中华眼镜蛇毒金属蛋白酶诱导人肥大细胞释放组胺的作用

目的： 探索中华眼镜蛇毒金属蛋白酶(MT)对人肥大细胞释放组胺的诱导作用及其机制。 方法： 用肝素凝胶和Superdex75亲和力凝胶纯化MT。将手术切除的人肺、大肠和扁桃体组织在37 ℃用Ⅰ型胶原酶和Ⅰ型透明质酸酶消化, 悬浮细胞均匀地加入含MT、刺激剂或缓冲液的试管中,进行激发实验并用荧光显色法测量上清液的组胺水平。 结果： 纯化后的MT在SDS-PAGE上呈1条带。MT能诱导人肺、大肠和扁桃体肥大细胞发生剂量依赖性组胺释放。浓度低至0.03 mg/L时, MT就能诱导大肠肥大细胞显著性的组胺释放, 但对于肺和扁桃体的肥大细胞，MT的最低有效浓度分别为0.3 mg/L和30 mg/L。 MT诱导大肠和肺肥大细胞组胺释放，12 min时达高峰，而扁桃体肥大细胞的组胺释放则在8 min时达高峰。人大肠、肺和扁桃体肥大细胞经代谢抑制剂和百日咳毒素预处理后，MT诱导其释放组胺的作用明显减弱。缺乏外源性钙、镁离子时，MT诱导肥大细胞释放组胺的能力也明显减弱。 结论： MT可能通过G蛋白偶联受体途径激活人肥大细胞，从而参与毒蛇咬伤人体后所发生的病理生理过程。     

眼镜蛇毒抗补体因子的研究

眼镜蛇毒抗补体因子 ,即眼镜蛇毒因子 (ｃｏｂｒａｖｅｎｏｍｆａｃｔｏｒ,ＣＶＦ) ,是来源于眼镜蛇科蛇毒的一种旁路补体激活物。由于ＣＶＦ既能激活补体又能最终耗竭补体 ,且性质稳定 ,特异性高 ,因而最适宜作为研究补体的工具药。本文综述ＣＶＦ的研究进展。1　ＣＶＦ的来源与化学早在上世纪末 ,人类就已发现眼镜蛇毒具有较强的抗补体活性 ,但直至六十年代后才分离提纯出来 ,并称为ＣＶＦ ,开始其理化性质、酶学特征和作用于补体系统的分子机理的研究。目前 ,ＣＶＦ的分子结构已查明 ,它是一种球状酸性糖蛋白 ,分子量因来源于不同的蛇种…     

Structure of the major oligosaccharide of cobra venom factor.

Cobra venom factor (CVF), the complement-activating glycoprotein in cobra venom, contains three or possibly four N-linked oligosaccharide chains per molecule and is devoid of O-linked saccharides. Analysis by lectin-affinity staining revealed the presence of complex-type oligosaccharides containing non-reducing terminal alpha-galactosyl residues and fucose residues linked to the proximal N-acetylglucosamine. Sialic acid residues could not be detected. For their structural analysis, the oligosaccharides were released by hydrazinolysis and fractionated on Bio-Gel P-4. Approximately 80% of the eluted oligosaccharides have a size equivalent of 17 +/- 2 glucose units. The major oligosaccharide representing about 45% of the total carbohydrate present in CVF was purified to homogeneity by MicroPak AX-5 HPLC and its structure was analyzed by sequential exoglycosidase digestion. The positions of the glycosidic linkages of the sugar residues were established by methylation analysis of CVF-derived glycopeptides. The data of these analyses indicated that the major oligosaccharide has a symmetrical fucosylated biantennary complex-type structure terminating with unusual alpha-galactosyl residues.     

Purification of cobra venom factor from phospholipase A contaminant.

It has been demonstrated that cobra venom factor prepared by the usual combination of ion exchange chromatography and sephadex gel filtration is contaminated by substantial amounts of a 'heavy' phospholipase A. The two activities may be separated by isoelectric focusing. Cobra venom factor focuses at pH between 5-75 and 6-75 whereas the phospholipase is all found at pH below 7-75. In certain test systems, particularly in vitro, and particularly where albumin concentrations are low, the contaminating phospholipase may produce effects that have been attributed to complement activation.     

Inhibition of bleomycin-induced pulmonary fibrosis by cobra venom factor.

A single dose of cobra venom factor, a known, potent depletor of serum complement, has recently been reported to be highly effective in inhibiting lung collagen deposition at 7 days after bleomycin treatment. The present study shows that this is associated with suppression of the bleomycin-induced increase in collagen synthesis, down to virtually normal levels. There is also a concomitant reduction in tissue free proline pool size. No significant effects are noted in noncollagenous protein synthetic rates. This inhibitory effect is not seen at 30 days after bleomycin and is correlated with a return to normal levels of serum complement hemolytic activity. These data suggest an important role for complementary activity in the lung's fibrotic response to bleomycin.     

