Effect of calcium on porcine ICSI embryos expressing EGFP is related to activation of ooplasmic DNase I

Several reliable methods to produce transgenic animals use the male genome. After penetration into oocytes, sperm DNA undergoes dramatic conformational changes that might represent an opportunity for exogenous DNA to integrate into the zygote genome. A nuclease,DNase I, with Ca2+/Mg2+dependent activity and Zn2+inhibition, is one of the enzymes responsible for sperm DNA remodeling. To date, the effect of different calcium concentrations in manipulation media on porcine intracytoplasmic sperm injection has not been fully investigated.The present study was conducted to examine the effect of calcium in the surrounding media, and we found that the number of embryos expressing green fluorescent protein(EGFP) was increased in media containing Ca2+. However, the number did not change over Ca2+concentrations from 2 to 8 mmol$L–1(P 0.05). Moreover, free Ca2+concentrations in the media were found to affect the efficiency which is porcine intracytoplasmic sperm injection(ICSI) embryos expressing EGFP protein, which was related to the activation of ooplasmic DNase I. These findings reveal a mechanism and pathway involving EGFP expression in ICSI embryos.     

烟草细胞凋亡过程中核酸酶活性研究

以烟草为材料 ,以 2 -甲基萘醌为诱导物 ,研究了植物细胞凋亡过程中核酸酶活性 ,结果表明 :2 -甲基萘醌诱导的烟草原生质体使鸡血红细胞发生了典型的凋亡现象 ,加入Zn2 +、EDTA、EGTA、AC -DEVD -CHO的样品无“DNAladder”出现 ;加入Ca2 +、Mg2 +后 ,“DNAladder”重新形成。 2 -甲基萘醌诱导的胞浆提取物和细胞核与只加细胞色素C诱导的胞浆提取物在电泳图上出现相同的蛋白条带 ,分子量约 36KD。从而表明 :烟草细胞凋亡过程中有特异核酸酶被激活 ,该核酸酶活性依赖于Ca2 +、Mg2 +,被Zn2 +、AC -DEVD -CHO抑制 ,该酶活性与caspase凋亡通路有关     

Microbiology. Arresting features of bacterial toxins

Bacteria produce an arsenal of sophisticated toxins that disrupt the normal processes of the host cell, usually by modifying or inactivating host cell proteins. Now, as Coburn and Leong discuss in their Perspective, members of the cytolethal distending toxin (CDT) family have been identified as enzymes that attack DNA (and not protein) within the host cell (Lara-Tejero and Galán). By attacking DNA, perhaps during chromosomal replication, CDTs cause the host cell to halt in G2 phase of the cell cycle.     

F-2毒素对体外培养猪卵巢颗粒细胞的毒害及V-E的解毒效果

采用细胞体外培养的方法,在卵巢颗粒细胞对数生长期,对F-2毒素的繁殖遗传毒性和V-E对其的解毒作用进行了研究．结果表明,各毒素组细胞活性显著低于正常对照组,而丙二醛（MDA）含量显著低于正常对照组;各解毒组细胞活性均显著高于各毒素组,但明显低于正常对照组,MDA含量显著低于各毒素组,但明显高于正常对照组．这表明,F-2毒素可能是通过脂质过氧化作用,对颗粒细胞致毒,V-E对F-2毒素具有较好的解毒作用,能提高中毒卵巢颗粒细胞的抗过氧化能力．     

苏云金芽孢杆菌的概况及其研究进展

苏云金芽孢杆菌(Bacillus thuringiensis,Bt)杀虫剂是对人和环境友好的安全型细菌杀虫剂。目前Bt杀虫剂已被广泛应用,但因使用剂量较大,导致防治害虫时费用过高。若在生物防治中使用超高效的细菌杀虫剂,就可降低其使用剂量。Bt 00-50-5菌株是从美国引进的高效杀虫菌株,其晶体蛋白的特征分析尚未完成,其毒素的作用机理研究也未进行。本项目即将开展如下研究:(1)先用具有特异性杀虫功能的Bt 00-50-5菌株进行发酵,继续完成对防治中国害虫防治效果的评价;再分析细菌的编码杀虫毒素基因和毒素的生化特征;然后混合荧光标记毒素和中肠细胞,加入昆虫中肠蛋白酶(或单独加入氨肽酶、碱性磷酸酶、ATP酶、枯草杆菌蛋白酶、钠或钾离子代之)反应,用荧光显微镜观察细胞变化;最后阐明杀虫毒素对靶标细胞作用机制。(2)通过研究毒素分子与受体分子的作用关系,拟在分子水平上阐明毒素的作用机制。     

