The genetic regulatory architecture and epigenomic basis for age-related changes in rattlesnake venom.

Developmental phenotypic changes can evolve under selection imposed by age- and size-related ecological differences. Many of these changes occur through programmed alterations to gene expression patterns, but the molecular mechanisms and gene-regulatory networks underlying these adaptive changes remain poorly understood. Many venomous snakes, including the eastern diamondback rattlesnake (Crotalus adamanteus), undergo correlated changes in diet and venom expression as snakes grow larger with age, providing models for identifying mechanisms of timed expression changes that underlie adaptive life history traits. By combining a highly contiguous, chromosome-level genome assembly with measures of expression, chromatin accessibility, and histone modifications, we identified cis-regulatory elements and trans-regulatory factors controlling venom ontogeny in the venom glands of C. adamanteus. Ontogenetic expression changes were significantly correlated with epigenomic changes within genes, immediately adjacent to genes (e.g., promoters), and more distant from genes (e.g., enhancers). We identified 37 candidate transcription factors (TFs), with the vast majority being up-regulated in adults. The ontogenetic change is largely driven by an increase in the expression of TFs associated with growth signaling, transcriptional activation, and circadian rhythm/biological timing systems in adults with corresponding epigenomic changes near the differentially expressed venom genes. However, both expression activation and repression contributed to the composition of both adult and juvenile venoms, demonstrating the complexity and potential evolvability of gene regulation for this trait. Overall, given that age-based trait variation is common across the tree of life, we provide a framework for understanding gene-regulatory-network-driven life-history evolution more broadly.

Effects of the Heterodimeric Neurotoxic Phospholipase A2 from the Venom of Vipera nikolskii on the Contractility of Rat Papillary Muscles and Thoracic Aortas.

Phospholipases A2 (PLA2s) are a large family of snake toxins manifesting diverse biological effects, which are not always related to phospholipolytic activity. Snake venom PLA2s (svPLA2s) are extracellular proteins with a molecular mass of 13-14 kDa. They are present in venoms in the form of monomers, dimers, and larger oligomers. The cardiovascular system is one of the multiple svPLA2 targets in prey organisms. The results obtained previously on the cardiovascular effects of monomeric svPLA2s were inconsistent, while the data on the dimeric svPLA2 crotoxin from the rattlesnake Crotalus durissus terrificus showed that it significantly reduced the contractile force of guinea pig hearts. Here, we studied the effects of the heterodimeric svPLA2 HDP-1 from the viper Vipera nikolskii on papillary muscle (PM) contractility and the tension of the aortic rings (ARs). HDP-1 is structurally different from crotoxin, and over a wide range of concentrations, it produced a long-term, stable, positive inotropic effect in PMs, which did not turn into contractures at the concentrations studied. This also distinguishes HDP-1 from the monomeric svPLA2s, which at high concentrations inhibited cardiac function. HDP-1, when acting on ARs preconstricted with 10 µM phenylephrine, induced a vasorelaxant effect, similar to some other svPLA2s. These are the first indications of the cardiac and vascular effects of true vipers' heterodimeric svPLA2s.

Structural studies with crotoxin B from Crotalus durissus collilineatus venom suggest a heterodimeric assembly formed by two new isoforms.

In accidents involving Crotalus snakes, the crotoxin complex (CTX) plays lethal action due to its neurotoxic activity. On the other hand, CTX have potential biotechnological application due to its anti-tumoral, anti-inflammatory, antimicrobial, analgesic and immunomodulatory properties. CTX is a heterodimer composed of Crotoxin A (CA or crotapotin), the acidic nontoxic and non-enzymatic component and; Crotoxin B (CB), a basic, toxic and catalytic PLA2. Currently, there are two classes of CTX isoforms, whose differences in their biological activities have been attributed to features presented in CB isoforms. Here, we present the crystal structure of CB isolated from the Crotalus durissus collilineatus venom. It amino acid sequence was assigned using the SEQUENCE SLIDER software, which revealed that the crystal structure is a heterodimer composed of two new CB isoforms (colCB-A and colCB-B). Bioinformatic and biophysical analyses showed that the toxin forms a tetrameric assembly in solution similar to CB from Crotalus durissus terrificus venom, despite some differences observed at the dimeric interface. By the previously proposed classification, the colCB-B presents features of the class I isoforms while colCB-A cannot be classified into classes I and II based on its amino acid sequence. Due to similar features observed for other CB isoforms found in the NCBI database and the results obtained for colCB-A, we suggest that there are more than two classes of CTX and CB isoforms in crotalic venoms.

