[show abstract][hide abstract] ABSTRACT: Currently, Crotalus viridis was divided into two species: Crotalus viridis and Crotalus oreganus. The current classification divides "the old" Crotalus viridis into two new and independent species: Crotalus viridis (subspecies: viridis and nuntius) and Crotalus oreganus (subspecies: abyssus, lutosus, concolor, oreganus, helleri, cerberus, and caliginis). The analysis of a product from cDNA (E6d), derived from the gland of a specie Crotalus viridis viridis, was found to produce an acid phospholipase A 2 . In this study we isolated and characterized a PLA 2 (D49) from Crotalus oreganus abyssus venom. Our studies show that the PLA 2 produced from the cDNA of Crotalus viridis viridis (named E6d) is exactly the same PLA 2 primary sequence of amino acids isolated from the venom of Crotalus oreganus abyssus. Thus, the PLA 2 from E6d cDNA is actually the same PLA 2 presented in the venom of Crotalus oreganus abyssus and does not correspond to the venom from Crotalus viridis viridis. These facts highlight the importance of performing more studies on subspecies of Crotalus oreganus and Crotalus viridis, since the old classification may have led to mixed results or mistaken data.
BioMed Research International 02/2015; 2014:1-15. · 2.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: The Mediterranean flour moth (Anagasta kuehniella) is a pest insect that attacks stored foods. The difficulty in controlling this kind of pest promotes the development of alternatives for pest control, among them the use of proteins with insecticide effect. In this work, we evaluated the role of a trypsin inhibitor purified from Entada acaciifolia seeds (EATI) on the A. kuehniella development. Different concentrations of inhibitor were added to a diet to determine its effects on insect performance. At 0.4%, the EATI decreases the larval weight and survival rates by 54.6% and 15%, respectively; in addition to the extension of the life cycle of insect. The biochemical analysis showed that the inhibitor is refractory to the digestion by midgut proteases, and led to a reduction of 32% in general proteolytic activity. A detailed analysis of the enzymatic activity revealed a decrease of 50% in trypsin activity as the chymotrypsin activity increased by 12%; possibly to compensate the commitment of the digestive process. The trypsins from the EATI-fed group stayed sensitive to the inhibition by EATI, and based on kinetic assays no new trypsin enzymes were produced as adaptation attempt. The insecticides effects observed for the EATI against this pest encourage a more in depth study of its possible long-term use as a biotechnological tool.
Pesticide Biochemistry and Physiology 01/2014; · 2.11 Impact Factor
[show abstract][hide abstract] ABSTRACT: Until recently, Crotalus viridis was a classification used to define a large group of snakes, also named Western Rattlesnakes that inhabit the eastern region of the Rocky Mountains in the United States, from southern Canada to northern Mexico. Currently, Crotalus viridis is now divided into two new species: Crotalus viridis and Crotalus oreganus. Furthermore more, over the past decade or so, phylogenetic and mitochondrial DNA analyses of sequences of great numbers of snakes, classified as Crotalus viridis, have shown the enormous taxonomic and molecular variations that exist in these snakes. As such, the current classification divides “the old” Crotalus viridis into two new and independent species: Crotalus viridis (subspecies: viridis and nuntius) and Crotalus oreganus (subspecies: abyssus, lutosus, concorlor, oreganus, helleri, cerberus and caliginis). The analysis of a product from cDNA, named as E6d, derived from the gland of a specie classified as Crotalus viridis viridis was found to produce an acid phospholipase A2. In this study, and in order to further understand the biological diversity of the subspecies of C. oregnaus, we isolated and characterized a PLA2 (D49) from Crotalus oreganus abyssus venom. Our structural studies show that the PLA2 produced from the cDNA of Crotalus viridis viridis (named E6d) has exactly the same PLA2 primary sequence of amino acids as that isolated from the venom of Crotalus oreganus abyssus. Thus, our results indicate that PLA2 from E6d cDNA is actually the same PLA2 as that of the venom of Crotalus oreganus abyssus and does not correspond to the venom from Crotalus viridis viridis. These facts highlight the importance of performing more studies on subspecies of Crotalus oreganus and Crotalus viridis, since the old classification may have led to mixed results or mistaken data
BioMed Research International 01/2014; in press:in press. · 2.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: Plant-derived trypsin inhibitors have been shown to have potent anti-insect effects and are a promising alternative for the biological control of pests. In this work, we tested the anti-insect activity of Adenanthera pavonina trypsin inhibitor (ApTI) against Diatraea saccharalis larvae, a major insect pest in sugarcane. The addition of 0.1% ApTI in short-term assays resulted in 87% and 63% decreased trypsin and chymotrypsin activities respectively. ApTI was not digested after 60 h incubation with D. saccharalis midgut proteases. The chronic effects of ApTI on F0 and F1 generations of D. saccharalis were also analyzed. The larvae from the F0 generation showed 55% and 21% decreased larval and pupal viability, respectively. ApTI-fed larvae from the F1 generation showed a decrease of 33% in survival rate and 23% in the average larval weight. Moreover, ApTI treatment reduced trypsin and chymotrypsin activities in F1 larvae. Thus, the anti-insect effects of ApTI on consecutive generations (F0 and F1) of D. saccharalis larvae demonstrate its potential for long-term control of this pest.
