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.

Cloning of novel bombesin precursor cDNAs from skin of Bombina maxima

Amphibian skin is a rich resource of bioactive peptides like proline-rich bombesin from frog Bombina maxima. A novel cDNA clone encoding a precursor protein that comprises proline-rich bombesin and a novel peptide, designated as bombestatin, was isolated from a skin cDNA library of B. maxima. The predicted primary structure of the novel peptide is WEVLLNVALIRLELLSCRSSKDQDQKESCGMHSW, in which two cysteines form a disulfide bond. A BLAST search of databases did not detect sequences with significant similarity. Bombestatin possesses dose-dependent contractile activity on rat stomach strips. The differences between cDNAs encoding PR-bombesin plus bombestatin and PR-bombesin alone are due to fragment insertions located in 3'-coding region and 3'-untranslational region, respectively. (c) 2005 Elsevier B.V. All rights reserved.

A new species of Genus Triplophysa (Nemacheilinae: Balitoridae), Triplophysa longipectoralis sp nov, from Guangxi, China

A new species, Triplophysa longipectoralis, is described from Liujiang River, Guangxi, China. The new species is distinguished from other species of Triplophysa by the following combination of characters: pectoral fin highly developed, reaching beyond pel

Communities of gastrointestinal helminths of fish in historically connected habitats: habitat fragmentation effect in a carnivorous catfish Pelteobagrus fulvidraco from seven lakes in flood plain of the Yangtze River, China

Background: Habitat fragmentation may result in the reduction of diversity of parasite communities by affecting population size and dispersal pattern of species. In the flood plain of the Yangtze River in China, many lakes, which were once connected with the river, have become isolated since the 1950s from the river by the construction of dams and sluices, with many larger lakes subdivided into smaller ones by road embankments. These artificial barriers have inevitably obstructed the migration of fish between the river and lakes and also among lakes. In this study, the gastrointestinal helminth communities were investigated in a carnivorous fish, the yellowhead catfish Pelteobagrus fulvidraco, from two connected and five isolated lakes in the flood plain in order to detect the effect of lake fragmentation on the parasite communities. Results: A total of 11 species of helminths were recorded in the stomach and intestine of P. fulvidraco from seven lakes, including two lakes connected with the Yangtze River, i.e. Poyang and Dongting lakes, and five isolated lakes, i.e. Honghu, Liangzi, Tangxun, Niushan and Baoan lakes. Mean helminth individuals and diversity of helminth communities in Honghu and Dongting lakes was lower than in the other five lakes. The nematode Procamallanus fulvidraconis was the dominant species of communities in all the seven lakes. No significant difference in the Shannon-Wiener index was detected between connected lakes (0.48) and isolated lakes (0.50). The similarity of helminth communities between Niushan and Baoan lakes was the highest (0.6708), and the lowest was between Tangxun and Dongting lakes (0.1807). The similarity was low between Dongting and the other lakes, and the similarity decreased with the geographic distance among these lakes. The helminth community in one connected lake, Poyang Lake was clustered with isolated lakes, but the community in Dongting Lake was separated in the tree. Conclusion: The similarity in the helminth communities of this fish in the flood-plain lakes may be attributed to the historical connection of these habitats and to the completion of the life-cycles of this fish as well as the helminth species within the investigated habitats. The diversity and the digenean majority in the helminth communities can be related to the diet of this fish, and to the lacustrine and macrophytic characters of the habitats. The lake isolation from the river had little detectable effect on the helminth communities of the catfish in flood-plain lakes of the Yangtze River. The low similarities in helminth communities between the Dongting Lake and others may just be a reflection of its unique water environment and anthropogenic alterations or fragmentation in this lake.

