Metallothionein-2 gene from the mandarin fish Siniperca chuatsi: cDNA cloning, tissue expression, and immunohistochemical localization

The metallothionein-2 (MT-2) gene was isolated from the mandarin fish, one of the most important industrial aquatic animals in China, by using rapid amplification of cDNA ends (RACE). The deduced amino acid sequence of MT-2 comprised 60 amino acids and showed approximately 62.3% identity to human metallothionein. Its promoter region was amplified by thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR). The MT-2 gene consists of 3 exons and 2 introns, extending approximately 900 bp of genomic sequence. Phylogenetic analysis clearly demonstrated that MT-2 formed a clade with fish metallothionein. The promoter region contained 5 putative metal-regulatory elements (MREs) and 1 TATA box. Real-time quantitative RT-PCR analysis revealed that MT-2 transcripts were significantly increased in the brain and gills and were stable in the muscles, liver, and trunk kidney in Cd2+-stimulated fish. Western blotting analysis demonstrated that the protein of the MT-2 gene was expressed mainly in the gills, liver, heart, trunk kidney, muscle, and intestine; it was weakly detected in the brain and head kidney. Moreover, the MT-2 protein was immunohistochemically detected in the cytoplasm in the liver and trunk kidney. All the above results revealed that the mandarin fish MT-2 would be a useful biomarker for metal pollution. (C) 2008 Published by Elsevier Inc.

Expressional induction of Paralichthys olivaceus cathepsin B gene in response to virus, poly I:C and lipopolysaccharide

Cathepsin B is a lysosomal cysteine protease of the papain-like enzyme family with multiple biological functions. In this study, Paralichthys olivaceus cathepsin B (PoCatB) cDNA was isolated from flounder embryonic cells (FEC) treated with UV-inactivated grass carp hemorrhage virus (GCHV) and subsequently identified as a vitally induced gene. The full length cDNA of PoCatB is 1801 bp encoding 330-amino acids. The deduced protein has high homology to all known cathepsin B proteins, containing an N-terminal signal peptide, cysteine protease active sites, the occluding loop segment and a glycosylation site, all of which are conserved in the cathepsin B family. PoCatB transcription of FEC cells could be induced by turbot (Scophthalmus maximus) rhabdovirus (SMRV), UV-inactivated SMRV, UV-inactivated GCHV, poly I:C or lipopolysaccharide (LPS), and SMRV or poly I:C was revealed to be most effective among the five inducers. In normal flounder, PoCatB mRNA was detectable in all examined tissues. Moreover, SMRV infection could result in significant upregulation of PoCatB mRNA, predominantly in spleen, head kidney, posterior kidney, intestine, gill and muscle with 18.2,10.9, 24.7,12, 31.5 and 18 fold increases at 72 h post-infection respectively. These results provided the first evidence for the transcriptional induction of cathepsin B in fish by virus and LPS, indicating existence of a novel function in viral defense. (C) 2008 Elsevier Ltd. All rights reserved.

Analysis of heavy metals of muscle and intestine tissue in fish - in Banan section of Chongqing from Three Gorges Reservoir, China

The present study was carried out to investigate contamination of heavy metals in 19 fish species from the Banan section of Chongqing in the Three Gorges, Yangtze River. The results showed that the mean concentrations of heavy metals were higher in intestine than muscle, except zinc in upper strata. In the fish inhabiting the upper strata, there were significant differences between mean concentrations of As, Cr, Cu and Hg in muscle and intestine (P <0.05). There were also significant differences between mean concentrations of Cr and Cu in muscle and intestine in the fish inhabiting middle strata. However, significant differences between mean concentrations of As, Cd, Hg, Pb and Zn were measured in fish inhabiting bottom strata in both intestine and muscle tissues (P <0.05). For the fish inhabiting different strata, the concentrations of As, Cd, Cr, Cu, Hg and Ph in muscle and intestine of the fish from bottom strata (BS) were higher than those in both upper strata (US) and middle strata (MS); whereas a higher concentration of Zn was measured in muscle and intestine from fish inhabiting upper strata. Mean metal concentrations were found to be higher in age 11 than those in age I in Coreius heterodon (2- and 1-year odl fish respectively). The overall results indicated that fish muscle in the Banan section were slightly contaminated by heavy metals, but did not exceed Chinese food standards.

