Sergio Marangoni

University of Campinas, Conceição de Campinas, São Paulo, Brazil

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Publications (246)505.52 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: A myographic study was performed to compare the neuromuscular effects of venoms and crotoxin-like proteins from Crotalus durissus ruruima and Crotalus durissus cumanensis in mice phrenic-diaphragm preparation. It was concluded that both venoms present neurotoxic activity as a consequence of their crotoxin content. Furthermore, crotoxin from C.d. cumanensis is more potent than that from C.d. ruruima venom. At the concentration range in which both venoms express neurotoxic activity, only C.d. cumanensis venom also manifest a direct myotoxic effect that probably involves the synergic participation of other components than crotoxin. Copyright © 2015. Published by Elsevier Ltd.
    Toxicon 01/2015; 96. DOI:10.1016/j.toxicon.2015.01.006 · 2.58 Impact Factor
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    ABSTRACT: The presynaptic action of Bothriopsis bilineata smaragdina (forest viper) venom and Bbil-TX, an Asp49 PLA2 from this venom, was examined in detail in mouse phrenic nerve-muscle (PND) preparations in vitro and in a neuroblastoma cell line (SK-N-SK) in order to gain a better insight in to the mechanism of action of the venom and associated Asp49 PLA2. In low Ca2+ solution, venom (3 μg/ml) caused a quadriphasic response in PND twitch height whilst at 10 μg/ml the venom additionally induced an abrupt and marked initial contracture followed by neuromuscular facilitation, rhythmic oscillations of nerve-evoked twitches, alterations in baseline and progressive blockade. The venom slowed the relaxation phase of muscle twitches. In low Ca2+, Bbil-TX [210 nM (3 μg/ml)] caused a progressive increase in PND twitch amplitude but no change in the decay time constant. Venom (10 μg/ml) and Bbil-TX (210 nM) caused minor changes in the compound action potential (CAP) amplitude recorded from sciatic nerve preparations, with no significant effect on rise time and latency; tetrodotoxin (3.1 nM) blocked the CAP at the end of the experiments. In mouse triangularis sterni nerve-muscle preparations (TSn-m) preparations, venom (10 μg/ml) and Bbil-TX (210 nM) significantly reduced the perineural waveform associated with the outward K+ current while the amplitude of the inward Na+ current was not significantly affected. Bbil-TX (210 nM) caused a progressive increase in the quantal content of TSn-m preparations maintained in low Ca2+ solution. Venom (3 μg/ml) and toxin (210 nM) increased the calcium fluorescence in SK-N-SH neuroblastoma cells loaded with Fluo3 AM and maintained in low or normal Ca2+ solution. In normal Ca2+, the increase in fluorescence amplitude was accompanied by irregular and frequent calcium transients. In TSn-m preparations loaded with Fluo4 AM, venom (10 μg/ml) caused an immediate increase in intracellular Ca2+ followed by oscillations in fluorescence and muscle contracture; Bbil-TX did not change the calcium fluorescence in TSn-m preparations. Immunohistochemical analysis of toxin-treated PND preparations revealed labeling of junctional ACh receptors but a loss of the presynaptic proteins synaptophysin and SNAP25. Together, these data confirm the presynaptic action of Bbil-TX and show that it involves modulation of K+ channel activity and presynaptic protein expression.
