Isolation and characterization of nerve growth factor from Vipera lebetina (snake) venom

Nerve growth factor from Vipera lebetina venom was purified by gel filtration and ion exchange chromatography steps. The NGF preparation obtained is a glycoprotein with weak arginine esterase activity. It hydrolyzes benzoylarginine ethyl ester (BAEE). Vipera lebetina NGF consists of multiple forms of protein with pI in the interval 9-10.5. All isoforms have identical molecular weights of 32,500.     

Snake venomics of the Armenian mountain vipers Macrovipera lebetina obtusa and Vipera raddei

Venoms from the Armenian mountain vipers Macrovipera lebetina obtusa and Vipera raddei were analyzed by RP-HPLC, N-terminal sequencing, MALDI-TOF mass fingerprinting and CID-MS/MS. The venom proteins of M.l. obtusa and V. raddei belong to 9 and 11 families, respectively. The two mountain viper venoms share bradykinin-potentiating/C-natriuretic peptides, and proteins from the dimeric distegrin, DC-fragment, CRISP, PLA(2), serine proteinase, C-type lectin-like, L-amino acid oxidase, and Zn(2+)-dependent metalloproteinase families, albeit each species exhibits distinct relative abundances. M.l. obtusa and V. raddei venoms contain unique components, e.g. the short disintegrin obtustatin in M.l. obtusa, and Kunitz-type serine proteinase inhibitor and VEGF-like molecules in V. raddei. The toxin formulation of M.l. obtusa and V. raddei venoms may be related to their adaptation to rocky mountain ecosystems. On the other hand, the possibility that the VEGF-like proteins from V. raddei underlie the reported potential therapeutic value of V. raddei venom for regenerating damaged peripheral nerves deserves further investigations. Using a similarity coefficient, we estimate that the similarity of venom proteins between M. l. obtusa and M. l. transmediterranea is less than 4%. Although this result would support the classification of M.l. obtusa and M.l. transmediterranea as different species, additional detailed genomic analyses are also required.     

Isolation and characterization of nerve growth factor from Vipera berus berus (common viper) venom

Nerve growth factor from Vipera berus berus venom was purified by gel filtration on Sephadex G-100 (superfine), ion-exchange-chromatography on DEAE-Sephadex A-50 and chromatofocusing on PBE 118. The Vipera berus berus venom NGF consists of multiple molecular forms with pls in the interval 9.1-9.7. All isoforms have identical mol. wts approximately 35,000 +/- 3000 (in gel filtration) and 17,000 +/- 2000, 15,000 +/- 2000 (by SDS electrophoresis with beta-mercaptethanol). V. berus berus venom NGF reacted with monoclonal antibodies against Viper lebetina NGF and caused differentiation of pheochromocytoma PC12 cells.     

Comparative analysis of the venom proteomes of Vipera ammodytes ammodytes and Vipera ammodytes meridionalis

The venom proteomics of Vipera ammodytes ammodytes and Vipera ammodytes meridionalis, snakes of public health significance and the most poisonous reptiles in Europe, were analyzed by FPLC, 2-D electrophoresis, sequence analysis, and MS/MS. FPLC analysis showed the presence of l-amino acid oxidase, monomeric and heterodimeric phospholipases A2, C-type lectin protein, and proteinases in the venom of V. a. ammodytes. Representatives of the same protein families were found in the venom of the other subspecies, V. a. meridionalis. N-terminally identical PLA2 neurotoxins were identified in both venoms. Difference in the PLA2 compositions of the venoms was also observed: a monomeric protein with phospholipase A2 activity, identical in the first 20 amino acid residues to the catalitically inactive acidic component of the heterodimeric PLA2 present in both venoms, was found only in that of V. a. meridionalis. Probably, this protein represents an intermediate form of the two components of the heterodimer. 2-D electrophoresis and MS/MS analysis showed that the two venoms shared a number of protein families: monomeric and heterodimeric Group II PLA2s, serine proteinases, Group I, II, and III metalloproteinases, l-amino acid oxidases (LAAOs), cysteine-rich secretory proteins, disintegrins, and growth factors. Totally, 38 venom components of the V. a. ammodytes, belonging to 9 protein families, and 67 components of the V. a. meridionalis venom belonging to 8 protein families were identified. The venom proteome of V. a. ammodytes shows larger diversity of proteins (139) in comparison to that of V. a. meridionalis (104 proteins). Most of the proteins are homologues of known representatives of the respective protein families. The protein compositions explain clinical effects of the V. ammodytes snakebites, such as difficulties in the breathing, paralysis, apoptosis, cloting disorders, hemorrhage, and tissue necrosis. The lists of secreted proteins by the two vipers can be used for further study of structure-function relationships in the toxins and for prediction and treatment of snakebite consequences.     

