WorldWideScience

Sample records for single subunit type

  1. Compensatory expression of human -Acetylglucosaminyl-1-phosphotransferase subunits in mucolipidosis type III gamma

    OpenAIRE

    Pohl , Sandra; Tiede , Stephan; Castrichini , Monica; Cantz , Michael; Gieselmann , Volkmar; Braulke , Thomas

    2009-01-01

    Abstract The N-Acetylglucosaminyl-1-phosphotransferase plays a key role in the generation of mannose 6-phosphate (M6P) recognition markers essential for efficient transport of lysosomal hydrolases to lysosomes. The phosphotransferase is composed of six subunits (?2, ?2, ?2). The ?- and ?-subunits are catalytically active and encoded by a single gene, GNPTAB, whereas the ?-subunit encoded by GNPTG is proposed to recognize conformational structures common to lysosomal enzymes. Defects in GN...

  2. A single or multistage mycobacterium avium subsp. paratuberculosis subunit vaccine

    DEFF Research Database (Denmark)

    2014-01-01

    The present invention provides one or more immunogenic polypeptides for use in a preventive or therapeutic vaccine against latent or active infection in a human or animal caused by a Mycobacterium species, e.g. Mycobacterium avium subsp. paratuberculosis. Furthermore a single or multi-phase vaccine...... comprising the one or more immunogenic polypeptides is provided for administration for the prevention or treatment of infection with a Mycobacterium species, e.g. Mycobacterium avium subsp. paratuberculosis. Additionally, nucleic acid vaccines, capable of in vivo expression of the multi-phase vaccine...

  3. A single or multistage mycobacterium avium subsp. paratuberculosis subunit vaccine

    DEFF Research Database (Denmark)

    2014-01-01

    The present invention provides one or more immunogenic polypeptides for use in a preventive or therapeutic vaccine against latent or active infection in a human or animal caused by a Mycobacterium species, e.g. Mycobacterium avium subsp. paratuberculosis. Furthermore a single or multi-phase vaccine...... comprising the one or more immunogenic polypeptides is provided for administration for the prevention or treatment of infection with a Mycobacterium species, e.g. Mycobacterium avium subsp. paratuberculosis. Additionally, nucleic acid vaccines, capable of in vivo expression of the multi-phase vaccine...... comprising the one or more immunogenic polypeptides, is provided for prevention or treatment of infection with a Mycobacterium species, e.g. Mycobacterium avium subsp. paratuberculosis....

  4. Modulatory mechanisms and multiple functions of somatodendritic A-type K+ channel auxiliary subunits

    Directory of Open Access Journals (Sweden)

    Henry Hungtao Jerng

    2014-03-01

    Full Text Available Auxiliary subunits are non-conducting, modulatory components of the multi-protein ion channel complexes that underlie normal neuronal signaling. They interact with the pore-forming α-subunits to modulate surface distribution, ion conductance, and channel gating properties. For the somatodendritic subthreshold A-type potassium (ISA channel based on Kv4 α-subunits, two types of auxiliary subunits have been extensively studied: Kv channel-interacting proteins (KChIPs and dipeptidyl peptidase-like proteins (DPLPs. KChIPs are cytoplasmic calcium-binding proteins that interact with intracellular portions of the Kv4 subunits, whereas DPLPs are type II transmembrane proteins that associate with the Kv4 channel core. Both KChIPs and DPLPs genes contain multiple start sites that are used by various neuronal populations to drive the differential expression of functionally distinct N-terminal variants. In turn, these N-terminal variants generate tremendous functional diversity across the nervous system. Here, we focus our review on (1 the molecular mechanism underlying the unique properties of different N-terminal variants, (2 the shaping of native ISA properties by the concerted actions of KChIPs and DPLP variants, and (3 the surprising ways that KChIPs and DPLPs coordinate the activity of multiple channels to fine-tune neuronal excitability. Unlocking the unique contributions of different auxiliary subunit N-terminal variants may provide an important opportunity to develop novel targeted therapeutics to treat numerous neurological disorders.

  5. Self-Subunit Swapping Occurs in Another Gene Type of Cobalt Nitrile Hydratase

    Science.gov (United States)

    Xia, Yuanyuan; Cui, Youtian; Kobayashi, Michihiko; Zhou, Zhemin

    2012-01-01

    Self-subunit swapping is one of the post-translational maturation of the cobalt-containing nitrile hydratase (Co-NHase) family of enzymes. All of these NHases possess a gene organization of , which allows the activator protein to easily form a mediatory complex with the α-subunit of the NHase after translation. Here, we discovered that the incorporation of cobalt into another type of Co-NHase, with a gene organization of , was also dependent on self-subunit swapping. We successfully isolated a recombinant NHase activator protein (P14K) of Pseudomonas putida NRRL-18668 by adding a Strep-tag N-terminal to the P14K gene. P14K was found to form a complex [α(StrepP14K)2] with the α-subunit of the NHase. The incorporation of cobalt into the NHase of P. putida was confirmed to be dependent on the α-subunit substitution between the cobalt-containing α(StrepP14K)2 and the cobalt-free NHase. Cobalt was inserted into cobalt-free α(StrepP14K)2 but not into cobalt-free NHase, suggesting that P14K functions not only as a self-subunit swapping chaperone but also as a metallochaperone. In addition, NHase from P. putida was also expressed by a mutant gene that was designed with a order. Our findings expand the general features of self-subunit swapping maturation. PMID:23226397

  6. [Nature of the heterogeneity of the 30S ribosomal subunits in vitro. II. Two types of inactivation of the 30S subunits of Escherichia coli ribosomes].

    Science.gov (United States)

    Peshin, N N; Kirillov, S V

    1979-01-01

    The influence of concentration of monovalent cations on the binding constant of Phe-tRNAPhe to 30S.poly(U) complex was studied. Two types of inactivation of the 30S subunits by ammonium ions at the low magnesium concentration (1 mM) were found. The first type of inactivation was observed at high concentrations of NH4+ ions (from 0.5 to 1.5 M) and due to the dissociation of ribosomal proteins from 30S subunits. This inactivation only decreased the binding constant of Phe-tRNAPhe to 30S.poly(U) complex up to 50 times but all 30S subunits were equally achieved in Phe-tRNAPhe binding. This type of inactivation was reversible, addition of S-proteins restored the association constant to the original value. At low concentration of NH4+ ions (below 100 mM) about half of the 30S subunits is irreversibly inactivated (the binding constant of Phe-tRNAPhe decreased below detectable level) probably as a result of conformational changes in ribosomal RNA. Both types of inactivation of the 30S subunits can take place during the preparation of isolated subunits of ribosomes.

  7. The elusive third subunit IIa of the bacterial B-type oxidases: the enzyme from the hyperthermophile Aquifex aeolicus.

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    Laurence Prunetti

    Full Text Available The reduction of molecular oxygen to water is catalyzed by complicated membrane-bound metallo-enzymes containing variable numbers of subunits, called cytochrome c oxidases or quinol oxidases. We previously described the cytochrome c oxidase II from the hyperthermophilic bacterium Aquifex aeolicus as a ba(3-type two-subunit (subunits I and II enzyme and showed that it is included in a supercomplex involved in the sulfide-oxygen respiration pathway. It belongs to the B-family of the heme-copper oxidases, enzymes that are far less studied than the ones from family A. Here, we describe the presence in this enzyme of an additional transmembrane helix "subunit IIa", which is composed of 41 amino acid residues with a measured molecular mass of 5105 Da. Moreover, we show that subunit II, as expected, is in fact longer than the originally annotated protein (from the genome and contains a transmembrane domain. Using Aquifex aeolicus genomic sequence analyses, N-terminal sequencing, peptide mass fingerprinting and mass spectrometry analysis on entire subunits, we conclude that the B-type enzyme from this bacterium is a three-subunit complex. It is composed of subunit I (encoded by coxA(2 of 59000 Da, subunit II (encoded by coxB(2 of 16700 Da and subunit IIa which contain 12, 1 and 1 transmembrane helices respectively. A structural model indicates that the structural organization of the complex strongly resembles that of the ba(3 cytochrome c oxidase from the bacterium Thermus thermophilus, the IIa helical subunit being structurally the lacking N-terminal transmembrane helix of subunit II present in the A-type oxidases. Analysis of the genomic context of genes encoding oxidases indicates that this third subunit is present in many of the bacterial oxidases from B-family, enzymes that have been described as two-subunit complexes.

  8. Expression of gill vacuolar-type H+-ATPase B subunit, and Na+, K+-ATPase alpha- and beta- subunit messenger RNAs in smolting Salmo salar

    DEFF Research Database (Denmark)

    Seidelin, Michel; Madsen, Steffen; Cutler, Christopher P

    2001-01-01

    Changes in gill vacuolar-type H+-ATPase B subunit, and Na+,K+-ATPase alpha and beta subunit mRNA expression were examined during the course of smoltification in Salmo salar. We cloned and sequenced cDNA fragments of S. salar gill i) vacuolar-type H+-ATPase (V-H+-ATPase) B subunit, ii) Na+,K+-ATPase....... The peak smelt stage was, however, characterized by simultaneously elevated gill Na+,K+-ATPase expression and low V-H+-ATPase expression, and possibly ensures the complete transformation of the gill into a hypo-osmoregulatory organ and hence the development of optimal SW-tolerance of the smolt....... alpha (1) subunit, and iii) Na+,K+-ATPase beta (1) subunit, and used these as Northern blotting probes. During smoltification, the salmon showed a typical increase in gill Na+,K+-ATPase activity and improved hypo-osmoregulatory ability as judged by their ability to regulate plasma [Cl-] in a 24-hr...

  9. Single-dose monomeric HA subunit vaccine generates full protection from influenza challenge

    CSIR Research Space (South Africa)

    Mallajosyula, JK

    2014-03-01

    Full Text Available Recombinant subunit vaccines are an efficient strategy to meet the demands of a possible influenza pandemic, because of rapid and scalable production. However, vaccines made from recombinant hemagglutinin (HA) subunit protein are often of low...

  10. Recycling of protein subunits during DNA translocation and cleavage by Type I restriction-modification enzymes.

    Science.gov (United States)

    Simons, Michelle; Szczelkun, Mark D

    2011-09-01

    The Type I restriction-modification enzymes comprise three protein subunits; HsdS and HsdM that form a methyltransferase (MTase) and HsdR that associates with the MTase and catalyses Adenosine-5'-triphosphate (ATP)-dependent DNA translocation and cleavage. Here, we examine whether the MTase and HsdR components can 'turnover' in vitro, i.e. whether they can catalyse translocation and cleavage events on one DNA molecule, dissociate and then re-bind a second DNA molecule. Translocation termination by both EcoKI and EcoR124I leads to HsdR dissociation from linear DNA but not from circular DNA. Following DNA cleavage, the HsdR subunits appear unable to dissociate even though the DNA is linear, suggesting a tight interaction with the cleaved product. The MTases of EcoKI and EcoAI can dissociate from DNA following either translocation or cleavage and can initiate reactions on new DNA molecules as long as free HsdR molecules are available. In contrast, the MTase of EcoR124I does not turnover and additional cleavage of circular DNA is not observed by inclusion of RecBCD, a helicase-nuclease that degrades the linear DNA product resulting from Type I cleavage. Roles for Type I restriction endonuclease subunit dynamics in restriction alleviation in the cell are discussed.

  11. γ-Aminobutyric Acid Type B (GABAB) Receptor Internalization Is Regulated by the R2 Subunit*

    Science.gov (United States)

    Hannan, Saad; Wilkins, Megan E.; Dehghani-Tafti, Ebrahim; Thomas, Philip; Baddeley, Stuart M.; Smart, Trevor G.

    2011-01-01

    γ-Aminobutyric acid type B (GABAB) receptors are important for slow synaptic inhibition in the CNS. The efficacy of inhibition is directly related to the stability of cell surface receptors. For GABAB receptors, heterodimerization between R1 and R2 subunits is critical for cell surface expression and signaling, but how this determines the rate and extent of receptor internalization is unknown. Here, we insert a high affinity α-bungarotoxin binding site into the N terminus of the R2 subunit and reveal its dominant role in regulating the internalization of GABAB receptors in live cells. To simultaneously study R1a and R2 trafficking, a new α-bungarotoxin binding site-labeling technique was used, allowing α-bungarotoxin conjugated to different fluorophores to selectively label R1a and R2 subunits. This approach demonstrated that R1a and R2 are internalized as dimers. In heterologous expression systems and neurons, the rates and extents of internalization for R1aR2 heteromers and R2 homomers are similar, suggesting a regulatory role for R2 in determining cell surface receptor stability. The fast internalization rate of R1a, which has been engineered to exit the endoplasmic reticulum, was slowed to that of R2 by truncating the R1a C-terminal tail or by removing a dileucine motif in its coiled-coil domain. Slowing the rate of internalization by co-assembly with R2 represents a novel role for GPCR heterodimerization whereby R2 subunits, via their C terminus coiled-coil domain, mask a dileucine motif on R1a subunits to determine the surface stability of the GABAB receptor. PMID:21724853

  12. Expression of Gill Vacuolar-Type H^+-ATPase B Subunit, and Na^+, K^+-ATPase α_1 and β_1 Subunit Messenger RNAs in Smolting Salmo salar(Physiology)

    OpenAIRE

    Michel, Seidelin; Steffen S., Madsen; Christopher P., Cutler; Gordon, Cramb; Institute of Biology, University of Southern Denmark-Main Campus : Odense University:(Present address)Department of Life Sciences and Chemistry, Roskilde University; Institute of Biology, University of Southern Denmark-Main Campus : Odense University; School of Biomedical Sciences, Bute Medical Buildings, University of St. Andrews; School of Biomedical Sciences, Bute Medical Buildings, University of St. Andrews

    2001-01-01

    Changes in gill vacuolar-type HT-ATPase B subunit, and Na^+,K^+-ATPase α and β subunit mRNA expression were examined during the course of smoltification in Salmo salar. We cloned and sequenced cDNA fragments of S. salar gill i) vacuolar-type H^+-ATPase (V-H^+-ATPase) B subunit, ii) Na^+,K^+-ATPase α_1 subunit, and iii) Na^+,K^+-ATPase β_1 subunit, and used these as Northern blotting probes. During smoltification, the salmon showed a typical increase in gill Na^+,K^+-ATPase activity and improv...

  13. Purification, crystallization and preliminary crystallographic analysis of the vacuole-type ATPase subunit E from Pyrococcus horikoshii OT3

    International Nuclear Information System (INIS)

    Lokanath, Neratur K.; Ukita, Yoko; Sugahara, Mitsuaki; Kunishima, Naoki

    2004-01-01

    The E subunit of vacuole-type ATPase from P. horikoshii OT3 was overexpressed, purified and crystallized. The native crystals diffracted X-rays to 1.85 Å resolution. The vacuole-type ATPases in eukaryotic cells translocate protons across various biological membranes including the vacuolar membrane by consuming ATP molecules. The E subunit of the multisubunit complex V-ATPase from Pyrococcus horikoshii OT3, which has a molecular weight of 22.88 kDa, has been cloned, overexpressed in Escherichia coli, purified and crystallized by the microbatch method using PEG 4000 as a precipitant at 296 K. A data set to 1.85 Å resolution with 98.8% completeness and an R merge of 6.5% was collected from a single flash-cooled crystal using synchrotron radiation. The crystal belonged to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 52.196, b = 55.317, c = 77.481 Å, and is most likely to contain one molecule per asymmetric unit

  14. Specific detection of type II human chorionic gonadotropin beta subunit produced by trophoblastic and neoplastic cells.

    Science.gov (United States)

    Aldaz-Carroll, L; Richon, S; Dangles-Marie, V; Cocquebert, M; Fournier, T; Troalen, F; Stevens, D; Guery, B; Hersant, A-M; Guibourdenche, J; Nordor, A; Pecking, A; Bellet, D

    2015-04-15

    The sequence of the beta-subunit of human chorionic gonadotropin (hCGβ) varies depending on whether hCGβ is encoded by type I or type II genes. Type II genes are upregulated in trophoblast and cancer but hCGβ can be detected in the serum of nonpregnant women and healthy individuals. We aimed to determine whether monoclonal antibody (mAb) FBT11-II specifically detects hCGβ encoded by type II genes (type II hCGβ). Competitive inhibition assays with synthetic peptides, immunocytochemical and immunohistochemical studies, type II hCGβ dosing immunoassays and sequencing of CGB genes were performed. Competitive inhibition assays determined that mAb FBT11-II recognizes the type II hCGβ derived peptide. CGB mRNA sequencing of JEG-3 (trophoblastic) and T24 (bladder) cell lines confirmed that JEG-3 expresses type II genes while T24 expresses exclusively type I. FBT11-II only recognizes JEG-expressed hCGβ. Placenta immunohistochemical studies confirmed that type II hCGβ expression is restricted to the syncytiotrophoblast. Immunoassays detected type II hCGβ in serum of patients with either nontrophoblastic cancers or fetal Down syndrome. Type II gene expression can be detected using FBT11-II. This specific recognition could improve the clinical usefulness of assays aimed at either managing aggressive tumors or screening for Down syndrome. Copyright © 2015. Published by Elsevier B.V.

  15. Mitochondrial Genes of Dinoflagellates Are Transcribed by a Nuclear-Encoded Single-Subunit RNA Polymerase.

    Directory of Open Access Journals (Sweden)

    Chang Ying Teng

    Full Text Available Dinoflagellates are a large group of algae that contribute significantly to marine productivity and are essential photosynthetic symbionts of corals. Although these algae have fully-functioning mitochondria and chloroplasts, both their organelle genomes have been highly reduced and the genes fragmented and rearranged, with many aberrant transcripts. However, nothing is known about their RNA polymerases. We cloned and sequenced the gene for the nuclear-encoded mitochondrial polymerase (RpoTm of the dinoflagellate Heterocapsa triquetra and showed that the protein presequence targeted a GFP construct into yeast mitochondria. The gene belongs to a small gene family, which includes a variety of 3'-truncated copies that may have originated by retroposition. The catalytic C-terminal domain of the protein shares nine conserved sequence blocks with other single-subunit polymerases and is predicted to have the same fold as the human enzyme. However, the N-terminal (promoter binding/transcription initiation domain is not well-conserved. In conjunction with the degenerate nature of the mitochondrial genome, this suggests a requirement for novel accessory factors to ensure the accurate production of functional mRNAs.

  16. Subunits of highly Fluorescent Protein R-Phycoerythrin as Probes for Cell Imaging and Single-Molecule Detection

    Energy Technology Data Exchange (ETDEWEB)

    Isailovic, Dragan [Iowa State Univ., Ames, IA (United States)

    2005-01-01

    The purposes of our research were: (1) To characterize subunits of highly fluorescent protein R-Phycoerythrin (R-PE) and check their suitability for single-molecule detection (SMD) and cell imaging, (2) To extend the use of R-PE subunits through design of similar proteins that will be used as probes for microscopy and spectral imaging in a single cell, and (3) To demonstrate a high-throughput spectral imaging method that will rival spectral flow cytometry in the analysis of individual cells. We first demonstrated that R-PE subunits have spectroscopic and structural characteristics that make them suitable for SMD. Subunits were isolated from R-PE by high-performance liquid chromatography (HPLC) and detected as single molecules by total internal reflection fluorescence microscopy (TIRFM). In addition, R-PE subunits and their enzymatic digests were characterized by several separation and detection methods including HPLC, capillary electrophoresis, sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE) and HPLC-electrospray ionization mass spectrometry (ESI-MS). Favorable absorption and fluorescence of the R-PE subunits and digest peptides originate from phycoerythrobilin (PEB) and phycourobilin (PUB) chromophores that are covalently attached to cysteine residues. High absorption coefficients and strong fluorescence (even under denaturing conditions), broad excitation and emission fluorescence spectra in the visible region of electromagnetic spectrum, and relatively low molecular weights make these molecules suitable for use as fluorescence labels of biomolecules and cells. We further designed fluorescent proteins both in vitro and in vivo (in Escherichia coli) based on the highly specific attachment of PEB chromophore to genetically expressed apo-subunits of R-PE. In one example, apo-alpha and apo-beta R-PE subunits were cloned from red algae Polisiphonia boldii (P. boldii), and expressed in E. coli. Although expressed apo-subunits formed inclusion

  17. SINGLE CHAIN VARIABLE FRAGMENTS OF ANTIBODIES AGAINST DIPHTHERIA TOXIN B-SUBUNIT ISOLATED FROM PHAGE DISPLAY HUMAN ANTIBODY LIBRARY

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    Oliinyk O. S.

    2014-02-01

    Full Text Available Diphtheria toxin is an exoantigen of Corynebacterium diphtheriae that inhibits protein synthesis and kills sensitive cells. The aim of this study was to obtain human recombinant single-chain variable fragment (scFv antibodies against receptor-binding B subunit of diphtheria toxin. 12 specific clones were selected after three rounds of a phage display naїve (unimmunized human antibody library against recombinant B-subunit. scFv DNA inserts from these 12 clones were digested with MvaI, and 6 unique restriction patterns were found. Single-chain antibodies were expressed in Escherichia coli XL1-blue. The recombinant proteins were characterized by immunoblotting of bacterial extracts and detection with an anti-E-tag antibody. The toxin B-subunit-binding function of the single-chain antibody was shown by ELISA. The affinity constants for different clones were found to be from 106 to 108 М–1. Due to the fact, that these antibody fragments recognized epitopes in the receptor-binding Bsubunit of diphtheria toxin, further studies are interesting to evaluate their toxin neutralization properties and potential for therapeutic applications. Obtained scFv-antibodies can also be used for detection and investigation of biological properties of diphtheria toxin.

  18. Mice deleted for heart-type cytochrome c oxidase subunit 7a1 develop dilated cardiomyopathy.

    Science.gov (United States)

    Hüttemann, Maik; Klewer, Scott; Lee, Icksoo; Pecinova, Alena; Pecina, Petr; Liu, Jenney; Lee, Michael; Doan, Jeffrey W; Larson, Douglas; Slack, Elise; Maghsoodi, Bita; Erickson, Robert P; Grossman, Lawrence I

    2012-03-01

    Subunit 7a of mouse cytochrome c oxidase (Cox) displays a contractile muscle-specific isoform, Cox7a1, that is the major cardiac form. To gain insight into the role of this isoform, we have produced a new knockout mouse line that lacks Cox7a1. We show that homozygous and heterozygous Cox7a1 knockout mice, although viable, have reduced Cox activity and develop a dilated cardiomyopathy at 6 weeks of age. Surprisingly, the cardiomyopathy improves and stabilizes by 6 months of age. Cox7a1 knockout mice incorporate more of the "liver-type" isoform Cox7a2 into the cardiac Cox holoenzyme and, also surprisingly, have higher tissue ATP levels. Copyright © 2011 Elsevier B.V. and Mitochondria Research Society. All rights reserved. All rights reserved.

  19. Subunits VIIa,b,c of human cytochrome c oxidase. Identification of both 'heart-type' and 'liver-type' isoforms of subunit VIIa in human heart

    NARCIS (Netherlands)

    van Kuilenburg, A. B.; van Beeumen, J. J.; van der Meer, N. M.; Muijsers, A. O.

    1992-01-01

    The N-terminal amino acid sequences and the electrophoretic mobilities of the subunits VIIa, VIIb and VIIc of cytochrome c oxidase purified from human heart were investigated and compared with those from human skeletal muscle and from bovine heart. In purified human heart cytochrome c oxidase, both

  20. Subunit rotation in a single FoF1-ATP synthase in a living bacterium monitored by FRET

    Science.gov (United States)

    Seyfert, K.; Oosaka, T.; Yaginuma, H.; Ernst, S.; Noji, H.; Iino, R.; Börsch, M.

    2011-03-01

    FoF1-ATP synthase is the ubiquitous membrane-bound enzyme in mitochondria, chloroplasts and bacteria which provides the 'chemical energy currency' adenosine triphosphate (ATP) for cellular processes. In Escherichia coli ATP synthesis is driven by a proton motive force (PMF) comprising a proton concentration difference ΔpH plus an electric potential ΔΨ across the lipid membrane. Single-molecule in vitro experiments have confirmed that proton-driven subunit rotation within FoF1-ATP synthase is associated with ATP synthesis. Based on intramolecular distance measurements by single-molecule fluorescence resonance energy transfer (FRET) the kinetics of subunit rotation and the step sizes of the different rotor parts have been unraveled. However, these experiments were accomplished in the presence of a PMF consisting of a maximum ΔpH ~ 4 and an unknown ΔΨ. In contrast, in living bacteria the maximum ΔpH across the plasma membrane is likely 0.75, and ΔΨ has been measured between -80 and -140 mV. Thus the problem of in vivo catalytic turnover rates, or the in vivo rotational speed in single FoF1-ATP synthases, respectively, has to be solved. In addition, the absolute number of functional enzymes in a single bacterium required to maintain the high ATP levels has to be determined. We report our progress of measuring subunit rotation in single FoF1-ATP synthases in vitro and in vivo, which was enabled by a new labeling approach for single-molecule FRET measurements.

  1. Type III restriction endonuclease EcoP15I is a heterotrimeric complex containing one Res subunit with several DNA-binding regions and ATPase activity.

    Science.gov (United States)

    Wyszomirski, Karol H; Curth, Ute; Alves, Jürgen; Mackeldanz, Petra; Möncke-Buchner, Elisabeth; Schutkowski, Mike; Krüger, Detlev H; Reuter, Monika

    2012-04-01

    For efficient DNA cleavage, the Type III restriction endonuclease EcoP15I communicates with two inversely oriented recognition sites in an ATP-dependent process. EcoP15I consists of methylation (Mod) and restriction (Res) subunits forming a multifunctional enzyme complex able to methylate or to cleave DNA. In this study, we determined by different analytical methods that EcoP15I contains a single Res subunit in a Mod(2)Res stoichiometry. The Res subunit comprises a translocase (Tr) domain carrying functional motifs of superfamily 2 helicases and an endonuclease domain with a PD..D/EXK motif. We show that the isolated Tr domain retains ATP-hydrolyzing activity and binds single- and double-stranded DNA in a sequence-independent manner. To localize the regions of DNA binding, we screened peptide arrays representing the entire Res sequence for their ability to interact with DNA. We discovered four DNA-binding regions in the Tr domain and two DNA-binding regions in the endonuclease domain. Modelling of the Tr domain shows that these multiple DNA-binding regions are located on the surface, free to interact with DNA. Interestingly, the positions of the DNA-binding regions are conserved among other Type III restriction endonucleases.

  2. Fusion of Cholera toxin B subunit (ctxB with Shigella dysenteriae type I toxin B subunit (stxB, Cloning and Expression that in E. coli

    Directory of Open Access Journals (Sweden)

    2012-12-01

    Full Text Available Background and Objective: Shiga toxin (STx is the main virulence factor in Shigella Dysenteriae type I and is composed of an enzymatic subunit STxA monomer and a receptor-binding STxB homopentamer. Shigella toxin B subunit (STxB is a non-toxic homopentameric protein responsible for toxin binding and internalization into target cells by interacting with glycolipid (Gb3. Cholera toxin B subunit (CTB has been known as a mucosal adjuvant for vaccines and genetic fusions of CTB with several hetroantigens such as stxB and can increase humoral and mucosal immunity response.Materials and Methods: In this study, after primer designing, the ctxB and stxB genes were amplified by PCR and cloned into the pGEM-T vector. The stxB gene with a nonfurin linker was fused to the ctB gene in the pGEM vector via the restriction enzyme method and thereafter the fused genes of ctB-stxB were subcloned in the pET28a(+ as an expression vector. The expressed chimeric protein was induced with IPTG and evaluated via the SDS.PAGE and Western blot techniques. Result: The pET28a (+/ctxB-stxB expression vector was confirmed by endonuclease digestion, PCR, and sequence analysis. The CTB-STB fusion protein was confirmed by the SDS-PAGE and Western-blot. Conclusion: The CTB-STB recombinant protein can be used as a new and desirable mucosal vaccine for Shigella Dysenteriae type I.

  3. Sub-unit Specific Regulation of Type-A GABAergic Receptors during Post-Natal Development of the Auditory Cortex

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    Liisa A. Tremere

    2011-01-01

    Full Text Available The GABA-A receptor has been strongly implicated in the organization and function of cortical sensory circuits in the adult mammal. In the present work, changes in the expression patterns of select GABA-A subunits were examined as a function of development. The RNA expression profiles for three subunit types were studied, α1, β2/3 and δ at four developmental time points, (p0, p15, p30 and p90. The o1, β2/3 subunits were present at birth and following a modest increase early in life; mRNA expression for these subunits were found at stable levels throughout life. The expression pattern for the δ subunit showed the most dramatic changes in the number of positive cells as a function of age. In early life, p0 through p15 expression of mRNA for the δ subunit was quite low but increased in later life, p30 and p90. Together these data suggest that much of the potential for inhibitory connectivity is laid down in the pre and early post-natal periods.

  4. Interdomain communication in the endonuclease/motor subunit of type I restriction-modification enzyme EcoR124I.

    Science.gov (United States)

    Sinha, Dhiraj; Shamayeva, Katsiaryna; Ramasubramani, Vyas; Řeha, David; Bialevich, Vitali; Khabiri, Morteza; Guzanová, Alena; Milbar, Niv; Weiserová, Marie; Csefalvay, Eva; Carey, Jannette; Ettrich, Rüdiger

    2014-07-01

    Restriction-modification systems protect bacteria from foreign DNA. Type I restriction-modification enzymes are multifunctional heteromeric complexes with DNA-cleavage and ATP-dependent DNA translocation activities located on endonuclease/motor subunit HsdR. The recent structure of the first intact motor subunit of the type I restriction enzyme from plasmid EcoR124I suggested a mechanism by which stalled translocation triggers DNA cleavage via a lysine residue on the endonuclease domain that contacts ATP bound between the two helicase domains. In the present work, molecular dynamics simulations are used to explore this proposal. Molecular dynamics simulations suggest that the Lys-ATP contact alternates with a contact with a nearby loop housing the conserved QxxxY motif that had been implicated in DNA cleavage. This model is tested here using in vivo and in vitro experiments. The results indicate how local interactions are transduced to domain motions within the endonuclease/motor subunit.

  5. Involvement of proteasomal subunits zeta and iota in RNA degradation.

    Science.gov (United States)

    Petit, F; Jarrousse, A S; Dahlmann, B; Sobek, A; Hendil, K B; Buri, J; Briand, Y; Schmid, H P

    1997-01-01

    We have identified two distinct subunits of 20 S proteasomes that are associated with RNase activity. Proteasome subunits zeta and iota, eluted from two-dimensional Western blots, hydrolysed tobacco mosaic virus RNA, whereas none of the other subunits degraded this substrate under the same conditions. Additionally, proteasomes were dissociated by 6 M urea, and subunit zeta, containing the highest RNase activity, was isolated by anion-exchange chromatography and gel filtration. Purified subunit zeta migrated as a single spot on two-dimensional PAGE with a molecular mass of approx. 28 kDa. Addition of anti-(subunit zeta) antibodies led to the co-precipitation of this proteasome subunit and nuclease activity. This is the first evidence that proteasomal alpha-type subunits are associated with an enzymic activity, and our results provide further evidence that proteasomes may be involved in cellular RNA metabolism. PMID:9337855

  6. Purification and functional reconstitution of a seven-subunit mrp-type na+/h+ antiporter.

    Science.gov (United States)

    Morino, Masato; Suzuki, Toshiharu; Ito, Masahiro; Krulwich, Terry Ann

    2014-01-01

    Mrp antiporters and their homologues in the cation/proton antiporter 3 family of the Membrane Transporter Database are widely distributed in bacteria. They have major roles in supporting cation and cytoplasmic pH homeostasis in many environmental, extremophilic, and pathogenic bacteria. These antiporters require six or seven hydrophobic proteins that form hetero-oligomeric complexes, while most other cation/proton antiporters require only one membrane protein for their activity. The resemblance of three Mrp subunits to membrane-embedded subunits of the NADH:quinone oxidoreductase of respiratory chains and to subunits of several hydrogenases has raised interest in the evolutionary path and commonalities of their proton-translocating domains. In order to move toward a greater mechanistic understanding of these unusual antiporters and to rigorously demonstrate that they function as secondary antiporters, powered by an imposed proton motive force, we established a method for purification and functional reconstitution of the seven-subunit Mrp antiporter from alkaliphilic Bacillus pseudofirmus OF4. Na(+)/H(+) antiporter activity was demonstrated by a fluorescence-based assay with proteoliposomes in which the Mrp complex was coreconstituted with a bacterial FoF1-ATPase. Proton pumping by the ATPase upon addition of ATP generated a proton motive force across the membranes that powered antiporter activity upon subsequent addition of Na(+).

  7. Cloning, chromosomal localization, and functional expression of the alpha 1 subunit of the L-type voltage-dependent calcium channel from normal human heart

    NARCIS (Netherlands)

    Schultz, D; Mikala, G; Yatani, A; Engle, D B; Iles, D E; Segers, B; Sinke, R J; Weghuis, D O; Klöckner, U; Wakamori, M

    1993-01-01

    A unique structural variant of the cardiac L-type voltage-dependent calcium channel alpha 1 subunit cDNA was isolated from libraries derived from normal human heart mRNA. The deduced amino acid sequence shows significant homology to other calcium channel alpha 1 subunits. However, differences from

  8. Plant single-cell and single-cell-type metabolomics.

    Science.gov (United States)

    Misra, Biswapriya B; Assmann, Sarah M; Chen, Sixue

    2014-10-01

    In conjunction with genomics, transcriptomics, and proteomics, plant metabolomics is providing large data sets that are paving the way towards a comprehensive and holistic understanding of plant growth, development, defense, and productivity. However, dilution effects from organ- and tissue-based sampling of metabolomes have limited our understanding of the intricate regulation of metabolic pathways and networks at the cellular level. Recent advances in metabolomics methodologies, along with the post-genomic expansion of bioinformatics knowledge and functional genomics tools, have allowed the gathering of enriched information on individual cells and single cell types. Here we review progress, current status, opportunities, and challenges presented by single cell-based metabolomics research in plants. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Identification of the EcoKI and EcoR124I Type I restriction--modification enzyme subunits by non-equilibrium pH gradient two-dimensional gel electrophoresis.

    Science.gov (United States)

    Nguyen, L D; Cajthamlová, K; Nguyen, H T; Weiser, J; Holubová, I; Weiserová, M

    2002-01-01

    Effectively optimized and reproducible procedure for monitoring the composition of type I restriction-modification endonucleases EcoKI and EcoR124I by non-equilibrium pH gradient two-dimensional (2-D) gel electrophoresis is described. Three subunits of the enzyme complex, which widely differ from one another in their isoelectric points and molar mass, were identified in crude cell extracts of E. coli. For the first time all three subunits of both EcoKI and EcoR124I were detected as distinct spots on a single 2-D gel. A sensitive immunoblotting procedure was suggested suitable for routine use in determining the identity of individual subunits. Potential application of this method for detailed studies of regulation of the function and stoichiometry of the enzyme complexes is discussed.

  10. Single protein omission reconstitution studies of tetracycline binding to the 30S subunit of Escherichia coli ribosomes

    International Nuclear Information System (INIS)

    Buck, M.; Cooperman, B.S.

    1990-01-01

    In previous work the authors showed that on photolysis of Escherichia coli ribosomes in the presence of [ 3 H]tetracycline (TC) the major protein labeled is S7, and they presented strong evidence that such labeling takes place from a high-affinity site related to the inhibitory action of TC. In this work they use single protein omission reconstitution (SPORE) experiments to identify those proteins that are important for high-affinity TC binding to the 30S subunit, as measured by both cosedimentation and filter binding assays. With respect to both sedimentation coefficients and relative Phe-tRNA Phe binding, the properties of the SPORE particles they obtain parallel very closely those measured earlier, with the exception of the SPORE particle lacking S13. A total of five proteins, S3, S7, S8, S14, and S19, are shown to be important for TC binding, with the largest effects seen on omission of proteins S7 and S14. Determination of the protein compositions of the corresponding SPORE particles demonstrates that the observed effects are, for the most part, directly attributable to the omission of the given protein rather than reflecting an indirect effect of omitting one protein on the uptake of another. A large body of evidence supports the notion that four of these proteins, S3, S7, S14, and S19, are included, along with 16S rRNA bases 920-1,396, in one of the major domains of the 30S subunit. The results support the conclusion that the structure of this domain is important for the binding of TC and that, within this domain, TC binds directly to S7

  11. The alpha(1S) subunit of the L-type calcium channel is not a predisposition gene for thyrotoxic periodic paralysis.

    Science.gov (United States)

    Tang, Nelson L S; Chow, C C; Ko, Gary T C; Tai, Morris H L; Kwok, Rachel; Yao, X Q; Cockram, Clive S

    2007-02-01

    Thyrotoxic periodic paralysis (TTP) has been associated with genetic variations in the gene encoding the alpha 1 subunit of the L-type calcium channel (CACNA1S). Mutations in CACNA1S are known to account for the majority of cases of familial hypokalaemic periodic paralysis (HOKPP). In this study we have examined 48 genetic polymorphisms in the CACNA1S gene and genotyped a tagging set of representative polymorphisms to determine the role of this gene in TPP. A genetic association study was carried out with 98 TPP patients and 162 male thyrotoxic controls. Among 47 polymorphisms evaluated for linkage disequilibrium (LD) and the spectrum of haplotypes in the Chinese population, 31 were selected as tagging single-nucleotide polymorphisms (SNPs) for genotyping the whole sample. A new genotyping protocol was used to analyse an insertion/deletion (I/D) polymorphism. We studied the LD among 47 polymorphisms in the CACNA1S gene, which comprised a set of high-density markers with an average of one SNP every 2 kb. Subsequently, 31 tagSNPs were genotyped for all the samples. The gene is composed of three LD blocks. With this block structure, we were confident that variations of the gene were comprehensively covered by the tagSNPs. No significant association was found between the polymorphisms and TPP. We established the LD structure of this calcium channel subunit gene (CACNA1S) for the first time. However, its genetic variations are not associated with TPP in Chinese patients.

  12. Escherichia coli Single-Stranded DNA-Binding Protein: NanoESI-MS Studies of Salt-Modulated Subunit Exchange and DNA Binding Transactions

    Science.gov (United States)

    Mason, Claire E.; Jergic, Slobodan; Lo, Allen T. Y.; Wang, Yao; Dixon, Nicholas E.; Beck, Jennifer L.

    2013-02-01

    Single-stranded DNA-binding proteins (SSBs) are ubiquitous oligomeric proteins that bind with very high affinity to single-stranded DNA and have a variety of essential roles in DNA metabolism. Nanoelectrospray ionization mass spectrometry (nanoESI-MS) was used to monitor subunit exchange in full-length and truncated forms of the homotetrameric SSB from Escherichia coli. Subunit exchange in the native protein was found to occur slowly over a period of hours, but was significantly more rapid in a truncated variant of SSB from which the eight C-terminal residues were deleted. This effect is proposed to result from C-terminus mediated stabilization of the SSB tetramer, in which the C-termini interact with the DNA-binding cores of adjacent subunits. NanoESI-MS was also used to examine DNA binding to the SSB tetramer. Binding of single-stranded oligonucleotides [one molecule of (dT)70, one molecule of (dT)35, or two molecules of (dT)35] was found to prevent SSB subunit exchange. Transfer of SSB tetramers between discrete oligonucleotides was also observed and is consistent with predictions from solution-phase studies, suggesting that SSB-DNA complexes can be reliably analyzed by ESI mass spectrometry.

  13. A Single Residue Mutation in the Gαq Subunit of the G Protein Complex Causes Blindness in Drosophila

    Directory of Open Access Journals (Sweden)

    Jinguo Cao

    2018-01-01

    Full Text Available Heterotrimeric G proteins play central roles in many signaling pathways, including the phototransduction cascade in animals. However, the degree of involvement of the G protein subunit Gαq is not clear since animals with previously reported strong loss-of-function mutations remain responsive to light stimuli. We recovered a new allele of Gαq in Drosophila that abolishes light response in a conventional electroretinogram assay, and reduces sensitivity in whole-cell recordings of dissociated cells by at least five orders of magnitude. In addition, mutant eyes demonstrate a rapid rate of degeneration in the presence of light. Our new allele is likely the strongest hypomorph described to date. Interestingly, the mutant protein is produced in the eyes but carries a single amino acid change of a conserved hydrophobic residue that has been assigned to the interface of interaction between Gαq and its downstream effector, PLC. Our study has thus uncovered possibly the first point mutation that specifically affects this interaction in vivo.

  14. Structure of HsdS Subunit from Thermoanaerobacter tengcongensis Sheds Lights on Mechanism of Dynamic Opening and Closing of Type I Methyltransferase

    Science.gov (United States)

    Gao, Pu; Tang, Qun; An, XiaoMin; Yan, XiaoXue; Liang, DongCai

    2011-01-01

    Type I DNA methyltransferases contain one specificity subunit (HsdS) and two modification subunits (HsdM). The electron microscopy model of M.EcoKI-M2S1 methyltransferase shows a reasonable closed state of this clamp-like enzyme, but the structure of the open state is still unclear. The 1.95 Å crystal structure of the specificity subunit from Thermoanaerobacter tengcongensis (TTE-HsdS) shows an unreported open form inter-domain orientation of this subunit. Based on the crystal structure of TTE-HsdS and the closed state model of M.EcoKI-M2S1, we constructed a potential open state model of type I methyltransferase. Mutational studies indicated that two α-helices (aa30-59 and aa466-495) of the TTE-HsdM subunit are important inter-subunit interaction sites in the TTE-M2S1 complex. DNA binding assays also highlighted the importance of the C-terminal region of TTE-HsdM for DNA binding by the TTE-M2S1 complex. On the basis of structural analysis, biochemical experiments and previous studies, we propose a dynamic opening and closing mechanism for type I methyltransferase. PMID:21399684

  15. Inactivation of gating currents of L-type calcium channels. Specific role of the alpha 2 delta subunit.

    Science.gov (United States)

    Shirokov, R; Ferreira, G; Yi, J; Ríos, E

    1998-06-01

    In studies of gating currents of rabbit cardiac Ca channels expressed as alpha 1C/beta 2a or alpha 1C/beta 2a/alpha 2 delta subunit combinations in tsA201 cells, we found that long-lasting depolarization shifted the distribution of mobile charge to very negative potentials. The phenomenon has been termed charge interconversion in native skeletal muscle (Brum, G., and E. Ríos. 1987. J. Physiol. (Camb.). 387:489-517) and cardiac Ca channels (Shirokov, R., R. Levis, N. Shirokova, and E. Ríos. 1992. J. Gen. Physiol. 99:863-895). Charge 1 (voltage of half-maximal transfer, V1/2 approximately 0 mV) gates noninactivated channels, while charge 2 (V1/2 approximately -90 mV) is generated in inactivated channels. In alpha 1C/beta 2a cells, the available charge 1 decreased upon inactivating depolarization with a time constant tau approximately 8, while the available charge 2 decreased upon recovery from inactivation (at -200 mV) with tau approximately 0.3 s. These processes therefore are much slower than charge movement, which takes charge movement and that of changes in their availability, which was even wider in the presence of alpha 2 delta, implies that charges 1 and 2 originate from separate channel modes. Because clear modal separation characterizes slow (C-type) inactivation of Na and K channels, this observation establishes the nature of voltage-dependent inactivation of L-type Ca channels as slow or C-type. The presence of the alpha 2 delta subunit did not change the V1/2 of charge 2, but sped up the reduction of charge 1 upon inactivation at 40 mV (to tau approximately 2 s), while slowing the reduction of charge 2 upon recovery (tau approximately 2 s). The observations were well simulated with a model that describes activation as continuous electrodiffusion (Levitt, D. 1989. Biophys. J. 55:489-498) and inactivation as discrete modal change. The effects of alpha 2 delta are reproduced assuming that the subunit lowers the free energy of the inactivated mode.

  16. Coreceptor Tropism Can Be Influenced by Amino Acid Substitutions in the gp41 Transmembrane Subunit of Human Immunodeficiency Virus Type 1 Envelope Protein▿

    Science.gov (United States)

    Huang, Wei; Toma, Jonathan; Fransen, Signe; Stawiski, Eric; Reeves, Jacqueline D.; Whitcomb, Jeannette M.; Parkin, Neil; Petropoulos, Christos J.

    2008-01-01

    Many studies have demonstrated that the third variable region (V3) of the human immunodeficiency virus type 1 (HIV-1) envelope protein (Env) is a major determinant of coreceptor tropism. Other regions in the surface gp120 subunit of Env can modulate coreceptor tropism in a manner that is not fully understood. In this study, we evaluated the effect of env determinants outside of V3 on coreceptor usage through the analysis of (i) patient-derived env clones that differ in coreceptor tropism, (ii) chimeric env sequences, and (iii) site-directed mutants. The introduction of distinct V3 sequences from CXCR4-using clones into an R5-tropic env backbone conferred the inefficient use of CXCR4 in some but not all cases. Conversely, in many cases, X4- and dual-tropic env backbones containing the V3 sequences of R5-tropic clones retained the ability to use CXCR4, suggesting that sequences outside of the V3 regions of these CXCR4-using clones were responsible for CXCR4 use. The determinants of CXCR4 use in a set of dual-tropic env sequences with V3 sequences identical to those of R5-tropic clones mapped to the gp41 transmembrane (TM) subunit. In one case, a single-amino-acid substitution in the fusion peptide of TM was able to confer CXCR4 use; however, TM substitutions associated with CXCR4 use varied among different env sequences. These results demonstrate that sequences in TM can modulate coreceptor specificity and that env sequences other than that of V3 may facilitate efficient CXCR4-mediated entry. We hypothesize that the latter plays an important role in the transition from CCR5 to CXCR4 coreceptor use. PMID:18353956

  17. Identification of proteasome subunit beta type 6 (PSMB6 associated with deltamethrin resistance in mosquitoes by proteomic and bioassay analyses.

    Directory of Open Access Journals (Sweden)

    Linchun Sun

    Full Text Available Deltamethrin (DM insecticides are currently being promoted worldwide for mosquito control, because of the high efficacy, low mammalian toxicity and less environmental impact. Widespread and improper use of insecticides induced resistance, which has become a major obstacle for the insect-borne disease management. Resistance development is a complex and dynamic process involving many genes. To better understand the possible molecular mechanisms involved in DM resistance, a proteomic approach was employed for screening of differentially expressed proteins in DM-susceptible and -resistant mosquito cells. Twenty-seven differentially expressed proteins were identified by two-dimensional electrophoresis (2-DE and mass spectrometry (MS. Four members of the ubiquitin-proteasome system were significantly elevated in DM-resistant cells, suggesting that the ubiquitin-proteasome pathway may play an important role in DM resistance. Proteasome subunit beta type 6 (PSMB6 is a member of 20S proteasomal subunit family, which forms the proteolytic core of 26S proteasome. We used pharmaceutical inhibitor and molecular approaches to study the contributions of PSMB6 in DM resistance: the proteasome inhibitor MG-132 and bortezomib were used to suppress the proteasomal activity and siRNA was designed to block the function of PSMB6. The results revealed that both MG-132 and bortezomib increased the susceptibility in DM-resistant cells and resistance larvae. Moreover, PSMB6 knockdown decreased cellular viability under DM treatment. Taken together, our study indicated that PSMB6 is associated with DM resistance in mosquitoes and that proteasome inhibitors such as MG-132 or bortezomib are suitable for use as a DM synergist for vector control.

  18. Transspecies dimorphic allelic lineages of the proteasome subunit β-type 8 gene (PSMB8) in the teleost genus Oryzias

    Science.gov (United States)

    Miura, Fumi; Tsukamoto, Kentaro; Mehta, Ratnesh Bhai; Naruse, Kiyoshi; Magtoon, Wichian; Nonaka, Masaru

    2010-01-01

    The proteasome subunit β-type 8 (PSMB8) gene in the jawed vertebrate MHC genomic region encodes a catalytic subunit of the immunoproteasome involved in the generation of peptides to be presented by the MHC class I molecules. A teleost, the medaka (Oryzias latipes), has highly diverged dimorphic allelic lineages of the PSMB8 gene with only about 80% amino acid identity, termed “PSMB8d” and “PSMB8N,” which have been retained by most wild populations analyzed. To elucidate the evolutionary origin of these two allelic lineages, seven species of the genus Oryzias were analyzed for their PSMB8 allelic sequences using a large number of individuals from wild populations. All the PSMB8 alleles of these species were classified into one of these two allelic lineages based on their nucleotide sequences of exons and introns, indicating that the Oryzias PSMB8 gene has a truly dichotomous allelic lineage. Retention of both allelic lineages was confirmed except for one species. The PSMB8d lineage showed a higher frequency than the PSMB8N lineage in all seven species. The two allelic lineages showed curious substitutions at the 31st and 53rd residues of the mature peptide, probably involved in formation of the S1 pocket, suggesting that these allelic lineages show a functional difference in cleavage specificity. These results indicate that the PSMB8 dimorphism was established before speciation within the genus Oryzias and has been maintained for more than 30–60 million years under a strict and asymmetric balancing selection through several speciation events. PMID:21098669

  19. Molecular characterization of a Leishmania donovani cDNA clone with similarity to human 20S proteasome a-type subunit

    DEFF Research Database (Denmark)

    Christensen, C B; Jørgensen, L; Jensen, A T

    2000-01-01

    Using plasma from patients infected or previously infected with Leishmania donovanii, we isolated a L. donovanii cDNA clone with similarity to the proteasome a-type subunit from humans and other eukaryotes. The cDNA clone, designated LePa, was DNA sequenced and Northern blot analysis of L...

  20. Carboxyl terminus heterogeneity of type IV fimbrial subunit protein of Pasteurella multocida isolates.

    Science.gov (United States)

    Shivachandra, Sathish Bhadravati; Kumar, Abhinendra; Yogisharadhya, Revanaiah; Ramakrishnan, M A; Viswas, K N

    2013-12-01

    Pasteurella multocida, a Gram-negative bacterial pathogen, known to affect a wide range of domestic as well as wild animal and avian species throughout the world by causing either systemic or localized infections termed as 'pasteurellosis'. P. multocida isolates are known to possess type IV fimbriae (pili) as one of the major virulence factors based on their role in adhesion to host surfaces and subsequent pathogenesis. In the present study, ptfA gene of Indian P. multocida isolates (n = 8) originated from different animal (buffalo, sheep, goat, pig) and avian host species (chicken, turkey, duck, quail) were amplified, cloned, sequenced and compared with available ptfA/fimbrial protein sequences in GenBank/publications (n = 22) to understand its variability with respect to geography/host/serogroup/disease specific patterns. Multiple sequence alignment revealed highly conserved N-terminus α-1 helix region and heterogeneous C-terminus (68-137 aa) comprised of β-strand regions (β1, β2, β3, β4) with conserved two pairs of cysteine residues. Interestingly, an existence of absolute homogeneity among the P. multocida isolates that caused haemorrhagic septicaemia in bovines and septicaemic pasteurellosis in sheep and goats was noticed. Pig isolates had 99.3% homogeneity. On contrary, more diversity (35.8%) was observed among isolates that caused fowl cholera in avians irrespective of identical capsular/somatic serogroup and similar host species. Phylogenetic analysis based on nucleotide sequences of ptfA gene revealed formation of mixed clusters with isolates representing different disease conditions as well as serogroups irrespective of country of origin which indicated the possible role of cross-species transmission among different animal/avian species. The study indicated highly conserved and host specific fimbriae among animal species than relatively divergent fimbriae among avian species.

  1. Development of an enhanced bovine viral diarrhea virus subunit vaccine based on E2 glycoprotein fused to a single chain antibody which targets to antigen-presenting cells.

    Science.gov (United States)

    Pecora, Andrea; Malacari, Darío A; Pérez Aguirreburualde, María S; Bellido, Demian; Escribano, José M; Dus Santos, María J; Wigdorovitz, Andrés

    2015-01-01

    Bovine viral diarrhea virus (BVDV) is an important cause of economic losses worldwide. E2 is an immunodominant protein and a promising candidate to develop subunit vaccines. To improve its immunogenicity, a truncated E2 (tE2) was fused to a single chain antibody named APCH, which targets to antigen-presenting cells. APCH-tE2 and tE2 proteins were expressed in the baculovirus system and their immunogenicity was firstly compared in guinea pigs. APCH-tE2 vaccine was the best one to evoke a humoral response, and for this reason, it was selected for a cattle vaccination experiment. All the bovines immunized with 1.5 μg of APCH-tE2 developed high levels of neutralizing antibodies against BVDV up to a year post-immunization, demonstrating its significant potential as a subunit vaccine. This novel vaccine is undergoing scale-up and was transferred to the private sector. Nowadays, it is being evaluated for registration as the first Argentinean subunit vaccine for cattle. Copyright © 2014 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  2. Effect of GLP-1 on the expression of NADPH oxidase subunits in the kidney of type 1 diabetic rats

    Directory of Open Access Journals (Sweden)

    Jin-jin LIU

    2013-09-01

    Full Text Available Objective To observe the effect of exenatide, a glucagon-like peptide-1 (GLP-1 receptor agonist, on the expression of NADPH oxidase subunits NOX4 and p22phox and connective tissue growth factor (CTGF in the kidney of streptozotocin (STZ-induced type 1 diabetic rats, and explore the protective effects and mechanisms of exenatide on the kidney of diabetic rats. Methods Thirty male Sprague-Dawley (SD rats were divided into control group (group A, n=7 and diabetic model group (n=23. Type 1 diabetic model was reproduced by intraperitoneal injection of streptozotocin. It was successful in 19 rats. Diabetic rats were randomly divided into diabetic control group (group B, n=10 and diabetic with treatment of exenatide group (group C, n=9. Rats in group C were injected subcutaneously with exenatide in dose of 5μg/kg twice daily. Rats in group A and B were given equivalent volume of normal saline by subcutaneous injection. All rats were sacrificed after eight weeks. The mRNA expression of renal p22phox and NOX4 were detected by real-time fluorescence quantitative PCR. The protein expression of CTGF was detected by immunohistochemical staining. Results The levels of blood glucose, lipids, creatinine, and urea nitrogen, the albumin excretion rate, kidney index, the mRNA expressions of renal NOX4 and p22phox, and the protein expression of renal CTGF were significantly increased in group B compared with that in group A (P0.05. Conclusion Exenatide can decrease the expressions of renal NOX4, p22phox and CTGF, decline the index of urinary protein, and alleviate the kidney hypertrophy in type 1 diabetic rats, implying that exenatide exerted a protective effect on the kidney.

  3. Effects of telmisartan on the expression of NADPH oxidase subunits in the myocardium of type 2 diabetic rats

    Directory of Open Access Journals (Sweden)

    Jia-wei LI

    2011-10-01

    Full Text Available Objective To explore the effect of telmisartan on the expression of NADPH oxidase subunits p22phox and NOX4 in the myocardiam of type 2 diabetic rats.Methods Thirty-six male Wistar rats were randomly divided into two groups: normal control group(group A,n=10,diabetic model group(n=26.Type 2 diabetic model was established by high-fat and high-sugar diet followed by intraperitoneal injection of a low dose of streptozotocin(STZ.After the model was reproduced successfully,20 diabetic rats were randomly divided into diabetic subgroup(group B,n=10 and telmisartan-treated subgroup(group C,n=10.Rats in group C were orally administered telmisartan(5mg/kg/d,and rats in group A and B were given equivalent volume of normal saline.All rats were sacrificed 12 weeks after treatment.The mRNA expressions of myocardial p22phox and NOX4 were detected by real-time fluorescence quantitative PCR,and the protein expressions of myocardial connective tissue growth factor(CTGF and copper-zinc-superoxide dismutase(Cu-Zn-SOD were detected by immunohistochemical staining.Results Compared with group A,the ratio of heart/body weight,the mRNA expression of myocardial p22phox and NOX4,and the protein expression of myocardial CTGF increased significantly in group B,and the protein expression of myocardial Cu-Zn-SOD decreased significantly(P 0.05.Conclusions Telmisartan can down-regulate the over-expression of myocardial NOX4 and p22phox mRNA in type 2 diabetic rats,lessen the myocardial damage induced by oxidative stress,thus plays a protective role in the myocardium of diabetic rats.

  4. An Aromatic Cap Seals the Substrate Binding Site in an ECF-Type S Subunit for Riboflavin

    Energy Technology Data Exchange (ETDEWEB)

    Karpowich, Nathan K.; Song, Jinmei; Wang, Da-Neng

    2016-06-13

    ECF transporters are a family of active membrane transporters for essential micronutrients, such as vitamins and trace metals. Found exclusively in archaea and bacteria, these transporters are composed of four subunits: an integral membrane substrate-binding subunit (EcfS), a transmembrane coupling subunit (EcfT), and two ATP-binding cassette ATPases (EcfA and EcfA'). We have characterized the structural basis of substrate binding by the EcfS subunit for riboflavin from Thermotoga maritima, TmRibU. TmRibU binds riboflavin with high affinity, and the protein–substrate complex is exceptionally stable in solution. The crystal structure of riboflavin-bound TmRibU reveals an electronegative binding pocket at the extracellular surface in which the substrate is completely buried. Analysis of the intermolecular contacts indicates that nearly every available substrate hydrogen bond is satisfied. A conserved aromatic residue at the extracellular end of TM5, Tyr130, caps the binding site to generate a substrate-bound, occluded state, and non-conservative mutation of Tyr130 reduces the stability of this conformation. Using a novel fluorescence binding assay, we find that an aromatic residue at this position is essential for high-affinity substrate binding. Comparison with other S subunit structures suggests that TM5 and Loop5-6 contain a dynamic, conserved motif that plays a key role in gating substrate entry and release by S subunits of ECF transporters.

  5. Eudesmane-Type Sesquiterpene Lactones Inhibit Nuclear Translocation of the Nuclear Factor κB Subunit RelB in Response to a Lymphotoxin β Stimulation.

