Crotalus Adamanteus Research Articles

Isolation and characterisation of insulin-releasing compounds from Crotalus adamanteus, Crotalus vegrandis and Bitis nasicornis venom.

Crude venom from three venomous snakes, Crotalus adamanteus, Crotalus vegrandis and Bitis nasicornis was fractionated by gel filtration chromatography, and selected fractions screened for in-vitro insulinotropic activity using clonal pancreatic BRIN-BD11 cells. Nineteen fractions stimulated insulin secretion and the structural identity of bioactive compounds responsible was probed using MALDI-ToF MS and N-terminal Edman degradation sequencing. Partial N-terminal sequences were determined and their homology to existing sequences identified using BLAST searching. The main insulinotropic peptide families identified were made up of snake venom serine proteinases, phospholipases A2 (PLA2) and disintegrins. Snake venom constituents may have therapeutic potential for diabetes, with each of the three viper venoms tested providing insulinotropic compounds from a range of different toxin families.

Using multiscale spatial models to assess potential surrogate habitat for an imperiled reptile.

In evaluating conservation and management options for species, practitioners might consider surrogate habitats at multiple scales when estimating available habitat or modeling species’ potential distributions based on suitable habitats, especially when native environments are rare. Species’ dependence on surrogates likely increases as optimal habitat is degraded and lost due to anthropogenic landscape change, and thus surrogate habitats may be vital for an imperiled species’ survival in highly modified landscapes. We used spatial habitat models to examine a potential surrogate habitat for an imperiled ambush predator (eastern diamondback rattlesnake, Crotalus adamanteus; EDB) at two scales. The EDB is an apex predator indigenous to imperiled longleaf pine ecosystems (Pinus palustris) of the southeastern United States. Loss of native open-canopy pine savannas and woodlands has been suggested as the principal cause of the species’ extensive decline. We examined EDB habitat selection in the Coastal Plain tidewater region to evaluate the role of marsh as a potential surrogate habitat and to further quantify the species’ habitat requirements at two scales: home range (HR) and within the home range (WHR). We studied EDBs using radiotelemetry and employed an information-theoretic approach and logistic regression to model habitat selection as use vs. availability. We failed to detect a positive association with marsh as a surrogate habitat at the HR scale; rather, EDBs exhibited significantly negative associations with all landscape patches except pine savanna. Within home range selection was characterized by a negative association with forest and a positive association with ground cover, which suggests that EDBs may use surrogate habitats of similar structure, including marsh, within their home ranges. While our HR analysis did not support tidal marsh as a surrogate habitat, marsh may still provide resources for EDBs at smaller scales.

Molecular Modelling Approaches for Designing Inhibitors of L-Amino Acid Oxidase from Crotalus adamanteus Venom

L-amino acid oxidase (LAAO) from snake venom induces diverse toxicity into the victims, which is attributed to H 2 O 2 generated during the catalytic conversion of L-amino acids. In this study, homology model of LAAO from Crotalus adamanteus has been compared with the crystal structure of LAAO from Calloselasma rhodostoma . The root mean square deviations obtained from superposition of the FADbinding, substrate-binding and helical domains of LAAO from Crotalus adamanteus with those of LAAO from Calloselasma rhodostoma crystal structure confirmed a high degree of structural similarity between them. Based on the interactions of the substrate, L-phenylalanine and the reversible inhibitor, o -aminobenzoic acid with the catalytic residues of LAAO from Calloselasma rhodostoma , five probable inhibitors were designed. AutoDock Vina program was employed to perform automated molecular docking of these probable inhibitors. Two of them emerged as reversible inhibitors with IC 50 values of 1.6 and 3.3 μM respectively.

Synthesis of γ-Phosphate-Labeled and Doubly Labeled Adenosine Triphosphate Analogs.

