Enzyme-linked immunosorbent assay of changes in serum levels of growth hormone (cGH) in common carps (Cyprinus carpio)

The aim of the present study was to purify the common native carp growth hormone (ncGH), produce monoclonal antibodies (mAbs) to common native carp growth hormone (ncGH), and further enhance the sensitivity of enzyme-linked immunosorbent assays (ELISA) for ncGH. Additionally, we investigated changes in serum ncGH levels in carps raised in different environmental conditions. The recombinant grass carp (Ctenopharyngodon idella) growth hormone was purified and used as antigen to immunize the rabbit. The natural ncGH was isolated from the pituitaries of common carp. SDS-PAGE and Western blot utilizing the polyclonal anti-rgcGH antibody confirmed the purification of ncGH from pituitaries. Purified ncGH was then used as an immunogen in the B lymphocyte hybridoma technique. A total of 14 hybridoma cell lines (FMU-cGH 1-14) were established that were able to stably secrete mAbs against ncGH. Among them, eight clones (FMU-cGH1-6, 12 and 13) were successfully used for Western blot while nine clones (FMU-cGH 1-7, 9 and 10) were used in fluorescent staining and immunohistochemistry. Epitope mapping by competitive ELISA demonstrated that these mAbs recognized five different epitopes. A sensitive sandwich ELISA for detection of ncGH was developed using FMU-cGH12 as the coating mAb and FMU-cGH6 as the enzyme labeled mAb. This detection system was found to be highly stable and sensitive, with detection levels of 70 pg/mL. Additionally, we found that serum ncGH levels in restricted food group and in the net cage group increased 6.9-and 5.8-fold, respectively, when compared to controls, demonstrating differences in the GH stress response in common carp under different living conditions.

Development of novel EST-SSR markers in common carp by data mining from public EST sequences

Expressed sequence tags (ESTs) are a source for microsatellite development. In the present study, EST-derived microsatelltes (EST-SSRs) were generated and characterized in the common carp (Cyprinus carpio) by data mining from updated public EST databases and by subsequent testing for polymorphism. About 5.5% (555) of 10,088 ESTs contain repeat motifs of various types and lengths with CA being the most abundant dinucleotide one. Out of the 60 EST-SSRs for which PCR primers were designed, 25 loci showed polymorphism in a common carp population with the alleles per locus ranging from 3 to 17 (mean 7). The observed (H-O) and expected (HE) heterozygosities of these EST-SSRs were 0.13-1.00 and 0.12-0.91, respectively. Six EST-SSR loci significantly deviated from the Hardy-Weinberg equilibrium (HWE) expectation, and the remaining 19 loci were in HWE. Of the 60 primer sets, the rates of polymorphic EST-SSRs were 42% in common carp, 17% in crucian carp (Carassius auratus), and 5% in silver carp (Hypophthalmichthys molitrix), respectively. These new EST-SSR markers would provide sufficient polymorphism for population genetic studies and genome mapping of the common carp and its closely related fishes. (c) 2007 Published by Elsevier B.V.

The acute effects of microcystin LR on the transcription of nine glutathione S-transferase genes in common carp Cyprinus carpio L.

The glutathione S-transferases play important roles in the detoxification of microcystin. In this experiment, nine glutathione S-transferase genes including cytosolic GSTs (rho, mu, theta, alpha and pi), mitochondrial GST (kappa) and microsomal GSTs (mGST1, mGST2 and mGST3) were cloned from common carp Cyprinus carpio. The mRNA abundance of each carp GST isoform in liver was analyzed by real time PCR. The relative changes after stimulation with microcystin LR were also analyzed: increased levels of transcription of GST alpha, rho and mGST3 isoforms were detected at 6 h post stimulation; the transcription of mu, theta and mGST2 isoforms were relatively stable; and all the GST isoforms except GST kappa and rho recovered to original levels compared with controls at 72 h. It is suggested that MC-LR showed different effects on the transcription of nine carp GST isoforms. (c) 2006 Elsevier B.V. All rights reserved.

Cloning and expression analysis in mature individuals of two chicken type-II GnRH (cGnRH-II) genes in common carp (Cyprinus carpio)

