Biomedical subjects

D Fournier

Publications and source records attributed to D Fournier.

At least 19 recordsLinked to original sources

PubMed1999

Mechanism of an insect glutathione S-transferase: kinetic analysis supporting a rapid equilibrium random sequential mechanism with housefly I1 isoform.

The steady-state kinetics of glutathione S-transferase I1 (GST I1) from housefly Musca domestica expressed in Escherichia coli were investigated with glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). Concentrations of the varied substrates were from 0.03 to 1 mM for GSH and 0.05 to 1 mM for CDNB. Within this range, Michaelis-Menten behaviour was observed and convergent straight lines in double reciprocal plots excluded a ping-pong kinetic mechanism. Instead, data were consistent either with rapid-equilibrium random or with steady-state ordered sequential mechanisms because of abscissa convergence. Discrimination was achieved by studying the reaction with another electrophilic partner, p-nitrophenyl-acetate (PNPA). Concentrations of PNPA and GSH varied within the ranges 0.5 to 10 mM and 0.03 to 0.6 mM, respectively. The complete set of data supports the proposal of a rapid-equilibrium random-sequential model with strictly independent sites for GSH and CDNB or PNPA. Kinetic parameters are thus true dissociation equilibrium constants with values of 0.15 mM for GSH, 0.15 mM for CDNB, and 7 mM for PNPA. Analysis of the inhibition by the product (S-(2,4-dinitrophenyl)-glutathione, 10 to 100 microM), on the coupling reaction between GSH and CDNB with either GSH (0.05 to 0.5 mM, CDNB 0.2 mM) or CDNB (0.05 to 0.5 mM, GSH 0.2 mM) varied, consistent with the proposed mechanism. Binding of product to the free enzyme excludes GSH (competitive inhibition pattern with Kp = 12 microM) but only slightly hinders binding of CDNB. Binding free energies, together with the inhibition pattern, suggest that the non-peptidic moiety of product interacts with an alternative sub-site within the large open pocket accommodating the various electrophilic substrates. These results lead us to propose a model for intra-pocket shifting of the non-peptidic moiety upon product formation which contributes to the product release.

Animals↗

PubMed1999

Is the insect glutathione S-transferase I gene family intronless?

The genes coding for class I glutathione S-transferases in insects were believed to be intronless because the coding sequence was not interrupted by an intron. But sequences of the untranslated 5' end of transcripts revealed the presence of an intron in housefly and Drosophila genes suggesting that most insect GSTI genes are in fact interrupted.

Animals↗

PubMed1998

A putative kinetic model for substrate metabolisation by Drosophila acetylcholinesterase.

Insect acetylcholinesterase, an enzyme whose catalytic site is located at the bottom of a gorge, can metabolise its substrate in a wide range of concentrations (from 1 microM to 200 mM) since it is activated at low substrate concentrations. It also presents inhibition at high substrate concentrations. Among the various rival kinetic models tested to analyse the kinetic behaviour of the enzyme, the simplest able to explain all the experimental data suggests that there are two sites for substrate molecules on the protein. Binding on the catalytic site located at the bottom of the gorge seems to be irreversible, suggesting that each molecule of substrate which enters the active site gorge is metabolised. Reversible binding at the peripheral site of the free enzyme has high affinity (2 microM), suggesting that this binding increases the probability of the substrate entering the active site gorge. Peripheral site occupation decreases the entrance rate constant of the second substrate molecule to the catalytic site and strongly affects the catalytic activity of the enzyme. On the other hand, catalytic site occupation lowers the affinity of the peripheral site for the substrate (34 mM). These effects between the two sites result both in apparent activation at low substrate concentration and in general inhibition at high substrate concentration.

Acetylcholine↗

PubMed1998

Inhibition of acetylcholinesterase by an alkylpyridinium polymer from the marine sponge, Reniera sarai.

