Biomedical subjects

H Rochat

Publications and source records attributed to H Rochat.

At least 163 records · Page 9Linked to original sources

PubMed1987

In vivo synergy of cardiotoxin and phospholipase A2 from the elapid snake Naja mossambica mossambica.

The lethality of elapid snake venoms is due to toxic polypeptides which are alpha-neurotoxins, phospholipases A2 and cardiotoxins. In contrast to alpha-neurotoxins, less is known about the mode of action of phospholipases A2 and cardiotoxins at a cellular and molecular level, although it has been demonstrated that the hemolytic effect of cardiotoxin and its action on muscle can be accelerated in vitro by phospholipase A2. Here we show that when mice are injected i.v. with phospholipase A2, a significant decrease in survival time is observed if a sub-LD50 amount of cardiotoxin is injected simultaneously. Furthermore, the survival time is drastically reduced when phospholipase A2 (2.3 LD50) is injected first, followed 15 min later by doses of cardiotoxin as small as 0.18 LD50. These results strongly suggest a synergy between the two polypeptides.

Animals↗

PubMed1987

Purification of contracture-inducing insect toxins from Buthinae scorpion venoms by immunoaffinity and high pressure liquid chromatography.

A method of rapid and selective detection and purification of contracture-inducing insect toxins from Buthinae scorpion venoms is described in this paper. It consists of two main steps: the first one is specific as it uses immunoaffinity chromatography with antibodies against Androctonus australis Hector IT; the second, reverse phase high pressure liquid chromatography, allows the final separation of the different toxins present in each of the three venoms studied. Two, three and four insect toxins have been purified, respectively, from the venoms of Androctonus australis Hector, Buthus occitanus mardochei and Leiurus quinquestriatus quinquestriatus. This work demonstrates that, in Buthinae venoms, contracture-inducing insect toxins antigenically related to AaH IT, the first one purified, constitute the most important and, in some cases, the only toxins present.

Amino Acids↗

PubMed1987

Antigenicity of peptide 19-28 of toxin II from the scorpion Androctonus australis as measured by different solid-phase tests and characterization of specific antibodies purified by immunoaffinity on the peptide or the toxin.

The antigenicity of peptide 19-28, a model of one of the major antigenic regions of toxin II of the scorpion Androctonus australis Hector was tested in different solid phase radioimmunoassay systems. The type of the solid phase and the mode of binding the synthetic peptide to the phase had a considerable effect on the resulting antigenicity. Two subpopulations of anti-toxin II antibodies were purified by affinity chromatography, one on Sepharose-peptide 19-28, the other on sepharose-toxin. The native or chemically modified toxin bound in the same way to these subpopulations. Denatured toxin was only poorly recognized by these antibodies. This suggests that the antibodies purified on peptide 19-28 recognize the same conformation dependent antigenic surface as do total anti-toxin antibodies.

Antibodies↗

PubMed1987

Use of high performance liquid chromatography to demonstrate quantitative variation in components of venom from the scorpion Androctonus australis Hector.

Using reverse-phase high performance liquid chromatography (RP-HPLC), to resolve less than 1 mg of scorpion venom, quantitative variations in protein components were demonstrated in Androctonus australis Hector venoms obtained either by electric or manual stimulation. The results support polymorphism of scorpion venom components at an individual level.

Animals↗

PubMed1987

Amino acid sequence of toxin XI of the scorpion Buthus occitanus tunetanus. Evidence of a mutation having an important effect upon neurotoxic activity.

The complete amino acid sequence of toxin XI of the North African scorpion Buthus occitanus tunetanus has been elucidated by automatic sequencing of the reduced and alkylated toxin and of the peptides obtained after tryptic cleavage restricted to arginyl bonds. This toxin is structurally homologous to toxin II of Androctonus australis Hector, the most active among the alpha-toxins, but is far less potent, both in vivo and in vitro. This work points out 12 mutations, many of which are conservative. Nevertheless, the most striking difference is the replacement of the lysine residue at position 58, known to be important in the activity of AaH toxin II, by a valine residue. Thus, it seems that the presence of a positive charge at this location facilitates the interactions between the receptor on the sodium channel and the alpha-type toxins.

Amino Acid Sequence↗

PubMed1987

Reduction and reoxidation of the neurotoxin II from the scorpion Androctonus australis Hector.

