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PubMed2026

First insights into the role of evolutionary history in shaping venom composition of Vipera ammodytes.

Understanding intraspecific venom variation requires distinguishing the contributions of neutral population history from natural selection. This study aims to determine whether venom variation in Vipera ammodytes species complex is structured across eight phylogenetic groups. Despite a complex evolutionary history, venom composition did not differ among phylogenetic groups within the analytical framework used, suggesting that shared ancestry alone does not explain venom variation. Whether local adaptation to environmental conditions explains the observed variation remains an open question for future studies.

Animals↗

PubMed2005

Snake venomics: comparative analysis of the venom proteomes of the Tunisian snakes Cerastes cerastes, Cerastes vipera and Macrovipera lebetina.

The protein composition of the crude venoms of the three most important vipers of Tunisia was analyzed by RP-HPLC, N-terminal sequence analysis, MALDI-TOF mass determination, and in-gel tryptic digestion followed by PMF and CID-MS/MS of selected peptide ions in a quadrupole-linear IT instrument. Our results show that the venom proteomes of Cerastes cerastes, Cerastes vipera, and Macrovipera lebetina are composed of proteins belonging to a few protein families. However, each venom showed distinct degree of protein composition complexity. The three venoms shared a number of protein classes though the relative occurrence of these toxins was different in each snake species. On the other hand, the venoms of the Cerastes species and Macrovipera lebetina each contained unique components. The comparative proteomic analysis of Tunisian snake venoms provides a comprehensible catalogue of secreted proteins, which may contribute to a deeper understanding of the biological effects of the venoms, and may also serve as a starting point for studying structure-function correlations of individual toxins.

Amino Acid Sequence↗

PubMed1996

Complete primary structure of the subunits of heterodimeric phospholipase A2 from Vipera a. zinnikeri venom.

Molecular weight determination of dimeric phospholipase A2 from Vipera aspis zinnikeri venom (PLA2-I) was performed with electrospray ionization mass spectrometry (ESI-MS). PLA2-I consists of an acidic and a basic subunit (subunit A and B), which bind noncovalently and dissociate under highly acidic conditions. The protonated molecular ions of subunit A and B were measured to be 13,655.9 and 13,842.6, respectively. The complete amino acid sequence was also determined by Edman sequencing of the S-pyridylethylated derivative and its peptides derived from enzymatic digestion. Both subunit A and B consist of 122 amino acid residues and contain 7 disulfide bonds. The theoretical molecular mass calculated from the primary structure completely agree with the ESI-MS data. THe sequential homology between subunit A and B was 63.9%; however, subunit A lacks enzymatic and biological activities that are characteristic for phospholipase A2. Although the amino acid residues essential for calcium binding (Tyr28, Gly30, Gly32, and Asp49) and catalysis (Asp92) were preserved, replacement of functionally important residue (His48) for catalysis with a Gln was found in subunit A. In addition, substitution of acidic amino acid residues for basic ones and hydrophilic residues for hydrophobic ones were observed in subunit A. Presumably, these changes in the primary structure of subunit A resulted in the loss of enzymatic activity and an increase in the binding ability with subunit B.

Amino Acid Sequence↗

PubMed1996

cDNA cloning and deduced amino acid sequence of fibrinolytic enzyme (lebetase) from Vipera lebetina snake venom.

The complete amino acid sequence of lebetase is deduced from the nucleotide sequence of a cDNA clone isolated by screening a venomous gland c DNA library of Central Asian Vipera lebetina snake. The cDNA sequence with 2011 basepairs encodes an open reading frame of 478 amino acids which includes an 18 amino acid signal peptide, plus an 175 amino acid segment of zymogen-like propeptide, a mature protein of 204 amino acids, a spacer of 18 amino acids and a disintegrin-like peptide of 63 amino acids. The mature protein lebetase as isolated from the crude venom has the molecular weight of approximately 23.7 kD and, thus, lebetase as well as several other snake venom metalloproteinases is translated as a precursor protein, which may be processed posttranslationally. The lebetase proprotein has a "cysteine switch" motif (PKMCGV) similar to that involved in the activation of matrix metalloproteinase zymogens. The mature protein (residues 223-427) shows the strongest similarity with fibrolase (63% identity), fibrinolytic enzyme from Agkistrodon contortrix contortrix venom. The metalloproteinase domain has a typical zinc-chelating sequence (HEXXHXXGXXH). In the disintegrin-like domain of protein, the RGD sequence is replaced by VGD.

