An architectural framework that may lie at the core of the postsynaptic density

The postsynaptic density (PSD) is a complex assembly of proteins associated with the postsynaptic membrane that organizes neurotransmitter receptors, signaling pathways, and regulatory elements within a cytoskeletal matrix. Here we show that the sterile alpha motif domain of rat Shank3/ProSAP2, a master scaffolding protein located deep within the PSD, can form large sheets composed of helical fibers stacked side by side. Zn2+, which is found in high concentrations in the PSD, binds tightly to Shank3 and may regulate assembly. Sheets of the Shank protein could form a platform for the construction of the PSD complex.     

高抗白粉病小麦-山羊草新种质TA002的创制和遗传研究

【目的】小麦白粉病是世界范围内对小麦危害最严重的病害之一。培育和推广抗病品种是防治小麦白粉病最经济、有效、安全的途径。偏凸山羊草和柱穗山羊草是普通小麦的近缘物种,蕴藏着丰富的抗病、抗虫、抗逆和优质等优异基因。利用远缘杂交途径,将偏凸山羊草和柱穗山羊草的抗白粉病基因导入普通小麦,培育抗白粉病小麦新种质,为小麦白粉病抗性遗传改良提供新抗源。【方法】通过细胞学分析和结实率调查,明确TA002及其与普通小麦杂种的细胞学稳定性和育性特点。利用十二烷基磺酸钠聚丙烯酰氨凝胶电泳（odium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE）及酸性聚丙烯酰胺凝胶电泳（acid polyacrylamide gel electrophoresis,A-PAGE）方法,分析TA002的高分子量麦谷蛋白亚基（high molecular weight glutenin subunit,HMW-GS）、低分子量麦谷蛋白亚基（low molecular weight glutenin subunit,LMW-GS）及醇溶蛋白亚基组成;通过白粉病菌分小种接种鉴定,明确TA002对小麦白粉病的抗性及其遗传特点;利用基因组原位杂交（genomic in situ hybridization,GISH）、多色原位杂交（multicolor genomic in situ hybridization,mc-GISH）、多色荧光原位杂交（multicolor fluorescence in situ hybridization,mc-FISH）及分子标记技术,分析明确TA002的染色体组成特点。【结果】TA002染色体数目为42条,它及其与普通小麦的杂种F₁减数第一次分裂中期细胞（pollen mother cells at metaphase I,PMCs MI）均含有21个二价体,减数第一次分裂后期细胞（pollen mother cells at anaphase I,PMCs AI）中的染色体分离均衡,结实率正常。在成株期,TA002、SDAU18及其双亲偏凸山羊草和柱穗山羊草对白粉病均具有良好抗性,而烟农15对白粉病表现髙感。TA002/辉县红F₁、TA002/铭贤169F₁对白粉病菌种E09表现免疫,F₂单株对E09的抗感分离比例符合3﹕1。种子贮藏蛋白分析结果表明,在TA002的醇溶蛋白和谷蛋白中均含有SDAU18的特有亚基,其醇溶蛋白还含有双亲没有的新型亚基。分别以单芒山羊草和尾状山羊草的基因组总DNA为探针,烟农15的基因组总DNA为封阻,对TA002的根尖细胞染色体进行基因组原位杂交,均未检测到杂交信号。同时以不同荧光素标记的乌拉尔图小麦和粗山羊草基因组总DNA为探针,拟斯卑尔脱山羊草基因组总DNA为封阻,对TA002的根尖细胞染色体进行mc-GISH,并利用以不同荧光素标记的寡核苷酸pSc119.2和pTa535对TA002的根尖细胞染色体进行mc-FISH,发现TA002具有完整的A、B和D基因组,但其4A、5A、6B、7B和5D染色体在FISH带型上与亲本烟农15的对应染色体具有显著差异。分子标记检测结果表明,TA002含有来源于偏凸山羊草和柱穗山羊草的遗传物质。【结论】TA002是一个细胞学稳定、农艺性状和育性良好的小麦-山羊草渐渗系,它含有偏凸-柱穗山羊草双二倍体特有的贮藏蛋白亚基及其双亲没有的新亚基,且高抗小麦白粉病,其白粉病抗性受显性单基因控制,该基因可能是来自偏凸山羊草或柱穗山羊草的一个新的白粉病抗性基因。     

