St14/TaqⅠRFLPs在甲型血友病基因探测与产前基因诊断中的应用

<正> 甲型血友病是最常见的遗传性凝血疾病,是由于凝血因子Ⅷ(FVⅢ)基因缺陷所致。目前对此病尚无满意的治疗方法。利用DNA分析技术进行甲型血友病基因检测与产前基因诊断对开展优生优育有重要的实际应用价值。由于FⅧ基因组织结构庞大,分子病理改变复杂,目前多采用DNA限制酶片段长度多态性(RFLPs)为遗传标志对有缺陷的FⅧ基因进行连锁分析。St 14(DXS52)是人X染色体长臂远端的一段基因外DNA序列,与FⅧ基因紧密连锁。国外已将St14/Taq Ⅰ RFLPs用于甲型血友病基因携带者的检测,但尚未见到用于产前基因诊断的报道。我们利用引进的St14片段为探针,采用简化的低脂奶粉杂交体系和DNA凝胶原     

转铁蛋白结构与功能的研究——骆驼血清转铁蛋自的分离纯化及其含单一铁结合部位的结构域的制备和鉴定

分离纯化获得的骆驼血清转铁蛋白由分子量为73,000和63,000两个组分组成。两者至少N-端五肽顺序相同(Met-Pro-Asp-Lys-Thr)。骆驼血清转铁蛋白在生理pH下不能与人胎盘转铁蛋白受体结合。用胰蛋白酶酶解骆驼转铁蛋白可以同时得到两个合单一铁结合部位的结构域,分别来自转铁蛋白分子的N-端称N-端结构域(分子量34,700和40,700)和C-端称C-端结构域(分子量35,100)。在上述结果的基础上指出并讨论了反刍动物转铁蛋白在结构和功能上存在更多的共同性,而与其它哺乳动物的转铁蛋白有着明显的区别。     

固定化酶的微环境效应—载体离子基团性质的影响

作者合成了阴离子型和阳离子型葡聚糖,以此为载体,用CNBr活化其剩余羟基,固定化了葡萄糖淀粉酶和葡萄糖异构酶。就离子型载体对固定化酶的蛋白载量、最适pH和热稳定性等的影响做了考察。发现固定化酶的蛋白载量不仅与载体的电性质有关,也与酶分子自身的电性质有关。当载体电性质与酶蛋白电性质相反时,固定化酶的蛋白载量增加,热稳定性提高、载体电性质与酶蛋白电性质相同时,固定化酶的蛋白载量不变或下降,其热稳定性不变。作者还发现当离子型载体孔度和体系缓冲液浓度一定时,酶分子能否进入多孔性载体内部,对其最适pH是否变化影响极大。若酶分子仅被连接在载体的外表层,其最适pH不发生变化,反之亦然。作者还观察到当多糖类载体引入氨基或羧基后,大大增强了其抵抗微生物侵蚀的能力。     

A confined variable region confers ligand specificity on fibroblast growth factor receptors: implications for the origin of the immunoglobulin fold.

Binding of cellular growth factors to their receptors constitutes a highly specific interaction and the basis for cell and tissue-type specific growth and differentiation. A unique feature of fibroblast growth factor (FGF) receptors is the multitude of structural variants and an unprecedented degree of cross-reactivity between receptors and their various ligands. To examine receptor-ligand specificity within these families of growth factors and receptors, we used genetic engineering to substitute discrete regions between Bek/FGFR2 and the closely related keratinocyte growth factor receptor (KGFR). We demonstrate that a confined, 50 amino acid, variable region within the third immunoglobulin-like domain of Bek and KGFR exclusively determines their ligand binding specificities. Replacing the variable region of Bek/FGFR2 with the corresponding sequence of KGFR resulted in a chimeric receptor which bound KGF and had lost the capacity to bind basic FGF. We present evidence that the two variable sequences are encoded by two distinct exons that map close together in the mouse genome and follow a constant exon, suggesting that the two receptors were derived from a common gene by mutually exclusive alternative mRNA splicing. These results identify the C-terminal half of the third immunoglobulin-like domain of FGF receptors as a major determinant for ligand binding and present a novel genetic mechanism for altering receptor-ligand specificity and generating receptor diversity.     

