红瘰疣螈皮肤的显微结构观察

对我国Ⅱ级重点保护野生动物红瘰疣螈(Tylototriton shanjing)成体的皮肤结构进行了显微观察。观察结果表明,其皮肤厚薄、微血管、色素分布,以及腺体的分布和大小等方面在身体的不同部位存在差异:头和背部的皮肤厚于腹部;黑色素细胞多分布于真皮浅层,以腹部及背侧无瘰粒处多见;真皮层富含黏液腺和颗粒腺,但分布不均匀;头嵴棱部及背部瘰粒下方颗粒腺的数量多、体积大,并充满分泌物;表皮下腺体外突,其表皮细胞层数减少,形成分泌物有效通道。同时,将红瘰疣螈皮肤与其他两栖类相比可知,其以上皮肤结构特征均体现了红瘰疣螈对山区阴湿环境中陆栖生活的重要适应。     

十一种无尾两栖类物种皮肤和肺显微结构

无尾两栖类动物的呼吸方式为肺呼吸和皮肤辅助呼吸,为探究两种呼吸器官的显微结构,本文采用解剖学和组织学的方法,对大蹼铃蟾(Bombina maxima)、腺角蟾(Megophrys glandulosa)、中华蟾蜍(Bufo gargarizans)、华西雨蛙(Hyla gongshanensis)、昭觉林蛙(Rana chaochiaoensis)、滇蛙(Dianrana pleuraden)、双团棘胸蛙(Gynandropaa yunnanensis)、贡山树蛙(Rhacophorus gongshanensis)、斑腿泛树蛙(Polypedates megacephalus)、饰纹姬蛙(Microhyla fissipes)、多疣狭口蛙(Kaloula verrucosa)的皮肤和肺的形态及显微结构进行观察。结果显示,背部和腹部皮下可见血管交错成网状,皮肤由表皮层和真皮层构成。除华西雨蛙外,其余10种均有分布于真皮疏松层与致密层间的钙化层;色素细胞位于疏松层上层,体背色素层较发达。肺囊状,中空密布血管,分为大小相当的左右肺叶,两肺叶相通并与心粘连,无气管和支气管。肺由肺囊壁、隔膜、毛细血管、肺泡细胞等结构组成。肺囊壁分为胸膜层、中间层和内层:胸膜层由扁平细胞构成,中间层由结缔组织构成,内层由肺细胞和毛细血管组成,隔膜由毛细血管和结缔组织构成,游离隔膜向中部靠拢可形成半封闭腔室,结合隔膜与肺囊壁形成封闭小腔室。在这11个物种中,肺较发达的个体,其皮肤结构中黏液腺的数量就会相对较少,组织形态学特征表现出与环境适应性关系较大,而受到系统发育关系影响较小。     

铃蟾肽

铃蟾肽赖仞冉永禄（中国科学院昆明动物研究所,昆明６５０２２３）关键词铃蟾肽两栖类动物皮肤中富含多种与哺乳类动物调节有关的活性肽,是获取多肽的巨大储存库。铃蟾肽（Ｂｏｍｂｅｓｉｎ,ＢＮ）是１９７１年由Ｅｒｓｐａｍｅｒ和Ａｎａｓｔａｓｉ等从欧洲铃蟾Ｂ．ｂ...     

非洲爪蟾血清白蛋白的分离纯化及胰蛋白酶抑制活性

通过凝胶过滤层析及两步阴离子交换层析,从非洲爪蟾(Xenopus laevis)的血清中获得了其68kDa的血清白蛋白。与大蹼铃蟾血清白蛋白相似,非洲爪蟾血清白蛋白也具有抑制胰蛋白酶的活性,但其抑制活力相对较低,180nmol/L的非洲爪蟾血清白蛋白能抑制84%的胰蛋白酶活性(30nmol/L)。经表面等离子共振法获得了其与胰蛋白酶的结合动力学常数,解离平衡常数KD=1.44×10-6mol/L。经Western blot分析发现,非洲爪蟾的皮肤中也分布有血清白蛋白。推测两栖类动物血清白蛋白具有的胰蛋白酶抑制活性可能是其抵御天敌捕食的一种防御措施。     

