RESUMO
Tetrodotoxin (TTX) is a potent neurotoxin that was first identified in pufferfish but has since been isolated from an array of taxa that host TTX-producing bacteria. However, determining its origin, ecosystem roles, and biomedical applications has challenged researchers for decades. Recognized as a poison and for its lethal effects on humans when ingested, TTX is primarily a powerful sodium channel inhibitor that targets voltage-gated sodium channels, including six of the nine mammalian isoforms. Although lethal doses for humans range from 1.5-2.0 mg TTX (blood level 9 ng/mL), when it is administered at levels far below LD50, TTX exhibits therapeutic properties, especially to treat cancer-related pain, neuropathic pain, and visceral pain. Furthermore, TTX can potentially treat a variety of medical ailments, including heroin and cocaine withdrawal symptoms, spinal cord injuries, brain trauma, and some kinds of tumors. Here, we (i) describe the perplexing evolution and ecology of tetrodotoxin, (ii) review its mechanisms and modes of action, and (iii) offer an overview of the numerous ways it may be applied as a therapeutic. There is much to be explored in these three areas, and we offer ideas for future research that combine evolutionary biology with therapeutics. The TTX system holds great promise as a therapeutic and understanding the origin and chemical ecology of TTX as a poison will only improve its general benefit to humanity.
Assuntos
Tetrodotoxina/toxicidade , Tetrodotoxina/uso terapêutico , Animais , Resistência a Medicamentos , Ecologia , Humanos , Neurotoxinas/uso terapêutico , Neurotoxinas/toxicidade , Filogenia , Venenos/uso terapêutico , Venenos/toxicidade , Bloqueadores dos Canais de Sódio/uso terapêutico , Bloqueadores dos Canais de Sódio/toxicidadeRESUMO
Extensive activation of glial cells during a latent period has been well documented in various animal models of epilepsy. However, it remains unclear whether activated glial cells contribute to epileptogenesis, i.e., the chronically persistent process leading to epilepsy. Particularly, it is not clear whether interglial communication between different types of glial cells contributes to epileptogenesis, because past literature has mainly focused on one type of glial cell. Here, we show that temporally distinct activation profiles of microglia and astrocytes collaboratively contributed to epileptogenesis in a drug-induced status epilepticus model. We found that reactive microglia appeared first, followed by reactive astrocytes and increased susceptibility to seizures. Reactive astrocytes exhibited larger Ca2+ signals mediated by IP3R2, whereas deletion of this type of Ca2+ signaling reduced seizure susceptibility after status epilepticus. Immediate, but not late, pharmacological inhibition of microglial activation prevented subsequent reactive astrocytes, aberrant astrocyte Ca2+ signaling, and the enhanced seizure susceptibility. These findings indicate that the sequential activation of glial cells constituted a cause of epileptogenesis after status epilepticus. Thus, our findings suggest that the therapeutic target to prevent epilepsy after status epilepticus should be shifted from microglia (early phase) to astrocytes (late phase).
Assuntos
Astrócitos/metabolismo , Epilepsia/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Microglia/metabolismo , Estado Epiléptico/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Sinalização do Cálcio , Modelos Animais de Doenças , Progressão da Doença , Suscetibilidade a Doenças , Epilepsia/induzido quimicamente , Epilepsia/patologia , Epilepsia/fisiopatologia , Gliose/metabolismo , Interleucina-1beta/metabolismo , Camundongos , Microglia/efeitos dos fármacos , Microglia/patologia , Agonistas Muscarínicos/toxicidade , Compostos Orgânicos/farmacologia , Pilocarpina/toxicidade , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Bloqueadores dos Canais de Sódio/toxicidade , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/patologia , Estado Epiléptico/fisiopatologia , Tetrodotoxina/toxicidade , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Ribosome inactivating proteins (RIPs) as family of anti-cancer drugs recently received much attention due to their interesting anti-cancer mechanism. In spite of small drugs, RIPs use the large-size effect (LSE) to prevent the efflux process governed by drug resistance transporters (DRTs) which prevents inside of the cells against drug transfection. There are many clinical translation obstacles that severely restrict their applications especially their delivery approach to the tumor cells. As the main goal of this review, we will focus on trichosanthin (TCS) and gelonin (Gel) and other types, especially scorpion venom-derived RIPs to clarify that they are struggling with what types of bio-barriers and these challenges could be solved in cancer therapy science. Then, we will try to highlight recent state-of-the-arts in delivery of RIPs for cancer therapy.
Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Neoplasias/tratamento farmacológico , Proteínas Inativadoras de Ribossomos Tipo 1/toxicidade , Tricosantina/toxicidade , Animais , Antineoplásicos Fitogênicos/toxicidade , Humanos , Proteínas Inativadoras de Ribossomos Tipo 1/uso terapêutico , Venenos de Escorpião/uso terapêutico , Venenos de Escorpião/toxicidade , Bloqueadores dos Canais de Sódio/uso terapêutico , Bloqueadores dos Canais de Sódio/toxicidade , Tricosantina/uso terapêuticoRESUMO
Indoxacarb, a commercialized oxadiazine insecticide, nearly irreversibly blocks open/inactivated, but not resting sodium channels. The structure-activity relationships showed that the substituents at the position of the chiral atom in the oxadiazine ring are very important to the biological activity of oxadiazine insecticide. Here we synthesized a series of tricyclic oxadiazine 4a-methyl ester derivatives. The chiral atom in the oxadiazine ring has been epimerized and substituted with either pyrethric acid or cinnamic acid derivatives. Benzene ring in the tricyclic moiety was substituted with a chlorine, fluorine, or bromine atom, and nitrogen-linked benzene ring was substituted with a trifluoromethyl or trifluoromethoxy group. Toxicity of these compounds against Spodoptera litura F. was evaluated. Diastereoisomers of most toxic compounds J7 and J9 with pyrethric acid moiety were separated by flash column chromatography. The more polar diastereoisomers, J7-L-Rf and J9-L-Rf, and compounds J24 and J26 with cinnamic acid moiety exhibited highest insecticidal activities. We further used Monte Carlo energy minimizations to dock compound J7 and J24 in the NavMs-based homology model of the open cockroach sodium channel. In the low-energy binding modes, the compound interacted with residues in the inner pore and domain interfaces, which previously were proposed to contribute to receptors of pyrethroids and sodium channel blocker insecticides. Our results define compound J7 and J24 as a potentially useful optimized hit for the development of multiple sites sodium channel blocker or modulator.
Assuntos
Inseticidas/química , Inseticidas/toxicidade , Oxazinas/química , Oxazinas/toxicidade , Bloqueadores dos Canais de Sódio/química , Bloqueadores dos Canais de Sódio/toxicidade , Animais , Baratas/efeitos dos fármacos , Baratas/metabolismo , Descoberta de Drogas , Ésteres/química , Ésteres/farmacologia , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Modelos Moleculares , Canais de Sódio/química , Canais de Sódio/metabolismo , Spodoptera/efeitos dos fármacos , Spodoptera/metabolismo , Relação Estrutura-AtividadeRESUMO
The scorpionism in Panama is notorious for the confluence and coexistence of buthid scorpions from the genera Centruroides and Tityus. This communication describes an overview of the larger representative toxic venom fractions from eight dangerous buthid scorpion species of Panama: Centruroides (C. granosus, C. bicolor, C. limbatus and C. panamensis) and Tityus (T. (A.) asthenes, T. (A.) festae, T. (T.) cerroazul and T. (A.) pachyurus). Their venoms were separated by HPLC and the corresponding sub-fractions were tested for lethality effects on mice and insects. Many fractions toxic to either mice or insects, or both, were found and have had their molecular masses determined by mass spectrometry analysis. The great majority of the lethal components had a molecular mass close to 7000 Da, assumed to be peptides that recognize Na+-channels, responsible for the toxicity symptoms observed in other buthids scorpion venoms. A toxic peptide isolated from the venom of T. pachyurus was sequenced by Edman degradation, allowing the synthesis of nucleotide probe for cloning the correspondent gene. The mature toxin based on the cDNA sequencing has the C-terminal residue amidated, contains 62 amino acid packed by 4 disulfide linkages, with molecular mass of 7099.1 Da. This same toxic peptide seems to be present in scorpions of the species T. pachyurus collected in 5 different regions of Panama, although the overall HPLC profile is quite different. The most diverse neurotoxic venom components from the genus Centruroides were found in the species C. panamensis, whereas T. cerroazul was the one from the genus Tityus. The most common neurotoxins were observed in the venoms of T. festae, T. asthenes and T. pachyurus with closely related molecular masses of 7099.1 and 7332 Da. The information reported here is considered very important for future generation of a neutralizing antivenom against scorpions from Panama. Furthermore, it will contribute to the growing interest in using bioactive toxins from scorpions for drug discovery purposes.
