一株鲢鱼肠道中嗜水气单胞菌拮抗菌株的筛选、鉴定及特性

从鲢鱼肠道中筛选嗜水气单胞菌的拮抗菌株,并对生长培养条件进行研究,以期为鲢鱼养殖过程中嗜水气单胞菌病的生物防治提供理论依据。通过对鲢鱼肠道中嗜水气单胞菌拮抗菌株的分离、筛选,并对其中拮抗作用较稳定的菌株进行生理生化、分子鉴定以及生长培养特性优化。经过多次筛选得到了15株拮抗效果较好的目标菌株,其中拮抗作用较稳定的S4为气单胞菌属,是偏碱性革兰氏阴性短杆菌;通过单因素和正交实验表明,S4最佳生长培养条件为1.5 g/d L淀粉,1.5 g/d L牛肉膏,1 g/d L氯化钙,32℃,pH 8.5,接种体积分数3%。研究结果为嗜水气单胞菌的防治及拮抗菌S4的应用提供理论依据。

Connective tissue growth factor hammerhead ribozyme attenuates human hepatic stellate cell function

AIM： To determine the effect of hammerhead ribozyme targeting connective tissue growth factor （CCN2） on human hepatic stellate cell （HSC） function.METHODS： CCN2 hammerhead ribozyme cDNA plus two self-cleaving sequences were inserted into pTriEx2 to produce pTriCCN2-Rz. Each vector was individually transfected into cultured LX-2 human HSCs, which were then stimulated by addition of transforming growth factor （TGF）-β1 to the culture medium. Semiquantitative RT-PCR was used to determine mRNA levels for CCN2 or collagen I, while protein levels of each molecule in cell /ysates and conditioned medium were measured by ELISA. Cell-cycle progression of the transfected cells was assessed by flow cytometry.RESULTS： In pTriEx2-transfected LX-2 cells, TGF-β1 treatment caused an increase in the mRNA level for CCN2 or collagen I, and an increase in produced and secreted CCN2 or extracellular collagen I protein levels, pTriCCN2-Rz-transfected LX-2 cells showed decreased basal CCN2 or collagen mRNA levels, as well as produced and secreted CCN2 or collagen I protein. Furthermore, the TGF-β1-induced increase in mRNA or protein for CCN2 or collagen I was inhibited partially in pTriCCN2-Rz-transfected LX-2 cells. Inhibition of CCN2 using hammerhead ribozyme cDNA resulted in fewer of the cells transitioning into S phase.CONCLUSION： Endogenous CCN2 is a mediator of basal or TGF-β1-induced collagen I production in human HSCs and regulates entry of the cells into S phase.

Molecular identification and culture trials of Eutreptiella gymnastica (Eutreptiales, Euglenophyceae)

During a survey for marine microalgal resources, we isolated a rare marine euglenoid from the coastal waters of Qingdao, China in 2009, and established a pure culture. Electron microscopic and molecular phylogenetic （18S rDNA and 16S rDNA sequences） analyses revealed a close affinity with Eutreptiella gymnastica, a bloom-forming species. Different culture conditions were monitored to understand optimal E. gymnastica growth characteristics. The optimal growth conditions in a batch culture of this isolate were 20C, 160 gmol photons/（mE.s） of white light, and a salinity of 10-31. Nutrient experiments demonstrated that growth increased dramatically with a phosphorus concentration greater than 72 gmol/L. Understanding the effect of culture conditions on E. gymnastica may help understanding the blooming mechanism of this alga in its natural environment.

Feeder-free maintenance of hESCs in mesenchymal stem cell-conditioned media： distinct requirements for TGF-β and IGF-Ⅱ

A paracrine regulation was recently proposed in human embryonic stem cells （hESCs） grown in mouse embryonic fibroblast （MEF）-conditioned media （MEF-CM）, where hESCs spontaneously differentiate into autologous fibroblastlike cells to maintain culture homeostasis by producing TGF-β and insulin-like growth factor-lI （IGF-Ⅱ） in response to basic fibroblast growth factor （bFGF）. Although the importance of TGF-β family members in the maintenance of pluripotency of hESCs is widely established, very little is known about the role of IGF-Ⅱ. In order to ease hESC cul- ture conditions and to reduce xenogenic components, we sought （i） to determine whether hESCs can be maintained stable and pluripotent using CM from human foreskin fibroblasts （HFFs） and human mesenchymal stem cells （hM- SCs） rather than MEF-CM, and （ii） to analyze whether the cooperation of bFGF with TGF-β and IGF-Ⅱ to maintain hESCs in MEF-CM may be extrapolated to hESCs maintained in allogeneic mesenchymal stem cell （MSC）-CM and HFF-CM. We found that MSCs and HFFs express all FGF receptors （FGFR1-4） and specifically produce TGF-β in response to bFGF. However, HFFs but not MSCs secrete IGF-Ⅱ. Despite the absence of IGF-Ⅱ in MSC-CM, hESC pluripotency and culture homeostasis were successfully maintained in MSC-CM for over 37 passages. Human ESCs derived on MSCs and hESCs maintained in MSC-CM retained hESC morphology, euploidy, expression of surface markers and transcription factors linked to pluripotency and displayed in vitro and in vivo multilineage developmental potential, suggesting that IGF-Ⅱ may be dispensable for hESC pluripotency. In fact, IGF-Ⅱ blocking had no effect on the homeostasis of hESC cultures maintained either on HFF-CM or on MSC-CM. These data indicate that hESCs are successfully maintained feeder-free with IGF-Ⅱ-lacking MSC-CM, and that the previously proposed paracrine mechanism by which bFGF cooperates with TGF-β and IGF-Ⅱ in the maintenance of hESCs in MEF-CM may not be fully extrapolated to hESCs maintained in CM from human MSCs.

