Publications by authors named "Fernanda C V Portaro"

26 Publications

  • Page 1 of 1

Targeting Loxosceles spider Sphingomyelinase D with small-molecule inhibitors as a potential therapeutic approach for loxoscelism.

J Enzyme Inhib Med Chem 2019 Dec;34(1):310-321

a Immunochemistry Laboratory , Butantan Institute , São Paulo , SP , Brazil.

Loxosceles spiders' venoms consist of a mixture of proteins, including the sphingomyelinases D (SMases D), which are the main toxic components responsible for local and systemic effects in human envenomation. Herein, based on the structural information of SMase D from Loxosceles laeta spider venom and virtual docking-based screening approach, three benzene sulphonate compounds (named 1, 5 and 6) were identified as potential Loxosceles SMase D inhibitors. All compounds inhibited the hydrolysis of the sphingomyelin substrate by both recombinant and native SMases D. Compounds 5 and 6 acted as SMases D uncompetitive inhibitors with Ki values of 0.49 µM and 0.59 µM, respectively. Compound 1 is a mixed type inhibitor, and presented a Ki value of 0.54 µM. In addition, the three compounds inhibited the binding of SMases D to human erythrocytes and the removal of glycophorin C from the cell surface, which are important events in the complement-dependent haemolysis induced by Loxosceles venom. Moreover, compounds 5 and 6 reduced the binding of SMases to human keratinocytes membrane and the venom induced cell death. Importantly, compounds 5 and 6 also controlled the development of the necrotic lesion in an in vivo model of loxoscelism. Together, our findings indicate that the novel SMase D inhibitors presented here are able to suppress both local and systemic reactions induced by Loxosceles venoms. Since the number of Loxosceles envenomation accidents is currently growing worldwide, our results indicate that both inhibitors are promising scaffolds for the rational design of new drugs targeting SMases D from these spiders.
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http://dx.doi.org/10.1080/14756366.2018.1546698DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6327989PMC
December 2019

Kn-Ba: a novel serine protease isolated from snake venom with fibrinogenolytic and kinin-releasing activities.

J Venom Anim Toxins Incl Trop Dis 2018 13;24:38. Epub 2018 Dec 13.

1Immunochemistry Laboratory, Butantan Institute, São Paulo, 05503-900 Brazil.

Background: is a venomous snake found in sub-Saharan Africa and in parts of Morocco and Saudi Arabia. The envenomation is characterized by local and systemic reactions including pain, blistering, edema and tissue damage, besides hemostatic and cardiovascular disturbances, which can cause death or permanent disabilities in its victims. However, the action mechanisms that provoke these effects remain poorly understood, especially the activities of purified venom components. Therefore, in order to elucidate the molecular mechanisms that make the venom so potent and harmful to human beings, this study reports the isolation and biochemical characterization of a snake venom serine protease (SVSP).

Methods: Solubilized venom was fractionated by molecular exclusion chromatography and the proteolytic activity was determined using fluorescent substrates. The peaks that showed serine protease activity were determined by blocking the proteolytic activity with site-directed inhibitors. In sequence, the fraction of interest was submitted to another cycle of molecular exclusion chromatography. The purified serine protease was identified by mass spectrometry and characterized biochemically and immunochemically.

Results: A serine protease of 33 kDa with fibrinogen-degrading and kinin-releasing activities was isolated, described, and designated herein as Kn-Ba. The experimental Butantan Institute antivenom produced against venom inhibited the Kn-Ba activity.

Conclusions: The in vitro activities of Kn-Ba can be correlated with the capacity of the venom to provoke bleeding and clotting disorders as well as hypotension, which are common symptoms presented by envenomed victims. Obtaining satisfactory Kn-Ba inhibition through the experimental antivenom is important, given the WHO's recommendation of immunotherapy in cases of human accidents with venomous snakes.
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http://dx.doi.org/10.1186/s40409-018-0176-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6293559PMC
December 2018

Tb II-I, a Fraction Isolated from Scorpion Venom, Alters Cytokines': Level and Induces Seizures When Intrahippocampally Injected in Rats.

Toxins (Basel) 2018 06 19;10(6). Epub 2018 Jun 19.

Laboratory of Pharmacology, Butantan Institute, Av. Dr. Vital Brazil 1500, São Paulo 05503-900, Brazil.

Scorpion venoms are composed of several substances with different pharmacological activities. Neurotoxins exert their effects by targeting ion channels resulting in toxic effects to mammals, insects and crustaceans. Tb II-I, a fraction isolated from scorpion venom, was investigated for its ability to induce neurological and immune-inflammatory effects. Two putative β-sodium channel toxins were identified in this fraction, Tb2 II and Tb 4, the latter having been completely sequenced by mass spectrometry. Male Wistar rats, stereotaxically implanted with intrahippocampal cannulas and electrodes, were injected with Tb II-I (2 µg/2 µL) via the intrahippocampal route. The behavior, electrographic activity and cellular integrity of the animals were analyzed and the intracerebral level of cytokines determined. Tb II-I injection induced seizures and damage in the hippocampus. These alterations were correlated with the changes in the level of the cytokines tumoral necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Therefore, the binding of Tb II-I to its target in the central nervous system may induce inflammation resulting in neuropathological and behavioral alterations.
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http://dx.doi.org/10.3390/toxins10060250DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6024361PMC
June 2018

Erratum to: Angiotensin-converting enzyme inhibitors of Bothrops jararaca snake venom affect the structure of mice seminiferous epithelium.

