Biomedical subjects

C Lecomte

Publications and source records attributed to C Lecomte.

At least 37 records · Page 2Linked to original sources

PubMed2000

Characterization and developmental expression of xSim, a Xenopus bHLH/PAS gene related to the Drosophila neurogenic master gene single-minded.

We have isolated a novel gene from Xenopus, denominated xSim, which encodes a protein of 760 amino acids containing a basic helix-loop-helix (bHLH) motif contiguous to a PAS domain characteristic of an emerging family of transcriptional regulators so called bHLH/PAS. xSim shares a strong amino acid sequence identity with the Drosophila Single-minded (dSim) and with the murine Sim1 and Sim2 proteins. Phylogenetic analysis reveals that xSim gene is an ortholog gene of the mSim2 gene. Spatio-temporal analysis shows a maternal and a zygotic expression of xSim throughout early Xenopus development. In situ hybridization assays reveal that the transcripts are enriched in the animal hemisphere until blastula stage and extend to the marginal zone at early gastrula stage. As development proceeds, xSim is mainly restricted to the central nervous system.

Amino Acid Sequence↗

PubMed2000

Maurotoxin and the Kv1.1 channel: voltage-dependent binding upon enantiomerization of the scorpion toxin disulfide bridge Cys31-Cys34.

Maurotoxin (MTX) is a 34-amino acid polypeptide cross-linked by four disulfide bridges that has been isolated from the venom of the scorpion Scorpio maurus palmatus and characterized. Maurotoxin competed with radiolabeled apamin and kaliotoxin for binding to rat brain synaptosomes and blocked K+ currents from Kv1 channel subtypes expressed in Xenopus oocytes. Structural characterization of the synthetic toxin identified half-cystine pairings at Cys3-Cys24, Cys9-Cys29, Cys13-Cys19 and Cys31-Cys34 This disulfide bridge pattern is unique among known scorpion toxins, particularly the existence of a C-terminal '14-membered disulfide ring' (i.e. cyclic domain 31-34), We therefore studied structure-activity relationships by investigating the structure and pharmacological properties of synthetic MTX peptides either modified at the C-terminus ¿i.e. MTX(1-29), [Abu31,34]-MTX and [Cys31,34, Tyr32]D-MTX) or mimicking the cyclic C-terminal domain [i.e. MTX(31-34)]. Unexpectedly, the absence of a disulfide bridge Cys31-Cys34 in [Abu 31,34]-MTX and MTX(1-29) resulted in MTX-unrelated half-cystine pairings of the three remaining disulfide bridges for the two analogs, which is likely to be responsible for their inactivity against Kv1 channel subtypes. Cyclic MTX(31-34) was also biologically inactive. [Cys31,34, Tyr32]D-MTX, which had a 'native', MTX-related, disulfide bridge organization, but a D-residue-induced reorientation of the C-terminal disulfide bridge, was potent at blocking the Kv1.1 channel. This peptide-induced Kv1.1 blockage was voltage-dependent (a property not observed for MTX), maximal in the low depolarization range and associated with on-rate changes in ligand binding. Thus, the cyclic C-terminal domain of MTX seems to be crucial for recognition of Kv1.3, and to a lesser extent, Kv1.2 channels and it may contribute to the stabilization and strength of the interaction between the toxin and the Kv1.1 channel.

Amino Acid Sequence↗

PubMed2000

Chemical synthesis and characterization of Pi1, a scorpion toxin from Pandinus imperator active on K+ channels.

