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1.
PLoS One ; 17(9): e0274488, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36094937

RESUMO

Naja sumatrana and Naja kaouthia are medically important elapids species found in Southeast Asia. Snake bite envenoming caused by these species may lead to morbidity or mortality if not treated with the appropriate antivenom. In this study, the in vitro neurotoxic and myotoxic effects N. sumatrana and N. kaouthia venoms from Malaysian specimens were assessed and compared. In addition, the neutralizing capability of Cobra Antivenom (CAV), King Cobra Antivenom (KCAV) and Neuro Polyvalent Antivenom (NPAV) from Thailand were compared. Both venoms produced concentration-dependent neurotoxic and myotoxic effects in the chick biventer cervicis nerve-muscle preparation. Based on the time to cause 90% inhibition of twitches (i.e. t90) N. kaouthia venom displayed more potent neurotoxic and myotoxic effects than N. sumatrana venom. All three of the antivenoms significantly attenuated venom-induced twitch reduction of indirectly stimulated tissues when added prior to venom. When added after N. sumatrana venom, at the t90 time point, CAV and NPAV partially restored the twitch height but has no significant effect on the reduction in twitch height caused by N. kaouthia venom. The addition of KCAV, at the t90 time point, did not reverse the attenuation of indirectly stimulated twitches caused by either venom. In addition, none of the antivenoms, when added prior to venom, prevented attenuation of directly stimulated twitches. Differences in the capability of antivenoms, especially NPAV and CAV, to reverse neurotoxicity and myotoxicity indicate that there is a need to isolate and characterize neurotoxins and myotoxins from Malaysian N. kaouthia and N. sumatrana venoms to improve neutralization capability of the antivenoms.


Assuntos
Antivenenos , Síndromes Neurotóxicas , Animais , Antivenenos/farmacologia , Miotoxicidade , Naja , Naja naja , Neurotoxinas/toxicidade , Tailândia
2.
Toxins (Basel) ; 14(8)2022 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-36006183

RESUMO

The Equatorial Spitting Cobra (Naja sumatrana) is a medically important venomous snake species in Southeast Asia. Its wide geographical distribution implies potential intra-specific venom variation, while there is no species-specific antivenom available to treat its envenoming. Applying a protein-decomplexing proteomic approach, the study showed that three-finger toxins (3FTX), followed by phospholipases A2 (PLA2), were the major proteins well-conserved across N. sumatrana venoms of different locales. Variations were noted in the subtypes and relative abundances of venom proteins. Of note, alpha-neurotoxins (belonging to 3FTX) are the least in the Penang specimen (Ns-PG, 5.41% of total venom proteins), compared with geographical specimens from Negeri Sembilan (Ns-NS, 14.84%), southern Thailand (Ns-TH, 16.05%) and Sumatra (Ns-SU, 10.81%). The alpha-neurotoxin abundance, in general, correlates with the venom's lethal potency. The Thai Naja kaouthia Monovalent Antivenom (NkMAV) was found to be immunoreactive toward the N. sumatrana venoms and is capable of cross-neutralizing N. sumatrana venom lethality to varying degrees (potency = 0.49-0.92 mg/mL, interpreted as the amount of venom completely neutralized per milliliter of antivenom). The potency was lowest against NS-SU venom, implying variable antigenicity of its lethal alpha-neurotoxins. Together, the findings suggest the para-specific and geographical utility of NkMAV as treatment for N. sumatrana envenoming in Southeast Asia.


Assuntos
Antivenenos , Naja , Animais , Antivenenos/farmacologia , Venenos Elapídicos/toxicidade , Elapidae , Indonésia , Malásia , Naja naja , Neurotoxinas , Proteômica , Tailândia
3.
Toxicon ; 216: 157-168, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35868411

