Expression, purification and virucidal activity of two recombinant isoforms of phospholipase A2 from Crotalus durissus terrificus venom.

The global emergence and re-emergence of arthropod-borne viruses (arboviruses) over the past four decades have become a public health crisis of international concern, especially in tropical and subtropical countries. A limited number of vaccines against arboviruses are available for use in humans; therefore, there is an urgent need to develop antiviral compounds. Snake venoms are rich sources of bioactive compounds with potential for antiviral prospection. The major component of Crotalus durissus terrificus venom is a heterodimeric complex called crotoxin, which is constituted by an inactive peptide (crotapotin) and a phospholipase A2 (PLA2-CB). We showed previously the antiviral effect of PLA2-CB against dengue virus, yellow fever virus and other enveloped viruses. The aims of this study were to express two PLA2-CB isoforms in a prokaryotic system and to evaluate their virucidal effects. The sequences encoding the PLA2-CB isoforms were optimized and cloned into a plasmid vector (pG21a) for recombinant protein expression. The recombinant proteins were expressed in the E. coli BL21(DE3) strain as insoluble inclusion bodies; therefore, the purification was performed under denaturing conditions, using urea for protein solubilization. The solubilized proteins were applied to a nickel affinity chromatography matrix for binding. The immobilized recombinant proteins were subjected to an innovative protein refolding step, which consisted of the application of a decreasing linear gradient of urea and dithiothreitol (DTT) concentrations in combination with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS) as a protein stabilizer. The refolded recombinant proteins showed phospholipase activity and virucidal effects against chikungunya virus, dengue virus, yellow fever virus and Zika virus.

Sacha inchi seeds from sub-tropical cultivation: effects of roasting on antinutrients, antioxidant capacity and oxidative stability.

Due to the strong bitter taste, sacha inchi seeds are usually consumed after roasting, which also contributes to the elimination of antinutrients. Sacha inchi plants fully adapted to cultivation under sub-tropical climate conditions were produced in southeastern Brazil. Our main goal was to evaluate the effect of dry heating (roasting) on the antinutrient content of these seeds. We also investigated the effects of the applied roasting treatments on the antioxidant activity, proximate composition and oxidative stability of the seeds. To the best of our knowledge, this is the first report on antinutrients of sacha inchi seeds cultivated under sub-tropical conditions, outside their native tropical environment. Except for saponins, which are not heat-labile compounds, the contents of all assessed antinutrients continually reduced with the increase in roasting temperature. Roasting improved antioxidant activity and phenolic content in the seeds at the highest temperature. Oxidation changes occurred in the seed oil, and they increased with temperature. However, maximum peroxide value was within the acceptable consumption limits. As a conclusion, roasting treatments can be applied to minimize the antinutrient potential in sacha inchi seeds. Knowledge on the composition and proper processing of sacha inchi cultivated under sub-tropical conditions may support future efforts focused on the development of new production areas.

Fraction of C. d. collilineatus venom containing crotapotin protects PC12 cells against MPP + toxicity by activating the NGF-signaling pathway.

Background: Parkinson's disease (PD) is the second most prevalent neurodegenerative disease. There is no effective treatment for neurodegenerative diseases. Snake venoms are a cocktail of proteins and peptides with great therapeutic potential and might be useful in the treatment of neurodegenerative diseases. Crotapotin is the acid chain of crotoxin, the major component of Crotalus durissus collilineatus venom. PD is characterized by low levels of neurotrophins, and synaptic and axonal degeneration; therefore, neurotrophic compounds might delay the progression of PD. The neurotrophic potential of crotapotin has not been studied yet. Methods: We evaluated the neurotrophic potential of crotapotin in untreated PC12 cells, by assessing the induction of neurite outgrowth. The activation of the NGF signaling pathway was investigated through pharmacological inhibition of its main modulators. Additionally, its neuroprotective and neurorestorative effects were evaluated by assessing neurite outgrowth and cell viability in PC12 cells treated with the dopaminergic neurotoxin MPP+ (1-methyl-4-phenylpyridinium), known to induce Parkinsonism in humans and animal models. Results: Crotapotin induced neuritogenesis in PC12 cells through the NGF-signaling pathway, more specifically, by activating the NGF-selective receptor trkA, and the PI3K/Akt and the MAPK/ERK cascades, which are involved in neuronal survival and differentiation. In addition, crotapotin had no cytotoxic effect and protected PC12 cells against the inhibitory effects of MPP+ on cell viability and differentiation. Conclusion: These findings show, for the first time, that crotapotin has neurotrophic/neuroprotective/neurorestorative potential and might be beneficial in Parkinson's disease. Additional studies are necessary to evaluate the toxicity of crotapotin in other cell models.

