Comparative map between the domestic pig and dog

Cross-species chromosome painting with probes derived from flow-sorted dog and human chromosomes was used to construct a high-resolution comparative map for the pig. In total 98 conserved autosomal segments between pig and dog were detected by probes specific for the 38 autosomes and X Chromosome of the dog. Further integration of our results with the published human-dog and cat-dog comparative maps, and with data from comparative gene mapping, increases the resolution of the current pig-human comparative map. It allows for the conserved syntenies detected in the pig, human, and cat to be aligned against the putative ancestral karyotype of eutherian mammals and for the history of karyotype evolution of the pig lineage to be reconstructed. Fifteen fusions, 17 fissions, and 23 inversions are required to convert the ancestral mammalian karyotype into the extant karyotype of the pig.

Cross-species chromosome painting in the golden mole and elephant-shrew: support for the mammalian clades Afrotheria and Afroinsectiphillia but not Afroinsectivora

Cross-species painting (fluorescence in situ hybridization) with 23 human (Homo sapiens (HSA)) chromosome-specific painting probes (HSA 1-22 and the X) was used to delimit regions of homology on the chromosomes of the golden mole (Ghrysochloris asiaticus) and elephant-shrew (Elephantulus rupestris). A cladistic interpretation of our data provides evidence of two unique associations, HSA 1/19p and 5/21/3, that support Afrotheria. The recognition of HSA 5/3/21 expands on the 3/21 synteny originally designated as an ancestral state for all eutherians. We have identified one adjacent segment combination (HSA2/8p/4) that is supportive of Afroinsectiphillia (aardvark, golden mole, elephant-shrew). Two segmental combinations (HSA 10q/17 and HSA 3/20) unite the aardvark and elephant-shrews as sister taxa. The finding that segmental syntenies in evolutionarily distant taxa can improve phylogenetic resolution suggests that they may be useful for testing sequence-based phylogenies of the early eutherian mammals. They may even suggest clades that sequence trees are not recovering with any consistency and thus encourage the search for additional rare genomic changes among afrotheres.

云南绵羊线粒体DNA遗传多态性研究

该研究用12种识别六碱基的限制性内切酶对来自云南省3个地方绵羊品种11个样本的线粒体DNA(mtDNA)进行了限制性片段长度多态性分析(RFLP)，结果表明，云南绵羊群体平均核苷歧异度为0.086%，平均遗传距离为0.004，说明mtDNA变异度很低，遗传多样性贫乏，提示云南绵羊可能起源于同一个共同祖先;昭通绵羊与德钦绵羊距离较远，大约分歧于30万年前，德钦绵羊与腾冲绵羊大约分歧于11万年前，而腾冲绵羊与昭通绵羊也于18万年前发生分歧。

懒猴属的核糖体DNA变异及其种间分化关系

用15种限制性内切酶和人28S、18S rDNA探针构建了懒猴属各物种核糖体DNA重复单位的限制性内切酶图谱。在进化速率较高的非转录间隔区, 在大、中、小獭猴中分别定位了23、24、24个酶切位点。结果表明, 懒猴属内似只有两个有效物种, 即大懒猴和小懒猴, 中懒猴与小懒猴的分化至多到半种级别。

猕猴属6个种的rDNA变异及其系统进化关系

以人28S,18S DNA为探针,用15种限制性内切酶构建了猕猴属6个种(M.mulatta、M.facsicularis、M.arctoides、M.assamensis、M.thibetana、M.nemestrina)和滇金丝猴(Rhinopithecus bieti),白颊长臂猿(Hylobates leucogenys)核糖体DNA重复单位的限制性内切酶图谱。红面猴(M.arctoides)与熊猴(M.assamensis)拥有完全相同的限制性内切酶图谱。基于内切酶图谱得到了68个信息位点并计算了各种rDNA重复型间的遗传距离。用PHYLIP version 3.5c软件包中的NEIGHBOR和RESTML程序,以滇金丝猴和白颊长臂猿为外群,构建了NJ树和最大似然树。两棵树的拓扑结构不完全一致,但恒河猴( (M.mulatta)和食蟹猴(M.facsicularis)总是位于树的基部。熊猴-红面猴(M.assamensis-M.arctoides)虽然与藏猴(M.thibetana)共享的限制性位点数更多,在NJ树上两类动物也最为接近,但在最大似然树中熊猴-红面猴却与平顶猴(M.nemestrina)聚在一起。因此,rDNA变异的数据尚不能对猕猴类动物进行有效的分组。

Functional consequences of new exon acquisition in mammalian chromodomain Y-like (CDYL) genes

The origin of new exons is an important mechanism for proteome diversity. Here, we report the recurrent origination of new exons in mammalian chromodomain Y-like (CDYL) genes and the functional consequences associated with the acquisition of the new exons