Gonadotropin-releasing hormone (GnRH) is a conservative neurodecapeptide family, which plays a crucial role in regulating the gonad development and in controlling the final sexual maturation in vertebrate. Two differing cGnRH-II cDNAs of common carp, namely cGnRH-II cDNA1 and cDNA2, were firstly cloned from the brain by rapid amplification of cDNA end (RACE) and reverse transcription- polymerase chain reaction (RT-PCR). The length of cGnRH-II cDNA1 and cDNA2 was 622 and 578 base pairs (bp), respectively. The cGnRH-II precursors encoded by two cDNAs consisted of 86 amino acids, including a signal peptide, cGnRH-II decapeptide and a GnRH-associated peptide (GAP) linked by a Gly-Lys-Arg proteolytic site. The results of intron trapping and Southern blot showed that two differing cGnRH-II genes in common carp genome were further identified, and that two genes might exist as a single copy. The multi-gene coding of common carp cGnRH-II gene offered novel evidence for gene duplication hypothesis. Using semi-quantitative RT-PCR, expression and relative expression levels of cGnRH-II genes were detected in five dissected brain regions, pituitary and gonad of common carp. With the exception of no mRNA2 in ovary, two cGnRH-II genes could be expressed in all the detected tissues. However, expression levels showed an apparent difference in different brain regions, pituitary and gonad. According to the expression characterization of cGnRH-II genes in brain areas, it was presumed that cGnRH-II might mainly work as the neurotransmitter and neuromodulator and also operate in the regulation for the GnRH releasing. Then, the expression of cGnRH-II genes in pituitary and gonad suggested that cGnRH-II might act as the autocrine or paracrine regulator.

Growth hormone gene transfer in common carp

The first successful case of transgenic fish was achieved in 1984. It is in a model system that the integration and expression of recombinant human growth hormone (hGH) in host red common carp (Cyprinus carpio, red var.) have been thoroughly studied. Recently, the integration sites have been recovered and characterized. Compared with non-transgenic peers, hGH-transgenic fish are prior in dietary utilization and growth performance. In view of bio-safety and bio-ethics, an "all-fish" construct CAgcGH, grass carp growth hormone fused with common carp P-actin promoter, has been generated and transferred into Yellow River carp (C carpio, local strain in Yellow River) fertilized eggs. Under middle-scale trial, CAgcGH-transgenics show higher growth rate and food conversion efficiency than the controls, which is consistent to laboratory findings. To avoid the potential impact of transgenic fish on the environment, a sterile strain of transgenic triploid fish has been successfully produced. The "all-fish" transgenic common carp is also approved safe enough as daily food, according to a test based on the pathological principles of new medicines issued by the Ministry of Health of China. The "all-fish" transgenic common carp with growth enhancement is now ready for market, but looking for governmental authorization. (C) 2003 Editions scientifiques et medicales Elsevier SAS and Ifremer/IRD/Inra/Cemagref. All rights reserved.

Transgene for growth hormone in common carp (Cyprinus carpio L.) promotes thymus development

The transgenic carp were produced by micro-injection of CAgcGHc into the fertilized eggs. Observation of the thymus development between the transgenics and non-transgenic controls was carried out. The thymus of one-year-old transgenics F1 showed a great increase in both size and weight. The unilateral thymus of the transgenics weighed from 190 to 295 mg with average 218.6 mg, whereas the unilateral thymus of the controls weighed 20-81 mg with average 42.5 mg; i.e. the thymus weight in the transgenics was 5.14 fold over that in the controls. The index of thymus/body weight in the transgenics was 2.97 fold over the controls. Light microscopy observation indicated that the thymus of the transgenics; well developed with the thickened outer region and compactly arranged thymocytes, while the thymus in the controls were degenerating with the thinned outer region, scattered thymocytes and groups of fatty cells. Further analysis with the electron microscopy revealed that pro-liferous cells in the transgenics; were mainly small lymphocytes and no pathological changes were found. The results confirmed that the "All-fish" GH-transgene promotes thymus development and thymocyte proliferation, and retards thymus degeneration. The study has laid a foundation for further analysis of the immunobiological function in GH-transgenic carp.

Effects of the “all-fish” GH (growth hormone) transgene expression on resistance to Ichthyophthirius multifiliis infections in common carp, Cyprinus carpio L.

A study was undertaken on the susceptibility of the F-4 generation of "all-fish" growth hormone transgenic carp, Cyprinus carpio L, against Ichthyophthirius multifiliis infections. When 1-year old, transgenic carp, with non-transgenic carp and non-manipulated carp (controls) were split into three batches, and experimental infections were performed throughout the 3-month period. All 72 fish were successfully infected. It was shown that there was a significant difference (P<0.01) on infection level between transgenics and non-transgenics, and transgenics and controls. It possibly resulted from transgenics that had stronger non-specific immune functions. In addition, fish surface area affected significantly infection level (P<0.001). Carp with larger surface area harboured more parasites for each type of fish, but transgenic with larger surface area than non-transgenics and controls (P<0.01), loaded fewer parasites than others. Besides, the time of infection also greatly influenced (P<0.001) infection level. Results showed that there was a significant decline in parasite infectivity through October to November (P<0.001). It was likely to suggest that there existed senescence resulted in failure of any I. multifiliis isolate maintenance. Significant difference in infectivity between isolate G from grass carp and isolate H from gold fish suggested that different parasite strains may exist. (C) 2009 Elsevier B.V. All rights reserved.

