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Marius Ptak - One of the best experts on this subject based on the ideXlab platform.
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1h nmr derived secondary structure and the overall fold of the potent anti mammal and anti Insect Toxin iii from the scorpion leiurus quinquestriatus quinquestriatus
FEBS Journal, 1996Co-Authors: Celine Landon, Herve Rochat, Bruno Cornet, Jean-marc Bonmatin, Francoise Vovelle, Charles Kopeyan, Marius PtakAbstract:We describe the secondary structure and the overall fold of Toxin III from the venom of the scorpion Leiurus quinquestriatus quinquestriatus determined using two-dimensional-1H-NMR spectroscopy. This protein, which contains 64 amino acids and 4 disulfide bridges, belongs to the long-chain Toxin category and is highly toxic to both mammals and Insects. The overall fold was determined on the basis of 1208 inter-proton-distance restraints derived from NOE measurements and 90 Ψ, Φ dihedral-angle restraints derived from NOE connectivities and 3JNH-αH coupling constants using the HABAS program. This fold, which mainly consists of an α-helix packed against a small antiparallel three-stranded β-sheet, and of several turns and loops, is similar to that of other long-chain scorpion Toxins. Aromatic and non-polar residues form several patches on the surface of the protein which alternate with patches of charged and polar residues. Such a topology should be important in the interactions of Toxin III with sodium channels in membranes. Two weakly constrained loops introduce some flexibility to the structure which could be related to the activity of this Toxin. The central core of Toxin III is compared with the cysteine-stabilized αβ motif (an α-helix connected to a β-sheet through two disulfide bridges) found in Insect defensins and plant thionins. Defensins and thionins are small proteins (≈40–50 amino acid residues) containing three or four disulfide bridges, respectively. This comparison confirms that the cysteine-stabilized αβ motif is a common core to a number of small proteins from different origins and having different activities.
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1H‐NMR‐Derived Secondary Structure and the Overall Fold of the Potent Anti‐Mammal and Anti‐Insect Toxin III from the Scorpion Leiurus quinquestriatus quinquestriatus
FEBS Journal, 1996Co-Authors: Celine Landon, Herve Rochat, Bruno Cornet, Jean-marc Bonmatin, Francoise Vovelle, Charles Kopeyan, Marius PtakAbstract:We describe the secondary structure and the overall fold of Toxin III from the venom of the scorpion Leiurus quinquestriatus quinquestriatus determined using two-dimensional-1H-NMR spectroscopy. This protein, which contains 64 amino acids and 4 disulfide bridges, belongs to the long-chain Toxin category and is highly toxic to both mammals and Insects. The overall fold was determined on the basis of 1208 inter-proton-distance restraints derived from NOE measurements and 90 Ψ, Φ dihedral-angle restraints derived from NOE connectivities and 3JNH-αH coupling constants using the HABAS program. This fold, which mainly consists of an α-helix packed against a small antiparallel three-stranded β-sheet, and of several turns and loops, is similar to that of other long-chain scorpion Toxins. Aromatic and non-polar residues form several patches on the surface of the protein which alternate with patches of charged and polar residues. Such a topology should be important in the interactions of Toxin III with sodium channels in membranes. Two weakly constrained loops introduce some flexibility to the structure which could be related to the activity of this Toxin. The central core of Toxin III is compared with the cysteine-stabilized αβ motif (an α-helix connected to a β-sheet through two disulfide bridges) found in Insect defensins and plant thionins. Defensins and thionins are small proteins (≈40–50 amino acid residues) containing three or four disulfide bridges, respectively. This comparison confirms that the cysteine-stabilized αβ motif is a common core to a number of small proteins from different origins and having different activities.
