Unraveiling the correlation among neurodevelopmental and inflammatory biomarkers in patients with chronic schizophrenia.

INTRODUCTION: Nuclear distribution element like-1 (Ndel1) is a cytosolic oligopeptidase, which was suggested as a potential biomarker of aberrant neurodevelopment and early stage of schizophrenia (SCZ). The involvement of Ndel1 in neurite outgrowth, neuronal migration and neurodevelopment was demonstrated. Moreover, Ndel1 cleaves neuropeptides, including the endogenous antipsychotic peptide neurotensin, and lower Ndel1 activity was reported in SCZ patients compared with healthy controls (HCs). Changes in brain-derived neurotrophic factor (BDNF) and inflammatory cytokines levels were also implicated in SCZ. OBJECTIVE: This preliminary study aimed to investigate the interactions between these immune and neurodevelopmental/neurotrophic biomarkers, namely BDNF and the recently identified SCZ biomarker Ndel1. RESULTS: We observed lower Ndel1 activity and IL-4 levels, and higher BDNF levels, in plasma of SCZ (N = 23) compared with HCs (N = 29). Interestingly, significant correlation between Ndel1 activity and IL-4 levels was observed in SCZ, while no correlation with any other evaluated interleukins (namely IL-2, IL-8, IL-10 and IL-17A) or BDNF levels was noticed. CONCLUSION: Although this hypothesis needs to be further explored for a better understanding of the mechanisms by which these altered pathways are associated to each other in SCZ, we suggest that Ndel1 and the inflammatory marker IL-4 are directly correlated.

Targeting Ca2+ and Mitochondrial Homeostasis by Antipsychotic Thioridazine in Leukemia Cells.

Mitochondria have pivotal roles in cellular physiology including energy metabolism, reactive oxygen species production, Ca2+ homeostasis, and apoptosis. Altered mitochondrial morphology and function is a common feature of cancer cells and the regulation of mitochondrial homeostasis has been identified as a key to the response to chemotherapeutic agents in human leukemias. Here, we explore the mechanistic aspects of cytotoxicity produced by thioridazine (TR), an antipsychotic drug that has been investigated for its anticancer potential in human leukemia cellular models. TR exerts selective cytotoxicity against human leukemia cells in vitro. A PCR array provided a general view of the expression of genes involved in cell death pathways. TR immediately produced a pulse of cytosolic Ca2+, followed by mitochondrial uptake, resulting in mitochondrial permeabilization, caspase 9/3 activation, endoplasmic reticulum stress, and apoptosis. Ca2+ chelators, thiol reducer dithiothreitol, or CHOP knockdown prevented TR-induced cell death. TR also exhibited potent cytotoxicity against BCL-2/BCL-xL-overexpressing leukemia cells. Additionally, previous studies have shown that TR exhibits potent antitumor activity in vivo in different solid tumor models. These findings show that TR induces a Ca2+-mediated apoptosis with involvement of mitochondrial permeabilization and ER stress in leukemia and it emphasizes the pharmacological potential of TR as an adjuvant in antitumor chemotherapy.

A Native CPP from Rattlesnake with Therapeutic and Theranostic Properties.

The cell-penetrating peptides (CPPs) are characterized by the ability of internalization into cells in vitro and in vivo, and the ability of these peptides can rely on a high content of positive charges, as it is the case of the native CPP crotamine. Crotamine is a polypeptide of about 42 amino acid residues with high content of basic residues as Arg and Lys. Although most of known CPPs are linear peptides, native crotamine from the venom of a South American rattlesnake has a well-defined 3D structure stabilized by three disulfide bonds which guarantee the exposure of side chains of basic amino acids. This 3D structure also protects this amphipathic polypeptide from the degradation even if administered by oral route, therefore, protecting also the biological activities of crotamine. As several different biological properties of crotamine are dependent of cell penetration, the methods mainly employed for analyzing crotamine properties as anthelminthic and antimalarial activities, antimicrobial and antitumor activities, with a unique selective cytotoxic property against actively proliferating cells, as tumor cells, were chosen based on crotamine ability of internalization mediated by its positive charge. This native cationic polypeptide is also able to efficiently carry, with no need of covalent linkage with the cargo, genetic material into cells in vitro and in vivo, suggesting its use in gene therapy. Moreover, the possibility of decorating gold nanoparticles keeping the ability of transfecting cells was demonstrated. More recently, the ability of crotamine to interfere in animal metabolism, inducing browning of adipose tissue and increasing the energy expenditure, and its application in renal therapy was demonstrated. As crotamine also accumulates specifically in tumor cells in vivo, and the potential utility of crotamine as a theranostic agent was then suggested. Therefore, diverse methodologies employed for the characterization and exploration of the therapeutic applications of this promising native CPP for remediation of several pathogenic conditions are presented here.