First description of a novel Weissella species as an opportunistic pathogen for rainbow trout Oncorhynchus mykiss (Walbaum) in China

Six strains of Gram-positive, catalase-negative, non-motile, irregular short rod-shaped Weissella bacteria, with width and length of 0.5-0.6 and 1.2-2.7 mu m were isolated from diseased rainbow trout Oncorhynchus mykiss (Walbaum) in winter of 2007 at a commercial fishery in Jingmen, Hubei province, China. The diseased rainbow trout exhibited hemorrhage in eyes, anal region, intestine and abdomen wall, petechia of liver, some fish with hydrocele in stomach. Six isolates had identical biochemical reactions, phylogenetic analysis of 16S rDNA sequences, amplified ribosomal DNA restriction analysis (ARDRA), enzymatic profile analysis and antimicrobial susceptibility results, indicating as a single clonal outbreak. But all were different from any other validated twelve Weissella species in the term of physiological and biochemical characters. It is indicated that isolates are phylogenetically closer to Weissella halotolerans, Weissella viridescens and Weissella minor on 16S rDNA phylogenetic analysis result, than to W halotolerans and W viridescens on the result of ARDRA study and enzymatic profile analysis. Antimicrobial susceptibility testing was used to scan effective drugs for the therapy of this disease. Experimental infection assays with one isolate were conducted and pathogenicity (by intraperitoneal injection) was demonstrated in rainbow trout O. mykiss (Walbaum) and crucian carp (Carassius auratus gibelio) fingerlings. Because no Weissella was detected in fish feedstuffs and pond water, the source of this pathogen remains unknown, and Weissella isolates were regarded as an opportunistic pathogen for rainbow trout. This is the first report of Weissella strains which can cause disease of cultured fish in the world. (C) 2009 Elsevier B.V. All rights reserved.

Distribution of microcystins in various organs (heart, liver, intestine, gonad, brain, kidney and lung) of Wistar rat via intravenous injection

The distribution of microcystins (MCs) in various tissues of Wistar rats was studied under laboratory conditions. Rats were injected intravenously (i.v.) with extracted MCs at a dose of 80 mu g MC-LRequivalent/kg body weight. MCs concentrations in various tissues were detected at 1, 2. 4, 6, 12 and 24 h post-injection using liquid chromatography-mass spectrometry (LC-MS). The highest concentration of MCs was found in kidney (0.034-0.295 mu g/g dry weight), followed by lung (0.007-0.067 mu g/g dry weight), stomach (0.010-0.058 mu g/g dry weight) and liver (0.003-0.052 mu g/g dry weight). The maximum MCs content in the whole body of rat, 2.9% of the injected dose, was observed at 2 h post-injection. MCs concentration was higher in kidney than in liver during the experiment, and two peaks of MCs concentration (at 2 and 24 h, respectively) were observed in kidney, indicating that MCs can be excreted directly via kidney of rat. Though heart, intestine, spleen, brain, gonad and stomach contained less than 0.2% of injected MCs during the whole experiment stage, the presence of MCs in these tissues represents potential damage to them. (c) 2008 Elsevier Ltd. All Fights reserved.

Triplophysa lixianensis, a new nemacheiline loach species (Pisces : Balitoridae) from the upper Yangtze River drainage in Sichuan Province, South China

Triplophysa lixianensis, a new nemacheiline loach species, is described from the Min Jiang of the upper Yangtze River drainage in Sichuan Province, South China. It can be separated from all other species of Triplophysa by having a unique combination of the following characters: posterior chamber of gas bladder greatly reduced or absent; caudal peduncle columnar with a roughly round cross- section at its beginning; anterior edge of lower jaw completely exposed or uncovered by lower lip; intestine short, forming a zigzag loop below stomach; dorsal- fin origin closer to caudal- fin base than to snout tip; pelvic fin inserted anterior to dorsal- fin origin; snout length 50.6 - 57.5 % of head length; eye diameter 12.3 15.4 % of head length; caudal peduncle length 25.1 - 27.1 % of standard length; anal fin with five branched rays; lower lip greatly furrowed with two thick lateral lobes; and body smooth or scaleless.

