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Abstract: The crystal structure of dimeric Lys49-phospholipase A2 myotoxin-II from Bothrops moojeni (MjTX-II) co-crystallized with stearic acid (C18H36O2) has been determined at a resolution of 1.8 Å. The electron density maps permitted the unambiguous inclusion of six stearic acid molecules in the refinement. Two stearic acid molecules could be located in the substrate-binding cleft of each monomer in positions, which favor the interaction of their carboxyl groups with active site residues. The way of binding of stearic acids to this Lys49-PLA2s is analogous to phospholipids and transition state analogues to catalytically active PLA2s. Two additional stearic acid molecules were located at the dimer interface region, defining a hitherto unidentified acyl-binding site on the protein surface. The strictly conserved Lys122 for Lys49-PLA2s may play a fundamental role for stabilization of legend-protein complex. The comparison of MjTX-II/satiric acid complex with other Lys-PLA2s structures whose putative fatty acids were located at their active site is also analysed. Molecular details of the stearic acid/protein interactions provide insights to binding in group I/II PLA2s, and to the possible interactions of Lys49-PLA2s with target membranes. [Copyright &y& Elsevier]

Abstract: Bothropstoxin-II (Bthtx-II), an Asp-49 phospholipase A2 (D-PLA2) isolated from Bothrops jararacussu snake venom is able to induce platelet aggregation in a concentration-dependent manner. This effect was not due to the release of ADP from platelets since the aggregation was not suppressed by ADP scavenger systems. PMSF and PPACK were unable to inhibit Bthtx-II-induced platelet aggregation. Thus, a thrombin-like proaggregating activity of Bthtx-II can be excluded as its mechanism of action. On the other hand, indomethacin at low concentrations inhibited more markedly the ATP-release reaction than the aggregation induced by Bthtx-II, indicating that generation of cyclooxigenase products is not the most important event for the platelet aggregation reaction. It was also found that staurosporine and genistein suppressed both platelet aggregation and ATP-release reactions, but not the platelet shape-change induced by Bthtx-II. Substances that either directly activates adenylyl cyclase enzyme (forskolin and PGE1) or cell-permeant increasing agents (dibutyril-cAMP) inhibited in a concentration-dependent fashion, the platelet aggregation effects induced by the protein. It is concluded that Bthtx-II induces platelet aggregation and secretion through multiple signal transduction pathways. [Copyright &y& Elsevier]

Bothrops moojeni crude venom (MjCV) and its two major toxins, namely myotoxin I (MjTX-I) and myotoxin II (MjTX-II) were alkylated by p-bromophenacyl bromide (BPB). After alkylation the i.p. LD50 (mice) of MjCV and MjTX-I/II increased from 6.0 to 15.7 mg/kg and from 8.0 to 45.0 mg/kg, respectively. In addition, doses of 5× LD50 of alkylated MjTX-I did not cause a single death in mice and no myonecrosis was detected for the alkylated toxins, although both proteins still induced edema. Antibodies to native and modified crude venom or myotoxins cross-reacted with 12 purified class II myotoxic phospholipases A2 found in snake venoms of the genus Bothrops. Myotoxic PLA2s from class I and class III were not recognized by the above antibodies. These results suggest that the overall antigenic structure is conserved among class II myotoxic PLA2s, despite differences in their amino acid sequences. Anti-MjTX-I-BPB and anti-MjTX-II-BPB rabbit serum, obtained against the modified myotoxins, were apparently more efficient than those obtained against the native myotoxins. In neutralization experiments, pre-incubation of crude venom or isolated myotoxins with antibodies raised against the native or modified toxins inhibited their PLA2 and myotoxic activities. Therefore, alkylation of His48 by BPB strongly reduces the local tissue damage induced by B. moojeni venom or isolated myotoxins while retaining antigenicity, which suggests a promising procedure for an enhanced antiophidian serum production for practical purposes. [Copyright &y& Elsevier]

Phospholipases A2 (PLA2s) constitute major components of snake venoms and have been extensively investigated not only because they are very abundant in these venoms but mainly because they display a wide range of biological effects, including neurotoxic, myotoxic, cytotoxic, edema-inducing, artificial membrane disrupting, anti-coagulant, platelet aggregation inhibiting, hypotensive, bactericidal, anti-HIV, anti-tumoral, anti-malarial and anti-parasitic. Due to this functional diversity, these structurally similar proteins aroused the interest of many researchers as molecular models for study of structure–function relationships. One of the main experimental strategies used for the study of myotoxic PLA2s is the traditional chemical modification of specific amino acid residues (His, Met, Lys, Tyr, Trp and others) and examination of the consequent effects upon the enzymatic, toxic and pharmacological activities. This line of research has provided useful insights into the structural determinants of the action of these enzymes and, together with additional strategies, supports the concept of the presence of ‘pharmacological sites’ distinct from the catalytic site in snake venom myotoxic PLA2s. [Copyright &y& Elsevier]

An acidic (pI∼4.5) phospholipase A2 (BthA-I-PLA2) was isolated from Bothrops jararacussu snake venom by ion-exchange chromatography on a CM-Sepharose column followed by reverse phase chromatography on an RP-HPLC C-18 column. It is an ∼13.7 kDa single chain Asp49 PLA2 with approximately 122 amino acid residues, 7 disulfide bridges, and the following N-terminal sequence: 1SLWQFGKMINYVM–GESGVLQYLSYGCYCGLGGQGQPTDATDRCCFVHDCC51. Crystals of this acidic protein diffracted beyond 2.0 A˚ resolution. These crystals are monoclinic and have unit cell dimensions of a=33.9, b=63.8, c=49.1 A˚, and β=104.0°. Although not myotoxic, cytotoxic, or lethal, the protein was catalytically 3–4 times more active than BthTX-II, a basic D49 myotoxic PLA2 from the same venom and other Bothrops venoms. Although it showed no toxic activity, it was able to induce time-independent edema, this activity being inhibited by EDTA. In addition, BthA-I-PLA2 caused a hypotensive response in the rat and inhibited platelet aggregation. Catalytic, antiplatelet and other activities were abolished by chemical modification with 4-bromophenacyl bromide, which is known to covalently bind to His48 of the catalytic site. Antibodies raised against crude B. jararacussu venom recognized this acidic PLA2, while anti-Asp49-BthTX-II recognized it weakly and anti-Lys49-BthTX-I showed the least cross-reaction. These data confirm that myotoxicity does not necessarily correlate with catalytic activity in native PLA2 homologues and that either of these two activities may exist alone. BthA-I-PLA2, in addition to representing a relevant molecular model of catalytic activity, is also a promising hypotensive agent and platelet aggregation inhibitor for further studies. [Copyright &y& Elsevier]