Revisiting the potential of South American rattlesnake Crotalus durissus terrificus toxins as therapeutic, theranostic and/or biotechnological agents.

The potential biotechnological and biomedical applications of the animal venom components are widely recognized. Indeed, many components have been used either as drugs or as templates/prototypes for the development of innovative pharmaceutical drugs, among which many are still used for the treatment of human diseases. A specific South American rattlesnake, named Crotalus durissus terrificus, shows a venom composition relatively simpler compared to any viper or other snake species belonging to the Crotalus genus, although presenting a set of toxins with high potential for the treatment of several still unmet human therapeutic needs, as reviewed in this work. In addition to the main toxin named crotoxin, which is under clinical trials studies for antitumoral therapy and which has also anti-inflammatory and immunosuppressive activities, other toxins from the C. d. terrificus venom are also being studied, aiming for a wide variety of therapeutic applications, including as antinociceptive, anti-inflammatory, antimicrobial, antifungal, antitumoral or antiparasitic agent, or as modulator of animal metabolism, fibrin sealant (fibrin glue), gene carrier or theranostic agent. Among these rattlesnake toxins, the most relevant, considering the potential clinical applications, are crotamine, crotalphine and gyroxin. In this narrative revision, we propose to organize and present briefly the updates in the accumulated knowledge on potential therapeutic applications of toxins collectively found exclusively in the venom of this specific South American rattlesnake, with the objective of contributing to increase the chances of success in the discovery of drugs based on toxins.

Impact of Reck expression and promoter activity in neuronal in vitro differentiation.

Reck (REversion-inducing Cysteine-rich protein with Kazal motifs) tumor suppressor gene encodes a multifunctional glycoprotein which inhibits the activity of several matrix metalloproteinases (MMPs), and has the ability to modulate the Notch and canonical Wnt pathways. Reck-deficient neuro-progenitor cells undergo precocious differentiation; however, modulation of Reck expression during progression of the neuronal differentiation process is yet to be characterized. In the present study, we demonstrate that Reck expression levels are increased during in vitro neuronal differentiation of PC12 pheochromocytoma cells and P19 murine teratocarcinoma cells and characterize mouse Reck promoter activity during this process. Increased Reck promoter activity was found upon induction of differentiation in PC12 cells, in accordance with its increased mRNA expression levels in mouse in vitro models. Interestingly, Reck overexpression, prior to the beginning of the differentiation protocol, led to diminished efficiency of the neuronal differentiation process. Taken together, our findings suggest that increased Reck expression at early stages of differentiation diminishes the number of neuron-like cells, which are positive for the beta-3 tubulin marker. Our data highlight the importance of Reck expression evaluation to optimize in vitro neuronal differentiation protocols.

Evaluation of NDEL1 oligopeptidase activity in blood and brain in an animal model of schizophrenia: effects of psychostimulants and antipsychotics.

Nuclear distribution element-like 1 (NDEL1) enzyme activity is important for neuritogenesis, neuronal migration, and neurodevelopment. We reported previously lower NDEL1 enzyme activity in blood of treated first episode psychosis and chronic schizophrenia (SCZ) compared to healthy control subjects, with even lower activity in treatment resistant chronic SCZ patients, implicating NDEL1 activity in SCZ. Herein, higher NDEL1 activity was observed in the blood and several brain regions of a validated animal model for SCZ at baseline. In addition, long-term treatment with typical or atypical antipsychotics, under conditions in which SCZ-like phenotypes were reported to be reversed in this animal model for SCZ, showed a significant NDEL1 activity reduction in blood and brain regions which is in line with clinical data. Importantly, these results support measuring NDEL1 enzyme activity in the peripheral blood to predict changes in NDEL1 activity in the CNS. Also, acute administration of psychostimulants, at levels reported to induce SCZ-like phenotype in normal rat strains, increased NDEL1 enzyme activity in blood. Therefore, alterations in NDEL1 activity after treatment with antipsychotics or psychostimulants may suggest a possible modulation of NDEL1 activity secondary to neurotransmission homeostasis and provide new insights into the role of NDEL1 in SCZ pathophysiology.

