Biomedical subjects

H Rochat

Publications and source records attributed to H Rochat.

At least 127 records · Page 7Linked to original sources

PubMed1991

Binding and toxicity of apamin. Characterization of the active site.

The structural features of apamin, a natural octadecapeptide from bee venom, enabling binding to its receptor and the expression of toxicity in mice, have been delineated by studying the effects on binding and toxicity of chemical modifications and amino acid substitutions in synthetic analogues. The results obtained indicate that the only hydrophobic residue, leucine at position 10, can be changed to alanine without a significant decrease in the specific activity. The need for a correct conformation has been established and also the importance of Gln-17 and the side chains of Arg-13 and Arg-14 (besides the charge effects). The interaction of apamin with its receptor, a calcium-activated potassium channel, is thus mediated by a precise topology around these three residues. Due to the ability to detect very low specific activities for some of the analogues, it has been shown that, individually, none of these interactions constitute an essential criteria for binding per se, but that their presence is necessary for the high specific activity of the toxin.

Amino Acid Sequence↗

PubMed1991

An anti-insect toxin purified from the scorpion Androctonus australis Hector also acts on the alpha- and beta-sites of the mammalian sodium channel: sequence and circular dichroism study.

A new anti-insect neurotoxin, AaH IT4, has been isolated from the venom of the North African scorpion Androctonus australis Hector. This polypeptide has a toxic effect on insects and mammals and is capable of competing with anti-insect scorpion toxins for binding to the sodium channel of insects; it also modulates the binding of alpha-type and beta-type anti-mammal scorpion toxins to the mammal sodium channel. This is the first report of a scorpion toxin able to exhibit these three kinds of activity. The molecule is composed of 65 amino acid residues and lacks methionine and, more unexpectedly, proline, which until now has been considered to play a role in the folded structure of all scorpion neurotoxins. The primary structure showed a poor homology with the sequences of other scorpion toxins; however, it had features in common with beta-type toxins. In fact, radioimmunoassays using antibodies directed to scorpion toxins representative of the main structural groups showed that there is a recognition of AaH IT4 via anti-beta-type toxin antibodies only. A circular dichroism study revealed a low content of regular secondary structures, particularly in beta-sheet structures, when compared to other scorpion toxins. This protein might be the first member of a new class of toxins to have ancestral structural features and a wide toxic range.

Amino Acid Sequence↗

PubMed1991

In vivo protection against scorpion toxins by liposomal immunization.

The possibility of raising a humoral immune response able to induce protection from the lethal effects of scorpion toxins was evaluated in the mouse model. A toxic fraction from the venom of the scorpion Tityus serrulatus was entrapped in sphingomyelin-cholesterol liposomes which yielded a conveniently detoxified immunogen. After three injections of this immunogen, all but three of a group of 18 mice developed an IgG response which was shown to be both specific and of good affinity for the toxic antigen. In vitro neutralization assays indicated that pre-incubation of a lethal dose of the toxic fraction with immune sera strongly diminished its toxicity. In vivo protection assays showed that mice with the highest levels of circulating anti-toxin antibodies could resist the challenge by double the normal LD50 of the toxic fraction, which killed all control non-immune mice. The protection was, however, found to be limited both in its duration and its effectiveness against higher amounts of toxin.

Animals↗

PubMed1991

Evidence for neurotoxic activity of tat from human immunodeficiency virus type 1.

The human immunodeficiency virus (HIV) genome codes for a trans-activating regulatory protein, tat. Using chemically synthesized tat, it was found that 125I-tat and 125I-tat38-86 specifically bound to rat brain synaptosomal membranes with moderate affinity (K0.5 = 3 microM). Interaction of tat with nerve cells was also revealed by flow cytometry, which showed its binding to rat glioma and murine neuroblastoma cells, using both direct fluorescence with fluorescein isothiocyanate-labeled tat and indirect immunofluorescence assays. This interaction was investigated with electrophysiology using isolated excitable frog muscle fibers and cockroach giant interneuron synapses. tat acted on the cell membrane and induced a large depolarization, accompanied by a decrease in membrane resistance, thereby modifying cell permeability. The neurotoxicity of tat was further demonstrated in vitro, on glioma and neuroblastoma cell growth, as well as by a 51Cr release assay in both tumor cell lines. Interestingly, no hemolytic activity of tat for human erythrocytes was found even when tat was tested at its highly neurotoxic concentration. Experiments in vivo showed that synthetic tat is a potent and lethal neurotoxic agent in mice. The use of tat peptide derivatives showed that basic region from 49 to 57 is necessary and sufficient for binding to cell membranes and toxicity.

