Isolation of a <Emphasis Type="Italic">Latimeria menadoensis</Emphasis><Emphasis Type="Italic">heat shock protein 70</Emphasis> (<Emphasis Type="Italic">Lmhsp70</Emphasis>) that has all the features of an inducible gene and encodes a functional molecular chaperone

Molecular chaperones facilitate the correct folding of other proteins, and heat shock proteins form one of the major classes of molecular chaperones. Heat shock protein 70 (Hsp70) has been extensively studied, and shown to be critically important for cellular protein homeostasis in almost all prokaryotic and eukaryotic systems studied to date. Since there have been very limited studies conducted on coelacanth chaperones, the main objective of this study was to genetically and biochemically characterize a coelacanth Hsp70. We have successfully isolated an Indonesian coelacanth (L. menadoensis) hsp70 gene, Lmhsp70, and found that it contained an intronless coding region and a potential upstream regulatory region. Lmhsp70 encoded a typical Hsp70 based on conserved structural and functional features, and the predicted upstream regulatory region was found to contain six potential promoter elements, and three potential heat shock elements (HSEs). The intronless nature of the coding region and the presence of HSEs suggested that Lmhsp70 was stress-inducible. Phylogenetic analyses provided further evidence that Lmhsp70 was probably inducible, and that it branched as a clade intermediate between bony fish and tetrapods. Recombinant LmHsp70 was successfully overproduced, purified and found to be functional using ATPase activity assays. Taken together, these data provide evidence for the first time that the coelacanth encodes a functional molecular chaperone system. K. W. Modisakeng and M. Jiwaji contributed equally to this study.     

Analysis of oligomeric proteins during unfolding by pH and temperature

During thermal transition and variation of pH, structural properties of 35 proteins and their complexes (bound with substrate and co-factor) were analyzed in detail. During pH alteration, these proteins were shown to have substantial differences in conformations. pH conformers were analyzed in detail. Free energy and other energy parameters were also estimated for these proteins at various pH and temperatures. Detailed structural analysis and binding interfaces of various substrates, inhibitors and cofactor of these proteins were also investigated using docking and molecular dynamic simulation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Proteomic analysis of membrane proteins expressed specifically in pluripotent murine embryonic stem cells

Embryonic stem cells (ESCs) are established from the inner cell mass of preimplantation embryos, are capable of self‐renewal, and exhibit pluripotency. Given these unique properties, ESCs are expected to have therapeutic potential in regenerative medicine and as a powerful tool for in vitro differentiation studies of stem cells. Various growth factors and extracellular matrix components regulate the pluripotency and differentiation of ESC progenies. Thus, the cell surface receptors that bind these regulatory factors are crucial for the precise regulation of stem cells. To identify membrane proteins that are involved in the regulation of pluripotent stem cells, the membrane proteins of murine ESCs cultured with or without leukemia inhibitory factor (LIF) were purified and analyzed by quantitative proteomics. 2‐D PAGE‐based analysis using fluorescently labeled proteins and shotgun‐based analysis with isotope‐labeled peptides identified 338 proteins, including transmembrane, membrane‐binding, and extracellular proteins, which were expressed specifically in pluripotent or differentiated murine ESCs. Functions of the identified proteins revealed cell adhesion molecules, channels, and receptors, which are expected to play important roles in the maintenance of murine ESC pluripotency. Membrane proteins that are expressed in pluripotent ESCs but not in differentiated cells such as Slc16a1 and Bsg could be useful for the selection of the stem cells in vitro.     

Analysis of low molecular weight plasma proteins using ultrafiltration and large gel two‐dimensional electrophoresis

In this study, various solvent systems were applied to obtain a high and consistent recovery rate of low molecular weight plasma proteins (LMPP) from human plasma. A buffer system containing 7 M urea, 2 M thiourea, 25 mM NH₄HCO₃ + 20% ACN (pH 8.2) produced the highest recovery rate of LMPP. To validate the recovery of cut off membrane (COM) obtained using the urea buffer system, 27 different 30 kDa COMs were used to prepare the LMPP sample which were then subjected to 1‐D SDS‐PAGE. Statistical analysis showed that the buffer system with COM produced a consistent the recovery of LMPP. In addition, 2‐DE analysis was also conducted to determine the relative intensity of each protein spot. When molecular weight ranges over 30 kDa and under 30 kDa were evaluated, 953 and 587 protein spots were observed in the gels, respectively, resulting in a total of 1540 protein spots being resolved. Identification of the major proteins were then performed using a nano‐LC/MS system comprised of an HPLC system and an ESI‐quadrupole IT MS equipped with a nano‐ESI source.     