J Venom Anim Toxins Incl Trop Dis 2015 26;21:33. Epub 2015 Aug 26.

Center for Applied Toxinology, Butantan Institute, São Paulo, SP Brazil.

[This corrects the article DOI: 10.1186/s40409-015-0030-y.].
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http://dx.doi.org/10.1186/s40409-015-0032-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4550052PMC
August 2015

Angiotensin-converting enzyme inhibitors of Bothrops jararaca snake venom affect the structure of mice seminiferous epithelium.

J Venom Anim Toxins Incl Trop Dis 2015 4;21:27. Epub 2015 Aug 4.

Center for Applied Toxinology, Butantan Institute, São Paulo, SP Brazil.

Background: Considering the similarity between the testis-specific isoform of angiotensin-converting enzyme and the C-terminal catalytic domain of somatic ACE as well as the structural and functional variability of its natural inhibitors, known as bradykinin-potentiating peptides (BPPs), the effects of different synthetic peptides, BPP-10c (
Methods: The adult animals received either one of the synthetic peptides or captopril (120 nmol/dose per testis) via injection into the testicular parenchyma. After seven days, the mice were sacrificed, and the testes were collected for histopathological evaluation.

Results: BPP-10c and BPP-AP showed an intense disruption of the epithelium, presence of atypical multinucleated cells in the lumen and high degree of seminiferous tubule degeneration, especially in BPP-AP-treated animals. In addition, both synthetic peptides led to a significant reduction in the number of spermatocytes and round spermatids in stages I, V and VII/VIII of the seminiferous cycle, thickness of the seminiferous epithelium and diameter of the seminiferous tubule lumen. Interestingly, no morphological or morphometric alterations were observed in animals treated with captopril or BPP-11e.

Conclusions: The major finding of the present study was that the demonstrated effects of BPP-10c and BPP-AP on the seminiferous epithelium are dependent on their primary structure and cannot be extrapolated to other BPPs.
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http://dx.doi.org/10.1186/s40409-015-0030-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4524108PMC
August 2015

Homology modeling, vasorelaxant and bradykinin-potentiating activities of a novel hypotensin found in the scorpion venom from Tityus stigmurus.

Toxicon 2015 Jul 28;101:11-8. Epub 2015 Apr 28.

Laboratório de Tecnologia e Biotecnologia Farmacêutica, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil; Programa de Pós-Graduação em Bioquímica, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil. Electronic address:

In a recent work by our group involving a transcriptomics approach applied to the venom glands from Tityus stigmurus we identified a new family of peptides called Hypotensins (TSTI0006C) (Almeida et al., 2012). The cluster TSTI0006C was analyzed in the main 25 amino acid residues and named T. stigmurus Hypotensin (TistH), showing a molecular mass of 2.7 kDa, an absence of cysteines and the presence of two C-terminal proline residues, which are a bradykinin-potentiating peptide (BPP) signature. Here, we describe the homology modeling of the three-dimensional structure of TistH. In addition, we evaluated the cardiovascular effects elicited by TistH in normotensive rats. Firstly, TistH showed no cytotoxic effect on horse erythrocyte. Furthermore, in normotensive rats TistH was able to potentiate the hypotensive action of bradykinin (BK) and induced a vasorelaxant effect in mesenteric artery rings by endothelium-dependent release of nitric oxide (NO) and demonstrated independent inhibition of angiotensin converting enzyme (ACE). Our data can contribute to a better understanding of the structural and functional characteristics of TistH and suggest its potential use in cardiovascular diseases.
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http://dx.doi.org/10.1016/j.toxicon.2015.04.003DOI Listing
July 2015

African adders: partial characterization of snake venoms from three Bitis species of medical importance and their neutralization by experimental equine antivenoms.

PLoS Negl Trop Dis 2015 Feb 2;9(2):e0003419. Epub 2015 Feb 2.

Immunochemistry Laboratory, Butantan Institute, São Paulo, São Paulo, Brazil.

Background: An alarming number of fatal accidents involving snakes are annually reported in Africa and most of the victims suffer from permanent local tissue damage and chronic disabilities. Envenomation by snakes belonging to the genus Bitis, Viperidae family, are common in Sub-Saharan Africa. The accidents are severe and the victims often have a poor prognosis due to the lack of effective specific therapies. In this study we have biochemically characterized venoms from three different species of Bitis, i.e., Bitis arietans, Bitis gabonica rhinoceros and Bitis nasicornis, involved in the majority of the human accidents in Africa, and analyzed the in vitro neutralizing ability of two experimental antivenoms.

Methodology/principal Findings: The data indicate that all venoms presented phospholipase, hyaluronidase and fibrinogenolytic activities and cleaved efficiently the FRET substrate Abz-RPPGFSPFRQ-EDDnp and angiotensin I, generating angiotensin 1-7. Gelatinolytic activity was only observed in the venoms of B. arietans and B. nasicornis. The treatment of the venoms with protease inhibitors indicated that Bitis venoms possess metallo and serinoproteases enzymes, which may be involved in the different biological activities here evaluated. Experimental antivenoms produced against B. arietans venom or Bitis g. rhinoceros plus B. nasicornis venoms cross-reacted with the venoms from the three species and blocked, in different degrees, all the enzymatic activities in which they were tested.