The complete nucleotide sequence of a nerve growth factor precursor from Bothrops jararacussu snake (Bj-NGF) was determined by DNA sequencing of a clone from cDNA library prepared from the poly(A) + RNA of the venom gland of B. jararacussu. cDNA encoding Bj-NGF precursor contained 723 bp in length, which encoded a prepro-NGF molecule with 241 amino acid residues. The mature Bj-NGF molecule was composed of 118 amino acid residues with theoretical pI and molecular weight of 8.31 and 13,537, respectively. Its amino acid sequence showed 97%, 96%, 93%, 86%, 78%, 74%, 76%, 76% and 55% sequential similarities with NGFs from Crotalus durissus terrificus, Agkistrodon halys pallas, Daboia (Vipera) russelli russelli, Bungarus multicinctus, Naja sp., mouse, human, bovine and cat, respectively. Phylogenetic analyses based on the amino acid sequences of 15 NGFs separate the Elapidae family (Naja and Bungarus) from those Crotalidae snakes (Bothrops, Crotalus and Agkistrodon). The three-dimensional structure of mature Bj-NGF was modeled based on the crystal structure of the human NGF. The model reveals that the core of NGF, formed by a pair of β-sheets, is highly conserved and the major mutations are both at the three β-hairpin loops and at the reverse turn. [Copyright &y& Elsevier]

Bothropstoxin-I (BthTX-I), a myotoxic Lys49 phospholipase A2 (PLA2) homologue isolated from Bothrops jararacussu snake venom, causes a range of biological effects, including myonecrosis, mouse paw edema, irreversible neuromuscular blockade and lysis of cell cultures. Among eight divalent cations assayed, Mn2+ was the most effective in reducing mouse paw edema induced by BthTX-I (25 μg). Preincubating BthTX-I with Mn2+ (1.0 mM) reduced mouse paw edema by 70% and myotoxicity by 60% in mice injected i.m. with 50 μg toxin. Mn2+ (50 μl of a 1 mM solution) administered within 1 min at the site of toxin injection was still but less effective in antagonising BthTX-I-induced myotoxicity. Mn2+ (1.0 mM) completely prevented BthTX-I (1.4 μM)-induced neuromuscular blockade in the mouse phrenic-nerve diaphragm preparation. Mn2+ (0.25 mM) protected about 85% of NB41A3 cells from lysis when coincubated with BthTX-I (1.0 μM) for 25 h. Preincubation with 0.25 mM Mn2+ increased the sensitivity of the cells to subsequent lysis by BthTX-I in the absence of Mn2+. BthTX-I (1 μM) caused extensive fatty acid release (from 0.8% of the total radiolabeled lipid in control cells to 56% with toxin) when incubated with the NB41A3 cell line for 25 h. PLA2 activity observed in cell cultures after addition of BthTX-I was considerably reduced by 0.25 mM Mn2+. Mn2+ ions constitute a promising agent to assess the action mechanism and the effects of enzymatic inhibition on the pharmacological activity of Lys49 PLA2 homologues. [Copyright &y& Elsevier]

Prof. Dr. José R. Giglio (1934-2014) made a highly significant contribution to the field of Toxinology. During 48 years devoted to research and teaching Prof Giglio published more than 160 articles, with more than 4400 citations, in international journals, trained a vast amount of graduate and undergraduate students, and developed an international network of collaborators. Throughout these years, he worked with dedication and deep commitment to science, leaving an immortalized legacy. During his professional career he contributed mostly in the isolation, and biochemical and functional characterization of various protein toxins derived from animal venoms such as snakes, scorpions and spiders, in addition to his studies searching for alternative therapies for poisoning. Even after his departure, his presence and influence remains among his former students and in the outstanding legacy of his scientific contributions. [ABSTRACT FROM AUTHOR]

Varespladib (LY315920) is a potent inhibitor of human group IIA phospholipase A 2 (PLA 2) originally developed to control inflammatory cascades of diseases associated with high or dysregulated levels of endogenous PLA 2. Recently, varespladib was also found to inhibit snake venom PLA 2 and PLA 2 -like toxins. Herein, ex vivo neuromuscular blocking activity assays were used to test the inhibitory activity of varespladib. The binding affinity between varespladib and a PLA 2 -like toxin was quantified and compared with other potential inhibitors for this class of proteins. Crystallographic and bioinformatic studies showed that varespladib binds to PrTX-I and BthTX-I into their hydrophobic channels, similarly to other previously characterized PLA 2 -like myotoxins. However, a new finding is that an additional varespladib binds to the MDiS region, a particular site that is related to muscle cell disruption by these toxins. The present results further advance the characterization of the molecular interactions of varespladib with PLA 2 -like myotoxins and provide additional evidence for this compound as a promising inhibitor candidate for different PLA 2 and PLA 2 -like toxins. [Display omitted] • An ex vivo assay was used to test the inhibitory activity of varespladib. • Affinity assays demonstrate that inhibitor interacts with the toxin. • Varespaldib binds into hydrophobic channel. • An additional varespladib binds to muscle cell disruption site (MDiS). • An alternative inhibitory mechanism by varespladib is proposed. [ABSTRACT FROM AUTHOR]

Snake envenomation is an ongoing global health problem and tropical neglected disease that afflicts millions of people each year. The only specific treatment, antivenom, has several limitations that affects its proper distribution to the victims and its efficacy against local effects, such as myonecrosis. The main responsible for this consequence are the phospholipases A 2 (PLA 2) and PLA 2 -like proteins, such as BthTX-I from Bothrops jararacussu. Folk medicine resorts to plants such as Tabernaemontana catharinensis to palliate these and other snakebite effects. Here, we evaluated the effect of its root bark extract and one of its isolated compounds, 12-methoxy-4-methyl-voachalotine (MMV), against the in vitro paralysis and muscle damage induced by BthTX-I. Secondary and quaternary structures of BthTX-I were not modified by the interaction with MMV. Instead, this compound interacted in an unprecedented way with the region inside the toxin hydrophobic channel and promoted a structural change in Val31, loop 58–71 and Membrane Disruption Site. Thus, we hypothesize that MMV inhibits PLA 2 -like proteins by preventing entrance of fatty acid into the hydrophobic channel. These data may explain the traditional use of T. catharinensis extract and confirm MMV as a promising candidate to complement antivenom or a structural guide to develop more effective inhibitors. [Display omitted] • Alkaloid MMV attenuates the neuromuscular blockade promoted by BthTX-I. • MMV does not alter BthTX-I secondary and quaternary structure. • MMV changes BthTX-I structure on Val31 and region 58-71. • MMV inhibits BthTX-I preventing entrance of fatty acids in the hydrophobic channel. [ABSTRACT FROM AUTHOR]

