汉滩病毒核蛋白与热休克蛋白GRP94、HSP27的相互作用

为研究汉滩病毒(Hantaan virus,HTNV)感染诱导乳鼠脑组织热休克蛋白GRP94、HSP27与病毒蛋白的相互关系,选出生2—3d的昆明乳鼠实验性感染汉滩病毒,取8d后发病乳鼠脑组织部分制石蜡切片,用免疫组化结合共聚焦显微镜检测组织中病毒抗原及GRP94、HSP27的表达,部分制匀浆液,用ELISA、免疫共沉淀方法分析病毒抗原和GRP94、HSP27的关系。结果示汉滩病毒感染诱导乳鼠脑组织神经细胞高表达GRP94且与细胞内病毒抗原有共定位关系,但未见HSP27诱导高表达;免疫共沉淀显示汉滩病毒核心抗原(HINV—NP)与GRP94、HSP27呈非共价复合物形式存在。该结果为进一步探讨HSPs在病毒感染复制中的作用以及抗病毒感染方面提供了有意义的实验资料。     

新型大肠杆菌高效表达载体pHsh的构建与应用

在现代生物学和生物技术研究中,通过基因的重组表达获得目标蛋白是一种应用最广泛的方法。因其培养简单、操作方便、遗传背景清楚、克隆表达系统成熟完善,大肠杆菌表达系统通常是人们表达重组蛋白的首选,而表达载体在重组蛋白的生产中起决定作用。pHsh及其衍生质粒是近年发展起来的新型大肠杆菌表达载体,其调控外源基因表达的原理不同于所有其他表达系统,并且具有表达水平高、成本低廉等特点。介绍大肠杆菌表达系统的组成和常用表达载体,并对由pHsh系列载体组成的Hsh表达体系的构建策略、表达调控机制及其使用方法进行综述。Hsh表达体系的建立和发展有望从一个不同的角度帮助解决基因的重组表达中常见的表达水平低、诱导剂成本高、包涵体形成等问题。     

Differences in the heat-shock response between thermotolerant and thermosusceptible cultivars of hexaploid wheat

Summary Heat-shock protein (HSP) gene expression in two wheat lines cv Mustang (heat-tolerant) and cv Sturdy (heat-susceptible) were analyzed to determine if wheat genotypes differing in heat tolerance also differ in in-vitro HSP synthesis (translatable HSP mRNAs) and steady-state levels of HSP mRNA. Several sets of mRNA were isolated from seedling leaf tissues which had been heat-stressed at 37 °C for various time intervals. These mRNAs were hybridized with HSP cDNA or genomic DNA probes (HSP17, 26, 70, 98, and ubiquitin). Protein profiles were compared using in-vitro translation and 2-D gels. The Northern slot-blot data from the heat-stress treatment provide evidence that the heat-tolerant cv Mustang synthesized low molecular weight (LMW) HSP mRNA earlier during exposure to heat shock and at a higher level than did the heat-susceptible cv Sturdy. This was especially true for the chloroplast-localized HSP. The protein profiles shown by 2-D gel analysis revealed that there were not only quantitative differences of individual HSPs between the two wheat lines, but also some unique HSPs which were only found in the Mustang HSP profiles. The high level of RFLP between the two wheat lines was revealed by Southern blot hybridization utilizing a HSP17 probe. These data provide a molecular basis for further genetic analysis of the role of HSP genes in thermal tolerance in wheat.     

