Biomedical subjects

D Fournier

Publications and source records attributed to D Fournier.

At least 37 records · Page 2Linked to original sources

PubMed2000

Improved multianalyte detection of organophosphates and carbamates with disposable multielectrode biosensors using recombinant mutants of Drosophila acetylcholinesterase and artificial neural networks.

Engineered variants of Drosophila melanogaster acetylcholinesterase (AChE) were used as biological receptors of AChE-multisensors for the simultaneous detection and discrimination of binary mixtures of cholinesterase-inhibiting insecticides. The system was based on a combination of amperometric multielectrode biosensors with chemometric data analysis of sensor outputs using artificial neural networks (ANN). The multisensors were fully manufactured by screen-printing, including enzyme immobilisation. Two types of multisensors were produced that consisted of four AChE variants each. The AChE mutants were selected in order to obtain high resolution, enhanced sensitivity and minimal assay time. This task was successfully achieved using multisensor I equipped with wild-type Drosophila AChE and mutants Y408F, F368L, and F368H. Each of the AChE variants was selected on the basis of displaying an individual sensitivity pattern towards the target analytes. For multisensor II, the inclusion of F368W, which had an extremely diminished paraoxon sensitivity, increased the sensor's capacity even further. Multisensors I and II were both used for inhibition analysis of binary paraoxon and carbofuran mixtures in a concentration range 0-5 microg/l, followed by data analysis using feed-forward ANN. The two analytes were determined with prediction errors of 0.4 microg/l for paraoxon and 0.5 microg/l for carbofuran. A complete biosensor assay and subsequent ANN evaluation was completed within 40 min. In addition, multisensor II was also investigated for analyte discrimination in real water samples. Finally, the properties of the multisensors were confirmed by simultaneous detection of binary organophosphate mixtures. Malaoxon and paraoxon in composite solutions of 0-5 microg/l were discriminated with predication errors of 0.9 and 1.6 microg/l, respectively.

Acetylcholinesterase↗

PubMed2000

Severe cognitive impairment in DMD: obvious clinical indication for Dp71 isoform point mutation screening.

Duchenne muscular dystrophy is associated with variable degrees of selective cognitive defect with lower scores for verbal intelligence and reading abilities. A number of findings have shown that rearrangements located in the second part of the gene seem to be preferentially associated with cognitive impairment. Several dystrophin transcripts are expressed in the brain. The more distal of them, Dp71, is predominant. We have carried out a mutational analysis of Dp71 transcript in 12 DMD patients severely, mildly or not retarded, all without detectable deletion or duplication. We have detected five point mutations causing Dp71 premature translation termination. All were found among the more severely mentally retarded patients of this group (VIQ < 50 and/or no reading acquisition).

Adolescent↗

PubMed1999

Local and sustained delivery of 5-fluorouracil from biodegradable microspheres for the radiosensitization of glioblastoma: a pilot study.

BACKGROUND: The authors have developed a new method of drug delivery into the brain using implantable biodegradable microspheres. In this study, this method was used to provide localized and sustained delivery of 5-fluorouracil (5-FU) after the surgical resection of glioblastoma. This antimetabolite and radiosensitizing drug was selected in an attempt to decrease the rate of local recurrence of the tumor. METHODS: Eight patients with newly diagnosed glioblastoma were included in the study and 2 increasing amounts of 5-FU were studied (70 mg and 132 mg). After surgical resection of the tumor, poly(D-L lactide-co-glycolide) 5-FU-loaded microspheres with an average dimension of 45 microm were implanted in the wall of the surgical bed. External beam radiation (59.4 grays) was initiated before the seventh postsurgical day. Patients were followed by clinical examination, magnetic resonance imaging, and 5-FU assays in the blood and cerebrospinal fluid (CSF). RESULTS: 5-FU assays confirmed sustained concentrations in the CSF for at least 1 month. Concentrations of 5-FU in the blood were lower and transitory. Systemic tolerance to the treatment was good; one case of recurrent brain swelling was observed at the higher dose studied. At the time of last follow-up the overall median survival time was 98 weeks from the time of implantation and 2 patients had achieved disease remission at 139 and 153 weeks, respectively. CONCLUSIONS: This study demonstrates that biodegradable microspheres are efficient systems for drug delivery into the brain and may have future application in the treatment of brain tumors. Further studies are needed to confirm the potential of 5-FU-loaded microspheres for the radiosensitization of glioblastoma. [Please see editorial on pages 197-9, this issue].

Adolescent↗

PubMed1999

Effect of tetramethylammonium, choline and edrophonium on insect acetylcholinesterase: test of a kinetic model.