This unit describes the synthesis of γ-phosphate-labeled and doubly labeled adenosine triphosphate (ATP) analogs and their characterization using the phosphodiesterase I from Crotalus adamanteus (snake venom phosphodiesterase; SVPD). In the key step of the synthesis, ATP or an ATP analog, bearing a linker containing a trifluoroacetamide group attached to the nucleoside, are modified with an azide-containing linker at the terminal phosphate using an alkylation reaction. Subsequently, different labels are introduced to the linkers by transformation of one functional group to an amine and coupling to an N-hydroxysuccinimide ester. Specifically, the Staudinger reaction of the azide is employed as a straightforward means to obtain an amine in the presence of various labels. Furthermore, the fluorescence characteristics of a fluorogenic, doubly labeled ATP analog are investigated following enzymatic cleavage by SVPD.

Early significant ontogenetic changes in snake venoms.

Snake venom plays a critical role in food acquisition, digestion, and defense. Venoms are known to change throughout the life of some snake species, but nothing is known about the venom composition of hatchling/neonate snakes prior to and just after their first shedding cycle, despite this being a critical time in the life of the snake. Using a cohort of Crotalus horridus and two cohorts of Crotalus adamanteus, we showed for the first time that snakes undergo significant changes in venom composition after the postnatal shedding event. The number of changes among cohorts ranged widely and there was wide variation in the direction of protein regulation, which appeared to be on a locus-specific level rather than protein-family level. These significant venom composition changes that take place in the first few weeks of life most likely play critical roles in venom economy and resource conservation and may partially explain the rare, post-birth maternal care found in some venomous species.

Contrasting modes and tempos of venom expression evolution in two snake species.

Selection is predicted to drive diversification within species and lead to local adaptation, but understanding the mechanistic details underlying this process and thus the genetic basis of adaptive evolution requires the mapping of genotype to phenotype. Venom is complex and involves many genes, but the specialization of the venom gland toward toxin production allows specific transcripts to be correlated with specific toxic proteins, establishing a direct link from genotype to phenotype. To determine the extent of expression variation and identify the processes driving patterns of phenotypic diversity, we constructed genotype-phenotype maps and compared range-wide toxin-protein expression variation for two species of snake with nearly identical ranges: the eastern diamondback rattlesnake (Crotalus adamanteus) and the eastern coral snake (Micrurus fulvius). We detected significant expression variation in C. adamanteus, identified the specific loci associated with population differentiation, and found that loci expressed at all levels contributed to this divergence. Contrary to expectations, we found no expression variation in M. fulvius, suggesting that M. fulvius populations are not locally adapted. Our results not only linked expression variation at specific loci to divergence in a polygenic, complex trait but also have extensive conservation and biomedical implications. C. adamanteus is currently a candidate for federal listing under the Endangered Species Act, and the loss of any major population would result in the irrevocable loss of a unique venom phenotype. The lack of variation in M. fulvius has significant biomedical application because our data will assist in the development of effective antivenom for this species.

Wound Healing Activity and Mechanisms of Action of an Antibacterial Protein from the Venom of the Eastern Diamondback Rattlesnake (Crotalus adamanteus)

Basic phospholipase A2 was identified from the venom of the eastern diamondback rattlesnake. The Crotalus adamanteus toxin-II (CaTx-II) induced bactericidal effects (7.8 µg/ml) on Staphylococcus aureus, while on Burkholderia pseudomallei (KHW), and Enterobacter aerogenes were killed at 15.6 µg/ml. CaTx-II caused pore formation and membrane damaging effects on the bacterial cell wall. CaTx-II was not cytotoxic on lung (MRC-5), skin fibroblast (HEPK) cells and in mice. CaTx-II-treated mice showed significant wound closure and complete healing by 16 days as compared to untreated controls (**P<0.01). Histological examination revealed enhanced collagen synthesis and neovascularization after treatment with CaTx-II versus 2% Fusidic Acid ointment (FAO) treated controls. Measurement of tissue cytokines revealed that interleukin-1 beta (IL-1β) expression in CaTx-II treated mice was significantly suppressed versus untreated controls. In contrast, cytokines involved in wound healing and cell migration i.e., monocyte chemotactic protein-1 (MCP-1), fibroblast growth factor-basic (FGF-b), chemokine (KC), granulocyte-macrophage colony-stimulating factor (GM-CSF) were significantly enhanced in CaTx-II treated mice, but not in the controls. CaTx-II also modulated nuclear factor-kappa B (NF-κB) activation during skin wound healing. The CaTx-II protein highlights distinct snake proteins as a potential source of novel antimicrobial agents with significant therapeutic application for bacterial skin infections.

