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Sample records for agkistrodon piscivorus leucostoma

  1. Histological analysis of spermatogenesis and the germ cell development strategy within the testis of the male Western Cottonmouth Snake, Agkistrodon piscivorus leucostoma.

    PubMed

    Gribbins, Kevin M; Rheubert, Justin L; Collier, Matthew H; Siegel, Dustin S; Sever, David M

    2008-11-20

    Cottonmouth (Agkistrodon piscivorus leucostoma) testes were examined histologically to determine the germ cell development strategy employed during spermatogenesis. Testicular tissues from Cottonmouths were collected monthly from swamps around Hammond, Louisiana. Pieces of testis were fixed in Trump's fixative, dehydrated in ethanol, embedded in Spurr's plastic, sectioned with an ultramicrotome, and stained with toluidine blue and basic fuchsin. Spermatogenesis within Cottonmouths occurs in two independent events within a single calendar year. The testes are active during the months of March-June and August-October with spermiation most heavily observed during April-May and October. To our knowledge, this is the first study that describes bimodal spermatogenesis occurring in the same year within the subfamily Crotalinae. During spermatogenesis, no consistent spatial relationships are observed between germ cell generations. Typically, either certain cell types were missing (spermatocytes) or the layering of 3-5 spermatids and/or spermatocytes within the same cross-section of seminiferous tubule prevented consistent spatial stages from occurring. This temporal pattern of sperm development is different from the spatial development found within birds and mammals, being more reminiscent of that seen in amphibians, and has now been documented within every major clade of reptile (Chelonia, Serpentes, Sauria, Crocodylia). This primitive-like sperm development, within a testis structurally similar to mammals and birds, may represent an intermediate testicular model within the basally positioned (phylogenetically) reptiles that may be evolutionarily significant.

  2. Exploring and understanding the functional role, and biochemical and structural characteristics of an acidic phospholipase A2, AplTx-I, purified from Agkistrodon piscivorus leucostoma snake venom.

    PubMed

    Resende, L M; Almeida, J R; Schezaro-Ramos, R; Collaço, R C O; Simioni, L R; Ramírez, D; González, W; Soares, A M; Calderon, L A; Marangoni, S; da Silva, S L

    2017-03-01

    Phospholipases A2 (PLA2s) constitute a class of extensively studied toxins, isolated from snake venoms. Basic PLA2 isoforms mediate various toxicological effects, while the acidic isoforms generally have higher enzymatic activities, but do not promote evident toxic effects. The functions of these acidic isoforms in snake venoms are still not completely understood and more studies are needed to characterize the biological functions and diversification of acidic toxins in order to justify their abundant presence in these secretions. Recently, Lomonte and collaborators demonstrated, in a proteomic and toxicological study, high concentrations of PLA2s in the venom of Agkistrodon piscivorus leucostoma. We have, herein, purified and characterized an acidic PLA2 from this snake venom, denominated AplTx-I, in order to better understand its biochemical and structural characteristics, as well as its biological effects. AplTx-I was purified using two chromatographic steps, in association with enzymatic and biological assays. The acidic toxin was found to be one of the most abundant proteins in the venom of A. p. leucostoma; the protein was monomeric with a molecular mass of 13,885.8 Da, as identified by mass spectrometry ESI-TOF and electrophoresis. The toxin has similar primary and tridimensional structures to those of other acidic PLA2s, a theoretical and experimental isoelectric point of ≈5.12, and a calcium-dependent enzyme activity of 25.8985 nM/min/mg, with maximum values at 37 °C and pH 8.0. Despite its high enzymatic activity on synthetic substrate, AplTx-I did not induce high or significant myotoxic, coagulant, anticoagulant, edema, neuromuscular toxicity in mouse phrenic nerve-diaphragm preparations or antibacterial activities. Interestingly, AplTx-I triggered a high and selective neuromuscular toxicity in chick biventer cervicis preparations. These findings are relevant to provide a deeper understanding of the pharmacology, role and diversification of acidic

  3. Agkistrodon piscivorus conanti (Florida cottonmouth) Diet

    USGS Publications Warehouse

    Grajal-Puche, Alejandro; Josimovich, Jillian; Falk, Bryan; Reed, Robert

    2016-01-01

    Agkistrodon piscivorus is a generalist predator that feeds on a variety of prey, including snakes (Gloyd and Conant 1990. Snakes of the Agkistrodon Complex: A Monographic Review. Society for the Study of Amphibians and Reptiles, Oxford, Ohio. 614 pp.; Lillywhite et al. 2002. Herpetol. Rev. 33:259–260; Hill and Beaupre 2008. Copeia 2008:105–114). Cemophora coccinea (Scarletsnake) is not known as one of the 26 species of snakes consumed by A. piscivorus (Ernst and Ernst 2011. Venomous Reptiles of the United States, Canada, and Northern Mexico: Volume 1. Johns Hopkins University Press, Baltimore, Maryland. 193 pp.). On 16 June 2015, at 2210 h, we found a dead-on-road A. piscivorus (total length [TL] = 51.0 cm) in Everglades National Park on Main Park Road, 1.88 km S Pa-hay-okee, Miami-Dade Co., Florida, USA (25.414085°N, 80.78183146°W, WGS84; elev. 3 m). The snake had been killed by a vehicle and some internal organs were exposed. Visible stomach contents included a small (TL ca. 15 cm) C. coccinea. Photographic vouchers of the A. piscivorus (UF-Herpetology 177194) and C. coccinea (UF-Herpetology 177195) were deposited in the Division of Herpetology, Florida Museum of Natural History, University of Florida. Despite the fact that these species are sympatric over large areas of the southeastern United States, this is the first known documented predation of C. coccinea by A. piscivorus.

  4. Innate immune performance and steroid hormone profiles of pregnant versus nonpregnant cottonmouth snakes (Agkistrodon piscivorus).

    PubMed

    Graham, Sean P; Earley, Ryan L; Guyer, Craig; Mendonça, Mary T

    2011-12-01

    Squamates (lizards and snakes) have independently evolved viviparity over 100 times, and exhibit a wide range of maternal investment in developing embryos from the extremes of lecithotrophic oviparity to matrotrophic viviparity. This group therefore provides excellent comparative opportunities for studying endocrine and immune involvement during pregnancy, and their possible interactions. We studied the cottonmouth (Agkistrodon piscivorus), since they exhibit limited placentation (e.g., ovoviviparity), allowing comparison with squamate species hypothesized to require considerable maternal immune modulation due to the presence of a more extensive placental connection. Furthermore, the cottonmouth's biennial reproductive cycle provides an opportunity for simultaneously comparing pregnant and non-pregnant females in the wild. We document significantly elevated concentrations of progesterone (P4) and significantly lower concentrations of estradiol (E2) in pregnant females relative to non-pregnant females. Pregnant females had lower plasma bacteria lysis capacity relative to non-pregnant females. This functional measure of innate immunity is a proxy for complement performance, and we also determined significant correlations between P4 and decreased complement performance in pregnant females. These findings are consistent with studies that have determined P4's role in complement modulation during pregnancy in mammals, and thus this study joins a growing number of studies that have demonstrated convergent and/or conserved physiological mechanisms regulating viviparous reproduction in vertebrates.

  5. Do warning displays predict striking behavior in a viperid snake, the cottonmouth (Agkistrodon piscivorus)?

    SciTech Connect

    Glaudas, X. and C.T. Winne

    2007-01-01

    Warning displays are defined as signals designed to intimidate predators or indicate a proclivity to fight. However, support for the idea that warning behaviors signal an intent to fight is largely based on anecdotes and isolated observations, and a complete understanding of antipredator behavior will only be achieved if specific hypotheses are experimentally tested. Herein, we tested in a North American viperid snake, the cottonmouth (Agkistrodon piscivorus), the hypothesis that warning displays serve as a reliable signal to potential predators that a snake will strike. The cottonmouth exhibits two stereotypical warning displays during predator confrontation, i.e., mouth gaping and tail vibrations, making it an ideal study organism to experimentally test the relationship between warning displays and defensive striking. To test this idea, we recorded the sequence of defensive behavior--gaping, tail vibrating, and striking--of cottonmouths towards a standardized predatory stimulus in the laboratory. As predicted, snakes that gaped during the trials were subsequently more likely to strike than snakes that did not. In contrast, striking behavior was independent of the occurrence of tail vibrations. Our results suggest that gaping behavior--but not tail-vibrating behavior--may provide an honest signal to would-be predators.

  6. Ultrastructure of spermiogenesis in the Cottonmouth, Agkistrodon piscivorus (Squamata: Viperidae: Crotalinae).

    PubMed

    Gribbins, Kevin M; Rheubert, Justin L; Anzalone, Marla L; Siegel, Dustin S; Sever, David M

    2010-03-01

    To date multiple studies exist that examine the morphology of spermatozoa. However, there are limited numbers of data detailing the ontogenic characters of spermiogenesis within squamates. Testicular tissues were collected from Cottonmouths (Agkistrodon piscivorus) and tissues from spermiogenically active months were analyzed ultrastructurally to detail the cellular changes that occur during spermiogenesis. The major events of spermiogenesis (acrosome formation, nuclear elongation/DNA condensation, and flagellar development) resemble that of other squamates; however, specific ultrastructural differences can be observed between Cottonmouths and other squamates studied to date. During acrosome formation vesicles from the Golgi apparatus fuse at the apical surface of the nuclear membrane prior to making nuclear contact. At this stage, the acrosome granule can be observed in a centralized location within the vesicle. As elongation commences the acrosome complex becomes highly compartmentalized and migrates laterally along the nucleus. Parallel and circum-cylindrical microtubules (components of the manchette) are observed with parallel microtubules outnumbering the circum-cylindrical microtubules. Flagella, displaying the conserved 9 + 2 microtubule arrangement, sit in nuclear fossae that have electron lucent shoulders juxtaposed on either side of the spermatids basal plates. This study aims to provide developmental characters for squamates in the subfamily Crotalinae, family Viperidae, which may be useful for histopathological studies on spermatogenesis in semi-aquatic species exposed to pesticides. Furthermore, these data in the near future may provide morphological characters for spermiogenesis that can be added to morphological data matrices that may be used in phylogenetic analyses.

  7. The reproductive biology of male cottonmouths (Agkistrodon piscivorus): do plasma steroid hormones predict the mating season?

    PubMed

    Graham, Sean P; Earley, Ryan L; Hoss, Shannon K; Schuett, Gordon W; Grober, Matthew S

    2008-01-01

    To better understand the proximate causation of the two major types of mating seasons described for North American pitvipers, we conducted a field study of the cottonmouth (Agkistrodon piscivorus) in Georgia from September 2003 to May 2005 that included an extensive observational regime and collection of tissues for behavioral, anatomical, histological, and hormone analysis. Enzyme immunoassays (EIA) of plasma samples and standard histological procedures were conducted on reproductive tissues. Evidence from the annual testosterone (T) and sexual segment of the kidney (SSK) cycle and their relationship to the spermatogenic cycle provide correlative evidence of a unimodal mating pattern in this species of pitviper, as these variables consistently predict the mating season in all snake species previously examined under natural conditions. In most reptiles studied to date, high plasma levels of T and corticosterone (CORT) coincide during the mating period, making the cottonmouth an exception to this trend; we suggest two possible explanations for increased CORT during spring (regulation of a spring basking period), and decreased CORT during summer (avoiding reproductive behavioral inhibition), in this species.

  8. Stressed snakes strike first: Hormone levels and defensive behavior in free ranging cottonmouths (Agkistrodon piscivorus).

    PubMed

    Herr, Mark W; Graham, Sean P; Langkilde, Tracy

    2017-03-01

    Stress is believed to be an important factor mediating animal behavior. Here we explore the relationship between concentrations of a stress hormone and defensive behavior of a snake. The cottonmouth (Agkistrodon piscivorus) is an abundant, large-bodied pitviper that is well known for its intense defensive behaviors. The defensive behaviors and hormonal ecology of cottonmouths have been studied extensively, but the interaction between these is not well understood. We conducted field trials, recording the snake's behavior and obtaining blood samples to quantify plasma CORT concentrations, both upon first encountering a snake and after a 30min standardized confinement stressor. We found that snakes with elevated levels of baseline CORT at first encounter were more likely to strike than exhibit a threat display when approached in the field. However, this behavior was not related to the magnitude of the snake's CORT increase following confinement, suggesting that more stress-prone snakes are not more defensive. Post-stressor antipredator behavior was also not related to any of our CORT measures. This study suggests that baseline CORT levels can be important correlates of defensive behavior. If this is a causative relationship, environmental challenges that increase baseline stress levels of populations may elevate cottonmouth defensive behavior. This would increase costs associated with defensive behavior (energetic, lost opportunity, etc.) and have important consequences for animal-human interactions.

