General biochemical and immunological characteristics of the venom from Peruvian scorpion Hadruroides lunatus

This communication describes the general biochemical properties and some immunological characteristics of the venom from the Peruvian scorpion Hadruroides lunatus, which is the most medically relevant species in Peru. The soluble venom of this scorpion is toxic to mice, the LD50 determined was 0.1 mg/kg and 21.55 mg/kg when the venom was injected intracranial or intraperitoneally, respectively. The soluble venom displayed proteolytic, hyaluronidasic, phospholipasic and cardiotoxic activities. High performance liquid chromatography of the soluble venom resulted in the separation of 20 fractions. Two peptides with phospholipasic activity were isolated to homogeneity and their molecular masses determined by mass spectrometry (MALDI TOF). Anti-H. lunatus venom sera were produced in rabbits. Western blotting analysis showed that most of the protein content of this venom is immunogenic. H. lunatus anti-venom displayed consistent cross-reactivity with venom antigens from the new World-scorpions Tityus serrulatus and Centruroides sculpturatus venoms; however, a weaker reactivity was observed against the venom antigens from the old World-scorpion Androctonus australis Hector. (C) 2012 Elsevier Ltd. All rights reserved.

Degradation of [D-Leu]-Microcystin-LR by solar heterogeneous photocatalysis (TiO2)

The present study investigates the use of solar heterogeneous photocatalyis (TiO2) for the destruction of [D-Leu]-Microcystin-LR, powerful toxin of widespread occurrence within cyanobacteria blooms. We extracted [D-Leu]-Microcystin-LR from a culture of Microcystis spp. and used a flat plate glass reactor coated with TiO2 (Degussa, P25) for the degradation studies. The irradiance was measured during the experiments with the aid of a spectroradiometer. After the degradation experiments, toxin concentrations were determined by HPLC and mineralization by TOC analyses. Acute and chronic toxicities were, quantified using mice and phosphatase inhibition in vitro assays, respectively. According to the performed experiments, 150 min were necessary to reduce the toxin concentration to the WHO's guideline for drinking water (from 10 to 1 mu g L-1) and to mineralize 90% of the initial carbon content. Another important finding is that solar heterogeneous photocatalysis was a destructive process indeed, not only for the toxin, but also for the other extract components and degradation products generated. Moreover, toxicity tests using mice have shown that the acute effect caused by the initial sample was removed. However, tests using the phosphatase enzyme indicated that it may be formed products capable of inducing chronic effects on mammals. The performed experiments indicate the feasibility of using solar heterogeneous photocatalysis for treating contaminated water with [D-Leu]-Microcystin-LR, not only due to its destruction, but also to the significant removal of organic matter and acute toxicity that can be achieved. (C) 2012 Elsevier Ltd. All rights reserved.

Combined snake venomics and venom gland transcriptomic analysis of Bothropoides pauloensis

Unraveling the repertoire of venom toxins of Bothropoides pauloensis was assessed by snake venomics and venom gland transcriptomic surveys. Both approaches yielded converging overall figures, pointing to metalloproteinases (similar to 37%), PLA(2)s (26-32%), and vasoactive (bradykinin-potentiating) peptides (12-17%) as the major toxin classes. The high occurrence of SVMPs, PLA(2) molecules, vasoactive peptides, along with serine proteinases, explains the local and systemic effects observed in envenomations by B. pauloensis. Minor (<3%) C-type lectin, serine proteinase, L-amino acid oxidase, nerve growth factor, and CRISP molecules were also identified in the transcriptome and the proteome. Low abundance (0.3%) EST singletons coding for vascular endothelial growth factor (svVEGF), ohanin, hyaluronidase, and 5' nucleotidase were found only in the venom gland cDNA library. At the molecular level, the transcriptomic and proteomic datasets display low compositional concordance. In particular, although there is good agreement between transcriptome and proteome in the identity of BPPs, PLA(2) molecules and L-amino acid oxidase, both datasets strongly depart in their C-type lectin and SVMP complements. These data support the view that venom composition is influenced by transcriptional and translational mechanisms and emphasize the value of combining proteomic and transcriptomic approaches to acquire a more complete understanding of the toxinological profile and natural history of the snake venom. (C) 2012 Elsevier B.V. All rights reserved.

