Hsp90 as a capacitor of phenotypic variation

Heat-shock protein 90 (Hsp90) chaperones the maturation of many regulatory proteins and, in the fruitfly Drosophila melanogaster, buffers genetic variation in morphogenetic pathways. Levels and patterns of genetic variation differ greatly between obligatorily outbreeding species such as fruitflies and self-fertilizing species such as the plant Arabidopsis thaliana. Also, plant development is more plastic, being coupled to environmental cues. Here we report that, in Arabidopsis accessions and recombinant inbred lines, reducing Hsp90 function produces an array of morphological phenotypes, which are dependent on underlying genetic variation. The strength and breadth of Hsp90's effects on the buffering and release of genetic variation suggests it may have an impact on evolutionary processes. We also show that Hsp90 influences morphogenetic responses to environmental cues and buffers normal development from destabilizing effects of stochastic processes. Manipulating Hsp90's buffering capacity offers a tool for harnessing cryptic genetic variation and for elucidating the interplay between genotypes, environments and stochastic events in the determination of phenotype.     

基于Hsp90 ATPase活性的抑制剂筛选模型研究

热休克蛋白90(Heat shock protein,Hsp90)作为分子伴侣,参与调控肿瘤细胞多条信号通路,对肿瘤细胞生长有重要作用.以pGEX-4T-1为载体,构建了Hsp90表达载体于宿主大肠杆菌中表达.通过孔雀绿磷钼酸铵-无机磷检测法,测定异源表达的Hsp90 ATPase活性.实验得出Hsp90对ATP的米氏常数(Km)为369.1 μmol/L,在Hsp90和ATP浓度分别为0.18,1 mmol/L时转换效率常数(kcat)为1.6 min-1,最大反应速率(Vmax)为1.0 μmol/(L·min).基于该方法,建立Hsp90 ATPase活性抑制剂筛选模型,以Geldanamycin和Radicicol为阳性对照,发现Rifamycin、Rugulosin及MycoE也有较强的Hsp90 ATPase抑制活性.该方法可用于筛选抗肿瘤候选化合物的研究,同时也可为具有ATPase活性蛋白的抑制剂筛选提供参考.     

Hsp90 stress potentiates rapid cellular adaptation through induction of aneuploidy

Aneuploidy--the state of having uneven numbers of chromosomes--is a hallmark of cancer and a feature identified in yeast from diverse habitats. Recent studies have shown that aneuploidy is a form of large-effect mutation that is able to confer adaptive phenotypes under diverse stress conditions. Here we investigate whether pleiotropic stress could induce aneuploidy in budding yeast (Saccharomyces cerevisae). We show that whereas diverse stress conditions can induce an increase in chromosome instability, proteotoxic stress, caused by transient Hsp90 (also known as Hsp82 or Hsc82) inhibition or heat shock, markedly increased chromosome instability to produce a cell population with high karyotype diversity. The induced chromosome instability is linked to an evolutionarily conserved role for the Hsp90 chaperone complex in kinetochore assembly. Continued growth in the presence of an Hsp90 inhibitor resulted in the emergence of drug-resistant colonies with chromosome XV gain. This drug-resistance phenotype is a quantitative trait involving copy number increases of at least two genes located on chromosome XV. Short-term exposure to Hsp90 stress potentiated fast adaptation to unrelated cytotoxic compounds by means of different aneuploid chromosome stoichiometries. These findings demonstrate that aneuploidy is a form of stress-inducible mutation in eukaryotes, capable of fuelling rapid phenotypic evolution and drug resistance, and reveal a new role for Hsp90 in regulating the emergence of adaptive traits under stress.     

Crystal structure of an Hsp90-nucleotide-p23/Sba1 closed chaperone complex

Hsp90 (heat shock protein of 90 kDa) is a ubiquitous molecular chaperone responsible for the assembly and regulation of many eukaryotic signalling systems and is an emerging target for rational chemotherapy of many cancers. Although the structures of isolated domains of Hsp90 have been determined, the arrangement and ATP-dependent dynamics of these in the full Hsp90 dimer have been elusive and contentious. Here we present the crystal structure of full-length yeast Hsp90 in complex with an ATP analogue and the co-chaperone p23/Sba1. The structure reveals the complex architecture of the 'closed' state of the Hsp90 chaperone, the extensive interactions between domains and between protein chains, the detailed conformational changes in the amino-terminal domain that accompany ATP binding, and the structural basis for stabilization of the closed state by p23/Sba1. Contrary to expectations, the closed Hsp90 would not enclose its client proteins but provides a bipartite binding surface whose formation and disruption are coupled to the chaperone ATPase cycle.     