Novel fluid grid and voidage calculation techniques for a discrete element model of a 3D cylindrical fluidized bed

A discrete element model (DEM) combined with computational fluid dynamics (CFD) was developed to model particle and fluid behaviour in 3D cylindrical fluidized beds. Novel techniques were developed to (1) keep fluid cells, defined in cylindrical coordinates, at a constant volume in order to ensure the conditions for validity of the volume-averaged fluid equations were satisfied and (2) smoothly and accurately measure voidage in arbitrarily shaped fluid cells. The new technique for calculating voidage was more stable than traditional techniques, also examined in the paper, whilst remaining computationally-effective. The model was validated by quantitative comparison with experimental results from the magnetic resonance imaging of a fluidised bed analysed to give time-averaged particle velocities. Comparisons were also made between theoretical determinations of slug rise velocity in a tall bed. It was concluded that the DEM-CFD model is able to investigate aspects of the underlying physics of fluidisation not readily investigated by experiment. © 2014 The Authors.

Characterization of complete genome sequence of the spring viremia of carp virus isolated from common carp (Cyprinus carpio) in China

The complete genome of spring viraemia of carp virus (SVCV) strain A-1 isolated from cultured common carp (Cyprinus carpio) in China was sequenced and characterized. Reverse transcription-polymerase chain reaction (RT-PCR) derived clones were constructed and the DNA was sequenced. It showed that the entire genome of SVCV A-1 consists of 11,100 nucleotide base pairs, the predicted size of the viral RNA of rhabdoviruses. However, the additional insertions in bp 4633-4676 and bp 4684-4724 of SVCV A-1 were different from the other two published SVCV complete genomes. Five open reading frames (ORFs) of SVCV A-1 were identified and further confirmed by RT-PCR and DNA sequencing of their respective RT-PCR products. The 5 structural proteins encoded by the viral RNA were ordered 3'-N-P-M-G-L-5'. This is the first report of a complete genome sequence of SVCV isolated from cultured carp in China. Phylogenetic analysis indicates that SVCV A-1 is closely related to the members of the genus Vesiculovirus, family Rhabdoviridae.

Toxicity of microcystins in the isolated hepatocytes of common carp (Cyprinus carpio L.)

The toxicity of hepatotoxic microcystins produced mainly by Microcystis aeruginosa in mammals and fishes was well studied in recent years. However, there were scarcely reports in toxic effects of microcystins on isolated hepatocytes of fishes, especially investigation of microcystin-induced apoptosis and/or necrosis in carp hepatocytes. In the present study, the isolated hepatocytes of common carp were exposed to various concentrations of microcystins (0.01, 0.1, 1, 10, 100, 1000 mu g L-1) for 2, 4, 8, 16 and 24 h, respectively, and cytotoxicity of microcystins in the toxin-treated cells was determined. Results of this study showed that cytotoxicity of microcystins on carp hepatocytes was time and dose-dependent, and the approximate LC50 of microcystins in carp hepatocytes was 169.2 mu g L-1. The morphological changes typical of apoptosis, such as blebbing of cell membrane, condensation and fragmentation of cell nucleus were observed in the hepatocytes exposed to microcystins (1, 10 and 100 mu g L-1) using fluorescence and differential interference contrast microscopy. Agarose gel electrophoresis of DNA demonstrated a typical apoptotic "ladder pattern" in microcystin-treated hepatocytes after 16 h of exposure. Results of the present study indicated that the form of cell death in microcystin-treated hepatocytes depend on the exposure dose of toxin. When lower concentration of microcystins (10 and 100 mu g L-1) was used for exposure, carp hepatocytes died in apoptosis while, when higher one used (1000 mu g L-1), they died in the form of necrosis. (C) 2006 Elsevier Inc. All rights reserved.

