Analgesic effects of Phalfa1 beta toxin: a review of mechanisms of action involving pain pathways

Phα1ß is a neurotoxin purified from spider venom that acts as a high-voltage-activated (HVA) calcium channel blocker. This spider peptide has shown a high selectivity for N-type HVA calcium channels (NVACC) and an analgesic effect in several animal models of pain. Its activity was associated with a reduction in calcium transients, glutamate release, and reactive oxygen species production from the spinal cord tissue and dorsal ganglia root (DRG) in rats and mice. It has been reported that intrathecal (i.t.) administration of Phα1ß to treat chronic pain reverted opioid tolerance with a safer profile than ω-conotoxin MVIIA, a highly selective NVACC blocker. Following a recent development of recombinant Phα1ß (CTK 01512-2), a new molecular target, TRPA1, the structural arrangement of disulphide bridges, and an effect on glial plasticity have been identified. CTK 01512-2 reproduced the antinociceptive effects of the native toxin not only after the intrathecal but also after the intravenous administration. Herein, we review the Phα1ß antinociceptive activity in the most relevant pain models and its mechanisms of action, highlighting the impact of CTK 01512-2 synthesis and its potential for multimodal analgesia.

Analgesic effects of Phα1ß toxin: a review of mechanisms of action involving pain pathways

Phα1ß is a neurotoxin purified from spider venom that acts as a high-voltage-activated (HVA) calcium channel blocker. This spider peptide has shown a high selectivity for N-type HVA calcium channels (NVACC) and an analgesic effect in several animal models of pain. Its activity was associated with a reduction in calcium transients, glutamate release, and reactive oxygen species production from the spinal cord tissue and dorsal ganglia root (DRG) in rats and mice. It has been reported that intrathecal (i.t.) administration of Phα1ß to treat chronic pain reverted opioid tolerance with a safer profile than ω-conotoxin MVIIA, a highly selective NVACC blocker. Following a recent development of recombinant Phα1ß (CTK 01512-2), a new molecular target, TRPA1, the structural arrangement of disulphide bridges, and an effect on glial plasticity have been identified. CTK 01512-2 reproduced the antinociceptive effects of the native toxin not only after the intrathecal but also after the intravenous administration. Herein, we review the Phα1ß antinociceptive activity in the most relevant pain models and its mechanisms of action, highlighting the impact of CTK 01512-2 synthesis and its potential for multimodal analgesia.

Chemical and Pharmacological Screening of Rhinella icterica (Spix 1824) Toad Parotoid Secretion in Avian Preparations.

The biological activity of Rhinella icterica parotoid secretion (RIPS) and some of its chromatographic fractions (RI18, RI19, RI23, and RI24) was evaluated in the current study. Mass spectrometry of these fractions indicated the presence of sarmentogenin, argentinogenin, (5ß,12ß)-12,14-dihydroxy-11-oxobufa-3,20,22-trienolide, marinobufagin, bufogenin B, 11α,19-dihydroxy-telocinobufagin, bufotalin, monohydroxylbufotalin, 19-oxo-cinobufagin, 3α,12ß,25,26-tetrahydroxy-7-oxo-5ß-cholestane-26-O-sulfate, and cinobufagin-3-hemisuberate that were identified as alkaloid and steroid compounds, in addition to marinoic acid and N-methyl-5-hydroxy-tryptamine. In chick brain slices, all fractions caused a slight decrease in cell viability, as also seen with the highest concentration of RIPS tested. In chick biventer cervicis neuromuscular preparations, RIPS and all four fractions significantly inhibited junctional acetylcholinesterase (AChE) activity. In this preparation, only fraction RI23 completely mimicked the pharmacological profile of RIPS, which included a transient facilitation in the amplitude of muscle twitches followed by progressive and complete neuromuscular blockade. Mass spectrometric analysis showed that RI23 consisted predominantly of bufogenins, a class of steroidal compounds known for their cardiotonic activity mediated by a digoxin- or ouabain-like action and the blockade of voltage-dependent L-type calcium channels. These findings indicate that the pharmacological activities of RI23 (and RIPS) are probably mediated by: (1) inhibition of AChE activity that increases the junctional content of Ach; (2) inhibition of neuronal Na+/K+-ATPase, leading to facilitation followed by neuromuscular blockade; and (3) blockade of voltage-dependent Ca2+ channels, leading to stabilization of the motor endplate membrane.

