Immobilized cultivation of Dictyostelium discoideum with polyurethane foam

研究了聚氨酯泡沫应用于固定化盘基网柄菌的可行性,发现以简单处理过的聚氨酯泡沫为载体,能够高效实现盘基网柄菌的固定化培养。考察了载体粒径大小、载体量和摇床转速等对固定化培养的影响,在优化的培养条件和固定化条件下,盘基网柄菌的最大细胞密度是悬浮培养的2~4倍。The immobilization of D.discoideum with polyurethane foam(PUF) was proposed and the effects of PUF's properties on the immobilization was examined.The results showed that PUF was a suitable carrier for the efficient imoblization of D.discoideum.The effects of PUF addition and culture conditions were optimized to attain high density of D.discoideum.Under optimized culture conditions,high cell density of D.discoideum(4.56×107 cells/mL) has been achieved in the shake flasks,which was 2～4 times higher than that of free-cell culture in the axenic medium.国家自然科学基金资助项目(30370039

Improving the Expression Level of Soluble Human Fas Ligand by Cultivating Recombinant Dictyostelium discoideum in CMC-ALG Microcapsules

首先研究了应用于液芯羧甲基纤维素钠-海藻酸钙(CMC-ALG)微胶囊制备中的各种化学组分对重组盘基网柄菌生长的影响,然后考察不同组分浓度制备而成的各种CMC-ALG微胶囊内重组盘基网柄菌的生长情况,从而得到较适的微胶囊制备的组分配比(CMC12g·L-1,SA8g·L-1,CaCl2100g·L-1)。结果表明,在以上较适条件下制备的微胶囊内重组盘基网柄菌的生长得到了极大的改善,最大的细胞密度比游离培养时提高了4倍,达到了8.0×107mL-1;相应的人类可溶性Fas配体(FasL)的表达水平也提高了1.5倍,达到了315μg·L-1。最后,开展了微胶囊化重组盘基网柄菌的二次重复发酵FasL的研究,结果表明,经过二次重复批次培养,最大细胞密度可达到1.24×108mL-1,为游离培养的8～10倍,而且FasL的表达水平还能维持高水平(280μg·L-1),为游离培养时的2倍。The biocompatibility between the growth of Dictyostelium discoideum AX3-pLu8 and chemical components used for the preparation of carboxy methyl cellulose-alginate(CMC-ALG)microcapsules was evaluated at first,and then the effects of the concentrations of each component forming the capsule on the growth behaviors of D.discoideum cultivated in it were studied.For microencapsulated cultivating D.discoideum,it was found that the most suitable capsule components are 12 g?L?1 of CMC,8 g?L?1 of sodium alginate(SA)and 100 g?L?1 of CaCl2.Using the microcapsules with mentioned composition,the cell density in it can reach 8.0×107 mL?1,this value is about 5 times that could be observed in suspension culture,and correspondently,in the capsules a high concentration(315 μg?L?1)of soluble human Fas ligand(shFasL)was detected,which is about 2.5 times higher than that obtained in suspension culture under the same culture condition.In addition,the immobilized cells could be used effectively for repeated batch cultivation and in the second repeated batch cultivation,a very high cell density(up to 1.24×108 mL?1)and still a high expression of FasL(280 μg?L?1)could be obtained.国家自然科学基金资助(20306025,30370039

Expression of the Soluble Human Fas Ligand in Dictyostelium discoideum

［中文文摘］用PCR扩增从激活的人中性粒细胞中得到的编码可溶性Fas配体胞外区中第14 1个到第2 81个氨基酸的cDNA ,将其与hCG_β信号肽片段融合到质粒MB12neo中,随后导入到盘基网柄菌AX3细胞中,得到分泌性表达hFasL的重组菌AX3_H3。为提高shFasL的表达量,对质粒pMB12neo作了改造,得到衍生质粒pMB74。利用质粒pMB74克隆表达shFasL ,得到高通量表达shFasL的重组菌AX3_pLu8。在复杂培养基HL_5C中,重组菌的细胞密度可达(1 5～2 )×10 7 mL ,AX3_H3及AX3_pLu8分泌的shFasL浓度分别为2 3 5 μg L及2 0 6 μg L。利用合成培养基SIH培养重组菌AX3_H3及AX3_pLu8,细胞密度均达到(4～5 )×10 7 mL ,shFasL浓度则分别达到111μg L和4 2 0 μg L。［英文文摘］An expression system is described for high-yield production of recombinant soluble human FasL (shFasL) in Dictyostelium discoideum cells. DNA encoding amino acids 141～281 of hFasL was PCR amplified from cDNA derived from activated human neutrophils. The resulting product was fused with a DNA fragment encoding hCG-β signal peptide and cloned in the expression vector pMB12neo. Dictyostelium strain AX3 was transfected with this plasmid, yielding a recombinant strain called AX3-pCESFL95-H3. In order to mprove the shFasL expression level , pMB12neo was optimized by replacing its transcriptional terminatorPpolyadenylation segment of the 2H3 gene with an actin8 terminatorPpolyadenylation segment , yielding derived expression vector pMB741 The recombinant Dictyostelium strain called AX3-pLu8 was generated with this new plasmid. When the recombinant cells were cultivated in a complex HL-5C medium, a cell density of (115～2) ×107PmL was reached , and the shFasL level expressed by strains AX3-pCESFL95-H3 and AX3-pLu8 was 2315 μgPL and 206μgPL , respectively. By using a newly developed synthetic medium called SIH as culture medium, higher cell density of (4～5) ×107PmL was achieved. Correspondently, 111 μgPL and 420 μgPL shFasL were secreted by recombinant strains AX3-pCESFL95-H3 and AX3-pLu8 ,respectively.国家自然科学基金资助项目(No.20306025,30370039

