Activating protein factor binds in vitro to upstream control sequences in heat shock gene chromatin

DNA sequences, important for the control of Drosophila heat shock gene expression, are packaged in chromatin in a nuclease hypersensitive configuration. Recently, two protein-binding (exonuclease-resistant) sites which cover the TATA box sequence and an upstream control element were shown to occur in vivo amidst the 5' terminal hypersensitive regions of several heat shock genes. Protein-binding at the TATA box is independent of heat shock, but the binding at the upstream element is heat shock dependent, and it was proposed that a heat shock activator protein, HAP, positively regulates the genes. Here, I report the detection of HAP activity in heat shocked cell extracts by reconstituting specific binding to hsp82 gene chromatin in vitro. Inhibition of the binding by free DNA from the 5' region of heat shock genes implies a coordinate regulation of the gene family through HAP interaction with the upstream heat shock consensus sequence. Furthermore, the special ease of induction of the hsp82 gene over other heat shock genes can be explained in molecular terms by the higher affinity of HAP for the hsp82 binding site, which contains a 28 base sequence with almost perfect dyad symmetry, GAAGCCTCTAGAAG/TTTCTAGAGACTTC.     

Irreversible association of peptides with class II MHC molecules in living cells.

Functional, morphological and biochemical evidence indicates that class II major histocompatibility complex (MHC) molecules associate with processed peptides during biosynthesis. Peptide/MHC complexes in living cells have been reported to be less stable than similar complexes generated in vitro, which has led to the suggestion that there may be a peptide exchange mechanism operating in vivo. Although this could increase the capacity for binding incoming antigens, it would reduce the efficacy of processed antigenic peptides by exchanging these for self peptides. Here we measure the half-life of peptide/class II complexes in human antigen-presenting cells and find that it is very similar to the half-life of class II molecules themselves, indicating that peptides are bound irreversibly under physiological conditions. Thus class II MHC retains long-term 'memory' of past encounters with antigen to maximize the opportunity for T cell/antigen-presenting cell interaction.     

辣椒热激转录因子家族在驯化过程中的演变

以拟南芥HSFs基因家族的已知信息为基础,挖掘辣椒栽培品种及其野生祖先品系基因组中HSFs基因,并对这些基因进行系统的比对和功能分析。在栽培辣椒遵辣一号基因组中共鉴定出25个HSFs基因,其野生祖先品系Chiltepin中鉴定出26个HSFs基因。从HSFs基因的染色体位置看,与其野生祖先品系Chiltepin比较,遵辣一号HSFs基因家族基本是一致的,在驯化过程中没有发生大的变化。与拟南芥一样,辣椒HSFs基因家族分成了A/B/C3个亚家族。从系统发育树中,可以清晰的看到辣椒HSFs基因在拟南芥中的同源基因。与拟南芥抗逆重要基因HSFA2基因同源的是遵辣一号的Capana08g000497与Chiltepin野生辣椒品系的Capang01g003145基因。这一重要的抗逆基因在驯化过程中,由Chiltepin一号染色体移动到遵辣一号的八号染色体。本研究为分析辣椒HSFs基因家族成员的功能提供了有价值的信息,为辣椒品种多种抗逆能力的改善,积累基因资源。     

Apoptosis in suspension culture of tobacco cells induced by heat shock

The induction of apoptosis in suspension culture of tobacco cells by heat shock is reported for the first time. Heat treatment (48℃ for 4 h) of tobacco cells led to the appearance of typical hallmarks of apoptosis. It was demonstrated by DNA laddering analysis that the cells treated with heat shock at 48℃ for 4 h had a serious degradation of nuclear DNA into multi-nu-cleosomal sizes, suggesting that heat shock activated endogenous nuclease which led to DNA cleavage at the linkage sites between the nucleosomes, but ladders were very faint for DNA from 2 and 9 h heat-treated cells. The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling (TUNEL) detection also showed that most of these treated cells (48℃ for 4 h) displayed positive reactions, indicating a serious DNA 3'-OH cleavage in their nuclei. Moreover, some other cytological changes in apoptotic cells, such as cell shrinkage, chromatin aggregation, nucleus collapse, have also been observed by 4', 6'-diamidino-2-phenyl-indole (DAPI) staining.     

基因转录的活细胞成像研究技术

转录是基因表达过程中的一个关键调节步骤,也是人类发育和疾病的基础。最近,用活细胞成像方法研究基因转录引起了人们广泛的兴趣．综述了此领域当前的技术进展,并讨论了将来的发展趋势．     

Mapping complex disease loci in whole-genome association studies

Identification of the genetic polymorphisms that contribute to susceptibility for common diseases such as type 2 diabetes and schizophrenia will aid in the development of diagnostics and therapeutics. Previous studies have focused on the technique of genetic linkage, but new technologies and experimental resources make whole-genome association studies more feasible. Association studies of this type have good prospects for dissecting the genetics of common disease, but they currently face a number of challenges, including problems with multiple testing and study design, definition of intermediate phenotypes and interaction between polymorphisms.     

