@article {454, title = {Mechanism of apoptosis induction in human breast cancer MCF-7 cell by Ruviprase, a small peptide from Daboia russelii russelii venom.[Mass Spectrometry]}, journal = {Chem Biol Interact}, volume = {258}, year = {2016}, month = {2016 Oct 25}, pages = {297-304}, abstract = {

Ruviprase, a 4.4~kDa peptide isolated from Daboia russelii russelii venom demonstrated antiproliferative activity against EMT6/AR1, U-87MG, HeLa and MCF-7 cancer cells with an IC50 value of 23.0, 8.8, 5.8 and 4.0~μg ml(-1), respectively. However, it was nontoxic to non-cancerous human embryonic kidney cell and human peripheral blood lymphocytes. Flow-cytometric analysis confirmed the apoptosis induction in MCF-7 cells by Ruviprase where it induced DNA condensation but did not cause mitotic blockage or chromosomal aberration in treated-cells. Immunofluorescence microscopic analysis indicated Ruviprase induced apoptosis in MCF-7 cells through p53 and p21-mediated pathways. Ruviprase generated reactive oxygen species (ROS), altered the mitochondrial transmembrane potential, and significantly decreased the cellular glutathione (GSH) content of MCF-7 cells. Immunoblotting and quantitative real-time PCR (qRT-PCR) analyses suggested that Ruviprase down-regulated the expression of anti-apoptotic protein Bcl-2, increased cleavage of poly (ADP-ribose) polymerase (PARP) protein, and up-regulated the expression of pro-apoptotic protein Bax, as well as executer protein caspase-7 to induced apoptosis in MCF-7 cells via intrinsic pathway. This is the first report on the characterization of the anticancer potential of a small, non-toxic and anticoagulant peptide purified from Russell{\textquoteright}s viper venom.

}, issn = {1872-7786}, doi = {10.1016/j.cbi.2016.09.004}, author = {Thakur, Rupamoni and Kini, Sudarshan and Kurkalang, Sillarine and Banerjee, Atanu and Chatterjee, Purba and Chanda, Abhishek and Chatterjee, Anupam and Panda, Dulal and Mukherjee, Ashis K} } @article {515, title = {Biochemical and pharmacological characterization of a toxic fraction and its cytotoxin-like component isolated from Russell{\textquoteright}s viper (Daboia russelii russelii) venom. (Mass Spectrometry)}, journal = {Comp Biochem Physiol C Toxicol Pharmacol}, volume = {168}, year = {2015}, month = {2015 Feb}, pages = {55-65}, abstract = {

The pathophysiological significance of a toxic fraction (GF-VI DEAE-II) isolated from Russell{\textquoteright}s viper venom (RVV) is characterized. GF-VI DEAE-II represents 1.6\% of the total RVV protein and it comprises of a 27.6kDa minor component (RP-I) (0.04\%, w/w) and a major 6.6kDa non-enzymatic peptide (1.11\%, w/w), named Rusvitoxin. The LC-MS/MS analysis of RP-I showed its identity to snake venom serine proteases, whereas Rusvitoxin demonstrated its close identity with snake venom three finger toxins, cytotoxins and cardiotoxins particularly from Naja sp. GF-VI DEAE-II was found to be non-cytotoxic to the tested mammalian cancer cells and non-hemolytic; nevertheless, it demonstrated α-fibrin(ogen)ase activity and in vivo toxicity in BALB/c mice with an LD50 (i.p.) of 2.3mg/kg. GF-VI DEAE-II induced lethargy and hind-leg paralysis in mice within 10min of i.p. injection. GF-VI DEAE-II induced hyperfibrinogenomia, and significantly altered (p\<0.05) the plasma levels of factor X, pro- and anti-inflammatory cytokines viz. TNF-α, IL-6 and IL-10 in treated mice. Histological observations of tissues and biochemical properties of serum from GF-VI DEAE-II-treated mice suggested multiple organ dysfunctions. Conversely, Rusvitoxin at a dose of 5mg/kg did not induce toxicity in BALB/c mice. At 1:15 (antigen: antivenom, w/w) ratio, commercially polyvalent and monovalent antivenoms neutralized more than 80\% of the fibrinolytic and anticoagulant activities of GF-VI DEAE-II. The present study suggests the significant role of GF-VI DEAE-II in RVV-induced pathogenesis in victim/prey.

}, keywords = {Animals, Anticoagulants, Antivenins, Cytotoxins, Fibrinolytic Agents, Interleukin-10, Interleukin-6, Mice, Mice, Inbred BALB C, Mollusk Venoms, Russell{\textquoteright}s Viper, Tumor Necrosis Factor-alpha, Viper Venoms}, issn = {1532-0456}, doi = {10.1016/j.cbpc.2014.12.001}, author = {Thakur, Rupamoni and Chattopadhyay, Pronobesh and Mukherjee, Ashis K} } @article {514, title = {Elucidation of procoagulant mechanism and pathophysiological significance of a new prothrombin activating metalloprotease purified from Daboia russelii russelii venom. (Mass Spectrometry)}, journal = {Toxicon}, volume = {100}, year = {2015}, month = {2015 Jun 15}, pages = {1-12}, abstract = {