青海海北发现一例淡黄色高原林蛙

<正>高原林蛙（Rana kukunoris）隶属于两栖纲（Amphibia）无尾目（Anura）蛙科（Ranidae）林蛙属,是青藏高原特有的两栖类动物（谢锋等2000）。高原林蛙背部多为灰褐色或棕红色,疣粒色略浅,其周围褐黑色,体侧散有黑色或红色斑,四肢具黑色横纹,雄蛙腹面多为粉红色或黄白色,雌蛙为红棕色,多无斑（费梁等2012）。在青藏高原高海拔、缺氧、强紫外线环境中,为维持个体生存及栖息地适应,高原林蛙皮肤结构产生了一系列特异性变化（米志平等2016,张湑泽等2018）。     

两栖类动物皮肤分泌物及其生物学适应意义--大蹼铃蟾皮肤分泌物蛋白质多肽组的启示

以大蹼铃蟾(Bombina maxima)为代表性物种,揭示了两栖类皮肤分泌物蛋白质多肽组丰富的分子和功能多样性。目前在大蹼铃蟾已发现的分子包括3类不同的抗菌肽、缓激肽、缓激肽增强肽和缓激肽拮抗肽、缓激肽基因相关肽、富含脯氨酸铃蟾肽及其基因相关肽、神经调节素U、Bv8肽、三叶型蛋白和蛋白酶抑制剂等。抗菌肽分子多样性及其形成机制、缓激肽及其基因相关肽功能和表达模式的多样性都较好地揭示了在多样的生态条件下,两栖类皮肤活性肽环境适应的分子基础及生物合成的调控机制。富含血红素辅基的白蛋白广泛分布在大蹼铃蟾皮肤外皮层细胞膜上和真皮海绵层内,表明它在皮肤的生理功能,如在呼吸、物质交换和渗透压调节中有重要作用。两栖类皮肤分泌物蛋白质多肽组,由于其分子结构的多样性、新颖性和哺乳类同系物的存在,在生物医学研究和天然药物开发中具有独特和不可替代的价值;同时,两栖类皮肤功能基因组具有多样性丰富、快速重组突变的特征,是探讨生物适应的基因基础、基因形成机制和进化特性等生物学基本问题的优秀模型。     

贵州北部四种丙栖类乳酸脱氢酶同工酶的比较研究

本文采用聚丙烯酰胺凝胶电泳方法,研究了贵州北部的四种两栖类动物：细痣疣螈、棘指角蟾、绿臭蛙和小弧斑姬蛙的心肝、肾等组织的乳酸脱氢酶（LDH）同工酶,发现LDH具有明显的种族特异性和组织特异性,不同种的动物和同种动物的不同组织都有各自的LDH同酶谱型,可以用它们作为识别物种的附加指标。本文初步探讨了生化分类学与系统分类学的关系。     

贵州两栖类新纪录及蛙属一新种

贵州两栖类动物过去曾有过调查,但局限于少数地区。为此,我们从1974年到1979年进行了比较全面的调查,其范围包括贵定、龙里、惠水、平塘、德江、江口、松桃、赤水、仁怀、湄潭、绥阳、遵义、务川、金沙、正安、雷山、榕江、从江、毕节、威宁、望谟、册亨、兴义及荔波等24县,获得两类动物标本一万二千余号,共计有50种(包括亚种),隶属于2目10科16属,其中有一新种及6种贵州新纪录,后者为: 1.黄斑小鲵Hynobius flavomaculatus Fei et Ye(绥阳) 2.大绿蛙Rana livida(Blyth)(江口) 3.棘胸蛙Rana spinosa David(江口、松桃、贵定、龙里、雷山及望谟)     

