The synthetic peptide PnPP-19 induces peripheral antinociception via activation of NO/cGMP/KATP pathway: Role of eNOS and nNOS.

BACKGROUND: and purpose: The peptide PnPP-19, derived from the spider toxin PnTx2-6 (renamed as δ-CNTX-Pn1c), potentiates erectile function by activating the nitrergic system. Since NO has been studied as an antinociceptive molecule and PnPP-19 is known to induce peripheral antinociception, we intended to evaluate whether PnPP-19 could induce peripheral antinociception through activation of this pathway. EXPERIMENTAL APPROACH: Nociceptive thresholds were measured by paw pressure test. PGE2 (2 µg/paw) was administered intraplantarly together with PnPP-19 and inhibitors/blockers of NOS, guanylyl cyclase and KATP channels. The nitrite concentration was accessed by Griess test. The expression and phosphorylation of eNOS and nNOS were determined by western blot. KEY RESULTS: PnPP-19 (5, 10 and 20 µg/paw) induced peripheral antinociception in rats. Administration of NOS inhibitor (L-NOarg), selective nNOS inhibitor (L-NPA), guanylyl cyclase inhibitor (ODQ) and the blocker of KATP (glibenclamide) partially inhibited the antinociceptive effect of PnPP-19 (10 µg/paw). Tissue nitrite concentration increased after PnPP-19 (10 µg/paw) administration. Expression of eNOS and nNOS remained the same in all tested groups, however the phosphorylation of nNOS Ser852 (inactivation site) increased and phosphorylation of eNOS Ser1177 (activation site) decreased after PGE2 injection. Administration of PnPP-19 reverted this PGE2-induced effect. CONCLUSIONS AND IMPLICATIONS: The peripheral antinociceptive effect induced by PnPP-19 is resulting from activation of NO-cGMP-KATP pathway. Activation of eNOS and nNOS might be required for such effect. Our results suggest PnPP-19 as a new drug candidate to treat pain and reinforce the importance of nNOS and eNOS activation, as well as endogenous NO release, for induction of peripheral antinociception.

PnPP-19, a spider toxin peptide, induces peripheral antinociception through opioid and cannabinoid receptors and inhibition of neutral endopeptidase.

BACKGROUND AND PURPOSE: The synthetic peptide PnPP-19 has been studied as a new drug candidate to treat erectile dysfunction. However, PnTx2-6, the spider toxin from which the peptide was designed, induces hyperalgesia. Therefore, we intended to investigate the role of PnPP-19 in the nociceptive pathway. EXPERIMENTAL APPROACH: Nociceptive thresholds were measured by paw pressure test. PnPP-19 was administered intraplantarly alone or with selective cannabinoid or opioid receptor antagonists. The hydrolysis of PnPP-19 by neutral endopeptidase (NEP) (EC 3.4.24.11), an enzyme that cleaves enkephalin, was monitored by HPLC and the cleavage sites were deduced by LC-MS. Inhibition by PnPP-19 and Leu-enkephalin of NEP enzyme activity was determined spectrofluorimetrically. KEY RESULTS: PnPP-19 (5, 10 and 20 µg per paw) induced peripheral antinociception in rats. Specific antagonists of µ opioid receptors (clocinnamox), δ opioid receptors (naltrindole) and CB1 receptors (AM251) partly inhibited the antinociceptive effect of PnPP-19. Inhibition of fatty acid amide hydrolase by MAFP or of anandamide uptake by VDM11 enhanced PnPP-19-induced antinociception. NEP cleaved PnPP-19 only after a long incubation, and Ki values of 35.6 ± 1.4 and 14.6 ± 0.44 µmol·L(-1) were determined for PnPP-19 and Leu-enkephalin respectively as inhibitors of NEP activity. CONCLUSIONS AND IMPLICATIONS: Antinociception induced by PnPP-19 appears to involve the inhibition of NEP and activation of CB1, µ and δ opioid receptors. Our data provide a greater understanding of the antinociceptive effects of PnPP-19. This peptide could be useful as a new antinociceptive drug candidate.

