Structures And Functions Of Three Neurotoxins From The Chinese Bird Spider Ornithoctonus Huwena

The venom from the Chinese bird spider Selenocosmia huwena, which is distributed in the hilly areas of Yunan and Guangxi in the south of China, contains a mixture of compounds with different types of biological activities. Three novel peptide neurotoxins were isolated and purified from the venom. The complete amino acid sequences were established by direct automated Edman degradation, and the biological functions of these toxins have been investigated on several experimental models. As the last part pf this thesis, the acutely dissociation and shortly culture of dorsal unpaired median (DUM) neuron from the cockroach Periplaneta Americana were primarily studied.(1) Purification and characterization of huwentoxin-VII and Huwentoxin-VIII. Two novel insecticidal neurotoxins, Huwentoxin-VII and Huwentoxin-VIII were isolated by the combination of ion exchange and reverse-phase HPLC. The masses of the two toxins were determined by MALDTI-TOF MS as 3,981.02 and 4,171.12 respectively. The sequences of the two toxins were determined and found to consist of 35 and 36 amino acid residues respectively, including six Cys residues, which form three disulphide bonds. The sequences of the two toxins are very similar to each other except for six residues. The two toxins share the similar biological activities. They can paralyze locust and cause death of mouse by injection to central nervous system (CNS),with high dosage. But with low dosage Huwentoxin-VIII can cause convulsion-reaction, vhile Huwentoxin-VII cannot. They can also block neuromuscular transmission in an isolated mouse phrenic nerve diaphragm preparation, and act cooperativelyto the activity of Huwentoxin-I.(2) Isolation and pharmacological characterization of Huwentoxin-X, a novel blocker of high-voltage-activated calcium channel from the spider Selenocosmia huwena. A novel toxin, named Huwentoxin-X, was purified and determined from the venom of the spider Selenocosmia huwena. The complete amino acid sequence of Huwentoxin-X is NH2-KCLPPGKPCYGATQKIPCCGVCSHNKCT-COOH, determined by direct automated Edman degradation. The sequence contains 6 Cys forming 3 disulfide bonds, and the theoretical molecular weight of 2, 931. 34 Da is perfectly agreement with that determined by mass spectrometry. The toxin shows less than 30% homologous with other Huwentoxins while more than 50% with Ptul and w-conotoxin SVIA, two calcium channelblockers. It blocks neuromuscular transmission in an isolated mouse phrenic nerve diaphragm preparation faintly, paralyzes mice when injected i.c.v. Under whole-cell voltage-clamp conditions, Huwentoxin-X has blocking effect on high-voltage-activated (HVA) calcium channels, N-type calcium currents in rat dorsal root ganglion neurons, but not L-type. When injected i.v., .it has no toxic to locust.(3) Acutely dissociation and shortly culture of the cockroach Periplaneta Americana dorsal unpaired median(DIM) neurons isolated from the terminal abdominal1ganglion(TAG). Isolation of adult DUM neurons was performed under sterile conditions using enzymatic digestion and mechanical dissociation of the median parts of the TAG from the nerve cord of the cockroach Periplaneta Americana.