Functional, immunological characterization, and anticancer activity of BaMtx: A new Lys49- PLA2 homologue isolated from the venom of Peruvian Bothrops atrox snake (Serpentes: Viperidae).

Bothorps atrox is responsible for most of the ophidism cases in Perú. As part of the envenoming, myotoxicity is one of the most recurrent and destructive effects. In this study, a myotoxin, named BaMtx, was purified from B. atrox venom to elucidate its biological, immunological, and molecular characteristics. BaMtx was purified using CM-Sephadex-C-25 ion-exchange resin and SDS-PAGE analysis showed a unique protein band of 13 kDa or 24 kDa under reducing or non-reducing conditions, respectively. cDNA sequence codified a 122-aa mature protein with high homology with other Lys49-PLA2s; modeled structure showed a N-terminal helix, a ß-wing region, and a C-terminal random coil. This protein has a poor phospholipase A2 enzymatic activity. BaMtx has myotoxic (DMM = 12.30 ± 0.95 µg) and edema-forming (DEM = 26.00 ± 1.15 µg) activities. Rabbit immunization with purified enzyme produced anti-BaMtx antibodies that reduced 50.28 ± 10.15% of myotoxic activity and showed significant cross-reactivity against B. brazili and B pictus venoms. On the other hand, BaMtx exhibits mild anti-proliferative and anti-migratory effects on breast cancer cells, affecting the ROS and NADH levels, which may reduce mitochondrial respiration. These results contribute to the understanding of B. atrox Lys49-PLA2 effects and establish the anticancer potential de BaMtx.

Mutagenic and genotoxic activities of Phospholipase A2 Bothropstoxin-I from Bothrops jararacussu in Drosophila melanogaster and human cell lines.

Phospholipase A2 Bothropstoxin-I (PLA2 BthTX-I) is a myotoxic Lys49-PLA2 from Bothrops jararacussu snake venom. In order to evaluate the DNA damage caused by BthTX-I, we used the Somatic Mutation and Recombination Test (SMART) in Drosophila melanogaster and Comet assay in HUVEC and DU-145 cells. For SMART, different concentrations of BthTX-I (6.72 to 430 µg/mL) were used and no significant changes in the survival rate were observed. Significant frequency of mutant spots was observed for the ST cross at the highest concentration of BthTX-I due to recombinogenic activity. In the HB cross, BthTX-I increased the number of mutant spots at intermediate concentrations, being 53.75 µg/mL highly mutagenic and 107.5 µg/mL predominantly recombinogenic. The highest concentrations were neither mutagenic nor recombinogenic, which could indicate cytotoxicity in the wing cells of D. melanogaster. In vitro, all BthTX-I concentrations (1 to 50 µg/mL) induced decrease in HUVEC cell viability, as well as in DU-145 cells at concentrations of 10, 25, and 50 µg/mL. The comet assay showed that in HUVEC and DU-145 cells, all BthTX-I concentrations promoted increase of DNA damage. Further studies should be performed to elucidate the mechanism of action of PLA2 BthTX-I and its possible use in therapeutic strategies against cancer.

Inflammasome Activation Induced by a Snake Venom Lys49-Phospholipase A2 Homologue.

BACKGROUND: Snake venom phospholipases A2 (PLA2s) have hemolytic, anticoagulant, myotoxic, oedematogenic, bactericidal, and inflammatory actions. BthTX-I, a Lys49-PLA2 isolated from Bothrops jararacussu venom, is an example of Lys49-PLA2 that presents such actions. NLRP3 is a cytosolic receptor from the NLR family responsible for inflammasome activation via caspase-1 activation and IL-1ß liberation. The study of NLRs that recognize tissue damage and activate the inflammasome is relevant in envenomation. METHODS: Male mice (18-20 g) received an intramuscular injection of BthTX-I or sterile saline. The serum was collected for creatine-kinase (CK), lactate dehydrogenase (LDH), and interleukin-1ß (IL-1ß) assays, and muscle was removed for inflammasome activation immunoblotting and qRT-PCR expression for nucleotide and oligomerization domain, leucine-rich repeat-containing protein family, pyrin-containing domain 3 receptor (NLRP3) inflammasome components. RESULTS: BthTX-I-induced inflammation and myonecrosis, shown by intravital microscope, and LDH and CK release, respectively. Mouse treatment with A438079, a P2X7 receptor antagonist, did not modify these effects. BthTX-I induced inflammasome activation in muscle, but P2X7R participation in this effect was not observed. CONCLUSION: Together, the results showed for the first time that BthTX-I in gastrocnemius muscle induces inflammation and consequently, inflammasome activation via NLRP3 with caspase-1 activation and IL-1ß liberation.

