Two Draft Genome Sequences of Chromobacterium violaceum Isolates from the Rio Negro.

The draft genome sequences of two Chromobacterium violaceum strains isolated from the Rio Negro are reported here. These bacteria carry most genetic systems associated with the production of bioactive compounds, but unlike other C. violaceum strains, they lack a dedicated operon for arsenic resistance.

The density and biomass of mesozooplankton and ichthyoplankton in the Negro and the Amazon Rivers during the rainy season: the ecological importance of the confluence boundary.

The boundary zone between two different hydrological regimes is often a biologically enriched environment with distinct planktonic communities. In the center of the Amazon River basin, muddy white water of the Amazon River meets with black water of the Negro River, creating a conspicuous visible boundary spanning over 10 km along the Amazon River. Here, we tested the hypothesis that the confluence boundary between the white and black water rivers concentrates prey and is used as a feeding habitat for consumers by investigating the density, biomass and distribution of mesozooplankton and ichthyoplankton communities across the two rivers during the rainy season. Our results show that mean mesozooplankton density (2,730 inds. m-3) and biomass (4.8 mg m-3) were higher in the black-water river compared to the white-water river (959 inds. m-3; 2.4 mg m-3); however an exceptionally high mesozooplankton density was not observed in the confluence boundary. Nonetheless we found the highest density of ichthyoplankton in the confluence boundary (9.7 inds. m-3), being up to 9-fold higher than in adjacent rivers. The confluence between white and black waters is sandwiched by both environments with low (white water) and high (black water) zooplankton concentrations and by both environments with low (white water) and high (black water) predation pressures for fish larvae, and may function as a boundary layer that offers benefits of both high prey concentrations and low predation risk. This forms a plausible explanation for the high density of ichthyoplankton in the confluence zone of black and white water rivers.

Biogeochemical typing of paddy field by a data-driven approach revealing sub-systems within a complex environment--a pipeline to filtrate, organize and frame massive dataset from multi-omics analyses.

We propose the technique of biogeochemical typing (BGC typing) as a novel methodology to set forth the sub-systems of organismal communities associated to the correlated chemical profiles working within a larger complex environment. Given the intricate characteristic of both organismal and chemical consortia inherent to the nature, many environmental studies employ the holistic approach of multi-omics analyses undermining as much information as possible. Due to the massive amount of data produced applying multi-omics analyses, the results are hard to visualize and to process. The BGC typing analysis is a pipeline built using integrative statistical analysis that can treat such huge datasets filtering, organizing and framing the information based on the strength of the various mutual trends of the organismal and chemical fluctuations occurring simultaneously in the environment. To test our technique of BGC typing, we choose a rich environment abounding in chemical nutrients and organismal diversity: the surficial freshwater from Japanese paddy fields and surrounding waters. To identify the community consortia profile we employed metagenomics as high throughput sequencing (HTS) for the fragments amplified from Archaea rRNA, universal 16S rRNA and 18S rRNA; to assess the elemental content we employed ionomics by inductively coupled plasma optical emission spectroscopy (ICP-OES); and for the organic chemical profile, metabolomics employing both Fourier transformed infrared (FT-IR) spectroscopy and proton nuclear magnetic resonance (1H-NMR) all these analyses comprised our multi-omics dataset. The similar trends between the community consortia against the chemical profiles were connected through correlation. The result was then filtered, organized and framed according to correlation strengths and peculiarities. The output gave us four BGC types displaying uniqueness in community and chemical distribution, diversity and richness. We conclude therefore that the BGC typing is a successful technique for elucidating the sub-systems of organismal communities with associated chemical profiles in complex ecosystems.

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.

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.

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.

Kinetic characterization of gyroxin, a serine protease from Crotalus durissus terrificus venom.

