Inhibition of acute nociceptive responses in rats after i.c.v. injection of Thr6-bradykinin, isolated from the venom of the social wasp, Polybia occidentalis

BACKGROUND AND PURPOSE: In this work, a neuroactive peptide from the venom of the neotropical wasp Polybia occidentalis was isolated and its anti-nociceptive effects were characterized in well-established pain induction models. EXPERIMENTAL APPROACH: Wasp venom was analysed by reverse-phase HPLC and fractions screened for anti-nociceptive activity. The structure of the most active fraction was identified by electron-spray mass spectrometry (ESI-MS/MS) and it was further assessed in two tests of anti-nociceptive activity in rats: the hot plate and tail flick tests. KEY RESULTS: The most active fraction contained a peptide whose structure was Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg-OH, which corresponds to that of Thr(6)-BK, a bradykinin analogue. This peptide was given by i.c.v. injection to rats. In the tail flick test, Thr(6)-BK induced anti-nociceptive effects, approximately twice as potent as either morphine or bradykinin also given i.c.v. The anti-nociceptive activity of Thr(6)-BK peaked at 30 min after injection and persisted for 2 h, longer than bradykinin. The primary mode of action of Thr(6)-BK involved the activation of B(2) bradykinin receptors, as anti-nociceptive effects of Thr(6)-BK were antagonized by a selective B(2) receptor antagonist. CONCLUSIONS AND IMPLICATIONS: Our data indicate that Thr(6)-BK acts through B(2) bradykinin receptors in the mammalian CNS, evoking antinociceptive behaviour. This activity is remarkably different from that of bradykinin, despite the structural similarities between both peptides. In addition, due to the increased metabolic stability of Thr(6)-BK, relative to that of bradykinin, this peptide could provide a novel tool in the investigation of kinin pathways involved with pain.     

Structural and biological characterization of two novel peptides from the venom of the neotropical social wasp Agelaia pallipes pallipes.

The venom of the neotropical social wasp Agelaia pallipes pallipes was fractionated by RP-HPLC resulting in the elution of seven fractions; the last two were re-fractionated under RP-HPLC by using isocratic elution conditions and the purity of the fractions were confirmed by using ESI-MS analysis. Both fractions are constituted of peptide components, which were sequenced by Edman degradation chemistry, resulting in the following sequences: Protonectin I-L-G-T-I-L-G-L-L-K-G-L-NH(2). Agelaia-MP I-N-W-L-K-L-G-K-A-I-I-D-A-L-NH(2). Both peptides are manually synthesized on solid-phase and functionally characterized by using Wistar rats cells. Protonectin is a non-hemolytic chemotactic peptide for polymorphonucleated leukocytes (PMNL), presenting some mast cell degranulating activity and potent antimicrobial action both against Gram-positive and Gram-negative bacteria. Agelaia-MP was characterized as a hemolytic mast cell degranulator toxin, presenting a poor antimicrobial action and no chemotaxis for PMNL.     

Conformation and lytic activity of eumenine mastoparan: a new antimicrobial peptide from wasp venom

Abstract: Eumenine mastoparan‐AF (EMP‐AF) is a novel membrane active tetradecapeptide recently isolated from the venom of solitary wasp, Anterhynchium flavomarginatum micado. It was reported previously that EMP‐AF peptide presented low cytolytic activities in human erythrocytes and in RBL‐2H3 mast cells. In the present work, we observed that this peptide is able to permeate anionic liposomes, and in less extension also the neutral ones. We present evidences showing that the permeation ability is well correlated with the amount of helical conformation assumed by the peptides in these environments. This peptide also showed a broad‐spectrum inhibitory activity against Gram‐positive and Gram‐negative bacteria. The permeability of liposomes and the antibiotic effect showed a significant reduction when C‐terminus was deamidated (in acidic form). The removal of the three first amino acid residues from the N‐terminus rendered the peptide inactive both in liposomes and in bacteria. The results suggest that the mechanism of action involves a threshold in the accumulation of the peptide at level of cell membrane.     

Structural and biological characterization of three novel mastoparan peptides from the venom of the neotropical social wasp Protopolybia exigua (Saussure).