The best of both worlds? Rattlesnake hybrid zones generate complex combinations of divergent venom phenotypes that retain high toxicity.

Studying the consequences of hybridization between closely related species with divergent traits can reveal patterns of evolution that shape and maintain extreme trophic adaptations. Snake venoms are an excellent model system for examining the evolutionary and ecological patterns that underlie highly selected polymorphic traits. Here we investigate hybrid venom phenotypes that result from natural introgression between two rattlesnake species that express highly divergent venom phenotypes: Crotalus o. concolor and C. v. viridis. Though not yet documented, interbreeding between these species may lead to novel venom phenotypes with unique activities that break the typical trends of venom composition in rattlesnakes. The characteristics of these unusual phenotypes could unveil the roles of introgression in maintaining patterns of venom composition and variation, including the near ubiquitous dichotomy between neurotoxic or degradative venoms observed across rattlesnakes. We use RADseq data to infer patterns of gene flow and hybrid ancestry between these diverged lineages and link these genetic data with analyses of venom composition, biological activity, and whole animal model toxicity tests to understand the impacts of introgression on venom composition. We find that introgressed populations express admixed venom phenotypes that do not sacrifice biological activity (lethal toxicity) or overall abundance of dominant toxins compared to parental venoms. These hybridized venoms therefore do not represent a trade-off in functionality between the typical phenotypic extremes but instead represent a unique combination of characters whose expression appears limited to the hybrid zone.

Proteomic Profiling of Extracellular Vesicles Isolated from Plasma and Peritoneal Exudate in Mice Induced by Crotalus scutulatus scutulatus Crude Venom and Its Purified Cysteine-Rich Secretory Protein (Css-CRiSP).

Increased vascular permeability is a frequent outcome of viperid snakebite envenomation, leading to local and systemic complications. We reported that snake venom cysteine-rich secretory proteins (svCRiSPs) from North American pit vipers increase vascular permeability both in vitro and in vivo. They also induce acute activation of several adhesion and signaling molecules that may play a critical role in the pathophysiology of snakebites. Extracellular vesicles (EVs) have gained interest for their diverse functions in intercellular communication, regulating cellular processes, blood-endothelium interactions, vascular permeability, and immune modulation. They also hold potential as valuable biomarkers for diagnosing, predicting, and monitoring therapeutic responses in different diseases. This study aimed to identify proteins in peritoneal exudate and plasma EVs isolated from BALB/c mice following a 30 min post-injection of Crotalus scutulatus scutulatus venom and its purified CRiSP (Css-CRiSP). EVs were isolated from these biofluids using the EVtrap method. Proteomic analysis of exudate- and plasma-derived EVs was performed using LC-MS/MS. We observed significant upregulation or downregulation of proteins involved in cell adhesion, cytoskeleton rearrangement, signal transduction, immune responses, and vesicle-mediated transports. These findings suggest that svCRiSPs play a crucial role in the acute effects of venom and contribute to the local and systemic toxicity of snakebites.

Single-Cell Heterogeneity in Snake Venom Expression Is Hardwired by Co-Option of Regulators from Progressively Activated Pathways.

The ubiquitous cellular heterogeneity underlying many organism-level phenotypes raises questions about what factors drive this heterogeneity and how these complex heterogeneous systems evolve. Here, we use single-cell expression data from a Prairie rattlesnake (Crotalus viridis) venom gland to evaluate hypotheses for signaling networks underlying snake venom regulation and the degree to which different venom gene families have evolutionarily recruited distinct regulatory architectures. Our findings suggest that snake venom regulatory systems have evolutionarily co-opted trans-regulatory factors from extracellular signal-regulated kinase and unfolded protein response pathways that specifically coordinate expression of distinct venom toxins in a phased sequence across a single population of secretory cells. This pattern of co-option results in extensive cell-to-cell variation in venom gene expression, even between tandemly duplicated paralogs, suggesting this regulatory architecture has evolved to circumvent cellular constraints. While the exact nature of such constraints remains an open question, we propose that such regulatory heterogeneity may circumvent steric constraints on chromatin, cellular physiological constraints (e.g., endoplasmic reticulum stress or negative protein-protein interactions), or a combination of these. Regardless of the precise nature of these constraints, this example suggests that, in some cases, dynamic cellular constraints may impose previously unappreciated secondary constraints on the evolution of gene regulatory networks that favors heterogeneous expression.