Articular inflammation induced by an enzymatically-inactive Lys49 phospholipase A2: activation of endogenous phospholipases contributes to the pronociceptive effect.

BACKGROUND: Arthritis is a set of inflammatory conditions that induce aching, stiffness, swelling, pain and may cause functional disability with severe consequences to the patient's lives. These are multi-mediated pathologies that cannot be effectively protected and/or treated. Therefore, the aim of this study was to establish a new model of acute arthritis, using a Lys49-PLA2 (Bothrops asper myotoxin II; MT-II) to induce articular inflammation. METHODS: The articular inflammation was induced by MT-II (10 µg/joint) injection into the left tibio-tarsal or femoral-tibial-patellar joints. Cellular influx was evaluated counting total and differential cells that migrated to the joint. The plasma extravasation was determined using Evans blue dye. The edematogenic response was evaluated measuring the joint thickness using a caliper. The articular hypernociception was determined by a dorsal flexion of the tibio-tarsal joint using an electronic pressure-meter test. The mediators involved in the articular hypernociception were evaluated using receptor antagonists and enzymatic inhibitors. RESULTS: Plasma extravasation in the knee joints was observed 5 and 15 min after MT-II (10 µg/joint) injection. MT-II also induced a polymorphonuclear cell influx into the femoral-tibial-patellar joints observed 8 h after its injection, a period that coincided with the peak of the hyperalgesic effect. Hyperalgesia was inhibited by the pretreatment of the animals with cyclooxygenase inhibitor indomethacin, with type-2 cyclooxygenase inhibitor celecoxib, with AACOCF3 and PACOCF3, inhibitors of cytosolic and Ca2+-independent PLA2s, respectively, with bradykinin B2 receptor antagonist HOE 140, with antibodies against TNFα, IL-1ß, IL-6 and CINC-1 and with selective ET-A (BQ-123) and ET-B (BQ-788) endothelin receptors antagonists. The MT-II-induced hyperalgesia was not altered by the lipoxygenase inhibitor zileuton, by the bradykinin B1 receptor antagonist Lys-(Des-Arg9,Leu8)-bradykinin, by the histamine and serotonin antagonists promethazine and methysergide, respectively, by the nitric oxide inhibitor LNMMA and by the inhibitor of matrix 1-, 2-, 3-, 8- and 9- metalloproteinases GM6001 (Ilomastat). CONCLUSION: These results demonstrated the multi-mediated characteristic of the articular inflammation induced by MT-II, which demonstrates its relevance as a model for arthritis mechanisms and treatment evaluation.

Articular inflammation induced by an enzymatically-inactive Lys49 phospholipase A2: activation of endogenous phospholipases contributes to the pronociceptive effect

Background Arthritis is a set of inflammatory conditions that induce aching, stiffness, swelling, pain and may cause functional disability with severe consequences to the patient's lives. These are multi-mediated pathologies that cannot be effectively protected and/or treated. Therefore, the aim of this study was to establish a new model of acute arthritis, using a Lys49-PLA2 (Bothrops asper myotoxin II; MT-II) to induce articular inflammation. Methods The articular inflammation was induced by MT-II (10 μg/joint) injection into the left tibio-tarsal or femoral-tibial-patellar joints. Cellular influx was evaluated counting total and differential cells that migrated to the joint. The plasma extravasation was determined using Evans blue dye. The edematogenic response was evaluated measuring the joint thickness using a caliper. The articular hypernociception was determined by a dorsal flexion of the tibio-tarsal joint using an electronic pressure-meter test. The mediators involved in the articular hypernociception were evaluated using receptor antagonists and enzymatic inhibitors. Results Plasma extravasation in the knee joints was observed 5 and 15 min after MT-II (10 μg/joint) injection. MT-II also induced a polymorphonuclear cell influx into the femoral-tibial-patellar joints observed 8 h after its injection, a period that coincided with the peak of the hyperalgesic effect. Hyperalgesia was inhibited by the pretreatment of the animals with cyclooxygenase inhibitor indomethacin, with type-2 cyclooxygenase inhibitor celecoxib, with AACOCF3 and PACOCF3, inhibitors of cytosolic and Ca2+-independent PLA2s, respectively, with bradykinin B2 receptor antagonist HOE 140, with antibodies against TNFα, IL-1β, IL-6 and CINC-1 and with selective ET-A (BQ-123) and ET-B (BQ-788) endothelin receptors antagonists. The MT-II-induced hyperalgesia was not altered by the lipoxygenase inhibitor zileuton, by the bradykinin B1 receptor antagonist Lys-(Des-Arg9,Leu8)-bradykinin, by the histamine and serotonin antagonists promethazine and methysergide, respectively, by the nitric oxide inhibitor LNMMA and by the inhibitor of matrix 1-, 2-, 3-, 8- and 9- metalloproteinases GM6001 (Ilomastat). Conclusion These results demonstrated the multi-mediated characteristic of the articular inflammation induced by MT-II, which demonstrates its relevance as a model for arthritis mechanisms and treatment evaluation.(AU)

