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PubMed2004

Standard and digestive metabolism in the banded water snake, Nerodia fasciata fasciata.

Estimating energy costs by respirometry is fundamental to many studies of the ecology, behavior and evolution of reptiles. However, traditional respirometry procedures seldom incorporate objective techniques for removal of outliers from estimates of metabolic parameters. We demonstrate how computer-automated respirometry equipment, which records many respiratory measurements over short intervals, can be coupled with mathematical procedures to produce robust estimates of pre- and post-prandial metabolism in banded water snakes (Nerodia fasciata fasciata). Standard metabolic rate of N. f. fasciata was estimated to be 1.21 ml O2/h (mass = 30.21 +/- 0.74 g) at 25 degrees C. After ingestion of a fish equaling 20% of their body mass, snakes exhibited a fivefold increase in metabolic rate with peak O2 consumption rate (VO2) reaching 6.5 ml O2/h. Total cost of digestion was 5.44 kJ, equivalent to approximately 21% of the energy in the meal. Repeated measurements of metabolism in the same individuals revealed that our methods yielded similar results, even when individuals exhibited different patterns of VO2 variation between respiratory trials. Our results underscore the importance of obtaining many VO2 measurements, coupled with objective removal of outlier values from estimates of metabolic rate, especially when metabolic values are to be interpreted in a comparative context.

Animals↗
PubMed2004

Sources and timing of calcium mobilization during embryonic development of the corn snake, Pantherophis guttatus.

Embryos of oviparous Reptilia (=turtles, lepidosaurs, crocodilians and birds) extract calcium for growth and development from reserves in the yolk and eggshell. Yolk provides most of the calcium to embryos of lizards and snakes. In contrast, the eggshell supplies most of the calcium for embryonic development of turtles, crocodilians and birds. The yolk sac and chorioallantoic membrane of birds recover and transport calcium from the yolk and eggshell and homologous membranes of squamates (lizards and snakes) probably transport calcium from these two sources as well. We studied calcium mobilization by embryos of the snake Pantherophis guttatus during the interval of greatest embryonic growth and found that the pattern of calcium transfer was similar to other snakes. Calcium recovery from the yolk is relatively low until the penultimate embryonic stage. Calcium removal from the eggshell begins during the same embryonic stage and total eggshell calcium drops in each of the final 2 weeks prior to hatching. The eggshell supplies 28% of the calcium of hatchlings. The timing of calcium transport from the yolk and eggshell is coincident with the timing of growth of the yolk sac and chorioallantoic membrane and expression of the calcium binding protein, calbindin-D28K, in these tissues as reported in previous studies. In the context of earlier work, our findings suggest that the timing and mechanism of calcium transport from the yolk sac of P. guttatus is similar to birds, but that both the timing and mechanism of calcium transport by the chorioallantoic membrane differs. Based on the coincident timing of eggshell calcium loss and embryonic calcium accumulation, we also conclude that recovery of eggshell calcium in P. guttatus is regulated by the embryo.

Animals↗
PubMed2005

Intraspecific differences in thermal tolerance of the diamondback watersnake (Nerodia rhombifer): effects of ontogeny, latitude, and sex.

Ontogenetic shifts in microhabitat use are widespread among taxa and can result in drastic shifts in thermal habitat among age classes. Likewise, geographic variation in climate along latitudinal gradients can cause differences in thermal environments among populations of a species. Using a common garden design, we examined four populations of a single species of semi-aquatic snake, Nerodia rhombifer, to determine whether ontogenetic shifts in habitat use (and/or body size) and latitudinal differences in ambient temperature have resulted in evolutionary changes in thermal tolerance. We found ontogenetic differences in thermal tolerance for all populations, with neonates tolerating temperatures 2 degrees C higher than adults, a pattern that is consistent with ontogenetic shifts in body size and microhabitat use in this species. There were differences in thermal tolerance among latitudes in neonates, suggesting genetic differences among populations, but adults showed no latitudinal differences. In combination, the increased thermal tolerance of neonates and the age-specific response to latitude suggest individuals may be most sensitive to selection on thermal tolerance as neonates. Although latitudinal differences exist in neonates, their tolerances were not ranked according to latitude, suggesting the effects of some other local factor (e.g., microclimate) may be important. Lastly, among neonates, females tolerate higher temperatures than males.

Adaptation, Physiological↗
PubMed2004

Feeding behavior and venom toxicity of coral snake Micrurus nigrocinctus (Serpentes: Elapidae) on its natural prey in captivity.