Reoxidation of the totally reduced scorpion neurotoxin II from Androctonus australis Hector (four disulfide bridges) has been investigated. The totally reduced toxin was highly insoluble in neutral and alkaline conditions, which prevented the use of the usual air oxidation process for renaturation. We tested a new method in which the reduced molecules were first solubilized in 10% (v/v) acetic acid and then oxidized by air through dialysis against a series of buffers with a slow pH gradient from 2.2 to 7.0 or 8.0. In this system, up to 95% of the protein was recovered in solution. Addition of reduced and oxidized glutathione accelerated refolding and also permitted a better recovery of fully active peptide as measured by both toxicity to mice and ability to displace 125I radiolabeled toxin II from its binding site on rat brain synaptosomal fractions. The reoxidation reaction could also be monitored directly by high pressure liquid chromatography. A strong effect of guanidine hydrochloride concentration as well as the temperature was observed both on the solubility of the reoxidation intermediates and on the refolding pathway. Finally, the method used, i.e. dialysis reoxidation with a pH gradient from 2.2 to 8.0 in 0.1 M sodium phosphate, 0.1 M sodium chloride, 20 mM guanidine hydrochloride, 1 mM oxidized and reduced glutathione allowed regeneration in high yield (70%) of a reoxidized toxin form indistinguishable from the native toxin. A minor stable and inactive molecular species (about 30%) showing a difference in mobility by electrophoresis was also detected.

Animals↗

PubMed1986

Immunochemistry of sea anemone toxins: structure-antigenicity relationships and toxin-receptor interactions probed by antibodies specific for one antigenic region.

Two antibody subpopulations directed against Anemonia sulcata toxin I or II have been purified by immunoaffinity chromatography. These antibodies are specific for a single antigenic region and were used in a structure-antigenicity relationship study using homologous toxins and chemically modified derivatives of A. sulcata toxin II. Asp-7 and/or Asp-9 and Gln-47 of toxin II were found to be implicated in the antigenic region recognized by the two antibody subpopulations. On the contrary, Arg-14, Lys-35, -36, and -46, and alpha-NH2 of the glycine residue of A. sulcata toxin II are not involved in the corresponding antigenic region. When assayed for interaction with the sodium channel, the antigenic region of toxin II, including Asp-9 and Gln-47, appeared fully accessible to its specific antibodies, suggesting that it is not involved in the binding of the toxin to its receptor.

Amino Acid Sequence↗

PubMed1986

Characterization of elapidae snake venom components using optimized reverse-phase high-performance liquid chromatographic conditions and screening assays for alpha-neurotoxin and phospholipase A2 activities.

The vast majority of Elapidae snake venoms, genus Naja, includes three classes of toxic polypeptides: alpha-neurotoxins, phospholipases A2, and cardiotoxins. A new experimental approach using reverse-phase high-performance liquid chromatography in particular has been developed, allowing their respective resolution, identification, and quantitation from milligram quantities of venom. First, definition of optimal chromatographic conditions for Naja mossambica mossambica toxins has been ascertained. Different column packing and solvent systems were compared for their efficiency, with particular attention to the ionic strength of the aqueous solvent. A medium-chain alkyl support (octyl) in conjunction with a volatile ammonium formate (0.15 M, pH 2.70)/acetonitrile solvent system was found to be particularly effective. All the components known until now from this venom could be resolved in a single step, and the elution order was alpha-neurotoxins, phospholipases A2, and cardiotoxins with a total recovery of absorbance and toxicity. Then, with these suitable conditions, we describe a new major cardiotoxin molecule in this venom by hydrophobic and not ionic-charge discrimination. Second, specific assays were designed to detect alpha-neurotoxin and phospholipase A2 activities in chromatographic fractions: alpha-neurotoxin activity was determined by competition for the binding of a radiolabeled alpha-neurotoxin to the acetylcholine receptor of the ray electric organ, and phospholipase A2 activity was defined by the enzymatic activity of these toxins with a fluorescent phospholipid as substrate. Finally, the applicability of these new methods to study other Naja snake venoms was demonstrated.

Animals↗

PubMed1986

Use of antibodies specific to defined regions of scorpion alpha-toxin to study its interaction with its receptor site on the sodium channel.

Five antibody populations selected by immunoaffinity chromatography for their specificity toward various regions of toxin II of the scorpion Androctonus australis Hector were used to probe the interaction of this protein with its receptor site on the sodium channel. These studies indicate that two antigenic sites, one located around the disulfide bridge 12-63 and one encompassing residues 50-59, are involved in the molecular mechanisms of toxicity neutralization. Fab fragments specific to the region around disulfide bridge 12-63 inhibit binding of the 125I-labeled toxin to its receptor site. Also, these two antigenic regions are inaccessible to their antibodies when the toxin is bound to its receptor site. In contrast, the two other antigenic sites encompassing the only alpha-helix region (residues 23-32) and a beta-turn structure (residues 32-35) are accessible to their respective antibodies when the toxin is bound to its receptor. Together, these data support the recent proposal that a region made of residues that are conserved in the scorpion toxin family is involved in the binding of the toxin to the receptor.