Biochemical characterization of the interaction between HspA1A and phospholipids

Seventy-kilodalton heat shock proteins (Hsp70s) are molecular chaperones essential for maintaining cellular homeostasis. Apart from their indispensable roles in protein homeostasis, specific Hsp70s localize at the plasma membrane and bind to specific lipids. The interaction of Hsp70s with lipids has direct physiological outcomes including lysosomal rescue, microautophagy, and promotion of cell apoptosis. Despite these essential functions, the Hsp70-lipid interactions remain largely uncharacterized. In this study, we characterized the interaction of HspA1A, an inducible Hsp70, with five phospholipids. We first used high concentrations of potassium and established that HspA1A embeds in membranes when bound to all anionic lipids tested. Furthermore, we found that protein insertion is enhanced by increasing the saturation level of the lipids. Next, we determined that the nucleotide-binding domain (NBD) of the protein binds to lipids quantitatively more than the substrate-binding domain (SBD). However, for all lipids tested, the full-length protein is necessary for embedding. We also used calcium and reaction buffers equilibrated at different pH values and determined that electrostatic interactions alone may not fully explain the association of HspA1A with lipids. We then determined that lipid binding is inhibited by nucleotide-binding, but it is unaffected by protein-substrate binding. These results suggest that the HspA1A lipid-association is specific, depends on the physicochemical properties of the lipid, and is mediated by multiple molecular forces. These mechanistic details of the Hsp70-lipid interactions establish a framework of possible physiological functions as they relate to chaperone regulation and localization.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-015-0636-6) contains supplementary material, which is available to authorized users.     

Heat-shock proteins during pollen development in maize

In contrast to sporophytic tissues, mature pollen of higher plants does not synthesize the typical set of heat-shock proteins (HSPs) in response to a marked temperature upshift. Immature grains, however, seem able to do so, at least partially. We investigated the characteristics of HSP synthesis throughout the male gametophytic phase in maize and compared gametophytic and sporophytic heat-shock responses. One-dimensional Sodium dodecyl sulfate-polyacryl-amide gel electrophoresis technique (SDS-PAGE) of newly synthesized proteins revealed that immature pollen synthesizes HSPs, some of which are not induced in sporophytic tissues. The heat-shock response appeared to be related to microgametophytic developmental stages. The strongest response was found in uninucleate microspores: at this stage, in addition to the sporophytic 102, 84, 72, and 18 kD HSPs, three other polypeptides of 74, 56, and 46 kD were observed. In the binucleate and trinucleate stages, only a reduced synthesis of few HSPs could be induced, and differences between genotypes were observed. In germinating pollen, HSP synthesis was not induced under a voriety of heat-stress conditions; however, the consti-tutive synthesis of two polypeptides of the same molecular weight, 72 and 64 kD, as two HSPs was observed. The biological significance of these results is discussed.     

Fine structure and distribution of contractile layers inAmoeba proteus preincubated at high temperature

Summary Ultrastructural and immunocytochemical studies allow the localization and identification of a microfilament cortex in heat-shockedAmoeba proteus at different stages of recovery to room temperature. Immediately after heating the cortex is in close contact with the cytoplasmic face of the plasma membrane; however, during cooling it detaches from the membrane and shifts toward the cell centre thus separating a region of peripheral hyaloplasm from central granuloplasm. After polymerization of a new submembrane cortex several detachment and reformation cycles rhythmically repeated for 2–3 hours until a multitude of stratified layers has been formed in the hyaloplasm.Electron micrographs reveal that the cortical layer at the plasma membrane is merely composed of a network of actin filaments, whereas the retracted contractile layers in the hyaloplasm and at the granuloplasmic border contain both, thick and thin filaments often arranged in bundles. The heat-shock induced activities of the microfilament cortex are based on the highly contractile properties of this system in conjunction with controlled displacements in the equilibrium between F- and G-actin.     

Supraoptimal growth-temperature reduces the heat-shock sensitivity of the chrysophycean flagellatePoterioochromonas malhamensis

Summary The chrysophycean flagellatePoterioochromonas malhamensis shows some characteristic alterations after a sublethal heat-shock (42 C, 16 minutes). Most cells round up and retract their cytoplasmic tail. The lorica formation is affected and, in nearly all cells, flagellar activity. Most membrane systems, especially the dictyosome, are severely damaged. In contrast the flagellar root apparatus and the cortical microtubules seem to be unaffected.After growth at a supraoptimal temperature of 33 C and a subsequent heat-shock (42 C, 16 minutes) the cells do not show the heat injuries reported above. The ability to form the lorica becomes also adapted to higher temperatures.Possible adaptation mechanisms are discussed.     

The expression of a heat-inducible chimeric gene in transgenic tobacco plants

Summary A chimeric gene under the control of the hsp70 promoter of Drosophila is heat regulated in roots, stems and leaves, but not in pollen of transgenic tobacco plants. For these and other parameters, it behaves similarly to plant heat-shock genes.