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1h nmr derived secondary structure and the overall fold of the potent anti mammal and anti Insect Toxin iii from the scorpion leiurus quinquestriatus quinquestriatus
FEBS Journal, 1996Co-Authors: Celine Landon, Bruno Cornet, Jean-marc Bonmatin, Francoise Vovelle, Charles Kopeyan, Marius Ptak, H RochatAbstract:We describe the secondary structure and the overall fold of Toxin III from the venom of the scorpion Leiurus quinquestriatus quinquestriatus determined using two-dimensional-1H-NMR spectroscopy. This protein, which contains 64 amino acids and 4 disulfide bridges, belongs to the long-chain Toxin category and is highly toxic to both mammals and Insects. The overall fold was determined on the basis of 1208 inter-proton-distance restraints derived from NOE measurements and 90 psi, phi dihedral-angle restraints derived from NOE connectivities and 3JNH-alphaH coupling constants using the HABAS program. This fold, which mainly consists of an alpha-helix packed against a small antiparallel three-stranded beta-sheet, and of several turns and loops, is similar to that of other long-chain scorpion Toxins. Aromatic and non-polar residues form several patches on the surface of the protein which alternate with patches of charged and polar residues. Such a topology should be important in the interactions of Toxin III with sodium channels in membranes. Two weakly constrained loops introduce some flexibility to the structure which could be related to the activity of this Toxin. The central core of Toxin III is compared with the cysteine-stabilized alpha beta motif (an alpha-helix connected to a beta-sheet through two disulfide bridges) found in Insect defensins and plant thionins. Defensins and thionins are small proteins (approximately 40--50 amino acid residues) containing three or four disulfide bridges, respectively. This comparison confirms that the cysteine-stabilized alpha beta motif is a common core to a number of small proteins from different origins and having different activities.
Herve Rochat - One of the best experts on this subject based on the ideXlab platform.
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Covalent structure of the Insect Toxin of the North African scorpion Androctonus australis Hector.
International journal of peptide and protein research, 2009Co-Authors: Hervé Darbon, Charles Kopeyan, E. Zlotkin, J. Van Rietschoten, Herve RochatAbstract:The complete covalent structure of the Insect Toxin purified from the venom of the North-African scorpion Androctonus australis Hector was described. Its amino acid sequence was established by phenylisothiocyanate degradation of several protein derivatives and proteolytic fragments in a liquid protein sequencer using either a "protein" or a "peptide" program. The position of the four disulfide bridges were deduced by analysis of proteolytic peptides before and after diperformic oxidation, and by partial labeling of the half cystine residues with [14C]-iodoacetic acid and determining the specific radioactivities of the S-[14C]-carboxymethylated phenylthiohydantoin cysteines. The sequences of the Insect and mammal Toxins from scorpions can be aligned with homology with the positions of seven half-cystine residues as registers. The mammal and Insect Toxins have three disulfide bridges at homologous positions. The mammal and Insect Toxins have three disulfide bridges at homologous positions. The fourth bridge is different in that Cys12 in mammal Toxin II is replaced by Cys38 in the Insect Toxin. It is likely that the position of the disulfide bridges is the same for all scorpion neuroToxins active on mammals. We believe that the shift of one half-cystine residue in the Insect Toxin may induce a conformational change in the structure of the protein, which, in turn, may partially account for the total specificity of this Toxin for Insect nervous system.
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BotIT6: a potent depressant Insect Toxin from Buthus occitanus tunetanus venom.
