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Biomedical subjects

M Leduc

Publications and source records attributed to M Leduc.

At least 19 recordsLinked to original sources
PubMed2006

Development of a kinetic metabolic model: application to Catharanthus roseus hairy root.

A kinetic metabolic model describing Catharanthus roseus hairy root growth and nutrition was developed. The metabolic network includes glycolysis, pentose-phosphate pathway, TCA cycle and the catabolic reactions leading to cell building blocks such as amino acids, organic acids, organic phosphates, lipids and structural hexoses. The central primary metabolic network was taken at pseudo-steady state and metabolic flux analysis technique allowed reducing from 31 metabolic fluxes to 20 independent pathways. Hairy root specific growth rate was described as a function of intracellular concentration in cell building blocks. Intracellular transport and accumulation kinetics for major nutrients were included. The model uses intracellular nutrients as well as energy shuttles to describe metabolic regulation. Model calibration was performed using experimental data obtained from batch and medium exchange liquid cultures of C. roseus hairy root using a minimal medium in Petri dish. The model is efficient in estimating the growth rate.

Catharanthus↗
PubMed2006

Accurate determination of the scattering length of metastable helium atoms using dark resonances between atoms and exotic molecules.

We present a new measurement of the s-wave scattering length a of spin-polarized helium atoms in the 2(3)S1 metastable state. Using two-photon photoassociation spectroscopy and dark resonances, we measure the energy E(nu)=14= -91.35+/- 0.06 MHz of the least-bound state nu = 14 in the interaction potential of the two atoms. We deduce a value of a=7.512+/-0.005 nm, which is at least 100 times more precise than the best previous determinations and is in disagreement with some of them. This experiment also demonstrates the possibility to create exotic molecules binding two metastable atoms with a lifetime of the order of 1 micros.

Journal Article↗
PubMed2003

Giant helium dimers produced by photoassociation of ultracold metastable atoms.

We produce giant, purely long-range helium dimers by photoassociation of metastable helium atoms in a magnetically trapped, ultracold cloud. The photoassociation laser is detuned close to the atomic 2(3)S1-2(3)P0 line and produces strong heating of the sample when resonant with molecular bound states. The temperature of the cloud serves as an indicator of the molecular spectrum. We report good agreement between our spectroscopic measurements and our calculations of the five bound states belonging to a 0(+)(u) purely long-range potential well. These previously unobserved states have classical inner turning points of about 150a(0) and outer turning points as large as 1150a(0).

Journal Article↗
PubMed2002

Transgenic goats produced by DNA pronuclear microinjection of in vitro derived zygotes.

This study was undertaken to investigate various factors affecting the outcomes of in vitro fertilization (IVF) of oocytes retrieved by laparoscopic ovum pick-up (LOPU) technique from prepubertal and adult goats, as well as to evaluate the developmental competence of in vitro produced embryos. Oocyte-cumulus complexes recovered by LOPU from donors stimulated with gonadotrophins were matured in vitro. Fresh semen was used for IVF following various capacitation treatments. In vitro produced zygotes were either cultured to assess in vitro development or were transferred into recipients for full term development. The results indicated that successful IVF of the goat oocytes was affected by factors such as sperm capacitation treatment, oocyte quality, and abundance of cumulus cells on zona pellucida. Oocytes from both prepubertal and adult goats demonstrated similar full term developmental competence despite the fact that in vitro developmental rates were lower for prepubertal goats. The births of transgenic offspring demonstrated that the established LOPU-IVF technology combined with pronuclear microinjection can be successfully used to produce transgenic goats.

Animals↗
PubMed2001

Bose-Einstein condensation of metastable helium.

We have observed a Bose-Einstein condensate in a dilute gas of 4He in the (3)2S(1) metastable state. We find a critical temperature of (4.7+/-0.5) microK and a typical number of atoms at the threshold of 8 x 10(6). The maximum number of atoms in our condensate is about 5 x 10(5). An approximate value for the scattering length a = (16+/-8) nm is measured. The mean elastic collision rate at threshold is then estimated to be about 2 x 10(4) s(-1), indicating that we are deeply in the hydrodynamic regime. The typical decay time of the condensate is 2 s, which places an upper bound on the rate constants for two-body and three-body inelastic collisions.

Journal Article↗
PubMed2001

Generation of dwarf goat (Capra hircus) clones following nuclear transfer with transfected and nontransfected fetal fibroblasts and in vitro-matured oocytes.

