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Geraldo Magalhães
Immunopathology Laboratory, Instituto Butantan, São Paulo, SP, Brazil

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Journal article
Published: 17 February 2020 in AMB Express
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Commercially available preparations of methionyl-human BMP-2 and CHO-derived hBMP-2, which belongs to the transforming growth factor β (TGF-β) superfamily, were used for a complete characterization. This protein is an extremely efficient osteoinductor that plays an important role during bone regeneration and embryonic development. Characterization was carried out via SDS-PAGE and Western blotting, followed by reversed-phase HPLC, size-exclusion HPLC and MALDI-TOF-MS. The classical in vitro bioassay, based on the induction of alkaline phosphatase activity in C2C12 cells, confirmed that hBMP-2 biological activity is mostly related to the dimeric form, being ~ 4-fold higher for the CHO-derived glycosylated form when compared with the E. coli counterpart. The E. coli-derived met-hBMP-2 has shown, by MALDI-TOF-MS, a large presence of the bioactive dimer. A more complex molecular mass (MM) distribution was found for the CHO-derived product, whose exact MM has never been reported because of its variable glycosylation. A method based on RP-HPLC was set up, allowing a quantitative and qualitative hBMP-2 determination even directly on ongoing culture media. Considering that hBMP-2 is highly unstable, presenting moreover an extremely high aggregate value, we believe that these data pave the way to a necessary characterization of this important factor when synthesized by DNA recombinant techniques in different types of hosts.

ACS Style

Miriam Fussae Suzuki; João Ezequiel Oliveira; Renata Damiani; Eliana Rosa Lima; Kleicy Cavalcante Amaral; Anderson Maikon De Souza Santos; Geraldo Santana Magalhães; Leonardo Perez Faverani; Luis Antonio Violin Dias Pereira; Fabiana Medeiros Silva; Paolo Bartolini. Human bone morphogenetic protein-2 (hBMP-2) characterization by physical–chemical, immunological and biological assays. AMB Express 2020, 10, 1 -10.

AMA Style

Miriam Fussae Suzuki, João Ezequiel Oliveira, Renata Damiani, Eliana Rosa Lima, Kleicy Cavalcante Amaral, Anderson Maikon De Souza Santos, Geraldo Santana Magalhães, Leonardo Perez Faverani, Luis Antonio Violin Dias Pereira, Fabiana Medeiros Silva, Paolo Bartolini. Human bone morphogenetic protein-2 (hBMP-2) characterization by physical–chemical, immunological and biological assays. AMB Express. 2020; 10 (1):1-10.

Chicago/Turabian Style

Miriam Fussae Suzuki; João Ezequiel Oliveira; Renata Damiani; Eliana Rosa Lima; Kleicy Cavalcante Amaral; Anderson Maikon De Souza Santos; Geraldo Santana Magalhães; Leonardo Perez Faverani; Luis Antonio Violin Dias Pereira; Fabiana Medeiros Silva; Paolo Bartolini. 2020. "Human bone morphogenetic protein-2 (hBMP-2) characterization by physical–chemical, immunological and biological assays." AMB Express 10, no. 1: 1-10.

Journal article
Published: 25 June 2019 in Toxicon
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Venoms of spiders and snakes contain toxins extremely active and, thus, provide a natural source for the development of new biotechnological tools. Among the diversity of toxins present in the venom of spiders from genus Loxosceles, the phospholipases D (PLDs) show high hydrolytic activity upon lysophosphatidylcholine (LPC) and sphingomyelin (SM), generating bioactive phospholipids such as cyclic phosphatidic acid (cPA). Since this mediator has been shown to play a major role in complex signaling pathways, including inhibition of tumor cells, the PLDs may hold the key to learn how toxins could be used for therapeutic purposes. However, the strong platelet aggregation of PLDs and their lack of selectivity impose a major limitation. On the other hand, disintegrins present in the venoms of Viperidae snakes are a potent inhibitor of platelet aggregation and possess high affinity and specificity to molecules called integrins that are highly expressed in some tumor cells, such as murine melanoma B16F10. Therefore, disintegrins might be suitable molecules to carry the PLDs to the malignant cells, so both toxins may work synergistically to eliminate these cells. Thus, in this work, a recombinant PLD from Loxosceles gaucho spider was recombinantly fused to a disintegrin from Echis carinatus snake to form a hybrid toxin called Rechistatin. This recombinant toxin was successfully expressed in bacteria, showed binding activity in B16F10 murine melanoma cells and exerted a synergistic cytotoxicity effect on these cells. Therefore, the approach presented in this work may represent a new strategy to explore new potential applications for spider PLDs.

