Danilo Menaldo - Academia.edu (original) (raw)

Papers by Danilo Menaldo

In the present study, experiments were carried out to evaluate the mutagenic potential and genoto... more In the present study, experiments were carried out to evaluate the mutagenic potential and genotoxic effects of Crotalus durissus terrificus snake venom and its isolated toxins on human lymphocytes, using the micronucleus and comet assays. Significant damage to DNA was observed for crotoxin and crotapotin (CA). Basic phospholipase A 2 (CB) and crotamine did not present any mutagenic potential when evaluated by the micronucleus test. C. d. terrificus crude venom was able to induce the formation of micronuclei, similarly to the mutagenic drug used as a positive control. In the comet assay, all the toxins tested (crotamine, crotoxin, CB and CA) and C. d. terrificus venom presented genotoxic activity. Studies on the cytogenetic toxicology of animal venoms and their isolated proteins are still very scarce in the literature, which emphasizes the importance of the present work for the identification and characterization of potential therapeutic agents, as well as for the better understanding of the mechanisms of action of toxins on the human body.

Toxicon : official journal of the International Society on Toxinology, Jan 15, 2008

Phospholipase A2 (PLA2, EC 3.1.1.4), a major component of snake venoms, specifically catalyzes th... more Phospholipase A2 (PLA2, EC 3.1.1.4), a major component of snake venoms, specifically catalyzes the hydrolysis of fatty acid ester bonds at position 2 of 1,2-diacyl-sn-3-phosphoglycerides in the presence of calcium. This article reports the purification and biochemical/functional characterization of BmooTX-I, a new myotoxic acidic phospholipase A2 from Bothrops moojeni snake venom. The purification of the enzyme was carried out through three chromatographic steps (ion-exchange on DEAE-Sepharose, molecular exclusion on Sephadex G-75 and hydrophobic chromatography on Phenyl-Sepharose). BmooTX-I was found to be a single-chain protein of 15,000 Da and pI 4.2. The N-terminal sequence revealed a high homology with other acidic Asp49 PLA2s from Bothrops snake venoms. It displayed a high phospholipase activity and platelet aggregation inhibition induced by collagen or ADP. Edema and myotoxicity in vivo were also induced by BmooTX-I. Analysis of myotoxic activity was carried out by optical an...

Peptides, 2008

p e p t i d e s 2 9 ( 2 0 0 8 ) 1 6 4 5 -1 6 5 6 Microbicide Bothrops brazili Synthetic peptides ... more p e p t i d e s 2 9 ( 2 0 0 8 ) 1 6 4 5 -1 6 5 6 Microbicide Bothrops brazili Synthetic peptides Phospholipases A 2 Myotoxins Snake venom a b s t r a c t This paper reports the purification and biochemical/pharmacological characterization of two myotoxic phospholipases A 2 (PLA 2 s) from Bothrops brazili venom, a native snake from Brazil. Both myotoxins (MTX-I and II) were purified by a single chromatographic step on a CM-Sepharose ion-exchange column up to a high purity level, showing M r $ 14,000 for the monomer and 28,000 Da for the dimer. The N-terminal and internal peptide amino acid sequences showed similarity with other myotoxic PLA 2 s from snake venoms, MTX-I belonging to Asp49 PLA 2 class, enzymatically active, and MTX-II to Lys49 PLA 2 s, catalytically inactive. Treatment of MTX-I with BPB and EDTA reduced drastically its PLA 2 and anticoagulant activities, corroborating the importance of residue His48 and Ca 2+ ions for the enzymatic catalysis. Both PLA 2 s induced myotoxic activity and dose-time dependent edema similar to other isolated snake venom toxins from Bothrops and Crotalus genus. The results also demonstrated that MTXs and cationic synthetic peptides derived from their 115-129 Cterminal region displayed cytotoxic activity on human T-cell leukemia (JURKAT) lines and microbicidal effects against Escherichia coli, Candida albicans and Leishmania sp. Thus, these PLA 2 proteins and C-terminal synthetic peptides present multifunctional properties that might be of interest in the development of therapeutic strategies against parasites, bacteria and cancer.

Journal of Venomous Animals and Toxins including Tropical Diseases, 2014

The L-amino acid oxidases (LAAOs) constitute a major component of snake venoms and have been wide... more The L-amino acid oxidases (LAAOs) constitute a major component of snake venoms and have been widely studied due to their widespread presence and various effects, such as apoptosis induction, cytotoxicity, induction and/or inhibition of platelet aggregation, hemorrhage, hemolysis, edema, as well as antimicrobial, antiparasitic and anti-HIV activities. The isolated and characterized snake venom LAAOs have become important research targets due to their potential biotechnological applications in pursuit for new drugs of interest in the scientific and medical fields. The current study discusses the antitumor effects of snake venom LAAOs described in the literature to date, highlighting the mechanisms of apoptosis induction proposed for this class of proteins.

