Biomedical subjects

H Rochat

Publications and source records attributed to H Rochat.

At least 109 records · Page 6Linked to original sources

PubMed1993

Synthesis and characterization of kaliotoxin. Is the 26-32 sequence essential for potassium channel recognition?

Kaliotoxin (KTX), a scorpion toxin characterized as a 37-residue inhibitor of the neuronal high conductance Ca(2+)-activated K+ channels (KCa channels), has been chemically synthetized. Differences were observed between natural toxin and the two peptides, KTX(1-37) and KTX(1-37)-amide. Re-examination of the KTX sequence showed that an extra lysine residue was present at the C-terminal end. The 38-residue synthetic peptide was found identical with natural toxin. All three peptides had comparable activities, with LD50 values of 6-9 pmol/mouse after intracerebroventricular injection, and Kd = 2-8 nM for blockage of the whole cell and unitary molluscan KCa currents. Pairing of the disulfide bonds in synthetic KTX corresponded to that in charybdotoxin and iberiotoxin. A competition assay between 125I-KTX(1-37) and different toxins (KTX, dendrotoxin, charybdotoxin, MCD peptide, and iberiotoxin) for binding to rat brain synaptosomal membranes suggested that KTX interacts also with voltage-gated K+ channels. Shorter peptides, KTX(25-35)-amide and KTX(26-32)-amide, expressed no KTX activity, but were able to compete in binding. They were further shown to antagonize KTX in both its toxicity and blocking activity. The (26-32) sequence of KTX, which is a highly conserved region, may contain a low affinity binding subsite essential for potassium channel recognition.

Amino Acid Sequence↗

PubMed1993

Characterization of a new leiurotoxin I-like scorpion toxin. PO5 from Androctonus mauretanicus mauretanicus.

Three novel peptide inhibitors of the SKCa channels were purified to homogeneity from the venom of the scorpion Androctonus mauretanicus mauretanicus using one step of RP-HPLC and competition assays with [125I]apamin to rat brain synaptosomes. PO1, PO2 and PO5 have K0.5 of 100, 100 and 0.02 nM, respectively, for the apamin binding site. The sequence of PO5 was established and compared to that of other scorpion toxins active on K+ channels: it contains 31 residues and has a free carboxyl end. it shares sequence similarity with apamin and leiurotoxin I.

Amino Acid Sequence↗

PubMed1993

P05, a new leiurotoxin I-like scorpion toxin: synthesis and structure-activity relationships of the alpha-amidated analog, a ligand of Ca(2+)-activated K+ channels with increased affinity.

The venom of the scorpion Androctonus mauretanicus mauretanicus contains a toxin, P05, which is structurally and functionally similar to scorpion leiurotoxin I (87% sequence identity), a blocker of the apamin-sensitive Ca(2+)-activated K+ channels. It is a 31-residue polypeptide cross-linked by three disulfide bridges. A C-terminal carboxyl-amidated analog of P05 (sP05-NH2) was chemically synthesized by the solid-phase technique and fully characterized. Toxicity assays in vivo established that sP05-NH2, like native P05, is a potent and lethal neurotoxic agent in mice (LD50 of 20 ng per mouse). Pharmacological assays in vitro however showed that, unlike P05 which has a binding affinity of 2 x 10(-11) M, sP05-NH2 apparently binds irreversibly to the apamin receptor. Iodination at the C-terminal His gave diiodo-sP05-NH2, which had a binding affinity similar to that of native P05. The disulfide bridge pairings were chemically determined for sP05-NH2 and thereby deduced for P05 and leiurotoxin I: linkages were between Cys3 and Cys21, Cys8 and Cys26, and Cys12 and Cys28. Molecular dynamics refinement of P05 also using data from leiurotoxin I suggests that P05 is mainly composed of a double-stranded, antiparallel beta-sheet (from Leu18 to Val29) linked to an alpha-helix (from Arg6 to Gly16) by two disulfides (Cys8-Cys26 and Cys12-Cys28) and to an extended fragment (from Thr1 to Leu5) by the third disulfide (Cys3-Cys21). In agreement with the model, circular dichroism analysis of sP05-NH2 showed that the toxin structure is highly rigid.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

Cytotoxic effect on lymphocytes of Tat from human immunodeficiency virus (HIV-1).

