Rupamoni Thakur

Bafibrinase: A non-toxic, non-hemorrhagic, direct-acting fibrinolytic serine protease from Bacillus sp. strain AS-S20-I exhibits in vivo anticoagulant activity and thrombolytic potency

A non-toxic, direct-acting fibrinolytic serine protease (Bafibrinase) demonstrating thrombolytic and anticoagulant properties was purified from Bacillus sp. strain AS-S20-I. Bafibrinase was monomeric, with a molecular mass of 32.3 kDa. The peptide mass fingerprinting of Bafibrinase revealed only 8.3% sequence coverage, suggesting it was a novel fibrinolytic enzyme. However, two of the tryptic digested de novo peptide sequences of Bafibrinase demonstrated good similarity with endopeptidases possessing serine in their catalytic triad. Further, catalytic activity of Bafibrinase was inhibited by serine protease inhibitor reinforcing this is a subtilisin-like serine protease. The apparent K(m) and V(max) values of Bafibrinase towards fibrin were determined as 0.24 μM and 2.8 μmol/min, respectively. It showed a K(m) value of 0.139 mM towards a chromogenic substrate for plasmin (D-Val-Leu-Lys-p-Nitroanilide dihydrochloride) and optimum activity at physiological conditions (37 °C and pH 7.4). Based on the cleavage pattern of fibrin and fibrinogen, Bafibrinase may be classified as an α,β-fibrinogenase. Bafibrinase could not degrade collagen and was non-cytotoxic to HT29 cells or mammalian erythrocytes. Further, Bafibrinase at a dose of 2 mg/kg was devoid of toxicity as well as hemorrhagic activity on BALB/c mouse model, supporting its suitability for the development of a better and safer thrombolytic drug. Bafibrinase was also superior to human plasmin in degrading in vitro thrombus. The in vivo anticoagulant nature of Bafibrinase is being explored for the treatment and prevention of thrombosis and other cardiovascular diseases.

Pathogenic Variation in the Pearl Millet Blast Pathogen Magnaporthe grisea and Identification of Resistance to Diverse Pathotypes

Blast, also known as leaf spot, caused by Pyricularia grisea (teleomorph: Magnaporthe grisea), has emerged as a serious disease affecting both forage and grain production in pearl millet in India. Pathogenic variation was studied in a greenhouse using 25 M. grisea isolates collected from four major pearl-millet-growing states in India (Rajasthan, Haryana, Maharashtra, and Uttar Pradesh) on 10 pearl millet genotypes (ICMB 02444, ICMB 02777, ICMB 06444, ICMB 93333, ICMB 96666, ICMB 97222, ICMB 99444, 863B, ICMR 06222, and ICMB 95444). Differential reactions to the test isolates were recorded on ICMB 02444, ICMB 93333, ICMB 97222, 863B, and ICMR 06222. The 25 isolates were grouped into five different pathotypes based on their reaction types (virulent = score ≥ 4 and avirulent = score ≤ 3 on a 1-to-9 scale). For the identification of resistance sources, a pearl millet mini-core comprising 238 accessions was evaluated under greenhouse conditions against five M. grisea isolates (Pg118, Pg119, Pg56, Pg53, and Pg45) representing the five pathotypes. Of 238 accessions, 32 were found to be resistant to at least one pathotype. Resistance to multiple pathotypes (two or more) was recorded in several accessions, while three accessions (IP 7846, IP 11036, and IP 21187) exhibited resistance to four of the five pathotypes. Four early-flowering (≤50 days) blast-resistant mini-core accessions (IP 7846, IP 4291, IP 15256, and IP 22449) and four accessions (IP 5964, IP 11010, IP 13636, and IP 20577) having high scores (≥7) for grain and green fodder yield potential and overall plant aspect were found to be promising for utilization in pearl millet improvement programs. Identification of five pathotypes of M. grisea and sources of resistance to these pathotypes will provide a foundation for breeding for blast resistance in pearl millet in India.

Resistance to Foliar Diseases in a Mini-Core Collection of Sorghum Germplasm

Anthracnose, leaf blight, and rust are important biotic constraints to grain and forage sorghum production worldwide and are best managed through host plant resistance. A sorghum mini-core collection, consisting of 242 germplasm accessions developed from a core collection of 2,246 landrace accessions originating from 58 countries, was evaluated to identify sources of resistance to foliar diseases. The mini-core accessions were evaluated in anthracnose- and leaf-blight-screening nurseries under artificial inoculation in the rainy and late rainy seasons, respectively, during 2009 and 2010. For rust resistance, screening was done under artificial inoculation in the greenhouse as well as in the field under natural infection. In all, 13 accessions were found resistant (score ≤3.0 on a 1-to-9 scale) to anthracnose and 27 to leaf blight in both 2009 and 2010. Six accessions exhibited resistance to rust in both the greenhouse and the field. In the resistant accessions, a wide range of diversity was observed for agronomic traits such as days to 50% flowering, plant height, and grain yield/plant, and morphological characteristics such as grain or glume color, glume coverage, endosperm texture, and panicle type (ear head compactness). Three mini-core accessions (IS 473, IS 23684, and IS 23521) exhibited resistance to all three diseases. These accessions with multiple disease resistance will be useful in sorghum disease resistance breeding programs.

Characterization of a pro-angiogenic, novel peptide from Russell's viper (Daboia russelii russelii) venom.