  9. Pharmacokinetics of nebulized and subcutaneously implanted terbinafine in cottonmouths (Agkistrodon piscivorus).

    PubMed

    Kane, L P; Allender, M C; Archer, G; Leister, K; Rzadkowska, M; Boers, K; Souza, M; Cox, S

    2017-04-06

    Ophidiomyces ophiodiicola, the causative agent of snake fungal disease, is proposed as a serious threat to the conservation of several snake populations. The objective of this study was to determine the pharmacokinetic parameters of terbinafine administered through nebulization and a sustained subcutaneous implant as potential treatments of Ophidiomyces in reptiles. Seven adult cottonmouths (Agkistrodon piscivorus) were used in single-dose trials. Each snake was nebulized with terbinafine (2 mg/ml) for 30 min and had blood collected before nebulization and up to 12 hr after nebulization. Following a 5-month washout, the same snakes were administered a subcutaneous implant containing 24.5 mg terbinafine; blood was collected at baseline, 1 day postimplant placement, and then once weekly for 9 weeks. Plasma for both studies was analyzed by high-performance liquid chromatography. The mean plasma concentrations of nebulized terbinafine peaked between 0.5 and 4 hr. The subcutaneously implanted terbinafine reached therapeutic concentrations on day 1 and maintained therapeutic for over 6 weeks. These methods and doses are recommended as potential treatment options for snake fungal disease in reptiles.

  10. Phylogenetic Analysis of Bacterial Communities in Different Regions of the Gastrointestinal Tract of Agkistrodon piscivorus, the Cottonmouth Snake

    PubMed Central

    Colston, Timothy J.; Noonan, Brice P.; Jackson, Colin R.

    2015-01-01

    Vertebrates are metagenomic organisms in that they are composed not only of their own genes but also those of their associated microbial cells. The majority of these associated microorganisms are found in the gastrointestinal tract (GIT) and presumably assist in processes such as energy and nutrient acquisition. Few studies have investigated the associated gut bacterial communities of non-mammalian vertebrates, and most rely on captive animals and/or fecal samples only. Here we investigate the gut bacterial community composition of a squamate reptile, the cottonmouth snake, Agkistrodon piscivorus through pyrosequencing of the bacterial 16S rRNA gene. We characterize the bacterial communities present in the small intestine, large intestine and cloaca. Many bacterial lineages present have been reported by other vertebrate gut community studies, but we also recovered unexpected bacteria that may be unique to squamate gut communities. Bacterial communities were not phylogenetically clustered according to GIT region, but there were statistically significant differences in community composition between regions. Additionally we demonstrate the utility of using cloacal swabs as a method for sampling snake gut bacterial communities. PMID:26039313

  11. Venomics of New World pit vipers: Genus-wide comparisons of venom proteomes across Agkistrodon

    PubMed Central

    Lomonte, Bruno; Tsai, Wan-Chih; Ureña-Diaz, Juan Manuel; Sanz, Libia; Mora-Obando, Diana; Sánchez, Elda E.; Fry, Bryan G.; Gutiérrez, José María; Gibbs, H. Lisle; Sovic, Michael G.; Calvete, Juan J.

    2015-01-01

    We report a genus-wide comparison of venom proteome variation across New World pit vipers in the genus Agkistrodon. Despite the wide variety of habitats occupied by this genus and that all its taxa feed on diverse species of vertebrates and invertebrate prey, the venom proteomes of copperheads, cottonmouths, and cantils are remarkably similar, both in the type and relative abundance of their different toxin families. The venoms from all the eleven species and subspecies sampled showed relatively similar proteolytic and PLA2 activities. In contrast, quantitative differences were observed in hemorrhagic and myotoxic activities in mice. The highest myotoxic activity was observed with the venoms of A. b. bilineatus, followed by A. p. piscivorus, whereas the venoms of A. c. contortrix and A. p. leucostoma induced the lowest myotoxic activity. The venoms of Agkistrodon bilineatus subspecies showed the highest hemorrhagic activity and A. c. contortrix the lowest. Compositional and toxicological analyses agree with clinical observations of envenomations by Agkistrodon in the USA and Central America. A comparative analysis of Agkistrodon shows that venom divergence tracks phylogeny of this genus to a greater extent than in Sistrurus rattlesnakes, suggesting that the distinct natural histories of Agkistrodon and Sistrurus clades may have played a key role in molding the patterns of evolution of their venom protein genes. Biological significance A deep understanding of the structural and functional profiles of venoms and of the principles governing the evolution of venomous systems is a goal of venomics. Isolated proteomics analyses have been conducted on venoms from many species of vipers and pit vipers. However, making sense of these large inventories of data requires the integration of this information across multiple species to identify evolutionary and ecological trends. Our genus-wide venomics study provides a comprehensive overview of the toxic arsenal across

  12. Management of Tissue Loss After Agkistrodon Snakebite: Appropriate Use of Crotalidae-Fab Antivenin.

    PubMed

    Larson, Kenneth W; Schaefer, Keith R; Austin, Cindy; Norton, Rhy; Finley, Phillip J

    2016-01-01

    Although initially created for the treatment of rattlesnake (genus: Crotalus) bites, Crotalidae-Fab antivenin is used to treat many different pit viper envenomations. However, the efficacy of Crotalidae-Fab in preventing tissue loss from copperhead (Agkistrodon contortrix) or cottonmouth (Agkistrodon piscivorus) snakebites remains unclear. Recent reports show that Agkistrodon-related bites rarely require treatment beyond simple observation and pain control. The purpose of this study was to examine the amount of tissue loss in patients who received Crotalidae-Fab compared with those who did not after an Agkistrodon bite. After institutional review board approval, a retrospective study was completed at a Level 1 trauma center. Between 2009 and 2013, a total of 57 snakebites were identified. Of the 57 bites, the snake species was documented in 36 cases including 31 copperheads, 1 cottonmouth, and 4 rattlesnakes. The other 21 bites were from unknown or nonvenomous species. Of the 32 Agkistrodon-related bites, 15 patients received Crotalidae-Fab (average of 3 vials administered) and 17 did not receive Crotalidae-Fab. None of the 32 patients, regardless of treatment option, had tissue loss or required surgical interventions. Only 1 patient received Crotalidae-Fab and debridement of a vesicle associated with the bite. No clinically significant differences were observed between the groups. These findings support previous literature that failed to show added benefit of Crotalidae-Fab treatment for Agkistrodon bites beyond patient comfort and pain control. Evaluation of current protocols for Agkistrodon envenomations is warranted. Snakebite wound education in trauma physicians and nurses may decrease unnecessary use of antivenom medication.

  13. Non-lethal sampling of liver tissue for toxicologic evaluation of Florida cottonmouths snakes, Agkistrodon piscivorus conanti.

    PubMed

    Quesada, Rolando J; McCleary, Ryan J R; Heard, Darryl J; Lillywhite, Harvey B

    2014-01-01

    Due to their longevity, strong site tenure, poikilothermic metabolism, and low-energy specializations, reptiles might serve as excellent environmental sentinels. Cottonmouth snakes are generalist predators and scavengers, and as such, may have higher exposure to persistent environmental contaminants as a result of bioaccumulation. Traditionally, assessment and monitoring of contaminant exposure in reptiles have involved lethal sampling techniques. In this paper, we describe a non-destructive technique for sampling liver tissue in live anesthetized Florida cottonmouths. Wild-caught snakes (n = 21) were anesthetized with propofol, and a liver wedge biopsy was obtained by clamping the edge of the organ with two small hemostatic mosquito forceps via right-sided coeliotomy incision. A minimum required tissue sample weighing >100 mg was harvested from all except one of the animals. No mortalities occurred during the procedures or recovery from anesthesia, and all snakes were released back into the field after the animal had consumed prey and defecated, usually within 2 weeks following surgery. Hemorrhage was a minor complication in most snakes, especially those with friable discolored livers. The procedure appeared to have no short-term deleterious effects, and two biopsied individuals were captured after being released into the field and appeared to be normal and healthy. However, follow-up studies and recapture of more snakes are needed to assess long-term survivability. Our non-destructive liver sampling technique might be implemented in toxicological studies of other squamates and could help to minimize the lethal sampling of threatened species.

  14. [Plasminogen activator from Agkistrodon halys halys venom].

    PubMed

    Karbovs'kyĭ, V L; Levkiv, M Iu; Savchuk, O M; Hornyts'ka, O V; Volkov, H L; Bukhan, Ts

    2006-01-01

    Plasminogen activator "Ahh-32" from Agkistrodon halys halys venom has been isolated and purified using affinity and ion-exchange chromatography. The purified enzyme consists of the single peptide-chain with molecular weigth of 32 kDa. It can convert free plasminogen into active form--plasmin. "Ahh-32" was inhibited by DFP and benzamidine. Besides, the enzyme influences significantly the activation of plasminogen by streptokinase without having effect on analogical process in case of usage of tissue tipe plasminogen activator. The obtained protein can be used as an instrument under investigation of protein-protein interactions in haemostasis system.

  15. Envenomation by a juvenile Malayan pit viper (Agkistrodon rhodostoma).

    PubMed

    Vest, D K; Kardong, K V

    1980-05-01

    Following an accidental bite inflicted by a juvenile Malayan pit viper (Agkistrodon rhodostoma), the progress of envenomation was carefully monitored and subsequent laboratory work performed to determine the amount and quality of venom injected. Even a very small amount of venom from this species is capable of inducing noticeable local symptoms including edema, subcutaneous bleeding, and throbbing. Constitutional symptoms were present but minimal. The extent of inflammation present at any given time following envenomation was found to be a more accurate diagnostic signal than the speed with which it developed. This case is similar to that of bites by juveniles of other species of viperines and crotalines and may serve to aid in the diagnosis and treatment of bites by juvenile specimens of serpents in these families.

  16. [Extraction, purification and identification of type II collagen from Agkistrodon acutus].

    PubMed

    Gu, Heng-Cun; Hu, Jin-Bo; Ding, Zhi-Shan; Fan, Yong-Sheng; Ding, Xing-Hong

    2013-11-01

    The object of the research was to extract, purify and identify the type II collagen of Agkistrodon acutus. Type II collagen of A. acutus was extracted by enzyme decomposition method, and purified by ion exchange column chromatography. It was characterized by SDS-PAGE gel electrophoresis, ultraviolet spectrophotometry, infrared absorption spectroscopy and mass spectroscopy. The results showed that the size of C II was about 130 kDa. It absorbed at 223 nm. IR spectrum obtained showed that the triple helical domains of amino-acid sequences were characterized by the repetition of triplets Gly-X-Y. The MS spectrum graphically stated that C II extracted from cow and A. acutus have the similar peptides. The C II of A. acutus was obtained by extraction and purification. Appraisal analysis by SDS-PAGE, UV, IR and MS, C II of A. acutus was consistent with the standard C II of cow. It was proved that the extracted protein was C II.

  17. Ribosomal DNA sequence divergence and group I introns within the Leucostoma species L. cinctum, L. persoonii, and L. parapersoonii sp. nov., ascomycetes that cause Cytospora canker of fruit trees.

    PubMed

    Adams, Gerard C; Surve-Iyer, Rupa S; Iezzoni, Amy F

    2002-01-01

    Leucostoma species that are the causal agents of Cytospora canker of stone and pome fruit trees were studied in detail. DNA sequence of the internal transcribed spacer regions and the 5.8S of the nuclear ribosomal DNA operon (ITS rDNA) supplied sufficient characters to assess the phylogenetic relationships among species of Leucostoma, Valsa, Valsella, and related anamorphs in Cytospora. Parsimony analysis of the aligned sequence divided Cytospora isolates from fruit trees into clades that generally agreed with the morphological species concepts, and with some of the phenetic groupings (PG 1-6) identified previously by isozyme analysis and cultural characteristics. Phylogenetic analysis inferred that isolates of L. persoonii formed two well-resolved clades distinct from isolates of L. cinctum. Phylogenetic analysis of the ITS rDNA, isozyme analysis, and cultural characteristics supported the inference that L. persoonii groups PG 2 and PG 3 were populations of a new species apparently more genetically different from L. persoonii PG 1 than from isolates representative of L. massariana, L. niveum, L. translucens, and Valsella melastoma. The new species, L. parapersoonii, was described. A diverse collection of isolates of L. cinctum, L. persoonii, and L. parapersoonii were examined for genetic variation using restriction fragment length polymorphism (RFLP) analysis of the ITS rDNA and the five prime end of the large subunit of the rDNA (LSU rDNA). HinfI and HpaII endonucleases were each useful in dividing the Leucostoma isolates into RFLP profiles corresponding to the isozyme phenetic groups, PG 1-6. RFLP analysis was more effective than isozyme analysis in uncovering variation among isolates of L. persoonii PG 1, but less effective within L. cinctum populations. Isolates representative of seven of the L. persoonii formae speciales proposed by G. Défago in 1935 were found to be genetically diverse isolates of PG 1. Two large insertions, 415 and 309 nucleotides long, in

  18. A novel plasminogen activator from Agkistrodon blomhoffii Ussurensis venom (ABUSV-PA): purification and characterization.