The human immunodeficiency virus type 1 (HIV-1) genome codes for trans-activator Tat, an 86-residue protein whose expression is critical for viral replication. Full-length Tat and Tat peptides from HIV-1 were chemically synthesized using optimized solid phase technique. Synthetic Tat2-86 was found not only to inhibit antigen-induced human peripheral blood lymphocyte (PBL) proliferation in vitro, as described by Viscidi et al. [1989, Science 246, 1606-1608], but also mitogen-induced PBL proliferation, with 50% inhibition obtained at 0.9 and 8 microM, respectively. To assess the mechanism by which Tat exert its inhibitory effect, we analysed its interaction and effect on CD4(+)-cells. Direct fluorescence and indirect immunofluorescence assays analysed by flow cytometry showed that fluorescein isothiocyanate-labeled and -unlabeled Tat interact (> 0.2 microM) with CD4-expressing lymphoid cells (CEM cell line). Experiments of chromium-51 release and Trypan blue exclusion on these tumor cells in vitro have demonstrated the capacity of Tat to modify cellular membrane permeability and cell viability, in a dose-dependent manner. The use of Tat peptides revealed that those containing the Tat basic region from 49 to 57 were able to bind to the cell membrane and to exhibit a cytotoxic activity on lymphocytes. Together, the data suggest that the potential cytotoxicity of Tat on lymphocytes could be directly implicated in virus-induced immune dysfunction observed in HIV-1 infected patients.

Amino Acid Sequence↗

PubMed1993

Characterization of toxin III of the scorpion Leiurus quinquestriatus quinquestriatus: a new type of alpha-toxin highly toxic both to mammals and insects.

The primary structure of toxin III of Leiurus quinquestriatus quinquestriatus (Lqq III) was elucidated by automatic Edman degradation of the reduced and S-carboxymethylated protein and derived tryptic peptides. Like other scorpion toxins that are active on sodium channels, Lqq III, consisting of 64 amino acids, is a 7 kDa single-chain polypeptide crosslinked by four disulfide bridges. It belongs to the alpha-toxin group, as judged by competition experiments with 125I AaH II for binding to rat brain synaptosomes (K0.5 = 7 x 10(-7) M). Lqq III is the first alpha-toxin to be characterized that is highly toxic to mice [LD50 = 50 micrograms (7.1 nmol)/kg body wt], by subcutaneous injection, insects Blatella germanica [LD50 = 60 ng (8.5 pmol)/g body wt.] and Musca domestica [LD50 = 120 ng (17 pmol)/g body wt]. When tested via the intracerebroventricular route, the toxicity for mice [55 micrograms (8 nmol)/kg] was of the same order as that found by subcutaneous injection, indicating that Lqq III has a higher affinity for peripheral sodium channels that for those of the central nervous system. There are three differences between the sequences of Lqq III and Lqh alpha IT, an alpha-toxin isolated from the venom of Leiurus quinquestriatus hebraeus. These substitutions are found at positions 20, 24, and 64 (Ser-->Ala,Asp-->Glu and His-->Arg, respectively). Surprisingly Lqh alpha IT is only weakly active in mice [LD50 = 5 mg (0.7 mumol)/kg], while in insects its toxicity is similar to that of Lqq III [140 ng (20 pmol)/g body wt blowfly larvae]. These observations are relevant to the definition of scorpion toxin structure-activity relationships.

Amino Acid Sequence↗

PubMed1993

Utilization of aspirin, quinine and verapamil in the prevention and treatment of scorpion venom intoxication.

