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Biomedical subjects

C Blanchet

Publications and source records attributed to C Blanchet.

At least 19 recordsLinked to original sources
PubMed2001

Epilepsy, hyperalgesia, impaired memory, and loss of pre- and postsynaptic GABA(B) responses in mice lacking GABA(B(1)).

GABA(B) (gamma-aminobutyric acid type B) receptors are important for keeping neuronal excitability under control. Cloned GABA(B) receptors do not show the expected pharmacological diversity of native receptors and it is unknown whether they contribute to pre- as well as postsynaptic functions. Here, we demonstrate that Balb/c mice lacking the GABA(B(1)) subunit are viable, exhibit spontaneous seizures, hyperalgesia, hyperlocomotor activity, and memory impairment. Upon GABA(B) agonist application, null mutant mice show neither the typical muscle relaxation, hypothermia, or delta EEG waves. These behavioral findings are paralleled by a loss of all biochemical and electrophysiological GABA(B) responses in null mutant mice. This demonstrates that GABA(B(1)) is an essential component of pre- and postsynaptic GABA(B) receptors and casts doubt on the existence of proposed receptor subtypes.

Animals↗
PubMed2001

The "catalytic" triad of isocitrate dehydrogenase kinase/phosphatase from E. coli and its relationship with that found in eukaryotic protein kinases.

The isocitrate dehydrogenase kinase/phosphatase (IDHK/P) of E. coli is a bifunctional enzyme responsible for the reversible phosphorylation of isocitrate dehydrogenase (IDH) on a seryl residue. As such, it belongs to the serine/threonine protein kinase family. However, only a very limited homology with the well-characterized eukaryotic members of that family was identified so far in its primary structure. In this report, a new region of amino acids including three putative residues involved in the kinase activity of IDHK/P was identified by sequence comparison with eukaryotic protein kinases. In IDHK/P, these residues are Asp-371, Asn-377, and Asp-403. Their counterpart eukaryotic residues have been shown to be involved in either catalysis (former residue) or magnesium binding (the two latter residues). Site-directed mutagenesis was performed on these three IDHK/P residues, and also on the Glu-439 residue equivalent to that of the Ala-Pro-Glu motif found in the eukaryotic protein kinases. Mutations of Asp-371 into either Ala, Glu, or Gln residues drastically lowered the yield and the quality of the purification. Nevertheless, the recovered mutant enzymes were barely able to phosphorylate IDH either in vitro or after expression in an aceK (-) mutant strain. In contrast, mutation of either Asn-377, Asp-403, or Glu-439 into an Ala residue altered neither the yield of purification nor the maximal phosphorylating capacity of the enzyme. However, when IDH was phosphorylated in the presence of increasing concentrations of magnesium ions, the two former mutants displayed a much lower affinity for this cation, with a K(m) value of 0.6 or 0.8 mM, respectively, as compared to 0.1 mM for the wild-type enzyme. On the other hand, the Glu439Ala mutant has an affinity for magnesium essentially unaffected. Therefore, and in contrast to the current opinion, our results suggest that the catalytic mechanism of IDHK/P exhibits some similarities with that found in the eukaryotic members of the protein kinase family.

Amino Acid Sequence↗
PubMed2001

ANTHEPROT: an integrated protein sequence analysis software with client/server capabilities.

Programs devoted to the analysis of protein sequences exist either as stand-alone programs or as Web servers. However, stand-alone programs can hardly accommodate for the analysis that involves comparisons on databanks, which require regular updates. Moreover, Web servers cannot be as efficient as stand-alone programs when dealing with real-time graphic display. We describe here a stand-alone software program called ANTHEPROT, which is intended to perform protein sequence analysis with a high integration level and clients/server capabilities. It is an interactive program with a graphical user interface that allows handling of protein sequence and data in a very interactive and convenient manner. It provides many methods and tools, which are integrated into a graphical user interface. ANTHEPROT is available for Windows-based systems. It is able to connect to a Web server in order to perform large-scale sequence comparison on up-to-date databanks. ANTHEPROT is freely available to academic users and may be downloaded at http://pbil.ibcp.fr/ANTHEPROT.

Algorithms↗
PubMed2001

A common mechanism for ATP hydrolysis in ABC transporter and helicase superfamilies.

