Structural, biochemical and immunochemical characterization of an acidic phospholipase A2 from Lachesis acrochorda (Viperidae: Crotalinae) venom.

Viperids of the genus Lachesis, also known as bushmasters, are capable of injecting great amounts of venom that cause severe envenomation incidents. Since phospholipases type A2 are mainly involved in edema and myonecrosis within the snakebite sites, in this work, the isolation, amino acid sequence and biochemical characterization of the first phospholipase type A2 from the venom of Lachesis acrochorda, named Lacro_PLA2, is described. Lacro_PLA2 is an acidic aspartic 49 calcium-dependent phospholipase A2 with 93% similarity to the L. stenophrys phospholipase. Lacro_PLA2 has a molecular mass of 13,969.7 Da and an experimental isoelectric point around 5.3. A combination of N-terminal Edman degradation and MS/MS spectrometry analyses revealed that Lacro_PLA2 contains 122 residues including 14 cysteines that form 7 disulfide bridges. A predicted 3D model shows a high resemblance to other viperid phospholipases. Nevertheless, immunochemical and phospholipase neutralization tests revealed a notorious level of immunorecognition of the isolated protein by two polyclonal antibodies from viperids from different genus, which suggest that Lacro_PLA2 resembles more to bothropic phospholipases. Lacro_PLA2 also showed significantly high edema activity when was injected into mice; so, it could be an alternative antigen in the development of antibodies against toxins of this group of viperids, seeking to improve commercial polyclonal antivenoms.

An overview of some enzymes from buthid scorpion venoms from Colombia: Centruroides margaritatus, Tityus pachyurus, and Tityus n. sp. aff. metuendus

Background: In Colombia, several species of Buthidae scorpions belonging to the genera Centruroides and Tityus coexist, and their stings are considered life-threatening to humans because of their venom neurotoxins. Despite previous studies focusing on neurotoxins from these scorpion genera, little is known about the enzymes present in their venoms and their relationship with whole venom toxicity. Methods: Here, using proteomic and biochemical protocols the enzymatic activities of the venoms of three Colombian scorpion species, C. margaritatus, T. pachyurus, and T. n. sp. aff. metuendus, were compared to establish the presence and absence of enzymes such as phospholipases, hyaluronidases, and proteases that could be related to venom toxicity. Results: C. margaritatus was positive for hyaluronidases, T. n. sp. aff. metuendus for proteases, and T. pachyurus exhibited activity for all three mentioned enzymes. Conclusion: This information provides valuable insights into the specific enzyme diversity of each species' venom and their potential role in venom toxicity, which could contribute to the development of better treatments and prevention strategies for scorpion envenomation.

Heterologous Expression of an Insecticidal Peptide Obtained from the Transcriptome of the Colombian Spider Phoneutria depilate.

Spider venoms are composed, among other substances, of peptide toxins whose selectivity for certain physiological targets has made them powerful tools for applications such as bioinsecticides, analgesics, antiarrhythmics, antibacterials, antifungals and antimalarials, among others. Bioinsecticides are an environmentally friendly alternative to conventional agrochemicals. In this paper, the primary structure of an insecticidal peptide was obtained from the venom gland transcriptome of the ctenid spider Phoneutria depilata (Transcript ID PhdNtxNav24). The peptide contains 53 amino acids, including 10 Cys residues that form 5 disulfide bonds. Using the amino acid sequence of such peptide, a synthetic gene was constructed de novo by overlapping PCRs and cloned into an expression vector. A recombinant peptide, named delta-ctenitoxin (rCtx-4), was obtained. It was expressed, folded, purified and validated using mass spectrometry (7994.61 Da). The insecticidal activity of rCtx-4 was demonstrated through intrathoracic injection in crickets (LD50 1.2 µg/g insect) and it was not toxic to mice. rCtx-4 is a potential bioinsecticide that could have a broad spectrum of applications in agriculture.

A proteomic overview of the major venom components from Tityus championi from Panama.