    PubMed

    Liu, Shuqing; Sun, Ming-Zhong; Greenaway, Frederick T

    2006-10-06

    A plasminogen activator with arginine ester hydrolysis activity (ABUSV-PA) has been identified and purified to homogeneity from Chinese Agkistrodon blomhoffii Ussurensis snake venom. ABUSV-PA, a monomeric protein with molecular mass of 27815.2 Da, was purified 180-fold with 0.02% recovery for protein and 3.6% recovery for esterase activity. ABUSV-PA reacts optimally with its substrate N(alpha)-tosyl-l-arginine-methyl ester (TAME) at approximately pH 7.5 and at 51 degrees C. Measurement from inductively coupled plasma-atomic emission spectroscopy (ICP-AES) reveals that ABUSV-PA is a Zn(2+)-containing protein with a stoichiometry of 1:1 [Zn(2+)]:[ABUSV-PA]. Analyses of esterase hydrolysis and UV absorption and CD spectra indicate that Zn(2+) plays an important role in maintaining the structural integrity rather than the esterase activity of ABUSV-PA. Divalent metal ions, including Ca(2+), Mg(2+), Cu(2+), Ni(2+), Mn(2+), and Co(2+), increase the TAME hydrolysis activity of ABUSV-PA. A red-shift of the emission wavelengths of the synchronous fluorescence of ABUSV-PA, compared to those of free Tyr and Trp, indicates a conformation where the Tyr and Trp residues are in exposed hydrophilic environments. The presence of zinc increases the hydrophobicity of the conformational environments surrounding the Trp residues of ABUSV-PA and affects the secondary structure of ABUSV-PA, as proved by UV absorption and CD spectroscopy.

  19. Purification and characterization of a novel antinociceptive peptide from venom of Agkistrodon halys Pallas.

    PubMed

    Ye, Yong; Li, Mingyang; Chen, Xuelan

    2013-04-01

    Venom of Agkistrodon halys Pallas can control severe pain such as cancer pain and neuropathic pain, but it is made up of complicated components. Aim of this study is to separate major analgesic fraction from venom of A. halys Pallas, and to reveal its biochemical and pharmacological properties. Three steps with ion exchange column first and molecular sieve columns next were used to separate and purify the fractions of venom. Analgesic effects were evaluated by hot plate tests and writhing tests in mice. The molecular weight (MW), isoelectric point, amino acid sequence, purity were respectively determined by SDS-PAGE electrophoresis, isoelectric focusing, Edman degradation and HPLC. The dependence and tolerance were observed by withdrawal test in rats, and analgesic effects were observed in mice during 7 days administration. Fourteen fractions were obtained by separation; the best analgesic fraction named Pallanalgesin was selected by ED50 and LD50. It had single band in electrophoresis, relative purity 92.16 %, MW 16.6 kDa, isoelectric point 8.8, and former sequence of ten amino acids H-L-L-Q-F-R-K-M-I-K. It showed significant analgesic effect without tolerance and dependence. As a novel analgesic, Pallanalgesin has been found to explain the function of venom of A. halys Pallas on severe pain control in traditional uses.

  20. Development of Snake Fungal Disease after Experimental Challenge with Ophidiomyces ophiodiicola in Cottonmouths (Agkistrodon piscivorous).

    PubMed

    Allender, Matthew C; Baker, Sarah; Wylie, Daniel; Loper, Daniel; Dreslik, Michael J; Phillips, Christopher A; Maddox, Carol; Driskell, Elizabeth A

    2015-01-01

    Snake fungal disease (SFD) is a clinical syndrome associated with dermatitis, myositis, osteomyelitis, and pneumonia in several species of free-ranging snakes in the US. The causative agent has been suggested as Ophidiomyces ophiodiicola, but other agents may contribute to the syndrome and the pathogenesis is not understood. To understand the role of O. ophiodiicola in SFD, a cottonmouth snake model of SFD was designed. Five cottonmouths (Agkistrodon piscivorous) were experimentally challenged by nasolabial pit inoculation with a pure culture of O. ophiodiicola. Development of skin lesions or facial swelling at the site of inoculation was observed in all snakes. Twice weekly swabs of the inoculation site revealed variable presence of O. ophiodiicola DNA by qPCR in all five inoculated snakes for 3 to 58 days post-inoculation; nasolabial flushes were not a useful sampling method for detection. Inoculated snakes had a 40% mortality rate. All inoculated snakes had microscopic lesions unilaterally on the side of the swabbed nasolabial pit, including erosions to ulcerations and heterophilic dermatitis. All signs were consistent with SFD; however, the severity of lesions varied in individual snakes, and fungal hyphae were only observed in 3 of 5 inoculated snakes. These three snakes correlated with post-mortem tissue qPCR evidence of O. ophiodiicola. The findings of this study conclude that O. ophiodiicola inoculation in a cottonmouth snake model leads to disease similar to SFD, although lesion severity and the fungal load are quite variable within the model. Future studies may utilize this model to further understand the pathogenesis of this disease and develop management strategies that mitigate disease effects, but investigation of other models with less variability may be warranted.

  1. A novel plasminogen activator from Agkistrodon blomhoffii Ussurensis venom (ABUSV-PA): Purification and characterization

    SciTech Connect

    Liu Shuqing; Sun Mingzhong . E-mail: ming-zhong.sun@case.edu; Greenaway, Frederick T.

    2006-10-06

    A plasminogen activator with arginine ester hydrolysis activity (ABUSV-PA) has been identified and purified to homogeneity from Chinese Agkistrodon blomhoffii Ussurensis snake venom. ABUSV-PA, a monomeric protein with molecular mass of 27815.2 Da, was purified 180-fold with 0.02% recovery for protein and 3.6% recovery for esterase activity. ABUSV-PA reacts optimally with its substrate N {sub {alpha}}-tosyl-L-arginine-methyl ester (TAME) at {approx}pH 7.5 and at 51 {sup o}C. Measurement from inductively coupled plasma-atomic emission spectroscopy (ICP-AES) reveals that ABUSV-PA is a Zn{sup 2+}-containing protein with a stoichiometry of 1:1 [Zn{sup 2+}]:[ABUSV-PA]. Analyses of esterase hydrolysis and UV absorption and CD spectra indicate that Zn{sup 2+} plays an important role in maintaining the structural integrity rather than the esterase activity of ABUSV-PA. Divalent metal ions, including Ca{sup 2+}, Mg{sup 2+}, Cu{sup 2+}, Ni{sup 2+}, Mn{sup 2+}, and Co{sup 2+}, increase the TAME hydrolysis activity of ABUSV-PA. A red-shift of the emission wavelengths of the synchronous fluorescence of ABUSV-PA, compared to those of free Tyr and Trp, indicates a conformation where the Tyr and Trp residues are in exposed hydrophilic environments. The presence of zinc increases the hydrophobicity of the conformational environments surrounding the Trp residues of ABUSV-PA and affects the secondary structure of ABUSV-PA, as proved by UV absorption and CD spectroscopy.

  2. Determination of hemocoagulase agkistrodon in a pharmaceutical preparation by high-performance liquid chromatography with pre-column derivatization and fluorescence detection.

    PubMed

    Cheng, Suyuan; Wang, Chaozhong; Li, Jing; Liang, Chenggang; Wang, Fengshan

    2013-07-01

    Currently, there is no analytical method for the quantification of hemocoagulase agkistrodon (HCA) in pharmaceutical preparations. This study presents a pre-column derivatization method for the quantification of HCA, a compound extracted from the venom of Agkistrodon acutus, in a pharmaceutical preparation (trade name Suling). In the proposed method, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate was used to tag the HCA substrate, and the derivatives were analyzed by high-performance liquid chromatography with fluorescence detection. Complete and homogeneous derivatization of HCA was confirmed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry analysis. The specificity of the method was validated by forced degradation, and interference was assessed using a placebo. Under the optimum chromatographic conditions, the calibration curve was linear over a range of 10 to 500 ng/mL, featuring a correlation coefficient of 0.9999. The limits of detection and quantification of the method were 0.57 and 1.6 ng/mL, respectively. The percentage recovery of HCA in quality control samples ranged from 97.49 to 99.15%. Overall, this novel method can be applied to the quantitative determination of HCA in pharmaceutical preparations.

  3. MANAGEMENT OF OSTEOMYELITIS CAUSED BY SALMONELLA ENTERICA SUBSP. HOUTENAE IN A TAYLOR'S CANTIL (AGKISTRODON BILINEATUS TAYLORI) USING AMIKACIN DELIVERED VIA OSMOTIC PUMP.

    PubMed

    Clancy, Meredith M; Newton, Alisa L; Sykes, John M

    2016-06-01

    An adult female Taylor's cantil (Agkistrodon bilineatus taylori) presented with marked spinal and mandibular osteomyelitis that cultured positive for Salmonella enterica subsp. houtenae, serovar IV 43:z4,z32:-. Progression of osteomyelitis was arrested by treatment using amikacin (0.026 mg/kg per hour) delivered via subcutaneous osmotic pump for 10 mo, replacing the pump every 4 wk. No adverse effects on renal function were appreciated throughout the course of therapy. Amikacin therapy was discontinued after improvement with treatment, but 5 mo later, bony lesions worsened, and an additional abscess formed at the previous pump site. The animal's condition declined and euthanasia was elected. Postmortem examination confirmed marked osteomyelitis with Salmonella infection of same serovar as the initial biopsy. This report highlights the pathogenicity of the S. enterica subsp. houtenae serovar and the ability to deliver effective amikacin dosage via osmotic pump to arrest osteomyelitis due to salmonellosis in a venomous snake.

  4. Biological and biochemical characterization of venom from the broad-banded copperhead (Agkistrodon contortrix laticinctus): isolation of two new dimeric disintegrins

    PubMed Central

    Rodríguez-Acosta, Alexis; Lucena, Sara; Alfonso, Andrea; Goins, Amber; Walls, Robert; Guerrero, Belsy; Suntravat, Montamas; Sánchez, Elda E.

    2016-01-01

    Disintegrins represent a family of effective cell-cell and cell-matrix inhibitors by binding to integrin receptors. Integrins are heterodimeric, transmembrane receptors that are the bridges for these cell interactions. Disintegrins have been shown to have many therapeutic implications for the treatment of strokes, heart attacks, and cancer. Two novel heterodimeric disintegrins were isolated from the venom of the broad-banded copperhead (Agkistrodon contortrix laticinctus). Crude venom separated by cation-exchange chromatography resulted in several fractions possessing hemorrhagic, fibrinolytic, gelatinase, and platelet activities. Venom fractions 2–3 and 17–19 showed fibrinolytic activity. Fractions 2–6, 8–11, and 16–21 had hemorrhagic activity. Gelatinase activity was found in fractions 3, 11, and 19. The isolation of laticinstatins 1 and 2 was accomplished by fractionating crude venom using reverse phase chromatography. Data from both SDS-PAGE and N-terminal sequencing determined that laticinstatins 1 and 2 were heterodimeric disintegrins, and both were assayed for their ability to inhibit platelet aggregation in human whole blood. Future functional evaluation of snake venom disintegrins shows considerable promise for elucidating the biochemical mechanisms of integrin-ligand interactions that will allow the development of adequate medications for hemostatic pathologies such as thrombosis, stroke, and cerebral and cardiac accidents. In this study, we are presenting the first report of the purification, and partial characterization of two new dimeric disintegrins isolated from the venom of broad-banded copperhead snakes. PMID:28090197

  5. Annual cycle of plasma testosterone in male copperheads, Agkistrodon contortrix (Serpentes, Viperidae): relationship to timing of spermatogenesis, mating, and agonistic behavior.