Identification of C-type isolectins in the venom of the scorpionfish Scorpaena plumieri.

Chemical analyses of the hemagglutinating fraction from Scorpaena plumieri venom revealed that it contains five components (Sp-CL 1-5) with similar chromatographic elution profiles (35-38% of acetonitrile), molecular masses (16,800-17,000 Da) and N-terminal sequences, suggesting that they are isoforms of the same protein. The amino acid sequence of Sp-CL4 was determined and shown to have homology with fish C-type lectins. These data demonstrate for the first time the presence of C-type isolectins in a scorpionfish venom.

Bmoo FIBMP-I: A New Fibrinogenolytic Metalloproteinase from Bothrops moojeni Snake Venom.

A new fibrinogenolytic metalloproteinase (Bmoo FIBMP-I) was purified from Bothrops moojeni snake venom. This enzyme was isolated through a combination of three chromatographic steps (ion-exchange, molecular exclusion, and affinity chromatography). Analyses by reverse phase chromatography, followed by mass spectrometry, showed the presence of enzyme isoforms with average molecular mass of 22.8 kDa. The SDS-PAGE analyses showed a single chain of 27.6 kDa, in the presence and absence of reducing agent. The protein has a blocked N-terminal. One of the peptides obtained by enzymatic digestion of a reduced and S-alkylated isoform was completely sequenced by mass spectrometry (MS/MS). Bmoo FIBMP-I showed similarity with hemorrhagic factor and several metalloproteinases (MP). This enzyme degraded Aα-chain faster than the Bß-chain and did not affect the γ-chain of bovine fibrinogen. The absence of proteolytic activity after treatment with EDTA, together with the observed molecular mass, led us to suggest that Bmoo FIBMP-I is a member of the P-I class of the snake venom MP family. Bmoo FIBMP-I showed pH-dependent proteolytic activity on azocasein, but was devoid of coagulant, defibrinating, or hemorrhagic activities. The kinetic parameters of proteolytic activity in azocasein were determined (V max = 0.4596 Uh(-1)nmol(-1) ± 0.1031 and K m = 14.59 mg/mL ± 4.610).

A potent vasoactive cytolysin isolated from Scorpaena plumieri scorpionfish venom.

A new vasoactive cytolytic toxin, referred to as Sp-CTx, has been purified from the venom of the scorpionfish Scorpaena plumieri by a combination of gel filtration and anion exchange chromatographies. An estimation of Sp-CTx native molecular mass, performed by size exclusion chromatography, demonstrated that it is a 121 kDa protein. Further physicochemical studies revealed its glycoproteic nature and dimeric constitution, comprising subunits of approximately 65 kDa (MALDI-TOF-MS). Such protein has proved to possess a potent hemolytic activity on washed rabbit erythrocytes (EC(50) 0.46 nM), whose effect was strongly reduced after treatment with antivenom raised against stonefish venom -Synanceja trachynis (SFAV). This cross-reactivity has been confirmed by western blotting. Like S. plumieri whole venom (100 microg/mL), Sp-CTx (1-50 nM) caused a biphasic response on phenylephrine pre-contracted rat aortic rings, characterized by an endothelium- and dose-dependent relaxation phase followed by a contractile phase. The vasorelaxant activity has been abolished by l-NAME, demonstrating the involvement of nitric oxide on the response. We report here the first isolation of a cytolytic/vasoactive protein from scorpionfish venom and the data provided suggest structural and functional similarities between Sp-CTx and previously published stonefish hemolytic toxins.

Functional expression of a recombinant toxin - rPnTx2-6 - active in erectile function in rat.

