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Biomedical subjects

E Carlier

Publications and source records attributed to E Carlier.

At least 19 recordsLinked to original sources
PubMed2006

Diagnosis and prognosis of overt disseminated intravascular coagulation in a general hospital -- meaning of the ISTH score system, fibrin monomers, and lipoprotein-C-reactive protein complex formation.

The meaning, the utility, and the prognostic significance of the International Society of Thrombosis and Hemostasis overt disseminated intravascular coagulation (DIC) score and other parameters of coagulation activation including soluble fibrin monomer complexes (SFMC), antithrombin and protein C consumption, and formation of lipoprotein-C-reactive protein (LP-CRP) complexes (MDA slope 1 and flag A2) were evaluated in 165 inpatients from a general hospital for whom DIC testing was required by the attending physicians. Of these 165 patients, 148 had an underlying disease that clearly justified the laboratory request from our systematic post hoc review of the clinical charts. Of these 148 patients, 28 had a positive overt DIC score, 19 had an A2 flag, and 4 had both. The DIC score was strongly related to several major markers of coagulation activation such as D-dimers, thrombin-antithrombin complexes, and soluble fibrin and was inversely related to antithrombin and protein C levels, which began to fall from DIC score 4 or higher. The formation of LP-CRP complexes was only related to Gram-negative sepsis and these patients had a strong inflammatory reaction. Independent risk factors for death were high creatininemia, positive overt DIC score, and/or presence of SFMC. In patients with positive DIC score, SFMC positivity and low levels of antithrombin and/or protein C were additional risk factors. The ISTH overt DIC score proves useful and adequate as a marker for clinically significant DIC. Illness severity is further defined by SFMC, antithrombin, and protein C levels. LP-CRP complexes are related to sepsis but not to actual overt DIC and lethal prognosis.

Biomarkers↗
PubMed2001

Disulfide bridge reorganization induced by proline mutations in maurotoxin.

Maurotoxin (MTX) is a 34-residue toxin that has been isolated from the venom of the chactidae scorpion Scorpio maurus palmatus, and characterized. Together with Pi1 and HsTx1, MTX belongs to a family of short-chain four-disulfide-bridged scorpion toxins acting on potassium channels. However, contrary to other members of this family, MTX exhibits an uncommon disulfide bridge organization of the type C1-C5, C2-C6, C3-C4 and C7-C8, versus C1-C5, C2-C6, C3-C7 and C4-C8 for both Pi1 and HsTx1. Here, we report that the substitution of MTX proline residues located at positions 12 and/or 20, adjacent to C3 (Cys(13)) and C4 (Cys(19)), results in conventional Pi1- and HsTx1-like arrangement of the half-cystine pairings. In this case, this novel disulfide bridge arrangement is without obvious incidence on the overall three-dimensional structure of the toxin. Pharmacological assays of this structural analog, [A(12),A(20)]MTX, reveal that the blocking activities on Shaker B and rat Kv1.2 channels remain potent whereas the peptide becomes inactive on rat Kv1.3. These data indicate, for the first time, that discrete point mutations in MTX can result in a marked reorganization of the half-cystine pairings, accompanied with a novel pharmacological profile for the analog.

Amino Acid Sequence↗
PubMed2001

A novel Xenopus oocyte expression system based on cytoplasmic coinjection of T7-driven plasmids and purified T7-RNA polymerase.

The Xenopus laevis South African frog oocyte is a well suited and widely used system for protein biochemistry and functional studies. So far, two methods are commonly in use for the expression of exogenous proteins in this system. Investigators have the choice between cytoplasmic injections of in vitro synthesized cRNA or nuclear injections of cDNA. Here, we describe a new method for ion channel expression in oocytes, which consists of a coinjection of T7-driven cDNA and T7-RNA polymerase directly into the cytoplasm. This technique uses very limited amounts of purified enzyme and is also applicable to SP6 polymerase. Commercially available polymerases can also conveniently substitute for self-purified enzymes. The technique can be used for electrophysiological and biochemical analysis. In particular, high level expressions have been achieved for potassium (Shaker B, Kv1.2 and Kv1.3) and sodium (P mu 1.2) channels, and we also demonstrate efficient metabolic labeling of the calcium channel auxiliary beta 3 subunit. The properties of the channels expressed by this technique are indistinguishable from those of the channels expressed by classical methods. Expression of multi-subunit proteins was also achieved illustrating that the technique can be used for structure-function analyses. Moreover, this novel expression technique avoids many drawbacks of the two former techniques. It clearly bypasses the costly and time-consuming step of cRNA synthesis in vitro, prevents delicate cRNA manipulation and is easier to perform and more reliable than nuclear injection. Finally, it does not affect cell survival rate. These data indicate that the T7-RNA polymerase expression technique could be widely used in the future for the expression of exogenous proteins in the Xenopus oocyte system.