Conclusion: These results suggest that the venoms of the three Bitis species, involved in accidents with humans in the Sub-Saharan Africa, contain a mixture of various enzymes that may act in the generation and development of some of the clinical manifestations of the envenomations. We also demonstrated that horse antivenoms produced against B. arietans or B. g. rhinoceros plus B. nasicornis venoms can blocked some of the toxic activities of these venoms.
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http://dx.doi.org/10.1371/journal.pntd.0003419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4340965PMC
February 2015

P-I snake venom metalloproteinase is able to activate the complement system by direct cleavage of central components of the cascade.

PLoS Negl Trop Dis 2013 31;7(10):e2519. Epub 2013 Oct 31.

Laboratório de Imunoquímica, Instituto Butantan, São Paulo, Brazil.

Background: Snake Venom Metalloproteinases (SVMPs) are amongst the key enzymes that contribute to the high toxicity of snake venom. We have recently shown that snake venoms from the Bothrops genus activate the Complement system (C) by promoting direct cleavage of C-components and generating anaphylatoxins, thereby contributing to the pathology and spread of the venom. The aim of the present study was to isolate and characterize the C-activating protease from Bothrops pirajai venom.

Results: Using two gel-filtration chromatography steps, a metalloproteinase of 23 kDa that activates Complement was isolated from Bothrops pirajai venom. The mass spectrometric identification of this protein, named here as C-SVMP, revealed peptides that matched sequences from the P-I class of SVMPs. C-SVMP activated the alternative, classical and lectin C-pathways by cleaving the α-chain of C3, C4 and C5, thereby generating anaphylatoxins C3a, C4a and C5a. In vivo, C-SVMP induced consumption of murine complement components, most likely by activation of the pathways and/or by direct cleavage of C3, leading to a reduction of serum lytic activity.

Conclusion: We show here that a P-I metalloproteinase from Bothrops pirajai snake venom activated the Complement system by direct cleavage of the central C-components, i.e., C3, C4 and C5, thereby generating biologically active fragments, such as anaphylatoxins, and by cleaving the C1-Inhibitor, which may affect Complement activation control. These results suggest that direct complement activation by SVMPs may play a role in the progression of symptoms that follow envenomation.
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http://dx.doi.org/10.1371/journal.pntd.0002519DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3814341PMC
June 2014

Enzymatic properties of venoms from Brazilian scorpions of Tityus genus and the neutralisation potential of therapeutical antivenoms.

Toxicon 2013 Jul 15;69:180-90. Epub 2013 Mar 15.

Immunology Laboratory, State University of Londrina, Londrina, PR, Brazil.

Tityus scorpion stings are an important public health problem in Brazil, where the incidence of such stings exceeds the incidence of the health problems caused by other venomous animals, including snakes. In this study, we have analysed specific enzymatic activities of the venom from the Brazilian scorpions of Tityus genus, i.e., Tityus serrulatus, Tityus bahiensis and Tityus stigmurus. The data presented here revealed that Tityus spp. venoms exhibited significant hyaluronidase activity but no phospholipase activity. All the venom samples exhibited the ability to hydrolyse Abz-FLRRV-EDDnp and dynorphin 1-13 substrates. These activities were inhibited by 1,10-phenanthroline but not by PMSF, indicating the presence of metalloproteinases in the Tityus spp. venoms. The venom peptidase activity on Abz-FLRRV-EDDnp and on dynorphin 1-13 was partially inhibited by therapeutic Brazilian anti-scorpion and anti-arachnidic antivenoms. Dynorphin 1-13 (YGGFLRRIRPKLK) contains two scissile bonds between the residues Leu-Arg and Arg-Arg that are susceptible to cleavage by the Tityus venom metallopeptidase(s). Their cleavage releases leu-enkephalin, an important bioactive peptide. The detection of metalloproteinase(s) with specificity for both dynorphin 1-13 degradation and leu-enkephalin releasing can be important for the mechanistic understanding of hypotension and bradycardia induction in cases of scorpion stings, whereas hyaluronidases might contribute to the diffusion of the toxins present in these venoms. Furthermore, the limited inhibition of the toxic enzymatic activities by commercial antivenoms illustrates the necessity of improvements in current antivenom preparation.
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http://dx.doi.org/10.1016/j.toxicon.2013.02.012DOI Listing
July 2013

Premolis semirufa (Walker, 1856) envenomation, disease affecting rubber tappers of the Amazon: searching for caterpillar-bristles toxic components.

PLoS Negl Trop Dis 2012 28;6(2):e1531. Epub 2012 Feb 28.

Immunochemistry Laboratory, Butantan Institute, São Paulo, São Paulo, Brazil.

Background: The caterpillar of the moth Premolis semirufa (Lepidoptera: Arctiidae), commonly named Pararama, is endemic of the Amazon basin. Accidental contact with these caterpillar bristles causes local symptoms such as intense heat, pain, edema and itching which last for three to seven days; however, after multiples contacts, it may induce joint-space narrowing and bone alteration, as well as degeneration of the articular cartilage and immobilization of the affected joints. Specific treatment for this disease does not exist, but corticosteroids are frequently administered. Despite of the public health hazard of Premolis semirufa caterpillar poisoning, little is known about the nature of the toxic components involved in the induction of the pathology.

