Biomedical subjects

H Rochat

Publications and source records attributed to H Rochat.

At least 145 records · Page 8Linked to original sources

PubMed1989

Accessibility of the highly conserved amino- and carboxy-terminal regions from HIV-1 external envelope glycoproteins.

Amino- and carboxy-terminal extremities of the envelope external glycoproteins are regions that have remained highly conserved between human immunodeficiency viruses HIV-1 and HIV-2. The corresponding peptides have been synthesized and their structure and function analyzed. Circular dichroism spectra showed evidence of alpha helical conformation when the peptides were dissolved in the nonpolar solvent trifuoroethanol. These two regions are indeed exposed on the molecule because they were accessible to their respective specific antibodies on the native gp160 precursor or processed gp120 glycoproteins of HIV-1. Neither the peptides nor rabbit or human antibodies directed against the N- and C-terminal peptides interfered with the interaction between HIV-1 external glycoprotein gp120 and its CD4 cellular receptor. Taken together, these results indicate that N- and C-terminal regions of gp120 are accessible on the quaternary structure of the virion as well as on the soluble form of gp120 and that these regions are not directly or indirectly involved in the binding of gp120 to CD4.

Animals↗

PubMed1989

Use of synthetic peptides for the detection of antibodies against the nef regulating protein in sera of HIV-infected patients.

Human sera were tested for the presence of anti-nef antibodies by radioimmunoassay (RIA), with recombinant radiolabelled nef expressed in E. coli. Of the 300 HIV-positive sera tested by RIA, 70 +/- 5.3% were found to be anti-nef positive. Anti-nef antibodies bound to nef with a high affinity (K 0.5 = 2.2 x 10(-9) M). In 31 of the sera, the specificity of anti-nef antibodies was further analysed by enzyme-linked immunosorbent assay (ELISA) with large synthetic peptides ranging from 31 to 66 amino acid residues and spanning the total sequence of nef from HIV-1. The results obtained showed that the immunodominant antigenic sites of nef were located close to the N- and C-terminal regions of the molecule.

Acquired Immunodeficiency Syndrome↗

PubMed1988

Monoclonal antibodies to scorpion toxins. Characterization and molecular mechanisms of neutralization.

Two mAb specific for the potent toxin II of the scorpion Androctonus australis Hector have been produced. One of them shows both high affinity binding to the toxin (Kd) = 0.8 nM) and in vivo and in vitro neutralizing properties. The mechanism by which the antibody neutralizes toxin binding to its receptor was shown to be of the competitive type, the epitope overlapping or being close to the receptor-binding region of the toxin. Several residues of the toxin clustered in the C-terminal region were shown likely to be part of the discontinuous epitope recognized by the antibody. The positive charge of the N epsilon-Lys-58 seems to play a pivotal role in the binding of the toxin to both the mAb and the sodium channel receptor.

Acetylation↗

PubMed1988

Localization of the toxic site of Naja mossambica cardiotoxins: small synthetic peptides express an in vivo lethality.

Cardiotoxins are small basic proteins which cause heart failure when they are injected in vivo. In order to better understand their molecular mode of action, short peptides designed on the model of the first loop of the molecule of cardiotoxin IV from Naja mossambica mossambica venom have been synthetized by the solid-phase procedure of Merrifield. These peptides express lethality in mouse when they are injected intravenously. Taking into account the respective molecular weights, they are 3.5 to 5% as toxic as the cardiotoxin. Furthermore, the symptomatology they induce is undistinguishable from that induced by cardiotoxins. These results strongly support our previous hypothesis that the first loop of the molecule is the toxic site of cardiotoxins.

Amino Acid Sequence↗

PubMed1988

Photoaffinity labeling of scorpion toxin receptors associated with insect synaptosomal Na+ channels.

Photoreactive and radioiodinated derivatives of several scorpion toxins acting on insect Na+ channels were prepared without loss of their pharmacological activities. Photoaffinity experiments were carried out on a synaptosomal fraction from the nerve cord of the cockroach Periplaneta americana: with all toxin derivatives, a single specifically labeled band was obtained with a molecular weight of 188,000 +/- 12,000 (n = 17). These results indicate for the first time the molecular weight of the scorpion toxin receptor from the insect nervous system which is probably associated with voltage sensitive Na+ channels. One of these toxins, toxin VII from Tityus serrulatus venom, has been previously shown to be active both in mammals and in insects, in rat brain synaptosomes this toxin labeled a Mr = 31,000 +/- 4,000 band in contrast, to observations in the insect preparation.