Bothrops venom contains a high amount of secreted phospholipase A 2 (sPLA 2 s) enzymes responsible for the inflammatory reaction and activation of leukocytes in cases of envenoming. PLA 2 s are proteins that have enzymatic activity and can hydrolyze phospholipids at the sn-2 position, thereby releasing fatty acids and lysophospholipids precursors of eicosanoids, which are significant mediators of inflammatory conditions. Whether these enzymes have a role in the activation and function of peripheral blood mononuclear cells (PBMCs) is not known. Here we show for the first time how two secreted PLA 2 s (BthTX-I and BthTX-II) isolated from the venom of Bothrops jararacussu affect the function and polarization of PBMCs. Neither BthTX-I nor BthTX-II exhibited significant cytotoxicity to isolated PBMCs compared with the control at any of the time points studied. RT-qPCR and enzyme-linked immunosorbent assays were used to determine changes in gene expression and the release of pro-inflammatory (TNF-α, IL-6, and IL-12) and anti-inflammatory (TGF-β and IL-10) cytokines, respectively, during the cell differentiation process. Lipid droplets formation and phagocytosis were also investigated. Monocytes/macrophages were labeled with anti-CD14, -CD163, and -CD206 antibodies to assay cell polarization. Both toxins caused a heterogeneous morphology (M1 and M2) on days 1 and 7 based on immunofluorescence analysis, revealing the considerable flexibility of these cells even in the presence of typical polarization stimuli. Thus, these findings indicate that the two sPLA 2 s trigger both immune response profiles in PBMCs indicating a significant degree of cell plasticity, which may be crucial for understanding the consequences of snake envenoming. [Display omitted] • BthTX-I and BthTX-II do not interfere with cell viability and cytotoxicity. • BthTX-I and BthTX-II led to cytoplasm morphological changes with pseudopods formation. • BthTX-I and BthTX-II induce phagocytosis and lipid bodies formation. • BthTX-I and BthTX-II induce the expression of both M1 and M2 cell markers phenotypes. • BthTX-I and BthTX-II induce pro and anti-inflammatory mediator's release. [ABSTRACT FROM AUTHOR]

To investigate the in vitro activity, synergism, cytotoxicity and cellular immunological response, as well as the molecular affinity between amphotericin B (AmB) and crotamine (CTA), derived from Crotalus durissus terrificus venom against Leishmania amazonensis. This study performed the inhibition of promastigotes and amastigotes' growth under different concentrations of the drug and pharmacological combinations (AmB + CTA) based on the Berimbaum method (synergism study). The lactate dehydrogenase (LDH) quantification method was used to determine the cytotoxicity of the drug and combinations employing four cell lines (J774, HepG2, VERO, and C2C12). Following, the levels of Tumour Necrose Factor-alpha (TNF-α) and Interleukin-12 (IL-12) cytokines, using enzyme-linked immunosorbent assay (ELISA) and nitrites, as an indirect measure of Nitric Oxide (NO), using the Griess reaction were assessed in the supernatants of infected macrophages. In silico approach (molecular docking and dynamics) and binding affinity (surface plasmon resonance) between the drug and toxin were also investigated. CTA enhanced AmB effect against promastigote and amastigote forms of L. amazonensis , decreased the drug toxicity in different cell lines and induced the production of important Th1-like cytokines and NO by infected macrophages. The pharmacological combination also displayed consistent molecular interactions with low energy of coupling and a concentration-dependent profile. Our data suggest that this pharmacological approach is a promising alternative treatment against L. amazonensis infection due to the improved activity (synergistic effect) achieved against the parasites' forms and to the decreased cytotoxic effect. [Display omitted] • Amphotericin B combined with crotamine displays a synergistic effect against promastigotes of L. amazonensis. • Amphotericin B cytotoxicity was decreased in different cell lines when combined with crotamine. • The pharmacological combination increases cytokines and nitric oxide productions by L. amazonensis -infected macrophages. • The combination between amphotericin B and crotamine displays consistent molecular interaction and binding affinit. [ABSTRACT FROM AUTHOR]

Abstract: Lys49 phospholipase A2 (PLA2) homologues are myotoxic proteins devoid of catalytic activity. Their toxic determinants map to the C-terminal region 115–129, which plays an effector role in membrane damage. The dimeric state was reported to be essential for a Lys49 PLA2 which lost its liposome-disrupting activity after dissociating into monomers at pH 5.0. This study, evaluated the effects of a pH-induced dissociation on the toxicity of four Lys49 PLA2s, using biological targets instead. Both their cytolytic and myotoxic activities were lower at pH 5.0 than at pH 7.2. However, in contrast with experiments using artificial bilayers, toxic effects upon biological targets were not abolished at pH 5.0. Importantly, C-terminal synthetic peptides of two Lys49 PLA2s also showed lower cytolytic action at pH 5.0 than at pH 7.2, indicating that factors other than the dimeric/monomeric state of the proteins may also be involved in these differences of toxicity. Results support the view that the dimeric state of Lys49 PLA2s could play an enhancing, although not essential role, in their C-terminal region-mediated mechanism of myotoxicity. [Copyright &y& Elsevier]

Snake venom glands are a rich source of bioactive molecules such as peptides, proteins and enzymes that show important pharmacological activity leading to in local and systemic effects as pain, edema, bleeding and muscle necrosis. Most studies on pharmacologically active peptides and proteins from snake venoms have been concerned with isolation and structure elucidation through methods of classical biochemistry. As an attempt to examine the transcripts expressed in the venom gland of Bothrops jararacussu and to unveil the toxicological and pharmacological potential of its products at the molecular level, we generated 549 expressed sequence tags (ESTs) from a directional cDNA library. Sequences obtained from single-pass sequencing of randomly selected cDNA clones could be identified by similarities searches on existing databases, resulting in 197 sequences with significant similarity to phospholipase A2 (PLA2), of which 83.2% were Lys49-PLA2 homologs (BOJU-I), 0.1% were basic Asp49-PLA2s (BOJU-II) and 0.6% were acidic Asp49-PLA2s (BOJU-III). Adjoining this very abundant class of proteins we found 88 transcripts codifying for putative sequences of metalloproteases, which after clustering and assembling resulted in three full-length sequences: BOJUMET-I, BOJUMET-II and BOJUMET-III; as well as 25 transcripts related to C-type lectin like protein including a full-length cDNA of a putative galactose binding C-type lectin and a cluster of eight serine-proteases transcripts including a full-length cDNA of a putative serine protease. Among the full-length sequenced clones we identified a nerve growth factor (Bj-NGF) with 92% identity with a human NGF (NGHUBM) and an acidic phospholipase A2 (BthA-I-PLA2) displaying 85–93% identity with other snake venom toxins. Genetic distance among PLA2s from Bothrops species were evaluated by phylogenetic analysis. Furthermore, analysis of full-length putative Lys49-PLA2 through molecular modeling showed conserved structural domains, allowing the characterization of those proteins as group II PLA2s. The constructed cDNA library provides molecular clones harboring sequences that can be used to probe directly the genetic material from gland venom of other snake species. Expression of complete cDNAs or their modified derivatives will be useful for elucidation of the structure–function relationships of these toxins and peptides of biotechnological interest. [Copyright &y& Elsevier]

