In-Depth Venome of the Brazilian Rattlesnake Crotalus durissus terrificus: An Integrative Approach Combining Its Venom Gland Transcriptome and Venom Proteome.

Snake venoms are complex mixtures mainly composed of proteins and small peptides. Crotoxin is one of the most studied components from Crotalus venoms, but many other components are less known due to their low abundance. The venome of Crotalus durissus terrificus, the most lethal Brazilian snake, was investigated by combining its venom gland transcriptome and proteome to create a holistic database of venom compounds unraveling novel toxins. We constructed a cDNA library from C. d. terrificus venom gland using the Illumina platform and investigated its venom proteome through high resolution liquid chromotography-tandem mass spectrometry. After integrating data from both data sets, more than 30 venom components classes were identified by the transcriptomic analysis and 15 of them were detected in the venom proteome. However, few of them (PLA2, SVMP, SVSP, and VEGF) were relatively abundant. Furthermore, only seven expressed transcripts contributed to ∼82% and ∼73% of the abundance in the transcriptome and proteome, respectively. Additionally, novel venom proteins are reported, and we highlight the importance of using different databases to perform the data integration and discuss the structure of the venom components-related transcripts identified. Concluding, this research paves the way for novel investigations and discovery of future pharmacological agents or targets in the antivenom therapy.

Immunosuppressive evidence of Tityus serrulatus toxins Ts6 and Ts15: insights of a novel K(+) channel pattern in T cells.

The voltage-gated potassium channel Kv1.3 is a novel target for immunomodulation of autoreactive effector memory T cells, which play a major role in the pathogenesis of autoimmune diseases. In this study, the Ts6 and Ts15 toxins isolated from Tityus serrulatus (Ts) were investigated for their immunosuppressant roles on CD4(+) cell subsets: naive, effector (TEF ), central memory (TCM) and effector memory (TEM). The electrophysiological assays confirmed that both toxins were able to block Kv1.3 channels. Interestingly, an extended Kv channel screening shows that Ts15 blocks Kv2.1 channels. Ts6 and Ts15 significantly inhibit the proliferation of TEM cells and interferon-γ production; however, Ts15 also inhibits other CD4(+) cell subsets (naive, TEF and TCM). Based on the Ts15 inhibitory effect of proliferation of all CD4(+) cell subsets, and based on its blocking effect on Kv2.1, we investigated the Kv2.1 expression in T cells. The assays showed that CD4(+) and CD8(+) cells express the Kv2.1 channels mainly extracellularly with TCM cells expressing the highest number of Kv2.1 channels. We also provide in vivo experimental evidence to the protective effect of Ts6 and Ts15 on delayed-type hypersensitivity reaction. Altogether, this study presents the immunosuppressive behaviour of Ts6 and Ts15 toxins, indicating that these toxins could be promising candidates for autoimmune disease therapy. Moreover, this is the first report illustrating the involvement of a novel K(+) channel subtype, Kv2.1, and its distribution in T-cell subsets.

Moojenactivase, a novel pro-coagulant PIIId metalloprotease isolated from Bothrops moojeni snake venom, activates coagulation factors II and X and induces tissue factor up-regulation in leukocytes.

Coagulopathies following snakebite are triggered by pro-coagulant venom toxins, in which metalloproteases play a major role in envenomation-induced coagulation disorders by acting on coagulation cascade, platelet function and fibrinolysis. Considering this relevance, here we describe the isolation and biochemical characterization of moojenactivase (MooA), a metalloprotease from Bothrops moojeni snake venom, and investigate its involvement in hemostasis in vitro. MooA is a glycoprotein of 85,746.22 Da, member of the PIIId group of snake venom metalloproteases, composed of three linked disulfide-bonded chains: an N-glycosylated heavy chain, and two light chains. The venom protease induced human plasma clotting in vitro by activating on both blood coagulation factors II (prothrombin) and X, which in turn generated α-thrombin and factor Xa, respectively. Additionally, MooA induced expression of tissue factor (TF) on the membrane surface of peripheral blood mononuclear cells (PBMC), which led these cells to adopt pro-coagulant characteristics. MooA was also shown to be involved with production of the inflammatory mediators TNF-α, IL-8 and MCP-1, suggesting an association between MooA pro-inflammatory stimulation of PBMC and TF up-regulation. We also observed aggregation of washed platelets when in presence of MooA; however, the protease had no effect on fibrinolysis. Our findings show that MooA is a novel hemostatically active metalloprotease, which may lead to the development of coagulopathies during B. moojeni envenomation. Moreover, the metalloprotease may contribute to the development of new diagnostic tools and pharmacological approaches applied to hemostatic disorders.

