Biomedical subjects

H Rochat

Publications and source records attributed to H Rochat.

At least 73 records · Page 4Linked to original sources

PubMed1996

Anti-HIV activity of multibranched peptide constructs derived either from the cleavage sequence or from the transmembrane domain (gp41) of the human immunodeficiency virus type 1 envelope.

Multibranched peptides (SPCs) derived either from the fusion protein (gp41) sequence or from the cleavage sequence of the human immunodeficiency virus type 1 envelope were chemically synthesized and tested for their ability to inhibit both syncytium formation and HIV production in CD4+ cells. The gp41-derived SPCs had no effect. In contrast, an SPC encompassing the envelope cleavage sites strongly inhibited both HIV Env-induced syncytium formation and viral production.

Amino Acid Sequence↗

PubMed1996

Synthesis and characterization of leiurotoxin I analogs lacking one disulfide bridge: evidence that disulfide pairing 3-21 is not required for full toxin activity.

Leiurotoxin I (Lei-NH2), a toxin isolated from the venom of the scorpion Leiurus quinquestriatus hebraeus, is a blocker of the apamin-sensitive Ca(2+)-activated K+ channels. It is a 31-residue polypeptide cross-linked by three disulfide bridges which are presumably between Cys3-Cys21, Cys8-Cys26, and Cys12-Cys28. To investigate the role of these disulfides, analogs of Lei-NH2 lacking one disulfide bridge (i.e., [Abu3,21]Lei-NH2, [Abu8,26]Lei-NH2, and [Abu12,28]Lei-NH2) were chemically synthesized by selective replacement of each pair of half-cystines forming a bridge by two alpha-aminobutyrate (Abu) residues. The two disulfide pairings of the main folded form of the synthetic analogs were established by enzymatic proteolysis. They were as expected between Cys8-Cys26 and Cys12-Cys28 for [Abu3,21]Lei-NH2 but were unexpectedly between Cys3-Cys12 and Cys21-Cys28 for [Abu8,26]Lei-NH2 and between Cys3-Cys8 and Cys21-Cys26 for [Abu12,28]Lei-NH2. The synthetic peptides were tested in vitro for their capacity to compete with the binding of [125I]apamin to rat brain synaptosomes and in vivo for their neurotoxicity in mice. In both assays, [Abu3,21]Lei-NH2 exhibited full Lei-NH2-like activity whereas [Abu8,26]Lei-NH2 and [Abu12,28]-Lei-NH2 possessed only residual activities (< 2% native toxin activity). This suggests that disulfide bridge Cys3-Cys21 is not essential per se for high toxin activity. Circular dichroism (CD) spectroscopy of the three analogs showed that only [Abu3,21]Lei-NH2 exhibited a CD spectrum similar to that of Lei-NH2, suggesting they both adopt closely related conformations, in agreement with the pharmacological data. Structural models of the analogs were constructed on the basis of the disulfide pairing assignment and compared with that of Lei-NH2.

Amino Acid Sequence↗

PubMed1996

Novel anti-platelet aggregation polypeptides from Vipera lebetina venom: isolation and characterization.

Lebetins 1 and Lebetins 2, two polypeptide groups that inhibit platelet aggregation, were isolated from Vipera lebetina venom by gel filtration and reverse phase chromatography. Amino acid sequencing indicated that the first group contains two major polypeptides of 13 and 12 residues; their molecular weight was determined by electrospray mass spectrometry. The second was composed of two peptides of 38 and 37 residues, each with one disulfide bond. Sequence analysis revealed neither RGD sequence nor homology with other proteins including known snake or tick polypeptides. Lebetins 1 were Pro and Lys rich peptides and their sequences were identical to the N-terminus of Lebetins 2. Lebetins inhibited platelet aggregation induced by thrombin, collagen and PAF-acether. The 50% concentration that inhibited human and rabbit platelet aggregation induced by thrombin was 590 nM and 125 nM for Lebetins 1 and 100 nM and 8 nM for Lebetins 2, respectively. Lebetins 1 and Lebetins 2 also inhibited fibrinogen-induced aggregation of alpha-chymotrypsin-treated platelets as well as in vivo collagen-induced thrombocytopenia in rats with half effective doses of 2 nmol/kg and 4.2 nmol/kg, respectively. Lebetins were not toxic after intravenous injection into mice and rats. These polypeptides form novel platelet inhibitors that could be used to delineate further the mechanisms of platelet aggregation and serve as a model for developing antithrombotic agents.