ACS Style

Raquel A.G.B. Siqueira; Paula A.L. Calabria; Maria C. Caporrino; Bianca C.L.F. Tavora; Katia C. Barbaro; Eliana L. Faquim-Mauro; Maisa S. Della-Casa; Geraldo S. Magalhães. When spider and snake get along: Fusion of a snake disintegrin with a spider phospholipase D to explore their synergistic effects on a tumor cell. Toxicon 2019, 168, 40 -48.

AMA Style

Raquel A.G.B. Siqueira, Paula A.L. Calabria, Maria C. Caporrino, Bianca C.L.F. Tavora, Katia C. Barbaro, Eliana L. Faquim-Mauro, Maisa S. Della-Casa, Geraldo S. Magalhães. When spider and snake get along: Fusion of a snake disintegrin with a spider phospholipase D to explore their synergistic effects on a tumor cell. Toxicon. 2019; 168 ():40-48.

Chicago/Turabian Style

Raquel A.G.B. Siqueira; Paula A.L. Calabria; Maria C. Caporrino; Bianca C.L.F. Tavora; Katia C. Barbaro; Eliana L. Faquim-Mauro; Maisa S. Della-Casa; Geraldo S. Magalhães. 2019. "When spider and snake get along: Fusion of a snake disintegrin with a spider phospholipase D to explore their synergistic effects on a tumor cell." Toxicon 168, no. : 40-48.

Journal article
Published: 12 February 2019 in Toxins
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Human accidents with spiders of the genus Loxosceles are an important health problem affecting thousands of people worldwide. Patients evolve to severe local injuries and, in many cases, to systemic disturbances as acute renal failure, in which cases antivenoms are considered to be the most effective treatment. However, for antivenom production, the extraction of the venom used in the immunization process is laborious and the yield is very low. Thus, many groups have been exploring the use of recombinant Loxosceles toxins, particularly phospholipases D (PLDs), to produce the antivenom. Nonetheless, some important venom activities are not neutralized by anti-PLD antibodies. Astacin-like metalloproteases (ALMPs) are the second most expressed toxin acting on the extracellular matrix, indicating the importance of its inclusion in the antigen’s formulation to provide a better antivenom. Here we show the construction of a hybrid recombinant immunogen, called LgRec1ALP1, composed of hydrophilic regions of the PLD and the ALMP toxins from Loxosceles gaucho. Although the LgRec1ALP1 was expressed as inclusion bodies, it resulted in good yields and it was effective to produce neutralizing antibodies in mice. The antiserum neutralized fibrinogenolytic, platelet aggregation and dermonecrotic activities elicited by L. gaucho, L. laeta, and L. intermedia venoms, indicating that the hybrid recombinant antigen may be a valuable source for the production of protective antibodies against Loxosceles ssp. venoms. In addition, the hybrid recombinant toxin approach may enrich and expand the alternative antigens for antisera production for other venoms.

ACS Style

Paula A. L. Calabria; Lhiri Hanna A. L. Shimokawa-Falcão; Monica Colombini; Ana M. Moura-Da-Silva; Katia C. Barbaro; Eliana L. Faquim-Mauro; Geraldo S. Magalhaes. Design and Production of a Recombinant Hybrid Toxin to Raise Protective Antibodies against Loxosceles Spider Venom. Toxins 2019, 11, 108 .

AMA Style

Paula A. L. Calabria, Lhiri Hanna A. L. Shimokawa-Falcão, Monica Colombini, Ana M. Moura-Da-Silva, Katia C. Barbaro, Eliana L. Faquim-Mauro, Geraldo S. Magalhaes. Design and Production of a Recombinant Hybrid Toxin to Raise Protective Antibodies against Loxosceles Spider Venom. Toxins. 2019; 11 (2):108.

Chicago/Turabian Style

Paula A. L. Calabria; Lhiri Hanna A. L. Shimokawa-Falcão; Monica Colombini; Ana M. Moura-Da-Silva; Katia C. Barbaro; Eliana L. Faquim-Mauro; Geraldo S. Magalhaes. 2019. "Design and Production of a Recombinant Hybrid Toxin to Raise Protective Antibodies against Loxosceles Spider Venom." Toxins 11, no. 2: 108.