Action Potentials↗

PubMed1990

Crystallization and preliminary X-ray study of AaH IT2, and insect-specific toxin from the scorpion Androctonus australis Hector.

An insect-specific toxin from the venom of the scorpion Androctonus australis Hector has been crystallized. The crystals are orthorhombic, space groups P2(1)2(1)2(1), with cell dimensions a = 66.4 A, b = 52.5 A and c = 36.1 A. Calculations based on the unit cell volume and toxin molecular mass suggest that there are two molecules in the asymmetric unit.

Animals↗

PubMed1990

Delineation of the functional site of a snake venom cardiotoxin: preparation, structure, and function of monoacetylated derivatives.

Toxin gamma, a cardiotoxin from the venom of the cobra Naja nigricollis, was modified with acetic anhydride, and the derivatives were separated by cation-exchange and reverse-phase chromatography. Nine monoacetylated derivatives were obtained, and those modified at positions 1, 2, 12, 23, and 35 were readily identified by automated sequencing. The overall structure of toxin gamma, composed of three adjacent loops (I, II, and III) rich in beta-sheet, was not affected by monoacetylation as revealed by circular dichroic analysis. Trp-11, Tyr-22, and Tyr-51 fluorescence intensities were not affected by modifications at Lys-12 and Lys-35, whereas Trp-11 fluorescence intensity slightly increased when Lys-1 and Lys-23 were modified. The cytotoxic activity of toxin gamma to FL cells in culture was unchanged after modification at positions 1 and 2, whereas it was 3-fold lower after modification at Lys-23 and Lys-35. The derivative modified at Lys-12 was 10-fold less active than native toxin. Using two isotoxins, we found that substitutions at positions 28, 30, 31, and 57 did not change the cytotoxic potency of toxin gamma. A good correlation between cytotoxicity, lethality, and, to some extent, depolarizing activity on cultured skeletal muscle cells was found. In particular, the derivative modified at Lys-12 always had the lowest potency. Our data show that the site responsible for cytotoxicity, lethality, and depolarizing activity is not diffuse but is well localized on loop I and perhaps at the base of loop II. This site is topographically different from the AcChoR binding site of the structurally similar snake neurotoxins.

Acetylation↗

PubMed1990

Primary structure of scorpion anti-insect toxins isolated from the venom of Leiurus quinquestriatus quinquestriatus.

The amino acid sequences of insect-selective scorpion toxins, purified from the venom of Leiurus quinquestriatus quinquestriatus, have been determined by automatic phenyl isothiocyanate degradation of the S-carboxymethylated proteins and derived proteolytic peptides. The excitatory toxin Lqq IT1 and Lqq IT1' (70 residues) show the shift of one half-cystine from an external position, which is characteristic of anti-mammal toxins, to an internal sequence position. Lqq IT2 (61 residues) displays the half-cystine residue in position 12, common to the sequence of all known anti-mammal toxins; it induces flaccid paralysis on insects but is non-toxic for the mouse. Lqq IT2 structurally defines a new type of anti-insect toxins from scorpion venoms. CD spectra and immunological data are in agreement with this finding.

Amino Acid Sequence↗

PubMed1990

Neurotoxins active on insects: amino acid sequences, chemical modifications, and secondary structure estimation by circular dichroism of toxins from the scorpion Androctonus australis Hector.

Two scorpion neurotoxins active only on insects, the insect toxins AaH IT1 and AaH IT2, were purified from the venom of scorpions Androctonus australis Hector collected in Tozeur (Tunisia) and characterized. AaH IT2 was sequenced and found to differ in four amino acid positions from AaH IT, the single previously sequenced insect toxin [Darbon, H., Zlotkin, E., Kopeyan, C., Van Rietschoten, J., & Rochat, H. (1982) Int. J. Pept. Protein Res. 20, 320-330] which possessed an equal potential for paralyzing fly larvae. The basic amino acid residues of AaH IT1, which differs from AaH IT by one amino acid residue, were selectively chemically modified. Six derivatives were characterized. Their toxicity toward fly larvae and cockroach was determined, and their affinity for the AaH IT1 synaptosomal receptor from cockroach nerve cord was measured. Modification of His-30, Lys-34, and Arg-60 showed no significant effect on biological activity. However, the modification of Lys-28 or Lys-51 demonstrated that these two amino acids are important for toxicity. Furthermore, simultaneous modifications of both Lys-28 and Lys-51 led to a cumulative decrease in biological activity. AaH IT1 and AaH IT2 show similar CD spectra. The secondary structures content of AaH IT2 was estimated from circular dichroism data. Results showed that this class of toxin should possess an additional alpha-helical region and a beta-sheet strand, not found in toxins active on mammals. Attempts to localize these secondary structural features in the amino acid sequence of AaH IT2 indicated that these two regions would be located within the last 20 C-terminal amino acid residues. From these studies on secondary structures, it is possible to consider that toxins active on insects are more structurally constrained than those active on mammals; a decreased molecular flexibility may be, at least partially, responsible for the observed specificity of these toxins for the insect sodium channel. Furthermore, the two alpha-helices found in insect toxins enclosed the two conserved Lys-28 and Lys-51 and might thus be implicated in the toxic site of insect toxins.

