American Rattlesnake Crotalus Durissus Terrificus Research Articles

Intrahippocampal infusion of crotamine isolated from Crotalus durissus terrificus alters plasma and brain biochemical parameters.

Crotamine is one of the main constituents of the venom of the South American rattlesnake Crotalus durissus terrificus. Here we sought to investigate the inflammatory and toxicological effects induced by the intrahippocampal administration of crotamine isolated from Crotalus whole venom. Adult rats received an intrahippocampal infusion of crotamine or vehicle and were euthanized 24 h or 21 days after infusion. Plasma and brain tissue were collected for biochemical analysis. Complete blood count, creatinine, urea, glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), creatine-kinase (CK), creatine kinase-muscle B (CK-MB) and oxidative parameters (assessed by DNA damage and micronucleus frequency in leukocytes, lipid peroxidation and protein carbonyls in plasma and brain) were quantified. Unpaired and paired t-tests were used for comparisons between saline and crotamine groups, and within groups (24 h vs. 21 days), respectively. After 24 h crotamine infusion promoted an increase of urea, GOT, GPT, CK, and platelets values (p ≤ 0.01), while red blood cells, hematocrit and leukocytes values decreased (p ≤ 0.01). Additionally, 21 days after infusion crotamine group showed increased creatinine, leukocytes, TBARS (plasma and brain), carbonyl (plasma and brain) and micronucleus compared to the saline-group (p ≤ 0.01). Our findings show that crotamine infusion alter hematological parameters and cardiac markers, as well as oxidative parameters, not only in the brain, but also in the blood, indicating a systemic pro-inflammatory and toxicological activity. A further scientific attempt in terms of preserving the beneficial activity over toxicity is required.

Blood oxygen affinity increases during digestion in the South American rattlesnake, Crotalus durissus terrificus

Digesting snakes experience massive increases in metabolism that can last for many days and are accompanied by adjustments in the oxygen transport cascade. Accordingly, we examined the oxygen-binding properties of the blood in the South American rattlesnake (Crotalus durissus terrificus) during fasting and 24 and 48h after the snakes have ingested a rodent meal corresponding to 15% (±2%) of its own body mass. In general, oxygen–hemoglobin (Hb–O2) affinity was significantly increased 24h post-feeding, and then returned toward fasting values within 48h post-feeding. Content of organic phosphates ([NTP] and [NTP]/[Hb]), hemoglobin cooperativity (Hill's n), and Bohr Effect (ΔlogP50/ΔpH) were not affected by feeding. The postprandial increase in Hb–O2 affinity in the South American rattlesnake can be almost entirely ascribed by the moderate alkaline tide that follows meal ingestion. In general, digesting snakes were able to regulate blood metabolites at quite constant levels (e.g., plasma osmolality, lactate, glucose, and total protein levels). The level of circulating lipids, however, was considerably increased, which may be related to their mobilization, since lipids are known to be incorporated by the enterocytes after snakes have fed. In conclusion, our results indicate that the exceptional metabolic increment exhibited by C. d. terrificus during meal digestion is entirely supported by the aerobic pathways and that among the attending cardiorespiratory adjustments, pulmonary Hb–O2 loading is likely improved due to the increment in blood O2 affinity.

Crotoxin induces apoptosis and autophagy in human lung carcinoma cells in vitro via activation of the p38MAPK signaling pathway.

Crotoxin (CrTX) is the primary toxin in South American rattlesnake (Crotalus durissus terrificus) venom, and exhibits antitumor and other pharmacological actions in vivo and in vitro. Here, we investigated the molecular mechanisms of the antitumor action of CrTX in human lung carcinoma cells in vitro. Human lung squamous carcinoma SK-MES-1 cells were tested. The cytotoxicity of CrTX was evaluated in both MTT and colony formation assays. Cell cycle was investigated with flow cytometry. Cell apoptosis was studied with Hoechst 33258 and Annexin V-FITC staining. The levels of relevant proteins were analyzed using Western blot assays. CrTX (25, 50, 100 μmol/L) inhibited the growth and colony formation of SK-MES-1 cells in dose- and time-dependent manners. CrTX increased the proportion of S phase cells and dose-dependently induced cell apoptosis, accompanied by down-regulating the expression of proliferating cell nuclear antigen (PCNA), and increasing the level of cleaved caspase-3. Furthermore, CrTX dose-dependently increased the expression of autophagy-related proteins LC3-II and beclin 1, and decreased the level of p62 in the cells. Moreover, CrTX (50 μmol/L) significantly increased p38MAPK phosphorylation in the cells. Pretreatment of the cells with SB203580, a specific inhibitor of p38MAPK, blocked the inhibition of CrTX on cell proliferation, as well as CrTX-induced apoptosis and cleaved caspase-3 expression. The p38MAPK signaling pathway mediates CrTX-induced apoptosis and autophagy of human lung carcinoma SK-MES-1 cells in vitro.

