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At least 19 recordsLinked to original sources
PubMed1993

[Isolation and characteristics of alpha-specific thrombin-like enzymes from venoms of the common pit viper (Agkistrodon halys halys) and the eastern pit viper (the central Asian subspecies Agkistrodon halys blomhoffii)].

Ancistron-H and Ancistron-B--two novel thrombin-like serine proteinases--have been purified 188- and 194-fold with a 95% recovery from the venoms of two Middle Asian subspecies of the pit viper--Agkistrodon halys halys and Agkistrodon halys Blomhoffii, using one-step affinity chromatography on agarose with an immobilized dye--Cibacron Blue F3GA (Blue-Sepharose 6B CL). The purified enzymes are one-chain glycoproteins with molecular masses of 34 and 29 kDa, pI of 6.6 and 6.3 and specific activities of 410 and 110 NIH units/mg protein, respectively. Their major amino acids are Gly, Val, Ser and Asp for Ancistron-H and Glu, Gly, Ser and Asp for Ancistron-B. During incubation with fibrinogen the enzymes cleave only the fibrinopeptide A from the A alpha-chain, leaving the B beta- and gamma-chains intact. Both enzymes hydrolyse arginine esters and thrombin-specific chromogenic peptide substrates, and display a weak caseinolytic activity but no fibrinolytic activity.

Agkistrodon↗
PubMed1990

HPLC-based two-step purification of fibrinolytic enzymes from the venom of Agkistrodon contortrix contortrix and Agkistrodon piscivorus conanti.

In investigations aimed at characterizing snake venom blood clot-dissolving enzymes, we have developed a rapid two-step high-performance chromatography method for the isolation of these fibrinolytic enzymes from the venoms of Agkistrodon contortrix contortrix and Agkistrodon piscivorus conanti. The first step consisted of hydrophobic interaction chromatography on a propyl-aspartamide column. Fractions containing the fibrinolytic activity were then concentrated and applied to a hydroxylapatite column. The resulting preparation, assessed for purity by reverse-phase chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was homogeneous. The molecular weight of both venom fibrinolytic enzymes was approximately 23,000 and amino acid analysis, immunological cross-reaction, cyanogen bromide, and tryptic digestion indicate a significant degree of structural similarity. However, the general proteolytic activity of the A. p. conanti venom enzyme was significantly lower than the corresponding activity of the A. c. contortrix venom, whereas their fibrinolytic activities were quite similar.

Amino Acids↗
PubMed1982

Thrombin-like and fibrinolytic enzymes in the venoms from the Gaboon viper (Bitis gabonica), eastern cottonmouth moccasin (Agkistrodon p. piscivorus) and southern copperhead (Agkistrodon c. contortrix) snakes.

Crude venom from B. gabonica contained weak fibrinogen clotting activity but no visible fibrinolytic activity, whereas venoms from A. p. piscivorus and A. c. contortrix exhibited fibrinolytic activity (by fibrin plate assay) but no thrombin-like activity. These snake venoms were fractionated on Sephadex G-100 with the following results. Thrombin-like activity in B. gabonica venom was eluted in a single protein peak with a molecular weight of 40,000. Agkistrodon p. piscivorus venom contained a single peak of fibrinolytic activity with a molecular weight of 34,000. Interestingly, venom from A. c. contortrix, which showed no thrombin-like activity in crude venom, contained both thrombin-like and fibrinolytic activities in fractions with molecular weights of 73,000 and 25,000 respectively. No plasminogen activation activity was observed in any of the crude venoms or venom fractions eluted from G-100. In view of the possible clinical potential of these enzymes as defibrinogenating or thrombolytic agents, it will be of great interest to further purify and characterize them.

Animals↗
PubMed1984

Classification of Agkistrodon species in China.

The wide geographical distribution of Agkistrodon and the slight morphological differences among the snakes of the genus Agkistrodon in China have posed a problem to taxonomists. We have employed polyacrylamide gel electrophoresis and immunological diffusion techniques for comparison of the venoms of different species and subspecies of Agkistrodon from various localities. The electrophoretic patterns of the proteins of the venoms were different from each other, but showed certain relations within species and subspecies. We used Ouchterlony double diffusion of a rabbit antiserum against the purified "neurotoxin" from the venom of Agkistrodon blomhoffii brevicaudaus (from the Zhejiang Province of China) on the various venoms of Agkistrodon. Precipitin lines formed with immunological identity between the same species, partial identity between closely related species and no precipitin line between different species. Combining experimental data, morphological characteristics and geographical distribution, we propose that the genus Agkistrodon (sensu stricto) in China consists of seven species and subspecies: (1) Agkistrodon blomhoffii brevicaudus Stejneger, (2) A. b ussuriensis Emelianov, (3) A. intermedius (Strauch), (4) A. saxatilis Emelianov, (5) A. shedaoensis Zhao, (6) A. strauchii Bedriaga, (7) A. monticola Werner. Agkistrodon acutus (Guenther) has recently been changed to a new genus, Deinagkitrodon, established by Gloyd in 1978.

