Toxinology in the proteomics era: a review on arachnid venom proteomics

The word venomics was coined to acknowledge the studies that use omics to investigate venom proteins and peptides. Venomics has evolved considerably over the last 20 years. The first works on scorpion or spider venomics were published in the early 2000's. Such studies relied on peptide mass fingerprinting (PMF) to characterize venom complexity. After the introduction of new mass spectrometers with higher resolution, sensitivity and mass accuracy, and the next-generation nucleotide sequencing, the complexity of data reported in research on scorpion and spider venomics increased exponentially, which allowed more comprehensive studies. In the present review article, we covered key publications on scorpion venomics and spider venomics, presenting historical grounds and implemented technologies over the last years. The literature presented in this review was selected after searching the PubMed database using the terms "(scorpion venom) AND (proteome)" for scorpion venomics, and "(spider venom) AND (proteome)" for publications on spider venomics. We presented the key aspects related to proteomics in the covered papers including, but not restricted to, the employed proteomic strategy (i.e., PMF, two-dimensional gel electrophoresis, shotgun/bottom-up and/or top-down/peptidome), and the type of mass spectrometer used. Some conclusions can be drawn from the present study. For example, the scorpion genus Tityus is the most studied concerning venomics, followed by Centruroides; whereas for spiders the studied genera were found more equally distributed. Another interesting conclusion is the lack of high throughput studies on post-translational modifications (PTMs) of scorpion and spider proteins. In our opinion, PTMs should be more studied as they can modulate the activity of scorpion and spider toxins.(AU)

Comparative venomic profiles of three spiders of the genus Phoneutria

Spider venoms induce different physio-pharmacological effects by binding with high affinity on molecular targets, therefore being of biotechnological interest. Some of these toxins, acting on different types of ion channels, have been identified in the venom of spiders of the genus Phoneutria, mainly from P. nigriventer. In spite of the pharmaceutical potential demonstrated by P. nigriventer toxins, there is limited information on molecules from venoms of the same genus, as their toxins remain poorly characterized. Understanding this diversity and clarifying the differences in the mechanisms of action of spider toxins is of great importance for establishing their true biotechnological potential. This prompted us to compare three different venoms of the Phoneutria genus: P. nigriventer (Pn-V), P. eickstedtae (Pe-V) and P. pertyi (Pp-V). Methods: Biochemical and functional comparison of the venoms were carried out by SDS-PAGE, HPLC, mass spectrometry, enzymatic activities and electrophysiological assays (whole-cell patch clamp). Results: The employed approach revealed that all three venoms had an overall similarity in their components, with only minor differences. The presence of a high number of similar proteins was evident, particularly toxins in the mass range of ~6.0 kDa. Hyaluronidase and proteolytic activities were detected in all venoms, in addition to isoforms of the toxins Tx1 and Tx2-6. All Tx1 isoforms blocked Nav1.6 ion currents, with slight differences. Conclusion: Our findings showed that Pn-V, Pe-V and Pp-V are highly similar concerning protein composition and enzymatic activities, containing isoforms of the same toxins sharing high sequence homology, with minor modifications. However, these structural and functional variations are very important for venom diversity. In addition, our findings will contribute to the comprehension of the molecular diversity of the venoms of the other species from Phoneutria genus, exposing their biotechnological potential as a source for searching for new active molecules.(AU)

Antimicrobial activity and partial chemical structure of acylpolyamines isolated from the venom of the spider Acanthoscurria natalensis