Articular inflammation induced by an enzymatically-inactive Lys49 phospholipase A2: activation of endogenous phospholipases contributes to the pronociceptive effect

Abstract Background Arthritis is a set of inflammatory conditions that induce aching, stiffness, swelling, pain and may cause functional disability with severe consequences to the patients lives. These are multi-mediated pathologies that cannot be effectively protected and/or treated. Therefore, the aim of this study was to establish a new model of acute arthritis, using a Lys49-PLA2 (Bothrops asper myotoxin II; MT-II) to induce articular inflammation. Methods The articular inflammation was induced by MT-II (10 g/joint) injection into the left tibio-tarsal or femoral-tibial-patellar joints. Cellular influx was evaluated counting total and differential cells that migrated to the joint. The plasma extravasation was determined using Evans blue dye. The edematogenic response was evaluated measuring the joint thickness using a caliper. The articular hypernociception was determined by a dorsal flexion of the tibio-tarsal joint using an electronic pressure-meter test. The mediators involved in the articular hypernociception were evaluated using receptor antagonists and enzymatic inhibitors. Results Plasma extravasation in the knee joints was observed 5 and 15 min after MT-II (10 g/joint) injection. MT-II also induced a polymorphonuclear cell influx into the femoral-tibial-patellar joints observed 8 h after its injection, a period that coincided with the peak of the hyperalgesic effect. Hyperalgesia was inhibited by the pretreatment of the animals with cyclooxygenase inhibitor indomethacin, with type-2 cyclooxygenase inhibitor celecoxib, with AACOCF3 and PACOCF3, inhibitors of cytosolic and Ca2+-independent PLA2s, respectively, with bradykinin B2 receptor antagonist HOE 140, with antibodies against TNF, IL-1, IL-6 and CINC-1 and with selective ET-A (BQ-123) and ET-B (BQ-788) endothelin receptors antagonists. The MT-II-induced hyperalgesia was not altered by the lipoxygenase inhibitor zileuton, by the bradykinin B1 receptor antagonist Lys-(Des-Arg9,Leu8)-bradykinin, by the histamine and serotonin antagonists promethazine and methysergide, respectively, by the nitric oxide inhibitor LNMMA and by the inhibitor of matrix 1-, 2-, 3-, 8- and 9- metalloproteinases GM6001 (Ilomastat). Conclusion These results demonstrated the multi-mediated characteristic of the articular inflammation induced by MT-II, which demonstrates its relevance as a model for arthritis mechanisms and treatment evaluation.

Ultrastructural aspects of mouse nerve-muscle preparation exposed to Bothrops jararacussu and Bothrops bilineatus venoms and their toxins BthTX-I and Bbil-TX: Unknown myotoxic effects.

Bites by Bothrops snakes normally induce local pain, haemorrhage, oedema and myonecrosis. Mammalian isolated nerve-muscle preparations exposed to Bothrops venoms and their phospholipase A2 toxins (PLA2 ) can exhibit a neurotoxic pattern as increase in frequency of miniature end-plate potentials (MEPPs) as well as in amplitude of end-plate potentials (EPPs); neuromuscular facilitation followed by complete and irreversible blockade without morphological evidence for muscle damage. In this work, we analysed the ultrastructural damage induced by Bothrops jararacussu and Bothrops bilineatus venoms and their PLA2 toxins (BthTX-I and Bbil-TX) in mouse isolated nerve-phrenic diaphragm preparations (PND). Under transmission electron microscopy (TEM), PND preparations previously exposed to B. jararacussu and B. bilineatus venoms and BthTX-I and Bbil-TX toxins showed hypercontracted and loosed myofilaments; unorganized sarcomeres; clusters of edematous sarcoplasmic reticulum and mitochondria; abnormal chromatin distribution or apoptotic-like nuclei. The principal affected organelles, mitochondria and sarcoplasmic reticulum, were those related to calcium buffering and, resulting in sarcomeres and myofilaments hypercontraction. Schwann cells were also damaged showing edematous axons and mitochondria as well as myelin sheath alteration. These ultrastructural changes caused by both of Bothrops venoms and toxins indicate that the neuromuscular blockade induced by them in vitro can also be associated with nerve and muscle degeneration.

