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Determination of in vivo toxicity and in vitro cytotoxicity of venom from the Cypriot blunt-nosed viper Macrovipera lebetina lebetina and antivenom production
Nalbantsoy, A;Karabay-Yavasoglu, NU;Sayim, F;Deliloglu-Gurhan, I;Gocmen, B;Arikan, H;Yildiz, MZ;
Journal of Venomous Animals and Toxins including Tropical Diseases , 2012, DOI: 10.1590/S1678-91992012000200011
Abstract: the venomous levantine viper, macrovipera lebetina lebetina is endemic to cyprus. the objective of this study was to investigate in vitro cytotoxicity, in vivo lethality, and antivenom production followed by a re-immunization schedule in mice against macrovipera lebetina lebetina venom. the ld50 value was estimated as 7.58 mg/kg within 24 hours by different venom doses administrated intraperitoneally in mice. freund's complete and incomplete adjuvants were used for first and second immunization of mice in antivenom production. a cell-based assay was performed to determine the effects of macrovipera lebetina lebetina venom and antivenom neutralizing potency on l929 cell viability. the snake venom toxicity and cytotoxicity were examined and comparison of results showed good correlation, the ld50 value was tenfold higher than the ic50 value. the ic50 value was 0.62 ± 0.18 ml after 48 hours treatment while the calculated value was 1.62 ± 0.25 ml for the culture media totally refreshed after two hours treatment with venom. the in vitro efficacy of antivenom against macrovipera lebetina lebetina venom was found to be low. this is the first report that describes the in vivo and in vitro toxic effects of macrovipera lebetina lebetina venom and antivenom production against this species.
Interactions of PLA2-s from Vipera lebetina, Vipera berus berus and Naja naja oxiana Venom with Platelets, Bacterial and Cancer Cells  [PDF]
Mari Samel,Heiki Vija,Imbi Kurvet,Kai Künnis-Beres,Katrin Trummal,Juhan Subbi,Anne Kahru,Jüri Siigur
Toxins , 2013, DOI: 10.3390/toxins5020203
Abstract: Secretory phospholipasesA 2 (sPLA 2s) form a large family of structurally related enzymes widespread in nature. Herein, we studied the inhibitory effects of sPLA 2s from Vipera lebetina (VLPLA 2), Vipera berus berus (VBBPLA 2), and Naja naja oxiana (NNOPLA 2) venoms on (i) human platelets, (ii) four different bacterial strains (gram-negative Escherichia coli and Vibrio fischeri; gram-positive Staphylococcus aureus and Bacillus subtilis) and (iii) five types of cancer cells (PC-3, LNCaP, MCF-7, K-562 and B16-F10) in vitro. sPLA 2s inhibited collagen-induced platelet aggregation: VBBPLA 2 IC 50 = 0.054, VLPLA 2 IC 50 = 0.072, NNOPLA 2 IC 50 = 0.814 μM. p-Bromophenacylbromide-inhibited sPLA 2 had no inhibitory action on platelets. 36.17 μM VBBPLA 2 completely inhibited the growth of gram-positive Bacillus subtilis whereas no growth inhibition was observed towards gram-negative Escherichia coli. The inhibitory action of sPLA 2s (~0.7 μM and ~7 μM) towards cancer cells depended on both venom and cell type. VBBPLA 2 (7.2 μM) inhibited significantly the viability of K-562 cells and the cell death appeared apoptotic. The sPLA 2s exhibited no inhibitory effect towards LNCaP cells and some effect (8%–20%) towards other cells. Thus, already sub-μM concentrations of sPLA 2s inhibited collagen-induced platelet aggregation and from the current suite of studied svPLA 2s and test cells, VBBPLA 2 was the most growth inhibitory towards Bacillus subtilis and K-562 cells.
