Two peptides, TsAP-1 and TsAP-2, from the venom of the Brazilian yellow scorpion, Tityus serrulatus: evaluation of their antimicrobial and anticancer activities

Here we report two novel 17-mer amidated linear peptides (TsAP-1 and TsAP-2) whose structures were deduced from cDNAs cloned from a venom-derived cDNA library of the Brazilian yellow scorpion, Tityus serrulatus. Both mature peptides were structurally-characterised following their location in chromatographic fractions of venom and synthetic replicates of each were subjected to a range of biological assays. The peptides were each active against model test micro-organisms but with different potencies. TsAP-1 was of low potency against all three test organisms (MICs 120-160µM), whereas TsAP-2 was of high potency against the Gram-positive bacterium, Staphylococcus aureus (MIC 5µM) and the yeast, Candida albicans (10µM). Haemolytic activity of TsAP-1 was low (4% at 160µM) and in contrast, that of TsAP-2 was considerably higher (18% at 20µM). Substitution of four neutral amino acid residues with Lys residues in each peptide had dramatic effects on their antimicrobial potencies and haemolytic activities, particularly those of TsAP-1. The MICs of the enhanced cationic analogue (TsAP-S1) were 2.5µM for S.aureus/C.albicans and 5µM for E.coli but with an associated large increase in haemolytic activity (30% at 5µM). The same Lys residue substitutions in TsAP-2 produced a dramatic effect on its MIC for E.coli lowering this from >320µM to 5µM. TsAP-1 was ineffective against three of the five human cancer cell lines tested while TsAP-2 inhibited the growth of all five. Lys residue substitution of both peptides enhanced their potency against all five cell lines with TsAp-S2 being the most potent with IC50 values ranging between 0.83 and 2.0 µM. TsAP-1 and TsAP-2 are novel scorpion venom peptides with broad spectrum antimicrobial and anticancer cell activities the potencies of which can be significantly enhanced by increasing their cationicity.

Renal effects and vascular reactivity induced by Tityus serrulatus venom

Tityus serrulatus, popularly known as yellow scorpion, is one of the most studied scorpion species in South America and its venom has supplied some highly active molecules. The effects of T. serrulatus venom upon the renal physiology in human showed increased renal parameters, urea and creatinine. However, in perfused rat kidney the effects were not tested until now. Isolated kidneys from Wistar rats, weighing 240-280 g, were perfused with Krebs-Henseleit solution containing 6% (g weight) of previously dialysed bovine serum albumin. The effects of T. serrulatus venom were studied on the perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF), glomerular filtration rate (GFR), sodium tubular transport (%TNa+), potassium tubular transport (%TK+) and chloride tubular transport (%TCl-). Tityus serrulatus venom (TsV; 10 mu g/mL) was added to the system 30 min after the beginning of each experiment (n = 6). This 30 min period was used as an internal control. The mesenteric bed was perfused with Krebs solution kept warm at 37 T by a constant flow (4 mL/min), while the variable perfusion pressure was measured by means of a pressure transducer. The direct vascular effects of TsV (10 mu g/mL/min; n=6), infused at a constant rate (0.1 mL/min), were examined and compared to the infusion of the vehicle alone at the same rate. TsV increased PP (PP30'= 127.8 +/- 0.69 vs PP60' = 154.2 +/- 14 mmHg*, *p < 0.05) and RVR (RVR30' = 6.29 +/- 0.25 vs RVR60' = 8.03 +/- 0.82 mmHg/mL g(-1) min(-1)*, *p < 0.05), decreased GFR (GFR(30') =0.58 +/- 0.02 vs GFR(60') = 0.46 +/- 0.01 mL g(-1) min(-1)*, *p < 0.05) and UF (UF30' = 0.135 +/- 0.001 vs UF60' = 0.114 +/- 0.003 mL g(-1)min(-1)*, *p < 0.05). Tubular transport was not affected during the whole experimental period (120 min). on the other hand, the infusion of TsV (10 mu g/mL/min) increased the basal perfusion pressure of isolated arteriolar mesenteric bed (basal pressure: 74.17 +/- 3.42 vs TsV 151.8 +/- 17.82 mmHg*, *p < 0.05). TsV affects renal haemodynamics probably by a direct vasoconstrictor action leading to decreased renal flow. (c) 2005 Elsevier Ltd. All rights reserved.