    PubMed

    Schuett, G W; Harlow, H J; Rose, J D; Van Kirk, E A; Murdoch, W J

    1997-03-01

    Reproductive cycles of the majority of squamate reptiles remain undescribed. Few studies are available on seasonal patterns of circulating steroid hormones in snakes. The goal of this study was to document the annual cycle of plasma testosterone (T) in male copperheads Agkistrodon contortrix, a North American pitviper (Serpentes, Viperidae). Two experimental conditions were used in this laboratory study. One condition (repeat-test group) consisted of 10 adult males that were sampled once each month for 11 months. The other condition (single-test groups) consisted of 10 groups each with 5 males (N = 50), and each male was tested a single time. The single-test condition was used to evaluate whether or not repeated handling and sampling affected T levels. The study was conducted from February-December, 1992. A well-defined seasonal pattern of plasma T levels was detected; patterns were similar under both experimental conditions with the exception that the repeat-test group had slightly lower levels. Levels of T were lowest (baseline) in April-May, increased in early summer (June), and were highest in late summer (August). Thereafter, T levels declined up to the time of hibernation (early November) and changed little during hibernation (November-January). Upon emergence from hibernation in late winter (February), T levels increased sharply from February to March and then decreased from March to April. The results are discussed in the context of timing of spermatogenesis, mating, and male agonistic behavior.

  6. Detection of eastern equine encephalomyelitis virus RNA in North American snakes.

    PubMed

    Bingham, Andrea M; Graham, Sean P; Burkett-Cadena, Nathan D; White, Gregory S; Hassan, Hassan K; Unnasch, Thomas R

    2012-12-01

    The role of non-avian vertebrates in the ecology of eastern equine encephalomyelitis virus (EEEV) is unresolved, but mounting evidence supports a potential role for snakes in the EEEV transmission cycle, especially as over-wintering hosts. To determine rates of exposure and infection, we examined serum samples from wild snakes at a focus of EEEV in Alabama for viral RNA using quantitative reverse transcription polymerase chain reaction. Two species of vipers, the copperhead (Agkistrodon contortrix) and the cottonmouth (Agkistrodon piscivorus), were found to be positive for EEEV RNA using this assay. Prevalence of EEEV RNA was more frequent in seropositive snakes than seronegative snakes. Positivity for the quantitative reverse transcription polymerase chain reaction in cottonmouths peaked in April and September. Body size and sex ratios were not significantly different between infected and uninfected snakes. These results support the hypothesis that snakes are involved in the ecology of EEEV in North America, possibly as over-wintering hosts for the virus.

  7. Crystal forms of a lysine-49 phospholipase A 2 from the eastern cottonmouth snake

    NASA Astrophysics Data System (ADS)

    Clancy, Laura L.; Rydel, Timothy J.; Muchmore, Steven W.; Holland, Debra R.; Watenpaugh, Keith D.; Finzel, Barry C.; Einspahr, Howard M.

    1990-09-01

    As part of an effort to determine the structure of a lysine-49 variant phospholipase A 2 from the venom of a North American pit viper, the eastern cottonmouth ( Agkistrodon piscivorus piscivorus), we have produced five different crystal forms grown under a variety of crystallization conditions. They include an orthorthombic form (P2 12 12 1 or P2 12 12; a=87.8(3) Å, b=76.2(3) Å, c=57.4(4) Å), an hexagonal form (space group P6 122 or its enantiomer; a= b=62.22(3) Å, c=137.1(3) Å) and two tetragonal forms - a neutral pH form (space group P4 12 12 or its enantiomer; a = b = 81.99(3) Å) and high pH form (space group P4 12 12; a = b = 71.5(1) Å, c = 57.6(2) Å one molecule per asymmetric unit) — the latter of which was used for structure determination.

  8. Facultative parthenogenesis in a critically endangered wild vertebrate.

    PubMed

    Fields, Andrew T; Feldheim, Kevin A; Poulakis, Gregg R; Chapman, Demian D

    2015-06-01

    Facultative parthenogenesis - the ability of sexually reproducing species to sometimes produce offspring asexually - is known from a wide range of ordinarily sexually reproducing vertebrates in captivity, including some birds, reptiles and sharks [1-3]. Despite this, free-living parthenogens have never been observed in any of these taxa in the wild, although two free-living snakes were recently discovered each gestating a single parthenogen - one copperhead (Agkistrodon contortrix) and one cottonmouth (Agkistrodon piscivorus) [1]. Vertebrate parthenogens are characterized as being of the homogametic sex (e.g., females in sharks, males in birds) and by having elevated homozygosity compared to their mother [1-3], which may reduce their viability [4]. Although it is unknown if either of the parthenogenetic snakes would have been carried to term or survived in the wild, facultative parthenogenesis might have adaptive significance [1]. If this is true, it is reasonable to hypothesize that parthenogenesis would be found most often at low population density, when females risk reproductive failure because finding mates is difficult [5]. Here, we document the first examples of viable parthenogens living in a normally sexually reproducing wild vertebrate, the smalltooth sawfish (Pristis pectinata). We also provide a simple approach to screen any microsatellite DNA database for parthenogens, which will enable hypothesis-driven research on the significance of vertebrate parthenogenesis in the wild.

  9. Facultative parthenogenesis discovered in wild vertebrates

    PubMed Central

    Booth, Warren; Smith, Charles F.; Eskridge, Pamela H.; Hoss, Shannon K.; Mendelson, Joseph R.; Schuett, Gordon W.

    2012-01-01

    Facultative parthenogenesis (FP)—asexual reproduction by bisexual species—has been documented in a variety of multi-cellular organisms but only recently in snakes, varanid lizards, birds and sharks. Unlike the approximately 80 taxa of unisexual reptiles, amphibians and fishes that exist in nature, FP has yet to be documented in the wild. Based on captive documentation, it appears that FP is widespread in squamate reptiles (snakes, lizards and amphisbaenians), and its occurrence in nature seems inevitable, yet the task of detecting FP in wild individuals has been deemed formidable. Here we show, using microsatellite DNA genotyping and litter characteristics, the first cases of FP in wild-collected pregnant females and their offspring of two closely related species of North American pitviper snakes—the copperhead (Agkistrodon contortrix) and cottonmouth (Agkistrodon piscivorus). Our findings support the view that non-hybrid origins of parthenogenesis, such as FP, are more common in squamates than previously thought. With this confirmation, FP can no longer be viewed as a rare curiosity outside the mainstream of vertebrate evolution. Future research on FP in squamate reptiles related to proximate control of induction, reproductive competence of parthenogens and population genetics modelling is warranted. PMID:22977071

  10. Facultative parthenogenesis discovered in wild vertebrates.

    PubMed

    Booth, Warren; Smith, Charles F; Eskridge, Pamela H; Hoss, Shannon K; Mendelson, Joseph R; Schuett, Gordon W

    2012-12-23

    Facultative parthenogenesis (FP)-asexual reproduction by bisexual species-has been documented in a variety of multi-cellular organisms but only recently in snakes, varanid lizards, birds and sharks. Unlike the approximately 80 taxa of unisexual reptiles, amphibians and fishes that exist in nature, FP has yet to be documented in the wild. Based on captive documentation, it appears that FP is widespread in squamate reptiles (snakes, lizards and amphisbaenians), and its occurrence in nature seems inevitable, yet the task of detecting FP in wild individuals has been deemed formidable. Here we show, using microsatellite DNA genotyping and litter characteristics, the first cases of FP in wild-collected pregnant females and their offspring of two closely related species of North American pitviper snakes-the copperhead (Agkistrodon contortrix) and cottonmouth (Agkistrodon piscivorus). Our findings support the view that non-hybrid origins of parthenogenesis, such as FP, are more common in squamates than previously thought. With this confirmation, FP can no longer be viewed as a rare curiosity outside the mainstream of vertebrate evolution. Future research on FP in squamate reptiles related to proximate control of induction, reproductive competence of parthenogens and population genetics modelling is warranted.

  11. Environmental contaminants in Texas, USA, wetland reptiles: Evaluation using blood samples

    USGS Publications Warehouse

    Clark, D.R.; Bickham, J.W.; Baker, D.L.; Cowman, D.F.

    2000-01-01

    Four species of reptiles (diamondback water snake [Nerodia rhombifer], blotched water snake [N. erythrogaster], cottonmouth [Agkistrodon piscivorus], and red-eared slider [Trachemys scripta]) were collected at two contaminated and three reference sites in Texas, USA. Old River Slough has received intensive applications of agricultural chemicals since the 1950s. Municipal Lake received industrial arsenic wastes continuously from 1940 to 1993. Blood samples were analyzed for organochlorines, potentially toxic elements, genetic damage, and plasma cholinesterase (ChE). Dichlorodiphenyldichloroethylene (DDE) concentrations reached as high as 3.0 ppm (wet weight) in whole blood of a diamondback water snake at Old River Slough, a level probably roughly equivalent to the maximum concentration found in plasma of peregrine falcons (Falco peregrinus) in 1978 to 1979 when DDE peaked in this sensitive species. Possible impacts on diamondback water snakes are unknown, but at least one diamondback water snake was gravid when captured, indicating active reproduction. Arsenic was not found in red-eared sliders (only species sampled) from Municipal Lake. Red-eared sliders of both sexes at Old River Slough showed declining levels of ChE with increasing mass, suggesting a life-long decrease of ChE levels. Possible negative population consequences are unknown, but no evidence was found in body condition (mass relative to carapace length) that red-eared sliders at either contaminated site were harmed.

  12. IDENTIFICATION OF REPTILIAN AND AMPHIBIAN BLOOD MEALS FROM MOSQUITOES IN AN EASTERN EQUINE ENCEPHALOMYELITIS VIRUS FOCUS IN CENTRAL ALABAMA

    PubMed Central

    CUPP, EDDIE W.; ZHANG, DUNHUA; YUE, XIN; CUPP, MARY S.; GUYER, CRAIG; SPRENGER, TONYA R.; UNNASCH, THOMAS R.

    2005-01-01

    Uranotaenia sapphirina, Culex erraticus, and Cx. peccator were collected in an enzootic eastern equine encephalomyelitis (EEE) virus focus in central Alabama (Tuskegee National Forest) from 2001 to 2003 and analyzed for virus as well as host selection. EEE virus was detected in each species every year except 2003, when pools of Cx. peccator were negative. Most (97%) of the 130 Cx. peccator blood meals identified were from ectothermic hosts; 3% were from birds. Among blood meals from reptiles (approximately 75% of the total), 81% were from Agkistrodon piscivorus (cottonmouth); all amphibian blood meals (approximately 25%) were from Rana spp. with > 50% taken from the bullfrog R. catesbeiana. Host identifications were made from 131 of 197 Cx. erraticus, but only 3 (2%) were derived from ectothermic species. Identification of Ur. sapphirina blood meals proved difficult and only 2 of 35 hosts were determined. Both were from R. catesbeiana. Ectothermic species are possible EEE virus reservoirs in the southeastern United States where species such as Cx. peccator and Ur. sapphirina occur with large, diverse reptilian, amphibian, and avian populations such as those at the Tuskegee site. PMID:15381805

  13. Proteomic Analyses of Agkistrodon contortrix contortrix Venom Using 2D Electrophoresis and MS Techniques.

    PubMed

    Bocian, Aleksandra; Urbanik, Małgorzata; Hus, Konrad; Łyskowski, Andrzej; Petrilla, Vladimír; Andrejčáková, Zuzana; Petrillová, Monika; Legáth, Jaroslav

    2016-12-13

    Snake venom is a complex mixture of proteins and peptides which in the Viperidae is mainly hemotoxic. The diversity of these components causes the venom to be an extremely interesting object of study. Discovered components can be used in search for new pharmaceuticals used primarily in the treatment of diseases of the cardiovascular system. In order to determine the protein composition of the southern copperhead venom, we have used high resolution two dimensional electrophoresis and MALDI ToF/ToF MS-based identification. We have identified 10 groups of proteins present in the venom, of which phospholipase A₂ and metalloprotease and serine proteases constitute the largest groups. For the first time presence of 5'-nucleotidase in venom was found in this group of snakes. Three peptides present in the venom were also identified. Two of them as bradykinin-potentiating agents and one as an inhibitor.