    Science.gov (United States)

    Quach, Hue Tu; Kondo, Tetsuya; Watanabe, Megumi; Tamura, Ryuichi; Yajima, Yoshiki; Sayama, Shinsei; Ando, Masayoshi; Kataoka, Takao

    2017-01-01

    The transcription factor nuclear factor κB (NF-κB) regulates various biological processes, including inflammatory responses. We previously reported that eudesmane-type sesquiterpene lactones inhibited multiple steps in the canonical NF-κB signaling pathway induced by tumor necrosis factor-α and interleukin-1α. In contrast, the biological activities of eudesmane-type sesquiterpene lactones on the non-canonical NF-κB signaling pathway remain unclear. In the present study, we found that (11S)-2α-bromo-3-oxoeudesmano-12,6α-lactone, designated santonin-related compound 2 (SRC2), inhibited NF-κB luciferase reporter activity induced by lymphotoxin β (LTβ) in human lung carcinoma A549 cells. Although SRC2 did not prevent the processing of the NF-κB subunit p100 induced by LTβ, it inhibited the nuclear translocation of RelB and p52 in response to the LTβ stimulation. In contrast to (-)-dehydroxymethylepoxyquinomicin, SRC2 inhibited the LTβ-induced nuclear translocation of the RelB (C144S) mutant in a manner similar to wild-type RelB. While eudesmane derivatives possessing an α-bromoketone moiety or α,β-unsaturated carbonyl moieties inhibited LTβ-induced NF-κB luciferase reporter activity, eudesmane derivatives possessing an α-bromoketone moiety exhibited stronger inhibitory activity on the LTβ-induced nuclear translocation of RelB than those possessing a single α-methylene-γ-lactone moiety. The results of the present study revealed that SRC2 inhibits the nuclear translocation of RelB in the non-canonical NF-κB signaling pathway induced by LTβ.

  6. A Single Point Mutation within the Coding Sequence of Cholera Toxin B Subunit Will Increase Its Expression Yield

    OpenAIRE

    Bakhshi, Bita; Boustanshenas, Mina; Ghorbani, Masoud

    2014-01-01

    Background: Cholera toxin B subunit (CTB) has been extensively considered as an immunogenic and adjuvant protein, but its yield of expression is not satisfactory in many studies. The aim of this study was to compare the expression of native and mutant recombinant CTB (rCTB) in pQE vector. Methods: ctxB fragment from Vibrio cholerae O1 ATCC14035 containing the substitution of mutant ctxB for amino acid S128T was amplified by PCR and cloned in pGETM-T easy vector. It was then transformed to E. ...

  7. Embryonic type Na+ channel β-subunit, SCN3B masks the disease phenotype of Brugada syndrome.

    Science.gov (United States)

    Okata, Shinichiro; Yuasa, Shinsuke; Suzuki, Tomoyuki; Ito, Shogo; Makita, Naomasa; Yoshida, Tetsu; Li, Min; Kurokawa, Junko; Seki, Tomohisa; Egashira, Toru; Aizawa, Yoshiyasu; Kodaira, Masaki; Motoda, Chikaaki; Yozu, Gakuto; Shimojima, Masaya; Hayashiji, Nozomi; Hashimoto, Hisayuki; Kuroda, Yusuke; Tanaka, Atsushi; Murata, Mitsushige; Aiba, Takeshi; Shimizu, Wataru; Horie, Minoru; Kamiya, Kaichiro; Furukawa, Tetsushi; Fukuda, Keiichi

    2016-09-28

    SCN5A is abundant in heart and has a major role in I Na . Loss-of-function mutation in SCN5A results in Brugada syndrome (BrS), which causes sudden death in adults. It remains unclear why disease phenotype does not manifest in the young even though mutated SCN5A is expressed in the young. The aim of the present study is to elucidate the timing of the disease manifestation in BrS. A gain-of-function mutation in SCN5A also results in Long QT syndrome type 3 (LQTS3), leading to sudden death in the young. Induced pluripotent stem cells (iPSCs) were generated from a patient with a mixed phenotype of LQTS3 and BrS with the E1784K SCN5A mutation. Here we show that electrophysiological analysis revealed that LQTS3/BrS iPSC-derived cardiomyocytes recapitulate the phenotype of LQTS3 but not BrS. Each β-subunit of the sodium channel is differentially expressed in embryonic and adult hearts. SCN3B is highly expressed in embryonic hearts and iPSC-derived cardiomyocytes. A heterologous expression system revealed that I Na of mutated SCN5A is decreased and SCN3B augmented I Na of mutated SCN5A. Knockdown of SCN3B in LQTS3/BrS iPSC-derived cardiomyocytes successfully unmasked the phenotype of BrS. Isogenic control of LQTS3/BrS (corrected-LQTS3/BrS) iPSC-derived cardiomyocytes gained the normal electrophysiological properties.

  8. A novel mechanism of V-type zinc inhibition of glutamate dehydrogenase results from disruption of subunit interactions necessary for efficient catalysis.

    Science.gov (United States)

    Bailey, Jaclyn; Powell, Lakeila; Sinanan, Leander; Neal, Jacob; Li, Ming; Smith, Thomas; Bell, Ellis

    2011-09-01

    Bovine glutamate dehydrogenase is potently inhibited by zinc and the major impact is on V(max) suggesting a V-type effect on catalysis or product release. Zinc inhibition decreases as glutamate concentrations decrease suggesting a role for subunit interactions. With the monocarboxylic amino acid norvaline, which gives no evidence of subunit interactions, zinc does not inhibit. Zinc significantly decreases the size of the pre-steady state burst in the reaction but does not affect NADPH binding in the enzyme-NADPH-glutamate complex that governs the steady state turnover, again suggesting that zinc disrupts subunit interactions required for catalytic competence. While differential scanning calorimetry suggests zinc binds and induces a slightly conformationally more rigid state of the protein, limited proteolysis indicates that regions in the vicinity of the antennae regions and the trimer-trimer interface become more flexible. The structures of glutamate dehydrogenase bound with zinc and europium show that zinc binds between the three dimers of subunits in the hexamer, a region shown to bind novel inhibitors that block catalytic turnover, which is consistent with the above findings. In contrast, europium binds to the base of the antenna region and appears to abrogate the inhibitory effect of zinc. Structures of various states of the enzyme have shown that both regions are heavily involved in the conformational changes associated with catalytic turnover. These results suggest that the V-type inhibition produced with glutamate as the substrate results from disruption of subunit interactions necessary for efficient catalysis rather than by a direct effect on the active site conformation. © 2011 The Authors Journal compilation © 2011 FEBS.

  9. A Novel Mechanism of V Type Zinc Inhibition of Glutamate Dehydrogenase Results from Disruption of Subunit Interactions Necessary for Efficient Catalysis

    Science.gov (United States)

    Bailey, Jaclyn; Powell, Lakeila; Sinanan, Leander; Neal, Jacob; Li, Ming; Smith, Thomas; Bell, Ellis

    2011-01-01

    SUMMARY Bovine Glutamate Dehydrogenase is potently inhibited by zinc and the major impact is on Vmax suggesting a V-type effect on catalysis or product release. Zinc inhibition decreases as glutamate concentrations decrease suggesting a role for subunit interactions. With the monocarboxylic amino acid, norvaline, which gives no evidence of subunit interactions zinc does not inhibit. Zinc significantly decreases the size of the pre-steady state burst in the reaction but does not affect NADPH binding in the Enzyme-NADPH-glutamate complex that governs the steady state turn-over again suggesting that zinc disrupts subunit interactions required for catalytic competence. While differential scanning calorimetry suggests zinc binds and induces a slightly conformationally more rigid state of the protein, limited proteolysis indicate regions in the vicinity of the antennae regions and the trimer-trimer interface become more flexible. The structures of GDH bound with zinc and europium show that zinc binds between the three dimers of subunits in the hexamer, a region shown to bind novel inhibitors that block catalytic turnover and is consistent with the above findings. In contrast, europium binds to the base of the antenna region and appears to abrogate the inhibitory effect of zinc. Structures of various states of the enzyme have shown that both regions are heavily involved in the conformational changes associated with catalytic turnover. These results suggest that the V-type inhibition produced with glutamate as the substrate results from disruption of subunit interactions necessary for efficient catalysis rather than by a direct effect on the active site conformation. PMID:21749647

  10. Regulation of the subunit composition of plastidic glutamine synthetase of the wild-type and of the phytochrome-deficient aurea mutant of tomato by blue/UV-A- or by UV-B-light

    International Nuclear Information System (INIS)

    Migge, A.; Carrayol, E.; Hirel, B.; Lohmann, M.; Meya, G.; Becker, T.W.

    1998-01-01

    The photomorphogenetic aurea mutant of tomato severely deficient in spectrophotometrically active phytochromes was used to study the light-regulation of the single-copy nuclear gene encoding plastidic glutamine synthetase (GS-2; EC 6.1.3.2). The de-etiolation of dark-grown aurea mutant seedling cotyledons showed an obligatory dependency on blue light. A limited red light-responsiveness of etiolated aurea cotyledons is, however, retained as seen by the stimulation of both the GS-2 transcript and protein level in the cotyledons of aurea seedlings during growth in red light. The subunits of the octameric GS-2 enzyme were represented by polypeptides with similar electrophoretic mobilities (polypeptides a) in etiolated wild-type or aurea mutant cotyledons. GS-2 proteins with similar apparent molecular masses were also seen in the cotyledons of red light-grown aurea mutant seedlings. In contrast, GS-2 polypeptides with different apparent molecular masses (polypeptides a and b) were detected in the cotyledons of wild-type seedlings grown in red light. This difference indicates that the (post-translational) modification of tomato GS-2 subunit composition is mediated by the photoreceptor phytochrome. The illumination of etiolated wild-type or aurea cotyledons with UV-A- or UV-B-light light resulted in an increase in both the GS-2 transcript and protein level. Following illumination of etiolated wild-type seedlings with UV-A-light, the relative proportion of the GS-2 polypeptides a and b was similar than upon irradiation with blue light but different than after exposure to UV-B- or red light. This result suggests the involvement of a blue/ UV-A-light-specific photoreceptor in the regulation of tomato GS-2 subunit composition. (author)

  11. Regulation of BK channels by auxiliary γ subunits

    Directory of Open Access Journals (Sweden)

    Jiyuan eZhang

    2014-10-01

    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.

  12. Diverse functional consequences of mutations in the Na+/K+-ATPase alpha2-subunit causing familial hemiplegic migraine type 2.

    NARCIS (Netherlands)

    Tavraz, N.N.; Friedrich, T.; Durr, K.L.; Koenderink, J.B.; Bamberg, E.; Freilinger, T.; Dichgans, M.

    2008-01-01

    Mutations in ATP1A2, the gene coding for the Na(+)/K(+)-ATPase alpha(2)-subunit, are associated with both familial hemiplegic migraine and sporadic cases of hemiplegic migraine. In this study, we examined the functional properties of 11 ATP1A2 mutations associated with familial or sporadic

  13. A single dose of the novel chimeric subunit vaccine E2-CD154 confers early full protection against classical swine fever virus.

    Science.gov (United States)

    Suárez, Marisela; Sordo, Yusmel; Prieto, Yanet; Rodríguez, María P; Méndez, Lídice; Rodríguez, Elsa M; Rodríguez-Mallon, Alina; Lorenzo, Elianet; Santana, Elaine; González, Nemecio; Naranjo, Paula; Frías, María Teresa; Carpio, Yamila; Estrada, Mario Pablo

    2017-08-03

    Classical swine fever is an economically important, highly contagious disease of swine worldwide. Subunit vaccines are a suitable alternative for the control of classical swine fever. However, such vaccines have as the main drawback the relatively long period of time required to induce a protective response, which hampers their use under outbreak conditions. In this work, a lentivirus-based gene delivery system is used to obtain a stable recombinant HEK 293 cell line for the expression of E2-CSFV antigen fused to porcine CD154 as immunostimulant molecule. The E2-CD154 chimeric protein was secreted into the medium by HEK293 cells in a concentration around 50mg/L in suspension culture conditions using spinner bottles. The E2-CD154 immunized animals were able to overcome the challenge with a high virulent CSF virus strain performed 7days after a unique dose of the vaccine without clinical manifestations of the disease. Specific anti-CSFV neutralizing antibodies and IFN-γ were induced 8days after challenge equivalent to 14days post-vaccination. The present work constitutes the first report of a subunit vaccine able to confer complete protection by the end of the first week after a single vaccination. These results suggest that the E2-CD154 antigen could be potentially used under outbreak conditions to stop CSFV spread and for eradication programs in CSF enzootic areas. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Sublingual tolerance induction with antigen conjugated to cholera toxin B subunit induces Foxp3+CD25+CD4+ regulatory T cells and suppresses delayed-type hypersensitivity reactions.

    Science.gov (United States)

    Sun, J-B; Cuburu, N; Blomquist, M; Li, B-L; Czerkinsky, C; Holmgren, J

    2006-09-01

    Although sublingual (s.l.) immunotherapy with selected allergens is safe and often effective for treating patients with allergies, knowledge of the immunological mechanisms involved remains limited. Can s.l. administration of antigen (Ag) induce peripheral immunological tolerance and also suppress delayed-type hypersensitivity (DTH) responses? To what extent can s.l.-induced tolerance be explained by the generation of Foxp3+CD25+CD4+ regulatory T cells (T(reg))? This study addressed these questions in mice and compared the relative efficacy of administering ovalbumin (OVA) conjugated to cholera toxin B (CTB) subunit with administration of the same Ag alone. We found that s.l. administration of a single or even more efficiently three repeated 40-mug doses of OVA/CTB conjugate suppressed T-cell proliferative responses to OVA by cervical lymph node (CLN), mesenteric lymph node (MLN) and spleen cells and concurrently strongly increased the frequency of Ag-specific T(reg) in CLN, MLN and spleen and also transforming growth factor-beta (TGF-beta) levels in serum. The CLN and splenic cells from OVA/CTB-treated BALB/c mice efficiently suppressed OVA-specific T-cell receptor (TCR) transgenic (DO11.10) CD25-CD4+ effector T-cell proliferation in vitro. Further, s.l. treatment with OVA/CTB completely suppressed OVA-specific DTH responses in vivo and T-cell proliferative responses in mice immunized subcutaneously with OVA in Freund's complete adjuvant. The intracellular expression of Foxp3 was strongly increased in OVA-specific (KJ1-26+) CD4+ T cells from OVA/CTB-treated mice. Thus, s.l. administration of CTB-conjugated Ag can efficiently induce peripheral T-cell tolerance associated with strong increases in serum TGF-beta levels and in Ag-specific Foxp3+CD25+CD4+ T(reg) cells.

  15. A single point mutation within the coding sequence of cholera toxin B subunit will increase its expression yield.

    Science.gov (United States)

    Bakhshi, Bita; Boustanshenas, Mina; Ghorbani, Masoud

    2014-07-01

    Cholera toxin B subunit (CTB) has been extensively considered as an immunogenic and adjuvant protein, but its yield of expression is not satisfactory in many studies. The aim of this study was to compare the expression of native and mutant recombinant CTB (rCTB) in pQE vector. ctxB fragment from Vibrio cholerae O1 ATCC14035 containing the substitution of mutant ctxB for amino acid S128T was amplified by PCR and cloned in pGETM-T easy vector. It was then transformed to E. coli Top 10F' and cultured on LB agar plate containing ampicillin. Sequence analysis confirmed the mature ctxB gene sequence and the mutant one in both constructs which were further subcloned to pQE-30 vector. Both constructs were subsequently transformed to E. coli M15 (pREP4) for expression of mature and mutant rCTB. SDS-PAGE analysis showed the maximum expression of rCTB in both systems at 5 hours after induction and Western-blot analysis confirmed the presence of rCTB in blotting membranes. The expression of mutant rCTB was much higher than mature rCTB, which may be the result of serine-to-threonine substitution at position 128 of mature rCTB amino acid sequence created by PCR mutagenesis. The mutant rCTB retained pentameric stability and its ability to bind to anti- cholera toxin IgG antibodies. Point mutation in ctxB sequence resulted in over-expression of rCTB, probably due to the increase of solubility of produced rCTB. Consequently, this expression system can be used to produce rCTB in high yield.

  16. Differential neutralizing activities of a single domain camelid antibody (VHH specific for ricin toxin's binding subunit (RTB.

    Directory of Open Access Journals (Sweden)

    Cristina Herrera

    Full Text Available Ricin, a member of the A-B family of ribosome-inactivating proteins, is classified as a Select Toxin by the Centers for Disease Control and Prevention because of its potential use as a biothreat agent. In an effort to engineer therapeutics for ricin, we recently produced a collection of alpaca-derived, heavy-chain only antibody VH domains (VHH or "nanobody" specific for ricin's enzymatic (RTA and binding (RTB subunits. We reported that one particular RTB-specific VHH, RTB-B7, when covalently linked via a peptide spacer to different RTA-specific VHHs, resulted in heterodimers like VHH D10/B7 that were capable of passively protecting mice against a lethal dose challenge with ricin. However, RTB-B7 itself, when mixed with ricin at a 1 ∶ 10 toxin:antibody ratio did not afford any protection in vivo, even though it had demonstrable toxin-neutralizing activity in vitro. To better define the specific attributes of antibodies associated with ricin neutralization in vitro and in vivo, we undertook a more thorough characterization of RTB-B7. We report that RTB-B7, even at 100-fold molar excess (toxin:antibody was unable to alter the toxicity of ricin in a mouse model. On the other hand, in two well-established cytotoxicity assays, RTB-B7 neutralized ricin with a 50% inhibitory concentration (IC50 that was equivalent to that of 24B11, a well-characterized and potent RTB-specific murine monoclonal antibody. In fact, RTB-B7 and 24B11 were virtually identical when compared across a series of in vitro assays, including adherence to and neutralization of ricin after the toxin was pre-bound to cell surface receptors. RTB-B7 differed from both 24B11 and VHH D10/B7 in that it was relatively less effective at blocking ricin attachment to receptors on host cells and was not able to form high molecular weight toxin:antibody complexes in solution. Whether either of these activities is important in ricin toxin neutralizing activity in vivo remains to be determined.

  17. First multi-epitope subunit vaccine against extraintestinal pathogenic Escherichia coli delivered by a bacterial type-3 secretion system (T3SS).

    Science.gov (United States)

    Wieser, Andreas; Magistro, Giuseppe; Nörenberg, Dominik; Hoffmann, Christiane; Schubert, Sören

    2012-01-01

    Infections due to extraintestinal pathogenic E. coli (ExPEC) are very common in humans as well as in animals. In humans ExPEC infections include urinary tract infections (UTI), septicemia, and wound infections, which result in significant morbidity, mortality, and substantial healthcare costs. In view of the increasing number of ExPEC infections caused by more and more resistant strains, effective prevention would be desirable. Given the rising treatment costs, a vaccine may be cost-effective in selected patient groups, such as women with recurrent UTI, patients with neurologic disorders impairing bladder function and men with prostate hyperplasia. Previous vaccine studies used single target proteins or whole inactivated ExPEC cells. Here, we describe a vaccine system for oral application based on artificial multiple subunit vaccine proteins. Those multi-epitope proteins are composed of predicted epitopes derived from ExPEC virulence-associated proteins. As ExPEC are known to form intracellular biofilms in the urothelium and can also resist killing by non-activated macrophages, T-cell responses are supposed to be an important measure to counteract these stages of ExPEC during infection. Therefore, a live bacterial antigen delivery system based upon the Salmonella type-III secretion system (T3SS) was used in this study to directly deliver the vaccine proteins into the cytoplasm of the host cells. Epitope-rich domains of the proteins FyuA, IroN, ChuA, IreA, Iha, and Usp were expressed in an attenuated Salmonella enterica serovar Typhimurium strain and translocated into target cells for extended periods of time inducing a strong T-cell response. No significant antibody titre increase against the secreted vaccine proteins could be detected in vaginal wash or serum. Despite that, one of the vaccine proteins was able to significantly reduce bacterial load in the challenge model of intraperitoneal sepsis. This study shows that a vaccine encompassing distinct epitopes of

  18. Single bovine sperm sex typing by amelogenin nested PCR.

    Science.gov (United States)

    Colley, A; Buhr, M; Golovan, S P

    2008-10-01

    Sex-sorted bovine semen has become a valuable tool in animal production for sex preselection. Development of novel sperm sexing technologies, or evaluation of the quality of existing methods, often requires a single-sperm, sex-typing method that is reliable and easy to perform. In the present study, we report the development, validation, and application of a simple, reliable, and cost-effective method for single-sperm sex typing using nested polymerase chain reaction (PCR), based on the amelogenin gene. Several hundred single sperm were isolated using a simple manual technique, or a high-speed flow-sorter, and were successfully sex-typed using the amelogenin nested PCR. Based on the pooled results of individual sperm, there was no significant difference in the semen sex ratio of unsorted (44.6% X-sperm and 55.4% Y-sperm) or X/Y-sorted semen (91.4% X-sperm and 94.0% Y-sperm), as compared to the expected ratio in unsorted semen or the post-sorting reanalysis data, respectively. The amelogenin single-sperm sexing method was an adaptable, accurate, and reliable tool for single-sperm sex typing.

  19. A novel role of the L-type calcium channel α1D subunit as a gatekeeper for intracellular zinc signaling: zinc wave.

    Directory of Open Access Journals (Sweden)

    Satoru Yamasaki

    Full Text Available Recent studies have shown that zinc ion (Zn can behave as an intracellular signaling molecule. We previously demonstrated that mast cells stimulated through the high-affinity IgE receptor (FcεRI rapidly release intracellular Zn from the endoplasmic reticulum (ER, and we named this phenomenon the "Zn wave". However, the molecules responsible for releasing Zn and the roles of the Zn wave were elusive. Here we identified the pore-forming α(1 subunit of the Cav1.3 (α(1D L-type calcium channel (LTCC as the gatekeeper for the Zn wave. LTCC antagonists inhibited the Zn wave, and an agonist was sufficient to induce it. Notably, α(1D was mainly localized to the ER rather than the plasma membrane in mast cells, and the Zn wave was impaired by α(1D knockdown. We further found that the LTCC-mediated Zn wave positively controlled cytokine gene induction by enhancing the DNA-binding activity of NF-κB. Consistent with this finding, LTCC antagonists inhibited the cytokine-mediated delayed-type allergic reaction in mice without affecting the immediate-type allergic reaction. These findings indicated that the LTCC α(1D subunit located on the ER membrane has a novel function as a gatekeeper for the Zn wave, which is involved in regulating NF-κB signaling and the delayed-type allergic reaction.

  20. Concholepas concholepas Ferritin H-like subunit (CcFer): Molecular characterization and single nucleotide polymorphism associated to innate immune response.

    Science.gov (United States)

    Chávez-Mardones, Jacqueline; Valenzuela-Muñoz, Valentina; Núñez-Acuña, Gustavo; Maldonado-Aguayo, Waleska; Gallardo-Escárate, Cristian

    2013-09-01

    Ferritin has been identified as the principal protein of iron storage and iron detoxification, playing a pivotal role for the cellular homeostasis in living organisms. However, recent studies in marine invertebrates have suggested its association with innate immune system. In the present study, one Ferritin subunit was identified from the gastropod Concholepas concholepas (CcFer), which was fully characterized by Rapid Amplification of cDNA Ends technique. Simultaneously, a challenge test was performed to evaluate the immune response against Vibrio anguillarum. The full length of cDNA Ccfer was 1030 bp, containing 513 bp of open reading frame that encodes to 170 amino acid peptide, which was similar to the Ferritin H subunit described in vertebrates. Untranslated Regions (UTRs) were identified with a 5'UTR of 244 bp that contains iron responsive element (IRE), and a 3'UTR of 273 bp. The predicted molecular mass of deduced amino acid of CcFer was 19.66 kDa and isoelectric point of 4.92. Gene transcription analysis revealed that CcFer increases against infections with V. anguillarum, showing a peak expression at 6 h post-infection. Moreover, a single nucleotide polymorphism was detected at -64 downstream 5'UTR sequence (SNP-64). Quantitative real time analysis showed that homozygous mutant allele (TT) was significantly associated with higher expression levels of the challenged group compared to wild (CC) and heterozygous (CT) variants. Our findings suggest that CcFer is associated to innate immune response in C. concholepas and that the presence of SNPs may involve differential transcriptional expression of CcFer. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. Molecular characterization of a Leishmania donovani cDNA clone with similarity to human 20S proteasome a-type subunit

    DEFF Research Database (Denmark)

    Christensen, C B; Jørgensen, L; Jensen, A T

    2000-01-01

    Using plasma from patients infected or previously infected with Leishmania donovanii, we isolated a L. donovanii cDNA clone with similarity to the proteasome a-type subunit from humans and other eukaryotes. The cDNA clone, designated LePa, was DNA sequenced and Northern blot analysis of L...... out of 25 patients with visceral leishmaniasis and four out of 18 patients with cutaneous leishmaniasis contained IgG antibodies which reacted with the purified LePa fusion protein as evaluated in an ELISA. The LePa DNA sequence was inserted into an eukaryotic expression vector and Balb/c mice were...

  2. Single-cell LEP-type cavity on measurement stand

    CERN Multimedia

    CERN PhotoLab

    1982-01-01

    A single-cell cavity, made of copper, with tapered connectors for impedance measurements. It was used as a model of LEP-type superconducting cavities, to investigate impedance and higher-order modes and operated at around 600 MHz (the LEP acceleration frequency was 352.2 MHz). See 8202500.

  3. Origin of new Brassica types from a single intergeneric hybrid ...

    Indian Academy of Sciences (India)

    Origin of new Brassica types from a single intergeneric hybrid between B. rapa and Orychophragmus violaceus by rapid chromosome evolution and introgression ... The lines with high productivity showed not only a wide spectrum of phenotypes but also obvious variations in fatty acid profiles of seed oil and glucosinolate ...

  4. Gene structure and mutations of glutaryl-coenzyme A dehydrogenase: impaired association of enzyme subunits that is due to an A421V substitution causes glutaric acidemia type I in the Amish.

    Science.gov (United States)

    Biery, B J; Stein, D E; Morton, D H; Goodman, S I

    1996-11-01

    The structure of the human glutaryl coenzyme A dehydrogenase (GCD) gene was determined to contain 11 exons and to span approximately 7 kb. Fibroblast DNA from 64 unrelated glutaric acidemia type I (GA1) patients was screened for mutations by PCR amplification and analysis of SSCP. Fragments with altered electrophoretic mobility were subcloned and sequenced to detect mutations that caused GA1. This report describes the structure of the GCD gene, as well as point mutations and polymorphisms found in 7 of its 11 exons. Several mutations were found in more than one patient, but no one prevalent mutation was detected in the general population. As expected from pedigree analysis, a single mutant allele causes GA1 in the Old Order Amish of Lancaster County, Pennsylvania. Several mutations have been expressed in Escherichia coli, and all produce diminished enzyme activity. Reduced activity in GCD encoded by the A421V mutation in the Amish may be due to impaired association of enzyme subunits.

  5. Gene knockout of the alpha6 subunit of the gamma-aminobutyric acid type A receptor: lack of effect on responses to ethanol, pentobarbital, and general anesthetics.

    Science.gov (United States)

    Homanics, G E; Ferguson, C; Quinlan, J J; Daggett, J; Snyder, K; Lagenaur, C; Mi, Z P; Wang, X H; Grayson, D R; Firestone, L L

    1997-04-01

    The alpha6 subunit of the gamma-aminobutyric acid type A receptor (GABA(A)-R) has been implicated in mediating the intoxicating effects of ethanol and the motor ataxic effects of general anesthetics. To test this hypothesis, we used gene targeting in embryonic stem cells to create mice lacking a functional alpha6 gene. Homozygous mice are viable and fertile and have grossly normal cerebellar cytoarchitecture. Northern blot and reverse transcriptase-polymerase chain reaction analyses demonstrated that the targeting event disrupted production of functional alpha6 mRNA. Autoradiography of histological sections of adult brains demonstrated that diazepam-insensitive binding of [3H]Ro15-4513 to the cerebellar granule cell layer of wild-type mice was completely absent in homozygous mice. Cerebellar GABA(A)-R density was unchanged in the mutant mice; however, the apparent affinity for muscimol was markedly reduced. Sleep time response to injection of ethanol after pretreatment with vehicle or Ro15-4513 did not differ between genotypes. Sleep time response to injection of pentobarbital and loss of righting reflex and response to tail clamp stimulus in mice anesthetized with volatile anesthetics also did not differ between genotypes. Thus, the alpha6 subunit of the GABA(A)-R is not required for normal development, viability, and fertility and does not seem to be a critical or unique component of the neuronal pathway mediating the hypnotic effect of ethanol and its antagonism by Ro15-4513 in mice. Similarly, the alpha6 subunit does not seem to be involved in the behavioral responses to general anesthetics or pentobarbital.

  6. Mutagenesis Study of the Cytochrome c Subunit Responsible for the Direct Electron Transfer-Type Catalytic Activity of FAD-Dependent Glucose Dehydrogenase

    Directory of Open Access Journals (Sweden)

    Yuki Yamashita

    2018-03-01

    Full Text Available The FAD-dependent glucose dehydrogenase from Burkholderia cepacia (FADGDH is a hetero-oligomeric enzyme that is capable of direct electron transfer (DET with an electrode. The cytochrome c (cyt c subunit, which possesses three hemes (heme 1, heme 2, and heme 3, from the N-terminal sequence, is known to enable DET; however, details of the electron transfer pathway remain unknown. A mutagenesis investigation of the heme axial ligands was carried out to elucidate the electron transfer pathway to the electron mediators and/or the electrode. The sixth axial ligand for each of the three heme irons, Met109, Met263, and Met386 were substituted with His. The catalytic activities of the wild-type (WT and mutant enzymes were compared by investigating their dye-mediated dehydrogenase activities and their DET abilities toward the electrode. The results suggested that (1 heme 1 with Met109 as an axial ligand is mainly responsible for the electron transfer with electron acceptors in the solution, but not for the DET with the electrode; (2 heme 2 with Met263 is responsible for the DET-type reaction with the electrode; and (3 heme 3 with Met386 seemed to be the electron acceptor from the catalytic subunit. From these results, two electron transfer pathways were proposed depending on the electron acceptors. Electrons are transferred from the catalytic subunit to heme 3, then to heme 2, to heme 1 and, finally, to electron acceptors in solution. However, if the enzyme complex is immobilized on the electrode and is used as electron acceptors, electrons are passed to the electrode from heme 2.

  7. The sedative but not the memory-blocking properties of ethanol are modulated by α5-subunit-containing γ-aminobutyric acid type A receptors.

    Science.gov (United States)

    Martin, Loren J; Zurek, Agnieszka A; Bonin, Robert P; Oh, Gabriel H T; Kim, John H; Mount, Howard T J; Orser, Beverley A

    2011-03-01

    The precise mechanisms underlying the memory-blocking properties of ethanol are unknown, in part because ethanol targets a wide array of neurotransmitter receptors and transporters. The aim of this study was to determine whether the memory loss caused by ethanol is mediated, in part, by α5 subunit-containing γ-aminobutyric acid subtype A receptors. These receptors have been implicated in learning and memory processes and are targets for a variety of neurodepressive drugs. Also, since these receptors generate a tonic inhibitory current in hippocampal pyramidal neurons, we examined whether concentrations of ethanol that block memory in vivo increased the tonic current using whole-cell patch-clamp recordings in hippocampal neurons. Null mutant mice lacking the α5 subunit (Gabra5-/-) and wild-type mice were equally impaired in contextual fear conditioning by moderate (1mg/kg) and high (1.5mg/kg) doses of ethanol. The higher dose of ethanol also reduced auditory delay fear conditioning to the same extent in the two genotypes. Interestingly, wild-type mice were more sensitive than Gabra5-/- mice to the sedative effects of low (0.5mg/kg) and moderate (1mg/kg) doses of ethanol in the open-field task. Concentrations of ethanol that impaired memory performance in vivo did not increase the amplitude of the tonic current. Together, the results suggest that the α5-subunit containing γ-aminobutyric acid subtype A receptors are not direct targets for positive modulation by ethanol nor do they contribute to ethanol-induced memory loss. In contrast, these receptors may contribute to the sedative properties of ethanol. Copyright © 2010 Elsevier B.V. All rights reserved.

  8. 3D-localization of the a-subunit in F 0F I-ATP synthase by time resolved single-molecule FRET

    Science.gov (United States)

    Düser, Monika G.; Zarrabi, Nawid; Bi, Yumin; Zimmermann, Boris; Dunn, Stanley D.; Börsch, Michael

    2006-02-01

    F °F I-ATP synthases catalyze the ATP formation from ADP and phosphate in the membranes of mitochondria, chloroplasts and bacteria. Internal rotation of subunits couples the chemical reaction at the F I part to the proton translocation through the F ° part. In these enzymes, the membrane-embedded a-subunit is part of the non-rotating 'stator' subunits and provides the proton channel of the F ° motor. At present, the relative position of the a-subunit is not known. We examined the rotary movements of the ɛ-subunit with respect to the non-rotating a-subunit by time resolved singlemolecule fluorescence resonance energy transfer (FRET) using a novel pulsed laser diode. Rotation of the ɛ-subunit during ATP hydrolysis was divided into three major steps. The stopping positions of ɛ resulted in three distinct FRET efficiency levels and FRET donor lifetimes. From these FRET efficiencies the position of the FRET donor at the asubunit was calculated. Different populations of the three resting positions of ɛ, which were observed previously, enabled us to scrutinize the models for the position of the a-subunit in the F ° part.

  9. N-linked glycans are required on epithelial Na+ channel subunits for maturation and surface expression.

    Science.gov (United States)

    Kashlan, Ossama B; Kinlough, Carol L; Myerburg, Michael M; Shi, Shujie; Chen, Jingxin; Blobner, Brandon M; Buck, Teresa M; Brodsky, Jeffrey L; Hughey, Rebecca P; Kleyman, Thomas R

    2018-03-01

    Epithelial Na + channel (ENaC) subunits undergo N-linked glycosylation in the endoplasmic reticulum where they assemble into an αβγ complex. Six, 13, and 5 consensus sites (Asn-X-Ser/Thr) for N-glycosylation reside in the extracellular domains of the mouse α-, β-, and γ-subunits, respectively. Because the importance of ENaC N-linked glycans has not been fully addressed, we examined the effect of preventing N-glycosylation of specific subunits on channel function, expression, maturation, and folding. Heterologous expression in Xenopus oocytes or Fischer rat thyroid cells with αβγ-ENaC lacking N-linked glycans on a single subunit reduced ENaC activity as well as the inhibitory response to extracellular Na + . The lack of N-linked glycans on the β-subunit also precluded channel activation by trypsin. However, channel activation by shear stress was N-linked glycan independent, regardless of which subunit was modified. We also discovered that the lack of N-linked glycans on any one subunit reduced the total and surface levels of cognate subunits. The lack of N-linked glycans on the β-subunit had the largest effect on total levels, with the lack of N-linked glycans on the γ- and α-subunits having intermediate and modest effects, respectively. Finally, channels with wild-type β-subunits were more sensitive to limited trypsin proteolysis than channels lacking N-linked glycans on the β-subunit. Our results indicate that N-linked glycans on each subunit are required for proper folding, maturation, surface expression, and function of the channel.

  10. Localization of a new type of X-linked liver glycogenosis to the chromosomal region Xp22 containing the liver {alpha}-subunit of phosphorylase kinase (PHKA2)

    Energy Technology Data Exchange (ETDEWEB)

    Hendrickx, J.; Coucke, P.; Willems, P.J. [Univ. of Antwerp (Belgium)] [and others

    1994-06-01

    The authors describe here a new type of X-linked liver glycogen storage disease. The main symptoms include liver enlargement and growth retardation. The clinical and biochemical abnormalities of this glycogenosis are similar to those of classical X-linked liver glycogenosis due to phosphorylase kinase deficiency (XLG). However, in constrast to patients with XLG, the patients described here have no reduced phosphorylase kinase activity in erythrocytes and leukocytes, and no enzyme deficiency could be found. Linkage analysis of four families with this X-linked type of liver glycogenosis assigned the disease gene to Xp22. Lod scores obtained with the markers DXS987, DXS207, and DXS999 were 3.97, 2.71, and 2.40, respectively, all at 0% recombination. Multipoint linkage analysis localized the disease gene between DXS143 and DXS989 with a maximum lod score of 4.70 at {theta}=0, relative to DXS987. As both the classical XLG gene and the liver {alpha}-subunit of PHK (PHKA2) are also located in Xp22, this variant type of XLG may be allelic to classical XLG, and both diseases may be caused by mutations in PHKA2. Therefore, they propose to classify XLG as XLG type I (the classical type of XLG) and XLG type II (the variant type of XLG). 28 refs., 2 figs., 3 tabs.

  11. Single cell transcriptional analysis reveals novel innate immune cell types

    Directory of Open Access Journals (Sweden)

    Linda E. Kippner

    2014-06-01

    Full Text Available Single-cell analysis has the potential to provide us with a host of new knowledge about biological systems, but it comes with the challenge of correctly interpreting the biological information. While emerging techniques have made it possible to measure inter-cellular variability at the transcriptome level, no consensus yet exists on the most appropriate method of data analysis of such single cell data. Methods for analysis of transcriptional data at the population level are well established but are not well suited to single cell analysis due to their dependence on population averages. In order to address this question, we have systematically tested combinations of methods for primary data analysis on single cell transcription data generated from two types of primary immune cells, neutrophils and T lymphocytes. Cells were obtained from healthy individuals, and single cell transcript expression data was obtained by a combination of single cell sorting and nanoscale quantitative real time PCR (qRT-PCR for markers of cell type, intracellular signaling, and immune functionality. Gene expression analysis was focused on hierarchical clustering to determine the existence of cellular subgroups within the populations. Nine combinations of criteria for data exclusion and normalization were tested and evaluated. Bimodality in gene expression indicated the presence of cellular subgroups which were also revealed by data clustering. We observed evidence for two clearly defined cellular subtypes in the neutrophil populations and at least two in the T lymphocyte populations. When normalizing the data by different methods, we observed varying outcomes with corresponding interpretations of the biological characteristics of the cell populations. Normalization of the data by linear standardization taking into account technical effects such as plate effects, resulted in interpretations that most closely matched biological expectations. Single cell transcription

  12. Identification of Plasmodium falciparum translation initiation eIF2β subunit: direct interaction with Protein Phosphatase type 1

    Directory of Open Access Journals (Sweden)

    Géraldine eTellier

    2016-05-01

    Full Text Available Protein phosphatase 1 (PP1c is one of the main phosphatases whose function is shaped by many regulators to confer a specific location and a selective function for this enzyme. Here, we report that eukaryotic initiation factor 2 of P. falciparum (PfeIF2β is an interactor of PfPP1c. Sequence analysis of PfeIF2 revealed a deletion of 111 amino acids when compared to its human counterpart and the presence of two potential binding motifs to PfPP1 (29FGEKKK34, 103KVAW106. As expected, we showed that PfeIF2 binds PfeIF2 and PfeIF5, confirming its canonical interaction with partners of the translation complex. Studies of the PfeIF2-PfPP1 interaction using wild-type, single and double mutated versions of PfeIF2β revealed that both binding motifs are critical. We next showed that PfeIF2 is able to induce Germinal Vesicle BreakDown (GVBD when expressed in Xenopus oocytes, an indicator of its capacity to regulate PP1. Only combined mutations of both binding motifs abolished the interaction with PP1 and the induction of GVBD. In P. falciparum, although the locus is accessible for genetic manipulation, PfeIF2 seems to play an essential role in intraerythrocytic cycle as no viable knockout parasites were detectable. Interestingly, as for PfPP1, the subcellular fractionation of P. falciparum localized PfeIF2β in cytoplasm and nuclear extracts, suggesting a potential effect on PfPP1 in both compartments and raising the question of a non-canonical function of PfeIf2 in the nucleus. Hence, the role played by PfeIF2 in blood stage parasites could occur at multiple levels involving the binding to proteins of the translational complex and to PfPP1.

  13. Identification of snails within the Bulinus africanus group from East Africa by multiplex SNaPshotäanalysis of single nucleotide polymorphisms within the cytochrome oxidase subunit I

    Directory of Open Access Journals (Sweden)

    Stothard JR

    2002-01-01

    Full Text Available Identification of populations of Bulinus nasutus and B. globosus from East Africa is unreliable using characters of the shell. In this paper, a molecular method of identification is presented for each species based on DNA sequence variation within the mitochondrial cytochrome oxidase subunit I (COI as detected by a novel multiplexed SNaPshotTM assay. In total, snails from 7 localities from coastal Kenya were typed using this assay and variation within shell morphology was compared to reference material from Zanzibar. Four locations were found to contain B. nasutus and 2 locations were found to contain B. globosus. A mixed population containing both B. nasutus and B. globosus was found at Kinango. Morphometric variation between samples was considerable and UPGMA cluster analysis failed to differentiate species. The multiplex SNaPshotTM assay is an important development for more precise methods of identification of B. africanus group snails. The assay could be further broadened for identification of other snail intermediate host species.

  14. Effects of heterotropic allosteric effectors on the equilibrium and kinetics of the reaction of a single ligand molecule with an alpha or beta subunit of deoxygenated HbA.

    Science.gov (United States)

    Karasik, Ellen; Kwiatkowski, Laura D; Hui, Hilda L; Colby, Judith F; Noble, Robert W

    2004-06-22

    Symmetrical FeZn hybrids of human HbA have been used to measure K(1)(alpha) and K(1)(beta), the dissociation constants for the binding of a single molecule of oxygen to unliganded HbA at an alpha subunit and at a beta subunit, respectively. The kinetic constants, l(1)'(alpha) and l(1)'(beta), for the combination of the first CO molecule to unliganded HbA at an alpha or a beta subunit, respectively, were also measured. Measurements were carried out between pH 6 and pH 8 in the presence and absence of inositol hexaphosphate (IHP). Both equilibrium constants exhibit a significant Bohr effect in the absence of IHP. The addition of IHP to a concentration of 0.1 mM increases both dissociation constants in a pH-dependent manner with the result that both Bohr effects are greatly reduced. These results require a negative thermodynamic linkage between the binding of a single oxygen at either an alpha or a beta subunit and the binding of IHP to the T quaternary structure of HbA. Although the beta hemes are relatively near the IHP binding site, a linkage between that site and the alpha hemes, such that the binding of a single oxygen molecule to the heme of one alpha subunit reduces the affinity of the T state for IHP, requires communication across the molecule. l(1)'(alpha) exhibits a very slight pH dependence, with a maximum variation of 20%, while l(1)'(beta) varies with pH three times as much. IHP has no effect on the pH dependence of either rate constant but reduces l(1)'(alpha) marginally, 20%, and l(1)'(beta) by 2-fold at all pH values.

  15. A Trp474Cys mutation in the alpha-subunit of beta-hexosaminidase causes a subacute encephalopathic form of G{sub M2} gangliosidosis, type 1

    Energy Technology Data Exchange (ETDEWEB)

    Petroulakis, E.; Cao, Z.; Salo, T. [Univ. of Manitoba, Winnipeg (Canada)] [and others

    1994-09-01

    Mutations in the HEXA gene that encodes the {alpha}-subunit of the heterodimeric lysosomal enzyme {beta}-hexosaminidase A, or Hex A ({alpha}{beta}), cause G{sub M2} gangliosidosis, type 1. The infantile form (Tay-Sachs disease) results when there is no residual Hex A activity, while less severe and more variable clinical phenotypes result when residual Hex A activity is present. A non-Jewish male who presented with an acute psychotic episode at age 16 was diagnosed with a subacute encephalopathic form of G{sub M2} gangliosidosis. At age 19, chronic psychosis with intermittent acute exacerbations remains the most disabling symptom in this patient and his affected brother although both exhibit some ataxia and moderately severe dysarthria. We have found a 4 bp insertion (+TATC 1278) associated with infantile Tay-Sachs disease on one allele; no previously identified mutation was found on the second allele. SSCP analysis detected a shift in exon 13 and sequencing revealed a G1422C mutation in the second allele that results in a Trp474Cys substitution. The presence of the mutation was confirmed by the loss of HaeIII and ScrFI sites in exon 13 PCR products from the subjects and their father. The mutation was introduced into the {alpha}-subunit cDNA and Hex S ({alpha}{alpha}) and Hex A ({alpha}{beta}) were transiently expressed in monkey COS-7 cells. The Trp474Cys mutant protein had approximately 5% and 12% of wild-type Hex S and Hex A activity, respectively. Western blot analysis revealed a small amount of residual mature {alpha}-subunit and a normal level of precursor protein. We conclude that the Trp474Cys mutation is the cause of the Hex A deficiency associated with a subacute (juvenile-onset) phenotype in this patient. Like other mutations in exon 13 of HEXA, it appears to affect intracellular processing. Studies of the defect in intracellular processing are in progress.

  16. Aluminum-rich mesoporous MFI - type zeolite single crystals

    DEFF Research Database (Denmark)

    Kustova, Marina; Kustov, Arkadii; Christensen, Christina Hviid

    2005-01-01

    are characterized by X-ray powder diffraction (XRD), scanning electron microscopy (SEM), ammonia temperature programmed desorption (NH3-TPD), and N-2 adsorption measurements. The obtained zeolites combine the high crystallinity and the characteristic micropores of zeolites with an intracrystalline mesopore system....... With this technique, only zeolites with relatively low Al contents were reported (Si/Al ratio about 100). In this work, the preparation of aluminum-rich mesoporous MFI-type zeolite single crystals (Si/Al similar to 16-50) using aluminum isopropoxide as the aluminum Source is reported for the first time. All samples...

  17. Single electron transistor with P-type sidewall spacer gates.

    Science.gov (United States)

    Lee, Jung Han; Li, Dong Hua; Lee, Joung-Eob; Kang, Kwon-Chil; Kim, Kyungwan; Park, Byung-Gook

    2011-07-01

    A single-electron transistor (SET) is one of the promising solutions to overcome the scaling limit of the Metal-Oxide-Semiconductor Field Effect Transistor (MOSFET). Up to now, various kinds of SETs are being proposed and SETs with a dual gate (DG) structure using an electrical potential barrier have been demonstrated for room temperature operation. To operate DG-SETs, however, extra bias of side gates is necessary. It causes new problems that the electrode for side gates and the extra bias for electrical barrier increase the complexity in circuit design and operation power consumption, respectively. For the reason, a new mechanism using work function (WF) difference is applied to operate a SET at room temperature by three electrodes. Its structure consists of an undoped active region, a control gate, n-doped source/drain electrodes, and metal/silicide or p-type silicon side gates, and a SET with metal/silicide gates or p-type silicon gates forms tunnel barriers induced by work function between an undoped channel and grounded side gates. Via simulation, the effectiveness of the new mechanism is confirmed through various silicide materials that have different WF values. Furthermore, by considering the realistic conditions of the fabrication process, SET with p-type sidewall spacer gates was designed, and its brief fabrication process was introduced. The characteristics of its electrical barrier and the controllability of its control gate were also confirmed via simulation. Finally, a single-hole transistor with n-type sidewall spacer gates was designed.

  18. Differences in the phenotypic effects of mutations in homologous MrpA and MrpD subunits of the multi-subunit Mrp-type Na+/H+antiporter.

    Science.gov (United States)

    Morino, Masato; Ogoda, Shinichiro; Krulwich, Terry Ann; Ito, Masahiro

    2017-01-01

    Mrp antiporters are the sole antiporters in the Cation/Proton Antiporter 3 family of transporter databases because of their unusual structural complexity, 6-7 hydrophobic proteins that function as a hetero-oligomeric complex. The two largest and homologous subunits, MrpA and MrpD, are essential for antiport activity and have direct roles in ion transport. They also show striking homology with proton-conducting, membrane-embedded Nuo subunits of respiratory chain complex I of bacteria, e.g., Escherichia coli. MrpA has the closest homology to the complex I NuoL subunit and MrpD has the closest homology to the complex I NuoM and N subunits. Here, introduction of mutations in MrpD, in residues that are also present in MrpA, led to defects in antiport function and/or complex formation. No significant phenotypes were detected in strains with mutations in corresponding residues of MrpA, but site-directed changes in the C-terminal region of MrpA had profound effects, showing that the MrpA C-terminal region has indispensable roles in antiport function. The results are consistent with a divergence in adaptations that support the roles of MrpA and MrpD in secondary antiport, as compared to later adaptations supporting homologs in primary proton pumping by the respiratory chain complex I.

  19. Difference in Perseverative Errors during a Visual Attention Task with Auditory Distractors in Alpha-9 Nicotinic Receptor Subunit Wild Type and Knock-Out Mice

    Directory of Open Access Journals (Sweden)

    Pascal Jorratt

    2017-11-01

    Full Text Available The auditory efferent system is a neural network that originates in the auditory cortex and projects to the cochlear receptor through olivocochlear (OC neurons. Medial OC neurons make cholinergic synapses with outer hair cells (OHCs through nicotinic receptors constituted by α9 and α10 subunits. One of the physiological functions of the α9 nicotinic receptor subunit (α9-nAChR is the suppression of auditory distractors during selective attention to visual stimuli. In a recent study we demonstrated that the behavioral performance of alpha-9 nicotinic receptor knock-out (KO mice is altered during selective attention to visual stimuli with auditory distractors since they made less correct responses and more omissions than wild type (WT mice. As the inhibition of the behavioral responses to irrelevant stimuli is an important mechanism of the selective attention processes, behavioral errors are relevant measures that can reflect altered inhibitory control. Errors produced during a cued attention task can be classified as premature, target and perseverative errors. Perseverative responses can be considered as an inability to inhibit the repetition of an action already planned, while premature responses can be considered as an index of the ability to wait or retain an action. Here, we studied premature, target and perseverative errors during a visual attention task with auditory distractors in WT and KO mice. We found that α9-KO mice make fewer perseverative errors with longer latencies than WT mice in the presence of auditory distractors. In addition, although we found no significant difference in the number of target error between genotypes, KO mice made more short-latency target errors than WT mice during the presentation of auditory distractors. The fewer perseverative error made by α9-KO mice could be explained by a reduced motivation for reward and an increased impulsivity during decision making with auditory distraction in KO mice.

  20. Difference in Perseverative Errors during a Visual Attention Task with Auditory Distractors in Alpha-9 Nicotinic Receptor Subunit Wild Type and Knock-Out Mice.

    Science.gov (United States)

    Jorratt, Pascal; Delano, Paul H; Delgado, Carolina; Dagnino-Subiabre, Alexies; Terreros, Gonzalo

    2017-01-01

    The auditory efferent system is a neural network that originates in the auditory cortex and projects to the cochlear receptor through olivocochlear (OC) neurons. Medial OC neurons make cholinergic synapses with outer hair cells (OHCs) through nicotinic receptors constituted by α9 and α10 subunits. One of the physiological functions of the α9 nicotinic receptor subunit (α9-nAChR) is the suppression of auditory distractors during selective attention to visual stimuli. In a recent study we demonstrated that the behavioral performance of alpha-9 nicotinic receptor knock-out (KO) mice is altered during selective attention to visual stimuli with auditory distractors since they made less correct responses and more omissions than wild type (WT) mice. As the inhibition of the behavioral responses to irrelevant stimuli is an important mechanism of the selective attention processes, behavioral errors are relevant measures that can reflect altered inhibitory control. Errors produced during a cued attention task can be classified as premature, target and perseverative errors. Perseverative responses can be considered as an inability to inhibit the repetition of an action already planned, while premature responses can be considered as an index of the ability to wait or retain an action. Here, we studied premature, target and perseverative errors during a visual attention task with auditory distractors in WT and KO mice. We found that α9-KO mice make fewer perseverative errors with longer latencies than WT mice in the presence of auditory distractors. In addition, although we found no significant difference in the number of target error between genotypes, KO mice made more short-latency target errors than WT mice during the presentation of auditory distractors. The fewer perseverative error made by α9-KO mice could be explained by a reduced motivation for reward and an increased impulsivity during decision making with auditory distraction in KO mice.

  1. The beta subunit of the type I Fcepsilon receptor is a target for peptides inhibiting IgE-mediated secretory response of mast cells.

    Science.gov (United States)

    Andrásfalvy, Márton; Péterfy, Hajna; Tóth, Gábor; Matkó, János; Abramson, Jakub; Kerekes, Krisztina; Vámosi, György; Pecht, Israel; Erdei, Anna

    2005-09-01

    Peptides originally derived from complement component C3a were earlier shown to inhibit the type I FcepsilonR (FcepsilonRI)-mediated degranulation of mucosal type mast cells. In the present study, we show that C3a7, a peptide with a natural sequence, and its modified derivative, C3a9, are powerful inhibitors of the above response of both serosal and mucosal type mastocytes. We demonstrate that these peptides inhibit FcepsilonRI-induced membrane proximal events, suppress phosphorylation of the FcepsilonRI beta subunit, the protein tyrosine kinase Lyn, as well as the transient rise in free cytosolic Ca2+ level. The late phase of cellular response was also inhibited, as demonstrated by the reduced TNF-alpha secretion. Experiments using two independent methods provided evidence that the interaction site of complement-derived peptides is the FcepsilonRI beta-chain. This was further supported by fluorescence confocal microscopic colocalization and resonance energy transfer measurements. Taken together, these results suggest the presence of distinct "activating" and "inhibitory" motifs in the C3a sequence. Response to both is in balance under physiologic conditions. Furthermore, present data predict that such inhibitory peptides may serve as potent agents for future therapeutic intervention.

  2. Identification of Plasmodium falciparum Translation Initiation eIF2β Subunit: Direct Interaction with Protein Phosphatase Type 1

    Czech Academy of Sciences Publication Activity Database

    Tellier, G.; Lenne, A.; Cailliau-Maggio, K.; Cabezas-Cruz, A.; Valdés, James J.; Martoriati, A.; Aliouat, El M.; Gosset, P.; Delaire, B.; Fréville, A.; Pierrot, C.; Khalife, J.

    2016-01-01

    Roč. 7, MAY 26 (2016), č. článku 777. ISSN 1664-302X Institutional support: RVO:60077344 Keywords : Plasmodium falciparum * Protein Phosphatase type1 * eIF2b * protein-protein interaction * translation complex Subject RIV: EE - Microbiology, Virology Impact factor: 4.076, year: 2016

  3. Glucocorticoids specifically enhance L-type calcium current amplitude and affect calcium channel subunit expression in the mouse hippocampus

    NARCIS (Netherlands)

    Chameau, P.; Qin, Y.; Spijker, S.; Smit, A.B.; Joels, M.

    2007-01-01

    Previous studies have shown that corticosterone enhances whole cell calcium currents in CA1 pyramidal neurons, through a pathway involving binding of glucocorticoid receptor homodimers to the DNA. We examined whether glucocorticoids show selectivity for L- over N-type of calcium currents. Moreover,

  4. Glucocorticoids specifically enhance L-type calcium current amplitude and affect calcium channel subunit expression in the mouse hippocampus.

    NARCIS (Netherlands)

    Chameau, P.J.P.; Qin, Y.J.; Smit, G.; Joëls, M.

    2007-01-01

    Previous studies have shown that corticosterone enhances whole cell calcium currents in CA1 pyramidal neurons, through a pathway involving binding of glucocorticoid receptor homodimers to the DNA. We examined whether glucocorticoids show selectivity for L- over N-type of calcium currents. Moreover,

  5. Phenotypic consequences of deletion of the {gamma}{sub 3}, {alpha}{sub 5}, or {beta}{sub 3} subunit of the type A {gamma}-aminobutyric acid receptor in mice

    Energy Technology Data Exchange (ETDEWEB)

    Culia, C.T.; Stubbs, L.J.; Montgomery, C.S.; Russell, L.B.; Rinchik, E.M. [Oak Ridge National Lab., TN (United States)

    1994-03-29

    Three genes (Gabrg3, Gabra5, and Gabrb3) encoding the {gamma}{sub 3}, {alpha}{sub 5}, and {beta}{sub 3} subunits of the type A {gamma}-aminobutyric acid receptor, respectively, are known to map near the pink-eyed dilution (p) locus in mouse chromosome 7. This region shares homology with a segment of human chromosome 15 that is implicated in Angelman syndrome, an inherited neurobehavioral disorder. By mapping Gabrg3-Gabra5-Gabrb3-telomere. Like Gabrb3, neither the Gabra5 nor Gabrg3 gene is functionally imprinted in adult mouse brain. Mice deleted for all three subunits die at birth with a cleft palate, although there are rare survivors ({approximately} 5%) that do not have a cleft palate but do exhibit a neurological abnormality characterized by tremor, jerky gait, and runtiness. The authors have previously suggested that deficiency of the {beta}{sub 3} subunit may be responsible for the clefting defect. Most notably, however, in this report they describe mice carrying two overlapping, complementing p deletions that fail to express the {gamma}{sub 3} transcript, as well as mice from another line that express neither the {gamma}{sub 3} nor {alpha}{sub 5} transcripts. Surprisingly, mice from both of these lines are phenotypically normal and do not exhibit any of the neurological symptoms characteristic of the rare survivors that are deleted for all three ({gamma}{sub 3}, {alpha}{sub 5}, and {beta}{sub 3}) subunits. These mice therefore provide a whole-organism type A {gamma}-aminobutyric-acid receptor background that is devoid of any receptor subtypes that normally contain the {gamma}{sub 3} and/or {alpha}{sub 5} subunits. The absence of an overt neurological phenotype in mice lacking the {gamma}{sub 3} and/or {alpha}{sub 5} subunits also suggests that mutations in these genes are unlikely to provide useful animal models for Angelman syndrome in humans.