Identification and characterization of a novel splice variant of gonadotropin alpha subunit in the common carp Cyprinus carpio

In this study, an alternative splicing transcript GtH-alpha 291 was identified by RT-PCR, which is 291 nt and exists not only in the pituitary but also in the ovary in common carp Cyprinus carpio. The analysis of GtH-alpha 291 amino acid sequence by the SignalP server predicted that the 'missing segment' might characterize as a signal peptide. In the secretion experiment, GtH-alpha 357 subunit could be secreted out of HeLa cells while GtH-alpha 291 could not, which confirmed the prediction. Co-immunoprecipitation assay proved that GtH-alpha 291 subunit is able to interact with both FSH-beta and LH-beta as GtH-alpha 357 does. This is the first report concerning an alternative splicing transcript of a GtH alpha subunit. Further studies are necessary to elucidate the specific role of this variant in the regulation of gonadal development and sexual maturation. (c) 2007 The Authors.

Responses of antioxidant systems in the hepatocytes of common carp (Cyprinus carpio L.) to the toxicity of microcystin-LR

The freshwater, bloom-forming cyanobacterium (blue-green alga) Microcystis aeruginosa produces a peptide hepatotoxin, which causes the damage of animal liver. Recently, toxic Microcystis blooms frequently occur in the eutrophic Dianchi Lake (300 km(2) and located in the South-Westem of China). Microcystin-LR from Microcystis in Dianchi was isolated and purified by high performance liquid chromatography (HPLC) and its toxicity to mouse and fish liver was studied (Li et al., 2001). In this study, six biochemical parameters (reactive oxygen species, glutathione, superoxide dismutase, catalase, glutathione peroxide and glutathione S-transferase) were determined in common carp hepatocytes when the cells were exposed to 10 mug microcystin-LR per litre. The results showed that reactive oxygen species (ROS) contents increased by more than one-time compared with the control after 6 h exposure to the toxin. In contrast, glutathione (GSH) levels in the hepatocytes exposed to microcystin-LR decreased by 47% compared with the control. The activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxide (GSH-Px) increased significantly after 6 h exposure to microcystin-LR, but glutathione S-transferase (GST) activity showed no difference from the control. These results suggested that the toxicity of microcystin-LR caused the increase of ROS contents and the depletion of GSH in hepatocytes exposed to the toxin and these changes led to oxidant shock in hepatocytes. Increases of SOD, CAT and GSH-Px activities revealed that these three kinds of antioxidant enzymes might play important roles in eliminating the excessive ROS. This paper also examined the possible toxicity mechanism of microcystin-LR on the fish hepatocytes and the results were similar to those with mouse hepatocytes. (C) 2003 Elsevier Science Ltd. All rights reserved.

Feasibility of biodegradable PLGA common bile duct stents: An in vitro and in vivo study

The current study investigates the feasibility of using a biodegradable polymeric stent in common bile duct (CBD) repair and reconstruction. Here, poly(l-lactide-co-glycolide) (PLGA, molar ratio LA/GA = 80/20) was processed into a circular tube- and dumbbell-shaped specimens to determine the in vitro degradation behavior in bile. The morphology, weight loss, and molecular weight changes were then investigated in conjunction with evaluations of the mechanical properties of the specimen. Circular tube-shaped PLGA stents with X-ray opacity were subsequently used in common bile duct exploration (CBDE) and primary suturing in canine models. Next, X-ray images of CBD stents in vivo were compared and levels of serum liver enzymes and a histological analysis were conducted after stent transplantation. The results showed that the PLGA stents exhibited the required biomedical properties and spontaneously disappeared from CBDs in 4-5 weeks. The degradation period and function match the requirements in repair and reconstruction of CBDs to support the duct, guide bile drainage, and reduce T-tube-related complications.

Dissection of rye B chromosomes, and nondisjunction properties of the dissected segments in a common wheat background

The rye B chromosome is a supernumerary chromosome that increases in number in its host by directed postmeiotic drive. Two types of rye B chromosomes that had been introduced into common wheat were dissected into separate segments by the gametocidal system to produce a number of rearranged B chromosomes, such as telosomes, terminal deletions and translocations with wheat chromosomes. A total of 13 dissected B chromosomes were isolated in common wheat, and were investigated for their nondisjunction. properties. Rearranged B chromosomes, separated from their B-specific repetitive sequences on the distal part of the long arm, did not undergo nondisjunction, and neither did a translocated wheat chromosome carrying a long-arm distal segment containing the B-specific repetitive sequences. However, such rearranged B chromosomes, missing their B-specific sequences could undergo nondisjunction when they coexisted with the standard B chromosome or a wheat chromosome carrying the B-specific sequences. Deficiencies of the short arm did not completely abolish the nondisjunction properties of the B chromosome, but did reduce the frequency of nondisjunction. These results confirmed previous suggestions that the directed nondisjunction of the rye B chromosome is controlled by two elements, pericentromeric sticking sites and a trans-acting element carried at the distal region of the long arm of the B chromosome. Additionally, it is now shown that the distal region of the long arm of the B chromosome which provides this function is that which carries the B-specific repetitive sequences.