Anti-snake venom activities of extracts and fractions from callus cultures of Sapindus saponaria

Context: Sapindus saponaria L. (Sapindaceae) bark, root, and fruits are used as sedatives and to treat gastric ulcer and also demonstrate diuretic and expectorant effects. Objective: The anti-snake venom properties of callus of S. saponaria are investigated here for the first time. Materials and methods: In vitro cultivated callus of Sapindus saponaria were lyophilized, and the extracts were prepared with different solvents, before submitting to phytochemical studies and evaluation of the anti-ophidian activity. Crude extracts were fractionated by liquid-liquid partition and the fractions were monitored by thin layer chromatography (TLC). Subsequently, anti-ophidian activities were analyzed toward Bothrops jararacussu Lacerda (Viperidae), B. moojeni Hoge (Viperidae), B. alternates Dumeril (Viperidea) and Crotalus durissus terrificus Lineu (Viperidae) venoms and isolated myotoxins and phospholipase A(2) (PLA(2)). Results: Fractions A1, A2 and the extract in MeOH:H2O (9:1) significantly inhibited the toxic and pharmacological activities induced by snake venoms and toxins, when compared to other extracts and fractions. The lethal, clotting, phospholipase, edema-inducing, hemorrhagic and myotoxic activities were partially inhibited by the different extracts and fractions. TLC profiles of the crude extracts (B and C) and fractions (A1 and A2) showed beta-sitosterol and stigmasterol as their main compounds. Stigmasterol exhibited inhibitory effects on enzymatic and myotoxic activities of PLA(2). Discussion and conclusion: Sapindus saponaria extracts and fractions presented anti-ophidian activity and could be used as an adjuvant to serum therapy or for its supplementation, and in addition, as a rich source of potential inhibitors of enzymes involved in several pathophysiological human and animal diseases.

Crotamine Pharmacology Revisited: Novel Insights Based on the Inhibition of K-V Channels

Crotamine, a 5-kDa peptide, possesses a unique biological versatility. Not only has its cell-penetrating activity become of clinical interest but, moreover, its potential selective antitumor activity is of great pharmacological importance. In the past, several studies have attempted to elucidate the exact molecular target responsible for the crotamine-induced skeletal muscle spasm. The aim of this study was to investigate whether crotamine affects voltage-gated potassium (K-V) channels in an effort to explain its in vivo effects. Crotamine was studied on ion channel function using the two-electrode voltage clamp technique on 16 cloned ion channels (12 K-V channels and 4 Na-V channels), expressed in Xenopus laevis oocytes. Crotamine selectively inhibits K-V 1.1, K-V 1.2, and K-V 1.3 channels with an IC50 of similar to 300 nM, and the key amino acids responsible for this molecular interaction are suggested. Our results demonstrate for the first time that the symptoms, which are observed in the typical crotamine syndrome, may result from the inhibition of K-V channels. The ability of crotamine to inhibit the potassium current through K-V channels unravels it as the first snake peptide with the unique multifunctionality of cell-penetrating and antitumoral activity combined with K-V channel-inhibiting properties. This new property of crotamine might explain some experimental observations and opens new perspectives on pharmacological uses.

Identification of novel components of NAD-utilizing metabolic pathways and prediction of their biochemical functions

Nicotinamide adenine dinucleotide (NAD) is a ubiquitous cofactor participating in numerous redox reactions. It is also a substrate for regulatory modifications of proteins and nucleic acids via the addition of ADP-ribose moieties or removal of acyl groups by transfer to ADP-ribose. In this study, we use in-depth sequence, structure and genomic context analysis to uncover new enzymes and substrate-binding proteins in NAD-utilizing metabolic and macromolecular modification systems. We predict that Escherichia coli YbiA and related families of domains from diverse bacteria, eukaryotes, large DNA viruses and single strand RNA viruses are previously unrecognized components of NAD-utilizing pathways that probably operate on ADP-ribose derivatives. Using contextual analysis we show that some of these proteins potentially act in RNA repair, where NAD is used to remove 2'-3' cyclic phosphodiester linkages. Likewise, we predict that another family of YbiA-related enzymes is likely to comprise a novel NAD-dependent ADP-ribosylation system for proteins, in conjunction with a previously unrecognized ADP-ribosyltransferase. A similar ADP-ribosyltransferase is also coupled with MACRO or ADP-ribosylglycohydrolase domain proteins in other related systems, suggesting that all these novel systems are likely to comprise pairs of ADP-ribosylation and ribosylglycohydrolase enzymes analogous to the DraG-DraT system, and a novel group of bacterial polymorphic toxins. We present evidence that some of these coupled ADP-ribosyltransferases/ribosylglycohydrolases are likely to regulate certain restriction modification enzymes in bacteria. The ADP-ribosyltransferases found in these, the bacterial polymorphic toxin and host-directed toxin systems of bacteria such as Waddlia also throw light on the evolution of this fold and the origin of eukaryotic polyADP-ribosyltransferases and NEURL4-like ARTs, which might be involved in centrosomal assembly. We also infer a novel biosynthetic pathway that might be involved in the synthesis of a nicotinate-derived compound in conjunction with an asparagine synthetase and AMPylating peptide ligase. We use the data derived from this analysis to understand the origin and early evolutionary trajectories of key NAD-utilizing enzymes and present targets for future biochemical investigations.

