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The membrane disruption mechanism of pandinin 1 (pin1), an antimicrobial peptide isolated from the venom of the African scorpion, was studied using 31P, 13C, 1H solid-state and multidimensional solution-state NMR spectroscopy. A high-resolution NMR solution structure of pin1 showed that the two distinct α-helical regions move around the central hinge region, which contains Pro19. 31P NMR spectra of lipid membrane in the presence of pin1, at various temperatures, showed that pin1 induces various lipid phase behaviors depending on the acyl chain length and charge of phospholipids. Notably, it was found that pin1 induced formation of the cubic phase in shorter lipid membranes above Tm. Further, the 13C NMR spectra of pin1 labeled at Leu28 under magic angle spinning (MAS) indicated that the motion of pin1 bound to the lipid bilayer was very slow, with a correlation time of the order of 10−3s. 31P NMR spectra of dispersions of four saturated phosphatidyl-cholines in the presence of three types of pin1 derivatives, [W4A, W6A, W15A]-pin1, pin1(1-18), and pin1(20-44), at various temperatures demonstrated that all three pin1 derivatives have a reduced ability to trigger the cubic phase. 13C chemical shift values for pin1(1-18) labeled at Val3, Ala10, or Ala11 under static or slow MAS conditions indicate that pin1(1-18) rapidly rotates around the average helical axis, and the helical rods are inclined at ∼30° to the lipid long axis. 13C chemical shift values for pin1(20-44) labeled at Gly25, Leu28, or Ala31 under static conditions indicate that pin1(20-44) may be isotropically tumbling. 1H MAS chemical shift measurements suggest that pin1 is located at the membrane-water interface approximately parallel to the bilayer surface. Solid-state NMR results correlated well with the observed biological activity of pin1 in red blood cells and bacteria.

The Om-toxins are short peptides (23–27 amino acids) purified from the venom of the scorpion Opisthacanthus madagascariensis. Their pharmacological targets are thought to be potassium channels. Like Csα/β (cystine-stabilized α/β) toxins, the Om-toxins alter the electrophysiological properties of these channels; however, they do not share any sequence similarity with other scorpion toxins. We herein demonstrate by electrophysiological experiments that Om-toxins decrease the amplitude of the K+ current of the rat channels Kv1.1 and Kv1.2, as well as human Kv1.3. We also determine the solution structure of three of the toxins by use of two-dimensional proton NMR techniques followed by distance geometry and molecular dynamics. The structures of these three peptides display an uncommon fold for ion-channel blockers, Csα/α (cystine-stabilized α-helix–loop–helix), i.e. two α-helices connected by a loop and stabilized by two disulphide bridges. We compare the structures obtained and the dipole moments resulting from the electrostatic anisotropy of these peptides with those of the only other toxin known to share the same fold, namely κ-hefutoxin1.

A new class of compounds, acylpolyamine has been isolated from spider venom constituents. Recent advances in highly sensitive mass spectrometric techniques have been applied successfully to characterize these acylpolyamines even with the use of a single venom gland. This has been achieved, in part, by improvements in fast atom bombardment mass spectrometry (FAB), continuous flow (FRIT) FAB-MS combined with reversed-phase high performance liquid chromatography (HPLC), matrix assisted laser desorptionionization (MALDI) and high energy collision induced dissociation (CID) tandem MSMS. Crude venom analysis without chromatographic separation can be realized directly by MALDI-MS. A charge-remote fragmentation method has provided abundant structure-related product ions and have reduced the quantity of required venom for the structure analysis of acylpolyamines. These mass spectrometric methods were proved to be useful for the analysis of complex constituents of spiders and other arthropod venom glands.

The orientation and pore-forming mechanisms of pandinin 2 (pin2), an antimicrobial peptide isolated from venom of the African scorpion Pandinus imperator, bound to magnetically oriented lipid bilayers were examined by 31P and 13C solid-state, and 15N liquid-state NMR spectroscopy. 31P NMR measurements at various temperatures, under neutral and acidic conditions, showed that membrane lysis occurred only under acidic conditions, and at temperatures below the liquid crystal-gel phase transition of the lipid bilayers, after incubation for two days in the magnet. Differential scanning calorimetry measurements showed that pin2 induced negative curvature strain in lipid bilayers. The 13C chemical shift values of synthetic pin2 labeled at Gly3, Gly8, Leu12, Phe17, or Ser18 under static or slow magic-angle spinning conditions, indicate that pin2 penetrates the membrane with its average helical axis perpendicular to the membrane surface. Furthermore, amide H-D exchange experiments of 15N-Ala4, Gly8, and Ala9 triply-labeled pin2 suggest that this peptide forms oligomers and confirms that the N-terminal region creates membrane pores.

In order to elucidate the structure activity relationships of the spider toxin termed NPTX-594, eleven toxin analogs were designed and synthesized, and their paralytic activities against cricket were tested. As a result of the present study, it was clarified that the Lys residue binding to the 1-amino group of 4,8-diaza-1,12-dodecanediamine (Dada) in the molecule of NPTX-594 is not an essential requisite for toxicity, and can be replaced with neutral or basic amino acids without any considerable loss of the activity. However, the replacement of the Lys residue with acidic amino acid residues, such as Asp or Glu, resulted in an extreme loss of the biological activity.