Animals↗
PubMed2000

Maurotoxin versus Pi1/HsTx1 scorpion toxins. Toward new insights in the understanding of their distinct disulfide bridge patterns.

Maurotoxin (MTX) is a scorpion toxin acting on several K(+) channel subtypes. It is a 34-residue peptide cross-linked by four disulfide bridges that are in an "uncommon" arrangement of the type C1-C5, C2-C6, C3-C4, and C7-C8 (versus C1-C5, C2-C6, C3-C7, and C4-C8 for Pi1 or HsTx1, two MTX-related scorpion toxins). We report here that a single mutation in MTX, in either position 15 or 33, resulted in a shift from the MTX toward the Pi1/HsTx1 disulfide bridge pattern. This shift is accompanied by structural and pharmacological changes of the peptide without altering the general alpha/beta scaffold of scorpion toxins.

Amino Acid Sequence↗
PubMed2000

Na(+) channel regulation by calmodulin kinase II in rat cerebellar granule cells.

The effects of specific CaM kinase II inhibitors were investigated on Na(+) channels from rat cerebellar granule cells. A maximal effect of KN-62 was observed at 20 microM and consisted of an 80% reduction of the peak Na(+) current after only a 10-min application. A hyperpolarizing shift of 8 mV in the steady-state inactivation was also observed. KN-04 (20 microM), an inactive analog, had no detectable effect. KN-62 was however inactive on Na(+) currents recorded from Chinese hamster ovary cells expressing the type II A alpha subunit. We have also analyzed the inhibitory effects of CaM kinase II 296-311 and CaM kinase II 281-309 peptides. Both peptides (75 microM) induced a maximum peak Na(+) current reduction within 30 min. Under similar conditions, a truncated peptide CaM kinase II 284-302 was ineffective. These results demonstrate that CaM kinase II acts as a modulator of Na(+) channel activity in cerebellar granule cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2000

Synthesis, 1H NMR structure, and activity of a three-disulfide-bridged maurotoxin analog designed to restore the consensus motif of scorpion toxins.

Maurotoxin (MTX) is a 34-residue toxin that has been isolated from the venom of the chactidae scorpion Scorpio maurus palmatus. The toxin displays an exceptionally wide range of pharmacological activity since it binds onto small conductance Ca(2+)-activated K(+) channels and also blocks Kv channels (Shaker, Kv1.2 and Kv1.3). MTX possesses 53-68% sequence identity with HsTx1 and Pi1, two other K(+) channel short chain scorpion toxins cross-linked by four disulfide bridges. These three toxins differ from other K(+)/Cl(-)/Na(+) channel scorpion toxins cross-linked by either three or four disulfide bridges by the presence of an extra half-cystine residue in the middle of a consensus sequence generally associated with the formation of an alpha/beta scaffold (an alpha-helix connected to an antiparallel beta-sheet by two disulfide bridges). Because MTX exhibits an uncommon disulfide bridge organization among known scorpion toxins (C1-C5, C2-C6, C3-C4, and C7-C8 instead of C1-C4, C2-C5, and C3-C6 for three-disulfide-bridged toxins or C1-C5, C2-C6, C3-C7, and C4-C8 for four-disulfide-bridged toxins), we designed and chemically synthesized an MTX analog with three instead of four disulfide bridges ([Abu(19),Abu(34)]MTX) and in which the entire consensus motif of scorpion toxins was restored by the substitution of the two half-cystines in positions 19 and 34 (corresponding to C4 and C8) by two isosteric alpha-aminobutyrate (Abu) derivatives. The three-dimensional structure of [Abu(19), Abu(34)]MTX in solution was solved by (1)H NMR. This analog adopts the alpha/beta scaffold with now conventional half-cystine pairings connecting C1-C5, C2-C6, and C3-C7 (with C4 and C8 replaced by Abu derivatives). This novel arrangement in half-cystine pairings that concerns the last disulfide bridge results mainly in a reorientation of the alpha-helix regarding the beta-sheet structure. In vivo, [Abu(19),Abu(34)]MTX remains lethal in mice as assessed by intracerebroventricular injection of the peptide (LD(50) value of 0. 25 microg/mouse). The structural variations are also accompanied by changes in the pharmacological selectivity of the peptide, suggesting that the organization pattern of disulfide bridges should affect the three-dimensional presentation of certain key residues critical to the blockage of K(+) channel subtypes.