RESUMO

Cobra (Naja spp.) envenoming is a life-threatening medical emergency, and a correct diagnosis is crucial to initiating timely and appropriate antivenom treatment. However, snakebite diagnostics remain unavailable in Southeast Asia. This study, therefore, developed an immunodetection assay with a potential diagnostic application for cobra envenoming. The cytotoxin of Naja kaouthia (Thai Monocled Cobra) (Nk-CTX) was purified from its venom to produce CTX-specific antibodies in rabbits and chickens. A double-antibody sandwich enzyme-linked immunosorbent assay was developed using the purified anti-Nk-CTX antibodies (immunoglobulin G and immunoglobulin Y), and its selectivity, specificity, and sensitivity for the venoms of five major cobra species in Southeast Asia (N. kaouthia, Naja sumatrana, Naja sputatrix, Naja siamensis, and Naja philippinensis) were studied. The results showed the immunoassay discriminates cobra venoms from other species commonly implicated in snakebites in Southeast Asia, i.e., the Malayan Krait, Many-banded Krait, King Cobra, Eastern Russell's Viper, Malayan Pit Viper and White-lipped Pit Viper. The immunoassay has a high sensitivity for the five cobra venoms, with detection limits (LoD) ranging from 0.6 to 2.6 ng/ml. Together, the findings suggest the potential diagnostic application of the cytotoxin immunoassay for cobra envenoming. The immunoassay was found to exhibit high immunoreactivity toward ten Asiatic cobra venoms (absorbance > 1.5), in contrast to African cobra venoms with low immunoreactivity (absorbance < 0.9). Considering the varying CTX antigenicity between Asiatic and African cobras, the immunoassay for African cobras should utilize antibodies produced specifically from the cytotoxins of African cobra venoms.


Assuntos
Elapidae , Mordeduras de Serpentes , Animais , Antivenenos , Bungarus , Galinhas , Citotoxinas , Venenos Elapídicos , Naja , Coelhos , Mordeduras de Serpentes/diagnóstico
4.
Toxins (Basel) ; 14(5)2022 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-35622581

RESUMO

Envenoming by cobras (Naja spp.) often results in extensive local tissue necrosis when optimal treatment with antivenom is not available. This study investigated the cytotoxicity of venoms and purified cytotoxins from the Monocled Cobra (Naja kaouthia), Taiwan Cobra (Naja atra), and Equatorial Spitting Cobra (Naja sumatrana) in a mouse fibroblast cell line, followed by neutralization of the cytotoxicity by three regional antivenoms: the Thai Naja kaouthia monovalent antivenom (NkMAV), Vietnamese snake antivenom (SAV) and Taiwanese Neuro bivalent antivenom (NBAV). The cytotoxins of N. atra (NA-CTX) and N. sumatrana (NS-CTX) were identified as P-type cytotoxins, whereas that of N. kaouthia (NK-CTX) is S-type. All venoms and purified cytotoxins demonstrated varying concentration-dependent cytotoxicity in the following trend: highest for N. atra, followed by N. sumatrana and N. kaouthia. The antivenoms moderately neutralized the cytotoxicity of N. kaouthia venom but were weak against N. atra and N. sumatrana venom cytotoxicity. The neutralization potencies of the antivenoms against the cytotoxins were varied and generally low across NA-CTX, NS-CTX, and NK-CTX, possibly attributed to limited antigenicity of CTXs and/or different formulation of antivenom products. The study underscores the need for antivenom improvement and/or new therapies in treating local tissue toxicity caused by cobra envenomings.


Assuntos
Antivenenos , Naja naja , Animais , Antivenenos/farmacologia , Citotoxinas/toxicidade , Venenos Elapídicos/toxicidade , Elapidae , Camundongos , Naja , Taiwan , Tailândia , Vietnã
5.
Toxins (Basel) ; 14(4)2022 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-35448886