In recent years, morbidity caused by scorpion sting of the species Tityus championi has increased in Panama. Therefore, the LD50 was determined by intravenous injection in 2.9 mg/kg and the venom of T. championi was separated using a HPLC system and their fractions were tested for biological activities in mice to identify the most toxic fractions to mammals. In addition, the venom fractions were also tested against invertebrates to look for insect-specific toxin peptides. The most toxic fractions were analyzed by MS/MS spectrometry. The primary structures of T. championi venom peptides with the most relevant activity were obtained, and the primary structure of one of most neurotoxic peptides was found at least in other four species of Tityus from Panama. This neurotoxin is quite important to be used as a protein target to be neutralized if developing antivenoms against the sting of this Panamanian scorpion or other relevant species of genera Tityus in the country.

Immunomodulatory Responses of Two Synthetic Peptides against Salmonella Typhimurium Infection.

In vitro assays of phagocytic activity showed that the peptide Pin2[G] stimulates phagocytosis in BMDM cells from 0.15 to 1.25 µg/mL, and in RAW 264.7 cells at 0.31 µg/mL. In the same way, the peptide FA1 induced phagocytosis in BMDM cells from 1.17 to 4.69 µg/mL and in RAW 264.7 cells at 150 µg/mL. Cytokine profiles of uninfected RAW 264.7 showed that Pin2[G] increased liberation TNF (from 1.25 to 10 µg/mL) and MCP-1 (10 µg/mL), and FA1 also increased the release of TNF (from 18.75 to 75 µg/mL) but did not increase the liberation of MCP-1. In RAW 264.7 macrophages infected with Salmonella enterica serovar Typhimurium, the expression of TNF increases with Pin2[G] (1.25-10 µg/mL) or FA1 (18.75-75 µg/mL). In these cells, FA1 also increases the expression of IL-12p70, IL-10 and IFN-γ when applied at concentrations of 37.5, 75 and 150 µg/mL, respectively. On the other hand, stimulation with 1.25 and 10 µg/mL of Pin2[G] promotes the expression of MCP-1 and IL-12p70, respectively. Finally, peptides treatment did not resolve murine gastric infection, but improves their physical condition. Cytokine profiles showed that FA1 reduces IFN-γ and MCP-1 but increases IL-10, while Pin2[G] reduces IFN-γ but increases the liberation of IL-6 and IL-12p70. This data suggests a promising activity of FA1 and Pin2[G] as immunomodulators of gastric infections in S. Typhimurium.

Identification of a crocodylian ß-defensin variant from Alligator mississippiensis with antimicrobial and antibiofilm activity.

ß-defensin host defense peptides are important components of the innate immune system of vertebrates. Although evidence of their broad antimicrobial, antibiofilm and immunomodulatory activities in mammals have been presented, ß-defensins from other vertebrate species, like crocodylians, remain largely unexplored. In this study, five new crocodylian ß-defensin variants from Alligator mississippiensis and Crocodylus porosus were selected for synthesis and characterization based on their charge and hydrophobicity values. Linear peptides were synthesized, folded, purified and then evaluated for their antimicrobial and antibiofilm activities against the bacterial pathogens, Salmonella enterica serovar Typhimurium, Staphylococcus aureus, Enterobacter cloacae and Acinetobacter baumannii. The Am23SK variant (SCRFSGGYCIWNWERCRSGHFLVALCPFRKRCCK) from A. mississippiensis displayed promising activity against both planktonic cells and bacterial biofilms, outperforming the human ß-defensin 3 under the experimental conditions. Moreover, Am23SK exhibited no cytotoxicity towards mammalian cells and exerted immunomodulatory effects in vitro, moderately suppressing the production of proinflammatory mediators from stimulated human bronchial epithelial cells. Overall, our results have expanded the activity landscape of crocodylian and reptilian ß-defensin in general.

Reptilian ß-defensins: Expanding the repertoire of known crocodylian peptides.