中华硬蜱血小板集聚抑制剂的分离纯化与活性研究

目的研究蜱类抗血小板集聚的生物活性成分, 了解其与其宿主相互作用的分子机制。方法用葡聚糖 Sephadex G-50 凝胶过滤及高效液相从半饱吸血的中华硬蜱(Ixodes sinensis) 唾液腺中分离纯化具有血小板集聚抑制活性 的蛋白质, 用飞行质谱测定该抑制剂的分子量, 并用兔富血小板血浆研究其血小板集聚抑制活性。结果通过液相分离, 从中华硬蜱唾液腺中得到了一种血小板集聚抑制剂, 用飞行质谱测定该抑制剂的相对分子质量(M r ) 为8 065。该抑制 剂对二磷酸腺苷(ADP) 诱导的血小板集聚表现强烈的抑制活性, 在浓度为10 μg/mL 时, 可以抑制90% 以上的血小板集 聚。结论首次分离获得中华硬蜱唾液腺血小板集聚抑制剂, 该抑制剂对硬蜱顺利获取宿主血餐至关重要。

蛇毒丝氨酸蛋白酶

蛇毒丝氨酸蛋白酶属于糜蛋白酶样丝氨酸蛋白酶。蛇毒丝氨酸蛋白酶几乎可作用于哺乳 动物凝血, 纤溶系统的各个步骤而对凝血、纤溶、血小板功能、血管完整性以及出血等发生影响。蛇 毒丝氨酸蛋白酶具有高度的序列相似性一, 但底物专一性却具较大的分化另一方面, 它们对生理性丝氨酸蛋白酶抑制剂有较强的耐受性及较长的体内半衰期, 这些特性使之成为蛋白 酶结构与功能研究的优良模型, 同时也具广阔的药物开发前景和作为药理研究的工具。

六种常见两栖类动物皮肤分泌物的生物活性比较

对6种常见两栖类动物皮肤水溶性分泌物的生物学活性与酶学活性进行了研究。其中，红瘰疣螈（Tylototriton verrucosus）、大蹼铃蟾（Bombina maxima）、华西蟾蜍（Bufo andrewsi）皮肤分泌物对小白鼠具有致死毒性，对小白鼠腹腔注射的半数致死剂量分别为11.5、18.8和264mg/kg。而沼哇（Rana nigromaculata）、泽蛙（Rana guentheri）、黑斑蛙（Rana limnocharis）的皮肤分泌物在小白鼠腹腔注射剂量达到500 mg/kg时仍不显示致死毒性。红瘰疣螈、大蹼铃蟾皮肤分泌物的毒性成分为3～60 kDa的多肽与蛋白质。6种两栖类动物皮肤分泌物都具有蛋白酶水解活性与胰蛋白酶蛋白酶抑制活性。红瘰疣螈、大蹼铃蟾和华西蟾蜍皮肤分泌物具有广谱抗菌活性。大蹼铃蟾、华西蟾蜍皮肤分泌物具有肿瘤细胞细胞毒活性。6种皮肤分泌物对哺乳类血液凝固系统无显著影响。仅发现红瘰疣螈皮肤分泌物具有磷脂酶A_2活性。6种皮肤分泌物均无乙酰胆碱脂酶活性。

两栖类动物皮肤活性物质的利用兼论中国两栖类资源开发的策略

着眼于疾病治疗因子的筛选和生物活性分子的发现,对中国部分两栖类动物进行了系统的筛选研究,发现了一系列的生物活性分子和可能具有临床应用价值的生物因子,并结合中国及世界两栖类资源开发利用的现状,主要从药物开发和资源保护的角度对中国两栖类资源的开发利用提出几点建议。

Antimicrobial peptides from skin secretions of Chinese red belly toad Bombina maxima

Two groups of antimicrobial peptides have been isolated from skin secretions of Bombina maxima. Peptides in the first group, named maximins 1, 2, 3, 4 and 5, are structurally related to bombinin-like peptides (BLPs). Unlike BLPs, sequence variations in maximins occurred all through the molecules. In addition to the potent antimicrobial activity, cytotoxicity against tumor cells and spermicidal action of maximins, maximin 3 possessed a significant anti-HIV activity. Maximins 1 and 3 were toxic to mice with LD50 values of 8.2 and 4.3 mg/kg, respectively. Peptides in the second group, termed maximins H1, H2, H3 and H4, are homologous with bombinin H peptides. cDNA sequences revealed that one maximin peptide plus one maximin H peptide derived from a common larger protein. (C) 2002 Elsevier Science Inc. All rights reserved.