Assuntos
Venenos de Escorpião/química , Escorpiões/classificação , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Gryllidae , Espectrometria de Massas , Camundongos , Panamá , Peptídeos/química , Venenos de Escorpião/genética , Venenos de Escorpião/toxicidade , Bloqueadores dos Canais de Sódio/química , Bloqueadores dos Canais de Sódio/toxicidade , Especificidade da EspécieRESUMO
We report the detailed chemical, immunological and pharmacological characterization of the α-toxin Bot IX from the Moroccan scorpion Buthus occitanus tunetanus venom. Bot IX, which consists of 70 amino acids, is a highly atypical toxin. It carries a unique N-terminal sequence extension and is highly lethal in mice. Voltage clamp recordings on oocytes expressing rat Nav1.2 or insect BgNav1 reveal that, similar to other α-like toxins, Bot IX inhibits fast inactivation of both variants. Moreover, Bot IX belongs to the same structural/immunological group as the α-like toxin Bot I. Remarkably, radioiodinated Bot IX competes efficiently with the classical α-toxin AaH II from Androctonus australis, and displays one of the highest affinities for Nav channels.
Assuntos
Venenos de Escorpião/toxicidade , Bloqueadores dos Canais de Sódio/toxicidade , Animais , Linhagem Celular , Baratas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Canal de Sódio Disparado por Voltagem NAV1.2/metabolismo , Domínios Proteicos , Ratos , Venenos de Escorpião/química , Venenos de Escorpião/farmacologia , Bloqueadores dos Canais de Sódio/química , Bloqueadores dos Canais de Sódio/farmacologia , XenopusRESUMO
Members of arachnida, such as spiders and scorpions, commonly produce venom with specialized venom glands, paralyzing their prey with neurotoxins that specifically target ion channels. Two well-studied motifs, the disulfide-directed hairpin (DDH) and the inhibitor cystine knot motif (ICK), are both found in scorpion and spider toxins. As arachnids, ticks inject a neurotoxin-containing cocktail from their salivary glands into the host to acquire a blood meal, but peptide toxins acting on ion channels have not been observed in ticks. Here, a new neurotoxin (ISTX-I) that acts on sodium channels was identified from the hard tick Ixodes scapularis and characterized. ISTX-I exhibits a potent inhibitory function with an IC50 of 1.6 µM for sodium channel Nav1.7 but not other sodium channel subtypes. ISTX-I adopts a novel structural fold and is distinct from the canonical ICK motif. Analysis of the ISTX-I, DDH and ICK motifs reveals that the new ISTX-I motif might be an intermediate scaffold between DDH and ICK, and ISTX-I is a clue to the evolutionary link between the DDH and ICK motifs. These results provide a glimpse into the convergent evolution of neurotoxins from predatory and blood-sucking arthropods.
Assuntos
Venenos de Artrópodes , Evolução Molecular , Ixodes/química , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Dobramento de Proteína , Bloqueadores dos Canais de Sódio , Motivos de Aminoácidos , Animais , Venenos de Artrópodes/química , Venenos de Artrópodes/toxicidade , Células HEK293 , Humanos , Masculino , Ratos , Ratos Sprague-Dawley , Bloqueadores dos Canais de Sódio/química , Bloqueadores dos Canais de Sódio/toxicidadeRESUMO
The toxin PnTx4(5-5) from the spider Phoneutria nigriventer is extremely toxic/lethal to insects but has no macroscopic behavioral effects observed in mice after intracerebral injection. Nevertheless, it was demonstrated that it inhibits the N-methyl-d-aspartate (NMDA) - subtype of glutamate receptors of cultured rat hippocampal neurons. PnTx4(5-5) has 63% identity to PnTx4(6-1), another insecticidal toxin from P. nigriventer, which can slow down the sodium current inactivation in insect central nervous system, but has no effect on Nav1.2 and Nav1.4 rat sodium channels. Here, we have cloned and heterologous expressed the toxin PnTx4(5-5) in Escherichia coli. The recombinant toxin rPnTx4(5-5) was tested on the sodium channel NavBg from the cockroach Blatella germanica and on mammalian sodium channels Nav1.2-1.6, all expressed in Xenopus leavis oocytes. We showed that the toxin has different affinity and mode of action on insect and mammalian sodium channels. The most remarkable effect was on NavBg, where rPnTx4(5-5) strongly slowed down channel inactivation (EC50 = 212.5 nM), and at 1 µM caused an increase on current peak amplitude of 105.2 ± 3.1%. Interestingly, the toxin also inhibited sodium current on all the mammalian channels tested, with the higher current inhibition on Nav1.3 (38.43 ± 8.04%, IC50 = 1.5 µM). Analysis of activation curves on Nav1.3 and Nav1.5 showed that the toxin shifts channel activation to more depolarized potentials, which can explain the sodium current inhibition. Furthermore, the toxin also slightly slowed down sodium inactivation on Nav1.3 and Nav1.6 channels. As far as we know, this is the first araneomorph toxin described which can shift the sodium channel activation to more depolarized potentials and also slows down channel inactivation.