  6. Inhibitory function of adapter-related protein complex 2 alpha 1 subunit in the process of nuclear translocation of human immunodeficiency virus type 1 genome

    International Nuclear Information System (INIS)

    Kitagawa, Yukiko; Kameoka, Masanori; Shoji-Kawata, Sanae; Iwabu, Yukie; Mizuta, Hiroyuki; Tokunaga, Kenzo; Fujino, Masato; Natori, Yukikazu; Yura, Yoshiaki; Ikuta, Kazuyoshi

    2008-01-01

    The transfection of human cells with siRNA against adapter-related protein complex 2 alpha 1 subunit (AP2α) was revealed to significantly up-regulate the replication of human immunodeficiency virus type 1 (HIV-1). This effect was confirmed by cell infection with vesicular stomatitis virus G protein-pseudotyped HIV-1 as well as CXCR4-tropic and CCR5-tropic HIV-1. Viral adsorption, viral entry and reverse transcription processes were not affected by cell transfection with siRNA against AP2α. In contrast, viral nuclear translocation as well as the integration process was significantly up-regulated in cells transfected with siRNA against AP2α. Confocal fluorescence microscopy revealed that a subpopulation of AP2α was not only localized in the cytoplasm but was also partly co-localized with lamin B, importin β and Nup153, implying that AP2α negatively regulates HIV-1 replication in the process of nuclear translocation of viral DNA in the cytoplasm or the perinuclear region. We propose that AP2α may be a novel target for disrupting HIV-1 replication in the early stage of the viral life cycle

  7. Proteolytic processing of the L-type Ca2+ channel alpha11.2 subunit in neurons [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Olivia R. Buonarati

    2017-07-01

    Full Text Available Background: The L-type Ca2+ channel Cav1.2 is a prominent regulator of neuronal excitability, synaptic plasticity, and gene expression. The central element of Cav1.2 is the pore-forming α11.2 subunit. It exists in two major size forms, whose molecular masses have proven difficult to precisely determine. Recent work suggests that α11.2 is proteolytically cleaved between the second and third of its four pore-forming domains (Michailidis et al,. 2014. Methods: To better determine the apparent molecular masses (MRof the α11.2 size forms, extensive systematic immunoblotting of brain tissue as well as full length and C-terminally truncated α11.2 expressed in HEK293 cells was conducted using six different region–specific antibodies against α11.2. Results: The full length form of α11.2 migrated, as expected, with an apparent MR of ~250 kDa. A shorter form of comparable prevalence with an apparent MR of ~210 kDa could only be detected in immunoblots probed with antibodies recognizing α11.2 at an epitope 400 or more residues upstream of the C-terminus. Conclusions: The main two size forms of α11.2 are the full length form and a shorter form, which lacks ~350 distal C-terminal residues. Midchannel cleavage as suggested by Michailidis et al. (2014 is at best minimal in brain tissue.

  8. Gene structure and mutations of glutaryl-coenzyme A dehydrogenase: Impaired association of enzyme subunits that is due to an A421V substitution causes glutaric acidemia type I in the Amish

    Energy Technology Data Exchange (ETDEWEB)

    Biery, B.J.; Stein, D.E.; Goodman, S.I. [Univ. of Colorado School of Medicine, Denver, CO (United States)] [and others

    1996-11-01

    The structure of the human glutaryl coenzyme A dehydrogenase (GCD) gene was determined to contain 11 exons and to span {approximately}7 kb. Fibroblast DNA from 64 unrelated glutaric academia type I (GA1) patients was screened for mutations by PCR amplification and analysis of SSCP. Fragments with altered electrophoretic mobility were subcloned and sequenced to detect mutations that caused GA1. This report describes the structure of the GCD gene, as well as point mutations and polymorphisms found in 7 of its 11 exons. Several mutations were found in more than one patient, but no one prevalent mutation was detected in the general population. As expected from pedigree analysis, a single mutant allele causes GA1 in the Old Order Amish of Lancaster County, Pennsylvania. Several mutations have been expressed in Escherichia coli, and all produce diminished enzyme activity. Reduced activity in GCD encoded by the A421V mutation in the Amish may be due to impaired association of enzyme subunits. 13 refs., 5 figs., 3 tabs.

  9. Early continuous white noise exposure alters l-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunit glutamate receptor 2 and gamma-aminobutyric acid type a receptor subunit beta3 protein expression in rat auditory cortex.

    Science.gov (United States)

    Xu, Jinghong; Yu, Liping; Zhang, Jiping; Cai, Rui; Sun, Xinde

    2010-02-15

    Auditory experience during the postnatal critical period is essential for the normal maturation of auditory function. Previous studies have shown that rearing infant rat pups under conditions of continuous moderate-level noise delayed the emergence of adult-like topographic representational order and the refinement of response selectivity in the primary auditory cortex (A1) beyond normal developmental benchmarks and indefinitely blocked the closure of a brief, critical-period window. To gain insight into the molecular mechanisms of these physiological changes after noise rearing, we studied expression of the AMPA receptor subunit GluR2 and GABA(A) receptor subunit beta3 in the auditory cortex after noise rearing. Our results show that continuous moderate-level noise rearing during the early stages of development decreases the expression levels of GluR2 and GABA(A)beta3. Furthermore, noise rearing also induced a significant decrease in the level of GABA(A) receptors relative to AMPA receptors. However, in adult rats, noise rearing did not have significant effects on GluR2 and GABA(A)beta3 expression or the ratio between the two units. These changes could have a role in the cellular mechanisms involved in the delayed maturation of auditory receptive field structure and topographic organization of A1 after noise rearing. Copyright 2009 Wiley-Liss, Inc.

  10. Lack of Association Between Type 2 Diabetes and the 3673G / A and 9041G / A Gene Variants of Vitamin K Epoxide Reductase Complex Subunit 1 (VKORC1).

    Science.gov (United States)

    Ozbayer, Cansu; Kurt, Hulyam; Nur Kebapci, Medine; Turgut Cosan, Didem; Colak, Ertugrul; Veysi Gunes, Hasan; Degirmenci, Irfan

    2018-02-22

    The vitamin K epoxide reductase complex subunit 1 (VKORC1) gene is expressed in many tissue types, and encodes the VKORC1 protein, which is a key enzyme in the vitamin K cycle. Although researchers have focused on the effects of vitamin K on glucose metabolism, and on its role in the development of type 2 diabetes (T2DM), no consensus has yet been reached. Therefore, here we aimed to investigate the association between VKORC1 variants and the risk of T2DM. The 3673G / A (rs9923231) and 9041G / A (rs7294) VKORC1 variants were investigated by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 100 control individuals and 100 patients with T2DM. The genomic regions were amplified by PCR; amplicons were digested using the AciI and NciI enzymes and visualized by agarose gel electrophoresis. The genotype frequencies of the 3673G / A variants were GG (22%), GA (56%), and AA (22%) in the control group and GG (19%), GA (52%), and AA (29%) in patients with T2DM (p > 0.05). The genotype frequencies of the 9041G / A variants were GG (37%), GA (53%), and AA (10%) in the control group and GG (46%), GA (45%), and AA (9%) in patients with T2DM (p > 0.05). In conclusion, we found no significant correlation between the control group and patients with T2DM, with regard to the different genetic models of the 3673G / A and 9041G / A variants. These data suggest that these VKORC1 gene variants may not be linked to T2DM.

  11. Characterization of the regulatory subunit of Yarrowia lipolytica cAMP-dependent protein kinase. Evidences of a monomeric protein.

    Science.gov (United States)

    Kronberg, Florencia; Giacometti, Romina; Ruiz-Herrera, José; Passeron, Susana

    2011-05-01

    cAMP-dependent protein kinase (PKA) catalytic (C) and regulatory (R) subunits from Yarrowia lipolytica are encoded by single genes, TPK1 and RKA1, respectively. Here we performed the heterologous expression, purification and characterization of the R subunit from Y. lipolytica yeast cells, and explored the main biochemical features of the PKA. The purified recombinant R, active and capable to interact with C subunit was used to prepare highly specific polyclonal antiserum. Sucrose-gradient centrifugation and gel filtration analysis of both recombinant and native R revealed the monomeric nature of this subunit. Hydrodynamic parameters of the holoenzyme indicated that Y. lipolytica PKA is a dimer of 90 kDa composed of an R subunit of 42 kDa and a C subunit of 39 kDa. The identification of the N-terminal sequence was carried out by mass spectrometry analysis of the purified native R subunit. The differences between N-terminal sequences of R subunits from Y. lipolytica and other organisms, particularly a short linker that spans the inhibitory site, were discussed as the possible cause of the lack of dimerization. R was identified as a type II subunit since our results indicated that it was phosphorylated in vivo by C at S124 identified by anti-phospho-PKA substrate (RRXS/T) antibody. Copyright © 2011 Elsevier Inc. All rights reserved.

  12. Subunit heterogeneity in the lima bean lectin.

    Science.gov (United States)

    Roberts, D D; Etzler, M E; Goldstein, I J

    1982-08-10

    Three forms of lectin (components I, II, and III) from lima beans (Phaseolus lunatus) have been purified on an affinity support containing the synthetic type A blood group trisaccharide alpha-D-GalNAc-(1 leads to 3)-[alpha-L-Fuc-(1 leads to 2)]-beta-D-Gal-(1 leads to). Conversion of components I and II to component III has been achieved by reduction in 10(-2) M dithiothreitol. Isoelectric focusing of lima bean lectin in the presence of 8 M urea and beta-mercaptoethanol revealed charge heterogeneity of the lectin subunits. Three major subunit classes of apparent pI 7.05, 6.65, and 6.45, designated alpha, beta, and alpha', respectively, were identified; they occur in a relative abundance of 2:5:3. Green lima beans harvested before maturity lacked the alpha' subunit (pI 6.45) which appears to accumulate during seed maturation. The three subunits are glycoproteins of identical size and immunochemical reactivity. Identical NH2-terminal sequences were found for the three subunits. Amino acid analysis and tryptic peptide mapping indicated that the observed charge heterogeneity is probably due to differences in the primary structure of the subunits. Studies of subunit composition of charge isolectins provided evidence of nonrandom subunit assembly. A model is proposed involving pairing of a pI 6.65 subunit with either a pI 7.06 or 6.45 subunit to form dimeric units. Possible roles for subunit heterogeneity and ordered subunit assembly in determining the metal and sugar binding properties of lima bean lectin are discussed.

  13. Evaluation of Zinc-alpha-2-Glycoprotein and Proteasome Subunit beta-Type 6 Expression in Prostate Cancer Using Tissue Microarray Technology.

    LENUS (Irish Health Repository)

    2010-07-23

    Prostate cancer (CaP) is a significant cause of illness and death in males. Current detection strategies do not reliably detect the disease at an early stage and cannot distinguish aggressive versus nonaggressive CaP leading to potential overtreatment of the disease and associated morbidity. Zinc-alpha-2-glycoprotein (ZAG) and proteasome subunit beta-Type 6 (PSMB-6) were found to be up-regulated in the serum of CaP patients with higher grade tumors after 2-dimensional difference gel electrophoresis analysis. The aim of this study was to investigate if ZAG and PSMB-6 were also overexpressed in prostatic tumor tissue of CaP patients. Immunohistochemical analysis was performed on CaP tissue microarrays with samples from 199 patients. Confirmatory gene expression profiling for ZAG and PSMB-6 were performed on 4 cases using Laser Capture Microdissection and TaqMan real-time polymerase chain reaction. ZAG expression in CaP epithelial cells was inversely associated with Gleason grade (benign prostatic hyperplasia>G3>G4\\/G5). PSMB-6 was not expressed in either tumor or benign epithelium. However, strong PSMB-6 expression was noted in stromal and inflammatory cells. Our results indicate ZAG as a possible predictive marker of Gleason grade. The inverse association between grade and tissue expression with a rising serum protein level is similar to that seen with prostate-specific antigen. In addition, the results for both ZAG and PSMB-6 highlight the challenges in trying to associate the protein levels in serum with tissue expression.

  14. Voltage-Gated Sodium Channel β1/β1B Subunits Regulate Cardiac Physiology and Pathophysiology

    Directory of Open Access Journals (Sweden)

    Nnamdi Edokobi

    2018-04-01

    Full Text Available Cardiac myocyte contraction is initiated by a set of intricately orchestrated electrical impulses, collectively known as action potentials (APs. Voltage-gated sodium channels (NaVs are responsible for the upstroke and propagation of APs in excitable cells, including cardiomyocytes. NaVs consist of a single, pore-forming α subunit and two different β subunits. The β subunits are multifunctional cell adhesion molecules and channel modulators that have cell type and subcellular domain specific functional effects. Variants in SCN1B, the gene encoding the Nav-β1 and -β1B subunits, are linked to atrial and ventricular arrhythmias, e.g., Brugada syndrome, as well as to the early infantile epileptic encephalopathy Dravet syndrome, all of which put patients at risk for sudden death. Evidence over the past two decades has demonstrated that Nav-β1/β1B subunits play critical roles in cardiac myocyte physiology, in which they regulate tetrodotoxin-resistant and -sensitive sodium currents, potassium currents, and calcium handling, and that Nav-β1/β1B subunit dysfunction generates substrates for arrhythmias. This review will highlight the role of Nav-β1/β1B subunits in cardiac physiology and pathophysiology.

  15. Complex Dynamic Scene Perception: Effects of Attentional Set on Perceiving Single and Multiple Event Types

    Science.gov (United States)

    Sanocki, Thomas; Sulman, Noah

    2013-01-01

    Three experiments measured the efficiency of monitoring complex scenes composed of changing objects, or events. All events lasted about 4 s, but in a given block of trials, could be of a single type (single task) or of multiple types (multitask, with a total of four event types). Overall accuracy of detecting target events amid distractors was…

  16. Effects of Traumatic Stress Induced in the Juvenile Period on the Expression of Gamma-Aminobutyric Acid Receptor Type A Subunits in Adult Rat Brain

    Directory of Open Access Journals (Sweden)

    Cui Yan Lu

    2017-01-01

    Full Text Available Studies have found that early traumatic experience significantly increases the risk of posttraumatic stress disorder (PTSD. Gamma-aminobutyric acid (GABA deficits were proposed to be implicated in development of PTSD, but the alterations of GABA receptor A (GABAAR subunits induced by early traumatic stress have not been fully elucidated. Furthermore, previous studies suggested that exercise could be more effective than medications in reducing severity of anxiety and depression but the mechanism is unclear. This study used inescapable foot-shock to induce PTSD in juvenile rats and examined their emotional changes using open-field test and elevated plus maze, memory changes using Morris water maze, and the expression of GABAAR subunits (γ2, α2, and α5 in subregions of the brain in the adulthood using western blotting and immunohistochemistry. We aimed to observe the role of GABAAR subunits changes induced by juvenile trauma in the pathogenesis of subsequent PTSD in adulthood. In addition, we investigated the protective effects of exercise for 6 weeks and benzodiazepine (clonazepam for 2 weeks. This study found that juvenile traumatic stress induced chronic anxiety and spatial memory loss and reduced expression of GABAAR subunits in the adult rat brains. Furthermore, exercise led to significant improvement as compared to short-term BZ treatment.

  17. Subunit compensation and plasticity of synaptic GABAA receptors induced by ethanol in α4 subunit knockout mice

    Directory of Open Access Journals (Sweden)

    Asha eSuryanarayanan

    2011-09-01

    Full Text Available There is considerable evidence that ethanol (EtOH potentiates γ-aminobutyric acid type A receptor (GABAAR action, but only GABAARs containing δ subunits appear sensitive to low mM EtOH. The α4 and δ subunits co-assemble into GABAARs which are relatively highly expressed at extrasynaptic locations in the dentate gyrus where they mediate tonic inhibition. We previously demonstrated reversible- and time-dependent changes in GABAAR function and subunit composition in rats after single-dose EtOH intoxication. We concluded that early tolerance to EtOH occurs by over-activation and subsequent internalization of EtOH-sensitive extrasynaptic α4βδ-GABAARs. Based on this hypothesis, any highly EtOH-sensitive GABAARs should be subject to internalization following exposure to suitably high EtOH doses. To test this, we studied the GABAARs in mice with a global deletion of the α4 subunit (KO. The dentate granule cells (DGCs of these mice exhibited greatly reduced tonic currents and greatly reduced potentiation by acutely applied EtOH, whereas synaptic currents showed heightened sensitivity to low EtOH concentrations. The hippocampus of naive KO mice showed reduced δ subunit protein levels, but increased α2, and γ2 levels compared to wild-type (WT controls, suggesting at least partial compensation by these subunits in synaptic, highly EtOH-sensitive GABAARs of KO mice. In WT mice, cross-linking and Western blot analysis at 1 h after an EtOH challenge (3.5 g/kg, i.p. revealed increased intracellular fraction of the α1, α4 and δ, but not α2, α5 or γ2 subunits. By contrast, we observed significant internalization of α1, α2, δ, and γ2 subunits after a similar EtOH challenge in KO mice. Synaptic currents from naïve KO mice were more sensitive to potentiation by zolpidem (0.3 μM, requiring α1/α2, inactive at α4/5 GABAARs than those from naïve WT mice. At 1 h after EtOH, synaptic currents of WT mice were unchanged, whereas those of KO mice

  18. 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

  19. Synthesis of single wall carbon nanotubes from a lamellar type ...

    Indian Academy of Sciences (India)

    Wintec

    These nanotubes are applicable to store more hydrogen. Keywords. AlPO4-L; single wall carbon nanotubes. 1. Introduction. Carbon nanotubes (Iijima 1991) are nano-scale structures formed by self assembly. They possess excellent chemical and physical properties (Rodney and Donald 1995; Chen et al 1998) that make ...

  20. Full HPV typing by a single restriction enzyme.

    Science.gov (United States)

    Santiago, Enrique; Camacho, Lucía; Junquera, Maria Luisa; Vázquez, Fernando

    2006-09-01

    Restriction fragment length polymorphism (RFLP) methods for genotyping genital human papillomavirus (HPV) are considered labor consuming and constrained by the reduced set of restriction enzymes capable of detecting specific mutations. However, we think that these methods have not taken full advantage of the high diversity of the known restriction enzymes. We have set out to find the best restriction enzyme for HPV typing. An extensive search for enzymes was carried out by combining statistical methods and database information. The search maximized the discrimination between high- and low-risk types by examining the sequence of the L1 gene flanked by primers MY09/11. Different electrophoretic resolutions and two variations of the RFLP method were considered. HpyCH4V is the best enzyme for discriminating between risk types. Moreover, HpyCH4V generates different patterns for virtually all the HPV types. The typical pattern consists of two or three fragments, which facilitates typing in mixed infections. The typing of a set of clinical samples confirmed the expectations. This result illustrates the possibilities of statistical methods to exploit the high diversity of restriction enzymes in order to classify samples in a pre-established hierarchy of types for which DNA sequences are known.

  1. Dissecting interdomain communication within cAPK regulatory subunit type IIβ using enhanced amide hydrogen/deuterium exchange mass spectrometry (DXMS)

    Science.gov (United States)

    Zawadzki, Kerri M.; Hamuro, Yoshitomo; Kim, Jack S.; Garrod, Siv; Stranz, David D.; Taylor, Susan S.; Woods, Virgil L.

    2003-01-01

    cAMP-dependent protein kinase (cAPK) is a heterotetramer containing a regulatory (R) subunit dimer bound to two catalytic (C) subunits and is involved in numerous cell signaling pathways. The C-subunit is activated allosterically when two cAMP molecules bind sequentially to the cAMP-binding domains, designated A and B (cAB-A and cAB-B, respectively). Each cAMP-binding domain contains a conserved Arg residue that is critical for high-affinity cAMP binding. Replacement of this Arg with Lys affects cAMP affinity, the structural integrity of the cAMP-binding domains, and cAPK activation. To better understand the local and long-range effects that the Arg-to-Lys mutation has on the dynamic properties of the R-subunit, the amide hydrogen/deuterium exchange in the RIIβ subunit was probed by electrospray mass spectrometry. Mutant proteins containing the Arg-to-Lys substitution in either cAMP-binding domain were deuterated for various times and then, prior to mass spectrometry analysis, subjected to pepsin digestion to localize the deuterium incorporation. Mutation of this Arg in cAB-A (Arg230) causes an increase in amide hydrogen exchange throughout the mutated domain that is beyond the modest and localized effects of cAMP removal and is indicative of the importance of this Arg in domain organization. Mutation of Arg359 (cAB-B) leads to increased exchange in the adjacent cAB-A domain, particularly in the cAB-A domain C-helix that lies on top of the cAB-B domain and is believed to be functionally linked to the cAB-B domain. This interdomain communication appears to be a unidirectional pathway, as mutation of Arg230 in cAB-A does not effect dynamics of the cAB-B domain. PMID:12930997

  2. 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.

  3. lemmingA encodes the Apc11 subunit of the APC/C in Drosophila melanogaster that forms a ternary complex with the E2-C type ubiquitin conjugating enzyme, Vihar and Morula/Apc2

    Directory of Open Access Journals (Sweden)

    Nagy Olga

    2012-03-01

    Full Text Available Abstract Background Ubiquitin-dependent protein degradation is a critical step in key cell cycle events, such as metaphase-anaphase transition and mitotic exit. The anaphase promoting complex/cyclosome (APC/C plays a pivotal role in these transitions by recognizing and marking regulatory proteins for proteasomal degradation. Its overall structure and function has been elucidated mostly in yeasts and mammalian cell lines. The APC/C is, however, a multisubunit assembly with at least 13 subunits and their function and interaction within the complex is still relatively uncharacterized, particularly in metazoan systems. Here, lemming (lmg mutants were used to study the APC/C subunit, Apc11, and its interaction partners in Drosophila melanogaster. Results The lmg gene was initially identified through a pharate adult lethal P element insertion mutation expressing developmental abnormalities and widespread apoptosis in larval imaginal discs and pupal abdominal histoblasts. Larval neuroblasts were observed to arrest mitosis in a metaphase-like state with highly condensed, scattered chromosomes and frequent polyploidy. These neuroblasts contain high levels of both cyclin A and cyclin B. The lmg gene was cloned by virtue of the lmg03424 P element insertion which is located in the 5' untranslated region. The lemming locus is transcribed to give a 2.0 kb mRNA that contains two ORFs, lmgA and lmgB. The lmgA ORF codes for a putative protein with more than 80% sequence homology to the APC11 subunit of the human APC/C. The 85 amino acid protein also contains a RING-finger motif characteristic of known APC11 subunits. The lmgA ORF alone was sufficient to rescue the lethal and mitotic phenotypes of the lmg138 null allele and to complement the temperature sensitive lethal phenotype of the APC11-myc9 budding yeast mutant. The LmgA protein interacts with Mr/Apc2, and they together form a binding site for Vihar, the E2-C type ubiquitin conjugating enzyme. Despite

  4. Synthesis of single wall carbon nanotubes from a lamellar type ...

    Indian Academy of Sciences (India)

    Wintec

    circular disc of fine copper mesh covered with collodion. Images were taken using a JEOL JSM-2000 EX electron microscope operated at an acceleration voltage of 200 kV. 3. Results and discussion. Figure 1 shows the as-synthesized AlPO4-L which was found to be of short range ordered lamellar type molecular ...

  5. Development of n- and p-type Doped Perovskite Single Crystals Using Solid-State Single Crystal Growth (SSCG) Technique

    Science.gov (United States)

    2017-10-09

    tried. Among them “n- and p-type doped” BaTiO3 single crystals have been successfully fabricated . And their bi- crystals containing a twin or twist...boundary are also fabricated using diffusion bonding process of two single crystal plates. These results demonstrate that the SSCG (solid-state...or Bridgman method have critical limitations; high production cost and compositional inhomogeneity throughout the crystal. These limitations result

  6. Single particle analysis of thylakoid proteins from Thermosynechococcus elongatus and Synechocystis 6803 : Localization of the CupA subunit of NDH-1

    NARCIS (Netherlands)

    Folea, I. Mihaela; Zhang, Pengpeng; Nowaczyk, Marc M.; Ogawa, Teruo; Aro, Eva-Marl; Boekema, Egbert J.; Aro, Eva-Mari

    The larger protein complexes of the cyanobacterial photosynthetic membrane of Thermosynechoccus elongatus and Synechocystis 6803 were studied by single particle electron microscopy after detergent solubilization, without any purification steps. Besides the "standard" L-shaped NDH-1L complex, related

  7. The porphyrin-iron hybrid hemoglobins. Absence of the Fe-His bonds in one type of subunits favors a deoxy-like structure with low oxygen affinity.

    Science.gov (United States)

    Fujii, M; Hori, H; Miyazaki, G; Morimoto, H; Yonetani, T

    1993-07-25

    Protoporphyrin-protoheme hybrid hemoglobins (Hb), in which the protohemes (Fe) in either the alpha- or beta-subunits were substituted with protoporphyrins IX (PP) alpha(PP)2 beta(Fe)2 and alpha(Fe)2 beta(PP)2 have been prepared. The structural and functional properties of these hybrid Hbs were investigated by measuring oxygen equilibrium curves and proton nuclear magnetic resonance spectra. The equilibrium constants of the first ligand, K1, observed for alpha(PP)2 beta(Fe)2 were much smaller than K1 values of HbA. The effects of pH and inositol hexaphosphate on K1 were substantially diminished. On the other hand, K1 values of alpha(Fe)2 beta(PP)2 were similar to those of HbA, including the pH and inositol hexaphosphate effects. The deoxy forms of alpha(PP)2 beta(Fe)2 and alpha(Fe)2 beta(PP)2 showed exchangeable proton resonances at 11 and 14 parts/million arising from the hydrogen bonds at the alpha 1 beta 2 contact in a deoxy-like structure. In the liganded form, these signals were dependent upon solution conditions. As K1 became larger, the reduction in the intensity of these signals was observed for both liganded forms. The resonance position of E11 Val originating from the beta subunits of alpha(PP)2 beta(Fe-CO)2 also varied in accordance with K1. We compare properties of PP-Fe hybrids with those of Co-Fe and Ni-Fe hybrids and conclude that the first oxygen binding to the beta heme may be linked to the metal-proximal His interaction in the alpha subunits. However, the first oxygen binding to the alpha heme is linked minimally to the metal-proximal His interaction in the beta subunits but may be correlated instead to the position of E11 Val relative to the porphyrin plane in the beta subunits.

  8. Molecular cloning and analysis of zebrafish voltage-gated sodium channel beta subunit genes: implications for the evolution of electrical signaling in vertebrates

    Directory of Open Access Journals (Sweden)

    Zhong Tao P

    2007-07-01

    Full Text Available Abstract Background Action potential generation in excitable cells such as myocytes and neurons critically depends on voltage-gated sodium channels. In mammals, sodium channels exist as macromolecular complexes that include a pore-forming alpha subunit and 1 or more modulatory beta subunits. Although alpha subunit genes have been cloned from diverse metazoans including flies, jellyfish, and humans, beta subunits have not previously been identified in any non-mammalian species. To gain further insight into the evolution of electrical signaling in vertebrates, we investigated beta subunit genes in the teleost Danio rerio (zebrafish. Results We identified and cloned single zebrafish gene homologs for beta1-beta3 (zbeta1-zbeta3 and duplicate genes for beta4 (zbeta4.1, zbeta4.2. Sodium channel beta subunit loci are similarly organized in fish and mammalian genomes. Unlike their mammalian counterparts, zbeta1 and zbeta2 subunit genes display extensive alternative splicing. Zebrafish beta subunit genes and their splice variants are differentially-expressed in excitable tissues, indicating tissue-specific regulation of zbeta1-4 expression and splicing. Co-expression of the genes encoding zbeta1 and the zebrafish sodium channel alpha subunit Nav1.5 in Chinese Hamster Ovary cells increased sodium current and altered channel gating, demonstrating functional interactions between zebrafish alpha and beta subunits. Analysis of the synteny and phylogeny of mammalian, teleost, amphibian, and avian beta subunit and related genes indicated that all extant vertebrate beta subunits are orthologous, that beta2/beta4 and beta1/beta3 share common ancestry, and that beta subunits are closely related to other proteins sharing the V-type immunoglobulin domain structure. Vertebrate sodium channel beta subunit genes were not identified in the genomes of invertebrate chordates and are unrelated to known subunits of the para sodium channel in Drosophila. Conclusion The

  9. Structural domains of the human GABAA receptor β3 subunit involved in the actions of pentobarbital

    Science.gov (United States)

    Serafini, Ruggero; Bracamontes, John; Steinbach, Joe Henry

    2000-01-01

    This study was conducted to search for the residues of the β3 subunit which affect pentobarbital action on the γ-aminobutyric acid type A (GABAA) receptor. Three chimeras were constructed by joining the GABAA receptor β3 subunit to the ρ1 subunit. For each chimera, the N-terminal sequence was derived from the β3 subunit and the C-terminal sequence from the ρ1 subunit, with junctions located between the membrane-spanning regions M2 and M3, in the middle of M2, or in M1, respectively.In receptors obtained by the coexpression of α1 with the chimeric subunits, in contrast with those obtained by the coexpression of α1 and β3, pentobarbital exhibited lower potentiation of GABA-evoked responses, and in the direct gating of Cl− currents, an increase in the EC50 together with a marked decrease in the relative maximal efficacy compared with that of GABA.Estimates of the channel opening probability through variance analysis and single-channel recordings of one chimeric subunit showed that the reduced relative efficacy for gating largely resulted from an increase in gating by GABA, with little change in efficacy of pentobarbital.A fit of the time course of the response by the predictions of a class of reaction schemes is consistent with the conclusion that the change in the concentration dependence of activation by pentobarbital is due to a change in pentobarbital affinity for the receptor. Therefore, the data suggest that residues of the β3 subunit involved in pentobarbital binding to GABAA receptors are located downstream from the middle of the M2 region. PMID:10790149

  10. Single Microwave-Photon Detector using an Artificial Lambda-type Three-Level System

    Science.gov (United States)

    2016-01-11

    Single microwave -photon detector using an artificial Λ-type three-level system Kunihiro Inomata,1∗†, Zhirong Lin,1†, Kazuki Koshino,2, William D...are those of the author and are not necessarily endorsed by the United States Government. Single microwave -photon detector using an artificial Λ-type...in both the optical and the microwave domains. However, the energy of mi- crowave quanta are four to five orders of magnitude less than their optical

  11. Estimating Intervention Effects across Different Types of Single-Subject Experimental Designs: Empirical Illustration

    Science.gov (United States)

    Moeyaert, Mariola; Ugille, Maaike; Ferron, John M.; Onghena, Patrick; Heyvaert, Mieke; Beretvas, S. Natasha; Van den Noortgate, Wim

    2015-01-01

    The purpose of this study is to illustrate the multilevel meta-analysis of results from single-subject experimental designs of different types, including AB phase designs, multiple-baseline designs, ABAB reversal designs, and alternating treatment designs. Current methodological work on the meta-analysis of single-subject experimental designs…

  12. In vivo and in vitro adjuvant activities of the B subunit of Type IIb heat-labile enterotoxin (LT-IIb-B5) from Escherichia coli

    Science.gov (United States)

    Liang, Shuang; Hosur, Kavita B.; Nawar, Hesham F.; Russell, Michael W.; Connell, Terry D.; Hajishengallis, George

    2009-01-01

    The pentameric B subunit of the Escherichia coli LT-IIb enterotoxin (LT-IIb-B5) activates TLR2 signaling in macrophages. Herein we demonstrate that LT-IIb-B5, in contrast to a TLR2-nonbinding point mutant, induces functional activation of bone marrow-derived dendritic cells and stimulates CD4+ T cell proliferation, activities which suggested that LT-IIb-B5 might function as an adjuvant in vivo. Indeed, in an intranasal mouse immunization model, LT-IIb-B5 augmented specific mucosal and serum antibody responses to a co-administered immunogen, at levels which were almost comparable to those induced by intact LT-IIb holotoxin, a potent but toxic adjuvant. Therefore, LT-IIb-B5 displays useful adjuvant properties which, combined with lack of enterotoxicity and relative stability against degradation, may find application in mucosal vaccines. PMID:19450646

  13. Heterogeneity of L-type calcium channel alpha 1 subunits: stereoselective discrimination of different populations by the novel 1,4-dihydropyridine B 874-67.

    Science.gov (United States)

    Lakitsch, M; Knaus, H G; Topar, G; Romanin, C; Boer, R; Flockerzi, D; Striessnig, J; Schindler, H; Hoeltje, H D; Glossmann, H

    1993-02-01

    The basic (pKa = 8.49) 1,4-dihydropyridine B 874-67 [[3-(C1R,2S)- 2-methylamino-1-phenylpropyl]-5-methyl-1,4-dihydro-2,6-dimethyl-(4R)-4-( 3-nitrophenyl)pyridine-3,5-dicarboxylate hydrochloride] has unique properties; it can discriminate two populations of alpha 1 subunits in 1,4-dihydropyridine-sensitive calcium channels labeled with the neutral 1,4-dihydropyridine (+)-[3H]PN 200-110. The two populations, which occur in proportions of approximately 2:1 in rabbit skeletal muscle membranes and highly purified calcium channel preparations, differ approximately 20-fold in their affinity. The corresponding diastereomer, B 874-66, and other 1,4-dihydropyridines (neutral, basic, or permanently charged) do not share this property. The two populations were observed at 2 degrees, 22 degrees, and 37 degrees in similar proportions. Heterogeneity was also observed for guinea pig heart membrane calcium channels labeled with (+)-[3H]PN 200-110. Heterotropic allosteric regulators, Ca2+, and Mg2+, but not Ba2+ and Ni2+, abolished the discriminatory activity of B 874-67 at 2 degrees and 22 degrees, regardless of whether binding of the neutral 1,4-dihydropyridine was stimulated or inhibited. It is proposed that the two alpha 1 subunit populations differ with respect to their 1,4-dihydropyridine binding domain. The structural basis for the two populations is unclear but may relate to the functional heterogeneity of membrane-bound and highly purified calcium channel preparations previously observed by others.

  14. An antisense oligodeoxynucleotide targeted against the type IIβ regulatory subunit mRNA of protein kinase inhibits cAMP-induced differentiation in HL-60 leukemia cells without affecting phorbol ester effects

    International Nuclear Information System (INIS)

    Tortora, G.; Clair, T.; Cho-Chung, Y.S.

    1990-01-01

    The type II β regulatory subunit of cAMP-dependent protein kinase (RII β ) has been hypothesized to play an important role in the growth inhibition and differentiation induced by site-selective cAMP analogs in human cancer cells, but direct proof of this function has been lacking. To address this tissue, HL-60 human promyelocytic leukemia cells were exposed to RII β antisense synthetic oligodeoxynucleotide, and the effects on cAMP-induced growth regulation were examined. Exposure of these cells to RII β antisense oligodeoxynucleotide resulted in a decrease in cAMP analog-induced growth inhibition and differentiation without apparent effect on differentiation induced by phorbol esters. This loss in cAMP growth regulatory function correlated with a decrease in basal and induced levels of RII β protein. Exposure to RII β sense, RI α and RII α antisense, or irrelevant oligodeoxynucleotides had no such effect. These results show that the RII β regulatory subunit of protein kinase plays a critical role in the cAMP-induced growth regulation of HL-60 leukemia cells

  15. Glycine receptor subunits expression in the developing rat retina.

    Science.gov (United States)

    Sánchez-Chávez, Gustavo; Velázquez-Flores, Miguel Ángel; Ruiz Esparza-Garrido, Ruth; Salceda, Rocío

    2017-09-01

    Glycine receptor (GlyR) consists of two α (1-4) and three β subunits. Considerable evidence indicates that the adult retina expresses the four types of α subunits; however, the proportion of these subunits in adult and immature retina is almost unknown. In this report we have studied mRNA and the protein expression of GlyR subunits in the retina during postnatal rat development by Real-Time qRT-PCR and western blot. mRNA and protein expression indicated a gradual increase of the α1, α3, α4 and β GlyR subunits during postnatal ages tested. The mRNA β subunit showed higher expression levels (∼3 fold) than those observed for the α1 and α3 subunits. Very interestingly, the α2 GlyR subunit had the highest expression in the retina, even in the adult. These results revealed the expression of GlyR at early postnatal ages, supporting its role in retina development. In addition, our results indicated that the adult retina expressed a high proportion of the α2 subunit, suggesting the expression of monomeric and/or heteromeric receptors. A variety of studies are needed to further characterize the role of the specific subunits in both adult and immature retina. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Rapid single step subcloning procedure by combined action of type II and type IIs endonucleases with ligase

    Directory of Open Access Journals (Sweden)

    Klingenspor Martin

    2007-11-01

    Full Text Available Abstract Background The subcloning of a DNA fragment from an entry vector into a destination vector is a routinely performed task in molecular biology labs. Results We here present a novel benchtop procedure to achieve rapid recombination into any destination vector of choice with the sole requirement of an endonuclease recognition site. The method relies on a specifically designed entry vector and the combined action of type II and type IIs endonucleases with ligase. The formulation leads to accumulation of a single stable cloning product representing the desired insert carrying destination vector. Conclusion The described method provides a fast single step procedure for routine subcloning from an entry vector into a series of destination vectors with the same restriction enzyme recognition site.

  17. Rapid single step subcloning procedure by combined action of type II and type IIs endonucleases with ligase.

    Science.gov (United States)

    Fromme, Tobias; Klingenspor, Martin

    2007-11-26

    The subcloning of a DNA fragment from an entry vector into a destination vector is a routinely performed task in molecular biology labs. We here present a novel benchtop procedure to achieve rapid recombination into any destination vector of choice with the sole requirement of an endonuclease recognition site. The method relies on a specifically designed entry vector and the combined action of type II and type IIs endonucleases with ligase. The formulation leads to accumulation of a single stable cloning product representing the desired insert carrying destination vector. The described method provides a fast single step procedure for routine subcloning from an entry vector into a series of destination vectors with the same restriction enzyme recognition site.

  18. Use of an α3-β4 nicotinic acetylcholine receptor subunit concatamer to characterize ganglionic receptor subtypes with specific subunit composition reveals species-specific pharmacologic properties

    Science.gov (United States)

    Stokes, Clare; Papke, Roger L.

    2012-01-01

    Drug development for nicotinic acetylcholine receptors (nAChR) is challenged by subtype diversity arising from variations in subunit composition. On-target activity for neuronal heteromeric receptors is typically associated with CNS receptors that contain α4 and other subunits, while off-target activity could be associated with ganglionic-type receptors containing α3β4 binding sites and other subunits, including β4, β2, α5, or α3 as a structural subunit in the pentamer. Additional interest in α3 β4 α5-containing receptors arises from genome-wide association studies linking these genes, and a single nucleotide polymorphism (SNP) in α5 in particular, to lung cancer and heavy smoking. While α3 and β4 readily form receptors in expression system such as the Xenopus oocyte, since α5 is not required for function, simple co-expression approaches may under-represent α5-containing receptors. We used a concatamer of human α3 and β4 subunits to form ligand-binding domains, and show that we can force the insertions of alternative structural subunits into the functional pentamers. These α3β4 variants differ in sensitivity to ACh, nicotine, varenicline, and cytisine. Our data indicated lower efficacy for varenicline and cytisine than expected for β4-containing receptors, based on previous studies of rodent receptors. We confirm that these therapeutically important α4 receptor partial agonists may present different autonomic-based side-effect profiles in humans than will be seen in rodent models, with varenicline being more potent for human than rat receptors and cytisine less potent. Our initial characterizations failed to find functional effects of the α5 SNP. However, our data validate this approach for further investigations. PMID:22580377

  19. Sodium channel β subunits: emerging targets in channelopathies.

    Science.gov (United States)

    O'Malley, Heather A; Isom, Lori L

    2015-01-01

    Voltage-gated sodium channels (VGSCs) are responsible for the initiation and propagation of action potentials in excitable cells. VGSCs in mammalian brain are heterotrimeric complexes of α and β subunits. Although β subunits were originally termed auxiliary, we now know that they are multifunctional signaling molecules that play roles in both excitable and nonexcitable cell types and with or without the pore-forming α subunit present. β subunits function in VGSC and potassium channel modulation, cell adhesion, and gene regulation, with particularly important roles in brain development. Mutations in the genes encoding β subunits are linked to a number of diseases, including epilepsy, sudden death syndromes like SUDEP and SIDS, and cardiac arrhythmia. Although VGSC β subunit-specific drugs have not yet been developed, this protein family is an emerging therapeutic target.

  20. The use of a single volar locking plate for AO C3-type distal radius fractures.

    Science.gov (United States)

    Earp, Brandon E; Foster, Brian; Blazar, Philip E

    2015-12-01

    A single volar locking plate (VLP) is now frequently used for open reduction and internal fixation (ORIF) of many types of distal radius fractures. Comminuted intra-articular distal radius fractures (AO C3-type) are typically the most challenging to surgically treat. No studies directly address the adequacy of a VLP alone for maintaining reduction of AO C-type fractures. We hypothesized that a single VLP provides an effective method for maintaining reduction for these fractures. We retrospectively evaluated radiographs of a series of AO C-type fractures. Seventy-seven patients with 77 AO C3-type fractures were identified from billing records and were eligible for the study. All patients were treated by fellowship-trained hand surgeons. Radiographs at the time of union were compared to those from immediately postoperatively. Sixty-nine of 77 (89.6 %) fractures treated with VLP fixation alone for AO C3-type distal radius fractures united without loss of reduction. Eight of 77 (10.4 %) patients treated with VLP for AO C3 fractures lost reduction. The most common fracture fragment to lose reduction was the lunate fossa (5 of 8); loss of reduction of the scaphoid fossa die-punch fragment (2 of 8) and the radial styloid (1 of 8) were also seen. The majority (89.6 %) of AO C3-type fractures treated with a single volar locking plate come to union without loss of reduction. Level IV.

  1. Proteome analysis reveals phosphorylation of ATP synthase beta -subunit in human skeletal muscle and proteins with potential roles in type 2 diabetes

    DEFF Research Database (Denmark)

    Højlund, Kurt; Wrzesinski, Krzysztof; Larsen, Peter Mose

    2003-01-01

    quantitate a large number of proteins and their post-translational modifications simultaneously and is a powerful tool to study polygenic diseases like type 2 diabetes. Using this approach on human skeletal muscle biopsies, we have identified eight potential protein markers for type 2 diabetes in the fasting...

  2. Structure and Subunit Arrangement of the A-type ATP Synthase Complex from the Archaeon Methanococcus jannaschii Visualized by Electron Microscopy

    NARCIS (Netherlands)

    Coskun, Ünal; Chaban, Yuriy L.; Lingl, Astrid; Müller, Volker; Keegstra, Wilko; Boekema, Egbert J.; Grüber, Gerhard; Gruber, 27460

    2004-01-01

    In Archaea, bacteria, and eukarya, ATP provides metabolic energy for energy-dependent processes. It is synthesized by enzymes known as A-type or F-type ATP synthase, which are the smallest rotatory engines in nature. Here, we report the first projected structure of an intact A1A0 ATP synthase from

  3. Immune Response in Calves Vaccinated with Type Three Secretion System Antigens and Shiga Toxin 2B Subunit of Escherichia coli O157:H7.

    Directory of Open Access Journals (Sweden)

    Luisina Martorelli

    Full Text Available Ruminants are the primary reservoir of Shiga-toxin producing Escherichia coli (STEC O157:H7 and the main source of infection for humans. The aim of this study was to assess the immunogenic properties of a candidate vaccine consisting on the recombinant proteins of E. coli O157:H7 IntiminC280, the carboxy-terminal fraction of Intimin γ, EspB and the fusion protein between the B subunit of Stx2 and Brucella Lumazine Synthase (BLS(BLS-Stx2B, in Holstein Fresian calves.To accomplish this goal we vaccinated calves with two doses of different vaccine formulations: 2 antigens (IntiminC280, EspB, 3 antigens (IntiminC280, EspB, BLS-Stx2B, BLS-Stx2B alone and a control non-vaccinated group. All antigens were expressed as recombinant proteins in E. coli. Specific IgG titres increased in vaccinated calves and the inclusion of BLS-Stx2B in the formulation seems to have a stimulatory effect on the humoral response to IntiminC280 and EspB after the booster. The neutralizing activity of antibodies against these two antigens was assessed in Red Blood Cell lysis assays and adherence to Hep-2 cells as a correlate of T3SS activity. Both sera from animals vaccinated with 2 or 3 antigens inhibited both virulence properties. Serological response to Stx2 was observed in animals vaccinated only with BLS-Stx2B and with 3 antigens and neutralization of Stx2 cytotoxicity was also observed in both groups. In conclusion, immunization of calves with BLS-Stx2B, IntiminC280 and EspB elicited a potent humoral response able to neutralize Shiga toxin 2 cytotoxity and the T3SS virulence properties in vitro. These results suggest that this formulation is a good candidate vaccine to reduce STEC shedding in cattle and needs to be further assessed in vivo.

  4. Immune Response in Calves Vaccinated with Type Three Secretion System Antigens and Shiga Toxin 2B Subunit of Escherichia coli O157:H7.

    Science.gov (United States)

    Martorelli, Luisina; Garbaccio, Sergio; Vilte, Daniel A; Albanese, Adriana A; Mejías, María P; Palermo, Marina S; Mercado, Elsa C; Ibarra, Cristina E; Cataldi, Angel A

    2017-01-01

    Ruminants are the primary reservoir of Shiga-toxin producing Escherichia coli (STEC) O157:H7 and the main source of infection for humans. The aim of this study was to assess the immunogenic properties of a candidate vaccine consisting on the recombinant proteins of E. coli O157:H7 IntiminC280, the carboxy-terminal fraction of Intimin γ, EspB and the fusion protein between the B subunit of Stx2 and Brucella Lumazine Synthase (BLS)(BLS-Stx2B), in Holstein Fresian calves.To accomplish this goal we vaccinated calves with two doses of different vaccine formulations: 2 antigens (IntiminC280, EspB), 3 antigens (IntiminC280, EspB, BLS-Stx2B), BLS-Stx2B alone and a control non-vaccinated group. All antigens were expressed as recombinant proteins in E. coli. Specific IgG titres increased in vaccinated calves and the inclusion of BLS-Stx2B in the formulation seems to have a stimulatory effect on the humoral response to IntiminC280 and EspB after the booster. The neutralizing activity of antibodies against these two antigens was assessed in Red Blood Cell lysis assays and adherence to Hep-2 cells as a correlate of T3SS activity. Both sera from animals vaccinated with 2 or 3 antigens inhibited both virulence properties. Serological response to Stx2 was observed in animals vaccinated only with BLS-Stx2B and with 3 antigens and neutralization of Stx2 cytotoxicity was also observed in both groups. In conclusion, immunization of calves with BLS-Stx2B, IntiminC280 and EspB elicited a potent humoral response able to neutralize Shiga toxin 2 cytotoxity and the T3SS virulence properties in vitro. These results suggest that this formulation is a good candidate vaccine to reduce STEC shedding in cattle and needs to be further assessed in vivo.

  5. Ultrastructure and Large Subunit rDNA-Based Phylogeny of Sphaerodinium cracoviense, an Unusual Freshwater Dinoflagellate with a Novel Type of Eyespot

    DEFF Research Database (Denmark)

    Craveiro, S.C.; Moestrup, Øjvind; Daugbjerg, N.

    2010-01-01

    Sphaerodinium cracoviense was collected near Cracow, Poland, and analysed by light microscopy, scanning electron microscopy, and serial-section transmission electron microscopy. Thecae showed a peridinioid type of plate arrangement with unusual numbers in the anterior intercalary and postcingular...

  6. Inactivation of single-chain urokinase-type plasminogen activator by thrombin in human subjects

    NARCIS (Netherlands)

    Braat, E. A.; Levi, M. [=Marcel M.; Bos, R.; Haverkate, F.; Lassen, M. R.; de Maat, M. P.; Rijken, D. C.

    1999-01-01

    Thrombin cleaves single-chain urokinase-type plasminogen activator (scu-PA) into a virtually inactive two-chain form (tcu-PA/T), a process that may protect a blood clot from early fibrinolysis. It is not known under what circumstances tcu-PA/T can be generated in vivo. We have studied the occurrence

  7. Identifying types of physical activity with a single accelerometer: Evaluating laboratory trained algorithms in daily life

    NARCIS (Netherlands)

    Cuba Gyllensten, I.; Bonomi, A.G.

    2011-01-01

    Accurate identification of physical activity types has been achieved in laboratory conditions using single-site accelerometers and classification algorithms. This methodology is then applied to free-living subjects to determine activity behaviour. This study aimed at analysing the reproducibility of

  8. Expression profile of G-protein βγ subunit gene transcripts in the mouse olfactory sensory epithelia

    Directory of Open Access Journals (Sweden)

    Aaron eSathyanesan

    2013-06-01

    Full Text Available Heterotrimeric G-proteins mediate a variety of cellular functions, including signal transduction in sensory neurons of the olfactory system. Whereas the Gα subunits in these neurons are well characterized, the gene transcript expression profile of Gβγ subunits is largely missing. Here we report our comprehensive expression analysis to identify Gβ and Gγ subunit gene transcripts in the mouse main olfactory epithelium (MOE and the vomeronasal organ (VNO. Our reverse transcriptase PCR (RT-PCR and realtime qPCR analyses of all known Gβ(β1,2,3,4,5 and Gγ(γ1,2,2t,3,4,5,7,8,10,11,12,13 subunits indicate presence of multiple Gβ and Gγ subunit gene transcripts in the MOE and the VNO at various expression levels. These results are supported by our RNA in situ hybridization (RISH experiments, which reveal the expression patterns of two Gβ subunits and four Gγ subunits in the MOE as well as one Gβ and four Gγ subunits in the VNO. Using double-probe fluorescence RISH and line intensity scan analysis of the RISH signals of two dominant Gβγ subunits, we show that Gγ13 is expressed in mature olfactory sensory neurons (OSNs, while Gβ1 is present in both mature and immature OSNs. Interestingly, we also found Gβ1 to be the dominant Gβsubunit in the VNO and present throughout the sensory epithelium. In contrast, we found diverse expression of Gγ subunit gene transcripts with Gγ2, Gγ3 and Gγ13 in the Gαi2-expressing neuronal population, while Gγ8 is expressed in both layers. Further, we determined the expression of these Gβγ gene transcripts in three post-natal developmental stages (p0, 7 and 14 and found their cell-type specific expression remains largely unchanged, except the transient expression of Gγ2 in a single basal layer of cells in the MOE during P7 and P14. Taken together, our comprehensive expression analyses reveal cell-type specific gene expression of multiple Gβ and Gγ in sensory neurons of the olfactory system.

  9. Single-Cell DNA barcoding using sequences from the small subunit rRNA and internal transcribed spacer region identifies new species of Trichonympha and Trichomitopsis from the hindgut of the termite Zootermopsis angusticollis.

    Directory of Open Access Journals (Sweden)

    Vera Tai

    Full Text Available To aid in their digestion of wood, lower termites are known to harbour a diverse community of prokaryotes as well as parabasalid and oxymonad protist symbionts. One of the best-studied lower termite gut communities is that of Zootermopsis angusticollis which has been known for almost 100 years to possess 3 species of Trichonympha (T. campanula, T. collaris, and T. sphaerica, 1 species of Trichomitopsis (T. termopsidis, as well as smaller flagellates. We have re-assessed this community by sequencing the small subunit (SSU rRNA gene and the internal transcribed spacer (ITS region from a large number of single Trichonympha and Trichomitopsis cells for which morphology was also documented. Based on phylogenetic clustering and sequence divergence, we identify 3 new species: Trichonympha postcylindrica, Trichomitopsis minor, and Trichomitopsis parvus spp. nov. Once identified by sequencing, the morphology of the isolated cells for all 3 new species was re-examined and found to be distinct from the previously described species: Trichonympha postcylindrica can be morphologically distinguished from the other Trichonympha species by an extension on its posterior end, whereas Trichomitopsis minor and T. parvus are smaller than T. termopsidis but similar in size to each other and cannot be distinguished based on morphology using light microscopy. Given that Z. angusticollis has one of the best characterized hindgut communities, the near doubling of the number of the largest and most easily identifiable symbiont species suggests that the diversity of hindgut symbionts is substantially underestimated in other termites as well. Accurate descriptions of the diversity of these microbial communities are essential for understanding hindgut ecology and disentangling the interactions among the symbionts, and molecular barcoding should be a priority for these systems.

  10. Single-Cell DNA barcoding using sequences from the small subunit rRNA and internal transcribed spacer region identifies new species of Trichonympha and Trichomitopsis from the hindgut of the termite Zootermopsis angusticollis.

    Science.gov (United States)

    Tai, Vera; James, Erick R; Perlman, Steve J; Keeling, Patrick J

    2013-01-01

    To aid in their digestion of wood, lower termites are known to harbour a diverse community of prokaryotes as well as parabasalid and oxymonad protist symbionts. One of the best-studied lower termite gut communities is that of Zootermopsis angusticollis which has been known for almost 100 years to possess 3 species of Trichonympha (T. campanula, T. collaris, and T. sphaerica), 1 species of Trichomitopsis (T. termopsidis), as well as smaller flagellates. We have re-assessed this community by sequencing the small subunit (SSU) rRNA gene and the internal transcribed spacer (ITS) region from a large number of single Trichonympha and Trichomitopsis cells for which morphology was also documented. Based on phylogenetic clustering and sequence divergence, we identify 3 new species: Trichonympha postcylindrica, Trichomitopsis minor, and Trichomitopsis parvus spp. nov. Once identified by sequencing, the morphology of the isolated cells for all 3 new species was re-examined and found to be distinct from the previously described species: Trichonympha postcylindrica can be morphologically distinguished from the other Trichonympha species by an extension on its posterior end, whereas Trichomitopsis minor and T. parvus are smaller than T. termopsidis but similar in size to each other and cannot be distinguished based on morphology using light microscopy. Given that Z. angusticollis has one of the best characterized hindgut communities, the near doubling of the number of the largest and most easily identifiable symbiont species suggests that the diversity of hindgut symbionts is substantially underestimated in other termites as well. Accurate descriptions of the diversity of these microbial communities are essential for understanding hindgut ecology and disentangling the interactions among the symbionts, and molecular barcoding should be a priority for these systems.

  11. Esthetic outcome of single implant crowns following type 1 and type 3 implant placement: a systematic review.

    Science.gov (United States)

    Shi, Jun-Yu; Wang, Ren; Zhuang, Long-Fei; Gu, Ying-Xin; Qiao, Shi-Chong; Lai, Hong-Chang

    2015-07-01

    The aim of the current review was to systematically appraise the esthetic outcome of soft tissue around single implant crowns following type 1 and type 3 implants placement in published dental literature. A PubMed, Embase, and the Cochrane Central Register of Controlled Trials search up to March 2013 was conducted for articles published in the dental literature and limited to human trials with no language restricted. Furthermore, the reference lists of related articles were systematically screened, and additional manual searches were also performed. The primary outcome was pink esthetics score (PES). The electronic search in the database of PubMed, Embase, and the Cohrane Central Register of Controlled Trials resulted in the identification of 463 titles. These titles were initially screened by the two independent reviewers for possible inclusion. Screening the abstracts and titles led to 28 articles for future full-text consideration. From these articles, 18 studies were excluded. Manual search identified one article. After quality assessment, eight studies were included in this review. This review showed that no significant difference of PES index could be found between type 1 and type 3 implant placement. According to the current evidence, short-term esthetic outcomes of peri-implant soft tissue did not show significant difference following type 1 and type 3 implants placement with well-selected patients. However, caution should be taken for clinicians to extrapolate this result to all types of patients, as more randomized clinical trials are needed for long-term soft-tissue esthetic outcome in patients with high esthetic risk following type 1 implant placement. PES frequency, peri-implant condition and other risk factors for peri-implantitis are recommended to be reported for future studies. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  12. Scheduling of hybrid types of machines with two-machine flowshop as the first type and a single machine as the second type

    Science.gov (United States)

    Hsiao, Ming-Chih; Su, Ling-Huey

    2018-02-01

    This research addresses the problem of scheduling hybrid machine types, in which one type is a two-machine flowshop and another type is a single machine. A job is either processed on the two-machine flowshop or on the single machine. The objective is to determine a production schedule for all jobs so as to minimize the makespan. The problem is NP-hard since the two parallel machines problem was proved to be NP-hard. Simulated annealing algorithms are developed to solve the problem optimally. A mixed integer programming (MIP) is developed and used to evaluate the performance for two SAs. Computational experiments demonstrate the efficiency of the simulated annealing algorithms, the quality of the simulated annealing algorithms will also be reported.

  13. Cell type discovery and representation in the era of high-content single cell phenotyping.

    Science.gov (United States)

    Bakken, Trygve; Cowell, Lindsay; Aevermann, Brian D; Novotny, Mark; Hodge, Rebecca; Miller, Jeremy A; Lee, Alexandra; Chang, Ivan; McCorrison, Jamison; Pulendran, Bali; Qian, Yu; Schork, Nicholas J; Lasken, Roger S; Lein, Ed S; Scheuermann, Richard H

    2017-12-21

    A fundamental characteristic of multicellular organisms is the specialization of functional cell types through the process of differentiation. These specialized cell types not only characterize the normal functioning of different organs and tissues, they can also be used as cellular biomarkers of a variety of different disease states and therapeutic/vaccine responses. In order to serve as a reference for cell type representation, the Cell Ontology has been developed to provide a standard nomenclature of defined cell types for comparative analysis and biomarker discovery. Historically, these cell types have been defined based on unique cellular shapes and structures, anatomic locations, and marker protein expression. However, we are now experiencing a revolution in cellular characterization resulting from the application of new high-throughput, high-content cytometry and sequencing technologies. The resulting explosion in the number of distinct cell types being identified is challenging the current paradigm for cell type definition in the Cell Ontology. In this paper, we provide examples of state-of-the-art cellular biomarker characterization using high-content cytometry and single cell RNA sequencing, and present strategies for standardized cell type representations based on the data outputs from these cutting-edge technologies, including "context annotations" in the form of standardized experiment metadata about the specimen source analyzed and marker genes that serve as the most useful features in machine learning-based cell type classification models. We also propose a statistical strategy for comparing new experiment data to these standardized cell type representations. The advent of high-throughput/high-content single cell technologies is leading to an explosion in the number of distinct cell types being identified. It will be critical for the bioinformatics community to develop and adopt data standard conventions that will be compatible with these new

  14. INTRINSIC REGULATION OF HEMOGLOBIN EXPRESSION BY VARIABLE SUBUNIT INTERFACE STRENGTHS

    Science.gov (United States)

    Manning, James M.; Popowicz, Anthony M.; Padovan, Julio C.; Chait, Brian T.; Manning, Lois R.

    2012-01-01

    SUMMARY The expression of the six types of human hemoglobin subunits over time is currently considered to be regulated mainly by transcription factors that bind to upstream control regions of the gene (the “extrinsic” component of regulation). Here we describe how subunit pairing and further assembly to tetramers in the liganded state is influenced by the affinity of subunits for one another (the “intrinsic” component of regulation). The adult hemoglobin dimers have the strongest subunit interfaces and the embryonic hemoglobins are the weakest with fetal hemoglobins of intermediate strength, corresponding to the temporal order of their expression. These variable subunit binding strengths and the attenuating effects of acetylation contribute to the differences with which these hemoglobin types form functional O2-binding tetramers consistent with gene switching. PMID:22129306

  15. Finite Element Analysis of Doorframe Structure of Single Oblique Pole Type in Container Crane

    Science.gov (United States)

    Cheng, X. F.; Wu, F. Q.; Tang, G.; Hu, X.