ABC (ATP-binding cassette) transporters and helicases are large superfamilies of seemingly unrelated proteins, whose functions depend on the energy provided by ATP hydrolysis. Comparison of the 3D structures of their nucleotide-binding domains reveals that, besides two well-characterized ATP-binding signatures, the folds of their nucleotide-binding sites are similar. Furthermore, there are striking similarities in the positioning of residues thought to be important for ATP binding or hydrolysis. Interestingly, structures have recently been obtained for two ABC proteins that are not involved in transport activities, but that have a function related to DNA modification. These ABC proteins, which contain a nucleotide-binding site akin to those of typical ABC transporters, might constitute the missing link between the two superfamilies.

ATP-Binding Cassette Transporters↗
PubMed2001

Identification of related proteins with weak sequence identity using secondary structure information.

Molecular modeling of proteins is confronted with the problem of finding homologous proteins, especially when few identities remain after the process of molecular evolution. Using even the most recent methods based on sequence identity detection, structural relationships are still difficult to establish with high reliability. As protein structures are more conserved than sequences, we investigated the possibility of using protein secondary structure comparison (observed or predicted structures) to discriminate between related and unrelated proteins sequences in the range of 10%-30% sequence identity. Pairwise comparison of secondary structures have been measured using the structural overlap (Sov) parameter. In this article, we show that if the secondary structures likeness is >50%, most of the pairs are structurally related. Taking into account the secondary structures of proteins that have been detected by BLAST, FASTA, or SSEARCH in the noisy region (with high E: value), we show that distantly related protein sequences (even with <20% identity) can be still identified. This strategy can be used to identify three-dimensional templates in homology modeling by finding unexpected related proteins and to select proteins for experimental investigation in a structural genomic approach, as well as for genome annotation.

Algorithms↗
PubMed2000

Gentamicin blocks ACh-evoked K+ current in guinea-pig outer hair cells by impairing Ca2+ entry at the cholinergic receptor.

Aminoglycoside antibiotics such as gentamicin are known to block the medial olivocochlear efferent system. In order to determine whether this inhibition takes place at the postsynaptic cholinergic receptors in outer hair cells (OHCs), we studied the effects of these polycationic molecules on cholinergic currents evoked in isolated guinea-pig OHCs. The cholinergic response of OHCs involves nicotinic-like receptors (nAChRs) permeable to Ca2+ ions that activate nearby Ca2+-sensitive K+ channels (KCa(ACh) channels). The extracellular application of gentamicin and neomycin reversibly blocked ACh-evoked K+ current (IK(ACh)) with IC50 values of 5.5 and 3.2 microM, respectively. The results showed that the blocking mechanism of IK(ACh) was due to inhibition of Ca2+ influx via nAChRs. Our study also provides interesting insights into the functional coupling between nAChRs and KCa(ACh) channels in OHCs. By directly recording the cation current flowing through nAChRs (In(ACh)) using an intracellular solution containing 10 mM BAPTA, we measured an EC50 near 110 microM for ACh-evoked In(ACh). This EC50 for ACh is one order of magnitude higher than that measured indirectly on IK(ACh). This reveals a rather low affinity of ACh for its receptor but a very efficient coupling between nAChRs and KCa(ACh) channels. We also show that a high external Ca2+ concentration reverts the gentamicin inhibition of IK(ACh) and that gentamicin directly alters the cation current flowing through the nAChRs of OHCs. We propose that gentamicin acts as a non-competitive cholinergic blocker by displacing Ca2+ from specific binding sites at the nAChRs. This block of the nAChRs at the level of the postsynaptic membrane in OHCs could explain the inhibitory effect of gentamicin reported on the crossed medial olivocochlear efferent system in vivo.

Acetylcholine↗
PubMed2000

Magnocellular neurons of the rat supraoptic nucleus are endowed with functional nicotinic acetylcholine receptors.

