Sample records for large subunits assemble

  1. The DEAD box protein Mrh4 functions in the assembly of the mitochondrial large ribosomal subunit. (United States)

    De Silva, Dasmanthie; Fontanesi, Flavia; Barrientos, Antoni


    Proteins in a cell are universally synthesized by ribosomes. Mitochondria contain their own ribosomes, which specialize in the synthesis of a handful of proteins required for oxidative phosphorylation. The pathway of mitoribosomal biogenesis and factors involved are poorly characterized. An example is the DEAD box proteins, widely known to participate in the biogenesis of bacterial and cytoplasmic eukaryotic ribosomes as either RNA helicases or RNA chaperones, whose mitochondrial counterparts remain completely unknown. Here, we have identified the Saccharomyces cerevisiae mitochondrial DEAD box protein Mrh4 as essential for large mitoribosome subunit biogenesis. Mrh4 interacts with the 21S rRNA, mitoribosome subassemblies, and fully assembled mitoribosomes. In the absence of Mrh4, the 21S rRNA is matured and forms part of a large on-pathway assembly intermediate missing proteins Mrpl16 and Mrpl39. We conclude that Mrh4 plays an essential role during the late stages of mitoribosome assembly by promoting remodeling of the 21S rRNA-protein interactions.

  2. Time course of large ribosomal subunit assembly in E. coli cells overexpressing a helicase inactive DbpA protein. (United States)

    Gentry, Riley C; Childs, Jared J; Gevorkyan, Jirair; Gerasimova, Yulia V; Koculi, Eda


    DbpA is a DEAD-box RNA helicase implicated in Escherichia coli large ribosomal subunit assembly. Previous studies have shown that when the ATPase and helicase inactive DbpA construct, R331A, is expressed in E. coli cells, a large ribosomal subunit intermediate accumulates. The large subunit intermediate migrates as a 45S particle in a sucrose gradient. Here, using a number of structural and fluorescent assays, we investigate the ribosome profiles of cells lacking wild-type DbpA and overexpressing the R331A DbpA construct. Our data show that in addition to the 45S particle previously described, 27S and 35S particles are also present in the ribosome profiles of cells overexpressing R331A DbpA. The 27S, 35S, and 45S independently convert to the 50S subunit, suggesting that ribosome assembly in the presence of R331A and the absence of wild-type DbpA occurs via multiple pathways.

  3. Clofarabine targets the large subunit (α) of human ribonucleotide reductase in live cells by assembly into persistent hexamers. (United States)

    Aye, Yimon; Brignole, Edward J; Long, Marcus J C; Chittuluru, Johnathan; Drennan, Catherine L; Asturias, Francisco J; Stubbe, JoAnne


    Clofarabine (ClF) is a drug used in the treatment of leukemia. One of its primary targets is human ribonucleotide reductase (hRNR), a dual-subunit, (α(2))(m)(β(2))(n), regulatory enzyme indispensable in de novo dNTP synthesis. We report that, in live mammalian cells, ClF targets hRNR by converting its α-subunit into kinetically stable hexamers. We established mammalian expression platforms that enabled isolation of functional α and characterization of its altered oligomeric associations in response to ClF treatment. Size exclusion chromatography and electron microscopy documented persistence of in-cell-assembled-α(6). Our data validate hRNR as an important target of ClF, provide evidence that in vivo α's quaternary structure can be perturbed by a nonnatural ligand, and suggest small-molecule-promoted, persistent hexamerization as a strategy to modulate hRNR activity. These studies lay foundations for documentation of RNR oligomeric state within a cell.

  4. Rubisco oligomers composed of linked small and large subunits assemble in tobacco plastids and have higher affinities for CO2 and O2. (United States)

    Whitney, Spencer Michael; Kane, Heather Jean; Houtz, Robert L; Sharwood, Robert Edward


    Manipulation of Rubisco within higher plants is complicated by the different genomic locations of the large (L; rbcL) and small (S; RbcS) subunit genes. Although rbcL can be accurately modified by plastome transformation, directed genetic manipulation of the multiple nuclear-encoded RbcS genes is more challenging. Here we demonstrate the viability of linking the S and L subunits of tobacco (Nicotiana tabacum) Rubisco using a flexible 40-amino acid tether. By replacing the rbcL in tobacco plastids with an artificial gene coding for a S40L fusion peptide, we found that the fusions readily assemble into catalytic (S40L)8 and (S40L)16 oligomers that are devoid of unlinked S subunits. While there was little or no change in CO2/O2 specificity or carboxylation rate of the Rubisco oligomers, their Kms for CO2 and O2 were reduced 10% to 20% and 45%, respectively. In young maturing leaves of the plastome transformants (called ANtS40L), the S40L-Rubisco levels were approximately 20% that of wild-type controls despite turnover of the S40L-Rubisco oligomers being only slightly enhanced relative to wild type. The reduced Rubisco content in ANtS40L leaves is partly attributed to problems with folding and assembly of the S40L peptides in tobacco plastids that relegate approximately 30% to 50% of the S40L pool to the insoluble protein fraction. Leaf CO2-assimilation rates in ANtS40L at varying pCO2 corresponded with the kinetics and reduced content of the Rubisco oligomers. This fusion strategy provides a novel platform to begin simultaneously engineering Rubisco L and S subunits in tobacco plastids.

  5. Heteromeric assembly of P2X subunits

    Directory of Open Access Journals (Sweden)

    Anika eSaul


    Full Text Available Transcripts and/or proteins of P2X receptor (P2XR subunits have been found in virtually all mammalian tissues. Generally more than one of the seven known P2X subunits have been identified in a given cell type. Six of the seven cloned P2X subunits can efficiently form functional homotrimeric ion channels in recombinant expression systems. This is in contrast to other ligand-gated ion channel families, such as the Cys-loop or glutamate receptors, where homomeric assemblies seem to represent the exception rather than the rule. P2XR mediated responses recorded from native tissues rarely match exactly the biophysical and pharmacological properties of heterologously expressed homomeric P2XRs. Heterotrimerization of P2X subunits is likely to account for this observed diversity. While the existence of heterotrimeric P2X2/3Rs and their role in physiological processes is well established, the composition of most other P2XR heteromers and/or the interplay between distinct trimeric receptor complexes in native tissues is not clear. After a description of P2XR assembly and the structure of the intersubunit ATP-binding site, this review summarizes the distribution of P2XR subunits in selected mammalian cell types and the biochemically and/or functionally characterized heteromeric P2XRs that have been observed upon heterologous co-expression of P2XR subunits. We further provide examples where the postulated heteromeric P2XRs have been suggested to occur in native tissues and an overview of the currently available pharmacological tools that have been used to discriminate between homo- and heteromeric P2XRs

  6. Assembly processes in oligomers containing structurally distinct subunits. [Hemoglobin, Hemocyanin

    Energy Technology Data Exchange (ETDEWEB)

    Bonaventura, C. (Duke Univ. Marine Laboratory, Beaufort, NC); Bonaventura, J.; Brouwer, M.


    There are two major classes of oxygen carrying proteins: the hemoglobins and the hemocyanins. Thetrameric hemoglobin is an oxygen carrier that has long served as a model in the analysis of allostery in proteins. In assembly processes as well, the oxygen carrying proteins appear to be good model systems which illustrate the distinct roles played by structurally diverse subunits. Thetrameric human hemoglobin shows definite differences in assembly and tetrameric stability depending on alpha-beta, alpha-alpha, beta-beta, alpha-gamma, etc., interactions. The blue-colored hemocyanins are found in the hemolymph of many molluscs and arthropods. In these molecules, oxygen binds at dimeric copper centers. Te reactivity toward oxygen is typically modulated by external factors such as pH and sodium chloride. Because of their extremely large size and subunit diversity, the hemocyanins may be particularly useful as assembly models.

  7. Accessory subunits are integral for assembly and function of human mitochondrial complex I. (United States)

    Stroud, David A; Surgenor, Elliot E; Formosa, Luke E; Reljic, Boris; Frazier, Ann E; Dibley, Marris G; Osellame, Laura D; Stait, Tegan; Beilharz, Traude H; Thorburn, David R; Salim, Agus; Ryan, Michael T


    Complex I (NADH:ubiquinone oxidoreductase) is the first enzyme of the mitochondrial respiratory chain and is composed of 45 subunits in humans, making it one of the largest known multi-subunit membrane protein complexes. Complex I exists in supercomplex forms with respiratory chain complexes III and IV, which are together required for the generation of a transmembrane proton gradient used for the synthesis of ATP. Complex I is also a major source of damaging reactive oxygen species and its dysfunction is associated with mitochondrial disease, Parkinson's disease and ageing. Bacterial and human complex I share 14 core subunits that are essential for enzymatic function; however, the role and necessity of the remaining 31 human accessory subunits is unclear. The incorporation of accessory subunits into the complex increases the cellular energetic cost and has necessitated the involvement of numerous assembly factors for complex I biogenesis. Here we use gene editing to generate human knockout cell lines for each accessory subunit. We show that 25 subunits are strictly required for assembly of a functional complex and 1 subunit is essential for cell viability. Quantitative proteomic analysis of cell lines revealed that loss of each subunit affects the stability of other subunits residing in the same structural module. Analysis of proteomic changes after the loss of specific modules revealed that ATP5SL and DMAC1 are required for assembly of the distal portion of the complex I membrane arm. Our results demonstrate the broad importance of accessory subunits in the structure and function of human complex I. Coupling gene-editing technology with proteomics represents a powerful tool for dissecting large multi-subunit complexes and enables the study of complex dysfunction at a cellular level.

  8. Stepwise and dynamic assembly of the earliest precursors of small ribosomal subunits in yeast. (United States)

    Zhang, Liman; Wu, Chen; Cai, Gaihong; Chen, She; Ye, Keqiong


    The eukaryotic ribosomal RNA (rRNA) is associated cotranscriptionally with numerous factors into an enormous 90S preribosomal particle that conducts early processing of small ribosomal subunits. The assembly pathway and structure of the 90S particle is poorly understood. Here, we affinity-purified and analyzed the constituents of yeast 90S particles that were assembled on a series of plasmid-encoded 3'-truncated pre-18S RNAs. We determined the assembly point of 65 proteins and the U3, U14, and snR30 small nucleolar RNAs (snoRNAs), revealing a stepwise and dynamic assembly map. The 5' external transcribed spacer (ETS) alone can nucleate a large complex. When the 18S rRNA is nearly complete, the 90S structure undergoes a dramatic reorganization, releasing U14, snR30, and 14 protein factors that bind earlier. We also identified a reference state of 90S that is fully assembled yet has not undergone 5'ETS processing. The assembly map present here provides a new framework to understand small subunit biogenesis.

  9. Tropomyosin diffusion over actin subunits facilitates thin filament assembly

    Directory of Open Access Journals (Sweden)

    Stefan Fischer


    Full Text Available Coiled-coil tropomyosin binds to consecutive actin-subunits along actin-containing thin filaments. Tropomyosin molecules then polymerize head-to-tail to form cables that wrap helically around the filaments. Little is known about the assembly process that leads to continuous, gap-free tropomyosin cable formation. We propose that tropomyosin molecules diffuse over the actin-filament surface to connect head-to-tail to partners. This possibility is likely because (1 tropomyosin hovers loosely over the actin-filament, thus binding weakly to F-actin and (2 low energy-barriers provide tropomyosin freedom for 1D axial translation on F-actin. We consider that these unique features of the actin-tropomyosin interaction are the basis of tropomyosin cable formation.

  10. Tropomyosin diffusion over actin subunits facilitates thin filament assembly (United States)

    Fischer, Stefan; Rynkiewicz, Michael J.; Moore, Jeffrey R.; Lehman, William


    Coiled-coil tropomyosin binds to consecutive actin-subunits along actin-containing thin filaments. Tropomyosin molecules then polymerize head-to-tail to form cables that wrap helically around the filaments. Little is known about the assembly process that leads to continuous, gap-free tropomyosin cable formation. We propose that tropomyosin molecules diffuse over the actin-filament surface to connect head-to-tail to partners. This possibility is likely because (1) tropomyosin hovers loosely over the actin-filament, thus binding weakly to F-actin and (2) low energy-barriers provide tropomyosin freedom for 1D axial translation on F-actin. We consider that these unique features of the actin-tropomyosin interaction are the basis of tropomyosin cable formation. PMID:26798831

  11. Assembly of the 30S ribosomal subunit: positioning ribosomal protein S13 in the S7 assembly branch. (United States)

    Grondek, Joel F; Culver, Gloria M


    Studies of Escherichia coli 30S ribosomal subunit assembly have revealed a hierarchical and cooperative association of ribosomal proteins with 16S ribosomal RNA; these results have been used to compile an in vitro 30S subunit assembly map. In single protein addition and omission studies, ribosomal protein S13 was shown to be dependent on the prior association of ribosomal protein S20 for binding to the ribonucleoprotein particle. While the overwhelming majority of interactions revealed in the assembly map are consistent with additional data, the dependency of S13 on S20 is not. Structural studies position S13 in the head of the 30S subunit > 100 A away from S20, which resides near the bottom of the body of the 30S subunit. All of the proteins that reside in the head of the 30S subunit, except S13, have been shown to be part of the S7 assembly branch, that is, they all depend on S7 for association with the assembling 30S subunit. Given these observations, the assembly requirements for S13 were investigated using base-specific chemical footprinting and primer extension analysis. These studies reveal that S13 can bind to 16S rRNA in the presence of S7, but not S20. Additionally, interaction between S13 and other members of the S7 assembly branch have been observed. These results link S13 to the 3' major domain family of proteins, and the S7 assembly branch, placing S13 in a new location in the 30S subunit assembly map where its position is in accordance with much biochemical and structural data.

  12. Exact Length Distribution of Filamentous Structures Assembled from a Finite Pool of Subunits. (United States)

    Harbage, David; Kondev, Jané


    Self-assembling filamentous structures made of protein subunits are ubiquitous in cell biology. These structures are often highly dynamic, with subunits in a continuous state of flux, binding to and falling off of filaments. In spite of this constant turnover of their molecular parts, many cellular structures seem to maintain a well-defined size over time, which is often required for their proper functioning. One widely discussed mechanism of size regulation involves the cell maintaining a finite pool of protein subunits available for assembly. This finite pool mechanism can control the length of a single filament by having assembly proceed until the pool of free subunits is depleted to the point when assembly and disassembly are balanced. Still, this leaves open the question of whether the same mechanism can provide size control for multiple filamentous structures that are assembled from a common pool of protein subunits, as is often the case in cells. We address this question by solving the steady-state master equation governing the stochastic assembly and disassembly of multifilament structures made from a shared finite pool of subunits. We find that, while the total number of subunits within a multifilament structure is well-defined, individual filaments within the structure have a wide, power-law distribution of lengths. We also compute the phase diagram for two multifilament structures competing for the same pool of subunits and identify conditions for coexistence when both have a well-defined size. These predictions can be tested in cell experiments in which the size of the subunit pool or the number of filament nucleators is tuned.

  13. Tracing human mitochondrial complex I assembly by use of GFP-tagged subunits

    NARCIS (Netherlands)

    Dieteren, C.E.J.; Koopman, W.J.H.; Nijtmans, L.G.J.


    Disturbances in the assembly of mitochondrial complex I (CI) are a frequent cause of mitochondrial disorders. Several lines of evidence hint at a semi-sequential assembly pathway, in which the 45 individual subunits that form the holoenzyme are pieced together by means of smaller intermediates. To u

  14. rRNA maturation in yeast cells depleted of large ribosomal subunit proteins.

    Directory of Open Access Journals (Sweden)

    Gisela Pöll

    Full Text Available The structural constituents of the large eukaryotic ribosomal subunit are 3 ribosomal RNAs, namely the 25S, 5.8S and 5S rRNA and about 46 ribosomal proteins (r-proteins. They assemble and mature in a highly dynamic process that involves more than 150 proteins and 70 small RNAs. Ribosome biogenesis starts in the nucleolus, continues in the nucleoplasm and is completed after nucleo-cytoplasmic translocation of the subunits in the cytoplasm. In this work we created 26 yeast strains, each of which conditionally expresses one of the large ribosomal subunit (LSU proteins. In vivo depletion of the analysed LSU r-proteins was lethal and led to destabilisation and degradation of the LSU and/or its precursors. Detailed steady state and metabolic pulse labelling analyses of rRNA precursors in these mutant strains showed that LSU r-proteins can be grouped according to their requirement for efficient progression of different steps of large ribosomal subunit maturation. Comparative analyses of the observed phenotypes and the nature of r-protein-rRNA interactions as predicted by current atomic LSU structure models led us to discuss working hypotheses on i how individual r-proteins control the productive processing of the major 5' end of 5.8S rRNA precursors by exonucleases Rat1p and Xrn1p, and ii the nature of structural characteristics of nascent LSUs that are required for cytoplasmic accumulation of nascent subunits but are nonessential for most of the nuclear LSU pre-rRNA processing events.

  15. Structure–Function Relationships in Fungal Large-Subunit Catalases

    Energy Technology Data Exchange (ETDEWEB)

    Diaz, A.; Valdez, V; Rudino-Pinera, E; Horjales, E; Hansberg, W


    Neurospora crassa has two large-subunit catalases, CAT-1 and CAT-3. CAT-1 is associated with non-growing cells and accumulates particularly in asexual spores; CAT-3 is associated with growing cells and is induced under different stress conditions. It is our interest to elucidate the structure-function relationships in large-subunit catalases. Here we have determined the CAT-3 crystal structure and compared it with the previously determined CAT-1 structure. Similar to CAT-1, CAT-3 hydrogen peroxide (H{sub 2}O{sub 2}) saturation kinetics exhibited two components, consistent with the existence of two active sites: one saturated in the millimolar range and the other in the molar range. In the CAT-1 structure, we found three interesting features related to its unusual kinetics: (a) a constriction in the channel that conveys H{sub 2}O{sub 2} to the active site; (b) a covalent bond between the tyrosine, which forms the fifth coordination bound to the iron of the heme, and a vicinal cysteine; (c) oxidation of the pyrrole ring III to form a cis-hydroxyl group in C5 and a cis-{gamma}-spirolactone in C6. The site of heme oxidation marks the starts of the central channel that communicates to the central cavity and the shortest way products can exit the active site. CAT-3 has a similar constriction in its major channel, which could function as a gating system regulated by the H{sub 2}O{sub 2} concentration before the gate. CAT-3 functional tyrosine is not covalently bonded, but has instead the electron relay mechanism described for the human catalase to divert electrons from it. Pyrrole ring III in CAT-3 is not oxidized as it is in other large-subunit catalases whose structure has been determined. Different in CAT-3 from these enzymes is an occupied central cavity. Results presented here indicate that CAT-3 and CAT-1 enzymes represent a functional group of catalases with distinctive structural characteristics that determine similar kinetics.

  16. Dynamics of Human Telomerase Holoenzyme Assembly and Subunit Exchange across the Cell Cycle. (United States)

    Vogan, Jacob M; Collins, Kathleen


    Human telomerase acts on telomeres during the genome synthesis phase of the cell cycle, accompanied by its concentration in Cajal bodies and transient colocalization with telomeres. Whether the regulation of human telomerase holoenzyme assembly contributes to the cell cycle restriction of telomerase function is unknown. We investigated the steady-state levels, assembly, and exchange dynamics of human telomerase subunits with quantitative in vivo cross-linking and other methods. We determined the physical association of telomerase subunits in cells blocked or progressing through the cell cycle as synchronized by multiple protocols. The total level of human telomerase RNA (hTR) was invariant across the cell cycle. In vivo snapshots of telomerase holoenzyme composition established that hTR remains bound to human telomerase reverse transcriptase (hTERT) throughout all phases of the cell cycle, and subunit competition assays suggested that hTERT-hTR interaction is not readily exchangeable. In contrast, the telomerase holoenzyme Cajal body-associated protein, TCAB1, was released from hTR in mitotic cells coincident with TCAB1 delocalization from Cajal bodies. This telomerase holoenzyme disassembly was reversible with cell cycle progression without any change in total TCAB1 protein level. Consistent with differential cell cycle regulation of hTERT-hTR and TCAB1-hTR protein-RNA interactions, overexpression of hTERT or TCAB1 had limited if any influence on hTR assembly of the other subunit. Overall, these findings revealed a cell cycle regulation that disables human telomerase association with telomeres while preserving the co-folded hTERT-hTR ribonucleoprotein catalytic core. Studies here, integrated with previous work, led to a unifying model for telomerase subunit assembly and trafficking in human cells.

  17. The Role of Disordered Ribosomal Protein Extensions in the Early Steps of Eubacterial 50 S Ribosomal Subunit Assembly

    Directory of Open Access Journals (Sweden)

    Youri Timsit


    Full Text Available Although during the past decade research has shown the functional importance of disorder in proteins, many of the structural and dynamics properties of intrinsically unstructured proteins (IUPs remain to be elucidated. This review is focused on the role of the extensions of the ribosomal proteins in the early steps of the assembly of the eubacterial 50 S subunit. The recent crystallographic structures of the ribosomal particles have revealed the picture of a complex assembly pathway that condenses the rRNA and the ribosomal proteins into active ribosomes. However, little is know about the molecular mechanisms of this process. It is thought that the long basic r-protein extensions that penetrate deeply into the subunit cores play a key role through disorder-order transitions and/or co-folding mechanisms. A current view is that such structural transitions may facilitate the proper rRNA folding. In this paper, the structures of the proteins L3, L4, L13, L20, L22 and L24 that have been experimentally found to be essential for the first steps of ribosome assembly have been compared. On the basis of their structural and dynamics properties, three categories of extensions have been identified. Each of them seems to play a distinct function. Among them, only the coil-helix transition that occurs in a phylogenetically conserved cluster of basic residues of the L20 extension appears to be strictly required for the large subunit assembly in eubacteria. The role of a helix-coil transitions in 23 S RNA folding is discussed in the light of the calcium binding protein calmodulin that shares many structural and dynamics properties with L20.

  18. rRNA maturation as a "quality" control step in ribosomal subunit assembly in Dictyostelium discoideum. (United States)

    Mangiarotti, G; Chiaberge, S; Bulfone, S


    In Dictyostelium discoideum, newly assembled ribosomal subunits enter polyribosomes while they still contain immature rRNA. rRNA maturation requires the engagement of the subunits in protein synthesis and leads to stabilization of their structure. Maturation of pre-17 S rRNA occurs only after the newly formed 40 S ribosomal particle has entered an 80 S ribosome and participated at least in the formation of one peptide bond or in one translocation event; maturation of pre-26 S rRNA requires the presence on the 80 S particle of a peptidyl-tRNA containing at least 6 amino acids. Newly assembled particles that cannot fulfill these requirements for structural reasons are disassembled into free immature rRNA and ribosomal proteins.

  19. Self-Assembly of Large Amyloid Fibers (United States)

    Ridgley, Devin M.

    Functional amyloids found throughout nature have demonstrated that amyloid fibers are potential industrial biomaterials. This work introduces a new "template plus adder" cooperative mechanism for the spontaneous self-assembly of micrometer sized amyloid fibers. A short hydrophobic template peptide induces a conformation change within a highly alpha-helical adder protein to form beta-sheets that continue to assemble into micrometer sized amyloid fibers. This study utilizes a variety of proteins that have template or adder characteristics which suggests that this mechanism may be employed throughout nature. Depending on the amino acid composition of the proteins used the mixtures form amyloid fibers of a cylindrical ( 10 mum diameter, 2 GPa Young's modulus) or tape (5- 10 mum height, 10-20 mum width and 100-200 MPa Young's modulus) morphology. Processing conditions are altered to manipulate the morphology and structural characteristics of the fibers. Spectroscopy is utilized to identify certain amino acid groups that contribute to the self-assembly process. Aliphatic amino acids (A, I, V and L) are responsible for initiating conformation change of the adder proteins to assemble into amyloid tapes. Additional polyglutamine segments (Q-blocks) within the protein mixtures will form Q hydrogen bonds to reinforce the amyloid structure and form a cylindrical fiber of higher modulus. Atomic force microscopy is utilized to delineate the self-assembly of amyloid tapes and cylindrical fibers from protofibrils (15-30 nm width) to fibers (10-20 mum width) spanning three orders of magnitude. The aliphatic amino acid content of the adder proteins' alpha-helices is a good predictor of high density beta-sheet formation within the protein mixture. Thus, it is possible to predict the propensity of a protein to undergo conformation change into amyloid structures. Finally, Escherichia coli is genetically engineered to express a template protein which self-assembles into large amyloid

  20. Exploring assembly energetics of the 30S ribosomal subunit using an implicit solvent approach. (United States)

    Trylska, Joanna; McCammon, J Andrew; Brooks Iii, Charles L


    To explore the relationship between the assembly of the 30S ribosomal subunit and interactions among the constituent components, 16S RNA and proteins, relative binding free energies of the T. thermophilus 30S proteins to the 16S RNA were studied based on an implicit solvent model of electrostatic, nonpolar, and entropic contributions. The late binding proteins in our assembly map were found not to bind to the naked 16S RNA. The 5' domain early kinetic class proteins, on average, carry the highest positive charge, get buried the most upon binding to 16S RNA, and show the most favorable binding. Some proteins (S10/S14, S6/S18, S13/S19) have more stabilizing interactions while binding as dimers. Our computed assembly map resembles that of E. coli; however, the central domain path is more similar to that of A. aeolicus, a hyperthermophilic bacteria.

  1. Assembly of ROMK1 (Kir 1.1a) inward rectifier K+ channel subunits involves multiple interaction sites. (United States)

    Koster, J C; Bentle, K A; Nichols, C G; Ho, K


    The ROMK1 (Kir 1.1a) channel is formed by a tetrameric complex of subunits, each characterized by cytoplasmic N- and C-termini and a core region of two transmembrane helices flanking a pore-forming segment. To delineate the general regions mediating the assembly of ROMK1 subunits we constructed epitope-tagged N-terminal, C-terminal, and transmembrane segment deletion mutants. Nonfunctional subunits with N-terminal, core region, and C-terminal deletions had dominant negative effects when coexpressed with wild-type ROMK1 subunits in Xenopus oocytes. In contrast, coexpression of these nonfunctional subunits with Kv 2.1 (DRK1) did not suppress Kv 2.1 currents in control oocytes. Interactions between epitope-tagged mutant and wild-type ROMK1 subunits were studied in parallel by immunoprecipitating [35S]-labeled oocyte membrane proteins. Complexes containing both wild-type and mutant subunits that retained H5, M2, and C-terminal regions were coimmunoprecipitated to a greater extent than complexes consisting of wild-type and mutant subunits with core region and/or C-terminal deletions. The present findings are consistent with the hypothesis that multiple interaction sites located in the core region and cytoplasmic termini of ROMK1 subunits mediate homomultimeric assembly.

  2. Binding of the Covalent Flavin Assembly Factor to the Flavoprotein Subunit of Complex II. (United States)

    Maklashina, Elena; Rajagukguk, Sany; Starbird, Chrystal A; McDonald, W Hayes; Koganitsky, Anna; Eisenbach, Michael; Iverson, Tina M; Cecchini, Gary


    Escherichia coli harbors two highly conserved homologs of the essential mitochondrial respiratory complex II (succinate:ubiquinone oxidoreductase). Aerobically the bacterium synthesizes succinate:quinone reductase as part of its respiratory chain, whereas under microaerophilic conditions, the quinol:fumarate reductase can be utilized. All complex II enzymes harbor a covalently bound FAD co-factor that is essential for their ability to oxidize succinate. In eukaryotes and many bacteria, assembly of the covalent flavin linkage is facilitated by a small protein assembly factor, termed SdhE in E. coli. How SdhE assists with formation of the covalent flavin bond and how it binds the flavoprotein subunit of complex II remain unknown. Using photo-cross-linking, we report the interaction site between the flavoprotein of complex II and the SdhE assembly factor. These data indicate that SdhE binds to the flavoprotein between two independently folded domains and that this binding mode likely influences the interdomain orientation. In so doing, SdhE likely orients amino acid residues near the dicarboxylate and FAD binding site, which facilitates formation of the covalent flavin linkage. These studies identify how the conserved SdhE assembly factor and its homologs participate in complex II maturation.

  3. Translation and Assembly of Radiolabeled Mitochondrial DNA-Encoded Protein Subunits from Cultured Cells and Isolated Mitochondria. (United States)

    Formosa, Luke E; Hofer, Annette; Tischner, Christin; Wenz, Tina; Ryan, Michael T


    In higher eukaryotes, the mitochondrial electron transport chain consists of five multi-subunit membrane complexes responsible for the generation of cellular ATP. Of these, four complexes are under dual genetic control as they contain subunits encoded by both the mitochondrial and nuclear genomes, thereby adding another layer of complexity to the puzzle of respiratory complex biogenesis. These subunits must be synthesized and assembled in a coordinated manner in order to ensure correct biogenesis of different respiratory complexes. Here, we describe techniques to (1) specifically radiolabel proteins encoded by mtDNA to monitor the rate of synthesis using pulse labeling methods, and (2) analyze the stability, assembly, and turnover of subunits using pulse-chase methods in cultured cells and isolated mitochondria.

  4. Chaperonin Structure - The Large Multi-Subunit Protein Complex

    Directory of Open Access Journals (Sweden)

    Irena Roterman


    Full Text Available The multi sub-unit protein structure representing the chaperonins group is analyzed with respect to its hydrophobicity distribution. The proteins of this group assist protein folding supported by ATP. The specific axial symmetry GroEL structure (two rings of seven units stacked back to back - 524 aa each and the GroES (single ring of seven units - 97 aa each polypeptide chains are analyzed using the hydrophobicity distribution expressed as excess/deficiency all over the molecule to search for structure-to-function relationships. The empirically observed distribution of hydrophobic residues is confronted with the theoretical one representing the idealized hydrophobic core with hydrophilic residues exposure on the surface. The observed discrepancy between these two distributions seems to be aim-oriented, determining the structure-to-function relation. The hydrophobic force field structure generated by the chaperonin capsule is presented. Its possible influence on substrate folding is suggested.

  5. Nanoparticle self-assembly by a highly stable recombinant spider wrapping silk protein subunit. (United States)

    Xu, Lingling; Tremblay, Marie-Laurence; Orrell, Kathleen E; Leclerc, Jérémie; Meng, Qing; Liu, Xiang-Qin; Rainey, Jan K


    Artificial spider silk proteins may form fibers with exceptional strength and elasticity. Wrapping silk, or aciniform silk, is the toughest of the spider silks, and has a very different protein composition than other spider silks. Here, we present the characterization of an aciniform protein (AcSp1) subunit named W1, consisting of one AcSp1 199 residue repeat unit from Argiope trifasciata. The structural integrity of recombinant W1 is demonstrated in a variety of buffer conditions and time points. Furthermore, we show that W1 has a high thermal stability with reversible denaturation at ∼71°C and forms self-assembled nanoparticle in near-physiological conditions. W1 therefore represents a highly stable and structurally robust module for protein-based nanoparticle formation. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  6. Functional inositol 1,4,5-trisphosphate receptors assembled from concatenated homo- and heteromeric subunits. (United States)

    Alzayady, Kamil J; Wagner, Larry E; Chandrasekhar, Rahul; Monteagudo, Alina; Godiska, Ronald; Tall, Gregory G; Joseph, Suresh K; Yule, David I


    Vertebrate genomes code for three subtypes of inositol 1,4,5-trisphosphate (IP3) receptors (IP3R1, -2, and -3). Individual IP3R monomers are assembled to form homo- and heterotetrameric channels that mediate Ca(2+) release from intracellular stores. IP3R subtypes are regulated differentially by IP3, Ca(2+), ATP, and various other cellular factors and events. IP3R subtypes are seldom expressed in isolation in individual cell types, and cells often express different complements of IP3R subtypes. When multiple subtypes of IP3R are co-expressed, the subunit composition of channels cannot be specifically defined. Thus, how the subunit composition of heterotetrameric IP3R channels contributes to shaping the spatio-temporal properties of IP3-mediated Ca(2+) signals has been difficult to evaluate. To address this question, we created concatenated IP3R linked by short flexible linkers. Dimeric constructs were expressed in DT40-3KO cells, an IP3R null cell line. The dimeric proteins were localized to membranes, ran as intact dimeric proteins on SDS-PAGE, and migrated as an ∼1100-kDa band on blue native gels exactly as wild type IP3R. Importantly, IP3R channels formed from concatenated dimers were fully functional as indicated by agonist-induced Ca(2+) release. Using single channel "on-nucleus" patch clamp, the channels assembled from homodimers were essentially indistinguishable from those formed by the wild type receptor. However, the activity of channels formed from concatenated IP3R1 and IP3R2 heterodimers was dominated by IP3R2 in terms of the characteristics of regulation by ATP. These studies provide the first insight into the regulation of heterotetrameric IP3R of defined composition. Importantly, the results indicate that the properties of these channels are not simply a blend of those of the constituent IP3R monomers.

  7. Primary mirror assemblies for large space telescopes (United States)

    Malamed, Evgeny R.; Sokolsky, M. N.


    In this report are considered the basic problems which relate to developemnt, manufacture, experimental trying out, and usage of primary mirrors (PM) of the large space telescopes intended to perform distant sounding of the Earth. Attention is concentrated on development of weight-reduced passive mirrors which ensure more reliable operation of the telescope as a whole. In the report we expressed the opinion that it is quite possible to manufacture a passive weight-reduced PM if its diameter is equal approximately to 3 m. Materials which may be used for the manufacturing of PM are beryllium and silicon carbide, physical and mechanical parameters of which are the most preferable ones. But it should be taken into consideration that this is the glass ceramic of CO115M brand which has been mastered by the industry of Russia in the greatest extent. It was confirmed that parameters of this material remain unchanged during a long period of time. Constructions of the PM, made of glass ceramic, as well as constructions of holders intended to fix the mirror, are presented in this report. A holder is used first of all to prevent lowering of a PM surface quality after a mirror has been removed from a machine and fixed in a primary mirror assembly (PMA). At present two-layer construction of a PM is preferable. This construction consists of thick base including weight reduction structure, which is in a radius which is optimum from the standpoint of deformation of a mirror operating surface. In the process of manufacture a mirror is deprived of its weight with the use of special pneumatic off-loading elements. PMA is erected in vertical plane by means of using an interferometric inspection system. In the end of this report we expressed the views on an approach to engineering of a PM by taking into account potentialities both of space ships and of carrier rockets.

  8. LEGO-NMR spectroscopy: a method to visualize individual subunits in large heteromeric complexes. (United States)

    Mund, Markus; Overbeck, Jan H; Ullmann, Janina; Sprangers, Remco


    Seeing the big picture: Asymmetric macromolecular complexes that are NMR active in only a subset of their subunits can be prepared, thus decreasing NMR spectral complexity. For the hetero heptameric LSm1-7 and LSm2-8 rings NMR spectra of the individual subunits of the complete complex are obtained, showing a conserved RNA binding site. This LEGO-NMR technique makes large asymmetric complexes accessible to detailed NMR spectroscopic studies.

  9. Discs-large (DLG is clustered by presynaptic innervation and regulates postsynaptic glutamate receptor subunit composition in Drosophila

    Directory of Open Access Journals (Sweden)

    Featherstone David E


    Full Text Available Abstract Background Drosophila discs-large (DLG is the sole representative of a large class of mammalian MAGUKs, including human DLG, SAP 97, SAP102, and PSD-95. MAGUKs are thought to be critical for postsynaptic assembly at glutamatergic synapses. However, glutamate receptor cluster formation has never been examined in Drosophila DLG mutants. The fly neuromuscular junction (NMJ is a genetically-malleable model glutamatergic synapse widely used to address questions regarding the molecular mechanisms of synapse formation and growth. Here, we use immunohistochemistry, confocal microscopy, and electrophysiology to examine whether fly NMJ glutamate receptor clusters form normally in DLG mutants. We also address the question of how DLG itself is localized to the synapse by testing whether presynaptic innervation is required for postsynaptic DLG clustering, and whether DLG localization requires the presence of postsynaptic glutamate receptors. Results There are thought to be two classes of glutamate receptors in the Drosophila NMJ: 1 receptors that contain the subunit GluRIIA, and 2 receptors that contain the subunit GluRIIB. In DLG mutants, antibody staining for the glutamate receptor subunit GluRIIA is normal, but antibody staining for the glutamate receptor subunit GluRIIB is significantly reduced. Electrophysiological analysis shows an overall loss of functional postsynaptic glutamate receptors, along with changes in receptor biophysical properties that are consistent with a selective loss of GluRIIB from the synapse. In uninnervated postsynaptic muscles, neither glutamate receptors nor DLG cluster at synapses. DLG clusters normally in the complete absence of glutamate receptors. Conclusions Our results suggest that DLG controls glutamate receptor subunit composition by selectively stabilizing GluRIIB-containing receptors at the synapse. We also show that DLG, like glutamate receptors, is localized only after the presynaptic neuron contacts the

  10. Assembly of the 30S subunit from Escherichia coli ribosomes occurs via two assembly domains which are initiated by S4 and S7. (United States)

    Nowotny, V; Nierhaus, K H


    A protein which initiates assembly of ribosomes is defined as a protein which binds to the respective rRNA without cooperativity (i.e., without the help of other proteins) during the onset of assembly and is essential for the formation of active ribosomal subunits. The number of proteins binding without cooperativity was determined by monitoring the reconstitution output of active particles at various inputs of 16S rRNA, in the presence of constant amounts of 30S-derived proteins (TP30): This showed that only two of the proteins of the 30S subunit are assembly-initiator proteins. These two proteins are still present on a LiCl core particle comprising 16S rRNA and 12 proteins (including minor proteins). The 12 proteins were isolated, and a series of reconstitution experiments at various levels of rRNA excess demonstrated that S4 and S7 are the initiator proteins. Pulse-chase experiments performed during the early assembly with 14C- and 3H-labeled TP30 and the determination of the 14C/3H ratio of the individual proteins within the assembled particles revealed a bilobal structure of the 30S assembly: A group of six proteins headed by S4 (namely, S4, S20, S16, S15, S6, and S18) resisted the chasing most efficiently (S4 assembly domain). None of the proteins depending on S7 during assembly were found in this group but rather in a second group with intermediate chasing stability [S7 assembly domain; consisting of S7, S9, (S8), S19, and S3]. A number of proteins could be fully chased during the early assembly and therefore represent "late assembly proteins" (S10, S5, S13, S2, S21, S1). These findings fit well with the 30S assembly map.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. Slow-dissociation effect of common signaling subunit beta c on IL5 and GM-CSF receptor assembly. (United States)

    Ishino, Tetsuya; Harrington, Adrian E; Zaks-Zilberman, Meirav; Scibek, Jeffery J; Chaiken, Irwin


    Receptor activation by IL5 and GM-CSF is a sequential process that depends on their interaction with a cytokine-specific subunit alpha and recruitment of a common signaling subunit beta (betac). In order to elucidate the assembly dynamics of these receptor subunits, we performed kinetic interaction analysis of the cytokine-receptor complex formation by a surface plasmon resonance biosensor. Using the extracellular domains of receptor fused with C-terminal V5-tag, we developed an assay method to co-anchor alpha and betac subunits on the biosensor surface. We demonstrated that dissociation of the cytokine-receptor complexes was slower when both subunits were co-anchored on the biosensor surface than when alpha subunit alone was anchored. The slow-dissociation effect of betac had a similar impact on GM-CSF receptor stabilization to that of IL5. The effects were abolished by alanine replacement of either Tyr18 or Tyr344 residue in betac, which together constitute key parts of a cytokine binding epitope. The data argue that betac plays an important role in preventing the ligand-receptor complexes from rapidly dissociating. This slow-dissociation effect of betac explains how, when multiple betac cytokine receptor alpha subunits are present on the same cell surface, selective betac usage can be controlled by sequestration in stabilized cytokine-alpha-betac complexes.

  12. Structural insights into the assembly of the 30S ribosomal subunit in vivo: functional role of S5 and location of the 17S rRNA precursor sequence. (United States)

    Yang, Zhixiu; Guo, Qiang; Goto, Simon; Chen, Yuling; Li, Ningning; Yan, Kaige; Zhang, Yixiao; Muto, Akira; Deng, Haiteng; Himeno, Hyouta; Lei, Jianlin; Gao, Ning


    The in vivo assembly of ribosomal subunits is a highly complex process, with a tight coordination between protein assembly and rRNA maturation events, such as folding and processing of rRNA precursors, as well as modifications of selected bases. In the cell, a large number of factors are required to ensure the efficiency and fidelity of subunit production. Here we characterize the immature 30S subunits accumulated in a factor-null Escherichia coli strain (∆rsgA∆rbfA). The immature 30S subunits isolated with varying salt concentrations in the buffer system show interesting differences on both protein composition and structure. Specifically, intermediates derived under the two contrasting salt conditions (high and low) likely reflect two distinctive assembly stages, the relatively early and late stages of the 3' domain assembly, respectively. Detailed structural analysis demonstrates a mechanistic coupling between the maturation of the 5' end of the 17S rRNA and the assembly of the 30S head domain, and attributes a unique role of S5 in coordinating these two events. Furthermore, our structural results likely reveal the location of the unprocessed terminal sequences of the 17S rRNA, and suggest that the maturation events of the 17S rRNA could be employed as quality control mechanisms on subunit production and protein translation.

  13. Two Arabidopsis ADP-glucose pyrophosphorylase large subunits (APL1 and APL2) are catalytic. (United States)

    Ventriglia, Tiziana; Kuhn, Misty L; Ruiz, Ma Teresa; Ribeiro-Pedro, Marina; Valverde, Federico; Ballicora, Miguel A; Preiss, Jack; Romero, José M


    ADP-glucose (Glc) pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed step in starch biosynthesis. Higher plant ADP-Glc PPase is a heterotetramer (alpha(2)beta(2)) consisting of two small and two large subunits. There is increasing evidence that suggests that catalytic and regulatory properties of the enzyme from higher plants result from the synergy of both types of subunits. In Arabidopsis (Arabidopsis thaliana), two genes encode small subunits (APS1 and APS2) and four large subunits (APL1-APL4). Here, we show that in Arabidopsis, APL1 and APL2, besides their regulatory role, have catalytic activity. Heterotetramers formed by combinations of a noncatalytic APS1 and the four large subunits showed that APL1 and APL2 exhibited ADP-Glc PPase activity with distinctive sensitivities to the allosteric activator (3-phosphoglycerate). Mutation of the Glc-1-P binding site of Arabidopsis and potato (Solanum tuberosum) isoforms confirmed these observations. To determine the relevance of these activities in planta, a T-DNA mutant of APS1 (aps1) was characterized. aps1 is starchless, lacks ADP-Glc PPase activity, APS1 mRNA, and APS1 protein, and is late flowering in long days. Transgenic lines of the aps1 mutant, expressing an inactivated form of APS1, recovered the wild-type phenotype, indicating that APL1 and APL2 have catalytic activity and may contribute to ADP-Glc synthesis in planta.

  14. Two Arabidopsis ADP-Glucose Pyrophosphorylase Large Subunits (APL1 and APL2) Are Catalytic1 (United States)

    Ventriglia, Tiziana; Kuhn, Misty L.; Ruiz, Ma Teresa; Ribeiro-Pedro, Marina; Valverde, Federico; Ballicora, Miguel A.; Preiss, Jack; Romero, José M.


    ADP-glucose (Glc) pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed step in starch biosynthesis. Higher plant ADP-Glc PPase is a heterotetramer (α2β2) consisting of two small and two large subunits. There is increasing evidence that suggests that catalytic and regulatory properties of the enzyme from higher plants result from the synergy of both types of subunits. In Arabidopsis (Arabidopsis thaliana), two genes encode small subunits (APS1 and APS2) and four large subunits (APL1–APL4). Here, we show that in Arabidopsis, APL1 and APL2, besides their regulatory role, have catalytic activity. Heterotetramers formed by combinations of a noncatalytic APS1 and the four large subunits showed that APL1 and APL2 exhibited ADP-Glc PPase activity with distinctive sensitivities to the allosteric activator (3-phosphoglycerate). Mutation of the Glc-1-P binding site of Arabidopsis and potato (Solanum tuberosum) isoforms confirmed these observations. To determine the relevance of these activities in planta, a T-DNA mutant of APS1 (aps1) was characterized. aps1 is starchless, lacks ADP-Glc PPase activity, APS1 mRNA, and APS1 protein, and is late flowering in long days. Transgenic lines of the aps1 mutant, expressing an inactivated form of APS1, recovered the wild-type phenotype, indicating that APL1 and APL2 have catalytic activity and may contribute to ADP-Glc synthesis in planta. PMID:18614708

  15. Next-generation sequencing and large genome assemblies. (United States)

    Henson, Joseph; Tischler, German; Ning, Zemin


    The next-generation sequencing (NGS) revolution has drastically reduced time and cost requirements for sequencing of large genomes, and also qualitatively changed the problem of assembly. This article reviews the state of the art in de novo genome assembly, paying particular attention to mammalian-sized genomes. The strengths and weaknesses of the main sequencing platforms are highlighted, leading to a discussion of assembly and the new challenges associated with NGS data. Current approaches to assembly are outlined and the various software packages available are introduced and compared. The question of whether quality assemblies can be produced using short-read NGS data alone, or whether it must be combined with more expensive sequencing techniques, is considered. Prospects for future assemblers and tests of assembly performance are also discussed.

  16. Next-generation sequencing and large genome assemblies


    Henson, Joseph; Tischler, German; Ning, Zemin


    The next-generation sequencing (NGS) revolution has drastically reduced time and cost requirements for sequencing of large genomes, and also qualitatively changed the problem of assembly. This article reviews the state of the art in de novo genome assembly, paying particular attention to mammalian-sized genomes. The strengths and weaknesses of the main sequencing platforms are highlighted, leading to a discussion of assembly and the new challenges associated with NGS data. Current approaches ...

  17. Mass distributions of a macromolecular assembly based on electrospray ionization mass spectrometric masses of the constituent subunits

    Indian Academy of Sciences (India)

    Leonid Hanin; Brian Green; Franck Zal; Serge Vinogradov


    Macromolecular assemblies containing multiple protein subunits and having masses in the megadalton (MDa) range are involved in most of the functions of a living cell. Because of variation in the number and masses of subunits, macromolecular assemblies do not have a unique mass, but rather a mass distribution. The giant extracelular erythrocruorins (Ers), ∼ 3.5 MDa, comprized of at least 180 polypeptide chains, are one of the best characterized assemblies. Three-dimensional reconstructions from cryoelectron microscopic images show them to be hexagonal bilayer complexes of 12 subassemblies, each comprised of 12 globin chains, anchored to a subassembly of 36 nonglobin linker chains. We have calculated the most probable mass distributions for Lumbricus and Riftia assemblies and their globin and linker subassemblies, based on the Lumbricus Er stoichiometry and using accurate subunit masses obtained by electrospray ionization mass spectrometry. The expected masses of Lumbricus and Riftia Ers are 3.517 MDa and 3.284 MDa, respectively, with a possible variation of ∼ 9% due to the breadth of the mass distributions. The Lumbricus Er mass is in astonishingly good agreement with the mean of 23 known masses, 3.524 ± 0.481 MDa.

  18. The BRCT domain from the large subunit of human Replication Factor C

    NARCIS (Netherlands)

    Kobayashi, Masakazu


    The work described in this thesis deals with characterization of DNA binding by the BRCT domain of the large subunit of RFC. Replication Factor C (RFC) is a five protein complex involved in initiating and regulating new DNA synthesis. The first half of the thesis describes region of the RFC and stru

  19. High resolution structure of the large ribosomal subunit from a Mesophilic Eubacterium

    Energy Technology Data Exchange (ETDEWEB)

    Harms, Joerg; Schluenzen, Frank; Zarivach, Raz; Bashan, Anat; Gat, Sharon; Agmon, Ilana; Bartels, Heike; Franceschi, Francois; Yonath, Ada (Weizmann Inst Israel); (Mac Planck Germany); (Max Planck Germany)


    We describe the high resolution structure of the large ribosomal subunit from Deinococcus radiodurans (D50S), a gram-positive mesophile suitable for binding of antibiotics and functionally relevant ligands. The over-all structure of D50S is similar to that from the archae bacterium Haloarcula marismortui (H50S); however, a detailed comparison revealed significant differences, for example, in the orientation of nucleotides in peptidyl transferase center and in the structures of many ribosomal proteins. Analysis of ribosomal features involved in dynamic aspects of protein biosynthesis that are partially or fully disordered in H50S revealed the conformations of intersubunit bridges in unbound subunits, suggesting how they may change upon subunit association and how movements of the L1-stalk may facilitate the exit of tRNA.

  20. Tagging ribosomal protein S7 allows rapid identification of mutants defective in assembly and function of 30 S subunits. (United States)

    Fredrick, K; Dunny, G M; Noller, H F


    Ribosomal protein S7 nucleates folding of the 16 S rRNA 3' major domain, which ultimately forms the head of the 30 S ribosomal subunit. Recent crystal structures indicate that S7 lies on the interface side of the 30 S subunit, near the tRNA binding sites of the ribosome. To map the functional surface of S7, we have tagged the protein with a Protein Kinase A recognition site and engineered alanine substitutions that target each exposed, conserved residue. We have also deleted conserved features of S7, using its structure to guide our design. By radiolabeling the tag sequence using Protein Kinase A, we are able to track the partitioning of each mutant protein into 30 S, 70 S, and polyribosome fractions in vivo. Overexpression of S7 confers a growth defect, and we observe a striking correlation between this phenotype and proficiency in 30 S subunit assembly among our collection of mutants. We find that the side chain of K35 is required for efficient assembly of S7 into 30 S subunits in vivo, whereas those of at least 17 other conserved exposed residues are not required. In addition, an S7 derivative lacking the N-terminal 17 residues causes ribosomes to accumulate on mRNA to abnormally high levels, indicating that our approach can yield interesting mutant ribosomes.

  1. Effect of the delta subunit on assembly and proton permeability of the F0 proton channel of Escherichia coli F1F0 ATPase.



    During the assembly of the Escherichia coli proton-translocating ATPase, the subunits of F1 interact with F0 to increase the proton permeability of the transmembrane proton channel. We tested the involvement of the delta subunit in this process by partially and completely deleting uncH (delta subunit) from a plasmid carrying the genes for the F0 subunits and delta and testing the effects of those F0 plasmids on the growth of unc+ and unc mutant E. coli strains. We found that the delta subunit...

  2. Large-scale assembly of colloidal particles (United States)

    Yang, Hongta

    This study reports a simple, roll-to-roll compatible coating technology for producing three-dimensional highly ordered colloidal crystal-polymer composites, colloidal crystals, and macroporous polymer membranes. A vertically beveled doctor blade is utilized to shear align silica microsphere-monomer suspensions to form large-area composites in a single step. The polymer matrix and the silica microspheres can be selectively removed to create colloidal crystals and self-standing macroporous polymer membranes. The thickness of the shear-aligned crystal is correlated with the viscosity of the colloidal suspension and the coating speed, and the correlations can be qualitatively explained by adapting the mechanisms developed for conventional doctor blade coating. Five important research topics related to the application of large-scale three-dimensional highly ordered macroporous films by doctor blade coating are covered in this study. The first topic describes the invention in large area and low cost color reflective displays. This invention is inspired by the heat pipe technology. The self-standing macroporous polymer films exhibit brilliant colors which originate from the Bragg diffractive of visible light form the three-dimensional highly ordered air cavities. The colors can be easily changed by tuning the size of the air cavities to cover the whole visible spectrum. When the air cavities are filled with a solvent which has the same refractive index as that of the polymer, the macroporous polymer films become completely transparent due to the index matching. When the solvent trapped in the cavities is evaporated by in-situ heating, the sample color changes back to brilliant color. This process is highly reversible and reproducible for thousands of cycles. The second topic reports the achievement of rapid and reversible vapor detection by using 3-D macroporous photonic crystals. Capillary condensation of a condensable vapor in the interconnected macropores leads to the

  3. Chromatin assembly factor 1, subunit A (P150 facilitates cell proliferation in human hepatocellular carcinoma

    Directory of Open Access Journals (Sweden)

    Xu M


    Full Text Available Meng Xu, Yuli Jia, Zhikui Liu, Linglong Ding, Run Tian, Hua Gu, Yufeng Wang, Hongyong Zhang, Kangsheng Tu, Qingguang Liu Department of Hepatobiliary Surgery, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, People’s Republic of China Abstract: Several studies have revealed that the abnormal expression of chromatin assembly factor 1, subunit A (P150 (CHAF1A was involved in the development of some types of malignant tumors. However, CHAF1A expression and its role in hepatocellular carcinoma (HCC remain poorly characterized. In this study, we first investigated CHAF1A expression in six cell lines and 116 pairs of HCC and matched normal tumor-adjacent tissues to evaluate the clinicopathological characteristics of CHAF1A in HCC. Then, we detected the proliferation and apoptosis in HCC cells. In addition, a subcutaneous tumor model in nude mice was performed to evaluate tumor growth in vivo. We found that the expression of CHAF1A was significantly higher in HCC tissues than that in adjacent nontumor tissues (P<0.01. Clinical analysis indicated that CHAF1A expression was significantly correlated with the tumor–node–metastasis stage, tumor number, and tumor differentiation in HCC tissues (P<0.05, respectively. We also found that CHAF1A may potentially function as a poor prognostic indicator for 5-year overall and disease-free survival in patients with HCC (P<0.05, respectively. The elevated expression of CHAF1A was also observed in HCC cell lines compared with that in normal LO2 hepatic cell line (P<0.01. HCC cancer cells exhibited inhibition of cell growth, reduction in colony-formation ability, increased cell apoptosis rate, and impaired tumorigenicity in nude mice after CHAF1A knockdown. Collectively, we propose that CHAF1A by potentially mediating cancer cell proliferation plays an important role in promoting the development of HCC and may serve as a potential therapeutic target in HCC. Keywords: CHAF1A, hepatocellular

  4. A telerobotic system for automated assembly of large space structures (United States)

    Rhodes, Marvin D.; Will, Ralph W.; Wise, Marion A.


    Future space missions such as polar platforms and antennas are anticipated to require large truss structures as their primary support system. During the past several years considerable research has been conducted to develop hardware and construction techniques suitable for astronaut assembly of truss structures in space. A research program has recently been initiated to develop the technology and to demonstrate the potential for automated in-space assembly of large erectable structures. The initial effort will be focused on automated assembly of a tetrahedral truss composed of 2-meter members. The facility is designed as a ground based system to permit evaluation of assembly concepts and was not designed for space qualification. The system is intended to be used as a tool from which more sophisticated procedures and operations can be developed. The facility description includes a truss structure, motionbases and a robot arm equipped with an end effector. Other considerations and requirements of the structural assembly describe computer control systems to monitor and control the operations of the assembly facility.

  5. Large-Scale Self-Assembled Ag Nanotubes

    Institute of Scientific and Technical Information of China (English)

    WEI Guodan; NAN Cewen; YU Dapeng


    A high yield of silver nanotubes with large aspect ratio were conveniently synthesized via an organic-assist solvothermal preparation technique using polyvinyl pyrrolidone (PVP) as a capping reagent and architecture soft-template. The molecular ratio between the repeating unit of PVP and AgNO3 plays a crucial role in determining the geometric shape of the product. Such novel-type Ag nanotubes were self-assembled by Ag nanoparticles, which had largely similar crystallographic orientation, forming a texture. The fact that nanoparticles without anisotropic crystal structures can form such superstructures by self-assembly may open a window for understanding a range of nanotube formation processes.

  6. Mapping of a conformational epitope on the cashew allergen Ana o 2: a discontinuous large subunit epitope dependent upon homologous or heterologous small subunit association. (United States)

    Xia, Lixin; Willison, LeAnna N; Porter, Lauren; Robotham, Jason M; Teuber, Suzanne S; Sathe, Shridhar K; Roux, Kenneth H


    The 11S globulins are members of the cupin protein superfamily and represent an important class of tree nut allergens for which a number of linear epitopes have been mapped. However, specific conformational epitopes for these allergens have yet to be described. We have recently reported a cashew Ana o 2 conformational epitope defined by murine mAb 2B5 and competitively inhibited by a subset of patient IgE antibodies. The 2B5 epitope appears to reside on the large (acidic) subunit, is dependent upon small (basic) subunit association for expression, and is highly susceptible to denaturation. Here we fine map the epitope using a combination of recombinant chimeric cashew Ana o 2-soybean Gly m 6 chimeras, deletion and point mutations, molecular modeling, and electron microscopy of 2B5-Ana o 2 immune complexes. Key residues appear confined to a 24 amino acid segment near the N-terminus of the large subunit peptide, a portion of which makes direct contact with the small subunit. These data provide an explanation for both the small subunit dependence and the structurally labile nature of the epitope.

  7. Off-pathway assembly of fimbria subunits is prevented by chaperone CfaA of CFA/I fimbriae from enterotoxigenic E. coli. (United States)

    Bao, Rui; Liu, Yang; Savarino, Stephen J; Xia, Di


    The assembly of the class 5 colonization factor antigen I (CFA/I) fimbriae of enterotoxigenic E. coli was proposed to proceed via the alternate chaperone-usher pathway. Here, we show that in the absence of the chaperone CfaA, CfaB, the major pilin subunit of CFA/I fimbriae, is able to spontaneously refold and polymerize into cyclic trimers. CfaA kinetically traps CfaB to form a metastable complex that can be stabilized by mutations. Crystal structure of the stabilized complex reveals distinctive interactions provided by CfaA to trap CfaB in an assembly competent state through donor-strand complementation (DSC) and cleft-mediated anchorage. Mutagenesis indicated that DSC controls the stability of the chaperone-subunit complex and the cleft-mediated anchorage of the subunit C-terminus additionally assist in subunit refolding. Surprisingly, over-stabilization of the chaperone-subunit complex led to delayed fimbria assembly, whereas destabilizing the complex resulted in no fimbriation. Thus, CfaA acts predominantly as a kinetic trap by stabilizing subunit to avoid its off-pathway self-polymerization that results in energetically favorable trimers and could serve as a driving force for CFA/I pilus assembly, representing an energetic landscape unique to class 5 fimbria assembly. Published 2016. This article is a U.S. Government work and is in the public domain in the USA. Molecular Microbiology published by John Wiley & Sons Ltd.

  8. SWAP-Assembler 2: Optimization of De Novo Genome Assembler at Large Scale

    Energy Technology Data Exchange (ETDEWEB)

    Meng, Jintao; Seo, Sangmin; Balaji, Pavan; Wei, Yanjie; Wang, Bingqiang; Feng, Shengzhong


    In this paper, we analyze and optimize the most time-consuming steps of the SWAP-Assembler, a parallel genome assembler, so that it can scale to a large number of cores for huge genomes with the size of sequencing data ranging from terabyes to petabytes. According to the performance analysis results, the most time-consuming steps are input parallelization, k-mer graph construction, and graph simplification (edge merging). For the input parallelization, the input data is divided into virtual fragments with nearly equal size, and the start position and end position of each fragment are automatically separated at the beginning of the reads. In k-mer graph construction, in order to improve the communication efficiency, the message size is kept constant between any two processes by proportionally increasing the number of nucleotides to the number of processes in the input parallelization step for each round. The memory usage is also decreased because only a small part of the input data is processed in each round. With graph simplification, the communication protocol reduces the number of communication loops from four to two loops and decreases the idle communication time. The optimized assembler is denoted as SWAP-Assembler 2 (SWAP2). In our experiments using a 1000 Genomes project dataset of 4 terabytes (the largest dataset ever used for assembling) on the supercomputer Mira, the results show that SWAP2 scales to 131,072 cores with an efficiency of 40%. We also compared our work with both the HipMER assembler and the SWAP-Assembler. On the Yanhuang dataset of 300 gigabytes, SWAP2 shows a 3X speedup and 4X better scalability compared with the HipMer assembler and is 45 times faster than the SWAP-Assembler. The SWAP2 software is available at

  9. Studies on Differential Nuclear Translocation Mechanism and Assembly of the Three Subunits of the Arabidopsis thaliana Transcription Factor NF-Y

    Institute of Scientific and Technical Information of China (English)

    Dieter Hackenberg; Yanfang Wu; Andrea Voigt; Robert Adams; Peter Schramm; Bernhard Grimm


    The eukaryotic transcription factor NF-Y consists of three subunits(A,B,and C),which are encoded in Arabidopsis thaliana in multigene families consisting of 10,13,and 13 genes,respectively.In principle,all potential combinations of the subunits are possible for the assembly of the heterotrimeric complex.We aimed at assessing the probability of each subunit to participate in the assembly of NF-Y.The evaluation of physical interactions among all members of the NF-Y subunit families indicate a strong requirement for NF-YB/NF-YC heterodimerization before the entire complex can be accomplished.By means of a modified yeast two-hybrid system assembly of all three subunits to a heterotrimeric complex was demonstrated.Using GFP fusion constructs,NF-YA and NF-YC localization in the nucleus was demonstrated,while NFYB is solely imported into the nucleus as a NF-YC-associated heterodimer NF-YC.This piggyback transport of the two Arabidopsis subunits differs from the import of the NF-Y heterotrimer of heterotrophic organisms.Based on a peptide structure model of the histone-fold-motifs,disulfide bonding among intramolecular conserved cysteine residues of NF-YB,which is responsible for the redox-regulated assembly of NF-YB and NF-YC in human and Aspergillus nidulans,can be excluded for Arabidopsis NF-YB.

  10. RNA tertiary interactions in the large ribosomal subunit: The A-minor motif

    Energy Technology Data Exchange (ETDEWEB)

    Nissen, Poul; Ippolito, Joseph A.; Ban, Nenad; Moore, Peter B.; Steitz, Thomas A. (Yale University); (Yale University); (Yale Unversity)


    Analysis of the 2.4-{angstrom} resolution crystal structure of the large ribosomal subunit from Haloarcula marismortui reveals the existence of an abundant and ubiquitous structural motif that stabilizes RNA tertiary and quaternary structures. This motif is termed the A-minor motif, because it involves the insertion of the smooth, minor groove edges of adenines into the minor groove of neighboring helices, preferentially at C-G base pairs, where they form hydrogen bonds with one or both of the 2' OHs of those pairs. A-minor motifs stabilize contacts between RNA helices, interactions between loops and helices, and the conformations of junctions and tight turns. The interactions between the 3' terminal adenine of tRNAs bound in either the A site or the P site with 23S rRNA are examples of functionally significant A-minor interactions. The A-minor motif is by far the most abundant tertiary structure interaction in the large ribosomal subunit; 186 adenines in 23S and 5S rRNA participate, 68 of which are conserved. It may prove to be the universally most important long-range interaction in large RNA structures.

  11. Assembly of the adenosine triphosphatase complex in Escherichia coli: assembly of F0 is dependent on the formation of specific F1 subunits. (United States)

    Cox, G B; Downie, J A; Langman, L; Senior, A E; Ash, G; Fayle, D R; Gibson, F


    A strain of Escherichia coli (AN1007) carrying the polar uncD436 allele which affects the operon coding for the F1-F0 adenosine triphosphatase (ATPase) complex was isolated and characterized. The uncD436 allele affected the two genes most distal to the operon promoter, i.e., uncD and uncC. Although the genes coding for the F0 portion of the ATPase complex were not affected in strains carrying this mutant allele, the lack of reconstitution of washed membranes by normal F1 ATPase suggested that a functional F0 might not be formed. This conclusion was supported by the observation that the 18,000-molecular-weight F0 subunit, coded for by the uncF gene, was absent from the membranes. Plasmid pAN36 (uncD+C+), when inserted into a strain carrying the uncD436 allele, resulted in the incorporation of the 18,000-molecular-weight F0 subunit into the membrane. A further series of experiments with Mu-induced polarity mutants, with and without plasmid pAN36, showed that the formation of both the alpha- and beta-subunits of F1 ATPase was an essential prerequisite to the incorporation into the membrane of the 18,000-molecular-weight F0 subunit and to the formation of a functional F0. Examination of the polypeptide composition of membranes from various unc mutants allowed a sequence for the normal assembly of the F1-F0 ATPase complex to be proposed.

  12. Independent in vitro assembly of all three major morphological parts of the 30S ribosomal subunit of Thermus thermophilus. (United States)

    Agalarov, S C; Selivanova, O M; Zheleznyakova, E N; Zheleznaya, L A; Matvienko, N I; Spirin, A S


    Fragments of the 16S rRNA of Thermus thermophilus representing the 3' domain (nucleotides 890-1515) and the 5' domain (nucleotides 1-539) have been prepared by transcription in vitro. Incubation of these fragments with total 30S ribosomal proteins of T. thermophilus resulted in formation of specific RNPs. The particle assembled on the 3' RNA domain contained seven proteins corresponding to Escherichia coli ribosomal proteins S3, S7, S9, S10, S13, S14, and S19. All of them have previously been shown to interact with the 3' domain of the 16S RNA and to be localized in the head of the 30S ribosomal subunit. The particle formed on the 5' RNA domain contained five ribosomal proteins corresponding to E. coli proteins S4, S12, S17, S16, and S20. These proteins are known to be localized in the main part of the body of the 30S subunit. Both types of particle were compact and had sedimentation coefficients of 15.5 S and 13 S, respectively. Together with our recent demonstration of the reconstitution of the RNA particle representing the platform of the T. thermophilus 30S ribosomal subunit [Agalarov, S.C., Zheleznyakova, E.N., Selivanova, O.M., Zheleznaya, L.A., Matvienko, N.I., Vasiliev, V.D. & Spirin, A.S. (1998) Proc. Natl Acad. Sci. USA 95, 999-1003], these experiments establish that all three main structural lobes of the small ribosomal subunit can be reconstituted independently of each other and prepared in the individual state.

  13. A separable domain of the p150 subunit of human chromatin assembly factor-1 promotes protein and chromosome associations with nucleoli



    Chromatin assembly factor-1 (CAF-1) is a three-subunit protein complex conserved throughout eukaryotes that deposits histones during DNA synthesis. Here we present a novel role for the human p150 subunit in regulating nucleolar macromolecular interactions. Acute depletion of p150 causes redistribution of multiple nucleolar proteins and reduces nucleolar association with several repetitive element–containing loci. Of note, a point mutation in a SUMO-interacting motif (SIM) within p150 abolishe...

  14. Chronic flumazenil alters GABA(A) receptor subunit mRNA expression, translation product assembly and channel function in neuronal cultures. (United States)

    Zheng, T M; Caruncho, H J; Zhu, W J; Vicini, S; Ikonomovic, S; Grayson, D R; Costa, E


    Flumazenil competitively blocks the pharmacological effects of both positive and negative allosteric modulators acting at the benzodiazepine binding sites of gamma-aminobutyric acid (GABA(A)) receptors. Using quantitative reverse transcription polymerase chain reaction, label-fracture immunocytochemistry and whole-cell patch-clamp recordings, we determined changes in the contents of selected GABA(A) receptor subunit mRNA(s), in their translation products and in the electrophysiological characteristics of the receptor channels in cultured cerebellar granule cells treated daily with flumazenil (10 microM) for 4 days in vitro. The contents of the alpha1 and alpha6 receptor subunit mRNAs were significantly increased in the flumazenil-treated group as compared with the dimethyl sulfoxide vehicle-treated control group, whereas there were no significant differences in the absolute amounts of the beta2, beta3, gamma2S, gamma2L++ + and delta receptor subunit mRNAs. The gold immunolabeling densities of the alpha1 and delta receptor subunits were significantly increased, whereas those of the alpha6, beta2/beta3 and gamma2 receptor subunits were decreased. Double-immunolabeling experiments using 5- and 10-nm gold particles suggest that after chronic flumazenil treatment, receptor subunit assemblies containing the alpha1/gamma2 and alpha6/delta subunits may be replaced by a receptor assembly containing the alpha1/delta subunits. The GABA potency in eliciting Cl- channel activity decreased significantly, as indicated by the elevated EC50 values, and the positive modulation of GABA action by diazepam also decreased. These results suggest that flumazenil, perhaps by blocking the action of endogenous allosteric modulators of GABA(A) receptors, may trigger a change in the expression and assembly of the subunits of the GABA(A) receptor. This implies that there might be a dynamic state in the regulation of GABA(A) receptor structure.

  15. Fluorescently tagged laminin subunits facilitate analyses of the properties, assembly and processing of laminins in live and fixed lung epithelial cells and keratinocytes. (United States)

    Hopkinson, Susan B; DeBiase, Phillip J; Kligys, Kristina; Hamill, Kevin; Jones, Jonathan C R


    Recent analyses of collagen, elastin and fibronectin matrix assembly, organization and remodeling have been facilitated by the use of tagged proteins that can be visualized without the need for antibody labeling. Here, we report the generation of C-terminal tagged, full-length and "processed" (alpha3DeltaLG4-5) human alpha3 as well as C-terminal tagged, full-length human beta3 laminin subunits in adenoviral vectors. Human epidermal keratinocytes (HEKs) and human bronchial epithelial (BEP2D) cells, which assemble laminin-332-rich matrices, as well as primary rat lung alveolar type II (ATII) cells, which elaborate a fibrous network rich in laminin-311, were infected with adenovirus encoding the tagged human laminin subunits. In HEKs and BEP2D cells, tagged, full-length alpha3, alpha3DeltaLG4-5 and beta3 laminin subunits incorporate into arrays of matrix organized into patterns that are comparable to those observed when such cells are stained using laminin-332 subunit antibody probes. Moreover, HEKs and BEP2Ds move over these tagged, laminin-332-rich matrix arrays. We have also used the tagged beta3 laminin subunit-containing matrices to demonstrate that assembled laminin-332 arrays influence laminin matrix secretion and/or assembly. In the case of rat ATII cells, although tagged alpha3 laminin subunits are not detected in the matrix of rat ATII cells infected with virus encoding full-length human alpha3 laminin protein, processed human alpha3 laminin subunits are incorporated into an extracellular fibrous array. We discuss how these novel laminin reagents can be used to study the organization, processing and assembly of laminin matrices and how they provide new insights into the potential functional importance of laminin fragments.

  16. Cholera toxin B subunits assemble into pentamers--proposition of a fly-casting mechanism.

    Directory of Open Access Journals (Sweden)

    Jihad Zrimi

    Full Text Available The cholera toxin B pentamer (CtxB(5, which belongs to the AB(5 toxin family, is used as a model study for protein assembly. The effect of the pH on the reassembly of the toxin was investigated using immunochemical, electrophoretic and spectroscopic methods. Three pH-dependent steps were identified during the toxin reassembly: (i acquisition of a fully assembly-competent fold by the CtxB monomer, (ii association of CtxB monomer into oligomers, (iii acquisition of the native fold by the CtxB pentamer. The results show that CtxB(5 and the related heat labile enterotoxin LTB(5 have distinct mechanisms of assembly despite sharing high sequence identity (84% and almost identical atomic structures. The difference can be pinpointed to four histidines which are spread along the protein sequence and may act together. Thus, most of the toxin B amino acids appear negligible for the assembly, raising the possibility that assembly is driven by a small network of amino acids instead of involving all of them.

  17. Interferon-γ-induced upregulation of immunoproteasome subunit assembly overcomes bortezomib resistance in human hematological cell lines. (United States)

    Niewerth, Denise; Kaspers, Gertjan J L; Assaraf, Yehuda G; van Meerloo, Johan; Kirk, Christopher J; Anderl, Janet; Blank, Jonathan L; van de Ven, Peter M; Zweegman, Sonja; Jansen, Gerrit; Cloos, Jacqueline


    polyubiquitinated proteins. This sensitization was abrogated by siRNA silencing of β5i but not by β1i silencing, prior to pulse exposure to interferon-γ. Downregulation of β5i subunit expression is a major determinant in acquisition of bortezomib-resistance and enhancement of its proteasomal assembly after induction by interferon-γ facilitates restoration of sensitivity in bortezomib-resistant leukemia cells towards bortezomib and next generation (immuno) proteasome inhibitors.

  18. Cloning and characterization of GST fusion tag stabilized large subunit of Escherichia coli acetohydroxyacid synthase I. (United States)

    Li, Heng; Liu, Nan; Wang, Wen-Ting; Wang, Ji-Yu; Gao, Wen-Yun


    There are three acetohydroxyacid synthase (AHAS, EC isozymes (I, II, and III) in the enterobacteria Escherichia coli among which AHAS I is the most active. Its large subunit (LSU) possesses full catalytic machinery, but is unstable in the absence of the small subunit (SSU). To get applicable LSU of AHAS I, we prepared and characterized in this study the polypeptide as a His-tagged (His-LSU) and a glutathione S-transferase (GST)-tagged (GST-LSU) fusion protein, respectively. The results showed that the His-LSU is unstable, whereas the GST-LSU displays excellent stability. This phenomenon suggests that the GST polypeptide fusion tag could stabilize the target protein when compared with histidine tag. It is the first time that the stabilizing effect of the GST tag was observed. Further characterization of the GST-LSU protein indicated that it possesses the basic functions of AHAS I with a specific activity of 20.8 μmol min(-1) mg(-1) and a Km value for pyruvate of 0.95 mM. These observations imply that introduction of the GST fusion tag to LSU of AHAS I does not affect the function of the protein. The possible reasons that the GST fusion tag could make the LSU stable are initially discussed.

  19. The large subunit of bacteriophage lambda's terminase plays a role in DNA translocation and packaging termination. (United States)

    Duffy, Carol; Feiss, Michael


    The DNA packaging enzyme of bacteriophage lambda, terminase, is a heteromultimer composed of a small subunit, gpNu1, and a large subunit, gpA, products of the Nu1 and A genes, respectively. The role of terminase in the initial stages of packaging involving the site-specific binding and cutting of the DNA has been well characterized. While it is believed that terminase plays an active role in later post-cleavage stages of packaging, such as the translocation of DNA into the head shell, this has not been demonstrated. Accordingly, we undertook a generalized mutagenesis of lambda's A gene and found ten lethal mutations, nine of which cause post-cleavage packaging defects. All were located in the amino-terminal two-thirds of gpA, separate from the carboxy-terminal region where mutations affecting the protein's endonuclease activity have been found. The mutants fall into five groups according to their packaging phenotypes: (1) two mutants package part of the lambda chromosome, (2) one mutant packages the entire chromosome, but very slowly compared to wild-type, (3) two mutants do not package any DNA, (4) four mutants, though inviable, package the entire lambda chromosome, and (5) one mutant may be defective in both early and late stages of DNA packaging. These results indicate that gpA is actively involved in late stages of packaging, including DNA translocation, and that this enzyme contains separate functional domains for its early and late packaging activities.

  20. In vitro import and assembly of the nucleus-encoded mitochondrial subunit III of cytochrome c oxidase (Cox3). (United States)

    Vázquez-Acevedo, Miriam; Rubalcava-Gracia, Diana; González-Halphen, Diego


    The cox3 gene, encoding subunit III of cytochrome c oxidase (Cox3) is in mitochondrial genomes except in chlorophycean algae, where it is localized in the nucleus. Therefore, algae like Chlamydomonas reinhardtii, Polytomella sp. and Volvox carteri, synthesize the Cox3 polypeptide in the cytosol, import it into mitochondria, and integrate it into the cytochrome c oxidase complex. In this work, we followed the in vitro internalization of the Cox3 precursor by isolated, import-competent mitochondria of Polytomella sp. In this colorless alga, the precursor Cox3 protein is synthesized with a long, cleavable, N-terminal mitochondrial targeting sequence (MTS) of 98 residues. In an import time course, a transient Cox3 intermediate was identified, suggesting that the long MTS is processed more than once. The first processing step is sensitive to the metalo-protease inhibitor 1,10-ortophenantroline, suggesting that it is probably carried out by the matrix-located Mitochondrial Processing Protease. Cox3 is readily imported through an energy-dependent import pathway and integrated into the inner mitochondrial membrane, becoming resistant to carbonate extraction. Furthermore, the imported Cox3 protein was assembled into cytochrome c oxidase, as judged by the presence of a labeled band co-migrating with complex IV in Blue Native Electrophoresis. A model for the biogenesis of Cox3 in chlorophycean algae is proposed. This is the first time that the in vitro mitochondrial import of a cytosol-synthesized Cox3 subunit is described.

