Thermoregulation effects of Phoneutria nigriventer isolated toxins in rats

Body temperature is primarily regulated by the hypothalamus, ensuring proper metabolic function. Envenomation by Phoneutria nigriventer can cause symptoms such as hypothermia, hyperthermia, sweating, and shivering, all related to thermoregulation. This study aims to analyze and identify components of the venom that affect thermoregulation and to evaluate possible mechanisms. Rats were used for thermoregulation analysis, venom fractionation by gel filtration and reverse-phase chromatography (C18), and sequencing by Edman degradation. The venom exhibited hypothermic effects in rats, while its fractions demonstrated both hypothermic (pool II) and hyperthermic (pool III) effects. Further separations of the pools with C18 identified specific peaks responsible for these effects. However, as the peaks were further purified, their effects became less significant. Tests on U87 human glioblastoma cells showed no toxicity. Sequencing of the most active peaks revealed masses similar to those of the Tachykinin and Ctenotoxin families, both known to act on the nervous system. The study concludes that molecules derived from venom can act synergistically or antagonistically. Additionally, toxins that affect thermoregulation are poorly studied and require further characterization. These toxins could potentially serve as sources for the development of new thermoregulatory drugs.

PnPP-15, a Synthetic Peptide Derived from a Toxin from Phoneutria nigriventer Spider Venom, Alleviates Diabetic Neuropathic Pain and Acts Synergistically with Pregabalin in Mice.

Diabetic neuropathic pain is one of the complications that affect a wide variety of the diabetic population and is often difficult to treat. Only a small number of patients experience pain relief, which usually comes with onerous side effects and low levels of satisfaction. The search for new analgesic drugs is necessary, given the limitations that current drugs present. Combining drugs to treat neuropathic pain has been attracting interest to improve their efficacy compared to single-drug monotherapies while also reducing dose sizes to minimize side effects. The aim of our study was to verify the antinociceptive effect of a synthetic peptide, PnPP-15, alone and combined with pregabalin, in male Swiss diabetic mice using the von Frey method. PnPP-15 is a synthetic peptide derived from PnPP19, a peptide representing a discontinuous epitope of the primary structure of the toxin PnTx2-6 from the venom of the spider Phoneutria nigriventer. The antinociceptive activity of both compounds was dose-dependent and showed synergism, which was verified by isobolographic analysis. Treatment with PnPP-15 did not cause spontaneous or forced motor changes and did not cause any damage or signs of toxicity in the analyzed organs (pancreas, lung, heart, kidney, brain, or liver). In conclusion, PnPP-15 is a great candidate for an analgesic drug against neuropathic pain caused by diabetes and exerts a synergistic effect when combined with pregabalin, allowing for even more efficient treatment.

Heterologous Expression of an Insecticidal Peptide Obtained from the Transcriptome of the Colombian Spider Phoneutria depilate.

Spider venoms are composed, among other substances, of peptide toxins whose selectivity for certain physiological targets has made them powerful tools for applications such as bioinsecticides, analgesics, antiarrhythmics, antibacterials, antifungals and antimalarials, among others. Bioinsecticides are an environmentally friendly alternative to conventional agrochemicals. In this paper, the primary structure of an insecticidal peptide was obtained from the venom gland transcriptome of the ctenid spider Phoneutria depilata (Transcript ID PhdNtxNav24). The peptide contains 53 amino acids, including 10 Cys residues that form 5 disulfide bonds. Using the amino acid sequence of such peptide, a synthetic gene was constructed de novo by overlapping PCRs and cloned into an expression vector. A recombinant peptide, named delta-ctenitoxin (rCtx-4), was obtained. It was expressed, folded, purified and validated using mass spectrometry (7994.61 Da). The insecticidal activity of rCtx-4 was demonstrated through intrathoracic injection in crickets (LD50 1.2 µg/g insect) and it was not toxic to mice. rCtx-4 is a potential bioinsecticide that could have a broad spectrum of applications in agriculture.

Isolated Peptide from Spider Venom Modulates Dendritic Cells In Vitro: A Possible Application in Oncoimmunotherapy for Glioblastoma.