The feeding behavior and venom toxicity of the coral snake Micrurus nigrocinctus (Serpentes: Elapidae) on its natural prey in captivity were investigated. Coral snakes searched for their prey (the colubrid snake Geophis godmani) in the cages. Once their preys were located, coral snakes stroke them with a rapid forward movement, biting predominantly in the anterior region of the body. In order to assess the role of venom in prey restraint and ingestion, a group of coral snakes was 'milked' in order to drastically reduce the venom content in their glands. Significant differences were observed between snakes with venom, i.e., 'nonmilked' snakes, and 'milked' snakes regarding their behavior after the bite. The former remained hold to the prey until paralysis was achieved, whereas the latter, in the absence of paralysis, moved their head towards the head of the prey and bit the skull to achieve prey immobilization by mechanical means. There were no significant differences in the time of ingestion between these two groups of coral snakes. Susceptibility to the lethal effect of coral snake venom greatly differed in four colubrid species; G. godmani showed the highest susceptibility, followed by Geophis brachycephalus, whereas Ninia psephota and Ninia maculata were highly resistant to this venom. In addition, the blood serum of N. maculata, but not that of G. brachycephalus, prolonged the time of death of mice injected with 2 LD(50)s of M. nigrocinctus venom, when venom and blood serum were incubated before testing. Subcutaneous injection of coral snake venom in G. godmani induced neurotoxicity and myotoxicity, without causing hemorrhage and without affecting heart and lungs. It is concluded that (a) M. nigrocinctus venom plays a role in prey immobilization, (b) venom induces neurotoxic and myotoxic effects in colubrid snakes which comprise part of their natural prey, and (c) some colubrid snakes of the genus Ninia present a conspicuous resistance to the toxic action of M. nigrocinctus venom.

Animals↗
PubMed2005

A case of fatal progressive neuropathy. Delayed consequence of multiple bites of a non-venomous snake?

Neurotoxicity is a common feature of the acute syndrome of systemic snake envenomation. In this report a patient with delayed onset of neurological manifestations after multiple bites of a non-venomous snake is described. The neurological cascade consisted of segmental painful muscle spasms presented several days after the snake bites, which gradually subsided and within a month was followed by muscle weakness in the legs. The latter progressed to flaccid quadriplegia with additional respiratory and autonomic failure leading to death after total disease duration of 5.5 months. The possibility that this unique neurological syndrome of fatal progressive neuropathy could be caused by either tetanus or an immune reaction initiated by snakebites is discussed.

Aged↗
PubMed2005

Organochlorine detection in the shed skins of snakes.

Lizards and snakes eliminate heavy metals in their shed skins. There are no data with regard to reptilian skin as a depuration route for organochlorine (OC) compounds; however, birds deposit OCs in feathers. Corn snakes (Elaphe guttata) were therefore fed thawed mice that had been injected with a mixture of alpha-chlordane, Aroclor 1254 (a polychlorinated biphenyl (PCB) mixture), and lindane (gamma-hexachlorocyclohexane) at 2, 8, and 4 mg/kg, respectively. Feeding of contaminated mice occurred on the first weekly feeding of each month, with remaining weekly feedings consisting of noncontaminated mice, and was continued for 6 months. Shed skins were evaluated in a multiresidue OC scan by gas chromatography. All three chemical contaminants were readily detected in the shed skins: chlordane, 0.155-0.213 ppm; PCB, 3.49-7.01 ppm; lindane: 0.028-0.042 ppm. These data suggest that the shed skin of snakes may serve as an elimination route for OC contaminants and as such may have utility as a noninvasive, nondestructive indicator tissue for assessing environmental contamination.

Animal Feed↗
PubMed2006

Excretion of three heavy metals in the shed skin of exposed corn snakes (Elaphe guttata).

Three heavy metals of regional environmental concern-lead, cadmium, and mercury-are frequently evaluated as part of wildlife bioaccumulation and health monitoring studies. Shed skins of snakes may represent a useful, nonlethal biomarker tissue for such determinations. In the present study, these three heavy metals were injected into thawed mice that were fed to corn snakes (Elaphe guttata) such that snakes received 2mg/kg of each metal/month. Shed skins were collected over a period of 34 weeks, and levels of the heavy metals were determined in skins by atomic absorption methods. Each heavy metal was readily detected in the shed skins, with mean levels as follows: lead 234 ppb, cadmium 806 ppb, and mercury 403 ppb. Control snakes that were fed nonmetal-injected mice produced shed skins that contained mean levels of 6, 12, and 20 ppb lead, cadmium, and mercury, respectively. These detection levels corresponded closely to expected trace metal contents of mouse chow and tap water. These results demonstrate that dietary metal exposure can be readily detected in shed snake skins, including at trace levels of exposure that may be consistent with, or below, environmental exposures.

