Bioactive peptides (cryptides) obtained by Bothrops jararaca serine peptidases action on myoglobin.

Serine peptidases and metallopeptidases are the primary toxins found in Bothrops snakes venoms, which act on proteins in the tissues of victims or prey, and release of peptides formed through proteolytic activity. Various studies have indicated that these peptides, released by the proteolytic activity of heterologous enzymes, generate molecules with unidentified functions, referred to as cryptids. To address this, we purified serine peptidases from Bothrops jararaca venom using molecular exclusion chromatography and then incubated them with the endogenous substrate myoglobin. As a control, we also incubated the substrate with trypsin. The resulting proteolytic fragments were analyzed, separated, and collected via HPLC. These fractions were then tested on cell cultures, the active fractions were sequenced (ALELFR and TGHPETLEK) and synthesized. After confirming their activity, the peptides underwent sequencing and synthesis for additional cell tests, including the increase of cell viability, cycle phases, proliferation, signaling, growth kinetics, angiogenesis, and migration. The results revealed that the synthesized peptides exhibited cellular repair properties, suggesting a potential role in tissue repair in the range of 0.05-5 µ M. Additionally, the effects of fragments resulting from myoglobin degradation isolated (ALELFR and TGHPETLEK) revealed a regenerative action on tissue.

Bacterial toxins: an overview on bacterial proteases and their action as virulence factors.

Bacterial pathogenicity is a result of a combination of factors, including resistance to environmental threats and to the host's defenses, growth capability, localization in the host, tissue specificity, resource obtaining mechanisms and the bacterium's own defenses to aggression. A variety of bacterial components, often specific to each strain, are involved in the microorganism's survival, adhesion and growth in the host. Many of them are harmful and, therefore, are called virulence factors. The effects caused by the virulence factors determine the degree of aggressivity of the strain. In many cases the virulence factors are secreted proteins or enzymes, sometimes performing very specific functions. The enzymatic activity is directed to specific proteins from cell membranes, synaptic vesicle fusion proteins, among other important targets. One of the most toxic bacterial proteins is secreted by Clostridium botulinum, targeted to synaptic vesicle fusion proteins, cleaving them with a zinc-metalloprotease activity, which results in severe neurotoxic effects with a lethal dose as low as eight nanograms per kilogram of body weight. The tetanus neurotoxin acts in a similar way but is less active and Bacillus anthracis also presents a potent metalloprotease activity. In this work we describe a selection of these specially interesting and important bacterial proteins and proteases, stressing their relevance in the pathological process and in medical studies.

Phospholipases A2 isolated from Micrurus lemniscatus coral snake venom: behavioral, electroencephalographic, and neuropathological aspects.

The present study evaluated four phospholipase A2 (PLA2) (Mlx-8, Mlx-9, Mlx-11 and Mlx-12) isolated from Micrurus lemniscatus snake venom (Elapidae). The effects of intrahippocampal administration of these toxins have been determined on behavior, electroencephalography, and neuronal degeneration in rats. These four PLA2 toxins induced motor and EEG alterations in a dose-dependent manner. Behavioral convulsions were characterized by clonic movements and were often accompanied by EEG alterations. Mlx toxins were convulsive but weakly epileptogenic, since low rates of seizure discharges were observed in EEG records. Neuronal injury seemed to depend on the dose of the toxin used. The highest doses of toxins caused severe intoxication and death of some animals. The injury of hippocampal cells was characterized by massive neuronal loss and hippocampal gliosis. A high occurrence of compulsive scratching was observed. Moreover, the onset of seizures induced by Mlx toxins was markedly delayed. The similarities between the effects of Mlx PLA2s and those isolated from other Elapidae snakes venoms suggest that their toxicity are probably due to their specific binding to neuronal membranes and to the catalysis of phospholipid hydrolysis, producing lysophospholipids and fatty acids. These compounds likely disturb the membrane conformation, causing a marked increase in the release of neurotransmitters and concurrent inhibition of vesicle fission and recycling. These toxins can be a useful tool to investigate properties of endogenous secretory PLA2s and therefore may be important both to study mechanisms involved in neurotransmitter release at nerve terminals and to explain the convulsive properties of PLA2s toxins.