Journal of Venomous Animals and Toxins , 1999, DOI: 10.1590/S0104-79301999000100006
Abstract: the immunochemical reactivity and neutralizing capacity of polyvalent vipera antivenom (vipera ammodytes, vipera aspis, vipera berus, vipera lebetina, and vipera xanthina) were tested on the enzymatic and biological activities of crotalus durissus terrificus and the following bothrops venoms from argentina (bothrops alternatus, bothrops ammodytoides, bothrops neuwiedii, bothrops jararaca, bothrops jararacussu, and bothrops moojeni). the vipera antivenom reacted weakly when tested by double immunoprecipitation (dip) and reacted with all the venoms when tested by elisa. several components in all the venoms studied were recognized in western blots. vipera antivenom deactivated to different degrees in vitro procoagulant, (indirect) hemolytic, and proteolytic activities in all the venoms studied. preincubation of bothrops alternatus venom with vipera antivenom neutralized a lethal potency of 4.5 ld50 in mice with an ed50 of 1.25 ± 0.25 ml per mg of venom, and with 1.0 ml/mg inhibited 54% of the hemorragic activity and 48% of necrotic activity. vipera antivenom (2.0 ml per mg toxin) inhibited the phospholipase a2 activity of purified crotoxin and decreased its lethal potency by 60%, while the neutralizing capacity on the lethal potency of crude crotalus durissus terrificus venom was poor even at a level of 5.0 ml/mg of venom.
Reappraisal of Vipera aspis Venom Neurotoxicity  [PDF]
Elisabeth Ferquel, Luc de Haro, Virginie Jan, Isabelle Guillemin, Sabine Jourdain, Alexandre Teynié, Jacques d'Alayer, Valérie Choumet
PLOS ONE , 2007, DOI: 10.1371/journal.pone.0001194
Abstract: Background The variation of venom composition with geography is an important aspect of intraspecific variability in the Vipera genus, although causes of this variability remain unclear. The diversity of snake venom is important both for our understanding of venomous snake evolution and for the preparation of relevant antivenoms to treat envenomations. A geographic intraspecific variation in snake venom composition was recently reported for Vipera aspis aspis venom in France. Since 1992, cases of human envenomation after Vipera aspis aspis bites in south-east France involving unexpected neurological signs were regularly reported. The presence of genes encoding PLA2 neurotoxins in the Vaa snake genome led us to investigate any neurological symptom associated with snake bites in other regions of France and in neighboring countries. In parallel, we used several approaches to characterize the venom PLA2 composition of the snakes captured in the same areas. Methodology/Principal Findings We conducted an epidemiological survey of snake bites in various regions of France. In parallel, we carried out the analysis of the genes and the transcripts encoding venom PLA2s. We used SELDI technology to study the diversity of PLA2 in various venom samples. Neurological signs (mainly cranial nerve disturbances) were reported after snake bites in three regions of France: Languedoc-Roussillon, Midi-Pyrénées and Provence-Alpes-C?te d'Azur. Genomes of Vipera aspis snakes from south-east France were shown to contain ammodytoxin isoforms never described in the genome of Vipera aspis from other French regions. Surprisingly, transcripts encoding venom neurotoxic PLA2s were found in snakes of Massif Central region. Accordingly, SELDI analysis of PLA2 venom composition confirmed the existence of population of neurotoxic Vipera aspis snakes in the west part of the Massif Central mountains. Conclusions/Significance The association of epidemiological studies to genetic, biochemical and immunochemical analyses of snake venoms allowed a good evaluation of the potential neurotoxicity of snake bites. A correlation was found between the expression of neurological symptoms in humans and the intensity of the cross-reaction of venoms with anti-ammodytoxin antibodies, which is correlated with the level of neurotoxin (vaspin and/or ammodytoxin) expression in the venom. The origin of the two recently identified neurotoxic snake populations is discussed according to venom PLA2 genome and transcriptome data.