Toxicon : official journal of the International Society on Toxinology, 2003Co-Authors: Thouraya Mejri, Herve Rochat, Sandrine Cestele, Marcel Pelhate, Lamia Borchani, Najet Srairi-abid, Rym Benkhalifa, Imed Regaya, Habib Karoui, Mohamed El AyebAbstract:A new depressant Insect Toxin Buthus occitanus tunetanus Insect-Toxin 6 (BotIT6) was purified by high-performance liquid chromatography from Buthus occitanus tunetanus (Bot) venom. BotIT6 is very active against Blatella germanica (LD50=10ng/100mg body mass) thus being one of the most potent anti-Insect Toxin so far characterised. When compared to other Insect Toxin sequences, BotIT6 present high similarities with depressant Insect Toxins with an additional arginine residue at the C-terminus and a methionine at position 27. The calculated net charge of BotIT6 is positive (+3) whereas it is negative for classical depressant Toxins: this might be associated with its high toxicity. Voltage current clump studies show that BotIT6 is not a very potent depressant Insect Toxin despite its high toxicity in vivo. BotIT6 is able to fully inhibit the specific binding of 125I AaHIT and 125I-BotIT2 on Periplaneta americana synaptosomal membrane vesicles with high affinities. Despite its higher toxicity BotIT6 is a weaker competitor with 125I AaHIT and 125I BotIT2 as compared to the other beta Toxins.Altogether, these results may suggest that BotIT6 probably defines a novel sub-group of depressant anti-Insect Toxins for which the receptor site can be overlapping, but not identical to that for classical depressant Insect Toxins.
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PnTx4-3, a new Insect Toxin from Phoneutria nigriventer venom elicits the glutamate uptake inhibition exhibited by PhTx4 toxic fraction.
Toxicon : official journal of the International Society on Toxinology, 2003Co-Authors: Leida Calegário Oliveira, Herve Rochat, Pascal Mansuelle, Marta N. Cordeiro, Maria Elena De Lima, Adriano Marcal Pimenta, M. Richardson, Suely G. FigueiredoAbstract:Several pools of neurotoxic peptides obtained from fractionated Phoneutria nigriventer venom induce different toxicological effects. One of them, PhTx4, is highly toxic towards Insects and displays only a slight toxicity when injected in mice. Also, this fraction contains a class of peptides that are able to inhibit glutamate uptake in preparations of mammalian central nervous systems (CNS). In this work a new Toxin called PnTx4-3 was isolated from the PhTx4 fraction by reverse phase and anion exchange steps using high performance liquid chromatography (HPLC). Edman sequencing of PnTx4-3 revealed that it was a polypeptide of 48 amino acid residues, containing 10 cysteines cross-linked by five disulfide bridges. The molecular mass measured by ES-Q-TOF mass spectrometry was 5199.49+/-0.64 Da, which is very close to the calculated mass from amino acid sequence (5199.99 Da). This Toxin induces immediate excitatory effects when injected intrathoracically in house flies and cockroaches. Intracerebroventricular injections of 30 microg of PnTx4-3 in mice resulted in no apparent signs of intoxication. In order to make an orthologous comparison, pharmacological characterisation were carried out in rat brain synaptosomes by using [3H]-L-glutamate, showed that the whole PhTx4 fraction as well as the pure Toxins PnTx4-3, Tx4(6-1) and Tx4(5-5) obtained of this fraction, were able to inhibit the glutamate uptake in the micromolar concentration range. PnTx4-3 inhibits the glutamate uptake in a dose dependent manner, with an IC50 of approximately 1 microM. PnTx4-3 is highly homologous to the Tx4(6-1) and Tx4(5-5) Toxins previously described from the same fraction.
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biochemical and pharmacological characterization of a depressant Insect Toxin from the venom of the scorpion buthacus arenicola
FEBS Journal, 1997Co-Authors: Sandrine Cestele, R Oughideni, Pascal Mansuelle, Charles Kopeyan, Claude Granier, Herve RochatAbstract:: A depressant Toxin active on Insects, Buthacus arenicola IT2, was isolated from the venom of the North African scorpion B. arenicola and its structural and pharmacological properties were investigated. B. arenicola IT2 is a single polypeptide of 61 amino acid residues, including 8 half-cystines but no methionine and histidine, with a molecular mass of 6835 Da. Its amino acid sequence is 79-95% identical to other depressant Toxins from scorpions. When injected into the cockroach Blatella germanica, B. arenicola IT2 induced a slow depressant flaccid paralysis with a LD50 of 175 ng. B. arenicola IT2 has two non-interacting binding sites in cockroach neuronal membranes: one of high affinity (Kd1 = 0.11 +/- 0.04 nM) and low capacity (Bmax1 = 2.2 +/- 0.6 pmol/mg), and one of low affinity (Kd2 = 24 +/- 7 nM) and high capacity (Bmax2 = 226 +/- 92 pmol/mg). Its binding to these two sites was completely inhibited by Leiurus quinquestriatus quinquestriatus IT2, a depressant Toxin from L. quinquestriatus quinquestriatus. Reciprocal-binding experiments between B. arenicola IT2 and the excitatory Insect-Toxin A. australis Hector IT revealed competition between the two Toxins for the high-affinity sites of B. arenicola IT2. B. arenicola IT2 has a higher affinity than L. quinquestriatus hebraeus IT2, a depressant Toxin from L. quinquestriatus hebraeus. Thus, B. arenicola IT2 represents an interesting tool to study the receptor site for depressant Toxins on Insect sodium channels.