  1. Type IV pilus assembly proficiency and dynamics influence pilin subunit phospho-form macro- and microheterogeneity in Neisseria gonorrhoeae.

    Directory of Open Access Journals (Sweden)

    Åshild Vik

    Full Text Available The PilE pilin subunit protein of the gonococcal Type IV pilus (Tfp colonization factor undergoes multisite, covalent modification with the zwitterionic phospho-form modification phosphoethanolamine (PE. In a mutant lacking the pilin-like PilV protein however, PilE is modified with a mixture of PE and phosphocholine (PC. Moreover, intrastrain variation of PilE PC modification levels have been observed in backgrounds that constitutively express PptA (the protein phospho-form transferase A required for both PE and PC modification. The molecular basis underlying phospho-form microheterogeneity in these instances remains poorly defined. Here, we examined the effects of mutations at numerous loci that disrupt or perturb Tfp assembly and observed that these mutants phenocopy the pilV mutant vis a vis phospho-form modification status. Thus, PC modification appears to be directly or indirectly responsive to the efficacy of pilin subunit interactions. Despite the complexity of contributing factors identified here, the data favor a model in which increased retention in the inner membrane may act as a key signal in altering phospho-form modification. These results also provide an alternative explanation for the variation in PilE PC levels observed previously and that has been assumed to be due to phase variation of pptA. Moreover, mass spectrometry revealed evidence for mono- and di-methylated forms of PE attached to PilE in mutants deficient in pilus assembly, directly implicating a methyltransferase-based pathway for PC synthesis in N. gonorrhoeae.

  2. Fragmentation of the large subunit ribosomal RNA gene in oyster mitochondrial genomes

    Directory of Open Access Journals (Sweden)

    Milbury Coren A


    Full Text Available Abstract Background Discontinuous genes have been observed in bacteria, archaea, and eukaryotic nuclei, mitochondria and chloroplasts. Gene discontinuity occurs in multiple forms: the two most frequent forms result from introns that are spliced out of the RNA and the resulting exons are spliced together to form a single transcript, and fragmented gene transcripts that are not covalently attached post-transcriptionally. Within the past few years, fragmented ribosomal RNA (rRNA genes have been discovered in bilateral metazoan mitochondria, all within a group of related oysters. Results In this study, we have characterized this fragmentation with comparative analysis and experimentation. We present secondary structures, modeled using comparative sequence analysis of the discontinuous mitochondrial large subunit rRNA genes of the cupped oysters C. virginica, C. gigas, and C. hongkongensis. Comparative structure models for the large subunit rRNA in each of the three oyster species are generally similar to those for other bilateral metazoans. We also used RT-PCR and analyzed ESTs to determine if the two fragmented LSU rRNAs are spliced together. The two segments are transcribed separately, and not spliced together although they still form functional rRNAs and ribosomes. Conclusions Although many examples of discontinuous ribosomal genes have been documented in bacteria and archaea, as well as the nuclei, chloroplasts, and mitochondria of eukaryotes, oysters are some of the first characterized examples of fragmented bilateral animal mitochondrial rRNA genes. The secondary structures of the oyster LSU rRNA fragments have been predicted on the basis of previous comparative metazoan mitochondrial LSU rRNA structure models.

  3. Differential roles of the COOH termini of AAA subunits of PA700 (19 S regulator) in asymmetric assembly and activation of the 26 S proteasome. (United States)

    Gillette, Thomas G; Kumar, Brajesh; Thompson, David; Slaughter, Clive A; DeMartino, George N


    The 26 S proteasome is an energy-dependent protease that degrades proteins modified with polyubiquitin chains. It is assembled from two multi-protein subcomplexes: a protease (20 S proteasome) and an ATPase regulatory complex (PA700 or 19 S regulatory particle) that contains six different AAA family subunits (Rpt1 to -6). Here we show that binding of PA700 to the 20 S proteasome is mediated by the COOH termini of two (Rpt2 and Rpt5) of the six Rpt subunits that constitute the interaction surface between the subcomplexes. COOH-terminal peptides of either Rpt2 or Rpt5 bind to the 20 S proteasome and activate hydrolysis of short peptide substrates. Simultaneous binding of both COOH-terminal peptides had additive effects on peptide substrate hydrolysis, suggesting that they bind to distinct sites on the proteasome. In contrast, only the Rpt5 peptide activated hydrolysis of protein substrates. Nevertheless, the COOH-terminal peptide of Rpt2 greatly enhanced this effect, suggesting that proteasome activation is a multistate process. Rpt2 and Rpt5 COOH-terminal peptides cross-linked to different but specific subunits of the 20 S proteasome. These results reveal critical roles of COOH termini of Rpt subunits of PA700 in the assembly and activation of eukaryotic 26 S proteasome. Moreover, they support a model in which Rpt subunits bind to dedicated sites on the proteasome and play specific, nonequivalent roles in the asymmetric assembly and activation of the 26 S proteasome.

  4. Large-scale parallel genome assembler over cloud computing environment. (United States)

    Das, Arghya Kusum; Koppa, Praveen Kumar; Goswami, Sayan; Platania, Richard; Park, Seung-Jong


    The size of high throughput DNA sequencing data has already reached the terabyte scale. To manage this huge volume of data, many downstream sequencing applications started using locality-based computing over different cloud infrastructures to take advantage of elastic (pay as you go) resources at a lower cost. However, the locality-based programming model (e.g. MapReduce) is relatively new. Consequently, developing scalable data-intensive bioinformatics applications using this model and understanding the hardware environment that these applications require for good performance, both require further research. In this paper, we present a de Bruijn graph oriented Parallel Giraph-based Genome Assembler (GiGA), as well as the hardware platform required for its optimal performance. GiGA uses the power of Hadoop (MapReduce) and Giraph (large-scale graph analysis) to achieve high scalability over hundreds of compute nodes by collocating the computation and data. GiGA achieves significantly higher scalability with competitive assembly quality compared to contemporary parallel assemblers (e.g. ABySS and Contrail) over traditional HPC cluster. Moreover, we show that the performance of GiGA is significantly improved by using an SSD-based private cloud infrastructure over traditional HPC cluster. We observe that the performance of GiGA on 256 cores of this SSD-based cloud infrastructure closely matches that of 512 cores of traditional HPC cluster.

  5. Interdependence of Pes1, Bop1, and WDR12 controls nucleolar localization and assembly of the PeBoW complex required for maturation of the 60S ribosomal subunit. (United States)

    Rohrmoser, Michaela; Hölzel, Michael; Grimm, Thomas; Malamoussi, Anastassia; Harasim, Thomas; Orban, Mathias; Pfisterer, Iris; Gruber-Eber, Anita; Kremmer, Elisabeth; Eick, Dirk


    The PeBoW complex is essential for cell proliferation and maturation of the large ribosomal subunit in mammalian cells. Here we examined the role of PeBoW-specific proteins Pes1, Bop1, and WDR12 in complex assembly and stability, nucleolar transport, and pre-ribosome association. Recombinant expression of the three subunits is sufficient for complex formation. The stability of all three subunits strongly increases upon incorporation into the complex. Only overexpression of Bop1 inhibits cell proliferation and rRNA processing, and its negative effects could be rescued by coexpression of WDR12, but not Pes1. Elevated levels of Bop1 induce Bop1/WDR12 and Bop1/Pes1 subcomplexes. Knockdown of Bop1 abolishes the copurification of Pes1 with WDR12, demonstrating Bop1 as the integral component of the complex. Overexpressed Bop1 substitutes for endogenous Bop1 in PeBoW complex assembly, leading to the instability of endogenous Bop1. Finally, indirect immunofluorescence, cell fractionation, and sucrose gradient centrifugation experiments indicate that transport of Bop1 from the cytoplasm to the nucleolus is Pes1 dependent, while Pes1 can migrate to the nucleolus and bind to preribosomal particles independently of Bop1. We conclude that the assembly and integrity of the PeBoW complex are highly sensitive to changes in Bop1 protein levels.

  6. Large organized surface domains self-assembled from nonpolar amphiphiles. (United States)

    Krafft, Marie Pierre


    For years, researchers had presumed that Langmuir monolayers of small C(n)F(2n+1)C(m)H(2m+1) (FnHm) diblock molecules (such as F8H16) consisted of continuous, featureless films. Recently we have discovered that they instead form ordered arrays of unusually large (~30-60 nm), discrete self-assembled surface domains or hemimicelles both at the surface of water and on solid substrates. These surface micelles differ in several essential ways from all previously reported or predicted molecular surface aggregates. They self-assemble spontaneously, even at zero surface pressure, depending solely on a critical surface concentration. They are very large (~100 times the length of the diblock) and involve thousands of molecules (orders of magnitude more than classical micelles). At the same time, the surface micelles are highly monodisperse and self-organize in close-packed hexagonal patterns (two-dimensional crystals). Their size is essentially independent from pressure, and they do not coalesce and are unexpectedly sturdy for soft matter (persisting even beyond surface film collapse). We and other researchers have observed large surface micelles for numerous diblocks, using Langmuir-Blodgett (LB) transfer, spin-coating and dip-coating techniques, or expulsion from mixed monolayers, and on diverse supports, establishing that hemimicelle formation and ordering are intrinsic properties of (perfluoroalkyl)alkanes. Notably, they involve "incomplete" surfactants with limited amphiphilic character, which further illustrates the outstanding capacity for perfluoroalkyl chains to promote self-assembly and interfacial film structuring. Using X-ray reflectivity, we determined a perfluoroalkyl-chain-up orientation. Theoretical investigations assigned self-assembly and hemimicelle stability to electrostatic dipole-dipole interactions at the interface between Fn- and Hm-sublayers. Grazing-incidence small-angle X-ray scattering (GISAXS) data collected directly on the surface of water

  7. 13-Deoxytedanolide, a marine sponge-derived antitumor macrolide, binds to the 60S large ribosomal subunit. (United States)

    Nishimura, Shinichi; Matsunaga, Shigeki; Yoshida, Minoru; Hirota, Hiroshi; Yokoyama, Shigeyuki; Fusetani, Nobuhiro


    13-Deoxytedanolide is a potent antitumor macrolide isolated from the marine sponge Mycale adhaerens. In spite of its remarkable activity, the mode of action of 13-deoxytedanolide has not been elucidated. [11-3H]-(11S)-13-Deoxydihydrotedanolide derived from the macrolide was used for identifying the target molecule from the yeast cell lysate. Fractionation of the binding protein revealed that the labeled 13-deoxytedanolide derivative strongly bound to the 80S ribosome as well as to the 60S large subunit, but not to the 40S small subunit. In agreement with this observation, 13-deoxytedanolide efficiently inhibited the polypeptide elongation. Interestingly, competition studies demonstrated that 13-deoxytedanolide shared the binding site on the 60S large subunit with pederin and its marine-derived analogues. These results indicate that 13-deoxytedanolide is a potent protein synthesis inhibitor and is the first macrolide to inhibit the eukaryotic ribosome.

  8. Degradation of the Large Subunit of Ribulose-1, 5-Bisphosphate Carboxylase/Oxygenase in Wheat Leaves

    Institute of Scientific and Technical Information of China (English)

    Lie-Feng ZHANG; Qi RUI; Lang-Lai XU


    The degradation of the large subunit (LSU) of ribulose- 1, 5-bisphosphate carboxylase/oxygenase (Rubisco; EC in wheat (Triticum aestivum L. cv. Yangmai 158) leaves was investigated. A 50 kDa fragment, a portion of the LSU of Rubisco, was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with antibody against tobacco Rubisco in crude enzyme extract of young wheat leaves. The appearance of the 50 kDa fragment was most obvious at 30-35 ℃ and pH 5.5. The LSU and its 50 kDa fragment both existed when the crude enzyme extract was incubated for 60 min. The amount of LSU decreased with incubation time from 0 to 3 h in crude enzyme extract. However, the 50 kDa fragment could not be found any pH from 4.5 to 8.5 in chloroplast lysates of young wheat leaves. In addition,through treatment with various inhibitors, reactions were inhibited by cysteine proteinase inhibitor E-64 or leupeptin.

  9. Combined large and small subunit ribosomal RNA phylogenies support a basal position of the acoelomorph flatworms. (United States)

    Telford, Maximilian J; Lockyer, Anne E; Cartwright-Finch, Chloë; Littlewood, D Timothy J


    The phylogenetic position of the phylum Platyhelminthes has been re-evaluated in the past decade by analysis of diverse molecular datasets. The consensus is that the Rhabditophora + Catenulida, which includes most of the flatworm taxa, are not primitively simple basal bilaterians but are related to coelomate phyla such as molluscs. The status of two other groups of acoelomate worms, Acoela and Nemertodermatida, is less clear. Although many characteristics unite these two groups, initial molecular phylogenetic studies placed the Nemertodermatida within the Rhabditophora, but placed the Acoela at the base of the Bilateria, distant from other flatworms. This contradiction resulted in scepticism about the basal position of acoels and led to calls for further data. We have sequenced large subunit ribosomal RNA genes from 13 rhabditophorans + catenulids, three acoels and one nemertodermatid, tripling the available data. Our analyses strongly support a basal position of both acoels and nemertodermatids. Alternative hypotheses are significantly less well supported by the data. We conclude that the Nemertodermatida and Acoela are basal bilaterians and, owing to their unique body plan and embryogenesis, should be recognized as a separate phylum, the Acoelomorpha.

  10. Generation of an attenuated Tiantan vaccinia virus by deletion of the ribonucleotide reductase large subunit. (United States)

    Kan, Shifu; Jia, Peng; Sun, Lili; Hu, Ningning; Li, Chang; Lu, Huijun; Tian, Mingyao; Qi, Yanxin; Jin, Ningyi; Li, Xiao


    Attenuation of the virulence of vaccinia Tiantan virus (VTT) underlies the strategy adopted for mass vaccination campaigns. This strategy provides advantages of safety and efficacy over traditional vaccines and is aimed at minimization of adverse health effects. In this study, a mutant form of the virus, MVTT was derived from VTT by deletion of the ribonucleotide reductase large subunit (R1) (TI4L). Compared to wild-type parental (VTT) and revertant (VTT-rev) viruses, virulence of the mutant MVTT was reduced by 100-fold based on body weight reduction and by 3,200-fold based on determination of the intracranial 50% lethal infectious dose. However, the immunogenicity of MVTT was equivalent to that of the parental VTT. We also demonstrated that the TI4L gene is not required for efficient replication. These data support the conclusion that MVTT can be used as a replicating virus vector or as a platform for the development of vaccines against infectious diseases and for cancer therapy.

  11. Unexpected Diagnosis of Cerebral Toxoplasmosis by 16S and D2 Large-Subunit Ribosomal DNA PCR and Sequencing

    DEFF Research Database (Denmark)

    Kruse, Alexandra Yasmin Collin; Kvich, Lasse Andersson; Eickhardt-Dalbøge, Steffen Robert;


    The protozoan parasite Toxoplasma gondii causes severe opportunistic infections. Here, we report an unexpected diagnosis of cerebral toxoplasmosis. T. gondii was diagnosed by 16S and D2 large-subunit (LSU) ribosomal DNA (rDNA) sequencing of a cerebral biopsy specimen and confirmed by T. gondii......-specific PCR and immunohistochemistry. The patient was later diagnosed with HIV/AIDS....

  12. Construction of diverse supramolecular assemblies of dimetal subunits differing in coordinated water molecules via strong hydrogen bonding interactions: Synthesis, crystal structures and spectroscopic properties

    Indian Academy of Sciences (India)

    Sadhika Khullar; Sanjay K Mandal


    Three new supramolecular assemblies (constructed through strong hydrogen bonding) of [Co2(bpta)2(adc)(H2O)4](ClO4)2.2H2O (1), [Cu2(bpta)2(fum)(H2O)2](ClO4)2 (2) and [Cu2(bpta)2(tdc)(H2O) (ClO4)](ClO4).3H2O (3), which are synthesised by one pot self-assembly of the metal salt, bpta ligand and the corresponding dicarboxylate under the same reaction conditions, are reported (where adc = acetylene dicarboxylate, fum = fumarate, tdc = 2,5-thiophenedicarboxylate and bpta = N,N'-bis(2-pyridylmethyl)-tertbutylamine). These compounds have varying degrees of coordinatedwater molecules per dimetal subunits (four for 1, two for 2 and one for 3, respectively). Furthermore, the orientation of the coordinated water molecules in 1 and 2, with respect to the mono (carboxylato)-bridged dimetal subunit, is different (cis and trans, respectively). On the other hand, there is a coordinated perchlorate ion in 3 making the two metal centers inequivalent. Unlike 1 and 3, there are no lattice water molecules in 2. This difference in the dimetal subunit in 1-3 and the presence or absence of the lattice water molecules are the keys to forming the diverse supramolecular assemblies. In 1 and 3, the involvement of lattice water molecules in the construction of such assemblies is distinctly different. In case of 2, the formation of supramolecular assembly depends on the coordinated water molecule (trans to each other) and thus a ladder shaped supramolecular assembly is the result. The strength of hydrogen bonding observed in the networks of 1-3 is indicated in the O…O distances (2.596 Å to 3.160 Å) and the OH…O angles 124° to 176°. All are characterised by elemental analysis, FTIR spectroscopy and single crystal X-ray diffraction studies.

  13. Development of a large area microstructure photomultiplier assembly (LAMPA) (United States)

    Clifford, E. T. H.; Dick, M.; Facina, M.; Wakeford, D.; Andrews, H. R.; Ing, H.; Best, D.; Baginski, M. J.


    Large area (> m2) position-sensitive readout of scintillators is important for passive/active gamma and neutron imaging for counter-terrorism applications. The goal of the LAMPA project is to provide a novel, affordable, large-area photodetector (8" x 8") by replacing the conventional dynodes of photomultiplier tubes (PMTs) with electron multiplier microstructure boards (MSBs) that can be produced using industrial manufacturing techniques. The square, planar format of the LAMPA assemblies enables tiling of multiple units to support large area applications. The LAMPA performance objectives include comparable gain, noise, timing, and energy resolution relative to conventional PMTs, as well as spatial resolution in the few mm range. The current LAMPA prototype is a stack of 8" x 8" MSBs made commercially by chemical etching of a molybdenum substrate and coated with hydrogen-terminated boron-doped diamond for high secondary emission yield (SEY). The layers of MSBs are electrically isolated using ceramic standoffs. Field-shaping grids are located between adjacent boards to achieve good transmission of electrons from one board to the next. The spacing between layers and the design of the microstructure pattern and grids were guided by simulations performed using an electro-optics code. A position sensitive anode board at the back of the stack of MSBs provides 2-D readout. This presentation discusses the trade studies performed in the design of the MSBs, the measurements of SEY from various electro-emissive materials, the electro-optics simulations conducted, the design of the 2-D readout, and the mechanical aspects of the LAMPA design, in order to achieve a gain of > 104 in an 8-stage stack of MSBs, suitable for use with various scintillators when coupled to an appropriate photocathode.

  14. Sequence Planning for On-Orbit Assembly of Large Space Truss Structures in a Multirobot Environment

    Institute of Scientific and Technical Information of China (English)

    GUO Jifeng; WANG Ping; CUI Naigang


    An approach to sequence planning for on-orbit assembly of large space truss structures in a multirobot environment is presented. A hierarchical representation of large space truss structures at the structural volume element level and strut level is adopted. The representation of connectivity matrix and directed graph is respectively presented at the strut level and SVE level. The multirobot environment that consists of autonomous space robots and struts is supposed. Then the multirobot serial assembly strategy, assembly states, assembly tasks and assembly sequences are described. The assembly sequence planning algorithms at the strut level and SVE level are respectively discussed. The results of the simulations show that this approach is feasible and efficient. Two extensions of this approach include more accurate assessment of the efficiency representation and improvements in planning algorithm. In the future, the assembly sequence planning of more large space truss structures and complex multirobot environments and assembly tasks will be considered.

  15. The catalytic properties of hybrid Rubisco comprising tobacco small and sunflower large subunits mirror the kinetically equivalent source Rubiscos and can support tobacco growth. (United States)

    Sharwood, Robert Edward; von Caemmerer, Susanne; Maliga, Pal; Whitney, Spencer Michael


    Plastomic replacement of the tobacco (Nicotiana tabacum) Rubisco large subunit gene (rbcL) with that from sunflower (Helianthus annuus; rbcL(S)) produced tobacco(Rst) transformants that produced a hybrid Rubisco consisting of sunflower large and tobacco small subunits (L(s)S(t)). The tobacco(Rst) plants required CO(2) (0.5% v/v) supplementation to grow autotrophically from seed despite the substrate saturated carboxylation rate, K(m), for CO(2) and CO(2)/O(2) selectivity of the L(s)S(t) enzyme mirroring the kinetically equivalent tobacco and sunflower Rubiscos. Consequently, at the onset of exponential growth when the source strength and leaf L(s)S(t) content were sufficient, tobacco(Rst) plants grew to maturity without CO(2) supplementation. When grown under a high pCO(2), the tobacco(Rst) seedlings grew slower than tobacco and exhibited unique growth phenotypes: Juvenile plants formed clusters of 10 to 20 structurally simple oblanceolate leaves, developed multiple apical meristems, and the mature leaves displayed marginal curling and dimpling. Depending on developmental stage, the L(s)S(t) content in tobacco(Rst) leaves was 4- to 7-fold less than tobacco, and gas exchange coupled with chlorophyll fluorescence showed that at 2 mbar pCO(2) and growth illumination CO(2) assimilation in mature tobacco(Rst) leaves remained limited by Rubisco activity and its rate (approximately 11 micromol m(-2) s(-1)) was half that of tobacco controls. (35)S-methionine labeling showed the stability of assembled L(s)S(t) was similar to tobacco Rubisco and measurements of light transient CO(2) assimilation rates showed L(s)S(t) was adequately regulated by tobacco Rubisco activase. We conclude limitations to tobacco(Rst) growth primarily stem from reduced rbcL(S) mRNA levels and the translation and/or assembly of sunflower large with the tobacco small subunits that restricted L(s)S(t) synthesis.

  16. Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit epsilon N-methyltransferase (United States)

    Houtz, Robert L.


    The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .sup..epsilon. N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the .epsilon.-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed.

  17. Assembly of the central domain of the 30S ribosomal subunit: roles for the primary binding ribosomal proteins S15 and S8. (United States)

    Jagannathan, Indu; Culver, Gloria M


    Assembly of the 30S ribosomal subunit occurs in a highly ordered and sequential manner. The ordered addition of ribosomal proteins to the growing ribonucleoprotein particle is initiated by the association of primary binding proteins. These proteins bind specifically and independently to 16S ribosomal RNA (rRNA). Two primary binding proteins, S8 and S15, interact exclusively with the central domain of 16S rRNA. Binding of S15 to the central domain results in a conformational change in the RNA and is followed by the ordered assembly of the S6/S18 dimer, S11 and finally S21 to form the platform of the 30S subunit. In contrast, S8 is not part of this major platform assembly branch. Of the remaining central domain binding proteins, only S21 association is slightly dependent on S8. Thus, although S8 is a primary binding protein that extensively contacts the central domain, its role in assembly of this domain remains unclear. Here, we used directed hydroxyl radical probing from four unique positions on S15 to assess organization of the central domain of 16S rRNA as a consequence of S8 association. Hydroxyl radical probing of Fe(II)-S15/16S rRNA and Fe(II)-S15/S8/16S rRNA ribonucleoprotein particles reveal changes in the 16S rRNA environment of S15 upon addition of S8. These changes occur predominantly in helices 24 and 26 near previously identified S8 binding sites. These S8-dependent conformational changes are consistent with 16S rRNA folding in complete 30S subunits. Thus, while S8 binding is not absolutely required for assembly of the platform, it appears to affect significantly the 16S rRNA environment of S15 by influencing central domain organization.

  18. Programmed Nanomaterial Assemblies in Large Scales: Applications of Synthetic and Genetically- Engineered Peptides to Bridge Nano-Assemblies and Macro-Assemblies

    Energy Technology Data Exchange (ETDEWEB)

    Matsui, Hiroshi


    Work is reported in these areas: Large-scale & reconfigurable 3D structures of precise nanoparticle assemblies in self-assembled collagen peptide grids; Binary QD-Au NP 3D superlattices assembled with collagen-like peptides and energy transfer between QD and Au NP in 3D peptide frameworks; Catalytic peptides discovered by new hydrogel-based combinatorial phage display approach and their enzyme-mimicking 2D assembly; New autonomous motors of metal-organic frameworks (MOFs) powered by reorganization of self-assembled peptides at interfaces; Biomimetic assembly of proteins into microcapsules on oil-in-water droplets with structural reinforcement via biomolecular recognition-based cross-linking of surface peptides; and Biomimetic fabrication of strong freestanding genetically-engineered collagen peptide films reinforced by quantum dot joints. We gained the broad knowledge about biomimetic material assembly from nanoscale to microscale ranges by coassembling peptides and NPs via biomolecular recognition. We discovered: Genetically-engineered collagen-like peptides can be self-assembled with Au NPs to generate 3D superlattices in large volumes (> μm{sup 3}); The assembly of the 3D peptide-Au NP superstructures is dynamic and the interparticle distance changes with assembly time as the reconfiguration of structure is triggered by pH change; QDs/NPs can be assembled with the peptide frameworks to generate 3D superlattices and these QDs/NPs can be electronically coupled for the efficient energy transfer; The controlled assembly of catalytic peptides mimicking the catalytic pocket of enzymes can catalyze chemical reactions with high selectivity; and, For the bacteria-mimicking swimmer fabrication, peptide-MOF superlattices can power translational and propellant motions by the reconfiguration of peptide assembly at the MOF-liquid interface.

  19. Construction and engineering of large biochemical pathways via DNA assembler. (United States)

    Shao, Zengyi; Zhao, Huimin


    DNA assembler enables rapid construction and engineering of biochemical pathways in a one-step fashion by exploitation of the in vivo homologous recombination mechanism in Saccharomyces cerevisiae. It has many applications in pathway engineering, metabolic engineering, combinatorial biology, and synthetic biology. Here we use two examples including the zeaxanthin biosynthetic pathway and the aureothin biosynthetic gene cluster to describe the key steps in the construction of pathways containing multiple genes using the DNA assembler approach. Methods for construct design, pathway assembly, pathway confirmation, and functional analysis are shown. The protocol for fine genetic modifications such as site-directed mutagenesis for engineering the aureothin gene cluster is also illustrated.

  20. Structural motifs and potential sigma homologies in the large subunit of human general transcription factor TFIIE. (United States)

    Ohkuma, Y; Sumimoto, H; Hoffmann, A; Shimasaki, S; Horikoshi, M; Roeder, R G


    The general transcription factor TFIIE has an essential role in eukaryotic transcription initiation together with RNA polymerase II and other general factors. Human TFIIE consists of two subunits of relative molecular mass 57,000 (TFIIE-alpha) and 34,000 (TFIIE-beta) and joins the preinitiation complex after RNA polymerase II and TFIIF. Here we report the cloning and structure of a complementary DNA encoding a functional human TFIIE-alpha. TFIIE-alpha is necessary for transcription initiation together with TFIIE-beta, and recombinant TFIIE-alpha can fully replace the natural subunit in an in vitro transcription assay. The sequence contains several interesting structural motifs (leucine repeat, zinc finger and helix-turn-helix) and sequence similarities to bacterial sigma factors that suggest direct involvement in the regulation of transcription initiation.

  1. The carB gene encoding the large subunit of carbamoylphosphate synthetase from Lactococcus lactis is transcribed monocistronically

    DEFF Research Database (Denmark)

    Martinussen, Jan; Hammer, Karin


    The biosynthesis of carbamoylphosphate is catalysed by the heterodimeric enzyme carbamoylphosphate synthetase (CPSase). The genes encoding the two subunits in procaryotes are normally transcribed as an operon, whereas in Lactococcus lactis, the gene encoding the large subunit (carB) is shown...... to be an isolated transcriptional unit. Carbamoylphosphate is a precursor in the biosynthesis of both pyrimidine nucleotides and arginine. By mutant analysis L. lactis is shown to possess only one carB gene; the same gene product is thus required for both biosynthetic pathways. Furthermore, arginine may satisfy...... the requirement for carbamoylphosphate in pyrimidine biosynthesis through degradation by the arginine deiminase pathway. The expression of the carB gene is subject to regulation at the level of transcription by pyrimidines most probably by an attenuator mechanism. Upstream of the carB gene, an open reading frame...

  2. Structure and expression analysis of genes encoding ADP-glucose pyrophosphorylase large subunit in wheat and its relatives. (United States)

    Zhang, Xiao-Wei; Li, Si-Yu; Zhang, Ling-Ling; Yang, Qiang; Jiang, Qian-Tao; Ma, Jian; Qi, Peng-Fei; Li, Wei; Chen, Guo-Yue; Lan, Xiu-Jin; Deng, Mei; Lu, Zhen-Xiang; Liu, Chunji; Wei, Yu-Ming; Zheng, You-Liang


    ADP-glucose pyrophosphorylase (AGP), which consists of two large subunits (AGP-L) and two small subunits (AGP-S), controls the rate-limiting step in the starch biosynthetic pathway. In this study, a full-length open reading frame (ORF) of AGP-L gene (named as Agp2) in wheat and a series of Agp2 gene sequences in wheat relatives were isolated. The coding region of Agp2 contained 15 exons and 14 introns including a full-length ORF of 1566 nucleotides, and the deduced protein contained 522 amino acids (57.8 kDa). Generally, the phylogenetic tree of Agp2 indicated that sequences from A- and D-genome donor species were most similar to each other and sequences from B-genome donor species contained more variation. Starch accumulation and Agp2 expression in wheat grains reached their peak at 21 and 15 days post anthesis (DPA), respectively.

  3. A separable domain of the p150 subunit of human chromatin assembly factor-1 promotes protein and chromosome associations with nucleoli. (United States)

    Smith, Corey L; Matheson, Timothy D; Trombly, Daniel J; Sun, Xiaoming; Campeau, Eric; Han, Xuemei; Yates, John R; Kaufman, Paul D


    Chromatin assembly factor-1 (CAF-1) is a three-subunit protein complex conserved throughout eukaryotes that deposits histones during DNA synthesis. Here we present a novel role for the human p150 subunit in regulating nucleolar macromolecular interactions. Acute depletion of p150 causes redistribution of multiple nucleolar proteins and reduces nucleolar association with several repetitive element-containing loci. Of note, a point mutation in a SUMO-interacting motif (SIM) within p150 abolishes nucleolar associations, whereas PCNA or HP1 interaction sites within p150 are not required for these interactions. In addition, acute depletion of SUMO-2 or the SUMO E2 ligase Ubc9 reduces α-satellite DNA association with nucleoli. The nucleolar functions of p150 are separable from its interactions with the other subunits of the CAF-1 complex because an N-terminal fragment of p150 (p150N) that cannot interact with other CAF-1 subunits is sufficient for maintaining nucleolar chromosome and protein associations. Therefore these data define novel functions for a separable domain of the p150 protein, regulating protein and DNA interactions at the nucleolus.

  4. Role of the low-molecular-weight subunits PetL, PetG, and PetN in assembly, stability, and dimerization of the cytochrome b6f complex in tobacco. (United States)

    Schwenkert, Serena; Legen, Julia; Takami, Tsuneaki; Shikanai, Toshiharu; Herrmann, Reinhold G; Meurer, Jörg


    The cytochrome b(6)f (Cyt b(6)f) complex in flowering plants contains nine conserved subunits, of which three, PetG, PetL, and PetN, are bitopic plastid-encoded low-molecular-weight proteins of largely unknown function. Homoplastomic knockout lines of the three genes have been generated in tobacco (Nicotiana tabacum 'Petit Havana') to analyze and compare their roles in assembly and stability of the complex. Deletion of petG or petN caused a bleached phenotype and loss of photosynthetic electron transport and photoautotrophy. Levels of all subunits that constitute the Cyt b(6)f complex were faintly detectable, indicating that both proteins are essential for the stability of the membrane complex. In contrast, DeltapetL plants accumulate about 50% of other Cyt b(6)f subunits, appear green, and grow photoautotrophically. However, DeltapetL plants show increased light sensitivity as compared to wild type. Assembly studies revealed that PetL is primarily required for proper conformation of the Rieske protein, leading to stability and formation of dimeric Cyt b(6)f complexes. Unlike wild type, phosphorylation levels of the outer antenna of photosystem II (PSII) are significantly decreased under state II conditions, although the plastoquinone pool is largely reduced in DeltapetL, as revealed by measurements of PSI and PSII redox states. This confirms the sensory role of the Cyt b(6)f complex in activation of the corresponding kinase. The reduced light-harvesting complex II phosphorylation did not affect state transition and association of light-harvesting complex II to PSI under state II conditions. Ferredoxin-dependent plastoquinone reduction, which functions in cyclic electron transport around PSI in vivo, was not impaired in DeltapetL.

  5. Role of the Low-Molecular-Weight Subunits PetL, PetG, and PetN in Assembly, Stability, and Dimerization of the Cytochrome b6f Complex in Tobacco1[C (United States)

    Schwenkert, Serena; Legen, Julia; Takami, Tsuneaki; Shikanai, Toshiharu; Herrmann, Reinhold G.; Meurer, Jörg


    The cytochrome b6f (Cyt b6f) complex in flowering plants contains nine conserved subunits, of which three, PetG, PetL, and PetN, are bitopic plastid-encoded low-molecular-weight proteins of largely unknown function. Homoplastomic knockout lines of the three genes have been generated in tobacco (Nicotiana tabacum ‘Petit Havana’) to analyze and compare their roles in assembly and stability of the complex. Deletion of petG or petN caused a bleached phenotype and loss of photosynthetic electron transport and photoautotrophy. Levels of all subunits that constitute the Cyt b6f complex were faintly detectable, indicating that both proteins are essential for the stability of the membrane complex. In contrast, ΔpetL plants accumulate about 50% of other Cyt b6f subunits, appear green, and grow photoautotrophically. However, ΔpetL plants show increased light sensitivity as compared to wild type. Assembly studies revealed that PetL is primarily required for proper conformation of the Rieske protein, leading to stability and formation of dimeric Cyt b6f complexes. Unlike wild type, phosphorylation levels of the outer antenna of photosystem II (PSII) are significantly decreased under state II conditions, although the plastoquinone pool is largely reduced in ΔpetL, as revealed by measurements of PSI and PSII redox states. This confirms the sensory role of the Cyt b6f complex in activation of the corresponding kinase. The reduced light-harvesting complex II phosphorylation did not affect state transition and association of light-harvesting complex II to PSI under state II conditions. Ferredoxin-dependent plastoquinone reduction, which functions in cyclic electron transport around PSI in vivo, was not impaired in ΔpetL. PMID:17556510

  6. AFM imaging reveals the assembly of a P2X receptor complex containing P2X2, P2X4 and P2X6 subunits



    Seven P2X purinergic receptor subunits have been identified: P2X1-P2X7. All except P2X6 assemble as homotrimers, and six heteromeric receptors (P2X1/2, P2X1/4, P2X1/5, P2X2/3, P2X2/6 and P2X4/6) have been described. In addition, P2X4 homomers associate with P2X2 or P2X7 homomers as dimers of trimers. The various P2X receptors show individual functional properties, suggesting distinct physiological roles. The overlapping expression of P2X2, P2X4 and P2X6 subunits has been shown in different ce...

  7. Isolated spinach ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit .sup..epsilon. N-methyltransferase and method of inactivating ribulose-1,5-bisphosphatase carboxylase/oxygenase large subunit .sup..epsilon. N-methyltransferase activity (United States)

    Houtz, Robert L.


    The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .sup..epsilon. N-methyltransferase (protein methylase III or Rubisco LSMT) from a plant which has a des(methyl) lysyl residue in the LS is disclosed. In addition, the full-length cDNA clones for Rubisco LSMT are disclosed. Transgenic plants and methods of producing same which have the Rubisco LSMT gene inserted into the DNA are also provided. Further, methods of inactivating the enzymatic activity of Rubisco LSMT are also disclosed.


    Directory of Open Access Journals (Sweden)

    Ahyar Ahmad


    Full Text Available Chromatin assembly factor-1 (CAF-1, a protein complex consisting of three subunits, p150, p60, and p48, is highly conserved from yeast to humans and facilitated nucleosome assembly of newly replicated DNA. The p48 subunit, CAF-1p48 (p48, with seven WD (Trp-Asp repeat motifs, is a member of the WD protein family. The immunoprecipitation experiment revealed that ß-propeller structure of p48 was less stringent for it's binding to HDAC-1, but more stringent for its binding to both histones H4 and CAF-1p60 but not to ASF-1, indicating that the proper ß-propeller structure of p48 is essential for the binding to these two proteins histone H4 and CAF-1p60. Complementation experiments, involving missense and truncated mutants of FLAG-tagged p48, revealed that mutations of every of seven WD dipeptide motifs, like both the N-terminal and C-terminal truncated mutations, could not rescue for the tet-induced lethality. These results indicate not only that p48 is essential for the viability of vertebrate cells, although the yeast p48 homolog is nonessential, but also that all the seven WD dipeptide motifs are necessary for the maintenance of the proper structure of p48 that is fundamentally important for cell viability.   Keywords: Chromatin assembly factor-1, complementation experiments, viability

  9. Promoter Structure of the RNA Polymerase II Large Subunit Gene in Caenorhabditis elegans and C. briggsae. (United States)

    Bird, D M; Kaloshian, I; Molinari, S


    The 5'-end of the Caenorhabditis elegans ama-1 gene transcript, which encodes the largest subunit of RNA polymerase II, was cloned. Sequencing revealed that the message is trans-spliced. To characterize the Ce-ama-1 promoter, DNA sequence spanning 3 kb upstream from the initiation codon was determined. Typical elements, such as TATA and Spl sites, were absent. The homologue of ama-1 in C. briggsae, Cb-ama-1, was isolated and its 5' flanking sequence compared with that of Ce-ama-1, revealing only limited similarity, although both sequences included a potential initiator-class transcriptional regulator and phased repeats of an ATC motif. The latter elements are postulated to facilitate DNA bending and may play a role in transcription regulation.

  10. Requirement of subunit co-assembly and ankyrin-G for M-channel localization at the axon initial segment

    DEFF Research Database (Denmark)

    Rasmussen, Hanne B; Frøkjaer-Jensen, Christian; Jensen, Camilla Stampe;


    The potassium channel subunits KCNQ2 and KCNQ3 are believed to underlie the M current of hippocampal neurons. The M-type potassium current plays a key role in the regulation of neuronal excitability; however, the subcellular location of the ion channels underlying this regulation has been...... controversial. We report here that KCNQ2 and KCNQ3 subunits are localized to the axon initial segment of pyramidal neurons of adult rat hippocampus and in cultured hippocampal neurons. We demonstrate that the localization of the KCNQ2/3 channel complex to the axon initial segment is favored by co......-expression of the two channel subunits. Deletion of the ankyrin-G-binding motif in both the KCNQ2 and KCNQ3 C-terminals leads to the disappearance of the complex from the axon initial segment, albeit the channel complex remains functional and still reaches the plasma membrane. We further show that although heteromeric...

  11. The small and large subunits of carbamoyl-phosphate synthase exhibit diverse contributions to pathogenicity in Xanthomonas citri subsp. citri

    Institute of Scientific and Technical Information of China (English)

    Guo Jing; SonG Xue; Zou Li-fang; Zou Hua-song; CHen Gong-you


    Carbamoyl-phosphate synthase plays a vital role in the carbon and nitrogen metabolism cycles. In Xanthomonas citri subsp. citri, carA and carB encode the smal and large subunits of carbamoyl-phosphate synthase, respectively. The deletion mutation of the coding regions revealed that carA did not affect any of the phenotypes, while carB played multiple roles in pathogenicity. The deletion of carB rendered the loss of pathogenicity in host plants and the ability to induce a hyper-sensitive reaction in the non-hosts. Quantitative reverse transcription-PCR assays indicated that 11 hrp genes coding the type III secretion system were suppressed when interacting with citrus plants. The mutation in carB also affected bacterial utilization of several carbon and nitrogen resources in minimal medium MMX and extracel ular enzyme activities. These data demonstrated that only the large subunit of carbamoyl-phosphate synthase was essential for canker development by X. citri subsp. citri.

  12. Large Scale Assembly and Characterization of BI-2223 HTS Conductors

    CERN Document Server

    Ballarino, Amalia; Mathot, Serge; Taylor, T; Brambilla, R


    The powering of the LHC machine requires more than 1000 High Temperature Superconducting (HTS) current leads. These leads contain, at their cold end, HTS conductors made of stacks of Bi-2223 tape with gold-doped silver matrix. CERN specified and purchased 31 km of this material, which was delivered on spools in unit lengths of 100 to 300 m. On reception the tape was inspected, cut into short length and vacuum soldered into stacks. All stacks were characterized in liquid nitrogen using a measuring procedure and set-up specifically developed for this purpose. Contact resistance values and critical currents at different electric field criteria were measured, from which the n-values have been extrapolated from the experimental V-I characteristics. This paper reports on the assembly and electrical characterization (up to 800 A) of more than ten thousand Bi-2223 stacks. Three types of stack were made from tape from two manufacturers. The assembly and soldering procedures and the set-up for the series electrical cha...

  13. Characterization of cDNA for the large subunit of the transcription initiation factor TFIIF. (United States)

    Aso, T; Vasavada, H A; Kawaguchi, T; Germino, F J; Ganguly, S; Kitajima, S; Weissman, S M; Yasukochi, Y


    At least six chromatographically resolvable general transcription factors may participate in accurate initiation by RNA polymerase II in HeLa cell-derived systems. TFIIF (also termed FC, RAP30/74 and beta/gamma) can bind directly to RNA polymerase II in solution and decrease the affinity of RNA polymerase II for nonspecific DNA. From studies on the kinetics of transcription initiation, on the composition of transcription initiation complexes fractionated by acrylamide gel electrophoresis, and on template competition experiments, TFIIF is known to act at an intermediate stage in initiation complex formation. It acts after TFIID firmly associates with DNA, but coincidentally with or immediately after RNA polymerase II binding to DNA, and before the recruitment of factor TFIIE. TFIIF may or may not have DNA helicase activity. The small subunit (RAP30) of TFIIF has been cloned and shows some amino-acid sequence homology to bacterial sigma factors. We have partially sequenced the RAP74 protein from purified HeLa cells, cloned its complementary DNA and shown that its translation product can interact with RAP30 in vitro as well as in vivo. The cDNA predicts an amino-acid sequence that lacks obvious DNA or RNA helicase motifs. It has regions rich in charged amino acids, including segments containing a higher content of acidic amino acids than are found in strong transcriptional activators such as VP16.

  14. Parallelized short read assembly of large genomes using de Bruijn graphs

    Directory of Open Access Journals (Sweden)

    Liu Yongchao


    Full Text Available Abstract Background Next-generation sequencing technologies have given rise to the explosive increase in DNA sequencing throughput, and have promoted the recent development of de novo short read assemblers. However, existing assemblers require high execution times and a large amount of compute resources to assemble large genomes from quantities of short reads. Results We present PASHA, a parallelized short read assembler using de Bruijn graphs, which takes advantage of hybrid computing architectures consisting of both shared-memory multi-core CPUs and distributed-memory compute clusters to gain efficiency and scalability. Evaluation using three small-scale real paired-end datasets shows that PASHA is able to produce more contiguous high-quality assemblies in shorter time compared to three leading assemblers: Velvet, ABySS and SOAPdenovo. PASHA's scalability for large genome datasets is demonstrated with human genome assembly. Compared to ABySS, PASHA achieves competitive assembly quality with faster execution speed on the same compute resources, yielding an NG50 contig size of 503 with the longest correct contig size of 18,252, and an NG50 scaffold size of 2,294. Moreover, the human assembly is completed in about 21 hours with only modest compute resources. Conclusions Developing parallel assemblers for large genomes has been garnering significant research efforts due to the explosive size growth of high-throughput short read datasets. By employing hybrid parallelism consisting of multi-threading on multi-core CPUs and message passing on compute clusters, PASHA is able to assemble the human genome with high quality and in reasonable time using modest compute resources.

  15. RelA NF-κB subunit activation as a therapeutic target in diffuse large B-cell lymphoma (United States)

    Manyam, Ganiraju C.; Visco, Carlo; Tzankov, Alexandar; Wang, Jing; Montes-Moreno, Santiago; Dybkaer, Karen; Chiu, April; Orazi, Attilio; Zu, Youli; Bhagat, Govind; Richards, Kristy L.; Hsi, Eric D.; Choi, William W.L.; Han van Krieken, J.; Huh, Jooryung; Ponzoni, Maurilio; Ferreri, Andrés J.M.; Møller, Michael B.; Parsons, Ben M.; Winter, Jane N.; Piris, Miguel A.; Jeffrey Medeiros, L.; Pham, Lan V.; Young, Ken H.


    It has been well established that nuclear factor kappa-B (NF-κB) activation is important for tumor cell growth and survival. RelA/p65 and p50 are the most common NF-κB subunits and involved in the classical NF-κB pathway. However, the prognostic and biological significance of RelA/p65 is equivocal in the field. In this study, we assessed RelA/p65 nuclear expression by immunohistochemistry in 487 patients with de novo diffuse large B-cell lymphoma (DLBCL), and studied the effects of molecular and pharmacological inhibition of NF-κB on cell viability. We found RelA/p65 nuclear expression, without associations with other apparent genetic or phenotypic abnormalities, had unfavorable prognostic impact in patients with stage I/II DLBCL. Gene expressionprofiling analysis suggested immune dysregulation and antiapoptosis may be relevant for the poorer prognosis associated with p65 hyperactivation in germinal center B-cell–like (GCB) DLBCL and in activated B-cell–like (ABC) DLBCL, respectively. We knocked down individual NF-κB subunits in representative DLBCL cells in vitro, and found targeting p65 was more effective than targeting other NF-κB subunits in inhibiting cell growth and survival. In summary, RelA/p65 nuclear overexpression correlates with significant poor survival in early-stage DLBCL patients, and therapeutic targeting RelA/p65 is effective in inhibiting proliferation and survival of DLBCL with NF-κB hyperactivation. PMID:27941215

  16. Large Conductance Ca2+-Activated K+ Channel (BKCa) α-Subunit Splice Variants in Resistance Arteries from Rat Cerebral and Skeletal Muscle Vasculature


    Zahra Nourian; Min Li; M Dennis Leo; Jaggar, Jonathan H.; Braun, Andrew P.; Hill, Michael A.


    Previous studies report functional differences in large conductance Ca2+ activated-K+ channels (BKCa) of smooth muscle cells (VSMC) from rat cerebral and cremaster muscle resistance arteries. The present studies aimed to determine if this complexity in BKCa activity may, in part, be due to splice variants in the pore-forming α-subunit. BKCa variants in the intracellular C terminus of the α-subunit, and their relative expression to total α-subunit, were examined by qPCR. Sequencing of RT-PCR p...

  17. Evaluating hypotheses of basal animal phylogeny using complete sequences of large and small subunit rRNA

    Energy Technology Data Exchange (ETDEWEB)

    Medina, Monica; Collins, Allen G.; Silberman, Jeffrey; Sogin, Mitchell L.


    We studied the evolutionary relationships among basal metazoan lineages by using complete large subunit (LSU) and small subunit (SSU) ribosomal RNA sequences for 23 taxa. After identifying competing hypotheses, we performed maximum likelihood searches for trees conforming to each hypothesis. Kishino-Hasegawa tests were used to determine whether the data (LSU, SSU, and combined) reject any of the competing hypotheses. We also conducted unconstrained tree searches, compared the resulting topologies, and calculated bootstrap indices. Shimodaira-Hasegawa tests were applied to determine whether the data reject any of the topologies resulting from the constrained and unconstrained tree searches. LSU, SSU, and the combined data strongly contradict two assertions pertaining to sponge phylogeny. Hexactinellid sponges are not likely to be the basal lineage of amonophyletic Porifera or the sister group to all other animals. Instead, Hexactinellida and Demospongia form a well-supported clade of siliceous sponges, Silicea. It remains unclear, on the basis of these data alone, whether the calcarean sponges are more closely related to Silicea or to nonsponge animals. The SSU and combined data reject the hypothesis that Bilateria is more closely related to Ctenophora than it is to Cnidaria, whereas LSU data alone do not refute either hypothesis. LSU and SSU data agree in supporting the monophyly of Bilateria, Cnidaria, Ctenophora, and Metazoa. LSU sequence data reveal phylogenetic structure in a data set with limited taxon sampling. Continued accumulation of LSU sequences should increase our understanding of animal phylogeny.

  18. The carB Gene Encoding the Large Subunit of Carbamoylphosphate Synthetase from Lactococcus lactis Is Transcribed Monocistronically (United States)

    Martinussen, Jan; Hammer, Karin


    The biosynthesis of carbamoylphosphate is catalyzed by the heterodimeric enzyme carbamoylphosphate synthetase. The genes encoding the two subunits of this enzyme in procaryotes are normally transcribed as an operon, but the gene encoding the large subunit (carB) in Lactococcus lactis is shown to be transcribed as an isolated unit. Carbamoylphosphate is a precursor in the biosynthesis of both pyrimidine nucleotides and arginine. By mutant analysis, L. lactis is shown to possess only one carB gene; the same gene product is thus required for both biosynthetic pathways. Furthermore, arginine may satisfy the requirement for carbamoylphosphate in pyrimidine biosynthesis through degradation by means of the arginine deiminase pathway. The expression of the carB gene is subject to regulation at the level of transcription by pyrimidines, most probably by an attenuator mechanism. Upstream of the carB gene, an open reading frame showing a high degree of similarity to those of glutathione peroxidases from other organisms was identified. PMID:9721272

  19. Preparation of Polyclonal Antibodies of Rubisco Large and Small Subunits and Their Application in the Functional Analysis of the Genes

    Institute of Scientific and Technical Information of China (English)

    Peng-Da MA; Tian-Cheng LU; Xiao-Fu ZHOU; Xiao-Juan ZHU; Xing-Zhi WANG


    Spinach Rubisco (ribulose-l,5-bisphosphate carboxylase/oxygenase) large (rbcL) and small (rbcS) subunits were separated by SDS-PAGE, and protein amount and purity were determined by Bradford assay. Polyclonal antibodies against rbcL and rbcS subunit were generated in female BALB/c mice and had no cross-reaction with each other. A total of 81 μg antigens were used and 0.3 ml anti-sera with titer of 1:5000were yielded. The antibodies were also applicable to study rbcL and rbcS in tobacco plant Nicotiana benthamiana. Potato virus X vector pGR107 induced silencing of rbcS gene by Agrobacterium in Nicotiana benthaniana was performed. The expression level ofrbcL and rbcS was lower in rbcS silenced plants than that in control plants as detected by the corresponding antibodies. This implied that the expression of rbcL was regulated by rbcS.

  20. G protein betagamma subunits interact with alphabeta- and gamma-tubulin and play a role in microtubule assembly in PC12 cells. (United States)

    Montoya, Valentina; Gutierrez, Christina; Najera, Omar; Leony, Denisse; Varela-Ramirez, Armando; Popova, Juliana; Rasenick, Mark M; Das, Siddhartha; Roychowdhury, Sukla


    The betagamma subunit of G proteins (Gbetagamma) is known to transfer signals from cell surface receptors to intracellular effector molecules. Recent results suggest that Gbetagamma also interacts with microtubules and is involved in the regulation of the mitotic spindle. In the current study, the anti-microtubular drug nocodazole was employed to investigate the mechanism by which Gbetagamma interacts with tubulin and its possible implications in microtubule assembly in cultured PC12 cells. Nocodazole-induced depolymerization of microtubules drastically inhibited the interaction between Gbetagamma and tubulin. Gbetagamma was preferentially bound to microtubules and treatment with nocodazole suggested that the dissociation of Gbetagamma from microtubules is an early step in the depolymerization process. When microtubules were allowed to recover after removal of nocodazole, the tubulin-Gbetagamma interaction was restored. Unlike Gbetagamma, however, the interaction between tubulin and the alpha subunit of the Gs protein (Gsalpha) was not inhibited by nocodazole, indicating that the inhibition of tubulin-Gbetagamma interactions during microtubule depolymerization is selective. We found that Gbetagamma also interacts with gamma-tubulin, colocalizes with gamma-tubulin in centrosomes, and co-sediments in centrosomal fractions. The interaction between Gbetagamma and gamma-tubulin was unaffected by nocodazole, suggesting that the Gbetagamma-gamma-tubulin interaction is not dependent on assembled microtubules. Taken together, our results suggest that Gbetagamma may play an important and definitive role in microtubule assembly and/or stability. We propose that betagamma-microtubule interaction is an important step for G protein-mediated cell activation. These results may also provide new insights into the mechanism of action of anti-microtubule drugs.

  1. A preliminary phylogenetic analysis of the Capsalidae (Platyhelminthes: Monogenea: Monopisthocotylea) inferred from large subunit rDNA sequences. (United States)

    Whittington, I D; Deveney, M R; Morgan, J A T; Chisholm, L A; Adlard, R D


    Phylogenetic relationships within the Capsalidae (Monogenea) were examined using large subunit ribosomal DNA sequences from 17 capsalid species (representing 7 genera, 5 subfamilies), 2 outgroup taxa (Monocotylidae) plus Udonella caligorum (Udonellidae). Trees were constructed using maximum likelihood, minimum evolution and maximum parsimony algorithms. An initial tree, generated from sequences 315 bases long, suggests that Capsalinae, Encotyllabinae, Entobdellinae and Trochopodinae are monophyletic, but that Benedeniinae is paraphyletic. Analyses indicate that Neobenedenia, currently in the Benedeniinae, should perhaps be placed in a separate subfamily. An additional analysis was made which omitted 3 capsalid taxa (for which only short sequences were available) and all outgroup taxa because of alignment difficulties. Sequence length increased to 693 bases and good branch support was achieved. The Benedeniinae was again paraphyletic. Higher-level classification of the Capsalidae, evolution of the Entobdellinae and issues of species identity in Neobenedenia are discussed.

  2. Elucidating the mechanisms of assembly and subunit interaction of the cellulose synthase complex of Arabidopsis secondary cell walls. (United States)

    Atanassov, Ivan I; Pittman, Jon K; Turner, Simon R


    Cellulose is the most abundant biopolymer in nature; however, questions relating to the biochemistry of its synthesis including the structure of the cellulose synthase complex (CSC) can only be answered by the purification of a fully functional complex. Despite its importance, this goal remains elusive. The work described here utilizes epitope tagging of cellulose synthase A (CESA) proteins that are known components of the CSC. To avoid problems associated with preferential purification of CESA monomers, we developed a strategy based on dual epitope tagging of the CESA7 protein to select for CESA multimers. With this approach, we used a two-step purification that preferentially selected for larger CESA oligomers. These preparations consisted solely of the three known secondary cell wall CESA proteins CESA4, CESA7, and CESA8. No additional CESA isoforms or other proteins were identified. The data are consistent with a model in which CESA protein homodimerization occurs prior to formation of larger CESA oligomers. This suggests that the three different CESA proteins undergo dimerization independently, but the presence of all three subunits is required for higher order oligomerization. Analysis of purified CESA complex and crude extracts suggests that disulfide bonds and noncovalent interactions contribute to the stability of the CESA subunit interactions. These results demonstrate that this approach will provide an excellent framework for future detailed analysis of the CSC.

  3. Hierarchical assembly may be a way to make large information-rich structures

    CERN Document Server

    Whitelam, Stephen


    Self-assembly in the laboratory can now yield `information-rich' nanostructures in which each component is of a distinct type and has a defined spatial position. Ensuring the thermodynamic stability of such structures requires inter-component interaction energies to increase logarithmically with structure size, in order to counter the entropy gained upon mixing component types in solution. However, self-assembly in the presence of strong interactions results in general in kinetic trapping, so suggesting a limit to the size of an (equilibrium) structure that can be self-assembled from distinguishable components. Here we study numerically a two-dimensional hierarchical assembly scheme already considered in experiment. We show that this scheme is immune to the kinetic traps associated with strong `native' interactions (interactions designed to stabilize the intended structure), and so, in principle, offers a way to make large information-rich structures. In this scheme the size of an assembled structure scales e...

  4. Pharmacological characterisation of α6β4* nicotinic acetylcholine receptors assembled from three different α6/α3 subunit chimeras in tsA201 cells

    DEFF Research Database (Denmark)

    Jensen, Anne Bjørnskov; Hoestgaard-Jensen, Kirsten; Jensen, Anders A.


    by their inefficient functional expression in vitro. In the present study we have characterized and compared the pharmacological properties displayed by α6β4 and α6β4β3 nicotinic acetylcholine receptors assembled in tsA201 cells from the classical α6/α3 chimera (C1) and two novel α6/α3 chimeras (C6F223L and C16F223L...... should be made keeping the molecular modifications in the α6 surrogate subunits in mind, this study sheds light on the pharmacological properties of α6β4⁎ nicotinic acetylcholine receptors and demonstrates the applicability of the C6F223L and C16F223L chimeras for studies of these receptors....

  5. Rrp12 and the Exportin Crm1 participate in late assembly events in the nucleolus during 40S ribosomal subunit biogenesis. (United States)

    Moriggi, Giulia; Nieto, Blanca; Dosil, Mercedes


    During the biogenesis of small ribosomal subunits in eukaryotes, the pre-40S particles formed in the nucleolus are rapidly transported to the cytoplasm. The mechanisms underlying the nuclear export of these particles and its coordination with other biogenesis steps are mostly unknown. Here we show that yeast Rrp12 is required for the exit of pre-40S particles to the cytoplasm and for proper maturation dynamics of upstream 90S pre-ribosomes. Due to this, in vivo elimination of Rrp12 leads to an accumulation of nucleoplasmic 90S to pre-40S transitional particles, abnormal 35S pre-rRNA processing, delayed elimination of processing byproducts, and no export of intermediate pre-40S complexes. The exportin Crm1 is also required for the same pre-ribosome maturation events that involve Rrp12. Thus, in addition to their implication in nuclear export, Rrp12 and Crm1 participate in earlier biosynthetic steps that take place in the nucleolus. Our results indicate that, in the 40S subunit synthesis pathway, the completion of early pre-40S particle assembly, the initiation of byproduct degradation and the priming for nuclear export occur in an integrated manner in late 90S pre-ribosomes.

  6. The human Arp2/3 complex is composed of evolutionarily conserved subunits and is localized to cellular regions of dynamic actin filament assembly. (United States)

    Welch, M D; DePace, A H; Verma, S; Iwamatsu, A; Mitchison, T J


    The Arp2/3 protein complex has been implicated in the control of actin polymerization in cells. The human complex consists of seven subunits which include the actin related proteins Arp2 and Arp3, and five others referred to as p41-Arc, p34-Arc, p21-Arc, p20-Arc, and p16-Arc (p omplex). We have determined the predicted amino acid sequence of all seven subunits. Each has homologues in diverse eukaryotes, implying that the structure and function of the complex has been conserved through evolution. Human Arp2 and Arp3 are very similar to family members from other species. p41-Arc is a new member of the Sop2 family of WD (tryptophan and aspartate) repeat-containing proteins and may be posttranslationally modified, suggesting that it may be involved in regulating the activity and/or localization of the complex. p34-Arc, p21-Arc, p20-Arc, and p16-Arc define novel protein families. We sought to evaluate the function of the Arp2/3 complex in cells by determining its intracellular distribution. Arp3, p34-Arc, and p21-Arc were localized to the lamellipodia of stationary and locomoting fibroblasts, as well to Listeria monocytogenes assembled actin tails. They were not detected in cellular bundles of actin filaments. Taken together with the ability of the Arp2/3 complex to induce actin polymerization, these observations suggest that the complex promotes actin assembly in lamellipodia and may participate in lamellipodial protrusion.

  7. Sequence diversity in the large subunit of RNA polymerase I contributes to Mefenoxam insensitivity in Phytophthora infestans. (United States)

    Randall, Eva; Young, Vanessa; Sierotzki, Helge; Scalliet, Gabriel; Birch, Paul R J; Cooke, David E L; Csukai, Michael; Whisson, Stephen C


    Phenylamide fungicides have been widely used for the control of oomycete-incited plant diseases for over 30 years. Insensitivity to this chemical class of fungicide was recorded early in its usage history, but the precise protein(s) conditioning insensitivity has proven difficult to determine. To determine the genetic basis of insensitivity and to inform strategies for the cloning of the gene(s) responsible, genetic crosses were established between Mefenoxam sensitive and intermediate insensitive isolates of Phytophthora infestans, the potato late blight pathogen. F1 progeny showed the expected semi-dominant phenotypes for Mefenoxam insensitivity and suggested the involvement of multiple loci, complicating the positional cloning of the gene(s) conditioning insensitivity to Mefenoxam. Instead, a candidate gene strategy was used, based on previous observations that the primary effect of phenylamide compounds is to inhibit ribosomal RNA synthesis. The subunits of RNA polymerase I (RNApolI) were sequenced from sensitive and insensitive isolates and F1 progeny. Single nucleotide polymorphisms (SNPs) specific to insensitive field isolates were identified in the gene encoding the large subunit of RNApolI. In a survey of field isolates, SNP T1145A (Y382F) showed an 86% association with Mefenoxam insensitivity. Isolates not showing this association belonged predominantly to one P. infestans genotype. The transfer of the 'insensitive' allele of RPA190 to a sensitive isolate yielded transgenic lines that were insensitive to Mefenoxam. These results demonstrate that sequence variation in RPA190 contributes to insensitivity to Mefenoxam in P. infestans.

  8. Global eukaryote phylogeny: Combined small- and large-subunit ribosomal DNA trees support monophyly of Rhizaria, Retaria and Excavata. (United States)

    Moreira, David; von der Heyden, Sophie; Bass, David; López-García, Purificación; Chao, Ema; Cavalier-Smith, Thomas


    Resolution of the phylogenetic relationships among the major eukaryotic groups is one of the most important problems in evolutionary biology that is still only partially solved. This task was initially addressed using a single marker, the small-subunit ribosomal DNA (SSU rDNA), although in recent years it has been shown that it does not contain enough phylogenetic information to robustly resolve global eukaryotic phylogeny. This has prompted the use of multi-gene analyses, especially in the form of long concatenations of numerous conserved protein sequences. However, this approach is severely limited by the small number of taxa for which such a large number of protein sequences is available today. We have explored the alternative approach of using only two markers but a large taxonomic sampling, by analysing a combination of SSU and large-subunit (LSU) rDNA sequences. This strategy allows also the incorporation of sequences from non-cultivated protists, e.g., Radiozoa (=radiolaria minus Phaeodarea). We provide the first LSU rRNA sequences for Heliozoa, Apusozoa (both Apusomonadida and Ancyromonadida), Cercozoa and Radiozoa. Our Bayesian and maximum likelihood analyses for 91 eukaryotic combined SSU+LSU sequences yielded much stronger support than hitherto for the supergroup Rhizaria (Cercozoa plus Radiozoa plus Foraminifera) and several well-recognised groups and also for other problematic clades, such as the Retaria (Radiozoa plus Foraminifera) and, with more moderate support, the Excavata. Within opisthokonts, the combined tree strongly confirms that the filose amoebae Nuclearia are sisters to Fungi whereas other Choanozoa are sisters to animals. The position of some bikont taxa, notably Heliozoa and Apusozoa, remains unresolved. However, our combined trees suggest a more deeply diverging position for Ancyromonas, and perhaps also Apusomonas, than for other bikonts, suggesting that apusozoan zooflagellates may be central for understanding the early evolution of

  9. Patterns and drivers of fish community assembly in a large marine ecosystem

    DEFF Research Database (Denmark)

    Pécuchet, Lauréne; Törnroos, Anna; Lindegren, Martin


    shaping community composition. However, community composition in the eastern part, an area beyond the steep decline in salinity, was characterized by fewer species with largely different trait characteristics, indicating that community assembly is also affected by biotic interactions. Our results add......The presence and survival of the species in a community depend on their abilities to maximize fitness in a given environment. The study of the processes that control survival and co‑existence, termed ‘assembly rules’, follows various mechanisms, primarily related to biotic or abiotic factors....... To determine assembly rules, ecological similarities of co-occurring species are often investigated. This can be evaluated using trait-based indices summarizing the species’ niches in a given community. In order to investigate the underlying processes shaping community assembly in marine ecosystems, we...

  10. Cloning, sequence analysis, and expression of the large subunit of the human lymphocyte activation antigen 4F2

    Energy Technology Data Exchange (ETDEWEB)

    Lumadue, J.A.; Glick, A.B.; Ruddle, F.H.


    Among the earliest expressed antigens on the surface of activated human lymphocytes is the surface antigen 4F2. The authors have used DNA-mediated gene transfer and fluorescence-activated cell sorting to obtain cell lines that contain the gene encoding the large subunit of the human 4F2 antigen in a mouse L-cell background. Human DNAs cloned from these cell lines were subsequently used as hybridization probes to isolate a full-length cDNA clone expressing 4F2. Sequence analysis of the coding region has revealed an amino acid sequence of 529 residues. Hydrophobicity plotting has predicted a probable structure for the protein that includes an external carboxyl terminus, an internal leader sequence, a single hydrophobic transmembrane domain, and two possible membrane-associated domains. The 4F2 cDNA detects a single 1.8-kilobase mRNA in T-cell and B-cell lines. RNA gel blot analysis of RNA derived from quiescent and serum-stimulated Swiss 3T3 fibroblasts reveals a cell-cycle modulation of 4F2 gene expression: the mRNA is present in quiescent fibroblasts but increases 8-fold 24-36 hr after stimulation, at the time of maximal DNA synthesis.

  11. Factors that affect large subunit ribosomal DNA amplicon sequencing studies of fungal communities: classification method, primer choice, and error.

    Directory of Open Access Journals (Sweden)

    Teresita M Porter

    Full Text Available Nuclear large subunit ribosomal DNA is widely used in fungal phylogenetics and to an increasing extent also amplicon-based environmental sequencing. The relatively short reads produced by next-generation sequencing, however, makes primer choice and sequence error important variables for obtaining accurate taxonomic classifications. In this simulation study we tested the performance of three classification methods: 1 a similarity-based method (BLAST + Metagenomic Analyzer, MEGAN; 2 a composition-based method (Ribosomal Database Project naïve bayesian classifier, NBC; and, 3 a phylogeny-based method (Statistical Assignment Package, SAP. We also tested the effects of sequence length, primer choice, and sequence error on classification accuracy and perceived community composition. Using a leave-one-out cross validation approach, results for classifications to the genus rank were as follows: BLAST + MEGAN had the lowest error rate and was particularly robust to sequence error; SAP accuracy was highest when long LSU query sequences were classified; and, NBC runs significantly faster than the other tested methods. All methods performed poorly with the shortest 50-100 bp sequences. Increasing simulated sequence error reduced classification accuracy. Community shifts were detected due to sequence error and primer selection even though there was no change in the underlying community composition. Short read datasets from individual primers, as well as pooled datasets, appear to only approximate the true community composition. We hope this work informs investigators of some of the factors that affect the quality and interpretation of their environmental gene surveys.

  12. Replication Factor C1, the Large Subunit of Replication Factor C, Is Proteolytically Truncated in Hutchinson-Gilford Progeria Syndrome (United States)

    Tang, Hui; Hilton, Benjamin; Musich, Phillip R.; Fang, Ding Zhi; Zou, Yue


    Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder due to a LMNA gene mutation which produces a mutant lamin A protein (progerin). Progerin also has been correlated to physiological aging and related diseases. However, how progerin causes the progeria remains unknown. Here we report that the large subunit (RFC1) of replication factor C is cleaved in HGPS cells, leading to the production of a truncated RFC1 of ~75 kDa which appears to be defective in loading PCNA and pol δ onto DNA for replication. Interestingly, the cleavage can be inhibited by a serine protease inhibitor, suggesting that RFC1 is cleaved by a serine protease. Due to the crucial role of RFC in DNA replication our findings provide a mechanistic interpretation for the observed replicative arrest and premature aging phenotypes of HPGS, and may lead to novel strategies in HGPS treatment. Furthermore, this unique truncated form of RFC1 may serve as a potential marker for HGPS. PMID:22168243

  13. Added resolution among ordinal level relationships of tapeworms (Platyhelminthes: Cestoda) with complete small and large subunit nuclear ribosomal RNA genes. (United States)

    Waeschenbach, Andrea; Webster, Bonnie L; Bray, Rodney A; Littlewood, D T J


    The addition of large subunit ribosomal DNA (lsrDNA) to small subunit ribosomal DNA (ssrDNA) has been shown to add resolution to phylogenies at various taxonomic levels for a diversity of phyla. We added nearly complete lsrDNA (4057-4593bp) sequences to ssrDNA (1940-2228bp) for 26 ingroup and 3 outgroup taxa in an attempt to provide an improved ordinal phylogeny for the Cestoda. Ten lsrDNA and seven ssrDNA sequences were generated from new taxa and 13 existing partial lsrDNA sequences were sequenced to completion. The majority of phylogenetic signal in the combined analysis came from lsrDNA (69.6% of parsimonious informative sites, as opposed to 30.4% obtained from ssrDNA), resulting in almost identical topologies for lsrDNA and lsr+ssrDNA (pairwise symmetric distance=6) in model-based analyses. Topology testing found trees based on partial lsrDNA (domains D1-D3)+ssrDNA and complete lsr+ssrDNA to differ significantly; the addition of lsrDNA domains D4-D12 had a significant effect on topology. Overall nodal support was greatest in the combined analysis and weakest for ssrDNA only. Our molecular phylogenies differed significantly from those based on morphology alone. Acetabulate lineages form a monophyletic group, with the Tetraphyllidea being paraphyletic. Support for the combined data was high for the following topology: (Litobothriidea (Lecanicephalidea (Rhinebothrium/Rhodobothrium (Clistobothrium (Pachybothrium(Acanthobothrium Proteocephalidea) (Mesocestoididae, Nippotaeniidea, Cyclophyllidea, Tetrabothriidea)))))); all genus names refer to tetraphyllidean lineages. Although the interrelationships among the four most derived taxa remain uncertain, overall ambiguity of the acetabulate interrelationships was reduced. The Pseudophyllidea were recovered as polyphyletic, with support for a sister-group relationship between Diphyllobothriidae and Haplobothriidea. The monophyly of the Trypanorhyncha was recovered for the first time based on molecular data. The positions

  14. N-terminal isoforms of the large-conductance Ca²⁺-activated K⁺ channel are differentially modulated by the auxiliary β1-subunit. (United States)

    Lorca, Ramón A; Stamnes, Susan J; Pillai, Meghan K; Hsiao, Jordy J; Wright, Michael E; England, Sarah K


    The large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel is essential for maintaining the membrane in a hyperpolarized state, thereby regulating neuronal excitability, smooth muscle contraction, and secretion. The BK(Ca) α-subunit has three predicted initiation codons that generate proteins with N-terminal ends starting with the amino acid sequences MANG, MSSN, or MDAL. Because the N-terminal region and first transmembrane domain of the α-subunit are required for modulation by auxiliary β1-subunits, we examined whether β1 differentially modulates the N-terminal BK(Ca) α-subunit isoforms. In the absence of β1, all isoforms had similar single-channel conductances and voltage-dependent activation. However, whereas β1 did not modulate the voltage-activation curve of MSSN, β1 induced a significant leftward shift of the voltage activation curves of both the MDAL and MANG isoforms. These shifts, of which the MDAL was larger, occurred at both 10 μM and 100 μM Ca(2+). The β1-subunit increased the open dwell times of all three isoforms and decreased the closed dwell times of MANG and MDAL but increased the closed dwell times of MSSN. The distinct modulation of voltage activation by the β1-subunit may be due to the differential effect of β1 on burst duration and interburst intervals observed among these isoforms. Additionally, we observed that the related β2-subunit induced comparable leftward shifts in the voltage-activation curves of all three isoforms, indicating that the differential modulation of these isoforms was specific to β1. These findings suggest that the relative expression of the N-terminal isoforms can fine-tune BK(Ca) channel activity in cells, highlighting a novel mechanism of BK(Ca) channel regulation.

  15. Electron tunneling through alkanedithiol self-assembled monolayers in large-area molecular junctions

    NARCIS (Netherlands)

    Akkerman, Hylke B.; Naber, Ronald C. G.; Jongbloed, Bert; van Hal, Paul A.; Blom, Paul W. M.; de Leeuw, Dago M.; de Boer, Bert


    The electrical transport through self-assembled monolayers of alkanedithiols was studied in large-area molecular junctions and described by the Simmons model [Simmons JIG (1963) J Appi Phys 34:1793-1803 and 2581-2590] for tunneling through a practical barrier, i.e., a rectangular barrier with the im

  16. Restoration of IFNγR subunit assembly, IFNγ signaling and parasite clearance in Leishmania donovani infected macrophages: role of membrane cholesterol.

    Directory of Open Access Journals (Sweden)

    Subha Sen


    Full Text Available Despite the presence of significant levels of systemic Interferon gamma (IFNγ, the host protective cytokine, Kala-azar patients display high parasite load with downregulated IFNγ signaling in Leishmania donovani (LD infected macrophages (LD-MØs; the cause of such aberrant phenomenon is unknown. Here we reveal for the first time the mechanistic basis of impaired IFNγ signaling in parasitized murine macrophages. Our study clearly shows that in LD-MØs IFNγ receptor (IFNγR expression and their ligand-affinity remained unaltered. The intracellular parasites did not pose any generalized defect in LD-MØs as IL-10 mediated signal transducer and activator of transcription 3 (STAT3 phosphorylation remained unaltered with respect to normal. Previously, we showed that LD-MØs are more fluid than normal MØs due to quenching of membrane cholesterol. The decreased rigidity in LD-MØs was not due to parasite derived lipophosphoglycan (LPG because purified LPG failed to alter fluidity in normal MØs. IFNγR subunit 1 (IFNγR1 and subunit 2 (IFNγR2 colocalize in raft upon IFNγ stimulation of normal MØs, but this was absent in LD-MØs. Oddly enough, such association of IFNγR1 and IFNγR2 could be restored upon liposomal delivery of cholesterol as evident from the fluorescence resonance energy transfer (FRET experiment and co-immunoprecipitation studies. Furthermore, liposomal cholesterol treatment together with IFNγ allowed reassociation of signaling assembly (phospho-JAK1, JAK2 and STAT1 in LD-MØs, appropriate signaling, and subsequent parasite killing. This effect was cholesterol specific because cholesterol analogue 4-cholestene-3-one failed to restore the response. The presence of cholesterol binding motifs [(L/V-X(1-5-Y-X(1-5-(R/K] in the transmembrane domain of IFNγR1 was also noted. The interaction of peptides representing this motif of IFNγR1 was studied with cholesterol-liposome and analogue-liposome with difference of two orders of

  17. Enzymatic assembly of epothilones: the EpoC subunit and reconstitution of the EpoA-ACP/B/C polyketide and nonribosomal peptide interfaces. (United States)

    O'Connor, Sarah E; Chen, Huawei; Walsh, Christopher T


    The biosynthesis of epothilones, a family of hybrid polyketide (PK)/nonribosomal peptide (NRP) antitumor agents, provides an ideal system to study a hybrid PK/NRP natural product with significant biomedical value. Here the third enzyme involved in epothilone production, the five domain 195 kDa polyketide synthase (PKS) EpoC protein, has been expressed and purified from Escherichia coli. EpoC was combined with the first two enzymes of the epothilone biosynthesis pathway, the acyl carrier protein (ACP) domain of EpoA and EpoB, to reconstitute the early steps in epothilone biosynthesis. The acyltransferase (AT) domain of EpoC transfers the methylmalonyl moiety from methylmalonyl-CoA to the holo HS-acyl carrier protein (ACP) in an autoacylation reaction. The ketosynthase (KS) domain of EpoC decarboxylates the methylmalonyl-S-EpoC acyl enzyme to generate the carbon nucleophile that reacts with methylthiazolylcarboxyl-S-EpoB. The resulting condensation product can be reduced in the presence of NADPH by the ketoreductase (KR) domain of EpoC and then dehydrated by the dehydratase (DH) domain to produce the methylthiazolylmethylacrylyl-S-EpoC acyl enzyme intermediate that serves as the acyl donor for subsequent elongation of the epothilone chain. The acetyl-CoA donor can be replaced with propionyl-CoA, isobutyryl-CoA, and benzoyl-CoA and the acyl chains accepted by both EpoB and EpoC subunits to produce ethyl-, isopropyl-, and phenylthiazolylmethylacrylyl-S-EpoC acyl enzyme intermediates, suggesting that future combinatorial biosynthetic variations in epothilone assembly may be feasible. These results demonstrate in vitro reconstitution of both the PKS/NRPS interface (EpoA-ACP/B) and the NRPS/PKS interface (EpoB/C) in the assembly line for this antitumor natural product.

