Abstract Background Cathepsin D (CatD) is a lysosomal proteolytic enzyme expressed in almost all tissues and organs. This protease is a multifunctional enzyme responsible for essential biological processes such as cell cycle regulation, differentiation, migration, tissue remodeling, neuronal growth, ovulation, and apoptosis. The overexpression and hypersecretion of CatD have been correlated with cancer aggressiveness and tumor progression, stimulating cancer cell proliferation, fibroblast growth, and angiogenesis. In addition, some studies report its participation in neurodegenerative diseases and inflammatory processes. In this regard, the search for new inhibitors from natural products could be an alternative against the harmful effects of this enzyme. Methods An investigation was carried out to analyze CatD interaction with snake venom toxins in an attempt to find inhibitory molecules. Interestingly, human CatD shows the ability to bind strongly to snake venom phospholipases A2 (svPLA2), forming a stable muti-enzymatic complex that maintains the catalytic activity of both CatD and PLA2. In addition, this complex remains active even under exposure to the specific inhibitor pepstatin A. Furthermore, the complex formation between CatD and svPLA2 was evidenced by surface plasmon resonance (SPR), two-dimensional electrophoresis, enzymatic assays, and extensive molecular docking and dynamics techniques. Conclusion The present study suggests the versatility of human CatD and svPLA2, showing that these enzymes can form a fully functional new enzymatic complex.

Resumo: O objetivo deste trabalho foi avaliar a atividade antibacteriana in vitro de venenos e toxinas purificadas de serpentes sobre a bactéria fitopatogênica Ralstonia solanacearum. As avaliações foram realizadas em 17 venenos brutos (13 de Bothrops, 3 de Crotalus e 1 de Lachesis) e sete toxinas (1 de Bothrops e 6 de Crotalus). A atividade antibacteriana foi avaliada em meio MB1 que continha os tratamentos solubilizados (1 μL mL-1). Utilizou-se o total de 100 μL de suspensão bacteriana (8,4 x 109 UFC mL-1). Após incubação a 28°C, avaliou-se o número de colônias bacterianas às 24, 48 e 72 horas após a inoculação. O gel SDS-PAGE a 15% foi usado para analisar o perfil proteico das amostras, tendo-se utilizado 5 μg de proteína no ensaio. Além disso, os valores de concentração inibitória mínima (CIM) e concentração letal (CL50) foram determinados pelo método Probit. Os venenos e as toxinas foram capazes de reduzir mais de 90% do crescimento de R. solanacearum. Esses resultados foram ou equivalentes aos do controle positivo cloranfenicol ou até melhores. Enquanto os valores de CIM variaram de 4,0 a 271,5 μg mL-1, a CL50 variou de 28,5 μg mL-1 a 4,38 mg mL-1. Dez venenos brutos (7 de Bothrops e 3 de Crotalus) e duas toxinas (giroxina e crotamina) são abordagens promissoras para o controle da bactéria fitopatogênica R. solanacearum.

Abstract: The objective of this work was to evaluate the in vitro antibacterial activity of snake venoms and purified toxins on the phytopathogenic bacterium Ralstonia solanacearum. The evaluations were performed with 17 crude venoms (13 from Bothrops, 3 from Crotalus, and 1 from Lachesis) and seven toxins (1 from Bothrops and 6 from Crotalus). Antibacterial activity was assessed in MB1 medium containing solubilized treatments (1 μL mL-1). A total of 100 μL bacterial suspension (8.4 x 109 CFU mL-1) was used. After incubation at 28°C, the number of bacterial colonies at 24, 48, and 72 hours after inoculation was evaluated. SDS-PAGE gel at 15% was used to analyze the protein patterns of the samples, using 5 μg protein of each sample in the assay. Furthermore, the minimum inhibitory concentration (MIC) and lethal concentration (LC50) values were determined by the Probit method. Venoms and toxins were able to reduce more than 90% of R. solanacearum growth. These results were either equivalent to those of the positive control chloramphenicol or even better. While MIC values ranged from 4.0 to 271.5 μg mL-1, LC50 ranged from 28.5 μg mL-1 to 4.38 mg mL-1. Ten crude venoms (7 from Bothrops and 3 from Crotalus) and two purified toxins (gyroxin and crotamine) are promising approaches to control the phytopathogenic bacterium R. solanacearum.