RESUMO

Despite antivenoms being the only established specific treatment for neuromuscular paralysis arising from snake envenoming, their ability to reverse the post-synaptic neurotoxicity in snake envenoming is poorly understood. We investigated the ability of five commercial antivenoms i.e., King cobra monovalent, Thai cobra monovalent, Thai neuro polyvalent, Indian polyvalent and Australian polyvalent antivenoms to reverse neurotoxicity induced by the venoms of King cobra (Ophiophagus hannah, 3 µg/mL), Indian cobra (Naja naja, 5 µg/mL) and Thai cobra (Naja kaouthia, 3 µg/mL) using the in vitro chick-biventer cervicis nerve-muscle preparation. All three venoms displayed post-synaptic neurotoxicity, which was prevented by all tested antivenoms (40 µL/mL) added to the bath prior to venom. All antivenoms partially reversed the established post-synaptic neuromuscular block after the addition of the three venoms during a 180 min observation period, but to varying degrees and at different rates. The neurotoxic effects of O. hannah venom recovered to a greater magnitude (based on twitch height restoration) and faster than the neurotoxicity of N. kaouthia venom, which recovered to a lower magnitude more slowly. The recovery of post-synaptic neurotoxicity by N. naja venom was hindered due to the likely presence of cytotoxins in the venom, which cause direct muscle damage. The observations made in this study provide further evidence that the commercial antivenoms are likely to actively reverse established α-neurotoxin-mediated neuromuscular paralysis in snake envenoming, and there is cross-neutralisation with different antivenoms.


Assuntos
Síndromes Neurotóxicas , Mordeduras de Serpentes , Animais , Antivenenos/farmacologia , Austrália , Venenos Elapídicos/toxicidade , Elapidae , Naja , Naja naja , Síndromes Neurotóxicas/etiologia , Paralisia , Mordeduras de Serpentes/tratamento farmacológico , Venenos de Serpentes
6.
Toxins (Basel) ; 14(4)2022 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-35448894

RESUMO

Antivenom immunotherapy is the mainstay of treatment for snakebite envenoming. Most parts of the world affected by snakebite envenoming depend on broad-spectrum polyspecific antivenoms that are known to contain a low content of case-specific efficacious immunoglobulins. Thus, advances in toxin-specific antibodies production hold much promise in future therapeutic strategies of snakebite envenoming. We report anti-3FTxs monoclonal antibodies developed against N. ashei venom in mice. All the three test mAbs (P4G6a, P6D9a, and P6D9b) were found to be IgG antibodies, isotyped as IgG1. SDS-PAGE analysis of the test mAbs showed two major bands at approximately 55 and 29 kDa, suggestive of immunoglobulin heavy and light chain composition, respectively. The immunoaffinity-purified test mAbs demonstrated higher binding efficacy to the target antigen compared to negative control. Similarly, a cocktail of the test mAbs was found to induce a significantly higher inhibition (p-value < 0.0001) compared to two leading commercial brands of antivenoms on the Kenyan market, implying a higher specificity for the target antigen. Both the test mAbs and 3FTxs polyclonal antibodies induced comparable inhibition (p-value = 0.9029). The inhibition induced by the 3FTxs polyclonal antibodies was significantly different from the two antivenoms (p-value < 0.0001). Our results demonstrate the prospects of developing toxin-specific monoclonal-based antivenoms for snakebite immunotherapy.


Assuntos
Antineoplásicos Imunológicos , Mordeduras de Serpentes , Animais , Anticorpos Monoclonais/farmacologia , Antivenenos/uso terapêutico , Venenos Elapídicos , Imunoglobulina G , Quênia , Camundongos , Naja/metabolismo , Mordeduras de Serpentes/tratamento farmacológico , Toxinas Três Dedos
7.
Sci Rep ; 12(1): 6394, 2022 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-35430620

RESUMO

African trypanosomes, such as Trypanosoma brucei, are flagellated protozoa which proliferate in mammals and cause a variety of diseases in people and animals. In a mammalian host, the external face of the African trypanosome plasma membrane is covered by a densely packed coat formed of variant surface glycoprotein (VSG), which counteracts the host's adaptive immune response by antigenic variation. The VSG is attached to the external face of the plasma membrane by covalent attachment of the C-terminus to glycosylphosphatidylinositol. As the trypanosome grows, newly synthesised VSG is added to the plasma membrane by vesicle fusion to the flagellar pocket, the sole location of exo- and endocytosis. Snake venoms contain dozens of components, including proteases and phospholipases A2. Here, we investigated the effect of Naja nigricollis venom on T. brucei with the aim of describing the response of the trypanosome to hydrolytic attack on the VSG. We found no evidence for VSG hydrolysis, however, N. nigricollis venom caused: (i) an enlargement of the flagellar pocket, (ii) the Rab11 positive endosomal compartments to adopt an abnormal dispersed localisation, and (iii) cell cycle arrest prior to cytokinesis. Our results indicate that a single protein family, the phospholipases A2 present in N. nigricollis venom, may be necessary and sufficient for the effects. This study provides new molecular insight into T. brucei biology and possibly describes mechanisms that could be exploited for T. brucei targeting.