  18. Deletion of Marek's disease virus large subunit of ribonucleotide reductase impairs virus growth in vitro and in vivo. (United States)

    Sun, Aijun; Lee, Lucy F; Khan, Owais A; Heidari, Mohammad; Zhang, Huanmin; Lupiani, Blanca; Reddy, Sanjay M


    Marek's disease virus (MDV), a highly cell-associated lymphotropic alphaherpesvirus, is the causative agent of a neoplastic disease in domestic chickens called Marek's disease (MD). In the unique long (UL) region of the MDV genome, open reading frames UL39 and UL40 encode the large and small subunits of the ribonucleotide reductase (RR) enzyme, named RR1 and RR2, respectively. MDV RR is distinguishable from that present in chicken and duck cells by monoclonal antibody T81. Using recombinant DNA technology we have generated a mutant MDV (Md5deltaRR1) in which RR1 was deleted. PCR amplification of the RR gene in Md5deltaRR1-infected duck embryo fibroblasts (DEF) confirmed the deletion of the 2.4 kb RR1 gene with a resultant amplicon of a 640-bp fragment. Restriction enzyme digests with SalI confirmed a UL39 deletion and the absence of gross rearrangement. The biologic characteristics of Md5deltaRR1 virus were studied in vitro and in vivo. The Md5deltaRR1 replicated in DEF, but significantly slower than parental Md5-BAC, suggesting that RR is important but not essential for replication in fibroblasts. In vivo studies, however, showed that the RR1 deletion virus was impaired for its ability to replicate in chickens. Inoculation of specific-pathogen-free (SPF) chickens with Md5deltaRR1 showed the mutant virus is nonpathogenic and does not induce MD in birds. A revertant virus, Md5deltaRR1/R, was generated with the restored phenotype of the parental Md5-BAC in vivo, indicating that RR is essential for replication of the virus in chickens. Protection studies in SPF chickens indicated that the Md5deltaRR1 virus is not a candidate vaccine against MD.

  19. Large pore volume mesoporous aluminum oxide synthesized via nano-assembly

    Institute of Scientific and Technical Information of China (English)


    A new nano-assembly approach has been proposed for the preparation of macropore volume mesoporous aluminum oxide supports. Secondary nano-assembly and a frame structure mechanism for large pore volume mesoporous supports have been proposed. In a primary nano-assembly supersoluble micelle,aluminum hydroxide nanoparticles were precipitated in situ in surfactants with a volume balance (VB) less than 1,followed by secondary nano-assembly in linear and cylindrical shapes. The secondary nano-assembly of cylindrical aluminum hydroxides was calcined to form nano cylindrical aluminum oxides. For the formation of macropore volume mesoporous supports,we utilized a frame structure mechanism of mesoporous support,in which the exterior surface of the carrier may not be continuous. This macropore volume support has been used for the hydrotreatment of a residual oil catalyst,which possesses the following physical characteristics:pore volume 1.8―2.7 mL·g-1,specific surface area 180―429 m2·g-1,average pore diameter 17―57 nm,average pore diameter more than 10 nm (81%―94%),porosity 87%―93%,and crush strength 7.7―25 N·mm-1.

  20. Large conductance Ca2+-activated K+ channel (BKCa α-subunit splice variants in resistance arteries from rat cerebral and skeletal muscle vasculature.

    Directory of Open Access Journals (Sweden)

    Zahra Nourian

    Full Text Available Previous studies report functional differences in large conductance Ca2+ activated-K+ channels (BKCa of smooth muscle cells (VSMC from rat cerebral and cremaster muscle resistance arteries. The present studies aimed to determine if this complexity in BKCa activity may, in part, be due to splice variants in the pore-forming α-subunit. BKCa variants in the intracellular C terminus of the α-subunit, and their relative expression to total α-subunit, were examined by qPCR. Sequencing of RT-PCR products showed two α-subunit variants, ZERO and STREX, to be identical in cremaster and cerebral arteries. Levels of STREX mRNA expression were, however, significantly higher in cremaster VSMCs (28.9±4.2% of total α-BKCa compared with cerebral vessels (16.5±0.9%. Further, a low level of BKCa SS4 α-subunit variant was seen in cerebral arteries, while undetectable in cremaster arteries. Protein biotinylation assays, in expression systems and arterial preparations, were used to determine whether differences in splice variant mRNA expression affect surface membrane/cytosolic location of the channel. In AD-293 and CHO-K1 cells, rat STREX was more likely to be located at the plasma membrane compared to ZERO, although the great majority of channel protein was in the membrane in both cases. Co-expression of β1-BKCa subunit with STREX or ZERO did not influence the dominant membrane expression of α-BKCa subunits, whereas in the absence of α-BKCa, a significant proportion of β1-subunit remained cytosolic. Biotinylation assays of cremaster and cerebral arteries showed that differences in STREX/ZERO expression do not alter membrane/cytosolic distribution of the channel under basal conditions. These data, however, revealed that the amount of α-BKCa in cerebral arteries is approximately 20X higher than in cremaster vessels. Thus, the data support the major functional differences in BKCa activity in cremaster, as compared to cerebral VSMCs, being related to total

  1. Dendronization-induced phase-transfer, stabilization and self-assembly of large colloidal Au nanoparticles (United States)

    Malassis, Ludivine; Jishkariani, Davit; Murray, Christopher B.; Donnio, Bertrand


    The phase-transfer of CTAB-coated aqueous, spherical gold nanoparticles, with metallic core diameters ranging from ca. 27 to 54 nm, into organic solvents by exchanging the primitive polar bilayer with lipophilic, disulfide dendritic ligands is reported. The presence of such a thick nonpolar organic shell around these large nanoparticles enhances their stabilization against aggregation, in addition to enabling their transfer into a variety of solvents such as chloroform, toluene or tetrahydrofuran. Upon the slow evaporation of a chloroform suspension deposited on a solid support, the dendronized hybrids were found to self-assemble into ring structures of various diameters. Moreover, their self-assembly at the liquid-air interface affords the formation of fairly long-range ordered monolayers, over large areas, that can then be entirely transferred onto solid substrates.The phase-transfer of CTAB-coated aqueous, spherical gold nanoparticles, with metallic core diameters ranging from ca. 27 to 54 nm, into organic solvents by exchanging the primitive polar bilayer with lipophilic, disulfide dendritic ligands is reported. The presence of such a thick nonpolar organic shell around these large nanoparticles enhances their stabilization against aggregation, in addition to enabling their transfer into a variety of solvents such as chloroform, toluene or tetrahydrofuran. Upon the slow evaporation of a chloroform suspension deposited on a solid support, the dendronized hybrids were found to self-assemble into ring structures of various diameters. Moreover, their self-assembly at the liquid-air interface affords the formation of fairly long-range ordered monolayers, over large areas, that can then be entirely transferred onto solid substrates. Electronic supplementary information (ESI) available: TEM microscope images. See DOI: 10.1039/c6nr03404g

  2. The role of humans and robots in the assembly of large infrared observatories (United States)

    Friedman, Edward J.; Espero, Tracey


    Many authors have endorsed the concept of assembly of large optics in space and have pointed out the technology needs for astronauts, infrastructure, robots and the observatories themselves. In this paper, we consider the technical issues associated with the integration and test in space of large optics during the next 15 years or so, when human activity is largely confined to low Earth orbit (LEO). We identify technical areas that need development and define a first version of the processes that might be used to create successful telescope missions that are tested in space. We identify a pathway that supports scalable solutions for very large systems necessary for imaging planets in other solar systems and other magnificent science. The investment in space integration and testing technology will return important dividends to designers of large space optics of the future. This approach to space optics testing is attractive because it overcomes the limits of ground testing associated with large test chambers, star simulators and the effects of gravity. It also directly benefits from, and supports, the technology and infrastructure investments about to be made by the new NASA Exploration Systems Enterprise, allowing both observatories and exploration missions to be assembled.

  3. NdhP is an exclusive subunit of large complex of NADPH dehydrogenase essential to stabilize the complex in Synechocystis sp. strain PCC 6803. (United States)

    Zhang, Jingsong; Gao, Fudan; Zhao, Jiaohong; Ogawa, Teruo; Wang, Quanxi; Ma, Weimin


    Two major complexes of NADPH dehydrogenase (NDH-1) have been identified in cyanobacteria. A large complex (NDH-1L) contains NdhD1 and NdhF1, which are absent in a medium size complex (NDH-1M). They play important roles in respiration, cyclic electron transport around photosystem I, and CO2 acquisition. Two mutants sensitive to high light for growth and impaired in NDH-1-mediated cyclic electron transfer were isolated from Synechocystis sp. strain PCC 6803 transformed with a transposon-bearing library. Both mutants had a tag in sml0013 encoding NdhP, a single transmembrane small subunit of the NDH-1 complex. During prolonged incubation of the wild type thylakoid membrane with n-dodecyl β-d-maltoside (DM), about half of the NDH-1L was disassembled to NDH-1M and the rest decomposed completely without forming NDH-1M. In the ndhP deletion mutant (ΔndhP), disassembling of NDH-1L to NDH-1M occurred even on ice, and decomposition to a small piece occurred at room temperature much faster than in the wild type. Deletion of the C-terminal tail of NdhP gave the same result. The C terminus of NdhP was tagged by YFP-His6. Blue native gel electrophoresis of the DM-treated thylakoid membrane of this strain and Western analysis using the antibody against GFP revealed that NdhP-YFP-His6 was exclusively confined to NDH-1L. During prolonged incubation of the thylakoid membrane of the tagged strain with DM at room temperature, NDH-1L was partially disassembled to NDH-1M and the 160-kDa band containing NdhP-YFP-His6 and possibly NdhD1 and NdhF1. We therefore conclude that NdhP, especially its C-terminal tail, is essential to assemble NdhD1 and NdhF1 and stabilize the NDH-1L complex. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. RelA NF-κB subunit activation as a therapeutic target in diffuse large B-cell lymphoma

    DEFF Research Database (Denmark)

    Zhang, Mingzhi; Xu-Monette, Zijun Y; Li, Ling;


    It has been well established that nuclear factor kappa-B (NF-κB) activation is important for tumor cell growth and survival. RelA/p65 and p50 are the most common NF-kB subunits and involved in the classical NF-kB pathway. However, the prognostic and biological significance of RelA/p65 is equivoca...

  5. Topography and stoichiometry of acidic proteins in large ribosomal subunits from Artemia salina as determined by crosslinking

    Energy Technology Data Exchange (ETDEWEB)

    Uchiumi, T.; Wahba, A.J.; Traut, R.R.


    The 60S subunits isolated from Artemia salina ribosomes were treated with the crosslinking reagent 2-iminothiolane under mild conditions. Proteins were extracted and fractions containing crosslinked acidic proteins were obtained by stepwise elution from CM-cellulose. Each fraction was analyzed by diagonal (two-dimensional nonreducing-reducing) NaDodSO/sub 4//polyacrylamide gel electrophoresis. Crosslinked proteins below the diagonal were radioiodinated and identified by two-dimensional acidic urea-NaDodSO/sub 4/ gel electrophoresis. Each of the acidic proteins P1 and P2 was crosslinked individually to the same third protein, PO. The fractions containing acidic proteins were also analyzed by two-dimensional nonequilibrium isoelectric focusing-NaDodSO/sub 4//polyacrylamide gel electrophoresis. Two crosslinked complexes were observed that coincide in isoelectric positions with monomeric P1 and P2, respectively. Both P1 and P2 appear to form crosslinked homodimers. These results suggest the presence in the 60S subunit of (P1)/sub 2/ and (P2)/sub 2/ dimers, each of which is anchored to PO. Protein PO appears to play the same role as L10 in Escherichia coli ribosomes and may form a pentameric complex with the two dimers in the 60S subunits.

  6. Bax assembles into large ring-like structures remodeling the mitochondrial outer membrane in apoptosis. (United States)

    Große, Lena; Wurm, Christian A; Brüser, Christian; Neumann, Daniel; Jans, Daniel C; Jakobs, Stefan


    The Bcl-2 family proteins Bax and Bak are essential for the execution of many apoptotic programs. During apoptosis, Bax translocates to the mitochondria and mediates the permeabilization of the outer membrane, thereby facilitating the release of pro-apoptotic proteins. Yet the mechanistic details of the Bax-induced membrane permeabilization have so far remained elusive. Here, we demonstrate that activated Bax molecules, besides forming large and compact clusters, also assemble, potentially with other proteins including Bak, into ring-like structures in the mitochondrial outer membrane. STED nanoscopy indicates that the area enclosed by a Bax ring is devoid of mitochondrial outer membrane proteins such as Tom20, Tom22, and Sam50. This strongly supports the view that the Bax rings surround an opening required for mitochondrial outer membrane permeabilization (MOMP). Even though these Bax assemblies may be necessary for MOMP, we demonstrate that at least in Drp1 knockdown cells, these assemblies are not sufficient for full cytochrome c release. Together, our super-resolution data provide direct evidence in support of large Bax-delineated pores in the mitochondrial outer membrane as being crucial for Bax-mediated MOMP in cells. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

  7. MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph. (United States)

    Li, Dinghua; Liu, Chi-Man; Luo, Ruibang; Sadakane, Kunihiko; Lam, Tak-Wah


    MEGAHIT is a NGS de novo assembler for assembling large and complex metagenomics data in a time- and cost-efficient manner. It finished assembling a soil metagenomics dataset with 252 Gbps in 44.1 and 99.6 h on a single computing node with and without a graphics processing unit, respectively. MEGAHIT assembles the data as a whole, i.e. no pre-processing like partitioning and normalization was needed. When compared with previous methods on assembling the soil data, MEGAHIT generated a three-time larger assembly, with longer contig N50 and average contig length; furthermore, 55.8% of the reads were aligned to the assembly, giving a fourfold improvement.

  8. Strain of Synechocystis PCC 6803 with Aberrant Assembly of Photosystem II Contains Tandem Duplication of a Large Chromosomal Region. (United States)

    Tichý, Martin; Bečková, Martina; Kopečná, Jana; Noda, Judith; Sobotka, Roman; Komenda, Josef


    Cyanobacterium Synechocystis PCC 6803 represents a favored model organism for photosynthetic studies. Its easy transformability allowed construction of a vast number of Synechocystis mutants including many photosynthetically incompetent ones. However, it became clear that there is already a spectrum of Synechocystis "wild-type" substrains with apparently different phenotypes. Here, we analyzed organization of photosynthetic membrane complexes in a standard motile Pasteur collection strain termed PCC and two non-motile glucose-tolerant substrains (named here GT-P and GT-W) previously used as genetic backgrounds for construction of many photosynthetic site directed mutants. Although, both the GT-P and GT-W strains were derived from the same strain constructed and described by Williams in 1988, only GT-P was similar in pigmentation and in the compositions of Photosystem II (PSII) and Photosystem I (PSI) complexes to PCC. In contrast, GT-W contained much more carotenoids but significantly less chlorophyll (Chl), which was reflected by lower level of dimeric PSII and especially trimeric PSI. We found that GT-W was deficient in Chl biosynthesis and contained unusually high level of unassembled D1-D2 reaction center, CP47 and especially CP43. Another specific feature of GT-W was a several fold increase in the level of the Ycf39-Hlip complex previously postulated to participate in the recycling of Chl molecules. Genome re-sequencing revealed that the phenotype of GT-W is related to the tandem duplication of a large region of the chromosome that contains 100 genes including ones encoding D1, Psb28, and other PSII-related proteins as well as Mg-protoporphyrin methylester cyclase (Cycl). Interestingly, the duplication was completely eliminated after keeping GT-W cells on agar plates under photoautotrophic conditions for several months. The GT-W strain without a duplication showed no obvious defects in PSII assembly and resembled the GT-P substrain. Although, we do not exactly

  9. Large-scale chemical assembly of atomically thin transistors and circuits (United States)

    Zhao, Mervin; Ye, Yu; Han, Yimo; Xia, Yang; Zhu, Hanyu; Wang, Siqi; Wang, Yuan; Muller, David A.; Zhang, Xiang


    Next-generation electronics calls for new materials beyond silicon, aiming at increased functionality, performance and scaling in integrated circuits. In this respect, two-dimensional gapless graphene and semiconducting transition-metal dichalcogenides have emerged as promising candidates due to their atomic thickness and chemical stability. However, difficulties with precise spatial control during their assembly currently impede actual integration into devices. Here, we report on the large-scale, spatially controlled synthesis of heterostructures made of single-layer semiconducting molybdenum disulfide contacting conductive graphene. Transmission electron microscopy studies reveal that the single-layer molybdenum disulfide nucleates at the graphene edges. We demonstrate that such chemically assembled atomic transistors exhibit high transconductance (10 µS), on-off ratio (˜106) and mobility (˜17 cm2 V-1 s-1). The precise site selectivity from atomically thin conducting and semiconducting crystals enables us to exploit these heterostructures to assemble two-dimensional logic circuits, such as an NMOS inverter with high voltage gain (up to 70).

  10. Self-assembly and nanosphere lithography for large-area plasmonic patterns on graphene. (United States)

    Lotito, Valeria; Zambelli, Tomaso


    Plasmonic structures on graphene can tailor its optical properties, which is essential for sensing and optoelectronic applications, e.g. for the enhancement of photoresponsivity of graphene photodetectors. Control over their structural and, hence, spectral properties can be attained by using electron beam lithography, which is not a viable solution for the definition of patterns over large areas. For the fabrication of large-area plasmonic nanostructures, we propose to use self-assembled monolayers of nanospheres as a mask for metal evaporation and etching processes. An optimized approach based on self-assembly at air/water interface with a properly designed apparatus allows the attainment of monolayers of hexagonally closely packed patterns with high long-range order and large area coverage; special strategies are devised in order to protect graphene against damage resulting from surface treatment and further processing steps such as reactive ion etching, which could potentially impair graphene properties. Therefore we demonstrate that nanosphere lithography is a cost-effective solution to create plasmonic patterns on graphene. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Directed self-assembly of large scaffold-free multi-cellular honeycomb structures

    Energy Technology Data Exchange (ETDEWEB)

    Tejavibulya, Nalin; Youssef, Jacquelyn; Bao, Brian; Ferruccio, Toni-Marie; Morgan, Jeffrey R, E-mail: [Department of Molecular Pharmacology, Physiology and Biotechnology, Center for Biomedical Engineering, Brown University, G-B 393, Biomed Center, 171 Meeting St, Providence, RI 02912 (United States)


    A significant challenge to the field of biofabrication is the rapid construction of large three-dimensional (3D) living tissues and organs. Multi-cellular spheroids have been used as building blocks. In this paper, we create large multi-cellular honeycomb building blocks using directed self-assembly, whereby cell-to-cell adhesion, in the context of the shape and obstacles of a micro-mold, drives the formation of a 3D structure. Computer-aided design, rapid prototyping and replica molding were used to fabricate honeycomb-shaped micro-molds. Nonadhesive hydrogels cast from these micro-molds were equilibrated in the cell culture medium and seeded with two types of mammalian cells. The cells settled into the honeycomb recess were unable to attach to the nonadhesive hydrogel and so cell-to-cell adhesion drove the self-assembly of a large multi-cellular honeycomb within 24 h. Distinct morphological changes occurred to the honeycomb and its cells indicating the presence of significant cell-mediated tension. Unlike the spheroid, whose size is constrained by a critical diffusion distance needed to maintain cell viability, the overall size of the honeycomb is not limited. The rapid production of the honeycomb building unit, with its multiple rings of high-density cells and open lumen spaces, offers interesting new possibilities for biofabrication strategies.

  12. Self-assembled fluids with order-parameter- dependent mobility: The large- limit

    Indian Academy of Sciences (India)

    N P Rapapa; N B Maliehe


    The effect of the order-parameter-dependent mobility, $ (\\vec{}) ∝ \\left( 1 - g \\dfrac{\\vec{}^{2}}{N} \\right)^{}$, on phase-ordering dynamics of self-assembled fluids is studied analytically within the large- limit. The study is for quenching from an uncorrelated high temperature state into the Lifshitz line within the microemulsion phase. In the later stage of the ordering process, the structure factor exhibits multiscaling behavior with characteristic length scale (/ ln )1/2(2+3). The order-parameter-dependent mobility is found to slow down the rate of coarsening.

  13. The First Assembly Line of Large-longitudinally-welded Steel Pipe in China Went into Operation

    Institute of Scientific and Technical Information of China (English)

    Li Bing


    @@ On July 27, the first assembly line to produce JCOE large diameter Longitudinally-submerged-arc-welded steel pipe in China, Which is the key homemade equipment project of "West-East Gas Transmission"project, was put into production. Chen Gen, vice general manager of CNPC; Xie Zhiqiang and Liu Haisheng, assistant chief manager of CNPC; Shi Xingquan, vice president of PetroChina; and the president of Itochu-Marubeni Steel & iron Co., Ltd.of Japan; attended the opening ceremony and cut the ribbon.

  14. A proposal for a collecting mirror assembly for large divergence x-ray sources. (United States)

    Ichimaru, Satoshi; Hatayama, Masatoshi; Ohchi, Tadayuki; Oku, Satoshi


    We propose a new type of collecting mirror assembly (CMA) for x rays, which will enable us to build a powerful optical system for collecting x rays from large divergence sources. The CMA consists of several mirror sections connected in series. The angle of each section is designed so that the x rays reflected from it are parallel to the x rays directly incident on the following sections. A simplified CMA structure is designed and applied to the Al-Kα emission line. It is estimated that by using the CMA the number of x rays detected could be increased by a factor of about 2.5.

  15. Dynamic regulation of β1 subunit trafficking controls vascular contractility. (United States)

    Leo, M Dennis; Bannister, John P; Narayanan, Damodaran; Nair, Anitha; Grubbs, Jordan E; Gabrick, Kyle S; Boop, Frederick A; Jaggar, Jonathan H


    Ion channels composed of pore-forming and auxiliary subunits control physiological functions in virtually all cell types. A conventional view is that channels assemble with their auxiliary subunits before anterograde plasma membrane trafficking of the protein complex. Whether the multisubunit composition of surface channels is fixed following protein synthesis or flexible and open to acute and, potentially, rapid modulation to control activity and cellular excitability is unclear. Arterial smooth muscle cells (myocytes) express large-conductance Ca(2+)-activated potassium (BK) channel α and auxiliary β1 subunits that are functionally significant modulators of arterial contractility. Here, we show that native BKα subunits are primarily (∼95%) plasma membrane-localized in human and rat arterial myocytes. In contrast, only a small fraction (∼10%) of total β1 subunits are located at the cell surface. Immunofluorescence resonance energy transfer microscopy demonstrated that intracellular β1 subunits are stored within Rab11A-postive recycling endosomes. Nitric oxide (NO), acting via cGMP-dependent protein kinase, and cAMP-dependent pathways stimulated rapid (≤1 min) anterograde trafficking of β1 subunit-containing recycling endosomes, which increased surface β1 almost threefold. These β1 subunits associated with surface-resident BKα proteins, elevating channel Ca(2+) sensitivity and activity. Our data also show that rapid β1 subunit anterograde trafficking is the primary mechanism by which NO activates myocyte BK channels and induces vasodilation. In summary, we show that rapid β1 subunit surface trafficking controls functional BK channel activity in arterial myocytes and vascular contractility. Conceivably, regulated auxiliary subunit trafficking may control ion channel activity in a wide variety of cell types.

  16. Efficient assembly of de novo human artificial chromosomes from large genomic loci

    Directory of Open Access Journals (Sweden)

    Stromberg Gregory


    Full Text Available Abstract Background Human Artificial Chromosomes (HACs are potentially useful vectors for gene transfer studies and for functional annotation of the genome because of their suitability for cloning, manipulating and transferring large segments of the genome. However, development of HACs for the transfer of large genomic loci into mammalian cells has been limited by difficulties in manipulating high-molecular weight DNA, as well as by the low overall frequencies of de novo HAC formation. Indeed, to date, only a small number of large (>100 kb genomic loci have been reported to be successfully packaged into de novo HACs. Results We have developed novel methodologies to enable efficient assembly of HAC vectors containing any genomic locus of interest. We report here the creation of a novel, bimolecular system based on bacterial artificial chromosomes (BACs for the construction of HACs incorporating any defined genomic region. We have utilized this vector system to rapidly design, construct and validate multiple de novo HACs containing large (100–200 kb genomic loci including therapeutically significant genes for human growth hormone (HGH, polycystic kidney disease (PKD1 and ß-globin. We report significant differences in the ability of different genomic loci to support de novo HAC formation, suggesting possible effects of cis-acting genomic elements. Finally, as a proof of principle, we have observed sustained ß-globin gene expression from HACs incorporating the entire 200 kb ß-globin genomic locus for over 90 days in the absence of selection. Conclusion Taken together, these results are significant for the development of HAC vector technology, as they enable high-throughput assembly and functional validation of HACs containing any large genomic locus. We have evaluated the impact of different genomic loci on the frequency of HAC formation and identified segments of genomic DNA that appear to facilitate de novo HAC formation. These genomic loci

  17. Genomic characterization of large heterochromatic gaps in the human genome assembly.

    Directory of Open Access Journals (Sweden)

    Nicolas Altemose


    Full Text Available The largest gaps in the human genome assembly correspond to multi-megabase heterochromatic regions composed primarily of two related families of tandem repeats, Human Satellites 2 and 3 (HSat2,3. The abundance of repetitive DNA in these regions challenges standard mapping and assembly algorithms, and as a result, the sequence composition and potential biological functions of these regions remain largely unexplored. Furthermore, existing genomic tools designed to predict consensus-based descriptions of repeat families cannot be readily applied to complex satellite repeats such as HSat2,3, which lack a consistent repeat unit reference sequence. Here we present an alignment-free method to characterize complex satellites using whole-genome shotgun read datasets. Utilizing this approach, we classify HSat2,3 sequences into fourteen subfamilies and predict their chromosomal distributions, resulting in a comprehensive satellite reference database to further enable genomic studies of heterochromatic regions. We also identify 1.3 Mb of non-repetitive sequence interspersed with HSat2,3 across 17 unmapped assembly scaffolds, including eight annotated gene predictions. Finally, we apply our satellite reference database to high-throughput sequence data from 396 males to estimate array size variation of the predominant HSat3 array on the Y chromosome, confirming that satellite array sizes can vary between individuals over an order of magnitude (7 to 98 Mb and further demonstrating that array sizes are distributed differently within distinct Y haplogroups. In summary, we present a novel framework for generating initial reference databases for unassembled genomic regions enriched with complex satellite DNA, and we further demonstrate the utility of these reference databases for studying patterns of sequence variation within human populations.

  18. Assembling Large, Multi-Sensor Climate Datasets Using the SciFlo Grid Workflow System (United States)

    Wilson, B.; Manipon, G.; Xing, Z.; Fetzer, E.


    NASA's Earth Observing System (EOS) is an ambitious facility for studying global climate change. The mandate now is to combine measurements from the instruments on the "A-Train" platforms (AIRS, AMSR-E, MODIS, MISR, MLS, and CloudSat) and other Earth probes to enable large-scale studies of climate change over periods of years to decades. However, moving from predominantly single-instrument studies to a multi-sensor, measurement-based model for long-duration analysis of important climate variables presents serious challenges for large-scale data mining and data fusion. For example, one might want to compare temperature and water vapor retrievals from one instrument (AIRS) to another instrument (MODIS), and to a model (ECMWF), stratify the comparisons using a classification of the "cloud scenes" from CloudSat, and repeat the entire analysis over years of AIRS data. To perform such an analysis, one must discover & access multiple datasets from remote sites, find the space/time "matchups" between instruments swaths and model grids, understand the quality flags and uncertainties for retrieved physical variables, assemble merged datasets, and compute fused products for further scientific and statistical analysis. To meet these large-scale challenges, we are utilizing a Grid computing and dataflow framework, named SciFlo, in which we are deploying a set of versatile and reusable operators for data query, access, subsetting, co-registration, mining, fusion, and advanced statistical analysis. SciFlo is a semantically-enabled ("smart") Grid Workflow system that ties together a peer-to-peer network of computers into an efficient engine for distributed computation. The SciFlo workflow engine enables scientists to do multi-instrument Earth Science by assembling remotely-invokable Web Services (SOAP or http GET URLs), native executables, command-line scripts, and Python codes into a distributed computing flow. A scientist visually authors the graph of operation in the Viz

  19. Large Area Fabrication of Semiconducting Phosphorene by Langmuir-Blodgett Assembly (United States)

    Kaur, Harneet; Yadav, Sandeep; Srivastava, Avanish. K.; Singh, Nidhi; Schneider, Jörg J.; Sinha, Om. P.; Agrawal, Ved V.; Srivastava, Ritu


    Phosphorene is a recently new member of the family of two dimensional (2D) inorganic materials. Besides its synthesis it is of utmost importance to deposit this material as thin film in a way that represents a general applicability for 2D materials. Although a considerable number of solvent based methodologies have been developed for exfoliating black phosphorus, so far there are no reports on controlled organization of these exfoliated nanosheets on substrates. Here, for the first time to the best of our knowledge, a mixture of N-methyl-2-pyrrolidone and deoxygenated water is employed as a subphase in Langmuir-Blodgett trough for assembling the nanosheets followed by their deposition on substrates and studied its field-effect transistor characteristics. Electron microscopy reveals the presence of densely aligned, crystalline, ultra-thin sheets of pristine phosphorene having lateral dimensions larger than hundred of microns. Furthermore, these assembled nanosheets retain their electronic properties and show a high current modulation of 104 at room temperature in field-effect transistor devices. The proposed technique provides semiconducting phosphorene thin films that are amenable for large area applications. PMID:27671093

  20. Artificial intelligence approach to planning the robotic assembly of large tetrahedral truss structures (United States)

    Homemdemello, Luiz S.


    An assembly planner for tetrahedral truss structures is presented. To overcome the difficulties due to the large number of parts, the planner exploits the simplicity and uniformity of the shapes of the parts and the regularity of their interconnection. The planning automation is based on the computational formalism known as production system. The global data base consists of a hexagonal grid representation of the truss structure. This representation captures the regularity of tetrahedral truss structures and their multiple hierarchies. It maps into quadratic grids and can be implemented in a computer by using a two-dimensional array data structure. By maintaining the multiple hierarchies explicitly in the model, the choice of a particular hierarchy is only made when needed, thus allowing a more informed decision. Furthermore, testing the preconditions of the production rules is simple because the patterned way in which the struts are interconnected is incorporated into the topology of the hexagonal grid. A directed graph representation of assembly sequences allows the use of both graph search and backtracking control strategies.

  1. The Caenorhabditis elegans protein SAS-5 forms large oligomeric assemblies critical for centriole formation. (United States)

    Rogala, Kacper B; Dynes, Nicola J; Hatzopoulos, Georgios N; Yan, Jun; Pong, Sheng Kai; Robinson, Carol V; Deane, Charlotte M; Gönczy, Pierre; Vakonakis, Ioannis


    Centrioles are microtubule-based organelles crucial for cell division, sensing and motility. In Caenorhabditis elegans, the onset of centriole formation requires notably the proteins SAS-5 and SAS-6, which have functional equivalents across eukaryotic evolution. Whereas the molecular architecture of SAS-6 and its role in initiating centriole formation are well understood, the mechanisms by which SAS-5 and its relatives function is unclear. Here, we combine biophysical and structural analysis to uncover the architecture of SAS-5 and examine its functional implications in vivo. Our work reveals that two distinct self-associating domains are necessary to form higher-order oligomers of SAS-5: a trimeric coiled coil and a novel globular dimeric Implico domain. Disruption of either domain leads to centriole duplication failure in worm embryos, indicating that large SAS-5 assemblies are necessary for function in vivo.

  2. The Human Arp2/3 Complex Is Composed of Evolutionarily Conserved Subunits and Is Localized to Cellular Regions of Dynamic Actin Filament Assembly


    Welch, Matthew D.; Angela H. DePace; Verma, Suzie; Iwamatsu, Akihiro; Mitchison, Timothy J.


    The Arp2/3 protein complex has been implicated in the control of actin polymerization in cells. The human complex consists of seven subunits which include the actin related proteins Arp2 and Arp3, and five others referred to as p41-Arc, p34-Arc, p21-Arc, p20-Arc, and p16-Arc (Arp complex). We have determined the predicted amino acid sequence of all seven subunits. Each has homologues in diverse eukaryotes, implying that the structure and function of the complex has been conserved through evol...

  3. Association of polymorphisms in calpain 1, (mu/I) large subunit, calpastatin, and cathepsin D genes with meat quality traits in double-muscled Piemontese cattle. (United States)

    Ribeca, Cinzia; Bonfatti, Valentina; Cecchinato, Alessio; Albera, Andrea; Maretto, Fabio; Gallo, Luigi; Carnier, Paolo


    Five single-nucleotide polymorphisms (SNPs) located in the calpain 1, (mu/I) large subunit (CAPN1), calpastatin (CAST), and cathepsin D (CTSD) genes were analyzed in a large sample of Piemontese cattle. The aim of this study was to evaluate allele and genotype frequencies of these SNPs and to investigate associations of CAPN1, CAST, and CTSD gene variants with meat quality traits. Minor allele frequencies ranged from 30 to 48%. The presence of the A allele at CAPN530 increased yellowness and drip loss. The CAST282 G allele was associated with an increased drip loss compared to the C allele, and the CAST2959 A allele decreased redness compared to the G allele.

  4. Assembling large genomes with single-molecule sequencing and locality-sensitive hashing. (United States)

    Berlin, Konstantin; Koren, Sergey; Chin, Chen-Shan; Drake, James P; Landolin, Jane M; Phillippy, Adam M


    Long-read, single-molecule real-time (SMRT) sequencing is routinely used to finish microbial genomes, but available assembly methods have not scaled well to larger genomes. We introduce the MinHash Alignment Process (MHAP) for overlapping noisy, long reads using probabilistic, locality-sensitive hashing. Integrating MHAP with the Celera Assembler enabled reference-grade de novo assemblies of Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster and a human hydatidiform mole cell line (CHM1) from SMRT sequencing. The resulting assemblies are highly continuous, include fully resolved chromosome arms and close persistent gaps in these reference genomes. Our assembly of D. melanogaster revealed previously unknown heterochromatic and telomeric transition sequences, and we assembled low-complexity sequences from CHM1 that fill gaps in the human GRCh38 reference. Using MHAP and the Celera Assembler, single-molecule sequencing can produce de novo near-complete eukaryotic assemblies that are 99.99% accurate when compared with available reference genomes.

  5. Clofarabine 5'-di and -triphosphates inhibit human ribonucleotide reductase by altering the quaternary structure of its large subunit. (United States)

    Aye, Yimon; Stubbe, Joanne


    Human ribonucleotide reductases (hRNRs) catalyze the conversion of nucleotides to deoxynucleotides and are composed of α- and β-subunits that form active α(n)β(m) (n, m = 2 or 6) complexes. α binds NDP substrates (CDP, UDP, ADP, and GDP, C site) as well as ATP and dNTPs (dATP, dGTP, TTP) allosteric effectors that control enzyme activity (A site) and substrate specificity (S site). Clofarabine (ClF), an adenosine analog, is used in the treatment of refractory leukemias. Its mode of cytotoxicity is thought to be associated in part with the triphosphate functioning as an allosteric inhibitor of hRNR. Studies on the mechanism of inhibition of hRNR by ClF di- and triphosphates (ClFDP and ClFTP) are presented. ClFTP is a reversible inhibitor (K(i) = 40 nM) that rapidly inactivates hRNR. However, with time, 50% of the activity is recovered. D57N-α, a mutant with an altered A site, prevents inhibition by ClFTP, suggesting its A site binding. ClFDP is a slow-binding, reversible inhibitor ( K(i)*; t(1/2) = 23 min). CDP protects α from its inhibition. The altered off-rate of ClFDP from E•ClFDP* by ClFTP (A site) or dGTP (S site) and its inhibition of D57N-α together implicate its C site binding. Size exclusion chromatography of hRNR or α alone with ClFDP or ClFTP, ± ATP or dGTP, reveals in each case that α forms a kinetically stable hexameric state. This is the first example of hexamerization of α induced by an NDP analog that reversibly binds at the active site.

  6. Large-scale self-assembly of uniform submicron silver sulfide material driven by precise pressure control (United States)

    Qi, Juanjuan; Chen, Ke; Zhang, Shuhao; Yang, Yun; Guo, Lin; Yang, Shihe


    The controllable self-assembly of nanosized building blocks into larger specific structures can provide an efficient method of synthesizing novel materials with excellent properties. The self-assembly of nanocrystals by assisted means is becoming an extremely active area of research, because it provides a method of producing large-scale advanced functional materials with potential applications in the areas of energy, electronics, optics, and biologics. In this study, we applied an efficient strategy, namely, the use of ‘pressure control’ to the assembly of silver sulfide (Ag2S) nanospheres with a diameter of approximately 33 nm into large-scale, uniform Ag2S sub-microspheres with a size of about 0.33 μm. More importantly, this strategy realizes the online control of the overall reaction system, including the pressure, reaction time, and temperature, and could also be used to easily fabricate other functional materials on an industrial scale. Moreover, the thermodynamics and kinetics parameters for the thermal decomposition of silver diethyldithiocarbamate (Ag(DDTC)) are also investigated to explore the formation mechanism of the Ag2S nanosized building blocks which can be assembled into uniform sub-micron scale architecture. As a method of producing sub-micron Ag2S particles by means of the pressure-controlled self-assembly of nanoparticles, we foresee this strategy being an efficient and universally applicable option for constructing other new building blocks and assembling novel and large functional micromaterials on an industrial scale.

  7. Large-scale self-assembly of uniform submicron silver sulfide material driven by precise pressure control. (United States)

    Qi, Juanjuan; Chen, Ke; Zhang, Shuhao; Yang, Yun; Guo, Lin; Yang, Shihe


    The controllable self-assembly of nanosized building blocks into larger specific structures can provide an efficient method of synthesizing novel materials with excellent properties. The self-assembly of nanocrystals by assisted means is becoming an extremely active area of research, because it provides a method of producing large-scale advanced functional materials with potential applications in the areas of energy, electronics, optics, and biologics. In this study, we applied an efficient strategy, namely, the use of 'pressure control' to the assembly of silver sulfide (Ag2S) nanospheres  with a diameter of approximately 33 nm into large-scale, uniform Ag2S sub-microspheres with a size of about 0.33 μm. More importantly, this strategy realizes the online control of the overall reaction system, including the pressure, reaction time, and temperature, and could also be used to easily fabricate other functional materials on an industrial scale. Moreover, the thermodynamics and kinetics parameters for the thermal decomposition of silver diethyldithiocarbamate (Ag(DDTC)) are also investigated to explore the formation mechanism of the Ag2S nanosized building blocks which can be assembled into uniform sub-micron scale architecture. As a method of producing sub-micron Ag2S particles by means of the pressure-controlled self-assembly of nanoparticles, we foresee this strategy being an efficient and universally applicable option for constructing other new building blocks and assembling novel and large functional micromaterials on an industrial scale.

  8. Mutational analysis of the prohead binding domain of the large subunit of terminase, the bacteriophage lambda DNA packaging enzyme. (United States)

    Yeo, A; Feiss, M


    Terminase, the DNA packaging enzyme of bacteriophage lambda, is made up of two subunits, gpNul and gpA, the products of the Nu1 and A genes. The activities of terminase include DNA binding, cos cleavage and prohead binding. Specificity domains within the structure of terminase have previously been defined by genetic studies of lambda-21 hybrids. The prohead binding domain of terminase is localized to the last 32 amino acid residues of gpA. Mutations in the prohead binding domain of gpA were constructed by introducing the corresponding amino acids from gp2, the gpA analog of bacteriophage 21. The last five residues of gpA can be replaced with little effect on the burst size of lambda. A phage with a replacement of the last six residues of gpA with the corresponding residues of gp2 was unable to form plaques, indicating that the sixth-to-last residues of gpA is crucial for prohead binding. Site-specific mutagenesis of the sixth-to-last position of gpA indicated that the sixth-to-last residue of gpA must be hydrophobic, of the seven amino acids tested, only isoleucine and valine can substitute for leucine at this position. Although the last five residues of gp2 were functional when they replaced the last five residues of gpA, two results indicated that the last five residues of gpA functioned better than the corresponding residues of gp2. First, the presence of a valine residue at the sixth-to-last position of gpA allowed plaque formation, whereas replacement of the last six residues of gpA with those of gp2, which substitutes a valine residue at the sixth-to-last position, was lethal. The second set of results indicating that the last five residues of gpA function better than the gp2 residues were obtained by study of revertants of lethal substitution mutations. In constructing the replacement mutations, a short linker was inserted into the C terminus of the A gene; this insertion created a short duplication of the end of the A gene, so that the normal C

  9. An assembly model for simulation of large-scale ground water flow and transport. (United States)

    Huang, Junqi; Christ, John A; Goltz, Mark N


    When managing large-scale ground water contamination problems, it is often necessary to model flow and transport using finely discretized domains--for instance (1) to simulate flow and transport near a contamination source area or in the area where a remediation technology is being implemented; (2) to account for small-scale heterogeneities; (3) to represent ground water-surface water interactions; or (4) some combination of these scenarios. A model with a large domain and fine-grid resolution will need extensive computing resources. In this work, a domain decomposition-based assembly model implemented in a parallel computing environment is developed, which will allow efficient simulation of large-scale ground water flow and transport problems using domain-wide grid refinement. The method employs common ground water flow (MODFLOW) and transport (RT3D) simulators, enabling the solution of almost all commonly encountered ground water flow and transport problems. The basic approach partitions a large model domain into any number of subdomains. Parallel processors are used to solve the model equations within each subdomain. Schwarz iteration is applied to match the flow solution at the subdomain boundaries. For the transport model, an extended numerical array is implemented to permit the exchange of dispersive and advective flux information across subdomain boundaries. The model is verified using a conventional single-domain model. Model simulations demonstrate that the proposed model operated in a parallel computing environment can result in considerable savings in computer run times (between 50% and 80%) compared with conventional modeling approaches and may be used to simulate grid discretizations that were formerly intractable.

  10. Galaxy And Mass Assembly (GAMA): The large scale structure of galaxies and comparison to mock universes

    CERN Document Server

    Alpaslan, Mehmet; Driver, Simon; Norberg, Peder; Baldry, Ivan; Bauer, Amanda E; Bland-Hawthorn, Joss; Brown, Michael; Cluver, Michelle; Colless, Matthew; Foster, Caroline; Hopkins, Andrew; Van Kampen, Eelco; Kelvin, Lee; Lara-Lopez, Maritza A; Liske, Jochen; Lopez-Sanchez, Angel R; Loveday, Jon; McNaught-Roberts, Tamsyn; Merson, Alexander; Pimbblet, Kevin


    From a volume limited sample of 45,542 galaxies and 6,000 groups with $z \\leq 0.213$ we use an adapted minimal spanning tree algorithm to identify and classify large scale structures within the Galaxy and Mass Assembly (GAMA) survey. Using galaxy groups, we identify 643 filaments across the three equatorial GAMA fields that span up to 200 $h^{-1}$ Mpc in length, each with an average of 8 groups within them. By analysing galaxies not belonging to groups we identify a secondary population of smaller coherent structures composed entirely of galaxies, dubbed `tendrils' that appear to link filaments together, or penetrate into voids, generally measuring around 10 $h^{-1}$ Mpc in length and containing on average 6 galaxies. Finally we are also able to identify a population of isolated void galaxies. By running this algorithm on GAMA mock galaxy catalogues we compare the characteristics of large scale structure between observed and mock data; finding that mock filaments reproduce observed ones extremely well. This p...

  11. Loss-of-function mutations of retromer large subunit genes suppress the phenotype of an Arabidopsis zig mutant that lacks Qb-SNARE VTI11. (United States)

    Hashiguchi, Yasuko; Niihama, Mitsuru; Takahashi, Tetsuya; Saito, Chieko; Nakano, Akihiko; Tasaka, Masao; Morita, Miyo Terao


    Arabidopsis thaliana zigzag (zig) is a loss-of-function mutant of Qb-SNARE VTI11, which is involved in membrane trafficking between the trans-Golgi network and the vacuole. zig-1 exhibits abnormalities in shoot gravitropism and morphology. Here, we report that loss-of-function mutants of the retromer large subunit partially suppress the zig-1 phenotype. Moreover, we demonstrate that three paralogous VPS35 genes of Arabidopsis have partially overlapping but distinct genetic functions with respect to zig-1 suppression. Tissue-specific complementation experiments using an endodermis-specific SCR promoter show that expression of VPS35B or VPS35C cannot complement the function of VPS35A. The data suggest the existence of functionally specialized paralogous VPS35 genes that nevertheless share common functions.

  12. Loss-of-Function Mutations of Retromer Large Subunit Genes Suppress the Phenotype of an Arabidopsis zig Mutant That Lacks Qb-SNARE VTI11[C][W (United States)

    Hashiguchi, Yasuko; Niihama, Mitsuru; Takahashi, Tetsuya; Saito, Chieko; Nakano, Akihiko; Tasaka, Masao; Morita, Miyo Terao


    Arabidopsis thaliana zigzag (zig) is a loss-of-function mutant of Qb-SNARE VTI11, which is involved in membrane trafficking between the trans-Golgi network and the vacuole. zig-1 exhibits abnormalities in shoot gravitropism and morphology. Here, we report that loss-of-function mutants of the retromer large subunit partially suppress the zig-1 phenotype. Moreover, we demonstrate that three paralogous VPS35 genes of Arabidopsis have partially overlapping but distinct genetic functions with respect to zig-1 suppression. Tissue-specific complementation experiments using an endodermis-specific SCR promoter show that expression of VPS35B or VPS35C cannot complement the function of VPS35A. The data suggest the existence of functionally specialized paralogous VPS35 genes that nevertheless share common functions. PMID:20086190

  13. Efficient expression of functional (α6β22β3 AChRs in Xenopus oocytes from free subunits using slightly modified α6 subunits.

    Directory of Open Access Journals (Sweden)

    Carson Kai-Kwong Ley

    Full Text Available Human (α6β2(α4β2β3 nicotinic acetylcholine receptors (AChRs are essential for addiction to nicotine and a target for drug development for smoking cessation. Expressing this complex AChR is difficult, but has been achieved using subunit concatamers. In order to determine what limits expression of α6* AChRs and to efficiently express α6* AChRs using free subunits, we investigated expression of the simpler (α6β22β3 AChR. The concatameric form of this AChR assembles well, but is transported to the cell surface inefficiently. Various chimeras of α6 with the closely related α3 subunit increased expression efficiency with free subunits and produced pharmacologically equivalent functional AChRs. A chimera in which the large cytoplasmic domain of α6 was replaced with that of α3 increased assembly with β2 subunits and transport of AChRs to the oocyte surface. Another chimera replacing the unique methionine 211 of α6 with leucine found at this position in transmembrane domain 1 of α3 and other α subunits increased assembly of mature subunits containing β3 subunits within oocytes. Combining both α3 sequences in an α6 chimera increased expression of functional (α6β22β3 AChRs to 12-fold more than with concatamers. This is pragmatically useful, and provides insights on features of α6 subunit structure that limit its expression in transfected cells.

  14. Fluorescence enhancement in large-scale self-assembled gold nanoparticle double arrays

    Energy Technology Data Exchange (ETDEWEB)

    Chekini, M.; Bierwagen, J.; Cunningham, A.; Bürgi, T., E-mail: [Département de Chimie Physique, Université de Genève, 1211 Genève (Switzerland); Filter, R. [Institute of Condensed Matter Theory and Solid State Optics, Abbe Center of Photonics, Friedrich-Schiller-Universität Jena, D-07743 Jena (Germany); Rockstuhl, C. [Institute of Nanotechnology, Karlsruhe Institute of Technology, D-76021 Karlsruhe (Germany); Institute of Theoretical Solid State Physics, Karlsruhe Institute of Technology, D-76131 Karlsruhe (Germany)


    Localized surface plasmon resonances excited in metallic nanoparticles confine and enhance electromagnetic fields at the nanoscale. This is particularly pronounced in dimers made from two closely spaced nanoparticles. When quantum emitters, such as dyes, are placed in the gap of those dimers, their absorption and emission characteristics can be modified. Both processes have to be considered when aiming to enhance the fluorescence from the quantum emitters. This is particularly challenging for dimers, since the electromagnetic properties and the enhanced fluorescence sensitively depend on the distance between the nanoparticles. Here, we use a layer-by-layer method to precisely control the distances in such systems. We consider a dye layer deposited on top of an array of gold nanoparticles or integrated into a central position of a double array of gold nanoparticles. We study the effect of the spatial arrangement and the average distance on the plasmon-enhanced fluorescence. We found a maximum of a 99-fold increase in the fluorescence intensity of the dye layer sandwiched between two gold nanoparticle arrays. The interaction of the dye layer with the plasmonic system also causes a spectral shift in the emission wavelengths and a shortening of the fluorescence life times. Our work paves the way for large-scale, high throughput, and low-cost self-assembled functionalized plasmonic systems that can be used as efficient light sources.

  15. Mutation of Gly195 of the ChlH Subunit of Mg-chelatase Reduces Chlorophyll and Further Disrupts PS II Assembly in a Ycf48-Deficient Strain of Synechocystis sp. PCC 6803 (United States)

    Crawford, Tim S.; Eaton-Rye, Julian J.; Summerfield, Tina C.


    Biogenesis of the photosystems in oxygenic phototrophs requires co-translational insertion of chlorophyll a. The first committed step of chlorophyll a biosynthesis is the insertion of a Mg2+ ion into the tetrapyrrole intermediate protoporphyrin IX, catalyzed by Mg-chelatase. We have identified a Synechocystis sp. PCC 6803 strain with a spontaneous mutation in chlH that results in a Gly195 to Glu substitution in a conserved region of the catalytic subunit of Mg-chelatase. Mutant strains containing the ChlH Gly195 to Glu mutation were generated using a two-step protocol that introduced the chlH gene into a putative neutral site in the chromosome prior to deletion of the native gene. The Gly195 to Glu mutation resulted in strains with decreased chlorophyll a. Deletion of the PS II assembly factor Ycf48 in a strain carrying the ChlH Gly195 to Glu mutation did not grow photoautotrophically. In addition, the ChlH-G195E:ΔYcf48 strain showed impaired PS II activity and decreased assembly of PS II centers in comparison to a ΔYcf48 strain. We suggest decreased chlorophyll in the ChlH-G195E mutant provides a background to screen for the role of assembly factors that are not essential under optimal growth conditions. PMID:27489555

  16. Association of Common Polymorphisms in the Nicotinic Acetylcholine Receptor Alpha4 Subunit Gene with an Electrophysiological Endophenotype in a Large Population-Based Sample.

    Directory of Open Access Journals (Sweden)

    A Mobascher

    Full Text Available Variation in genes coding for nicotinic acetylcholine receptor (nAChR subunits affect cognitive processes and may contribute to the genetic architecture of neuropsychiatric disorders. Single nucleotide polymorphisms (SNPs in the CHRNA4 gene that codes for the alpha4 subunit of alpha4/beta2-containing receptors have previously been implicated in aspects of (mostly visual attention and smoking-related behavioral measures. Here we investigated the effects of six synonymous but functional CHRNA4 exon 5 SNPs on the N100 event-related potential (ERP, an electrophysiological endophenotype elicited by a standard auditory oddball. A total of N = 1,705 subjects randomly selected from the general population were studied with electroencephalography (EEG as part of the German Multicenter Study on nicotine addiction. Two of the six variants, rs1044396 and neighboring rs1044397, were significantly associated with N100 amplitude. This effect was pronounced in females where we also observed an effect on reaction time. Sequencing of the complete exon 5 region in the population sample excluded the existence of additional/functional variants that may be responsible for the observed effects. This is the first large-scale population-based study investigation the effects of CHRNA4 SNPs on brain activity measures related to stimulus processing and attention. Our results provide further evidence that common synonymous CHRNA4 exon 5 SNPs affect cognitive processes and suggest that they also play a role in the auditory system. As N100 amplitude reduction is considered a schizophrenia-related endophenotype the SNPs studied here may also be associated with schizophrenia outcome measures.

  17. Exploring the Transferability of Large Supramolecular Assemblies to the Vacuum-Solid Interface

    DEFF Research Database (Denmark)

    Xu, W.; Dong, M. D.; Gersen, H.;


    We present an interplay of high-resolution scanning tunneling microscopy imaging and the corresponding theoretical calculations based on elastic scattering quantum chemistry techniques of the adsorption of a gold-functionalized rosette assembly and its building blocks on a Au(111) surface...... from those formed by the individual molecular building blocks of the rosette assembly, suggesting that the assembly itself can be transferred intact to the surface by in situ thermal sublimation. This unanticipated result will open up new perspectives for growth of complex 3-D supramolecular...

  18. Gut microbiota dysbiosis and bacterial community assembly associated with cholesterol gallstones in large-scale study. (United States)

    Wu, Tao; Zhang, Zhigang; Liu, Bin; Hou, Dezhi; Liang, Yun; Zhang, Jie; Shi, Peng


    Elucidating gut microbiota among gallstone patients as well as the complex bacterial colonization of cholesterol gallstones may help in both the prediction and subsequent lowered risk of cholelithiasis. To this end, we studied the composition of bacterial communities of gut, bile, and gallstones from 29 gallstone patients as well as the gut of 38 normal individuals, examining and analyzing some 299, 217 bacterial 16S rRNA gene sequences from 120 samples. First, as compared with normal individuals, in gallstone patients there were significant (P gut bacterial phylum Proteobacteria and decreases of three gut bacterial genera, Faecalibacterium, Lachnospira, and Roseburia. Second, about 70% of gut bacterial operational taxonomic units (OTUs) from gallstone patients were detectable in the biliary tract and bacteria diversity of biliary tract was significantly (P gut. Third, analysis of the biliary tract core microbiome (represented by 106 bacteria OTUs) among gallstone patients showed that 33.96% (36/106) of constituents can be matched to known bacterial species (15 of which have publicly available genomes). A genome-wide search of MDR, BSH, bG, and phL genes purpotedly associated with the formation of cholesterol gallstones showed that all 15 species with known genomes (e.g., Propionibacterium acnes, Bacteroides vulgates, and Pseudomonas putida) contained at least contained one of the four genes. This finding could potentially provide underlying information needed to explain the association between biliary tract microbiota and the formation of cholesterol gallstones. To the best of our knowledge, this is the first study to discover gut microbiota dysbiosis among gallstone patients, the presence of which may be a key contributor to the complex bacteria community assembly linked with the presence of cholesterol gallstones. Likewise, this study also provides the first large-scale glimpse of biliary tract microbiota potentially associated with cholesterol gallstones. Such

  19. Large-scale self-assembled zirconium phosphate smectic layers via a simple spray-coating process. (United States)

    Wong, Minhao; Ishige, Ryohei; White, Kevin L; Li, Peng; Kim, Daehak; Krishnamoorti, Ramanan; Gunther, Robert; Higuchi, Takeshi; Jinnai, Hiroshi; Takahara, Atsushi; Nishimura, Riichi; Sue, Hung-Jue


    The large-scale assembly of asymmetric colloidal particles is used in creating high-performance fibres. A similar concept is extended to the manufacturing of thin films of self-assembled two-dimensional crystal-type materials with enhanced and tunable properties. Here we present a spray-coating method to manufacture thin, flexible and transparent epoxy films containing zirconium phosphate nanoplatelets self-assembled into a lamellar arrangement aligned parallel to the substrate. The self-assembled mesophase of zirconium phosphate nanoplatelets is stabilized by epoxy pre-polymer and exhibits rheology favourable towards large-scale manufacturing. The thermally cured film forms a mechanically robust coating and shows excellent gas barrier properties at both low- and high humidity levels as a result of the highly aligned and overlapping arrangement of nanoplatelets. This work shows that the large-scale ordering of high aspect ratio nanoplatelets is easier to achieve than previously thought and may have implications in the technological applications for similar materials.


    Directory of Open Access Journals (Sweden)

    Ahyar Ahmad


    Full Text Available We cloned and sequenced cDNA encoding p48 subunit of the chicken CAF-1, chCAF-1p48, and histone acetyltransferase-1, chHAT-1 from chicken DT40 cell lines. We showed that the p48 subunit of CAF-1 tightly binds to two regions of chicken histone deacetylase 2, chHDAC-2, located between amino acid residues 82-180 and 245-314, respectively. We also established that two N-terminal, two C-terminal, or one N-terminal and one C-terminal WD repeat motif of chCAF-1p48 are required for this interaction. The GST pulldown assay, involving truncated and missense mutants of chCAF-1p48, revealed not only that a region containing the seventh WD dipeptide motif of chCAF-1p48, comprising amino acids 376-405, binds to chHAT-1 in vitro, but also that mutation of the motif has no influence on the in vitro interaction. We also established that the region, which is located between amino acids 380-408 of chHAT-1 and contains a leucine zipper motif, is required for its in vitro interaction with chCAF-1p48. Mutation on each of four Leu residues in the leucine zipper motif of chHAT-1 causes the disappearance of the interaction with chCAF-1p48. These results should be useful information for understanding the participation of chCAF-1p48 protein as histones chaperone in DNA-utilizing processes, such as replication, recombination, repair and gene expression in DT40 chicken B cell.

  1. Phylogenetic analysis of Tilletia and allied genera in order Tilletiales (Ustilaginomycetes; Exobasidiomycetidae) based on large subunit nuclear rDNA sequences. (United States)

    Castlebury, Lisa A; Carris, Lori M; Vinky, Kálmán


    The order Tilletiales (Ustilaginomycetes, Basidiomycota) includes six genera (Conidiosporomyces, Erratomyces, Ingoldiomyces, Neovossia, Oberwinkleria and Tilletia) and approximately 150 species. All members of Tilletiales infect hosts in the grass family Poaceae with the exception of Erratomyces spp., which occur on hosts in the Fabaceae. Morphological features including teliospore ornamentation, number and nuclear condition of primary basidiospores and ability of primary basidiospores to conjugate and form an infective dikaryon were studied in conjunction with sequence analysis of the large subunit nuclear rDNA gene (nLSU). Analysis based on nLSU data shows that taxa infecting hosts in the grass subfamily Pooideae form one well supported lineage. This lineage comprises most of the reticulate-spored species that germinate to form a small number of rapidly conjugating basidiospores and includes the type species Tilletia tritici. Two tuberculate-spored species with a large number of nonconjugating basidiospores, T. indica and T. walkeri, and Ingoldiomyces hyalosporus are also included in this lineage. Most of the species included in the analysis with echinulate, verrucose or tuberculate teliospores that germinate to form a large number (>30) of nonconjugating basidiospores infect hosts in the subfamilies Panicoideae, Chloridoideae, Arundinoideae and Ehrhartoideae. This group of species is more diverse than the pooid-infecting taxa and in general do not form well supported clades corresponding to host subfamily. The results of this work suggest that morphological characters used to segregate Neovossia, Conidiosporomyces and Ingoldiomyces from Tilletia are not useful generic level characters and that all included species can be accommodated in the genus Tilletia.

  2. Synthesis of Nitrogen-Doped Mesoporous Carbon Spheres with Extra-Large Pores through Assembly of Diblock Copolymer Micelles

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Jing [Waseda University, Tokyo, Japan; Liu, Jiang [Curtin University of Technology, Perth, Australia; Li, Cuiling [KEK, Tsukuba, Ibaraki, Japan; Li, Yunqi [Waseda University, Tokyo, Japan; Tade, Moses O. [Curtin University of Technology, Perth, Australia; Dai, Sheng [ORNL; Yamauchi, Yusuke [Waseda University, Tokyo, Japan


    In this study, the synthesis of highly nitrogen-doped mesoporous carbon spheres (NMCS) is reported. The large pores of the NMCS were obtained through self-polymerization of dopamine (DA) and spontaneous co-assembly of diblock copolymer micelles. The resultant narrowly dispersed NMCS possess large mesopores (ca. 16 nm) and small particle sizes (ca. 200 nm). Lastly, the large pores and small dimensions of the N-heteroatom-doped carbon spheres contribute to the mass transportation by reducing and smoothing the diffusion pathways, leading to high electrocatalytic activity.

  3. Expression of the gene for large subunit of m-calpain is elevated in skeletal muscle from Duchenne muscular dystrophy patients

    Indian Academy of Sciences (India)

    Tajamul Hussain; Harleen Mangath; C. Sundaram; M. P. J. S. Anandaraj


    Calpain is an intracellular nonlysosomal protease involved in essential regulatory or processing functions of the cell, mediated by physiological concentrations of Ca2+. However, in an environment of abnormal intracellular calcium, such as that seen in Duchenne muscular dystrophy (DMD), calpain is suggested to cause degeneration of muscle owing to enhanced activity. To test whether the reported increase in calpain activity in DMD results from de novo synthesis of the protease, we have assessed the quantitative changes in mRNA specific for m-calpain. mRNA isolated from DMD and control muscle was analysed by dot blot hybridization using a cDNA probe for the large subunit of m-calpain. Compared to control a four-fold increase in specific mRNAwas observed in dystrophic muscle. This enhanced expression of the m-calpain gene in dystrophic condition suggests that the reported increase in m-calpain activity results from de novo synthesis of protease and underlines the important role of m-calpain in DMD.

  4. Restriction Fragment Length Polymorphism Analysis of Large Subunit rDNA of Symbiotic Dinoflagellates from Scleractinian Corals in the Zhubi Coral Reef of the Nansha Islands

    Institute of Scientific and Technical Information of China (English)


    Zooxanthellae are very important for the coral reef ecosystem. The diversity of coral hosts is high in the South China Sea, but the diversity of zooxanthellae has not yet been investigated. We chose the Zhubi Coral Reef of the Nansha Islands as the region to be surveyed in the present study because it represents a typical tropical coral reef of the South China Sea and we investigated zooxanthellae diversity in 10 host scleractinian coral species using polymerase chain reaction (PCR) of the large subunit rRNA and restriction fragment length polymorphism (RFLP) patterns. Pocillopora verrucosa, Acropora pelifera, Acropora millepora, Fungia fungites, Galaxea fascicularis, and Acropora pruinosa harbor Clade C, Goniastrea aspera harbors Clade D, and Acropora formosa harbors Clades D and C. Therefore, the Clade C is the dominant type in the Zhubi Coral Reef of the NanshaIslands. Furthermore, the results of the present also disprove what has been widely accepted, namely that one coral host harbors only one algal symbiont. The coral-algal symbiosis is flexible, which may be an important mechanism for surviving coral bleaching. Meanwhile, on the basis of the results of the present study, we think that Symbiodinium Clade D may be more tolerant to stress than Symbiodinium Clade C.

  5. The mitochondrial ribosomal protein of the large subunit, Afo1p, determines cellular longevity through mitochondrial back-signaling via TOR1. (United States)

    Heeren, Gino; Rinnerthaler, Mark; Laun, Peter; von Seyerl, Phyllis; Kössler, Sonja; Klinger, Harald; Hager, Matthias; Bogengruber, Edith; Jarolim, Stefanie; Simon-Nobbe, Birgit; Schüller, Christoph; Carmona-Gutierrez, Didac; Breitenbach-Koller, Lore; Mück, Christoph; Jansen-Dürr, Pidder; Criollo, Alfredo; Kroemer, Guido; Madeo, Frank; Breitenbach, Michael


    Yeast mother cell-specific aging constitutes a model of replicative aging as it occurs in stem cell populations of higher eukaryotes. Here, we present a new long-lived yeast deletion mutation,afo1 (for aging factor one), that confers a 60% increase in replicative lifespan. AFO1/MRPL25 codes for a protein that is contained in the large subunit of the mitochondrial ribosome. Double mutant experiments indicate that the longevity-increasing action of the afo1 mutation is independent of mitochondrial translation, yet involves the cytoplasmic Tor1p as well as the growth-controlling transcription factor Sfp1p. In their final cell cycle, the long-lived mutant cells do show the phenotypes of yeast apoptosis indicating that the longevity of the mutant is not caused by an inability to undergo programmed cell death. Furthermore, the afo1 mutation displays high resistance against oxidants. Despite the respiratory deficiency the mutant has paradoxical increase in growth rate compared to generic petite mutants. A comparison of the single and double mutant strains for afo1 and fob1 shows that the longevity phenotype of afo1 is independent of the formation of ERCs (ribosomal DNA minicircles). AFO1/MRPL25 function establishes a new connection between mitochondria, metabolism and aging.

  6. Isolation and phylogenetic relationship of orchid-mycorrhiza from Spathoglottis plicata of Papua using mitochondrial ribosomal large subunit (mt-Ls DNA

    Directory of Open Access Journals (Sweden)



    Full Text Available Sufaati S, Agustini V, Suharno. 2012. Isolation and phylogenetic relationship of orchid-mycorrhiza from Spathoglottis plicata of Papua using mitochondrial ribosomal large subunit (mt-Ls DNA. Biodiversitas 13: 59-64. All terrestrial mycorrhiza have mutual symbiotic with mycorrhizal fungi in order to gain nutrient from surrounding environment. This study was done to isolate and to identify mycorrhiza orchid that associates with Spathoglottis plicata and were collected from Cagar Alam Pegunungan Cycloops (CAPC, Jayapura. Isolation of mycorrhizal orchid came after the modified method of Manoch and Lohsomboon (1991. The result showed that based on the morphological characteristic, there was presumably 14 isolations. However, only 2 isolations have been known, namely Rhizoctonia sp. and Tulasnella sp., while the rest were not identified yet. Among them, the DNA of the 11 isolations were able to be extracted for further analysis. The constructed phylogenetic tree performed that those species could be grouped into 4 major clusters. Two species, Rhizoctonia sp. and Tulasnella sp. were in different clusters.

  7. Chlamydophila pneumoniae HflX belongs to an uncharacterized family of conserved GTPases and associates with the Escherichia coli 50S large ribosomal subunit. (United States)

    Polkinghorne, Adam; Ziegler, Urs; González-Hernández, Yanela; Pospischil, Andreas; Timms, Peter; Vaughan, Lloyd


    Predicted members of the HflX subfamily of phosphate-binding-loop guanosine triphosphatases (GTPases) are widely distributed in the bacterial kingdom but remain virtually uncharacterized. In an attempt to understand mechanisms used for regulation of growth and development in the chlamydiae, obligate intracellular and developmentally complex bacteria, we have begun investigations into chlamydial GTPases; we report here what appears to be the first analysis of a HflX family GTPase using a predicted homologue from Chlamydophila pneumoniae. In agreement with phylogenetic predictions for members of this GTPase family, purified recombinant Cp. pneumoniae HflX was specific for guanine nucleotides and exhibited a slow intrinsic GTPase activity when incubated with [gamma-(32)P]GTP. Using HflX-specific monoclonal antibodies, HflX could be detected by Western blotting and high-resolution confocal microscopy throughout the vegetative growth cycle of Cp. pneumoniae and, at early time points, appeared to partly localize to the membrane. Ectopic expression of Cp. pneumoniae HflX in Escherichia coli revealed co-sedimentation of HflX with the E. coli 50S large ribosomal subunit. The results of this work open up some intriguing possibilities for the role of GTPases belonging to this previously uncharacterized family of bacterial GTPases. Ribosome association is a feature shared by other important conserved GTPase families and more detailed investigations will be required to delineate the role of HflX in bacterial ribosome function.

  8. Phylogenetic diversity of ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit genes of bacterioplankton in the East China Sea

    Institute of Scientific and Technical Information of China (English)

    ZENG Yonghui; JIAO Nianzhi; CAI Haiyuan; CHEN Xihan; WEI Chaoling


    Phylogenetic diversity of Form I and Form Ⅱ ribulose-1, 5-bisphosphate carboxylase/oxygenase (RubisCO) large subunit (rbcL) genes in the inshore and offshore areas of the East China Sea were investigated. Two new primer sets were designed for amplifying partial sequences of rbcL genes from Proteobacteria. Four rbcL gene clone libraries were constructed by amplification and cloning of approximately 640~800 bp sequences of bacterioplankton populations.The method of screening library by denaturing gradient gel electrophoresis (DGGE) was introduced. The results show that the diversity of Form I is higher in offshore waters with higher salinity and lower productivity, while that of Form Ⅱ is higher at the inshore station where salinity is lower and productivity is higher. Several clusters of sequences obtained are deeply rooted and show low similarity (60%~78%) to the known rbcL in existing databases.The degree of diversity of rbcL genes is directly related to environmental variables, including temperature, salinity,pH, dissolved oxygen, etc. These results indicate that rbcL gene can be used as an effective indicator for genetic diversity and population variability of bacterioplankton with the ability of carbon dioxide fixation in the sea.

  9. Expression of fission yeast cdc25 driven by the wheat ADP-glucose pyrophosphorylase large subunit promoter reduces pollen viability and prevents transmission of the transgene in wheat. (United States)

    Chrimes, D; Rogers, H J; Francis, D; Jones, H D; Ainsworth, C


    Cell number was to be measured in wheat (Triticum aestivum) endosperm expressing Spcdc25 (a fission yeast cell-cycle regulator) controlled by a supposedly endosperm-specific promoter, AGP2 (from the large subunit of ADP glucose pyrophosphorylase). Wheat was transformed by biolistics either with AGP2::GUS or AGP2::Spcdc25. PCR and RT-PCR checked integration and expression of the transgene, respectively. In cv. Chinese Spring, AGP2::GUS was unexpectedly expressed in carpels and pollen, as well as endosperm. In cv. Cadenza, three AGP2::Spcdc25 plants, AGP2::Spcdc25.1, .2 and .3, were generated. Spcdc25 expression was detected in mature leaves of AGP2::Spcdc25.1/.3 which exhibited abnormal spikes, 50% pollen viability and low seed set per plant; both were small compared with the nonexpressing and normal AGP2::Spcdc25.2. Spcdc25 was not transmitted to the T(1) in AGP2::Spcdc25.1 or .3, which developed normally. Spcdc25 was PCR-positive in AGP2::Spcdc25.2, using primers for a central portion, but not with primers for the 5' end, of the ORF, indicating a rearrangement; Spcdc25 was not expressed in either T(0) or T(1). The AGP2 promoter is not tissue-specific and Spcdc25 expression disrupted reproduction.

  10. Shared Subunits of Tetrahymena Telomerase Holoenzyme and Replication Protein A Have Different Functions in Different Cellular Complexes. (United States)

    Upton, Heather E; Chan, Henry; Feigon, Juli; Collins, Kathleen


    In most eukaryotes, telomere maintenance relies on telomeric repeat synthesis by a reverse transcriptase named telomerase. To synthesize telomeric repeats, the catalytic subunit telomerase reverse transcriptase (TERT) uses the RNA subunit (TER) as a template. In the ciliate Tetrahymena thermophila, the telomerase holoenzyme consists of TER, TERT, and eight additional proteins, including the telomeric repeat single-stranded DNA-binding protein Teb1 and its heterotrimer partners Teb2 and Teb3. Teb1 is paralogous to the large subunit of the general single-stranded DNA binding heterotrimer replication protein A (RPA). Little is known about the function of Teb2 and Teb3, which are structurally homologous to the RPA middle and small subunits, respectively. Here, epitope-tagging Teb2 and Teb3 expressed at their endogenous gene loci enabled affinity purifications that revealed that, unlike other Tetrahymena telomerase holoenzyme subunits, Teb2 and Teb3 are not telomerase-specific. Teb2 and Teb3 assembled into other heterotrimer complexes, which when recombinantly expressed had the general single-stranded DNA binding activity of RPA complexes, unlike the telomere-specific DNA binding of Teb1 or the TEB heterotrimer of Teb1, Teb2, and Teb3. TEB had no more DNA binding affinity than Teb1 alone. In contrast, heterotrimers reconstituted with Teb2 and Teb3 and two other Tetrahymena RPA large subunit paralogs had higher DNA binding affinity than their large subunit alone. Teb1 and TEB, but not RPA, increased telomerase processivity. We conclude that in the telomerase holoenzyme, instead of binding DNA, Teb2 and Teb3 are Teb1 assembly factors. These findings demonstrate that Tetrahymena telomerase holoenzyme and RPA complexes share subunits and that RPA subunits have distinct functions in different heterotrimer assemblies. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Expression and assembly of active human cardiac troponin in Escherichia coli. (United States)

    Lassalle, Michael W


    Cardiomyopathy-related mutations in human cardiac troponin subunits, including troponin C (hcTnC), troponin I (hcTnI), and troponin T (hcTnT), are well-documented. Recently, it has been recognised that human cardiac troponin (hcTn) is a sophisticated allosteric system. Therefore, the effect of drugs on this protein complex should be studied with assembled hcTn rather than a short fragment of a subunit or the subunit itself. Here, we describe the expression and assembly of active hcTn in Escherichia coli, a novel method that is rapid and simple, and produces large amounts of functional hcTn.

  12. Molecular identification of veterinary yeast isolates by use of sequence-based analysis of the D1/D2 region of the large ribosomal subunit. (United States)

    Garner, Cherilyn D; Starr, Jennifer K; McDonough, Patrick L; Altier, Craig


    Conventional methods of yeast identification are often time-consuming and difficult; however, recent studies of sequence-based identification methods have shown promise. Additionally, little is known about the diversity of yeasts identified from various animal species in veterinary diagnostic laboratories. Therefore, in this study, we examined three methods of identification by using 109 yeast samples isolated during a 1-year period from veterinary clinical samples. Comparison of the three methods-traditional substrate assimilation, fatty acid profile analysis, and sequence-based analysis of the region spanning the D1 and D2 regions (D1/D2) of the large ribosomal subunit-showed that sequence analysis provided the highest percent identification among the three. Sequence analysis identified 87% of isolates to the species level, whereas substrate assimilation and fatty acid profile analysis identified only 54% and 47%, respectively. Less-stringent criteria for identification increased the percentage of isolates identified to 98% for sequence analysis, 62% for substrate assimilation, and 55% for fatty acid profile analysis. We also found that sequence analysis of the internal transcribed spacer 2 (ITS2) region provided further identification for 36% of yeast not identified to the species level by D1/D2 sequence analysis. Additionally, we identified a large variety of yeast from animal sources, with at least 30 different species among the isolates tested, and with the majority not belonging to the common Candida spp., such as C. albicans, C. glabrata, C. tropicalis, and the C. parapsilosis group. Thus, we determined that sequence analysis of the D1/D2 region was the best method for identification of the variety of yeasts found in a veterinary population.

  13. Self-assembled block copolymer membranes: From basic research to large-scale manufacturing

    KAUST Repository

    Nunes, Suzana Pereira


    Order and porosity of block copolymer membranes have been controlled by solution thermodynamics, self-assembly, and macrophase separation. We have demonstrated how the film manufacture with long-range order can be up-scaled with the use of conventional membrane production technology.

  14. Non-equivalent roles of two periplasmic subunits in the function and assembly of triclosan pump TriABC from Pseudomonas aeruginosa. (United States)

    Weeks, Jon W; Nickels, Logan M; Ntreh, Abigail T; Zgurskaya, Helen I


    In Gram-negative bacteria, multidrug efflux transporters function in complexes with periplasmic membrane fusion proteins (MFPs) that enable antibiotic efflux across the outer membrane. In this study, we analyzed the function, composition and assembly of the triclosan efflux transporter TriABC-OpmH from Pseudomonas aeruginosa. We report that this transporter possesses a surprising substrate specificity that encompasses not only triclosan but the detergent SDS, which are often used together in antibacterial soaps. These two compounds interact antagonistically in a TriABC-dependent manner and negate antibacterial properties of each other. Unlike other efflux pumps that rely on a single MFP for their activities, two different MFPs, TriA and TriB, are required for triclosan/SDS resistance mediated by TriABC-OpmH. We found that analogous mutations in the α-helical hairpin and membrane proximal domains of TriA and TriB differentially affect triclosan efflux and assembly of the complex. Furthermore, our results show that TriA and TriB function as a dimer, in which TriA is primarily responsible for stabilizing interactions with the outer membrane channel, whereas TriB is important for the stimulation of the transporter. We conclude that MFPs are engaged into complexes as asymmetric dimers, in which each protomer plays a specific role.

