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1.
PLoS Negl Trop Dis ; 14(2): e0008054, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32032357

RESUMO

Naja atra envenomation is one of the most significant clinical snakebite concerns in Taiwan. Taiwanese freeze-dried neurotoxic antivenom (FNAV) is currently used clinically for the treatment of cobra snakebite, and has been shown to limit the mortality of cobra envenomation to less than 1%. However, more than half of victims (60%) require surgery because of local tissue necrosis, a major problem in patients with cobra envenomation. Although the importance of evaluating the neutralizing effect of FNAV on this pathology is recognized, whether FNAV is able to prevent the local necrosis extension induced by N. atra venom has not been investigated in detail. Cytotoxins (CTXs) are considered as the major components of N. atra venom that cause necrosis. In the current study, we isolated CTXs from whole cobra venom and used both whole venom and purified CTXs to develop animal models for assessing the neutralization potential of FNAV against venom necrotizing activity. Local necrotic lesions were successfully produced in mice using CTXs in place of whole N. atra venom. FNAV was able to rescue mice from a subcutaneously injected lethal dose of cobra venom; however, it was unable to prevent CTX-induced dermo-necrosis. Furthermore, using the minimal necrosis dose (MND) of CTXs and venom proteome data, we found a dose of whole N. atra venom suitable for FNAV and developed a workable protocol for inducing local necrosis in rodent models that successfully imitated the clinical circumstance of cobra envenoming. This information provides a more comprehensive understanding of the pathophysiology of N. atra envenomation, and serves as a guide for improving current antivenom strategies and advancing clinical snakebite management in Taiwan.


Assuntos
Antivenenos/uso terapêutico , Venenos Elapídicos/toxicidade , Naja naja , Necrose/induzido quimicamente , Animais , Citotoxinas/química , Citotoxinas/toxicidade , Venenos Elapídicos/química , Camundongos , Camundongos Endogâmicos ICR , Taiwan
2.
J Med Virol ; 92(4): 433-440, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31967321

RESUMO

The current outbreak of viral pneumonia in the city of Wuhan, China, was caused by a novel coronavirus designated 2019-nCoV by the World Health Organization, as determined by sequencing the viral RNA genome. Many initial patients were exposed to wildlife animals at the Huanan seafood wholesale market, where poultry, snake, bats, and other farm animals were also sold. To investigate possible virus reservoir, we have carried out comprehensive sequence analysis and comparison in conjunction with relative synonymous codon usage (RSCU) bias among different animal species based on the 2019-nCoV sequence. Results obtained from our analyses suggest that the 2019-nCoV may appear to be a recombinant virus between the bat coronavirus and an origin-unknown coronavirus. The recombination may occurred within the viral spike glycoprotein, which recognizes a cell surface receptor. Additionally, our findings suggest that 2019-nCoV has most similar genetic information with bat coronovirus and most similar codon usage bias with snake. Taken together, our results suggest that homologous recombination may occur and contribute to the 2019-nCoV cross-species transmission.


Assuntos
Betacoronavirus/genética , Quirópteros/virologia , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/virologia , Reservatórios de Doenças , Pneumonia Viral/transmissão , Pneumonia Viral/virologia , Serpentes/virologia , Glicoproteína da Espícula de Coronavírus/genética , Animais , Betacoronavirus/classificação , Betacoronavirus/fisiologia , Bungarus/genética , Bungarus/virologia , Quirópteros/genética , Uso do Códon , Infecções por Coronavirus/epidemiologia , Surtos de Doenças , Evolução Molecular , Genoma Viral , Recombinação Homóloga , Especificidade de Hospedeiro , Humanos , Naja naja/genética , Naja naja/virologia , Filogenia , Pneumonia Viral/epidemiologia , Serpentes/genética , Zoonoses
3.
Nat Rev Genet ; 21(3): 133, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31992867
4.
Nat Genet ; 52(1): 106-117, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31907489

RESUMO

Snakebite envenoming is a serious and neglected tropical disease that kills ~100,000 people annually. High-quality, genome-enabled comprehensive characterization of toxin genes will facilitate development of effective humanized recombinant antivenom. We report a de novo near-chromosomal genome assembly of Naja naja, the Indian cobra, a highly venomous, medically important snake. Our assembly has a scaffold N50 of 223.35 Mb, with 19 scaffolds containing 95% of the genome. Of the 23,248 predicted protein-coding genes, 12,346 venom-gland-expressed genes constitute the 'venom-ome' and this included 139 genes from 33 toxin families. Among the 139 toxin genes were 19 'venom-ome-specific toxins' (VSTs) that showed venom-gland-specific expression, and these probably encode the minimal core venom effector proteins. Synthetic venom reconstituted through recombinant VST expression will aid in the rapid development of safe and effective synthetic antivenom. Additionally, our genome could serve as a reference for snake genomes, support evolutionary studies and enable venom-driven drug discovery.


