Venom of the giant ant Dinoponera quadriceps attenuates inflammatory pain in mouse cutaneous wound healing model

Arthropod venoms are potential sources of bioactive substances, providing tools for the validation of popular use and new drugs design. Ants belonging to the genus Dinoponera are used in the folk medicine to treat inflammatory conditions. It was previously demonstrated that the venom of the giant ant Dinoponera quadriceps (DqV), containing a mixture of polypeptides, elicit antinociceptive effect in mice models of chemical, mechanical and thermal nociception. The aim of this study was to evaluate DqV antiinflammatory and antihypernociceptive effects in a mice model of traumatic cutaneous wound. Colonies of D. quadriceps were collected in the ‘‘Serra de Maranguape’’ (State of Ceará, northeastern Brazil), a small mountain range located on the coastal zone, and the venom secreted by the ant glands was extracted with capillary tubes, further lyophilized and maintained at -20 ± 1ºC until use. Wounds were performed in the dorsum of Swiss mice. Animals received intravenous (i.v.) injection of DqV (50 µg -1kg day-1) during 3 days for evaluation of inflammatory parameters present in the wounds: hypernociception, leukocyte infiltrate, myeloperoxidase activity, nitrite nitrate-1 content. Data was tested by two-way ANOVA and Bonferroni’s post-hoc test. DqV reduced (2.7 folds) hypernociception at 48 hours, leukocyte infiltration by 65% at 6 hours and myeloperoxidase activity by 60% at 0.5 hour after wound induction. In conclusion, the venom extracted from D. quadriceps glands attenuates inflammation and hypernociception in mice cutaneous wounds.

Natural Occurrence in Venomous Arthropods of Antimicrobial Peptides Active against Protozoan Parasites.

Arthropoda is a phylum of invertebrates that has undergone remarkable evolutionary radiation, with a wide range of venomous animals. Arthropod venom is a complex mixture of molecules and a source of new compounds, including antimicrobial peptides (AMPs). Most AMPs affect membrane integrity and produce lethal pores in microorganisms, including protozoan pathogens, whereas others act on internal targets or by modulation of the host immune system. Protozoan parasites cause some serious life-threatening diseases among millions of people worldwide, mostly affecting the poorest in developing tropical regions. Humans can be infected with protozoan parasites belonging to the genera Trypanosoma, Leishmania, Plasmodium, and Toxoplasma, responsible for Chagas disease, human African trypanosomiasis, leishmaniasis, malaria, and toxoplasmosis. There is not yet any cure or vaccine for these illnesses, and the current antiprotozoal chemotherapeutic compounds are inefficient and toxic and have been in clinical use for decades, which increases drug resistance. In this review, we will present an overview of AMPs, the diverse modes of action of AMPs on protozoan targets, and the prospection of novel AMPs isolated from venomous arthropods with the potential to become novel clinical agents to treat protozoan-borne diseases.

A Dipteran's Novel Sucker Punch: Evolution of Arthropod Atypical Venom with a Neurotoxic Component in Robber Flies (Asilidae, Diptera).

Predatory robber flies (Diptera, Asilidae) have been suspected to be venomous due to their ability to overpower well-defended prey. However, details of their venom composition and toxin arsenal remained unknown. Here, we provide a detailed characterization of the venom system of robber flies through the application of comparative transcriptomics, proteomics and functional morphology. Our results reveal asilid venoms to be dominated by peptides and non-enzymatic proteins, and that the majority of components in the crude venom is represented by just ten toxin families, which we have named Asilidin1-10. Contrary to what might be expected for a liquid-feeding predator, the venoms of robber flies appear to be rich in novel peptides, rather than enzymes with a putative pre-digestive role. The novelty of these peptides suggests that the robber fly venom system evolved independently from hematophagous dipterans and other pancrustaceans. Indeed, six Asilidins match no other venom proteins, while three represent known examples of peptide scaffolds convergently recruited to a toxic function. Of these, members of Asilidin1 closely resemble cysteine inhibitor knot peptides (ICK), of which neurotoxic variants occur in cone snails, assassin bugs, scorpions and spiders. Synthesis of one of these putative ICKs, U-Asilidin1-Mar1a, followed by toxicity assays against an ecologically relevant prey model revealed that one of these likely plays a role as a neurotoxin involved in the immobilization of prey. Our results are fundamental to address these insights further and to understand processes that drive venom evolution in dipterans as well as other arthropods.

