ISOLATION AND PROPERTIES OF A BLOOD-COAGULATION FACTOR-X ACTIVATOR FROM THE VENOM OF KING COBRA (OPHIOPHAGUS HANNAH)

A specific blood coagulation factor X activator was purified from the venom of Ophiophagus hannah by gel filtration and two steps of FPLC Mono-Q column ion-exchange chromatography. It showed a single protein band both in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and alkaline polyacrylamide gel electrophoresis. The mol. wt was estimated to be 62,000 in non-reducing conditions and 64,500 in reducing conditions by SDS-PAGE. The isoelectric point was found to be pH 5.6. The enzyme had weak amidolytic activities toward CBS 65-25, but it showed no activities on S-2266, S-2302, thrombin substrate S-2238, plasmin substrate S-2251 or factor Xa substrate S-2222. It had no arginine esterase activity toward substrate benzoylarginine ethylester (BAEE). The enzyme activated factor X in vitro and the effect was absolutely Ca2+ dependent, with a Hill coefficient of 6.83. It could not activate prothrombin nor had any effect on fibrinogen and thus appeared to act specifically on factor X. The procoagulant activity of the enzyme was almost completely inhibited by serine protease inhibitors like PMSF, TPCK and soybean trypsin inhibitor; partially inhibited by L-cysteine. Metal chelator EDTA did not inhibit its procoagulant activity. These results suggest that the factor X activator from O. hannah venom is a serine protease.

Trimeresurus stejnegeri snake venom plasminogen activator - Site-directed mutagenesis and molecular modeling

The specific plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA) is a serine proteinase presenting 23% sequence identity with the proteinase domain of tissue type plasminogen activator, and 63% with batroxobin, a fibrinogen clotting enzyme from Bothrops atrox venom that does not activate plasminogen. TSV-PA contains six disulfide bonds and has been successfully overexpressed in Escherichia coli (Zhang, Y., Wisner, A., Xiong, Y. L,, and Bon, C, (1995) J. Biol. Chem. 270, 10246-10255), To identify the functional domains of TSV-PA, we focused on three short peptide fragments of TSV-PA showing important sequence differences with batroxobin and other venom serine proteinases. Molecular modeling shows that these sequences are located in surface loop regions, one of which is next to the catalytic site, When these sequences were replaced in TSV-PA by the equivalent batroxobin residues none generated either fibrinogen-clotting or direct fibrinogenolytic activity, Two of the replacements had little effect in general and are not critical to the specificity of TSV-PA for plasminogen. Nevertheless, the third replacement, produced by the conversion of the sequence DDE 96a-98 to NVI, significantly increased the K-m for some tripeptide chromogenic substrates and resulted in undetectable plasminogen activation, indicating the key role that the sequence plays in substrate recognition by the enzyme.

Isolation and cDNA cloning of cholecystokinin from the skin of Rana nigrovittata

Many neuroendocrine peptides that are distributed in amphibian gastrointestinal tract and central nervous system are also found in amphibian skins, and these peptides are classified into skin-gut-brain triangle peptides, such as bombesins, gastrin-releasi

Isolation and preliminary characterization of a 22-kDa protein with trypsin inhibitory activity from toad Bufo andrewsi skin

A novel trypsin inhibitor termed BATI was purified to homogeneity from the skin extracts of toad Bufo andrewsi by successive ion-exchange, gel-filtration and reverse-phase chromatography. BATI is basic single chain glycoprotein, with apparent molecular weight of 22 kDa in SDS-PAGE. BATI is a thermal stable competitive inhibitor and effectively inhibits trypsin's catalytic activity on peptide substrate with the inhibitor constant (K-i) value of 14 nM and shows no inhibitory effect on chymotrypsin, thrombin and elastase. The N-terminal sequence of BATI is EKDSITD, which shows no similarity with other known trypsin inhibitors. (c) 2005 Elsevier Ltd. All rights reserved.

Isolation and cloning of a metalloproteinase from king cobra snake venom

A 50 kDa fibrinogenolytic protease, ohagin, from the venom of Ophiophagus hannah was isolated by a combination of gel filtration, ion-exchange and heparin affinity chromatography. Ohagin specifically degraded the alpha-chain of human fibrinogen and the pr

Isolation, expression and characterization of a novel dual serine protease inhibitor, OH-TCI, from king cobra venom

Snake venom Kunitz/BPTI members are good tools for understanding of structure-functional relationship between serine proteases and their inhibitors. A novel dual Kunitz/BPTI serine proteinase inhibitor named OH-TCI (trypsin- and chymotrypsin-dual inhibito

Bioactivity of epidermal secretion of two marine catfish from Mumbai

Crude mucus and its partially purified fractions from two marine catfish from Mumbai, Arius dussumieri and Osteogeneiosus militaris were assayed for their crinotoxicity through assays for hemolysis and haemagglutination of chicken erythrocytes, formation of paw edema in mice, and antibacterial activity against one gram-positive and four gram-negative bacteria. Assays were also done to block the edema using Phineramine maleate, Piroxicam, and Atropine sulfate. Crude toxin as well as their fractions from both the fishes exhibited haemolytic and haemagglutinating activities on chicken blood, besides edematous activity in mice models. The edematous activity was blocked by Phineramine maleate and Piroxicam but enhanced by Atropine sulfate; however, all these activities, either blocking or enhancing, were statistically insignificant. Antibacterial activity was absent in all the extracts tested.