澜沧江漫湾电站水库地区两栖动物的考察研究

漫湾电站地区共有两栖类２ 目８ 科１８ 属３８ 种。区系特点是： （１） 物种很丰富, 占中国两栖类种数１３－６２ ％ , 超过２０ 个省区, 超过云南红河州, 而与西双版纳相等, 是中国两栖类最丰富的地区之一, 也是云南种类最多的地区;（２） 全部属东洋界种类; （３） 西南区种类占绝对优势, 有２６ 种, 占６８－４２％ ; （４） 华中区及华中华南种类很少, 共３ 种; （５） 锄足蟾科种类特多, 有１２ 种; （６） 特有种很多, 有漫湾特有７ 种, 云南特有１６ 种,中国特有１３ 种以上。垂直分布可分为河谷低山带, 海拔８００ － １ ５００ｍ ; 中山带, 海拔１ ５００ － ２ ２００ｍ ; 高山带,２ ２００ －２ ８００ｍ ;２ ８００３ ２９１ｍ 的山顶带。泽蛙、双团棘胸蛙及云南小狭口蛙为本地区优势种。稀有种多, 应加强保护; 红瘰疣螈为国家Ⅱ级保护动物。     

长白山国家级自然保护区两栖类动物道路致死特征分析

为了解旅游道路交通导致的两栖类致死效应,于2007~2014年在环绕长白山国家级自然保护区的环长白山旅游公路(环区公路)、头道环区公路以及白山公路分别选取1条样线,采用直接计数法统计了交通致死的两栖类种类、数量,以及致死个体处于交通路段的位置,共统计257次,累计调查样带总长2 441 km。道路致死两栖类种类有5种,累计致死个体数为7 910只,平均致死率为3.34只/km。最容易致死的两栖类动物是中国林蛙(Rana chensinensi)、东方铃蟾(Bombina orientalis)、中华蟾蜍(Bufo gargariza),这3种动物占到致死总数的96.36%;经Moses Test检验,白山公路的致死率、头道公路的致死率及环区公路致死率均与对照土路致死率差异显著(白山公路:n=67,P=0.000;头道公路:n=76,P=0.000;环区公路:n=164,P=0.000)。白山公路与头道公路两栖类致死率差异显著(n=93,P=0.010),白山公路与环区公路的两栖类道路致死率差异显著(n=181,P=0.000);头道公路与环区公路的两栖类道路致死率差异显著(n=190,P=0.000)。两栖类道路致死高峰期在8月份,其次较集中出现在4~6月的繁殖期。从公路年度致死变化来看,2007~2014年期间除了中国林蛙和中华蟾蜍致死率呈小幅波动外,其他种类致死率总体呈下降趋势,只有日本树蟾(Hyla japonica)年度致死率差异显著(Kruskal Wallis Test:χ2=18.031,df=7,P=0.012);不同月份中华蟾蜍(Pearson:R=0.939,n=7,P=0.002)、中国林蛙(Pearson:R=0.912,n=7,P=0.004)、日本树蟾(Pearson:R=0.904,n=7,P=0.005)致死率与交通量显著相关。本研究认为,两栖类的道路致死率与两栖类种群大小、种群密度、生活史及交通量等都存在显著相关性。     

Purification of a lysozyme from skin secretions of Bufo andrewsi

A novel toad lysozyme (named BA-lysozyme) was purified from skin secretions of Bufo andrewsi by a three-step chromatography procedure. BA-lysozyme is a single chain protein and the apparent molecular weight is about 15 kDa as judged by SDS-PAGE. The specific lytic activity against Micrococcus lysodeikticus of BA-lysozyme is 2.7 x 10(5) units/mg, indicating that it is a potent lysozyme. It displayed potent bactericidal activity against Staphylococcus aureus and Escherichia coli with minimal inhibitory concentrations (MIC) of 1 and 8 microM, respectively. The deduced primary structure of BA-lysozyme from cloned cDNA was confirmed by N-terminal sequencing and peptide mass fingerprinting. Its amino acid sequence shares 56.5% identity with that of chicken egg-white lysozyme. Phylogenetic analysis indicates that B. andrewsi lysozyme is closely related to that of turtle. This is the first report on the isolation and primary structure determination of amphibian lysozyme.     

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 LD(50) 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.     

Comparison of the toxicities of patulin and patulin adducts formed with cysteine.

