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Purificação, caracterização bioquímica e eletrofisiológica da toxina Mic6c7NTX da Peçonha da Serpente Micrurus ibiboboca (Merrem, 1820) / Purification, Biochemical and Electrophysiological Characterization of the Toxin Mic6c7NTX from the Micrurus ibiboboca (Merrem, 1820)Donato, Micheline Freire 29 August 2008 (has links)
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Previous issue date: 2008-08-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Snake venoms contain a complex arsenal of protein bio-active components,
many of these being neurotoxins (NTXs). These snakes have high neurotoxic
activity venom, corresponding to the Elapidae family, which includes coral
snakes (Micrurus) whose venom contains circa 90-95% of low molecular mass
protein components. Among these, several are postsynaptic neurotoxins or α-
NTXs (MM = 6-9 kDa). The Micrurus ibiboboca (Merren, 1820) is a snake of the
Elapidae family witch is quite common in the Northeast of Brazil. In spite of the
great diversity of species of Micrurus, scarce works involving the nervous
system with isolated and pure toxins of those serpents has been developed in
level biochemical, pharmacological and electrophysiological. The aim of this
study was to purify the toxin Mic6c7NTX of the Micrurus ibiboboca venom,
characterize to biochemically and electrophysiologically the toxin Mic6c7NTX in
the peripheral nervous system (PNS) of rats, evaluating alterations in the record
of the Compound Action Potential (CAP) of the isolate nerve and the toxin
activity on the voltage-dependent sodium channels (Nav) in the neurons of the
dorsal root ganglion (DRG). The venom was extracted from the Micrurus
ibiboboca collected in Paraiba State (Brazil). Initially, electrophysiological tests
(current clamp method) using the single sucrose gap technique were
accomplished with crude venom (100μg/mL). It was observed that in this
concentration the crude venom caused reduction in the CAP amplitude (25%).
This neurotoxity led into an intriguing question: what components of the venom
would promote to reduction in the excitability of the nerve? Based upon this
question, I decided to purify the venom throughout the Liquid Chromatography
of the High Performance (HPLC) of the Cation Exchange Chromatography
(CIEX) and the Reverse Phase Chromatography (RPC). The molecular mass
(MM) of the raw toxin was determined by mass-spectrometry (MALDI-QTOF/
MS) and N-terminal sequence by means of Edman s Degradation. The
search for similarity with other toxins was accomplished against proteomic data
bank. The CIEX profile showed 19 fractions and the highest peak fraction was
used for the second dimension. The toxin Mic6c7NTX obtained by RPC showed
elution in 26.7%of the acetonitrile (ACN) and MM 7.047.56Da. The obtained
partial N-terminal sequence showed 31 aminoacid residues. The search for
similarity of structure and function showed great similarity (65%) with other short
chain α-NTXs Australian elapids snakes. The electrophysiological studies
(single sucrose gap technique) showed that the toxin Mic6c7NTX (1 μM)
reduced the excitability of the isolate nerve similarly to the reduction observed in
the crude venom about 21%. Other CAP parameters such as despolarization
speed (DSCAP), repolarization time (τCAP) and peak of time (PTCAP) did not show
alterations. This suggests that the toxin may be affecting the Nav channels. For
the confirmation of that hypothesis experiments were accomplished with whole
cell patch-clamp technique in DRG neurons. This results showed that the toxin
Mic6c7NTX (1 WM) abolished completely the current of Nav channels sensitive
the tetrodotoxin (TTX-S). Also the Nav channels TTX resistant (TTX-R) were
investigated in the presence of the Mic6c7NTX toxin previously using TTX (100
nM). This results showed that the toxin Mic6c7NTX (100 nM) abolished
completely the current of Nav channels TTX-R and IC50 = 30nM. However,
reversion of this blocking was not observed. The present study biochemically
and electrophysiologically characterized an α-NTX of the Micrurus ibiboboca
elapid snake. Furthermore, it showed a potent toxin with affinity Nav channels
TTX-S and TTX-R of the PNS. This is the first α-NTX isolated and identified of
the venom from the Micrurus ibiboboca (Merrem, 1820) snake. / As serpentes da família Elapidae possuem uma peçonha com alta atividade
neurotóxica e capacidade de letalidade. Fazem parte dessa família as
serpentes corais americanas (gênero Micrurus) com suas peçonhas contendo
cerca de 90-95% de componentes protéicos, sendo na sua maior parte
neurotoxinas com baixa massa molecular (6-8 kDa), podendo ser destacadas
as neurotoxinas com ação pós-sinápticas ou α-Neurotoxinas (α-NTX). A
Micrurus ibiboboca (Merrem, 1820) é uma serpente da família Elapidae, comum
na região Nordeste. Apesar da grande diversidade de espécies do gênero
Micrurus sp., escassos trabalhos envolvendo atividade de toxinas isoladas e
puras destas peçonhas e sistema nervoso têm sido desenvolvidos em nível
bioquímico, farmacológico ou eletrofisiológico. O objetivo desse estudo foi
purificar a toxina Mic6c7NTX da peçonha de M. ibiboboca, caracterizar
bioquímicamente e investigar com ferramentas eletrofisiológicas a ação da
toxina no Sistema Nervoso Periférico (SNP) de ratos avaliando alterações no
Potencial de Ação Composto (PAC) do nervo isquiático isolado e a atividade da
toxina nos canais para sódio dependentes de voltagem (Nav) em neurônios do
gânglio da raiz dorsal (DRG). A peçonha da M. ibiboboca foi extraída de
serpentes coletadas no Estado da Paraíba (Brasil). Inicialmente, ensaios
eletrofisiológicos com o método de current clamp utilizando a técnica de single
sucrose gap foram realizados com a peçonha bruta (100 Wg/mL). Os
resultados mostraram que a peçonha bruta nessa concentração promoveu
redução na amplitude do PAC (25%). Esse efeito da toxina na excitabilidade do
nervo levantou o questionamento: Que componentes da peçonha estariam
causando essa diminuição da excitabilidade? A peçonha foi purificada por meio
de Cromatografia Líquida de Alta Performance (HPLC), de troca catiônica
(CIEX) e fase reversa (RPC). Na sequência, os picos da CIEX foram
submetidos à RPC e posteriormente analisados por espectrometria de massas
(MALDI-TOF/MS) que detectou a massa molecular da toxina Mic6c7NTX de
7.047,56 Da. Em seguida, foi determinado o seu N-terminal por Degradação de
Edman que apresentou 31 resíduos de aminoácidos e serviu de estudo para a
bioinformática na busca por similaridade em banco de dados proteômicos com
outras toxinas protéicas, demonstrando que a toxina Mic6c7NTX apresentou
similaridade (65%) com α-NTXs de cadeia curta de serpentes elapídicas
australianas. Posteriormente, foi investigado o efeito da toxina isolada no SNP.