In vivo effects of the association of the psychoactive phenotiazine thioridazine on antitumor activity and hind limb paralysis induced by the native polypeptide crotamine.

Crotamine is a cationic polypeptide composed by 42 amino acid residues with several pharmacological and biological properties, including the selective ability to enter and kill actively proliferating tumour cells, which led us to propose its use as a theranostic agent for cancer therapy. At the moment, the improvement of crotamine antitumoral efficacy by association with chemotherapeutic adjuvants is envisioned. In the present work, we evaluated the association of crotamine with the antitumoral adjuvant phenotiazine thioridazine (THD). In spite of the clear efficacy of these both compounds as anticancer agents in long-term in vivo treatment of animal model bearing implanted xenograph melanoma tumor, the expected mutual potentiation of the antitumor effects was not observed here. Moreover, this association revealed for the first time the influence of THD on crotamine ability to trigger the hind limb paralysis in mice, and this discovery may represent the first report suggesting the potential involvement of the CNS in the action of this snake polypeptide on the skeletal muscle paralysis, which was classically believed to be essentially limited to a direct action in peripheral tissues as the skeletal muscle. This is also supported by the observed ability of crotamine to potentiate the sedative effects of THD which action was consistently demonstrated to be based on its central action. The better characterization of crotamine properties in CNS may certainly bring important insights for the knowledge needed to pave the way toward the use of this molecule as a theranostic compound in human diseases as cancer.

Crotamine Cell-Penetrating Nanocarriers: Cancer-Targeting and Potential Biotechnological and/or Medical Applications.

Crotamine is a basic, 42-residue polypeptide from snake venom that has been shown to possess cell-penetrating properties. Here we describe the preparation, purification, biochemical and biophysical analysis of venom-derived, recombinant, chemically synthesized, and fluorescent-labeled crotamine. We also describe the formation and characterization of crotamine-DNA and crotamine-RNA nanoparticles; and the delivery of these nanoparticles into cells and animals. Crotamine forms nanoparticles with a variety of DNA and RNA molecules, and crotamine-plasmid DNA nanoparticles are selectively delivered into actively proliferating cells in culture or in living organisms such as mice, Plasmodium, and worms. As such, these nanoparticles could form the basis for a nucleic acid drug-delivery system. We also describe here the design and characterization of crotamine-functionalized gold nanoparticles, and the delivery of these nanoparticles into cells. We also evaluated the viability of using the combination of crotamine with silica nanoparticles in animal models, aiming to provide slow delivery, and to decrease the crotamine doses needed for the biological effects. In addition, the efficacy of administering crotamine orally was also demonstrated.

Neuroinflammation and glial cell activation in mental disorders.

Mental disorders (MDs) are highly prevalent and potentially debilitating complex disorders which causes remain elusive. Looking into deeper aspects of etiology or pathophysiology underlying these diseases would be highly beneficial, as the scarce knowledge in mechanistic and molecular pathways certainly represents an important limitation. Association between MDs and inflammation/neuroinflammation has been widely discussed and accepted by many, as high levels of pro-inflammatory cytokines were reported in patients with several MDs, such as schizophrenia (SCZ), bipolar disorder (BD) and major depression disorder (MDD), among others. Correlation of pro-inflammatory markers with symptoms intensity was also reported. However, the mechanisms underlying the inflammatory dysfunctions observed in MDs are not fully understood yet. In this context, microglial dysfunction has recently emerged as a possible pivotal player, as during the neuroinflammatory response, microglia can be over-activated, and excessive production of pro-inflammatory cytokines, which can modify the kynurenine and glutamate signaling, is reported. Moreover, microglial activation also results in increased astrocyte activity and consequent glutamate release, which are both toxic to the Central Nervous System (CNS). Also, as a result of increased microglial activation in MDs, products of the kynurenine pathway were shown to be changed, influencing then the dopaminergic, serotonergic, and glutamatergic signaling pathways. Therefore, in the present review, we aim to discuss how neuroinflammation impacts on glutamate and kynurenine signaling pathways, and how they can consequently influence the monoaminergic signaling. The consequent association with MDs main symptoms is also discussed. As such, this work aims to contribute to the field by providing insights into these alternative pathways and by shedding light on potential targets that could improve the strategies for pharmacological intervention and/or treatment protocols to combat the main pharmacologically unmatched symptoms of MDs, as the SCZ.