Amino Acid Sequence↗

PubMed1996

Tetrodotoxin reverses brevetoxin allosteric inhibition of scorpion alpha-toxin binding on rat brain sodium channels.

Voltage-sensitive sodium channels are responsible for the initiation of action potentials in many excitable cells. Several neurotoxins bind to distinct receptor sites on sodium channels and reveal strong allosteric interactions among them. Scorpion alpha toxins, which inhibit sodium channel inactivation by binding to receptor site 3, have been very important tools to study sodium channel structure and function. Recently, we have shown that brevetoxin induce a strong negative allosteric modulation on scorpion alpha-toxin binding on rat brain sodium channels, in contrast to previously published studies. In this report we have examined the reasons for this discrepancy and found new, unexpected allosteric interactions between the tetrodotoxin and brevetoxin receptor sites, using scorpion alpha-toxin as sensitive probe for subtle conformational changes on sodium channels. Tetrodotoxin reverses the negative modulation induced by brevetoxin on scorpion alpha-toxin binding, revealing new dynamic interactions in sodium channel structure.

Allosteric Site↗

PubMed1996

Characterization of a new peptide from Tityus serrulatus scorpion venom which is a ligand of the apamin-binding site.

A new ligand (Ts kappa) of the apamin binding site on rat brain synaptosomes (K0.5 = 300 pM) was purified and characterized from the venom of Tityus serrulatus. It is a polypeptide toxin of 35 amino acid residues, with three disulfide bridges. Its cDNA was amplified from a venom gland cDNA library and the nucleotide sequence determined. A model of Ts kappa was constructed by amino acid replacement using charybdotoxin structure as determined by 1H nuclear magnetic resonance as starting model.

Amino Acid Sequence↗

PubMed1996

Characterization of alpha-neurotoxin and phospholipase A2 activities from Micrurus venoms. Determination of the amino acid sequence and receptor-binding ability of the major alpha-neurotoxin from Micrurus nigrocinctus nigrocinctus.

New World elapids are coral snakes that belong to the genus Micrurus, and for which the venom biochemistry is mostly unknown. Analysis has been difficult because the coral snakes produce small quantities of venom. Clinical observations following bites show mainly neurotoxic effects. Experimentally, cardiotoxic, haemolytic and myotoxic activities are also reported. An experimental approach, using reverse-phase high-performance liquid chromatography and specific assays for alpha-neurotoxin and phospholipase A2 activities, was conducted on milligram quantities of venoms from three Micrurus species from Costa Rica; M. nigrocinctus nigrocinctus, M. alleni yatesi and M. multifasciatus. Neurotoxicity was determined by competition binding experiments with the Torpedo marmorata acetylcholine receptor. Phospholipase A2 activity was measured by fluorimetry using a pyrene lipid substrate. In this way, we purified and characterized seven alpha-neurotoxins, five phospholipases A2 and four toxin homologs. The amino acid sequence of the major alpha-neurotoxin from M. nigrocinctus nigrocinctus venom was fully determined and compared to Old Word representatives. Distance matrix data were generated to set up phylogeny relationships among elapid short-chain alpha-neurotoxins, which proved to be in accordance with the taxonomic classification and geographical distribution of snake species.

Americas↗

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↗

PubMed1996

1H-NMR-derived secondary structure and the overall fold of the potent anti-mammal and anti-insect toxin III from the scorpion Leiurus quinquestriatus quinquestriatus.

We describe the secondary structure and the overall fold of toxin III from the venom of the scorpion Leiurus quinquestriatus quinquestriatus determined using two-dimensional-1H-NMR spectroscopy. This protein, which contains 64 amino acids and 4 disulfide bridges, belongs to the long-chain toxin category and is highly toxic to both mammals and insects. The overall fold was determined on the basis of 1208 inter-proton-distance restraints derived from NOE measurements and 90 psi, phi dihedral-angle restraints derived from NOE connectivities and 3JNH-alphaH coupling constants using the HABAS program. This fold, which mainly consists of an alpha-helix packed against a small antiparallel three-stranded beta-sheet, and of several turns and loops, is similar to that of other long-chain scorpion toxins. Aromatic and non-polar residues form several patches on the surface of the protein which alternate with patches of charged and polar residues. Such a topology should be important in the interactions of toxin III with sodium channels in membranes. Two weakly constrained loops introduce some flexibility to the structure which could be related to the activity of this toxin. The central core of toxin III is compared with the cysteine-stabilized alpha beta motif (an alpha-helix connected to a beta-sheet through two disulfide bridges) found in insect defensins and plant thionins. Defensins and thionins are small proteins (approximately 40--50 amino acid residues) containing three or four disulfide bridges, respectively. This comparison confirms that the cysteine-stabilized alpha beta motif is a common core to a number of small proteins from different origins and having different activities.