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Purification, structure and activity of three Insect Toxins from Buthus occitanus tunetanus venom
Toxicon : official journal of the International Society on Toxinology, 1997Co-Authors: Lamia Borchani, Herve Rochat, Pascal Mansuelle, Sandrine Cestele, Charles Kopeyan, Marcel Pelhate, Habib Karoui, Maria Stankiewicz, Riadh Kharrat, Mohamed El AyebAbstract:One contractive and two depressant Toxins active on Insect were purified by high-performance liquid chromatography from the venom of Buthus occitanus tunetanus (Bot). The two depressant Toxins, BotIT4 and BotIT5, differ only at position 6 (Arg for Lys) and are equally toxic to Insects (ld50 to Blatella germanica = 110ng/100mg body weight). They show a strong antigenic cross-reaction with a depressive Toxin from Leiurus quinquestriatus quinquestriatus (LqqIT2). The two Toxins are able to inhibit with high affinity (K0.5 between 2 and 3 nM) the specific binding of the radioiodinated excitatory Insect Toxin (125I-AaHIT) on its receptor site on Periplaneta americana synaptosomal membranes. These Toxins depolarize the cockroach axon, irreversibly block the action potential, and slow down and very progressively block the transmembrane transient Na+ current. The contracturant Toxin BotIT1 is highly toxic to B. germanica (ld50 = 60 ng/ 100 mg body weight) and barely toxic to mice (ld50 = 1 μg/20 g body weight) when injected intracerebroventricularly. It does not compete with 125I-AaHIT for its receptor site on P. americana synaptosomal membranes. On cockroach axon, BotIT1 develops plateau potentials and slows down the inactivation mechanism of the Na+ channels. Thus, BotIT1 belongs to the group of a Insect-selective Toxins and shows a strong sequence identity (> 90%) with LqhαIT and LqqIII, two Insect α-Toxins previously purified from the venom of L. q. hebraeus and L. q. quinquestriatus, respectively.
Anil K Lala - One of the best experts on this subject based on the ideXlab platform.
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purification and characterization of a short Insect Toxin from the venom of the scorpion buthus tamulus
FEBS Letters, 2002Co-Authors: Ritu Dhawan Dhawan, Anurag Sethi, Suresh K Joseph, Anil K LalaAbstract:A short chain peptide has been isolated from the venom of a red scorpion of Indian origin, Buthus tamulus. This peptide was purified using ion exchange and reverse phase chromatography and was characterized by molecular weight determination and amino acid sequence. The primary structure analysis shows that BtITx3 is a short peptide of 35 amino acid residues having a molecular weight of 3796 Da. The Toxin shows toxicity towards the Lepidopteran species of Insect Helicoverpa armigera causing flaccid paralysis and even death within 24 h. It shows more than 50% homology with the short InsectoToxins having four disulfide bridges, which suggests that the Toxin belongs to the class of short chain Toxins blocking the chloride ion channels. This sequence homology study has also helped to bring out the structure–function relationship between the various short Toxins. Homology modeling done by using template structure of a known Toxin indicated that this Toxin consists of a similar α/β scaffold, as present in other scorpion Toxins.