Acetylcholine can stimulate the release of vasopressin. In organ-cultured hypothalamo-neurohypophyseal systems, acetylcholine enhanced vasopressin release by acting in or near the supraoptic nucleus Extracellular recordings suggested that acetylcholine can increase supraoptic neuron excitability. These effects could be mimicked, in part, by nicotine or blocked by nicotinic antagonists, suggesting that they might be mediated by nicotinic acetylcholine receptors. Autoradiography indicated that alpha-bungarotoxin binding sites are present in the supraoptic nucleus; however, neither acetylcholine nor nicotine binding sites could be detected. Thus, the existence, let alone the nature, of nicotinic receptors in the supraoptic nucleus has so far remained elusive. The present work attempts to determine: (i) whether functional nicotinic receptors are present in this nucleus; (ii) whether they are located on neurosecretory magnocellular cells or at presynaptic sites; (iii) what their pharmacological and biophysical properties are; (iv) whether they influence the activity of all or only part of supraoptic neurons. Whole-cell recordings were performed in hypothalamic slices or in acutely dissociated supraoptic neurons and the effect of nicotinic agonists was tested under voltage-clamp conditions. Autoradiography was done in coronal hypothalamic sections, using [3H]epibatidine and [125I]alpha-bungarotoxin as ligands. Our results indicate that supraoptic neurons possess functional nicotinic receptors containing the alpha7 subunit.

Acetylcholine↗
PubMed2000

MPSA: integrated system for multiple protein sequence analysis with client/server capabilities.

UNLABELLED: MPSA is a stand-alone software intended to protein sequence analysis with a high integration level and Web clients/server capabilities. It provides many methods and tools, which are integrated into an interactive graphical user interface. It is available for most Unix/Linux and non-Unix systems. MPSA is able to connect to a Web server (e.g. http://pbil.ibcp.fr/NPSA) in order to perform large-scale sequence comparison on up-to-date databanks. AVAILABILITY: Free to academic http://www.ibcp.fr/mpsa/ CONTACT: c.blanchet@ibcp.fr

Computing Methodologies↗
PubMed2000

The association between heel ultrasound and hormone replacement therapy is modulated by a two-locus vitamin D and estrogen receptor genotype.

Evidence supports the role of estrogen deprivation in the process of bone remodeling and increased risk of fracture in postmenopausal women but little is known about the genetic basis of individual differences in response to therapy. In a cross-sectional study, 425 ambulatory postmenopausal French-Canadian women from Quebec (age range, 42-85 years old) were genotyped for a common Bsm I polymorphism at the vitamin D receptor (VDR) gene as well as a Pvu II polymorphism in the estrogen receptor (ESR1) gene. Heel ultrasound was determined by right calcaneal quantitative ultrasound (QUS) and results were expressed as an age-and-weight-adjusted stiffness index (heel SI z score). Our aim was to investigate the interaction between hormone-replacement therapy (HRT) and receptor genotypes in an effect on heel SI. Notably, a two-locus genotype (VDR-bb/ESR-PP) present in 9.5% of women was responsible for over 30% of the total HRT-related heel SI difference in the whole sample. Women bearing this combined VDR/ESR1 genotype who received HRT for more than 5 years had a 21% (1.25 SD) greater heel SI (p = 0.002) than those bearing the same genotype but who received HRT for <5 years. This may translate into a 2- to 3-fold difference in the risk of fracture. Although follow-up studies are needed, our findings suggest that QUS of the heel in postmenopausal women taking HRT is affected by variation in VDR and ESR1 loci, jointly.

Adult↗
PubMed2000

[Respiratory manifestations after exposure to sulfurous anhydride in wine-cellar workers: 6 case reports].

Sulfite treatment of wine [a process exploiting the biocidal and anti-oxidant properties of sulfur dioxide (SO2)] involves the use of liquified gas, aqueous solutions or bisulfites, i.e. the salts of sulfurous acid which slowly release SO2. This procedure can result in repeated exposures of operators to significant amounts of SO2. However, risks associated with the use of SO2 are greatly under-estimated by wine producers and wine-cellar workers. We report on 6 cases of respiratory symptoms attributable to SO2 identified during a survey of wine-cellars in the French Beaujolais district. Their pathogenesis is discussed after an overview of the occupational toxicology of SO2.

Adult↗
PubMed1999

Molecular model, calcium sensitivity, and disease specificity of a conformational thyroperoxidase B-cell epitope.