Crotoxin and phospholipases A(2) from Crotalus durissus terrificus showed antiviral activity against dengue and yellow fever viruses

Dengue is the most important arbovirus in the world with an estimated of 50 million dengue infections occurring annually and approximately 2.5 billion people living in dengue endemic countries. Yellow fever is a viral hemorrhagic fever with high mortality that is transmitted by mosquitoes. Effective vaccines against yellow fever have been available for almost 70 years and are responsible for a significant reduction of occurrences of the disease worldwide; however, approximately 200,000 cases of yellow fever still occur annually, principally in Africa. Therefore, it is a public health priority to develop antiviral agents for treatment of these virus infections. Crotalus durissus terrificus snake, a South American rattlesnake, presents venom with several biologically actives molecules. In this study, we evaluated the antiviral activity of crude venom and isolated toxins from Crotalus durissus terrificus and found that phospholipases A(2) showed a high inhibition of Yellow fever and dengue viruses in VERO E6 cells. (C) 2011 Elsevier Ltd. All rights reserved.

Serrumab: A human monoclonal antibody that counters the biochemical and immunological effects of Tityus serrulatus venom

In Brazil, the species Tityus serrulatus is responsible for the most severe cases of scorpion envenomation. There is currently a need for new scorpion anti-venoms that are more effective and less harmful. This study attempted to produce human monoclonal antibodies capable of inhibiting the activity of T. serrulatus venom (TsV), using the Griffin.1 library of human single-chain fragment-variable (scFv) phage antibodies. Four rounds of phage antibody selection were performed, and the round with the highest phage antibody titer was chosen for the production of monoclonal phage antibodies and for further analysis. The scFv 2A, designated serrumab, was selected for the production and purification of soluble antibody fragments. In a murine peritoneal macrophage cell line (J774.1), in vitro assays of the cytokines interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, and IL-10 were performed. In male BALB/c mice, in vivo assays of plasma urea, creatinine, aspartate transaminase, and glucose were performed, as well as of neutrophil recruitment and leukocyte counts. It was found that serrumab inhibited the TsV-induced increases in the production of IL-6, TNF alpha, and IL-10 in J774.1 cells. The in vivo inhibition assay showed that serrumab also prevented TsV-induced increases in the plasma levels of urea, creatinine, aspartate transaminase, and glucose, as well as preventing the TsV-induced increase in neutrophil recruitment. The results indicate that the human monoclonal antibody serrumab is a candidate for inclusion in a mixture of specific antibodies to the various toxins present in TsV. Therefore, serrumab shows promise for use in the production of new anti-venom.

Development of a high sensitive automatic setup for screening of microcystins in surface waters by employing a LED-based photometric detector

In this manuscript, an automatic setup for screening of microcystins in surface waters by employing photometric detection is described. Microcystins are toxins delivered by cyanobacteria within an aquatic environment, which have been considered strongly poisonous for humans. For that reason, the World Health Organization (WHO) has proposed a provisional guideline value for drinking water of 1 mu g L-1. In this work, we developed an automated equipment setup, which allows the screening of water for concentration of microcystins below 0.1 mu g V. The photometric method was based on the enzyme-linked immunosorbent assay (ELISA) and the analytical signal was monitored at 458 nm using a homemade LED-based photometer. The proposed system was employed for the detection of microcystins in rivers and lakes waters. Accuracy was assessed by processing samples using a reference method and applying the paired t-test between results. No significant difference at the 95% confidence level was observed. Other useful features including a linear response ranging from 0.05 up to 2.00 mu g L-1 (R-2 =0.999) and a detection limit of 0.03 mu g L-1 microcystins were achieved. (C) 2011 Elsevier B.V. All rights reserved.