Snakebite envenoming is the cause of an ongoing health crisis in several regions of the world, particularly in tropical and neotropical countries. This scenario creates an urgent necessity for new practical solutions to address the limitations of current therapies. The current study investigated the isolation, phytochemical characterization, and myotoxicity inhibition mechanism of gallic acid (GA), a myotoxin inhibitor obtained from Anacardium humile. The identification and isolation of GA was achieved by employing analytical chromatographic separation, which exhibited a compound with retention time and nuclear magnetic resonance spectra compatible with GA's commercial standard and data from the literature. GA alone was able to inhibit the myotoxic activity induced by the crude venom of Bothrops jararacussu and its two main myotoxins, BthTX-I and BthTX-II. Circular dichroism (CD), fluorescence spectroscopy (FS), dynamic light scattering (DLS), and interaction studies by molecular docking suggested that GA forms a complex with BthTX-I and II. Surface plasmon resonance (SPR) kinetics assays showed that GA has a high affinity for BthTX-I with a K D of 9.146 × 10−7 M. Taken together, the two-state reaction mode of GA binding to BthTX-I, and CD, FS and DLS assays, suggest that GA is able to induce oligomerization and secondary structure changes for BthTX-I and -II. GA and other tannins have been shown to be effective inhibitors of snake venoms' toxic effects, and herein we demonstrated GA's ability to bind to and inhibit a snake venom PLA 2 , thus proposing a new mechanism of PLA 2 inhibition, and presenting more evidence of GA's potential as an antivenom compound. [ABSTRACT FROM AUTHOR]

Given the magnitude of the global snakebite crisis, strategies to ensure the quality of antivenom, as well as the availability and sustainability of its supply are under development by several research groups. Recombinant DNA technology has allowed the engineering of monoclonal antibodies and recombinant fragments as alternatives to conventional antivenoms. Besides having higher therapeutic efficacy, with broad neutralization capacity against local and systemic toxicity, novel antivenoms need to be safe and cost-effective. Due to the biological and physical chemical properties of camelid single-domain antibodies, with high volume of distribution to distal tissue, their modular format, and their versatility, their biotechnological application has grown considerably in recent decades. This article presents the most up-to-date developments concerning camelid single-domain-based antibodies against major toxins from snake venoms, the main venomous animals responsible for reported envenoming cases and related human deaths. A brief discussion on the composition, challenges, and perspectives of antivenoms is presented, as well as the road ahead for next-generation antivenoms based on single-domain antibodies. [ABSTRACT FROM AUTHOR]

Photobiomodulation using light-emitting diode (LED) treatment has analgesic and anti-inflammatory effects which can be an effective therapeutic associated with serum therapy for local treatment of snakebites. Here we explored the effects of LED treatment on isolated macrophage under Bothrops jararacussu venom. Results showed that LED induced IL-6 and TNF-α genes down-regulation and, TGF and ARG1 genes up-regulation which indicates a polarization of macrophages to an M2 phenotype contributing to both tissue repair and resolution of inflammation. [Display omitted] • Light Emitting Diode treatment induced the macrophage polarization toward a M2 phenotype under B. jararacussu venom action. • Light Emitting Diode treatment did not interfere with phagocytosis by macrophages under B. jararacussu venom action. • Light Emitting Diode treatment did not impede with macrophages lipid droplets formation under B. jararacussu venom action. [ABSTRACT FROM AUTHOR]

A basic sPLA 2 (D49) from the venom of snake Agkistrodon piscivorus leucostoma (AplTX-II) was isolated, purified and characterized. We determined the enzymatic and pharmacological profiles of this toxin. AplTX-II was isolated with a high level of purity through reverse phase chromatography and molecular exclusion. The enzyme showed pI 9.48 and molecular weight of 14,003 Da. The enzymatic activity of the AplTX-II depended on Ca2+ pH and temperature. The comparison of the primary structure with other sPLA 2 s revealed that AplTX-II presented all the structural reasons expected for a basic sPLA 2 s. Additionally, we have resolved its structure with the docked synthetic substrate NOBA (4-nitro-3-octanoyloxy benzoic acid) by homology modeling, and performed MD simulations with explicit solvent. Structural similarities were found between the enzyme's modeled structure and other snake sPLA 2 X-Ray structures, available in the PDB database. NOBA and active-site water molecules spontaneously adopted stable positions and established interactions in full agreement with the reaction mechanism, proposed for the physiological substrate, suggesting that NOBA hydrolysis is an excellent model to study phospholipid hydrolysis. [ABSTRACT FROM AUTHOR]

Studies have shown that inhibition of Plasmodium falciparum Purine Nucleoside Phosphorylase (Pf PNP) blocks the purine salvage pathway in vitro and in vivo. In this study, P f PNP was evaluated as a model in the search for new inhibitors using surface plasmon resonance (SPR). Its expression, purification, oligomeric state, kinetic constants, calorimetric parameters and kinetic mechanisms were obtained. P f PNP was immobilized on a CM5 sensor chip and sensorgrams were produced through binding the enzyme to the substrate MESG and interactions between molecules contained in 10 fractions of natural extracts. The oligomeric state showed that recombinant P f PNP is a hexamer. The true steady-state kinetic parameters for the substrate inosine were: K M 17 μM, k cat 1.2 s−1, V Max 2.2 U/mg and k cat /K M 7 × 10−4; for MESG they were: K M 131 μM, k cat 2.4 s−1, V Max 4.4 U/mg and k cat /K M 1.8 × 10−4. The thermodynamic parameters for the substrate Phosphate were: Δ G − 5.8 cal mol−1, Δ H − 6.5 cal mol−1 and Δ S − 2.25 cal mol−1/degree. The ITC results demonstrated that the binding of phosphate to free P f PNP led to a significant change in heat and association constants and thermodynamic parameters. A sequential ordered mechanism was proposed as the kinetic mechanism. Three plant extracts contained molecules capable of interacting with P f PNP, showing different levels of affinity. The identification of plant extract fractions containing molecules that interact with recombinant P f PNP using SRP validates this target as a model in the search for new inhibitors. In this study, we showed for the first time the true steady-state kinetic parameters for reactions catalyzed by P f PNP and a model using P f PNP as a target for High-throughput Screening for new inhibitors through SPR. This knowledge will allow for the development of more efficient research methods in the search for new drugs against malaria. [ABSTRACT FROM AUTHOR]