Animals↗
PubMed1993

Cross-reactivities of monoclonal antibodies to a myotoxin from the venom of the broad-banded copperhead (Agkistrodon contortrix laticinctus).

A panel of murine monoclonal antibodies (MAbs) specific for a 14,500 mol. wt myotoxin of Agkistrodon contortrix laticinctus (broad-banded copperhead) venom was produced by cell fusion using the purified toxin as immunogen. The MAbs were used in immunoblotting and ELISA experiments to determine their immunological cross-reactivities. The three MAbs used in this study showed different immunoblotting and ELISA patterns when they were tested against 43 related and unrelated venom samples, indicating that they are specific for three different epitopes. MAb-42a specifically reacted with 50% of Agkistrodon venoms tested without cross-reacting with venoms of closely related species. This MAb reacted with venoms collected from Agkistrodon specimens from Florida, Kansas and Texas, but did not react with those from Louisiana, South Carolina, Ohio, New York or Mexico. MAb-6a showed similar reactivity but did react with venoms of snakes from Mexico. These results clearly indicate a geographical variation of the epitopes present in the myotoxin. Another antibody, MAb-18a, recognized an epitope present in the 14,500 mol. wt protein of all Agkistrodon venoms tested, but also reacted weakly with non-14,500 proteins from some Crotalus and Bothrops venoms tested. Both MAb-6a and MAb-18a neutralized the toxin-induced myonecrosis, but MAb-42a did not. Based on these results, the 14,500 mol. wt protein could serve as a general marker for venoms from snakes in the genus Agkistrodon found in North America.

Animals↗
PubMed1994

Development of an enzyme-linked immunosorbent assay (ELISA) for identification of venoms from snakes in the Agkistrodon genus.

An enzyme-linked immunosorbent assay (ELISA) using a purified myotoxin from the venom of Agkistrodon contortrix laticintus (broad-banded copperhead) as immunogen was developed for potential use in the identification of envenomation by snakes belonging to the genus Agkistrodon native to North America. The specificity of the assay was tested using a total of 43 venom samples from snakes of diverse geographic locations. Venom samples used for cross-reactivity determination represent eight snake families including 14 species from the genus Crotalus. The assay detected venom from all Agkistrodon species tested without significant cross-reactivity with other venoms except for samples from two species of Bothrops which do not occur naturally north of Southern Mexico. The detection limit of the assay was 2 ng/ml for homologous crude venom dissolved in normal human serum. The assay was highly accurate in correlating optical densities with venom concentrations (r = 0.997). The presence of the antigen in experimental envenomations was readily detected by the assay at an i.m. injection dosage of 0.1 microgram/g. This ELISA is a promising test for identification of envenomations by species of Agkistrodon found in most of North America. It can also be used to study the kinetics of the myotoxin in experimental envenomations.

Agkistrodon↗
PubMed2004

[Purification and characterization of platelet aggregation inhibitor component from venom of agkistrodon halys pallas].

Snake venom proteins,particularly from the viper and elapid families, have been known to contain a number of platelet active components including what cause platelet aggregation or inhibit platelet aggregation. Some of them have potential clinical usefulness for the treatment of human hemorrhagic or thrombotic disease. Agkistrodon halys pallas belonging to viper family is only growing in China. The aim of this study was to purify a human platelet aggregation inhibitor from venom of Agkistrodon halys pallas and determine its biochemical character. Whether a component could inhibit human platelet aggregation was act as a method to follow the tracks of the protein. Crude venom of Agkistrodon halys pallas was loaded onto a DEAE-Sepharose CL-6B chromatography column could gain 6 peaks. A platelet inhibitor with molecular mass of 65 kD on SDS-PAGE, was purified from peak 2 by Sephadex G-75 gel filtration and SP-Sepharose, Mono Q on FPLC. It could inhibit human platelet aggregation induced by ADP, collagen without activities of phospholipase A2, esterase, fibrinogenolytic. It is concluded that a platelet inhibitor can be isolated and purified from venom of Agkistrodon halys pallas and its inhibition of platelet aggregation is does-dependent.

Crotalid Venoms↗
PubMed2006

A catalog for transcripts in the venom gland of the Agkistrodon acutus: identification of the toxins potentially involved in coagulopathy.