Effects of the Natural Peptide Crotamine from a South American Rattlesnake on Candida auris, an Emergent Multidrug Antifungal Resistant Human Pathogen.

Invasive Candida infections are an important growing medical concern and treatment options are limited to a few antifungal drug classes, with limited efficacies depending on the infecting organism. In this scenario, invasive infections caused by multiresistant Candida auris are emerging in several places around the world as important healthcare-associated infections. As antimicrobial peptides (AMPs) exert their activities primarily through mechanisms involving membrane disruption, they have a lower chance of inducing drug resistance than general chemical antimicrobials. Interestingly, we previously described the potent candicidal effect of a rattlesnake AMP, crotamine, against standard and treatment-resistant clinical isolates, with no hemolytic activity. We evaluated the antifungal susceptibility of several Candida spp. strains cultured from different patients by using the Clinical and Laboratory Standards Institute (CLSI) microdilution assay, and the antifungal activity of native crotamine was evaluated by a microbial growth inhibition microdilution assay. Although all Candida isolates evaluated here showed resistance to amphotericin B and fluconazole, crotamine (40-80 µM) exhibited in vitro activity against most isolates tested. We suggest that this native polypeptide from the South American rattlesnake Crotalus durissus terrificus has potential as a structural model for the generation of a new class of antimicrobial compounds with the power to fight against multiresistant Candida spp.

Long term safety of targeted internalization of cell penetrating peptide crotamine into renal proximal tubular epithelial cells in vivo.

Activated proximal tubular epithelial cells (PTECs) play a crucial role in progressive tubulo-interstitial fibrosis in native and transplanted kidneys. Targeting PTECs by non-viral delivery vectors might be useful to influence the expression of important genes and/or proteins in order to slow down renal function loss. However, no clinical therapies that specifically target PTECs are available at present. We earlier showed that a cationic cell penetrating peptide isolated from South American rattlesnake venom, named crotamine, recognizes cell surface heparan sulfate proteoglycans and accumulates in cells. In healthy mice, crotamine accumulates mainly in kidneys after intraperitoneal (ip) injection. Herein we demonstrate for the first time, the overall safety of acute or long-term treatment with daily ip administrated crotamine for kidneys functions. Accumulation of ip injected crotamine in the kidney brush border zone of PTECs, and its presence inside these cells were observed. In addition, significant lower in vitro crotamine binding, uptake and reporter gene transport and expression could be observed in syndecan-1 deficient HK-2 PTECs compared to wild-type cells, indicating that the absence of syndecan-1 impairs crotamine uptake into PTECs. Taken together, our present data show the safety of in vivo long-term treatment with crotamine, and its preferential uptake into PTECs, which are especially rich in HSPGs such as syndecan-1. In addition to the demonstrated in vitro gene delivery mediated by crotamine in HK-2 cells, the potential applicability of crotamine as prototypic non-viral (gene) delivery nanocarrier to modulate PTEC gene and/or protein expression was confirmed.

Crotamine induces browning of adipose tissue and increases energy expenditure in mice.

Crotamine, originally isolated from rattlesnake venom, has been extensively studied due to its pleiotropic biological properties, and special attention has been paid to its antitumor activity. However, long-term treatment with crotamine was accompanied by a reduction in animal body weight gain and by increases in glucose tolerance. As cancer is commonly associated with cachexia, to preclude the possible cancer cachexia-like effect of crotamine, herein this polypeptide was administered in healthy wild-type C57/BL6 mice by the oral route daily, for 21 days. Reduced body weight gain, in addition to decreased white adipose tissue (WAT) and increased brown adipose tissue (BAT) mass were observed in healthy animals in the absence of tumor. In addition, we observed improved glucose tolerance and increased insulin sensitivity, accompanied by a reduction of plasma lipid levels and decreased levels of biomarkers of liver damage and kidney disfunctions. Importantly, long-term treatment with crotamine increased the basal metabolic rate in vivo, which was consistent with the increased expression of thermogenic markers in BAT and WAT. Interestingly, cultured brown adipocyte cells induced to differentiation in the presence of crotamine also showed increases in some of these markers and in lipid droplets number and size, indicating increased brown adipocyte maturation.

Design and characterization of crotamine-functionalized gold nanoparticles.