Background: Acylpolyamines are one of the main non-peptide compounds present in spider venom and represent a promising alternative in the search for new molecules with antimicrobial action. Methods: The venom of Acanthoscurria natalensis spider was fractionated by reverse-phase liquid chromatography (RP-HPLC) and the antimicrobial activity of the fractions was tested using a liquid growth inhibition assay. The main antimicrobial fraction containing acylpolyamines (ApAn) was submitted to two additional chromatographic steps and analyzed by MALDI-TOF. Fractions of interest were accumulated for ultraviolet (UV) spectroscopy and ESI-MS/MS analysis and for minimum inhibitory concentration (MIC) and hemolytic activity determination. Results: Five acylpolyamines were isolated from the venom with molecular masses between 614 Da and 756 Da, being named ApAn728, ApAn614a, ApAn614b, ApAn742 and ApAn756. The analysis of UV absorption profile of each ApAn and the fragmentation pattern obtained by ESI-MS/MS suggested the presence of a tyrosyl unit as chromophore and a terminal polyamine chain consistent with structural units PA43 or PA53. ApAn presented MIC between 128 µM and 256 µM against Escherichia coli and Staphylococcus aureus, without causing hemolysis against mouse erythrocytes. Conclusion: The antimicrobial and non-hemolytic properties of the analyzed ApAn may be relevant for their application as possible therapeutic agents and the identification of an unconventional chromophore for spider acylpolyamines suggests an even greater chemical diversity.(AU)

Tarantula cubensis extract (Theranekron®) inhibits inflammation in carrageenan-induced acute paw edema in rats / [Extrato de tarântula cubensis (Theranekron®) inibe a inflamação no edema induzido por algas vermelhas na pata de ratos]

The aim of this study was to investigate the anti-inflammatory effect of alcoholic extract of Tarantula cubensis alcoholic extract (TCAE) in experimentally induced inflammation in rats. Fifty-four adult Sprague-Dawley male rats were randomly divided into nine groups. Paw edema was induced by 0.2mL subplantar (s.p.) injection of 1% carrageenan (CAR) into the right hind paw. Rats were treated with the nonsteroidal anti-inflammatory drug (NSAID) indomethacin (INDO) (10mg/kg, p.o.) or TCAE at different doses (1, 10 or 100µg/kg) injected s.c. for systemic or s.p. for local anti-inflammatory effect. Saline was used as control. Changes in paw thickness, volume, and weight were calculated as percentages. Formalin-fixed paws were used for histopathological examination. We detected that TCAE applied s.c. at 10µg/kg and 100µg/kg doses resulted in thinner paw thickness, lower paw volume, and lower paw weights four hours after the induction of inflammation when compared with the INDO group (p<0.05). The paw edema inhibitory effect of TCAE applied at a dose of 10µg/kg, s.c. was 68% when compared with the INDO which had an inhibitory effect of 56%. These results were verified with similar histopathological findings. The anti-inflammatory feature of 10µg/kg of TCAE given systematically was similar to the effects of INDO. Our results suggest that TCAE has anti-inflammatory effects by reducing edema and decreasing inflammatory reaction. These results may be attributed to the inhibition of the production of proinflammatory mediators. Thus, TCAE may be considered as a potential anti-inflammatory agent for treating acute inflammatory conditions.

O objetivo deste estudo foi investigar o efeito anti-inflamatório do extrato alcoólico de Tarantula cubensis (TCAE) na inflamação induzida experimentalmente em ratos. Cinqüenta e quatro ratos Sprague-Dawley adultos machos foram divididos aleatoriamente em nove grupos. O edema da pata foi induzido pela injeção de 0,2mL de subplantar (s.p.) de 1% de carragena (CAR) na pata traseira direita. Ratos foram tratados com o medicamento antiinflamatório não esteróide (NSAID) indometacina (INDO) (10mg/kg, p.o.) ou TCAE em doses diferentes (1, 10 ou 100µg/kg) injetado s.c. para efeito sistêmico ou s.p. para efeito antiinflamatório local. A soro fisiológico foi usado como controle. As mudanças na espessura da pata, volume e peso foram calculadas como porcentagens. As patas fixadas com fórmalina foram usadas para exame histopatológico. Detectamos que o TCAE aplicado s.c. em doses de 10µg/kg e 100µg/kg resultou em menor espessura da pata, menor volume da pata e menor peso da pata quatro horas após a indução da inflamação quando comparado com o grupo INDO (p<0,05). O efeito inibidor do edema da pata de TCAE aplicado na dose de 10µg/kg, s.c. foi de 68% quando comparado com o INDO que teve um efeito inibidor de 56%. Estes resultados foram verificados com resultados histopatológicos semelhantes. A característica anti-inflamatória de 10µg/kg de TCAE dada sistematicamente foi semelhante aos efeitos do INDO. Nossos resultados sugerem que o TCAE tem efeitos anti-inflamatórios reduzindo o edema e diminuindo a reação inflamatória. Estes resultados podem ser atribuídos à inibição da produção de mediadores pró-inflamatórios. Assim, o TCAE pode ser considerado como um agente antiinflamatório potencial para o tratamento de condições inflamatórias agudas.