Amino Acid Sequence↗

PubMed1996

Protein and cDNA structures of an acidic phospholipase A2, the enzymatic part of an unusual, two-component toxin from Vipera palaestinae.

In the venom of Vipera palaestinae an unusual, two-component toxin was found. The two components of the toxin are an acidic phospholipase A2 (VpaPLA2) and a basic protein, both with an apparent molecular mass of about 15 kDa. Each component alone is not toxic; however, their mixture is lethal. We have determined the amino acid and cDNA sequences of VpaPLA2. The protein primary structure was solved by sequencing the peptides generated by chemical cleavage of the molecule using CNBr, formic acid and hydroxylamine-hydrochloride and by enzymatic fragmentation with trypsin and chymotrypsin. VpaPLA2 consists of 122 amino acid residues and has all the structural characteristics of subgroup IIA PLA2s. It shows the highest amino acid similarity to a non-toxic phospholipase A2 from Eristocophis macmahoni (82%), whereas the most similar toxic phospholipases A2 share about 70% of residues with VpaPLA2. The substitution of His20 for a hydrophobic residue (Leu) in VpaPLA2 might be one of the reasons that its complex with the basic protein could not be observed.

Amino Acid Sequence↗

PubMed2000

Purification and characterization of a growth factor-like which increases capillary permeability from Vipera lebetina venom.

We have investigated the effect of Vipera lebetina venom on capillary permeability and isolated an increasing capillary permeability protein (ICPP) which is devoid of arginine ester hydrolase and phospholipase A2 activities. This protein was purified with a yield of about 0.2% by fast protein liquid chromatography (FPLC) using successively Superose 12, Mono Q, and Mono S columns and by high-pressure liquid chromatography (HPLC) on a C8 reverse-phase column. The purified protein migrated on SDS-PAGE as a band of about 27 kDa under nonreducing conditions and as a band of about 16 kDa under reducing conditions. Chromatography on a C8 column of reduced and alkylated protein yielded a single peak suggesting that this protein is homodimeric. This protein was refractory to Edman degradation chemistry. We used successfully a chemical unblocking involving the incubation of the protein with HCl in anhydrous methanol. The N-terminal amino acid sequence clearly shows considerable similarity to that of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF).

Amino Acid Sequence↗

PubMed1993

Sexual activity and plasma levels of sex steroids in the aspic viper Vipera aspis L. (Reptilia, Viperidae).

Reproductive behavior and associated sexual activity was studied in individual male and female Vipera aspis over a 3-year period in western France in an attempt to correlate mating behavior with blood levels of gonadal sex steroids. Males had higher average levels of both testosterone (T) and 5 alpha-dihydrotestosterone (DHT) than females. Levels of progesterone (P) did not differ significantly between the two sexes but estradiol-17 beta (E2) concentrations were significantly higher in females during the season of mating. Spring mating behavior and copulation in males was associated with significantly increased levels of T and DHT, compared with postmating males; and a similar, but not significant trend, was evident with autumnal mating. The only statistically significant hormonal difference detected in males showing no sexual activity in autumn, was an elevated level of E2 at 0.52 +/- 0.20 ng/ml compared with 0.09 +/- 0.03 ng/ml in spring-breeding males (P = 0.05). Estrus in females is associated with increased levels of all four steroids but significant only for E2 and DHT. Levels of P were significantly reduced in females displaying seasonal anestrous in the spring immediately following reproduction. Females not displaying estrus in either spring or autumn had significantly lower plasma DHT and E2. Although mating behavior in males is associated statistically with elevated levels of T and DHT, a tight correlation is not obvious at the individual level, suggesting that increased concentrations of androgens are a necessary, but not sufficient, condition for mating. The data from V. aspis suggest that, as in a number of other reptilian species, high circulating levels of androgens function to "condition" or "organize" sexual behavior in males which may be displayed at some later time, well after actual levels have fallen, thus engendering the impression that reproductive behavior may be temporally dissociated from essential hormonal stimuli.

Animals↗

PubMed1994

The influence of body condition on 17-beta estradiol levels in relation to vitellogenesis in female Vipera aspis (Reptilia, Viperidae).