Assuntos
Biologia Computacional/métodos , Venenos Elapídicos/análise , Venenos Elapídicos/genética , Genoma , Naja naja/genética , Transcriptoma , Sequência de Aminoácidos , Animais , Perfilação da Expressão Gênica , Índia , Homologia de Sequência
5.
Toxicon ; 171: 78-85, 2019 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-31622638

RESUMO

Snakebite is one of the health concerns worldwide. Naja oxiana is one of the venomous snakes with a high mortality rate. Anti-serum therapy is the only treatment of the victims. However, in some cases, antiserum injection leads to some side effects in host like serum sickness and anaphylactic shock. It is crucial to develop a neutralizing agent with low side effects. The human antibody library (non-immunized library) was used to isolate specific antibodies against N.oxiana venom components. Four rounds of biopanning were performed to enrich scFv-displaying phages against the venom of N. oxiana. Enrichment of scFv-displaying phages against N. oxiana venom was analyzed by polyclonal Enzyme-Linked Immunosorbent Assay (ELISA). Specific antibody fragments against N. oxiana venom were selected through monoclonal ELISA, and were expressed in E. coli bacterial cells. Purification of the selected clones was performed by using nickel affinity chromatography. Neutralization and protective capacity of specific antibody fragments were analyzed in C57BL/6 mice (i.v. injection). Results of biopanning and polyclonal ELISA demonstrate a successful enrichment process. Five specific antibody fragments with the highest signal in monoclonal ELISA were selected, expressed, and purified. The purity of expressed antibody fragments was monitored by SDS-PAGE and Western blot. The selected antibody fragments were able to neutralize two LD50 of N. oxiana venom and protected all mice when injected 15 min post-envenomation. The data indicate that such selected antibodies are promising tools for further studies and in the development of novel protective agents against N. oxiana venom.


Assuntos
Venenos Elapídicos/imunologia , Região Variável de Imunoglobulina/imunologia , Naja naja , Animais , Antivenenos/imunologia , Ensaio de Imunoadsorção Enzimática , Escherichia coli , Humanos , Camundongos Endogâmicos C57BL , Mordeduras de Serpentes/terapia
6.
Dokl Biochem Biophys ; 487(1): 282-286, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31559598

RESUMO

The study of the influence of cobra Naja oxiana cardiotoxins on the contractility of the rat papillary muscles and its rhythmoinotropic characteristics has shown that the presence of toxins induces a slight contractility decrease in the stimulation frequency range up to 0.1 Hz. In the stimulation frequency range from 0.1 to 0.5 Hz, a positive inotropic effect is found. However, the positive inotropic effect is replaced by a negative one with further increase in the frequency up to 3 Hz. In the presence of cardiotoxins, the positive force-frequency relationship in the region of 1-3 Hz, characteristic of healthy rat myocardium, disappears and the relationship becomes completely negative. L-type calcium channel blocker nifedipine does not affect the changes induced by toxins, while a high concentration (10 mM) of calcium prevents the effects of cardiotoxins on the muscle. The results obtained show that the impairment of the force-frequency relationship occurs long before the development of irreversible damage in the myocardium and may be the first sign of the pathological action of cardiotoxins.


Assuntos
Proteínas Cardiotóxicas de Elapídeos/farmacologia , Coração/efeitos dos fármacos , Coração/fisiologia , Contração Miocárdica/efeitos dos fármacos , Naja naja , Animais , Relação Dose-Resposta a Droga , Ratos
7.
Mol Biol Rep ; 46(6): 6087-6098, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31502192