BnP1, a novel P-I metalloproteinase from Bothrops neuwiedi venom: biological effects benchmarking relatively to jararhagin, a P-III SVMP.

Snake venom metalloproteinases (SVMPs) have been extensively studied and their effects associated with the local bleeding observed in human accidents by viper snakes. Representatives of P-I and P-III classes of SVMPs similarly hydrolyze extracellular matrix proteins or coagulation factors while only P-III SVMPs induce significant hemorrhage in experimental models. In this work, the effects of P-I and P-III SVMPs on plasma proteins and cultures of muscle and endothelial cells were compared in order to enlighten the mechanisms involved in venom-induced hemorrhage. To reach this comparison, BnP1 was isolated from B. neuwiedi venom and used as a weakly hemorrhagic P-I SVMPs and jararhagin was used as a model of potently hemorrhagic P-III SVMP. BnP1 was isolated by size exclusion and anion-exchange chromatographies, showing apparent molecular mass of approximately 24kDa and sequence similarity with other members of SVMPs, which allowed its classification as a group P-I SVMP. The comparison of local effects induced by SVMPs showed that BnP1 was devoid of significant myotoxic and hemorrhagic activities and jararhagin presented only hemorrhagic activity. BnP1 and jararhagin were able to hydrolyze fibrinogen and fibrin, although the latter displayed higher activity in both systems. Using HUVEC primary cultures, we observed that BnP1 induced cell detachment and a decrease in the number of viable endothelial cells in levels comparable to those observed by treatment with jararhagin. Moreover, both BnP1 and jararhagin induced apoptosis in HUVECs while only a small increase in LDH supernatant levels was observed after treatment with jararhagin, suggesting that the major mechanism involved in endothelial cell death is apoptosis. Jararhagin and BnP1 induced little effects on C2C12 muscle cell cultures, characterized by a partial detachment 24h after treatment and a mild necrotic effect as evidenced by a small increase in the supernatants LDH levels. Taken together, our data show that P-I and P-III SVMPs presented comparable effects except for the hemorrhagic activity, suggesting that hydrolysis of coagulation factors or damage to endothelial cells are not sufficient for induction of local bleeding.

Ivermectin reduces motor coordination, serum testosterone, and central neurotransmitter levels but does not affect sexual motivation in male rats.

Ivermectin (IVM) is a macrocyclic lactone used for the treatment of parasitic infections and widely used in veterinary medicine as endectocide. In mammals, evidence indicates that IVM interacts with γ-aminobutyric acid (GABA)-mediated chloride channels. GABAergic system is involved in the manifestation of sexual behavior. We previously found that IVM at therapeutic doses did not alter sexual behavior in male rats, but at a higher dose, the appetitive phase of sexual behavior was impaired. Thus, we investigated whether the reduction of sexual behavior that was previously observed was a consequence of motor or motivational deficits that are induced by IVM. Data showed significant decrease in striatal dopaminergic system activity and lower testosterone levels but no effects on sexual motivation or penile erection. These findings suggest IVM may activate the GABAergic system and reduce testosterone levels, resulting in a reduction of motor coordination as consequence of the inhibition of striatal dopamine release.

Blockade of acetylcholine release at the motor endplate by a polypeptide from the venom of Phoneutria nigriventer.