  15. Assembly of the small outer capsid protein, Soc, on bacteriophage T4: a novel system for high density display of multiple large anthrax toxins and foreign proteins on phage capsid. (United States)

    Li, Qin; Shivachandra, Sathish B; Zhang, Zhihong; Rao, Venigalla B


    Bacteriophage T4 capsid is a prolate icosahedron composed of the major capsid protein gp23*, the vertex protein gp24*, and the portal protein gp20. Assembled on its surface are 810 molecules of the non-essential small outer capsid protein, Soc (10 kDa), and 155 molecules of the highly antigenic outer capsid protein, Hoc (39 kDa). In this study Soc, a "triplex" protein that stabilizes T4 capsid, is targeted for molecular engineering of T4 particle surface. Using a defined in vitro assembly system, anthrax toxins, protective antigen, lethal factor and their domains, fused to Soc were efficiently displayed on the capsid. Both the N and C termini of the 80 amino acid Soc polypeptide can be simultaneously used to display antigens. Proteins as large as 93 kDa can be stably anchored on the capsid through Soc-capsid interactions. Using both Soc and Hoc, up to 1662 anthrax toxin molecules are assembled on the phage T4 capsid under controlled conditions. We infer from the binding data that a relatively high affinity capsid binding site is located in the middle of the rod-shaped Soc, with the N and C termini facing the 2- and 3-fold symmetry axes of the capsid, respectively. Soc subunits interact at these interfaces, gluing the adjacent capsid protein hexamers and generating a cage-like outer scaffold. Antigen fusion does interfere with the inter-subunit interactions, but these interactions are not essential for capsid binding and antigen display. These features make the T4-Soc platform the most robust phage display system reported to date. The study offers insights into the architectural design of bacteriophage T4 virion, one of the most stable viruses known, and how its capsid surface can be engineered for novel applications in basic molecular biology and biotechnology.

  16. Nanostructured grating patterns over a large area fabricated by optically directed assembly (United States)

    Huang, Xiaoping; Chen, Kai; Qi, Mingxi; Li, Yu; Hou, Yumeng; Wang, Ying; Zhao, Qing; Luo, Xiangang; Xu, Qingyu


    Optical trapping and manipulation of nanoparticles (NPs) have been widely used in nanotechnology and biology. Here, we demonstrate an optically directed assembly (ODA) route for bottom-up fabrication of stable nanostructured grating patterns in solution using laser standing evanescent wave (LSEW) fields. The control mechanism is the intriguing cooperative action of the periodically line-centered attractive optical gradient force and the near field dipolar coupling force induced by LSEW, which leads to assembly of the colloidal silver NPs into robust grating patterns within minutes. The anisotropic polarization nature of the grating patterns was studied further by examining the morphology correlation of the surface-enhanced Raman scattering (SERS)-based signal amplification. We show the LSEW ODA method can optimize and stabilize the strongest dipolar coupling style among the NPs during pattern assembly. These results advance the further understanding of ODA of colloid NPs and might have many potential applications in SERS, catalysis, nanophotonics and nano-fabrication.Optical trapping and manipulation of nanoparticles (NPs) have been widely used in nanotechnology and biology. Here, we demonstrate an optically directed assembly (ODA) route for bottom-up fabrication of stable nanostructured grating patterns in solution using laser standing evanescent wave (LSEW) fields. The control mechanism is the intriguing cooperative action of the periodically line-centered attractive optical gradient force and the near field dipolar coupling force induced by LSEW, which leads to assembly of the colloidal silver NPs into robust grating patterns within minutes. The anisotropic polarization nature of the grating patterns was studied further by examining the morphology correlation of the surface-enhanced Raman scattering (SERS)-based signal amplification. We show the LSEW ODA method can optimize and stabilize the strongest dipolar coupling style among the NPs during pattern assembly

  17. Multicolor, large-area fluorescence sensing through oligothiophene-self-assembled monolayers. (United States)

    Melucci, Manuela; Zambianchi, Massimo; Favaretto, Laura; Palermo, Vincenzo; Treossi, Emanuele; Montalti, Marco; Bonacchi, Sara; Cavallini, Massimiliano


    We present a new strategy to realize self-assembled monolayers (SAMs) on quartz and silicon with a multicolour fluorescence pattern starting from a single, proton sensitive oligothiophene dye exposed at a defined pH. Fine tuning of the SAMs emission color over the entire visible range, including white, is demonstrated. Finally, integration of SAMs in patterned thin layer cells (TLCs) is exploited to demonstrate cation sensing potential in real devices.

  18. Cost effective flip chip assembly and interconnection technologies for large area pixel sensor applications

    Energy Technology Data Exchange (ETDEWEB)

    Fritzsch, T., E-mail: [Fraunhofer IZM, Gustav-Meyer-Allee 25, Berlin 13355 (Germany); Jordan, R.; Oppermann, H. [Fraunhofer IZM, Gustav-Meyer-Allee 25, Berlin 13355 (Germany); Ehrmann, O. [Berlin Institute of Technology (TUB), Berlin 10623 (Germany); Toepper, M.; Baumgartner, T.; Lang, K.-D. [Fraunhofer IZM, Gustav-Meyer-Allee 25, Berlin 13355 (Germany)


    Much of the cost of manufacturing pixel detectors is due to bumping and flip chip assembly of the readout chips onto sensor tiles, even if it is done on wafer level. To address this issue, Fraunhofer IZM investigated two new technological approaches, namely screen printing using dry film resist and chip-to-wafer assembly. In the first approach, solder bumps with diameters of 80 and 25 {mu}m in pitches of 110 and 60 {mu}m, respectively, were produced by screen-printing solder paste using a photo-structured dry film resist. Results indicated that the technology is a viable high yield and low cost bumping process. The second approach was developed to decrease the number of manual handling steps in pixel module manufacturing, which is critical for reducing processing time and cost. Here, chip designs on 200 mm readout chip (ROC) wafers and 150 mm sensor wafers were especially adapted for chip-to-wafer assembly and to ensure that the interconnection yield and reliability could be tested. After bumping and dicing of the readout chip wafer and UBM plating on the sensor wafer, individual dice were flip chip mounted on the pre-diced sensor wafer. This paper describes the technological steps, key processing parameters and first results for both technologies.

  19. Mutations affecting the high affinity ATPase center of gpA, the large subunit of bacteriophage lambda terminase, inactivate the endonuclease activity of terminase. (United States)

    Hwang, Y; Feiss, M


    Phage lambda terminase carries out the cos cleavage reaction that generates mature chromosomes from immature concatemeric DNA. The ATP-stimulated endonuclease activity of terminase is located in gpA, the large terminase subunit. There is a high affinity ATPase center in gpA, and a match to the conserved P-loop of known ATPases is found starting near residue 490. Changing the conserved P-loop lysine at residue 497 of gpA affects the high affinity ATPase activity of terminase. In the present work, mutations causing the gpA changes K497A and K497D were found to be lethal, and phages carrying these mutations were defective in cos cleavage, in vivo. Purified K497A and K497D enzymes cleaved cos in vitro at rates reduced from the wild-type rate by factors of 1000 and 2000, respectively. The strong defects in cos cleavage are sufficient to explain the lethality of the K497A and K497D defects. In in vitro packaging studies using mature (cleaved) phage DNA, the K497A enzyme was indistinguishable from the wild-type enzyme, and the K497D enzyme showed a mild packaging defect under limiting terminase conditions. In a purified DNA packaging system, the wild-type and K497D enzymes showed similar packaging activities that were stimulated to half-maximal levels at about 3 microM ATP, indicating that the K497D change does not affect DNA translocation. In sum, the work indicates that the high affinity ATPase center of gpA is involved in stimulation of the endonuclease activity of terminase.

  20. Dynamic-template-directed multiscale assembly for large-area coating of highly-aligned conjugated polymer thin films (United States)

    Mohammadi, Erfan; Zhao, Chuankai; Meng, Yifei; Qu, Ge; Zhang, Fengjiao; Zhao, Xikang; Mei, Jianguo; Zuo, Jian-Min; Shukla, Diwakar; Diao, Ying


    Solution processable semiconducting polymers have been under intense investigations due to their diverse applications from printed electronics to biomedical devices. However, controlling the macromolecular assembly across length scales during solution coating remains a key challenge, largely due to the disparity in timescales of polymer assembly and high-throughput printing/coating. Herein we propose the concept of dynamic templating to expedite polymer nucleation and the ensuing assembly process, inspired by biomineralization templates capable of surface reconfiguration. Molecular dynamic simulations reveal that surface reconfigurability is key to promoting template-polymer interactions, thereby lowering polymer nucleation barrier. Employing ionic-liquid-based dynamic template during meniscus-guided coating results in highly aligned, highly crystalline donor-acceptor polymer thin films over large area (>1 cm2) and promoted charge transport along both the polymer backbone and the π-π stacking direction in field-effect transistors. We further demonstrate that the charge transport anisotropy can be reversed by tuning the degree of polymer backbone alignment.

  1. Performance of cable isolators in the transport of large optical assemblies (United States)

    Good, John M.; Hill, Gary; Schroeder-Mrozinski, Emily; Lee, Hanshin; Kriel, Herman; Savage, Richard; Benjamin, Scott; Stone, Robert; Frater, Eric


    Following a 7-year, multi-million dollar effort to fabricate a 730 kg, 4 element Wide Field Corrector (WFC) for the Hobby-Eberly Telescope (HET) Wide Field Upgrade (WFU), it needed to be transported 820 km to its destination at the McDonald Observatory in West Texas. The final system optical test for the assembly required repeatability in the +/- 2μm range. Due to the size, mass, and ultimate destination of the payload, the only option available for transport was via roadway on a flat-bed trailer. While the route was primarily interstate highway, it presented a great variety of vibrational inputs due to poor paving conditions, and mountain roadways. Consideration also had be given to avoiding high ambient temperatures. Early in the design of the corrector assembly it was assumed that cable isolators would be the key element to isolate the payload from vibrational inputs, however, few documented references were available to provide the assurances required for transporting a load so key to the success of the telescope program. Tests were designed to simulate the load conditions, and inputs and outputs to the test load were measured for verification of the isolator performance. This was followed up with monitoring of vibration throughput during the actual shipment of the WFC. Upon arrival at the destination, the alignment of the assembly was checked and found to have no appreciable change in the alignment. Data and lessons learned are presented on the performance of air-ride trailers as well as the performance of cable isolators.

  2. ROSA-V large scale test facility (LSTF) system description for the third and fourth simulated fuel assemblies

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Mitsuhiro; Nakamura, Hideo; Ohtsu, Iwao [Japan Atomic Energy Research Inst., Tokai, Ibaraki (Japan). Tokai Research Establishment] [and others


    The Large Scale Test Facility (LSTF) is a full-height and 1/48 volumetrically scaled test facility of the Japan Atomic Energy Research Institute (JAERI) for system integral experiments simulating the thermal-hydraulic responses at full-pressure conditions of a 1100 MWe-class pressurized water reactor (PWR) during small break loss-of-coolant accidents (SBLOCAs) and other transients. The LSTF can also simulate well a next-generation type PWR such as the AP600 reactor. In the fifth phase of the Rig-of-Safety Assessment (ROSA-V) Program, eighty nine experiments have been conducted at the LSTF with the third simulated fuel assembly until June 2001, and five experiments have been conducted with the newly-installed fourth simulated fuel assembly until December 2002. In the ROSA-V program, various system integral experiments have been conducted to certify effectiveness of both accident management (AM) measures in beyond design basis accidents (BDBAs) and improved safety systems in the next-generation reactors. In addition, various separate-effect tests have been conducted to verify and develop computer codes and analytical models to predict non-homogeneous and multi-dimensional phenomena such as heat transfer across the steam generator U-tubes under the presence of non-condensable gases in both current and next-generation reactors. This report presents detailed information of the LSTF system with the third and fourth simulated fuel assemblies for the aid of experiment planning and analyses of experiment results. (author)

  3. Self-assembly of large-scale and ultrathin silver nanoplate films with tunable plasmon resonance properties. (United States)

    Zhang, Xiao-Yang; Hu, Anming; Zhang, Tong; Lei, Wei; Xue, Xiao-Jun; Zhou, Yunhong; Duley, Walt W


    We describe a rapid, simple, room-temperature technique for the production of large-scale metallic thin films with tunable plasmonic properties assembled from size-selected silver nanoplates (SNPs). We outline the properties of a series of ultrathin monolayer metallic films (8-20 nm) self-assembled on glass substrates in which the localized surface plasmon resonance can be tuned over a range from 500 to 800 nm. It is found that the resonance peaks of the films are strongly dependent on the size of the nanoplates and the refractive index of the surrounding dielectric. It is also shown that the bandwidth and the resonance peak of the plasmon resonance spectrum of the metallic films can be engineered by simply controlling aggregation of the SNP. A three-dimensional finite element method was used to investigate the plasmon resonance properties for individual SNPs in different dielectrics and plasmon coupling in SNP aggregates. A 5-17 times enhancement of scattering from these SNP films has been observed experimentally. Our experimental results, together with numerical simulations, indicate that this self-assembly method shows great promise in the production of nanoscale metallic films with enormous electric-field enhancements at visible and near-infrared wavelengths. These may be utilized in biochemical sensing, solar photovoltaic, and optical processing applications.

  4. 3D Point Cloud Data Basis Shape Management for Assembly of Modularized Large and Complicated Marine Structures

    Directory of Open Access Journals (Sweden)

    Deok-Hyun Yoon


    Full Text Available As global competition heats up, in order to improve the productivity, simulation-based methods are becoming increasingly dominant in shipyards. The advancement of the CAD-based production management process even allows verification of installability and functionality before beginning the actual construction. However, whether the ship has been exactly constructed as designed can still and only be manually verified for a limited area. Therefore, significant interblock and intermodule errors are inevitably present in assembly, resulting in costly, time-consuming inspections and modifications. If the construction errors and defects can be investigated and controlled in each shop before assembly of modules, the productivity will be considerably improved. In the installation simulation of large structures, early detection and correction of the errors in junction allow fast and efficient assembly and provide better quality product development even with distributed construction yards. This technique can promote interindustrial collaboration among companies of different sizes, resulting in a significant improvement in overall productivity. In this paper, 3D point cloud data basis shape management framework has been studied with several case studies in a shipyard.

  5. Nature-Inspired Interconnects for Self-Assembled Large-Scale Network-on-Chip Designs

    CERN Document Server

    Teuscher, Christof


    Future nano-scale electronics built up from an Avogadro number of components needs efficient, highly scalable, and robust means of communication in order to be competitive with traditional silicon approaches. In recent years, the Networks-on-Chip (NoC) paradigm emerged as a promising solution to interconnect challenges in silicon-based electronics. Current NoC architectures are either highly regular or fully customized, both of which represent implausible assumptions for emerging bottom-up self-assembled molecular electronics that are generally assumed to have a high degree of irregularity and imperfection. Here, we pragmatically and experimentally investigate important design trade-offs and properties of an irregular, abstract, yet physically plausible 3D small-world interconnect fabric that is inspired by modern network-on-chip paradigms. We vary the framework's key parameters, such as the connectivity, the number of switch nodes, the distribution of long- versus short-range connections, and measure the net...

  6. Folding and assembly of the large molecular machine Hsp90 studied in single-molecule experiments. (United States)

    Jahn, Markus; Buchner, Johannes; Hugel, Thorsten; Rief, Matthias


    Folding of small proteins often occurs in a two-state manner and is well understood both experimentally and theoretically. However, many proteins are much larger and often populate misfolded states, complicating their folding process significantly. Here we study the complete folding and assembly process of the 1,418 amino acid, dimeric chaperone Hsp90 using single-molecule optical tweezers. Although the isolated C-terminal domain shows two-state folding, we find that the isolated N-terminal as well as the middle domain populate ensembles of fast-forming, misfolded states. These intradomain misfolds slow down folding by an order of magnitude. Modeling folding as a competition between productive and misfolding pathways allows us to fully describe the folding kinetics. Beyond intradomain misfolding, folding of the full-length protein is further slowed by the formation of interdomain misfolds, suggesting that with growing chain lengths, such misfolds will dominate folding kinetics. Interestingly, we find that small stretching forces applied to the chain can accelerate folding by preventing the formation of cross-domain misfolding intermediates by leading the protein along productive pathways to the native state. The same effect is achieved by cotranslational folding at the ribosome in vivo.

  7. Results of a large scale neutron spectrometry and dosimetry comparison exercise at the Cadarache moderator assembly

    Energy Technology Data Exchange (ETDEWEB)

    Thomas, D.J.; Naismith, O.F.; Taylor, G.C. [National Physical Lab., Teddington (United Kingdom); Chartier, J.-L.; Posny, F. [CEA Centre d`Etudes de Fontenay-aux-Roses, 92 (France). Inst. de Protection et de Surete Nucleaire; Klein, H. [Physikalisch-Technische Bundesanstalt, Braunschweig (Germany)


    Eurados Working Group 7 recently organised a large-scale comparison of neutron spectrometry and dosimetry measurements at the IPSN/SDOS laboratory of the CEA Cadarache Research Centre in France. A large number of participants took part with a range of instruments including spectrometers, tissue-equivalent proportional counters, personal dosemeters, and survey instruments. The neutron field used for the exercise was a primarily low energy neutron spectrum similar to those which have been measured recently around nuclear facilities. This paper presents the results of the measurements and attempts to draw conclusions about the accuracy attainable with the various devices, their advantages and drawbacks, and potential problems. (author).

  8. Genomic libraries: II. Subcloning, sequencing, and assembling large-insert genomic DNA clones. (United States)

    Quail, Mike A; Matthews, Lucy; Sims, Sarah; Lloyd, Christine; Beasley, Helen; Baxter, Simon W


    Sequencing large insert clones to completion is useful for characterizing specific genomic regions, identifying haplotypes, and closing gaps in whole genome sequencing projects. Despite being a standard technique in molecular laboratories, DNA sequencing using the Sanger method can be highly problematic when complex secondary structures or sequence repeats are encountered in genomic clones. Here, we describe methods to isolate DNA from a large insert clone (fosmid or BAC), subclone the sample, and sequence the region to the highest industry standard. Troubleshooting solutions for sequencing difficult templates are discussed.

  9. Sequence assembly

    DEFF Research Database (Denmark)

    Scheibye-Alsing, Karsten; Hoffmann, S.; Frankel, Annett Maria


    Despite the rapidly increasing number of sequenced and re-sequenced genomes, many issues regarding the computational assembly of large-scale sequencing data have remain unresolved. Computational assembly is crucial in large genome projects as well for the evolving high-throughput technologies...

  10. Diverse roles for auxiliary subunits in phosphorylation-dependent regulation of mammalian brain voltage-gated potassium channels. (United States)

    Vacher, Helene; Trimmer, James S


    Voltage-gated ion channels are a diverse family of signaling proteins that mediate rapid electrical signaling events. Among these, voltage-gated potassium or Kv channels are the most diverse partly due to the large number of principal (or α) subunits and auxiliary subunits that can assemble in different combinations to generate Kv channel complexes with distinct structures and functions. The diversity of Kv channels underlies much of the variability in the active properties between different mammalian central neurons and the dynamic changes that lead to experience-dependent plasticity in intrinsic excitability. Recent studies have revealed that Kv channel α subunits and auxiliary subunits are extensively phosphorylated, contributing to additional structural and functional diversity. Here, we highlight recent studies that show that auxiliary subunits exert some of their profound effects on dendritic Kv4 and axonal Kv1 channels through phosphorylation-dependent mechanisms, either due to phosphorylation on the auxiliary subunit itself or by influencing the extent and/or impact of α subunit phosphorylation. The complex effects of auxiliary subunits and phosphorylation provide a potent mechanism to generate additional diversity in the structure and function of Kv4 and Kv1 channels, as well as allowing for dynamic reversible regulation of these important ion channels.

  11. Synthesis of Large Molecules in Cometary Ice Analogs: Physical Properties Related to Self-Assembly Processes (United States)

    Dworkin, Jason P.; Sandford, Scott A.; Deamer, David W.; Gillette, J. Seb; Zare, Richard N.; Allamandola, Louis J. (Technical Monitor)


    strikingly similar to those produced by extracts of the Murchison meteorite. Together, these results suggest a link between organic material photochemically synthesized on the cold grains in dense, interstellar molecular clouds and compounds that may have contributed to the organic inventory of the primitive Earth. For example, the amphiphilic properties of such compounds permit self-assembly into the membranous boundary structures that required for the first forms of cellular life.

  12. Very large photoconduction enhancement upon self-assembly of a new triindole derivative in solution-processed films

    Energy Technology Data Exchange (ETDEWEB)

    Gallego-Gomez, Francisco; Villalvilla, Jose M.; Quintana, Jose A.; Diaz-Garcia, Maria A. [Instituto Universitario de Materiales de Alicante and Dpto. Fisica Aplicada, Universidad de Alicante, 03080 Alicante (Spain); Garcia-Frutos, Eva M.; Gutierrez-Puebla, Enrique; Monge, Angeles; Gomez-Lor, Berta [Instituto de Ciencia de Materiales de Madrid, CSIC, C/Sor Juana Ines de la Cruz 3, 28049 Madrid (Spain)


    A new carbazole-related small molecule exhibiting self-assembly into ordered nanostructures in solution-processed cast films has been synthesized and its charge-photogeneration and -transport properties have been investigated. Large photoconductivity was measured in the amorphous state while an enormous improvement in the photoconduction properties was observed when the molecules spontaneously organized. Photocurrents increased upon self-assembly by up to four orders of magnitude, mostly due to the drastic enhancement of the charge photogeneration. A greatly favorable arrangement of the aromatic cores in the resulting nanostructures, which were characterized by X-ray analysis, may explain these improvements. Photocurrents of mA cm{sup -2}, on/off ratios of 10{sup 4} and quantum efficiencies of unity at low field and light intensity, which are among the best values reported to date, along with the simplicity of fabrication, give this readily-available organic system great potential for use in plastic optoelectronic devices. (Copyright copyright 2011 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  13. Transfer and assembly of large area TiO2 nanotube arrays onto conductive glass for dye sensitized solar cells (United States)

    Zhang, Jun; Li, Siqian; Ding, Hao; Li, Quantong; Wang, Baoyuan; Wang, Xina; Wang, Hao


    Highly ordered titanium oxide nanotube arrays are synthesized by a two-step anodic oxidation of pure titanium foil at constant voltage. It is found that the length of nanotube arrays firstly increased rapidly with the anodization time, and then the growth rate gradually slowed down with further increasing the anodization time. The mechanism of anodization time-dependent tube length growth is discussed. Large area free-standing TiO2 nanotube (TNT) arrays are detached from the underlying Ti foil and transferred onto the fluorine-doped tin oxide (FTO) conductive glass substrates to serve as the photoanodes of the dye-sensitized solar cells (DSSCs). The photoelectric performance of the DSSCs assembled by TNT/FTO films is strongly related to the tube length of titania and the surface treatment. For the photoanodes without any surface modification, the highest overall photovoltaic conversion efficiency (PCE) that can be achieved is 4.12% in the DSSC assembled with 33-μm-thick TNT arrays, while the overall PCE of DSSC based on the 33-μm-thick TNT arrays increases to 9.02% in response to the treatment with TiCl4.

  14. A 2D Fourier tool for the analysis of photo-elastic effect in large granular assemblies (United States)

    Leśniewska, Danuta


    Fourier transforms are the basic tool in constructing different types of image filters, mainly those reducing optical noise. Some DIC or PIV software also uses frequency space to obtain displacement fields from a series of digital images of a deforming body. The paper presents series of 2D Fourier transforms of photo-elastic transmission images, representing large pseudo 2D granular assembly, deforming under varying boundary conditions. The images related to different scales were acquired using the same image resolution, but taken at different distance from the sample. Fourier transforms of images, representing different stages of deformation, reveal characteristic features at the three (`macro-`, `meso-` and `micro-`) scales, which can serve as a data to study internal order-disorder transition within granular materials.

  15. Assembly of large purely inorganic Ce-stabilized/bridged selenotungstates: from nanoclusters to layers. (United States)

    Chen, Wei-Chao; Qin, Chao; Li, Yang-Guang; Zang, Hong-Ying; Shao, Kui-Zhan; Su, Zhong-Min; Wang, En-Bo


    A versatile one-pot strategy was used to synthesize two large, purely inorganic selenotungstates, nanocluster K(6)Na(16) [Ce(6)Se(6)W(67)O(230) (OH)(6) (H(2)O)(17)]⋅47 H(2)O (1) and layer K(9)Na(5) Ce(H(2)O)(4) [Ce(6)Se(10)W(51)O(187) (OH)(7) (H(2)O)(18)]⋅45H(2)O(2), by combining cerium centers and SeO(3) (2-) heteroanion templates. Compound 1 displays a Ce-stabilized hexameric nanocluster with one rhombus-like {W(4)O(15) (OH)(3)} unit in the center, whereas compound 2 is the first example of a Ce-bridged layer selenotungstate network based on linkage of the unusual {Ce(6)Se(10)W(51)O(187) (OH)(7) (H(2)O)(18)} clusters and additional Ce(H(2)O)(4) fragments via Ce-O-Se bridges. The compounds were characterized by elemental analyses, IR spectroscopy, thermogravimetric analyses, powder and single-crystal X-ray diffraction, and electrospray ionization mass spectrometry. Moreover, the electrochemical property of compound 1 was also investigated. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Self-assembled large-area annular cavity arrays with tunable cylindrical surface plasmons for sensing. (United States)

    Ni, Haibin; Wang, Ming; Shen, Tianyi; Zhou, Jing


    Surface plasmons that propagate along cylindrical metal/dielectric interfaces in annular apertures in metal films, called cylindrical surface plasmons (CSPs), exhibit attractive optical characteristics. However, it is challenging to fabricate these nanocoaxial structures. Here, we demonstrate a practical low-cost route to manufacture highly ordered, large-area annular cavity arrays (ACAs) that can support CSPs with great tunability. By employing a sol-gel coassembly method, reactive ion etching and metal sputtering techniques, regular, highly ordered ACAs in square-centimeter-scale with a gap width tunable in the range of several to hundreds of nanometers have been produced with good reproducibility. Ag ACAs with a gap width of 12 nm and a gap height of 635 nm are demonstrated. By finite-difference time-domain simulation, we confirm that the pronounced dips in the reflectance spectra of ACAs are attributable to CSP resonances excited in the annular gaps. By adjusting etching time and Ag film thickness, the CSP dips can be tuned to sweep the entire optical range of 360 to 1800 nm without changing sphere size, which makes them a promising candidate for forming integrated plasmonic sensing arrays. The high tunability of the CSP resonant frequencies together with strong electric field enhancement in the cavities make the ACAs promising candidates for surface plasmon sensors and SERS substrates, as, for example, they have been used in liquid refractive index (RI) sensing, demonstrating a sensitivity of 1505 nm/RIU and a figure of merit of 9. One of the CSP dips of ACAs with a certain geometry size is angle- (0-70 degrees) and polarization-independent and can be used as a narrow-band absorber. Furthermore, the nano annular cavity arrays can be used to construct solar cells, nanolasers and nanoparticle plasmonic tweezers.

  17. Assembly and structure of protein phosphatase 2A

    Institute of Scientific and Technical Information of China (English)

    SHI YiGong


    Protein phosphatase 2A (PP2A) represents a conserved family of important protein serinetthreonine phosphatases in species ranging from yeast to human. The PP2A core enzyme comprises a scaffold subunit and a catalytic subunit. The heterotrimeric PP2A holoenzyme consists of the core enzyme and a variable regulatory subunit. The catalytic subunit of PP2A is subject to reversible methylation, mediated by two conserved enzymes. Both the PP2A core and holoenzymes are regulated through interaction with a large number of cellular cofactors. Recent biochemical and structural investigation reveals critical insights into the assembly and function of the PP2A core enzyme as well as two families of holoenzyme. This review focuses on the molecular mechanisms revealed by these latest advances.

  18. Assembly and structure of protein phosphatase 2A

    Institute of Scientific and Technical Information of China (English)


    Protein phosphatase 2A (PP2A) represents a conserved family of important protein serine/threonine phosphatases in species ranging from yeast to human. The PP2A core enzyme comprises a scaffold subunit and a catalytic subunit. The heterotrimeric PP2A holoenzyme consists of the core enzyme and a variable regulatory subunit. The catalytic subunit of PP2A is subject to reversible methylation, medi-ated by two conserved enzymes. Both the PP2A core and holoenzymes are regulated through interac-tion with a large number of cellular cofactors. Recent biochemical and structural investigation reveals critical insights into the assembly and function of the PP2A core enzyme as well as two families of holoenzyme. This review focuses on the molecular mechanisms revealed by these latest advances.

  19. Patterning of self-assembled monolayers by phase-shifting mask and its applications in large-scale assembly of nanowires

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Fan; Zhang, Dakuan; Wang, Jianyu; Sheng, Yun; Wang, Xinran; Chen, Kunji; Zhou, Minmin [Key Laboratory of Advanced Photonic and Electronic Materials and School of Electronic Science and Engineering, Nanjing University, Nanjing 210093 (China); Yan, Shancheng [Key Laboratory of Advanced Photonic and Electronic Materials and School of Electronic Science and Engineering, Nanjing University, Nanjing 210093 (China); School of Geography and Biological Information, Nanjing University of Posts and Telecommunications, Nanjing 210046 (China); Shen, Jiancang; Pan, Lijia; Shi, Yi, E-mail: [Key Laboratory of Advanced Photonic and Electronic Materials and School of Electronic Science and Engineering, Nanjing University, Nanjing 210093 (China); Collaborative Innovation Center of Advanced Micro-structures, Nanjing University, Nanjing 210093 (China)


    A nonselective micropatterning method of self-assembled monolayers (SAMs) based on laser and phase-shifting mask (PSM) is demonstrated. Laser beam is spatially modulated by a PSM, and periodic SAM patterns are generated sequentially through thermal desorption. Patterned wettability is achieved with alternating hydrophilic/hydrophobic stripes on octadecyltrichlorosilane monolayers. The substrate is then used to assemble CdS semiconductor nanowires (NWs) from a solution, obtaining well-aligned NWs in one step. Our results show valuably the application potential of this technique in engineering SAMs for integration of functional devices.

  20. Paramyotonia congenita and hyperkalemic periodic paralysis associated with a Met 1592 Val substitution in the skeletal muscle sodium channel alpha subunit--a large kindred with a novel phenotype. (United States)

    Kelly, P; Yang, W S; Costigan, D; Farrell, M A; Murphy, S; Hardiman, O


    Paramyotonia congenita (PC) and Hyperkalemic periodic paralysis (HyperPP) are caused by amino acid substitutions in the alpha subunit of the human skeletal muscle sodium channel. One such substitution, methionine for valine at position 1592, has been associated with HyperPP with myotonia and cold sensitivity. We report clinical, electromyographic (EMG), genetic and pathological features of a large kindred with the Met1592Val substitution. Affected members were phenotypically heterogenous and had episodic potassium-sensitive paralysis, and stiffness and weakness induced by exercise and cold, which was confirmed by EMG studies. These features indicate a combined PC-HyperPP phenotype not previously described with this mutation.

  1. Isolated spinach ribulose-1,5-bisphosphate carboxylase/oxgenase large subunit .epsilon. n-methyltransferase and method of inactivating ribulose-1,5-bishosphatase .epsilon. n-methyltransferase activity (United States)

    Houtz, Robert L.


    The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .sup..epsilon. N-methyltansferase (protein methylase III or Rubisco LSMT) from a plant which has a des(methyl) lysyl residue in the LS is disclosed. In addition, the full-length cDNA clones for Rubisco LSMT are disclosed. Transgenic plants and methods of producing same which have the Rubisco LSMT gene inserted into the DNA are also provided. Further, methods of inactivating the enzymatic activity of Rubisco LSMT are also disclosed.

  2. Disease-associated mutations in the HSPD1 gene encoding the large subunit of the mitochondrial HSP60/HSP10 chaperonin complex

    Directory of Open Access Journals (Sweden)

    Peter Bross


    Full Text Available Heat shock protein 60 (HSP60 forms together with heat shock protein 10 (HSP10 double-barrel chaperonin complexes that are essential for folding to the native state of proteins in the mitochondrial matrix space. Two extremely rare monogenic disorders have been described that are caused by missense mutations in the HSPD1 gene that encodes the HSP60 subunit of the HSP60/HSP10 chaperonin complex. Investigations of the molecular mechanisms underlying these disorders have revealed that different degrees of reduced HSP60 function produce distinct neurological phenotypes. While mutations with deleterious or strong dominant negative effects are not compatible with life, HSPD1 gene variations found in the human population impair HSP60 function and depending on the mechanism and degree of HSP60 dys- and malfunction cause different phenotypes. We here summarize the knowledge on the effects of disturbances of the function of the HSP60/HSP10 chaperonin complex by disease-associated mutations.

  3. Linear and nonlinear optical characterization of self-assembled, large-area gold nanosphere metasurfaces with sub-nanometer gaps. (United States)

    Fontana, Jake; Maldonado, Melissa; Charipar, Nicholas; Trammell, Scott A; Nita, Rafaela; Naciri, Jawad; Pique, Alberto; Ratna, Banahalli; Gomes, Anderson S L


    We created centimeter-scale area metasurfaces consisting of a quasi-hexagonally close packed monolayer of gold nanospheres capped with alkanethiol ligands on glass substrates using a directed self-assembly approach. We experimentally characterized the morphology and the linear and nonlinear optical properties of metasurfaces. We show these metasurfaces, with interparticle gaps of 0.6 nm, are modeled well using a classical (without charge transfer) description. We find a large dispersion of linear refractive index, ranging from values less than vacuum, 0.87 at 600 nm, to Germanium-like values of 4.1 at 880 nm, determined using spectroscopic ellipsometry. Nonlinear optical characterization was carried out using femtosecond Z-scan and we observe saturation behavior of the nonlinear absorption (NLA) and nonlinear refraction (NLR). We find a negative NLR from these metasurfaces two orders of magnitude larger (nsub>2,satsub> = -7.94x10-9 cm2/W at Isub>sat,n2sub> = 0.43 GW/cm2) than previous reports on gold nanostructures at similar femtosecond time scales. We also find the magnitude of the NLA comparable to the largest values reported (βsub>2,satsub> = -0.90x105 cm/GW at Isub>sat,β2sub> = 0.34 GW/cm2). Precise knowledge of the index of refraction is of crucial importance for emerging dispersion engineering technologies. Furthermore, utilizing this directed self-assembly approach enables the nanometer scale resolution required to develop the unique optical response and simultaneously provides high-throughput for potential device realization.

  4. Protein phosphatase 2B (PP2B, calcineurin) in Paramecium: partial characterization reveals that two members of the unusually large catalytic subunit family have distinct roles in calcium-dependent processes. (United States)

    Fraga, D; Sehring, I M; Kissmehl, R; Reiss, M; Gaines, R; Hinrichsen, R; Plattner, H


    We characterized the calcineurin (CaN) gene family, including the subunits CaNA and CaNB, based upon sequence information obtained from the Paramecium genome project. Paramecium tetraurelia has seven subfamilies of the catalytic CaNA subunit and one subfamily of the regulatory CaNB subunit, with each subfamily having two members of considerable identity on the amino acid level (>or=55% between subfamilies, >or=94% within CaNA subfamilies, and full identity in the CaNB subfamily). Within CaNA subfamily members, the catalytic domain and the CaNB binding region are highly conserved and molecular modeling revealed a three-dimensional structure almost identical to a human ortholog. At 14 members, the size of the CaNA family is unprecedented, and we hypothesized that the different CaNA subfamily members were not strictly redundant and that at least some fulfill different roles in the cell. This was tested by selecting two phylogenetically distinct members of this large family for posttranscriptional silencing by RNA interference. The two targets resulted in differing effects in exocytosis, calcium dynamics, and backward swimming behavior that supported our hypothesis that the large, highly conserved CaNA family members are not strictly redundant and that at least two members have evolved diverse but overlapping functions. In sum, the occurrence of CaN in Paramecium spp., although disputed in the past, has been established on a molecular level. Its role in exocytosis and ciliary beat regulation in a protozoan, as well as in more complex organisms, suggests that these roles for CaN were acquired early in the evolution of this protein family.

  5. Protein Phosphatase 2B (PP2B, Calcineurin) in Paramecium: Partial Characterization Reveals That Two Members of the Unusually Large Catalytic Subunit Family Have Distinct Roles in Calcium-Dependent Processes▿‡ (United States)

    Fraga, D.; Sehring, I. M.; Kissmehl, R.; Reiss, M.; Gaines, R.; Hinrichsen, R.; Plattner, H.


    We characterized the calcineurin (CaN) gene family, including the subunits CaNA and CaNB, based upon sequence information obtained from the Paramecium genome project. Paramecium tetraurelia has seven subfamilies of the catalytic CaNA subunit and one subfamily of the regulatory CaNB subunit, with each subfamily having two members of considerable identity on the amino acid level (≥55% between subfamilies, ≥94% within CaNA subfamilies, and full identity in the CaNB subfamily). Within CaNA subfamily members, the catalytic domain and the CaNB binding region are highly conserved and molecular modeling revealed a three-dimensional structure almost identical to a human ortholog. At 14 members, the size of the CaNA family is unprecedented, and we hypothesized that the different CaNA subfamily members were not strictly redundant and that at least some fulfill different roles in the cell. This was tested by selecting two phylogenetically distinct members of this large family for posttranscriptional silencing by RNA interference. The two targets resulted in differing effects in exocytosis, calcium dynamics, and backward swimming behavior that supported our hypothesis that the large, highly conserved CaNA family members are not strictly redundant and that at least two members have evolved diverse but overlapping functions. In sum, the occurrence of CaN in Paramecium spp., although disputed in the past, has been established on a molecular level. Its role in exocytosis and ciliary beat regulation in a protozoan, as well as in more complex organisms, suggests that these roles for CaN were acquired early in the evolution of this protein family. PMID:20435698

  6. Role of the Rubisco Small Subunit

    Energy Technology Data Exchange (ETDEWEB)

    Spreitzer, Robert Joseph [Univ. of Nebraska, Lincoln, NE (United States)


    Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of CO2 fixation in photosynthesis. However, it is a slow enzyme, and O2 competes with CO2 at the active site. Oxygenation initiates the photorespiratory pathway, which also results in the loss of CO2. If carboxylation could be increased or oxygenation decreased, an increase in net CO2 fixation would be realized. Because Rubisco provides the primary means by which carbon enters all life on earth, there is much interest in engineering Rubisco to increase the production of food and renewable energy. Rubisco is located in the chloroplasts of plants, and it is comprised of two subunits. Much is known about the chloroplast-gene-encoded large subunit (rbcL gene), which contains the active site, but much less is known about the role of the nuclear-gene-encoded small subunit in Rubisco function (rbcS gene). Both subunits are coded by multiple genes in plants, which makes genetic engineering difficult. In the eukaryotic, green alga Chlamydomonas reinhardtii, it has been possible to eliminate all the Rubisco genes. These Rubisco-less mutants can be maintained by providing acetate as an alternative carbon source. In this project, focus has been placed on determining whether the small subunit might be a better genetic-engineering target for improving Rubisco. Analysis of a variable-loop structure (βA-βB loop) of the small subunit by genetic selection, directed mutagenesis, and construction of chimeras has shown that the small subunit can influence CO2/O2 specificity. X-ray crystal structures of engineered chimeric-loop enzymes have indicated that additional residues and regions of the small subunit may also contribute to Rubisco function. Structural dynamics of the small-subunit carboxyl terminus was also investigated. Alanine-scanning mutagenesis of the most-conserved small-subunit residues has identified a

  7. Large-scale, thick, self-assembled, nacre-mimetic brick-walls as fire barrier coatings on textiles (United States)

    Das, Paramita; Thomas, Helga; Moeller, Martin; Walther, Andreas


    Highly loaded polymer/clay nanocomposites with layered structures are emerging as robust fire retardant surface coatings. However, time-intensive sequential deposition processes, e.g. layer-by-layer strategies, hinders obtaining large coating thicknesses and complicates an implementation into existing technologies. Here, we demonstrate a single-step, water-borne approach to prepare thick, self-assembling, hybrid fire barrier coatings of sodium carboxymethyl cellulose (CMC)/montmorillonite (MTM) with well-defined, bioinspired brick-wall nanostructure, and showcase their application on textile. The coating thickness on the textile is tailored using different concentrations of CMC/MTM (1–5 wt%) in the coating bath. While lower concentrations impart conformal coatings of fibers, thicker continuous coatings are obtained on the textile surface from highest concentration. Comprehensive fire barrier and fire retardancy tests elucidate the increasing fire barrier and retardancy properties with increasing coating thickness. The materials are free of halogen and heavy metal atoms, and are sourced from sustainable and partly even renewable building blocks. We further introduce an amphiphobic surface modification on the coating to impart oil and water repellency, as well as self-cleaning features. Hence, our study presents a generic, environmentally friendly, scalable, and one-pot coating approach that can be introduced into existing technologies to prepare bioinspired, thick, fire barrier nanocomposite coatings on diverse surfaces.

  8. Self-assembly of large-scale aggregates of porphyrin from its dimers and their absorption and luminescence properties (United States)

    Udal'tsov, A. V.; Kazarin, L. A.; Sweshnikov, A. A.


    Properties of aggregates of protonated meso-tetraphenylporphine (TPP) dimers have been investigated by absorption and luminescence spectroscopies and scanning electron microscopy. It was found that the absorption and fluorescence spectra obtained at a low and several times higher concentration of porphyrin differ considerably. The changes in absorption spectra of TPP in the water-THF-glycerol (84:6:10, v/v) mixture in the presence of 0.4 N HCl with time and the appearance of a green precipitate after several days indicate aggregation of the porphyrin. The near IR emission at 1000 nm, which is assigned to the fluorescence of donor-acceptor water-porphyrin dimeric complex, is revealed in the fluorescence spectra of TPP in aqueous solution of THF in the presence of 0.4 N HCl at the low concentration of porphyrin on excitation at 465 nm. In contrast, the near IR emission is not observed in the solution with several times higher concentration of porphyrin, but a shoulder at ca 800 nm is appreciable in the corresponding spectrum. The large-scale aggregates of TPP with sizes approximately from 1 μm to several micrometers are found in thin films of the protonated porphyrin. It is proposed that the aggregates are formed as a result of self-assembly from different protonated porphyrin dimers and have an ordered structure.

  9. Facile synthesis of large-scale Ag nanosheet-assembled films with sub-10 nm gaps as highly active and homogeneous SERS substrates (United States)

    Li, Zhongbo; Meng, Guowen; Liang, Ting; Zhang, Zhuo; Zhu, Xiaoguang


    We report a facile low-cost synthetic approach to large-scale Ag nanosheet-assembled films with a high density of uniformly distributed sub-10 nm gaps between the adjacent nanosheets on Si substrates via galvanic cell reactions. The distribution density of Ag nanosheets on substrates could be tailored by tuning the duration of the HF-etching and the concentration of citric acid in the solution. Furthermore, in conjunction with a conventional photolithography, highly uniform patterned Ag nanosheet-assembled structures with different morphologies can be achieved on Si substrates via galvanic-cell-induced growth. By using rhodamine 6G as a standard test molecule, the large-scale Ag nanosheet-assembled films exhibit highly active and homogenous surface-enhanced Raman scattering (SERS) effect and also show promising potentials as reliable SERS substrates for rapid detection of trace polychlorinated biphenyls (PCBs).

  10. Radiation hybrid maps of D-genome of Aegilops tauschii and their application in sequence assembly of large and complex plant genomes (United States)

    The large and complex genome of bread wheat (Triticum aestivum L., ~17 Gb) requires high-resolution genome maps saturated with ordered markers to assist in anchoring and orienting BAC contigs/ sequence scaffolds for whole genome sequence assembly. Radiation hybrid (RH) mapping has proven to be an e...

  11. Large-scale self-assembled zirconium phosphate smectic layers via a simple spray-coating process

    National Research Council Canada - National Science Library

    Wong, Minhao; Ishige, Ryohei; White, Kevin L; Li, Peng; Kim, Daehak; Krishnamoorti, Ramanan; Gunther, Robert; Higuchi, Takeshi; Jinnai, Hiroshi; Takahara, Atsushi; Nishimura, Riichi; Sue, Hung-Jue


    .... Here we present a spray-coating method to manufacture thin, flexible and transparent epoxy films containing zirconium phosphate nanoplatelets self-assembled into a lamellar arrangement aligned parallel to the substrate...

  12. Accessory subunit NUYM (NDUFS4) is required for stability of the electron input module and activity of mitochondrial complex I. (United States)

    Kahlhöfer, Flora; Kmita, Katarzyna; Wittig, Ilka; Zwicker, Klaus; Zickermann, Volker


    Mitochondrial complex I is an intricate 1MDa membrane protein complex with a central role in aerobic energy metabolism. The minimal form of complex I consists of fourteen central subunits that are conserved from bacteria to man. In addition, eukaryotic complex I comprises some 30 accessory subunits of largely unknown function. The gene for the accessory NDUFS4 subunit of human complex I is a hot spot for fatal pathogenic mutations in humans. We have deleted the gene for the orthologous NUYM subunit in the aerobic yeast Yarrowia lipolytica, an established model system to study eukaryotic complex I and complex I linked diseases. We observed assembly of complex I which lacked only subunit NUYM and retained weak interaction with assembly factor N7BML (human NDUFAF2). Absence of NUYM caused distortion of iron sulfur clusters of the electron input domain leading to decreased complex I activity and increased release of reactive oxygen species. We conclude that NUYM has an important stabilizing function for the electron input module of complex I and is essential for proper complex I function.

  13. Impact of Defect Creation and Motion on the Thermodynamics and Large-Scale Reorganization of Self-Assembled Clathrin Lattices

    Energy Technology Data Exchange (ETDEWEB)

    Mehraeen, Shafigh


    We develop a theoretical model for the thermodynamics and kinetics of clathrin self-assembly. Our model addresses the behavior in two dimensions and can be easily extended to three dimensions, facilitating the study of membrane, surface, and bulk assembly. The clathrin triskelia are modeled as flexible pinwheels that form leg-leg associations and resist bending and stretching deformations. Thus, the pinwheels are capable of forming a range of ring structures, including 5-, 6-, and 7-member rings that are observed experimentally. Our theoretical model employs Brownian dynamics to track the motion of clathrin pinwheels at sufficiently long time scales to achieve complete assembly. Invoking theories of dislocation-mediated melting in two dimensions, we discuss the phase behavior for clathrin self-assembly as predicted by our theoretical model. We demonstrate that the generation of 5-7 defects in an otherwise perfect honeycomb lattice resembles creation of two dislocations with equal and opposite Burgers vectors. We use orientational- and translational-order correlation functions to predict the crystalline-hexatic and hexatic-liquid phase transitions in clathrin lattices. These results illustrate the pivotal role that molecular elasticity plays in the physical behavior of self-assembling and self-healing materials.

  14. From I-Deas to NX : Changing the Design Application and Creating Work Instructions for a Large Industrial Product Assembly


    Mäkinen, Harri


    The objective of this thesis was to provide Citec Oy Ab with information whether it is beneficial to switch from using Siemens I-Deas to using Siemens NX, as the CAD design and drafting application, within a specific product group of Citec’s customer, an industrial product company. To acquire this information a case project in NX was made. This was the main method of this project type thesis. The project work included building the products main top level assembly and creating an assembly draw...

  15. Divergent Evolution of Nuclear Localization Signal Sequences in Herpesvirus Terminase Subunits. (United States)

    Sankhala, Rajeshwer S; Lokareddy, Ravi K; Cingolani, Gino


    The tripartite terminase complex of herpesviruses assembles in the cytoplasm of infected cells and exploits the host nuclear import machinery to gain access to the nucleus, where capsid assembly and genome-packaging occur. Here we analyzed the structure and conservation of nuclear localization signal (NLS) sequences previously identified in herpes simplex virus 1 (HSV-1) large terminase and human cytomegalovirus (HCMV) small terminase. We found a monopartite NLS at the N terminus of large terminase, flanking the ATPase domain, that is conserved only in α-herpesviruses. In contrast, small terminase exposes a classical NLS at the far C terminus of its helical structure that is conserved only in two genera of the β-subfamily and absent in α- and γ-herpesviruses. In addition, we predicted a classical NLS in the third terminase subunit that is partially conserved among herpesviruses. Bioinformatic analysis revealed that both location and potency of NLSs in terminase subunits evolved more rapidly than the rest of the amino acid sequence despite the selective pressure to keep terminase gene products active and localized in the nucleus. We propose that swapping NLSs among terminase subunits is a regulatory mechanism that allows different herpesviruses to regulate the kinetics of terminase nuclear import, reflecting a mechanism of virus:host adaptation.

  16. Inventory control: cytochrome c oxidase assembly regulates mitochondrial translation. (United States)

    Mick, David U; Fox, Thomas D; Rehling, Peter


    Mitochondria maintain genome and translation machinery to synthesize a small subset of subunits of the oxidative phosphorylation system. To build up functional enzymes, these organellar gene products must assemble with imported subunits that are encoded in the nucleus. New findings on the early steps of cytochrome c oxidase assembly reveal how the mitochondrial translation of its core component, cytochrome c oxidase subunit 1 (Cox1), is directly coupled to the assembly of this respiratory complex.

  17. Complete modification maps for the cytosolic small and large subunit rRNAs of Euglena gracilis: functional and evolutionary implications of contrasting patterns between the two rRNA components. (United States)

    Schnare, Murray N; Gray, Michael W


    In the protist Euglena gracilis, the cytosolic small subunit (SSU) rRNA is a single, covalently continuous species typical of most eukaryotes; in contrast, the large subunit (LSU) rRNA is naturally fragmented, comprising 14 separate RNA molecules instead of the bipartite (28S+5.8S) eukaryotic LSU rRNA typically seen. We present extensively revised secondary structure models of the E. gracilis SSU and LSU rRNAs and have mapped the positions of all of the modified nucleosides in these rRNAs (88 in SSU rRNA and 262 in LSU rRNA, with only 3 LSU rRNA modifications incompletely characterized). The relative proportions of ribose-methylated nucleosides and pseudouridine (∼60% and ∼35%, respectively) are closely similar in the two rRNAs; however, whereas the Euglena SSU rRNA has about the same absolute number of modifications as its human counterpart, the Euglena LSU rRNA has twice as many modifications as the corresponding human LSU rRNA. The increased levels of rRNA fragmentation and modification in E. gracilis LSU rRNA are correlated with a 3-fold increase in the level of mispairing in helical regions compared to the human LSU rRNA. In contrast, no comparable increase in mispairing is seen in helical regions of the SSU rRNA compared to its homologs in other eukaryotes. In view of the reported effects of both ribose-methylated nucleoside and pseudouridine residues on RNA structure, these correlations lead us to suggest that increased modification in the LSU rRNA may play a role in stabilizing a 'looser' structure promoted by elevated helical mispairing and a high degree of fragmentation.

  18. Molecular determinants of desensitization and assembly of the chimeric GABA(A) receptor subunits (alpha1/gamma2) and (gamma2/alpha1) in combinations with beta2 and gamma2

    DEFF Research Database (Denmark)

    Elster, L; Kristiansen, U; Pickering, D S


    2 and the remainder of the gamma2 or alpha1 subunits, respectively, were expressed with beta2 and beta2gamma2 in Spodoptera frugiperda (Sf-9) cells using the baculovirus expression system. The (alpha1/gamma2)beta2 and (alpha1/gamma2)beta2gamma2 but not the (gamma2/alpha1)beta2 and (gamma2/alpha1......)beta2gamma2 subunit combinations formed functional receptor complexes as shown by whole-cell patch-clamp recordings and [3H]muscimol and [3H]flunitrazepam binding. Moreover, the surface immunofluorescence staining of Sf-9 cells expressing the (alpha1/gamma2)-containing receptors was pronounced...

  19. Ribosome-stalk biogenesis is coupled with recruitment of nuclear-export factor to the nascent 60S subunit. (United States)

    Sarkar, Anshuk; Pech, Markus; Thoms, Matthias; Beckmann, Roland; Hurt, Ed


    Nuclear export of preribosomal subunits is a key step during eukaryotic ribosome formation. To efficiently pass through the FG-repeat meshwork of the nuclear pore complex, the large pre-60S subunit requires several export factors. Here we describe the mechanism of recruitment of the Saccharomyces cerevisiae RNA-export receptor Mex67-Mtr2 to the pre-60S subunit at the proper time. Mex67-Mtr2 binds at the premature ribosomal-stalk region, which later during translation serves as a binding platform for translational GTPases on the mature ribosome. The assembly factor Mrt4, a structural homolog of cytoplasmic-stalk protein P0, masks this site, thus preventing untimely recruitment of Mex67-Mtr2 to nuclear pre-60S particles. Subsequently, Yvh1 triggers Mrt4 release in the nucleus, thereby creating a narrow time window for Mex67-Mtr2 association at this site and facilitating nuclear export of the large subunit. Thus, a spatiotemporal mark on the ribosomal stalk controls the recruitment of an RNA-export receptor to the nascent 60S subunit.

  20. Large work function shift of gold induced by a novel perfluorinated azobenzene-based self-assembled monolayer. (United States)

    Crivillers, Núria; Osella, Silvio; Van Dyck, Colin; Lazzerini, Giovanni M; Cornil, David; Liscio, Andrea; Di Stasio, Francesco; Mian, Shabbir; Fenwick, Oliver; Reinders, Federica; Neuburger, Markus; Treossi, Emanuele; Mayor, Marcel; Palermo, Vincenzo; Cacialli, Franco; Cornil, Jérôme; Samorì, Paolo


    Tune it with light! Self-assembled monolayers on gold based on a chemisorbed novel azobenzene derivative with a perfluorinated terminal phenyl ring are prepared. The modified substrate shows a significant work function increase compared to the bare metal. The photo-conversion between trans and cis isomers chemisorbed on the surface shows great perspectives for being an accessible route to tune the gold properties by means of light.

  1. Perturbation-based Markovian transmission model for probing allosteric dynamics of large macromolecular assembling: a study of GroEL-GroES.

    Directory of Open Access Journals (Sweden)

    Hsiao-Mei Lu


    Full Text Available Large macromolecular assemblies are often important for biological processes in cells. Allosteric communications between different parts of these molecular machines play critical roles in cellular signaling. Although studies of the topology and fluctuation dynamics of coarse-grained residue networks can yield important insights, they do not provide characterization of the time-dependent dynamic behavior of these macromolecular assemblies. Here we develop a novel approach called Perturbation-based Markovian Transmission (PMT model to study globally the dynamic responses of the macromolecular assemblies. By monitoring simultaneous responses of all residues (>8,000 across many (>6 decades of time spanning from the initial perturbation until reaching equilibrium using a Krylov subspace projection method, we show that this approach can yield rich information. With criteria based on quantitative measurements of relaxation half-time, flow amplitude change, and oscillation dynamics, this approach can identify pivot residues that are important for macromolecular movement, messenger residues that are key to signal mediating, and anchor residues important for binding interactions. Based on a detailed analysis of the GroEL-GroES chaperone system, we found that our predictions have an accuracy of 71-84% judged by independent experimental studies reported in the literature. This approach is general and can be applied to other large macromolecular machineries such as the virus capsid and ribosomal complex.

  2. Glutamate-119 of the large alpha-subunit is the catalytic base in the hydration of 2-trans-enoyl-coenzyme A catalyzed by the multienzyme complex of fatty acid oxidation from Escherichia coli. (United States)

    He, X Y; Yang, S Y


    Glu139 of the large alpha-subunit of the multienzyme complex of fatty acid oxidation from Escherichia coli was identified as the catalytic residue of enoyl-CoA hydratase [Yang, S.-Y., He, X.-Y., & Schulz, H. (1995) Biochemistry 34, 6441-6447]. To determine whether any of the other conserved protic residues is directly involved in the hydratase catalysis, the multienzyme complexes with either an alpha/Asp69 --> Asn or an alpha/Glu119 --> Gln mutation were overproduced and characterized. The catalytic properties of 3-ketoacyl-CoA thiolase and l-3-hydroxyacyl-CoA dehydrogenase of the mutant complexes were almost unaffected. The amidation of Asp69 and Glu119 caused a 7.6- and 88-fold decrease, respectively, in the kcat of enoyl-CoA hydratase without a significant change in the Km value of the hydratase as well as a 5.9- and 62-fold increase, respectively, in the Km of Delta3-cis-Delta2-trans-enoyl-CoA isomerase with a very small decrease in the kcat of the latter enzyme. The data suggest that the carboxyl group of Glu119 is particularly important to the catalytic activity of enoyl-CoA hydratase. Furthermore, the wild-type hydratase shows a bell-shaped pH dependence of the kcat/Km with pKa values of 5.9 and 9.2, whereas the Glu119 --> Gln mutant hydratase has only a single pKa of 9.5. A simple explanation for these observations is that a deprotonated Glu119 and a protonated Glu139 are required for the high kcat of the enoyl-CoA hydratase. The results of site-directed mutagenesis studies, together with the structural information about the spatial arrangement of two conserved glutamate residues of rat liver enoyl-CoA hydratase [Engel, C. K., Mathieu, M., Zeelen, J. P., Hiltunen, J. K., and Wierenga, R. K. (1996) EMBO J. 15, 5135-5145] to which Glu119 and Glu139 of the large alpha-subunit correspond, lead to the conclusion that the gamma-carboxyl group of Glu119 serves as the second general acid-base functional group in catalyzing the hydration of 2-trans-enoyl-CoA.

  3. Solid-state NMR on a large multidomain integral membrane protein: the outer membrane protein assembly factor BamA

    NARCIS (Netherlands)

    Renault, M.A.M.; Bos, M.P.; Tommassen, J.P.M.; Baldus, M.


    Multidomain proteins constitute a large part of prokaryotic and eukaryotic proteomes and play fundamental roles in various physiological processes. However, their structural characterization is challenging because of their large size and intrinsic flexibility. We show here that motional-filtered

  4. Diversity of heterotrimeric G-protein γ subunits in plants

    Directory of Open Access Journals (Sweden)

    Trusov Yuri


    Full Text Available Abstract Background Heterotrimeric G-proteins, consisting of three subunits Gα, Gβ and Gγ are present in most eukaryotes and mediate signaling in numerous biological processes. In plants, Gγ subunits were shown to provide functional selectivity to G-proteins. Three unconventional Gγ subunits were recently reported in Arabidopsis, rice and soybean but no structural analysis has been reported so far. Their relationship with conventional Gγ subunits and taxonomical distribution has not been yet demonstrated. Results After an extensive similarity search through plant genomes, transcriptomes and proteomes we assembled over 200 non-redundant proteins related to the known Gγ subunits. Structural analysis of these sequences revealed that most of them lack the obligatory C-terminal prenylation motif (CaaX. According to their C-terminal structures we classified the plant Gγ subunits into three distinct types. Type A consists of Gγ subunits with a putative prenylation motif. Type B subunits lack a prenylation motif and do not have any cysteine residues in the C-terminal region, while type C subunits contain an extended C-terminal domain highly enriched with cysteines. Comparative analysis of C-terminal domains of the proteins, intron-exon arrangement of the corresponding genes and phylogenetic studies suggested a common origin of all plant Gγ subunits. Conclusion Phylogenetic analyses suggest that types C and B most probably originated independently from type A ancestors. We speculate on a potential mechanism used by those Gγ subunits lacking isoprenylation motifs to anchor the Gβγ dimer to the plasma membrane and propose a new flexible nomenclature for plant Gγ subunits. Finally, in the light of our new classification, we give a word of caution about the interpretation of Gγ research in Arabidopsis and its generalization to other plant species.

  5. Analysis of iron- and sulfur-oxidizing bacteria in a treatment plant of acid rock drainage from a Japanese pyrite mine by use of ribulose-1, 5-bisphosphate carboxylase/oxygenase large-subunit gene. (United States)

    Kamimura, Kazuo; Okabayashi, Ai; Kikumoto, Mei; Manchur, Mohammed Abul; Wakai, Satoshi; Kanao, Tadayoshi


    Iron- and sulfur-oxidizing bacteria in a treatment plant of acid rock drainage (ARD) from a pyrite mine in Yanahara, Okayama prefecture, Japan, were analyzed using the gene (cbbL) encoding the large subunit of ribulose-1, 5-bisphosphate carboxylase/oxygenase (RubisCO). Analyses of partial sequences of cbbL genes from Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans and Acidithiobacillus caldus strains revealed the diversity in their cbbL gene sequences. In contrast to the presence of two copies of form I cbbL genes (cbbL1 and cbbL2) in A. ferrooxidans genome, A. thiooxidans and A. caldus had a single copy of form I cbbL gene in their genomes. A phylogenetic analysis based on deduced amino acid sequences from cbbL genes detected in the ARD treatment plant and their close relatives revealed that 89% of the total clones were affiliated with A. ferrooxidans. Clones loosely affiliated with the cbbL from A. thiooxidans NB1-3 or Thiobacillus denitrificans was also detected in the treatment plant. cbbL gene sequences of iron- or sulfur-oxidizing bacteria isolated from the ARD and the ARD treatment plant were not detected in the cbbL libraries from the treatment plant, suggesting the low frequencies of isolates in the samples. Copyright 2009 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  6. Structural dynamics and ssDNA binding activity of the three N-terminal domains of the large subunit of Replication Protein A from small angle X-ray scattering

    Energy Technology Data Exchange (ETDEWEB)

    Pretto, Dalyir I.; Tsutakawa, Susan; Brosey, Chris A.; Castillo, Amalchi; Chagot, Marie-Eve; Smith, Jarrod A.; Tainer, John A.; Chazin, Walter J.


    Replication Protein A (RPA) is the primary eukaryotic ssDNA binding protein utilized in diverse DNA transactions in the cell. RPA is a heterotrimeric protein with seven globular domains connected by flexible linkers, which enable substantial inter-domain motion that is essential to its function. Small angle X-ray scattering (SAXS) experiments on two multi-domain constructs from the N-terminus of the large subunit (RPA70) were used to examine the structural dynamics of these domains and their response to the binding of ssDNA. The SAXS data combined with molecular dynamics simulations reveal substantial interdomain flexibility for both RPA70AB (the tandem high affinity ssDNA binding domains A and B connected by a 10-residue linker) and RPA70NAB (RPA70AB extended by a 70-residue linker to the RPA70N protein interaction domain). Binding of ssDNA to RPA70NAB reduces the interdomain flexibility between the A and B domains, but has no effect on RPA70N. These studies provide the first direct measurements of changes in orientation of these three RPA domains upon binding ssDNA. The results support a model in which RPA70N remains structurally independent of RPA70AB in the DNA bound state and therefore freely available to serve as a protein recruitment module.

  7. The Arabidopsis gene DIG6 encodes a large 60S subunit nuclear export GTPase 1 that is involved in ribosome biogenesis and affects multiple auxin-regulated development processes

    KAUST Repository

    Zhao, Huayan


    The circularly permuted GTPase large subunit GTPase 1 (LSG1) is involved in the maturation step of the 60S ribosome and is essential for cell viability in yeast. Here, an Arabidopsis mutant dig6 (drought inhibited growth of lateral roots) was isolated. The mutant exhibited multiple auxin-related phenotypes, which included reduced lateral root number, altered leaf veins, and shorter roots. Genetic mapping combined with next-generation DNA sequencing identified that the mutation occurred in AtLSG1-2. This gene was highly expressed in regions of auxin accumulation. Ribosome profiling revealed that a loss of function of AtLSG1-2 led to decreased levels of monosomes, further demonstrating its role in ribosome biogenesis. Quantitative proteomics showed that the expression of certain proteins involved in ribosome biogenesis was differentially regulated, indicating that ribosome biogenesis processes were impaired in the mutant. Further investigations showed that an AtLSG1-2 deficiency caused the alteration of auxin distribution, response, and transport in plants. It is concluded that AtLSG1-2 is integral to ribosome biogenesis, consequently affecting auxin homeostasis and plant development.

  8. BCL::Fold--de novo prediction of complex and large protein topologies by assembly of secondary structure elements.

    Directory of Open Access Journals (Sweden)

    Mert Karakaş

    Full Text Available Computational de novo protein structure prediction is limited to small proteins of simple topology. The present work explores an approach to extend beyond the current limitations through assembling protein topologies from idealized α-helices and β-strands. The algorithm performs a Monte Carlo Metropolis simulated annealing folding simulation. It optimizes a knowledge-based potential that analyzes radius of gyration, β-strand pairing, secondary structure element (SSE packing, amino acid pair distance, amino acid environment, contact order, secondary structure prediction agreement and loop closure. Discontinuation of the protein chain favors sampling of non-local contacts and thereby creation of complex protein topologies. The folding simulation is accelerated through exclusion of flexible loop regions further reducing the size of the conformational search space. The algorithm is benchmarked on 66 proteins with lengths between 83 and 293 amino acids. For 61 out of these proteins, the best SSE-only models obtained have an RMSD100 below 8.0 Å and recover more than 20% of the native contacts. The algorithm assembles protein topologies with up to 215 residues and a relative contact order of 0.46. The method is tailored to be used in conjunction with low-resolution or sparse experimental data sets which often provide restraints for regions of defined secondary structure.

  9. Galaxy And Mass Assembly (GAMA): Trends in galaxy colours, morphology, and stellar populations with large scale structure, group, and pair environments

    CERN Document Server

    Alpaslan, Mehmet; Robotham, Aaron S G; Obreschkow, Danail; Andrae, Ellen; Cluver, Michelle; Kelvin, Lee S; Lange, Rebecca; Owers, Matt; Taylor, Edward N; Andrews, Stephen K; Bamford, Steven; Bland-Hawthorn, Joss; Brough, Sarah; Brown, Michael J I; Colless, Matthew; Davies, Luke J M; Eardley, Elizabeth; Grootes, Meiert W; Hopkins, Andrew M; Kennedy, Rebecca; Liske, Jochen; Lara-Lopez, Maritza A; Lopez-Sanchez, Angel R; Loveday, Jon; Madore, Barry F; Mahajan, Smriti; Meyer, Martin; Moffett, Amanda; Norberg, Peder; Penny, Samantha; Pimbblet, Kevin A; Popescu, Cristina C; Seibert, Mark; Tuffs, Richard


    We explore trends in galaxy properties with Mpc-scale structures using catalogues of environment and large scale structure from the Galaxy And Mass Assembly (GAMA) survey. Existing GAMA catalogues of large scale structure, group and pair membership allow us to construct galaxy stellar mass functions for different environmental types. To avoid simply extracting the known underlying correlations between galaxy properties and stellar mass, we create a mass matched sample of galaxies with stellar masses between $9.5 \\leq \\log{M_*/h^{-2} M_{\\odot}} \\leq 11$ for each environmental population. Using these samples, we show that mass normalised galaxies in different large scale environments have similar energy outputs, $u-r$ colours, luminosities, and morphologies. Extending our analysis to group and pair environments, we show galaxies that are not in groups or pairs exhibit similar characteristics to each other regardless of broader environment. For our mass controlled sample, we fail to see a strong dependence of S\\...

  10. Structure-function of proteins interacting with the alpha1 pore-forming subunit of high voltage-activated calcium channel

    Directory of Open Access Journals (Sweden)

    Alan eNeely


    Full Text Available Openings of high-voltage-activated calcium channels lead to a transient increase in calcium concentration that in turn activate a plethora of cellular functions, including muscle contraction, secretion and gene transcription. To coordinate all these responses calcium channels form supramolecular assemblies containing effectors and regulatory proteins that couple calcium influx to the downstream signal cascades and to feedback elements. According to the original biochemical characterization of skeletal muscle Dihydropyridine receptors, high-voltage-activated calcium channels are multi-subunit protein complexes consisting of a pore-forming subunit (α1 associated with four additional polypeptide chains β, α2, δ and γ, often referred to as accessory subunits. Twenty-five years after the first purification of a high-voltage calcium channel, the concept of a flexible stoichiometry to expand the repertoire of mechanisms that regulate calcium channel influx has emerged. Several other proteins have been identified that associate directly with the α1-subunit, including calmodulin and multiple members of the small and large GTPase family. Some of these proteins only interact with a subset of α1-subunits and during specific stages of biogenesis. More strikingly, most of the α1-subunit interacting proteins, such as the β-subunit and small GTPases, regulate both gating and trafficking through a variety of mechanisms. Modulation of channel activity covers almost all biophysical properties of the channel. Likewise, regulation of the number of channels in the plasma membrane is performed by altering the release of the α1-subunit from the endoplasmic reticulum, by reducing its degradation or enhancing its recycling back to the cell surface. In this review, we discuss the structural basis, interplay and functional role of selected proteins that interact with the central pore-forming subunit of high-voltage-activated calcium channels.

  11. Structure-function of proteins interacting with the α1 pore-forming subunit of high-voltage-activated calcium channels (United States)

    Neely, Alan; Hidalgo, Patricia


    Openings of high-voltage-activated (HVA) calcium channels lead to a transient increase in calcium concentration that in turn activate a plethora of cellular functions, including muscle contraction, secretion and gene transcription. To coordinate all these responses calcium channels form supramolecular assemblies containing effectors and regulatory proteins that couple calcium influx to the downstream signal cascades and to feedback elements. According to the original biochemical characterization of skeletal muscle Dihydropyridine receptors, HVA calcium channels are multi-subunit protein complexes consisting of a pore-forming subunit (α1) associated with four additional polypeptide chains β, α2, δ, and γ, often referred to as accessory subunits. Twenty-five years after the first purification of a high-voltage calcium channel, the concept of a flexible stoichiometry to expand the repertoire of mechanisms that regulate calcium channel influx has emerged. Several other proteins have been identified that associate directly with the α1-subunit, including calmodulin and multiple members of the small and large GTPase family. Some of these proteins only interact with a subset of α1-subunits and during specific stages of biogenesis. More strikingly, most of the α1-subunit interacting proteins, such as the β-subunit and small GTPases, regulate both gating and trafficking through a variety of mechanisms. Modulation of channel activity covers almost all biophysical properties of the channel. Likewise, regulation of the number of channels in the plasma membrane is performed by altering the release of the α1-subunit from the endoplasmic reticulum, by reducing its degradation or enhancing its recycling back to the cell surface. In this review, we discuss the structural basis, interplay and functional role of selected proteins that interact with the central pore-forming subunit of HVA calcium channels. PMID:24917826

  12. Large-area fabrication and characterisation of ultraviolet regime metamaterials manufactured using self-assembly techniques (Conference Presentation) (United States)

    Wardley, William P.; Nasir, Mazhar E.; Rodríguez Fortuño, Francisco J.; Vilain, Sébastien; Skov Campbell, Serena; Wurtz, Gregory A.; Zayats, Anatoly; Dickson, Wayne


    Metamaterials have a number of interesting and potentially useful applications in a variety of fields, such as chemical and biological sensing, enhancement of spontaneous emission, nonlinear optics and as substrates for use in surface enhanced Raman spectroscopy (SERS). However, to date the low-wavelength cutoff for the majority of work at the higher frequency end of the spectrum has been determined by use of the coinage metals, which intrinsically prohibit their implementation below a vacuum wavelength of approximately 500nm for gold and 350nm for silver. Producing nanostructured plasmonic media that exhibit metamaterial functionalities in the ultraviolet will have a number of benefits. Not only will working in a new range of the electromagnetic spectrum allow for higher energy photons to be controlled, but a number of other benefits arise from the behaviour of different materials in the ultraviolet. For instance, many biological molecules, including DNA, exhibit fluorescence in the UV range, allowing for label-free detection and analysis of biological material; the intrinsic electronic absorption can be used to increase this label-free bio-sensitivity as well as enable the possibility of SE(R)RS, a process further enhanced by the frequency dependence on the efficiency of this scattering process. Here, we demonstrate the fabrication and characterisation of metamaterials operating in the deep-near UV. By using alternatives to the coinage metals, including aluminium and gallium, we have measured optical responses in the system down to approximately 200 nm. Sample preparation utilises a self-assembly method, allowing for the production of macroscopic-sized assemblies (> 1 cm2) of nanometric elements (radius ~ 25 nm, separation ~ 100 nm). Careful control of the fabrication conditions allows fine control of the structural parameters, which in turn allows tunability of the optical properties over a wide range of wavelengths (> 200 nm). The structures produced include

  13. Determining the topology of virus assembly intermediates using ion mobility spectrometry-mass spectrometry. (United States)

    Knapman, Tom W; Morton, Victoria L; Stonehouse, Nicola J; Stockley, Peter G; Ashcroft, Alison E


    We have combined ion mobility spectrometry-mass spectrometry with tandem mass spectrometry to characterise large, non-covalently bound macromolecular complexes in terms of mass, shape (cross-sectional area) and stability (dissociation) in a single experiment. The results indicate that the quaternary architecture of a complex influences its residual shape following removal of a single subunit by collision-induced dissociation tandem mass spectrometry. Complexes whose subunits are bound to several neighbouring subunits to create a ring-like three-dimensional (3D) architecture undergo significant collapse upon dissociation. In contrast, subunits which have only a single neighbouring subunit within a complex retain much of their original shape upon complex dissociation. Specifically, we have determined the architecture of two transient, on-pathway intermediates observed during in vitro viral capsid assembly. Knowledge of the mass, stoichiometry and cross-sectional area of each viral assembly intermediate allowed us to model a range of potential structures based on the known X-ray structure of the coat protein building blocks. Comparing the cross-sectional areas of these potential architectures before and after dissociation provided tangible evidence for the assignment of the topologies of the complexes, which have been found to encompass both the 3-fold and the 5-fold symmetry axes of the final icosahedral viral shell. Such insights provide unique information about virus assembly pathways that could allow the design of anti-viral therapeutics directed at the assembly step. This methodology can be readily applied to the structural characterisation of many other non-covalently bound macromolecular complexes and their assembly pathways.

  14. SB-205384 Is a Positive Allosteric Modulator of Recombinant GABAA Receptors Containing Rat α3, α5, or α6 Subunit Subtypes Coexpressed with β3 and γ2 Subunits


    Heidelberg, Laura S.; Warren, James W.; Fisher, Janet L.


    Many drugs used to treat anxiety are positive modulators of GABAA receptors, which mediate fast inhibitory neurotransmission. The GABAA receptors can be assembled from a combination of at least 16 different subunits. The receptor’s subunit composition determines its pharmacologic and functional properties, and subunit expression varies throughout the brain. A primary goal for new treatments targeting GABAA receptors is the production of subunit-selective modulators acting upon a discrete popu...

  15. Phylogenetic relationships in the mushroom genus Coprinus and dark-spored allies based on sequence data from the nuclear gene coding for the large ribosomal subunit RNA: divergent domains, outgroups, and monophyly. (United States)

    Hopple, J S; Vilgalys, R


    Phylogenetic relationships were investigated in the mushroom genus Coprinus based on sequence data from the nuclear encoded large-subunit rDNA gene. Forty-seven species of Coprinus and 19 additional species from the families Coprinaceae, Strophariaceae, Bolbitiaceae, Agaricaceae, Podaxaceae, and Montagneaceae were studied. A total of 1360 sites was sequenced across seven divergent domains and intervening sequences. A total of 302 phylogenetically informative characters was found. Ninety-eight percent of the average divergence between taxa was located within the divergent domains, with domains D2 and D8 being most divergent and domains D7 and D10 the least divergent. An empirical test of phylogenetic signal among divergent domains also showed that domains D2 and D3 had the lowest levels of homoplasy. Two equally most parsimonious trees were resolved using Wagner parsimony. A character-state weighted analysis produced 12 equally most parsimonious trees similar to those generated by Wagner parsimony. Phylogenetic analyses employing topological constraints suggest that none of the major taxonomic systems proposed for subgeneric classification is able to completely reflect phylogenetic relationships in Coprinus. A strict consensus integration of the two Wagner trees demonstrates the problematic nature of choosing outgroups within dark-spored mushrooms. The genus Coprinus is found to be polyphyletic and is separated into three distinct clades. Most Coprinus taxa belong to the first two clades, which together form a larger monophyletic group with Lacrymaria and Psathyrella in basal positions. A third clade contains members of Coprinus section Comati as well as the genus Leucocoprinus, Podaxis pistillaris, Montagnea arenaria, and Agaricus pocillator. This third clade is separated from the other species of Coprinus by members of the families Strophariaceae and Bolbitiaceae and the genus Panaeolus.