One of the major families of host defense peptides (HDPs) in vertebrates are ß-defensins. They constitute important components of innate immunity and have remained an interesting topic of research for more than two decades. While many ß-defensin sequences in mammals and birds have been identified and their properties and functions characterized, ß-defensin peptides from other groups of vertebrates, particularly reptiles, are still largely unexplored. In this review, we focus on reptilian ß-defensins and summarize different aspects of their biology, such as their genomic organization, evolution, structure, and biological activities. Reptilian ß-defensin genes exhibit similar genomic organization to birds and their number and gene structure are variable among different species. During the evolution of reptiles, several gene duplication and deletion events have occurred and the functional diversification of ß-defensins has been mainly driven by positive selection. These peptides display broad antimicrobial activity in vitro, but a deeper understanding of their mechanisms of action in vivo, including their role as immunomodulators, is still lacking. Reptilian ß-defensins constitute unique polypeptide sequences to expand our current understanding of innate immunity in these animals and elucidate core biological functions of this family of HDPs across amniotes.

Venom gland transcriptome from Heloderma horridum horridum by high-throughput sequencing.

Lizards of the Helodermatidae (Anguimorpha) family consist of at least two well recognized species: Heloderma horridum horridum and Heloderma suspectum suspectum. They contain specialized glands in their jaws that produce venomous secretions that causes envenoming symptoms to bitten animals. One way to study proteins from such secretions is by RNA-seq; a powerful molecular tool to characterize the transcriptome of such specialized gland, and its protein secretions. The total RNA from venom gland tissues of H. horridum horridum was extracted and a cDNA library was constructed and sequenced. Overall, 114,172 transcripts were found, and 199 were annotated based on sequence similarities to previously described peptides/proteins. Transcripts coding for putative exendins, defensins, natriuretics and serine protease inhibitors were the most highly expressed. Transcripts that code for several putative serine proteases, phospholipases, metalloproteases, lipases, L-amino oxidase and nucleases were also found. Some of the novel identified transcripts were translationally controlled tumor proteins, venom factors, vespryns, waprins, lectins, cystatins and serine protease inhibitors. All these new protein structures may contribute to a better understanding of the venomous secretions of the Helodermatidae family.

Key amino acid residues involved in mammalian and insecticidal activities of Magi4 and Hv1b, cysteine-rich spider peptides from the δ-atracotoxin family.

δ-Atracotoxins, also known as δ-hexatoxins, are spider neurotoxic peptides, lethal to both vertebrates and insects. Their mechanism of action involves the binding to of the S3/S4 loop of the domain IV of the voltage-gated sodium channels (Nav). Because of the chemical difficulties of synthesizing folded synthetic δ-atracotoxins correctly, here we explore an expression system that is designed to produce biologically active recombinant δ-atracotoxins, and a number of variants, in order to establish certain amino acids implicated in the pharmacophore of this lethal neurotoxin. In order to elucidate and verify which amino acid residues play a key role that is toxic to vertebrates and insects, amino acid substitutes were produced by aligning the primary structures of several lethal δ-atracotoxins with those of δ-atracotoxins-Hv1b; a member of the δ-atracotoxin family that has low impact on vertebrates and is not toxic to insects. Our findings corroborate that the substitutions of the amino acid residue Y22 from δ-atracotoxin-Mg1a (Magi4) to K22 in δ-atracotoxin-Hv1b reduces its mammalian activity. Moreover, the substitutions of the amino acid residues Y22 and N26 from δ-atracotoxin-Mg1a (Magi4) to K22 and N26 in δ-atracotoxin-Hv1b reduces its insecticidal activity. Also, the basic residues K4 and R5 are important for keeping such insecticidal activity. Structural models suggest that such residues are clustered onto two bioactive surfaces, which share similar areas, previously reported as bioactive surfaces for scorpion α-toxins. Furthermore, these bioactive surfaces were also found to be similar to those found in related spider and anemone toxins, which affect the same Nav receptor, indicating that these motifs are important not only for scorpion but may be also for animal toxins that affect the S3/S4 loop of the domain IV of the Nav.

Enhanced Tolerance against a Fungal Pathogen and Insect Resistance in Transgenic Tobacco Plants Overexpressing an Endochitinase Gene from Serratia marcescens.

