Search PubMed⌕ Search

Biomedical subjects

D Bertrand

Publications and source records attributed to D Bertrand.

At least 109 records · Page 6Linked to original sources
PubMed1997

Only snake curaremimetic toxins with a fifth disulfide bond have high affinity for the neuronal alpha7 nicotinic receptor.

Long chain and short chain curaremimetic toxins from snakes possess 66-74 residues with five disulfide bonds and 60-62 residues with four disulfide bonds, respectively. Despite their structural differences all of these toxins bind with high affinity to the peripheral nicotinic acetylcholine receptors (AChR). Binding experiments have now revealed that long chain toxins only, like the neuronal kappa-bungarotoxin, have a high affinity for a chimeric form of the neuronal alpha7 receptor, with Kd values ranging from about 1 to 12 nM. In contrast, all other toxins bind to the chimeric alpha7 receptor with a low affinity, with Kd values ranging between 3 and 22 microM. These results are supported by electrophysiological recordings on both the wild-type and chimeric alpha7 receptors. Amino acid sequence analyses have suggested that high affinities for the neuronal receptor are associated with the presence of the fifth disulfide at the tip of the toxin second loop. In agreement with this conclusion, we show that a long chain toxin whose fifth disulfide is reduced and then dithiopyridylated has a low affinity (Kd = 12 microM) for the neuronal alpha7 receptor, whereas it retains a high affinity (Kd = 0.35 nM) for the peripheral AChR. Thus, a long chain curaremimetic toxin having a reduced fifth disulfide bond behaves like a short chain toxin toward both the peripheral and neuronal AChR. Therefore, functional classification of toxins that bind to AChRs should probably be done by considering their activities on both peripheral and neuronal receptors.

Amino Acid Sequence↗
PubMed1997

An insertion mutation of the CHRNA4 gene in a family with autosomal dominant nocturnal frontal lobe epilepsy.

Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is the first, and to date only, idiopathic epilepsy for which a specific mutation has been found. A missense mutation in the critical M2 domain of the alpha4 subunit of the neuronal nicotinic acetylcholine receptor (CHRNA4) has been recently identified in one large Australian pedigree. Here we describe a novel mutation in the M2 domain of the CHRNA4 gene in a Norwegian family. Three nucleotides (GCT) were inserted at nucleotide position 776 into the coding region for the C-terminal end of the M2 domain. Physiological investigations of the receptor reconstituted with the mutated CHRNA4 subunit reveal that this insertion does not prevent the receptor function but increases its apparent affinity for ACh. In addition, this mutant receptor shows a significantly lower calcium permeability that, at the cellular level, may correspond to a loss of function. Comparison of the two mutations identified so far in families with ADNFLE illustrates that different mutations can result in similar phenotypes.

Amino Acid Sequence↗
PubMed1997

A new device for in vivo measurement of nasal transepithelial potential difference in cystic fibrosis patients and normal subjects.

Measurement of transepithelial potential difference (PD) on the nasal mucosa has been proposed to test for defective ion transport in cystic fibrosis (CF), and its possible correction after gene therapy or other treatments. The "classical" method records nasal PD under the inferior turbinate, with the disadvantage that the tip of the electrode is not seen by the operator. We have developed a purpose-designed perfusion electrode for PD recording on the visible, medial/posterior aspect of the turbinate. We wanted to determine whether such PD recordings adequately discriminate between CF patients and normal subjects. Measurements of baseline PD and response to a standardized perfusion protocol were performed in 20 normal subjects and 12 CF patients. Solutions of amiloride, with or without low chloride buffer were applied for 3 min. Increased baseline PD and depolarization after amiloride discriminated CF patients from normal subjects. Only one CF patient overlapped with the normal range. Superfusion of low chloride buffer with amiloride and terbutaline caused repolarization in 18 out of 20 normal subjects (90%), consistent with physiological Cl- secretion process, but in none of the CF patients. We conclude that measurements of potential difference on the medial/posterior aspect of the turbinate can discriminate between cystic fibrosis patients and normal subjects. At this site, visual control of the measurement is possible, and the mucosa is easily accessible for subsequent cytological sampling or biopsy.