Assuntos
Escherichia coli/metabolismo , Neurotoxinas/toxicidade , Canais de Sódio/efeitos dos fármacos , Venenos de Aranha/toxicidade , Animais , Baratas , Escherichia coli/genética , Neurotoxinas/genética , Neurotoxinas/metabolismo , Bloqueadores dos Canais de Sódio/metabolismo , Bloqueadores dos Canais de Sódio/toxicidade , Canais de Sódio/metabolismo , Venenos de Aranha/química , Aranhas/genéticaRESUMO
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.
Assuntos
Cnidários , Venenos de Cnidários/toxicidade , Neurotoxinas/toxicidade , Peptídeos/toxicidade , Animais , Anti-Infecciosos/imunologia , Anti-Infecciosos/isolamento & purificação , Anti-Infecciosos/toxicidade , Venenos de Cnidários/imunologia , Venenos de Cnidários/isolamento & purificação , Humanos , Neurotoxinas/imunologia , Neurotoxinas/isolamento & purificação , Peptídeos/imunologia , Peptídeos/isolamento & purificação , Bloqueadores dos Canais de Sódio/imunologia , Bloqueadores dos Canais de Sódio/isolamento & purificação , Bloqueadores dos Canais de Sódio/toxicidadeRESUMO
Selenocosmia jiafu is a medium-sized theraphosid spider and an attractive source of venom, because it can be bred in captivity and it produces large amounts of venom. We performed reversed-phase high-performance liquid chromatography (RP-HPLC) and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analyses and showed that S. jiafu venom contains hundreds of peptides with a predominant mass of 3000-4500 Da. Patch clamp analyses indicated that the venom could inhibit voltage-gated Na+, K+ and Ca2+ channels in rat dorsal root ganglion (DRG) neurons. The venom exhibited inhibitory effects on tetrodotoxin-resistant (TTX-R) Na+ currents and T-type Ca2+ currents, suggesting the presence of antagonists to both channel types and providing a valuable tool for the investigation of these channels and for drug development. Intra-abdominal injection of the venom had severe toxic effects on cockroaches and caused death at higher concentrations. The LD50 was 84.24 µg/g of body weight in the cockroach. However, no visible symptoms or behavioral changes were detected after intraperitoneal injection of the venom into mice even at doses up to 10 mg/kg body weight. Our results provide a basis for further case-by-case investigations of peptide toxins from this venom.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Neurônios/efeitos dos fármacos , Neurotoxinas/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Venenos de Aranha/farmacologia , Animais , Bloqueadores dos Canais de Cálcio/química , Bloqueadores dos Canais de Cálcio/toxicidade , Canais de Cálcio/fisiologia , Cromatografia Líquida de Alta Pressão , Baratas/efeitos dos fármacos , Feminino , Gânglios Espinais/citologia , Gânglios Espinais/fisiologia , Dose Letal Mediana , Masculino , Camundongos , Neurônios/fisiologia , Neurotoxinas/química , Neurotoxinas/toxicidade , Peptídeos/química , Peptídeos/isolamento & purificação , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Ratos , Ratos Sprague-Dawley , Bloqueadores dos Canais de Sódio/química , Bloqueadores dos Canais de Sódio/toxicidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Venenos de Aranha/química , Venenos de Aranha/toxicidade , Aranhas , Canais de Sódio Disparados por Voltagem/fisiologiaRESUMO
Semicarbazones are synthesized by the condensation of semicarbazide and aldehyde/ketone. The literature survey revealed that semicarbazones had been emerged as compounds with diverse biological activities including anticonvulsant, antitubercular, anticancer, and antimicrobial activities. The anticonvulsant activity of semicarbazones is mainly attributed due to the presence of an aryl binding site with aryl/alkyl hydrophobic group, a hydrogen bonding domain and an electron donor group and they are suggested to act by inhibiting sodium ion (Na(+)) channel. Dimmock et al., reported an extensive series of semicarbazones and reported 4-(4-fluorophenoxy) benzaldehyde semicarbazone (C0102862, V102862) as lead molecule. In MES (oral) screening C0102862 showed protective index (PI > 315) more than carbamazepine (PI 101), phenytoin (PI > 21.6) and valproate (PI > 2.17). This review briefly describes the information available about semicarbazone analogs and their anticonvulsant activity.
