Disintegrin, hemorrhagic, and proteolytic activities of Mohave rattlesnake, Crotalus scutulatus scutulatus venoms lacking Mojave toxin

Venom from the Mohave rattlesnake, Crotalus scutulatus scutulatus, has been reported to be either: (1) neurotoxic; (2) hemorrhagic, or both (3) neurotoxic and hemorrhagic. In this study, 14 Mohave rattlesnakes from Arizona and Texas (USA) were analyzed for the presence of disintegrins and Mojave toxin. All venom samples were analyzed for the presence of hemorrhagic, proteolytic and disintegrin activities. The venoms were each chromatographed by reverse phase and their fractions tested for disintegrin activity. All specimens containing Mojave toxin were the most toxic and lacked proteolytic, hemorrhagic and disintegrin activities. In contrast, the venoms containing these activities lacked Mojave toxin. Two disintegrin genes, scutustatin and mojavestatin, were identified by PCR of genomic sequences. Scutustatin is a highly conserved disintegrin, while mojavestatin shows low conservation to other known disintegrins. Venoms with the highest LD50 measurements lacked both disintegrin genes, while the specimens with intermediate and low LD50 contained both genes. The intermediate LD50 group contained Mojave toxin and both disintegrin genes, but lacked hemorrhagic and disintegrin activity. Our results raise the possibility that scutustatin and mojavestatin are not expressed in the intermediate LD50 group, or that they may not be the same disintegrins responsible for the disintegrin activity found in the venom. Therefore, it is possible that Mohave rattlesnakes may produce more than two disintegrins.     

Comparison of the biodistribution of free or liposome-entrapped Crotalus durissus terrificus (South American rattlesnake) venom in mice

The local absorption rate, clearance and tissue distribution of Crotalus durissus terrificus venom, (Cdt) were examined using a two-antibody sandwich ELISA assay. We compared the biodistribution of both free or encapsulated Cdt in mice. Following subcutaneous injection of 10 microg/mouse of free Cdt (0.8 LD50), venom was detected in serum after 15 min, showed its highest level at 30 min (45+/-5 ng/ml) and was cleared from the circulation after 6 h. After 2 h of inoculation, venom was detected in the kidney (57+/-9 ng/g of tissue), spleen (18+/-4 ng/g of tissue) and brain (14+/-6 ng/g of tissue). For both subcutaneous or intravenous injection of free Cdt, venom was firstly detected in the kidney. No Cdt appeared either in the kidney, spleen, brain, or other tissues after subcutaneous inoculation of encapsulated venom even though a higher dose was used, 25 microg/mouse (2 LD50). Venom remained at the site of injection for a period of 1 week. Following intravenous injection of encapsulated venom (5 microg/mouse, 2 LD50), venom was detected in liver and spleen tissues. The biodistribution of encapsulated venom is discussed in relation to the effects of reduction of toxicity and increase of adjuvanticity.     

Isolation of two phospholipases A2 from Mojave rattlesnake (Crotalus scutulatus scutulatus) venom and variation of immunologically related venom proteins in different populations

Two phospholipases A2 of mol. wt 14,500 (P1) and 14,400 (P2) and pI 9.2 and 7.4 respectively were isolated from Crotalus scutulatus scutulatus venom. The two isoenzymes cross-reacted immunologically with phospholipase A2 from C. adamanteus and C, atrox, but not with Mojave toxin, excluding them as the basic subunit of the Mojave toxin complex. C. s. scutulatus venoms from Arizona had two common bands recognized by anti-P2 which were absent in most C. s. scutulatus venoms from Texas, suggesting two genetically different populations east and west of the Continental Divide.     