Dendritic cells (DCs) vaccine is a potential tool for oncoimmunotherapy. However, it is known that this therapeutic strategy has failed in solid tumors, making the development of immunoadjuvants highly relevant. Recently, we demonstrated that Phoneutria nigriventer spider venom (PnV) components are cytotoxic to glioblastoma (GB) and activate macrophages for an antitumor profile. However, the effects of these molecules on the adaptive immune response have not yet been evaluated. This work aimed to test PnV and its purified fractions in DCs in vitro. For this purpose, bone marrow precursors were collected from male C57BL6 mice, differentiated into DCs and treated with venom or PnV-isolated fractions (F1-molecules < 3 kDa, F2-3 to 10 kDa and F3->10 kDa), with or without costimulation with human GB lysate. The results showed that mainly F1 was able to activate DCs, increasing the activation-dependent surface marker (CD86) and cytokine release (IL-1ß, TNF-α), in addition to inducing a typical morphology of mature DCs. From the F1 purification, a molecule named LW9 was the most effective, and mass spectrometry showed it to be a peptide. The present findings suggest that this molecule could be an immunoadjuvant with possible application in DC vaccines for the treatment of GB.

Holistic profiling of the venom from the Brazilian wandering spider Phoneutria nigriventer by combining high-throughput ion channel screens with venomics.

Introduction: Spider venoms are a unique source of bioactive peptides, many of which display remarkable biological stability and neuroactivity. Phoneutria nigriventer, often referred to as the Brazilian wandering spider, banana spider or "armed" spider, is endemic to South America and amongst the most dangerous venomous spiders in the world. There are 4,000 envenomation accidents with P. nigriventer each year in Brazil, which can lead to symptoms including priapism, hypertension, blurred vision, sweating, and vomiting. In addition to its clinical relevance, P. nigriventer venom contains peptides that provide therapeutic effects in a range of disease models. Methods: In this study, we explored the neuroactivity and molecular diversity of P. nigriventer venom using fractionation-guided high-throughput cellular assays coupled to proteomics and multi-pharmacology activity to broaden the knowledge about this venom and its therapeutic potential and provide a proof-of-concept for an investigative pipeline to study spider-venom derived neuroactive peptides. We coupled proteomics with ion channel assays using a neuroblastoma cell line to identify venom compounds that modulate the activity of voltage-gated sodium and calcium channels, as well as the nicotinic acetylcholine receptor. Results: Our data revealed that P. nigriventer venom is highly complex compared to other neurotoxin-rich venoms and contains potent modulators of voltage-gated ion channels which were classified into four families of neuroactive peptides based on their activity and structures. In addition to the reported P. nigriventer neuroactive peptides, we identified at least 27 novel cysteine-rich venom peptides for which their activity and molecular target remains to be determined. Discussion: Our findings provide a platform for studying the bioactivity of known and novel neuroactive components in the venom of P. nigriventer and other spiders and suggest that our discovery pipeline can be used to identify ion channel-targeting venom peptides with potential as pharmacological tools and to drug leads.

Manifestaciones cardiovasculares y síndrome compartimental asociado a mordedura por Phoneutria spp: Reporte de un caso / Cardiovascular manifestations and compartment syndrome associated with bite by Phoneutria spp: case report / Manifestações cardiovasculares e síndrome compartimental associadas à mordida de Phoneutria spp: Relato de caso

Se describen las características sobre el phoneutrismo a propósito de un caso. El phoneutrismo es el término con el que se conoce al accidente ocasionado por la mordedura de la araña phoneutria spp, la cual tiene una relevancia clínica dada por las características tóxicas de su veneno. Se presenta un caso de mordedura de la araña phoneutria spp de un paciente atendido en un hospital de alta complejidad de la ciudad de Medellín, Colombia, con manifestaciones cardiovasculares y hallazgos compatibles con un síndrome compartimental, lo cual es inusual en este tipo de arañas, por lo que se necesitó vigilancia en unidad de alta dependencia y fasciotomía cutánea. En Colombia este tipo de accidentes no son de reporte obligatorio, por tanto existe un alto riesgo de subregistro. Lo llamativo de este caso está en las manifestaciones cardiovasculares y la presencia de síndrome compartimental que no se ha descrito en la literatura con este subespecie de arañas.

The characteristics of phoneutrism are described in relation to a case. Phoneutrism is the term with which the accident caused by the bite of the phoneutria spp spider is known, which has clinical relevance given by the toxic characteristics of its venom. We present a case of a bite by the phoneutria spp spider in a patient treated at a high-complexity hospital in the city of Medellín, Colombia, with cardiovascular manifestations and findings compatible with compartment syndrome, which is unusual in this type of spiders, and required surveillance in a high dependency unit and cutaneous fasciotomy. In Colombia reporting this type of accident is not mandatory; therefore, there is a high risk of underreporting. What is striking about this case is the cardiovascular manifestations and the presence of compartment syndrome that has not been described in the literatura with this genre of spiders.