Production and packaging of a biological arsenal: evolution of centipede venoms under morphological constraint.

Venom represents one of the most extreme manifestations of a chemical arms race. Venoms are complex biochemical arsenals, often containing hundreds to thousands of unique protein toxins. Despite their utility for prey capture, venoms are energetically expensive commodities, and consequently it is hypothesized that venom complexity is inversely related to the capacity of a venomous animal to physically subdue prey. Centipedes, one of the oldest yet least-studied venomous lineages, appear to defy this rule. Although scutigeromorph centipedes produce less complex venom than those secreted by scolopendrid centipedes, they appear to rely heavily on venom for prey capture. We show that the venom glands are large and well developed in both scutigerid and scolopendrid species, but that scutigerid forcipules lack the adaptations that allow scolopendrids to inflict physical damage on prey and predators. Moreover, we reveal that scolopendrid venom glands have evolved to accommodate a much larger number of secretory cells and, by using imaging mass spectrometry, we demonstrate that toxin production is heterogeneous across these secretory units. We propose that the differences in venom complexity between centipede orders are largely a result of morphological restrictions of the venom gland, and consequently there is a strong correlation between the morphological and biochemical complexity of this unique venom system. The current data add to the growing body of evidence that toxins are not expressed in a spatially homogenous manner within venom glands, and they suggest that the link between ecology and toxin evolution is more complex than previously thought.

Arthropod venom Hyaluronidases: biochemical properties and potential applications in medicine and biotechnology

Hyaluronidases are enzymes that mainly degrade hyaluronan, the major glycosaminoglycan of the interstitial matrix. They are involved in several pathological and physiological activities including fertilization, wound healing, embryogenesis, angiogenesis, diffusion of toxins and drugs, metastasis, pneumonia, sepsis, bacteremia, meningitis, inflammation and allergy, among others. Hyaluronidases are widely distributed in nature and the enzymes from mammalian spermatozoa, lysosomes and animal venoms belong to the subclass EC 3.2.1.35. To date, only five three-dimensional structures for arthropod venom hyaluronidases (Apis mellifera and Vespula vulgaris) were determined. Additionally, there are four molecular models for hyaluronidases fromMesobuthus martensii, Polybia paulista and Tityus serrulatus venoms. These enzymes are employed as adjuvants to increase the absorption and dispersion of other drugs and have been used in various off-label clinical conditions to reduce tissue edema. Moreover, a PEGylated form of a recombinant human hyaluronidase is currently under clinical trials for the treatment of metastatic pancreatic cancer. This review focuses on the arthropod venom hyaluronidases and provides an overview of their biochemical properties, role in the envenoming, structure/activity relationship, and potential medical and biotechnological applications.

Venomic and transcriptomic analysis of centipede Scolopendra subspinipes dehaani.

Centipedes have venom glands in their first pair of limbs, and their venoms contain a large number of components with different biochemical and pharmacological properties. However, information about the compositions and functions of their venoms is largely unknown. In this study, Scolopendra subspinipes dehaani venoms were systematically investigated by transcriptomic and proteomic analysis coupled with biological function assays. After random screening approximately 1500 independent clones, 1122 full length cDNA sequences, which encode 543 different proteins, were cloned from a constructed cDNA library using a pair of venom glands from a single centipede species. Neurotoxins, ion channel acting components and venom allergens were the main fractions of the crude venom as revealed by transcriptomic analysis. Meanwhile, 40 proteins/peptides were purified and characterized from crude venom of S. subspinipes dehaani. The N-terminal amino acid sequencing and mass spectrum results of 29 out of these 40 proteins or peptides matched well with their corresponding cDNAs. The purified proteins/peptides showed different pharmacological properties, including the following: (1) platelet aggregating activity; (2) anticoagulant activity; (3) phospholipase A(2) activity; (4) trypsin inhibiting activity; (5) voltage-gated potassium channel activities; (6) voltage-gated sodium channel activities; (7) voltage-gated calcium channel activities. Most of them showed no significant similarity to other protein sequences deposited in the known public database. This work provides the largest number of protein or peptide candidates with medical-pharmaceutical significance and reveals the toxin nature of centipede S. subspinipes dehaani venom.

Kallikrein-kinin system activation by Lonomia obliqua caterpillar bristles: involvement in edema and hypotension responses to envenomation.