Assuntos
Trypanosoma brucei brucei , Animais , Venenos Elapídicos/metabolismo , Endocitose , Humanos , Mamíferos/metabolismo , Naja , Fosfolipases A2/metabolismo , Trypanosoma brucei brucei/metabolismo , Glicoproteínas Variantes de Superfície de Trypanosoma/metabolismo
8.
Toxins (Basel) ; 13(12)2021 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-34941697

RESUMO

Phospholipase A2 (PLA2) toxins are one of the main toxin families found in snake venom. PLA2 toxins are associated with various detrimental effects, including neurotoxicity, myotoxicity, hemostatic disturbances, nephrotoxicity, edema, and inflammation. Although Naja sumatrana venom contains substantial quantities of PLA2 components, there is limited information on the function and activities of PLA2 toxins from the venom. In this study, a secretory PLA2 from the venom of Malaysian N. sumatrana, subsequently named A2-EPTX-Nsm1a, was isolated, purified, and characterized. A2-EPTX-Nsm1a was purified using a mass spectrometry-guided approach and multiple chromatography steps. Based on LC-MSMS, A2-EPTX-Nsm1a was found to show high sequence similarity with PLA2 from venoms of other Naja species. The PLA2 activity of A2-EPTX-Nsm1 was inhibited by 4-BPB and EDTA. A2-EPTX-Nsm1a was significantly less cytotoxic in a neuroblastoma cell line (SH-SY5Y) compared to crude venom and did not show a concentration-dependent cytotoxic activity. To our knowledge, this is the first study that characterizes and investigates the cytotoxicity of an Asp49 PLA2 isolated from Malaysian N. sumatrana venom in a human neuroblastoma cell line.


Assuntos
Venenos Elapídicos/enzimologia , Naja , Fosfolipases A2 Secretórias/química , Fosfolipases A2 Secretórias/toxicidade , Animais , Linhagem Celular Tumoral , Venenos Elapídicos/toxicidade , Humanos , Fosfolipases A2 Secretórias/isolamento & purificação
9.
Front Immunol ; 12: 752442, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34691069

RESUMO

African spitting cobras are unique among cobras for their potent anticoagulant venom activity arising from strong inhibition of Factor Xa. This anticoagulant effect is exerted by venom phospholipase A2 (Group I PLA2) toxins whose activity contributes to the lethality of these species. This anticoagulant toxicity is particularly problematic as it is not neutralized by current antivenoms. Previous work demonstrated this trait for Naja mossambica, N. nigricincta, N. nigricollis, and N. pallida. The present work builds upon previous research by testing across the full taxonomical range of African spitting cobras, demonstrating that N. ashei, N. katiensis, and N. nubiae are also potently anticoagulant through the inhibition of Factor Xa, and therefore the amplification of potent anticoagulant activity occurred at the base of the African spitting cobra radiation. Previous work demonstrated that the enzyme-inhibitor varespladib was able to neutralize this toxic action for N. mossambica, N. nigricincta, N. nigricollis, and N. pallida venoms. The current work demonstrates that varespladib was also able to neutralize N. ashei, N. katiensis, and N. nubiae. Thus varespladib is shown to have broad utility across the full range of African spitting cobras. In addition, we examined the cross-reactivity of the metalloprotease inhibitor prinomastat, which had been previously intriguingly indicated as being capable of neutralizing viperid venom PLA2 (Group II PLA2). In this study prinomastat inhibited the FXa-inhibiting PLA2 toxins of all the African spitting cobras at the same concentration at which it has been shown to inhibit metalloproteases, and thus was comparably effective in its cross-reactivity. In addition we showed that the metalloprotease-inhibitor marimastat was also able to cross-neutralize PLA2 but less effectively than prinomastat. Due to logistical (cold-chain requirement) and efficacy (cross-reactivity across snake species) limitations of traditional antivenoms, particularly in developing countries where snakebite is most common, these small molecule inhibitors (SMIs) might hold great promise as initial, field-based, treatments for snakebite envenoming as well as addressing fundamental limitations of antivenom in the clinical setting where certain toxin effects are unneutralized.