    2017-07-01

    Compared with the composite type, the single oblique pole type has more advantages, such as simple structure, thrift steel and high safe overhead clearance. The finite element model of the single oblique pole type is established in nodes by ANSYS, and more details are considered when the model is simplified, such as the section of Girder and Boom, torque in Girder and Boom occurred by Machinery house and Trolley, density according to the way of simplification etc. The stress and deformation of ten observation points are compared and analyzed, when the trolley is in nine dangerous positions. Based on the result of analysis, six dangerous points are selected to provide reference for the detection and evaluation of container crane.

  16. Single Muscle Fiber Proteomics Reveals Fiber-Type-Specific Features of Human Muscle Aging

    Directory of Open Access Journals (Sweden)

    Marta Murgia

    2017-06-01

    Full Text Available Skeletal muscle is a key tissue in human aging, which affects different muscle fiber types unequally. We developed a highly sensitive single muscle fiber proteomics workflow to study human aging and show that the senescence of slow and fast muscle fibers is characterized by diverging metabolic and protein quality control adaptations. Whereas mitochondrial content declines with aging in both fiber types, glycolysis and glycogen metabolism are upregulated in slow but downregulated in fast muscle fibers. Aging mitochondria decrease expression of the redox enzyme monoamine oxidase A. Slow fibers upregulate a subset of actin and myosin chaperones, whereas an opposite change happens in fast fibers. These changes in metabolism and sarcomere quality control may be related to the ability of slow, but not fast, muscle fibers to maintain their mass during aging. We conclude that single muscle fiber analysis by proteomics can elucidate pathophysiology in a sub-type-specific manner.

  17. Crystallization and preliminary X-ray analysis of the C-terminal fragment of PorM, a subunit of the Porphyromonas gingivalis type IX secretion system.

    Science.gov (United States)

    Stathopulos, Julien; Cambillau, Christian; Cascales, Eric; Roussel, Alain; Leone, Philippe

    2015-01-01

    PorM is a membrane protein involved in the assembly of the type IX secretion system (T9SS) from Porphyromonas gingivalis, a major bacterial pathogen responsible for periodontal disease in humans. The periplasmic domain of PorM was overexpressed in Escherichia coli and purified. A fragment of the purified protein was obtained by limited proteolysis. Crystals of this fragment belonged to the tetragonal space group P4(3)2(1)2. Native and MAD data sets were recorded to 2.85 and 3.1 Å resolution, respectively, using synchrotron radiation.

  18. Multicolor imaging of cancer cells with fluorophore-tagged aptamers for single cell typing.

    Science.gov (United States)

    Wang, Song; Kong, Hao; Gong, Xiaoyun; Zhang, Sichun; Zhang, Xinrong

    2014-08-19

    The discrimination of the type of cancer cells remains challenging due to the subtle differences in their expression of membrane receptors. In this work, we developed a multicolor cell imaging method for distinguishing the type of cancer cells with fluorophore-tagged aptamers. We found that the interaction between aptamers and cancer cells was affected by both of the sequence of aptamers and the labeled dyes. As the co-ownership of biomarkers for different cancer cell lines, the fluorophore-tagged aptamers interacted with different cancer cell lines in different degree, resulting in a distinct color to discriminate the type of cancer cells at single cell level. Taking advantage of the cross-reactive ability of the fluorophore-tagged aptamers, we could not only distinguish the cancerous cells quickly from large quantities of noncancerous cells, but also identify the type of the cancerous cells. This work has potential application for cancer diagnostic and therapy in the future.

  19. Structural modeling of dahlia-type single-walled carbon nanohorn aggregates by molecular dynamics.

    Science.gov (United States)

    Hawelek, L; Brodka, A; Dore, John C; Hannon, Alex C; Iijima, S; Yudasaka, M; Ohba, T; Kaneko, K; Burian, A

    2013-09-19

    The structure of dahlia-type single-walled carbon nanohorn aggregates has been modeled by classical molecular dynamics simulations, and the validity of the model has been verified by neutron diffraction. Computer-generated models consisted of an outer part formed from single-walled carbon nanohorns with diameters of 20-50 Å and a length of 400 Å and an inner turbostratic graphite-like core with a diameter of 130 Å. The diffracted intensity and the pair correlation function computed for such a constructed model are in good agreement with the neutron diffraction experimental data. The proposed turbostratic inner core explains the occurrence of the additional (002) and (004) graphitic peaks in the diffraction pattern of the studied sample and provides information about the interior structure of the dahlia-type aggregates.

  20. RADIAL VELOCITIES OF GALACTIC O-TYPE STARS. II. SINGLE-LINED SPECTROSCOPIC BINARIES

    International Nuclear Information System (INIS)

    Williams, S. J.; Gies, D. R.; Hillwig, T. C.; McSwain, M. V.; Huang, W.

    2013-01-01

    We report on new radial velocity measurements of massive stars that are either suspected binaries or lacking prior observations. This is part of a survey to identify and characterize spectroscopic binaries among O-type stars with the goal of comparing the binary fraction of field and runaway stars with those in clusters and associations. We present orbits for HDE 308813, HD 152147, HD 164536, BD–16°4826, and HDE 229232, Galactic O-type stars exhibiting single-lined spectroscopic variation. By fitting model spectra to our observed spectra, we obtain estimates for effective temperature, surface gravity, and rotational velocity. We compute orbital periods and velocity semiamplitudes for each system and note the lack of photometric variation for any system. These binaries probably appear single-lined because the companions are faint and because their orbital Doppler shifts are small compared to the width of the rotationally broadened lines of the primary.

  1. Comprehensive Identification and Spatial Mapping of Habenular Neuronal Types Using Single-Cell RNA-Seq.

    Science.gov (United States)

    Pandey, Shristi; Shekhar, Karthik; Regev, Aviv; Schier, Alexander F

    2018-04-02

    The identification of cell types and marker genes is critical for dissecting neural development and function, but the size and complexity of the brain has hindered the comprehensive discovery of cell types. We combined single-cell RNA-seq (scRNA-seq) with anatomical brain registration to create a comprehensive map of the zebrafish habenula, a conserved forebrain hub involved in pain processing and learning. Single-cell transcriptomes of ∼13,000 habenular cells with 4× cellular coverage identified 18 neuronal types and dozens of marker genes. Registration of marker genes onto a reference atlas created a resource for anatomical and functional studies and enabled the mapping of active neurons onto neuronal types following aversive stimuli. Strikingly, despite brain growth and functional maturation, cell types were retained between the larval and adult habenula. This study provides a gene expression atlas to dissect habenular development and function and offers a general framework for the comprehensive characterization of other brain regions. Copyright © 2018 Elsevier Ltd. All rights reserved.

  2. Charge collection measurements in single-type column 3D sensors

    International Nuclear Information System (INIS)

    Scaringella, M.; Polyakov, A.; Sadrozinski, H.F.-W.; Bruzzi, M.; Tosi, C.; Boscardin, M.; Piemonte, C.; Pozza, A.; Ronchin, S.; Zorzi, N.; Dalla Betta, G.-F.

    2007-01-01

    We report on charge collection studies on 3D silicon detectors of single-type column n-diffusions in p-substrate, configured either as strip or as pad detectors. The charge is generated by penetrating beta particles from a 90 Sr source which, together with a scintillation counter, serves as an electron telescope. The charge collection as a function of bias voltage is compared with the depletion thickness derived from the measured C-V characteristics

  3. Charge collection measurements with p-type Magnetic Czochralski silicon single pad detectors

    International Nuclear Information System (INIS)

    Tosi, C.; Bruzzi, M.; Macchiolo, A.; Scaringella, M.; Petterson, M.K.; Sadrozinski, H.F.-W.; Betancourt, C.; Manna, N.; Creanza, D.; Boscardin, M.; Piemonte, C.; Zorzi, N.; Borrello, L.; Messineo, A.

    2007-01-01

    The charge collected from beta source particles in single pad detectors produced on p-type Magnetic Czochralski (MCz) silicon wafers has been measured before and after irradiation with 26 MeV protons. After a 1 MeV neutron equivalent fluence of 1x10 15 cm -2 the collected charge is reduced to 77% at bias voltages below 900 V. This result is compared with previous results from charge collection measurements

  4. Identification of soybean proteins from a single cell type: The root hair

    Energy Technology Data Exchange (ETDEWEB)

    Brechenmacher, Laurent; Nguyen, Tran H.; Hixson, Kim K.; Libault, Marc; Aldrich, Joshua T.; Pasa-Tolic, Ljiljana; Stacey, Gary

    2012-11-01

    Root hairs are a terminally differentiated single cell type, mainly involved in water and nutrient uptake from the soil. The soybean root hair cell represents an excellent model for the study of single cell systems biology. In this study, we identified 5702 proteins, with at least two peptides, from soybean root hairs using an accurate mass and time tag approach, establishing the most comprehensive proteome reference map of this single cell type. We also showed that trypsin is the most appropriate enzyme for soybean proteomic studies by performing an in silico digestion of the soybean proteome database using different proteases. Although the majority of proteins identified in this study are involved in basal metabolism, the function of others are more related to root hair formation/function and include proteins involved in nutrient uptake (transporters) or vesicular trafficking (cytoskeleton and RAB proteins). Interestingly, some of these proteins appear to be specifically expressed in root hairs and constitute very good candidates for further studies to elucidate unique features of this single cell model.

  5. C-terminal splice variants of P/Q-type Ca2+ channel CaV2.1 α1 subunits are differentially regulated by Rab3-interacting molecule proteins.

    Science.gov (United States)

    Hirano, Mitsuru; Takada, Yoshinori; Wong, Chee Fah; Yamaguchi, Kazuma; Kotani, Hiroshi; Kurokawa, Tatsuki; Mori, Masayuki X; Snutch, Terrance P; Ronjat, Michel; De Waard, Michel; Mori, Yasuo

    2017-06-02

    Voltage-dependent Ca 2+ channels (VDCCs) mediate neurotransmitter release controlled by presynaptic proteins such as the scaffolding proteins Rab3-interacting molecules (RIMs). RIMs confer sustained activity and anchoring of synaptic vesicles to the VDCCs. Multiple sites on the VDCC α 1 and β subunits have been reported to mediate the RIMs-VDCC interaction, but their significance is unclear. Because alternative splicing of exons 44 and 47 in the P/Q-type VDCC α 1 subunit Ca V 2.1 gene generates major variants of the Ca V 2.1 C-terminal region, known for associating with presynaptic proteins, we focused here on the protein regions encoded by these two exons. Co-immunoprecipitation experiments indicated that the C-terminal domain (CTD) encoded by Ca V 2.1 exons 40-47 interacts with the α-RIMs, RIM1α and RIM2α, and this interaction was abolished by alternative splicing that deletes the protein regions encoded by exons 44 and 47. Electrophysiological characterization of VDCC currents revealed that the suppressive effect of RIM2α on voltage-dependent inactivation (VDI) was stronger than that of RIM1α for the Ca V 2.1 variant containing the region encoded by exons 44 and 47. Importantly, in the Ca V 2.1 variant in which exons 44 and 47 were deleted, strong RIM2α-mediated VDI suppression was attenuated to a level comparable with that of RIM1α-mediated VDI suppression, which was unaffected by the exclusion of exons 44 and 47. Studies of deletion mutants of the exon 47 region identified 17 amino acid residues on the C-terminal side of a polyglutamine stretch as being essential for the potentiated VDI suppression characteristic of RIM2α. These results suggest that the interactions of the Ca V 2.1 CTD with RIMs enable Ca V 2.1 proteins to distinguish α-RIM isoforms in VDI suppression of P/Q-type VDCC currents. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Empagliflozin/linagliptin single-pill combination therapy for patients with type 2 diabetes mellitus.

    Science.gov (United States)

    Jain, Rajeev Kumar

    2017-04-01

    Type 2 diabetes mellitus (T2DM) is typically progressive, with sequential addition of therapies often needed to address increasing hyperglycemia over the disease course. Using treatments in combination may be preferred to sequential addition, as a means of providing a more rapid clinical response and potentially avoiding clinical inertia. In such cases, a single-pill combination can help to reduce pill burden. Although various single-pill combinations of oral glucose-lowering agents are available, empagliflozin/linagliptin was the first approved combination of a sodium glucose co-transporter 2 (SGLT2) inhibitor with a dipeptidyl peptidase 4 (DPP-4) inhibitor in the United States. Areas covered: Two publications of the clinical trial investigating the efficacy and safety of single-pill combinations of empagliflozin/linagliptin in treatment-naive or metformin-treated patients with T2DM (NCT01422876) are reviewed, and their potential impact on clinical practice is discussed. Expert opinion: The study discussed provides evidence for the efficacy and safety of empagliflozin/linagliptin single pills. Addition of an empagliflozin/linagliptin single pill may be considered in patients with inadequate glycemic control on metformin, or as an alternative to first-line treatment with empagliflozin or linagliptin when metformin is not suitable, particularly in patients with very poor glycemic control, or those who need to achieve target more quickly.

  7. submitter Search for singly produced vector-like down-type quarks in single-lepton final states with ATLAS

    CERN Document Server

    Rehnisch, Laura

    A search for vector-like and excited down-type (Q = 1/3) quarks with the ATLAS detector is presented in this thesis. The existence of these quarks is predicted by various models beyond the Standard Model, motivated by some limitations of that theory. Quarks from two specific models are searched for in single production mode, as this is predicted to yield higher cross-sections than pair production in the investigated mass range. The search focuses on decays of the heavy quarks to a W boson and a top quark, the latter of which subsequently decays almost exclusively into another W boson and a bottom quark. Requiring one of the two W bosons to decay leptonically leads to final states containing exactly one lepton (electron or muon in this case), several jets, one of which can be identified as originating from a bottom quark, and some amount of missing energy in the transverse plane stemming from the neutrino of the leptonic W decay. The reconstructed mass of the heavy quark is used to discriminate between signal ...

  8. High glucose increases glomerular filtration barrier permeability by activating protein kinase G typesubunits in a Nox4-dependent manner.

    Science.gov (United States)

    Piwkowska, Agnieszka; Rogacka, Dorota; Audzeyenka, Irena; Angielski, Stefan; Jankowski, Maciej

    2014-01-01

    Hyperglycemia is a primary factor that disturbs podocyte function in the glomerular filtration process; this disturbance leads to the development of diabetic nephropathy, and ultimately, renal failure. Podocyte function may also be altered by biological agents that modify protein kinase activity, including the cGMP-activated protein kinase type Iα (PKGIα). We hypothesized that hyperglycemia-induced podocyte protein hyperpermeability was dependent on PKGIα activation, and that PKGIα was activated via dimerization induced by reactive oxygen species. This hypothesis was investigated in rat podocytes cultured in high glucose (HG, 30 mM). Protein expression was measured with Western blot and immunofluorescence. Podocyte permeability was measured with a transmembrane albumin flux assay. We found that HG increased podocyte permeability in long-term incubations (1, 3, and 5 days); permeability was increased by 66% on day 5. This effect was abolished with apocynin, a NAD(P)H inhibitor, and Rp-8-Br-cGMPS, a PKG inhibitor. It was also abolished by introducing small interfering RNAs (siRNAs) against Nox4 and PKGIα into cultured podocytes. Furthermore, HG increased PKGIα dimerization by 138% (0.23 ± 0.04 vs. 0.54 ± 0.09; Pfiltration barrier via Nox4-dependent PKGIα dimerization. © 2013 Published by Elsevier Inc.

  9. Association between ABO blood type and live-birth outcomes in single-embryo transfer cycles.

    Science.gov (United States)

    Pereira, Nigel; Patel, Hency H; Stone, Logan D; Christos, Paul J; Elias, Rony T; Spandorfer, Steven D; Rosenwaks, Zev

    2017-11-01

    To investigate the association between ABO blood type and live-birth outcomes in patients undergoing IVF with day 5 single-embryo transfer (SET). Retrospective cohort study. University-affiliated center. Normal responders, blood type and live birth, while controlling for confounders. Odds ratios (OR) with 95% confidence intervals (CI) for live birth were estimated. A total of 2,329 patients were included. The mean age of the study cohort was 34.6 ± 4.78 years. The distribution of blood types was as follows: A = 897 (38.5%); B = 397 (17.0%); AB = 120 (5.2%); and, O = 1,915 (39.3%) patients. There was no difference in the baseline demographics, ovarian stimulation, or embryo quality parameters between the blood types. The unadjusted ORs for live birth when comparing blood type A (referent) with blood types B, AB, and O were 0.96 (95% CI, 0.6-1.7), 0.72 (95% CI, 0.4-1.2), and 0.96 (95% CI. 0.6-1.7), respectively. The adjusted ORs for live birth remained not significant when comparing blood type A to blood types B, AB, and O individually. No difference in birth weight or gestational age at delivery was noted among the four blood types. Our findings suggest that ABO blood type is not associated with live-birth rate, birth weight, or gestational age at delivery in patients undergoing IVF with day 5 SET. Copyright © 2017 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  10. Altered learning, memory, and social behavior in type 1 taste receptor subunit 3 knock-out mice are associated with neuronal dysfunction.

    Science.gov (United States)

    Martin, Bronwen; Wang, Rui; Cong, Wei-Na; Daimon, Caitlin M; Wu, Wells W; Ni, Bin; Becker, Kevin G; Lehrmann, Elin; Wood, William H; Zhang, Yongqing; Etienne, Harmonie; van Gastel, Jaana; Azmi, Abdelkrim; Janssens, Jonathan; Maudsley, Stuart

    2017-07-07

    The type 1 taste receptor member 3 (T1R3) is a G protein-coupled receptor involved in sweet-taste perception. Besides the tongue, the T1R3 receptor is highly expressed in brain areas implicated in cognition, including the hippocampus and cortex. As cognitive decline is often preceded by significant metabolic or endocrinological dysfunctions regulated by the sweet-taste perception system, we hypothesized that a disruption of the sweet-taste perception in the brain could have a key role in the development of cognitive dysfunction. To assess the importance of the sweet-taste receptors in the brain, we conducted transcriptomic and proteomic analyses of cortical and hippocampal tissues isolated from T1R3 knock-out (T1R3KO) mice. The effect of an impaired sweet-taste perception system on cognition functions were examined by analyzing synaptic integrity and performing animal behavior on T1R3KO mice. Although T1R3KO mice did not present a metabolically disrupted phenotype, bioinformatic interpretation of the high-dimensionality data indicated a strong neurodegenerative signature associated with significant alterations in pathways involved in neuritogenesis, dendritic growth, and synaptogenesis. Furthermore, a significantly reduced dendritic spine density was observed in T1R3KO mice together with alterations in learning and memory functions as well as sociability deficits. Taken together our data suggest that the sweet-taste receptor system plays an important neurotrophic role in the extralingual central nervous tissue that underpins synaptic function, memory acquisition, and social behavior. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Microscopic mechanisms for long QT syndrome type 1 revealed by single-channel analysis of I(Ks) with S3 domain mutations in KCNQ1.

    Science.gov (United States)

    Eldstrom, Jodene; Wang, Zhuren; Werry, Daniel; Wong, Nathan; Fedida, David

    2015-02-01

    The slowly activating delayed rectifier current IKs participates in cardiac repolarization, particularly at high heart rates, and mutations in this K(+) channel complex underlie long QT syndrome (LQTS) types 1 and 5. The purpose of this study was to determine biophysical mechanisms of LQT1 through single-channel kinetic analysis of IKs carrying LQT1 mutations in the S3 transmembrane region of the pore-forming subunit KCNQ1. We analyzed cell-attached recordings from mammalian cells in which a single active KCNQ1 (wild type or mutant) and KCNE1 complex could be detected. The S3 mutants of KCNQ1 studied (D202H, I204F, V205M, and S209F), with the exception of S209F, all led to a reduction in channel activity through distinct kinetic mechanisms. D202H, I204F, and V205M showed decreased open probability (Po) compared with wild type (0.07, 0.04, and 0.12 vs 0.2); increased first latency from 1.66 to >2 seconds at +60 mV (I204F, V205M); variable-to-severe reductions in open dwell times (≥50% in V205M); stabilization of closed states (D202H); and an inability of channels to reach full conductance levels (V205M, I204F). S209F is a kinetic gain-of-function mutation with a high Po (0.40) and long open-state dwell times. S3 mutations in KCNQ1 cause diverse kinetic defects in I(Ks), affecting opening and closing properties, and can account for LQT1 phenotypes. Copyright © 2015 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

  12. Vascular smooth muscle cells express the alpha(1A) subunit of a P-/Q-type voltage-dependent Ca(2+)Channel, and It is functionally important in renal afferent arterioles

    DEFF Research Database (Denmark)

    Hansen, Pernille B. Lærkegaard; Jensen, Boye L.; Andreasen, D

    2000-01-01

    in rat aorta, brain, aortic smooth muscle cells (A7r5), VSMCs, and mesangial cells. Immunolabeling with an anti-alpha(1A) antibody was positive in acid-macerated, microdissected preglomerular vessels and in A7r5 cells. Patch-clamp experiments on aortic A7r5 cells showed 22+/-4% (n=6) inhibition of inward...... Ca(2+) current by omega-Agatoxin IVA (10(-8) mol/L), which in this concentration is a specific inhibitor of P-type VDCCs. Measurements of intracellular Ca(2+) in afferent arterioles with fluorescence-imaging microscopy showed 32+/-9% (n=10) inhibition of the K(+)-induced rise in Ca(2......+) in the presence of 10(-8) mol/L omega-Agatoxin IVA. In microperfused rabbit afferent arterioles, omega-Agatoxin IVA inhibited depolarization-mediated contraction with an EC(50) of 10(-17) mol/L and complete blockade at 10(-14) mol/L. We conclude that the alpha(1A) subunit is expressed in VSMCs from renal...

  13. Antibodies against the majority subunit of type IV Pili disperse nontypeable Haemophilus influenzae biofilms in a LuxS-dependent manner and confer therapeutic resolution of experimental otitis media.

    Science.gov (United States)

    Novotny, Laura A; Jurcisek, Joseph A; Ward, Michael O; Jordan, Zachary B; Goodman, Steven D; Bakaletz, Lauren O

    2015-04-01

    Despite resulting in a similar overall outcome, unlike antibodies directed against the DNABII protein, integration host factor (IHF), which induce catastrophic structural collapse of biofilms formed by nontypeable Haemophilus influenzae (NTHI), those directed against a recombinant soluble form of PilA [the majority subunit of Type IV pili (Tfp) produced by NTHI], mediated gradual 'top-down' dispersal of NTHI from biofilms. This dispersal occurred via a mechanism that was dependent upon expression of both PilA (and by inference, Tfp) and production of AI-2 quorum signaling molecules by LuxS. The addition of rsPilA to a biofilm-targeted therapeutic vaccine formulation comprised of IHF plus the powerful adjuvant dmLT and delivered via a noninvasive transcutaneous immunization route induced an immune response that targeted two important determinants essential for biofilm formation by NTHI. This resulted in significantly earlier eradication of NTHI from both planktonic and adherent populations in the middle ear, disruption of mucosal biofilms already resident within middle ears prior to immunization and rapid resolution of signs of disease in an animal model of experimental otitis media. These data support continued development of this novel combinatorial immunization approach for resolution and/or prevention of multiple diseases of the respiratory tract caused by NTHI. © 2015 John Wiley & Sons Ltd.

  14. Sequence of the human glycogen-associated regulatory subunit of type 1 protein phosphatase and analysis of its coding region and mRNA level in muscle from patients with NIDDM

    DEFF Research Database (Denmark)

    Chen, Y H; Hansen, L; Chen, Min

    1994-01-01

    Impaired insulin-stimulated glycogen synthesis of peripheral tissues is a characteristic feature of many patients with non-insulin-dependent diabetes mellitus (NIDDM) and their first-degree relatives with normal glucose tolerance, suggesting putative inherited defects in this metabolic pathway....... In previous studies, we have failed to reveal mutations in the coding regions of the muscle-specific glycogen synthase gene and the three genes that encode the catalytic subunits of protein phosphatase 1 (PP1) as frequent causes of insulin resistance. Because the glycogen-associated regulatory subunit...... of protein phosphatase 1 (PP1 G-subunit) plays a key role in the insulin stimulation of glycogen synthesis and the activity of PP1 is decreased in insulin-resistant subjects, we have now cloned the human G-subunit cDNA to search for abnormalities in the corresponding gene (designated PPP1R3 in the human...

  15. Synthesis and characterization of p-type boron-doped IIb diamond large single crystals

    International Nuclear Information System (INIS)

    Li Shang-Sheng; Li Xiao-Lei; Su Tai-Chao; Jia Xiao-Peng; Ma Hong-An; Huang Guo-Feng; Li Yong

    2011-01-01

    High-quality p-type boron-doped IIb diamond large single crystals are successfully synthesized by the temperature gradient method in a china-type cubic anvil high-pressure apparatus at about 5.5 GPa and 1600 K. The morphologies and surface textures of the synthetic diamond crystals with different boron additive quantities are characterized by using an optical microscope and a scanning electron microscope respectively. The impurities of nitrogen and boron in diamonds are detected by micro Fourier transform infrared technique. The electrical properties including resistivities, Hall coefficients, Hall mobilities and carrier densities of the synthesized samples are measured by a four-point probe and the Hall effect method. The results show that large p-type boron-doped diamond single crystals with few nitrogen impurities have been synthesized. With the increase of quantity of additive boron, some high-index crystal faces such as {113} gradually disappear, and some stripes and triangle pits occur on the crystal surface. This work is helpful for the further research and application of boron-doped semiconductor diamond. (cross-disciplinary physics and related areas of science and technology)

  16. Single-pill combination therapy for type 2 diabetes mellitus: linagliptin plus empagliflozin.

    Science.gov (United States)

    Aronson, Ronnie

    2015-05-01

    Treatment of type 2 diabetes mellitus invariably requires the use of multiple daily medications which can impact negatively on patient adherence. As a result, there is growing interest in the use of single-pill combinations that can reduce the pill burden. Many such formulations incorporate metformin, although this agent is not suitable for all patients. The single-pill combination of the dipeptidyl peptidase-4 inhibitor linagliptin with the sodium glucose co-transporter 2 inhibitor empagliflozin offers a new and attractive option, given their complementary mechanisms of action. Publications with titles containing the keywords 'linagliptin' or 'empagliflozin' were identified from a non-systematic search of PubMed without date restrictions, together with abstracts presented at the annual meetings of the American Diabetes Association and the European Association for the Study of Diabetes 2012-2014. ClinicalTrials.gov was searched for entries containing these two keywords. Additional references known to the author were included. The efficacy and safety of linagliptin and empagliflozin as monotherapy or in combination with other oral antidiabetic drugs has been established through extensive clinical trial programs. Studies specifically evaluating the efficacy/safety of a dipeptidyl peptidase-4 inhibitor/sodium glucose co-transporter 2 inhibitor in combination are limited, but do include two studies of linagliptin/empagliflozin of up to 52 weeks in duration. These studies show that the single-pill combination of linagliptin and empagliflozin produced clinical improvements in glycemic control that were generally superior to the improvements seen with linagliptin and empagliflozin alone, but with a safety profile comparable to that of the individual constituents. The single-pill combination of linagliptin and empagliflozin, with their complementary mechanisms of action, is a promising treatment option for patients with type 2 diabetes mellitus. It would reduce the daily

  17. Sequence of the human glycogen-associated regulatory subunit of type 1 protein phosphatase and analysis of its coding region and mRNA level in muscle from patients with NIDDM

    DEFF Research Database (Denmark)

    Chen, Y H; Hansen, L; Chen, Min

    1994-01-01

    . In previous studies, we have failed to reveal mutations in the coding regions of the muscle-specific glycogen synthase gene and the three genes that encode the catalytic subunits of protein phosphatase 1 (PP1) as frequent causes of insulin resistance. Because the glycogen-associated regulatory subunit...... of protein phosphatase 1 (PP1 G-subunit) plays a key role in the insulin stimulation of glycogen synthesis and the activity of PP1 is decreased in insulin-resistant subjects, we have now cloned the human G-subunit cDNA to search for abnormalities in the corresponding gene (designated PPP1R3 in the human...... genome nomenclature) in patients with NIDDM. The human cDNA was isolated from a skeletal muscle cDNA library and was found to encode a 126-kDa protein, which shows 73% amino acid identity to the rabbit PP1 G-subunit. The human G-subunit cDNA from 30 insulin-resistant NIDDM patients was analyzed...

  18. Single-channel L-type Ca2+ currents in chicken embryo semicircular canal type I and type II hair cells.

    Science.gov (United States)

    Zampini, Valeria; Valli, Paolo; Zucca, Giampiero; Masetto, Sergio

    2006-08-01

    Few data are available concerning single Ca channel properties in inner ear hair cells and particularly none in vestibular type I hair cells. By using the cell-attached configuration of the patch-clamp technique in combination with the semicircular canal crista slice preparation, we determined the elementary properties of voltage-dependent Ca channels in chicken embryo type I and type II hair cells. The pipette solutions included Bay K 8644. With 70 mM Ba(2+) in the patch pipette, Ca channel activity appeared as very brief openings at -60 mV. Ca channel properties were found to be similar in type I and type II hair cells; therefore data were pooled. The mean inward current amplitude was -1.3 +/- 0.1 (SD) pA at - 30 mV (n = 16). The average slope conductance was 21 pS (n = 20). With 5 mM Ba(2+) in the patch pipette, very brief openings were already detectable at -80 mV. The mean inward current amplitude was -0.7 +/- 0.2 pA at -40 mV (n = 9). The average slope conductance was 11 pS (n = 9). The mean open time and the open probability increased significantly with depolarization. Ca channel activity was still present and unaffected when omega-agatoxin IVA (2 microM) and omega-conotoxin GVIA (3.2 microM) were added to the pipette solution. Our results show that types I and II hair cells express L-type Ca channels with similar properties. Moreover, they suggest that in vivo Ca(2+) influx might occur at membrane voltages more negative than -60 mV.

  19. A new type of single-phase five-level inverter

    Science.gov (United States)

    Xu, Zhi; Li, Shengnan; Qin, Risheng; Zhao, Yanhang

    2017-11-01

    At present, Neutral Point Clamped (NPC) multilevel inverter is widely applied in new energy field. However, it has some disadvantages including low utilization rate of direct current (DC) voltage source and the unbalance of neutral potential. Therefore, a new single-phase five level inverter is proposed in this paper. It has two stage structure, the former stage is equivalent to three level DC/DC converter, and the back stage uses H bridge to realize inverter. Compared with the original central clamp type inverter, the new five level inverter can improve the utilization of DC voltage, and realize the neutral point potential balance with hysteresis comparator.

  20. High speed single nucleotide polymorphism typing of a hereditary haemochromatosis mutation with capillary array electrophoresis microplates.

    Science.gov (United States)

    Medintz, I; Wong, W W; Sensabaugh, G; Mathies, R A

    2000-07-01

    A single nucleotide polymorphism (SNP) typing assay is developed and evaluated on a microfabricated capillary array electrophoresis system. Using fluorescently labeled allele-specific primers, the S65C (193A-->T) substitution associated with hereditary haemochromatosis in the HFE gene is genotyped. The covalently labeled polymerase chain reaction (PCR) products are separated on a microfabricated radial capillary array electrophoresis microplate using nondenaturing gel media in under two minutes. Detection is accomplished with a laser-excited rotary confocal scanner. The Rox-labeled A-allele specific amplicon (211 bp) is differentiated from the R110-labeled T-allele specific amplicon (201 bp) by both size and color. This study demonstrates the feasibility of using allele-specific PCR with covalently labeled primers for high speed fluorescent SNP typing on microfabricated radial capillary array electrophoresis microplates.

  1. The small subunit 1 of the Arabidopsis isopropylmalate isomerase is required for normal growth and development and the early stages of glucosinolate formation.

    Science.gov (United States)

    Imhof, Janet; Huber, Florian; Reichelt, Michael; Gershenzon, Jonathan; Wiegreffe, Christoph; Lächler, Kurt; Binder, Stefan

    2014-01-01

    In Arabidopsis thaliana the evolutionary and functional relationship between Leu biosynthesis and the Met chain elongation pathway, the first part of glucosinolate formation, is well documented. Nevertheless the exact functions of some pathway components are still unclear. Isopropylmalate isomerase (IPMI), an enzyme usually involved in Leu biosynthesis, is a heterodimer consisting of a large and a small subunit. While the large protein is encoded by a single gene (isopropylmalate isomerase large subunit1), three genes encode small subunits (isopropylmalate isomerase small subunit1 to 3). We have now analyzed small subunit 1 (isopropylmalate isomerase small subunit1) employing artificial microRNA for a targeted knockdown of the encoding gene. Strong reduction of corresponding mRNA levels to less than 5% of wild-type levels resulted in a severe phenotype with stunted growth, narrow pale leaf blades with green vasculature and abnormal adaxial-abaxial patterning as well as anomalous flower morphology. Supplementation of the knockdown plants with leucine could only partially compensate for the morphological and developmental abnormalities. Detailed metabolite profiling of the knockdown plants revealed changes in the steady state levels of isopropylmalate and glucosinolates as well as their intermediates demonstrating a function of IPMI SSU1 in both leucine biosynthesis and the first cycle of Met chain elongation. Surprisingly the levels of free leucine slightly increased suggesting an imbalanced distribution of leucine within cells and/or within plant tissues.

  2. Laser microdissection and DNA typing of cells from single hair follicles.

    Science.gov (United States)

    Di Martino, D; Giuffrè, G; Staiti, N; Simone, A; Todaro, P; Saravo, L

    2004-12-02

    Isolation and identification of single cells from tissue samples or smears assume a great relevance in pathological and forensic applications; in this latter field, the possibility to identify a specific genetic profile can be obtained by short tandem repeat (STR) typing, allowing to achieve a scientific proof important in law courts. It is well known that DNA extraction may be performed from several tissue fragments, blood traces, spermatozoa as well as telogen hair. However, in the last case, few follicle cells are coupled to a great amount of keratin reducing the efficiency of DNA amplification. Recently, the introduction of laser microdissection technique has greatly improved the capability to select single cells without any cross-contamination. In the present report, we have performed a laser microdissection using a Leica AS LMD (Leica Microsystems, Germany), utilized on cutting the telogen hair in order to exclusively collect the lower part of the follicle and reduce keratin contamination. In this way we can accurately extract an adequate amount of DNA, successfully typed by STR profile.

  3. Structure of AscE and Induced Burial Regions in AscE and AscG upon Formation of the Chaperone Needle-subunit Complex of Type III Secretion System in Aeromonas Hydrophila

    Energy Technology Data Exchange (ETDEWEB)

    Tan, Y.; Yu, H; Leung, K; Sivaraman, J; Mok, Y

    2008-01-01

    In the type III secretion system (T3SS) of Aeromonas hydrophila, the putative needle complex subunit AscF requires both putative chaperones AscE and AscG for formation of a ternary complex to avoid premature assembly. Here we report the crystal structure of AscE at 2.7 A resolution and the mapping of buried regions of AscE, AscG, and AscF in the AscEG and AscEFG complexes using limited protease digestion. The dimeric AscE is comprised of two helix-turn-helix monomers packed in an antiparallel fashion. The N-terminal 13 residues of AscE are buried only upon binding with AscG, but this region is found to be nonessential for the interaction. AscE functions as a monomer and can be coexpressed with AscG or with both AscG and AscF to form soluble complexes. The AscE binding region of AscG in the AscEG complex is identified to be within the N-terminal 61 residues of AscG. The exposed C-terminal substrate-binding region of AscG in the AscEG complex is induced to be buried only upon binding to AscF. However, the N-terminal 52 residues of AscF remain exposed even in the ternary AscEFG complex. On the other hand, the 35-residue C-terminal region of AscF in the complex is resistant to protease digestion in the AscEFG complex. Site-directed mutagenesis showed that two C-terminal hydrophobic residues, Ile83 and Leu84, of AscF are essential for chaperone binding.

  4. Distinct circular single-stranded DNA viruses exist in different soil types.

    Science.gov (United States)

    Reavy, Brian; Swanson, Maud M; Cock, Peter J A; Dawson, Lorna; Freitag, Thomas E; Singh, Brajesh K; Torrance, Lesley; Mushegian, Arcady R; Taliansky, Michael

    2015-06-15

    The potential dependence of virus populations on soil types was examined by electron microscopy, and the total abundance of virus particles in four soil types was similar to that previously observed in soil samples. The four soil types examined differed in the relative abundances of four morphological groups of viruses. Machair, a unique type of coastal soil in western Scotland and Ireland, differed from the others tested in having a higher proportion of tailed bacteriophages. The other soils examined contained predominantly spherical and thin filamentous virus particles, but the Machair soil had a more even distribution of the virus types. As the first step in looking at differences in populations in detail, virus sequences from Machair and brown earth (agricultural pasture) soils were examined by metagenomic sequencing after enriching for circular Rep-encoding single-stranded DNA (ssDNA) (CRESS-DNA) virus genomes. Sequences from the family Microviridae (icosahedral viruses mainly infecting bacteria) of CRESS-DNA viruses were predominant in both soils. Phylogenetic analysis of Microviridae major coat protein sequences from the Machair viruses showed that they spanned most of the diversity of the subfamily Gokushovirinae, whose members mainly infect obligate intracellular parasites. The brown earth soil had a higher proportion of sequences that matched the morphologically similar family Circoviridae in BLAST searches. However, analysis of putative replicase proteins that were similar to those of viruses in the Circoviridae showed that they are a novel clade of Circoviridae-related CRESS-DNA viruses distinct from known Circoviridae genera. Different soils have substantially different taxonomic biodiversities even within ssDNA viruses, which may be driven by physicochemical factors. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  5. THE DISTRIBUTION OF DIFFERENT TYPES OF DIABETES IN CHILDHOOD: A SINGLE CENTER EXPERIENCE.

    Science.gov (United States)

    Haliloğlu, Belma; Abali, Saygın; Buğrul, Fuat; Çelik, Enes; Baş, Serpil; Atay, Zeynep; Güran, Tülay; Turan, Serap; Bereket, Abdullah

    2017-11-24

    Type 1 diabetes (T1D) is the most common cause of diabetes in childhood but type 2 diabetes (T2D) and maturity onset diabetes of the young (MODY) are emerging and noteworthy causes of diabetes in youths. The aim of the study is to determine the distribution, trends and clinical features of the different types of diabetes in childhood in a tertiary single-center. Children and adolescents aged 0-18 years who were diagnosed "diabetes/persistent hyperglycemia" between January 1999 and December 2016, were reviewed. Clinical and laboratory characteristics of the patients at the diagnosis and type of diabetes were recorded. The mean age of 835 patients (48.7% females) at diagnosis was 8.8±4.4 years. Eighty-four percent of the patients were diagnosed with T1D, 5.7% with T2D, 5.3% with clinical MODY and 5% with other types of diabetes. The frequency of all DKA and severe DKA in T1D were 48.4% and 11.6%, respectively. Fourteen patients (29.2%) with T2D presented with ketosis and 2 of them (4.2%) had DKA at diagnosis. Antibody positivity was 83.1% in T1D and 14.8% in T2D. A statistically significant increase in the frequency of T2D has clearly been demonstrated in recent years with a frequency of 1.9%, 2.4% and 7.9% in 1999-2004, 2005-2010 and 2011-2016, respectively (pchildhood, a trend towards increase in the frequency of T2D in recent years is notable in our population.

  6. Echinococcus granulosus Antigen B Structure: Subunit Composition and Oligomeric States

    Science.gov (United States)

    Monteiro, Karina M.; Cardoso, Mateus B.; Follmer, Cristian; da Silveira, Nádya P.; Vargas, Daiani M.; Kitajima, Elliot W.; Zaha, Arnaldo; Ferreira, Henrique B.

    2012-01-01

    Background Antigen B (AgB) is the major protein secreted by the Echinococcus granulosus metacestode and is involved in key host-parasite interactions during infection. The full comprehension of AgB functions depends on the elucidation of several structural aspects that remain unknown, such as its subunit composition and oligomeric states. Methodology/Principal Findings The subunit composition of E. granulosus AgB oligomers from individual bovine and human cysts was assessed by mass spectrometry associated with electrophoretic analysis. AgB8/1, AgB8/2, AgB8/3 and AgB8/4 subunits were identified in all samples analyzed, and an AgB8/2 variant (AgB8/2v8) was found in one bovine sample. The exponentially modified protein abundance index (emPAI) was used to estimate the relative abundance of the AgB subunits, revealing that AgB8/1 subunit was relatively overrepresented in all samples. The abundance of AgB8/3 subunit varied between bovine and human cysts. The oligomeric states formed by E. granulosus AgB and recombinant subunits available, rAgB8/1, rAgB8/2 and rAgB8/3, were characterized by native PAGE, light scattering and microscopy. Recombinant subunits showed markedly distinct oligomerization behaviors, forming oligomers with a maximum size relation of rAgB8/3>rAgB8/2>rAgB8/1. Moreover, the oligomeric states formed by rAgB8/3 subunit were more similar to those observed for AgB purified from hydatid fluid. Pressure-induced dissociation experiments demonstrated that the molecular assemblies formed by the more aggregative subunits, rAgB8/2 and rAgB8/3, also display higher structural stability. Conclusions/Significance For the first time, AgB subunit composition was analyzed in samples from single hydatid cysts, revealing qualitative and quantitative differences between samples. We showed that AgB oligomers are formed by different subunits, which have distinct abundances and oligomerization properties. Overall, our findings have significantly contributed to increase the

  7. Plasminogen Activator Inhibitor Type-1 Tag Single-Nucleotide Polymorphisms in Patients with Diabetes Mellitus Type 2 and Diabetic Retinopathy.

    Science.gov (United States)

    Siokas, Vasileios; Dardiotis, Efthimios; Sokolakis, Thomas; Kotoula, Maria; Tachmitzi, Sophia V; Chatzoulis, Dimitrios Z; Almpanidou, Pavlina; Stefanidis, Ioannis; Hadjigeorgiou, Georgios M; Tsironi, Evangelia E

    2017-07-01

    There is accumulating evidence for genetic susceptibility to the development of diabetic retinopathy (DR). The role of plasminogen activator inhibitor-1 (PAI-1) in DR risk remains controversial. The present study was designed to investigate possible influence of PAI-1 gene region polymorphisms on the risk of DR and on the risk of developing DR early vs late in the course of type 2 diabetes mellitus (T2DM). A total of 138 patients with DR, 107 patients with T2DM without DR, and 315 healthy controls were recruited. To cover the majority of the genetic variability across the extended region of PAI-1 gene, five tag single-nucleotide polymorphisms (SNPs) from the HapMap using a pairwise approach and an r 2 ≥ 0.8 and a minor allele frequency (MAF) of >0.05 were identified. Using logistic regression analyses, tag SNPs and haplotypes were tested for associations with DR risk and risk of DR development early or late in the course of T2DM. The generalized odds ratio (OR G ) was calculated to estimate the mutational load effect on DR development among all participants. Corrections for multiple comparisons were carried out (p-value < 0.01). A significant effect of rs2070682 on the risk of early DR onset was found in the codominant model of inheritance [odds ratio, OR (95% confidence interval, CI): 5.04 (1.47-17.28), p = 0.018]. However, this association marginally did not survive multiple testing corrections. No other significant association between PAI-1 tag-SNPs and haplotypes was revealed. Furthermore, no significant mutational load effect of PAI-1 tag SNPs on the risk of DR development in T2DM course was found. In conclusion, the present study does not provide any strong evidence that PAI-1 gene variants are implicated in the risk of DR or the development of DR during T2DM course.

  8. Study of the signal formation in single-type column 3D silicon detectors

    International Nuclear Information System (INIS)

    Piemonte, Claudio; Boscardin, Maurizio; Bosisio, Luciano; Dalla Betta, Gian-Franco; Pozza, Alberto; Ronchin, Sabina; Zorzi, Nicola

    2007-01-01

    Because of their superior radiation resistance, three-dimensional (3D) silicon sensors are receiving more and more interest for application in the innermost layers of tracker systems for experiments running in very high luminosity colliders. Their short electrode distance allows for both a low depletion voltage and a high charge collection efficiency even at extremely high radiation fluences. In order to fully understand the properties of a 3D detector, a thorough characterization of the signal formation mechanism is of paramount importance. In this work the shape of the current induced by localized and uniform charge depositions in a single-type column 3D detector is studied. A first row estimation is given applying the Ramo theorem, then a more complete TCAD simulation is used to provide a more realistic pulse shape

  9. High efficiency 40 K single-stage Stirling-type pulse tube cryocooler

    Science.gov (United States)

    Wu, X. L.; Chen, L. B.; Pan, C. Z.; Cui, C.; Wang, J. J.; Zhou, Y.

    2017-12-01

    A high efficiency single-stage Stirling-type coaxial pulse tube cryocooler (SPTC) operating at around 40 K has been designed, built and tested. The double-inlet and the inertance tubes together with the gas reservoir were adopted as the phase shifters. Under the conditions of 2.5 MPa charging pressure and 30 Hz operating frequency, the prototype has achieved a no-load temperature of 23.8 K with 330 W of electric input power at a rejection temperature of 279 K. When the input power increases to 400 W, it can achieve a cooling capacity of 4.7 W/40 K while rejecting heat at 279 K yielding an efficiency of 7.02% relative to Carnot. It achieves a cooling capacity of 5 W/40 K with an input power of 450 W. It takes 10 minutes for the SPTC to cool to its no-load temperature of 40 K from 295 K.

  10. Stochastic gravitational wave background from the single-degenerate channel of type Ia supernovae

    International Nuclear Information System (INIS)

    Falta, David; Fisher, Robert

    2011-01-01

    We demonstrate that the integrated gravitational wave signal of type Ia supernovae (SNe Ia) in the single-degenerate channel out to cosmological distances gives rise to a continuous background to spaceborne gravitational wave detectors, including the Big Bang Observer and Deci-Hertz Interferometer Gravitational wave Observatory planned missions. This gravitational wave background from SNe Ia acts as a noise background in the frequency range 0.1-10 Hz, which heretofore was thought to be relatively free from astrophysical sources apart from neutron-star and white-dwarf binaries, and therefore a key window in which to study primordial gravitational waves generated by inflation. While inflationary energy scales of > or approx. 10 16 GeV yield inflationary gravitational wave backgrounds in excess of our range of predicted backgrounds, for lower energy scales of ∼10 15 GeV, the inflationary gravitational wave background becomes comparable to the noise background from SNe Ia.

  11. The contribution of single photon emission computed tomography in the clinical assessment of Alzheimer type dementia

    International Nuclear Information System (INIS)

    Boudousq, V.; Collombier, L.; Kotzki, P.O.

    1999-01-01

    Interest of brain single-photon emission computed tomography to support clinical diagnosis of Alzheimer-type dementia is now established. Numerous studies have reported a decreased perfusion in the association cortex of the parietal lobe and the posterior temporal regions. In patients with mild cognitive complaints, the presence of focal hypoperfusion may increase substantially the probability of the disease. In addition, emission tomography emerges as a helpful tool in situation in which there is diagnostic doubt. In this case, the presence of temporo-parietal perfusion deficit associated with hippocampal atrophy on MRI or X-ray computed tomography contributes to diagnostic accuracy. However, some studies suggest that emission tomography may be useful for preclinical prediction of Alzheimer's disease and to predict cognitive decline. (author)

  12. Phonon-limited mobility in n-type single-layer MoS2 from first principles

    DEFF Research Database (Denmark)

    Kaasbjerg, Kristen; Thygesen, Kristian S.; Jacobsen, Karsten W.

    2012-01-01

    We study the phonon-limited mobility in intrinsic n-type single-layer MoS2 for temperatures T > 100 K. The materials properties including the electron-phonon interaction are calculated from first principles and the deformation potentials and Frohlich interaction in single-layer MoS2 are establish...

  13. Oscillations of a single Abrikosov vortex in hard type-II superconductors

    Science.gov (United States)

    Rusakov, V. F.; Chabanenko, V. V.; Nabiałek, A.; Chumak, O. M.

    2017-06-01

    During the last decade, detection and manipulation of single vortex lines in bulk superconductors have been achieved experimentally. Electrodynamic response of pinned vortices in the high-frequency range is instrumental in studying specific aspects of their behavior. The present paper reviews the state of the art in studies of the oscillations of a single Abrikosov vortex in type II superconductors. The equations for free and forced oscillations of a single elastic vortex line are analyzed taking into account different forces affecting its motion: pinning, elasticity, viscosity and the Lorenz force. The equations also account for the inertial properties of a vortex due to various mechanisms of massiveness. The nature and magnitude of the vortex effective mass caused by some of the mechanisms are discussed in the paper. The roles of each force and inertia in the free oscillation spectrum are thoroughly analyzed. For the De Gennes and Matricon mode (at about a megahertz) with parabolic dispersion and the pinning force taken into account, there is an activation threshold. Taking into account the effective vortex mass in the equation of motion leads to the occurrence of a high-frequency mode (at about a terahertz) in the oscillation spectrum which is also of the activation nature. Estimations of the characteristic frequencies for these modes are given for two common superconductors, NbTi and anisotropic YBaCuO. The paper also presents the features of the resonant behavior of an elastic massive vortex line arising under an external uniform harmonic driving force that decays into the bulk of the sample, taking into account all the above forces. The frequency and temperature dependences of the energy absorption by a vortex line are analyzed. Maximum absorption in the low-frequency mode corresponds to the threshold frequency, while that in the high-frequency mode corresponds to the vortex cyclotron frequency. Vortex manipulation experiments and vortex dynamics simulation

  14. A human RNA polymerase II subunit is encoded by a recently generated multigene family

    Directory of Open Access Journals (Sweden)

    Mattei Marie-Geneviève

    2001-11-01

    Full Text Available Abstract Background The sequences encoding the yeast RNA polymerase II (RPB subunits are single copy genes. Results While those characterized so far for the human (h RPB are also unique, we show that hRPB subunit 11 (hRPB11 is encoded by a multigene family, mapping on chromosome 7 at loci p12, q11.23 and q22. We focused on two members of this family, hRPB11a and hRPB11b: the first encodes subunit hRPB11a, which represents the major RPB11 component of the mammalian RPB complex ; the second generates polypeptides hRPB11bα and hRPB11bβ through differential splicing of its transcript and shares homologies with components of the hPMS2L multigene family related to genes involved in mismatch-repair functions (MMR. Both hRPB11a and b genes are transcribed in all human tissues tested. Using an inter-species complementation assay, we show that only hRPB11bα is functional in yeast. In marked contrast, we found that the unique murine homolog of RPB11 gene maps on chromosome 5 (band G, and encodes a single polypeptide which is identical to subunit hRPB11a. Conclusions The type hRPB11b gene appears to result from recent genomic recombination events in the evolution of primates, involving sequence elements related to the MMR apparatus.

  15. porphyrin and anthracene subunits

    Indian Academy of Sciences (India)

    Unknown

    rated to dryness under reduced pressure. The residue was dissolved in ≈50 .... spectrum of each of these axial-bonding type tin(IV) porphyrins showed only a low inten- sity peak due to the parent M+ ion. ..... However, this exercise clearly reveals that there is a near perpendicular juxtaposition of the porphyrin and the axial ...

  16. Single-donor islet transplantation in type 1 diabetes: patient selection and special considerations

    Directory of Open Access Journals (Sweden)

    Tatum JA

    2017-02-01

    Full Text Available Jacob A Tatum,* Max O Meneveau,* Kenneth L Brayman Department of Surgery, Division of Transplantation, The University of Virginia Health System, Charlottesville, VA, USA *These authors contributed equally to this work. Abstract: Type 1 diabetes mellitus is an autoimmune disorder of the endocrine pancreas that currently affects millions of people in the United States. Although the disease can be managed with exogenous insulin administration, the ultimate cure for the condition lies in restoring a patient’s ability to produce their own insulin. Islet cell allotransplantation provides a means of endogenous insulin production. Though far from perfected, islet transplants are now a proven treatment for type 1 diabetics. However, proper patient selection is critical for achieving optimal outcomes. Given the shortage of transplantable organs, selecting appropriate candidates for whom the procedure will be of greatest benefit is essential. Although many of those who receive islets do not retain insulin independence, grafts do play a significant role in preventing hypoglycemic episodes that can be quite detrimental to quality of life and potentially fatal. Additionally, islet transplant requires lifelong immunosuppression. Antibodies, both preformed and following islet infusion, may play important roles in graft outcomes. Finally, no procedure is without inherent risk and islet transfusions can have serious consequences for recipients’ livers in the form of both vascular and metabolic complications. Therefore, patient-specific factors that should be taken into account before islet transplantation include aims of therapy, sensitization, and potential increased risk for hepatic and portal-venous sequelae. Keywords: islet transplantation, diabetes mellitus type 1, brittle diabetes, single donor, patient

  17. Computational Analysis of Single Nucleotide Polymorphisms Associated with Altered Drug Responsiveness in Type 2 Diabetes

    Directory of Open Access Journals (Sweden)

    Valerio Costa

    2016-06-01

    Full Text Available Type 2 diabetes (T2D is one of the most frequent mortality causes in western countries, with rapidly increasing prevalence. Anti-diabetic drugs are the first therapeutic approach, although many patients develop drug resistance. Most drug responsiveness variability can be explained by genetic causes. Inter-individual variability is principally due to single nucleotide polymorphisms, and differential drug responsiveness has been correlated to alteration in genes involved in drug metabolism (CYP2C9 or insulin signaling (IRS1, ABCC8, KCNJ11 and PPARG. However, most genome-wide association studies did not provide clues about the contribution of DNA variations to impaired drug responsiveness. Thus, characterizing T2D drug responsiveness variants is needed to guide clinicians toward tailored therapeutic approaches. Here, we extensively investigated polymorphisms associated with altered drug response in T2D, predicting their effects in silico. Combining different computational approaches, we focused on the expression pattern of genes correlated to drug resistance and inferred evolutionary conservation of polymorphic residues, computationally predicting the biochemical properties of polymorphic proteins. Using RNA-Sequencing followed by targeted validation, we identified and experimentally confirmed that two nucleotide variations in the CAPN10 gene—currently annotated as intronic—fall within two new transcripts in this locus. Additionally, we found that a Single Nucleotide Polymorphism (SNP, currently reported as intergenic, maps to the intron of a new transcript, harboring CAPN10 and GPR35 genes, which undergoes non-sense mediated decay. Finally, we analyzed variants that fall into non-coding regulatory regions of yet underestimated functional significance, predicting that some of them can potentially affect gene expression and/or post-transcriptional regulation of mRNAs affecting the splicing.

  18. Submitochondrial distributions and stabilities of subunits 4, 5, and 6 of yeast cytochrome oxidase in assembly defective mutants.

    Science.gov (United States)

    Glerum, D M; Tzagoloff, A

    1997-08-04

    The concentration and submitochondrial distribution of the subunit polypeptides of cytochrome oxidase have been studied in wild type yeast and in different mutants impaired in assembly of this respiratory complex. All the subunit polypeptides of the enzyme are associated with mitochondrial membranes of wild type cells, except for a small fraction of subunits 4 and 6 that is recovered in the soluble protein fraction of mitochondria. Cytochrome oxidase mutants consistently display a severe reduction in the steady-state concentration of subunit 1 due to its increased turnover. As a consequence, most of subunit 4, which normally is associated with subunit 1, is found in the soluble fraction. A similar shift from membrane-bound to soluble subunit 6 is seen in mutants blocked in expression of subunit 5a. In contrast, null mutations in COX6 coding for subunit 6 promote loss of subunit 5a. The absence of subunit 5a in the cox6 mutant is the result of proteolytic degradation rather than regulation of its expression by subunit 6. The possible role of the ATP-dependent proteases Rca1p and Afg3p in proteolysis of subunits 1 and 5a has been assessed in strains with combined mutations in COX6, RCA1, and/or AFG3. Immunochemical assays indicate that another protease(s) must be responsible for most of the proteolytic loss of these proteins.

  19. Expression of the scaffolding subunit A of protein phosphatase 2A during rat testicular development

    NARCIS (Netherlands)

    van den Ham, R.; van Dissel-Emiliani, F. M. F.; van Pelt, A. M. M.

    2003-01-01

    Previously, we found that the poly(A)+ RNA of the scaffolding subunit A (alpha isoform) of protein phosphatase 2A (PP2A-Aalpha) was clearly expressed by fetal gonocytes but weakly expressed by adult single (As), paired (Apr), and aligned (Aal) A spermatogonia. The scaffolding subunit A of PP2A

  20. WHITE DWARF/M DWARF BINARIES AS SINGLE DEGENERATE PROGENITORS OF TYPE Ia SUPERNOVAE

    International Nuclear Information System (INIS)

    Wheeler, J. Craig

    2012-01-01

    Limits on the companions of white dwarfs in the single-degenerate scenario for the origin of Type Ia supernovae (SNe Ia) have gotten increasingly tight, yet igniting a nearly Chandrasekhar mass C/O white dwarf from a condition of near hydrostatic equilibrium provides compelling agreement with observed spectral evolution. The only type of non-degenerate stars that survive the tight limits, M V ∼> 8.4 on the SN Ia in SNR 0509-67.5 and M V ∼> 9.5 in the remnant of SN 1572, are M dwarfs. While M dwarfs are observed in cataclysmic variables, they have special properties that have not been considered in most work on the progenitors of SNe Ia: they have small but finite magnetic fields and they flare frequently. These properties are explored in the context of SN Ia progenitors. White dwarf/M dwarf pairs may be sufficiently plentiful to provide, in principle, an adequate rate of explosions even with slow orbital evolution due to magnetic braking or gravitational radiation. Even modest magnetic fields on the white dwarf and M dwarf will yield adequate torques to lock the two stars together, resulting in a slowly rotating white dwarf, with the magnetic poles pointing at one another in the orbital plane. The mass loss will be channeled by a 'magnetic bottle' connecting the two stars, landing on a concentrated polar area on the white dwarf. This enhances the effective rate of accretion compared to spherical accretion. Luminosity from accretion and hydrogen burning on the surface of the white dwarf may induce self-excited mass transfer. The combined effects of self-excited mass loss, polar accretion, and magnetic inhibition of mixing of accretion layers give possible means to beat the 'nova limit' and grow the white dwarf to the Chandrasekhar mass even at rather moderate mass accretion rates.