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Purification and characterization of a short Insect Toxin from the venom of the scorpion Buthus tamulus.
FEBS letters, 2002Co-Authors: Ritu Dhawan Dhawan, Suresh Joseph, Anurag Sethi, Anil K LalaAbstract:A short chain peptide has been isolated from the venom of a red scorpion of Indian origin, Buthus tamulus. This peptide was purified using ion exchange and reverse phase chromatography and was characterized by molecular weight determination and amino acid sequence. The primary structure analysis shows that BtITx3 is a short peptide of 35 amino acid residues having a molecular weight of 3796 Da. The Toxin shows toxicity towards the Lepidopteran species of Insect Helicoverpa armigera causing flaccid paralysis and even death within 24 h. It shows more than 50% homology with the short InsectoToxins having four disulfide bridges, which suggests that the Toxin belongs to the class of short chain Toxins blocking the chloride ion channels. This sequence homology study has also helped to bring out the structure-function relationship between the various short Toxins. Homology modeling done by using template structure of a known Toxin indicated that this Toxin consists of a similar alpha/beta scaffold, as present in other scorpion Toxins.
Marcel Pelhate - One of the best experts on this subject based on the ideXlab platform.
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exploration of the functional site of a scorpion alpha like Toxin by site directed mutagenesis
Biochemistry, 2003Co-Authors: Chunguang Wang, Marcel Pelhate, Nicolas Gilles, Alain Hamon, Frederic Le Gall, M Stankiewicz, Yumei Xiong, Dacheng Wang, Chengwu ChiAbstract:Scorpion alpha-neuroToxins can be classified into distinct subgroups according to their sequence and pharmacological properties. Using toxicity tests, binding studies, and electrophysiological recordings, BmK M1, a Toxin from the Asian scorpion Buthus martensi Karsch, was experimentally identified as an alpha-like Toxin. Being the first alpha-like Toxin available in a recombinant form, BmK M1 was then modified by site-directed mutagenesis for investigation of the molecular basis of its activity. The results suggested a functional site which protrudes from the molecular scaffold as a unique tertiary arrangement, constituted by the five-residue reverse turn 8-12 and the C-terminal segment. The C-terminal basic residues Lys62 and His64 together with Lys8 in the turn, which are critical for the bioactivities, may directly interact with the receptor site on the sodium channel. Residues Asn11 and Arg58, indispensable for the activities, are mainly responsible for stabilizing the distinct conformation of the putative bioactive site. Among others, His10 and His64 seem to be involved in the preference of BmK M1 for phylogenetically distinct target sites. The comparison of BmK M1 with Aah2 (classical alpha-Toxin) and LqhalphaIT (alpha-Insect Toxin) showed that the specific orientation of the C-terminus mediated by the reverse turn might be relevant to the preference of alpha-Toxin subgroups for phylogenetically distinct yet closely related receptor sites. The Y5G mutation indicated the "conserved hydrophobic surface" might be structurally important for stabilizing the beta-sheet in the alpha/beta-scaffold. The observations in this work shed light on the nature and roles of the residues possibly involved in the biological activity of a scorpion alpha-like Toxin.