Compartment syndrome after South American rattlesnake (Crotalus durissus terrificus) envenomation

Context. In order to report the outcome of a patient who developed compartment syndrome after South American rattlesnake (Crotalus durissus terrificus) envenomation, confirmed by subfascial pressure measurement and magnetic resonance imaging (MRI). Case details. A 63-year-old male was admitted 1 h after being bitten on the right elbow by a “large” snake, which was not brought for identification. Physical and laboratory features upon admission revealed two fang marks, local tense swelling, paresthesia, intense local pain, hypertension, coagulopathy, and CK = 1530 U/L (RV < 170 U/L). The case was initially treated with bothropic antivenom (80 mL, intravenously), with no improvement. Evolution within 13–14 h post-bite revealed generalized myalgia, muscle weakness, palpebral ptosis, and severe rhabdomyolysis (CK = 126,160 U/L) compatible with envenoming by C. d. terrificus. The patient was then treated with crotalic antivenom (200 mL, intravenously), fluid replacement, and urine alkalinization. Twenty-four–hour post-bite MRI showed marked muscular edema in the anterior compartment of the right forearm, with a high subfascial pressure (40 mmHg) being detected 1 h later. ELISA of a blood sample obtained upon admission, before antivenom infusion, revealed a high serum concentration of C. d. terrificus venom. No fasciotomy was performed and the patient was discharged seven days later without sequelae. Conclusion. Snakebite by C. d. terrificus with subfascial venom injection may lead to increased intracompartmental pressure.

Interaction of the rattlesnake toxin crotamine with model membranes.

Crotamine is one of the main constituents of the venom of the South American rattlesnake Crotalus durissus terrificus. A common gene ancestry and structural similarity with the antimicrobial β-defensins (identical disulfide bond pattern and highly positive net charge) suggested potential antimicrobial activities for this snake toxin. Although crotamine demonstrated low activity against both Gram-positive and Gram-negative bacteria, a pronounced antifungal activity was observed against Candida spp., Trichosporon spp., and Cryptococcus neoformans. Crotamine's selective antimicrobial properties, with no observable hemolytic activity, stimulated us to evaluate the potential applications of this polypeptide as an antiyeast or candicidal agent for medical and industrial application. Aiming to understand the mechanism(s) of action underlying crotamine antimicrobial activity and its selectivity for fungi, we present herein studies using membrane model systems (i.e., large unilamellar vesicles, LUVs, and giant unilamellar vesicles, GUVs), with different phospholipid compositions. We show here that crotamine presents a higher lytic activity on negatively charged membranes compared with neutral membranes, with or without cholesterol or ergosterol content. The vesicle burst was not preceded by membrane permeabilization as is generally observed for pore forming peptides. Although such a property of disrupting lipid membranes is very important to combat multiresistant fungi, no inhibitory activity was observed for crotamine against biofilms formed by several Candida spp. strains, except for a limited effect against C. krusei biofilm.