As características do fonutrismo são descritas em um relatório de um caso. Fonutrismo é o termo usado para descrever o acidente causado pela mordida da aranha Phoneutria spp, que é clinicamente relevante devido às características tóxicas de seu veneno. Apresentamos um caso de mordida de aranha por uma aranha Phoneutria em um paciente tratado em um hospital de alta complexidade na cidade de Medellín, Colômbia, com manifestações cardiovasculares e achados compatíveis com a síndrome compartimental, o que é incomum neste tipo de aranha, exigindo vigilância em uma unidade de alta de-pendência e fasciotomia cutânea. Na Colômbia, este tipo de acidente não é obrigatório, portanto, há um alto risco de subnotificação. O que é impressionante neste caso são as manifestações cardiovasculares e a presença da síndrome compartimental, que não foi descrita na literatura com esta subespécie de aranha.

Transcriptomic Analysis of the Venom Gland and Enzymatic Characterization of the Venom of Phoneutria depilata (Ctenidae) from Colombia.

The transcriptome of the venom glands of the Phoneutria depilata spider was analyzed using RNA-seq with an Illumina protocol, which yielded 86,424 assembled transcripts. A total of 682 transcripts were identified as potentially coding for venom components. Most of the transcripts found were neurotoxins (156) that commonly act on sodium and calcium channels. Nevertheless, transcripts coding for some enzymes (239), growth factors (48), clotting factors (6), and a diuretic hormone (1) were found, which have not been described in this spider genus. Furthermore, an enzymatic characterization of the venom of P. depilata was performed, and the proteomic analysis showed a correlation between active protein bands and protein sequences found in the transcriptome. The transcriptomic analysis of P. depilata venom glands show a deeper description of its protein components, allowing the identification of novel molecules that could lead to the treatment of human diseases, or could be models for developing bioinsecticides.

Comparative venomic profiles of three spiders of the genus Phoneutria.

Background: 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.

Diversity of intestinal protozoa and clinical signs associated in wild-caught Phoneutria nigriventer kept in captivity for the anti-arachnid serum production.

The phylum Arthropoda comprises approximately 85% of all described animal species. The class Arachnida includes some invertebrates of great importance as they are either involved in the transmission of diseases or poses a risk of human envenomation. Spiders belonging to the genus Phoneutria sp., are the arachnids exhibiting medical importance. These animals were quarantined and maintained in captivity at the Biotério de Artrópodes of the Instituto Butantan, São Paulo, Brazil, for the production of the anti-arachnid serum. A total 509 feces samples from different Phoneutria nigriventer were analyzed, and 131 (25.73%) samples were found to be positive for flagellates and ciliates. All positive samples were subjected to DNA extraction and amplification of 18S gene. A total of 16 sequences were obtained and analyzed using BLAST. Sequences were identified as Colpoda steiini, one as Colpoda aspera, one to Colpoda sp., and one as "ciliated". Four identified as Parabodo caudatus, two as Urostipulosphaera sp., one as Helkesimastix sp., and one as a Euglena-like. The presence of clinical signs was observed in 16 spiders. The intestinal protozoa that affect armed spiders were identified for the first time as an initial step for understanding the parasitic diseases in these organisms.

Análise do desenvolvimento e rendimento de ootecas de Phoneutria nigriventer (keyserlingi,1981) no Biotério de Artrópodes do Instituto Butantan-SP

The species Phoneutria nigriventer belongs to the class Arachnida and order aranae, it is described as a synanthropic animal, with characteristics of aggressiveness, high stress, wanderers and opportunists. In addition, it has a medical interest in public health, having a potential poison to cause injuries, not only in predatory prey, but also in humans. The developmental stages of the species are, in sequence: ootheca, egg, pre-larval, larval, young, juvenile, pre-adult and adult. In the present study, the phases of ootheca and, mainly, hatchlings were analyzed, through a creation in captivity with constant control of physical parameters (temperature and humidity), without significant variations. Thus, through follow-up, it is possible to understand aspects that influence development, to ensure the success of individuals until sexual maturation. As a sample number we obtained an N=50, among these, some arranged in the arthropod vivarium via the external environment and a large majority built internally by pregnant females. To effect the hatching of the oothecae, they were allocated in a nursery, with substrates and essential materials that mimic a natural, dark and humidified environment. Thus, 40 oothecae were designated internally, being thirty-three viable, six not viable and one partially viable, the remaining 10 came from the external environment with seven viable and three not viable. In general, the number of individualized individuals ranged from 9 to 1652, with hatching times ranging from 17 to 74 days and dates from hatching to the effective individualizing action from 14 to 45 days. The dimensioning of the volume and weight of the eggs was also defined through previously established formulas. Therefore, when comparing with other results, it was noticed that the number of individuals exceeded expectations and the hatching date fit the observed, with only one outline in the research sample.

