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PubMed2000

Respiratory diseases, diagnostics, and therapy in snakes.

The pathogenesis of respiratory tract disease in snakes is complex. A snake must first be exposed to an infectious agent. If the snake is immunocompetent, then it usually prevails over the disease. When conditions of captive husbandry fall far less than those encountered in nature, snakes begin a battle that they have a hard time winning. Preventive education, preventive medicine, and quarantine allow clients to know that a health partnership is essential to accomplishing every veterinarian's mission, namely, a healthy pet.

Animal Husbandry↗
PubMed2002

Rhythmic contractions in chick amnio-yolk sac and snake amnion during embryogenesis.

The rhythmic movements of fetal membranes in chick and reptile embryos were studied to explore the developmental role of the extra-embryonic motor activity. In the snakes Lamprophis fuliginosus and Elaphe radiata, rhythmic contractions of amnion inside the developing egg were recorded from the 11th incubation day until pre-hatching stages (ca. day 60-72). The duration of these contractions averaged 2.02+/-0.27 min. The frequency ranged from 2 to 6 per 10 min and averaged 4.61+/-0.57 per 10 min. A tendency of frequency to increase toward the end of embryogenesis was observed. Lowering the temperature from 28 to 20 degrees C significantly decreased the frequency of amnion contractions to 2.85+/-0.91 per 10 min. The isolated snake amnion retained its capacity for spontaneous contraction. Noradrenaline inhibited, acetylcholine stimulated and serotonin did not affect the rhythmic activity of the isolated snake amnion. Similar effects were found when these agents were applied into the snake amniotic cavity. In the chick, yolk sac rhythmic contractions were recorded from the fifth until the 12th incubation days. The duration of these contractions ranged from 15 to 60 s, their frequency averaged 11.8+/-3.18 per 10 min and depended on temperature. The low temperature threshold was approximately 30 degrees C. After surgical removal of the amnion and embryo, the yolk sac continued contracting inside the egg. The yolk sac rhythmic contractions likely participate in the space movement of the embryo inside the egg during embryogenesis.

Amnion↗
PubMed1992

Substitution of Torpedo acetylcholine receptor alpha 1-subunit residues with snake alpha 1- and rat nerve alpha 3-subunit residues in recombinant fusion proteins: effect on alpha-bungarotoxin binding.

A fusion protein consisting of the TrpE protein and residues 166-211 of the Torpedo acetylcholine receptor alpha 1 subunit was produced in Escherichia coli using a pATH10 expression vector. Residues in the Torpedo sequence were changed by means of oligonucleotide-directed mutagenesis to residues present in snake alpha 1 subunit and rat nerve alpha 3 subunit which do not bind alpha-bungarotoxin. The fusion protein of the Torpedo sequence bound 125I-alpha-bungarotoxin with high affinity (IC50 = 2.5 x 10(-8) M from competition with unlabeled toxin, KD = 2.3 x 10(-8) M from equilibrium saturation binding data). Mutation of three Torpedo residues to snake residues, W184F, K185W, and W187S, had no effect on binding. Conversion of two additional Torpedo residues to snake, T191S and P194L, reduced alpha-bungarotoxin binding to undetectable levels. The P194L mutation alone abolished toxin binding. Mutation of three Torpedo alpha 1 residues to neuronal alpha 3-subunit residues, W187E, Y189K, and T191N, also abolished detectable alpha-bungarotoxin binding. Conversion of Try-189 to Asn which is present in the snake sequence (Y189N) abolished toxin binding. It is concluded that in the sequence of the alpha subunit of Torpedo encompassing Cys-192 and Cys-193, Try-189 and Pro-194 are important determinants of alpha-bungarotoxin binding. Tyr-189 may interact directly with cationic groups or participate in aromatic-aromatic interactions while Pro-194 may be necessary to maintain a conformation conductive to neurotoxin binding.

Amino Acid Sequence↗
PubMed1990

A method to predict the percutaneous permeability of various compounds: shed snake skin as a model membrane.

Penetration of various compounds through shed snake skin was measured in vitro to examine the effect of lipophilicity and molecular size of a compound on permeability through this model membrane. The permeabilities were found to be controlled by the lipophilicity and the molecular size of the permeant. The smaller and the more lipophilic the compound, the greater the permeability. Equations have been developed to predict the permeability from the molecular weight and the distribution coefficient of a compound. Further, the lipophilicity of shed snake skin is similar to that of human skin and the response of shed snake skin to the molecular size of a permeant is more similar to human skin than to hairless mouse skin. Considering the similarities between shed snake skin and human stratum corneum in terms of structure, composition, and permeability characteristics, the same considerations may apply to permeability through human stratum corneum.

