ASGP受体介导的体内基因转移

我们针对肝细胞特异的ＡＳＧＰ（去唾液酸糖蛋白）受体,构建了一种具有肝细胞特异导向性的基因转移载体系统,该载体包括两种共价结合的功能成分：一种为ＡＳＧＰ,作为配体,与肝细胞表面特异的ＡＳＧＰ受体结合;另一种为多聚左旋赖氨酸（ｐｏｌｙ－Ｌ－ｌｙｓｉｎｅ）,与ＤＮＡ以强静电作用相结合。小鼠尾静脉注射３２Ｐ－ＤＮＡ－ｐｏｌｙ－Ｌ－ｌｙｓｉｎｅ－ＡＳＯＲ（去唾液酸ａｌ酸性糖蛋白）或等量的３２Ｐ－ＤＮＡ、ＡＳＯＲ、ｐｏｌｙ－ＤＬ－ｌｙｓｉｎｅ单体混合物,２０小时后,小鼠肝、脾、肾各组织的放射性计数结果表明该载体系统具有较强的肝组织特异性。     

用^32P标记cDNA探针进行核酸斑点杂交检测马铃薯卷叶病毒（PLRV）

利用克隆的马铃薯卷叶病毒（ＰＬＲＶ）外壳蛋白（ＣＰ）基因ｃＤＮＡ,用切口平移法制成＾３２Ｐ标记探针,通过核酸斑点杂交,对马铃薯卷叶病毒ＲＮＡ,提纯的马铃薯卷法病毒和感染ＰＬＲＶ的马铃薯茎、叶、声     

鱼类LZF—I DNA指纹探针的设备

利用Ｏｌｉｇｏ １０００ＤＮＡ合成仪合成了长庶４５ｂｐ的寡核苷酸单链,经纯化后用同位素γ－＾３２Ｐ－ＡＴＰ作５‘末端票房后,制备鱼类ＬＺＦ－Ｉ ＤＮＡ指纹探针。通过对鱼类的九体实验,亲子鉴定实验,组织细胞的稳定性实验和鱼类种类的适用范围实验后,测得：（１）ＬＺＦ－Ｉ ＤＮＡ指纹探针属多位点寡核苷酸探针;（２）ＬＺＦ－Ｉ ＤＮＡ指纹殖在鱼类种群中的临别机率为９．２３－１０＾０１６;（３）ＬＺＦ－Ｉ     

IL—2重组杆状病毒载体的构建及表达

将人白细胞介素２（ＩＬ－２）ｃＤＮＡ插入苜蓿银纹夜蛾核型多角体病毒（ＡｃＮ－ＰＶ）的转移载体ｐＶＬ１３９２中。经限制性酶切和ＤＮＡ杂交筛选、鉴定出重组转移载体ｐＶＬ１３９２－ＩＬ２。重组载体通过在昆虫细胞内共转染将ＩＬ－２ｃＤＮＡ转移到野生型ＡｃＮＰＶＤＮＡ中。经３２Ｐ标记探针鉴定出重组病毒Ａｃ１３９２－ＩＬ２。用重组病毒感染昆虫细胞,结果表达产物有明显刺激ＣＴＬＬ细胞生长,［３Ｈ］－ＴｄＲ掺入法检测ＩＬ－２活性为１５００ＩＵ／ｍ１。     

水稻蜡质基因5’端上游顺序中的核蛋白结合区

通过ｗｘ基因转录起始点上游２１２０ｂｐ长的ＤＮＡ序列的测定,用不同的限制性内切酶对此片段进行消化,获得１５个大小不同的ＤＮＡ片段并构建成不同的亚克隆。这些片段经３２Ｐ标记后分别做探针同水稻不同组织来源的核蛋白进行凝胶滞后实验和特异的竞争实验,表明ＨｉｎｆＩａ－２、ＨｉｎｆＩｃ－１、ＨｉｎｆＩｃ－２、ＨｉｎｆＩｄ、ＲｓａＩａ和ＲｓａＩｅ（ＲｓａＩａ片段与ＨｉｎｆＩａ、ＨｉｎｆＩｃ片段重叠）片段能与从未成熟的水稻种子中抽提的核蛋白结合。     

