棕点湍蛙皮肤分泌液中促胰岛素释放肽的分离纯化、基因克隆及活性分析

通过分离纯化棕点湍蛙(Amolops loloensis)皮肤分泌液中的生物活性物质,得到有促胰岛素释放活性的分离峰,并鉴定其结构.采用葡聚糖Sephadex G-50凝胶层析和反相高效液相(RP-HPLC)等手段对棕点湍蛙皮肤分泌液进行分离纯化,利用胰岛素释放实验进行活性检测,Edman降解法测定活性峰的氨基酸序列,反转录法构建cDNA文库并克隆其基因.得到一个具有显著的促胰岛素释放活性的十六肽,测得其氨基酸序列为:FMPIvGKsMSGLSGKL-NH2,命名为amolopin-1.由cDNA(开放阅读框为192bp)推导的氨基酸一级结构显示,其前体由64个氨基酸残基(aa)组成,包括高度保守的信号肽(22aa),酸性肽以及成熟肽.经过数据库序列比对,从棕点湍蛙皮肤中得到一个新的促胰岛素释放肽,进一步分析其作用机理和药代动力学,极有可能得到一个新的治疗糖尿病的降糖药物.     

A novel algesic peptide derived from skin secretions of the frog Amolops loloensis.

Several algesic agents including bradykinin and tachykinin have been identified from skin secretions of amphibians. They may act in defensive roles against aggressors. In this study, a novel peptide named Amolos with an amino acid sequence of FLPIVGAKL and isolated from skin secretion of the frog Amolops loloensis, is shown to strengthen nociceptive responses induced by inflammatory factors and strongly inhibit the contraction of isolated ileum. A synthetic peptide based on the sequence obtained showed characterization data identical to those of the isolated material, confirming its structure. These two types of responses seem to be a part of the defensive functions against predators or aggressors. The current results suggest that pharmacological molecules in amphibian skins not only act as innate defense mechanisms against microorganisms but also exert other defensive physiological functions against other aggressors.     

基于12S和16S rRNA序列的湍蛙属部分物种的系统发育关系

测定了湍蛙属 6个种共 10个种群 ,以及 4个外群种的线粒体 12S和 16SrRNA基因片段 ,比对后有94 0bp序列 ,发现 35 2个变异位点、 186个简约性位点。运用NJ法、MP法、ML法构建了系统关系树 ,各系统树一致表明内群为一单系群 ,分为两组 :第一组中 ,四川湍蛙两种群先聚合 ,再和棕点湍蛙聚为一支 ;第二组中 ,香港湍蛙和戴云湍蛙聚为一支 ,而香港大屿山离岛湍蛙种群首先与华南湍蛙相聚 ,再与武夷湍蛙构成姐妹支。研究结果表明 :香港地区增加 1种湍蛙分布 ;戴云湍蛙是一有效种 ;四川湍蛙的石棉和洪雅种群间遗传差异达到或超过其他种间的分歧水平。     

横断山区四种湍蛙的细胞遗传学研究

通过染色体组型分析,C带(BSG技术)分析及一种简便的Ag-NORs带分析,对四川湍蛙、理县湍蛙、棕点湍蛙和棘皮湍蛙的种间关系、染色体的演化及其性染色体等问题进行了初步探讨。结果表明:(1)四川湍蛙、理县湍蛙和棕点湍蛙之间的亲缘关系较近,而它们与棘皮湍蛙的亲缘关系较远;(2)在近缘种的分化中,染色体结构异染色质的变化和臂间倒位是重要的因素之一,这在小型染色体上表现得尤为突出;(三)四川湍蛙具有在形态上分化很明显的性染色体。C带分析表明,此性染色体主要由常染色质构成,但在其Y染色体的长臂上存在明显的中间C带,推测尚处于性染色体分化的初期阶段。     

云南景东地区四川湍蛙的核型,C—带和Ag—NORs的研究

本文报道了云南景东四川湍蛙的核型：２ｎ＝２６（２０Ｍ＋６ｓＭ），ＮＦ＝５２，５＋８模式，次缢痕以及Ａｇ－ＮＯＲｓ均位于６ｐｉｎｔｅｒ，Ｃ－带阳性分布于全部染色体的着丝点区域和８ｑｉｎｔｅｒ，但６ｐｉｎｔｅｒ１２ｐｔｅｒ和１３ｑｔｅｒ等处间或也显示Ｃ－带正染。没有发现与性别相关的异形染色体。与采自四川宝兴地区的居群作了比较，并讨论了细胞地理学问题。     

A new species of the genus Amolops (Anura: Ranidae) from high-altitude Sichuan,southwestern China,with a discussion on the taxonomic status of Amolops kangtingensis

A new species of the genus Amolops Cope,1865 is described from Xinduqiao,Kangding,Sichuan.It was previously identified as Amolops kangtingensis,which is synonymized to Amolops mantzorum in this study.The new species,Amolops xinduqiao sp.nov.,is distinguished from all other congeners by the following combination of characters:(1) medium body size,adult males SVL 41.2-47.5 mm (n=15,average 43.9 mm),adult females SVL 48.5-56.6 mm (n=15,average 52.5 mm);(2) head length equal to width or slightly wider than long;(3) tympanum small,but distinct;(4) vomerine teeth in two tiny rows,separated by a space about one vomerine teeth row;(5) bony projections on lower jaw absent;(6) dorsolateral folds usually absent;(7) tarsal folds or glands on tarsus absent;(8) circummarginal groove on disc of finger Ⅰ absent;(9) tibiotarsal articulation reaching nostril or beyond;(10) webs of toe Ⅳ reaching to distal articulation,other toes fully webbed to disc;and (11) vocal sac absent in males.     