Amino Acid Sequence↗

PubMed1990

Conformational flexibility of a scorpion toxin active on mammals and insects: a circular dichroism study.

Three scorpion toxins have been analyzed by circular dichroism in water and in 2,2,2-trifluoroethanol (TFE) solutions. These toxins were chosen because they are representative of three kinds of pharmacological activities: (1) toxin AaH IT2, an antiinsect toxin purified from the venom of Androctonus australis Hector, which is able to bind to insect nervous system preparation, (2) toxin Css II, from the venom of Centruroides suffusus suffusus, which is a beta-type antimammal toxin capable of binding to mammal nervous system preparation, and (3) the toxin Ts VII from the venom of Tityus serrulatus, which is able to bind to both types of nervous systems. In order to minimize bias, CD data were analyzed by a predictive algorithm to assess secondary structure content. Among the three molecules, Ts VII presented the most unordered secondary structure in water, but it gained in ordered forms when solubilized in TFE. These results indicated that the Ts VII backbone is the most flexible, which might result in a more pronounced tendency for this toxin molecule to undergo conformational changes. This is consistent with the fact that it competes with both antiinsect and beta-type antimammal toxins for the binding to the sodium channel.

Amino Acid Sequence↗

PubMed1990

Structure-activity relationships of scorpion alpha-neurotoxins: contribution of arginine residues.

The role of arginine residues in the structure-activity relationships of alpha-scorpion neurotoxins was studied. Toxins I and II from Androctonus australis Hector (north African scorpion), containing respectively 2 and 3 arginines, were modified by phenylglyoxal or p-hydroxyphenylglyoxal. Modified derivatives were purified by reverse-phase HPLC and/or ion exchange HPLC. Subsequent bioassays showed that toxin I (AaH I) derivatives with single modifications on Arg 2 and Arg 60 had low activity (25 and 14% of residual activity, assessed in receptor binding experiments). Doubly modified (Arg 2, Arg 60) AaH I had 7% residual activity while further derivatization of the alpha-amino group led to an almost inactive derivative. These results agree with the involvement of arginines 2 and 60, as well as the alpha-amino group, of AaH I in the toxin/receptor interaction, probably via electrostatic interactions. Consistent with the role of N-terminal residues, the selective removal of the N-terminal dipeptide Val-Arg of toxin III from the same scorpion resulted in low activity (7% residual activity). The arginine residue in position 56 of toxin II was important for bioactivity since the derivative modified by phenylglyoxal on Arg 56 exhibited low residual activity (20%). Arg 62 and Arg 18, on the other hand, can be modified without any great effect on the pharmacological activity of AaH II. These results furnish a more precise picture of those residues involved in the "toxic region", which appears to be composed of residues belonging to the conserved hydrophobic surface and to the C-terminal and N-terminal sequences.

Amino Acid Sequence↗

PubMed1990

Identification of antigenic residues on apamin recognized by polyclonal antibodies.

The structural requirements for antigenic recognition of apamin--an 18-amino acid, disulfide-bridged peptide--by rabbit antibodies were defined using a set of 18 apamin analogs in a competition liquid-phase radioimmunoassay. Some residues contribute considerably to antigenic recognition, e.g. Ala10, Arg13, and others to a lesser extent, e.g. Arg14, Glu7 and Thr8. The N- and C-terminal moieties of apamin are less antigenically important. These findings suggest that a good part of antibody specificities are directed to the central tightly folded part of the molecule. They are consistent with the observation that in saturating conditions, labeled apamin can, on average, bind one specific Fab fragment.

Amino Acid Sequence↗

PubMed1990

Epitope mapping of apamin by means of monoclonal antibodies raised against free or carrier-coupled peptide.