  16. Interaction of Ddc1 and RPA with single-stranded/double-stranded DNA junctions in yeast whole cell extracts: Proteolytic degradation of the large subunit of replication protein A in ddc1Δ strains. (United States)

    Sukhanova, Maria V; D'Herin, Claudine; Boiteux, Serge; Lavrik, Olga I


    To characterize proteins that interact with single-stranded/double-stranded (ss/ds) DNA junctions in whole cell free extracts of Saccharomyces cerevisiae, we used [(32)P]-labeled photoreactive partial DNA duplexes containing a 3'-ss/ds-junction (3'-junction) or a 5'-ss/ds-junction (5'-junction). Identification of labeled proteins was achieved by MALDI-TOF mass spectrometry peptide mass fingerprinting and genetic analysis. In wild-type extract, one of the components of the Ddc1-Rad17-Mec3 complex, Ddc1, was found to be preferentially photocrosslinked at a 3'-junction. On the other hand, RPAp70, the large subunit of the replication protein A (RPA), was the predominant crosslinking product at a 5'-junction. Interestingly, ddc1Δ extracts did not display photocrosslinking of RPAp70 at a 5'-junction. The results show that RPAp70 crosslinked to DNA with a 5'-junction is subject to limited proteolysis in ddc1Δ extracts, whereas it is stable in WT, rad17Δ, mec3Δ and mec1Δ extracts. The degradation of the RPAp70-DNA adduct in ddc1Δ extract is strongly reduced in the presence of the proteasome inhibitor MG 132. We also addressed the question of the stability of free RPA, using anti-RPA antibodies. The results show that RPAp70 is also subject to proteolysis without photocrosslinking to DNA upon incubation in ddc1Δ extract. The data point to a novel property of Ddc1, modulating the turnover of DNA binding proteins such as RPAp70 by the proteasome.

  17. Characterization of a gene from chromosome 1B encoding the large subunit of ADPglucose pyrophosphorylase from wheat: evolutionary divergence and differential expression of Agp2 genes between leaves and developing endosperm. (United States)

    Thorneycroft, David; Hosein, Felicia; Thangavelu, Madan; Clark, Joanna; Vizir, Igor; Burrell, Michael M; Ainsworth, Charles


    A full-length genomic clone containing the gene encoding the large subunit of the ADPglucose pyrophosphorylase (Agp2), was isolated from a genomic library prepared from etiolated shoots of hexaploid wheat (Triticum aestivum L., cv, Chinese Spring). The coding region of this gene is identical to one of the cDNA clones previously isolated from a developing wheat grain cDNA library and is therefore an actively transcribed gene. The sequence represented by the cDNA spans 4.8 kb of the genomic clone and contains 15 introns. 2852 bp of DNA flanking the transcription start site of the gene was cloned upstream of the GUS (beta-glucuronidase) reporter gene. This Agp2::GUS construct and promoter deletions were used to study the pattern of reporter gene expression in both transgenic tobacco and wheat plants. Histochemical analysis of GUS expression in transgenic tobacco demonstrated that the reporter gene was expressed in guard cells of leaves and throughout the seed. In transgenic wheat, reporter gene expression was confined to the endosperm and aleurone with no expression in leaves. The cloned Agp2 gene was located to chromosome 1B by gene-specific PCR with nullisomic-tetrasomic lines. Northern analysis demonstrated that the Agp2 genes are differentially expressed in leaves and developing endosperm; while all three classes of Agp2 genes are transcribed in developing wheat grain endosperm, only one is transcribed in leaves. The differences between the Agp2 genes are discussed in relation to the evolution of hexaploid wheat.

  18. MPV17L2 is required for ribosome assembly in mitochondria (United States)

    Dalla Rosa, Ilaria; Durigon, Romina; Pearce, Sarah F.; Rorbach, Joanna; Hirst, Elizabeth M.A.; Vidoni, Sara; Reyes, Aurelio; Brea-Calvo, Gloria; Minczuk, Michal; Woellhaf, Michael W.; Herrmann, Johannes M.; Huynen, Martijn A.; Holt, Ian J.; Spinazzola, Antonella


    MPV17 is a mitochondrial protein of unknown function, and mutations in MPV17 are associated with mitochondrial deoxyribonucleic acid (DNA) maintenance disorders. Here we investigated its most similar relative, MPV17L2, which is also annotated as a mitochondrial protein. Mitochondrial fractionation analyses demonstrate MPV17L2 is an integral inner membrane protein, like MPV17. However, unlike MPV17, MPV17L2 is dependent on mitochondrial DNA, as it is absent from ρ0 cells, and co-sediments on sucrose gradients with the large subunit of the mitochondrial ribosome and the monosome. Gene silencing of MPV17L2 results in marked decreases in the monosome and both subunits of the mitochondrial ribosome, leading to impaired protein synthesis in the mitochondria. Depletion of MPV17L2 also induces mitochondrial DNA aggregation. The DNA and ribosome phenotypes are linked, as in the absence of MPV17L2 proteins of the small subunit of the mitochondrial ribosome are trapped in the enlarged nucleoids, in contrast to a component of the large subunit. These findings suggest MPV17L2 contributes to the biogenesis of the mitochondrial ribosome, uniting the two subunits to create the translationally competent monosome, and provide evidence that assembly of the small subunit of the mitochondrial ribosome occurs at the nucleoid. PMID:24948607

  19. Transcriptome sequencing of lentil based on second-generation technology permits large-scale unigene assembly and SSR marker discovery

    Directory of Open Access Journals (Sweden)

    Materne Michael


    Full Text Available Abstract Background Lentil (Lens culinaris Medik. is a cool-season grain legume which provides a rich source of protein for human consumption. In terms of genomic resources, lentil is relatively underdeveloped, in comparison to other Fabaceae species, with limited available data. There is hence a significant need to enhance such resources in order to identify novel genes and alleles for molecular breeding to increase crop productivity and quality. Results Tissue-specific cDNA samples from six distinct lentil genotypes were sequenced using Roche 454 GS-FLX Titanium technology, generating c. 1.38 × 106 expressed sequence tags (ESTs. De novo assembly generated a total of 15,354 contigs and 68,715 singletons. The complete unigene set was sequence-analysed against genome drafts of the model legume species Medicago truncatula and Arabidopsis thaliana to identify 12,639, and 7,476 unique matches, respectively. When compared to the genome of Glycine max, a total of 20,419 unique hits were observed corresponding to c. 31% of the known gene space. A total of 25,592 lentil unigenes were subsequently annoated from GenBank. Simple sequence repeat (SSR-containing ESTs were identified from consensus sequences and a total of 2,393 primer pairs were designed. A subset of 192 EST-SSR markers was screened for validation across a panel 12 cultivated lentil genotypes and one wild relative species. A total of 166 primer pairs obtained successful amplification, of which 47.5% detected genetic polymorphism. Conclusions A substantial collection of ESTs has been developed from sequence analysis of lentil genotypes using second-generation technology, permitting unigene definition across a broad range of functional categories. As well as providing resources for functional genomics studies, the unigene set has permitted significant enhancement of the number of publicly-available molecular genetic markers as tools for improvement of this species.

  20. Classification of 2-dimensional array patterns: assembling many small neural networks is better than using a large one. (United States)

    Chen, Liang; Xue, Wei; Tokuda, Naoyuki


    In many pattern classification/recognition applications of artificial neural networks, an object to be classified is represented by a fixed sized 2-dimensional array of uniform type, which corresponds to the cells of a 2-dimensional grid of the same size. A general neural network structure, called an undistricted neural network, which takes all the elements in the array as inputs could be used for problems such as these. However, a districted neural network can be used to reduce the training complexity. A districted neural network usually consists of two levels of sub-neural networks. Each of the lower level neural networks, called a regional sub-neural network, takes the elements in a region of the array as its inputs and is expected to output a temporary class label, called an individual opinion, based on the partial information of the entire array. The higher level neural network, called an assembling sub-neural network, uses the outputs (opinions) of regional sub-neural networks as inputs, and by consensus derives the label decision for the object. Each of the sub-neural networks can be trained separately and thus the training is less expensive. The regional sub-neural networks can be trained and performed in parallel and independently, therefore a high speed can be achieved. We prove theoretically in this paper, using a simple model, that a districted neural network is actually more stable than an undistricted neural network in noisy environments. We conjecture that the result is valid for all neural networks. This theory is verified by experiments involving gender classification and human face recognition. We conclude that a districted neural network is highly recommended for neural network applications in recognition or classification of 2-dimensional array patterns in highly noisy environments.

  1. Dynein light chain 1 induces assembly of large Bim complexes on mitochondria that stabilize Mcl-1 and regulate apoptosis. (United States)

    Singh, Prafull Kumar; Roukounakis, Aristomenis; Frank, Daniel O; Kirschnek, Susanne; Das, Kushal Kumar; Neumann, Simon; Madl, Josef; Römer, Winfried; Zorzin, Carina; Borner, Christoph; Haimovici, Aladin; Garcia-Saez, Ana; Weber, Arnim; Häcker, Georg


    The Bcl-2 family protein Bim triggers mitochondrial apoptosis. Bim is expressed in nonapoptotic cells at the mitochondrial outer membrane, where it is activated by largely unknown mechanisms. We found that Bim is regulated by formation of large protein complexes containing dynein light chain 1 (DLC1). Bim rapidly inserted into cardiolipin-containing membranes in vitro and recruited DLC1 to the membrane. Bim binding to DLC1 induced the formation of large Bim complexes on lipid vesicles, on isolated mitochondria, and in intact cells. Native gel electrophoresis and gel filtration showed Bim-containing mitochondrial complexes of several hundred kilodaltons in all cells tested. Bim unable to form complexes was consistently more active than complexed Bim, which correlated with its substantially reduced binding to anti-apoptotic Bcl-2 proteins. At endogenous levels, Bim surprisingly bound only anti-apoptotic Mcl-1 but not Bcl-2 or Bcl-XL, recruiting only Mcl-1 into large complexes. Targeting of DLC1 by RNAi in human cell lines induced disassembly of Bim-Mcl-1 complexes and the proteasomal degradation of Mcl-1 and sensitized the cells to the Bcl-2/Bcl-XL inhibitor ABT-737. Regulation of apoptosis at mitochondria thus extends beyond the interaction of monomers of proapoptotic and anti-apoptotic Bcl-2 family members but involves more complex structures of proteins at the mitochondrial outer membrane, and targeting complexes may be a novel therapeutic strategy. © 2017 Singh et al.; Published by Cold Spring Harbor Laboratory Press.

  2. Chaperoning 5S RNA assembly. (United States)

    Madru, Clément; Lebaron, Simon; Blaud, Magali; Delbos, Lila; Pipoli, Juliana; Pasmant, Eric; Réty, Stéphane; Leulliot, Nicolas


    In eukaryotes, three of the four ribosomal RNAs (rRNAs)—the 5.8S, 18S, and 25S/28S rRNAs—are processed from a single pre-rRNA transcript and assembled into ribosomes. The fourth rRNA, the 5S rRNA, is transcribed by RNA polymerase III and is assembled into the 5S ribonucleoprotein particle (RNP), containing ribosomal proteins Rpl5/uL18 and Rpl11/uL5, prior to its incorporation into preribosomes. In mammals, the 5S RNP is also a central regulator of the homeostasis of the tumor suppressor p53. The nucleolar localization of the 5S RNP and its assembly into preribosomes are performed by a specialized complex composed of Rpf2 and Rrs1 in yeast or Bxdc1 and hRrs1 in humans. Here we report the structural and functional characterization of the Rpf2-Rrs1 complex alone, in complex with the 5S RNA, and within pre-60S ribosomes. We show that the Rpf2-Rrs1 complex contains a specialized 5S RNA E-loop-binding module, contacts the Rpl5 protein, and also contacts the ribosome assembly factor Rsa4 and the 25S RNA. We propose that the Rpf2-Rrs1 complex establishes a network of interactions that guide the incorporation of the 5S RNP in preribosomes in the initial conformation prior to its rotation to form the central protuberance found in the mature large ribosomal subunit. © 2015 Madru et al.; Published by Cold Spring Harbor Laboratory Press.

  3. Probing subunit-subunit interactions in the yeast vacuolar ATPase by peptide arrays.

    Directory of Open Access Journals (Sweden)

    Lee S Parsons

    Full Text Available BACKGROUND: Vacuolar (H(+-ATPase (V-ATPase; V(1V(o-ATPase is a large multisubunit enzyme complex found in the endomembrane system of all eukaryotic cells where its proton pumping action serves to acidify subcellular organelles. In the plasma membrane of certain specialized tissues, V-ATPase functions to pump protons from the cytoplasm into the extracellular space. The activity of the V-ATPase is regulated by a reversible dissociation mechanism that involves breaking and re-forming of protein-protein interactions in the V(1-ATPase - V(o-proton channel interface. The mechanism responsible for regulated V-ATPase dissociation is poorly understood, largely due to a lack of detailed knowledge of the molecular interactions that are responsible for the structural and functional link between the soluble ATPase and membrane bound proton channel domains. METHODOLOGY/PRINCIPAL FINDINGS: To gain insight into where some of the stator subunits of the V-ATPase associate with each other, we have developed peptide arrays from the primary sequences of V-ATPase subunits. By probing the peptide arrays with individually expressed V-ATPase subunits, we have identified several key interactions involving stator subunits E, G, C, H and the N-terminal domain of the membrane bound a subunit. CONCLUSIONS: The subunit-peptide interactions identified from the peptide arrays complement low resolution structural models of the eukaryotic vacuolar ATPase obtained from transmission electron microscopy. The subunit-subunit interaction data are discussed in context of our current model of reversible enzyme dissociation.

  4. Cooperative Subunit Refolding of a Light-Harvesting Protein through a Self-Chaperone Mechanism. (United States)

    Laos, Alistair J; Dean, Jacob C; Toa, Zi S D; Wilk, Krystyna E; Scholes, Gregory D; Curmi, Paul M G; Thordarson, Pall


    The fold of a protein is encoded by its amino acid sequence, but how complex multimeric proteins fold and assemble into functional quaternary structures remains unclear. Here we show that two structurally different phycobiliproteins refold and reassemble in a cooperative manner from their unfolded polypeptide subunits, without biological chaperones. Refolding was confirmed by ultrafast broadband transient absorption and two-dimensional electronic spectroscopy to probe internal chromophores as a marker of quaternary structure. Our results demonstrate a cooperative, self-chaperone refolding mechanism, whereby the β-subunits independently refold, thereby templating the folding of the α-subunits, which then chaperone the assembly of the native complex, quantitatively returning all coherences. Our results indicate that subunit self-chaperoning is a robust mechanism for heteromeric protein folding and assembly that could also be applied in self-assembled synthetic hierarchical systems.

  5. Albumin-NIR dye self-assembled nanoparticles for photoacoustic pH imaging and pH-responsive photothermal therapy effective for large tumors. (United States)

    Chen, Qian; Liu, Xiaodong; Zeng, Jianfeng; Cheng, Zhenping; Liu, Zhuang


    Real-time in vivo pH imaging in the tumor, as well as designing therapies responsive to the acidic tumor microenvironment to achieve optimized therapeutic outcomes have been of great interests in the field of nanomedicine. Herein, a pH-responsive near-infrared (NIR) croconine (Croc) dye is able to induce the self-assembly of human serum albumin (HSA) to form HSA-Croc nanoparticles useful not only for real-time ratiometric photoacoustic pH imaging of the tumor, but also for pH responsive photothermal therapy with unexpected great performance against tumors with relatively large sizes. Such HSA-Croc nanoparticles upon intravenous injection exhibit efficient tumor homing. As the decrease of pH, the absorption of Croc at 810 nm would increase while that at 680 nm would decrease, allowing real-time pH sensing in the tumor by double-wavelength ratiometric photoacoustic imaging, which reveals the largely decreased pH inside the cores of large tumors. Moreover, utilizing HSA-Croc as a pH-responsive photothermal agent, effective photothermal ablation of large tumors is realized, likely owing to the more evenly distributed intratumoral heating compared to that achieved by conventional pH-insensitive photothermal agents, which are effective mostly for tumors with small sizes.

  6. Self-assembly of highly fluorescent semiconductor nanorods into large scale smectic liquid crystal structures by coffee stain evaporation dynamics

    Energy Technology Data Exchange (ETDEWEB)

    Nobile, Concetta; Carbone, Luigi; Fiore, Angela; Cingolani, Roberto; Manna, Liberato; Krahne, Roman [National Nanotechnology Laboratory of CNR-INFM, Via Arnesano, 73100 Lecce (Italy)], E-mail:


    We deposit droplets of nanorods dispersed in solvents on substrate surfaces and let the solvent evaporate. We find that strong contact line pinning leads to dense nanorod deposition inside coffee stain fringes, where we observe large scale lateral ordering of the nanorods with the long axis of the rods oriented parallel to the contact line. We observe birefringence of these coffee stain fringes by polarized microscopy and we find the direction of the extraordinary refractive index parallel to the long axis of the nanorods.

  7. Assembling Resistance Against Large-Scale Land Deals: Challenges for Conflict Transformation in Bougainville, Papua New Guinea

    Directory of Open Access Journals (Sweden)

    Anne Hennings


    Full Text Available Responding to the academic void on the impact of socio-ecological conflicts on peacebuilding and conflict transformation, I turn to resistance against large-scale land acquisitions in post-war contexts. Promising in terms of reconstruction and economic prosperity, the recent rush on land may, however, entail risks for reconciliation processes and long-term peace prospects. With reference to post-war Bougainville – as yet an autonomous province of Papua New Guinea – the article aims to conceptualize the impact of resistance against large-scale land deals on conflict transformation processes. Applying assemblage theory thereby allows not only analyzing multilayered dynamics in post-conflict societies but also new perspectives on socio-ecological conflicts. The findings suggest increasing resistance, for example, advocacy politics, demonstrations or sit-ins, against land deals and state territorialization in Bougainville with resemblances to pre-war contentious politics against Panguna mine. Yet, the lasting war trauma, a high weapon prevalence, and growing social friction add to destructive deterritorialization processes that are currently slowed down by the upcoming independence referendum.

  8. Experimental and numerical investigation of ADP square crystal with large aperture in the new Final Optics Assembly under the non-critical phase matching (United States)

    Sun, Fuzhong; Zhang, Peng; Bai, Qingshun; Lu, Lihua; Xiang, Yong


    This paper presented a new Final Optics Assembly (FOA) of ammonium dihydrogen phosphate (ADP) square crystal with large aperture under the non-critical phase matching (NCPM), which controlled by the constant temperature water, and the temperature distribution was analyzed by simulation and experiment. Firstly, thermal analysis was carried out, as well as the temperature distribution of the cavity only heated under different velocities was analyzed. Then, the temperature distributions of ADP square crystal in the cavity were achieved using the Finite Volume Method (FVM), and this prediction was validated by the experiment results when the velocity is 0.1 m/s and 0.5 m/s. Finally, the optimal FHG conversion efficiency was obtained and the comparison of different heating methods was also highlighted.

  9. An STM study on nonionic fluorosurfactant zonyl FSN self-assembly on Au(111): large domains, few defects, and good stability. (United States)

    Tang, Yongan; Yan, Jiawei; Zhou, Xiaoshun; Fu, Yongchun; Mao, Bingwei


    Nonionic Fluorosurfactant Zonyl FSN self-assembly on Au(111) is investigated with scanning tunneling microscopy under ambient conditions. STM reveals that the FSN forms SAMs on Au(l11) with very large domain size and almost no defects. A (mean square root of 3 x mean square root of 3)R3 degree arrangement of the FSN SAM on Au(111) is observed. The SAMs show excellent chemical stability and last for at least a month in atmospheric conditions. The structure and stability of the FSN SAMs are compared with those of alkanethiols SAMs. It is expected that FSN may serve as a new kind of molecule to form SAMs for surface modification, which would benefit wider applications for various purposes.

  10. Self-assembly of large-scale crack-free gold nanoparticle films using a ‘drain-to-deposit’ strategy

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Guang; Hallinan, Daniel T.


    Gold nanoparticles are widely studied due to the ease of controlled synthesis, facile surface modification, and interesting physical properties. However, a technique for depositing large-area, crack-free monolayers on solid substrates is lacking. Herein is presented a method for accomplishing this. Spherical gold nanoparticles were synthesized as an aqueous dispersion. Assembly into monolayers and ligand exchange occurred simultaneously at an organic/aqueous interface. Then the monolayer film was deposited onto arbitrary solid substrates by slowly pumping out the lower, aqueous phase. This allowed the monolayer film (and liquid–liquid interface) to descend without significant disturbance, eventually reaching substrates contained in the aqueous phase. The resulting macroscopic quality of the films was found to be superior to films transferred by Langmuir techniques. The surface plasmon resonance and Raman enhancement of the films were evaluated and found to be uniform across the surface of each film.

  11. Self-assembly of large-scale crack-free gold nanoparticle films using a ‘drain-to-deposit’ strategy (United States)

    Yang, Guang; Hallinan, Daniel T., Jr.


    Gold nanoparticles are widely studied due to the ease of controlled synthesis, facile surface modification, and interesting physical properties. However, a technique for depositing large-area, crack-free monolayers on solid substrates is lacking. Herein is presented a method for accomplishing this. Spherical gold nanoparticles were synthesized as an aqueous dispersion. Assembly into monolayers and ligand exchange occurred simultaneously at an organic/aqueous interface. Then the monolayer film was deposited onto arbitrary solid substrates by slowly pumping out the lower, aqueous phase. This allowed the monolayer film (and liquid-liquid interface) to descend without significant disturbance, eventually reaching substrates contained in the aqueous phase. The resulting macroscopic quality of the films was found to be superior to films transferred by Langmuir techniques. The surface plasmon resonance and Raman enhancement of the films were evaluated and found to be uniform across the surface of each film.

  12. Allotopic Expression of a Gene Encoding FLAG Tagged-subunit 8 of Yeast Mitochondrial ATP Synthase

    Directory of Open Access Journals (Sweden)



    Full Text Available Subunit 8 of yeast mitochondrial ATP synthase is a polypeptide of 48 amino acids encoded by the mitochondrial ATP8 gene. A nuclear version of subunit 8 gene has been designed to encode FLAG tagged-subunit 8 fused with a mitochondrial signal peptide. The gene has been cloned into a yeast expression vector and then expressed in a yeast strain lacking endogenous subunit 8. Results showed that the gene was successfully expressed and the synthesized FLAG tagged-subunit 8 protein was imported into mitochondria. Following import, the FLAG tagged-subunit 8 protein assembled into functional mitochondrial ATP synthase complex. Furthermore, the subunit 8 protein could be detected using anti-FLAG tag monoclonal antibody.

  13. Asymmetric Assembly of Merkel Cell Polyomavirus Large T-Antigen Origin Binding Domains at the Viral Origin

    Energy Technology Data Exchange (ETDEWEB)

    C Harrison; G Meinke; H Kwun; H Rogalin; P Phelan; P Bullock; Y Chang; P Moore; A Bohm


    The double-stranded DNA polyomavirus Merkel cell polyomavirus (MCV) causes Merkel cell carcinoma, an aggressive but rare human skin cancer that most often affects immunosuppressed and elderly persons. As in other polyomaviruses, the large T-antigen of MCV recognizes the viral origin of replication by binding repeating G(A/G)GGC pentamers. The spacing, number, orientation, and necessity of repeats for viral replication differ, however, from other family members such as SV40 and murine polyomavirus. We report here the 2.9 {angstrom} crystal structure of the MCV large T-antigen origin binding domain (OBD) in complex with a DNA fragment from the MCV origin of replication. Consistent with replication data showing that three of the G(A/G)GGC-like binding sites near the center of the origin are required for replication, the crystal structure contains three copies of the OBD. This stoichiometry was verified using isothermal titration calorimetry. The affinity for G(A/G)GGC-containing double-stranded DNA was found to be {approx} 740 nM, approximately 8-fold weaker than the equivalent domain in SV40 for the analogous region of the SV40 origin. The difference in affinity is partially attributable to DNA-binding residue Lys331 (Arg154 in SV40). In contrast to SV40, a small protein-protein interface is observed between MCV OBDs when bound to the central region of the origin. This protein-protein interface is reminiscent of that seen in bovine papilloma virus E1 protein. Mutational analysis indicates, however, that this interface contributes little to DNA binding energy.

  14. Assembly of 500,000 inter-specific catfish expressed sequence tags and large scale gene-associated marker development for whole genome association studies

    Energy Technology Data Exchange (ETDEWEB)

    Catfish Genome Consortium; Wang, Shaolin; Peatman, Eric; Abernathy, Jason; Waldbieser, Geoff; Lindquist, Erika; Richardson, Paul; Lucas, Susan; Wang, Mei; Li, Ping; Thimmapuram, Jyothi; Liu, Lei; Vullaganti, Deepika; Kucuktas, Huseyin; Murdock, Christopher; Small, Brian C; Wilson, Melanie; Liu, Hong; Jiang, Yanliang; Lee, Yoona; Chen, Fei; Lu, Jianguo; Wang, Wenqi; Xu, Peng; Somridhivej, Benjaporn; Baoprasertkul, Puttharat; Quilang, Jonas; Sha, Zhenxia; Bao, Baolong; Wang, Yaping; Wang, Qun; Takano, Tomokazu; Nandi, Samiran; Liu, Shikai; Wong, Lilian; Kaltenboeck, Ludmilla; Quiniou, Sylvie; Bengten, Eva; Miller, Norman; Trant, John; Rokhsar, Daniel; Liu, Zhanjiang


    Background-Through the Community Sequencing Program, a catfish EST sequencing project was carried out through a collaboration between the catfish research community and the Department of Energy's Joint Genome Institute. Prior to this project, only a limited EST resource from catfish was available for the purpose of SNP identification. Results-A total of 438,321 quality ESTs were generated from 8 channel catfish (Ictalurus punctatus) and 4 blue catfish (Ictalurus furcatus) libraries, bringing the number of catfish ESTs to nearly 500,000. Assembly of all catfish ESTs resulted in 45,306 contigs and 66,272 singletons. Over 35percent of the unique sequences had significant similarities to known genes, allowing the identification of 14,776 unique genes in catfish. Over 300,000 putative SNPs have been identified, of which approximately 48,000 are high-quality SNPs identified from contigs with at least four sequences and the minor allele presence of at least two sequences in the contig. The EST resource should be valuable for identification of microsatellites, genome annotation, large-scale expression analysis, and comparative genome analysis. Conclusions-This project generated a large EST resource for catfish that captured the majority of the catfish transcriptome. The parallel analysis of ESTs from two closely related Ictalurid catfishes should also provide powerful means for the evaluation of ancient and recent gene duplications, and for the development of high-density microarrays in catfish. The inter- and intra-specific SNPs identified from all catfish EST dataset assembly will greatly benefit the catfish introgression breeding program and whole genome association studies.

  15. A comparison of the unfolding and dissociation of the large ribosome subunits from Rhodopseudomonas spheroides N.C.I.B. 8253 and Escherichia coli M.R.E. 600. (United States)

    Robinson, A; Sykes, J


    1. The behaviour of the large ribosomal subunit from Rhodopseudomonas spheroides (45S) has been compared with the 50S ribosome from Escherichia coli M.R.E. 600 (and E. coli M.R.E. 162) during unfolding by removal of Mg(2+) and detachment of ribosomal proteins by high univalent cation concentrations. The extent to which these processes are reversible with these ribosomes has also been examined. 2. The R. spheroides 45S ribosome unfolds relatively slowly but then gives rise directly to two ribonucleoprotein particles (16.6S and 13.7S); the former contains the intact primary structure of the 16.25S rRNA species and the latter the 15.00S rRNA species of the original ribosome. No detectable protein loss occurs during unfolding. The E. coli ribosome unfolds via a series of discrete intermediates to a single, unfolded ribonucleoprotein unit (19.1S) containing the 23S rRNA and all the protein of the original ribosome. 3. The two unfolded R. spheroides ribonucleoproteins did not recombine when the original conditions were restored but each simply assumed a more compact configuration. Similar treatments reversed the unfolding of the E. coli 50S ribosomes; replacement of Mg(2+) caused the refolding of the initial products of unfolding and in the presence of Ni(2+) the completely unfolded species (19.1S) again sedimented at the same rate as the original ribosomes (44S). 4. Ribosomal proteins (25%) were dissociated from R. spheroides 45S ribosomes by dialysis against a solution with a Na(+)/Mg(2+) ratio of 250:1. During this process two core particles were formed (21.2S and 14.2S) and the primary structures of the two original rRNA species were conserved. This dissociation was not reversed. With E. coli 50S approximately 15% of the original ribosomal protein was dissociated, a single 37.6S core particle was formed, the 23S rRNA remained intact and the ribosomal proteins would reassociate with the core particle to give a 50S ribosome. 5. The ribonuclease activities in R

  16. Study on Ericaceae Plants and Analysis of Rubisco large subunit (RbcL)%杜鹃花科植物及其 Rubisco 大亚基(RbcL)初探

    Institute of Scientific and Technical Information of China (English)

    周晓馥; 陈思霖; 武慧; 徐洪伟


    Ericaceae is a kind of woody plants widely distributed around the world .It is not only the landscape plants worldwide,but also has significant economic and medicinal value .Ribulose-1,5-bisphosphate carboxylase/oxygenase ( Rubisco ) is involved in the assimilation of CO2 and also closely related to photosynthesis and photorespiration in plants.The catalytic sites of Rubisco mainly located in the Rubisco large subunit (RbcL),so it has a great significance to study the structure and character of RbcL .The existing Ericaceae plants in China and the RbcL obtained from NCBI were classified in the present study .This study not only lay a solid foundation for further research into the structure and character of RbcL as well as the photosynthesis mechanism of Ericaceae ,but also has great significance in the conservation ,development and utilization of plant resources .%杜鹃花科( Ericaceae )植物为木本植物,在世界范围内分布十分广泛,其不仅是世界主要的观赏花卉,而且还具有重要的经济价值和药用价值。核酮糖-1,5-二磷酸羧化酶/加氧酶( Ribulose-1,5-bisphosphate carbox-ylase/oxygenase,Rubisco)参与CO2的同化,与植物的光合作用和光呼吸密切相关,而Rubisco具有催化活性的位点主要位于Rubisco大亚基(RbcL),因此对于RbcL的研究显得尤为重要。该研究对截至2016年5月1日,我国现存的杜鹃花科植物进行了分类,同时对从NCBI中获得的杜鹃花科RbcL条目进行了统计及分类。这不仅为更好地剖析杜鹃花科植物光合作用机理及RbcL结构和功能的研究奠定基础,同时对于珍惜植物资源的保护、开发和利用具有重要意义。

  17. Biogenesis of cytochrome oxidase-sophisticated assembly lines in the mitochondrial inner membrane. (United States)

    Herrmann, Johannes M; Funes, Soledad


    Biogenesis of the cytochrome oxidase complex in the mitochondrial inner membrane depends on the concerted action of a variety of proteins. Recent studies shed light on this biological assembly process revealing an astonishingly complex procedure by which the different subunits of the enzymes are put together and the required cofactors are supplied. In this review we present a hypothetical model for the assembly process of cytochrome oxidase based on the current knowledge of the functions of specific assembly factors. According to this model the two largest subunits of the complex are first equipped with their respective cofactors on independent assembly lines. Prior to their assembly with the residual subunits that complete the whole complex, these two subcomplexes remain bound to substrate-specific chaperones. We propose that these chaperones, Mss51 for subunit 1 and Cox20 for subunit 2, control the coordinate assembly process to prevent potentially harmful redox reactions of unassembled or misassembled subunits.

  18. Next Generation Very Large Array Memo No. 8 Science Working Group 3: Galaxy Assembly through Cosmic Time

    CERN Document Server

    Casey, Caitlin M; Lacy, Mark; Hales, Christopher A; Barger, Amy; Narayanan, Desika; Carilli, Chris; Alatalo, Katherine; da Cunha, Elisabete; Emonts, Bjorn; Ivison, Rob; Kimball, Amy; Kohno, Kotaro; Murphy, Eric; Riechers, Dominik; Sargent, Mark; Walter, Fabian


    The Next-Generation Very Large Array (ngVLA) will be critical for understanding how galaxies are built and evolve at the earliest epochs. The sensitivity and frequency coverage will allow for the detection of cold gas and dust in `normal' distant galaxies, including the low-J transitions of molecular gas tracers such as CO, HNC, and HCO+; synchrotron and free-free continuum emission; and even the exciting possibility of thermal dust emission at the highest (z~7) redshifts. In particular, by enabling the total molecular gas reservoirs to be traced to unprecedented sensitivities across a huge range of epochs simultaneously -- something no other radio or submillimeter facility will be capable of -- the detection of the crucial low-J transitions of CO in a diverse body of galaxies will be the cornerstone of ngVLA's contribution to high-redshift galaxy evolution science. The ultra-wide bandwidths will allow a complete sampling of radio SEDs, as well as the detection of emission lines necessary for spectroscopic co...

  19. Development of a composite large-size SiPM (assembled matrix) based modular detector cluster for MAGIC (United States)

    Hahn, A.; Mazin, D.; Bangale, P.; Dettlaff, A.; Fink, D.; Grundner, F.; Haberer, W.; Maier, R.; Mirzoyan, R.; Podkladkin, S.; Teshima, M.; Wetteskind, H.


    The MAGIC collaboration operates two 17 m diameter Imaging Atmospheric Cherenkov Telescopes (IACTs) on the Canary Island of La Palma. Each of the two telescopes is currently equipped with a photomultiplier tube (PMT) based imaging camera. Due to the advances in the development of Silicon Photomultipliers (SiPMs), they are becoming a widely used alternative to PMTs in many research fields including gamma-ray astronomy. Within the Otto-Hahn group at the Max Planck Institute for Physics, Munich, we are developing a SiPM based detector module for a possible upgrade of the MAGIC cameras and also for future experiments as, e.g., the Large Size Telescopes (LST) of the Cherenkov Telescope Array (CTA). Because of the small size of individual SiPM sensors (6 mm×6 mm) with respect to the 1-inch diameter PMTs currently used in MAGIC, we use a custom-made matrix of SiPMs to cover the same detection area. We developed an electronic circuit to actively sum up and amplify the SiPM signals. Existing non-imaging hexagonal light concentrators (Winston cones) used in MAGIC have been modified for the angular acceptance of the SiPMs by using C++ based ray tracing simulations. The first prototype based detector module includes seven channels and was installed into the MAGIC camera in May 2015. We present the results of the first prototype and its performance as well as the status of the project and discuss its challenges.

  20. Inventory control: cytochrome oxidase assembly regulates mitochondrial translation (United States)

    Mick, David U.; Fox, Thomas D.; Rehling, Peter


    Mitochondria maintain a genome and translation-machinery to synthesize a small subset of subunits of the oxidative phosphorylation system. These organellar gene products must assemble with imported subunits that are encoded in the nucleus to build up functional enzymes. New findings on the early steps in cytochrome oxidase assembly reveal how the mitochondrial translation of its core component Cox1 is directly coupled to the assembly of this respiratory complex. PMID:21179059

  1. Subunit organization in cytoplasmic dynein subcomplexes (United States)

    King, Stephen J.; Bonilla, Myriam; Rodgers, Michael E.; Schroer, Trina A.


    Because cytoplasmic dynein plays numerous critical roles in eukaryotic cells, determining the subunit composition and the organization and functions of the subunits within dynein are important goals. This has been difficult partly because of accessory polypeptide heterogeneity of dynein populations. The motor domain containing heavy chains of cytoplasmic dynein are associated with multiple intermediate, light intermediate, and light chain accessory polypeptides. We examined the organization of these subunits within cytoplasmic dynein by separating the molecule into two distinct subcomplexes. These subcomplexes were competent to reassemble into a molecule with dynein-like properties. One subcomplex was composed of the dynein heavy and light intermediate chains whereas the other subcomplex was composed of the intermediate and light chains. The intermediate and light chain subcomplex could be further separated into two pools, only one of which contained dynein light chains. The two pools had distinct intermediate chain compositions, suggesting that intermediate chain isoforms have different light chain–binding properties. When the two intermediate chain pools were characterized by analytical velocity sedimentation, at least four molecular components were seen: intermediate chain monomers, intermediate chain dimers, intermediate chain monomers with bound light chains, and a mixture of intermediate chain dimers with assorted bound light chains. These data provide new insights into the compositional heterogeneity and assembly of the cytoplasmic dynein complex and suggest that individual dynein molecules have distinct molecular compositions in vivo. PMID:11967380

  2. The diversity of GABA(A) receptor subunit distribution in the normal and Huntington's disease human brain. (United States)

    Waldvogel, H J; Faull, R L M


    GABA(A) receptors are assembled into pentameric receptor complexes from a total of 19 different subunits derived from a variety of different subunit classes (α1-6, β1-3, γ1-3, δ, ɛ, θ, and π) which surround a central chloride ion channel. GABA(A) receptor complexes are distributed heterogeneously throughout the brain and spinal cord and are activated by the extensive GABAergic inhibitory system. In this chapter, we describe the heterogeneous distribution of six of the most widely distributed subunits (α1, α2, α3, β2,3, and γ2) throughout the human basal ganglia. This review describes the studies we have carried out on the normal and Huntington's disease human basal ganglia using autoradiographic labeling and immunohistochemistry in the human basal ganglia. GABA(A) receptors are known to react to changing conditions in the brain in neurological disorders, especially in Huntington's disease and display a high degree of plasticity which is thought to compensate for loss of function caused by disease. In Huntington's disease, the variable loss of GABAergic medium spiny striatopallidal projection neurons is associated with a loss of GABA(A) receptor subunits in the striosome and/or the matrix compartments of the striatum. By contrast in the globus pallidus, a loss of the GABAergic striatal projection neurons results in a dramatic upregulation of subunits on the large postsynaptic pallidal neurons; this is thought to be a compensatory plastic mechanism resulting from the loss of striatal GABAergic input. Most interestingly, our studies have revealed that the subventricular zone overlying the caudate nucleus contains a variety of proliferating progenitor stem cells that possess a heterogeneity of GABA(A) receptor subunits which may play a role in human brain repair mechanisms. © 2015 Elsevier Inc. All rights reserved.

  3. The forkhead transcription factor Foxi1 is a master regulator of vacuolar H-ATPase proton pump subunits in the inner ear, kidney and epididymis.

    Directory of Open Access Journals (Sweden)

    Hilmar Vidarsson

    Full Text Available The vacuolar H(+-ATPase dependent transport of protons across cytoplasmic membranes in FORE (forkhead related cells of endolymphatic epithelium in the inner ear, intercalated cells of collecting ducts in the kidney and in narrow and clear cells of epididymis require expression of several subunits that assemble into a functional multimeric proton pump. We demonstrate that expression of four such subunits A1, B1, E2 and a4 all co-localize with the forkhead transcription factor Foxi1 in a subset of epithelial cells at these three locations. In cells, of such epithelia, that lack Foxi1 we fail to identify any expression of A1, B1, E2 and a4 demonstrating an important role for the transcription factor Foxi1 in regulating subunit availability. Promoter reporter experiments, electrophoretic mobility shift assays (EMSA and site directed mutagenesis demonstrate that a Foxi1 expression vector can trans-activate an a4-promoter reporter construct in a dose dependent manner. Furthermore, we demonstrate using chromatin immunoprecipitation (ChIP assays that Foxi1-dependent activation to a large extent depends on cis-elements at position -561/-547 in the a4 promoter. Thus, we provide evidence that Foxi1 is necessary for expression of at least four subunits in three different epithelia and most likely is a major determinant for proper assembly of a functional vacuolar H(+-ATPase complex at these locations.

  4. Assembly and activation of alternative complement components on endothelial cell-anchored ultra-large von Willebrand factor links complement and hemostasis-thrombosis.

    Directory of Open Access Journals (Sweden)

    Nancy A Turner

    Full Text Available BACKGROUND: Vascular endothelial cells (ECs express and release protein components of the complement pathways, as well as secreting and anchoring ultra-large von Willebrand factor (ULVWF multimers in long string-like structures that initiate platelet adhesion during hemostasis and thrombosis. The alternative complement pathway (AP is an important non-antibody-requiring host defense system. Thrombotic microangiopathies can be associated with defective regulation of the AP (atypical hemolytic-uremic syndrome or with inadequate cleavage by ADAMTS-13 of ULVWF multimeric strings secreted by/anchored to ECs (thrombotic thrombocytopenic purpura. Our goal was to determine if EC-anchored ULVWF strings caused the assembly and activation of AP components, thereby linking two essential defense mechanisms. METHODOLOGY/PRINCIPAL FINDINGS: We quantified gene expression of these complement components in cultured human umbilical vein endothelial cells (HUVECs by real-time PCR: C3 and C5; complement factor (CF B, CFD, CFP, CFH and CFI of the AP; and C4 of the classical and lectin (but not alternative complement pathways. We used fluorescent microscopy, monospecific antibodies against complement components, fluorescent secondary antibodies, and the analysis of >150 images to quantify the attachment of HUVEC-released complement proteins to ULVWF strings secreted by, and anchored to, the HUVECs (under conditions of ADAMTS-13 inhibition. We found that HUVEC-released C4 did not attach to ULVWF strings, ruling out activation of the classical and lectin pathways by the strings. In contrast, C3, FB, FD, FP and C5, FH and FI attached to ULVWF strings in quantitative patterns consistent with assembly of the AP components into active complexes. This was verified when non-functional FB blocked the formation of AP C3 convertase complexes (C3bBb on ULVWF strings. CONCLUSIONS/SIGNIFICANCE: AP components are assembled and activated on EC-secreted/anchored ULVWF multimeric

  5. Large-area one-step assembly of three-dimensional porous metal micro/nanocages by ethanol-assisted femtosecond laser irradiation for enhanced antireflection and hydrophobicity. (United States)

    Li, Guoqiang; Li, Jiawen; Zhang, Chenchu; Hu, Yanlei; Li, Xiaohong; Chu, Jiaru; Huang, Wenhao; Wu, Dong


    The capability to realize 2D-3D controllable metallic micro/nanostructures is of key importance for various fields such as plasmonics, electronics, bioscience, and chemistry due to unique properties such as electromagnetic field enhancement, catalysis, photoemission, and conductivity. However, most of the present techniques are limited to low-dimension (1D-2D), small area, or single function. Here we report the assembly of self-organized three-dimensional (3D) porous metal micro/nanocages arrays on nickel surface by ethanol-assisted femtosecond laser irradiation. The underlying formation mechanism was investigated by a series of femtosecond laser irradiation under exposure time from 5 to 30 ms. We also demonstrate the ability to control the size of micro/nanocage arrays from 0.8 to 2 μm by different laser pulse energy. This method features rapidness (∼10 min), simplicity (one-step process), and ease of large-area (4 cm(2) or more) fabrication. The 3D cagelike micro/nanostructures exhibit not only improved antireflection from 80% to 7% but also enhanced hydrophobicity from 98.5° to 142° without surface modification. This simple technique for 3D large-area controllable metal microstructures will find great potential applications in optoelectronics, physics, and chemistry.

  6. Composite x-ray image assembly for large-field digital mammography with one- and two-dimensional positioning of a focal plane array (United States)

    Halama, G.; McAdoo, J.; Liu, H.


    To demonstrate the feasibility of a novel large-field digital mammography technique, a 1024 x 1024 pixel Loral charge-coupled device (CCD) focal plane array (FPA) was positioned in a mammographic field with one- and two-dimensional scan sequences to obtain 950 x 1800 pixel and 3600 x 3600 pixel composite images, respectively. These experiments verify that precise positioning of FPAs produced seamless composites and that the CCD mosaic concept has potential for high-resolution, large-field imaging. The proposed CCD mosaic concept resembles a checkerboard pattern with spacing left between the CCDs for the driver and readout electronics. To obtain a complete x-ray image, the mosaic must be repositioned four times, with an x-ray exposure at each position. To reduce the patient dose, a lead shield with appropriately patterned holes is placed between the x-ray source and the patient. The high-precision motorized translation stages and the fiber-coupled-scintillating-screen-CCD sensor assembly were placed in the position usually occupied by the film cassette. Because of the high mechanical precision, seamless composites were constructed from the subimages. This paper discusses the positioning, image alignment procedure, and composite image results. The paper only addresses the formation of a seamless composite image from subimages and will not consider the effects of the lead shield, multiple CCDs, or the speed of motion.

  7. Characterization of heterosubunit complexes formed by the R1 and R2 subunits of herpes simplex virus 1 and equine herpes virus 4 ribonucleotide reductase. (United States)

    Sun, Y; Conner, J


    We report on the separate PCR cloning and subsequent expression and purification of the large (R1) and small (R2) subunits from equine herpes virus type 4 (EHV-4) ribonucleotide reductase. The EHV-4 R1 and R2 subunits reconstituted an active enzyme and their abilities to complement the R1 and R2 subunits from the closely related herpes simplex virus 1 (HSV-1) ribonucleotide reductase, with the use of subunit interaction and enzyme activity assays, were analysed. Both EHV-4 R1/HSV-1 R2 and HSV-1 R1/EHV-4 R2 were able to assemble heterosubunit complexes but, surprisingly, neither of these complexes was fully active in enzyme activity assays; the EHV-4 R1/HSV-1 R2 and HSV-1 R1/EHV-4 R2 enzymes had 50% and 5% of their respective wild-type activities. Site-directed mutagenesis was used to alter two non-conserved residues located within the highly conserved and functionally important C-termini of the EHV-4 and HSV-1 R1 proteins. Mutation of Pro-737 to Lys and Lys-1084 to Pro in EHV-4 and HSV-1 R1 respectively had no effects on subunit assembly. Mutation of Pro-737 to Lys in EHV-4 R1 decreased enzyme activity by 50%; replacement of Lys-1084 by Pro in HSV-1 R1 had no effect on enzyme activity. Both alterations failed to restore full enzyme activities to the heterosubunit enzymes. Therefore probably neither of these amino acids has a direct role in catalysis. However, mutation of the highly conserved Tyr-1111 to Phe in HSV-1 R1 inactivated enzyme activity without affecting subunit interaction.

  8. Supramolecular Assembly of Comb-like Macromolecules Induced by Chemical Reactions that Modulate the Macromolecular Interactions In Situ. (United States)

    Xia, Hongwei; Fu, Hailin; Zhang, Yanfeng; Shih, Kuo-Chih; Ren, Yuan; Anuganti, Murali; Nieh, Mu-Ping; Cheng, Jianjun; Lin, Yao


    Supramolecular polymerization or assembly of proteins or large macromolecular units by a homogeneous nucleation mechanism can be quite slow and require specific solution conditions. In nature, protein assembly is often regulated by molecules that modulate the electrostatic interactions of the protein subunits for various association strengths. The key to this regulation is the coupling of the assembly process with a reversible or irreversible chemical reaction that occurs within the constituent subunits. However, realizing this complex process by the rational design of synthetic molecules or macromolecules remains a challenge. Herein, we use a synthetic polypeptide-grafted comb macromolecule to demonstrate how the in situ modulation of interactions between the charged macromolecules affects their resulting supramolecular structures. The kinetics of structural formation was studied and can be described by a generalized model of nucleated polymerization containing secondary pathways. Basic thermodynamic analysis indicated the delicate role of the electrostatic interactions between the charged subunits in the reaction-induced assembly process. This approach may be applicable for assembling a variety of ionic soft matters that are amenable to chemical reactions in situ.

  9. CCAN Assembly Configures Composite Binding Interfaces to Promote Cross-Linking of Ndc80 Complexes at the Kinetochore. (United States)

    Pekgöz Altunkaya, Gülsah; Malvezzi, Francesca; Demianova, Zuzana; Zimniak, Tomasz; Litos, Gabriele; Weissmann, Florian; Mechtler, Karl; Herzog, Franz; Westermann, Stefan


    Partitioning of the genome requires kinetochores, large protein complexes that mediate dynamic attachment of chromosomes to the spindle. Kinetochores contain two supramolecular protein assemblies. The ten-protein KMN network harbors key microtubule-binding sites in the Ndc80 complex and mediates assembly of checkpoint complexes via the KNL-1/Spc105 protein [1, 2]. As KMN does not contact DNA directly, it relies on different centromere-binding proteins for recruitment and cell-cycle-dependent assembly. These proteins are collectively referred to as the CCAN (constitutive centromere-associated network) [2-4]. The molecular mechanisms by which CCAN subunits associate, however, have remained incompletely defined. In particular, it is unclear how CCAN subunits facilitate the assembly of a microtubule-binding interface that contains multiple Ndc80 molecules bound to different receptors [5]. Here, we dissect molecular mechanisms that underlie targeting of the CCAN subunit Cnn1/CENP-T to the sequence-determined point centromeres of budding yeast. Systematic quantitative mass spectrometry experiments reveal association dependencies within the yeast CCAN network. We show that evolutionarily conserved residues in the histone-fold domain of Cnn1 are required for the formation of a stable five-subunit CCAN subassembly with the Ctf3 complex. Cnn1 localizes in a Ctf3-dependent manner to the core of the yeast point centromere, overlapping with the yeast CENP-A protein Cse4. By arranging the N-terminal domains of the CCAN subunits Mcm16, Mcm22, and Cnn1 into close proximity, the Ctf3c-Cnn1-Wip1 complex configures a composite interaction site for two molecules of the Ndc80 complex. Our experiments show how cooperative assembly mechanisms organize the microtubule-binding interface of the kinetochore. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Complementation of Escherichia coli unc mutant strains by chloroplast and cyanobacterial F1-ATPase subunits. (United States)

    Lill, H; Burkovski, A; Altendorf, K; Junge, W; Engelbrecht, S


    The genes encoding the five subunits of the F1 portion of the ATPases from both spinach chloroplasts and the cyanobacterium Synechocystis sp. PCC 6803 were cloned into expression vectors and expressed in Escherichia coli. The recombinant subunits formed inclusion bodies within the cells. Each particular subunit was expressed in the respective unc mutant, each unable to grow on non-fermentable carbon sources. The following subunits restored growth under conditions of oxidative phosphorylation: alpha (both sources, cyanobacterial subunit more than spinach subunit), beta (cyanobacterial subunit only), delta (both spinach and Synechocystis), and epsilon (both sources), whereas no growth was achieved with the gamma subunits from both sources. Despite a high degree of sequence homology the large subunits alpha and beta of spinach and cyanobacterial F1 were not as effective in the substitution of their E. coli counterparts. On the other hand, the two smallest subunits of the E. coli ATPase could be more effectively replaced by their cyanobacterial or chloroplast counterparts, although the sequence identity or even similarity is very low. We attribute these findings to the different roles of these subunits in F1: The large alpha and beta subunits contribute to the catalytic centers of the enzyme, a function rendering them very sensitive to even minor changes. For the smaller delta and epsilon subunits it was sufficient to maintain a certain tertiary structure during evolution, with little emphasis on the conservation of particular amino acids.

  11. Accessory NUMM (NDUFS6) subunit harbors a Zn-binding site and is essential for biogenesis of mitochondrial complex I (United States)

    Kmita, Katarzyna; Wirth, Christophe; Warnau, Judith; Guerrero-Castillo, Sergio; Hunte, Carola; Hummer, Gerhard; Kaila, Ville R. I.; Zwicker, Klaus; Brandt, Ulrich; Zickermann, Volker


    Mitochondrial proton-pumping NADH:ubiquinone oxidoreductase (respiratory complex I) comprises more than 40 polypeptides and contains eight canonical FeS clusters. The integration of subunits and insertion of cofactors into the nascent complex is a complicated multistep process that is aided by assembly factors. We show that the accessory NUMM subunit of complex I (human NDUFS6) harbors a Zn-binding site and resolve its position by X-ray crystallography. Chromosomal deletion of the NUMM gene or mutation of Zn-binding residues blocked a late step of complex I assembly. An accumulating assembly intermediate lacked accessory subunit N7BM (NDUFA12), whereas a paralog of this subunit, the assembly factor N7BML (NDUFAF2), was found firmly bound instead. EPR spectroscopic analysis and metal content determination after chromatographic purification of the assembly intermediate showed that NUMM is required for insertion or stabilization of FeS cluster N4. PMID:25902503

  12. Comparison of sequencing the D2 region of the large subunit ribosomal RNA gene (MicroSEQ®) versus the internal transcribed spacer (ITS) regions using two public databases for identification of common and uncommon clinically relevant fungal species. (United States)

    Arbefeville, S; Harris, A; Ferrieri, P


    Fungal infections cause considerable morbidity and mortality in immunocompromised patients. Rapid and accurate identification of fungi is essential to guide accurately targeted antifungal therapy. With the advent of molecular methods, clinical laboratories can use new technologies to supplement traditional phenotypic identification of fungi. The aims of the study were to evaluate the sole commercially available MicroSEQ® D2 LSU rDNA Fungal Identification Kit compared to the in-house developed internal transcribed spacer (ITS) regions assay in identifying moulds, using two well-known online public databases to analyze sequenced data. 85 common and uncommon clinically relevant fungi isolated from clinical specimens were sequenced for the D2 region of the large subunit (LSU) of ribosomal RNA (rRNA) gene with the MicroSEQ® Kit and the ITS regions with the in house developed assay. The generated sequenced data were analyzed with the online GenBank and MycoBank public databases. The D2 region of the LSU rRNA gene identified 89.4% or 92.9% of the 85 isolates to the genus level and the full ITS region (f-ITS) 96.5% or 100%, using GenBank or MycoBank, respectively, when compared to the consensus ID. When comparing species-level designations to the consensus ID, D2 region of the LSU rRNA gene aligned with 44.7% (38/85) or 52.9% (45/85) of these isolates in GenBank or MycoBank, respectively. By comparison, f-ITS possessed greater specificity, followed by ITS1, then ITS2 regions using GenBank or MycoBank. Using GenBank or MycoBank, D2 region of the LSU rRNA gene outperformed phenotypic based ID at the genus level. Comparing rates of ID between D2 region of the LSU rRNA gene and the ITS regions in GenBank or MycoBank at the species level against the consensus ID, f-ITS and ITS2 exceeded performance of the D2 region of the LSU rRNA gene, but ITS1 had similar performance to the D2 region of the LSU rRNA gene using MycoBank. Our results indicated that the MicroSEQ® D2 LSU r

  13. 夏枯草RuBPCase大亚基基因的克隆与表达%Molecular Cloning and Expression Analysis of A Large Subunit Gene of RuBPCase ( PvrbcL ) from Prunella vulgaris

    Institute of Scientific and Technical Information of China (English)

    许锋; 蒋丽阳; 曹腾; 宁迎晶; 程水源


    In order to clone the large subunit gene (rbcL) of Rubisco-1,5-bisphosphate carboxylase/ oxylase (RuBPCase), one of the important enzyme involved in photosynthesis and photorespiration, from P. vulgaris, and so as to provide basic data for studying on the structure and function of RuBPCase and discussing the photosynthesis mechanism on the molecular level of P. vulgaris, a pair of specific primers was designed. An rbcL gene fragment, named PvrbcL., was cloned from P. vulgaris using genomic DNA as template. The results showed that the length of PvrbcL sequence was 837 bp, encoding a 278-amino-acid protein. The GenBank accession No. of PvrbcL gene was JN692563. The BLAST results showed that the homology of the PvrbcL nucleotide sequence with rbcL genes from Prunella grandiflora , Salvia pachyphylla , Nicotiana tabacum , Glycine max , Oryza sativa , Phaseolus vulgaris , Vitis vinifer, and Brassica napus was 86%~100%, and the homology of amino acid sequences was 92//o~ 100%. Northern blot analysis revealed that PvrbcL gene expressed in leaves and stems, and the highest level was detected in leaves. Developmental expression patter indicated that the expression level of PvrbcL gene was increased along with the growth of the leaves. The isolation and expression of PvrbcL gene could provide the basis for further studying the structure and function of RuBPCase in P. vulgaris.%为从夏枯草中克隆参与光合作用和光呼吸代谢的关键酶RuBPcase大亚基基因rbcL,为今后研究夏枯草RuBPcase的结构和功能以及从分子水平探讨夏枯草的光合作用机理提供必要的基础数据,以夏枯草基因组DNA为模板,采用PCR法对夏枯草RuBPCase大亚基基因(PvrbcL)的克隆与表达进行研究.结果表明:PCR法扩增出夏枯草RuBPcase大亚基基因片段PvrbcL,基因长837bp,编码278个氨基酸,GenBank登录号为JN692563;经BLAST序列比较,夏枯草PvrbcL基因核苷酸序列与大花夏枯草、鼠尾草、烟草、大豆、水稻

  14. An example of deep crustal brines effecting large-scale chemical modification of the crust during assembly of the Paleoproterozoic Columbia supercontinent (United States)

    Glassley, W. E.; Korstgard, J. A.; Sørensen, K.


    The Nagssugtoqidian Mobile Belt (NMB) is a 300 km wide, 800 km long orogenic zone in central Greenland. It is part of the >20,000 km long complex of orogenic belts that formed between 1.7 and 2.0 Gya during assembly of the first supercontinent, Columbia,. The NMB is composed of several major crustal-scale shear zones that intervene between coherent blocks of high-grade metamorphic rocks. By combining detailed field, laboratory and aeromagnetic studies along the most northern of the shear zones (the Nordre Stromfjord Shear Zone - NSSZ), we have been able to document large scale chemical modification of the crust caused by the invasion of brines generated during continent-continent collision. The tectonic framework for this process is the following: 1. At 1,923 +/- 20 Mya: Emplacement of a calc-alkaline complex within and on ca. 2.8 Gya continental crust. The basal cumulate portion of the complex is well preserved and records invasion of multiple magma pulses. Coeval pillow basalts and cogenetic porphyritic mafics are also present. 2. 1,900 to 1,800 Mya: Tectonic emplacement of mafic and ultramafic rocks under high pressure (>2.5 GPa), eclogite facies conditions. The emplacement of these lenses probably occurred prior to or during thrust stacking associated with continent-continent collision. The age of the high pressure metamorphism makes these the oldest known eclogite facies metamorphic rocks in the world. 3. 1760 to 1720 Mya: Development of the transcurrent NSSZ, with displacements in excess of a hundred kilometers. Profound chemical enrichment of potassium and phosphorus along the entire length of the NSSZ unequivocally demonstrates that the shear zone was the focus of massive fluid movement. Detailed analysis of phase relationships documents a P-T path identical in form to that of Alpine metamorphic rocks, but displaced toward higher temperatures. These observations provide compelling evidence that assembly of this segment of Columbia involved subduction of

  15. Regulation of BK channels by auxiliary γ subunits

    Directory of Open Access Journals (Sweden)

    Jiyuan eZhang


    Full Text Available The large-conductance, calcium- and voltage-activated potassium (BK channel has the largest single-channel conductance among potassium channels and can be activated by both membrane depolarization and increases in intracellular calcium concentration. BK channels consist of pore-forming, voltage- and calcium-sensing α subunits, either alone or in association with regulatory subunits. BK channels are widely expressed in various tissues and cells including both excitable and non-excitable cells and display diverse biophysical and pharmacological characteristics. This diversity can be explained in part by posttranslational modifications and alternative splicing of the α subunit, which is encoded by a single gene, KCNMA1, as well as by tissue-specific β subunit modulation. Recently, a leucine-rich repeat-containing membrane protein, LRRC26, was found to interact with BK channels and cause an unprecedented large negative shift (~-140 mV in the voltage dependence of the BK channel activation. LRRC26 allows BK channels to open even at near-physiological calcium concentration and membrane voltage in non-excitable cells. Three LRRC26-related proteins, LRRC52, LRRC55, and LRRC38, were subsequently identified as BK channel modulators. These LRRC proteins are structurally and functionally distinct from the BK channel β subunits and were designated as γ subunits. The discovery of the γ subunits adds a new dimension to BK channel regulation and improves our understanding of the physiological functions of BK channels in various tissues and cell types. Unlike BK channel β subunits, which have been intensively investigated both mechanistically and physiologically, our understanding of the γ subunits is very limited at this stage. This article reviews the structure, modulatory mechanisms, physiological relevance, and potential therapeutic implications of γ subunits as they are currently understood.

  16. Optical Space Telescope Assembly Project (United States)

    National Aeronautics and Space Administration — The Optical Space Telescope Assembly (OSTA) task is to demonstrate the technology readiness of assembling large space telescopes on orbit in 2015. This task is an...

  17. Efficient reconstitution of functional Escherichia coli 30S ribosomal subunits from a complete set of recombinant small subunit ribosomal proteins. (United States)

    Culver, G M; Noller, H F


    Previous studies have shown that the 30S ribosomal subunit of Escherichia coli can be reconstituted in vitro from individually purified ribosomal proteins and 16S ribosomal RNA, which were isolated from natural 30S subunits. We have developed a 30S subunit reconstitution system that uses only recombinant ribosomal protein components. The genes encoding E. coli ribosomal proteins S2-S21 were cloned, and all twenty of the individual proteins were overexpressed and purified. Reconstitution, following standard procedures, using the complete set of recombinant proteins and purified 16S ribosomal RNA is highly inefficient. Efficient reconstitution of 30S subunits using these components requires sequential addition of proteins, following either the 30S subunit assembly map (Mizushima & Nomura, 1970, Nature 226:1214-1218; Held et al., 1974, J Biol Chem 249:3103-3111) or following the order of protein assembly predicted from in vitro assembly kinetics (Powers et al., 1993, J MoI Biol 232:362-374). In the first procedure, the proteins were divided into three groups, Group I (S4, S7, S8, S15, S17, and S20), Group II (S5, S6, S9, Sll, S12, S13, S16, S18, and S19), and Group III (S2, S3, S10, S14, and S21), which were sequentially added to 16S rRNA with a 20 min incubation at 42 degrees C following the addition of each group. In the second procedure, the proteins were divided into Group I (S4, S6, S11, S15, S16, S17, S18, and S20), Group II (S7, S8, S9, S13, and S19), Group II' (S5 and S12) and Group III (S2, S3, S10, S14, and S21). Similarly efficient reconstitution is observed whether the proteins are grouped according to the assembly map or according to the results of in vitro 30S subunit assembly kinetics. Although reconstitution of 30S subunits using the recombinant proteins is slightly less efficient than reconstitution using a mixture of total proteins isolated from 30S subunits, it is much more efficient than reconstitution using proteins that were individually isolated

  18. Ultrahigh Molecular Weight Linear Block Copolymers: Rapid Access by Reversible-Deactivation Radical Polymerization and Self- Assembly into Large Domain Nanostructures

    Energy Technology Data Exchange (ETDEWEB)

    Mapas, Jose Kenneth D.; Thomay, Tim; Cartwright, Alexander N.; Ilavsky, Jan; Rzayev, Javid


    Block copolymer (BCP) derived periodic nanostructures with domain sizes larger than 150 nm present a versatile platform for the fabrication of photonic materials. So far, the access to such materials has been limited to highly synthetically involved protocols. Herein, we report a simple, “user-friendly” method for the preparation of ultrahigh molecular weight linear poly(solketal methacrylate-b-styrene) block copolymers by a combination of Cu-wire-mediated ATRP and RAFT polymerizations. The synthesized copolymers with molecular weights up to 1.6 million g/mol and moderate dispersities readily assemble into highly ordered cylindrical or lamella microstructures with domain sizes as large as 292 nm, as determined by ultra-small-angle x-ray scattering and scanning electron microscopy analyses. Solvent cast films of the synthesized block copolymers exhibit stop bands in the visible spectrum correlated to their domain spacings. The described method opens new avenues for facilitated fabrication and the advancement of fundamental understanding of BCP-derived photonic nanomaterials for a variety of applications.

  19. PS-b-PEO/Silica Films with Regular and Reverse Mesostructures of Large Characteristic Length Scales Prepared by Solvent Evaporation-Induced Self-Assembly

    Energy Technology Data Exchange (ETDEWEB)



    Since the discovery of surfactant-templated silica by Mobil scientists in 1992, mesostructured silica has been synthesized in various forms including thin films, powders, particles, and fibers. In general, mesostructured silica has potential applications, such as in separation, catalysis, sensors, and fluidic microsystems. In respect to these potential applications, mesostructured silica in the form of thin films is perhaps one of the most promising candidates. The preparation of mesostructured silica films through preferential solvent evaporation-induced self-assembly (EISA) has recently received much attention in the laboratories. However, no amphiphile/silica films with reverse mesophases have ever been made through this EISA procedure. Furthermore, templates employed to date have been either surfactants or poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) triblock copolymers, such as pluronic P-123, both of which are water-soluble and alcohol-soluble. Due to their relatively low molecular weight, the templated silica films with mesoscopic order have been limited to relatively small characteristic length scales. In the present communication, the authors report a novel synthetic method to prepare mesostructured amphiphilic/silica films with regular and reverse mesophases of large characteristic length scales. This method involves evaporation-induced self-assembly (EISA) of amphiphilic polystyrene-block-poly(ethylene oxide) (PS-b-PEO) diblock copolymers. In the present study, the PS-b-PEO diblocks are denoted as, for example, PS(215)-b-PEO(100), showing that this particular sample contains 215 S repeat units and 100 EO repeat units. This PS(215)-b-PEO(100) diblock possesses high molecular weight and does not directly mix with water or alcohol. To the authors knowledge, no studies have reported the use of water-insoluble and alcohol-insoluble amphiphilic diblocks as structure-directing agents in the synthesis of mesostructured silica films through

  20. Proteomic characterization of the small subunit of Chlamydomonas reinhardtii chloroplast ribosome: identification of a novel S1 domain-containing protein and unusually large orthologs of bacterial S2, S3, and S5. (United States)

    Yamaguchi, Kenichi; Prieto, Susana; Beligni, María Verónica; Haynes, Paul A; McDonald, W Hayes; Yates, John R; Mayfield, Stephen P


    To understand how chloroplast mRNAs are translated into functional proteins, a detailed understanding of all of the components of chloroplast translation is needed. To this end, we performed a proteomic analysis of the plastid ribosomal proteins in the small subunit of the chloroplast ribosome from the green alga Chlamydomonas reinhardtii. Twenty proteins were identified, including orthologs of Escherichia coli S1, S2, S3, S4, S5, S6, S7, S9, S10, S12, S13, S14, S15, S16, S17, S18, S19, S20, and S21 and a homolog of spinach plastid-specific ribosomal protein-3 (PSRP-3). In addition, a novel S1 domain-containing protein, PSRP-7, was identified. Among the identified proteins, S2 (57 kD), S3 (76 kD), and S5 (84 kD) are prominently larger than their E. coli or spinach counterparts, containing N-terminal extensions (S2 and S5) or insertion sequence (S3). Structural predictions based on the crystal structure of the bacterial 30S subunit suggest that the additional domains of S2, S3, and S5 are located adjacent to each other on the solvent side near the binding site of the S1 protein. These additional domains may interact with the S1 protein and PSRP-7 to function in aspects of mRNA recognition and translation initiation that are unique to the Chlamydomonas chloroplast.

  1. Conducting Automated Test Assembly Using the Premium Solver Platform Version 7.0 with Microsoft Excel and the Large-Scale LP/QP Solver Engine Add-In (United States)

    Cor, Ken; Alves, Cecilia; Gierl, Mark J.


    This review describes and evaluates a software add-in created by Frontline Systems, Inc., that can be used with Microsoft Excel 2007 to solve large, complex test assembly problems. The combination of Microsoft Excel 2007 with the Frontline Systems Premium Solver Platform is significant because Microsoft Excel is the most commonly used spreadsheet…

  2. The subunit composition and function of mammalian cytochrome c oxidase. (United States)

    Kadenbach, Bernhard; Hüttemann, Maik


    Cytochrome c oxidase (COX) from mammals and birds is composed of 13 subunits. The three catalytic subunits I-III are encoded by mitochondrial DNA, the ten nuclear-coded subunits (IV, Va, Vb, VIa, VIb, VIc, VIIa, VIIb, VIIc, VIII) by nuclear DNA. The nuclear-coded subunits are essentially involved in the regulation of oxygen consumption and proton translocation by COX, since their removal or modification changes the activity and their mutation causes mitochondrial diseases. Respiration, the basis for ATP synthesis in mitochondria, is differently regulated in organs and species by expression of tissue-, developmental-, and species-specific isoforms for COX subunits IV, VIa, VIb, VIIa, VIIb, and VIII, but the holoenzyme in mammals is always composed of 13 subunits. Various proteins and enzymes were shown, e.g., by co-immunoprecipitation, to bind to specific COX subunits and modify its activity, but these interactions are reversible, in contrast to the tightly bound 13 subunits. In addition, the formation of supercomplexes with other oxidative phosphorylation complexes has been shown to be largely variable. The regulatory complexity of COX is increased by protein phosphorylation. Up to now 18 phosphorylation sites have been identified under in vivo conditions in mammals. However, only for a few phosphorylation sites and four nuclear-coded subunits could a specific function be identified. Research on the signaling pathways leading to specific COX phosphorylations remains a great challenge for understanding the regulation of respiration and ATP synthesis in mammalian organisms. This article reviews the function of the individual COX subunits and their isoforms, as well as proteins and small molecules interacting and regulating the enzyme.

  3. Genetic mapping of the LMP2 proteasome subunit gene to the BoLA class IIb region

    Energy Technology Data Exchange (ETDEWEB)

    Shalhevet, D.; Da, Y.; Beever, J.E.; Eijk, M.J.T. van; Ma, R.; Lewin, H.A.; Gaskins, H.R. [Univ. of Illinois, Champaign, IL (United States)


    Recent identification of four tightly-linked genes within the class II region of the major histocompatibility complex (MHC) in humans and rodents has led to a better understanding of class I antigen processing mechanisms. Two of these genes, LMP2 and LMP7, encode subunits of a low molecular mass poypeptide (LMP) complex. Several observations suggest that the LMP complex may be the proteolytic system responsible for generating the size-restricted peptides required for MHC class I assembly. For example, the LMP complex is a large cytoplasmic structure that is antigenically and biochemically related to the proteasome, a proteolytic complex that mediates degradation of ubiquitinated substrates. Data regarding proteolytic specificity indicates that the LMP complex may specifically produce nonamers, the appropriate peptide size for class I binding. In addition, similar to all components of the class I assembly process, intra-MHC LMP genes are regulated by IFN{gamma}. 26 refs., 2 figs., 1 tab.

  4. Impaired folding and subunit assembly as disease mechanism

    DEFF Research Database (Denmark)

    Bross, P; Andresen, B S; Gregersen, N


    mutations. Characterization of the effect of these mutations is particularly important in order to establish that they are disease causing and to estimate their severity. We use the experiences with investigation of medium-chain acyl-CoA dehydrogenase deficiency as an example to illustrate that (i) impaired......Rapid progress in DNA technology has entailed the possibility of readily detecting mutations in disease genes. In contrast to this, techniques to characterize the effects of mutations are still very time consuming. It has turned out that many of the mutations detected in disease genes are missense...... folding is a common effect of missense mutations occurring in genetic diseases, (ii) increasing the level of available chaperones may augment the level of functional mutant protein in vivo, and (iii) one mutation may have multiple effects. The interplay between the chaperones assisting folding...

  5. Conservation of helical bundle structure between the exocyst subunits.

    Directory of Open Access Journals (Sweden)

    Nicole J Croteau

    Full Text Available BACKGROUND: The exocyst is a large hetero-octomeric protein complex required for regulating the targeting and fusion of secretory vesicles to the plasma membrane in eukaryotic cells. Although the sequence identity between the eight different exocyst subunits is less than 10%, structures of domains of four of the subunits revealed a similar helical bundle topology. Characterization of several of these subunits has been hindered by lack of soluble protein for biochemical and structural studies. METHODOLOGY/PRINCIPAL FINDINGS: Using advanced hidden Markov models combined with secondary structure predictions, we detect significant sequence similarity between each of the exocyst subunits, indicating that they all contain helical bundle structures. We corroborate these remote homology predictions by identifying and purifying a predicted domain of yeast Sec10p, a previously insoluble exocyst subunit. This domain is soluble and folded with approximately 60% alpha-helicity, in agreement with our predictions, and capable of interacting with several known Sec10p binding partners. CONCLUSIONS/SIGNIFICANCE: Although all eight of the exocyst subunits had been suggested to be composed of similar helical bundles, this has now been validated by our hidden Markov model structure predictions. In addition, these predictions identified protein domains within the exocyst subunits, resulting in creation and characterization of a soluble, folded domain of Sec10p.

  6. NOX Activation by Subunit Interaction and Underlying Mechanisms in Disease (United States)

    Rastogi, Radhika; Geng, Xiaokun; Li, Fengwu; Ding, Yuchuan


    Nicotinamide adenine dinucleotide phosphate (NAPDH) oxidase (NOX) is an enzyme complex with the sole function of producing superoxide anion and reactive oxygen species (ROS) at the expense of NADPH. Vital to the immune system as well as cellular signaling, NOX is also involved in the pathologies of a wide variety of disease states. Particularly, it is an integral player in many neurological diseases, including stroke, TBI, and neurodegenerative diseases. Pathologically, NOX produces an excessive amount of ROS that exceed the body’s antioxidant ability to neutralize them, leading to oxidative stress and aberrant signaling. This prevalence makes it an attractive therapeutic target and as such, NOX inhibitors have been studied and developed to counter NOX’s deleterious effects. However, recent studies of NOX have created a better understanding of the NOX complex. Comprised of independent cytosolic subunits, p47-phox, p67-phox, p40-phox and Rac, and membrane subunits, gp91-phox and p22-phox, the NOX complex requires a unique activation process through subunit interaction. Of these subunits, p47-phox plays the most important role in activation, binding and translocating the cytosolic subunits to the membrane and anchoring to p22-phox to organize the complex for NOX activation and function. Moreover, these interactions, particularly that between p47-phox and p22-phox, are dependent on phosphorylation initiated by upstream processes involving protein kinase C (PKC). This review will look at these interactions between subunits and with PKC. It will focus on the interaction involving p47-phox with p22-phox, key in bringing the cytosolic subunits to the membrane. Furthermore, the implication of these interactions as a target for NOX inhibitors such as apocynin will be discussed as a potential avenue for further investigation, in order to develop more specific NOX inhibitors based on the inhibition of NOX assembly and activation. PMID:28119569

  7. Dynamic pathways for viral capsid assembly

    Energy Technology Data Exchange (ETDEWEB)

    Hagan, Michael F.; Chandler, David


    We develop a class of models with which we simulate the assembly of particles into T1 capsid-like objects using Newtonian dynamics. By simulating assembly for many different values of system parameters, we vary the forces that drive assembly. For some ranges of parameters, assembly is facile, while for others, assembly is dynamically frustrated by kinetic traps corresponding to malformed or incompletely formed capsids. Our simulations sample many independent trajectories at various capsomer concentrations, allowing for statistically meaningful conclusions. Depending on subunit (i.e., capsomer) geometries, successful assembly proceeds by several mechanisms involving binding of intermediates of various sizes. We discuss the relationship between these mechanisms and experimental evaluations of capsid assembly processes.

  8. Subunit Composition and Substrate Specificity of a MOF-containing Histone Acetyltransferase Distinct from the Male-specific Lethal (MSL) Complex* (United States)

    Cai, Yong; Jin, Jingji; Swanson, Selene K.; Cole, Michael D.; Choi, Seung Hyuk; Florens, Laurence; Washburn, Michael P.; Conaway, Joan W.; Conaway, Ronald C.


