TRAIL in the mandarin fish Siniperca chuatsi: Gene and its apoptotic effect in HeLa cells

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is one of the TNF superfamily members, participating in many biological processes including cell proliferation and apoptotic death. In this study, a TRAIL gene was cloned from a perciform fish, the mandarin fish Siniperca chuatsi, a major cultured fish in China's aquaculture, and is named as SCTRAIL for S. chuatsi TRAIL. The full-length cDNA of SCTRAIL is 1359 bp, encoding a 283-amino-acid protein. This deduced protein contains the CYS231, a 23-mer fragment of transmembrane region, a glycosylation site and a TNF family signature, all of which are conserved among TRAIL members. SCTRAIL gene consists of six exons, with five intervening introns, spaced over approximately 9 kb of genomic sequence. Southern blotting demonstrated that the SCTRAIL gene is present as a single copy in mandarin fish genome. A 620 bp promoter region obtained by genome walking contains a number of putative transcription factor binding sites, such as Oct-1, Sp-1, NF-1, RAP-1, C/EBPaLp, NF-kappa B and AP-1. The SCTRAIL is constitutively expressed in all the analyzed tissues, as revealed by RT-PCR, which is confirmed by Western blotting analysis using polyclonal antibody against bacteria-derived recombinant SCTRAIL protein. As an apoptosis-inducing ligand, the overexpression of SCTRAIL but not the mutant SCTRAIL-C203S in HeLa cells induced changes characteristic of apoptosis, including chromatin condensation, nucleus fragmentation, DNA ladder, and increase of sub-G0/G1 cells in FACS analysis. (c) 2007 Elsevier Ltd. All rights reserved.

Seasonal variations of estrogenic compounds and their estrogenicities in influent and effluent from a municipal sewage treatment plant in China

The seasonal variations of estrogenic compounds and the estrogenicities of influent and effluent were investigated by OF chemical analysis and in vitro assay in a municipal sewage treatment plant in Wuhan (China). The levels of eight estrogenic compounds, including 17 beta-estradiol (E-2) estrone (E-1), estriol (E-3) diethylstilbestrol (DES), 17 alpha-ethinylestradiol, nonylphenol (NP), 4-tert-octylphenol (OP), and bisphenol A (BPA), were measured by gas chromatography-mass spectrometry. Total estrogenic activity of sewage was quantitatively assessed using primary cultured hepatocytes of male Megalobrama amblycephala Yih using vitellogenin as a biomarker. The E-2 equivalents (EEQs) obtained from the chemical analysis were consistent with those measured by bioassay. The natural (E-1, E-2, and E-3) and synthetic (DES) estrogens, as well as NP, were the main contributors of the total EEQs of influent and effluent in the present study. The levels of natural estrogens E-1 and E-3 in the influent and effluent were higher in winter than in summer, whereas the situation for NP and OP was the reverse. The levels of E-2, DES, and BPA varied little among different seasons. 17 alpha-Ethinylestradiol was not detected in the influent and effluent. The estrogenicities of the influent and of the primary and secondary effluents were all higher in summer than in winter. Estrogenic activities in winter mainly originated from natural (E-1, E-2, and E-3) and synthetic (DES) estrogens, whereas the increase of EEQs in summer was contributed by NP The results from chemical analysis and bioassay demonstrate that estrogenic compounds cannot be entirely removed by the existing sewage treatment process, which should be further improved to protect aquatic ecosystems and human health.

Apoptosis induction on human hepatoma cells Hep G2 of decabrominated diphenyl ether (PBDE-209)

Polybrominated diphenyl ethers (PBDEs) are an important class of halogenated organic brominated flame retardants. Because of their presence in abiotic and biotic environments widely and their structural similarity to polychlorinated biphenyls (PCBs), concern has been raised on their possible adverse health effects to humans. This study was designed to determine the anti-proliferative, apoptotic properties of decabrominated diphenyl ether (PBDE-209), using a human hepatoma Hep G2 line as a model system. Hep G2 cells were cultured in the presence of PBDE-209 at various concentrations (1.0-100.0 mu mol/L) for 72 h and the percentage of cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The results showed that PBDE-209 inhibited the cells viability in time and concentration-dependent characteristics at concentrations (10.0-100.0 mu mol/L). We found that anti-proliferative effect of PBDE-209 was associated with apoptosis on Hep G2 cells by determinations of morphological changes, cell cycle and apoptosis. Mechanism study showed that PBDE-209 could increase the generation of intracellular reactive oxygen species (ROS) concentration-dependently. Antioxidant N-acetylcyteine partially inhibited the increase of ROS. The mechanism for its hepatoma-inhibitory effects was the induction of cellular apoptosis through ROS generation. In addition, activity of lactate dehydrogenase (LDH) release increased when the cells incubated with PBDE-209 at various concentrations and times. These results suggested that PBDE-209 had the toxicity activity of anti-proliferation and induction of apoptosis in tumor cells in vitro. (c) 2007 Elsevier Ireland Ltd. All rights reserved.

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.