Agkistrodon acutus is a special agkistrodon halys, only distributed in Southern China, with a few exceptions in Vietnam. It is a cherished element used in traditional Chinese medicine. In order to produce a global panorama of gene expression in the Agkistrodon acutus venom gland, a non-normalized cDNA library was constructed, and 8696 high quality 5' end expressed sequenced tags (ESTs) were sequenced and analyzed. The initial sequences were assembled into 2855 clusters. Of these clusters, only 45.60% clusters matched known sequence and 54.40% had no match to any known sequence in GenBank. Except for putative cellular proteins (1184 clusters), the remaining 118 clusters (40.16% of all ESTs) corresponded to sequences associated with diverse toxin function. According to expression abundance, the major toxin components were metalloproteinases (32.08%) and C-type lectin (5.22%), and other components including bradykinin-potentiating peptide (0.90%), serine proteases (0.51%), nucleotidase and nuclease (0.41%), phospholipase A2 (0.30%), disintegrin (0.05%), cytokine-like molecules (0.06%), and other proteins (0.63%). The majority of these components are thought to be responsible for coagulopathy after A. acutus bites. We have therefore generated a comprehensive catalog of the A. acutus venom gland described so far. Gene expression from the very specialized secretory tissue, especially for those involved in coagulopathy, can be surveyed and provide important information in finding novel toxins.

Agkistrodon↗
PubMed1998

The first discovery of larval Gnathostoma hispidum (Nematoda: Gnathostomidae) from a snake host, Agkistrodon brevicaudus.

The present study was performed to observe the infection status of several kind of animals with indigenous Gnathostoma in Korea, and morphological characteristics of gnathostome larvae detected from pit-viper, Agkistrodon brevicaudus, for the species identification. To know the existence of Gnathostoma in Korea, 3,450 loaches, 24 bullfrogs, several kinds of snakes, i.e., 55 Elaphe rufodorsata, 2 Dinodon rufozonatum rufozonatum, 62 Rhabdophis tigrinus tigrinus and 87 Agkistrodon spp., and 438 cats were examined. A total of 21 larval gnathostomes was detected from 12 pit-vipers, A. brevicaudus. They were 2,233 x 0.343 mm in average size and covered with about 210 transverse rows of minute cuticular spines. Their characteristic head bulbs were provided with 4 rows of hooklets of which average numbers in each row were 36.8, 39.0, 41.7 and 44.3, posteriorly. In the cross sections of midgut level, the intestinal wall consisted of a single layer of 19-25 elongate epithelial cells with a single nucleus. SEM observation of the larvae revealed unique features of head bulb, cuticular spines on transverse striations and a cervical papilla. On the basis of above morphological characteristics, they were identified as the advanced third-stage larvae of Gnathostoma hispidum. It was first confirmed that the pit-viper, Agkistrodon brevicaudus is the snake intermediate host of G. hispidum.

Agkistrodon↗
PubMed1999

Expression and Biochemical Characterization of Acidic Phospholipase A(2)I from Agkistrodon acutus.

A cDNA encoding acidic phospholipase A(2)I(A.aAPLA(2)I)from Agkistrodon acutus was inserted into a bacterial expression vector and effectively expressed in E.coli RR1. The protein was produced as insoluble inclusion bodies. After partial purification by washing the inclusion bodies with Triton X-100, denaturing and refolding, the renatured recombinant protein was purified by FPLC column Superose(TM)12. The enzymatic acti-vity and platelet aggregation inhibiting effect of the expressed A.aAPLA(2)I is close to those of denatured-refolded native acidic PLA(2) from Agkistrodon halys Pallas, and has the same hemolytic activity as denatured-refolded basic phospholipase A(2) from Agkistrodon halys Pallas. The roles of various amino acid residues in the enzymatic activity and pharmacological activities of phospholipase A2 are discussed.

Journal Article↗
PubMed1999

Cloning and Sequencing of Genes Encoding Phospholipase A(2) from Agkistrodon acutus.

Synthetic oligonucleotides were used to amplify phospholipase A(2) (PLA(2)) gene by RT-PCR from total RNA of snake Agkistrodon acutus venom gland. The PCR products were subcloned and positive clones were screened with acidic PLA(2) gene from Agkistrodon halys Pallas. Finally, four cDNAs of PLA(2) isoenzymes were isolated. Their complete sequences were determined by bidirectional sequencing and their amino acid sequences were deduced. They were designated as A.aAPLA(2)I A.aAPLA(2)II A.aBPLA(2) and A.aLys(49)-PLA(2) according to their isoelectric points calculated by computer and special structure characteristics respectively. The amino acid sequence of 1 10 residues of A.aAPLA(2)I deduced from the cDNA is identical to that of acidic PLA(2) which had been isolated from Agkistrodon acutus. A.aLys(49)-PLA(2) is unique because of the usual Asp(49) is replaced by Lys(49), which may lower its enzymatic activity. Their similarity scores were calculated and compared by computer. The successful cloning of these isoenzymes genes may provide more information for the study on structure-function relationship of PLA(2) family.