Methodology/principal Findings: Here we have investigated the biological and immunochemical characteristics of the caterpillar's bristles components. Analysis of the bristles extract in in vitro assays revealed the presence of proteolytic and hyaluronidase activities but no phospholipase A(2) activity. In vivo, it was observed that the bristles extract is not lethal but can induce an intense inflammatory process, characterized by the presence of neutrophils in the paw tissues of injected mice. Furthermore, the bristles components stimulated an intense and specific antibody response but autoantibodies such as anti-DNA or anti-collagen type II were not detected.

Conclusion: The results suggest that Premolis semirufa caterpillar bristles secretion contains a mixture of different enzymes that may act together in the generation and development of the clinical manifestations of the Pararama envenomation. Moreover, the high immunogenicity of the caterpillar bristles components, as shown by the generation of high antibody titers, may also contribute to the induction and establishment of the inflammatory disease.
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http://dx.doi.org/10.1371/journal.pntd.0001531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3289609PMC
June 2012

Diversity of Micrurus snake species related to their venom toxic effects and the prospective of antivenom neutralization.

PLoS Negl Trop Dis 2010 Mar 9;4(3):e622. Epub 2010 Mar 9.

Immunochemistry Laboratory, Butantan Institute, São Paulo, SP, Brazil.

Background: Micrurus snake bites can cause death by muscle paralysis and respiratory arrest, few hours after envenomation. The specific treatment for coral snake envenomation is the intravenous application of heterologous antivenom and, in Brazil, it is produced by horse immunization with a mixture of M. corallinus and M. frontalis venoms, snakes that inhabit the South and Southeastern regions of the country. However, this antivenom might be inefficient, considering the existence of intra- and inter-specific variations in the composition of the venoms. Therefore, the aim of the present study was to investigate the toxic properties of venoms from nine species of Micrurus: eight present in different geographic regions of Brazil (M. frontalis, M. corallinus, M. hemprichii, M. spixii, M. altirostris, M. surinamensis, M. ibiboboca, M. lemniscatus) and one (M. fulvius) with large distribution in Southeastern United States and Mexico. This study also analyzed the antigenic cross-reactivity and the neutralizing potential of the Brazilian coral snake antivenom against these Micrurus venoms.

Methodology/principal Findings: Analysis of protein composition and toxicity revealed a large diversity of venoms from the nine Micrurus species. ELISA and Western blot assays showed a varied capability of the therapeutic antivenom to recognize the diverse species venom components. In vivo and in vitro neutralization assays indicated that the antivenom is not able to fully neutralize the toxic activities of all venoms.

Conclusion: These results indicate the existence of a large range of both qualitative and quantitative variations in Micrurus venoms, probably reflecting the adaptation of the snakes from this genus to vastly dissimilar habitats. The data also show that the antivenom used for human therapy in Brazil is not fully able to neutralize the main toxic activities present in the venoms from all Micrurus species occurring in the country. It suggests that modifications in the immunization scheme, with the inclusion of other venoms in the antigenic mixture, should occur in order to generate effective therapeutic coral snake antivenom.
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http://dx.doi.org/10.1371/journal.pntd.0000622DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2834742PMC
March 2010

Administration of Mycobacterium leprae rHsp65 aggravates experimental autoimmune uveitis in mice.

PLoS One 2009 Nov 19;4(11):e7912. Epub 2009 Nov 19.

Department of Immunology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.

The 60 kDa heat shock protein family, Hsp60, constitutes an abundant and highly conserved class of molecules that are highly expressed in chronic-inflammatory and autoimmune processes. Experimental autoimmune uveitis [EAU] is a T cell mediated intraocular inflammatory disease that resembles human uveitis. Mycobacterial and homologous Hsp60 peptides induces uveitis in rats, however their participation in aggravating the disease is poorly known. We here evaluate the effects of the Mycobacterium leprae Hsp65 in the development/progression of EAU and the autoimmune response against the eye through the induction of the endogenous disequilibrium by enhancing the entropy of the immunobiological system with the addition of homologous Hsp. B10.RIII mice were immunized subcutaneously with interphotoreceptor retinoid-binding protein [IRBP], followed by intraperitoneally inoculation of M. leprae recombinant Hsp65 [rHsp65]. We evaluated the proliferative response, cytokine production and the percentage of CD4(+)IL-17(+), CD4(+)IFN-gamma(+) and CD4(+)Foxp3(+) cells ex vivo, by flow cytometry. Disease severity was determined by eye histological examination and serum levels of anti-IRBP and anti-Hsp60/65 measured by ELISA. EAU scores increased in the Hsp65 group and were associated with an expansion of CD4(+)IFN-gamma(+) and CD4(+)IL-17(+) T cells, corroborating with higher levels of IFN-gamma. Our data indicate that rHsp65 is one of the managers with a significant impact over the immune response during autoimmunity, skewing it to a pathogenic state, promoting both Th1 and Th17 commitment. It seems comprehensible that the specificity and primary function of Hsp60 molecules can be considered as a potential pathogenic factor acting as a whistleblower announcing chronic-inflammatory diseases progression.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0007912PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2775913PMC
November 2009

SMase II, a new sphingomyelinase D from Loxosceles laeta venom gland: molecular cloning, expression, function and structural analysis.

Toxicon 2009 Jun 26;53(7-8):743-53. Epub 2009 Feb 26.