Hsp90抑制剂木犀草苷对非小细胞肺癌的体外抑制活性及作用机理研究

本文利用荧光偏振法(FP)构建分子伴侣Hsp90的筛选平台,筛选得到黄酮类化合物木犀草苷,并用热稳定实验验证了它与蛋白的结合作用;评价了木犀草苷对14株癌细胞的体外增殖抑制作用,发现木犀草苷对非小细胞肺癌有良好的选择性抑制活性;利用细胞克隆形成、western blot以及流式细胞检测技术进一步深入研究其可能的作用机制,发现木犀草苷能剂量依赖性地抑制非小细胞肺癌的增殖;下调Hsp90的客户蛋白Raf-1、HER2的表达;将H460细胞阻滞在G2/M期;诱导细胞凋亡,并且引起Bax的表达上调。实验结果表明,黄酮类化合物木犀草苷可以通过抑制Hsp90的活性进而降解肿瘤细胞内的客户蛋白,诱导H460细胞G2/M期阻滞,上调Bax表达诱导细胞凋亡来发挥抗肿瘤活性。     

A tumour suppressor network relying on the polyamine-hypusine axis

Tumour suppressor genes encode a broad class of molecules whose mutational attenuation contributes to malignant progression. In the canonical situation, the tumour suppressor is completely inactivated through a two-hit process involving a point mutation in one allele and chromosomal deletion of the other. Here, to identify tumour suppressor genes in lymphoma, we screen a short hairpin RNA library targeting genes deleted in human lymphomas. We functionally identify those genes whose suppression promotes tumorigenesis in a mouse lymphoma model. Of the nine tumour suppressors we identified, eight correspond to genes occurring in three physically linked 'clusters', suggesting that the common occurrence of large chromosomal deletions in human tumours reflects selective pressure to attenuate multiple genes. Among the new tumour suppressors are adenosylmethionine decarboxylase 1 (AMD1) and eukaryotic translation initiation factor 5A (eIF5A), two genes associated with hypusine, a unique amino acid produced as a product of polyamine metabolism through a highly conserved pathway. Through a secondary screen surveying the impact of all polyamine enzymes on tumorigenesis, we establish the polyamine-hypusine axis as a new tumour suppressor network regulating apoptosis. Unexpectedly, heterozygous deletions encompassing AMD1 and eIF5A often occur together in human lymphomas and co-suppression of both genes promotes lymphomagenesis in mice. Thus, some tumour suppressor functions can be disabled through a two-step process targeting different genes acting in the same pathway.     

基于 AT90S8535的炉温控制器设计

介绍了以AT90S8535为控制芯片的电炉炉温控制系统的设计.该控制器由AT90S853单片机、键盘/显示、热电偶、温度变送器、可控硅触发电路等组成.控制器采用了PID控制算法.由AT90S8535单片机构成的炉温控制器具有高效、控制精度高、外围电路简单、可靠性好、成本低等优点,因此它具有很好的市场推行价值.     

Dependence of single-molecule junction conductance on molecular conformation

Since it was first suggested that a single molecule might function as an active electronic component, a number of techniques have been developed to measure the charge transport properties of single molecules. Although scanning tunnelling microscopy observations under high vacuum conditions can allow stable measurements of electron transport, most measurements of a single molecule bonded in a metal-molecule-metal junction exhibit relatively large variations in conductance. As a result, even simple predictions about how molecules behave in such junctions have still not been rigorously tested. For instance, it is well known that the tunnelling current passing through a molecule depends on its conformation; but although some experiments have verified this effect, a comprehensive mapping of how junction conductance changes with molecular conformation is not yet available. In the simple case of a biphenyl--a molecule with two phenyl rings linked by a single C-C bond--conductance is expected to change with the relative twist angle between the two rings, with the planar conformation having the highest conductance. Here we use amine link groups to form single-molecule junctions with more reproducible current-voltage characteristics. This allows us to extract average conductance values from thousands of individual measurements on a series of seven biphenyl molecules with different ring substitutions that alter the twist angle of the molecules. We find that the conductance for the series decreases with increasing twist angle, consistent with a cosine-squared relation predicted for transport through pi-conjugated biphenyl systems.     