Aside from serotherapy, the treatment of scorpion venom intoxication is symptomatic. The aim of this study was to compare the efficacy of drugs usually used in scorpion venom intoxications (atropine, propranolol) to that of other compounds, chosen in light of the pathophysiology of scorpion venom intoxication: dipyridamole, doxapram, quinine formate, lysine-acetyl-salicylate, valproate and verapamil. Using mice, the parameters evaluated were the preventive and therapeutic effects of drugs during experimental venom intoxication by Androctonus australis Hector and one of its toxins AaH 1, and by Buthus occitanus and Tityus serrulatus tunetanus on the other hand. It was found that although most of the drugs used could prolong the survival of the animals, the administration of verapamil and more so that of aspirin or quinine formate led to a 50 to 100% loss of venom and toxin toxicity, depending on the drug and the origin of the venom. In the case of propranolol, doxapram, atropine, dipyridamole and valproate, no or little protection were observed. If these results are confirmed in humans, the systematic use of these drugs could be a simple means for treating scorpion venom intoxication. The problem of scorpion venom intoxication poses a health problem both in the North African Maghreb and in the Americas. As a result of considerable information campaigns, the number of scorpion venom intoxications in Tunisia has dropped from 3000 in 1967 to 1000 per year in the 1980s. Serotherapy has reduced mortality to 0.35%, most deaths occurring in underweight children. In light of the large number of countries in which there is a risk of scorpion venom intoxication in the summertime, however, its prevention and treatment remain a major problem.

Animals↗

PubMed1993

Evaluation of structure-antigenicity relationship of peptides from human immunodeficiency virus type 1 (HIV-1) p18 protein by circular dichroism.

The antigenicity of Human Immunodeficiency Virus type 1 (HIV-1) matrix p18 protein was evaluated by analyzing the specificity of anti-p18 antibodies elicited either in HIV-1 infected humans, or in HIV-1 infected or immunized chimpanzees, against a panel of long and short overlapping synthetic peptides [from 12 to 46 amino acid (aa) residues] covering the entire sequence of p18. The relationship between peptide structure and antigenicity was further investigated by probing the secondary structures of the peptides by circular dichroism. The results obtained clearly showed the immunodominance of the N-terminal region mimicked by peptide P1 (aa 2-45), which reacted with 52 and 100% of human and chimpanzee anti-p18 sera, respectively. In contrast smaller 15 aa long peptides C1, C2, C3, C4 and P3 which cover the entire sequence of immunodominant peptide P1, showed only weak or no reactivity. In contrast to widely accepted hypotheses, circular dichroism analysis of both small and large peptides secondary structures did not show any obvious correlation between antigenicity and the ability of peptides to adopt an ordered conformation.

AIDS Vaccines↗

PubMed1993

Central analgesic effect of valproate in patients with epilepsy.

Clinical practitioners have often observed in the course of their daily work that the pain thresholds of epileptic patients seem to differ from those of healthy subjects. These patients can suffer from quite severe traumatic lesions without apparently experiencing any pain. Previous studies have shown that the absence of pain is due to treatment, since most antiepileptic drugs also have analgesic effects. In the present study, it was proposed to assess the pain thresholds of 15 epileptic patients (with tonic-clonic seizures generalized at outset) treated with valproate, by measuring the leg flexion nociceptive reflex (or RIII reflex) threshold: the stimulation threshold at which this reflex is triggered is known to be correlated with the pain threshold. The results were compared with 15 control subjects. The nociceptive threshold of the patients with valproate treatment was significantly higher than that of the control population. The nociceptive threshold was also in good correlation with the valproate plasma level. These data are discussed from the point of view of the gaba-ergic system and mechanisms possibly involved.

Adolescent↗

PubMed1993

Multi-branched peptides based on the HIV-1 V3 loop consensus motif inhibit HIV-1 and HIV-2 infection in CD4+ and CD4- cells.

The V3 loop is an hypervariable region of the HIV-1 surface envelope glycoprotein gp120 that is able to generate neutralizing antibodies. These antibodies are generally type-specific. They inhibit the interaction between the V3 loop and the membrane molecules involved in virus-cell and cell-cell fusion. Synthetic peptides based on the V3 loop sequence have been tentatively used to block the fusion process, but the results were unsuccessful. In this report, we confirm that monomeric V3 peptides are devoid of any anti-HIV activity. However, we found that, when assembled in a synthetic polymeric construction (SPC), these peptides have been able to inhibit the infection of human CD4+ lymphocytes and macrophages as well as CD4- epithelial intestinal cells by distantly related isolates of HIV-1 and HIV-2. V3 SPCs, based on the consensus HIV-1 sequence, may therefore represent a new class of therapeutically useful anti-HIV agents able to neutralize a wide range of viral isolates in both CD4+ and CD4- susceptible cells.

