Chaperone-Like Activity of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> Hsp16.3 Does Not Require Its Intact (Native) Structures

Small heat shock proteins (sHsps) were found to exhibit efficient chaperone-like activities under stress conditions although their native structures are severely disturbed. Here, using an alternative approach (site-directed mutagenesis), we obtained two structurally and functionally distinct Mycobacterium tuberculosis Hsp16.3 single-site mutant proteins. The G59W mutant protein (with Gly59 substituted by Trp) is capable of exhibiting efficient chaperone-like activity even under non-stress conditions although its secondary, tertiary, and quaternary structures are very different from that of the wild type protein. By contrast, the G59A mutant protein (with Gly59 substituted by Ala) resembles with the wild type protein in structure and function. These observations suggest that the Gly59 of the Hsp16.3 protein is critical for its folding and assembly. In particular, we propose that the exhibition of chaperone-like activity for Hsp16.3 does not require its intact (native) structures but requires the disturbance of its native structures (i.e., the native structure-disturbed Hsp16.3 retains its chaperone-like activity or even becomes more active). In addition, the behavior of such an active mutant protein (G59W) also strongly supports our previous suggestion that Hsp16.3 exhibits chaperone-like activity via oligomeric dissociation.     

Plasmodial heat shock proteins: targets for chemotherapy

Heat shock proteins act as molecular chaperones, facilitating protein folding in cells of living organisms. Their role is particularly important in parasites because environmental changes associated with their life cycles place a strain on protein homoeostasis. Not surprisingly, some heat shock proteins are essential for the survival of the most virulent malaria parasite, Plasmodium falciparum . This justifies the need for a greater understanding of the specific roles and regulation of malarial heat shock proteins. Furthermore, heat shock proteins play a major role during invasion of the host by the parasite and mediate in malaria pathogenesis. The identification and development of inhibitor compounds of heat shock proteins has recently attracted attention. This is important, given the fact that traditional antimalarial drugs are increasingly failing, as a consequence of parasite increasing drug resistance. Heat shock protein 90 (Hsp90), Hsp70/Hsp40 partnerships and small heat shock proteins are major malaria drug targets. This review examines the structural and functional features of these proteins that render them ideal drug targets and the challenges of targeting these proteins towards malaria drug design. The major antimalarial compounds that have been used to inhibit heat shock proteins include the antibiotic, geldanamycin, deoxyspergualin and pyrimidinones. The proposed mechanisms of action of these molecules and the pathways they inhibit are discussed.     

火烧高温对泥炭藓孢子萌发力影响的模拟实验

作为生态系统稳定性维持的一个重要因素,火对泥炭地优势植物泥炭藓(Sphagnum)孢子库的影响尚不清楚.以采自长白山区泥炭地的泥炭土和3种泥炭藓的成熟孢子为实验材料,室内模拟火烧,以此设置不同温度水平(20、40、60或100℃,持续0.5、1、2、4或10 min),对泥炭藓孢子进行热激处理,经萌发实验后,研究火烧高温对孢子萌发率的影响.结果显示,火烧期间各层土温随深度而递减,表层泥炭可达300℃的极端高温,而1 cm深温度仅为70℃,体现出泥炭土良好的热缓冲性;40℃的热激可使锈色泥炭藓(S.fuscum)与中位泥炭藓(S.magellanicum)孢子萌发率提高20%与50%;60℃的热激使尖叶泥炭藓(S.capillifolium)孢子的萌发率提高1倍;100℃热激对3种泥炭藓孢子萌发则有强烈的抑制作用.研究表明,泥炭藓孢子耐受高温的能力有限,但土壤中的孢子凭借泥炭的良好热缓冲性,可以躲避火烧高温造成的致命伤害,适度的热激甚至能提高其萌发力,对其在火后的建植及种群的长存可能有重要意义.     

毕氏海蓬子类囊体膜与卵磷脂重组脂质体的耐热性研究

采用卵磷脂(PC)构建脂质体,然后将毕氏海蓬子类囊体膜蛋白复合物重组到脂质体中.分析不同温度(25℃、35℃、45℃和55℃)处理后蛋白脂质体的电子传递活性、吸收光谱和荧光光谱的变化,以探讨膜脂与膜蛋白在高温胁迫下的交互作用.结果显示:蛋白脂质体光系统Ⅱ(PSⅡ)的放氧活性和光系统Ⅰ(PSⅠ)的耗氧活性随着PC比例的提高而增加,在PC与类囊体膜比例为4∶1(Lipid∶Chl,w/w)时达到最高,同时蛋白脂质体的吸收光谱和荧光光谱也呈上升趋势;在PC与类囊体膜重组比例为4∶1条件下,高温处理后的蛋白脂质体的PSⅡ放氧活性和PSⅠ耗氧活性显著大于未经重组的,其吸收光谱和荧光光谱峰值下降幅度低于未经重组的,且峰位基本没有变化.研究表明,PC可能通过增加结合天线的大小来促进蛋白脂质体对光能的吸收和能量从外周天线到PSⅡ和PSⅠ核心复合物的传递;在脂质体中,PC与类囊体膜的交互作用提高了PSⅡ和PSⅠ在高温胁迫下的光化学效率,增强了PSⅡ和PSⅠ的耐热性.     