The toxicities of patulin and of the patulin adducts formed with cysteine were compared using the mutation-sensitive strain Escherichia coli W3110 thy polA1 and its polA1+ revertant. The acute toxicities of patulin and of the adduct mixture were also compared using NMRI mice. The adduct mixture was shown by thin-layer chromatography to consist of one ninhydrin-positive, one ninhydrin- and MBTH (3-methyl-2-benzothiazolinone hydrazone)-positive, three MBTH-positive, and two ninhydrin- and MBTH-negative components. The results showed that patulin was over 100 times more toxic to E. coli than the adduct complex. Neither patulin nor the adduct mixture was found to induce the repair effect in E. coli. In the mouse feeding tests, the oral 50% lethal dose for patulin was 29 mg/kg, while that of the adduct mixture was greater than 2,370 mg/kg.     

Comparison of the toxicities of patulin and patulin adducts formed with cysteine.

The toxicities of patulin and of the patulin adducts formed with cysteine were compared using the mutation-sensitive strain Escherichia coli W3110 thy polA1 and its polA1+ revertant. The acute toxicities of patulin and of the adduct mixture were also compared using NMRI mice. The adduct mixture was shown by thin-layer chromatography to consist of one ninhydrin-positive, one ninhydrin- and MBTH (3-methyl-2-benzothiazolinone hydrazone)-positive, three MBTH-positive, and two ninhydrin- and MBTH-negative components. The results showed that patulin was over 100 times more toxic to E. coli than the adduct complex. Neither patulin nor the adduct mixture was found to induce the repair effect in E. coli. In the mouse feeding tests, the oral 50% lethal dose for patulin was 29 mg/kg, while that of the adduct mixture was greater than 2,370 mg/kg.     

Anti-inflammatory activity of aqueous extracts of five Costa Rican medicinal plants in Sprague-Dawley rats

The anti-inflammatory properties of Loasa speciosa and Loasa triphylla (Loasaceae), Urtica leptuphylla and Urera baccifera (Urticaceae), and Chaptalia nutans (Asteracene) were studied using the carregeenan induced rat paw edema model. Aqueous extracts of each plant were made according to the ethnobotanical use. The hippocratic assay was made with female rats; the dose used was 500 mg/kg i.p. and the control group received 0.5 ml of n.s.s.. All the animals treated showed hypothermia, and those treated with the extracts of Chaptalia nutans, Urera baccifera and Urtica leptuphylla showed an increased colinergic activity. Acute toxicities of the aqueous extracts were studied in mice an the mean lethal doses ranged between 1.0226 and 1.2022 g/kg. The extracts of Urera baccifera, Chaptalia nutans, Loasa speciosa and Loasa triphylla (500 mg/kg i.p.) showed an anti-inflammatory activity comparable with that of indomethacin. The extracts of U. baccifera and C. nutans, which showed the greatest anti-inflammatory activity, did not show it when used orally (500 mg/kg p.o.).     

Isolation, toxicity and amino terminal sequences of three major neurotoxins in the venom of Malayan krait (Bungarus candidus) from Thailand

We isolated the most lethal toxins in the venom of the Malayan krait (Bungarus candidus), one of the medically most important snake species in southeast Asia. Three beta-BTx like basic neurotoxins, T1-1, T1-2, and T2, with PLA2 activity were isolated from pooled venom of eight B. candidus from southern Thailand by cation-exchange chromatography, followed by adsorption chromatography on hydroxylapatite and RP-HPLC, with 14-, 16-, and 4-fold increases in toxicity compared to crude venom. The LDs50 determined in mice weighing 18-20 g were 0.26, 0.22, and 0.84 micro g per mouse with i.v. injection. T1-1 and T1-2 possessed comparable lethal toxicities to those of beta1-BTx, the most toxic neurotoxin in B. multicinctus venom, and the major neurotoxin in B. flaviceps venom. The apparent molecular weights of the native toxins were approximately 25-25.5 kDa. They consist of two polypeptide chains with apparent molecular weights of 15.5-16.5 and 8-8.5 kDa, respectively. The amino terminal sequences of the two chains of each of the toxins determined by Edman degradation exhibited considerable similarity with those of the A-chains and B-chains of beta-BTxs in the venom of Bungarus multicinctus.     

