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.

Genetic diversity: Geographical distribution and toxin profiles of Microcystis strains (Cyanobacteria) in China

Twenty strains of Microcystis Kutz were isolated from different freshwater bodies in China to analyze the diversity, geographical distribution and toxin profiles. Based on whole-cell polymerase chain reaction of cpcBA-IGS nucleotide sequence, the derived neighbor-joining (NJ) and maximum parsimony (MP) trees indicate that these strains of Microcystis can be divided into four clusters. The strains from south, middle and north region of China formed distinct lineages, suggesting high diversity and a geographical distribution from south to north locations. Moreover, the results being indicating high variable genotypes of the strains of the Microcystis strains from the same lake show that there is high diversity of Microcystis within a water bloom population. Comparing the results of the present study with those reported for compared with 43 strains of Microcystis from other locations, also reveals Chinese strains have high similarity with those from regions in the North Hemispherical. This suggests that the Microcystis strains in the world might have a geographical distribution. Analysis of 30 strains using the primers MCF/TER and TOX2P/TOX2M showed that there was no correlation between the gene of cpcBA-IGS and the presence of mcy. Toxic strains were founded to be predominant in different water bodies throughout China.

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.

Nitrate and phosphate supplementation to increase toxin production by the marine dinoflagellate Alexandrium tamarense

Alexandrium tamarense toxins have great value in biotechnology research as well as important in connection with shellfish poisoning. The influence of nitrate or nitrate and phosphate supplementation on cell biomass and toxin content were investigated in batch cultures. When cultures at low nitrate (88.2 mu M NaNO3) Were supplemented with 793.8 mu M NaNO3 at day 10 the cell density and cellular toxin contents were increased by 6-29% and 20-76%, respectively, compared with controls, and maximal values were 43,600 cells/ml (day 38) and 0.91 pg/cell (day 31). Supplementation with nitrate at day 14 or with nitrate and phosphate at day 10/14 to the cultures did not increase the cell density compared with the non-supplemented middle nitrate or high phosphate (108 mu M NaH2PO4) cultures, respectively, but increased the cellular toxin contents by an average of 52%. The results showed that supplementation with nitrate or with nitrate and phosphate at different growth phases of the cultures increased toxin yield by an average of 46%. Supplementation with nitrate at selected times to maintain continuous low level of nitrate might contribute to the effective increase of toxin yield of A. tamarense. (c) 2005 Elsevier Ltd. All rights reserved.

Improvement in growth and toxin production of Alexandrium tamarense by two-step culture method

The growth and toxin content of the dinoflagellate Alexandrium tamarense ATHK was markedly affected by culture methods. In early growth phase at lower cell density static or mild agitation methods were beneficial to growth, but continuous agitation or aeration, to some extent, had an adverse effect on cell growth. Static culture in 2 L Erlenmeyer flasks had the highest growth rate (0.38 d(-1)) but smaller cell size compared with other culture conditions. Cells grown under aerated conditions possessed low nitrogen and phosphorus cell yields, namely high N and P cell-quota. At day 18, cells grown in continuous agitated and 1 h aerated culture entered the late stationary phase and their cellular toxin contents were higher (0.67 and 0.54 pg cell(-1)) compared with cells grown by other culture methods (0.27-0.49 pg cell(-1)). The highest cell density and cellular toxin content were 17190 cells mL(-1) and 1.26 pg cell(-1) respectively in an airlift photobioreactor with two-step culture. The results indicate that A. tamarense could be grown successfully in airlift photobioreactor by a two-step culture method, which involved cultivating the cells statically for 4 days and then aerating the medium. This provides an efficient way to enhance cell and toxin yield of A. tamarense.