Brazilian Theraphosidae: a toxicological point of view

The Theraphosidae family includes the largest number of species of the Mygalomorphae infraorder, with hundreds of species currently catalogued. However, there is a huge lack on physiologic and even ecologic information available, especially in Brazil, which is the most biodiverse country in the world. Over the years, spiders have been presented as a source of multiple biologically active compounds with basic roles, such as primary defense against pathogenic microorganisms or modulation of metabolic pathways and as specialized hunters. Spider venoms also evolved in order to enable the capture of prey by interaction with a diversity of molecular targets of interest, raising their pharmaceutical potential for the development of new drugs. Among the activities found in compounds isolated from venoms and hemocytes of Brazilian Theraphosidae there are antimicrobial, antifungal, antiparasitic and antitumoral, as well as properties related to proteinase action and neuromuscular blockage modulated by ionic voltage-gated channel interaction. These characteristics are present in different species from multiple genera, which is strong evidence of the important role in spider survival. The present review aims to compile the main results of studies from the last decades on Brazilian Theraphosidae with special focus on results obtained with the crude venom or compounds isolated from both venom and hemocytes, and their physiological and chemical characterization.(AU)

Brown spider venom toxins: what are the functions of astacins, serine proteases, hyaluronidases, allergens, TCTP, serpins and knottins?

Accidents caused by the bites of brown spiders (Loxosceles) generate a clinical condition that often includes a threatening necrotic skin lesion near the bite site along with a remarkable inflammatory response. Systemic disorders such as hemolysis, thrombocytopenia, and acute renal failure may occur, but are much less frequent than the local damage. It is already known that phospholipases D, highly expressed toxins in Loxosceles venom, can induce most of these injuries. However, this spider venom has a great range of toxins that probably act synergistically to enhance toxicity. The other protein classes remain poorly explored due to the difficulty in obtaining sufficient amounts of them for a thorough investigation. They include astacins (metalloproteases), serine proteases, knottins, translationally controlled tumor proteins (TCTP), hyaluronidases, allergens and serpins. It has already been shown that some of them, according to their characteristics, may participate to some extent in the development of loxoscelism. In addition, all of these toxins present potential application in several areas. The present review article summarizes information regarding some functional aspects of the protein classes listed above, discusses the directions that could be taken to materialize a comprehensive investigation on each of these toxins as well as highlights the importance of exploring the full venom repertoire.(AU)

Brown spider venom toxins: what are the functions of astacins, serine proteases, hyaluronidases, allergens, TCTP, serpins and knottins?

Accidents caused by the bites of brown spiders (Loxosceles) generate a clinical condition that often includes a threatening necrotic skin lesion near the bite site along with a remarkable inflammatory response. Systemic disorders such as hemolysis, thrombocytopenia, and acute renal failure may occur, but are much less frequent than the local damage. It is already known that phospholipases D, highly expressed toxins in Loxosceles venom, can induce most of these injuries. However, this spider venom has a great range of toxins that probably act synergistically to enhance toxicity. The other protein classes remain poorly explored due to the difficulty in obtaining sufficient amounts of them for a thorough investigation. They include astacins (metalloproteases), serine proteases, knottins, translationally controlled tumor proteins (TCTP), hyaluronidases, allergens and serpins. It has already been shown that some of them, according to their characteristics, may participate to some extent in the development of loxoscelism. In addition, all of these toxins present potential application in several areas. The present review article summarizes information regarding some functional aspects of the protein classes listed above, discusses the directions that could be taken to materialize a comprehensive investigation on each of these toxins as well as highlights the importance of exploring the full venom repertoire.(AU)