A novel Kunitz-type neurotoxin peptide identified from skin secretions of the frog Amolops loloensis.

A novel Kunitz-type neurotoxin peptide that inhibited voltage-gated sodium channel was purified and characterized from the skin secretions of rufous-spotted torrent frog, Amolops loloensis. It has a 240-bp cDNA encoding an 79-amino acid residue (aa) precursor protein containing 6 half-cysteines. The precursor was proven to release a 57-aa mature peptide with amino acid sequence, DRNPICNLPPKEGFCLWMMRRSFFNPSKGRCDTFGYRGCGGNKNNFETPRACKEACG. The mature was named amotoxin. Amotoxin shares sequence homology with other Kunitz-type toxins and also has three cysteine bridges. Amotoxin showed an inhibitory ability against trypsin with an inhibitory constant (Ki) of 0.087 µM. To the best of our knowledge, this is the first gene-encoded neurotoxin found in Amolops loloensis. Recombinant amotoxin showed similar functional properties as the native amotoxin. The functional properties of amotoxin may provide insights into the ecological adaptation of amphibians and deepen our understanding about the biological function spectrum of amphibian skin peptides.

Structure-Function Elucidation of a New α-Conotoxin, MilIA, from Conus milneedwardsi.

The a-Conotoxins are peptide toxins that are found in the venom of marine cone snails and they are potent antagonists of various subtypes of nicotinic acetylcholine receptors (nAChRs). Because nAChRs have an important role in regulating transmitter release, cell excitability, and neuronal integration, nAChR dysfunctions have been implicated in a variety of severe pathologies. We describe the isolation and characterization of α-conotoxin MilIA, the first conopeptide from the venom of Conus milneedwardsi. The peptide was characterized by electrophysiological screening against several types of cloned nAChRs that were expressed in Xenopus laevis oocytes. MilIA, which is a member of the α3/5 family, is an antagonist of muscle type nAChRs with a high selectivity for muscle versus neuronal subtype nAChRs. Several analogues were designed and investigated for their activity in order to determine the key epitopes of MilIA. Native MilIA and analogues both showed activity at the fetal muscle type nAChR. Two single mutations (Met9 and Asn10) allowed for MilIA to strongly discriminate between the two types of muscle nAChRs. Moreover, one analogue, MilIA [∆1,M2R, M9G, N10K, H11K], displayed a remarkable enhanced potency when compared to native peptide. The key residues that are responsible for switching between muscle and neuronal nAChRs preference were elucidated. Interestingly, the same analogue showed a preference for α9α10 nAChRs among the neuronal types.

µ-TRTX-Ca1a: a novel neurotoxin from Cyriopagopus albostriatus with analgesic effects.

Human genetic and pharmacological studies have demonstrated that voltage-gated sodium channels (VGSCs) are promising therapeutic targets for the treatment of pain. Spider venom contains many toxins that modulate the activity of VGSCs. To date, only 0.01% of such spider toxins has been explored, and thus there is a great potential for discovery of novel VGSC modulators as useful pharmacological tools or potential therapeutics. In the current study, we identified a novel peptide, µ-TRTX-Ca1a (Ca1a), in the venom of the tarantula Cyriopagopus albostriatus. This peptide consisted of 38 residues, including 6 cysteines, i.e. IFECSISCEIEKEGNGKKCKPKKCKGGWKCKFNICVKV. In HEK293T or ND7/23 cells expressing mammalian VGSCs, this peptide exhibited the strongest inhibitory activity on Nav1.7 (IC50 378 nM), followed by Nav1.6 (IC50 547 nM), Nav1.2 (IC50 728 nM), Nav1.3 (IC50 2.2 µM) and Nav1.4 (IC50 3.2 µM), and produced negligible inhibitory effect on Nav1.5, Nav1.8, and Nav1.9, even at high concentrations of up to 10 µM. Furthermore, this peptide did not significantly affect the activation and inactivation of Nav1.7. Using site-directed mutagenesis of Nav1.7 and Nav1.4, we revealed that its binding site was localized to the DIIS3-S4 linker region involving the D816 and E818 residues. In three different mouse models of pain, pretreatment with Cala (100, 200, 500 µg/kg) dose-dependently suppressed the nociceptive responses induced by formalin, acetic acid or heat. These results suggest that Ca1a is a novel neurotoxin against VGSCs and has a potential to be developed as a novel analgesic.