Many kinds of venomous principles modulate physiological responses of mammalian signal transduction systems, on which they act selectively as enhancers, inhibitors or some other kind of effectors. These toxins have become useful tools for physiological research. We have characterized paralyzing toxins from the venom of spiders, scorpions, insects, jellyfishes and sea anemones in the subtropical region including the Ryukyu Islands. Venom profiles are screened by MALDITOF fingerprinting analysis prior to purification of the venomous components, then marked target toxins of small molecular mass 1000–5000 are characterized directly by means of mass spectrometric techniques such as FritFAB MSMS, PSDCIDTOF MS, Capil. HPLCQTOF MSMS etc. The proteinous toxins of jellyfish or sea anemone are characterized by RTPCR technique by the information of the cleaved peptides after the protein was hydrolyzed by appropriate peptidase and the sequence of the cleaved peptide was determined by conventional methods. The venom of Araneid spider is mainly composed of a mixture of closely related acylpolyamines. More than 90 polyamine toxins were identified from one venom sac of the Madagascan spider, Nephilengys borbonica, by FritFab MSMS employing charge remote fragmentation technique. A novel polyamine toxin was also found from the rare wondering spider, Macrothele gigasfrom Iriomote Island. The structure of the toxin is an analog of polyamine toxin found in trapdoor spiders. Many kinds of cystinerich peptides showing various types of ion channel antagonism have been isolated from spiders. A series of toxins possessing the same mode of cystine knots was recently isolated from the saliva of assassin bugs, Peirates turpis, Isyndus obscurus, Agriophodrus dohrni. These toxins act as calcium channel blocker. Most of the scorpion toxins reported are from scorpions hazardous to humans, and they belong to the major super family Buthoidea. Among them are the wellknown genera, such as Buthus, Androctonus, Centruroides, Leiurus, or Tytius. We have investigated the minor group of scorpions from the super family Chactoidea Scorpionidae, Ishnuridae. The venoms of these scorpions, involving the genera Heterometrus, Pandinus, Opisthacanthus, and Isometrus, contain different kinds of peptide toxins. Fingerprinting peptide analysis of the toxin profiles for these scorpions showed some difference from the profiles of Buthoidea scorpions. These venoms contain linear poreforming peptides and 2cystinebridged toxins in addition to 4cystinebridged toxins. The most hazardous jellyfish in Okinawa, Chiropsalmus quadrigatus, and the related box jellyfishes, Carybdea rastoni, C. alata, contain quite labile proteinaceous toxins, CqTX, CrTX and CaTX, respectively. The toxins were inactivated by adding an organic solvent such as methanol or acetonitrile, by changing the pH of the toxin solution, dialyzing the toxin solution, storing the toxin in a refrigerator, or by lyophilizing the toxin solution. However, the toxic activity was retained in the presence of sodium chloride. We purified the jellyfish toxins by adding sodium chloride through all steps of the purification procedure and finally obtained the whole primary amino acid sequence of the toxin by RTPCR method. The toxic protein CqTX is homologous to the other box jelly fish toxin, CrTX and CaTX. These toxins belong to a new class of proteins since they show no homology to known proteins. Another notorious and dangerous specimen in the Ryukyu Islands is Phyllodiscus semori. The venom is composed of three kinds of proteins PsTX20A, PsTX60A, PsTX60B. PsTX20A shows homology to the proteinaceous toxin actinoporin, a cytolytic protein isolated from the genus Actinia, but PsTX60s has no homology to any ever cloned proteins. Further elucidation of the mechanism of toxic action of these Coelenterates is in progress.

A Caenorhabditis eleganscDNA encoding a new insulin/IGF-like peptide was cloned and examined. The predicted peptide shows significant sequence similarity with the peptide Ceinsulin-1 reported previously and contains a characteristic insertion consisting of three residues in the putative B domain as with the Ceinsulin-1. The gene expression pattern during development is almost identical to that of Ceinsulin-1. The predicted tertiary structure of the peptide is quite similar to that of Ceinsulin-1, and their predicted receptor-recognition surfaces also closely match. These facts suggest that both peptides could recognize the same receptor.

Five amphipathic peptides with antimicrobial, hemolytic, and insecticidal activity were isolated from the crude venom of the wolf spider Oxyopes kitabensis. The peptides, named oxyopinins, are the largest linear cationic amphipathic peptides from the venom of a spider that have been chemically characterized at present. According to their primary structure Oxyopinin 1 is composed of 48 amino acid residues showing extended sequence similarity to the ant insecticidal peptide ponericinL2 and to the frog antimicrobial peptide dermaseptin. Oxyopinins 2a, 2b, 2c, and 2d have highly similar sequences. At least 27 out of 37 amino acid residues are conserved. They also show a segment of sequence similar to ponericinL2. Circular dichroism analyses showed that the secondary structure of the five peptides is essentially α-helical. Oxyopinins showed disrupting activities toward both biological membranes and artificial vesicles, particularly to those rich in phosphatidylcholine. Electrophysiological recordings performed on insect cells (Sf9) showed that the oxyopinins produce a drastic reduction of cell membrane resistance by opening non-selective ion channels. Additionally, a new paralytic neurotoxin named Oxytoxin 1 was purified from the same spider venom. It contains 69 amino acid residue cross-linked by five disulfide bridges. Application of mixtures containing oxyopinins and Oxytoxin 1 to insect larvae showed a potentiation phenomenon, by which an increase lethality effect is observed. These results suggest that the linear amphipathic peptides in spider venoms and neuropeptides cooperate to capture insects efficiently.

A method incorporating nested collision‐induced dissociation/post‐source decay (CID/PSD) combined with endopeptidase digestion is described as an approach to determine the sequence of N‐terminally modified peptides. The information from immonium and related ions observed in the CID/PSD spectrum was used for the selection of a suitable endopeptidase for the digestion of peptides. Rapid and reliable assignment of peptide sequence was performed by the comparison of CID/PSD spectra of both intact and endopeptidese‐digested peptide fragments, since the assignments of the observed fragment ions to either N‐ or C‐terminal ions can thus be carried out unambiguously. This nested CID/PSD method was applied to the sequence determination of two peptides from the solitary wasps Anoplius samariensisand Batozonellus maculifrons(pompilid wasps), which could not be sequenced by the Edman method due to N‐terminal modification. Copyright © 2002 John Wiley & Sons, Ltd.