Widow spiders in the New World: a review on Latrodectus Walckenaer, 1805 (Theridiidae) and latrodectism in the Americas

Humankind has always been fascinated by venomous animals, as their toxic substances have transformed them into symbols of power and mystery. Over the centuries, researchers have been trying to understand animal venoms, unveiling intricate mixtures of molecules and their biological effects. Among venomous animals, Latrodectus Walckenaer, 1805 (widow spiders) have become feared in many cultures worldwide due to their extremely neurotoxic venom. The Latrodectus genus encompasses 32 species broadly spread around the globe, 14 of which occur in the Americas. Despite the high number of species found in the New World, the knowledge on these spiders is still scarce. This review covers the general knowledge on Latrodectus spp. from the Americas. We address widow spiders' taxonomy; geographical distribution and epidemiology; symptoms and treatments of envenomation (latrodectism); venom collection, experimental studies, proteome and transcriptome; and biotechnological studies on these Latrodectus spp. Moreover, we discuss the main challenges and limitations faced by researchers when trying to comprehend this neglected group of medically important spiders. We expect this review to help overcome the lack of information regarding widow spiders in the New World.(AU)

Pain-related toxins in scorpion and spider venoms: a face to face with ion channels

Pain is a common symptom induced during envenomation by spiders and scorpions. Toxins isolated from their venom have become essential tools for studying the functioning and physiopathological role of ion channels, as they modulate their activity. In particular, toxins that induce pain relief effects can serve as a molecular basis for the development of future analgesics in humans. This review provides a summary of the different scorpion and spider toxins that directly interact with pain-related ion channels, with inhibitory or stimulatory effects. Some of these toxins were shown to affect pain modalities in different animal models providing information on the role played by these channels in the pain process. The close interaction of certain gating-modifier toxins with membrane phospholipids close to ion channels is examined along with molecular approaches to improve selectivity, affinity or bioavailability in vivo for therapeutic purposes.(AU)

Neuroactive venom compounds obtained from Phlogiellus bundokalbo as potential leads for neurodegenerative diseases: insights on their acetylcholinesterase and beta-secretase inhibitory activities in vitro

Spider venom is a rich cocktail of neuroactive compounds designed to prey capture and defense against predators that act on neuronal membrane proteins, in particular, acetylcholinesterases (AChE) that regulate synaptic transmission through acetylcholine (ACh) hydrolysis - an excitatory neurotransmitter - and beta-secretases (BACE) that primarily cleave amyloid precursor proteins (APP), which are, in turn, relevant in the structural integrity of neurons. The present study provides preliminary evidence on the therapeutic potential of Phlogiellus bundokalbo venom against neurodegenerative diseases. Methods Spider venom was extracted by electrostimulation and fractionated by reverse-phase high-performance liquid chromatography (RP-HPLC) and characterized by matrix-assisted laser desorption ionization-time flight mass spectrometry (MALDI-TOF-MS). Neuroactivity of the whole venom was observed by a neurobehavioral response from Terebrio molitor larvae in vivo and fractions were screened for their inhibitory activities against AChE and BACE in vitro. Results The whole venom from P. bundokalbo demonstrated neuroactivity by inducing excitatory movements from T. molitor for 15 min. Sixteen fractions collected produced diverse mass fragments from MALDI-TOF-MS ranging from 900-4500 Da. Eleven of sixteen fractions demonstrated AChE inhibitory activities with 14.34% (± 2.60e-4) to 62.05% (± 6.40e-5) compared with donepezil which has 86.34% (± 3.90e-5) inhibition (p > 0.05), while none of the fractions were observed to exhibit BACE inhibition. Furthermore, three potent fractions against AChE, F1, F3, and F16 displayed competitive and uncompetitive inhibitions compared to donepezil as the positive control. Conclusion The venom of P. bundokalbo contains compounds that demonstrate neuroactivity and anti-AChE activities in vitro, which could comprise possible therapeutic leads for the development of cholinergic compounds against neurological diseases.(AU)