Preliminary hazard assessment of polychlorinated biphenyls, polybrominated diphenyl ethers, and polychlorinated dibenzo-p-dioxins and dibenzofurans to Yangtze finless porpoise in Dongting Lake, China

Yangtze finless porpoise (Neophocaena phocaenoides asiaeorientalis), a protected endangered species, is the sole freshwater subspecies of finless porpoise, living only in the middle and lower reaches of the Yangtze River, China, and its appended lakes. Its population has decreased sharply to 1,400 because of human activities, including environmental contamination. In the present study, polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), and polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) were determined in the blubber, liver, kidney, stomach, small intestine, and brains of five individual Yangtze finless porpoises collected from 1998 to 2004. The results showed PCB concentrations ranged from 0.06 to 1.89 mu g/g lipid weight in the organs and consisted mainly of penta-, hexa-. and decachlorinated biphenyls. The PBDE concentrations were between 5.32 and 72.76 ng/g lipid weight. Tetra-, penta-, and hexabrominated diphenyl ethers were the major homologues. The PCDD/F concentrations ranged from 65 to 1,563 pg/g lipid weight, and their predominant homologues were penta- and hexachlorinated dibenzofurans and hepta- and octachlorinated dibenzo-p-dioxins. The hazard quotients (HQs) based on toxic equivalency were determined to be greater than one in all individuals for PCBs, for PCDD/Fs, and for PCBs and PCDD/Fs In addition, HQs would be higher if PBDEs were included. The results suggest that reduction of environmental contamination may contribute greatly to protecting this highly endangered species.

Size-related shifts in reliance on benthic and pelagic food webs by lake anchovy

This paper reports large variations in stable carbon and nitrogen isotope ratios of lake anchovy (Coilia ectenes taihuensis) from Lake Chaohu, China. The lake anchovy exhibited a significant C-13- and N-15- enrichment in relation to increasing fish length, and the isotopic compositions of small lake anchovy (<= 130 mm) were significantly more enriched than those of large lake anchovy (> 130 mm). The significant differences in the isotopic compositions of small and large lake anchovy suggested that their assimilated diets differed over a period of time and reflected the size-related diet shift of this fish. Bellamya aeruginosa and Corbicula fluminea were used to establish the baseline carbon signal of benthic and pelagic food webs, and these data were used to parameterize a 2-source mixing model to estimate in consumers the contribution of carbon derived from benthic versus pelagic food webs. Mixing models showed that small lake anchovy derived only 37% of their carbon from benthic food web, indicating increased reliance on pelagic prey, whereas benthic prey contributed 71% of large lake anchovy diet, suggesting greater use of benthic sources. These data indicate that there was a change in lake anchovy feeding strategy related to their size, suggesting a role in dynamic coupling between pelagic and benthic food chains. The trophic position of small lake anchovy averaged 3.0, indicating a zooplankton-based diet, compared with 3.6 in large lake anchovy, indicative of an increase in piscivorous diet. Overlap in the isotopic compositions of small and large lake anchovy probably indicated that these fish occasionally shared common diets, as suggested by stomach content studies, and/or resulted from the differences in the rate of isotopic turnover depending on differences in growth rate and metabolic turnover between small and large anchovy during diet shift from pelagic to benthic food webs. This study presents the contributions of benthic and pelagic food webs supporting lake anchovy and indicates that the intraspecific isotopic dynamic should be considered when applying stable isotope analyses to infer trophic interactions in aquatic ecosystems.

