小麦叶片蛋白质双向电泳的改良方法

以普通小麦叶片为实验材料，在原有双向电泳基础上通过对样品制备、样品溶解、等电聚焦电泳、SDS-聚丙烯酰胺凝胶电泳以及染色方法等关键步骤进行改进和优化，在小麦叶片可溶性蛋白的分析中获得了满意的双向电泳图谱。     

苎麻生长素结合蛋白BnABP1与GFP融合在烟草中的表达

运用已克隆的苎麻生长素结合蛋白BnABP1基因cDNA及植物表达载体pCAMBIA1300 GFP,构建了35S启动子控制的苎麻BnABP1基因编码区段与绿色荧光蛋白(GFP)基因融合表达重组体(pCAMBIA1300 GFP BnABP1)。通过根癌农杆菌介导法将其转化烟草WS38,经抗性筛选和PCR检测获得了转基因烟草。对转基因烟草细胞进行荧光显微镜观察,发现在细胞质膜和内膜系统上都有较强烈的荧光信号,进一步证明苎麻生长素结合蛋白ABP1已结合在细胞的膜系统上。     

Tim50 maintains the permeability barrier of the mitochondrial inner membrane

Transport of metabolites across the mitochondrial inner membrane is highly selective, thereby maintaining the electrochemical proton gradient that functions as the main driving force for cellular adenosine triphosphate synthesis. Mitochondria import many preproteins via the presequence translocase of the inner membrane. However, the reconstituted Tim23 protein constitutes a pore remaining mainly in its open form, a state that would be deleterious in organello. We found that the intermembrane space domain of Tim50 induced the Tim23 channel to close. Presequences overcame this effect and activated the channel for translocation. Thus, the hydrophilic cis domain of Tim50 maintains the permeability barrier of mitochondria by closing the translocation pore in a presequence-regulated manner.     

Bacteriophage M13 procoat protein inserts into the plasma membrane as a loop structure

The major coat protein of bacteriophage M13 is synthesized as a precursor, the procoat, with a typical leader (signal) sequence of 23 residues at its NH2-terminus. A fusion protein that contains the NH2-terminal 141 residues of cytoplasmic ribulokinase and all but the first ten residues of M13 procoat was made. The fusion protein inserts into the plasma membrane of Escherichia coli and is processed by leader peptidase to give rise to a leader peptide of 155 residues and the mature coat protein of 50 residues. The NH2-terminus of the leader peptide remains in the cytoplasm and is protected from protease added to the medium outside of the cell. This indicates that M13 procoat inserts into the membrane as a loop structure and that the NH2-terminus of a leader peptide remains within the cytoplasm during membrane insertion.     

Long unfolded linkers facilitate membrane protein import through the nuclear pore complex

Active nuclear import of soluble cargo involves transport factors that shuttle cargo through the nuclear pore complex (NPC) by binding to phenylalanine-glycine (FG) domains. How nuclear membrane proteins cross through the NPC to reach the inner membrane is presently unclear. We found that at least a 120-residue-long intrinsically disordered linker was required for the import of membrane proteins carrying a nuclear localization signal for the transport factor karyopherin-α. We propose an import mechanism for membrane proteins in which an unfolded linker slices through the NPC scaffold to enable binding between the transport factor and the FG domains in the center of the NPC.     

高吸水性树脂包膜尿素的结构特征及养分控/缓释性能

 【目的】研究高吸水性树脂包膜尿素的结构特征及养分控/缓释性能。【方法】以不同类型和吸水倍率的高吸水性树脂为包膜材料，以大颗粒尿素和改性矿物包膜尿素为原料肥料，研制出高吸水性树脂单层包膜尿素、高吸水性树脂复式包膜尿素系列产品，利用电子扫描电镜研究了其结构特征，并以水中溶出率法和模拟土柱淋溶法研究其养分释放特性。【结果】高吸水性树脂包膜尿素水中24 h氮素溶出率在18.22%～83.87%之间，土壤中4周氮素累积淋出率在40.91%～65.38%之间，较尿素降低5.21%～40.68%。控/缓释性能与高吸水性树脂的类型、吸水倍率及用量有关。聚丙烯酰胺型高吸水性树脂包膜尿素控/缓释效果好于交联聚丙烯酸盐型高吸水性树脂包膜尿素。随着高吸水性树脂用量的增加，高吸水性树脂包膜尿素的水中溶出率和土壤淋出率均明显下降；扫描电镜图显示高吸水性树脂复式包膜尿素的包膜效果明显优于高吸水性树脂单层包膜尿素，其控/缓释效果还与内膜材料有关。高吸水性树脂包膜尿素的内外膜均是由大小不一形状不规则的包膜材料微粒无序紧密堆积，并由胶粘剂填充空隙胶联而成，包膜叠层间和叠层内有微小孔隙,它们是尿素溶出的通道。【结论】高吸水性树脂包膜尿素兼具吸水、保水和养分控/缓释性能，是具有较好开发和应用前景的保水型控/缓释肥料。     

Point mutational inactivation of the retinoblastoma antioncogene

The retinoblastoma (Rb) antioncogene encodes a nuclear phosphoprotein, p105-Rb, that forms protein complexes with the adenovirus E1A and SV40 large T oncoproteins. A novel, aberrant Rb protein detected in J82 bladder carcinoma cells was not able to form a complex with E1A and was less stable than p105-Rb. By means of a rapid method for the detection of mutations in Rb mRNA, this defective Rb protein was observed to result from a single point mutation within a splice acceptor sequence in J82 genomic DNA. This mutation eliminates a single exon and 35 amino acids from its encoded protein product.     