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botit6 a potent depressant Insect Toxin from buthus occitanus tunetanus venom
Toxicon, 2003Co-Authors: Thouraya Mejri, Sandrine Cestele, Marcel Pelhate, Lamia Borchani, Rym Benkhalifa, Imed Regaya, Habib Karoui, Najet Srairiabid, H RochatAbstract:Abstract A new depressant Insect Toxin Buthus occitanus tunetanus Insect-Toxin 6 (BotIT6) was purified by high-performance liquid chromatography from Buthus occitanus tunetanus (Bot) venom. BotIT6 is very active against Blatella germanica (LD50=10 ng/100 mg body mass) thus being one of the most potent anti-Insect Toxin so far characterised. When compared to other Insect Toxin sequences, BotIT6 present high similarities with depressant Insect Toxins with an additional arginine residue at the C-terminus and a methionine at position 27. The calculated net charge of BotIT6 is positive (+3) whereas it is negative for classical depressant Toxins: this might be associated with its high toxicity. Voltage current clump studies show that BotIT6 is not a very potent depressant Insect Toxin despite its high toxicity in vivo. BotIT6 is able to fully inhibit the specific binding of 125 I AaHIT and 125 I-BotIT2 on Periplaneta americana synaptosomal membrane vesicles with high affinities. Despite its higher toxicity BotIT6 is a weaker competitor with 125 I AaHIT and 125 I BotIT2 as compared to the other β Toxins. Altogether, these results may suggest that BotIT6 probably defines a novel sub-group of depressant anti-Insect Toxins for which the receptor site can be overlapping, but not identical to that for classical depressant Insect Toxins.
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BotIT6: a potent depressant Insect Toxin from Buthus occitanus tunetanus venom.
Toxicon : official journal of the International Society on Toxinology, 2003Co-Authors: Thouraya Mejri, Herve Rochat, Sandrine Cestele, Marcel Pelhate, Lamia Borchani, Najet Srairi-abid, Rym Benkhalifa, Imed Regaya, Habib Karoui, Mohamed El AyebAbstract:A new depressant Insect Toxin Buthus occitanus tunetanus Insect-Toxin 6 (BotIT6) was purified by high-performance liquid chromatography from Buthus occitanus tunetanus (Bot) venom. BotIT6 is very active against Blatella germanica (LD50=10ng/100mg body mass) thus being one of the most potent anti-Insect Toxin so far characterised. When compared to other Insect Toxin sequences, BotIT6 present high similarities with depressant Insect Toxins with an additional arginine residue at the C-terminus and a methionine at position 27. The calculated net charge of BotIT6 is positive (+3) whereas it is negative for classical depressant Toxins: this might be associated with its high toxicity. Voltage current clump studies show that BotIT6 is not a very potent depressant Insect Toxin despite its high toxicity in vivo. BotIT6 is able to fully inhibit the specific binding of 125I AaHIT and 125I-BotIT2 on Periplaneta americana synaptosomal membrane vesicles with high affinities. Despite its higher toxicity BotIT6 is a weaker competitor with 125I AaHIT and 125I BotIT2 as compared to the other beta Toxins.Altogether, these results may suggest that BotIT6 probably defines a novel sub-group of depressant anti-Insect Toxins for which the receptor site can be overlapping, but not identical to that for classical depressant Insect Toxins.
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Purification, structure and activity of three Insect Toxins from Buthus occitanus tunetanus venom.
Toxicon, 1997Co-Authors: Lamia Borchani, Pascal Mansuelle, Sandrine Cestele, Charles Kopeyan, Marcel Pelhate, M Stankiewicz, Habib Karoui, H Rochat, Riadh Kharrat, Mohamed El AyebAbstract:One contractive and two depressant Toxins active on Insect were purified by high-performance liquid chromatography from the venom of Buthus occitanus tunetanus (Bot). The two depressant Toxins, BotIT4 and BotIT5, differ only at position 6 (Arg for Lys) and are equally toxic to Insects (LD50 to Blatella germanica = 110 ng/100 mg body weight). They show a strong antigenic cross-reaction with a depressive Toxin from Leiurus quinquestriatus quinquestriatus (LqqIT2). The two Toxins are able to inhibit with high affinity (K0.5 between 2 and 3 nM) the specific binding of the radioiodinated excitatory Insect Toxin (125I-AaHIT) on its receptor site on Periplaneta americana synaptosomal membranes. These Toxins depolarize the cockroach axon, irreversibly block the action potential, and slow down and very progressively block the transmembrane transient Na+ current. The contracturant Toxin BotIT1 is highly toxic to B. germanica (LD50 = 60 ng/ 100 mg body weight) and barely toxic to mice (LD50 = 1 microgram/20 g body weight) when injected intracerebroventricularly. It does not compete with 125I-AaHIT for its receptor site on P. americana synaptosomal membranes. On cockroach axon, BotIT1 develops plateau potentials and slows down the inactivation mechanism of the Na+ channels. Thus, BotIT1 belongs to the group of alpha Insect-selective Toxins and shows a strong sequence identity (\textgreater 90%) with Lqh alpha IT and LqqIII, two Insect alpha-Toxins previously purified from the venom of L. q. hebraeus and L. q. quinquestriatus. respectively.