Effects of Tityus serrulatus scorpion venom on lung mechanics and inflammation in mice

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Aplicação de nanopartículas de quitosana com potencial de adjuvante na produção de soro contra o venneno do escorpião Tityus serrulatus

In Brazil, several species of scorpions are known to cause accidents which can lead to death, which are mainly belonging to the genus Tityus. The scorpion Tityus serrulatus is the main responsible for more severe cases. Anti-scorpion serums are routinely produced by various institutions, despite their effectiveness, quality and action depends on how quickly treatment is started. Studies have been developed in the search for appropriate technologies to encapsulate and release recombinant or natives proteins capable of inducing antibody production. In this context, chitosan copolymer which can be obtained from the partial deacetylation of chitin or in some microorganisms and it is biocompatible and biodegradable has been widely used for this purpose. This study aimed to search for a system release from chitosan nanoparticles for peptide / protein of the venom of the scorpion T. serrulatus, able to provide a new model of immunization in animals, in order to obtain a potential novel polyclonal serum, anti-venom T. serrulatus. The chitosan nanoparticles were prepared by ionic gelation with polyanion tripolyphosphate (TPP). After standardizing the concentrations of TPP and chitosan was evaluated the efficiency of incorporation of bovine serum albumin (BSA) and scorpion venom, showed particle size compatible with the intended purpose. The particles showed adequate size around 200nm. The crosslinking was confirmed by absorption spectroscopy in the infrared. After verified the high encapsulation efficiency (EE) for acid bicinconínico method (BCA) protein assay and the particle size distribution, the success of the technique was proven and the potential for in vivo application of nanoparticles. The experimental animals were vaccinated and the antibodies measured by ELISA

AMINO-ACID-SEQUENCE OF TSTX-V, AN ALPHA-TOXIN FROM TITYUS-SERRULATUS SCORPION-VENOM, AND ITS EFFECT ON K+ PERMEABILITY OF BETA-CELLS FROM ISOLATED RAT ISLETS OF LANGERHANS

Highly purified Tityustoxin V (TsTX-V), an alpha-toxin isolated from the venom of the Brazilian scorpion Tityus serrulatus, was obtained by ion exchange chromatography on carboxymethylcellulose-52. It was shown to be homogeneous by reverse phase high performance liquid chromatography, N-terminal sequencing (first 39 residues) of the reduced and alkylated protein and by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate and tricine. Following enzymatic digestion, the complete amino acid sequence (64 residues) was determined. The sequence showed higher homology with the toxins from the venoms of the North African than with those of the North and South American scorpions. Using the rate of Rb-86(+) release from depolarized rat pancreatic beta-cells as a measure of K+ permeability changes, TsTX-V (5.6 mu g/ml) was found to increase by 2.0-2.4-fold the rate of marker outflow in the presence of 8.3 mM glucose. This effect was persistent and slowly reversible, showing similarity to that induced by 100 mu-M veratridine, an agent that increases the open period of Na+ channels, delaying their inactivation. It is suggested that, by extending the depolarized period, TsTX-V indirectly affects beta-cell voltage-dependent K+ channels, thus increasing K+ permeability.

Karyotype Conservation in 2 Populations of the Parthenogenetic Scorpion Tityus serrulatus (Buthidae): rDNA and Its Associated Heterochromatin Are Concentrated on Only One Chromosome

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Supersensitivity of the isolated guinea-pig vas deferens induced by a toxic fraction of scorpion venom (Tityus serrulatus)

1. Tityustoxin (TsTx), a toxic fraction of Tityus serrulatus venom, was studied on the isolated guinea-pig vas deferens. It increased significantly the maximal response of the preparation to both norepinephrine and acetylcholine and decreased the effective median dose of norepinephrine. 2. The effect of TsTx on norepinephrine median dose was unchanged when atropinized or pharmacologically 'denervated' preparations were used but was abolished when both procedures were associated. 3. Atropinization of pharmacologically denervated muscles almost never modify the TsTx-induced increase in the maximal response to norepinephrine. 4. On denervated or phentolamine-treated muscles TsTx-induced increase in the maximal response to acetylcholine was abolished. 5. It was concluded that toxin predominantly induces adrenergic postsynaptic supersensitivity. 6. Of minor significance, it also induces presynaptic cholinergic and adrenergic supersensitivity. 7. Comparison of these results with those of crude venom indicates that TsTx effects may result from the sum of the effects of subcomponents not demonstrated by the chemical procedures here utilized.

Programa de controle de surto de escorpião Tityus serrulatus, Lutz e Mello 1922, no município de Aparecida, SP (Scorpiones, Buthidae).