Affinity Labels↗

PubMed1988

1H nuclear-magnetic-resonance studies of the three-dimensional structure of the cardiotoxin CTXIIb from Naja mossambica mossambica in aqueous solution and comparison with the crystal structures of homologous toxins.

Using the previously reported sequence-specific 1H-NMR assignments, structural constraints for the cardiotoxin CTXIIb from Naja mossambica mossambica were collected. These include distance constraints from nuclear Overhauser enhancement measurements both in the laboratory and in the rotating frame, dihedral angle constraints derived from spin-spin coupling constants, and constraints from hydrogen bonds and disulfide bridges. Structure calculations with the distance geometry program DISMAN confirmed the presence of the previously identified antiparallel beta-sheets formed by residues 1-5 and 10-14, and by 20-27, 35-39 and 49-55, and established the nature of the connections between the individual beta-strands. These include a right-handed crossover between the two peripheral strands in the triple-stranded beta-sheet, and a type I tight turn immediately preceding the beta-strand 49-55. The spatial arrangement of the polypeptide backbone in the solution structure of CTXIIb is closely similar to that in the crystal structure of the homologous cardiotoxin VII4 from the same species. In an Appendix the origin of the large pH dependence of two amide proton chemical shifts in CTXIIb is explained.

Animals↗

PubMed1988

Preparation of a polyvalent antivenom against various Mexican scorpion Centruroides species.

Antisera were obtained from rabbits injected with four different immunogens from the Mexican scorpion Centruroides suffusus suffusus i.e. the crude venom, a telson extract, a toxic fraction obtained from this telson extract by gel filtration and the same toxic fraction subjected to acetylation. The neutralizing capacity of these antisera are compared: it appears that a telson extract can be used instead of the crude venom to produce an efficient antiserum. The immunological properties of ground telsons obtained from three other species of the Mexican scorpion Centruroides (Centruroides noxius, Centruroides limpidus limpidus, Centruroides limpidus tecomanus) are studied with the antisera raised against Centruroides suffusus suffusus immunogens: an almost total cross-neutralization is observed.

Animals↗

PubMed1988

Orthorhombic crystals and three-dimensional structure of the potent toxin II from the scorpion Androctonus australis Hector.

Orthorhombic crystals (space group P212121, a = 45.94 A, b = 40.68 A, c = 29.93 A) of the potent scorpion alpha-toxin II from Androctonus australis Hector were grown using sterile techniques. The structure was solved by a combination of heavy-atom and model phasing. Subsequently, it was refined at 1.8 A resolution by a fast-Fourier restrained least-squares procedure. The crystallographic R factor is 0.152 for data with 7.0 A greater than d greater than 1.8 A and F greater than 2.5 sigma (F) and 0.177 when all data are considered. Eighty-nine solvent molecules have been incorporated into the model. The dense core formed by the alpha-helical and antiparallel beta-sheet moieties and three of the four disulfide bridges is similar in variant 3, a toxin purified from the North American scorpion Centruroides sculpturatus, and in toxin II. However, the two molecules differ markedly in the orientation of loops protruding from the core. Toxin II seems to contain several highly ordered solvent molecules. Eight of them occupy a cavity consisting of the C-terminal region and a loop found only in scorpion alpha-toxins. The highly reactive and pharmacologically important Lys-58 is found at one of the extremes of this cavity, where it establishes a series of hydrogen bonds with protein and solvent atoms. The reactivities of the five lysine residues of toxin II are highly correlated with the formation of hydrogen bonds, hydrophobic interactions, and salt links.

Amino Acid Sequence↗

PubMed1988

Immunochemistry of scorpion toxins. Synthesis and antigenic properties of a model of a loop region specific to alpha-toxins.

A region of the toxin II of the scorpion Androctonus australis Hector, possessing a loop structure, is shown to be antigenic. Some clear hints for the probable antigenic character of this region were obtained by the protruding properties of the loop region, as assessed by accessibility computations using atomic coordinates of the toxin and Lee-Richards algorithm. A synthetic replica of the loop region was obtained in a linear and cyclised form. Within the total anti-toxin antibody population, we have found and isolated those that recognize the model peptides. A high affinity binding of these specific antibodies to the parent toxin was demonstrated, affording experimental evidence for the antigenic properties of the loop region.

Amino Acid Sequence↗

PubMed1987

Monitoring the purification by high-performance liquid chromatography of cardiotoxins from Naja mossambica mossambica using phase-sensitive two-dimensional nuclear magnetic resonance.