Animal venoms are important sources of novel pharmacological tools, useful in biochemical characterization of their receptors. Venom quality control, batch‐to‐batch homogeneity and high reproducibility of venom fractionation and toxin purification are crucial issues for biochemical and pharmacological studies. To address these issues, a study of the variability of tarantula spider venom samples was undertaken. Venom profiles of samples collected from individuals of different age and sex, and from sibling spiders of the same species, were generated by high‐performance liquid chromatography (HPLC) and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOFMS) and analyzed to assess venom variability and method accuracy. Sex‐linked venom variation was studied on eight species. Clear qualitative differences were observed for six out of eight species, as well as quantitative differences. Age‐related variation studied in Poecilotheria rufilatashowed essentially age‐related quantitative differences between adults of both sexes and immature juveniles. The venoms of nine siblings and three wild‐collected Pterinochilus murinuswere studied for individual variation, showing only very minor quantitative differences. On the same samples, the quality of MALDI‐TOFMS venom fingerprinting was demonstrated to be highly reproducible. Our results show that tarantula venom peptide fingerprinting is a highly reliable identification method, that pooled batches of venom from several animals can be used for venom purification, that venom composition does not appear to be qualitatively related to ontogenesis in the spiders studied, and that qualitative sex‐linked variation occurs across most species and may be important in activity studies. Copyright © 2002 John Wiley & Sons, Ltd.

The deadly box jellyfish (Sea Wasp, Habu-kurage in Japanese) Chiropsalmus quadrigatusHaeckel (Cubozoa) is distributed widely in the tropical Pacific region. In Japan, three fatal cases due to stings from this species have been reported officially. We successfully isolated C. quadrigatustoxin-A (CqTX-A, 44 kDa), a major proteinaceous toxin, for the first time, from the nematocysts of C. quadrigatus. CqTX-A showed lethal toxicity to crayfish when administered via intraperitoneal injection (LD50=80 μg/kg) and hemolytic activity toward 0.8% sheep red blood cells (ED50= 160 ng/ml). Furthermore, we sequenced the cDNA encoding CqTX-A. The deduced amino acid sequence of CqTX-A (462 amino acids) showed 25.2% and 21.6% sequence similarity to Carybdea rastonitoxins (CrTXs) and Carybdea alatatoxin-A (CrTX-A), respectively, which are Cubozoan jellyfish toxins.

The venomous sea anemone Phyllodiscus semonicauses cases of severe stinging. We isolated Phyllodiscus semonitoxin 20A (PsTX-20A), a hemolytic and lethal polypeptide (20 kDa), from the nematocyst venom of this species for the first time. Furthermore, we sequenced the cDNA encoding PsTX-20A. The deduced amino acid sequence of PsTX-20A showed that this toxin was a new member of the actinoporin family, which consists of several cytolytic polypeptides originating from sea anemones. PsTX-20A showed lethal toxicity to the shrimp Palaemon paucidenswhen administered viaintraperitoneal injection (LD50, 50 μg/kg) and hemolytic activity toward 0.8% sheep red blood cells (ED50, 80 ng/ml).

Two novel antimicrobial peptides have been identified and characterized from venom of the African scorpion Pandinus imperator. The peptides, designated pandinin 1 and 2, are α-helical polycationic peptides, with pandinin 1 belonging to the group of antibacterial peptides previously described from scorpions, frogs and insects, and pandinin 2 to the group of short magainin-type helical peptides from frogs. Both peptides demonstrated high antimicrobial activity against a range of Gram-positive bacteria (2.4–5.2μM), but were less active against Gram-negative bacteria (2.4–38.2μM), and only pandinin 2 affected the yeast Candida albicans. Pandinin 2 also demonstrated strong haemolytic activity (11.1–44.5μM) against sheep erythrocytes, in contrast with pandinin 1, which was not haemolytic. CD studies and a high-resolution structure of pandinin 2 determined by NMR, showed that the two peptides are both essentially helical, but differ in their overall structure. Pandinin 2 is composed of a single α-helix with a predominantly hydrophobic N-terminal sequence, whereas pandinin 1 consists of two distinct α-helices separated by a coil region of higher flexibility. This is the first report of magainin-type polycationic antimicrobial peptides in scorpion venom. Their presence brings new insights into the mode of action of scorpion venom and also opens new avenues for the discovery of novel antibiotic molecules from arthropod venoms.

The insecticidal neurotoxins AaIT, LqhIT2, and miew-agaIV from venom of Arachnida were rapidly re-purified from their respective crude venoms using mass spectrometry analyses and two high pressure chromatographic steps. The purity of each toxin was confirmed by capillary zone electrophoresis. Each toxin was identified by mass spectrometry and their biological activity confirmed by bioassays against tobacco cutworms and mice. In addition, AaIT and LqhIT2 were partially sequenced using post-source decay mass spectrometry analysis. The spider neurotoxin miew-agaIV was identified from other isomassic peptide by fingerprint mass analysis of its tryptic fragments.

Using a methodology based on HPLC fractionation, mass spectrometry analysis, and microinjection guide-bioassays, a peptide with toxicity to both insects and mammals was isolated from the venom of the scorpion Leiurus quinquestriatus hebraeus, and named LqhIV (after LqhII and LqhIII[1]). The primary sequence, circular dichroism spectrum and three-dimensional structure simulation of LqhIV indicate that it contains similar structure to that of the group of alpha scorpion toxins.

A novel spider toxin, NPTX-594, comprised of four constituents: i.e., 2,4-dihydroxyphenylacetic acid (Dhpa), asparagine, 4,8-diaza-1,12-dodecanediamine (Dada), and lysine, was chemically synthesized. The synthetic compound was completely identical with the natural product as regards 1H NMR and mass spectra. The structure of NPTX-594 was thus determined synthetically to be N12-(Dhpa-Asn)-N1-Lys-Dada, as proposed by spectrometric elucidation.