Scale-up of NaA zeolite membranes on α-Al_2O_3 hollow fibers by a secondary growth method with vacuum seeding

NaA zeolite membranes were prepared by secondary growth method on the outer surface of α-Al2O3 hollow fiber supports. Vacuum seeding method was used for planting zeolite seeds on the support surfaces. Hydrothermal crystallization was then carried out in a synthesis solution with molar ratio of Al2O3：SiO2：Na2O：H2O = 1：2：2：120 at 100 ℃ for 4 h. Effects of seeding conditions on preparation of hollow fiber NaA zeolite membranes were extensively investigated. Moreover, hollow fiber membrane modules with packing membrane areas of ca. 0.1 and 0.2 m2 were fabricated to separate ethanol/water mixture. It is found that the thickness of seed layer is obviously affected by seed suspension concentration, coating time and vacuum degree. Close-packing seed layer is required to obtain high-quality membranes. The optimized seeding conditions （seed suspension mass concentration of 0.5%-0.7% coating time of 5 s and vacuum degree of 10 kPa） lead to dense NaA zeolite layer with a thickness of 6-8 gin. Typically, an as-synthesized hollow fiber NaA zeolite membrane exhibits good pervaporation performance with a permeation flux of 7.02 kg· m^- 2· h^- 1 and separation factor 〉 10000 for sepa- ration of 90%; （by mass） ethanol/water mixture at 75 ℃ High reproducibility has been achieved for batch-scale production of hollow fiber NaA zeolite membranes by the hydrothermal synthesis approach.

Effect of Plant Hormones on Callus Induction from Fruit and Seedling Explants of Chilli Pepper （Capsicum annuum L.）

This experiment was conducted to optimize the culture conditions to induce calli from chilli pepper （Capsicum annuum L.） local cultivar. The mature seeds were surface sterilized for one min with 70% Ethanol followed by 20 min with （0%, 2%, 4% or 6%） NaOCI and were germinated on MS medium with 2 mg/L GA3. Seedlings and mature fruits were used as explants source. The placenta, pericarp, hypocotyls, cotyledonal leaves, shoot tips and roots were cultured on MS media supplemented with Kinetin （0.0 mg/L, 0.5 mg/L, 1.0 mg/L, 2.0 mg/L） and IAA （0.0 mg/L, 1.0 mg/L, 2.0 mg/L） in different combinations or NAA or 2,4-D （0.0 mg/L, 1.0 mg/L, 2.0 mg/L, 4.0 mg/L）. Callus fresh weight was recorded after 4 weeks in culture. The results showed that the best sterilizing method was with 70% Ethanol followed by 20 mints with （4% or 6%） NaOCI, however 6% NaOCI reduced seed＇s viability. Callus was induced from all explants cultured on MS media supplemented with IAA and Kinetin except the placenta and the pericarp. The results showed that the hypocotyls surpass all other explants in the mean callus fresh weight which was 160.58 mg compared with 147.81 mg, 134.95 mg, and 122.33 mg for cotyledonal leaves, shoot tips and roots respectively. Moreover the analysis of the interaction between the growth regulators and the explants showed that 2 mg/L IAA and Kinetin had significant effect on callus mean fresh weight which was （309.74, 339.14, 358.48, and 284.64） mg for the shoot tips, cotyledonal leaves, hypocotyls and roots, respectively. On the other hand, 2 mg/L 2,4-D or NAA was the best concentration for callus induction from the placenta and the pericarp. The pericarp gave a mean fresh weight of 276.90 mg in the presence of 2,4-D compared with 253.60 mg for the placenta. Moreover the pericarps gave significantly higher fresh weight than the placenta with an average of 210.3mg and 184.9 mg respectively in the presence of 2 mg/L NAA. In conclusion the best sterilization method of chilli pepper seeds is by 70% ethanol for one minute followed by 20 min in 4% （NaOCI）. The best explants for callus induction only is the Hypocotyls grown on MS medium supplemented with 2 mg/L oflAA and Kinetin under the conditions of the current experiment.

Effect of Growing Conditions of Rice Donor Plants on Anther Culture in Vitro

An experiment was conducted to find the effect of three types of donor plants growing conditions （growth chamber, open space ＋ growth chambers and open space） on callus induction and subsequent plant regeneration and productive shoot regeneration from the anthers culture in vitro of four rice hybrid （1-2, 2-1, 7-1, 13-3） developed by Primorsky Scientific Research Institute of Agriculture. Five variants of N6 medium （N6-I, N6-2, N6-3, Mix-l, N6-4） were used as basal medium. Mean value of callus induction frequency on three types of conditions ranged from 5.68% to 9.44% and the difference was non-significantly. In general, callus derived from donor plants grown on condition of open space ＋ growth chambers showed significantly better performances for plant regeneration （0.23 green regenerants on anther and 3.77 green regenerants on callus） and productive shoot regeneration （0.06 productive regenerants on anther and 0.56 productive regenerants on callus）. Favourable conditions for donor plant growth in open space positively affect on callus induction and regenaration. It is possible to get assured results on many hybrids, but not the highest. In growth chamber, frequency of callus induction can be the maximal only on some samples, few hybrids are resulted in deficiency of callus induction.