Animals↗
PubMed2005

Differential swimming performance of two natricine snakes exposed to a cholinesterase-inhibiting pesticide.

Environmental contaminants have direct effects on organisms at the molecular, cellular, and tissue levels, but the net results of these sub-organismal effects are only consequential to exposed populations if they alter organism-level traits that ultimately influence fitness (e.g., growth, locomotor performance, reproduction, and survival). Here, we explore the possibility that the swimming performance of neonate black swamp snakes (Seminatrix pygaea) and diamondback water snakes (Nerodia rhombifer) may be affected by exposure to carbaryl (2.5 and 5.0 mg/L). The highest concentration of carbaryl caused greater reductions in swim velocity in S. pygaea than in N. rhombifer. Most individuals recovered from the effects of carbaryl on swimming performance within 96 h, but recovery was significantly slower in S. pygaea than in N. rhombifer. We hypothesize that the sensitivity of S. pygaea may arise from its highly permeable integument compared to other natricines. Our findings suggest that performance can serve as an ecologically relevant response to contaminant exposure in reptiles and warrants further study.

Animals↗
PubMed2005

Chemoarchitecture and afferent connections of the "olfactostriatum": a specialized vomeronasal structure within the basal ganglia of snakes.

The olfactostriatum, a portion of the striatal complex of snakes, is the major tertiary vomeronasal structure in the ophidian brain, receiving substantial afferents from the nucleus sphericus, the primary target of accessory olfactory bulb efferents. In the present study, we have characterized the olfactostriatum of garter snakes (Thamnophis sirtalis) on the basis of chemoarchitecture (distribution of serotonin, neuropeptide Y and tyrosine hydroxylase) and hodology (afferent connections). The olfactostriatum is densely immunoreactive for serotonin and neuropeptide Y and shows moderate-to-weak immunoreactivity for tyrosine hydroxylase. In addition to afferents from the nucleus sphericus, the olfactostriatum receives inputs from the dorsal and lateral cortices, nucleus of the accessory olfactory tract, external and dorsolateral amygdalae, dorsomedial thalamic nucleus, ventral tegmental area and raphe nuclei. Double labeling experiments demonstrated that the distribution of serotonin and neuropeptide Y in this area almost completely overlaps the terminal field of projections from the nucleus sphericus. Also, serotonergic and dopaminergic innervation of the olfactostriatum likely arise, respectively, from the raphe nuclei and the ventral tegmental area, whereas local circuit neurons originate the neuropeptide Y immunoreactivity. These results indicate that the olfactostriatum of snakes could be a portion of the nucleus accumbens, with features characteristic of the accumbens shell, devoted to processing vomeronasal information. Comparative data suggest that a similar structure is present in the ventral striatum of amphibians and mammals.

Afferent Pathways↗
PubMed2005

Efferent connections of the "olfactostriatum": a specialized vomeronasal structure within the basal ganglia of snakes.

The olfactostriatum is a portion of the basal ganglia of snakes that receives substantial vomeronasal afferents through projections from the nucleus sphericus. In a preceding article, the olfactostriatum of garter snakes (Thamnophis sirtalis) was characterized on the basis of chemoarchitecture (distribution of serotonin, neuropeptide Y and tyrosine hydroxylase) and pattern of afferent connections [Martinez-Marcos, A., Ubeda-Banon, I., Lanuza, E., Halpern, M., 2005. Chemoarchitecture and afferent connections of the "olfactostriatum": a specialized vomeronasal structure within the basal ganglia of snakes. J. Chem. Neuroanat. 29, 49-69]. In the present study, its efferent connections have been investigated. The olfactostriatum projects to the main and accessory olfactory bulbs, lateral cortex, septal complex, ventral pallidum, external, ventral anterior and dorsolateral amygdalae, bed nucleus of the stria terminalis, preoptic area, lateral posterior hypothalamic nucleus, ventral tegmental area, substantia nigra and raphe nuclei. Tracer injections in the nucleus accumbens proper, a structure closely associated with the olfactostriatum, result in a similar pattern of efferent connections with the exception of those reaching the main and accessory olfactory bulbs, lateral cortex, external, ventral anterior and dorsolateral amygdalae and bed nucleus of the stria terminalis. These data, therefore, help to characterize the olfactostriatum, an apparently specialized area of the nucleus accumbens. Double labeling experiments after tracer injections in the nucleus sphericus and the lateral posterior hypothalamic nucleus demonstrate a pathway between these two structures through the olfactostriatum. Injections in the olfactostriatum and in the medial amygdala show parallel projections to the lateral posterior hypothalamic nucleus. Since this hypothalamic nucleus has been previously described as projecting to the hypoglossal nucleus, both, the medial amygdala and the olfactostriatum may mediate vomeronasal influence on tongue-flick behavior.