Degradation of 17 alpha-ethinylestradiol in aqueous solution by ozonation

This study investigates the ozonation of 17 alpha-ethinylestradiol (EE2) in aqueous solution. The affecting factors on the degradation of EE2 were studied and described in details, such as initial EE2 concentration, initial pH value and ozone concentration. In addition, some parameters such as pH. electrical conductivity, mineralization efficiency and degradation products were monitored during the process. The mineralization efficiency of EE2 could reach 53.9%. During the ozonation process the rapid decrease of pH and the sharp increase of electrical conductivity indicated the fort-nation of acidic by-products, small fragments and ions which were confirmed by high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GUMS) analysis. Results showed that there were intermediate products of smaller molecule with higher polarity produced during the course of EE2 degradation. Then a possible reaction pathway for EE2 degradation involving all intermediates detected is proposed. During the ozonation process EE2 was first oxidized into hydroxyl-semiquinone isomers which were subsequently degraded into low molecular weight compounds such as oxalic acid, malonate, glutarate, and so on. Furthermore. these organic acids are easily oxidized by ozone into carbon dioxide (CO2). This work shows that ozonation process is promising for the removal of EE2. The results can provide some useful information for the potential treatment of EE2 by ozonation in aqueous solution. (c) 2005 Elsevier B.V. All rights reserved.

In vivo studies on toxin accumulation in liver and ultrastructural changes of hepatocytes of the phytoplanktivorous bighead carp i.p.-injected with extracted microcystins

Phytoplanktivorous bighead carp were injected i.p. with extracted microcystins (mainly MC-RR and -LR) at two doses, 200 and 500 MC-LReq. mu g kg(-1) bw, and the changes in extractable MCs in liver and in the ultrastructure of hepatocytes were studied at 1, 3, 12, 24 and 48 h after injection. Quantitative and qualitative determinations of MCs in the liver were conducted by HPLC and LC-MS, respectively. MC concentration in the liver reached the maxima at 12 It (2.89 mu g MCs g(-1) dry weight at the lower dose) or at 3 h (5.43 mu g MCs g(-1) dry weight at the higher dose) post-injection, followed by sharp declines afterwards, whereas the ultrastructural changes of hepatocytes in both dose groups suggest progressive increases in severity toward the directions of apoptosis and necrosis from I to 24 h, respectively. There were two new findings in fish: widening of intercellular spaces was among the early ultrastructural changes induced by MCs and ultrastructural recovery of hepatocytes was evident at 48 h post-injection in both dose groups. Both the present and previous studies suggest that with in vivo or in vitro exposure to microcystins, hepatocyte damage in fish tends to proceed toward the direction of apoptosis at lower MC concentrations but toward the direction of necrosis at high MC concentrations. The temporal dynamics of MCs in the liver suggest that bighead carp may have a mechanism to degrade or bind MC-LR actively after it enters the blood system. (c) 2005 Elsevier Ltd. All rights reserved.

Molecular and expression characterization of three gonadotropin subunits common alpha, FSH beta and LH beta in groupers

A SMART cDNA plasmid library was constructed from protogyous greasy grouper (Epinephelus coioides) pituitary, and the full-length cDNAs of three gonadotropin (GTH) subunits common alpha, FSH beta and LH beta were cloned and sequenced from the library. The nucleotide sequences of common alpha, FSH beta and LH beta subunit cDNAs are 647, 594 and 574 bp in length, and encode for mature peptides of 94, 99 and 115 aa, respectively. High homology was observed by amino acid sequence alignment and identity comparison of the grouper mature peptides of common alpha, FSH beta and LH beta with that of other fishes. Phylogenetic tree analyses of the three GTH mature subunits revealed similar phylogeny relationships among the studied fish species. Three polyclonal antibodies were prepared from the in vitro expressed common alpha, FSH beta and LH beta mature proteins, respectively. Western blot analysis and immunofluoresence localization were performed on two typical stages of ovarian development stages in red-spotted grouper. Significant differences in protein expression levels of three gonadotropin subunits were revealed between the two ovarian development stages. In the individuals with resting ovary, common alpha was almost not detected in pituitaries, and FSH beta and LH beta expression levels were very low. While in the individuals with developing ovary, the expression of all three gonadotropin subunits reached to a high level. Immunofluoresence localization indicated that the grouper FSH beta cells mainly distributed in the middle area of PPD, while the LH beta cells distributed more widely, including in the area similar to the FSH beta cells and at the external periphery of pituitary near to the PI side. The common alpha might be expressed in both FSH beta and LH beta cells. Double immunofluoresence localization further demonstrated FSH beta and LH beta expression in distinct cells in the PPD area, although the FSH beta and LH beta cells were detected in the identical area of PPD. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

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.