The developmental potential of caprine fetal fibroblast nuclei after in vitro transfection and nuclear transfer (NT) into enucleated, in vitro-matured oocytes was evaluated. Fetal fibroblasts were isolated from Day 27 to Day 30 fetuses from a dwarf breed of goat (BELE: breed early lactate early). Cells were transfected with constructs containing the enhanced green fluorescent protein (eGFP) and neomycin resistance genes and were selected with G418. Three eGFP lines and one nontransfected line were used as donor cells in NT. Donor cells were cultured in Dulbecco minimum Eagle medium plus 0.5% fetal calf serum for 4-8 days prior to use in NT. Immature oocytes were recovered by laparoscopic ovum pick-up and matured for 24 h prior to enucleation and NT. Reconstructed embryos were transferred as cleaved embryos into synchronized recipients. A total of 27 embryos derived from transgenic cells and 70 embryos derived from nontransgenic cells were transferred into 13 recipients. Five recipients (38%) were confirmed pregnant at Day 35 by ultrasound. Of these, four recipients delivered five male kids (7.1% of embryos transferred) derived from the nontransfected line. One recipient delivered a female kid derived from an eGFP line (7.7% of embryos transferred for that cell line). Presence of the eGFP transgene was confirmed by polymerase chain reaction, Southern blotting, and fluorescent in situ hybridization analyses. Nuclear transfer derivation from the donor cells was confirmed by single-strand confirmation polymorphism analysis. These results demonstrate that both in vitro-transfected and nontransfected caprine fetal fibroblasts can direct full-term development following NT.

Animals↗
PubMed2000

Interaction of linker for activation of T cells with multiple adapter proteins in platelets activated by the glycoprotein VI-selective ligand, convulxin.

The snake venom toxin convulxin activates platelets through the collagen receptor glycoprotein VI (GPVI)/Fc receptor gamma-chain (FcR gamma-chain) complex leading to tyrosine phosphorylation and activation of the tyrosine Syk and phospholipase Cgamma2 (PLCgamma2). In the present study, we demonstrate that convulxin is a considerably more powerful agonist than collagen or the GPVI-selective collagen-related peptide (CRP). Confirmation that the response to convulxin is mediated solely via Syk was provided by studies on Syk-deficient platelets. The increase in phosphorylation of the FcR gamma-chain is associated with marked increases in tyrosine phosphorylation of downstream proteins including Syk, linker for activation of T cells (LAT), SLP-76, and PLCgamma2. The transmembrane adapter LAT coprecipitates with SLP-76 and PLCgamma2, as well as with a number of other adapter proteins, some of which have not been previously described in platelets, including Cbl, Grb2, Gads, and SKAP-HOM. Gads is constitutively associated with SLP-76 and is probably the protein bridging its association with LAT. There was no detectable association between Grb2 and SLP-76 in control or stimulated cells, suggesting that the interaction of LAT with Grb2 is present in a separate complex to that of LAT-Gads-SLP-76. These results show that the trimeric convulxin stimulates a much greater phosphorylation of the FcR gamma-chain and subsequent downstream responses relative to CRP and collagen, presumably because of its ability to cause a greater degree of cross-linking of GPVI. The adapter LAT appears to play a critical role in recruiting a number of other adapter proteins to the surface membrane in response to activation of GPVI, presumably at sites of glycolipid-enriched microdomains, enabling an organized signaling cascade that leads to platelet activation.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Expression of the collagen receptor glycoprotein VI during megakaryocyte differentiation.

This study examined the expression of the platelet collagen receptor glycoprotein VI (GPVI) in megakaryocyte cell lines and primary megakaryocytes by reverse transcriptase-polymerase chain reaction and by flow cytometry and ligand blotting using the snake venom toxin convulxin. Expression of GPVI is increased in the megakaryoblastic cell lines HEL and CMK on differentiation with the phorbol ester phorbol 12-myristate 13-acetate (PMA), along with the Fc receptor gamma-chain (FcR gamma-chain). The increase in GPVI expression is associated with marked potentiation of tyrosine phosphorylation and Ca(++) elevation in response to convulxin. Syk, linker for activated T cells, and phospholipase C gamma 2 (PLC gamma 2) are among the proteins tyrosine phosphorylated on convulxin stimulation in PMA-differentiated HEL cells. Studies on primary murine megakaryocytes grown in vitro confirmed that GPVI is up-regulated in parallel with functional activation, assessed by measurement of [Ca(++)](i), during differentiation. The results demonstrate that expression of GPVI is up-regulated along with the FcR gamma-chain during differentiation of megakaryocytes. (Blood. 2000;96:2740-2745)

Animals↗
PubMed2000

Collagen, convulxin, and thrombin stimulate aggregation-independent tyrosine phosphorylation of CD31 in platelets. Evidence for the involvement of Src family kinases.