小鼠胞内氯离子通道5基因启动子核心功能区域鉴定及转录调控分析

为研究胞内氯离子通道5基因(Chloride intracellular channel 5,CLIC5)广泛参与调节细胞内的各项生理活动与生化反应,并探讨该基因自身的表达调控机制,以小鼠基因组序列为模板,利用PCR技术扩增小鼠CLIC5基因5′上游调控序列,将其插入荧光素酶报告基因表达载体(pGL3-Basic)中,同时采用5′侧翼区缺失的方法构建了7个缺失不同DNA片段的荧光素酶表达载体。重组质粒与海肾荧光素酶载体(phRL-TK)共同瞬时转染HEK-293细胞,经双荧光素酶报告基因活性分析后,确定CLIC5基因的核心启动子区。利用生物信息学方法预测其中转录因子结合位点及启动子区甲基化状况。结果表明,CLIC5基因启动子缺乏TATA盒,但含有典型的GC盒及其他潜在转录因子结合位点;双荧光素酶报告基因活性分析表明,CLIC5基因-329～+1、-624～+1、-917～+1和-2 230～+1区域的启动子活性较高,其中-624～+1区域的启动子活性最强。进一步分析表明,启动子区-624～-329存在负性调控元件,预测存在转录因子结合位点RXR heterodimer binding sites与GC-Box factorsSp1/GC,-420～-283范围内存在CpG岛位点。     

木霉菌ECT-01-2对人参锈腐病菌的拮抗作用

以木霉菌株ECT-01(Trichoderma sp.)为出发菌株,孢子经紫外线照射处理后获得1株高拮抗活性的突变体ECT-01-2。通过对峙培养与发酵液处理,研究突变体ECT-01-2对人参锈腐菌的拮抗作用。结果表明,ECT-01-2对人参锈腐菌的抑制率高达83.68%。发酵液对人参锈腐菌丝和孢子都有一定的抑制作用,初步明确了ECT-01-2对人参锈腐菌的拮抗机理以竞争、重寄生、抗生作用及溶菌作用为主。     

莲子草假隔链格孢毒素的致病机理研究

就莲子草假隔链格孢(Nimbya alternantherae)毒素Vuculic acid对空心莲子草(Alligaror alternanthera)的致病机理进行了研究.分别用不同含量的毒素溶液处理空心莲子草离体叶片,测定其对叶片组织细胞膜透性、丙二醛(MDA)含量、细胞内Na 、K 渗漏量以及过氧化物酶(POD)、抗坏血酸过氧化物酶(APX)和过氧化氢酶(CAT)活性的影响.结果表明,空心莲子草叶片组织浸出液的相对电导率随毒素含量的增高和处理时间的增加而增大;Na 的渗漏量高于对照;叶片组织MDA的含量也高于对照;空心莲子草叶片组织内CAT和POD活性在处理前、中期(1～6 h),均随时间的延长呈上升趋势,但酶活性都低于对照;APX活性高于对照.     

Direct visualization of horizontal gene transfer

Conjugation allows bacteria to acquire genes for antibiotic resistance, novel virulence attributes, and alternative metabolic pathways. Using a fluorescent protein fusion, SeqA-YFP, we have visualized this process in real time and in single cells of Escherichia coli. We found that the F pilus mediates DNA transfer at considerable cell-to-cell distances. Integration of transferred DNA by recombination occurred in up to 96% of recipients; in the remaining cells, the transferred DNA was fully degraded by the RecBCD helicase/nuclease. The acquired integrated DNA was tracked through successive replication rounds and was found to occasionally split and segregate with different chromosomes, leading to the inheritance of different gene clusters within the cell lineage. The incidence of DNA splitting corresponds to about one crossover per cell generation.     

