尖吻蝮蛇毒促凝组分的血栓形成研究

尖吻蝮蛇毒促凝组分的血栓形成研究刘敏涓，管锦霞，周立红，刘泽霖我们观察了尖吻蝮蛇毒类凝血酶组分对凝血酶凝固作用及时血栓弹力图的影响。报道如下。材料和方法１血液标本：正常人静脉血与０．１３ｍｏｌ／Ｌ枸橼酸钠以９：１混和。２试剂：尖吻蝮蛇毒促凝组分系尖吻...     

尖吻蝮蛇凝血酶的止血作用及其作用机制的研究

本研究旨在探讨从国产尖吻蝮蛇蛇毒中分离的尖吻蝮蛇凝血酶（HCA）的止血作用及其作用机制。以兔全血凝固时间和小鼠断尾出血时间为指标评价HCA的促凝与止血作用；以全血凝块溶解和纤维蛋白原裂解为指标探讨HCA的止血作用机制。结果表明，HCA能显著缩短兔全血凝固时间和小鼠断尾出血时间，有明显的剂量依赖关系．其作用与阳性对照药立止血（repitilase）相似。HCA使纤维蛋白原α亚基裂解，其诱导形成的血浆凝块能在尿素溶液中溶解。结论：HCA通过水解纤维蛋白原的α亚基释放出A肽，形成纤维蛋白聚合体而产生止血作用．但不引起凝血因子Ⅷ的释放。     

头孢米诺钠冻干粉和尖吻蝮蛇血凝酶注射剂存在配伍禁忌

正头孢米诺钠是一种可溶于水的白色粉末,具有快速杀灭革兰氏阳性菌以及阴性菌的作用,达到杀菌目的[1]。可用0.9%氯化钠注射液溶解后静脉滴注或静脉注射。尖吻蝮蛇血凝酶注射剂是一种从尖吻蝮蛇毒液中提取分离出的蛇毒类凝血酶,具有类凝血酶样作用,能促进血管破损部位的血小板聚集[2]。主要作用于纤维蛋白原促进凝血,常规用于外科手术浅表创面止血、消化道出血及胃肠息肉切除术后止血[3]。一般用0.9%氯化钠注射液溶     

扇贝防御素基因改良5′cDNA末端快速扩增聚合酶链反应筛选方法的研究

目的应用改良5′cDNA末端快速扩增聚合酶链反应(RACE)筛选扇贝抗菌肽基因,寻找基于基因序列同源性的双壳类抗菌肽(AMP)的筛选方法。方法 TRIzol法提取扇贝血淋巴中总RNA。根据贻贝抗菌肽的保守序列设计基因特异性简并引物,进行改良5′RACE钓取可能防御素基因cDNA的5′端序列、AT克隆、DNA测序和同源性分析。结果采用改良5′RACE从扇贝血淋巴RNA中得到多个未知基因的cDNA 5′端序列,挑选600bp以下的基因片段进行AT克隆和DNA测序后得到3个插入片段序列,其长度分别为257、275和511bp。通过BLAST程序搜索GenBank核酸数据库,未发现与之高度同源的基因。结论从扇贝血淋巴中获得的3个5′端cDNA序列可能属于新的基因。应用改良5′RACE能钓取含防御素保守序列的新基因片段,由此表明此方案具有可行性。     

蕲蛇酶治疗缺血性脑血管病58例临床观察

蕲蛇酶治疗缺血性脑血管病５８例临床观察福建建宁县医院（３５４５００）黄建隆郑世文徐美英蕲蛇酶是从中药——蕲蛇（五步蛇、尖吻蝮蛇）蛇毒中分离、纯化的一种凝血酶样酶的制剂［１］，能预防和治疗动、静脉血栓形成。我院自１９９３年始应用该药治疗缺血性脑血管病５...     