This work describes for the first time the characterization of the enzymatic features of gyroxin, a serine protease from Crotalus durissus terrificus venom, capable to induce barrel rotation syndrome in rodents. Measuring the hydrolysis of the substrate ZFR-MCA, the optimal pH for proteolytic cleavage of gyroxin was found to be at pH 8.4. Increases in the hydrolytic activity were observed at temperatures from 25 °C to 45 °C, and increases of NaCl concentration up to 1 M led to activity decreases. The preference of gyroxin for Arg residues at the substrate P1 position was also demonstrated. Taken together, this work describes the characterization of substrate specificity of gyroxin, as well as the effects of salt and pH on its enzymatic activity.

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 toxicity and efficacy in mouse models of melanoma.

OBJECTIVES: Selective anticancer cell activity for both cell-penetrating and cationic antimicrobial peptides has previously been reported. As crotamine possesses activities similar to both of these, this study investigates crotamine's anticancer toxicity in vitro and in vivo. RESEARCH DESIGN AND METHODS: In vitro cancer cell viability was evaluated after treatment with 1 and 5 µg/ml of crotamine. In vivo crotamine cytotoxic effects in C57Bl/6J mice bearing B16-F10 primary cutaneous melanoma were tested, with two groups each containing 35 mice. The crotamine-treated group received 1 µg/day of crotamine per animal, subcutaneously which was well tolerated; the untreated group received a placebo. RESULTS: Crotamine at 5 µg/ml was lethal to B16-F10, Mia PaCa-2 and SK-Mel-28 cells and inoffensive to normal cells. In vivo crotamine treatment over 21 days significantly delayed tumor implantation, inhibited tumor growth and prolonged the lifespan of the mice. Mice in the crotamine-treated group survived at significantly higher rates (n = 30/35) than those in the untreated group (n = 7/35) (significance calculated with the Kaplan-Meier estimator). The average tumor weight in the untreated group was 4.60 g but was only about 0.27 g in the crotamine-treated mice, if detectable. CONCLUSIONS: These data warrant further exploration of crotamine as a tumor inhibition compound.

Transcriptome analysis of the Amazonian viper Bothrops atrox venom gland using expressed sequence tags (ESTs).

Bothrops atrox is a highly dangerous pit viper in the Brazilian Amazon region. We produced a global catalogue of gene transcripts to identify the main toxin and other protein families present in the B. atrox venom gland. We prepared a directional cDNA library, from which a set of 610 high quality expressed sequence tags (ESTs) were generated by bioinformatics processing. Our data indicated a predominance of transcripts encoding mainly metalloproteinases (59% of the toxins). The expression pattern of the B. atrox venom was similar to Bothrops insularis, Bothrops jararaca and Bothrops jararacussu in terms of toxin type, although some differences were observed. B. atrox showed a higher amount of the PIII class of metalloproteinases which correlates well with the observed intense hemorrhagic action of its toxin. Also, the PLA2 content was the second highest in this sample compared to the other three Bothrops transcriptomes. To our knowledge, this work is the first transcriptome analysis of an Amazonian rain forest pit viper and it will contribute to the body of knowledge regarding the gene diversity of the venom gland of members of the Bothrops genus. Moreover, our results can be used for future studies with other snake species from the Amazon region to investigate differences in gene patterns or phylogenetic relationships.

Transcriptome analysis of the Amazonian viper Bothrops atrox venomgland using expressed sequence tags (ESTs)

Bothrops atrox is a highly dangerous pit viper in the Brazilian Amazon region.We produced a global catalogue of gene transcripts to identify the main toxin and other protein families present in the B. atrox venom gland. We prepared a directional cDNA library, from which a set of 610 high quality expressed sequence tags (ESTs) were generated by bioinformatics processing. Our data indicated a predominance of transcripts encoding mainly metalloproteinases(59% of the toxins). The expression pattern of the B. atrox venom was similar to Bothrops insularis, Bothrops jararaca and Bothrops jararacussu in terms of toxin type, although some differences were observed. B. atrox showed a higher amount of the PIII classof metalloproteinases which correlates well with the observed intense hemorrhagic action of its toxin. Also, the PLA2 content was the second highest in this sample compared to theother three Bothrops transcriptomes. To our knowledge, this work is the first transcriptome analysis of an Amazonian rain forest pit viper and it will contribute to the body of knowledge regarding the gene diversity of the venom gland of members of the Bothropsgenus. Moreover, our results can be used for future studies with other snake species from the Amazon region to investigate differences in gene patterns or phylogenetic relationships.