Assuntos
Anticonvulsivantes/uso terapêutico , Epilepsia Parcial Complexa/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Semicarbazonas/uso terapêutico , Animais , Anticonvulsivantes/síntese química , Anticonvulsivantes/química , Anticonvulsivantes/toxicidade , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Eletrochoque , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Estrutura Molecular , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/toxicidade , Pentilenotetrazol/toxicidade , Ratos , Teste de Desempenho do Rota-Rod , Semicarbazonas/síntese química , Semicarbazonas/química , Semicarbazonas/toxicidade , Bloqueadores dos Canais de Sódio/síntese química , Bloqueadores dos Canais de Sódio/química , Bloqueadores dos Canais de Sódio/uso terapêutico , Bloqueadores dos Canais de Sódio/toxicidadeRESUMO
Invasive Indo-Pacific lionfish (Pterois volitans) have rapidly expanded in the Western Atlantic over the past decade and have had a significant negative impact on reef fish biodiversity, habitat, and community structure, with lionfish out-competing native predators for resources. In an effort to reduce this population explosion, lionfish have been promoted for human consumption in the greater Caribbean region. This study examined whether the geographical expansion of the lionfish into a known ciguatera-endemic region can pose a human health threat for ciguatera fish poisoning (CFP). More than 180 lionfish were collected from waters surrounding the US Virgin Islands throughout 2010 and 2011. Ciguatoxin testing included an in vitro neuroblastoma cytotoxicity assay for composite toxicity assessment of sodium-channel toxins combined with confirmatory liquid chromatography tandem mass spectrometry. A 12% prevalence rate of ciguatoxic lionfish exceeding the FDA guidance level of 0.1 µg/kg C-CTX-1 equivalents was identified in fish from the U.S. Virgin Islands, highlighting a potential consumption risk in this region. This study presents the first evidence that the invasive lionfish, pose a direct human health risk for CFP and highlights the need for awareness and research on this food safety hazard in known endemic areas.
Assuntos
Ciguatera/epidemiologia , Peixes/fisiologia , Biologia Marinha , Alimentos Marinhos/efeitos adversos , Animais , Oceano Atlântico , Biodiversidade , Região do Caribe , Cromatografia Líquida de Alta Pressão , Ciguatoxinas/química , Ecossistema , Inocuidade dos Alimentos , Humanos , Indicadores e Reagentes , Toxinas Marinhas/toxicidade , Carne/análise , Carne/toxicidade , Neuroblastoma/patologia , Comportamento Predatório , Bloqueadores dos Canais de Sódio/toxicidade , Espectrometria de Massas em Tandem , Testes de Toxicidade , Ilhas Virgens AmericanasRESUMO
Jellyfish are efficient predators which prey on crabs, fish larvae, and small fish. Their venoms consist of various toxins including neurotoxins that paralyse prey organisms immediately. One possible mode of action of neurotoxins is the blockage of voltage-gated sodium (Na(v)) channels. A novel polypeptide with Na(v) channel blocking activity was isolated from the northern Scyphozoa Cyanea capillata (L., 1758). For that purpose, a bioactivity-guided multidimensional liquid chromatographic purification method has been developed. A neurotoxic activity of resulting chromatographic fractions was demonstrated by a bioassay, which based on the mouse neuroblastoma cell line Neuro2A. The purification process yielded one fraction containing a single polypeptide with proven activity. The molecular weight of 8.22 kDa was determined by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-ToF MS). Utilising Laser Microdissection and Pressure Catapulting (LMPC) for the separation of different nematocyst types in combination with direct MALDI-ToF MS analysis of the intact capsules, the neurotoxin was found to be present in all types of fishing tentacle isorhizas (A-isorhizas, a-isorhizas, O-isorhizas) of C. capillata medusae.