シガ イカ ダイガク シカ コウクウ ゲカガク コウザ ニ オケル コツギンコウ ノ ゲンジョウ ： ムキン ダッカイ カッセイコツ ヲ モチイタ ガクコツ サイケン ノ リンショウ セイセキ

Our department established a bone bank using autolyzed, antigen extracted, allogeneic bone (AAA bone) in which the immunogenicity is markedly reduced by chemical treatment, and has started its clinical application to reconstruction of bone defects. An outline of the bone bank and the clinical results are reported. The graft used AAA bone prepared by chemically treating (e.g. decalcification using hydrochloric acid) cortical bone of human long tubular bones of the upper and lower limbs. In 1994, we first decided to transport AAA bone from the Federal Republic Germany to Japan for clinical application. We also recruited domestic donors by asking neighboring hospitals to provide excess bone fragments resected in plastic surgery of the jaw and lower limbs amputated from severely diabetic patients due to peripheral necrosis. These procedures were carried out with the approval by the Safety Measures Division, Drug Safety Bureau, Ministry of Health and Welfare and the Ethical Committee of our University. The donors were selected according to the donor qualification criteria of the Japan Orthopedic Society and with sufficient precaution against infection of Creutzfeldt-Jakob disease. Four limbs (of 4 patients) have been provided to date, prepared into AAA bone by chemical treatments at our department, and used clinically. AAA bone implantation has been made in 16 patients to date. They were 12 males and 4 females aged 15 to 65 years. Their diseases were mandibular cysts in 13, odontoma in 1, fracture of the mandible in 1, and malignant tumor of the mandible in 1. Postoperative panoramic radiograms were obtained in 6 patients with mandibular cysts 2 years or longer after surgery, and the state of bone formation at the implantation site was observed. The oral cavity was also examined for abnormalities, and the percent efficacy was calculated from the results of both examinations. The usefulness rating was very useful in 4 and useful in 3, with a percent efficacy of 100%. The results were satisfactory also in patients followed up for a shorter period after surgery. AAA bone was removed due to infection in only 1 patient, but none of the recipients developed a serious complication. The maximum followed-up period to date is 4 years and 7 months. The postoperative course has been uneventful in all patients, with none showing abnormalities. From these results, AAA bone is considered to have the following merits: (1) By sterilization with EO gas and vacuum packing after preparation, AAA bone can be preserved at room temperature, be used at any time, and is easy to handle. (2) It is highly plastic and can be readily adjusted to various forms during surgery. (3) Implanted AAA bone is completely replaced by the recipient bone. (4) The degree of bone formation at the implantation site can be easily evaluated by radiography

Damage to mangroves from extreme cold in early 2008 in southern China

通讯作者Author for correspondence (E-mail: [email protected])2008年初,我国南方19个省经历了50年一遇的持续低温雨雪冰冻天气。极端气候对华南沿海各省的红树林区造成不同程度的危害。2008年3月,在我国南方各省红树林区的10个代表性地点,对这次寒害造成的红树植物伤害程度进行了系统的调查。结果表明:冬季低温对红树林的影响极为显著,特别是在低纬度的海南、广西和广东湛江,由于极端低温正值夜间退潮,对红树林的影响更为显著;在纬度较高的福建,本地红树种类秋茄(Kandelia obovata)、桐花树(Aegiceras corniculatum)和白骨壤(Avicennia marina)及引种的木榄(Bruguiera gymnorrhiza)等,由于长期适应于冬季较低的气温或在种植前经过抗寒锻炼,具有较强的抗寒能力。各地主要红树植物中,广布种秋茄、桐花树和白骨壤最为耐寒,其耐寒性均大于红树科的木榄、海莲(Bruguiera sexangula)和红海榄(Rhizophora stylosa)。海桑(Sonneratia caseolaris)对温度的敏感性最强,抗寒能力最低,因此,即使在其原产地海南也受到较为严重的寒害,在纬度更高的引种地出现大面积受害甚至全部死亡,而从孟加拉国引种的无瓣海桑(Sonneratia apetala)却显示出一定的抗寒能力。同一地点的红树植物幼苗的抗寒能力低于大树。此次寒害也造成了苗圃场的种苗大量死亡,成熟的植株提前落花落果,这势必会影响后继一两年内红树林的自然更新和人工造林。因此,在未来红树林造林或人工引种中,一定要考虑到红树植物的抗寒能力。国家自然科学基金(30671646和30700092);厦门大学“闽江学者”启动基

