A novel bradykinin-related peptide from skin secretions of toad Bombina maxima and its precursor containing six identical copies of the final product

Amphibian skin contains rich bradykinin-related peptides, but the mode of biosynthesis of these peptides is unknown. In the present study, a novel bradykinin-related peptide, termed bombinakinin M, was purified from skin secretions of the Chinese red bell

Biological activities of skin secretions of the salamander Tylototriton verrucosus

Water-soluble skin secretions of salamander Tylototriton venucosus, first described by Anderson in 1871, were studied for their biological and enzymatic activities. They were found to be toxic to mice with an intraperitoneal LD50 of 11.5 mg/kg. Using Sephadex G-75 gel filtration, it was proven that the toxic components of the secretions are proteins with molecular weights ranging from 30,000 to 50,000 Da. The secretions of T. venucosus display a wide spectrum of antimicrobial activities and also contain both proteolytic activity and trypsin inhibitory activity. In contrast, neither hemolytic nor hemorrhagic activities were found. The secretions were determined to have phospholipase A(2) activity; however, no acetylcholine esterase activity was detectable under the assay conditions.

ISOLATION AND PROPERTIES OF A BLOOD-COAGULATION FACTOR-X ACTIVATOR FROM THE VENOM OF KING COBRA (OPHIOPHAGUS HANNAH)

A specific blood coagulation factor X activator was purified from the venom of Ophiophagus hannah by gel filtration and two steps of FPLC Mono-Q column ion-exchange chromatography. It showed a single protein band both in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and alkaline polyacrylamide gel electrophoresis. The mol. wt was estimated to be 62,000 in non-reducing conditions and 64,500 in reducing conditions by SDS-PAGE. The isoelectric point was found to be pH 5.6. The enzyme had weak amidolytic activities toward CBS 65-25, but it showed no activities on S-2266, S-2302, thrombin substrate S-2238, plasmin substrate S-2251 or factor Xa substrate S-2222. It had no arginine esterase activity toward substrate benzoylarginine ethylester (BAEE). The enzyme activated factor X in vitro and the effect was absolutely Ca2+ dependent, with a Hill coefficient of 6.83. It could not activate prothrombin nor had any effect on fibrinogen and thus appeared to act specifically on factor X. The procoagulant activity of the enzyme was almost completely inhibited by serine protease inhibitors like PMSF, TPCK and soybean trypsin inhibitor; partially inhibited by L-cysteine. Metal chelator EDTA did not inhibit its procoagulant activity. These results suggest that the factor X activator from O. hannah venom is a serine protease.

An anionic antimicrobial peptide from toad Bombina maxima

Amphibian skin is a rich resource of antimicrobial peptides like maximins and maximins H from toad Bombina maxima. A novel cDNA clone encoding a precursor protein that comprises maximin 3 and a novel peptide. named maximin H5. was isolated from a skin cDNA library of B. maxima. The predicted primary structure of maximin H5 is ILGPVLGLVSDTLDDVLGIL-NH2,. Containing three aspartate residues and no basic amino acid residues. maximin H5 is characterized by an anionic property. Different from cationic maximin H peptides. only Gram-positive strain Staphylococcus aureus was sensitive to maximin H5. while the other bacteria] and fungal strains tested ere resistant to it. The presence of metal ions. like Zn2+ and Mg2+, did not increase its antimicrobial potency. Maximin H5 represents the first example of potential anionic antimicrobial peptides from amphibians, The results provide the first evidence that. together kith cationic antimicrobial peptides. anionic antimicrobial peptides may also exist naturally as part of the innate defense system. (C), 2002 Elsevier Science (USA). All rights reserved.

猕猴胚胎干细胞的分离培养和鉴定

目的　从体外培养成熟囊胚中分离并鉴定猕猴胚胎干细胞(embryonic stem cell , ES cell) 。方法　猕猴卵母细 胞经体外成熟培养、体外受精和早期胚胎体外成熟培养后,获得猕猴囊胚。当囊胚由透明带自然孵出后,用细玻璃针剥离 囊胚中的内细胞团(inner cell mass , ICM) 并与饲养细胞进行共培养。由ICM分离,培养并鉴定胚胎干细胞集落。结果　由 4 只FSH 超排猕猴中共取得92 个处于GV 期的猕猴卵母细胞,选取其中的22 个用HECM210 培养基培养后,获得6 个高质 量的囊胚,由此6 个囊胚中分离得到3 个内细胞团,并由此最终获得1 株猕猴ES 细胞,即RS5 细胞。RS5 细胞具高比例核P 质比,核仁多,其细胞集落边缘平整,其内各单个细胞清晰。经约5 个月的连续传代后,仍保持了正常二倍体的核型,其染 色体数目为42 条。碱性磷酸酶细胞组织化学染色为阳性,说明RS5 细胞为未分化态的胚胎干细胞。经高密度和长时间 培养后,RS5 细胞可进一步分化为多种类型细胞。结论　RS5 细胞株具有自我更新能力和多分化潜能,属于胚胎干细胞。