Assuntos
Neurotoxinas/isolamento & purificação , Cifozoários/patogenicidade , Bloqueadores dos Canais de Sódio/isolamento & purificação , Animais , Linhagem Celular Tumoral , Camundongos , Peso Molecular , Neurotoxinas/toxicidade , Bloqueadores dos Canais de Sódio/toxicidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Scorpion toxins targeting voltage-gated sodium (Na(V)) channels are peptides that comprise 60-76 amino acid residues cross-linked by four disulfide bridges. These toxins can be divided in two groups (α and ß toxins), according to their binding properties and mode of action. The scorpion α-toxin Ts2, previously described as a ß-toxin, was purified from the venom of Tityus serrulatus, the most dangerous Brazilian scorpion. In this study, seven mammalian Na(V) channel isoforms (rNa(V)1.2, rNa(V)1.3, rNa(V)1.4, hNa(V)1.5, mNa(V)1.6, rNa(V)1.7 and rNa(V)1.8) and one insect Na(V) channel isoform (DmNa(V)1) were used to investigate the subtype specificity and selectivity of Ts2. The electrophysiology assays showed that Ts2 inhibits rapid inactivation of Na(V)1.2, Na(V)1.3, Na(V)1.5, Na(V)1.6 and Na(V)1.7, but does not affect Na(V)1.4, Na(V)1.8 or DmNa(V)1. Interestingly, Ts2 significantly shifts the voltage dependence of activation of Na(V)1.3 channels. The 3D structure of this toxin was modeled based on the high sequence identity (72%) shared with Ts1, another T. serrulatus toxin. The overall fold of the Ts2 model consists of three ß-strands and one α-helix, and is arranged in a triangular shape forming a cysteine-stabilized α-helix/ß-sheet (CSαß) motif.
Assuntos
Ativação do Canal Iônico/efeitos dos fármacos , Neurotoxinas/química , Neurotoxinas/toxicidade , Venenos de Escorpião/química , Venenos de Escorpião/toxicidade , Canais de Sódio/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Células Cultivadas , Eletrofisiologia , Feminino , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Oócitos/citologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Ratos , Escorpiões/metabolismo , Homologia de Sequência de Aminoácidos , Bloqueadores dos Canais de Sódio/química , Bloqueadores dos Canais de Sódio/toxicidade , Canais de Sódio/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Xenopus laevis/metabolismoRESUMO
Toxins such as tetraethylammonium (TEA) and tetrodotoxin (TTX) may reduce the number of working potassium and sodium ion channels by poisoning and making them blocked, respectively. In this paper, we study how channel blocking (CB) affects the time delay-induced multiple coherence resonance (MCR), i.e., a phenomenon that the spiking of neuronal networks intermittently reaches the most ordered state, in stochastic Hodgkin-Huxley neuron networks. It is found that potassium and sodium CB have distinct effects. For potassium CB, the MCR occurs more frequently as the CB develops, but for sodium CB the MCR is badly impaired and only the first coherence resonance (CR) holds and, consequently, the MCR evolves into a single CR as sodium CB develops. We found for sodium CB the spiking becomes disordered at larger delay lengths, which may be the reason for the destruction of the MCR. The underlying mechanism is briefly discussed in terms of distinct effects of potassium and sodium CB on the spiking activity. These results show that potassium CB can increase the frequency of MCR with time delay, but sodium CB may suppress and even destroy the delay-induced MCR. These findings may help to understand the joint effects of CB and time delay on the spiking coherence of neuronal networks.