Biomonitoring genotoxicity and cytotoxicity of Microcystis aeruginosa (Chroococcales, Cyanobacteria) using the Allium cepa test

Water pollution caused by toxic cyanobacteria is a problem worldwide, increasing with eutrophication. Due to its biological significance, genotoxicity should be a focus for biomonitoring pollution owing to the increasing complexity of the toxicological environment in which organisms are exposed. Cyanobacteria produce a large number of bioactive compounds, most of which lack toxicological data. Microcystins comprise a class of potent cyclic heptapeptide toxins produced mainly by Microcystis aeruginosa. Other natural products can also be synthesized by cyanobacteria, such as the protease inhibitor, aeruginosin. The hepatotoxicity of microcystins has been well documented, but information on the genotoxic effects of aeruginosins is relatively scarce. In this study, the genotoxicity and ecotoxicity of methanolic extracts from two strains of M. aeruginosa NPLJ-4, containing high levels of microcystin, and M. aeruginosa NPCD-1, with high levels of aeruginosin, were evaluated. Four endpoints, using plant assays in Allium cepa were applied: rootlet growth inhibition, chromosomal aberrations, mitotic divisions, and micronucleus assays. The microcystin content of M. aeruginosa NPLJ-4 was confirmed through ELISA, while M. aeruginosa NPCD-1 did not produce microcystins. The extracts of M. aeruginosa NPLJ-4 were diluted at 0.01, 0.1, 1 and 10 ppb of microcystins: the same procedure was used to dilute M. aeruginosa NPCD-1 used as a parameter for comparison, and water was used as the control. The results demonstrated that both strains inhibited root growth and induced rootlet abnormalities. The strain rich in aeruginosin was more genotoxic, altering the cell cycle, while microcystins were more mitogenic. These findings indicate the need for future research on non-microcystin producing cyanobacterial strains. Understanding the genotoxicity of M. aeruginosa extracts can help determine a possible link between contamination by aquatic cyanobacteria and high risk of primary liver cancer found in some areas as well as establish water level limits for compounds not yet studied. (C) 2012 Elsevier B.V. All rights reserved.

Looking over Toxin-K+ Channel Interactions. Clues from the Structural and Functional Characterization of alpha-KTx Toxin Tc32, a Kv1.3 Channel Blocker

alpha-KTx toxin Tc32, from the Amazonian scorpion Tityus cambridgei, lacks the dyad motif; including Lys27, characteristic of the family and generally associated with channel blockage. The toxin has been cloned and expressed for the first time. Electrophysiological experiments, by showing that the recombinant form blocks Kv1.3 channels of olfactory bulb periglomerular cells like the natural Tc32 toxin, when tested on the Kv1.3 channel of human T lymphocytes, confirmed it is in an active fold. The nuclear magnetic resonance-derived structure revealed it exhibits an alpha/beta scaffold typical of the members of the alpha-KTx family. TdK2 and TdK3, all belonging to the same alpha-KTx 18 subfamily, share significant sequence identity with Tc32 but diverse selectivity and affinity for Kv1.3 and Kv1.1 channels. To gain insight into the structural features that may justify those differences, we used the recombinant Tc32 nuclear magnetic resonance-derived structure to model the other two toxins, for which no experimental structure is available. Their interaction with Kv1.3 and Kv1.1 has been investigated by means of docking simulations. The results suggest that differences in the electrostatic features of the toxins and channels, in their contact surfaces, and in their total dipole moment orientations govern the affinity and selectivity of toxins. In addition, we found that, regardless of whether the dyad motif is present, it is always a Lys side chain that physically blocks the channels, irrespective of its position in the toxin sequence.

Colibacillosis in lambs is associated to type I heat-stable enterotoxin in a farm in Sao Paulo State, Brazil

Twenty seven (48.2%) culture supernatants of 56 Escherichia coli isolated from diarrheic lamb feces (7 to 10 days old) in Sao Paulo State, Brazil, presented positive results to suckling mice assay (fluid accumulation) but none caused cytopathic effects on Vero and CHO cells, indicating that these strains did not produced LT or VT toxins. PCR assays showed that these 27 E. coli strains harbored estA, that codifies for STa, but not for stx1, stx2 or cnf genes. The positive STa strains were checked for genes that codify for F41, F17 and K99 fimbriae, wich are considered colonization factors in ETEC. Only F17 was detect in two samples (7.4%). Twelve of 27 STa positive carried hlyA gene and presented hemolytic activity in blood Agar. Presence of rotavirus was not detected among the diarrheic feces. These data suggests that STa must be an important diarrheagenic factor to small ruminants in Sao Paulo State.

