RESUMO
BACKGROUND: Agkistrodon acutus, a traditional Chinese medicine, clinically used in the treatment of rheumatism, tumor, and cardiovascular and cerebrovascular diseases. Due to the unique medicinal value and the difficulty of artificial breeding of Agkistrodon acutus, the supply of Agkistrodon acutus on the market exceeds the demand, and a large number of its adulterants are found on the market. In this study, the cytb gene sequences of Agkistrodon acutus and 9 snakes were compared and analyzed, specific primers were designed, and specific PCR methods were established to detect Agkistrodon acutus medicinal samples on the market. RESULTS: This method was successfully applied to distinguish the snake from other adulterated species, and tested 18 Agkistrodon acutus samples randomly purchased from six cities. Twelve samples were counterfeit and six were genuine. The standard reference material of Agkistrodon acutus was cloned by molecular cloning and sequencing, and the gene sequence difference with other species was significant. It shows that the region could be used as the fingerprint region of the target species. CONCLUSIONS: The proposed method can be used as a species-specific marker and can be highly distinguished from other adulterated snake species, which is helpful to effectively avoid the problem of false sale of Agkistrodon acutus.
Assuntos
Animais , Reação em Cadeia da Polimerase/métodos , Agkistrodon/genética , Citocromos b/genética , Mitocôndrias/genética , Serpentes , Especificidade da Espécie , DNA/análise , Clonagem Molecular , Medicina Tradicional ChinesaRESUMO
Protein C activation initiated by the thrombin-thrombomodulin complex forms the major physiological anticoagulant pathway. Agkistrodon contortrix contortrix protein C activator, a glycosylated single-chain serine proteinase, activates protein C without relying on thrombomodulin. The crystal structures of native and inhibited Agkistrodon contortrix contortrix protein C activator determined at 1.65 and 1.54 A resolutions, respectively, indicate the pivotal roles played by the positively charged belt and the strategic positioning of the three carbohydrate moieties surrounding the catalytic site in protein C recognition, binding, and activation. Structural changes in the benzamidine-inhibited enzyme suggest a probable function in allosteric regulation for the anion-binding site located in the C-terminal extension, which is fully conserved in snake venom serine proteinases, that preferentially binds Cl(1-) instead of SO(4)(2-).
Assuntos
Ancrod/química , Ancrod/metabolismo , Venenos de Crotalídeos/química , Venenos de Crotalídeos/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Proteína C/metabolismo , Agkistrodon/genética , Agkistrodon/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Ancrod/antagonistas & inibidores , Ancrod/genética , Animais , Benzamidinas/farmacologia , Domínio Catalítico , Venenos de Crotalídeos/antagonistas & inibidores , Venenos de Crotalídeos/genética , Cristalografia por Raios X , Hemostasia , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intercelular , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/antagonistas & inibidores , Peptídeos/genética , Conformação Proteica , Homologia de Sequência de Aminoácidos , Inibidores de Serina Proteinase/farmacologia , Eletricidade Estática , Trombomodulina/metabolismoRESUMO
ACL myotoxin (ACLMT) is a K49 phospholipase A(2)-like protein isolated from the venom of the snake Agkistrodon contortrix laticinctus (broad-banded copperhead) that induces necrosis of skeletal muscle. We have previously cloned and sequenced the cDNA coding for ACLMT from a venom gland cDNA library. In order to perform structure and function studies, we have developed an expression system for production of ACLMT as a fusion protein with maltose binding protein (MBP) from the periplasm of bacteria, using the pMAL-p2 expression vector. The cDNA coding for the mature toxin without the signal peptide was amplified by PCR and subcloned into the pMAL-p2 vector. The new plasmid (pMAL-MT) was used to transform BL21(DE3) E. coli cells. Culture of transformed cells induced with IPTG led to the expression of a 60 kDa fusion protein which strongly reacts with anti-native ACLMT antibodies. The fusion protein was purified from the bacterial periplasm by affinity chromatography in an amylose column and by gel filtration. The purified fusion protein (MBP-rACLMT) was able to induce necrosis of skeletal muscle of mice very similar to that caused by the native myotoxin.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Agkistrodon/genética , Proteínas de Bactérias/genética , Venenos de Crotalídeos/enzimologia , Proteínas de Escherichia coli , Vetores Genéticos , Isopropiltiogalactosídeo/metabolismo , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Proteínas de Transporte de Monossacarídeos , Neurotoxinas/metabolismo , Fosfolipases A/genética , Fosfolipases A/toxicidade , Plasmídeos/genética , Proteínas Recombinantes de Fusão , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes/genética , Toxinas Biológicas/isolamento & purificação , Agkistrodon/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Transporte/química , Cromatografia de Afinidade , Cromatografia em Gel , Venenos de Crotalídeos/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Biblioteca Gênica , Injeções Intramusculares , Lisina/química , Maltose/isolamento & purificação , Proteínas Ligantes de Maltose , Proteínas de Membrana/química , Proteínas de Membrana/isolamento & purificação , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Necrose , Neurotoxinas/toxicidade , Fosfolipases A/isolamento & purificação , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Toxinas Biológicas/metabolismo , Toxinas Biológicas/toxicidade , Transformação GenéticaRESUMO
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.