LRP16基因启动子活性分析

本研究的目的在于分析LRP16基因不同启动子区域的活性，为深入研究LRP16基因的表达调控机制奠定基础。在NCBI的人类基因组数据库中截取并下载LRP16基因转录起始位点5侧翼区2．7kb的基因组序列，设计PCR引物，从健康外周血单个核细胞中克隆和亚克隆了LRP16基因的启动子分子。对各个长度相差400bp左右的亚克隆启动子片段调控荧光素酶表达的作用强度进行比较分析。结果获得了与GeneBank序列一致、长度为2．6kb的LRP16基因启动子DNA序列，其主要调控序列在LRP16基因转录起始位点5侧翼区的-200至-600bp。结论：LRP16基因启动子是一个典型的Ⅱ型启动子，其启动子活性在-200至-600bp区域最强。     

从单个细胞扩增靶基因片段的技术

目的 建立从人的单个细胞扩增特定靶基因片段进行基因诊断的技术。方法 利用显微操作技术挑取单个纤维母细胞，各置于0.2ml簿壁反应管的裂解液中，合成针对抑癌基因P53第5－9外显子（e）的引物，运用15mer高度随机引物或一个特异引物进行单个细胞的整修基因组DNA或特异靶片段的预扩增，再以其为模板进行巢式聚合酶链反应（PCR）扩增P53基因第e5-9的1900bp片段及含第5，6外显子的580bp片段，并用ABI－377测序仪测定其序列。结果 运用稀释至0.01ng的基因组DNA扩增1900bp片段，优化PCR的条件，分别从300个单个纤维母细胞扩增上述1900bp片段，10个获得1900bp片段，成功率为33％。拉坟580bp片段，则阳性率可达605。序列分析证明确为P53基因第5，6外显子序列。结论 运用本实验室建立的技术可从人的单个细胞扩增特定靶基因片段，并测定其序列，作出基因诊断。     

尖吻蝮蛇毒抗凝与纤溶组分对动物血栓的溶栓效应

目的：观察尖吻蝮蛇毒类凝血酶与纤溶酶单用与合用对动物血栓的溶栓作用。 方法：①实验于2005-01／06在广西医科大学药理学实验室完成。选用新西兰兔120只，雌雄不拘。②取家兔96只，随机分为4组：对照组，纤溶酶组，类凝血酶组，纤溶酶＋类凝血酶组，每组24只，每组于实验30，60，120，240min4个时间点各6只。③建立家兔肺栓塞模型：麻醉后固定，耳缘静脉取血1mL，并与凝血酶水溶液混合制备血栓，分一侧颈外静脉，将制好的血栓注入，然后加注20mL生理盐水，于30,60,120,240min后分别将纤溶酶0，7mg／kg，类凝血酶4μg／kg，纤溶酶0．7mg／kg＋凝血酶4μg／kg耳缘静脉注射于纤溶酶组。类凝血酶组，纤溶酶＋类凝血酶组。24h后处死兔，取出肺中栓子称重计算相对溶栓率[（溶前血栓湿重-溶后血栓湿重）／溶前血栓湿重x100％1。④将其余24只家兔随机分为4组：对照组，纤溶酶组，类凝血酶组，纤溶酶＋类凝血酶组，每组6只。建立家兔动脉血栓模型：麻醉家兔后，分离左侧的颈总动脉和右侧颈外静脉，取一端长7cm的聚乙烯管，中间内置一根6cm已称重的4号手术线。以肝素（5&;#215;100^4／L）浸湿管壁后插入左颈总动脉和右颈外静脉之间。开放血流，15min后，取出手术线称重，减去原线质量即为药前血栓湿重。然后将纤溶酶O．7mg／kg，类凝血酶2μg／kg，纤溶酶0．7mg／kg＋凝血酶2μg／kg分别静脉注射于纤溶酶组，类凝血酶组，纤溶酶＋类凝血酶组，于给药后15，30，60，120min分别测定聚乙烯管中的手术线质量，血栓湿重=总质量-丝线干重。③计量资料差异比较采用t检验。结果：新西兰兔120只均进入结果分析。①家兔肺栓塞溶栓率：纤溶酶组，类凝血酶组，纤溶酶＋类凝血酶组明显高于对照组（t=16．895-35．441，P〈0．01）。②家兔动脉血栓湿重：纤溶酶组，类凝血酶组，纤溶酶＋类凝血酶组明显低于对照组（t=2．899-10．561，P〈0．01）。 结论：尖吻蝮蛇毒类凝血酶与纤溶酶有协同溶栓作用。     