  1. Designing Kerr interactions using multiple superconducting qubit types in a single circuit

    Science.gov (United States)

    Elliott, Matthew; Joo, Jaewoo; Ginossar, Eran

    2018-02-01

    The engineering of Kerr interactions is of great interest for processing quantum information in multipartite quantum systems and for investigating many-body physics in a complex cavity-qubit network. We study how coupling multiple different types of superconducting qubits to the same cavity modes can be used to modify the self- and cross-Kerr effects acting on the cavities and demonstrate that this type of architecture could be of significant benefit for quantum technologies. Using both analytical perturbation theory results and numerical simulations, we first show that coupling two superconducting qubits with opposite anharmonicities to a single cavity enables the effective self-Kerr interaction to be diminished, while retaining the number splitting effect that enables control and measurement of the cavity field. We demonstrate that this reduction of the self-Kerr effect can maintain the fidelity of coherent states and generalised Schrödinger cat states for much longer than typical coherence times in realistic devices. Next, we find that the cross-Kerr interaction between two cavities can be modified by coupling them both to the same pair of qubit devices. When one of the qubits is tunable in frequency, the strength of entangling interactions between the cavities can be varied on demand, forming the basis for logic operations on the two modes. Finally, we discuss the feasibility of producing an array of cavities and qubits where intermediary and on-site qubits can tune the strength of self- and cross-Kerr interactions across the whole system. This architecture could provide a way to engineer interesting many-body Hamiltonians and be a useful platform for quantum simulation in circuit quantum electrodynamics.

  2. Diagnostic accuracy of single photon emission CT in Alzheimer-type dementia

    International Nuclear Information System (INIS)

    Hanyu, Haruki; Abe, Shinei; Arai, Hisayuki; Asano, Tetsuichi; Iwamoto, Toshihiko; Takasaki, Masaru; Suzuki, Takanari

    1992-01-01

    To determine the diagnostic accuracy of single photon emission computed tomography (SPECT) with 123 I-IMP in Alzheimer-type dementia (ATD), we studied 46 ATD patients and 23 healthy controls. The patients fulfilled the NINCDS-ADRDA criteria for probable or definite ATD and were classified as having mild, moderate, and severe ATD by neuropsychological examinations. To assess regional cerebral blood flow, we performed qualitative SPECT image analysis without any knowledge of the subject's clinical classification. The image was regarded as abnormal if cerebral blood flow was reduced in the unlilateral or bilateral temporoparietal association areas, with or without any reduction of flow in other brain regions. The diagnostic sensitivity (abnormal image/ patient) of 123 I-IMP SPECT in mild, moderate and severe ATD was 67%, 86% and 92%, because an abnormal image was found in only 2/23 healthy controls. Eight ATD patients without reduced temporoparietal perfusion showed normal perfusion or frontal hypoperfusion. These results suggest that 123 I-IMP SPECT may provide an accurate and sensitive diagnostic marker for ATD. The detection of these characteristic abnormalities of cerebral perfusion could well be applied to the clinical diagnosis of ATD. (author)

  3. Modes of Operation of the BKCa Channel β2 Subunit

    Science.gov (United States)

    Savalli, Nicoletta; Kondratiev, Andrei; de Quintana, Sarah Buxton; Toro, Ligia; Olcese, Riccardo

    2007-01-01

    The β2 subunit of the large conductance Ca2+- and voltage-activated K+ channel (BKCa) modulates a number of channel functions, such as the apparent Ca2+/voltage sensitivity, pharmacological and kinetic properties of the channel. In addition, the N terminus of the β2 subunit acts as an inactivating particle that produces a relatively fast inactivation of the ionic conductance. Applying voltage clamp fluorometry to fluorescently labeled human BKCa channels (hSlo), we have investigated the mechanisms of operation of the β2 subunit. We found that the leftward shift on the voltage axis of channel activation curves (G(V)) produced by coexpression with β2 subunits is associated with a shift in the same direction of the fluorescence vs. voltage curves (F(V)), which are reporting the voltage dependence of the main voltage-sensing region of hSlo (S4-transmembrane domain). In addition, we investigated the inactivating mechanism of the β2 subunits by comparing its properties with the ones of the typical N-type inactivation process of Shaker channel. While fluorescence recordings from the inactivated Shaker channels revealed the immobilization of the S4 segments in the active conformation, we did not observe a similar feature in BKCa channels coexpressed with the β2 subunit. The experimental observations are consistent with the view that the β2 subunit of BKCa channels facilitates channel activation by changing the voltage sensor equilibrium and that the β2-induced inactivation process does not follow a typical N-type mechanism. PMID:17591990

  4. Modes of operation of the BKCa channel beta2 subunit.

    Science.gov (United States)

    Savalli, Nicoletta; Kondratiev, Andrei; de Quintana, Sarah Buxton; Toro, Ligia; Olcese, Riccardo

    2007-07-01

    The beta(2) subunit of the large conductance Ca(2+)- and voltage-activated K(+) channel (BK(Ca)) modulates a number of channel functions, such as the apparent Ca(2+)/voltage sensitivity, pharmacological and kinetic properties of the channel. In addition, the N terminus of the beta(2) subunit acts as an inactivating particle that produces a relatively fast inactivation of the ionic conductance. Applying voltage clamp fluorometry to fluorescently labeled human BK(Ca) channels (hSlo), we have investigated the mechanisms of operation of the beta(2) subunit. We found that the leftward shift on the voltage axis of channel activation curves (G(V)) produced by coexpression with beta(2) subunits is associated with a shift in the same direction of the fluorescence vs. voltage curves (F(V)), which are reporting the voltage dependence of the main voltage-sensing region of hSlo (S4-transmembrane domain). In addition, we investigated the inactivating mechanism of the beta(2) subunits by comparing its properties with the ones of the typical N-type inactivation process of Shaker channel. While fluorescence recordings from the inactivated Shaker channels revealed the immobilization of the S4 segments in the active conformation, we did not observe a similar feature in BK(Ca) channels coexpressed with the beta(2) subunit. The experimental observations are consistent with the view that the beta(2) subunit of BK(Ca) channels facilitates channel activation by changing the voltage sensor equilibrium and that the beta(2)-induced inactivation process does not follow a typical N-type mechanism.

  5. Low Temperature Characterization of PMOS-type Gate-all-around Silicon nanowire FETs as single-hole-transistors

    Science.gov (United States)

    Hong, B. H.; Hwang, S. W.; Lee, Y. Y.; Son, M. H.; Ahn, D.; Cho, K. H.; Yeo, K. H.; Kim, D.-W.; Jin, G. Y.; Park, D.

    2011-12-01

    We report the single hole tunneling characteristics observed from a PMOS-type gate-all-around silicon nanowire field-effect-transistor with the radius 5 nm and the length 44 nm. The total capacitance of the quantum dot obtained from the measured Coulomb oscillations and Coulomb diamonds matches with the ideal capacitance of the silicon cylinder. It suggests that the observed single hole tunneling is originated from the fabricated structure.

  6. Stable incorporation versus dynamic exchange of β subunits in a native Ca2+ channel complex.

    Science.gov (United States)

    Campiglio, Marta; Di Biase, Valentina; Tuluc, Petronel; Flucher, Bernhard E

    2013-05-01

    Voltage-gated Ca(2+) channels are multi-subunit membrane proteins that transduce depolarization into cellular functions such as excitation-contraction coupling in muscle or neurotransmitter release in neurons. The auxiliary β subunits function in membrane targeting of the channel and modulation of its gating properties. However, whether β subunits can reversibly interact with, and thus differentially modulate, channels in the membrane is still unresolved. In the present study we applied fluorescence recovery after photobleaching (FRAP) of GFP-tagged α1 and β subunits expressed in dysgenic myotubes to study the relative dynamics of these Ca(2+) channel subunits for the first time in a native functional signaling complex. Identical fluorescence recovery rates of both subunits indicate stable interactions, distinct recovery rates indicate dynamic interactions. Whereas the skeletal muscle β1a isoform formed stable complexes with CaV1.1 and CaV1.2, the non-skeletal muscle β2a and β4b isoforms dynamically interacted with both α1 subunits. Neither replacing the I-II loop of CaV1.1 with that of CaV2.1, nor deletions in the proximal I-II loop, known to change the orientation of β relative to the α1 subunit, altered the specific dynamic properties of the β subunits. In contrast, a single residue substitution in the α interaction pocket of β1aM293A increased the FRAP rate threefold. Taken together, these findings indicate that in skeletal muscle triads the homologous β1a subunit forms a stable complex, whereas the heterologous β2a and β4b subunits form dynamic complexes with the Ca(2+) channel. The distinct binding properties are not determined by differences in the I-II loop sequences of the α1 subunits, but are intrinsic properties of the β subunit isoforms.

  7. Characterization of ryanodine receptor type 1 single channel activity using "on-nucleus" patch clamp.

    Science.gov (United States)

    Wagner, Larry E; Groom, Linda A; Dirksen, Robert T; Yule, David I

    2014-08-01

    In this study, we provide the first description of the biophysical and pharmacological properties of ryanodine receptor type 1 (RyR1) expressed in a native membrane using the on-nucleus configuration of the patch clamp technique. A stable cell line expressing rabbit RyR1 was established (HEK-RyR1) using the FLP-in 293 cell system. In contrast to untransfected cells, RyR1 expression was readily demonstrated by immunoblotting and immunocytochemistry in HEK-RyR1 cells. In addition, the RyR1 agonists 4-CMC and caffeine activated Ca(2+) release that was inhibited by high concentrations of ryanodine. On nucleus patch clamp was performed in nuclei prepared from HEK-RyR1 cells. Raising the [Ca(2+)] in the patch pipette resulted in the appearance of a large conductance cation channel with well resolved kinetics and the absence of prominent subconductance states. Current versus voltage relationships were ohmic and revealed a chord conductance of ∼750pS or 450pS in symmetrical 250mM KCl or CsCl, respectively. The channel activity was markedly enhanced by caffeine and exposure to ryanodine resulted in the appearance of a subconductance state with a conductance ∼40% of the full channel opening with a Po near unity. In total, these properties are entirely consistent with RyR1 channel activity. Exposure of RyR1 channels to cyclic ADP ribose (cADPr), nicotinic acid adenine dinucleotide phosphate (NAADP) or dantrolene did not alter the single channel activity stimulated by Ca(2+), and thus, it is unlikely these molecules directly modulate RyR1 channel activity. In summary, we describe an experimental platform to monitor the single channel properties of RyR channels. We envision that this system will be influential in characterizing disease-associated RyR mutations and the molecular determinants of RyR channel modulation. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. A new single nucleotide polymorphisms typing method and device by bioluminometric assay coupled with a photodiode array

    Science.gov (United States)

    Kamahori, Masao; Harada, Kunio; Kambara, Hideki

    2002-11-01

    Easy and inexpensive single nucleotide polymorphisms (SNPs) typing systems are required for the practice of genetic testing as well as genetic medicine. Most of the SNPs typing systems use laser-induced fluorescence detection coupled with fluorophore tagging on DNA, which are expensive. A new simple and inexpensive SNPs typing system is presented. It uses a bioluminometric assay coupled with modified primer extension reactions and an inexpensive photodiode array for the luminometric detection. The reagents consumed in the assay are also inexpensive. Although the system is very small, simple and inexpensive, it gives enough sensitivity for detecting target DNAs as small as 10 fmol, which is good enough for SNPs typing.

  9. Laser characterisation of a 3D single-type column p-type prototype module read out with ATLAS SCT electronics

    International Nuclear Information System (INIS)

    Ehrich, T.; Kuehn, S.; Boscardin, M.; Dalla Betta, G.-F.; Eckert, S.; Jakobs, K.; Maassen, M.; Parzefall, U.; Piemonte, C.; Pozza, A.; Ronchin, S.; Zorzi, N.

    2007-01-01

    In this paper measurements of a 3D single-type column (3D-stc) microstrip silicon device are shown. The 3D-stc sensor has n-type columns in p-type substrate. It has been connected to an ATLAS SCT ABCD3T chip and is readout with ATLAS SCT electronics at 40 MHz. Spatial measurements were done with a laser setup to investigate the expected low field region in 3D devices. An influence of the p-stops on the collected charge has been observed

  10. Laser characterisation of a 3D single-type column p-type prototype module read out with ATLAS SCT electronics

    Energy Technology Data Exchange (ETDEWEB)

    Ehrich, T. [Physikalisches Institut, Universitaet Freiburg, Hermann-Herder Str. 3, 79104 Freiburg (Germany); Kuehn, S. [Physikalisches Institut, Universitaet Freiburg, Hermann-Herder Str. 3, 79104 Freiburg (Germany)], E-mail: susanne.kuehn@physik.uni-freiburg.de; Boscardin, M.; Dalla Betta, G.-F. [ITC-irst Trento, Microsystems Division, via Sommarive, 18 38050 Povo di Trento (Italy); Eckert, S.; Jakobs, K.; Maassen, M.; Parzefall, U. [Physikalisches Institut, Universitaet Freiburg, Hermann-Herder Str. 3, 79104 Freiburg (Germany); Piemonte, C.; Pozza, A.; Ronchin, S.; Zorzi, N. [ITC-irst Trento, Microsystems Division, via Sommarive, 18 38050 Povo di Trento (Italy)

    2007-12-11

    In this paper measurements of a 3D single-type column (3D-stc) microstrip silicon device are shown. The 3D-stc sensor has n-type columns in p-type substrate. It has been connected to an ATLAS SCT ABCD3T chip and is readout with ATLAS SCT electronics at 40 MHz. Spatial measurements were done with a laser setup to investigate the expected low field region in 3D devices. An influence of the p-stops on the collected charge has been observed.

  11. Dynamic properties of motor proteins with two subunits

    International Nuclear Information System (INIS)

    Kolomeisky, Anatoly B; III, Hubert Phillips

    2005-01-01

    The dynamics of motor protein molecules consisting of two subunits is investigated using simple discrete stochastic models. Exact steady-state analytical expressions are obtained for velocities and dispersions for any number of intermediate states and conformations between the corresponding binding states of proteins. These models enable us to provide a detailed description and comparison of two different mechanisms of the motion of motor proteins along the linear tracks: the hand-over-hand mechanism, when the motion of subunits alternate; and the inchworm mechanism, when one subunit is always trailing another one. It is shown that the proteins in the hand-over-hand mechanism move faster and fluctuate more than the molecules in the inchworm mechanism. The effect of external forces on dynamic properties of motor proteins is also discussed. Finally, a quantitative method, based on experimental observations for single motor proteins, is proposed for distinguishing between two mechanisms of motion

  12. Probing the functional subunits of the tonoplast H+-ATPase

    International Nuclear Information System (INIS)

    Randall, S.K.; Lai, S.; Sze, H.

    1986-01-01

    The tonoplast ATPase of oat roots is composed of at least three polypeptides of 72, 60, and 16 kDa. The 16 kDA polypeptide covalently binds N,N'-dicyclohexylcarbodiimide and is postulated to be a component of the proton channel. Initial studies to identify other subunits indicate that both the 72 and 60 kDa subunits covalently bind 14 C]-7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and [ 14 C]N-ethylamleimide, inhibitors of the tonoplast ATPase. ATP prevents binding of these inhibitors suggesting that both the 72 and 60 kDa subunits are involved in substrate binding. Polyclonal antibody has been made to the 72 kDa subunit. Western blot analysis of tonoplast vesicles reveals single reactive polypeptide (72 kDa). The antibody shows no cross-reactivity towards either the mitochondrial F 1 -ATPase or the plasma membrane ATPase. This antibody specifically inhibits ATP hydrolysis and ATP-dependent H + pumping in native tonoplast vesicles. The authors conclude that the 72 kDa subunit is intimately associated with the catalytic (or ATP-binding) site

  13. Factors associated with serum B-type natriuretic peptide in infants with single ventricles.

    Science.gov (United States)

    Butts, Ryan J; Zak, Victor; Hsu, Daphne; Cnota, James; Colan, Steven D; Hehir, David; Kantor, Paul; Levine, Jami C; Margossian, Renee; Richmond, Marc; Szwast, Anita; Williams, Derek; Williams, Richard; Atz, Andrew M

    2014-06-01

    Data regarding the value of B-type natriuretic peptide (BNP) measurements in infants with a single-ventricle (SV) physiology are lacking. This analysis aimed to describe the BNP level changes in infants with an SV physiology before and after superior cavopulmonary connection (SCPC) surgery. Levels of BNP were measured by a core laboratory before SCPC (at 5.0 ± 1.6 months) and at the age of 14 months during a multicenter trial of angiotensin-converting enzyme inhibition therapy for infants with SV. Multivariable longitudinal analysis was used to model the associations between BNP levels and three sets of grouped variables (echocardiography, catheterization, growth). Multivariable analysis was performed to assess associations with patient characteristics at both visits. Associations between BNP levels and neurodevelopmental variables were investigated at the 14 month visit because neurodevelopmental assessment was performed only at this visit. The BNP level was significantly higher before SCPC (n = 173) than at the age of 14 months (n = 134). The respective median levels were 80.8 pg/ml (interquartile range [IQR], 35-187 pg/ml) and 34.5 pg/ml (IQR, 17-67 pg/ml) (p SCPC and in 21 subjects (16 %) at the age of 14 months. In the 117 patients who had BNP measurements at both visits, the median BNP level decreased 32 pg/ml (IQR, 1-79 pg/ml) (p SCPC surgery (p SCPC surgery (p = 0.04), and a lower Bayley psychomotor developmental index (p = 0.02). The levels of BNP decreases in infants with SV from the pre-SCPC visit to the age of 14 months. A higher BNP level is associated with increased ventricular dilation in systole, increased AV valve regurgitation, impaired growth, and poorer neurodevelopmental outcomes. Therefore, BNP level may be a useful seromarker for identifying infants with SV at risk for worse outcomes.

  14. Interaction of iron status with single nucleotide polymorphisms on incidence of type 2 diabetes.

    Directory of Open Access Journals (Sweden)

    Jihye Kim

    Full Text Available The objective of this study is to find single nucleotide polymorphisms (SNPs associated with a risk of Type 2 diabetes (T2D in Korean adults and to investigate the longitudinal association between these SNPs and T2D and the interaction effects of iron intake and average hemoglobin level. Data from the KoGES_Ansan and Ansung Study were used. Gene-iron interaction analysis was conducted using a two-step approach. To select candidate SNPs associated with T2D, a total of 7,935 adults at baseline were included in genome-wide association analysis (step one. After excluding T2D prevalent cases, prospective analyses were conducted with 7,024 adults aged 40-69 (step two. The association of selected SNPs and iron status with T2D and their interaction were determined using a Cox proportional hazard model. A total of 3 SNPs [rs9465871 (CDKAL1, rs10761745 (JMJD1C, and rs163177 (KCNQ1] were selected as candidate SNPs related to T2D. Among them, rs10761745 (JMJD1C and rs163177 (KCNQ1 were prospectively associated with T2D. High iron intake was also prospectively associated with the risk of T2D after adjusting for covariates. Average hemoglobin level was positively associated with T2D after adjusting for covariates in women. We also found significant interaction effects between rs10761745 (JMJD1C and average hemoglobin levels on the risk of T2D among women with normal inflammation and without anemia at baseline. In conclusion, KCNQ1 and JMJD1C may prospectively contribute to the risk of T2D incidence among adults over the age of 40 and JMJD1C, but CDKAL1 may not, and iron status may interactively contribute to T2D incidence in women.

  15. Surface properties of alumino-silicate single-walled nanotubes of the imogolite type.

    Science.gov (United States)

    Bonelli, Barbara; Armandi, Marco; Garrone, Edoardo

    2013-08-28

    An IR spectroscopy study is reported on the nature and accessibility of external and internal surfaces of single-walled alumino-silicate nanotubes (NTs) of the imogolite type. NTs form bundles with hexagonal symmetry, in which three kinds of surfaces may be figured out: surface A is the inner surface of NTs; surface B is that between three aligned NTs in the hexagonal packing; and surface C arises from slit mesopores between bundles. Two materials were considered: proper imogolite (IMO, (OH)3Al2O3SiOH) and its methylated analogue, (Me-IMO, (OH)3Al2O3SiCH3). The chemical nature of the outer surface of NTs is the same in both materials, i.e. a curved gibbsite sheet with both Al-OH-Al and Al-O-Al groups and an amphoteric character. The inner surface is very hydrophilic in IMO NTs, lined by closely packed silanols, and hydrophobic in Me-IMO, all silanols being replaced by -SiCH3 groups. The change in chemical composition is accompanied by an increment in pore size, about 1.0 nm in IMO, and ca. 2.0 nm in Me-IMO, which implies a change in the accessibility of the B surface, not available to any molecule in IMO, and accessible in Me-IMO to small molecules like water, due to larger pores between NTs. Aluminol species at the B surface display an acidic nature, in contrast with that of the same species at surface C, because of a confinement effect.

  16. Single cell-type comparative metabolomics of epidermal bladder cells from the halophyte Mesembryanthemum crystallinum.

    Directory of Open Access Journals (Sweden)

    Bronwyn Jane Barkla

    2015-06-01

    Full Text Available One of the remarkable adaptive features of the halophyte and facultative CAM plant Mesembryathemum crystallinum are the specialized modified trichomes called epidermal bladder cells (EBC which cover the leaves, stems, and peduncle of the plant. They are present from an early developmental stage but upon salt stress rapidly expand due to the accumulation of water and sodium. This particular plant feature makes it an attractive system for single cell type studies, with recent proteomics and transcriptomics studies of the EBC establishing that these cells are metabolically active and have roles other than sodium sequestration. To continue our investigation into the function of these unusual cells we carried out a comprehensive global analysis of the metabolites present in the EBC extract by gas chromatography Time-of-Flight mass spectrometry (GC-TOF and identified 194 known and 722 total molecular features. Statistical analysis of the metabolic changes between control and salt-treated samples was used to identify 352 significantly differing metabolites (268 after correction for FDR. Principal components analysis provided an unbiased evaluation of the data variance structure. Biochemical pathway enrichment analysis suggested significant perturbations in 13 biochemical pathways as defined in KEGG. More than 50% of the metabolites that show significant changes in the EBC, can be classified as compatible solutes and include sugars, sugar alcohols, protein and non-protein amino acids, and organic acids, highlighting the need to maintain osmotic homeostasis to balance the accumulation of Na and Cl ions. Overall, the comparison of metabolic changes in salt treated relative to control samples suggest large alterations in Mesembryanthemum crystallinum epidermal bladder cells.

  17. The beta subunit of casein kinase II

    DEFF Research Database (Denmark)

    Boldyreff, B; Piontek, K; Schmidt-Spaniol, I

    1991-01-01

    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.......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....

  18. Typing of 49 autosomal SNPs by single base extension and capillary electrophoresis for forensic genetic testing

    DEFF Research Database (Denmark)

    Børsting, Claus; Tomas Mas, Carmen; Morling, Niels

    2012-01-01

    We describe a method for simultaneous amplification of 49 autosomal single nucleotide polymorphisms (SNPs) by multiplex PCR and detection of the SNP alleles by single base extension (SBE) and capillary electrophoresis. All the SNPs may be amplified from only 100 pg of genomic DNA and the length...

  19. The inactivation of single-chain urokinase-type plasminogen activator by thrombin in a plasma milieu : effect of thrombomodulin

    NARCIS (Netherlands)

    Braat, E.A.M.; Los, P.; Rijken, D.C.

    1998-01-01

    Thrombin cleaves single-chain urokinase-type plasminogen activator (scu- PA) into a virtually inactive two-chain form (tcu-PA/T), a process which may contribute to the maintenance of a fresh blood clot. We have examined the inactivation of scu-PA by thrombin in a plasma milieu to get more insight in

  20. Types and Dynamics of Gendered Space: A Case of Emirati Female Learners in a Single-Gender Context

    Science.gov (United States)

    Alzeer, Gergana

    2018-01-01

    This article is concerned with gendered spaces as they emerge from exploring Emirati female learners' spatiality in a single-gender context. By conducting ethnographic research and utilising Lefebvre's triad of perceived, conceived and lived space for the analysis and categorisation of students' spaces, three types of gendered spaces emerged:…

  1. The influence of the type of embryo culture medium on neonatal birthweight after single embryo transfer in IVF

    NARCIS (Netherlands)

    Vergouw, C.G.; Kostelijk, E.H.; Doejaaren, E.; Hompes, P.G.A.; Lambalk, C.B.; Schats, R.

    2012-01-01

    STUDY QUESTION Does the type of medium used to culture fresh and frozenthawed embryos influence neonatal birthweight after single embryo transfer (SET) in IVF? SUMMARY ANSWER A comparison of two commercially available culture media showed no significant influence on mean birthweight and mean

  2. Human heart cytochrome c oxidase subunit VIII. Purification and determination of the complete amino acid sequence

    NARCIS (Netherlands)

    van Kuilenburg, A. B.; Muijsers, A. O.; Demol, H.; Dekker, H. L.; van Beeumen, J. J.

    1988-01-01

    Subunit VIII was purified from a preparation of the human heart cytochrome c oxidase and its complete amino acid sequence was determined. The sequence proved to be much more related to that of the bovine liver oxidase subunit VIII than to that found in bovine heart. Our finding of a 'liver-type'

  3. Lion (Panthera leo) and caracal (Caracal caracal) type IIx single muscle fibre force and power exceed that of trained humans.

    Science.gov (United States)

    Kohn, Tertius A; Noakes, Timothy D

    2013-03-15

    This study investigated for the first time maximum force production, shortening velocity (Vmax) and power output in permeabilised single muscle fibres at 12°C from lion, Panthera leo (Linnaeus 1758), and caracal, Caracal caracal (Schreber 1776), and compared the values with those from human cyclists. Additionally, the use and validation of previously frozen tissue for contractile experiments is reported. Only type IIx muscle fibres were identified in the caracal sample, whereas type IIx and only two type I fibres were found in the lion sample. Only pure type I and IIa, and hybrid type IIax fibres were identified in the human samples - there were no pure type IIx fibres. Nevertheless, compared with all the human fibre types, the lion and caracal fibres were smaller (Plion: 3008±151 μm(2), caracal: 2583±221 μm(2)). On average, the felid type IIx fibres produced significantly greater force (191-211 kN m(-2)) and ~3 times more power (29.0-30.3 kN m(-2) fibre lengths s(-1)) than the human IIax fibres (100-150 kN m(-2), 4-11 kN m(-2) fibre lengths s(-1)). Vmax values of the lion type IIx fibres were also higher than those of human type IIax fibres. The findings suggest that the same fibre type may differ substantially between species and potential explanations are discussed.

  4. Nocturnal Glucose Metabolism in Type 1 Diabetes: A Study Comparing Single Versus Dual Tracer Approaches.

    Science.gov (United States)

    Mallad, Ashwini; Hinshaw, Ling; Dalla Man, Chiara; Cobelli, Claudio; Basu, Rita; Lingineni, Ravi; Carter, Rickey E; Kudva, Yogish C; Basu, Ananda

    2015-08-01

    Understanding the effect size, variability, and underlying physiology of the dawn phenomenon is important for next-generation closed-loop control algorithms for type 1 diabetes (T1D). We used an iterative protocol design to study 16 subjects with T1D on individualized insulin pump therapy for two successive nights. Endogenous glucose production (EGP) rates at 3 a.m. and 7 a.m. were measured with [6,6-(2)H(2)]glucose as a single tracer, infused from midnight to 7 a.m. in all subjects. To explore possibility of tracer recycling due to prolonged [6,6-(2)H(2)]glucose infusion, which was highly probable after preplanned interim data analyses, we infused a second tracer, [6-(3)H]glucose, from 4 a.m. to 7 a.m. in the last seven subjects to measure EGP at 7 a.m. Cortisol concentrations increased during both nights, but changes in glucagon and insulin concentration were inconsistent. Although the plasma glucose concentrations rose from midnight to 7 a.m. during both nights, EGP measured with [6,6-(2)H(2)]glucose between 3 a.m. and 7 a.m. did not differ during Night 1 but fell in Night 2. However, EGP measured with [6-(3)H]glucose at 7 a.m. was higher than that measured with [6,6-(2)H(2)]glucose during both nights, thereby suggesting tracer recycling probably underestimating EGP calculated at 7 a.m. with [6,6-(2)H(2)]glucose. Likewise, EGP was higher at 7 a.m. with [6-(3)H]glucose than at 3 a.m. with [6,6-(2)H(2)]glucose during both nights. The data demonstrate a consistent overnight rise in glucose concentrations through increased EGP, mediated likely by rising cortisol concentrations. The observations with the dual tracer approach imply significant tracer recycling leading to underestimation of EGP measured by longer-duration tracer infusion.

  5. Improved crystallization of Escherichia coli ATP synthase catalytic complex (F1) by introducing a phosphomimetic mutation in subunit

    International Nuclear Information System (INIS)

    Roy, Ankoor; Hutcheon, Marcus L.; Duncan, Thomas M.; Cingolani, Gino

    2012-01-01

    A phosphomimetic mutation in subunit ∊ dramatically increases reproducibility for crystallization of Escherichia coli ATP synthase catalytic complex (F 1 ) (subunit composition α 3 β 3 γ∊). Diffraction data were collected to ∼3.15 Å resolution using synchrotron radiation. The bacterial ATP synthase (F O F 1 ) of Escherichia coli has been the prominent model system for genetics, biochemical and more recently single-molecule studies on F-type ATP synthases. With 22 total polypeptide chains (total mass of ∼529 kDa), E. coli F O F 1 represents nature’s smallest rotary motor, composed of a membrane-embedded proton transporter (F O ) and a peripheral catalytic complex (F 1 ). The ATPase activity of isolated F 1 is fully expressed by the α 3 β 3 γ ‘core’, whereas single δ and ∊ subunits are required for structural and functional coupling of E. coli F 1 to F O . In contrast to mitochondrial F 1 -ATPases that have been determined to atomic resolution, the bacterial homologues have proven very difficult to crystallize. In this paper, we describe a biochemical strategy that led us to improve the crystallogenesis of the E. coli F 1 -ATPase catalytic core. Destabilizing the compact conformation of ∊’s C-terminal domain with a phosphomimetic mutation (∊S65D) dramatically increased crystallization success and reproducibility, yielding crystals of E. coli F 1 that diffract to ∼3.15 Å resolution

  6. Only one ATP-binding DnaX subunit is required for initiation complex formation by the Escherichia coli DNA polymerase III holoenzyme.

    Science.gov (United States)

    Wieczorek, Anna; Downey, Christopher D; Dallmann, H Garry; McHenry, Charles S

    2010-09-17

    The DnaX complex (DnaX(3)δδ'χ psi) within the Escherichia coli DNA polymerase III holoenzyme serves to load the dimeric sliding clamp processivity factor, β(2), onto DNA. The complex contains three DnaX subunits, which occur in two forms: τ and the shorter γ, produced by translational frameshifting. Ten forms of E. coli DnaX complex containing all possible combinations of wild-type or a Walker A motif K51E variant τ or γ have been reconstituted and rigorously purified. DnaX complexes containing three DnaX K51E subunits do not bind ATP. Comparison of their ability to support formation of initiation complexes, as measured by processive replication by the DNA polymerase III holoenzyme, indicates a minimal requirement for one ATP-binding DnaX subunit. DnaX complexes containing two mutant DnaX subunits support DNA synthesis at about two-thirds the level of their wild-type counterparts. β(2) binding (determined functionally) is diminished 12-30-fold for DnaX complexes containing two K51E subunits, suggesting that multiple ATPs must be bound to place the DnaX complex into a conformation with maximal affinity for β(2). DNA synthesis activity can be restored by increased concentrations of β(2). In contrast, severe defects in ATP hydrolysis are observed upon introduction of a single K51E DnaX subunit. Thus, ATP binding, hydrolysis, and the ability to form initiation complexes are not tightly coupled. These results suggest that although ATP hydrolysis likely enhances β(2) loading, it is not absolutely required in a mechanistic sense for formation of functional initiation complexes.

  7. Flagella-generated forces reveal gear-type motor in single cells of the green alga, Chlamydomonas reinhardtii.

    Science.gov (United States)

    D'Souza, Jacinta S; Gudipati, Mohanram; Dharmadhikari, Jayashree A; Dharmadhikari, Aditya K; Kashyap, Abhishek; Sivaramakrishnan, Manaswini; Aiyer, Manaswini; Rao, Usha; Mathur, Deepak; Rao, Basuthkar J

    2009-03-06

    Optically trapped single cells of the biflagellated, green alga, Chlamydomonas reinhardtii, rotate. The rotational dynamics of trapped wild-type and mutant cells show that functional flagella play a decisive role: the entire flagellar apparatus (central microtubules, radial spokes, and dynein arms) is involved. Any aberration in this apparatus leads to non-functionality, indicating a gear-type mechanism. The translational and rotational motions of the wild-type and mutant cells do not differ significantly. Optical forces alone do not play a vital role in the rotational dynamics of this cellular motor, making them useful as probes of the internal dynamics without external influence.

  8. Sensitivity of the Single Particle Soot Photometer to different black carbon types

    Science.gov (United States)

    Laborde, M.; Mertes, P.; Zieger, P.; Dommen, J.; Baltensperger, U.; Gysel, M.

    2012-05-01

    Black carbon (BC) is now mainly of anthropogenic origin. It is the dominant light absorbing component of atmospheric aerosols, playing an important role in the earth's radiative balance and therefore relevant to climate change studies. In addition, BC is known to be harmful to human beings making it relevant to policy makers. Nevertheless, the measurement of BC remains biased by the instrument-based definition of BC. The Single Particle Soot Photometer (SP2), allows the measurement of the refractory BC (rBC) mass of individual particles using laser-induced incandescence. However, the SP2 needs an empirical calibration to retrieve the rBC mass from the incandescence signal and the sensitivity of the SP2 differs between different BC types. Ideally, for atmospheric studies, the SP2 should be calibrated using ambient particles containing a known mass of ambient rBC. However, such "ambient BC" calibration particles cannot easily be obtained and thus commercially available BC particles are commonly used for SP2 calibration instead. In this study we tested the sensitivity of the SP2 to different BC types in order to characterize the potential error introduced by using non-ambient BC for calibration. The sensitivity of the SP2 was determined, using an aerosol particle mass analyzer, for rBC from thermodenuded diesel exhaust, wood burning exhaust and ambient particles as well as for commercially available products: Aquadag® and fullerene soot. Thermodenuded, fresh diesel exhaust has been found to be ideal for SP2 calibration for two reasons. First, the small amount of non-BC matter upon emission reduces the risk of bias due to incomplete removal of non-BC matter and second, it is considered to represent atmospheric rBC in urban locations where diesel exhaust is the main source of BC. The SP2 was found to be up to 16% less sensitive to rBC from thermodenuded ambient particles (≤15 fg) than rBC from diesel exhaust, however, at least part of this difference can be explained

  9. Somatosensory neuron types identified by high-coverage single-cell RNA-sequencing and functional heterogeneity

    Science.gov (United States)

    Li, Chang-Lin; Li, Kai-Cheng; Wu, Dan; Chen, Yan; Luo, Hao; Zhao, Jing-Rong; Wang, Sa-Shuang; Sun, Ming-Ming; Lu, Ying-Jin; Zhong, Yan-Qing; Hu, Xu-Ye; Hou, Rui; Zhou, Bei-Bei; Bao, Lan; Xiao, Hua-Sheng; Zhang, Xu

    2016-01-01

    Sensory neurons are distinguished by distinct signaling networks and receptive characteristics. Thus, sensory neuron types can be defined by linking transcriptome-based neuron typing with the sensory phenotypes. Here we classify somatosensory neurons of the mouse dorsal root ganglion (DRG) by high-coverage single-cell RNA-sequencing (10 950 ± 1 218 genes per neuron) and neuron size-based hierarchical clustering. Moreover, single DRG neurons responding to cutaneous stimuli are recorded using an in vivo whole-cell patch clamp technique and classified by neuron-type genetic markers. Small diameter DRG neurons are classified into one type of low-threshold mechanoreceptor and five types of mechanoheat nociceptors (MHNs). Each of the MHN types is further categorized into two subtypes. Large DRG neurons are categorized into four types, including neurexophilin 1-expressing MHNs and mechanical nociceptors (MNs) expressing BAI1-associated protein 2-like 1 (Baiap2l1). Mechanoreceptors expressing trafficking protein particle complex 3-like and Baiap2l1-marked MNs are subdivided into two subtypes each. These results provide a new system for cataloging somatosensory neurons and their transcriptome databases. PMID:26691752

  10. Targeting the Immunogenetic Diseases with the Appropriate HLA Molecular Typing: Critical Appraisal on 2666 Patients Typed in One Single Centre

    Directory of Open Access Journals (Sweden)

    M. Guarene

    2013-01-01

    Full Text Available We compared the immunogenetic data from 2666 patients affected by HLA-related autoimmune diseases with those from 4389 ethnically matched controls (3157 cord blood donors CBD, 1232 adult bone marrow donors BMD, to verify the appropriateness of HLA typing requests received in the past decade. The frequency of HLA-B*27 phenotype was 10.50% in 724 ankylosing spondylitis, 16.80% in 125 uveitis (3.41% BMD, 4.24% CBD, P<0.0001; HLA-B*51 allele was 15.57% in 212 Behçet’s disease (12.91% BMD, 9.88% CBD, P<0.0001; the HLA-DRB1-rheumatoid arthritis (RA shared epitope was 13.72% in 554 RA (10.85% BMD, 13.48% CBD, P=0.016; the carriers of almost one of HLA-DQB1 susceptibility alleles were 84.91% in 795 celiac disease (CD and 59.37% in 256 insulin-dependent diabetes mellitus (IDDM (46.06% in 875 CBD, 42.75% in 662 BMD P<0.0001. Overall, our results show that the HLA marker frequencies were higher in patients than controls, but lower than expected from the literature data (excluding CD and IDDM and demonstrate that, in complex immunogenetic conditions, a substantial number of genetic analyses are redundant and inappropriate, burdening to the public health costs. For this reason, we suggest the Italian Scientific Society of Immunogenetics to establish guidelines to improve the appropriateness of typing requests.

  11. Role of Asp544 in subunit I for Na+ pumping by Vitreoscilla cytochrome bo

    International Nuclear Information System (INIS)

    Chung, Yeon T.; Stark, Benjamin C.; Webster, Dale A.

    2006-01-01

    The conserved Glu540 in subunit I of Escherichia coli cytochrome bo (a H + pump) is replaced by Asp544 in the Vitreoscilla enzyme (a Na + pump). Site-directed mutagenesis of the Vitreoscilla cytochrome bo operon changed this Asp to Glu, and both wild type and mutant cyo's were transformed into E. coli strain GV100, which lacks cytochrome bo. Compared to the wild type transformant the Asp544Glu transformant had decreased ability to pump Na + as well as decreased stimulation in respiratory activity in the presence of Na + . Preliminary experiments indicated that this mutant also had increased ability to pump protons, suggesting that this single change may provide cation pumping specificity in this group of enzymes

  12. Common-Ground-Type Tansformerless Inverters for Single-Phase Solar Photovoltaic Systems

    DEFF Research Database (Denmark)

    Siwakoti, Yam Prasad; Blaabjerg, Frede

    2018-01-01

    This paper proposes a family of novel flying capacitor transformerless inverters for single-phase photovoltaic (PV) systems. Each of the new topologies proposed is based on a flying capacitor principle and requires only four power switches and/or diodes, one capacitor, and a small filter at the o......This paper proposes a family of novel flying capacitor transformerless inverters for single-phase photovoltaic (PV) systems. Each of the new topologies proposed is based on a flying capacitor principle and requires only four power switches and/or diodes, one capacitor, and a small filter...

  13. Type-II second-harmonic-generation properties of YCOB and GdCOB single crystals.

    Science.gov (United States)

    Liu, Yanqing; Qi, Hongwei; Lu, Qingming; Yu, Fapeng; Wang, Zhengping; Xu, Xinguang; Zhao, Xian

    2015-02-09

    As excellent nonlinear optical (NLO) crystals, YCa(4)O(BO(3))(3) (YCOB) and GdCa(4)O(BO(3))(3) (GdCOB) have been paid much attention since their first appearance in 1990's. From that time to now, almost all of related researches and applications have focused on their type-I phase-matching (PM) configurations which possess large effective NLO coefficient (d(eff)). In this paper, type-II second-harmonic-generation (SHG) properties of these two crystals are reported, including PM curve, d(eff), angular acceptance and walk-off angle. Both of the type-II SHG experiments for 1064 and 1320 nm have indicated that the optimum directions which have maximum d(eff) locate in the second octant, i.e. (90° crystal, the largest type-II SHG conversion efficiency of a 1064 nm Nd:YAG pico-second laser is 55%, which reaches the same level of the optimum type-I sample. To our knowledge this is the first time that type-II SHG performance of YCOB and GdCOB crystals is investigated intensively. Our research has shown that the smaller d(eff) of type-II PM can be compensated by its larger angular acceptance and less beam walk-off. The same level SHG conversion efficiency implies for such type crystals the type-II components have the potential to replace type-I ones and obtain important NLO applications in the future.

  14. Analysis of the entire ryanodine receptor type 1 and alpha 1 subunit of the dihydropyridine receptor (CACNA1S) coding regions for variants associated with malignant hyperthermia in Australian families.

    Science.gov (United States)

    Gillies, R L; Bjorksten, A R; Du Sart, D; Hockey, B M

    2015-03-01

    Defects in the genes coding for the skeletal muscle ryanodine receptor (RYR1) and alpha 1 subunit of the dihydropyridine receptor (CACNA1S) have been identified as causative for malignant hyperthermia (MH). Sixty-two MH susceptible individuals presenting to the same diagnostic centre had copy deoxyribonucleic acid, derived from muscle ribonucleic acid, sequenced to identify variants with the potential to be responsible for the MH phenotype in both RYR1 and CACNA1S. These genetic findings were combined with clinical episode details and in vitro contracture test results to improve our understanding of the Australian MH cohort. Twelve novel variants were identified in RYR1 and six in CACNA1S. Known RYR1 causative mutations were identified in six persons and novel variants in RYR1 and CACNA1S in a further 17 persons. Trends indicated higher mutation identification in those with more definitive clinical episodes and stronger in vitro contracture test responses.

  15. Isolation and characterization of recombinant human casein kinase II subunits alpha and beta from bacteria

    DEFF Research Database (Denmark)

    Grankowski, N; Boldyreff, B; Issinger, O G

    1991-01-01

    cDNA encoding the casein kinase II (CKII) subunits alpha and beta of human origin were expressed in Escherichia coli using expression vector pT7-7. Significant expression was obtained with E. coli BL21(DE3). The CKII subunits accounted for approximately 30% of the bacterial protein; however, most...... of the expressed proteins were produced in an insoluble form. The recombinant CKII alpha subunit was purified by DEAE-cellulose chromatography, followed by phosphocellulose and heparin-agarose chromatography. The recombinant CKII beta subunit was extracted from the insoluble pellet and purified in a single step...... at a 1:1 ratio of both subunits. Using a synthetic peptide (RRRDDDSDDD) as a substrate, the maximum protein kinase stimulation observed was fourfold under the conditions used. The Km of the reconstituted enzyme for the synthetic peptide (80 microM) was comparable to the mammalian enzyme (40-60 micro...

  16. Single microwave-photon detector using an artificial Λ-type three-level system

    Science.gov (United States)

    Inomata, Kunihiro; Lin, Zhirong; Koshino, Kazuki; Oliver, William D.; Tsai, Jaw-Shen; Yamamoto, Tsuyoshi; Nakamura, Yasunobu

    2016-07-01

    Single-photon detection is a requisite technique in quantum-optics experiments in both the optical and the microwave domains. However, the energy of microwave quanta are four to five orders of magnitude less than their optical counterpart, making the efficient detection of single microwave photons extremely challenging. Here we demonstrate the detection of a single microwave photon propagating through a waveguide. The detector is implemented with an impedance-matched artificial Λ system comprising the dressed states of a driven superconducting qubit coupled to a microwave resonator. Each signal photon deterministically induces a Raman transition in the Λ system and excites the qubit. The subsequent dispersive readout of the qubit produces a discrete `click'. We attain a high single-photon-detection efficiency of 0.66+/-0.06 with a low dark-count probability of 0.014+/-0.001 and a reset time of ~400 ns. This detector can be exploited for various applications in quantum sensing, quantum communication and quantum information processing.

  17. Alternative types of molecule-decorated atomic chains in Au–CO–Au single-molecule junctions

    Directory of Open Access Journals (Sweden)

    Zoltán Balogh

    2015-06-01

    Full Text Available We investigate the formation and evolution of Au–CO single-molecule break junctions. The conductance histogram exhibits two distinct molecular configurations, which are further investigated by a combined statistical analysis. According to conditional histogram and correlation analysis these molecular configurations show strong anticorrelations with each other and with pure Au monoatomic junctions and atomic chains. We identify molecular precursor configurations with somewhat higher conductance, which are formed prior to single-molecule junctions. According to detailed length analysis two distinct types of molecule-affected chain-formation processes are observed, and we compare these results to former theoretical calculations considering bridge- and atop-type molecular configurations where the latter has reduced conductance due to destructive Fano interference.

  18. Moisture supply in Danish single-family houses – the influence of occupant behaviour and type of room

    DEFF Research Database (Denmark)

    Møller, Eva B.; Hansen, Ernst Jan de Place

    2017-01-01

    . The study focuses on the dependency of number and age of occupants, occupants’ time spent in the house, square meters living space, ventilation habits and type of room. Moisture supply depends on the type of room; bathroom, basement and living room have the highest values. None of the other parameters seems......According to ISO 13788, the internal moisture supply in dwellings can be described by humidity classes defined by outdoor temperature, occupancy and ventilation. Hygrothermal measurements in indoor air in 500 Danish single-family houses were made to investigate if this corresponds with reality...

  19. Development and evaluation of one step single tube multiplex RT-PCR for rapid detection and typing of dengue viruses

    OpenAIRE

    Parida Manmohan; Shrivastava Ambuj; Santhosh SR; Dash Paban; Saxena Parag; Rao PV

    2008-01-01

    Abstract Background Dengue is emerging as a major public health concern in many parts of the world. The development of a one-step, single tube, rapid, and multiplex reverse transcription polymerase chain reaction (M-RT-PCR) for simultaneous detection and typing of dengue virus using serotype specific primers during acute phase of illness is reported. Results An optimal assay condition with zero background was established having no cross-reaction with closely related members of flavivirus (Jap...

  20. Effect of ligand substitution on the exchange interactions in {Mn12}-type single-molecule magnets

    OpenAIRE

    Boukhvalov, D. W.; Dobrovitski, V. V.; Kögerler, P.; Al-Saqer, M.; Katsnelson, M. I.; Lichtenstein, A. I.; Harmon, B. N.

    2010-01-01

    We investigate how the ligand substitution affects the intra-molecular spin exchange interactions, studying a prototypal family of single-molecule magnets comprising dodecanuclear cluster molecules [Mn12O12(COOR)16]. We identify a simple scheme based on accumulated Pauling electronegativity numbers (a.e.n.) of the carboxylate ligand groups (R). The redistribution of the electron density, controlled by a.e.n. of a ligand, changes the degree of hybridization between 3d electrons of manganese an...

  1. Facilitation by the beta2a subunit of pore openings in cardiac Ca2+ channels.

    Science.gov (United States)

    Costantin, J; Noceti, F; Qin, N; Wei, X; Birnbaumer, L; Stefani, E

    1998-02-15

    1. Single channel recordings were performed on the cardiac calcium channel (alpha1C) in order to study the effect of coexpression of the accessory beta2a subunit. On-cell patch clamp recordings were performed after expression of these channels in Xenopus oocytes. 2. The alpha1C subunit, when expressed alone, had similar single channel properties to native cardiac channels. Slow transitions between low and high open probability (Po) gating modes were found as well as fast gating transitions between the open and closed states. 3. Coexpression of the beta2a subunit caused changes in the fast gating during high Po mode. In this mode, open time distributions reveal at least three open states and the beta2a subunit favours the occupancy of the longest, 10-15 ms open state. No effect of the beta2a subunit was found when the channel was gating in the low Po mode. 4. Slow gating transitions were also affected by the beta2a subunit. The high Po mode was maintained for the duration of the depolarizing pulse in the presence of the beta2a subunit; while the alpha1C channel when expressed alone, frequently switched into and out of the high Po mode during the course of a sweep. 5. The beta2a subunit also affected mode switching that occurred between sweeps. Runs analysis revealed that the alpha1C subunit has a tendency toward non-random mode switching. The beta2a subunit increased this tendency. A chi2 analysis of contingency tables indicated that the beta2a subunit caused the alpha1C channel to gain 'intrinsic memory', meaning that the mode of a given sweep can be non-independent of the mode of the previous sweep. 6. We conclude that the beta2a subunit causes changes to the alpha1C channel in both its fast and slow gating behaviour. The beta2a subunit alters fast gating by facilitating movement of the channel into an existing open state. Additionally, the beta2a subunit decreases the slow switching between low and high Po modes.

  2. Subunit conformational variation within individual GroEL oligomers resolved by Cryo-EM.

    Science.gov (United States)

    Roh, Soung-Hun; Hryc, Corey F; Jeong, Hyun-Hwan; Fei, Xue; Jakana, Joanita; Lorimer, George H; Chiu, Wah

    2017-08-01

    Single-particle electron cryo-microscopy (cryo-EM) is an emerging tool for resolving structures of conformationally heterogeneous particles; however, each structure is derived from an average of many particles with presumed identical conformations. We used a 3.5-Å cryo-EM reconstruction with imposed D7 symmetry to further analyze structural heterogeneity among chemically identical subunits in each GroEL oligomer. Focused classification of the 14 subunits in each oligomer revealed three dominant classes of subunit conformations. Each class resembled a distinct GroEL crystal structure in the Protein Data Bank. The conformational differences stem from the orientations of the apical domain. We mapped each conformation class to its subunit locations within each GroEL oligomer in our dataset. The spatial distributions of each conformation class differed among oligomers, and most oligomers contained 10-12 subunits of the three dominant conformation classes. Adjacent subunits were found to more likely assume the same conformation class, suggesting correlation among subunits in the oligomer. This study demonstrates the utility of cryo-EM in revealing structure dynamics within a single protein oligomer.

  3. A single mutation in Taiwanese H6N1 influenza hemagglutinin switches binding to human-type receptors

    Energy Technology Data Exchange (ETDEWEB)

    de Vries, Robert P.; Tzarum, Netanel; Peng, Wenjie; Thompson, Andrew J.; Ambepitiya Wickramasinghe, Iresha N.; de la Pena, Alba T. Torrents; van Breemen, Marielle J.; Bouwman, Kim M.; Zhu, Xueyong; McBride, Ryan; Yu, Wenli; Sanders, Rogier W.; Verheije, Monique H.; Wilson, Ian A.; Paulson, James C.

    2017-07-10

    In June 2013, the first case of human infection with an avian H6N1 virus was reported in a Taiwanese woman. Although this was a single non-fatal case, the virus continues to circulate in Taiwanese poultry. As with any emerging avian virus that infects humans, there is concern that acquisition of human-type receptor specificity could enable transmission in the human population. Despite mutations in the receptor-binding pocket of the human H6N1 isolate, it has retained avian-type (NeuAcα2-3Gal) receptor specificity. However, we show here that a single nucleotide substitution, resulting in a change from Gly to Asp at position 225 (G225D), completely switches specificity to human-type (NeuAcα2-6Gal) receptors. Significantly, G225D H6 loses binding to chicken trachea epithelium and is now able to bind to human tracheal tissue. Structural analysis reveals that Asp225 directly interacts with the penultimate Gal of the human-type receptor, stabilizing human receptor binding.

  4. Size-controllable DNA nanoribbons assembled from three types of reusable brick single-strand DNA tiles.

    Science.gov (United States)

    Shi, Xiaolong; Chen, Congzhou; Li, Xin; Song, Tao; Chen, Zhihua; Zhang, Zheng; Wang, Yanfeng

    2015-11-21

    Precise control of nanostructure is a significant goal shared by supramolecular chemistry, nanotechnology and materials science. In DNA nanotechnology, methods of constructing desired DNA nanostructures using programmable DNA strands have been studied extensively and have become a promising branch of research, but developing universal and low-cost (in the sense of using fewer types of DNA strands) methods remains a challenge. In this work, we propose a novel approach to assemble size-controllable DNA nanoribbons with three types of reusable brick SSTs (single-stranded DNA tiles), where the control of ribbon size is achieved by regulating the concentration ratio between manipulative strands and packed single-stranded DNA tiles. In our method, three types of brick SSTs are sufficient in assembling DNA nanoribbons of different sizes, which is much less than the number of types of unique tile-programmable assembling strategy, thus achieving a universal and low-cost method. The assembled DNA nanoribbons are observed and analyzed by atomic force microscopy (AFM). Experimental observations strongly suggest the feasibility and reliability of our method.

  5. Study shows colon and rectal tumors constitute a single type of cancer

    Science.gov (United States)

    The pattern of genomic alterations in colon and rectal tissues is the same regardless of anatomic location or origin within the colon or the rectum, leading researchers to conclude that these two cancer types can be grouped as one, according to The Cancer

  6. Demographic and Clinical Characteristics of Type 1 Diabetes Mellitus in Omani Children - Single Center Experience

    Directory of Open Access Journals (Sweden)

    Saif Al-Yaarubi

    2014-03-01

    Full Text Available Objectives: To describe the demographic characteristics and clinical presentation of Omani children with type 1 diabetes mellitus at Sultan Qaboos University Hospital, Muscat, Oman. Methods: A retrospective analysis of all children with type 1 diabetes mellitus attending the Pediatric Endocrine Unit at Sultan Qaboos University Hospital, Oman from June 2006 to May 2013. Results: One hundred and forty-four patients were included in the study. The mean±SD of age at diagnosis was 6.7 ± 3.7 years. The median duration of symptoms was 10 days (IQR; 5-14. The most commonly reported presenting symptoms were polyuria (94%, polydipsia (82%, and weight loss (59%. Diabetic ketoacidosis at initial presentation was diagnosed in 31% of the patients. Different insulin regimens were prescribed: multiple daily injections in 109 (76% patients, twice daily insulin regimen in 23 (16% patients, and insulin pump therapy in 12 (8% patients. Family history of type 1 diabetes mellitus was present in 31 (22% patients. There were no significant differences in presenting complaints (polyuria, p=0.182; polydipsia, p=0.848, duration of symptoms (p=0.331, reported weight loss (p=0.753, or diabetic ketoacidosis at presentation (p=0.608 between patients with and without family history of type 1 diabetes mellitus. Conclusion: Polyuria, polydipsia and weight loss are the most common presenting symptoms. Family history of type 1 diabetes mellitus is highly prevalent among the studied patients. Diabetic ketoacidosis was found to be less common in Oman compared to other diabetes centers in the Middle East.

  7. Improving assessment of daily energy expenditure by identifying types of physical activity with a single accelerometer.

    Science.gov (United States)

    Bonomi, A G; Plasqui, G; Goris, A H C; Westerterp, K R

    2009-09-01

    Accelerometers are often used to quantify the acceleration of the body in arbitrary units (counts) to measure physical activity (PA) and to estimate energy expenditure. The present study investigated whether the identification of types of PA with one accelerometer could improve the estimation of energy expenditure compared with activity counts. Total energy expenditure (TEE) of 15 subjects was measured with the use of double-labeled water. The physical activity level (PAL) was derived by dividing TEE by sleeping metabolic rate. Simultaneously, PA was measured with one accelerometer. Accelerometer output was processed to calculate activity counts per day (AC(D)) and to determine the daily duration of six types of common activities identified with a classification tree model. A daily metabolic value (MET(D)) was calculated as mean of the MET compendium value of each activity type weighed by the daily duration. TEE was predicted by AC(D) and body weight and by AC(D) and fat-free mass, with a standard error of estimate (SEE) of 1.47 MJ/day, and 1.2 MJ/day, respectively. The replacement in these models of AC(D) with MET(D) increased the explained variation in TEE by 9%, decreasing SEE by 0.14 MJ/day and 0.18 MJ/day, respectively. The correlation between PAL and MET(D) (R(2) = 51%) was higher than that between PAL and AC(D) (R(2) = 46%). We conclude that identification of activity types combined with MET intensity values improves the assessment of energy expenditure compared with activity counts. Future studies could develop models to objectively assess activity type and intensity to further increase accuracy of the energy expenditure estimation.

  8. Optimal strategy for a single-qubit gate and the trade-off between opposite types of decoherence

    Science.gov (United States)

    Alicki, Robert; Horodecki, Michał; Horodecki, Paweł; Horodecki, Ryszard; Jacak, Lucjan; Machnikowski, Paweł

    2004-07-01

    We study reliable quantum-information processing (QIP) under two different types of environment. The first type is Markovian exponential decay, and the appropriate elementary strategy of protection of qubit is to apply fast gates. The second one is strongly non-Markovian and occurs solely during operations on the qubit. The best strategy is then to work with slow gates. If the two types are both present, one has to optimize the speed of gate. We show that such a trade-off is present for a single-qubit operation in a semiconductor quantum dot implementation of QIP, where recombination of exciton (qubit) is Markovian, while phonon dressing gives rise to the non-Markovian contribution.

  9. Optimal strategy for a single-qubit gate and the trade-off between opposite types of decoherence

    International Nuclear Information System (INIS)

    We study reliable quantum-information processing (QIP) under two different types of environment. The first type is Markovian exponential decay, and the appropriate elementary strategy of protection of qubit is to apply fast gates. The second one is strongly non-Markovian and occurs solely during operations on the qubit. The best strategy is then to work with slow gates. If the two types are both present, one has to optimize the speed of gate. We show that such a trade-off is present for a single-qubit operation in a semiconductor quantum dot implementation of QIP, where recombination of exciton (qubit) is Markovian, while phonon dressing gives rise to the non-Markovian contribution

  10. Single-Particle Tracking of Human Immunodeficiency Virus Type 1 Productive Entry into Human Primary Macrophages.

    Science.gov (United States)

    Li, Qin; Li, Wei; Yin, Wen; Guo, Jia; Zhang, Zhi-Ping; Zeng, Dejun; Zhang, Xiaowei; Wu, Yuntao; Zhang, Xian-En; Cui, Zongqiang

    2017-04-25

    Macrophages are one of the major targets of human immunodeficiency virus (HIV-1), but the viral entry pathway remains poorly understood in these cells. Noninvasive virus labeling and single-virus tracking are effective tools for studying virus entry. Here, we constructed a quantum dot (QD)-encapsulated infectious HIV-1 particle to track viral entry at a single-particle level in live human primary macrophages. QDs were encapsulated in HIV-1 virions by incorporating viral accessory protein Vpr-conjugated QDs during virus assembly. With the HIV-1 particles encapsulating QDs, we monitored the early phase of viral infection in real time and observed that, during infection, HIV-1 was endocytosed in a clathrin-mediated manner; the particles were translocated into Rab5A-positive endosomes, and the core was released into the cytoplasm by viral envelope-mediated endosomal fusion. Drug inhibition assays verified that endosome fusion contributes to HIV-1 productive infection in primary macrophages. Additionally, we observed that a dynamic actin cytoskeleton is critical for HIV-1 entry and intracellular migration in primary macrophages. HIV-1 dynamics and infection could be blocked by multiple different actin inhibitors. Our study revealed a productive entry pathway in macrophages that requires both endosomal function and actin dynamics, which may assist in the development of inhibitors to block the HIV entry in macrophages.

