Affinity purification of PSD-95-containing postsynaptic complexes

A widely used method for the preparation of postsynaptic density (PSD) fractions consists of treatment of synaptosomal membranes with Triton X-100 and further purification by density gradient centrifugation. In the present study, the purity of this preparation was assessed by electron microscopic analysis. Thin-section and rotary shadow immuno-electron microscopy of the Triton X-100-derived PSD fraction shows many PSD-95-positive structures that resemble in situ PSDs in shape and size. However, the fraction also includes contaminants such as CaMKII clusters, spectrin filaments and neurofilaments. We used magnetic beads coated with an antibody against PSD-95 to further purify PSD-95-containing complexes from the Triton-derived PSD fraction. Biochemical analysis of the affinity-purified material shows a substantial reduction in the astrocytic marker glial fibrillary acidic protein and electron microscopic analysis shows mostly individual PSDs attached to magnetic beads. This preparation was used to assess the association of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-type glutamate receptors with the PSD-95-containing complex. AMPA receptors are demonstrated by immunoblotting to be present in the complex, although they do not co-purify exclusively with PSD-95, suggesting the existence of two pools of receptors, one associated with the PSD-95 scaffold and the other not. Of the AMPA receptor-anchoring proteins tested, SAP-97 is present in the affinity-purified preparation whereas GRIP is found only in trace amounts. These results imply that a subpopulation of AMPA receptors is anchored to the PSD-95-containing scaffold through interaction of GluR1 with SAP-97.     

Chemical modification of the polymer surface and consequences on electrooptical properties of liquid crystal—polymer composites

In order to improve our understanding of the mechanisms involved in the electrooptical effects of liquid crystal—polymer composites, and to identify the relevant parameters of these complex systems, we have investigated the influence of the chemical nature of the polymer surface on the anchoring of the liquid crystal. Our method consists in removing the liquid crystal from the composite by dissolution followed by supercritical drying of the solvent. The surface of the polymer is then modified by different reagents and the liquid crystal is reintroduced to the cell.Comparison of the results shows that for a cyanobiphenyl liquid crystal, fluorinated or hydroxylated chemical moieties on the surface of the polymer induce a stronger anchoring than alkyl moieties. This phenomenon seems to be due to the enhanced chemical compatibility between the liquid crystal and the surface of the polymer in the case of alkyl moieties. Finally, the study has shown that a reduction of anchoring of the liquid crystal is responsible for a decrease in driving voltage, rise time and hysteresis and an increase in decay time and off-state transmission.     

Pollen and plant macrofossil analyses from the Gedesby ship — a medieval shipwreck from Falster,Denmark

In 1988, a well-preserved shipwreck was discovered during work on drainage channels in Bøtø Nor, a former arm of the Baltic Sea on the island of Falster. The ship was built in the Nordic style with a single central mast. It was radiocarbon dated to the second half of the 13th century A.D. and is thus Scandinavia's best-preserved medieval ship. The ship's hull was lined with wattle, over which lay a thick layer of organic material, interpreted as animal dung. Microscopic examination revealed a tangle of well-preserved stems, lengths of straw, leaf fragments, lengths of cereal rachis, crushed grains and numerous other seeds and fruits. The ship had apparently been used to transport animals and the wattle had served to protect the hull and give the animals a firm footing during the voyage. Prior to the voyage the animals had grazed various habitats in the vicinity of the coast — coastal pasture, other grasslands, open woodland, newly-harvested cornfields, fallow land and marshy areas; they had drunk water from ponds. The fact that all the seeds in the material, including the cereals, were mature and well-developed, suggests that the dung comes from animals which had grazed in the late summer or early autumn. The archaeobotanical investigations suggest a location in southeastern Denmark or more generally the southern Baltic.     

NMR Assignment of the mTOR Domain Responsible for Rapamycin Binding

A fully automated, NOE-based NMR structure determination of a uniformly ¹³C,¹⁵N-labeled protein was achieved in crude cell-extract, without purification of the overexpressed protein. Essentially complete sequence-specific assignments were obtained using triple resonance experiments, based on the high intensity of the resonances from the overexpressed protein relative to those of the background. For the collection of NOE distance constraints, efficient discrimination between NOE cross peaks from the target protein and background signals was achieved using the programs ATNOS and CANDID. In the iterative ATNOS/CANDID procedure, the identification of the desired protein NOEs is initially guided by the self-consistency of the protein NOE-network. Although the intensities of the signals in this network vary over a wide range, and are in many instances comparable to or smaller than those of the background, the first cycle of calculations resulted in the correct global polypeptide fold, and the structure was then refined in six subsequent cycles using the intermediate NMR structures for additional guidance. The experience gained with this work demonstrates that the ATNOS/CANDID procedure for automatic protein structure determination is highly robust and reliable in the presence of intense background signals, and might thus also represent a platform for future protein structure determinations in physiological fluids.     

Related protein-protein interaction modules present drastically different surface topographies despite a conserved helical platform

The subcellular localization of cAMP-dependent protein kinase (PKA) occurs through interaction with A-Kinase Anchoring Proteins (AKAPs). AKAPs bind to the PKA regulatory subunit dimer of both type Ialpha and type IIalpha (RIalpha and RIIalpha). RIalpha and RIIalpha display characteristic localization within different cell types, which is maintained by interaction of AKAPs with the N-terminal dimerization and docking domain (D/D) of the respective regulatory subunit. Previously, we reported the solution structure of RIIa D/D module, both free and bound to AKAPs. We have now solved the solution structure of the dimerization and docking domain of the type Ialpha regulatory dimer subunit (RIalpha D/D). RIalpha D/D is a compact docking module, with unusual interchain disulfide bonds that help maintain the AKAP interaction surface. In contrast to the shallow hydrophobic groove for AKAP binding across the surface of the RIIalpha D/D dimeric interface, the RIalpha D/D module presents a deep cleft for proposed AKAP binding. RIalpha and RIIalpha D/D interaction modules present drastically differing dimeric topographies, despite a conserved X-type four-helix bundle structure.     

