Elevated ability to compete for limited food resources by 'all-fish' growth hormone transgenic common carp Cyprinus carpio

Food consumption, number of movements and feeding hierarchy of juvenile transgenic common carp Cyprinus carpio and their size-matched non-transgenic conspecifics were measured under conditions of limited food supply. Transgenic fish exhibited 73 center dot 3% more movements as well as a higher feeding order, and consumed 1 center dot 86 times as many food pellets as their non-transgenic counterparts. After the 10 day experiment, transgenic C. carpio had still not realized their higher growth potential, which may be partly explained by the higher frequency of movements of transgenics and the 'sneaky' feeding strategy used by the non-transgenics. The results indicate that these transgenic fish possess an elevated ability to compete for limited food resources, which could be advantageous after an escape into the wild. It may be that other factors in the natural environment (i.e. predation risk and food distribution), however, would offset this advantage. Thus, these results need to be assessed with caution.

Detection of microcystins in environmental samples using surface plasmon resonance biosensor

An indirect inhibitive surface plasmon resonance (SPR) immunoassay was developed for the microcystins (MCs) detection. The bioconjugate of MC-LR and bovine serum albumin (BSA) was immobilized on a CM5 sensor chip. A serial premixture of MC-LR standards (or samples) and monoclonal antibody (mAb) were injected over the functional sensor surface, and the subsequent specific immunoreaction was monitored on the BIAcore 3000 biosensor and generated a signal with an increasing intensity in response to the decreasing MCs concentration. The developed SPR immunoassay has a wide quantitative range in 1-100 mu g L-1. Although not as sensitive as conventional enzyme-linked immunosorbent assay (ELISA), the SPR biosensor offered unique advantages: (I) the sensor chip could be reusable without any significant loss in its binding activity after 50 assay-regeneration cycles, (2) one single assay could be accomplished in 50 min (including 30-min preincubation and 20-min BIAcore analysis), and (3) this method did not require multiple steps. The SPR biosensor was also used to detect MCs in environmental samples, and the results compared well with those obtained by ELISA. We conclude that the SPR biosensor offers outstanding advantages for the MCs detection and may be further developed as a field-portable sensor for real-time monitoring of MCs on site in the near future. (C) 2009 Published by Elsevier B.V.

RESPONSES OF Elodea nuttallii AND Ceratophyllum demersum TO HIGH TEMPERATURE

There were large losses of exotic species Elodea nuttallii during summer in eutrophic lakes of the middle and lower reaches of the Yanatze River, China. To investigate the main causes, the heat tolerance of E. nuttallii was studied and compared with that of native species Ceratopkyllum demersum by using an aquaria system in the laboratory. Under 4500 lx light intensity and 12-h L/12-h D cycle, E. nuttallii cultured in 1/5 Hoaglands solution at 39 degrees C showed a positive growth rate during the first 15 days, and the growth rate was higher than that at 35 degrees C. But after 15 days, the growth rates became negative for those cultured both at 39 and 35 degrees C. However, the growth rate was positive for more than 20 days for those cultured at 25 degrees C. Under the same conditions, the growth rate, productivity and chlorophyll content of E. nuttallii were significantly higher than that of C. demersum. Heat tolerance of E. nuttallii was also stronger than that of C. demersum. The optimal temperature for the growth of the two plants depended on the experimental period: both plants grew at an optimal rate at higher temperature if the experimental period was short; nevertheless the plants achieved optimal growth at a lower temperature if the experiment was conducted for a longer period. At the same light intensity, the heat tolerance of C. demersum in tap water with sediment was markedly stronger than that of E. nuttallii at 39 degrees C. Average growth rate of C. demersum was 4.5 times higher than that of E. nuttallii within 25 days. The positive growth period lasted for less than 25 days for E. nuttallii and for more than 25 days for C. demersum. When they were cultured in 1/5 Hoaglands solution and in tap water with sediment, the growth rate of C. demersum increased from 0.4 to 79.4 mg/d.g fresh weight (FW) within 20 days. E. nuttallii increased from 8.3 to 24.4 mg/d-g FW within 20 days. Both grew better in tap water with sediment than in 1/5 Hoaglands solution. The results demonstrated that the nutritional status of the water other than the high temperature affected the heat tolerance of E. nuttallii during summer. E. nuttallii has great ecological safe risk in China.