抑制钙信号转导对柔嫩艾美耳球虫入侵细胞的影响

 【目的】探讨柔嫩艾美耳球虫（Eimeria tenella）入侵细胞与钙信号转导之间的关系,揭示鸡柔嫩艾美耳球虫病的发生机制。【方法】采用体外狗肾细胞（madin-darby canine kidney cell,MDCK细胞）培养技术,检测了细胞外缺钙、Ca2+内流阻断剂（硝苯地平）和钙调蛋白抑制剂（三氟拉嗪）对E. tenella子孢子入侵率的影响,并测定了培养细胞上清液中乳酸脱氢酶（LDH）和细胞活性。【结果】E. tenella子孢子入侵细胞的抑制率随着细胞外Ca2+浓度的降低而升高。钙离子浓度降低到600 μmol•L-1时,入侵率（23.33%）均极显著低于对照组（P＜0.01）;细胞外无钙时,入侵抑制率高达53.18%;硝苯地平和三氟拉嗪均能极显著抑制子孢子的入侵（P＜0.01）,其中10 μmol•L-1的硝苯地平和50 μmol•L-1三氟拉嗪分别对子孢子的入侵抑制率达71.41%和97.13%,二者合用入侵抑制率可达98.59%。在E. tenella子孢子入侵细胞的过程中,MDCK细胞的活性均在90%以上,与对照组差异不显著（P＞0.05）,接种E. tenella子孢子的MDCK细胞培养液中LDH的活性与未接种的活性差异不显著（P＞0.05）。【结论】细胞外钙缺乏、钙通道阻断剂硝苯地平和钙调蛋白抑制剂三氟拉嗪对E.tenella子孢子入侵MDCK细胞均有抑制作用,但子孢子入侵对MDCK细胞的活性无明显影响。     

红细胞裂解法及不同培养条件对兔骨髓间充质干细胞体外增殖的影响

研究采用红细胞裂解液法从兔骨髓中分离BMSCs,相差显微镜观察其形态,四甲基偶氮唑盐法(MTT法)筛选基础培养液和血清浓度,绘制生长曲线,用流式细胞仪检测细胞周期,免疫细胞化学鉴定表面抗原。分离的细胞在形态学观察与生长动力学上均符合BMSCs特征;在实验范围内DMEM/F12+20%FBS是体外培养兔BMSCs的最佳体系;细胞周期结果显示,BMSCs平均82%处于G0/G1期,18%处于S+G2期;免疫细胞化学结果显示所分离的BMSCsCD90、CD44阳性表达,CD34阴性表达。本研究结果表明,通过红细胞裂解液法分离的兔BMSCs,其具有体外增殖和多向分化的潜能,可以作为组织工程的种子细胞。     

两种细胞株滴定重组猪α干扰素效价的比较研究

采用水泡性口炎病毒（VSV）作为攻击病毒,以微量细胞病变抑制法对同批次不同浓度的8组重组猪α干扰素（rPoIFN-α）样品进行了效价检测,以中国药品生物制品检定所指定使用的标化重组人α干扰素为效价滴定的参比标准,对在牛肾细胞（MDBK）株和人喉癌上皮细胞（HEp-2）株所得结果进行比较,并对细胞与待测样品按＂一步法＂和先加细胞,待细胞长满单层再加待测样品的＂两步法＂所得的rPoIFN-α抗病毒效价进行比较。结果表明：MDBK细胞株与HEp-2细胞株对于rPoIFN-α抗病毒活性检测结果无显著性差异,均可用于rPoIFN-α抗病毒活性的检测,重组人α干扰素滴定系统可用于rPoIFN-α效价检测。两种加样方法所得结果基本相同,推荐使用＂一步法＂。     

外源DNA完全酶解成分在水稻浸胚法导入中的诱变作用

外源ＤＮＡ以ＤＮＡ酶（ＤＮａｓｅⅠ）完全酶解后，浸泡处理灿型中优早２号糙米，研究了外源ＤＮＡ酶解成分在水稻浸胚法导入中的诱变作用。处理当代即表现了一定程度的变异，表明浸胚法外源ＤＮＡ导入过程中确存在着完全降解成分的诱变作用。     

玉米弯孢叶斑病菌毒素除草活性的研究

以杂草种子萌发和幼苗生长受阻为指标,测定了玉米弯孢叶斑病菌毒素的除草活性.结果表明:毒素对马唐种子的萌发有明显的抑制作用,抑制率可达100%,但对稗草、狗尾草种子萌发的抑制作用较小.在供试植物出苗3 d后喷施毒素,发现对马唐幼苗有致萎活性,但对出苗7 d的幼苗没有作用;对稗草、狗尾草幼苗无毒杀作用.安全性试验结果表明:...     