Laboratório de Imunoquímica, Instituto Butantan, São Paulo, Brazil.

Sphingomyelinase D (SMase D) present in the venoms of Loxosceles spiders is the principal component responsible for local and systemic effects observed in the loxoscelism. By using "expressed sequencing tag", it was possible to identify, in a L. laeta venom gland library, clones containing inserts coding for proteins with similarity to SMase D. One of these clones was expressed and the recombinant protein compared with the previously characterized SMase I from L. laeta, in terms of their biological, biochemical and structural properties. The new recombinant protein, SMase II, possesses all the biological properties ascribed to the whole venom and SMase I. SMase II shares 40% and 77% sequence similarity with SMase I and Lb3, respectively; the latter, a SMase D isoform from L. boneti, catalytically inactive. Molecular modeling and molecular dynamics simulations were employed to understand the structural basis, especially the presence of an additional disulfide bridge, in an attempt to account for the observed differences in SMases D activity.
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http://dx.doi.org/10.1016/j.toxicon.2009.02.013DOI Listing
June 2009

Ctenus medius and Phoneutria nigriventer spiders venoms share noxious proinflammatory activities.

J Med Entomol 2009 Jan;46(1):58-66

Laboratório de Imunoquímica, Instituto Butantan, Av. Prof. Vital Brazil, 1500, CEP 05503-900, São Paulo, Brazil.

Ctenus medius Keyserling, 1891 (Araneae: Ctenidae) co-occurs in various microhabitats of the Brazilian Atlantic Forest and can be easily misidentified as the medically important spider Phoneutria nigriventer Keyserling, 1981 (Ctenidae). Despite being phylogenetically close to Phoneutria, no data are available about the toxic potential of Ctenus medius venom. Here we show that, although presenting different profile of protein composition, C. medius venom displays some of the toxic properties exhibited by P. nigriventer venom, including proteolytic, hyaluronidasic and phospholipasic activities, as well as the ability of causing hyperalgesia and edema. Moreover, C. medius venom interferes in the activation of the complement system in concentrations that P. nigriventer venom is inactive. Thus, these data show that venoms of spiders from Ctenidae family share important proinflammatory properties and suggest that the C. medius bite may have an important noxious effect in human accidents.
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http://dx.doi.org/10.1603/033.046.0108DOI Listing
January 2009

Interspecific variation in venom composition and toxicity of Brazilian snakes from Bothrops genus.

Toxicon 2008 Dec 17;52(8):842-51. Epub 2008 Oct 17.

Immunochemistry Laboratory, Butantan Institute, Av. Prof. Vital Brazil, 1500, CEP 05503-900, São Paulo, Brazil.

The genus Bothrops spp. is responsible for 90% of envenomation by snakes in Brazil, and the standard treatment for snakebites is the antivenom therapy. The anti-bothropic serum produced by Butantan Institute is prepared by the hyperimmunization of horses with a pool of venoms from Bothrops alternatus, Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni and Bothrops neuwiedi. In this study, the biochemical and biological characteristics of the venoms from nineteen snakes of the genus Bothrops, responsible for human accidents in Brazil, were analysed. Venoms, particularly from Crotalidae and Viperidae snakes, are rich sources of serine proteases and metalloproteases and the ability of the Brazilian anti-bothropic serum to neutralize the proteolytic activity of these venoms were also tested. The results obtained here show the existence of a large range of variation in the composition and activities in Bothrops spp. toxins and demonstrate that the anti-bothropic serum is not able to fully neutralize the toxic activities of all analysed venoms. These suggest that for the preparation of a fully effective therapeutic anti-bothropic serum, other venoms should be included in the immunization mixture.
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http://dx.doi.org/10.1016/j.toxicon.2008.10.002DOI Listing
December 2008

Proteomics of the neurotoxic fraction from the sea anemone Bunodosoma cangicum venom: Novel peptides belonging to new classes of toxins.

Comp Biochem Physiol Part D Genomics Proteomics 2008 Sep 26;3(3):219-25. Epub 2008 Apr 26.

Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, Rua do Matão, Travessa 14, 321, CEP 05508-900 São Paulo-SP, Brazil.

In contrast to the many studies on the venoms of scorpions, spiders, snakes and cone snails, up to now there has been no report of the proteomic analysis of sea anemones venoms. In this work we report for the first time the peptide mass fingerprint and some novel peptides in the neurotoxic fraction (Fr III) of the sea anemone Bunodosoma cangicum venom. Fr III is neurotoxic to crabs and was purified by rp-HPLC in a C-18 column, yielding 41 fractions. By checking their molecular masses by ESI-Q-Tof and MALDI-Tof MS we found 81 components ranging from near 250 amu to approximately 6000 amu. Some of the peptidic molecules were partially sequenced through the automated Edman technique. Three of them are peptides with near 4500 amu belonging to the class of the BcIV, BDS-I, BDS-II, APETx1, APETx2 and Am-II toxins. Another three peptides represent a novel group of toxins (~3200 amu). A further three molecules (~ approximately 4900 amu) belong to the group of type 1 sodium channel neurotoxins. When assayed over the crab leg nerve compound action potentials, one of the BcIV- and APETx-like peptides exhibits an action similar to the type 1 sodium channel toxins in this preparation, suggesting the same target in this assay. On the other hand one of the novel peptides, with 3176 amu, displayed an action similar to potassium channel blockage in this experiment. In summary, the proteomic analysis and mass fingerprint of fractions from sea anemone venoms through MS are valuable tools, allowing us to rapidly predict the occurrence of different groups of toxins and facilitating the search and characterization of novel molecules without the need of full characterization of individual components by broader assays and bioassay-guided purifications. It also shows that sea anemones employ dozens of components for prey capture and defense.
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http://dx.doi.org/10.1016/j.cbd.2008.04.002DOI Listing
September 2008

Administration of M. leprae Hsp65 interferes with the murine lupus progression.