Journal article
Published: 13 June 2017 in Toxins
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Spider envenomation, from the genus Loxosceles, is frequently reported as a cause of necrotic lesions in humans around the world. Among the many components found in the venom of Loxosceles genus, phospholipases D (PLDs) are the most investigated, since they can cause a massive inflammatory response, dermonecrosis, hemolysis and platelet aggregation, among other effects. Even though the PLDs induce strong platelet aggregation, there are no studies showing how the PLDs interact with platelets to promote this effect. Since many agonists must interact with specific receptors on the platelet membrane to induce aggregation, it is reasonable to expect that the PLDs may, in some way, also interact with platelets, to induce this activity. Therefore, to address this possibility, in this work, a recombinant PLD, called LgRec1, from L. gaucho was fused to enhanced green fluorescent protein (EGFP) and used as a probe to detect the interaction of LgRec1 to platelets, by fluorescence-activated cell sorter (FACS) and confocal microscopy. The preservation of biological activities of this chimera toxin was also analyzed. As a first, the results show that LgRec1 does not require plasma components to bind to platelets, although these components are necessary to LgRec1 to induce platelet aggregation. Also, the attachment of LgRec1 to human platelets’ cell membranes suggests that the exposure of phosphatidylserine (PS) may act as a scaffold for coagulation factors. Therefore, the results add new information about the binding of Loxosceles PLDs to platelets, which may help unravel how these toxins promote platelet aggregation.

ACS Style

Daniel A. Fukuda; Maria C. Caporrino; Katia C. Barbaro; Maisa S. Della-Casa; Eliana L. Faquim-Mauro; Geraldo S. Magalhaes. Recombinant Phospholipase D from Loxosceles gaucho Binds to Platelets and Promotes Phosphatidylserine Exposure. Toxins 2017, 9, 191 .

AMA Style

Daniel A. Fukuda, Maria C. Caporrino, Katia C. Barbaro, Maisa S. Della-Casa, Eliana L. Faquim-Mauro, Geraldo S. Magalhaes. Recombinant Phospholipase D from Loxosceles gaucho Binds to Platelets and Promotes Phosphatidylserine Exposure. Toxins. 2017; 9 (6):191.

Chicago/Turabian Style

Daniel A. Fukuda; Maria C. Caporrino; Katia C. Barbaro; Maisa S. Della-Casa; Eliana L. Faquim-Mauro; Geraldo S. Magalhaes. 2017. "Recombinant Phospholipase D from Loxosceles gaucho Binds to Platelets and Promotes Phosphatidylserine Exposure." Toxins 9, no. 6: 191.

Journal article
Published: 27 February 2017 in Toxins
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Many animal toxins may target the same molecules that need to be controlled in certain pathologies; therefore, some toxins have led to the formulation of drugs that are presently used, and many other drugs are still under development. Nevertheless, collecting sufficient toxins from the original source might be a limiting factor in studying their biological activities. Thus, molecular biology techniques have been applied in order to obtain large amounts of recombinant toxins into Escherichia coli. However, most animal toxins are difficult to express in this system, which results in insoluble, misfolded, or unstable proteins. To solve these issues, toxins have been fused with tags that may improve protein expression, solubility, and stability. Among these tags, the SUMO (small ubiquitin-related modifier) has been shown to be very efficient and can be removed by the Ulp1 protease. However, removing SUMO is a labor- and time-consuming process. To enhance this system, here we show the construction of a bicistronic vector that allows the expression of any protein fused to both the SUMO and Ulp1 protease. In this way, after expression, Ulp1 is able to cleave SUMO and leave the protein interest-free and ready for purification. This strategy was validated through the expression of a new phospholipase D from the spider Loxosceles gaucho and a disintegrin from the Bothrops insularis snake. Both recombinant toxins showed good yield and preserved biological activities, indicating that the bicistronic vector may be a viable method to produce proteins that are difficult to express.

ACS Style

Lhiri H. A. L. Shimokawa-Falcão; Maria C. Caporrino; Katia C. Barbaro; Maisa S. Della-Casa; Geraldo S. Magalhães. Toxin Fused with SUMO Tag: A New Expression Vector Strategy to Obtain Recombinant Venom Toxins with Easy Tag Removal inside the Bacteria. Toxins 2017, 9, 82 .

AMA Style

Lhiri H. A. L. Shimokawa-Falcão, Maria C. Caporrino, Katia C. Barbaro, Maisa S. Della-Casa, Geraldo S. Magalhães. Toxin Fused with SUMO Tag: A New Expression Vector Strategy to Obtain Recombinant Venom Toxins with Easy Tag Removal inside the Bacteria. Toxins. 2017; 9 (3):82.

