The Experts below are selected from a list of 537 Experts worldwide ranked by ideXlab platform

Igor Križaj - One of the best experts on this subject based on the ideXlab platform.

  • the procoagulant snake venom serine protease potentially having a dual blood coagulation factor v and x activating activity
    Toxins, 2020
    Co-Authors: Zorica Latinovic, Adrijana Leonardi, Jože Pungercar, Manjunatha R Kini, Cho Yeow Koh, Alenka Trampus Bakija, Igor Križaj
    Abstract:

    A procoagulant snake venom serine protease was isolated from the venom of the nose-horned viper (Vipera ammodytes ammodytes). This 34 kDa glycoprotein, termed VaaSP-VX, possesses five kDa N-linked carbohydrates. Amino acid sequencing showed VaaSP-VX to be a chymotrypsin-like serine protease. Structurally, it is highly homologous to VaaSP-6 from the same venom and to nikobin from the venom of Vipera nikolskii, neither of which have known functions. VaaSP-VX does not affect platelets. The specific proteolysis of blood coagulation factors X and V by VaaSP-VX suggests that its blood-coagulation-inducing effect is due to its ability to activate these two blood coagulation factors, which following activation, combine to form the prothrombinase complex. VaaSP-VX may thus represent the first example of a serine protease with such a dual activity, which makes it a highly suitable candidate to replace diluted Russell's viper venom in lupus anticoagulant testing, thus achieving greater reliability of the analysis. As a blood-coagulation-promoting substance that is resistant to serpin inhibition, VaaSP-VX is also interesting from the therapeutic point of view for treating patients suffering from hemophilia.

  • Purification and characterisation of two hemorrhagic metalloproteinases from the venom of the long-nosed viper, Vipera ammodytes ammodytes
    Toxicon, 2017
    Co-Authors: Igor Križaj, Franc Gubenšek, Adrijana Leonardi
    Abstract:

    Two hemorrhagic proteins, VaH1 and VaH2, have been purified from Vipera ammodytes ammodytes venom. They are monomeric glycoproteins of an apparent molecular mass of 70 kDa and multiple isoelectric points around pH 5.5. Both molecules are proteolytically active against azocasein as substrate. VaH1, which was characterised in detail, showed maximum activity at pH 7.5. Ethylenediaminetetraacetic acid eliminated the proteolyticas well as the hemorrhagic activity of VaH1 while iodoacetamide, phenylmethylsulfonyl fluoride and pepstatin A, inhibitors of cysteine, serine and aspartic proteinases respectively, had no effect. VaH1 is therefore a metalloproteinase whose hemorrhagic activity is very likely the result of its proteolytic activity. VaH1 is a fibrinogenase, hydrolysing exclusively the A-chain of fibrinogen. In the B-chain of insulin it cleaved with a high preference the bond between Ala14 and Leu15. Based on its molecular mass, VaH1 (as well as VaH2) is a Class P-III metalloproteinase. Partial amino acid sequences of its CNBr fragments demonstrated a high level of identity with the reprolysin subfamily of zinc-metalloproteinases.

  • Vipera ammodytes bites treated with antivenom Viperatab a case series with pharmacokinetic evaluation
    Clinical Toxicology, 2017
    Co-Authors: Miran Brvar, Igor Križaj, Maja Lang Balija, Tihana Kurtovic, Damjan Grenc, Beata Halassy
    Abstract:

    AbstractContext: In clinical practice it is difficult to differentiate between V. berus and V. ammodytes venomous bites. In the past this was not a concern, but due to the current shortage in Viperfav™ and European viper venom antiserum availability, V. a. ammodytes venomous bites have recently been treated with ViperaTAb®, which is a pharmaceutical formulation containing a monospecific ovine Fab fragments against the venom of V. berus.Objective: To evaluate ViperaTAb® in V. a. ammodytes envenomations.Materials and methods: This is a prospective case series of three consecutive patients envenomed by V. a. ammodytes snakebite treated with ViperaTAb®. V. ammodytes venom, neurotoxic ammodytoxins, and Fab fragment levels were determined in serum samples and a pharmacokinetic analysis of the antivenom Fab fragments was carried out.Results: Three patients bitten by V. a. ammodytes with extensive local swelling, neurological symptoms and recurrent thrombocytopenia were treated with ViperaTAb®. V. ammodytes venom...

  • Expression of fully active ammodytoxin A, a potent presynaptically neurotoxic phospholipase A2, in Escherichia coli
    FEBS letters, 2016
    Co-Authors: Igor Križaj, Franc Gubenšek, Borut Strukelj, Jože Pungercar, N. S. Liang
    Abstract:

    A cDNA encoding the most presynaptically neurotoxic phospholipase A2, ammodytoxin A, from the venom of the long-nosed viper (Vipera ammodytes ammodytes) has been expressed in Escherichia coli. Ammodytoxin A was produced as a fusion protein with the 81 N-terminal residues of adenylate kinase followed by the tetrapeptide recognition site for factor Xa (IEGR) just preceding the first amino acid residue of the toxin. The fusion protein was expressed under the control of tac promoter without IPTG induction in the form of insoluble inclusion bodies. It was dissolved in guanidine hydrochloride, S-sulfonated and refolded in a reoxidation mixture including a reduced/oxidized glutathione redox couple. Ammodytoxin A was fully activated by limited hydrolysis with trypsin that preferentially cleaves the fusion protein at the factor Xa recognition site and purified by cation-exchange chromatography. The correct N-terminus was confirmed by protein sequencing. Recombinant ammodytoxin A has been proved to be indistinguishable from the native toxin in its enzymatic activity and toxicity. © 1993.