Subcellular localization and characterization of G protein-coupled receptor homolog from lymphocystis disease virus isolated in China

G protein-coupled receptors (GPCRs) constitute a large superfamily involved in various types of signal transduction pathways, and play an important role in coordinating the activation and migration of leukocytes to sites of infection and inflammation. Viral GPCRs, on the other hand, can help the virus to escape from host immune surveillance and contribute to viral pathogenesis. Lymphocystis disease virus isolated in China (LCDV-C) contains a putative homolog of cellular GPCRs, LCDV-C GPCR. In this paper, LCDV-C GPCR was cloned, and the subcellular localization and characterization of GPCR protein were investigated in fish cells. LCDV-C GPCR encoded a 325-amino acid peptide, containing a typical seven-transmembrane domain characteristic of the chemokine receptors and a conserved DRY motif that is usually essential for receptor activation. Transient transfection of GPCR-EGFP in fathead minnow (FHM) cells and epithelioma papulosum cyprini (EPC) cells indicated that LCDV-C GPCR was expressed abundantly in both the cytoplasm and nucleoplasm. Transient overexpression of GPCR in these two cells cannot induce obvious apoptosis. FHM cells stably expressing GPCR showed enhanced cell proliferation and significant anchorage-independent growth. The effects of GPCR protein on external apoptotic stimuli were examined. Few apoptotic bodies were observed in cells expressing GPCR treated with actinomycin D (ActD). Quantitative analysis of apoptotic cells indicated that a considerable decrease in the apoptotic fraction of cells expressing GPCR, compared with. the control cells, was detected after exposure to ActD and cycloheximide. These data suggest that LCDV-C GPCR may inhibit apoptosis as part of its potential mechanism in mediating cellular transformation.

Hydrodynamics in an expanded bed of large size ion-exchange resin, and natural product adsorption

Successful applications of expanded bed adsorption (EBA) technology have been widely reported in the literature for protein purification. Little has been reported on the recovery of natural products and active components of Chinese herbal preparations using EBA technology. In this study, the hydrodynamic behavior in an expanded bed of cation resin, 001 x 7 Styrene-DVB, was investigated. Ephedrine hydrochloride (EH) was used as a model natural product to test the dynamic binding capacity (DBC) in the expanded bed. EBA of EH directly from a feedstock containing powdered herbs has also been investigated. These particles are different from commercially available expanded bed adsorbents by virtue of their large size (20S to 1030 gm). When the adsorbent bed is expanded to approximately 1.3 to 1.5 times its settled bed height, the axial liquid-phase dispersion coefficient was found to be of the order 10(-5) m(2) s(-1), which falls into the range 1.0 x 10(-6) to 1.0 X 10(-5) m(2) s(-1) observed previously in protein purification. Because of the favorable column efficiency (low axial dispersion coefficient), the recovery yield and purification factor values of EH directly from a feedstock reached 86.5% and 18, respectively. The results suggest that EBA technology holds promise for the recovery of natural products and active components of Chinese herbal preparations.

Parentage determination of an isolated Yangtze finless porpoise population Neophocaena phocaenoides asiaeorientalis in the Yangtze Tian-e-Zhou Baiji National Natural Reserve based on molecular data

Reproductive behaviors are poorly known for the Yangtze finless porpoise Neophocaena phocaenoides asiaeorientalis. In this study, the parentage of an isolated Yangtze finless porpoise population inhabiting the Yangtze Tian-e-Zhou Baiji National Natural Reserve is determined by analysis of microsatellite loci and mitochondrial DNA (mtDNA) control region sequences, and the porpoise's reproductive behaviors are studied. Overall 4 full parentage assignments and additional 3 single parentage assignments were determined for the population of 23 individuals. The analysis shows that their estimated reproductive cycle is shorter than that reported previously and there probably exists an overlapping between gestation and lactation period. The Study also shows that the female does not show fidelity to a particular male for breeding and vice versa, the oldest males did not monopolize mating and the dominance rank could not be so strict for the porpoise society. Moreover, the porpoise's mating pattern and relatedness among candidate parents are discussed here. These results provide important information for making guidelines of management and conservation for this protected population.

