人结核分枝杆菌特异性核酸探针制备及结核病PCR检测技术的初步临床应用

采用人结核分枝杆菌(Mycobacterium tuberculosis TB)染色体DNA为模板,选择位于插入片段IS6110中884～865和568～588碱基对处的两个片段为引物,扩增出317bp的特异性片段．将其克隆进pUCl9载体。酶切图谱分析和DNA序列测定证实为目的片段。该片段经DIG标记,分别与11种分枝杆菌DNA进行Southern杂交,结果证明只与人型复合分枝杆菌发生杂交反应。利用该对引物建立的PcR检测拄术对74份结核病痰液标本进行检测,并与临床细菌快速培养结果相比较,发现48份临床阳性均为PcR阳性,在26份临床阴性标本中亦发现11份PCR检测阳性。将标本PCR产物与克隆探针进行杂交,显示两者结果完全一致。说明PCR检测体系结果可靠,其灵敏度明显高于目前临床所采用的方法,可作为一种常规技术用于结核病的临床检测。     

DNA生物传感器进展

近几年来,酶传感器、免疫传感器及微生物传感器等发展较为成熟,而DNA生物传感器的研究相对较少.文章从核酸杂交的原理出发介绍了DNA生物传感器的工作原理,举例说明了电化学、光学和声学等几种典型的DNA生物传感器,指出了其固有的优缺点,肯定了DNA传感器发展前景.     

The prevalence of Fasciola hepatica in its snail intermediate host determined by DNA probe assay

Accurate snail intermediate host infection prevalence data have the potential to be extremely useful in determining seasonal transmission dynamics of Fasciola hepatica. Because the microscopic techniques currently used lack the sensitivity and specificity necessary to obtain meaningful infection prevalence data, we developed a highly accurate and efficient DNA probe assay. The assay has a sensitivity of 100%, a specificity of >99%, easily detects a single miracidia and does not cross-hybridize with DNA of Fascioloides magna, Paramphistomum liorchis or Heterobilharzia americana, trematodes that share the same intermediate host and enzootic range as Fasciola hepatica. Using this assay, we determined the prevalence of F. hepatica in its snail intermediate host, Fossaria cubensis, during the second year of a 2-year study on the epizootiology of Fasciola hepatica in Florida. The overall infection prevalence of snails assayed in this study (n = 5246) was 1.5% and ranged from 0.1% to 3.1% for individual cattle ranches. Additionally, infection prevalence differed significantly for successive size groupings of snails, varying from 0% for 1-mm snails to 18.5% for 9- and 10-mm snails. The accuracy and efficiency of the DNA probe assay reported here for determining snail infection prevalence offers an inexpensive alternative to tracer animal studies for determining the epizootiology of F. hepatica.     

鱼类LZF-IDNA指纹探针的制备

利用Ｏｌｉｇｏ１０００ＤＮＡ合成仪（Ｂｅｃｋｍａｎ）合成了长度为４５ｂｐ的寡核苷酸单链,经纯化后用同位素γ－３２Ｐ－ＡＴＰ作５′末端标记后,制备成鱼类ＬＺＦ－ＩＤＮＡ指纹探针。通过对鱼类的群体实验、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－ＩＤＮＡ指纹探针属多位点寡核苷酸探针;（２）ＬＺＦ－ＩＤＮＡ指纹探针在鱼类种群中的鉴别机率为９．２３×１０－１６;（３）ＬＺＦ－ＩＤＮＡ指纹探针在鱼类亲子鉴定实验中的父系概率为０．９９９９６２;（４）ＬＺＦ－ＩＤＮＡ指纹探针,是一种稳定的,既具有个体识别能力,又具有一定种属特异性的、适用于鱼类ＤＮＡ指纹图研究的基因指纹探针。     

脑内微透析采样技术及其在神经科学中的应用

作为一种新的在体化学采样技术,脑内微透析引起了神经科学家的关注。它与迅速发展起来的高灵敏度的微量分析技术相结合,实现了对体内细胞外环境中化学物质变化的动态监测,从而在神经科学领域获得应用。本系统地介绍了这一新技术的原理和方法,并扼要地介绍了一这一技术在神经科学中的应用及其取得的新进展,并结合本实验室的工作经验,对该技术存在的一些问题进行了讨论。     

编码天麻抗真菌蛋白cDNA的分子克隆

用重组ＤＮＡ 技术研究了编码天麻抗真菌蛋白（ＧＡＦＰ）的基因,从天麻（Ｇａｓｔｒｏｄｉａ ｅｌａｔａＢｌ．）块茎中提取Ｐｏｌｙ（Ａ） ｍ ＲＮＡ 后合成ｃＤＮＡ,构建成表达型ｃＤＮＡ 文库,用纯化的蛋白质探针通过免疫筛选找出对应的ｃＤＮＡ 克隆。在进一步证明所选用的ｃＤＮＡ 克隆含有重组的λ－ｐｈａｇｅ ＤＮＡ 后,提取和纯化含有插入片段重组子的ＤＮＡ,用Ｅｃｏ ＲＩ酶切分析可见插入片段。已分离出编码天麻抗真菌蛋白的基因     