Hyperglycosylation impairs the inhibitory activity of rCdtPLI2, the first recombinant beta-phospholipase A2 inhibitor.

Crotoxin, a phospholipase A2 (PLA2) complex and the major Crotalus venom component, is responsible for the main symptoms described in crotalic snakebite envenomings and a key target for PLA2 inhibitors (PLIs). PLIs comprise the alpha, beta and gamma families, and, due to a lack of reports on beta-PLIs, this study aimed to heterologously express CdtPLI2 from Crotalus durissus terrificus venom gland to improve the knowledge of the neglected beta-PLI family. Thereby, recombinant CdtPLI2 (rCdtPLI2) was produced in the eukaryotic Pichia pastoris system to keep some native post-translational modifications. rCdtPLI2 (~41 kDa) presents both N- and O-linked glycans. Alpha-mannosidase digested-rCdtPLI2 (1 mol) strongly inhibited (73%) CB-Cdc catalytic activity (5 moles), demonstrating that glycosylations performed by P. pastoris affect rCdtPLI2 action. Digested-rCdtPLI2 also inhibited PLA2s from diverse Brazilian snake venoms. Furthermore, rCdtPLI2 (1 mol) abolished the catalytic activity of Lmr-PLA2 (5 moles) and reduced the CTx-Cdc (5 moles) enzyme activity by 65%, suppressing basic and acidic snake venom PLA2s. Additionally, crotalic antivenom did not recognize rCdtPLI2, suggesting a lack of neutralization by antivenom antibodies. These findings demonstrate that studying snake venom components may reveal interesting novel molecules to be studied in the snakebite treatment and help to understand these underexplored inhibitors.

The complex repertoire of Tityus spp. venoms: Advances on their composition and pharmacological potential of their toxins.

Animal venoms are a rich and complex source of components, including peptides (such as neurotoxins, anionic peptides and hypotensins), lipids, proteins (such as proteases, hyaluronidases and phospholipases) and inorganic compounds, which affect all biological systems of the envenoming victim. Their action may result in a wide range of clinical manifestations, including tachy/bradycardia, hyper/hypotension, disorders in blood coagulation, pain, edema, inflammation, fever, muscle paralysis, coma and even death. Scorpions are one of the most studied venomous animals in the world and interesting bioactive molecules have been isolated and identified from their venoms over the years. Tityus spp. are among the scorpions with high number of accidents reported in the Americas, especially in Brazil. Their venoms have demonstrated interesting results in the search for novel agents with antimicrobial, anti-viral, anti-parasitic, hypotensive, immunomodulation, anti-insect, antitumor and/or antinociceptive activities. Furthermore, other recent activities still under investigation include drug delivery action, design of anti-epileptic drugs, investigation of sodium channel function, treatment of erectile disfunction and priapism, improvement of scorpion antivenom and chelating molecules activity. In this scenario, this paper focuses on reviewing advances on Tityus venom components mainly through the modern omics technologies as well as addressing potential therapeutic agents from their venoms and highlighting this abundant source of pharmacologically active molecules with biotechnological application.

Do Antibiotics Potentiate Proteases in Hemotoxic Snake Venoms?

Antibiotics are often administered with antivenom following snakebite envenomings in order to avoid secondary bacterial infections. However, to this date, no studies have evaluated whether antibiotics may have undesirable potentiating effects on snake venom. Herein, we demonstrate that four commonly used antibiotics affect the enzymatic activities of proteolytic snake venom toxins in two different in vitro assays. Similar findings in vivo could have clinical implications for snakebite management and require further examination.

Identification of cross-reactive human single-chain variable fragments against phospholipases A2 from Lachesis muta and Bothrops spp venoms.