    Human MOF (MYST1), a member of the MYST (Moz-Ybf2/Sas3-Sas2-Tip60) family of histone acetyltransferases (HATs), is the human ortholog of the Drosophila males absent on the first (MOF) protein. MOF is the catalytic subunit of the male-specific lethal (MSL) HAT complex, which plays a key role in dosage compensation in the fly and is responsible for a large fraction of histone H4 lysine 16 (H4K16) acetylation in vivo. MOF was recently reported to be a component of a second HAT complex, designated the non-specific lethal (NSL) complex (Mendjan, S., Taipale, M., Kind, J., Holz, H., Gebhardt, P., Schelder, M., Vermeulen, M., Buscaino, A., Duncan, K., Mueller, J., Wilm, M., Stunnenberg, H. G., Saumweber, H., and Akhtar, A. (2006) Mol. Cell 21, 811–823). Here we report an analysis of the subunit composition and substrate specificity of the NSL complex. Proteomic analyses of complexes purified through multiple candidate subunits reveal that NSL is composed of nine subunits. Two of its subunits, WD repeat domain 5 (WDR5) and host cell factor 1 (HCF1), are shared with members of the MLL/SET family of histone H3 lysine 4 (H3K4) methyltransferase complexes, and a third subunit, MCRS1, is shared with the human INO80 chromatin-remodeling complex. In addition, we show that assembly of the MOF HAT into MSL or NSL complexes controls its substrate specificity. Although MSL-associated MOF acetylates nucleosomal histone H4 almost exclusively on lysine 16, NSL-associated MOF exhibits a relaxed specificity and also acetylates nucleosomal histone H4 on lysines 5 and 8. PMID:20018852

  9. RNA-controlled assembly of tobacco mosaic virus-derived complex structures: from nanoboomerangs to tetrapods (United States)

    Eber, Fabian J.; Eiben, Sabine; Jeske, Holger; Wege, Christina


    The in vitro assembly of artificial nanotubular nucleoprotein shapes based on tobacco mosaic virus-(TMV-)-derived building blocks yielded different spatial organizations of viral coat protein subunits on genetically engineered RNA molecules, containing two or multiple TMV origins of assembly (OAs). The growth of kinked nanoboomerangs as well as of branched multipods was determined by the encapsidated RNAs. A largely simultaneous initiation at two origins and subsequent bidirectional tube elongation could be visualized by transmission electron microscopy of intermediates and final products. Collision of the nascent tubes' ends produced angular particles with well-defined arm lengths. RNAs with three to five OAs generated branched multipods with a maximum of four arms. The potential of such an RNA-directed self-assembly of uncommon nanotubular architectures for the fabrication of complex multivalent nanotemplates used in functional hybrid materials is discussed.

  10. F1-dependent translation of mitochondrially encoded Atp6p and Atp8p subunits of yeast ATP synthase


    Rak, Malgorzata; Tzagoloff, Alexander


    The ATP synthase of yeast mitochondria is composed of 17 different subunit polypeptides. We have screened a panel of ATP synthase mutants for impaired expression of Atp6p, Atp8p, and Atp9p, the only mitochondrially encoded subunits of ATP synthase. Our results show that translation of Atp6p and Atp8p is activated by F1 ATPase (or assembly intermediates thereof). Mutants lacking the α or β subunits of F1, or the Atp11p and Atp12p chaperones that promote F1 assembly, have normal levels of the b...

  11. Insights into the composition and assembly of the membrane arm of plant complex I through analysis of subcomplexes in Arabidopsis mutant lines. (United States)

    Meyer, Etienne H; Solheim, Cory; Tanz, Sandra K; Bonnard, Géraldine; Millar, A Harvey


    NADH-ubiquinone oxidoreductase (Complex I, EC is the largest complex of the mitochondrial respiratory chain. In eukaryotes, it is composed of more than 40 subunits that are encoded by both the nuclear and mitochondrial genomes. Plant Complex I differs from the enzyme described in other eukaryotes, most notably due to the large number of plant-specific subunits in the membrane arm of the complex. The elucidation of the assembly pathway of Complex I has been a long-standing research aim in cellular biochemistry. We report the study of Arabidopsis mutants in Complex I subunits using a combination of Blue-Native PAGE and immunodetection to identify stable subcomplexes containing Complex I components, along with mass spectrometry analysis of Complex I components in membrane fractions and two-dimensional diagonal Tricine SDS-PAGE to study the composition of the largest subcomplex. Four subcomplexes of the membrane arm of Complex I with apparent molecular masses of 200, 400, 450, and 650 kDa were observed. We propose a working model for the assembly of the membrane arm of Complex I in plants and assign putative roles during the assembly process for two of the subunits studied.

  12. The Subfamily-Specific Interaction between Kv2.1 and Kv6.4 Subunits Is Determined by Interactions between the N- and C-termini


    Elke Bocksteins; Evy Mayeur; Abbi Van Tilborg; Glenn Regnier; Jean-Pierre Timmermans; Snyders, Dirk J


    The "silent" voltage-gated potassium (KvS) channel subunit Kv6.4 does not form electrically functional homotetramers at the plasma membrane but assembles with Kv2.1 subunits, generating functional Kv2.1/Kv6.4 heterotetramers. The N-terminal T1 domain determines the subfamily-specific assembly of Kv1-4 subunits by preventing interactions between subunits that belong to different subfamilies. For Kv6.4, yeast-two-hybrid experiments showed an interaction of the Kv6.4 N-terminus with the Kv2.1 N-...

  13. Structure and assembly of scalable porous protein cages (United States)

    Sasaki, Eita; Böhringer, Daniel; van de Waterbeemd, Michiel; Leibundgut, Marc; Zschoche, Reinhard; Heck, Albert J. R.; Ban, Nenad; Hilvert, Donald


    Proteins that self-assemble into regular shell-like polyhedra are useful, both in nature and in the laboratory, as molecular containers. Here we describe cryo-electron microscopy (EM) structures of two versatile encapsulation systems that exploit engineered electrostatic interactions for cargo loading. We show that increasing the number of negative charges on the lumenal surface of lumazine synthase, a protein that naturally assembles into a ~1-MDa dodecahedron composed of 12 pentamers, induces stepwise expansion of the native protein shell, giving rise to thermostable ~3-MDa and ~6-MDa assemblies containing 180 and 360 subunits, respectively. Remarkably, these expanded particles assume unprecedented tetrahedrally and icosahedrally symmetric structures constructed entirely from pentameric units. Large keyhole-shaped pores in the shell, not present in the wild-type capsid, enable diffusion-limited encapsulation of complementarily charged guests. The structures of these supercharged assemblies demonstrate how programmed electrostatic effects can be effectively harnessed to tailor the architecture and properties of protein cages.

  14. Ferritin Assembly in Enterocytes of Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Abraham Rosas-Arellano


    Full Text Available Ferritins are protein nanocages that accumulate inside their cavity thousands of oxidized iron atoms bound to oxygen and phosphates. Both characteristic types of eukaryotic ferritin subunits are present in secreted ferritins from insects, but here dimers between Ferritin 1 Heavy Chain Homolog (Fer1HCH and Ferritin 2 Light Chain Homolog (Fer2LCH are further stabilized by disulfide-bridge in the 24-subunit complex. We addressed ferritin assembly and iron loading in vivo using novel transgenic strains of Drosophila melanogaster. We concentrated on the intestine, where the ferritin induction process can be controlled experimentally by dietary iron manipulation. We showed that the expression pattern of Fer2LCH-Gal4 lines recapitulated iron-dependent endogenous expression of the ferritin subunits and used these lines to drive expression from UAS-mCherry-Fer2LCH transgenes. We found that the Gal4-mediated induction of mCherry-Fer2LCH subunits was too slow to effectively introduce them into newly formed ferritin complexes. Endogenous Fer2LCH and Fer1HCH assembled and stored excess dietary iron, instead. In contrast, when flies were genetically manipulated to co-express Fer2LCH and mCherry-Fer2LCH simultaneously, both subunits were incorporated with Fer1HCH in iron-loaded ferritin complexes. Our study provides fresh evidence that, in insects, ferritin assembly and iron loading in vivo are tightly regulated.

  15. 基于关键装配特性的大型零部件最佳装配位姿多目标优化算法%Multiple-objective Optimization Algorithm Based on Key Assembly Characteristics to Posture Best Fit for Large Component Assembly

    Institute of Scientific and Technical Information of China (English)

    朱绪胜; 郑联语


    To control the key assembly characteristics and attain the best fit of posture with the aid of large-scale measurement, a multiple-objective optimization algorithm is proposed based on key assembly characteristics to posture best fit for large component assembly. This approach regards the posture best fit, which is a key activity of large component assembly in measurement-assisted assembly (MAA), as a two-phase optimal problem. In the first phase, the measurement coordinate system and the global coordinate system are unified with minimum error based on singular value decomposition, and the current posture of the components being assembled is optimally solved in terms of the minimum variation of all the reference points. In the second phase, the synthetic error requirement of assembly is computed according to the importance of every key assembly characteristic to the whole product, and the best posture of the movable components are optimally determined by minimizing the synthetic error. Then the optimal model and the process procedures for the two phases based on particle swarm optimization are proposed. In this model, each posture to be calculated is modeled as a six dimensional particle (with three movement and three rotation parameters). Finally, an assembly example about the joining of two cabin sections of a satellite mainframe structure is performed to verify the effectiveness of the proposed approach and process algorithm. The experiment result shows that the algorithm is robust and effective to control every key assembly characteristic and improve the assembly quality.%为了控制装配过程中的关键装配特性,以大尺寸测量技术为辅助,实现大型零部件最优位姿装配,提出基于关键装配特性的大型零部件最佳装配位姿多目标优化算法.该方法将测量辅助装配(MAA)中的关键环节——最佳装配位姿拟合问题分为两步:第1步利用基于奇异值分解的解析方法将测量坐标系与装配现场

  16. Crystallization of the Nonameric Small Terminase Subunit of bacteriophage P22

    Energy Technology Data Exchange (ETDEWEB)

    A Roy; A Bhardwaj; G Cingoloni


    The packaging of viral genomes into preformed empty procapsids is powered by an ATP-dependent genome-translocating motor. This molecular machine is formed by a heterodimer consisting of large terminase (L-terminase) and small terminase (S-terminase) subunits, which is assembled into a complex of unknown stoichiometry, and a dodecameric portal protein. There is considerable confusion in the literature regarding the biologically relevant oligomeric state of terminases, which, like portal proteins, form ring-like structures. The number of subunits in a hollow oligomeric protein defines the internal diameter of the central channel and the ability to fit DNA inside. Thus, knowledge of the exact stoichiometry of terminases is critical to decipher the mechanisms of terminase-dependent DNA translocation. Here, the gene encoding bacteriophage P22 S-terminase in Escherichia coli has been overexpressed and the protein purified under native conditions. In the absence of detergents and/or denaturants that may cause disassembly of the native oligomer and formation of aberrant rings, it was found that P22 S-terminase assembles into a concentration-independent nonamer of {approx}168 kDa. Nonameric S-terminase was crystallized in two different crystal forms at neutral pH. Crystal form I belonged to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 144.2, b = 144.2, c = 145.3 {angstrom}, and diffracted to 3.0 {angstrom} resolution. Crystal form II belonged to space group P2{sub 1}, with unit-cell parameters a = 76.48, b = 100.9, c = 89.95 {angstrom}, {beta} = 93.73{sup o}, and diffracted to 1.75 {angstrom} resolution. Preliminary crystallographic analysis of crystal form II confirms that the S-terminase crystals contain a nonamer in the asymmetric unit and are suitable for high-resolution structure determination.

  17. Crystallization of the Nonameric Small Terminase Subunit of Bacteriophage P22

    Energy Technology Data Exchange (ETDEWEB)

    A Roy; A Bhardwaj; G Cingolani


    The packaging of viral genomes into preformed empty procapsids is powered by an ATP-dependent genome-translocating motor. This molecular machine is formed by a heterodimer consisting of large terminase (L-terminase) and small terminase (S-terminase) subunits, which is assembled into a complex of unknown stoichiometry, and a dodecameric portal protein. There is considerable confusion in the literature regarding the biologically relevant oligomeric state of terminases, which, like portal proteins, form ring-like structures. The number of subunits in a hollow oligomeric protein defines the internal diameter of the central channel and the ability to fit DNA inside. Thus, knowledge of the exact stoichiometry of terminases is critical to decipher the mechanisms of terminase-dependent DNA translocation. Here, the gene encoding bacteriophage P22 S-terminase in Escherichia coli has been overexpressed and the protein purified under native conditions. In the absence of detergents and/or denaturants that may cause disassembly of the native oligomer and formation of aberrant rings, it was found that P22 S-terminase assembles into a concentration-independent nonamer of {approx}168 kDa. Nonameric S-terminase was crystallized in two different crystal forms at neutral pH. Crystal form I belonged to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 144.2, b = 144.2, c = 145.3 {angstrom}, and diffracted to 3.0 {angstrom} resolution. Crystal form II belonged to space group P2{sub 1}, with unit-cell parameters a = 76.48, b = 100.9, c = 89.95 {angstrom}, {beta} = 93.73{sup o}, and diffracted to 1.75 {angstrom} resolution. Preliminary crystallographic analysis of crystal form II confirms that the S-terminase crystals contain a nonamer in the asymmetric unit and are suitable for high-resolution structure determination.

  18. The Robust Assembly of Small Symmetric Nanoshells. (United States)

    Wagner, Jef; Zandi, Roya


    Highly symmetric nanoshells are found in many biological systems, such as clathrin cages and viral shells. Many studies have shown that symmetric shells appear in nature as a result of the free-energy minimization of a generic interaction between their constituent subunits. We examine the physical basis for the formation of symmetric shells, and by using a minimal model, demonstrate that these structures can readily grow from the irreversible addition of identical subunits. Our model of nanoshell assembly shows that the spontaneous curvature regulates the size of the shell while the mechanical properties of the subunit determine the symmetry of the assembled structure. Understanding the minimum requirements for the formation of closed nanoshells is a necessary step toward engineering of nanocontainers, which will have far-reaching impact in both material science and medicine.

  19. Study on Auto Assembly Technology for Large Array Antenna of Mobile Radar%机动雷达大型天线自动对接技术的研究

    Institute of Scientific and Technical Information of China (English)



    The large array antenna of mobile radar is assembled in segments with crane,which takes much time for professional operators to assemble the radar and then limits the maneuverability of large radar. Through studying on the automatic docking technology of large antenna,the automatic docking technology of large parts used in the indoor is applied in the assembly of large antenna in the field to realize the rapid assem-bly of the large antenna of mobile radar.The engineering principles and the key technologies are discussed in this paper,including position and pose tracking measurement,automatic adj ustment of the position and pose of large an-tenna,and auto-control system.The implementations of the main functions are also studied.%机动雷达大型天线阵面常采用多块天线子阵面吊装拼接的架设方式,架设时受架设效率低、吊装操作存在不确定因素等的制约,使得大型雷达的机动性较差。通过对大型天线阵面自动对接技术的研究,将目前已有的室内大型零部件自动对接技术应用到大型天线装配技术中,以实现机动雷达大型天线的快速装配。主要论述了机动雷达大型天线自动对接的工程原理和所需攻克的关键技术,其中包括数字化自动测量技术、天线位姿自动调节技术和伺服控制技术等;并对其主要功能的实现方法进行了研究。

  20. 某大型风电齿轮箱弹性销轴的过盈装配%Interference Fit Assembly of Flexible Pin in A Large Wind Turbine Gearbox

    Institute of Scientific and Technical Information of China (English)



      介绍了一种新型风电齿轮箱中小直径、大过盈量的高精度装配工艺设计。着重分析了装配件的结构特性、技术难点,经过分析计算结合并试验,形成了一套可行的工艺方案。%This article introduces a process design for high precision assembly of parts with small and medium size connected by large value of interference which are used in a new type wind power gearbox ,it focuses on analyzing the structure characteristics of matching parts and technology difficulties in assembly ,and finally gets a feasible process solution through calculation combining with tests .

  1. Mapping of the Mouse Actin Capping Protein Beta Subunit Gene

    Directory of Open Access Journals (Sweden)

    Cooper John A


    Full Text Available Abstract Background Capping protein (CP, a heterodimer of α and β subunits, is found in all eukaryotes. CP binds to the barbed ends of actin filaments in vitro and controls actin assembly and cell motility in vivo. Vertebrates have three isoforms of CPβ produced by alternatively splicing from one gene; lower organisms have one gene and one isoform. Results We isolated genomic clones corresponding to the β subunit of mouse CP and identified its chromosomal location by interspecies backcross mapping. Conclusions The CPβ gene (Cappb1 mapped to Chromosome 4 between Cdc42 and D4Mit312. Three mouse mutations, snubnose, curly tail, and cribriform degeneration, map in the vicinity of the β gene.

  2. A new genus of athecate interstitial dinoflagellates, Togula gen. nov., previously encompassed within Amphidinium sensu lato: Inferred from light and electron microscopy and phylogenetic analyses of partial large subunit ribosomal DNA sequences

    DEFF Research Database (Denmark)

    Jørgensen, Mårten Flø; Murray, Shauna; Daugbjerg, Niels


    and scanning electron microscopy did not reveal significant differences between the vegetative motile cells; however, cells about to undergo mitosis developed longitudinal grooves on the hypocone in one of the clades but not in the other. Both clades differed substantially from A. britannicum in partial large...

  3. Inner/Outer nuclear membrane fusion in nuclear pore assembly: biochemical demonstration and molecular analysis. (United States)

    Fichtman, Boris; Ramos, Corinne; Rasala, Beth; Harel, Amnon; Forbes, Douglass J


    Nuclear pore complexes (NPCs) are large proteinaceous channels embedded in double nuclear membranes, which carry out nucleocytoplasmic exchange. The mechanism of nuclear pore assembly involves a unique challenge, as it requires creation of a long-lived membrane-lined channel connecting the inner and outer nuclear membranes. This stabilized membrane channel has little evolutionary precedent. Here we mapped inner/outer nuclear membrane fusion in NPC assembly biochemically by using novel assembly intermediates and membrane fusion inhibitors. Incubation of a Xenopus in vitro nuclear assembly system at 14°C revealed an early pore intermediate where nucleoporin subunits POM121 and the Nup107-160 complex were organized in a punctate pattern on the inner nuclear membrane. With time, this intermediate progressed to diffusion channel formation and finally to complete nuclear pore assembly. Correct channel formation was blocked by the hemifusion inhibitor lysophosphatidylcholine (LPC), but not if a complementary-shaped lipid, oleic acid (OA), was simultaneously added, as determined with a novel fluorescent dextran-quenching assay. Importantly, recruitment of the bulk of FG nucleoporins, characteristic of mature nuclear pores, was not observed before diffusion channel formation and was prevented by LPC or OA, but not by LPC+OA. These results map the crucial inner/outer nuclear membrane fusion event of NPC assembly downstream of POM121/Nup107-160 complex interaction and upstream or at the time of FG nucleoporin recruitment.

  4. Subcellular distribution of the V-ATPase complex in plant cells, and in vivo localisation of the 100 kDa subunit VHA-a within the complex

    Directory of Open Access Journals (Sweden)

    Kluge Christoph


    Full Text Available Abstract Background Vacuolar H+-ATPases are large protein complexes of more than 700 kDa that acidify endomembrane compartments and are part of the secretory system of eukaryotic cells. They are built from 14 different (VHA-subunits. The paper addresses the question of sub-cellular localisation and subunit composition of plant V-ATPase in vivo and in vitro mainly by using colocalization and fluorescence resonance energy transfer techniques (FRET. Focus is placed on the examination and function of the 95 kDa membrane spanning subunit VHA-a. Showing similarities to the already described Vph1 and Stv1 vacuolar ATPase subunits from yeast, VHA-a revealed a bipartite structure with (i a less conserved cytoplasmically orientated N-terminus and (ii a membrane-spanning C-terminus with a higher extent of conservation including all amino acids shown to be essential for proton translocation in the yeast. On the basis of sequence data VHA-a appears to be an essential structural and functional element of V-ATPase, although previously a sole function in assembly has been proposed. Results To elucidate the presence and function of VHA-a in the plant complex, three approaches were undertaken: (i co-immunoprecipitation with antibodies directed to epitopes in the N- and C-terminal part of VHA-a, respectively, (ii immunocytochemistry approach including co-localisation studies with known plant endomembrane markers, and (iii in vivo-FRET between subunits fused to variants of green fluorescence protein (CFP, YFP in transfected cells. Conclusions All three sets of results show that V-ATPase contains VHA-a protein that interacts in a specific manner with other subunits. The genomes of plants encode three genes of the 95 kDa subunit (VHA-a of the vacuolar type H+-ATPase. Immuno-localisation of VHA-a shows that the recognized subunit is exclusively located on the endoplasmic reticulum. This result is in agreement with the hypothesis that the different isoforms of VHA

  5. Thermodynamic Interrogation of the Assembly of a Viral Genome Packaging Motor Complex. (United States)

    Yang, Teng-Chieh; Ortiz, David; Nosaka, Lyn'Al; Lander, Gabriel C; Catalano, Carlos Enrique


    Viral terminase enzymes serve as genome packaging motors in many complex double-stranded DNA viruses. The functional motors are multiprotein complexes that translocate viral DNA into a capsid shell, powered by a packaging ATPase, and are among the most powerful molecular motors in nature. Given their essential role in virus development, the structure and function of these biological motors is of considerable interest. Bacteriophage λ-terminase, which serves as a prototypical genome packaging motor, is composed of one large catalytic subunit tightly associated with two DNA recognition subunits. This protomer assembles into a functional higher-order complex that excises a unit length genome from a concatemeric DNA precursor (genome maturation) and concomitantly translocates the duplex into a preformed procapsid shell (genome packaging). While the enzymology of λ-terminase has been well described, the nature of the catalytically competent nucleoprotein intermediates, and the mechanism describing their assembly and activation, is less clear. Here we utilize analytical ultracentrifugation to determine the thermodynamic parameters describing motor assembly and define a minimal thermodynamic linkage model that describes the effects of salt on protomer assembly into a tetrameric complex. Negative stain electron microscopy images reveal a symmetric ring-like complex with a compact stem and four extended arms that exhibit a range of conformational states. Finally, kinetic studies demonstrate that assembly of the ring tetramer is directly linked to activation of the packaging ATPase activity of the motor, thus providing a direct link between structure and function. The implications of these results with respect to the assembly and activation of the functional packaging motor during a productive viral infection are discussed.

  6. Operon Gene Order Is Optimized for Ordered Protein Complex Assembly. (United States)

    Wells, Jonathan N; Bergendahl, L Therese; Marsh, Joseph A


    The assembly of heteromeric protein complexes is an inherently stochastic process in which multiple genes are expressed separately into proteins, which must then somehow find each other within the cell. Here, we considered one of the ways by which prokaryotic organisms have attempted to maximize the efficiency of protein complex assembly: the organization of subunit-encoding genes into operons. Using structure-based assembly predictions, we show that operon gene order has been optimized to match the order in which protein subunits assemble. Exceptions to this are almost entirely highly expressed proteins for which assembly is less stochastic and for which precisely ordered translation offers less benefit. Overall, these results show that ordered protein complex assembly pathways are of significant biological importance and represent a major evolutionary constraint on operon gene organization.

  7. The helical domain of the EcoR124I motor subunit participates in ATPase activity and dsDNA translocation

    Directory of Open Access Journals (Sweden)

    Vitali Bialevich


    Full Text Available Type I restriction-modification enzymes are multisubunit, multifunctional molecular machines that recognize specific DNA target sequences, and their multisubunit organization underlies their multifunctionality. EcoR124I is the archetype of Type I restriction-modification family IC and is composed of three subunit types: HsdS, HsdM, and HsdR. DNA cleavage and ATP-dependent DNA translocation activities are housed in the distinct domains of the endonuclease/motor subunit HsdR. Because the multiple functions are integrated in this large subunit of 1,038 residues, a large number of interdomain contacts might be expected. The crystal structure of EcoR124I HsdR reveals a surprisingly sparse number of contacts between helicase domain 2 and the C-terminal helical domain that is thought to be involved in assembly with HsdM. Only two potential hydrogen-bonding contacts are found in a very small contact region. In the present work, the relevance of these two potential hydrogen-bonding interactions for the multiple activities of EcoR124I is evaluated by analysing mutant enzymes using in vivo and in vitro experiments. Molecular dynamics simulations are employed to provide structural interpretation of the functional data. The results indicate that the helical C-terminal domain is involved in the DNA translocation, cleavage, and ATPase activities of HsdR, and a role in controlling those activities is suggested.

  8. The helical domain of the EcoR124I motor subunit participates in ATPase activity and dsDNA translocation (United States)

    Shamayeva, Katsiaryna; Guzanova, Alena; Řeha, David; Csefalvay, Eva; Carey, Jannette; Weiserova, Marie


    Type I restriction-modification enzymes are multisubunit, multifunctional molecular machines that recognize specific DNA target sequences, and their multisubunit organization underlies their multifunctionality. EcoR124I is the archetype of Type I restriction-modification family IC and is composed of three subunit types: HsdS, HsdM, and HsdR. DNA cleavage and ATP-dependent DNA translocation activities are housed in the distinct domains of the endonuclease/motor subunit HsdR. Because the multiple functions are integrated in this large subunit of 1,038 residues, a large number of interdomain contacts might be expected. The crystal structure of EcoR124I HsdR reveals a surprisingly sparse number of contacts between helicase domain 2 and the C-terminal helical domain that is thought to be involved in assembly with HsdM. Only two potential hydrogen-bonding contacts are found in a very small contact region. In the present work, the relevance of these two potential hydrogen-bonding interactions for the multiple activities of EcoR124I is evaluated by analysing mutant enzymes using in vivo and in vitro experiments. Molecular dynamics simulations are employed to provide structural interpretation of the functional data. The results indicate that the helical C-terminal domain is involved in the DNA translocation, cleavage, and ATPase activities of HsdR, and a role in controlling those activities is suggested. PMID:28133570

  9. iAssembler: a package for de novo assembly of Roche-454/Sanger transcriptome sequences

    Directory of Open Access Journals (Sweden)

    Zheng Yi


    Full Text Available Abstract Background Expressed Sequence Tags (ESTs have played significant roles in gene discovery and gene functional analysis, especially for non-model organisms. For organisms with no full genome sequences available, ESTs are normally assembled into longer consensus sequences for further downstream analysis. However current de novo EST assembly programs often generate large number of assembly errors that will negatively affect the downstream analysis. In order to generate more accurate consensus sequences from ESTs, tools are needed to reduce or eliminate errors from de novo assemblies. Results We present iAssembler, a pipeline that can assemble large-scale ESTs into consensus sequences with significantly higher accuracy than current existing assemblers. iAssembler employs MIRA and CAP3 assemblers to generate initial assemblies, followed by identifying and correcting two common types of transcriptome assembly errors: 1 ESTs from different transcripts (mainly alternatively spliced transcripts or paralogs are incorrectly assembled into same contigs; and 2 ESTs from same transcripts fail to be assembled together. iAssembler can be used to assemble ESTs generated using the traditional Sanger method and/or the Roche-454 massive parallel pyrosequencing technology. Conclusion We compared performances of iAssembler and several other de novo EST assembly programs using both Roche-454 and Sanger EST datasets. It demonstrated that iAssembler generated significantly more accurate consensus sequences than other assembly programs.

  10. Association-dissociation process with aging subunits: Recursive solution (United States)

    Niedermayer, Thomas; Lipowsky, Reinhard


    The coupling of stochastic growth and shrinkage of one-dimensional structures to random aging of the constituting subunits defines the simple association-dissociation-aging process which captures the essential features of the nonequilibrium assembly of cytoskeletal filaments. Because of correlations, previously employed mean-field methods fail to correctly describe filament growth. We study an alternative formulation of the full master equation of the stochastic process. An ansatz for the steady-state solution leads to a recursion relation which allows for the calculation of all emergent quantities with increasing accuracy and in excellent agreement with stochastic simulations.

  11. Exchangeability of the b subunit of the Cl(-)-translocating ATPase of Acetabularia acetabulum with the beta subunit of E. coli F1-ATPase: construction of the chimeric beta subunits and complementation studies. (United States)

    Ikeda, M; Kadowaki, H; Ikeda, H; Moritani, C; Kanazawa, H


    The gene encoding the b subunit of the Cl(-)-translocating ATPase (aclB) was isolated from total RNA and poly(A)+ RNA of Acetabularia acetabulum and sequenced (total nucleotides of 3038 bp and an open reading frame with 478 amino acids). The deduced amino acid sequence showed high similarity to the beta subunit of the F type ATPases, but was different in the N-terminal 120 amino acids. The role of the N-terminal region was investigated using an F -ATPase beta-less mutant of E. coli, JP17. The JP17 strain expressing the aclB could not grow under conditions permitting oxidative phosphorylation, although ACLB was detected in the membrane fraction. The beta subunit was divided into three portions: amino acid position from 1 to 95 (portion A), 96 to 161 (portion B) and 162 to the C-terminus (portion C). The corresponding regions of ACLB were designated as portions A' (from 1 to 106), B' (from 107 to 172) and C' (from 173 to 478). Chimeric proteins with combinations of A-B'-C', A-B-C' and A'-B-C restored the function as the beta subunit in E. coli F0F1-complex, but those with combinations of A'-B'-C and A-B'-C had no function as the beta subunit. These findings suggested that portion B plays an important role in the assembly and function of the beta subunit in the F0F1-complex, while portion B' of ACLB exhibited inhibitory effects on assembly and function. In addition, portion A was also important for interaction of the beta subunit with the alpha subunit in E. coli F0F1-complex. These findings also suggested that the b subunit of the Cl(-)-translocating ATPase of A. acetabulum has a different function in the Cl(-)-translocating ATPase complex, although the primary structure resembled to the beta subunit of the F1-ATPase.

  12. Dengue vaccine: an update on recombinant subunit strategies. (United States)

    Martin, J; Hermida, L


    Dengue is an increasing public health problem worldwide, with the four serotypes of the virus infecting over 390 million people annually. There is no specific treatment or antiviral drug for dengue, and prevention is largely limited to controlling the mosquito vectors or disrupting the human-vector contact. Despite the considerable progress made in recent years, an effective vaccine against the virus is not yet available. The development of a dengue vaccine has been hampered by many unique challenges, including the need to ensure the absence of vaccine-induced enhanced severity of disease. Recombinant protein subunit vaccines offer a safer alternative to other vaccine approaches. Several subunit vaccine candidates are presently under development, based on different structural and non-structural proteins of the virus. Novel adjuvants or immunopotentiating strategies are also being tested to improve their immunogenicity. This review summarizes the current status and development trends of subunit dengue vaccines.

  13. AP1000核电站CA20大型结构模块组装技术探讨%Discussion on assembly technology of AP1000 nuclear power plant CA20 large structural module

    Institute of Scientific and Technical Information of China (English)

    田利民; 王杰; 陈尚锐


    Large module assembly is critical and difficult in AP1000 nuclear power unit modularization construction, and CA20 structural module is the first module to be put in place in AP1000 nuclear island and it is a typical large-scale structural module with large dimension and heavy weight. This paper mainly introduces the assembly process of one Nuclear Power Plant 1#NI CA20 structural module and analyzes its emphases and difficulties, which provides reference and guidance for AP1000 nuclear power unit modularization construction in the future.%大型模块组装是AP1000核电机组模块化施工的重点难点,CA20结构模块是AP1000核岛中第一个就位的模块,其尺寸大、重量重,是典型的大型结构模块。本文主要介绍某核电站1#核岛CA20结构模块组装过程,分析其重点和难点,为以后AP1000核电机组模块化施工提供借鉴和指导。

  14. Role of the Rubisco small subunit. Final report for period May 1, 1997--April 30,2000

    Energy Technology Data Exchange (ETDEWEB)

    Spreitzer, Robert J.


    CO{sub 2} and O{sub 2} are mutually competitive at the active site of ribulose-1,5-biphosphate (RuBP) carboxylase/oxygenase (Rubisco). Rubisco contains two subunits, each present in eight copies. The 15-kD small subunit is coded by a family of nuclear RbcS genes. Until now, the role of the small subunit in Rubisco structure or catalytic efficiency is not known. Because of other work in eliminating the two RbcS genes in the green algo Chlamydomonas reinhardtii, it is now possible to address questions about the structure-function relationships of the eukaryotic small subunit. There are three specific aims in this project: (1) Alanine scanning mutagenesis is being used to dissect the importance of the {beta}A/{beta}B loop, a feature unique to the eukaryotic small subunit. (2) Random mutagenesis is being used to identify additional residues or regions of the small subunit that are important for holoenzyme assembly and function. (3) Attempts are being made to express foreign small subunits in Chlamydomonas to examine the contribution of small subunits to holoenzyme assembly, catalytic efficiency, and CO{sub 2}/O{sub 2} specificity.

  15. Application of Digital Simulation Pre-assembling Technology in Large-scale Steel Structure%数字模拟预拼装在大型钢结构工程中的应用

    Institute of Scientific and Technical Information of China (English)



    The components in super-high-rise trusses or large-span stadium are complex at present and the components have some spatial correlations, so the requirements on manufacture are more and more strictly for the interface of components. Only controlling the precision of single component can t meet the requirements of on-site installation, so pre-assembly in factory is always necessary. However, integral pre-assembly technology can be limited by site, hoisting equipments and time. Thus digital simulation pre-assembling technology is needed to solve the problems mentioned above. This paper introduces simulation pre-assembling process of trusses in some area of Shanghai Tower. The simulation pre-assembling technology and entity pre-assembling technology are comparatively analyzed. Then the application conditions of digital simulation pre-assembling technology are obtained on this base. Thus corresponding control measures are suggested. The solution for difficulties during coordinate collection of pre-assembling is demonstrated, as well as the feasibility of digital simulation pre-assembling technology in the on-site installation.%目前,超高层桁架层、大跨度场馆等钢结构的构件形式比较复杂,且构件之间具有空间关联性,因此对构件间接口的制作精度要求很高,有时仅靠控制单体构件精度无法满足现场安装要求,因此对于复杂的构件,通常要求在加工厂进行预拼装.由于场地、吊装设备、时间周期等方面的限制,有时不具备整体预拼装的条件,数字模拟预拼装方法的出现能够较好地解决这一问题,但这一方法并未在钢结构中普及.本文以上海中心大厦某区桁架层为例,详细介绍模拟预拼装的工艺原理,并将部分构件的模拟拼装与实体拼装效果进行比较分析,在此基础上分析数字模拟预拼装的应用条件,提出对应的控制措施,并论证了有关预拼装坐标采集这一难点的应对措施及数字模拟预

  16. Light extraction improvement of InGaN light-emitting diodes with large-area highly ordered ITO nanobowls photonic crystal via self-assembled nanosphere lithography

    Directory of Open Access Journals (Sweden)

    Kui Wu


    Full Text Available The InGaN multiple quantum well light-emitting diodes (LEDs with different sizes of indium-tin-oxide (ITO nanobowl photonic crystal (PhC structure has been fabricated using self-assembled monolayer nanosphere lithography. The light output power (LOP of PhC LEDs (at 350 mA has been enhanced by 63.5% and the emission divergence exhibits a 28.8° reduction compared to conventional LEDs without PhC structure. Current-Voltage curves have shown that these PhC structures on ITO layer will not degrade the LED electrical properties. The finite-difference time-domain simulation (FDTD has also been performed for light extraction and emission characteristics, which is consistent with the experimental results.

  17. Invited article: a test-facility for large-area microchannel plate detector assemblies using a pulsed sub-picosecond laser. (United States)

    Adams, Bernhard; Chollet, Matthieu; Elagin, Andrey; Oberla, Eric; Vostrikov, Alexander; Wetstein, Matthew; Obaid, Razib; Webster, Preston


    The Large Area Picosecond Photodetector Collaboration is developing large-area fast photodetectors with time resolution tests on bare 8 in.-square MCP plates or into a smaller chamber for tests on 33-mm circular substrates. We present the experimental setup, detector calibration, data acquisition, analysis tools, and typical results demonstrating the performance of the test facility.

  18. Modular assembly of yeast cytochrome oxidase. (United States)

    McStay, Gavin P; Su, Chen Hsien; Tzagoloff, Alexander


    Previous studies of yeast cytochrome oxidase (COX) biogenesis identified Cox1p, one of the three mitochondrially encoded core subunits, in two high-molecular weight complexes combined with regulatory/assembly factors essential for expression of this subunit. In the present study we use pulse-chase labeling experiments in conjunction with isolated mitochondria to identify new Cox1p intermediates and place them in an ordered pathway. Our results indicate that before its assimilation into COX, Cox1p transitions through five intermediates that are differentiated by their compositions of accessory factors and of two of the eight imported subunits. We propose a model of COX biogenesis in which Cox1p and the two other mitochondrial gene products, Cox2p and Cox3p, constitute independent assembly modules, each with its own complement of subunits. Unlike their bacterial counterparts, which are composed only of the individual core subunits, the final sequence in which the mitochondrial modules associate to form the holoenzyme may have been conserved during evolution.

  19. Proteomic investigations of complex I composition: How to define a subunit?

    Directory of Open Access Journals (Sweden)

    Etienne H Meyer


    Full Text Available Complex I is present in almost all aerobic species. Being the largest complex of the respiratory chain, it has a central role in energizing biological membranes and is essential for many organisms. Bacterial complex I is composed of 14 subunits that are sufficient to achieve the respiratory functions. Eukaryotic enzymes contain orthologs of the 14 bacterial subunits and around 30 additional subunits. This complexity suggests either that complex I requires more stabilizing subunits in mitochondria or that it fulfills additional functions. In many organisms recent work on complex I concentrated on the determination of its exact composition. This review summarizes the work done to elucidate complex I composition in the model plant Arabidopsis and proposes a model for the organization of its 44 confirmed subunits. The comparison of the different studies investigating the composition of complex I across species identifies sample preparation for the proteomic analysis as critical to differentiate between true subunits, assembly factors or proteins associated with complex I. Coupling comparative proteomics with biochemical or genetic studies is thus required to define a subunit and its function within the complex.

  20. Phorbol-induced surface expression of NR2A subunit homologues in HEK293 cells

    Institute of Scientific and Technical Information of China (English)

    Chan-ying ZHENG; Xiu-juan YANG; Zhan-yan FU; Jian-hong LUO


    Aim: N-methyl-D-aspartate receptors (NMDAR) are heteromeric complexes primarily assembled from NR1 and NR2 subunits. In normal conditions, NR2 sub-units assemble into homodimers in the endoplasmic reticulum (ER). These homodimers remain in the ER until they coassemble with NR1 dimers and are trafficked to the cell surface. However, it still remains unclear whether functional homomeric NMDAR exist in physiological or pathological conditions. Methods: We transfected GFP-NR2A alone into HEK293 cells, treated the cells with PKC activator 12-myristate-13 acetate (PMA), and then detected surface NR2A sub-units with a live cell immunostaining method. We also used a series of NR2A mutants with a partial deletion of its C-terminus to identify the regions that are involved in the PMA-mediated surface expression of NR2A subunits. Results: NR2A subunits were expressed on the cell membrane after incubation with PMA (200 nmol/L,30 min), although no functional NMDA channels were detected after PMA-induced membrane trafficking. Immunostaining with an ER marker also revealed that NR2A subunits were exported from the ER after PMA treatment. Furthermore, the deletion of amino acids between 1149-1347 or 1354-1464 of NR2A inhibited PMA-induced surface expression of NR2A subunits. Conclusion: First, our data suggests that PMA treatment can induce the surface expression of homomeric NR2A subunits. Furthermore, this process is probably mediated by the NR2A C-terminal region between positions 1149 and 1464.

  1. Large Scale Explorative Oligonucleotide Probe Selection for Thousands of Genetic Groups on a Computing Grid: Application to Phylogenetic Probe Design Using a Curated Small Subunit Ribosomal RNA Gene Database

    Directory of Open Access Journals (Sweden)

    Faouzi Jaziri


    Full Text Available Phylogenetic Oligonucleotide Arrays (POAs were recently adapted for studying the huge microbial communities in a flexible and easy-to-use way. POA coupled with the use of explorative probes to detect the unknown part is now one of the most powerful approaches for a better understanding of microbial community functioning. However, the selection of probes remains a very difficult task. The rapid growth of environmental databases has led to an exponential increase of data to be managed for an efficient design. Consequently, the use of high performance computing facilities is mandatory. In this paper, we present an efficient parallelization method to select known and explorative oligonucleotide probes at large scale using computing grids. We implemented a software that generates and monitors thousands of jobs over the European Computing Grid Infrastructure (EGI. We also developed a new algorithm for the construction of a high-quality curated phylogenetic database to avoid erroneous design due to bad sequence affiliation. We present here the performance and statistics of our method on real biological datasets based on a phylogenetic prokaryotic database at the genus level and a complete design of about 20,000 probes for 2,069 genera of prokaryotes.

  2. Polymer Directed Protein Assemblies

    Directory of Open Access Journals (Sweden)

    Patrick van Rijn


    Full Text Available Protein aggregation and protein self-assembly is an important occurrence in natural systems, and is in some form or other dictated by biopolymers. Very obvious influences of biopolymers on protein assemblies are, e.g., virus particles. Viruses are a multi-protein assembly of which the morphology is dictated by poly-nucleotides namely RNA or DNA. This “biopolymer” directs the proteins and imposes limitations on the structure like the length or diameter of the particle. Not only do these bionanoparticles use polymer-directed self-assembly, also processes like amyloid formation are in a way a result of directed protein assembly by partial unfolded/misfolded biopolymers namely, polypeptides. The combination of proteins and synthetic polymers, inspired by the natural processes, are therefore regarded as a highly promising area of research. Directed protein assembly is versatile with respect to the possible interactions which brings together the protein and polymer, e.g., electrostatic, v.d. Waals forces or covalent conjugation, and possible combinations are numerous due to the large amounts of different polymers and proteins available. The protein-polymer interacting behavior and overall morphology is envisioned to aid in clarifying protein-protein interactions and are thought to entail some interesting new functions and properties which will ultimately lead to novel bio-hybrid materials.

  3. Hierarchical RNA Processing Is Required for Mitochondrial Ribosome Assembly

    Directory of Open Access Journals (Sweden)

    Oliver Rackham


    Full Text Available The regulation of mitochondrial RNA processing and its importance for ribosome biogenesis and energy metabolism are not clear. We generated conditional knockout mice of the endoribonuclease component of the RNase P complex, MRPP3, and report that it is essential for life and that heart and skeletal-muscle-specific knockout leads to severe cardiomyopathy, indicating that its activity is non-redundant. Transcriptome-wide parallel analyses of RNA ends (PARE and RNA-seq enabled us to identify that in vivo 5′ tRNA cleavage precedes 3′ tRNA processing, and this is required for the correct biogenesis of the mitochondrial ribosomal subunits. We identify that mitoribosomal biogenesis proceeds co-transcriptionally because large mitoribosomal proteins can form a subcomplex on an unprocessed RNA containing the 16S rRNA. Taken together, our data show that RNA processing links transcription to translation via assembly of the mitoribosome.

  4. Stoichiometry of δ subunit containing GABAA receptors (United States)

    Patel, B; Mortensen, M; Smart, T G


    Background and Purpose Although the stoichiometry of the major synaptic αβγ subunit-containing GABAA receptors has consensus support for 2α:2β:1γ, a clear view of the stoichiometry of extrasynaptic receptors containing δ subunits has remained elusive. Here we examine the subunit stoichiometry of recombinant α4β3δ receptors using a reporter mutation and a functional electrophysiological approach. Experimental Approach Using site-directed mutagenesis, we inserted a highly characterized 9′ serine to leucine mutation into the second transmembrane (M2) region of α4, β3 and δ subunits that increases receptor sensitivity to GABA. Whole-cell, GABA-activated currents were recorded from HEK-293 cells co-expressing different combinations of wild-type (WT) and/or mutant α4(L297S), β3(L284S) and δ(L288S) subunits. Key Results Recombinant receptors containing one or more mutant subunits showed increased GABA sensitivity relative to WT receptors by approximately fourfold, independent of the subunit class (α, β or δ) carrying the mutation. GABA dose–response curves of cells co-expressing WT subunits with their respective L9′S mutants exhibited multiple components, with the number of discernible components enabling a subunit stoichiometry of 2α, 2β and 1δ to be deduced for α4β3δ receptors. Varying the cDNA transfection ratio by 10-fold had no significant effect on the number of incorporated δ subunits. Conclusions and Implications Subunit stoichiometry is an important determinant of GABAA receptor function and pharmacology, and δ subunit-containing receptors are important mediators of tonic inhibition in several brain regions. Here we demonstrate a preferred subunit stoichiometry for α4β3δ receptors of 2α, 2β and 1δ. PMID:24206220

  5. Spatially confined assembly of nanoparticles. (United States)

    Jiang, Lin; Chen, Xiaodong; Lu, Nan; Chi, Lifeng


    The ability to assemble NPs into ordered structures that are expected to yield collective physical or chemical properties has afforded new and exciting opportunities in the field of nanotechnology. Among the various configurations of nanoparticle assemblies, two-dimensional (2D) NP patterns and one-dimensional (1D) NP arrays on surfaces are regarded as the ideal assembly configurations for many technological devices, for example, solar cells, magnetic memory, switching devices, and sensing devices, due to their unique transport phenomena and the cooperative properties of NPs in assemblies. To realize the potential applications of NP assemblies, especially in nanodevice-related applications, certain key issues must still be resolved, for example, ordering and alignment, manipulating and positioning in nanodevices, and multicomponent or hierarchical structures of NP assemblies for device integration. Additionally, the assembly of NPs with high precision and high levels of integration and uniformity for devices with scaled-down dimensions has become a key and challenging issue. Two-dimensional NP patterns and 1D NP arrays are obtained using traditional lithography techniques (top-down strategies) or interfacial assembly techniques (bottom-up strategies). However, a formidable challenge that persists is the controllable assembly of NPs in desired locations over large areas with high precision and high levels of integration. The difficulty of this assembly is due to the low efficiency of small features over large areas in lithography techniques or the inevitable structural defects that occur during the assembly process. The combination of self-assembly strategies with existing nanofabrication techniques could potentially provide effective and distinctive solutions for fabricating NPs with precise position control and high resolution. Furthermore, the synergistic combination of spatially mediated interactions between nanoparticles and prestructures on surfaces may play

  6. Thiamine diphosphate binds to intermediates in the assembly of adenovirus fiber knob trimers in Escherichia coli. (United States)

    Schulz, Ryan; Zhang, Yian-Biao; Liu, Chang-Jun; Freimuth, Paul


    Assembly of the adenovirus (Ad) homotrimeric fiber protein is nucleated by its C-terminal knob domain, which itself can trimerize when expressed as a recombinant protein fragment. The non-interlocked, globular structure of subunits in the knob trimer implies that trimers assemble from prefolded monomers through a dimer intermediate, but these intermediates have not been observed and the mechanism of assembly therefore remains uncharacterized. Here we report that expression of the Ad serotype 2 (Ad2) knob was toxic for thi- strains of Escherichia coli, which are defective in de novo synthesis of thiamine (vitamin B1). Ad2 knob trimers isolated from a thi+ strain copurified through multiple chromatography steps with a small molecule of mass equivalent to that of thiamine diphosphate (ThDP). Mutant analysis did not implicate any specific site for ThDP binding. Our results suggest that ThDP may associate with assembly intermediates and become trapped in assembled trimers, possibly within one of several large cavities that are partially solvent-accessible or buried completely within the trimer interior.

  7. Plant mitochondrial Complex I composition and assembly: A review. (United States)

    Subrahmanian, Nitya; Remacle, Claire; Hamel, Patrice Paul


    In the mitochondrial inner membrane, oxidative phosphorylation generates ATP via the operation of several multimeric enzymes. The proton-pumping Complex I (NADH:ubiquinone oxidoreductase) is the first and most complicated enzyme required in this process. Complex I is an L-shaped enzyme consisting of more than 40 subunits, one FMN molecule and eight Fe-S clusters. In recent years, genetic and proteomic analyses of Complex I mutants in various model systems, including plants, have provided valuable insights into the assembly of this multimeric enzyme. Assisted by a number of key players, referred to as "assembly factors", the assembly of Complex I takes place in a sequential and modular manner. Although a number of factors have been identified, their precise function in mediating Complex I assembly still remains to be elucidated. This review summarizes our current knowledge of plant Complex I composition and assembly derived from studies in plant model systems such as Arabidopsis thaliana and Chlamydomonas reinhardtii. Plant Complex I is highly conserved and comprises a significant number of subunits also present in mammalian and fungal Complexes I. Plant Complex I also contains additional subunits absent from the mammalian and fungal counterpart, whose function in enzyme activity and assembly is not clearly understood. While 14 assembly factors have been identified for human Complex I, only two proteins, namely GLDH and INDH, have been established as bona fide assembly factors for plant Complex I. This article is part of a Special Issue entitled Respiratory complex I, edited by Volker Zickermann and Ulrich Brandt.

  8. Fast Assembly of Gold Nanoparticles in Large-Area 2D Nanogrids Using a One-Step, Near-Infrared Radiation-Assisted Evaporation Process. (United States)

    Utgenannt, André; Maspero, Ross; Fortini, Andrea; Turner, Rebecca; Florescu, Marian; Jeynes, Christopher; Kanaras, Antonios G; Muskens, Otto L; Sear, Richard P; Keddie, Joseph L


    When fabricating photonic crystals from suspensions in volatile liquids using the horizontal deposition method, the conventional approach is to evaporate slowly to increase the time for particles to settle in an ordered, periodic close-packed structure. Here, we show that the greatest ordering of 10 nm aqueous gold nanoparticles (AuNPs) in a template of larger spherical polymer particles (mean diameter of 338 nm) is achieved with very fast water evaporation rates obtained with near-infrared radiative heating. Fabrication of arrays over areas of a few cm(2) takes only 7 min. The assembly process requires that the evaporation rate is fast relative to the particles' Brownian diffusion. Then a two-dimensional colloidal crystal forms at the falling surface, which acts as a sieve through which the AuNPs pass, according to our Langevin dynamics computer simulations. With sufficiently fast evaporation rates, we create a hybrid structure consisting of a two-dimensional AuNP nanoarray (or "nanogrid") on top of a three-dimensional polymer opal. The process is simple, fast, and one-step. The interplay between the optical response of the plasmonic Au nanoarray and the microstructuring of the photonic opal results in unusual optical spectra with two extinction peaks, which are analyzed via finite-difference time-domain method simulations. Comparison between experimental and modeling results reveals a strong interplay of plasmonic modes and collective photonic effects, including the formation of a high-order stopband and slow-light-enhanced plasmonic absorption. The structures, and hence their optical signatures, are tuned by adjusting the evaporation rate via the infrared power density.

  9. An alternating GluN1-2-1-2 subunit arrangement in mature NMDA receptors.

    Directory of Open Access Journals (Sweden)

    Morgane Riou

    Full Text Available NMDA receptors (NMDARs form glutamate-gated ion channels that play a critical role in CNS physiology and pathology. Together with AMPA and kainate receptors, NMDARs are known to operate as tetrameric complexes with four membrane-embedded subunits associating to form a single central ion-conducting pore. While AMPA and some kainate receptors can function as homomers, NMDARs are obligatory heteromers composed of homologous but distinct subunits, most usually of the GluN1 and GluN2 types. A fundamental structural feature of NMDARs, that of the subunit arrangement around the ion pore, is still controversial. Thus, in a typical NMDAR associating two GluN1 and two GluN2 subunits, there is evidence for both alternating 1/2/1/2 and non-alternating 1/1/2/2 arrangements. Here, using a combination of electrophysiological and cross-linking experiments, we provide evidence that functional GluN1/GluN2A receptors adopt the 1/2/1/2 arrangement in which like subunits are diagonal to one another. Moreover, based on the recent crystal structure of an AMPA receptor, we show that in the agonist-binding and pore regions, the GluN1 subunits occupy a "proximal" position, closer to the central axis of the channel pore than that of GluN2 subunits. Finally, results obtained with reducing agents that differ in their membrane permeability indicate that immature (intracellular and functional (plasma-membrane inserted pools of NMDARs can adopt different subunit arrangements, thus stressing the importance of discriminating between the two receptor pools in assembly studies. Elucidating the quaternary arrangement of NMDARs helps to define the interface between the subunits and to understand the mechanism and pharmacology of these key signaling receptors.

  10. A protein inventory of human ribosome biogenesis reveals an essential function of exportin 5 in 60S subunit export. (United States)

    Wild, Thomas; Horvath, Peter; Wyler, Emanuel; Widmann, Barbara; Badertscher, Lukas; Zemp, Ivo; Kozak, Karol; Csucs, Gabor; Lund, Elsebet; Kutay, Ulrike


    The assembly of ribosomal subunits in eukaryotes is a complex, multistep process so far mostly studied in yeast. In S. cerevisiae, more than 200 factors including ribosomal proteins and trans-acting factors are required for the ordered assembly of 40S and 60S ribosomal subunits. To date, only few human homologs of these yeast ribosome synthesis factors have been characterized. Here, we used a systematic RNA interference (RNAi) approach to analyze the contribution of 464 candidate factors to ribosomal subunit biogenesis in human cells. The screen was based on visual readouts, using inducible, fluorescent ribosomal proteins as reporters. By performing computer-based image analysis utilizing supervised machine-learning techniques, we obtained evidence for a functional link of 153 human proteins to ribosome synthesis. Our data show that core features of ribosome assembly are conserved from yeast to human, but differences exist for instance with respect to 60S subunit export. Unexpectedly, our RNAi screen uncovered a requirement for the export receptor Exportin 5 (Exp5) in nuclear export of 60S subunits in human cells. We show that Exp5, like the known 60S exportin Crm1, binds to pre-60S particles in a RanGTP-dependent manner. Interference with either Exp5 or Crm1 function blocks 60S export in both human cells and frog oocytes, whereas 40S export is compromised only upon inhibition of Crm1. Thus, 60S subunit export is dependent on at least two RanGTP-binding exportins in vertebrate cells.

  11. A Novel Method for Efficient Preparation of Mucosal Adjuvant Escherichia coli Heat-Labile Enterotoxin Mutant (LTm) by Artificially Assisted Self-Assembly In Vitro. (United States)

    Liu, Di; Zhang, Na; Zheng, Wenyun; Guo, Hua; Wang, Xiaoli; Wang, Tianwen; Wang, Ping; Ma, Xingyuan


    As well-known powerful mucosal adjuvant proteins, Escherichia coli heat-labile enterotoxin (LT) and its non-toxic or low-toxic mutants (LTm) are capable of promoting strong mucosal immune responses to co-administered antigens in various types of vaccines. However, due to the complex composition and special structure, the yield of LTm directly from the recombinant genetic engineering strains is quite low. Here, we put forward a novel method to prepare LTm protein which designed, expressed, and purified three kinds of component subunits respectively and assembled them into a hexamer structure in vitro by two combination modes. In addition, by simulated in vivo environment of polymer protein assembly, the factors of the protein solution system which include environment temperature, pH, ionic strength of the solution, and ratio between each subunit were taken into consideration. Finally, we confirmed the optimal conditions of two assembly strategies and prepared the hexamer holotoxin in vitro. These results are not only an important significance in promoting large-scale preparation of the mucosal adjuvant LTm but also an enlightening to produce other multi-subunit proteins.

  12. Sabot assembly

    Energy Technology Data Exchange (ETDEWEB)

    Bzorgi, Fariborz


    A sabot assembly includes a projectile and a housing dimensioned and configured for receiving the projectile. An air pressure cavity having a cavity diameter is disposed between a front end and a rear end of the housing. Air intake nozzles are in fluid communication with the air pressure cavity and each has a nozzle diameter less than the cavity diameter. In operation, air flows through the plurality of air intake nozzles and into the air pressure cavity upon firing of the projectile from a gun barrel to pressurize the air pressure cavity for assisting in separation of the housing from the projectile upon the sabot assembly exiting the gun barrel.

  13. Elongated polyproline motifs facilitate enamel evolution through matrix subunit compaction.

    Directory of Open Access Journals (Sweden)

    Tianquan Jin


    Full Text Available Vertebrate body designs rely on hydroxyapatite as the principal mineral component of relatively light-weight, articulated endoskeletons and sophisticated tooth-bearing jaws, facilitating rapid movement and efficient predation. Biological mineralization and skeletal growth are frequently accomplished through proteins containing polyproline repeat elements. Through their well-defined yet mobile and flexible structure polyproline-rich proteins control mineral shape and contribute many other biological functions including Alzheimer's amyloid aggregation and prolamine plant storage. In the present study we have hypothesized that polyproline repeat proteins exert their control over biological events such as mineral growth, plaque aggregation, or viscous adhesion by altering the length of their central repeat domain, resulting in dramatic changes in supramolecular assembly dimensions. In order to test our hypothesis, we have used the vertebrate mineralization protein amelogenin as an exemplar and determined the biological effect of the four-fold increased polyproline tandem repeat length in the amphibian/mammalian transition. To study the effect of polyproline repeat length on matrix assembly, protein structure, and apatite crystal growth, we have measured supramolecular assembly dimensions in various vertebrates using atomic force microscopy, tested the effect of protein assemblies on crystal growth by electron microscopy, generated a transgenic mouse model to examine the effect of an abbreviated polyproline sequence on crystal growth, and determined the structure of polyproline repeat elements using 3D NMR. Our study shows that an increase in PXX/PXQ tandem repeat motif length results (i in a compaction of protein matrix subunit dimensions, (ii reduced conformational variability, (iii an increase in polyproline II helices, and (iv promotion of apatite crystal length. Together, these findings establish a direct relationship between polyproline tandem

  14. A large-scale in vivo analysis reveals that TALENs are significantly more mutagenic than ZFNs generated using context-dependent assembly


    Chen, Shijia; Oikonomou, Grigorios; Chiu, Cindy N.; Niles, Brett J.; Liu, Justin; Lee, Daniel A.; Antoshechkin, Igor; Prober, David A.


    Zinc-finger nucleases (ZFNs) and TAL effector nucleases (TALENs) have been shown to induce targeted mutations, but they have not been extensively tested in any animal model. Here, we describe a large-scale comparison of ZFN and TALEN mutagenicity in zebrafish. Using deep sequencing, we found that TALENs are significantly more likely to be mutagenic and induce an average of 10-fold more mutations than ZFNs. We observed a strong correlation between somatic and germ-line mutagenicity, and identi...

  15. The subunit delta-subunit b domain of the Escherichia coli F1F0 ATPase. The B subunits interact with F1 as a dimer and through the delta subunit. (United States)

    Rodgers, A J; Wilkens, S; Aggeler, R; Morris, M B; Howitt, S M; Capaldi, R A


    The delta and b subunits are both involved in binding the F1 to the F0 part in the Escherichia coli ATP synthase (ECF1F0). The interaction of the purified delta subunit and the isolated hydrophilic domain of the b subunit (bsol) has been studied here. Purified delta binds to bsol weakly in solution, as indicated by NMR studies and protease protection experiments. On F1, i.e. in the presence of ECF1-delta, delta, and bsol interact strongly, and a complex of ECF1.bsol can be isolated by native gel electrophoresis. Both delta subunit and bsol are protected from trypsin cleavage in this complex. In contrast, the delta subunit is rapidly degraded by the protease when bound to ECF1 when bsol is absent. The interaction of bsol with ECF1 involves the C-terminal domain of delta as delta(1-134) cannot replace intact delta in the binding experiments. As purified, bsol is a stable dimer with 80% alpha helix. A monomeric form of bsol can be obtained by introducing the mutation A128D (Howitt, S. M., Rodgers, A. J.,W., Jeffrey, P. D., and Cox, G. B. (1996) J. Biol. Chem. 271, 7038-7042). Monomeric bsol has less alpha helix, i.e. only 58%, is much more sensitive to trypsin cleavage than dimer, and unfolds at much lower temperatures than the dimer in circular dichroism melting studies, indicating a less stable structure. The bsol dimer, but not monomer, binds to delta in ECF1. To examine whether subunit b is a monomor or dimer in intact ECF1F0, CuCl2 was used to induce cross-link formation in the mutants bS60C, bQ104C, bA128C, bG131C, and bS146C. With the exception of bS60C, CuCl2 treatment resulted in formation of b subunit dimers in all mutants. Cross-linking yield was independent of nucleotide conditions and did not affect ATPase activity. These results show the b subunit to be dimeric for a large portion of the C terminus, with residues 124-131 likely forming a pair of parallel alpha helices.

  16. Lateral assembly of oxidized graphene flakes into large-scale transparent conductive thin films with a three-dimensional surfactant 4-sulfocalix[4]arene (United States)

    Sundramoorthy, Ashok K.; Wang, Yilei; Wang, Jing; Che, Jianfei; Thong, Ya Xuan; Lu, Albert Chee W.; Chan-Park, Mary B.


    Graphene is a promising candidate material for transparent conductive films because of its excellent conductivity and one-carbon-atom thickness. Graphene oxide flakes prepared by Hummers method are typically several microns in size and must be pieced together in order to create macroscopic films. We report a macro-scale thin film fabrication method which employs a three-dimensional (3-D) surfactant, 4-sulfocalix[4]arene (SCX), as a lateral aggregating agent. After electrochemical exfoliation, the partially oxidized graphene (oGr) flakes are dispersed with SCX. The SCX forms micelles, which adsorb on the oGr flakes to enhance their dispersion, also promote aggregation into large-scale thin films under vacuum filtration. A thin oGr/SCX film can be shaved off from the aggregated oGr/SCX cake by immersing the cake in water. The oGr/SCX thin-film floating on the water can be subsequently lifted from the water surface with a substrate. The reduced oGr (red-oGr) films can be as thin as 10−20 nm with a transparency of >90% and sheet resistance of 890 ± 47 kΩ/sq. This method of electrochemical exfoliation followed by SCX-assisted suspension and hydrazine reduction, avoids using large amounts of strong acid (unlike Hummers method), is relatively simple and can easily form a large scale conductive and transparent film from oGr/SCX suspension. PMID:26040436

  17. Assembling consumption

    DEFF Research Database (Denmark)

    Assembling Consumption marks a definitive step in the institutionalisation of qualitative business research. By gathering leading scholars and educators who study markets, marketing and consumption through the lenses of philosophy, sociology and anthropology, this book clarifies and applies...... societies. This is an essential reading for both seasoned scholars and advanced students of markets, economies and social forms of consumption....

  18. Mutation of C20orf7 Disrupts Complex I Assembly and Causes Lethal Neonatal Mitochondrial Disease

    NARCIS (Netherlands)

    Sugiana, Canny; Pagliarini, David J.; McKenzie, Matthew; Kirby, Denise M.; Salemi, Renato; Abu-Amero, Khaled K.; Dahl, Hans-Henrik M.; Hutchison, Wendy M.; Vascotto, Katherine A.; Smith, Stacey M.; Newbold, Robert F.; Christodoulou, John; Calvo, Sarah; Mootha, Vamsi K.; Ryan, Michael T.; Thorburn, David R.


    Complex I (NADH:ubiquinone oxidoreductase) is the first and largest multimeric complex of the mitochondrial respiratory chain. Human complex I comprises seven Subunits encoded by mitochondrial DNA and 38 nuclear-encoded subunits that are assembled together in a process that is only partially

  19. Association of condensin with chromosomes depends on DNA binding by its HEAT-repeat subunits. (United States)

    Piazza, Ilaria; Rutkowska, Anna; Ori, Alessandro; Walczak, Marta; Metz, Jutta; Pelechano, Vicent; Beck, Martin; Haering, Christian H


    Condensin complexes have central roles in the three-dimensional organization of chromosomes during cell divisions, but how they interact with chromatin to promote chromosome segregation is largely unknown. Previous work has suggested that condensin, in addition to encircling chromatin fibers topologically within the ring-shaped structure formed by its SMC and kleisin subunits, contacts DNA directly. Here we describe the discovery of a binding domain for double-stranded DNA formed by the two HEAT-repeat subunits of the Saccharomyces cerevisiae condensin complex. From detailed mapping data of the interfaces between the HEAT-repeat and kleisin subunits, we generated condensin complexes that lack one of the HEAT-repeat subunits and consequently fail to associate with chromosomes in yeast and human cells. The finding that DNA binding by condensin's HEAT-repeat subunits stimulates the SMC ATPase activity suggests a multistep mechanism for the loading of condensin onto chromosomes.

  20. Subtype-independent near full-length HIV-1 genome sequencing and assembly to be used in large molecular epidemiological studies and clinical management

    Directory of Open Access Journals (Sweden)

    Sebastian Grossmann


    Full Text Available Introduction: HIV-1 near full-length genome (HIV-NFLG sequencing from plasma is an attractive multidimensional tool to apply in large-scale population-based molecular epidemiological studies. It also enables genotypic resistance testing (GRT for all drug target sites allowing effective intervention strategies for control and prevention in high-risk population groups. Thus, the main objective of this study was to develop a simplified subtype-independent, cost- and labour-efficient HIV-NFLG protocol that can be used in clinical management as well as in molecular epidemiological studies. Methods: Plasma samples (n=30 were obtained from HIV-1B (n=10, HIV-1C (n=10, CRF01_AE (n=5 and CRF01_AG (n=5 infected individuals with minimum viral load >1120 copies/ml. The amplification was performed with two large amplicons of 5.5 kb and 3.7 kb, sequenced with 17 primers to obtain HIV-NFLG. GRT was validated against ViroSeqTM HIV-1 Genotyping System. Results: After excluding four plasma samples with low-quality RNA, a total of 26 samples were attempted. Among them, NFLG was obtained from 24 (92% samples with the lowest viral load being 3000 copies/ml. High (>99% concordance was observed between HIV-NFLG and ViroSeqTM when determining the drug resistance mutations (DRMs. The N384I connection mutation was additionally detected by NFLG in two samples. Conclusions: Our high efficiency subtype-independent HIV-NFLG is a simple and promising approach to be used in large-scale molecular epidemiological studies. It will facilitate the understanding of the HIV-1 pandemic population dynamics and outline effective intervention strategies. Furthermore, it can potentially be applicable in clinical management of drug resistance by evaluating DRMs against all available antiretrovirals in a single assay.

  1. Radiation Effects Simulation of Fuel Assemblies

    Institute of Scientific and Technical Information of China (English)

    CUI; Yao


    Due to a large number of photons irradiated by the fuel assemblies after radiation in the reactor,the data acquisition and image reconstruction will be interfered seriously for the nuclear fuel assembly non-destructive testing system.Therefore,in process of the fuel assembly NDT system

  2. Dynamic assembly of polymer nanotube networks via kinesin powered microtubule filaments (United States)

    Paxton, Walter F.; Bouxsein, Nathan F.; Henderson, Ian M.; Gomez, Andrew; Bachand, George D.


    We describe for the first time how biological nanomotors may be used to actively self-assemble mesoscale networks composed of diblock copolymer nanotubes. The collective force generated by multiple kinesin nanomotors acting on a microtubule filament is large enough to overcome the energy barrier required to extract nanotubes from polymer vesicles comprised of poly(ethylene oxide-b-butadiene) in spite of the higher force requirements relative to extracting nanotubes from lipid vesicles. Nevertheless, large-scale polymer networks were dynamically assembled by the motors. These networks displayed enhanced robustness, persisting more than 24 h post-assembly (compared to 4-5 h for corresponding lipid networks). The transport of materials in and on the polymer membranes differs substantially from the transport on analogous lipid networks. Specifically, our data suggest that polymer mobility in nanotubular structures is considerably different from planar or 3D structures, and is stunted by 1D confinement of the polymer subunits. Moreover, quantum dots adsorbed onto polymer nanotubes are completely immobile, which is related to this 1D confinement effect and is in stark contrast to the highly fluid transport observed on lipid tubules.We describe for the first time how biological nanomotors may be used to actively self-assemble mesoscale networks composed of diblock copolymer nanotubes. The collective force generated by multiple kinesin nanomotors acting on a microtubule filament is large enough to overcome the energy barrier required to extract nanotubes from polymer vesicles comprised of poly(ethylene oxide-b-butadiene) in spite of the higher force requirements relative to extracting nanotubes from lipid vesicles. Nevertheless, large-scale polymer networks were dynamically assembled by the motors. These networks displayed enhanced robustness, persisting more than 24 h post-assembly (compared to 4-5 h for corresponding lipid networks). The transport of materials in and on

  3. Molecular architecture of the yeast Elongator complex reveals an unexpected asymmetric subunit arrangement. (United States)

    Setiaputra, Dheva T; Cheng, Derrick Th; Lu, Shan; Hansen, Jesse M; Dalwadi, Udit; Lam, Cindy Hy; To, Jeffrey L; Dong, Meng-Qiu; Yip, Calvin K


    Elongator is a ~850 kDa protein complex involved in multiple processes from transcription to tRNA modification. Conserved from yeast to humans, Elongator is assembled from two copies of six unique subunits (Elp1 to Elp6). Despite the wealth of structural data on the individual subunits, the overall architecture and subunit organization of the full Elongator and the molecular mechanisms of how it exerts its multiple activities remain unclear. Using single-particle electron microscopy (EM), we revealed that yeast Elongator adopts a bilobal architecture and an unexpected asymmetric subunit arrangement resulting from the hexameric Elp456 subassembly anchored to one of the two Elp123 lobes that form the structural scaffold. By integrating the EM data with available subunit crystal structures and restraints generated from cross-linking coupled to mass spectrometry, we constructed a multiscale molecular model that showed the two Elp3, the main catalytic subunit, are located in two distinct environments. This work provides the first structural insights into Elongator and a framework to understand the molecular basis of its multifunctionality.

  4. Self-assembly of flagellin on Au(111) surfaces. (United States)

    González Orive, Alejandro; Pissinis, Diego E; Diaz, Carolina; Miñán, Alejandro; Benítez, Guillermo A; Rubert, Aldo; Daza Millone, Antonieta; Rumbo, Martin; Hernández Creus, Alberto; Salvarezza, Roberto C; Schilardi, Patricia L


    The adsorption of flagellin monomers from Pseudomonas fluorescens on Au(111) has been studied by Atomic Force Microscopy (AFM), Scanning Tunneling Microscopy (STM), X-ray Photoelectron Spectroscopy (XPS), Surface Plasmon Resonance (SPR), and electrochemical techniques. Results show that flagellin monomers spontaneously self-assemble forming a monolayer thick protein film bounded to the Au surface by the more hydrophobic subunit and exposed to the environment the hydrophilic subunit. The films are conductive and allow allocation of electrochemically active cytochrome C. The self-assembled films could be used as biological platforms to build 3D complex molecular structures on planar metal surfaces and to functionalize metal nanoparticles.

  5. Integrin alpha(3)-subunit expression modulates alveolar epithelial cell monolayer formation. (United States)

    Lubman, R L; Zhang, X L; Zheng, J; Ocampo, L; Lopez, M Z; Veeraraghavan, S; Zabski, S M; Danto, S I; Borok, Z


    We investigated expression of the alpha(3)-integrin subunit by rat alveolar epithelial cells (AECs) grown in primary culture as well as the effects of monoclonal antibodies with blocking activity against the alpha(3)-integrin subunit on AEC monolayer formation. alpha(3)-Integrin subunit mRNA and protein were detectable in AECs on day 1 and increased with time in culture. alpha(3)- and beta(1)-integrin subunits coprecipitated in immunoprecipitation experiments with alpha(3)- and beta(1)-subunit-specific antibodies, consistent with their association as the alpha(3)beta(1)-integrin receptor at the cell membrane. Treatment with blocking anti-alpha(3) monoclonal antibody from day 0 delayed development of transepithelial resistance, reduced transepithelial resistance through day 5 compared with that in untreated AECs, and resulted in large subconfluent patches in monolayers viewed by scanning electron microscopy on day 3. These data indicate that alpha(3)- and beta(1)-integrin subunits are expressed in AEC monolayers where they form the heterodimeric alpha(3)beta(1)-integrin receptor at the cell membrane. Blockade of the alpha(3)-integrin subunit inhibits formation of confluent AEC monolayers. We conclude that the alpha(3)-integrin subunit modulates formation of AEC monolayers by virtue of the key role of the alpha(3)beta(1)-integrin receptor in AEC adhesion.

  6. Regulation of Voltage-Activated K(+) Channel Gating by Transmembrane β Subunits. (United States)

    Sun, Xiaohui; Zaydman, Mark A; Cui, Jianmin


    Voltage-activated K(+) (K(V)) channels are important for shaping action potentials and maintaining resting membrane potential in excitable cells. K(V) channels contain a central pore-gate domain (PGD) surrounded by four voltage-sensing domains (VSDs). The VSDs will change conformation in response to alterations of the membrane potential thereby inducing the opening of the PGD. Many K(V) channels are heteromeric protein complexes containing auxiliary β subunits. These β subunits modulate channel expression and activity to increase functional diversity and render tissue specific phenotypes. This review focuses on the K(V) β subunits that contain transmembrane (TM) segments including the KCNE family and the β subunits of large conductance, Ca(2+)- and voltage-activated K(+) (BK) channels. These TM β subunits affect the voltage-dependent activation of K(V) α subunits. Experimental and computational studies have described the structural location of these β subunits in the channel complexes and the biophysical effects on VSD activation, PGD opening, and VSD-PGD coupling. These results reveal some common characteristics and mechanistic insights into K(V) channel modulation by TM β subunits.

  7. Regulation of KV channel voltage-dependent activation by transmembrane β subunits

    Directory of Open Access Journals (Sweden)

    Xiaohui eSun


    Full Text Available Voltage-activated K+ (KV channels are important for shaping action potentials and maintaining resting membrane potential in excitable cells. KV channels contain a central pore-gate domain (PGD surrounded by four voltage-sensing domains (VSD. The VSDs will change conformation in response to alterations of the membrane potential thereby inducing the opening of the PGD. Many KV channels are heteromeric protein complexes containing auxiliary β subunits. These β subunits modulate channel expression and activity to increase functional diversity and render tissue specific phenotypes. This review focuses on the KV β subunits that contain transmembrane (TM segments including the KCNE family and the β subunits of large conductance, Ca2+- and voltage-activated K+ (BK channels. These TM β subunits affect the voltage-dependent activation of KV α subunits. Experimental and computational studies have described the structural location of these β subunits in the channel complexes and the biophysical effects on VSD activation, PGD opening and VSD-PGD coupling. These results reveal some common characteristics and mechanistic insights into KV channel modulation by TM β subunits.

  8. A large-scale superhydrophobic surface-enhanced Raman scattering (SERS) platform fabricated via capillary force lithography and assembly of Ag nanocubes for ultratrace molecular sensing. (United States)

    Tan, Joel Ming Rui; Ruan, Justina Jiexin; Lee, Hiang Kwee; Phang, In Yee; Ling, Xing Yi


    An analytical platform with an ultratrace detection limit in the atto-molar (aM) concentration range is vital for forensic, industrial and environmental sectors that handle scarce/highly toxic samples. Superhydrophobic surface-enhanced Raman scattering (SERS) platforms serve as ideal platforms to enhance detection sensitivity by reducing the random spreading of aqueous solution. However, the fabrication of superhydrophobic SERS platforms is generally limited due to the use of sophisticated and expensive protocols and/or suffers structural and signal inconsistency. Herein, we demonstrate a high-throughput fabrication of a stable and uniform superhydrophobic SERS platform for ultratrace molecular sensing. Large-area box-like micropatterns of the polymeric surface are first fabricated using capillary force lithography (CFL). Subsequently, plasmonic properties are incorporated into the patterned surfaces by decorating with Ag nanocubes using the Langmuir-Schaefer technique. To create a stable superhydrophobic SERS platform, an additional 25 nm Ag film is coated over the Ag nanocube-decorated patterned template followed by chemical functionalization with perfluorodecanethiol. Our resulting superhydrophobic SERS platform demonstrates excellent water-repellency with a static contact angle of 165° ± 9° and a consequent analyte concentration factor of 59-fold, as compared to its hydrophilic counterpart. By combining the analyte concentration effect of superhydrophobic surfaces with the intense electromagnetic "hot spots" of Ag nanocubes, our superhydrophobic SERS platform achieves an ultra-low detection limit of 10(-17) M (10 aM) for rhodamine 6G using just 4 μL of analyte solutions, corresponding to an analytical SERS enhancement factor of 10(13). Our fabrication protocol demonstrates a simple, cost- and time-effective approach for the large-scale fabrication of a superhydrophobic SERS platform for ultratrace molecular detection.

  9. Facilitative-competitive interactions in an old-growth forest: the importance of large-diameter trees as benefactors and stimulators for forest community assembly.