Ouabain resistance conferred by expression of the cDNA for a murine Na+, K+-ATPase alpha subunit

The molecular basis for the marked difference between primate and rodent cells in sensitivity to the cardiac glycoside ouabain has been established by genetic techniques. A complementary DNA encoding the entire alpha 1 subunit of the mouse Na+- and K+-dependent adenosine triphosphatase (ATPase) was inserted into the expression vector pSV2. This engineered DNA molecule confers resistance against 10(-4) M ouabain to monkey CV-1 cells. Deletion of sequences encoding the carboxyl terminus of the alpha 1 subunit abolish the activity of the complementary DNA. The ability to assay the biological activity of this ATPase in a transfection protocol permits the application of molecular genetic techniques to the analysis of structure-function relationships for the enzyme that establishes the internal Na+/K+ environment of most animal cells. The full-length alpha 1 subunit complementary DNA will also be useful as a dominant selectable marker for somatic cell genetic studies utilizing ouabain-sensitive cells.     

草菇培养料中微生物总DNA的高效提取

比较了化学裂解与酶裂解相结合法、氯化苄法、CTAB法、改良CTAB法、MP土壤试剂盒法和植物试剂盒法6种提取草菇培养料总DNA的方法.结果表明:不同方法的A260/A280比值有较大差异,其中化学裂解与酶裂解相结合法提取DNA的产量最高,而新型植物基因组DNA提取试剂盒提取培养料DNA的产量最低,分别为(2372.72...     

小鼠TLR3基因双敲除打靶载体构建及巨噬细胞RAW264.7TLR3-/-细胞系的建立

[目的]建立Toll样受体3(TLR3)基因缺失的小鼠巨噬细胞RAW264.7细胞系,为探索狂犬病毒感染机体过程中TLR3在固有免疫反应中的作用机制提供理论依据.[方法]采用Golden Gate Kit试剂盒组装转录激活样效应因子核酸酶(TALEN)打靶载体pTALEN-TLR3,经酶切和测序验证其连接正确后,通过脂质体瞬时转染RAW264.7细胞,转染后提取细胞DNA,用T7核酸内切酶酶切验证TALEN质粒剪切活性.[结果]TALENs左右臂分两部分连接,首先完成A、B部分的各自连接,然后分别将T1LA与T1LB、T1RA与T1RB、T2LA与T2LB、T2RA与T2RB连接,TALEN模块经过两次连接后的PCR鉴定结果显示,T1L、T1R和T2L的4个克隆均呈阳性,T2R有3个克隆呈阳性.T2L和T2R质粒共转染RAW264.7细胞后提取DNA为模板,经PCR扩增后用T7核酸内切酶进行酶切,酶切后的DNA电泳结果显示TALEN2剪切活性较强,共获得3条条带(931、555和376 bp).TALEN打靶载体pTALEN-TLR3转染RAW264.7细胞24 h后用胰酶进行消化,并加入800μg/mL G418进行筛选,7 d后获得细胞单克隆;挑选阳性细胞克隆进行T7核酸内切酶酶切鉴定及测序,结果发现4-1和4-40号细胞克隆为双敲细胞系,均缺失7 bp的核苷酸碱基,为非3整数倍碱基缺失,可造成后续基因移码突变,使细胞基因功能失活.[结论]通过TALEN技术可成功构建TLR3基因双敲除的小鼠巨噬细胞RAW264.7TLR3-/-细胞系,且可用于狂犬病毒感染细胞后细胞因子和TLR3间的关系研究.     