Journal of insect physiology 12/2013; · 2.24 Impact Factor
[show abstract][hide abstract] ABSTRACT: Bothrops brazili is a snake found in the forests of the Amazonian region whose commercial therapeutic anti-bothropic serum has low efficacy for local myotoxic effects, resulting in an important public health problem in this area. Catalytically inactive phospholipases A2-like (Lys49-PLA2s) are among the main components from Bothrops genus venoms and are capable to cause drastic myonecrosis. Several studies have shown that the C-terminal region of these toxins, which includes a variable combination of positively charged and hydrophobic residues, is responsible for their activity. In this work we describe the crystal structures of two Lys49-PLA2s (BbTX-II and MTX-II) from Bothrops brazili venom and a comprehensive structural comparison with several Lys49-PLA2s. Based on these results, it was identified two independent sites of interaction between protein and membrane which leads to the proposition of a new myotoxic mechanism for bothropic Lys49-PLA2s composed by five different steps. This proposition is able to fully explain the action of these toxins and may be useful to develop efficient inhibitors for complement the conventional antivenom administration.
Biochimica et Biophysica Acta 10/2013; · 4.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: A thrombin-like enzyme named TLBbar was isolated from Bothrops barnetti snake venom and its biochemical and pharmacological characteristics were determined. TLBbar was purified using size exclusion chromatography and reverse phase HPLC, showing molecular mass of 28750.7 Da determined by mass spectrometry. TLBbar serine protease is basic (pI 7.4) and its structure shows similarity with other serine proteases of snake venom. Optimal proteolytic activity was at 37°C and pH 8; this activity was strongly inhibited by PMSF and Leupeptin, however; heparin, and soybean trypsin inhibitor (SBT-I) were ineffective. Kinetic studies on BApNA chromogenic substrate have revealed that TLBbar presents a Michaelis-Menten kinetics, with values of and of 0.433 mM and 0.42 nmol/min, respectively. TLBbar showed high clotting activity upon bovine and human plasma, presenting IC of 125 and minimum dose coagulant (MDC) of 2.23 μg/μL. TLBbar cleavages the Aα chain of bovine fibrinogen, with maximal efficiency at 30–40°C in the presence of calcium after two hours incubation; this fibronogenolityc activity was inhibited by PMSF and Leupeptin, confirming its classification in the group of serine proteases. In addition, TLBbar is capable of aggregating platelets in the same way that thrombin in concentrations of 2.5 μg/μL.