Linking the transcriptome and proteome to characterize the venom of the eastern diamondback rattlesnake (Crotalus adamanteus)

Understanding the molecular basis of the phenotype is key to understanding adaptation, and the relationship between genes and specific traits is represented by the genotype-phenotype map. The specialization of the venom-gland towards toxin production enables the use of transcriptomics to identify a large number of loci that contribute to a complex phenotype (i.e., venom), while proteomic techniques allow verification of the secretion of the proteins produced by these loci, creating a genotype-phenotype map. We used the extensive database of mRNA transcripts generated by the venom-gland transcriptome of Crotalus adamanteus along with proteomic techniques to complete the genotype-phenotype map for the C. adamanteus venom system. Nanospray LC/MS(E) analysis of a whole venom sample identified evidence for 52 of the 78 unique putative toxin transcript clusters, including 44 of the 50 most highly expressed transcripts. Tandem mass spectrometry and SDS-PAGE of reversed-phase high-performance liquid chromatography fractions identified 40 toxins which clustered into 20 groups and represented 10 toxin families, creating a genotype-phenotype map. By using the transcriptome to understand the proteome we were able to achieve locus-specific resolution and provide a detailed characterization of the C. adamanteus venom system. Identifying the mechanisms by which genetic variation presents itself to the sieve of selection at the phenotypic level is key to understanding the molecular basis of adaptation, and the first step in understanding this relationship is to identify the genetic basis of the phenotype through the construction of a genotype-phenotype map. We used the high-throughput venom-gland transcriptomic characterization of the eastern diamondback rattlesnake (C. adamanteus) and proteomic techniques to complete and confirm the genotype-phenotype map, providing a detailed characterization of the C. adamanteus venom system.

Synthesis and Stability of a 2′‐O‐[N‐(Aminoethyl)carbamoyl]methyladenosine‐Containing Dinucleotide

AbstractWorking towards the synthesis of 2′‐O‐[N‐(aminoethyl)carbamoyl]methyl‐modified di‐ and oligonucleotides, we have synthesised a protected 2′‐O‐[N‐(aminoethyl)carbamoyl]methyl‐modified adenosine where the modification is introduced in a convenient one‐pot three‐step procedure. The corresponding H‐phosphonate building block was also synthesised, and from this intermediate, a 2′‐O‐[N‐(aminoethyl)carbamoyl]methyl‐containing dinucleotide could be made. We also performed studies on the chemical and enzymatic stability of this dinucleotide. The dinucleotide was subjected to different ammonolysis and other basic conditions, and HPLC analysis showed that the modification was intact to most conditions, but that there was some minor hydrolysis when NH3 (concd. aq.) was used at 55 °C. Under several other sets of conditions, including saturated NH3 in methanol, and ethylenediamine, the amide remained intact. Treatment of the dinucleotide with Phosphodiesterase I from Crotalus adamanteus venom and Phosphodiesterase II from bovine spleen showed that the N‐(aminoethyl)carbamoylmethyl moiety gives the phosphodiester linkage substantial protection against enzymatic degradation; the phosphodiester was not degraded by PDE II at all after seven days.

Snake co‐occurrence patterns are best explained by habitat and hypothesized effects of interspecific interactions

Snakes often occur in species-rich assemblages, and sympatry is thought to be facilitated primarily by low diet overlap, not interspecific interactions. We selected, a priori, three species pairs consisting of species that are morphologically and taxonomically similar and may therefore be likely to engage in interspecific, consumptive competition. We then examined a large-scale database of snake detection/nondetection data and used occupancy modelling to determine whether these species occur together more or less frequently than expected by chance while accounting for variation in detection probability among species and incorporating important habitat categories in the models. For some snakes, we obtained evidence that the probabilities that habitat patches are used are influenced by the presence of potentially competing congeneric species. Specifically, timber rattlesnakes (Crotalus horridus) were less likely than expected by chance to use areas that also contained eastern diamond-backed rattlesnakes (Crotalus adamanteus) when the proportion of evergreen forest was relatively high. Otherwise, they occurred together more often than expected by chance. Complex relationships were revealed between habitat use, detection probabilities and occupancy probabilities of North American racers (Coluber constrictor) and coachwhips (Coluber flagellum) that indicated the probability of competitive exclusion increased with increasing area of grassland habitat, although there was some model uncertainty. Cornsnakes (Pantherophis guttatus or Pantherophis slowinskii) and ratsnakes (Pantherophis alleghaniensis, Pantherophis spiloides, or Pantherophis obsoletus) exhibited differences in habitat selection, but we obtained no evidence that patterns of use for this species pair were influenced by current interspecific interactions. Overall, our results are consistent with the hypothesis that competitive interactions influence snake assemblage composition; the strength of these effects was affected by landscape-scale habitat features. Furthermore, we suggest that current interspecific interactions may influence snake occupancy, challenging the paradigm that contemporary patterns of snake co-occurrence are largely a function of diet partitioning that arose over evolutionary time.