PLoS One 2008 Aug 21;3(8):e3025. Epub 2008 Aug 21.

Immunochemistry Laboratory, Instituto Butantan, São Paulo, Brazil.

The heat shock protein [Hsp] family guides several steps during protein synthesis, are abundant in prokaryotic and eukaryotic cells, and are highly conserved during evolution. The Hsp60 family is involved in assembly and transport of proteins, and is expressed at very high levels during autoimmunity or autoinflammatory phenomena. Here, the pathophysiological role of the wild type [WT] and the point mutated K(409)A recombinant Hsp65 of M. leprae in an animal model of Systemic Lupus Erythematosus [SLE] was evaluated in vivo using the genetically homogeneous [NZBxNZW]F(1) mice. Anti-DNA and anti-Hsp65 antibodies responsiveness was individually measured during the animal's life span, and the mean survival time [MST] was determined. The treatment with WT abbreviates the MST in 46%, when compared to non-treated mice [p<0.001]. An increase in the IgG2a/IgG1 anti-DNA antibodies ratio was also observed in animals injected with the WT Hsp65. Incubation of BALB/c macrophages with F(1) serum from WT treated mice resulted in acute cell necrosis; treatment of these cells with serum from K(409)A treated mice did not cause any toxic effect. Moreover, the involvement of WT correlates with age and is dose-dependent. Our data suggest that Hsp65 may be a central molecule intervening in the progression of the SLE, and that the point mutated K(409)A recombinant immunogenic molecule, that counteracts the deleterious effect of WT, may act mitigating and delaying the development of SLE in treated mice. This study gives new insights into the general biological role of Hsp and the significant impact of environmental factors during the pathogenesis of this autoimmune process.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0003025PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2515089PMC
August 2008

Characterization of urinary metabolites from four synthetic bradykinin potentiating peptides (BPPs) in mice.

Toxicon 2008 Sep 4;52(3):501-7. Epub 2008 Jul 4.

Center for Applied Toxinology CAT-CEPID, Instituto Butantan, Av. Vital Brasil, 1500, São Paulo, SP 05503-900, Brazil.

BPPs have been identified in the venom of the Bothrops jararaca snake, or deduced from precursor proteins expressed either in the venom gland or in the brain of the snake. Their potentiating activity on bradykinin (Bk) is assumed to occur through a somatic angiotensin-converting enzyme (sACE) inhibitory mechanism. We have demonstrated that synthetic BPPs show remarkable functional differences, despite their high amino acid sequence similarities. Recently, we demonstrated that BPP-10c, after i.p. administration, was found in its intact form and in the form of a unique metabolite (des-Pro(10) BPP-10c) in mouse urine. Given this finding, we selected a number of BPPs with different structure-activities - BPP-5a (
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http://dx.doi.org/10.1016/j.toxicon.2008.06.024DOI Listing
September 2008

A novel bradykinin potentiating peptide isolated from Bothrops jararacussu venom using catallytically inactive oligopeptidase EP24.15.

FEBS J 2008 May 8;275(10):2442-54. Epub 2008 Apr 8.

Laboratório Especial de Toxinologia Aplicada-CAT/CEPID, Instituto Butantan, São Paulo, Brazil.

Characterization of the peptide content of venoms has a number of potential benefits for basic research, clinical diagnosis, development of new therapeutic agents, and production of antiserum. Here, we use a substrate-capture assay that employs a catalytically inactive mutant of thimet oligopeptidase (EC 3.4.24.15; EP24.15) to identify novel bioactive peptides in Bothrops jararacussu venom. Of the peptides captured with inactive EP24.15 and identified by mass spectrometry, three were previously identified bradykinin-potentiating peptides (BPP),
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http://dx.doi.org/10.1111/j.1742-4658.2008.06389.xDOI Listing
May 2008

Tissue distribution in mice of BPP 10c, a potent proline-rich anti-hypertensive peptide of Bothrops jararaca.

Toxicon 2008 Mar 17;51(4):515-23. Epub 2007 Nov 17.

Center for Applied Toxinology CAT-CEPID, Instituto Butantan, Av. Vital Brasil 1500, 05503-900 São Paulo, SP, Brazil.