Enzyme specificity and effects of gyroxin, a serine protease from the venom of the South American rattlesnake Crotalus durissus terrificus, on protease-activated receptors

Gyroxin is a serine protease displaying a thrombin-like activity found in the venom of the South American rattlesnake Crotalus durissus terrificus. Typically, intravenous injection of purified gyroxin induces a barrel rotation syndrome in mice. The serine protease thrombin activates platelets aggregation by cleaving and releasing a tethered N-terminus peptide from the G-protein-coupled receptors, known as protease-activated receptors (PARs). Gyroxin also presents pro-coagulant activity suggested to be dependent of PARs activation. In the present work, the effects of these serine proteases, namely gyroxin and thrombin, on PARs were comparatively studied by characterizing the hydrolytic specificity and kinetics using PARs-mimetic FRET peptides. We show for the first time that the short (sh) and long (lg) peptides mimetizing the PAR-1, -2, -3, and -4 activation sites are all hydrolyzed by gyroxin exclusively after the Arg residues. Thrombin also hydrolyzes PAR-1 and -4 after the Arg residue, but hydrolyzes sh and lg PAR-3 after the Lys residue. The kcat/KM values determined for gyroxin using sh and lg PAR-4 mimetic peptides were at least 2150 and 400 times smaller than those determined for thrombin, respectively. For the sh and lg PAR-2 mimetic peptides the kcat/KM values determined for gyroxin were at least 6500 and 2919 times smaller than those determined for trypsin, respectively. The kcat/KM values for gyroxin using the PAR-1 and -3 mimetic peptides could not be determined due to the extreme low hydrolysis velocity. Moreover, the functional studies of the effects of gyroxin on PARs were conducted in living cells using cultured astrocytes, which express all PARs. Despite the ability to cleavage the PAR-1, -2, -3, and -4 peptides, gyroxin was unable to activate the PARs expressed in astrocytes as determined by evaluating the cytosolic calcium mobilization. On the other hand, we also showed that gyroxin is able to interfere with the activation of PAR-1 by thrombin or by synthetic PAR-1 agonist in cultured astrocytes. Taken together, the data presented here allow us showing that gyroxin cleaves PARs-mimetic peptides slowly and it does not induce activation of PARs in astrocytes. Although gyroxin does not mobilize calcium it was shown to interfere with PARs activation by thrombin and PAR-1 agonist. The determination of gyroxin enzymatic specificity and kinetics on PAR-1, -2, -3, and -4 will potentially help to fill the gap in the knowledge in this field, as the PARs are still believed to have a key role for the gyroxin biological effects.

Does the rattle of Crotalus durissus terrificus reveal its dietary history?

BackgroundEnvironmental devastation threatens the survival of many species, including venomous snakes such as the South American rattlesnake Crotalus durissus terrificus. This observation is based on the decrease of snakes collected and donated to Brazilian research institutes. Nevertheless, some individuals have managed to survive and procreate. The question is how these snakes are adapting in these new environmental conditions.MethodsTo answer it, the carbon-13 level of rattlesnakes and their feed (either laboratory or wild mice) was evaluated by isotope-ratio mass spectrometry. Thus, rattle segments from 16 adults and 15 offspring of captive snakes, and of three wild newborn C. d. terrificus were evaluated as well as 17 Mus musculus mice captured in traps, four live feeder mice and the ration offered to mice at animal houses.ResultsThe isotopic exchange time of the captive adult snakes (n = 16) varied between 33 and 37 months and of captive-born animals (n = 15), until reaching a plateau of equilibrium, varied from 18 to 24 months. Regarding the captured Mus musculus (n = 17), 88.23% (n = 15) were from a C4 environment. Of the six rattle rings from offspring of captured C. d. terrificus, five were from a C4 environment, whereas of the 170 rattle rings studied, 60% originated from a C3 environment and 40% from a C4. The same carbon-13 values were found in captive snakes.ConclusionsBased on the present results, it can be inferred that most C. d. terrificus snakes (60%) fed animals from a C3 environment; birds consist of an alimentary alternative for snakes, as well as rodents, small reptiles and amphibians; different venom compositions among snakes from the same region may be related to the food type; the primary rattle of offspring reflects the maternal diet during gestation; and, finally, the different rattle rings indicate the alimentary history of these animals.Electronic supplementary materialThe online version of this article (doi:10.1186/1678-9199-20-53) contains supplementary material, which is available to authorized users.