Biochimie, 2014

Venomous and non-venomous snakes possess phospholipase A2 (PLA2) inhibitory proteins (PLIs) in th... more Venomous and non-venomous snakes possess phospholipase A2 (PLA2) inhibitory proteins (PLIs) in their blood serum. This study shows the expression and biochemical and functional characterization of a recombinant alpha inhibitor from Bothrops alternatus snake, named rBaltMIP. Its expression was performed in Pichia pastoris heterologous system, resulting in an active recombinant protein. The expressed inhibitor was tested regarding its ability to inhibit the phospholipase activity of different PLA2s, showing slight inhibitions especially at the molar ratios of 1:1 and 1:3 (PLA2:PLI). rBaltMIP was also effective in decreasing the myotoxic activity of the tested toxins at molar ratios greater than 1:0.4 (myotoxin:PLI). The inhibition of the myotoxic activity of different Asp49 (BthTX-II and PrTX-III) and Lys49 (BthTX-I and PrTX-I) myotoxins was also performed without the prior incubation of myotoxins/inhibitor in order to analyze the real possibility of using snake plasma inhibitors or recombinant inhibitors as therapeutic agents for treating envenomations. As a result, rBaltMIP was able to significantly inhibit the myotoxicity of Lys49 myotoxins. Histopathological analysis of the gastrocnemius muscles of mice showed that the myotoxins are able to induce severe damage to the muscle fibers of experimental animals by recruiting a large number of leukocyte infiltrates, besides forming an intense accumulation of intercellular fluid, leading to local edema. When those myotoxins were incubated with rBaltMIP, a reduction of the damage site could be observed. Furthermore, the cytotoxic activity of Asp49 PLA2s and Lys49 PLA2-like enzymes on C2C12 cell lines was decreased, as shown by the higher cell viabilities after preincubation with rBaltMIP. Heterologous expression would enable large-scale obtainment of rBaltMIP, thus allowing further investigations for the elucidation of possible mechanisms of inhibition of snake PLA2s, which have not yet been fully clarified.

Toxicon, 2008

Phospholipase A 2 (PLA 2 , EC 3.1.1.4), a major component of snake venoms, specifically catalyzes... more Phospholipase A 2 (PLA 2 , EC 3.1.1.4), a major component of snake venoms, specifically catalyzes the hydrolysis of fatty acid ester bonds at position 2 of 1,2-diacyl-sn-3phosphoglycerides in the presence of calcium. This article reports the purification and biochemical/functional characterization of BmooTX-I, a new myotoxic acidic phospholipase A 2 from Bothrops moojeni snake venom. The purification of the enzyme was carried out through three chromatographic steps (ion-exchange on DEAE-Sepharose, molecular exclusion on Sephadex G-75 and hydrophobic chromatography on Phenyl-Sepharose). BmooTX-I was found to be a single-chain protein of 15,000 Da and pI 4.2. The N-terminal sequence revealed a high homology with other acidic Asp49 PLA 2 s from Bothrops snake venoms. It displayed a high phospholipase activity and platelet aggregation inhibition induced by collagen or ADP. Edema and myotoxicity in vivo were also induced by BmooTX-I. Analysis of myotoxic activity was carried out by optical and ultrastructural microscopy, demonstrating high levels of leukocytary infiltrate. Previous treatment of BmooTX-I with BPB reduced its enzymatic and myotoxic activities, as well as the effect on platelet aggregation. Acidic myotoxic PLA 2 s from Bothrops snake venoms have been little explored and the knowledge of its structural and functional features will be able to contribute for a better understanding of their action mechanism regarding enzymatic and toxic activities.

Toxicon, 2009

In the present study, a thrombin-like enzyme named BpSP-I was isolated from Bothrops pauloensis s... more In the present study, a thrombin-like enzyme named BpSP-I was isolated from Bothrops pauloensis snake venom and its biochemical, enzymatic and pharmacological characteristics were determined. BpSP-I is a glycoprotein that contains both N-linked carbohydrates and sialic acid in its structure, with M r ¼ 34,000 under reducing conditions and pI w 6.4. The N-terminal sequence of the enzyme (VIGGDECDINEHPFL) showed high similarity with other thrombin-like enzymes from snake venoms. BpSP-I showed high clotting activity upon bovine and human plasma and was inhibited by PMSF, benzamidine and leupeptin. Moreover, this enzyme showed stability when examined at different temperatures (À70 to 37 C), pH values (3-9) or in the presence of divalent metal ions (Ca 2þ , Mg 2þ , Zn 2þ and Mn 2þ ). BpSP-I showed high catalytic activity upon substrates, such as fibrinogen, TAME, S-2238 and S-2288. It also showed kallikrein-like activity, but was unable to act upon factor Xa and plasmin substrates. Indeed, the enzyme did not induce hemorrhage, myotoxicity or edema. Taken together, our data showed that BpSP-I is in fact a thrombin-like enzyme isoform isolated from Bothrops pauloensis snake venom.

Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 2011

Journal of Proteomics, 2013

Bothrops pirajai snake venom was analyzed by a proteomic strategy. Proteins were separated by RP-... more Bothrops pirajai snake venom was analyzed by a proteomic strategy. Proteins were separated by RP-HPLC, followed by SDS-PAGE, in-gel tryptic digestion, identification by MALDI-TOF/TOF mass spectrometry, and assignment to known protein families by similarity. Proteins belonging to six families were found in B. pirajai venom, including abundant PLA2s and metalloproteinases, with the remaining proteins distributed among l-amino acid oxidase, serine proteinase, disintegrin and lectin-like families. A P-I class metalloproteinase, named BpirMP, was isolated from this venom by three chromatographic steps. The enzyme has a molecular mass of 23.1kDa, as determined by mass spectrometry. Its proteolytic activity on azocasein was inhibited by chelating and reducing agents, with optimum activity at higher pH values and 37°C. BpirMP presented weak hemorrhagic activity, with an MHD of 50μg, and was able to hydrolyze basement membrane components in vivo and in vitro. The toxin cleaved both Aα and Bβ chains of fibrinogen and was also able to degrade fibrin and blood clots in vitro. The primary sequence analysis indicates that BpirMP contains a zinc ligand motif and a CVM motif that is associated with a Met-turn structure. These results demonstrate that BpirMP is a zinc-dependent hemorrhagic metalloproteinase with fibrin(ogen)olytic and thrombolytic activities. This manuscript describes the diversity of protein components present in the venom of Bothops pirajai, a threatened snake species from northeastern Brazil, as well as the isolation and biochemical properties of a PI-SVMP. The results showed distinct mechanisms of action that should contribute in the elucidation of the differences in the hemorrhagic potential of SVMPs, allowing a better understanding of this class of enzymes and of the biology of Bothrops pirajai species.

Peptides, 2008

This paper reports the purification and biochemical/pharmacological characterization of two myoto... more This paper reports the purification and biochemical/pharmacological characterization of two myotoxic phospholipases A2 (PLA2s) from Bothrops brazili venom, a native snake from Brazil. Both myotoxins (MTX-I and II) were purified by a single chromatographic step on a CM-Sepharose ion-exchange column up to a high purity level, showing Mr∼14,000 for the monomer and 28,000Da for the dimer. The N-terminal and internal

Current Topics in Medicinal Chemistry, 2007

Phospholipases A 2 (PLA 2 s) are commonly found in snake venoms from Viperidae, Hydrophidae and E... more Phospholipases A 2 (PLA 2 s) are commonly found in snake venoms from Viperidae, Hydrophidae and Elaphidae families and have been extensively studied due to their pharmacological and physiopathological effects in living organisms. This article reports a review on natural and artificial inhibitors of enzymatic, toxic and pharmacological effects induced by snake venom PLA 2 s. These inhibitors act on PLA 2 s through different mechanisms, most of them still not completely understood, including binding to specific domains, denaturation, modification of specific amino acid residues and others. Several substances have been evaluated regarding their effects against snake venoms and isolated toxins, including plant extracts and compounds from marine animals, mammals and snakes serum plasma, in addition to poly or monoclonal antibodies and several synthetic molecules. Research involving these inhibitors may be useful to understand the mechanism of action of PLA 2 s and their role in envenomations caused by snake bite. Furthermore, the biotechnological potential of PLA 2 inhibitors may provide therapeutic molecular models with antiophidian activity to supplement the conventional serum therapy against these multifunctional enzymes.

Blood Coagulation & Fibrinolysis, 2010

Previous studies have shown that venoms of social wasps and bees exhibit strong anticoagulant act... more Previous studies have shown that venoms of social wasps and bees exhibit strong anticoagulant activity. The present study describes the anticoagulant and fibrinogen-degrading pharmacological properties of the venom of Polybia occidentalis social wasp. The results demonstrated that this venom presented anticoagulant effect, inhibiting the coagulation at different steps of the clotting pathway (intrinsic, extrinsic and common pathway). The venom inhibited platelet aggregation and degraded plasma fibrinogen, possibly containing metal-dependent metalloproteases that specifically cleave the Bb-chain of fibrinogen. In conclusion, fibrinogenolytic and anticoagulant properties of this wasp venom find a potential application in drug development for the treatment of thrombotic disorders.