Assuntos
Antivenenos/farmacologia , Inibidores Enzimáticos/farmacologia , Mordeduras de Serpentes/terapia , Venenos de Serpentes/antagonistas & inibidores , Acetatos/farmacologia , África , Animais , Ácidos Hidroxâmicos/farmacologia , Técnicas In Vitro , Indóis/farmacologia , Cetoácidos/farmacologia , Naja , Compostos Orgânicos/farmacologia
10.
Int J Biol Macromol ; 184: 776-786, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-34174307

RESUMO

Naja sumatrana venom cytotoxin (sumaCTX) is a basic protein which belongs to three-finger toxin family. It has been shown to induce caspase-dependent, mitochondrial-mediated apoptosis in MCF-7 cells at lower concentrations. This study aimed to investigate the alteration of secretome in MCF-7 cells following membrane permeabilization by high concentrations of sumaCTX, using label-free quantitative (LFQ) approach. The degree of membrane permeabilization of sumaCTX was determined by lactate dehydrogenase (LDH) assay and calcein-propidium iodide (PI) assays. LDH and calcein-PI assays revealed time-dependent membrane permeabilization within a narrow concentration range. However, as toxin concentrations increased, prolonged exposure of MCF-7 cells to sumaCTX did not promote the progression of membrane permeabilization. The secretome analyses showed that membrane permeabilization was an event preceding the release of intracellular proteins. Bioinformatics analyses of the LFQ secretome revealed the presence of 105 significantly distinguished proteins involved in metabolism, structural supports, inflammatory responses, and necroptosis in MCF-7 cells treated with 29.8 µg/mL of sumaCTX. Necroptosis was presumably an initial stress response in MCF-7 cells when exposed to high sumaCTX concentration. Collectively, sumaCTX-induced the loss of membrane integrity in a concentration-dependent manner, whereby the cell death pattern of MCF-7 cells transformed from apoptosis to necroptosis with increasing toxin concentrations.


Assuntos
Neoplasias da Mama/metabolismo , Proteínas Neurotóxicas de Elapídeos/farmacologia , Naja/metabolismo , Proteômica/métodos , Animais , Neoplasias da Mama/tratamento farmacológico , Permeabilidade da Membrana Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida , Relação Dose-Resposta a Droga , Venenos Elapídicos/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Espectrometria de Massas em Tandem , Fatores de Tempo
11.
Toxicon ; 199: 12-19, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34010665

RESUMO

Many advanced snakes possess a unique venom delivery system which they utilise to subdue prey and for defence. Despite extensive efforts, the evolutionary differences in this key system between advanced snake families remains enigmatic. The current study has investigated the development of the venom delivery system using two oviparous Elapidae models, Naja siamensis and Oxyuranus microlepidotus. The development stages of the embryos in both models were detailed using previously standardised characterisation. Variations in the days post-oviposition between these stages was observed, despite a continuous development trajectory. These differences also translated to the development of the venom delivery system.


Assuntos
Elapidae , Naja , Animais , Venenos Elapídicos
12.
Front Immunol ; 12: 652242, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33936074

RESUMO

Systemic complement activation drives a plethora of pathological conditions, but its role in snake envenoming remains obscure. Here, we explored complement's contribution to the physiopathogenesis of Naja annulifera envenomation. We found that N. annulifera venom promoted the generation of C3a, C4a, C5a, and the soluble Terminal Complement Complex (sTCC) mediated by the action of snake venom metalloproteinases. N. annulifera venom also induced the release of lipid mediators and chemokines in a human whole-blood model. This release was complement-mediated, since C3/C3b and C5a Receptor 1 (C5aR1) inhibition mitigated the effects. In an experimental BALB/c mouse model of envenomation, N. annulifera venom promoted lipid mediator and chemokine production, neutrophil influx, and swelling at the injection site in a C5a-C5aR1 axis-dependent manner. N. annulifera venom induced systemic complementopathy and increased interleukin and chemokine production, leukocytosis, and acute lung injury (ALI). Inhibition of C5aR1 with the cyclic peptide antagonist PMX205 rescued mice from these systemic reactions and abrogated ALI development. These data reveal hitherto unrecognized roles for complement in envenomation physiopathogenesis, making complement an interesting therapeutic target in envenomation by N. annulifera and possibly by other snake venoms.