In this study we cloned a chitinase gene (SmchiC), from Serratia marcescens isolated from the corpse of a Diatraea magnifactella lepidopteran, which is an important sugarcane pest. The chitinase gene SmchiC amplified from the S. marcescens genome was cloned into the transformation vector p2X35SChiC and used to transform tobacco (Nicotiana tabacum L. cv Petit Havana SR1). The resistance of these transgenic plants to the necrotrophic fungus Botrytis cinerea and to the pest Spodoptera frugiperda was evaluated: both the activity of chitinase as well as the resistance against B. cinerea and S. frugiperda was significantly higher in transgenic plants compared to the wild-type.

Pore-forming spider venom peptides show cytotoxicity to hyperpolarized cancer cells expressing K+ channels: A lentiviral vector approach.

Recent studies demonstrated the upregulation of K+ channels in cancer cells. We have previously found that a pore-forming peptide LaFr26, purified from the venom of the Lachesana sp spider, was selectively incorporated into K+ channel expressing hyperpolarized cells. Therefore, it is expected that this peptide would have selective cytotoxicity to hyperpolarized cancer cells. Here we have tested whether LaFr26 and its related peptide, oxyopinin-2b, are selectively cytotoxic to K+ channel expressing cancer cells. These peptides were cytotoxic to the cells, of which resting membrane potential was hyperpolarized. The vulnerabilities of K+ channel-expressing cell lines correlated with their resting membrane potential. They were cytotoxic to lung cancer cell lines LX22 and BEN, which endogenously expressed K+ current. Contrastingly, these peptides were ineffective to glioblastoma cell lines, U87 and T98G, of which membrane potentials were depolarized. Peptides have a drawback, i.e. poor drug-delivery, that hinders their potential use as medicine. To overcome this drawback, we prepared lentiviral vectors that can express these pore-forming peptides and tested the cytotoxicity to K+ channel expressing cells. The transduction with these lentiviral vectors showed autotoxic activity to the channel expressing cells. Our study provides the basis for a new oncolytic viral therapy.

cDNA cloning, heterologous expression, protein folding and immunogenic properties of a phospholipase A2 from Bothrops ammodytoides venom.

A mRNA transcript that codes for a phospholipase (PLA2) was isolated from a single venom gland of the Bothrops ammodytoides viper. The PLA2 transcript was cloned onto a pCR®2.1-TOPO vector and subsequently expressed heterologously in the E. coli strain M15, using the pQE30 vector. The recombinant phospholipase was named rBamPLA2_1, and is composed of an N-terminal fusion protein of 16 residues, along with 122 residues from the mature protein that includes 14 cysteines that form 7 disulfide bonds. Following bacterial expression, rBamPLA2_1 was obtained from inclusion bodies and extracted using a chaotropic agent. rBamPLA2_1 had an experimental molecular mass of 15,692.5 Da that concurred with its theoretical molecular mass. rBamPLA2_1 was refolded in in vitro conditions and after refolding, three main protein fractions with similar molecular masses, were identified. Although, the three fractions were considered to represent different oxidized cystine isoforms, their secondary structures were comparable. All three recombinant isoforms were active on egg-yolk phospholipid and recognized similar cell membrane phospholipids to be native PLA2s, isolated from B. ammodytoides venom. A mixture of the three rBamPLA2_1 cystine isoforms was used to immunize a horse in order to produce serum antibodies (anti-rBamPLA2_1), which partially inhibited the indirect hemolytic activity of B. ammodytoides venom. Although, anti-rBamPLA2_1 antibodies were not able to recognize crotoxin, a PLA2 from the venom of a related but different viper genus, Crotalus durissus terrificus, they recognized PLA2s in other venoms from regional species of Bothrops.

Short-chain consensus alpha-neurotoxin: a synthetic 60-mer peptide with generic traits and enhanced immunogenic properties.