Pi1 is a 35-residue toxin cross-linked by four disulfide bridges that has been isolated from the venom of the chactidae scorpion Pandinus imperator. Due to its very low abundance in the venom, we have chemically synthesized this toxin in order to study its biological activity. Enzyme-based proteolytic cleavage of the synthetic Pi1 (sPi1) demonstrates half-cystine pairings between Cys4-Cys25, Cys10-Cys30, Cys14-Cys32 and Cys20-Cys35, which is in agreement with the disulfide bridge organization initially reported on the natural toxin. In vivo, intracerebroventricular injection of sPi1 in mice produces lethal effects with an LD50 of 0.2 microgram per mouse. In vitro, the application of sPi1 induces drastic inhibition of Shaker B (IC50 of 23 nM) and rat Kv1.2 channels (IC50 of 0.44 nM) heterologously expressed in Xenopus laevis oocytes. No effect was observed on rat Kv1.1 and Kv1.3 currents upon synthetic peptide application. Also, sPi1 is able to compete with 125I-labeled apamin for binding onto rat brain synaptosomes with an IC50 of 55 pM. Overall, these results demonstrate that sPi1 displays a large spectrum of activities by blocking both SK- and Kv1-types of K+ channels; a selectivity reminiscent of that of maurotoxin, another structurally related four disulfide-bridged scorpion toxin that exhibits a different half-cystine pairing pattern.

Amino Acid Sequence↗

PubMed2000

Virus neutralizing antibodies against a panel of 18 BVDV isolates in calves vaccinated with Rispoval RS-BVD.

Seven of nine colostrum-deprived calves, free from infection with bovine virus diarrhoea virus (BVDV), were vaccinated with Rispoval RS-BVD on two occasions, 21 days apart, while the other two were kept as BVDV infection controls. The virus neutralizing (VN) serum antibodies induced by vaccination were tested for their ability to neutralize 18 European BVDV isolates, including laboratory reference strains and recent field isolates, both cytopathic and non-cytopathic biotypes as well as genotypes I and II. The strains were isolated in Belgium, France, Germany and the United Kingdom. While there were large variations in the vaccine-induced VN titres of the individual calves against all the strains, e.g. the titres against Osloss NCP, the European reference strain ranged from 1.7 to 6.7 (1:log2), serum from each animal was capable of neutralizing between nine and all 18 of the strains tested. Nevertheless, from the results of this study, it can be concluded that in colostrum-deprived BVDV seronegative calves, Rispoval RS-BVD can stimulate the production of VN antibodies capable of neutralizing a wide range of antigenically diverse European isolates of BVDV, including genotypes I and II.

Animals↗

PubMed2000

Electron density and electrostatic properties of two peptide molecules: tyrosyl-glycyl-glycine monohydrate and glycyl-aspartic acid dihydrate.

The electron density and electrostatic properties of Tyr-Gly-Gly and Gly-Asp molecules have been determined from high-resolution X-ray diffraction data at 123 K. Topological properties of the charge distribution are discussed and compared with those derived from other experimental studies on peptide molecules, and the characteristics of the (3,-1) critical points of the C=O, C-N, C-C bonds are analysed. Crystal data for Tyr-Gly-Gly: C13H17N3O5.H2O, Mr = 313, orthorhombic, P2(1)2(1)2(1), Z = 4, T = 123 +/- 2 K; lattice parameters: a = 7.984 (2), b = 9.535 (3), c = 18.352 (5) A, V= 1397.1 (6) A3, Dx = 1.49 g cm(-3), mu = 1.2 cm(-1) for lambdaMo = 0.7107 A. Crystal data for Gly-Asp: C6H10N2O5.2H2O, Mr = 212, orthorhombic, P2(1)2(1)2(1), Z = 4, T = 123 +/- 2 K; lattice parameters: a = 9.659 (1), b = 9.672 (1), c = 10.739 (1) A, V= 1003.3 (4) A3, Dx = 1.40 g cm(-3), mu = 1.3 cm(-1) for lambda(Mo)= 0.7107 A.

Crystallography, X-Ray↗

PubMed2000

Experimental charge density and electrostatic potential of glycyl-L-threonine dihydrate.

The experimental electron density distribution in glycyl-L-threonine dihydrate has been investigated using single-crystal X-ray diffraction data at 110 K to a resolution of (sin theta/lambda) = 1.2 A(-1). Multipolar pseudo-atom refinement was carried out against 5417 observed data and the molecular electron density was analyzed using topological methods. The experimental electrostatic potential around the molecule is discussed in terms of molecular interactions. Crystal data: C6H12N2O4. 2H2O, Mr = 212.2, orthorhombic, P2(1)2(1)2(1), Z = 4, F(000) = 456 e, T = 110 K, a = 9.572 (3), b = 10.039 (3), c = 10.548 (2) A, V = 1013.6 (4) A3, Dx = 1.3 g cm(-3), mu = 1.2 cm(-1) for lambdaMo = 0.7107 A.

