2-D-NMR study of the conformation characteristics of toxin 3 from Naja naja siamensis venom

The spatial structure of "long" toxin 3 Naja naja siamensis in solution has been studied by methods of two-dimensional (2D) 1H NMR spectroscopy. The individual signal assignments for 67 out of 71 residues and analysis of nuclear Overhauser effects between distinct protons of the molecule allowed the comparison of the toxin 3 conformations at different pH values and temperatures. It was shown that the deprotonated imidazole ring of His22 residue (at pH greater than or equal to 7,5) is surrounded by the side chains of Cys17, Pro18, Val23, Cys24, Cys45, Ala46 and Thr48 residues. On the contrary, the protonated imidazole ring of His22 (at pH less than 4,0) is exposed into solvent. Ionization of His22 is accompanied by a change in the Tyr25 aromatic ring orientation and affects the conformational mobility of the Cys17, His22, Cys45 and Ala47 side chains. The revealed conformational features of toxin 3 in solution are discussed in connection with the differences between "long" and "short" neurotoxins in the kinetics of their binding to acetylcholine receptor.     

Screening of plants containing Naja naja siamensis cobra venom inhibitory activity using modified ELISA technique

Enzyme-linked immunosorbent assay (ELISA) has been modified for screening plants with antagonistic activity to Naja naja siamensis cobra venom. Aqueous extracts from plants were investigated for their inhibitory effects on the binding of anti-cobra venom antibody to antigen, cobra venom, fixed onto 96-well microtiter plates. Ingredients in extracts were allowed to react with immobilized venom before the subsequent addition of antivenom antibody. Venom components affected by exposure to the extracts, unable to interact with their specific antibody, were predicted to be unable to bind to their native destinations or natural receptors. Curcuma cf. zedoaria, an old Thai medicinal plant, showed clear inhibitory activity in the ELISA test. Neurotoxin and protein degradative enzymes, major components in venom, were identified as targets of this extract in Western immunoblotting analysis. Ingredients in the extract showed high affinity to the toxin in competition assay by immunoprecipitation. The extract attenuated toxin activity by extending contraction time of diaphragm muscle after envenomation and had a potency to protect cellular proteins from venom degradative enzymes. Curcuma parviflora, with less activity in ELISA, exhibited acceptable results in two experiments but negative results in two experiments, whereas Curcuma longa, having low activity in the ELISA test, never showed any favorable results. Screening of 36 samples could classify plants into an inhibition range of 0 to 86%. This modified ELISA is recommended as a preliminary screening method for inhibitors with a large number of samples.     

Purification of a post-synaptic neurotoxic phospholipase A2 from Naja naja venom and its inhibition by a glycoprotein from Withania somnifera

A post-synaptic neurotoxic phospholipase A(2) (PLA(2)) has been purified from Indian cobra Naja naja venom. It was associated with a peptide in the venom. The association was disrupted using 8 M urea. It is denoted to be a basic protein by its behavior on both ion exchange chromatography and electrophoresis. It is toxic to mice, LD(50) 1.9 mg/kg body weight (ip). It is proved to be post-synaptic PLA(2) by chymographic experiment using frog nerve-muscle preparation. A glycoprotein, (WSG) was isolated from a folk medicinal plant Withania somnifera. The WSG inhibited the phospholipase A(2) activity of NN-XIa-PLA(2,) isolated from the cobra venom, completely at a mole-to-mole ratio of 1:2 (NN-XIa-PLA(2): WSG) but failed to neutralize the toxicity of the molecule. However, it reduced the toxicity as well as prolonged the death time of the experimental mice approximately 10 times when compared to venom alone. The WSG also inhibited several other PLA(2) isoforms from the venom to varying extent. The interaction of the WSG with the PLA(2) is confirmed by fluorescence quenching and gel-permeation chromatography. Chemical modification of the active histidine residue of PLA(2) using p-brophenacyl bromide resulted in the loss of both catalytic activity as well as neurotoxicity of the molecule. These findings suggest that the venom PLA(2) has multiple sites on it; perhaps some of them are overlapping. Application of the plant extract on snakebite wound confirms the medicinal value associated with the plant.     

Dihydropyridine effects on skeletal muscle fatigue.

1. The purpose of this investigation was to determine if alterations in extracellular calcium (Ca2+) influx by the dihydropyridine derivatives Bay K 8644 and nifedipine affected skeletal muscle fatigue. 2. Tetanic contractions (80 Hz, 100 msec) of frog sartorius muscles were evoked every sec for 3 min. Muscles were fatigued in normal Ringer's solution (NR), in NR containing 1 microM nifedipine of 10 microM Bay K 8644 or in low Ca2+ Ringer's. 3. In each case, the experimental conditions increased the rate and magnitude of fatigue. Rate constants of fatigue obtained during Bay K 8644, nifedipine and low Ca2+ conditions (-.0122 +/- .0016, -.0397 +/- 0022 and 0.0169 +/- .0064 sec-1, respectively) were significantly greater than NR (-.0104 +/- .0006 sec-1, p less than .05). In addition, tetanic forces developed at the end of the stimulation period under the experimental conditions (3.90 +/- 0.81, 1.21 +/- 1.40 and 2.04 +/- 1.10% of initial) were significantly less than NR (7.18 +/- 1.27%, p less than .05). 4. Caffeine contracture forces (10 mM) evoked immediately after stimulation were not significantly different between conditions. 5. These results suggest that alterations in sarcolemmal Ca2+ exchange has some influence on the fatigue process.     