Structural basis for specific substrate recognition by the chloroplast signal recognition particle protein cpSRP43

Secretory and membrane proteins carry amino-terminal signal sequences that, in cotranslational targeting, are recognized by the signal recognition particle protein SRP54 without sequence specificity. The most abundant membrane proteins on Earth are the light-harvesting chlorophyll a/b binding proteins (LHCPs). They are synthesized in the cytoplasm, imported into the chloroplast, and posttranslationally targeted to the thylakoid membrane by cpSRP, a heterodimer formed by cpSRP54 and cpSRP43. We present the 1.5 angstrom crystal structure of cpSRP43 characterized by a unique arrangement of chromodomains and ankyrin repeats. The overall shape and charge distribution of cpSRP43 resembles the SRP RNA, which is absent in chloroplasts. The complex with the internal signal sequence of LHCPs reveals that cpSRP43 specifically recognizes a DPLG peptide motif. We describe how cpSPR43 adapts the universally conserved SRP system to posttranslational targeting and insertion of the LHCP family of membrane proteins.     

Tracking SNARE complex formation in live endocrine cells

Syntaxin, synaptosome-associated protein of 25 kD (SNAP25), and vesicle-associated membrane protein/synaptobrevin are collectively called SNAP receptor (SNARE) proteins, and they catalyze neuronal exocytosis by forming a "core complex." The steps in core complex formation are unknown. Here, we monitored SNARE complex formation in vivo with the use of a fluorescent version of SNAP25. In PC12 cells, we found evidence for a syntaxin-SNAP25 complex that formed with high affinity, required only the amino-terminal SNARE motif of SNAP25, tolerated a mutation that blocks formation of other syntaxin-SNAP25 complexes, and assembled reversibly when Ca2+ entered cells during depolarization. The complex may represent a precursor to the core complex formed during a Ca2+-dependent priming step of exocytosis.     

SOCS2真核表达载体的构建及在3T3 L1前体脂肪细胞中的表达

细胞因子信号抑制蛋白(SOCS)是细胞因子信号通路的重要负调控因子.构建SOCS2真核表达载体并转染3T3-L1前体脂肪细胞,为研究SOCS2对不同细胞因子调控脂代谢奠定基础.取小鼠皮下脂肪提总RNA进行RT-PCR,克隆获得pMDl8-SOCS2质粒载体,双酶切目的基因,并克隆至质粒载体pcDNA3.1,成功构建了重组质粒载体pcDNA3.1-SOCS2;重组质粒瞬时转染3T3-L1前体脂肪细胞系,以未转染和空载体转染作为对照,RT-PCR和Westem blotting检测重组质粒转染组SOCS2核酸和蛋白表达,表达水平均显著高于对照组,为进一步研究SOCS2基因对不同细胞因子调控脂肪代谢提供基础.     

Host cell invasion by apicomplexan parasites: insights from the co-structure of AMA1 with a RON2 peptide

Apicomplexan parasites such as Toxoplasma gondii and Plasmodium species actively invade host cells through a moving junction (MJ) complex assembled at the parasite-host cell interface. MJ assembly is initiated by injection of parasite rhoptry neck proteins (RONs) into the host cell, where RON2 spans the membrane and functions as a receptor for apical membrane antigen 1 (AMA1) on the parasite. We have determined the structure of TgAMA1 complexed with a RON2 peptide at 1.95 angstrom resolution. A stepwise assembly mechanism results in an extensive buried surface area, enabling the MJ complex to resist the mechanical forces encountered during host cell invasion. Besides providing insights into host cell invasion by apicomplexan parasites, the structure offers a basis for designing therapeutics targeting these global pathogens.     