Intracellular Zinc Trafficking during Crotalus atrox Venom Wound Development.

In this study, we examined zinc trafficking in human umbilical vein endothelial cells (HUVEC) stimulated with Crotalus atrox (CA venom) snake venom. We utilized MTS cytotoxicity assays to monitor the cytotoxic range of CA venom. HUVEC monolayers stimulated with 10 µg/mL CA venom for 3 h displayed cellular retraction, which coincided with 53.0 ± 6.5 percent viability. In contrast, venom concentrations of 100 µg/mL produced a complete disruption of cellular adherence and viability decreased to 36.6 ± 1.0. The zinc probe Fluozin-3AM was used to detect intracellular zinc in non-stimulated controls, HUVEC stimulated with 10 µg/mL CA venom or HUVEC preincubated with TPEN for 2 h then stimulated with 10 µg/mL CA venom. Fluorescent intensity analysis returned values of 1434.3 ± 197.4 for CA venom demonstrating an increase of about two orders of magnitude in labile zinc compared to non-stimulated controls. Endothelial response to CA venom induced a 96.1 ± 3.0- and 4.4 ± 0.41-fold increase in metallothionein 1X (MT1X) and metallothionein 2A (MT2A) gene expression. Zinc chelation during CA venom stimulation significantly increased cell viability, suggesting that the maintenance of zinc homeostasis during envenomation injury improves cell survival.

Venom comparisons of endemic and micro-endemic speckled rattlesnakes Crotalus mitchellii, C. polisi and C. thalassoporus from Baja California Peninsula.

A high diversity of rattlesnake species can be found in the Baja California peninsula and the island of the Gulf of California, nevertheless, their venom has been poorly evaluated. The aim of this work was to present the first characterization of endemic Crotalus mitchellii, micro endemic C. polisi and C. thalassoporus venoms. All samples provoke human plasma coagulation showing doses in the rank of 2.3-41.0 µg and also produce rapid hydrolysis of the alpha chain of bovine fibrinogen while the beta chain is attacked at larger incubation periods by C. polisi and especially by C. thalassoporus. Phospholipase activity ranging from 23.2 to 173.8 U/mg. The venoms of C. thalassoporus and C. polisi show very high hemorrhagic activity (from 0.03 to 0.31 µg). A total of 130 toxin-related proteins were identified and classified into ten families. Crotalus mitchellii venom was characterized by high abundance of crotoxin-like and other phospholipase proteins (34.5%) and serine proteinases (29.8%). Crotalus polisi showed a similar proportion of metalloproteinases (34%) and serine proteinases (22.8%) components with important contribution of C-type lectins (14.3%) and CRiSP (14.0%) proteins. Venom of C. thalassoporus is dominated by metalloproteases that amount to more than 66% of total toxin proteins. These results provide a foundation for comprehending the biological, ecological and evolutionary significance of venom composition of speckled rattlesnake from the Baja California peninsula.

Formyl peptide receptors are involved in CTX-induced impairment of lymphocyte functions.

Crotoxin (CTX) is a neurotoxin that is isolated from the venom of Crotalus durissus terrificus, which displays immunomodulatory, anti-inflammatory, and anti-tumoral effects. Previous research has demonstrated that CTX promotes the adherence of leukocytes to the endothelial cells in blood microcirculation and the high endothelial venules of lymph nodes, which reduces the number of blood cells and lymphocytes. Studies have also shown that these effects are mediated by lipoxygenase-derived mediators. However, the exact lipoxygenase-derived eicosanoid involved in the CTX effect on lymphocytes is yet to be characterized. As CTX stimulates lipoxin-derived mediators from macrophages and lymphocyte effector functions could be modulated by activating formyl peptide receptors, we aimed to investigate whether these receptors were involved in CTX-induced redistribution and functions of lymphocytes in rats. We used male Wistar rats treated with CTX to demonstrate that Boc2 (butoxycarbonyl-Phe-Leu-Phe-Leu-Phe), an antagonist of formyl peptide receptors, prevented CTX-induced decrease in the number of circulating lymphocytes and increased the expression of the lymphocyte adhesion molecule LFA1. CTX reduced the T and B lymphocyte functions, such as lymphocyte proliferation in response to the mitogen Concanavalin A and antibody production in response to BSA immunization, respectively, which was prevented by the administration of Boc2. Importantly, mesenteric lymph node lymphocytes from CTX-treated rats showed an increased release of 15-epi-LXA4. These results indicate that formyl peptide receptors mediate CTX-induced redistribution of lymphocytes and that 15-epi-LXA4 is a key mediator of the immunosuppressive effects of CTX.