Amino Acid Sequence↗
PubMed2000

The I-II loop of the Ca2+ channel alpha1 subunit contains an endoplasmic reticulum retention signal antagonized by the beta subunit.

The auxiliary beta subunit is essential for functional expression of high voltage-activated Ca2+ channels. This effect is partly mediated by a facilitation of the intracellular trafficking of alpha1 subunit toward the plasma membrane. Here, we demonstrate that the I-II loop of the alpha1 subunit contains an endoplasmic reticulum (ER) retention signal that severely restricts the plasma membrane incorporation of alpha1 subunit. Coimmunolabeling reveals that the I-II loop restricts expression of a chimera CD8-I-II protein to the ER. The beta subunit reverses the inhibition imposed by the retention signal. Extensive deletion of this retention signal in full-length alpha1 subunit facilitates the cell surface expression of the channel in the absence of beta subunit. Our data suggest that the beta subunit favors Ca2+ channel plasma membrane expression by inhibiting an expression brake contained in beta-binding alpha1 sequences.

Animals↗
PubMed2000

Effect of maurotoxin, a four disulfide-bridged toxin from the chactoid scorpion Scorpio maurus, on Shaker K+ channels.

Maurotoxin is a 34-residue toxin isolated from the venom of the Tunisian chactoid scorpion Scorpio maurus palmatus and contains four disulfide bridges that are normally found in long-chain toxins of 60-70 amino acid residues, which affect voltage-gated sodium channels. However, despite the unconventional disulfide-bridge pattern of maurotoxin, the conformation of this toxin remains similar to that of other toxins acting on potassium channels. Here, we analyzed the effects of synthetic maurotoxin on voltage-gated Shaker potassium channels (ShB) expressed in Xenopus oocytes. Maurotoxin produces a strong, but reversible, inhibition of the ShB K+ current with an IC50 of 2 nM. Increasing concentrations of the toxin induce a progressively higher block at saturating concentrations. At nonsaturating concentrations of the toxin (5-20 nM), the channel block appears slightly more pronounced at threshold potentials suggesting that the toxin may have a higher affinity for the closed state of the channel. At the single channel level, the toxin does not modify the unitary current amplitude, but decreases ensemble currents by increasing the number of depolarizing epochs that failed to elicit any opening. A point mutation of Lys23 to alanine in maurotoxin produces a 1000-fold reduction in the IC50 of block by the toxin suggesting the importance of this charged residue for the interaction with the channel. Maurotoxin does not affect K+ currents carried by Kir2.3 channels in oocytes or Na+ currents carried by the alphaIIa channel expressed in CHO cells.

Amino Acid Motifs↗
PubMed2000

Ion channel activation by SPC3, a peptide derived from the HIV-1 gp120 V3 loop.

SPC3 is a multibranched peptide containing eight identical GPGRAF motifs which are derived from the human immunodeficiency virus (HIV)-1 gp120 V3 loop consensus sequence. This molecule was reported to prevent the infection of CD4+ cells by various HIV-1 and HIV-2 strains. However, the molecular mode of action of SPC3 remains unclear. Here, we investigated the possibility that SPC3 could interact with alpha/beta-chemokine receptors following observations that, first, the V3 loop is likely to be involved in alpha/beta-chemokine receptor-dependent HIV entry and, second, natural ligands of these receptors are potent inhibitors of cell infection. To address this point, we examined the effects of SPC3 on Xenopus oocytes either uninjected or expressing exogenous human CXCR4 alpha-chemokine receptors. Extracellular applications of micromolar concentrations of SPC3 onto Xenopus oocytes trigger potent inward chloride currents which can be inhibited by increasing extracellular Ca2+ concentration. This effect can be blocked by chloride channel antagonists and is highly specific to SPC3 as it is not triggered by structural analogs of SPC3. The SPC3-induced chloride conductance in oocytes is alpha/beta-chemokine receptor dependent because: (i) SPC3 alters the sensitivity of this channel to external applications of human recombinant MIP-1alpha, a natural ligand of human CCR5 receptor, and (ii) the amplitude of the inward current could be increased by the expression of exogenous human CXCR4 chemokine receptor. The effect of SPC3 appears to rely on the activation of a phospholipase A2 signaling pathway, but is not affected by changes in cytosolic Ca2+ concentration, or by alterations in Gi/Go protein, adenylate cyclase, phospholipase C or protein kinase C activity. Altogether, the data indicate that SPC3 is capable of activating a surface alpha/beta-chemokine-like receptor-mediated signaling pathway in competent cells, thereby triggering, either directly or indirectly, a Ca2+-inactivated chloride conductance.