  11. Insertion sequence element single nucleotide polymorphism typing provides insights into the population structure and evolution of Mycobacterium ulcerans across Africa.

    Science.gov (United States)

    Vandelannoote, Koen; Jordaens, Kurt; Bomans, Pieter; Leirs, Herwig; Durnez, Lies; Affolabi, Dissou; Sopoh, Ghislain; Aguiar, Julia; Phanzu, Delphin Mavinga; Kibadi, Kapay; Eyangoh, Sara; Manou, Louis Bayonne; Phillips, Richard Odame; Adjei, Ohene; Ablordey, Anthony; Rigouts, Leen; Portaels, Françoise; Eddyani, Miriam; de Jong, Bouke C

    2014-02-01

    Buruli ulcer is an indolent, slowly progressing necrotizing disease of the skin caused by infection with Mycobacterium ulcerans. In the present study, we applied a redesigned technique to a vast panel of M. ulcerans disease isolates and clinical samples originating from multiple African disease foci in order to (i) gain fundamental insights into the population structure and evolutionary history of the pathogen and (ii) disentangle the phylogeographic relationships within the genetically conserved cluster of African M. ulcerans. Our analyses identified 23 different African insertion sequence element single nucleotide polymorphism (ISE-SNP) types that dominate in different areas where Buruli ulcer is endemic. These ISE-SNP types appear to be the initial stages of clonal diversification from a common, possibly ancestral ISE-SNP type. ISE-SNP types were found unevenly distributed over the greater West African hydrological drainage basins. Our findings suggest that geographical barriers bordering the basins to some extent prevented bacterial gene flow between basins and that this resulted in independent focal transmission clusters associated with the hydrological drainage areas. Different phylogenetic methods yielded two well-supported sister clades within the African ISE-SNP types. The ISE-SNP types from the "pan-African clade" were found to be widespread throughout Africa, while the ISE-SNP types of the "Gabonese/Cameroonian clade" were much rarer and found in a more restricted area, which suggested that the latter clade evolved more recently. Additionally, the Gabonese/Cameroonian clade was found to form a strongly supported monophyletic group with Papua New Guinean ISE-SNP type 8, which is unrelated to other Southeast Asian ISE-SNP types.

  12. Role of the Rubisco Small Subunit

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-11-05

    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

  13. Nondiabetic kidney disease in type 2 diabetic patients: A single center experience

    Science.gov (United States)

    Das, U.; Dakshinamurty, K. V.; Prayaga, A.; Uppin, M. S.

    2012-01-01

    Nondiabetic renal disease (NDRD) is seen as a cause of proteinuria and renal failure in type 2 diabetes mellitus (DM). The clinical differences between NDRD and diabetic glomerulosclerosis (DGS) are not clear. This study was done to find the spectrum of NDRD in type 2 DM patients and differences in clinical profile between NDRD and DGS patients. Data of patients with type 2 DM who underwent renal biopsy in this institute from 1990 to 2008 were analyzed retrospectively. Patients were categorized as isolated NDRD, NDRD with DGS, and isolated DGS. A total of 75 patients were included. Mean age was 45 ± 10.2 years, male to female ratio was 3.1 : 1, median duration of DM was 12 months (range, 1 year-15 years), proteinuria was 4.2 ± 3.4 g/day, and serum creatinine was 4.3 ± 3.9 mg/dl. Hypertension was observed in 63 (84%) cases and microscopic hematuria in 24 (32%) cases. Nephrotic syndrome (38.7%) was the commonest clinical presentation. Forty-eight (64%) cases had NDRD and 27 (36%) had DGS. The commonest NDRD was minimal change disease (12.5%). Three (6.3%) patients had lupus nephritis. Tubulointerstitial nephritis has been observed in 10.4% patients. No significant differences between NDRD and DGS patients were found except hypertension which was significantly high in the DGS group. Acute kidney injury and nephritic syndrome were not observed in the DGS group. In conclusion, the incidence of biopsy-proven NDRD in type 2 DM in this study was high. Kidney biopsy aided in the detection of NDRD in clinically suspected patients. PMID:23326046

  14. Bacterial cellulose biosynthesis: diversity of operons, subunits, products and functions

    Science.gov (United States)

    Römling, Ute; Galperin, Michael Y.

    2015-01-01

    Summary Recent studies of bacterial cellulose biosynthesis, including structural characterization of a functional cellulose synthase complex, provided the first mechanistic insight into this fascinating process. In most studied bacteria, just two subunits, BcsA and BcsB, are necessary and sufficient for the formation of the polysaccharide chain in vitro. Other subunits – which differ among various taxa – affect the enzymatic activity and product yield in vivo by modulating expression of biosynthesis apparatus, export of the nascent β-D-glucan polymer to the cell surface, and the organization of cellulose fibers into a higher-order structure. These auxiliary subunits play key roles in determining the quantity and structure of the resulting biofilm, which is particularly important for interactions of bacteria with higher organisms that lead to rhizosphere colonization and modulate virulence of cellulose-producing bacterial pathogens inside and outside of host cells. Here we review the organization of four principal types of cellulose synthase operons found in various bacterial genomes, identify additional bcs genes that encode likely components of the cellulose biosynthesis and secretion machinery, and propose a unified nomenclature for these genes and subunits. We also discuss the role of cellulose as a key component of biofilms formed by a variety of free-living and pathogenic bacteria and, for the latter, in the choice between acute infection and persistence in the host. PMID:26077867

  15. Masked Visual Analysis: Minimizing Type I Error in Visually Guided Single-Case Design for Communication Disorders.

    Science.gov (United States)

    Byun, Tara McAllister; Hitchcock, Elaine R; Ferron, John

    2017-06-10

    Single-case experimental designs are widely used to study interventions for communication disorders. Traditionally, single-case experiments follow a response-guided approach, where design decisions during the study are based on participants' observed patterns of behavior. However, this approach has been criticized for its high rate of Type I error. In masked visual analysis (MVA), response-guided decisions are made by a researcher who is blinded to participants' identities and treatment assignments. MVA also makes it possible to conduct a hypothesis test assessing the significance of treatment effects. This tutorial describes the principles of MVA, including both how experiments can be set up and how results can be used for hypothesis testing. We then report a case study showing how MVA was deployed in a multiple-baseline across-subjects study investigating treatment for residual errors affecting rhotics. Strengths and weaknesses of MVA are discussed. Given their important role in the evidence base that informs clinical decision making, it is critical for single-case experimental studies to be conducted in a way that allows researchers to draw valid inferences. As a method that can increase the rigor of single-case studies while preserving the benefits of a response-guided approach, MVA warrants expanded attention from researchers in communication disorders.

  16. 28 CFR 51.6 - Political subunits.

    Science.gov (United States)

    2010-07-01

    ... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Political subunits. 51.6 Section 51.6 Judicial Administration DEPARTMENT OF JUSTICE (CONTINUED) PROCEDURES FOR THE ADMINISTRATION OF SECTION 5 OF THE VOTING RIGHTS ACT OF 1965, AS AMENDED General Provisions § 51.6 Political subunits. All political...

  17. Transcriptomes of major renal collecting duct cell types in mouse identified by single-cell RNA-seq.

    Science.gov (United States)

    Chen, Lihe; Lee, Jae Wook; Chou, Chung-Lin; Nair, Anil V; Battistone, Maria A; Păunescu, Teodor G; Merkulova, Maria; Breton, Sylvie; Verlander, Jill W; Wall, Susan M; Brown, Dennis; Burg, Maurice B; Knepper, Mark A

    2017-11-14

    Prior RNA sequencing (RNA-seq) studies have identified complete transcriptomes for most renal epithelial cell types. The exceptions are the cell types that make up the renal collecting duct, namely intercalated cells (ICs) and principal cells (PCs), which account for only a small fraction of the kidney mass, but play critical physiological roles in the regulation of blood pressure, extracellular fluid volume, and extracellular fluid composition. To enrich these cell types, we used FACS that employed well-established lectin cell surface markers for PCs and type B ICs, as well as a newly identified cell surface marker for type A ICs, c-Kit. Single-cell RNA-seq using the IC- and PC-enriched populations as input enabled identification of complete transcriptomes of A-ICs, B-ICs, and PCs. The data were used to create a freely accessible online gene-expression database for collecting duct cells. This database allowed identification of genes that are selectively expressed in each cell type, including cell-surface receptors, transcription factors, transporters, and secreted proteins. The analysis also identified a small fraction of hybrid cells expressing aquaporin-2 and anion exchanger 1 or pendrin transcripts. In many cases, mRNAs for receptors and their ligands were identified in different cells (e.g., Notch2 chiefly in PCs vs. Jag1 chiefly in ICs), suggesting signaling cross-talk among the three cell types. The identified patterns of gene expression among the three types of collecting duct cells provide a foundation for understanding physiological regulation and pathophysiology in the renal collecting duct.

  18. Diabetes complications in 1952 type 2 diabetes mellitus patients managed in a single institution

    International Nuclear Information System (INIS)

    Alwakeel, Jamal S.; Suliman, R.; Tarif, N.; Al-Suwaida, A.; Hammad, D.; Al-Asaad, H.; Al-Harbi, A.; Al-Mohaya, S.; Alam, A.

    2008-01-01

    Because there is no recent update on the state of diabetes and its concomitant applications in Saudi Arabia, we undertook a study of the prevalence of health complications in patients with type 2 diabetes mellitus admitted to our institution. We conducted a retrospective review of medical results of adult Saudi patients with type 2 diabetes who were seen in clinics or admitted to the Security Forces Hospital, Riyadh, Saudi Arabia, between January 1989 and January 2004. Of 1952, 943 (48.3%) were males. For the whole study population the mean age at enrollment was 58.4+-14.2 years, the mean age at the onset of diabetes was 48.1+-12.8 years, the mean duration of diabetes was 10.4+-7.5 years, and the mean duration of follow-up was 7.9+-4.6 years. Nephropathy was the most prevalent complication, occurring in 626 patients (32.1%). Acute coronary syndrome occurred in 451 (23.1%), cataracts in 447 (22.9$), retinopathy in 326 (16.7%), and myocardial infarction in 279 (14.3%), Doubling of serum ceartinine was seen in 250 (12.8%) and 79 (4.0%) went into dialysis. Hypertension was present in 1524 (78.1%) dyslipidemia in 764 (39.1%). Overall mortality was 8.2%. Multiple complications were frequent. Males had higher prevalence of complications than females (P<.05). Mortality was significantly higher in males 92 (9.8%) than females 69 (6.8%) (P=.024). The prevalence of complications significantly increased with duration of diabetes and age (P<.05). Among Saudis, the prevalence of concomitant diabetic complications is high, with cardiovascular and renal complications the most frequent. Many patients had multiple complications. Early and frequent screening in patients with type 2 diabetes is desirable to identify patients at high risk for concomitant complications and to prevent disabilities. (author)

  19. Fabrication and Characterization of a Single Hole Transistor in p-type GaAs/AlGaAs Heterostructures

    Energy Technology Data Exchange (ETDEWEB)

    Tracy, Lisa A [Sandia National Laboratories (SNL-NM), Albuquerque, NM (United States); Reno, John L. [Sandia National Laboratories (SNL-NM), Albuquerque, NM (United States); Hargett, Terry W. [Sandia National Laboratories (SNL-NM), Albuquerque, NM (United States)

    2015-09-01

    Most spin qubit research to date has focused on manipulating single electron spins in quantum dots. However, hole spins are predicted to have some advantages over electron spins, such as reduced coupling to host semiconductor nuclear spins and the ability to control hole spins electrically using the large spin-orbit interaction. Building on recent advances in fabricating high-mobility 2D hole systems in GaAs/AlGaAs heterostructures at Sandia, we fabricate and characterize single hole transistors in GaAs. We demonstrate p-type double quantum dot devices with few-hole occupation, which could be used to study the physics of individual hole spins and control over coupling between hole spins, looking towards eventual applications in quantum computing. Intentionally left blank

  20. Growth of large size lithium niobate single crystals of high quality by tilting-mirror-type floating zone method

    Energy Technology Data Exchange (ETDEWEB)

    Sarker, Abdur Razzaque, E-mail: razzaque_ru2000@yahoo.com [Department of Physics, University of Rajshahi (Bangladesh)

    2016-05-15

    Large size high quality LiNbO{sub 3} single crystals were grown successfully by tilting-mirror-type floating zone (TMFZ) technique. The grown crystals were characterized by X-ray diffraction, etch pits density measurement, Impedance analysis, Vibrating sample magnetometry (VSM) and UV-Visible spectrometry. The effect of mirror tilting during growth on the structural, electrical, optical properties and defect density of the LiNbO{sub 3} crystals were investigated. It was found that the defect density in the crystals reduced for tilting the mirror in the TMFZ method. The chemical analysis revealed that the grown crystals were of high quality with uniform composition. The single crystals grown by TMFZ method contains no low-angle grain boundaries, indicating that they can be used for high efficiency optoelectronic devices. (author)

  1. Single and multi-subject clustering of flow cytometry data for cell-type identification and anomaly detection.

    Science.gov (United States)

    Pouyan, Maziyar Baran; Jindal, Vasu; Birjandtalab, Javad; Nourani, Mehrdad

    2016-08-10

    Measurement of various markers of single cells using flow cytometry has several biological applications. These applications include improving our understanding of behavior of cellular systems, identifying rare cell populations and personalized medication. A common critical issue in the existing methods is identification of the number of cellular populations which heavily affects the accuracy of results. Furthermore, anomaly detection is crucial in flow cytometry experiments. In this work, we propose a two-stage clustering technique for cell type identification in single subject flow cytometry data and extend it for anomaly detection among multiple subjects. Our experimentation on 42 flow cytometry datasets indicates high performance and accurate clustering (F-measure > 91 %) in identifying main cellular populations. Furthermore, our anomaly detection technique evaluated on Acute Myeloid Leukemia dataset results in only <2 % false positives.

  2. Variation of power generation at different buffer types and conductivities in single chamber microbial fuel cells

    KAUST Repository

    Nam, Joo-Youn

    2010-01-15

    Microbial fuel cells (MFCs) are operated with solutions containing various chemical species required for the growth of electrochemically active microorganisms including nutrients and vitamins, substrates, and chemical buffers. Many different buffers are used in laboratory media, but the effects of these buffers and their inherent electrolyte conductivities have not been examined relative to current generation in MFCs. We investigated the effect of several common buffers (phosphate, MES, HEPES, and PIPES) on power production in single chambered MFCs compared to a non-buffered control. At the same concentrations the buffers produced different solution conductivities which resulted in different ohmic resistances and power densities. Increasing the solution conductivities to the same values using NaCl produced comparable power densities for all buffers. Very large increases in conductivity resulted in a rapid voltage drop at high current densities. Our results suggest that solution conductivity at a specific pH for each buffer is more important in MFC studies than the buffer itself given relatively constant pH conditions. Based on our analysis of internal resistance and a set neutral pH, phosphate and PIPES are the most useful buffers of those examined here because pH was maintained close to the pKa of the buffer, maximizing the ability of the buffer to contribute to increase current generation at high power densities. © 2009 Elsevier B.V. All rights reserved.

  3. Variation of power generation at different buffer types and conductivities in single chamber microbial fuel cells.

    Science.gov (United States)

    Nam, Joo-Youn; Kim, Hyun-Woo; Lim, Kyeong-Ho; Shin, Hang-Sik; Logan, Bruce E

    2010-01-15

    Microbial fuel cells (MFCs) are operated with solutions containing various chemical species required for the growth of electrochemically active microorganisms including nutrients and vitamins, substrates, and chemical buffers. Many different buffers are used in laboratory media, but the effects of these buffers and their inherent electrolyte conductivities have not been examined relative to current generation in MFCs. We investigated the effect of several common buffers (phosphate, MES, HEPES, and PIPES) on power production in single chambered MFCs compared to a non-buffered control. At the same concentrations the buffers produced different solution conductivities which resulted in different ohmic resistances and power densities. Increasing the solution conductivities to the same values using NaCl produced comparable power densities for all buffers. Very large increases in conductivity resulted in a rapid voltage drop at high current densities. Our results suggest that solution conductivity at a specific pH for each buffer is more important in MFC studies than the buffer itself given relatively constant pH conditions. Based on our analysis of internal resistance and a set neutral pH, phosphate and PIPES are the most useful buffers of those examined here because pH was maintained close to the pK(a) of the buffer, maximizing the ability of the buffer to contribute to increase current generation at high power densities. Copyright 2009 Elsevier B.V. All rights reserved.

  4. Large-scale separation of single-walled carbon nanotubes by electronic type using click chemistry

    Science.gov (United States)

    Um, Jo-Eun; Song, Sun Gu; Yoo, Pil J.; Song, Changsik; Kim, Woo-Jae

    2018-01-01

    Single-walled carbon nanotubes (SWCNTs) can be either metallic or semiconducting, making their separation critical for applications in nanoelectronics, biomedical materials, and solar cells. Herein, we investigate a novel solution-phase separation method based on click chemistry (azide-alkyne Huisgen cycloaddition) and determine its efficiency and scalability. In this method, metallic SWCNTs in metallic/semiconducting SWCNT mixtures are selectively functionalized with alkyne groups by being reacted with 4-propargyloxybenezenediazonium tetrafluoroborate. Subsequently, silica nanoparticles are functionalized with azide groups and reacted with alkyne-bearing metallic SWCNTs in the SWCNT mixture in the presence of a Cu catalyst. As a result, metallic SWCNTs are anchored on silica powder, whereas non-functionalized semiconducting SWCNTs remain in solution. Low-speed centrifugation effectively removes the silica powder with attached metallic SWCNTs, furnishing a solution of highly pure semiconducting SWCNTs, as confirmed by Raman and UV-vis/near-infrared absorption measurements. This novel separation scheme exhibits the advantage of simultaneously separating both metallic and semiconducting SWCNTs from their mixtures, being cost-effective and therefore applicable at an industrial scale.

  5. Unexpected Binding Mode of a Potent Indeno[1,2-b]indole-Type Inhibitor of Protein Kinase CK2 Revealed by Complex Structures with the Catalytic Subunit CK2α and Its Paralog CK2α′

    Directory of Open Access Journals (Sweden)

    Jennifer Hochscherf

    2017-12-01

    Full Text Available Protein kinase CK2, a member of the eukaryotic protein kinase superfamily, is associated with cancer and other human pathologies and thus an attractive drug target. The indeno[1,2-b]indole scaffold is a novel lead structure to develop ATP-competitive CK2 inhibitors. Some indeno[1,2-b]indole-based CK2 inhibitors additionally obstruct ABCG2, an ABC half transporter overexpressed in breast cancer and co-responsible for drug efflux and resistance. Comprehensive derivatization studies revealed substitutions of the indeno[1,2-b]indole framework that boost either the CK2 or the ABCG2 selectivity or even support the dual inhibition potential. The best indeno[1,2-b]indole-based CK2 inhibitor described yet (IC50 = 25 nM is 5-isopropyl-4-(3-methylbut-2-enyl-oxy-5,6,7,8-tetrahydroindeno[1,2-b]indole-9,10-dione (4p. Herein, we demonstrate the membrane permeability of 4p and describe co-crystal structures of 4p with CK2α and CK2α′, the paralogs of human CK2 catalytic subunit. As expected, 4p occupies the narrow, hydrophobic ATP site of CK2α/CK2α′, but surprisingly with a unique orientation: its hydrophobic substituents point towards the solvent while its two oxo groups are hydrogen-bonded to a hidden water molecule. An equivalent water molecule was found in many CK2α structures, but never as a critical mediator of ligand binding. This unexpected binding mode is independent of the interdomain hinge/helix αD region conformation and of the salt content in the crystallization medium.

  6. Protein kinase A regulatory subunit distribution in medulloblastoma

    International Nuclear Information System (INIS)

    Mucignat-Caretta, Carla; Denaro, Luca; Redaelli, Marco; D'Avella, Domenico; Caretta, Antonio

    2010-01-01

    Previous studies showed a differential distribution of the four regulatory subunits of cAMP-dependent protein kinases inside the brain, that changed in rodent gliomas: therefore, the distribution of these proteins inside the brain can give information on the functional state of the cells. Our goal was to examine human brain tumors to provide evidence for a differential distribution of protein kinase A in different tumors. The distribution of detergent insoluble regulatory (R1 and R2) and catalytic subunits of cAMP dependent kinases was examined in pediatric brain tumors by immunohistochemistry and fluorescent cAMP analogues binding. R2 is organized in large single dots in medulloblastomas, while it has a different appearance in other tumors. Fluorescent cAMP labelling was observed only in medulloblastoma. A different distribution of cAMP dependent protein kinases has been observed in medulloblastoma

  7. Analysis of Horse Myostatin Gene and Identification of Single Nucleotide Polymorphisms in Breeds of Different Morphological Types

    OpenAIRE

    Dall'Olio, Stefania; Fontanesi, Luca; Nanni Costa, Leonardo; Tassinari, Marco; Minieri, Laura; Falaschini, Adalberto

    2010-01-01

    Myostatin (MSTN) is a negative modulator of muscle mass. We characterized the horse (Equus caballus) MSTN gene and identified and analysed single nucleotide polymorphisms (SNPs) in breeds of different morphological types. Sequencing of coding, untranslated, intronic, and regulatory regions of MSTN gene in 12 horses from 10 breeds revealed seven SNPs: two in the promoter, four in intron 1, and one in intron 2. The SNPs of the promoter (GQ183900:g.26T > C and GQ183900:g.156T > C, the latter loc...

  8. Plutonium (TRU) transmutation and {sup 233}U production by single-fluid type accelerator molten-salt breeder (AMSB)

    Energy Technology Data Exchange (ETDEWEB)

    Furukaw, Kazuo [Tokai Univ., Kanagawa (Japan); Kato, Yoshio [Japan Atom. Ene. Res. Inst., Ibaraki (Japan); Chigrinov, Sergey E. [Academy of Science, Minsk (Belarus)

    1995-10-01

    For practical/industrial disposition of Pu(TRU) by accelerator facility, not only physical soundness and safety but also the following technological rationality should be required: (1) few R&D items including radiation damage, heat removal and material compatibility; (2) few operation/maintenance/processing works: (3) few reproduction of radioactivity; (4) effective energy production in parallel. This will be achieved by the new modification of Th-fertilizing Single-Fluid type Accelerator Molten-Salt Breeder (AMSB), by which a global nuclear energy strategy for next century might be prepared.

  9. Development of a single multi-locus sequence typing scheme for Taylorella equigenitalis and Taylorella asinigenitalis.

    Science.gov (United States)

    Duquesne, Fabien; Hébert, Laurent; Breuil, Marie-France; Matsuda, Motoo; Laugier, Claire; Petry, Sandrine

    2013-12-27

    We describe here the development of a multilocus sequence typing (MLST) scheme for Taylorella equigenitalis, the causative agent of contagious equine metritis (CEM), and Taylorella asinigenitalis, a nonpathogenic bacterium. MLST was performed on a set of 163 strains collected in several countries over 35 years (1977-2012). The MLST data were analyzed using START2, MEGA 5.05 and eBURST, and can be accessed at http://pubmlst.org/taylorella/. Our results revealed a clonal population with 39 sequence types (ST) and no common ST between the two Taylorella species. The eBURST analysis grouped the 27 T. equigenitalis STs into four clonal complexes (CC1-4) and five unlinked STs. The 12 T. asinigenitalis STs were grouped into three clonal complexes (CC5-7) and five unlinked STs, among which CC1 (68.1% of the 113 T. equigenitalis) and CC5 (58.0% of the 50 T. asinigenitalis) were dominants. The CC1, still in circulation in France, contains isolates from the first CEM outbreaks that simultaneously emerged in several countries in the late 1970s. The emergence in different countries (e.g. France, Japan, and United Arab Emirates) of STs without any genetic relationship to CC1 suggests the existence of a natural worldwide reservoir that remains to be identified. T. asinigenitalis appears to behave same way since the American, Swedish and French isolates have unrelated STs. This first Taylorella sp. MLST is a powerful tool for further epidemiological investigations and population biology studies of the Taylorella genus. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. Wegner estimate and localization for alloy-type models with sign-changing exponentially decaying single-site potentials

    Science.gov (United States)

    Leonhardt, Karsten; Peyerimhoff, Norbert; Tautenhahn, Martin; Veselić, Ivan

    2015-05-01

    We study Schrödinger operators on L2(ℝd) and ℓ2(ℤd) with a random potential of alloy-type. The single-site potential is assumed to be exponentially decaying but not necessarily of fixed sign. In the continuum setting, we require a generalized step-function shape. Wegner estimates are bounds on the average number of eigenvalues in an energy interval of finite box restrictions of these types of operators. In the described situation, a Wegner estimate, which is polynomial in the volume of the box and linear in the size of the energy interval, holds. We apply the established Wegner estimate as an ingredient for a localization proof via multiscale analysis.

  11. Proton Pumping and Slippage Dynamics of a Eukaryotic P-Type ATPase Studied at the Single-Molecule Level

    DEFF Research Database (Denmark)

    Veshaguri, Salome

    In all eukaryotes the plasma membrane potential and secondary transport systems are energized by P-type ATPases whose regulation however remains poorly understood. Here we monitored at the single-molecule level the activity of the prototypic proton pumping P-type ATPase Arabidopsis thaliana isoform...... pumping rates remained constant. Titration of ATP down to ~1% of apparent Km for ATPase activity exclusively affected the distributions of the durations the pump spends in active and inactive states. The dramatic consequence of our findings is that ATP reduction decreased ATP/H+ stoichiometry of the pump....... We propose that variable ATP/H+ stoichiometry emerges as a novel mechanism for adaptation when challenged with depletion of ATP that is likely relevant for other ATPases. Such measurements will provide indispensable insights into the mechanisms of function and regulation of many other ion...

  12. Single assay for simultaneous detection and differential identification of human and avian influenza virus types, subtypes, and emergent variants.

    Directory of Open Access Journals (Sweden)

    David Metzgar

    Full Text Available For more than four decades the cause of most type A influenza virus infections of humans has been attributed to only two viral subtypes, A/H1N1 or A/H3N2. In contrast, avian and other vertebrate species are a reservoir of type A influenza virus genome diversity, hosting strains representing at least 120 of 144 combinations of 16 viral hemagglutinin and 9 viral neuraminidase subtypes. Viral genome segment reassortments and mutations emerging within this reservoir may spawn new influenza virus strains as imminent epidemic or pandemic threats to human health and poultry production. Traditional methods to detect and differentiate influenza virus subtypes are either time-consuming and labor-intensive (culture-based or remarkably insensitive (antibody-based. Molecular diagnostic assays based upon reverse transcriptase-polymerase chain reaction (RT-PCR have short assay cycle time, and high analytical sensitivity and specificity. However, none of these diagnostic tests determine viral gene nucleotide sequences to distinguish strains and variants of a detected pathogen from one specimen to the next. Decision-quality, strain- and variant-specific pathogen gene sequence information may be critical for public health, infection control, surveillance, epidemiology, or medical/veterinary treatment planning. The Resequencing Pathogen Microarray (RPM-Flu is a robust, highly multiplexed and target gene sequencing-based alternative to both traditional culture- or biomarker-based diagnostic tests. RPM-Flu is a single, simultaneous differential diagnostic assay for all subtype combinations of type A influenza viruses and for 30 other viral and bacterial pathogens that may cause influenza-like illness. These other pathogen targets of RPM-Flu may co-infect and compound the morbidity and/or mortality of patients with influenza. The informative specificity of a single RPM-Flu test represents specimen-specific viral gene sequences as determinants of virus type, A

  13. Elevated serum triglycerides is the strongest single indicator for the presence of metabolic syndrome in patients with type 2 diabetes

    Directory of Open Access Journals (Sweden)

    Dimou Eleni

    2006-10-01

    Full Text Available Abstract Background Patients with diabetes already fulfill one diagnostic criterion for MS according to the existing classifications. Our aim was to identify one single clinical parameter, which could effectively predict the presence of MS in patients with type 2 diabetes. Methods We studied all patients with type 2 diabetes who attended our Diabetes Outpatient Clinic during a three-month period. Waist circumference, blood pressure and serum lipids were measured. Establishment of MS diagnosis was based a on National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP III criteria and b on International Diabetes Federation (IDF criteria. Receiver operating characteristic (ROC analysis was applied in order to identify the clinical parameter with the highest predictive capability for MS. Among the 500 participating patients (231 males, 269 females, MS was diagnosed in 364 patients (72.8% according to the NCEP ATP III criteria and in 408 patients (81.6% according to the IDF criteria. Results For the NCEP ATP III classification, serum triglycerides (in the overall population, waist and HDL (in female population demonstrated the highest predictive capability for MS (AUCs:0.786, 0.805 and 0.801, respectively. For the IDF classification, no single parameter reached an AUC > 0.800 in the overall population. In females, HDL displayed a satisfactory predictive capability for MS with an AUC which was significantly higher than the one in males (0.785 vs. 0.676, respectively, p Conclusion Elevated serum triglycerides strongly indicate the presence of MS in patients with type 2 diabetes. In female patients with type 2 diabetes, central obesity was the second stronger predictor of MS besides hypertriglyceridemia.

  14. A single dose of sodium nitrate does not improve oral glucose tolerance in patients with type 2 diabetes mellitus.

    Science.gov (United States)

    Cermak, Naomi M; Hansen, Dominique; Kouw, Imre W K; van Dijk, Jan-Willem; Blackwell, Jamie R; Jones, Andrew M; Gibala, Martin J; van Loon, Luc J C

    2015-08-01

    Dietary nitrate (NO3(-)) supplementation has been proposed as an emerging treatment strategy for type 2 diabetes. We hypothesized that ingestion of a single bolus of dietary NO3(-) ingestion improves oral glucose tolerance in patients with type 2 diabetes. Seventeen men with type 2 diabetes (glycated hemoglobin, 7.3% ± 0.2%) participated in a randomized crossover experiment. The subjects ingested a glucose beverage 2.5 hours after consumption of either sodium NO3(-) (0.15 mmol NaNO3(-) · kg(-1)) or a placebo solution. Venous blood samples were collected before ingestion of the glucose beverage and every 30 minutes thereafter during a 2-hour period to assess postprandial plasma glucose and insulin concentrations. The results show that plasma NO3(-) and nitrite levels were increased after NaNO3(-) as opposed to placebo ingestion (treatment-effect, P = .001). Despite the elevated plasma NO3(-) and nitrite levels, ingestion of NaNO3(-) did not attenuate the postprandial rise in plasma glucose and insulin concentrations (time × treatment interaction, P = .41 for glucose, P = .93 for insulin). Despite the lack of effect on oral glucose tolerance, basal plasma glucose concentrations measured 2.5 hours after NaNO3(-) ingestion were lower when compared with the placebo treatment (7.5 ± 0.4 vs 8.3 ± 0.4 mmol/L, respectively; P = .04). We conclude that ingestion of a single dose of dietary NO3(-) does not improve subsequent oral glucose tolerance in patients with type 2 diabetes. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. ESTIMATION OF THE SCALE PARAMETER FROM THE RAYLEIGH DISTRIBUTION FROM TYPE II SINGLY AND DOUBLY CENSORED DATA

    Directory of Open Access Journals (Sweden)

    Ahmad Saeed Akhter

    2009-01-01

    Full Text Available As common as the normal distribution is the Rayleigh distribution which occurs in works on radar, properties of sine wave plus-noise, etc. Rayleigh (1880 derived it from the amplitude of sound resulting from many important sources. The Rayleigh distribution is widely used in communication engineering, reliability analysis and applied statistics. Since the Rayleigh distribution has linearly increasing rate, it is appropriate for components which might not have manufacturing defects but age rapidly with time. Several types of electro-vacum devices have this feature. It is connected with one dimension and two dimensions random walk and is some times referred to as a random walk frequency distribution. It is a special case of Weibull distribution (1951 of wide applicability. It can be easily derived from the bivariate normal distribution with and p = 0. For further application of Rayleigh distribution, we refer to Johnson and Kotz (1994. Adatia (1995 has obtained the best linear unbiased estimator of the Rayleigh scale parameter based on fairly large censored samples. Dyer and Whisend (1973 obtained the BLUE of scale parameter based on type II censored samples for small N = 2(15. With the advance of computer technology it is now possible to obtain BLUE for large samples. Hirai (1978 obtained the estimate of the scale parameter from the Rayleigh distribution singly type II censored from the left side and right side and variances of the scale parameter. In this paper, we estimate the scale parameter of type II singly and doubly censored data from the Rayleigh distribution using Blom’s (1958 unbiased nearly best estimates and compare the efficiency of this estimate with BLUE and MLE.

  16. Induction of specific neuron types by overexpression of single transcription factors.

    Science.gov (United States)

    Teratani-Ota, Yusuke; Yamamizu, Kohei; Piao, Yulan; Sharova, Lioudmila; Amano, Misa; Yu, Hong; Schlessinger, David; Ko, Minoru S H; Sharov, Alexei A

    2016-10-01

    Specific neuronal types derived from embryonic stem cells (ESCs) can facilitate mechanistic studies and potentially aid in regenerative medicine. Existing induction methods, however, mostly rely on the effects of the combined action of multiple added growth factors, which generally tend to result in mixed populations of neurons. Here, we report that overexpression of specific transcription factors (TFs) in ESCs can rather guide the differentiation of ESCs towards specific neuron lineages. Analysis of data on gene expression changes 2 d after induction of each of 185 TFs implicated candidate TFs for further ESC differentiation studies. Induction of 23 TFs (out of 49 TFs tested) for 6 d facilitated neural differentiation of ESCs as inferred from increased proportion of cells with neural progenitor marker PSA-NCAM. We identified early activation of the Notch signaling pathway as a common feature of most potent inducers of neural differentiation. The majority of neuron-like cells generated by induction of Ascl1, Smad7, Nr2f1, Dlx2, Dlx4, Nr2f2, Barhl2, and Lhx1 were GABA-positive and expressed other markers of GABAergic neurons. In the same way, we identified Lmx1a and Nr4a2 as inducers for neurons bearing dopaminergic markers and Isl1, Fezf2, and St18 for cholinergic motor neurons. A time-course experiment with induction of Ascl1 showed early upregulation of most neural-specific messenger RNA (mRNA) and microRNAs (miRNAs). Sets of Ascl1-induced mRNAs and miRNAs were enriched in Ascl1 targets. In further studies, enrichment of cells obtained with the induction of Ascl1, Smad7, and Nr2f1 using microbeads resulted in essentially pure population of neuron-like cells with expression profiles similar to neural tissues and expressed markers of GABAergic neurons. In summary, this study indicates that induction of transcription factors is a promising approach to generate cultures that show the transcription profiles characteristic of specific neural cell types.

  17. Modal analysis and dispersion curves of an elliptical W-type single mode fiber

    Science.gov (United States)

    Prajapati, Y.; Maurya, Jitendra Bahadur; Singh, Vivek; Saini, J. P.

    2015-05-01

    In this paper, the propagation of electromagnetic waves in W-type elliptical dielectric optical fiber having various cladding layers is presented. The presented fiber has concentric core and cladding which have elliptical cross section and the refractive index of one of the inner cladding (the cladding between first clad and last clad) maximum. Using elliptic cylindrical coordinates, boundary conditions are derived and longitudinal field components for the even and odd modes are obtained. The characteristic equation for the fiber to be studied is determined by solving the Mathieu ( q > 0) and the modified Mathieu functions ( q < 0). In order to study the fundamental mode, the modal index m is put as m = 1. Finally, the cutoff frequencies for several lower order modes have been calculated and their dispersion characteristics are plotted. The effects of elliptical eccentricity e on the mode cutoff frequencies and mode transmissions are also addressed. The analysis shows that one can control the propagation property of optical fiber by increasing the number of inner claddings. These claddings provide additional degree of freedom to control the modes.

  18. Pregnancy outcomes and nutritional indices after 3 types of bariatric surgery performed at a single institution.

    Science.gov (United States)

    Mead, Nancy C; Sakkatos, Panagiotis; Sakellaropoulos, George C; Adonakis, George L; Alexandrides, Theodore K; Kalfarentzos, Fotis

    2014-01-01

    Nutritional status during pregnancy and the effects of nutritional deficiencies on pregnancy outcomes after bariatric surgery is an important issue that warrants further study. The objective of this study was to investigate pregnancy outcomes and nutritional indices after restrictive and malabsorptive procedures. We investigated pregnancy outcomes of 113 women who gave birth to 150 children after biliopancreatic diversion (BPD), Roux-en-Y gastric bypass (RYGB), and sleeve gastrectomy (SG) between June 1994 and December 2011. Biochemical indices and pregnancy outcomes were compared among the different types of surgery and to overall 20-year hospital data, as well as to 56 presurgery pregnancies in 36 women of the same group. Anemia was observed in 24.2% and 15.6% of pregnancies after BPD and RYGB, respectively. Vitamin B12 levels decreased postoperatively in all groups, with no further decrease during pregnancy; however, low levels were observed not only after BPD (11.7%) and RYGB (15.6%), but also after SG (13.3%). Folic acid levels increased. Serum albumin levels decreased in all groups during pregnancy, but hypoproteinemia was seen only after BPD. Neonates after BPD had significantly lower average birth weight without a higher frequency of low birth weight defined asnutritional supplement guidelines are followed. Closer monitoring is required in pregnancies after malabsorptive procedures especially regarding protein nutrition. Copyright © 2014 American Society for Bariatric Surgery. Published by Elsevier Inc. All rights reserved.

  19. Cellulose synthesis in two secondary cell wall processes in a single cell type.

    Science.gov (United States)

    Mendu, Venugopal; Stork, Jozsef; Harris, Darby; DeBolt, Seth

    2011-11-01

    Plant cells have a rigid cell wall that constrains internal turgor pressure yet extends in a regulated and organized manner to allow the cell to acquire shape. The primary load-bearing macromolecule of a plant cell wall is cellulose, which forms crystalline microfibrils that are organized with respect to a cell's function and shape requirements. A primary cell wall is deposited during expansion whereas secondary cell wall is synthesized post expansion during differentiation. A complex form of asymmetrical cellular differentiation occurs in Arabidopsis seed coat epidermal cells, where we have recently shown that two secondary cell wall processes occur that utilize different cellulose synthase (CESA) proteins. One process is to produce pectinaceous mucilage that expands upon hydration and the other is a radial wall thickening that reinforced the epidermal cell structure. Our data illustrate polarized specialization of CESA5 in facilitating mucilage attachment to the parent seed and CESA2, CESA5 and CESA9 in radial cell wall thickening and formation of the columella. Herein, we present a model for the complexity of cellulose biosynthesis in this highly differentiated cell type with further evidence supporting each cellulosic secondary cell wall process.

  20. Fast and Slow Inhibition in the Visual Thalamus Is Influenced by Allocating GABAA Receptors with Different γ Subunits

    Directory of Open Access Journals (Sweden)

    Zhiwen Ye

    2017-04-01

    Full Text Available Cell-type specific differences in the kinetics of inhibitory postsynaptic conductance changes (IPSCs are believed to impact upon network dynamics throughout the brain. Much attention has focused on how GABAA receptor (GABAAR α and β subunit diversity will influence IPSC kinetics, but less is known about the influence of the γ subunit. We have examined whether GABAAR γ subunit heterogeneity influences IPSC properties in the thalamus. The γ2 subunit gene was deleted from GABAARs selectively in the dorsal lateral geniculate nucleus (dLGN. The removal of the γ2 subunit from the dLGN reduced the overall spontaneous IPSC (sIPSC frequency across all relay cells and produced an absence of IPSCs in a subset of relay neurons. The remaining slower IPSCs were both insensitive to diazepam and zinc indicating the absence of the γ2 subunit. Because these slower IPSCs were potentiated by methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM, we propose these IPSCs involve γ1 subunit-containing GABAAR activation. Therefore, γ subunit heterogeneity appears to influence the kinetics of GABAAR-mediated synaptic transmission in the visual thalamus in a cell-selective manner. We suggest that activation of γ1 subunit-containing GABAARs give rise to slower IPSCs in general, while faster IPSCs tend to be mediated by γ2 subunit-containing GABAARs.

  1. Clinical and molecular characterization of 112 single-center patients with Neurofibromatosis type 1.

    Science.gov (United States)

    Corsello, Giovanni; Antona, Vincenzo; Serra, Gregorio; Zara, Federico; Giambrone, Clara; Lagalla, Luca; Piccione, Maria; Piro, Ettore

    2018-04-04

    The aim of this retrospective study was to define clinical and molecular characteristics of a large sample of neurofibromatosis type 1 (NF1) patients, as well as to evaluate mutational spectrum and genotype-phenotype correlation. NF1 is a relatively common neurogenetic disorder (1:2500-1:3000 individuals). It is caused by mutations of the NF1 gene on chromosome 17ql1.2, with autosomal dominant pattern of inheritance and wide phenotypical variability. Café-au-lait spots (CALs), cutaneous and/or subcutaneous neurofibromas (CNFs/SCNFs), skinfold freckling, skeletal abnormalities, Lisch nodules of the iris and increased risk of learning and intellectual disabilities, as well as tumors of the nervous system and other organs are its main clinical features. The preliminary group collected 168 subjects with clinical suspicion of NF1. They were evaluated following the National Institutes of Health (NIH) criteria for NF1, revised by Gutmann et al. 1997, integrated for 67 of them by molecular testing. According to these references, 112 of 168 patients were diagnosed as NF1. The sample was characterized by an equal sex ratio (57 males, 55 females) and age distribution ranging from 10 days to 60 years of age (mean age, 13 years). A wide spectrum of clinical features has been observed in our patients. Mutational analysis resulted positive in 51 cases (76%). Twenty-four mutations detected in our cohort have not been reported to date. This study may contribute to a better definition of genotypic and phenotypic features of NF1 patients, with respect to further insights into the clinical characterization of the disease. In addition, an amplification of the spectrum of mutations in the NF1 gene has been documented.

  2. Glycogen storage disease type III in Egyptian children: a single centre clinico-laboratory study.

    Science.gov (United States)

    El-Karaksy, Hanaa; Anwar, Ghada; El-Raziky, Mona; Mogahed, Engy; Fateen, Ekram; Gouda, Amr; El-Mougy, Fatma; El-Hennawy, Ahmed

    2014-06-01

    Glycogen storage disease type III (GSD III) is an autosomal recessive disorder caused by deficiency of glycogen debrancher enzyme and is characterised by clinical variability. We herein describe the clinical and laboratory findings in 31 Egyptian patients with GSD III presenting to the Paediatric Hepatology Unit, Cairo University, Egypt. Eighteen patients (58%) were males. Their ages ranged between 6 months to 12 years. The main presenting complaint was progressive abdominal distention in 55%. Twelve patients (38.7%) had a history of recurrent attacks of convulsions; four had an erroneous diagnosis of hypocalcaemia and epilepsy. Doll-like facies was noted in 90%. Abdominal examination of all cases revealed abdominal distention and soft hepatomegaly which had bright echogenicity by ultrasound. Hypertriglyceridaemia was present in 93.6%, hyperlactacidaemia in 51.6% and hyperuricaemia in 19.4%. Liver biopsy showed markedly distended hepatocytes with well distinct cytoplasmic boundaries and 32% had macrovesicular fatty changes. Serum creatine kinase was elevated in 64.6% of patients and correlated positively and significantly with age (r=0.7 and P=<0.001), while serum triglycerides correlated negatively with age (r=-0.4 and P=0.05). Blood glucose assessment and search for hepatomegaly in an infant with recurrent seizures may prevent delay in the diagnosis. A huge soft liver reaching the left midclavicular line that appears echogenic on ultrasonography is characteristic of GSD III. A distended hepatocyte with rarified cytoplasm is pathognomonic but not diagnostic. Hypertriglyceridaemia correlates negatively with age, in contrary to CK level. Copyright © 2014 Arab Journal of Gastroenterology. Published by Elsevier Ltd. All rights reserved.

  3. Sensing interrogation technique for fiber-optic interferometer type of sensors based on a single-passband RF filter.

    Science.gov (United States)

    Chen, Hao; Zhang, Shiwei; Fu, Hongyan; Zhou, Bin; Chen, Nan

    2016-02-08

    In this paper, a sensing interrogation system for fiber-optic interferometer type of sensors by using a single-passband radio-frequency (RF) filter has been proposed and experimentally demonstrated. The fiber-optic interferometer based sensors can give continuous optical sampling, and along with dispersive medium a single-passband RF frequency response can be achieved. The sensing parameter variation on the fiber-optic interferometer type of sensors will affect their free spectrum range, and thus the peak frequency of the RF filter. By tracking the central frequency of the passband the sensing parameter can be demodulated. As a demonstration, in our experiment a fiber Mach-Zehnder interferometer (FMZI) based temperature sensor has been interrogated. By tracking the peak frequency of the passband the temperature variation can be monitored. In our experiment, the sensing responsivity of 10.5 MHz/°C, 20.0 MHz/°C and 41.2 MHz/°C, when the lengths of sensing fiber are 1 m, 2 m and 4 m have been achieved.

  4. Analytical and semi-analytical inverse kinematics of SSRMS-type manipulators with single joint locked failure

    Science.gov (United States)

    Xu, Wenfu; She, Yu; Xu, Yangsheng

    2014-12-01

    Redundant space manipulators, including Space Station Remote Manipulator System (SSRMS), Special Purpose Dexterous Manipulator (SPDM) and European Robotic Arm (ERA), have been playing important roles in the construction and maintenance of International Space Station (ISS). They all have 7 revolute joints arranged in similar configurations, and are referred to as SSRMS-type manipulators. When a joint is locked in an arbitrary position due to some failures, a 7R manipulator degrades to a 6R manipulator. Without a spherical wrist or three consecutive parallel joints, the inverse kinematics of the 6R manipulator is very complex. In this paper, we propose effective methods to resolve the inverse kinematics for different cases of any joint locked in an arbitrary position. Firstly, configuration characteristics of the SSRMS-type redundant manipulators are analyzed. Then, an existing of closed-form inverse kinematics is discussed for locking different joints. Secondly, D-H frames and corresponding D-H parameters of the new 6-DOF manipulator formed by locking a joint in an arbitrary position are re-constructed. A unified table is then created to describe the kinematics for all possible cases of single joint locking failure. Thirdly, completely analytical and semi-analytical methods are presented to solve the inverse kinematics equations, and the former is used for locking joint 1, 2, 6 or 7 while the latter for locking joint 3, 4 or 5. Finally, typical cases for single joint locking are studied. The results verify the proposed methods.

  5. Simultaneous isolation of emm89-type Streptococcus pyogenes strains with a wild-type or mutated covS gene from a single streptococcal toxic shock syndrome patient.

    Science.gov (United States)

    Masuno, Katsuaki; Okada, Ryo; Zhang, Yan; Isaka, Masanori; Tatsuno, Ichiro; Shibata, Shinichiro; Hasegawa, Tadao

    2014-04-01

    Streptococcal toxic shock syndrome (STSS) is a re-emerging infectious disease in many developed countries. Recent studies have suggested that mutations in CovRS, a two-component regulatory system in Streptococcus pyogenes, play important roles in the pathogenesis of STSS. However, in vivo evidence of the significance of CovRS in human infections has not been fully demonstrated. We investigated five S. pyogenes strains isolated simultaneously from the pharynx, sputum, knee joint, cerebrospinal fluid and blood of a single STSS patient. All were emm89-type strains, and multilocus sequence typing (MLST) analysis revealed that the strains of pharynx and blood were isogenic. The growth rates of the strains from pharynx and sputum were faster than those of the other strains. Protein profiles of the culture supernatants of strains from the pharynx and sputum were also different from those of the other strains. Sequence analyses revealed that strains from the knee joint, cerebrospinal fluid and blood contained a single nucleotide difference in the covS coding region, resulting in one amino acid change, compared with the other strains. Introduction of a plasmid containing the covS gene from the pharynx strain to the blood strain increased the production of SpeB protein. This suggests that the one amino acid alteration in CovS was relevant to pathogenesis. This report supports the idea that mutated CovS plays important roles in vivo in the dissemination of S. pyogenes from the upper respiratory tract of human to aseptic tissues such as blood and cerebrospinal fluid.

  6. ENDOPLASMIC RETICULUM-MEDIATED QUALITY-CONTROL OF TYPE-I COLLAGEN PRODUCTION BY CELLS FROM OSTEOGENESIS IMPERFECTA PATIENTS WITH MUTATIONS IN THE PRO-ALPHA-1(I) CHAIN CARBOXYL-TERMINAL PROPEPTIDE WHICH IMPAIR SUBUNIT ASSEMBLY

    NARCIS (Netherlands)

    LAMANDE, [No Value; CHESSLER, SD; GOLUB, SB; BYERS, PH; CHAN, D; COLE, WG; SILLENCE, DO; BATEMAN, JF

    1995-01-01

    A heterozygous single base change in exon 49 of COL1A1, which converted the codon for pro alpha 1(I) carboxyl-terminal propeptide residue 94 from tryptophan (TGG) to cysteine (TGT) was identified in a baby with lethal osteogenesis imperfecta (OI64). The C-propeptide mutations in OI64 and in another

  7. Clinicopathological study of nondiabetic renal disease in type 2 diabetic patients: A single center experience from India

    Directory of Open Access Journals (Sweden)

    Kamal V Kanodia

    2017-01-01

    Full Text Available Diabetic nephropathy (DN is a major complication of diabetes mellitus (DM, leading to chronic kidney disease/end-stage renal disease. Wide spectrum of nondiabetic renal diseases (NDRD is reported in type-2 diabetes (type-2 DM. We carried out this single-center study to find clinical, laboratory, and histological features of NDRD in type-2 DM patients and to assess the prevalence of NDRD in India. A single-center retrospective study which included analysis of renal biopsies from patients with type-2 DM, performed between January 2008 and September 2016. Biopsy findings were categorized into three groups, Group-I (isolated NDRD; Group-II (NDRD superimposed on underlying DN; and Group-III (isolated DN. Out of 152 diabetic patients (111 males and 41 females, 35 (23.03% patients were of Group-I (isolated NDRD, 35 (23.03% of Group-II (NDRD superimposed on underlying DN, and 82 (53.95% of Group-III (isolated DN. The mean age (in years was 55.08 ± 10.71, 55.65 ± 8.71, and 54.45 ± 9.01 respectively in Group-I, II, and III. Nephrotic syndrome (NS was the most common clinical presentation in all groups. Duration of DM was significantly shorter in Group-I than in Group-II. Diabetic retinopathy was absent in Group-I. Proteinuria was more in Group-III than Group-I. Low serum C3 and/or C4 levels was observed in five (14.29% cases of Group-I and Group-II each and two (2.43% cases of Group-III. Nearly, 70 (46.05% patients were found to have NDRD either in isolated form or as combined lesions. The most common histological types of NDRD were acute tubulointerstitial nephritis (38.57% followed by benign nephrosclerosis (15.72%, membranous nephropathy (10%, IgA nephropathy (7.14%, and membranoproliferative glomerulonephritis (7.14%. The incidence of NDRD (with/without DN in type-2 DM is very high. Shorter duration of diabetes, hematuria, absence of retinopathy, low serum complement levels, and nephrotic range proteinuria are predictors of NDRD.

  8. Distinct Subunit Domains Govern Synaptic Stability and Specificity of the Kainate Receptor

    Directory of Open Access Journals (Sweden)

    Christoph Straub

    2016-07-01

    Full Text Available Synaptic communication between neurons requires the precise localization of neurotransmitter receptors to the correct synapse type. Kainate-type glutamate receptors restrict synaptic localization that is determined by the afferent presynaptic connection. The mechanisms that govern this input-specific synaptic localization remain unclear. Here, we examine how subunit composition and specific subunit domains contribute to synaptic localization of kainate receptors. The cytoplasmic domain of the GluK2 low-affinity subunit stabilizes kainate receptors at synapses. In contrast, the extracellular domain of the GluK4/5 high-affinity subunit synergistically controls the synaptic specificity of kainate receptors through interaction with C1q-like proteins. Thus, the input-specific synaptic localization of the native kainate receptor complex involves two mechanisms that underlie specificity and stabilization of the receptor at synapses.

  9. The Type and Impact of Evidence Review Group Exploratory Analyses in the NICE Single Technology Appraisal Process.

    Science.gov (United States)

    Carroll, Christopher; Kaltenthaler, Eva; Hill-McManus, Daniel; Scope, Alison; Holmes, Michael; Rice, Stephen; Rose, Micah; Tappenden, Paul; Woolacott, Nerys

    2017-06-01

    As part of the UK National Institute for Health and Care Excellence (NICE) single technology appraisal process, independent evidence review groups (ERGs) critically appraise a company's submission relating to a specific technology and indication. To explore the type of additional exploratory analyses conducted by ERGs and their impact on the recommendations made by NICE. The 100 most recently completed single technology appraisals with published guidance were selected for inclusion. A content analysis of relevant documents was undertaken to identify and extract relevant data, and narrative synthesis was used to rationalize and present these data. The types of exploratory analysis conducted in relation to companies' models were fixing errors, addressing violations, addressing matters of judgment, and the provision of a new, ERG-preferred base case. Ninety-three of the 100 ERG reports contained at least one of these analyses. The most frequently reported type of analysis in these 93 ERG reports related to the category "Matters of judgment," which was reported in 83 reports (89%). At least one of the exploratory analyses conducted and reported by an ERG is mentioned in 97% of NICE appraisal consultation documents and 94% of NICE final appraisal determinations, and had a clear influence on recommendations in 72% of appraisal consultation documents and 47% of final appraisal determinations. These results suggest that the additional analyses undertaken by ERGs in the appraisal of company submissions are highly influential in the policy-making and decision-making process. Copyright © 2017 International Society for Pharmacoeconomics and Outcomes Research (ISPOR). Published by Elsevier Inc. All rights reserved.

  10. Kinetic pathway of 40S ribosomal subunit recruitment to hepatitis C virus internal ribosome entry site.

    Science.gov (United States)

    Fuchs, Gabriele; Petrov, Alexey N; Marceau, Caleb D; Popov, Lauren M; Chen, Jin; O'Leary, Seán E; Wang, Richard; Carette, Jan E; Sarnow, Peter; Puglisi, Joseph D

    2015-01-13

    Translation initiation can occur by multiple pathways. To delineate these pathways by single-molecule methods, fluorescently labeled ribosomal subunits are required. Here, we labeled human 40S ribosomal subunits with a fluorescent SNAP-tag at ribosomal protein eS25 (RPS25). The resulting ribosomal subunits could be specifically labeled in living cells and in vitro. Using single-molecule Förster resonance energy transfer (FRET) between RPS25 and domain II of the hepatitis C virus (HCV) internal ribosome entry site (IRES), we measured the rates of 40S subunit arrival to the HCV IRES. Our data support a single-step model of HCV IRES recruitment to 40S subunits, irreversible on the initiation time scale. We furthermore demonstrated that after binding, the 40S:HCV IRES complex is conformationally dynamic, undergoing slow large-scale rearrangements. Addition of translation extracts suppresses these fluctuations, funneling the complex into a single conformation on the 80S assembly pathway. These findings show that 40S:HCV IRES complex formation is accompanied by dynamic conformational rearrangements that may be modulated by initiation factors.

  11. Exploring single nucleotide polymorphisms previously related to obesity and metabolic traits in pediatric-onset type 2 diabetes.

    Science.gov (United States)

    Miranda-Lora, América Liliana; Cruz, Miguel; Aguirre-Hernández, Jesús; Molina-Díaz, Mario; Gutiérrez, Jorge; Flores-Huerta, Samuel; Klünder-Klünder, Miguel

    2017-07-01

    To evaluate the association of 64 obesity-related polymorphisms with pediatric-onset type 2 diabetes and other glucose- and insulin-related traits in Mexican children. Case-control and case-sibling designs were followed. We studied 99 patients with pediatric-onset type 2 diabetes, their siblings (n = 101) without diabetes, 83 unrelated pediatric controls and 137 adult controls. Genotypes were determined for 64 single nucleotide polymorphisms, and a possible association was examined between those genotypes and type 2 diabetes and other quantitative traits, after adjusting for age, sex and body mass index. In the case-pediatric control and case-adult control analyses, five polymorphisms were associated with increased likelihood of pediatric-onset type 2 diabetes; only one of these polymorphisms (CADM2/rs1307880) also showed a consistent effect in the case-sibling analysis. The associations in the combined analysis were as follows: ADORA1/rs903361 (OR 1.9, 95% CI 1.2; 3.0); CADM2/rs13078807 (OR 2.2, 95% CI 1.2; 4.0); GNPDA2/rs10938397 (OR 2.2, 95% CI 1.4; 3.7); VEGFA/rs6905288 (OR 1.4, 95% CI 1.1; 2.1) and FTO/rs9939609 (OR 1.8, 95% CI 1.0; 3.2). We also identified 16 polymorphisms nominally associated with quantitative traits in participants without diabetes. ADORA/rs903361, CADM2/rs13078807, GNPDA2/rs10938397, VEGFA/rs6905288 and FTO/rs9939609 are associated with an increased risk of pediatric-onset type 2 diabetes in the Mexican population.

  12. Single nucleotide polymorphism typing of Mycobacterium ulcerans reveals focal transmission of buruli ulcer in a highly endemic region of Ghana.