Snake venom disintegrins update: insights about new findings.

Snake venom disintegrins are low molecular weight, non-enzymatic proteins rich in cysteine, present in the venom of snakes from the families Viperidae, Crotalidae, Atractaspididae, Elapidae, and Colubridae. This family of proteins originated in venom through the proteolytic processing of metalloproteinases (SVMPs), which, in turn, evolved from a gene encoding an A Disintegrin And Metalloprotease (ADAM) molecule. Disintegrins have a recognition motif for integrins in their structure, allowing interaction with these transmembrane adhesion receptors and preventing their binding to proteins in the extracellular matrix and other cells. This interaction gives disintegrins their wide range of biological functions, including inhibition of platelet aggregation and antitumor activity. As a result, many studies have been conducted in an attempt to use these natural compounds as a basis for developing therapies for the treatment of various diseases. Furthermore, the FDA has approved Tirofiban and Eptifibatide as antiplatelet compounds, and they are synthesized from the structure of echistatin and barbourin, respectively. In this review, we discuss some of the main functional and structural characteristics of this class of proteins and their potential for therapeutic use.

Isolation and characterization of the first phosphodiesterase (Bj-PDE) from the venom of Bothrops jararacussu snake.

Phosphodiesterases are exonucleases that sequentially hydrolyse phosphodiester bonds of polynucleotides from the 3'-end and release 5-mononucleotides. After more than one decade without any advance in the study of Bothropic phosphodiesterases, we described here the isolation of the first phosphodiesterase from Bothrops jararacussu, which we named Bj-PDE. A five-step column chromatography procedure (size exclusion, hydrophobic interaction, cation exchange, lentil lectin affinity, and blue sepharose affinity) enabled isolation of Bj-PDE with preserved and stable enzymatic activity (bis(p-nitrophenyl) phosphate substrate), Km = 6.9 mM (± 0.7 mM), kcat/Km = 1.7 × 104 M-1 s-1 (± 0.2 × 104 M-1 s-1), MW = 116 kDa (SDS-PAGE), optimum activity around 45 °C at pH 8.0, and stability for 81 days at different storage temperatures (8, -20, and - 80 °C). Ca2+ and Mg2+ ions positively influenced Bj-PDE activity, while EDTA had the opposite action. Zn2+ restored >50 % of enzyme activity after its inhibition by EDTA. The Bj-PDE partial sequence identified by mass spectrometry was very similar to the sequence of BATXPDE1 from Bothrops atrox, which was evolutionarily close to this new PDE. Therefore, our study represents an important progress on the isolation of this minor toxin and sheds new lights on the properties and bioprospection of bothropic phosphodiesterases.

Effect of proteins isolated from Brazilian snakes on enterovirus A71 replication cycle: An approach against hand, foot and mouth disease.

Enterovirus A71 (EVA71) belongs to the Picornaviridae family and is the main etiological agent of hand, foot, and mouth disease (HFMD). There is no approved antiviral against EVA71, and therefore the search for novel anti-EVA71 therapeutics is essential. In this context, the antiviral activity of proteins isolated from snake venoms has been reported against a range of viruses. Here, the proteins CM10 and CM14 isolated from Bothrops moojeni, and Crotamin and PLA2CB isolated from Crotalus durissus terrificus were investigated for their antiviral activity against EVA71 infection. CM14 and Crotamin possessed a selective index (SI) of 170.8 and 120.4, respectively, while CM10 and PLA2CB had an SI of 67.4 and 12.5, respectively. CM14 inhibited all steps of viral replication (protective effect: 76 %; virucidal: 99 %; and post-entry: 99 %). Similarly, Crotamin inhibited up to 99 % of three steps. In contrast, CM10 and PLA2CB impaired one or two steps of EVA71 replication, respectively. Further dose-response assays using increasing titres of EVA71 were performed and CM14 and Crotamin retained functionality with high concentrations of EVA71 (up to 1000 TCID50). These data demonstrate that proteins isolated from snake venom are potent inhibitors of EVA71 and could be used as scaffolds for future development of novel antivirals.