A viral-fusion-peptide-like molecular switch drives membrane insertion of botulinum neurotoxin A1.

Botulinum neurotoxin (BoNT) delivers its protease domain across the vesicle membrane to enter the neuronal cytosol upon vesicle acidification. This process is mediated by its translocation domain (HN), but the molecular mechanism underlying membrane insertion of HN remains poorly understood. Here, we report two crystal structures of BoNT/A1 HN that reveal a novel molecular switch (termed BoNT-switch) in HN, where buried α-helices transform into surface-exposed hydrophobic ß-hairpins triggered by acidic pH. Locking the BoNT-switch by disulfide trapping inhibited the association of HN with anionic liposomes, blocked channel formation by HN, and reduced the neurotoxicity of BoNT/A1 by up to ~180-fold. Single particle counting studies showed that an acidic environment tends to promote BoNT/A1 self-association on liposomes, which is partly regulated by the BoNT-switch. These findings suggest that the BoNT-switch flips out upon exposure to the acidic endosomal pH, which enables membrane insertion of HN that subsequently leads to LC delivery.

Novel Kunitz-like Peptides Discovered in the Zoanthid Palythoa caribaeorum through Transcriptome Sequencing.

Palythoa caribaeorum (class Anthozoa) is a zoanthid that together jellyfishes, hydra, and sea anemones, which are venomous and predatory, belongs to the Phyllum Cnidaria. The distinguished feature in these marine animals is the cnidocytes in the body tissues, responsible for toxin production and injection that are used majorly for prey capture and defense. With exception for other anthozoans, the toxin cocktails of zoanthids have been scarcely studied and are poorly known. Here, on the basis of the analysis of P. caribaeorum transcriptome, numerous predicted venom-featured polypeptides were identified including allergens, neurotoxins, membrane-active, and Kunitz-like peptides (PcKuz). The three predicted PcKuz isotoxins (1-3) were selected for functional studies. Through computational processing comprising structural phylogenetic analysis, molecular docking, and dynamics simulation, PcKuz3 was shown to be a potential voltage gated potassium-channel inhibitor. PcKuz3 fitted well as new functional Kunitz-type toxins with strong antilocomotor activity as in vivo assessed in zebrafish larvae, with weak inhibitory effect toward proteases, as evaluated in vitro. Notably, PcKuz3 can suppress, at low concentration, the 6-OHDA-induced neurotoxicity on the locomotive behavior of zebrafish, which indicated PcKuz3 may have a neuroprotective effect. Taken together, PcKuz3 figures as a novel neurotoxin structure, which differs from known homologous peptides expressed in sea anemone. Moreover, the novel PcKuz3 provides an insightful hint for biodrug development for prospective neurodegenerative disease treatment.

Defensin-neurotoxin dyad in a basally branching metazoan sea anemone.

Recent studies suggest that vertebrate and invertebrate defensins have evolved from two independent ancestors, and that both defensins could share origins with animal toxins. Here, we purified novel sea anemone neurotoxin (BDS)-like antimicrobial peptides (AMPs)-Crassicorin-I and its putative homolog (Crassicorin-II)-from the pharynx extract of an anthozoan sea anemone (Urticina crassicornis). Based on structural analyses and cDNA cloning, mature Crassicorin-I represents a cationic AMP likely generated from a precursor and comprising 40 amino acid residues, including six cysteines forming three intramolecular disulfide bonds. Recombinant Crassicorin-I produced in a heterologous bacterial-expression system displayed antimicrobial activity against both a gram-positive bacterium (Bacillus subtilis) and gram-negative bacteria (Escherichia coli and Salmonella enterica). The Crassicorin-I transcript was upregulated by immune challenge, suggesting its involvement in defense mechanisms against infectious pathogens in sea anemone. Sequence alignment and three-dimensional molecular modeling revealed that Crassicorin-I exhibits high degrees of structural similarity to sea anemone neurotoxins that share ß-defensin fold which is found in vertebrate defensins and invertebrate big-defensins. Consistent with its structural similarity to neurotoxins, Crassicorin-I exhibited paralytic activity toward a crustacean. These findings motivated our investigation and subsequent discovery of antimicrobial activity from other known sea anemone neurotoxins, such as APETx1 and ShK. Collectively, our work signified that Crassicorin-I is the first AMP identified from a sea anemone and provided evidence of a functional linkage between AMPs and neurotoxins in a basally branching metazoan.