RESUMO

The complete genome sequence provides the opportunity for genome-wide and coding region analysis of SSRs in the king cobra and for cross-species identification of microsatellite markers in the Chinese cobra. In the Ophiophagus hannah genome, tetranucleotide repeats (38.03%) were the most abundant category, followed by dinucleotides (23.03%), pentanucleotides (13.07%), mononucleotides (11.78%), trinucleotides (11.49%) and hexanucleotides (2.6%). Twenty predominant motifs in the O. hannah genome were (A)n (C)n, (AC)n, (AG)n, (AT)n, (AGG)n, (AAT)n, (AAG)n, (AAC)n, (ATG)n, (ATAG)n, (AAGG)n, (ATCT)n, (CCTT)n, (ATTT)n, (AAAT)n, (AATAG)n, (ATTCT)n, (ATATGT)n, (AGATAT)n. In total, 4344 SSRs were found in coding sequences (CDSs). Tetranucleotides (52.79%) were the most abundant microsatellite type in CDS, followed by trinucleotides (28.50%), dinucleotides (11.02%), pentanucleotides (4.42%), mononucleotides (1.77%), and hexanucleotides (1.50%). A total of 984 CDSs containing microsatellites were assigned 11152 Gene Ontology (GO) functional terms. Gene Ontology (GO) analysis demonstrated that cellular process, cell and binding were the most frequent GO terms in biological process, cellular component and molecular function, respectively. Thirty-two novel highly polymorphic (PIC > 0.5) SSR markers for Naja atra were developed from cross-species amplification based on the tetranucleotide microsatellite sequences in the king cobra genome. The number of alleles (NA) per locus had between 3 and 11 alleles with an average of 6.5, the polymorphism information content (PIC) value ranged from 0.521 to 0.858 (average = 0.707), the observed heterozygosity (Ho) of 32 microsatellite loci ranged from 0.292 to 0.875 (mean = 0.678), the expected heterozygosity (HE) ranged from 0.561 to 0.889 (average = 0.761), and 3 microsatellite loci exhibited statistically significant departure from Hardy-Weinberg equilibrium (HWE) after Bonferroni correction (p < 0.003).


Assuntos
Repetições de Microssatélites/genética , Naja naja/genética , Ophiophagus hannah/genética , Alelos , Animais , Loci Gênicos/genética , Marcadores Genéticos/genética , Estudo de Associação Genômica Ampla/métodos , Polimorfismo Genético/genética , Análise de Sequência de DNA/métodos
8.
Int J Biol Macromol ; 136: 512-520, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31199971

RESUMO

Snake venom cardiotoxins (CTXs) present diverse pharmacological functions. Previous studies have reported that CTXs affect the activity of some serine proteases, namely, chymotrypsin, subtilisin, trypsin, and acetylcholinesterase. To elucidate the mode of action of CTXs, the interaction of CTXs with chymotrypsin was thus investigated. It was found that Naja atra CTX isotoxins concentration-dependently enhanced chymotrypsin activity. The capability of CTX1 and CTX5 in increasing chymotrypsin activity was higher than that of CTX2, CTX3, and CTX4. Removal of the molecular beacon-bound CTXs by chymotrypsin, circular dichroism measurement, and acrylamide quenching of Trp fluorescence indicated that CTXs bound to chymotrypsin. Chemical modification of Lys, Arg, or Met residues of CTX1 attenuated its capability to enhance chymotrypsin activity without impairing their bond with chymotrypsin. Catalytically inactive chymotrypsin retained the binding affinity for native and modified CTX1. CTX1 and chemically modified CTX1 differently altered the global conformation of chymotrypsin and inactivated chymotrypsin. Moreover, CTX1 did not reduce the interaction of 2-(p-toluidino)-naphthalene-6-sulfonate (TNS) with chymotrypsin and inactivated chymotrypsin. Together with previous results revealing that TNS can bind at the hydrophobic region of active site in chymotrypsin, our data suggest that CTXs can enhance chymotrypsin activity by binding to the region outside the enzyme's active site.


Assuntos
Cardiotoxinas/farmacologia , Quimotripsina/metabolismo , Naja naja , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cardiotoxinas/química , Cardiotoxinas/metabolismo , Quimotripsina/química , Simulação de Acoplamento Molecular , Conformação Proteica/efeitos dos fármacos
9.
Toxicon ; 164: 31-43, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30953661