While studying the humoral mechanisms involved in thyroid autoimmunity, we located a B-cell autoepitope in the extracellular C-terminal region of human thyroperoxidase. Structural modeling showed that this region encompasses both a Sushi-like and an epidermal growth factor-like domain, the flexible arrangement of which was putatively stabilized by calcium. The recombinant peptide was found to contain the previously identified conformational thyroperoxidase autoepitope. The occurrence of a calcium-induced conformational change was confirmed using a recombinant peptide monoclonal antibody, the decrease of which in binding to calcium-saturated thyroperoxidase was reversed by a chelating agent. The disease specificity of recombinant peptide, which was more frequently recognized by Hashimoto's than by Graves' patients, adds to its potential value as a diagnostic and preventive tool in the context of B-cell autoimmunity.

Amino Acid Sequence↗
PubMed1999

A scorpion alpha-like toxin that is active on insects and mammals reveals an unexpected specificity and distribution of sodium channel subtypes in rat brain neurons.

Several scorpion toxins have been shown to exert their neurotoxic effects by a direct interaction with voltage-dependent sodium channels. Both classical scorpion alpha-toxins such as Lqh II from Leiurus quiquestratus hebraeus and alpha-like toxins as toxin III from the same scorpion (Lqh III) competitively interact for binding on receptor site 3 of insect sodium channels. Conversely, Lqh III, which is highly toxic in mammalian brain, reveals no specific binding to sodium channels of rat brain synaptosomes and displaces the binding of Lqh II only at high concentration. The contrast between the low-affinity interaction and the high toxicity of Lqh III indicates that Lqh III binding sites distinct from those present in synaptosomes must exist in the brain. In agreement, electrophysiological experiments performed on acute rat hippocampal slices revealed that Lqh III strongly affects the inactivation of voltage-gated sodium channels recorded either in current or voltage clamp, whereas Lqh II had weak, or no, effects. In contrast, Lqh III had no effect on cultured embryonic chick central neurons and on sodium channels from rat brain IIA and beta1 subunits reconstituted in Xenopus oocytes, whereas sea anemone toxin ATXII and Lqh II were very active. These data indicate that the alpha-like toxin Lqh III displays a surprising subtype specificity, reveals the presence of a new, distinct sodium channel insensitive to Lqh II, and highlights the differences in distribution of channel expression in the CNS. This toxin may constitute a valuable tool for the investigation of mammalian brain function.

Animals↗
PubMed1998

Bone mineral density in French Canadian women.

This cross-sectional study investigated bone mineral density (BMD) at the lumbar spine (L2-4) and femoral neck in French Canadian women residing in the Quebec city area. Data collection was initiated in 1988 and completed in 1994. A total of 747 French Canadian Caucasian women (16-79 years of age) with no metabolic bone disease were evaluated. BMD measurements were obtained using dual-photon absorptiometry (DPA) or dual-energy X-ray absorptiometry (DXA). Anthropometric measures such as weight, height and body mass index (BMI) were recorded. Medical files provided information on demographic characteristics, hormonal profile and lifestyle habits. Results show a curvilinear trend of BMD with aging. Furthermore, the peak BMD at the lumbar spine (L2-4) was reached at 29 years followed by a stable phase until 35 years, after which BMD started to decrease. The pattern of bone evolution at the femoral neck was different, peak BMD being achieved earlier, at 21 years, while after age 26 years a significant decrease was already observed. Women older than 60 years showed the lowest BMD. Regression analysis showed that age, weight and height are determinants of BMD at the lumbar spine and explained 33.9% of inter-individual variation. At the femoral neck, 29.1% of variation was explained by age and height only. In conclusion, our data suggest that French Canadian women have a different pattern of bone loss at the femoral neck compared with the lumbar spine, according to their mean BMD values.

Adolescent↗
PubMed1997

Protein structure prediction. Implications for the biologist.

Recent improvements in the prediction of protein secondary structure are described, particularly those methods using the information contained into multiple alignments. In this respect, the prediction accuracy has been checked and methods that take into account multiple alignments are 70% correct for a three-state description of secondary structure. This quality is obtained by a 'leave-one out' procedure on a reference database of proteins sharing less than 25% identity. Biological applications such as 'protein domain design' and structural phylogeny are given. The biologist's point of view is also considered and joint predictions are encouraged in order to derive an amino acid based accuracy. All the tools described in this paper are available for biologists on the Web (http/www.ibcp.fr/predict.html).