The three-fingered toxin family and more precisely short-chain α-neurotoxins (also known as Type I α-neurotoxins) are crucial in defining the elapid envenomation process, but paradoxically, they are barely neutralized by current elapid snake antivenoms. This work has been focused on the primary structural identity among Type I neurotoxins in order to create a consensus short-chain α-neurotoxin with conserved characteristics. A multiple sequence alignment considering the twelve most toxic short-chain α-neurotoxins reported from the venoms of the elapid genera Acanthophis, Oxyuranus, Walterinnesia, Naja, Dendroaspis and Micrurus led us to propose a short-chain consensus α-neurotoxin, here named ScNtx. The synthetic ScNtx gene was de novo constructed and cloned into the expression vector pQE30 containing a 6His-Tag and an FXa proteolytic cleavage region. Escherichia coli Origami cells transfected with the pQE30/ScNtx vector expressed the recombinant consensus neurotoxin in a soluble form with a yield of 1.5 mg/L of culture medium. The 60-amino acid residue ScNtx contains canonical structural motifs similar to α-neurotoxins from African elapids and its LD50 of 3.8 µg/mice is similar to the most toxic short-chain α-neurotoxins reported from elapid venoms. Furthermore, ScNtx was also able to antagonize muscular, but not neuronal, nicotinic acetylcholine receptors (nAChR). Rabbits immunized with ScNtx were able to immune-recognize short-chain α-neurotoxins within whole elapid venoms. Type I neurotoxins are difficult to isolate and purify from natural sources; therefore, the heterologous expression of molecules such ScNtx, bearing crucial motifs and key amino acids, is a step forward to create common immunogens for developing cost-effective antivenoms with a wider spectrum of efficacy, quality and strong therapeutic value.

Cloning and sequencing of three-finger toxins from the venom glands of four Micrurus species from Mexico and heterologous expression of an alpha-neurotoxin from Micrurus diastema.

The three-finger toxins (3FTxs) represent an extremely diverse protein family in elapid venoms, where the short chain α-neurotoxins are the most relevant toxin group from the clinical point of view. Essentially, the 3FTxs variability and the low proportions of α-neurotoxins in the venoms of North American coral snakes make it difficult to obtain effective elapid antivenoms against the envenomation symptoms caused mainly by these α-neurotoxins. In this work, thirty 3FTx transcript sequences were obtained from the venom glands of four coral snake species from Mexico (M. diastema, M. laticollaris, M. browni and M. tener). The transcripts were mined using a forward oligonucleotide based on the highly conserved signal peptide from the 3FTxs, and four of these transcripts, named MlatA1, B.D, B.E and D.H, encoded for short-chain α-neurotoxins. Additionally, one isoform of the D.H α-neurotoxin transcript was identified in the venom of M. diastema. The mature α-neurotoxin coded in the D.H transcript was heterologously expressed, and it was found soluble (4.2 mg/l) in the cytoplasm of a bacterial system. The recombinant D.H (rD.H) had an IC50 value of 31.5 ±â€¯4.4 nM on nicotinic acetylcholine receptors of the muscular type expressed in rhabdomyosarcoma cells (TE671). The rDH also had an LD50 of 0.15 mg/kg mice, and it was evaluated as a potential immunogen in New Zealand rabbits. The protective capacity of rabbit sera was tested against two native coral snake α-neurotoxins, and against rD.H. One of the anti-rD.H rabbit sera was able to neutralize the lethality of all three neurotoxins when tested in groups of CD1 mice. This work contributes to the increasing understanding of the high diversity of 3FTxs, and shows that recombinant coral snake α-neurotoxins are promising supplements for hyperimmunization protocols for coral snake antivenom production.

Molecular dynamics simulation of the membrane binding and disruption mechanisms by antimicrobial scorpion venom-derived peptides.