Os estudos eletrofisiológicos em single sucrose gap demonstraram que a
toxina Mic6c7NTX (1 WM) reduziu a excitabilidade do nervo isolado de forma
similar à observada pela peçonha bruta. Não foram observadas alterações
significantes em outros parâmetros do PAC, como velocidade de
despolarização (VDPAC), tempo de repolarização (τPAC) e tempo de pico
(PTPAC), sugerindo que a toxina atuasse num sítio de ligação específico dos
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canais Nav no SNP. Para a confirmação dessa hipótese foram realizados
experimentos de voltage clamp com a técnica de whole cell patch-clamp em
cultura primária de neurônios DRG da medula espinhal de ratos. Os resultados
mostraram que a toxina Mic6c7NTX (1 WM) aboliu completamente as correntes
dos canais Nav sensíveis à tetrodotoxina (TTX-S). Também foi investigado o
efeito da toxina sobre a população de canais Nav resistentes à TTX (TTX-R),
utilizando previamente TTX (100 nM) para bloquear os canais Nav TTX-S. Os
registros com a toxina Mic6c7NTX (100 nM) demonstraram um bloqueio total
da corrente nos canais Nav TTX-R dos DRGs e uma IC50 da toxina em torno de
30 nM. Também foi observado que essa toxina se liga aos canais Nav de forma
lenta e irreversível. O presente estudo caracterizou bioquímica e
eletrofisiologicamente uma α-NTX da serpente elapídica Micrurus ibiboboca.
Farmacologicamente, trata-se de uma potente toxina com afinidade aos canais
Nav TTX-S e TTX-R do SNP. Essa é a primeira α-NTX isolada e caracterizada
da peçonha da serpente Micrurus ibiboboca (Merrem, 1820).
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Evolutionary Genetics of Tetrodotoxin (TTX) Resistance in Snakes: Tracking a Feeding Adaptation from Populations Through CladesFeldman, Chris R. 01 December 2008 (has links)
Understanding the nature of adaptive evolution has been the recent focus of research detailing the genetic basis of adaptation and theoretical work describing the mechanics of adaptive evolution. Nevertheless, key questions regarding the process of adaptive evolution remain. Ultimately, a detailed description of the ecological context, evolutionary history, and genetic basis of adaptations is required to advance our understanding of adaptive evolution. To address some of the contemporary issues surrounding adaptive evolution, I examine phenotypic and genotypic changes in a snake feeding adaptation. Adaptations can arise through fixation of novel mutations or recruitment of existing variation. Some populations of the garter snakes Thamnophis sirtalis, T. couchii, and T. atratus possess elevated resistance to tetrodotoxin (TTX), the lethal toxin of their newt prey. I show that TTX resistance has evolved independently through amino acid changes at critical sites in a voltage-gated sodium channel protein (Nav1.4) targeted by TTX. Thus, adaptive evolution has occurred multiple times in garter snakes via de novo acquisition of beneficial mutations. Detailing the genetic basis of adaptive variation in natural populations is the first step towards understanding the tempo and mode of adaptive evolution. I evaluate the contribution of Nav1.4 alleles to TTX resistance in two garter snake species from central coastal California. Allelic variation in Nav1.4 explains 29% and 98% of the variation in TTX resistance in T. atratus and T. sirtalis, respectively, demonstrating that Nav1.4 is a major effect locus. The simple genetic architecture of TTX resistance in garter snakes may significantly impact the dynamics of trait change and coevolution. Patterns of convergent evolution are cited as some of the most compelling examples of the strength of natural selection in shaping organismal diversity. Yet repeated patterns may tell us as much about the constraints that restrict evolution as about the importance of natural selection. I present data on convergent molecular adaptations in parallel arms races between diverse snakes and amphibians from across the globe. Six snake species that prey on TTX bearing amphibians have independently acquired amino acid changes in Nav1.4. The derived mutations are clustered in two portions of the gene, often involving the same sites and substitutions. While a number of amino acid changes can make Nav1.4 insensitive to TTX, most of these negatively impact or abolish the ion-conducting function of the protein. Thus, intramolecular pleiotropy likely prevents most replacements from becoming fixed and imposes limits on protein evolution.
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