Comparative study of three short-chain neurotoxins from the venom of Naja kaouthia (Yunnan, China)

Three short-chain neurotoxins named NT-I, NT-II, and NT-III were purified from the venom of Naja kaouthia, a snake distributed throughout the south of Yunnan province, China, by a series of chromatographic steps, including an FPLC Resource S column. Their molecular weights, determined by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS, were 6952.19 Da, 6854.92 Da, and 6828.80 Da, respectively. NT-I consisted of 62 amino acid residues, and the other two consisted of 61 amino acid residues, including 8 cysteines. After hydrolysis by endoproteinase Glu-C, their primary sequences were determined. A test of their activities demonstrated that they effectively inhibited muscle contractions induced by electric stimulation. Furthermore, the extent of inhibition caused by NT-II and NT-III was less than that of NT-I. The IC(50)s were 0.04 mug/ml, 0.20 mug/ml, and 0.23 mug/ml for NT-I, NT-II, and NT-III, respectively. Compared with NT-II and NT-III, the higher activity of NT-I may be a result of the amino acid residue substitution Ile36 to Arg36.

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.

L-amino acid oxidase from Naja atra venom activates and binds to human platelets

An L-amino acid oxidase (LAAO), NA-LAAO, was purified from the venom of Naja atra. Its N-terminal sequence shows great similarity with LAAOs from other snake venoms. NA-LAAO dose-dependently induced aggregation of washed human platelets. However, it had n

A nerve growth factor from the venom of Chinese cobra (Naja naja atra) and its effects on male reproductive system in rats

A nerve growth factor (NGF) was isolated from the venom of Chinese cobra (Naja naja ntr a) by ion exchange chromatography, gel filtration and fast protein liquid chromatography (FPLC). The N-terminal sequence of 22 amino acid residues was identical with other NGFs previously purified from the venom of the same genus. The NGF monomer molecular weight was estimated to be 13 500 by reducing SDS-PAGE and the isoelectric point was determined to be 7.2 by isoelectric focusing electrophoresis. NGF improved the epididymal sperm motility of male rats and increased the pregnancy rate and fetus number of mated female rats. The serum levels of luteinizing hormone (LH) and follicle stimulating hormone (FSH) of male rats administrated NGF + gossypol was lower than that of male rats administrated gossypol. Histological sections of testes and epididymides showed that NGF reduced the destructive effects of gossypol on rat testes. (C) 1999 Elsevier Science Inc. All rights reserved.

A highly active anticomplement factor from the venom of Naja kaouthia

A highly active anticomplement factor (cobra venom factor) from the venom of Naja kaouthia in South Yunnan, China was isolated by sequential column chromatography (SP-Sephadex-C25, Q Sepharose HP and Sephadex G-150). It displays strong anticomplement acti

Purification and cloning of cysteine-rich proteins from Trimeresurus jerdonii and Naja atra venoms

Three 26 kDa proteins, named as TJ-CRVP, NA-CRVP1 and NA-CRVP2, were isolated from the venoms of Trimeresurus jerdonii and Naja atra, respectively. The N-terminal sequences of TJ-CRVP and NA-CRVPs were determined. These components were devoid of the enzymatic activities tested, such as phospholipase A(2), arginine esterase, proteolysis, L-amino acid oxidase, 5' nucleotidase, acetylcholinesterase. Furthermore, these three components did not have the following biological activities: coagulant and anticoagulant activities, lethal activity, myotoxicity, hemorrhagic activity, platelet aggregation and platelet aggregation-inhibiting activities. These proteins are named as cysteine-rich venom protein (CRVP) because their sequences showed high level of similarity with mammalian cysteine-rich secretory protein (CRISP) family. Recently, some CRISP-like proteins were also isolated from several different snake venoms, including Agkistrodon blomhoffi, Trimeresurus flavoviridis, Lanticauda semifascita and king cobra. We presumed that CRVP might be a common component in snake venoms. Of particular interest, phylogenetic analysis and sequence alignment showed that NA-CRVP1 and ophanin, both from elapid snakes, share higher similarity with CRVPs from Viperidae snakes. (C) 2003 Elsevier Ltd. All rights reserved.

Effect of electric stimulation on heart beat and body muscle in fish

The effect of AC and DC electric stimulations on the heart-rate and the entire body of Heteropneustis fossillis, Tilapia mossambica and Macrobrachium rosenbergii were studied and presented in kymograph tracings. The reaction of spinal cord in Puntius ticto, Heteropneustis fossilis and Tilapia mossambica to D. C. field was observed to find out its role in electric shocks. A test-check of the electrical resistance of a few species was also conducted. The effect of D. C. and A.C. on the body muscle was found to be the same as that in the case of frog. Different degrees of cardiac slowing were observed in AC and DC. Unbalanced galvanotropic movements were also noticed in spinal fishes.