Biologicals, 2009

The present work evaluates both in vitro and in vivo antitumor activity of BPB-modified BthTX-I a... more The present work evaluates both in vitro and in vivo antitumor activity of BPB-modified BthTX-I and its cationic synthetic peptide derived from the 115e129 C-terminal region. BPB-BthTX-I presented cytotoxicity of 10e40% on different tumor cell lines, which were also susceptible to the lytic action of the synthetic peptide. Injection of the modified protein or the peptide in mice, 5 days after transplantation of S180 tumor cells, reduced 30 and 36% of the tumor size on day 14th and 76 and 79% on day 60th, respectively, when compared to the untreated control group. Thus, these antitumor properties might be of interest in the development of therapeutic strategies against cancer. .br (A.M. Soares), veridiana@ ingeb.ufu.br (V.M. Rodrigues).

Biochimie, 2012

This study reports the isolation and biochemical characterization of two different serine proteas... more This study reports the isolation and biochemical characterization of two different serine proteases from Bothrops pirajai snake venom, thus providing a comparative analysis of the enzymes. The isolation process consisted of three consecutive chromatographic steps (Sephacryl S-200, Benzamidine Sepharose and C2/C18), resulting in two serine proteases, named BpirSP27 and BpirSP41 after their molecular masses by mass spectrometry 121 and 40,639 Da, respectively). Estimation by SDS-PAGE under denaturing conditions showed that, when deglycosylated with PNGase F, BpirSP27 and BpirSP41 had their molecular masses reduced by approximately 15 and 42%, respectively. Both are acidic enzymes, with pI of approximately 4.7 for BpirSP27 and 3.7 for BpirSP41, and their N-terminal amino acid sequences showed 57% identity to each other, with high similarity to the sequences of other snake venom serine proteases (SVSPs). The enzymes showed different actions on bovine fibrinogen, with BpirSP27 acting preferentially on the Bb chain and BpirSP41 on both Aa and Bb chains. The two serine proteases were also able to degrade fibrin and blood clots in vitro depending on the doses and incubation periods, with higher results for BpirSP41. Both enzymes coagulated the human plasma in a dose-dependent manner, and BpirSP41 showed a higher coagulant potential, with minimum coagulant dose (MCD) of w3.5 mg versus 20 mg for BpirSP27. The enzymes were capable of hydrolyzing different chromogenic substrates, including S-2238 for thrombin-like enzymes, but only BpirSP27 acted on the substrate S-2251 for plasmin. They also showed high stability against variations of temperature and pH, but their activities were significantly reduced after preincubation with Cu 2þ ion and specific serine protease inhibitors. In addition, BpirSP27 induced aggregation of washed platelets to a greater extent than BpirSP41. The results showed significant structural and functional differences between B. pirajai serine proteases, providing interesting insights into the structureefunction relationship of SVSPs.

Biochimie, 2009

Crotalus durissus rattlesnakes are responsible for the most lethal cases of snakebites in Brazil.... more Crotalus durissus rattlesnakes are responsible for the most lethal cases of snakebites in Brazil. Crotalus durissus collilineatus subspecies is related to a great number of accidents in Southeast and Central West regions, but few studies on its venom composition have been carried out to date. In an attempt to describe the transcriptional profile of the C. durissus collilineatus venom gland, we generated a cDNA library and the sequences obtained could be identified by similarity searches on existing databases. Out of 673 expressed sequence tags (ESTs) 489 produced readable sequences comprising 201 singletons and 47 clusters of two or more ESTs. One hundred and fifty reads (60.5%) produced significant hits to known sequences. The results showed a predominance of toxin-coding ESTs instead of transcripts coding for proteins involved in all cellular functions. The most frequent toxin was crotoxin, comprising 88% of toxincoding sequences. Crotoxin B, a basic phospholipase A 2 (PLA 2 ) subunit of crotoxin, was represented in more variable forms comparing to the non-enzymatic subunit (crotoxin A), and most sequences coding this molecule were identified as CB1 isoform from Crotalus durissus terrificus venom. Four percent of toxin-related sequences in this study were identified as growth factors, comprising five sequences for vascular endothelial growth factor (VEGF) and one for nerve growth factor (NGF) that showed 100% of identity with C. durissus terrificus NGF. We also identified two clusters for metalloprotease from PII class comprising 3% of the toxins, and two for serine proteases, including gyroxin (2.5%). The remaining 2.5% of toxin-coding ESTs represent singletons identified as homologue sequences to cardiotoxin, convulxin, angiotensin-converting enzyme inhibitor and C-type natriuretic peptide, Ohanin, crotamin and PLA 2 inhibitor. These results allowed the identification of the most common classes of toxins in C. durissus collilineatus snake venom, also showing some unknown classes for this subspecies and even for C. durissus species, such as cardiotoxins and VEGF.