Isolation and characterization of Scophthalmus maximus rhabdovirus

A rhabdovirus associated with a lethal hemorrhagic disease in cultured turbot Scophthalm us maximus Linnaeus was isolated. The virus induced typical cytopathogenic effects (CPE) in 9 of 15 fish cell lines examined and was then propagated and isolated from infected carp leucocyte cells (CLC). Electron microscopy observations revealed that the negatively stained virions had a typical bullet-shaped morphology with one rounded end and one flat base end. The bullet-shaped morphology was more obvious and clear in ultrathin sections of infected cells. Experimental infections also indicated that the S. maximus rhabdovirus (SMRV) was not only a viral pathogen for cultured turbot, but also had the ability to infect other fish species, such as freshwater grass carp. A partial nucleotide sequence of the SMRV polymerase gene was determined by RT-PCR using 2 pairs of degenerate primers designed according to the conserved sequences of rhabdovirus polymerase genes. Homology analysis, amino acid sequence alignment, and phylogenetic relationship analysis of the partial SMRV polymerase sequence indicated that SMRV was genetically distinct from other rhabdoviruses. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified SMRV revealed 5 major structural proteins, and their molecular masses were estimated to be about 250, 58, 47, 42, and 28 kDa. Significant serological reactivity differences were also observed between SMRV and its nearest neighbor, spring viremia of carp virus (SVCV). The data suggest that SMRV is likely a novel fish rhabdovirus, although it is closely related to rhabdoviruses in the genus Vesiculovirus.

Phycobilisome-Deficient Strains of Synechocystis sp. PCC 6803 Have Reduced Size and Require Carbon-Limiting Conditions to Exhibit Enhanced Productivity.

Reducing excessive light harvesting in photosynthetic organisms may increase biomass yields by limiting photoinhibition and increasing light penetration in dense cultures. The cyanobacterium Synechocystis sp. PCC 6803 harvests light via the phycobilisome, which consists of an allophycocyanin core and six radiating rods, each with three phycocyanin (PC) discs. Via targeted gene disruption and alterations to the promoter region, three mutants with two (pcpcT→C) and one (ΔCpcC1C2:pcpcT→C) PC discs per rod or lacking PC (olive) were generated. Photoinhibition and chlorophyll levels decreased upon phycobilisome reduction, although greater penetration of white light was observed only in the PC-deficient mutant. In all strains cultured at high cell densities, most light was absorbed by the first 2 cm of the culture. Photosynthesis and respiration rates were also reduced in the ΔCpcC1C2:pcpcT→C and olive mutants. Cell size was smaller in the pcpcT→C and olive strains. Growth and biomass accumulation were similar between the wild-type and pcpcT→C under a variety of conditions. Growth and biomass accumulation of the olive mutant were poorer in carbon-saturated cultures but improved in carbon-limited cultures at higher light intensities, as they did in the ΔCpcC1C2:pcpcT→C mutant. This study shows that one PC disc per rod is sufficient for maximal light harvesting and biomass accumulation, except under conditions of high light and carbon limitation, and two or more are sufficient for maximal oxygen evolution. To our knowledge, this study is the first to measure light penetration in bulk cultures of cyanobacteria and offers important insights into photobioreactor design.

Molecular cloning and stress-induced expression of paralichthys olivaceus heme-regulated initiation factor 2 alpha kinase

The heme-regulated initiation factor 2 alpha kinase (HRI) is acknowledged to play an important role in translational shutoff in reticulocytes in response to various cellular stresses. In this study, we report its homologous cDNA cloning and characterization from cultured flounder embryonic cells (FEC) after treatment with UV-inactivated grass carp haemorrhagic virus (GCHV). The full-length cDNA of Paralichthys olivaceus HRI homologue (PoHRI) has 2391 bp and encodes a protein of 651 amino acids. The putative PoHRI protein exhibits high identity with all members of eIF2 alpha kinase family. It contains 12 catalytic subdomains located within the C-terminus of all Ser/Thr protein kinases, a unique kinase insertion of 136 amino acids between subdomains IV and V, and a relatively conserved N-terminal domain (NTD). Upon heat shock, virus infection or Poly PC treatment, PoHRI mRNA and protein are significantly upregulated in FEC cells but show different expression patterns in response to different stresses. In healthy flounders, PoHRI displays a wide tissue distribution at both the mRNA and protein levels. These results indicate that PoHRI is a ubiquitous eIF2a kinase and might play an important role in translational control over nonheme producing FEC cells under different stresses. (c) 2006 Elsevier Ltd. All rights reserved.