Abstract INTRODUCTION: Crotalus envenomations cause serious complications and can be fatal without appropriate treatment. Venom isoforms present and inter/intraspecific variations in the venom composition can result in different symptoms presented by bites by snakes from the same species but from different geographical regions. We comparatively evaluated the local and systemic effects caused by Crotalus durissus terrificus (Cdt), C.d. collilineatus (Cdcolli), and C.d. cascavella (Cdcasc) envenomation. METHODS: Venom chromatography was performed. Proteolytic, phospholipase, and LAAO activities were analyzed. Edema, myotoxicity, hepatotoxicity, nephrotoxicity, and coagulation alterations were evaluated. RESULTS: The venom SDS-PAGE analyses found the presence of convulxin, gyroxin, crotoxin, and crotamine in Cdt and Cdcolli venoms. Crotamine was not present in the Cdcasc venom. Cdt, Cdcollli, and Cdcasc venoms had no proteolytic activity. Only Cdcasc and Cdt venoms had phospholipase activity. LAAO activity was observed in Cdcolli and Cdcasc venoms. Cdcolli and Cdcasc venoms caused 36.7% and 13.3% edema increases, respectively. Cdt venom caused a 10% edema induction compared to those by other venoms. All venoms increased TOTAL-CK, MB-CK, and LDH levels (indicating muscle injury) and ALT, AST, GGT, and ALP levels (markers of liver damage) and were able to induce a neuromuscular blockade. Urea and creatinine levels were also altered in both plasma and urine, indicating kidney damage. Only Cdcolli and Cdcasc venoms increased TAPP and TAP. CONCLUSIONS: Together, these results allow us to draw a distinction between local and systemic effects caused by Crotalus subspecies, highlighting the clinical and biochemical effects produced by their respective venoms.

Abstract INTRODUCTION Brazil has the largest number of snakebite cases in South America, of which the large majority is concentrated in the Midwest and North. METHODS In this descriptive observational study, we assessed the epidemiological and clinical snakebite cases referred to the Centro de Medicina Tropical de Rondônia from September 2008 to September 2010. RESULTS We followed up 92 cases from admission until discharge, namely 81 (88%) men and 11 (12%) women, with a mean age of 37 years, and mainly from rural areas (91.3%). The snakebites occurred while performing work activities (63%) during the Amazon rainy season (78.3%). The vast majority of individuals presented from the Porto Velho microregion (84.7%). Approximately 95.6% of the snakebites were caused by snakes of the genus Bothrops, followed by two lachetics and two elapidics cases. Surgery was performed in 10 cases (9 fasciotomies in the lower limb and 1 amputation). No deaths were reported in this study, but 4 cases (4.3%) developed sequelae in the lower limb. CONCLUSIONS This study can contribute to a better understanding of envenomation in the state of Rondônia and thus can be useful for identifying real conditions that can increase the incidence of snakebites in this region. Moreover, the study results can serve as a basis for improving educational campaigns designed to prevent these types of snakebites, as well as for preserving snakes.

Abstract Background: Snake venoms are a complex mixture of proteins, organic and inorganic compounds. Some of these proteins, enzymatic or non-enzymatic ones, are able to interact with platelet receptors, causing hemostatic disorders. The possible therapeutic potential of toxins with antiplatelet properties may arouse interest in the pharmacological areas. The present study aimed to purify and characterize an antiplatelet DC protein from Bothrops alternatus snake venom. Methods: The protein, called BaltDC (DC protein from B. alternatus snake venom), was purified by a combination of ion-exchange chromatography on DEAE-Sephacel column and gel filtration on Sephadex G-75. The molecular mass was estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). The amino acid sequence of the N-terminal region was carried out by Edman degradation method. Platelet aggregation assays were performed in human platelet-rich plasma (PRP). Infrared (IR) spectroscopy was used in order to elucidate the interactions between BaltDC and platelet membrane. Results: BaltDC ran as a single protein band on SDS-PAGE and showed apparent molecular mass of 32 kDa under reducing or non-reducing conditions. The N-terminal region of the purified protein revealed the amino acid sequence IISPPVCGNELLEVGEECDCGTPENCQNECCDA, which showed identity with other snake venom metalloproteinases (SVMPs). BaltDC was devoid of proteolytic, hemorrhagic, defibrinating or coagulant activities, but it showed a specific inhibitory effect on platelet aggregation induced by ristocetin and epinephrine in PRP. IR analysis spectra strongly suggests that PO 3 2 − groups, present in BaltDC, form hydrogen bonds with the PO 2 − groups present in the non-lipid portion of the membrane platelets. Conclusions: BaltDC may be of medical interest since it was able to inhibit platelet aggregation.