鸡VCP蛋白全长cDNA的克隆及序列分析

目的 获得鸡含缬酪肽蛋白（Valosin-containing protein,VCP）的cDNA和氨基酸序列,为进一步研究VCP和视网膜发育及病变的关系提供理论依据。方法 根据已发表的鸡ATP酶p97的基因序列片段设计引物,从白来杭鸡视网膜cDNA文库中获得VCP全基因序列,并进行序列分析和比较。结果 获得了VCP的全长cDNA序列,此基因cD-NA全长为有2 916 bp,并有2 421 bp的完整开放阅读框,编码806个氨基酸。鸡VCP的基因及其对应的氨基酸序列与人、小鼠VCP基因及其对应的氨基酸序列高度同源,并具有ATP酶结合位点的高度保守AAA序列。结论 获得了全长cDNA序列已在Genbank中登录（AB195711）,为从基因及蛋白质水平研究VCP和视网膜发育及病变的关系奠定了基础。     

PCR法分子克隆及序列分析广西产眼镜王蛇蛇毒α-神经毒素基因

目的 为了获得眼镜王蛇(ophiophagus hannah，Oh)蛇毒a-神经毒素(a-Neurotoxin．a-NT)的基因序列。方法 我们通过比较了基因库中已知眼镜蛇科不同种类毒蛇来源的a-NT基因，发现它们有较高的同源性，特别是5’和3’非翻译区及引导肽部分高度保守，据此设计了包括翻译起始点的上游引物，以及为了得到3’端较完整非编码部分而设计了基本上属于d(T)的反意链下游RACE-PCR引物。为了克服引物所带来的模糊扩增，还在蛋白编码部分再设计一对上下游引物，由此组成P1、P2、P3和P4四对引物。采用Nacleospin RNA Kit法分别从3条活Oh蛇毒腺中提取mRNA，以3’端引物合成eDNA的第一链，并以此作为模板，分别用四对引物进行PCR扩增反应，得到了目的基因不同长度的PCR产物，产物经精制后进行测序，对比分析其结果。结果 获得了全长474bp的Oh．eDNA的基因核苷酸序列，包括5’端60bp，信号肽伴启动子ATG63bp，蛋白质密码部分216bp和3’端186bp并含有TGA终止码。经基因库信息计算机分析其信号肽与眼镜蛇树属(Pseudonnaja textilis，Pt．)海蛇(Laticauda semufasciata，Ls)100％同源，96.8％与眼镜蛇南洋亚种(Naja sputatrix，Ns)和银环蛇(Bungarus multicinctus，Bm)同源．蛋白密码部分83.3％与Ns，79.2％与Pt，76.4％与Ls和74.1％与Bm同源．氨基酸顺序分析信号肽后紧接着的72个氨基酸90.3％与已发现的眼镜王蛇毒长链a—NT’toxina同源，大约73．6％Toxinb、69．7％Oh-4、66.7％Oh-5、56.9％Oh-6A和6B同源，并与a-银环蛇毒素54.2％同源。结论 新发现的Oh-eDNA属于长链a-NT的基因。     

Helodermatine, a kallikrein-like, hypotensive enzyme from the venom of Heloderma horridum horridum (Mexican beaded lizard)

We have purified and characterized the major N-benzoyl-L-arginine ethyl ester hydrolase from the venom of Heloderma horridum horridum. The enzyme belongs to the serine proteinase family, and its activity vs. peptide amide substrates and human high-molecular-weight kininogen suggests a similarity to the family of kallikreins. This interpretation is corroborated by its reactivity with the natural inhibitors soybean trypsin inhibitor and Kunitz-type bovine pancreatic trypsin inhibitor (aprotinin). Injection of the enzyme (2-16 micrograms/kg) into anesthetized rabbits leads to a rapid dose-dependent transient decrease of the arterial blood pressure. Like glandular kallikrein it specifically converts single-chain tissue type plasminogen activator into its double chain form. In contrast to other kallikrein-like enzymes from snake venoms it shows no thrombin-like or plasminogen activator activity. The enzyme is a single-chain glycoprotein (Mr 63,000). The N-terminal sequence revealed significant homology to pig pancreatic kallikrein and to kallikrein like enzymes from Crotalus atrox and Crotalus adamanteus venom. This enzyme, which we name Helodermatine, is the first purified from Sauria with kallikrein-like properties.     