HIFU治疗下生物体焦域温度分布的研究与应用

高强度聚焦超声(HIFU)为无创治疗肿瘤带来了新的途径,本文对HIFU治疗下生物体焦域热分布和热传导模型的理论进行了初步的介绍,并在理论的指导下进行不同治疗剂量的实验研究,结果表明组织内具有热波传热效应,治疗时可以根据治疗剂量的需要选取不同的参数,以达到最佳治疗效果。     

Identification of bis-ANS binding sites in Mycobacterium tuberculosis small heat shock protein Hsp16.3: evidences for a two-step substrate-binding mechanism

Small heat shock proteins (sHSPs), as one important subclass of molecular chaperones, are able to specifically bind to denatured substrate proteins rather than to native proteins, of which their substrate-binding sites are far from clear. Our previous study showed an overlapping nature of the sites for both hydrophobic probe 1,1'-Bi(4-anilino)naphthalene-5,5'-disulfonic acid (bis-ANS) binding and substrate binding in Mycobacterium tuberculosis Hsp16.3 [X. Fu, H. Zhang, X. Zhang, Y. Cao, W. Jiao, C. Liu, Y. Song, A. Abulimiti, Z. Chang, A dual role for the N-terminal region of M. tuberculosis Hsp16.3 in self-oligomerization and binding denaturing substrate proteins, J. Biol. Chem. 280 (2005) 6337-6348]. In this work, two bis-ANS binding sites in Hsp16.3 were identified by a combined use of reverse phase HPLC, mass spectroscopy and N-terminal protein sequencing. One site is in the N-terminal region and the other one in the N-terminus of alpha-crystallin domain, both of which are similar to those identified so far in sHSPs. However, accumulating data suggest that these two sites differentially function in binding substrate proteins. With regard to this difference, we proposed a two-step mechanism by which Hsp16.3 binds substrate proteins, i.e., substrate proteins are recognized and initially captured by the N-terminal region that is exposed in the dissociated Hsp16.3 oligomers, and then the captured substrate proteins are further stabilized in the complex by the subsequent binding of the N-terminus of alpha-crystallin domain.     

庙岛群岛南五岛灌木群落结构及其对环境因子的响应

该研究对山东省庙岛群岛南五岛灌木植被及环境因子进行调查,运用TWINSPAN等级划分法和多元分析技术研究调查区域内灌木的植被分布、群落结构及其与环境因子间的关系。结果显示:(1)调查期间庙岛群岛南五岛共发现灌木27种,隶属于15科23属,荆条、扁担木、酸枣为优势种。(2)灌木群丛主要被划分为6个群丛,即桑群丛、刺槐-柘树群丛、酸枣群丛、扁担木群丛、荆条群丛、紫穗槐群丛。(3)应用典范对应分析(CCA)并采用向前引入法对环境因子进行逐步筛选,Monte Carlo置换检验结果显示,乔木层盖度、土壤pH是影响庙岛群岛南五岛灌木空间分布的关键环境因子。研究认为,CCA二维排序结果与TWINSPAN分类结果较为一致,庙岛群岛南五岛森林灌木群落主要分布于乔木层盖度适中、偏酸性土壤的环境下。     

热暴露大鼠心肌细胞钙稳态及其调节机制的研究

本文观察了离体成年大鼠心室肌肌质网、线粒体Ｃａ＾２＋－ＡＴＰ酶活性和总钙含是到及原代培养乳腺心肌细胞＾４６Ｃａ摄取、活性钙调素相对含量、胞浆内游离钙浓度在不同温度热暴露４０ｍｉｎ后的变化。结果表明：热暴露后,心肌奖浆、肌质网及线粒体中的Ｃａ＾２＋－ＡＴＰ酶活发表 所下降,心肌肌质网及线粒体中的总钙含量亦有降低趋势,心肌细胞活性钙调素相对含量显著下降,＾４６Ｃａ摄取量及胞浆内游离浓度显著升高。提示,