A espécie Phoneutria nigriventer enquadra-se na classe Arachnida e ordem aranae, ela é descrita como um animal sinantrópico, com características de agressividade, elevado estresse, errantes e oportunistas. Além disso, possui interesse médico na saúde pública, tendo um veneno potencial para causar injúrias, não apenas em presas-predadores, como também nos seres humanos. Os estágios de desenvolvimento da espécie são, em sequência: ooteca, ovo, pré-larval, larval, filhotes, jovens, pré- adulto e adulto. No presente estudo, foram analisados as fases de ootecas e, principalmente, filhotes, através de uma criação em cativeiro com controle constante de parâmetros físicos (temperatura e umidade), sem variações significativas. Assim, pelo acompanhamento pode-se compreender aspectos que influenciam o desenvolvimento, para garantir o sucesso dos indivíduos até maturação sexual. Como número amostral obtivemos um N=50, dentre essas, algumas dispostas no biotério de artrópodes via meio externo e, uma grande maioria construídas internamente por fêmeas prenhas. Para efetivação da eclosão das ootecas, estas foram alocadas em um viveiro, com substratos e materiais essenciais que mimetizem um ambiente natural, escuro e umidificado. Dessa forma, 40 ootecas foram designadas internamente, sendo trinta e três viáveis, seis inviáveis e uma parcialmente viável, as 10 restantes vieram de meio externo com sete viáveis e três inviáveis. De modo geral, o número de indivíduos individualizados variou de 9 a 1652, com tempos de eclosão no intervalo de 17 a 74 dias e datas desde a eclosão até a efetiva ação de individualizar de 14 a 45 dias. Definiu-se, também, o dimensionamento de volume e peso dos ovos através de fórmulas estabelecidas previamente. Portanto, ao comparar com outros resultados, percebeu-se que o número de indivíduos superou as expectativas e, a data de eclosão encaixou-se no observado, com apenas um outline na amostragem da pesquisa.

Diversity of intestinal protozoa and clinical signs associated in wild-caught Phoneutria nigriventer kept in captivity for the anti-arachnid serum production

The phylum Arthropoda comprises approximately 85% of all described animal species. The class Arachnida includes some invertebrates of great importance as they are either involved in the transmission of diseases or poses a risk of human envenomation. Spiders belonging to the genus Phoneutria sp., are the arachnids exhibiting medical importance. These animals were quarantined and maintained in captivity at the Biotério de Artrópodes of the Instituto Butantan, São Paulo, Brazil, for the production of the anti-arachnid serum. A total 509 feces samples from different Phoneutria nigriventer were analyzed, and 131 (25.73%) samples were found to be positive for flagellates and ciliates. All positive samples were subjected to DNA extraction and amplification of 18S gene. A total of 16 sequences were obtained and analyzed using BLAST. Sequences were identified as Colpoda steiini, one as Colpoda aspera, one to Colpoda sp., and one as “ciliated”. Four identified as Parabodo caudatus, two as Urostipulosphaera sp., one as Helkesimastix sp., and one as a Euglena-like. The presence of clinical signs was observed in 16 spiders. The intestinal protozoa that affect armed spiders were identified for the first time as an initial step for understanding the parasitic diseases in these organisms.

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)

Comparative venomic profiles of three spiders of the genus Phoneutria

Abstract Background: 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.

Components from spider venom activate macrophages against glioblastoma cells: new potential adjuvants for anticancer immunotherapy.

Immunomodulation has been considered an important approach in the treatment of malignant tumours. However, the modulation of innate immune cells remains an underexplored tool. Studies from our group demonstrated that the Phoneutria nigriventer spider venom (PnV) administration increased the infiltration of macrophage in glioblastoma, in addition to decreasing the tumour size in a preclinical model. The hypothesis that PnV would be modulating the innate immune system led us to the main objective of the present study: to elucidate the effects of PnV and its purified fractions on cultured macrophages. Results showed that PnV and the three fractions activated macrophages differentiated from bone marrow precursors. Further purification generated 23 subfractions named low weight (LW-1 to LW-12) and high weight (HW-1 to HW-11). LW-9 presented the best immunomodulatory effect. Treated cells were more phagocytic, migrated more, showed an activated morphological profile and induced an increased cytotoxic effect of macrophages on tumour cells. However, while M1-controls (LPS) increased IL-10, TNF-alpha and IL-6 release, PnV, fractions and subfractions did not alter any cytokine, with the exception of LW-9 that stimulated IL-10 production. These findings suggest that molecules present in LW-9 have the potential to be used as immunoadjuvants in the treatment of cancer.