A Continuum theory for the prediction of lateral and rotational positioning of α-helices in membrane proteins: Bacteriorhodopsin

We have developed a new method for the prediction of the lateral and the rotational positioning of transmembrane helices, based upon the present status of knowledge about the dominant interaction of the tertiary structure formation. The basic assumption about the interaction is that the interhelix binding is due to the polar interactions and that very short extramembrane loop segments restrict the relative position of the helices. Another assumption is made for the simplification of the prediction that a helix may be regarded as a continuum rod having polar interaction fields around it. The polar interaction field is calculated by a probe helix method, using a copolymer of serine and alanine as probe helices. The lateral position of helices is determined by the strength of the interhelix binding estimated from the polar interaction field together with the length of linking loop segments. The rotational positioning is determined by the polar interaction field, assuming the optimum lateral configuration. The structural change due to the binding of a prosthetic group is calculated, fixing the rotational freedom of a helix that is connected to the prosthetic group. Applying this method to bacteriorhodopsin, the optimum lateral and rotational positioning of transmembrane helices that are very similar to the experimental configuration was obtained. This method was implemented by a software system, which was developed for this work, and automatic calculation became possible for membrane proteins comprised of several transmembrane helices. © 1995 Wiley-Liss, Inc.     

Genome characterization of glucose-isomerase-producingStreptomyces sp. NCIM 2730: detection of sequences homologous to a rice repeat sequence

Restriction analysis of the genomic DNA from a high glucose/xylose-isomerase-yieldingStreptomyces sp. NCIM 2730 revealed a number of distinct bands on a background smear, indicating the occurrence of repeated DNA sequences in the genome. Optical renaturation analysis indicated that 25% of the genome comprised rapidly reannealing sequences with a copy number of 50 and a kinetic complexity of 3×10³. Hybridization of theStreptomyces genomic library with theStreptomyces DNA, supported the estimate of the repetitive DNA content derived from the re-association kinetics of the DNA. Hybridization of DNA from three differentStreptomyces species with a rice repetitive DNA probe revealed the presence of homologous sequences, which is a unique finding.M.S. Ghatge was and V.V. Deshpande and P.K. Ranjekar are with the Division of Biochemical Sciences, National Chemical Laboratory, Pune -41108, India; M.S. Ghatge is now with the Department of Microbiology & Molecular Biology, The University of Kansas Medical Center, 36th and Rainbow Blvd, Kansas City, Kansas - 66103, USA.     

荧光探针在大鼠胃粘膜表面疏水性研究中的应用

采用１－萘胺－８－磺酸（ＡＮＳ）为疏水探针,对大鼠胃粘膜表面疏水性作了研究,结果表明：以ＡＮＳ（２５μｍｏｌ／Ｌ）与胃粘膜表面刮取物（胃粘液凝胶层）混合后的萤光强度（正常为１．２３±０．１９ＲＦＵ／胃）可代表该粘液层的疏水性;以不同浓度ＡＮＳ与胃粘液混合后的萤光强度呈饱和趋势,可用Ｓｃａｔｃｈａｒｄ作图法求得粘液中ＡＮＳ的最大萤光强度（２．４６７±０．６３８ＲＦＵ／胃）和相对亲和系数（０．０３２±０．０１６）,它们可分别代表胃粘液中疏水基团的总量和单个基团的疏水性,从而可阐明胃粘膜被盐酸损伤后凝胶层粘液的ＡＮＳ萤光减弱,系其疏水基团总量减少,而非单个基团的疏水性改变所致。     

(Na + K)-ATPase activity of crude homogenates of rat skeletal muscle as estimated from their K-dependent 3-O-methylfluorescein phosphatase activity

A highly sensitive fluorimetric assay using 3-O-methylfluorescein phosphate as substrate was used in the determination of K⁺-dependent phosphatase activity in preparations of rat skeletal muscle. The gastrocnemius muscle was chosen because of mixed fibre composition. Crude, detergent treated homogenate was used so as to avoid loss of activity during purification. K⁺-dependent phosphatase activities in the range 0.19–0.37 μmol · (g wet weight)⁻¹ · min⁻¹ were obtained, the value decreasing with age and K⁺-deficiency. Complete inhibition of the K⁺-dependent phosphatase was obtained with 10⁻³ M ouabain. Using a KSCN-extracted muscle enzyme the intimate relation between K⁺-dependent phosphatase activity and (Na⁺ + K⁺)-activated ATP hydrolysis could be demonstrated. A molecular activity of 620 min⁻¹ was estimated from simultaneous determination of K⁺-dependent phosphatase activity and [³H]ouabain binding capacity using the partially purified enzyme preparation. The corresponding enzyme concentration in the crude homogenates was calculated and corresponded well with the number of [³H]ouabain binding sites measured in intact muscles or biopsies hereof.