ABSTRACT
Background: Naja atra is a venomous snake species medically relevant in China. In the current study, we evaluated the composition and toxicological profile of venom collected from farm-raised N. atra. Methods: Venom was collected from third-generation captive bred N. atra on a snake farm in Hunan Province, China. The venom was analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis and nano-liquid chromatography with electrospray ionization tandem mass spectrometry. In addition, hemolytic activity, median lethal dose, serum biochemical and histopathological parameters were accessed. Results: N. atra venom proteome was dominated by phospholipase A2 (46.5%) and three-finger toxins (41.4 %), and a set of common low relative abundance proteins, including cysteine-rich secretory proteins (4.7%), NGF-beta (2.4%), snake venom metalloproteinase (1.5%), glutathione peroxidase (0.6%), vespryn (0.3%), and 5ʹ-nucleotidases (0.2%) were also found. Furthermore, the venom exhibited direct hemolytic activity, neurotoxicity, myotoxicity, and high lethal potency in mice, with a subcutaneous median lethal dose of 1.02 mg/kg. Histopathological analysis and serum biochemical tests revealed that venom caused acute hepatic, pulmonary and renal injury in mice. Conclusion: This study revealed the composition and toxicity of venom collected from farm-raised N. atra, thereby providing a reference for the analysis of venom samples collected from captive-born venomous snakes in the future.(AU)
Subject(s)
Animals , Snake Venoms/toxicity , Phospholipases A2 , Naja naja , Myotoxicity , NucleotidasesABSTRACT
Abstract Background: Naja atra is a venomous snake species medically relevant in China. In the current study, we evaluated the composition and toxicological profile of venom collected from farm-raised N. atra. Methods: Venom was collected from third-generation captive bred N. atra on a snake farm in Hunan Province, China. The venom was analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis and nano-liquid chromatography with electrospray ionization tandem mass spectrometry. In addition, hemolytic activity, median lethal dose, serum biochemical and histopathological parameters were accessed. Results: N. atra venom proteome was dominated by phospholipase A2 (46.5%) and three-finger toxins (41.4 %), and a set of common low relative abundance proteins, including cysteine-rich secretory proteins (4.7%), NGF-beta (2.4%), snake venom metalloproteinase (1.5%), glutathione peroxidase (0.6%), vespryn (0.3%), and 5-nucleotidases (0.2%) were also found. Furthermore, the venom exhibited direct hemolytic activity, neurotoxicity, myotoxicity, and high lethal potency in mice, with a subcutaneous median lethal dose of 1.02 mg/kg. Histopathological analysis and serum biochemical tests revealed that venom caused acute hepatic, pulmonary and renal injury in mice. Conclusion: This study revealed the composition and toxicity of venom collected from farm-raised N. atra, thereby providing a reference for the analysis of venom samples collected from captive-born venomous snakes in the future.
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Objective: To prepare Naja atra neurotoxin (NT) loaded dissolving microneedles (DMNs-NT), and investigate the physicochemical properties and in vitro transdermal properties. Methods: DMNs-NT was prepared by a two-step centrifugation method. The ratio of CS and PVP K30, the water content of the matrix solution, and the backing layer material were optimized by the indexes of formability and mechanical strength of the microneedles and flexibility of the backing layer. The drug loading content was determined by HPLC, and the morphological characteristics were observed under an optical microscope, and the stability was also examined. Franz diffusion cell was used to investigate its in vitro skin permeation characteristics. Results: Through the single-factor exploration, we confirmed that the optimal prescription technique for DMNs-NT preparation was a 1:1 ratio of CS and PVP k30, a 5:4 ratio of matrix material and water, with CMC as the backing layer material. The DMNs-NT had a pyramidal shape with a smooth surface and a length of approximately 500 μm. The drug loading content of per tablet was (15.4 ± 0.5) μg. The drug was located in the upper part of the needle. DMNs-NT had good stability within 3 months. The results of in vitro skin permeation assay showed that the cumulative penetration of NT in DMNs-NT could reach 95.8% in 4 h, while NT solution barely penetrated the skin, which proved that it had a good promoting effect on NT transdermal delivery. Conclusion: In this study, DMNs-NT had good mechanical properties and good skin penetration, which realized the transdermal drug delivery of macromolecular drugs.