Complement: substitution of the terminal component in immune hemolysis by 1,10-phenanthroline

The participation of metal ions in the terminal step of immune cytolysis was investigated with chelating agents. 1,10-Phenanthroline induced lysis of sensitized sheep erythrocytes which had reacted with the first eight components of human complement. Hemolysis caused by 1,10-phenanthroline resembled lysis produced by the ninth component of complement in dependence on cell-bound eighth component and on temperature and in inhibition by 0.09M ethylenediaminetetraacetic acid. Bivalent metal ions reduced the hemolytic capacity of 1,10-phenanthroline, and Fe(++) inhibited the activity of the ninth component. Since trivalent iron had no such effects, it is postulated that the hemolytic activity of 1,10-phenanthroline and the ninth component of complement is a function of their affinity for Fe(++).     

氧氟沙星与Cu2+复合物对非洲绿猴肾细胞(Vero)的毒性效应

抗生素氧氟沙星(Ofloxacin,OFL)与重金属铜离子(Cu~(2+))复合污染对离体培养的非洲绿猴肾细胞(Vero)的生长抑制作用及毒性效应,是值得关注的热点问题。通过模拟试验,研究了抗生素OFL与Cu~(2+)及其复合物对非洲绿猴肾活体细胞(Vero)的毒性效应。在正常体细胞环境下培养非洲绿猴肾细胞(Vero)3 d,加入OFL浓度为0.63、1.25、2.5、5、10 mg·L~(-1),加入Cu~(2+)浓度为2.75、6.88mg·L~(-1),反应24 h,并通过MTT法检测OFL与Cu~(2+)及其复合物对非洲绿猴肾细胞生长的抑制率。结果表明:OFL与Cu~(2+)及其复合物会导致细胞形态变化,细胞破碎,贴壁率降低。随着Cu~(2+)浓度增加,Vero细胞生长抑制率逐渐上升;OFL对Vero细胞生长有显著的影响。在2.75 mg·L~(-1)Cu~(2+)浓度时,OFL与Cu~(2+)的复合物对细胞的抑制率在30%到36%之间;当OFL浓度为2.5、5、10 mg·L~(-1)时,OFL与Cu~(2+)的复合物对细胞的抑制率与OFL、Cu~(2+)单一抑制率之和有显著差异,但随OFL浓度的变化没有显著差异;在6.88 mg·L~(-1)Cu~(2+)浓度处理时,OFL浓度为1.25、2.5 mg·L~(-1)时,OFL与Cu~(2+)复合物对细胞抑制率的实测值与OFL、Cu~(2+)单一抑制率之和之间没有显著差异;而当OFL浓度为0.63、5、10 mg·L~(-1)时,OFL与Cu~(2+)的复合物对细胞抑制率的实测值显著低于OFL、Cu~(2+)单一抑制率之和;OFL与Cu~(2+)的复合物对细胞的抑制率随着OFL浓度的增加而增加。OFL与Cu~(2+)的复合物会对Vero细胞产生毒性效应,并表现为抑制细胞生长,对细胞产生了复合污染;OFL与Cu~(2+)的复合物对细胞生长的抑制率小于OFL与Cu~(2+)单一对细胞生长抑制率之和,OFL与Cu~(2+)对细胞生长的毒性具有拮抗作用。     

Isolation and Structural Indentification of Herbicidal Toxin Fractions Produced by Pythium aphanidermatum

In order to understand the compsition and structure of herbicidal component of Pythium aphanidermatum,the isolation and structural indentification were researched.The culture filtrate was extracted by ethyl acetate,petroleum,and chloroform with the same volume respectively and the activity of the crude toxin was bioassayed.The toxin was separated by using the method of thin layer chromatography（TLC）,then the main fraction was separated by HPLC,and the structure was analyzed by the sepctrum of IR,13C-NMR and 1HNMR.The results showed that the ethyl acetate extracts had the strongest herbicidal activity.Using the method of TLC,the bioassay results showed that the extracts with Rf 0.19 had the strongest effect on weeds and the inhibition to Digitaria sanguinalis and Amaranthus retroflexus reached five levels,and the component was proved to be dimethyl o-phthalate from the spectrum of IR,13C-NMR and 1HNMR,which was one of the components from the toxin,and it had herbicidal activity.