RESUMO

Naja kaouthia is one of the most prevalent medically important snakes of North East India and Bangladesh responsible for most of the bite cases. In this study, an attempt was made to decipher venom variation of Naja kaouthia venom from North East India and Bangladesh. Using multidimensional methods including reverse phase HPLC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D-PAGE) and two-dimensional gel electrophoresis (2D-PAGE), the quantitative differences in venom composition have been revealed. Moreover, tested in-vitro biochemical and biological activities also exhibited differences which could be due to venom variability. Furthermore, neutralization efficacy of commercially available Indian polyvalent antivenoms (Vins, Bharat Serum, Haffkine) was evaluated and the results displayed significant differences in neutralizing efficacy between the antivenoms. Immunoblotting experiments showed antivenom molecules cross reacted with high molecular mass components while poorly reacted towards low molecular mass proteins. Immuno-depletion study demonstrated that Vins polyvalent antivenom was poor in immunocapturing the venom proteins of both North East Indian and Bangladesh origin Naja kaouthia at the ratio of 1:16 (venom: antivenom).


Assuntos
Antivenenos/imunologia , Venenos Elapídicos/química , Venenos Elapídicos/imunologia , Naja naja , Animais , Antivenenos/farmacologia , Bangladesh , Reações Cruzadas , Humanos , Índia , Células MCF-7 , Masculino , Camundongos , Testes de Neutralização
10.
Toxicon ; 165: 110-115, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31029638

RESUMO

BACKGROUND: Cytotoxin 1 (CTX1) purified from Naja atra Cantor venom could inhibit cancer cell proliferation, but the mechanism is not clear. This study aimed to investigate the mechanism by which leukemia cells are killed by CTX1. MATERIALS AND METHODS: HL-60 and KG1a cells were treated with CTX1 and the cell death was detected. RESULTS: The viability of HL-60 and KG1a cells decreased in a dose- and time-dependent manner after treatment with CTX1. CTX1 mainly induced late apoptosis and necrosis. The cell death induced by CTX1 could be rescued by specific necroptosis inhibitor Nec-1 but not by caspase inhibitor Z-VAD-fmk in HL-60 cells. In addition, CTX1 increased lysosome membrane permeability (LMP) and release of cathepsin B. CONCLUSION: CTX1 could induce necroptosis in leukemia cells, and it is related to LMP increase and cathepsin release. CTX1 could be a promising anti-cancer drug for leukemia therapy.


Assuntos
Antineoplásicos/farmacologia , Citotoxinas/farmacologia , Venenos Elapídicos/farmacologia , Necrose/induzido quimicamente , Animais , Antineoplásicos/isolamento & purificação , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Citotoxinas/isolamento & purificação , Venenos Elapídicos/química , Células HL-60 , Humanos , Leucemia , Naja naja
11.
J Biol Chem ; 294(18): 7324-7334, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-30804211

RESUMO

The voltage-gated sodium channel Nav1.8 is preferentially expressed in peripheral nociceptive neurons and contributes to inflammatory and neuropathic pain. Therefore, Nav1.8 has emerged as one of the most promising analgesic targets for pain relief. Using large-scale screening of various animal-derived toxins and venoms for Nav1.8 inhibitors, here we identified µ-EPTX-Na1a, a 62-residue three-finger peptide from the venom of the Chinese cobra (Naja atra), as a potent inhibitor of Nav1.8, exhibiting high selectivity over other voltage-gated sodium channel subtypes. Using whole-cell voltage-clamp recordings, we observed that purified µ-EPTX-Na1a blocked the Nav1.8 current. This blockade was associated with a depolarizing shift of activation and repolarizing shift of inactivation, a mechanism distinct from that of any other gating modifier toxin identified to date. In rodent models of inflammatory and neuropathic pain, µ-EPTX-Na1a alleviated nociceptive behaviors more potently than did morphine, indicating that µ-EPTX-Na1a has a potent analgesic effect. µ-EPTX-Na1a displayed no evident cytotoxicity and cardiotoxicity and produced no obvious adverse responses in mice even at a dose 30-fold higher than that producing a significant analgesic effect. Our study establishes µ-EPTX-Na1a as a promising lead for the development of Nav1.8-targeting analgesics to manage pain.