The snake venom proline-rich peptide BPP 10c is an active somatic angiotensin-converting enzyme (sACE) inhibitors. Recently we demonstrated that the anti-hypertensive effect of BPP 10c is not related to the inhibition of sACE alone, thus suggesting that this enzyme is not its only target for blood pressure reduction. In the present work, a biodistribution study in Swiss mice of [(125)I]-BPP 10c in the absence or in the presence of a saturating concentration of captopril, a selective active-site inhibitor of sACE, demonstrated that: (1) [(125)I]-BPP 10c was present in several organs and the renal absorption was significantly high; (2) [(125)I]-BPP 10c showed a clear preference for the kidney, maintaining a high concentration in this organ in the presence of captopril for at least 3h; (3) The residual amount of [(125)I]-BPP 10c in the kidney of animals simultaneously treated with captopril suggest that the peptide can interact with other targets different from sACE in this organ. We also showed that Cy3-labeled BPP 10c was internalized by human embryonic kidney cells (HEK-293T). Taken together, these results suggest that sACE inhibition by captopril affects the tissue distribution of [(125)I]-BPP 10c and that the anti-hypertensive effects of BPP 10c are not only dependent on sACE inhibition.
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http://dx.doi.org/10.1016/j.toxicon.2007.11.003DOI Listing
March 2008

Caissarolysin I (Bcs I), a new hemolytic toxin from the Brazilian sea anemone Bunodosoma caissarum: purification and biological characterization.

Biochim Biophys Acta 2006 Mar 17;1760(3):453-61. Epub 2006 Jan 17.

Laboratório de Imunoquímica, Instituto Butantan, Av. Prof. Vital Brazil, 1500, CEP 05508-900, São Paulo, SP, Brazil.

Two cationic proteins, C1 and C3, were purified to homogeneity from the hemolytic fraction of the venom of Bunodosoma caissarum sea anemone. The purification processes employed gel filtration followed by ion exchange chromatography, being the purity and molecular mass confirmed by SDS-PAGE and mass spectrometry. Protein C1 represented the second major peak of the hemolytic fraction and was previously believed to be a cytolysin belonging to a new class of hemolysins. The C1 protein has a molecular mass of 15495 Da and was assayed for hemolysis, PLA2 activity and acute toxicity in crabs and mice, showing no activity in these assays. It has an amino terminal with no similarity to all known hemolysins and, therefore, should not be considered a toxin, being its function completely unknown. The protein C3 (19757 Da), that also lacks PLA2 activity, was recognized by antiserum against Eqt II and presented high hemolytic activity to human erythrocytes (ED50 of 0.270 microg/ml), being named Caissarolysin I (Bcs I). Its activity was inhibited by pre-incubation with sphingomyelin (SM) and also when in presence of erythrocytes pre-treated with the SMase P2, a phospholipase D from the brown spider Loxosceles intermedia, indicating that SM is the main target of Bcs I. Caissarolysin I is the first hemolysin purified from a sea anemone belonging to the genus Bunodosoma and belongs to the Actinoporin family of sea anemone hemolysins.
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http://dx.doi.org/10.1016/j.bbagen.2005.12.018DOI Listing
March 2006

Cloning and characterization of the human and rabbit NUDEL-oligopeptidase promoters and their negative regulation.

Biochim Biophys Acta 2005 Jul;1730(1):77-84

Laboratory for Applied Toxinology, Butantan Institute, São Paulo, 05503-900, Brazil.

NUDEL-oligopeptidase is a cytosolic cysteine peptidase, active towards oligopeptides and involved in the conversion and inactivation of a number of bioactive peptides. This protein interacts with neuronal proteins and is essential for brain development and cortical organization during embryogenesis. In this study, 5'-flanking sequences of the human and rabbit NUDEL-oligopeptidase gene were cloned into the pGL3 reporter gene vector and the promoter activity of the full-length fragment and deletions series was measured in transient transfection assays using two different cell lines, namely, C6 rat glioma and NH15 human neuroblastoma. Overall, a very similar pattern of promoter activity was obtained for both rabbit and human NUDEL-oligopeptidase promoter sequences, and their respective serial deletion constructs upon transient transfection into these cell lines. The only exception was for the longest rabbit upstream sequence that displayed about 1.8-fold higher luciferase expression upon transfection into NH15 neuronal cells than that observed upon transfection into C6 glioma cells. On the other hand, no significant difference was observed for the human longest sequence. These results are in good agreement with the expression pattern of NUDEL-oligopeptidase in human and rabbit tissues.
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http://dx.doi.org/10.1016/j.bbaexp.2005.06.001DOI Listing
July 2005

Inhibition of NUDEL (nuclear distribution element-like)-oligopeptidase activity by disrupted-in-schizophrenia 1.

Proc Natl Acad Sci U S A 2005 Mar 23;102(10):3828-33. Epub 2005 Feb 23.

Center for Applied Toxinology, Laboratories of Immunogenetics and Immunochemistry, Butantan Institute, and Laboratory of Neurosciences, Universidade Cidade de São Paulo, SP 05503-900, São Paulo, Brazil.

Recently, nuclear distribution element-like (NUDEL) has been implicated to play a role in lissencephaly and schizophrenia through interactions with the lissencephaly gene 1 (Lis1) and disrupted-in-schizophrenia 1 (DISC1) products, respectively. Interestingly, NUDEL is the same protein as endooligopeptidase A (EOPA), a thiol-activated peptidase involved in conversion and inactivation of a number of bioactive peptides. In this study, we have cloned EOPA from the human brain and have confirmed that it is equivalent to NUDEL, leading us to suggest a single name, NUDEL-oligopeptidase. In the brain, the monomeric form of NUDEL-oligopeptidase is responsible for the peptidase activity whose catalytic mechanism is likely to involve a reactive cysteine, because mutation of Cys-273 fully abolished NUDEL-oligopeptidase activity without disrupting the protein's secondary structure. Cys-273 is very close to the DISC1-binding site on NUDEL-oligopeptidase. Intriguingly, DISC1 inhibits NUDEL-oligopeptidase activity in a competitive fashion. We suggest that the activity of NUDEL-oligopeptidase is under tight regulation through protein-protein interactions and that disruption of these interactions, as postulated in a Scottish DISC1 translocation schizophrenia cohort, may lead to aberrant regulation of NUDEL-oligopeptidase, perhaps providing a substrate for the pathology of schizophrenia.
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http://dx.doi.org/10.1073/pnas.0500330102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC553309PMC
March 2005

Mass spectrometric and high performance liquid chromatography profiling of the venom of the Brazilian vermivorous mollusk Conus regius: feeding behavior and identification of one novel conotoxin.