CD4 Antigens↗

PubMed1992

Molecular cloning and nucleotide sequence analysis of a cDNA encoding the main beta-neurotoxin from the venom of the South American scorpion Tityus serrulatus.

A cDNA encoding the main Tityus serrulatus beta-neurotoxin was isolated from a venom gland cDNA library by using an oligonucleotide probe. The amino acid sequence deduced from the cDNA nucleotide sequence indicated that the toxin is the processed product of a precursor containing: (i) a signal peptide of 20 residues; (ii) the amino acid sequence of the mature toxin; and (iii) an extra Gly-Lys-Lys tail at the C-terminal end before the termination codon. Thus, in addition to the removal of the signal peptide by a signal peptidase, the generation of the mature toxin requires both a post-translational cleavage by a carboxypeptidase specific for basic residues and the action of an alpha-amidating enzyme. These results also show that the biosynthetic pathway for beta-toxins of 'New World' scorpion venoms is similar to that already described for alpha-toxins of 'Old World' scorpion venoms.

Amino Acid Sequence↗

PubMed1992

Kaliotoxin, a novel peptidyl inhibitor of neuronal BK-type Ca(2+)-activated K+ channels characterized from Androctonus mauretanicus mauretanicus venom.

A peptidyl inhibitor of the high conductance Ca(2+)-activated K+ channels (KCa) has been purified to homogeneity from the venom of the scorpion Androctonus mauretanicus mauretanicus. The peptide has been named kaliotoxin (KTX). It is a single 4-kDa polypeptide chain. Its complete amino acid sequence has been determined. KTX displays sequence homology with other scorpion-derived inhibitors of Ca(2+)-activated or voltage-gated K+ channels: 44% homology with charybdotoxin (CTX), 52% with noxiustoxin (NTX), and 44% with iberiotoxin (IbTX). Electrophysiological experiments performed in identified nerve cells from the mollusc Helix pomatia showed that KTX specifically suppressed the whole cell Ca(2+)-activated K+ current. KTX had no detectable effects on voltage-gated K+ current (delayed rectifier and fast transient A current) or on L-type Ca2+ currents. KTX interacts in a one-to-one way with KCa channels with a Kd of 20 nM. Single channel experiments were performed on high conductance KCa channels excised from the above Helix neurons and from rabbit coeliac ganglia sympathetic neurons. KTX acted exclusively at the outer face of the channel. KTX applied on excised outside-out KCa channels induced a transient period of fast-flicker block followed by a persistent channel blockade. The KTX-induced block was not voltage-dependent which suggests differences in the blockade of KCa channels by KTX and by CTX. Comparison of KTX and CTX sequences leads to the identification of a short amino acid sequence (26-33) which may be implicated in the toxin-channel interaction. KTX therefore appears to be a useful tool for elucidating the molecular pharmacology of the high conductance Ca(2+)-activated K+ channel.

Amino Acid Sequence↗

PubMed1992

The amino acid sequence of toxin IV from the Androctonus australis scorpion: differing effects of natural mutations in scorpion alpha-toxins on their antigenic and toxic properties.

The complete amino acid sequence (64 residues) of the AaH IV toxin from the scorpion Androctonus australis Hector was determined by automated Edman degradation and was compared with the sequences of other Androctonus toxins. AaH IV was also tested by radioimmunoassay for binding to antisera raised against other toxins of the same species. The results indicated that AaH IV shares some of the antigenic properties of AaH I and AaH III toxins, but does not cross-react with anti-AaH II antibodies. The structural basis for the observed antigenic relationships can be found in the high degree of homology displayed by AaH IV with regard to AaH I and III, the changes in amino acid residues equally affecting regions included or excluded from the main predicted antigenic sites of AaH IV. The lower biological potency of AaH IV is presumably the result of some of the sequence differences. In particular, substitution affecting the charge and bulkiness of residue 61 could account for the poor receptor binding and consequential weak toxic properties of this molecule.

