Novel mastoparan and protonectin analogs isolated from a solitary wasp, Orancistrocerus drewseni drewseni.

Two novel biologically active peptides, Eumenine mastoparan-OD and Orancis-Protonectin, were isolated from a solitary wasp, Orancistrocerus drewseni drewseni (Eumeninae, Vespidae). MALDI-TOF MS analysis of a small amount of the crude venom gave two intensive molecular-related ion peaks at m/z 1269.9 and 1552.

Biochemical characterization of cysteine-rich peptides from Oxyopes sp. venom that block calcium ion channels.

Oxytoxins (OxyTx1 and OxyTx2) are disulfide-rich peptides isolated from the venom of the spider Oxyopes lineatus that block voltage-sensitive calcium ion channels (VSCCs). OxyTx1 was identified previously and isolated from the related spider Oxyopes kitabensis; however, its pharmacology was unknown. OxyTx1 and OxyTx2 contain 69 and 55 amino acid residues with molecular masses of 8058.

Solution structure of agelenin, an insecticidal peptide isolated from the spider Agelena opulenta, and its structural similarities to insect-specific calcium channel inhibitors.

Agelenin, isolated from the Agelenidae spider Agelena opulenta, is a peptide composed of 35 amino acids. We determined the three-dimensional structure of agelenin using two-dimensional NMR spectroscopy. The structure is composed of a short antiparallel beta-sheet and four beta-turns, which are stabilized by three disulfide bonds.

Novel biologically active peptides from the venom of Polistes rothneyi iwatai.

Four novel peptides, polistes-mastoparan-R1, 2, 3, and polistes-protonectin, were isolated from the venom of a paper wasp, Polistes rothneyi iwatai. MALDI-TOF MS analysis of a small amount of the crude venom gave six molecular-related ion peaks. Among them, m/z 1565 was expected to be a novel peptide.

Nanoanalysis of the arthropod neuro-toxins.

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 become useful tools for physiological research. We have employed and characterized paralyzing toxins from the venom of spiders, insects and scorpions with a limited supply.

Eumenitin, a novel antimicrobial peptide from the venom of the solitary eumenine wasp Eumenes rubronotatus.

A novel antimicrobial peptide, eumenitin, was isolated from the venom of the solitary eumenine wasp Eumenes rubronotatus. The sequence of eumenitin, Leu-Asn-Leu-Lys-Gly-Ile-Phe-Lys-Lys-Val-Ala-Ser-Leu-Leu-Thr, was mostly analyzed by mass spectrometry together with Edman degradation, and corroborated by solid-phase synthesis. This peptide has characteristic features of cationic linear alpha-helical antimicrobial peptides, and therefore, can be predicted to adopt an amphipathic alpha-helix secondary structure.

Induction of morphological changes in model lipid membranes and the mechanism of membrane disruption by a large scorpion-derived pore-forming peptide.

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 alpha-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.

Molecular components and toxicity of the venom of the solitary wasp, Anoplius samariensis.

The solitary spider wasp, Anoplius samariensis, is known to exhibit a unique long-term, non-lethal paralysis in spiders that it uses as a food source for its larvae. However, neither detailed venom components nor paralytic compounds have ever been characterized. In this study, we examined the components in the low molecular weight fraction of the venom and the paralytic activity of the high molecular weight fraction.

A novel L-glutamate transporter inhibitor reveals endogenous D-aspartate homeostasis in rat pheochromocytoma MPT1 cells.

We previously reported for the first time that D-aspartate (D-Asp) is biosynthesized by cultured mammalian cells such as pheochromocytoma (PC)12 cells and its subclone MPT1 (FEBS Lett. 434 (1998) 231, Arch. Biochem.

A spider toxin that induces a typical effect of scorpion alpha-toxins but competes with beta-toxins on binding to insect sodium channels.

Delta-palutoxins from the spider Paracoelotes luctuosus (Araneae: Amaurobiidae) are 36-37 residue long peptides that show preference for insect sodium channels (NaChs) and modulate their function. Although they slow NaCh inactivation in a fashion similar to that of receptor site 3 modifiers, such as scorpion alpha-toxins, they actually bind with high affinity to the topologically distinct receptor site 4 of scorpion beta-toxins. To resolve this riddle, we scanned by Ala mutagenesis the surface of delta-PaluIT2, a delta-palutoxin variant with the highest affinity for insect NaChs, and compared it to the bioactive surface of a scorpion beta-toxin.

An unusual fold for potassium channel blockers: NMR structure of three toxins from the scorpion Opisthacanthus madagascariensis.

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 Csalpha/beta (cystine-stabilized alpha/beta) toxins, the Om-toxins alter the electrophysiological properties of these channels; however, they do not share any sequence similarity with other scorpion toxins.

Solution structure of IsTX. A male scorpion toxin from Opisthacanthus madagascariensis (Ischnuridae).

The novel sex-specific potassium channel inhibitor IsTX, a 41-residue peptide, was isolated from the venom of male Opisthacanthus madagascariensis. Two-dimensional NMR techniques revealed that the structure of IsTX contains a cysteine-stabilized alpha/beta-fold. IsTX is classified, based on its sequential and structural similarity, in the scorpion short toxin family alpha-KTx6.

Pore formation of phospholipid membranes by the action of two hemolytic arachnid peptides of different size.