Journal Article↗
PubMed2005

Crystallization and preliminary X-ray crystallographic studies of Protac, a commercial protein C activator isolated from Agkistrodon contortrix contortrix venom.

The protein C pathway plays an important role in the control and regulation of the blood coagulation cascade and prevents the propagation of the clotting process on the endothelium surface. In physiological systems, protein C activation is catalyzed by thrombin, which requires thrombomodulin as a cofactor. The protein C activator from Agkistrodon contortrix contortrix acts directly on the zymogen of protein C converting it into the active form, independently of thrombomodulin. Suitable crystals of the protein C activator from Agkistrodon contortrix contortrix were obtained from a solution containing 2 M ammonium sulfate as the precipitant and these crystals diffracted to 1.95 A resolution at a synchrotron beamline. The crystalline array belongs to the monoclinic space group C2 with unit cell dimensions a=80.4, b=63.3 and c=48.2 A, alpha=gamma=90.0 degrees and beta=90.8 degrees.

Agkistrodon↗
PubMed2004

Effect of a recombinant Lys49PLA2 myotoxin and Lys49PLA2-derived synthetic peptides from Agkistrodon species on membrane permeability to water.

The aim of this work was to study the effect of recombinant ACL myotoxin, a Lys49PLA2 from Agkistrodon contortrix laticinctus snake venom and Lys49PLA2-derived synthetic peptides corresponding to the region 115-129 of venom of the two different Agkistrodon species on water permeability in the toad urinary bladder. The water flow through the membrane was measured gravimetrically in bag preparations of the bladder. The addition of recombinant ACL myotoxin-MBP (maltose binding protein) fusion protein (10 nM) to the bathing solution significantly increased (above 60%) the water transport compared with the control hemibladders. The addition of the Lys49PLA2-derived synthetic peptides in several concentrations to the bathing solution did not affect the water transport across membrane. These results suggest that the ACL myotoxin effect on water transport is not related to the cytotoxic C-terminal region.

Agkistrodon↗
PubMed2003

Structures of cadmium-binding acidic phospholipase A2 from the venom of Agkistrodon halys Pallas at 1.9A resolution.

Phospholipase A(2) coordinates Ca(2+) ion through three carbonyl oxygen atoms of residues 28, 30, and 32, two carboxyl oxygen atoms of residue Asp49, and two (or one) water molecules, forming seven (or six) coordinate geometry of Ca(2+) ligands. Two crystal structures of cadmium-binding acidic phospholipase A(2) from the venom of Agkistrodon halys Pallas (i.e., Agkistrodon blomhoffii brevicaudus) at different pH values (5.9 and 7.4) were determined to 1.9A resolution by the isomorphous difference Fourier method. The well-refined structures revealed that a Cd(2+) ion occupied the position expected for a Ca(2+) ion, and that the substitution of Cd(2+) for Ca(2+) resulted in detectable changes in the metal-binding region: one of the carboxyl oxygen atoms from residue Asp49 was farther from the metal ion while the other one was closer and there were no water molecules coordinating to the metal ion. Thus the Cd(2+)-binding region appears to have four coordinating oxygen ligands. The cadmium binding to the enzyme induced no other significant conformational change in the enzyme molecule elsewhere. The mechanism for divalent cadmium cation to support substrate binding but not catalysis is discussed.

Agkistrodon↗
PubMed2001

Purification and characterization of calobin II, a second type of thrombin-like enzyme from Agkistrodon caliginosus (Korean viper).