Assuntos
Potenciais de Ação/efeitos dos fármacos , Modelos Neurológicos , Neurônios/metabolismo , Neurotoxinas/toxicidade , Bloqueadores dos Canais de Potássio/toxicidade , Bloqueadores dos Canais de Sódio/toxicidade , Simulação por Computador , Processos Estocásticos , Fatores de TempoRESUMO
Diversity among Conus toxins mirrors the high species diversity in the Indo-Pacific region, and evolution of both is thought to stem from feeding-niche specialization derived from intra-generic competition. This study focuses on Conus californicus, a phylogenetic outlier endemic to the temperate northeast Pacific. Essentially free of congeneric competitors, it preys on a wider variety of organisms than any other cone snail. Using molecular cloning of cDNAs and mass spectrometry, we examined peptides isolated from venom ducts to elucidate the sequences and post-translational modifications of two eight-cysteine toxins (cal12a and cal12b of type 12 framework) that block voltage-gated Na(+) channels. Based on homology of leader sequence and mode of action, these toxins are related to the O-superfamily, but differ significantly from other members of that group. Six of the eight cysteine residues constitute the canonical framework of O-members, but two additional cysteine residues in the N-terminal region define an O+2 classification within the O-superfamily. Fifteen putative variants of Cal12.1 toxins have been identified by mRNAs that differ primarily in two short hypervariable regions and have been grouped into three subtypes (Cal12.1.1-3). This unique modular variation has not been described for other Conus toxins and suggests recombination as a diversity-generating mechanism. We propose that these toxin isoforms show specificity for similar molecular targets (Na(+) channels) in the many species preyed on by C. californicus and that individualistic utilization of specific toxin isoforms may involve control of gene expression.
Assuntos
Caramujo Conus/química , Venenos de Moluscos/genética , Peptídeos/genética , Bloqueadores dos Canais de Sódio/toxicidade , Animais , Sequência de Bases , California , Clonagem Molecular , Primers do DNA/genética , Eletrofisiologia , Biblioteca Gênica , Espectrometria de Massas , Dados de Sequência Molecular , Venenos de Moluscos/análise , Venenos de Moluscos/classificação , Oceano Pacífico , Peptídeos/análise , Peptídeos/classificação , Análise de Sequência de DNARESUMO
Brevetoxins (PbTx) are sodium channel neurotoxins produced by the marine dinoflagellate Karenia brevis during red tide blooms. Inhalation of PbTx in normal individuals and individuals with pre-existing airways disease results in adverse airway symptoms including bronchoconstriction. In animal models of allergic inflammation, inhalation of PbTx results in a histamine H1-mediated bronchoconstriction suggestive of mast cell activation. How mast cells would respond directly to PbTx is unknown. We thus explored the activation of mouse bone marrow-derived mast cells (BMMCs) following exposure to purified PbTx-2. Following in vitro exposure to PbTx-2, we examined cellular viability, mast cell degranulation (ß-hexosaminidase release), intracellular Ca²+ and Na+ flux, and the production of inflammatory mediators (IL-6). PbTx-2 induced significant cellular toxicity within 24 h as measured by LDH release and Annexin-V staining. However, within 1 h of exposure, PbTx-2 induced BMMC degranulation and an increase in IL-6 mRNA expression independent of the high-affinity IgE receptor (FcεRI) stimulation. Activation of BMMCs by PbTx-2 was associated with altered intracellular Ca²+ and Na+ levels. Brevenal, a naturally produced compound that antagonizes the activity of PbTx, prevented changes in intracellular Na+ levels but did not alter activation of BMMCs by PbTx-2. These findings demonstrate that PbTx-2 activates mast cells independent of FcεRI providing insight into critical events in the pathogenesis and a potential therapeutic target in brevetoxin-induced airway symptoms.