Phenotypic integration in the feeding system of the eastern diamondback rattlesnake (Crotalus adamanteus)

Selection can vary geographically across environments and temporally over the lifetime of an individual. Unlike geographic contexts, where different selective regimes can act on different alleles, age‐specific selection is constrained to act on the same genome by altering age‐specific expression. Snake venoms are exceptional traits for studying ontogeny because toxin expression variation directly changes the phenotype; relative amounts of venom components determine, in part, venom efficacy. Phenotypic integration is the dependent relationship between different traits that collectively produce a complex phenotype and, in venomous snakes, may include traits as diverse as venom, head shape and fang length. We examined the feeding system of the eastern diamondback rattlesnake (Crotalus adamanteus) across environments and over the lifetime of individuals and used a genotype–phenotype map approach, protein expression data and morphological data to demonstrate that: (i) ontogenetic effects explained more of the variation in toxin expression variation than geographic effects, (ii) both juveniles and adults varied geographically, (iii) toxin expression variation was a result of directional selection and (iv) different venom phenotypes covaried with morphological traits also associated with feeding in temporal (ontogenetic) and geographic (functional) contexts. These data are the first to demonstrate, to our knowledge, phenotypic integration between multiple morphological characters and a biochemical phenotype across populations and age classes. We identified copy number variation as the mechanism driving the difference in the venom phenotype associated with these morphological differences, and the parallel mitochondrial, venom and morphological divergence between northern and southern clades suggests that each clade may warrant classification as a separate evolutionarily significant unit.     

Structure and in vitro activities of a Copper II-chelating anionic peptide from the venom of the scorpion Tityus stigmurus

Anionic Peptides are molecules rich in aspartic acid (Asp) and/or glutamic acid (Glu) residues in the primary structure. This work presents, for the first time, structural characterization and biological activity assays of an anionic peptide from the venom of the scorpion Tityus stigmurus, named TanP. The three-dimensional structure of TanP was obtained by computational modeling and refined by molecular dynamic (MD) simulations. Furthermore, we have performed circular dichroism (CD) analysis to predict TanP secondary structure, and UV–vis spectroscopy to evaluate its chelating activity. CD indicated predominance of random coil conformation in aqueous medium, as well as changes in structure depending on pH and temperature. TanP has chelating activity on copper ions, which modified the peptide’s secondary structure. These results were corroborated by MD data. The molar ratio of binding (TanP:copper) depends on the concentration of peptide: at lower TanP concentration, the molar ratio was 1:5 (TanP:Cu²⁺), whereas in concentrated TanP solution, the molar ratio was 1:3 (TanP:Cu²⁺). TanP was not cytotoxic to non-neoplastic or cancer cell lines, and showed an ability to inhibit the in vitro release of nitric oxide by LPS-stimulated macrophages. Altogether, the results suggest TanP is a promising peptide for therapeutic application as a chelating agent.     

Evaluation of models for the evolution of protein sequences and functions under structural constraint

In the field of evolutionary structural genomics, methods are needed to evaluate why genomes evolved to contain the fold distributions that are observed. In order to study the effects of population dynamics in the evolved genomes we need fast and accurate evolutionary models which can analyze the effects of selection, drift and fixation of a protein sequence in a population that are grounded by physical parameters governing the folding and binding properties of the sequence. In this study, various knowledge-based, force field, and statistical methods for protein folding have been evaluated with four different folds: SH2 domains, SH3 domains, Globin-like, and Flavodoxin-like, to evaluate the speed and accuracy of the energy functions. Similarly, knowledge-based and force field methods have been used to predict ligand binding specificity in SH2 domain. To demonstrate the applicability of these methods, the dynamics of evolution of new binding capabilities by an SH2 domain is demonstrated.     

Hemorrhagic toxins from Western diamondback rattlesnake (Crotalus atrox) venom: isolation and characterization of five toxins and the role of zinc in hemorrhagic toxin e.