AN ACTIVATOR OF BLOOD-COAGULATION FACTOR-X FROM THE VENOM OF BUNGARUS-FASCIATUS

A specific activator of blood coagulation factor X was purified from the venom of Bungarus fasciatus by gel filtration and by ion-exchange chromatography on a Mono-Q column (FPLC). It consisted of a single polypeptide chain, with a mel. wt of 70,000 in reducing and non-reducing conditions. The enzyme had an amidolytic activity towards the chromogenic substrates S-2266 and S-2302 but it did not hydrolyse S-2238, S2251 or S-2222, which are specific substrates for thrombin, plasmin and factor Xa, respectively. The enzyme activated factor X in vitro and the effect was Ca2+ dependent with a Hill coefficient of 7.9. As with physiological activators, the venom activator cleaves the heavy chain of factor X, producing the activated factor Xa alpha. The purified factor X activator from B. fasciatus venom did not activate prothrombin, nor did it cleave or clot purified fibrinogen. The amidolytic activity and the factor X activation activity of the factor X activator from B. fasciatus venom were readily inhibited by serine protease inhibitors such as diisopropyl fluorophosphate (DFP), phenylmethanesulfonyl fluoride (PMSF), benzamidine and by soybean trypsin inhibitor but not by EDTA. These observations suggest that the factor X activator from B. fasciatus venom is a serine protease. It therefore differs from those of activators obtained from Vipera russelli and Bothrops atrox venoms, which are metalloproteinases.

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.

猕猴精浆纤溶酶原激活因子的来源及在精子获能中的作用

猕猴附睾、前列腺和精囊均表达tPA、uPA和PAI-1 mRNAs。加入uPA能维持精子的活力,使精子产生超激活运动,诱导顶体反应的发生,并使精子获得激活卵子的能力。这说明猕猴精浆PA除来源于睾丸外,可能主要来源于附睾及附性腺;在体外,uPA,而不是tPA,可能诱导精子获能。

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。

Identification and cloning of a trypsin inhibitor from skin secretions of Chinese red-belly toad Bombina maxima

A novel trypsin inhibitor was identified and purified from skin secretions of Chinese red-belly toad Bombina maxima. The partial N-terminal 29 amino acid residues of the peptide, named BMTI, were determined by automated Edman degradation. This allowed the cloning of a full-length cDNA encoding BMTI from a cDNA library prepared from the toad skin. The deduced complete amino acid sequence of BMTI indicates that mature BMTI is composed of 60 amino acids. A FASTA search in the databanks revealed that BMTI exhibits 81.7% sequence identity with BSTI, a trypsin/thrombin inhibitor from European toad Bombina bombina skin secretions. Sequence differences between BMTI and BSTI were due to 11 substitutions at positions 2, 9, 25, 27, 36-37, 39, 41-42, 50 and 56. BMTI potently inhibited trypsin with a K-i value of 0.06 muM, similar to that of BSTI. However, unlike BSTI, which also inhibited thrombin with a K-i value of 1 muM, no inhibitory effect of BMTI on thrombin was observed under the assay conditions. (C) 2002 Elsevier Science Inc. All rights reserved.

灵长类卵母细胞体外发育潜能的调控

灵长类卵母细胞细胞质成熟调控研究较为滞后, 使得体外成熟的灵长类卵母细胞的胚胎发育潜能十 分低下。提高其潜能对治疗人类不育症有重要的应用价值, 并可推动灵长类胚胎发育的研究。基于猕猴卵母细 胞质成熟调控研究, 讨论了无血清成熟培养基中雌激素和孕激素、能量物质、氨基酸对卵母细胞质成熟的影 响, 以及动物年龄和生殖周期与发育潜能的关系。从分子水平进一步解释这些因素的作用, 阐明卵母细胞质成 熟的机理将是今后工作的方向。