Pin2 and Oxki1 are cationic amphipathic peptides that permeate lipid membranes through formation of pores. Their mechanism of binding to phosphocholine (PC) membranes differs. Spin-probe experiments showed that both Pin2 and Oxki1 penetrate the lipid membrane of small unilamellar vesicles (SUVs).

Orientation and pore-forming mechanism of a scorpion pore-forming peptide bound to magnetically oriented lipid bilayers.

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.

Synthesis of carbamate-type caged derivatives of a novel glutamate transporter blocker.

L-threo-beta-Benzyloxyaspartate (L-TBOA) and (2S,3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (L-TFB-TBOA) are potent nontransportable blockers for glutamate transporters. We synthesized a carbamate-type coumarin derivative of L-TBOA 3a as a caged blocker and compared 3a with the corresponding ester-type analogs 1. The carbamate 3a was less sensitive to photolysis than the ester 1 but was more stable in the aqueous solution.

Characterization of novel L-threo-beta-benzyloxyaspartate derivatives, potent blockers of the glutamate transporters.

Nontransportable blockers of the glutamate transporters are important tools for investigating mechanisms of synaptic transmission. DL-threo-beta-Benzyloxyaspartate (DL-TBOA) is a potent blocker of all subtypes of the excitatory amino acid transporters (EAATs). We characterized novel L-TBOA analogs possessing a substituent on their respective benzene rings.

L-Glutamate in the extracellular space regulates endogenous D-aspartate homeostasis in rat pheochromocytoma MPT1 cells.

In previous studies [FEBS Lett. 434 (1998) 231, Arch. Biochem.

Prepro-tachykinin gene expression in the brain of the honeybee Apis mellifera.

We have recently identified a tachykinin-related peptide (AmTRP) from the mushroom bodies (MBs) of the brain of the honeybee Apis mellifera L. by using direct matrix-assisted laser desorption/ionization with time-of-flight mass spectometry and have isolated its cDNA. Here, we have examined prepro-AmTRP gene expression in the honeybee brain by using in situ hybridization.

Cloning and characterization of a Caenorhabditis elegans cDNA encoding a new insulin/IGF-like peptide.

A Caenorhabditis elegans cDNA 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.

Identification of GYRKPPFNGSIFamide (crustacean-SIFamide) in the crayfish Procambarus clarkii by topological mass spectrometry analysis.

A new concept relating to the purification protocol for biological proteins and peptides has been designed as "topological mass spectrometry analysis," in combination with MALDI-TOF MS using slices of tissues, chromatographic purification from the extract of tissues, molecular cloning for the determination of the precursor structure, and capillary LC-MS/MS analysis for elucidation of its posttranslational modifications. In an actual application, we identified an alpha-amidated neuropeptide from the red swamp crayfish (Procambarus clarkii) brain. Initially, an MS number of around m/z 1382 was found by the direct MALDI-TOF MS analysis with slices of the accessory lobe of the brain.

Solution structure of ADO1, a toxin extracted from the saliva of the assassin bug, Agriosphodrus dohrni.

ADO1 is a toxin purified from the saliva of the assassin bug, Agriosphodrus dohrni. Because of its similarity in sequence to Ptu1 from another assassin bug, we did not assess its pharmacologic target. Here, we demonstrate by electrophysiologic means that ADO1 targets the P/Q-type voltage-sensitive calcium channel.

The hemolytic activity of six arachnid cationic peptides is affected by the phosphatidylcholine-to-sphingomyelin ratio in lipid bilayers.

The hemolytic activity of six cationic amphipathic peptides (Oxki1, Oxki2, Pin1, Pin2, IsCT1 and IsCT2) from arachnids strongly depends on the source of red blood cells. The hemolytic activity of the amphipathic peptides was correlated to the phosphocholine-to-sphingomyelin ratio (PC/SM) content, the potency order of which on mammal erythrocytes ranked as follows Guinea pig>pig>sheep. The spider peptides, Oxki1 and Oxki2, prefer small unilamellar vesicles (SUV) composed of PC, but they could not disrupt SUVs made of SM only.

Distinct primary structures of the major peptide toxins from the venom of the spider Macrothele gigas that bind to sites 3 and 4 in the sodium channel.

Six peptide toxins (Magi 1-6) were isolated from the Hexathelidae spider Macrothele gigas. The amino acid sequences of Magi 1, 2, 5 and 6 have low similarities to the amino acid sequences of known spider toxins. The primary structure of Magi 3 is similar to the structure of the palmitoylated peptide named PlTx-II from the North American spider Plectreurys tristis (Plectreuridae).

Synthesis and photoreactivity of caged blockers for glutamate transporters.

L-TBOA (L-threo-beta-benzyloxyaspartate) is, so far, the most potent non-transportable blocker for glutamate transporters. We synthesized alpha-CMCM-L-TBOA (1a) possessing [7-(carboxymethoxy)coumarin-4-yl]methyl ester as a caging group. alpha-CMCM-L-TBOA (1a) is biologically inactive until UV irradiation and the photolysis of 1a immediately released L-TBOA to show glutamate uptake inhibition.

A new polypeptide toxin from the nematocyst venom of an Okinawan sea anemone Phyllodiscus semoni (Japanese name "unbachi-isoginchaku").

The venomous sea anemone Phyllodiscus semoni causes cases of severe stinging. We isolated Phyllodiscus semoni toxin 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.