Adult↗
PubMed1997

[Effects of the improvement in the quality of care in a university hospital].

In charge with the development of quality assurance in a university hospital, the authors have recorded actions realised in medical departments to improve quality. A first questionnaire was sent to every physician (340) and every chief nurse (170). The 126 answers allowed to pick 467 actions out and to identify 72 professionals who were involved in quality actions. These last ones have been asked during semi-directive interviews in view to describe those actions, their results and difficulties encountered. The main actions consisted in writing procedures (197), animating working groups (84) or department reunions (54), realising studies (54) or measuring indicators (33). This study shows the professionals' dynamism who spontaneously develop actions to improve the quality of care. It also shows the limits of these actions because of a lack of specific resources and methods.

Communication↗
PubMed1996

Human alpha4beta2 neuronal nicotinic acetylcholine receptor in HEK 293 cells: A patch-clamp study.

The cloning and expression of genes encoding for the human neuronal nicotinic acetylcholine receptors (nAChRs) has opened new possibilities for investigating their physiological and pharmacological properties. Cells (HEK 293) stably transfected with two of the major brain subunits, alpha4 and beta2, were characterized electrophysiologically using the patch-clamp technique. Fast application of the natural ligand ACh can evoke currents up to 3500 pA, with an apparent affinity (EC50) of 3 microM and a Hill coefficient of 1.2. The rank order of potency of four nAChR ligands to activate human alpha4beta2 receptors is (-)-nicotine > ACh > (-)-cytisine > ABT-418. At saturating concentrations, the efficacy of these ligands is ABT-418 >> (-)-nicotine > ACh >> (-)-cytisine > GTS-21 (previously named DMXB). Coapplication of 1 microM ACh with known nAChR inhibitors such as dihydro-beta-erythroidine and methyllycaconitine reversibly reduces the current evoked by the agonist with respective IC50 values of 80 nM and 1.5 microM. The current-voltage relationship of human alpha4beta2 displays a strong rectification at positive potentials. Experiments of ionic substitutions suggest that human alpha4beta2 nAChRs are permeable to sodium and potassium ions. In the "outside-out" configuration, ACh evokes unitary currents (main conductance 46 pS) characterized by a very fast rundown. Potentiation of the ACh-evoked currents is observed when the extracellular calcium concentration is increased from 0.2 to 2 mM. In contrast, however, a reduction of the evoked currents is observed when calcium concentration is elevated above 2 mM.

Calcium↗
PubMed1996

Identification of calcium binding sites that regulate potentiation of a neuronal nicotinic acetylcholine receptor.

The divalent cation calcium potentiates the physiological response of neuronal nicotinic receptors to agonists by enhancing ionic current amplitudes, apparent agonist affinity and cooperativity. Here we show that mutations in several consensus Ca2+ binding sequences from the N-terminal domain of the neuronal alpha 7 nicotinic acetylcholine receptor alter Ca2+ potentiation of the alpha 7-V201-5HT3 chimera. Mutations E18Q or E44Q abolish calcium-enhanced agonist affinity but preserve the calcium increase of plateau current amplitudes and cooperativity. On the other hand, mutations of amino acids belonging to the 12 amino acid canonical domain (alpha 7 161-172) alter all features of potentiation by enhancing (D163, S169), reducing (E161, S165, Y167) or abolishing (E172) calcium effects on ionic current amplitudes and agonist affinity. Introduction of the alpha 7 161-172 domain in the calcium insensitive 5-hydroxytryptamine (5HT3) serotoninergic receptor results in a receptor activated by 5HT and potentiated by calcium. In vitro terbium fluorescence studies with an alpha 7 160-174 peptide further show that mutation E172Q also alters in vitro calcium binding. Data are consistent with the occurrence of distinct categories of regulatory calcium binding sites, among which the highly conserved (alpha 7 161-172) domain may simultaneously contribute to calcium and agonist binding.

Amino Acid Sequence↗
PubMed1996

[Identification of victims of violence and torture: the practitioner's role].