A Review of Rattlesnake Venoms.

Venom components are invaluable in biomedical research owing to their specificity and potency. Many of these components exist in two genera of rattlesnakes, Crotalus and Sistrurus, with high toxicity and proteolytic activity variation. This review focuses on venom components within rattlesnakes, and offers a comparison and itemized list of factors dictating venom composition, as well as presenting their known characteristics, activities, and significant applications in biosciences. There are 64 families and subfamilies of proteins present in Crotalus and Sistrurus venom. Snake venom serine proteases (SVSP), snake venom metalloproteases (SVMP), and phospholipases A2 (PLA2) are the standard components in Crotalus and Sistrurus venom. Through this review, we highlight gaps in the knowledge of rattlesnake venom; there needs to be more information on the venom composition of three Crotalus species and one Sistrurus subspecies. We discuss the activity and importance of both major and minor components in biomedical research and drug development.

Biological and Medical Aspects Related to South American Rattlesnake Crotalus durissus (Linnaeus, 1758): A View from Colombia.

In Colombia, South America, there is a subspecies of the South American rattlesnake Crotalus durissus, C. d. cumanensis, a snake of the Viperidae family, whose presence has been reduced due to the destruction of its habitat. It is an enigmatic snake from the group of pit vipers, venomous, with large articulated front fangs, special designs on its body, and a characteristic rattle on its tail. Unlike in Brazil, the occurrence of human envenomation by C. durisus in Colombia is very rare and contributes to less than 1% of envenomation caused by snakes. Its venom is a complex cocktail of proteins with different biological effects, which evolved with the purpose of paralyzing the prey, killing it, and starting its digestive process, as well as having defense functions. When its venom is injected into humans as the result of a bite, the victim presents with both local tissue damage and with systemic involvement, including a diverse degree of neurotoxic, myotoxic, nephrotoxic, and coagulopathic effects, among others. Its biological effects are being studied for use in human health, including the possible development of analgesic, muscle relaxant, anti-inflammatory, immunosuppressive, anti-infection, and antineoplastic drugs. Several groups of researchers in Brazil are very active in their contributions in this regard. In this work, a review is made of the most relevant biological and medical aspects related to the South American rattlesnake and of what may be of importance for a better understanding of the snake C. d. cumanensis, present in Colombia and Venezuela.

Myotoxin-3 from the Pacific Rattlesnake Crotalus oreganus oreganus Venom Is a New Microtubule-Targeting Agent.

Microtubule targeting agents (MTA) are anti-cancer molecules that bind tubulin and interfere with the microtubule functions, eventually leading to cell death. In the present study, we used an in vitro microtubule polymerization assay to screen several venom families for the presence of anti-microtubule activity. We isolated myotoxin-3, a peptide of the crotamine family, and three isoforms from the venom of the Northern Pacific rattlesnake Crotalus oreganus oreganus, which was able to increase tubulin polymerization. Myotoxin-3 turned out to be a cell-penetrating peptide that slightly diminished the viability of U87 glioblastoma and MCF7 breast carcinoma cells. Myotoxin 3 also induced remodeling of the U87 microtubule network and decreased MCF-7 microtubule dynamic instability. These effects are likely due to direct interaction with tubulin. Indeed, we showed that myotoxin-3 binds to tubulin heterodimer with a Kd of 5.3 µM and stoichiometry of two molecules of peptide per tubulin dimer. Our results demonstrate that exogenous peptides are good candidates for developing new MTA and highlight the richness of venoms as a source of pharmacologically active molecules.