A panel of 20 monoclonal antibodies raised against the bee-venom peptide apamin (18 residues, 2 disulfide bridges) was prepared. Nine monoclonal antibodies (mAb) were obtained from a mouse immunized with free apamin and 11 from a mouse immunized with a mixture of free and carrier-coupled peptide. Using a panel of 11 synthetic apamin analogs, we examined the fine antigenic specificity of each antibody. The mAb generated against free apamin preferentially bound to the central part of the peptide and less frequently recognized the N- and C-terminal regions. However, monoclonal antibodies obtained by immunization with carrier-bound apamin showed a broader range of specificities, consistent with the possibility of the entire surface of this small antigen becoming immunogenic upon coupling to the carrier.

Amino Acid Sequence↗

PubMed1990

Large fragments of nef-protein and gp110 envelope glycoprotein from HIV-1. Synthesis, CD analysis and immunoreactivity.

Chemical synthesis of large peptide fragments (from 18 to 66 amino acid residues long) of the gp110 envelope glycoprotein and of nef-protein from HIV-1 was achieved by the solid phase method. Stepwise assembling of the peptide chains was carried out automatically on 4-(oxymethyl)-phenylacetamidomethyl resin using the N-alpha-butyloxycarbonyl amino acids with benzyl-based side chain protecting groups. Two elongation protocols were used depending on the peptide chain length: a standard cycle, mainly characterized by a single coupling step (Boc-amino acid symmetrical anhydride in dimethylformamide), and an optimized one for large peptides, based on a double coupling strategy (Boc-amino acid symmetrical anhydride first in dimethylformamide, then in dichloromethane). Final cleavage of the peptide from the solid support was carried out by anhydrous hydrogen fluoride and crude peptides were purified by C18 reverse phase medium pressure liquid chromatography after molecular filtration. Characterization of the purified peptides was done by analytical HPLC, amino acid content determination, and circular dichroism analysis both in polar (H2O) and in non-polar (TFE) environments. Immunoreactivity of anti-nef positive sera from HIV-1 infected patients by ELISA with the synthetic peptides was investigated. The results showed four major antigenic regions of nef-protein and mainly the immunodominance of the N- and C-termini of the molecule. Several of these peptides should prove to be useful for both diagnosis and vaccination purposes.

Amino Acid Sequence↗

PubMed1989

Precursors of Androctonus australis scorpion neurotoxins. Structures of precursors, processing outcomes, and expression of a functional recombinant toxin II.

From a cDNA library made from telsons of scorpions of the species Androctonus australis, full-length cDNAs of about 370 nucleotides encoding precursors of toxins active on mammals or on insects have been isolated using oligonucleotide probes. Sequence analysis of the cDNAs revealed that the precursors contain signal peptides of about 20 amino acid residues. In addition, precursors of toxins active on mammals have extensions at their COOH-terminal ends: Arg or Gly-Arg. The processing steps required to generate toxins from their respective precursors are thus not identical for all of them. Southern blot analysis performed at the genomic level with a cDNA encoding toxin II suggested a single copy gene having a minimum size of 2800 base pairs. Finally, in an attempt to successfully express an animal toxin, monkey kidney COS-7 cells transfected with a plasmid harboring a cDNA encoding toxin II transiently expressed a recombinant toxin having the immunological and biological properties of toxin II.

Amino Acid Sequence↗

PubMed1989

Structure/activity relationships of scorpion alpha-toxins. Multiple residues contribute to the interaction with receptors.

Chemical modifications of tyrosine and tryptophan residues of scorpion alpha-neurotoxins II and III from Androctonus australis Hector were performed as well as modification of the two arginines and the alpha-amino group of toxin I. The pharmacological potencies of each derivative were assessed in vivo by LD50 measurement and in vitro by competition experiments with 125I-toxin for synaptosomal receptors. Arginine residues in positions 2 and 60 and the alpha-amino group of Androctonus toxin I were derivatized by p-hydroxyphenylglyoxal; the corresponding modified toxins exhibit low pharmacological potencies. Tryptophan 38 of toxin II and tryptophan 45 of toxin III were modified by nitrophenylsulfenyl chloride, leading respectively to a poorly and a fully active derivative. The tetranitromethane modification of tyrosine residues in positions 60, 5 and 14 of toxin III induced respectively 60%, 40% and 30% of loss of biological activity. Circular dichroic analysis indicated that for every derivative, except the nitrophenylsulfenyl derivative of Trp-45 of AaH III, the conformation of the toxin was not altered by derivatization. Conformational integrity was also confirmed by full activity of the derivatives in radioimmunoassays. Taken together, the results suggest that aromatic residues belonging to the conserved hydrophobic surface, to the C-terminal and to the loop region 37-44 are involved in the molecular mechanisms by which scorpion alpha-toxins act. Charged residues in the N-terminal and C-terminal also contribute to the high efficacy of the binding process. It appears that all important residues are clustered on one face of the toxin, suggesting a multipoint interaction with the proteins of the sodium channel.