Assuntos
Venenos Elapídicos/química , Canal de Sódio Disparado por Voltagem NAV1.8/efeitos dos fármacos , Bloqueadores dos Canais de Sódio/farmacologia , Analgésicos/efeitos adversos , Analgésicos/farmacologia , Analgésicos/uso terapêutico , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Camundongos , Naja naja , Neuralgia/tratamento farmacológico , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley
12.
Toxins (Basel) ; 11(1)2019 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-30658470

RESUMO

Native disulfide formation is crucial to the process of disulfide-rich protein folding in vitro. As such, analysis of the disulfide bonds can be used to track the process of the folding reaction; however, the diverse structural isomers interfere with characterization due to the non-native disulfide linkages. Previously, a mass spectrometry (MS) based platform coupled with peptide demethylation and an automatic disulfide bond searching engine demonstrated the potential to screen disulfide-linked peptides for the unambiguous assignment of paired cysteine residues of toxin components in cobra venom. The developed MS-based platform was evaluated to analyze the disulfide bonds of structural isomers during the folding reaction of synthetic cardiotoxin A3 polypeptide (syn-CTX A3), an important medical component in cobra venom. Through application of this work flow, a total of 13 disulfide-linked peptides were repeatedly identified across the folding reaction, and two of them were found to contain cysteine pairings, like those found in native CTX A3. Quantitative analysis of these disulfide-linked peptides showed the occurrence of a progressive disulfide rearrangement that generates a native disulfide bond pattern on syn-CTX A3 folded protein. The formation of these syn-CTX A3 folded protein reaches a steady level in the late stage of the folding reaction. Biophysical and cell-based assays showed that the collected syn-CTX A3 folded protein have a ß-sheet secondary structure and cytotoxic activity similar to that of native CTX A3. In addition, the immunization of the syn-CTX A3 folded proteins could induce neutralization antibodies against the cytotoxic activity of native CTX A3. In contrast, these structure activities were poorly observed in the other folded isomers with non-native disulfide bonds. The study highlights the ability of the developed MS platform to assay isomers with heterogeneous disulfide bonds, providing insight into the folding mechanism of the bioactive protein generation.


Assuntos
Proteínas Cardiotóxicas de Elapídeos/química , Dissulfetos/química , Peptídeos/química , Animais , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida , Proteínas Cardiotóxicas de Elapídeos/farmacologia , Células HL-60 , Humanos , Isomerismo , Espectrometria de Massas , Naja naja , Peptídeos/farmacologia , Dobramento de Proteína , Estrutura Secundária de Proteína
13.
Expert Rev Proteomics ; 15(11): 949-961, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30345852

RESUMO

BACKGROUND: Cobra bite is frequently reported across the Indian subcontinent and is associated with a high rate of death and morbidity. In eastern India (EI) Naja naja and Naja kaouthia are reported to be the two most abundant species of cobra. RESEARCH DESIGN AND METHODS: The venom proteome composition of N. naja (NnV) and N. kaouthia (NkV) from Burdwan districts of EI were compared by separation of venom proteins by 1D-SDS-PAGE followed by LC-MS/MS analysis of protein bands. The potency of commercial polyantivenom (PAV) was assessed by neutralization, ELISA, immuno-blot and venom-PAV immunoaffinity chromatography studies. RESULTS: Proteomic analysis identified 52 and 55 proteins for NnV and NkV, respectively, when searched against the Elapidae database. A small quantitative difference in venom composition between these two species of cobra was observed. PAVs exhibited poor cross-reactivity against low molecular mass toxins (<20 kDa) of both cobra venoms, which was substantiated by a meager neutralization of their phospholipase A2 activity. Phospholipase A2 and 3FTx, the two major classes of nonenzymatic and enzymatic proteins, respectively, were partially recognized by PAVs. CONCLUSIONS: Efforts must be made to improve immunization protocols and supplement existing antivenoms with antibodies raised against the major toxins of these venoms.


Assuntos
Antivenenos/imunologia , Venenos Elapídicos/imunologia , Naja , Proteoma/análise , Animais , Antivenenos/farmacologia , Cromatografia Líquida , Reações Cruzadas , Venenos Elapídicos/análise , Venenos Elapídicos/toxicidade , Eletroforese em Gel de Poliacrilamida , Enzimas/imunologia , Enzimas/metabolismo , Humanos , Índia , Naja naja , Proteômica/métodos , Mordeduras de Serpentes/mortalidade , Mordeduras de Serpentes/fisiopatologia , Especificidade da Espécie , Espectrometria de Massas em Tandem
14.
Toxins (Basel) ; 10(10)2018 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-30248928