Platelet endothelial cell adhesion molecule-1 (CD31) is a 130-kDa glycoprotein receptor present on the surface of platelets, neutrophils, monocytes, certain T-lymphocytes, and vascular endothelial cells. CD31 is involved in adhesion and signal transduction and is implicated in the regulation of a number of cellular processes. These include transendothelial migration of leukocytes, integrin regulation, and T-cell function, although its function in platelets remains unclear. In this study, we demonstrate the ability of the platelet agonists collagen, convulxin, and thrombin to induce tyrosine phosphorylation of CD31. Furthermore, we show that this event is independent of platelet aggregation and secretion and is accompanied by an increase in surface expression of CD31. A kinase capable of phosphorylating CD31 was detected in CD31 immunoprecipitates, and its activity was increased following activation of platelets. CD31 tyrosine phosphorylation was reduced or abolished by the Src family kinase inhibitor PP2, suggesting a role for these enzymes. In accordance with this, each of the Src family members expressed in platelets, namely Fyn, Lyn, Src, Yes, and Hck, was shown to co-immunoprecipitate with CD31. The involvement of Src family kinases in this process was confirmed through the study of mouse platelets deficient in Fyn.

Animals↗
PubMed2000

Convulxin binding to platelet receptor GPVI: competition with collagen related peptides.

Convulxin (CVX), a potent platelet aggregating protein from the venom of the snake Crotalus durissus terrificus, is known to bind to the platelet collagen receptor, glycoprotein VI (GPVI). CVX binding to human platelets was investigated by flow cytometry, using fluorescein labeled convulxin (FITC-CVX). Scatchard analysis indicated high and low affinity binding sites with Kd values of 0.6 and 4 nM and Bmax values of 1200 and 2000 binding sites per platelet. FITC-CVX binding was inhibited by collagen related peptides (CRPs) comprising a repeated GPO sequence, namely GCO(GPO)(10)GCOGNH(2) and GKO(GPO)(10)GKOGNH(2), which also bind to receptor GPVI. These peptides (monomeric or cross-linked forms) gave a high affinity inhibition of 10-20% for concentrations between 10 ng/ml and 5 microg/ml, followed by a second phase of inhibition at concentrations greater than 5 microg/ml. It was shown also that the inhibition of FITC-CVX binding by CRPs was independent on the time of preincubation of platelets with CRPs, and the same percentage of inhibition was seen with various concentrations of convulxin. Confocal microscopy of the distribution of FITC-CVX binding sites on platelets showed an homogeneous distribution of FITC-CVX bound to GPVI, although some limited clustering may exist.

Binding, Competitive↗
PubMed1998

Cloning of subunits of convulxin, a collagen-like platelet-aggregating protein from Crotalus durissus terrificus venom.

Convulxin (CVX) is a potent platelet-aggregating glycoprotein from the venom of the snake Crotalus durissus terrificus. It consists of two subunits, alpha and beta, joined by disulphide bridges in a hexameric structure. A cDNA library from venom gland was constructed in the vector pT3T7. The cloned cDNAs encoding the two chains of CVX were sequenced. Both are preceded by an identical 23-amino acid peptide signal sequence and encode sequences of 135 amino acids for the alpha chain and 125 amino acids for the beta chain. These polypeptides include a carbohydrate-recognition domain (CRD) in which some of the specific amino acids required for binding Ca2+ and galactose or mannose are absent. The presence of such a domain means that CVX can be included in the family of C-type lectins along with other snake venom proteins, although it is not a true lectin. Assuming that the localization of intracatenary disulphide bridges of each CVX chain is similar to that of the CRD and that an intercatenary bridge between the alpha and beta chains is similar to that of the C-type lectin botrocetin, we postulate the existence of an additional intercatenary bridge, which explains the tridimeric structure (alphabeta)3 of CVX.

Amino Acid Sequence↗
PubMed1998

Convulxin-induced platelet adhesion and aggregation: involvement of glycoproteins VI and IaIIa.

The interaction of convulxin (Cvx), a 72-kDa glycoprotein isolated from the venom of Crotalus durissus terrificus with human platelets has been studied. Cvx at low concentrations (below 100 pM) induced platelet aggregation, dense body secretion and intracellular calcium mobilization which indicates that Cvx is a potent activator of human platelets. Cvx-induced platelet aggregation and secretion was inhibited by 6Fl an anti-integrin alpha2beta1 monoclonal antibody that was without effect on calcium mobilization. Anti-GPVI Fab fragments inhibited aggregation, secretion and calcium mobilization triggered by Cvx. In addition, immobilized Cvx was found to induce divalent cation-independent platelet adhesion in a static system. Platelet adhesion to Cvx was inhibited by anti-GPVI Fab fragments but not by anti-integrin alpha2beta1 . Cvx was shown to bind to a 57,000 Dalton protein that was identified as GPVI. Altogether, these results indicate that GPVI behaves as a receptor for Cvx, while integrin alpha2beta1 could play a regulatory role in Cvx-induced platelet aggregation. Cvx and collagen interaction with platelets, thus appears to share some characteristics but to also have specific properties.