    Directory of Open Access Journals (Sweden)

    Katharina Röltgen

    Full Text Available Buruli ulcer (BU is an emerging necrotizing disease of the skin and subcutaneous tissue caused by Mycobacterium ulcerans. While proximity to stagnant or slow flowing water bodies is a risk factor for acquiring BU, the epidemiology and mode of M. ulcerans transmission is poorly understood. Here we have used high-throughput DNA sequencing and comparisons of the genomes of seven M. ulcerans isolates that appeared monomorphic by existing typing methods. We identified a limited number of single nucleotide polymorphisms (SNPs and developed a real-time PCR SNP typing method based on these differences. We then investigated clinical isolates of M. ulcerans on which we had detailed information concerning patient location and time of diagnosis. Within the Densu river basin of Ghana we observed dominance of one clonal complex and local clustering of some of the variants belonging to this complex. These results reveal focal transmission and demonstrate, that micro-epidemiological analyses by SNP typing has great potential to help us understand how M. ulcerans is transmitted.

  13. Analysis of horse myostatin gene and identification of single nucleotide polymorphisms in breeds of different morphological types.

    Science.gov (United States)

    Dall'Olio, Stefania; Fontanesi, Luca; Nanni Costa, Leonardo; Tassinari, Marco; Minieri, Laura; Falaschini, Adalberto

    2010-01-01

    Myostatin (MSTN) is a negative modulator of muscle mass. We characterized the horse (Equus caballus) MSTN gene and identified and analysed single nucleotide polymorphisms (SNPs) in breeds of different morphological types. Sequencing of coding, untranslated, intronic, and regulatory regions of MSTN gene in 12 horses from 10 breeds revealed seven SNPs: two in the promoter, four in intron 1, and one in intron 2. The SNPs of the promoter (GQ183900:g.26T>C and GQ183900:g.156T>C, the latter located within a conserved TATA-box like motif) were screened in 396 horses from 16 breeds. The g.26C and the g.156C alleles presented higher frequency in heavy (brachymorphic type) than in light breeds (dolichomorphic type such as Italian Trotter breed). The significant difference of allele frequencies for the SNPs at the promoter and analysis of molecular variance (AMOVA) on haplotypes indicates that these polymorphisms could be associated with variability of morphology traits in horse breeds.

  14. A novel high-resolution single locus sequence typing scheme for mixed populations of Propionibacterium acnes in vivo.

    Directory of Open Access Journals (Sweden)

    Christian F P Scholz

    Full Text Available The Gram-positive anaerobic bacterium Propionibacterium acnes is a prevalent member of the normal skin microbiota of human adults. In addition to its suspected role in acne vulgaris it is involved in a variety of opportunistic infections. Multi-locus sequence-typing (MLST schemes identified distinct phylotypes associated with health and disease. Being based on 8 to 9 house-keeping genes these MLST schemes have a high discriminatory power, but their application is time- and cost-intensive. Here we describe a single-locus sequence typing (SLST scheme for P. acnes. The target locus was identified with a genome mining approach that took advantage of the availability of representative genome sequences of all known phylotypes of P. acnes. We applied this SLST on a collection of 188 P. acnes strains and demonstrated a resolution comparable to that of existing MLST schemes. Phylogenetic analysis applied to the SLST locus resulted in clustering patterns identical to a reference tree based on core genome sequences. We further demonstrate that SLST can be applied to detect multiple phylotypes in complex microbial communities by a metagenomic pyrosequencing approach. The described SLST strategy may be applied to any bacterial species with a basically clonal population structure to achieve easy typing and mapping of multiple phylotypes in complex microbiotas. The P. acnes SLST database can be found at http://medbac.dk/slst/pacnes.

  15. Analysis of Horse Myostatin Gene and Identification of Single Nucleotide Polymorphisms in Breeds of Different Morphological Types

    Directory of Open Access Journals (Sweden)

    Stefania Dall'Olio

    2010-01-01

    Full Text Available Myostatin (MSTN is a negative modulator of muscle mass. We characterized the horse (Equus caballus MSTN gene and identified and analysed single nucleotide polymorphisms (SNPs in breeds of different morphological types. Sequencing of coding, untranslated, intronic, and regulatory regions of MSTN gene in 12 horses from 10 breeds revealed seven SNPs: two in the promoter, four in intron 1, and one in intron 2. The SNPs of the promoter (GQ183900:g.26T>C and GQ183900:g.156T>C, the latter located within a conserved TATA-box like motif were screened in 396 horses from 16 breeds. The g.26C and the g.156C alleles presented higher frequency in heavy (brachymorphic type than in light breeds (dolichomorphic type such as Italian Trotter breed. The significant difference of allele frequencies for the SNPs at the promoter and analysis of molecular variance (AMOVA on haplotypes indicates that these polymorphisms could be associated with variability of morphology traits in horse breeds.

  16. Properties and local environment of p-type and photoluminescent rare earths implanted into ZnO single crystals

    CERN Document Server

    Rita, EMC; Wahl, U; Soares, JC

    This thesis presents an experimental study of the local environment of p-type and Rare- Earth dopants implanted in ZnO single-crystals (SCs). Various nuclear and bulk property techniques were combined in the following evaluations: Implantation damage annealing was evaluated in ZnO SCs implanted with Fe, Sr and Ca. P-type dopants Cu and Ag implanted ZnO SCs were studied revealing that the solubility of Cu in substituting Zn is considerably higher than that of Ag. These results are discussed within the scope of the ZnO p-type doping problematic with these elements. Experimental proofs of the As “anti-site” behavior in ZnO were for the first time attained, i.e., the majority of As atoms are substitutional at the Zn site (SZn), possibly surrounded by two Zn vacancies (VZn). This reinforces the theoretical prediction that As acts as an acceptor in ZnO via the AsZn-2VZn complex formation. The co-doping of ZnO SC with In (donor) and As (acceptor) was addressed. The most striking result is the possible In-As “p...

  17. Effect of separator and inoculum type on electricity generation and microbial community in single-chamber microbial fuel cells.

    Science.gov (United States)

    Yu, Jaecheul; Park, Younghyun; Lee, Taeho

    2014-04-01

    Single-chamber microbial fuel cell (SMFC)-I consisted of 4 separator-electrode assemblies (SEAs) with two types of cation exchange membrane (CEM: Nafion and CMI 7000) and an anion exchange membrane (AEM: AMI 7001). SMFC-II consisted of 4 SEAs with Nafion and three types of nonwoven fabric. SMFC-I and -II were inoculated with anaerobic digested and activated sludge, respectively, and operated under fed-batch mode. In SMFC I, AEM-SEA showed a maximum power density (PDmax). Nafion-SEA showed a PDmax in SMFC II, which was similar to that of Nafion-SEA of SMFC I. Although different bacteria were developed in SMFC-I (Deltaproteobacteria and Firmicutes) and SMFC-II (Gammaproteobacteria, Betaproteobacteria and Bacteroidetes), the inoculum type little affects electricity generation. Variations of pH and oxygen in biofilm have influenced microbial community structure and electricity generation according to the electrode and separator material. Although the electricity generation of non-woven fabric-SEA was less than that of Nafion-SEA, the use of non-woven fabrics is expected to reduce the construction and operating costs of MFCs.

  18. Development and evaluation of one step single tube multiplex RT-PCR for rapid detection and typing of dengue viruses

    Directory of Open Access Journals (Sweden)

    Parida Manmohan

    2008-01-01

    Full Text Available Abstract Background Dengue is emerging as a major public health concern in many parts of the world. The development of a one-step, single tube, rapid, and multiplex reverse transcription polymerase chain reaction (M-RT-PCR for simultaneous detection and typing of dengue virus using serotype specific primers during acute phase of illness is reported. Results An optimal assay condition with zero background was established having no cross-reaction with closely related members of flavivirus (Japanese encephalitis, West Nile, Yellow fever and alphavirus (Chikungunya. The feasibility of M-RT-PCR assay for clinical diagnosis was validated with 620 acute phase dengue patient sera samples of recent epidemics in India. The comparative evaluation vis a vis conventional virus isolation revealed higher sensitivity. None of the forty healthy serum samples screened in the present study revealed any amplification, thereby establishing specificity of the reported assay for dengue virus only. Conclusion These findings clearly suggested that M-RT-PCR assay reported in the present study is the rapid and cost-effective method for simultaneous detection as well as typing of the dengue virus in acute phase patient serum samples. Thus, the M-RT-PCR assay developed in this study will serve as a very useful tool for rapid diagnosis and typing of dengue infections in endemic areas.

  19. Genetic Variations in the Kir6.2 Subunit (KCNJ11 of Pancreatic ATP-Sensitive Potassium Channel Gene Are Associated with Insulin Response to Glucose Loading and Early Onset of Type 2 Diabetes in Childhood and Adolescence in Taiwan

    Directory of Open Access Journals (Sweden)

    Yi-Der Jiang

    2014-01-01

    Full Text Available To investigate the role of E23K polymorphism of the KCNJ11 gene on early onset of type 2 diabetes in school-aged children/adolescents in Taiwan, we recruited 38 subjects with type 2 diabetes (ages 18.6 ± 6.6 years; body mass index percentiles 83.3 ± 15.4 and 69 normal controls (ages 17.3 ± 3.8 years; body mass index percentiles 56.7 ± 29.0 from a national surveillance for childhood/adolescent diabetes in Taiwan. We searched for the E23K polymorphism of the KCNJ11 gene. We found that type 2 diabetic subjects had higher carrier rate of E23K polymorphism of KCNJ11 gene than control subjects (P = 0.044. After adjusting for age, gender, body mass index percentiles, and fasting plasma insulin, the E23K polymorphism contributed to an increased risk for type 2 diabetes (P = 0.047. K23-allele-containing genotypes conferring increased plasma insulin level during OGTT in normal subjects. However, the diabetic subjects with the K23-allele-containing genotypes had lower fasting plasma insulin levels after adjustment of age and BMI percentiles. In conclusion, the E23K variant of the KCNJ11 gene conferred higher susceptibility to type 2 diabetes in children/adolescents. Furthermore, in normal glucose-tolerant children/adolescents, K23 allele carriers had a higher insulin response to oral glucose loading.

  20. Acetylcholine Receptor: Complex of Homologous Subunits

    Science.gov (United States)

    Raftery, Michael A.; Hunkapiller, Michael W.; Strader, Catherine D.; Hood, Leroy E.

    1980-06-01

    The acetylcholine receptor from the electric ray Torpedo californica is composed of five subunits; two are identical and the other three are structurally related to them. Microsequence analysis of the four polypeptides demonstrates amino acid homology among the subunits. Further sequence analysis of both membrane-bound and Triton-solubilized, chromatographically purified receptor gave the stoichiometry of the four subunits (40,000:50,000:60,000:65,000 daltons) as 2:1:1:1, indicating that this protein is a pentameric complex with a molecular weight of 255,000 daltons. Genealogical analysis suggests that divergence from a common ancestral gene occurred early in the evolution of the receptor. This shared ancestry argues that each of the four subunits plays a functional role in the receptor's physiological action.

  1. Downregulation of the nuclear-encoded subunits of the complexes III and IV disrupts their respective complexes but not complex I in procyclic Trypanosoma brucei.

    Science.gov (United States)

    Horváth, Anton; Horáková, Eva; Dunajcíková, Petra; Verner, Zdenek; Pravdová, Eliska; Slapetová, Iveta; Cuninková, L'udmila; Lukes, Julius

    2005-10-01

    The function, stability and mutual interactions of selected nuclear-encoded subunits of respiratory complexes III and IV were studied in the Trypanosoma brucei procyclics using RNA interference (RNAi). The growth rates and oxygen consumption of clonal cell lines of knock-downs for apocytochrome c1 (apoc1) and the Rieske Fe-S protein (Rieske) of complex III, and cytochrome c oxidase subunit 6 (cox6) of complex IV were markedly decreased after RNAi induction. Western analysis of mitochondrial lysates using specific antibodies confirmed complete elimination of the targeted proteins 4-6 days after induction. The Rieske protein was reduced in the apoc1 knock-down and vice versa, indicating a mutual interdependence of these components of complex III. However, another subunit of complex IV remained at the wild-type level in the cox6 knock-down. As revealed by two-dimensional blue native/SDS-PAGE electrophoresis, silencing of a single subunit resulted in the disruption of the respective complex, while the other complex remained unaffected. Membrane potential was reproducibly decreased in the knock-downs and the activities of complex III and/or IV, but not complex I, were drastically reduced, as measured by activity assays and histochemical staining. Using specific inhibitors, we have shown that in procyclics with depleted subunits of the respiratory complexes the flow of electrons was partially re-directed to the alternative oxidase. The apparent absence in T. brucei procyclics of a supercomplex composed of complexes I and III may represent an ancestral state of the respiratory chain.

  2. β-Subunits of the SnRK1 Complexes Share a Common Ancestral Function Together with Expression and Function Specificities; Physical Interaction with Nitrate Reductase Specifically Occurs via AKINβ1-Subunit1[C][OA

    Science.gov (United States)

    Polge, Cécile; Jossier, Mathieu; Crozet, Pierre; Gissot, Lionel; Thomas, Martine

    2008-01-01

    The SNF1/AMPK/SnRK1 kinases are evolutionary conserved kinases involved in yeast, mammals, and plants in the control of energy balance. These heterotrimeric enzymes are composed of one α-type catalytic subunit and two γ- and β-type regulatory subunits. In yeast it has been proposed that the β-type subunits regulate both the localization of the kinase complexes within the cell and the interaction of the kinases with their targets. In this work, we demonstrate that the three β-type subunits of Arabidopsis (Arabidopsis thaliana; AKINβ1, AKINβ2, and AKINβ3) restore the growth phenotype of the yeast sip1Δsip2Δgal83Δ triple mutant, thus suggesting the conservation of an ancestral function. Expression analyses, using AKINβ promoter∷β-glucuronidase transgenic lines, reveal different and specific patterns of expression for each subunit according to organs, developmental stages, and environmental conditions. Finally, our results show that the β-type subunits are involved in the specificity of interaction of the kinase with the cytosolic nitrate reductase. Together with previous cell-free phosphorylation data, they strongly support the proposal that nitrate reductase is a real target of SnRK1 in the physiological context. Altogether our data suggest the conservation of ancestral basic function(s) together with specialized functions for each β-type subunit in plants. PMID:18768910

  3. Separation of ribosomal subunits of Escherichia coli by Sepharose chromatography using reverse salt gradient.

    Science.gov (United States)

    Kirillov, S V; Makhno, V I; Peshin, N N; Semenkov, Y P

    1978-11-01

    A mixture of 30 S and 50 S subunits quantitatively absorbs on a column of Sepharose--4B from the buffer: 0.02 M Tris--HCl, pH 7.5, containing 1.5 M (NH4)2SO4. During elution by reverse gradient of ammonium sulphate (1.5--0.05 M) the subunits are eluted at different salt concentrations. Complete separation of subunits is attained in the absence of Mg2+ ions. The 30 S subunits prepared from 70 S ribosomes according to this procedure are fully active in the codon--dependent binding of a specific aminoacyl--tRNA. After their reassociation with 50 S subunits isolated by zonal centrifugation, the resulting 70 S ribosomes are active in polypeptide synthesis at the same degree as control 70 S ribosomes in which both types of subunits were prepared by zonal centrifugation. The initial 70 S ribosomes for the chromatographic separation into subunits can be obtained by their pelleting from a crude extract with subsequent washing with concentrated solutions of NH4Cl in the ultracentrifuge, or by salt fractionation of the crude extract according to a slightly modified procedure of Kurland.

  4. Regulatory T Cell Responses in Participants with Type 1 Diabetes after a Single Dose of Interleukin-2: A Non-Randomised, Open Label, Adaptive Dose-Finding Trial.

    Directory of Open Access Journals (Sweden)

    John A Todd

    2016-10-01

    Full Text Available Interleukin-2 (IL-2 has an essential role in the expansion and function of CD4+ regulatory T cells (Tregs. Tregs reduce tissue damage by limiting the immune response following infection and regulate autoreactive CD4+ effector T cells (Teffs to prevent autoimmune diseases, such as type 1 diabetes (T1D. Genetic susceptibility to T1D causes alterations in the IL-2 pathway, a finding that supports Tregs as a cellular therapeutic target. Aldesleukin (Proleukin; recombinant human IL-2, which is administered at high doses to activate the immune system in cancer immunotherapy, is now being repositioned to treat inflammatory and autoimmune disorders at lower doses by targeting Tregs.To define the aldesleukin dose response for Tregs and to find doses that increase Tregs physiologically for treatment of T1D, a statistical and systematic approach was taken by analysing the pharmacokinetics and pharmacodynamics of single doses of subcutaneous aldesleukin in the Adaptive Study of IL-2 Dose on Regulatory T Cells in Type 1 Diabetes (DILT1D, a single centre, non-randomised, open label, adaptive dose-finding trial with 40 adult participants with recently diagnosed T1D. The primary endpoint was the maximum percentage increase in Tregs (defined as CD3+CD4+CD25highCD127low from the baseline frequency in each participant measured over the 7 d following treatment. There was an initial learning phase with five pairs of participants, each pair receiving one of five pre-assigned single doses from 0.04 × 106 to 1.5 × 106 IU/m2, in order to model the dose-response curve. Results from each participant were then incorporated into interim statistical modelling to target the two doses most likely to induce 10% and 20% increases in Treg frequencies. Primary analysis of the evaluable population (n = 39 found that the optimal doses of aldesleukin to induce 10% and 20% increases in Tregs were 0.101 × 106 IU/m2 (standard error [SE] = 0.078, 95% CI = -0.052, 0.254 and 0.497

  5. Risk capital allocation with autonomous subunits

    DEFF Research Database (Denmark)

    Hougaard, Jens Leth; Smilgins, Aleksandrs

    2016-01-01

    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...... fairness tests related directly to the problem of risk capital allocation and show that the Lorenz set satisfies all three tests in contrast to other well-known coherent methods. Finally, we discuss how to deal with non-uniqueness of the Lorenz set.......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 than...... the sum of the risks of the individual subunits. The question is how to allocate the risk capital of the group among the subunits in a fair way. In this paper we propose to use the Lorenz set as an allocation method. We show that the Lorenz set is operational and coherent. Moreover, we propose three...

  6. Network type sp3 boron-based single-ion conducting polymer electrolytes for lithium ion batteries

    Science.gov (United States)

    Deng, Kuirong; Wang, Shuanjin; Ren, Shan; Han, Dongmei; Xiao, Min; Meng, Yuezhong

    2017-08-01

    Electrolytes play a vital role in modulating lithium ion battery performance. An outstanding electrolyte should possess both high ionic conductivity and unity lithium ion transference number. Here, we present a facile method to fabricate a network type sp3 boron-based single-ion conducting polymer electrolyte (SIPE) with high ionic conductivity and lithium ion transference number approaching unity. The SIPE was synthesized by coupling of lithium bis(allylmalonato)borate (LiBAMB) and pentaerythritol tetrakis(2-mercaptoacetate) (PETMP) via one-step photoinitiated in situ thiol-ene click reaction in plasticizers. Influence of kinds and content of plasticizers was investigated and the optimized electrolytes show both outstanding ionic conductivity (1.47 × 10-3 S cm-1 at 25 °C) and high lithium transference number of 0.89. This ionic conductivity is among the highest ionic conductivity exhibited by SIPEs reported to date. Its electrochemical stability window is up to 5.2 V. More importantly, Li/LiFePO4 cells with the prepared single-ion conducting electrolytes as the electrolyte as well as the separator display highly reversible capacity and excellent rate capacity under room temperature. It also demonstrates excellent long-term stability and reliability as it maintains capacity of 124 mA h g-1 at 1 C rate even after 500 cycles without obvious decay.

  7. Single locus typing of MHC class I and class II B loci in a population of red jungle fowl.

    Science.gov (United States)

    Worley, K; Gillingham, M; Jensen, P; Kennedy, L J; Pizzari, T; Kaufman, J; Richardson, D S

    2008-05-01

    In species with duplicated major histocompatibility complex (MHC) genes, estimates of genetic variation often rely on multilocus measures of diversity. It is possible that such measures might not always detect more detailed patterns of selection at individual loci. Here, we describe a method that allows us to investigate classical MHC diversity in red jungle fowl (Gallus gallus), the wild ancestor of the domestic chicken, using a single locus approach. This is possible due to the well-characterised gene organisation of the 'minimal essential' MHC (BF/BL region) of the domestic chicken, which comprises two differentially expressed duplicated class I (BF) and two class II B (BLB) genes. Using a combination of reference strand-mediated conformation analysis, cloning and sequencing, we identify nine BF and ten BLB alleles in a captive population of jungle fowl. We show that six BF and five BLB alleles are from the more highly expressed locus of each gene, BF2 and BLB2, respectively. An excess of non-synonymous substitutions across the jungle fowl BF/BL region suggests that diversifying selection has acted on this population. Importantly, single locus screening reveals that the strength of selection is greatest on the highly expressed BF2 locus. This is the first time that a population of red jungle fowl has been typed at the MHC region, laying the basis for further research into the underlying processes acting to maintain MHC diversity in this and other species.

  8. The influence of the type of embryo culture medium on neonatal birthweight after single embryo transfer in IVF.

    Science.gov (United States)

    Vergouw, Carlijn G; Kostelijk, E Hanna; Doejaaren, Els; Hompes, Peter G A; Lambalk, Cornelis B; Schats, Roel

    2012-09-01

    Does the type of medium used to culture fresh and frozen-thawed embryos influence neonatal birthweight after single embryo transfer (SET) in IVF? A comparison of two commercially available culture media showed no significant influence on mean birthweight and mean birthweight adjusted for gestational age, gender and parity (z-scores) of singletons born after a fresh or frozen-thawed SET. Furthermore, we show that embryo freezing and thawing cycles may lead to a significantly higher mean birthweight. Animal studies have shown that culture media constituents are responsible for changes in birthweight of offspring. In human IVF, there is still little knowledge of the effect of medium type on birthweight. Until now, only a small number of commercially available culture media have been investigated (Vitrolife, Cook(®) Medical and IVF online medium). Our study adds new information: it has a larger population of singleton births compared with the previously published studies, it includes outcomes of other media types (HTF and Sage(®)), not previously analysed, and it includes data on frozen-thawed SETs. This study was a retrospective analysis of birthweights of singleton newborns after fresh (Day 3) or frozen-thawed (Day 5) SET cycles, using embryos cultured in either of two different types of commercially available culture media, between 2008 and 2011. Before January 2009, a single-step culture medium was used: human tubal fluid (HTF) with 4 mg/ml human serum albumin. From January 2009 onwards, a commercially available sequential medium was introduced: Sage(®), Quinn's advantage protein plus medium. Singletons born after a fresh SET (99 embryos cultured in HTF and 259 in Sage(®)) and singletons born after a frozen-thawed SET (32 embryos cultured in HTF only, 41 in HTF and Sage(®) and 86 in Sage(®) only) were analysed. Only patients using autologous gametes without the use of a gestational carrier were considered. Also excluded were (vanishing) twins, triplets

  9. The [Fe(III)[Fe(III)(L1)2]3] star-type single-molecule magnet.

    Science.gov (United States)

    Saalfrank, Rolf W; Scheurer, Andreas; Bernt, Ingo; Heinemann, Frank W; Postnikov, Andrei V; Schünemann, Volker; Trautwein, Alfred X; Alam, Mohammad S; Rupp, Holger; Müller, Paul

    2006-06-21

    Star-shaped complex [Fe(III)[Fe(III)(L1)2]3] (3) was synthesized starting from N-methyldiethanolamine H2L1 (1) and ferric chloride in the presence of sodium hydride. For 3, two different high-spin iron(III) ion sites were confirmed by Mössbauer spectroscopy at 77 K. Single-crystal X-ray structure determination revealed that 3 crystallizes with four molecules of chloroform, but, with only three molecules of dichloromethane. The unit cell of 3.4CHCl3 contains the enantiomers (delta)-[(S,S)(R,R)(R,R)] and (lambda)-[(R,R)(S,S)(S,S)], whereas in case of 3.3CH2Cl2 four independent molecules, forming pairs of the enantiomers [lambda-(R,R)(R,R)(R,R)]-3 and [lambda-(S,S)(S,S)(S,S)]-3, were observed in the unit cell. According to SQUID measurements, the antiferromagnetic intramolecular coupling of the iron(III) ions in 3 results in a S = 10/2 ground state multiplet. The anisotropy is of the easy-axis type. EPR measurements enabled an accurate determination of the ligand-field splitting parameters. The ferric star 3 is a single-molecule magnet (SMM) and shows hysteretic magnetization characteristics below a blocking temperature of about 1.2 K. However, weak intermolecular couplings, mediated in a chainlike fashion via solvent molecules, have a strong influence on the magnetic properties. Scanning tunneling microscopy (STM) and scanning tunneling spectroscopy (STS) were used to determine the structural and electronic properties of star-type tetranuclear iron(III) complex 3. The molecules were deposited onto highly ordered pyrolytic graphite (HOPG). Small, regular molecule clusters, two-dimensional monolayers as well as separated single molecules were observed. In our STS measurements we found a rather large contrast at the expected locations of the metal centers of the molecules. This direct addressing of the metal centers was confirmed by DFT calculations.

  10. Gene structure and mutations of glutaryl-coenzyme A dehydrogenase: impaired association of enzyme subunits that is due to an A421V substitution causes glutaric acidemia type I in the Amish.

    OpenAIRE

    Biery, B. J.; Stein, D. E.; Morton, D. H.; Goodman, S. I.

    1996-01-01

    The structure of the human glutaryl coenzyme A dehydrogenase (GCD) gene was determined to contain 11 exons and to span approximately 7 kb. Fibroblast DNA from 64 unrelated glutaric acidemia type I (GA1) patients was screened for mutations by PCR amplification and analysis of SSCP. Fragments with altered electrophoretic mobility were subcloned and sequenced to detect mutations that caused GA1. This report describes the structure of the GCD gene, as well as point mutations and polymorphisms fou...

  11. Intron retention in mRNA encoding ancillary subunit of insect voltage-gated sodium channel modulates channel expression, gating regulation and drug sensitivity.

    Directory of Open Access Journals (Sweden)

    Céline M Bourdin

    Full Text Available Insect voltage-gated sodium (Nav channels are formed by a well-known pore-forming α-subunit encoded by para-like gene and ancillary subunits related to TipE from the mutation "temperature-induced-paralysis locus E." The role of these ancillary subunits in the modulation of biophysical and pharmacological properties of Na(+ currents are not enough documented. The unique neuronal ancillary subunit TipE-homologous protein 1 of Drosophila melanogaster (DmTEH1 strongly enhances the expression of insect Nav channels when heterologously expressed in Xenopus oocytes. Here we report the cloning and functional expression of two neuronal DmTEH1-homologs of the cockroach, Periplaneta americana, PaTEH1A and PaTEH1B, encoded by a single bicistronic gene. In PaTEH1B, the second exon encoding the last 11-amino-acid residues of PaTEH1A is shifted to 3'UTR by the retention of a 96-bp intron-containing coding-message, thus generating a new C-terminal end. We investigated the gating and pharmacological properties of the Drosophila Nav channel variant (DmNav1-1 co-expressed with DmTEH1, PaTEH1A, PaTEH1B or a truncated mutant PaTEH1Δ(270-280 in Xenopus oocytes. PaTEH1B caused a 2.2-fold current density decrease, concomitant with an equivalent α-subunit incorporation decrease in the plasma membrane, compared to PaTEH1A and PaTEH1Δ(270-280. PaTEH1B positively shifted the voltage-dependences of activation and slow inactivation of DmNav1-1 channels to more positive potentials compared to PaTEH1A, suggesting that the C-terminal end of both proteins may influence the function of the voltage-sensor and the pore of Nav channel. Interestingly, our findings showed that the sensitivity of DmNav1-1 channels to lidocaine and to the pyrazoline-type insecticide metabolite DCJW depends on associated TEH1-like subunits. In conclusion, our work demonstrates for the first time that density, gating and pharmacological properties of Nav channels expressed in Xenopus oocytes can be

  12. 229 nm UV LEDs on aluminum nitride single crystal substrates using p-type silicon for increased hole injection

    Science.gov (United States)

    Liu, Dong; Cho, Sang June; Park, Jeongpil; Seo, Jung-Hun; Dalmau, Rafael; Zhao, Deyin; Kim, Kwangeun; Gong, Jiarui; Kim, Munho; Lee, In-Kyu; Albrecht, John D.; Zhou, Weidong; Moody, Baxter; Ma, Zhenqiang

    2018-02-01

    AlGaN based 229 nm light emitting diodes (LEDs), employing p-type Si to significantly increase hole injection, were fabricated on single crystal bulk aluminum nitride (AlN) substrates. Nitride heterostructures were epitaxially deposited by organometallic vapor phase epitaxy and inherit the low dislocation density of the native substrate. Following epitaxy, a p-Si layer is bonded to the heterostructure. LEDs were characterized both electrically and optically. Owing to the low defect density films, large concentration of holes from p-Si, and efficient hole injection, no efficiency droop was observed up to a current density of 76 A/cm2 under continuous wave operation and without external thermal management. An optical output power of 160 μW was obtained with the corresponding external quantum efficiency of 0.03%. This study demonstrates that by adopting p-type Si nanomembrane contacts as a hole injector, practical levels of hole injection can be realized in UV light-emitting diodes with very high Al composition AlGaN quantum wells, enabling emission wavelengths and power levels that were previously inaccessible using traditional p-i-n structures with poor hole injection efficiency.

  13. Strain typing of Zygosaccharomyces yeast species using a single molecular method based on polymorphism of the intergenic spacer region (IGS).

    Science.gov (United States)

    Wrent, Petra; Rivas, Eva-María; Peinado, José M; de Silóniz, María-Isabel

    2010-08-15

    Unlike previously reported methods that need a combination of several typing techniques, we have developed a single method for strain typing of the Zygosaccharomyces bailii, Z. mellis and Z. rouxii spoilage species. Strains belonging to other species have also been included for comparison. We have demonstrated that the IGS-PCR RFLP method has a high discriminative power. Considering the three endonucleases used in this work, we have obtained a variability of 100% for Z. mellis and Z. rouxii strains and up to 70% for Z. bailii. We have also detected two misidentified Z. mellis strains (CBS 711 and CBS 7412) which have RFLP patterns with a set of bands characteristic of Z. rouxii strains. Sequencing of 26S rDNA D1/D2 domains and the 5.8-ITS rDNA region confirmed these strains as Z. rouxii. The method also groups three certified hybrid strains of Zygosaccharomyces in a separate cluster. Copyright 2010 Elsevier B.V. All rights reserved.

  14. Effects of bovine cytochrome P450 single-nucleotide polymorphism, forage type and body condition on production traits in cattle.

    Science.gov (United States)

    Sales, M A; Larson, M J; Reiter, S T; Brown, A H; Brown, M A; Looper, M L; Coffey, K P; Rosenkrans, C F

    2012-08-01

    Relating single-nucleotide polymorphisms (SNP) to cows with acceptable productivity could benefit cattle breeders in areas where tall fescue is the predominant forage. This study aimed to (i) identify SNPs in bovine cytochrome P450 3A28 (CYP3A28) and (ii) determine the associations between SNP genotype, forage and cow body condition (BC). Genotype (CC, CG or GG) and forage [Kentucky-31 wild-type endophyte-infected tall fescue (KY+) vs. bermudagrass] effects on milk volume and quality were determined in Herd 1 cows (123 cows); in Herd 2 (99 cows), genotype and BC (low vs. moderate) effects on ovarian follicle size, calving date and calving per cent were determined; and in Herd 3 (114 cows), effects of genotype and fescue cultivar [KY+ vs. non-toxic endophyte-infected tall fescue (HiMag4)] were related to calving per cent, calving date and weaning weights of both cow and her calf. A cytosine (C) to guanine (G) transversion at base 994 (C994G) in CYP3A28 was identified. There was a genotype × forage type interaction (p productivity in cows. © 2011 Blackwell Verlag GmbH.

  15. A Single Bout of High-Intensity Interval Training Reduces Awareness of Subsequent Hypoglycemia in Patients With Type 1 Diabetes.

    Science.gov (United States)

    Rooijackers, Hanne M; Wiegers, Evita C; van der Graaf, Marinette; Thijssen, Dick H; Kessels, Roy P C; Tack, Cees J; de Galan, Bastiaan E

    2017-07-01

    High-intensity interval training (HIIT) has gained increasing popularity in patients with diabetes. HIIT acutely increases plasma lactate levels. This may be important, since the administration of lactate during hypoglycemia suppresses symptoms and counterregulation while preserving cognitive function. We tested the hypothesis that, in the short term, HIIT reduces awareness of hypoglycemia and attenuates hypoglycemia-induced cognitive dysfunction. In a randomized crossover trial, patients with type 1 diabetes and normal awareness of hypoglycemia (NAH), patients with impaired awareness of hypoglycemia (IAH), and healthy participants ( n = 10 per group) underwent a hyperinsulinemic-hypoglycemic (2.6 mmol/L) clamp, either after a HIIT session or after seated rest. Compared with rest, HIIT reduced symptoms of hypoglycemia in patients with NAH but not in healthy participants or patients with IAH. HIIT attenuated hypoglycemia-induced cognitive dysfunction, which was mainly driven by changes in the NAH subgroup. HIIT suppressed cortisol and growth hormone responses, but not catecholamine responses to hypoglycemia. The present findings demonstrate that a single HIIT session rapidly reduces awareness of subsequent hypoglycemia in patients with type 1 diabetes and NAH, but does not in patients with IAH, and attenuates hypoglycemia-induced cognitive dysfunction. The role of exercise-induced lactate in mediating these effects, potentially serving as an alternative fuel for the brain, should be further explored. © 2017 by the American Diabetes Association.

  16. A new type of FCT martensite phase in single-crystalline Fe{sub 3}Pt Invar alloy

    Energy Technology Data Exchange (ETDEWEB)

    Yamamoto, Masataka; Sekida, Sayaka [Department of Materials Science and Engineering, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, Suita, Osaka 565-0871 (Japan); Fukuda, Takashi, E-mail: fukuda@mat.eng.osaka-u.ac.jp [Department of Materials Science and Engineering, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, Suita, Osaka 565-0871 (Japan); Kakeshita, Tomoyuki [Department of Materials Science and Engineering, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, Suita, Osaka 565-0871 (Japan); Takahashi, Kohki; Koyama, Keiichi; Nojiri, Hiroyuki [High-Field Laboratory for Superconducting Materials, Institute for Materials Research, Tohoku University, 2-1-1 Sendai Katahira, Aoba-ku, Sendai, Miyagi 980-8577 (Japan)

    2011-08-25

    Highlights: {center_dot} TEM observations at room temperature reveal that the specimen is a single phase of L1{sub 2}-type structure. {center_dot} The spontaneous magnetization and magnetic susceptibility curves of Fe{sub 3}Pt with S = 0.88 have bend points at 60 K. {center_dot} XRD measurements reveal that this alloy exhibits a martensitic transformation to FCT and the transformation temperature is 60 K. {center_dot} The tetragonality c/a is larger than unity in the FCT martensite. {center_dot} This tetragonality is in contrast to the tetragonality c/a < 1generally observed in the FCT martensite with S < 0.8. - Abstract: Martensitic transformation in a highly ordered Fe{sub 3}Pt has been investigated by magnetization and X-ray diffraction measurements. We confirmed that a new type of face-centered tetragonal (FCT) martensite phase appears below 60 K in Fe{sub 3}Pt with a degree of order S = 0.88. The tetragonality c/a gradually increases with decreasing temperature, and is approximately 1.005 at 10 K. This is in contrast to the tetragonality c/a < 1 generally observed in the FCT martensite with a degree of order less than 0.8. The spontaneous magnetization increases in association with the transformation.

  17. Relating proton pumps with gap junctions: colocalization of ductin, the channel-forming subunit c of V-ATPase, with subunit a and with innexins 2 and 3 during Drosophila oogenesis.

    Science.gov (United States)

    Lautemann, Julia; Bohrmann, Johannes

    2016-07-13

    Ion-transport mechanisms and gap junctions are known to cooperate in creating bioelectric phenomena, like pH gradients, voltage gradients and ion fluxes within single cells, tissues, organs, and whole organisms. Such phenomena have been shown to play regulatory roles in a variety of developmental and regenerative processes. Using Drosophila oogenesis as a model system, we aim at characterizing in detail the mechanisms underlying bioelectric phenomena in order to reveal their regulatory functions. We, therefore, investigated the stage-specific distribution patterns of V-ATPase components in relation to gap-junction proteins. We analysed the localization of the V-ATPase components ductin (subunit c) and subunit a, and the gap-junction components innexins 2 and 3, especially in polar cells, border cells, stalk cells and centripetally migrating cells. These types of follicle cells had previously been shown to exhibit characteristic patterns of membrane channels as well as membrane potential and intracellular pH. Stage-specifically, ductin and subunit a were found either colocalized or separately enriched in different regions of soma and germ-line cells. While ductin was often more prominent in plasma membranes, subunit a was more prominent in cytoplasmic and nuclear vesicles. Particularly, ductin was enriched in polar cells, stalk cells, and nurse-cell membranes, whereas subunit a was enriched in the cytoplasm of border cells, columnar follicle cells and germ-line cells. Comparably, ductin and both innexins 2 and 3 were either colocalized or separately enriched in different cellular regions. While ductin often showed a continuous membrane distribution, the distribution of both innexins was mostly punctate. Particularly, ductin was enriched in polar cells and stalk cells, whereas innexin 2 was enriched in the oolemma, and innexin 3 in centripetally migrating follicle cells. In lateral follicle-cell membranes, the three proteins were found colocalized as well as

  18. Application of AERONET Single Scattering Albedo and Absorption Angstrom Exponent to Classify Dominant Aerosol Types during DRAGON Campaigns

    Science.gov (United States)

    Giles, D. M.; Holben, B. N.; Eck, T. F.; Schafer, J.; Crawford, J. H.; Kim, J.; Sano, I.; Liew, S.; Salinas Cortijo, S. V.; Chew, B. N.; Lim, H.; Smirnov, A.; Sorokin, M.; Kenny, P.; Slutsker, I.

    2013-12-01

    Aerosols can have major implications on human health by inducing respiratory diseases due to inhalation of fine particles from biomass burning smoke or industrial pollution and on radiative forcing whereby the presence of absorbing aerosol particles (e.g., black carbon) increases atmospheric heating. Aerosol classification techniques have utilized aerosol loading and aerosol properties derived from multi-spectral and multi-angle observations by ground-based (e.g., AERONET) and satellite instrumentation (e.g., MISR). Aerosol Robotic Network (AERONET) data have been utilized to determine aerosol types by implementing various combinations of measured aerosol optical depth or retrieved size and absorption aerosol properties (e.g., Gobbi et al., 2007; Russell et al., 2010). Giles et al. [2012] showed single scattering albedo (SSA) relationship with extinction Angstrom exponent (EAE) can provide an estimate of the general classification of dominant aerosol types (i.e., desert dust, urban/industrial pollution, biomass burning smoke, and mixtures) based on data from ~20 AERONET sites located in known aerosol source regions. In addition, the absorption Angstrom exponent relationship with EAE can provide an indication of the dominant absorbing aerosol type such as dust, black carbon, brown carbon, or mixtures of them. These classification techniques are applied to the AERONET Level 2.0 quality assured data sets collected during Distributed Regional Aerosol Gridded Observational Network (DRAGON) campaigns in Maryland (USA), Japan, South Korea, Singapore, Penang (Malaysia), and California (USA). An analysis of aerosol type classification for DRAGON sites is performed as well as an assessment of the spatial variability of the aerosol types for selected DRAGON campaigns. Giles, D. M., B. N. Holben, T. F. Eck, A. Sinyuk, A. Smirnov, I. Slutsker, R. R. Dickerson, A. M. Thompson, and J. S. Schafer (2012), An analysis of AERONET aerosol absorption properties and classifications

  19. Isolated single coronary artery (RII-B type presenting as an inferior wall myocardial infarction: A rare clinical entity

    Directory of Open Access Journals (Sweden)

    Ankur C. Thummar

    2014-09-01

    Full Text Available Isolated single coronary artery without other congenital cardiac anomalies is very rare among the different variations of anomalous coronary patterns. The prognosis in patients with single coronary varies according to the anatomic distribution and associated coronary atherosclerosis. If the left main coronary artery travels between the aorta and pulmonary arteries, it may be a cause of sudden cardiac death. We present multimodality images of a single coronary artery, in which the whole coronary system originated by a single trunk from the right sinus of Valsalva with inter-arterial course of left main coronary artery. This rare type of single coronary artery was classified as RII-B type according to Lipton's scheme of classification. A significant flow-limiting lesions were found in the right coronary artery that was successfully treated with percutaneous coronary intervention.

  20. Pharmacokinetics and pharmacodynamics of saruplase, an unglycosylated single-chain urokinase-type plasminogen activator, in patients with acute myocardial infarction

    NARCIS (Netherlands)

    Koster, R. W.; Cohen, A. F.; Hopkins, G. R.; Beier, H.; Günzler, W. A.; van der Wouw, P. A.

    1994-01-01

    We examined in patients with acute myocardial infarction (AMI) the pharmacokinetics of saruplase, an unglycosylated, single chain, urokinase-type plasminogen activator (rscu-PA) by measuring urokinase-type plasminogen activator (u-PA) antigen and total u-PA activity, its conversion to active

  1. Characterization of a novel mutant KCNQ1 channel subunit lacking a large part of the C-terminal domain.

    Science.gov (United States)

    Kimoto, Katsuya; Kinoshita, Koshi; Yokoyama, Tomoki; Hata, Yukiko; Komatsu, Takuto; Tsushima, Eikichi; Nishide, Kohki; Yamaguchi, Yoshiaki; Mizumaki, Koichi; Tabata, Toshihide; Inoue, Hiroshi; Nishida, Naoki; Fukurotani, Kenkichi

    2013-10-18

    A mutation of KCNQ1 gene encoding the alpha subunit of the channel mediating the slow delayed rectifier K(+) current in cardiomyocytes may cause severe arrhythmic disorders. We identified KCNQ1(Y461X), a novel mutant gene encoding KCNQ1 subunit whose C-terminal domain is truncated at tyrosine 461 from a man with a mild QT interval prolongation. We made whole-cell voltage-clamp recordings from HEK-293T cells transfected with either of wild-type KCNQ1 [KCNQ1(WT)], KCNQ1(Y461X), or their mixture plus KCNE1 auxiliary subunit gene. The KCNQ1(Y461X)-transfected cells showed no delayed rectifying current. The cells transfected with both KCNQ1(WT) and KCNQ1(Y461X) showed the delayed rectifying current that is thought to be mediated largely by homomeric channel consisting of KCNQ1(WT) subunit because its voltage-dependence of activation, activation rate, and deactivation rate were similar to the current in the KCNQ1(WT)-transfected cells. The immunoblots of HEK-293T cell-derived lysates showed that KCNQ1(Y461X) subunit cannot form channel tetramers by itself or with KCNQ1(WT) subunit. Moreover, immunocytochemical analysis in HEK-293T cells showed that the surface expression level of KCNQ1(Y461X) subunit was very low with or without KCNQ1(WT) subunit. These findings suggest that the massive loss of the C-terminal domain of KCNQ1 subunit impairs the assembly, trafficking, and function of the mutant subunit-containing channels, whereas the mutant subunit does not interfere with the functional expression of the homomeric wild-type channel. Therefore, the homozygous but not heterozygous inheritance of KCNQ1(Y461X) might cause major arrhythmic disorders. This study provides a new insight into the structure-function relation of KCNQ1 channel and treatments of cardiac channelopathies. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. Functional regulation of BK potassium channels by γ1 auxiliary subunits.

    Science.gov (United States)

    Gonzalez-Perez, Vivian; Xia, Xiao-Ming; Lingle, Christopher J

    2014-04-01

    Many K(+) channels are oligomeric complexes with intrinsic structural symmetry arising from the homo-tetrameric core of their pore-forming subunits. Allosteric regulation of tetramerically symmetric proteins, whether by intrinsic sensing domains or associated auxiliary subunits, often mirrors the fourfold structural symmetry. Here, through patch-clamp recordings of channel population ensembles and also single channels, we examine regulation of the Ca(2+)- and voltage-activated large conductance Ca(2+)-activated K(+) (BK) channel by associated γ1-subunits. Through expression of differing ratios of γ1:α-subunits, the results reveal an all-or-none functional regulation of BK channels by γ-subunits: channels either exhibit a full gating shift or no shift at all. Furthermore, the γ1-induced shift exhibits a state-dependent labile behavior that recapitulates the fully shifted or unshifted behavior. The γ1-induced shift contrasts markedly to the incremental shifts in BK gating produced by 1-4 β-subunits and adds a new layer of complexity to the mechanisms by which BK channel functional diversity is generated.

  3. A molecular breadboard: Removal and replacement of subunits in a hepatitis B virus capsid.

    Science.gov (United States)

    Lee, Lye Siang; Brunk, Nicholas; Haywood, Daniel G; Keifer, David; Pierson, Elizabeth; Kondylis, Panagiotis; Wang, Joseph Che-Yen; Jacobson, Stephen C; Jarrold, Martin F; Zlotnick, Adam

    2017-11-01

    Hepatitis B virus (HBV) core protein is a model system for studying assembly and disassembly of icosahedral structures. Controlling disassembly will allow re-engineering the 120 subunit HBV capsid, making it a molecular breadboard. We examined removal of subunits from partially crosslinked capsids to form stable incomplete particles. To characterize incomplete capsids, we used two single molecule techniques, resistive-pulse sensing and charge detection mass spectrometry. We expected to find a binomial distribution of capsid fragments. Instead, we found a preponderance of 3 MDa complexes (90 subunits) and no fragments smaller than 3 MDa. We also found 90-mers in the disassembly of uncrosslinked HBV capsids. 90-mers seem to be a common pause point in disassembly reactions. Partly explaining this result, graph theory simulations have showed a threshold for capsid stability between 80 and 90 subunits. To test a molecular breadboard concept, we showed that missing subunits could be refilled resulting in chimeric, 120 subunit particles. This result may be a means of assembling unique capsids with functional decorations. © 2017 The Protein Society.

  4. The 2.3 {angstrom} crystal structure of cholera toxin B subunit pentamer: Choleragenoid

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Rong-Guang; Westbrook, M.L. [Argonne National Lab., IL (United States); Maulik, P.R.; Reed, R.A.; Shipley, G. [Boston Univ., MA (United States). School of Medicine; Westbrook, E.M. [Argonne National Lab., IL (United States)]|[Northwestern Univ., Evanston, IL (United States); Scott, D.L.; Otwinowski, Z. [Yale Univ., New Haven, CT (United States)

    1996-02-01

    Cholera toxin, a heterohexameric AB{sub 5} enterotoxin released by Vibrio cholera, induces a profuse secretory diarrhea in susceptible hosts. Choleragenoid, the B subunit pentamer of cholera toxin, directs the enzymatic A subunit to its target by binding to GM{sub 1} gangliosides exposed on the luminal surface of intestinal epithelial cells. We have solved the crystal structure of choleragenoid at 2.3 {Angstrom} resolution by combining single isomorphous replacement with non-crystallographic symmetry averaging. The structure of the B subunits, and their pentameric arrangement, closely resembles that reported for the intact holotoxin (choleragen), the heat-labile enterotoxin from E. coli, and for a choleragenoid-GM{sub 1} pentasaccharide complex. In the absence of the A subunit the central cavity of the B pentamer is a highly solvated channel. The binding of the A subunit or the receptor pentasaccharide to choleragenoid has only a modest effect on the local stereochemistry and does not perceptibly alter the subunit interface.

  5. The Subunit Principle in Scar Face Revision.

    Science.gov (United States)

    Elshahat, Ahmed; Lashin, Riham

    2017-06-01

    Facial scaring is considered one of the most difficult cosmetic problems for any plastic surgeon to solve. The condition is more difficult if the direction of the scar is not parallel to relaxed skin tension lines. Attempts to manage this difficult situation included revisions using geometric designs, Z plasties or W plasties to camouflage the straight line visible scaring. The use of long-lasting resorbable sutures was tried too. Recently, the use of botulinum toxin during revision improved the results. Fractional CO2 lasers, microfat grafts, and platelet-rich plasma were added to the armamentarium. The scar is least visible if placed in the junction between the facial subunits. The aim of this study is to investigate the use of the subunit principle to improve the results of scar revision. Four patients were included in this study. Tissue expansion of the intact part of the subunit allowed shifting the scar to the junction between the affected subunit and the adjacent one. Tissue expansion, delivery of the expanders, and advancement of the flaps were successful in all patients. The fact that this is a 2-stage procedure and sacrifices some of the intact skin from the affected facial subunit, makes this technique reserved to patients with ugly facial scars who are ambitious to improve their appearance.

  6. Pharmacological consequences of the coexpression of BK channel α and auxiliary β subunits

    Science.gov (United States)

    Torres, Yolima P.; Granados, Sara T.; Latorre, Ramón

    2014-01-01

    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

  7. Factors associated with anxiety and depression among type 2 diabetes outpatients in Malaysia: a descriptive cross-sectional single-centre study

    OpenAIRE

    Ganasegeran, Kurubaran; Renganathan, Pukunan; Manaf, Rizal Abdul; Al-Dubai, Sami Abdo Radman

    2014-01-01

    Objective To determine the prevalence and factors associated with anxiety and depression among type 2 diabetes outpatients in Malaysia. Design Descriptive, cross-sectional single-centre study with universal sampling of all patients with type 2 diabetes. Setting Endocrinology clinic of medical outpatient department in a Malaysian public hospital. Participants All 169 patients with type 2 diabetes (men, n=99; women, n=70) aged between 18 and 90 years who acquired follow-up treatment from the en...

  8. Construction of a hybrid β-hexosaminidase subunit capable of forming stable homodimers that hydrolyze GM2 ganglioside in vivo.

    Science.gov (United States)

    Tropak, Michael B; Yonekawa, Sayuri; Karumuthil-Melethil, Subha; Thompson, Patrick; Wakarchuk, Warren; Gray, Steven J; Walia, Jagdeep S; Mark, Brian L; Mahuran, Don

    2016-01-01

    Tay-Sachs or Sandhoff disease result from mutations in either the evolutionarily related HEXA or HEXB genes encoding respectively, the α- or β-subunits of β-hexosaminidase A (HexA). Of the three Hex isozymes, only HexA can interact with its cofactor, the GM2 activator protein (GM2AP), and hydrolyze GM2 ganglioside. A major impediment to establishing gene or enzyme replacement therapy based on HexA is the need to synthesize both subunits. Thus, we combined the critical features of both α- and β-subunits into a single hybrid µ-subunit that contains the α-subunit active site, the stable β-subunit interface and unique areas in each subunit needed to interact with GM2AP. To facilitate intracellular analysis and the purification of the µ-homodimer (HexM), CRISPR-based genome editing was used to disrupt the HEXA and HEXB genes in a Human Embryonic Kidney 293 cell line stably expressing the µ-subunit. In association with GM2AP, HexM was shown to hydrolyze a fluorescent GM2 ganglioside derivative both in cellulo and in vitro. Gene transfer studies in both Tay-Sachs and Sandhoff mouse models demonstrated that HexM expression reduced brain GM2 ganglioside levels.

  9. Extricating Manual and Non-Manual Features for Subunit Level Medical Sign Modelling in Automatic Sign Language Classification and Recognition.

    Science.gov (United States)

    R, Elakkiya; K, Selvamani

    2017-09-22

    Subunit segmenting and modelling in medical sign language is one of the important studies in linguistic-oriented and vision-based Sign Language Recognition (SLR). Many efforts were made in the precedent to focus the functional subunits from the view of linguistic syllables but the problem is implementing such subunit extraction using syllables is not feasible in real-world computer vision techniques. And also, the present recognition systems are designed in such a way that it can detect the signer dependent actions under restricted and laboratory conditions. This research paper aims at solving these two important issues (1) Subunit extraction and (2) Signer independent action on visual sign language recognition. Subunit extraction involved in the sequential and parallel breakdown of sign gestures without any prior knowledge on syllables and number of subunits. A novel Bayesian Parallel Hidden Markov Model (BPaHMM) is introduced for subunit extraction to combine the features of manual and non-manual parameters to yield better results in classification and recognition of signs. Signer independent action aims in using a single web camera for different signer behaviour patterns and for cross-signer validation. Experimental results have proved that the proposed signer independent subunit level modelling for sign language classification and recognition has shown improvement and variations when compared with other existing works.

  10. Construction of a hybrid β-hexosaminidase subunit capable of forming stable homodimers that hydrolyze GM2 ganglioside in vivo

    Directory of Open Access Journals (Sweden)

    Michael B Tropak

    2016-01-01

    Full Text Available Tay-Sachs or Sandhoff disease result from mutations in either the evolutionarily related HEXA or HEXB genes encoding respectively, the α- or β-subunits of β-hexosaminidase A (HexA. Of the three Hex isozymes, only HexA can interact with its cofactor, the GM2 activator protein (GM2AP, and hydrolyze GM2 ganglioside. A major impediment to establishing gene or enzyme replacement therapy based on HexA is the need to synthesize both subunits. Thus, we combined the critical features of both α- and β-subunits into a single hybrid µ-subunit that contains the α-subunit active site, the stable β-subunit interface and unique areas in each subunit needed to interact with GM2AP. To facilitate intracellular analysis and the purification of the µ-homodimer (HexM, CRISPR-based genome editing was used to disrupt the HEXA and HEXB genes in a Human Embryonic Kidney 293 cell line stably expressing the µ-subunit. In association with GM2AP, HexM was shown to hydrolyze a fluorescent GM2 ganglioside derivative both in cellulo and in vitro. Gene transfer studies in both Tay-Sachs and Sandhoff mouse models demonstrated that HexM expression reduced brain GM2 ganglioside levels.

  11. Construction of a hybrid β-hexosaminidase subunit capable of forming stable homodimers that hydrolyze GM2 ganglioside in vivo

    Science.gov (United States)

    Tropak, Michael B; Yonekawa, Sayuri; Karumuthil-Melethil, Subha; Thompson, Patrick; Wakarchuk, Warren; Gray, Steven J; Walia, Jagdeep S; Mark, Brian L; Mahuran, Don

    2016-01-01

    Tay-Sachs or Sandhoff disease result from mutations in either the evolutionarily related HEXA or HEXB genes encoding respectively, the α- or β-subunits of β-hexosaminidase A (HexA). Of the three Hex isozymes, only HexA can interact with its cofactor, the GM2 activator protein (GM2AP), and hydrolyze GM2 ganglioside. A major impediment to establishing gene or enzyme replacement therapy based on HexA is the need to synthesize both subunits. Thus, we combined the critical features of both α- and β-subunits into a single hybrid µ-subunit that contains the α-subunit active site, the stable β-subunit interface and unique areas in each subunit needed to interact with GM2AP. To facilitate intracellular analysis and the purification of the µ-homodimer (HexM), CRISPR-based genome editing was used to disrupt the HEXA and HEXB genes in a Human Embryonic Kidney 293 cell line stably expressing the µ-subunit. In association with GM2AP, HexM was shown to hydrolyze a fluorescent GM2 ganglioside derivative both in cellulo and in vitro. Gene transfer studies in both Tay-Sachs and Sandhoff mouse models demonstrated that HexM expression reduced brain GM2 ganglioside levels. PMID:26966698

  12. Thermostable cross-protective subunit vaccine against Brucella species.

    Science.gov (United States)

    Cherwonogrodzky, John W; Barabé, Nicole D; Grigat, Michelle L; Lee, William E; Poirier, Robert T; Jager, Scott J; Berger, Bradley J

    2014-12-01

    A subunit vaccine candidate was produced from Brucella suis 145 (biovar 4; expressing both the A antigen of Brucella abortus and the M antigen of Brucella melitensis). The preparation consisted mostly of polysaccharide (PS; >90% [wt/wt]; both cell-associated PS and exo-PS were combined) and a small amount of protein (1 to 3%) with no apparent nucleic acids. Vaccinated mice were protected (these had a statistically significant reduction in bacterial colonization compared to that of unvaccinated controls) when challenged with representative strains of three Brucella species most pathogenic for humans, i.e., B. abortus, B. melitensis, and B. suis. As little as 1 ng of the vaccine, without added adjuvant, protected mice against B. suis 145 infection (5 × 10(5) CFU), and a single injection of 1 μg of this subunit vaccine protected mice from B. suis 145 challenge for at least 14 months. A single immunization induced a serum IgG response to Brucella antigens that remained elevated for up to 9 weeks. The use of heat (i.e., boiling-water bath, autoclaving) in the vaccine preparation showed that it was thermostable. This method also ensured safety and security. The vaccine produced was immunogenic and highly protective against multiple strains of Brucella and represents a promising candidate for further evaluation. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  13. C-terminal interactors of the AMPA receptor auxiliary subunit Shisa9.

    NARCIS (Netherlands)

    Karataeva, A.R.; Klaassen, R.V.; Ruiperez-Alonso, M.; Hjorth, J.; van Nierop, P.; Spijker, S.; Mansvelder, H.D.; Smit, A.B.

    2014-01-01

    Shisa9 (initially named CKAMP44) has been identified as auxiliary subunit of the AMPA-type glutamate receptors and was shown to modulate its physiological properties. Shisa9 is a type-I transmembrane protein and contains a C-terminal PDZ domain that potentially interacts with cytosolic proteins. In

  14. Oxidative stress in patients with type 1 diabetes mellitus: is it affected by a single bout of prolonged exercise?

    Science.gov (United States)

    Francescato, Maria Pia; Stel, Giuliana; Geat, Mario; Cauci, Sabina

    2014-01-01

    Presently, no clear-cut guidelines are available to suggest the more appropriate physical activity for patients with type 1 diabetes mellitus due to paucity of experimental data obtained under patients' usual life conditions. Accordingly, we explored the oxidative stress levels associated with a prolonged moderate intensity, but fatiguing, exercise performed under usual therapy in patients with type 1 diabetes mellitus and matched healthy controls. Eight patients (4 men, 4 women; 49±11 years; Body Mass Index 25.0±3.2 kg·m(-2); HbA1c 57±10 mmol·mol(-1)) and 14 controls (8 men, 6 women; 47±11 years; Body Mass Index 24.3±3.3 kg·m(-2)) performed a 3-h walk at 30% of their heart rate reserve. Venous blood samples were obtained before and at the end of the exercise for clinical chemistry analysis and antioxidant capacity. Capillary blood samples were taken at the start and thereafter every 30 min to determine lipid peroxidation. Patients showed higher oxidative stress values as compared to controls (95.9±9.7 vs. 74.1±12.2 mg·L(-1) H2O2; pexercise (p = NS), while oxidative defence increased significantly at the end of exercise (pdiabetes exercising under their usual life conditions (i.e. usual therapy and diet). Specifically, we found that the oxidative stress was not exacerbated due to a single bout of prolonged moderate intensity aerobic exercise, a condition simulating several outdoor leisure time physical activities. Oxidative defence increased in both patients and controls, suggesting beneficial effects of prolonged aerobic fatiguing exercise.