Cytotoxic effects of crotamine are mediated through lysosomal membrane permeabilization.

Crotamine, one of the main toxic components of Crotalus durissus terrificus venom, is a small non-enzymatic basic polypeptide, which causes hind limb paralysis and necrosis of muscle cells. It is well-known that several toxins penetrate into the cytosol through endocytosis, although in many cases the mechanism by which this occurs has not been fully investigated. Recently, using low concentrations of crotamine, we demonstrated the uptake of this toxin into actively proliferative cells via endocytosis, an event that ensues crotamine binding to cell membrane heparan sulfate proteoglycans. Thus, crotamine can be regarded as a cell-penetrating peptide that, additionally, has been shown to be able of delivering some biologically active molecules into various cells. Herein, we investigate one of the mechanisms by which crotamine exerts its cytotoxic effects by following its uptake into highly proliferative cells, as CHO-K1 cells. Crotamine accumulation in the acidic endosomal/lysosomal vesicles was observed within 5 in after treatment of these cells with a cytotoxic concentration of this toxin, a value determined here by classical MTT assay. This accumulation caused disruption of lysosomal vesicles accompanied by the leakage of these vesicles contents into the cytosol. This lysosomal lysis also promoted the release of cysteine cathepsin and an increase of caspase activity in the cytoplasm. This chain of events seems to trigger a cell death process. Overall, our data suggest that lysosomes are the primary targets for crotamine cytotoxicity, a proposal corroborated by the correlation between both the kinetics and concentration-dependence of crotamine accumulation in lysosome compartments and the cytotoxic effects of this protein in CHO-K1 cells. Although crotamine is usually regarded as a myotoxin, we observed that intraperitoneal injection of fluorescently labeled crotamine in living mice led to significant and rapid accumulation of this toxin in the cell cytoplasm of several tissues, suggesting that crotamine cytotoxicity might not be restricted to muscle cells.

Identification of novel bradykinin-potentiating peptides (BPPs) in the venom gland of a rattlesnake allowed the evaluation of the structure-function relationship of BPPs.

Aiming to extend the knowledge about the diversity of bradykinin-potentiating peptides (BPPs) and their precursor proteins, a venom gland cDNA library from the South American rattlesnake (Crotalus dursissus terrificus, Cdt) was screened. Two novel homologous cDNAs encoding the BPPs precursor protein were cloned. Their sequence contain only one single longer BPP sequence with the typical IPP-tripeptide, and two short potential BPP-like molecules, revealing a unique structural organization. Several peptide sequences structurally similar to the BPPs identified in the precursor protein from Cdt and also from others snakes, were chemically synthesized and were bioassayed both in vitro and in vivo, by means of isolated smooth muscle preparations and by measurements of blood pressure in anaesthetized rats, respectively. We demonstrate here that a pyroglutamyl residue at the N-terminus with a high content of proline residues, even with the presence of a IPP moiety characteristic of typical BPPs, are not enough to determine a bradykinin-potentiating activity to these peptides. Taken together, our results indicate that the characterization of the BPPs precursor proteins and identification of characteristic glutamine residues followed by proline-rich peptide sequences are not enough to predict if these peptides, even with a pyroglutamyl residue at the N-terminus, will present the typical pharmacological activities described for the BPPs.

Crotamine mediates gene delivery into cells through the binding to heparan sulfate proteoglycans.