    Toxicon 01/2015; 96. DOI:10.1016/j.toxicon.2015.01.001 · 2.58 Impact Factor
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    ABSTRACT: A new PLA 2 (Bp-13) was purified from Bothrops pauloensis snake venom after a single chromatographic step of RP-HPLC on í µí¼‡-Bondapak C-18. Amino acid analysis showed a high content of hydrophobic and basic amino acids and 14 half-cysteine residues. The N-terminal sequence showed a high degree of homology with basic Asp49 PLA 2 myotoxins from other Bothrops venoms. Bp-13 showed allosteric enzymatic behavior and maximal activity at pH 8.1, 36 ∘ –45 ∘ C. Full Bp-13 PLA 2 activity required Ca 2+ ; its PLA 2 activity was inhibited by Mg 2+ , Mn 2+ , Sr 2+ , and Cd 2+ in the presence and absence of 1 mM Ca 2+. In the mouse phrenic nerve-diaphragm (PND) preparation, the time for 50% paralysis was concentration-dependent (í µí±ƒ < 0.05). Both the replacement of Ca 2+ by Sr 2+ and temperature lowering (24 ∘ C) inhibited the Bp-13 PLA 2-induced twitch-tension blockade. Bp-13 PLA 2 inhibited the contractile response to direct electrical stimulation in curarized mouse PND preparation corroborating its contracture effect. In biventer cervicis preparations, Bp-13 induced irreversible twitch-tension blockade and the KCl evoked contracture was partially, but significantly, inhibited (í µí±ƒ > 0.05). The main effect of this new Asp49 PLA 2 of Bothrops pauloensis venom is on muscle fiber sarcolemma, with avian preparation being less responsive than rodent preparation. The study enhances biochemical and pharmacological characterization of B. pauloensis venom.
    01/2015; 2015. DOI:10.1155/2015/826059
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    ABSTRACT: This paper describes the characterization of a trypsin inhibitor from Poincianella pyramidalis seeds (PpyTI). The partial sequencing of PpyTI revealed homology to Kunitz inhibitors, clustered as a member of Family I03 in MEROPS database. PpyTI has a single polypeptide chain of 19,042 Da and presents stability at high temperatures (up to 70 °C) and a wide range of pH. In vitro assays showed that disulfide bridges have an important stabilization role of reactive site in PpyTI, a characteristic shared among several Kunitz inhibitors. Bioassays carried out with the Mediterranean flour moth (Anagasta kuehniella) revealed a significant decrease in both larval weight and survival of PpyTI-fed larvae, besides a larval stage extension. Through biochemical analysis, we demonstrated that the PpyTI insecticide effects were triggered by digestion process commitment, through the inhibition of trypsin and chymotrypsin activities, the major digestive enzymes in this species. The insecticide effects and biochemical characterization of PpyTI encourage further studies using this inhibitor for insect pest control. Copyright © 2014 Elsevier Inc. All rights reserved.
    Pesticide Biochemistry and Physiology 12/2014; 118. DOI:10.1016/j.pestbp.2014.12.001 · 2.11 Impact Factor
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    ABSTRACT: A monomeric basic PLA2 (PhTX-II) of 14149.08 Da molecular weight was purified to homogeneity from Porthidium hyoprora venom. Amino acid sequence by in tandem mass spectrometry revealed that PhTX-II belongs to Asp49 PLA2 enzyme class and displays conserved domains as the catalytic network, Ca2+-binding loop and the hydrophobic channel of access to the catalytic site, reflected in the high catalytic activity displayed by the enzyme. Moreover, PhTX-II PLA2 showed an allosteric behavior and its enzymatic activity was dependent on Ca2+. Examination of PhTX-II PLA2 by CD spectroscopy indicated a high content of alpha-helical structures, similar to the known structure of secreted phospholipase IIA group suggesting a similar folding. PhTX-II PLA2 causes neuromuscular blockade in avian neuromuscular preparations with a significant direct action on skeletal muscle function, as well as, induced local edema and myotoxicity, in mice. The treatment of PhTX-II by BPB resulted in complete loss of their catalytic activity that was accompanied by loss of their edematogenic effect. On the other hand, enzymatic activity of PhTX-II contributes to this neuromuscular blockade and local myotoxicity is dependent not only on enzymatic activity. These results show that PhTX-II is a myotoxic Asp49 PLA2 that contributes with toxic actions caused by P. hyoprora venom.