Chicago/Turabian Style

Lhiri H. A. L. Shimokawa-Falcão; Maria C. Caporrino; Katia C. Barbaro; Maisa S. Della-Casa; Geraldo S. Magalhães. 2017. "Toxin Fused with SUMO Tag: A New Expression Vector Strategy to Obtain Recombinant Venom Toxins with Easy Tag Removal inside the Bacteria." Toxins 9, no. 3: 82.

Journal article
Published: 01 September 2015 in Toxicon
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Disintegrins are cysteine-rich toxins containing the RGD motif exposed in a loop that binds integrins such as αIIbβ3, α5β1 and αvβ3. The flexibility of the RGD loop, controlled by the profile of the cysteine pairs and the residues flanking the RGD sequence, are key structural features for the functional activity of these molecules. Recently, our group reported a transcript in the venom gland of Bothrops neuwiedi corresponding to a new P-II SVMP precursor, BnMPIIx, in which the RGD-binding loop includes many substituted residues and unique cysteine residues at the C-terminal. In this paper, we obtained the recombinant disintegrin domain of BnMPIIx, Neuwiedin, which inhibited ADP-induced platelet aggregation, endothelial cell adhesion to fibrinogen and tube formation in Matrigel with no particular selectivity to αIIbβ3 or endothelial cell integrins. This value was also comparable to the inhibition observed with other recombinant disintegrins with conserved cysteine positions and residues in RGD loop. In this regard, Neuwiedin is an important component to understand the functional relevance of the diversity generated by accelerated evolution of venom toxins as well as to find out eventual new disintegrin-dependent targets that may be approached with disintegrins.

ACS Style

I. Lima-Dos-Santos; M.S. Della-Casa; J.A. Portes-Junior; P.A.L. Calabria; G.S. Magalhães; A.M. Moura-Da-Silva. Characterization of Neuwiedin, a new disintegrin from Bothrops neuwiedi venom gland with distinct cysteine pattern. Toxicon 2015, 104, 57 -64.

AMA Style

I. Lima-Dos-Santos, M.S. Della-Casa, J.A. Portes-Junior, P.A.L. Calabria, G.S. Magalhães, A.M. Moura-Da-Silva. Characterization of Neuwiedin, a new disintegrin from Bothrops neuwiedi venom gland with distinct cysteine pattern. Toxicon. 2015; 104 ():57-64.

Chicago/Turabian Style

I. Lima-Dos-Santos; M.S. Della-Casa; J.A. Portes-Junior; P.A.L. Calabria; G.S. Magalhães; A.M. Moura-Da-Silva. 2015. "Characterization of Neuwiedin, a new disintegrin from Bothrops neuwiedi venom gland with distinct cysteine pattern." Toxicon 104, no. : 57-64.

Journal article
Published: 01 September 2014 in Toxicon
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BaP1 is a P-I class snake venom metalloproteinase (SVMP) relevant in the local tissue damage associated with envenomings by Bothrops asper, a medically important snake species in Central America and parts of South and North America. The main treatment for these accidents is the passive immunotherapy using antibodies raised in horses. In order to obtain more specific and batch-to-batch consistent antivenons, recombinant antibodies are considered a good option compared to animal immunization. We constructed a recombinant single chain variable fragment (scFv) from a monoclonal antibody against BaP1 (MABaP1) formerly secreted by a hybridoma clone. This recombinant antibody was cloned into pMST3 vector in fusion with SUMO protein and contains VH and VL domains linked by a flexible (G4S)3 polypeptide (scFvBaP1). The aim of this work was to produce scFvBaP1 and to evaluate its potential concerning the neutralization of biologically important activities of BaP1. The cytoplasmic expression of this construct was successfully achieved in C43 (DE3) bacteria. Our results showed that scFvBaP1-SUMO fusion protein presented an electrophoretic band of around 43 kDa from which SUMO alone corresponded to 13.6 kDa, and only the scFv was able to recognize BaP1 as well as the whole venom by ELISA. In contrast, neither an irrelevant scFv anti-LDL nor its MoAb partner recognized it. BaP1-induced fibrinolysis was significantly neutralized by scFvBaP1, but not by SUMO, in a concentration-dependent manner. In addition, scFvBaP1, as well as MaBaP1, completely neutralized in vivo hemorrhage, muscle necrosis, and inflammation induced by the toxin. Docking analyses revealed possible modes of interaction of the recombinant antibody with BaP1. Our data showed that scFv recognized BaP1 and whole B. asper venom, and neutralized biological effects of this SVMP. This scFv antibody can be used for understanding the molecular mechanisms of neutralization of SVMPs, and for exploring the potential of recombinant antibody fragments for improving the neutralization of local tissue damage in snakebite envenoming.Fundação de Amparo à Pesquisa do Estado de São Paulo/[2012/01028-3]/FAPESP/BrasilFundação de Amparo à Pesquisa do Estado de São Paulo/[PIPE/FAPESP 2004/08297-3]/FAPESP/BrasilConselho Nacional de Desenvolvimento Científico e Tecnológico//CNPq/BrasilUniversidad de Costa Rica//UCR/Costa RicaUCR::Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