A novel cytotoxic linear peptide, IsCT, was characterized from scorpion Opisthacanthus madagascariensis. It is a linear peptide with a molecular weight of 1501.9 Da composed of 13 amino acid residues without cysteines. MS/MS analysis showed that its C-terminal is amidated. The identity of IsCT is re-confirmed by comparing the chemical synthesized peptide with the natural one. IsCT demonstrated antimicrobial activity against both gram-positive and gram-negative bacteria and hemolytic activity to sheep red blood cells. Also, it can release histamine from rat peritoneal mast cells. The CD absorption suggested that IsCT had an α-helix configuration in aqueous TFE. IsCT is one of the shortest natural cytotoxic peptides described, and it will be a suitable model for studying peptide-lipid interactions.

A new scorpion toxin (3751.8 Da) was isolated from the Buthus martensivenom, sequenced and chemically synthesized (sBmTX3). The A‐type current of striatum neurons in culture completely disappeared when 1 μM sBmTX3 was applied (Kd=54 nM), whereas the sustained K+current was unaffected. 125I‐sBmTX3 specifically bound to rat brain synaptosomes (maximum binding=14 fmol mg−1of protein, Kd=0.21 nM). A panel of toxins yet described as specific ligands for K+channels were unable to compete with 125I‐sBmTX3. A high density of 125I‐sBmTX3 binding sites was found in the striatum, hippocampus, superior colliculus, and cerebellum in the adult rat brain.

Three novel peptides were isolated from the venomous saliva of predatory reduviids. They were identified by mass spectrometry and HPLC analysis and consist of 34–36 amino acid residues. They are relatively homologous to the calcium channel blockers ω‐conotoxins from marine cone snails and belong to the four‐loop Cys scaffold structural class. Ptu1, the shortest peptide, was chemically synthesized (sPtu1) and co‐eluted with its native form. Circular dichroism spectra of the sPtu1 showed a high content of β‐turns similar to that of ω‐conotoxins GVIA and MVIIA. Electrophysiological experiments demonstrated that sPtu1 reversibly blocks the N‐type calcium channels expressed in BHK cells.

A new scorpion toxin (3751.8 Da) was isolated from the Buthus martensivenom, sequenced and chemically synthesized (sBmTX3). The A-type current of striatum neurons in culture completely disappeared when 1 μM sBmTX3 was applied ( Kd=54 nM), whereas the sustained K +current was unaffected. 125I-sBmTX3 specifically bound to rat brain synaptosomes (maximum binding=14 fmol mg −1of protein, Kd=0.21 nM). A panel of toxins yet described as specific ligands for K +channels were unable to compete with 125I-sBmTX3. A high density of 125I-sBmTX3 binding sites was found in the striatum, hippocampus, superior colliculus, and cerebellum in the adult rat brain.

Three novel peptides were isolated from the venomous saliva of predatory reduviids. They were identified by mass spectrometry and HPLC analysis and consist of 34–36 amino acid residues. They are relatively homologous to the calcium channel blockers ω-conotoxins from marine cone snails and belong to the four-loop Cys scaffold structural class. Ptu1, the shortest peptide, was chemically synthesized (sPtu1) and co-eluted with its native form. Circular dichroism spectra of the sPtu1 showed a high content of β-turns similar to that of ω-conotoxins GVIA and MVIIA. Electrophysiological experiments demonstrated that sPtu1 reversibly blocks the N-type calcium channels expressed in BHK cells.

The mechanism of Na+channel block by three macrocyclic polyamine derivatives and two spider toxins was studied with voltage clamp and internal perfusion method in squid axons.All these chemicals specifically block Na+channels in the open state only from the internal surface, and do not affect K+channels.The blocking effect is enhanced as the depolarizing pulse becomes larger. Blocked channels are unable to shift to the inactivated state.In the case of cyclam and guanidyl‐side armed cyclam (G‐cyclam), quick release of these chemicals from the binding sites is proven by the increase in the tail current and prolongation of the time course of the off gating current. On the other hand, in the presence of N‐4 and the spider toxins, their detachment was delayed significantly.Molecular requirements for the block of Na+channels by these molecules are the presence of positive charge and hydrophobicity.

1 The mechanism of Na+ channel block by three macrocyclic polyamine derivatives and two spider toxins was studied with voltage clamp and internal perfusion method in squid axons. 2 All these chemicals specifically block Na+ channels in the open state only from the internal surface, and do not affect K+ channels. 3 The blocking effect is enhanced as the depolarizing pulse becomes larger. Blocked channels are unable to shift to the inactivated state. 4 In the case of cyclam and guanidyl-side armed cyclam (G-cyclam), quick release of these chemicals from the binding sites is proven by the increase in the tail current and prolongation of the time course of the off gating current. On the other hand, in the presence of N-4 and the spider toxins, their detachment was delayed significantly. 5 Molecular requirements for the block of Na+ channels by these molecules are the presence of positive charge and hydrophobicity. British Journal of Pharmacology (2001) 132, 63 – 72

The two insecticidal peptides Bm32‐VI and Bm33‐I, isolated from the venom of the Chinese scorpion Buthus martensiinduce paralytical symptoms typical of insect contractive toxins. They show, respectively, 74% and 77% homology with AaIT from Androctonus australis, comparable insecticidal activity and no vertebrate toxicity. Under voltage‐clamp conditions, both toxins induced (1) an increased fast Na+current, (2) a shift in voltage dependence of Na+current activation, (3) the occurrence of a delayed current, and (4) a slow development of a holding current. Increased Na+conductance at negative potential values is responsible for axonal hyperexcitability and the contractive paralysis of insect prey.