Antibacterial Properties of Crotoxin from Crotalus durissus terrificus-Insight into the Mechanism of Action.

The growing problem of antibiotic resistance among bacteria requires searching for new therapeutic agents with bacteriostatic and/or bactericidal properties. Crotoxin is a ß-neurotoxin from the venom of the Crotalus durissus terrificus. It is composed of two subunits: CA (non-active) and CB (with phospholipase A2 activity). It has already been shown that the isolated CB, but not the CA, subunit of crotoxin exhibits an antibacterial activity towards a variety of Gram-positive and Gram-negative bacterial species. However, no studies on the whole crotoxin complex have been carried out so far. We tested the antibacterial properties of crotoxin, as well as its isolated CB subunit, towards Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 6535, Micrococcus luteus ATCC 10240, Escherichia coli ATCC 25922, Escherichia coli ATCC 8739, and Pseudomonas aeruginosa ATCC 10145. Both toxins exhibited antibacterial properties only against Micrococcus luteus ATCC 10240. Crotoxin showed only bacteriostatic activity with a MIC of 46 µM, while the CB subunit acted as both a bacteriostatic and bactericidal agent with a MIC = MBC = 0.21 µM. The bacteriostatic effect of the toxins was independent of the enzymatic activity of the CB subunit. Bactericidal properties, however, require phospholipase A2 activity. Both toxins reduced bacteria viability at the MIC by 72% and 85% for crotoxin- and CB-treated bacteria, respectively. The membrane permeability increased approximately three times within the first hour of incubation with toxins; afterwards, either no significant changes or a decrease of membrane permeability, compared to the control cells, were observed. We isolated a single, approximately 30 kDa bacterial wall protein which belongs to the NlpC/P60 family that interacts with crotoxin leading to the inhibition of bacterial growth. Neither crotoxin nor the CB subunit showed any cytotoxic properties to human fibroblasts at the MIC during the three-day incubation.

Antileishmanial activity, cytotoxicity and cellular response of amphotericin B in combination with crotamine derived from Crotalus durissus terrificus venom using in vitro and in silico approaches.

OBJECTIVE: To investigate the in vitro activity, synergism, cytotoxicity and cellular immunological response, as well as the molecular affinity between amphotericin B (AmB) and crotamine (CTA), derived from Crotalus durissus terrificus venom against Leishmania amazonensis. METHODS: This study performed the inhibition of promastigotes and amastigotes' growth under different concentrations of the drug and pharmacological combinations (AmB + CTA) based on the Berimbaum method (synergism study). The lactate dehydrogenase (LDH) quantification method was used to determine the cytotoxicity of the drug and combinations employing four cell lines (J774, HepG2, VERO, and C2C12). Following, the levels of Tumour Necrose Factor-alpha (TNF-α) and Interleukin-12 (IL-12) cytokines, using enzyme-linked immunosorbent assay (ELISA) and nitrites, as an indirect measure of Nitric Oxide (NO), using the Griess reaction were assessed in the supernatants of infected macrophages. In silico approach (molecular docking and dynamics) and binding affinity (surface plasmon resonance) between the drug and toxin were also investigated. RESULTS: CTA enhanced AmB effect against promastigote and amastigote forms of L. amazonensis, decreased the drug toxicity in different cell lines and induced the production of important Th1-like cytokines and NO by infected macrophages. The pharmacological combination also displayed consistent molecular interactions with low energy of coupling and a concentration-dependent profile. CONCLUSION: Our data suggest that this pharmacological approach is a promising alternative treatment against L. amazonensis infection due to the improved activity (synergistic effect) achieved against the parasites' forms and to the decreased cytotoxic effect.

Crotoxin modulates metabolism and secretory activity of peritoneal macrophages from Walker 256 tumor-bearing rats.