Bushmasters (Lachesis spp) and lancehead vipers (Bothrops spp) are two of the most dangerous snakes found in Latin America. Victims of envenoming by these snakes require urgent administration of antivenom. Here, we report the identification of a small set of broadly neutralizing human monoclonal single-chain variable fragment (scFv) antibodies targeting key phospholipases A2 from Lachesis and Bothrops spp using phage display technology and demonstrate their in vitro efficacy using a hemolysis assay.

Scorpion Venom: Detriments and Benefits.

Scorpion venom may cause severe medical complications and untimely death if injected into the human body. Neurotoxins are the main components of scorpion venom that are known to be responsible for the pathological manifestations of envenoming. Besides neurotoxins, a wide range of other bioactive molecules can be found in scorpion venoms. Advances in separation, characterization, and biotechnological approaches have enabled not only the development of more effective treatments against scorpion envenomings, but have also led to the discovery of several scorpion venom peptides with interesting therapeutic properties. Thus, scorpion venom may not only be a medical threat to human health, but could prove to be a valuable source of bioactive molecules that may serve as leads for the development of new therapies against current and emerging diseases. This review presents both the detrimental and beneficial properties of scorpion venom toxins and discusses the newest advances within the development of novel therapies against scorpion envenoming and the therapeutic perspectives for scorpion toxins in drug discovery.

Unity Makes Strength: Exploring Intraspecies and Interspecies Toxin Synergism between Phospholipases A2 and Cytotoxins.

Toxin synergism is a complex biochemical phenomenon, where different animal venom proteins interact either directly or indirectly to potentiate toxicity to a level that is above the sum of the toxicities of the individual toxins. This provides the animals possessing venoms with synergistically enhanced toxicity with a metabolic advantage, since less venom is needed to inflict potent toxic effects in prey and predators. Among the toxins that are known for interacting synergistically are cytotoxins from snake venoms, phospholipases A2 from snake and bee venoms, and melittin from bee venom. These toxins may derive a synergistically enhanced toxicity via formation of toxin complexes by hetero-oligomerization. Using a human keratinocyte assay mimicking human epidermis in vitro, we demonstrate and quantify the level of synergistically enhanced toxicity for 12 cytotoxin/melittin-PLA2 combinations using toxins from elapids, vipers, and bees. Moreover, by utilizing an interaction-based assay and by including a wealth of information obtained via a thorough literature review, we speculate and propose a mechanistic model for how toxin synergism in relation to cytotoxicity may be mediated by cytotoxin/melittin and PLA2 complex formation.

Pioneering Study on Rhopalurus crassicauda Scorpion Venom: Isolation and Characterization of the Major Toxin and Hyaluronidase.

Scorpionism is responsible for most accidents involving venomous animals in Brazil, which leads to severe symptoms that can evolve to death. Scorpion venoms consist of complexes cocktails, including peptides, proteins, and non-protein compounds, making separation and purification procedures extremely difficult and time-consuming. Scorpion toxins target different biological systems and can be used in basic science, for clinical, and biotechnological applications. This study is the first to explore the venom content of the unexplored scorpion species Rhopalurus crassicauda, which inhabits exclusively the northernmost state of Brazil, named Roraima, and southern region of Guyana. Here, we pioneer the fractionation of the R. crassicauda venom and isolated and characterized a novel scorpion beta-neurotoxin, designated Rc1, and a monomeric hyaluronidase. R. crassicauda venom and Rc1 (6,882 Da) demonstrated pro-inflammatory activities in vitro and a nociceptive response in vivo. Moreover, Rc1 toxin showed specificity for activating Nav1.4, Nav1.6, and BgNav1 voltage-gated ion channels. This study also represents a new perspective for the treatment of envenomings in Roraima, since the Brazilian scorpion and arachnid antivenoms were not able to recognize R. crassicauda venom and its fractions (with exception of hyaluronidase). Our work provides useful insights for the first understanding of the painful sting and pro-inflammatory effects associated with R. crassicauda envenomings.

EP80317 Restrains Inflammation and Mortality Caused by Scorpion Envenomation in Mice.