RESUMO

Traditional, horse-derived antivenin is currently the most efficient treatment against snake bites. However, it is costly and has unpredictable side effects. Thus, alternative, cost-effective strategies for producing antivenin are needed. In this study, we immunized hens with inactivated NNA venom proteins from the cobra Naja naja atra (NNA). Purified yolk IgY antibodies showed specific anti-NNA binding activity comparable to that of the equine-derived antivenin. We used phage display technology to generate two antibody libraries containing 9.0 × 108 and 8.4 × 108 clones with a short or long linker, respectively. The phage ELISA indicated that anti-NNA clones displaying single-chain variable fragments (scFv) were significantly enriched after biopanning. The nucleotide sequences of the light and heavy chain genes of 30 monoclonal scFv antibodies were determined and classified into six groups with the short linker and nine groups with the long linker. These scFv clones specifically bound to NNA proteins but not to venom proteins from other snakes. Their binding affinities were further determined by competitive ELISA. Animal model studies showed that anti-NNA IgY antibodies exhibited complete protective effects, while a combination of scFv antibodies raised the survival rates and times of mice challenged with lethal doses of NNA venom proteins.


Assuntos
Antivenenos/imunologia , Venenos Elapídicos/imunologia , Naja naja , Anticorpos de Cadeia Única/imunologia , Animais , Galinhas/imunologia , Feminino , Imunoglobulinas/imunologia , Camundongos Endogâmicos ICR , Proteínas de Répteis/imunologia , Mordeduras de Serpentes/diagnóstico , Mordeduras de Serpentes/terapia
15.
J Proteomics ; 187: 59-68, 2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-29929037

RESUMO

Deinagkistrodon acutus, Trimeresurus stejnegeri, Protobothrops mucrosquamatus, Daboia russelii siamensis, Bungarus multicinctus and Naja atra are the six medically important venomous snake species in Taiwan. In this study, we characterized and compared their venom protein profiles using proteomic approaches. The major snake venom proteins were identified by GeLC-MS/MS and the total venom proteome was characterized by in-solution digestion coupled with LC-MS/MS. A total of 27-52 proteins, categorized into 23 protein families, were identified in each snake's venom. The major venom components found in Viperidae species (D. acutus, T. stejnegeri, P. mucrosquamatus and D. russelii) were C-type lectin, snake venom serine proteinase, venom metalloproteinase and phospholipase A2, whereas three-finger toxin and phospholipase A2 were the major components detected in the venom of Elapidae snakes (B. multicinctus and N. atra). This study also provided the first demonstration of some low-abundance proteins in these six snake venoms, including 5'-nucleotidase, glutaminyl-peptide cyclotransferase and phosphodiesterase, among others. Furthermore, we found that cobra venom factor (CVF) is a cobra-specific protein. We produced anti-peptide antibodies against CVF and used it to develop a highly sensitive SISCAPA-MRM assay for quantifying CVF. The limit of detection and lower limit of quantification were 3.2 and 9.6 attomoles, respectively. This assay was used to precisely quantify CVF in 1 µg crude venom proteins from three Naja species and king cobra. The amount of CVF varied from 0.9 to 54.36 femtomoles (equivalent to 0.16-10.03 mg/g of venom protein). BIOLOGICAL SIGNIFICANCE: There are six medically significant venomous snakes in Taiwan. The venoms of the four Viperidae species (Deinagkistrodon acutus, Trimeresurus stejnegeri, Protobothrops mucrosquamatus and Daboia russelii siamensis) cause local tissue swelling; this symptom is also seen in N. atra envenomation in humans, potentially complicating the differential diagnosis of envenomation by N. atra and Viperidae species. Thus, characterization of the venom proteomes of the six Taiwanese snakes, including the relative abundance of the major components and species-specific protein(s) in each venom type, could be useful for future venom research, including the development of new assay(s) for detecting snake species-specific venom protein(s) and new type(s) of antivenom.


Assuntos
Venenos Elapídicos/análise , Espectrometria de Massas/métodos , Proteoma/análise , Proteômica/métodos , Venenos de Serpentes/análise , Animais , Anticorpos/análise , Anticorpos/química , Antivenenos/análise , Antivenenos/química , Bungarus , Cromatografia Líquida , Venenos Elapídicos/química , Venenos Elapídicos/metabolismo , Elapidae , Marcação por Isótopo/métodos , Naja naja , Proteoma/química , Proteoma/metabolismo , Venenos de Serpentes/química , Venenos de Serpentes/metabolismo , Especificidade da Espécie , Taiwan , Espectrometria de Massas em Tandem/métodos , Viperidae
16.
Sci Rep ; 8(1): 9716, 2018 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-29946111