Unravelling the Diversity of the Cyclopiazonic Acid Family of Mycotoxins in Aspergillus flavus by UHPLC Triple-TOF HRMS.

Cyclopiazonic acid (α-cyclopiazonic acid, α-CPA) is an indole-hydrindane-tetramic acid neurotoxin produced by various fungal species, including the notorious food and feed contaminant Aspergillus flavus. Despite its discovery in A. flavus cultures approximately 40 years ago, its contribution to the A. flavus mycotoxin burden is consistently minimized by our focus on the more potent carcinogenic aflatoxins also produced by this fungus. Here, we report the screening and identification of several CPA-type alkaloids not previously found in A. flavus cultures. Our identifications of these CPA-type alkaloids are based on a dereplication strategy involving accurate mass high resolution mass spectrometry data and a careful study of the α-CPA fragmentation pattern. In total, 22 CPA-type alkaloids were identified in extracts from the A. flavus strains examined. Of these metabolites, 13 have been previously reported in other fungi, though this is the first report of their existence in A. flavus. Two of our metabolite discoveries, 11,12-dehydro α-CPA and 3-hydroxy-2-oxo CPA, have never been reported for any organism. The conspicuous presence of CPA and its numerous derivatives in A. flavus cultures raises concerns about the long-term and cumulative toxicological effects of these fungal secondary metabolites and their contributions to the entire A. flavus mycotoxin problem.

Old Weapons for New Wars: Bioactive Molecules From Cnidarian Internal Defense Systems.

The renewed interest in the study of genes of immunity in Cnidaria has led to additional information to the scenario of the first stages of immunity evolution revealing the cellular processes involved in symbiosis, in the regulation of homeostasis and in the fight against infections. The recent study with new molecular and functional approach on these organisms have therefore contributed with unexpected information on the knowledge of the stages of capturing activities and defense mechanisms strongly associated with toxin production. Cnidarians are diblastic aquatic animals with radial symmetry; they represent the ancestral state of Metazoa, they are the simplest multicellular organisms that have reached the level of tissue organization.The Cnidaria phylum has evolved using biotoxins as defense or predation mechanisms for ensure survival in hostile and competitive environments such as the seas and oceans. From benthic and pelagic species a large number of toxic compounds that have been determined can have an active role in the development of various antiviral, anticancer and antibacterial functions. Although the immune defense response of these animals is scarcely known, the tissues and the mucus produced by cnidarians are involved in immune defense and contain a large variety of peptides such as sodium and potassium channel neurotoxins, cytolysins, phospholipase A2 (PLA2), acid-sensing ion channel peptide toxins (ASICs) and other toxins, classified following biochemical and pharmacological studies on the basis of functional, molecular and structural parameters. These basal metazoan in fact, are far from "simple" in the range of methods at their disposal to deal with potential prey but also invading microbes and pathogens. They could also take advantage of the multi-functionality of some of their toxins, for example, some bioactive molecules have characteristics of toxicity associated with a potential antimicrobial activity. The interest in cnidarians was not only directed to the study of toxins and venom, but also to the fact these animals have been suggested as source of new molecules potentially relevant for biotechnology and pharmaceutical applications. Here, we review the cnidarian type of toxins regarding their multifunctional role and the future possibility of drawing important applications in fields ranging from biology to pharmacology.

Tetrodotoxin, an Extremely Potent Marine Neurotoxin: Distribution, Toxicity, Origin and Therapeutical Uses.