Outer membrane active transport: structure of the BtuB:TonB complex

In Gram-negative bacteria, the import of essential micronutrients across the outer membrane requires a transporter, an electrochemical gradient of protons across the inner membrane, and an inner membrane protein complex (ExbB, ExbD, TonB) that couples the proton-motive force to the outer membrane transporter. The inner membrane protein TonB binds directly to a conserved region, called the Ton-box, of the transporter. We solved the structure of the cobalamin transporter BtuB in complex with the C-terminal domain of TonB. In contrast to its conformations in the absence of TonB, the Ton-box forms a beta strand that is recruited to the existing beta sheet of TonB, which is consistent with a mechanical pulling model of transport.     

Global topology analysis of the Escherichia coli inner membrane proteome

The protein complement of cellular membranes is notoriously resistant to standard proteomic analysis and structural studies. As a result, membrane proteomes remain ill-defined. Here, we report a global topology analysis of the Escherichia coli inner membrane proteome. Using C-terminal tagging with the alkaline phosphatase and green fluorescent protein, we established the periplasmic or cytoplasmic locations of the C termini for 601 inner membrane proteins. By constraining a topology prediction algorithm with this data, we derived high-quality topology models for the 601 proteins, providing a firm foundation for future functional studies of this and other membrane proteomes. We also estimated the overexpression potential for 397 green fluorescent protein fusions; the results suggest that a large fraction of all inner membrane proteins can be produced in sufficient quantities for biochemical and structural work.     

叶绿体运输蛋白的转运方式和运输机制研究

叶绿体的空间结构有6个区隔：外膜、膜间隙、内膜、基质、类囊体膜和类囊体腔。重点介绍了运输蛋白的跨外膜转运和跨内膜转运的运输机制,分别在叶绿体外膜易位子TOC和内膜易位子TIC的协助下实现跨膜转运。同时,介绍了分子伴侣的作用,它可提供前体蛋白运输的动力。     

苹果三倍体和二倍体细胞超微结构观察

对３个苹果品种的二倍体和２个苹果品种三倍体只峙细胞超微结构的观察发现,三倍体细胞超微结构与二倍体明显不同。三倍体细胞,细胞核线粒体、叶绿体的膜结构多存在一定程度的损伤,细胞叶绿体片层膜或者肿长,或者表现稀疏。     

Dynamic nuclear actin assembly by Arp2/3 complex and a baculovirus WASP-like protein

Diverse bacterial and viral pathogens induce actin polymerization in the cytoplasm of host cells to facilitate infection. Here, we describe a pathogenic mechanism for promoting dynamic actin assembly in the nucleus to enable viral replication. The baculovirus Autographa californica multiple nucleopolyhedrovirus induced nuclear actin polymerization by translocating the host actin-nucleating Arp2/3 complex into the nucleus, where it was activated by p78/83, a viral Wiskott-Aldrich syndrome protein (WASP)-like protein. Nuclear actin assembly by p78/83 and Arp2/3 complex was essential for viral progeny production. Recompartmentalizing dynamic host actin may represent a conserved mode of pathogenesis and reflect viral manipulation of normal functions of nuclear actin.     

饥饿胁迫下大鳞副泥鳅外周红细胞的形态变化

[目的]为研究饥饿胁迫下鱼类的生理生化变化提供参考。[方法]采用传统瑞氏染色法研究饥饿胁迫下大鳞副泥鳅外周红细胞的形态变化。[结果]大鳞副泥鳅的成熟红细胞逐渐发生变化,先出现核膜溶解,然后出现细胞形变,接着出现核质扩散和细胞膜溶解,最后整个成熟红细胞膜完全溶解,核染色反应由紫黑色变为紫红色最后变为粉红色。大鳞副泥鳅的幼稚红细胞则明显增多。[结论]在饥饿胁迫下大鳞副泥鳅外周红细胞出现核异常,主要表现为核膜溶解和核质扩散,因此红细胞核异常可以作为饥饿胁迫的指标。     