Mercury concentrations in Yangtze finless porpoises (Neophocaena phocaenoides asiaeorientalis) from Eastern Dongting Lake, China

Mercury concentrations in some key tissues (liver, kidney, small intestine, stomach, blubber and brain) of five Yangtze finless porpoises (Neophocaena phocaenoides asiaeorientalis), accidentally died in the Eastern Dongting Lake of China between 1998 and 2004, were investigated using Atomic Fluorescent Spectrometry (AFS). Total mercury (T-Hg) concentrations ranged between 0.17 to 181 mu g g wet weight. and varied significantly in different tissues. Liver is the most preferable target organ for mercury accumulation. although the highest T-Hg concentrations were not found exclusively in liver, but also in kidney and small intestine in some individuals. Moreover, it was demonstrated that mercury concentrations increased positively with age of the animals. However, the highest T-Hg concentrations in liver and kidney were both found in a 2-month-old calf, which implied that the transfer efficiency of mercury from mothers to babies in Yangtze finless porpoise is remarkable. And what is more important, it seems that the Yangtze finless porpoise in Eastern Donating Lake had much higher T-Hg levels than those reported for other Phocoenidae species. To be noticed, the T-Hg was accumulated tremendously from aquatic environment to the Yangtze finless porpoise's liver, reaching a bioaccumulation factor (BAF) of 4.3x10(5) in the Eastern Donating Lake ecosystem.

Variation in stable isotope signatures of seston and a zooplanktivorous fish in a eutrophic Chinese lake

Temporal and spatial changes in delta(13)C and delta(15)N of seston (mainly phytoplankton) and isotopic relationship between seston and the lake anchovy (Coilia ectenes) were studied in the large eutrophic freshwater Lake Chaohu in China. Much of the spatial and temporal variation in delta(13)C of lake anchovies was explained by variation in seston, indicating a strong link between pelagic primary production and higher order consumers. Because the lake is shallow, there were no significant differences in delta(13)C and delta(15)N of seston between surface and overlying waters. Spatially, the relatively high delta(13)C and delta(15)N of seston in the western part of the lake might be due to high levels of anthropogenically derived N and C introduced from the surrounding cities through sewage drainage systems. The trophic position of the lake anchovy in the food web of Lake Chaohu was estimated to be 2.9-4.1 (3.5 +/- 0.4), which agrees well with the previous stomach content analysis suggesting that the lake anchovy fed both on zooplankton and small planktivorous fishes.