  14. Proteomic Analyses of Agkistrodon contortrix contortrix Venom Using 2D Electrophoresis and MS Techniques

    PubMed Central

    Bocian, Aleksandra; Urbanik, Małgorzata; Hus, Konrad; Łyskowski, Andrzej; Petrilla, Vladimír; Andrejčáková, Zuzana; Petrillová, Monika; Legáth, Jaroslav

    2016-01-01

    Snake venom is a complex mixture of proteins and peptides which in the Viperidae is mainly hemotoxic. The diversity of these components causes the venom to be an extremely interesting object of study. Discovered components can be used in search for new pharmaceuticals used primarily in the treatment of diseases of the cardiovascular system. In order to determine the protein composition of the southern copperhead venom, we have used high resolution two dimensional electrophoresis and MALDI ToF/ToF MS-based identification. We have identified 10 groups of proteins present in the venom, of which phospholipase A2 and metalloprotease and serine proteases constitute the largest groups. For the first time presence of 5′-nucleotidase in venom was found in this group of snakes. Three peptides present in the venom were also identified. Two of them as bradykinin-potentiating agents and one as an inhibitor. PMID:27983581

  15. Practical application of the protein C activator Protac from Agkistrodon contortrix venom.

    PubMed

    Stocker, K; Fischer, H; Meier, J

    1988-01-01

    The protein C activator Protac from A. contortrix venom is being investigated as a potential antithrombotic agent and as a tool for the preparation of activated protein C. Its established major application is the zymogen activation in functional protein C determinations based on either a clotting assay or a chromogenic substrate technique. The sensitivity of the activated partial thromboplastin time as an indicator reaction for Protac activated protein C depends on the contact activator component of the reagent. Protein C dose-response increased in the following order: kaolin greater than ellagic acid greater than sulfatide. This phenomenon is due to a competition of molecular affinities between Protac, plasma components and the different activating surfaces.

  16. A Novel Direct Factor Xa Inhibitory Peptide with Anti-Platelet Aggregation Activity from Agkistrodon acutus Venom Hydrolysates

    PubMed Central

    Chen, Meimei; Ye, Xiaohui; Ming, Xin; Chen, Yahui; Wang, Ying; Su, Xingli; Su, Wen; Kong, Yi

    2015-01-01

    Snake venom is a natural substance that contains numerous bioactive proteins and peptides, nearly all of which have been identified over the last several decades. In this study, we subjected snake venom to enzymatic hydrolysis to identify previously unreported bioactive peptides. The novel peptide ACH-11 with the sequence LTFPRIVFVLG was identified with both FXa inhibition and anti-platelet aggregation activities. ACH-11 inhibited the catalytic function of FXa towards its substrate S-2222 via a mixed model with a Ki value of 9.02 μM and inhibited platelet aggregation induced by ADP and U46619 in a dose-dependent manner. Furthermore, ACH-11 exhibited potent antithrombotic activity in vivo. It reduced paralysis and death in an acute pulmonary thrombosis model by 90% and attenuated thrombosis weight in an arterio-venous shunt thrombosis model by 57.91%, both at a dose of 3 mg/kg. Additionally, a tail cutting bleeding time assay revealed that ACH-11 did not prolong bleeding time in mice at a dose of 3 mg/kg. Together, our results reveal that ACH-11 is a novel antithrombotic peptide exhibiting both FXa inhibition and anti-platelet aggregation activities, with a low bleeding risk. We believe that it could be a candidate or lead compound for new antithrombotic drug development. PMID:26035670

  17. X-ray absorption near edge structure study on Acutolysin-C, a zinc-metalloproteinase from Agkistrodon acutus venom: Insight into the acid-inactive mechanism

    NASA Astrophysics Data System (ADS)

    Zhao, Wei; Chu, Wangsheng; Li, Shujun; Liu, Yiwei; Gao, Bin; Niu, Liwen; Teng, Maikun; Benfatto, Maurizio; Hu, Tiandou; Wu, Ziyu

    2007-11-01

    Acutolysin-C, a snake-venom zinc metalloproteinase, displays a distinct pH-dependent proteolytic activity, which has been tentatively assigned to a structural change of the zinc-containing catalytic center. In this work we compare X-ray absorption near-edge structure (XANES) experimental spectra at the Zn K-edge and theoretical calculations of solutions at different pH values. The experimental data show clear differences confirmed by a best fit using the MXAN procedure. The results show that, when pH decreases from pH 8.0 to pH 3.0, the zinc-coordinating catalytic water molecule moves far from the Glu143 residue that is considered to play an essential role in the proteolytic process. Data suggests that this is the possible mechanism that deactivates the metalloproteinase.

  18. Fungi Associated with Softening of Bisulfite-Brined Cherries.

    PubMed

    Lewis, J C; Pierson, C F; Powers, M J

    1963-03-01

    Softening of sound, calcium bisulfite-brined cherries was induced fairly quickly by brining them with cherries rotted by Aspergillus niger, Cytospora leucostoma, and Penicillium expansum, but not with cherries rotted by a variety of other microorganisms, including Alternaria sp., Aspergillus oryzae, Aureobasidium pullulans, Botrytis cinerea, Cladosporium sp., Mucor racemosus, Rhizopus stolonifer, and Sclerotinia fructicola. Rapid softening was correlated with the presence of a bisulfite-stable polygalacturonase, as demonstrated by a cup-plate test. A survey of naturally rotted cherries suggests the involvement of a bark-canker fungus, C. leucostoma, in softening of commercially brined cherries in the Pacific Northwest.

  19. Two new Cytospora species, Cytospora vinacea sp. nov. and Cytospora viticola sp. nov., from declining vineyards in eastern North America

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cytospora species are ubiquitous pathogens of woody plants, causing branch dieback and wood cankers in numerous perennial hosts, including agronomic crops (e.g., Prunus), timber trees (e.g., Eucalyptus), and riparian hosts (e.g., Salix). Cytospora chrysosperma, C. cincta, and C. leucostoma have been...

  20. Characterization of Cytospora isolates from wood cankers of declining grapevine in North America, with the descriptions of two new Cytospora species

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cytospora species are ubiquitous pathogens of woody plants, causing dieback and wood cankers in numerous perennial hosts, including agronomic crops (e.g., Prunus), timber trees (e.g., Eucalyptus), and riparian hosts (e.g., Salix). Cytospora chrysosperma, C. cincta, and C. leucostoma have been report...

  1. Unusual snail species involved in the transmission of Fasciola hepatica in watercress beds in central France.

    PubMed

    Dreyfuss, G; Vignoles, P; Abrous, M; Rondelaud, D

    2002-06-01

    Four freshwater pulmonate species (Lymnaea ovata, L. stagnalis, Physa acuta, Planorbis leucostoma) were living in several watercress beds known for their relationships with human cases of fasciolosis, whereas L. truncatula was never found. The aims of these studies were to determine the prevalence of natural infections with Fasciola hepatica in snails and to verify if these species might ensure the full larval development of this trematode (with cercarial shedding) when they were experimentally subjected to F. hepatica only, or to co-infections with an other trematode species. Investigations were so carried out in six snail populations living in watercress beds (including three for P. acuta) and in four others originating from three brooks or a pond (as controls). Snails naturally infected with F. hepatica were found in two watercress beds inhabited by L. ovata (prevalence of infection: 1.4%) and P. leucostoma (0.1%), respectively. The L. ovata from the watercress bed could be infected at a higher size than those from the control population and the prevalence of this infection was greater in the bed population. Similar findings were noted for L. stagnalis. Despite single or dual infections, the results obtained with the four populations of P. acuta were unsuccessful. In contrast, the co-infections of young P. leucostoma with Paramphistomum daubneyi and F. hepatica resulted in the shedding of some F. hepatica cercariae. According to the authors, the occurrence of fasciolosis in these watercress beds would be the consequence of frequent natural encounters between parasite and snails (L. ovata, L. stagnalis), or of co-infections with P. daubneyi and F. hepatica (P. leucostoma). In watercress beds only colonized by P. acuta, a lymnaeid species would have ensured the larval development of F. hepatica but it would have been eliminated by P. acuta, as this last species was known to be invasive and could colonize open drainage ditches on siliceous soil.

  2. Identification of single copy nuclear DNA markers for North American pit vipers.

    PubMed

    Gibbs, H Lisle; Diaz, Jose

    2010-01-01

    We describe 18 single copy nuclear DNA loci (10 loci cloned from a Sistrurus catenatus catenatus genomic library and eight intron-based loci amplified using conserved primers) that detect sequence variation in species from all genera (Sistrurus, Agkistrodon and Crotalus) of North American pit viper snakes. These loci (mean size in bp ± SE: 433 ± 51) show large numbers of phylogenetically informative sites across species (mean + SE: 10.2 ± 1.5), but limited variation within subspecies suggesting that they will be most useful for multilocus species-level phylogenetic analyses in these snakes.

  3. [Application of rapid PCR to authenticate medicinal snakes].

    PubMed

    Chen, Kang; Jiang, Chao; Yuan, Yuan; Huang, Lu-Qi; Li, Man

    2014-10-01

    To obtained an accurate, rapid and efficient method for authenticate medicinal snakes listed in Chinese Pharmacopoeia (Zaocysd humnades, Bungarus multicinctus, Agkistrodon acutus), a rapid PCR method for authenticate snakes and its adulterants was established based on the classic molecular authentication methods. DNA was extracted by alkaline lysis and the specific primers were amplified by two-steps PCR amplification method. The denatured and annealing temperature and cycle numbers were optimized. When 100 x SYBR Green I was added in the PCR product, strong green fluorescence was visualized under 365 nm UV whereas adulterants without. The whole process can complete in 30-45 minutes. The established method provides the technical support for authentication of the snakes on field.

  4. Anadromous alewives, Alosa pseudoharengus, as prey for white perch, Morone americana

    USGS Publications Warehouse

    Moring, J.R.; Mink, L.H.

    2002-01-01

    The reintroduction of anadromous alewives, Alosa pseudoharengus, to their historic habitats in the inland waters of the United States and Canada, has prompted concerns about possible interactions with a popular sport fish, white perch, Morone americana. Both species are now widely distributed in northeastern North America. Diets of white perch in Lake George, Maine, U.S.A., where alewives were absent, were monitored and compared with those of white perch populations that were sympatric with anadromous alewives in two coastal Maine lakes, Biscay Pond and North Pond. In the presence of introduced alewives, the diet of adult white perch became almost exclusively juvenile alewives by late summer in ponds where both species were present. White perch that were sympatric with alewives were more piscivorus than were Lake George white perch, which primarily consumed Cladocera. Not only were alewives the principal prey item in the diet of white perch in Biscay and North ponds, but adult alewives were largely cannibalistic by August. Thus, success of reintroducing anadromous alewives in waters containing white perch may be negatively impacted by predation as well as cannibalism.

  5. Optimum temperature for growth and preferred temperatures of age-0 lake trout

    USGS Publications Warehouse

    Edsall, Thomas A.; Cleland, Joshua

    2000-01-01

    This study was performed to determine the thermal preferences and optimum temperature for growth of age-0 lake trout Salvelinus namaycush to help predict the thermal habitat they select when they leave the spawning grounds and to assess the risk posed to them in the Great Lakes by piscivorus, nonnative fishes whose thermal habitat preferences are known. The test fish were hatched in the laboratory from eggs taken from wild fish, acclimated to 5, 10, 15, and 18°C, and fed to excess with commercial trout food for 47 d. The test fish grew at all of the temperatures, and the specific growth rate was highest at about 12.5°C (3.8% wet body weight/d). Fish used in the growth study were also tested in a vertical thermal gradient tank and had a final thermal preferendum between 10.1°C and 10.2°C. These results, which generally agreed with those of an earlier laboratory study of the temperature preference of age-1 lake trout and the limited information on thermal habitat use by age-0 lake trout in the Great Lakes, indicated age-0 lake trout would tend to seek temperatures near 10°C, or as high as 12.5°C, during summer if food was abundant. Published information on thermal habitat use of age-1 and adult alewives Alosa pseudoharengus and rainbow smelt Osmerus mordax indicated they would be expected to co-occur with age-0 lake trout during much of the time when the lake trout were small enough to be eaten by these two introduced piscivores.

  6. Thousands of microsatellite loci from the venomous coralsnake (Micrurus fulvius) and variability of select loci across populations and related species

    PubMed Central

    Castoe, Todd A.; Streicher, Jeffrey W.; Meik, Jesse M.; Ingrasci, Matthew J.; Poole, Alexander W.; de Koning, A.P. Jason; Campbell, Jonathan A.; Parkinson, Christopher L.; Smith, Eric N.; Pollock, David D.