Amygdala↗
PubMed2003

Effects of ultraviolet B on epidermal morphology, shedding, lipid peroxide, and antioxidant enzymes in Cope's rat snake (Elaphe taeniura).

Cope's rat snakes (Elaphe taeniura) favor to expose under sunlight in order to increase their body temperature simultaneously increasing the risk of skin damage by ultraviolet B (UVB) irradiation. We have investigated the effects of UVB irradiation on their skin. Results show that the UVB transmission of the keratinous layer was only 5.1+/-0.36%. The peak of epidermal damage and malondialdehyde (MDA) content, a product of lipid peroxidation, simultaneously occurred 72-96, 48 or 24 h after exposure to 300, 500 and 800 mJ/cm2 of UVB radiation, respectively. Superoxide dismutase (SOD) activity was inhibited by UVB and the lowest activity occurred 24, 48, 12 and 12 h after exposure to 110, 300, 500 and 800 mJ/cm2 of UVB, respectively. SOD activity recovered later to some extent but mostly remained below control level. After exposure to different doses of UVB radiation, catalase (CAT) activity was inhibited immediately, and then gradually recovered and even increased to peak levels above control level. The highest CAT levels accompanied the most serious damage of skin morphology. Later on, CAT activity decreased and recovered again close to or below control level, which was accompanied by shedding off the damaged epidermal complex. This indicated that the epidermal damage induced by UVB is closely related to lipid peroxidation, where CAT acts as a primary antioxidant enzyme. Moreover, the keratinous layer protects the viable cell layer against UVB damage as well.

Animals↗
PubMed2006

Isolation and characterization of rufoxin, a novel protein exhibiting neurotoxicity from venom of the psammophiine, Rhamphiophis oxyrhynchus (Rufous beaked snake).

Colubrid snake venoms potentially represent a vast source of novel biological actives and structural motifs owing to their diverse phylogeny. The present study describes the identification of rufoxin, a neurotoxin from the venom of Rhamphiophis oxyrhynchus (Rufous beaked snake) which is a member of the African colubrid lineage, the psammophiines. Rufoxin (1 microM) displayed reversible post-synaptic neurotoxic activity as evidenced by significant inhibition of indirect twitches and responses to exogenous nicotinic agonists in the chick biventer cervicis nerve-muscle preparation. Rufoxin (0.1-1.0 microM) also caused a rightward parallel shift of cumulative concentration-response curves to carbachol (CCh; 0.6-80 microM) without a significant depression of the maximum response, suggestive of classical competitive antagonism at the skeletal muscle nicotinic receptor. Rufoxin lacks NH(2)-terminal sequence homology to previously identified snake venom toxins. This work indicates a wider distribution of neurotoxins across the advanced snake superfamily than previously described.

Analysis of Variance↗
PubMed2006

Characterization of novel DNA-binding proteins expressed in snake oocyte cDNA library.

DNA-binding proteins play pivotal roles in transcription, DNA-replication, recombination/repair and determine cell-fate in all physiological conditions of differentiation, development and disease. As they are present in extremely small amounts in cells, their isolation/identification, particularly from scarce tissues is impracticable. We cloned the cDNA pool of snake (Ptyas mucosus) oocytes (a scarce tissue) in bacteria, overexpressed total library, purified and identified DNA-binding proteins expressed in the library. Although snake databases do not exist, we identified 23 DNA-binding proteins, obtained 10-15 amino acids internal sequence tags of six of them and succeeded in PCR amplification of the cDNAs of five proteins. We employed electro spray ionization mass spectrometry, matrix assisted laser desorption/ionization time of flight and analyzed the results by peptide mass fingerprint (PMF) and various sequence BLAST analyses. Proteins identified were largely unanimous between the PMF and BLAST analyses. We expect these proteins to play important roles in snake embryonic development and differentiation. We arrived at homologous mouse proteins to some of the identified snake proteins and are working towards characterizing their structure and physiological function. Similar approaches shall prove valuable in isolation and identification of important factors from scarce carcinoma tissues, mammalian oocytes and early embryos, which might be involved in important functions like nuclear reprogramming, embryonic development and differentiation.

