Analysis of serine/threonine-linked oligosaccharides derived by alkaline-borohydride treatment of mucin glycoproteins electroblotted onto membranes: comparison of the saccharide profiles of the 390 kDa and 350 kDa forms of epitectin

Alkaline borohydride treatment is widely used for the release of carbohydrate moieties from O-glycosylated glycoproteins and mucins. We have adapted this procedure to micro quantities of glycoproteins blotted on membranes. After electrophoresis and transfer to nitrocellulose, nylon or polyvinylidene difluoride membrane, alkaline borohydride treatment was done directly on glycoprotein containing areas of membrane which were cut out with the aid of guide strips stained with Coomassie Blue or lectin-digoxigenin. In combination with standard saccharide fractionation techniques, this procedure can be used to characterize the oligosaccharides of mucins or mucin-type glycoproteins that are separated by gel electrophoresis from crude sources. Using this approach we have characterized the saccharides derived from the two species of epitectin, a malignancy-associated mucin type glycoprotein, isolated from metabolically labelled H.Ep2 cells.     

甘蔗和烟草幼叶原生质体的核酸含量与核酸酶活性及其影响因素

与幼叶组织相比,酶法新鲜分离的甘蔗和烟草幼叶原生质体内的ＲＮＡ、ＤＮＡ及总核酸含量均降低。其原因可能是刚游离的原生质体内酸性和碱性ＲＮＡ酶与ＤＮＡ酶等活性提高所致。甘蔗叶原生质体内的核酸降低量和ＲＮＡ酶与ＤＮＡ酶活性的增加程度均高于烟草。随用作渗透压稳定剂的甘露醇浓度增加,甘蔗和烟草叶原生质体的ＲＮＡ酶和ＤＮＡ酶活性均相应提高。其中以甘蔗叶原生质体的核酸酶活性增加水平较明显。在细胞壁降解产物的作用下,除了甘蔗原生质体内的ＲＮＡ酶活性略被促进外,其ＤＮＡ酶和烟草叶原生质体内的核酸酶均不受影响     

Characterization of the oriC region of Mycobacterium smegmatis.

A 3.5-kb DNA fragment containing the dnaA region of Mycobacterium smegmatis has been hypothesized to be the chromosomal origin of replication or oriC (M. Rajagopalan et al., J. Bacteriol. 177:6527-6535, 1995). This region included the rpmH gene, the dnaA gene, and a major portion of the dnaN gene as well as the rpmH-dnaA and dnaA-dnaN intergenic regions. Deletion analyses of this region revealed that a 531-bp DNA fragment from the dnaA-dnaN intergenic region was sufficient to exhibit oriC activity, while a 495-bp fragment from the same region failed to exhibit oriC activity. The oriC activities of plasmids containing the 531-bp sequence was less than the activities of those containing the entire dnaA region, suggesting that the regions flanking the 531-bp sequence stimulated oriC activity. The 531-bp region contained several putative nine-nucleotide DnaA-protein recognition sequences [TT(G/C)TCCACA] and a single 11-nucleotide AT-rich cluster. Replacement of adenine with guanine at position 9 in five of the putative DnaA boxes decreased oriC activity. Mutations at other positions in two of the DnaA boxes also decreased oriC activity. Deletion of the 11-nucleotide AT-rich cluster completely abolished oriC activity. These data indicate that the designated DnaA boxes and the AT-rich cluster of the M. smegmatis dnaA-dnaN intergenic region are essential for oriC activity. We suggest that M. smegmatis oriC replication could involve interactions of the DnaA protein with the putative DnaA boxes as well as with the AT-rich cluster.     

长萼木通属——木通科一新属

缺瓣牛姆瓜Holboellia apetala Q．Xia,J．Z.Suen et Z．X．Peng在一系列特征上与牛姆瓜属Holboellia植物不同,不应隶属于该属,而代表了一个未被描述过的新属—长萼木通属。新属在茎、叶、果实和花的大部分特征上与木通属Akebia相似,但花萼特征又与野木瓜属Stauntonia接近,其系统位置介于这两个属之间,并偏于前者。     

Site-specific cleavage of chromosomes in vitro through Cre-lox recombination.

Site-specific recombination systems are useful tools for chromosome engineering in vivo and site-specific DNA cleavage methods have applications in genome analysis and gene isolation. Here, we report a new method to fragment chromosomes in vitro using the Cre-lox site-specific recombination system. Two lox sites were targeted into the 5.7 Mb chromosomes I of Schizosaccharomyces pombe. In vitro recombination between chromosomal lox sites and exogenously provided lox oligonucleotides 'cleaved' the chromosome at the defined lox sequences. Site-specific cleavage of lox sites in the tobacco genome was also demonstrated. This recombination-based cleavage method provides a novel approach for structural and functional analyses of eukaryotic chromosomes as it allows direct isolation of chromosome regions that correspond to phenotypes revealed through Cre-lox mediated chromosome rearrangements in vivo. Moreover, recombination with end-labeled lox oligonucleotides would permit the specific end-labeling of chromosome segments to facilitate the long range mapping of chromosomes.     

