Effects of Arg26 and Lys27 mutation on the bioactivity of HNTX-Ⅳ

Hainantoxin-Ⅳ (HNTX-Ⅳ)was isolated from the Chinese bird spider Ornithoctorcs hainana and identified as a novel antagonist of tetrodotoxin-sensitive (TTX-S)sodium channels.As revealed by the solution structure of HNTX-Ⅳ solved by two-dimensional nuclear magnetic resonance (2D-NMR),HNTX-Ⅳ adopts an inhibitor cystine knot motif.To check the role of basic residues during HNTX-Ⅳ's interaction with TTX-S sodium channels,R26A and K27A mutants of HNTX-Ⅳ were constructed by solid-phase chemical synthesis.The synthesized peptides were purified and refolded under optimized oxidation conditions.Correct synthesis and folding were confirmed by MALDI-TOF mass spectrometry and NMR spectroscopy,respectively.Using the whole-cell patch-clamp technique,Lys27 but not Arg26 was identified as a key residue for HNTX-Ⅳ's bioactivity against TTX-S sodium channels,because R26A-HNTX-Ⅳ showed slightly reduced activity and K27A-HNTX-Ⅳ showed almost no inhibition.     

重组泛素连接酶SH2-U—box／RING的构建与表达

目的构建重组泛素连接酶SH2-U—box、SH2-RING,并克隆进入pFlag—CMV4真核表达载体,为研究靶向降解慢性粒细胞白血病（chronic myelocytic leukemia,CML）患者瘤细胞中过度活化的BCR／ABL,抑制肿瘤细胞的生长提供基础。方法设计引物,扩增接头分子Grb2的SH2结构域以及E3泛素连接酶CHIP的U—box、Cb1的RING结构域,通过重组PCR,将SH2分别与U—box、RING进行融合,融合片段双酶切之后插入真核表达载体pFlag—CMV4,经过酶切鉴定及测序后,转染HEK293T细胞,Western印迹验证重组质粒的表达。结果PCR结果提示SH2-U—box条带大小888bp,SH2一RING大小为633bp,重组质粒酶切鉴定和测序结果均正确,转染后可见融合蛋白的表达。结论成功构建真核重组表达载体pFlag—CMV4-SH2-U—box和pFlag—CMV4-SH2-RING,转染HEK293T细胞后能够正确表达,为后续研究奠定了基础。     

曲古抑菌素A对体外培养牛成纤维细胞组蛋白乙酰化和甲基化的影响

Trichostatin A(TSA)是一种特异的组蛋白去乙酰化酶抑制剂。研究显示,TSA可以特异地抑制组蛋白去乙酰化酶活性,提高细胞的组蛋白乙酰化水平,激活基因的表达。但是,目前还不是很清楚TSA处理是否对组蛋白甲基化产生影响。本研究以成纤维细胞为研究对象,利用免疫细胞化学技术及激光共聚焦显微镜,探讨了TSA处理体细胞对其组蛋白乙酰化及甲基化修饰的影响。结果显示,随TSA浓度增加,体细胞形态发生明显的改变,细胞变得扁平且核区较大,处理后组蛋白H4K8位点的乙酰化水平随着TSA浓度的增加明显提高。检测组蛋白H3上两个甲基化位点发现,随组蛋白乙酰化水平的增加,H3K4位点的三甲基化（H3K4me3）水平也显著提高。但是,对于H3K9的二甲基化水平（H3K9me2）则没有明显变化。以上结果显示,TSA的处理不仅可以提高体细胞的组蛋白乙酰化水平,同时也增加了与基因表达激活相关组蛋白修饰位点的甲基化水平,但是对于与沉默基因相关的组蛋白修饰位点则没有明显的影响。     

两种菌株来源的glyA基因的克隆、表达及酶活性检测

采用PCR方法,分别从大肠杆菌和嗜热链球菌基因组DNA中扩增获得glyA基因,分别克隆入载体pET-28 a(+)中并进行表达,分离和纯化得到两种不同来源的SHMT,分别检测两种SHMT的逆向酶活。比较来源于大肠杆菌K12与嗜热链球菌AS1.2471中的glyA基因表达的丝氨酸羟甲基转移酶(SHMT)的活性,以获得高活性的SHMT。结果成功获得两种菌中的glyA基因,并表达出具有较高活性的SHMT,其中嗜热链球菌中glyA基因表达出的SHMT的酶活性大约为大肠杆菌的两倍。从嗜热链球菌中克隆表达的SHMT具有更高的催化活性及良好的工业应用前景。     

PTEN在人食管上皮永生化细胞株和食管癌细胞株中的表达

目的:探讨人胚食管上皮永生化细胞株SHEE、SHEEMT、食管癌细胞株EC8712中PTEN表达的差,判断食管上皮细胞在恶性转化过程中是否有PTEN缺失现象的发生;方法:培养三种细胞株,采用免疫组织化学、激光共聚焦显微镜、流式细胞仪和Westernblot等检测方法对三种细胞株中PTEN的表达进行检测。结果:三种细胞株均有PTEN的表达,表达强度与分化程度有关,PTEN在三种细胞株中表达强弱顺序为SHEE>SHEEMT>EC8712,差异有统计学意义(P<0.01);结论:PTEN在SHEE、SHEEMT和EC8712三种来源于食管鳞状上皮但分化程度不同的细胞株均中表达,表达强度与分化程度相关,分化程度越高,表达越强。     