[show abstract][hide abstract] ABSTRACT: The neuromuscular activity of Bbil-TX, a PLA2 with catalytic activity isolated from Bothriopsis bilineata smargadina venom, was examined in chick biventer cervicis (BC) and mouse phrenic nerve-diaphragm (PND) preparations. In BC preparations, Bbil-TX (0.5-10 μg/ml) caused time- and concentration-dependent blockade that was not reversed by washing; the times for 50% blockade were 87±7, 41±7 and 19±2 min (mean±SEM; n=4-6) for 1, 5 and 10 μg/ml, respectively. Muscle contractures to exogenous ACh and KCl were unaffected. The toxin (10 μg/ml) also did not affect the twitch-tension of directly-stimulated, curarized (10 μg/ml) BC preparations. However, Bbil-TX (10 μg/ml) produced mild morphological alterations (edematous and/or hyperchromic fibers) in BC; there was also a progressive release of CK (from 116±17 IU/ml (basal) to 710±91 IU/ml after 45 min). Bbil-TX (5 μg/ml)-induced blockade was markedly inhibited at 22-24 °C and pretreatment with p-bromophenacyl bromide (p-BPB) abolished the neuromuscular blockade. Bbil-TX (3-30 μg/ml, n=4-6) caused partial time- and concentration-dependent blockade in PND preparations (52±2% at the highest concentration). Bbil-TX (30 μg/ml) also markedly reduced the MEPPs frequency [from 26±2.5 (basal) to 10±1 after 60 min; n=5; p<0.05] and the quantal content of PND preparations [from 94±14 (basal) to 24±3 after 60 min; n=5; p<0.05] but caused only minor depolarization of the membrane resting potential [from -80±1 mV (basal) to -66±2 mV after 120 min; n=5; p<0.05], with no significant change in the depolarizing response to exogenous carbachol. These results show that Bbil-TX is a presynaptic PLA2 that contributes to the neuromuscular blockade caused by B. b. smargadina venom.
[show abstract][hide abstract] ABSTRACT: The fall armyworm (Spodoptera frugiperda) is an important pest insect due to high degree of polyphagia. In order to better understand its adaptation mechanism against plant protease inhibitors, bioassays were carried supplementing diet with the Kunitz trypsin inhibitor from Entada acaciifolia seeds (EATI). In vitro assays showed an increase of proteolytic activity in EATI-fed larvae midgut. Moreover, the trypsin enzymes showed insensitivity to inhibition with EATI. In order to understand what genes were overexpressed after chronic exposition to EATI, quantitative RT-PCR analyses were performed and revealed an increase in transcription of two trypsin genes, suggesting its participation in insensitivity of midgut trypsins. Another important result was the expression of one chymotrypsin gene, which is not expressed in control fed-larvae but induced in EATI-fed larvae. New regions of higher molecular weight showing proteolytic activity were visualized in inhibitor-fed larvae by zymography gel electrophoresis, proposing that the new enzymes expressed in response of inhibitor dietary would be formatting oligomers. This is a characteristic also observed in other pest insects that adapt to feed in plant protease inhibitors diet. Additional assays revealed that trypsins from EATI-fed larvae also became insensitive against Kunitz and Bowman-Birk inhibitors from soybean. This result suggests a possible involvement of the same S. frugiperda genes in adaptation against Kunitz and Bowman-Birk inhibitors in their host plants.
Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology 03/2013; · 1.61 Impact Factor
[show abstract][hide abstract] ABSTRACT: Anagasta kuehniella is a polyphagous pest that causes economic losses worldwide. This species produces serine proteases as its major enzymes for protein digestion. In this study, a new serine-protease inhibitor was isolated from Acacia polyphylla seeds (AcKI).Further analysis revealed that AcKI is formed by two polypeptide chains with a relative molecular mass of ∼20 kDa. The effects of AcKI on the development, survival, and enzymatic activity of Anagasta kuehniella larvae were evaluated, by incorporating AcKI in an artificial diet. Bioassays revealed a reduction in larval weight of ∼50% with the lower concentration of AcKI used in the study (0.5%). Although additionalassays showed an increase in endogenous trypsin and chymotrypsin activities, with a degree of AcKI-insensivity, AcKI produces an anti nutritional effect on A. kuehniella, indicating AcKI as a promising bioinsecticide protein for engineering plants that are resistant to insect pests.