Many asylum seekers and refugees arriving in Switzerland have been exposed to major trauma such as war, incarceration or torture. A growing number of Swiss doctors treat patients who are victims of violence and torture, and may need to draw up medical reports on sequelae of such trauma. These are often difficult to detect, as patients do not bring up the subject spontaneously. A request for a medical report offers an opportunity to recognize and explore the patient's past traumatic experiences and can be a first step in the therapeutic process. The experience gained in this field at the Travel and Migration Medicine Unit, Geneva, is reported in this article. Analysis of 82 cases treated between June 1991 and December 1993 documents the types of trauma alleged and illustrates the patients' complaints and the clinical signs observed.

Adolescent↗
PubMed1996

Nicotinic acetylcholine receptors in the nematode Caenorhabditis elegans.

Two cDNAs (Ce21 and Ce13) were isolated from a Caenorhabditis elegans library screened with a probe encoding conserved domains of the avian alpha5 neuronal nicotinic acetylcholine receptor (nAChR). Alignments to all nAChR subunits in the EMBL/Swissprot data base demonstrate that the Ce21 protein most resembles the vertebrate alpha7 subunit, whereas Ce13 is closest to the ARD subunit of Drosophila. The corresponding genes were isolated and hybridized to YAC grids: Ce21 maps on chromosome V near the his-23 gene, and Ce13 on chromosome I very near or at unc-29. The structure of the Ce21 gene was compared with that of other vertebrate and invertebrate nAChR genes and found to share by far the largest number of conserved splice sites with the vertebrate alpha7 gene. Upon expression in the Xenopus oocyte system, the Ce21 subunit assembled into a functional homomeric nAChR, whose properties were compared with those of the chicken alpha7 receptor. The anthelmintic nicotinic agonist levamisole is unable to activate the Ce21 and alpha7 receptors, but efficiently antagonises their responses to ACh. Both receptors desensitise quickly upon agonist application, are more sensitive to nicotine than to acetylcholine, and are efficiently blocked by dihydro-beta-erythroidine. Unlike the alpha7 receptor, however, the Ce21 receptor is relatively insensitive to methyllycaconitine and to alpha-bungarotoxin. The similarities in protein sequence, gene structure and physiological properties between alpha7 and Ce21 suggest a very ancient lineage for the alpha7 class of nAChR subunits.

Amino Acid Sequence↗
PubMed1996

Neuronal nicotinic alpha 7 receptor expressed in Xenopus oocytes presents five putative binding sites for methyllycaconitine.

1. The recently isolated compound methyllycaconitine (MLA) is a plant toxin which is a competitive inhibitor of nicotinic acetylcholine receptors (nAChRs). We found that homomeric alpha 7 receptors display a very high sensitivity to MLA with an IC50 in the picomolar range. 2. The competitive nature of the alpha 7 MLA blockade was reinforced by the observation that this compound has no action on wild-type serotoninergic receptors (5-HT3), whereas it is a powerful antagonist of chimaeric receptors alpha 7-5-HT3. 3. The time course of MLA inhibition of the wild-type (WT) alpha 7 follows a monotonic exponential decay whose time constant is proportional to the MLA concentration and could be described by a bimolecular mechanism with a forward rate constant (k+) of 2.7 x 10(7) S-1 M-1. In contrast, recovery from MLA inhibition displays an S-shaped time course that is incompatible with a simple bimolecular reaction. 4. Given the pentameric nature of the neuronal nicotinic receptors, a linear chain model, including five putative MLA binding sites corresponding to the homomeric nature of alpha 7, is proposed. 5. Both onset and recovery data obtained on the alpha 7 wild-type receptor are adequately described by this model assuming that a single MLA molecule is sufficient to block receptor function. 6. Analysis of MLA blockade and recovery of reconstituted heteromeric alpha 4 beta 2 receptors reveals, as expected, a time course compatible with only two binding sites for the toxin and, thus, further supports the validity of our model.

Aconitine↗
PubMed1996

A kinetic mechanism for nicotinic acetylcholine receptors based on multiple allosteric transitions.