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Refined electrophysiological analysis suggests that a depressant Toxin is a sodium channel opener rather than a blocker.
Life sciences, 1997Co-Authors: Rym Benkhalifa, Noam Zilberberg, Michael Gurevitz, Bruno Lapied, Maria Stankiewicz, Michael Turkov, Marcel PelhateAbstract:The effects of a recombinant depressant Insect Toxin from Leiurus quinquestriatus hebraeus, Lqh IT2-r, have been studied in current and voltage-clamp conditions on the isolated axonal and DUM neuron preparations of the cockroach Periplaneta americana. Lqh IT2-r depolarizes the axon, blocks the evoked action potentials, and modifies the amplitude and the kinetics of the sodium current. The inward transient peak current is greatly decreased and is followed by a maintained slow activating-deactivating sodium current. The slow component develops at membrane potentials more negative than the control, and has a time constant of activation of several tens of milliseconds. The flaccid properties of Lqh IT2-r do not correspond to a blockage of the Na+ channels, but may be attributed to modified Na+ channels which open at more negative potential, activate slowly and do not inactivate normally.
Aihua Liang - One of the best experts on this subject based on the ideXlab platform.
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AcMNPV-mediated expression of BmK IT promotes the apoptosis of Sf9 cells and replication of AcMNPV.
Sheng li xue bao : [Acta physiologica Sinica], 2015Co-Authors: Jie Zhao, Aihua LiangAbstract:Chinese scorpion Buthus martensii Karsch (BmK) venom is a rich source of neuroToxins which bind to various ion channels with high affinity and specificity and thus widely used as compounds to modulate channel gating or channel currents. To promote the Insecticidal effects of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), the gene encoding an excitatory Insect Toxin, BmK IT, was inserted into the genome of AcMNPV to construct a recombinant baculovirus, AcMNPV-BmK IT. Spodopter frugiperda 9 (Sf9) cells were infected with AcMNPV and AcMNPV-BmK IT respectively for 24 h. Results from the MTT assay, TUNEL assay, analysis of the expression level of apoptosis-related proteins (c-Myc, cleaved-Caspase3, Bcl-2 and Bax) of Sf9 cells, the transcription level of key genes (38K, C42, P78, F) of AcMNPV, and viral propagation assay demonstrated that AcMNPV-mediated expression of BmK IT promoted the apoptosis of Sf9 cells and replication of AcMNPV. The results laid a foundation for further structural and functional analysis of BmK IT.
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Effects of recombinant baculovirus AcMNPV-BmK IT on the formation of early cables and nuclear polymerization of actin in Sf9 cells
Cytotechnology, 2015Co-Authors: Taotao Lin, Aihua LiangAbstract:Autographa californica nuclearpoly hedrosis virus (AcMNPV) is one of the most important baculoviridae. However, the application of AcMNPV as a biocontrol agent has been limited. Previously, we engineered Buthus martensii Karsch Insect Toxin (BmK IT) gene into the genome of AcMNPV. The bioassay data indicated that the recombinant baculovirus AcMNPV-BmK IT significantly enhanced the anti-Insect efficacy of the virus. The actin cytoskeleton is the major component beneath the surface of eukaryotic cells. In this report, the effects of AcMNPV-BmK IT on the formation of early cables of actin and nuclear filamentous-actin (F-actin) were studied. The results indicated that these baculovirus induced rearrangement of the actin cytoskeleton of host cells during infection and actin might participate in the transportation of baculovirus from cytoplasm to the nuclei. AcMNPV-BmK IT delayed the formation of early cables of actin and nuclear F-actin and accelerated the clearance of actin in the nuclei.