Amino Acid Sequence↗

PubMed1996

Two neurotoxins (BmK I and BmK II) from the venom of the scorpion Buthus martensi Karsch: purification, amino acid sequences and assessment of specific activity.

Two neurotoxins, BmK I and BmK II, were purified from the venom of the Chinese scorpion Buthus martensi Karsch. The complete amino acid sequences of both toxins, each containing 64 amino acid residues, were determined by the automatic sequencing of reduced and S-carboxymethylated toxins and their peptides, obtained after cleavage with TPCK-treated trypsin and Staphylococcus aureus V8 protease, respectively. Toxicity as minimum lethal dose tested by i.c.v. injection in mice showed that BmK I was six times more potent than BmK II. Only two amino acid replacements were found: at position 59 Val in BmK I was replaced by Ile in BmK II, and at position 62 a basic Lys residue in BmK I was substituted by a neutral Asn residue in BmK II. These features suggest that the positively charged residue (Lys or Arg) in the C-terminal position 62 (or 61 or 63) may also play an important role in facilitating the interaction between scorpion neurotoxins and the receptor on sodium channels. The effects of BmK I on nerve excitability were examined with the crayfish axon using intracellular recording and voltage-clamp conditions. The results indicate that BmK I preferentially blocks the sodium channel inactivation process. Thus, functional and structural similarities suggest that BmK I and BmK II belong to group 3 of scorpion alpha-type toxins.

Amino Acid Sequence↗

PubMed1996

Adenosine and neuropathic pain.

Recent studies have reported the possibilities of relieving neuropathic pain by administering adenosine or its analogs. In order to determine if there exists a metabolic anomaly of this nucleoside in patients with neuropathic pain, circulating adenosine levels were compared in three patient groups. The first was composed of individuals suffering from neuropathic pain, the second of patients with nervous system lesions in the absence of pain, and the third was composed of patients suffering from pain resulting from excessive nociception. The adenosine blood levels of these patients were compared to those of a control group. Finally, adenosine in the cerebrospinal fluid (CSF) of some patients was also assayed. The results show that there are reduced levels of blood and CSF adenosine in patients with neuropathic pain. This adenosine deficiency could explain the potential therapeutic effects of administering adenosine or its analogs.

Adenosine↗

PubMed1996

Activity-induced internalization and rapid degradation of sodium channels in cultured fetal neurons.

A regulatory mechanism for neuronal excitability consists in controlling sodium channel density at the plasma membrane. In cultured fetal neurons, activation of sodium channels by neurotoxins, e.g., veratridine and alpha-scorpion toxin (alpha-ScTx) that enhance the channel open state probability induced a rapid down-regulation of surface channels. Evidence that the initial step of activity-induced sodium channel down-regulation is mediated by internalization was provided by using 125I-alpha-ScTx as both a channel probe and activator. After its binding to surface channels, the distribution of 125I-alpha-ScTx into five subcellular compartments was quantitatively analyzed by EM autoradiography. 125I-alpha-ScTx was found to accumulate in tubulovesicular endosomes and disappear from the cell surface in a time-dependent manner. This specific distribution was prevented by addition of tetrodotoxin (TTX), a channel blocker. By using a photoreactive derivative to covalently label sodium channels at the surface of cultured neurons, we further demonstrated that they are degraded after veratridine-induced internalization. A time-dependent decrease in the amount of labeled sodium channel alpha subunit was observed after veratridine treatment. After 120 min of incubation, half of the alpha subunits were cleaved. This degradation was prevented totally by TTX addition and was accompanied by the appearance of an increasing amount of a 90-kD major proteolytic fragment that was already detected after 45-60 min of veratridine treatment. Exposure of the photoaffinity-labeled cells to amphotericin B, a sodium ionophore, gave similar results. In this case, degradation was prevented when Na+ ions were substituted by choline ions and not blocked by TTX. After veratridine- or amphotericin B-induced internalization of sodium channels, breakdown of the labeled alpha subunit was inhibited by leupeptin, while internalization was almost unaffected. Thus, cultured fetal neurons are capable of adjusting sodium channel density by an activity-dependent endocytotic process that is triggered by Na+ influx.