ALGAL GROWTH POTENTIAL AND NUTRIENT LIMITATION IN SPRING IN THREE-GORGES RESERVOIR, CHINA

In China, especially in Three-Gorges Reservoir, our knowledge of the algal growth potential and nutrient limitation was still limited. In the spring of 2006, the water column ratios of total nitrogen/total phosphorus were investigated and algal bioassays performed to determine algal growth potential of waters and nutrient limitation of mainstream and Xiangxi Bay of Three-Gorges Reservoir. The results showed sampling sites in mainstream were co-limited by N and P or P-limited alone, and sites in Xiangxi Bay were N-limited alone. Fe likely played an important role in determining the appearance and disappearance of algal blooms of Three-Gorges Reservoir. Native algae, Pseudokirchneriella subcapitata and Cyclotella meneghiniana, had high growth potential in Three-Gorges Reservoir.

Extraction of genomic DNA using a new amino silica monolithic column

A new amino silica monolithic column was developed for DNA extraction in a miniaturized format. The monolithic column was prepared in situ by polymerization of tetraethoxysilane (TEOS) and N-(beta-aminoethyl)-gamma-aminopropylmethyldimethoxysilane (AEAPMDMS). DNA was loaded in 50 mM tris(hydroxylmethyl)aminomethane-EDTA buffer at pH 7.0 and eluted with 300 mM potassium phosphate solution at pH 10.0. Under optimal condition, a 6.0-cm monolithic column provided a capacity of 56 ng DNA with an extraction efficiency of 71 +/- 5.2% (X +/- RSD). When the amino silica monolithic column was applied to extract genomic DNA from the whole blood of crucian carp, an extraction efficiency of 52 +/- 5.6% (X +/- SD) was obtained by three extractions. Since the chaotropic-based sample loading and organic solvent wash steps were avoided in this procedure, the purified DNA was suitable for downstream processes such as PCR. This amino silica monolithic column was demonstrated to allow rapid and efficient DNA purification in microscale.

Enhanced expression of antifreeze protein genes drives the development of freeze tolerance in an Antarctica isolate of Chlorella vulgaris

In adaptation to new environments, organisms may accumulate mutations within encoding sequences to modify protein characteristics or acquire mutations within regulatory sequences to alter gene expression levels. With the development of antifreeze capability as the example, this study presents the evidence that change in gene expression level is probably the most important mechanism for adaptive evolution in a green alga Chlorella vulgaris. C. vulgaris NJ-7, an isolate from Antarctica, possesses an 18S rRNA sequence identical to that of a temperate isolate, SAG211-11b/UTEX259, but shows much higher freeze tolerance than the later isolate. The chromosomal DNA/cDNA of four antifreeze genes, namely hiC6, hiC12, rpl10a and hsp70, from the two isolates of C. vulgaris were cloned and sequenced, and very few variations of deduced amino acid sequences were found. In contrast, the transcription of hiC6, hiC12 and rpl10a was greatly intensified in NJ-7 compared to that in UTEX259, which is correlated to the significantly enhanced freeze tolerance of the Antarctica isolate. (C) 2009 National Natural Science Foundation of China and Chinese Academy of Sciences. Published by Elsevier Limited and Science in China Press. All rights reserved.