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Chinese Cobra (Naja naja atra) bite is one of the leading causes of snake-bite mortality in China. The traditional anti-cobra venom serum therapy was found to be expensive and with high frequency of side effects. Therefore attempts were made to generate a high titer immunoglobulin from egg yolk (IgY) of crude cobra-venom immunized Leghorn hens, and to standardize an effective method for producing avian antivenom in relatively pure form. The IgY was isolated first by water dilution method to remove the lipid, then extracted by ammonium sulfate precipitation, and purified through anion exchange chromatogram. The different purities of IgY from different isolating stages were submitted to enzyme-linked immunosorbent assay and SDS-PAGE to determine their titers. Immunoblotting showed that the purified IgY (ion exchange chromatography fraction, IECF) recognized several antigenic fractions of cobra venom, and presented with the character of polyclonal antibody. IECF on SDS-PAGE under reducing conditions migrated as a 65 kDa heavy chain and a 35 kDa light chain, respectively. The LD50 of the N. naja atra venom was 0.62 mg/kg body weight in mice. Four times the LD50 dose of venom was selected as challenge dose, and the ED50 of IgY was 3.04 mg IECF/mg venom. The results indicate that the activity of anti-snake venom IgY could be obviously elevated by ion exchange chromatography, thus possessing therapeutic significance for snakebite envenomation.
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Chinese cobra (Naja naja atra) cardiotoxins are three-fingered family with 60～62 amino acids bind by four disulfide bonds. CardiotoxinⅢ (CTXⅢ) is one of the major toxic component which can cause hemolysis and cytotoxicity. However, there is no report on the fusion expression of CTXⅢ in soluble form so far. The cloning, expression and purification of recombinant CTX Ⅲ (rCTXⅢ) from Naja naja atra in E. coli and in yeast Pichia pastoris were reported here. CTXⅢ gene, fused with enterokinase in E.coli His-patch Thioredoxin expression system, were expressed in soluble form and released by osmotic-shock treatment. CTX Ⅲ gene was also cloned and expressed in the methylotropic yeast Pichia pastoris pPIC9K expression vector in the first time. The yield of the secretion level was 9.5 mg/L. Using straightforward one-step chromatography procedure, the rCTXⅢ, with three additional amino acids (GYT) at the N-terminal site, was purified to a purity of more than 90% and recovery yield of 65%. The purified rCTX Ⅲ was further characterized by cytotoxic assay with IC50 4.66μg/ml. An effective expression and purification system for recombinant CTXs in P. pastoris was developed, this system will permit us the ready isolation of active cardiotoxins. This protocol can also be easily used for the production of the toxin in a larger scale with low cost.
ABSTRACT
Chinese cobra (Naja naja atra) cardiotoxins are three-fingered family with 60～62 amino acids bind by four disulfide bonds. CardiotoxinⅢ (CTXⅢ) is one of the major toxic component which can cause hemolysis and cytotoxicity. However, there is no report on the fusion expression of CTXⅢ in soluble form so far. The cloning, expression and purification of recombinant CTX Ⅲ (rCTXⅢ) from Naja naja atra in E. coli and in yeast Pichia pastoris were reported here. CTXⅢ gene, fused with enterokinase in E.coli His-patch Thioredoxin expression system, were expressed in soluble form and released by osmotic-shock treatment. CTX Ⅲ gene was also cloned and expressed in the methylotropic yeast Pichia pastoris pPIC9K expression vector in the first time. The yield of the secretion level was 9.5 mg/L. Using straightforward one-step chromatography procedure, the rCTXⅢ, with three additional amino acids (GYT) at the N-terminal site, was purified to a purity of more than 90% and recovery yield of 65%. The purified rCTX Ⅲ was further characterized by cytotoxic assay with IC50 4.66μg/ml. An effective expression and purification system for recombinant CTXs in P. pastoris was developed, this system will permit us the ready isolation of active cardiotoxins. This protocol can also be easily used for the production of the toxin in a larger scale with low cost.