Biodegradation of poly(ε-caprolactone) (PCL) by a new Penicillium oxalicum strain DSYD05-1

In this study, fungi isolated from soil were screened for their ability to form clear zones on agar plates with emulsified poly(ε-caprolactone) (PCL). The most active strain, designated as DSYD05, was identified as Penicillium oxalicum on the basis of morphological characteristics and phylogenetic analysis. Mutant DSYD05-1, obtained by ultraviolet-light mutagenesis from strain DSYD05, was more effective in PCL degradation. In liquid cultures of the mutant strain with PCL emulsion, DSYD05-1 showed the highest PCL-degrading activity after 4?days of cultivation. The products of PCL degradation were analysed by mass spectrometry; the results indicated that 6-hydroxyhexanoic acid was produced and assimilated during cultivation. The degradation of PCL film by DSYD05-1 was observed by scanning electron microscopy, and was indicative of a three-stage degradation process. The degradation of amorphous parts of the film preceded that of the crystalline center and then the peripheral crystalline regions. In addition, DSYD05-1 showed a wide range of substrate specificity, with capability to degrade PCL, poly(β-hydroxybutyrate), and poly(butylene succinate), but not poly(lactic acid), indicating that the strain could have potential for application in the treatment or recycling of bio-plastic wastes.     

电针诱导心肌缺血大鼠延髓头端腹外侧区nNOS和iNOS差异表达

许多研究表明,延髓头端腹外侧区(rostral ventrolateml medulla,RVLM)的NO/NOS系统参与心血管活动的中枢调节.本实验以结扎Wistar大鼠左冠状动脉前降支法建立急性心肌缺血(acute myocardial ischemia,AMI)动物模型,观察针刺"内关"穴改善AMI大鼠的心功能作用,同时检测大鼠RVLM区神经元型一氧化氮合酶(neuronal nitric oxide synthase,nNOS)和诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)表达的变化,进而探讨针刺治疗AMI的中枢机制.实验观察显示,AMI大鼠心功能各项指标减弱,伴随外周血去甲肾上腺素(norepinephrine,NE)和脑钠肽(brain natriuretic peptide,BNP)水平显著升高,同时RVLM区nNOS阳性神经元数和nNOS mRNA表达升高,而iNOS水平则降低.针刺"内关"穴(Pe 6)(每天30 min,连续5天)改善心功能,降低AMI大鼠血清中NE和BNP的水平,同时升高iNOS并降低nNOS在RVLM的表达.以上结果提示,针刺治疗心肌缺血的同时可以调节iNOS/NO和nNOS/NO在RVLM的变化,这可能与针刺通过调节RVLM区的NO含量进而降低交感传出,从而改善AMI大鼠的心功能有关.     

应用微卫星标记对雌核发育银鲫的遗传多样性初探

利用Crooijmans et al.(1997)分离的包含CA重复单元的普通鲤鱼（Cyprinus carpino.L)的8个微卫星DNA标记,对银鲫（Carassius auratus gibelio Bloch)的5个不同雌核发育系的24尾个体进行PCR扩增。分析电泳结果发现,除MFW28未能在银鲫中稳定地扩增出相应的同源序列,其余的7对引物扩增的重复性和稳定性都很好,随引物不同,各等位基因数为1-14个,大小在100-506bp。在MFW1、MFW4、MFW19、MFW20、MFW23和MFW246个微卫星的扩增图谱中,不同的雌核发育系扩增出各自独特的图谱,而同一系内的不同个体间具有高度的遗传同质性,但仍然在个别个体中检测到少量的多态片段。不同系间的扩增图谱呈现出高度的遗传异质性,共鉴定出23个可以用于有效区分5个不同雌核发育系的分子标记。这5个微卫星标记反映了银鲫5个雌核发育系间的相互亲缘关系,其中P和A系同属一个雌核发育系,F系起源于E系,A、D和E系可能分别独立地起源于不同的杂交事件,鉴定的微卫星分子标记为进行银鲫群体遗传学和进化遗传学研究,以及银鲫的分子标记育种和进行基因组作图提供了理想的工具。     

含硫多肽类抗生素那西肽的菌种诱变及产量提高

那西肽（Ｎｏｓｉｈｅｐｔｉｄｅ）产生菌Ｓｔｒｅｐｔｏｍｙｃｅｓ ａｃｔｕｏｓｕｓ Ｚ１１,经ＮＴＧ处理和对Ｃｕ＾２＋,Ｍｎ＾２＋耐受选育,获得突变株Ｚ１５,其可耐受Ｃｕ＾２＋为３００μｇ／ｍｌ,耐Ｍｎ＾２＋１０００μｇ／ｍｌ,产生那西肽可达１５００μｇ／ｍｌ是Ｚ１１活力的１．６倍。