In the current study, the putative cDNA for PnTx2-6 toxin of the Phoneutria nigriventer spider venom was cloned and expressed as tioredoxin fusion protein in the cytoplasm of Escherichia coli. The fusion protein was purified from the bacterial extracts by combination of immobilized Ni-ion affinity and gel filtration chromatographies. Then, it was cleaved by enterokinase and the generated recombinant PnTx2-6 (rPnTx2-6) was further purified by reverse-phase HPLC. Likewise the native toxin purified from the spider venom, rPnTx2-6 potentiates the erectile function when injected in rats. This result indicates that the production of functional recombinant PnTx2-6 might be an alternative to provide this basic and valuable tool for study, as well as for further understanding such complex physiological system, including its correlation with the central nervous system and local tissue factors.

LyeTx I, a potent antimicrobial peptide from the venom of the spider Lycosa erythrognatha.

LyeTx I, an antimicrobial peptide isolated from the venom of Lycosa erythrognatha, known as wolf spider, has been synthesised and its structural profile studied by using the CD and NMR techniques. LyeTx I has shown to be active against bacteria (Escherichia coli and Staphylococcus aureus) and fungi (Candida krusei and Cryptococcus neoformans) and able to alter the permeabilisation of L: -alpha-phosphatidylcholine-liposomes (POPC) in a dose-dependent manner. In POPC containing cholesterol or ergosterol, permeabilisation has either decreased about five times or remained unchanged, respectively. These results, along with the observed low haemolytic activity, indicated that antimicrobial membranes, rather than vertebrate membranes seem to be the preferential targets. However, the complexity of biological membranes compared to liposomes must be taken in account. Besides, other membrane components, such as proteins and even specific lipids, cannot be discarded to be important to the preferential action of the LyeTx I to the tested microorganisms. The secondary structure of LyeTx I shows a small random-coil region at the N-terminus followed by an alpha-helix that reached the amidated C-terminus, which might favour the peptide-membrane interaction. The high activity against bacteria together with the moderate activity against fungi and the low haemolytic activity have indicated LyeTx I as a good prototype for developing new antibiotic peptides.

A new family of small (4kDa) neurotoxins from the venoms of spiders of the genus Phoneutria.

A family of 4kDa neurotoxic peptides was purified from venoms of Phoneutria spiders. All have six cysteine residues, and low similarity with other neurotoxins. Three toxins caused moderate inhibition of L-type Ca(2+) channels. The structure of toxin PRTx27C3 was modeled and compared with toxin ADO1. The importance of four residues is suggested.

Tityus serrulatus Hypotensins: a new family of peptides from scorpion venom.

Using a proteomic approach, a new structural family of peptides was put in evidence in the venom of the yellow scorpion Tityus serrulatus. Tityus serrulatus Hypotensins (TsHpt) are random-coiled linear peptides and have a similar bradykinin-potentiating peptide (BPP) amino acid signature. TsHpt-I (2.7kDa), the first member of this family, was able to potentiate the hypotensive effects of bradykinin (BK) in normotensive rats. Using the C-terminal of this peptide as a template, a synthetic analog peptide (TsHpt-I([17-25])) was designed to held the BK-potentiating effect. A relevant hypotensive effect, independent on BK, was also observed on both TsHpt (native and synthetic). To better evaluate this hypotensive effect, we examined the vasorelaxation of aortic rings from male Wistar rats and the peptides were able to induce endothelium-dependent vasorelaxation dependent on NO release. Both TsHpt could not inhibit ACE activity. These peptides appear to exert their anti-hypertensive effect through NO-dependent and ACE-independent mechanisms.

Tx2-6 toxin of the Phoneutria nigriventer spider potentiates rat erectile function.