Phosphorus in suspended matter and sediments of a hypertrophic lake. A case study: Lake Dianchi, China

From June 2004 to December 2004, Lake Dianchi, which had large scale of cyanobacterial blooms was investigated in order to study P-fractionation in the suspended matter and the sediment. The investigation improves our understanding of phosphorus in Lake Dianchi and the relationship between phosphorus and cyanobacterial blooms. It contributes to the available literature on the behavior of P in hypertrophic lakes. The distribution of P-fractions in Lake Dianchi was not uniform from northwest to south, but was closely related to the trophic status of the whole lake. The concentrations of total phosphorus, labile P (NH4Cl-P), Organic P (NaOH-NRP) and loss on ignition in suspended matter were positively correlated with the strength of cyanobacterial blooms. Total phosphorus in suspended matter was relatively stable for almost half an year and closely related to Chl. a concentration. The main content of organic phosphorus is in the cyanobacterial blooms. The concentrations of phosphorus bound to metal oxides and carbonates (NaOH-SRP and HCl-P) in sediment were similar to NaOH-SRP and HCl-P in the corresponding suspended matter. The latter two forms of P in suspended matter were not affected by cyanobacterial blooms, indicating that the inorganic phosphorus is derived from the sediment after resuspension from the sediment due to wind and wave action. The contribution of the different P-fractions to TP in sediment and in suspended matter indicates that NH4Cl-P in the suspended matter is an important buffer for maintaining dissolved phosphorus in water.

Zebrafish eaf1 and eaf2/u19 Mediate Effective Convergence and Extension Movements through the Maintenance of wnt11 and wnt5 Expression

Studies have attributed several functions to the Eaf family, including tumor suppression and eye development. Given the potential association between cancer and development, we set forth to explore Eaf1 and Eaf2/U19 activity in vertebrate embryogenesis, using zebrafish. In situ hybridization revealed similar eaf1 and eaf2/u19 expression patterns. Morpholino-mediated knockdown of either eaf1 or eaf2/u19 expression produced similar morphological changes that could be reversed by ectopic expression of target or reciprocal-target mRNA. However, combination of Eaf1 and Eaf2/U19 (Eafs)-morpholinos increased the severity of defects, suggesting that Eaf1 and Eaf2/U19 only share some functional redundancy. The Eafs knockdown phenotype resembled that of embryos with defects in convergence and extension movements. Indeed, knockdown caused expression pattern changes for convergence and extension movement markers, whereas cell tracing experiments using kaeda mRNA showed a correlation between Eafs knockdown and cell migration defects. Cardiac and pancreatic differentiation markers revealed that Eafs knockdown also disrupted midline convergence of heart and pancreatic organ precursors. Noncanonical Wnt signaling plays a key role in both convergence and extension movements and midline convergence of organ precursors. We found that Eaf1 and Eaf2/U19 maintained expression levels of wnt11 and wnt5. Moreover, wnt11 or wnt5 mRNA partially rescued the convergence and extension movement defects occurring in eafs morphants. Wnt11 and Wnt5 converge on rhoA, so not surprisingly, rhoA mRNA more effectively rescued defects than either wnt11 or wnt5 mRNA alone. However, the ectopic expression of wnt11 and wnt5 did not affect eaf1 and eaf2/u19 expression. These data indicate that eaf1 and eaf2/u19 act upstream of noncanonical Wnt signaling to mediate convergence and extension movements.

Feasibility of cyanobacterial inoculation for biological soil crusts formation in desert area

Practical testing of the feasibility of cyanobacterial inoculation to speed up the recovery of biological soil crusts in the field was conducted in this experiment. Results showed that cyanobacterial and algal cover climbed up to 48.5% and a total of 14 cyanobacterial and algal species were identified at the termination of inoculation experiment; biological crusts' thickness, compressive and chlorophyll a content increased with inoculation time among 3 years; moss species appeared in the second year; cyanobacterial inoculation increased organic carbon and total nitrogen of the soil; total salt, calcium carbonate and electrical conductivity in the soil also increased after inoculation. Diverse vascular plant communities composed of 10 and 9 species are established by cyanobacterial inoculation on the windward and leeward surface of the dunes, respectively, after 3 years. The Simpson index for the above two communities are 0.842 and 0.852, while the Shannon-Weiner index are 2.097 and 2.053, respectively. In conclusion, we suggest that cyanobacterial inoculation would be a suitable and effective technique to recover biological soil crusts, and may further restore the ecological system. (C) 2008 Published by Elsevier Ltd.