1. The mechanisms underlying the muscle relaxation effect of a fraction (PF3) isolated from the Phoneutria nigriventer spider venom were assessed on mouse diaphragm and chick biventer cervicis muscle preparations. 2. PF3 (0.25-4 micrograms ml-1) produced a concentration-dependent blockade of the nerve-elicited muscle twitch of the mouse diaphragm (IC50 = 0.8 micrograms ml-1) without affecting the directly induced muscle twitch. In similar preparations, the crude venom (1-10 micrograms ml-1) produced muscle contracture and blocked both the direct and indirectly induced muscle twitches. 3. In the chick biventer cervicis muscle, PF3 (1-5 micrograms ml-1) blocked the nerve stimulated muscle twitch (IC50 = 1.26 micrograms ml-1), but did not alter the postjunctional response to exogenous acetylcholine (ACh, 10 microM-10 mM). 4. PF3 (2-8 micrograms ml-1) reduced the frequency of miniature endplate potentials (m.e.p.ps) recorded intracellularly from the mouse diaphragm muscle fibers by 58 to 64%, and diminished the amplitude of m.e.p.ps by 20 to 40% of control. The relationship between log m.e.p.p. frequency and log [Ca2+]o was shifted rightwards in the presence of 4 micrograms ml-1 PF3. 5. Raising the frequency of m.e.p.ps with high K+ medium or theophylline (3 mM) did not prevent the toxin-induced depression of spontaneous ACh release. 6. The quantal content of e.p.ps (m), determined in cut-diaphragm muscle fibres, was reduced by 53% and 77% of control by 1 and 4 micrograms ml-1 PF3, respectively. At 1 microgram ml-1 the toxin shifted the relationship between log m and log [Ca2+]o towards higher values without apparent change of the slope. 7. E.p.p. trains elicited at 10 to 50 Hz in the presence of PF3 (1 microgram ml-1) exhibited irregular amplitudes and facilitation related to the frequency of nerve stimulation. 8. It is concluded that PF3 blocks neuromuscular transmission by acting prejunctionally and reducing the nerve-evoked transmitter release. The effect was related to a diminished Ca2+ entry into the nerve terminal associated with inhibition of exocytosis.

Pharmacological effects and metabolism of neurotensin and bradykinin in the isolated rat uterus.

Neurotensin (NT) and bradykinin (BK) were found to cause contractions of isolated rat uterus preparations. In 97% of the experiments, acute tachyphylaxis followed soon after the initial administration of NT. Interrelation between the oxytocic effects of NT and BK was not observed. Among the NT fragments studied, only NT-(9-13) had an oxytocic effect (less than 1.0%). All NT fragments tested induced tachyphylaxis to NT regardless of their efficacy. Using HPLC analysis, NT but not BK was found to be degraded by the intact rat uterus. A major involvement of a carboxydipeptidase cleaving at Tyr11-Ile12 is suggested. Carboxyl-blocked neurotensinamide (NT-NH2) was found to be resistent to proteolysis and not develop tachyphylaxis. No cross-tachyphylaxis was observed with NT or NT-(1-11). The results suggest the existence of different receptors for NT and BK in the uterus, as well as the existence of different receptors or receptor states that interact with NT or NT-NH2 in the rat uterus.

A new bradykinin-potentiating peptide (peptide P) isolated from the venom of Bothrops jararacussu (jararacuçu tapete, urutu dourado).

Several bradykinin potentiators were identified in the venom of Bothrops jararacussu by chromatographic techniques and biological assays. One of them which was isolated inhibited the angiotensin-converting enzyme in vitro and potentiated the bradykinin-induced lowering of the arterial pressure in the rat.

Behavioral, electroencephalographic, and histopathologic effects of a neuropeptide isolated from Tityus serrulatus scorpion venom in rats.

The effects of intrahippocampal administration of a neuropeptide (TS-8F toxin) isolated from Tityus serrulatus scorpion venom have been determined on behavior, limbic seizures, and neuronal degeneration in rats. Behavioral observation showed orofacial automatism, wet dog shakes, and myoclonus. Concomitantly, the electroencephalographic record showed high-frequency and high-voltage spikes that evolved to seizure activity in the hippocampus and cortex. Seven days after TS-8F toxin microinjection, neuronal damage was observed in CA1 and CA2 pyramidal cells and in granular cells of the dentate gyrus. The results suggest that TS-8F toxin may be responsible, at least in part, by the epileptic effects observed with the crude venom. Thus, this toxin may be a useful tool in the study of some neurobiological process.

Evidence for a role of beta-endorphin in activity of nigrostriatal neurons in the rat.

The effects of icv administration of beta-endorphin on secretory activity of dopaminergic neurons is described. Homovanillic and dihydroxyphenyl acetic acid levels in cerebrospinal fluid and extracts of brain tissue were determined after administration of beta-endorphin to animals pretreated or not with naloxone. The results suggest that beta-endorphin interferes with formation of dopaminergic metabolites by acting on opioid receptors.