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Functional study of active residues scorpion Insect Toxin BmK IT from Buthus martensii Karsch
Biotechnology and Bioprocess Engineering, 2014Co-Authors: Renjia Yang, Wujian Chen, Aihua LiangAbstract:Chinese scorpion Buthus martensii Karsch (BmK) venom is a rich source of neuroToxins which bind to various ion channels with high affinity and specificity and thus widely used as compounds to modulate channel gating. An excitatory Insect Toxin, BmK IT, is not conserved with a glutamate residue at the preceding position of the third Cys residue, and is a Toxin with a non-glutamate residue at the relevant position in the excitatory scorpion β-Toxin subfamily. In this study, the mutants of recombinant BmK IT (BmK IT (I25E), BmK IT (E15G), BmK IT C-terminal (TKSYCDVQIN) truncated) were achieved by site-directed mutagenesis. Biological activity of BmK IT and its mutants confirmed these residues or peptides played key roles in BmK IT. BmK IT (I25E) could increase the sensitivity of BmK IT, but BmK IT(E15G) could decrease the sensitivity of BmK IT on Sf9 cells. BmK IT truncated C-terminal hydrophobic amino acids could cross the species boundaries and was effective on mammalian C6 cells. To date, several excitatory Insect Toxins have been isolated and identified from the venom of Buthus martensii Karsch. However, no functional data are available and therefore its classification in the family of excitatory Insect Toxins remains putative and is just based on its high similarity with the other Toxins of this family. These results verified I25, E15 and C-terminal (TKSYCDVQIN) in BmK IT played key roles in the interaction of the BmK IT and its receptor- sodium channels on the surface of Insect cells and laid a foundation for further structural and functional analysis of BmK IT.
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Effect of location of the His-tag on the production of soluble and functional Buthus martensii Karsch Insect Toxin.
Protein expression and purification, 2008Co-Authors: Xiao-jun Fan, Aihua LiangAbstract:The low yield and poor folding efficiency in vivo of soluble and active recombinant cysteine-rich proteins expressed in Escherichia coli are a major challenge for large-scale protein production and purification. Expression vectors containing Buthus martensii Karsch Insect Toxin (BmK IT) fused to the C terminus of the intein Ssp DnaB were constructed in an attempt to overcome this problem. Following purification and intein self-cleavage, the fusion protein His(6)-intein-IT produced insoluble BmK IT, while intein-IT-His(6) generated soluble and properly folded BmK IT. This result indicated that the positioning of the His(6) tag has a key role in the production of soluble and functional BmK IT.
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Transgenic cotton expressing synthesized scorpion Insect Toxin AaHIT gene confers enhanced resistance to cotton bollworm (Heliothis armigera) larvae.
Biotechnology letters, 2007Co-Authors: Xiaoli Luo, Aihua Liang, Zhian Wang, Yingchuan Tian, Yi SunAbstract:A synthetic scorpion Hector Insect Toxin (AaHIT) gene, under the control of a CaMV35S promoter, was cloned into cotton via Agrobacterium tumefaciens-mediated transformation. Southern blot analyses indicated that integration of the transgene varied from one to more than three estimated copies per genome; seven homozygous transgenic lines with one copy of the T-DNA insert were then selected by PCR and Southern blot analysis. AaHIT expression was from 0.02 to 0.43% of total soluble protein determined by western blot. These homozygous transgenic lines killed larvae of cotton bollworm (Heliothis armigera) by 44-98%. The AaHIT gene could used therefore an alternative to Bt Toxin and proteinase inhibitor genes for producing transgenic cotton crops with effective control of bollworm.