RESUMO

In order to facilitate/expedite the production of effective and affordable snake antivenoms, a novel in vitro potency assay was previously developed. The assay is based on an antiserum's ability to bind to postsynaptic neurotoxin (PSNT) and thereby inhibit the PSNT binding to the nicotinic acetylcholine receptor (nAChR). The assay was shown to work well with antiserum against Thai Naja kaouthia which produces predominantly the lethal PSNTs. In this work, the assay is demonstrated to work well with antiserum/antivenom against Bungarus candidus (BC), which also produces lethal presynaptic neurotoxins, as well as antivenom against Sri Lankan Naja naja (NN), which produces an abundance of cytotoxins. The in vitro and in vivo median effective ratios (ER50s) for various batches of antisera against BC showed a correlation (R2) of 0.8922 (p < 0.001) while the corresponding value for the anti-NN antivenom was R2 = 0.7898 (p < 0.01). These results, together with the known toxin profiles of various genera of elapids, suggest that this in vitro assay could be used with antisera against other species of Bungarus and Naja and possibly other neurotoxic snake venoms worldwide. The assay should significantly save numerous lives of mice and accelerate production of life-saving antivenoms.


Assuntos
Antivenenos/metabolismo , Antivenenos/farmacologia , Bungarus/metabolismo , Naja naja/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Camundongos , Ligação Proteica
17.
Pak J Pharm Sci ; 31(2(Suppl.)): 685-689, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29625942

RESUMO

Recent recognition about snake bite envenomation on June, 2017 as neglected tropical disease under category-A by World Health Organization advocated again its undeniable importance. Present circumstances reasoned to work on a neglected subspecies of Naja naja, i.e., Naja naja karachiensis (N. n. karachensis) has been documented for frequent deaths in Pakistan. In this study median lethal toxic dose (LD50) was determined intraperitoneally in Swiss albino mice and was found to be 2.0µg/g (2.0mg/kg) equal in potency to Naja pallida (red spitting African cobra). Total protein contents (188±0.011µg / 200µg of dry weight) were high enough (94%) to represent an arsenal of proteins. Furthermore, 99mTc was labeled 99.9% with venom and didn't find to alter hemolytic activity of venom in dose dependent manner at 125µg/ml (p>0.5), 250 µg/ml (p>0.1) and 500 µg/ml (p>0.1) when compared with its crude form. Present work will pave the way for proteomics study in effective production of antidote against specific species of snakes as dare demand of it has been felt since long period of time in Pakistan.


Assuntos
Venenos Elapídicos/química , Venenos Elapídicos/toxicidade , Hemolíticos/farmacologia , Naja naja , Proteínas/análise , Animais , Relação Dose-Resposta a Droga , Hemólise/efeitos dos fármacos , Hemolíticos/química , Dose Letal Mediana , Masculino , Camundongos , Tecnécio/química
18.
Toxins (Basel) ; 10(3)2018 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-29495566

RESUMO

Naja naja atra venom (NNAV) is composed of various proteins, peptides, and enzymes with different biological and pharmacological functions. A number of previous studies have reported that NNAV exerts potent analgesic effects on various animal models of pain. The clinical studies using whole venom or active components have confirmed that NNAV is an effective and safe medicine for treatment of chronic pain. Furthermore, recent studies have demonstrated that NNAV has anti-inflammatory and immune regulatory actions in vitro and in vivo. In this review article, we summarize recent studies of NNAV and its components on inflammation and immunity. The main new findings in NNAV research show that it may enhance innate and humoral immune responses while suppressing T lymphocytes-mediated cellular immunity, thus suggesting that NNAV and its active components may have therapeutic values in the treatment of inflammatory and autoimmune diseases.


Assuntos
Anti-Inflamatórios , Venenos Elapídicos , Fatores Imunológicos , Naja naja , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Venenos Elapídicos/química , Venenos Elapídicos/farmacologia , Venenos Elapídicos/uso terapêutico , Humanos , Fatores Imunológicos/química , Fatores Imunológicos/farmacologia , Fatores Imunológicos/uso terapêutico
19.
Int J Dermatol ; 57(5): 605-610, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29460967