A rare case of multiple rattlesnake bites

The rattlesnake (Crotalus adamanteus) is a venomous viper inhabiting the southeastern parts of the United States. It is not found in the Balkans and Europe habitats. Subjects of the species are grown and seen in museums, exhibitions and terrariums, and sometimes in private collections. This may generate potentially toxic exposures to the venom in accidental contact. Acute poisoning with rattlesnake poison in Bulgaria is exotic, rare and even casuistic. The venom of the rattlesnake exhibits neuropathic, proteolytic and hemolytic activities. Antivenom is not currently easily available in Bulgaria--it is not usually stored in hospitals because it is very rarely used and therefore rather expensive. We present a case of multiple envenomation (two different occasions) of one and the same person who kept rattlesnakes in a private terrarium. Local toxic syndrome was observed with burning and stinging pain at bite site combined with limited hemorrhage and necrosis. The hemolytic reaction and the local toxic results were successfully managed without resorting to any specific antidotal therapy.

Life history constraints contribute to the vulnerability of a declining North American rattlesnake

Delayed maturation and age-biased dispersal work in concert under past selection to maximize individual fitness and contribute to population viability; however, these life history attributes can become dysfunctional in a dynamic, anthropogenic landscape when important population demographics cannot redistribute in response to novel landscape change. We used long-term monitoring data to estimate age at maturity, potential longevity, survival, and fidelity for the declining eastern diamondback rattlesnake (EDB), Crotalus adamanteus. We used radio telemetry data and known-fate models to examine adult survival, and we combined mark-recapture and radio telemetry data to examine survival and fidelity using a combined recapture/recovery model. Monthly adult survival was higher during the active season (April–November; 99.5%) as compared to the inactive season (December–March; 96.3%), despite a higher probability of detecting human-caused mortalities during the active season. Rattlesnakes matured in 7.1years and potential longevity exceeded 20years. Fidelity estimates indicated mature EDBs had a low probability of dispersing from the study area, while younger, sexually immature individuals were more likely to emigrate. The combination of a slow life history and an ontogenetic shift in emigration suggests EDB life history limits the species’ ability to respond to landscape change, shedding further light on EDB imperilment. Management efforts will benefit from activities that maintain high adult survival. Furthermore, EDB fidelity should be considered in conservation plans, particularly in light of the species’ longevity, as these characteristics suggest that mature EDBs may not readily redistribute at the landscape scale in response to habitat degradation.

CDNA cloning of a snake venom metalloproteinase from the eastern diamondback rattlesnake (Crotalus adamanteus), and the expression of its disintegrin domain with anti-platelet effects