Assessing the Complex Formation between Crotamine – A Natural Cellpenetrating Peptide – and DNA Using High Sensitive Fluorescence Exclusion Assay

Crotamine is a natural cationic polypeptide isolated from the venom of South American rattlesnake (Crotalus durissus terrificus) capable of forming complexes with DNA molecules by means of electrostatic interactions and translocating biological membranes. With such properties, crotamine can act as a transfection agent for several cell types but mainly in a stage of proliferation. However, to improve the biological use of this peptide, it is interesting to better characterize the formation and stability of crotamina-DNA complex. Thus, the present study aimed to analyze the stoichiometry, formation kinetics and protease stability of crotamina-EGFP complex using high sensitive fluorescent methodology, based on fluorophore exclusion assay. The best DNA:crotamine mass proportion for complex formation ranges between 1:10 to 1:40 as mass ratio. In 1:10 group, crotamine was able to complex about 75% of the available DNA as fast as in 20 seconds, and after 10 min of reaction crotamine reached its maximum complexation capacity, being able to complex 80% of total amount of DNA available. Under these conditions, we have determined that 1μg of crotamine can complex 80 μg of circular plasmid DNA (~4700 bp). Crotamine-DNA complex was resistant to proteinase K degradation even after twelve hours of enzymatic incubation. Additionally, the complex was sufficiently stable to trypsin digestion for a long incubation time: more than 73% of crotamine-DNA condensates maintained the integrity after 60 min, whereas 55% was intact after 180 min. Crotamine-DNA complexes were resistant to physiological temperatures, as well as stable for 15 days at 4°C and for two months when stored at -20°C. These results lead us to think that, in the best conditions of preparation proposed here, crotamine-DNA condensates can be used in an efficient and standardized manner as a potential agent of transfection in a transgenesis programme.

Antinociceptive activity of crotoxin in the central nervous system: A functional Magnetic Resonance Imaging study

Crotoxin, the main neurotoxic component of the venom of South American rattlesnake (Crotalus durissus terrificus), is reported to have potent antinociceptive activity. Several authors have shown mainly in behavioral pain models that crotoxin induces antinociceptive effects, supposed to be mediated by actions on the central nervous system. The antinociceptive effects of crotoxin (45 μg/kg ip) in rats were verified in this study by increased response latencies in a Hargreaves test and tail flick test. In addition, it was demonstrated that crotoxin does not lead to motor impairments during a rotarod test and open field test. The main objective, carried out by blood oxygen level dependent functional Magnetic Resonance Imaging (BOLD fMRI) in anesthetized rats, was to determine which specific brain structures are involved in these antinociceptive effects. Moreover, potential antihyperalgesic effects were investigated by inducing a local hyperalgesia on the left hind paw. Therefore, antinociceptive effects (right paw) and antihyperalgesic effects (left paw) of crotoxin were able to be differentiated. As a result, crotoxin exhibited dominant antihyperalgesic but also antinociceptive effects during pain stimulation. Reductions of BOLD signal already occurred in brain input structures but were most prominent in primary and secondary somatosensory cortices. In conclusion, BOLD fMRI in anesthetized rats proved to be a helpful tool in toxinology, particularly in unraveled mechanisms of modulating nociception in the central nervous system by (potential) analgesics like crotoxin.

Coagulopathy as the main systemic manifestation after envenoming by a juvenile South American rattlesnake (Crotalus durissus terrificus): Case report

Context. Rattlesnake bites in Brazil are generally caused by adult individuals, with most of the envenomed patients showing systemic manifestations that include varying degrees of neurotoxicity (acute myasthenia), rhabdomyolysis and coagulopathy, with only mild or no local manifestations. We report a case of envenoming by a juvenile South American rattlesnake (Crotalus durissus terrificus) that involved coagulopathy as the main systemic manifestation. Case details. A 19-year-old male was admitted to our Emergency Department with coagulopathy (incoagulable PT, APTT and INR), no remarkable local manifestations and no signs/symptoms of myasthenia or rhabdomyolysis (serum CK, LDH, ALT and AST within reference levels) 5 days after being bitten by a small snake that was described as a rattlesnake but was not brought for identification at admission. The patient had already been treated in another Emergency Department with i.v. bothropic antivenom (AV) 1 h and 4 days post-bite. Based on the possibility of an unusual rattlesnake bite, crotalic AV was administered i.v., which improved the coagulation (9 h post-CroAV, INR = 2.11; 36 h post-CroAV, INR = 1.42). During hospitalization, relatives brought the snake that caused the bite, which was identified as a 38-cm long C. d. terrificus. Discussion. Little is known about the clinical manifestations after bites by juvenile C. d. terrificus. This case shows that systemic envenoming by juvenile C. d. terrificus may result in coagulopathy as the main systemic manifestation, without neuromyotoxic features normally associated with bites by adult specimens. Despite the delayed administration, crotalic AV was effective in improving the blood coagulation.