Five previously unknown hemorrhagic proteins, designated hemorrhagic toxins a,b,c,d, and e, were isolated from the venom of the western diamondback rattlesnake (Crotalus atrox). Molecular weights of hemorrhagic toxins a-e were determined to be 68 000, 24 000, 24 000, 24 000, and 25 700, respectively, by sodium dodecyl sulfate-phosphate gel electrophoresis using various polyacrylamide gel concentrations. Amino acid composition showed a total of 636, 200, 213, 214, and 219 amino acids for hemorrhagic toxins a-e, respectively. All the hemorrhagic toxins were found to lose their hemorrhagic activities with the metal chelators ethylenediaminetetraacetic acid and 1, 10-phenanthroline. All the hemorrhagic toxins were found to contain approximately 1 mol of zinc/mol of toxin, and they were all demonstrated to be proteolytic when dimethylcasein and dimethylhemoglobin were used as substrates. When zinc was removed from hemorrhagic toxin e with 1,10-phenanthroline, both both the proteolytic and hemorrhagic activities were equally inhibited. When the apohemorrhagic toxin e thus produced was incubated with zinc, the hemorrhagic and proteolytic activities were regenerated to the same extent. CD, UV, and Raman spectroscopy were used to study the structure of native hemorrhagin toxin e as well as the structural changes caused by zinc removal. From CD spectroscopy the native toxin was estimated to consist of 23% alpha helix, 6% beta structure, and 71% random-coil conformation. When over 90% of the zinc was removed, the alpha-helix content dropped from 23 to 7%.     

Utility of a set of microsatellite primers developed for the massasauga rattlesnake (Sistrurus catenatus) for population genetic studies of the timber rattlesnake (Crotalus horridus)

I tested six microsatellite DNA primer pairs developed for the massasauga rattlesnake (Sistrurus catenatus) on a sample population of the timber rattlesnake (Crotalus horridus). It had been speculated in a previous publication that cross‐species amplification would not be worthwhile across the two rattlesnake genera. However, for this primer set (the only one currently published for the genus Sistrurus), successful amplification at each locus was accomplished for all loci with an annealing temperature of 57 °C and locus‐specific buffer conditions. Each locus was polymorphic, with the number of alleles per locus ranging from two to 12. Significant heterozygote deficits were detected for three loci (Scu01, Scu05 and Scu07). For Scu01, all individuals were homozygous for the same allele except one female who was homozygous for a different allele. This same female was also homozygous for a rare allele at Scu07. When this female was removed from the data set, the number of observed heterozygotes at Scu01 and Scu07 did not differ significantly from random expectations. However, a large heterozygote deficit persisted at Scu05 (despite subsampling), suggesting that this locus may not be useful for population genetic studies of timber rattlesnakes. Despite some limitations, this set of primers may be a useful complement to those already developed for the genus Crotalus. Moreover, the results of this study seem to provide new justification for further studies of cross‐species amplification of microsatellite loci across the two rattlesnake genera.     

Isolation and characterization of hemorrhagic factors a and b from the venom of the Chinese habu snake (Trimeresurus mucrosquamatus)

Hemorrhagic factors a and b were isolated from Trimeresurus mucrosquamatus venom by Sephadex G-100, CM-Sephadex C-50 and DEAE-Sephacel column chromatographies. The hemorrhagic factors were homogeneous, as established by a single band on acrylamide gel electrophoresis, isoelectric focusing and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Molecular weights of 15 000 and 27 000 were found for hemorrhagic factors a and b, respectively. Factor a possesses proteolytic activity hydrolyzing the His(10)-Leu(11), Tyr(16)-Leu(17) and Arg(22)-Gly(23) bonds of oxidized insulin B chain, whereas, factor b hydrolyzed only the Ala(14)-Leu(15) bond. Hemorrhagic activity of these hemorrhagic factors was inhibited by ethylenediaminetetraacetic acid, 1,10-phenanthroline or p-chloromercuribenzoate, but not by soybean trypsin inhibitor or diisopropyl fluorophosphate. The hemorrhagic factors were injected into the skin of the back of albino rabbits, and the minimum hemorrhagic dose of factors a and b was 1.7 and 2.3 μg, respectively. These purified hemorrhagic factors were not lethal at 15 μg/g in mice. Factor a hydrolyzed the Bβ chain of fibrinogen, while factor b hydrolyzed the Aα chain. Hemorrhagic factor a was shown to differ immunologically from factor b. Factors a and b produced systemic hemorrhage in internal organs such as the heart and stomach of mice. Moreover, factor b produced hemorrhage in the liver.     