Amphotericin B↗

PubMed1996

Adenosine and the nervous system: clinical implications.

After a review of the metabolism and pharmacology of adenosine, this work will examine the various therapeutic possibilities involving the use of agonists or antagonists of adenosine A1 or A2 receptors in neurological disorders. Promising preclinical results have been obtained with epilepsy, cerebral ischemia, alcoholism, and pain.

Adenosine↗

PubMed1996

Characterization of PO1, a new peptide ligand of the apamin-sensitive Ca2+ activated K+ channel.

A new peptide ligand of the small conductance Ca2+ activated K+ channels has been purified from the venom (obtained by manual rather than electrical stimulation of the scorpion Androctonus mauretanicus mauretanicus), by following the inhibition of the 125I-apamin binding to its receptor on rat brain synaptosomes. Only one step on a C18 reversed-phase high-performance liquid chromatography column was necessary to obtain PO1. Its K0.5 for the apamin binding site was 100 nM. The amino acid sequence of PO1 is different from those of leiurotoxin and PO5. For the first time the same peptide was also purified from the venoms of two other species of North African scorpions, Androctonus australis and Buthus occitanus tunetanus. PO1 was chemically synthesized by the solid-phase technique and fully characterized. A model of PO1 was constructed by amino acid replacement using PO5 nuclear magnetic resonance studies as the starting model. Structure-activity relationships between these toxins and their receptor are discussed.

Animals↗

PubMed1995

Recombinant and chemical derivatives of apamin. Implication of post-transcriptional C-terminal amidation of apamin in biological activity.

The use of the colicin A lysis protein to direct the extracellular release of a fusion protein from Escherichia coli was investigated as an approach for the preparation of recombinant animal toxins. Apamin, a bee venom neurotoxin, was used as the model toxin. It is reticulated by two disulfide bridges and interacts with small conductance Ca(2+)-activated K+ channels. Substantial amounts of free recombinant apamin were obtained by CNBr cleavage of the fusion protein [col-(1-171)-apa] and HPLC purification. It was recognized by conformation-dependent monoclonal antibodies with a K0.5 value close to that for natural apamin, indicating that folding was correct. In toxicity and binding experiments, the recombinant apamin displayed low activity. The recombinant and natural molecules differed by the amidation of the C-terminal histidine residue. Previous structure/activity relationship studies do not implicate this C-terminal residue in activity but the role of its amidation was not investigated. An apamin analog with a non-amidated C-terminal residue was then chemically synthesized. The biological properties of both recombinant and chemical molecules were determined. Amidation of the C-terminal alpha-carboxyl of apamin appears to be essential for full expression of its biological activity.

Amides↗

PubMed1995

Correlation of antiviral activity with beta-turn types for V3 synthetic multibranched peptides from HIV-1 gp120.

SPC3 is a synthetic multibranched peptide containing eight HIV-1 gp120 V3 loop GPGRAF motifs. SPC3 inhibits HIV-1 infection in human lymphocytes and macrophages, while the monomer counterpart of SPC3, i.e., the GPGRAF peptide, has no effect. Circular dichroism (CD) of these molecules in phosphate buffer, pH 7, and in a water solution containing 50% trifluoroethanol (TFE) showed significant differences. In TFE, the inactive monomer has a CD spectrum associated to type II beta-turn (class B spectrum), while SPC3 has a class C CD spectrum associated to type I beta-turn. To investigate the structure--function relationship, SPC3 analogs were built in solid-phase synthesis, and their activity and structures were compared to SPC3. Analogs having respectively two and four GPGRAF motifs show that polymerization is associated with these structural changes. Analogs with eight motifs but differing in their sequence show also that the sequence is important to stabilize a type I beta-turn structure. The activity tests of these analogs show a remarkable correlation between the antiviral activity and their ability to exhibit a class C CD spectrum associated to type I beta-turn. Taking in account CD results, a model was made using energy minimization and dynamics, which shows that, for SPC3, a model with motifs in a type I beta-turn structure is favored compared to one with a type II beta-turn. These data suggest that the SPC3 antiviral activity is related to the structure of the GPGRAF motif in the polymer, with special emphasis on the presence of a type I beta-turn structure.