Dipeptides↗

PubMed2000

Towards the charge-density study of proteins: a room-temperature scorpion-toxin structure at 0.96 A resolution as a first test case.

The number of protein structures refined at a resolution higher than 1.0 A is continuously increasing. Subatomic structures may deserve a more sophisticated model than the spherical atomic electron density. In very high resolution structural studies (d < 0.5 A) of small peptides, a multipolar atom model is used to describe the valence electron density. This allows a much more accurate determination of the anisotropic thermal displacement parameters and the estimate of atomic charges. This information is of paramount importance in the understanding of biological processes involving enzymes and metalloproteins. The structure of the scorpion Androctonus australis Hector toxin II has been refined at 0.96 A resolution using synchrotron diffraction data collected at room temperature. Refinement with a multipolar electron-density model in which the multipole populations are transferred from previous peptide studies led to the observation of valence electrons on covalent bonds of the most ordered residues. The refined net charges of the peptide-bond atoms were of the correct sign but were underestimated. Such protein-structure refinements against higher resolution data collected at cryogenic temperature will enable the calculation of experimental atomic charges and properties such as electrostatic potentials.

Crystallography, X-Ray↗

PubMed2000

Perception of "appropriate" age for retirement among young adults, middle-aged adults, and elderly people.

The "appropriate" age for retirement as it is perceived by young adults, middle-aged adults, and elderly people has been studied. No respondents were surprised or had trouble expressing an opinion about the minimum and maximum "appropriate" ages for retirement. Representations of the "appropriate" retirement age vary primarily as a function of the perceived physical constraints involved in the occupation, and also depend on the age of the person being questioned; the younger the respondent, and lower the perceived "appropriate" minimum age. There was no tendency among the young adults to prolong the work life of older individuals. Nor was there a tendency to associate aging with the loss of intellectual capacities likely to lead to early retirement.

Adult↗

PubMed1999

The use of CCD area detectors in charge-density research. Application to a mineral compound: the alpha-spodumene LiAl(SiO3)2.

X-ray diffraction data sets collected on both Nonius and Siemens (Bruker) goniometers equipped with charge-coupled device (CCD) area detectors have been tested for the electron-density determination of the aluminosilicate mineral compound alpha-spodumene LiAl(SiO(3))(2), aluminium lithium silicon oxide. Data collection strategies, reflection intensity peak integration methods and experimental error estimates are different for the two instruments. Therefore, the consistency and quality of the two types of CCD measurements have been carefully compared to each other and to high-resolution data collected on a conventional CAD-4 point-detector diffractometer. Multipole density model refinements were carried out against the CCD data and the statistical factors analysed in terms of experimental weighting schemes based on the standard uncertainties of the diffraction intensities derived by the Nonius and Siemens software programs. Consistent experimental electron-density features in the Si-O-Si and Si-O-Al bridges were found from both CCD data sets. The net atomic charges obtained from the kappa refinements against each CCD data set are also in good agreement and quite comparable with the results of the conventional CAD-4 experiment.

Journal Article↗

PubMed1999

Electron density in ammonium dihydrogen phosphate: non-uniqueness of the multipolar model in simple inorganic structures.

X-ray and neutron diffraction data of a single crystal of ammonium dihydrogen phosphate have been used for the determination of the electron density using multipolar expansion of the density around each nucleus. As the ammonium group was found to be nearly neutral from unconstrained multipole refinement, constrained refinements have been performed with the charge of the ammonium group ranging from zero to one. On the other hand, the expansion of the radial functions of the phosphorus atom was varied. All refinements led to almost the same agreement factors and residual densities. The consequences of such uncertainties on the topology of the electron density are discussed, namely the topology of the P-O bond critical point.

Journal Article↗

PubMed1999

Topological analysis of the electron density in hydrogen bonds.