Present report shows for the first time on the induction of in vitro angiogenesis by a 3.9 kDa novel peptide (RVVAP) purified from Russells viper venom. Secondary structure of RVVAP is made up of 36.8% α-helix, 33.3% β pleated sheets and 29.9% turns. Optimum angiogenesis and significant elevation in endothelial migration were observed at 50 ng/ml of RVVAP treatment; above this concentration, progressive decrease in wound healing was noted. RVVAP (1.0 μg/ml) was non-cytotoxic to U87-MG, HeLa and HT-29 cells; however, increasing the RVVAP concentration above 500 ng/ml resulted in induction of chromosomal aberrations and delay in cell cycle kinetics of Chinese hamster ovary cells.

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.

Background The monocled cobra (Naja kaouthia) is responsible for snakebite fatality in Indian subcontinent and in south-western China. Phospholipase A2 (PLA2; EC 3.1.1.4) 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/Ca2+. 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.

Identification of Blast Resistance in a Core Collection of Foxtail Millet Germplasm

Blast, also known as leaf spot, caused by Pyricularia grisea (teleomorph: Magnaporthe grisea), is a serious disease affecting both forage and grain production in foxtail millet in India. For the identification of new and diverse sources of blast resistance, a foxtail millet core collection comprising 155 accessions was evaluated against the Patancheru isolate (Fx 57) of M. grisea. In a field screen during 2009 and 2010, 21 accessions were identified with neck and head blast resistance against Fx 57. In a greenhouse screen, 11 of the 155 accessions exhibited seedling leaf blast resistance to the same isolate. Further evaluation of the selected 28 accessions (found resistant to neck and head blast under field conditions during 2009 and 2010 or leaf blast in the greenhouse screen) against four M. grisea isolates (Fx 57, Fx 58, Fx 60, and Fx 62 from Patancheru, Nandyal, Vizianagaram, and Mandya, respectively) led to the identification of 16 accessions with leaf, sheath, neck, and head blast resistance to at least one isolate. Two accessions (ISe 1181 and ISe 1547) were free from head blast infection and showed resistance to leaf (score ≤3.0 on a 1-to-9 scale), neck, and sheath blast (score ≤2.0 on a 1-to-5 scale) against all four isolates. In addition, ISe 1067 and ISe 1575 also exhibited high levels of blast resistance. Blast-resistant accessions with superior agronomic and nutritional quality traits can be evaluated in multilocation yield trials before releasing them for cultivation to farmers.

Biochemical and pharmacological characterization of a toxic fraction and its cytotoxin-like component isolated from Russell's viper (Daboia russelii russelii) venom.

The pathophysiological significance of a toxic fraction (GF-VI DEAE-II) isolated from Russells 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.

A Brief Appraisal on Russell’s Viper Venom (Daboia russelii russelii) Proteinases

Russell’s viper (Daboia russelii) is an important member of the Viperidae family which is widely distributed across Southern Asia including India. Russell’s viper venom (RVV) is indisputably a potent mixture of various toxic and nontoxic components that have evolved to interfere with vital physiological processes like coagulation and fibrinolysis. Many of these components are metalloor serine proteinases. Venoms from the snakes of Viperidae family are reported to be rich in R. Thakur Microbial Biotechnology and Protein Research Laboratory, Department of Molecular Biology and Biotechnology, School of Sciences, Tezpur University, Tezpur, Assam, India e-mail: rupat@tezu.ernet.in A.K. Mukherjee (*) Department of Molecular Biology and Biotechnology, Research and Development, ONGC-Centre for Petroleum Biotechnology, DBT Nodal Centre for Medical Colleges and Biomedical Research Institutes of North-East India (Unit of Department of Biotechnology, Govt. of India), Tezpur University, Tezpur, Assam, India e-mail: editor_tsi@yahoo.in; akm@tezu.ernet.in # Springer Science+Business Media Dordrecht 2017 P. Gopalakrishnakone et al. (eds.), Snake Venoms, Toxinology, DOI 10.1007/978-94-007-6410-1_19 123 proteinases which contributes to hemostatic alterations in the victims and is also responsible for tissue-specific hemorrhagic effects. However, till date, not many proteinases have been reported from RVV. In this brief review, the currently available data on the structural and functional attributes of RVV proteinases have been focused and summarized. The pathophysiological significance and the therapeutic/ diagnostic application(s) of the identified RVV proteinases have also been discussed. It is relevant to note that a more detailed analysis of the entire complexity of RVV proteinases will contribute to the better understanding of their role in Russell’s viper envenomation and subsequent antivenom therapy. Most importantly, novel RVV proteinases can also be explored for their usefulness in the development of diagnostic/therapeutic agents to deal with lifethreatening diseases.

Pathophysiological significance and therapeutic applications of snake venom protease inhibitors

ABSTRACT Protease inhibitors are important constituents of snake venom and play important roles in the pathophysiology of snakebite. Recently, research on snake venom protease inhibitors has provided valuable information to decipher the molecular details of various biological processes and offer insight for the development of some therapeutically important molecules from snake venom. The process of blood coagulation and fibrinolysis, in addition to affecting platelet function, are well known as the major targets of several snake venom protease inhibitors. This review summarizes the structure‐functional aspects of snake venom protease inhibitors that have been described to date. Because diverse biological functions have been demonstrated by protease inhibitors, a comparative overview of their pharmacological and pathophysiological properties is also highlighted. In addition, since most snake venom protease inhibitors are non‐toxic on their own, this review evaluates the different roles of individual protease inhibitors that could lead to the identification of drug candidates and diagnostic molecules.