轴对称零件高温热冲击动态热应力分析

采用泛函分析法，导出非定常温度场热弹性轴对称问题的线性时空有限元基本方程，并开发了预测轴对称零件承受高温热冲击载荷时热弹性动态应力分布的分析软件。在传热方程中不考虑热耦合项的情况下，建立了轴对称零件承受高温热冲击载荷时的有限元分析理论和离散模型。     

细胞凋亡过程中热休克蛋白的调节

热休克蛋白(heatshockproteins,HSPs)是一类进化上高度保守,广泛存在于自然界原核、真核细胞中的蛋白质。HSPs在正常细胞中呈基础性表达,维持细胞的基本形态和功能;它在应激环境下的细胞中,可参与细胞的损伤和修复,发挥应激保护作用。新近的研究发现,HSPs在细胞凋亡中发挥重要作用。     

Sustained oscillations in living cells

Glycolytic oscillations in yeast have been studied for many years simply by adding a glucose pulse to a suspension of cells and measuring the resulting transient oscillations of NADH. Here we show, using a suspension of yeast cells, that living cells can be kept in a well defined oscillating state indefinitely when starved cells, glucose and cyanide are pumped into a cuvette with outflow of surplus liquid. Our results show that the transitions between stationary and oscillatory behaviour are uniquely described mathematically by the Hopf bifurcation. This result characterizes the dynamical properties close to the transition point. Our perturbation experiments show that the cells remain strongly coupled very close to the transition. Therefore, the transition takes place in each of the cells and is not a desynchronization phenomenon. With these two observations, a study of the kinetic details of glycolysis, as it actually takes place in a living cell, is possible using experiments designed in the framework of nonlinear dynamics. Acetaldehyde is known to synchronize the oscillations. Our results show that glucose is another messenger substance, as long as the glucose transporter is not saturated.     

Effects of metal ions and heat shock on the expression of a metallothionein-like gene htMT2 in Helianthus tuberosus

The expression of htMT2 in tissues of Helianthus tuberosus treated with metal ions and heat shock is studied by Northern blot hybridization. The results indicate that htMT2 is not expressed in roots. The expression in leaves and stems can be reduced by Cu2+ and induced by low concentration of Zn2+. However, the expression of htMT2 can be suppressed by Zn2+ when its concentrations are above 500 μmol/L. The effects of Ca2+ on the expression of htMT2 in leaves are similar to that of Zn2+. But Ca2+ induces the expression of htMT2 in stems. The expression levels of htMT2 in various tissues are not significantly affected by heat shock. The results above demonstrate that the expression of htMT2 can be regulated by metal ions and confirm that htMT2 is a new metallothionein-like gene.     

BCG 65KD热休克蛋白的纯化

目的：研究卡介苗（BCG）65KD热休克蛋白的提纯方法。方法：BCG经37℃恒温振荡培养两周,42℃热休克处理2h,培养液经离心、抽滤、盐析沉淀、SDS-PAGE分离、电泳洗脱等步聚纯化。结果：提纯产物经SDS－PAGE分析,其电泳区带在65KD位置,经抑制试验表明具有较高抗原活性结论：流程方法简单、实用,有助于对IDDM的诊断及其深入研究。     

Glycoproteins form mixed disulphides with oxidoreductases during folding in living cells

The formation of intra- and interchain disulphide bonds constitutes an integral part of the maturation of most secretory and membrane-bound proteins in the endoplasmic reticulum. Evidence indicates that members of the protein disulphide isomerase (PDI) superfamily are part of the machinery needed for proper oxidation and isomerization of disulphide bonds. Models based on in vitro studies predict that the formation of mixed disulphide bonds between oxidoreductase and substrate is intermediate in the generation of the native intrachain disulphide bond in the substrate polypeptide. Whether this is how thiol oxidoreductases work inside the endoplasmic reticulum is not clear. Nor has it been established which of the many members of the PDI superfamily interacts directly with newly synthesized substrate proteins, because transient mixed disulphides have never been observed in the mammalian endoplasmic reticulum during oxidative protein folding. Here we describe the mechanisms involved in co- and post-translational protein oxidation in vivo. We show that the endoplasmic-reticulum-resident oxidoreductases PDI and ERp57 are directly involved in disulphide oxidation and isomerization, and, together with the lectins calnexin and calreticulin, are central in glycoprotein folding in the endoplasmic reticulum of mammalian cells.