A 5′ truncated snake venom metalloproteinase was identified from a cDNA library constructed from venom glands of an eastern diamondback rattlesnake (Crotalus adamanteus). The 5′-rapid amplification of cDNA ends (RACE) was used to obtain the 1865 bp full-length cDNA sequence of a snake venom metalloproteinase (CamVMPII). CamVMPII encodes an open reading frame of 488 amino acids, which includes a signal peptide, a pro-domain, a metalloproteinase domain, a spacer, and an RGD-disintegrin domain. The predicted amino acid sequence of CamVMPII showed a 91%, 90%, 83%, and 82% sequence homology to the P-II class enzymes of C. adamanteus metalloproteinase 2, Crotalus atrox CaVMP-II, Gloydius halys agkistin, and Protobothrops jerdonii jerdonitin, respectively. Disintegrins are potent inhibitors of both platelet aggregation and integrin-dependent cell adhesion. Therefore, the disintegrin domain (Cam-dis) of CamVMPII was amplified by PCR, cloned into a pET-43.1a vector, and expressed in Escherichia coli BL21. Affinity purified recombinantly modified Cam-dis (r-Cam-dis) with a yield of 8.5 mg/L culture medium was cleaved from the fusion tags by enterokinase cleavage. r-Cam-dis was further purified by two-step chromatography consisting of HiTrap™ Benzamidine FF column, followed by Talon Metal affinity column with a final yield of 1 mg/L culture. r-Cam-dis was able to inhibit all three processes of platelet thrombus formation including platelet adhesion with an estimated IC50 of 1 nM, collagen- and ADP-induced platelet aggregation with the estimated IC50s of 18 and 6 nM, respectively, and platelet function on clot retraction. It is a potent anti-platelet inhibitor, which should be further investigated for drug discovery to treat stroke patients or patients with thrombotic disorders.

Irreversible inactivation of snake venom l-amino acid oxidase by covalent modification during catalysis of l-propargylglycine

Snake venom l-amino acid oxidase (SV-LAAO, a flavor-enzyme) has attracted considerable attention due to its multifunctional nature, which is manifest in diverse clinical and biological effects such as inhibition of platelet aggregation, induction of cell apoptosis and cytotoxicity against various cells. The majority of these effects are mediated by H2O2 generated during the catalytic conversion of l-amino acids. The substrate analog l-propargylglycine (LPG) irreversibly inhibited the enzyme from Crotalus adamanteus and Crotalus atrox in a dose- and time-dependent manner. Inactivation was irreversible which was significantly protected by the substrate l-phenylalanine. A Kitz–Wilson replot of the inhibition kinetics suggested formation of reversible enzyme–LPG complex, which occurred prior to modification and inactivation of the enzyme. UV–visible and fluorescence spectra of the enzyme and the cofactor strongly suggested formation of covalent adduct between LPG and an active site residue of the enzyme. A molecular modeling study revealed that the FAD-binding, substrate-binding and the helical domains are conserved in SV-LAAOs and both His223 and Arg322 are the important active site residues that are likely to get modified by LPG. Chymotrypsin digest of the LPG inactivated enzyme followed by RP-HPLC and MALDI mass analysis identified His223 as the site of modification. The findings reported here contribute towards complete inactivation of SV-LAAO as a part of snake envenomation management.

The genesis of an exceptionally lethal venom in the timber rattlesnake (Crotalus horridus) revealed through comparative venom-gland transcriptomics

BackgroundSnake venoms generally show sequence and quantitative variation within and between species, but some rattlesnakes have undergone exceptionally rapid, dramatic shifts in the composition, lethality, and pharmacological effects of their venoms. Such shifts have occurred within species, most notably in Mojave (Crotalus scutulatus), South American (C. durissus), and timber (C. horridus) rattlesnakes, resulting in some populations with extremely potent, neurotoxic venoms without the hemorrhagic effects typical of rattlesnake bites.ResultsTo better understand the evolutionary changes that resulted in the potent venom of a population of C. horridus from northern Florida, we sequenced the venom-gland transcriptome of an animal from this population for comparison with the previously described transcriptome of the eastern diamondback rattlesnake (C. adamanteus), a congener with a more typical rattlesnake venom. Relative to the toxin transcription of C. adamanteus, which consisted primarily of snake-venom metalloproteinases, C-type lectins, snake-venom serine proteinases, and myotoxin-A, the toxin transcription of C. horridus was far simpler in composition and consisted almost entirely of snake-venom serine proteinases, phospholipases A2, and bradykinin-potentiating and C-type natriuretic peptides. Crotalus horridus lacked significant expression of the hemorrhagic snake-venom metalloproteinases and C-type lectins. Evolution of shared toxin families involved differential expansion and loss of toxin clades within each species and pronounced differences in the highly expressed toxin paralogs. Toxin genes showed significantly higher rates of nonsynonymous substitution than nontoxin genes. The expression patterns of nontoxin genes were conserved between species, despite the vast differences in toxin expression.ConclusionsOur results represent the first complete, sequence-based comparison between the venoms of closely related snake species and reveal in unprecedented detail the rapid evolution of snake venoms. We found that the difference in venom properties resulted from major changes in expression levels of toxin gene families, differential gene-family expansion and loss, changes in which paralogs within gene families were expressed at high levels, and higher nonsynonymous substitution rates in the toxin genes relative to nontoxins. These massive alterations in the genetics of the venom phenotype emphasize the evolutionary lability and flexibility of this ecologically critical trait.