点带石斑鱼脑组织和视网膜中神经型一氧化氮合成酶的分布与定位

【目的】研究点带石斑鱼脑组织和视网膜中的一氧化氮（NO）含量及神经型一氧化氮合成酶（nNOS）分布与活性,为深入揭示NO在点带石斑鱼神经系统中的作用机制打下基础。【方法】采用NADPH-d组织化学染色法、免疫组织化学法、蛋白免疫印迹（Western blotting）及Griess试剂法,对点带石斑鱼脑组织和视网膜中nNOS的分布与活性及NO含量进行分析。【结果】经NADPH-d组织化学染色后,一氧化氮合成酶（NOS）阳性物质呈蓝色。在点带石斑鱼大脑中,NOS阳性物质位于神经元和神经纤维;在小脑中,NOS分布在颗粒层的颗粒细胞、分子层的神经纤维和浦肯野细胞层的浦肯野细胞;在视网膜中,NOS分布于色素上皮层、视锥视杆层、外核层、外网层、内核层、内网层、节细胞层和视神经纤维层。经免疫组织化学染色后,nNOS阳性物质呈深棕色。在大脑中,nNOS存在于神经元和神经纤维;在小脑中,nNOS分布在颗粒层、分子层和浦肯野细胞层;在视网膜中,nNOS分布在色素上皮层、视锥视杆层、外核层、外网层、内核层、内网层、节细胞层和视神经纤维层。Western blotting检测结果显示,点带石斑鱼脑组织和视网膜中的nNOS在显色后的硝酸纤维素膜上均出现3条免疫印迹条带,对应分子量分别为80、120和130kD。NO含量在点带石斑鱼脑组织和视网膜中分别为17.601±1.743和13.624±1.249μmol/L,nNOS活性在脑组织和视网膜中分别为38.070±3.047和23.748±2.860U/mg。【结论】在点带石斑鱼脑组织和视网膜中均存在nNOS,推测nNOS在点带石斑鱼脑神经及视神经系统生理活动中发挥重要作用。脑组织中的NO含量和nNOS活性均显著高于视网膜,是由于脑组织作为重要的中枢神经,对机体生理功能起重要调节作用。     

Membrane structure: some general principles

The arrangement of lipids and some proteins in the erythrocyte membrane has been discussed. The conclusions from this are listed here as a set of general guidelines for the structure of membranes of higher organisms: some of these rules may be wrong. But at this stage it seems useful to sharpen our thoughts in this way and thereby focus attention on various specific points. 1) The basis of a membrane is a lipid bilayer with (i) choline phospholipids and glycolipids in the external half and (ii) amino (and possibly some choline) phospholipids in the cytoplasmic half. There is effectively no lipid exchange across the bilayer (unless enzymatically catalyzed) (68). 2) Some proteins extend across the bilayer. Where this is so, they will in general have carbohydrate on their surface remote from the cytoplasm. This carbohydrate may prevent the protein diffusing out of the membrane into the cytoplasm; it acts as a lock on the protein. 3) Just as lipids do not flip-flop, proteins do not rotate across the membrane. Lateral motion or rotation of lipids and proteins in the plane of the bilayer may be expected. 4) Most membrane protein is associated with the inner, cytoplasmic, urface of the membrane. Proteins are not usually associated exclusively with the outer half of the lipid bilayer. 5) Membrane proteins are a special class of cytoplasmic proteins, not of secreted proteins.     

The function of a leader peptide in translocating charged amino acyl residues across a membrane

Insertion of bacteriophage coat proteins into the membrane of infected bacterial cells can be studied as a model system of protein translocation across membranes. The coat protein of the filamentous bacteriophage Pf3--which infects Pseudomonas aeruginosa--is 44 amino acids in length and has the same basic structure as the coat protein of bacteriophage M13, which infects Escherichia coli. However, unlike the Pf3 coat protein, the M13 coat protein is synthesized as a precursor (procoat) with a typical leader (signal) sequence, which is cleaved after membrane insertion. Nevertheless, when the gene encoding the Pf3 coat protein is expressed in E. coli, the protein is translocated across the membrane. Hybrid M13 and Pf3 coat proteins were constructed in an attempt to understand how the Pf3 coat protein is translocated without a leader sequence. These studies demonstrated that the extracellular regions of the proteins determined their cellular location. When three charged residues in this region were neutralized, the leader-free M13 coat protein was also inserted into the membrane. Differences in the water shell surrounding these residues may account for efficient membrane insertion of the protein without a leader sequence.     

Atrial natriuretic peptide inhibits a cation channel in renal inner medullary collecting duct cells

The patch-clamp technique was used to examine the effects of atrial natriuretic peptide (ANP) and its second messenger guanosine 3',5'-monophosphate (cGMP) on an amiloride-sensitive cation channel in the apical membrane of renal inner medullary collecting duct cells. Both ANP (10(-11) M) and dibutyryl guanosine 3',5'-monophosphate (10(-4) M) inhibited the channel in cell-attached patches, and cGMP (10(-5) M) inhibited the channel in inside-out patches. The inner medullary collecting duct is the first tissue in which ANP, via its second messenger cGMP, has been shown to regulate single ion channels. The results suggest that the natriuretic action of ANP is related in part to cGMP-mediated inhibition of electrogenic Na+ absorption by the inner medullary collecting duct.