The venom of the spider Phoneutria nigriventer contains several toxins that have bioactivity in mammals and insects. Accidents involving humans are characterized by various symptoms including penile erection. Here we investigated the action of Tx2-6, a toxin purified from the P. nigriventer spider venom that causes priapism in rats and mice. Erectile function was evaluated through changes in intracavernosal pressure/mean arterial pressure ratio (ICP/MAP) during electrical stimulation of the major pelvic ganglion (MPG) of normotensive and deoxycorticosterone-acetate (DOCA)-salt hypertensive rats. Nitric oxide (NO) release was detected in cavernosum slices with fluorescent dye (DAF-FM) and confocal microscopy. The effect of Tx2-6 was also characterized after intracavernosal injection of a non-selective nitric oxide synthase (NOS) inhibitor, L-NAME. Subcutaneous or intravenous injection of Tx2-6 potentiated the elevation of ICP/MAP induced by ganglionic stimulation. L-NAME inhibited penile erection and treatment with Tx2-6 was unable to reverse this inhibition. Tx2-6 treatment induced a significant increase of NO release in cavernosum tissue. Attenuated erectile function of DOCA-salt hypertensive rats was fully restored after toxin injection. Tx2-6 enhanced erectile function in normotensive and DOCA-salt hypertensive rats, via the NO pathway. Our studies suggest that Tx2-6 could be important for development of new pharmacological agents for treatment of erectile dysfunction.

Peptides of arachnid venoms with insecticidal activity targeting sodium channels.

Arachnids have a venom apparatus and secrete a complex chemical mixture of low molecular mass organic molecules, enzymes and polypeptide neurotoxins designed to paralyze or kill their prey. Most of these toxins are specific for membrane voltage-gated sodium channels, although some may also target calcium or potassium channels and other membrane receptors. Scorpions and spiders have provided the greatest number of the neurotoxins studied so far, for which, a good number of primary and 3D structures have been obtained. Structural features, comprising a folding that determines a similar spatial distribution of charged and hydrophobic side chains of specific amino acids, are strikingly common among the toxins from spider and scorpion venoms. Such similarities are, in turn, the key feature to target and bind these proteins to ionic channels. The search for new insecticidal compounds, as well as the study of their modes of action, constitutes a current approach to rationally design novel insecticides. This goal tends to be more relevant if the resistance to the conventional chemical products is considered. A promising alternative seems to be the biotechnological approach using toxin-expressing recombinant baculovirus. Spider and scorpion toxins having insecticidal activity are reviewed here considering their structures, toxicities and action mechanisms in sodium channels of excitable membranes.

Development of a tele-stethoscope and its application in pediatric cardiology.

Over the years, many attempts have been made to develop special stethoscopes for the teaching of auscultation. The objective of this article is to report on the experience with the development and implementation of an electronic stethoscope and a virtual library of cardiac sounds. There were four stages to this project: (1) the building of the prototype to acquire, filter and amplify the cardiac sounds, (2) the development of a software program to record, reproduce and visualize them, (3) the testing of the prototype in a clinical scenario, and (4) the development of an internet site, to store and display the sounds collected. The first two stages are now complete. The prototype underwent an initial evaluation in a clinical scenario within the Unit and during virtual out-patient clinical sessions. One hundred auscultations were recorded during these tests. They were reviewed and discussed on-line by a panel of experience cardiologists during the sessions. Although the sounds were considered "satisfactory" for diagnostic purposes by the cardiology team, they identified some qualitative differences in the electronic recorded auscultations, such as a higher pitch of the recorded sounds. Prospective clinical studies are now being conducted to further evaluate the interference of the electronic device in the physicians' capability to diagnose different cardiac conditions. An internet site (www.caduceusvirtual.com.br/ auscultaped) was developed to host these cardiac auscultations. It is set as a library of cardiac sounds, catalogued by pathologies and already contains examples from auscultations of the majority of common congenital heart lesions, such as septal defects and valvar lesions.

Molecular characterization of a neutralizing murine monoclonal antibody against Tityus serrulatus scorpion venom.