Roles of Bothrops jararacussu toxins I and II: Antiviral findings against Zika virus.

Zika virus is the etiologic agent of Zika fever, and has been previously associated with cases of microcephaly, drawing the attention of the health authorities worldwide. However, no vaccine or antiviral are currently available. Phospholipases A2 (PLA2) isolated from snake venoms have demonstrated antiviral activity against several viruses. Here we demonstrated the anti-ZIKV activity of bothropstoxins-I and II (BthTX-I and II) isolated from Bothrops jararacussu venom. Vero E6 cells were infected with ZIKVPE243 in the presence of compounds for 72 h, when virus titers were evaluated. BthTX-I and II presented strong dose-dependent inhibition of ZIKV, with a SI of 149.1 and 1.44 × 105, respectively. These toxins mainly inhibited the early stages of the replicative cycle, such as during the entry of ZIKV into host cells, as shown by the potent virucidal effect, suggesting the action of these toxins on the virus particles. Moreover, BthTX-I and II presented significant activity towards post-entry stages of the ZIKV replicative cycle. Molecular docking analyses showed that BthTX-I and II potentially interact with DII and DIII domains from ZIKV Envelope protein. Our findings show that these PLA2s could be used as useful templates for the development of future antiviral candidate drugs against Zika fever.

Inflammatory Profile Associated with Secondary Infection from Bothrops atrox Snakebites in the Brazilian Amazon.

Bothrops snakebite envenomation (SBE) is consider an important health problem in Brazil, where Bothrops atrox is mainly responsible in the Brazilian Amazon. Local effects represent a relevant clinical issue, in which inflammatory signs and symptoms in the bite site represent a potential risk for short and long-term disabilities. Among local complications, secondary infections (SIs) are a common clinical finding during Bothrops atrox SBE and are described by the appearance of signs such as abscess, cellulitis or necrotizing fasciitis in the affected site. However, the influence of SI in the local events is still poorly understood. Therefore, the present study describes for the first time the impact of SBE wound infection on local manifestations and inflammatory response from patients of Bothrops atrox SBE in the Brazilian Amazon. This was an observational study carried out at the Fundação de Medicina Tropical Dr. Heitor Vieira Dourado, Manaus (Brazil), involving victims of Bothrops SBE. Clinical and laboratorial data were collected along with blood samples for the quantification of circulating cytokines and chemokines before antivenom administrations (T0) and 24 h (T1), 48 h (T2), 72 h (T3) and 7 days after (T4). From the 94 patients included in this study, 42 presented SI (44.7%) and 52 were without SI (NSI, 55.3%). Patients classified as moderate envenoming presented an increased risk of developing SI (OR = 2.69; CI 95% = 1.08-6.66, p = 0.033), while patients with bites in hands showed a lower risk (OR = 0.20; CI 95% = 0.04-0.96, p = 0.045). During follow-up, SI patients presented a worsening of local temperature along with a sustained profile of edema and pain, while NSI patients showed a tendency to restore and were highlighted in patients where SI was diagnosed at T2. As for laboratorial parameters, leukocytes, erythrocyte sedimentation ratio, fibrinogen and C-reactive protein were found increased in patients with SI and more frequently in patients diagnosed with SI at T3. Higher levels of circulating IL-2, IL-10, IL-6, TNF, INF-γ and CXCL-10 were observed in SI patients along with marked correlations between these mediators and IL-4 and IL-17, showing a plurality in the profile with a mix of Th1/Th2/Th17 response. The present study reports for the first time the synergistic effects of local infection and envenoming on the inflammatory response represented by local manifestations, which reflected on laboratorial parameters and inflammatory mediators and thus help improve the clinical management of SI associated to Bothrops SBE.

Microbial transformation of the sesquiterpene lactone tagitinin C by the fungus Aspergillus terreus.