A conserved family of enzymes that phosphorylate inositol hexakisphosphate

Inositol pyrophosphates are a diverse group of high-energy signaling molecules whose cellular roles remain an active area of study. We report a previously uncharacterized class of inositol pyrophosphate synthase and find it is identical to yeast Vip1 and Asp1 proteins, regulators of actin-related protein-2/3 (ARP 2/3) complexes. Vip1 and Asp1 acted as enzymes that encode inositol hexakisphosphate (IP6) and inositol heptakisphosphate (IP7) kinase activities. Alterations in kinase activity led to defects in cell growth, morphology, and interactions with ARP complex members. The functionality of Asp1 and Vip1 may provide cells with increased signaling capacity through metabolism of IP6.     

Requirement for coronin 1 in T lymphocyte trafficking and cellular homeostasis

The evolutionarily conserved actin-related protein (Arp2/3) complex is a key component of actin filament networks that is dynamically regulated by nucleation-promoting and inhibitory factors. Although much is known about actin assembly, the physiologic functions of inhibitory proteins are unclear. We generated coronin 1-/- mice and found that coronin 1 exerted an inhibitory effect on cellular steady-state F-actin formation via an Arp2/3-dependent mechanism. Whereas coronin 1 was required for chemokine-mediated migration, it was dispensable for T cell antigen receptor functions in T cells. Moreover, actin dynamics, through a mitochondrial pathway, was linked to lymphocyte homeostasis.     

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.     

Essential cytoplasmic translocation of a cytokine receptor-assembled signaling complex

Cytokine signaling is thought to require assembly of multicomponent signaling complexes at cytoplasmic segments of membrane-embedded receptors, in which receptor-proximal protein kinases are activated. Indeed, CD40, a tumor necrosis factor receptor (TNFR) family member, forms a complex containing adaptor molecules TRAF2 and TRAF3, ubiquitin-conjugating enzyme Ubc13, cellular inhibitor of apoptosis proteins 1 and 2 (c-IAP1/2), IkappaB kinase regulatory subunit IKKgamma (also called NEMO), and mitogen-activated protein kinase (MAPK) kinase kinase MEKK1 upon ligation. TRAF2, Ubc13, and IKKgamma were required for complex assembly and activation of MEKK1 and MAPK cascades. However, these kinases were not activated unless the multicomponent signaling complex translocated from CD40 to the cytosol upon c-IAP1/2-induced degradation of TRAF3. This two-stage signaling mechanism may apply to other innate immune receptors, accounting for spatial and temporal separation of MAPK and IKK signaling.     

A mitochondrial pyruvate carrier required for pyruvate uptake in yeast, Drosophila, and humans

Pyruvate constitutes a critical branch point in cellular carbon metabolism. We have identified two proteins, Mpc1 and Mpc2, as essential for mitochondrial pyruvate transport in yeast, Drosophila, and humans. Mpc1 and Mpc2 associate to form an ~150-kilodalton complex in the inner mitochondrial membrane. Yeast and Drosophila mutants lacking MPC1 display impaired pyruvate metabolism, with an accumulation of upstream metabolites and a depletion of tricarboxylic acid cycle intermediates. Loss of yeast Mpc1 results in defective mitochondrial pyruvate uptake, and silencing of MPC1 or MPC2 in mammalian cells impairs pyruvate oxidation. A point mutation in MPC1 provides resistance to a known inhibitor of the mitochondrial pyruvate carrier. Human genetic studies of three families with children suffering from lactic acidosis and hyperpyruvatemia revealed a causal locus that mapped to MPC1, changing single amino acids that are conserved throughout eukaryotes. These data demonstrate that Mpc1 and Mpc2 form an essential part of the mitochondrial pyruvate carrier.     