We have examined the applicability of the ‘nested’ collision induced dissociation/post-source decay (CID/PSD) method to the sequencing of novel peptides from solitary wasps which have neurotoxic venom for paralyzing other insects. The CID/PSD spectrum of a ladder peptide derived from an exopeptidase digest was compared with that of the intact peptide. The mass peaks observed only in the CID/PSD spectrum of a ladder peptide were extracted as C-terminal fragment ions. Assignment of C-terminal fragment ions enabled calculation of N-terminal fragment masses, leading to differentiation between N-terminal fragment ions and internal fragment ions. This methodology allowed rapid and sensitive identification by removing ambiguity in the assignment of the fragment ions, and proved useful for sequencing unknown peptides, in particular those available as natural products with a limited supply. Copyright © 2000 John Wiley & Sons, Ltd.

We have examined the applicability of the ‘nested’ collision induced dissociation/post‐source decay (CID/PSD) method to the sequencing of novel peptides from solitary wasps which have neurotoxic venom for paralyzing other insects. The CID/PSD spectrum of a ladder peptide derived from an exopeptidase digest was compared with that of the intact peptide. The mass peaks observed only in the CID/PSD spectrum of a ladder peptide were extracted as C‐terminal fragment ions. Assignment of C‐terminal fragment ions enabled calculation of N‐terminal fragment masses, leading to differentiation between N‐terminal fragment ions and internal fragment ions. This methodology allowed rapid and sensitive identification by removing ambiguity in the assignment of the fragment ions, and proved useful for sequencing unknown peptides, in particular those available as natural products with a limited supply. Copyright © 2000 John Wiley & Sons, Ltd.

During summer and autumn, the box jellyfish (sea wasp) Carybdea rastoni is one of the most bothersome stinging pests to swimmers and bathers on the Japanese coast. Two labile but potent hemolytic toxins from the tentacles of Carybdea rastoni were isolated in their active forms using newly developed purification methods. The molecular masses of the isolated C. rastoni toxin-A and toxin-B (CrTX-A and CrTX-B) are 43 and 46 kDa, respectively, as calculated from SDS–PAGE. In the present study, we sequenced the full-length cDNA (1600 bp), which encodes both CrTX-A and CrTX-B. The deduced 450 amino acid sequence of the CrTXs, showed no significant homology with any known protein. This report presents the first complete sequence of a proteinaceous jellyfish toxin. Furthermore, it was revealed that CrTX-A was primarily localized in the nematocyst, whereas CrTX-B was detected only in the tentacle. Because the nematocyst is the organ responsible for the cnidarian sting, the remainder of the study focused on the toxicity of CrTX-A. We found that CrTX-A was fatally toxic to mice at 20 μg/kg (i.v.) and crayfish at 5 μg/kg (i.p.). Subcutaneously injected CrTX-A (0.1 μg) caused inflammation of mouse skin. These results showed that CrTX-A is responsible for the cutaneous inflammation observed in humans stung by C. rastoni.

The box jellyfish (sea wasp) Carybdea alata Reynaud, 1830 (Cubozoa) is distributed widely in the tropics. The sting of C. alata causes severe pain and cutaneous inflammation in humans. We successfully isolated C. alata toxin-A (CaTX-A, 43 kDa) and -B (CaTX-B, 45 kDa) for the first time from the tentacle of C. alata collected at a site along the Hawaiian shore. The experimental results showed that CaTX-A, but not CaTX-B, is present in the nematocyst, the organ responsible for stinging. Both CaTX-A and -B showed potent hemolytic activity, with CaTX-A being lethally toxic to crayfishes when administered via intraperitoneal injection. Furthermore, we sequenced the cDNA encoding CaTX-A. The deduced amino acid sequence of CaTX-A (463 amino acids) showed 43.7% homology to Carybdea rastoni toxins (CrTXs) but not with any other known proteins. Therefore, these jellyfish toxins potentially represent a novel class of bioactive proteins. Secondary structure analysis of CaTX-A and CrTXs suggested the presence of amphiphilic α-helices, which are also seen in several known hemolytic or cytolytic protein toxins, including peptide toxins.

Diapause, aging, and fat accumulation in Caenorhabditis elegans are regulated by DAF-2, a homolog of mammalian insulin/insulin-like growth factor-I (IGF-I) receptors. We have cloned and characterized a C. elegans gene encoding a new insulin/IGF-like peptide. The gene containing three exons encodes a precursor protein 95 residue long. Although the putative precursor contains a signal peptide, B chain, C peptide, and A chain like the preproinsulin, the mature peptide consists of one polypeptide-like IGF. The predicted tertiary structure seems similar to crystal structure of insulin. Therefore, the peptide may be a hybrid molecule of insulin and IGF. The peptide expression was detected at the embryonic and several larval stages. Disruption of the peptide production led to an extended life span like the daf-2 mutation, suggesting that the peptide should be one of the ligands of the DAF-2. This is the first description of the peptide that mediates animal longevity.

BmKTX is a toxin recently purified from the venom of Buthus Martensi, which belongs to the kaliotoxin family. We have determined its solution structure by use of conventional two-dimensional NMR techniques followed by distance-geometry and energy minimization. The calculated structure is composed of a short α-helix (residues 14 to 20) connected by a tight turn to a two-stranded antiparallel β-sheet (sequences 25–27 and 32–34). The β-turn connecting these strands belongs to type I. The N-terminal segment (sequence 1 to 8) runs parallel to the β-sheet although it cannot be considered as a third strand. Comparison of the conformation of BmKTX and toxins of the kaliotoxin familyclearly demonstrates that they are highly related. Therefore, analysis of the residues belonging to the interacting surface of those toxins allows us to propose a functional map of BmKTX slightly different from the one of KTX and AgTX2, which may explain the variations in affinities of these toxins towardsthe Kv1.3 channels. Proteins 2000;38:70–78. © 2000 Wiley-Liss, Inc.