Computer Communication Networks↗
PubMed1997

Fast, slow, and steady-state effects of contralateral acoustic activation of the medial olivocochlear efferent system in awake guinea pigs: action of gentamicin.

The function of the medial olivocochlear efferent system was observed in awake guinea pigs by recording, in the absence of ipsilateral external acoustic stimulation, the ensemble background activity (EBA) of the VIIIth nerve from an electrode chronically implanted on the round window of one ear. The EBA was measured by calculating the power value of the round window signal in the 0.5- to 2.5-kHz band after digital or analog (active) filtering. This EBA was compared with and without the addition of a low-level broadband noise to the opposite ear. The contralateral broadband noise (CLBN, 55 dB SPL) induced, via the efferent system, a decrease (suppression) of this EBA. With the use of noise bursts of different durations, two components in this suppression could be observed. After the onset of a 1-s CLBN, the power value of the EBA decreased rapidly by 38.0 +/- 4.2% (mean +/- SD, n = 3), with a latency of <10 ms and a decay time constant of 13.1 +/- 1.0 ms (fast effect). At the offset of the 1-s CLBN, EBA came back to prestimulation values with a similar latency and a time constant of 15.5 +/- 2.9 ms. During longer CLBN stimulation (>/=1 min), EBA presented, after the fast decrease, an additional, slower decrease of 15.6 +/- 3.1%, with a delay of 9.8 +/- 1.3 s and a decay time constant of 16.1 +/- 5.0 s (n = 12, slow effect), and then remained remarkably constant for as long as observed, i.e., >2 h (steady state). The average global suppression was thus up to 47.8 +/- 5.8% of the basal, pre-CLBN-stimulation EBA value. At the offset of the CLBN, EBA returned to pre-CLBN level with fast and slow phases, with, for the slow phase, no delay and a time constant of 32.1 +/- 8.1 s. Fast and slow changes in EBA power values were observed after a single injection of gentamicin (GM) at different doses (150, 200, and 250 mg/kg). At 150 and 200 mg/kg, GM progressively and reversibly blocked the rapid effect, but the slow component of the efferent medial suppression remained remarkably unchanged. However, at higher doses both the fast and slow suppressions were totally yet still reversibly blocked. These observations indicate that the medial olivocochlear efferent system exerts sustained influences on outer hair cells and that this effect develops in two different steps that may have different basic cellular mechanisms.

Acoustic Stimulation↗
PubMed1997

[Amylin].

Explore the source record for details and available documents.

Amyloid↗
PubMed1996

Acetylcholine-induced potassium current of guinea pig outer hair cells: its dependence on a calcium influx through nicotinic-like receptors.

The cholinergic efferent inhibition of mammalian outer hair cells (OHCs) is mediated by a hyperpolarizing K+ current. We have made whole-cell tight-seal recordings from single OHCs isolated from the guinea pig cochlea to characterize the mechanism by which acetylcholine (ACh) activates K+ channels. After ACh application, OHCs exhibited a biphasic response: an early depolarizing current preceding the predominant hyperpolarizing K+ current. The current-voltage (I-V) relationship of the ACh-induced response displayed an N-shape, suggesting the involvement of Ca2+ influx. When whole-cell recording was combined with confocal calcium imaging, we simultaneously observed the ACh-induced K+ current (IK(ACh)) and a Ca2+ response restricted to the synaptic area of the cell. This IK(ACh) could be prevented by loading OHCs with 10 mM of the fast Ca2+ buffer bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid (or BAPTA), therefore allowing the observation of the ACh-induced early current in isolation. This early current revealed nicotinic features because it activated with an intrinsic delay in the millisecond range, reversed nearly in between potassium and sodium equilibrium potentials, and was blocked by curare. However, it was strongly reduced in the absence of external Ca2+, and its I-V relationship displayed an unusual outward rectification at positive membrane potentials and an inward rectification below -60 mV. The results indicate that the cholinergic response of mammalian OHCs involves a "nicotinic-like" nonspecific cation channel through which Ca2+ enters and triggers activation of nearby Ca2+-dependent K+ channels.

Acetylcholine↗