Mutations of acetylcholinesterase which confer insecticide resistance in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> populations

Background

Organophosphate and carbamate insecticides irreversibly inhibit acetylcholinesterase causing death of insects. Resistance-modified acetylcholinesterases(AChEs) have been described in many insect species and sequencing of their genes allowed several point mutations to be described. However, their relative frequency and their cartography had not yet been addressed.

Results

To analyze the most frequent mutations providing insecticide resistance in Drosophila melanogaster acetylcholinesterase, the Ace gene was cloned and sequenced in several strains harvested from different parts of the world. Sequence comparison revealed four widespread mutations, I161V, G265A, F330Y and G368A. We confirm here that mutations are found either isolated or in combination in the same protein and we show that most natural populations are heterogeneous, composed of a mixture of different alleles. In vitro expression of mutated proteins showed that combining mutations in the same protein has two consequences: it increases resistance level and provides a wide spectrum of resistance.

Conclusion

The presence of several alleles in natural populations, offering various resistance to carbamate and organophosphate compounds will complicate the establishment of resistance management programs.

     

A signal transfer system through three compartments transduces the plant cell contact-dependent signal controlling Ralstonia solanacearum hrp genes

Ralstonia solanacearum hrp genes encode a type III secretion system required for disease development in host plants and for hypersensitive response elicitation on non-hosts. hrp genes are expressed in the presence of plant cells through the HrpB regulator. This activation, which requires physical interaction between the bacteria and the plant cell, is sensed by the outer membrane receptor PrhA. PrhA transduces the plant cell contact-dependent signal through a complex regulatory cascade integrated by the PrhJ, HrpG, and HrpB regulators. In this study, we have identified two genes, named prhI and prhR, that belong to the hrp gene cluster and whose predicted products show homology with extracytoplasmic function sigma factors and transmembrane proteins, respectively. Strains carrying a mutation in prhIR show a delayed pathogenic phenotype toward host plants. PrhIR control the plant cell contact-dependent activation of hrp genes. prhIR gene expression is induced by a signal present in the plant cell coculture that is not PrhA-dependent. Genetic evidence shows that PrhIR act upstream of PrhJ in the regulatory cascade, likely transducing the signal sensed by PrhA through the periplasm as described for signal transfer systems through three compartments. This is the first report of such a surface signaling mechanism activating pathogenicity determinants in response to a nondiffusible plant cell wall signal.     

Failure of the interaction between presenilin 1 and the substrate of gamma-secretase to produce Abeta in insect cells

Aggregates of beta-amyloid peptide (Abeta) are the major component of the amyloid core of the senile plaques observed in Alzheimer's disease (AD). Abeta results from the amyloidogenic processing of its precursor, the amyloid precursor protein (APP), by beta- and gamma-secretase activities. If beta-secretase has recently been identified and termed BACE, the identity of gamma-secretase is still obscure. Studies with knock-out mice showed that presenilin 1 (PS1), of which mutations are known to be the first cause of inherited AD, is mandatory for the gamma-secretase activity. However, the proteolytic activity of PS1 remains a matter of debate. Here we used transfected Sf9 insect cells, a cellular model lacking endogenous beta- and/or gamma-secretase activities, to characterize the role of BACE and PS1 in the amyloidogenic processing of human APP. We show that, in Sf9 cells, BACE performs the expected beta-secretase cleavage of APP, generating C99. We also show that C99, which is a substrate of gamma-secretase, tightly binds to the human PS1. Despite this interaction, Sf9 cells still do not produce Abeta. This strongly argues against a direct proteolytic activity of PS1 in APP processing, and points toward an implication of PS1 in trafficking/presenting its substrate to the gamma-secretase.     

Un nouveau gisement à ambre insectifère et à végétaux (Albien terminal probable) : Archingeay (Charente-Maritime, France)

A layer of clay interbedded in sandstone, which age is likely uppermost Albian, yielded a new deposit with amber and fossil plants in Charente-Maritime (south-west France). A survey of the arthropods found in amber, the xylologic and palynologic determinations and the sedimentologic study are in progress. We already have datas to propose a palaeoenvironmental reconstruction of the coast of the northern Aquitain basin at the end of lower Cretaceous : an estuarine area under warm and wet climate.     