Crotoxin (CTX), the major toxin of Crotalus durissus terrificus snake venom, induces an inhibitory effect on tumor development and modulates the functions of macrophages (MØs), which play a key role as a defense mechanism against tumor growth. In early tumor progression stage, MØs are avidly phagocytic (inflammatory cell), releasing reactive nitrogen intermediates-RNI/ROI and cytokines TNF-α, IL-1ß, and IL-6. However, when the tumor has been developed, tumor-associated MØ (angiogenic cell) presents a decrease in the mentioned activities. We reported that CTX stimulates H2O2 release, NO production and secretion of cytokines by peritoneal MØs obtained from non-tumor-bearing rats. Considering that the mentioned mediators control tumor growth, it is mandatory to investigate whether CTX stimulates the production of these mediators by MØs obtained from tumor-bearing animals. The aim of this work was then to evaluate the CTX effect on metabolism and functions of peritoneal MØs obtained from Walker 256 tumor-bearing rats. For this purpose, male Wistar rats were subcutaneously inoculated in the right flank with 1 mL sterile suspension of 2 × 107 Walker 256 tumor cells. CTX (18 µg per animal) was subcutaneously administered in two protocols: a) on the 1st day of tumor cell injection and b) on the 4th day of tumor cell inoculation. In both protocols, MØs were obtaining on the 14th day of tumor cell inoculation to evaluate the release of H2O2, NO, and pro-inflammatory cytokines (IL-1ß, TNFα, and IL-6); maximal activity of hexokinase, glucose-6-phosphate dehydrogenase, citrate synthase, and 14CO2 production from [U-14C]-glucose and [U-14C]-glutamine. The treatment with CTX stimulated the release of NO, H2O2, and cytokines, and glucose and glutamine metabolism. Metabolic and functional changes induced by CTX were accompanied by a decrease of tumor growth as indicated by tumor fresh weight and diameter. These results indicate CTX not only as a scientific tool to investigate changes in metabolism and functions of peritoneal MØs but also for a better understanding of the mechanisms involved in tumor growth.

State-of-the-art review of snake venom phosphodiesterases (svPDEs).

Phosphodiesterases (PDEs) constitute an enzyme group able to hydrolyze nucleic acids as well as some second messengers. Due to this ability and their expression in several human tissues and organs, PDEs can control a gamut of physiological processes. They are also involved in some pathological conditions, such as Alzheimer's disease and erectile dysfunction. PDEs are also expressed in snake venom glands, being called snake venoms phosphodiesterases, or simply svPDEs. The occurrence of these enzymes has already been reported in crotalid, elapid and viperid venoms, such as Crotalus, Naja and Trimeresurus, respectively, but not all of them have been characterized concerning their structure, activity and function. In this review, we are addressing general characteristics of svPDEs, in addition to their structural, biochemical and functional characteristics, and we also report some potential applications of svPDEs.

The anti-ovarian carcinoma activity of L-amino acid oxidase from Crotalus adamanteus venom in vivo and in vitro.

Snake venom L-Amino-acid oxidase (svLAAO) has become a critical research target in molecular biology and medical science since its widespread presence and diverse biological roles, including antitumor application. Our research confirmed that Crotalus adamanteus (C. adamanteus) venom LAAO exhibited potential anti-ovarian cancer activity both in vivo and in vitro. C. adamanteus venom LAAO significantly reduced viability of ovarian cancer cells and caused morphological changes that preceded cell death. The results of molecular biology experiments showed that C. adamanteus venom LAAO caused expression changes of genes related to apoptotic pathways either intrinsically or extrinsically in ovarian cancer cells. Animal experiments and histological analysis also proved that C. adamanteus venom LAAO could effectively inhibit the tissue damage caused by ovarian cancer, and animals treated with C. adamanteus venom LAAO showed higher survival time. Catalase blocked the major apoptosis induction of C. adamanteus venom LAAO on ovarian cancer cells, suggesting that the cytotoxicity of C. adamanteus venom LAAO on ovarian cancer cells was mainly mediated by H2O2. These results infer that C. adamanteus venom LAAO will have some advantages in new drug research and antitumor drug development in future.

Crotoxin B: Heterologous Expression, Protein Folding, Immunogenic Properties, and Irregular Presence in Crotalid Venoms.