Scientific advances in nanotechnology and nanoscience have enabled stability optimization and signal amplification in immunoassays by taking advantage of unique properties of nanomaterials. Biosensors based on antibodies and their fragments, also called immunosensors, are compact tools capable of providing refined antigen detection capacity. Different immunoassays that utilize these molecules for biorecognition have been used as diagnostic tools. In this regard, camelid single domain antibodies fulfill several requirements, such as nanometric size, high affinity, specificity, solubility, stability, biotechnological versatility, and low cost of production, constituting an important source for the development of immunodiagnostic devices. In this review, the main technological advances involving this specific class of molecules, as well as their major biotechnological applications will be addressed, with emphasis on their use as biosensors applied to diagnostics in human health. [ABSTRACT FROM AUTHOR]

A comparative venom proteomic analysis of the Brazilian southern coral snake, M. frontalis , the Amazon coral snake M. spixii spixii , and the aquatic coral snake M. surinamensis is reported. Venoms from M. frontalis and M. s. spixii were composed mainly (>90% of the total venom proteome) by 3FTxs and PLA 2 s in different proportions, and minor proteins from 2 to 5 protein families. Conversely, the aquatic coral snake expressed a streamlined (95%) 3FTx venom with low abundance (4.2%) of PLA 2 molecules. A compositional-lethal activity for natural prey correlation analysis suggests that M. surinamensis venom may has evolved under strong pressure to quickly immobilize aquatic prey. On the other hand, venoms from M. frontalis and M. s. spixii , whose diet consist mainly of amphisbaenians and colubrid snakes, may have been shaped through balancing selection. Our work provides strong evidence for the occurrence in M. frontalis venom, but not in those from M. s. spixi and M. surinamensis , of a KUN-PLA 2 complex homologue to heterodimeric venom toxins from some long-tailed monadal coral snakes that target acid-sensing receptors ASIC1a/2 evoking pain. The M. frontalis protein would represent the first example of a KUN-PLA 2 heterodimer in a South American short-tailed triadal coral snake venom. Image 1 • Comparative venomics showed that Micrurus frontalis and M. s. spixii exhibited balanced (3FTx ~ PLA 2) venom phenotypes. • The aquatic coral snake, M. surinamensis , expressed a streamlined (95%) 3FTx venom. • The three coral snakes investigated exhibited high evolvability, sharing only a few ESI-MS toxin masses among them. • Balanced and streamlined venom phenotypes may have evolved under different selective regimes. • The first KUN-PLA 2 complex in South American short-tailed triadal coral snakes was identified in M. frontalis venom. [ABSTRACT FROM AUTHOR]

Among the activities triggered by Crotalus durissus terrificus snake venom, coagulation is intriguing and contradictory since the venom contains both coagulant and anticoagulant precursor proteins. This work describes the in vitro effects of crude venom and purified proteins from snake Crotalus durissus terrificus as they affect coagulation factors of clotting pathways. Coagulant and/or anticoagulant activities of crude venom, and purified proteins were all analyzed directly in human plasma. Clots formed by crude venom and Gyroxin presented as flexible hyaline masses in punctiform distribution. Clot formation time evaluation of isolated proteins with PT and APTT assays made it possible to infer that these proteins interfere in all coagulation pathways. However, regarding ophidism by C. d. terrificus , Gyroxin acts directly, breaking down fibrinogen to fibrin and increasing the amount plasminogen activator, which results in the formation of thrombi. Crotoxin complex, Crotoxin A and Crotoxin B proteins can act in prothrombinase complex formation; Crotoxin B can inhibit prothrombinase complex formation by direct interaction with Factor Xa. Crotamine interacts with negatively charged regions of differing coagulation factors in all coagulation pathways, and possesses a whole set of activities causing dysfunction, activation and/or inhibition of natural anticoagulants and disturbing hemostasis. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the in vivo anti- Leishmania amazonensis activity of a Phospholipase A 2 (Asp49-PLA 2 ), isolated from Bothrops jararacussu venom, encapsulated in liposomes as a modified toxin release system. The activity of the liposomes was evaluated in BALB/c mice, previously infected with 1 × 10 5 of the parasite's promastigotes. The size of the paw lesion in Asp49-PLA 2 -liposomal-treated animals, after 21 days, was observed as decreasing by 16% relative to the untreated control group and 12% by the Glucantime®-treated animals, which was used as a reference drug. At the end of the treatment, the animals were sacrificed and the paw and lymph node tissues were collected. Part of the collection was used to recover amastigotes and another to quantify cytokines and nitrites. In the group treated with Asp49-PLA 2 -liposomes the parasitic load was observed to be reduced by 73.5% in the macerated lymph node, compared to the control group. Comparatively, in the paw tissue was observed a reduction of 57.1%. The infected groups treated with Asp49-PLA 2 -liposomes showed significant production in TNF-α measured in lymph nodes and paw (43.73 pg/mL ± 2.25 and 81.03 pg/mL ± 5.52, respectively) and nitrite levels (31.28 μM ± 0.58 and 35.64 μM ± 5.08) also measured in lymph nodes and paw tissues, respectively, compared to untreated groups. These results indicate that the Asp49-PLA 2 -loaded liposomes were able to activate the production of some cellular components of the protective T H 1 response during the infection, constituting a promising tool for inducing the microbicidal activity of the Leishmania -infected macrophages. [ABSTRACT FROM AUTHOR]

This report describes the effect of photobiomodulation (PBM) on edema formation, leukocyte influx, prostaglandin E 2 (PGE 2 ) biosynthesis and cytotoxicity caused by bothropstoxin-I (BthTX-I), a phospholipase A 2 (PLA 2 ) homologue isolated from Bothrops jararacussu snake venom. Swiss mice or C2C12 cells were irradiated with low-level laser (LLL) at 685 nm wavelength, an energy density of 4.6 J/cm 2 and an irradiation time of 13 s. To evaluate the effect on edema formation and leukocyte influx, LLL was applied to the site of inoculation 30 min and 3 h post-injection. C2C12 cells were exposed to BthTX-I and immediately irradiated. PBM significantly reduced paw edema formation, peritoneal leukocyte influx and PGE 2 synthesis, but increased the viability of C2C12 muscle cells after BthTX-I incubation. These findings demonstrate that PBM attenuated the inflammatory events induced by BthTX-I. The attenuation of PGE 2 synthesis could be an important factor in the reduced inflammatory response caused by laser irradiation. The ability of LLL irradiation to protect muscle cells against the deleterious effects of BthTX-I may indicate preservation of the plasma membrane. [ABSTRACT FROM AUTHOR]