Landscape‐level influences of terrestrial snake occupancy within the southeastern United States

Habitat loss and degradation are thought to be the primary drivers of species extirpations, but for many species we have little information regarding specific habitats that influence occupancy. Snakes are of conservation concern throughout North America, but effective management and conservation are hindered by a lack of basic natural history information and the small number of large-scale studies designed to assess general population trends. To address this information gap, we compiled detection/nondetection data for 13 large terrestrial species from 449 traps located across the southeastern United States, and we characterized the land cover surrounding each trap at multiple spatial scales (250-, 500-, and 1000-m buffers). We used occupancy modeling, while accounting for heterogeneity in detection probability, to identify habitat variables that were influential in determining the presence of a particular species. We evaluated 12 competing models for each species, representing various hypotheses pertaining to important habitat features for terrestrial snakes. Overall, considerable interspecific variation existed in important habitat variables and relevant spatial scales. For example, kingsnakes (Lampropeltis getula) were negatively associated with evergreen forests, whereas Louisiana pinesnake (Pituophis ruthveni) occupancy increased with increasing coverage of this forest type. Some species were positively associated with grassland and scrub/shrub (e.g., Slowinski's cornsnake, Elaphe slowinskii) whereas others, (e.g., copperhead, Agkistrodon contortrix, and eastern diamond-backed rattlesnake, Crotalus adamanteus) were positively associated with forested habitats. Although the species that we studied may persist in varied landscapes other than those we identified as important, our data were collected in relatively undeveloped areas. Thus, our findings may be relevant when generating conservation plans or restoration goals. Maintaining or restoring landscapes that are most consistent with the ancestral habitat preferences of terrestrial snake assemblages will require a diverse habitat matrix over large spatial scales.

Enzymatic measurement of phosphatidylserine in cultured cells

Phosphatidylserine (PS) is a quantitatively minor membrane phospholipid involved in diverse cellular functions. In this study, we developed a new fluorometric method for measuring PS using combinations of specific enzymes and Amplex Red. The calibration curve for PS measurement was linear and hyperbolic at low (0-50 µM) and high (50-1000 µM) concentrations, respectively, and the detection limit was 5 µM (50 pmol in the reaction mixture). This assay quantified PS regardless of the chain length and the number of double bonds. We applied this new method to the determination of PS content in HEK293 cells, which was validated by a recovery study and comparison with TLC-phosphorus assay. We showed that the PS content was high in sparse cells. The overexpression of PS synthase 1 elevated not only the cellular PS content but also the phosphatidylcholine (PC) and phosphatidylethanolamine (PE) contents, suggesting the conversion of PS into PE and the enhancement of PC production. This new assay for PS measurement is simple, specific, sensitive, and high throughput, and it will be useful to clarify the metabolism and biological functions of PS.

The venom-gland transcriptome of the eastern diamondback rattlesnake (Crotalus adamanteus)