Nicotinic acetylcholine receptors are transmembrane oligomeric proteins that mediate interconversions between open and closed channel states under the control of neurotransmitters. Fast in vitro chemical kinetics and in vivo electrophysiological recordings are consistent with the following multi-step scheme. Upon binding of agonists, receptor molecules in the closed but activatable resting state (the Basal state, B) undergo rapid transitions to states of higher affinities with either open channels (the Active state, A) or closed channels (the initial Inactivatable and fully Desensitized states, I and D). In order to represent the functional properties of such receptors, we have developed a kinetic model that links conformational interconversion rates to agonist binding and extends the general principles of the Monod-Wyman-Changeux model of allosteric transitions. The crucial assumption is that the linkage is controlled by the position of the interconversion transition states on a hypothetical linear reaction coordinate. Application of the model to the peripheral nicotine acetylcholine receptor (nAChR) accounts for the main properties of ligand-gating, including single-channel events, and several new relationships are predicted. Kinetic simulations reveal errors inherent in using the dose-response analysis, but justify its application under defined conditions. The model predicts that (in order to overcome the intrinsic stability of the B state and to produce the appropriate cooperativity) channel activation is driven by an A state with a Kd in the 50 nM range, hence some 140-fold stronger than the apparent affinity of the open state deduced previously. According to the model, recovery from the desensitized states may occur via rapid transit through the A state with minimal channel opening, thus without necessarily undergoing a distinct recovery pathway, as assumed in the standard 'cycle' model. Transitions to the desensitized states by low concentration 'pre-pulses' are predicted to occur without significant channel opening, but equilibrium values of IC50 can be obtained only with long pre-pulse times. Predictions are also made concerning allosteric effectors and their possible role in coincidence detection. In terms of future developments, the analysis presented here provides a physical basis for constructing more biologically realistic models of synaptic modulation that may be applied to artificial neural networks.

Allosteric Site↗
PubMed1996

Male-killing bacterium in a fifth ladybird beetle, Coleomegilla maculata (Coleoptera:Coccinellidae).

Inherited symbionts which selectively cause the death of male hosts are found widely across the Insecta. Previous studies have shown a single, but different micro-organism to be responsible for male-killing in each taxonomic group studied. We here produce evidence that within a group of insects, the Coccinellidae, there is more than one causal agent of male lethality. We report a novel observation of a male-killing trait in the species Coleomegilla maculata. Six of 26 crosses were found to produce a female-biased sex ratio associated with a low egg hatch-rate. The trait was matrilinearly inherited and was observed to be tetracycline-sensitive. However, tests which indicate the presence of a Rickettsia, previously found to cause male-killing in another member of the Coccinellidae, Adalia bipunctata, proved negative. We therefore conclude that the phenomenon of male-killing is multicausal, within, as well as between, taxonomic groups of the Insecta.

Animals↗
PubMed1996

Ethanol inhibition of nicotinic acetylcholine type alpha 7 receptors involves the amino-terminal domain of the receptor.

Recent studies have suggested that alcohols can affect the function of neurotransmitter-gated ion channels by a direct interaction with the receptor protein. However, the molecular region of the receptor protein that mediates the alcohol action is not known. To address this question, we studied the effect of ethanol on the function of recombinant nicotinic acetylcholine type alpha 7 (nACh alpha 7) receptors, 5-hydroxytryptamine (serotonin) type 3 (5-HT3) receptors, and a chimeric receptor constructed from these two receptors. The receptors were expressed in Xenopus oocytes and their function was studied using the two-electrode voltage-clamp technique. Ethanol inhibited the response of nACh alpha 7 receptors in a concentration-dependent manner over the concentration range of 5-100 mM; the EC50 for this inhibition was 33 mM ethanol. Ethanol decreased the maximal amplitude (Emax) of the nACh alpha 7 receptor agonist concentration-response curve, without significantly affecting the EC50. In contrast, ethanol potentiated 5-HT3 receptor-mediated responses at low agonist concentrations. The potentiation was concentration-dependent over the concentration range of 10-100 mM; the EC50 for this potentiation was 57 mM ethanol. The magnitude of the ethanol potentiation of 5-HT3 receptor-mediated responses decreased with increasing agonist concentration. The chimeric receptor had the amino-terminal domain from the nACh alpha 7 receptor and the transmembrane and carboxyl-terminal domains from the 5-HT3 receptor. Ethanol was found to inhibit the function of this chimeric receptor in a manner similar to that of nACh alpha 7 receptors. Because the inhibition transfers with the amino-terminal domain of the receptor, the observations suggest that the amino-terminal domain of the receptor is involved in the inhibition.