涂丝型聚氯乙烯膜谷氨酸根选择电极的研制与应用

[目的]研究制备一种以谷氨酸与邻二氮菲合铁配离子形成的缔合物为电活性物的新型全固态聚氯乙烯(PVC)膜谷氨酸涂丝选择电极。[方法]以谷氨酸与邻二氮菲合铁配离子形成的缔合物为电活性物,研究全固态涂丝型PVC谷氨酸根选择电极的制备,并利用该电极对谷氨酸根的响应,测定血清中谷氨酸的含量,将其结果与高效液相色谱法进行对比,分析该方法的实用性。[结果]电极的线性响应范围为1.0×10-1～1.0×10-5mol/L,级差32 mV/pC,检测限为8×10-6mol/L。[结论]试验中所制备电极响应迅速,重现性好,利用其测定谷氨酸含量方法简便,结果与高效液相法相符,其是一种易于微型化的载体动态检测方法。     

抗磷酸酪氨酸抗体的制备

目的；制备抗磷酸酪氨酸抗体。方法：以EDC（碳化二亚胺）交联法合成磷酸酪氨酸牛血清白蛋白（P－Tyr－BSA）；以P－Tyr－BSA免疫新西兰免，抗血清经硫酸该盐析和DEAE纤维素柱层析纯化；以琼脂糖双扩法测定抗体效价。结果：得到抗血清效价为1：32，抗血清与牛血清蛋白（BSA）交叉反应的效价为1：2。纯化的抗体与磷酸酪氨酸牛血清白蛋白、磷酸酪氨酸卵清蛋白、磷酸丝氨酸牛血清白蛋白、磷酸苏氨酸牛血清白蛋白反应效价分别为1：32、1：16、1：2、1：2，所得抗体蛋白量为120mg。纯化的抗体在醋酸纤维素薄膜上电泳为单一区带。结论：用此法制备得的抗体专一性高，交叉反应低，抗体量大。     

Generation and Identification of Monoclonal Antibody Against Porcine Adipocyte Plasma Membrane Proteins

Production of monoclonal antibody against porcine adipocyte plasma membrane proteins to explore a new way of controlling body fat deposition and improving carcass quality is discussed in this article. Membrane proteins of pig adipocyte plasma membrane proteins were extracted with the help of sucrose density gradient centrifugation, and two kinds of proteins were obtained. The monoclonal antibody （designated 3B2 and 3F3） of IgG1 and IgG2b subclass against adipocyte membrane proteins were produced by immunization, with adipocyte membrane proteins as an antigen, and its titer was 1：105 detected by enzyme-linked immunoadsorbent assay （ELISA）. The cell strains were identified by analyzing the number of chromosomes, the heat stability, the acid and alkali, the types and subtypes of immnoglobulin, and its peculiarities and affinities. Through identification, the chromosome number of hybridoma cell strains was from 80 to 100 and the strains formed good hybridomas colonies. The strains＇ affinity constants were 4.63 × 10^9 and 3.75 × 10^9 （mol L^-1）-1, respectively. At the same time, the McAb secreted was stable to environmental factors, such as, temperature, acid, alkali and so on. The monoclonal antibodies had been obtained and their specificity to porcine adipocyte plasma membrane proteins had been identified.     

Removal of shelterin reveals the telomere end-protection problem

The telomere end-protection problem is defined by the aggregate of DNA damage signaling and repair pathways that require repression at telomeres. To define the end-protection problem, we removed the whole shelterin complex from mouse telomeres through conditional deletion of TRF1 and TRF2 in nonhomologous end-joining (NHEJ) deficient cells. The data reveal two DNA damage response pathways not previously observed upon deletion of individual shelterin proteins. The shelterin-free telomeres are processed by microhomology-mediated alternative-NHEJ when Ku70/80 is absent and are attacked by nucleolytic degradation in the absence of 53BP1. The data establish that the end-protection problem is specified by six pathways [ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3 related) signaling, classical-NHEJ, alt-NHEJ, homologous recombination, and resection] and show how shelterin acts with general DNA damage response factors to solve this problem.     