Consumption of fa cai Nostoc soup: A Potential for BMAA exposure from Nostoc cyanobacteria in China?

Grown in arid regions of western China the cyanobacterium Nostoc flagelliforme - called fa cai in Mandarin and fat choy in Cantonese - is wild-harvested and used to make soup consumed during New Year's celebrations. High prices, up to $125 USD/kg, led to overharvesting in Inner Mongolia, Ningxia, Gansu, Qinghai, and Xinjiang. Degradation of arid ecosystems, desertification, and conflicts between Nostoc harvesters and Mongol herdsman concerned the Chinese environmental authorities, leading to a government ban of Nostoc commerce. This ban stimulated increased marketing of a substitute made from starch. We analysed samples purchased throughout China as well as in Chinese markets in the United States and the United Kingdom. Some were counterfeits consisting of dyed starch noodles. A few samples from California contained Nostoc flagelliforme but were adulterated with starch noodles. Other samples, including those from the United Kingdom, consisted of pure Nostoc flagelliforme. A recent survey of markets in Cheng Du showed no real Nostoc flagelliforme to be marketed. Real and artificial fa cai differ in the presence of beta-N-methylamino-L-alanine (BMAA). Given its status as a high-priced luxury food, the government ban on collection and marketing, and the replacement of real fa cai with starch substitutes consumed only on special occasions, it is anticipated that dietary exposure to BMAA from fa cai will be reduced in the future in China.

Simultaneous determination of microcystin contaminations in various vertebrates (fish, turtle, duck and water bird) from a large eutrophic Chinese lake, Lake Taihu, with toxic Microcystis blooms

This is the first to conduct Simultaneous determination of microcystin (MC) contaminations in multi-groups of vertebrates (fish, turtle, duck and water bird) from Lake Taihu with Microcystis blooms. MCs (-RR, -YR, -LR) in Microcystis scum was 328 mu g g(-1) DW. MCs reached 235 mu g g(-1) DW in intestinal contents of phytoplanktivorous silver carp, but never exceeded 0.1 mu g g(-1) DW in intestinal contents of other animals. The highest MC content in liver of fish was in Carassius auratus (150 ng g(-1) DW), followed by silver carp and Culter ilishaeformis, whereas the lowest was in common carp (3 ng g(-1) DW). In livers of turtle, duck and water bird, MC content ranged from 18 to 30 ng g(-1) DW. High MC level was found in the gonad, egg yolk and egg white of Nycticorax nycticorax and Anas platyrhynchos, suggesting the potential effect of MCs on water bird and duck embryos. High MC contents were identified for the first time in the spleens of N. nycticorax and A. platyrhynchos (6.850 and 9.462 ng g(-1) DW, respectively), indicating a different organotropism of MCs in birds. Lakes with deaths of turtles or water birds in the literatures had a considerably higher MC content in both cyanobacteria and wildlife than Lake Taihu, indicating that toxicity of cyanobacteria may determine accumulation level of MCs and consequently fates of aquatic wildlife. (C) 2009 Elsevier B.V. All rights reserved.

Transfer, distribution and bioaccumulation of microcystins in the aquatic food web in Lake Taihu, China, with potential risks to human health

In this paper, accumulation and distribution of microcystins (MCs) was examined monthly in six species of fish with different trophic levels in Meiliang Bay, Lake Taihu, China, from June to November 2005, Microcystins were analyzed by liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS). Average recoveries of spiked fish samples were 67.7% for MC-RR, 85.3% for MC-YR, and 88.6% for MC-LR. The MCs (MC-RR+MC-YR+MC-LR) concentration in liver and gut content was highest in phytoplanktivorous fish, followed by omnivorous fish, and was lowest in carnivorous fish; while MCs concentration in muscle was highest in omnivorous fish, followed by phytoplanktivorous fish, and was lowest in carnivorous fish. This is the first study reporting MCs accumulation in the gonad of fish in field. The main uptake of MC-YR in fish seems to be through the gills from the dissolved MCs. The WHO limit for tolerable daily intake was exceeded only in common carp muscle. (C) 2008 Elsevier B.V. All rights reserved.