    Toxins 11/2014; 6(11):3077-97. DOI:10.3390/toxins6113077 · 2.48 Impact Factor
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    ABSTRACT: Crotamine is one of the main constituents of the venom of the South American rattlesnake Crotalus durissus terrificus. Here we sought to investigate the inflammatory and toxicological effects induced by the intrahippocampal administration of crotamine isolated from Crotalus whole venom. Adult rats received an intrahippocampal infusion of crotamine or vehicle and were euthanized 24 h or 21 days after infusion. Plasma and brain tissue were collected for biochemical analysis. Complete blood count, creatinine, urea, glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), creatine-kinase (CK), creatine kinase-muscle B (CK-MB) and oxidative parameters (assessed by DNA damage and micronucleus frequency in leukocytes, lipid peroxidation and protein carbonyls in plasma and brain) were quantified. Unpaired and paired t-tests were used for comparisons between saline and crotamine groups, and within groups (24 h vs. 21 days), respectively. After 24 h crotamine infusion promoted an increase of urea, GOT, GPT, CK, and platelets values (p ≤ 0.01), while red blood cells, hematocrit and leukocytes values decreased (p ≤ 0.01). Additionally, 21 days after infusion crotamine group showed increased creatinine, leukocytes, TBARS (plasma and brain), carbonyl (plasma and brain) and micronucleus compared to the saline-group (p ≤ 0.01). Our findings show that crotamine infusion alter hematological parameters and cardiac markers, as well as oxidative parameters, not only in the brain, but also in the blood, indicating a systemic pro-inflammatory and toxicological activity. A further scientific attempt in terms of preserving the beneficial activity over toxicity is required.
    International Journal of Environmental Research and Public Health 11/2014; 11(11):11438-11449. DOI:10.3390/ijerph111111438 · 1.99 Impact Factor
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    ABSTRACT: Previous research has shown that crotamine, a toxin isolated from the venom of Crotalus durissus terrificus, induces the release of acetylcholine and dopamine in the central nervous system of rats. Particularly, these neurotransmitters are important modulators of memory processes. Therefore, in this study we investigated the effects of crotamine infusion on persistence of memory in rats. We verified that the intrahippocampal infusion of crotamine (1 μg/μl; 1μl/side) improved the persistence of object recognition and aversive memory. By other side, the intrahippocampal infusion of the toxin did not alter locomotor and exploratory activities, anxiety or pain threshold. These results demonstrate a future prospect of using crotamine as potential pharmacological tool to treat diseases involving memory impairment, although it is still necessary more researches to better elucidate the crotamine effects on hippocampus and memory.
    Toxicon 05/2014; DOI:10.1016/j.toxicon.2014.04.017 · 2.58 Impact Factor
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    ABSTRACT: Although the hydrozoan Olindias sambaquiensis is the most common jellyfish associated with human envenomation in southeastern and southern Brazil, information about the composition of its venom is rare. Thus, the present study aimed to analyze pharmacological aspects of O. sambaquiensis venom as well as clinical manifestations observed in affected patients. Crude protein extracts were prepared from the tentacles of animals; peptides and proteins were sequenced and submitted to circular dichroism spectroscopy. Creatine kinase, cytotoxicity and hemolytic activity were evaluated by specific methods. We identified two novel cytolysins denominated oshem 1 and oshem 2 from the tentacles of this jellyfish. The cytolysins presented the amino acid sequences NEGKAKCGNTAGSKLTFKSADECTKTGQK (oshem 1) and NNSKAKCGDLAGWSKLTFKSADECTKTGQKS (oshem 2) with respective molecular masses of 3.013 kDa and 3.375 kDa. Circular dichroism revealed that oshem 1 has random coils and small alpha-helix conformation as main secondary structure whereas oshem 2 presents mainly random coils as its main secondary structure probably due to the presence of W (13) in oshem 2. The hemolysis levels induced by oshem 1 and oshem 2 using a peptide concentration of 0.2 mg/mL were, respectively, 51.7 +/- 6.5% and 32.9 +/- 8.7% (n = 12 and p <= 0.05). Oshem 1 and oshem 2 showed significant myonecrotic activity, evaluated by respective CK level measurements of 1890.4 +/- 89 and 1212.5 +/- 103 (n = 4 and p <= 0.05). In addition, myonecrosis was also evaluated by cell survival, which was measured at 72.4 +/- 8.6% and 83.5 +/- 6.7% (n = 12 and p <= 0.05), respectively. The structural analysis showed that both oshem 1 and oshem 2 should be classified as a small basic hemolytic peptide. The amino acid sequences of two peptides were highly similar while the primary amino acid sequence analysis revealed W (22th) as the most important mutation. Finally oshem 1 and oshem 2 are the first cytolytic peptides isolated from the Olindias sambaquiensis and should probably represent a novel class of cytolytic peptides.