ZapA, a possible virulence factor from Proteus mirabilis exhibits broad protease substrate specificity.

The opportunistic bacterium Proteus mirabilis secretes a metalloprotease, ZapA, considered to be one of its virulence factors due to its IgA-degrading activity. However, the substrate specificity of this enzyme has not yet been fully characterized. In the present study we used fluorescent peptides derived from bioactive peptides and the oxidized beta-chain of insulin to determine the enzyme specificity. The bradykinin- and dynorphin-derived peptides were cleaved at the single bonds Phe-Ser and Phe-Leu, with catalytic efficiencies of 291 and 13 mM/s, respectively. Besides confirming already published cleavage sites, a novel cleavage site was determined for the beta-chain of insulin (Val-Asn). Both the natural and the recombinant enzyme displayed the same broad specificity, demonstrated by the presence of hydrophobic, hydrophilic, charged and uncharged amino acid residues at the scissile bonds. Native IgA, however, was resistant to hydrolysis by ZapA.

Development of an operational substrate for ZapA, a metalloprotease secreted by the bacterium Proteus mirabilis.

The protease ZapA, secreted by Proteus mirabilis, has been considered to be a virulence factor of this opportunistic bacterium. The control of its expression requires the use of an appropriate methodology, which until now has not been developed. The present study focused on the replacement of azocasein with fluorogenic substrates, and on the definition of enzyme specificity. Eight fluorogenic substrates were tested, and the peptide Abz-Ala-Phe-Arg-Ser-Ala-Ala-Gln-EDDnp was found to be the most convenient for use as an operational substrate for ZapA. A single peptide bond (Arg-Ser) was cleaved with a Km of 4.6 microM, a k cat of 1.73 s-1, and a catalytic efficiency of 376 (mM s)-1. Another good substrate for ZapA was peptide 6 (Abz-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Gln-EDDnp) which was cleaved at a single bond (Phe-Ser) with a Km of 13.6 microM, a k cat of 3.96 s-1 and a catalytic efficiency of 291 (mM s)-1. The properties of the amino acids flanking the scissile bonds were also evaluated, and no clear requirement for the amino acid residue at P1 was found, although the enzyme seems to have a preference for a hydrophobic residue at P2.

Characterization of two vasoactive peptides isolated from the plasma of the snake Crotalus durissus terrificus.

Incubation of plasma from the snake Crotalus durissus terrificus (CDTP) with trypsin generated two hypotensive peptides. The primary structure of the peptides was established for two sequences as: (Ser-Ile-Pro-Gln-Ala-Pro-Thr-Ser-Asn-Leu-Ile-Glu-Ala-Thr-Lys) and (Lys-Pro-Asp-Ala-Asn-Gln-Val-Leu-Ile-Gln-Val-Ile-Gly-Val). These peptides display homology with fragments of albumin from Trimeresurus flavoviridis. Bolus intra-arterial injection of the purified or the synthetic peptide produced a strong and sustained vasopressor response in the anaesthetized snake (CDT) and rats (Wistar); this hypotensive effect was also potentiated by captopril-an angiotensin-converting (0.1 mg/kg) enzyme inhibitor.

Effects of three vasoactive peptides isolated from the plasma of the snake Bothrops jararaca.

Incubation of plasma from the snake Bothrops jararaca (BJP) with trypsin generated two hypotensive peptides. The primary structure of the peptides was established for three sequences as: Asn-Pro-Phe-Val-Asp-Ala (fraction 13), Ser-Lys-Pro-Asn-Met-Ser-Asp-Glu-Ser-Leu-Ala-Val-Ala-Ile (fraction 14), Asn-Pro-Phe- Val-Asp-Ala (fraction 15). These peptides display homology with fragments of albumin from Trimeresurus flavoviridis. A bolus intra-arterial injection of the purified or the synthetic peptide produced a strong and sustained vasopressor response in the anaesthetized snake B. jararaca and Wistar rats; this hypotensive effect was also potentiated by captopril, an angiotensin-converting enzyme inhibitor (0.1 mg/kg). The natural concentrations of these peptides in plasma need to be determined and could play a physiological role in snake blood pressure regulation.