猪I型补体受体与C3b活性片段相互结合的体外检测

【目的】检测猪红细胞类补体受体I型（Complement receptor 1-like,CR1-like）与C3b活性片段能否发生结合,以期为阐明猪红细胞发挥免疫粘附功能的分子机理提供科学数据。【方法】利用前期已构建的CR1-like_(3-6)、CR1-like_(8-11)功能域片段的重组质粒建立酵母双杂交检测体系,运用酵母共转化的方法将诱饵质粒（重组pGBKT7-CR1-like）与捕获质粒（重组pGADT7-C3b）共同转入Y2HGold酵母细胞中,分别利用一缺平板SD/-Leu、SD/-Trp和二缺平板SD/-Leu/-Trp（DDO）严格筛选共转化成功的酵母细胞,再根据报告因子是否表达来鉴别转化子在SD/-Leu/-Trp/X-α-Gal（DDO/X）、SD/-Leu/-Trp/X-α-Gal/Aba（DDO/X/A）二缺培养板上的生长情况,并结合菌落的颜色变化现象综合判定CR1-like活性片段与补体C3b在酵母细胞中是否发生相互结合;然后运用免疫沉淀技术分离酵母细胞中CR1-like与C3b结合复合物,并对该复合物的特异性进行Western blot鉴定。【结果】试验成功将pGBKT7-CR1-like与pGADT7-C3b基因共转入Y2HGold酵母细胞。共转化的酵母克隆在SD/-Leu、SD/-Trp、DDO平板上能够正常生长,在DDO/X、DDO/X/A平板上正常生长且菌落呈现蓝色,由此表明,试验中酵母双杂交系统建立成功,并通过试验获得了阳性酵母克隆。共同转化了pGBKT7-CR1-like和pGADT7-C3b质粒的酵母菌落PCR反向鉴定结果显示,在共转化的酵母菌中含有目的基因CR1-like_(3-6)和CR1-like_(8-11),共转化组的质粒酶切后出现C3b基因片段,与设计大小一致,说明重组质粒成功共转化入酵母细胞中。免疫沉淀试验中应用pGBKT7载体的标签抗体c-Myc沉淀酵母细胞中的融合蛋白,以c-Myc为一抗进行Western blot检测发现,单独转化了pGBKT7-CR1-like_(3-6)和pGBKT7-CR1-like_(8-11)的融合蛋白在50 kD处出现特异性条带;共转化pGBKT7-CR1-like_(3-6) + pGADT7-C3b和共转化pGBKT7-CR1-like_(8-11) + pGADT7-C3b的酵母融合蛋白在83 kD处出现特异性条带;以HA单克隆抗体为一抗进行Western blot检测时,在pGBKT7-CR1-like_(3-6)和pGBKT7-CR1-like_(8-11)融合蛋白中没有出现特异性条带,只有3、4泳道中共转化的酵母融合蛋白在83 kD处出现特异性条带,表明在Y2HGold酵母细胞中存在CR1-like与C3b识别结合的复合物。使用CR1-like单克隆抗体沉淀酵母细胞中的融合蛋白,以CR1-like单克隆抗体为一抗进行Western blot检测发现,单独转化了pGBKT7-CR1-like_(3-6)和pGBKT7-CR1-like_(8-11)的融合蛋白在50 kD处出现特异性条带;共转化pGBKT7-CR1-like_(3-6) + pGADT7-C3b和共转化pGBKT7-CR1-like_(8-11) + pGADT7-C3b的酵母融合蛋白在83 kD处出现特异性条带;以C3单克隆抗体为一抗进行Western blot检测发现,在pGBKT7-CR1-like_(3-6)和pGBKT7-CR1-like_(8-11)融合蛋白中没有出现特异性条带,泳道3、4所示只有共转化的酵母融合蛋白在83 kD处出现特异性条带,表明在Y2HGold酵母细胞中存在具有生物活性的CR1-like与C3b识别结合的复合物。通过多个单克隆抗体杂交结果,可看出诱饵质粒的表达产物CR1-like_(3-6)、CR1-like_(8-11)片段与捕获质粒的表达产物C3b片段可在酵母细胞内发生结合。【结论】猪红细胞CR1-like发挥免疫粘附功能的识别配体为C3b,为猪红细胞CR1-like功能域分子结构的进一步解析提供了重要数据依据。