The venom of the Neotropical social wasp Protopolybia exigua(Saussure) was fractionated by RP-HPLC resulting in the elution of 20 fractions. The homogeneity of the preparations were checked out by using ESI-MS analysis and the fractions 15, 17 and 19 (eluted at the most hydrophobic conditions) were enough pure to be sequenced by Edman degradation chemistry, resulting in the following sequences: Protopolybia MPI I-N-W-L-K-L-G-K-K-V-S-A-I-L-NH2 Protopolybia-MP II I-N-W-K-A-I-I-E-A-A-K-Q-A-L-NH2 Protopolybia-MP III I-N-W-L-K-L-G-K-A-V-I-D-A-L-NH2 All the peptides were manually synthesized on-solid phase and functionally characterized. Protopolybia-MP I is a hemolytic mastoparan, probably acting on mast cells by assembling in plasma membrane, resulting in pore formation; meanwhile, the peptides Protopolybia-MP II and -MP III were characterized as a non-hemolytic mast cell degranulator toxins, which apparently act by virtue of their binding to G-protein receptor, activating the mast cell degranulation.     

Two families of antimicrobial peptides from wasp (Vespa magnifica) venom.

The hornet possesses highly toxic venom, which is rich in toxin, enzymes and biologically active peptides. Many bioactive substances were identified from wasp venom. Two families of antimicrobial peptides were purified and characterized from the venom of the wasp, Vespamagnifica (Smith). The primary structures of these peptides are homologous to those of chemotactic peptides and mastoparans isolated from other vespid venoms. They also share similarity to temporins which are amphibian antimicrobial peptides identified from the skin of the frog, Ranaboylii. These peptides show antimicrobial activities against bacteria and fungi. However, they show little hemolytic activity against human blood red cells.     

Myotoxic effects of mastoparan from Polybia paulista (Hymenoptera, Epiponini) wasp venom in mice skeletal muscle.

In a previous study, we showed that the Polybia paulista wasp venom causes strong myonecrosis. This study was undertaken to characterize the myotoxic potency of mastoparan (Polybia-MPII) isolated from venom (0.25 microg/microl) and injected in the tibial anterior (TA) muscle (i.m.) of Balb/c mice. The time course of the changes was followed at muscle degenerative (3 and 24h) and regenerative (3, 7, and 21 days) periods (n=6) after injection and compared to matched controls by calculation of the percentage of cross-sectional area affected and determination of creatine kinase (CK) activity (n=10). The results showed that although MP was strongly myotoxic, its capacity for regeneration was maintained high. Since the extent of tissue damage was not correlated with the CK serum levels, which remained very low, we raised the hypothesis that the enzyme underwent denaturation by the peptide. Evidence suggested that MP induced the death of TA fibers by necrosis and apoptosis and had the sarcolemma as its primordial target. Given its amphiphilic polycationic nature and based on the vast spectrum of functions attributed to the peptide, we suggest that MP interaction with cell membrane impaired the phosphorylation of dystrophin essential for sarcolemma mechanical stability, and disturbed Ca2+ mobilization with obvious implications on sarcoplasmic reticulum and mitochondrial functioning.     

Comprehensive Analysis and Biological Characterization of Venom Components from Solitary Scoliid Wasp Campsomeriella annulata annulata

Venoms of solitary wasps are utilized for prey capture (insects and spiders), paralyzing them with a stinger injection to be offered as food for their larvae. Thus, the identification and characterization of the components of solitary wasp venoms can have biotechnological application. In the present study, the venom components profile of a solitary scoliid wasp, Campsomeriella annulata annulata, was investigated through a comprehensive analysis using LC-MS and -MS/MS. Online mass fingerprinting revealed that the venom extract contains 138 components, and MS/MS analysis identified 44 complete sequences of the peptide components. The peptides are broadly divided into two classes: bradykinin-related peptides, and linear α-helical peptides. Among the components of the first class, the two main peptides, α-campsomerin (PRLRRLTGLSPLR) and β-campsomerin (PRLRRLTGLSPLRAP), had their biological activities evaluated. Both peptides had no effects on metallopeptidases [human neprilysin (NEP) and angiotensin-converting enzyme (ACE)] and acetylcholinesterase (AChE), and had no cytotoxic effects. Studies with PC12 neuronal cells showed that only α-campsomerin was able to enhance cell viability, while β-campsomerin had no effect. It is noteworthy that the only difference between the primary structures from these peptides is the presence of the AP extension at the C-terminus of β-campsomerin, compared to α-campsomerin. Among the linear α-helical peptides, annulatin (ISEALKSIIVG-NH₂) was evaluated for its biological activities. Annulatin showed histamine releasing activity from mast cells and low hemolytic activity, but no antimicrobial activities against all microbes tested were observed. Thus, in addition to providing unprecedented information on the whole components, the three peptides selected for the study suggest that molecules present in solitary scoliid wasp venoms may have interesting biological activities.     