Biochemical Pharmacology, 2008

... sequence Carolina D. Sant Ana a , Danilo L. Menaldo a ,Ta ssia R. Costa a , Harryson Godoy a ... more ... sequence Carolina D. Sant Ana a , Danilo L. Menaldo a ,Ta ssia R. Costa a , Harryson Godoy a ... Biochem Biophys Res Commun 2007;355:302 6. [2] Sta beli RG, Sant Ana CD, Ribeiro PH, Costa TR, Ticli ... 7] Deolindo P, Teixeira-Ferreira AS, Melo EJ, Arnholdt AC, Souza W, Alves ...

Basic & Clinical Pharmacology & Toxicology, 2009

We genetically modified Eclipta alba using Agrobacterium rhizogenes LBA 9402, with the aim of pro... more We genetically modified Eclipta alba using Agrobacterium rhizogenes LBA 9402, with the aim of producing secondary metabolites with pharmacological properties against phospholipase A 2 and the myotoxic activities of snake venom. Extracts from in natura aerial parts and roots, both native and genetically modified ( in vitro ), were prepared and analysed by high-performance liquid chromatography. In natura materials showed the coumestan wedelolactone at higher concentration in the aerial parts, while demethylwedelolactone appeared at higher concentration in roots. Among the modified roots, clone 19 showed higher concentrations of these coumestans. Our results show that the in natura extracts of plants collected from Botucatu and Ribeirão Preto were efficient in inhibiting snake venom phospholipase A 2 activity. Regarding in vitro material, the best effect against Crotalus durissus terrificus venom was that of clone 19. Clone 19 and isolated coumestans (wedelolactone and demethylwedelolactone) inhibited the myotoxic activity induced by basic phospholipases A 2 isolated from the venoms of Crotalus durissus terrificus (CB) and Bothrops jararacussu (BthTX-I and II). The search for antivenom is justified by the need of finding active principles that are more efficient in neutralizing snake venoms and also as an attempt to complement serum therapy. LUCIANA C. DIOGO ET AL.

International Immunopharmacology, 2015

Snake venom metalloproteinases have been described as responsible for several inflammatory effect... more Snake venom metalloproteinases have been described as responsible for several inflammatory effects. In this study, we investigated the edema and hyperalgesia induced in rats by Batroxase, a P-I metalloproteinase from Bothrops atrox venom, along with possible inflammatory mediators involved in these responses. Batroxase or sterile saline was injected into rat paws and the edema and hyperalgesic effects were evaluated for 6h by using a plethysmometer and a Von Frey system, respectively. Batroxase induced significant edematogenic and hyperalgesic peak responses in the first hours after administration. The inflammatory mediators involved in these responses were assayed by pretreatment of animals with synthesis inhibitors or receptor antagonists. Peak responses were significantly reduced by administration of the glucocorticoid dexamethasone, the H1 receptor antagonist diphenhydramine and the FLAP inhibitor MK-886. Rat paws injected with compound 48/80, a mast cell degranulating agent, followed by Batroxase injection resulted in significant reduction of the edema and hyperalgesia. However, Batroxase itself induced minor degranulation of RBL-2H3 mast cells in vitro. Additionally, the inflammatory responses did not seem to be related to prostaglandins, bradykinin or nitric oxide. Our results indicate a major involvement of histamine and leukotrienes in the edema and hyperalgesia induced by Batroxase, which could be related, at least in part, to mast cell degranulation.

Toxicon : official journal of the International Society on Toxinology, Jan 15, 2008

Peptides, 2008

Journal of Venomous Animals and Toxins including Tropical Diseases, 2014

Biochimie, 2014

Toxicon, 2008

Toxicon, 2009

Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 2011

Journal of Proteomics, 2013

Peptides, 2008

Current Topics in Medicinal Chemistry, 2007

Blood Coagulation & Fibrinolysis, 2010

Biologicals, 2009

Biochimie, 2012

Biochimie, 2009

Biochemical Pharmacology, 2008

Basic & Clinical Pharmacology & Toxicology, 2009

International Immunopharmacology, 2015