High-resolution phase-sensitive two-dimensional proton nuclear magnetic resonance was used to monitor the preparation by high-performance liquid chromatography of homogeneous proteins from the venom of Naja mossambica mossambica. This resulted in the characterization of a heterogeneous protein preparation VII2, which had been used in earlier structural studies by NMR, as well as a homogeneous protein CTXIIb and a nearly homogeneous protein fraction CTXIIa, which are now both subject to further investigations of their solution conformations.

Amino Acids↗

PubMed1987

Sequence-specific 1H-NMR assignments and determination of the secondary structure in aqueous solution of the cardiotoxins CTXIIa and CTXIIb from Naja mossambica mossambica.

Sequence-specific assignments of the 1H-nuclear magnetic resonance (NMR) spectra of the cardiotoxins CTXIIa and CTXIIb from Naja mossambica mossambica were obtained using two-dimensional NMR experiments at 500 MHz and the independently determined amino acid sequences. Assignments were obtained from data at 25 degrees C and 45 degrees C for all but one backbone proton of the 60 residues in each protein. Complete or partial assignments are also reported for the side-chain protons. These assignments supercede those published previously for the toxin preparation VII2 [Hosur, R. V., Wider, G. & Wüthrich K. (1983) Eur. J. Biochem. 130, 497-508]. The 1H/2H-exchange kinetics were measured in 2H2O at 20 degrees C for the amide protons and the N-terminal amino group. These and additional NMR data enabled the determination of the secondary structure in aqueous solution, which is virtually identical in CTXIIa and CTXIIb. Both proteins contain a short double-stranded antiparallel beta-sheet comprising the residues 2-4 and 11-13, and a triple-stranded antiparallel beta-sheet consisting of the residues 20-26, 35-39, and 49-55. The two peripheral strands of the triple-stranded beta-structure were found to be connected by a right-handed cross-over, and the locations of several tight turns were also identified.

Amino Acid Sequence↗

PubMed1987

Isolation of two 67 kDa calcium-binding proteins from pig lung differing in affinity for phospholipids and in anti-phospholipase A2 activity.

Two 67 kDa proteins adsorbed to membranes in the presence of Ca2+ have been purified to homogeneity from pig lung using conventional procedures, followed by calcium-dependent affinity chromatography on polyacrylamide-immobilized phosphatidylserine. The two proteins were, respectively, excluded (67E) and retained (67R) on the column in the presence of Ca2+. On the basis of amino acid composition and isoelectric point, 67R was identified as 67 kDa calelectrin/calcimedin, whereas 67E could be differentiated from albumin, calregulin, 67 kDa fragment of protein kinase C and surfactant-associated proteins. Only 67R was slightly phosphorylated by protein kinase C, reacted with an antibody raised against 32.5 kDa endonexin and inhibited pig pancreas phospholipase A2 in a way similar to that of lipocortin or endonexin. These data bring further support to the view that inhibition of phospholipase A2 by lipocortin or other related proteins involves interaction with the lipid/water interface. They also provide evidence for a new kind of Ca2+-binding protein (67E), whose role still remains to be determined.

Amino Acids↗

PubMed1987

Immunochemistry of scorpion toxins. Immunogenicity of peptide 19-28 a model of an accessible and relatively rigid region.

Peptide 19-28, a model of an antigenic region of Androctonus australis Hector toxin II, has a rigid alpha-helix structure in the native protein. It was used as immunogen either in the free form, or bound to bovine serum albumin (BSA) or linked to its synthesis support (macroporous polyacrylamide resin). Only the anti-(peptide-BSA) and the anti-(peptide-resin) antibodies recognized the native toxin. The use of short synthetic analogues of peptide 19-28 suggests a specificity difference in the two antipeptides. Anti-(peptide-BSA) recognizes probably two determinants localized at the C and N terminals of the peptide chain. Anti-(peptide-resin) preferentially recognizes the N-terminal extremity. Finally we showed that the alpha-helix region remains accessible to antipeptide 19-28 when the toxin is bound to its receptor.

Amino Acid Sequence↗

PubMed1987

Lipocortin-like anti-phospholipase A2 activity of endonexin.

Endonexin (protein II, 32.5 kDa) has been purified to homogeneity from bovine liver in the following steps: selective extraction by EGTA from membranes precipitated with Triton X-100/calcium; chromatography on DEAF-TSK 545 at pH 7.0, endonexin being eluted at 0.1 M NaCl; affinity chromatography on polyacrylamide-immobilized phosphatidylserine; gel filtration on TSK 3000. The amino acid composition was essentially similar to that previously reported. Using [3H]oleic acid-labelled Escherichia coli membranes as substrate, endonexin inhibited phospholipase A2 from pig pancreas. Maximal inhibition was 55 and 70%, whereas 50% inhibition occurred at 480 and 120 nM endonexin and lipocortin II, respectively. These data could be related to common features shared by both lipocortins/calpactins and endonexin, i.e. the presence of a consensus sequence and the ability to bind to anionic phospholipids in a calcium-dependent manner.