Purification, sequencing and structural characterization of the phospholipase A1 from the venom of the social wasp Polybia paulista (Hymenoptera, Vespidae).

The biochemical and functional characterization of wasp venom toxins is an important prerequisite for the development of new tools both for the therapy of the toxic reactions due to envenomation caused by multiple stinging accidents and also for the diagnosis and therapy of allergic reactions caused by this type of venom. PLA(1) was purified from the venom of the neotropical social wasp Polybia paulista by using molecular exclusion and cation exchange chromatographies; its amino acid sequence was determined by using automated Edman degradation and compared to the sequences of other vespid venom PLA(1)'s. The enzyme exists as a 33,961.40 Da protein, which was identified as a lipase of the GX class, liprotein lipase superfamily, pancreatic lipases (ab20.3) homologous family and RP2 sub-group of phospholipase. P. paulista PLA(1) is 53-82% identical to the phospholipases from wasp species from Northern Hemisphere. The use restrained-based modeling permitted to describe the 3-D structure of the enzyme, revealing that its molecule presents 23% alpha-helix, 28% beta-sheet and 49% coil. The protein structure has the alpha/beta fold common to many lipases; the core consists of a tightly packed beta-sheet constituted of six-stranded parallel and one anti-parallel beta-strand, surrounded by four alpha-helices. P. paulista PLA(1) exhibits direct hemolytic action against washed red blood cells with activity similar to the Cobra cardiotoxin from Naja naja atra. In addition to this, PLA(1) was immunoreactive to specific IgE from the sera of P. paulista-sensitive patients.     

Agelaia MP-I: A peptide isolated from the venom of the social wasp, Agelaia pallipes pallipes, enhances insulin secretion in mice pancreatic islets

Peptides isolated from animal venoms have shown the ability to regulate pancreatic beta cell function. Characterization of wasp venoms is important, since some components of these venoms present large molecular variability, and potential interactions with different signal transduction pathways. For example, the well studied mastoparan peptides interact with a diversity of cell types and cellular components and stimulate insulin secretion via the inhibition of ATP dependent K⁺ (K_ATP) channels, increasing intracellular Ca²⁺ concentration. In this study, the insulin secretion of isolated pancreatic islets from adult Swiss mice was evaluated in the presence of synthetic Agelaia MP-I (AMP-I) peptide, and some mechanisms of action of this peptide on endocrine pancreatic function were characterized. AMP-I was manually synthesized using the Fmoc strategy, purified by RP-HPLC and analyzed using ESI-IT-TOF mass spectrometry. Isolated islets were incubated at increasing glucose concentrations (2.8, 11.1 and 22.2 mM) without (Control group: CTL) or with 10 μM AMP-I (AMP-I group). AMP-I increased insulin release at all tested glucose concentrations, when compared with CTL (P < 0.05). Since molecular analysis showed a potential role of the peptide interaction with ionic channels, insulin secretion was also analyzed in the presence of 250 μM diazoxide, a K_ATP channel opener and 10 μM nifedipine, a Ca²⁺ channel blocker. These drugs abolished insulin secretion in the CTL group in the presence of 2.8 and 11.1 mM glucose, whereas AMP-I also enhanced insulin secretory capacity, under these glucose conditions, when incubated with diazoxide and nifedipine. In conclusion, AMP-I increased beta cell secretion without interfering in K_ATP and L-type Ca²⁺ channel function, suggesting a different mechanism for this peptide, possibly by G protein interaction, due to the structural similarity of this peptide with Mastoparan-X, as obtained by modeling.     