Amino Acid Sequence↗

PubMed1992

The beta-type toxin Ts II from the scorpion Tityus serrulatus: amino acid sequence determination and assessment of biological and antigenic properties.

The toxin Ts II from the venom of the Brazilian scorpion Tityus serrulatus was purified in two successive chromatographic steps. The amino acid sequence was then determined by automated Edman degradation of the reduced and S-carboxymethylated protein and of proteolytic peptides derived from it. This sequence appears to differ from that of previously characterized toxins found in this venom. However, it is identical to the recently published sequence of protein III-8 from the same venom [Possani et al., J Biol Chem 266:3178-3185, 1991], except that the C-terminus was found to be amidated. Homologies were found between the sequence of Ts II and that of other toxins from Tityus; in particular, the amino acid sequence of Ts II displays 72% sequence identity with Ts VII (also called Titx gamma). Consistent with this structural similarity, some biological properties of Ts II were found to be similar to those of Ts VII: Ts II has an intracerebroventricular LD50 of 6 ng, as compared to 0.6 ng for Ts VII; in a receptor binding assay Ts II, like Ts VII, was found to behave as a beta-type toxin and to inhibit the binding of the reference labelled toxin with a K0.5 of 5 x 10(-9) M, as compared to 7 x 10(-11) M for Ts VII. Nevertheless, Ts II is unable to bind to anti-Ts VII antibodies in radioimmunoassay experiments, indicating the non-conservation between the two toxins of at least some antigenically important residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1992

Toxin III of the scorpion Androctonus australis Hector: proton nuclear magnetic resonance assignments and secondary structure.

1H NMR has been applied to a 3.5 mM, pH 5.4, solution of toxin III (64 amino acids) from venom of the scorpion Androctonus australis Hector. The resonance assignment strategy began by applying a generalized main-chain directed method for rapid identification and resonance assignments of secondary structures. The remaining resonances were assigned by the sequential method. Major structural features include a helix of 2 1/2 turns (residues 20-28) which is linked by two disulfide bridges to the central strand of a triple-stranded anti-parallel beta-sheet. Turns were identified at residues 15-17, 47-49 and also at residues 51-53. Numerous NOEs have been observed between hydrophobic residues which suggest the presence of a hydrophobic core; these include Leu37, Leu23, Val47, Tyr14, Trp45 and Tyr5. The Trp45 and Tyr5 rings lie orthogonal to one another. No crystal structure has been solved for this AaH III toxin. Comparisons are made with other members of the scorpion toxin family.

Amino Acid Sequence↗

PubMed1992

Specificity of anti-P25 antibodies produced against whole HIV-1 particles or soluble forms of the protein.

Specificity of anti-p25 antibodies produced against either whole Human Immunodeficiency Virus type 1 (HIV-1) particles in humans and chimpanzees, or against soluble forms of the protein in chimpanzees and rabbits was analyzed by ELISA using a panel of 37 long (> or = 30 residues) or shorter (9-21 residues) overlapping peptides covering the entire p25 sequence. Antibodies elicited by intact virions presented similar reactivity patterns in HIV-1-infected humans and in HIV-1-infected or immunized chimpanzees and recognized only a limited region mostly the C-terminus of the molecule. Moreover, 8 of the human sera (36%), which nonetheless reacted with high titers and avidity with native p25, did not bind to any long or short peptide. These results suggest that the majority of antibodies elicited by viral particles are presumably directed to conformational epitopes. In contrast, antibodies raised against soluble forms of p25 could react against all long peptides but one (residues 211-245) and against some short peptides, indicating that most of p25 sequence may be immunogenic under these conditions. These results suggest that the reactivity spectrum of anti p25 antibodies is rather different if they are produced against intact HIV-1 particles or the soluble protein. They also indicate that it may be possible to manipulate the specificity of the humoral immune response by using either intact virions or purified proteins.

