (mean (SD) difference -0.21 (0.68), coefficient of variation 4.58%) with direct measurement in 74 patients (NO range 1-69 ppb).Flow and pressure controlled collection of exhaled NO into a reservoir with silica gel provides values identical to the direct measurement and may be used to monitor asthma at home and where analysers are not on site.

Reaction Kinetics, Molecular Action, and Mechanisms of Cellulolytic Proteins

Cellulolytic proteins form a complex of enzymes that work together to depolymerize cellulose to the soluble products cellobiose and glucose. Fundamental studies on their molecular mechanisms have been facilitated by advances in molecular biology. These studies have shown homology between cellulases from different microorganisms, and common mechanisms between enzymes whose modes of action have sometimes been viewed as being different, as suggested by the distribution of soluble products. A more complete picture of the cellulolytic action of these proteins has emerged and combines the physical and chemical characteristics of solid cellulose substrates with the specialized structure and function of the cellulases that break it down. This chapter combines the fundamentals of cellulose structure with enzyme function in a manner that relates the cellulose binding and biochemical kinetics at the catalytic site of the proteins to the macroscopic behavior of cellulase enzyme systems.