Effects of nitrogen forms on the production of cyanobacterial toxin microcystin-LR by an isolated Microcystis aeruginosa

A cyanobacterial strain, which produced high content of microcystin-LR (MC-LR) but no rnicrocystin-RR (MC-RR), was isolated from the hypertrophic Dianchi Lake in China and identified as Microcystis aeruginosa DC-1. Effects of nitrogen containing chemicals and trace elements on the growth and the production of MC-LR by this strain were Studied. In the presence of bicine, compared with urea and ammonium, nitrate greatly promoted the growth and the production of MC-LR. However, leucine and arginine, which were the constitutional components in the molecular structure of MC-LR or RR, inhibited the production of MC-LR. Iron and silicon up to 10mg/L had little effects on the growth of M. aeruginosa DC-1, but the production of MC-LR was apparently enhanced. Under all conditions studied here, only MC-LR but no RR was detected within the cells of M. aeruginosa DC-1. Thus, chemical forms of nitrogen, rather than the usually concerned the total nitrogen, Lind trace elements played important roles in the production of MC toxins during cyanobacterial blooms.

Complete genome sequence of lymphocystis disease virus isolated from China

Lymphocystis diseases in fish throughout the world have been extensively described. Here we report the complete genome sequence of lymphocystis disease virus isolated in China (LCDV-C), an LCDV isolated from cultured flounder (Paralichthys olivaceus) with lymphocystis disease in China. The LCDV-C genome is 186,250 bp, with a base composition of 27.25% G+C. Computer-assisted analysis revealed 240 potential open reading frames (ORFs) and 176 nonoverlapping putative viral genes, which encode polypeptides ranging from 40 to 1,193 amino acids. The percent coding density is 67%, and the average length of each ORF is 702 bp. A search of the GenBank database using the 176 individual putative genes revealed 103 homologues to the corresponding ORFs of LCDV-1 and 73 potential genes that were not found in LCDV-1 and other iridoviruses. Among the 73 genes, there are 8 genes that contain conserved domains of cellular genes and 65 novel genes that do not show any significant homology with the sequences in public databases. Although a certain extent of similarity between putative gene products of LCDV-C and corresponding proteins of LCDV-1 was revealed, no colinearity was detected when their ORF arrangements and coding strategies were compared to each other, suggesting that a high degree of genetic rearrangements between them has occurred. And a large number of tandem and overlapping repeated sequences were observed in the LCDV-C genome. The deduced amino acid sequence of the major capsid protein (MCP) presents the highest identity to those of LCDV-1 and other iridoviruses among the LCDV-C gene products. Furthermore, a phylogenetic tree was constructed based on the multiple alignments of nine MCP amino acid sequences. Interestingly, LCDV-C and LCDV-1 were clustered together, but their amino acid identity is much less than that in other clusters. The unexpected levels of divergence between their genomes in size, gene organization, and gene product identity suggest that LCDV-C and LCDV-1 shouldn't belong to a same species and that LCDV-C should be considered a species different from LCDV-1.

Infection and propagation of lymphocystis virus isolated from the cultured flounder Paralichthys olivaceus in grass carp cell lines

The causative agent of lymphocystis disease that frequently occurs in cultured flounder Paralichthys olivaceus in China is lymphocystis virus (LV). In this study, 13 fish cell lines were tested for their susceptibility to LV. Of these, 2 cell lines derived from the freshwater grass carp Ctenopharyngodon idellus proved susceptible to the LV, and 1 cell line, GCO (grass carp ovary), was therefore used to replicate and propagate the virus. An obvious cytopathic effect (CPE) was first observed in cell monolayers at 1 d post-inoculation, and at 3 d this had extended to about 75% of the cell monolayer. However, no further CPE extension was observed after 4 d. Cytopathic characteristics induced by the LV were detected by Giemsa staining and fluorescence microscopic observation with Hoechst 33258 staining. The propagated virus particles were also observed by electron microscopy. Ultrastructure analysis revealed several distinct cellular changes, such as chromatin compaction and margination, vesicle formation, cell-surface convolution, nuclear fragmentation and the occurrence of characteristic 'blebs' and cell fusion. This study provides a detailed report of LV infection and propagation in a freshwater fish cell line, and presents direct electron microscopy evidence for propagation of the virus in infected cells. A possible process by which the CPEs are controlled is suggested.

