Antioxidant enzyme activities of Microcystis aeruginosa in response to nonylphenols and degradation of nonylphenols by M-aeruginosa

The aim of this study was to examine the effects of chemical nonylphenols (NPs) on the antioxidant system of Microcystis aeruginosa strains. The degradation and sorption of NPs by M. aeruginosa were also evaluated. High concentrations of NPs (1 and 2 mg/l) were found to cause increases in superoxidase dismutase (SOD) and glutathione-S-transferase (GST) activities and in glutathione (GSH) levels. These results suggest that toxic stress manifested by elevated SOD and GST levels and GSH contents may be responsible for the toxicity of NPs to M. aeruginosa and that the algal cells could improve their antioxidant and detoxification ability through the enhancement of enzymatic and nonenzymatic prevention substances. The observed elevations in GSH levels and GST activities were relatively higher than those in SOD activities, indicating that GSH and GST contributed more in eliminating toxic effects than SOD. Low concentrations of NPs (0.05-0.2 mg/l) enhanced cell growth and decreased GST activity in algal cells of M. aeruginosa, suggesting that NPs may have acted as a protecting factor, such as an antioxidant. The larger portion of the NPs (> 60%) disappeared after 12 days of incubation, indicating the strong ability of M. aeruginosa to degrade the moderate persistent NP compounds. The sorption ratio of M. aeruginosa after a 12-day exposure to low nominal concentrations of NPs (0.02-0.5 mg/l) was relatively high (> 30%). The fact that M. aeruginosa effectively resisted the toxic effects of NPs and strongly degraded these pollutants indicate that M. aeruginosa cells have a strong ability to adapt to variations in environmental conditions and that low and moderate concentrations of organic compounds may favor its survival. Further studies are needed to provide detailed information on the fate of persistent organic pollutants and the survival of algae and to determine the possible role of organic pollutants in the occurrence of water blooms in eutrophic lakes.

Characterization and application of monoclonal antibodies against turbot (Scophthalmus maximus) rhabdovirus

Five monoclonal antibodies (mAbs), 1G8, 1H9, 2D2, 2D3, and 2F5, against Scophthalmus maximus rhabdovirus (SMRV) were prepared. Characterization of the mAbs included indirect enzyme-linked immunosorbent assay, isotyping, viral inhibition assay, immunofluorescence staining of virus-infected cell cultures, and Western blot analysis. Isotyping revealed that 1G8 and 1H9 were of the IgG2b subclass and that the other three were IgM. 2D2, 2D3, and 2F5 partially inhibited SMRV infection in epithelioma. papulosum cyprinid (EPC) cell culture. Western blotting showed that all five mAbs could react with two SMRV proteins with molecular masses of approximately 30 kDa (P) and 26 kDa (M). These two proteins were localized within the cytoplasm of SMRV-infected EPC cells by immunofluorescence assay. Also, progressive foci of viral replication in cell cultures were monitored from 6 to 24 h, using mAb 2D3 as the primary antibody. A flow cytometry procedure was used to detect and quantify SMRV-infected (0.01 PFU/cell) EPC cells with mAb 2D3, and 10.8% of cells could be distinguished as infected 36 h postinfection. Moreover, mAb 2D3 was successfully applied for the detection of viral antigen in cryosections from flounder tissues by immunohistochemistry tests.

An enzyme-linked immunosorbent assay for rare minnow (Gobiocypris rarus) vitellogenin and comparison of vitellogenin responses in rare minnow and zebrafish (Danio rerio)

A competitive enzyme-linked immunosorbent assay (ELISA) was developed to determine vitellogenin (Vtg) in rare minnow (Gobiocypris rarus) based on the separation and purification of rare minnow Vtg (r-Vtg) as well as the production of polyclonal antibody against r-Vtg in rabbits. Three different ELISAs for measuring r-Vtg were then compared: (1) indirect ELISA with the antibody against carp (Cyprinus carpio) Vtg (c-Vtg) (IC-ELISA); (2) competitive ELISA with the antibody against c-Vtg, and using r-Vtg for coating the plates and preparing standard curve (CC-ELISA); (3) competitive ELISA with the antibody against r-Vtg, and using r-Vtg for coating the plates and preparing standard curve (CR-ELISA). The result showed that the homologous CR-ELISA was the most sensitive among the three assays for quantifying r-Vtg. The sensitivities to 17 alpha-ethinylestradiol (EE2) Of rare minnow and zebrafish (Danio rerio) were compared upon the establishment of homologous competitive ELISA. The lowest observed effect concentrations (LOECs) to induce Vtg were found to be 0.8 ng EE2 l(-1) for rare minnow and 4 ng EE2 l(-1) for zebrafish respectively. Afterwards, CR-ELISA was applied to measure Vtg concentration in whole body homogenate (WBH) of juvenile rare minnow fed by three diets (tubifex from wastewater treatment plant, Artemia nauplii and commercial pellet food), and the agreements between bioassay and GC-MS analysis demonstrated that rare minnow was a sensitive fish model for assessing estrogenic effects of endocrine disrupting compounds in aquatic environment. (c) 2006 Elsevier B.V. All rights reserved.

Effects of age and dietary protein level on digestive enzyme activity and gene expression of Pelteobagrus fulvidraco larvae

The present research studied the effects of age and dietary protein level on pepsin, trypsin and amylase activity and their mRNA level in Petteobagrus fulvidraco larvae from 3 to 26 days after hatch (DAH). Three DAH larvae were fed three isoenergetic diets, containing 42.8% (CP 43), 47.3% (CP 47) and 52.8% (CP 53) crude protein. Live food (newly hatched Artemia, unenriched) was included as a control. The effects of age on enzyme activity and mRNA were as follows: pepsin and trypsin activity in all treatment groups showed a significant (P < 0.05) increase at the beginning and decrease later although the timing of decrease was not the same among treatment groups and between the digestive enzymes. Pepsin and trypsin mRNA level followed the pattern of their respective enzyme changes. Age significantly affected amylase activity (P < 0.05) while age had no effect on amylase mRNA during the experimental period. The four diets significantly (P < 0.05) affected activity and mRNA level of pepsin and trypsin. Diets did not affect amylase activity or mRNA level. These results suggest that the effects of age on pepsin and trypsin gene expressions are at the transcriptional level. Dietary protein level does affect pepsin and trypsin gene expression in the early life of P. fulvidraco. There were no transcriptional effects on amylase gene expression. (c) 2005 Elsevier B.V. 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.

A new enzyme immunoassay for PCDD/F TEQ screening in environmental samples: Comparison to micro-EROD assay and to chemical analysis

A new Enzyme ImmunoAssay (EIA) for PCDD/F TEQ measurement in extracts of environmental samples was described. The bioassay TEQ which derived from EIA and EROD were compared with each other and with results from chemical analysis. For all environmental samples, the EROD-TEQ is higher than the value from chemical analysis. However, the EIA-TEQ is much more identical with the value from chemical analysis. Our results indicate that the EIA assay is a complementary method to the EROD assay and should be useful as a rapid and sensitive screening tool for environmental samples in many situations. (C) 1999 Elsevier Science Ltd. All rights reserved