Reading, writing and drawing disorder due to right brain damage.

The author studies the written language in a series of right-handed patients with a right side cerebral lesion. Reduplications and omission of strokes and of letters are noted on writing. The cause of these disorders is attributed by the author to an impairment of visual and kinesthetic control of the act of writing, and not to a truly aphasic alteration. This impairment is part of a more general deficit, namely a distorted interpretation of spatial data. The author proposes to call this writing disorder afferent dysgraphia.

TsTx toxin isolated from Tityus serrulatus scorpion venom-induced spontaneous recurrent seizures and mossy fiber sprouting.

PURPOSE: TsTx is a scorpion alpha-type toxin that binds to site 3 of the Na+ channels in a voltage-dependent mode, slowing or blocking the inactivation mechanism of these channels (Possani et al., Eur J Biochem 1999). This binding increases depolarization time of the channel and consequently induces excessive neurotransmitter release. Previously we reported that hippocampal injection of TsTx induces clonic convulsions, electrographic seizures, and hippocampal damage. This investigation was designed to characterize the long-term behavioral, electroencephalographic (EEG), and histopathologic features after a single TsTx injection into the rat hippocampus. METHODS: Cannulas and electrodes were stereotaxically implanted in the CA1 dorsal hippocampus of rats. Three days after surgery, the animals were injected into the hippocampus with 1 microl of TsTx, 2 microg (n = 9) or 0.1 M phosphate buffer (n = 5). After injection, EEG records and behavioral observations were made during 10 h. For a period of 4 months, the animals were observed through direct visual observation for 8 h/day, 5 days/week for occurrence of convulsive seizures. At the end of the experiment, the animals were processed for histologic analyses. Sections 40- and 20-microm thick were stained according to neo-Timm or Nissl methods, respectively. Cell counts in the cresyl violet-stained sections were performed within the hippocampal pyramidal cell layers (CA1, CA3, and CA4) and granule cell of the dentate gyrus. RESULTS: Fifteen minutes after TsTx injection, facial automatisms, rearing, masticatory jaw movements, sniffing, and wet-dog shakes were observed. In approximately 1 h, limbic convulsions characterized by forelimb clonus, rearing, and falling after generalized clonic convulsion with jumping, wild running, and falling were observed. EEG showed isolated spikes and clusters of spikes that started in the hippocampus and evolved to the cortex, isolated seizures, and epileptic discharges delayed 1-2 min. These recurred repeatedly characterizing the status epilepticus (SE). SE was characterized in 77.5% of animals. Seizures were no longer observed 24 h after the injection. Spontaneous recurrent seizures (SRSs) occurred 31-49 days after TsTx injection. All animals that showed SE in the acute period developed SRSs. Facial myoclonus, generalized clonus, forelimb clonus, rearing, and falling characterized the seizures. The seizure frequency was 1-2 per animal per week. All rats injected with TsTx had significantly fewer cells in CA1, CA3, and CA4 subfield of the hippocampal formation compared with the animals of the control group (p < 0.05, analysis of variance, followed by Tukey test). Neo-Timm-positive granules, normally absent in the supragranular layer, were present in dentate gyrus of rats with TsTx-induced SRSs. CONCLUSIONS: Our results suggest that SRSs observed in this study may be a consequence of the TsTx-induced SE. All animals injected with the toxin showed massive neuronal loss in the hippocampal subfields CA1, CA3, and CA4, but only those that had SRSs showed mossy fiber sprouting in the supragranular layer of the dentate gyrus. This shows synaptic reorganization that also is observed in human epileptogenic tissue. These results indicate that TsTx toxin may be a useful tool for studies on neuronal lesions and/or experimental epilepsy. Studies on the mechanisms involved are under investigation.

Biochemical and pharmacological studies on a lethal neurotoxic polypeptide from Phoneutria nigriventer spider venom.