ACS Style

J.M.A. Castro; T.S. Oliveira; Caio Silveira; M.C. Caporrino; Dunia Rodriguez; A.M. Moura-Da-Silva; O.H.P. Ramos; A. Rucavado; J.M. Gutiérrez; G.S. Magalhães; E.L. Faquim-Mauro; I. Fernandes. A neutralizing recombinant single chain antibody, scFv, against BaP1, A P-I hemorrhagic metalloproteinase from Bothrops asper snake venom. Toxicon 2014, 87, 81 -91.

AMA Style

J.M.A. Castro, T.S. Oliveira, Caio Silveira, M.C. Caporrino, Dunia Rodriguez, A.M. Moura-Da-Silva, O.H.P. Ramos, A. Rucavado, J.M. Gutiérrez, G.S. Magalhães, E.L. Faquim-Mauro, I. Fernandes. A neutralizing recombinant single chain antibody, scFv, against BaP1, A P-I hemorrhagic metalloproteinase from Bothrops asper snake venom. Toxicon. 2014; 87 ():81-91.

Chicago/Turabian Style

J.M.A. Castro; T.S. Oliveira; Caio Silveira; M.C. Caporrino; Dunia Rodriguez; A.M. Moura-Da-Silva; O.H.P. Ramos; A. Rucavado; J.M. Gutiérrez; G.S. Magalhães; E.L. Faquim-Mauro; I. Fernandes. 2014. "A neutralizing recombinant single chain antibody, scFv, against BaP1, A P-I hemorrhagic metalloproteinase from Bothrops asper snake venom." Toxicon 87, no. : 81-91.

Journal article
Published: 01 September 2013 in Biochimie
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Loxosceles venom comprises a mixture of diverse toxins that induces intense local inflammatory reaction, dermonecrotic injury, platelet aggregation, hemolytic anemia and acute renal failure. Among several toxins in the venom, phospholipases D (PLDs), also called dermonecrotic toxins, are the most important and best studied, since they account for the main effects observed in loxoscelism. Despite their importance, biological analysis of PLDs is hampered by the minute amounts normally purified from the venom, and therefore many efforts have been made to clone those toxins. However, to date, no PLD from Loxosceles gaucho has been obtained in a heterologous system. Thus, in this work we show the cloning of a PLD from L. gaucho venom gland, named LgRec1, which was successfully expressed in a bacterial system. LgRec1 evoked local reaction (edema, erythema, ecchymosis, and paleness), dermonecrosis and hemolysis. It was also able to hydrolyze sphingomyelin and promote platelet aggregation. ELISA and Western blot analysis showed that LgRec1 was recognized by an anti-L. gaucho venom serum, a commercial arachnidic antivenom as well as a monoclonal antibody raised against the dermonecrotic fraction of L. gaucho venom. In addition, LgRec1 demonstrated to be highly immunogenic and antibodies raised against this recombinant toxin inhibited local reaction (~65%) and dermonecrosis (~100%) elicited by L. gaucho whole venom. Since PLDs are considered the major components accounting for the local and systemic envenomation effects caused by spiders from genus Loxosceles, the information provided here may help to understand the mechanisms behind clinical symptomatology.

ACS Style

Geraldo S. Magalhães; Maria C. Caporrino; Maisa S. Della-Casa; Louise Kimura; José Pedro Prezotto Neto; Daniel A. Fukuda; José A. Portes-Junior; Ana Gisele Da Costa Neves Ferreira; Marcelo L. Santoro; Katia C. Barbaro. Cloning, expression and characterization of a phospholipase D from Loxosceles gaucho venom gland. Biochimie 2013, 95, 1773 -1783.