Over 1 million cases of scorpion stings are estimated every year, whereas current treatment is limited to antivenom serum combined with supportive therapy. Tityus serrulatus scorpion venom (TsV) is composed of diverse molecules, including toxins that induce a catecholamine storm and mediate classical symptoms of scorpion envenomation. However, the same toxins promote an intense inflammatory response coordinated by innate immune cells, such as macrophages, contributing significantly to the lung edema and mortality caused by TsV injection. Macrophages sense TsV via innate immune receptors, including TLR2, TLR4, and CD14 that promote inflammation and mortality via PGE2/cAMP/PKA/NF-κB/IL-1ß axis. The scavenger receptor CD36 also recognizes TsV, but in contrast to the other receptors, it drives the production of leukotriene B4 (LTB4). This lipid mediator operates via BLT1 receptor to reduce cAMP production and consequently IL-1ß release, which results in resistance to fatal outcomes of experimental scorpion envenomation. EP80317 is an hexapeptide that serves as a ligand for CD36 and features protective effects under conditions such as atherosclerosis and vascular inflammation. In this study, we evaluated the effects of EP80317 treatment during experimental scorpion envenomation. EP80317 treatment suppressed mouse peritoneal macrophage production of IL-1ß, IL-6, tumor necrosis factor (TNF-α), CCL3, and PGE2 in vitro. EP80317 treatment also boosted the production of LTB4 and IL-10 in response to TsV. Importantly, EP80317 restrained lung inflammation and mortality caused by TsV in vivo. Taken together, these data indicate a strong therapeutic potential of EP80317 as a supportive treatment to control inflammation induced by scorpion envenomation.

Subproteome of Lachesis muta rhombeata venom and preliminary studies on LmrSP-4, a novel snake venom serine proteinase.

BACKGROUND: Lachesis muta rhombeata is one of the venomous snakes of medical importance in Brazil whose envenoming is characterized by local and systemic effects which may produce even shock and death. Its venom is mainly comprised of serine and metalloproteinases, phospholipases A2 and bradykinin-potentiating peptides. Based on a previously reported fractionation of L. m. rhombeata venom (LmrV), we decided to perform a subproteome analysis of its major fraction and investigated a novel component present in this venom. METHODS: LmrV was fractionated through molecular exclusion chromatography and the main fraction (S5) was submitted to fibrinogenolytic activity assay and fractionated by reversed-phase chromatography. The N-terminal sequences of the subfractions eluted from reversed-phase chromatography were determined by automated Edman degradation. Enzyme activity of LmrSP-4 was evaluated upon chromogenic substrates for thrombin (S-2238), plasma kallikrein (S-2302), plasmin and streptokinase-activated plasminogen (S-2251) and Factor Xa (S-2222) and upon fibrinogen. All assays were carried out in the presence or absence of possible inhibitors. The fluorescence resonance energy transfer substrate Abz-KLRSSKQ-EDDnp was used to determine the optimal conditions for LmrSP-4 activity. Molecular mass of LmrSP-4 was determined by MALDI-TOF and digested peptides after trypsin and Glu-C treatments were analyzed by high resolution MS/MS using different fragmentation modes. RESULTS: Fraction S5 showed strong proteolytic activity upon fibrinogen. Its fractionation by reversed-phase chromatography gave rise to 6 main fractions (S5C1-S5C6). S5C1-S5C5 fractions correspond to serine proteinases whereas S5C6 represents a C-type lectin. S5C4 (named LmrSP-4) had its N-terminal determined by Edman degradation up to the 53rd amino acid residue and was chosen for characterization studies. LmrSP-4 is a fibrinogenolytic serine proteinase with high activity against S-2302, being inhibited by PMSF and benzamidine, but not by 1,10-phenantroline. In addition, this enzyme exhibited maximum activity within the pH range from neutral to basic and between 40 and 50 °C. About 68% of the LmrSP-4 primary structure was covered, and its molecular mass is 28,190 Da. CONCLUSIONS: Novel serine proteinase isoforms and a lectin were identified in LmrV. Additionally, a kallikrein-like serine proteinase that might be useful as molecular tool for investigating bradykinin-involving process was isolated and partially characterized.

Scorpion toxins targeting Kv1.3 channels: insights into immunosuppression.