Amino Acid Sequence↗

PubMed1989

On the inhibition of [Na+,K+]-ATPases by the components of Naja mossambica mossambica venom: evidence for two distinct rat brain [Na+,K+]-ATPase activities.

We have compared the effects of highly purified preparations of cardiotoxins and phospholipases A2 from Naja mossambica mossambica venom on rat brain [Na+,K+]-ATPase activity. The results were the following: (i) micromolar concentrations of cardiotoxin preparations were required to inhibit [Na+,K+]-ATPase activity to the extent achieved by picomolar concentrations of phospholipases A2; i.e., the inhibitory effect of cardiotoxins appeared to be related to the contamination of the preparations by trace amounts of phospholipase A2; (ii) comparing phospholipases A2 from varied origins, a correlation was observed between [Na+,K+]-ATPase inhibition, isoelectric point, and toxicity for mice; (iii) when rat brain membranes were used, incubation for extended times with the most basic N. mossambica mossambica phospholipase A2 resulted in a biphasic [Na+,K+]-ATPase inhibition, suggesting that two distinct [Na+,K+]-ATPases were affected differentially. In contrast, incubation of rat brain membranes with either porcine pancreatic phospholipase A2, notexin, or beta-bungarotoxin and also incubation of erythrocyte membranes with the most basic N. mossambica mossambica phospholipase A2 produced monophasic [Na+,K+]-ATPase inhibitions. We discuss a possible specific action of toxic, basic phospholipase A2 on one of the [Na+,K+]-ATPase isoforms of excitable membranes.

Animals↗

PubMed1989

Specificity of antibodies to sea anemone toxin III and immunogenicity of the pharmacological site of anemone and scorpion toxins.

Toxin III (ATX III) of the sea anemone (Anemonia sulcata) is a polypeptide containing 27 amino acid residues. It has no sequence similarity with other toxins (ATX I and II) from the same species, or with scorpion toxins, although they apparently act in a similar manner by prolonging action potentials. The specificity of ATX III antibodies was characterized using ATX III, ATX I, native and chemically modified ATX II, and scorpion alpha-toxins. The results obtained suggest that a region of ATX III, partially or totally overlapping the pharmacological site shared with ATX I and ATX II, is immunogenic. It includes a guanidino and at least two carboxylate groups. The corresponding region is not immunogenic in ATX I and ATX II. Anti-(ATX III) antibodies recognize the similar regions of ATX I and ATX II and apparently do not recognize scorpion toxins.

Action Potentials↗

PubMed1989

Changes in Na channel properties of frog and rat skeletal muscles induced by the AaH II toxin from the scorpion Androctonus australis.

The effects of the mammal toxin II isolated from the venom of the scorpion Androctonus australis Hector (AaH II) were studied under current and voltage clamp conditions in frog (semitendinosus) and rat (fast e.d.l. and slow soleus) skeletal twitch muscle fibres. In both species, AaH II induced a dose-dependent prolongation of the action potential (AP) leading at saturating concentration to APs with long plateaus of about 1.5 s in frog and 5 s in rat e.d.l. and soleus fibres. The concentrations to induce 50% of the maximal effect (K0.5) were 9.1 x 10(-9) M in the frog and 1.4 x 10(-9) M in the rat. AaH II increased the time constants of inactivation of the peak Na current and induced a maintained Na current that was greater in rat e.d.l. and soleus (31.6% of peak current amplitude at -30 mV; K0.5 = 0.8 x 10(-9) M) than in frog (16.5%; K0.5 = 15.5 x 10(-9) M) muscles. Peak and maintained Na currents were TTX-sensitive and had identical threshold and reversal potentials. The half-maximum maintained permeability occurred at a potential 20 mV more positive than the peak permeability. Recovery from inactivation and steady-state inactivation of the inactivating Na current remained unchanged. The maintained current deactivated with normal fast kinetics. The action of the toxin reversed poorly on washout but could be largely removed by conditioning depolarizations more positive than the reversal potential of the Na current. Our results suggest that, in vertebrate skeletal muscle fibres, AaH II affects all the Na channels and are consistent with the hypothesis that the maintained current originates from a reopening of previously inactivated Na channels.

Action Potentials↗