Characterization of three Arabidopsis homologs of human RING membrane anchor E3 ubiquitin ligase

Ubiquitination affects diverse physiological processes in eukaryotic cells. AtRMA1 was previously identified as an Arabidopsis homolog of human RING membrane-anchor E3 ubiquitin (Ub) ligase. Here, we identified two additional AtRMA homologs, AtRMA2 and AtRMA3. The predicted AtRMA proteins contain a RING motif and a trans-membrane domain in their N-terminal and extreme C-terminal regions, respectively. Bacterially expressed AtRMAs exhibited E3 ligase activity in vitro, which was abrogated by mutation of the conserved cysteine residue in their RING domains. In vivo targeting experiments using an Arabidopsis protoplast-transfection system showed that all three AtRMAs are localized to the ER. Although RT-PCR analysis indicated that AtRMA mRNAs were expressed constitutively in all tissues examined, their promoter activities were differentially detected in a tissue-specific fashion in AtRMA-promoter::GUS transgenic Arabidopsis plants. The AtRMA1 and AtRMA3 genes are predominantly expressed in major tissues, such as cotyledons, leaves, shoot–root junction, roots, and anthers, while AtRMA2 expression is restricted to the root tips and leaf hydathodes. We suggest that a ubiquitnation pathway involving these AtRMA E3 Ub ligases may play a role in the growth and development of Arabidopsis.     

Two-point anchoring of a lanthanide-binding peptide to a target protein enhances the paramagnetic anisotropic effect

Paramagnetic lanthanide ions fixed in a protein frame induce several paramagnetic effects such as pseudo-contact shifts and residual dipolar couplings. These effects provide long-range distance and angular information for proteins and, therefore, are valuable in protein structural analysis. However, until recently this approach had been restricted to metal-binding proteins, but now it has become applicable to non-metalloproteins through the use of a lanthanide-binding tag. Here we report a lanthanide-binding peptide tag anchored via two points to the target proteins. Compared to conventional single-point attached tags, the two-point linked tag provides two to threefold stronger anisotropic effects. Though there is slight residual mobility of the lanthanide-binding tag, the present tag provides a higher anisotropic paramagnetic effect. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genome resources for climate‐resilient cowpea,an essential crop for food security

Cowpea (Vigna unguiculata L. Walp.) is a legume crop that is resilient to hot and drought‐prone climates, and a primary source of protein in sub‐Saharan Africa and other parts of the developing world. However, genome resources for cowpea have lagged behind most other major crops. Here we describe foundational genome resources and their application to the analysis of germplasm currently in use in West African breeding programs. Resources developed from the African cultivar IT97K‐499‐35 include a whole‐genome shotgun (WGS) assembly, a bacterial artificial chromosome (BAC) physical map, and assembled sequences from 4355 BACs. These resources and WGS sequences of an additional 36 diverse cowpea accessions supported the development of a genotyping assay for 51 128 SNPs, which was then applied to five bi‐parental RIL populations to produce a consensus genetic map containing 37 372 SNPs. This genetic map enabled the anchoring of 100 Mb of WGS and 420 Mb of BAC sequences, an exploration of genetic diversity along each linkage group, and clarification of macrosynteny between cowpea and common bean. The SNP assay enabled a diversity analysis of materials from West African breeding programs. Two major subpopulations exist within those materials, one of which has significant parentage from South and East Africa and more diversity. There are genomic regions of high differentiation between subpopulations, one of which coincides with a cluster of nodulin genes. The new resources and knowledge help to define goals and accelerate the breeding of improved varieties to address food security issues related to limited‐input small‐holder farming and climate stress.     

GFP-SA融合蛋白的表达纯化及其锚定修饰肿瘤细胞的研究

目的　GFP（绿色荧光蛋白）-SA（链亲和素）双功能融合蛋白的制备及其鉴定研究,以展示我们建立的技术平台,即用含链亲和素的双功能融合蛋白对生物素化的细胞表面进行高效的锚定修饰。方法　构建原核表达载体pET24d/GFP-SA转化大肠杆菌BL21(DE3)。用IPTG诱导重组蛋白的表达,用镍金属螯合（Ni-NTA）层析柱进行纯化。用制备的GFP-SA双功能融合蛋白,对B16肿瘤细胞已生物素化的细胞表面进行修饰,经荧光显微镜和流式细胞仪进行修饰效率分析。此外,用MTT法检测细胞表面修饰对肿瘤细胞活力及其生长情况的影响。结果　GFP-SA重组融合蛋白在大肠杆菌实现了高效表达（约占细菌总蛋白的20%）,通过纯化和复性制备的GFP-SA双功能融合蛋白具有双重活性,即：链亲和素介导的、对生物素高效特异的结合活性,和GFP发射绿色荧光的活性,并能高效修饰表面已生物素化的肿瘤细胞。此外,GFP-SA双功能融合蛋白的细胞表面修饰对细胞的活力及其生长无显著影响。结论　GFP-SA融合蛋白能高效修饰表面已生物素化的肿瘤细胞,可用作肿瘤疫苗研究的示踪蛋白及实验对照体系。