Phoneutria nigriventer Tx3-3 peptide toxin reduces fibromyalgia symptoms in mice.

Fibromyalgia is characterized by the amplification of central nervous system pain with concomitant fatigue, sleep, mood disorders, depression, and anxiety. It needs extensive pharmacological therapy. In the present study, Swiss mice were treated with reserpine (0.25 mg/kg, s.c.) over three consecutive days, in order to reproduce the pathogenic process of fibromyalgia. On day 4, the administrations of the Tx3-3 toxin produced significant antinociception in the mechanical allodynia (87.16% ±12.7%) and thermal hyperalgesia (49.46% ± 10.6%) tests when compared with the PBS group. The effects produced by the classical analgesics (duloxetine 30 mg/kg, pramipexole 1 mg/kg, and pregabalin 30 mg/kg, p.o., respectively) in both of the tests also demonstrated antinociception. The administrations were able to increase the levels of the biogenic amines (5-HTP and DE) in the brain. The treatments with pramipexole and pregabalin, but not duloxetine, decreased the immobility time in the FM-induced animals that were submitted to the forced swimming test; however, the Tx3-3 toxin (87.45% ± 4.3%) showed better results. Taken together, the data has provided novel evidence of the ability of the Tx3-3 toxin to reduce painful and depressive symptoms, indicating that it may have significant potential in the treatment of FM.

Spider venom components decrease glioblastoma cell migration and invasion through RhoA-ROCK and Na+/K+-ATPase ß2: potential molecular entities to treat invasive brain cancer.

BACKGROUND: Glioblastoma (GB) cells have the ability to migrate and infiltrate the normal parenchyma, leading to the formation of recurrent tumors often adjacent to the surgical extraction site. We recently showed that Phoneutria nigriventer spider venom (PnV) has anticancer effects mainly on the migration of human GB cell lines (NG97 and U-251). The present work aimed to investigate the effects of isolated components from the venom on migration, invasiveness, morphology and adhesion of GB cells, also evaluating RhoA-ROCK signaling and Na+/K+-ATPase ß2 (AMOG) involvement. METHODS: Human (NG97) GB cells were treated with twelve subfractions (SFs-obtained by HPLC from PnV). Migration and invasion were evaluated by scratch wound healing and transwell assays, respectively. Cell morphology and actin cytoskeleton were shown by GFAP and phalloidin labeling. The assay with fibronectin coated well plate was made to evaluate cell adhesion. Western blotting demonstrated ROCK and AMOG levels and a ROCK inhibitor was used to verify the involvement of this pathway. Values were analyzed by the GraphPad Prism software package and the level of significance was determinate using one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test. RESULTS: Two (SF1 and SF11) of twelve SFs, decreased migration and invasion compared to untreated control cells. Both SFs also altered actin cytoskeleton, changed cell morphology and reduced adhesion. SF1 and SF11 increased ROCK expression and the inhibition of this protein abolished the effects of both subfractions on migration, morphology and adhesion (but not on invasion). SF11 also increased Na+/K+-ATPase ß2. CONCLUSION: All components of the venom were evaluated and two SFs were able to impair human glioblastoma cells. The RhoA effector, ROCK, was shown to be involved in the mechanisms of both PnV components. It is possible that AMOG mediates the effect of SF11 on the invasion. Further investigations to isolate and biochemically characterize the molecules are underway.

Intravitreal injection of peptides PnPa11 and PnPa13, derivatives of Phoneutria nigriventer spider venom, prevents retinal damage.