The biotransformation of the sesquiterpene lactone tagitinin C by the fungus Aspergillus terreus MT 5.3 yielded a rare derivative that was elucidated by spectrometric methods. The fungus led to the formation of a different product through an unusual epoxidation reaction between C4 and C5, formation of a C3,C10 ether bridge, and a methoxylation of the C1 of tagitinin C. The chemical structure of the product, namely 1ß-methoxy-3α-hydroxy-3,10ß-4,5α-diepoxy-8ß-isobutyroyloxygermacr-11(13)-en-6α,12-olide, is the same as that of a derivative that was recently isolated from the flowers of a Brazilian population of Mexican sunflower (Tithonia diversifolia), which is the source of the substrate tagitinin C. The in vitro cytotoxic activity of the substrate and the biotransformed product were evaluated in HL-60 cells using an MTT assay, and both compounds were found to be cytotoxic. We show that soil fungi may be useful in the biotransformation of sesquiterpene lactones, thereby leading to unusual changes in their chemical structures that may preserve or alter their biological activities, and may also mimic plant biosynthetic pathways for production of secondary metabolites.

A rational protocol for the successful crystallization of L-amino-acid oxidase from Bothrops atrox.

Despite the valuable contributions of robotics and high-throughput approaches to protein crystallization, the role of an experienced crystallographer in the evaluation and rationalization of a crystallization process is still crucial to obtaining crystals suitable for X-ray diffraction measurements. In this work, the difficult task of crystallizing the flavoenzyme L-amino-acid oxidase purified from Bothrops atrox snake venom was overcome by the development of a protocol that first required the identification of a non-amorphous precipitate as a promising crystallization condition followed by the implementation of a methodology that combined crystallization in the presence of oil and seeding techniques. Crystals were obtained and a complete data set was collected to 2.3 Šresolution. The crystals belonged to space group P2(1), with unit-cell parameters a = 73.64, b = 123.92, c = 105.08 Å, ß = 96.03°. There were four protein subunits in the asymmetric unit, which gave a Matthews coefficient V(M) of 2.12 Å(3) Da(-1), corresponding to 42% solvent content. The structure has been solved by molecular-replacement techniques.

Isolation, functional, and partial biochemical characterization of galatrox, an acidic lectin from Bothrops atrox snake venom.

Snake venom lectins have been studied in regard to their chemical structure and biological functions. However, little is known about lectins isolated from Bothrops atrox snake venom. We report here the isolation and partial functional and biochemical characterization of an acidic glycan-binding protein called galatrox from this venom. This lectin was purified by affinity chromatography using a lactosyl-sepharose column, and its homogeneity and molecular mass were evaluated by high-performance liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. The purified galatrox was homogeneous and characterized as an acidic protein (pI 5.2) with a monomeric and dimeric molecular mass of 16.2 and 32.5 kDa, respectively. Alignment of N-terminal and internal amino acid sequences of galatrox indicated that this protein exhibits high homology to other C-type snake venom lectins. Galatrox showed optimal hemagglutinating activity at a concentration of 100 µg/ml and this effect was drastically inhibited by lactose, ethylenediaminetetraacetic acid, and heating, which confirmed galatrox's lectin activity. While galatrox failed to induce the same level of paw edema or mast cell degranulation as B. atrox crude venom, galatrox did alter cellular viability, which suggested that galatrox might contribute to venom toxicity by directly inducing cell death.

Synephrine and caffeine combination promotes cytotoxicity, DNA damage and transcriptional modulation of apoptosis-related genes in human HepG2 cells.

The abusive consumption of thermogenic supplements occurs worldwide and deserves special attention due to their use to stimulate weight loss and prevent obesity. Thermogenic formulations usually contain Synephrine (SN) and Caffeine (CAF), stimulating compounds extracted from natural sources, but no genetic toxicology studies have predicted this hazardous combination potential. This study examined the toxicogenomic responses induced by SN and CAF, either alone or in combination, in the human hepatic cell line HepG2 in vitro. SN (0.03-30 µM) and CAF (0.6-600 µM) alone did neither decrease cell viability nor induce DNA damage, as assessed using the MTT and comet assays, respectively. SN (3 µM) and CAF (30-600 µM) were combined at concentrations similar to those found in commercial dietary supplements. SN/CAF at 3:90 and 3:600 µM ratios significantly decreased cell viability and increased DNA damage levels in HepG2 cells. CAF (600 µM) and the SN/CAF association at 3:60, 3:90, and 3:600 µM ratios promoted cell death by apoptosis, as demonstrated by flow cytometry. Similar results were observed in gene expression (RT-qPCR): SN/CAF up-regulated the expression of apoptosis- (BCL-2 and CASP9) and DNA repair-related (XPC) genes. SN/CAF at 3:90 µM also downregulated the expression of cell cycle control (CDKN1A) genes. In conclusion, the SN/CAF combination reduces cell viability by inducing apoptosis, damages DNA, and modulates the transcriptional expression of apoptosis-, cell cycle-, and DNA repair-related genes in human hepatic (HepG2) cells in vitro. These effects can be worrisome to consumers of thermogenic supplements.