BmKTX is a toxin recently purified from the venom of Buthus Martensi, which belongs to the kaliotoxin family. We have determined its solution structure by use of conventional two‐dimensional NMR techniques followed by distance‐geometry and energy minimization. The calculated structure is composed of a short α‐helix (residues 14 to 20) connected by a tight turn to a two‐stranded antiparallel β‐sheet (sequences 25–27 and 32–34). The β‐turn connecting these strands belongs to type I. The N‐terminal segment (sequence 1 to 8) runs parallel to the β‐sheet although it cannot be considered as a third strand. Comparison of the conformation of BmKTX and toxins of the kaliotoxin familyclearly demonstrates that they are highly related. Therefore, analysis of the residues belonging to the interacting surface of those toxins allows us to propose a functional map of BmKTX slightly different from the one of KTX and AgTX2, which may explain the variations in affinities of these toxins towardsthe Kv1.3 channels. Proteins 2000;38:70–78. © 2000 Wiley‐Liss, Inc.

BmKTX is a toxin recently purified from the venom of Buthus Martensi, which belongs to the kaliotoxin family. We have determined its solution structure by use of conventional two-dimensional NMR techniques followed by distance-geometry and energy minimization. The calculated structure is composed of a short α-helix (residues 14 to 20) connected by a tight turn to a two-stranded antiparallel β-sheet (sequences 25–27 and 32–34). The β-turn connecting these strands belongs to type I. The N-terminal segment (sequence 1 to 8) runs parallel to the β-sheet although it cannot be considered as a third strand. Comparison of the conformation of BmKTX and toxins of the kaliotoxin familyclearly demonstrates that they are highly related. Therefore, analysis of the residues belonging to the interacting surface of those toxins allows us to propose a functional map of BmKTX slightly different from the one of KTX and AgTX2, which may explain the variations in affinities of these toxins towardsthe Kv1.3 channels. Proteins 2000;38:70–78. © 2000 Wiley-Liss, Inc.

BmKTX is a toxin recently purified from the venom of Buthus Martensi, which belongs to the kaliotoxin family. We have determined its solution structure by use of conventional two-dimensional NMR techniques followed by distance-geometry and energy minimization. The calculated structure is composed of a short α-helix (residues 14 to 20) connected by a tight turn to a two-stranded antiparallel β-sheet (sequences 25–27 and 32–34). The β-turn connecting these strands belongs to type I. The N-terminal segment (sequence 1 to 8) runs parallel to the β-sheet although it cannot be considered as a third strand. Comparison of the conformation of BmKTX and toxins of the kaliotoxin familyclearly demonstrates that they are highly related. Therefore, analysis of the residues belonging to the interacting surface of those toxins allows us to propose a functional map of BmKTX slightly different from the one of KTX and AgTX2, which may explain the variations in affinities of these toxins towardsthe Kv1.3 channels. Proteins 2000;38:70–78. © 2000 Wiley-Liss, Inc.

The search for novel pharmacological tools in spider venoms involves the need for precise and reproducible species identification methods. As an addition to morphological analysis, we have developed venom fingerprinting by reversed‐phase chromatography and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOFMS) as an efficient and precise venom identification tool. In order to compare the possible use of liquid chromatography electrospray ionization mass spectrometry (LC/ESI‐MS) as an additional venom characterization tool, we have applied both methodologies to the study of several tarantula venom samples in the Pterinochilus murinusgroup. These species possess highly active venoms yet their taxonomy remains difficult. We demonstrate that both methodologies can be successfully applied to tarantula venom characterization. MALDI‐TOFMS and ESI‐MS gave similar overall profiles and allowed fine discrimination of samples. At least one venom sample was proven to belong to a completely different venom group. Coupling of ESI‐MS with HPLC separation afforded a new dimension in venom analysis, with clear discrimination between components of similar Mr and gave a finer picture of venom composition, number of molecular species and molecular weight distribution. Copyright © 1999 John Wiley & Sons, Ltd.

The search for novel pharmacological tools in spider venoms involves the need for precise and reproducible species identification methods. As an addition to morphological analysis, we have developed venom fingerprinting by reversed-phase chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) as an efficient and precise venom identification tool. In order to compare the possible use of liquid chromatography electrospray ionization mass spectrometry (LC/ESI-MS) as an additional venom characterization tool, we have applied both methodologies to the study of several tarantula venom samples in the Pterinochilus murinus group. These species possess highly active venoms yet their taxonomy remains difficult. We demonstrate that both methodologies can be successfully applied to tarantula venom characterization. MALDI-TOFMS and ESI-MS gave similar overall profiles and allowed fine discrimination of samples. At least one venom sample was proven to belong to a completely different venom group. Coupling of ESI-MS with HPLC separation afforded a new dimension in venom analysis, with clear discrimination between components of similar Mr and gave a finer picture of venom composition, number of molecular species and molecular weight distribution. Copyright © 1999 John Wiley & Sons, Ltd.

The conformational change upon binding with phospholipid membrane has been studied of mastoparan from wasp venom, a tetradecapeptide causing the degranulation of mast cells. The 270-MHz 1H-NMR spectra and CD spectra indicate that the mastoparan molecule takes the α-helical conformation in methanol solution, but a much less ordered form in aqueous solution. On binding with phospholipid membrane, the α-helical conformation is formed even in aqueous medium. Such a conformational change is primarily due to the interaction between the aliphatic side chains of mastoparan and the hydrophobic interior of phospholipid membrane, in contrast to the case of melittin from bee venom.