Pandinin 2 (Pin2) is an alpha-helical polycationic peptide, identified and characterized from venom of the African scorpion Pandinus imperator with high antimicrobial activity against Gram-positive bacteria and less active against Gram-negative bacteria, however it has demonstrated strong hemolytic activity against sheep red blood cells. In the chemically synthesized Pin2GVG analog, the GVG motif grants it low hemolytic activity while keeping its antimicrobial activity. In this work, we performed 12 µs all-atom molecular dynamics simulation of the antimicrobial peptides (AMPs) Pin2 and Pin2GVG to explore their adsorption mechanism and the role of their constituent amino acid residues when interacting with pure POPC and pure POPG membrane bilayers. Starting from an α-helical conformation, both AMPs are attracted at different rates to the POPC and POPG bilayer surfaces due to the electrostatic interaction between the positively charged amino acid residues and the charged moieties of the membranes. Since POPG is an anionic membrane, the PAMs adhesion is stronger to the POPG membrane than to the POPC membrane and they are stabilized more rapidly. This study reveals that, before the insertion begins, Pin2 and Pin2GVG remained partially folded in the POPC surface during the first 300 and 600 ns, respectively, while they are mostly unfolded in the POPG surface during most of the simulation time. The unfolded structures provide for a large number of intermolecular hydrogen bonds and stronger electrostatic interactions with the POPG surface. The results show that the aromatic residues at the N-terminus of Pin2 initiate the insertion process in both POPC and POPG bilayers. As for Pin2GVG in POPC the C-terminus residues seem to initiate the insertion process while in POPG this process seems to be slowed down due to a strong electrostatic attraction. The membrane conformational effects upon PAMs binding are measured in terms of the area per lipid and the contact surface area. Several replicas of the systems lead to the same observations.

Venoms of Centruroides and Tityus species from Panama and their main toxic fractions.

The scorpionism in Panama is notorious for the confluence and coexistence of buthid scorpions from the genera Centruroides and Tityus. This communication describes an overview of the larger representative toxic venom fractions from eight dangerous buthid scorpion species of Panama: Centruroides (C. granosus, C. bicolor, C. limbatus and C. panamensis) and Tityus (T. (A.) asthenes, T. (A.) festae, T. (T.) cerroazul and T. (A.) pachyurus). Their venoms were separated by HPLC and the corresponding sub-fractions were tested for lethality effects on mice and insects. Many fractions toxic to either mice or insects, or both, were found and have had their molecular masses determined by mass spectrometry analysis. The great majority of the lethal components had a molecular mass close to 7000 Da, assumed to be peptides that recognize Na+-channels, responsible for the toxicity symptoms observed in other buthids scorpion venoms. A toxic peptide isolated from the venom of T. pachyurus was sequenced by Edman degradation, allowing the synthesis of nucleotide probe for cloning the correspondent gene. The mature toxin based on the cDNA sequencing has the C-terminal residue amidated, contains 62 amino acid packed by 4 disulfide linkages, with molecular mass of 7099.1 Da. This same toxic peptide seems to be present in scorpions of the species T. pachyurus collected in 5 different regions of Panama, although the overall HPLC profile is quite different. The most diverse neurotoxic venom components from the genus Centruroides were found in the species C. panamensis, whereas T. cerroazul was the one from the genus Tityus. The most common neurotoxins were observed in the venoms of T. festae, T. asthenes and T. pachyurus with closely related molecular masses of 7099.1 and 7332 Da. The information reported here is considered very important for future generation of a neutralizing antivenom against scorpions from Panama. Furthermore, it will contribute to the growing interest in using bioactive toxins from scorpions for drug discovery purposes.

Proteomic and transcriptomic analysis of saliva components from the hematophagous reduviid Triatoma pallidipennis.

Species belonging to the Triatominae subfamily are commonly associated with Chagas disease, as they are potential vectors of the parasite Trypanosoma cruzi. However, their saliva contains a cocktail of diverse anti-hemostatic proteins that prevent blood coagulation, vasodilation and platelet aggregation of blood; components with indisputable therapeutic potential. We performed a transcriptomic and proteomic analyses of salivary glands and protein spots from 2DE gels of milked saliva, respectively, from the Mexican Triatoma pallidipennis. Massive sequencing techniques were used to reveal this protein diversity. A total of 78 out of 233 transcripts were identified as proteins in the saliva, divided among 43 of 55 spots from 2DE gels of saliva, identified by LC-MS/MS analysis. Some of the annotated transcripts putatively code for anti-hemostatic proteins, which share sequence similarities with proteins previously described for South American triatomines. The most abundant as well as diverse transcripts and proteins in the saliva were the anti-hemostatic triabins. For the first time, a transcriptomic analysis uncovered other unrelated but relevant components in triatomines, including antimicrobial and thrombolytic polypeptides. Likewise, unique proteins such as the angiotensin-converting enzyme were identified not just in the salivary gland transcriptome but also at saliva proteome of this North American bloodsucking insect. BIOLOGICAL SIGNIFICANCE: This manuscript is the first report of the correlation between proteome and transcriptome of Triatoma pallidipennis, which shows for the first time the presence of proteins in this insect that have not been characterized in other species of this family. This information contributes to a better understanding of the multiple host defense mechanisms that are being affected at the moment of blood ingestion by the insect. Furthermore, this report gives a repertoire of possible therapeutic proteins.