Lonomia obliqua envenomation induces an intense burning sensation at the site of contact and severe hemorrhage followed by edema and hypotension, and after few days death can occur usually due to acute renal failure. In order to understand more about the envenomation syndrome, the present study investigates the role played by kallikrein-kinin system (KKS) in edematogenic and hypotensive responses to the envenomation by L. obliqua. The incubation of L. obliqua caterpillar bristles extract (LOCBE) with plasma results in kallikrein activation, measured by cromogenic assay using the kallikrein synthetic substrate S-2302 (H-D-Pro-Phe-Arg-pNA). It was also showed that LOCBE was able to release kinins from low-molecular weight kininogen (LMWK). Moreover, it was demonstrated that previous administration of a kallikrein inhibitor (aprotinin) or bradykinin B2 receptor antagonist (HOE-140) significantly reduces the edema and hypotension in response to LOCBE, using mouse paw edema bioassay and mean arterial blood pressure analysis, respectively. The results demonstrate a direct involvement of the KKS in the edema formation and in the fall of arterial pressure that occur in the L. obliqua envenomation syndrome.

Diagnosis of Hymenoptera venom allergy.

The purpose of diagnostic procedure is to classify a sting reaction by history, identify the underlying pathogenetic mechanism, and identify the offending insect. Diagnosis of Hymenoptera venom allergy thus forms the basis for the treatment. In the central and northern Europe vespid (mainly Vespula spp.) and honeybee stings are the most prevalent, whereas in the Mediterranean area stings from Polistes and Vespula are more frequent than honeybee stings; bumblebee stings are rare throughout Europe and more of an occupational hazard. Several major allergens, usually glycoproteins with a molecular weight of 10-50 kDa, have been identified in venoms of bees, vespids. and ants. The sequences and structures of the majority of venom allergens have been determined and several have been expressed in recombinant form. A particular problem in the field of cross-reactivity are specific immunoglobulin E (IgE) antibodies directed against carbohydrate epitopes, which may induce multiple positive test results (skin test, in vitro tests) of still unknown clinical significance. Venom hypersensitivity may be mediated by immunologic mechanisms (IgE-mediated or non-IgE-mediated venom allergy) but also by nonimmunologic mechanisms. Reactions to Hymenoptera stings are classified into normal local reactions, large local reactions, systemic toxic reactions, systemic anaphylactic reactions, and unusual reactions. For most venom-allergic patients an anaphylactic reaction after a sting is very traumatic event, resulting in an altered health-related quality of life. Risk factors influencing the outcome of an anaphylactic reaction include the time interval between stings, the number of stings, the severity of the preceding reaction, age, cardiovascular diseases and drug intake, insect type, elevated serum tryptase, and mastocytosis. Diagnostic tests should be carried out in all patients with a history of a systemic sting reaction to detect sensitization. They are not recommended in subjects with a history of large local reaction or no history of a systemic reaction. Testing comprises skin tests with Hymenoptera venoms and analysis of the serum for Hymenoptera venom-specific IgE. Stepwise skin testing with incremental venom concentrations is recommended. If diagnostic tests are negative they should be repeated several weeks later. Serum tryptase should be analyzed in patients with a history of a severe sting reaction.

Purification and characterization of two forms of antigen 5 from polybia scutellaris venom.

Two polypeptides from the venom of Polybia scutellaris were purified to homogeneity by RP-HPLC. They differ very slightly in mol. wt (both are about 23,000) and hydrophobicity, and have isoelectric points greater than 9. Amino acid analyzes show close similarity between them and with antigen 5 of vespids from different species. The two polypeptides have an identical N-terminal sequence (18 amino acids) which shows a high degree of homology with those of other vespids. Owing to the fact that the venom of this species is non-allergenic, the data for the mol. wt, isoelectric point, amino acid composition and N-terminal sequence allow us to identify the isolated polypeptides as two forms of antigen 5. Amino acids at positions 5 and 11 in P. scutellaris antigen 5 differ from those of the previously known sequences for antigen 5, suggesting that one or other might be responsible for the lack of allergenicity of the P. scutellaris venom.

The purification and amino acid sequence of the lethal neurotoxin Tx1 from the venom of the Brazilian 'armed' spider Phoneutria nigriventer.