Peripheral kappa and delta opioid receptors are involved in the antinociceptive effect of crotalphine in a rat model of cancer pain

Cancer pain is an important clinical problem and may not respond satisfactorily to the current analgesic therapy. We have characterized a novel and potent analgesic peptide, crotalphine, from the venom of the South American rattlesnake Crotalus durissus terrificus. In the present work, the antinociceptive effect of crotalphine was evaluated in a rat model of cancer pain induced by intraplantar injection of Walker 256 carcinoma cells. Intraplantar injection of tumor cells caused the development of hyperalgesia and allodynia, detected on day 5 after tumor cell inoculation. Crotalphine (6μg/kg), administered p.o., blocked both phenomena. The antinociceptive effect was detected 1h after treatment and lasted for up to 48h. Intraplantar injection of nor-binaltorphimine (50g/paw), a selective antagonist of κ-opioid receptors, antagonized the antinociceptive effect of the peptide, whereas N,N-diallyl-Tyr-Aib-Phe-Leu (ICI 174,864, 10μg/paw), a selective antagonist of δ-opioid receptors, partially reversed this effect. On the other hand, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr amide (CTOP, 20g/paw), an antagonist of μ-opioid receptors, did not modify crotalphine-induced antinociception. These data indicate that crotalphine induces a potent and long lasting opioid-mediated antinociception in cancer pain.

Crotoxin in humans: analysis of the effects on extraocular and facial muscles

Crotoxin is the main neurotoxin of South American rattlesnake Crotalus durissus terrificus. The neurotoxic action is characterized by a presynaptic blockade. The purpose of this research is to assess the ability of crotoxin to induce temporary paralysis of extraocular and facial muscles in humans. Doses of crotoxin used ranged from 2 to 5 units (U), each unit corresponding to one LD50. We first applied 2U of crotoxin in one of the extraocular muscles of 3 amaurotic individuals to be submitted to ocular evisceration. In the second stage, we applied crotoxin in 12 extraocular muscles of 9 patients with strabismic amblyopia. In the last stage, crotoxin was used in the treatment of blepharospasm in another 3 patients. No patient showed any systemic side effect or change in vision or any eye structure problem after the procedure. The only local side effects observed were slight conjunctival hyperemia, which recovered spontaneously. In 2 patients there was no change in ocular deviation after 2U crotoxin application. Limitation of the muscle action was observed in 8 of the 12 applications. The change in ocular deviation after application of 2U of crotoxin (9 injections) was in average 15.7 prism diopters (PD). When the dose was 4U (2 applications) the change was in average 37.5 PD and a single application of 5U produced a change of 16 PD in ocular deviation. This effect lasted from 1 to 3 months. Two of the 3 patients with blepharospasm had the hemifacial spasm improved with crotoxin, which returned after 2 months. This study provides data suggesting that crotoxin may be a useful new therapeutic option for the treatment of strabismus and blepharospasm. We expect that with further studies crotoxin could be an option for many other medical areas.

DNA-Interactive Properties of Crotamine, a Cell-Penetrating Polypeptide and a Potential Drug Carrier