Evolution of the expression of the Gld gene in the reproductive tract of Drosophila.

During the preadult development of Drosophila melanogaster, the GLD (glucose dehydrogenase) gene (Gld) is expressed in a variety of tissues, including the immature reproductive tract. At the adult stage the expression of Gld becomes largely restricted to the reproductive tract of males and females. We examined the expression of GLD in the adult reproductive tract of 50 species in the genus Drosophila, as well as in those of a few representative species from four other closely related genera. GLD exhibits considerable organ-specific diversity in the reproductive tract of males and females. Among these species, five male GLD phenotypes and six female GLD phenotypes were found. In contrast, the preadult expression of GLD in representative species from each distinct adult pattern type was determined and found to be highly conserved in both the immature reproductive tract and non-reproductive organs. Moreover, the set of reproductive organs that express GLD during preadult development is equivalent to the sum of the five male and six female adult GLD phenotypes. To initially define the contribution of cis- versus trans-acting factors responsible for differences in adult GLD expression between two of these species--D. melanogaster and D. pseudoobscura--we transferred the D. pseudoobscura Gld to the genome of D. melanogaster and investigated its expression. GLD expression patterns of these transformants displayed characteristics that are unique to both species, suggesting the presence of both cis- and trans-acting differences between these two species.     

Calcium-activated potassium channels expressed from cloned complementary DNAs.

Calcium-activated potassium channels were expressed in Xenopus oocytes by injection of RNA transcribed in vitro from complementary DNAs derived from the slo locus of Drosophila melanogaster. Many cDNAs were found that encode closely related proteins of about 1200 aa. The predicted sequences of these proteins differ by the substitution of blocks of amino acids at five identified positions within the putative intracellular region between residues 327 and 797. Excised inside-out membrane patches showed potassium channel openings only with micromolar calcium present at the cytoplasmic side; activity increased steeply both with depolarization and with increasing calcium concentration. The single-channel conductance was 126 pS with symmetrical potassium concentrations. The mean open time of the channels was clearly different for channels having different substituent blocks of amino acids. The results suggest that alternative splicing gives rise to a large family of functionally diverse, calcium-activated potassium channels.     

Resolution and stability of oxazepam enantiomers

A solvent mixture containing dioxane, acetonitrile, and hexane was found to be suitable as a mobile phase to resolve oxazepam enantiomers by chiral stationary phase high performance liquid chromatography using covalent Pirkle columns. The resolved oxazepam enantiomers in this solvent mixture had a racemization half-life greater than 3 days at 23°C. When desiccated at 0°C as dried residue, OX enantiomers were stable for at least 50 days with less than 2% racemization. The conditions which stabilized OX enantiomers significantly facilitated the determination of racemization half-lives of OX enantiomers in a variety of aqueous and nonaqueous solvents and at different temperatures. © 1992 Wiley-Liss, Inc.     

Gastric effects of thyrotropin-releasing hormone microinjected into the dorsal vagal nucleus in cats

We investigated the gastric acid secretory and motility responses to microinjection of thyrotropin-releasing hormone (TRH) into the dorsal motor nucleus of the vagus (DMV) in anesthetized cats. Gastric acid output was collected every 15 min through a gastric cannula after saline flush and titrated to pH 7.0. Antral and corpus contractions were continuously recorded by extraluminal force transducers. TRH dissolved in 200 nl of saline and microinjected unilaterally into the DMV induced a dose-dependent (50-200 ng) increase in gastric acid secretion. The acid secretory response began in the first 15 min collection and lasted 45 min. TRH frequently increased the force of contractions of the antrum and corpus within one minute of microinjection. The minimal effective dose for eliciting increased motility was lower than for inducing acid secretion. These results demonstrate that TRH acts in the DMV of cats to stimulate gastric acid secretion and contractions.