Monoclonal antibodies (mAbs) against Tityus serrulatus venom were obtained by the fusion of SP2/0 murine myeloma cells and spleen cells from BALB/c mice immunized with a toxic fraction (TstFG50) of the Tityus venom (this G50 chromatography fraction represents most of the toxicity of the crude venom) conjugated to bovine serum albumin (BSA) with glutaraldehyde. From the initial screening of over 200 hybridoma fusion wells, a panel of 9 anti-TstFG50 secreting hybridomas was established. The capacity of mAbs to neutralize the TstFG50 toxic fraction toxic was determined by in vitro neutralization assays and by inhibition of the binding of 125I-TsVII to its site on rat brain synaptosomes. Only mAbTs1 neutralized 50% of the toxic effects produced by scorpion venom and showed 35% inhibition of the binding of 125I-TsVII at 10(-7) M. To map the epitope recognized by the protective mAbTs1, we prepared a comprehensive series of overlapping 15-mer synthetic peptides covering the amino acid sequences of the four Tityus proteins. MAbTs1 reacted with peptide 26 of TsIV (KKSKDKKADSGYSYW), peptide 30 of TsVII (KKGSSGYSAWPASYS) and peptide 31 of TsNTxP (KKGSSGYSAWPASYS). MAbTs1 was not reactive with any peptide from TsII. The N-terminal lysine residue from the epitope was found to be critical for mAbTs1 binding. The epitope was positioned on the available three-dimensional structure of TsVII together with the recently identified residues from the pharmacophore of beta-scorpion toxins. The neutralizing properties of mAbTs1 might be explained by spatial vicinity of epitope residues with pharmacophore residues.

Neutralizing properties of Musa paradisiaca L. (Musaceae) juice on phospholipase A2, myotoxic, hemorrhagic and lethal activities of crotalidae venoms.

The use of plants as medicine has been referred to since ancient peoples, perhaps as early as Neanderthal man. Plants are a source of many biologically active products and nowadays they are of great interest to the pharmaceutical industry. The study of how people of different culture use plants in particular ways has led to the discovery of important new medicines. In this work, we verify the possible activity of Musa paradisiaca L. (Musaceae) against the toxicity of snake venoms. Musa paradisiaca, an important source of food in the world, has also been reported to be popularly used as an anti-venom. Interaction of Musa paradisiaca extract (MsE) with snake venom proteins has been examined in this study. Phospholipase A2 (PLA2), myotoxic and hemorrhagic activities, including lethality in mice, induced by crotalidae venoms were significantly inhibited when different amounts of MsE were mixed with these venoms before assays. On the other hand, mice that received MsE and venoms without previous mixture or by separated routes were not protected against venom toxicity. Partial chemical characterization of MsE showed the presence of polyphenols and tannins and they are known to non-specifically inactivate proteins. We suggest that these compounds can be responsible for the in vitro inhibition of the toxic effects of snake venoms. In conclusion, according to our results, using mice as experimental model, MsE does not show protection against the toxic effects of snake venoms in vivo, but if was very effective when the experiments were done in vitro.

Enzymes with gelatinolytic activity can be found in Tityus bahiensis and Tityus serrulatus venoms.

Enzymes with gelatinolytic activity were detected in Tityus bahiensis and Tityus serrulatus venom. Their activity was optimal at pH 8.0 in SDS-PAGE-gelatin. They were inhibited by PMSF but not by iodoacetamide, pepstatin or phenantrolin in the assay conditions used. This suggests that these enzymes are serine proteases. The presence of metal ions did not affect the proteolytic activity of these enzymes. Several possible functions may be envisaged for these enzymes: in tissue permeabilization, pancreatitis and toxin processing.

Regulation of the glutamate uptake by extracellular calcium.

To determine whether [Ca(2+)](e) modulates glutamate re-uptake, we studied the uptake mechanism into rat cerebrocortical synaptosomes. The removal of extracellular Ca(2+) caused a negative modulation in the uptake mechanism. The calculated K(50) value was 0.185 +/- 0.019 mM (n = 4). The Michaelis-Menten data analysis indicate that absence of Ca(2+) diminished the V(max) kinetic parameter by about 60% without changing significantly the K(m) suggesting a non-competitive mechanism. We also tested the involvement of intracellular Ca(2+) in this phenomenon by trapping BAPTA into the synaptosomal vesicles to control the Ca(2+) concentration. Our results suggest that intracellular Ca(2+) changes have a less predominant role on the glutamate uptake than do extracellular Ca(2+). These findings argue in favor of an important role of extracellular [Ca(2+)] in maintaining the L-glutamate re-uptake mechanism in the mammalian central nervous system.