Tetrodotoxin (TTX) is a potent neurotoxin responsible for many human intoxications and fatalities each year. The origin of TTX is unknown, but in the pufferfish, it seems to be produced by endosymbiotic bacteria that often seem to be passed down the food chain. The ingestion of contaminated pufferfish, considered the most delicious fish in Japan, is the usual route of toxicity. This neurotoxin, reported as a threat to human health in Asian countries, has spread to the Pacific and Mediterranean, due to the increase of temperature waters worldwide. TTX, for which there is no known antidote, inhibits sodium channel producing heart failure in many cases and consequently death. In Japan, a regulatory limit of 2 mg eq TTX/kg was established, although the restaurant preparation of "fugu" is strictly controlled by law and only chefs qualified are allowed to prepare the fish. Due to its paralysis effect, this neurotoxin could be used in the medical field as an analgesic to treat some cancer pains.

Jingzhaotoxin-35, a novel gating-modifier toxin targeting both Nav1.5 and Kv2.1 channels.

Jingzhaotoxin-35 (JZTX-35), a 36-residue polypeptide, was purified from the venom of the Chinese tarantula Chilobrachys jingzhao. JZTX-35 inhibited Nav1.5 and Kv2.1 currents with the IC50 value of 1.07 µM and 3.62 µM, respectively, but showed no significant effect on either Na(+) currents or Ca(2+) currents evoked in hippocampal neurons. It shifted the activation of the Nav1.5 and Kv2.1 channels to more depolarized voltages, and markedly shifted the steady-state inactivation of Nav1.5 currents toward more hyperpolarized potentials. Moreover, JZTX-35 can bind to a close state of Nav1.5 and Kv2.1 channels. These results indicate that JZTX-35 is a new gating modifier toxin. JZTX-35 shares high sequence similarity with Jingzhaotoxins (JZTXs) targeting Nav1.5 or Kv2.1 channels, but they showed different ion channel selectivity. Structure-function analysis in this study would provide important clues for the exploration of ion channel selectivity of JZTXs.

A novel neurotoxin from venom of the spider, Brachypelma albopilosum.

Spiders have evolved highly selective toxins for insects. There are many insecticidal neurotoxins in spider venoms. Although a large amount of work has been done to focus on neurotoxicity of spider components, little information, which is related with effects of spider toxins on tumor cell proliferation and cytotoxicity, is available for Brachypelma albopilosum venom. In this work, a novel spider neurotoxin (brachyin) was identified and characterized from venoms of the spider, Brachypelma albopilosum. Brachyin is composed of 41 amino acid residues with the sequence of CLGENVPCDKDRPNCCSRYECLEPTGYGWWYASYYCYKKRS. There are six cysteines in this sequence, which form three disulfided bridges. The serine residue at the C-terminus is amidated. Brachyin showed strong lethal effects on American cockroaches (Periplaneta americana) and Tenebrio molitor (common mealbeetle). This neurotoxin also showed significant analgesic effects in mice models including abdominal writhing induced by acetic acid and formalin-induced paw licking tests. It was interesting that brachyin exerted marked inhibition on tumor cell proliferation.

Detection of a novel neurotoxic metabolite of Parkinson's disease-related neurotoxin, 1-benzyl-1,2,3,4- tetrahydroisoquinoline.

Naturally occurring low-molecular weight compounds with a chemical structure like that of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, such as 1-benzyl-1,2,3,4-tetrahydroisoquinoline(1BnTIQ), are candidates for the endogenous neurotoxins that cause Parkinson's disease (PD). 1BnTIQ is an endogenous amine in human CSF and increases in the CSF of patients with PD. It inhibits complex Iand elicits PD-like behavioral abnormalities in monkey and mouse. In this study, we searched metabolites of 1BnTIQ by rat liver S9 using liquid chromatography-tandem mass spectrometry, and identified a dehydrated metabolite, 1-benzyl-3,4-dihydroisoquinoline (1BnDIQ). 1BnDIQ was identified by corresponding mass spectra and precursor ion scans in authentic and complete enzyme samples. Multiple reaction monitoring analysis showed microsome-dependent 1BnDIQ production. We previously reported that 1BnDIQ is more toxic than 1BnTIQ in cytotoxicity study in SH-SY5Y neuroblastoma cells. In addition, 1BnTIQ is reported to pass through the blood-brain barrier of the rat brain, and 1BnDIQ is supposed to be more lipophilic than 1BnTIQ. 1BnDIQ may easily reach the brain, and it might contribute to PD-related neurotoxicity.