Analysis of expression of the binary toxin genes from Bacillus sphaericus in Anabaena and the potential in mosquito control

Anabaena strains expressing the binary toxin genes of Bacillus sphaericus produce high larvicidal activity with living cells. Western blot analysis showed that the 51-kDa and 42-kDa toxin proteins were stable in Anabaena. When a DNA fragment upstream of the 51-kDa protein gene was deleted, the toxicity was reduced by over a hundred-fold, whereas deletions at the coding regions showed that the cooperation of the two proteins expressed in Anabaena is essential for the larvicidal activity. Outdoor tests showed that the genetically altered Anabaena could keep containers with natural water from being inhabited by Culex larvae for over 2 months.

Effect of feeding strategy and carbohydrate source on carbohydrate utilization by white sturgeon (Acipenser transmontanus) and hybrid tilapia (Oreochromis niloticus X O-aureus)

Two 8-week growth trials were conducted to determine the effect of continuous (CF) versus 2 meals day(-1) (MF) feeding and 30% starch versus 30% glucose diets on the carbohydrate utilization of 9.0-g white sturgeon and 0.56-g hybrid tilapia. The two trials were conducted under similar conditions except that sturgeon were kept at 18.5 degrees C in a flow-through system and tilapia were kept at 26 degrees C in a recirculating system. Significantly (P less than or equal to 0.05) higher specific growth rate (SGR), feed efficiency (FE), protein efficiency ratio (PER), body lipid content and liver glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) activities were observed in the CF than MF sturgeon. Only SGR, FE and PER were higher in sturgeon fed the starch than the glucose diets. Only higher liver G6PDH and malic enzyme (ME) activities were observed in the CF than MF tilapia but higher SGR, FE, PER and liver G6PDH, 6PGDH and ME activities were observed in tilapia fed the starch diet than those fed the glucose diet. This suggested that carbohydrate utilization by sturgeon was more affected by feeding strategy whereas tilapia was more affected by carbohydrate source. Furthermore, white sturgeon can utilize carbohydrates better than hybrid tilapia regardless of feeding strategy and carbohydrate source.

Metal catalysis-free, direction-controlled planar growth of single-crystalline alpha-Si3N4 nanowires on Si(100) substrate

Single-crystalline alpha-Si3N4 nanowires are controlled to grow perpendicular to the wet-etched trenches in the SiO0.94 film on the plane of the Si substrate without metal catalysis. A detailed characterization is carried out by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The photoluminescence at 600 nm from alpha-Si3N4 nanowires is attributed to the recombination at the defect state formed by the Si dangling bond N3 equivalent to Si-center dot. The growth mechanism is considered to be related to the catalysis and nitridation of SiO nanoclusters preferably re-deposited around the inner corner of the trenches, as well as faster Si diffusion along the slanting side walls of the trenches. This simple direction-controlled growth method is compatible with the CMOS process, and could facilitate the fabrication of alpha-Si3N4 nanoelectronic or nanophotonic devices on the Si platform.

Dielectric response and its light-induced change in undoped alpha-Si : H films below 13 MHz

The low frequency (<13 MHz) dielectric response and its light-induced change in undoped a-Si:H were investigated in detail. The dielectric constant epsilon (the real part) in this range decreases with illumination time: following a stretched exponential law similar to that found for other light-induced changes. The saturation relative change was about 0.1-0.2 % for the measured samples. The change is fading away either after repeated illumination-annealing training or by aging at room temperature. The present results indicate some rearrangement of the whole Si network caused by light soaking.

Alpha OSEK:一个基于OSEK/VDX标准实现的嵌入式实时操作系统

文章介绍了基于OSEK标准实现的嵌入式实时操作系统-AlphaOSEK。该操作系统适于深嵌入、小内存、有强实时需求的环境。介绍了操作系统的各组成模块及相关特征,还分析了AlphaOSEK对OSEK标准的优化。