手术后结合90Sr-90Y敷贴治疗增生性瘢痕及瘢痕疙瘩的疗效观察

目的：探讨^90Sr-^90Y敷贴治疗对增生性瘢痕及瘢痕疙瘩的疗效．方法：对收治的112例瘢痕疙瘩和增生性瘢痕患者，采用^90Sr-^90Y敷贴治疗与手术切除后结合^90Sr-^90Y敷贴综合治疗，均采用多次小剂量法照射，每疗程30-50Gy，一般2～3个疗程．结果：采用单纯敷贴治疗75例患者。治愈48例（占64％），显效18例（占24％），综合治疗组37例患者，治愈33例（占89．2％），显效2例（占5．4％），两组治愈率比较（P〈0．05）。差异有显著性．表明综合治疗治愈率高于单纯敷贴治疗．结论：^90Sr-^90Y敷贴治疗是一种简便有效的治疗方法，尤其是手术后结合敷贴综合治疗疗效更加显著．     

A new class of angiotensin-converting enzyme inhibitors

Much current attention focuses on the renin-angiotensin system in relation to mechanisms controlling blood pressure and renal function. Recent demonstrations (ref. 1, ref. 2 and refs therein) that angiotensin-converting enzyme inhibitors show promising clinical antihypertensive properties have been of particular interest. We now report on the design of a novel series of substituted N-carboxymethyl-dipeptides which are active in inhibiting angiotensin-converting enzyme at nanomolar levels. We suggest that these compounds are transition-state inhibitors and that extensions of this design to other metalloendopeptidases merit further study.     

结核分枝杆菌Hsp16．3对感染巨噬细胞凋亡的影响

为研究结核分枝杆菌Hsp16．3对感染巨噬细胞凋亡的影响。分别用结核分枝杆菌国际标准强毒株H37Rv株（H37Rv）、结核分枝杆菌国际标准强毒株H37Rv株Hsp16．3基因缺失突变株（△H37Rv）、卡介苗（BCG）和卡介苗Hsp16．3基因缺失突变株（△BCG）感染RAW264．7巨噬细胞株,使用流式细胞技术于感染1、3、6及12h检测各组感染巨噬细胞的凋亡率,并分析其变化。结果显示,巨噬细胞感染结核分枝杆菌后凋亡率升高,其中在感染1、3、12h,BCG组与H37Rv组比较差异具有统计学意义（P〈O．05）;在感染1、3h,△BCG组与AH37Rv组比较差异具有统计学意义（P〈0．05）;在感染3、6、12h,△H37Rv组与H37Rv组比较差异具有统计学意义（P〈0．05）,△BCG组与BCG组比较差异具有统计学意义（P〈0．05）。由此可知,结核分枝杆菌Hspl6．3可抑制感染巨噬细胞的凋亡。     

Localization of the binding site for the human high-affinity Fc receptor on IgG

A major pathway in the clearance of pathogens involves the coating of the pathogen with specific antibodies, and the binding of the antibody Fc region to cell receptors. This can trigger engulfment of the pathogen by phagocytes or lysis by killer cells. By oligonucleotide site-directed mutagenesis we have engineered a single amino acid change in a mouse IgG2b antibody (Glu 235----Leu) which now enables the antibody to bind to the FcRI (high affinity) receptor on human monocytes with a 100-fold improvement in affinity. This indicates that Leu 235 is a major determinant in the binding of antibody to FcRI and that the receptor may interact directly with the region linking the CH2 domain to the hinge. Tailoring the affinity of antibodies for cell receptors could help dissect their role in clearing pathogen.     

采油污水回注系统高效缓蚀阻垢剂

针对辽河油田某采油厂采油污水回注系统腐蚀结垢的严重情况,分析了注水系统水质各项指标,明确其腐蚀结垢原因.通过实验室动态模拟实验、现场试验等方法,筛选复配适合用于采油污水的缓蚀阻垢剂.实验结果表明,在药剂质量浓度45mg/L时,注水系统腐蚀速率为0.05mm/a,阻垢率达98%以上,能同时实现腐蚀与结垢的有效控制.该药剂投加剂量小,成本低,缓蚀阻垢效果优良,具有较好的推广应用价值.     

建筑群的雷击选择性分析及直击雷防护

以电气几何模型的滚球法为理论依据,分析建筑物雷击点的选择性,定性推导建筑群之间的相互屏蔽性,总结建筑群直击雷防护的一些措施,为直击雷的防护提供更加合理和有效的方法.     

基于DHT90的智能湿度计研制

讨论了基于DHT90温湿度传感器的测量原理,介绍了基于DHT90的智能湿度计的接口、温湿度数据传输以及硬件与软件设计.智能湿度计控制以C8051F330单片机为核心,采用非线性校正和温度补偿算法,实现了湿度的智能化、快速准确测量.该仪器测量精确度高、响应速度快、结构简单,在石油化工等行业有广泛的应用价值.     