Purification and amino-acid sequence of a nerve growth factor from the venom of Vipera russelli russelli.

Nerve growth factor (NGF) was purified from the venom of Vipera russelli russelli by Sephadex G-50 gel filtration, S-Sepharose column chromatography and Blue-Sepharose CL-6B column chromatography. The purified NGF was found to be a glycoprotein, whose apparent molecular mass was estimated to be about 17.5 kDa by SDS-PAGE. The amino-acid sequence was determined by a combination of conventional methods. The V. r. russelli NGF was composed of 117 amino-acid residues with one residue, Asn-21, being N-linked glycosylated and the molecular mass of its protein portion was calculated to be 13,280 Da.     

Biphasic effect on platelet aggregation by phospholipase a purified from Vipera russellii snake venom

A basic phospholipase A was isolated from Vipera russellii snake venom. It induced a biphasic effect on washed rabbit platelets suspended in Tyrode's solution. The first phase was a reversible aggregation which was dependent on stirring and extracellular calcium. The second phase was an inhibitory effect on platelet aggregation, occurring 5 min after the addition of the venom phospholipase A without stirring or after a recovery from the reversible aggregation. The aggregating phase could be inhibited by indomethacin, tetracaine, papaverine, creatine phosphate/creatine phosphokinase, mepacrine, verapamil, sodium nitroprusside, prostaglandin E1 or bovine serum albumin. The venom phospholipase A released free fatty acids from synthetic phosphatidylcholine and intact platelets. p-Bromophenacyl bromide-modified venom phospholipase A lost its phospholipase A enzymatic and platelet-aggregating activities, but protected platelets from the aggregation induced by the native enzyme. The second phase of the venom phospholipase A action showed a different degree of inhibition on platelet aggregation induced by some activators in following order: arachidonic acid greater than collagen greater than thrombin greater than ionophore A23187. The longer the incubation time or the higher the concentration of the venom phospholipase A, the more pronounced was the inhibitory effect. The venom phospholipase A did not affect the thrombin-induced release reaction which was caused by intracellular Ca2+ mobilization in the presence of EDTA, but inhibited collagen-induced release reaction which was caused by Ca2+ influx from extracellular medium. The inhibitory effect of the venom phospholipase A and also lysophosphatidylcholine or arachidonic acid could be antagonized or reversed by bovine serum albumin. It was concluded that the first stimulatory phase of the venom phospholipase A action might be due to arachidonate liberation from platelet membrane. The second phase of inhibition of platelet aggregation and the release of ATP might be due to the inhibitory action of the split products produced by this venom phospholipase A.     

Neuropharmacological studies on the venom of Vipera russelli.

Neuropharmacological studies have been conducted on the venom of V. russelli on experimental animals. The venom was found to produce alteration in general behaviour pattern, reduction in spontaneous motility, hypothermia, potentiation of pentobarbitone hypnosis, analgesia, reduction in exploratory behaviour pattern, muscle relaxant action, and suppression of aggressive behaviour. The venom caused a significant increase in brain GABA content in mice. The observations are suggestive of a potent CNS-depressant action of V. russelli venom.     

Ammodytoxin content of Vipera ammodytes ammodytes venom as a prognostic factor for venom immunogenicity

Venoms are complex mixtures of proteins, peptides and other compounds whose biochemical and biological variability has been clearly demonstrated. These molecules have been used as antigens for immunization of anti-venom-producing animals (horses or sheep). Ammodytoxins (Atx) are potently neurotoxic compounds, and the most toxic compounds isolated so far from the Vipera ammodytes ammodytes (Vaa) venom. Recently we have shown that the level of antibodies specific to Vaa venom's most toxic component, ammodytoxin A (AtxA), (anti-AtxA IgG) in Vaa venom immunized rabbit sera highly correlated to the venom toxicity–neutralization potential of these sera. Here we investigated whether Atx content of Vaa venom could influence the outcome of immunization procedure. The novel ELISA was developed for precise determination of Atx content and Atx was quantified in venom samples used for immunization of rabbits. We clearly showed that animals immunized with the venom containing lower amount of Atx produced sera with significantly lower venom toxicity–neutralizing power and, vice versa, animals immunized with venoms containing higher amount of Atx produced sera with higher venom toxicity–neutralizing ability. Thus, the content of Atx in Vaa venom is a relevant parameter of its suitability in the production of highly protective Vaa anti-venom.     