Selected scorpion toxin exposures induce cytokine release in human peripheral blood mononuclear cells.

A cytokine screening on human peripheral blood mononuclear cells (PBMCs) stimulated with selected scorpion toxins (ScTx's) was performed in order to evaluate their effect on human immune cells. The ScTx's chosen for this report were three typical buthid scorpion venom peptides, one with lethal effects on mammals Centruroides suffussus suffusus toxin II (CssII), another, with lethal effects on insects and crustaceans Centruroides noxius toxin 5 (Cn5), and one more without lethal effects Tityus discrepans toxin (Discrepin). A Luminex multiplex analysis was performed in order to determine the amounts chemokines and cytokines IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12-p40, IL-13, interferon alpha (IFN-α), interferon gamma (IFN-γ), tumor necrosis factor alpha TNF-α, and interferon-inducible protein-10 (IP-10) secreted from human PBMCs exposed to these toxins. Although, the ScTx Cn5 is not lethal for mammals, it was able to induce the secretion of cytokines IL-1ß, IL-6, and TNF-α, IL-10 and IP-10 in comparison to the lethal CssII, which was able to induce only IP-10 secretion. Discrepin also was able to induce only IP-10. Interestingly, only low amounts of interferons α and ß were induced in the presence of the ScTx's assayed. In a synergic experiment, the combination of Discrepin and Cn5 displayed considerable reverse effects on induction of IL-1ß, IL-6, IL-10 and TNF-α, but they had a slight synergic effect on IP-10 cytokine production in comparison with the single effect obtained with the Cn5 alone. Thus, the results obtained suggest that the profile of secreted cytokines promoted by ScTx Cn5 is highly related with a cytokine storm event, and also it suggests that the mammalian lethal neurotoxins are not solely responsible of the scorpion envenomation symptomatology.

Panusin represents a new family of ß-defensin-like peptides in invertebrates.

Beta_defensin have been solely found in vertebrates until ß-defensin-like peptides were described as transcript isoforms in two species of Panulirus genus. They were considered as putative antimicrobials since their biological activity have not been demonstrated. Here we purified and characterized a defensin-like peptide from the hemocytes of spiny lobster P. argus, hereafter named panusin. Structurally, panusin presents a cysteine-stabilized α/ß motif, and is prone to form homodimers. Biological activity of panusin showed broad-spectrum antimicrobial activity, characterized for being strikingly salt-resistant. Panusin did not showed hemolytic activity but was demonstrated its binding capacity to different lipid membrane models, indicating amphipathicity of ß-sheet core as driving force for its antimicrobial activity. Panusin is considered a new kind of arthropod defensin which share structural and biological features with beta-defensin from vertebrates. The presence of beta-defensin like peptides in crustacean might suggest the emergence of the evolutionary relationship of ß-defensins from vertebrates.

Synthetic peptide antigens derived from long-chain alpha-neurotoxins: Immunogenicity effect against elapid venoms.

Three-finger toxins (3FTXs), especially α-neurotoxins, are the most poorly neutralized elapid snake toxins by current antivenoms. In this work, the conserved structural similarity and motif arrangements of long-chain α-neurotoxins led us to design peptides with consensus sequences. Eight long-chain α-neurotoxins (also known as Type II) were used to generate a consensus sequence from which two peptides were chemically synthesized, LCP1 and LCP2. Rabbit sera raised against them were able to generate partially-neutralizing antibodies, which delayed mice mortality in neutralization assays against Naja haje, Dendrospis polylepis and Ophiophagus hannah venoms.