ABSTRACT
Chinese cobra (Naja naja atra) cardiotoxins are three-fingered family with 60～62 amino acids bind by four disulfide bonds. CardiotoxinⅢ (CTXⅢ) is one of the major toxic component which can cause hemolysis and cytotoxicity. However, there is no report on the fusion expression of CTXⅢ in soluble form so far. The cloning, expression and purification of recombinant CTX Ⅲ (rCTXⅢ) from Naja naja atra in E. coli and in yeast Pichia pastoris were reported here. CTXⅢ gene, fused with enterokinase in E.coli His-patch Thioredoxin expression system, were expressed in soluble form and released by osmotic-shock treatment. CTX Ⅲ gene was also cloned and expressed in the methylotropic yeast Pichia pastoris pPIC9K expression vector in the first time. The yield of the secretion level was 9.5 mg/L. Using straightforward one-step chromatography procedure, the rCTXⅢ, with three additional amino acids (GYT) at the N-terminal site, was purified to a purity of more than 90% and recovery yield of 65%. The purified rCTX Ⅲ was further characterized by cytotoxic assay with IC50 4.66μg/ml. An effective expression and purification system for recombinant CTXs in P. pastoris was developed, this system will permit us the ready isolation of active cardiotoxins. This protocol can also be easily used for the production of the toxin in a larger scale with low cost.
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A novel fibrinogenolytic protease,named atrase A,has been purified from the venom of Naja atra by sequential chromatography.Atrase A is a single chain glycoprotein with a molecular weight of 64.6 kD,an isoelectric point of pH 9.6 and a neutral sugar content of 4.16%.Atrase A specifically and slowly degraded α-chain of fibrinogen.This fibrinogenolytic activity Was inhibited by chelating agents(EDTA,EGTA and 1,10-phenanthroline)and DTY,and partially inhibited by PMSF,but not by soybean trypsin inhibitor,indicating it is a metalloproteinase.Atrase A showed edema-inducing activity and bactericidal activity against Staphylococcusa aureus.Atrase A did not show cytotoxicity on A549 and K562 cells in MTT assay,but detached adherent A549 cells from the substrate.Atrase A did not show significant inhibition of platelet aggregation induced by ADP or collagen,and did not exhibit proteolytic activities towards fibrin,azocasein and BAEE.It also did not show hemorrhage activity when injected subcutaneously into mice.
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Cardiotoxin III (CTX III), a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, has been reported to have anticancer activity. CTX III-induced K562 cell apoptosis was confirmed by DNA fragmentation (DNA ladder, sub-G1 formation) and phosphatidylserine (PS) externalization with an IC50 value of 1.7 mug/ml at 48 h. A mechanistic analysis demonstrated that CTX III-induced apoptotic cell death was accompanied by up-regulation of both Bax and endonuclease G (Endo G), and downregulation of Bcl-X(L). CTX III had no effect on the levels of Bcl-2, Bid, XIAP survivin, and AIF proteins. CTX III treatment caused loss of the mitochondrial membrane potential (delta psi m), release of mitochondrial cytochrome c to the cytosol, and activation of both caspase-9 and -3. CTX III-induced apoptosis was significantly blocked by the broad-spectrum caspase inhibitor Z-VAD-FMK. However, CTX III did not generate reactive oxygen species (ROS) and antioxidants, including N-acetylcysteine and catalase, did not block CTX III-induced apoptosis in K562 cells. Modulation of Bax, Bcl-X(L), and the Endo G proteins, release of mitochondrial cytochome c, and activation of caspase-3 and -9 all are involved in the CTX III-triggered apoptotic process in human leukemia K562 cells.
Subject(s)
Humans , bcl-X Protein/metabolism , bcl-2-Associated X Protein/metabolism , Up-Regulation/drug effects , Reactive Oxygen Species/metabolism , Mitochondrial Proteins/metabolism , Mitochondrial Membranes/drug effects , Membrane Potentials/drug effects , K562 Cells , Inhibitor of Apoptosis Proteins/metabolism , Endodeoxyribonucleases/metabolism , Down-Regulation/drug effects , Cobra Cardiotoxin Proteins/pharmacology , Cytochromes c/metabolism , Cell Proliferation/drug effects , Caspases/metabolism , Apoptosis/drug effectsABSTRACT
Aim To purify L-amino acid oxidase(LAAO) from the venom of Naja atra and study its effect on endothelial cells.Methods The NAV-LAAO was purified by ion-exchange chromatography and affinity chromatography.The MTT assay and Western blot were used to detect the viability and apoptosis of HUVEC.The tubule-forming was used to study the angiogenesis of cells.Results The NAV-LAAO was purified successfully from the venom of Naja atra.The molecular weight of NAV-LAAO was determined to be 58 ku by SDS-PAGE.NAV-LAAO effectively inhibited the growth and tubule-forming of HUVEC,and the 50% inhibitory concentration(IC50) was 21.42 mg?L-1.Compared with control,the levels of caspase-3 and caspase-8 increased in HUVEC treated with NAV-LAAO.Conclusions The NAV-LAAO is purified successfully from Naja atra venom by ion-exchange chromatography and affinity chromatography.The NAV-LAAO inhibits the growth and tubule-forming capacity of HUVEC in a dose-dependent manner.