Towards toxin PEGylation: The example of rCollinein-1, a snake venom thrombin-like enzyme, as a PEGylated biopharmaceutical prototype.

PEGylation was firstly described around 50 years ago and has been used for more than 30 years as a strategy to improve the drugability of biopharmaceuticals. However, it remains poorly employed in toxinology, even though it may be a promising strategy to empower these compounds in therapeutics. This work reports the PEGylation of rCollinein-1, a recombinant snake venom serine protease (SVSP), able to degrade fibrinogen and inhibit the hEAG1 potassium channel. We compared the functional, structural, and immunogenic properties of the non-PEGylated (rCollinein-1) and PEGylated (PEG-rCollinein-1) forms. PEG-rCollinein-1 shares similar kinetic parameters with rCollinein-1, maintaining its capability of degrading fibrinogen, but with reduced activity on hEAG1 channel. CD analysis revealed the maintenance of protein conformation after PEGylation, and thermal shift assays demonstrated similar thermostability. Both forms of the enzyme showed to be non-toxic to peripheral blood mononuclear cells (PBMC). In silico epitope prediction indicated three putative immunogenic peptides. However, immune response on mice showed PEG-rCollinein-1 was devoid of immunogenicity. PEGylation directed rCollinein-1 activity towards hemostasis control, broadening its possibilities to be employed as a defibrinogenant agent.

A comparative study on the leishmanicidal activity of the L-amino acid oxidases BjussuLAAO-II and BmooLAAO-II isolated from Brazilian Bothrops snake venoms.

This study aims to examine whether two L-amino acid oxidases isolated from Bothrops snake venom (SV-LAAOs) were cytotoxic to Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis, two causative agents of leishmaniasis, which is an endemic disease in tropical and subtropical countries. The SV-LAAOs BjussuLAAO-II and BmooLAAO-II were isolated from Bothrops jararacussu and Bothrops moojeni venom, respectively, through a three-step chromatography process that used molecular exclusion, hydrophobic interaction, and affinity columns. BmooLAAO-II is a new SV-LAAO isoform that we isolated in this study. The purified BjussuLAAO-II and BmooLAAO-II had high L-amino acid oxidase-specific activity: 3481.17 and 4924.77 U/mg/min, respectively. Both SV-LAAOs were strongly cytotoxic to the two Leishmania species, even at low concentrations. At the same concentration, BjussuLAAO-II and BmooLAAO-II exerted different cytotoxic effects on the parasites. We reported for the first time that the SV-LAAOs suppressed cell proliferation and altered the mitochondrial membrane potential of the two Leishmania species. Surprisingly, BjussuLAAO-II increased the intracellular reactive oxygen species production only in L. (L.) amazonensis, while BmooLAAO-II increased the intracellular reactive oxygen species production only in L. (V.) braziliensis, indicating that these SV-LAAOs had a certain specificity of action.

Chikungunya virus entry is strongly inhibited by phospholipase A2 isolated from the venom of Crotalus durissus terrificus.