Cytotoxicity of recombinant tamapin and related toxin-like peptides on model cell lines.

The scorpion toxin tamapin displays the most potent and selective blockage against KCa2.2 channels known to date. In this work, we report the biosynthesis, three-dimensional structure, and cytotoxicity on cancer cell lines (Jurkat E6-1 and human mammary breast cancer MDA-MB-231) of recombinant tamapin and five related peptides bearing mutations on residues (R6A,R7A, R13A, R6A-R7A, and GS-tamapin) that were previously suggested to be important for tamapin's activity. The indicated cell lines were used as they constitutively express KCa2.2 channels. The studied toxin-like peptides displayed lethal responses on Jurkat T cells and breast cancer cells; their effect is dose- and time-dependent with IC50 values in the nanomolar range. The order of potency is r-tamapin>GS-tamapin>R6A>R13A>R6A-R7A>R7A for Jurkat T cells and r-tamapin>R7A for MDA-MB-231 breast cancer cells. Our structural determination by NMR demonstrated that r-tamapin preserves the folding of the αKTx5 subfamily and that neither single nor double alanine mutations affect the three-dimensional structure of the wild-type peptide. In contrast, our activity assays show that changes in cytotoxicity are related to the chemical nature of certain residues. Our results suggest that the toxic activity of r-tamapin on Jurkat and breast cancer cells could be mediated by the interaction of charged residues in tamapin with KCa2.2 channels via the apoptotic cell death pathway.

Effects of Cylindrospermopsis raciborskii strains (Woloszynska, 1912) Senayya & Subba Raju on the mobility of Daphnia laevis (Cladocera, Daphniidae) / Efeitos de cepas de Cylindrospermopsis raciborskii (Woloszynska, 1912), Senayya & Subba Raju na mobilidade de Daphnia laevis (Cladocera, Daphniidae)

Cylindrospermopsis raciborskii is a cyanobacterium distributed worldwide that is known to produce cyanotoxins. Some of the Brazilian strains can produce saxitoxins (STXs), which are classified as neurotoxins and can paralyze cladocerans .Daphnia laevis is a cladoceran with a wide distribution in the Americas and has been studied as a possible test-organism in toxicity bioassays. The present work tested the acute effect on D laevis mobility when fed a saxitoxin-producing (STX and neoSTX) C. raciborskii strain, CYRF-01, and compared the results with the effects of a non-toxic strain (NPCS-1). Neonates (6-24 hours after birth) were exposed to concentrations of C. raciborskii varying from 102 to 106 cells·mL–1 of each strain for up to three hours. The cladocerans were then transferred to a medium without toxic filaments for 24 hours. Only the organisms exposed to the STX-producing strain showed signs of the immobilization of swimming movements, confirming the effects of the toxins. There was a linear correlation between the time required to induce stopping the swimming movement, with a shorter time to needed to induce immobilization at a higher the concentration; this correlation was inverse to the time required to recover the swimming movements (longer at higher concentrations, p < 0.1). D. laevis is a tropical and subtropical species with great potential for use in toxicity tests for the detection of STXs, despite being native to and found in a great array of freshwater bodies. This is the first assay testing STX-producing and non-producing C. raciborskii strains on D. laevis, species that are both found in Brazilian ecosystems.

Cylindrospermopsis raciborskii é uma espécie de cianobactéria, difundida mundialmente, conhecida como produtora de cianotoxinas. Algumas linhagens brasileiras são conhecidas como produtoras de saxitoxinas (STXs), as quais são classificadas como neurotoxinas e podem induzir a paralização dos movimentos natatórios em cladóceros. Daphnia laevis é um cladócero de ampla distribuição nas Américas e tem sido estudado como um organismo teste para uso em ensaios de toxicidade. Neste sentido, o presente trabalho avaliou os efeitos agudos de C. raciborskii, de uma cepa produtora de STXs (STX e neoSTX) - CYRF-01 e outra não produtora de cianotoxinas (NPCS-1) sobre a mobilidade de D. laevis. Para tanto, neonatas entre 6-24 horas de idade foram submetidas a concentrações de C. raciborskii variando de 102 a 106 céls.mL–1 de ambas as cepas, por um período de 3 horas, e transferidos posteriormente para um meio isento de filamentos tóxicos por 24horas. Apenas os organismos expostos à cepa produtora de STXs apresentaram paralisia dos movimentos natatórios, confirmando o efeito da cianotoxina. Houve uma correlação linear entre o tempo de paralização e as concentrações, isto é, nas concentrações mais altas, os organismos paralisaram num curto período de tempo, e conseqüentemente, levaram mais tempo para se recuperar (p < 0,1). D.laevis é uma espécie de região tropical e subtropical com potencial uso em bioensaios para detecção de STXs. Trata-se dos primeiros relatos acerca da resposta de D.laevis quando submetida às cepas de C. raciborskii produtora e não produtora de STXs, ambas isoladas de ecossistemas brasileiros.