New expression vector system for glycoprotein production——Dictyostelium discoideum

近年来,盘基网柄菌作为异源重组糖蛋白表达载体的研究受到了学术界的重视,已经有多种具有生物活性的复杂糖蛋白成功地得到了表达。通过对表达产物的研究发现,盘基网柄菌具有各种翻译后加工机制,例如磷酸化、酰基化及形成GPI(糖基磷脂酰基醇)锚点等,具有类似于高等动物的糖基化修饰能力。与哺乳动物细胞表达载体相比较,盘基网柄菌具有培养成本低廉、细胞生长迅速及易于大规模培养的优势。盘基网柄菌有可能发展成为一种有重要应用前景的糖蛋白表达载体系统。A new expression system for glycoprotein production,Dictyostelium discoideum had attracted academic interests in recent years.A number of complicated glycoproteins with biological activity were expressed successfully by Dictyostelium discoideum.Based on the study of the expressed products,it was found that Dictyostelium discoideum was capable to perform post-translational modifications of protein,including glycosylation,phosphorylation,acylation and formation of glycosylphosphatidylinositol(GPI) anchors,etc.,which was similar with mammalian cells.The results showed the advantages of Dictyostelium discoideum expression system over those of Prokaryote and Eucaryote microorganisms.Comparing with mammalian cell,Dictyostelium discoideum expression system was cheap in culture medium,fast in cell growth and easy to scale up.Dictyostelium discoideum would be developed into an important expression system for glycoprotein production.国家自然科学基金(NO30370039)资

Machine for setting multi-rod screw

一种多轴螺栓拧紧机，是在拧紧机的基座上设置两组多根扳手轴，并在一组多轴扳手轴上附设一切换装置，通过操作切换装置改变扳手轴伸出的长度，使两组多根扳手轴分别进行作业，可对两种不同（或相同）型的螺栓和／或不同分布的螺栓进行拧紧操作，它为一种多轴回转工具，在本机末端工具是可更换的，当在输出端装上丝锥或钻头时，还可用于进行攻丝或钻孔等作业。这种有切换功能的螺栓拧紧机的结构设计合理，操作方便，功能齐全，解决了一机多用，提高了装配效率

Multi-axle bolt tighteming machine

一种多轴螺栓拧紧机,是在拧紧机的基座上设置两组套筒扳手轴,每组套筒扳手轴有2～10个套筒扳手,其中一组滑动套筒扳手轴由套筒扳手、与其前端插接的方轴和通过轴套安装在方轴后端的花键轴构成,且滑动套筒扳手轴上附设一由切换手柄、滑轴和拨叉组成的切换装置,其中拨叉一端通过与花键轴啮合的、可在其上滑动的轴套和花键轴连接,由切换手柄控制的可在滑轴上左右滑动的拨叉的另一端通过托座套安装在滑轴上。它具有功能多,操作方便,装配效率高等优点

Genetic diversity of the Rhizobia and screening of highefficient growth-promoting strains isolated from Sesbania cannabina in Rudong County

【目的】研究江苏如东沿海滩涂田菁根瘤菌的遗传多样性和系统发育,筛选高效促生田菁根瘤菌。【方法】采用16S rRNA 基因、持家基因(recA、atpD、glnⅡ)、共生基因(nodA、nifH)的系统发育分析,并进行温室盆栽接种试验筛选高效菌株。【结果】分离到的32株田菁根瘤菌分布于Ensifer、Neorhizobium、 Rhizobium,并与E. meliloti、N. huautlense、E. sesbaniae、R. pusense 亲缘关系最近。共生基因nodA、nifH 的系统进化分析结果基本一致,均与E. saheli 最为相近。7株代表菌株均能耐受5% 的NaCl(W/V)浓度,其中 YIC5082耐盐性最强,能够耐受6%的NaCl。温室盆栽试验表明7株代表菌株均能与田菁有效结瘤固氮,其中6株菌显著提高了植株的株高和鲜重。【结论】江苏如东沿海滩涂田菁根瘤菌具有较丰富的遗传多样性,其中N. huautlense、E. meliloti 为优势种。该地区的田菁根瘤菌多数具有较好的共生有效性及耐盐性, YIC5077促生效果最为突出,并表现出较强的固氮及结瘤能力,具有良好的应用前景