This paper describes the development of a facile and environmentally friendly strategy for supporting crotamine on gold nanoparticles (GNPs). Our approach was based on the covalent binding interaction between the cell penetrating peptide crotamine, which is a snake venom polypeptide with preference to penetrate dividing cells, and a polyethylene glycol (PEG) ligand, which is a nontoxic, water-soluble and easily obtainable commercial polymer. Crotamine was derivatized with ortho-pyridyldisulfide-polyethyleneglycol-N-hydroxysuccinimide (OPSS-PEG-SVA) cross-linker to produce OPSS-PEG-crotamine as the surface modifier of GNP. OPSS-PEG-SVA can serve not only as a surface modifier, but also as a stabilizing agent for GNPs. The successful PEGylation of the nanoparticles was demonstrated using different physicochemical techniques, while the grafting densities of the PEG ligands and crotamine on the surface of the nanoparticles were estimated using a combination of electron microscopy and mass spectrometry analysis. In vitro assays confirmed the internalization of these GNPs, into living HeLa cells. The results described herein suggest that our approach may serve as a simple platform for the synthesis of GNPs decorated with crotamine with well-defined morphologies and uniform dispersion, opening new roads for crotamine biomedical applications.

Oral treatment with a rattlesnake native polypeptide crotamine efficiently inhibits the tumor growth with no potential toxicity for the host animal and with suggestive positive effects on animal metabolic profile.

The efficacy of crotamine as antitumoral was first demonstrated by daily intraperitoneal (IP) injections of low doses of this toxin in an animal model bearing melanoma tumors. Significant inhibition of tumor growth and increased lifespan of mice bearing tumor was also noticed after 21 consecutive days of this daily IP administration of crotamine. However, due to the limited acceptance of treatments by IP route in clinical conditions, herein, we evaluated the antitumor effect of this native polypeptide employing the oral route. The efficacy of crotamine in inhibiting the melanoma growth in vivo, even after passing through the gastrointestinal tract of the animal, was confirmed here. In addition, biochemical biomarkers and also histopathological analysis showed both the absence of any potential toxic effects in tissues or organs of the animal in which the highest accumulation of crotamine is expected. Interestingly, a reduction of weight gain was observed mainly in animals with tumor treated with crotamine by IP route, but not by oral administration. Albeit, oral administered crotamine was able to significantly decrease the body weight gain of healthy animals without tumor. Taking advantage of this same experimental animal models receiving crotamine by oral route, it was possible to show metabolic changes as the increased capacity of glucose clearance, which was accompanied by a reduction of the total cholesterol, and by increased high-density lipoprotein levels, both observed mainly in the absence of tumor. Triglycerides and low-density lipoprotein were also significantly decreased, but only in the absence of tumor. Taken together, these data suggest a clear trend for metabolic positive effects and mischaracterize unhealthy condition of animals, with or without tumors, treated with crotamine for 21 days. In addition, this study confirmed the efficacy of crotamine administered by oral route as antitumor agent, which besides the additional advantage of administration convenience and decreased risk of toxic effects, allowed the serendipitous observation of several positive metabolic effects on treated animals.

NDE1 and NDEL1 from genes to (mal)functions: parallel but distinct roles impacting on neurodevelopmental disorders and psychiatric illness.

NDE1 (Nuclear Distribution Element 1, also known as NudE) and NDEL1 (NDE-Like 1, also known as NudEL) are the mammalian homologues of the fungus nudE gene, with important and at least partially overlapping roles for brain development. While a large number of studies describe the various properties and functions of these proteins, many do not directly compare the similarities and differences between NDE1 and NDEL1. Although sharing a high degree structural similarity and multiple common cellular roles, each protein presents several distinct features that justify their parallel but also unique functions. Notably both proteins have key binding partners in dynein, LIS1 and DISC1, which impact on neurodevelopmental and psychiatric illnesses. Both are implicated in schizophrenia through genetic and functional evidence, with NDE1 also strongly implicated in microcephaly, as well as other neurodevelopmental and psychiatric conditions through copy number variation, while NDEL1 possesses an oligopeptidase activity with a unique potential as a biomarker in schizophrenia. In this review, we aim to give a comprehensive overview of the various cellular roles of these proteins in a "bottom-up" manner, from their biochemistry and protein-protein interactions on the molecular level, up to the consequences for neuronal differentiation, and ultimately to their importance for correct cortical development, with direct consequences for the pathophysiology of neurodevelopmental and mental illness.