Convergent evolution of [D-Leucine1] microcystin-LR in taxonomically disparate cyanobacteria

Abstract Background Many important toxins and antibiotics are produced by non-ribosomal biosynthetic pathways. Microcystins are a chemically diverse family of potent peptide toxins and the end-products of a hybrid NRPS and PKS secondary metabolic pathway. They are produced by a variety of cyanobacteria and are responsible for the poisoning of humans as well as the deaths of wild and domestic animals around the world. The chemical diversity of the microcystin family is attributed to a number of genetic events that have resulted in the diversification of the pathway for microcystin assembly. Results Here, we show that independent evolutionary events affecting the substrate specificity of the microcystin biosynthetic pathway have resulted in convergence on a rare [D-Leu1] microcystin-LR chemical variant. We detected this rare microcystin variant from strains of the distantly related genera Microcystis, Nostoc, and Phormidium. Phylogenetic analysis performed using sequences of the catalytic domains within the mcy gene cluster demonstrated a clear recombination pattern in the adenylation domain phylogenetic tree. We found evidence for conversion of the gene encoding the McyA2 adenylation domain in strains of the genera Nostoc and Phormidium. However, point mutations affecting the substrate-binding sequence motifs of the McyA2 adenylation domain were associated with the change in substrate specificity in two strains of Microcystis. In addition to the main [D-Leu1] microcystin-LR variant, these two strains produced a new microcystin that was identified as [Met1] microcystin-LR. Conclusions Phylogenetic analysis demonstrated that both point mutations and gene conversion result in functional mcy gene clusters that produce the same rare [D-Leu1] variant of microcystin in strains of the genera Microcystis, Nostoc, and Phormidium. Engineering pathways to produce recombinant non-ribosomal peptides could provide new natural products or increase the activity of known compounds. Our results suggest that the replacement of entire adenylation domains could be a more successful strategy to obtain higher specificity in the modification of the non-ribosomal peptides than point mutations.

Sandflies (Diptera, Psychodidae) from forest areas in Botucatu municipality, central western São Paulo State, Brazil

Abstract Background The study of the distribution and ecology of sandfly species is essential for epidemiological surveillance and estimation of the transmission risk of Leishmania spp. infection. Findings In the present study, sandflies were captured in native fragmented forest areas in Rubião Júnior district, Botucatu municipality, São Paulo state, Brazil, between September 2001 and January 2005. A minimum of two automatic light traps were installed per night from 6 pm to 8 am, in different months, resulting in approximately 900 collecting hours. During this period, 216 sandfly specimens of sixteen species were captured. Pintomyia monticola and Brumptomyia guimaraesi were the most abundant with 56 specimens (25.93%) captured per species, followed by Pintomyia fischeri 28 (12.96%) and Psathyromyia pascalei 18 (8.33%). Other captured species were Lutzomyia amarali, Sciopemyia sordellii, Psathyromyia aragaoi, Nyssomyia whitmani, Migonemyia migonei, Pintomyia bianchigalatiae, Pintomyia misionensis, Brumptomyia carvalheiroi, Brumptomyia cardosoi, Brumptomyia cunhai, Brumptomyia nitzulescui, Brumptomyia brumpti and Brumptomyia spp. represented by 58 (26.85%) specimens. Conclusions Although less frequently found, the presence of Pintomyia fischeri, Nyssomyia whitmani and Migonemyia migonei, known vectors of Leishmania braziliensis, indicates risk of American cutaneous leishmaniasis occurrence. Moreover, the absence of Lutzomyia longipalpis-the main vector of Leishmania infantum chagasi, which is the agent of American visceral leishmaniasis-suggests that there is no risk of introduction and establishment of this disease in the studied area.

Molecular typing of Clostridium perfringens isolated from swine in slaughterhouses from São Paulo State, Brazil

Clostridium perfringens is an anaerobic Gram-positive bacterium known as common pathogen for humans, for domestic and wildlife animals. Although infections caused by C. perfringens type C and A in swine are well studied, just a few reports describe the genetic relationship among strains in the epidemiological chain of swine clostridioses, as well as the presence of the microorganism in the slaughterhouses. The aim of the present study was to isolate C. perfringens from feces and carcasses from swine slaughterhouses, characterize the strains in relation to the presence of enterotoxin, alpha, beta, epsilon, iota and beta-2 toxins genes, using polymerase chain reaction (PCR) and comparing strains by means of Pulsed field gel electrophoresis (PFGE). Clostridium perfringens isolation frequencies in carcasses and finishing pig intestines were of 58.8% in both types of samples. According to the polymerase chain reaction assay, only alfa toxin was detected, being all isolates also negative to enterotoxin and beta2 toxin. Through PFGE technique, the strains were characterized in 35 pulsotypes. In only one pulsotype, the isolate from carcass sample was grouped with fecal isolate of the same animal, suggesting that the risk of cross-contamination was low. Despite the high prevalence of C. perfringens in swine carcasses from the slaughterhouses assessed, the risk of food poisoning to Brazilian pork consumers is low, since all strains were negative to cpe-gene, codifying enterotoxin.