A scorpion control program was proposed for the town of Aparecida (SP), an endemic region of Tityus serrulatus. Clusters of scorpions in urban and rural areas, environmental degradation of the town's outskirts and new scorpion procreation and dispersal habitats were studied. In addition, infrastructure problems such as the disposal and collection of residential and municipal refuse, sanitation (sewage and storm sewer), condition of vacant lots and constructions in the urban area were evaluated. After an epidemiological study, educational measures such as the distribution of pamphlets, cleaning group work, visits to residences and cooperation from High School teachers and students were also suggested. Chemical control was indicated in high-risk sites, especially those of near-school buildings. Furthermore, the use of natural predators was also mentioned within the present sanitation regulations for urban areas. The authors assert that these procedures must be integrated and continued uninterruptedly for several years. They also suggest a collaborative work with those responsible for the dengue eradication program, as well as the institution of the scorpion study week, which would greatly contribute to the education of the population, to preventive programs and to scorpion control.

Selection of environmental temperature by the yellow scorpion Tityus serrulatus Lutz & Mello, 1922 (Scorpiones, Buthidae)

The preferred temperature of the yellow scorpion Tityus serrulatus was investigated since its dispersion is a matter of concern. Adult T. serrulatus, weighing 1.24 ± 0.20 g (mean + sd) and with a standard length of 59.3 ± 2.5 mm, were used. A metallic corridor (120 cm long, 5 cm large and 10 cm high) with thermal gradient ranging from 0°C to 40°C was used. Tityus serrulatus chose and stayed in temperatures ranging from 14° C to 38°C when safe conditions were offered (dark and thigmotactic stimuli). The number of animals that remained in the 11°C-20°C, 21°C-30°C, and 31°C-40°C temperature zones were 8, 8, and 9, respectively. The chi-square test (degree of freedom = 2) showed that differences were not significant (p>0.05). Some animals moved to lower temperature areas (less than 8°C) when the corridor was completely illuminated and thigmotactic stimuli were absent, which led the animals to present a torpor state. It is concluded that T. serrulatus does not select a specific environmental temperature. Associated with the capacity of temporally surviving at low temperatures, this species seems to be highly adaptable to different thermal zones.

Isolation, homology modeling and renal effects of a C-type natriuretic peptide from the venom of the Brazilian yellow scorpion (Tityus serrulatus)

Mammalian natriuretic peptides (NPs) have been extensively investigated for use as therapeutic agents in the treatment of cardiovascular diseases. Here, we describe the isolation, sequencing and tridimensional homology modeling of the first C-type natriuretic peptide isolated from scorpion venom. In addition, its effects on the renal function of rats and on the mRNA expression of natriuretic peptide receptors in the kidneys are delineated. Fractionation of Tityusserrulatus venom using chromatographic techniques yielded a peptide with a molecular mass of 2190.64Da, which exhibited the pattern of disulfide bridges that is characteristic of a C-type NP (TsNP, T. serrulatus Natriuretic Peptide). In the isolated perfused rat kidney assay, treatment with two concentrations of TsNP (0.03 and 0.1μg/mL) increased the perfusion pressure, glomerular filtration rate and urinary flow. After 60min of treatment at both concentrations, the percentages of sodium, potassium and chloride transport were decreased, and the urinary cGMP concentration was elevated. Natriuretic peptide receptor-A (NPR-A) mRNA expression was down regulated in the kidneys treated with both concentrations of TsNP, whereas NPR-B, NPR-C and CG-C mRNAs were up regulated at the 0.1μg/mL concentration. In conclusion, this work describes the isolation and modeling of the first natriuretic peptide isolated from scorpion venom. In addition, examinations of the renal actions of TsNP indicate that its effects may be related to the activation of NPR-B, NPR-C and GC-C. © 2013 Elsevier Ltd.

Uma abordagem peptidômica do venedo do escorpião Tityus serrulatus

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Agressividade relacionada ao contexto alimentar em escorpião-amarelo Tityus Serrulatus (Lutz & Mello, 1922)(Scorpiones, Buthidae)

Scorpions are among the oldest living groups of animals, and are present in almost every continent. Although many studies are made about the toxins present in their venom, little is known about their behavior. The aim of this paper is to investigate the expression of agressiveness in the brazilian yellow scorpion Tityus serrulatus, specifically the relation between agressiveness, motor activity and feeding condition. The study was divided in two parts. First, we used 16 adult individuals, placed in two terrarium, to establish an ethogram. Each group was observed for 36h, and the behaviors displayed were described and categorized as agonistic or non-agonistic. In the second part of the experiment, we used 32 adult animals in three different nutritional states: Feeding (still ingesting food), Sated (1 to 4 days since last meal) and Deprived (14 to 25 days since last meal). The individuals were paired, each pair placed in a terrarium and observed for 30min. Behaviors displayed were timed in seconds. Our results show that only Feeding individuals displayed agonism towards others, and no relation was observed between motor activity and feeding conditions

Síntese e caracterização de pequenos peptídeos lineares do veneno de Tityus serrulatus (Buthidae)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)