Toxicon 2005 Jan;45(1):113-22

Department of Physiology, Bioscience Institute, University of São Paulo, São Paulo, SP, Brazil.

Carnivorous mollusks belonging to the genus Conus paralyze their prey by injecting a rich mixture of biologically active peptides. Conus regius is a vermivorous member of this genus that inhabits Brazilian tropical waters. Inter-, intra-species and individual variations of cone snail venom have been previously reported. In order to investigate intra-specific differences in C. regius venom, its feeding behavior and the correlation between these two factors, animals were pooled according to gender, size and season of collection, and their venom composition was compared by high performance liquid chromatography (HPLC). Both the whole venom and one specific peak were monitored by HPLC. Chromatographic profiles revealed no significant differences in their peak areas, indicating that the venom composition, based solely in the presence or absence of the major peaks, is stable regardless of season, gender and size. Therefore, analysis of one given toxin, eluting in one of the major peaks, is representative among the population. Moreover, this work presents the identification of one novel conotoxin (rg11a), which amino acid sequence was deduced by mass spectrometry.
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http://dx.doi.org/10.1016/j.toxicon.2004.09.018DOI Listing
January 2005

The C-type natriuretic peptide precursor of snake brain contains highly specific inhibitors of the angiotensin-converting enzyme.

J Neurochem 2003 May;85(4):969-77

Center for Applied Toxinology--CAT/CEPID, Instituto Butantan, Avenue.Vital Brazil 15600, São Paulo, SP 05530-900, Brazil.

The bradykinin-potentiating peptides from Bothrops jararaca venom are the most potent natural inhibitors of the angiotensin-converting enzyme. The biochemical and biological features of these peptides were crucial to demonstrate the pivotal role of the angiotensin-converting enzyme in blood pressure regulation. In the present study, seven bradykinin-potentiating peptides were identified within the C-type natriuretic peptide precursor cloned from snake brain. The bradykinin-potentiating peptides deduced from the B. jararaca brain precursor are strong in vitro inhibitors of the angiotensin-converting enzyme (nanomolar range), and also potentiate the bradykinin effects in ex vivo and in vivo experiments. Two of these peptides are novel bradykinin-potentiating peptides, one of which displays high specificity toward the N-domain active site of the somatic angiotensin-converting enzyme. In situ hybridization studies revealed the presence of the bradykinin-potentiating peptides precursor mRNAs in distinct regions of the B. jararaca brain, such as the ventromedial hypothalamus, the paraventricular nuclei, the paraventricular organ, and the subcommissural organ. The biochemical and pharmacological properties of the brain bradykinin-potentiating peptides, their presence within the neuroendocrine regulator C-type natriuretic peptide precursor, and their expression in regions of the snake brain correlated to neuroendocrine functions, strongly suggest that these peptides belong to a novel class of endogenous vasoactive peptides.
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http://dx.doi.org/10.1046/j.1471-4159.2003.01743.xDOI Listing
May 2003

The Mycobacterium leprae hsp65 displays proteolytic activity. Mutagenesis studies indicate that the M. leprae hsp65 proteolytic activity is catalytically related to the HslVU protease.

Biochemistry 2002 Jun;41(23):7400-6

Center for Applied Toxinology, Butantan Institute, CAT/CEPID, Rua Murajuba 125, São Paulo, SP 05467-010, Brazil.

The present study reports, for the first time, that the recombinant hsp65 from Mycobacterium leprae (chaperonin 2) displays a proteolytic activity toward oligopeptides. The M. leprae hsp65 proteolytic activity revealed a trypsin-like specificity toward quenched fluorescence peptides derived from dynorphins. When other peptide substrates were used (beta-endorphin, neurotensin, and angiotensin I), the predominant peptide bond cleavages also involved basic amino acids in P(1), although, to a minor extent, the hydrolysis involving hydrophobic and neutral amino acids (G and F) was also observed. The amino acid sequence alignment of the M. leprae hsp65 with Escherichia coli HslVU protease suggested two putative threonine catalytic groups, one in the N-domain (T(136), K(168), and Y(264)) and the other in the C-domain (T(375), K(409), and S(502)). Mutagenesis studies showed that the replacement of K(409) by A caused a complete loss of the proteolytic activity, whereas the mutation of K(168) to A resulted in a 25% loss. These results strongly suggest that the amino acid residues T(375), K(409), and S(502) at the C-domain form the catalytic group that carries out the main proteolytic activity of the M. leprae hsp65. The possible pathophysiological implications of the proteolytic activity of the M. leprae hsp65 are now under investigation in our laboratory.
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http://dx.doi.org/10.1021/bi011999lDOI Listing
June 2002