AMA Style

Geraldo S. Magalhães, Maria C. Caporrino, Maisa S. Della-Casa, Louise Kimura, José Pedro Prezotto Neto, Daniel A. Fukuda, José A. Portes-Junior, Ana Gisele Da Costa Neves Ferreira, Marcelo L. Santoro, Katia C. Barbaro. Cloning, expression and characterization of a phospholipase D from Loxosceles gaucho venom gland. Biochimie. 2013; 95 (9):1773-1783.

Chicago/Turabian Style

Geraldo S. Magalhães; Maria C. Caporrino; Maisa S. Della-Casa; Louise Kimura; José Pedro Prezotto Neto; Daniel A. Fukuda; José A. Portes-Junior; Ana Gisele Da Costa Neves Ferreira; Marcelo L. Santoro; Katia C. Barbaro. 2013. "Cloning, expression and characterization of a phospholipase D from Loxosceles gaucho venom gland." Biochimie 95, no. 9: 1773-1783.

Journal article
Published: 01 November 2012 in Toxicon
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Snake venom metalloproteinases (SVMP) are abundant toxins in venoms of viper snakes and play a relevant role in the complex and multifactorial tissue damage characteristic of Viperidae envenoming. Jararhagin, a SVMP isolated from Bothrops jararaca venom, induces a fast onset hemorrhagic lesions acting directly on the capillary vessels, which are disrupted by toxin adhesion and degradation of extracellular matrix proteins like collagen IV. Jararhagin also triggers inflammatory response, where endothelial cells are activated, resulting in the enhanced rolling of circulating leukocytes, nitric oxide generation, prostacyclin production and pro-inflammatory cytokines release. Jararhagin also decreases endothelial cells viability inducing apoptosis (in vitro studies). In the present study we attempted to correlate the effect of sub-apoptotic doses of jararhagin on human umbilical vein endothelial cells (HUVECs) and gene expression of pro-inflammatory mediators, using microarray assay, real time PCR and detection of specific proteins on HUVEC surface or released in the medium. Jararhagin was effective in activate and up-regulate the gene expression of different mediators such as E-selectin, VCAM-1, IL-8, CD69, Ang-2 and MMP-10. Despite the increase in expression of genes coding for such molecules, jararhagin did not induce increased concentrations of E-selectin, VCAM-1 and IL-8 produced or released by endothelial cells. In conclusion, jararhagin is able to activate pro-inflammatory gene transcription on endothelial cells however this stimulus is not sufficient to result in the consequent expression of pro-inflammatory effectors molecules like E-selectin, VCAM-1 and IL-8. The time courses of these events, as well as the doses of jararhagin are important points to be addressed herein

ACS Style

Daiana S. Lopes; Eliana Faquim-Mauro; Geraldo S. Magalhães; Iara C. Lima; Cristiani Baldo; Jay W. Fox; Ana Maria Moura-Da-Silva; Patricia B. Clissa. Gene expression of inflammatory mediators induced by jararhagin on endothelial cells. Toxicon 2012, 60, 1072 -1084.

AMA Style

Daiana S. Lopes, Eliana Faquim-Mauro, Geraldo S. Magalhães, Iara C. Lima, Cristiani Baldo, Jay W. Fox, Ana Maria Moura-Da-Silva, Patricia B. Clissa. Gene expression of inflammatory mediators induced by jararhagin on endothelial cells. Toxicon. 2012; 60 (6):1072-1084.

Chicago/Turabian Style

Daiana S. Lopes; Eliana Faquim-Mauro; Geraldo S. Magalhães; Iara C. Lima; Cristiani Baldo; Jay W. Fox; Ana Maria Moura-Da-Silva; Patricia B. Clissa. 2012. "Gene expression of inflammatory mediators induced by jararhagin on endothelial cells." Toxicon 60, no. 6: 1072-1084.

Journal article
Published: 01 January 2011 in BMC Genetics
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Snake venom metalloproteinases (SVMPs) are widely distributed in snake venoms and are versatile toxins, targeting many important elements involved in hemostasis, such as basement membrane proteins, clotting proteins, platelets, endothelial and inflammatory cells. The functional diversity of SVMPs is in part due to the structural organization of different combinations of catalytic, disintegrin, disintegrin-like and cysteine-rich domains, which categorizes SVMPs in 3 classes of precursor molecules (PI, PII and PIII) further divided in 11 subclasses, 6 of them belonging to PII group. This heterogeneity is currently correlated to genetic accelerated evolution and post-translational modifications.