In our previous report, we purified and cloned the gene of a thrombin-like enzyme, calobin, from the venom of Agkistrodon caliginosus (Hahn, B.S., Yang, K.Y., Park, E.M., Chang, I.M., Kim, Y. S., 1996. Purification and molecular cloning of calobin, a thrombin-like enzyme from Agkistrodon caliginosus (Korean viper). J. Biochem. 119, 835-843.). During the purification of calobin, a second type of thrombin-like protease was found and it was purified using Affi-Gel Blue and Mono-S cation-exchange chromatography. It was identified as a serine protease with a molecular weight of 41, 000 on SDS-PAGE and its isoelectric point was determined to be 7.4 by isoelectric focusing. It showed little azocaseinolytic and fibrinolytic activity. However, this enzyme acted on fibrinogen to form fibrin with a specific activity of 7,587 NIH equivalent units and also exhibited arginine esterase activity. Amino acid sequencing of the N-terminal region established a primary structure composed of Val-Ile-Gly-Gly-Asp-Glu-Cys-Asn-Ile-Asn-Glu-His-Arg-Phe-Leu-Val-Ala-X -Tyr. This sequence was entirely consistent with that of calobin and showed a high homology with other thrombin-like enzymes, such as ancrod, batroxobin and gyroxin. Based on the biochemical properties such as molecular weight and isoelectric point, we can demonstrate a second thrombin-like protein showing a high potent clotting activity from the venom of Korean viper.

Agkistrodon↗
PubMed2004

[Exotic snakes in Europe. A case of Mexican Moccasin (Agkistrodon bilineatus) snakebite].

INTRODUCTION: In the last years exotic snakebite envenomations are increasingly reported. These cases are difficult to manage because of the limited experience of European physicians in the treatment of bites from such venomous snakes; moreover, specific antivenoms are unevenly stocked and they are difficult to find in case of a medical emergency. OBSERVATION: A 39-year-old herpetologist was bitten in his right hand by a mexican moccasin (Agkistrodon bilineatus) at the workplace, and presented in the Emergency Department 19 hours later. At admission, clinical evaluation showed local necrosis, swelling involving the entire limb up to the trunk, and severe pain. The specific antidote, not stocked in Italy, was sought abroad; its finding and routeing up to spot delivery required 12 hours. The antivenom, given 32 hours after the bite with no adverse reactions, was only partially effective. The clinical course was characterized by extensive edema with rhabdomyolysis. The necrotic wound at the bite site required after several days surgical debridement, and eventually skin graft. At 3 months follow-up, motor impairment of his right hand fingers with functional disability was still present. COMMENTS: The envenomation by Agkistrodon bilineatus has some clinical aspects in common with that by European viper species, although crotalid venom usually causes more severe manifestations. The antivenom supply from a foreign country may delay its administration. A specific legislation aimed to simplify antidotes importing procedures for professional snake handlers may improve antivenoms availability and allow their timely use, as soon as clinically indicated.

Adult↗
PubMed2006

[Three-step column chromatographic method for separation and purification of thrombin-like enzyme from venom of Agkistrodon halys pallas].

A three-step column chromatographic method, utilizing CM Sepharose CL-6B, DEAE Sepharose Fast Flow and Sephadex G-75 in sequence, for the separation and purification of thrombin-like enzyme from crude venom of Agkistrodon halys pallas was developed. Based on the separation results on lab-scale chromatographic column, this isolation and purification process was amplified according to the diameter of the chromatographic columns used in labscale. It was found that when the amount of the sample loaded on the large-scale columns was 25 times as that loaded on the lab-scale columns, the quality level of the thrombin-like enzyme obtained with the large-scale columns was almost the same as that obtained with lab-scale columns. The relative molecular mass of thrombin-like enzyme obtained was about 33500, and its purity was about 96%. When using this method to isolate thrombin-like enzyme from crude venom of Agkistrodon halys pallas, the total protein yield and total activity yield of thrombin-like enzyme were about 0.3% and 64% respectively, and specific activity was more than 2000 U/mg.

Agkistrodon↗
PubMed2000

Phylogeography of the pitviper clade Agkistrodon: historical ecology, species status, and conservation of cantils.

We used mitochondrial DNA sequences from three gene regions and two tRNAs (ND4, tRNA-HIS-SER, 12S, and 16S rDNA) to investigate the historical ecology of the New World pitviper clade Agkistrodon, with emphasis on the disjunct subspecies of the cantil, A. bilineatus. We found strong evidence that the copperhead (A. contortrix) is basal to its congeners, and that the cottonmouth (A. piscivorus) is basal to cantils. Phylogeography and natural history of the living terminal taxa imply that Agkistrodon primitively occupied relatively temperate habitats, with subsequent evolution of tropicality in ancestral A. bilineatus. Our best supported phylogeny rejects three gulf arc scenarios for the biogeography of A. bilineatus. We find significant statistical support for an initial divergence between populations on the east and west coasts of México and subsequent occupancy of the Yucatán Peninsula, by way of subhumid corridors in northern Central America. Based on phylogenetic relationships, morphological and molecular divergence, and allopatry we elevate A. b. taylori of northeastern México to species status. Taylor's cantil is likely threatened by habitat destruction and small geographical range, and we offer recommendations for its conservation and management.

Agkistrodon↗