First report of aphantoxins in China - waterblooms of toxigenic Aphanizomenon flos-aquae in Lake Dianchi

The oligohaline cyanobacterium Aphanizomenon flos-aquae (L.) Ralfs (A. flos-aquae) has been reported in several countries to produce paralytic shellfish poisons (PSPs) or protracted toxic effects. In the past years, A. flos-aquae blooms have occurred annually in the eutrophic Lake Dianchi (300 km(2) in area, located in southwestern China). Material from natural blooms dominated by A. flosaquae was collected and lyophilized. Acute toxicity testing was performed by mouse bioassay using extracts from the lyophilized material. Clear symptoms of PSPs, intoxications were observed. To confirm the production of PSPs, a strain of A. flos-aquae (DC-1) was isolated and maintained in culture. Histopathological effects were studied by examining the organ damages using transmission electron microscopy (TEM). Slight hepatocytic damage with swollen mitochondria was found. The ultrastructural pulmonary lesions were characterized by distortied nuclei and indenting of karyotheca, together with degeneration and tumefaction of mitochondria and endoplasmic reticulum. Control animals injected with acetic acid did not exhibit histopathological damage in any organ. Toxic effects of cultured algal cells on enzymatic systems in the mouse were studied using sublethal doses of extracts. Significant glutathione-S-transferase (GST) and lactate dehydrogenase (LDH) increases, together with decrease of the glutathione (GSH) level, were measured. These results indicated a potential role of PSPs intoxicating and metabolizing in the test animals. HPLC-FLD and LC/MS analysis of extracts from cultured material demonstrated the PSP toxins produced by A. flos-aquae bloom. To the best of our knowledge, this is the first study reporting chemically and toxicologically confirmed PSP toxins related to A. flosaquae in China. (c) 2005 Elsevier Inc. All rights reserved.

Population growth and physiological characteristics of microalgae in a miniaturized bioreactor during space flight

A strain of microalgae (Anabaena siamensis) had been cultured in a miniaturized bioreactor during a retrievable satellite flight for 15 days. By means of remote sensing equipment installed in the satellite, we gained the growth curve of microalgae population in space every day in real time. The curve indicated that the growth of microalgae in space was slower than the control on ground. Inoculation of the retrieved microalgae culture showed that the growth rate was distinctively higher than ground control. But after several generations, both cultures indicated similar growth rates. Those data showed that algae, can adapt to space environment easily which may be valuable for designing more complex bioreactor and controlled ecological life support system in future experiment. (C) 2006 Elsevier Ltd. All rights reserved.

Gene cloning and functional analysis of glycosaminoglycan-degrading enzyme chondroitin AC lyase from Flavobacterium columnare G(4)

The chondroitin AC lyase gene, cslA, was cloned for the first time from the fish bacterial pathogen F. columnare G(4). From the first transcription initiation site, the cslA extends 2620 nucleotides to the end of the 3' region. The open reading frame of cslA transcript has 2286 nucleotides encoding 762 amino acids with a 16 residues long signal peptide at the N-terminus. The gene, cslA was then successfully expressed in Escherichia coli and recombinant chondroitin AC lyase, rChonAC was purified, with its lytic activity analyzed. Zymography analysis copolymerized with chondroitin sulphate revealed the lytic activity of rChonAC and also the crude native ChonAC isolated from periplamic space of cultured F. columnare G(4). The low level of lytic activity observed in crude native ChonAC may be due possibly to the low level of expression of this gene in the cultured condition. The expression and the role of this virulence factor is of interest for further research on the pathogenesis of F. columnare.

Effects of solar UV radiation on morphology and photosynthesis of filamentous cyanobacterium Arthrospira platensis

To study the impact of solar UV radiation (UVR) (280 to 400 nm) on the filamentous cyanobacterium Arthrospira (Spirulina) platensis, we examined the morphological changes and photosynthetic performance using an indoor-grown strain (which had not been exposed to sunlight for decades) and an outdoor-grown strain (which had been grown under sunlight for decades) while they were cultured with three solar radiation treatments: PAB (photosynthetically active radiation [PAR] plus UVR; 280 to 700 nm), PA (PAR plus UV-A; 320 to 700 nm), and P (PAR only; 400 to 700 nm). Solar UVR broke the spiral filaments of A. platensis exposed to full solar radiation in short-term low-cell-density cultures. This breakage was observed after 2 h for the indoor strain but after 4 to 6 h for the outdoor strain. Filament breakage also occurred in the cultures exposed to PAR alone; however, the extent of breakage was less than that observed for filaments exposed to full solar radiation. The spiral filaments broke and compressed when high-cell-density cultures were exposed to full solar radiation during long-term experiments. When UV-B was screened off, the filaments initially broke, but they elongated and became loosely arranged later (i.e., there were fewer spirals per unit of filament length). When UVR was filtered out, the spiral structure hardly broke or became looser. Photosynthetic 0, evolution in the presence of UVR was significantly suppressed in the indoor strain compared to the outdoor strain. UVR-induced inhibition increased with exposure time, and it was significantly lower in the outdoor strain. The concentration of UV-absorbing compounds was low in both strains, and there was no significant change in the amount regardless of the radiation treatment, suggesting that these compounds were not effectively used as protection against solar UVR. Self-shading, on the other hand, produced by compression of the spirals over adaptive time scales, seems to play an important role in protecting this species against deleterious UVR. Our findings suggest that the increase in UV-B irradiance due to ozone depletion not only might affect photosynthesis but also might alter the morphological development of filamentous cyanobacteria during acclimation or over adaptive time scales.