Recently we have shown that crotamine, a toxin from the South American rattlesnake Crotalus durissus terrificus venom, belongs to the family of cell-penetrating peptides. Moreover, crotamine was demonstrated to be a marker of centrioles, of cell cycle, and of actively proliferating cells. Herein we show that this toxin at non-toxic concentrations is also capable of binding electrostatically to plasmid DNA forming DNA-peptide complexes whose stabilities overcome the need for chemical conjugation for carrying nucleic acids into cells. Interestingly, crotamine demonstrates cell specificity and targeted delivery of plasmid DNA into actively proliferating cells both in vitro and in vivo, which distinguishes crotamine from other known natural cell-penetrating peptides. The mechanism of crotamine penetration and cargo delivery into cells was also investigated, showing the involvement of heparan sulfate proteoglycans in the uptake phase, which is followed by endocytosis and peptide accumulation within the acidic endosomal vesicles. Finally, the permeabilization of endosomal membranes induced by crotamine results in the leakage of the vesicles contents to the cell cytosol.

Crotacetin, a novel snake venom C-type lectin homolog of convulxin, exhibits an unpredictable antimicrobial activity.

Snake venom (sv) C-type lectins encompass a group of hemorrhagic toxins that are capable of interfering with blood stasis. A very well-studied svC-type lectin is the heterodimeric toxin, convulxin (CVX), from the venom of South American rattlesnake Crotalus durissus terrificus. CVX is able to activate platelets and induce their aggregation by acting via p62/GPVI collagen receptor. By using polymerase chain reaction homology screening, we have cloned several cDNA precursors of CVX subunit homologs. One of them, named crotacetin (CTC) beta-subunit, predicts a polypeptide with a topology very similar to the tridimensional conformations of other subunits of CVX-like snake toxins, as determined by computational analysis. Using gel permeation and reverse-phase high-performance liquid chromatography, CTC was purified from C. durissus venoms. CTC can be isolated from the venom of several C. durissus subspecies, but its quantitative predominance is in the venom of C. durissus cascavella. Functional analysis indicates that CTC induces platelet aggregation, and, importantly, exhibits an antimicrobial activity against Gram-positive and -negative bacteria, comparable with CVX.

Properties of cell penetrating peptides (CPPs).

Different approaches have been developed for the introduction of macromolecules, proteins and DNA into target cells. Viral (retroviruses, lentiviruses, etc.) and nonviral (liposomes, bioballistics etc.) vectors as well as lipid particles have been tested as DNA delivery systems. However, all of them share several undesirable effects that are difficult to overcome, such as unwanted immunoresponse and limited cell targeting. The discovery of the cell penetrating peptides (CPPs) showing properties of macromolecules carriers and enhancers of viral vectors, opened new opportunities for the delivery of biologically active cargos, including therapeutically relevant genes into various cells and tissues. This review summarizes recent data about the best characterized CPPs as well as those sharing cell-penetrating and cargo delivery properties despite differing in the primary sequence. The putative mechanisms of CPPs penetration into cells and interaction with intracellular structures such as chromosomes, cytoskeleton and centrioles are addressed. We further discuss recent developments in overcoming the lack of cells specificity, one of the main obstacles for CPPs application in gene therapy. In particular, we review a newly discovered affinity of CPPs to actively proliferating cells.

Automated NMR structure determination and disulfide bond identification of the myotoxin crotamine from Crotalus durissus terrificus.