Acquired Immunodeficiency Syndrome↗

PubMed1992

Influence of carbohydrate moieties on the immunogenicity of human immunodeficiency virus type 1 recombinant gp160.

The role of carbohydrates in the immunogenicity of human immunodeficiency virus type 1 (HIV-1) glycoproteins (gp160 and gp120) remains poorly understood. We have analyzed the specificity and neutralizing capacity of antibodies raised against native gp160 or against gp160 deglycosylated by either endo F-N glycanase, neuraminidase, or alpha-mannosidase. Rabbits immunized with these immunogens produced antibodies that recognized recombinant gp160 (rgp160) from HIV-1 in a radioimmunoassay and in an enzyme-linked immunosorbent assay. Antibodies elicited by the different forms of deglycosylated gp160 were analyzed for their reactivity against a panel of synthetic peptides. Compared with anti-native gp160 antisera, serum reactivity to most peptides remained unchanged, or it could increase (peptide P41) or decrease. Only antibodies raised against mannosidase-treated gp160 failed to react with a synthetic peptide (peptide P29) within the V3 loop of gp120. Rabbits immunized with desialylated rgp160 generated antibodies which recognized not only rgp160 from HIV-1 but also rgp140 from HIV-2 at high titers. Although all antisera produced against glycosylated or deglycosylated rgp160 could prevent HIV-1 binding to CD4-positive cells in vitro, only antibodies raised against native or desialylated gp160 neutralized HIV-1 infectivity and inhibited syncytium formation between HIV-1-infected cells and noninfected CD4-positive cells, whereas antibodies raised against alpha-mannosidase-treated gp160 inhibited neither virus replication nor syncytium formation. These findings indicate that the carbohydrate moieties of gp160 can modulate the specificity and the protective efficiency of the antibody response to the molecule.

Amino Acid Sequence↗

PubMed1991

Lethal neurotoxicity in mice of the basic domains of HIV and SIV Rev proteins. Study of these regions by circular dichroism.

We have recently reported a basic domain-mediated neurotoxic activity of HIV-1 Tat [1991, J. Virol. 65, 961-965]. Here we have tested the neurotoxicity in vivo of several Rev-related synthetic peptides and found that only those mimicking the basic regions of Rev from HIV-1, HIV-2 and SIV were lethal to mice. In contrast, the homologous domain of HTLV-1 Rex was found to be inactive for lethal activity. Analysis of the tropism of these peptides for phospholipids has demonstrated a direct interaction of the basic domain-containing peptides, except Rex, with acidic--but not neutral--phospholipids. As determined by circular dichroism, a possible correlation between the conformation of the basic regions and the toxicity is discussed.

Amino Acid Sequence↗

PubMed1991

Activating region of HIV-1 Tat protein: vacuum UV circular dichroism and energy minimization.

Tat protein is a trans-acting transcriptional activator of the human immunodeficiency virus type 1 and is essential for viral transcription. By homology with other transcriptional activators, Tat is expected to possess a nucleic acid binding region and a separate adjacent activating region. In order to localize the activating region of Tat, we have synthesized the sequences 2-23 and 38-60 of the protein. These two peptides contain the two candidates for the activating regions proposed from mutation experiments in previous studies: sequence 1-13 and sequence 38-45. The argument advanced to justify the location of the activating region within the sequence 1-13 was the periodicity of acidic, polar, and hydrophobic residues consistent with that of an amphipathic alpha-helix, similar to the activating region of many eukaryotic transcriptional activators. We have monitored by vacuum UV circular dichroism the ability of each peptide to adopt an alpha-helical conformation under conditions that strongly favor the formation of secondary structures. Only peptide 38-60 adopts an alpha-helical conformation in these conditions, in keeping with Chou-Fasman prediction. Energy minimization and molecular dynamics were carried out for several possible conformations of sequences 1-14 and 38-60. Our results indicate that only the sequence 38-45 is able to form an alpha-helix with amphipathic characteristics.

Amino Acid Sequence↗