Evaluation of the effect of gamma rays on the venom of Vipera lebetina by biochemical study

Snake bites represent a serious public health problem in many areas of the world. In Algeria, two widespread snakes are Vipera lebetina and Cerastes cerastes. Vipera lebetina venom causes local hemorrhage and necrosis, and it may lead to permanent limb loss. The principal causes of mortality after snakebites are acute renal failure and hemorrhage, which occur not only locally, at the site of the bite, but also systemically, contributing to the cardiovascular shock characteristic of severe envenomation. Gamma radiation has been shown to be effective for attenuating venom toxicity. Vipera lebetina venom was irradiated with two doses of gamma rays (1 and 2 kGy) from a 60Co source, and the venom's toxic, enzymatic, and structural properties were analyzed. Intraperitoneal injection of the irradiated venoms (100-500 microg/20 g mouse body mass) revealed a significant decrease of the toxicity. Irradiated venoms with 1 and 2 kGy doses were four and nine times less toxic, respectively, than the native venom. A biochemical characterization of in vitro enzymatic activities was performed. Vipera lebetina displayed in vitro caseinolytic, amidolytic, esterasic, coagulant, and phospholipase A2 activities. Caseinolytic, amidolytic, esterasic, and coagulative activities were reduced for the irradiated venoms; only phospholipase A2 activity was abolished in the irradiated venom with a dose of 2 kGy. The native and irradiated venoms were separated by gel filtration and electrophoresis. Chromatographic and electrophoretic profiles were drastically changed as compared with the native venom. Vipera lebetina venom detoxified by gamma rays was used for active immunization, and the presence of antibody in the immune sera was detected by ELISA. The immunogenic properties were preserved and the antisera obtained with the irradiated venoms could cross-react. Antisera were able to neutralize the toxic effect of V. lebetina native venom. These results indicate that irradiation of V. lebetina venom with a dose of 2 kGy can promote a significant detoxification, keeping the immunological properties intact.     

Properties of phosphodiesterase from Vipera lebetina venom immobilized on Agarose

Preparations of phosphodiesterase from Vipera lebetina venom immobilized on agarose were obtained. The kinetic properties for the hydrolyses of various substrates of soluble and immobilized phosphodiesterase, e. g. effects of pH, temperature, substrate concentrations, etc., were compared. The values of Km, V and activation energy for the substrate hydrolyses were determined.     

Isolation and characterization of a neurotoxin from Vipera palestinae venom

A neurotoxic protein, denoted viperotoxin, was isolated from the venom of Vipera palestinae. Viporotoxin is homogeneous by ultracentrifugal, electrophoretic and immunochemical criteria. It contains 108 amino acid residues and has a molecular weight of about 11600 (ultracentrifuge). Viperotoxin is composed of one polypeptide chain cross-linked intramolecularly by three disulfide bridges. Lysine is in the amino-terminal position, and proline in the carboxy-terminal position of the viperotoxin chain.     

Protective effect of xanthohumol against age-related brain damage

It has been recently shown that xanthohumol, a flavonoid present in hops, possesses antioxidant, anti-inflammatory and chemopreventive properties. However, its role in the aging brain has not been addressed so far. Therefore, this study aimed to investigate the possible neuroprotective activity of xanthohumol against age-related inflammatory and apoptotic brain damage in male senescence-accelerated prone mice (SAMP8). Animals were divided into 4 groups: Untreated young mice, untreated old mice and old mice treated either with 1 mg kg⁻¹ day⁻¹ or 5 mg kg⁻¹ day⁻¹ xanthohumol. Young and old senescence accelerated resistant mice (SAMR1) were used as controls. After 30 days of treatment, animals were sacrificed and their brains were collected and immediately frozen in liquid nitrogen. mRNA (GFAP, TNF-α, IL-1β, AIF, BAD, BAX, XIAP, NAIP and Bcl-2) and protein (GFAP, TNF-α, IL-1β, AIF, BAD, BAX, BDNF, synaptophysin and synapsin) expressions were measured by RT-PCR and Western blotting, respectively. Significant increased levels of pro-inflammatory (TNF-α, IL-1β) and pro-apoptotic (AIF, BAD, BAX) markers were observed in both SAMP8 and SAMR1 old mice compared to young animals (P<.05) and also in SAMP8 untreated old mice compared to SAMR1 (P<.05). These alterations were significantly less evident in animals treated with both doses of xanthohumol (P<.05). Also, a reduced expression of synaptic markers was observed in old mice compared to young ones (P<.05) but it significantly recovered with 5 mg kg⁻¹ day⁻¹ xanthohumol treatment (P<.05). In conclusion, xanthohumol treatment modulated the inflammation and apoptosis of aged brains, exerting a protective effect on damage induced by aging.