Angiotensin converting enzyme activity is positively associated with IL-17a levels in patients with schizophrenia.

Previous studies of our group showed increased plasmatic Angiotensin-I Converting Enzyme (ACE) activity in schizophrenia (SCZ) patients compared to healthy controls, which was also associated to poor cognitive functioning. The ACE main product angiotensin II (Ang-II) has pro-inflammatory properties. Activated immune-inflammatory responses in SCZ and their association with disease progression and cognitive impairments are also well-described. Therefore, we examined here the association of plasma ACE activity and inflammatory mediators in 33 SCZ patients and 92 healthy controls. Non-parametric correlations were used to investigate the association of the enzyme activity and the peripheral levels of immune inflammatory markers as interleukins, tumor necrosis factor (TNF-α), and interferon (IFN-γ). Although no significant correlations could be observed for ACE activity and measured cytokines levels in healthy controls, a significant positive correlation for ACE enzymatic activity and IL-17a levels was observed in SCZ patients. Correcting for gender did not change these results. Moreover, a significant association for ACE activity and IFN-γ levels was also observed. To our knowledge, this is the first study to show a significant association between higher ACE activity and the levels of cytokines, namely IL-17a and IFN-γ, in patients with SCZ.

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 ß-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.

State of the art in the studies on crotamine, a cell penetrating peptide from South American rattlesnake.

Animal venoms comprise a naturally selected cocktail of bioactive peptides/proteins and other molecules, each of which playing a defined role thanks to the highly specific interactions with diverse molecular targets found in the prey. Research focused on isolation, structural, and functional characterizations of novel natural biologics (bioactive peptides/proteins from natural sources) has a long way to go through from the basic science to clinical applications. Herein, we overview the structural and functional characteristics of the myoneurotoxin crotamine, firstly isolated from the South American rattlesnake venom. Crotamine is the first venom peptide classified as a natural cell penetrating and antimicrobial peptide (CPP and AMP) with a more pronounced antifungal activity. In contrast to other known natural CPPs and AMPs, crotamine demonstrates a wide spectrum of biological activities with potential biotechnological and therapeutic values. More recent studies have demonstrated the selective in vitro anticancer activity of crotamine. In vivo, using a murine melanoma model, it was shown that crotamine delays tumor implantation, inhibits tumor cells proliferation, and also increases the survival of mice engrafted with subcutaneous melanoma. The structural and functional properties and also the possible biotechnological applications of minimized molecules derived from crotamine are also discussed.

Unraveling the antifungal activity of a South American rattlesnake toxin crotamine.

Crotamine is a highly basic peptide from the venom of Crotalus durissus terrificus rattlesnake. Its common gene ancestry and structural similarity with the ß-defensins, mainly due to an identical disulfide bond pattern, stimulated us to assess the antimicrobial properties of native, recombinant, and chemically synthesized crotamine. Antimicrobial activities against standard strains and clinical isolates were analyzed by the colorimetric microdilution method showing a weak antibacterial activity against both Gram-positive and Gram-negative bacteria [MIC (Minimum Inhibitory Concentration) of 50->200 µg/mL], with the exception of Micrococcus luteus [MIC ranging from 1 to 2 µg/mL]. No detectable activity was observed for the filamentous fungus Aspergillus fumigatus and Trichophyton rubrum at concentrations up to 125 µg/mL. However, a pronounced antifungal activity against Candida spp., Trichosporon spp., and Cryptococcus neoformans [12.5-50.0 µg/mL] was observed. Chemically produced synthetic crotamine in general displayed MIC values similar to those observed for native crotamine, whereas recombinant crotamine was overridingly more potent in most assays. On the other hand, derived short linear peptides were not very effective apart from a few exceptions. Pronounced ultrastructure alteration in Candida albicans elicited by crotamine was observed by electron microscopy analyses. The peculiar specificity for highly proliferating cells was confirmed here showing potential low cytotoxic effect of crotamine against nontumoral mammal cell lines (HEK293, PC12, and primary culture astrocyte cells) compared to tumoral B16F10 cells, and no hemolytic activity was observed. Taken together these results suggest that, at low concentration, crotamine is a potentially valuable anti-yeast or candicidal agent, with low harmful effects on normal mammal cells, justifying further studies on its mechanisms of action aiming medical and industrial applications.

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.