Seventy-six wild Vipera aspis females were caught over 3 years and placed in outdoor enclosures; 39 reproduced and 37 did not. Almost all the reproductive females had a body condition index (BCI) greater than 0.70 when vitellogenesis began. Monthly blood samples were taken by cardiac puncture. The main plasma parameters of vitellogenesis were measured by spectrophotometry: total plasma calcium, phosphorus, phospholipids, cholesterol, triglycerides, proteins, and albumin. Plasma 17-beta estradiol levels were determined by RIA. Vitellogenesis started soon after hibernation in reproductive females with very high 17-beta estradiol concentrations (average of 4.00 ng/ml) and there was a marked mobilization of maternal reserves (fat bodies, liver, and vertebral bone) associated with very high values of plasma calcium, phosphorus, phospholipids, cholesterol, triglycerides, and proteins. The kinetics of the main plasma components were described throughout the vitellogenesis period (from March to early June), when all plasma parameters differed markedly between reproductive and nonreproductive females. After ovulation, the differences between the two groups of females disappeared except in the case of albumin, which remained at a very low level in reproductive females for 6 months. All nonreproductive females had low 17-beta estradiol plasma levels during vitellogenesis (average of 0.08 ng/ml) and there was no suggestion of mobilization of maternal reserves. After vitellogenesis plasma concentrations of estradiol were low in reproductive (an average of 0.08 ng/ml) and in nonreproductive animals (0.06 ng/ml). Five nonreproductive females kept in the laboratory were estrogenized by 17-beta estradiol silastic implants. The 17-beta estradiol concentrations were close to those measured in reproductive females during vitellogenesis. Maternal reserves were mobilized, with almost all metabolic parameters exhibiting the vitellogenic pattern. When the silastic implants were removed, 17-beta estradiol concentrations dropped sharply to a basal level, but the other components were maintained near the vitellogenic values for several months. In contrast to previous studies on viviparous snakes, these results suggest that in V. aspis 17-beta estradiol levels are linked strictly to vitellogenesis.

Animals↗

PubMed2001

Changes in plasma progesterone in relation to vitellogenesis and gestation in the viviparous snake Vipera aspis.

Viviparity has arisen from oviparity on more than 100 independent occasions in squamate reptiles, providing an unique opportunity to investigate the ways in which endocrine control of gestation length has been modified by natural selection during this major transition in reproductive modes. Intuitively, the evolution of viviparity might be expected to involve an increasingly important role for the steroid hormone progesterone, rather than estradiol. Unfortunately, published data on this topic in snakes are scarce and often contradictory. Females of the viviparous snake Vipera aspis reproduce with a lower than annual frequency, providing the opportunity to examine steroid profiles simultaneously in vitellogenic, pregnant, and postparturient versus nonreproductive females. From 1990 to 1994, more than 500 blood samples were collected from more than 100 females. Progesterone, estradiol-17beta, and several plasma metabolites were assayed by radioimmunoassay and spectrophotometry. In contrast to earlier studies, we found significant differences between plasma progesterone levels in reproducing (10.5 +/- 9.1 ng ml(-1), N = 168) and nonreproducing (5.1 +/- 4.2 ng ml(-1), N = 121) females. Estradiol reached high levels during vitellogenesis (4.8 +/- 4.0 ng ml(-1), N = 16), and progesterone levels increased during gestation (from 3.7 ng ml(-1) before gestation to 18.7 ng ml(-1) at midgestation). However, experimental elevation of plasma progesterone levels with implants (up to 44.4 ng ml(-1)) did not modify plasma metabolite levels or delay parturition.

Animals↗

PubMed2002

Fat is sexy for females but not males: the influence of body reserves on reproduction in snakes (Vipera aspis).

Reproduction is energetically expensive for both sexes, but the magnitude of expenditure and its relationship to reproductive success differ fundamentally between males and females. Males allocate relatively little to gamete production and, thus, can reproduce successfully with only minor energy investment. In contrast, females of many species experience high fecundity-independent costs of reproduction (such as migration to nesting sites), so they need to amass substantial energy reserves before initiating reproductive activity. Thus, we expect that the relationship between energy reserves and the intensity of reproductive behavior involves a threshold effect in females, but a gradual (or no) effect in males. We tested this prediction using captive vipers (Vipera aspis), dividing both males and females into groups of high versus low body condition. Snakes from each group were placed together and observed for reproductive behavior; sex-steroid levels were also measured. As predicted, females in below-average body condition had very low estradiol levels and did not show sexual receptivity, whereas males of all body condition indices had significant testosterone levels and displayed active courtship. Testosterone levels and courtship intensity increased gradually (i.e., no step function) with body condition in males, but high estradiol levels and sexual receptivity were seen only in females with body reserves above a critical threshold.