A lethal neurotoxic polypeptide of Mr 8 kDa was purified from the venom of the South American 'armed' or wandering spider Phoneutria nigriventer by centrifugation, gel filtration on Superose 12, and reverse phase FPLC on columns of Pharmacia PepRPC and ProRPC. The purified neurotoxin Tx1 had an LD50 of 0.05 mg/kg in mice following intracerebroventricular injection. The complete amino acid sequence of the neurotoxin was determined by automated Edman degradation of the native and S-carboxymethylated protein in pulsed liquid and dual phase sequencers, and by the manual DABITC/PITC double coupling method applied to fragments obtained after digestions with the S. aureus V8 protease and trypsin. The neurotoxin Tx1 consists of a single chain of 77 amino acid residues, which contains a high proportion of cysteine. The primary structure showed no homology to other identified spider toxins.

Tarantula (Eurypelma californicum) venom, a multicomponent system.

The venom of the tarantula Eurypelma californicum was analysed biochemically, the components were isolated and characterized. The pH value of the crude venom is 5.3 +/- 0.3. After dilution with distilled water, UV-absorption spectra showed a single maximum at 258 nm (pH ca. 7.0). A second maximum at 328 nm emerged above pH 8.0. Protein concentration of the venom is ca. 65 mg/ml. After Coomassie staining SDS-PAGE patterns show three major bands with apparent molecular masses around 40 kDa, 4.3 kDa and 1.3 kDa besides some weak high molecular protein bands. The following low-molecular mass constituents were determined in the crude venom: ATP, ADP, AMP, glutamic acid, aspartic acid, gamma-aminobutyric acid, glucose and the ions potassium, sodium, calcium, magnesium and chloride; the osmolality was 361 micro0smol/ml. The LD50 value for female cockroaches was 0.15 microliters venom per g body weight and for male cockroaches 0.4 microliters venom per g body weight. Separation of the crude venom by gel chromatography yielded four elution peaks. Peak I contains the enzyme hyaluronidase. The activity is 200-900 U/microliters. Peak II contains a mixture of toxic peptides. Peak III contains the 1.3-kDa components of SDS-PAGE and peak IV mainly contains ATP. Venom proteins including the enzyme hyaluronidase were precipitated by 5% trichloroacetic acid. The supernatant was separated by HPLC into 13 fractions. Fraction 1 contains glutamic acid, aspartic acid, gamma-aminobutyric acid and ATP; fraction 2 contains ATP, ADP and AMP as well as a component 2' visible in SDS-PAGE as 1.3-kDa band and consisting of spermine and tryptophan; fraction 3 contains ATP and an unknown component 3'; fractions 4-6 also show a 1.3-kDa band in SDS-PAGE, fraction 4 being tyrosylspermine and fractions 5 and 6 containing compounds of spermine and aromatic molecules; fraction 7 contains a peptide which lacks aromatic amino acids, it was sequenced from the N-terminus; fractions 8-13 contain very similar toxic peptides. The peptides in fractions 11 and 12, labeled ESTX for Eurypelma spider toxin, were cleaved with different enzymes and sequenced. They differ in one amino acid in position 26. Homologies with scorpion toxins and with a toxin of the spider Segestria florentina were found.

Comparative studies on the protein composition of hymenopteran venom reservoirs.

The proteins of venom reservoirs from 25 hymenopteran species from 21 genera were investigated with regard to their protein composition and immunological similarities. It was found that low mol. wt proteins and polypeptides are typical for the venoms of ants, social wasps and bees. Six species of ichneumonoid parasitic wasps lack these low mol. wt proteins. Except for bee venoms none of the low mol. wt proteins contain mannose. The venoms of ichneumonoid parasites and ants contained primarily acidic proteins, whereas those of social wasps and bees contained abundant neutral and basic proteins. Immunological analysis showed that a number of venom proteins from parasitic braconid (Chelonus) wasps shared conserved immunoreactive determinants with the venom proteins of higher Hymenoptera, including some not of parasitic habit. Implications of phylogenetic relationships among hymenopteran species are discussed.

Algunas características de los venenos y secreciones digestivas de Aphonopelma seemanni y Sphaerobothria hoffmanni (Araneae: Theraphosidae) de Costa Rica / Characteristics of the venoms and digestive secretions of Aphonopelma seemanni and Sphaerobothria hoffmanni (Araneae: Theraphosidae) of Costa Rica

A comparison of some components of the venoms of two Costa Rican tarantulas, Aphonopelma seeamanni (Cambridge) and Sphaerobothria hoffmanni (Karsch) by polyacrylamide gel electrophoresis shows patterns similar to those of Dugesiella hentzi (Girard), a North American tarantula. The digestive secretions have proteins that do not enter the 15% gels; thus no bands are obsorved. The method used by the tarantulas to consume their prey involves the action of both the venom and the digestive secretions. The percent protein, pH, proteolytic activity and hemolytic activity of venom and digestive secretions of both species were determined, and a high proteolytic activity for digestive secretions was found.