Salt tolerance of Microcoleus vaginatus Gom., a cyanobacterium isolated from desert algal crust, was enhanced by exogenous carbohydrates

Microcoleus vaginatus isolated from a desert algal crust of Shapotou was cultured in BG-11 medium containing 0.2mol l(-1) NaCl or 0.2mol l(-1) NaCl plus 100mg l(-1) sucrose, extracellular polymeric substances (EPS) or hot water-soluble polysaccharides (HWP), respectively. Photosynthetic oxygen evolution rates, photosystem 11 activity (Fv/Fm) and dark respiration of NaCl-stressed cells were enhanced significantly by the added sucrose or EPS under salt stress conditions (0.2mol l(-1) NaCl). Compared with cells treated with salt alone, sodium contents in cells reduced significantly; the content of cellular total carbohydrate did not change, and intracellular sucrose, water-soluble sugar increased significantly following the addition of exogenous carbohydrates. Sucrose synthase (SS) activity of NaCl-stressed cells increased following the addition of sucrose, and sucrose phosphate synthase (SPS) activity of NaCl-stressed cells increased following the addition of exogenous sucrose, EPS or HWP compared with cells stressed with NaCl only. The results suggested that the extruded EPS might be re-absorbed by cells of M. vaginatus as carbon source, they could increase salt tolerance of M. vaginatus through the changes of carbohydrate metabolism and the selective uptake of sodium ions. (C) 2003 Elsevier Science Ltd. All rights reserved.

Myxobolus lentisuturalis sp n. (Myxozoa : Myxobolidae), a new muscle-infecting species from the Prussian carp, Carassius gibelio from China

A new highly pathogenic muscle-infecting species of the genus Myxobolus Butschli, 1882 is described from the Prussian carp, Carassius gibelio (Bloch, 1782) using spore morphology and SSU rDNA sequence data. Phylogenetic analyses elucidated relationship of the newly described Myxobolus lentisuturalis to other Myxobolus species and supported its position of an independent species.

Characterisation of a pathogenic virus isolated from marine threadfin fish (Eleutheronema tetradactylus) during a disease outbreak

An unknown virus was isolated from massive mortality of cultured threadfin (Eleutheronema tetradactylus) fingerlings. The virus replicated in BF-2 fish cell line and produced a plaque-like cytopathic effect. Electron micrographs revealed non-enveloped, icosahedral particles approximately 70-80 nm in diameter composed of a double capsid layer. Viroplasms and subviral particles approximately 30 run in diameter and complete particles of 70 nm in diameter were also observed in the infected BF-2 tissue culture cells. The virus was resistant upon pH 3 to 11 and ether treatment. It is also stable to heat treatment (3 h at 56 T). Replication was not inhibited by 5-iododeoxyuridine (5-IUdR). Acridine orange stain revealed typical reovirus-like cytoplasmic inclusion bodies. Electrophoresis of purified virus revealed 11 segments of double-stranded RNA and five major structural polypeptides of approximately 136, 132, 71, 41 and 33 kDa. Based on these findings, the virus isolated was identified to belong to the genus Aquareovirus and was designated as threadfin reovirus. This virus differed from a majority of other aquareovirus by its increase in virus infectivity upon exposure to various treatments such as high and low pH, heat (56 degreesC), ether and 5-IUdR. The RNA and virion protein banding pattern of the threadfin reovirus was shown to differ from another Asian isolate, the grass carp hemorrhage reovirus (GCV). Artificial injection of the threadfin reovirus into threadfin fingerlings resulted in complete mortality, whereas sea bass (Lates calcarifer) fingerlings infected via bath route showed severe mortality within a week after exposure. These results indicate that the threadfin virus is another pathogenic Asian aquareovirus isolate that could cross-infect into another marine fish, the sea bass. (C) 2002 Elsevier Science B.V. All rights reserved.