5-氟尿嘧啶-β-环糊精结肠靶向前体药物的研究

5-氟尿嘧啶(5-Fluorouracil， 5-FU)是一种抗代谢药物，广泛用于临床治疗结直肠癌、胃癌、乳腺癌等多种癌症，但其首过代谢显著、亲脂性较低，选择性差、毒副作用大。为克服这些缺点人们对5-FU进行了大量的修饰工作，包括小分子修饰以及与各种载体形成微球、微囊、纳米粒、共价前药等。 环糊精(Cyclodextrin，简称CD)，可被结肠中的糖苷酶特异性地降解成小分子糖，而胃和小肠中由于缺乏相应的酶而使环糊精不被降解，这一特性在结肠药物的靶向输送及释放中有重要应用价值。环糊精中含有丰富的羟基，易进行化学修饰，将药物与环糊精通过共价键结合制成前药，使其在胃和小肠中不降解，而在盲结肠中被特异性的酶降解释出药物，达到结肠靶向释药的目的。研究表明，环糊精作为一种前药载体为结肠靶向释药和缓释、控释系统提供了一种有效的手段。 本工作选择5-氟尿嘧啶为模型药物、β-环糊精作为载体，通过中间体5-FU羧酸衍生物的制备及其与β-环糊精的偶联，合成了系列5-FU-β-CD前体药物，并利用紫外、红外、质谱、核磁、元素分析、热分析等手段对其进行结构表征。同时，还研究了前体药物的体外释药性质。具体内容包括： 1. 含有羧基的5-FU衍生物中间体的合成：(5-氟尿嘧啶-1-基)-乙酸(FUAC)、3-(5-氟尿嘧啶-1-基)-丙酸(FUPC)、5-(5-氟尿嘧啶-1-基)-戊酸(FUVC)的合成。 2. 中间体5-FU的羧酸衍生物与β-CD的偶联：分别通过以6-OTs-β-CD为中间体的取代法和活化酯法，合成了第一面取代和第二面取代的5-FU-β-CD大分子前体药物。在二面取代的前体药物制备中，通过改变原料的比例，合成了系列不同取代度(DS)的2-[(5-氟尿嘧啶-1-基)-乙酰基] -β-环糊精结合物。 3. 对上述前体药物进行体外释放研究：分别考察了前体药物在不同pH缓冲溶液中的水解行为及其在小鼠胃肠道人工体液中的酶解行为，并通过UV-Vis及HPLC对前体药物释放情况进行检测分析。 5-Fluorouracil(5-Fu), commonly known as a broad-spectrum antineoplastic drug, has been widely used in the treatment of various kinds of cancer including colon cancer for 40 years. However, this antitumor agent exhibits serious adverse effects, such as their marrow toxicity, gastrointestinal reaction and low selectivity in their clinical use. In order to improve its antitumor activity and reduce its toxicity, the compound was modified in various ways, including the formation of conjugated prodrugs with kinds of carrier, microsphere and nanoparticles etc. Cyclodextrins(CDs) are known to be barely capable of being hydrolyzed and only slightly absorbed in passing through the stomach and small intestine; however they are fermented into small saccharides by colonic microflora and thus absorbed as small saccharides in the large intestine. This biodegradation property of CDs may be useful as a colon-targeting carrier, and thus CD prodrugs may serve as a source of site-specific delivery of drugs to colon. It was demonstrated that prodrugs of CDs can provide a versatile means for construction of not only colon targeted delivery systems, but also delayed release systems. 5-Fluorouracil was taken as a model drug and β-CD as the carrier in this study. Series prodrugs of 5-FU was prepared through the preparation of reactive 5-FU derivatives containing carboxyl group and coupling to hydroxyl groups of CD. The structures of the conjugates were charactered by using IR, UV–vis, ESI-MS, 1H, 13C-NMR spectra, elemental analyses, and thermal analysis. In vitro hydrolysis behavior in aqueous solution and in rat gastrointestinal tract contents of the conjugates were also investigated. The main content of this dissertation includes following aspects: 1. The preparation of 5-FU derivatives containing carboxyl group: 5-Fluorouracil- acetic acid(FUAC)、3-(5-FU-1)-propionic acid (FUPC)、and 5-(5-FU-1)-valeric acid(FUVC). 2. The coupling of 5-FU derivatives to β-CD: 5-FU was selectively conjugated onto the primary or secondary hydroxyl groups of β-CD through an ester linkage, by the substitution of 6-OTs-β-CD and the activated ester method respectively. For the secondary face conjugation, the degree of substitution(DS) can be controlled by changing the mole ratio of the starting materials(FUAC and β-CD). 3. In vitro release behavior of the conjugates in aqueous solution and in rat gastro- intestinal tract contents of the conjugates were investigated, and the reaction was monitored and analyzed by using UV-Vis and HPLC methods.

Three tetraspanins from Chinese shrimp, Fenneropenaeus chinensis, may play important roles in WSSV infection

Three members of the tetraspanin/TM4SF superfamily were cloned from Chinese shrimp, Fenneropenaeus chinensis. The deduced amino acid sequences of the three proteins have typical motifs of the tetraspanin/TM4SF superfamily. Phylogenetic analysis of the proteins, together with the known tetraspanins of invertebrates and vertebrates, revealed that they belong to different tetraspanin subfamilies: CD9, CD63 and tetraspanin-3. The three cloned genes of CD9, CD63 and tetraspanin-3 showed apparently different tissue distributions. The CD9 gene (FcCD9) was specifically expressed in the hepatopancreas. While for the CD63 gene (FcCD63), the highest expression was detected in nerves, epidermis and heart, with low expression in haemocytes, ovary, gill, hepatopancreas and stomach and no expression in intestine, muscle and lymphoid organ. Compared with FcCD9 and FcCD63, the tetraspanin-3 gene (FcTetraspanin-3) was more broadly expressed and its highest expression was detected in the intestine. Its expression in nerves was lower than in the intestine, but was higher than in other tissues. Expression in haemocytes, ovary and muscle was much lower than in other tissues. The expression profiles of FcCD9, FcCD63 and FcTetraspanin-3 in different tissues, including haemocytes, lymphoid organ and hepatopancreas, were compared by real-time PCR when shrimp were challenged by live white spot syndrome virus (WSSV) and heat-inactivated WSSV. All three tetraspanins were markedly up-regulated in the live WSSV-challenged shrimp tissues. The data suggested that the three cloned members of TM4SF superfamily in Chinese shrimp may play a key role in the route of WSSV infection.