Phospholipases A 2 (PLA 2 s) are important enzymes present in snake venoms and are related to a wide spectrum of pharmacological effects, however the toxic potential and therapeutic effects of acidic isoforms have not been fully explored and understood. Due to this, the present study describes the isolation and biochemical characterization of two new acidic Asp49-PLA 2 s from Bothrops brazili snake venom, named Braziliase-I and Braziliase-II. The venom was fractionated in three chromatographic steps: ion exchange, hydrophobic interaction and reversed phase. The isoelectric point (pI) of the isolated PLA 2 s was determined by two-dimensional electrophoresis, and 5.2 and 5.3 pIs for Braziliase-I and II were observed, respectively. The molecular mass was determined with values ​​of 13,894 and 13,869 Da for Braziliase-I and II, respectively. Amino acid sequence by Edman degradation and mass spectrometry completed 87% and 74% of the sequences, respectively for Braziliase-I and II. Molecular modeling of isolated PLA 2 s using acid PLA 2 BthA-I-PLA 2 from B. jararacussu template showed high quality. Both acidic PLA 2 s showed no significant myotoxic activity, however they induced significant oedematogenic activity. Braziliase-I and II (100 μg/mL) showed 31.5% and 33.2% of cytotoxicity on Trypanosoma cruzi and 26.2% and 19.2% on Leishmania infantum, respectively. Braziliase-I and II (10 μg) inhibited 96.98% and 87.98% of platelet aggregation induced by ADP and 66.94% and 49% induced by collagen, respectively. The acidic PLA 2 s biochemical and structural characterization can lead to a better understanding of its pharmacological effects and functional roles in snakebites pathophysiology, as well as its possible biotechnological applications as research probes and drug leads. [ABSTRACT FROM AUTHOR]

Crotalus Neutralizing Factor (CNF) is an inhibitor of phospholipase A 2 (PLA 2 ), present in the blood plasma of Crotalus durissus terrificus snake. This inhibitor neutralizes the lethal and enzymatic activity of crotoxin, the main neurotoxin from this venom. In this study, we investigated the effects of CNF on the functionality of human peripheral blood mononuclear cells (PBMCs) and human neutrophils. The following parameters were evaluated: viability and proliferation, chemotaxis, cytokines and LTB 4 production, cytosolic PLA 2 s activity, myeloperoxidase (MPO) and superoxide anion (O 2 − ) production. CNF showed no toxicity on PBMCs or neutrophils, and acts by stimulating the release of TNF-α and LTB 4 , but neither stimulates IL-10 and IL-2 nor affects PBMCs proliferation and O 2 − release. In neutrophils, CNF induces chemotaxis but does not induce the release of both MPO and O 2 − . However, it induces LTB 4 and IL-8 production. These data show the influence of CNF on PBMCs’ function by inducing TNF-α and LTB 4 production, and on neutrophils, by stimulating chemotaxis and LTB 4 production, via cytosolic PLA 2 activity, and IL-8 release. The inflammatory profile produced by CNF is shown for the first time. Our present results suggest that CNF has a role in activation of leukocytes and exert proinflammatory effects on these cell. [ABSTRACT FROM AUTHOR]

Phospholipases A 2 inhibitors (PLIs) produced by venomous and non-venomous snakes play essential role in this resistance. These endogenous inhibitors may be classified by their fold in PLIα, PLIβ and PLIγ. Phospholipases A 2 (PLA 2 s) develop myonecrosis in snake envenomation, a consequence that is not efficiently neutralized by antivenom treatment. This work aimed to identify and characterize two PLIs from Amazonian snake species, Bothrops atrox and Micrurus lemniscatus . Liver tissues RNA of specimens from each species were isolated and amplified by RT-PCR using PCR primers based on known PLIγ gene sequences, followed by cloning and sequencing of amplified fragments. Sequence similarity studies showed elevated identity with inhibitor PLIγ gene sequences from other snake species. Molecular models of translated inhibitors' gene sequences resemble canonical three finger fold from PLIγ and support the hypothesis that the decapeptide (residues 107–116) may be responsible for PLA 2 inhibition. Structural studies and action mechanism of these PLIs may provide necessary information to evaluate their potential as antivenom or as complement of the current ophidian accident treatment. [ABSTRACT FROM AUTHOR]

Animal venoms and their chemical compounds have aroused both empirical and scientific attention for ages. However, there has been a significant increase in scientific investigations in recent decades, allowing the production of various formulations that are helping in the development of many important tools for biotechnological, diagnostic, or therapeutic use, both in human and animal health, as well as in plants. Venoms are composed of biomolecules and inorganic compounds that may have physiological and pharmacological activities that are not related to their principal actions (prey immobilization, digestion, and defense). Snake venom toxins, mainly enzymatic and non-enzymatic proteins, and peptides have been identified as potential prototypes for new drugs and/or models for the development of pharmacologically active structural domains for the treatment of cancer, cardiovascular diseases, neurodegenerative and autoimmune diseases, pain, and infectious-parasitic diseases. This minireview aims to provide an overview of the biotechnological potential of animal venoms, with a focus on snakes, and to introduce the reader to the fascinating world of Applied Toxinology, where animal biodiversity can be used to develop therapeutic and diagnostic applications for humans. [ABSTRACT FROM AUTHOR]

Snake venoms contain various proteins, especially phospholipases A 2 (PLA 2 s), which present potential applications in diverse areas of health and medicine. In this study, a new basic PLA 2 from Bothrops marajoensis with parasiticidal activity was purified and characterized biochemically and biologically. B. marajoensis venom was fractionated through cation exchange followed by reverse phase chromatographies. The isolated toxin, BmajPLA 2 -II, was structurally characterized with MALDI-TOF (Matrix-assisted laser desorption/ionization-time of flight) mass spectrometry, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by two-dimensional electrophoresis, partial amino acid sequencing, an enzymatic activity assay, circular dichroism, and dynamic light scattering assays. These structural characterization tests presented BmajPLA 2 -II as a basic Lys49 PLA 2 homologue, compatible with other basic snake venom PLA 2 s (svPLA 2 ), with a tendency to form aggregations. The in vitro anti-parasitic potential of B. marajoensis venom and of BmajPLA 2 -II was evaluated against Leishmania infantum promastigotes and Trypanosoma cruzi epimastigotes, showing significant activity at a concentration of 100 μg/mL. The venom and BmajPLA 2 -II presented IC 50 of 0.14 ± 0.08 and 6.41 ± 0.64 μg/mL, respectively, against intraerythrocytic forms of Plasmodium falciparum with CC 50 cytotoxicity values against HepG2 cells of 43.64 ± 7.94 and >150 μg/mL, respectively. The biotechnological potential of these substances in relation to leishmaniasis, Chagas disease and malaria should be more deeply investigated. [ABSTRACT FROM AUTHOR]