Animals↗
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↗
PubMed1997

Direct block of voltage-sensitive sodium channels by genistein, a tyrosine kinase inhibitor.

Genistein, an isoflavone inhibitor of tyrosine-specific protein kinases, was shown to specifically block the 22Na+ influx through voltage-sensitive Na+ channels in cultured rat brain neurons, whereas other tyrosine kinase antagonists such as lavendustin A, compound 5, tyrphostin A47 and an erbstatin analog were inactive at concentrations known to block kinase activity in other neuronal systems. Dose-response curves for genistein indicated a half-maximum effect at 60 microM. Daidzein, an inactive analog of genistein, had a similar inhibitory effect on the 22Na+ influx with a half-maximum effect at 195 microM. The time course of genistein action was rapid, because maximum effect on 22Na+ influx was obtained in less than 20 s at 100 microM. Analysis of Na+ currents by the whole-cell recording technique showed that 20 microM genistein reduced the sodium current and shifted the voltage dependence of both activation and inactivation curves. No competition with [3H]saxitoxin binding was observed, whereas the binding of [3H]batrachotoxinin A 20-alpha-benzoate to rat brain synaptosomal membranes was partially inhibited, which suggested a direct or allosteric interaction with neurotoxin binding site 2. These data taken together clearly indicate that the inhibition of voltage-sensitive sodium channels by genistein is not mediated by tyrosine kinase inhibition.

Animals↗
PubMed1996

Scorpion toxins affecting sodium current inactivation bind to distinct homologous receptor sites on rat brain and insect sodium channels.

Sodium channels posses receptor sites for many neurotoxins, of which several groups were shown to inhibit sodium current inactivation. Receptor sites that bind alpha- and alpha-like scorpion toxins are of particular interest since neurotoxin binding at these extracellular regions can affect the inactivation process at intramembranal segments of the channel. We examined, for the first time, the interaction of different scorpion neurotoxins, all affecting sodium current inactivation and toxic to mammals, with alpha-scorpion toxin receptor sites on both mammalian and insect sodium channels. As specific probes for rat and insect sodium channels, we used the radiolabeled alpha-scorpion toxins AaH II and LqhalphaIT, the most active alpha-toxins on mammals and insect, respectively. We demonstrate that the different scorpion toxins may be classified to several groups, according to their in vivo and in vitro activity on mammalian and insect sodium channels. Analysis of competitive binding interaction reveal that each group may occupy a distinct receptor site on sodium channels. The alpha-mammal scorpion toxins and the anti-insect Lqh alphaIT bind to homologous but not identical receptor sites on both rat brain and insect sodium channels. Sea anemone toxin ATX II, previously considered to share receptor site 3 with alpha-scorpion toxins, is suggested to bind to a partially overlapping receptor site with both AaH II and Lqh alphaIT. Competitive binding interactions with other scorpion toxins suggest the presence of a putative additional receptor site on sodium channels, which may bind a unique group of these scorpion toxins (Bom III and IV), active on both mammals and insects. We suggest the presence of a cluster of receptor sites for scorpion toxins that inhibit sodium current inactivation, which is very similar on insect and rat brain sodium channels, in spite of the structural and pharmacological differences between them. The sea anemone toxin ATX II is also suggested to bind within this cluster.

Amino Acid Sequence↗
PubMed1995

Synergistic action of corticosterone on kainic acid-induced electrophysiological alterations in the hippocampus.

The present study investigates the effect of overexposure to high doses of the stress hormone corticosterone (CORT) on the electrophysiological changes produced in the hippocampus after local microinjection of KA. Extracellular recordings were performed in the CA1 area of mouse hippocampal slices prepared after a 7-day recovery period following KA microinfusion alone or combined with 3 days overexposure to CORT. The results showed that CORT shifts the KA response profile approximately 40-fold, since animals treated with a non-toxic dose of 0.01 microgram KA and CORT exhibited epileptic activity and a shift on the paired-pulse response similar to that observed in animals treated with high doses of KA (0.4 microgram). This synergistic action of CORT on the electrophysiological changes induced by KA was antagonized by the antiglucocorticoid RU486 whereas the antimineralocorticoid spironolactone was ineffective. These results suggest that CORT may play an important role in modulating the severity of KA-induced seizures in the hippocampal structure probably by GR-receptor mediated action.