ACS Style

Ana M Moura-Da-Silva; Maria Stella Furlan; Maria Cristina Caporrino; Kathleen F Grego; José Antonio Portes-Junior; Patrícia B Clissa; Richard H Valente; Geraldo S Magalhães. Diversity of metalloproteinases in Bothrops neuwiedi snake venom transcripts: evidences for recombination between different classes of SVMPs. BMC Genetics 2011, 12, 94 -94.

AMA Style

Ana M Moura-Da-Silva, Maria Stella Furlan, Maria Cristina Caporrino, Kathleen F Grego, José Antonio Portes-Junior, Patrícia B Clissa, Richard H Valente, Geraldo S Magalhães. Diversity of metalloproteinases in Bothrops neuwiedi snake venom transcripts: evidences for recombination between different classes of SVMPs. BMC Genetics. 2011; 12 (1):94-94.

Chicago/Turabian Style

Ana M Moura-Da-Silva; Maria Stella Furlan; Maria Cristina Caporrino; Kathleen F Grego; José Antonio Portes-Junior; Patrícia B Clissa; Richard H Valente; Geraldo S Magalhães. 2011. "Diversity of metalloproteinases in Bothrops neuwiedi snake venom transcripts: evidences for recombination between different classes of SVMPs." BMC Genetics 12, no. 1: 94-94.

Journal article
Published: 01 January 2011 in Toxicon
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Insularin (INS) was obtained from Bothrops insularis venom by reversed-phase high-performance liquid chromatography using a C(18) column and characterized as a disintegrin by peptide mass fingerprint and inhibition of ADP-induced platelet aggregation. A cDNA coding for P-II a metalloproteinase/disintegrin was cloned from a cDNA library from B. insularis venom glands. The deduced protein sequence possesses 73 amino acid residues, including the N-terminal, internal peptides of native insularin, the ARGDNP-sequence and 12 cysteines in a conserved alignment. This cDNA fragment was subcloned in the pGEX-4T-1 vector and expressed in a prokaryotic expression system as a fusion protein with glutathione S-transferase (GST-INS). Both native and recombinant insularin inhibited ADP-induced platelet aggregation and endothelial cells (HUVEC) adhesion with similar activities indicating that GST-INS folded correctly and preserved the integrin-binding loop. Insularin may be a tool in studies that involve platelets and endothelial cell adhesion dependent on alphaIIbeta3 and alphavbeta3 integrins.

ACS Style

Maisa Splendore Della-Casa; Inacio Junqueira-De-Azevedo; Diego Butera; Patricia Clissa; Daiana Lopes; Solange M T Serrano; Daniel Pimenta; Geraldo S. Magalhães; Paulo Lee Ho; Ana Maria Moura-Da-Silva. “Insularin, a disintegrin from Bothrops insularis venom: Inhibition of platelet aggregation and endothelial cell adhesion by the native and recombinant GST-insularin proteins”. Toxicon 2011, 57, 125 -133.

AMA Style

Maisa Splendore Della-Casa, Inacio Junqueira-De-Azevedo, Diego Butera, Patricia Clissa, Daiana Lopes, Solange M T Serrano, Daniel Pimenta, Geraldo S. Magalhães, Paulo Lee Ho, Ana Maria Moura-Da-Silva. “Insularin, a disintegrin from Bothrops insularis venom: Inhibition of platelet aggregation and endothelial cell adhesion by the native and recombinant GST-insularin proteins”. Toxicon. 2011; 57 (1):125-133.

Chicago/Turabian Style

Maisa Splendore Della-Casa; Inacio Junqueira-De-Azevedo; Diego Butera; Patricia Clissa; Daiana Lopes; Solange M T Serrano; Daniel Pimenta; Geraldo S. Magalhães; Paulo Lee Ho; Ana Maria Moura-Da-Silva. 2011. "“Insularin, a disintegrin from Bothrops insularis venom: Inhibition of platelet aggregation and endothelial cell adhesion by the native and recombinant GST-insularin proteins”." Toxicon 57, no. 1: 125-133.

Journal article
Published: 24 June 2010 in Molecular Biotechnology
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Deficiency of the lysosomal glucocerebrosidase (GCR) enzyme results in Gaucher's disease, the most common inherited storage disorder. Treatment consists of enzyme replacement therapy by the administration of recombinant GCR produced in Chinese hamster ovary cells. The production of anti-GCR antibodies has already been described with placenta-derived human GCR that requires successive chromatographic procedures. Here, we report a practical and efficient method to obtain anti-GCR polyclonal antibodies against recombinant GCR produced in Escherichia coli and further purified by a single step through nickel affinity chromatography. The purified GCR was used to immunize BALB/c mice and the induction of anti-GCR antibodies was evaluated by enzyme-linked immunosorbent assay. The specificity of the antiserum was also evaluated by western blot analysis against recombinant GCR produced by COS-7 cells or against endogenous GCR of human cell lines. GCR was strongly recognized by the produced antibodies, either as cell-associated or as secreted forms. The detected molecular masses of 59-66 kDa are in accordance to the expected size for glycosylated GCR. The GCR produced in E. coli would facilitate the production of polyclonal (shown here) and monoclonal antibodies and their use in the characterization of new biosimilar recombinant GCRs coming in the near future.

