Snake venom probes of platelet adhesion receptors and their ligands

Snake venom proteins that modulate platelet adhesive interactions are chiefly from either of two main structural families: the C-type lectin-like family, or the metalloproteinase-disintegrins. Snake venom probes from both families selectively target platelet adhesion receptors, including glycoprotein (GP) Ib-IX-V, GP VI, 2β1 and IIbβ3 (GP IIb-IIIa). These receptors act together to mediate platelet adhesion, activation and aggregation (thrombus formation) under hydrodynamic shear stress in flowing blood. The receptors are members of the leucine-rich repeat family (GP Ib-IX-V), the immunoglobulin superfamily (GP VI), or integrins (2β1, IIbβ3). In addition, adhesive glycoproteins in matrix and/or plasma such as von Willebrand factor (that binds GP Ib and IIbβ3), collagen (that binds GP V, GP VI and 2β1), or fibrinogen (that binds IIbβ3), are also targeted by C-type lectin family or metalloproteinase-disintegrin snake venom proteins. Emerging structural and functional evidence is beginning to explain how interactions between the conserved structural module-domains that make up these mammalian and snake proteins are regulated. Whether homologous adhesion/counter-receptors on platelets and other vascular cells are also potential snake venom targets is as yet largely unexplored.     

Jararhagin, a snake venom metalloproteinase-disintegrin, stimulates epithelial cell migration in an in vitro restitution model

The snake venom metalloproteinase-disintegrin jararhagin (JG) has no chemotactic activity but stimulates the migration of neutrophils in vivo through a mechanism still unclear. In this study we investigated the effects of jararhagin on epithelial cell adhesion and migration in vitro. F-actin arrangement and the distribution of laminin, fibronectin, several integrins and phosphorylated Focal Adhesion Kinase (FAK) were studied using rhodamine–phalloidin and immunofluorescence. Maximum stimulation of migration (about 100%) was obtained with 5 μg/ml JG, with about 38% inhibition of cellular adhesion. In migratory cells the toxin stimulated the formation of filopodia, lamellipodia and stress fibers. The pericellular fibronectin matrix was lost in migrating cells, while laminin was less affected. The toxin stimulated FAK phosphorylation and the recruitment of v-containing integrins to focal contacts, whereas integrins containing the 2 subunit were reduced in these junctions. Inactivation of the toxin with 1,10 phenanthroline showed that the catalytic activity is important for the effect of jararhagin on cell migration, FAK phosphorylation and for the recruitment of v, but not as much for the anti-adhesive effect. In conclusion, jararhagin stimulates the migration of epithelial cells in vitro through a mechanism that involves its proteolytic activity, qualitative changes in cellular adhesion and the formation of actin-rich cellular processes.     

Structural and functional characterization of myotoxin I, a Lys49 phospholipase A2 homologue from the venom of the snake Bothrops atrox.

A new myotoxin was isolated from the venom of Bothrops atrox from Colombia. B. atrox myotoxin I is a homodimer, with a subunit molecular mass of 13,826, and a pI of 8.9. Its complete nucleotide sequence was obtained by cDNA cloning, indicating a mature product of 122 residues that belongs to the family of Lys49 phospholipase A(2) (PLA(2)) homologues, a subgroup of catalytically inactive proteins within the group IIA. Accordingly, the toxin was devoid of phospholipase and anticoagulant activities, in vitro. In mice, it induced conspicuous local myonecrosis, edema, and a systemic interleukin-6 response. In vitro, it was cytolytic upon myoblasts, and weakly bactericidal. The toxin showed highest homology with other Lys49 PLA(2)s, both in its primary and three-dimensional modeled structure, although with an evident difference in the C-terminal region. Unlike Lys49 proteins of American crotalids having 121 residues, this toxin presents an insertion (Asn) between positions 118 and 119. Despite several substitutions within the C-terminal region 115-129 between B. atrox myotoxin I and B. asper myotoxin II, antibodies against synthetic peptide 115-129 of the latter were strongly cross-reactive to the former, indicating the antigenic conservation of this site, known to be critical for the membrane-damaging activities of Lys49 myotoxins.     