Topological analysis of the experimental electron density rho(r) in hydrogen-bonding regions has been carried out for a large number of organic compounds using different multipole models and techniques. Relevant systematic relationships between topological properties at the critical points and the usual geometric parameters are pointed out. Results involving X-ray data only and joint X-ray and neutron data, as well as special hydrogen bonding cases (symmetric, bifurcated, peptide bonds, etc.) are included and analysed in the same framework. A new classification of hydrogen bonds using the positive curvature of the electron density at the critical point [lambda(3)(r(CP))] is proposed.

Journal Article↗

PubMed1999

Molecular fragment electric moments derived from the fit of the experimental electrostatic potential. Application to the water molecule.

The electrostatic potential is a multicenter property that can be expressed as a sum of the contributions of electric moments located at each atomic site of a molecule. Independently of the model used to generate the electrostatic potential around the system, these atomic moments can be accurately obtained by the fit of this physical property outside the van der Waals envelop. However, the larger the system, the greater the number of parameters. In this study a way is proposed to reduce the number of centers in the representation of the electrostatic potential which becomes a sum of fragment contributions rather than atomic ones. A sample of six water molecules in different crystal environments was chosen to discuss the derived values of the electric moments referred to the molecular center of mass.

Journal Article↗

PubMed1999

Charge density study of N-acetyl-L-tyrosine ethyl ester monohydrate derived from CCD area detector data.

The crystal structure, thermal vibrations and electron density of the peptide N-acetyl-L-tyrosine ethyl ester monohydrate, C(13)H(17)NO(4).H(2)O, have been analysed using single-crystal X-ray diffraction data collected at 110 K with Mo Kalpha radiation to a resolution of (sinstraight theta/lambda)(max) = 1.1 Å(-1). A CCD area detector was used to collect 98 393 data during one week. A multipolar atom density model was fitted against the 10 189 unique data with I > 2sigma(I) [R(F) = 0.027, wR(F) = 0.020, g.o.f. = 0.65] in order to map the valence electron distribution. These deformation densities compare very well with those obtained from conventional diffractometers equipped with scintillation detectors. This work shows that area detectors permit charge density studies in a more routine way than is possible with conventional diffractometers.

Journal Article↗

PubMed1999

Chemical synthesis and structure-activity relationships of Ts kappa, a novel scorpion toxin acting on apamin-sensitive SK channel.

Tityus kappa (Ts kappa), a novel toxin from the venom of the scorpion Tityus serrulatus, is a 35-residue polypeptide cross-linked by three disulphide bridges and acts on small-conductance calcium-activated potassium channels (SK channels). Ts K was chemically synthesized using the solid-phase method and characterized. The synthetic product, sTs kappa, was indistinguishable from the natural toxin when tested in vitro in competition assay with radiolabelled apamin for binding to rat brain synaptosomes (IC50 = 3 nM). The sTs kappa was further tested in vivo for lethal activity to mice following intracerebroventricular inoculation (LD50 = 70 ng per mouse). The half-cystine pairings were formerly established by enzyme-based cleavage of sTs kappa; they were between Cys7-Cys28, Cys13-CyS33 and Cys17-Cys35, which is a disulphide bridge pattern similar to that of other short scorpion toxins. According to previous studies on SK channel-acting toxins, the putative influence of certain basic residues of Ts kappa (i.e. Arg6, Arg9, Lys18, Lys19) in its pharmacological activity was investigated using synthetic point-mutated analogues of the toxin with an Ala substitution at these positions. Data from binding assay, together with conformational analysis of the synthetic analogues by 1H-NMR, suggest that Arg6, and to a lesser extent Arg9, are important residues for an high-affinity interaction of this toxin with SK channels; interestingly these residues are located outside the alpha-helical structure, whereas the pharmacologically important basic residues from other SK channel-specific toxins had been located inside the alpha-helix.

Amino Acid Sequence↗

PubMed1999

Luteinizing hormone measurement in polycystic ovary syndrome: a practical approach.