Reaction of fishes to the underwater A.C. field

A comparative study on the effect of A.C. field on Puntius ticto, Heteropneustis fossilis and Tilapta mossambica was carried out using a slowly rising field intensity. Well defined reactions appeared in the species of fish with slight specific variations, depending on their orientation in the electrical field, on reaching the field intensity to specific value. These reactions can be distinguished as first reaction, when the fish perceive the surrounding field, jerky swimming when parallel to the current lines (longitudinal oscillotaxis), the static position finally adopted by the fish sooner or latter depending on the potential gradient (transverse oscillotaxis), and a state of muscular rigidity (tetanus). After switching off the current, a hypnotic condition prevailed in the treated fishes before returning to their normal swimming condition. The orientation of fish body in the field had an important bearing on the behaviour reactions and current thresholds necessary for those reactions. Initial reaction, jerky swimming between electrodes and hypnosis after stoppage of current appeared in fishes earlier when the fish body was in parallel to the current lines, whereas fishes responded to transverse oscillotaxis quickly when perpendicular to current lines.

Response of fishes to the underwater D.C. field

Response to external electric field (D. C.) of three different varieties of fish namely Puntius ticto, Heteropneustis fossilis and Tilapia mossambica having different anatomical and behavioural characteristics were studied. Clearly distinguished reactions occurred one after another m all the varieties of fish with the increase in field intensity with minor specific variations. These reactions can be broadly classified into initial start (first reaction), forced swimming (galvanotaxis), slackening of body muscle (galvanonarcosis) and state of muscular rigidity (tetanus). The orientation of the organism (projection of nervous element) to the surrounding field has been found to have important bearing on the behaviour reactions. Clearly differentiated anodic taxis and true narcosis set in when fish body axis was parallel to the lines of current conduction. But with greater angle between the body axis and the current lines, fish did not show well marked reactions. Fish body, when perpendicular to current lines responded for anodic curvature and off balance swimming followed by tetanus.

The preservation of some Indian freshwater fish

Quality changes in Labeo rohita, Cirrhinus mrigala and Tilapia mossambica were studied during storage in ice and at ambient temperature (29-32°C). After 10h at ambient temperature L. rohita and T. mossambica were completely spoiled, while C. mrigala became unacceptable within 11h. Organoleptic examination, total volatile base nitrogen and total bacterial counts showed that L. rohita (0.5 kg and above) and C. mrigala (0.5 kg and above) were acceptable up to 7 to 8 days of storage in ice in popular container (Bamboo basket) lined with palmyrah mat) at ambient temperature while T. mossambica (30-40 g) was acceptable up to 6 days. Influence of size of fish on shelf life in ice studied with reference to L. rohita showed that fishes below 500 g were acceptable up to 6 days of storage in ice in popular container at ambient temperature while those above 1000 g size were acceptable up to 8 days. Provision of an alkathene lining to the popular container extended the storage life of L. rohita from 6 days to 8 days. Pre-chilled and iced L. rohita had a longer shel flife (9 days) than merely iced sample (7 days).

Structure-function relationship of three neurotoxins from the venom of Naja kaouthia: a comparison between the NMR-derived structure of NT2 with its homologues, NT1 and NT3

Three homologous short-chain neurotoxins, named NT1, NT2 and NT3, were purified from the venom of Naja kaouthia. NT1 has an identical amino acid sequence to cobrotoxin from Naja naja atra [Biochemistry 32 (1993) 2131]. NT3 shares the same sequence with cobrotoxin b [J. Biochem. (Tokyo) 122 (1997) 1252], whereas NT2 is a novel 6 1 -residue neurotoxin. Tests of their physiological functions indicate that NT1 shows a greater inhibition of muscle contraction induced by electrical stimulation of the nerve than do NT2 and NT3. Homonuclear proton two-dimensional NMR methods were utilized to study the solution tertiary structure of NT2. A homology model-building method was employed to predict the structure of NT3. Comparison of the structures of these three toxins shows that the surface conformation of NT1 facilitates the substituted base residues, Arg28, Arg30, and Arg36, to occupy the favorable spatial location in the central region of loop 11, and the cation groups of all three arginines face out of the molecular surface of NT1 This may contribute greatly to the higher binding of NT1 with AchR compared to NT2 and NT3. (C) 2002 Elsevier Science B,V. All rights reserved.

Purification, characterization and primary structure of a chymotrypsin inhibitor from Naja atra venom

A chymotrypsin inhibitor, designated NA-CI, was isolated from the venom of the Chinese cobra Naja atra by three-step chromatography. It inhibited bovine (x-chymotrypsin with a K-i of 25 nM. The molecular mass of NA-CI was determined to be 6403.8 Da by matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) analysis. The complete amino acid sequence was determined after digestion of S-carboxymethylated inhibitor with Staphylococcus aureus V8 protease and porcine trypsin. NA-CI was a single polypeptide chain composed of 57 amino acid residues. The main contact site with the protease (PI) has a Phe, showing the specificity of the inhibitor. NA-CI shared great similarity with the chymotrypsin inhibitor from Naja naja venom (identities = 89.5%) and other snake venom protease inhibitors. (C) 2003 Elsevier Inc. All rights reserved.

莫桑比克非罗鱼胚胎人工孚化试验

<正> 一、前言莫桑比克罗非鱼(Tilapia mossambica)是一种属口孚化的鱼类,雌鱼把受精卵含在口腔中进行孚化,待仔鱼卵黄囊全部消失,并能独立生活,这时,雌鱼才把它们从口中放出(一般需要5—7天孚化,半个月左右从口中放出)。因此,它们的孚化率和成活率都很高。若将其受精卵从雌鱼口中取出,用自来水进行人工孚化时,则几乎都于孚化前死亡。我们曾用奠桑比克罗非鱼的胚胎做材料,研究其遗传变异问题,由于人工孚化问