Fractionation of Phoneutria nigriventer spider venom by gel filtration and HPLC yielded a few fractions that induced different effects when administered intraperitoneally in mice. One of these fractions, PF3, was chemically characterized as a cysteine-rich polypeptide of approximately 8360 MW. Administered at 0.1 mg/kg, i.p., PF3 induced a progressive paralysis and death of mice within 30 minutes. Partial sequence analysis of PF3 revealed certain homologies with other spider toxins already described, particularly omega-AGAIIA (60%) from Agelenopsis aperta. Pharmacological characterization carried out in superfused chopped rat striatal tissues preloaded with [3H]-Dopamine ([3H]-DA) showed that PF3 (0.1 microgram/ml) decreased the [3H]-DA release induced by 20 mM K+ or 100 microM glutamate without changing the basal release. At 1 microgram/ml, PF3 inhibited 33% of the basal release of [3H]-DA; the transmitter release stimulated by K+ or by glutamate was reduced by respectively, 87% and 77% of corresponding control values. PF3 (0.1 micrograms/ml) altered the dose-response curves of glutamate (1 microM-10 mM), by reducing by 36% of its maximal effect. Naloxone (1 microM) did not influence the effect of PF3. The results indicate that PF3 inhibits the [3H]-DA release induced by membrane depolarization or that mediated by NMDA glutamate receptors. These data suggest that the mechanism of action of PF3 may involve a blockade of Ca2+ channels as well as a direct effect on the exocytotic machinery.

Isolation and properties of a new kallikrein inhibitor from Tityus serrulatus venom.

The kallikrein inhibitor-peptide content of Tityus serrulatus scorpion crude venom was purified by Sephadex G-50 and Sephadex G-25 fine gel filtration chromatographies, followed by two steps of reverse-phase column on HPLC. The isolated inhibitor peptide was homogeneous in its N-terminal and partial amino acid sequence, showing a molecular weight of 4.489 Da by mass spectrometry and amino acid analysis. The peptide was tested with rat plasma and urine kallikrein, which resulting in an inhibition with similar affinity to both enzymes, showing an IC50 of 14.3 microM after 13 and 8 min, respectively, using kininogen as substrate on the isolated guinea-pig ileum bioassay. The porcine pancreatic kallikrein showed after 10 min an IC50 value of 12.6 microM with H-D-Val-Leu-Arg-pNA HCl as substrate. In addition, the isolated peptide significantly inhibited porcine pancreatic kallikrein with values in the range of apparent or absolute calculated peptide Ki = 2.5 microM. The inhibitor was heat resistant and stable at pH values less than 5.

Lack of involvement of the kallikrein-kinin system in the depressor effect of alpha-methyldopa in normotensive rats.

1. We evaluated the involvement of circulating kinins in the hypotensive effect of the antihypertensive drug alpha-methyldopa (alpha-MD) in normotensive rats. 2. The alpha-MD effects were more pronounced in urethane-anesthetized rats than in unanesthetized animals. 3. Treatment with alpha-MD did not affect the circulating levels of kininogen, the bradykinin precursor. 4. Pretreatment with captopril that potentiates the depressor effects of bradykinin did not potentiate the hypotension to alpha-MD. 5. The lack of changes in kininogen levels and the failure of captopril to potentiate the depressor effects of alpha-MD rules out an involvement of the circulating kallikrein-kinin system in the response to alpha-MD.

Neurotoxic effects of three fractions isolated from Tityus serrulatus scorpion venom.

Scorpion venoms contain low molecular weight basic polypeptides, neurotoxins, that are the principal toxic agents. These toxins act on ion channels, promoting a derangement that may result in an abnormal release of neurotransmitters. In the present study we investigated some of the effects of the F, H and J fractions isolated from Tityus serrulatus scorpion venom on the central nervous system of rodents. The venom was partially purified by gel filtration chromatography. The neurotoxic effect of these fractions was studied on convulsive activity after intravenous injection, and on electrographic activity and neuronal integrity of rat hippocampus when injected directly into this brain area. The results showed that intravenous injection of the F and H fractions induced convulsions, and intrahippocampal injection caused electrographic seizures in rats and neuronal damage in specific hippocampal areas. Fraction J injected intravenously reduced the general activity of mice in the open field but induced no changes when injected into the brain. These results suggest that scorpion toxins are able to act directly on the central nervous system promoting behavioural, electrographic and histological modifications.