Amino Acid Sequence↗

PubMed1995

Alpha-scorpion toxins binding on rat brain and insect sodium channels reveal divergent allosteric modulations by brevetoxin and veratridine.

At least six topologically separated neurotoxin receptor sites have been identified on sodium channels that reveal strong allosteric interactions among them. We have studied the allosteric modulation induced by veratridine, binding to receptor site 2, and brevetoxin PbTx-1, occupying receptor site 5, on the binding of alpha-scorpion toxins at receptor site 3, on three different neuronal sodium channels: rat brain, locust, and cockroach synaptosomes. We used 125I-AaH II, the most active alpha-scorpion toxin on vertebrates, and 125I-Lqh alpha IT, shown to have high activity on insects, as specific probes for receptor site 3 in rat brain and insect sodium channels. Our results reveal that brevetoxin PbTx-1 generates three types of effects at receptor site 3:1) negative allosteric modulation in rat brain sodium channels, 2) positive modulation in locust sodium channels, and 3) no effect on cockroach sodium channel. However, PbTx-1 activates sodium channels in cockroach axon similarly to its activity in other preparation. Veratridine positively modulates both rat brain and locust sodium channels but had no effect on alpha-toxin binding in cockroach. The dramatic differences in allosteric modulations in each sodium channel subtype suggest structural differences in receptor sites for PbTx-1 and/or at the coupling regions with alpha-scorpion toxin receptor sites in the different sodium channels, which can be detected by combined application of specific channel modifiers and may elucidate the dynamic gating activity and the mechanism of allosteric interactions among various neurotoxin receptors.

Allosteric Regulation↗

PubMed1995

SPC3, a synthetic peptide derived from the V3 domain of human immunodeficiency virus type 1 (HIV-1) gp120, inhibits HIV-1 entry into CD4+ and CD4- cells by two distinct mechanisms.

The third variable region (V3 loop) of gp120, the HIV-1 surface envelope glycoprotein, plays a key role in HIV-1 infection and pathogenesis. Recently, we reported that a synthetic multibranched peptide (SPC3) containing eight V3-loop consensus motifs (GPGRAF) inhibited HIV-1 infection in both CD4+ and CD4- susceptible cells. In the present study, we investigated the mechanisms of action of SPC3 in these cell types--i.e., CD4+ lymphocytes and CD4- epithelial cells expressing galactosylceramide (GalCer), an alternative receptor for HIV-1 gp120. We found that SPC3 was a potent inhibitor of HIV-1 infection in CD4+ lymphocytes when added 1 h after initial exposure of the cells to HIV-1, whereas it had no inhibitory effect when present only before and/or during the incubation with HIV-1. These data suggested that SPC3 did not inhibit the binding of HIV-1 to CD4+ lymphocytes but interfered with a post-binding step necessary for virus entry. In agreement with this hypothesis, SPC3 treatment after HIV-1 exposure dramatically reduced the number of infected cells without altering gp120-CD4 interaction or viral gene expression. In contrast, SPC3 blocked HIV-1 entry into CD4-/GalCer+ human colon epithelial cells when present in competition with HIV-1 but had no effect when added after infection. Accordingly, SPC3 was found to inhibit the binding of gp120 to the GalCer receptor. Thus, the data suggest that SPC3 affects HIV-1 infection by two distinct mechanisms: (i) prevention of GalCer-mediated HIV-1 attachment to the surface of CD4-/GalCer+ cells and (ii) post-binding inhibition of HIV-1 entry into CD4+ lymphocytes.

Antigens, CD↗

PubMed1995

Characterization and ultrastructural localization of annexin VI from mitochondria.

Annexin VI, a member of a family of related intracellular proteins that associate reversibly with membrane phospholipids in a Ca(2+)-dependent manner, has been purified from bovine liver mitochondria and characterized. Moreover, biochemical and immunocytochemical lines of evidence are presented which strongly suggest that annexin VI is closely associated with the cristae in the inner membrane of mitochondria. These findings are consistent with a calcium channel activity of annexin VI in mitochondria.

Amino Acid Sequence↗