    Journal of Venomous Animals and Toxins including Tropical Diseases 03/2014; 20(1):10. DOI:10.1186/1678-9199-20-10 · 0.43 Impact Factor
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    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; 108. DOI:10.1016/j.pestbp.2013.12.007 · 2.11 Impact Factor
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    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. · 2.71 Impact Factor
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    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 A2. In this study we isolated and characterized a PLA2 (D49) from Crotalus oreganus abyssus venom. Our studies show that the PLA2 produced from the cDNA of Crotalus viridis viridis (named E6d) is exactly the same PLA2 primary sequence of amino acids isolated from the venom of Crotalus oreganus abyssus. Thus, the PLA2 from E6d cDNA is actually the same PLA2 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.
    01/2014; 2014:654170. DOI:10.1155/2014/654170
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    ABSTRACT: Previous research has shown that crotamine, a toxin isolated from the venom of Crotalus durissus terrificus, induces the release of acetylcholine and dopamine in the central nervous system of rats. Particularly, these neurotransmitters are important modulators of memory processes. Therefore, in this study we investigated the effects of crotamine infusion on persistence of memory in rats. We verified that the intrahippocampal infusion of crotamine (1 μg/μl; 1μl/side) improved the persistence of object recognition and aversive memory. By other side, the intrahippocampal infusion of the toxin did not alter locomotor and exploratory activities, anxiety or pain threshold. These results demonstrate a future prospect of using crotamine as potential pharmacological tool to treat diseases involving memory impairment, although it is still necessary more researches to better elucidate the crotamine effects on hippocampus and memory.
    Toxicon 01/2014; · 2.58 Impact Factor
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    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.
    12/2013; 2013:943520. DOI:10.1155/2013/943520
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    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; DOI:10.1016/j.jinsphys.2013.11.012 · 2.24 Impact Factor
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    ABSTRACT: The fall armyworm (Spodoptera frugiperda) is an important pest insect due to high degree of polyphagia. Inorder to better understand its adaptation mechanism against plant protease inhibitors, bioassays were carriedsupplementing dietwith the Kunitz trypsin inhibitor fromEntada acaciifolia seeds (EATI). In vitro assays showed an increase of proteolytic activity in EATI-fed larvaemidgut.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. Newregions of higher molecular weight showing proteolytic activity were visualized in inhibitor-fed larvae by zymography gel electrophoresis, proposing that the newenzymes expressed in response of inhibitor dietarywould 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 fromEATI-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.
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    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; DOI:10.1016/j.bbapap.2013.10.009 · 4.66 Impact Factor
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    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 09/2013; 2013:153045. DOI:10.1155/2013/153045 · 2.71 Impact Factor
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    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).
    08/2013; 2013:903292. DOI:10.1155/2013/903292
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    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.
    07/2013; 2013. DOI:10.1155/2013/207170

Publication Stats

4k Citations
505.52 Total Impact Points

Institutions

  • 1993–2015
    • University of Campinas
      • • Institute of Biology (IB)
      • • Departamento de Bioquímica
      • • Departamento de Farmacologia
      Conceição de Campinas, São Paulo, Brazil
  • 2009
    • Max Planck Institute of Psychiatry
      München, Bavaria, Germany
  • 2003–2009
    • Universidade Federal de Mato Grosso do Sul
      • • Departamento de Tecnologia de Alimentos e Saúde Pública (DTA)
      • • Departamento de Ciências Naturais (DCN) (CPTL)
      • • Centre for Biological and Health Sciences
      Campo Grande, Estado de Mato Grosso do Sul, Brazil
    • University of São Paulo
      San Paulo, São Paulo, Brazil
  • 2001–2006
    • Matrix Biology Institute
      Эджуатэ, New Jersey, United States
  • 1998–2003
    • Laboratório Nacional de Luz Síncrotron
      Conceição de Campinas, São Paulo, Brazil
  • 1999
    • Instituto Agronômico de Campinas
      Conceição de Campinas, São Paulo, Brazil
  • 1998–1999
    • CEP America
      Емеривил, California, United States
  • 1996
    • Instituto de Biologia Molecular do Paraná
      Curityba, Paraná, Brazil