Amino Acid Sequence↗

PubMed1986

Tityus serrulatus toxin VII bears pharmacological properties of both beta-toxin and insect toxin from scorpion venoms.

Some beta-toxins from the South American scorpion Tityus serrulatus (e.g. Ts VII) are highly toxic both for mouse and fly larva. Radioiodinated Ts VII and the insect toxin from the North African scorpion Androctonus australis Hector (AaH IT) bind to the same site on a house fly head synaptosomal fraction. These results reinforce the hypothesis about the existence of a correlated series of scorpion toxins as previously defined by amino acid compositions and sequences, and immunological and circular dichroism studies, in suggesting that Ts VII constitutes a link which may fill the pharmacological gap existing between beta-toxins and insect toxins such as AaH IT.

Animals↗

PubMed1986

Specificity and neutralizing capacity of antibodies elicited by a synthetic peptide of scorpion toxin.

Polyclonal antibodies raised against a synthetic peptide (sequence 50-59) of Androctonus australis Hector toxin II can neutralize the effects of toxin II in vivo. The antigenic specificities of anti-peptide and anti-toxin antibodies were compared by competitive aqueous phase radioimmunoassay by using 125I-toxin II, chemically modified or homologous toxins, and the synthetic peptide 50-59, either free or bound to bovine serum albumin (BSA). The antipeptide and anti-toxin antibodies had a comparable high affinity for the native toxin, but anti-peptide antibodies exhibited a lower binding capacity. Anti-peptide antibodies had a higher affinity for native toxin than for the peptide 50-59 bound to BSA, used as immunogen, and were unable to recognize the free peptide. These results suggest that it is necessary to restrict the conformational freedom of the immunizing peptide in order to obtain anti-peptide antibodies with a high affinity for the toxin. The lysine residue at position 58 of toxin II, essential for toxicity, appears to be immunogenic when immunization is with peptide 50-59 bound to BSA and not with the native toxin. This residue is antigenic in the native toxin, however, as shown by the anti-peptide antibodies.

Animals↗

PubMed1986

Differential effects of defined chemical modifications on antigenic and pharmacological activities of scorpion alpha and beta toxins.

Specific chemical modifications of scorpion alpha and beta toxins have been used to study the involvement of particular residues in both the pharmacological and the antigenic sites of these toxins. Modification by 1,2-cyclohexanedione of arginine-27 of a beta toxin, Centruroides suffusus suffusus toxin II, drastically decrease the antigenic activity without any influence on the pharmacological activity. Conversely, modification by the same reagent of arginine-2 of an alpha toxin, Androctonus australis Hector toxin III, led to a 100-times less pharmacologically potent derivative and did not induce a significant loss of antigenic activity. Excision of the N-terminal pentapeptide of another alpha toxin, Buthus occitanus mardochei toxin III, by pepsin digestion led to a non-toxic derivative retaining full antigenic activity. Thus, the N-terminal part of the conserved hydrophobic surface of the toxin is highly implicated in the pharmacological activity, whereas the region of arginine-27, located in the alpha helix situated on the back surface, opposite the conserved hydrophobic region, is fully implicated in the antigenic activity and is far from the pharmacological site. These results are good arguments in favor of the idea that in scorpion toxins the surfaces implicated in the pharmacological and the antigenic activities do not overlap. Since the antigenic sites are present in highly variable sequence the development of an efficient polyvalent serotherapy is questionable.

Amino Acids↗

PubMed1986

Conformational variations amongst scorpion toxins.

Circular dichroism spectra were obtained for ten scorpion neurotoxins (representing five species of scorpion) in order to provide an understanding of their relative conformations in solution. Despite a high degree of amino acid sequence homology, the toxins clearly differ from each other in terms of CD-detectable structure. When superimposed, the CD spectra suggest that the toxins form a series of related conformational variants. Since the resemblances amongst the individual CD spectra can be correlated to degrees of sequence resemblance and pharmacological specificity, conformational balance could be an important factor in both toxin evolution and target recognition.

Animals↗

PubMed1986

Large scale purification of toxins from the venom of the scorpion Androctonus australis Hector.

A large scale procedure for purification of the toxins in the venom of the North African scorpion Androctonus australis has been developed. This procedure optimizes the sequence of the steps, leading to better yields of toxins and shortening of the time. It is possible to use 10-15 g batches of venom and also prevent cross-contamination of the toxins. High pressure liquid chromatography has been used to separate AaH I and I', two isotoxins which differ by one amino acid residue in position 17: valine or isoleucine. The procedure allows the characterization of a new toxin active in mice, AaH IV, which represents 0.6 and 2.5% of the venom weight and toxicity, respectively. This new component has been characterized as an alpha toxin made up of 61 amino acid residues and having a neutral isoelectric point.