BackgroundSnake venoms have significant impacts on human populations through the morbidity and mortality associated with snakebites and as sources of drugs, drug leads, and physiological research tools. Genes expressed by venom-gland tissue, including those encoding toxic proteins, have therefore been sequenced but only with relatively sparse coverage resulting from the low-throughput sequencing approaches available. High-throughput approaches based on 454 pyrosequencing have recently been applied to the study of snake venoms to give the most complete characterizations to date of the genes expressed in active venom glands, but such approaches are costly and still provide a far-from-complete characterization of the genes expressed during venom production.ResultsWe describe the de novo assembly and analysis of the venom-gland transcriptome of an eastern diamondback rattlesnake (Crotalus adamanteus) based on 95,643,958 pairs of quality-filtered, 100-base-pair Illumina reads. We identified 123 unique, full-length toxin-coding sequences, which cluster into 78 groups with less than 1% nucleotide divergence, and 2,879 unique, full-length nontoxin coding sequences. The toxin sequences accounted for 35.4% of the total reads, and the nontoxin sequences for an additional 27.5%. The most highly expressed toxin was a small myotoxin related to crotamine, which accounted for 5.9% of the total reads. Snake-venom metalloproteinases accounted for the highest percentage of reads mapping to a toxin class (24.4%), followed by C-type lectins (22.2%) and serine proteinases (20.0%). The most diverse toxin classes were the C-type lectins (21 clusters), the snake-venom metalloproteinases (16 clusters), and the serine proteinases (14 clusters). The high-abundance nontoxin transcripts were predominantly those involved in protein folding and translation, consistent with the protein-secretory function of the tissue.ConclusionsWe have provided the most complete characterization of the genes expressed in an active snake venom gland to date, producing insights into snakebite pathology and guidance for snakebite treatment for the largest rattlesnake species and arguably the most dangerous snake native to the United States of America, C. adamanteus. We have more than doubled the number of sequenced toxins for this species and created extensive genomic resources for snakes based entirely on de novo assembly of Illumina sequence data.

Stability of a 2′‐O‐(Carbamoylmethyl)adenosine‐Containing Dinucleotide

AbstractIn the interest of pursuing the synthesis of 2′‐O‐carbamoylmethyl‐modified oligonucleotides from building blocks already containing the modification, we have performed studies on the chemical and enzymatic stability of a 2′‐O‐carbamoylmethyl‐modified dinucleotide. The dinucleotide was subjected to ammonolysis and other basic conditions such as treatment with ethylenediamine and sodium hydroxide at different temperatures. The amide of the carbamoylmethyl group partially hydrolyzed under standard deprotection conditions for oligonucleotide synthesis (conc. aq. NH3 at 55 °C). However, under several other conditions, including saturated NH3 in methanol, the amide remained intact. Treatment of the dinucleotide with Phosphodiesterase I from Crotalus adamanteus venom and Phosphodiesterase II from Bovine spleen, showed that the carbamoylmethyl moiety gives substantial protection of the phosphodiester towards enzymatic degradation.

The influence of aeration on activity and operational stability of two snake venom amino acid oxidases

Two l-amino acid oxidases from snake venom Crotalus adamanteus and Crotalus atrox were used as catalysts in the biotransformation of l-methionine to 2-oxo-4-methylthiobutyric acid. Catalase was present in all reactions to remove the hydrogen peroxide that evolves in the reaction, to prevent its reaction with an α-keto acid, and potential negative influence on amino acid oxidases. Batch reactor experiments without and with aeration were carried out with the two enzymes. Michaelis constants for oxygen were estimated from the initial reaction rate experiments and were found to be 0.030 and 0.053 mM for l-AAO from C. adamanteus and C. atrox, respectively. It was found that aeration increases the reaction rate and decreases the time necessary to achieve 100% l-methionine conversion due to the increase of oxygen concentration in the solution. It was also found that the increase of the air flow rate causes faster enzyme operational stability decay rate. The operational stability decay rate constant for C. adamanteus enzyme was estimated to be 6.29 x 10 −4 min −1 in the experiment without aeration. It increased in the experiment with aeration at 10 L h −1 to 3.75 × 10 −4 min −1, and was practically the same at higher air flow rate. Experiments without l-methionine presence proved that oxygen concentration was the reason for the decrease in enzyme activity for this enzyme and not the gas bubbles itself. In these experiments operational stability decay rate constants were one order of magnitude higher than when l-methionine was oxidized. It was found for l-amino acid oxidase from C. atrox that the operational stability decay rate constant linearly depends on the air flow rate. This dependence can be described by the following equation: k d = 3.1 × 10 −4 × qv + 3.4 × 10 −4 min −1. The operational stability decay rate constants values estimated from the experiments carried out at different air flow rates in the batch reactor were found to be similar to the ones estimated for l-AAO from C. adamanteus.