An initiator codon mutation in ornithine-delta-aminotransferase causing gyrate atrophy of the choroid and retina.

Gyrate atrophy of the choroid and retina (GA) is an autosomal recessive chorioretinal degeneration caused by deficiency of the mitochondrial matrix enzyme, ornithine-delta-aminotransferase (OAT). To study the molecular basis of the mutations causing GA, we cloned and sequenced the human OAT cDNA and determined the intron-exon arrangement of the structural gene. Using the cDNA template, we synthesized antisense RNA probes and performed RNase A protection experiments with RNA from four Lebanese GA patients. We found a probe-target mismatch at the 5' end of the first coding exon and amplified this region of the patients' genomic DNA using the polymerase chain reaction. Sequence analysis showed a G----A transition, changing the initiator ATG (methionine) codon to ATA. This mutation segregates with the GA allele in both pedigrees. Initiation of translation at the closest in-frame methionine codon would truncate OAT by 138 amino acids, eliminating the entire mitochondrial leader sequence and 113 amino acids of the mature peptide.     

cDNA cloning of human plasminogen activator-inhibitor from endothelial cells.

Full-length cDNA for plasminogen activator inhibitor (PAI-1) was isolated from a human umbilical vein endothelial cell (HUVEC) lambda gt11 cDNA library. Three overlapping clones were identified by immunologic screening of 10(6) recombinant phage using a rabbit anti-human fibrosarcoma PAI-1 antiserum. The fusion proteins encoded by these three clones also react strongly with a monoclonal mouse anti-human fibrosarcoma PAI-1 antibody. By nucleotide sequence analysis, PAI-1 cDNA encodes a protein containing 402 amino acids with a predicted, nonglycosylated molecular mass of 45 kD. Identity of this material as authentic PAI-1 was confirmed by the presence of high level homology with the primary amino acid sequence of an internal peptide prepared from purified rat hepatoma PAI-1. The predicted amino acid sequence also reveals extensive homology with other members of the serine protease inhibitor gene family. Cultured HUVECs contain two PAI-1 mRNA species, both encoded by a single gene, differing by 1 kb in the 3' untranslated region. The PAI-1 gene is located on human chromosome 7.     

Pharmacokinetics and pharmacodynamics of the myotoxic venom of Pseudechis australis (mulga snake) in the anesthetised rat

Context. Myotoxicity is a common clinical effect of snake envenoming and results from either local or systemic myotoxins in snake venoms. Although numerous myotoxins have been isolated from snake venoms, there has been limited study on the relationship between the time course of venom concentrations (pharmacokinetics) and the time course of muscle injury measured as a rise in creatine kinase (CK) (pharmacodynamics). Objective. The aim of this study was to develop an in vivo model of myotoxicity to investigate the time course of myotoxicity and the effect of antivenom. Materials and methods. Anesthetised rats were administered Pseudechis australis (mulga snake) venom either through i.v., i.m. or s.d. route, including a range of doses (5–100 μg/kg). Serial blood samples were collected for measurement of venom using enzyme immunoassay and measurement of CK and creatinine. Antivenom was administered before, 1 and 6 h after venom administration to investigate its effect on muscle injury. Plots of venom and CK versus time were made and the area under the curve (AUC) was calculated. Results. There was a significant dose-dependent increase in CK concentration after administration of P. australis venom, which was greatest for i.v. administration. Timed measurement of venom concentrations showed a rapid absorption through s.d. and i.m. routes and a delayed rise in CK concentrations following any route. Antivenom prevented myotoxicity shown by a decrease in the CK AUC, which was most effective if given earliest. There was a rise in creatinine following i.v. venom administration. Conclusion. The study shows the delayed relationship between venom absorption and the rise in CK, consistent with the delayed onset of myotoxicity in human envenoming. Antivenom prevented myotoxicity more effectively if given earlier.     