  • venomics of Vipera berus berus to explain differences in pathology elicited by Vipera ammodytes ammodytes envenomation therapeutic implications
    Journal of Proteomics, 2016
    Co-Authors: Zorica Latinović, Jernej Šribar, Adrijana Leonardi, Beata Halassy, Tamara Sajevic, Alenka Trampusbakija, Robert Frangež, Monika Cecilija žužek, Jože Pungercar, Igor Križaj
    Abstract:

    Abstract Vipera berus berus (Vbb) is the most widely distributed and Vipera ammodytes ammodytes (Vaa) the most venomous viper in Europe. In particular areas of the Old continent their toxic bites constitute a considerable public health problem. To make the current envenomation therapy more effective we have analysed the proteome of Vbb venom and compared it with that of Vaa. We found the proteome of Vbb to be much less complex and to contain smaller levels of particularly snaclecs and sPLA2s. Snaclecs are probably responsible for thrombocytopenia. The neurotoxic sPLA2s, ammodytoxins, are responsible for the most specific feature of the Vaa venom poisoning − induction of signs of neurotoxicity in patients. These molecules were not found in Vbb venom. Both venoms induce haemorrhage and coagulopathy in man. As Vaa and Vbb venoms possess homologous P-III snake venom metalloproteinases, the main haemorrhagic factors, the severity of the haemorrhage is dictated by concentration and specific activity of these molecules. The much greater anticoagulant effect of Vaa venom than that of Vbb venom lies in its higher extrinsic pathway coagulation factor-proteolysing activity and content of ammodytoxins which block the prothrombinase complex formation. Biological significance Envenomations by venomous snakes constitute a considerable public health problem worldwide, and also in Europe. In the submitted work we analysed the venom proteome of Vipera berus berus (Vbb), the most widely distributed venomous snake in Europe and compared it with the venom proteome of the most venomous viper in Europe, Vipera ammodytes ammodytes (Vaa). We have offered a possible explanation, at the molecular level, for the differences in clinical pictures inflicted by the Vbb and Vaa venoms. We have provided an explanation for the effectiveness of treatment of Vbb envenomation by Vaa antiserum and explained why full protection of Vaa venom poisoning by Vbb antiserum should not be always expected, especially not in cases of severe poisoning. The latter makes a strong case for Vaa antiserum production as we are faced with its shortage due to ceasing of production of two most frequently used products.

Beata Halassy - One of the best experts on this subject based on the ideXlab platform.

  • Vipera ammodytes bites treated with antivenom Viperatab a case series with pharmacokinetic evaluation
    Clinical Toxicology, 2017
    Co-Authors: Miran Brvar, Igor Križaj, Maja Lang Balija, Tihana Kurtovic, Damjan Grenc, Beata Halassy
    Abstract:

    AbstractContext: In clinical practice it is difficult to differentiate between V. berus and V. ammodytes venomous bites. In the past this was not a concern, but due to the current shortage in Viperfav™ and European viper venom antiserum availability, V. a. ammodytes venomous bites have recently been treated with ViperaTAb®, which is a pharmaceutical formulation containing a monospecific ovine Fab fragments against the venom of V. berus.Objective: To evaluate ViperaTAb® in V. a. ammodytes envenomations.Materials and methods: This is a prospective case series of three consecutive patients envenomed by V. a. ammodytes snakebite treated with ViperaTAb®. V. ammodytes venom, neurotoxic ammodytoxins, and Fab fragment levels were determined in serum samples and a pharmacokinetic analysis of the antivenom Fab fragments was carried out.Results: Three patients bitten by V. a. ammodytes with extensive local swelling, neurological symptoms and recurrent thrombocytopenia were treated with ViperaTAb®. V. ammodytes venom...

  • venomics of Vipera berus berus to explain differences in pathology elicited by Vipera ammodytes ammodytes envenomation therapeutic implications
    Journal of Proteomics, 2016
    Co-Authors: Zorica Latinović, Jernej Šribar, Adrijana Leonardi, Beata Halassy, Tamara Sajevic, Alenka Trampusbakija, Robert Frangež, Monika Cecilija žužek, Jože Pungercar, Igor Križaj
    Abstract:

    Abstract Vipera berus berus (Vbb) is the most widely distributed and Vipera ammodytes ammodytes (Vaa) the most venomous viper in Europe. In particular areas of the Old continent their toxic bites constitute a considerable public health problem. To make the current envenomation therapy more effective we have analysed the proteome of Vbb venom and compared it with that of Vaa. We found the proteome of Vbb to be much less complex and to contain smaller levels of particularly snaclecs and sPLA2s. Snaclecs are probably responsible for thrombocytopenia. The neurotoxic sPLA2s, ammodytoxins, are responsible for the most specific feature of the Vaa venom poisoning − induction of signs of neurotoxicity in patients. These molecules were not found in Vbb venom. Both venoms induce haemorrhage and coagulopathy in man. As Vaa and Vbb venoms possess homologous P-III snake venom metalloproteinases, the main haemorrhagic factors, the severity of the haemorrhage is dictated by concentration and specific activity of these molecules. The much greater anticoagulant effect of Vaa venom than that of Vbb venom lies in its higher extrinsic pathway coagulation factor-proteolysing activity and content of ammodytoxins which block the prothrombinase complex formation. Biological significance Envenomations by venomous snakes constitute a considerable public health problem worldwide, and also in Europe. In the submitted work we analysed the venom proteome of Vipera berus berus (Vbb), the most widely distributed venomous snake in Europe and compared it with the venom proteome of the most venomous viper in Europe, Vipera ammodytes ammodytes (Vaa). We have offered a possible explanation, at the molecular level, for the differences in clinical pictures inflicted by the Vbb and Vaa venoms. We have provided an explanation for the effectiveness of treatment of Vbb envenomation by Vaa antiserum and explained why full protection of Vaa venom poisoning by Vbb antiserum should not be always expected, especially not in cases of severe poisoning. The latter makes a strong case for Vaa antiserum production as we are faced with its shortage due to ceasing of production of two most frequently used products.

  • A Single Dose of Viperfav TM May Be Inadequate for Vipera ammodytes Snake Bite: A Case Report and Pharmacokinetic Evaluation
    Toxins, 2016
    Co-Authors: Tihana Kurtović, Igor Križaj, Maja Lang Balija, Miran Brvar, Damjan Grenc, Beata Halassy
    Abstract:

    ViperfavTM is a commercial F(ab’)2 antivenom prepared against European vipers venom. It is safe and effective for treating envenomation caused by Vipera aspis and Vipera berus. Therapeutic efficacy for treating Vipera ammodytes ammodytes (V. a. ammodytes) envenoming has not been yet described, although protective efficacy has been demonstrated in preclinical studies. We report on a 32-year-old man bitten by V. a. ammodytes who was treated with Viperfav™. Viperfav™ promptly reduced local extension and improved systemic pathological signs, but 24 h after the incident a recurrence of thrombocytopenia occurred despite a favorable pharmacokinetic profile with systemic clearance (1.64 (mL·h−1)·kg−1) and elimination half-life (97 h) among the highest ever reported. The recommended dose of Viperfav™ for V. aspis and V. berus bites may be inadequate for serious V. a. ammodytes envenomations. Following V. a. ammodytes bite, serial blood counts and coagulation profiles should be performed to help guide Viperfav™ treatment, along with supplemental administration as indicated.

  • Identification of proteins interacting with ammodytoxins in Vipera ammodytes ammodytes venom by immuno-affinity chromatography
    Analytical and Bioanalytical Chemistry, 2014
    Co-Authors: Marija Brgles, Lidija Kovačič, Igor Križaj, Gunter Allmaier, Tihana Kurtović, Miloš Barut, Martina Marchetti-deschmann, Maja Lang-balija, Beata Halassy
    Abstract:

    In order to perform their function, proteins frequently interact with other proteins. Various methods are used to reveal protein interacting partners, and affinity chromatography is one of them. Snake venom is composed mostly of proteins, and various protein complexes in the venom have been found to exhibit higher toxicity levels than respective components separately. Complexes can modulate envenomation activity of a venom and/or potentiate its effect. Our previous data indicate that the most toxic components of the Vipera ammodytes ammodytes ( Vaa ) venom isolated so far—ammodytoxins (Atxs)—are contributing to the venom’s toxicity only moderately; therefore, we aimed to explore whether they have some interacting partner(s) potentiating toxicity. For screening of possible interactions, immuno-affinity chromatography combined with identification by mass spectrometry was used. Various chemistries (epoxy, carbonyldiimidazole, ethylenediamine) as well as protein G functionality were used to immobilize antibodies on monolith support, a Convective Interaction Media disk. Monoliths have been demonstrated to better suit the separation of large biomolecules. Using such approach, several proteins were indicated as potential Atx-binding proteins. Among these, the interaction of Atxs with a Kunitz-type inhibitor was confirmed by far-Western dot-blot and surface plasmon resonance measurement. It can be concluded that affinity chromatography on monolithic columns combined with mass spectrometry identification is a successful approach for screening of protein interactions and it resulted with detection of the interaction of Atx with Kunitz-type inhibitor in Vaa venom for the first time.

  • Paraspecificity of Vipera a. ammodytes-specific antivenom towards MontiVipera raddei and MacroVipera lebetina obtusa venoms.
    Toxicon : official journal of the International Society on Toxinology, 2013
    Co-Authors: Tihana Kurtović, Maja Lang Balija, Naira M Ayvazyan, Beata Halassy
    Abstract:

    Abstract Antivenom raised against the venom of nose-horned viper, Vipera ammodytes (V. a.) ammodytes (European viper venom antiserum, Zagreb antivenom), contains neutralising equine F(ab′)2 fragments that are clinically successful against homologous venom, but also against the venoms of several others medically important European snakes due to its paraspecific action. In this work we demonstrated that Zagreb antivenom is preclinically effective in neutralising lethal toxicity and hemorrhagicity of venoms of Armenian mountain snakes – MontiVipera raddei and MacroVipera lebetina obtusa as well. In order to better understand the biochemical basis of the observed paraspecificity, the ability of anti-V. a. ammodytes serum to recognise and neutralise proteinases of the two venoms was also investigated. Anti-V. a. ammodytes serum showed surprisingly low capacity to inhibit metalloproteinases of both venoms included in the study, probably due to weak immunorecognition of their P-I representatives. Also, it completely failed to abolish enzymatic action of serine proteinases from MacroVipera lebetina obtusa venom. Relevance of such finding is yet to be established.