    Directory of Open Access Journals (Sweden)

    Andreas Fichtner

    Full Text Available The role of competition in tree communities is increasingly well understood, while little is known about the patterns and mechanisms of the interplay between above- and belowground competition in tree communities. This knowledge, however, is crucial for a better understanding of community dynamics and developing adaptive near-natural management strategies. We assessed neighbourhood interactions in an unmanaged old-growth European beech (Fagus sylvatica forest by quantifying variation in the intensity of above- (shading and belowground competition (crowding among dominant and co-dominant canopy beech trees during tree maturation. Shading had on average a much larger impact on radial growth than crowding and the sensitivity to changes in competitive conditions was lowest for crowding effects. We found that each mode of competition reduced the effect of the other. Increasing crowding reduced the negative effect of shading, and at high levels of shading, crowding actually had a facilitative effect and increased growth. Our study demonstrates that complementarity in above- and belowground processes enable F. sylvatica to alter resource acquisition strategies, thus optimising tree radial growth. As a result, competition seemed to become less important in stands with a high growing stock and tree communities with a long continuity of anthropogenic undisturbed population dynamics. We suggest that growth rates do not exclusively depend on the density of potential competitors at the intraspecific level, but on the conspecific aggregation of large-diameter trees and their functional role for regulating biotic filtering processes. This finding highlights the potential importance of the rarely examined relationship between the spatial aggregation pattern of large-diameter trees and the outcome of neighbourhood interactions, which may be central to community dynamics and the related forest ecosystem services.

  10. In vitro assembly of catalase. (United States)

    Baureder, Michael; Barane, Elisabeth; Hederstedt, Lars


    Most aerobic organisms contain catalase, which functions to decompose hydrogen peroxide. Typical catalases are structurally complex homo-tetrameric enzymes with one heme prosthetic group buried in each subunit. It is not known how catalase in the cell is assembled from its constituents. The bacterium Enterococcus faecalis cannot synthesize heme but can acquire it from the environment to form a cytoplasmic catalase. We have in E. faecalis monitored production of the enzyme polypeptide (KatA) depending on the availability of heme and used our findings to devise a procedure for the purification of preparative amounts of in vivo-synthesized apocatalase. We show that fully active catalase can be obtained in vitro by incubating isolated apoprotein with hemin. We have characterized features of the assembly process and describe a temperature-trapped hemylated intermediate of the enzyme maturation process. Hemylation of apocatalase does not require auxiliary cell components, but rapid assembly of active enzyme seemingly is assisted in the cell. Our findings provide insight about catalase assembly and offer new experimental possibilities for detailed studies of this process.

  11. The beta subunit of casein kinase II

    DEFF Research Database (Denmark)

    Boldyreff, B; Piontek, K; Schmidt-Spaniol, I;


    cDNAs encoding the beta subunit of pig and mouse CKII were isolated. The porcine cDNA was expressed as a fusion protein in Escherichia coli and used for the production of anti-CKII-beta subunit specific antibodies....

  12. Risk capital allocation with autonomous subunits

    DEFF Research Database (Denmark)

    Hougaard, Jens Leth; Smilgins, Aleksandrs


    Risk capital allocation problems have been widely discussed in the academic literature. We consider a set of independent subunits collaborating in order to reduce risk: that is, when subunit portfolios are merged a diversification benefit arises and the risk of the group as a whole is smaller tha...

  13. Molecular architecture of the human Mediator-RNA polymerase II-TFIIF assembly.

    Directory of Open Access Journals (Sweden)

    Carrie Bernecky


    Full Text Available The macromolecular assembly required to initiate transcription of protein-coding genes, known as the Pre-Initiation Complex (PIC, consists of multiple protein complexes and is approximately 3.5 MDa in size. At the heart of this assembly is the Mediator complex, which helps regulate PIC activity and interacts with the RNA polymerase II (pol II enzyme. The structure of the human Mediator-pol II interface is not well-characterized, whereas attempts to structurally define the Mediator-pol II interaction in yeast have relied on incomplete assemblies of Mediator and/or pol II and have yielded inconsistent interpretations. We have assembled the complete, 1.9 MDa human Mediator-pol II-TFIIF complex from purified components and have characterized its structural organization using cryo-electron microscopy and single-particle reconstruction techniques. The orientation of pol II within this assembly was determined by crystal structure docking and further validated with projection matching experiments, allowing the structural organization of the entire human PIC to be envisioned. Significantly, pol II orientation within the Mediator-pol II-TFIIF assembly can be reconciled with past studies that determined the location of other PIC components relative to pol II itself. Pol II surfaces required for interacting with TFIIB, TFIIE, and promoter DNA (i.e., the pol II cleft are exposed within the Mediator-pol II-TFIIF structure; RNA exit is unhindered along the RPB4/7 subunits; upstream and downstream DNA is accessible for binding additional factors; and no major structural re-organization is necessary to accommodate the large, multi-subunit TFIIH or TFIID complexes. The data also reveal how pol II binding excludes Mediator-CDK8 subcomplex interactions and provide a structural basis for Mediator-dependent control of PIC assembly and function. Finally, parallel structural analysis of Mediator-pol II complexes lacking TFIIF reveal that TFIIF plays a key role in

  14. Carrier subunit of plasma membrane transporter is required for oxidative folding of its helper subunit. (United States)

    Rius, Mònica; Chillarón, Josep


    We study the amino acid transport system b(0,+) as a model for folding, assembly, and early traffic of membrane protein complexes. System b(0,+) is made of two disulfide-linked membrane subunits: the carrier, b(0,+) amino acid transporter (b(0,+)AT), a polytopic protein, and the helper, related to b(0,+) amino acid transporter (rBAT), a type II glycoprotein. rBAT ectodomain mutants display folding/trafficking defects that lead to type I cystinuria. Here we show that, in the presence of b(0,+)AT, three disulfides were formed in the rBAT ectodomain. Disulfides Cys-242-Cys-273 and Cys-571-Cys-666 were essential for biogenesis. Cys-673-Cys-685 was dispensable, but the single mutants C673S, and C685S showed compromised stability and trafficking. Cys-242-Cys-273 likely was the first disulfide to form, and unpaired Cys-242 or Cys-273 disrupted oxidative folding. Strikingly, unassembled rBAT was found as an ensemble of different redox species, mainly monomeric. The ensemble did not change upon inhibition of rBAT degradation. Overall, these results indicated a b(0,+)AT-dependent oxidative folding of the rBAT ectodomain, with the initial and probably cotranslational formation of Cys-242-Cys-273, followed by the oxidation of Cys-571-Cys-666 and Cys-673-Cys-685, that was completed posttranslationally.

  15. Carrier Subunit of Plasma Membrane Transporter Is Required for Oxidative Folding of Its Helper Subunit* (United States)

    Rius, Mònica; Chillarón, Josep


    We study the amino acid transport system b0,+ as a model for folding, assembly, and early traffic of membrane protein complexes. System b0,+ is made of two disulfide-linked membrane subunits: the carrier, b0,+ amino acid transporter (b0,+AT), a polytopic protein, and the helper, related to b0,+ amino acid transporter (rBAT), a type II glycoprotein. rBAT ectodomain mutants display folding/trafficking defects that lead to type I cystinuria. Here we show that, in the presence of b0,+AT, three disulfides were formed in the rBAT ectodomain. Disulfides Cys-242-Cys-273 and Cys-571-Cys-666 were essential for biogenesis. Cys-673-Cys-685 was dispensable, but the single mutants C673S, and C685S showed compromised stability and trafficking. Cys-242-Cys-273 likely was the first disulfide to form, and unpaired Cys-242 or Cys-273 disrupted oxidative folding. Strikingly, unassembled rBAT was found as an ensemble of different redox species, mainly monomeric. The ensemble did not change upon inhibition of rBAT degradation. Overall, these results indicated a b0,+AT-dependent oxidative folding of the rBAT ectodomain, with the initial and probably cotranslational formation of Cys-242-Cys-273, followed by the oxidation of Cys-571-Cys-666 and Cys-673-Cys-685, that was completed posttranslationally. PMID:22493502

  16. Significance of a two-domain structure in subunits of phycobiliproteins revealed by the normal mode analysis. (United States)

    Kikuchi, H; Wako, H; Yura, K; Go, M; Mimuro, M


    Phycobiliproteins are basic building blocks of phycobilisomes, a supra-molecular assembly for the light-capturing function of photosynthesis in cyanobacteria and red algae. One functional form of phycobiliproteins is a trimeric form consisting of three identical units having C(3) symmetry, with each unit composed of two kinds of subunits, the alpha-subunit and beta-subunit. These subunits have similar chain folds and can be divided into either globin-like or X-Y helices domains. We studied the significance of this two-domain structure for their assembled structures and biological function (light-absorption) using a normal mode analysis to investigate dynamic aspects of their three-dimensional structures. We used C-phycocyanin (C-PC) as an example, and focused on the interactions between the two domains. The normal mode analysis was carried out for the following two cases: 1) the whole subunit, including the two domains; and 2) the globin-like domain alone. By comparing the dynamic properties, such as correlative movements between residues and the fluctuations of individual residues, we found that the X-Y helices domain plays an important role not only in the C(3) symmetry assemblies of the subunits in phycobiliproteins, but also in stabilizing the light absorption property by suppressing the fluctuation of the specific Asp residues near the chromophore. Interestingly, the conformation of the X-Y helices domain corresponds to that of a module in pyruvate phosphate dikinase (PPDK). The module in PPDK is involved in the interactions of two domains, just as the X-Y helices domain is involved in the interactions of two subunits. Finally, we discuss the mechanical construction of the C-PC subunits based on the normal mode analysis.

  17. Dump assembly (United States)

    Goldmann, Louis H.


    A dump assembly having a fixed conduit and a rotatable conduit provided with overlapping plates, respectively, at their adjacent ends. The plates are formed with openings, respectively, normally offset from each other to block flow. The other end of the rotatable conduit is provided with means for securing the open end of a filled container thereto. Rotation of the rotatable conduit raises and inverts the container to empty the contents while concurrently aligning the conduit openings to permit flow of material therethrough.

  18. Definition of the nuclear encoded protein composition of bovine heart mitochondrial complex I. Identification of two new subunits. (United States)

    Carroll, Joe; Shannon, Richard J; Fearnley, Ian M; Walker, John E; Hirst, Judy


    Mitochondrial NADH:ubiquinone oxidoreductase (complex I) from bovine heart is a complicated multisubunit, membrane-bound assembly. Seven subunits are encoded by mitochondrial DNA, and the sequences of 36 nuclear encoded subunits have been described. The subunits of complex I and two subcomplexes (Ialpha and Ibeta) were resolved on one- and two-dimensional gels and by reverse-phase high performance liquid chromatography. Mass spectrometric analysis revealed two previously unknown subunits in complex I, named B14.7 and ESSS, one in each subcomplex. Coding sequences for each protein were identified in data bases and were confirmed by cDNA cloning and sequencing. Subunit B14.7 has an acetylated N terminus, no presequence, and contains four potential transmembrane helices. It is homologous to subunit 21.3b from complex I in Neurospora crassa and is related to Tim17, Tim22, and Tim23, which are involved in protein translocation across the inner membrane. Subunit ESSS has a cleaved mitochondrial import sequence and one potential transmembrane helix. A total of 45 different subunits of bovine complex I have now been characterized.

  19. Spectroscopic properties of Callinectes sapidus hemocyanin subunits (United States)

    Stoeva, Stanka; Dolashka, Pavlina; Bankov, Banko; Voelter, Wolfgang; Salvato, Benedeto; Genov, Nicolay


    The two major subunits of the Callinectes sapidus hemocyanin were isolated and characterized by spectroscopic techniques. They consist of 641 and 652 residues, respectively. Circular dichroism spectra showed that the structural integrity of the isolated polypeptide chains is preserved. Tryptophan fluorescence parameters were determined for the hemocyanin aggregates and for the subunits Cs1 and Cs2. The emitting tryptophyl fluorophores in the native hemocyanin are deeply buried in hydrophobic regions and are shielded from the solvent by the quaternary structure of the protein aggregates. In two subunits, obtained after dissociation of the aggregates, these residues become "exposed". It is concluded that the tryptophyl side chains in Cs1 and Cs2 are located in subunit interfaces (contact regions) in a negatively charged environment when the polypeptide chains are aggregated. Most probably they participate in hydrophobic protein-protein interactions. The environment of these fluorophores is more negatively charged after the dissociation of the aggregates to subunits.

  20. General Assembly

    CERN Multimedia

    Staff Association


    5th April, 2016 – Ordinary General Assembly of the Staff Association! In the first semester of each year, the Staff Association (SA) invites its members to attend and participate in the Ordinary General Assembly (OGA). This year the OGA will be held on Tuesday, April 5th 2016 from 11:00 to 12:00 in BE Auditorium, Meyrin (6-2-024). During the Ordinary General Assembly, the activity and financial reports of the SA are presented and submitted for approval to the members. This is the occasion to get a global view on the activities of the SA, its financial management, and an opportunity to express one’s opinion, including taking part in the votes. Other points are listed on the agenda, as proposed by the Staff Council. Who can vote? Only “ordinary” members (MPE) of the SA can vote. Associated members (MPA) of the SA and/or affiliated pensioners have a right to vote on those topics that are of direct interest to them. Who can give his/her opinion? The Ordinary General Asse...

  1. Self-assembling protein nanoparticles in the design of vaccines

    Directory of Open Access Journals (Sweden)

    Jacinto López-Sagaseta


    Full Text Available For over 100 years, vaccines have been one of the most effective medical interventions for reducing infectious disease, and are estimated to save millions of lives globally each year. Nevertheless, many diseases are not yet preventable by vaccination. This large unmet medical need demands further research and the development of novel vaccines with high efficacy and safety. Compared to the 19th and early 20th century vaccines that were made of killed, inactivated, or live-attenuated pathogens, modern vaccines containing isolated, highly purified antigenic protein subunits are safer but tend to induce lower levels of protective immunity. One strategy to overcome the latter is to design antigen nanoparticles: assemblies of polypeptides that present multiple copies of subunit antigens in well-ordered arrays with defined orientations that can potentially mimic the repetitiveness, geometry, size, and shape of the natural host-pathogen surface interactions. Such nanoparticles offer a collective strength of multiple binding sites (avidity and can provide improved antigen stability and immunogenicity. Several exciting advances have emerged lately, including preclinical evidence that this strategy may be applicable for the development of innovative new vaccines, for example, protecting against influenza, human immunodeficiency virus, and respiratory syncytial virus. Here, we provide a concise review of a critical selection of data that demonstrate the potential of this field. In addition, we highlight how the use of self-assembling protein nanoparticles can be effectively combined with the emerging discipline of structural vaccinology for maximum impact in the rational design of vaccine antigens.

  2. Developmental and Subcellular Organization of Single-Cell C₄ Photosynthesis in Bienertia sinuspersici Determined by Large-Scale Proteomics and cDNA Assembly from 454 DNA Sequencing. (United States)

    Offermann, Sascha; Friso, Giulia; Doroshenk, Kelly A; Sun, Qi; Sharpe, Richard M; Okita, Thomas W; Wimmer, Diana; Edwards, Gerald E; van Wijk, Klaas J


    Kranz C4 species strictly depend on separation of primary and secondary carbon fixation reactions in different cell types. In contrast, the single-cell C4 (SCC4) species Bienertia sinuspersici utilizes intracellular compartmentation including two physiologically and biochemically different chloroplast types; however, information on identity, localization, and induction of proteins required for this SCC4 system is currently very limited. In this study, we determined the distribution of photosynthesis-related proteins and the induction of the C4 system during development by label-free proteomics of subcellular fractions and leaves of different developmental stages. This was enabled by inferring a protein sequence database from 454 sequencing of Bienertia cDNAs. Large-scale proteome rearrangements were observed as C4 photosynthesis developed during leaf maturation. The proteomes of the two chloroplasts are different with differential accumulation of linear and cyclic electron transport components, primary and secondary carbon fixation reactions, and a triose-phosphate shuttle that is shared between the two chloroplast types. This differential protein distribution pattern suggests the presence of a mRNA or protein-sorting mechanism for nuclear-encoded, chloroplast-targeted proteins in SCC4 species. The combined information was used to provide a comprehensive model for NAD-ME type carbon fixation in SCC4 species.

  3. Minimus: a fast, lightweight genome assembler

    Directory of Open Access Journals (Sweden)

    Salzberg Steven L


    Full Text Available Abstract Background Genome assemblers have grown very large and complex in response to the need for algorithms to handle the challenges of large whole-genome sequencing projects. Many of the most common uses of assemblers, however, are best served by a simpler type of assembler that requires fewer software components, uses less memory, and is far easier to install and run. Results We have developed the Minimus assembler to address these issues, and tested it on a range of assembly problems. We show that Minimus performs well on several small assembly tasks, including the assembly of viral genomes, individual genes, and BAC clones. In addition, we evaluate Minimus' performance in assembling bacterial genomes in order to assess its suitability as a component of a larger assembly pipeline. We show that, unlike other software currently used for these tasks, Minimus produces significantly fewer assembly errors, at the cost of generating a more fragmented assembly. Conclusion We find that for small genomes and other small assembly tasks, Minimus is faster and far more flexible than existing tools. Due to its small size and modular design Minimus is perfectly suited to be a component of complex assembly pipelines. Minimus is released as an open-source software project and the code is available as part of the AMOS project at Sourceforge.

  4. A Two-Piece Derivative of a Group I Intron RNA as a Platform for Designing Self-Assembling RNA Templates to Promote Peptide Ligation

    Directory of Open Access Journals (Sweden)

    Takahiro Tanaka


    Full Text Available Multicomponent RNA-peptide complexes are attractive from the viewpoint of artificial design of functional biomacromolecular systems. We have developed self-folding and self-assembling RNAs that serve as templates to assist chemical ligation between two reactive peptides with RNA-binding capabilities. The design principle of previous templates, however, can be applied only to limited classes of RNA-binding peptides. In this study, we employed a two-piece derivative of a group I intron RNA from the Tetrahymena large subunit ribosomal RNA (LSU rRNA as a platform for new template RNAs. In this group I intron-based self-assembling platform, modules for the recognition of substrate peptides can be installed independently from modules holding the platform structure. The new self-assembling platform allows us to expand the repertoire of substrate peptides in template RNA design.

  5. Design of a hyperstable 60-subunit protein icosahedron (United States)

    Hsia, Yang; Bale, Jacob B.; Gonen, Shane; Shi, Dan; Sheffler, William; Fong, Kimberly K.; Nattermann, Una; Xu, Chunfu; Huang, Po-Ssu; Ravichandran, Rashmi; Yi, Sue; Davis, Trisha N.; Gonen, Tamir; King, Neil P.; Baker, David


    The icosahedron is the largest of the Platonic solids, and icosahedral protein structures are widely used in biological systems for packaging and transport. There has been considerable interest in repurposing such structures for applications ranging from targeted delivery to multivalent immunogen presentation. The ability to design proteins that self-assemble into precisely specified, highly ordered icosahedral structures would open the door to a new generation of protein containers with properties custom-tailored to specific applications. Here we describe the computational design of a 25-nanometre icosahedral nanocage that self-assembles from trimeric protein building blocks. The designed protein was produced in Escherichia coli, and found by electron microscopy to assemble into a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 molar guanidine hydrochloride at up to 80 degrees Celsius, and undergo extremely abrupt, but reversible, disassembly between 2 molar and 2.25 molar guanidinium thiocyanate. The icosahedron is robust to genetic fusions: one or two copies of green fluorescent protein (GFP) can be fused to each of the 60 subunits to create highly fluorescent ‘standard candles’ for use in light microscopy, and a designed protein pentamer can be placed in the centre of each of the 20 pentameric faces to modulate the size of the entrance/exit channels of the cage. Such robust and customizable nanocages should have considerable utility in targeted drug delivery, vaccine design and synthetic biology.

  6. Cox26 is a novel stoichiometric subunit of the yeast cytochrome c oxidase. (United States)

    Levchenko, Maria; Wuttke, Jan-Moritz; Römpler, Katharina; Schmidt, Bernhard; Neifer, Klaus; Juris, Lisa; Wissel, Mirjam; Rehling, Peter; Deckers, Markus


    The cytochrome c oxidase (COX) is the terminal enzyme of the respiratory chain. The complex accepts electrons from cytochrome c and passes them onto molecular oxygen. This process contributes to energy capture in the form of a membrane potential across the inner membrane. The enzyme complex assembles in a stepwise process from the three mitochondria-encoded core subunits Cox1, Cox2 and Cox3, which associate with nuclear-encoded subunits and cofactors. In the yeast Saccharomyces cerevisiae, the cytochrome c oxidase associates with the bc1-complex into supercomplexes, allowing efficient energy transduction. Here we report on Cox26 as a protein found in respiratory chain supercomplexes containing cytochrome c oxidase. Our analyses reveal Cox26 as a novel stoichiometric structural subunit of the cytochrome c oxidase. A loss of Cox26 affects cytochrome c oxidase activity and respirasome organization.

  7. Cloning and expression of the human N-methyl-D-aspartate receptor subunit NR3A

    DEFF Research Database (Denmark)

    Eriksson, Maria; Nilsson, Anna; Froelich-Fabre, Susanne


    Native N-methyl-D-aspartate (NMDA) receptors are heteromeric assemblies of four or five subunits. The NMDA receptor subunits, NR1, NR2A, NR2B, NR2C, and NR2D have been cloned in several species, including man. The NR3A subunit, which in rodents is predominantly expressed during early development......, seems to function by reducing the NMDA receptor response. The human homologue to the rat NR3A, however, had not been cloned. In order to study the functions of the human NR3A (hNR3A), we have cloned and sequenced the hNR3A. It was found to share 88% of the DNA sequence with the rat gene, corresponding...

  8. Cloning and expression of the human N-methyl-D-aspartate receptor subunit NR3A

    DEFF Research Database (Denmark)

    Eriksson, Maria; Nilsson, Anna; Froelich-Fabre, Susanne


    Native N-methyl-D-aspartate (NMDA) receptors are heteromeric assemblies of four or five subunits. The NMDA receptor subunits, NR1, NR2A, NR2B, NR2C, and NR2D have been cloned in several species, including man. The NR3A subunit, which in rodents is predominantly expressed during early development......, seems to function by reducing the NMDA receptor response. The human homologue to the rat NR3A, however, had not been cloned. In order to study the functions of the human NR3A (hNR3A), we have cloned and sequenced the hNR3A. It was found to share 88% of the DNA sequence with the rat gene, corresponding...

  9. Subunit sequences of the 4 x 6-mer hemocyanin from the golden orb-web spider, Nephila inaurata. (United States)

    Averdam, Anne; Markl, Jürgen; Burmester, Thorsten


    The transport of oxygen in the hemolymph of many arthropod and mollusc species is mediated by large copper-proteins that are referred to as hemocyanins. Arthropod hemocyanins are composed of hexamers and oligomers of hexamers. Arachnid hemocyanins usually form 4 x 6-mers consisting of seven distinct subunit types (termed a-g), although in some spider taxa deviations from this standard scheme have been observed. Applying immunological and electrophoretic methods, six distinct hemocyanin subunits were identified in the red-legged golden orb-web spider Nephila inaurata madagascariensis (Araneae: Tetragnathidae). The complete cDNA sequences of six subunits were obtained that corresponded to a-, b-, d-, e-, f- and g-type subunits. No evidence for a c-type subunit was found in this species. The inclusion of the N. inaurata hemocyanins in a multiple alignment of the arthropod hemocyanins and the application of the Bayesian method of phylogenetic inference allow, for the first time, a solid reconstruction of the intramolecular evolution of the chelicerate hemocyanin subunits. The branch leading to subunit a diverged first, followed by the common branch of the dimer-forming b and c subunits, while subunits d and f, as well as subunits e and g form common branches. Assuming a clock-like evolution of the chelicerate hemocyanins, a timescale for the evolution of the Chelicerata was obtained that agrees with the fossil record.

  10. Subunit mass analysis for monitoring antibody oxidation. (United States)

    Sokolowska, Izabela; Mo, Jingjie; Dong, Jia; Lewis, Michael J; Hu, Ping


    Methionine oxidation is a common posttranslational modification (PTM) of monoclonal antibodies (mAbs). Oxidation can reduce the in-vivo half-life, efficacy and stability of the product. Peptide mapping is commonly used to monitor the levels of oxidation, but this is a relatively time-consuming method. A high-throughput, automated subunit mass analysis method was developed to monitor antibody methionine oxidation. In this method, samples were treated with IdeS, EndoS and dithiothreitol to generate three individual IgG subunits (light chain, Fd' and single chain Fc). These subunits were analyzed by reversed phase-ultra performance liquid chromatography coupled with an online quadrupole time-of-flight mass spectrometer and the levels of oxidation on each subunit were quantitated based on the deconvoluted mass spectra using the UNIFI software. The oxidation results obtained by subunit mass analysis correlated well with the results obtained by peptide mapping. Method qualification demonstrated that this subunit method had excellent repeatability and intermediate precision. In addition, UNIFI software used in this application allows automated data acquisition and processing, which makes this method suitable for high-throughput process monitoring and product characterization. Finally, subunit mass analysis revealed the different patterns of Fc methionine oxidation induced by chemical and photo stress, which makes it attractive for investigating the root cause of oxidation.

  11. Tabulation as a high-resolution alternative to coarse-graining protein interactions: Initial application to virus capsid subunits (United States)

    Spiriti, Justin; Zuckerman, Daniel M.


    Traditional coarse-graining based on a reduced number of interaction sites often entails a significant sacrifice of chemical accuracy. As an alternative, we present a method for simulating large systems composed of interacting macromolecules using an energy tabulation strategy previously devised for small rigid molecules or molecular fragments [S. Lettieri and D. M. Zuckerman, J. Comput. Chem. 33, 268-275 (2012); J. Spiriti and D. M. Zuckerman, J. Chem. Theory Comput. 10, 5161-5177 (2014)]. We treat proteins as rigid and construct distance and orientation-dependent tables of the interaction energy between them. Arbitrarily detailed interactions may be incorporated into the tables, but as a proof-of-principle, we tabulate a simple α-carbon Gō-like model for interactions between dimeric subunits of the hepatitis B viral capsid. This model is significantly more structurally realistic than previous models used in capsid assembly studies. We are able to increase the speed of Monte Carlo simulations by a factor of up to 6700 compared to simulations without tables, with only minimal further loss in accuracy. To obtain further enhancement of sampling, we combine tabulation with the weighted ensemble (WE) method, in which multiple parallel simulations are occasionally replicated or pruned in order to sample targeted regions of a reaction coordinate space. In the initial study reported here, WE is able to yield pathways of the final ˜25% of the assembly process.

  12. The structure of the COPII transport-vesicle coat assembled on membranes. (United States)

    Zanetti, Giulia; Prinz, Simone; Daum, Sebastian; Meister, Annette; Schekman, Randy; Bacia, Kirsten; Briggs, John A G


    Coat protein complex II (COPII) mediates formation of the membrane vesicles that export newly synthesised proteins from the endoplasmic reticulum. The inner COPII proteins bind to cargo and membrane, linking them to the outer COPII components that form a cage around the vesicle. Regulated flexibility in coat architecture is essential for transport of a variety of differently sized cargoes, but structural data on the assembled coat has not been available. We have used cryo-electron tomography and subtomogram averaging to determine the structure of the complete, membrane-assembled COPII coat. We describe a novel arrangement of the outer coat and find that the inner coat can assemble into regular lattices. The data reveal how coat subunits interact with one another and with the membrane, suggesting how coordinated assembly of inner and outer coats can mediate and regulate packaging of vesicles ranging from small spheres to large tubular carriers. DOI:

  13. 基于PLC的大型船体分段合拢对接控制系统%Automatic Assembling Control System of Large Ship Body Section Based on PLC

    Institute of Scientific and Technical Information of China (English)

    郑雄胜; 芮晓松


    An advanced automatic hydraulic assembling system on PLC for large ship was developed. It can move and lift the different parts of big ship, adjust their postures, and finally fit all of them together automatically. It can reduce the large crane occupied time. The system has some advantages as high safety, powerful, high accuracy and low operating cost.%设计一种基于PLC的大型船体分段液压自动对中合拢控制系统,能够快速准确地进行船体分段的顶升、平移、位姿调整及行走对接,从而实现了船体分段合拢对接作业的自动化,可减少大型吊车的占用时间,提高作业效率,并具有安全、可靠、低作业成本等优点.

  14. The paracaspase MALT1 cleaves the LUBAC subunit HOIL1 during antigen receptor signaling. (United States)

    Douanne, Tiphaine; Gavard, Julie; Bidère, Nicolas


    Antigen-receptor-mediated activation of lymphocytes relies on a signalosome comprising CARMA1 (also known as CARD11), BCL10 and MALT1 (the CBM complex). The CBM activates nuclear factor κB (NF-κB) transcription factors by recruiting the 'linear ubiquitin assembly complex' (LUBAC), and unleashes MALT1 paracaspase activity. Although MALT1 enzyme shapes NF-κB signaling, lymphocyte activation and contributes to lymphoma growth, the identity of its substrates continues to be elucidated. Here, we report that the LUBAC subunit HOIL1 (also known as RBCK1) is cleaved by MALT1 following antigen receptor engagement. HOIL1 is also constitutively processed in the 'activated B-cell-like' (ABC) subtype of diffuse large B-cell lymphoma (DLBCL), which exhibits aberrant MALT1 activity. We further show that the overexpression of MALT1-insensitive HOIL1 mitigates T-cell-receptor-mediated NF-κB activation and subsequent cytokine production in lymphocytes. Thus, our results unveil HOIL1 as a negative regulator of lymphocyte activation cleaved by MALT1. This cleavage could therefore constitute an appealing therapeutic target for modulating immune responses.

  15. Divergence of RNA polymerase α subunits in angiosperm plastid genomes is mediated by genomic rearrangement (United States)

    Blazier, J. Chris; Ruhlman, Tracey A.; Weng, Mao-Lun; Rehman, Sumaiyah K.; Sabir, Jamal S. M.; Jansen, Robert K.


    Genes for the plastid-encoded RNA polymerase (PEP) persist in the plastid genomes of all photosynthetic angiosperms. However, three unrelated lineages (Annonaceae, Passifloraceae and Geraniaceae) have been identified with unusually divergent open reading frames (ORFs) in the conserved region of rpoA, the gene encoding the PEP α subunit. We used sequence-based approaches to evaluate whether these genes retain function. Both gene sequences and complete plastid genome sequences were assembled and analyzed from each of the three angiosperm families. Multiple lines of evidence indicated that the rpoA sequences are likely functional despite retaining as low as 30% nucleotide sequence identity with rpoA genes from outgroups in the same angiosperm order. The ratio of non-synonymous to synonymous substitutions indicated that these genes are under purifying selection, and bioinformatic prediction of conserved domains indicated that functional domains are preserved. One of the lineages (Pelargonium, Geraniaceae) contains species with multiple rpoA-like ORFs that show evidence of ongoing inter-paralog gene conversion. The plastid genomes containing these divergent rpoA genes have experienced extensive structural rearrangement, including large expansions of the inverted repeat. We propose that illegitimate recombination, not positive selection, has driven the divergence of rpoA. PMID:27087667

  16. Cleft Lip Repair: The Hybrid Subunit Method. (United States)

    Tollefson, Travis T


    The unilateral cleft lip repair is one of the most rewarding and challenging of plastic surgery procedures. Surgeons have introduced a variety of straight line, geometric, and rotation-advancement designs, while in practice the majority of North American surgeons have been using hybrids of the rotation-advancement techniques. The anatomic subunit approach was introduced in 2005 by Fisher and has gained popularity, with early adopters of the design touting its simplicity and effectiveness. The objectives of this article are to summarize the basic tenets of respecting the philtral subunit, accurate measurement and planning, and tips for transitioning to this subunit approach.

  17. Directed Assembly of Gold Nanoparticles

    DEFF Research Database (Denmark)

    Westerlund, Axel Rune Fredrik; Bjørnholm, Thomas


    As a complement to common "top-down" lithography techniques, "bottom-up" assembly techniques are emerging as promising tools to build nanoscale structures in a predictable way. Gold nanoparticles that are stable and relatively easy to synthesize are important building blocks in many such structures...... due to their useful optical and electronic properties. Programmed assembly of gold nanoparticles in one, two, and three dimensions is therefore of large interest. This review focuses on the progress from the last three years in the field of directed gold nanoparticle and nanorod assembly using...

  18. The four canonical tpr subunits of human APC/C form related homo-dimeric structures and stack in parallel to form a TPR suprahelix. (United States)

    Zhang, Ziguo; Chang, Leifu; Yang, Jing; Conin, Nora; Kulkarni, Kiran; Barford, David


    The anaphase-promoting complex or cyclosome (APC/C) is a large E3 RING-cullin ubiquitin ligase composed of between 14 and 15 individual proteins. A striking feature of the APC/C is that only four proteins are involved in directly recognizing target proteins and catalyzing the assembly of a polyubiquitin chain. All other subunits, which account for >80% of the mass of the APC/C, provide scaffolding functions. A major proportion of these scaffolding subunits are structurally related. In metazoans, there are four canonical tetratricopeptide repeat (TPR) proteins that form homo-dimers (Apc3/Cdc27, Apc6/Cdc16, Apc7 and Apc8/Cdc23). Here, we describe the crystal structure of the N-terminal homo-dimerization domain of Schizosaccharomyces pombe Cdc23 (Cdc23(Nterm)). Cdc23(Nterm) is composed of seven contiguous TPR motifs that self-associate through a related mechanism to those of Cdc16 and Cdc27. Using the Cdc23(Nterm) structure, we generated a model of full-length Cdc23. The resultant "V"-shaped molecule docks into the Cdc23-assigned density of the human APC/C structure determined using negative stain electron microscopy (EM). Based on sequence conservation, we propose that Apc7 forms a homo-dimeric structure equivalent to those of Cdc16, Cdc23 and Cdc27. The model is consistent with the Apc7-assigned density of the human APC/C EM structure. The four canonical homo-dimeric TPR proteins of human APC/C stack in parallel on one side of the complex. Remarkably, the uniform relative packing of neighboring TPR proteins generates a novel left-handed suprahelical TPR assembly. This finding has implications for understanding the assembly of other TPR-containing multimeric complexes.

  19. General Assembly

    CERN Multimedia

    Staff Association


    Mardi 5 mai à 11 h 00 Salle 13-2-005 Conformément aux statuts de l’Association du personnel, une Assemblée générale ordinaire est organisée une fois par année (article IV.2.1). Projet d’ordre du jour : 1- Adoption de l’ordre du jour. 2- Approbation du procès-verbal de l’Assemblée générale ordinaire du 22 mai 2014. 3- Présentation et approbation du rapport d’activités 2014. 4- Présentation et approbation du rapport financier 2014. 5- Présentation et approbation du rapport des vérificateurs aux comptes pour 2014. 6- Programme 2015. 7- Présentation et approbation du projet de budget 2015 et taux de cotisation pour 2015. 8- Pas de modifications aux Statuts de l'Association du personnel proposée. 9- Élections des membres de la Commission é...

  20. General Assembly

    CERN Multimedia

    Staff Association


    Mardi 5 avril à 11 h 00 BE Auditorium Meyrin (6-2-024) Conformément aux statuts de l’Association du personnel, une Assemblée générale ordinaire est organisée une fois par année (article IV.2.1). Projet d’ordre du jour : Adoption de l’ordre du jour. Approbation du procès-verbal de l’Assemblée générale ordinaire du 5 mai 2015. Présentation et approbation du rapport d’activités 2015. Présentation et approbation du rapport financier 2015. Présentation et approbation du rapport des vérificateurs aux comptes pour 2015. Programme de travail 2016. Présentation et approbation du projet de budget 2016 Approbation du taux de cotisation pour 2017. Modifications aux Statuts de l'Association du personnel proposée. Élections des membres de la Commissio...

  1. General assembly

    CERN Multimedia

    Staff Association


    Mardi 5 mai à 11 h 00 Salle 13-2-005 Conformément aux statuts de l’Association du personnel, une Assemblée générale ordinaire est organisée une fois par année (article IV.2.1). Projet d’ordre du jour : Adoption de l’ordre du jour. Approbation du procès-verbal de l’Assemblée générale ordinaire du 22 mai 2014. Présentation et approbation du rapport d’activités 2014. Présentation et approbation du rapport financier 2014. Présentation et approbation du rapport des vérificateurs aux comptes pour 2014. Programme 2015. Présentation et approbation du projet de budget 2015 et taux de cotisation pour 2015. Pas de modifications aux Statuts de l'Association du personnel proposée. Élections des membres de la Commission électorale. &am...

  2. General Assembly

    CERN Multimedia

    Staff Association


    Conformément aux statuts de l’Association du personnel, une Assemblée générale ordinaire est organisée une fois par année (article IV.2.1). Projet d’ordre du jour : Adoption de l’ordre du jour. Approbation du procès-verbal de l’Assemblée générale ordinaire du 5 avril 2016. Présentation et approbation du rapport d’activités 2016. Présentation et approbation du rapport financier 2016. Présentation et approbation du rapport des vérificateurs aux comptes pour 2016. Programme de travail 2017. Présentation et approbation du projet de budget 2017 Approbation du taux de cotisation pour 2018. Modifications aux Statuts de l'Association du personnel proposées. Élections des membres de la Commission électorale. Élections des vérifica...

  3. MR-1S Interacts with PET100 and PET117 in Module-Based Assembly of Human Cytochrome c Oxidase. (United States)

    Vidoni, Sara; Harbour, Michael E; Guerrero-Castillo, Sergio; Signes, Alba; Ding, Shujing; Fearnley, Ian M; Taylor, Robert W; Tiranti, Valeria; Arnold, Susanne; Fernandez-Vizarra, Erika; Zeviani, Massimo


    The biogenesis of human cytochrome c oxidase (COX) is an intricate process in which three mitochondrial DNA (mtDNA)-encoded core subunits are assembled in a coordinated way with at least 11 nucleus-encoded subunits. Many chaperones shared between yeast and humans are involved in COX assembly. Here, we have used a MT-CO3 mutant cybrid cell line to define the composition of assembly intermediates and identify new human COX assembly factors. Quantitative mass spectrometry analysis led us to modify the assembly model from a sequential pathway to a module-based process. Each module contains one of the three core subunits, together with different ancillary components, including HIGD1A. By the same analysis, we identified the short isoform of the myofibrillogenesis regulator 1 (MR-1S) as a new COX assembly factor, which works with the highly conserved PET100 and PET117 chaperones to assist COX biogenesis in higher eukaryotes.

  4. X-linked primary ciliary dyskinesia due to mutations in the cytoplasmic axonemal dynein assembly factor PIH1D3 (United States)

    Olcese, Chiara; Patel, Mitali P.; Shoemark, Amelia; Kiviluoto, Santeri; Legendre, Marie; Williams, Hywel J.; Vaughan, Cara K.; Hayward, Jane; Goldenberg, Alice; Emes, Richard D.; Munye, Mustafa M.; Dyer, Laura; Cahill, Thomas; Bevillard, Jeremy; Gehrig, Corinne; Guipponi, Michel; Chantot, Sandra; Duquesnoy, Philippe; Thomas, Lucie; Jeanson, Ludovic; Copin, Bruno; Tamalet, Aline; Thauvin-Robinet, Christel; Papon, Jean- François; Garin, Antoine; Pin, Isabelle; Vera, Gabriella; Aurora, Paul; Fassad, Mahmoud R.; Jenkins, Lucy; Boustred, Christopher; Cullup, Thomas; Dixon, Mellisa; Onoufriadis, Alexandros; Bush, Andrew; Chung, Eddie M. K.; Antonarakis, Stylianos E.; Loebinger, Michael R.; Wilson, Robert; Armengot, Miguel; Escudier, Estelle; Hogg, Claire; Al-Turki, Saeed; Anderson, Carl; Antony, Dinu; Barroso, Inês; Beales, Philip L.; Bentham, Jamie; Bhattacharya, Shoumo; Carss, Keren; Chatterjee, Krishna; Cirak, Sebahattin; Cosgrove, Catherine; Allan, Daly; Durbin, Richard; Fitzpatrick, David; Floyd, Jamie; Foley, A. Reghan; Franklin, Chris; Futema, Marta; Humphries, Steve E.; Hurles, Matt; McCarthy, Shane; Muddyman, Dawn; Muntoni, Francesco; Parker, Victoria; Payne, Felicity; Plagnol, Vincent; Raymond, Lucy; Savage, David B.; Scambler, Peter J.; Schmidts, Miriam; Semple, Robert; Serra, Eva; Stalker, Jim; van Kogelenberg, Margriet; Vijayarangakannan, Parthiban; Walter, Klaudia; Amselem, Serge; Sun, Zhaoxia; Bartoloni, Lucia; Blouin, Jean-Louis; Mitchison, Hannah M.


    By moving essential body fluids and molecules, motile cilia and flagella govern respiratory mucociliary clearance, laterality determination and the transport of gametes and cerebrospinal fluid. Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder frequently caused by non-assembly of dynein arm motors into cilia and flagella axonemes. Before their import into cilia and flagella, multi-subunit axonemal dynein arms are thought to be stabilized and pre-assembled in the cytoplasm through a DNAAF2–DNAAF4–HSP90 complex akin to the HSP90 co-chaperone R2TP complex. Here, we demonstrate that large genomic deletions as well as point mutations involving PIH1D3 are responsible for an X-linked form of PCD causing disruption of early axonemal dynein assembly. We propose that PIH1D3, a protein that emerges as a new player of the cytoplasmic pre-assembly pathway, is part of a complementary conserved R2TP-like HSP90 co-chaperone complex, the loss of which affects assembly of a subset of inner arm dyneins. PMID:28176794

  5. Gene targeting of CK2 catalytic subunits. (United States)

    Seldin, David C; Lou, David Y; Toselli, Paul; Landesman-Bollag, Esther; Dominguez, Isabel


    Protein kinase CK2 is a highly conserved and ubiquitous serine-threonine kinase. It is a tetrameric enzyme that is made up of two regulatory CK2beta subunits and two catalytic subunits, either CK2alpha/CK2alpha, CK2alpha/CK2alpha', or CK2alpha'/CK2alpha'. Although the two catalytic subunits diverge in their C termini, their enzymatic activities are similar. To identify the specific function of the two catalytic subunits in development, we have deleted them individually from the mouse genome by homologous recombination. We have previously reported that CK2alpha' is essential for male germ cell development, and we now demonstrate that CK2alpha has an essential role in embryogenesis, as mice lacking CK2alpha die in mid-embryogenesis, with cardiac and neural tube defects.

  6. Gene targeting of CK2 catalytic subunits (United States)

    Lou, David Y.; Toselli, Paul; Landesman-Bollag, Esther; Dominguez, Isabel


    Protein kinase CK2 is a highly conserved and ubiquitous serine–threonine kinase. It is a tetrameric enzyme that is made up of two regulatory CK2β subunits and two catalytic subunits, either CK2α/CK2α, CK2α/ CK2α′, or CK2α′/CK2α′. Although the two catalytic subunits diverge in their C termini, their enzymatic activities are similar. To identify the specific function of the two catalytic subunits in development, we have deleted them individually from the mouse genome by homologous recombination. We have previously reported that CK2α′is essential for male germ cell development, and we now demonstrate that CK2α has an essential role in embryogenesis, as mice lacking CK2α die in mid-embryogenesis, with cardiac and neural tube defects. PMID:18594950

  7. Insights into the subunit in-teractions of the chloroplast ATP synthase

    Institute of Scientific and Technical Information of China (English)


    Subunit interactions of the chloroplast F0F1- ATP synthase were studied using the yeast two-hybrid system. The coding sequences of all the nine subunits of spinach chloroplast ATP synthase were cloned in two-hybrid vectors. The vectors were transformed into the yeast strains HF7c and SFY526 by various pairwise combinations, and the protein interactions were analyzed by measuring the yeast growth on minimal SD medium without serine, lucine and histidine. Interactions of γ Subunit with wild type or two truncated mutants of γ sununit, △εN21 and △εC45, which lose their abilities to inhibit the ATP hydrolysis, were also detected by in vitro and in vivo binding assay. The present results are largely accordant to the common structure model of F0F1-ATP synthase. Different from that in the E. Coli F0F1-ATP synthase, the δ subunit of chloroplast ATP syn- thase could interact with β,γ,ε and all the CF0 subunits in the two-hybrid system. These results suggested that though the chloroplast ATP synthase shares the similar structure and composition of subunits with the enzyme from E. Coli, it may be different in the subunit interactions and con- formational change during catalysis between these two sources of ATP synthase. Based on the present results and our knowledge of structure model of E. Coli ATP synthase, a deduced structure model of chloroplast ATP synthase was proposed.

  8. An ER-resident membrane protein complex regulates nicotinic acetylcholine receptor subunit composition at the synapse (United States)

    Almedom, Ruta B; Liewald, Jana F; Hernando, Guillermina; Schultheis, Christian; Rayes, Diego; Pan, Jie; Schedletzky, Thorsten; Hutter, Harald; Bouzat, Cecilia; Gottschalk, Alexander


    Nicotinic acetylcholine receptors (nAChRs) are homo- or heteropentameric ligand-gated ion channels mediating excitatory neurotransmission and muscle activation. Regulation of nAChR subunit assembly and transfer of correctly assembled pentamers to the cell surface is only partially understood. Here, we characterize an ER transmembrane (TM) protein complex that influences nAChR cell-surface expression and functional properties in Caenorhabditis elegans muscle. Loss of either type I TM protein, NRA-2 or NRA-4 (nicotinic receptor associated), affects two different types of muscle nAChRs and causes in vivo resistance to cholinergic agonists. Sensitivity to subtype-specific agonists of these nAChRs is altered differently, as demonstrated by whole-cell voltage-clamp of dissected adult muscle, when applying exogenous agonists or after photo-evoked, channelrhodopsin-2 (ChR2) mediated acetylcholine (ACh) release, as well as in single-channel recordings in cultured embryonic muscle. These data suggest that nAChRs desensitize faster in nra-2 mutants. Cell-surface expression of different subunits of the ‘levamisole-sensitive' nAChR (L-AChR) is differentially affected in the absence of NRA-2 or NRA-4, suggesting that they control nAChR subunit composition or allow only certain receptor assemblies to leave the ER. PMID:19609303

  9. Chaperone-assisted assembly of the proteasome core particle. (United States)

    Matias, Ana C; Ramos, Paula C; Dohmen, R Jürgen


    The 26S proteasome is a non-lysosomal protease in the cytosol and nucleus of eukaryotic cells. Its main function is to mediate ubiquitin-dependent proteolysis. The 26S proteasome is a multimeric complex composed by the 20S proteasome CP (core particle) and the 19S RPs (regulatory particles). Although the atomic structure of the 26S proteasome has not yet been determined, high-resolution structures are available for its CP. Studies on the complicated assembly pathway of the proteasome have revealed that it involves an unprecedented number of dedicated chaperones. Assembly of the CP alone involves three conserved proteasome-assembly chaperones [PAC1-PAC2, PAC3-PAC4 and UMP1 (ubiquitin-mediated proteolysis 1)]. Whereas the two heterodimeric PACs have been implicated in the formation of rings of the seven distinct alpha subunits, UMP1 is important for the formation and dimerization of proteasome precursor complexes containing beta subunits. Dimerization coincides with the incorporation of the last beta subunit (beta7). Additional modules important for the assembly of precursor complexes and their dimerization reside in the beta subunits themselves, either as transient or as permanent extensions. Particularly important domains are the propeptide of beta5 and the C-terminal extensions of beta2 and beta7. Upon maturation of the active sites by autocatalytic processing, UMP1 is degraded by the native proteasome.

  10. Assembling the archaeal ribosome: roles for translation-factor-related GTPases

    NARCIS (Netherlands)

    Blombach, F.; Brouns, S.J.J.; Oost, van der J.


    The assembly of ribosomal subunits from their individual components (rRNA and ribosomal proteins) requires the assistance of a multitude of factors in order to control and increase the efficiency of the assembly process. GTPases of the TRAFAC (translation-factor-related) class constitute a major typ

  11. Functional characterization of Kv channel beta-subunits from rat brain. (United States)

    Heinemann, S H; Rettig, J; Graack, H R; Pongs, O


    1. The potassium channel beta-subunit from rat brain, Kv beta 1.1, is known to induce inactivation of the delayed rectifier channel Kv1.1 and Kv1.4 delta 1-110. 2. Kv beta 1.1 was co-expressed in Xenopus oocytes with various other potassium channel alpha-subunits. Kv beta 1.1 induced inactivation in members of the Kv1 subfamily with the exception of Kv 1.6; no inactivation of Kv 2.1, Kv 3.4 delta 2-28 and Kv4.1 channels could be observed. 3. The second member of the beta-subunit subfamily, Kv beta 2, had a shorter N-terminal end, accelerated inactivation of the A-type channel Kv 1.4, but did not induce inactivation when co-expressed with delayed rectifiers of the Kv1 channel family. 4. To test whether this subunit co-assembles with Kv alpha-subunits, the N-terminal inactivating domains of Kv beta 1.1 and Kv beta 3 were spliced to the N-terminus of Kv beta 2. The chimaeric beta-subunits (beta 1/ beta 2 and beta 3/ beta 2) induced fast inactivation of several Kv1 channels, indicating that Kv beta 2 associates with these alpha-subunits. No inactivation was induced in Kv 1.3, Kv 1.6, Kv2.1 and Kv3.4 delta 2-28 channels. 5. Kv beta 2 caused a voltage shift in the activation threshold of Kv1.5 of about -10 mV, indicating a putative physiological role. Kv beta 2 had a smaller effect on Kv 1.1 channels. 6. Kv beta 2 accelerated the activation time course of Kv1.5 but had no marked effect on channel deactivation.

  12. Interactions between subunits of Saccharomyces cerevisiae RNase MRP support a conserved eukaryotic RNase P/MRP architecture. (United States)

    Aspinall, Tanya V; Gordon, James M B; Bennett, Hayley J; Karahalios, Panagiotis; Bukowski, John-Paul; Walker, Scott C; Engelke, David R; Avis, Johanna M


    Ribonuclease MRP is an endonuclease, related to RNase P, which functions in eukaryotic pre-rRNA processing. In Saccharomyces cerevisiae, RNase MRP comprises an RNA subunit and ten proteins. To improve our understanding of subunit roles and enzyme architecture, we have examined protein-protein and protein-RNA interactions in vitro, complementing existing yeast two-hybrid data. In total, 31 direct protein-protein interactions were identified, each protein interacting with at least three others. Furthermore, seven proteins self-interact, four strongly, pointing to subunit multiplicity in the holoenzyme. Six protein subunits interact directly with MRP RNA and four with pre-rRNA. A comparative analysis with existing data for the yeast and human RNase P/MRP systems enables confident identification of Pop1p, Pop4p and Rpp1p as subunits that lie at the enzyme core, with probable addition of Pop5p and Pop3p. Rmp1p is confirmed as an integral subunit, presumably associating preferentially with RNase MRP, rather than RNase P, via interactions with Snm1p and MRP RNA. Snm1p and Rmp1p may act together to assist enzyme specificity, though roles in substrate binding are also indicated for Pop4p and Pop6p. The results provide further evidence of a conserved eukaryotic RNase P/MRP architecture and provide a strong basis for studies of enzyme assembly and subunit function.

  13. Condensin HEAT subunits required for DNA repair, kinetochore/centromere function and ploidy maintenance in fission yeast.

    Directory of Open Access Journals (Sweden)

    Xingya Xu

    Full Text Available Condensin, a central player in eukaryotic chromosomal dynamics, contains five evolutionarily-conserved subunits. Two SMC (structural maintenance of chromosomes subunits contain ATPase, hinge, and coiled-coil domains. One non-SMC subunit is similar to bacterial kleisin, and two other non-SMC subunits contain HEAT (similar to armadillo repeats. Here we report isolation and characterization of 21 fission yeast (Schizosaccharomyces pombe mutants for three non-SMC subunits, created using error-prone mutagenesis that resulted in single-amino acid substitutions. Beside condensation, segregation, and DNA repair defects, similar to those observed in previously isolated SMC and cnd2 mutants, novel phenotypes were observed for mutants of HEAT-repeats containing Cnd1 and Cnd3 subunits. cnd3-L269P is hypersensitive to the microtubule poison, thiabendazole, revealing defects in kinetochore/centromere and spindle assembly checkpoints. Three cnd1 and three cnd3 mutants increased cell size and doubled DNA content, thereby eliminating the haploid state. Five of these mutations reside in helix B of HEAT repeats. Two non-SMC condensin subunits, Cnd1 and Cnd3, are thus implicated in ploidy maintenance.


    Mazzaferro, Simone; Bermudez, Isabel; Sine, Steven M


    Acetylcholine receptors comprising α4 and β2 subunits are the most abundant class of nicotinic acetylcholine receptor in the brain. They contribute to cognition, reward, mood, and nociception and are implicated in a range of neurological disorders. Previous measurements of whole-cell macroscopic currents showed that α4 and β2 subunits assemble in two predominant pentameric stoichiometries, which differ in their sensitivity to agonists, antagonists, and allosteric modulators. Here we compare agonist-elicited single channel currents from receptors assembled with an excess of either the α4 or β2 subunit, forming receptor populations biased toward one or the other stoichiometry, with currents from receptors composed of five concatemeric subunits in which the subunit stoichiometry is predetermined. Our results associate each subunit stoichiometry with a unique single channel conductance, mean open channel lifetime, and sensitivity to the allosteric potentiator 3-[3-(3-pyridinyl)-1,2,4-oxadiazol-5-yl]benzonitrile (NS-9283). Receptors with the composition (α4β2)2α4 exhibit high single channel conductance, brief mean open lifetime, and strong potentiation by NS-9283, whereas receptors with the composition (α4β2)2β2 exhibit low single channel conductance and long mean open lifetime and are not potentiated by NS-9283. Thus single channel current measurements reveal bases for the distinct functional and pharmacological properties endowed by different stoichiometries of α4 and β2 subunits and establish pentameric concatemers as a means to delineate interactions between subunits that confer these properties. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Oms1 associates with cytochrome c oxidase assembly intermediates to stabilize newly synthesized Cox1. (United States)

    Bareth, Bettina; Nikolov, Miroslav; Lorenzi, Isotta; Hildenbeutel, Markus; Mick, David U; Helbig, Christin; Urlaub, Henning; Ott, Martin; Rehling, Peter; Dennerlein, Sven


    The mitochondrial cytochrome c oxidase assembles in the inner membrane from subunits of dual genetic origin. The assembly process of the enzyme is initiated by membrane insertion of the mitochondria-encoded Cox1 subunit. During complex maturation, transient assembly intermediates, consisting of structural subunits and specialized chaperone-like assembly factors, are formed. In addition, cofactors such as heme and copper have to be inserted into the nascent complex. To regulate the assembly process, the availability of Cox1 is under control of a regulatory feedback cycle in which translation of COX1 mRNA is stalled when assembly intermediates of Cox1 accumulate through inactivation of the translational activator Mss51. Here we isolate a cytochrome c oxidase assembly intermediate in preparatory scale from coa1Δ mutant cells, using Mss51 as bait. We demonstrate that at this stage of assembly, the complex has not yet incorporated the heme a cofactors. Using quantitative mass spectrometry, we define the protein composition of the assembly intermediate and unexpectedly identify the putative methyltransferase Oms1 as a constituent. Our analyses show that Oms1 participates in cytochrome c oxidase assembly by stabilizing newly synthesized Cox1.

  16. Ribosomal small subunit domains radiate from a central core (United States)

    Gulen, Burak; Petrov, Anton S.; Okafor, C. Denise; Vander Wood, Drew; O’Neill, Eric B.; Hud, Nicholas V.; Williams, Loren Dean


    The domain architecture of a large RNA can help explain and/or predict folding, function, biogenesis and evolution. We offer a formal and general definition of an RNA domain and use that definition to experimentally characterize the rRNA of the ribosomal small subunit. Here the rRNA comprising a domain is compact, with a self-contained system of molecular interactions. A given rRNA helix or stem-loop must be allocated uniquely to a single domain. Local changes such as mutations can give domain-wide effects. Helices within a domain have interdependent orientations, stabilities and interactions. With these criteria we identify a core domain (domain A) of small subunit rRNA. Domain A acts as a hub, linking the four peripheral domains and imposing orientational and positional restraints on the other domains. Experimental characterization of isolated domain A, and mutations and truncations of it, by methods including selective 2′OH acylation analyzed by primer extension and circular dichroism spectroscopy are consistent with our architectural model. The results support the utility of the concept of an RNA domain. Domain A, which exhibits structural similarity to tRNA, appears to be an essential core of the small ribosomal subunit. PMID:26876483

  17. Ribosomal small subunit domains radiate from a central core (United States)

    Gulen, Burak; Petrov, Anton S.; Okafor, C. Denise; Vander Wood, Drew; O'Neill, Eric B.; Hud, Nicholas V.; Williams, Loren Dean


    The domain architecture of a large RNA can help explain and/or predict folding, function, biogenesis and evolution. We offer a formal and general definition of an RNA domain and use that definition to experimentally characterize the rRNA of the ribosomal small subunit. Here the rRNA comprising a domain is compact, with a self-contained system of molecular interactions. A given rRNA helix or stem-loop must be allocated uniquely to a single domain. Local changes such as mutations can give domain-wide effects. Helices within a domain have interdependent orientations, stabilities and interactions. With these criteria we identify a core domain (domain A) of small subunit rRNA. Domain A acts as a hub, linking the four peripheral domains and imposing orientational and positional restraints on the other domains. Experimental characterization of isolated domain A, and mutations and truncations of it, by methods including selective 2‧OH acylation analyzed by primer extension and circular dichroism spectroscopy are consistent with our architectural model. The results support the utility of the concept of an RNA domain. Domain A, which exhibits structural similarity to tRNA, appears to be an essential core of the small ribosomal subunit.

  18. Design and Checking Calculation of a Large-Type Assembled Walking Floating Gantry%拼组式大型行走浮龙门设计与检算

    Institute of Scientific and Technical Information of China (English)



    Aiming at solving the problems of hoisting operations in the construction of existing bridge foundation, piers and a- butments, and with the foundation construction of Pier 7 of the Gutianxi Mega Bridge as a practical example,the design of the assembled large-type walking floating gantry is studied and performed in the paper, with the draft of the floating body of the lateral beam, longitudinal beam and that of the floating body under the key conditions, the stability, the inclination angle, etc. check-calculated. It may serve as a useful reference for the research on and the design of similar above-water equipments.%针对水库深水既有桥梁基础、墩台施工吊装难题,以古田溪特大桥7号墩基础施工为背景,研究设计了拼组式大型行走浮龙门,并对其横梁、纵梁及关键工况下的浮体吃水、稳定性、倾斜角等进行了检算,为类似水上装备研究设计提供参考。

  19. Biosynthesis of the Torpedo californica Acetylcholine Receptor α Subunit in Yeast (United States)

    Fujita, Norihisa; Nelson, Nathan; Fox, Thomas D.; Claudio, Toni; Lindstrom, Jon; Riezman, Howard; Hess, George P.


    Yeast cells were transformed with a plasmid containing complementary DNA encoding the α subunit of the Torpedo californica acetylcholine receptor. These cells synthesized a protein that had the expected molecular weight, antigenic specificity, and ligand-binding properties of the α subunit. The subunit was inserted into the yeast plasma membrane, demonstrating that yeast has the apparatus to express a membrane-bound receptor protein and to insert such a foreign protein into its plasma membrane. The α subunit constituted approximately 1 percent of the total yeast membrane proteins, and its density was about the same in the plasma membrane of yeast and in the receptor-rich electric organ of Electrophorus electricus. In view of the available technology for obtaining large quantities of yeast proteins, it may now be possible to obtain amplified amounts of interesting membrane-bound proteins for physical and biochemical studies.

  20. Splice variants of Na(V1.7 sodium channels have distinct β subunit-dependent biophysical properties.

    Directory of Open Access Journals (Sweden)

    Clare Farmer

    Full Text Available Genes encoding the α subunits of neuronal sodium channels have evolutionarily conserved sites of alternative splicing but no functional differences have been attributed to the splice variants. Here, using Na(V1.7 as an exemplar, we show that the sodium channel isoforms are functionally distinct when co-expressed with β subunits. The gene, SCN9A, encodes the α subunit of the Na(V1.7 channel, and contains both sites of alternative splicing that are highly conserved. In conditions where the intrinsic properties of the Na(V1.7 splice variants were similar when expressed alone, co-expression of β1 subunits had different effects on channel availability that were determined by splicing at either site in the α subunit. While the identity of exon 5 determined the degree to which β1 subunits altered voltage-dependence of activation (P = 0.027, the length of exon 11 regulated how far β1 subunits depolarised voltage-dependence of inactivation (P = 0.00012. The results could have a significant impact on channel availability, for example with the long version of exon 11, the co-expression of β1 subunits could lead to nearly twice as large an increase in channel availability compared to channels containing the short version. Our data suggest that splicing can change the way that Na(V channels interact with β subunits. Because splicing is conserved, its unexpected role in regulating the functional impact of β subunits may apply to multiple voltage-gated sodium channels, and the full repertoire of β subunit function may depend on splicing in α subunits.

  1. New disguises for an old channel: MaxiK channel beta-subunits. (United States)

    Orio, Patricio; Rojas, Patricio; Ferreira, Gonzalo; Latorre, Ramón


    Ca(2+)-activated K(+) channels of large conductance (MaxiK or BK channels) control a large variety of physiological processes, including smooth muscle tone, neurosecretion, and hearing. Despite being coded by a single gene (Slowpoke), the diversity of MaxiK channels is great. Regulatory b-subunits, splicing, and metabolic regulation create this diversity fundamental to the adequate function of many tissues.

  2. Pharmacological consequences of the coexpression of BK channel α and auxiliary β subunits (United States)

    Torres, Yolima P.; Granados, Sara T.; Latorre, Ramón


    Coded by a single gene (Slo1, KCM) and activated by depolarizing potentials and by a rise in intracellular Ca2+ concentration, the large conductance voltage- and Ca2+-activated K+ channel (BK) is unique among the superfamily of K+ channels. BK channels are tetramers characterized by a pore-forming α subunit containing seven transmembrane segments (instead of the six found in voltage-dependent K+ channels) and a large C terminus composed of two regulators of K+ conductance domains (RCK domains), where the Ca2+-binding sites reside. BK channels can be associated with accessory β subunits and, although different BK modulatory mechanisms have been described, greater interest has recently been placed on the role that the β subunits may play in the modulation of BK channel gating due to its physiological importance. Four β subunits have currently been identified (i.e., β1, β2, β3, and β4) and despite the fact that they all share the same topology, it has been shown that every β subunit has a specific tissue distribution and that they modify channel kinetics as well as their pharmacological properties and the apparent Ca2+ sensitivity of the α subunit in different ways. Additionally, different studies have shown that natural, endogenous, and synthetic compounds can modulate BK channels through β subunits. Considering the importance of these channels in different pathological conditions, such as hypertension and neurological disorders, this review focuses on the mechanisms by which these compounds modulate the biophysical properties of BK channels through the regulation of β subunits, as well as their potential therapeutic uses for diseases such as those mentioned above. PMID:25346693

  3. Study on Horizontal Docking Assembly Method for Segment Thin-walled Solid Rocket Motor with Large Opening%薄壁大开口分段固体发动机卧式对接装配研究

    Institute of Scientific and Technical Information of China (English)

    彭莎莎; 刘永盛; 宗路航; 罗玲莉; 苏昌银


    对薄壁大开口分段固体发动机卧式对接装配进行研究,应用ANSYS软件对燃烧室在重力作用下的应力、应变云图进行分析,提出变形识别与安全校正是解决结构件形变的一种方法。采用径向圆周10点均分校正法,采集燃烧室对接径向U型件边沿的变形量,制定了燃烧室变形安全校正值,经校正后中段与前段燃烧室对接径向边沿的10个点之间距离最大值为0.02mm。实现了Φ2m/分段式大型发动机对接装配。经Φ2m/分段式发动机地面试车获得成功证实校正值合理。%In this paper, the horizontal docking assembly of segment thin-walled large opening solid rocket motor is analyzed. The stress and strain of combustion chamber under the gravity is analyzed by ANSYS software and then deformation recognition and safe correction are proposed to prevent deformation of structural parts. The radial circular 10 average points correction method is used to collect the deformation amount of docking radial direction of edge of the U-shaped part, and to devise the safety correction value of deformation of single and double combustion chamber. Finally it is proved that the biggest gap of docking radial edge of 10 points between front and middle chamber is 0.02mm using different angle correction. Docking assembly of 2m/ segment solid rocket motor is realized and the correction value is proved reasonable after the success of ground fire test of 2m/segment solid rocket motor.

  4. Mitochondrial ribosome assembly in health and disease. (United States)

    De Silva, Dasmanthie; Tu, Ya-Ting; Amunts, Alexey; Fontanesi, Flavia; Barrientos, Antoni


    The ribosome is a structurally and functionally conserved macromolecular machine universally responsible for catalyzing protein synthesis. Within eukaryotic cells, mitochondria contain their own ribosomes (mitoribosomes), which synthesize a handful of proteins, all essential for the biogenesis of the oxidative phosphorylation system. High-resolution cryo-EM structures of the yeast, porcine and human mitoribosomal subunits and of the entire human mitoribosome have uncovered a wealth of new information to illustrate their evolutionary divergence from their bacterial ancestors and their adaptation to synthesis of highly hydrophobic membrane proteins. With such structural data becoming available, one of the most important remaining questions is that of the mitoribosome assembly pathway and factors involved. The regulation of mitoribosome biogenesis is paramount to mitochondrial respiration, and thus to cell viability, growth and differentiation. Moreover, mutations affecting the rRNA and protein components produce severe human mitochondrial disorders. Despite its biological and biomedical significance, knowledge on mitoribosome biogenesis and its deviations from the much-studied bacterial ribosome assembly processes is scarce, especially the order of rRNA processing and assembly events and the regulatory factors required to achieve fully functional particles. This article focuses on summarizing the current available information on mitoribosome assembly pathway, factors that form the mitoribosome assembly machinery, and the effect of defective mitoribosome assembly on human health.

  5. Design of a hyperstable 60-subunit protein icosahedron (United States)

    Hsia, Yang; Bale, Jacob B.; Gonen, Shane; Shi, Dan; Sheffler, William; Fong, Kimberly K.; Nattermann, Una; Xu, Chunfu; Huang, Po-Ssu; Ravichandran, Rashmi; Yi, Sue; Davis, Trisha N.; Gonen, Tamir; King, Neil P.; Baker, David


    The icosahedron and the dodecahedron are the largest of the Platonic solids, and icosahedral protein structures are widely utilized in biological systems for packaging and transport1,2. There has been considerable interest in repurposing such structures3–5, for example, virus-like particles for the targeted delivery and vaccine design. The ability to design proteins that self assemble into precisely specified, highly ordered icosahedral structures would open the door to a new generation of protein 'containers' that could exhibit properties custom-made for various applications. In this manuscript, we describe the computational design of an icosahedral nano-cage that self-assembles from trimeric building blocks. Electron microscopy images of the designed protein expressed in E. coli reveals a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 M guanidine hydrochloride at up to 80 °C, and undergo extremely abrupt, but reversible, disassembly between 2 M and 2.25 M guanidinium thiocyanate. The icosahedron is robust to genetic fusions: one or two copies of superfolder GFP can be fused to each of the 60 subunits to create highly fluorescent standard candles for light microscopy, and a designed protein pentamer can be placed in the center of each of the twenty pentameric faces to potentially gate macromolecule access to the nanocage interior. Such robust designed nanocages should have considerable utility for targeted drug delivery6, vaccine design7, and synthetic biology8. PMID:27309817

  6. The F13 residue is critical for interaction among the coat protein subunits of papaya mosaic virus. (United States)

    Laliberté Gagné, M E; Lecours, K; Gagné, S; Leclerc, D


    Papaya mosaic virus (PapMV) coat protein (CP) in Escherichia coli was previously showed to self-assemble in nucleocapsid-like particles (NLPs) that were similar in shape and appearance to the native virus. We have also shown that a truncated CP missing the N-terminal 26 amino acids is monomeric and loses its ability to bind RNA. It is likely that the N-terminus of the CP is important for the interaction between the subunits in self-assembly into NLPs. In this work, through deletion and mutation analysis, we have shown that the deletion of 13 amino acids is sufficient to generate the monomeric form of the CP. Furthermore, we have shown that residue F13 is critical for self-assembly of the CP subunits into NLPs. The replacement of F13 with hydrophobic residues (L or Y) generated mutated forms of the CP that were able to self-assemble into NLPs. However, the replacement of F13 by A, G, R, E or S was detrimental to the self-assembly of the protein into NLPs. We concluded that a hydrophobic interaction at the N-terminus is important to ensure self-assembly of the protein into NLPs. We also discuss the importance of F13 for assembly of other members of the potexvirus family.

  7. Deciphering the rules governing assembly order of mammalian septin complexes. (United States)

    Sellin, Mikael E; Sandblad, Linda; Stenmark, Sonja; Gullberg, Martin


    Septins are conserved GTP-binding proteins that assemble into lateral diffusion barriers and molecular scaffolds. Vertebrate genomes contain 9-17 septin genes that encode both ubiquitous and tissue-specific septins. Expressed septins may assemble in various combinations through both heterotypic and homotypic G-domain interactions. However, little is known regarding assembly states of mammalian septins and mechanisms directing ordered assembly of individual septins into heteromeric units, which is the focus of this study. Our analysis of the septin system in cells lacking or overexpressing selected septins reveals interdependencies coinciding with previously described homology subgroups. Hydrodynamic and single-particle data show that individual septins exist solely in the context of stable six- to eight-subunit core heteromers, all of which contain SEPT2 and SEPT6 subgroup members and SEPT7, while heteromers comprising more than six subunits also contain SEPT9. The combined data suggest a generic model for how the temporal order of septin assembly is homology subgroup-directed, which in turn determines the subunit arrangement of native heteromers. Because mammalian cells normally express multiple members and/or isoforms of some septin subgroups, our data also suggest that only a minor fraction of native heteromers are arranged as perfect palindromes.

  8. Using Markov State Models to Study Self-Assembly

    CERN Document Server

    Perkett, Matthew R


    Markov state models (MSMs) have been demonstrated to be a powerful method for computationally studying intramolecular processes such as protein folding and macromolecular conformational changes. In this article, we present a new approach to construct MSMs that is applicable to modeling a broad class of multi-molecular assembly reactions. Distinct structures formed during assembly are distinguished by their undirected graphs, which are defined by strong subunit interactions. Spatial inhomogeneities of free subunits are accounted for using a recently developed Gaussian-based signature. Simplifications to this state identification are also investigated. The feasibility of this approach is demonstrated on two different coarse-grained models for virus self-assembly. We find good agreement between the dynamics predicted by the MSMs and long, unbiased simulations, and that the MSMs can reduce overall simulation time by orders of magnitude.

  9. GABA B receptor subunit expression in glia. (United States)

    Charles, K J; Deuchars, J; Davies, C H; Pangalos, M N


    GABA(B) receptor subunits are widely expressed on neurons throughout the CNS, at both pre- and postsynaptic sites, where they mediate the late, slow component of the inhibitory response to the major inhibitory neurotransmitter GABA. The existence of functional GABA(B) receptors on nonneuronal cells has been reported previously, although the molecular composition of these receptors has not yet been described. Here we demonstrate for the first time, using immunohistochemistry the expression of GABA(B1a), GABA(B1b), and GABA(B2) on nonneuronal cells of the rat CNS. All three principle GABA(B) receptor subunits were expressed on these cells irrespective of whether they had been cultured or found within brain tissue sections. At the ultrastructural level GABA(B) receptor subunits were expressed on astrocytic processes surrounding both symmetrical and assymetrical synapses in the CA1 subregion of the hippocampus. In addition, GABA(B1a), GABA(B1b), and GABA(B2) receptor subunits were expressed on activated microglia in culture but were not found on myelin forming oligodendrocytes in the white matter of rat spinal cord. Together these data demonstrate that the obligate subunits of functional GABA(B) receptors are expressed in astrocytes and microglia in the rat CNS.

  10. Atomic structure and handedness of the building block of a biological assembly. (United States)

    Loquet, Antoine; Habenstein, Birgit; Chevelkov, Veniamin; Vasa, Suresh Kumar; Giller, Karin; Becker, Stefan; Lange, Adam


    Noncovalent supramolecular assemblies possess in general several unique subunit-subunit interfaces.The basic building block of such an assembly consists of several subunits and contains all unique interfaces. Atomic-resolution structures of monomeric subunits are typically accessed by crystallography or solution NMR and fitted into electron microscopy density maps. However, the structure of the intact building block in the assembled state remains unknown with this hybrid approach. Here, we present the solid-state NMR atomic structure of the building block of the type III secretion system needle. The building block structure consists of a homotetrameric subunit complex with three unique supramolecular interfaces. Side-chain positions at the interfaces were solved at atomic detail. The high-resolution structure reveals unambiguously the helical handedness of the assembly, determined to be right-handed for the type III secretion system needle.Additionally, the axial rise per subunit could be extracted from the tetramer structure and independently validated by mass-per-length measurements.

  11. Complementation of the Fo c Subunit of Escherichia coli with That of Streptococcus mutans and Properties of the Hybrid FoF1 ATP Synthase



    The c subunit of Streptococcus mutans ATP synthase (FoF1) is functionally exchangeable with that of Escherichia coli, since E. coli with a hybrid FoF1 is able to grow on minimum succinate medium through oxidative phosphorylation. E. coli F1 bound to the hybrid Fo with the S. mutans c subunit showed N,N′-dicyclohexylcarbodiimide-sensitive ATPase activity similar to that of E. coli FoF1. Thus, the S. mutans c subunit assembled into a functional Fo together with the E. coli a and b subu...

  12. Auxiliary KCNE subunits modulate both homotetrameric Kv2.1 and heterotetrameric Kv2.1/Kv6.4 channels


    Jens-Peter David; Stas, Jeroen I.; Nicole Schmitt; Elke Bocksteins


    The diversity of the voltage-gated K+ (Kv) channel subfamily Kv2 is increased by interactions with auxiliary β-subunits and by assembly with members of the modulatory so-called silent Kv subfamilies (Kv5-Kv6 and Kv8-Kv9). However, it has not yet been investigated whether these two types of modulating subunits can associate within and modify a single channel complex simultaneously. Here, we demonstrate that the transmembrane β-subunit KCNE5 modifies the Kv2.1/Kv6.4 current extensively, whereas...

  13. Balancing parallel assembly lines with disabled workers


    Araújo, Felipe F. B.; Costa,Alysson M.; Miralles, Cristóbal


    We study an assembly line balancing problem that occurs in sheltered worker centers for the disabled, where workers with very different characteristics are present. We are interested in the situation in which parallel assembly lines are allowed and name the resulting problem as parallel assembly line worker assignment and balancing problem. We present a linear mixed-integer formulation and a four-stage heuristic algorithm. Computational results with a large set of instances recently proposed ...

  14. Henry Ford vs. assembly line balancing


    Wilson, J M


    Ford’s Assembly Line at Highland Park is one of the most influential conceptualizations of a production system. New data reveal Ford’s operations were adaptable to strongly increasing and highly variable demand. These analyses show Ford’s assembly line was used differently than modern ones and their production systems were more flexible than previously recognized. Assembly line balancing theory largely ignores earlier practice. It will be shown that Ford used multiple lines flexibly to cope w...