    2012-01-01

    Studies of population genetics increasingly use next-generation DNA sequencing to identify microsatellite loci in non-model organisms. There are, however, relatively few studies that validate the feasibility of transitioning from marker development to experimental application across populations and species. North American coralsnakes of the Micrurus fulvius species complex occur in the United States and Mexico, and little is known about their population structure and phylogenetic relationships. This absence of information and population genetics markers is particularly concerning because they are highly venomous and have important implications on human health. To alleviate this problem in coralsnakes, we investigated the feasibility of using 454 shotgun sequences for microsatellite marker development. First, a genomic shotgun library from a single individual was sequenced (~7.74 megabases; 26,831 reads) to identify potentially amplifiable microsatellite loci (PALs). We then hierarchically sampled 76 individuals from throughout the geographic distribution of the species complex and examined whether PALs were amplifiable and polymorphic. Approximately half of the loci tested were readily amplifiable from all individuals, and 80% of the loci tested for variation were variable and thus informative as population genetic markers. To evaluate the repetitive landscape characteristics across multiple snakes, we also compared microsatellite content between the coralsnake and two other previously sampled snakes, the venomous copperhead (Agkistrodon contortrix) and Burmese python (Python molurus). PMID:22938699

  7. Poisonous snakebite in central Texas. Possible indicators for antivenin treatment.

    PubMed Central

    White, R R; Weber, R A

    1991-01-01

    Sixty-seven patients hospitalized for poisonous snakebite between 1975 and 1990 were managed by elevation, tetanus prophylaxis, intravenous fluids and antibiotics, and often by a limited excision of the bite site in the Emergency Department, with sequential laboratory studies as needed. Antivenin was used for systemic envenomation, and 23 of the 67 patients (34%) received 133 vials. Thirteen of the twenty-three patients (56%) had adverse reactions to the antivenin. Two significant observations arose. First age was an indicator. Eleven of eighteen patients 12 years or younger (61%) received antivenin, whereas 12 of 49 patients older than 12 years (24%) received antivenin (p = 0.0085, Fisher's exact test). Second species of snake was an indicator. Sixty-two snakes were identified (93%). Of 39 rattlesnake (Crotalus and Sistrurus) bites, 20 patients received antivenin (53%), but of 23 copperhead and water moccasin (Agkistrodon) bites, only three patients (12.5%) received antivenin (p = 0.0025). Antivenin may be indicated for use in systemic rattlesnake envenomation, especially in younger patients. PMID:2025067

  8. Infrared imaging in vipers: differential responses of crotaline and viperine snakes to paired thermal targets.

    PubMed

    Safer, Adam B; Grace, Michael S

    2004-09-23

    Pit vipers use infrared-sensitive pit organs to accurately target homeothermic prey even in the absence of visual cues. It has been suggested that other vipers, including large ambush predators of the genus Bitis, also may use radiant infrared information for predatory targeting. We compared behavioral responses of pit vipers and snakes of the viperine genus Bitis to paired targets of different temperatures (i.e. cool and warm balloons), some scented with rodent odors. The rates of tongue flicking, head turning and approaches by pit vipers (Agkistrodon contortrix and Crotalus atrox) were significantly higher toward warm targets than toward cool ones. Moreover, they all were significantly higher in pit vipers than in the vipers Bitis arietans, Bitis gabonica, and Bitis nasicornis. Bitis sp. exhibited no significant differences in their behaviors toward warm versus cool targets. Pit vipers often struck at targets (always the warm target even when paired with a prey-scented cool target), but Bitis sp. never struck at either warm or cool targets. These results show that the behavioral correlates of infrared-based predatory and/or defensive targeting differ significantly between these two groups of viperid snakes, and suggest that the neural substrates of infrared imaging in crotaline snakes are either absent or functionally distinct in viperine snakes of the genus Bitis.

  9. Identification and functional analysis of a novel bradykinin inhibitory peptide in the venoms of New World Crotalinae pit vipers.

    PubMed

    Graham, Robert Leslie James; Graham, Ciaren; McClean, Stephen; Chen, Tianbao; O'Rourke, Martin; Hirst, David; Theakston, David; Shaw, Chris

    2005-12-23

    A novel undecapeptide has been isolated and structurally characterized from the venoms of three species of New World pit vipers from the subfamily, Crotalinae. These include the Mexican moccasin (Agkistrodon bilineatus), the prairie rattlesnake (Crotalus viridis viridis), and the South American bushmaster (Lachesis muta). The peptide was purified from all three venoms using a combination of gel permeation chromatography and reverse-phase HPLC. Automated Edman degradation sequencing and MALDI-TOF mass spectrometry established its peptide primary structure as: Thr-Pro-Pro-Ala-Gly-Pro-Asp-Val-Gly-Pro-Arg-OH, with a non-protonated molecular mass of 1063.18 Da. A synthetic replicate of the peptide was found to be an antagonist of bradykinin action at the rat vascular B2 receptor. This is the first bradykinin inhibitory peptide isolated from snake venom. Database searching revealed the peptide to be highly structurally related (10/11 residues) with a domain residing between the bradykinin-potentiating peptide and C-type natriuretic peptide domains of a recently cloned precursor from tropical rattlesnake (Crotalus durissus terrificus) venom gland. BIP thus represents a novel biological entity from snake venom.

  10. Effect of iron and carbon monoxide on fibrinogenase-like degradation of plasmatic coagulation by venoms of four Crotalus species.

    PubMed

    Nielsen, Vance G; Redford, Daniel T; Boyle, Patrick K

    2017-01-01

    Annually, thousands suffer poisonous snake bite, often from defibrinogenating species. Iron and carbon monoxide (CO) improve coagulation kinetics by modulation of fibrinogen as demonstrated in various Agkistrodon species and Crotalus atrox. Thus, we sought to determine whether pretreatment of plasma with iron and CO could attenuate venom-mediated catalysis of fibrinogen obtained from four common Crotalus species with known fibrinogenase activity. Human plasma was pretreated with ferric chloride (0-10 μmol/l) and CO-releasing molecule-2 (0-100 μmol/l) prior to exposure to venom from a Northern Pacific rattlesnake, Arizona black rattlesnake, prairie rattlesnake, or red diamond rattlesnake. The concentration of venom used decreased coagulation function of one or more kinetic parameters by at least 50% of normal values. Coagulation kinetics were determined with thrombelastography.Three snake venoms significantly degraded plasmatic coagulation kinetics, prolonging the onset to clot formation, diminishing velocity of clot growth and decreasing clot strength. However, red diamond rattlesnake venom exposure resulted in mixed coagulation kinetics, significantly decreasing the time to onset of coagulation without decreasing the velocity of clot growth. Iron and CO attenuated these coagulation kinetic changes in a species-specific manner. Further in vitro investigation of other fibrinogenolytic venoms is indicated to determine if iron and CO can attenuate venom compromised coagulation.

  11. Ability of wedelolactone, heparin, and para-bromophenacyl bromide to antagonize the myotoxic effects of two crotaline venoms and their PLA2 myotoxins.

    PubMed

    Melo, P A; Ownby, C L

    1999-01-01

    We examined the ability of wedelolactone, heparin and para-bromophenacyl bromide to antagonize the myotoxic activity in mice of venoms from Crotalus viridis viridis and Agkistrodon contortrix laticinctus and two phospholipase A2 myotoxins, CVV myotoxin and ACL myotoxin, isolated from them. Myotoxicity was measured by the increase in plasma creatine kinase (CK) activity at two hours and histological changes in extensor digitorum longus muscle (EDL) at three hours after injection of the test solution. Both heparin and wedelolactone independently reduced the myotoxic effect of both crude venoms and both myotoxins, but wedelolactone was more effective. Wedelolactone plus heparin reduced the myotoxic effect of CVV myotoxin more than either antagonist alone. The PLA2 inhibitor, para-bromophenacyl bromide (pBPB), reduced the myotoxic effect of both myotoxins more than either wedelolactone or heparin. On the other hand, the myotoxic effect of polylysine was not reduced by either wedelolactone or para-bromophenacyl bromide, but it was reduced by heparin. These results indicate that wedelolactone, para-bromophenacyl bromide and heparin are antagonists of these two phospholipase A2 myotoxins, and that antagonism by the first two compounds may be due to a more specific interaction with these proteins than that by the latter.

  12. Purification and cloning of cysteine-rich proteins from Trimeresurus jerdonii and Naja atra venoms.

    PubMed

    Jin, Yang; Lu, Qiumin; Zhou, Xingding; Zhu, Shaowen; Li, Rui; Wang, Wanyu; Xiong, Yuliang

    2003-10-01

    Three 26 kDa proteins, named as TJ-CRVP, NA-CRVP1 and NA-CRVP2, were isolated from the venoms of Trimeresurus jerdonii and Naja atra, respectively. The N-terminal sequences of TJ-CRVP and NA-CRVPs were determined. These components were devoid of the enzymatic activities tested, such as phospholipase A(2), arginine esterase, proteolysis, L-amino acid oxidase, 5'nucleotidase, acetylcholinesterase. Furthermore, these three components did not have the following biological activities: coagulant and anticoagulant activities, lethal activity, myotoxicity, hemorrhagic activity, platelet aggregation and platelet aggregation-inhibiting activities. These proteins are named as cysteine-rich venom protein (CRVP) because their sequences showed high level of similarity with mammalian cysteine-rich secretory protein (CRISP) family. Recently, some CRISP-like proteins were also isolated from several different snake venoms, including Agkistrodon blomhoffi, Trimeresurus flavoviridis, Lanticauda semifascita and king cobra. We presumed that CRVP might be a common component in snake venoms. Of particular interest, phylogenetic analysis and sequence alignment showed that NA-CRVP1 and ophanin, both from elapid snakes, share higher similarity with CRVPs from Viperidae snakes.

  13. AhV_aPA-induced vasoconstriction involves the IP₃Rs-mediated Ca²⁺ releasing.

    PubMed

    Zeng, Fuxing; Zou, Zhisong; Niu, Liwen; Li, Xu; Teng, Maikun

    2013-08-01

    AhV_aPA, the acidic PLA₂ purified from Agkistrodon halys pallas venom, was previously reported to possess a strong enzymatic activity and can remarkably induce a further contractile response on the 60 mM K⁺-induced contraction with an EC₅₀ in 369 nM on mouse thoracic aorta rings. In the present study, we found that the p-bromo-phenacyl-bromide (pBPB), which can completely inhibit the enzymatic activity of AhV_aPA, did not significantly reduce the contractile response on vessel rings induced by AhV_aPA, indicating that the vasoconstrictor effects of AhV_aPA are independent of the enzymatic activity. The inhibitor experiments showed that the contractile response induced by AhV_aPA is mainly attributed to the Ca²⁺ releasing from Ca²⁺ store, especially sarcoplasmic reticulum (SR). Detailed studies showed that the Ca²⁺ release from SR is related to the activation of inositol trisphosphate receptors (IP₃Rs) rather than ryanodine receptors (RyRs). Furthermore, the vasoconstrictor effect could be strongly reduced by pre-incubation with heparin, indicating that the basic amino acid residues on the surface of AhV_aPA may be involved in the interaction between AhV_aPA and the molecular receptors. These findings offer new insights into the functions of snake PLA₂ and provide a novel pathogenesis of A. halys pallas venom.

  14. Genetic Identification of Spirometra decipiens Plerocercoids in Terrestrial Snakes from Korea and China.

    PubMed

    Jeon, Hyeong-Kyu; Park, Hansol; Lee, Dongmin; Choe, Seongjun; Kim, Kyu-Heon; Sohn, Woon-Mok; Eom, Keeseon S

    2016-04-01

    Human sparganosis is a zoonotic disease caused by infection with larval forms (procercoid/plerocercoid) of Spirometra spp. The purpose of this study was to identify Spirometra spp. of infected snakes using a multiplex PCR assay and phylogenetic analysis of mitochondrial DNA sequence data from the spargana of terrestrial snakes obtained from Korea and China. A total of 283 snakes were obtained that included 4 species of Colubridae comprising Rhabdophis tigrinus tigrinus (n=150), Dinodon rufozonatum rufozonatum (n=64), Elaphe davidi (n=2), and Elaphe schrenkii (n=7), and 1 species of Viperidae, Agkistrodon saxatilis (n=60). The snakes were collected from the provinces of Chungbuk, Chungnam, and Gyeongbuk in Korea (n=161), and from China (n=122). The overall infection rate with spargana was 83% (235/283). The highest was recorded for D. rufozonatum rufozonatum (100%), followed by A. saxatilis (85%) and R. tigrinus tigrinus (80%), with a negative result for E. davidi (0%) and E. schrenkii (0%). The sequence identities between the spargana from snakes (n=50) and Spirometra erinaceieuropaei (KJ599680) or S. decipiens (KJ599679) control specimens were 90.8% and 99.2%, respectively. Pairwise genetic distances between spargana (n=50) and S. decipiens ranged from 0.0080 to 0.0107, while those between spargana and S. erinaceieuropaei ranged from 0.1070 to 0.1096. In this study, all of the 904 spargana analyzed were identified as S. decipiens either by a multiplex PCR assay (n=854) or mitochondrial cox1 sequence analysis (n=50).