Effects of the cationic antimicrobial peptide eumenitin from the venom of solitary wasp Eumenes rubronotatus in planar lipid bilayers: surface charge and pore formation activity.

Eumenitin, a novel cationic antimicrobial peptide from the venom of solitary wasp Eumenes rubronotatus, was characterized by its effects on black lipid membranes of negatively charged (azolectin) and zwitterionic (1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) or DPhPC-cholesterol) phospholipids: surface potential changes, single-channel activity, ion selectivity, and pore size were studied. We found that eumenitin binds preferentially to charged lipid membranes as compared with zwitterionic ones. Eumenitin is able to form pores in azolectin (G1=118.00+/-3.67pS or G2=160.00+/-7.07pS) and DPhPC membranes (G=61.13+/-7.57pS). Moreover, cholesterol addition to zwitterionic DPhPC membranes inhibits pore formation activity but does not interfere with the binding of peptide. Open pores presented higher cation (K+) over anion (Cl-) selectivity. The pore diameter was estimated at between 8.5and 9.8 angstroms in azolectin membranes and about 4.3 angstroms in DPhPC membranes. The results are discussed based on the toroidal pore model for membrane pore-forming activity and ion selectivity.     

An anticoagulant serine protease from the wasp venom of Vespa magnifica.

Wasp is an important venomous animal that can induce human fatalities. Coagulopathy is a clinical symptom after massive wasp stings, but the reason leading to the envenomation manifestation is still not known. In this paper, a toxin protein is purified and characterized by Sephadex G-75 gel filtration, CM-Sephadex C-25 cationic exchange and fast protein liquid chromatography (FPLC) from the venom of the wasp, Vespa magnifica (Smith). This protein, named magnvesin, contains serine protease-like activity and inhibits blood coagulation. The cDNA encoding magnvesin is cloned from the venom sac cDNA library of the wasp. The deduced protein from the cDNA is composed of 305 amino acid residues. Magnvesin shares 52% identity with allergen serine protease from the wasp Polistes dominulus. Magnvesin exerted its anti-coagulant function by hydrolyzing coagulant factors TF, VII, VIII, IX and X.     

舟山眼镜蛇毒蕈碱样多肽MP的部分理化性质测定

目的对舟山眼镜蛇毒蕈碱样多肽MP成分进行纯化及部分理化性质测定。方法采用色谱技术纯化蛇毒MP成分,质谱仪测定其相对分子质量(Mr);Edman降解法分析部分氨基酸序列,与已知成分比对;以卵磷脂为底物测定MP组分的磷脂酶A2活性;采用Bliss法测定MP对小鼠的急性毒性。结果 MP的Mr为13 260,其N端16位氨基酸序列为NLYQFKNMIQCTVPSR,与已知蛇毒磷脂酶A2基本一致,并具有较强的磷脂酶A2活性;腹腔注射MP对小鼠的LD50值为14.3 mg/kg。结论 MP为一种眼镜蛇毒磷脂酶A2。     

Protonectin (1-6): A novel chemotactic peptide from the venom of the social wasp Agelaia pallipes pallipes

Peptides constitute the largest group of Hymenoptera venom toxins; some of them interact with GPCR, being involved with the activation of different types of leukocytes, smooth muscle contraction and neurotoxicity. Most of these toxins vary from dodecapeptides to tetradecapeptides, amidated at their C-teminal amino acid residue. The venoms of social wasps can also contains some tetra-, penta-, hexa- and hepta-peptides, but just a few of them have been structurally and functionally characterized up to now. Protonectin (ILGTILGLLKGL-NH₂) is a polyfunctional peptide, presenting mast cell degranulation, release of lactate dehydrogenase (LDH) from mast cells, antibiosis against Gram-positive and Gram-negative bacteria and chemotaxis for polymorphonucleated leukocytes (PMNL), while Protonectin (1-6) (ILGTIL-NH₂) only presents chemotaxis for PMNL. However, the mixture of Protonectin (1-6) with Protonectin in the molar ratio of 1:1 seems to potentiate the biological activities dependent of the membrane perturbation caused by Protonectin, as observed in the increasing of the activities of mast cell degranulation, LDH releasing from mast cells, and antibiosis. Despite both peptides are able to induce PMNL chemotaxis, the mixture of them presents a reduced activity in comparison to the individual peptides. Apparently, when mixed both peptides seems to form a supra-molecular structure, which interact with the receptors responsible for PMNL chemotaxis, disturbing their individual docking with these receptors. In addition to this, a comparison of the sequences of both peptides suggests that the sequence ILGTIL is conserved, suggesting that it must constitute a linear motif for the structural recognition by the specific receptor which induces leukocytes migration.     