Toxicity and toxins of natural blooms and isolated strains of Microcystis spp. (Cyanobacteria) and improved procedure for purification of cultures

All samples of cyanobacterial blooms collected from 1986 to 1989 from Lake Kasumigaura, Ibaraki Prefecture, Japan, were hepatotoxic. The 50% lethal doses (LD50s) of the blooms to mice ranged from 76 to 556 mg/kg of body weight. Sixty-eight Microcystis cell clones (67 Microcystis aeruginosa and 1 M. viridis) were isolated from the blooms. Twenty-three strains (including the M. viridis strain) were toxic. However, the ratio of toxic to nontoxic strains among the blooms varied (6 to 86%). Microcystins were examined in six toxic strains. Five toxic strains produced microcystin-RR, -YR, and -LR, with RR being the dominant toxin in these strains. Another strain produced 7-desmethylmicrocystin-LR and an unknown microcystin. This strain showed the highest toxicity. Establishment of axenic strains from the Microcystis cells exhibiting extracellularly mucilaginous materials was successful by using a combination of the agar plate technique and two-step centrifugation.     

Toxicity and toxins of natural blooms and isolated strains of Microcystis spp. (Cyanobacteria) and improved procedure for purification of cultures.

All samples of cyanobacterial blooms collected from 1986 to 1989 from Lake Kasumigaura, Ibaraki Prefecture, Japan, were hepatotoxic. The 50% lethal doses (LD50s) of the blooms to mice ranged from 76 to 556 mg/kg of body weight. Sixty-eight Microcystis cell clones (67 Microcystis aeruginosa and 1 M. viridis) were isolated from the blooms. Twenty-three strains (including the M. viridis strain) were toxic. However, the ratio of toxic to nontoxic strains among the blooms varied (6 to 86%). Microcystins were examined in six toxic strains. Five toxic strains produced microcystin-RR, -YR, and -LR, with RR being the dominant toxin in these strains. Another strain produced 7-desmethylmicrocystin-LR and an unknown microcystin. This strain showed the highest toxicity. Establishment of axenic strains from the Microcystis cells exhibiting extracellularly mucilaginous materials was successful by using a combination of the agar plate technique and two-step centrifugation.     

Characterization of naturally occurring peptides in the skin secretion of Rana pipiens frog reveal pipinin-1 as the novel insulin-releasing agent.

Naturally occurring insulinotropic peptides were isolated from the skin secretions of Rana pipiens frogs. Crude secretions (50 mg; 5-10 frogs) obtained by mild electrical stimulation of the dorsal skin surface were purified by reversed-phase high-performance liquid chromatography (HPLC) yielding 80 fractions. In acute incubations with glucose-responsive BRIN-BD11 cells, fractions 40-47 (band 1) and fractions 60-65 (band 2) showed significant 1.7-6.7-fold increases in insulin-releasing activity (P < 0.001) compared with 5.6 mm glucose alone. Pooled fractions in bands 1 and 2 were rechromatographed yielding a total of seven peaks capable of subsequent 1.2-1.8-fold stimulation of insulin release. Final purification by HPLC to single homogenous peaks revealed one prominent peptide (peak 4.1) with insulin-releasing activity which lacked effects on cell viability. Electrospray mass spectrometric analysis of this peptide indicated molecular mass of 2562.6 Da. Determination of the primary amino acid sequence of this peptide revealed a 24-amino acid sequence: FLPIIAGVAAKVFPKIFCAISKKC. Database search showed a 100% homology to histamine-releasing pipinin-1. In conclusion, this study revealed the skin secretions of Rana pipiens to be a rich source of insulin-releasing peptides. The discovery of insulinotropic activity for pipinin-1, initially characterized as an antimicrobial is interesting and merits further investigation.     

Bolevenine, a toxic protein from the Japanese toadstool Boletus venenatus

A toxic protein, called bolevenine, was isolated from the toxic mushroom Boletus venenatus based on its lethal effects on mice. On SDS-PAGE, in either the presence or absence of 2-mercaptoethanol, this protein showed a single band of approximately 12 kDa. In contrast, based on gel filtration and MALDI-TOFMS, its relative molecular mass was estimated to be approximately 30 kDa and approximately 33 kDa, respectively, indicating that the protein consists of three identical subunits. This toxin exhibited its lethal activity following injection at 10mg/kg into mice. The N-terminal amino acid sequence was determined up to 18, and found to be similar to the previously reported bolesatine, a toxic compound isolated from Boletus satanas.