Chikungunya virus (CHIKV) is the etiologic agent of Chikungunya fever, a globally spreading mosquito-borne disease. There is no approved antiviral or vaccine against CHIKV, highlighting an urgent need for novel therapies. In this context, snake venom proteins have demonstrated antiviral activity against several viruses, including arboviruses which are relevant to public health. In particular, the phospholipase A2CB (PLA2CB), a protein isolated from the venom of Crotalus durissus terrificus was previously shown to possess anti-inflammatory, antiparasitic, antibacterial and antiviral activities. In this study, we investigated the multiple effects of PLA2CB on the CHIKV replicative cycle in BHK-21 cells using CHIKV-nanoluc, a marker virus carrying nanoluciferase reporter. The results demonstrated that PLA2CB possess a strong anti-CHIKV activity with a selectivity index of 128. We identified that PLA2CB treatment protected cells against CHIKV infection, strongly impairing virus entry by reducing adsorption and post-attachment stages. Moreover, PLA2CB presented a modest yet significant activity towards post-entry stages of CHIKV replicative cycle. Molecular docking calculations indicated that PLA2CB may interact with CHIKV glycoproteins, mainly with E1 through hydrophobic interactions. In addition, infrared spectroscopy measurements indicated interactions of PLA2CB and CHIKV glycoproteins, corroborating with data from in silico analyses. Collectively, this data demonstrated the multiple antiviral effects of PLA2CB on the CHIKV replicative cycle, and suggest that PLA2CB interacts with CHIKV glycoproteins and that this interaction blocks binding of CHIKV virions to the host cells.

Unraveling the structure and function of CdcPDE: A novel phosphodiesterase from Crotalus durissus collilineatus snake venom.

This study reports the isolation, structural, biochemical, and functional characterization of a novel phosphodiesterase from Crotalus durissus collilineatus venom (CdcPDE). CdcPDE was successfully isolated from whole venom using three chromatographic steps and represented 0.7% of total protein content. CdcPDE was inhibited by EDTA and reducing agents, demonstrating that metal ions and disulfide bonds are necessary for its enzymatic activity. The highest enzymatic activity was observed at pH 8-8.5 and 37 °C. Kinetic parameters indicated a higher affinity for the substrate bis(p-nitrophenyl) phosphate compared to others snake venom PDEs. Its structural characterization was done by the determination of the protein primary sequence by Edman degradation and mass spectrometry, and completed by the building of molecular and docking-based models. Functional in vitro assays showed that CdcPDE is capable of inhibiting platelet aggregation induced by adenosine diphosphate in a dose-dependent manner and demonstrated that CdcPDE is cytotoxic to human keratinocytes. CdcPDE was recognized by the crotalid antivenom produced by the Instituto Butantan. These findings demonstrate that the study of snake venom toxins can reveal new molecules that may be relevant in cases of snakebite envenoming, and that can be used as molecular tools to study pathophysiological processes due to their specific biological activities.

A Representative GIIA Phospholipase A2 Activates Preadipocytes to Produce Inflammatory Mediators Implicated in Obesity Development.

Adipose tissue secretes proinflammatory mediators which promote systemic and adipose tissue inflammation seen in obesity. Group IIA (GIIA)-secreted phospholipase A2 (sPLA2) enzymes are found to be elevated in plasma and adipose tissue from obese patients and are active during inflammation, generating proinflammatory mediators, including prostaglandin E2 (PGE2). PGE2 exerts anti-lipolytic actions and increases triacylglycerol levels in adipose tissue. However, the inflammatory actions of GIIA sPLA2s in adipose tissue cells and mechanisms leading to increased PGE2 levels in these cells are unclear. This study investigates the ability of a representative GIIA sPLA2, MT-III, to activate proinflammatory responses in preadipocytes, focusing on the biosynthesis of prostaglandins, adipocytokines and mechanisms involved in these effects. Our results showed that MT-III induced biosynthesis of PGE2, PGI2, MCP-1, IL-6 and gene expression of leptin and adiponectin in preadipocytes. The MT-III-induced PGE2 biosynthesis was dependent on cytosolic PLA2 (cPLA2)-α, cyclooxygenases (COX)-1 and COX-2 pathways and regulated by a positive loop via the EP4 receptor. Moreover, MT-III upregulated COX-2 and microsomal prostaglandin synthase (mPGES)-1 protein expression. MCP-1 biosynthesis induced by MT-III was dependent on the EP4 receptor, while IL-6 biosynthesis was dependent on EP3 receptor engagement by PGE2. These data highlight preadipocytes as targets for GIIA sPLA2s and provide insight into the roles played by this group of sPLA2s in obesity.

Role of crotoxin in coagulation: novel insights into anticoagulant mechanisms and impairment of inflammation-induced coagulation.