BaltLAAO-I, an L-amino acid oxidase isolated from Bothrops alternatus, is a glycoprotein enzyme with a pI–5.3, 15% sugar and a related molecular mass of 66,000 Da in its monomeric form, and 123,000 Da in its dimeric form. The objective of this study is to describe the cytotoxicity activity induced by BaltLAAO-I isolated from Bothrops alternatus venom and its possible mechanism of action on tumor cells. Our results clearly depict that BaltLAAO-I has a strong selective cytotoxic activity on tumor cell lines (JURKAT, SK-BR-3 and B16F10). On the other hand, the results show low cytotoxicity on human peripheral blood mononuclear cells. Furthermore, our findings demonstrate that BaltLAAO-I induces the apoptosis of tumor cell lines through a cytotoxic activity exerted by a generation of reactive oxygen intermediates. All in all, the data indicate that LAAOs exert a selective cytotoxic role on tumor cells, demonstrating a great potential for future use in clinical therapy. [ABSTRACT FROM AUTHOR]

The action of LAAO, an L-amino acid oxidase isolated from Calloselasma rhodosthoma snake venom, on isolated human neutrophil function was investigated. Cr-LAAO showed no toxicity on neutrophils. Cr-LAAO in its native form induced the neutrophil chemotaxis, suggesting that its primary structure is essential for stimulation the cell. p38 MAPK and PI3K have a role as signaling pathways of CR-LAAO induced chemotaxis. This toxin also induced the production of hydrogen peroxide and stimulated phagocytosis in neutrophils. Furthermore, Cr-LAAO was able to stimulate neutrophils to release IL-6, IL-8, MPO, LTB 4 and PGE 2 . Together, the data showed that the Cr-LAAO triggers relevant proinflammatory events. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate in vitro anti- Leishmania amazonensis activity of a Phospholipase A 2 (Asp49-PLA 2 ) isolated from the venom of Bothrops jararacussu and its encapsulated form. Asp49-PLA 2 (2 mg/mL) was added to a lipid mixture, solubilized in chloroform and dried under nitrogen flow. The lipid vesicles were formed homogeneously using the extrusion method, physicochemically characterized by their diameters, zeta potentials, encapsulation rate and also submitted to a molecular docking in silico analysis. The activity of Asp49-liposomes was evaluated in vitro against promastigote forms of L. amazonensis and J774 macrophages. Parasite and macrophage viabilities using MTT method were assessed after 48 h incubation. L. amazonensis -infected macrophages were also incubated with encapsulated Asp49 and in solution form. The amastigote forms were counted inside the infected macrophages and the culture supernatants were collected for nitrites and TNF-α quantifications. Asp49-PLA 2 in solution form displayed an anti - Leishmania concentration-dependent effect. Asp49-liposomes were able to reduce 78% of promastigote forms and preserved 82% of J774 macrophages' viability. After 48 h of incubation with nanoencapsulated Asp49-PLA 2 there was a significant reduction in the number of amastigotes (55%; p < 0.05) compared to the control group. When the macrophages were infected and incubated with Asp49-liposomes a significant increase in the production of nitrites and TNF-α was observed when compared to infected cells alone. The results indicated that the liposomal system achieved in this study is a promising tool to enhance microbicidal activity of the infected macrophages, conferring a biotechnological therapeutic approach against experimental leishmaniasis. [ABSTRACT FROM AUTHOR]

Snake venom is a complex mixture of active compounds consisting of 80–90% proteins and peptides that exhibit a variety of biological actions that are not completely clarified or identified. Of these, phospholipase A 2 is one of the molecules that has shown great biotechnological potential. The objectives of this study were to isolate, biochemically and biologically characterize a Lys49 phospholipase A 2 homologue from the venom of Bothrops neuwiedi urutu . The protein was purified after two chromatographic steps, anion exchange and reverse phase. The purity and relative molecular mass were assessed by SDS-PAGE, observing a molecular weight typical of PLA 2 s, subsequently confirmed by mass spectrometry obtaining a mass of 13,733 Da. As for phospholipase activity, the PLA 2 proved to be enzymatically inactive. The analyses by Edman degradation and sequencing of the peptide fragments allowed for the identification of 108 amino acid residues; this sequence showed high identity with other phospholipases A 2 from Bothrops snake venoms, and identified this molecule as a novel PLA 2 isoform from B . neuwiedi urutu venom, called BnuTX-I. In murine models, both BnuTX-I as well as the venom induced edema and myotoxic responses. The cytotoxic effect of BnuTX-I in murine macrophages was observed at concentrations above 12 μg/mL. BnuTX-I also presented antimicrobial activity against gram-positive and negative bacterial strains, having the greatest inhibitory effect on Pseudomonas aeruginosa . The results allowed for the identification of a new myotoxin isoform with PLA 2 structure with promising biotechnological applications. [ABSTRACT FROM AUTHOR]

Snake venom toxins are related not only in detention, death and the promotion of initial digestion of prey but also due to their different biochemical, structural and pharmacological effects they can result in new drugs. Among these toxins snake venom serine proteases (SVSPs) should be highlighted because they are responsible for inducing changes in physiological functions such as blood coagulation, fibrinolysis, and platelet aggregation. This article presents the first serine protease (SP) isolated from Bothrops brazili : BbrzSP-32. The new SP showed 36 kDa of relative molecular mass and its absolute mass was confirmed by mass spectrometry as 32,520 Da. It presents 79.48% identity when compared to other SVSPs and was able to degrade the α-chain of fibrinogen, in in vitro models, because of this it is considered a SVTLE-A. It showed dose-dependent activity in the process of degradation of fibrin networks demonstrating greater specificity for this activity when compared to its thrombolytic action. BbrzSP-32 demonstrated proteolytic activity on gelatin and chromogenic substrates for serine proteases and thrombin-like enzymes (S-2288 and S-2238 respectively), besides having coagulant activity on human plasma. After pre-incubation with PMSF and benzamidine the coagulant and proteolytic activities on the S-2288 and S-2238 substrates were reduced. BbrzSP-32 shows stability against pH and temperature variations, demonstrating optimum activity between 30 and 40 °C and in the pH range 7.5 to 8.5. A new SP with potential biotechnological application was isolated. [ABSTRACT FROM AUTHOR]