Large polymeric 3-alkylpyridinium salts have been isolated from the marine sponge Reniera sarai. They are composed of N-butyl(3-butylpyridinium) repeating subunits, polymerized head-to-tail, and exist as a mixture of two main polymers with molecular weights without counterion of about 5520 and 18900. The monomer analogue of the inhibitor, N-butyl-3-butylpyridinium iodide has been synthesized. This molecule shows mixed reversible inhibition of acetylcholinesterase. The polymers also act as acetylcholinesterase inhibitors and show an unusual inhibition pattern. We tentatively describe it as quick initial reversible binding, followed by slow binding or irreversible inhibition of the enzyme. This kinetics suggests that there are several affinity binding sites on the acetylcholinesterase molecule where the polymer can bind. The first binding favors binding to other sites which leads to an apparently irreversibly linked enzyme-inhibitor complex.

Animals↗

PubMed1998

Engineering sensitive acetylcholinesterase for detection of organophosphate and carbamate insecticides.

High quantities of various acetylcholinesterases can now be produced following in vitro expression and it is possible to use them as biosensors to detect organophosphates and carbamates insecticides. In order to check the potentialities of acetylcholinesterase from various sources, we have studied enzyme from bovine erythrocyte, Electrophorus electricus, Drosophila melanogaster, Torpedo californica and Caenorhabditis elegans. It appears that insect acetylcholinesterase is more susceptible to a broad range of organophosphates and carbamates insecticides than the other tested enzymes. D. melanogaster is 8-fold more sensitive than E. electricus enzyme and this sensitivity has been increased to 12-fold by introducing a mutation at position 408.

Acetylcholinesterase↗

PubMed1998

Two invertebrate acetylcholinesterases show activation followed by inhibition with substrate concentration.

In vertebrates there are two cholinesterases, with differences in catalytic behaviour with respect to substrate concentration: butyrylcholinesterase displays an increased activity at low substrate concentrations, whereas acetylcholinesterase displays inhibition by excess substrate. In two invertebrates, Drosophila melanogaster and Caenorhabditis elegans, we found cholinesterases that showed both kinetic complexities: substrate activation at low substrate concentrations followed by inhibition at higher concentrations. These triphasic kinetics can be explained by the presence of two enzymes with different kinetic behaviours or more probably by the existence of a single enzyme regulated by the substrate concentration.

Acetylcholinesterase↗

PubMed1998

Drosophila acetylcholinesterase: effect of post-translational [correction of post-traductional] modifications on the production in the baculovirus system and substrate metabolization.

Acetylcholinesterase cDNAs from Drosophila melanogaster modified on its primary sequence were cloned into baculovirus and were expressed in Sf9 cells with the aim to identify a mutant form that produces the enzyme at a high level. Directed mutagenesis was used in order to independently knockout different sites of post-translational modifications: exchange of the C-terminal hydrophobic peptide for a glycolipid molecule, dimerization by disulfide bridge, N-linked glycosylation at the five accessible sites, and subunit formation by proteolytic cleavage of a hydrophilic peptide found in the precursor. Another mutation involved the elimination of a free cysteine in the mature protein. All mutations involving post-translational modifications resulted in lower recoveries, suggesting that they are useful for maintaining high amounts of protein in the synapse. By contrast, elimination of a free cysteine in the mature protein permitted an increase in the level of production of the enzyme. These mutations did not affect specific activity of the enzyme at substrate concentrations ranging from 3 microM to 200 mM, suggesting that activation and inhibition of the enzyme activity does not originate from a polymorphism in post-translational modifications.

Acetylcholine↗

PubMed1998

Stabilization of recombinant Drosophila acetylcholinesterase.