Assuntos
Toxinas Marinhas/toxicidade , Mastócitos/efeitos dos fármacos , Mastócitos/fisiologia , Neurotoxinas/toxicidade , Animais , Apoptose/efeitos dos fármacos , Células da Medula Óssea , Sinalização do Cálcio , Degranulação Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Éteres/farmacologia , Imunoglobulina E/metabolismo , Interleucina-6/metabolismo , Transporte de Íons/efeitos dos fármacos , Toxinas Marinhas/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos C57BL , Neurotoxinas/antagonistas & inibidores , Oxocinas , Polímeros/farmacologia , RNA Mensageiro/metabolismo , Receptores de IgE/agonistas , Sódio/metabolismo , Bloqueadores dos Canais de Sódio/antagonistas & inibidores , Bloqueadores dos Canais de Sódio/toxicidadeRESUMO
New advances in the use of NG108-15 cells for marine toxins detection and quantification are presented. We have established modifications on ouabain and veratridine proportion to obtain good toxins dose-response curves on this cell line for neurotoxins acting on voltage gated sodium channel (VGSC). Interesting, differences in the toxic response were observed between two VGSC activating toxins, brevetoxin-3 and pacific ciguatoxin-1. For non-VGSC acting lipophilic toxins, several factors that may influence toxin detection and quantification were analyzed. One hour cultures and forty-eight hours of exposure time, compared with 24h of culture and 24h of exposure, would increase NG108-15 cell maximal yessotoxin (YTX) and azaspiracid-1 (AZA-1) toxic response whereas no change was observed for okadaic acid (OA), dinophysistoxin-1 and pectenotoxin-2. Dose-response curves obtained for YTX or AZA-1 showed variability according to the day of the experiment while good reproducibility was obtained for OA. Evaporation time of toxin solutions before cell exposure could be an important source of variability in AZA-1 toxic response evaluation.
Assuntos
Glioma/patologia , Células Híbridas/patologia , Toxinas Marinhas/toxicidade , Neuroblastoma/patologia , Neurotoxinas/toxicidade , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Toxinas Marinhas/química , Neurotoxinas/química , Reprodutibilidade dos Testes , Bloqueadores dos Canais de Sódio/toxicidadeRESUMO
Pyrethroid insecticides are classified as type I or type II based on their distinct symptomology and effects on sodium channel gating. Structurally, type II pyrethroids possess an alpha-cyano group at the phenylbenzyl alcohol position, which is lacking in type I pyrethroids. Both type I and type II pyrethroids inhibit deactivation consequently prolonging the opening of sodium channels. However, type II pyrethroids inhibit the deactivation of sodium channels to a greater extent than type I pyrethroids inducing much slower decaying of tail currents upon repolarization. The molecular basis of a type II-specific action, however, is not known. Here we report the identification of a residue G(1111) and two positively charged lysines immediately downstream of G(1111) in the intracellular linker connecting domains II and III of the cockroach sodium channel that are specifically involved in the action of type II pyrethroids, but not in the action of type I pyrethroids. Deletion of G(1111), a consequence of alternative splicing, reduced the sodium channel sensitivity to type II pyrethroids, but had no effect on channel sensitivity to type I pyrethroids. Interestingly, charge neutralization or charge reversal of two positively charged lysines (Ks) downstream of G(1111) had a similar effect. These results provide the molecular insight into the type II-specific interaction of pyrethroids with the sodium channel at the molecular level.
Assuntos
Baratas/genética , Baratas/metabolismo , Inseticidas/toxicidade , Piretrinas/toxicidade , Bloqueadores dos Canais de Sódio/toxicidade , Canais de Sódio/efeitos dos fármacos , Canais de Sódio/genética , Sequência de Aminoácidos , Animais , DNA/genética , Ativação do Canal Iônico/efeitos dos fármacos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Nitrilas/toxicidade , Oócitos/metabolismo , RNA Complementar/biossíntese , RNA Complementar/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Two new polypeptide toxins named Hm-1 and Hm-2 were isolated from the venom of the crab spider Heriaeus melloteei. These toxins consist of 37 and 40 amino acid residues, respectively, contain three intramolecular disulfide bonds, and presumably adopt the inhibitor cystine knot motif. Hm-1 is C-terminally amidated and shows a low degree of homology to spider toxins agelenin and micro-agatoxin-II, whereas Hm-2 has no relevantly related peptide sequences. Hm-1 and Hm-2 were found to act on mammalian voltage-gated Na(+) channels. Both toxins caused a strong decrease of Na(+) current peak amplitude, with IC(50) values of 336.4 and 154.8 nM, respectively, on Na(V)1.4. Hm-1 and Hm-2 did not shift the voltage-dependence of activation, nor did they change the kinetics of fast inactivation of the Na(+) currents. Interestingly, both toxins negatively shifted the steady-state inactivation process, which might have important functional consequences in vivo. However, this hyperpolarizing shift cannot by itself explain the observed inhibition of the Na(+) current, indicating that the two presented toxins could provide important structural information about the interaction of polypeptide inhibitors with voltage-gated Na(+) channels.