Scorpion venoms are natural sources of molecules that have, in addition to their toxic function, potential therapeutic applications. In this source the neurotoxins can be found especially those that act on potassium channels. Potassium channels are responsible for maintaining the membrane potential in the excitable cells, especially the voltage-dependent potassium channels (Kv), including Kv1.3 channels. These channels (Kv1.3) are expressed by various types of tissues and cells, being part of several physiological processes. However, the major studies of Kv1.3 are performed on T cells due its importance on autoimmune diseases. Scorpion toxins capable of acting on potassium channels (KTx), mainly on Kv1.3 channels, have gained a prominent role for their possible ability to control inflammatory autoimmune diseases. Some of these toxins have already left bench trials and are being evaluated in clinical trials, presenting great therapeutic potential. Thus, scorpion toxins are important natural molecules that should not be overlooked in the treatment of autoimmune and other diseases.

Insights into the structure, function and stability of bordonein-L, the first L-amino acid oxidase from Crotalus durissus terrificus snake venom.

Snake venom L-amino acid oxidases (svLAAOs) are an interesting class of enzymes with important biological activities. Their participation in key metabolic processes, including pathological disorders, suggest that svLAAOs are potential lead compounds in drug discovery. However, their short-term stability defies their applications. This paper describes the stability studies together with functional and structural characterization of the LAAO bordonein-L. It has 498 amino acid residues, one N-glycosylation site and two disulfide bonds, revealed by high-resolution MS/MS. Molecular modeling approach showed its monomer folds into three conserved domains: FAD, substrate and helical domains. Differential scanning fluorimetry showed the enzyme tends to destabilize from neutral to basic pHs and in presence of mono/bivalent ions and it is highly stabilized by acid pHs and its substrates. However, high concentrations of L-amino acids decrease bordonein-L enzyme activity. Dynamic light scattering revealed bordonein-L remains in the dimeric and monodisperse form, so aggregation does not cause the rapidly decrease of enzyme activity. In vitro, the enzyme exhibited cytotoxicity against fibroblast cell line and killed Leishmania amazonensis promastigotes, intensified by substrate addition. Concluding, our results provide biochemistry and biophysical insights to improve LAAOs stability and better approaches to long-term storage. Moreover, our study emphasizes the importance of proper buffers choice mainly in cell-based assays.

A new acidic myotoxic, anti-platelet and prostaglandin I2 inductor phospholipase A2 isolated from Bothrops moojeni snake venom.

Phospholipase A2 (PLA2, EC 3.1.1.4), a major component of snake venoms, specifically catalyzes the hydrolysis of fatty acid ester bonds at position 2 of 1,2-diacyl-sn-3-phosphoglycerides in the presence of calcium. This article reports the purification and biochemical/functional characterization of BmooTX-I, a new myotoxic acidic phospholipase A2 from Bothrops moojeni snake venom. The purification of the enzyme was carried out through three chromatographic steps (ion-exchange on DEAE-Sepharose, molecular exclusion on Sephadex G-75 and hydrophobic chromatography on Phenyl-Sepharose). BmooTX-I was found to be a single-chain protein of 15,000 Da and pI 4.2. The N-terminal sequence revealed a high homology with other acidic Asp49 PLA2s from Bothrops snake venoms. It displayed a high phospholipase activity and platelet aggregation inhibition induced by collagen or ADP. Edema and myotoxicity in vivo were also induced by BmooTX-I. Analysis of myotoxic activity was carried out by optical and ultrastructural microscopy, demonstrating high levels of leukocytary infiltrate. Previous treatment of BmooTX-I with BPB reduced its enzymatic and myotoxic activities, as well as the effect on platelet aggregation. Acidic myotoxic PLA2s from Bothrops snake venoms have been little explored and the knowledge of its structural and functional features will be able to contribute for a better understanding of their action mechanism regarding enzymatic and toxic activities.

CD36 Shunts Eicosanoid Metabolism to Repress CD14 Licensed Interleukin-1ß Release and Inflammation.

