Cloning and expression of surface antigens from occult chronic hepatitis B virus infections and their recognition by commercial detection assays

Occult hepatitis B virus (HBV) infections show little or no serological markers of viral infection, including the absence of hepatitis B surface antigen (HBsAg) which is the main marker of ongoing HBV infection. Such infections can be important in the context of blood and/or organ donations. To study whether mutations contribute to HBsAg seronegativity, S gene sequences from such patients were amplified and cloned. Sequencing revealed 12 clones from seven different patients which contained potentially important mutations. The sequences were subcloned into an expression vector and mutant HBsAgs were expressed in cell culture. The capacity of three HBsAg detection assays to recognise the mutant HBsAgs was studied. Three categories were found: mutant HBsAgs that are not recognised by the assays, those that are recognised as well as wild-type (WT) antigen and an intermediate category where detection of the mutant HBsAgs is reduced with respect to WT. Most of the isolates fall into the second category. Mutations can therefore contribute to HBsAg seronegativity in occult HBV infections, but in most cases the explanation is probably the low level of viral replication.     

Human growth hormone 1 (GH1) gene expression: complex haplotype-dependent influence of polymorphic variation in the proximal promoter and locus control region

The proximal promoter region of the human pituitary expressed growth hormone (GH1) gene is highly polymorphic, containing at least 15 single nucleotide polymorphisms (SNPs). This variation is manifest in 40 different haplotypes, the high diversity being explicable in terms of gene conversion, recurrent mutation, and selection. Functional analysis showed that 12 haplotypes were associated with a significantly reduced level of reporter gene expression whereas 10 haplotypes were associated with a significantly increased level. The former tend to be more prevalent in the general population than the latter (p<0.01), possibly as a consequence of selection. Although individual SNPs contributed to promoter strength in a highly interactive and non-additive fashion, haplotype partitioning was successful in identifying six SNPs as major determinants of GH1 gene expression. The prediction and functional testing of hitherto unobserved super-maximal and sub-minimal promoter haplotypes was then used to test the efficacy of the haplotype partitioning approach. Electrophoretic mobility shift assays demonstrated that five SNP sites exhibit allele-specific protein binding. An association was noted between adult height and the mean in vitro expression value corresponding to an individual's GH1 promoter haplotype combination (p=0.028) although only 3.3% of the variance of adult height was found to be explicable by reference to this parameter. Three additional SNPs, identified within sites I and II of the upstream locus control region (LCR), were ascribed to three distinct LCR haplotypes. A series of LCR-GH1 proximal promoter constructs were used to demonstrate that 1) the LCR enhanced proximal promoter activity by up to 2.8-fold depending upon proximal promoter haplotype, and that 2) the activity of a given proximal promoter haplotype was also differentially enhanced by different LCR haplotypes. The genetic basis of inter-individual differences in GH1 gene expression thus appears to be extremely complex.     

PBX1基因剪切体表达与SLE的相关研究

了解PBX1基因各种剪切体的表达在SLE患者和正常人中是否存在差异 ,探讨PBX1的表达与SLE发病的相关性。通过PCR扩增及毛细管芯片电泳 ,确证剪切体h、k、l存在于人体 ;通过实时荧光定量PCR技术 ,对剪切体h、k、l分别进行SLE患者组和正常组的mRNA表达定量比较。结果发现这 3种剪切体在患者组中的表达较正常人明显降低 ,正常人的表达是SLE的 9～ 12倍。重度患者的k、l剪切体与轻中度的病人相比表达明显降低 ,并发狼疮性肾炎的病人k剪切体的表达较无肾累及的病人显著降低。说明PBX1基因剪切体h、k、l在SLE患者中mRNA表达水平下降 ,并与SLE活动度及肾累及有关。提示机体通过PBX1的表达量的调节可能参与SLE的发病     

Diagnosis of squamous-cell carcinoma of the lung by sputum cytology: with special reference to correlation of diagnostic accuracy with size and proximal extent of resected tumor

Sputum cytology was performed in 179 cases of squamous-cell carcinoma of the lung; 134 cases were diagnosed as positive. There were no significant differences in diagnostic accuracy of sputum cytology between tumors sizes. In cases with tumors extending proximally into the main, lobar, or segmental bronchi, the diagnostic accuracy of sputum cytology was significantly higher than in cases where the proximal invasion of tumor was limited to the peripheral bronchi. In cases with tumors 3 cm or less in diameter, when tumors extended proximally into main, lobar, or segmental bronchi, the diagnostic accuracy of sputum cytology was significantly higher than in cases with tumors extending proximally into subsegmental or subsubsegmental bronchi. In peripherally located squamous-cell carcinoma, in cases in which the tumor arose in subsegmental or subsubsegmental bronchi, carcinoma could be detected by sputum cytology even when it was roentgenographically occult.     

转录调控蛋白PrfA对两组新近发现的单核细胞增生李斯特菌基因的体外转录作用的研究

目的研究转录调控蛋白PrfA对两组新近发现的单核细胞增生李斯特菌基因的体外转录作用。方法利用本室近年来建立的体外转录系统,对两组基于转录基因组体内研究发现的5个可能的受PrfA不同调节的单核细胞增生李斯特菌基因进行了体外转录活性的研究。结果第一组中的hpt基因的体外转录活性受PrfA正调节,而其它4个基因既不被PrfA正调节也不被负调节。结论除hpt基因外,其它4个基因体外转录结果与体内实验不相一致,说明PrfA在体内可能通过复杂多样的非直接方式、或者还需要一些目前未知的因子来调控这些新近发现的基因的表达。     

KyoT2 结合蛋白KBP1对RBP-J介导的转录活性的影响

目的 :研究KyoT2与KyoT2结合蛋白 1(KyoT2 bindingprotein1,KBP1)之间的物理相互作用 ,以及这种相互作用对重组信号结合蛋白J (recombinationsignalbindingprotein Jκ ,RBP J)介导的转录活性的影响。方法 :体外GST pulldown、体内免疫共沉淀、哺乳动物细胞双杂交实验和报告基因实验证实 ,KBP1与KyoT2之间存在物理和功能上的相互作用。结果 :体内和体外证实 ,KBP1与KyoT2之间均存在相互作用。转录活性分析实验显示 ,过表达KBP1可拮抗RING1对RBP J介导的转录的抑制效应。结论 :KBP1可通过与RING1竞争结合KyoT2而间接参与对Notch信号途径的调控     

中药心康口服液抗柯萨奇B3病毒RNA复制作用的研究

目的研究探讨中药心康口服液对感染心肌中柯萨奇B3病毒RNA复制的影响。方法小鼠感染柯萨奇病毒B3(CVB3)诱发急性病毒性心肌炎,于急性期治疗后提取鼠心肌总RNA,用逆转录-聚合酶链反应技术检测心肌CVB3RNA含量,经琼脂糖凝胶电泳后,在凝胶成像系统下观察。结果中药心康口服液能明显降低心肌CVB3-RNA的含量,与病毒对照组比较差异有统计学意义,P<0.01;感染期不同阶段心肌病毒RNA含量的检测显示,心康口服液2个治疗组的病毒含量的检出,并没有随着用药时间的延长而显著降低。结论中药心康口服液能有效地影响心肌CVB3RNA的复制,其对病毒的作用是抑制复制而非杀灭。