人绒毛膜促性腺激素α、β亚基cDNA的筛选与序列分析

构建３周龄人胚ｃＤＮＡ 文库,用［α－３２Ｐ］ｄＣＴＰ标记的一链ｃＤＮＡ 为探针从中筛选高度表达的克隆子并测序,得到人绒毛膜促性腺激素（ＨＣＧ）的α和β亚基ｃＤＮＡ 克隆,序列同源性分析表明：α亚基编码区的ｃＤＮＡ 序列与已报道的完全同源,β亚基编码区第３０位碱基Ｇ→Ａ,该碱基的改变不引起氨基酸的改变．另外,在α和β亚基非编码区发现有多处碱基的改变．     

抗真菌药——瞄准着DNA拓扑异构酶

抗真菌药──瞄准着ＤＮＡ拓扑异构酶徐钤（第一军医大学生化教研室，广州５１０５１５）关键词抗真菌药，ＤＮＡ拓扑异构酶１、ＤＮＡ拓扑异构酶的功能ＤＮＡ拓扑异构酶（ＤＮＡｔｏｐｏｉｓｏｍｅｒａｓｅ，ＴＯＰＯ）能改变（或调整）ＤＮＡ的拓扑学结构。ＴＯＰＯｓ可...     

中国5种珍稀绢蝶非损伤取样的mtDNA序列及系统进化

应用非损伤性取样ＤＮＡ测序技术测定了４种来自云南白马雪山和１种来自新疆天山的５种珍稀绢蝶的线粒体ＤＮＡ细胞色素ｂ基因部分ＤＮＡ序列。在获得的４３３ｂｐ的序列中,Ａ＋Ｔ约占７５．４％,其中４０个核苷酸位点存在变异（约９．２４％）。ＤＮＡ一级序列数据显示,该５种绢蝶间ＤＮＡ序列变异丰富。ＰＡＵＰ３．１．１（简约法）数据分析软件构建该５个种绢蝶的分子系统树显示,爱珂绢蝶和巴裔绢蝶的亲缘关系比较接近,阿波     

荧光素标记JH12.6探针进行的人DNA指纹图分析

采用荧光素标记和鲁米诺化学发光技术,建立了ＤＮＡ指纹图的分析方法。用该方法标记ＪＨ１２．６探针获得了清晰易读、分辨率高的人ＤＮＡ指纹图。经对云南省７８名无关个体进行ＤＮＡ指纹图分析,计算出无关个体的相关机率为７．４×１０（－１１）,平均等位基因频率为０．０９。比较同位素（３２）Ｐ标记方法表明,用荧光素标记ＪＨ１２．６探针进行人ＤＮＡ指纹图分析,具有简便、快速、安全、经济的特点,可在法医学鉴定及其它领域里推广应用。     

一个新的受雄激素诱导的胞浆蛋白的组织分布

本文以３２Ｐ－５＇末端标记ＰＳＢＰ－Ｃ３基因启动子的３１ｂｐＤＮＡ片段作探针，用凝胶阻滞分析法检测了大鼠、小鼠、兔和猴各组织以及人病理组织的胞浆中与此ＤＮＡ片段特异结合的新蛋白质（Ｃ３Ｐ４），以期对其功能的探讨提供必要的线索。结果显示：（１）Ｃ３Ｐ４蛋白以不同的含量存在于４种动物的雄性器官中；（２）４种动物的脑组织均含有此蛋白质，其他组织中很难检测到，且其结果因种族而异：（３）雌性动物中此蛋白质的组织分布除性器官外，与雄性动物相同；（４）在所测定的８种人器官的几种病理组织中，初步观察到胃癌中含量较高，乳房的良性和恶性肿瘤中都有Ｃ２Ｐ４蛋白存在，７例前列腺肥大中有３例比较明显。     

Complement component 5a (C5a)

The 74 amino acid glycoprotein, complement component 5a (C5a), is a potent pro-inflammatory mediator cleaved enzymatically from its precursor, C5, upon activation of the complement cascade. C5a is quickly metabolised by carboxypeptidases, forming the less potent C5adesArg. Acting via a classical G protein-coupled receptor, CD88, C5a and C5adesArg exert a number of effects essential to the innate immune response, while their actions at the more recently discovered non-G protein-coupled receptor, C5L2 (or GPR77), remain unclear. The widespread expression of C5a receptors throughout the body allows C5a to elicit a broad range of effects. Thus, C5a has been found to be a significant pathogenic driver in a number of immuno-inflammatory diseases, making C5a inhibition an attractive therapeutic strategy.     