The uses of pure and stable acetylcholinesterase can range from simple basic research to applications in environment quality assessment. In order to satisfy some of these needs its recombinant expression is routinely performed. Affinity-purified recombinant Drosophila melanogaster acetylcholinesterase proved to be instable; an apparent cause of this seemed to be the presence of contaminants with protease activity as evidenced by SDS-PAGE. The elimination of these accompanying products was achieved by anion-exchange, hydrophobic interaction, and cibacron blue affinity chromatography applied downstream from procainamide affinity chromatography. The utilization of a parallel affinity acting via an engineered histidine tail permitted the elimination of the copurified proteases as well. Despite the elimination of the contaminants, the apparently pure extracts were still unstable. It is shown that such instability can be counterbalanced by provoking protein-protein interactions, either between enzyme molecules or with other molecules such as bovine serum albumin. Another way to reduce instability is the addition of a reversible inhibitor or polyethylene glycol 3350.

Acetylcholinesterase↗

PubMed1998

A new attempt to assess the effect of learning processes on the cholinergic system: studies on fruitflies and honeybees.

The effect of training on the functioning of the cholinergic system was investigated in fruitflies and in honeybees. Drosophila were submitted to a passive avoidance conditioning of the proboscis extension response (PER). Flies had to learn to suppress the sugar-induced PER to avoid an aversive quinine reinforcement. In a yoked control group, the punishment was administered with no relation to the response displayed. Honeybees underwent a five-trial olfactory conditioning of the PER elicited by an antennal gustatory stimulation. In the control group, olfactory and gustatory stimulations were unpaired to prevent a learning process from developing. Immediately at the end of the learning session, acetylcholinesterase (AChE) activity was individually measured on the whole animal for Drosophila and on the head for the honeybee in experimental and in control groups. In fruitflies and honeybees, the AChE rate did not differ between the experimental group and its respective control group. Moreover, no significant correlation could be found individually between the learning performance and the AChE rate in either Drosophila or in honeybees. This experiment did not reveal any modulatory effect of the learning acquisition level on the AChE activity in insects as was previously reported in honeybees.

Acetylcholinesterase↗

PubMed1997

[Thoracic ultrasound].

Ultrasonography is the best imaging modality for detection of pleural effusions and the presence of septations, but is not accepted as a reliable diagnostic tool for other thoracic problems. However, the technique is useful for analysis of parietal lesions and pulmonary masses arising in the periphery of the lung. Biopsy of such lesions and drainage of pleural effusions are often easier under sonographic guidance than with CT. Because of its advantages, sonography should be used more often as a diagnostic and therapeutic tool in thoracic imaging.

Humans↗

PubMed1997

Cholinesterases from the common oyster (Crassostrea gigas). Evidence for the presence of a soluble acetylcholinesterase insensitive to organophosphate and carbamate inhibitors.

Marine bivalves such as oysters and mussels are widely used as bioindicators of contamination in the monitoring of pollutant effects. As filter feeders, these species are known to be good general indicators of chemical contamination. However, the efficient use of decreased acetylcholinesterase activity in the oyster as a biomarker of exposure to neurotoxic compounds requires a definition of the different types of cholinesterases coexisting in this mollusk. This study reports the partial purification, separation and characterization of two cholinesterases extracted from the oyster Crassostrea gigas. Differences in apparent molecular weight, type of glycosylation and hydrophobicity, and sensitivity to inhibitors suggest that they are encoded by two different genes. 'A' cholinesterase (apparent molecular weight 200 kDa) is anchored to the membrane via a glycolipid, is not glycosylated but sensitive to organophosphate and carbamate inhibitors. 'B' cholinesterase (molecular weight 330 kDa) is hydrophilic, glycosylated and highly resistant to organophosphate and carbamate inhibitors. The kinetic properties of these two cholinesterases were compared with those of other invertebrate cholinesterases. The presence of a cholinesterase insensitive to insecticides suggests that a significant improvement in the use of oyster cholinesterases as biomarkers of pollutant effects could be achieved by simple separation of the two forms.

Acetylcholinesterase↗

PubMed1997

Cloning and localization of a glutathione S-transferase class I gene from Anopheles gambiae.