Cholinesterases display a non-Michaelian behaviour with respect to substrate concentration. With the insect enzyme, there is an activation at low substrate concentrations and an inhibition at high concentrations. Previous studies allow us to propose a kinetic model involving a secondary non-productive binding site for the substrate. Unexpectedly, this secondary site has a very high affinity for the substrate when the enzyme is free. On the contrary, when the catalytic site of the enzyme is occupied a strong decrease of this affinity was observed. Moreover, a substrate molecule bound to the peripheral site results in a global decrease of the acylation and/or the deacylation step. Kinetic studies with three reversible inhibitors, tetramethylammonium, edrophonium and choline supported the kinetic model and enable its further refinement.

Acetylcholinesterase↗

PubMed1999

Inhibition of Drosophila acetylcholinesterase by 7-(methylethoxyphosphinyloxy)1-methyl-quinolinium iodide.

The kinetic behaviour of Drosophila melanogaster acetylcholinesterase toward its substrate shows, in comparison with classic Michaelis-Menten kinetics, an apparent homotropic cooperative double activation-inhibition pattern. In order to construct an appropriate kinetic model and obtain further information on the mechanism of the catalytic action of this enzyme, the hydrolysis of acetylthiocholine in the absence and presence of different concentrations of synthetic quaternary methylphosphonate, 7-(methylethoxyphosphinyloxy)1-methyl-quinolinium iodide (MEPQ), was followed on a stopped-flow apparatus. The reaction at low substrate concentrations was followed until the change of the absorbance became negligible and at high concentrations only initial parts were recorded. A simultaneous analysis of the progress curves using numerical integration showed that the powerful organophosphonate inhibitor binds and compete with the substrate for the same binding sites. The results are also in accordance with the hypothesis that virtually every substrate or quasi-substrate molecule that enters into the gorge of active site is hydrolysed.

Acetylcholinesterase↗

PubMed1999

High-resolution ultrasound evaluation of internal jugular venous valves.

Although the presence of valves in the distal portion of the internal jugular veins has been widely described by anatomists and pathologists, their existence remains underscored in the medical literature. We set out to demonstrate their sonographic aspect and to evaluate their morphology, location and prevalence. Seventy-five cadavers were evaluated for the presence and aspect of such valves through neck dissections. Concurrent evaluation of the sonographic character of these structures was performed in another cohort of 75 adults. Valves of the distal portion of the internal jugular veins were found in 93 % of the cadaveric studies. Ultrasonography easily demonstrated the valve leaflets in 87 % of patients; all were observed in the distal portion of the internal jugular (IJ) veins. Sixty percent were bilateral with the majority of unilateral veins found on the right side. High-resolution ultrasonography easily demonstrates valves in the distal portion of the IJ veins which are present in the great majority of persons. Their evaluation may be of use in percutaneous procedures using this venous access.

Aged↗

PubMed1999

Ventral transdural herniation of the thoracic spinal cord: surgical treatment in four cases and review of literature.

BACKGROUND: A specific cause of progressive Brown-Sequard syndrome has been identified: a ventral herniation of the thoracic spinal cord through the dural sleeve on one side. METHOD: Four female patients who were affected by a progressive Brown Sequard syndrome related to a transdural spinal cord herniation have been investigated and were submitted to surgery and postoperative evaluation. FINDINGS: The MRI scan showed atrophy and forward displacement of the spinal cord on one side and adhesion of the spinal cord to the dura mater. CT myelography demonstrated the disappearance of the premedullar rim at the level of the herniation and the shadow of the extradural herniation. Surgical treatment consisted in the excision of the arachnoid cyst when there was one, section of the dentate ligament, release of the adhesions, detachment of the spinal cord from the hernial orifice, and lastly suture of the dural tear or placement by a patch. Follow-up examination showed motor improvement with persistent sensory deficit in two cases and stabilisation in two cases. INTERPRETATION: The cause of the dural tear, either traumatic or congenital could not be confirmed in the four cases. Symptoms probably occur when herniation fills the orifice and strangulation happens which explains the late appearance and progressive evolution of this myelopathy. Mobilisation of the herniated spinal cord back into the intradural space can be achieved by surgery and may stop the evolution of the symptoms and signs.

Adult↗

PubMed1999

Cholinesterases from the marine mussels Mytilus galloprovincialis Lmk. and M. edulis L. from the freshwater bivalve Corbicula fluminea Müller.