Journal of Agricultural and Food Chemistry 02/2013; · 2.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: Bleu TX-III was isolated from Bothrops leucurus snake venom on one-step analytical chromatography reverse phase HPLC, was homogeneous on SDS-PAGE, and was confirmed by Q-Tof Ultima API ESI/MS (TOF MS mode) mass spectrometry in 14243.8 Da. Multiple alignments of Bleu TX-III show high degree of homology with basic PLA2 myotoxins from other Bothrops venoms. Our studies on local and systemic myotoxicity "in vivo" reveal that Bleu TX-III is myotoxin with local but not systemic action due to the decrease in the plasmatic CK levels when Bleu TX-III is administrated by intravenous route in mice (dose 1 and 5 μ g). And at a dose of 20 μ g myotoxin behaves like a local and systemic action. Bleu TX-III induced moderate marked paw edema, evidencing the local increase in vascular permeability. The inflammatory events induced in the mice (I.M.) were investigated. The increase in the levels of IL-1, IL-6, and TNF- α was observed in the plasma. It is concluded that Bleu TX-III induces inflammatory events in this model. The enzymatic phospholipid hydrolysis may be relevant to these phenomena. Bothrops leucurus venom is still not extensively explored, and the knowledge of its toxins separately through the study of structure/function will contribute for a better understanding of its action mechanism.
BioMed research international. 01/2013; 2013:941467.
[show abstract][hide abstract] ABSTRACT: We recently described the isolation of a basic PLA2 (PhTX-I) from Porthidium hyoprora snake venom. This toxin exhibits high catalytic activity, induces in vivo myotoxicity, moderates footpad edema, and causes in vitro neuromuscular blockade. Here, we describe the chemical modifications of specific amino acid residues (His, Tyr, Lys, and Trp), performed in PhTX-I, to study their effects on the structural, enzymatic, and pharmacological properties of this myotoxin. After chemical treatment, a single His, 4 Tyr, 7 Lys, and one Trp residues were modified. The secondary structure of the protein remained unchanged as measured by circular dichroism; however other results indicated the critical role played by Lys and Tyr residues in myotoxic, neurotoxic activities and mainly in the cytotoxicity displayed by PhTX-I. His residue and therefore catalytic activity of PhTX-I are relevant for edematogenic, neurotoxic, and myotoxic effects, but not for its cytotoxic activity. This dissociation observed between enzymatic activity and some pharmacological effects suggests that other molecular regions distinct from the catalytic site may also play a role in the toxic activities exerted by this myotoxin. Our observations supported the hypothesis that both the catalytic sites as the hypothetical pharmacological sites are relevant to the pharmacological profile of PhTX-I.
BioMed research international. 01/2013; 2013:103494.
[show abstract][hide abstract] ABSTRACT: The neuroprotection induced by Hypericum brasiliense Choisy extract (HBE) and its main active polyphenol compound quercetin, against Crotalus durissus terrificus (Cdt) venom and crotoxin and crotamine, was enquired at both central and peripheral mammal nervous system. Cdt venom (10 μ g/mL) or crotoxin (1 μ g/mL) incubated at mouse phrenic nerve-diaphragm preparation (PND) induced an irreversible and complete neuromuscular blockade, respectively. Crotamine (1 μ g/mL) only induced an increase of muscle strength at PND preparations. At mouse brain slices, Cdt venom (1, 5, and 10 μ g/mL) decreased cell viability. HBE (100 μ g/mL) inhibited significantly the facilitatory action of crotamine (1 μ g/mL) and was partially active against the neuromuscular blockade of crotoxin (1 μ g/mL) (data not shown). Quercetin (10 μ g/mL) mimicked the neuromuscular protection of HBE (100 μ g/mL), by inhibiting almost completely the neurotoxic effect induced by crotoxin (1 μ g/mL) and crotamine (1 μ g/mL). HBE (100 μ g/mL) and quercetin (10 μ g/mL) also increased cell viability in mice brain slices. Quercetin (10 μ g/mL) was more effective than HBE (100 μ g/mL) in counteracting the cell lysis induced by Cdt venom (1 and 10 μ g/mL, resp.). These results and a further phytochemical and toxicological investigations could open new perspectives towards therapeutic use of Hypericum brasiliense standardized extract and quercetin, especially to counteract the neurotoxic effect induced by snake neurotoxic venoms.