The primary structure of a major polypeptide component from the venom of Naja melanoleuca.

The venom of the forest cobra, Naja melanoleuca, contains a number of homologous polypeptides containing between 60 and 71 amino acid resides. The primary structure of a major component (approx. 10% by weight of the crude venom) has been determined unambiguously. The molecule contians 61 amino acid residues and four disulphide bridges. It has not effect on neuromuscular transmission or the excitatory or inhibitory responses to acetylcholine of molluscan neurons. The molecule is similar to, but not identical with, the so-called cytotoxins VII2 and VII3 isolated, by others, from the same venom but reported to be minor components.     

眼镜蛇毒纤维蛋白原溶解因子的纯化及理化性质的研究

经Heparin-SepharoseCL-6B亲和层析和SephadexG-150分子筛层析,从中国眼镜蛇毒(Najanajaatra)中纯化出一种可水解纤维蛋白原Aα链的蛋白酶——组分F.SDS-PAGE测得分子量为47100,为一含2%中性己糖的单肽链糖蛋白;它对纤维蛋白、酪蛋白和苯甲酰-L-精氨酸乙酯(BAEE)均无水解作用,也无激活纤溶酶原的作用;100μg皮下或皮内注射,未见出血反应。组分F对酸和热均不稳定。EDTA,EGTA,PMSF和肝素等可抑制其纤维蛋白原溶解活性,benzami-dine,aprotinin和大豆胰蛋白酶抑制因子(SBTI)则无抑制作用。     

Amino-acid sequences of four cytotoxins (cytotoxins I, II, III and IV) purified from the venom of the Thailand cobra, Naja naja siamensis

Four cytotoxins, designated as cytotoxins I, II, III and IV, were isolated from the venom of the Thailand cobra (Naja naja siamensis) by gel filtration on Sephadex G-75 followed by CM-cellulose chromatography. The amino-acid sequences were determined by a combination of conventional methods. Cytotoxins I, II, III and IV were each composed of 60 amino-acid residues and their molecular weights were calculated to be 6693, 6646, 6709 and 6739, respectively. The amino-acid sequences were compared with those of cytotoxins from other cobra venoms already sequenced.     

Hemodynamic effects of nicotine in canine skeletal muscle.

Studies were conducted in 36 artificially ventilated, anesthetized dogs to clarify hemodynamic effects of nicotine in resting gracilis muscle. In Series 1, effects of intravenous nicotine (36 micrograms/kg/min) were evaluated in (i) intact muscles (Condition 1), (ii) denervated muscles (Condition 2), (iii) denervated muscles following local alpha-adrenergic blockade (Condition 3), (iv) denervated muscles following combined local alpha- and beta-adrenergic blockade (Condition 4), and (v) intact muscles with aortic pressure maintained constant (Condition 5). In Series 2, nicotine was infused directly into the gracilis artery at a rate of 3.6 micrograms/kg/min. Muscle blood flow was obtained with an electromagnetic flowmeter and used to calculate vascular resistance and oxygen consumption (Fick equation). Plasma catecholamine levels were determined with a radioenzymatic method. Intravenous nicotine doubled mean aortic pressure under Conditions 1-4. In intact and denervated muscles (Conditions 1 and 2) proportional increases in vascular resistance, reflective of vasoconstriction, held blood flow constant. Muscle oxygen consumption was unchanged. alpha-Adrenergic blockade with phenoxybenzamine following denervation (Condition 3) converted muscle vasoconstriction to vasodilation during nicotine infusion. Additional beta-adrenergic blockade (Condition 4) restored muscle vasoconstriction. Nicotine-induced muscle vasoconstriction was maintained under controlled pressure (Condition 5). Intravenous nicotine significantly increased plasma catecholamine levels. Intra-arterial infusions of nicotine (Series 2) caused no hemodynamic changes in muscle. In conclusion, intravenous nicotine caused vasoconstriction in muscle, which was not due to reduced metabolic demand, pressure-flow autoregulation, or a direct [corrected] effect on vascular smooth muscle, but to stimulation of alpha-adrenergic receptors. Following denervation, this vasoconstriction was maintained by elevated plasma catecholamines. alpha-Adrenergic blockade unmasked nicotine-induced vasodilation mediated by beta-adrenergic receptors, whereas combined alpha- and beta-adrenergic blockade unmasked nicotine-induced vasoconstriction by a nonadrenergic mechanism.     

Basic phospholipase from the venom of Naja nigricollis is cytotoxic

The basic phospholipase A2 purified from the venom of Naja nigricollis (Institut Pasteur), possesses an intense cytotoxic activity toward fetal cells from FL strain. At a concentration equal to 1.6 x 10(-6) M, the PLA2 lyses 50% of the cells present in a suspension containing 3.5 x 10(6) cells per millilitre. Other PLA2 from various origins do not exhibit such cytotoxic activity.