In order to improve the molecular basis for the use of bivalve cholinesterases as a reliable biomarker for aquatic pollution, the polymorphism and characterization of these enzymes in Mytilus edulis, Mytilus galloprovincialis and Corbicula fluminea were investigated. All results are consistent with the presence of only one pharmacological form of cholinesterase in each species. The molecular masses were 180 kDa for the two marine mussels and 240 kDa for C. fluminea. The cholinesterases are anchored to the membrane by a glycosyl inositol phosphate like the Ga form (type I) described in vertebrates. Surprisingly, these cholinesterases were poorly inhibited by organophosphorous compounds compared to enzymes from other sources. This suggests that these bivalves could be used as a biomarker for acute rather than chronic contaminations by anticholinesterase insecticides.

Animals↗

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

Interaction of 3-alkylpyridinium polymers from the sea sponge Reniera sarai with insect acetylcholinesterase.

3-Alkylpyridinium polymers (poly-APS), composed of 29 or 99 N-butyl-3-butyl pyridinium units, were isolated from the marine sponge Reniera sarai. They act as potent cholinesterase inhibitors. The inhibition kinetics pattern reveals several successive phases ending in irreversible inhibition of the enzyme. To provide more information on mechanism of inhibition, interaction of poly-APS and N-butyl-3-butyl pyridinium iodide (NBPI) with soluble dimeric and monomeric insect acetylcholinesterase (AChE) was studied by using enzyme intrinsic fluorescence and light scattering, conformational probes ANS and trypsin, and SDS-PAGE. Poly-APS quenched tryptophan fluorescence emission of AChE more extensively than NBPI. Both inhibitors exhibited a pseudo-Lehrer type of quenching. Interaction of poly-APS with dimeric AChE did not induce significant changes of the enzyme conformation as assayed by using the hydrophobic probe ANS and trypsin digestion. In contrast to NBPI, titration of both monomeric and dimeric AChE with poly-APS resulted in the appearance of large complexes detected by measuring light scattering. An excess of poly-APS produced AChE precipitation as proved on SDS-PAGE. None of the effects were observed with trypsin as a control. It was concluded that AChE aggregation and precipitation rather than the enzyme conformational changes accounted for the observed irreversible component of poly-APS inhibition.

Acetylcholinesterase↗

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↗

PubMed1999

Subclavian steal syndrome and flow-related aneurysm. Another reason to treat.

A 48-year-old woman presented with a symptomatic right subclavian steal syndrome due to proximal subclavian artery stenosis. Anatomically the innominate artery was absent. Collateral circulation followed the vertebro-vertebral pathway with reversal of blood flow in the ipsilateral vertebral artery. There was also multiple dilated intervertebral collaterals and an associated saccular aneurysm on one of them. Surgical carotid-subclavian transposition permitted relief of clinical symptoms, disappearance of collateral circulation and subtotal regression of the aneurysm. This spontaneous evolution confirmed the role of high-flow in the pathogenesis of some aneurysms and the habitually good prognosis of flow-related aneurysms with correction of the cause. Arteriography still appears essential in diagnosis, pretherapeutic assessment and sometimes post-treatment evaluation in subclavian steal syndrome.

Aneurysm↗

PubMed1999

[Spontaneous spinal cord herniation: a little-known cause of Brown-Sequard syndrome. Report of two cases and review of the literature].

We report two cases of spinal cord herniation. The first one concerns a 28 year-old female who presented with an eighteen-month past history of spasticity of the right lower limb associated with somesthetic sensorial disturbances of the left side with a T4 level, related to a T3-T4 spinal cord herniation. The second case concerned a 58 year-old female with a past history of a left lower limb pyramidal syndrome associated with somesthetic and proprioceptive sensibilities disturbances of the right side with a T5 level related to a T4-T5 spinal cord herniation. Spinal cord herniation is an uncommon and underestimated cause of a progressive Brown-Sequard syndrome. The diagnosis is now easy on the MRI, showing a ventral attraction of the cord. The outcome is usually good, following the surgical reduction of the herniation.

Adult↗

PubMed1999

[Post-traumatic intracranial arterial aneurysm. Two cases with review of the literature].

Post-traumatic intracranial aneurysms are rare entity, which can appear following even benign head injuries. Their clinical manifestations are various, from a typical SAH to an uncommon intracerebral hematoma. Medical history runs usually from a couple of days to several years. Two cases of intracranial aneurysm have been observed from 1995 to 1998. The first case presented as a cortical aneurysm in a cerebral contusion following a week of evolution. The second case presented as a cortical hematoma with a head trauma 10 years before. All patients were operated on with uneventful follow-up. Post-traumatic intracranial aneurysms can exhibit an increase in size or a spontaneous thrombosis. Diagnosis is made by angiography, which has to be repeated if treatment has to be delayed. Best results are obtained by exclusion of the lesion, if allowed by the patient's condition.

Adult↗

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↗