Animals↗
PubMed1995

Methylene blue administration in septic shock: a clinical trial.

OBJECTIVE: A release of nitric oxide has been incriminated in the cardiovascular alterations of septic shock. Since guanylate cyclase is the target enzyme in the endothelium-dependent relaxation mediated by nitric oxide, we studied the acute effects of methylene blue, a potent inhibitor of guanylate cyclase in patients with septic shock. DESIGN: Prospective clinical trial. SETTING: Medical-surgical intensive care unit in a university hospital. PATIENTS: Fourteen patients with severe septic shock requiring adrenergic therapy. INTERVENTIONS: Short-term intravenous infusion of methylene blue. MEASUREMENTS AND MAIN RESULTS: Hemodynamic measurements were obtained at baseline, and 30, 60, and 90 mins after the infusion of 2 mg/kg of methylene blue. Methylene blue administration was followed by a progressive increase in mean arterial pressure (from 61.1 +/- 7.6 to 71.7 +/- 12.0 mm Hg at 60 mins, p < .01). Pulmonary arterial pressure, cardiac filling pressures, cardiac output oxygen delivery, and oxygen consumption were not significantly affected. Left ventricular stroke work increased from 42.5 +/- 17.9 to 48.9 +/- 14.5 g.m after 60 mins (p < .05). Arterial lactate concentration decreased from 3.4 +/- 1.4 to 2.7 +/- 1.3 mmol/L (p < .05). Since these effects were transient, a second dose of methylene blue was administered 90 mins later to six patients and was followed by a similar response. No adverse effect was observed. CONCLUSIONS: In septic shock patients, the administration of methylene blue results in a transient and reproducible increase in arterial pressure, associated with an improvement in cardiac function, but does not increase cellular oxygen availability. The significant reduction in blood lactate concentration is probably related to the reductor effect of methylene blue, rather than to an improvement in tissue oxygenation.

Adult↗
PubMed1994

Sodium channel internalization in developing neurons.

Neurotoxin-induced activation of voltage-dependent Na+ channels provoked rapid (t1/2 = 15-20 min) channel down-regulation in cultured rat brain neurons, resulting in a 50%-70% decrease in [3H]saxitoxin and 125I-alpha-scorpion toxin binding capacities as well as a decrease in Na+ peak current. Experiments using 125I-alpha-scorpion toxin as both a Na+ channel activator and a surface channel probe showed that a fraction of the bound toxin was internalized, since it was not releasable by acidic washing. Internalization was inhibited by tetrodotoxin, abolished in Na(+)-free medium, and induced by amphotericin B, a Na+ ionophore. Moreover, down-regulation occurred only in immature neuronal tissue, either cultured fetal neurons or postnatal hippocampal slices, but was absent in adult brain. These observations indicate that Na+ channel internalization is triggered by Na+ influx into neurons and may be involved in the control of electrical activity during development.

Aging↗
PubMed1994

Corticosterone effects on long-term potentiation in mouse hippocampal slices.

Corticosterone (CORT) can alter several electrophysiological properties in the hippocampus. Few studies have reported the effects of CORT on hippocampal long-term potentiation (LTP). In the CA1 region of the hippocampal slice, we studied the relative increase in population spike (PS) amplitude after inducing tetanus (PS-LTP) in control conditions and in the presence of CORT (0.5 nM or 5 microM). Two perifusion mediums were used which differed in calcium concentration (M1:Ca 2.5 mM; M2: Ca 3.1 mM). In the control slices, the PS-LTP amplitude was greater in the M1 medium. With the low CORT concentration (0.5 nM), the PS-LTP amplitude was greater than in control conditions in the M2 medium only. A similar PS-LTP amplitude increase was also observed in the M2 medium after application of 0.5 nM aldosterone (type I receptor analog). With the high CORT concentration (5 microM), PS-LTP amplitude decreased in the two conditions. This effect was reversed by co-application of RU486 (type II receptor antagonist). These results demonstrate that CORT can modulate PS-LTP via its two types of hippocampal CORT receptors, depending on the concentration applied. They also showed that the extracellular calcium concentration may contribute to the functional change induced by CORT.

Aldosterone↗