Crotoxin complex CA/CB and crotamine are the main toxins associated with Crotalus envenomation besides the enzymatic activities of phospholipases (PLA2) and proteases. The neutralization at least of the crotoxin complex by neutralizing the subunit B could be a key in the production process of antivenoms against crotalids. Therefore, in this work, a Crotoxin B was recombinantly expressed to evaluate its capacity as an immunogen and its ability to produce neutralizing antibodies against crotalid venoms. A Crotoxin B transcript from Crotalus tzabcan was cloned into a pCR®2.1-TOPO vector (Invitrogen, Waltham, MA, USA) and subsequently expressed heterologously in bacteria. HisrCrotoxin B was extracted from inclusion bodies and refolded in vitro. The secondary structure of HisrCrotoxin B was comparable to the secondary structure of the native Crotoxin B, and it has PLA2 activity as the native Crotoxin B. HisrCrotoxin B was used to immunize rabbits, and the obtained antibodies partially inhibited the activity of PLA2 from C. tzabcan. The anti-HisrCrotoxin B antibodies neutralized the native Crotoxin B and the whole venoms from C. tzabcan, C. s. salvini, and C. mictlantecuhtli. Additionally, anti-HisrCrotoxin B antibodies recognized native Crotoxin B from different Crotalus species, and they could discriminate venom in species with high or low levels of or absence of Crotoxin B.

Evaluation of crotamine based probes as intracellular targeted contrast agents for magnetic resonance imaging.

Crotamine is a lysine and cysteine rich 42 amino acids long bio-active polypeptide, isolated from the venom of a South American rattlesnake, that can also be used as cell penetrating peptide. A facile synthetic scheme for coupling cargo molecules like fluorophores (carboxyfluorescein) or MRI probes (Gd-DO3A-based macrocycle) is presented. The toxicity, cellular internalization and steady-state accumulation after long-term incubation for 18 h, as well as magnetic resonance relaxivities and cellular relaxation rates of crotamine based probes were evaluated and compared to its shorter synthetic fragment CyLoP-1. The longitudinal relaxivity (r1) of the conjugates of CyLoP-1 and crotamine is significantly lower in medium than in water indicating to the lower contrast enhancement efficacy of DO3A-based probes in biological samples. Carboxyfluorescein labeled crotamine did not exhibit toxicity up to a concentration of 2.5 µM. CyLoP-1 accumulated about four times better within the cells compared to crotamine. Fluorescence microscopy suggests different predominant uptake mechanisms for crotamine and CyLoP-1 in 3T3 cells. While crotamine is predominantly localized in vesicular structures (most likely endosomes and lysosomes) within the cell, CyLoP-1 is mainly homogeneously distributed in the cytosol. The cellular relaxation rate (R1, cell) of the crotamine based probe was not significantly increased whereas the corresponding CyLoP-1-derivative showed a slightly elevated R1, cell. This study indicates the potential of crotamine and in particular the shorter fragment CyLoP-1 to be useful for an efficient transmembrane delivery of agents directed to intracellular (cytosolic) targets. However, the applicability of the conjugates synthesized here as contrast agents in MR imaging is limited. Further improvement is needed to prepare more efficient probes for MRI applications, i.e., by replacing the DO3A- with a DOTA-based chelate.

Augmented anandamide signalling in the substantia nigra pars reticulata mediates panicolytic-like effects in mice confronted by Crotalus durissus terrificus pit vipers.

RATIONALE: The endocannabinoid modulation of fear and anxiety due to the on-demand synthesis and degradation is supported by a large body of research. Although it has been proposed that anandamide (AEA) in the substantia nigra pars reticulata (SNpr) seems to be important for the organisation of innate fear-related behaviours, a role for endogenous AEA has yet to be clarified. METHODS: Mice were treated with the fatty acid amide hydrolase (FAAH) selective inhibitor URB597 at different concentrations (0.01, 0.1, 1 nmol/0.1 µL) in the SNpr and confronted by rattlesnakes (Crotalus durissus terrificus). The most effective dose of URB597 (1 nmol) was also preceded by microinjections of the CB1 receptor antagonist AM251 (0.1 nmol) into the SNpr, and mice were then confronted by the venomous snake. RESULTS: URB597 (0.1 and 1 nmol) in the SNpr decreased the expression of defensive behaviours such as defensive attention, escape, and time spent inside the burrow of mice confronted by rattlesnakes. Moreover, pretreatment of SNpr with AM251 suppressed these antiaversive effects of URB597 in this midbrain structure. CONCLUSION: Overall, these data clearly indicate that the panicolytic consequences of endogenous AEA enhancement in the SNpr are mediated by CB1 receptor signalling.