BioMed research international. 01/2013; 2013:943520.
[show abstract][hide abstract] ABSTRACT: Ophidian envenomation is an important health problem in Brazil and other South American countries. In folk medicine, especially in developing countries, several vegetal species are employed for the treatment of snakebites in communities that lack prompt access to serum therapy. However, the identification and characterization of the effects of several new plants or their isolated compounds, which are able to inhibit the activities of snake venom, are extremely important and such studies are imperative. Snake venom contains several organic and inorganic compounds; phospholipases A2 (PLA2s) are one of the principal toxic components of venom. PLA2s display a wide variety of pharmacological activities, such as neurotoxicity, myotoxicity, cardiotoxicity, anticoagulant, hemorrhagic, and edema-inducing effects. PLA2 inhibition is of pharmacological and therapeutic interests as these enzymes are involved in several inflammatory diseases. This review describes the results of several studies of plant extracts and their isolated active principles, when used against crude snake venoms or their toxic fractions. Isolated inhibitors, such as steroids, terpenoids, and phenolic compounds, are able to inhibit PLA2s from different snake venoms. The design of specific inhibitors of PLA2s might help in the development of new pharmaceutical drugs, more specific antivenom, or even as alternative approaches for treating snakebites.
BioMed Research International 01/2013; 2013:153045. · 2.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: BrTX-I, a PLA2, was purified from Bothrops roedingeri venom after only one chromatographic step using reverse-phase HPLC on μ -Bondapak C-18 column. A molecular mass of 14358.69 Da was determined by MALDI-TOF mass spectrometry. Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The total amino acid sequence was obtained using SwissProt database and showed high amino acid sequence identity with other PLA2 from snake venom. The amino acid composition showed that BrTX-I has a high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical of a basic PLA2. BrTX-I presented PLA2 activity and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0, 35-45°C, and required Ca(2+). In vitro, the whole venom and BrTX-I caused a neuromuscular blockade in biventer cervicis preparations in a similar way to other Bothrops species. BrTX-I induced myonecrosis and oedema-forming activity analyzed through injection of the purified BrTX-I in mice. Since BrTX-I exerts a strong proinflammatory effect, the enzymatic phospholipid hydrolysis might be relevant for these phenomena; incrementing levels of IL-1, IL-6, and TNF α were observed at 15 min, 30 min, one, two, and six hours postinjection, respectively.
BioMed research international. 01/2013; 2013:591470.
[show abstract][hide abstract] ABSTRACT: We report a rapid purification method using one-step chromatography of SVSP Rhombeobin (LMR-47) from Lachesis muta rhombeata venom and its procoagulant activities and effects on platelet aggregation. The venom was fractionated by a single chromatographic step in RP-HPLC on a C8 Discovery BIO Wide Pore, showing high degree of molecular homogeneity with molecular mass of 47035.49 Da. Rhombeobin showed amidolytic activity upon BA ρ NA, with a broad optimum pH (7-10) and was stable in solution up to 60°C. The amidolytic activity was inhibited by serine proteinase inhibitors and reducing agents, but not chelating agents. Rhombeobin showed high coagulant activity on mice plasma and bovine fibrinogen. The deduced amino acid sequence of Rhombeobin showed homology with other SVSPs, especially with LM-TL (L. m. muta) and Gyroxin (C. d. terrificus). Rhombeobin acts, in vitro, as a strong procoagulant enzyme on mice citrated plasma, shortening the APTT and PT tests in adose-dependent manner. The protein showed, "ex vivo", a strong defibrinogenating effect with 1 µg/animal. Lower doses activated the intrinsic and extrinsic coagulation pathways and impaired the platelet aggregation induced by ADP. Thus, this is the first report of a venom component that produces a venom-induced consumptive coagulopathy (VICC).
BioMed research international. 01/2013; 2013:903292.