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Objective To investigate the effects of CTX-d from venoms of cobra (Naja naja atra) on inducing NB4 apoptosis and its mechanism. Methods MTT was used to detect the antitumor effect of CTX-d in vitro; Electron microscope and flow cytometry were used to observe the apoptotic inducing effect of CTX-d in NB4 cells; Mitochondrial transmembrane potential change (??m) was analyzed by flow cytometry; The levels of caspase-9, caspase-3, and cytochrome C in the cytosol fraction were analyzed by Western blotting. Results The IC50 values of CTX-d affected on NB4 cell for 6 and 12 h were 1.8 and 1.35 ?g/mL, respectively. CTX-d could induce morphological changes, such as condensed chromatin and swelling mitochondria in NB4 cells. Analyzed by flow cytometry, CTX-d induced apoptosis in NB4 cells evidenced by increasing sub G1 cell population in a dose- and time-dependent manner. The mitochondrial membrane potential of NB4 cells had already decreased when incubated with CTX-d (1.0 ?g/mL) for 0.5 h, and cytochrome C in the cytosol was detected simultaneously, which indicated the release of cytochrome C from mitochondria to cytosol. The caspase-9 was activated initially at 1 h after 1.0 ?g/mL CTX-d treatment, whereas the cleavage of caspase-3 was detected at 0.5 h. This suggested that some other mechanism may be involved in caspase-3 activation. Conclusion The results suggest that the loss of mitochondrial membrane potential and the release of cytochrome C from the mitochondria into the cytosol are the early events of CTX-d on NB4 apoptosis. Once release into the cytosol, cytochrome C precedes the activation of caspase-9 and -3 to leading to the apoptosis and there are maybe some other mechanism involved in caspase-3 activation.
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AIM To purify cardiotoxin from Naja atra venom and investigate the relationship between cardiotoxicity of cardiotoxin and coronary artery spasm induced by cardiotoxin. METHODS Cardio toxin 13 (CTX 13) was fractionated and purified by chromatography and gel filtration from Chinese cobra (Naja atra) venom. The cardiotoxicity were observed in rat in situ, its isolated heart preparation and papillary muscle preparations. RESULTS Ion exchange chromatography of lyophilized cobra venom on SP Sephadex C 50 yielded 15 fractions, of thses fractions, cardiotoxic activities were found in fraction 11, 12, 13, and 14. Gel filtration and Ion chromatography of fraction 13 on Sephadex G 50 and SP Sephadex C 25 were performed consecutively and CTX 13 was obtained. It was homogeneous on polyacrylamide gel electrophoresis with MW= 7 769 ku, and 60 amino acid residues. The iv LD 50 in mice was 0 756 mg?kg -1 . CTX 13 increased the coronary resistance and reduced the contractility of rat Langendorff heart preparations. Systolic standstill finally occurred. When the heart preparations were pretreated with nitrendipine, an calcium channel blocker, the resistance seldom increased. The contractility slightly decreased at the beginning and then significantly increased. The tonus of contraction did not occurred. CTX 13 induced dose dependent contraction of pig coronary artery ring segments. Nitrendipine inhibited the action of CTX 13 on the coronary ring segments. However, nitrendipine had no effects on the action of CTX 13 in the rat papillary muscle preparations. The MLD of CTX 13 by venoclysis was changed from (444 7?28 5) ?g?kg -1 to (541 1?23 2) ?g?kg -1 in anaesthetized rats while the rats were pretreated with nitrendipine. CONCLUTION The coronary artery spasm may be one of the causes of death due to CTX 13.