Animals↗
PubMed2006

Adaptive evolution of tetrodotoxin resistance in animals.

Tetrodotoxin (TTX), first isolated from pufferfish (tetraodontids), is a highly potent neurotoxin that selectively binds to voltage-gated sodium channels (Na(v)) in muscle and nerve tissues causing paralysis and death. Saxitoxin (STX) is a TTX-related neurotoxin produced by dinoflagellates. Recent investigations have implicated diverse substitutions in the P-loop regions of skeletal muscle and neuronal Na(v) channels in the convergent evolution of neurotoxin resistance in pufferfish, garter snakes and softshell clams, which has enabled them to feed on TTX- and STX-bearing organisms.

Amino Acid Sequence↗
PubMed2004

Muscle and skin necrotizing and edema-forming activities of Duvernoy's gland secretion of the xenodontine colubrid snake Philodryas patagoniensis from the north-east of Argentina.

Philodryas patagoniensis is a colubrid snake spread by all South America, but very little is known about the composition and biological activities of its Duvernoy's gland secretion. In order to characterize it, we studied edematogenic, dermonecrotic and myonecrotic activities. For edematogenic activity, solutions containing different amounts of secretion were injected s.c. in the right foot pad of mice, both feet were subsequently cut off and weighed individually. For myonecrotic activity, mice were injected i.m. with solutions containing 40 microg of secretion, and at various time intervals mice were bled to determine serum creatine kinase activity and gastrocnemius muscles were removed for microscopic examination (Hematoxylin-Eosin stain). For dermonecrotic activity, solutions containing different amounts of secretion were injected into the shaved dorsal skin of mice; the necrotic lesion was measured on the inner surface of the skin and trimmed for microscopic examination (Hematoxylin-Eosin stain). Phospholipase A(2) activity was evaluated using a kinetic method. Results showed that P. patagoniensis Duvernoy's gland secretion exhibits a high edematogenic activity and moderate myonecrotic and dermonecrotic activities, while lacking phospholipase A(2) effect. Regarding edema, a 30% increase in the weight was produced by injecting 0.26 microg of Duvernoy's gland secretion. Microscopically, myonecrosis reached its highest intensity 12 h after injection, which was also demonstrated by serum creatine kinase levels. Dermonecrosis was proportional to the amount of secretion injected, with a minimum necrotizing dose of 15.7 microg. Myonecrotic, edematogenic and dermonecrotic activities were inhibited when the secretion was pre-incubated with 1 mM Na(2)EDTA. This suggests that the enzymes responsible for those activities are mostly metalloproeinases. All the studies carried out up to now demonstrate the potential toxicity of P. patagoniensis Duvernoy's gland secretion (which inhabits the north-east region of Argentina) and that the local lesions caused by this colubrid snake are very similar to those found in bothropic accidents. This latter suggests a more careful evaluation of the victims when considering the medical treatment to be adopted.

Analysis of Variance↗
PubMed2005

A biochemical and pharmacological examination of Rhamphiophis oxyrhynchus (Rufous beaked snake) venom.

The venom of R. oxyrhynchus, a member of the psammophiine subfamily of the colubrid assemblage, was examined for biological activity using biochemical and pharmacological techniques. Venom displayed a high protein content, a complex electrophorectic profile and PLA2 activity but no detectable proteolytic or haematological activities. In the chick biventer cervicis nerve muscle preparation, venom (1-10 microg/ml) displayed postsynaptic neurotoxic activity as evidenced by inhibition of indirect (0.1 Hz, 0.2 ms, supramaximal V) twitches and responses to exogenous acetylcholine (1 mM) and carbachol (20 microM). This inhibitory effect was poorly reversible by washing. Venom (30-50 microg/ml) caused a rapid and readily reversible inhibition of direct (0.1 Hz, 2 ms, supramaximal V) twitches of the chick biventer cervicis nerve muscle preparation without morphological changes to the muscle fibers. Venom (30-100 microg/ml) inhibited electrically-evoked (0.2 Hz, 0.3 ms, 70-100 V) twitches of the prostatic segment of the rat vas deferens. This inhibitory effect was not significantly attenuated by 8-phenyltheophylline (8-PT; 20 microM), idazoxan (1 microM), a combination of ranitidine (0.2 microM) and thioperamide (10 microM) or capsazepine (10 microM). Venom (5 mg/kg) induced hypotension with subsequent cardiovascular collapse in the anaesthetised rat. The cardiovascular collapse was prevented by artificial respiration of the animals prior to venom administration. The biological activities demonstrated by R. oxyrhynchus venom may aid in prey envenomation strategies such as prey immobilisation. This study provides further evidence that colubrid venoms are comprised of multiple components which can display a variety of actions, some of which may be novel, therefore reinforcing the largely untapped potential of colubrid venoms.

