XMolecular Cloning and Sequence Analyses of cDNAs Encoding Seven C2type Lectin2like Protein Subunits from Daboia russellii siamensis

按照Promega 公司的mRNA 提取试剂盒操作手册, 从圆斑蝰蛇( Daboia russellii siamensis ) 的毒腺中提 取mRNA ; 利用RT2PCR 的方法进行体外扩增, 获得C - 型凝集素蛋白的基因, 克隆到pMD182T 载体中。随机挑 选14 个阳性克隆进行核酸测序, 获得7 个编码不同蛇毒C - 型凝集素样蛋白亚基的cDNA , 分别命名为DRS2L1 、 DRS2L2 、DRS2L3 、DRS2L4 、DRS2L5 、DRS2L6 和DRS2L7 。由基因序列推导出的氨基酸序列表明, 克隆到的7 个蛇 毒C - 型凝集素样蛋白的亚基中均有糖识别结构域存在。BLAST 分析显示, 仅有DRS2L1 的蛋白序列与目前已知 的蛇毒C - 型凝集素样蛋白的α亚基相似。序列同源性比较并结合半胱氨酸位点分析, 推测DRS2L1 和DRS2L2 可能分别是圆斑蝰蛇毒Ⅹ因子激活剂的轻链LC2 和LC1 。DRS2L3 和DRS2L4 可能是高分子量的蛇毒C - 型凝集 素样蛋白的β亚基, 而DRS2L5 和DRS2L6 可能是低分子量的蛇毒C - 型凝集素样蛋白的β亚基。DRS2L7 可能是 类似于血小板膜糖蛋白Ib 结合蛋白的β亚基。

曼蝽属一新种记述及对Menida salvazana Distant的重新描述(半翅目:蝽科)

描述了半翅目蝽科曼蝽属一新种--华美曼蝽Menida speciosa Zheng ＆ Xiong, sp. nov.,模式产地为云南思茅菜阳河保护区倮倮新寨山;以及根据模式标本重新描述了Menida salvazana Distant 1921。

CHARACTERIZATION OF OHS1, AN ARGININE/LYSINE AMIDASE FROM THE VENOM OF KING COBRA (OPHIOPHAGUS HANNAH)

In this paper, we present the results of purification and characterization of an arginine/lysine amidase from the venom of Ophiophagus hannah (OhS1). It was purified by Sephadex G-75 gel filtration and ion-exchange chromatography on DEAE-Sepharose CL-6B. It is a protein of about 43,000, consisting of a single polypeptide chain. It is a minor component in the venom. The purified enzyme was capable of hydrolysing several tripeptidyl-p-nitroanilide substrates having either arginine or lysine as the C-terminal residue. We studied the kinetic parameters of OhS1 on six these chromogenic substrates. OhS1 did not clot fibrinogen. Electrophoresis of fibrinogen degraded with OhS1 revealed the disappearance of the alpha- and beta-chains and the appearance of lower mel. wt fragments. OhS1 had no hemorrhagic activity. It did not hydrolyse casein, nor did it act on blood coagulation factor X, prothrombin and plasminogen. The activity of OhS1 was completely inhibited by NPGB, PMSF, DFP, benzamidine and soybean trypsin inhibitor, suggesting it is a serine protease. Metal chelator (EDTA) had no effect on it.

Prolonged cardiac xenograft survival in guinea pig-to-rat model by a highly active cobra venom factor

A highly active cobra venom factor (CVF) was isolated from the venom of Naja kaouthia by sequential column chromatography. It displays strong anticomplementary activity, and has 1515 U of anti complementary activity per mg protein. A single dose of 0.1 mg/kg CVF given i.v. to rats completely abrogated complement activity for nearly 5 days. Given 0.02 mg/kg of CVF. the complement activity of rats was reduced by more than 96.5% in 6 It. In guinea pig-to-rat heart transplant model, rats treated with a single dose of 0.05 mg/kg CVF had significantly prolonged xenograft survival (56.12 +/- 6.27 h in CVF-treated rats vs. 0.19 +/- 0.07 h in control rats, P < 0.001). (C) 2003 Elsevier Ltd. All rights reserved.

In vitro activities of small peptides from snake venom against clinical isolates of drug-resistant Mycobacterium tuberculosis

The recent re-emergence of tuberculosis, especially the multidrug-resistant cases, has highlighted the importance of screening effective novel drugs against Mycobacterium tuberculosis. In this study, the in vitro activities of small peptides isolated from snake venom were investigated against multidrug-resistant M. tuberculosis. Minimum inhibitory concentrations (MICs) were determined by the Bactec TB-460 radiometric method. A small peptide with the amino acid sequence ECYRKSDIVTCEPWQKFCYREVTFFPNHPVYLSGCASECTETNSKWCCTTDKCNRARGG (designated as vgf-1) from Naja atra (isolated from Yunnan province of China) venom had in vitro activity against clinically isolated multidrug-resistant strains of M. tuberculosis. The MIC was 8.5 mg/l. The antimycobacterial domain of this 60aa peptide is under investigation. (C) 2003 Elsevier Science B.V. and the International Society of Chemotherapy. All rights reserved.