RESUMO

BACKGROUND: Snake envenomation is a neglected global health problem. There is a need for a prehospital treatment of neurotoxic snakebite that prolongs survival and allows time for a victim to reach a hospital for antivenom therapy. Tumescent epinephrine consists of a large volume of dilute epinephrine (2 mg/l) injected subcutaneously. It functions as "contravenom" by causing capillary vasoconstriction and delaying venom absorption. METHODS: A murine model of neurotoxic envenomation using lidocaine as a surrogate for neurotoxic snake venom was first developed in a pilot study. A lethal dose of lidocaine was injected subcutaneously into control and treatment groups. Mice in the treatment group were then treated with a tumescent infiltration of dilute epinephrine in saline, while control mice either received no treatment or tumescent infiltration with saline alone. The experiment was repeated using lethal doses of neurotoxic Naja naja cobra venom. The main end-points were survival rate and survival time. RESULTS: None of the control mice survived a lethal (LD100 ) dosage of subcutaneous lidocaine. Mice given an LD100 of subcutaneous lidocaine and treated immediately with tumescent epinephrine had 80% survival. Following LD50 doses of Naja naja venom, 50% of control mice survived, while 94% survived when treated immediately with tumescent epinephrine (P < 0.01). All animals died following LD100 doses of Naja naja venom, but survival was significantly prolonged (P < 0.0001) by immediate tumescent epinephrine. CONCLUSIONS: Tumescent epinephrine, when given immediately after toxin injection, improves survival rates in mice following neurotoxic doses of lidocaine or Naja naja cobra venom.


Assuntos
Antivenenos/administração & dosagem , Epinefrina/administração & dosagem , Síndromes Neurotóxicas/prevenção & controle , Mordeduras de Serpentes/terapia , Venenos de Serpentes/toxicidade , Animais , Antivenenos/farmacologia , Modelos Animais de Doenças , Feminino , Humanos , Injeções Subcutâneas , Estimativa de Kaplan-Meier , Lidocaína , Camundongos , Naja naja , Síndromes Neurotóxicas/etiologia , Projetos Piloto , Distribuição Aleatória , Sensibilidade e Especificidade , Mordeduras de Serpentes/mortalidade
20.
PLoS One ; 13(1): e0190778, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29364903

RESUMO

Naja atra subsp. atra cardiotoxin 1 (CTX-1), produced by Chinese cobra snakes, belonging to Elapidae family, is included in the three-finger toxin family and exerts high cytotoxicity and antimicrobial activity too. Using as template mainly the tip and the subsequent ß-strand of the first "finger" of this toxin, different sequences of 20 amino acids linear peptides have been designed in order to avoid toxic effects but to maintain or even strengthen the partial antimicrobial activity already seen for the complete toxin. As a result, the sequence NCP-0 (Naja Cardiotoxin Peptide-0) was designed as ancestor and subsequently 4 other variant sequences of NCP-0 were developed. These synthesized variant sequences have shown microbicidal activity towards a panel of reference and field strains of Gram-positive and Gram-negative bacteria. The sequence named NCP-3, and its variants NCP-3a and NCP-3b, have shown the best antimicrobial activity, together with low cytotoxicity against eukaryotic cells and low hemolytic activity. Bactericidal activity has been demonstrated by minimum bactericidal concentration (MBC) assay at values below 10 µg/ml for most of the tested bacterial strains. This potent antimicrobial activity was confirmed even for unicellular fungi Candida albicans, Candida glabrata and Malassezia pachydermatis (MBC 50-6.3 µg/ml), and against the fast-growing mycobacteria Mycobacterium smegmatis and Mycobacterium fortuitum. Moreover, NCP-3 has shown virucidal activity on Bovine Herpesvirus 1 (BoHV1) belonging to Herpesviridae family. The bactericidal activity is maintained even in a high salt concentration medium (125 and 250 mM NaCl) and phosphate buffer with 20% Mueller Hinton (MH) medium against E. coli, methicillin resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa reference strains. Considering these in vitro obtained data, the search for active sequences within proteins presenting an intrinsic microbicidal activity could provide a new way for discovering a large number of novel and promising antimicrobial peptides families.


Assuntos
Anti-Infecciosos/farmacologia , Proteínas Cardiotóxicas de Elapídeos/química , Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Anti-Infecciosos/química , Candida/efeitos dos fármacos , Bovinos , Dicroísmo Circular , Hemólise/efeitos dos fármacos , Herpesvirus Bovino 1/efeitos dos fármacos , Malassezia/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Mycobacterium/efeitos dos fármacos , Naja naja , Peptídeos/química , Conformação Proteica , Ovinos , Staphylococcus aureus/efeitos dos fármacos
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