Widow spiders in the New World: a review on Latrodectus Walckenaer, 1805 (Theridiidae) and latrodectism in the Americas

Humankind has always been fascinated by venomous animals, as their toxic substances have transformed them into symbols of power and mystery. Over the centuries, researchers have been trying to understand animal venoms, unveiling intricate mixtures of molecules and their biological effects. Among venomous animals, Latrodectus Walckenaer, 1805 (widow spiders) have become feared in many cultures worldwide due to their extremely neurotoxic venom. The Latrodectus genus encompasses 32 species broadly spread around the globe, 14 of which occur in the Americas. Despite the high number of species found in the New World, the knowledge on these spiders is still scarce. This review covers the general knowledge on Latrodectus spp. from the Americas. We address widow spiders' taxonomy; geographical distribution and epidemiology; symptoms and treatments of envenomation (latrodectism); venom collection, experimental studies, proteome and transcriptome; and biotechnological studies on these Latrodectus spp. Moreover, we discuss the main challenges and limitations faced by researchers when trying to comprehend this neglected group of medically important spiders. We expect this review to help overcome the lack of information regarding widow spiders in the New World.(AU)

Neuroactive venom compounds obtained from Phlogiellus bundokalbo as potential leads for neurodegenerative diseases: insights on their acetylcholinesterase and beta-secretase inhibitory activities in vitro

Spider venom is a rich cocktail of neuroactive compounds designed to prey capture and defense against predators that act on neuronal membrane proteins, in particular, acetylcholinesterases (AChE) that regulate synaptic transmission through acetylcholine (ACh) hydrolysis - an excitatory neurotransmitter - and beta-secretases (BACE) that primarily cleave amyloid precursor proteins (APP), which are, in turn, relevant in the structural integrity of neurons. The present study provides preliminary evidence on the therapeutic potential of Phlogiellus bundokalbo venom against neurodegenerative diseases. Methods Spider venom was extracted by electrostimulation and fractionated by reverse-phase high-performance liquid chromatography (RP-HPLC) and characterized by matrix-assisted laser desorption ionization-time flight mass spectrometry (MALDI-TOF-MS). Neuroactivity of the whole venom was observed by a neurobehavioral response from Terebrio molitor larvae in vivo and fractions were screened for their inhibitory activities against AChE and BACE in vitro. Results The whole venom from P. bundokalbo demonstrated neuroactivity by inducing excitatory movements from T. molitor for 15 min. Sixteen fractions collected produced diverse mass fragments from MALDI-TOF-MS ranging from 900-4500 Da. Eleven of sixteen fractions demonstrated AChE inhibitory activities with 14.34% (± 2.60e-4) to 62.05% (± 6.40e-5) compared with donepezil which has 86.34% (± 3.90e-5) inhibition (p > 0.05), while none of the fractions were observed to exhibit BACE inhibition. Furthermore, three potent fractions against AChE, F1, F3, and F16 displayed competitive and uncompetitive inhibitions compared to donepezil as the positive control. Conclusion The venom of P. bundokalbo contains compounds that demonstrate neuroactivity and anti-AChE activities in vitro, which could comprise possible therapeutic leads for the development of cholinergic compounds against neurological diseases.(AU)

First record of the tarantula genus Bumba (Araneae: Theraphosidae) from Bolivia, with the description of a new species

ABSTRACT A new species of Bumba Pérez-Miles, Bonaldo & Miglio, 2014 is described and illustrated. This species comprises the first record of the genus for Bolivia. Bumba paunaka sp. nov. differs from the other species in the genus in the black longitudinal band on abdomen, in the long and slender embolus of the palpal bulb and in the thin and long subapical spine of the retrolateral branch of tibial apophysis. Females additionally differ in the spermathecal morphology. Finally, a distribution map for all known species of Bumba is presented.