Ayse Nalbantsoy - One of the best experts on this subject based on the ideXlab platform.

  • cytotoxicity of silica nanoparticles with transcaucasian nose horned viper Vipera ammodytes transcaucasiana venom on u87mg and shsy5y neuronal cancer cells
    Applied Biochemistry and Biotechnology, 2018
    Co-Authors: Cigdem Celen, Mert Karış, Bayram Göçmen, Ceren Kececiler, Ozlem Yesilceliktas, Ayse Nalbantsoy
    Abstract:

    Highly bioactive compounds of the snake venom make them particular sources for anticancer agent development. They contain very rich peptide-protein structures. Therefore, they are very susceptible to environmental conditions such as temperature, pH, and light. In this study, Vipera ammodytes transcaucasiana venom was encapsulated in PAMAM-G4 dendrimer by sol-gel method in order to prevent degradation of venom contents from the environmental conditions. For this purpose, nanoparticles were prepared by sol-gel methodology and SEM analyses were performed. U87MG and SHSY5Y neuronal cancer cell lines were treated with different concentrations of venom-containing nanoparticles and cytotoxicity was determined by MTT assay. IC50 values of nanoparticles with snake venom were calculated as 37.24 and 44.64 μg/ml for U87MG and SHSY5Y cells, respectively. The IC50 values of nanoparticles with snake venom were calculated as 10.07 and 7.9 μg/ml for U87MG and SHSY5Y cells, respectively. As a result, nanoparticles with V. a. transcaucasiana venom showed remarkably high cytotoxicity. Encapsulation efficiency of nanoparticles with 1 mg/ml snake venom was determined as %67 via BCA™ protein analysis. In conclusion, this method is found to be convenient and useful for encapsulating snake venom as well as being suitable for drug delivery systems.

  • Comparative Venomics of the Vipera ammodytes transcaucasiana and Vipera ammodytes montandoni from Turkey Provides Insights into Kinship
    Toxins, 2018
    Co-Authors: Benjamin-florian Hempel, Maik Damm, Mert Karış, Mehmet Anıl Oğuz, Bayram Göçmen, Ayse Nalbantsoy
    Abstract:

    The Nose-horned Viper (Vipera ammodytes) is one of the most widespread and venomous snakes in Europe, which causes high frequent snakebite accidents. The first comprehensive venom characterization of the regional endemic Transcaucasian Nose-horned Viper (Vipera ammodytes transcaucasiana) and the Transdanubian Sand Viper (Vipera ammodytes montandoni) is reported employing a combination of intact mass profiling and bottom-up proteomics. The bottom-up analysis of both subspecies identified the major snake protein families of viper venoms. Furthermore, intact mass profiling revealed the presence of two tripeptidic metalloprotease inhibitors and their precursors. While previous reports applied multivariate analysis techniques to clarify the taxonomic status of the subspecies, an accurate classification of Vipera ammodytes transcaucasiana is still part of the ongoing research. The comparative analysis of the viper venoms on the proteome level reveals a close relationship between the Vipera ammodytes subspecies, which could be considered to clarify the classification of the Transcaucasian Nose-horned Viper. However, the slightly different ratio of some venom components could be indicating interspecific variations of the two studied subspecies or intraspecies alternations based on small sample size. Additionally, we performed a bioactivity screening with the crude venoms against several human cancerous and non-cancerous cell lines, which showed interesting results against a human breast adenocarcinoma epithelial cell line. Several fractions of Vipera a. transcaucasiana demonstrated a strong cytotoxic effect on triple negative MDA MB 231 breast cancer cells.

  • Anadolu'da yayılış gösteren Vipera ammodytes(Burunlu Engerek) zehrinin sitotoksik, anti-anjiyogenik, anti-tümör ve antimikrobiyal aktivitelerinin taranması
    2016
    Co-Authors: Naşit İğci, Ayse Nalbantsoy, Leman Gizem Erkan, Gözde Yılmaz Akça, Hüsniye Yalçın Tansel, Murat Yalçın, Bayram Göçmen
    Abstract:

    Amaç: Bu çalışmada, Vipera ammodytes (Burunlu Engerek) ham zehrinin sitotoksik, antimikrobiyal, anti-anjiyojenik ve anti-tümör aktivitelerinin taranmasını amaçlanmıştır.Metod: PC3, HeLa, CaCo-2, U-87MG ve MCF-7 kanser hücre hatları ve bir normal hücre hattı (Vero) kullanılarak MTT testi ile sitotoksite taraması yapılmıştır. Antimikrobiyal aktivite broth seyreltme metodu kullanılarak hesaplanan minimum inhibitör konsantrasyon ile değerlendirilmiştir. Escherichia coli ATCC 25922, E. coli 0157:H7, Enterococcus faecalis 29212, Enterococcus faecium DSM 13590, Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228, Salmonella typhimirium CCM 5445, Proteus vulgaris ATCC 6957, Bacillus cereus ATCC 7064 ve Candida albicans ATCC 10239 türleri kullanılmıştır. Anti-anjiyogenik anti-tümör aktivite civciv koryoallantoik membran (CAM) modeli ile değerlendirilmiştir.Bulgular: V. ammodytes zehrinin 48  saat sonunda hücreler üzerindeki IC50 değeri, 1.8 ve 7.0 ?g/mL arasında değişmiştir. Zehir P. vulgaris, S. aureus, S. epidermidis ve C. albicans (en yüksek etki) üzerinde antimikrobiyal etki göstermiştir. CAM deney modelinde 2 ve 10 ?g/mL zehir dozlarında (sırasıyla 1.1 ve 2.0 skorları ile) anjiyogenezde doza bağlı baskılanma görülmüştür.Sonuç: Bu çalışmanın sonuçları Anadolu'da bulunan V. ammodytes zehrinin sahip olduğu biyolojik aktiviteleri ortaya koymuş ve ileride yapılacak biyoaktivite rehberli karakterizasyon çalışmaları için sahip olduğu potansiyeli göstermiştir.Objective: In the present study, we aimed to screen the cytotoxic, antimicrobial, anti-angiogenic and anti-tumorogenic activities of Anatolian Vipera ammodytes (Nosehorned Viper) crude venom.Material and methods: The cytotoxicity was screened against PC3, HeLa, CaCo-2, U-87MG, MCF-7 and Vero cells by using MTT assay. The antimicrobial activity on Escherichia coli ATCC 25922, E. coli 0157:H7, Enterococcus faecalis 29212, Enterococcus faecium DSM 13590, Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228, Salmonella typhimirium CCM 5445, Proteus vulgaris ATCC 6957, Bacillus cereus ATCC 7064 and Candida albicans ATCC 10239 was assayed by determining the minimum inhibitory concentration using the broth dilu-tion method. Anti-angiogenic and anti-tumorogenic activ-ity was assessed by using chick chorioallantoic membrane (CAM) assay.Results: The IC50 value of V. ammodytes venom on cul-tured cells varied from 1.8 to 7.0 ?g/mL after 48 h treat-ment. Venom showed antimicrobial activity on P. vulgaris, S. aureus, S. epidermidis, E. faecium and C. albicans (the highest activity). The venom exhibited dose-dependent anti-angiogenic activity on CAM model at 2 and 10 ?g/mL doses with scores of 1.1 and 2.0, respectively.Conclusion: The results of the present study contributed to the knowledge of the biological activities of Anatolian V. ammodytes venom and showed its potential for further bioactivity guided characterization studies

Bayram Göçmen - One of the best experts on this subject based on the ideXlab platform.

  • cytotoxicity of silica nanoparticles with transcaucasian nose horned viper Vipera ammodytes transcaucasiana venom on u87mg and shsy5y neuronal cancer cells
    Applied Biochemistry and Biotechnology, 2018
    Co-Authors: Cigdem Celen, Mert Karış, Bayram Göçmen, Ceren Kececiler, Ozlem Yesilceliktas, Ayse Nalbantsoy
    Abstract:

    Highly bioactive compounds of the snake venom make them particular sources for anticancer agent development. They contain very rich peptide-protein structures. Therefore, they are very susceptible to environmental conditions such as temperature, pH, and light. In this study, Vipera ammodytes transcaucasiana venom was encapsulated in PAMAM-G4 dendrimer by sol-gel method in order to prevent degradation of venom contents from the environmental conditions. For this purpose, nanoparticles were prepared by sol-gel methodology and SEM analyses were performed. U87MG and SHSY5Y neuronal cancer cell lines were treated with different concentrations of venom-containing nanoparticles and cytotoxicity was determined by MTT assay. IC50 values of nanoparticles with snake venom were calculated as 37.24 and 44.64 μg/ml for U87MG and SHSY5Y cells, respectively. The IC50 values of nanoparticles with snake venom were calculated as 10.07 and 7.9 μg/ml for U87MG and SHSY5Y cells, respectively. As a result, nanoparticles with V. a. transcaucasiana venom showed remarkably high cytotoxicity. Encapsulation efficiency of nanoparticles with 1 mg/ml snake venom was determined as %67 via BCA™ protein analysis. In conclusion, this method is found to be convenient and useful for encapsulating snake venom as well as being suitable for drug delivery systems.

  • Comparative Venomics of the Vipera ammodytes transcaucasiana and Vipera ammodytes montandoni from Turkey Provides Insights into Kinship
    Toxins, 2018
    Co-Authors: Benjamin-florian Hempel, Maik Damm, Mert Karış, Mehmet Anıl Oğuz, Bayram Göçmen, Ayse Nalbantsoy
    Abstract:

    The Nose-horned Viper (Vipera ammodytes) is one of the most widespread and venomous snakes in Europe, which causes high frequent snakebite accidents. The first comprehensive venom characterization of the regional endemic Transcaucasian Nose-horned Viper (Vipera ammodytes transcaucasiana) and the Transdanubian Sand Viper (Vipera ammodytes montandoni) is reported employing a combination of intact mass profiling and bottom-up proteomics. The bottom-up analysis of both subspecies identified the major snake protein families of viper venoms. Furthermore, intact mass profiling revealed the presence of two tripeptidic metalloprotease inhibitors and their precursors. While previous reports applied multivariate analysis techniques to clarify the taxonomic status of the subspecies, an accurate classification of Vipera ammodytes transcaucasiana is still part of the ongoing research. The comparative analysis of the viper venoms on the proteome level reveals a close relationship between the Vipera ammodytes subspecies, which could be considered to clarify the classification of the Transcaucasian Nose-horned Viper. However, the slightly different ratio of some venom components could be indicating interspecific variations of the two studied subspecies or intraspecies alternations based on small sample size. Additionally, we performed a bioactivity screening with the crude venoms against several human cancerous and non-cancerous cell lines, which showed interesting results against a human breast adenocarcinoma epithelial cell line. Several fractions of Vipera a. transcaucasiana demonstrated a strong cytotoxic effect on triple negative MDA MB 231 breast cancer cells.

  • Anadolu'da yayılış gösteren Vipera ammodytes(Burunlu Engerek) zehrinin sitotoksik, anti-anjiyogenik, anti-tümör ve antimikrobiyal aktivitelerinin taranması
    2016
    Co-Authors: Naşit İğci, Ayse Nalbantsoy, Leman Gizem Erkan, Gözde Yılmaz Akça, Hüsniye Yalçın Tansel, Murat Yalçın, Bayram Göçmen
    Abstract:

    Amaç: Bu çalışmada, Vipera ammodytes (Burunlu Engerek) ham zehrinin sitotoksik, antimikrobiyal, anti-anjiyojenik ve anti-tümör aktivitelerinin taranmasını amaçlanmıştır.Metod: PC3, HeLa, CaCo-2, U-87MG ve MCF-7 kanser hücre hatları ve bir normal hücre hattı (Vero) kullanılarak MTT testi ile sitotoksite taraması yapılmıştır. Antimikrobiyal aktivite broth seyreltme metodu kullanılarak hesaplanan minimum inhibitör konsantrasyon ile değerlendirilmiştir. Escherichia coli ATCC 25922, E. coli 0157:H7, Enterococcus faecalis 29212, Enterococcus faecium DSM 13590, Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228, Salmonella typhimirium CCM 5445, Proteus vulgaris ATCC 6957, Bacillus cereus ATCC 7064 ve Candida albicans ATCC 10239 türleri kullanılmıştır. Anti-anjiyogenik anti-tümör aktivite civciv koryoallantoik membran (CAM) modeli ile değerlendirilmiştir.Bulgular: V. ammodytes zehrinin 48  saat sonunda hücreler üzerindeki IC50 değeri, 1.8 ve 7.0 ?g/mL arasında değişmiştir. Zehir P. vulgaris, S. aureus, S. epidermidis ve C. albicans (en yüksek etki) üzerinde antimikrobiyal etki göstermiştir. CAM deney modelinde 2 ve 10 ?g/mL zehir dozlarında (sırasıyla 1.1 ve 2.0 skorları ile) anjiyogenezde doza bağlı baskılanma görülmüştür.Sonuç: Bu çalışmanın sonuçları Anadolu'da bulunan V. ammodytes zehrinin sahip olduğu biyolojik aktiviteleri ortaya koymuş ve ileride yapılacak biyoaktivite rehberli karakterizasyon çalışmaları için sahip olduğu potansiyeli göstermiştir.Objective: In the present study, we aimed to screen the cytotoxic, antimicrobial, anti-angiogenic and anti-tumorogenic activities of Anatolian Vipera ammodytes (Nosehorned Viper) crude venom.Material and methods: The cytotoxicity was screened against PC3, HeLa, CaCo-2, U-87MG, MCF-7 and Vero cells by using MTT assay. The antimicrobial activity on Escherichia coli ATCC 25922, E. coli 0157:H7, Enterococcus faecalis 29212, Enterococcus faecium DSM 13590, Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228, Salmonella typhimirium CCM 5445, Proteus vulgaris ATCC 6957, Bacillus cereus ATCC 7064 and Candida albicans ATCC 10239 was assayed by determining the minimum inhibitory concentration using the broth dilu-tion method. Anti-angiogenic and anti-tumorogenic activ-ity was assessed by using chick chorioallantoic membrane (CAM) assay.Results: The IC50 value of V. ammodytes venom on cul-tured cells varied from 1.8 to 7.0 ?g/mL after 48 h treat-ment. Venom showed antimicrobial activity on P. vulgaris, S. aureus, S. epidermidis, E. faecium and C. albicans (the highest activity). The venom exhibited dose-dependent anti-angiogenic activity on CAM model at 2 and 10 ?g/mL doses with scores of 1.1 and 2.0, respectively.Conclusion: The results of the present study contributed to the knowledge of the biological activities of Anatolian V. ammodytes venom and showed its potential for further bioactivity guided characterization studies

  • new locality records for four rare species of vipers reptilia viperidae in turkey
    Zoology in The Middle East, 2014
    Co-Authors: Bayram Göçmen, Mehmet Anıl Oğuz, Naşit İğci, Konrad Mebert, Bahadir Akman, Mehmet Zulfu Yildiz, Cagatay Altin
    Abstract:

    We report new localities and range extensions for four vipers from Turkey. The distribution of MontiVipera wagneri is extended substantially southwards into Mus province, showing that this species is not endemic to the Aras valley as previously stated in the literature. Similarly, an unverified photograph of MontiVipera albizona from Erzincan province, at its currently known eastern limit, was confirmed with new material, and its range is extended substantially southwards to the Anamos (Nur) Mountains in Hatay province. Smaller range extensions are reported for Vipera ammodytes transcaucasiana and V. darevskii.