Adipose Tissue↗

PubMed1993

Crystals of phospholipase A2 inhibitor. The non-toxic component of vipoxin from the venom of Bulgarian viper (Vipera ammodytes).

Phospholipase A2 inhibitor, the non-toxic, acidic component of vipoxin from the venom of Bulgarian viper (Vipera ammodytes), has been crystallized. The tetragonal crystals obtained, exhibit the symmetry of space group P4(1)22 (or 4(3)22) with unit cell dimensions a = b = 59.9 A; c = 141.1 A; alpha = beta = gamma = 90 degrees. For two molecules per asymmetric unit, this would give Vm = 2.3 A3/Da, indicating normal packing. The crystals diffract to 2.5 A and a native data set to 3.2 A resolution has been collected.

Animals↗

PubMed1987

Experimental transmission of Sarcocystis muriviperae n. sp. to laboratory mice by sporocysts from the Palestinian viper (Vipera palaestinae): a light and electron microscope study.

Sporocysts collected from the feces of a Palestinian viper (Vipera palaestinae) were administered orally to species of various rodent genera such as Mus, Microtus, Mastomys, Meriones and Oryctolagus. Infections developed only in laboratory mice (Mus musculus). This investigation established the life cycle of Sarcocystis muriviperae in the laboratory. S. muriviperae is described as a new species, based on light and electron microscopic observations and repeated transmission studies. Naturally and experimentally infected Palestinian vipers both excreted structurally identical sporocysts measuring 9.6 micron (8.8-10.5 microns) by 12.2 microns (11.7-12.9 microns). Sporulation inside the snakes' intestine is completed between 14 and 19 days post-inoculation (p.i.). Rosette-like schizogonic stages were found in the liver cells of laboratory mice 9-10 days after infection with sporulated sporocysts. Sarcocysts measured up to 1,000 microns in length on day 36 p.i. and were mainly filled with metrocytes. The septated sarcocysts found 136 or 165 days p.i. reached a length of 5-8 mm and a width of 150-400 microns. The primary sarcocyst wall formed cauliflower-like branched protrusions about 3.5 microns in length.

Animals↗

PubMed1994

Paternity determination in the adder (Vipera berus)--DNA fingerprinting or random amplified polymorphic DNA?

We performed breeding experiments with adders (Vipera berus) to determine whether multiple matings may result in multiple paternity. DNA fingerprinting of mothers, their offspring, and possible fathers using a polydinucleotide probe [(TG)n] gave a low overall similarity between unrelated individuals (0.18 +/- 0.07; SD) and an average of 17 bands that were male-specific. In no cases were there fewer than seven paternal-specific bands present in the fingerprint of an offspring, enabling us unambiguously to identify the biological father among five males. Multiple paternity was detected in the investigated broods with offspring sired exclusively by the captive males. PCR amplification of random amplified polymorphic DNA (RAPD) using 16 decamer primers gave 76 bands and an average similarity of 0.95 (+/- 0.01) between the males, which were collected at different, geographically well-separated localities. Although there were on average 8.3 (+/- 1.9) bands that differ between males in pairwise comparisons, there were only 1.9 (+/- 1.1) bands per male that are specific for a particular individual. Thus, RAPDs are adequate for paternity determination only in experiments with a low number of males, whereas DNA fingerprinting offers sufficient information to discriminate between large numbers of putative fathers.

Animals↗

PubMed1995

Effects of chlorpromazine on the enzymatic and toxic properties of the most basic phospholipase A2 from Vipera russelli snake venom.

The effect of chlorpromazine on various biological activities of phospholipase A2 VRV-PL-VIIIa from Vipera russelli snake venom was investigated. The drug inhibited the in vitro phospholipase A2 activity of the enzyme by 55%. The ID50 was found to be 1.15 microM. Increasing substrate concentration relieved the inhibition of phospholipase A2 activity by the drug indicating probable interaction with the substrate. The drug totally quenched the fluorescence intensity of the enzyme. Chlorpromazine increased the LD50 of the enzyme by 1.6 fold. The drug also inhibited hemolytic and anticoagulant potencies but failed to inhibit edema inducing activity and myotoxicity of the enzyme.