Assuntos
Ativação do Complemento/imunologia , Complemento C5a/imunologia , Complemento C5a/metabolismo , Receptor da Anafilatoxina C5a/metabolismo , Venenos de Serpentes/imunologia , Animais , Biomarcadores , Quimiocinas/metabolismo , Modelos Animais de Doenças , Humanos , Hidrólise , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Modelos Biológicos , Naja , Ligação Proteica , Transdução de Sinais , Mordeduras de Serpentes
13.
Toxins (Basel) ; 13(5)2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33922825

RESUMO

Bites from elapid snakes typically result in neurotoxic symptoms in snakebite victims. Neurotoxins are, therefore, often the focus of research relating to understanding the pathogenesis of elapid bites. However, recent evidence suggests that some elapid snake venoms contain anticoagulant toxins which may help neurotoxic components spread more rapidly. This study examines the effects of venom from the West African black-necked spitting cobra (Naja nigricollis) on blood coagulation and identifies potential coagulopathic toxins. An integrated RPLC-MS methodology, coupled with nanofractionation, was first used to separate venom components, followed by MS, proteomics and coagulopathic bioassays. Coagulation assays were performed on both crude and nanofractionated N. nigricollis venom toxins as well as PLA2s and 3FTx purified from the venom. Assays were then repeated with the addition of either the phospholipase A2 inhibitor varespladib or the snake venom metalloproteinase inhibitor marimastat to assess whether either toxin inhibitor is capable of neutralizing coagulopathic venom activity. Subsequent proteomic analysis was performed on nanofractionated bioactive venom toxins using tryptic digestion followed by nanoLC-MS/MS measurements, which were then identified using Swiss-Prot and species-specific database searches. Varespladib, but not marimastat, was found to significantly reduce the anticoagulant activity of N. nigricollis venom and MS and proteomics analyses confirmed that the anticoagulant venom components mostly consisted of PLA2 proteins. We, therefore, conclude that PLA2s are the most likely candidates responsible for anticoagulant effects stimulated by N. nigricollis venom.


Assuntos
Acetatos/farmacologia , Anticoagulantes/toxicidade , Venenos Elapídicos/toxicidade , Indóis/farmacologia , Fosfolipases A2/farmacologia , Animais , Coagulação Sanguínea/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Venenos Elapídicos/antagonistas & inibidores , Cromatografia Gasosa-Espectrometria de Massas , Ácidos Hidroxâmicos/farmacologia , Cetoácidos , Naja , Proteômica
14.
Molecules ; 26(8)2021 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-33918763

RESUMO

Three-finger toxins are naturally occurring proteins in Elapidae snake venoms. Nowadays, they are gaining popularity because of their therapeutic potential. On the other hand, these proteins may cause undesirable reactions inside the body's cells. A full assessment of the safety of Naja ashei venom components for human cell application is still unknown. The aim of the study was to determine the effect of the exogenous application of three-finger toxins on the cells of monocytes (U-937) and promyelocytes (HL-60), with particular emphasis on the modification of their membranes under the influence of various doses of 3FTx protein fraction (0-120 ng/mL). The fraction exhibiting the highest proportion of 3FTx proteins after size exclusion chromatography (SEC) separation was used in the experiments. The structural response of cell membranes was described on the basis of single-component and multi-component Langmuir monolayers that mimicked the native membranes. The results show that the mechanism of protein-lipid interactions depends on both the presence of lipid polar parts (especially zwitterionic type of lipids) and the degree of membrane saturation (the greatest-for unsaturated lipids). The biochemical indicators reflecting the tested cells (MDA, LDH, cell survival, induction of inflammation, LD50) proved the results that were obtained for the model.