Characterization of a new bradykinin-potentiating peptide (TmF) from Trimeresurus mucrosquamatus

A novel bradykinin-potentiating peptide (BPP), designated as TmF, has been purified to homogeneity from the venom of Trimeresurus mucrosquamatus by 70% cold methanol extraction, Sephadex G-15 gel filtration and reverse-phase high performance liquid chromatography (RP-HPLC). The amino acid sequence of TmF was determined to be pGlu-Gly-Arg-Pro-Leu-Gly-Pro-Pro-Ile-Pro-Pro (pGlu denotes pyroglutamic acid), which shared high homology with other BPPs. The molecular mass of TmF was 1.1107 kD as determinated by electrospray ionization-mass spectrometry (ESI-MS), which was in accordance with the calculated value of 1.1106 kD. The potentiating "unit" of TmF to bradykinin-induced (BK-induced) contraction on the guinea-pig ileum in vitro was (1.13 +/- 0.3) unit (mg/L), and TmF (5.0 x 10(-4) mg/kg) increased the pressure-lowering-effect of bradykinin (5.0 x 10(-5) mg/kg) with approximate descent value of (14 +/- 2) mmHg. In addition, TmF inhibited the conversion of angiotensin I to angiotensin 11, 2 x 10(-3) mg of TmF caused 50% inhibition (IC50) of angiotensin-converting enzyme (ACE) hydrolyzing activity to bradykinin.

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.

Improved suppression of circulating complement does not block acute vascular rejection of pig-to-rhesus monkey cardiac transplants

At present, acute vascular rejection (AVR) remains a primary obstacle inhibiting long-term graft survival in the pig-to-non-human primate transplant model. The present study was undertaken to determine whether repetitive injection of low dose Yunnan-cobra venom factor (Y-CVF), a potent complement inhibitor derived from the venom of Naja kaouthia can completely abrogate hemolytic complement activity and subsequently improve the results in a pig-to-rhesus monkey heterotopic heart transplant model. Nine adult rhesus monkeys received a heterotopic heart transplant from wild-type pigs and the recipients were allocated into two groups: group 1 (n = 4) received repetitive injection of low dose Y-CVF until the end of the study and group 2 (n = 5) did not receive Y-CVF. All recipients were treated with cyclosporine A (CsA), cyclophosphamide (CyP) and steroids. Repetitive Y-CVF treatment led to very dramatic fall in CH50 and serum C3 levels (CH50 < 3 units/C3 remained undetectable throughout the experiment) and successfully prevented hyperacute rejection (HAR), while three of five animals in group 2 underwent HAR. However, the continuous suppression of circulating complement did not prevent AVR and the grafts in group 1 survived from 8 to 13 days. Despite undetectable C3 in circulating blood, C3 deposition was present in these grafts. The venular thrombosis was the predominant histopathologic feature of AVR. We conclude that repetitive injection of low dose Y-CVF can be used to continuously suppress circulating complement in a very potent manner and successfully prevent HAR. However, this therapy did not inhibit complement deposition in the graft and failed to prevent AVR. These data suggest that using alternative pig donors [i.e. human decay accelerating factor (hDAF)-transgenic] in combination with the systemic use of complement inhibitors may be necessary to further control complement activation and improve survival in pig-to-non-human primate xenotransplant model.

TMVA, a novel GPIb-binding protein, significantly prevents platelet microthrombi formation and prolongs discordant cardiac xenograft survival

In xenotransplantation, donor endothelium is the first target of immunological attack. Activation of the endothelial cell by preformed natural antibodies leads to platelet binding via the interaction of the glycoprotein (GP) Ib and von Willebrand factor (vWF). TMVA is a novel GPIb-binding protein purified from the venom of Trimeresurus mucrosquamatus. In this study, the inhibitory effect of TMVA on platelet aggregation in rats and the effect on discordant guinea pig-to-rat cardiac xenograft survival were investigated. Three doses (8, 20 or 40 mug/kg) of TMVA were infused intravenously to 30 rats respectively. Platelet aggregation rate was assayed 0.5, 12, and 24 h after TMVA administration. Wister rats underwent guinea pig cardiac cervical heterotopic transplantation using single dosing of TMVA (20 mug/kg, i.v., 0.5 h before reperfusion). Additionally, levels of TXB2 and 6-keto-PGF(1alpha) within rejected graft tissues were determined by radioimmunoassay. Treatment with TMVA at a dose of 20 or 40 mug/kg resulted in complete inhibition of platelet aggregation 0.5 h after TMVA administration. Rats receiving guinea pig cardiac xenografts after TMVA therapy had significantly prolonged xenograft survival. Histologic and immunopathologic analysis of cardiac xenografts in TMVA treatment group showed no intragraft platelet microthrombi formation and fibrin deposition. Additionally, the ratio of 6-keto-PGF(1alpha) to TXB2 in TMVA treatment group was significantly higher than those in control group. We conclude that the use of this novel GPIb-binding protein was very effective in preventing platelet microthrombi formation and fibrin deposition in a guinea pig-to-rat model and resulted in prolongation of xenograft survival. The increased ratio of PGI(2)/TXA(2) in TMVA treatment group may protect xenografts from the endothelial cell activation and contribute to the prolongation of xenograft survival.