Animals↗
PubMed2004

Resistance of neonates and field-collected garter snakes (Thamnophis spp.) to tetrodotoxin.

Prior studies of tetrodotoxin (TTX) resistance in garter snakes (Thamnophis spp.) have used laboratory-reared neonates as subjects, but the use of field-caught individuals would reduce cost and effort. We compared estimates of TTX resistance in field-caught and laboratory-born garter snakes. We found that a mass-adjusted dose of TTX administered to field-caught garter snakes produces an estimate of a population 50% dose that is comparable and unbiased with respect to those previously reported using laboratory-born neonates. Dose-response curves estimated for three field-caught populations closely matched the curves estimated from neonate data. The method was tested using populations with levels of TTX resistance ranging between approximately 5-90 mass-adjusted mouse units for their respective 50% doses. The technique of using field-caught snakes as test subjects provides larger genetically independent data sets that are more easily obtained. Our results indicate that changes in mass during development parallel ontogenetic shifts in TTX resistance.

Anesthetics, Local↗
PubMed2003

Amino acid sequence of the alpha- and beta-globin chains of the Erabu sea snake (Laticaudia semifasciata).

We determined the complete amino acid sequences of the Erabu sea snake (Laticaudia semifasciata) hemoglobin by analyzing the intact globin chains, enzymatically digested fragments, and chemical cleavage fragments to clarify the molecular evolution and phylogenetic classification of the sea snake. The Erabu sea snake has two types of hemoglobin components, Hb-I and Hb-II, which contain different alpha- and beta-chains. This is the second report of the complete primary structure for hemoglobin of snakes. The sequences were compared with those of other reptilian hemoglobins. Amino acids at positions critical for the structure and physiological functions of hemoglobin were loosely conserved. The requirements for binding of ATP and of diphosphoglycerate as allosteric effectors of beta-globins seemed to be fulfilled.

Amino Acid Sequence↗
PubMed1993

Responses to escalating predatory threat in garter and ribbon snakes (Thamnophis).

Several models suggest that animals perform a stable sequence of responses during increasing threat from a potential predator. To test the comparative utility of such models, 45 garter and ribbon snakes from four species (genus Thamnophis) were given an antipredator behavior test in which the snakes were exposed to a series of five levels of escalating threat: (a) threat is out of view, (b) threat looms over arena, (c) threat is nonmoving human hand near snake's head, (d) threat is moving human hand, and (e) threat is experimenter's tapping the snake's body. Five different antipredator behaviors were measured. It was found that both qualitative and quantitative changes in behavior occurred between threat levels. However, species differences were also found in the occurrence and amount of every behavior recorded, which indicates that even closely related species may differ in behaviors exhibited in different phases of a predatory encounter.

Animals↗
PubMed1978

Garter snake trailing behavior: effects of varying prey-extract concentration and mode of prey-extract presentation.

In a multiple-choice maze, garter snakes were trained to follow earthworm-extract trails for worm bit rewards. In Experiment 1, they were tested for their abilities fo follow extract trails that had been dried or extract trails that were removed from direct lingual access by a perforated floor. Snakes were able to follow the dry trails and unable to follow removed trails. In Experiment 2, snakes were tested for their behavioral responses to different concentrations of extract trails. Snakes trailed more accurately, moved more slowly, and exhibited much higher tongue flick rates on the intense concentration trails. The results are interpreted in terms of the assumption that effective trails are perceived by the tongue flick delivery of odorants to the vomeronasal organs.

Animals↗
PubMed2003

A single amino acid substitution of Leu130Ile in snake DNases I contributes to the acquisition of thermal stability.

We purified pancreatic deoxyribonucleases I (DNases I) from three snakes, Elaphe quadrivirgata, Elaphe climacophora and Agkistrodon blomhoffii, and cloned their cDNAs. Each mature snake DNase I protein comprised 262 amino acids. Wild-type snake DNases I with Leu130 were more thermally unstable than wild-type mammalian and avian DNases I with Ile130. After substitution of Leu130Ile, the thermal stabilities of the snake enzymes were higher than those of their wild-type counterparts and similar to mammalian wild-type enzyme levels. Conversely, substituting Ile130Leu of mammalian DNases I made them more thermally unstable than their wild-type counterparts. Therefore, a single amino acid substitution, Leu130Ile, might be involved in an evolutionally critical change in the thermal stabilities of vertebrate DNases I. Amphibian DNases I have a Ser205 insertion in a Ca2+-binding site of mammalian and avian enzymes that reduces their thermal stabilities [Takeshita, H., Yasuda, T., Iida, R., Nakajima, T., Mori, S., Mogi, K., Kaneko, Y. & Kishi, K. (2001) Biochem. J.357, 473-480]. Thus, it is plausible that the thermally stable wild-type DNases I of the higher vertebrates, such as mammals and birds, have been generated by a single Leu130Ile substitution of reptilian enzymes through molecular evolution following Ser205 deletion from amphibian enzymes. This mechanism may reflect one of the evolutionary changes from cold-blooded to warm-blooded vertebrates.