Assuntos
Venenos Elapídicos/química , Venenos Elapídicos/toxicidade , Membranas Artificiais , Naja/metabolismo , Proteínas/toxicidade , Animais , Fracionamento Químico , Cromatografia em Gel , Feminino , Células HL-60 , Humanos , L-Lactato Desidrogenase/metabolismo , Dose Letal Mediana , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Malondialdeído/metabolismo , Membranas , Pressão , Temperatura , Células U937
15.
Toxins (Basel) ; 13(2)2021 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-33672715

RESUMO

Cobra venoms contain three-finger toxins (TFT) including α-neurotoxins efficiently binding nicotinic acetylcholine receptors (nAChRs). As shown recently, several TFTs block GABAA receptors (GABAARs) with different efficacy, an important role of the TFTs central loop in binding to these receptors being demonstrated. We supposed that the positive charge (Arg36) in this loop of α-cobratoxin may explain its high affinity to GABAAR and here studied α-neurotoxins from African cobra N. melanoleuca venom for their ability to interact with GABAARs and nAChRs. Three α-neurotoxins, close homologues of the known N. melanoleuca long neurotoxins 1 and 2, were isolated and sequenced. Their analysis on Torpedocalifornica and α7 nAChRs, as well as on acetylcholine binding proteins and on several subtypes of GABAARs, showed that all toxins interacted with the GABAAR much weaker than with the nAChR: one neurotoxin was almost as active as α-cobratoxin, while others manifested lower activity. The earlier hypothesis about the essential role of Arg36 as the determinant of high affinity to GABAAR was not confirmed, but the results obtained suggest that the toxin loop III may contribute to the efficient interaction of some long-chain neurotoxins with GABAAR. One of isolated toxins manifested different affinity to two binding sites on Torpedo nAChR.


Assuntos
Colinérgicos/farmacologia , Proteínas Neurotóxicas de Elapídeos/farmacologia , Venenos Elapídicos/metabolismo , Antagonistas de Receptores de GABA-A/farmacologia , Naja , Receptores de GABA/efeitos dos fármacos , Receptor Nicotínico de Acetilcolina alfa7/efeitos dos fármacos , Animais , Sítios de Ligação , Ligação Competitiva , Linhagem Celular Tumoral , Colinérgicos/metabolismo , Proteínas Neurotóxicas de Elapídeos/metabolismo , Antagonistas de Receptores de GABA-A/metabolismo , Potenciais da Membrana , Camundongos , Ligação Proteica , Conformação Proteica , Receptores de GABA/genética , Receptores de GABA/metabolismo , Relação Estrutura-Atividade , Torpedo , Xenopus laevis , Receptor Nicotínico de Acetilcolina alfa7/metabolismo
16.
Toxicon ; 197: 24-32, 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-33775665

RESUMO

Proteomics technologies enable a comprehensive study of complex proteins and their functions. The venom proteomes of three medically important Nigerian Elapidae snakes Naja haje, Naja katiensis and Naja nigricollis was studied using HILIC coupled with LC-MS/MS analysis. Results revealed a total of 57, 55, and 46 proteins in the venoms of N. haje, N. katiensis, and N. nigricollis, respectively, with molecular mass ranging between 5 and 185  kDa. These snakes have 38 common proteins in addition to 3 uncommon proteins: actiflagelin, cathelicidin, and cystatin identified in their venoms. The identified proteins belonged to 14 protein families in N. haje and N. katiensis, and 12 protein families in N. nigricollis. Of the total venom proteins, 3FTx was the most abundant protein family, constituting 52% in N. haje and N. katiensis, and 41% in N. nigricollis, followed by PLA2, constituting 37% in N. nigricollis, 26% in N. haje, and 24% in N. katiensis. Other protein families, including LAAO, CRISPs, VEGF, PLB, CVF, SVMP, SVH, AMP, PI, Globin, Actin, and C-type lectins, were also detected, although, at very low abundances. Quantification of the relative abundance of each protein revealed that alpha and beta fibrinogenase and PLA2, which constituted 18-26% of the total proteome, were the most abundant. The 3 uncommon proteins have no known function in snake venom. However, actiflagelin activates sperm motility; cystatin inhibits angiogenesis, while cathelicidin exerts antimicrobial effects. The three Nigerian Naja genus proteomes displayed 70% similarity in composition, which suggests the possibility of formulating antivenom that may cross-neutralise the venoms of cobra species found in Nigeria. These data provide insights into clinically relevant peptides/proteins present in the venoms of these snakes. Data are available via ProteomeXchange with identifier PXD024627.