  15. Quantitative investigation of two metallohydrolases by X-ray absorption spectroscopy near-edge spectroscopy

    NASA Astrophysics Data System (ADS)

    Zhao, W.; Chu, W. S.; Yang, F. F.; Yu, M. J.; Chen, D. L.; Guo, X. Y.; Zhou, D. W.; Shi, N.; Marcelli, A.; Niu, L. W.; Teng, M. K.; Gong, W. M.; Benfatto, M.; Wu, Z. Y.

    2007-09-01

    The last several years have witnessed a tremendous increase in biological applications using X-ray absorption spectroscopy (BioXAS), thanks to continuous advancements in synchrotron radiation (SR) sources and detector technology. However, XAS applications in many biological systems have been limited by the intrinsic limitations of the Extended X-ray Absorption Fine Structure (EXAFS) technique e.g., the lack of sensitivity to bond angles. As a consequence, the application of the X-ray absorption near-edge structure (XANES) spectroscopy changed this scenario that is now continuously changing with the introduction of the first quantitative XANES packages such as Minut XANES (MXAN). Here we present and discuss the XANES code MXAN, a novel XANES-fitting package that allows a quantitative analysis of experimental data applied to Zn K-edge spectra of two metalloproteins: Leptospira interrogans Peptide deformylase ( LiPDF) and acutolysin-C, a representative of snake venom metalloproteinases (SVMPs) from Agkistrodon acutus venom. The analysis on these two metallohydrolases reveals that proteolytic activities are correlated to subtle conformation changes around the zinc ion. In particular, this quantitative study clarifies the occurrence of the LiPDF catalytic mechanism via a two-water-molecules model, whereas in the acutolysin-C we have observed a different proteolytic activity correlated to structural changes around the zinc ion induced by pH variations.

  16. Identification and functional analysis of a novel bradykinin inhibitory peptide in the venoms of New World Crotalinae pit vipers

    SciTech Connect

    James Graham, Robert Leslie . E-mail: rl.graham@ulster.ac.uk; Graham, Ciaren; McClean, Stephen; Chen, Tianbao; O'Rourke, Martin; Hirst, David; Theakston, David; Shaw, Chris

    2005-12-23

    A novel undecapeptide has been isolated and structurally characterized from the venoms of three species of New World pit vipers from the subfamily, Crotalinae. These include the Mexican moccasin (Agkistrodon bilineatus), the prairie rattlesnake (Crotalus viridis viridis), and the South American bushmaster (Lachesis muta). The peptide was purified from all three venoms using a combination of gel permeation chromatography and reverse-phase HPLC. Automated Edman degradation sequencing and MALDI-TOF mass spectrometry established its peptide primary structure as: Thr-Pro-Pro-Ala-Gly-Pro-Asp-Val-Gly-Pro-Arg-OH, with a non-protonated molecular mass of 1063.18 Da. A synthetic replicate of the peptide was found to be an antagonist of bradykinin action at the rat vascular B2 receptor. This is the first bradykinin inhibitory peptide isolated from snake venom. Database searching revealed the peptide to be highly structurally related (10/11 residues) with a domain residing between the bradykinin-potentiating peptide and C-type natriuretic peptide domains of a recently cloned precursor from tropical rattlesnake (Crotalus durissus terrificus) venom gland. BIP thus represents a novel biological entity from snake venom.

  17. Tetrathyridia of Mesocestoides lineatus in Chinese snakes and their adults recovered from experimental animals.

    PubMed

    Cho, Shin-Hyeong; Kim, Tong-Soo; Kong, Yoon; Na, Byoung-Kuk; Sohn, Woon-Mok

    2013-10-01

    Morphological characteristics of Mesocestoides lineatus tetrathyridia collected from Chinese snakes and their adults recovered from experimental animals were studied. The tetrathyridia were detected mainly in the mesentery of 2 snake species, Agkistrodon saxatilis (25%) and Elaphe schrenckii (20%). They were 1.73 by 1.02 mm in average size and had an invaginated scolex with 4 suckers. Adult tapeworms were recovered from 2 hamsters and 1 dog, which were orally infected with 5-10 larvae each. Adults from hamsters were about 32 cm long and those from a dog were about 58 cm long. The scolex was 0.56 mm in average width with 4 suckers of 0.17 by 0.15 mm in average size. Mature proglottids measured 0.29 by 0.91 mm (av.). Ovaries and vitellaria bilobed and located in the posterior portion of proglottids. The cirrus sac was oval-shaped and located median. Testes were follicular, distributed in both lateral fields of proglottids, and 41-52 in number per proglottid. Gravid proglottids were 1.84 by 1.39 mm (av.) with a characteristic paruterine organ. Eggs were 35 by 27 µm in average size with a hexacanth embryo. These morphological characteristics of adult worms were identical with those of M. lineatus reported previously. Therefore, it has been confirmed that the tetrathyridia detected in 2 species of Chinese snakes are the metacestodes of M. lineatus, and 2 snake species, A. saxatilis and E. schrenckii, play the role of intermediate hosts.

  18. Stable isotope tracer reveals that viviparous snakes transport amino acids to offspring during gestation.

    PubMed

    Van Dyke, James U; Beaupre, Steven J

    2012-03-01

    Viviparity and placentation have evolved from oviparity over 100 times in squamate reptiles (lizards and snakes). The independent origins of placentation have resulted in a variety of placental morphologies in different taxa, ranging from simple apposition of fetal and maternal tissues to endotheliochorial implantation that is homoplasious with mammalian placentation. Because the eggs of oviparous squamates transport gases and water from the environment and calcium from the eggshell, the placentae of viviparous squamates are thought to have initially evolved to accomplish these functions from within the maternal oviduct. Species with complex placentae have also been shown to rely substantially, or even primarily, on placental transport of organic nutrients for embryonic nutrition. However, it is unclear whether species with only simple placentae are also capable of transporting organic nutrients to offspring. Among viviparous squamates, all of the snakes that have been studied thus far have been shown to have simple placentae. However, most studies of snake placentation are limited to a single lineage, the North American Natricinae. We tested the abilities of four species of viviparous snakes - Agkistrodon contortrix (Viperidae), Boa constrictor (Boidae), Nerodia sipedon (Colubridae: Natricinae) and Thamnophis sirtalis (Colubridae: Natricinae) - to transport diet-derived amino acids to offspring during gestation. We fed [(15)N]leucine to pregnant snakes, and compared offspring (15)N content with that of unlabeled controls. Labeled females allocated significantly more (15)N to offspring than did controls, but (15)N allocation did not differ among species. Our results indicate that viviparous snakes are capable of transporting diet-derived amino acids to their offspring during gestation, possibly via placentation.

  19. Purification, primary structures and evolution of coagulant proteases from Deinagkistrodon actus venom.

    PubMed

    Nikandrov, Nikolai N; Deshimaru, Masanobu; Tani, Ayako; Chijiwa, Takahito; Shibata, Hiroki; Chang, Chang-Chun; Fukumaki, Yasuyuki; Ito, Tatsumi; Ohno, Motonori

    2005-12-15

    Deinagkistrodon (formerly Agkistrodon) actus (Taiwan) snake venom was found to contain at least seven closely related coagulant proteases. One of them, named actibin, was purified to homogeneity by means of four chromatographic steps. Actibin acted on fibrinogen to form fibrin clots with extremely high specific activity of 1,630 NIH units/mg and preferentially released fibrinopeptide A. Actibin was an acidic glycoprotein (pI 3.4) with molecular weight of 41,000, which was reduced to 28,800 after deglycosylation with N-glycanase. The k(cat)/K(m) values of actibin for hydrolysis of tosyl-l-arginine methyl ester and benzoyl-l-arginine p-nitroanilide were one-third to a half those for thrombin, reflecting a high potency of actibin in fibrinogen clotting. The amidase activities of actibin and its family proteases were inhibited by 3,4-dichloroisocoumarin, a serine protease inhibitor, indicating that actibin and its family proteases are serine proteases. Four cDNAs, named DaP1 and DaP7-DaP9, encoding D. actus coagulant proteases were cloned. All cDNAs contain an open reading frame of 780 bp coding for 260 amino acid residues, including a signal peptide of 24 amino acid residues. Their amino acid sequences predicted are highly homologous to one another with one to five amino acid substitutions. When four D. actus protease cDNAs were compared with the cDNAs coding for Trimeresurus flavoviridis and T. gramineus venom serine proteases, accelerated evolution was clearly observed. Similarity of the nucleotide sequences of four D. actus protease cDNAs with no synonymous and one to five nonsynonymous substitutions seems not to be in direct conformity with accelerated evolution. This possibly suggests that they have evolved to a similar direction to enhance their clotting activity rather than to produce other physiological activities.

  20. Increase of the cytotoxic effect of Bothrops jararacussu venom on mouse extensor digitorum longus and soleus by potassium channel blockers and by Na(+)/K(+)-ATPase inhibition.

    PubMed

    Tomaz, Marcelo A; Fernandes, Fabrício F A; El-Kik, Camila Z; Moraes, Raphael A M; Calil-Elias, Sabrina; Saturnino-Oliveira, Jeison; Martinez, Ana Maria B; Ownby, Charlotte L; Melo, Paulo A

    2008-09-15

    We investigated the myotoxicity of Bothrops jararacussu crude venom and other cytolytic agents on mouse isolated extensor digitorum longus (EDL) and soleus (SOL) muscles, which present distinct properties: EDL is a fast-twitch, white muscle with predominantly glycolytic fibers, while SOL is slow-twitch, red muscle with predominantly oxidative fibers. Muscles were exposed to B. jararacussu crude venom (25 microg/ml) and other crotaline venoms (Agkistrodon contortrix laticinctus; Crotalus viridis viridis; Crotalus durissus terrificus) at the same concentration. Basal creatine kinase (CK) release to bathing solution was 0.43+/-0.06 for EDL and 0.29+/-0.06 for SOL (U g(-)(1) h(-)(1), n=36 for each muscle). Sixty minutes after exposure to B. jararacussu venom, EDL presented higher increase in the rate of CK release than SOL, respectively, 13.2+/-1.5 and 2.9+/-0.7 U g(-)(1)h(-)(1), n=10-12. Muscle denervation, despite decreasing CK content, did not affect sensitivities to B. jararacussu venom. Ouabain and potassium channel blockers (TEA; clotrimazole; glibenclamide) increased the rate of CK release by B. jararacussu in EDL and SOL muscles, decreasing and almost abolishing the different sensitivity. When we exposed EDL or SOL muscles to Naja naja, Apis mellifera venoms (25 microg/ml), or Triton X-100 (0.01%), they showed similar rate of CK release. Our present data suggest that a mechanism involving intracellular calcium regulation or potassium channels may participate in the different sensitivity of EDL and SOL to B. jararacussu venom.