1,1,1-Trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) resistance in both adults and larvae of Anopheles gambiae is mediated by stage-specific glutathione S-transferases (GSTs). On the basis of their biochemical characteristics the larval resistance-associated GSTs are likely to be insect class I GSTs. Aggst1-2, a class I GST gene, which is expressed in larvae, has been cloned from the malaria vector A. gambiae. The gene was inserted into a bacterial expression system, and the detection of 1-chloro-2,4-dinitrobenzene (CDNB) conjugating activity in Eschericia coli expressing the recombinant enzyme confirmed that aggst1-2 encodes a catalytically active GST. The gene encodes a 209 amino acid protein with 46% sequence similarity to a Drosophila melanogaster class I GST (GST-D1), 44% similarity with a Musca domestica class I GST (MdGST-1), but only low levels of homology with class II insect GSTs, including the adult specific AgGST2-1 from A. gambiae. Southern analysis of genomic DNA indicated that A. gambiae has multiple class I GSTs. In situ hybridization of class I genomic and cDNA clones to polytene chromosomes identified a single region of complementarity on chromosome 2R division 18B, suggesting that these class I GSTs in A. gambiae are arranged sequentially in the genome. Three positive overlapping recombinant clones were identified from an A. gambiae genomic library. Mapping and partial sequencing of these clones suggests that there are several GSTs and truncated GST pseudogenes within the 30kb of DNA that these clones span.

Amino Acid Sequence↗

PubMed1997

In vivo modulation of benzodiazepine receptor function after inhibition of endogenous gamma-aminobutyyric acid synthesis.

The influence of decreased endogenous gamma-aminobutyric acid (GABA) concentration on benzodiazepine receptor function was studied in the brain of living baboons. Positron emission tomography and the radiotracer [11C]flumazenil combined with electroencephalography were used to determine the pharmacological properties of two bezodiazepine receptors agonists, diazepam and bretazenil, in baboons pre-treated or not with DL-allylglycine (an inhibitor of GABA synthesis). Our results show that, in vivo, DL-allylglycine reduces the affinity of benzodiazepine receptors for their agonists without altering the intrinsic capability of agonists to allosterically modulate GABAergic transmission.

Allylglycine↗

PubMed1997

Transposition-mediated transcriptional overexpression as a mechanism of insecticide resistance.

It has been proposed that amplification of genes for esterase that provide resistance to insecticides may originate from transposition events. To test this hypothesis, we have constructed a minigene coding for a soluble acetylcholinesterase under the control of a nontissue-specific promoter (hsp70). When introduced into Drosophila, the gene is expressed in all tissues and the extra acetylcholinesterase produced confers a low level of insecticide resistance (twofold). The minigene was mobilized by crossing the initial transformant with a strain providing a source of P-element transposase. After 34 generations of exposure to the organophosphate parathion, we obtained a strain with a higher resistance (fivefold). This strain had only one extra Ace gene, which overexpressed acetylcholinesterase. Thus, following transposition, resistance resulted from the overexpression of a single copy of the gene and not from gene amplification.

Acetylcholinesterase↗

PubMed1997

Variation of dominance of newly arisen adaptive genes.

Newly arisen adaptive alleles such as insecticide resistance genes represent a good opportunity to investigate the theories put forth to explain the molecular basis of dominance and its possible evolution. Dominance levels of insecticide resistance conferred by insensitive alleles of the acetylcholinesterase gene were analyzed in five resistant strains of the mosquito Culex pipiens. Dominance levels were found to differ between strains, varying from partial recessivity to complete dominance. This variation was not explained by differences in catalytic properties of the enzyme, since four of the five resistant strains had identical inhibition properties for the insensitive acetylcholinesterase. Among these four laboratory strains and in individuals collected from natural populations, we found a correlation between increased acetylcholinesterase activities and higher dominance levels. We propose a molecular explanation for how variation in acetylcholinesterase activity may result in variation of dominance level. We also conjecture that the four resistant strains did not differ in their amino acid sequence in the catalytically active regions of acetylcholinesterase, but that the expression of the gene was regulated by either neighboring or distant sites, thereby modifying the dominance level. Under this interpretation, dominance levels may evolve in this system, since heritable variation in acetylcholinesterase activity was found.