BACKGROUND: Snake venom phospholipases A2 (svPLA2) are biologically active toxins, capable of triggering and modulating a wide range of biological functions. Among the svPLA2s, crotoxin (CTX) has been in the spotlight of bioprospecting research due to its role in modulating immune response and hemostasis. In the present study, novel anticoagulant mechanisms of CTX, and the modulation of inflammation-induced coagulation were investigated. METHODS: CTX anticoagulant activity was evaluated using platelet poor plasma (PPP) and whole blood (WB), and also using isolated coagulation factors and complexes. The toxin modulation of procoagulant and pro-inflammatory effects was evaluated using the expression of tissue factor (TF) and cytokines in lipopolysaccharide (LPS)-treated peripheral blood mononuclear cells (PBMC) and in WB. RESULTS: The results showed that CTX impaired clot formation in both PPP and WB, and was responsible for the inhibition of both intrinsic (TF/factor VIIa) and extrinsic (factor IXa/factor VIIIa) tenase complexes, but not for factor Xa and thrombin alone. In addition, the PLA2 mitigated the prothrombinase complex by modulating the coagulation phospholipid role in the complex. In regards to the inflammation-coagulation cross talk, the toxin was capable of reducing the production of the pro-inflammatory cytokines IL-1ß, IL-6 and TNF-α, and was followed by decreased levels of TF and procoagulant activity from LPS-treated PBMC either isolated or in WB. CONCLUSION: The results obtained in the present study recognize the toxin as a novel medicinal candidate to be applied in inflammatory diseases with coagulation disorders.

A Synthetic Snake-Venom-Based Tripeptide Protects PC12 Cells from the Neurotoxicity of Acrolein by Improving Axonal Plasticity and Bioenergetics.

The synthetic peptide p-BTX-I is based on the native peptide (formed by glutamic acid, valine and tryptophan) isolated from Bothrops atrox venom. We have previously demonstrated its neuroprotective and neurotrophic properties in PC12 cells treated with the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP+). Now, we have investigated the neuroprotective effects and mechanisms of p-BTX-I against the toxicity of acrolein in PC12 cells. Studies have demonstrated that acrolein might play an important role in the etiology of Alzheimer's disease (AD), which is characterized by neuronal and synaptic loss. Our results showed that not only acrolein reduced cell differentiation and cell viability, but also altered the expression of markers of synaptic communication (synapsin I), energy metabolism (AMPK-α, Sirt I and glucose uptake), and cytoskeleton (ß-III-tubulin). Treatment with p-BTX-I increased the percentage of differentiation in cells treated with acrolein and significantly attenuated cell viability loss, besides counteracting the negative effects of acrolein on synapsin I, AMPK-α, Sirt I, glucose uptake, and ß-III-tubulin. Additionally, p-BTX-I alone increased the expression of apolipoprotein E (apoE) gene, associated with the proteolytic degradation of ß-amyloid peptide aggregates, a hallmark of AD. Taken together, these findings demonstrate that p-BTX-I protects against acrolein-induced neurotoxicity and might be a tool for the development of novel drugs for the treatment of neurodegenerative diseases.

Crotoxin-Induced Mice Lung Impairment: Role of Nicotinic Acetylcholine Receptors and COX-Derived Prostanoids.

Respiratory compromise in Crotalus durissus terrificus (C.d.t.) snakebite is an important pathological condition. Considering that crotoxin (CTX), a phospholipase A2 from C.d.t. venom, is the main component of the venom, the present work investigated the toxin effects on respiratory failure. Lung mechanics, morphology and soluble markers were evaluated from Swiss male mice, and mechanism determined using drugs/inhibitors of eicosanoids biosynthesis pathway and autonomic nervous system. Acute respiratory failure was observed, with an early phase (within 2 h) characterized by enhanced presence of eicosanoids, including prostaglandin E2, that accounted for the increased vascular permeability in the lung. The alterations of early phase were inhibited by indomethacin. The late phase (peaked 12 h) was marked by neutrophil infiltration, presence of pro-inflammatory cytokines/chemokines, and morphological alterations characterized by alveolar septal thickening and bronchoconstriction. In addition, lung mechanical function was impaired, with decreased lung compliance and inspiratory capacity. Hexamethonium, a nicotinic acetylcholine receptor antagonist, hampered late phase damages indicating that CTX-induced lung impairment could be associated with cholinergic transmission. The findings reported herein highlight the impact of CTX on respiratory compromise, and introduce the use of nicotinic blockers and prostanoids biosynthesis inhibitors as possible symptomatic therapy to Crotalus durissus terrificus snakebite.