Nimmy Chacko*, Mohammed Ibrahim , Prerana Shetty and C.S. Shastry
International Journal of Pharmaceutical Sciences and Research , 2012,
Abstract: Ethnopharmacological relevance: Calotropis gigantea is used traditionally to treat common diseases such as fever, rheumatism, indigestion, cough, cold, eczema, asthma, elephantiasis, nausea, vomiting and diarrhoea, either alone or with other medicinesAim of the study: To evaluate the antivenom activity of Calotropis gigantea plant extract against Vipera russelli snake venomMaterials and methods: The lyophilized snake venom of Vipera Russelli was dissolved in saline and required concentrations were prepared. Lyophilized polyvalent snake venom antiserum was used as reference serum. The methanolic extract of Calotropis gigantea was evaluated for its efficacy to neutralize various actions of the venom like lethality, necrotizing activity, edema forming activity and haemorrahgic activity.Results: Oral administration of C. gigantea plant extract at dose levels 200 and 400 mg/kg body weight effectively neutralized the lethal effect of 2LD50 and 3LD50 of V. russelli venom in mice (in-vivo neutralization). In in-vitro studies, the plant extract at all dose levels, i.e. 100, 200 and 400mg/kg body weight effectively neutralized 2LD50 and 3 LD50 of Vipera russelli venom. Oral administration of the plant extract at various dose levels was found to effectively inhibit the induction of haemorrhage and necrosis by the venom. At doses 200 and 400 mg/kg, the antinecrotic effect of plant extract was significant. The effect of methanolic extract of C. gigantea against edema induced by viperid venom was studied at 60, 120, 180 and 240 minutes. Plant extract at dose levels 200mg/kg and 400mg/kg showed significant anti-inflammatory activity at 240 min, and effect was comparable with that produced by the antivenom. Conclusion: Present study confirms the anti snake venom activity of alcoholic extract of C. gigantea.
Pharmacological Aspects of Vipera xantina palestinae Venom  [PDF]
Tatjana Momic,Franziska T. Arlinghaus,Hadar Arien-Zakay,Jeoshua Katzhendler,Johannes A. Eble,Cezary Marcinkiewicz,Philip Lazarovici
Toxins , 2011, DOI: 10.3390/toxins3111420
Abstract: In Israel, Vipera xantina palestinae (V.x.p.) is the most common venomous snake, accounting for several hundred cases of envenomation in humans and domestic animals every year, with a mortality rate of 0.5 to 2%. In this review we will briefly address the research developments relevant to our present understanding of the structure and function of V.x.p. venom with emphasis on venom disintegrins. Venom proteomics indicated the presence of four families of pharmacologically active compounds: (i) neurotoxins; (ii) hemorrhagins; (iii) angioneurin growth factors; and (iv) different types of integrin inhibitors. Viperistatin, a α1β1selective KTS disintegrin and VP12, a α2β1 selective C-type lectin were discovered. These snake venom proteins represent promising tools for research and development of novel collagen receptor selective drugs. These discoveries are also relevant for future improvement of antivenom therapy towards V.x.p. envenomation.
Use of MALDI-TOF mass spectrometry for specificity studies of biomedically important proteases  [PDF]
Jüri Siigur,Katrin Trummal,Külli T nism gi,Mari Samel,Ene Siigur,Heikki Vija,Indrek Tammiste,Juhan Subbi
Spectroscopy: An International Journal , 2002, DOI: 10.1155/2002/204307
Abstract: Proteases play crucial role starting from fertilization until to cell death. Our studies of the two Viperidae venoms (Levantine viper Vipera lebetina, Common viper Vipera berus) have demonstrated the existence of biomedically important proteases, both coagulants and anticoagulants that may be useful as diagnostic tools or potential therapeutics. We showed that venoms of both snakes contain: (i) metalloproteases and serine proteases that degrade fibrinogen, but not fibrin; (ii) factor X activators (VLFXA, VBFXAE); (iii) bradykinin?releasing serine proteases. Additionally Vipera lebetina snake venom contains thrombolytic fibrin degrading metalloenzyme (lebetase), HUVEC cell apoptosis inducing metalloprotease (VLAIP), factor V activator (VLFVA), thermostable β-fibrinogenase and α-fibrinogenase which has no homolog among known serine proteases. We examined the activity of snake venom proteases against bradykinin, substance P, insulin B?chain and 6–10 amino acid residues containing peptides synthesized according to potential cleavage regions of fibrinogen, factor X, factor IX, factor V, α2?macroglobulin bait region and pregnancy zone protein (PZP). We used MALDI TOF mass spectrometry technique for the discovery and identification of peptides released by protease hydrolysis. The sensitive and quick MALDI-TOF mass spectrometry methodology allows us to obtain the primary information about the substrate specificity of different proteases against various peptides and proteins.