Distribution of 3α-hydroxysteroid dehydrogenase in rat brain and molecular cloning of multiple cDNAs encoding structurally related proteins in humans

3α-Hydroxysteroid dehydrogenase in the brain is responsible for production of neuroactive tetrahydrosteroids that interact with the major inhibitory gamma-aminobutyric acid receptor complexes. Distribution of 3α-hydroxysteroid dehydrogenase in different regions of the brain in rats was evaluated by activity assay and by Western immunoblotting using a monoclonal antibody against liver 3α-hydroxysteroid dehydrogenase as the probe. The olfactory bulb was found to contain the highest level of 3α-hydroxysteroid dehydrogenase activity, while moderate levels of the enzyme activity were found in other regions such as cerebellum, cerebral cortex, hypothalamus and pituitary. Some activity was found in the rest of the brain such as amygdala, brain stem, caudate putamen, cingulate cortex, hippocampus, midbrain, and thalamus. The protein levels of 3α-hydroxysteroid dehydrogenase in different regions of the brain as detected by Western immunoblotting are comparable to those of the enzyme activity. We used the rat cDNA as the probe to screen a human liver λ gt11 cDNA library. A total of four different cDNAs were identified and sequenced. One of the cDNAs is identical to that of the human chlordecone reductase cDNA except that our clone contains a much longer 5′-coding sequence than previously reported. The other three cDNAs display high degrees of sequence homology to those of both rat 3α-hydroxysteroid dehydrogenase and human chlordecone reductase. We are currently investigating the functional relationship between the enzymes encoded by these human cDNAs and 3α-hydroxysteroid dehydrogenase.     

细胞周期和调控因子

近期对细胞周期调控的研究获得了突破性进展.人们深入地研究了周期蛋白家族和pp³⁴蛋白家族在周期调控中的作用及二者之间的相互关系,同时还发现了很多与之相关的调控因子,它们彼此相互作用,形成了极为复杂的级联调控网络.     

乙型脑炎病毒NS2A-C60A位点突变对其生物学特性影响研究*

目的:旨在探索Ⅰ型日本乙型脑炎病毒传代致弱后基因组突变NS2A-C60A对乙脑病毒生物学特性的影响。方法:首先通过对传代致弱及原始乙脑毒株基因组序列进行测序比对、结构预测分析并利用Western blotting(WB)确定了目标研究位点NS2A-C60A;然后使用反向遗传定点突变技术构建拯救了包含NS2A-C60A单点突变的病毒株;最后利用噬斑形态观察、生长曲线、双萤光素酶分析,WB以及炎性因子检测和动物实验研究了该单点突变对于乙脑病毒生物学特性的影响。结果:首次研究发现Ⅰ型乙脑病毒传代致弱会导致NS1'蛋白表达的显著下降以及可能的相关位点NS2A-C60A,并成功拯救获得了NS2A-C60A单点突变毒株rJEV-C60A,研究发现NS2A-C60A突变对乙脑病毒的生长特性及噬斑形成没有显著影响,但是能够显著降低乙脑病毒NS1'蛋白的表达,并且该位点突变能够轻微阻碍乙脑病毒对细胞炎性因子表达的抑制,动物实验结果显示NS2A-C60A点突变病毒与原毒株具有相似的神经毒力,说明该位点突变不是影响乙脑病毒毒力致弱的关键位点。结论:新发现的NS2A-C60A位点突变能够显著减少乙脑病毒NS1'蛋白的表达,但是对其增殖、诱导炎症及神经毒力等生物学特性没有显著影响。     

人参对大鼠胃G细胞、D细胞影响的免疫组织化学观察

本文应用免疫组织化学PAP方法观察大鼠胃G细胞、D细胞的分布和形态特征,以及人参对胃G细胞、D细胞免疫细胞化学活性的影响。用细胞显微分光光度计检测了服用人参的G细胞、D细胞免疫反应阳性面积及光密度。检测结果表明,人参能使大鼠胃G细胞、D细胞增大,增加G细胞中胃泌素的含量和D细胞中生长抑素的含量。本文的结果提示,人参对大鼠胃G细胞和D细胞的形态及分泌活动有调节性影响。     

山楂叶螨、苹果全爪螨及其捕食性天敌生态位的研究Ⅰ——时间与空间生态位

统计分析表明两种叶螨间生态位重叠值较大、各自生态位宽度较大。两种叶螨的时动空间生态位与各自种群消长曲线相一致,显示了它们对资源有相似要求以及“逐步扩散整树危害”的取食危害方式。它们的生态位分离表现在叶片正、反面及叶片反面的某些小“领域”上。苹果全爪螨更喜欢分布在树冠上层和外层。统计和模糊分析都表明草蛉和小花螨与两种叶螨的时间同步和空间同域性较强,捕食螨与叶螨的时间生态位重叠较小,而空间生态位重叠较大。天敌与叶螨间的时空生态位重叠以及天敌食谱生态位宽度结合分析有助于合理评价天敌的控制作用。