A new toxin, named HsTX1, has been identified in the venom of Heterometrus spinnifer (Scorpionidae), on the basis of its ability to block the rat Kv1.3 channels expressed in Xenopus oocytes. HsTX1 has been purified and characterized as a 34-residue peptide reticulated by four disulphide bridges. HsTX1 shares 53% and 59% sequence identity with Pandinus imperator toxin1 (Pi1) and maurotoxin, two recently isolated four-disulphide-bridged toxins, whereas it is only 32-47% identical with the other scorpion K+ channel toxins, reticulated by three disulphide bridges. The amidated and carboxylated forms of HsTX1 were synthesized chemically, and identity between the natural and the synthetic amidated peptides was proved by mass spectrometry, co-elution on C18 HPLC and blocking activity on the rat Kv1.3 channels. The disulphide bridge pattern was studied by (1) limited reduction-alkylation at acidic pH and (2) enzymic cleavage on an immobilized trypsin cartridge, both followed by mass and sequence analyses. Three of the disulphide bonds are connected as in the three-disulphide-bridged scorpion toxins, and the two extra half-cystine residues of HsTX1 are cross-linked, as in Pi1. These results, together with those of CD analysis, suggest that HsTX1 probably adopts the same general folding as all scorpion K+ channel toxins. HsTX1 is a potent inhibitor of the rat Kv1.3 channels (IC50 approx. 12 pM). HsTX1 does not compete with 125I-apamin for binding to its receptor site on rat brain synaptosomal membranes, but competes efficiently with 125I-kaliotoxin for binding to the voltage-gated K+ channels on the same preparation (IC50 approx. 1 pM).

We have applied a recently proposed liquid matrix‐assisted laser desorption/ionization (LMALDI) method on a 4‐sector tandem mass spectrometer equipped with an array detector at MS‐2. Although the ionizing laser beam is pulsed, the use of a liquid matrix can prolong the analyte ion current over 20 min without moving the laser irradiation spot and provide an ion current that is stable enough to acquire both mass spectra and collision‐induced dissociation (CID) spectra. Product ions, which originate from chemical background overlapping the precursor ions, were not observed in the MALDI‐CID spectra of acylpolyamines (below m/z1000) unlike the case with FAB spectra. These LMALDI‐CID spectra are shown to provide detailed structural information of equivalent or higher equality compared with spectra obtained by using FAB‐high energy CID conditions on a 4‐sector tandem mass spectrometer. The advantages of the LMALDI‐CID method compared with FAB‐CID is discussed, together with the structure determination of new glutaminergic nerve blocker acylpolyamines obtained from Nephila madagascariencisspider venom.© 1997 John Wiley & Sons, Ltd.

The increased interest in spider venom toxins has made precise identification of venom samples necessary for reproducibility of biochemical and pharmacological studies. In the family Theraphosidae (Tarantulas), identification of specimens may be difficult and a new approach involving multidimensional biochemical analysis of venoms is proposed. Combined HPLC, capillary electrophoresis and matrix‐assisted laser desorption/ionization fingerprinting of venom peptides permits accurate and reproducible identification of venom samples, and can be correlated with morphological observations. This rapid and very accurate methodology can be applied to minute amounts of samples obtained from live animals, on a large scale, with high reproducibility. Application of the methodology to five separate examples demonstrates confirmation of sample identity, separation of closely related species on the basis of venom profiles, and solution of classification problems not easily solved by morphological examination. Discussion of sample variability and the use of a dual matrix system is presented. © 1998 John Wiley & Sons, Ltd.

Conformationally restricted analogs of serine possessing a 5- or 6-membered carbocyclic ring, 1 and 2, were synthesized in an optically active form in short steps. The syntheses were started with rac-1, 1-dimethoxy-2-cyclopentanol and rac-trans-1,2-cyclohexanediol, respectively. The key to the present syntheses was a diastereoselective construction of a chiral amino nitrile group onto the ketone group of 5 or 11. This was achieved by the use of an internal Strecker reaction to give optically pure amino nitriles 7a and 13a, respectively. In this reaction, the original chirality derived from phenylalanyl group was efficiently transplanted into both the ketone and α-hydroxyl groups via an imine-enamine equilibrium of the ketimine intermediate 6 or 12. Phenylalanyl moiety of the Strecker adducts was removed by oxidative and hydrolytic treatments of the amino nitriles to give the titled amino acids. Chirality 9:459–462, 1997. © 1997 Wiley-Liss, Inc.

A facile synthesis of 6-hydroxyindole-3-acetic acid 1a, which is the proposed aromatic subunit of NPTX-1∼6, is described. Radical cyclization of isonitrile 2successfully afforded 9in high yield. The aromatic subunit of NPTX-1∼6 was confirmed as 4-hydroxyindole-3-acetic acid 12by comparison of the 1H-NMR spectra with those of authentic 4- and 6-hydroxyindole-3-acetic acids.

Recent study has shown that the venom of some orb-web spiders contain potent blockers of the glutamate receptors. Joro spider toxin (JSTX) derived from Nephila clavata has been found to block excitatory postsynaptic potentials and glutamate-evoked responses in the neuromuscular synapse of crustacea, the squid giant synapse and the mammalian brain synapse. Structures of the toxins (JSTXs, NSTXs) of spiders belonging to the genus Nephila were determined and it was found that a unique 2,4-dihydroxyphenylacetyl asparaginyl cadaverine part was conserved between all toxins, indicating that this part is intimately involved in the blocking activity.Labeling of synthesized JSTX-3 was used for histological investigation of glutamate receptors. Using autoradiography 125I-JSTX-3 was found to bind at the lobster neuromuscular synapse. A histochemical study utilizing the interaction of biotinylated JSTX-3 with avidin showed specific binding of the toxin in rat hippocampus and cerebellum. JSTX-3 was used for isolation of glutamate receptors from brain. A crude synaptic membrane fraction from rat hippocampus and cerebellum was solubilized by Triton X-100. SDS-PAGE of the affinity purified JSTX-3 binding proteins showed at least 4 bands around 70 K daltons.