Amino acid sequence of versutoxin, a lethal neurotoxin from the venom of the funnel-web spider Atrax versutus.

The complete amino acid sequence of versutoxin, a lethal neurotoxic polypeptide isolated from the venom of male and female funnel-web spiders of the species Atrax versutus, was determined. Sequencing was performed in a gas-phase protein sequencer by automated Edman degradation of the S-carboxymethylated toxin and fragments of it produced by reaction with CNBr. Versutoxin consisted of a single chain of 42 amino acid residues. It was found to have a high proportion of basic residues and of cystine. The primary structure showed marked homology with that of robustoxin, a novel neurotoxin recently isolated from the venom of another funnel-web-spider species, Atrax robustus.

[Toxic components of the venom of the cellar spider Segestria florentina]. / Izuchenie toksicheskikh komponentov iada pogrebnogo pauka Segestria florentina.

Two neurotoxins and one insectotoxin have been isolated from venom of the cellar spider Segestria florentina, their homogeneity being proved by disk electrophoresis, isoelectric focusing, and analysis of N-terminal amino acid residues. The neurotoxins are polypeptides with molecular mass about 5000 D. For the insectotoxin, containing 35 amino acid residues with molecular mass 3988 D, the total primary structure is established.

Estudios electroforeticos y acción procoagulante del veneno de loxosceles laeta / Electrophoretics studies and procoagulant action of the loxosceles laeta venom

El veneno de arañas adultas Loxósceles laeta fue obtenido mediante electroestimulación y disección glandular. La distribución electroforética de las proteínas del veneno fue obtenida en gel de poliacrilamida a pH 8.6, obteniéndose 19 y 22 bandas proteicas respectivamente. Mediante electroforesis a pH 7.2 en presencia de dodecil sulfato de sodio de sodio se observaron tan solo 18 y 20 respectivamente. Las muestras estudiadas no producen coagulación del fibrinógeno humano, bovino ni plasma humano citratado. El veneno loxoscélico no hidrolizá los substratos BAPna, TAME ni Chromozym TH lo que demuestra la ausencia de Enzima similar a trombina. La presencia de acción procoagulante in vitro del veneno fue demostrada por la aceleración del tiempo de recalcificación del plasma humano. Se discute el rol de esta actividad en el envenenamiento loxoscélico

Complete amino acid sequence of a new type of lethal neurotoxin from the venom of the funnel-web spider Atrax robustus.

Robustoxin, the lethal neurotoxin isolated from the venom of the male Sydney funnel-web spider, Atrax robustus, is of unique structural type and physiological mode of action. The primary structure of this 42-residue peptide was determined to be H2N-Cys-Ala-Lys-Lys-Arg-Asn-Trp-Cys-Gly-Lys-Asn-Glu-Asp-Cys-Cys-Cys-Pro- Met-Lys-Cys-Ile-Tyr-Ala-Trp-Tyr-Asn-Gln-Gln-Gly-Ser-Cys-Gln-Thr-Thr-Ile- Thr-Gly-Leu-Phe-Lys-Lys-Cys-H. The disposition of disulphide-bridged cysteine residues at both the amino- and carboxy-termini and as a triplet at residues 14-16 appears to have no precedent amongst neurotoxins.

Isolation and partial characterization of a toxin from the venom of the East African orthognath spider Pterinochilus spec.

A minor basic polypeptide in the venom of the East African orthognath spider Pterinochilus spec. was the most toxic of 16 basic components in this venom. This toxin A4/4 was purified by gel permeation on Sephadex G-50 and ion exchange chromatography on CM-Sephadex C-25 with a yield of 1.3% of the total protein content of the crude venom. The polypeptide has an isoelectric point of 9.39 and molecular weight of 10,500. The s.c. LD50 in mice is 0.1 mg/kg. Death occurred due to respiratory paralysis. After administration of sublethal doses rapid reversibility of the symptoms and total recovery of the animals was observed.