A novel short neurotoxin, cobrotoxin c, from monocellate cobra (Naja kaouthia) venom: isolation and purification, primary and secondary structure determination, and tertiary structure modeling

A novel short neurotoxin, cobrotoxin c (CBT C) was isolated from the venom of monocellate cobra (Naja kaouthia) using a combination of ion-exchange chromatography and FPLC. Its primary structure was determined by Edman degradation. CBT C is composed of 61 amino acid residues. It differs from cobrotoxin b (CBT B) by only two amino acid substitutions, Thr/Ala11 and Arg/Thr56, which are not located on the functionally important regions by sequence similarity. However, the LD50 is 0.08 mg/g to mice, i.e. approximately five-fold higher than for CBT B. Strikingly, a structure-function relationship analysis suggests the existence of a functionally important domain on the outside of Loop III of CBT C. The functionally important basic residues on the outside of Loop III might have a pairwise interaction with alpha subunit, instead of gamma or delta subunits of the nicotinic acetylcholine receptor (nAChR). (C) 2002 Elsevier Science Inc. All rights reserved.

Physical Blocking Neural Tube Closure Affects Radial Intercalation and Neural Crest Midline-directed Migration in Xenopus Dorsal Explants

神经管闭合缺陷(NTDs)是一种严重的先天畸形疾病,在新生儿中有千分之一的发病率.神经管融合前后,多种组织参与形态发生运动.神经管一经融合,神经嵴细胞就会向背侧中线方向产生单极突出并向此方向迁移形成神经管的顶部.与此同时,神经管从腹侧开始发生辐射状切入以实现单层化.在此,我们在非洲爪蟾的移植体中机械阻断神经管的闭合以检测其细胞运动及随后的图式形成.结果显示神经管闭合缺陷的移植体不能形成单层化的神经管,并且神经嵴细胞滞留在侧面区域不能向背侧中线迁移,而对神经前体标记基因的检测显示神经管的背腹图式形成并未受到影响.以上结果表明神经管的融合对于辐射状切入和神经嵴细胞向背侧中线方向的迁移过程是必需的,而对于神经管的沿背腹轴方向的图式形成是非必需的.

Isolation of Salmonella larochelle for the first time in India

The isolation of a new serotype, S. larochelle (6, 7: eh:1, 2), is reported in this communication. This serotype has not so far been reported from any source in this country. The serotype was isolated on November 27th, 1979 at Bombay from a market sample of frozen frog legs intended for domestic consumption.

Studies on isolation of salmonella from sea foods 1. Comparison of enrichment and selective media for recovery of salmonellae from fish

Three enrichment broths and six plating media were compared for efficiency of detection Salmonella in the presence of numbers of Coliforms (10super(5)/ml) and proteus (10super(3)/ml) from artificially inoculated fish samples. Recovery experiments Salmonella anatum, S. typhimurium and S. enteritidis indicated that the two enrichment broths Dulcitol Selinite (DSE) and Selinite Cystine (SC) were equally efficient. Further, the viability of Salmonella, inoculated into fish muscle and kept at 4°C for 48 hours, was found to be not affected by the low temperature storage. Selective plating media like Xylose Lysine Deoxycholate agar (XLD), Brilliant Green Sulphadiazine agar (BGS) and Brilliant Green agar (BG) were found to be superior in performance to Salmonella-Shigella agar: (SS) and Bismuth Sui phite agar (BiS).

Utilization of prawn waste: isolation of chitin and its conversion to chitosan

process is described for the preparation of chitosan from prawn waste. The process involves extraction of protein using 0.5% sodium hydroxide solution, bleaching the protein free mass with bleach liquor containing 0.3-0.5% available chlorine followed by demineralisation with 1.25 N hydrochloric acid in the cold and deacetylation using 1:1 (w/w) sodium hydroxide solution at 100°C for 2 hours.

Chemical control of psychrophilic bacterial spoilage of fish 1. Isolation and identification of psychrophilic bacteria from marine fish

Making use of the streak plate technique and low temperature incubation, 28 cultures belonging to six genera namely, Achromobacter, Flavobacterium, Pseudomonas, Micrococcus, Vibrio and Alcaligenes were isolated from different varieties of marine fish. The growth studies indicated that all of them were able to grow between -5 and +5°C within a week's time and none of them showed growth at 37°C. The optimum temperature of growth for all these cultures was in the range 25-28°C. Among these only one, i.e., a Vibrio sp., was found to be an obligate psychrophile.