ACS Style

Juliana Branco Novo; Maria Leonor Sarno Oliveira; Geraldo Santana Magalhães; Ligia Morganti; Isaias Raw; Paulo Lee Ho. Generation of Polyclonal Antibodies Against Recombinant Human Glucocerebrosidase Produced in Escherichia coli. Molecular Biotechnology 2010, 46, 279 -286.

AMA Style

Juliana Branco Novo, Maria Leonor Sarno Oliveira, Geraldo Santana Magalhães, Ligia Morganti, Isaias Raw, Paulo Lee Ho. Generation of Polyclonal Antibodies Against Recombinant Human Glucocerebrosidase Produced in Escherichia coli. Molecular Biotechnology. 2010; 46 (3):279-286.

Chicago/Turabian Style

Juliana Branco Novo; Maria Leonor Sarno Oliveira; Geraldo Santana Magalhães; Ligia Morganti; Isaias Raw; Paulo Lee Ho. 2010. "Generation of Polyclonal Antibodies Against Recombinant Human Glucocerebrosidase Produced in Escherichia coli." Molecular Biotechnology 46, no. 3: 279-286.

Journal article
Published: 01 June 2010 in Toxicon
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SVMPs are multi-domain proteolytic enzymes in which disintegrin-like and cysteine-rich domains bind to cell receptors, plasma or ECM proteins. We have recently reported that jararhagin, a P-III class SVMP, binds to collagen with high affinity through an epitope located within the Da-disintegrin sub-domain. In this study, we evaluated the binding of jararhagin to alpha(2)beta(1) integrin (collagen receptor) using monoclonal antibodies and recombinant jararhagin fragments. In solid phase assays, binding of jararhagin to alpha(2)beta(1) integrin was detectable from concentrations of 20 nM. Using recombinant fragments of jararhagin, only fragment JC76 (residues 344-421), showed a significant binding to recombinant alpha(2)beta(1) integrin. The anti-jararhagin monoclonal antibody MAJar 3 efficiently neutralised binding of jararhagin to collagen, but not to recombinant alpha(2)beta(1) integrin nor to cell-surface-exposed alpha(2)beta(1) integrin (alpha(2)-K562 transfected cells and platelets). The same antibody neutralised collagen-induced platelet aggregation. Our data suggest that jararhagin binding to collagen and alpha(2)beta(1) integrin occurs by two independent motifs, which are located on disintegrin-like and cysteine-rich domains, respectively. Moreover, toxin binding to collagen appears to be sufficient to inhibit collagen-induced platelet aggregation.

ACS Style

Isabelle Tanjoni; Karla Evangelista; Maisa S. Della-Casa; Diego Butera; Geraldo S. Magalhães; Cristiani Baldo; Patrícia B. Clissa; Irene Fernandes; Johannes Eble; Ana M. Moura-Da-Silva. Different regions of the class P-III snake venom metalloproteinase jararhagin are involved in binding to α2β1 integrin and collagen. Toxicon 2010, 55, 1093 -1099.

AMA Style

Isabelle Tanjoni, Karla Evangelista, Maisa S. Della-Casa, Diego Butera, Geraldo S. Magalhães, Cristiani Baldo, Patrícia B. Clissa, Irene Fernandes, Johannes Eble, Ana M. Moura-Da-Silva. Different regions of the class P-III snake venom metalloproteinase jararhagin are involved in binding to α2β1 integrin and collagen. Toxicon. 2010; 55 (6):1093-1099.

Chicago/Turabian Style

Isabelle Tanjoni; Karla Evangelista; Maisa S. Della-Casa; Diego Butera; Geraldo S. Magalhães; Cristiani Baldo; Patrícia B. Clissa; Irene Fernandes; Johannes Eble; Ana M. Moura-Da-Silva. 2010. "Different regions of the class P-III snake venom metalloproteinase jararhagin are involved in binding to α2β1 integrin and collagen." Toxicon 55, no. 6: 1093-1099.