The pharmacological mechanisms of platelet aggregation induced by highly toxic proteins (CrTX-I, CrTX-II, and CrTX-III) obtained from tentacles of a jellyfish, Carybdea rastonii, were investigated. When the partially purified toxin (pCrTX) and CrTXs were added to the citrated platelet-rich plasma (PRP), aggregation was produced in a concentration-dependent manner. The activity of CrTXs was approximately 100 times more potent than pCrTX. The CrTXs-induced aggregation was little affected by indomethacin and quinacrine at concentrations sufficient to inhibit arachidonic acid- and collagen-induced aggregation. The CrTXs-induced aggregation in washed platelets was significantly augmented in the presence of Ca2+. The pretreatment with verapamil failed to modify this augmentation of aggregation. The concentration of cytoplasmic-free calcium ([Ca2+]i) of platelets was increased by CrTXs at the same concentrations that produced aggregation. This effect of CrTXs was again little affected by verapamil. CrTXs at the same concentrations as those that produced aggregation and increased [Ca2+]icaused depolarization of platelets, which was unchanged after pretreatment with sodium or potassium transport inhibitors. CrTX-I significantly increased the 22Na flux into platelets and this effect of CrTX-I was unaffected by tetrodotoxin. The CrTX-I-induced aggregation, depolarization, and increase in [Ca2+]iwere all significantly attenuated in the low Na+medium. These results suggest that CrTXs cause a massive depolarization by increasing cation permeability and this generalized depolarization permits an inward movement of Ca2+down its electrochemical gradient which, in turn, triggers platelet aggregation.

We cloned a cDNA for the glutamate transporter of the nematodeCaenorhabditis elegans.The nucleotide sequence and Northern blotting indicated that the glutamate transporter gene is transcribed as a polycistronic mRNA inC. elegansand that subsequenttrans-splicing yields two distinct monocistronic mRNAs for the glutamate transporter and the ATP synthase c subunit, respectively. The yields of these monocistronic mRNAs were quite different, suggesting that glutamate transport inC. elegansis regulated in the post-transcriptional phase. The glutamate transporter ofC. elegansshows about 50–60% sequence similarity with those of mammals. This is the first description of invertebrate glutamate transporters.

The first synthesis of Nephilatoxin-8 (NPTX-8), a new neurotoxin of Joro spider (Nephila clavata), has been achieved by employing two key azide intermediates for the incorporation of the characteristic polyamines of the toxin, cadaverine and spermidine.

Two neutrophil chemotactic factors were isolated from the culture filtrates of Streptococcus sanguisATCC 10556 and were chemically characterized as N-terminal blocked peptides of low molecular weight. One of the factors consisted of proline, valine, methionine, isoleucine and leucine and the other of methionine, isoleucine, leucine and phenylalanine. In both factors, methionine was detected as the sole N-terminal amino acid, but the amino group was blocked. The removal of N-terminal methionine yielded several N-terminal amino acids, suggesting that S. sanguisproduced several N-terminal blocked methionyl peptides, all of which could be chemotactically active.

The first total synthesis of NPTX-5 and NPTX-6, Joro spider ( Nephila clavata) toxins having a 4-hydroxyindole nucleus and a unique putreanine trimer and tetramer acylpolyamine chain, respectively, has been achieved by the iterative use of a key azide compound.

Precise identification of arthropod species is fundamental in venom research, particularly in groups where taxonomy problems remain unsolved. High-performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis of crude venoms of six tarantula species in the genus Brachypelma showed that the characteristic chromatographic and peptide ion profiles obtained can be used to discriminate amongst closely related species. This method permits rapid mass fingerprinting of large numbers of samples in a reproducible manner, and offers a powerful systematic tool in combination with morphological methods for the classification of tarantula species. The sensitivity and precision of the method may offer a way to solve complex taxonomic relationships not easily resolved by morphological measurements, in a non-destructive manner. Additionally, peptide mapping of crude venoms by MALDI-TOFMS will speed up the discovery of novel ligands of neuronal receptors, since major venom components of related species share a high sequence homology and are likely to possess similar pharmacological properties. © 1997 John Wiley & Sons, Ltd.

We have applied a recently proposed liquid matrix-assisted laser desorption/ionization (LMALDI) method on a 4-sector tandem mass spectrometer equipped with an array detector at MS-2. Although the ionizing laser beam is pulsed, the use of a liquid matrix can prolong the analyte ion current over 20 min without moving the laser irradiation spot and provide an ion current that is stable enough to acquire both mass spectra and collision-induced dissociation (CID) spectra. Product ions, which originate from chemical background overlapping the precursor ions, were not observed in the MALDI-CID spectra of acylpolyamines (below m/z 1000) unlike the case with FAB spectra. These LMALDI-CID spectra are shown to provide detailed structural information of equivalent or higher equality compared with spectra obtained by using FAB-high energy CID conditions on a 4-sector tandem mass spectrometer. The advantages of the LMALDI-CID method compared with FAB-CID is discussed, together with the structure determination of new glutaminergic nerve blocker acylpolyamines obtained from Nephila madagascariencis spider venom.© 1997 John Wiley & Sons, Ltd.

Specificity of Shore’s method for the histamine determination was investigated by examining the fluorescence spectrum of the extract from rat gastric mucosa, biopsy specimen of human gastric mucosa, human gastric juice and human whole blood. An almost identical spectrum to that of pure histamine was obtained with the extract from rat gastric mucosa and human whole blood, whereas the spectrum with the extract of human gastric biopsy specimen and human gastric juice was different from that of histamine. By means of high-performance liquid chromatography, an approximately three times more concentrated extract from human gastric mucosa gave a distinct peak of histarnine while a hundred times concentrated gastric juice gave no measurable peak of histamine.