Amino Acid Sequence↗

PubMed1987

Purification and chemical and biological characterizations of seven toxins from the Mexican scorpion, Centruroides suffusus suffusus.

Seven polypeptides highly toxic to mice were isolated from the venom of the scorpion, Centruroides suffusus suffusus (Css), and their chemical and toxic properties were characterized. It was shown that the most active toxins by intracerebroventricular injection are less active when injected subcutaneously. The complete amino acid sequence (66 residues) of toxin II (Css II) has been determined. The C-terminal end is amidated as found for most other scorpion toxins. Css II is a beta-type toxin, previously used to define the binding site for activation of the sodium channel. Using rat brain synaptosomes, we demonstrated that all Css toxins compete with 125I-Css II to bind to site 4 and should be considered as beta-scorpion toxins. Specific binding parameters for Css VI, one of the most active toxins, were determined: KD = 100 pM; capacity in binding sites, 2.2 pmol of toxin/mg of synaptosomal protein. Css VI was shown to inhibit gamma-aminobutyric acid uptake by synaptosomes: K 0.5 = 100 pM, which agrees with its KD. Competition experiments between the seven Css toxins and 125I-Css II for antiserum raised against Css II demonstrated that all these toxins have common antigenic properties.

Amino Acid Sequence↗

PubMed1987

Mixed phospholipid-cardiotoxin monomolecular films studied by intrinsic polarized surface fluorescence.

Surface fluorescence spectroscopy was used to study mixed phospholipid-cardiotoxin monomolecular films. Using polarized incident light we were able to detect the intrinsic fluorescence emission at a wavelength of 339 nm of the single Trp11 in the cardiotoxin molecule. Its fluorescence intensity increased continuously upon film compression up to a maximal value reached at 42 mN x m-1 in surface pressure. A relative polarization index can be used as an indication of the orientation of the indole ring. A transition at around 25 +/- 5 mN x m-1 in the orientation of the Trp11 relative to the plane of the interface was clearly evidenced during film compression, in agreement with our independent data showing a flipping of the whole toxin molecule (Bougis et al., (1981), Biochemistry 20, 4915-4920).

Cobra Cardiotoxin Proteins↗

PubMed1987

Characterization of six toxins from the venom of the Moroccan scorpion Buthus occitanus mardochei.

When the venom of the Moroccan scorpion Buthus occitanus mardochei was submitted to a combination of several chromatographic steps (including gel-filtration and ion-exchange chromatographies), seven proteins were obtained, six being lethal to mice. These proteins have been characterized by their chemical, immunological and toxic properties. The amino acid sequence (66 residues) of Bom III, the most noteworthy toxin of the venom as for its amino acid composition, is proposed following automatic sequencing of the reduced and S-methylated protein and of chymotryptic peptides. It was obvious that this sequence is somewhat different from those of toxins belonging to the same structural and immunological group (Bom III was found to be immunologically related to Buthus occitanus tunetanus toxins I and II which both share with it 56% of homology. Furthermore, Bom III was found to be unable to compete (as does Bot I) with toxin II of Androctonus australis Hector (an alpha-type toxin) for neurotoxin binding site 3 on the sodium channel of rat brain synaptosomes. Bom III was also unable to compete with toxin II of Centruroides suffusus suffusus (a beta-type toxin) to neurotoxin binding site 4 of the same channel.

Amino Acid Sequence↗

PubMed1987

Re-examination of the specificity of the scorpion Androctonus australis hector insect toxin towards arthropods.

The toxicity of the insect toxin from the venom of the scorpion Androctonus australis Hector has been studied in six species of insects, three species of crustaceans (two terrestrial and one aquatic) and in mice. Paralysing and lethal effects were observed not only in insects but also on the three crustacean species. For insects the LD50 varies from 2 to 1310 ng per 100 mg of insect, according to the species. On crustaceans doses up to 3-9 micrograms per 100 mg of animal induced a paralysing or lethal effect. In mice injections of 0.05 and 1 mg of the toxin intracerebroventricularly and s.c. resulted in no apparent symptoms of intoxication. These results demonstrate that the insect toxin of Androctonus australis Hector has no toxicity for the mouse, but also that it is not absolutely specific for insects.

Animals↗