Animals↗
PubMed2005

Pharmacological characterisation of a neurotoxin from the venom of Boiga dendrophila (mangrove catsnake).

In this study, we have pharmacologically characterised boigatoxin-A, a three finger toxin isolated from the venom of the colubrid, Boiga dendrophila (Mangrove catsnake). In the chick biventer cervicis nerve-muscle preparation boigatoxin-A (1 microM) displayed poorly reversible postsynaptic blockade as evidenced by the inhibition of indirect (0.1 Hz, 0.2 ms, supramaximal V) twitches and responses to exogenous acetylcholine (1 mM) and carbachol (20 microM). Boigatoxin-A (0.3-0.5 microM) caused a concentration-dependent depression of the maximum response of cumulative concentration response curves to CCh (0.6-80 microM). Boigatoxin-A (1 microM) induced readily reversible inhibition of electrically evoked (0.2 Hz, 0.3 ms, 70-100 V) twitches of the prostatic segment of the rat vas deferens. This inhibition was not significantly attenuated by 8-phenyltheophylline (20 microM) or idazoxan (1 microM). Boigatoxin-A (1 microM) did not affect alpha,beta-mATP (10 microM) or noradrenaline (25 microM) responses in unstimulated epididymal segments of the rat vas deferens. Our data suggests that this toxin has weak postsynaptic neurotoxicity in skeletal muscle and also prejunctional neurotoxic activity in the smooth muscle of the rat vas deferens to inhibit the release of neurotransmitter(s), but not via prejunctional purinergic or adrenergic receptors. This is the first report of such activity for a toxin isolated from snake venom and reinforces the largely untapped potential of colubrid venoms.

Animals↗
PubMed2005

Duvernoy's gland secretion of Philodryas patagoniensis from the northeast of Argentina: its effects on blood coagulation.

Duvernoy's gland secretion of Philodryas patagoniensis exhibits high hemorrhagic activity, containing enzymes that are able to degrade the vascular wall. In this work we aim to determine if the secretion can also affect the hemostatic system by causing changes in blood coagulation. Procoagulant and coagulant activities were evaluated on plasma and fibrinogen, respectively. The delay in the thrombin clotting time of fibrinogen previously incubated with the secretion was also determined. Specific hydrolysis of fibrinogen and fibrin incubated with the secretion at different time intervals was shown by electrophoresis on polyacrylamide gel. To determine the structural characteristics of the enzymes degrading fibrinogen and fibrin, secretion were incubated in the presence of 45 mM Na(2)EDTA, 40 mM Benzamidine, and/or 2 mM PMSF before the incubation with fibrinogen or fibrin, respectively. The effect in vivo was investigated in adult male rats injected with different dose of secretion, aliquots of blood were withdrawn at different time intervals, and the fibrinogen concentration was determined. Duvernoy's gland secretion of P. patagoniensis did not clot plasma or fibrinogen. It exhibited a potent fibrinogenolytic activity degrading the Aalpha-chain faster than the Bbeta-chain, whereas gamma-chain was resistant. This latter corresponded with a strong delay in the thrombin clotting time of fibrinogen (4 mg/ml) pre-incubated with the secretion, being 9.53 microg the amount of protein from Duvernoy's gland secretion that increased the thrombin clotting time from 20 to 60 s. In vivo, the loss of rat plasma fibrinogen was proportional to the amount of secretion injected. The secretion also hydrolyzed fibrin degrading the alpha-monomer. Inhibition studies with Na(2)EDTA, Benzamidine, and/or PMSF showed that metalloproteinases and serinoproteinases are the main enzymes responsible for the hydrolyzing activity on fibrinogen and fibrin. All these results demonstrate that Duvernoy's gland secretion of P. patagoniensis possesses enzymes able to hydrolyze plasma components playing a relevant role in the blood coagulation. These hydrolyzing activities and those acting on the wall of blood vessels let the secretion exhibit a high hemorrhagic activity, which may result in permanent sequelae or even cause the death of the victims bitten by this colubrid snake.

Analysis of Variance↗