Crotamine, a 42-residue polypeptide derived from the venom of the South American rattlesnake Crotalus durissus terrificus, has been shown to be a cell-penetrating protein that targets chromosomes, carries plasmid DNA into cells, and shows specificity for actively proliferating cells. Given this potential role as a nucleic acid-delivery vector, we have studied in detail the binding of crotamine to single- and double-stranded DNAs of different lengths and base compositions over a range of ionic conditions. Agarose gel electrophoresis and ultraviolet spectrophotometry analysis indicate that complexes of crotamine with long-chain DNAs readily aggregate and precipitate at low ionic strength. This aggregation, which may be important for cellular uptake of DNA, becomes less likely with shorter chain length. 25-mer oligonucleotides do not show any evidence of such aggregation, permitting the determination of affinities and size via fluorescence quenching experiments. The polypeptide binds non-cooperatively to DNA, covering about 5 nucleotide residues when it binds to single (ss) or (ds) double stranded molecules. The affinities of the protein for ss- vs. ds-DNA are comparable, and inversely proportional to salt levels. Analysis of the dependence of affinity on [NaCl] indicates that there are a maximum of ∼3 ionic interactions between the protein and DNA, with some of the binding affinity attributable to non-ionic interactions. Inspection of the three-dimensional structure of the protein suggests that residues 31 to 35, Arg-Trp-Arg-Trp-Lys, could serve as a potential DNA-binding site. A hexapeptide containing this sequence displayed a lower DNA binding affinity and salt dependence as compared to the full-length protein, likely indicative of a more suitable 3D structure and the presence of accessory binding sites in the native crotamine. Taken together, the data presented here describing crotamine-DNA interactions may lend support to the design of more effective nucleic acid drug delivery vehicles which take advantage of crotamine as a carrier with specificity for actively proliferating cells.

Study of crotoxin on the induction of paralysis in extraocular muscle in animal model

Crotoxin is the major toxin of the venom of the South American rattlesnake Crotalus durissus terrificus, capable of causing a blockade of the neurotransmitters at the neuromuscular junction. The objective of this study was to appraise the action and effectiveness of the crotoxin induced paralysis of the extraocular muscle and to compare its effects with the botulinum toxin type A (BT-A). The crotoxin, with LD50 of 1.5 µg, was injected into the superior rectus muscle in ten New Zealand rabbits. The concentration variance was 0.015 up to 150 µg. Two rabbits received 2 units of botulinum toxin type A for comparative analysis. The evaluation of the paralysis was performed using serial electromyography. After the functional recovery of the muscles, which occurred after two months, six rabbits were sacrificed for anatomopathology study. The animals did not show any evidence of systemic toxicity. Transitory ptosis was observed in almost every animal and remained up to fourteen days. These toxins caused immediate blockade of the electrical potentials. The recovery was gradual in the average of one month with regeneration signs evident on the electromyography. The paralysis effect of the crotoxin on the muscle was proportional to its concentration. The changes with 1.5 µg crotoxin were similar to those produced by the botulinum toxin type A. The histopathology findings were localized to the site of the injection. No signs of muscle fiber's necrosis were seen in any sample. The alterations induced by crotoxin were also proportional to the concentration and similar to botulinum toxin type A in concentration of 1.5 µg. Crotoxin was able to induce transitory paralysis of the superior rectus muscle. This effect was characterized by reduction of action potentials and non-specific signs of fibrillation. Crotoxin, in concentration of 1.5 µg was able to induce similar effects as botulinum toxin type A.

Envenomations by Bothrops and Crotalus Snakes Induce the Release of Mitochondrial Alarmins

Skeletal muscle necrosis is a common manifestation of viperid snakebite envenomations. Venoms from snakes of the genus Bothrops, such as that of B. asper, induce muscle tissue damage at the site of venom injection, provoking severe local pathology which often results in permanent sequelae. In contrast, the venom of the South American rattlesnake Crotalus durissus terrificus, induces a clinical picture of systemic myotoxicity, i.e., rhabdomyolysis, together with neurotoxicity. It is known that molecules released from damaged muscle might act as ‘danger’ signals. These are known as ‘alarmins’, and contribute to the inflammatory reaction by activating the innate immune system. Here we show that the venoms of B. asper and C. d. terrificus release the mitochondrial markers mtDNA (from the matrix) and cytochrome c (Cyt c) from the intermembrane space, from ex vivo mouse tibialis anterior muscles. Cyt c was released to a similar extent by the two venoms whereas B. asper venom induced the release of higher amounts of mtDNA, thus reflecting hitherto some differences in their pathological action on muscle mitochondria. At variance, injection of these venoms in mice resulted in a different time-course of mtDNA release, with B. asper venom inducing an early onset increment in plasma levels and C. d. terrificus venom provoking a delayed release. We suggest that the release of mitochondrial ‘alarmins’ might contribute to the local and systemic inflammatory events characteristic of snakebite envenomations.