本溪钓鱼台地貌的分布及成因

辽宁本溪钓鱼台地貌由晚元古代青白口纪石英砂岩所形成,在成景地层上是中国砂(砾)岩地貌最古老的类型之一.通过实地考察法和历史资料对比法,发现该地貌在成景岩石的性质、成景岩层时代、微造型地貌组合和沉积环境等方面,都有别于中国砂(砾)岩地貌类型,因此确定它是一种新的地貌景观类型,这对完善中国砂(砾)岩地貌的分类体系有着重要的科学意义.在理论上阐述了辽宁本溪钓鱼台地貌发育的自然条件、分布特征以及形成原因,并进一步论述了钓鱼台地貌的分布规律以及成因类型.鉴于本溪钓鱼台地貌具有古老性、科学性、奇特性和可观赏性等特点,具有重大的科研价值和旅游开发价值,其对本溪国家地质公园的进一步开发、推动本溪旅游业的发展有着重大的现实意义.     

CAN总线通信技术在LTU90A智能摊铺机中的应用

采用EPEC2000系列车用微处理器模块,开发了LTU90A智能摊铺机,实现筑路工程机械自动控制;在通讯硬件平台的支持下,便于筑路机群各机种间通讯和远程故障诊断,有利于提高摊铺机路面施工质量和效率;针对EPEC车用模块,分析了CODESYS编程软件的开发、用户程序的设计及系统函数库的调用;利用CAN总线通讯技术在通讯过程中具有的可靠性、实时性和灵活性的特点,采用CAN网络结构和CAN2.0B数据包的定义,将数据传送分为3种发送级别.由于摊铺机工作环境恶劣,为了提高系统通讯的可靠性,在CAN的MAC层已具有CRC校验的基础上,增加一级求和取模校验措施.实践结果表明,CANBUS系统可靠性高,便于扩充,实时性好.     

A comparison of inhibition in orientation and spatial frequency selectivity of cat visual cortex

Neurones in the visual cortex are highly selective for orientation and spatial frequency of visual stimuli. There is strong neurophysiological evidence that orientation selectivity is enhanced by inhibitory interconnections between columns in the cortex which have different orientation sensitivities, an idea which is supported by experiments using neuropharmacological manipulation or complex visual stimuli. It has also been proposed that selectivity for spatial frequency is mediated in part by a similar mechanism to that for orientation, although evidence for this is based on special use of visual stimuli, which hampers interpretation of the findings. We have therefore examined selectivity for both orientation and spatial frequency using a technique which allows direct inferences about inhibitory processes. Our method uses microiontophoresis of an excitatory amino acid to elevate maintained discharge of single neurones in the visual cortex. We then present visual stimuli both within and outside the range of orientations and spatial frequencies which cause a cell to respond with increased discharge. Our results show that orientations presented on either side of the responsive range usually produce clear suppression of maintained discharge. In marked contrast, spatial frequencies shown to either side of the responsive range have little or no effect on maintained activity. We conclude that there is an intracortical organization of inhibitory connections between cells tuned to different orientations but not different spatial frequencies.     

A second human interleukin-2 binding protein that may be a component of high-affinity interleukin-2 receptors

Although activated human T and B lymphocytes express both high-affinity and low-affinity membrane receptors for interleukin-2 (IL-2), the structural features that distinguish these receptors have remained unresolved. The high-affinity receptors appear to mediate IL-2 induced T cell growth and internalization of IL-2, whereas no function has yet been ascribed to the low-affinity receptors. The Tac antigen is an IL-2 binding protein of relative molecular mass 55,000 (Mr 55K) that participates in the formation of both high- and low-affinity receptors. But Tac complementary DNA transfection and membrane fusion studies have suggested that additional T-cell components are required to produce high-affinity IL-2 receptors. In this study, we report the identification of a second human IL-2 binding protein that (1) has an Mr of approximately 70K, (2) lacks reactivity with the anti-Tac antibody, (3) binds IL-2 with intermediate affinity and (4) is present on the surface of resting T cells, large granular lymphocytes (natural killer cells), and certain T and B cell lines in the absence of the Tac antigen. Chemical crosslinking of 125I-labelled IL-2 bound to high-affinity IL-2 receptors produces labelling of both the p70 protein and the Tac antigen and the anti-Tac antibody blocks the crosslink detection of both of these proteins. Expression of Tac cDNA in a T cell line expressing the p70 protein, but lacking both Tac and high-affinity receptors, results in the reconstitution of high-affinity IL-2 receptors in these cells. Together, these findings suggest that the high-affinity human IL-2 receptor may be a membrane complex composed of at least the p70 protein and Tac antigen.