  • an electrophoretic comparison of the venoms of a colubrid and various viperid snakes from turkey and cyprus with some taxonomic and phylogenetic implications
    Zootaxa, 2005
    Co-Authors: Huseyin Arikan, Ahmet Mermer, Bayram Göçmen, Hasan Bahar
    Abstract:

    Venom extracts obtained from a colubrid snake [Malpolon monspessulanus (Hermann)] and eight viperids [Vipera xanthina (Gray), Vipera ammodytes (Linnaeus), Vipera kaznakovi (Nikolsky), Vipera eriwanensis (Bonaparte), Vipera wagneri Nilson A in the colubrid sample the total protein fraction number was 8, while in viperid samples they number between 10 and 14, indicating the venom complexity in viperids is higher than that of colubrid snakes. Electrophoretic data support the phylogenetic argument previously outlined of the family Viperidae. Moreover, in the light of the differences of the venom electrophoretic patterns, it is concluded that the southern Anatolian population of MacroVipera lebetina should not be identified as the nominate subspecies M. l. lebetina, which lives in Cyprus.

Adrijana Leonardi - One of the best experts on this subject based on the ideXlab platform.

  • the procoagulant snake venom serine protease potentially having a dual blood coagulation factor v and x activating activity
    Toxins, 2020
    Co-Authors: Zorica Latinovic, Adrijana Leonardi, Jože Pungercar, Manjunatha R Kini, Cho Yeow Koh, Alenka Trampus Bakija, Igor Križaj
    Abstract:

    A procoagulant snake venom serine protease was isolated from the venom of the nose-horned viper (Vipera ammodytes ammodytes). This 34 kDa glycoprotein, termed VaaSP-VX, possesses five kDa N-linked carbohydrates. Amino acid sequencing showed VaaSP-VX to be a chymotrypsin-like serine protease. Structurally, it is highly homologous to VaaSP-6 from the same venom and to nikobin from the venom of Vipera nikolskii, neither of which have known functions. VaaSP-VX does not affect platelets. The specific proteolysis of blood coagulation factors X and V by VaaSP-VX suggests that its blood-coagulation-inducing effect is due to its ability to activate these two blood coagulation factors, which following activation, combine to form the prothrombinase complex. VaaSP-VX may thus represent the first example of a serine protease with such a dual activity, which makes it a highly suitable candidate to replace diluted Russell's viper venom in lupus anticoagulant testing, thus achieving greater reliability of the analysis. As a blood-coagulation-promoting substance that is resistant to serpin inhibition, VaaSP-VX is also interesting from the therapeutic point of view for treating patients suffering from hemophilia.

  • Purification and characterisation of two hemorrhagic metalloproteinases from the venom of the long-nosed viper, Vipera ammodytes ammodytes
    Toxicon, 2017
    Co-Authors: Igor Križaj, Franc Gubenšek, Adrijana Leonardi
    Abstract:

    Two hemorrhagic proteins, VaH1 and VaH2, have been purified from Vipera ammodytes ammodytes venom. They are monomeric glycoproteins of an apparent molecular mass of 70 kDa and multiple isoelectric points around pH 5.5. Both molecules are proteolytically active against azocasein as substrate. VaH1, which was characterised in detail, showed maximum activity at pH 7.5. Ethylenediaminetetraacetic acid eliminated the proteolyticas well as the hemorrhagic activity of VaH1 while iodoacetamide, phenylmethylsulfonyl fluoride and pepstatin A, inhibitors of cysteine, serine and aspartic proteinases respectively, had no effect. VaH1 is therefore a metalloproteinase whose hemorrhagic activity is very likely the result of its proteolytic activity. VaH1 is a fibrinogenase, hydrolysing exclusively the A-chain of fibrinogen. In the B-chain of insulin it cleaved with a high preference the bond between Ala14 and Leu15. Based on its molecular mass, VaH1 (as well as VaH2) is a Class P-III metalloproteinase. Partial amino acid sequences of its CNBr fragments demonstrated a high level of identity with the reprolysin subfamily of zinc-metalloproteinases.

  • venomics of Vipera berus berus to explain differences in pathology elicited by Vipera ammodytes ammodytes envenomation therapeutic implications
    Journal of Proteomics, 2016
    Co-Authors: Zorica Latinović, Jernej Šribar, Adrijana Leonardi, Beata Halassy, Tamara Sajevic, Alenka Trampusbakija, Robert Frangež, Monika Cecilija žužek, Jože Pungercar, Igor Križaj
    Abstract:

    Abstract Vipera berus berus (Vbb) is the most widely distributed and Vipera ammodytes ammodytes (Vaa) the most venomous viper in Europe. In particular areas of the Old continent their toxic bites constitute a considerable public health problem. To make the current envenomation therapy more effective we have analysed the proteome of Vbb venom and compared it with that of Vaa. We found the proteome of Vbb to be much less complex and to contain smaller levels of particularly snaclecs and sPLA2s. Snaclecs are probably responsible for thrombocytopenia. The neurotoxic sPLA2s, ammodytoxins, are responsible for the most specific feature of the Vaa venom poisoning − induction of signs of neurotoxicity in patients. These molecules were not found in Vbb venom. Both venoms induce haemorrhage and coagulopathy in man. As Vaa and Vbb venoms possess homologous P-III snake venom metalloproteinases, the main haemorrhagic factors, the severity of the haemorrhage is dictated by concentration and specific activity of these molecules. The much greater anticoagulant effect of Vaa venom than that of Vbb venom lies in its higher extrinsic pathway coagulation factor-proteolysing activity and content of ammodytoxins which block the prothrombinase complex formation. Biological significance Envenomations by venomous snakes constitute a considerable public health problem worldwide, and also in Europe. In the submitted work we analysed the venom proteome of Vipera berus berus (Vbb), the most widely distributed venomous snake in Europe and compared it with the venom proteome of the most venomous viper in Europe, Vipera ammodytes ammodytes (Vaa). We have offered a possible explanation, at the molecular level, for the differences in clinical pictures inflicted by the Vbb and Vaa venoms. We have provided an explanation for the effectiveness of treatment of Vbb envenomation by Vaa antiserum and explained why full protection of Vaa venom poisoning by Vbb antiserum should not be always expected, especially not in cases of severe poisoning. The latter makes a strong case for Vaa antiserum production as we are faced with its shortage due to ceasing of production of two most frequently used products.

  • vah3 one of the principal hemorrhagins in Vipera ammodytes ammodytes venom is a homodimeric p iiic metalloproteinase
    Biochimie, 2013
    Co-Authors: Tamara Sajevic, Lidija Kovačič, Adrijana Leonardi, Beata Halassy, Alenka Trampusbakija, Jože Pungercar, Tihana Kurtovic, Maja Langbalija, Igor Križaj
    Abstract:

    Abstract Hemorrhage is the most potent manifestation of envenomation by Vipera ammodytes ammodytes (V. a. ammodytes) venom in man. A detailed description of the venom components contributing to this effect is thus medically very important. We have characterized a novel component, termed here VaH3, as a potently hemorrhagic snake venom metalloproteinase (SVMP). Its proteolytic activity and overall stability depend on the presence of Zn2+ and Ca2+ ions. The molecular mass of this slightly acidic molecule, determined by MALDI/TOF analysis, is 104 kDa. Chemical reduction and S-carbamoylmethylation result in a single monomer of 53.7 kDa. N-deglycosylation decreased this mass by 4.6 kDa. The complete amino acid sequence of VaH3 was determined by protein and cDNA sequencing, showing that each of the identical glycoprotein subunits comprise a metalloproteinase, a disintegrin-like domain and a cysteine-rich domain, VaH3 belongs to the P-IIIc class of SVMPs. It shows strong sequence similarity to vascular endothelial cell apoptosis-inducing reprolysins. Anti-ammodytagin antibodies strongly cross-reacted with VaH3 and completely neutralized its hemorrhagic activity in rat, despite the fact that the two hemorrhagic P-III SVMPs from V. a. ammodytes venom do not share a very high degree of amino acid sequence identity. In spite of its narrow proteolytic specificity, VaH3 rapidly cleaved some basal membrane and extracellular matrix proteins, such as collagen IV, fibronectin and nidogen. Moreover, it also hydrolyzed plasma proteins involved in blood coagulation. It is an effective α-fibrinogenase that cleaves prothrombin and factor X without activating them. The degradation of these proteins likely contributes to the hemorrhagic activity of VaH3. A three-dimensional model of VaH3 was built to help explain structure–function relationships in ADAM/ADAMTS, a family of proteins having significant therapeutic potential and substantial sequence similarity to VaH3.

  • The standard mouse assay of anti-venom quality does not measure antibodies neutralising the haemorrhagic activity of Vipera ammodytes venom.
    Toxicon : official journal of the International Society on Toxinology, 2012
    Co-Authors: Tihana Kurtović, Adrijana Leonardi, Igor Križaj, Marija Brgles, Lidija Habjanec, Maja Lang-balija, Beata Halassy
    Abstract:

    The venom of Vipera ammodytes ammodytes (Vaa), like the venoms of other Viperinae snakes, is largely haemorrhagic and necrotising, and only to a lesser extent neurotoxic to humans. The components most extensively studied so far, and most probably involved in generating the observed pathologies, are haemorrhagins (H), members of the metalloproteinase group of enzymes, and neurotoxic ammodytoxins (Atxs), that belong to the secretory phospholipases A2. Rabbit antisera were prepared containing functional antibodies specific for each class of pathology-inducing venom constituents and for both classes together. The involvement of these antibodies in neutralising the toxicity of whole Vaa venom was assessed using the ED50 assay in mice. This assay is the only regulatorily approved assay for estimating anti-venom potency and as such has the task to quantify the active compound neutralising venom-induced pathology of the anti-venom. Fully functional anti-Atx antibodies were shown to be responsible for neutralising the portion of venom toxicity, while anti-H antibodies were not protective in this assay. Thus, the mouse ED50 assay, intended to measure the active principle of the anti-venom, does not measure antibodies specific for Vaa venom haemorrhagins, and consequently does not fulfil its primary task from the regulatory point of view.