景东湍蛙抗菌肽jindongenin-1d的分析和活性测定

新型抗菌肽研究有助于解决细菌对抗生素的耐药性问题。本研究用SMART技术构建了景东湍蛙Amolops jingdongensis皮肤的全长cDNA文库。通过单克隆和测序获得一个抗菌肽cDNA序列,序列比对结果表明其属于jindongenin-1家族,命名为jindongenin-1d。其cDNA序列全长321bp,编码含66个氨基酸残基的多肽。该多肽包括1个信号肽和1个前肽序列。成熟jindongenin-1d多肽包含24个氨基酸残基,理论分子量为2 709.38,等电点为9.24。对人工合成的jindongenin-1d蛋白进行了抗菌和溶血活性分析,结果表明jindongenin-1d对所选的革兰氏阴性菌、革兰氏阳性菌和真菌均有显著抑制作用,同时有弱溶血活性。本研究结果有助于进一步了解两栖动物皮肤分泌物活性物质的多态性和新型抗感染药物的设计。     

湖南壶瓶山国家级自然保护区华南湍蛙种群监测

2004年5～11月,在湖南壶瓶山国家级自然保护区,采用标志一重捕法对华南湍蛙种群进行了生态监测,结果发现,华南湍蛙出蛰以后种群开始增加,繁殖期过后种群开始下降。在壶瓶山当气温低于10℃以下时,华南湍蛙就开始进入冬眠期。种群的存活率从出蛰到再次冬眠期间基本上呈直线下降,在8月之前种群中均有新添加的数量,以后未发现。在整个过程中,雌雄性比一般大于1,在繁殖季节基本接近于1。     

同一噪音环境下武夷湍蛙与凹耳蛙的求偶鸣声特征比较及其适应策略

为了解同域分布的两种无尾两栖类动物武夷湍蛙(Amolops wuyiensis)和凹耳蛙(Odorrana tormotus)在高噪音环境下的求偶鸣声特征及其适应策略,该研究利用超声录音设备录制并分析了繁殖季节武夷湍蛙和凹耳蛙雄性个体在同一噪音环境下的求偶鸣声。结果显示,繁殖期武夷湍蛙在不同时段均能发出3~6个音节数不等的单一鸣声,每个音节由2~10个声脉冲组成,鸣声平均持续时间为2198.20ms,主频为2231.90Hz,信噪声强差为33.00dB,且鸣声不含超声组分,不具备超声通讯的基础。凹耳蛙在每天的18:00—21:00有集中鸣叫行为,鸣声平均持续时间为331.80ms,主频为6665.50Hz,信噪声强差为37.00dB,且鸣声谐波包含超声组分,与前人描述一致。经进一步分析发现,武夷湍蛙和凹耳蛙的鸣声主频和声强均高于背景噪音,噪音不会对其鸣声产生掩蔽作用。通过比较分析得知,武夷湍蛙鸣声主频率<凹耳蛙,推测其声信号传播距离相对后者更远,该蛙在噪音环境下有可能通过调整自身的发声策略(即采用多音节鸣叫声、增加鸣叫时长和鸣叫频次等)来完成种内通讯,并通过改变鸣声时长来体现雄性自身的品质,以便提高对雌性的吸引力。而相同噪音环境下的凹耳蛙则可能采用较为节约能量的方式提高声信号频率的通讯策略,完成种内竞争和交流。     

Characterization of diverse antimicrobial peptides in skin secretions of Chungan torrent frog Amolops chunganensis

We have cloned, synthesized, and characterized 11 novel antimicrobial peptides from a skin derived cDNA library of the Chungan torrent frog, Amolops chunganensis. Seven of the 11 antimicrobial peptides were present in authentic A. chunganensis skin secretions. Sequence analysis indicated that the 11 peptides belonged to the temporin, esculentin-2, palustrin-2, brevinin-1, and brevinin-2 families. The peptides displayed potent antimicrobial activities against several strains of microorganisms. One peptide, brevinin-1CG5, demonstrated antimicrobial activity against all tested Gram-positive and Gram-negative bacteria and fungi, and showed high antimicrobial potency (MIC = 0.6 μM) against Gram-positive bacterium Rhodococcus rhodochrous. Some peptides also demonstrated weak hemolytic activity against human erythrocytes in vitro. Phylogenetic analysis based on the amino acid sequences of brevinin-1, brevinin-2, and esculentin-2 peptides from family Ranidae confirmed that the current taxonomic status of A. chunganensis is correct.