Structure of the human B lymphocyte receptor for C3d and the Epstein- Barr virus and relatedness to other members of the family of C3/C4 binding proteins [published erratum appears in J Exp Med 1988 Nov 1;168(5):1953-4]

Human complement receptor type 2 (CR2) is the B lymphocyte receptor for C3d and the Epstein-Barr virus. This protein is also a member of a family of C3b/C4b binding proteins that regulate complement activation, comprise tandemly repeated 60-75 amino acid sequences, and whose genes map to band q32 on chromosome 1. Overlapping cDNA clones encoding the entire human CR2 protein have been isolated from a human tonsillar cDNA library. The derived amino acid sequence of 1,032 residues encodes a peptide of 112,716 mol wt. A signal peptide was identified, followed by 15 copies of the short consensus repeat (SCR) structure common to the C3/C4 binding protein family. The entire extracellular portion of the protein comprised SCRs, thus, the ligand binding sites both for C3d and the EBV protein gp350/220 are positioned within this structure. Immediately following the final SCR was a transmembrane sequence of 24 amino acids and a cytoplasmic region of 34 amino acids. One of five cDNA clones isolated contained an additional SCR, providing evidence for alternative mRNA splicing or gene products of different human alleles. The CR2 cDNAs were used to isolate CR2-specific genomic phage. The entire CR2 coding sequences were found within 20 kb of human DNA. Analysis of the CR2 cDNA sequence indicated that CR2 contained internally homologous regions and suggested that CR2 arose by duplication of a primordial gene sequence encoding four SCRs. Comparison of the CR2 peptide sequence with those of other members of the gene family has identified many regions highly homologous with human CR1, fewer with C4bp and decay accelerating factor, and very few with factor H, and suggested that CR2 and CR1 arose by duplication of the same ancestral gene sequence. The homology between CR2 and CR1 extended to the transmembrane and cytoplasmic regions, suggesting that these sequences were derived from a common membrane-bound precursor.     

Duplication of exon 3 in the glycophorin C gene gives rise to the Lsa blood group antigen

BACKGROUND: The Gerbich-related Lsa blood group antigen (Ge6) resides on the higher-molecular-weight forms of glycophorin C (GPC) and glycophorin D (GPD). Southern blot analysis has previously revealed an additional GPC exon 3 insert in the genomic DNA from an Ls(a+) individual. STUDY DESIGN AND METHODS: To confirm the duplication of exon 3 in the GPC mRNA, total RNA prepared from the Epstein-Barr virus- transformed lymphocytes of an Ls(a+) individual was used in the synthesis of first-strand cDNA. The first-strand cDNA served as a template for the amplification of GPC-related DNA by polymerase chain reaction. After subcloning, the polymerase chain reaction cDNA was sequenced with a kit. Hemagglutination inhibition of anti-Lsa sera with synthetic peptides was performed to identify the location of Lsa on the GPC.Lsa protein. RESULTS: Sequencing the GPC.Lsa cDNA showed an insert of 84 nucleotides, which corresponds to the entire sequence of exon 3 in the GPC gene (GYPC). Since the exon 3-duplicated exon 3 boundary of GYPC.Lsa encodes a novel amino acid sequence on GPC.Lsa and GPD.Lsa, synthetic peptides consisting of the amino acids flanking this junction were used to determine the amino acid sequence that is essential for expression of Lsa. The peptide DIVVIA/EPDPG was noninhibitory, while peptides with the sequence TPTIMDIVVIA/EPDPG or SPSVLDIVVIA/EPDPG inhibited anti-Lsa reactivity. CONCLUSION: Lsa is located within the sequence of amino acids encoded by the nucleotides at the exon 3- duplicated exon 3 boundary, and the conformation of this peptide sequence is important for recognition of Lsa by human anti-Lsa.     