The Essential Role of Endogenous Ghrelin in Growth Hormone Expression during Zebrafish Adenohypophysis Development

Ghrelin, a multifunctional hormone, including potent GH stimulation activity, has been suggested to be important during embryonic development. Expression of ghrelin has been confirmed in the zebrafish pancreas during embryonic stages. Interfering with ghrelin function using two specific antisense morpholino oligonucleotides causes defects during zebrafish embryonic development. In ghrelin morphants the expression of GH was abolished in zebrafish somatotropes, whereas the expression patterns of the other key molecules involved in hypothalamic-pituitary development and distinct pituitary hormones genes remain largely intact at the appropriate time during zebrafish adenohypophysis development. Effective rescue of the ghrelin morphants with exogenous ghrelin mRNA showed that the correct gene had been targeted. Moreover, by analyzing the efficiencies of the ghrelin morphants rescue experiments with various forms of exogenous mutant ghrelin mRNAs, we also demonstrated the essentiality of the form acyl-ghrelin on GH stimulation during zebrafish adenohypophysis development. Our in vivo experiments, for the first time, also provided evidence of the existence of functional obestatin in the C-terminal part of zebrafish proghrelin peptides. Our research here has demonstrated that zebrafish is a unique model for functional studies of endogenous ghrelin, especially during embryonic development. (Endocrinology 150: 2767-2774, 2009)

Effects of thiamine on treatment performance of textile wastewater

Textile wastewater is commonly treated with activated sludge process technology. However, its treatment performance has not been demonstrated to be very effective. In this study, the effects of micronutrient thiamine on removal efficiencies of dissolved organic carbon (DOC) and chemical oxygen demand (COD) of textile wastewater in a batch test, together with its effect on the oxygen uptake rate (OUR) of activated sludge, were evaluated. Significant improvements were observed in the removal rates of DOC, COD and OUR with 121%, 156% and 121% of those of the control, respectively, when 0.5-2.0 mg/L thiamine was added to the wastewater treatment system. Thiamine could be probably used to improve the treatment performance of textile wastewater.

A high throughput Nile red method for quantitative measurement of neutral lipids in microalgae

Isolation of high neutral lipid-containing microalgae is key to the commercial success of microalgae-based biofuel production. The Nile red fluorescence method has been successfully applied to the determination of lipids in certain microalgae, but has been unsuccessful in many others, particularly those with thick, rigid cell walls that prevent the penetration of the fluorescence dye. The conventional "one sample at a time" method was also time-consuming. In this study, the solvent dimethyl sulfoxide (DMSO) was introduced to microalgal samples as the stain carrier at an elevated temperature. The cellular neutral lipids were determined and quantified using a 96-well plate on a fluorescence spectrophotometer with an excitation wavelength of 530 nm and an emission wavelength of 575 run. An optimized procedure yielded a high correlation coefficient (R-2 = 0.998) with the lipid standard triolein and repeated measurements of replicates. Application of the improved method to several green algal strains gave very reproducible results with relative standard errors of 8.5%, 3.9% and 8.6%, 4.5% for repeatability and reproducibility at two concentration levels (2.0 mu g/mL and 20 mu g/mL), respectively. Moreover, the detection and quantification limits of the improved Nile red staining method were 0.8 mu g/mL and 2.0 mu g/mL for the neutral lipid standard triolein, respectively. The modified method and a conventional gravimetric determination method provided similar results on replicate samples. The 96-well plate-based Nile red method can be used as a high throughput technique for rapid screening of a broader spectrum of naturally-occurring and genetically-modified algal strains and mutants for high neutral lipid/oil production. (C) 2009 Published by Elsevier B.V.