Various conotoxin diversifications revealed by a venomic study of Conus flavidus.

Conotoxins are peptide neurotoxins produced by predatory cone snails. They are mostly cysteine-rich short peptides with remarkable structural diversity. The conserved signal peptide sequences of their mRNA-encoded precursors have enabled the grouping of known conotoxins into a limited number of superfamilies. However, the conotoxins within each superfamily often present variable sequences, cysteine frameworks, and post-translational modifications. To understand better how conotoxins are diversified, we performed a venomic study with C. flavidus, an uninvestigated vermivorous Conus species, by combining transcriptomic and proteomic analyses. In order to obtain the full-length conotoxin sequences, protease digestion was not performed with the venom extraction prior to spectra acquisition via tandem mass spectrometry (MS/MS). Because conotoxins are produced from mRNA-encoded precursors by means of proteolytic cleavage, nonspecific digestion of precursors was applied during the database search. Special attention was also paid in interpreting the MS/MS spectra. All together, these analyses identified 69 nonredundant cDNA sequences and 31 conotoxin components with confident MS/MS spectra. A new Q-superfamily was also identified. More importantly, this study revealed that conotoxin-encoding transcripts are diversified by hypermutation, fragment insertion/deletion, and mutation-induced premature termination, and that a single mRNA species can produce multiple toxin products through alternative post-translational modifications and alternative cleavages of the translated precursor. These multiple diversification strategies at different levels may explain, at least in part, the diversity of conotoxins, and provide the basis for further investigation.

Novel sesquiterpenoid glycosides and sesquiterpenes from the roots of Illicium henryi.

Seven new sesquiterpenoid glycosides, consisting of one thapsan-type (1), two capnellane-type (2 and 3), four floridanolide sesquiterpene glycosides (4-7), and one new azulene-type sesquiterpene (8), along with six known sesquiterpenes (9-14) were isolated from the roots of Illicium henryi. The structures of 1-8 were elucidated by extensive spectroscopic analyses and chemical methods. The absolute configurations of 1, 2, and 8 were determined based on Rh2(OCOCF3)4-induced CD, Mo2(OAc)4-induced CD data, and calculated electronic circular dichroism (ECD), respectively. Compound 1 was found to exhibit weak neurotoxicant activity.

Purification, characterization, and chemical modification of neurotoxic peptide from Daboia russelii snake venom of India.

Comprehensive knowledge of venom composition is very important for effective management of snake envenomation and antivenom preparation. Daboia russelii venom from the eastern region of India is the most neurotoxic among the four venom samples investigated. From the eastern D. russelii venom sample, neurotoxic peptide has been purified by combined method of ion exchange gel permeation chromatography and reversed phase high performance liquid chromatography. Molecular weight of Daboia neurotoxin III (DNTx-III) found to be 6,849 Da (as measured on matrix-assisted laser desorption/ionisation-time of flight mass spectrometer), and N-terminal amino acid sequences is I K C F I T P D U T S Q A. Approximate LD50 dosage was 0.24 mg/kg body weight. It produced concentration- and time-dependent inhibition of indirectly stimulated twitches of Rana hexadactyla sciatic nerve gastrocnemius muscle preparations. Chemical modification of DNTx-III tryptophan residue(s) reduced the twitch height inhibition property of toxin, signifying the importance of tryptophan residues for the neurotoxic function. This type of neurotoxic peptide is unique to east Indian regional D. russelii venom.