Bactericidal and neurotoxic activities of two myotoxic phospholipases A2 from Bothrops neuwiedi pauloensis snake venom.

Two basic myotoxic PLA(2)s, namely BnpTX-I and II, were isolated from Bothrops neuwiedi pauloensis snake venom through three chromatographic steps: ion-exchange chromatography on CM-Sepharose, gel filtration on Sephadex G-50 and reverse phase HPLC on a C18 column. Both PLA(2)s showed a M(r) around 14,000 for the monomer and 28,000 for the dimer (as estimated by SDS-PAGE), pI approximately 7.8 and approximately 121 amino acid residues cross-linked by seven disulfide bonds. The N-terminal sequences revealed significant homology with Asp49 basic myotoxic PLA(2)s from other snake venoms. The catalytic and anticoagulant activities of BnpTX-I were higher than those of BnpTX-II. Both were able to induce cytotoxicity in vitro, as well as, myotoxicity, edema and lethality in mice. BnpTX-I also induced neurotoxic effect on mouse neuromuscular preparations and bactericidal activity on Eschericia coli and Staphylococcus aureus. After chemical modification of BnpTX-I with BPB or incubation with EDTA or Mn(2+) ions, the catalytic activity was completely abolished, while the toxic and pharmacological activities were partially reduced. Interaction with heparin inhibited the cytotoxic and bactericidal effects. Anti-BthTX-I, anti-BthTX-II and anti-115-129-C terminal antibodies strongly recognize both BnpTX-I and II. It is shown that the neurotoxic effect induced by B. neuwiedi pauloensis venom is due to the presence of myotoxic PLA(2)s. The data also corroborate the hypothesis of a partial dissociation between toxic and enzymatic domains. In addition, BnpTX-I displays a heparin binding C-terminal region, which is probably responsible for the cytotoxic and bactericidal effects.     

Chemical modification of arginine residues of Notechis scutatus scutatus notexin.

Notexin, a presynaptic phospholipase A2 (PLA2) neurotoxin isolated from Notechis scutatus scutatus venom, was inactivated by arginine-specific reagents, phenylglyoxal and 1,2-cyclohexanedione. Kinetic analyses of the modification reaction revealed that the inactivation of notexin followed pseudo-first order kinetics and the loss of PLA2 activity was correlated with the incorporation of one molecule of modification reagent per toxin molecule. However, the results of amino acid analysis and sequence determination revealed that two arginine residues at positions 43 and 79 of notexin were modified simultaneously. Modification of the arginine residues was accompanied with a decrease in the ability to inhibit the indirectly evoked contraction of chick biventer cervicis muscle and bind with synaptic membranes. The secondary structure of the toxin molecule did not significantly change after modification with phenylglyoxal as revealed by the CD spectra. The modified derivative retained its affinity for Ca2+, indicating that the modified arginine residues did not participate in Ca2+ -binding. Together with the notion that Arg-43 and Arg-79 of notexin are located in the proximity of its catalytic site and toxic site, respectively, our results suggest that modification of Arg-43 and Arg-79 should differently contribute to the observed decrease in the PLA2 activity and neurotoxic effect of notexin.     

富血小板血浆治疗毒蛇咬伤难愈合性创面的疗效评价

目的:评价富血小板血浆(PRP)治疗毒蛇咬伤难愈合性创面的临床疗效。方法:回顾性分析PRP治疗7例毒蛇咬伤难愈合性创面患者的临床疗效。结果:经3～6次治疗,7例患者创面伤口平均愈合率达96.8%,治疗前创面平均面积为(14.44±14.697)cm²,治疗后平均面积为(1.245±3.174)cm²。结论:PRP治疗毒蛇咬伤难愈合性创面效果显著。     

