Clamp loading,unloading and intrinsic stability of the PCNA, β and gp45 sliding clamps of human,E. coli and T4 replicases

Background: The high speed and processivity of replicative DNA polymerases reside in a processivity factor which has been shown to be a ring-shaped protein. This protein (‘sliding clamp’) encircles DNA and tethers the catalytic unit to the template. Although in eukaryotic, prokaryotic and bacteriophage-T4 systems, the processivity factors are ring-shaped, they assume different oligomeric states. The Escherichia coli clamp (the β subunit) is active as a dimer while the eukaryotic and T4 phage clamps (PCNA and gp45, respectively) are active as trimers. The clamp can not assemble itself on DNA. Instead, a protein complex known as a clamp loader utilizes ATP to assemble the ring around the primer-template. This study compares properties of the human PCNA clamp with those of E. coli and T4 phage. Results: The PCNA ring is a stable trimer down to a concentration below 100 nm (K_d ≈ 21 nm ). On DNA, the PCNA clamp slides freely and dissociates from DNA slowly (t_1/2 ≈ 24 min). β is more stable in solution (K_d < 60 pm ) and on DNA (t_1/2 ≈ 1 h) than PCNA which may be explained by its simpler oligomeric state. The T4 gp45 clamp is a much less stable trimer than PCNA (K_d ≈ 250 nm ) and requires association with the polymerase to stabilize it on DNA as observed previously. The consequence of this cooperation between clamp and polymerase is that upon finishing a template and dissociation of the polymerase from DNA, the gp45 clamp spontaneously dissociates from DNA without assistance. However, the greater stability of the PCNA and β clamps on DNA necessitates an active process for their removal. The clamp loaders (RF-C and γ complex) were also capable of unloading their respective clamps from DNA in the presence of ATP. Conclusions: The stability of the different clamps in solution correlates with their stability on DNA. Thus, the low stability of the T4 clamp explains the inability to isolate gp45 on DNA. The stability of the PCNA and β clamps predicts they will require an unloading factor to recycle them on and off DNA during replication. The clamp loaders of PCNA and β double as clamp unloaders presumably for the purpose of clamp recycling.     

Predicted Near-Future Oceanic Warming Enhances Mercury Toxicity in Marine Copepods

Bulletin of Environmental Contamination and Toxicology - The effects of acute mercury exposure (118&nbsp;µg/L) on the marine copepod Tigriopus japonicus were examined at 22 and...     

小鼠补体受体Ⅱ型基因改造后感染EB病毒

通过对小鼠补体受体Ⅱ型基因（CR2）进行定点突变,然后将野生型和突变型小鼠CR2/1（MCR2/1）及人CR2（hCR2)用电穿孔方法导入小鼠SP2/0进行表达,采用免疫组织化学等方法鉴定阳性细胞系。用EB病毒对转染阳性细胞进行感染,EBER-1杂交结果显示,仅转染hCR2和突变型MCR2（mtMCR2)的SP2/0细胞感染了EB病毒,前者感染EB病毒的阳性率较后者高很多。这为进一步研究EB病毒进入细胞的机制及建立EB病毒相关的鼻咽癌动物模型奠定了良好的基础。     

基因芯片对PMA激活的血管内皮细胞早期反应基因的研究

目的 :用基因芯片研究 PMA激活的血管内皮细胞的早期反应基因 (imm ediate early response gene,ERG)。方法 :以包含 40 96条人类基因的 DNA芯片检测血管内皮细胞受代谢增强剂 PMA(phorbol myristate acetate)激活后早期的基因表达谱 ,并从中筛查出 ERG。结果 :血管内皮细胞受 PMA作用 6 h后 ,17条基因上调 ,11条下调。数据处理聚类分析表明 17条上调基因中多数 (13/ 17)属蛋白质磷酸酶和转录调控因子基因 ,而下调基因中多数 (9/ 11)为细胞分裂相关基因。结论 :证明PMA激活的人血管内皮细胞早期反应基因主要是转录调控因子和蛋白质磷酸酶基因。     

检测Ⅳ型胶原蛋白直接双抗夹心法的建立与评价

目的 :建立血清 型胶原蛋白 (COL - )浓度测定的直接双抗夹心法 ,为临床诊断肝纤维化提供一种新的指标。方法 :采用本科室自制的 COL - 单克隆酶标抗体、多克隆抗体建立的直接双抗夹心法 ,同时采用日本试剂盒测定 6 0份正常和肝脏病人血清 COL- 浓度做对照比较。结果 :两种方法无显著性差异 (P>0 .0 5 ,t=0 .16 6 ) ;两法相关分析 ,γ=0 .834,回归方程为 Y=0 .5 9X+ 70 .874。结论 :直接双抗夹心法测定血清 COL- 操作简便、特异、准确 ,可作为辅助诊断肝纤维化的可靠指标 ,并可在临床推广应用     

补肾健脾法治疗胎怯的临床研究

目的:研究补肾健脾法治疗胎怯的临床疗效。方法:以补肾健脾方药制成助长口服液为治疗组,以不用药为对照组,进行治疗胎怯的临床对照观察。结果:治疗组疗效(78%)显著优于对照组(52%),P<0.001;治疗组在改善体重、身长、头围、胸围、上臂围等方面显著优于对照组,P<0.05～0.01;治疗组治疗后血清T3升高,T4下降,与对照组相比,P<0.05;在降低患病率方面,治疗组(43%)优于对照组(72%),P<0.05;治疗组远期疗效优于对照组,P<0.05。结论:补肾健脾法治疗胎怯有显著的临床疗效。     

高晶体-高胶体渗透压溶液对受机械性损伤的海马神经细胞容积的影响

观察机械性损伤的海马神经细胞在不同浓度高晶体－ 高胶体渗透压溶液中容积的变化。取原代培养大白鼠胚胎的海马神经细胞,用超声波机械损伤细胞后暴露于含０．５ ｇ／Ｌ、１ ｇ／Ｌ和２．５ ｇ／Ｌ氯化钠的细胞培养基中１５ ｍ ｉｎ。结果显示机械性损伤的细胞明显肿胀（Ｐ＜ ０．０５）。当细胞暴露于不同浓度的高晶体－ 高胶体溶液１５ ｍ ｉｎ 后, 细胞容积与同一实验时间的对照值相比明显下降, 并保持至第７ ｄ （Ｐ＜ ０．０５）。表明受到机械性损伤的海马神经细胞在高晶体－ 高胶体渗透压环境中的容积调节功能丧失或减弱。     

腹腔镜下全子宫切除术的临床评估

目的：对腹腔腔镜下全宫切除的临床价值进行评估。方法：对４３ 例因诊断为子宫肌瘤（３３ 例） 、子宫腺肌症（６例） 及子宫内膜增殖症（４ 例） 的患者行腹腔镜下全子宫切除术,对同时期１２１ 例具有同样适应症的患者行腹式全子宫切除手术。比较两组病人术中术后情况。结果：两组病人的手术时间、术中出血量无显著性差异,腹腔镜下全子宫切除术的手术时间与子宫增大有关,术中出血量与手术时间及子宫大小无关;行腹腔镜下全子宫切除术的病人手术损伤及术后阴道残端出血发生率无增高,术后疼痛的发生率明显减少,术后使用抗菌素时间、术后住院时间及术后恢复正常活动的时间缩短。结论：腹腔镜下全子宫切除术虽不能完全代替腹式全宫切除术,却是一种安全、可靠,适于临床广泛开展的手术方式。