Immunodiagnosis of snake venom poisoning

Uncertainty as to the species diagnosis remains a serious problem in the management of snake venom poisoning. This is particularly so in areas inhabited by numerous species, the venoms of which elicit similar pharmacological effects and clinical symptoms and against which para-specific cross-neutralizing antivenom is not available. Attempts have been made to eliminate some of this ambiguity through the development of various immunodiagnostic tests. Tests based on ELISA are sensitive, specific and even quantitative and adaptable to field application. In the development of diagnostic tests for use in developing countries, however, practical consideration must be given to speed, cost, simplicity in terms of equipment and expertise, and stability to the climate and storage conditions. This may dictate further modification or selection of more suitable alternative methodologies. Furthermore, the test may have to allow more flexibility in accommodating local species distributions and to address probable complications of heterophile antibodies in test samples from rural people.     

Antibody conjugates with cobra venom factor. Synthesis and biochemical characterization

Immunoconjugates are semi-synthetic hybrid proteins which bear great promise to become a new generation of anti-tumor agents. While many immunoconjugates have been shown to be selectively cytotoxic in in vitro model systems, dramatic in vivo anti-tumor effects have not been reported. To improve the activity of immunoconjugates, careful structure-function analyses have to be performed. We report here such an analysis for immunoconjugates consisting of a monoclonal anti-tumor antibody (MoAb) and cobra venom factor (CVF), the complement-activating glycoprotein from cobra venom, synthesized with the heterobifunctional crosslinking reagent N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). It is shown that a reaction mixture after protein coupling contains free MoAb and CVF as well as hybrid proteins of different compositions (dimers (MoAb-CVF), trimers (MoAb2-CVF, MoAb-CVF2), tetramers (MoAb-CVF3, MoAb2-CVF2, MoAb3-CVF), and some higher oligomers). While free MoAb and CVF can be removed by size exclusion chromatography, separation of different oligomeric hybrid proteins is not possible by this method. From the biochemical characterization of the hybrid proteins, which included the determination of sedimentation coefficients, recording of circular dichroism spectra with subsequent determination of secondary structure, and ultrastructural analysis by transmission electron microscopy, it was concluded that the two proteins do not undergo major structural changes upon coupling, and that the coupling of the two proteins is random with no preferential relative orientation. The functional inactivation of CVF was substantial (approximately 70%) due to both derivatization with SPDP and subsequent conjugation to the MoAb, with conjugation being relatively more inactivating than derivatization. In contrast, the binding activity of the antibody was far less susceptible to inactivation. In conclusion, our data indicate that immunoconjugate synthesis with heterobifunctional crosslinking reagents results in a mixture of heterogeneous hybrid proteins and causes substantial functional inactivation. For successful in vivo anti-tumor activity of future immunoconjugates with CVF and other protein ligands better methods for immunoconjugate synthesis will have to be developed.     

Decomplementation by cobra venom factor suppresses Yersinia-induced arthritis in rats.

Lewis rats experimentally infected with Yersinia enterocolitica O8 develop Yersinia-induced arthritis (YIA), which resembles very much reactive arthritis in humans. To investigate the involvement of serum complement in induction and maintenance of YIA, we decomplementated Yersinia-infected Lewis rats by treatment with cobra venom factor starting on day 7 after infection (prearthritic state). Reduction of serum complement activity in vivo by cobra venom factor treatment coincided with suppression of YIA clinically and histomorphologically.     

Cell-mediated immunological responsiveness in mice decomplemented with cobra venom factor.

In vivo administration of cobra factor (CoF) the C3-activating protein of cobra venom, had no suppressive effect on the in vitro response of lymphocytes to PHA, LPS or allogeneic cells; nor did it affect the generation of cells cytotoxic to allogeneic target cells. HVG reactivity was inhibited by commercially available but not purified CoF, and the latter also failed to prolong skin allograft survival. It is concluded that in vivo complement depletion does not interfere with T cell responses, and that previous reports of prolonged survival of skin allografts and inhibition of cutaneous delayed hypersensitivity reactions following treatment with CoF may have been due to impurities in the preparations used.     

Effects of decomplementation with cobra venom factor on experimental vasculitis.