Amino Acid Sequence↗
PubMed2002

In vitro neuromuscular activity of snake venoms.

1. Snake venoms consist of a multitude of pharmacologically active components used for the capture of prey. Neurotoxins are particularly important in this regard, producing paralysis of skeletal muscles. These neurotoxins can be classified according to their site of action (i.e. pre- or post-synaptic). 2. Presynaptic neurotoxins, which display varying phospholipase A2 activities, have been identified in the venoms of the four major families of venomous snakes (i.e. Crotalidae, Elapidae, Hydrophiidae and Viperidae). The blockade of transmission produced by these toxins is usually characterized by a triphasic effect on acetylcholine release. Considerable work has been directed at identifying the binding site(s) on the presynaptic nerve terminal for these toxins, although their mechanism of action remains unclear. 3. Post-synaptic neurotoxins are antagonists of the nicotinic receptor on the skeletal muscle. Depending on their sequence, post-synaptic toxins are subdivided into short- and long-chain toxins. These toxins display different binding kinetics and different affinity for subtypes of nicotinic receptors. Post-synaptic neurotoxins have only been identified in venoms from the families Elapidae and Hydrophiidae. 4. Due to the high cost of developing new antivenoms and the reluctance of many companies to engage in this area of research, new methodologies are required to test the efficacy of existing antivenoms to ensure their optimal use. While chicken eggs have proven useful for the examination of haemorrhagic venoms, this procedure is not suited to venoms that primarily display neurotoxic activity. The chick biventer cervicis muscle has proven useful for this procedure, enabling the rapid screening of antivenoms against a range of venoms. 5. Historically, the lethality of snake venoms has been based on murine LD50 studies. Due to ethical reasons, these studies are being superseded by in vitro studies. Instead, the time taken to produce 90% inhibition of nerve-mediated twitches (i.e. t90) in skeletal muscle preparations can be determined. However, these two procedures result in different rank orders because they are measuring two different parameters. While murine LD50 determinations are based on "quantity", t90 values are based on how "quick" a venom acts. Therefore, knowledge of both parameters is still desirable. 6. In vitro neuromuscular preparations have proven to be invaluable tools in the examination of snake venoms and isolated neurotoxins. They will continue to play a role in further elucidating the mechanism of action of these highly potent toxins. Further study of these toxins may provide more highly specific research tools or lead compounds for pharmaceutical agents.

Animals↗
PubMed1986

Earthworms produce a collagen-like substance detected by the garter snake vomeronasal system.

Earthworms (Lumbricus terrestris) produce a chemical substance that is readily detected by and serves as an attractant for garter snakes (Thamnophis sirtalis). This chemoattractant is sensed by the vomeronasal system of snakes. Amino acid analysis of the chemoattractant revealed a high hydroxyproline/proline ratio and large amounts of serine and threonine. More than one-third of the residues were glycine. No hydroxylysine and no cysteine were present. Carbohydrate analyses revealed a high content of galactose (11% by weight) and smaller amounts of fucose, mannose, glucose, N-acetylglucosamine, and N-acetylgalactosamine. These results were very similar to results reported for the amino acid composition and carbohydrate content of earthworm cuticle collagen and gelatin. A preparation of purified earthworm cuticle collagen proved to be a potent source of chemoattractant for garter snakes. Further, it was not possible to prepare chemoattractant from decuticlized earthworms. These results strongly suggest that a component of the earthworm chemoattractant for snakes is structurally related to earthworm cuticle collagen.

Animals↗
PubMed1992

How the mongoose can fight the snake: the binding site of the mongoose acetylcholine receptor.