Secretory pancreatic stone protein messenger RNA. Nucleotide sequence and expression in chronic calcifying pancreatitis.

The pancreatic stone protein and its secretory form (PSP-S) are inhibitors of CaCO3 crystal growth, possibly involved in the stabilization of pancreatic juice. We have established the structure of PSP-S mRNA and monitored its expression in chronic calcifying pancreatitis (CCP). A cDNA encoding pre-PSP-S has been cloned from a human pancreatic cDNA library. Its nucleotide sequence revealed that it comprised all but the 5' end of PSP-S mRNA, which was obtained by sequencing the first exon of the PSP-S gene. The complete mRNA sequence is 775 nucleotides long, including 5'- and 3'- noncoding regions of 80 and 197 nucleotides, respectively, attached to a poly(A) tail of approximately 125 nucleotides. It encodes a preprotein of 166 amino acids, including a prepeptide of 22 amino acids. No overall sequence homology was found between PSP-S and other pancreatic proteins. Some homology with several serine proteases was observed in the COOH-terminal region, however. The mRNA levels of PSP-S, trypsinogen, chymotrypsinogen, and colipase in CCP and control pancreas were compared. PSP-S mRNA was three times lower in CCP than in control, whereas the others were not altered. It was concluded that PSP-S gene expression is specifically reduced in CCP patients.     

Spectrin Rouen (beta 220-218), a novel shortened beta-chain variant in a kindred with hereditary elliptocytosis. Characterization of the molecular defect as exon skipping due to a splice site mutation.

The molecular defect responsible for the shortened beta-spectrin chain variant, spectrin Rouen, was identified by analysis of cDNA and genomic DNA of affected individuals after amplification by the polymerase chain reaction. Peripheral blood reticulocyte RNA was transcribed into cDNA and amplified using primers corresponding to the 3' end of beta-spectrin cDNA. Agarose gel electrophoresis of cDNA amplification products from affected individuals revealed the expected band of 391 bp as well as a shortened band of 341 bp. Nucleotide sequencing of the shortened cDNA amplification product revealed that the sequences corresponding to the penultimate exon of the beta-spectrin gene (exon Y) were absent. This result was confirmed by hybridization of a Southern blot of amplification products with a labeled probe specific for exon Y. Nucleotide sequencing of the proband's amplified genomic DNA corresponding to this region of the beta-spectrin gene revealed a mutation in the 5' donor consensus splice site of the intron downstream of the Y exon, TGG/GTGAGT to TGG/GTTAGT, in one allele. We postulate that this mutation leads to the splicing out or skipping of exon Y, thus producing a shortened beta-spectrin chain. To our knowledge, this is the first documented example of exon skipping as the cause of a shortened beta-spectrin chain in a case of hereditary elliptocytosis. The exon skip results in the loss of the 17 amino acids of exon Y and creates a frameshift with the synthesis of 33 novel amino acids prior to premature chain termination 14 residues upstream of the normal carboxy terminus of the beta-spectrin chain, giving a mutant beta-spectrin chain that is 31 amino acids shorter than the normal chain.     

出血性蛇毒诱导血管内皮细胞凋亡的成分及作用机制研究进展

出血性蛇毒能专一性诱导血管内皮细胞(vascular endothelial cells，VEC)凋亡，研究人员已从中分离出5种VEC凋亡诱导成份，其中2种为L—aa氧化酶类，3种属于金属蛋白酶／解整联蛋白家族。研究证实前者可通过氧化VEC细胞膜上的L—leu产生H2O2而诱导其凋亡，后者则通过干扰膜整联蛋白与其配体的结合而使VEC凋亡。在由蛇毒诱导的VEC凋亡过程中，p53和bcl-2基因表达增加，且bcl-2的mRNA被剪辑成2条。已证实锚定依赖性信号分子αvβ3和磷脂信号分子PC-PLC参与该过程的信号转导。对该领域进一步研究，有望从蛇毒中纯化出或人工构建出专一地诱导肿瘤血管细胞凋亡的成分。本文总结了出血性蛇毒方面的研究进展。