First record of the tarantula genus Euathlus (Araneae: Theraphosidae: Theraphosinae) in Peru, with the description of a threatened new species

RESUMEN Se describe y diagnostica una nueva especie de Euathlus Ausserer, 1875 (Araneae: Theraphosidae: Theraphosinae), Euathlus vanessae sp. nov. El género es conocido en Chile y Argentina, y E. vanessae sp. nov. representa el primer registro oficial del género para el Perú, siendo el nuevo límite más septentrional de distribución conocida. Se proporcionan datos de distribución e historia natural, adicionalmente se discute el estado de conservación de la nueva especie y se propone como En Peligro (EN) según los criterios de la UICN.

ABSTRACT A new species of Euathlus Ausserer, 1875 (Araneae: Theraphosidae: Theraphosinae), Euathlus vanessae sp. nov. is described and diagnosed. The genus is known from Chile and Argentina, and E. vanessae sp. nov. represents the first record for the genus in Peru, being the new northernmost limit of its known distribution. Distribution and natural history data are provided. Additionally, the conservation status of the new species is discussed and it is proposed as Endangered (EN) according to IUCN criteria.

First record of the tarantula genus Euathlus (Araneae: Theraphosidae: Theraphosinae) in Peru, with the description of a threatened new species / Primer registro del género de tarántulas Euathlus (Araneae: Theraphosidae: Theraphosinae) en Perú, con la descripción de una nueva especie amenazada

A new species of Euathlus Ausserer, 1875 (Araneae: Theraphosidae: Theraphosinae), Euathlus vanessae sp. nov. is described and diagnosed. The genus is known from Chile and Argentina, and E. vanessae sp. nov. represents the first record for the genus in Peru, being the new northernmost limit of its known distribution. Distribution and natural history data are provided. Additionally, the conservation status of the new species is discussed and it is proposed as Endangered (EN) according to IUCN criteria.(AU)

Se describe y diagnostica una nueva especie de Euathlus Ausserer, 1875 (Araneae: Theraphosidae: Theraphosinae), Euathlus vanessae sp. nov. El género es conocido en Chile y Argentina, y E. vanessae sp. nov. representa el primer registro oficial del género para el Perú, siendo el nuevo límite más septentrional de distribución conocida. Se proporcionan datos de distribución e historia natural, adicionalmente se discute el estado de conservación de la nueva especie y se propone como En Peligro (EN) según los criterios de la UICN.(AU)

First record of the tarantula genus Euathlus (Araneae: Theraphosidae: Theraphosinae) in Peru, with the description of a threatened new species / Primer registro del género de tarántulas Euathlus (Araneae: Theraphosidae: Theraphosinae) en Perú, con la descripción de una nueva especie amenazada

A new species of Euathlus Ausserer, 1875 (Araneae: Theraphosidae: Theraphosinae), Euathlus vanessae sp. nov. is described and diagnosed. The genus is known from Chile and Argentina, and E. vanessae sp. nov. represents the first record for the genus in Peru, being the new northernmost limit of its known distribution. Distribution and natural history data are provided. Additionally, the conservation status of the new species is discussed and it is proposed as Endangered (EN) according to IUCN criteria.

Se describe y diagnostica una nueva especie de Euathlus Ausserer, 1875 (Araneae: Theraphosidae: Theraphosinae), Euathlus vanessae sp. nov. El género es conocido en Chile y Argentina, y E. vanessae sp. nov. representa el primer registro oficial del género para el Perú, siendo el nuevo límite más septentrional de distribución conocida. Se proporcionan datos de distribución e historia natural, adicionalmente se discute el estado de conservación de la nueva especie y se propone como En Peligro (EN) según los criterios de la UICN.