Insights into the role of oligomeric state on the biological activities of crotoxin: crystal structure of a tetrameric phospholipase A2 formed by two isoforms of crotoxin B from Crotalus durissus terrificus venom

Crotoxin B (CB or Cdt PLA(2)) is a basic Asp49-PLA(2) found in the venom of Crotalus durissus terrificus and it is one of the subunits that constitute the crotoxin (Cro). This heterodimeric toxin, main component of the C. d. terrificus venom, is completed by an acidic, nontoxic, and nonenzymatic component (crotoxin A, CA or crotapotin), and it is related to important envenomation effects such as neurological disorders, myotoxicity, and renal failure. Although Cro has been crystallized since 1938, no crystal structure of this toxin or its subunits is currently available. In this work, the authors present the crystal structure of a novel tetrameric complex formed by two dimers of crotoxin B isoforms (CB1 and CB2). The results suggest that these assemblies are stable in solution and show that Ser1 and Glu92 of CB1 and CB2, respectively, play an important role in the oligomerization. The tetrameric and dimeric conformations resulting from the association of the isoforms may increase the neurotoxicity of the toxin CB by the creation of new binding sites, which could improve the affinity of the molecular complexes to the presynaptic membrane.     

In vivo and in vitro activity of venom from the endoparasitic wasp Pimpla turionellae (L.) (Hymenoptera: Ichneumonidae)

The biological activity of venom from Pimpla turionellae L. (Hymenoptera: Ichneumonidae) was examined in vivo toward larvae and pupae of Galleriae mellonella L. (Lepidoptera: Pyralidae), and in vitro toward bacterial and fungal cultures, as well as cultured insect cells. Pupae of G. mellonella were far more susceptible to the venom than larvae. At low doses of venom [0.1 venom reservoir equivalents (VRE)], pupal abdominal mobility was inhibited within 30 min, and by 24 h, all pupae injected with venom concentrations >0.5 VRE were completely paralyzed. These same doses of venom resulted in an inhibition of adult emergence. Host larvae were far less sensitive to wasp venom as evidenced by all venom injected larvae remaining responsive to mechanical stimulation by 1 h post injection, even at concentrations equivalent to 1 venom reservoir. Eventually (>2 h at 25 degrees C), venom-injected larvae became immobile, then flaccid, and all died within 24 h post-injection. At lower concentrations of wasp venom, the onset of paralysis was delayed by comparison to that evoked by 1 VRE, and few host larvae were able to pupate. Development of host larvae to adult emergence was also reduced in a dose-dependent manner, with eclosion completely prevented at high concentrations (>0.5 VRE) of venom. Venom doses <0.5 VRE did not appear to induce paralysis or alter larval development. When venom was incubated with bacterial or fungal cultures, no antimicrobial activity was detected. However, wasp venom was found to be cytotoxic and cytolytic to cultured cells derived from the cabbage looper Trichoplusia ni Hubner (Lepidoptera: Noctuidae) and the yellow fever mosquito, Aedes aegypti (L.) (Diptera: Culcidae). Though both cell types displayed similar susceptibility in terms of LC50s, the lepidopteran cells responded much more rapidly with regard to the onset of morphological changes and the timing of cell death. A possible mode of action for the venom is discussed.     

Variation in oxygen consumption of the western diamondback rattlesnake (Crotalus atrox): implications for sexual size dimorphism

Variation in metabolism affects energy budgets of individuals and may serve as a mechanism that influences variation at whole organism or population levels. For example, sex differences in metabolic expenditure may contribute to bioenergetic sources of sexual size dimorphism. We measured oxygen consumption rates of 48 western diamondback rattlesnakes (Crotalus atrox) from a sexually dimorphic population and tested the effects of body mass, body temperature and time of day, in three groups of snakes: males, non-reproductive females, and vitellogenic females. Metabolic rates of male and non-reproductive female C. atrox were similar to rates reported for other rattlesnakes (mass exponents ranging from 0.645–0.670). Oxygen consumption was affected by body mass, body temperature and time of day, and was approximately 1.4 times greater in vitellogenic females than in non-reproductive females. No differences were found between males and non-reproductive females. Accordingly, differences in metabolic rate apparently do not contribute directly to sexual dimorphism in this population. Nevertheless, estimates of size-dependent maintenance expenditure lead us to hypothesize that adult female body size may represent a compromise between selection for increased litter size (accomplished by increasing body size), and selection for increased reproductive frequency (accomplished by decreasing body size, and, therefore inactive maintenance expenditure); this is a mechanistic scenario suggested previously for some endotherms. Accepted: 20 May 1998     