Dynamic Changes in Lipid Peroxidation and Antioxidant Level in Rat’s Tissues with Macrovipera lebetina obtusa and Montivipera raddei Venom Intoxication  [PDF]
N. A. Zaqaryan, N. A. Ghazaryan, N. M. Ayvazyan
Journal of Biophysical Chemistry (JBPC) , 2014, DOI: 10.4236/jbpc.2014.54017
Abstract: We investigated the balance of free radicals in different tissues (liver, heart, brain and muscle) of rats in course of in vivo and in vitro processing by Macrovipera lebetina obtusa (MLO) and Montivipera raddei (MR) snake venoms. Chemiluminescence (ChL) levels were examined in tissue assays after incubation (at 37 °C for a period of 10 min) with venom for in vitro experiments and in tissue assays isolated of 10 min after venom injection for in vivo experiments. The TBA-test was also performed to confirm the free radical expression. The activities of antioxidant enzymes (such as superoxide dismutase and glutathione peroxidase) in isolated tissues were detected by spectro-photometry. During the in vitro processing chemiluminescence levels of tissue homogenates significantly decreased, while in course of in vivo intoxication the level of ChL was elevated in brain and liver; lipid peroxidation also increased in brain tissue, but there was no significant balance change in other tissues; the activity of superoxide dismutase mainly correlated with changes of free radical balance during intoxication. On the contrary, the activity of glutathione peroxidase showed the reverse tendencies to change. We suggest that free radicals and their oxidative stresses may play a role in the early stage of intoxication causing the so-named “spreading-effect”, which is very characteristic for the venom of vipers.
New locality records for the adder (Vipera berus) in the Carpathian Corner, Romania  [PDF]
Alexandru Strugariu,Tibor Sos,Alexandru Sotek,Iulian Gherghel
Advances in Environmental Sciences , 2009,
Abstract: The Carpathian Corner region (Buz u and Vrancea Counties) is one of the Romanian areas inwhich data on the herpetofauna is most scarce. The adder (Vipera berus), a widespread but threatenedRomanian snake species, has been previously recorded in the Carpathian Corner area around 50 yearsago but its presence in the area has not been recently reconfirmed. Here we give the first locality recordfor the adder in Vrancea County and two new records for the species in Buz u County, by means ofpersonal observations made during the last decade.
Vixapatin (VP12), a C-Type Lectin-Protein from Vipera xantina palestinae Venom: Characterization as a Novel Anti-angiogenic Compound  [PDF]
Tatjana Momic,Gadi Cohen,Reuven Reich,Franziska T. Arlinghaus,Johannes A. Eble,Cezary Marcinkiewicz,Philip Lazarovici
Toxins , 2012, DOI: 10.3390/toxins4100862
Abstract: A C-type lectin-like protein (CTL), originally identified as VP12 and lately named Vixapatin, was isolated and characterized from Israeli viper Vipera xantina palestinae snake venom. This CTL was characterized as a selective α2β1 integrin inhibitor with anti-melanoma metastatic activity. The major aim of the present study was to prove the possibility that this protein is also a potent novel anti-angiogenic compound. Using an adhesion assay, we demonstrated that Vixapatin selectively and potently inhibited the α2 mediated adhesion of K562 over-expressing cells, with IC50 of 3 nM. 3 nM Vixapatin blocked proliferation of human dermal microvascular endothelial cells (HDMEC); 25 nM inhibited collagen I induced migration of human fibrosarcoma HT-1080 cells; and 50 nM rat C6 glioma and human breast carcinoma MDA-MB-231 cells. 1 μM Vixapatin reduced HDMEC tube formation by 75% in a Matrigel assay. Furthermore, 1 μM Vixapatin decreased by 70% bFGF-induced physiological angiogenesis, and by 94% C6 glioma-induced pathological angiogenesis, in shell-less embryonic quail chorioallantoic membrane assay. Vixapatin’s ability to inhibit all steps of the angiogenesis process suggest that it is a novel pharmacological tool for studying α2β1 integrin mediated angiogenesis and a lead compound for the development of a novel anti-angiogenic/angiostatic/anti-cancer drug.
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