The peripheral L-arginine–nitric oxide–cyclic GMP pathway and ATP-sensitive K+ channels are involved in the antinociceptive effect of crotalphine on neuropathic pain in rats

Crotalphine, a 14 amino acid peptide first isolated from the venom of the South American rattlesnake Crotalus durissus terrificus, induces a peripheral long-lasting and opioid receptor-mediated antinociceptive effect in a rat model of neuropathic pain induced by chronic constriction of the sciatic nerve. In the present study, we further characterized the molecular mechanisms involved in this effect, determining the type of opioid receptor responsible for this effect and the involvement of the nitric oxide-cyclic GMP pathway and of K⁺ channels. Crotalphine (0.2 or 5 μg/kg, orally; 0.0006 μg/paw), administered on day 14 after nerve constriction, inhibited mechanical hyperalgesia and low-threshold mechanical allodynia. The effect of the peptide was antagonized by intraplantar administration of naltrindole, an antagonist of δ-opioid receptors, and partially reversed by norbinaltorphimine, an antagonist of κ-opioid receptors. The effect of crotalphine was also blocked by 7-nitroindazole, an inhibitor of the neuronal nitric oxide synthase; by 1H-(1,2,4) oxadiazolo[4,3-a]quinoxaline-1-one, an inhibitor of guanylate cyclase activation; and by glibenclamide, an ATP-sensitive K⁺ channel blocker. The results suggest that peripheral δ-opioid and κ-opioid receptors, the nitric oxide-cyclic GMP pathway, and ATP-sensitive K⁺ channels are involved in the antinociceptive effect of crotalphine. The present data point to the therapeutic potential of this peptide for the treatment of chronic neuropathic pain.

The Natural Cell-Penetrating Peptide Crotamine Targets Tumor Tissue in Vivo and Triggers a Lethal Calcium-Dependent Pathway in Cultured Cells

Our goal was to demonstrate the in vivo tumor specific accumulation of crotamine, a natural peptide from the venom of the South American rattlesnake Crotalus durissus terrificus, which has been characterized by our group as a cell penetrating peptide with a high specificity for actively proliferating cells and with a concentration-dependent cytotoxic effect. Crotamine cytotoxicity has been shown to be dependent on the disruption of lysosomes and subsequent activation of intracellular proteases. In this work, we show that the cytotoxic effect of crotamine also involves rapid intracellular calcium release and loss of mitochondrial membrane potential as observed in real time by confocal microscopy. The intracellular calcium overload induced by crotamine was almost completely blocked by thapsigargin. Microfluorimetry assays confirmed the importance of internal organelles, such as lysosomes and the endoplasmic reticulum, as contributors for the intracellular calcium increase, as well as the extracellular medium. Finally, we demonstrate here that crotamine injected intraperitoneally can efficiently target remote subcutaneous tumors engrafted in nude mice, as demonstrated by a noninvasive optical imaging procedure that permits in vivo real-time monitoring of crotamine uptake into tumor tissue. Taken together, our data indicate that the cytotoxic peptide crotamine can be used potentially for a dual purpose: to target and detect growing tumor tissues and to selectively trigger tumor cell death.

Crotamine, a Small Basic Polypeptide Myotoxin from Rattlesnake Venom with Cell-Penetrating Properties

Crotamine, a low molecular weight cationic polypeptide from the venom of the South American rattlesnake Crotalus durissus terrificus is a natural cell-penetrating peptide with functional versatility. The presence of nine lysine residues and three disulfide bonds renders crotamine highly compact, stable and positively charged. Topologically, crotamine adopts an ancient β-defensin fold that is found in diverse families of endogenous and venom polypeptides dedicated to host defense. Crotamine is unique among several classes of bioactive peptides because it possesses both cell penetrating and antimicrobial activities and selective biological action toward some cell types at a given cell cycle phase. Because it can rapidly and efficiently translocate into actively proliferating cells, crotamine is being investigated for labeling highly replicating cells and for use as a chemotherapeutic adjuvant. Peptides derived from crotamine, nucleolar targeting peptides (NrTPs), have been designed and are being studied. NrTPs retain some crotamine properties, such as efficient cellular uptake and preferential nuclear localization whereas they improve upon other properties. For example, NrTPs are smaller than crotamine, show higher preferential nucleolar localization, and better facilitate ZIP-code localization of therapeutic proteins.