The biological diagnosis of polycystic ovary syndrome (PCO) remains questionable, and a single immunological hLH (ihLH) determination can be misleading. In order better to characterize these patients, we studied hLH pulsatility every 10 min for 4h using a radioimmunoassay and then compared the results with others we obtained with a biological method. Radioimmunological and biological profiles were similar in patients with PCO and in controls. We also studied pulsatility characteristics - frequency and amplitude - and calculated the area under the curve (AUC). There was no significant increase in frequency in our 10 patients with PCO but, as in other studies, increased amplitude of hLH pulses was observed. The most discriminating parameter was the AUC. For practical purposes, we propose that hLH in patients with PCO could be assessed efficiently by taking four samples every 10 min, with computerized calculation of the AUC.

Adolescent↗

PubMed1998

Transferability of multipole charge-density parameters: application to very high resolution oligopeptide and protein structures.

Crystallography at sub-atomic resolution permits the observation and measurement of the non-spherical character of the electron density (parameterized as multipoles) and of the atomic charges. This fine description of the electron density can be extended to structures of lower resolution by applying the notion of transferability of the charge and multipole parameters. A database of such parameters has been built from charge-density analysis of several peptide crystals. The aim of this study is to assess for which X-ray structures the application of transferability is physically meaningful. The charge-density multipole parameters have been transferred and the X-ray structure of a 310 helix octapeptide Ac-Aib2-L-Lys(Bz)-Aib2-L-Lys(Bz)-Aib2-NHMe refined subsequently, for which diffraction data have been collected to a resolution of 0.82 A at a cryogenic temperature of 100 K. The multipoles transfer resulted in a significant improvement of the crystallographic residual factors wR and wR free. The accumulation of electrons in the covalent bonds and oxygen lone pairs is clearly visible in the deformation electron-density maps at its expected value. The refinement of the charges for nine different atom types led to an additional improvement of the R factor and the refined charges are in good agreement with those of the AMBER molecular modelling dictionary. The use of scattering factors calculated from average results of charge-density work gives a negligible shift of the atomic coordinates in the octapeptide but induces a significant change in the temperature factors (DeltaB approximately 0.4 A2). Under the spherical atom approximation, the temperature factors are biased as they partly model the deformation electron density. The transfer of the multipoles thus improves the physical meaning of the thermal-displacement parameters. The contribution to the diffraction of the different components of the electron density has also been analyzed. This analysis indicates that the electron-density peaks are well defined in the dynamic deformation maps when the thermal motion of the atoms is moderate (B typically lower than 4 A2). In this case, a non-truncated Fourier synthesis of the deformation density requires that the diffraction data are available to a resolution better than 0.9 A.

Crystallography, X-Ray↗

PubMed1998

Persistently infected cattle stabilise bovine viral diarrhea virus leading to herd specific strains.

Animals persistently infected with BVDV are important in the epizootiology of the Bovine Viral Diarrhea (BVD) because they are a permanent source of contamination within a herd. These animals produce large quantities of virus and have, therefore, been proposed as responsible for generating antigenic variability. However, limited studies have failed to detect antigenic or genetic changes in viruses isolated at different time from persistently infected animals. One hypothesis to account for this stability is that the immunotolerance is accompanied by a selection against antigenic change. The presence of an immunotolerant persistently infected (IPI) animal in a herd would in turn lead to herd specific strains. To verify this hypothesis, we compared 17 BVDV strains isolated from IPI animals from 3 herds of Eastern Belgium. The comparison was based on the sequence of a 389 bp fragment of E2--a gene encoding for a highly variable glycoprotein. Sequences were strongly conserved within herds but were quite different between herds, indicating that BVDV herd-specific strains do exist and are associated with the presence of IPI animals.

Animals↗

PubMed1998

Synthetic peptides as tools to investigate the structure and pharmacology of potassium channel-acting short-chain scorpion toxins.

In the last decade, numerous polypeptide toxins acting on ion channels have been isolated and characterized from diverse scorpion venoms. These toxins are useful pharmacological probes to study ion-specific channel proteins because they interact selectively with these channels and modulate their activities. Since low amounts of natural toxins can be isolated from scorpion venoms, the chemical synthesis approach is extremely useful to produce larger quantities of toxins and toxin analogs. This report is a succinct overview of the possibilities offered by the chemical synthesis to investigate pharmacological and structural properties of these compounds.

Amino Acid Sequence↗