The ligand binding site of the nicotinic acetylcholine receptor (AcChoR) is within a short peptide from the alpha subunit that includes the tandem cysteine residues at positions 192 and 193. To elucidate the molecular basis of the binding properties of the AcChoR, we chose to study nonclassical muscle AcChoRs from animals that are resistant to alpha-neurotoxins. We have previously reported that the resistance of snake AcChoR to alpha-bungarotoxin (alpha-BTX) may be accounted for by several major substitutions in the ligand binding site of the receptor. In the present study, we have analyzed the binding site of AcChoR from the mongoose, which is also resistant to alpha-neurotoxins. It was shown that mongoose AcChoR does not bind alpha-BTX in vivo or in vitro. cDNA fragments of the alpha subunit of mongoose AcChoR corresponding to codons 122-205 and including the presumed ligand binding site were cloned, sequenced, and expressed in Escherichia coli. The expressed protein fragments of the mongoose, as well as of snake receptors, do not bind alpha-BTX. The mongoose fragment is highly homologous (greater than 90%) to the respective mouse fragment. Out of the seven amino acid differences between the mongoose and mouse in this region, five cluster in the presumed ligand binding site, close to cysteines 192 and 193. These changes are at positions 187 (Trp----Asn), 189 (Phe----Thr), 191 (Ser----Ala), 194 (Pro----Leu), and 197 (Pro----His). The mongoose like the snake AcChoR has a potential glycosylation site in the binding site domain. Sequence comparison between species suggests that substitutions at positions 187, 189, and 194 are important in determining the resistance of mongoose and snake AcChoR to alpha-BTX. In addition, it was shown that amino acid residues that had been reported to be necessary for acetylcholine binding are conserved in the toxin-resistant animals as well.

Amino Acid Sequence↗
PubMed1999

Prognostic factors for amputation in the case of envenoming by snakes of the Bothrops genus (Viperidae).

The prognostic factors for amputation following envenoming by snakes of the Bothrops genus were identified from the medical records of 3139 patients. Each of these patients had been bitten by Bothrops sp. and treated in the Hospital Vital Brazil, in São Paulo, Brazil, between 1981 and 1990. The 21 (0.67%) of the patients who had undergone amputation were compared with the 3118 who had not, with respect to the characteristics of the accident, the snake, the victim, the local and systemic manifestations of the envenoming and the treatment. There was an association between amputation and the month of the accident, the time of day when the accident happened, the length of the attacking snake, the anatomical region bitten, systemic bleeding and renal failure. Patients bitten in the fingers, during the cooler months, between 00.00 and 12.00 hours and/or by snakes > 60 cm in length, who developed blisters and abscesses at the site of the bite, systemic bleeding and/or renal failure underwent amputation more frequently than the others (P < 0.05 for each).

Adolescent↗
PubMed2002

Prothrombin activation in eastern tiger snake bite.

AIMS: To perform ex vivo studies in eastern tiger snake envenomation which define the haemostatic events associated with prothrombin activation. METHOD: Serial studies were performed on plasma from six individuals with evidence of eastern tiger snake envenomation. These analyses were particularly directed to fibrinogen levels, F1 + 2, TAT and evidence of fibrinolysis. RESULTS: There was a substantial rise in F1 + 2 and thrombin-antithrombin (TAT) complexes in all cases, even with minimal evenomation. In some cases the molar ratio of F1 + 2 and TAT was reduced from the relationship normally seen in vitro and ex vivo in clinical thrombosis. There was a dramatic fall in factor V and VIII levels which occurred 4-6 hours before the decline in prothrombin and AT3. This related in time to a fall in alpha2AP and plasminogen. Protein C levels also declined dramatically but many hours after presentation. CONCLUSIONS: F1 + 2 and TAT are sensitive markers of tiger snake evenomation. In some patients with massive prothrombin activation, the common mechanism for TAT clearance may be altered or overwhelmed. Conversely, the renal clearance of the smaller F1 + 2 may be enhanced. In the absence of thrombocytopaenia, which is a very sensitive marker of DIC, the fall in labile factors with tiger snake envenomation is significantly contributed to by proteolytic digestion of clotting factors.

Adolescent↗
PubMed2003

Identification and characterization of a serum protein homologous to alpha-type phospholipase A2 inhibitor (PLIalpha) from a nonvenomous snake, Elaphe quadrivirgata.