Acetylcholinesterase↗

PubMed1997

[Ultrasonography of the breast].

Mammography, which is the first-choice examination for breast disease, has some limitations despite recent technical improvement, which has lead to better image quality and reduced x-ray exposure. It is less accurate in dense breasts, which are sometimes difficult to read and in which a palpable lesion or an infraclinical cancer is often not recognized. In such cases, ultrasonography is very useful and should be used as the second examination technique. Breast sonography done by a skilled sonographer with a good equipment can raise the accuracy for benign or malignant breast lesions.

Breast Diseases↗

PubMed1997

[Prevention of hemorrhagic complications in the lateral retinacular section of the patella. A study of the lateral arteries of the knee applied to the prevention of knee hemarthrosis].

PURPOSE OF THE STUDY: Arthroscopic lateral retinacular release is one of the most employed procedures for patellar chondromalacia. A literature review show a complication rate of 10 to 18 p. 100 of postoperative hemarthrosis. This work aims to study the vessels anatomy of the lateral side of the knee in order to find anatomical landmarks allowing to avoid or coagulate them. MATERIAL AND METHODS: Thirty-three cadaver knees were dissected. Measures were made related to the lateral superior genicular artery and the lateral inferior genicular artery. A study using tracing-paper was also carried out. The main part of the work was more descriptive, studying anastomosis between the different arteries and veins location. RESULTS: Concerning the lateral superior genicular artery, measure analysis showed that this artery was always cut in lateral patellar retinaculum. This artery never runs more than one centimeter proximally to the base of patella, which is too insufficient to escape from lateral retinacular release. We noted the possibility of locating 90 p. 100 of lateral superior genicular arteries in a minimal distance of 15 mm, in front of the lateral proximal angle of the patella. Concerning the lateral inferior genicular artery, only a few arteries, protected in the meniscal wall in its early course, can remain intact. Two thirds of these arteries are very vulnerable running across the lateral side of the knee. The study of the tracing-papers confirmed topographic study measurements. The descriptive study emphasized the number and the importance of anastomoses between these different arteries. Each artery is flanked by two large satellite veins, which also attribute a veinous origin to a possible bleeding. DISCUSSION: The topographic study of the lateral inferior genicular vessels shows that their course varies. It seems necessary to avoid their division by performing the standard anterior lateral inferior arthroscopic portal proximally to these vessels. This can be realized at the beginning of the procedure through cutaneous transillumination. The lateral superior genicular vessels are always cut. They are nevertheless reachable through a small lateral incision of about 10 mm, distally to the lateral superior angle of the patella. 90 p. 100 at least of these arteries could be coagulated in such a way. We also emphasize the use of classical methods for the prevention of excessive venous bleeding, such as a compression dressing. CONCLUSION: Following this anatomical study, we suggest, as a supplementary precaution, a selective hemostasis of the lateral superior genicular vessels through a small incision associated with the location of the lateral inferior genicular vessels by cutaneous transillumination.

Arthroscopy↗

PubMed1997

[Intradural lumbar disk hernias. Apropos of 6 cases and review of the literature].

Transdural discal hernia is a rare complication of lumbar discal pathology. In this paper, we report five cases of transdural herniated disc and one intraradicular discal hernia. In the literature review, we found 97 cases since the first case described by Walter Dandy in 1942. The main risk in this exceptional location of discal hernia is to miss an intradural discal piece leading to a poor result. MRI or CT scan with intrathecal contrast can show an intradural lesion but they usually fail to indicate its precise nature.

Adult↗