Interleukin (IL)-1ß is a potential target for treatment of several inflammatory diseases, including envenomation by the scorpion Tityus serrulatus. In this context, bioactive lipids such as prostaglandin (PG)E2 and leukotriene (LT)B4 modulate the production of IL-1ß by innate immune cells. Pattern recognition receptors (PRRs) that perceive T. serrulatus venom (TsV), and orchestrate LTB4, PGE2, and cyclic adenosine monophosphate (cAMP) production to regulate IL-1ß release are unknown. Furthermore, molecular mechanisms driving human cell responses to TsV remain uncharacterized. Here, we identified that both CD14 and CD36 control the synthesis of bioactive lipids, inflammatory cytokines, and mortality mediated by TsV. CD14 induces PGE2/cAMP/IL-1ß release and inflammation. By contrast, CD36 shunts eicosanoid metabolism toward production of LTB4, which represses the PGE2/cAMP/IL-1ß axis and mortality. Of importance, the molecular mechanisms observed in mice strongly correlate with those of human cell responses to TsV. Overall, this study provides major insights into molecular mechanisms connecting CD14 and CD36 with differential eicosanoid metabolism and inflammation mediated by IL-1ß.

Platelet activating factor receptors drive CXC chemokine production, neutrophil influx and edema formation in the lungs of mice injected with Tityus serrulatus venom.

Lung injury is a common finding and a frequent cause of death in cases of severe human envenoming by scorpion sting. The present work investigated the effects of pretreatment with a platelet activation factor receptor (PAFR) antagonist and a CXCR2 inhibitor on the lung injury induced by subcutaneous injection of Tityus serrulatus venom (TsV) in mice. Lung injury was assessed by evaluating the extravasation of Evans blue dye, as an index of increased vascular permeability, the neutrophil accumulation (mieloperoxidase activity), the concentration of tumor necrosis factor-alpha (TNF-alpha) and the chemokine KC in the lung after TsV administration. Neutrophil influx was preceded by the production of KC and dependent on CXCR2, as shown by the ability of repertaxin, a CXCR2 inhibitor, to prevent an increase of MPO activity in the lung. Repertaxin had no effect on TsV-induced lethality. The PAFR antagonist (UK-74,505) significantly reduced TsV-induced vascular permeability changes and neutrophil influx in the lungs. The inhibition of neutrophil influx was associated with inhibition of the production of the CXCR2-active chemokine KC. UK-74,505 had no effect on the lethality induced by TsV. In conclusion, these results show that the influx of neutrophils in the lungs of mice injected with TsV is dependent on the activation of PAFR and on PAFR-dependent production of the chemokine KC as well as activation of CXCR2 on neutrophils. Although lung injury may contribute to late lethality after TsV envenoming, acute lethality is not modified by inhibitors of neutrophil influx.

Antifungal Activity against Filamentous Fungi of Ts1, a Multifunctional Toxin from Tityus serrulatus Scorpion Venom.

Antimicrobial peptides (AMPs) are ubiquitous and multipotent components of the innate immune defense arsenal used by both prokaryotic and eukaryotic organisms. The search for new AMPs has increased in recent years, due to the growing development of microbial resistance to therapeutical drugs. In this work, we evaluate the effects of Tityus serrulatus venom (Tsv), its fractions and its major toxin Ts1, a beta-neurotoxin, on fungi growth. The fractions were obtained by ion-exchange chromatography of Tsv. The growth inhibition of 11 pathogenic and non-pathogenic filamentous fungi (Aspergillus fumigatus, A. nidulans, A. niger, A. terreus, Neurospora crassa, Penicillium corylophilum, P. ochrochloron, P. verrucosum, P. viridicatum, P. waksmanii, and Talaromyces flavus) was evaluated by quantitative microplate reader assay. Tsv (100 and 500 µg/well, which correspond to 1 and 5 mg/mL, respectively, of total soluble protein) was active in inhibiting growth of A. nidulans, A. terreus, P. corylophilum, and P. verrucosum, especially in the higher concentration used and at the first 30 h. After this period, fungi might have used Tsv components as alternative sources of nutrients, and therefore, increased their growth tax. Only fractions IX, X, XI, XIIA, XIIB (3 and 7.5 µg/well, which correspond to 30 and 75 µg/mL, respectively, of total soluble protein) and Ts1 (1.5, 3, and 6 µg/well, which correspond to 2.18, 4.36, and 8.72 µM, respectively) showed antifungal activity. Ts1 showed to be a non-morphogenic toxin with dose-dependent activity against A. nidulans, inhibiting 100% of fungal growth from 3 µg/well (4.36 µM). The inhibitory effect of Ts1 against A. nidulans growth was accompanied by fungistatic effects and was not amended by 1 mM CaCl2 or tetrodotoxin (46.98 and 93.96 µM). The structural differences between Ts1 and drosomycin, a potent cysteine-rich antifungal peptide, are discussed here. Our results highlight the antifungal potential of the first cysteine-containing scorpion toxin. Since Ts1 is a multifunctional toxin, we suggest that it could be used as a template in the design of engineered scorpion AMPs and in the search for new mechanisms of action of antifungal drugs.