Mercuric chloride (HgCl2) induces autoimmunity in susceptible rat strains, with hyper-IgE, appearance of a number of autoantibodies, and widespread tissue injury, including necrotizing vasculitis in the gut. In the early phase of tissue injury there is granulocyte infiltration; later there is immunoglobulin deposition along basement membranes in vessels. We have analysed the role of complement in this model using cobra venom factor (CVF), which causes decomplementation lasting around 5 days. The characteristic time course when HgCl2 is given over 10 days is that tissue injury and autoantibody levels reach a peak at around day 15 (start of HgCl2 = day 0). We therefore gave CVF either early (day 0), intermediate (day 5) or late (day 10); a fourth group (controls) received HgCl2 but no CVF. At each time point, CVF caused complete decomplementation which lasted for at least 5 days. Serum IgE and autoantibody levels were similar in all four experimental groups. Tissue injury in the 'early' CVF group and in the 'late' CVF group was not significantly different from controls, but in the intermediate group tissue injury was significantly more severe than in controls. These data indicate that the complement system does not play a major role in the induction of autoantibodies by HgCl2, nor in the effector phase of tissue injury. We speculate that the exacerbation of tissue injury by CVF in the group given this agent at an intermediate stage of the model is explained by the presence of products of C3 activation which have proinflammatory effects during the phase of active granulocyte-mediated tissue injury.     

Host response toBothrops asper snake venom

As part of the characterization of the host reactivity to the venom ofBothrops asper, we investigated the inflammatory responses in the mouse footpad model. The subcutaneously injected venom induced a rapid increase of serum IL-6 concentration, which peaked between 3 and 6 h and returned to normal values at 12 h. In contrast, serum TNF- and IL-1 were not detectable at any time point studied. A myotoxic phospholipase A₂ isoform purified from this venom, myotoxin II, was also able to induce a systemic IL-6 release when injected into the footpad. Both venom and myotoxin induced local edema and a leukocyte infiltrate accumulating in the muscle and subdermal tissue within 6 h. The infiltrate consisted predominantly of neutrophils at 6 and 24 h, but at later times, mononuclear cells also appeared. The edema, leukocyte infiltration, and IL-6 responses did not depend on the hemorrhagic activity of venom, since all three effects were seen after injection of (1) preneutralized venom, devoid of hemorrhagic activity, and (2) purified myotoxin II. Circulating platelet numbers were significantly decreased 30 min after venom injection and returned to normal after 12 h. The venom also induced a rapid inversion in the ratio of neutrophils to lymphocytes in peripheral blood, which did not normalize until 12 h later. The present observations suggest that venom, besides its cytotoxic properties, induces early hematologic and immunologic alterations. These findings may be of relevance in future treatment modalities.     

Vipera lebetina venom contains all types of snake venom metalloproteases

Snake venoms contain four classes of metalloproteases that all have a typical zinc-chelating sequence (HEXXHGXXH). N-terminal sequences and internal sequences of different purified metalloproteases were determined using Edman sequencing and LC MS/MS technique. Oligonucleotides were designed and used as primers for cDNA cloning from Vipera lebetina venom gland cDNA library. We found that isoforms of fibrinolytic enzyme lebetase Le-4 and Le-3 are synthesized in different way: Le-4 is synthesized as P-I type metalloprotease, Le-3 is synthesized with disintegrin-like domain as P-II type protease and processed post-translationally. An endothelial cell apoptosis-inducing heterodimeric glycosylated metalloprotease, V. lebetina apoptosis-inducing protease (VLAIP), belongs to P-III type containing metalloprotease, disintegrin-like and cysteine-rich domains. All these enzymes hydrolyze the Aalpha-chain and more slowly the Bbeta-chain of fibrinogen. Treatment of HUVEC cells with VLAIP induces changes in the attachment of cells to the substrate and causes apoptosis. V. lebetina venom contains also P-IV type-specific coagulant factor X activator (VLFXA) that cleaves the Arg52-Ile53 bond in the heavy chain of human factor X. VLFXA is a glycoprotein composed of a heavy chain and two C-type lectin-like light chains linked by disulfide bonds. The heavy and light chains of VLFXA are synthesized from different genes.     

Dissociation of anticomplementary and adjuvant properties of proteins derived from cobra venom.

The ability of preparations of cobra factor (CoF) to convert C3 via the properdin pathway (anticomplementary activity) has been demonstrated to be unrelated to its ascribed ability to convert a tolerogenic signal into an immunogenic stimulus (adjuvant activity). It has been shown that CoF preparations which have become inactive with regard to anticomplementary activity still retain full adjuvant properties. Further, when both activities are present in preparations of CoF, they can be separated by gel filtration chromatography into a fraction with approximate molecular weight 150,000 which contains anticomplementary activity and a fraction with approximate molecular weight 20,000, which contains adjuvant properties.