Disruption of pupariation and eclosion behavior in the flesh fly, Sarcophaga bullata Parker (Diptera: Sarcophagidae), by venom from the ectoparasitic wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae)

The action of venom from the ectoparasitic wasp, Nasonia vitripennis, was monitored by examining alterations in patterned muscular movements characteristic of pupariation and eclosion behavior in the flesh fly, Sarcophaga bullata. Venom injected into larvae prior to pupariation caused a dose-dependent delay in pupariation. Eventually, such larvae did pupariate, but puparia were abnormally formed. Barographic records revealed that all elements of pupariation behavior were present in venom-injected larvae, but pupariation behavior was not well synchronized with tanning, thus implying that the venom caused disruption in the temporal organization of central motor programs. When larvae were ligated and injected with venom posterior to the ligature, no response was evident in the posterior region, suggesting that the venom does not directly stimulate muscles or neuromuscular junctions. Injection of exogenous ecdysteroid into venom-injected larvae restored some elements of pupariation behavior, consistent with ecdysone's role in stimulating the release of anterior retraction factor and puparium tanning factor, two factors that are released from the CNS to regulate pupariation. When the venom was injected into newly emerged imagoes, the duration of extrication behavior was shortened, whereas all phases of post-eclosion behavior were lengthened. These observations imply that the venom affects CNS centers that regulate the muscular systems engaged in extrication and post-eclosion behavior.     

Observations on the chemosensory responses of the midget faded rattlesnake (<Emphasis Type="Italic">Crotalus oreganus concolor</Emphasis>): discrimination of envenomated prey in a type II venom species

Rattlesnakes use prey chemical cues for ambush site selection and for relocating envenomated (E) prey following a predatory strike. The ability to discriminate between E and non-envenomated (NE) prey cues has been widely studied in rattlesnake species that produce type I venoms, which show high levels of snake venom metalloproteinase (SVMP) activity and low lethal toxicity [lethal dose which kills 50% of test animals (LD₅₀) >1.0 µg/g]. However, E vs. NE prey discrimination studies have not been conducted on rattlesnake species that produce a type II venom that consists of low SVMP activity and high lethal toxicity (LD₅₀ <1.0 µg/g). In the current study, long-term captive Crotalus oreganus concolor, which produce a type II venom, were tested for their ability to discriminate between chemical cues of natural (Sceloporus undulatus and Peromyscus maniculatus) and non-natural (Hemidactylus frenatus and Mus musculus) prey cues, as well as for their ability to discriminate between E and NE mouse carcasses, when prey envenomation occurred by a conspecific. Snakes showed significant levels of tongue flicking towards the chemical extracts of P. maniculatus and M. musculus, suggesting that C. oreganus concolor exhibit both innate and experience-based plasticity in response to prey chemical cues. In addition, C. oreganus concolor were able to discriminate between E and NE prey sources, when envenomation occurred by a conspecific, indicating that a type II venomous species can also discriminate between E and NE chemical cues.     

Molecular evolution and secondary structural conservation in the B-cell lymphoma leukemia 2 (bcl-2) family of proto-oncogene products