A phospholipase A1 platelet activator from the wasp venom of Vespa magnifica (Smith).

Wasp is an important venomous animal that can induce human fatalities. Aortic thrombosis and cerebral infarction are major clinical symptoms after massive wasp stings but the reason leading to the envenomation manifestation is still not known. In this paper, a toxin protein is purified and characterized by Sephadex G-75 gel filtration, CM-Sephadex C-25 cationic exchange and fast protein liquid chromatography (FPLC) from the venom of the wasp, Vespa magnifica (Smith). This protein, named magnifin, contains phospholipase-like activity and induces platelet aggregation. The cDNA encoding magnifin is cloned from the venom sac cDNA library of the wasp. The predicted protein was deduced from the cDNA with a sequence composed of 337 amino acid residues. Magnifin is very similar to other phospholipase A(1) (PLA(1)), especially to other wasp allergen PLA(1). Magnifin can activate platelet aggregation and induce thrombosis in vivo. The current results proved that PLA(1) in wasp venom could be contributable to aortic thrombosis after massive wasp stings.     

蝎毒多肽提取物对白血病细胞粘附力影响的实验研究

目的 观察蝎毒多肽提取物(PESV,防治白血病中药提取物)对抑制人白血病原代细胞粘附力的影响.方法 以人白血病原代细胞为研究对象,台盼蓝拒染法检测PESV对人白血病原代细胞的细胞毒作用;观察其对白血病原代细胞与人脐静脉内皮细胞(HUVEC)粘附的影响.结果 PESV在对细胞最佳作用浓度15μg·mL-1下,能够明显抑制人白血病原代细胞与HU-VEC的粘附,抑制率为(52.18±21.01)%;各浓度组与阳性对照组比较具有显著性差异(P<0.05).结论 PESV在对细胞最佳作用浓度下,对人白血病原代细胞的黏附力有明显抑制作用.     

白眉蝮蛇毒精氨酸酯酶Ames试验(英文)

目的　研究白眉蝮蛇 (Agkistrodonhalysussuriensis)毒中精氨酸酯酶的致突变作用。 方法　用鼠伤寒沙门细菌营养缺陷型突变株TA97,TA98,TA10 0和TA10 2 ,采用平皿掺入法进行Ames试验 ,将实验分为加和不加代谢激活系统S92组平行试验。精氨酸酯酶设 6个浓度 :10 .0× 10 -3 ,5 .0× 10 -3 ,2 .5× 10 -3 ,1.2 5× 10 -3 ,0 .6 2 5× 10 -3 和 0 .312× 10 -3 U/mL。结果　加和不加代谢激活系统S9两种条件下 ,精氨酸酯酶不诱发鼠伤寒沙门细菌营养缺陷型突株的回复突变。Ames试验结果为阴性。结论　从致突变角度考虑 ,精氨酸酯酶在高于“蝮蛇清栓酶”(主要成分为精氨酸酯酶 )临床治疗剂量约 10 0 0倍的条件下仍然较安全。     

Channel-forming activity in the venom of the cockroach-hunting wasp, Ampulex compressa.

The parasitoid solitary wasp Ampulex compressa uses the cockroach Periplaneta americana as a food supply for its larvae. To subdue its prey, the wasp injects a venom cocktail into the brain of the cockroach. We investigated channel activity of A. compressa venom by collecting venom and incorporating it into a planar lipid bilayer. The venom, reconstituted into the bilayer, showed ion channel activity, forming a fast-fluctuating channel with a small conductance of 20+/-0.1pS, with no voltage sensitivity. These channels were not observed when the venom was digested with proteases before application to the bilayer, but were not affected by exposure to protease after their incorporation into the bilayer, indicating that the active venom component is a peptide. The channels were found to be cation selective with similar selectivity for the monovalent cations K(+), Li(+) and Na(+), but showed high selectivity against anions (Cl(-)) and divalent cations (Ca(2+) and Mg(2+)). This study is the first demonstration and biophysical characterization of channel activity in the venom of A. compressa. The possible functional significance of this channel activity is discussed in light of the unusual nature of the effects of this wasp venom on the behavior of its prey.     