[show abstract][hide abstract] ABSTRACT: Bbil-TX, a PLA2, was purified from Bothriopsis bilineata snake venom after only one chromatographic step using RP-HPLC on μ -Bondapak C-18 column. A molecular mass of 14243.8 Da was confirmed by Q-Tof Ultima API ESI/MS (TOF MS mode) mass spectrometry. The partial protein sequence obtained was then submitted to BLASTp, with the search restricted to PLA2 from snakes and shows high identity values when compared to other PLA2s. PLA2 activity was presented in the presence of a synthetic substrate and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0 and 25-37°C. Maximum PLA2 activity required Ca(2+) and in the presence of Cd(2+), Zn(2+), Mn(2+), and Mg(2+) it was reduced in the presence or absence of Ca(2+). Crotapotin from Crotalus durissus cascavella rattlesnake venom and antihemorrhagic factor DA2-II from Didelphis albiventris opossum sera under optimal conditions significantly inhibit the enzymatic activity. Bbil-TX induces myonecrosis in mice. The fraction does not show a significant cytotoxic activity in myotubes and myoblasts (C2C12). The inflammatory events induced in the serum of mice by Bbil-TX isolated from Bothriopsis bilineata snake venom were investigated. An increase in vascular permeability and in the levels of TNF-a, IL-6, and IL-1 was was induced. Since Bbil-TX exerts a stronger proinflammatory effect, the phospholipid hydrolysis may be relevant for these phenomena.
BioMed research international. 01/2013; 2013:612649.
[show abstract][hide abstract] ABSTRACT: Native Inga laurina (Fabaceae) trypsin inhibitor (ILTI) was tested for anti-insect activity against Diatraea saccharalis and Heliothis virescens larvae. The addition of 0.1% ILTI to the diet of D. saccharalis did not alter larval survival but decreased larval weight by 51%. The H. virescens larvae that were fed a diet containing 0.5% ILTI showed an 84% decrease in weight. ILTI was not digested by the midgut proteinases of either species of larvae. The trypsin levels were reduced by 55.3% in the feces of D. saccharalis and increased by 24.1% in the feces of H. virescens. The trypsin activity in both species fed with ILTI was sensitive to the inhibitor, suggesting that no novel proteinase resistant to ILTI was induced. Additionally, ILTI exhibited inhibitory activity against the proteinases present in the larval midgut of different species of Lepidoptera. The organization of the ilti gene was elucidated by analyzing its corresponding genomic sequence. The recombinant ILTI protein (reILTI) was expressed and purified, and its efficacy was evaluated. Both native ILTI and reILTI exhibited a similar strong inhibitory effect on bovine trypsin activity. These results suggest that ILTI presents insecticidal properties against both insects and may thus be a useful tool in the genetic engineering of plants.
Comparative Biochemistry and Physiology Part C Toxicology & Pharmacology 08/2012; 156(3-4):148-58. · 2.71 Impact Factor
[show abstract][hide abstract] ABSTRACT: Two myotoxic and noncatalytic Lys49-phospholipases A(2) (braziliantoxin-II and MT-II) and a myotoxic and catalytic phospholipase A(2) (braziliantoxin-III) from the venom of the Amazonian snake Bothrops brazili were crystallized. The crystals diffracted to resolutions in the range 2.56-2.05 Å and belonged to space groups P3(1)21 (braziliantoxin-II), P6(5)22 (braziliantoxin-III) and P2(1) (MT-II). The structures were solved by molecular-replacement techniques. Both of the Lys49-phospholipases A(2) (braziliantoxin-II and MT-II) contained a dimer in the asymmetric unit, while the Asp49-phospholipase A(2) braziliantoxin-III contained a monomer in its asymmetric unit. Analysis of the quaternary assemblies of the braziliantoxin-II and MT-II structures using the PISA program indicated that both models have a dimeric conformation in solution. The same analysis of the braziliantoxin-III structure indicated that this protein does not dimerize in solution and probably acts as a monomer in vivo, similar to other snake-venom Asp49-phospholipases A(2).
Acta Crystallographica Section F Structural Biology and Crystallization Communications 08/2012; 68(Pt 8):935-8. · 0.55 Impact Factor