多曲方丝弓与直丝弓技术治疗成人开(牙合)的疗效对比

目的:比较使用多曲方丝弓技术和直丝弓技术(使用"摇椅形"弓丝)治疗前后的颌面部软、硬组织的变化,探讨成人开(牙合)畸形不同的矫治设计和矫治技术的选择.方法:选择28例(男性10例,女性18例),18～35岁前牙开(牙合)畸形患者,按照开(牙合)程度把28例病人分成2组.14例患者用多曲方丝弓技术矫治,另外14例患者用直丝弓技术矫治.两组开(牙合)程度OB值经独立样本t检验结果(P为O.462),表明两组间无差别,对矫治前后的X线头颅定位侧位片进行测量分析,采用配对t检验和独立样本t检验评价比较不同的矫治方法矫治前后的变化和矫治时间的差异.结果:多曲方丝组矫治后上、下颌切牙伸长,且上切牙内收,上、下磨牙直立并略压低,前牙达到覆(牙合)时间和矫治完成时间短于直丝技术组..直丝技术组矫治后上、下颌切牙伸长,未见内收,下颌磨牙直立.结论:矫治开牙合多曲方丝弓技术疗效优于直丝弓技术;疗程短于直丝弓技术.     

Molecular characterization of L-amino acid oxidase from king cobra venom.

An L-amino acid oxidase from Ophiophagus hannah snake venom (Oh-LAAO) was purified by successive gel filtration, ion-exchange and heparin chromatography. Oh-LAAO did not induce platelet aggregation; however, it had potent inhibitory activity on platelet aggregation induced by ADP and U46619, but showed no effect on platelet aggregation induced by thrombin, mucetin, ristocetin and stejnulxin. By RT-PCR and 5'-RACE methods, the complete Oh-LAAO cDNA was cloned from the venom gland total RNA preparations. The cDNA sequence contains an open-reading frame (ORF) of 1476-bp, which encodes a protein of 491 amino acids comprising a signal peptide of 25 amino acids and 466-residue mature protein. The predicted protein sequence of Oh-LAAO was confirmed by N-terminal and trypsin-digested internal peptides sequencing together with peptide mass fingerprinting. cDNAs encoding for ORF of LAAOs from Bungarus fasciatus and B. multicinctus were cloned and reported in this study. In addition, partial cDNA encoding for Naja atra LAAO was also reported. Oh-LAAO shared approximately 50% protein sequence identity with other known snake venom LAAOs. Phylogenetic analysis indicated that Oh-LAAO is evolutionary distant to other snake venom LAAOs.     

骨性Ⅲ类倾向青春期单颌拔牙患者二次矫治1例报告

<正>颅颌面生长发育过程中,因演化、遗传和环境等因素的影响,会导致颅颌面及(牙合)畸形,并影响生长发育,大多数错畸形是在生长发育期形成的^[1-2]。骨性Ⅰ类或有Ⅲ类倾向的青春期患者,上颌的单颌拔牙可能会导致上下颌矢状向不调,尤其是在骨性Ⅲ类倾向的患者中若医生只是关注到上颌牙列的拥挤,     

内镜腹腔镜联合治疗胆总管结石150例报告

目的:探讨内镜腹腔镜联合治疗胆总管结石的价值。方法:回顾性分析150例胆总管结石患者经内镜腹腔镜联合治疗的临床资料。结果:本组患者全部治愈。120例患者接受了十二指肠乳头括约肌切开术(endoscopic sphicterotomy,EST)和十二指肠逆行胰胆管造影术(endoscapic retrograde cholangiopancreatography,ERCP);30例患者接受了腹腔镜胆囊切除术(laparoscopic cholecystectomy,LC)+腹腔镜胆总管切开取石术(laparoscopic choledocholithotomy T-tube drainage,LCTD)。结论:内镜腹腔镜联合治疗胆总管结石优于传统手术,在具备较高内镜、腹腔镜技术水平的条件下是安全可行的,且可作为首选方式。