Metalloproteases play a fundamental role in snake venom envenomation inducing hemorrhagic, fibrigen(ogen)olytic and myotoxic effects in their victims. Several snake venoms, such as those from the Bothrops genus, present important local effects which are not efficiently neutralized by conventional serum therapy. Consequently, these accidents may result in permanent sequelae and disability, creating economic and social problems, especially in developing countries, leading the attention of the World Health Organization that considered ophidic envenomations a neglected tropical disease. Aiming to produce an efficient inhibitor against bothropic venoms, we synthesized different molecules classified as quinolinones – a group of low-toxic chemical compounds widely used as antibacterial and antimycobacterial drugs – and tested their inhibitory properties against hemorrhage caused by bothropic venoms. The results from this initial screening indicated the molecule 2-hydroxymethyl-6-methoxy-1,4-dihydro-4-quinolinone (Q8) was the most effective antihemorrhagic compound among all of the assayed synthetic quinolinones. Other in vitro and in vivo experiments showed this novel compound was able to inhibit significantly the hemorrhagic and/or proteolytic activities of bothropic crude venoms and isolated snake venom metalloproteases (SVMPs) even at lower concentrations. Docking and molecular dynamic simulations were also performed to get insights into the structural basis of Q8 inhibitory mechanism against proteolytic and hemorrhagic SVMPs. These structural studies demonstrated that Q8 may form a stable complex with SVMPs, impairing the access of substrates to the active sites of these toxins. Therefore, both experimental and structural data indicate that Q8 compound is an interesting candidate for antiophidic therapy, particularly for the treatment of the hemorrhagic and necrotic effects induced by bothropic venoms. [ABSTRACT FROM AUTHOR]

This study describes the biochemical and functional characterization of a new metalloproteinase named BbMP-1, isolated from Bothrops brazili venom. BbMP-1 was homogeneous on SDS-PAGE, presented molecular mass of 22,933Da and pI 6.4. The primary structure was partially elucidated with high identity with others metalloproteinases from Viperidae venoms. The enzymatic activity on azocasein was evaluated in different experimental conditions (pH, temperature). A significant reduction in enzyme activity after exposure to chelators of divalent cations (EDTA), reducing agents (DTT), pH less than 5.0 or temperatures higher than 45 °C was observed. BbMP-1 showed activity on fibrinogen degrading Aα chain quickly and to a lesser extent the Bβ chain. Also demostrated to be weakly hemorrhagic, presenting however, significant myotoxic and edematogenic activity. The in vitro activity of BbMP-1 against Plasmodium falciparum showed an IC 50 of 3.2 ± 2.0 μg/mL. This study may help to understand the pathophysiological effects induced by this group of toxin and their participation in the symptoms observed in cases of snake envenomation. Moreover, this result is representative for this group of proteins and shows the biotechnological potential of BbMP-1 by the demonstration of its antiplasmodial activity. [ABSTRACT FROM AUTHOR]

The Micrurus genus is the American representative of Elapidae family. Micrurus spixii is endemic of South America and northern states of Brazil. Elapidic venoms contain neurotoxins that promote curare-mimetic neuromuscular blockage. In this study, biochemical and functional characterizations of M. spixii crude venom were performed and a new neurotoxic phospholipase A 2 called MsPLA 2 -I was isolated. M. spixii crude venom caused severe swelling in the legs of tested mice and significant release of creatine kinase (CK) showing its myotoxic activity. Leishmanicidal activity against Leishmania amazonensis (IC 50 1.24 μg/mL) was also observed, along with antiplasmodial activity against Plasmodium falciparum , which are unprecedented for Micrurus venoms. MsPLA 2 -I with a Mr 12,809.4 Da was isolated from the crude venom of M. spixii . The N-terminal sequencing of a fragment of 60 amino acids showed 80% similarity with another PLA 2 from Micrurus altirostris . This toxin and the crude venom showed phospholipase activity. In a mouse phrenic nerve-diaphragm preparation, M. spixii venom and MsPLA 2 -I induced the blockage of both direct and indirect twitches. While the venom presented a pronounced myotoxic activity, MsPLA 2 -I expressed a summation of neurotoxic activity. The results of this study make M. spixii crude venom promising compounds in the exploration of molecules with microbicidal potential. [ABSTRACT FROM AUTHOR]

Abstract: The in vitro effects of LAAO, an l-amino acid oxidase isolated from Calloselasma rhodosthoma snake venom, on isolated human neutrophil function were investigated. LAAO showed no toxicity on neutrophils. At non-cytotoxic concentrations, LAAO induced the superoxide anion production by isolated human neutrophil. This toxin, in its native form, is also able to stimulate the production of hydrogen peroxide in neutrophils, suggesting that its primary structure is essential for stimulation the cell. Moreover, the incubation of LAAO and phenol red medium did not induce the production of hydrogen peroxide. Furthermore, LAAO was able to stimulate neutrophils to release proinflammatory mediators such as IL-8 and TNF-α as well as NETs liberation. Together, the data showed that the LAAO triggers relevant proinflammatory events. Particular regions of the molecule distinct from the LAAO catalytic site may be involved in the onset of inflammatory events. [Copyright &y& Elsevier]

Abstract: Crotoxin is a neurotoxin from Crotalus durissus terrificus venom that shows immunomodulatory, anti-inflammatory, antimicrobial, antitumor and analgesic activities. Structurally, this toxin is a heterodimeric complex composed by a toxic basic PLA2 (Crotoxin B or CB) non-covalently linked to an atoxic non-enzymatic and acidic component (Crotapotin, Crotoxin A or CA). Several CA and CB isoforms have been isolated and characterized, showing that the crotoxin venom fraction is, in fact, a mixture of different molecules derived from the combination of distinct subunit isoforms. Intercro (IC) is a protein from the same snake venom which presents high similarity in primary structure to CB, indicating that it could be an another isoform of this toxin. In this work, we compare IC to the crotoxin complex (CA/CB) and/or CB in order to understand its functional aspects. The experiments with IC revealed that it is a new toxin with different biological activities from CB, keeping its catalytic activity but presenting low myotoxicity and absence of neurotoxic activity. The results also indicated that IC is structurally similar to CB isoforms, but probably it is not able to form a neurotoxic active complex with crotoxin A as observed for CB. Moreover, structural and phylogenetic data suggest that IC is a new toxin with possible toxic effects not related to the typical CB neurotoxin. [Copyright &y& Elsevier]