A proposed 3D structure for crotamine based on homology building, molecular simulations and circular dichroism.

Crotamine, isolated from the venom of the South American rattlesnake Crotalus durissus terrificus is a strongly basic 42-amino acid polypeptide belonging to the small basic myotoxin family. As no tridimensional structure is available for this myotoxin subfamily, despite its important pharmacological interest, we propose in this paper a theoretical 3D model for crotamine. Starting from a homology modelling procedure, followed by intensive molecular dynamics (MD) simulations in water and complementary CD experiments, the designed 3D model is the first example of a tridimensional structure in this family of small basic myotoxins. Crotamine, therefore, belongs to a newly identified structural family presenting a common fold also found in beta-defensin and antopleurine-B. The proposed 3D model will be used for future calculations about crotamine aggregation and interaction with membranes.

Anti-PLA2 action test of Casearia sylvestris Sw.

Casearia sylvestris (Flacourtiaceae) is a plant which grows in the wild. The crude extract and pure substances from this plant induced partial inhibition of the PLA: (phospholipase A2) activity of snake venoms and some purified toxins. C. sylvestris extract efficiently neutralized the hemorrhagic and myotoxic activities caused by crude venoms and toxins.

The toxin Tx4(6-1) from the spider Phoneutria nigriventer slows down Na(+) current inactivation in insect CNS via binding to receptor site 3.

Tx4(6-1) a neurotoxic peptide from the venom of the aggressive South American 'armed' spider Phoneutria nigriventer, has been previously isolated and sequenced. It shows no detectable activity in mice but affects the peripheral nervous system of insects by stimulating glutamate release at the neuromuscular junction. Here we investigate possible interactions of the toxin with voltage-activated sodium channels (Na(v)). We confirm that it is ineffective on mammalian Na(v) channels, and establish that it competes with the alpha-like toxin 125I-Bom IV, for binding on the site 3 of insect Na(v) channel (IC(50) value around 25nM). The physiological consequences of this binding to the insect Na(v) channel are shown by electrophysiology: Tx4(6-1) prolongs evoked axonal action potentials (APs) (<500&mgr;s duration in control). Prolonged 8-10ms or 'plateau' 500-800ms APs accompanied by repetitive firing at 80-150Hz are recorded after 4-8min of toxin action. This modification of evoked activity is due to a slowing down of sodium current inactivation. Effects of Tx4(6-1) on sodium current are compared with those of a typical scorpion alpha-toxin and of some other spider toxins active on insect Na(v) channels. At the end of long voltage pulses, the maintained inward sodium current may represent 50% of the peak current after scorpion alpha-toxin but only about 8-10% after spider toxins. To understand the slight differences in the effects of alpha-scorpion and spider toxins on the insect Na(v) channel, structural studies of toxin-channels interactions would be necessary.

Purification, amino-acid sequence and partial characterization of two toxins with anti-insect activity from the venom of the South American scorpion Tityus bahiensis (Buthidae).

We report here the isolation by a two-step chromatographic procedure of two new toxins from the South American scorpion Tityus bahiensis. Their amino-acid sequences and some of their biological features were established. The two toxins have different biological properties. Toxin TbIT-I had almost no activity or pharmacological effects in vertebrate tissues whereas it was lethal to house flies (LD50 80.0 ng/house fly). In contrast, Tb2-II was active against both mammals (intracerebroventricular injection of 100 ng/mouse was lethal) and insects (LD50 40.0 ng/house fly). The amino-acid sequences of these toxins were established and found to be similar (60-95%) to previously described beta-toxins from the Tityus genus. Based on the available comparative information, this study attempts identify possible structure-function relationships that may be responsible for the differences in bioactivity displayed by these toxins.