The procoagulant proteases present in Russell{\textquoteright}s Viper venom (RVV) are responsible for promoting consumption coagulopathy in victims. In this study, a procoagulant metalloprotease (Rusviprotease) possessing prothrombin activating and α-fibrinogenase properties has been purified and characterized from RVV. Rusviprotease is a 26.8\ kDa glycoprotein which also exists in other multimeric forms. The peptide mass fingerprinting and secondary structure analyses of Rusviprotease revealed its similarity with snake venom prothrombin activators and metalloproteases. Similar to group A prothrombin activators, Rusviprotease cleaved prothrombin independent of any co-factor requirement generating meizothrombin which is further cleaved to form thrombin. The Km and Vmax values of Rusviprotease towards prothrombin were determined to be 1.73\ μM, and 153.5\ nM thrombin generated/min/μmoles of Rusviprotease, respectively. The Km and Vmax values of Rusviprotease towards fibrinogen were calculated to be 3.14\ μM and 78.7\ nmol/min, respectively. Spectrofluorometric study provided the evidence of interaction between Rusviprotease and factor Xa with a Kd value of\ 6.64\ nM. This interaction augmented the prothrombin activating property of the factor Xa-prothrombinase-Rusviprotease complex by 2.5 fold. Intravenous injection of Rusviprotease to BALB/c mice (0.1\ mg/kg) resulted in in\ vivo defibrinogenation rendering the blood incoagulable. In conclusion, Rusviprotease is the first example of a prothrombin activator with fibrinogenolytic property purified from Daboia russelii russelii venom.

}, keywords = {Animals, Blood Coagulation, Blood Platelets, Chemical Fractionation, Coagulants, Erythrocytes, Goats, Metalloproteases, Mice, Inbred BALB C, Plasma, Russell{\textquoteright}s Viper, Toxicity Tests, Acute, Viper Venoms}, issn = {1879-3150}, doi = {10.1016/j.toxicon.2015.03.019}, author = {Thakur, Rupamoni and Chattopadhyay, Pronobesh and Ghosh, Siddharth S and Mukherjee, Ashis K} } @article {517, title = {Two acidic, anticoagulant PLA2 isoenzymes purified from the venom of monocled cobra Naja kaouthia exhibit different potency to inhibit thrombin and factor Xa via phospholipids independent, non-enzymatic mechanism. (Mass Spectrometry - Proteomics)}, journal = {PLoS One}, volume = {9}, year = {2014}, month = {2014}, pages = {e101334}, abstract = {

BACKGROUND: The monocled cobra (Naja kaouthia) is responsible for snakebite fatality in Indian subcontinent and in south-western China. Phospholipase A2 (PLA2; EC is one of the toxic components of snake venom. The present study explores the mechanism and rationale(s) for the differences in anticoagulant potency of two acidic PLA2 isoenzymes, Nk-PLA2α (13463.91 Da) and Nk-PLA2β (13282.38 Da) purified from the venom of N. kaouthia.

PRINCIPAL FINDINGS: By LC-MS/MS analysis, these PLA2s showed highest similarity (23.5\% sequence coverage) with PLA2 III isolated from monocled cobra venom. The catalytic activity of Nk-PLA2β exceeds that of Nk-PLA2α. Heparin differentially regulated the catalytic and anticoagulant activities of these Nk-PLA2 isoenzymes. The anticoagulant potency of Nk-PLA2α was comparable to commercial anticoagulants warfarin, and heparin/antithrombin-III albeit Nk-PLA2β demonstrated highest anticoagulant activity. The anticoagulant action of these PLA2s was partially contributed by a small but specific hydrolysis of plasma phospholipids. The strong anticoagulant effect of Nk-PLA2α and Nk-PLA2β was achieved via preferential, non-enzymatic inhibition of FXa (Ki = 43 nM) and thrombin (Ki = 8.3 nM), respectively. Kinetics study suggests that the Nk-PLA2 isoenzymes inhibit their "pharmacological target(s)" by uncompetitive mechanism without the requirement of phospholipids/Ca(2+). The anticoagulant potency of Nk-PLA2β which is higher than that of Nk-PLA2α is corroborated by its superior catalytic activity, its higher capacity for binding to phosphatidylcholine, and its greater strength of thrombin inhibition. These PLA2 isoenzymes thus have evolved to affect haemostasis by different mechanisms. The Nk-PLA2β partially inhibited the thrombin-induced aggregation of mammalian platelets suggesting its therapeutic application in the prevention of unwanted clot formation.

CONCLUSION/SIGNIFICANCE: In order to develop peptide-based superior anticoagulant therapeutics, future application of Nk-PLA2α and Nk-PLA2β for the treatment and/or prevention of cardiovascular disorders are proposed.

}, keywords = {Anticoagulants, Blood Platelets, Chemical Fractionation, Chromatography, Liquid, Cobra Venoms, Factor Xa, Factor Xa Inhibitors, Isoenzymes, Kinetics, Phospholipases A2, Phospholipids, Sequence Analysis, Protein, Tandem Mass Spectrometry, Thrombin}, issn = {1932-6203}, doi = {10.1371/journal.pone.0101334}, author = {Mukherjee, Ashis K and Kalita, Bhargab and Thakur, Rupamoni} }