  15. Using concatenated subunits to investigate the functional consequences of heterotetrameric inositol 1,4,5-trisphosphate receptors. (United States)

    Chandrasekhar, Rahul; Alzayady, Kamil J; Yule, David I


    Inositol 1,4,5-trisphosphate receptors (IP3Rs) are a family of ubiquitous, ER localized, tetrameric Ca2+ release channels. There are three subtypes of the IP3Rs (R1, R2, R3), encoded by three distinct genes, that share ∼60-70% sequence identity. The diversity of Ca2+ signals generated by IP3Rs is thought to be largely the result of differential tissue expression, intracellular localization and subtype-specific regulation of the three subtypes by various cellular factors, most significantly InsP3, Ca2+ and ATP. However, largely unexplored is the notion of additional signal diversity arising from the assembly of both homo and heterotetrameric InsP3Rs. In the present article, we review the biochemical and functional evidence supporting the existence of homo and heterotetrameric populations of InsP3Rs. In addition, we consider a strategy that utilizes genetically concatenated InsP3Rs to study the functional characteristics of heterotetramers with unequivocally defined composition. This approach reveals that the overall properties of IP3R are not necessarily simply a blend of the constituent monomers but that specific subtypes appear to dominate the overall characteristics of the tetramer. It is envisioned that the ability to generate tetramers with defined wild type and mutant subunits will be useful in probing fundamental questions relating to IP3R structure and function.

  16. A cysteine in the repetitive domain of a high-molecular-weight glutenin subunit interferes with the mixing properties of wheat dough. (United States)

    Gao, Xin; Zhang, Qisen; Newberry, Marcus P; Chalmers, Ken J; Mather, Diane E


    The quality of wheat (Triticum aestivum L.) for making bread is largely due to the strength and extensibility of wheat dough, which in turn is due to the properties of polymeric glutenin. Polymeric glutenin consists of high- and low-molecular-weight glutenin protein subunits linked by disulphide bonds between cysteine residues. Glutenin subunits differ in their effects on dough mixing properties. The research presented here investigated the effect of a specific, recently discovered, glutenin subunit on dough mixing properties. This subunit, Bx7.1, is unusual in that it has a cysteine in its repetitive domain. With site-directed mutagenesis of the gene encoding Bx7.1, a guanine in the repetitive domain was replaced by an adenine, to provide a mutant gene encoding a subunit (MutBx7.1) in which the repetitive-domain cysteine was replaced by a tyrosine residue. Bx7.1, MutBx7.1 and other Bx-type glutenin subunits were heterologously expressed in Escherichia coli and purified. This made it possible to incorporate each individual subunit into wheat flour and evaluate the effect of the cysteine residue on dough properties. The Bx7.1 subunit affected dough mixing properties differently from the other subunits. These differences are due to the extra cysteine residue, which may interfere with glutenin polymerisation through cross-linkage within the Bx7.1 subunit, causing this subunit to act as a chain terminator.

  17. Optimized subunit vaccine protects against experimental leishmaniasis. (United States)

    Bertholet, Sylvie; Goto, Yasuyuki; Carter, Lauren; Bhatia, Ajay; Howard, Randall F; Carter, Darrick; Coler, Rhea N; Vedvick, Thomas S; Reed, Steven G


    Development of a protective subunit vaccine against Leishmania spp. depends on antigens and adjuvants that induce appropriate immune responses. We evaluated a second generation polyprotein antigen (Leish-110f) in different adjuvant formulations for immunogenicity and protective efficacy against Leishmania spp. challenges. Vaccine-induced protection was associated with antibody and T cell responses to Leish-110f. CD4 T cells were the source of IFN-gamma, TNF, and IL-2 double- and triple-positive populations. This study establishes the immunogenicity and protective efficacy of the improved Leish-110f subunit vaccine antigen adjuvanted with natural (MPL-SE) or synthetic (EM005) Toll-like receptor 4 agonists.

  18. Observations and Models of Galaxy Assembly Bias (United States)

    Campbell, Duncan A.


    The assembly history of dark matter haloes imparts various correlations between a halo’s physical properties and its large scale environment, i.e. assembly bias. It is common for models of the galaxy-halo connection to assume that galaxy properties are only a function of halo mass, implicitly ignoring how assembly bias may affect galaxies. Recently, programs to model and constrain the degree to which galaxy properties are influenced by assembly bias have been undertaken; however, the extent and character of galaxy assembly bias remains a mystery. Nevertheless, characterizing and modeling galaxy assembly bias is an important step in understanding galaxy evolution and limiting any systematic effects assembly bias may pose in cosmological measurements using galaxy surveys.I will present work on modeling and constraining the effect of assembly bias in two galaxy properties: stellar mass and star-formation rate. Conditional abundance matching allows for these galaxy properties to be tied to halo formation history to a variable degree, making studies of the relative strength of assembly bias possible. Galaxy-galaxy clustering and galactic conformity, the degree to which galaxy color is correlated between neighbors, are sensitive observational measures of galaxy assembly bias. I will show how these measurements can be used to constrain galaxy assembly bias and the peril of ignoring it.

  19. Assembly of MHC class I molecules within the endoplasmic reticulum. (United States)

    Zhang, Yinan; Williams, David B


    MHC class I molecules bind cytosolically derived peptides within the endoplasmic reticulum (ER) and present them at the cell surface to cytotoxic T cells. A major focus of our laboratory has been to understand the functions of the diverse proteins involved in the intracellular assembly of MHC class I molecules. These include the molecular chaperones calnexin and calreticulin, which enhance the proper folding and subunit assembly of class I molecules and also retain assembly intermediates within the ER; ERp57, a thiol oxidoreductase that promotes heavy chain disulfide formation and proper assembly of the peptide loading complex; tapasin, which recruits class I molecules to the TAP peptide transporter and enhances the loading of high affinity peptide ligands; and Bap31, which is involved in clustering assembled class I molecules at ER exit sites for export along the secretory pathway. This review describes our contributions to elucidating the functions of these proteins; the combined effort of many dedicated students and postdoctoral fellows.

  20. Silent S-Type Anion Channel Subunit SLAH1 Gates SLAH3 Open for Chloride Root-to-Shoot Translocation. (United States)

    Cubero-Font, Paloma; Maierhofer, Tobias; Jaslan, Justyna; Rosales, Miguel A; Espartero, Joaquín; Díaz-Rueda, Pablo; Müller, Heike M; Hürter, Anna-Lena; Al-Rasheid, Khaled A S; Marten, Irene; Hedrich, Rainer; Colmenero-Flores, José M; Geiger, Dietmar


    Higher plants take up nutrients via the roots and load them into xylem vessels for translocation to the shoot. After uptake, anions have to be channeled toward the root xylem vessels. Thereby, xylem parenchyma and pericycle cells control the anion composition of the root-shoot xylem sap [1-6]. The fact that salt-tolerant genotypes possess lower xylem-sap Cl(-) contents compared to salt-sensitive genotypes [7-10] indicates that membrane transport proteins at the sites of xylem loading contribute to plant salinity tolerance via selective chloride exclusion. However, the molecular mechanism of xylem loading that lies behind the balance between NO3(-) and Cl(-) loading remains largely unknown. Here we identify two root anion channels in Arabidopsis, SLAH1 and SLAH3, that control the shoot NO3(-)/Cl(-) ratio. The AtSLAH1 gene is expressed in the root xylem-pole pericycle, where it co-localizes with AtSLAH3. Under high soil salinity, AtSLAH1 expression markedly declined and the chloride content of the xylem sap in AtSLAH1 loss-of-function mutants was half of the wild-type level only. SLAH3 anion channels are not active per se but require extracellular nitrate and phosphorylation by calcium-dependent kinases (CPKs) [11-13]. When co-expressed in Xenopus oocytes, however, the electrically silent SLAH1 subunit gates SLAH3 open even in the absence of nitrate- and calcium-dependent kinases. Apparently, SLAH1/SLAH3 heteromerization facilitates SLAH3-mediated chloride efflux from pericycle cells into the root xylem vessels. Our results indicate that under salt stress, plants adjust the distribution of NO3(-) and Cl(-) between root and shoot via differential expression and assembly of SLAH1/SLAH3 anion channel subunits.

  1. Total Synthesis of Bryostatins. Development of Methodology for Atom-Economic and Stereoselective Synthesis of the C-ring Subunit (United States)

    Trost, Barry M.; Frontier, Alison J.; Thiel, Oliver R.; Yang, Hanbiao; Dong, Guangbin


    Bryostatins, a family of structurally complicated macrolides, exhibit an exceptional range of biological activities. The limited availability and structural complexity of these molecules makes development of an efficient total synthesis particularly important. This article describes our initial efforts towards the total synthesis of bryostatins, in which chemoselective and atom-economical methods for stereoselective assembly of the C-ring subunit were developed. A Pd-catalyzed tandem alkyne-alkyne coupling/6-endo-dig cyclization sequence was explored and successfully pursued in the synthesis of a dihydropyran ring system. Elaboration of this methodology ultimately led to a concise synthesis of the C-ring subunit of bryostatins. PMID:21793057

  2. Western blot analysis of BK channel β1-subunit expression should be interpreted cautiously when using commercially available antibodies. (United States)

    Bhattarai, Yogesh; Fernandes, Roxanne; Kadrofske, Mark M; Lockwood, Lizbeth R; Galligan, James J; Xu, Hui


    Large conductance Ca(2+)-activated K(+) (BK) channels consist of pore-forming α- and accessory β-subunits. There are four β-subunit subtypes (β1-β4), BK β1-subunit is specific for smooth muscle cells (SMC). Reduced BK β1-subunit expression is associated with SMC dysfunction in animal models of human disease, because downregulation of BK β1-subunit reduces channel activity and increases SMC contractility. Several anti-BK β1-subunit antibodies are commercially available; however, the specificity of most antibodies has not been tested or confirmed in the tissues from BK β1-subunit knockout (KO) mice. In this study, we tested the specificity and sensitivity of six commercially available antibodies from five manufacturers. We performed western blot analysis on BK β1-subunit enriched tissues (mesenteric arteries and colons) and non-SM tissue (cortex of kidney) from wild-type (WT) and BK β1-KO mice. We found that antibodies either detected protein bands of the appropriate molecular weight in tissues from both WT and BK β1-KO mice or failed to detect protein bands at the appropriate molecular weight in tissues from WT mice, suggesting that these antibodies may lack specificity for the BK β1-subunit. The absence of BK β1-subunit mRNA expression in arteries, colons, and kidneys from BK β1-KO mice was confirmed by RT-PCR analysis. We conclude that these commercially available antibodies might not be reliable tools for studying BK β1-subunit expression in murine tissues under the denaturing conditions that we have used. Data obtained using commercially available antibodies should be interpreted cautiously. Our studies underscore the importance of proper negative controls in western blot analyses. © 2014 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  3. Automated Solar-Array Assembly (United States)

    Soffa, A.; Bycer, M.


    Large arrays are rapidly assembled from individual solar cells by automated production line developed for NASA's Jet Propulsion Laboratory. Apparatus positions cells within array, attaches interconnection tabs, applies solder flux, and solders interconnections. Cells are placed in either straight or staggered configurations and may be connected either in series or in parallel. Are attached at rate of one every 5 seconds.

  4. Most neutralizing human monoclonal antibodies target novel epitopes requiring both Lassa virus glycoprotein subunits (United States)

    Robinson, James E.; Hastie, Kathryn M.; Cross, Robert W.; Yenni, Rachael E.; Elliott, Deborah H.; Rouelle, Julie A.; Kannadka, Chandrika B.; Smira, Ashley A.; Garry, Courtney E.; Bradley, Benjamin T.; Yu, Haini; Shaffer, Jeffrey G.; Boisen, Matt L.; Hartnett, Jessica N.; Zandonatti, Michelle A.; Rowland, Megan M.; Heinrich, Megan L.; Martínez-Sobrido, Luis; Cheng, Benson; de la Torre, Juan C.; Andersen, Kristian G.; Goba, Augustine; Momoh, Mambu; Fullah, Mohamed; Gbakie, Michael; Kanneh, Lansana; Koroma, Veronica J.; Fonnie, Richard; Jalloh, Simbirie C.; Kargbo, Brima; Vandi, Mohamed A.; Gbetuwa, Momoh; Ikponmwosa, Odia; Asogun, Danny A.; Okokhere, Peter O.; Follarin, Onikepe A.; Schieffelin, John S.; Pitts, Kelly R.; Geisbert, Joan B.; Kulakoski, Peter C.; Wilson, Russell B.; Happi, Christian T.; Sabeti, Pardis C.; Gevao, Sahr M.; Khan, S. Humarr; Grant, Donald S.; Geisbert, Thomas W.; Saphire, Erica Ollmann; Branco, Luis M.; Garry, Robert F.


    Lassa fever is a severe multisystem disease that often has haemorrhagic manifestations. The epitopes of the Lassa virus (LASV) surface glycoproteins recognized by naturally infected human hosts have not been identified or characterized. Here we have cloned 113 human monoclonal antibodies (mAbs) specific for LASV glycoproteins from memory B cells of Lassa fever survivors from West Africa. One-half bind the GP2 fusion subunit, one-fourth recognize the GP1 receptor-binding subunit and the remaining fourth are specific for the assembled glycoprotein complex, requiring both GP1 and GP2 subunits for recognition. Notably, of the 16 mAbs that neutralize LASV, 13 require the assembled glycoprotein complex for binding, while the remaining 3 require GP1 only. Compared with non-neutralizing mAbs, neutralizing mAbs have higher binding affinities and greater divergence from germline progenitors. Some mAbs potently neutralize all four LASV lineages. These insights from LASV human mAb characterization will guide strategies for immunotherapeutic development and vaccine design. PMID:27161536

  5. A computational investigation on the connection between dynamics properties of ribosomal proteins and ribosome assembly.

    Directory of Open Access Journals (Sweden)

    Brittany Burton

    Full Text Available Assembly of the ribosome from its protein and RNA constituents has been studied extensively over the past 50 years, and experimental evidence suggests that prokaryotic ribosomal proteins undergo conformational changes during assembly. However, to date, no studies have attempted to elucidate these conformational changes. The present work utilizes computational methods to analyze protein dynamics and to investigate the linkage between dynamics and binding of these proteins during the assembly of the ribosome. Ribosomal proteins are known to be positively charged and we find the percentage of positive residues in r-proteins to be about twice that of the average protein: Lys+Arg is 18.7% for E. coli and 21.2% for T. thermophilus. Also, positive residues constitute a large proportion of RNA contacting residues: 39% for E. coli and 46% for T. thermophilus. This affirms the known importance of charge-charge interactions in the assembly of the ribosome. We studied the dynamics of three primary proteins from E. coli and T. thermophilus 30S subunits that bind early in the assembly (S15, S17, and S20 with atomic molecular dynamic simulations, followed by a study of all r-proteins using elastic network models. Molecular dynamics simulations show that solvent-exposed proteins (S15 and S17 tend to adopt more stable solution conformations than an RNA-embedded protein (S20. We also find protein residues that contact the 16S rRNA are generally more mobile in comparison with the other residues. This is because there is a larger proportion of contacting residues located in flexible loop regions. By the use of elastic network models, which are computationally more efficient, we show that this trend holds for most of the 30S r-proteins.

  6. Organization of Mitochondrial Gene Expression in Two Distinct Ribosome-Containing Assemblies

    Directory of Open Access Journals (Sweden)

    Kirsten Kehrein


    Full Text Available Mitochondria contain their own genetic system that provides subunits of the complexes driving oxidative phosphorylation. A quarter of the mitochondrial proteome participates in gene expression, but how all these factors are orchestrated and spatially organized is currently unknown. Here, we established a method to purify and analyze native and intact complexes of mitochondrial ribosomes. Quantitative mass spectrometry revealed extensive interactions of ribosomes with factors involved in all the steps of posttranscriptional gene expression. These interactions result in large expressosome-like assemblies that we termed mitochondrial organization of gene expression (MIOREX complexes. Superresolution microscopy revealed that most MIOREX complexes are evenly distributed throughout the mitochondrial network, whereas a subset is present as nucleoid-MIOREX complexes that unite the whole spectrum of organellar gene expression. Our work therefore provides a conceptual framework for the spatial organization of mitochondrial protein synthesis that likely developed to facilitate gene expression in the organelle.

  7. Probe tip heating assembly (United States)

    Schmitz, Roger William; Oh, Yunje


    A heating assembly configured for use in mechanical testing at a scale of microns or less. The heating assembly includes a probe tip assembly configured for coupling with a transducer of the mechanical testing system. The probe tip assembly includes a probe tip heater system having a heating element, a probe tip coupled with the probe tip heater system, and a heater socket assembly. The heater socket assembly, in one example, includes a yoke and a heater interface that form a socket within the heater socket assembly. The probe tip heater system, coupled with the probe tip, is slidably received and clamped within the socket.

  8. In situ structure of trypanosomal ATP synthase dimer reveals a unique arrangement of catalytic subunits (United States)

    Mühleip, Alexander W.; Dewar, Caroline E.; Schnaufer, Achim; Kühlbrandt, Werner; Davies, Karen M.


    We used electron cryotomography and subtomogram averaging to determine the in situ structures of mitochondrial ATP synthase dimers from two organisms belonging to the phylum euglenozoa: Trypanosoma brucei, a lethal human parasite, and Euglena gracilis, a photosynthetic protist. At a resolution of 32.5 Å and 27.5 Å, respectively, the two structures clearly exhibit a noncanonical F1 head, in which the catalytic (αβ)3 assembly forms a triangular pyramid rather than the pseudo-sixfold ring arrangement typical of all other ATP synthases investigated so far. Fitting of known X-ray structures reveals that this unusual geometry results from a phylum-specific cleavage of the α subunit, in which the C-terminal αC fragments are displaced by ∼20 Å and rotated by ∼30° from their expected positions. In this location, the αC fragment is unable to form the conserved catalytic interface that was thought to be essential for ATP synthesis, and cannot convert γ-subunit rotation into the conformational changes implicit in rotary catalysis. The new arrangement of catalytic subunits suggests that the mechanism of ATP generation by rotary ATPases is less strictly conserved than has been generally assumed. The ATP synthases of these organisms present a unique model system for discerning the individual contributions of the α and β subunits to the fundamental process of ATP synthesis. PMID:28096380

  9. Genetic variations of glycinin subunit genes among cultivated and wild type soybean species

    Institute of Scientific and Technical Information of China (English)


    Glycinin is a predominant storage protein in most soybean accessions. It is a hexamer constituted by five major subunits, which can be classified into two groups. Group Ⅰ contains Gl, G2 and G3, and Group Ⅱ contains G4 and G5. The genes encoding these subunits have been designated from Gyl to Gy5, respectively. In the present study, Gyl genomic fragments were cloned from wild accessions of subgenera Glycine glycine, Glycine soja and a cultivar of Glycine max. Their sequences and the deduced amino acid sequences were compared. The residues critical for assembling of G1 subunits from the wild perennial accession were conservative. The Gy4 fragments were cloned from two wild perennial accessions and compared with that from subgenus Soja. The intron 3 of Gy4 had abundant variations between the subgenera G. Soja and G. Glycine as well as within the subgenus G. Glycine. Abundant variations existed in the disordered regions 3 and 4 of G4 subunits from two wild perennial accessions. The genomic organization of glycinin genes was analyzed in 19 accessions from subgenera Soja and Glycine. The hybridization patterns were identical among the accessions of subgenus Soja. On the contrary, abundant polymorphisms existed between the accessions from subgenus Glycine. These results indicated that glycinin genes have high degree of conservation within subgenus Soja but more variations within subgenus Glycine.

  10. Detailed analysis of the human mitochondrial contact site complex indicate a hierarchy of subunits. (United States)

    Ott, Christine; Dorsch, Eva; Fraunholz, Martin; Straub, Sebastian; Kozjak-Pavlovic, Vera


    Mitochondrial inner membrane folds into cristae, which significantly increase its surface and are important for mitochondrial function. The stability of cristae depends on the mitochondrial contact site (MICOS) complex. In human mitochondria, the inner membrane MICOS complex interacts with the outer membrane sorting and assembly machinery (SAM) complex, to form the mitochondrial intermembrane space bridging complex (MIB). We have created knockdown cell lines of most of the MICOS and MIB components and have used them to study the importance of the individual subunits for the cristae formation and complex stability. We show that the most important subunits of the MIB complex in human mitochondria are Mic60/Mitofilin, Mic19/CHCHD3 and an outer membrane component Sam50. We provide additional proof that ApoO indeed is a subunit of the MICOS and MIB complexes and propose the name Mic23 for this protein. According to our results, Mic25/CHCHD6, Mic27/ApoOL and Mic23/ApoO appear to be periphery subunits of the MICOS complex, because their depletion does not affect cristae morphology or stability of other components.

  11. Rcf1 mediates cytochrome oxidase assembly and respirasome formation, revealing heterogeneity of the enzyme complex. (United States)

    Vukotic, Milena; Oeljeklaus, Silke; Wiese, Sebastian; Vögtle, F Nora; Meisinger, Chris; Meyer, Helmut E; Zieseniss, Anke; Katschinski, Doerthe M; Jans, Daniel C; Jakobs, Stefan; Warscheid, Bettina; Rehling, Peter; Deckers, Markus


    The terminal enzyme of the mitochondrial respiratory chain, cytochrome oxidase, transfers electrons to molecular oxygen, generating water. Within the inner mitochondrial membrane, cytochrome oxidase assembles into supercomplexes, together with other respiratory chain complexes, forming so-called respirasomes. Little is known about how these higher oligomeric structures are attained. Here we report on Rcf1 and Rcf2 as cytochrome oxidase subunits in S. cerevisiae. While Rcf2 is specific to yeast, Rcf1 is a conserved subunit with two human orthologs, RCF1a and RCF1b. Rcf1 is required for growth in hypoxia and complex assembly of subunits Cox13 and Rcf2, as well as for the oligomerization of a subclass of cytochrome oxidase complexes into respirasomes. Our analyses reveal that the cytochrome oxidase of mitochondria displays intrinsic heterogeneity with regard to its subunit composition and that distinct forms of respirasomes can be formed by complex variants.

  12. Newnes electronics assembly handbook

    CERN Document Server

    Brindley, Keith


    Newnes Electronics Assembly Handbook: Techniques, Standards and Quality Assurance focuses on the aspects of electronic assembling. The handbook first looks at the printed circuit board (PCB). Base materials, basic mechanical properties, cleaning of assemblies, design, and PCB manufacturing processes are then explained. The text also discusses surface mounted assemblies and packaging of electromechanical assemblies, as well as the soldering process. Requirements for the soldering process; solderability and protective coatings; cleaning of PCBs; and mass solder/component reflow soldering are des

  13. Unraveling the complexity of mitochondrial complex I assembly: A dynamic process. (United States)

    Sánchez-Caballero, Laura; Guerrero-Castillo, Sergio; Nijtmans, Leo


    Mammalian complex I is composed of 44 different subunits and its assembly requires at least 13 specific assembly factors. Proper function of the mitochondrial respiratory chain enzyme is of crucial importance for cell survival due to its major participation in energy production and cell signaling. Complex I assembly depends on the coordination of several crucial processes that need to be tightly interconnected and orchestrated by a number of assembly factors. The understanding of complex I assembly evolved from simple sequential concept to the more sophisticated modular assembly model describing a convoluted process. According to this model, the different modules assemble independently and associate afterwards with each other to form the final enzyme. In this review, we aim to unravel the complexity of complex I assembly and provide the latest insights in this fundamental and fascinating process. This article is part of a Special Issue entitled Respiratory complex I, edited by Volker Zickermann and Ulrich Brandt.

  14. Structural and biochemical characterization of human PR70 in isolation and in complex with the scaffolding subunit of protein phosphatase 2A.

    Directory of Open Access Journals (Sweden)

    Rebecca Dovega

    Full Text Available Protein Phosphatase 2A (PP2A is a major Ser/Thr phosphatase involved in the regulation of various cellular processes. PP2A assembles into diverse trimeric holoenzymes, which consist of a scaffolding (A subunit, a catalytic (C subunit and various regulatory (B subunits. Here we report a 2.0 Å crystal structure of the free B''/PR70 subunit and a SAXS model of an A/PR70 complex. The crystal structure of B''/PR70 reveals a two domain elongated structure with two Ca2+ binding EF-hands. Furthermore, we have characterized the interaction of both binding partner and their calcium dependency using biophysical techniques. Ca2+ biophysical studies with Circular Dichroism showed that the two EF-hands display different affinities to Ca2+. In the absence of the catalytic C-subunit, the scaffolding A-subunit remains highly mobile and flexible even in the presence of the B''/PR70 subunit as judged by SAXS. Isothermal Titration Calorimetry studies and SAXS data support that PR70 and the A-subunit have high affinity to each other. This study provides additional knowledge about the structural basis for the function of B'' containing holoenzymes.

  15. Human COX20 cooperates with SCO1 and SCO2 to mature COX2 and promote the assembly of cytochrome c oxidase. (United States)

    Bourens, Myriam; Boulet, Aren; Leary, Scot C; Barrientos, Antoni


    Cytochrome c oxidase (CIV) deficiency is one of the most common respiratory chain defects in patients presenting with mitochondrial encephalocardiomyopathies. CIV biogenesis is complicated by the dual genetic origin of its structural subunits, and assembly of a functional holoenzyme complex requires a large number of nucleus-encoded assembly factors. In general, the functions of these assembly factors remain poorly understood, and mechanistic investigations of human CIV biogenesis have been limited by the availability of model cell lines. Here, we have used small interference RNA and transcription activator-like effector nucleases (TALENs) technology to create knockdown and knockout human cell lines, respectively, to study the function of the CIV assembly factor COX20 (FAM36A). These cell lines exhibit a severe, isolated CIV deficiency due to instability of COX2, a mitochondrion-encoded CIV subunit. Mitochondria lacking COX20 accumulate CIV subassemblies containing COX1 and COX4, similar to those detected in fibroblasts from patients carrying mutations in the COX2 copper chaperones SCO1 and SCO2. These results imply that in the absence of COX20, COX2 is inefficiently incorporated into early CIV subassemblies. Immunoprecipitation assays using a stable COX20 knockout cell line expressing functional COX20-FLAG allowed us to identify an interaction between COX20 and newly synthesized COX2. Additionally, we show that SCO1 and SCO2 act on COX20-bound COX2. We propose that COX20 acts as a chaperone in the early steps of COX2 maturation, stabilizing the newly synthesized protein and presenting COX2 to its metallochaperone module, which in turn facilitates the incorporation of mature COX2 into the CIV assembly line.

  16. N-linked glycosylation of a subunit isoforms is critical for vertebrate vacuolar H(+) -ATPase (V-ATPase) biosynthesis. (United States)

    Esmail, Sally; Kartner, Norbert; Yao, Yeqi; Kim, Joo Wan; Reithmeier, Reinhart A F; Manolson, Morris F


    The a subunit of the V0 membrane-integrated sector of human V-ATPase has four isoforms, a1-a4, with diverse and crucial functions in health and disease. They are encoded by four conserved paralogous genes, and their vertebrate orthologs have positionally conserved N-glycosylation sequons within the second extracellular loop, EL2, of the a subunit membrane domain. Previously, we have shown directly that the predicted sequon for the a4 isoform is indeed N-glycosylated. Here we extend our investigation to the other isoforms by transiently transfecting HEK 293 cells to express cDNA constructs of epitope-tagged human a1-a3 subunits, with or without mutations that convert Asn to Gln at putative N-glycosylation sites. Expression and N-glycosylation were characterized by immunoblotting and mobility shifts after enzymatic deglycosylation, and intracellular localization was determined using immunofluorescence microscopy. All unglycosylated mutants, where predicted N-glycosylation sites had been eliminated by sequon mutagenesis, showed increased relative mobility on immunoblots, identical to what was seen for wild-type a subunits after enzymatic deglycosylation. Cycloheximide-chase experiments showed that unglycosylated subunits were turned over at a higher rate than N-glycosylated forms by degradation in the proteasomal pathway. Immunofluorescence colocalization analysis showed that unglycosylated a subunits were retained in the ER, and co-immunoprecipitation studies showed that they were unable to associate with the V-ATPase assembly chaperone, VMA21. Taken together with our previous a4 subunit studies, these observations show that N-glycosylation is crucial in all four human V-ATPase a subunit isoforms for protein stability and ultimately for functional incorporation into V-ATPase complexes. © 2017 Wiley Periodicals, Inc.

  17. Thermodynamic and structural analysis of microtubule assembly: the role of GTP hydrolysis. (United States)

    Vulevic, B; Correia, J J


    that assembly with GTP/2 M glycerol and with taxol is consistent with conformational rearrangements in 3-6% of the total amino acids in the heterodimer. In addition, taxol binding contributes to the thermodynamics of the overall process by reducing the delta H degree and delta S degree for microtubule assembly. In the presence of GMPCPP or GMPCP, tubulin subunits associate with extensive conformational rearrangement, corresponding to 10% and 26% of the total amino acids in the heterodimer, respectively, which gives rise to a large loss of configurational entropy. An alternative, and probably preferable, interpretation of these data is that, especially with GMPCP-tubulin, additional isomerization or protonation events are induced by the presence of the methylene moiety and linked to microtubule assembly. Structural analysis shows that GTP hydrolysis is not required for sheet closure into a microtubule cylinder, but only increases the probability of this event occurring. Sheet extensions and sheet polymers appear to have a similar average length under various conditions, suggesting that the minimum cooperative unit for closure of sheets into a microtubule cylinder is approximately 400 nm long. Because of their low level of occurrence, sheets are not expected to significantly affect the thermodynamics of assembly.

  18. Architects at the bacterial surface - sortases and the assembly of pili with isopeptide bonds. (United States)

    Hendrickx, Antoni P A; Budzik, Jonathan M; Oh, So-Young; Schneewind, Olaf


    The cell wall envelope of Gram-positive bacteria can be thought of as a surface organelle for the assembly of macromolecular structures that enable the unique lifestyle of each microorganism. Sortases - enzymes that cleave the sorting signals of secreted proteins to form isopeptide (amide) bonds between the secreted proteins and peptidoglycan or polypeptides - function as the principal architects of the bacterial surface. Acting alone or with other sortase enzymes, sortase construction leads to the anchoring of surface proteins at specific sites in the envelope or to the assembly of pili, which are fibrous structures formed from many protein subunits. The catalysis of intermolecular isopeptide bonds between pilin subunits is intertwined with the assembly of intramolecular isopeptide bonds within pilin subunits. Together, these isopeptide bonds endow these sortase products with adhesive properties and resistance to host proteases.

  19. Structural Assembly Demonstration Experiment (SADE) experiment design (United States)

    Akin, D. L.; Bowden, M. L.


    The Structural Assembly Demonstration Experiment concept is to erect a hybrid deployed/assembled structure as an early space experiment in large space structures technology. The basic objectives can be broken down into three generic areas: (1) by performing assembly tasks both in space and in neutral buoyancy simulation, a mathematical basis will be found for the validity conditions of neutral buoyancy, thus enhancing the utility of water as a medium for simulation of weightlessness; (2) a data base will be established describing the capabilities and limitations of EVA crewmembers, including effects of such things as hardware size and crew restraints; and (3) experience of the M.I.T. Space Systems Lab in neutral buoyancy simulation of large space structures assembly indicates that the assembly procedure may create the largest loads that a structure will experience during its lifetime. Data obtained from the experiment will help establish an accurate loading model to aid designers of future space structures.

  20. Genetic exchange of the S2 and S3 subunits in pertussis toxin. (United States)

    Raze, Dominique; Veithen, Alex; Sato, Hiroko; Antoine, Rudy; Menozzi, Franco D; Locht, Camille


    Bordetella pertussis, the causative agent of whooping cough, produces a complex hetero-oligomeric exotoxin, named pertussis toxin (PTX), which is responsible for several of the clinical manifestations associated with whooping cough. The toxin is composed of five dissimilar subunits, named S1 through S5 and arranged in a hexameric structure with a 1S1:1S2:1S3:2S4:1S5 stoichiometry. Although S2 and S3 share 70% amino acid identity, these two subunits were previously thought not to be able to substitute for each other in toxin assembly/secretion and the biological activities of PTX. Here, we show that toxin analogues containing two S3 subunits and lacking S2 (PTXdeltaS2), or containing two S2 subunits and lacking S3 (PTXdeltaS3), can be produced, assembled and secreted by B. pertussis strains, in which the S2-encoding cistron or the S3-coding cistrons have been inactivated by internal in-frame deletions that avoid downstream effects. In fact, PTXdeltaS3 was produced in higher amounts in the bacterial culture supernatants than natural PTX, whereas PTXdeltaS2 was produced in lower amounts than PTX. The action of the toxin analogues on the clustering of Chinese Hamster Ovary cells was also affected differentially by the S2-S3 substitution. These toxin analogues constitute thus interesting probes for the study of cellular functions, in particular immune cell functions, for which natural PTX has already shown its usefulness.

  1. Mutation in mitochondrial complex IV subunit COX5A causes pulmonary arterial hypertension, lactic acidemia, and failure to thrive. (United States)

    Baertling, Fabian; Al-Murshedi, Fathiya; Sánchez-Caballero, Laura; Al-Senaidi, Khalfan; Joshi, Niranjan P; Venselaar, Hanka; van den Brand, Mariël Am; Nijtmans, Leo Gj; Rodenburg, Richard Jt


    COX5A is a nuclear-encoded subunit of mitochondrial respiratory chain complex IV (cytochrome c oxidase). We present patients with a homozygous pathogenic variant in the COX5A gene. Clinical details of two affected siblings suffering from early-onset pulmonary arterial hypertension, lactic acidemia, failure to thrive, and isolated complex IV deficiency are presented. We show that the variant lies within the evolutionarily conserved COX5A/COX4 interface domain, suggesting that it alters the interaction between these two subunits during complex IV biogenesis. In patient skin fibroblasts, the enzymatic activity and protein levels of complex IV and several of its subunits are reduced. Lentiviral complementation rescues complex IV deficiency. The monomeric COX1 assembly intermediate accumulates demonstrating a function of COX5A in complex IV biogenesis. A potential therapeutic lead is demonstrated by showing that copper supplementation leads to partial rescue of complex IV deficiency in patient fibroblasts.

  2. Complex control of GABA(A receptor subunit mRNA expression: variation, covariation, and genetic regulation.

    Directory of Open Access Journals (Sweden)

    Megan K Mulligan

    Full Text Available GABA type-A receptors are essential for fast inhibitory neurotransmission and are critical in brain function. Surprisingly, expression of receptor subunits is highly variable among individuals, but the cause and impact of this fluctuation remains unknown. We have studied sources of variation for all 19 receptor subunits using massive expression data sets collected across multiple brain regions and platforms in mice and humans. Expression of Gabra1, Gabra2, Gabrb2, Gabrb3, and Gabrg2 is highly variable and heritable among the large cohort of BXD strains derived from crosses of fully sequenced parents--C57BL/6J and DBA/2J. Genetic control of these subunits is complex and highly dependent on tissue and mRNA region. Remarkably, this high variation is generally not linked to phenotypic differences. The single exception is Gabrb3, a locus that is linked to anxiety. We identified upstream genetic loci that influence subunit expression, including three unlinked regions of chromosome 5 that modulate the expression of nine subunits in hippocampus, and that are also associated with multiple phenotypes. Candidate genes within these loci include, Naaa, Nos1, and Zkscan1. We confirmed a high level of coexpression for subunits comprising the major channel--Gabra1, Gabrb2, and Gabrg2--and identified conserved members of this expression network in mice and humans. Gucy1a3, Gucy1b3, and Lis1 are novel and conserved associates of multiple subunits that are involved in inhibitory signaling. Finally, proximal and distal regions of the 3' UTRs of single subunits have remarkably independent expression patterns in both species. However, corresponding regions of different subunits often show congruent genetic control and coexpression (proximal-to-proximal or distal-to-distal, even in the absence of sequence homology. Our findings identify novel sources of variation that modulate subunit expression and highlight the extraordinary capacity of biological networks to buffer

  3. PetG and PetN, but not PetL, are essential subunits of the cytochrome b6f complex from Synechocystis PCC 6803. (United States)

    Schneider, Dirk; Volkmer, Thomas; Rögner, Matthias


    The cytochrome b(6)f complex consists of four large core subunits and an additional four low molecular weight subunits, the function of which is elusive thus far. Here we sought to determine whether small subunits PetG, PetL, and PetN are essential for a cyanobacterial cytochrome b(6)f complex. We found that only PetL is dispensable, whereas PetG and PetN appear to be essential. Possible roles of the small cytochrome b(6)f complex subunits are discussed, and observations from our study are compared with previous findings.

  4. Folding, stability, and physical properties of the alpha subunit of bacterial luciferase. (United States)

    Noland, B W; Dangott, L J; Baldwin, T O


    Bacterial luciferase is a heterodimeric (alphabeta) enzyme composed of homologous subunits. When the Vibrio harveyi luxA gene is expressed in Escherichia coli, the alpha subunit accumulates to high levels. The alpha subunit has a well-defined near-UV circular dichroism spectrum and a higher intrinsic fluorescence than the heterodimer, demonstrating fluorescence quenching in the enzyme which is reduced in the free subunit [Sinclair, J. F., Waddle, J. J., Waddill, W. F., and Baldwin, T. O. (1993) Biochemistry 32, 5036-5044]. Analytical ultracentrifugation of the alpha subunit has revealed a reversible monomer to dimer equilibrium with a dissociation constant of 14.9 +/- 4.0 microM at 18 degrees C in 50 mM phosphate and 100 mM NaCl, pH 7.0. The alpha subunit unfolded and refolded reversibly in urea-containing buffers by a three-state mechanism. The first transition occurred over the range of 0-2 M urea with an associated free-energy change of 2.24 +/- 0.25 kcal/mol at 18 degrees C in 50 mM phosphate buffer, pH 7.0. The second, occurring between 2.5 and 3.5 M urea, comprised a cooperative transition with a free-energy change of 6.50 +/- 0.75 kcal/mol. The intermediate species, populated maximally at ca. 2 M urea, has defined near-UV circular dichroism spectral properties distinct from either the native or the denatured states. The intrinsic fluorescence of the intermediate suggested that, although the quantum yield had decreased, the tryptophanyl residues remained largely buried. The far-UV circular dichroism spectrum of the intermediate indicated that it had lost ca. 40% of its native secondary structure. N-Terminal sequencing of the products of limited proteolysis of the intermediate showed that the C-terminal region of the alpha subunit became protease labile over the urea concentration range at which the intermediate was maximally populated. These observations have led us to propose an unfolding model in which the first transition is the unfolding of a C

  5. Autonomous electrochromic assembly

    Energy Technology Data Exchange (ETDEWEB)

    Berland, Brian Spencer; Lanning, Bruce Roy; Stowell, Jr., Michael Wayne


    This disclosure describes system and methods for creating an autonomous electrochromic assembly, and systems and methods for use of the autonomous electrochromic assembly in combination with a window. Embodiments described herein include an electrochromic assembly that has an electrochromic device, an energy storage device, an energy collection device, and an electrochromic controller device. These devices may be combined into a unitary electrochromic insert assembly. The electrochromic assembly may have the capability of generating power sufficient to operate and control an electrochromic device. This control may occur through the application of a voltage to an electrochromic device to change its opacity state. The electrochromic assembly may be used in combination with a window.

  6. PKA regulatory subunit expression in tooth development. (United States)

    de Sousa, Sílvia Ferreira; Kawasaki, Katsushige; Kawasaki, Maiko; Volponi, Ana Angelova; Gomez, Ricardo Santiago; Gomes, Carolina Cavaliéri; Sharpe, Paul T; Ohazama, Atsushi


    Protein kinase A (PKA) plays critical roles in many biological processes including cell proliferation, cell differentiation, cellular metabolism and gene regulation. Mutation in PKA regulatory subunit, PRKAR1A has previously been identified in odontogenic myxomas, but it is unclear whether PKA is involved in tooth development. The aim of the present study was to assess the expression of alpha isoforms of PKA regulatory subunit (Prkar1a and Prkar2a) in mouse and human odontogenesis by in situ hybridization. PRKAR1A and PRKAR2A mRNA transcription was further confirmed in a human deciduous germ by qRT-PCR. Mouse Prkar1a and human PRKAR2A exhibited a dynamic spatio-temporal expression in tooth development, whereas neither human PRKAR1A nor mouse Prkar2a showed their expression in odontogenesis. These isoforms thus showed different expression pattern between human and mouse tooth germs. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. A model for the interaction of the G3-subdomain of Geobacillus stearothermophilus IF2 with the 30S ribosomal subunit

    NARCIS (Netherlands)

    Dongre, Ramachandra; Folkers, Gert E; Gualerzi, Claudio O; Boelens, Rolf; Wienk, Hans


    Bacterial translation initiation factor IF2 complexed with GTP binds to the 30S ribosomal subunit, promotes ribosomal binding of fMet-tRNA, and favors the joining of the small and large ribosomal subunits yielding a 70S initiation complex ready to enter the translation elongation phase. Within the I

  8. Versatile microrobotics using simple modular subunits (United States)

    Cheang, U. Kei; Meshkati, Farshad; Kim, Hoyeon; Lee, Kyoungwoo; Fu, Henry Chien; Kim, Min Jun


    The realization of reconfigurable modular microrobots could aid drug delivery and microsurgery by allowing a single system to navigate diverse environments and perform multiple tasks. So far, microrobotic systems are limited by insufficient versatility; for instance, helical shapes commonly used for magnetic swimmers cannot effectively assemble and disassemble into different size and shapes. Here by using microswimmers with simple geometries constructed of spherical particles, we show how magnetohydrodynamics can be used to assemble and disassemble modular microrobots with different physical characteristics. We develop a mechanistic physical model that we use to improve assembly strategies. Furthermore, we experimentally demonstrate the feasibility of dynamically changing the physical properties of microswimmers through assembly and disassembly in a controlled fluidic environment. Finally, we show that different configurations have different swimming properties by examining swimming speed dependence on configuration size.

  9. Versatile microrobotics using simple modular subunits. (United States)

    Cheang, U Kei; Meshkati, Farshad; Kim, Hoyeon; Lee, Kyoungwoo; Fu, Henry Chien; Kim, Min Jun


    The realization of reconfigurable modular microrobots could aid drug delivery and microsurgery by allowing a single system to navigate diverse environments and perform multiple tasks. So far, microrobotic systems are limited by insufficient versatility; for instance, helical shapes commonly used for magnetic swimmers cannot effectively assemble and disassemble into different size and shapes. Here by using microswimmers with simple geometries constructed of spherical particles, we show how magnetohydrodynamics can be used to assemble and disassemble modular microrobots with different physical characteristics. We develop a mechanistic physical model that we use to improve assembly strategies. Furthermore, we experimentally demonstrate the feasibility of dynamically changing the physical properties of microswimmers through assembly and disassembly in a controlled fluidic environment. Finally, we show that different configurations have different swimming properties by examining swimming speed dependence on configuration size.

  10. Self-assembly of [B-SbW9O33]9- subunit with transition metal ions (Mn2+, Cu2+, Co2+) in aqueous solution: syntheses, structures and magnetic properties of sandwich type polyoxometalates with Subvalent Sb(III) heteroatom. (United States)

    Wang, Jing-Ping; Ma, Peng-Tao; Li, Jie; Niu, Hong-Yu; Niu, Jing-Yang


    Rational self-assembly of Sb(2)O(3) and Na(2)WO(4), or (NH(4))(18)[NaSb(9)W(21)O(86)] with transition-metal ions (Mn(2+), Cu(2+), Co(2+)), in aqueous solution under controlled conditions yield a series of sandwich type complexes, namely, Na(2)H(2)[Mn(2.5)W(1.5)(H(2)O)(8)(B-beta-SbW(9)O(33))(2)]32 H(2)O (1), Na(4)H(7)[Na(3)(H(2)O)(6)Mn(3)(mu-OAc)(2)(B-alpha-SbW(9)O(33))(2)]20 H(2)O (OAc=acetate anion) (2), NaH(8)[Na(2)Cu(4)Cl(B-alpha-SbW(9)O(33))(2)]21 H(2)O (3), Na(8)K[Na(2)K(H(2)O)(2){Co(H(2)O)}(3)(B-alpha-SbW(9)O(33))(2)] 10 H(2)O (4), and Na(5)H[{Co(H(2)O)(2)}(3)W(H(2)O)(2)(B-beta-SbW(9)O(33))(2)]11.5 H(2)O (5). These structures are determined by using the X-ray diffraction technique and further characterized by obtaining IR spectra and performing elemental analysis. Structure analysis reveals that polyoxoanions in 1 and 5 comprise of two [B-beta-SbW(9)O(33)](9-) building units, whereas 2, 3, and 4 consist of two isomerous [B-alpha-SbW(9)O(33)](9-) building blocks, which are all linked by different transition-metal ions (Mn(2+), Cu(2+), or Co(2+)) with different quantitative nuclearity. It should be noted that compound 2 represents the first one-dimensional sinusoidal chain based on sandwich like tungstoantimonate building blocks through the carboxylate-bridging ligands. Additionally, 3 is constructed from sandwiched anions [Na(2)Cu(4)Cl(B-alpha-SbW(9)O(33))(2)](9-) linked to each other to form an infinitely extended 2D network, whereas 5 shows an interesting 3D framework built up from offset sandwich type polyoxoanion [{Co(H(2)O)(2)}(3)W(H(2)O)(2)(B-beta-SbW(9)O(33))(2)](6-) linked by Co(2+) and Na(+) ions. EPR studies performed at 110 K and room temperature reveal that the metal cations (Mn(2+), Cu(2+), Co(2+)) reside in a square-pyramidal geometry in 2, 3, and 4. The magnetic behavior of 1-4 suggests the presence of weak antiferromagnetic coupling interactions between magnetic metal centers with the exchange integral J=-0.552 cm(-1) in 2.

  11. The Brain-Specific Beta4 Subunit Downregulates BK Channel Cell Surface Expression


    Sonal Shruti; Joanna Urban-Ciecko; Fitzpatrick, James A.; Robert Brenner; Bruchez, Marcel P.; Alison L Barth


    The large-conductance K(+) channel (BK channel) can control neural excitability, and enhanced channel currents facilitate high firing rates in cortical neurons. The brain-specific auxiliary subunit β4 alters channel Ca(++)- and voltage-sensitivity, and β4 knock-out animals exhibit spontaneous seizures. Here we investigate β4's effect on BK channel trafficking to the plasma membrane. Using a novel genetic tag to track the cellular location of the pore-forming BKα subunit in living cells, we fi...

  12. Characteristics of an R-phycoerythrin with two γ subunits prepared from red macroalga Polysiphonia urceolata.

    Directory of Open Access Journals (Sweden)

    Lu Wang

    Full Text Available An R-phycoerythrin (R-PE was isolated by gel filtrations on Sepharose CL-4B and Sephadex G-150 from the phycobiliprotein extract of the marine red macroalga Polysiphonia urceolata Grev and further purified by ion exchange chromatography on DEAE-Sepharose Fast Flow. The purified R-PE showed three absorption peaks at 498 nm, 538 nm, 566 nm and one fluorescent emission maximum at 577 nm. Although the R-PE showed a single band on the examination by native PAGE, it exhibited two very close bands at pH about 4.7 in native isoelectric focusing (IEF. Polypeptide analysis of the R-PE demonstrated that it contained four chromophore-carrying subunits, α18.2, β20.6, γ31.6 (γ', γ34.6 (γ, and no colorless polypeptide; its subunit composition was 6α18.2:6β20.6:1 γ31.6:2γ34.6. The α and β subunits were distributed within a acidic pH range from 5.0 to 6.0 in denaturing IEF and the γ subunits were in a basic pH range from 7.6 to 8.1. These results reveal that the prepared R-PE may exist in two hexamers of γ (αβ3 γ (αβ3γ' and γ (αβ3 γ'(αβ3 γ and that the R-PE participate in the rod domain assembly of P. urceolata phycobilisomes by stacking each of its trimer (αβ3 face-to-face with the aid of one γ subunit (γ or γ'.

  13. Switch in glutamate receptor subunit gene expression in CA1 subfield of hippocampus following global ischemia in rats.


    Pellegrini-Giampietro, D E; Zukin, R.S.; Bennett, M V; Cho, S; Pulsinelli, W. A.


    Severe, transient global ischemia of the brain induces delayed damage to specific neuronal populations. Sustained Ca2+ influx through glutamate receptor channels is thought to play a critical role in postischemic cell death. Although most kainate-type glutamate receptors are Ca(2+)-impermeable, Ca(2+)-permeable kainate receptors have been reported in specific kinds of neurons and glia. Recombinant receptors assembled from GluR1 and/or GluR3 subunits in exogenous expression systems are permeab...

  14. Sequence analysis of β-subunit genes of the 20S proteasome in patients with relapsed multiple myeloma treated with bortezomib or dexamethasone

    NARCIS (Netherlands)

    D.I. Lichter (David); H. Danaee (Hadi); M.D. Pickard (Michael); O. Tayber (Olga); M. Sintchak (Michael); H. Shi (Hongliang); P.G. Richardson (Paul Gerard); J. Cavenagh (Jamie); J. Bladé (Joan); T. Facon (Thierry); R. Niesvizky; M. Alsina (Melissa); W. Dalton (William); P. Sonneveld (Pieter); S. Lonial (Sagar); H. van de Velde (Helgi); D. Ricci (Deborah); D.-L. Esseltine (Dixie-Lee); W.L. Trepicchio (William); G. Mulligan (George); K.C. Anderson (Kenneth Carl)


    textabstractVariations within proteasome β (PSMB) genes, which encode the β subunits of the 20S proteasome, may affect proteasome function, assembly, and/or binding of proteasome inhibitors. To investigate the potential association between PSMB gene variants and treatment-emergent resistance to bort

  15. Selective assembly of laminin variants by human carcinoma cells

    DEFF Research Database (Denmark)

    Wewer, U M; Wayner, E A; Hoffstrom, B G


    basement membranes, the pattern of production of various laminin subunits remains to be explored. EXPERIMENTAL DESIGN: The expression of laminin was examined in several human carcinoma cells using a panel of specific cDNA probes as well as polyclonal and chain specific monoclonal antibodies......BACKGROUND: The laminins are heterotrimeric basement membrane glycoproteins. Eight subunits that can be assembled into laminins have been characterized and are known as: A, B1, B2, S, M, K, B2t, B1k laminin chains. Although many neoplastic cells secrete laminins and some of them even assemble....... For this purpose a human laminin S chain 2 kb cDNA was isolated and characterized and used together with existing probes for laminin chains. RESULTS: All carcinoma cell lines had a high level of expression of three light chains (B1, S and B2) mRNA. In contrast, the heavy chains of laminin, A and M, were expressed...

  16. Na+ channel β subunits: Overachievers of the ion channel family

    Directory of Open Access Journals (Sweden)

    William J Brackenbury


    Full Text Available Voltage gated Na+ channels (VGSCs in mammals contain a pore-forming α subunit and one or more β subunits. There are five mammalian β subunits in total: β1, β1B, β2, β3, and β4, encoded by four genes: SCN1B-SCN4B. With the exception of the SCN1B splice variant, β1B, the β subunits are type I topology transmembrane proteins. In contrast, β1B lacks a transmembrane domain and is a secreted protein. A growing body of work shows that VGSC β subunits are multifunctional. While they do not form the ion channel pore, β subunits alter gating, voltage-dependence, and kinetics of VGSC α subunits and thus regulate cellular excitability in vivo. In addition to their roles in channel modulation, β subunits are members of the immunoglobulin (Ig superfamily of cell adhesion molecules (CAMs and regulate cell adhesion and migration. β subunits are also substrates for sequential proteolytic cleavage by secretases. An example of the multifunctional nature of β subunits is β1, encoded by SCN1B, that plays a critical role in neuronal migration and pathfinding during brain development, and whose function is dependent on Na+ current and γ-secretase activity. Functional deletion of SCN1B results in Dravet Syndrome, a severe and intractable pediatric epileptic encephalopathy. β subunits are emerging as key players in a wide variety of pathophysiologies, including epilepsy, cardiac arrhythmia, multiple sclerosis, Huntington’s disease, neuropsychiatric disorders, neuropathic and inflammatory pain, and cancer. β subunits mediate multiple signaling pathways on different timescales, regulating electrical excitability, adhesion, migration, pathfinding, and transcription. Importantly, some β subunit functions may operate independent of α subunits. Thus, β subunits perform critical roles during development and disease. As such, they may prove useful in disease diagnosis and therapy.

  17. First inactive conformation of CK2 alpha, the catalytic subunit of protein kinase CK2

    DEFF Research Database (Denmark)

    Raaf, Jennifer; Issinger, Olaf-Georg; Niefind, Karsten


    (EPKs). To function as regulatory key components, EPKs normally exist in inactive ground states and are activated only upon specific signals. Typically, this activation is accompanied by large conformational changes in helix alpha C and in the activation segment, leading to a characteristic arrangement......The Ser/Thr kinase casein kinase 2 (CK2) is a heterotetrameric enzyme composed of two catalytic chains (CK2alpha, catalytic subunit of CK2) attached to a dimer of two noncatalytic subunits (CK2beta, noncatalytic subunit of CK2). CK2alpha belongs to the superfamily of eukaryotic protein kinases...... of catalytic key elements. For CK2alpha, however, no strict physiological control of activity is known. Accordingly, CK2alpha was found so far exclusively in the characteristic conformation of active EPKs, which is, in this case, additionally stabilized by a unique intramolecular contact between the N...

  18. In Vitro Reassembly of Tobacco Ribulose-1,5-bisphosphate Carboxylase/ Oxygenase from Fully Denatured Subunits

    Institute of Scientific and Technical Information of China (English)

    Zhen-Hua YONG; Gen-Yun CHEN; Jiao-Nai SHI; Da-Quan XU


    It has been generally proved impossible to reassemble ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from fully denatured subunits in vitro in higher plant, because large subunit of fully denatured Rubisco is liable to precipitate when the denaturant is removed by common methods of direct dilution and one-step dialysis. In our experiment, the problem of precipitation was resolved by an improved gradual dialysis method, which gradually decreased the concentration of denaturant. However, fully denatured Rubisco subunits still could not be reassembled into holoenzyme using gradual dialysis unless chaperonin 60was added. The restored activity of reassembled Rubisco was approximately 8% of natural enzyme. The quantity of reassembled Rubisco increased greatly when heat shock protein 70 was present in the reassembly process. ATP and Mg2+ were unnecessary for in vitro reassembly of Rubisco, and Mg2+ inhibited the reassembly process. The reassembly was weakened when ATP, Mg2+ and K+ existed together in the reassembly process.

  19. Structure, dynamics, assembly, and evolution of protein complexes. (United States)

    Marsh, Joseph A; Teichmann, Sarah A


    The assembly of individual proteins into functional complexes is fundamental to nearly all biological processes. In recent decades, many thousands of homomeric and heteromeric protein complex structures have been determined, greatly improving our understanding of the fundamental principles that control symmetric and asymmetric quaternary structure organization. Furthermore, our conception of protein complexes has moved beyond static representations to include dynamic aspects of quaternary structure, including conformational changes upon binding, multistep ordered assembly pathways, and structural fluctuations occurring within fully assembled complexes. Finally, major advances have been made in our understanding of protein complex evolution, both in reconstructing evolutionary histories of specific complexes and in elucidating general mechanisms that explain how quaternary structure tends to evolve. The evolution of quaternary structure occurs via changes in self-assembly state or through the gain or loss of protein subunits, and these processes can be driven by both adaptive and nonadaptive influences.

  20. Functional Analysis of a Wheat AGPase Plastidial Small Subunit with a Truncated Transit Peptide

    Directory of Open Access Journals (Sweden)

    Yang Yang


    Full Text Available ADP-glucose pyrophosphorylase (AGPase, the key enzyme in starch synthesis, consists of two small subunits and two large subunits with cytosolic and plastidial isoforms. In our previous study, a cDNA sequence encoding the plastidial small subunit (TaAGPS1b of AGPase in grains of bread wheat (Triticum aestivum L. was isolated and the protein subunit encoded by this gene was characterized as a truncated transit peptide (about 50% shorter than those of other plant AGPS1bs. In the present study, TaAGPS1b was fused with green fluorescent protein (GFP in rice protoplast cells, and confocal fluorescence microscopy observations revealed that like other AGPS1b containing the normal transit peptide, TaAGPS1b-GFP was localized in chloroplasts. TaAGPS1b was further overexpressed in a Chinese bread wheat cultivar, and the transgenic wheat lines exhibited a significant increase in endosperm AGPase activities, starch contents, and grain weights. These suggested that TaAGPS1b subunit was targeted into plastids by its truncated transit peptide and it could play an important role in starch synthesis in bread wheat grains.

  1. Missing Links in Antibody Assembly Control

    Directory of Open Access Journals (Sweden)

    Tiziana Anelli


    Full Text Available Fidelity of the humoral immune response requires that quiescent B lymphocytes display membrane bound immunoglobulin M (IgM on B lymphocytes surface as part of the B cell receptor, whose function is to recognize an antigen. At the same time B lymphocytes should not secrete IgM until recognition of the antigen has occurred. The heavy chains of the secretory IgM have a C-terminal tail with a cysteine instead of a membrane anchor, which serves to covalently link the IgM subunits by disulfide bonds to form “pentamers” or “hexamers.” By virtue of the same cysteine, unassembled secretory IgM subunits are recognized and retained (via mixed disulfide bonds by members of the protein disulfide isomerase family, in particular ERp44. This so-called “thiol-mediated retention” bars assembly intermediates from prematurely leaving the cell and thereby exerts quality control on the humoral immune response. In this essay we discuss recent findings on how ERp44 governs such assembly control in a pH-dependent manner, shuttling between the cisGolgi and endoplasmic reticulum, and finally on how pERp1/MZB1, possibly as a co-chaperone of GRP94, may help to overrule the thiol-mediated retention in the activated B cell to give way to antibody secretion.

  2. Polymer Directed Protein Assemblies

    NARCIS (Netherlands)

    van Rijn, Patrick


    Protein aggregation and protein self-assembly is an important occurrence in natural systems, and is in some form or other dictated by biopolymers. Very obvious influences of biopolymers on protein assemblies are, e. g., virus particles. Viruses are a multi-protein assembly of which the morphology is

  3. Polymer Directed Protein Assemblies

    NARCIS (Netherlands)

    van Rijn, Patrick

    Protein aggregation and protein self-assembly is an important occurrence in natural systems, and is in some form or other dictated by biopolymers. Very obvious influences of biopolymers on protein assemblies are, e. g., virus particles. Viruses are a multi-protein assembly of which the morphology is

  4. Influence of magnesium and polyamines on the reactivity of individual ribosomal subunit proteins to lactoperoxidase-catalyzed iodination. (United States)

    Michalski, C J; Boyle, S M; Sells, B H


    30S and 50S subunits, in the presence of either 20 mM Mg2+ or 6 mM Mg2+ and 5mM spermidine plus 25 mM putrescine, were observed to completely associate to form 70S monosomes as monitored by sucrose gradient sedimentation. Subunits maintained under the above ionic conditions were compared with 30S and 50S particles at low (6 mM) magnesium concentration with respect to the reactivity of individual ribosomal proteins to lactoperoxidase-catalyzed iodination. Altered reactivity to enzymatic iodination of ribosomal proteins S4, S9, S10, S14, S17, S19, and S20 in the small subunit of ribosomal proteins, L2, L9, L11, L27, and L30 in the large subunit following incubation with high magnesium or magnesium and polyamines suggests that a conformation change in both subunits accompanies the formation of 70S monosomes. The results further demonstrate that the effect of Mg2+ on subunit conformation is mimicked when polyamines are substituted for magnesium necessary for subunit association.

  5. Coi1 is a novel assembly factor of the yeast complex III-complex IV supercomplex. (United States)

    Singhal, Ravi K; Kruse, Christine; Heidler, Juliana; Strecker, Valentina; Zwicker, Klaus; Düsterwald, Lea; Westermann, Benedikt; Herrmann, Johannes M; Wittig, Ilka; Rapaport, Doron


    The yeast bc1 complex (complex III) and cytochrome oxidase (complex IV) are mosaics of core subunits encoded by the mitochondrial genome and additional nuclear-encoded proteins imported from the cytosol. Both complexes build in the mitochondrial inner membrane various supramolecular assemblies. The formation of the individual complexes and their supercomplexes depends on the activity of dedicated assembly factors. We identified a so far uncharacterized mitochondrial protein (open reading frame YDR381C-A) as an important assembly factor for complex III, complex IV, and their supercomplexes. Therefore, we named this protein Cox interacting (Coi) 1. Deletion of COI1 results in decreased respiratory growth, reduced membrane potential, and hampered respiration, as well as slow fermentative growth at low temperature. In addition, coi1Δ cells harbour reduced steady-state levels of subunits of complexes III and IV as well as of the assembled complexes and supercomplexes. Interaction of Coi1 with respiratory chain subunits seems transient, as it appears to be a stoichiometric subunit neither of complex III nor of complex IV. Collectively, this work identifies a novel protein that plays a role in the assembly of the mitochondrial respiratory chain. © 2017 by The American Society for Cell Biology.

  6. GABAA receptor β3 subunit expression regulates tonic current in developing striatopallidal medium spiny neurons

    Directory of Open Access Journals (Sweden)

    Megan eJanssen


    Full Text Available The striatum is a key structure for movement control, but the mechanisms that dictate the output of distinct subpopulations of medium spiny projection neurons (MSNs, striatonigral projecting and dopamine D1 receptor- (D1+ or striatopallidal projecting and dopamine D2 receptor- (D2+ expressing neurons, remains poorly understood. GABA-mediated tonic inhibition largely controls neuronal excitability and action potential firing rates, and we previously suggested with pharmacological analysis that the GABAA receptor β3 subunit plays a large role in the basal tonic current seen in D2+ MSNs from young mice (Ade et al, 2008; Janssen et al, 2009. In this study, we demonstrated the essential role of the β3 GABAA receptor subunit in mediating MSN tonic currents using conditional β3 subunit knock-out (β3f/fDrd2 mice. Cre-lox genetics were used to generate conditional knock-out animals where Cre recombinase was expressed under the D2 receptor (Drd2 promoter. We show that while the wild-type MSN tonic current pattern demonstrates a high degree of variability, tonic current patterns from β3f/fDrd2 mice are narrow, suggesting that the β3 subunit is essential to striatal MSN GABA-mediated tonic current. Our data also suggest that a distinct population of synaptic receptors upregulate due to β3 subunit removal. Further, deletion of this subunit significantly decreases the D2+ MSN excitability. These results offer insight for target mechanisms in Parkinson’s disease, where symptoms arise due to the imbalance in striatal D1+ and D2+ MSN excitability and output.

  7. The conserved Bud20 zinc finger protein is a new component of the ribosomal 60S subunit export machinery. (United States)

    Bassler, Jochen; Klein, Isabella; Schmidt, Claudia; Kallas, Martina; Thomson, Emma; Wagner, Maria Anna; Bradatsch, Bettina; Rechberger, Gerald; Strohmaier, Heimo; Hurt, Ed; Bergler, Helmut


    The nuclear export of the preribosomal 60S (pre-60S) subunit is coordinated with late steps in ribosome assembly. Here, we show that Bud20, a conserved C(2)H(2)-type zinc finger protein, is an unrecognized shuttling factor required for the efficient export of pre-60S subunits. Bud20 associates with late pre-60S particles in the nucleoplasm and accompanies them into the cytoplasm, where it is released through the action of the Drg1 AAA-ATPase. Cytoplasmic Bud20 is then reimported via a Kap123-dependent pathway. The deletion of Bud20 induces a strong pre-60S export defect and causes synthetic lethality when combined with mutant alleles of known pre-60S subunit export factors. The function of Bud20 in ribosome export depends on a short conserved N-terminal sequence, as we observed that mutations or the deletion of this motif impaired 60S subunit export and generated the genetic link to other pre-60S export factors. We suggest that the shuttling Bud20 is recruited to the nascent 60S subunit via its central zinc finger rRNA binding domain to facilitate the subsequent nuclear export of the preribosome employing its N-terminal extension.

  8. Sensor mount assemblies and sensor assemblies (United States)

    Miller, David H [Redondo Beach, CA


    Sensor mount assemblies and sensor assemblies are provided. In an embodiment, by way of example only, a sensor mount assembly includes a busbar, a main body, a backing surface, and a first finger. The busbar has a first end and a second end. The main body is overmolded onto the busbar. The backing surface extends radially outwardly relative to the main body. The first finger extends axially from the backing surface, and the first finger has a first end, a second end, and a tooth. The first end of the first finger is disposed on the backing surface, and the tooth is formed on the second end of the first finger.

  9. Prefoldin Subunits Are Protected from Ubiquitin-Proteasome System-mediated Degradation by Forming Complex with Other Constituent Subunits* (United States)

    Miyazawa, Makoto; Tashiro, Erika; Kitaura, Hirotake; Maita, Hiroshi; Suto, Hiroo; Iguchi-Ariga, Sanae M. M.; Ariga, Hiroyoshi


    The molecular chaperone prefoldin (PFD) is a complex comprised of six different subunits, PFD1-PFD6, and delivers newly synthesized unfolded proteins to cytosolic chaperonin TRiC/CCT to facilitate the folding of proteins. PFD subunits also have functions different from the function of the PFD complex. We previously identified MM-1α/PFD5 as a novel c-Myc-binding protein and found that MM-1α suppresses transformation activity of c-Myc. However, it remains unclear how cells regulate protein levels of individual subunits and what mechanisms alter the ratio of their activities between subunits and their complex. In this study, we found that knockdown of one subunit decreased protein levels of other subunits and that transfection of five subunits other than MM-1α into cells increased the level of endogenous MM-1α. We also found that treatment of cells with MG132, a proteasome inhibitor, increased the level of transfected/overexpressed MM-1α but not that of endogenous MM-1α, indicating that overexpressed MM-1α, but not endogenous MM-1α, was degraded by the ubiquitin proteasome system (UPS). Experiments using other PFD subunits showed that the UPS degraded a monomer of PFD subunits, though extents of degradation varied among subunits. Furthermore, the level of one subunit was increased after co-transfection with the respective subunit, indicating that there are specific combinations between subunits to be stabilized. These results suggest mutual regulation of protein levels among PFD subunits and show how individual subunits form the PFD complex without degradation. PMID:21478150

  10. Liposome-Based Adjuvants for Subunit Vaccines: Formulation Strategies for Subunit Antigens and Immunostimulators

    DEFF Research Database (Denmark)

    Schmidt, Signe Tandrup; Foged, Camilla; Korsholm, Karen Smith;


    for which no effective vaccines exist. The subunit vaccine technology exploits pathogen subunits as antigens, e.g., recombinant proteins or synthetic peptides, allowing for highly specific immune responses against the pathogens. However, such antigens are usually not sufficiently immunogenic to induce......The development of subunit vaccines has become very attractive in recent years due to their superior safety profiles as compared to traditional vaccines based on live attenuated or whole inactivated pathogens, and there is an unmet medical need for improved vaccines and vaccines against pathogens...... been licensed for use in human vaccines, and they mainly stimulate humoral immunity. Thus, there is an unmet demand for the development of safe and efficient adjuvant systems that can also stimulate cell-mediated immunity (CMI). Adjuvants constitute a heterogeneous group of compounds, which can broadly...

  11. Soldering in electronics assembly

    CERN Document Server

    Judd, Mike


    Soldering in Electronics Assembly discusses several concerns in soldering of electronic assemblies. The book is comprised of nine chapters that tackle different areas in electronic assembly soldering. Chapter 1 discusses the soldering process itself, while Chapter 2 covers the electronic assemblies. Chapter 3 talks about solders and Chapter 4 deals with flux. The text also tackles the CS and SC soldering process. The cleaning of soldered assemblies, solder quality, and standards and specifications are also discussed. The book will be of great use to professionals who deal with electronic assem

  12. The 20S proteasome as an assembly platform for the 19S regulatory complex

    DEFF Research Database (Denmark)

    Hendil, Klaus Aksel Bjørner; Kriegenburg, Franziska; Tanaka, Keiji


    26S proteasomes consist of cylindrical 20S proteasomes with 19S regulatory complexes attached to the ends. Treatment with high concentrations of salt causes the regulatory complexes to separate into two sub-complexes, the base, which is in contact with the 20S proteasome, and the lid, which...... is the distal part of the 19S complex. Here, we describe two assembly intermediates of the human regulatory complex. One is a dimer of the two ATPase subunits, Rpt3 and Rpt6. The other is a complex of nascent Rpn2, Rpn10, Rpn11, Rpn13, and Txnl1, attached to preexisting 20S proteasomes. This early assembly...... complex does not yet contain Rpn1 or any of the ATPase subunits of the base. Thus, assembly of 19S regulatory complexes takes place on preexisting 20S proteasomes, and part of the lid is assembled before the base....

  13. Clean Industrial Room for Drift Tube Assembling

    CERN Document Server

    Glonti, GL; Evtoukhovitch, P G; Kroa, G; Manz, A; Potrap, I N; Rihter, P; Stoletov, G D; Tskhadadze, E G; Chepurnov, V F; Chirkov, A V; Shelkov, G A


    Description of a clean industrial room for assembly of drift tubes for the muon spectrometer of the ATLAS experiment is presented. High quality specifications on the detectors to be produced demanded creation of a workplace with stable temperature and humidity, as well as minimum quantity of dust in the room. Checking of parameters of intra-room air during long period of continuous work has been confirmed correctness of the designed characteristics of the climatic system installed in the clean room. The room large volum (\\sim 190 m^3), the powerful and flexible climatic system, and simplicity of service allow assembling of detectors with length up to 5 m. Subsequent checking of functionality of the assembled detectors has shown high quality of assembling (the amount of rejected tubes does not exceed 2 %). It demonstrates conformity to the assembling quality requirements for mass production of drift chambers for the muon spectrometer.

  14. Vector assembly of colloids on monolayer substrates (United States)

    Jiang, Lingxiang; Yang, Shenyu; Tsang, Boyce; Tu, Mei; Granick, Steve


    The key to spontaneous and directed assembly is to encode the desired assembly information to building blocks in a programmable and efficient way. In computer graphics, raster graphics encodes images on a single-pixel level, conferring fine details at the expense of large file sizes, whereas vector graphics encrypts shape information into vectors that allow small file sizes and operational transformations. Here, we adapt this raster/vector concept to a 2D colloidal system and realize `vector assembly' by manipulating particles on a colloidal monolayer substrate with optical tweezers. In contrast to raster assembly that assigns optical tweezers to each particle, vector assembly requires a minimal number of optical tweezers that allow operations like chain elongation and shortening. This vector approach enables simple uniform particles to form a vast collection of colloidal arenes and colloidenes, the spontaneous dissociation of which is achieved with precision and stage-by-stage complexity by simply removing the optical tweezers.

  15. The intrinsic factor-vitamin B12 receptor, cubilin, is assembled into trimers via a coiled-coil alpha-helix. (United States)

    Lindblom, A; Quadt, N; Marsh, T; Aeschlimann, D; Mörgelin, M; Mann, K; Maurer, P; Paulsson, M


    A large protein was purified from bovine kidney, using selective extraction with EDTA to solubilize proteins anchored by divalent cation-dependent interactions. An antiserum raised against the purified protein labeled the apical cell surface of the epithelial cells in proximal tubules and the luminal surface of small intestine. Ten peptide sequences, derived from the protein, all matched the recently published sequences for rat (Moestrup, S. K., Kozyraki, R., Kristiansen, M., Kaysen, J. H., Holm Rasmussen, H., Brault, D., Pontillon, F., Goda, F. O., Christensen, E. I., Hammond, T. G., and Verroust, P. J. (1998) J. Biol. Chem. 273, 5235-5242) and human cubilin, a receptor for intrinsic factor-vitamin B12 complexes, identifying the protein as bovine cubilin. In electron microscopy, a three-armed structure was seen, indicating an oligomerization of three identical subunits. This model was supported by the Mr values of about 1,500,000 for the intact protein and 440,000 for its subunits obtained by analytical ultracentrifugation. In a search for a potential assembly domain, we identified a region of heptad repeats in the N-terminal part of the cubilin sequence. Computer-assisted analysis supported the presence of a coiled-coil alpha-helix between amino acids 103 and 132 of the human cubilin sequence and predicted the formation of a triple coiled-coil. We therefore conclude that cubilin forms a noncovalent trimer of identical subunits connected by an N-terminal coiled-coil alpha-helix.

  16. Composite fan stator assembly

    Energy Technology Data Exchange (ETDEWEB)

    Donges, G.L.


    A composite fan stator assembly is described for a gas turbine engine having at least two fan rotor stages, the composite stator assembly comprising: an annular composite fan case assembly including an access port, the fan case assembly circumferentially disposed around first and second fan rotor stage locations, a composite fan stator stage supported by and extending radially inward of the fan case assembly and axially disposed between the two fan rotor stage locations, the fan stator stage includes at least one removable vane segment accessible for removal through the access port for assembly and reassembly, the composite fan case assembly including a separable composite forward fan case assembly and a separable composite aft fan case assembly spaced axially aft of the forward fan case assembly, the forward fan case assembly being bolted to the aft fan case assembly, wherein the composite fan stator stage is axially and radially trapped and supported by the forward and aft fan case assemblies. A composite stator vane assembly comprising: a composite inner shroud, a composite outer shroud disposed radially outward of the inner shroud, a plurality of vanes disposed between the shrouds, the vanes including a suction side and a pressure side and radially inner and outer roots, the roots extending through platforms of corresponding ones of the inner and outer shrouds, four box-type attachment elements corresponding to curved suction and pressure sides of the inner and outer roots, the box-type attachment elements having two connected legs angled with respect to each other, a first one of the legs extending along, conforming to the curve of, and bonded to a corresponding one of the airfoil root sides, and a second one of the legs extending along and bonded to a composite shroud surface.

  17. MspA nanopores from subunit dimers.

    Directory of Open Access Journals (Sweden)

    Mikhail Pavlenok

    Full Text Available Mycobacterium smegmatis porin A (MspA forms an octameric channel and represents the founding member of a new family of pore proteins. Control of subunit stoichiometry is important to tailor MspA for nanotechnological applications. In this study, two MspA monomers were connected by linkers ranging from 17 to 62 amino acids in length. The oligomeric pore proteins were purified from M. smegmatis and were shown to form functional channels in lipid bilayer experiments. These results indicated that the peptide linkers did not prohibit correct folding and localization of MspA. However, expression levels were reduced by 10-fold compared to wild-type MspA. MspA is ideal for nanopore sequencing due to its unique pore geometry and its robustness. To assess the usefulness of MspA made from dimeric subunits for DNA sequencing, we linked two M1-MspA monomers, whose constriction zones were modified to enable DNA translocation. Lipid bilayer experiments demonstrated that this construct also formed functional channels. Voltage gating of MspA pores made from M1 monomers and M1-M1 dimers was identical indicating similar structural and dynamic channel properties. Glucose uptake in M. smegmatis cells lacking porins was restored by expressing the dimeric mspA M1 gene indicating correct folding and localization of M1-M1 pores in their native membrane. Single-stranded DNA hairpins produced identical ionic current blockades in pores made from monomers and subunit dimers demonstrating that M1-M1 pores are suitable for DNA sequencing. This study provides the proof of principle that production of single-chain MspA pores in M. smegmatis is feasible and paves the way for generating MspA pores with altered stoichiometries. Subunit dimers enable better control of the chemical and physical properties of the constriction zone of MspA. This approach will be valuable both in understanding transport across the outer membrane in mycobacteria and in tailoring MspA for nanopore

  18. Photoinduced reduction of the medial FeS center in the hydrogenase small subunit HupS from Nostoc punctiforme. (United States)

    Raleiras, Patrícia; Hammarström, Leif; Lindblad, Peter; Styring, Stenbjörn; Magnuson, Ann


    The small subunit from the NiFe uptake hydrogenase, HupSL, in the cyanobacterium Nostoc punctiforme ATCC 29133, has been isolated in the absence of the large subunit (P. Raleiras, P. Kellers, P. Lindblad, S. Styring, A. Magnuson, J. Biol. Chem. 288 (2013) 18,345-18,352). Here, we have used flash photolysis to reduce the iron-sulfur clusters in the isolated small subunit, HupS. We used ascorbate as electron donor to the photogenerated excited state of Ru(II)-trisbipyridine (Ru(bpy)3), to generate Ru(I)(bpy)3 as reducing agent. Our results show that the isolated small subunit can be reduced by the Ru(I)(bpy)3 generated through flash photolysis. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. A Self-Assembled Electro-Active M8L4 Cage Based on Tetrathiafulvalene Ligands

    Directory of Open Access Journals (Sweden)

    Sébastien Goeb


    Full Text Available Two self-