Effects of tetrabrominated diphenyl ether and hexabromocyclododecanes in single and complex exposure to hepatoma HepG2 cells

This study was designed to determine cytotoxic effects of PBDE-47 and HBCDs individually or with a mixture of both compounds exposure to Hep G2 cells. The results showed PBDE-47 and HBCDs induced increase of nitric oxide synthase (NOS) activity, release of NO. dissipation of mitochondria membrane potential and cell apoptosis. Exposure to HBCDs induced ROS formation. Moreover, preincubation with PTIO (NO scavanger) and N-acetylcysteine (ROS scavanger) partially reversed cytotoxic effects of these compounds. The possible mechanism is that PBDE-47 and HBCDs could boost generation of NO and/or ROS, impact mitochondria, and result in start-ups of apoptosis program. Cells exposed to mixture of both compounds and each of them showed non-apoptotic rate significant difference, but the combination of them caused more adverse effects on cells. These results Suggest that PBDE-47 and HBCDs in single and complex exposure have the cytotoxic activity of anti-proliferation and induction of apoptosis in tumor cells in vitro. (C) 2008 Elsevier B.V. All rights reserved.

Structure, organization and expression of common carp (Cyprinus carpio L.) NKEF-B gene

Natural killer (NK) cell enhancing factor (NKEF) belongs to the newly defined peroxiredoxin (Prx) family. Its functions are to enhance NK cell cytotoxicity and to protect DNA and proteins from oxidative damage. In this study, a partial cDNA sequence of carp NKEF-B was isolated from thymus cDNA library. Subsequently, the full-length cDNA of carp NKEF-B was obtained by means of 3' and 5' RACE, respectively. The full-length cDNA of carp NKEF-B was 1022 bp, consisting of a 73 bp 5'-terminal untranslated region (UTR), a 355 bp T-terminal UTR, and a 594 bp open reading frame coding for a protein of 197 amino acids. Carp NKEF-B contained two consensus Val-Cys-Pro (VCP) motifs and three consensus cysteine (Cys-51, Cys-70 and Cys-172) residues. Sequence comparison showed that the deduced amino acid sequence of carp NKEF-B had an overall similarity of 74-96% to that of other species homologues. Phylogenetic analysis revealed that carp NKEF-B forms a cluster with other known teleost NKEF-Bs. Then, by PCR we obtained a 5.1 -k long genomic DNA of carp NKEF-B containing six exons and five introns. Realtime RT-PCR results showed that carp NKEF-B gene was predominantly detected in kidney and head kidney under un-infected conditions. Whereas under SVCV-infection condition, the expression of NKEF-B gene was significantly increased in blood cells, gill, intestine and spleen, but maintained in liver, and decreased significantly in kidney and head kidney. Finally, the rNKEF-B was constructed and expressed in Escherichia coli. By using an antibody against carp rNKEF-B, immunohistochemical study further indicated that NKEF-B positive cells are mainly some RBCs and a few epithelial cells in gill and intestine, and that under SVCV-infection condition, these positive cells or positive products in their cytoplasm were mainly increased in gill and spleen sections of carp. The results obtained in the present study will help to understand the function of NKEF-B in teleost innate immunity. (C) 2008 Elsevier Ltd. All rights reserved.

Identification of Differentially Expressed Genes Between Cloned and Zygote-Developing Zebrafish (Danio rerio) Embryos at the Dome Stage Using Suppression Subtractive Hybridization

Comparative analyses of differentially expressed genes between somatic cell nuclear transfer (SCNT) embryos and zygote-developing (ZD) embryos are important for understanding the molecular mechanism underlying the reprogramming processes. Herein, we used the suppression subtractive hybridization approach and from more than 2900 clones identified 96 differentially expressed genes between the SCNT and ZD embryos at the dome stage in zebrafish. We report the first database of differentially expressed genes in zebrafish SCNT embryos. Collectively, our findings demonstrate that zebrafish SCNT embryos undergo significant reprogramming processes during the dome stage. However, most differentially expressed genes are down-regulated in SCNT embryos, indicating failure of reprogramming. Based on Ensembl description and Gene Ontology Consortium annotation, the problems of reprogramming at the dome stage may occur during nuclear remodeling, translation initiation, and regulation of the cell cycle. The importance of regulation from recipient oocytes in cloning should not be underestimated in zebrafish.