Journal Article↗
PubMed1997

Convulxin, a potent platelet-aggregating protein from Crotalus durissus terrificus venom, specifically binds to platelets.

Convulxin, a very potent aggregating protein from rattlesnake venom, was purified by a new procedure and its heterodimeric structure alpha 3 beta 3 was confirmed. The polypeptide N-terminal sequences of convulxin subunits were determined by Edman degradation. They are very similar and appear homologous to botrocetin from Bothrops jararaca venom and to rattlesnake lectin from Crotalus atrox venom, both being classified among the C-type lectin family. The binding of 125I-labelled convulxin to blood platelets has also been analysed under equilibrium conditions. These studies indicated that convulxin binds to platelets with a high affinity (Kd = 30 pM) on a small number of binding sites (1000 binding sites per cell). The high-affinity binding of convulxin appears specific to platelets, since it is not observed on other cell types such as neutrophils and erythrocytes. Also, the high-affinity binding of convulxin to membranes platelet is not inhibited by alpha-thrombin, fibrinogen, collagen, laminin binding inhibitor, RGDS peptide, adenosine diphosphate, platelet-activating factor-acether, serotonin or epinephrine. This, together with the recent observation that platelet activation by convulxin is partially mediated by phospholipase C and involves other mechanisms as well, indicates that convulxin may interact with a specific platelet acceptor (receptor) protein which has yet to be characterized.

Amino Acid Sequence↗
PubMed1996

Nuclear magnetic resonance imaging with hyperpolarised helium-3.

BACKGROUND: Magnetic resonance imaging (MRI) relies on magnetisation of hydrogen nuclei (protons) of water molecules in tissue as source of the signal. This technique has been valuable for studying tissues that contain significant amounts of water, but biological settings with low proton content, notably the lungs, are difficult to image. We report use of spin-polarised helium-3 for lung MRI. METHODS: A volunteer inhaled hyperpolarised 3He to fill the lungs, which were imaged with a conventional MRI detector assembly. The nuclear spin polarisation of helium, and other noble gases, can be greatly enhanced by laser optical pumping and is about 10(5) times larger than the polarisation of water protons. This enormous gain in polarisation easily overcomes the loss in signal due to the lower density of the gas. FINDINGS: The in-vivo experiment was done in a whole-body MRI scanner. The 3He image showed clear demarcation of the lung against diaphragm, heart, chest wall, and blood vessels (which gave no signal). The signal intensity within the air spaces was greatest in lung regions that are preferentially ventilated in the supine position; less well ventilated areas, such as the apices, showed a weaker signal. INTERPRETATION: MRI with hyperpolarised 3He gas could be an alternative to established nuclear medicine methods. The ability to image air spaces offers the possibility of investigating physiological and pathophysiological processes in pulmonary ventilation and differences in its regional distribution.

Adult↗
PubMed1995

Inverse correlation between the proportion of salivary bacteria inhibiting Streptococcus mutans and the percentage of untreated carious teeth.

To evaluate the role of inhibitory substances produced by bacteria in the oral cavity, we estimated, by a deferred test on Todd-Hewitt agar enriched with hemin and vitamin K, the proportion of bacteria that inhibited or stimulated the growth of Streptococcus mutans and Porphyromonas gingivalis, from the saliva of 109 patients (54 males and 55 females) attending our dental clinics. The patients, aged from 8 to 75 years old (mean: 31 +/- 18 years), were randomly selected whatever the reason for their visit. The results, evaluated with the Spearman rank test, indicated that there was no statistically significant (P > 0.05) correlation between the proportion of salivary bacteria inhibiting or stimulating P. gingivalis with the Community Periodontal Index of Treatment Needs (CPITN), the number of carious, missing and filled teeth, or with the decayed, missing and filled teeth index (DMFT). Also, no statistically significant correlation was observed between the proportion of salivary bacteria stimulating the growth of S. mutans and the above mentioned health indexes. However, a statistically significant (P < 0.005) negative correlation was found between the percentage of cultivated bacteria that inhibit S. mutans and the percentage of untreated carious teeth as well as with the CPITN. The results thus indicate a possible role for inhibitory substances produced by bacteria in the maintenance of oral health.

Adolescent↗