First record of the tarantula genus Bumba (Araneae: Theraphosidae) from Bolivia, with the description of a new species

A new species of Bumba Pérez-Miles, Bonaldo & Miglio, 2014 is described and illustrated. This species comprises the first record of the genus for Bolivia. Bumba paunaka sp. nov. differs from the other species in the genus in the black longitudinal band on abdomen, in the long and slender embolus of the palpal bulb and in the thin and long subapical spine of the retrolateral branch of tibial apophysis. Females additionally differ in the spermathecal morphology. Finally, a distribution map for all known species of Bumba is presented.(AU)

First record of the tarantula genus Bumba (Araneae: Theraphosidae) from Bolivia, with the description of a new species

A new species of Bumba Pérez-Miles, Bonaldo & Miglio, 2014 is described and illustrated. This species comprises the first record of the genus for Bolivia. Bumba paunaka sp. nov. differs from the other species in the genus in the black longitudinal band on abdomen, in the long and slender embolus of the palpal bulb and in the thin and long subapical spine of the retrolateral branch of tibial apophysis. Females additionally differ in the spermathecal morphology. Finally, a distribution map for all known species of Bumba is presented.

Jingzhaotoxin-X, a gating modifier of Kv4.2 and Kv4. 3 potassium channels purified from the venom of the Chinese tarantula Chilobrachys jingzhao

The tarantula Chilobrachys jingzhao is one of the largest venomous spiders in China. In previous studies, we purified and characterized at least eight peptides from C. jingzhao venom. In this report, we describe the purification and characterization of Jingzhaotoxin-X (JZTX-X), which selectively blocks Kv4.2 and Kv4.3 potassium channels. Methods: JZTX-X was purified using a combination of cation-exchange HPLC and reverse-phase HPLC. The amino-acid sequence was determined by automated Edman degradation and confirmed by mass spectrometry (MS). Voltage-gated ion channel currents were recorded in HEK293t cells transiently transfected with a variety of ion channel constructs. In addition, the hyperalgesic activity of JZTX-X and the toxin´s effect on motor function were assessed in mice. Results: JZTX-X contained 31 amino acids, with six cysteine residues that formed three disulfide bonds within an inhibitory cysteine knot (ICK) topology. In whole-cell voltage-clamp experiments, JZTX-X inhibited Kv4.2 and Kv4.3 potassium channels in a concentration- and voltage-dependent manner, without affecting other ion channels (Kv1.1, 1.2, 1.3, 2.1, delayed rectifier potassium channels, high- and low-voltage-activated Ca2+ channels, and voltage-gated sodium channels Nav1.5 and 1.7). JZTX-X also shifted the voltage-dependent channel activation to more depolarized potentials, whereas extreme depolarization caused reversible toxin binding to Kv4.2 channels. JZTX-X shifted the Kv4.2 and Kv4.3 activities towards a resting state, since at the resting potential the toxin completely inhibited the channels, even in the absence of an applied physical stimulus. Intrathecal or intraplantar injection of JZTX-X caused a long-lasting decrease in the mechanical nociceptive threshold (hyperalgesia) but had no effect on motor function as assessed in the rotarod test. Conclusions: JZTX-X selectively suppresses Kv4.2 and Kv4.3 potassium channel activity in a concentration- and voltage-dependent manner and causes long-lasting mechanical hyperalgesia.(AU)

Gene sequence analysis of toxins from the spider Phoneutria nigriventer revealed an intronless feature

Phoneutria nigriventer spider venom contains several cysteine-rich peptide toxins that act on different ion channels. Despite extensive studies on its venom and description of cDNA sequences of several of its toxin precursors, the gene structure of these toxins remains unknown. Methods: Genomic regions encoding the precursors of three previously characterized P. nigriventer toxins - PnTx1, PnTx2-5 and PnTx4(5-5) - were amplified by PCR using specific primers. PCR fragments were cloned and sequenced. Obtained sequences were compared with their corresponding cDNA sequences. Results: The size of PCR fragments obtained and sequences corresponding to genomic regions encoding for the toxin precursors matched their cDNA sequences. Conclusions: Despite a few nucleotide substitutions in the genomic regions encoding for the toxin precursors when compared with cDNA sequences, the results of the present work indicate that P. nigriventer toxins do not contain introns in their genes sequences.(AU)