Role of a Novel Tetrodotoxin-Resistant Sodium Channel in the Nitrergic Relaxation of Corpus Cavernosum from the South American Rattlesnake Crotalus Durissus Terrificus

Coitus in snakes may last up to 28 hours; however, the mechanisms involved are unknown. To evaluate the relevance of the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP)-phosphodiesterase type 5 (PDE5) system in snake corpus cavernosum reactivity. Hemipenes were removed from anesthetized South American rattlesnakes (Crotalus durissus terrificus) and studied by light and scanning electronic microscopy. Isolated Crotalus corpora cavernosa (CCC) were dissected from the non-spiny region of the hemipenises, and tissue reactivity was assessed in organ baths. Cumulative concentration-response curves were constructed for acetylcholine (ACh), sodium nitroprusside (SNP), 5-cyclopropyl-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]pyrimidin-4-ylamine (BAY 41-2272), and tadalafil in CCC precontracted with phenylephrine. Relaxation induced by electrical field stimulation (EFS) was also done in the absence and presence of N(ω) nitro-L-arginine methyl ester (L-NAME; 100 µM), 1H-[1, 2, 4] oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 µM) and tetrodotoxin (TTX; 1 µM). The hemipenes consisted of two functionally concentric corpora cavernosa, one of them containing radiating bundles of smooth muscle fibers (confirmed by α-actin immunostaining). Endothelial and neural nitric oxide synthases were present in the endothelium and neural structures, respectively; whereas soluble guanylate cyclase and PDE5 were expressed in trabecular smooth muscle. ACh and SNP relaxed isolated CCC, with the relaxations being markedly reduced by L-NAME and ODQ, respectively. BAY 41-2272 and tadalafil caused sustained relaxations with potency (pEC(50) ) values of 5.84 ± 0.17 and 5.10 ± 0.08 (N=3-4), respectively. In precontracted CCC, EFS caused frequency-dependent relaxations that lasted three times longer than those in mammalian CC. Although these relaxations were almost abolished by either L-NAME or ODQ, they were unaffected by TTX. In contrast, EFS-induced relaxations in marmoset CC were abolished by TTX. Rattlesnake CC relaxation is mediated by the NO-cGMP-PDE5 pathway in a manner similar to mammals. The novel TTX-resistant Na channel identified here may be responsible for the slow response of smooth muscle following nerve stimulation and could explain the extraordinary duration of snake coitus.

Gyroxin increases blood-brain barrier permeability to Evans blue dye in mice

Gyroxin is a serine protease enzyme component of the South American rattlesnake (Crotalus durissus terrificus) venom. This toxin displays several activities, including the induction of blood coagulation (fibrinogenolytic activity), vasodilation and neurotoxicity, resulting in an effect called barrel rotation. The mechanisms involved in this neurotoxic activity are not well known. Because gyroxin is a member of a potentially therapeutic family of enzymes, including thrombin, ancrod, batroxobin, trypsin and kallicrein, the identification of the mechanism of gyroxin’s action is extremely important. In this study, gyroxin was isolated from crude venom by affinity and molecular exclusion chromatography. Analysis of the isolated gyroxin via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a single protein band with a molecular weight of approximately 28 kDa, confirming the identity of the molecule. Furthermore, intravenous administration of purified gyroxin (0.25 μg/g of body weight) to mice resulted in symptoms compatible with barrel rotation syndrome, confirming the neurotoxic activity of the toxin. Mice treated with gyroxin showed an increase in the concentration of albumin-Evans blue in brain extracts, indicating an increase in the blood-brain barrier (BBB) permeability. This gyroxin-induced increase in BBB permeability was time-dependent, reaching a peak within 15 min after exposure, similar to the time span in which the neurotoxic syndrome (barrel rotation) occurs. This work provides the first evidence of gyroxin’s capacity to temporarily alter the permeability of the BBB.