Amino Acids↗

PubMed1986

Immunochemistry of scorpion alpha-toxins: study with synthetic peptides of the antigenicity of four regions of toxin II of Androctonus australis Hector.

Sequences 19-29 and 28-39 of toxin II of the North African scorpion Androctonus australis Hector have been synthesized. These two peptides correspond to the highest peaks in the hydrophilicity profile of toxin II and were thus believed to account for a significant proportion of toxin antigenicity. Affinity chromatography of solid-phase-bonded peptides allowed us to purify two sub-populations from the total IgGs raised against the native toxin. They both still bound to 125I-toxin II and showed a restricted heterogeneity in their specificity. Solid-phase immunoassays confirmed the antigenicity of these synthetic peptides and also that of two other previously described synthetic replicates of the antigenic regions of toxin II: sequences (5-14) S-S (60-64) and 50-59. The location of the four antigenic regions relative to the postulated location of the receptor-binding site of the toxin is discussed.

Amino Acid Sequence↗

PubMed1985

Penetration of a cardiotoxin into cardiolipin model membranes and its implications on lipid organization.

The interaction of cardiotoxin II of Naja mossambica mossambica with cardiolipin model membranes was investigated by binding, fluorescence, resonance energy transfer, fluorescence quenching, 31P NMR, freeze-fracture, and small-angle X-ray experiments. An initially electrostatic binding appeared to be accompanied by a deep penetration, most likely into the acyl chain region of the phospholipids, indicating a hydrophobic contribution to the strong interaction (KD congruent to 5 X 10(-8) M). This binding results in a fusion of unilamellar vesicles as indicated by a fluorescence-based fusion assay, freeze-fracture, and X-ray diffraction. In these fused structures freeze-fracture electron microscopy reveals the appearance of particles, which is accompanied by the induction of an isotropic component in 31P NMR. The well-defined particles are interpreted as inverted micelles, and the localization of the cardiotoxin molecule in these structures is discussed.

Animals↗

PubMed1985

An excitatory and a depressant insect toxin from scorpion venom both affect sodium conductance and possess a common binding site.

Two insect selective toxins were purified by gel-permeation and ion-exchange chromatographies from the venom of the scorpion, Leiurus quinquestriatus quinquestriatus, and their chemical and pharmacological properties were studied. The first toxin (LqqIT1) induces a fast excitatory contraction paralysis of fly larvae and is about 40 times more toxic than the crude venom. It is a polypeptide composed of 71 amino acids, including 8 half-cystines and devoid of methionine and tryptophan, with an estimated molecular weight of 8189 and a pI value of 8.5. The second toxin (LqqIT2) induces a slow depressant, flaccid paralysis of fly larvae. It is composed of 72 amino acids, including 8 half-cystines, is devoid of proline methionine and histidine, and has an estimated molecular weight of 7990 and a pI value of 8.3. The contrasting symptomatology of these toxins is interpreted in terms of their effects on an isolated axonal preparation of the cockroach in current and voltage clamp conditions. LqqIT1 (0.5-4 microM) induced repetitive firing of the axon which was attributable to two changes in the sodium conductance, a small increase in the peak conductance and a slowing of its turning off. LqqIT2 (1-8 microM) caused a blockage of the evoked action potentials, attributable to both a strong depolarization of the axonal membrane and a progressive suppression of the sodium current. Neither toxin affected potassium conductance. The two toxins differ mainly in their opposite effects on the activatable sodium permeability. In binding assays to a preparation of insect synaptosomal membrane vesicles, the two toxins were shown to competitively displace the radioiodinated excitatory insect toxin derived from the venom of the scorpion, Androctonus australis [( 125I]AaIT), which strongly resembles, in its chemistry and action, the LqqIT1 toxin. The present two toxins have demonstrated a strong affinity closely resembling the AaIT, with KD values of 0.4, 1.9, and 1.0 nM for LqqIT1, LqqIT2, and AaIT, respectively. These data suggest the possibility that the excitatory and depressant insect toxins share a common binding site associated with sodium channels in insect neuronal membranes.

Amino Acids↗

PubMed1985

Polymorphism and quantitative variations of toxins in the venom of the scorpion Androctonus australis Hector.

Using highly specific radioimmunoassays for toxins I, II and III of the scorpion Androctonus australis Hector, the concentrations of these neurotoxins have been determined in pooled as well as in individual samples. Variations were found that support polymorphism of scorpion toxins at an individual level. Radioimmunoassays were also used to detect toxin I of Buthus occitanus tunetanus and toxin II of Androctonus australis Hector and also antigenically homologous toxins in the venoms of several North African scorpions. These results are interpreted in terms of scorpion serotherapy.

Animals↗