Crotamine is one of four major components of the venom of the South American rattlesnake Crotalus durissus terrificus. Similar to its counterparts in the family of the myotoxins, it induces myonecrosis of skeletal muscle cells. This paper describes a new NMR structure determination of crotamine in aqueous solution at pH 5.8 and 20 degrees C, using standard homonuclear 1H NMR spectroscopy at 900MHz and the automated structure calculation software ATNOS/CANDID/DYANA. The automatic NOESY spectral analysis included the identification of a most likely combination of the six cysteines into three disulfide bonds, i.e. Cys4-Cys36, Cys11-Cys30 and Cys18-Cys37; thereby a generally applicable new computational protocol is introduced to determine unknown disulfide bond connectivities in globular proteins. A previous NMR structure determination was thus confirmed and the structure refined. Crotamine contains an alpha-helix with residues 1-7 and a two-stranded anti-parallel beta-sheet with residues 9-13 and 34-38 as the only regular secondary structures. These are connected with each other and the remainder of the polypeptide chain by the three disulfide bonds, which also form part of a central hydrophobic core. A single conformation was observed, with Pro13 and Pro21 in the trans and Pro20 in the cis-form. The global fold and the cysteine-pairing pattern of crotamine are similar to the beta-defensin fold, although the two proteins have low sequence homology, and display different biological activities.

Protein mapping of the salivary complex from a hematophagous leech.

The salivary complex of leeches contains many components able to modulate physiological mechanisms, such as coagulation and fibrinolysis, and it is composed by the salivary glands and proboscis, encompassing two different proteomes. The bidimensional electrophoretic pattern of the salivary complex from the Haementeria depressa leech revealed a total of 352 spots, 103 in common with the muscular tissue and 249 exclusive from the salivary complex as detected by silver staining; these spots showed isoelectric points from 3.5 to 9.5 and covered an apparent molecular weight range from 10 to 105 kDa. The following isoforms of proteins were identified by mass spectrometry analysis: antiplatelet protein, myohemerythrin and carbonic anhydrase. Since the leeches were not fed for about 2-3 months to stimulate the secretion of proteins that facilitates the blood metabolism, these most abundant proteins in the salivary complex excised from leeches, are expected to play a role during feeding and might have some anti-hemostatic properties. Furthermore, by zymography, a gelatinolytic and a fibrinolytic protein were identified.

Mass spectrometric and high performance liquid chromatography profiling of the venom of the Brazilian vermivorous mollusk Conus regius: feeding behavior and identification of one novel conotoxin.

Carnivorous mollusks belonging to the genus Conus paralyze their prey by injecting a rich mixture of biologically active peptides. Conus regius is a vermivorous member of this genus that inhabits Brazilian tropical waters. Inter-, intra-species and individual variations of cone snail venom have been previously reported. In order to investigate intra-specific differences in C. regius venom, its feeding behavior and the correlation between these two factors, animals were pooled according to gender, size and season of collection, and their venom composition was compared by high performance liquid chromatography (HPLC). Both the whole venom and one specific peak were monitored by HPLC. Chromatographic profiles revealed no significant differences in their peak areas, indicating that the venom composition, based solely in the presence or absence of the major peaks, is stable regardless of season, gender and size. Therefore, analysis of one given toxin, eluting in one of the major peaks, is representative among the population. Moreover, this work presents the identification of one novel conotoxin (rg11a), which amino acid sequence was deduced by mass spectrometry.

Crotamine is a novel cell-penetrating protein from the venom of rattlesnake Crotalus durissus terrificus.

Herein we report that crotamine, a small lysine- and cysteine-rich protein from the venom of the South American rattlesnake, can rapidly penetrate into different cell types and mouse blastocysts in vitro. In vivo crotamine strongly labels cells from mouse bone marrow and spleen and from peritoneal liquid, as shown by fluorescent confocal laser-scanning microscopy. Nuclear localization of crotamine was observed in both fixed and unfixed cells. In the cytoplasm, crotamine specifically associates with centrosomes and thus allows us to follow the process of centriole duplication and separation. In the nucleus, it binds to the chromosomes at S/G2 phase, when centrioles start dividing. Moreover, crotamine appears as a marker of actively proliferating cells, as shown by 5-BrdU cell-proliferation assay. Crotamine in the micromolar range proved nontoxic to any of the cell cultures tested and did not affect the pluripotency of ES cells or the development of mouse embryos.