Identification of Lys49-PLA2 from crude venom of Crotalus atrox as a human neutrophil-calcium modulating protein.

We fortuitously observed a human neutrophil intracellular free-calcium concentration ([Ca(2+)]i) increasing activity in the commercially available phosphodiesterase I (PDE I), which is actually dried crude venom of Crotalus atrox. As this activity was not observed with another commercially available pure PDE I, we tried to find out the causative molecule(s) present in 'crude' PDE, and identified Lys49-phospholipase A2 (Lys49-PLA2 or K49-PLA2), a catalytically inactive protein which belongs to the phospholipase A2 family, by activity-driven three HPLC (reverse phase, size exclusion, reverse phase) steps followed by SDS-PAGE and LC-MS/MS. K49-PLA2 induced Ca(2+) infl ux in human neutrophils without any cytotoxic eff ect. Two calcium channel inhibitors, 2-aminoetoxydiphenyl borate (2-APB) (30 µM) and SKF-96365 (20 µM) signifi cantly inhibited K49-PLA2-induced [Ca(2+)]i increase. These results suggest that K49-PLA2 modulates [Ca(2+)]i in human neutrophils via 2-APB- and SKF-96365-sensitive calcium channels without causing membrane disruption.

Identification of Lys49-PLA2 from crude venom of Crotalus atrox as a human neutrophil-calcium modulating protein

We fortuitously observed a human neutrophil intracellular free-calcium concentration ([Ca2+]i) increasing activity in the commercially available phosphodiesterase I (PDE I), which is actually dried crude venom of Crotalus atrox. As this activity was not observed with another commercially available pure PDE I, we tried to find out the causative molecule(s) present in 'crude' PDE, and identified Lys49-phospholipase A2 (Lys49-PLA2 or K49-PLA2), a catalytically inactive protein which belongs to the phospholipase A2 family, by activity-driven three HPLC (reverse phase, size exclusion, reverse phase) steps followed by SDS-PAGE and LC-MS/MS. K49-PLA2 induced Ca2+ infl ux in human neutrophils without any cytotoxic eff ect. Two calcium channel inhibitors, 2-aminoetoxydiphenyl borate (2-APB) (30 microM) and SKF-96365 (20 microM) signifi cantly inhibited K49-PLA2-induced [Ca2+]i increase. These results suggest that K49-PLA2 modulates [Ca2+]i in human neutrophils via 2-APB- and SKF-96365-sensitive calcium channels without causing membrane disruption.

Synthesis and characterization of an antibacterial and non-toxic dimeric peptide derived from the C-terminal region of Bothropstoxin-I.

Infectious diseases are among the leading global causes of death, increasing the search for novel antibacterial agents. Among these, biologically active peptides are an excellent research tool. Using solid-phase peptide synthesis (SPPS), this work aimed to synthesize the peptide derived from the C-terminal region of Bothropstoxin-I (BthTX-I) (p-BthTX-I, sequence: KKYRYHLKPFCKK), and its disulfide-linked dimeric form, obtained via air oxidation (p-BthTX-I)2. Two other peptides were synthesized to evaluate the dimerization effect on antimicrobial activity. In both sequences, the cysteine (Cys) residue was replaced by the serine (Ser) residue, differing, however, in their C-terminus position. The antimicrobial activity of the peptides against gram-negative (Escherichia (E.) coli) and gram-positive (Staphylococcus (S.) aureus) bacteria and yeast (Candida (C.) albicans) was evaluated. Interestingly, only peptides containing the Cys residue showed antimicrobial activity, suggesting the importance of Cys residue and its dimerization for the observed activity. Apparently, p-BthTX-I and (p-BthTX-I)2 did not promote lysis or form pores and were not able to interact with membranes. Furthermore, they neither showed antifungal activity against C. albicans nor toxicity against erythrocytes, epithelial cells, or macrophages, indicating a potential specificity against prokaryotic cells.

Purification and preliminary crystallographic analysis of a new Lys49-PLA2 from B. Jararacussu.

BjVIII is a new myotoxic Lys49-PLA2 isolated from Bothrops jararacussu venom that exhibits atypical effects on human platelet aggregation. To better understand the mode of action of BjVIII, crystallographic studies were initiated. Two crystal forms were obtained, both containing two molecules in the asymmetric unit (ASU). Synchrotron radiation diffraction data were collected to 2.0 A resolution and 1.9 A resolution for crystals belonging to the space group P2(1)2(1)2(1) (a = 48.4 A, b = 65.3 A, c = 84.3 A) and space group P3(1)21 (a = b = 55.7 A, c = 127.9 A), respectively. Refinement is currently in progress and the refined structures are expected to shed light on the unusual platelet aggregation activity observed for BjVIII.