Purification and characterization of a novel short-chain insecticidal toxin with two disulfide bridges from the venom of the scorpion Liocheles australasiae.

Scorpion venoms contain a variety of peptides toxic to mammals, insects and crustaceans. Most of the scorpion toxins have been isolated from the venoms of scorpions in the family Buthidae, but little interest has been paid to non-Buthidae scorpions. In this study, we isolated a short-chain insecticidal toxin (LaIT1) from the venom of the scorpion Liocheles australasiae belonging to the Hemiscorpiidae family. This toxin showed insect toxicity against crickets at a dose of 1.0 microg/insect, but no toxicity was observed against mice even after injection of 1.0 microg of LaIT1 via the intracerebroventricular route, suggesting that the effect of the toxin is insect-selective. Edman sequencing and mass spectrometric analysis revealed that the toxin is composed of 36 amino acid residues and cross-linked by only two disulfide bridges. The pattern of the disulfide bridges was assigned by LC/MS analysis after enzymatic digestion. LaIT1 shows no sequence homology to any other known toxins, suggesting that this toxin represents a novel structural motif class.     

Isolation and in vitro partial characterization of hemolytic proteins from the nematocyst venom of the jellyfish Stomolophus meleagris

Jellyfish venom contains various toxins and can cause itching, edema, muscle aches, shortness of breath, blood pressure depression, shock or even death after being stung. Hemolytic protein is one of the most hazardous components in the venom. The present study investigated the hemolytic activity of the nematocyst venom from jellyfish Stomolophus meleagris. Anion exchange chromatography, DEAE Sepharose Fast Flow, and gel filtration chromatography, Superdex200 had been employed to isolate hemolytic proteins from the nematocyst venom of jellyfish S. meleagris. Hemolysis of chicken red blood cells was used to quantify hemolytic potency of crude nematocyst venom and chromatography fractions during the purification process. Native-PAGE profile displayed one protein band in the purified hemolytic protein (SmTX); however, two protein bands with apparent molecular weights of ～45 kDa and 52 kDa were observed in the reducing SDS–PAGE analysis. Approximately 70 μg/mL of SmTX caused 50% hemolysis (HU₅₀) of the erythrocyte suspension. The hemolytic activity of SmTX was shown to be temperature and pH dependent, with the optimum temperature and pH being 37 °C and pH 5.0. The present study is the first report of isolation and partial characterization of hemolytic proteins from the nematocyst venom of the jellyfish S. meleagris. The mechanism of the hemolytic activity of SmTX is not clear and deserves further investigation.     

Pharmacological characterization of Synoeca cyanea venom: An aggressive social wasp widely distributed in the Neotropical region

The venom of social wasps has been poorly studied so far, despite the high number of accidents in humans and assessment of the use of these wasps as a biological control of pests. The study of the pharmacological effects of the venom is of great importance since the poisoning is dangerous causing serious systemic effects, including death in the case of multiple attacks. In this study, the pharmacological activities of venom from the social wasp Synoeca cyanea were evaluated by the following assays: LD50 in mice, the behavioural effects and the hemorrhagic activity induced by the venom in mice, the oedematogenic activity in rat, the haemolysis in human blood, the stimulating effect on guinea-pig smooth muscle, and the antimicrobial activity. The aim was to determine the toxic effects of venom and to perform a comparative study with earlier work conducted with venom from other wasp species. Results showed that S. cyanea venom produced a potent dose-dependent oedema, as well as antibacterial and haemolytic activities, suggesting the presence of histamine, serotonin, kinins and other molecules related to increased vascular permeability and cytolytic activity in this venom. Despite previous studies with wasp venoms, S. cyanea venom presented a slight hemorrhagic effect. Data obtained in the smooth muscle assay also suggest the presence of BK or analogues in S. cyanea whole venom. The knowledge of symptoms and effects produced by S. cyanea venom is critical for health organizations, in order to improve clinical treatment in accidents caused by wasp stings.