新疆冬春麦区小麦地方品种贮藏蛋白遗传多样性研究

采用SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）对236份新疆小麦地方品种的高分子量麦谷蛋白亚基（HMW-GS）的组成进行了分析。结果表明：Glu-Ⅰ位点共有19种等位基因,其中Glu-Al位点3种,Glu-Bl位点7种,Glu—D1住点9种;亚基null、7＋8、2＋12在各自的位点上出现频率最高,分别达到91．95％、85．17％、80．93％;亚基组成类型共有21种,主要为null／7＋8／2＋12,频率达70．34％;同时筛选出33份含有1、2^＊、13＋16、14＋15、5＋10、1．5＋10、174-18等优质亚基的材料,可作为优质基因源。利用酸性聚丙烯酰胺凝胶电泳（A-PAGE）对其中的65份地方品种进行醇溶蛋白多样性分析。结果表明：电泳出现64条迁移率不同的谱带,构成65种组合,其中ω区出现的谱带最多,达17条;其次是β和γ区各16条,α区出现的谱带数最少,为15条。从每条谱带在65份材料中出现的频率看,总的变异范围为1．54％～93．85％;α、β、γ和ω4个分区多样性指数（H1）分别为0．498、0．386、0．523和0．348,表明新疆麦区小麦地方品种贮藏蛋白位点存在丰富的遗传多样性。     

通过断裂内含肽介导的反式剪接合成大的蛋白

大肠杆菌难以表达大的蛋白,毒性蛋白以及膜蛋白,“Npu DnaE内含肽表达系统“使这些蛋白的表达成为可能。该系统的基本原理是：在特定位点处将目标基因（编码T7 RNA聚合酶的基因）断裂成两部分,然后分别与Npu DnaE内含肽的N端,C端片段融合,两种融合基因分别表达纯化,在体外将两种融合蛋白等摩尔比混合即可产生有功能的T7 RNA聚合酶。理论上,该体系也可用于合成其他大的蛋白,毒性蛋白或膜蛋白。     

Small hairpin RNA targeting at Bcl-2 increases cytarabine-induced apoptosis in Raji cells

Bcl-2, a prominent member of the Bcl-2 family proteins, is responsible for the dysregulation of apoptosis and resistance to chemotherapy. In this study, we inves-tigated whether small hairpin RNA （shRNA） targeting at Bcl-2 mRNA could enhance cytarabine （Ara-C）- induced apoptosis in Raji cells. Bcl-2 shRNA was trans-fected into Raji cells and the expression levels of Bcl-2 mRNA and protein were assayed by RT-PCR and immunofluorescence. Cell proliferation was determined by MTT assay. Apoptosis was determined by morphological observation and flow cytometric analysis. Our results show that expression levels of Bcl-2 mRNA and protein from Raji cells transfected with Bcl-2 shRNA decreased, compared with either negative control shRNA group or untransfected cells group （P 〈 0.05）. Viability of cells transfected with Bcl-2 shRNA was less than the cells transfected with control shRNA and untransfected Raji cells, respectively （P〈 0.05）. Bcl-2 shRNA combined with Ara-C significantly inhibited the growth of cells （P 〈 0.05）. There was no difference in cell survival between control shRNA/Ara-C combination and cells treated with Ara-C alone. Using Giemsa staining, cells treated with Bcl-2 shRNA plus Ara-C at 48h displayed changes of apoptosis. Apoptotic rates of Raji cells treated with Bcl-2 shRNA combined with Ara-C significantly increased （P 〈 0.05）, compared with either control shRNA/Ara-C combi- nation or Ara-C-treated cells alone. Our results suggest that the shRNA against Bcl-2 mRNA could increase Ara-C-induced apoptosis in Raji cells.     

Plant-based strategies aimed at expressing HIV antigens and neutralizing antibodies at high levels. Nef as a case study

The first evidence that plants represent a valid, safe and cost-effective alternative to traditional expression systems for large-scale production of antigens and antibodies was described more than 10 years ago. Since then, considerable improvements have been made to increase the yield of plant-produced proteins. These include the use of signal sequences to target proteins to different cellular compartments, plastid transformation to achieve high transgene dosage, codon usage optimization to boost gene expression, and protein fusions to improve recombinant protein stability and accumulation. Thus, several HIV/SIV antigens and neutralizing anti-HIV antibodies have recently been successfully expressed in plants by stable nuclear or plastid transformation, and by transient expression systems based on plant virus vectors or Agrobacterium-mediated infection. The current article gives an overview of plant expressed HIV antigens and antibodies and provides an account of the use of different strategies aimed at increasing the expression of the accessory multifunctional HIV-1 Nef protein in transgenic plants.