A novel proline rich bombesin-related peptide (PR-bombesin) from toad Bombina maxima

A novel bombesin-related peptide was isolated from skin secretions of Chinese red belly toad Bombina maxima. Its primary structure was established as pGlu-Lys-Lys-Pro-Pro-Arg-Pro-Pro-Gln-Trp-Ala-Val-Gly-His-Phe-Met-NH2. The amino-terminal (N-terminal) 8-residue segment comprising four prolines and three basic residues is extensively different from bombesins from other Bombina species. The peptide was thus named proline rich bombesin (PR-bombesin). PR-bumbesin was found to elicit concentration-dependent contractile effects in the rat stomach strip, with both increased potency and intrinsic activity as compared with those of [Leu(13)]bombesin. Analysis of different bombesin cDNA structures revealed that an 8 to 14- nucleotide fragment replacement in the peptide coding region (TGGGGAAT in the cDNAs of multiple bombesin forms from Bombina orientalis and CACCCCGGCCACCC in the cDNA of PR-bombesin) resulted in an unusual Pro-Pro-Arg-Pro-Pro motif in the N-terminal part of PR-bombesin. (C) 2002 Elsevier Science Inc. All rights reserved.

A NOVEL PLASMINOGEN-ACTIVATOR FROM SNAKE-VENOM - PURIFICATION, CHARACTERIZATION, AND MOLECULAR-CLONING

A novel plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA) has been identified and purified to homogeneity. It is a single chain glycoprotein with an apparent molecular weight of 33,000 and an isoelectric point of pH 5.2. It specifically activates plasminogen through an enzymatic reaction. The activation of human native GIu-plasminogen by TSV-PA is due to a single cleavage of the molecule at the peptide bond Arg(561)-Val-(562). Purified TSV-PA, which catalyzes the hydrolysis of several tripeptide p-nitroanilide substrates, does not activate nor degrade prothrombin, factor X, or protein C and does not clot fibrinogen nor show fibrino(geno)lytic activity in the absence of plasminogen. The activity of TSV-PA was readily inhibited by phenylmethanesulfonyl fluoride and by p-nitrophenyl-p-guanidinobenzoate. Oligonucleotide primers designed on the basis of the N-terminal and the internal peptide sequences of TSV-PA were used for the amplification of cDNA fragments by polymerase chain reaction. This allowed the cloning of a full-length cDNA encoding TSV-PA from a cDNA library prepared from the venom glands. The deduced complete amino acid sequence of TSV-PA indicates that the mature TSV-PA protein is composed of 234 amino acids and contains a single potential N-gIycosylation site at Asn(1G1). The sequence of TSV-PA exhibits a high degree of sequence identity with other snake venom proteases: 66% with the protein C activator from Aghistrodon contortrix contortrix venom, 63% with batroxobin, and 60% with the factor V activator from Russell's viper venom. On the other hand, TSV-PA shows only 21-23% sequence similarity with the catalytic domains of u-PA and t-PA. Furthermore, TSV-PA lacks the sequence site that has been demonstrated to be responsible for the interaction of t-PA (KHRR) and u-PA (RRHR) with plasminogen activator inhibitor type 1.

纤溶酶原激活因子与猕猴精子运动力的关系及其在附睾和附性腺中的表达调节

原位杂交的结果表明11酸睾酮诱导少精子症和弱精子症,tPA mRNA的表达在附睾头、精囊及前列腺减少,而在附睾体升高,附睾尾表达基本无变化;uPA mRNA的表达在附睾头、附睾体、前列腺减少,而在精囊升高,附睾尾表达基本无变化;PAI-1 mRNA的表达在附睾头、附睾体、精囊下降,而在前列腺升高,附睾尾表达无显著变化。单侧隐睾手术不影响tPA、uPA和PAI-1 mRNA的表达。结果提示附睾头和附睾体分泌的uPA可能与精子前向运动能力的获得相关。tPA、uPA和PAI-1 mRNA在猕猴附睾头部和体部、前列腺和精囊中的表达可能受睾酮的调节,但不受睾丸分泌因子及温度的影响,且在不同部位睾酮的调节具不同的特征,而附睾尾tPA、uPA和PAI-1的表达则可能是组成性表达。

Trimeresurus stejnegeri snake venom plasminogen activator - Site-directed mutagenesis and molecular modeling

The specific plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA) is a serine proteinase presenting 23% sequence identity with the proteinase domain of tissue type plasminogen activator, and 63% with batroxobin, a fibrinogen clotting enzyme from Bothrops atrox venom that does not activate plasminogen. TSV-PA contains six disulfide bonds and has been successfully overexpressed in Escherichia coli (Zhang, Y., Wisner, A., Xiong, Y. L,, and Bon, C, (1995) J. Biol. Chem. 270, 10246-10255), To identify the functional domains of TSV-PA, we focused on three short peptide fragments of TSV-PA showing important sequence differences with batroxobin and other venom serine proteinases. Molecular modeling shows that these sequences are located in surface loop regions, one of which is next to the catalytic site, When these sequences were replaced in TSV-PA by the equivalent batroxobin residues none generated either fibrinogen-clotting or direct fibrinogenolytic activity, Two of the replacements had little effect in general and are not critical to the specificity of TSV-PA for plasminogen. Nevertheless, the third replacement, produced by the conversion of the sequence DDE 96a-98 to NVI, significantly increased the K-m for some tripeptide chromogenic substrates and resulted in undetectable plasminogen activation, indicating the key role that the sequence plays in substrate recognition by the enzyme.