Assuntos
Naja , Proteômica , Animais , Cromatografia Líquida , Venenos Elapídicos/toxicidade , Elapidae , Humanos , Masculino , Naja haje , Nigéria , Venenos de Serpentes , Motilidade Espermática , Espectrometria de Massas em Tandem
17.
Trans R Soc Trop Med Hyg ; 115(1): 78-84, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-32945886

RESUMO

BACKGROUND: The Philippine cobra (Naja philippinensis) and Samar cobra (Naja samarensis) are two WHO Category 1 medically important venomous snakes in the Philippines. Philippine cobra antivenom (PCAV) is the only antivenom available in the country, but its neutralization capacity against the venoms of N. philippinensis and hetero-specific N. samarensis has not been reported. This knowledge gap greatly hinders the optimization of antivenom use in the region. METHODS: This study examined the immunological binding and neutralization capacity of PCAV against the two cobra venoms using WHO-recommended protocols. RESULTS: In mice, both venoms were highly neurotoxic and lethal with a median lethal dose of 0.18 and 0.20 µg/g, respectively. PCAV exhibited strong and comparable immunoreactivity toward the venoms, indicating conserved venom antigenicity between the two allopatric species. In in vivo assay, PCAV was only moderately effective in neutralizing the toxicity of both venoms. Its potency was even lower against the hetero-specific N. samarensis venom by approximately two-fold compared with its potency against N. philippinensis venom. CONCLUSION: The results indicated that PCAV could be used to treat N. samarensis envenomation but at a higher dose, which might increase the risk of hypersensitivity and worsen the shortage of antivenom supply in the field. Antivenom manufacturing should be improved by developing a low-dose, high-efficacy product against cobra envenomation.


Assuntos
Antivenenos , Venenos Elapídicos , Animais , Camundongos , Naja , Naja naja , Filipinas
18.
Toxicon ; 189: 79-90, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33130187

RESUMO

Among the captivating world of venomous snakes, an outstanding group of cobras from the family Elapidae is characterized by a distinctive structure of proteroglyphous venom apparatus that allows the ejection of venom from the fangs and formation of aerosol particles. The venom of "spitting" cobras is innocuous when sprayed over the intact skin, but contact with the eye surface is followed by ophthalmia and a temporary blindness, that may remain permanent, unless immediate and adequate medical interventions are carried out. The aim of this work was to monitor and evaluate induced vasoactive effects as well as embryotoxic effects with the regard to the whole crude venom of four "spitting" cobra species (African species Naja ashei and Naja nigricollis, Asian species Naja siamensis and Naja sumatrana). Vasoactive effects were visualized using the Hen's Egg Test - Chorioallantoic membrane (HET-CAM) test. The Chick Embryotoxicity Screening Test (CHEST) was used to estimate embryotoxicity and the data were then processed using statistical analysis. The highest embryonic mortality rate was observed after administration of venom from Naja nigricollis among the whole crude venoms tested. All tested venoms induced fast spreading of pathological alterations in the blood vessels on the chorioallantoic membrane. Our study discloses a detailed insight into microscopic level processes in venom-induced changes observed on the chicken embryos and on the vascular network in their chorioallantoic membrane. This article also highlights the increasing importance of the role of the chicken embryos and the importance of observing changes in the chorioallantoic membrane applied for toxicological and medical research as an appropriate alternative animal model in relation to 3R's principles.


Assuntos
Bioensaio , Venenos Elapídicos/toxicidade , Naja , Animais , Embrião de Galinha , Membrana Corioalantoide , Elapidae
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