From a liver cDNA library prepared from a nonvenomous striated snake, Elaphe quadrivirgata, we isolated a cDNA encoding a novel protein, PLIalpha-like protein (PLIalpha-LP), having approximately 70% sequence identities with the alpha-type phospholipase A2 (PLA2) inhibitors (PLIalpha(s)) previously purified from the venomous snakes Agkistrodon blomhoffii siniticus and Trimeresurus flavoviridis. Since the PLI-LP with a highly conserved C-type lectin-like domain (CTLD) would be predicted to function as a PLA2 inhibitor, we purified this protein from E. quadrivirgata serum by sequential chromatography on Hi-trap Blue, Mono Q, and Superdex 200 columns. The purified 51-kDa protein with PLIalpha-like immunoreactivity was found to be a trimer of 18-kDa PLIalpha-LP, which was comparable to the trimeric structure of PLIalpha. But, unexpectedly, this protein did not show any inhibitory activity against various snake venom PLA2s. Furthermore, it did not inhibit the endogenous PLA2 activities in various tissue homogenates prepared from this snake. Lack of the inhibitory activity in PLIalpha-LP may provide important information concerning the structure-function relationships of PLIalpha.

Amino Acid Sequence↗
PubMed1985

Cross-hybridizing snake satellite, Drosophila, and mouse DNA sequences may have arisen independently.

Previous reports have interpreted hybridization between snake satellite DNA and DNA clones from a variety of distant taxonomic groups as evidence for evolutionary conservation, which implies common ancestry (homology) and/or convergence (analogy) to produce the cross-hybridizing sequences. We have isolated 11 clones from a genomic library of Drosophila melanogaster, using a cloned 2.5-kb snake satellite probe of known nucleotide sequence. We have also analysed published sequence data from snakes, mice, and Drosophila. These data show that (1) all of the cross-hybridization between the snake, fly, and mouse clones can be accounted for by the presence of either of two tandem repeats, [GATA]n and [GACA]n and (2) these tandem repeats are organized differently among the different species. We find no evidence that these sequences are homologous apart from the existence of the simple repeat itself, although their divergence from a common ancestral sequence cannot be ruled out. The sequences contain a variety of homogeneous clusters of tandem repeats of CATA, GA, TA, and CA, as well as GATA and GACA. We suggest that these motifs may have arisen by a self-accelerating process involving slipped-strand mispairing of DNA. Homogeneity of the clusters might simply be the result of a rate of accumulation of tandem repeats that exceeds that of other mutations.

Animals↗
PubMed1997

Foci of amino acid residue conservation in the 3D structures of the Kunitz BPTI proteinase inhibitors: how do variants from snake venom differ?

The Kunitz BPTI proteinase inhibitor family is divisible into subgroups based on source and bioactivity. Variants from snake venoms are of special interest because some show only weak inhibitory activity against the common proteinases while others are neurotoxic. We analysed the sequences for each subgrouping in the context of the common chain fold to predict the 3D location of interactive sites. The method used was an enhanced from of the previously devised 'regiovariation analysis.' This revealed the foci in 3D of amino acid side chain conservation in each subgroup. Locally high levels of side-chain conservation extending substantially in three dimensions can be associated more with the preservation of function than conformation, hence the foci probably reveal the most functionally relevant sites. For the inhibitor variants that do not originate from snake venom, regiovariation analysis gave an exact prediction of the antiproteinase site revealed by X-ray crystallography of inhibitor-enzyme complexes. However, this site is not the principal focus of evolutionary conservation in the inhibitors from snake venom, and other areas of the molecular surface are more prominent. The neurotoxic variants from snake venom (the dendrotoxins) have the principal focus of conservation near their C-terminal region, so this may be the origin of their special properties.

Amino Acid Sequence↗
PubMed1998

A study of an endothelin antagonist from a Chinese anti-snake venom medicinal herb.

Because it is well known that endothelin (ET) plays an important role in the pathogenesis of cardiovascular diseases, antagonists of ET for clinical use are very important. Because ET and some snake toxins have a homologous structure and similar biologic actions the effect of Chinese anti-snake venom herbal medicines on ET bioactivity was investigated both in vivo and in vitro. Hong Bei Si Chou [Cissus assamica (Laws.) Craib] is a herbal medicine used to treat snake bite in Guangxi province. It was found that all the different fractions of EtOH extraction, the EtOAc part of the EtOH extraction, and resverotrol (3,4'5-trihydroxytransstilbene) isolated from the EtOAc part could antagonize ET both in vivo and in vitro. These three fractions transiently relaxed ET-contracted isolated rat aortic ring in a dose-dependent manner. They also antagonized the lethal effects of ET-1 in mice and inhibited blood pressure elevation induced by ET-1. The results have shown that it is possible to find ET antagonists in Chinese anti-snake venom medicinal herbs. In the future, our work should shed new light on the treatment of cardiovascular diseases in which ET is involved.

Animals↗