Identification and molecular characterization of a peritrophin-like protein from fleshy prawn (Fenneropenaeus chinensis)

Peritrophin, one of the components of the peritrophic matrix, was first isolated from the intestine of insects. It is thought to protect insects from invasion of microorganisms and to stimulate digestion of food. Peritrophin-like proteins have also been found in crustaceans, as a component of the egg layer. In this study, one fragment of the peritrophin-like gene was obtained from fleshy prawn (Chinese shrimp) (Fenneropenaeus chinensis) by panning the T7 phage display library constructed with the shrimp hemocyte cDNA. The total sequence of the peritrophin cDNA was cloned by modified SMART cDNA and LD-PCR methods. The full cDNA is 1048 bp and the deduced protein is composed of 274 amino acids, including 21 amino acid signal peptide, and four peritrophin A domains and the latter three forming three chitin-binding domains. Similarity analysis results showed that the peritrophin-like protein from F chinensis has significant similarities with peritrophin-like and cortical rod proteins from other shrimp. It was inducing expression in hemocytes, heart, stomach, gut, and gills of the infected shrimp, and constitutive expression in the ovaries. No expression signal was detected in the hepatopancreas of either infected or noninfected shrimp. The recombinant peritrophin-like protein has the activity of binding Gram-negative bacteria and strong binding activity to chitin. Therefore, the bacteria and chitin binding activities of the peritrophin-like protein suggest that it may plays a role in immune defense and other physiological resposes. (c) 2005 Elsevier Ltd. All rights reserved.

Food utilization of adult flatfishes co-occurring in the Bohai Sea of China

Stomach contents were examined of 4527 adult individuals of 12 flatfish species collected during the 1982 - 1983 Bohai Sea Fisheries Resources Investigation. Their food habits, diet diversity, similarity of prey taxa, trophic niche breadth and diet overlap were systematically analysed. Ninety-seven prey species belonging to the Coelenterata, Nemertinea, Polychaeta, Mollusca, Crustacea, Echinodermata, Hemichordata and fish were found and five of them were considered to be principal prey for flatfishes: Alpheus japonicus, Oratosquilla oratoria, Alpheus distinguendus, Loligo japonicus and Crangon affinis. Among the flatfishes, Paralichthys olivaceus was piscivorous, whereas Pseodopleuronectes yokohamae and Pseudopleuronectes herzensteini both had polychaetes and molluscs as their main prey groups. Pleuronichthys cornutus was classified as a polychaete-mollusc eater, with a strong preference for crustaceans. Verasper variegatus, Cynoglossus semilaevis, Eopsetta grigorjewi and Cleisthenes herzensteini ate crustaceans. Kareius bicoloratus was classified as a mollusc-crustacean eater: Cynoglossus abbreviatus, Cynoglossus joyneri and Zebrias zebra were grouped as crustacean-fish eaters. However, Z. zebra also took polychaetes and C. abbreviatus and C. joyneri preyed on some molluscs. Trophic relationships among the flatfishes were complicated, but they occupied distinctive microhabitats in different seasons and selected their specific prey items, which was favourable to the stability of the flatfish community in the Bohai Sea.