Osmindo Rodrigues Pires

Occurrence of tetrodotoxin and its analogues in the Brazilian frog Brachycephalus ephippium (Anura: Brachycephalidae).

Brachycephalus ephippium is a diurnal frog, that shows aposematic colouration and inhabits Atlantic forest leaf litter in south-eastern Brazil. The presence of tetrodotoxin (TTX) in the skin, liver and ovaries of B. ephippium was demonstrated. The skin (260 M.U./g) exhibited the highest toxicity followed by liver (177 M.U./g). TTX and its analogues, tetrodonic acid, 4-epitetrodotoxin and 4,9 anhydrotetrodotoxin were isolated and identified by HPLC followed by fluorimetric analysis. TTX and 11-nortetrodotoxin-6(S)-ol had their presence confirmed by mass spectrometry (MALDI-TOF). The results confirm Brachycephalidae as a fourth family of anurans containing TTX.

The occurrence of 11-oxotetrodotoxin, a rare tetrodotoxin analogue, in the brachycephalidae frog Brachycephalus ephippium

11-oxoTTX is an analogue 4-5 times more toxic than TTX itself, been rare even in marine animals. Two ions at m/z 320 and 336 corresponding to TTX and 11-oxoTTX (M+H(+)), respectively, were detected in the Brachycephalidae frog Brachycephalus ephippium extracts. The fragment ion pattern of 11-oxoTTX is similar to that TTX, although its possible to verify some specific fragments.

Purification and primary structure determination of Tf4, the first bioactive peptide isolated from the venom of the Brazilian scorpion Tityus fasciolatus

In the present study Tityus fasciolatus crude venom toxicity was evaluated and we also report the purification and characterization of a 6.6 kDa neurotoxin isolated from T. fasciolatus venom. This new toxin, named Tf4, has a molecular mass of 6614Da and its primary structure is homologous to TbIT-I from T. bahiensis and TsTX-VI and TsNTxP from T. serrulatus. Tf4 delays frog sodium channel inactivation reversibly, but it is non-toxic to mammals or crustaceans. An attempt to identify the residues responsible for the partial loss of toxicity in Tf4 was carried out based on homology modeling and sequence comparison.

A Novel Vasoactive Proline-Rich Oligopeptide from the Skin Secretion of the Frog Brachycephalus ephippium.

Proline-rich oligopeptides (PROs) are a large family which comprises the bradykinin-potentiating peptides (BPPs). They inhibit the activity of the angiotensin I-converting enzyme (ACE) and have a typical pyroglutamyl (Pyr)/proline-rich structure at the N- and C-terminus, respectively. Furthermore, PROs decrease blood pressure in animals. In the present study, the isolation and biological characterization of a novel vasoactive BPP isolated from the skin secretion of the frog Brachycephalus ephippium is described. This new PRO, termed BPP-Brachy, has the primary structure WPPPKVSP and the amidated form termed BPP-BrachyNH2 inhibits efficiently ACE in rat serum. In silico molecular modeling and docking studies suggest that BPP-BrachyNH2 is capable of forming a hydrogen bond network as well as multiple van der Waals interactions with the rat ACE, which blocks the access of the substrate to the C-domain active site. Moreover, in rat thoracic aorta BPP-BrachyNH2 induces potent endothelium-dependent vasodilatation with similar magnitude as captopril. In DAF-FM DA-loaded aortic cross sections examined by confocal microscopy, BPP-BrachyNH2 was found to increase the release of nitric oxide (NO). Moreover, BPP-BrachyNH2 was devoid of toxicity in endothelial and smooth muscle cell cultures. In conclusion, the peptide BPP-BrachyNH2 has a novel sequence being the first BPP isolated from the skin secretion of the Brachycephalidae family. This opens for exploring amphibians as a source of new biomolecules. The BPP-BrachyNH2 is devoid of cytotoxicity and elicits endothelium-dependent vasodilatation mediated by NO. These findings open for the possibility of potential application of these peptides in the treatment of endothelial dysfunction and cardiovascular diseases.

Cm38: a new antimicrobial peptide active against Klebsiella pneumoniae is homologous to Cn11.

Antimicrobial peptides (AMPs) represent a large and ubiquitous group of peptides. The current crisis in antibiotic therapy has led to an intensified search for new antimicrobial agents. In this regard, scorpion venom constitutes a rich source of biologically active peptides including AMPs. In the present study, the purification of a novel peptide with antimicrobial activity against the Gram-negative bacteria Klebsiella pneumoniae is described. This antimicrobial peptide, named Cm38, was purified from Centruroides margaritatus scorpion venom using a two-step chromatographic strategy using C8 and C18 columns. This toxin inhibits the proliferation of the Gram-negative bacteria Klebsiella pneumoniae with a Minimal Inhibitory Concentration (MIC) of 64 μM. An analysis of the N-terminal sequence of Cm38 revealed a close structural relationship to Cn11, a Na+-channel modulator toxin previously isolated from Centruroides noxius scorpion venom. Therefore, to test Cm38 for effects on ion channels, we measured its effects on action potential firing in cultured dorsal root ganglion neurons. Cm38 depolarized and increased action potential firing in a subset of neurons tested. The present work reports a new peptide related to Cn11 with antimicrobial properties that is also active in neurons.