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.     

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.     

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。     

急性病毒性肝炎的发病率慢性肝炎患病率和肝病死亡率的研究

1984～1987年,在黑龙江、河北、河南、湖南、上海五个省市城乡10.08855人口中进行急性肝炎发病率、慢性肝炎患病率、与病毒性肝炎有关的肝病死亡率的研究。急性肝炎标化发病率为152.19/10万,主要发生在20～50岁组人群;因无甲肝暴发流行,除上海外各点季节发病率分布均衡。慢性肝炎标化患病率为158.25/10万(诊断标准为6个月前有明确急性肝炎病史,现有明显的临床症状或体征,肝功能异常,故实际慢肝患病率要高于此数字);与病毒性肝炎有关的肝病死亡(包括肝癌)标化率为22.65/10万,其中肝病为 13.14/10万。男性死亡率显著高于女性。     

视黄酸和亚硒酸钠对肝癌细胞某些表型逆转的加和作用

 人肝癌细胞株SMMC-7721经1μmol/L视黄酸和或2.5μmol/L亚硒酸钠处理后,膜上纤维连接蛋白沉着量逐日上升,且较相应天数的对照组细胞增加,而甲胎蛋白分泌量和~3H-TdR参入率被明显抑制。视黄酸和亚硒酸钠同时处理的联合组作用强度接近于两者单独使用时作用强度的加和。对以上结果和视黄酸及亚硒酸钠使肝癌细胞接触抑制恢复及表型逆转的关系作了讨论。     

Effect of 6-thioguanine on Chlamydia trachomatis growth in wild-type and hypoxanthine-guanine phosphoribosyltransferase-deficient cells.

Chlamydiae have evolved a biphasic life cycle to facilitate their survival in two discontinuous habitats. The unique growth cycle is represented by two alternating forms of the organism, the elementary body and the reticulate body. Chlamydiae have an absolute nutritional dependency on the host cell to provide ribonucleoside triphosphates and other essential intermediates of metabolism. This report describes the pleiotropic effects of the purine antimetabolite 6-thioguanine on chlamydial replication. In order to display cytotoxicity, 6-thioguanine must first be converted to the nucleotide level by the host cell enzyme hypoxanthine-guanine phosphoribosyltransferase. Our results show that 6-thioguanine is an effective inhibitor of chlamydial growth with either wild-type or hypoxanthine-guanine phosphoribosyltransferase-deficient cell lines as the host. Interestingly, the mechanism of 6-thioguanine-induced inhibition of chlamydial growth is different depending on which cell line is used. With wild-type cells as the host, the cytotoxic effects of 6-thioguanine on chlamydial growth are relatively fast and irreversible. Under these circumstances, cytotoxicity likely results from the combined effect of starving chlamydiae for purine ribonucleotides and incorporation of host-derived 6-thioguanine-containing nucleotides into chlamydial nucleic acids. With hypoxanthine-guanine phosphoribosyltransferase-deficient cells as the host, 6-thioguanine must be present at the start of the chlamydial infection cycle to be effective and the growth inhibition is reversible upon removal of the antimetabolite. These findings suggest that in hypoxanthine-guanine phosphoribosyltransferase-deficient cells, the free base 6-thioguanine may inhibit the differentiation of elementary bodies to reticulate bodies. With hypoxanthine-guanine phosphoribosyltransferase-deficient cells as the host, 6-thioguanine was used as a selective agent in culture to isolate a Chlamydia trachomatis isolate resistant to the effects of the drug. This drug resistant C. trachomatis isolate was completely resistant to 6-thioguanine in hypoxanthine-guanine phosphoribosyltransferase-deficient cells; however, it displayed wildtype sensitivity to 6-thioguanine when cultured in wild-type host cells.     

放射污染区古菌分离及多样性分析

【目的】研究放射污染区古菌多样性。【方法】放射污染区采集土样,采用甘油-精氨酸培养基(GJ)、甘油-天冬氨酸培养基(C1)、海藻糖-肌酸培养基(B7)、甘露醇-丙氨酸培养基(Z5)、干酪素-甘露醇培养基(CMKA)、壳聚糖-天冬酰胺培养基(F6)、甘露醇-酸水解酪蛋白培养基(GW1)、CM培养基、HP培养基和KC培养基10种分离培养基,采用梯度稀释法对古菌进行分离,将分离获得的菌株经形态特征,16S rRNA基因片段扩增及限制性内切酶酶切,选取酶切图谱中存在差异性的条带进行测序,最终通过序列比对,聚类分析,获得不同种类的古菌资源。【结果】从该土样中共获得了256株古菌,最终筛选出71株不同类型的古菌,这71株古菌均属于广古菌门,盐杆菌纲,盐杆菌目,盐杆菌科,分布于盐陆生菌属(Haloterrigena)、纳白菌属(Natrialba)、盐球菌属(Halococcus)、盐红菌属(Halorubrum)、盐长寿菌属(Halovivax)、纳线菌属(Natrinema)、盐碱球菌属(Natronococcus)、盐二型菌属(Halobiforma)、盐惰菌属(Halopiger)、盐池栖菌属(Halostagnicola)、富盐菌属(Haloferax)11个属,26个种,其中31株菌的16S rRNA基因序列与已有效发表菌株的序列相似性小于98%,Haloterrigena为该土样的优势菌属。对于分离效果较好的F6培养基采用了梯度营养成分的稀释,最终获得了19株古菌,这些菌株相互之间存在一定的差异性。【结论】本次分离获得了大量的古菌,表明放射污染区存在着较为丰富的古菌资源,其中蕴藏着多种新的物种类型,具有较大的研究价值。