Animals↗
PubMed1995

Stable expression and pharmacological properties of the human alpha 7 nicotinic acetylcholine receptor.

The alpha 7 neuronal nicotinic acetylcholine receptor subtype forms a Ca(2+)-permeable homooligomeric ion channel sensitive to alpha-bungarotoxin in Xenopus oocytes. In this study, we have stably and functionally expressed the human alpha 7 cDNA in a mammalian cell line, HEK-293 and examined its pharmacologic properties. [125I] alpha-Bungarotoxin bound to transfected cells with a Kd value of 0.7 nM and a Bmax value of 973 pmoL/mg protein. No specific binding was detected in untransfected cells. Specific binding could be displaced by unlabeled alpha-bungarotoxin (Ki = 0.5 nM) and an excellent correlation was observed between binding affinities of a series of nicotinic cholinergic ligands in transfected cells and those in the human neuroblastoma IMR-32 cell line. Additionally, cell surface expression of alpha 7 receptors was detected by fluorescein isothiocyanate-conjugated alpha-bungarotoxin in transfected cells. Whole cell currents sensitive to blockade by alpha-bungarotoxin, and with fast kinetics of activation and inactivation, were recorded from transfected cells upon rapid application of (-)-nicotine or acetylcholine with EC50 values of 49 microM and 155 microM respectively. We conclude that the human alpha 7 subunit when expressed alone can form functional ion channels and that the stably transfected HEK-293 cell line serves as a unique system for studying human alpha 7 nicotinic receptor function and regulation, and for examining ligand interactions.

Acetylcholine↗
PubMed1995

Inward rectification of neuronal nicotinic acetylcholine receptors investigated by using the homomeric alpha 7 receptor.

The strong inward rectification observed in neuronal nicotinic ACh receptors was examined by using alpha-bungarotoxin sensitive, homomeric alpha 7 neuronal nicotinic ACh receptors derived from chick brain. Receptors were expressed in Xenopus laevis oocytes and functionally assessed by the two electrode voltage clamp technique. Site directed mutagenesis of residues thought to line the putative ion pore revealed that negatively charged glutamate residues located at the inner mouth of the channel are essential for rectification. This finding was confirmed both for the active open state and for receptors mutated to conduct in one of the desensitized states. No outward tail relaxations were observed with voltage jumps to depolarizing potentials, suggesting that rectification was not due to intrinsic gating. For the wild type receptor, intracellular injection of CDTA, a chelating agent having a high affinity for Mg2+, reduced rectification in a dose dependent manner, suggesting that rectification originates, in part, from open channel block by internal free Mg2+. These findings support the hypothesis that charged residues at the inner mouth of the pore influence the Mg2+ affinity of the blocking site.

Amino Acid Sequence↗
PubMed1995

Identification of a new component of the agonist binding site of the nicotinic alpha 7 homooligomeric receptor.

Tryptophan 54 of the alpha 7 neuronal nicotinic homooligomeric receptor is homologous to gamma-Trp-55 and delta-Trp-57 of non-alpha subunits of Torpedo receptor labeled by d-tubocurarine. This residue was mutated on the alpha 7-V201-5-hydroxytryptamine (5HT)3 homooligomeric chimera, which displays alpha 7 nicotinic pharmacology, and for which both equilibrium binding studies and electrophysiological recordings could be carried out in parallel. Replacement of Trp-54 by a Phe, Ala, or His causes a progressive decrease both in binding affinity and in responses (EC50 or IC50) for acetylcholine, nicotine, and dihydro-beta-erythroidine, without significant modification in alpha-Bgtx binding. Except for Gln-56, comparatively small effects are observed when the other residues of the 52-58 region are mutated into alanine. These data support the participation of Trp-54 to ligand binding, and provide evidence for a new "complementary component" of the alpha 7 nicotinic binding site, distinct from its three-loop "principal component," and homologous to the "non-alpha component" present on gamma and delta subunits.

Amino Acid Sequence↗