Activation of 2-5A-dependent RNase by analogs of 2-5A (5'-O-triphosphoryladenylyl(2'----5')adenylyl(2'----5')adenosine ) using 2',5'-tetraadenylate (core)-cellulose

A variety of 2-5A (px(A2'p)nA; x = 2 or 3, n greater than or equal to 2) analogs were assayed for their abilities to activate murine 2-5A-dependent RNase (subsequently "the nuclease") using a recently developed method. This technique consists of immobilizing and partially purifying the nuclease using core-cellulose [A2'p)3A-cellulose) and then monitoring the breakdown of poly(U)-3'-[32P]Cp into acid-soluble fragments. Several 5'-adenosinecapped analogs of 2-5A (containing a tetra-, tri-, or diphosphate) were analyzed, and it was found that reducing the number of phosphoryl groups between the 5' to 5'-diadenosine linkages resulted in a progressive loss of activity. Because A5' pppp(A2'p)3A was a potent activator of the nuclease yet stable during the assay these results suggested that a free 5'-phosphoryl group may not be required for the activation of the nuclease. A number of 8-bromoadenosine-substituted analogs of 2-5A were also studied. Curiously, the brominations decreased the activities of the 5'-di- and triphosphorylated molecules while substantially increasing the activities of the 5'-monophosphorylated species. The results indicated that a tri- or diphosphate moiety on the 5'-end of 2-5A or the presence of ATP is not absolutely required for the nuclease to be active. Furthermore, the ATP analog, beta, gamma-methylene ATP, did not inhibit the activity of the nuclease. Finally, a 3',5'-phosphodiester linkage isomer of 2-5A and a 3'-deoxy (cordycepin) analog of 2-5A were tested, and both were found to be completely without activity.     

Structure of the Parainfluenza Virus 5 (PIV5) Hemagglutinin-Neuraminidase (HN) Ectodomain

Paramyxoviruses cause a wide variety of human and animal diseases. They infect host cells using the coordinated action of two surface glycoproteins, the receptor binding protein (HN, H, or G) and the fusion protein (F). HN binds sialic acid on host cells (hemagglutinin activity) and hydrolyzes these receptors during viral egress (neuraminidase activity, NA). Additionally, receptor binding is thought to induce a conformational change in HN that subsequently triggers major refolding in homotypic F, resulting in fusion of virus and target cell membranes. HN is an oligomeric type II transmembrane protein with a short cytoplasmic domain and a large ectodomain comprising a long helical stalk and large globular head domain containing the enzymatic functions (NA domain). Extensive biochemical characterization has revealed that HN-stalk residues determine F specificity and activation. However, the F/HN interaction and the mechanisms whereby receptor binding regulates F activation are poorly defined. Recently, a structure of Newcastle disease virus (NDV) HN ectodomain revealed the heads (NA domains) in a “4-heads-down” conformation whereby two of the heads form a symmetrical interaction with two sides of the stalk. The interface includes stalk residues implicated in triggering F, and the heads sterically shield these residues from interaction with F (at least on two sides). Here we report the x-ray crystal structure of parainfluenza virus 5 (PIV5) HN ectodomain in a “2-heads-up/2-heads-down” conformation where two heads (covalent dimers) are in the “down position,” forming a similar interface as observed in the NDV HN ectodomain structure, and two heads are in an “up position.” The structure supports a model in which the heads of HN transition from down to up upon receptor binding thereby releasing steric constraints and facilitating the interaction between critical HN-stalk residues and F.     

Adenosine(5')tetraphospho(5')adenosine-binding protein of calf thymus

An adenosine(5')tetraphospho(5')adenosine (Ap4A) binding protein has been purified from calf thymus. The protein is comprised of a single polypeptide of Mr 54000 and is capable of high-affinity (Kd = 13 microM) binding of Ap4A with great substrate specificity. The Ap4A binding protein has been isolated in two forms: a 'free', or non-polymerase-bound, form which predominates, and a similar form which copurifies with DNA polymerase alpha, but which can be resolved from it. The free form of Ap4A binding protein contains associated adenosine(5')tetraphospho(5')adenosine phosphohydrolase (Ap4Aase) activity, while the form resolved from DNA polymerase alpha contains no such activity. The Ap4Aase activity, which catalyzes the phosphohydrolysis of Ap4A to ATP and AMP, is strongly inhibited by low levels (50-100 microM) of Zn2+ without any effect on the Ap4A binding protein activity. This difference in associated Ap4Aase activity between free and polymerase-bound forms of the protein, plus the copurification mentioned above, indicate a specific association between Ap4A binding protein and DNA polymerase alpha.