The nature of the bcl-2 family of protooncogenes was analyzed by sequence alignment, secondary structure prediction, and phylogenetic techniques. Phylogenies were inferred from both the nucleic acid and amino acid sequences of the human, murine, rat, and chicken sequences for BCL-2 and BCL-X, human MCL1, murine A1, the nematode Caenorhabditis elegans and Caenorhabditis briggsiae ced-9 proteins, and the sequences BHRF1 from Epstein-Barr and LMW5-HL from African swine fever viruses. Both sequence alignment and secondary structure prediction techniques supported the conservation of both the overall secondary structure and the carboxy-terminal transmembrane domain in all members of the family. All the treeing methods employed (distance matrix, maximum likelihood, and parsimony) supported a tree in which the proapoptotic proteins BCL-2 and BCL-X represent the most recent additions to the group. All the trees also indicated that the viral proteins BHRF1 and LMW-HL arose from a common ancestor, an ancestor they shared in common with the pro-apoptotic control protein BAX, indicating that this function of BAX evolved only recently. The most ancient branches are represented by the nematode ced-9 protein and by the control genes MCL1 and A1, which in the treeing methods employed represent separate lineages within the most ancient grouping. These results demonstrate the evolution of a highly conserved family of developmental control genes from nematode to man—genes that encode proteins essential for normal development but which are highly conserved in terms of predicted structure and possible cellular localization. The evolutionary analysis also indicates that the family may be even larger than originally predicted and that other members are waiting to be discovered. Correspondence to: D. Lloyd Evans     

根田鼠几种觅食功能反应模型的比较

植物组织空间排列对植食性哺乳动物功能反应的作用,是觅食生态学的热点问题之一。以新鲜紫花苜蓿叶片为食物,改变苜蓿叶片大小调控根田鼠口量,改变叶片间距调控叶片密度,设置叶片空间异质性斑块。在空间异质性斑块上测定根田鼠的觅食行为及其参数,检验植食性哺乳动物4种功能反应模型的预测性。除复合模型参数Rmax、h和Vmax及口量模型参数Rmax的最大似然估计值与测定值近似外,其它模型参数的估计值与测定值均存在较大差异。根田鼠摄入率测定值与4种模型预测值的线性回归均显著(P0.01),但与复合模型预测值的线性拟合效果最佳。表明,复合模型能很好地解释根田鼠觅食与行走的竞争对摄入率调节的动态。根田鼠复合模型存在调节其功能反应机制转变的距离临界值(d)。叶片间距大于该值时,叶片密度调节摄入率;叶片间距小于该值时,口量调节摄入率。结果充分地验证了提出的特定假设:在植物密集条件下,植物大小能调节植食性小型哺乳动物根田鼠的摄入率;在植物稀疏条件下,植物密度调节其摄入率。     

Functional involvement of Lys-6 in the enzymatic activity of phospholipase A2 fromBungarus multicinctus (Taiwan banded krait) snake venom

Phospholipase A₂ (PLA₂) fromBungarus multicinctus snake venom was subjected to Lys modification with 4-chloro-3,5-dinitrobenzoate and trinitrobenzene sulfonic acid, and one major carboxydinitrophenylated (CDNP) PLA₂ and two trinitrophenylated (TNP) derivatives (TNP-1 and TNP-2) were separated by high-performance liquid chromatography. The results of amino acid analysis and sequence determination revealed that CDNP-PLA₂ and TNP-1 contained one modified Lys residue at position 6, and both Lys-6 and Lys-62 were modified in TNP-2. It seemed that the Lys-6 was more accessible to modified reagents than other Lys residues in PLA₂. Modification of Lys-6 caused a 94% drop in enzymatic activity as observed with CDNP-PLA₂ and TNP-1. Alternatively, the enzyme modified on both Lys-6 and Lys-62 retained little PLA₂ activity. Either carboxydinitrophenylation or trinitrophenylation did not significantly affect the secondary structure of the enzyme molecule as revealed by the CD spectra, and Ca²⁺ binding and antigenicity of Lys-6-modified PLA₂ were unaffected. Conversion of nitro groups to amino groups resulted in a partial restoration of enzymatic activity of CDNP-PLA₂ to 32% of that of PLA₂. It reflected that the positively charged side chain of Lys-6 might play an exclusive role in PLA₂ activity. The TNP derivatives could be regenerated with hydrazine hydrochloride. The biological activity of the regenerated PLA₂ is almost the same as that of native PLA₂. These results suggest that the intact Lys-6 is essential for the enzymatic activity of PLA₂, and that incorporation of a bulky CDNP or TNP group on Lys-6 might give rise to a distortion of the interaction between substrate and the enzyme molecule, and the active conformation of PLA₂.