An ABC transporter from Bacillus thuringiensis is essential for beta-exotoxin I production

beta-Exotoxin I is a nonspecific insecticidal metabolite secreted by some Bacillus thuringiensis strains. Several studies of B. thuringiensis strains that have lost the capacity to produce beta-exotoxin I have suggested that there is a strong correlation between high levels of beta-exotoxin I production and the ability to synthesize crystal proteins. In this study, we showed that a mutant strain, B. thuringiensis 407-1(Cry(-))(Pig(+)), with no crystal gene, produced considerable amounts of beta-exotoxin I together with a soluble brown melanin pigment. Therefore, beta-exotoxin I production can take place after a strain has lost the plasmids bearing the cry genes, which suggests that these curable plasmids probably contain determinants involved in the regulation of beta-exotoxin I production. Using a mini-Tn10 transposon, we constructed a library of strain 407-1(Cry(-))(Pig(+)) mutants. We screened for nonpigmented mutants with impaired beta-exotoxin I production and identified a genetic locus harboring two genes (berA and berB) essential for beta-exotoxin I production. The deduced amino acid sequence of the berA gene displayed significant similarity to the ATP-binding domains of the DRI (drug resistance and immunity) family of ATP-binding cassette (ABC) proteins involved in drug resistance and immunity to bacteriocins and lantibiotics. The berB gene encodes a protein with six putative transmembrane helices, which probably constitutes the integral membrane component of the transporter. The demonstration that berAB is required for beta-exotoxin I production and/or resistance in B. thuringiensis adds an adenine nucleotide analog to the wide range of substrates of the superfamily of ABC proteins. We suggest that berAB confers beta-exotoxin I immunity in B. thuringiensis, through active efflux of the molecule.     

Acceleration of Drosophila melanogaster acetylcholinesterase methanesulfonylation: peripheral ligand D-tubocurarine enhances the affinity for small methanesulfonylfluoride

D-Tubocurarine, a reversible peripheral inhibitor of cholinesterases accelerates methanesulfonylation of Drosophila melanogaster wild type and W359L mutant. The kinetic evaluation of the process was performed in a step-by-step analysis. The second order overall sulfonylation rate constants, determined from classical residual activity measurements, were used in the subsequent analysis of progress curves. The latter were obtained by measuring the hydrolysis of acetylthiocholine in a complex reaction system of enzyme, substrate, irreversible and reversible inhibitor. The underlying kinetic mechanisms, from such a complex data, could only be untangled by targeted inspection and successive incorporation of reaction steps for which experimental evidence existed. The study showed that the peripheral ligand D-tubocurarine, by binding at the entrance into the active site of the two investigated enzymes (Golicnik et al., Biochemistry 40 (2001) 1214), enhances the affinity for small methanesulfonylfluoride, rather to speeding up the formation of a stable covalent enzyme-inhibitor complex. The specific arrangements at the rim of the active site of each individual enzyme dictate the actual events which can be detected by kinetic means.     

Real-time MRI and articulatory coordination in speech

This paper describes the real-time MRI technique and its use for the study of speech production. The two major problems, (i) the simultaneous recording of the MR images and the speech signal and (ii) the synchronisation of the images and of the speech signal, are addressed. Measurement accuracy on real-time images is evaluated by comparison with similar measurements on static MR images.     

SIMS REIN: a multi-source information system for end-stage renal disease

In France, the prevalence of End-Stage Renal Disease (ESRD) is not precisely known. The sources of information are scattered and not coordinated. Consequently, care is ill adapted to meet the demand. The Multi-Source Information System is the basis of the Renal Epidemiology and Information Network (REIN). It is dedicated to improve and organise our medical and epidemiological knowledge of ESRD and to aid public health decision-making in this area. The proposed approach is based on the datawarehouses. This model allows a unified vision of scattered data into distinct databases, for a better management, be it particular (patient follow-up) or global (regional follow-up), with a finality of aid in decision-making. Several categories of problems were considered: the global conception of the information system, the organisation of the datawarehouse, which offers different viewpoints of the data, the integration of heterogeneous data coming from different sources, data exchange and definition of a specific ontology.     

Multiplicative genetic effects in scrapie disease susceptibility

Despite experimental evidence that scrapie is an infectious disease of sheep, variations of the occurrence of the natural disease suggest an influence of host genetic factors. It has been established that the genetic polymorphism of the prion protein (PrP) gene is correlated to the incidence of scrapie and to the survival time: five polymorphisms have been described by variations at amino-acid codons 136, 154 and 171. In this paper we study the effect on scrapie susceptibility of the pairing of the five allelic variants known to exist: we show that scrapie susceptibility is given by the produce of the elementary allelic factors. This first well-documented evidence of a multiplicative property of genetic risk factors could give hints on the underlying mechanisms of prion-induced neurodegenerative diseases.