BACKGROUND: PnPa11 and PnPa13 are synthetic peptides derived from Phoneutria nigriventer spider venom, which display antinociceptive and neuroprotective properties. In this work, we evaluated the safety of intravitreal use and the neuroprotective effect of these peptides. METHODS: The cytotoxicity and the antiangiogenic activity of these peptides were evaluated by the sulforhodamine-B method and chicken chorioallantoic membrane (CAM) assay, respectively. The in vivo safety was analyzed in Wistar rats that were intravitreally injected with different doses (0.50; 1.25; 2.50; 3.75 and 5.00 µg/mL) of these peptides (right eye, n = 6). The retinal function was assessed by electroretinography exams (ERG), intraocular pressure (IOP), and histological analyzes. In order to investigate the neuroprotective effect, Wistar rats received intravitreal injections (right eye, n = 6) of peptides at 1.25 µg/mL and then were exposed to blue LED light. In addition, the visual function and the retinal microstructure were verified. RESULTS: Cytotoxicity analyses demonstrated that the peptides did not present any toxicity over ARPE-19 (adult retinal pigmented epithelial) cell line and the antiangiogenic study highlighted that the peptides promoted the reduction of blood vessels. The intravitreal injection did not cause major changes, neither induced any irreversible damage. In the retinal degeneration assay, the ERG records demonstrated that the prior treatment with PnPa11 and PnPa13 protected the retina from damage. Morphological analyses confirmed the ERG findings. Immunoblotting analyses revealed that PnPa11 increased Erk1/2, NR2A, and NR2B retinal expression after the light stress model, but did not cause Akt1 activation, while PnPa13 prevented Erk1/2 and Akt1 dephosphorylation. CONCLUSIONS: The intraocular administration of these peptides was well tolerated and presented protective activity against retinal degeneration, suggesting the potential use of these peptides as neuroprotectors in the ophthalmological field.

Pain modulatory properties of Phoneutria nigriventer crude venom and derived peptides: A double-edged sword.

Phoneutria nigriventer venom (PNV) is a complex mixture of toxins exerting multiple pharmacological effects that ultimately result in severe local pain at the site of the bite. It has been proposed that the PNV-induced pain is mediated by both peripheral and central mechanisms. The nociception triggered by PNV is peripherally mediated by the activation of B2, 5-HT4, NMDA, AMPA, NK1, and NK2 receptors, as well as TTXS-Na+, ASIC, and TRPV1 channels. The activation of tachykinin, glutamate and CGRP receptors along with the production of inflammatory mediators are, at least partially, responsible for the central component of pain. Despite its well established pro-nociceptive properties, PNV contains some toxins with antinociceptive activity, which have been studied in the last few years. The toxins ω-CNTX-Pn4a, ω-CNTX-Pn2a, ω-CNTX-Pn3a, κ-CNTX-Pn1a, U7-CNTX-Pn1a, δ-CNTX-Pn1a, and Γ-CNTX-Pn1a from PNV, as well as the semi-synthetic peptide PnPP-19 have been tested in different experimental models of pain showing consistent antinociceptive properties. This review aims to discuss the pro- and antinociceptive actions of PNV and its toxins, highlighting possible mechanisms involved in these apparently dualistic properties.

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

BACKGROUND: 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.

Analgesic effects of the CTK 01512-2 toxin in different models of orofacial pain in rats.

BACKGROUND: Orofacial pain is clinically challenging, having therapeutic failures and side effects. This study evaluated the antinociceptive activities of the CTK 01512-2 toxin, the TRPA1 channel antagonist, and the selective inhibitor of the N-type voltage-gated calcium channels (N-type VGCC), in different pain models. MATERIALS AND METHODS: The trigeminal ganglia were stimulated in vitro with capsaicin. The in vivo models received subcutaneous (sc) injections of formalin into the upper lip of the rats, Freund's Complete Adjuvant (FCA) into the temporomandibular joint (TMJ), and infraorbital nerve constrictions (IONC). CTK 01512-2 at concentrations of 30, 100, and 300 pmol/site, intrathecally (ith), and MVIIA at 10, 30, and 100 pmol/site in the formalin test, guided the doses for the models. The glutamate levels in the CSF of the rats that were submitted to IONC were analyzed. RESULTS: CTK 01512-2 decreased the nociceptive behavior in the inflammatory phase of the formalin test (65.94 ± 7.35%) and MVIIA in the neurogenic phase (81.23 ± 3.36%). CTK 01512-2 reduced facial grooming with FCA in the TMJ (96.7 ± 1.6%), and in the IONC neuropathy model, it decreased heat hyperalgesia (100%) and cold hyperalgesia (81.61 ± 9.02%). The levels of glutamate in the trigeminal ganglia in vitro (81.40 ± 8.59%) and in the CSF in vivo (70.0 ± 9.2%) were reduced. CONCLUSIONS: The roles of TRPA1 in pain transduction and the performance of CTK 01512-2 in the inhibition of the N-type VGCCs were reinforced. This dual activity may represent an advantage in clinical treatments.