Investigating possible biological targets of Bj-CRP, the first cysteine-rich secretory protein (CRISP) isolated from Bothrops jararaca snake venom.

Cysteine-rich secretory proteins (CRISPs) are commonly described as part of the protein content of snake venoms, nevertheless, so far, little is known about their biological targets and functions. Our study describes the isolation and characterization of Bj-CRP, the first CRISP isolated from Bothrops jararaca snake venom, also aiming at the identification of possible targets for its actions. Bj-CRP was purified using three chromatographic steps (Sephacryl S-200, Source 15Q and C18) and showed to be an acidic protein of 24.6kDa with high sequence identity to other snake venom CRISPs. This CRISP was devoid of proteolytic, hemorrhagic or coagulant activities, and it did not affect the currents from 13 voltage-gated potassium channel isoforms. Conversely, Bj-CRP induced inflammatory responses characterized by increase of leukocytes, mainly neutrophils, after 1 and 4h of its injection in the peritoneal cavity of mice, also stimulating the production of IL-6. Bj-CRP also acted on the human complement system, modulating some of the activation pathways and acting directly on important components (C3 and C4), thus inducing the generation of anaphylatoxins (C3a, C4a and C5a). Therefore, our results for Bj-CRP open up prospects for better understanding this class of toxins and its biological actions.

Mediators involved in the febrile response induced by Tityus serrulatus scorpion venom in rats.

Tityus serrulatus venom (Tsv) was intraperitoneally (ip) injected at doses of 75, 150 and 300mug/kg and IL-1beta (2.0 microg/kg) was given intravenously (iv) to male Wistar rats. Rectal temperature was measured by radiotelemetry. Vagotomy was performed according to Bluthe et al. [1994. Lipopolysaccharide induces sickness behaviour in rats by a vagal mediated mechanism. C R Acad. Sci. 317(6), 499-503]. Cerebrospinal fluid (CSF) and peritoneal fluid (PF) levels of bradykinin (BK) were measured by ELISA. B(1) (des-Arg(9)-[Leu(8)]-BK; DALBK) and B(2) kinin receptor (icatibant) antagonists (1.0 mg/kg each), the induced nitric oxide synthase inhibitor aminoguanidine (50.0 mg/kg), the neuronal nitric oxide synthase inhibitor 7-nitroindazole (30.0 mg/kg), the dual cyclooxygenase inhibitor ibuprofen (10.0 mg/kg), the selective interleukin-1 receptor antagonist IL-ra (2.0 mg/kg) and dipyrone (120 mg/kg) were given ip. Celecoxib (5 mg/kg) was given per os (po). Tsv at doses of 75 microg/kg evoked no change in rectal temperature while at doses of 150 and 300 microg/kg it promoted long-lasting fever (2 degrees C+/-0.1). Tsv (150 microg/kg) increased by nearly 3 and 5 times, respectively BK concentration in the CSF and in the PF. Subdiaphragmatic vagotomy or 7-nitroindazole reduced, icatibant, DALBK, IL-1ra, aminoguanidine and dipyrone abolished, while ibuprofen and celecoxib failed to affect Tsv-induced fever. These results suggest that PGs do not play a relevant role, whereas, kinins via their B(1) and B(2) receptors, IL-1, nitric oxide and vagal neurotransmission are involved in Tsv-induced fever.