  1. Akbu-LAAO exhibits potent anti-tumor activity to HepG2 cells partially through produced H2O2 via TGF-β signal pathway

    PubMed Central

    Guo, Chunmei; Liu, Shuqing; Dong, Panpan; Zhao, Dongting; Wang, Chengyi; Tao, Zhiwei; Sun, Ming-Zhong

    2015-01-01

    Previously, we characterized the biological properties of Akbu-LAAO, a novel L-amino acid oxidase from Agkistrodon blomhoffii ussurensis snake venom (SV). Current work investigated its in vitro anti-tumor activity and underlying mechanism on HepG2 cells. Akbu-LAAO inhibited HepG2 growth time and dose-dependently with an IC50 of ~38.82 μg/mL. It could induce the apoptosis of HepG2 cells. Akbu-LAAO exhibited cytotoxicity by inhibiting growth and inducing apoptosis of HepG2 as it showed no effect on its cell cycle. The inhibition of Akbu-LAAO to HepG2 growth partially relied on enzymatic-released H2O2 as catalase only partially antagonized this effect. cDNA microarray results indicated TGF-β signaling pathway was linked to the cytotoxicity of Akbu-LAAO on HepG2. TGF-β pathway related molecules CYR61, p53, GDF15, TOB1, BTG2, BMP2, BMP6, SMAD9, JUN, JUNB, LOX, CCND1, CDK6, GADD45A, CDKN1A were deregulated in HepG2 following Akbu-LAAO stimulation. The presence of catalase only slightly restored the mRNA changes induced by Akbu-LAAO for differentially expressed genes. Meanwhile, LDN-193189, a TGF-β pathway inhibitor reduced Akbu-LAAO cytotoxicity on HepG2. Collectively, we reported, for the first time, SV-LAAO showed anti-tumor cell activity via TGF-β pathway. It provides new insight of SV-LAAO exhibiting anti-tumor effect via a novel signaling pathway. PMID:26655928

  2. Tetrathyridia of Mesocestoides lineatus in Chinese Snakes and Their Adults Recovered from Experimental Animals

    PubMed Central

    Cho, Shin-Hyeong; Kim, Tong-Soo; Kong, Yoon; Na, Byoung-Kuk

    2013-01-01

    Morphological characteristics of Mesocestoides lineatus tetrathyridia collected from Chinese snakes and their adults recovered from experimental animals were studied. The tetrathyridia were detected mainly in the mesentery of 2 snake species, Agkistrodon saxatilis (25%) and Elaphe schrenckii (20%). They were 1.73 by 1.02 mm in average size and had an invaginated scolex with 4 suckers. Adult tapeworms were recovered from 2 hamsters and 1 dog, which were orally infected with 5-10 larvae each. Adults from hamsters were about 32 cm long and those from a dog were about 58 cm long. The scolex was 0.56 mm in average width with 4 suckers of 0.17 by 0.15 mm in average size. Mature proglottids measured 0.29 by 0.91 mm (av.). Ovaries and vitellaria bilobed and located in the posterior portion of proglottids. The cirrus sac was oval-shaped and located median. Testes were follicular, distributed in both lateral fields of proglottids, and 41-52 in number per proglottid. Gravid proglottids were 1.84 by 1.39 mm (av.) with a characteristic paruterine organ. Eggs were 35 by 27 µm in average size with a hexacanth embryo. These morphological characteristics of adult worms were identical with those of M. lineatus reported previously. Therefore, it has been confirmed that the tetrathyridia detected in 2 species of Chinese snakes are the metacestodes of M. lineatus, and 2 snake species, A. saxatilis and E. schrenckii, play the role of intermediate hosts. PMID:24327778

  3. Genetic Identification of Spirometra decipiens Plerocercoids in Terrestrial Snakes from Korea and China

    PubMed Central

    Jeon, Hyeong-Kyu; Park, Hansol; Lee, Dongmin; Choe, Seongjun; Kim, Kyu-Heon; Sohn, Woon-Mok; Eom, Keeseon S.

    2016-01-01

    Human sparganosis is a zoonotic disease caused by infection with larval forms (procercoid/plerocercoid) of Spirometra spp. The purpose of this study was to identify Spirometra spp. of infected snakes using a multiplex PCR assay and phylogenetic analysis of mitochondrial DNA sequence data from the spargana of terrestrial snakes obtained from Korea and China. A total of 283 snakes were obtained that included 4 species of Colubridae comprising Rhabdophis tigrinus tigrinus (n=150), Dinodon rufozonatum rufozonatum (n=64), Elaphe davidi (n=2), and Elaphe schrenkii (n=7), and 1 species of Viperidae, Agkistrodon saxatilis (n=60). The snakes were collected from the provinces of Chungbuk, Chungnam, and Gyeongbuk in Korea (n=161), and from China (n=122). The overall infection rate with spargana was 83% (235/283). The highest was recorded for D. rufozonatum rufozonatum (100%), followed by A. saxatilis (85%) and R. tigrinus tigrinus (80%), with a negative result for E. davidi (0%) and E. schrenkii (0%). The sequence identities between the spargana from snakes (n=50) and Spirometra erinaceieuropaei (KJ599680) or S. decipiens (KJ599679) control specimens were 90.8% and 99.2%, respectively. Pairwise genetic distances between spargana (n=50) and S. decipiens ranged from 0.0080 to 0.0107, while those between spargana and S. erinaceieuropaei ranged from 0.1070 to 0.1096. In this study, all of the 904 spargana analyzed were identified as S. decipiens either by a multiplex PCR assay (n=854) or mitochondrial cox1 sequence analysis (n=50). PMID:27180576

  4. Discovery of highly divergent repeat landscapes in snake genomes using high-throughput sequencing.

    PubMed

    Castoe, Todd A; Hall, Kathryn T; Guibotsy Mboulas, Marcel L; Gu, Wanjun; de Koning, A P Jason; Fox, Samuel E; Poole, Alexander W; Vemulapalli, Vijetha; Daza, Juan M; Mockler, Todd; Smith, Eric N; Feschotte, Cédric; Pollock, David D

    2011-01-01

    We conducted a comprehensive assessment of genomic repeat content in two snake genomes, the venomous copperhead (Agkistrodon contortrix) and the Burmese python (Python molurus bivittatus). These two genomes are both relatively small (∼1.4 Gb) but have surprisingly extensive differences in the abundance and expansion histories of their repeat elements. In the python, the readily identifiable repeat element content is low (21%), similar to bird genomes, whereas that of the copperhead is higher (45%), similar to mammalian genomes. The copperhead's greater repeat content arises from the recent expansion of many different microsatellites and transposable element (TE) families, and the copperhead had 23-fold greater levels of TE-related transcripts than the python. This suggests the possibility that greater TE activity in the copperhead is ongoing. Expansion of CR1 LINEs in the copperhead genome has resulted in TE-mediated microsatellite expansion ("microsatellite seeding") at a scale several orders of magnitude greater than previously observed in vertebrates. Snakes also appear to be prone to horizontal transfer of TEs, particularly in the copperhead lineage. The reason that the copperhead has such a small genome in the face of so much recent expansion of repeat elements remains an open question, although selective pressure related to extreme metabolic performance is an obvious candidate. TE activity can affect gene regulation as well as rates of recombination and gene duplication, and it is therefore possible that TE activity played a role in the evolution of major adaptations in snakes; some evidence suggests this may include the evolution of venom repertoires.

  5. Traditional Chinese Medicine Induced Liver Injury

    PubMed Central

    2014-01-01

    Traditional Chinese Medicine (TCM) is popular around the world and encompasses many different practices with particular emphasis on herbal TCM. Using the PubMed database, a literature search was undertaken to assess the extent herbal TCM products exert rare hepatotoxicity. Analysis of reported cases revealed numerous specified herbal TCM products with potential hepatotoxicity. Among these were An Shu Ling, Bai Fang, Bai Xian Pi, Ban Tu Wan, Bo He, Bo Ye Qing Niu Dan, Bofu Tsu Sho San, Boh Gol Zhee, Cang Er Zi, Chai Hu, Chaso, Chi R Yun, Chuan Lian Zi, Ci Wu Jia, Da Chai Hu Tang, Da Huang, Du Huo, Gan Cao, Ge Gen, Ho Shou Wu, Hu Bohe You, Hu Zhang, Huang Qin, Huang Yao Zi, Hwang Geun Cho, Ji Gu Cao, Ji Ji, Ji Xue Cao, Jiguja, Jin Bu Huan, Jue Ming Zi, Kamishoyosan, Kudzu, Lei Gong Teng, Long Dan Xie Gan Tang, Lu Cha, Ma Huang, Mao Guo Tian Jie Cai, Onshido, Polygonum multiflorum, Qian Li Guang, Ren Shen, Sairei To, Shan Chi, Shen Min, Shi Can, Shi Liu Pi, Shou Wu Pian, Tian Hua Fen, White flood, Wu Bei Zi, Xi Shu, Xiao Chai Hu Tang, Yin Chen Hao, Zexie, Zhen Chu Cao, and various unclassified Chinese herbal mixtures. Causality was firmly established for a number of herbal TCM products by a positive reexposure test result, the liver specific scale of CIOMS (Council for International Organizations of Medical Sciences), or both. Otherwise, the quality of case data was mixed, especially regarding analysis of the herb ingredients because of adulteration with synthetic drugs, contamination with heavy metals, and misidentification. In addition, non-herbal TCM elements derived from Agaricus blazei, Agkistrodon, Antelope, Bombyx, Carp, Fish gallbladder, Phellinus, Scolopendra, Scorpio, and Zaocys are also known or potential hepatotoxins. For some patients, the clinical course was severe, with risks for acute liver failure, liver transplantation requirement, and lethality. In conclusion, the use of few herbal TCM products may rarely be associated with hepatotoxicity in some

  6. Annotated catalogue of the Tachinidae (Insecta, Diptera) of the Afrotropical Region, with the description of seven new genera

    PubMed Central

    O’Hara, James E.; Cerretti, Pierfilippo

    2016-01-01

    regalis Cerretti & O’Hara, sp. n. (South Africa), and Myxophryxe satanas Cerretti & O’Hara, sp. n. (South Africa) (Exoristinae, Goniini); and Stiremania Cerretti & O’Hara with type species Stiremania karoo Cerretti & O’Hara, gen. n. and sp. n. (South Africa), and Stiremania robusta Cerretti & O’Hara, sp. n. (South Africa) (Exoristinae, Goniini). Paraclara Bezzi, 1908 is transferred from the Cylindromyiini to the Hermyini, comb. n. Sarrorhina Villeneuve, 1936 is transferred from the Minthoini to the Graphogastrini, comb. n. Three genera are newly recorded from the Afrotropical Region: Madremyia Townsend, 1916 (Eryciini); Paratrixa Brauer & Bergenstamm, 1891 (Blondeliini); and Simoma Aldrich, 1926 (Goniini). Three genera previously recorded from the Afrotropical Region are no longer recognized from the region: Calozenillia Townsend, 1927 (Palaearctic, Oriental and Australasian regions); Eurysthaea Robineau-Desvoidy, 1863 (Palaearctic, Oriental and Australasian regions); and Trixa Meigen, 1824 (Palaearctic and Oriental regions). Two species are newly recorded from the Afrotropical Region: Amnonia carmelitana Kugler, 1971 (Ethiopia, Kenya); and Simoma grahami Aldrich, 1926 (Namibia). Three species previously recorded from the Afrotropical Region are no longer recognized from the region: Euthera peringueyi Bezzi, 1925 (Oriental Region); Hamaxia incongrua Walker, 1860 (Palaearctic, Oriental and Australasian regions); Leucostoma tetraptera (Meigen, 1824) (Palaearctic Region). New replacement names are proposed for five preoccupied names of Afrotropical species: Billaea rubida O’Hara & Cerretti for Phorostoma rutilans Villeneuve, 1916, preoccupied in the genus Billaea Robineau-Desvoidy, 1830 by Musca rutilans Fabricius, 1781, nom. n.; Cylindromyia braueri O’Hara & Cerretti for Ocyptera nigra Villeneuve, 1918, preoccupied in the genus Cylindromyia Meigen, 1803 by Glossidionophora nigra Bigot, 1885, nom. n.; Cylindromyia rufohumera O’Hara & Cerretti for Ocyptera

  7. Alfimeprase.

    PubMed

    2008-01-01

    Alfimeprase, a fibrolase derivative with thrombolytic activity produced by recombinant DNA technology, was discovered by Amgen and was in development with Nuvelo for the treatment of stroke and catheter occlusion. However, development has been discontinued. Fibrolase is a zinc-containing metalloendopeptidase that was first isolated from the venom of the Southern copperhead snake, Agkistrodon contortrix contortrix. Alfimeprase directly degrades fibrin to break down clots. Alfimeprase is infused directly into the thrombus (side-hole catheter pushed through the entire clot) via multiple manual pulsed infusions. Alfimeprase degrades fibrin directly and entrapped blood cells are freed. Excess alfimeprase is rapidly inactivated by alpha-2 macroglobulin through an irreversible, covalent interaction. Phase III development of alfimeprase for peripheral arterial occlusion was discontinued based on poor results from the NAPA-2 and SONOMA-2 trials; however, Nuvelo resumed development of alfimeprase in 2007 for stroke and catheter occlusion.Alfimeprase's thrombolytic activity appears to be localized to the site of delivery because it is rapidly inactivated by alpha-2 macroglobulin, a naturally occurring protein in the blood, as it moves away from the site of delivery and into general blood circulation. In January 2002, Amgen and Hyseq Pharmaceuticals (now Nuvelo) entered into a collaboration to develop and commercialize alfimeprase. Nuvelo is to develop the product through clinical trials and Amgen was to be responsible for its manufacture. Both companies were to participate in commercial activities; Amgen was to have the option to lead these. Full financial terms were not disclosed. Further to this agreement, in November 2004 Amgen granted Nuvelo worldwide rights to develop and commercialize alfimeprase in exchange for milestone and royalty payments. In August 2007, Nuvelo decided to focus on core development programmes that it believed would produce the nearest-term proof