Plant-antivenom is a computational Websystem about medicinal plants with anti-venom properties. The system consists of a database of these plants, including scientific publications on this subject and amino acid sequences of active principles from venomous animals. The system relates these data allowing their integration through different search applications. For the development of the system, the first surveys were conducted in scientific literature, allowing the creation of a publication database in a library for reading and user interaction. Then, classes of categories were created, allowing the use of tags and the organization of content. This database on medicinal plants has information such as family, species, isolated compounds, activity, inhibited animal venoms, among others. Provision is made for submission of new information by registered users, by the use of wiki tools. Content submitted is released in accordance to permission rules defined by the system. The database on biological venom protein amino acid sequences was structured from the essential information from National Center for Biotechnology Information (NCBI). Plant-antivenom's interface is simple, contributing to a fast and functional access to the system and the integration of different data registered on it. Plant-antivenom system is available on the Internet at http://gbi.fmrp.usp.br/plantantivenom.

Mandevilla velutina (Mart.) Woodson (Apocynaceae) é uma planta medicinal de espécie nativa de regiões do Cerrado brasileiro com propriedades anti-ofídicas. Devido a alta exploração e devastação deste bioma, M. velutina está em perigo de extinção. Como uma iniciativa para a conservação desta espécie ameaçada e economicamente importante, um protocolo de micropropagação foi desenvolvido e genótipos foram colocados no Banco de Germoplasma "Cerrado In vitro". Para a propagação in vitro de M. velutina, segmentos nodais foram inoculados no meio Murashige and Skoog (MS) suplementado com diferentes concentrações de BA, zeatina, 2ip, DTT e TDZ. A melhor resposta de multiplicação foi obtida em meio suplementado com 0.44 µM BA na razão 1: 6.7. Plântulas cultivadas em MS/2 suplementado com 26.85 µM NAA enraizaram com sucesso (50.5%). Quando plântulas enraizadas e não enraizadas foram aclimatizadas em condições de solo, grande perda foi observada entre plântulas não enraizadas, enquanto as enraizadas tiveram 100% de sobrevivência. Foi possível a manutenção de 43% de M. velutina em condições saudáveis por seis meses, sem subculturas usando meio MS suplementado com 2% de sacarose, 13.8 mM de espermidina, 2% de sorbitol e 2% de dextrose.

Mandevilla velutina (Mart.) Woodson (Apocynaceae) is a medicinal plant species with antivenom properties, native from Brazilian Savanna regions (Cerrado), which due to overexploitation and habitat deforestation is in danger of extinction. As an initiative for conserving this endangered but economically important plant species, a micropropagation protocol was developed and genotypes were stored in the Germplasm Bank "Cerrado In vitro". For the in vitro propagation of M. velutina, nodal segments were inoculated on Murashige and Skoog (MS) medium supplemented with different concentrations of BA, Zeatin, 2ip, DTT and TDZ. Best multiplication ratio was achieved when to the medium 0.44 µM BA, ranging 1: 6.7, were added. Plantlets cultured on MS/2 medium supplemented with 26.85 µM NAA rooted successfully (50.5%). Although rooted and un-rooted plantlets acclimatized to soil conditions, great losses were observed within un-rooted plantlets, while the rooted presented 100 % survival. It was possible to maintain 43% of the M. velutina germplasm under healthy conditions for six months, with no subcultures, using the MS medium supplemented with 2% sucrose, 13.8 mM spermidine, 2% sorbitol and 2% dextrose.