Animals↗

PubMed1985

Plasma aldosterone in newborn and adult Vipera aspis.

In the venomous viviparous snake Vipera aspis, plasma aldosterone concentration shows significant seasonal changes mainly related to temperature-dependent behavior. A difference is also present between newborns and adults. A correlation between sodium and potassium status and aldosterone plasma level in active and inactive life is suggested.

Aldosterone↗

PubMed1991

Structural changes of the periosteocytic area in Vipera aspis (L.) (Ophidia, Viperidae) bone tissue in various physiological conditions.

Microradiographs of ribs and vertebrae of the snake Vipera aspis, over an annual cycle, show a significant enlargement of the osteocytic lacunae in the winter months and, for the breeding females, during the period of embryo development. This enlargement is due to resorption of bone substance (periosteocytic osteolysis). The objection that such morphological findings could as well be explained by the formation of new, larger osteocytes derived from recent osteoblasts does not apply to the present animal model. No internal bone remodelling occurs during the annual seasonal cycle and therefore no new osteoblasts would have differentiated to osteocytes in the interior of the bone. In the vertebrae, an additional process is indicated as an area of decreased mineral density, termed demineralization halo, around the periosteocytic lacunae. An electron microscopy study suggests that this process of demineralization is not the first stage of periosteocytic resorption, but an additional process of demineralization. Thus, both osteolysis and demineralization halos in the perilacunar osteocytic region of the bone tissue represent reversible biological processes mediated by the osteocytes.

Animals↗

PubMed2001

Antagonization of TNF attenuates systemic hemodynamic manifestations of envenomation in a rat model of Vipera aspis snakebite.

OBJECTIVES: Tumor necrosis factor (TNF) has been reported as a mediator of local tissue injury following snake envenomation in an intact rat model. We investigated whether systemic release of TNF occurs following Vipera aspis envenomation. We further analyzed the possible connection between envenomation-related hemodynamic depression and TNF antagonization (TNF antibodies or soluble TNF receptor). DESIGN: A prospective, randomized, controlled experimental study using a rat model for snake envenomation. SETTINGS: A medical university hospital research laboratory. INTERVENTION: Eighty rats (300-400 g) were divided into four groups (n = 20): control and three experimental groups. Intramuscular injection of V. asis 500 microg/kg was administered to the three experimental groups: venom only (group 1), venom and 40 microg anti-TNF antibodies (group 2), venom and 250 microg soluble TNF receptor (p55-R; group 3). Hemodynamic parameters were monitored up to 4 h following venom injection. MEASUREMENTS AND RESULTS: A significant hemodynamic deterioration (reduction in heart rate and blood pressure) occurred 30 min following venom injection in group 1 compared to groups 2 and 3, where hemodynamic parameters remained stable throughout the 4 h observation period. Serum levels of TNF were detected 15 min after venom injection and peaked after 2 h at 485+/-12 pg/ml. CONCLUSIONS: The hemodynamic consequences of intramuscular injection of V. aspis venom can be blunted in a rat by systemic antagonization of TNF activity prior to venom injection. The poisonous hemodynamic effects of the V. aspis venom might be caused by systemic release of TNF.

Animals↗

PubMed2006

The postnatal development of the optic nerve of a reptile (Vipera aspis): A quantitative ultrastructural study.

The number of axons in the optic nerve of the ovoviviparous reptile Vipera aspis was estimated from electron micrographs taken during the first 5 weeks of postnatal life. One to two days after birth, the optic nerve contains about 170,000 fibres, of which about 9% are myelinated. At the end of the fifth postnatal week, the number of optic fibres has fallen to about 100,000, of which about 42% are myelinated. This fibre loss continues after the fifth postnatal week, since in the adult viper the nerve contains about 60,000 fibres, of which 85% are myelinated; overall, about 65% of the optic nerve fibres present at birth disappear before the number of axons stabilises at the adult level. This study shows, for the first time, that the mode of development of the visual axons of reptiles is not that of anamniote vertebrates but similar to that of birds and mammals.

Animals↗