  15. The PorX Response Regulator of the Porphyromonas gingivalis PorXY Two-Component System Does Not Directly Regulate the Type IX Secretion Genes but Binds the PorL Subunit

    Science.gov (United States)

    Vincent, Maxence S.; Durand, Eric; Cascales, Eric

    2016-01-01

    The Type IX secretion system (T9SS) is a versatile multi-protein complex restricted to bacteria of the Bacteriodetes phylum and responsible for the secretion or cell surface exposition of diverse proteins that participate to S-layer formation, gliding motility or pathogenesis. The T9SS is poorly characterized but a number of proteins involved in the assembly of the secretion apparatus in the oral pathogen Porphyromonas gingivalis have been identified based on genome substractive analyses. Among these proteins, PorY, and PorX encode typical two-component system (TCS) sensor and CheY-like response regulator respectively. Although the porX and porY genes do not localize at the same genetic locus, it has been proposed that PorXY form a bona fide TCS. Deletion of porX in P. gingivalis causes a slight decrease of the expression of a number of other T9SS genes, including sov, porT, porP, porK, porL, porM, porN, and porY. Here, we show that PorX and the soluble cytoplasmic domain of PorY interact. Using electrophoretic mobility shift, DNA-protein co-purification and heterologous host expression assays, we demonstrate that PorX does not bind T9SS gene promoters and does not directly regulate expression of the T9SS genes. Finally, we show that PorX interacts with the cytoplasmic domain of PorL, a component of the T9SS membrane core complex and propose that the CheY-like PorX protein might be involved in the dynamics of the T9SS. PMID:27630829

  16. The PorX response regulator of the Porphyromonas gingivalis PorXY two-component system does not directly regulate the Type IX secretion genes but binds the PorL subunit.

    Directory of Open Access Journals (Sweden)

    Maxence S Vincent

    2016-08-01

    Full Text Available The Type IX secretion system (T9SS is a versatile multi-protein complex restricted to bacteria of the Bacteriodetes phylum and responsible for the secretion of surface attachment of diverse proteins that participate to S-layer formation, gliding motility or pathogenesis. The T9SS is poorly characterized but a number of proteins involved in the assembly of the secretion apparatus in the oral pathogen Porphyromonas gingivalis have been identified based on genome substractive analyses. Among these proteins, PorY and PorX encode typical two-component system (TCS sensor and CheY-like response regulator respectively. Although the porX and porY genes do not localize at the same genetic locus, it has been proposed that PorXY form a bona fide TCS. Deletion of the porX in P. gingivalis causes a slight decrease of the expression of a number of other T9SS genes, including sov, porT, porP, porK, porL, porM, porN and porY. Here, we show that PorX and the soluble cytoplasmic domain of PorY interact. Using electrophoretic mobility shift, DNA-protein co-purification and heterologous host expression assays, we showed that PorX does not bind and does not directly regulate expression of the T9SS genes. Finally, we show that PorX interacts with the cytoplasmic domain of PorL, a component of the T9SS membrane core complex and propose that the CheY-like PorX protein might be involved in the dynamics of the T9SS.

  17. Conduction band mass determinations for n-type InGaAs/InAlAs single quantum wells

    Energy Technology Data Exchange (ETDEWEB)

    Jones, E.D.; Reno, J.L. [Sandia National Labs., Albuquerque, NM (United States); Kotera, Nobuo [Kyushu Inst. of Tech., Iizuka, Fukuoka (Japan); Wang, Y. [Florida State Univ., Tallahassee, FL (United States). National High Magnetic Field Lab.

    1998-05-01

    The authors report the measurement of the conduction band mass in n-type single 27-ML-wide InGaAs/InAlAs quantum well lattice matched to InP using two methods: (1) Magnetoluminescence spectroscopy and (2) far-infrared cyclotron resonance. The magnetoluminescence method utilizes Landau level transitions between 0 and 14 T at 1.4 K. The far infrared cyclotron resonance measurements were made at 4.2 K and to fields as large up to 18 T. The 2D-carrier density N{sub 2D} = 3 {times} 10{sup 11} cm{sup {minus}2} at low temperatures. The magnetoluminescence technique yielded an effective conduction-band mass of m{sub c} = 0.062m{sub 0} while the far infrared cyclotron resonance measurements gave m{sub c} = 0.056m{sub 0}, where m{sub 0} is the free electron mass. Both measurements show no evidence for any significant conduction-band nonparabolicity.

  18. A Modified Model Reference Adaptive Control for a Single Motor of Latch Type Control Element Drive Mechanism

    International Nuclear Information System (INIS)

    Park, Bae Jeong

    2016-01-01

    A modified Model Reference Adaptive Control (MRAC) for a single motor of latch type Control Element Drive Mechanism (CEDM) is described herein. The CEDM has complicated dynamic characteristics including electrical, mechanical, and magnetic effects. The previous control system has utilized a Proportional-Integral (PI) controller, and the control performance is limited according to nonlinear dynamic characteristics and environmental conditions. The modified MRAC using system identification (ID) technique improves the control performance in the operating condition such as model parameter variation and environmental condition change. The modified MRAC using the identified reference model with feed-forward gain and 180Hz noise reduction filter presents better performance under normal and/or abnormal condition. The simplified reference model can make H/W implementation more practical on the viewpoint of less computation and good performance. Actually, the CEDM controller shall be capable of controlling 101 control element assemblies (CEAs) individually in the nuclear power plant. Because the load conditions and the environmental condition around the 101 CEAs are all different minutely, the proposed modified MRAC can be a good practice. The modified MRAC controller will be applied in the real nuclear power plant later and this will overcome some weak point of PI controller

  19. Factors affecting the performance of a single-chamber microbial fuel cell-type biological oxygen demand sensor.

    Science.gov (United States)

    Yang, Gai-Xiu; Sun, Yong-Ming; Kong, Xiao-Ying; Zhen, Feng; Li, Ying; Li, Lian-Hua; Lei, Ting-Zhou; Yuan, Zhen-Hong; Chen, Guan-Yi

    2013-01-01

    Microbial fuel cells (MFCs) are devices that exploit microorganisms as biocatalysts to degrade organic matter or sludge present in wastewater (WW), and thereby generate electricity. We developed a simple, low-cost single-chamber microbial fuel cell (SCMFC)-type biochemical oxygen demand (BOD) sensor using carbon felt (anode) and activated sludge, and demonstrated its feasibility in the construction of a real-time BOD measurement system. Further, the effects of anodic pH and organic concentration on SCMFC performance were examined, and the correlation between BOD concentration and its response time was analyzed. Our results demonstrated that the SCMFC exhibited a stable voltage after 132 min following the addition of synthetic WW (BOD concentration: 200 mg/L). Notably, the response signal increased with an increase in BOD concentration (range: 5-200 mg/L) and was found to be directly proportional to the substrate concentration. However, at higher BOD concentrations (>120 mg/L) the response signal remained unaltered. Furthermore, we optimized the SCMFC using synthetic WW, and tested it with real WW. Upon feeding real WW, the BOD values exhibited a standard deviation from 2.08 to 8.3% when compared to the standard BOD5 method, thus demonstrating the practical applicability of the developed system to real treatment effluents.

  20. Oxidative stress in patients with type 1 diabetes mellitus: is it affected by a single bout of prolonged exercise?

    Directory of Open Access Journals (Sweden)

    Maria Pia Francescato

    Full Text Available Presently, no clear-cut guidelines are available to suggest the more appropriate physical activity for patients with type 1 diabetes mellitus due to paucity of experimental data obtained under patients' usual life conditions. Accordingly, we explored the oxidative stress levels associated with a prolonged moderate intensity, but fatiguing, exercise performed under usual therapy in patients with type 1 diabetes mellitus and matched healthy controls. Eight patients (4 men, 4 women; 49±11 years; Body Mass Index 25.0±3.2 kg·m(-2; HbA1c 57±10 mmol·mol(-1 and 14 controls (8 men, 6 women; 47±11 years; Body Mass Index 24.3±3.3 kg·m(-2 performed a 3-h walk at 30% of their heart rate reserve. Venous blood samples were obtained before and at the end of the exercise for clinical chemistry analysis and antioxidant capacity. Capillary blood samples were taken at the start and thereafter every 30 min to determine lipid peroxidation. Patients showed higher oxidative stress values as compared to controls (95.9±9.7 vs. 74.1±12.2 mg·L(-1 H2O2; p<0.001. In both groups, oxidative stress remained constant throughout the exercise (p = NS, while oxidative defence increased significantly at the end of exercise (p<0.02 from 1.16±0.13 to 1.19±0.10 mmol·L(-1 Trolox in patients and from 1.09±0.21 to 1.22±0.14 mmol·L(-1 Trolox in controls, without any significant difference between the two groups. Oxidative stress was positively correlated to HbA1c (p<0.005 and negatively related with uric acid (p<0.005. In conclusion, we were the first to evaluate the oxidative stress in patients with type 1 diabetes exercising under their usual life conditions (i.e. usual therapy and diet. Specifically, we found that the oxidative stress was not exacerbated due to a single bout of prolonged moderate intensity aerobic exercise, a condition simulating several outdoor leisure time physical activities. Oxidative defence increased in both patients and controls, suggesting

  1. A single nucleotide polymorphism within the acetyl-coenzyme A carboxylase beta gene is associated with proteinuria in patients with type 2 diabetes

    DEFF Research Database (Denmark)

    Maeda, Shiro; Kobayashi, Masa-aki; Araki, Shin-ichi

    2010-01-01

    It has been suggested that genetic susceptibility plays an important role in the pathogenesis of diabetic nephropathy. A large-scale genotyping analysis of gene-based single nucleotide polymorphisms (SNPs) in Japanese patients with type 2 diabetes identified the gene encoding acetyl-coenzyme A ca......It has been suggested that genetic susceptibility plays an important role in the pathogenesis of diabetic nephropathy. A large-scale genotyping analysis of gene-based single nucleotide polymorphisms (SNPs) in Japanese patients with type 2 diabetes identified the gene encoding acetyl...... among patients with type 2 diabetes and proteinuria. A meta-analysis revealed that rs2268388 was significantly associated with proteinuria in Japanese patients with type 2 diabetes (p = 5.35 x 10(-8), odds ratio = 1.61, 95% Cl: 1.35-1.91). Rs2268388 was also associated with type 2 diabetes...

  2. Contact stresses modeling at the Panda-type fiber single-layer winding and evaluation of their impact on the fiber optic properties

    Science.gov (United States)

    Lesnikova, Yu I.; Smetannikov, O. Yu; Trufanov, A. N.; Trufanov, N. A.

    2017-02-01

    The impact of contact transverse forces on the birefringence of the single-mode polarization-maintaining Panda-type fiber is numerically modeled. It has been established that with a single-row power winding on a cylindrical mandrel, the fiber tension at winding is the principal factor that influences birefringence. When coiling the fiber based on the local defect microbending, the birefringence at the microbending point differs from that of the free fiber by 1.3%.

  3. Association of single nucleotide polymorphisms in TCF2 with type 2 diabetes susceptibility in a Han Chinese population.

    Directory of Open Access Journals (Sweden)

    Xuelong Zhang

    Full Text Available Hepatocyte nuclear factor 1β (HNF1β, a transcription factor encoded by the transcription factor 2 gene (TCF2, plays a critical role in pancreatic cell formation and glucose homeostasis. It has been suggested that single nucleotide polymorphisms (SNPs of TCF2 are associated with susceptibility to type 2 diabetes (T2D. However, published results are inconsistent and inclusive. To further investigate the role of these common variants, we examined the association of TCF2 polymorphisms with the risk of T2D in a Han population in northeastern China. We genotyped five SNPs in 624 T2D patients and 630 healthy controls by using a SNaPshot method, and evaluated the T2D risk conferred by individual SNPs and haplotypes. In the single-locus analysis, we found that rs752010, rs4430796 and rs7501939 showed allelic differences between T2D patients and healthy controls, with an OR of 1.26 (95% CI 1.08-1.51, P = 0.003, an OR of 1.23 (95% CI 1.06-1.55, P = 0.001 and an OR of 1.28 (95% CI 1.10-1.61, P = 0.001, respectively. Genotype association analysis of each locus also revealed that the homozygous carriers of the at-risk allele had a significant increased T2D risk compared to homozygous carriers of the other allele (OR 1.78, 95% CI 1.20-2.64 for rs752010; OR 1.82, 95% CI 1.24-2.67 for rs4430796; OR 1.95, 95% CI 1.31-2.90 for rs7501939, even after Bonferroni correction for multiple comparisons. Besides, the haplotype-based analysis demonstrated that AGT in block rs752010-rs4430796-rs7501939 was associated with about 30% increase in T2D risk (OR 1.31, 95% CI 1.09-1.57, P = 0.01. Our findings suggested that TCF2 variants may be involved in T2D risk in a Han population of northeastern China. Larger studies with ethnically diverse populations are warranted to confirm the results reported in this investigation.

  4. Sequence analysis of dolphin ferritin H and L subunits and possible iron-dependent translational control of dolphin ferritin gene

    Directory of Open Access Journals (Sweden)

    Sasaki Yukako

    2008-10-01

    Full Text Available Abstract Background Iron-storage protein, ferritin plays a central role in iron metabolism. Ferritin has dual function to store iron and segregate iron for protection of iron-catalyzed reactive oxygen species. Tissue ferritin is composed of two kinds of subunits (H: heavy chain or heart-type subunit; L: light chain or liver-type subunit. Ferritin gene expression is controlled at translational level in iron-dependent manner or at transcriptional level in iron-independent manner. However, sequencing analysis of marine mammalian ferritin subunits has not yet been performed fully. The purpose of this study is to reveal cDNA-derived amino acid sequences of cetacean ferritin H and L subunits, and demonstrate the possibility of expression of these subunits, especially H subunit, by iron. Methods Sequence analyses of cetacean ferritin H and L subunits were performed by direct sequencing of polymerase chain reaction (PCR fragments from cDNAs generated via reverse transcription-PCR of leukocyte total RNA prepared from blood samples of six different dolphin species (Pseudorca crassidens, Lagenorhynchus obliquidens, Grampus griseus, Globicephala macrorhynchus, Tursiops truncatus, and Delphinapterus leucas. The putative iron-responsive element sequence in the 5'-untranslated region of the six different dolphin species was revealed by direct sequencing of PCR fragments obtained using leukocyte genomic DNA. Results Dolphin H and L subunits consist of 182 and 174 amino acids, respectively, and amino acid sequence identities of ferritin subunits among these dolphins are highly conserved (H: 99–100%, (99→98 ; L: 98–100%. The conserved 28 bp IRE sequence was located -144 bp upstream from the initiation codon in the six different dolphin species. Conclusion These results indicate that six different dolphin species have conserved ferritin sequences, and suggest that these genes are iron-dependently expressed.

  5. Immunochemical aspects of crotoxim and its subunits

    International Nuclear Information System (INIS)

    Nakazone, A.K.

    1979-01-01

    Crotamine and crotoxin with the subunits - phospholipase A and crotapotin - were obtained by purification from Crotalus durissus terrificus venom. Interaction studies of the subunits using crotalic antiserum, indicated that: crotoxin is formed of crotapotin and phospholipase A with the molar ratio of 1 to 1; using crotapotin 125 I the presence of a soluble complex was shown with the same antiserum. Immunological precipitation reactions demonstrated that crotapotin is antigenic: crotapotin and phospholipase A presented similar antigenic determinants; crotoxin antiserum reacted with each one of the submits; when the subunits are mixed to form synthetic crotoxin some antigenic determinants are masked in the process of interaction. Crotamine, interacted with crotapotin 1:1, without hidden antigenic determinants crotapotin antigenic site seems to be formed by, at least, one lysine. Enzimatical activity of phospholipase A apreared to be dependent on some reaction conditions when its arginine residues are blocked. Tyrosines of phospholipase A are more susceptible to labelling with 131 I than crotapotin. Gama irradiation of aqueous solutions of the subunits produced modifications in the ultraviolet spectra. A decrease of the enzymatic activity occured as a function of radiation dosis. Immunological activities of crotapotin and phospholipase A were not altered [pt

  6. Purification and characterization of the glycoprotein hormone α-subunit-like material secreted by HeLa cells

    International Nuclear Information System (INIS)

    Cox, G.S.; Rimerman, R.A.

    1988-01-01

    The protein secreted by HeLa cells that cross-reacts with antiserum developed against the α-subunit of human chorionic gonadotropin (hCG) has been purified approximately 30,000-fold from concentrated culture medium by organic solvent fractionation followed by ion exchange, gel filtration, and lectin affinity chromatography. The final preparation had a specific activity (by RIA) of 6.8 x 10 5 ng of α/mg of protein and appeared homogeneous by electrophoresis on reducing/denaturing polyacrylamide gels (SDS-PAGE). Amino acid analysis indicated that HeLa-α had a composition very similar to that of the urinary hCG α-subunit. However, comparison of hCG-α and HeLa-α demonstrated that the tumor-associated subunit was not identical with its normal counterpart. The purified tumor protein had an apparent molecular weight greater than that of the urinary α-subunit when analyzed by SDS-PAGE, and this difference was even greater when a partially purified preparation was examined by an immunoblot technique (Western). Isoelectric focusing of the HeLa and hCG subunits demonstrated that the tumor protein had a lower pI. Immunoprecipitation and electrophoresis of α-subunit from HeLa cultures labeled with [ 3 H]fucose indicated that the tumor subunit was fucosylated, whereas analysis of hCG-α hydrosylates by HPLC confirmed previous reports that the placental subunit does not contain fucose. The results indicate that, regardless of whether or not a single α-subunit gene is being expressed in both normal and neoplastic tissues, posttranslational modifications lead to a highly altered subunit in the tumor. The differences observed may be useful in diagnosing neoplastic vs hyperplastic conditions and may lend insight into the mechanism of ectopic hormone production by tumors

  7. Molecular cloning of the α subunit of human and guinea pig leukocyte adhesion glycoprotein Mo1: Chromosomal localization and homology to the α subunits of integrins

    International Nuclear Information System (INIS)

    Arnaout, M.A.; Remold-O'Donnell, E.; Pierce, M.W.; Harris, P.; Tenen, D.G.

    1988-01-01

    The cell surface-glycoprotein Mo1 is a member of the family of leukocyte cell adhesion molecules (Leu-CAMs) that includes lymphocyte function-associated antigen 1 (LFA-1) and p150,95. Each Leu-CAM is a heterodimer with a distinct α subunit noncovalently associated with a common β subunit. The authors describe the isolation and analysis of two partial cDNA clones encoding the α subunit of the Leu-CAM Mo1 in humans and guinea pigs. A monoclonal antibody directed against an epitope in the carboxyl-terminal portion of the guinea pig α chain was used for immunoscreening a λgt11 expression library. The sequence of a 378-base-pair insert from one immunoreactive clone revealed a single continuous open reading frame encoding 126 amino acids including a 26-amino acid tryptic peptide isolated from the purified guinea pig α subunit. A cDNA clone of identical size was isolated from a human monocyte/lymphocyte cDNA library by using the guinea pig clone as a probe. The human clone also encoded a 126-amino acid peptide including the sequence of an additional tryptic peptide present in purified human Mo1α chain. Southern analysis of DNA from hamster-human hybrids localized the human Mo1α chain to chromosome 16, which has been shown to contain the gene for the α chain of lymphocyte function-associated antigen 1. These data suggest that the α subunits of Leu-CAMs evolved by gene duplication from a common ancestral gene and strengthen the hypothesis that the α subunits of these heterodimeric cell adhesion molecules on myeloid and lymphoid cells, platelets, and fibroblasts are evolutionary related

  8. LA PLANTA ÚNICA COMO TIPO RESISTENTE A LA ESCALA / the single plan as a type resistant to scale

    Directory of Open Access Journals (Sweden)

    Silvia Colmenares Vilata

    2014-05-01

    Full Text Available RESUMEN El sistema capitalista ha generado dos de los modelos espaciales de indiferencia funcional más extremos: por un lado, el espacio tecnificado y repetido del edificio de oficinas en altura, encarnado por la ‘planta tipo’ y, por otro, el espacio diáfano y extenso del gran contendor, que responde a la lógica de lo que llamaremos ‘planta única’. En los edificios construidos para la industria automovilística y bélica americana se encuentra el germen de un tipo que servirá de modelo para los nuevos espacios de consumo de bienes y servicios de la ciudad post-industrial. En ella, la identificación del rascacielos con el centro y del contenedor con la periferia sigue vigente, porque la densidad necesaria del primero y la ocupación extensiva del segundo hacen que resulte imposible invertir los términos. Sin embargo, ambos representan el desarrollo estrictamente pragmático de los principios de la ‘planta libre’ como sistema operativo genérico, dando lugar a construcciones de gran tamaño que ponen a prueba la resistencia a la escala del propio tipo. Tomando como referencia la neutralidad característica de la ‘planta única’ el texto analiza el comportamiento frente al cambio de tamaño de sus dos configuraciones complementarias: la gran sala hipóstila y la gran sala diáfana. SUMMARY The capitalist system has generated two of the most extreme spatial models of functional indifference: on the one hand, the technified and repeated space of the high-rise office building, incarnated by the “typical plan” and, on the other, the diaphanous and extensive space of the large container, which responds to the logic of what we will call “single plan”. In the buildings constructed for the American automobile and military industries is the germ of a type that would serve as a model for the new spaces for consumption of goods and services of the post-industrial city. In that city, the identification of the skyscraper with the

  9. Single-crystalline M-type Sr Hexaferrites studied by {sup 57}Fe Mössbauer spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Nagasawa, Nobumoto, E-mail: rk15v002@stkt.u-hyogo.ac.jp; Ikeda, Shugo [University of Hyogo, Graduate School of Material Science and Center for Novel Material Science under Multi-extreme Conditions (Japan); Shimoda, Aiko; Waki, Takeshi; Tabata, Yoshikazu; Nakamura, Hiroyuki [Kyoto University, Department of Material Science and Engineering (Japan); Kobayashi, Hisao [University of Hyogo, Graduate School of Material Science and Center for Novel Material Science under Multi-extreme Conditions (Japan)

    2016-12-15

    The {sup 57}Fe Mössbauer spectra of the single crystalline and the finely ground Sr{sub 1−x}La{sub x}Fe{sub 12−y}Co{sub y}O{sub 19} (x = 0 : y = 0, x = 0.192 : y = 0.152 and x = 0.456 : y = 0.225) samples have been measured to investigate the La-Co substitution effects. All observed spectra at 150 K were well fitted using the five subspectra which correspond to the five crystallographical nonequivalent Fe sites in the M-type hexaferrite, indicating that the valence changes to Fe{sup 2+} ions in the Fe{sup 3+} ions were not observed in our Sr{sub 1−x}La{sub x}Fe{sub 12−y}Co{sub y}O{sub 19} samples. In SrFe{sub 12}O{sub 19}, the relative absorption intensities in the five subspectra show the large anisotropies in the recoilless fractions at the five Fe sites whereas these anisotropies were not observed in Sr{sub 0.544}La{sub 0.456}Fe{sub 11.775}Co{sub 0.225}O{sub 19}. These results indicate the chemical compositional dependence on the anisotropies of the recoilless fractions at the five Fe sites. The substitution of a Co{sup 2+} ion for the Fe{sup 3+} ion changes the center shifts of the Fe{sup 3+} ions near the Co{sup 2+} ion by the perturbation of the Fe-O-Co hybridizations. Therefore, the Co{sup 2+} ions occupy the 4f{sub 1} and the 4f{sub 2} sites due to the chemical compositional dependences of the refined magnetic hyperfine field and center shifts of the Fe{sup 3+} ions.

  10. Single base substitution causing the fragrant phenotype and development of a type-specific marker in aromatic coconut (Cocos nucifera).

    Science.gov (United States)

    Vongvanrungruang, A; Mongkolsiriwatana, C; Boonkaew, T; Sawatdichaikul, O; Srikulnath, K; Peyachoknagul, S

    2016-09-19

    The fragrance gene, betaine aldehyde dehydrogenase 2 (Badh2), has been well studied in many plant species. The objectives of this study were to clone Badh2 and compare the sequences between aromatic and non-aromatic coconuts. The complete coding region was cloned from cDNA of both aromatic and non-aromatic coconuts. The nucleotide sequences were highly homologous to Badh2 genes of other plants. Badh2 consisted of a 1512-bp open reading frame encoding 503 amino acids. A single nucleotide difference between aromatic and non-aromatic coconuts resulted in the conversion of alanine (non-aromatic) to proline (aromatic) at position 442, which was the substrate binding site of BADH2. The ring side chain of proline could destabilize the structure leading to a non-functional enzyme. Badh2 genomic DNA was cloned from exon 1 to 4, and from exon 5 to 15 from the two coconut types, except for intron 4 that was very long. The intron sequences of the two coconut groups were highly homologous. No differences in Badh2 expression were found among the tissues of aromatic coconut or between aromatic and non-aromatic coconuts. The amino acid sequences of BADH2 from coconut and other plants were compared and the genetic relationship was analyzed using MEGA 7.0. The phylogenetic tree reconstructed by the Bayesian information criterion consisted of two distinct groups of monocots and dicots. Among the monocots, coconut (Cocos nucifera) and oil palm (Elaeis guineensis) were the most closely related species. A marker for coconut differentiation was developed from one-base substitution site and could be successfully used.

  11. Cloning, expression, purification, crystallization and X-ray crystallographic analysis of CofB, the minor pilin subunit of CFA/III from human enterotoxigenic Escherichia coli.

    Science.gov (United States)

    Kawahara, Kazuki; Oki, Hiroya; Fukakusa, Shunsuke; Maruno, Takahiro; Kobayashi, Yuji; Motooka, Daisuke; Taniguchi, Tooru; Honda, Takeshi; Iida, Tetsuya; Nakamura, Shota; Ohkubo, Tadayasu

    2015-06-01

    Colonization factor antigen III (CFA/III) is one of the virulence factors of human enterotoxigenic Escherichia coli (ETEC) that forms the long, thin, proteinaceous fibres of type IV pili through assembly of its major and minor subunits CofA and CofB, respectively. The crystal structure of CofA has recently been reported; however, the lack of structural information for CofB, the largest among the known type IV pilin subunits, hampers a comprehensive understanding of CFA/III pili. In this study, constructs of wild-type CofB with an N-terminal truncation and the corresponding SeMet derivative were cloned, expressed, purified and crystallized. The crystals belonged to the rhombohedral space group R32, with unit-cell parameters a = b = 103.97, c = 364.57 Å for the wild-type construct and a = b = 103.47, c = 362.08 Å for the SeMet-derivatized form. Although the diffraction quality of these crystals was initially very poor, dehydration of the crystals substantially improved the resolution limit from ∼ 4.0 to ∼ 2.0 Å. The initial phase was solved by the single-wavelength anomalous dispersion (SAD) method using a dehydrated SeMet CofB crystal, which resulted in an interpretable electron-density map.

  12. Riboprinting of Naegleria spp.: small-subunit versus large-subunit rDNA.

    Science.gov (United States)

    De Jonckheere, J F

    1994-01-01

    The nonpathogenic amoeba Naegleria lovaniensis is closely related to the human pathogen N. fowleri. Both grow at a maximal temperature of 45 degrees C and, therefore, are often found together in the environment. As they are morphologically inseparable at the light-microscope level, refined techniques are necessary to separate the two species. I have used restriction-fragment-length polymorphism analysis of the polymerase chain reaction (PCR)-amplified ribosomal RNA gene, or riboprinting, to distinguish between the different Naegleria spp. Riboprints generated from the small subunit and the large subunit separate N. fowleri from N. lovaniensis. To examine the taxonomic relationships among all Naegleria spp., analysis of the large subunit has to be performed; the small subunit contains a 1.3-kb group I intron, which interferes with tree building based upon restriction sites.

  13. Type II restriction modification system methylation subunit of Alicyclobacillus acidocaldarius

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Brady D.; Newby, Deborah T.; Lacey, Jeffrey A.; Thompson, David N.; Thompson, Vicki S.; Apel, William A.; Roberto, Francisco F.; Reed, David W.

    2018-02-13

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods for modulating or altering recombination inside or outside of a cell using isolated and/or purified polypeptides and/or nucleic acid sequences from Alicyclobacillus acidocaldarius.

  14. Type II restriction modification system methylation subunit of Alicyclobacillus acidocaldarius

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Brady D.; Newby, Deborah T.; Lacey, Jeffrey A.; Thompson, David N.; Thompson, Vicki S.; Apel, William A.; Roberto, Francisco F.; Reed, David W.

    2017-02-14

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods for modulating or altering recombination inside or outside of a cell using isolated and/or purified polypeptides and/or nucleic acid sequences from Alicyclobacillus acidocaldarius.

  15. Masked Visual Analysis: Minimizing Type I Error in Visually Guided Single-Case Design for Communication Disorders

    Science.gov (United States)

    Byun, Tara McAllister; Hitchcock, Elaine R.; Ferron, John

    2017-01-01

    Purpose: Single-case experimental designs are widely used to study interventions for communication disorders. Traditionally, single-case experiments follow a response-guided approach, where design decisions during the study are based on participants' observed patterns of behavior. However, this approach has been criticized for its high rate of…

  16. Effects of Calorie Restriction and Fiber Type on Glucose Uptake and Abundance of Electron Transport Chain and Oxidative Phosphorylation Proteins in Single Fibers from Old Rats.

    Science.gov (United States)

    Wang, Haiyan; Arias, Edward B; Yu, Carmen S; Verkerke, Anthony R P; Cartee, Gregory D

    2017-11-09

    Calorie restriction (CR; reducing calorie intake by ~40% below ad libitum) can increase glucose uptake by insulin-stimulated muscle. Because skeletal muscle is comprised of multiple, heterogeneous fiber types, our primary aim was to determine the effects of CR (initiated at 14 weeks old) and fiber type on insulin-stimulated glucose uptake by single fibers of diverse fiber types in 23-26-month-old rats. Isolated epitrochlearis muscles from AL and CR rats were incubated with [3H]-2-deoxyglucose ± insulin. Glucose uptake and fiber type were determined for single fibers dissected from the muscles. We also determined CR-effects on abundance of several key metabolic proteins in single fibers. CR resulted in: (a) significantly (p glucose uptake by insulin-stimulated type I, IIA, IIB, IIBX, and IIX fibers; (b) significantly (p glucose uptake in each fiber type of rat skeletal muscle in the absence of upregulation of the abundance of hexokinase II or key mitochondrial ETC and OxPhos proteins. © The Author 2017. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  17. Radiation inactivation of glutamate dehydrogenase hexamer: lack of energy transfer between subunits

    International Nuclear Information System (INIS)

    Kempner, E.S.; Miller, J.H.

    1983-01-01

    The effects of ionizing radiation on glutamate dehydrogenase and on fluorescein isothiocyanate--tagged glutamate dehydrogenase were analyzed by target theory. Enzymatic activity, fluorescence, and the survival of the 56,000-dalton monomer subunit were determined on frozen samples irradiated at -135 degrees C and on lyophilized samples irradiated at either -135 degrees C or +30 degrees C. The effects of temperature were the same for all three parameters. Enzymatic activity was lost after small doses of high-energy electrons, whereas fluorescence and monomer subunits survived much larger doses of radiation. Target analysis revealed that the functional unit size for enzymatic activity was the hexamer, confirming both the earlier radiation study and conventional biochemical analyses. Target sizes obtained from fluorescence and subunit structure measurements were close to that of the monomer. These results indicate that the primary ionization caused by electron bombardment results in damage to a single polypeptide strand and that there is no massive transfer of radiation energy to other units in the hexamer. The large target size observed for enzymatic activity appears to be a structural requirement for the simultaneous presence of six intact subunits rather than the result of the spread of energy from the initial site to adjacent chains with consequent damage to other subunits

  18. Harnessing light energy with a planar transparent hybrid of graphene/single wall carbon nanotube/n-type silicon heterojunction solar cell

    DEFF Research Database (Denmark)

    Chen, Leifeng; Yu, Hua; Zhong, Jiasong

    2015-01-01

    The photovoltaic conversion efficiency of a solar cell fabricated by a simple electrophoretic method with a planar transparent hybrid of graphenes (GPs) and single wall carbon nanotubes (SCNTs)/n-type silicon heterojunction was significantly increased compared to GPs/n-Si and SCNTs/n-Si solar cells...

  19. Erratum : Critical Properties of Spin-1 Antiferromagnetic Heisenberg Chains with Bond Alternation and Uniaxial Single-Ion-Type Anisotropy (vol 69, pg 237, 2000)

    OpenAIRE

    Chen, Wei; 飛田, 和男; Sanctuary, Bryan C.

    2008-01-01

    Original Paper :Critical Properties of Spin-1 Antiferromagnetic Heisenberg Chains with Bond Alternation and Uniaxial Single-Ion-Type AnisotropyWei Chen, Kazuo Hida and Bryan Clifford Sanctuary Journal of the Physical Society of Japan 69 (2000) pp.237-241

  20. MASH test 3-11 of the TxDOT single slope bridge rail (type SSTR) on pan-formed bridge deck

    Science.gov (United States)

    2011-03-01

    The objective of this crash test was to determine whether the TxDOT Single Slope Traffic Rail (Type : SSTR) would perform acceptably on a pan-formed deck when tested according to the guidelines set forth in : Manual for Assessing Safety Hardware (MAS...

  1. Human mediator subunit MED15 promotes transcriptional activation.

    Science.gov (United States)

    Nakatsubo, Takuya; Nishitani, Saori; Kikuchi, Yuko; Iida, Satoshi; Yamada, Kana; Tanaka, Aki; Ohkuma, Yoshiaki

    2014-10-01

    In eukaryotes, the Mediator complex is an essential transcriptional cofactor of RNA polymerase II (Pol II). In humans, it contains up to 30 subunits and consists of four modules: head, middle, tail, and CDK/Cyclin. One of the subunits, MED15, is located in the tail module, and was initially identified as Gal11 in budding yeast, where it plays an essential role in the transcriptional regulation of galactose metabolism with the potent transcriptional activator Gal4. For this reason, we investigated the function of the human MED15 subunit (hMED15) in transcriptional activation. First, we measured the effect of hMED15 knockdown on cell growth in HeLa cells. The growth rate was greatly reduced. By immunostaining, we observed the colocalization of hMED15 with the general transcription factors TFIIE and TFIIH in the nucleus. We measured the effects of siRNA-mediated knockdown of hMED15 on transcriptional activation using two different transcriptional activators, VP16 and SREBP1a. Treatment with siRNAs reduced transcriptional activation, and this reduction could be rescued by overexpression of HA/Flag-tagged, wild-type hMED15. To investigate hMED15 localization, we treated human MCF-7 cells with the MDM2 inhibitor Nutlin-3, thus inducing p21 transcription. We found that hMED15 localized to both the p53 binding site and the p21 promoter region, along with TFIIE and TFIIH. These results indicate that hMED15 promotes transcriptional activation.

  2. Molecular and functional identification of cyclic AMP-sensitive BKCa potassium channels (ZERO variant) and L-type voltage-dependent calcium channels in single rat juxtaglomerular cells

    DEFF Research Database (Denmark)

    Friis, Ulla G; Jørgensen, Finn; Andreasen, Ditte

    2003-01-01

    This study aimed at identifying the type and functional significance of potassium channels and voltage-dependent calcium channels (Ca(v)) in single rat JG cells using whole-cell patch clamp. Single JG cells displayed outward rectification at positive membrane potentials and limited net currents......, respectively. Double immunofluorescence confirmed the presence of BKCa and renin in the same cell. cAMP increased the outward current by 1.6-fold, and this was inhibited by 74% with iberiotoxin. Expression of the cAMP-sensitive splice variant (ZERO) of BKCa was confirmed in single-sampled JG cells by RT...... no effect. We conclude that JG cells express functional cAMP-sensitive BKCa channels (the ZERO splice variant) and voltage-dependent L-type Ca2+ channels....

  3. Crystal structure of the bacterial luciferase/flavin complex provides insight into the function of the beta subunit.

    Science.gov (United States)

    Campbell, Zachary T; Weichsel, Andrzej; Montfort, William R; Baldwin, Thomas O

    2009-07-07

    Bacterial luciferase from Vibrio harveyi is a heterodimer composed of a catalytic alpha subunit and a homologous but noncatalytic beta subunit. Despite decades of enzymological investigation, structural evidence defining the active center has been elusive. We report here the crystal structure of V. harveyi luciferase bound to flavin mononucleotide (FMN) at 2.3 A. The isoalloxazine ring is coordinated by an unusual cis-Ala-Ala peptide bond. The reactive sulfhydryl group of Cys106 projects toward position C-4a, the site of flavin oxygenation. This structure also provides the first data specifying the conformations of a mobile loop that is crystallographically disordered in both prior crystal structures [(1995) Biochemistry 34, 6581-6586; (1996) J. Biol. Chem. 271, 21956 21968]. This loop appears to be a boundary between solvent and the active center. Within this portion of the protein, a single contact was observed between Phe272 of the alpha subunit, not seen in the previous structures, and Tyr151 of the beta subunit. Substitutions at position 151 on the beta subunit caused reductions in activity and total quantum yield. Several of these mutants were found to have decreased affinity for reduced flavin mononucleotide (FMNH(2)). These findings partially address the long-standing question of how the beta subunit stabilizes the active conformation of the alpha subunit, thereby participating in the catalytic mechanism.

  4. 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.

  5. Aerosol optical properties at Lampedusa (Central Mediterranean. 2. Determination of single scattering albedo at two wavelengths for different aerosol types

    Directory of Open Access Journals (Sweden)

    D. Meloni

    2006-01-01

    Full Text Available Aerosol optical properties were retrieved from direct and diffuse spectral irradiance measurements made by a multi-filter rotating shadowband radiometer (MFRSR at the island of Lampedusa (35.5° N, 12.6° E, in the Central Mediterranean, in the period July 2001–September 2003. In a companion paper (Pace et al., 2006 the aerosol optical depth (AOD and Ångström exponent were used together with airmass backward trajectories to identify and classify different aerosol types. The MFRSR diffuse-to-direct ratio (DDR at 415.6 nm and 868.7 nm for aerosol classified as 'biomass burning-urban/industrial', originating primarily from the European continent, and desert dust, originating from the Sahara, is used in this study to estimate the aerosol single scattering albedo (SSA. A detailed radiative transfer model is initialised with the measured aerosol optical depth; calculations are performed at the two wavelengths varying the SSA values until the modelled DDR matches the MFRSR observations. Sensitivity studies are performed to estimate how uncertainties on AOD, DDR, asymmetry factor (g, and surface albedo influence the retrieved SSA values. The results show that a 3% variation of AOD or DDR produce a change of about 0.02 in the retrieved SSA value at 415.6 and 868.7 nm; a ±0.06 variation of the asymmetry factor g produces a change of the estimated SSA of <0.04 at 415.6 nm, and <0.06 at 868.7 nm; finally, an increase of the assumed surface albedo of 0.05 causes very small changes (0.01–0.02 in the retrieved SSA. The calculations show that the SSA of desert dust (DD increases with wavelength, from 0.81±0.05 at 415.6 nm to 0.94±0.05 at 868.7 nm; on the contrary, the SSA of urban/industrial (UN aerosols decreases from 0.96±0.02 at 415.6 nm to 0.87±0.07 at 868.7 nm; the SSA of biomass burning (BB particles is 0.82±0.04 at 415.6 nm and 0.80±0.05 at 868.7 nm. Episodes of UN aerosols occur usually in June and July; long lasting BB aerosol episodes

  6. allelic variation of hmw glutenin subunits of ethiopian bread wheat ...

    African Journals Online (AJOL)

    journal

    reduced subunits of glutenin proteins bands are separated: the high molecular weight (HMW) and low molecular weight (LMW) subunits (Payne et al.,1980; Jackson et al., 1983). The HMW glutenin subunits (GS) of wheat protein are quantitatively minor, but functionally an important group of gluten proteins in the process of ...

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

    Directory of Open Access Journals (Sweden)

    Irena Roterman

    2009-03-01

    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.

  8. Escherichia coli F1Fo-ATP synthase with a b/δ fusion protein allows analysis of the function of the individual b subunits.

    Science.gov (United States)

    Gajadeera, Chathurada S; Weber, Joachim

    2013-09-13

    The "stator stalk" of F1Fo-ATP synthase is essential for rotational catalysis as it connects the nonrotating portions of the enzyme. In Escherichia coli, the stator stalk consists of two (identical) b subunits and the δ subunit. In mycobacteria, one of the b subunits and the δ subunit are replaced by a b/δ fusion protein; the remaining b subunit is of the shorter b' type. In the present study, it is shown that it is possible to generate a functional E. coli ATP synthase containing a b/δ fusion protein. This construct allowed the analysis of the roles of the individual b subunits. The full-length b subunit (which in this case is covalently linked to δ in the fusion protein) is responsible for connecting the stalk to the catalytic F1 subcomplex. It is not required for interaction with the membrane-embedded Fo subcomplex, as its transmembrane helix can be removed. Attachment to Fo is the function of the other b subunit which in turn has only a minor (if any at all) role in binding to δ. Also in E. coli the second b subunit can be shortened to a b' type.

  9. Putative nicotinic acetylcholine receptor subunits express differentially through the life cycle of codling moth, Cydia pomonella (Lepidoptera: Tortricidae).

    Science.gov (United States)

    Martin, Jessica A; Garczynski, Stephen F

    2016-04-01

    Nicotinic acetylcholine receptors (nAChRs) are the targets of neonicotinoids and spinosads, two insecticides used in orchards to effectively control codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae). Orchardists in Washington State are concerned about the possibility of codling moth field populations developing resistance to these two insecticides. In an effort to help mitigate this issue, we initiated a project to identify and characterize codling moth nAChR subunits expressed in heads. This study had two main goals; (i) identify transcripts from a codling moth head transcriptome that encode for nAChR subunits, and (ii) determine nAChR subunit expression profiles in various life stages of codling moth. From a codling moth head transcriptome, 24 transcripts encoding for 12 putative nAChR subunit classes were identified and verified by PCR amplification, cloning, and sequence determination. Characterization of the deduced protein sequences encoded by putative nAChR transcripts revealed that they share the distinguishing features of the cys-loop ligand-gated ion channel superfamily with 9 α-type subunits and 3 β-type subunits identified. Phylogenetic analysis comparing these protein sequences to those of other insect nAChR subunits supports the identification of these proteins as nAChR subunits. Stage expression studies determined that there is clear differential expression of many of these subunits throughout the codling moth life cycle. The information from this study will be used in the future to monitor for potential target-site resistance mechanisms to neonicotinoids and spinosads in tolerant codling moth populations. © 2014 Institute of Zoology, Chinese Academy of Sciences.

  10. DNA sequences, recombinant DNA molecules and processes for producing the A and B subunits of cholera toxin and preparations containing so-obtained subunit or subunits

    Energy Technology Data Exchange (ETDEWEB)

    Harford, N.; De Wilde, M.

    1987-05-19

    A recombinant DNA molecule is described comprising at least a portion coding for subunits A and B of cholera toxin, or a fragment or derivative of the portion wherein the fragment or derivative codes for a polypeptide have an activity which can induce an immune response to subunit A; can induce an immune response to subunit A and cause epithelial cell penetration and the enzymatic effect leading to net loss of fluid into the gut lumen; can bind to the membrane receptor for the B subunit of cholera toxin; can induce an immune response to subunit B; can induce an immune response to subunit B and bind to the membrane receptor; or has a combination of the activities.

  11. Reconstitution of normal and hyperactivated forms of casein kinase-2 by variably mutated beta-subunits

    DEFF Research Database (Denmark)

    Boldyreff, B; Meggio, F; Pinna, L A

    1993-01-01

    -terminal part (mutants beta delta 209-215, beta delta 194-215, beta delta 181-215, beta delta 171-215, beta delta 150-215) or have undergone Ala substitutions for the acidic and basic residues which are concentrated in the sequences 55-70 and 171-180, respectively. All these mutants have been examined...... for their ability to functionally replace the wild type beta-subunit. All substitutions and the deletions delta 1-4, delta 194-215, and delta 209-215 are compatible with effective binding of the catalytic alpha-subunit, as judged by sucrose density gradient analysis, stimulation of catalytic activity...

  12. The TFIIH subunit Tfb3 regulates cullin neddylation

    Science.gov (United States)

    Rabut, Gwenaël; Le Dez, Gaëlle; Verma, Rati; Makhnevych, Taras; Knebel, Axel; Kurz, Thimo; Boone, Charles; Deshaies, Raymond J.; Peter, Matthias

    2011-01-01

    Summary Cullin proteins are scaffolds for the assembly of multi-subunit ubiquitin ligases, which ubiquitylate a large number of proteins involved in widely-varying cellular functions. Multiple mechanisms cooperate to regulate cullin activity, including neddylation of their C-terminal domain. Interestingly, we found that the yeast Cul4-type cullin Rtt101 is not only neddylated but also ubiquitylated, and both modifications promote Rtt101 function in vivo. Surprisingly, proper modification of Rtt101 neither correlated with catalytic activity of the RING-domain of Hrt1 nor did it require the Nedd8 ligase Dcn1. Instead, ubiquitylation of Rtt101 was dependent on the ubiquitin-conjugating enzyme Ubc4, while efficient neddylation involves the RING-domain protein Tfb3, a subunit of the transcription factor TFIIH. Tfb3 also controls Cul3 neddylation and activity in vivo, and physically interacts with Ubc4 and the Nedd8-conjugating enzyme Ubc12 as well as the Hrt1/Rtt101 complex. Together, these results suggest that the conserved RING-domain protein Tfb3 controls activation of a subset of cullins. PMID:21816351

  13. The calcium channel β2 (CACNB2 subunit repertoire in teleosts

    Directory of Open Access Journals (Sweden)

    Mueller Rachel

    2008-04-01

    Full Text Available Abstract Background Cardiomyocyte contraction is initiated by influx of extracellular calcium through voltage-gated calcium channels. These oligomeric channels utilize auxiliary β subunits to chaperone the pore-forming α subunit to the plasma membrane, and to modulate channel electrophysiology 1. Several β subunit family members are detected by RT-PCR in the embryonic heart. Null mutations in mouse β2, but not in the other three β family members, are embryonic lethal at E10.5 due to defects in cardiac contractility 2. However, a drawback of the mouse model is that embryonic heart rhythm is difficult to study in live embryos due to their intra-uterine development. Moreover, phenotypes may be obscured by secondary effects of hypoxia. As a first step towards developing a model for contributions of β subunits to the onset of embryonic heart rhythm, we characterized the structure and expression of β2 subunits in zebrafish and other teleosts. Results Cloning of two zebrafish β2 subunit genes (β2.1 and β2.2 indicated they are membrane-associated guanylate kinase (MAGUK-family genes. Zebrafish β2 genes show high conservation with mammals within the SH3 and guanylate kinase domains that comprise the "core" of MAGUK proteins, but β2.2 is much more divergent in sequence than β2.1. Alternative splicing occurs at the N-terminus and within the internal HOOK domain. In both β2 genes, alternative short ATG-containing first exons are separated by some of the largest introns in the genome, suggesting that individual transcript variants could be subject to independent cis-regulatory control. In the Tetraodon nigrovidis and Fugu rubripes genomes, we identified single β2 subunit gene loci. Comparative analysis of the teleost and human β2 loci indicates that the short 5' exon sequences are highly conserved. A subset of 5' exons appear to be unique to teleost genomes, while others are shared with mammals. Alternative splicing is temporally and

  14. Goniometer-based femtosecond X-ray diffraction of mutant 30S ribosomal subunit crystals

    Directory of Open Access Journals (Sweden)

    E. Han Dao

    2015-07-01

    Full Text Available In this work, we collected radiation-damage-free data from a set of cryo-cooled crystals for a novel 30S ribosomal subunit mutant using goniometer-based femtosecond crystallography. Crystal quality assessment for these samples was conducted at the X-ray Pump Probe end-station of the Linac Coherent Light Source (LCLS using recently introduced goniometer-based instrumentation. These 30S subunit crystals were genetically engineered to omit a 26-residue protein, Thx, which is present in the wild-type Thermus thermophilus 30S ribosomal subunit. We are primarily interested in elucidating the contribution of this ribosomal protein to the overall 30S subunit structure. To assess the viability of this study, femtosecond X-ray diffraction patterns from these crystals were recorded at the LCLS during a protein crystal screening beam time. During our data collection, we successfully observed diffraction from these difficult-to-grow 30S ribosomal subunit crystals. Most of our crystals were found to diffract to low resolution, while one crystal diffracted to 3.2 Å resolution. These data suggest the feasibility of pursuing high-resolution data collection as well as the need to improve sample preparation and handling in order to collect a complete radiation-damage-free data set using an X-ray Free Electron Laser.

  15. Goniometer-based femtosecond X-ray diffraction of mutant 30S ribosomal subunit crystals.

    Science.gov (United States)

    Dao, E Han; Sierra, Raymond G; Laksmono, Hartawan; Lemke, Henrik T; Alonso-Mori, Roberto; Coey, Aaron; Larsen, Kevin; Baxter, Elizabeth L; Cohen, Aina E; Soltis, S Michael; DeMirci, Hasan

    2015-07-01

    In this work, we collected radiation-damage-free data from a set of cryo-cooled crystals for a novel 30S ribosomal subunit mutant using goniometer-based femtosecond crystallography. Crystal quality assessment for these samples was conducted at the X-ray Pump Probe end-station of the Linac Coherent Light Source (LCLS) using recently introduced goniometer-based instrumentation. These 30S subunit crystals were genetically engineered to omit a 26-residue protein, Thx, which is present in the wild-type Thermus thermophilus 30S ribosomal subunit. We are primarily interested in elucidating the contribution of this ribosomal protein to the overall 30S subunit structure. To assess the viability of this study, femtosecond X-ray diffraction patterns from these crystals were recorded at the LCLS during a protein crystal screening beam time. During our data collection, we successfully observed diffraction from these difficult-to-grow 30S ribosomal subunit crystals. Most of our crystals were found to diffract to low resolution, while one crystal diffracted to 3.2 Å resolution. These data suggest the feasibility of pursuing high-resolution data collection as well as the need to improve sample preparation and handling in order to collect a complete radiation-damage-free data set using an X-ray Free Electron Laser.

  16. Single-mode amplification in Yb-doped rod-type photonic crystal fibers for high brilliance lasers

    DEFF Research Database (Denmark)

    Poli, F.; Lægsgaard, Jesper; Passaro, D.

    2009-01-01

    to identify a proper ring characteristic that is width, position and refractive index. Then rod-type PCF designs have been optimized with a full-vector modal solver based on the finite-element method. Then, the amplification properties of the Yb-doped rod-type PCFs have been investigated by assuming a forward...

  17. 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

    2016-01-01

    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...

  18. Fc receptor gamma subunit polymorphisms and systemic lupus erythematosus

    International Nuclear Information System (INIS)

    Al-Ansari, Aliya; Ollier, W.E.; Gonzalez-Gay, Miguel A.; Gul, Ahmet; Inanac, Murat; Ordi, Jose; Teh, Lee-Suan; Hajeer, Ali H.

    2004-01-01

    To investigate the possible association between Fc receptor gamma polymorphisms and systemic lupus erythematosus (SLE). We have investigated the full FcR gamma gene for polymorphisms using polymerase chain reaction (PCR)-single strand confirmational polymorphisms and DNA sequencing .The polymorphisms identified were genotype using PCR-restriction fragment length polymorphism. Systemic lupus erythematosus cases and controls were available from 3 ethnic groups: Turkish, Spanish and Caucasian. The study was conducted in the year 2001 at the Arthritis Research Campaign, Epidemiology Unit, Manchester University Medical School, Manchester, United Kingdom. Five single nucleotide polymorphisms were identified, 2 in the promoter, one in intron 4 and, 2 in the 3'UTR. Four of the 5 single nucleotide polymorphisms (SNPs) were relatively common and investigated in the 3 populations. Allele and genotype frequencies of all 4 investigated SNPs were not statistically different cases and controls. fc receptor gamma gene does not appear to contribute to SLE susceptibility. The identified polymorphisms may be useful in investigating other diseases where receptors containing the FcR gamma subunit contribute to the pathology. (author)

  19. Linear viral load increase of a single HPV-type in women with multiple HPV infections predicts progression to cervical cancer.

    Science.gov (United States)

    Depuydt, Christophe E; Thys, Sofie; Beert, Johan; Jonckheere, Jef; Salembier, Geert; Bogers, Johannes J

    2016-11-01

    Persistent high-risk human papillomavirus (HPV) infection is strongly associated with development of high-grade cervical intraepithelial neoplasia or cancer (CIN3+). In single type infections, serial type-specific viral-load measurements predict the natural history of the infection. In infections with multiple HPV-types, the individual type-specific viral-load profile could distinguish progressing HPV-infections from regressing infections. A case-cohort natural history study was established using samples from untreated women with multiple HPV-infections who developed CIN3+ (n = 57) or cleared infections (n = 88). Enriched cell pellet from liquid based cytology samples were subjected to a clinically validated real-time qPCR-assay (18 HPV-types). Using serial type-specific viral-load measurements (≥3) we calculated HPV-specific slopes and coefficient of determination (R(2) ) by linear regression. For each woman slopes and R(2) were used to calculate which HPV-induced processes were ongoing (progression, regression, serial transient, transient). In transient infections with multiple HPV-types, each single HPV-type generated similar increasing (0.27copies/cell/day) and decreasing (-0.27copies/cell/day) viral-load slopes. In CIN3+, at least one of the HPV-types had a clonal progressive course (R(2)  ≥ 0.85; 0.0025copies/cell/day). In selected CIN3+ cases (n = 6), immunostaining detecting type-specific HPV 16, 31, 33, 58 and 67 RNA showed an even staining in clonal populations (CIN3+), whereas in transient virion-producing infections the RNA-staining was less in the basal layer compared to the upper layer where cells were ready to desquamate and release newly-formed virions. RNA-hybridization patterns matched the calculated ongoing processes measured by R(2) and slope in serial type-specific viral-load measurements preceding the biopsy. In women with multiple HPV-types, serial type-specific viral-load measurements predict the natural history of the

  20. Diversity in genomic organisation, developmental regulation and distribution of the murine PR72/B" subunits of protein phosphatase 2A

    Directory of Open Access Journals (Sweden)

    Janssens Veerle

    2008-08-01

    Full Text Available Abstract Background Protein phosphatase 2A (PP2A is a serine/threonine-specific phosphatase displaying vital functions in growth and development through its role in various signalling pathways. PP2A holoenzymes comprise a core dimer composed of a catalytic C and a structural A subunit, which can associate with a variable B-type subunit. The importance of the B-type subunits for PP2A regulation cannot be overestimated as they determine holoenzyme localisation, activity and substrate specificity. Three B-type subunit families have been identified: PR55/B, PR61/B' and PR72/B", of which the latter is currently the least characterised. Results We deduced the sequences and genomic organisation of the different murine PR72/B" isoforms: three genes encode nine isoforms, five of which are abundantly expressed and give rise to genuine PP2A subunits. Thereby, one novel subunit was identified. Using Northern blotting, we examined the tissue-specific and developmental expression of these subunits. All subunits are highly expressed in heart, suggesting an important cardiac function. Immunohistochemical analysis revealed a striated expression pattern of PR72 and PR130 in heart and skeletal muscle, but not in bladder smooth muscle. The subcellular localisation and cell cycle regulatory ability of several PR72/B" isoforms were determined, demonstrating differences as well as similarities. Conclusion In contrast to PR55/B and PR61/B', the PR72/B" family seems evolutionary more divergent, as only two of the murine genes have a human orthologue. We have integrated these results in a more consistent nomenclature of both human and murine PR72/B" genes and their transcripts/proteins. Our results provide a platform for the future generation of PR72/B" knockout mice.

  1. Kinetic mechanism of luciferase subunit folding and assembly.

    Science.gov (United States)

    Clark, A C; Raso, S W; Sinclair, J F; Ziegler, M M; Chaffotte, A F; Baldwin, T O

    1997-02-18

    The kinetic mechanism in vitro of the folding and assembly of the heterodimeric flavin monooxygenase bacterial luciferase has been defined by a unique set of rate constants which describe both the productive refolding pathway and competing off-pathway reactions in 50 mM phosphate, pH 7.0 at 18 degrees C. The individual alpha and beta subunits fold independently to form heterodimerization-competent species, alpha i and beta i. The alpha i beta i species can interact to form an inactive heterodimeric intermediate, [alpha beta ]i, which isomerizes to form the active alpha beta structure; the structure of the enzyme has been determined to 1.5 A resolution [Fisher, A. J., Thompson, T. B., Thoden, J. B., Baldwin, T. O., & Rayment, I. (1996) J. Biol. Chem. 271, 21956-21968]. In the absence of alpha i, beta i can form a kinetically trapped homodimer, beta 2, with a second-order rate constant of about 180 M-1 s-1 [Sinclair, J. F., Ziegler, M. M., & Baldwin, T. O. (1994) Nat. Struct. Biol. 1, 320-326]; the structure of beta 2 has recently been reported [Thoden. J. B., Holden, H. M., Fisher, A. J., Sinclair. J. F., Wesenberg, G., Baldwin, T.O., & Rayment, I. (1997) Protein Sci. 6, 13-23]. The beta i species, or some other form that precedes beta i on the refolding pathway, can also undergo a first-order conversion into a form (designated beta x) that cannot associate with alpha i to form the native enzyme. The rate constant for this process, assigned here, accounts well for the previously observed dependence of final yield on concentration of refolding species [Ziegler, M.M., Goldberg, M.E., Chaffotte, A. F., & Baldwin, T. O. (1993) J. Biol. Chem. 268, 10760-10765]. In simulations of the refolding reaction, all processes associated with the refolding of the individual subunits were combined into single first-order rate constants for each subunit which were consistent with the rate constants determined from stopped-flow circular dichroism studies. The first-order rate constant

  2. Synthesis of a wild-type and three mutant Cucurbita maxima trypsin inhibitor-encoding genes by a single-strand approach.

    Science.gov (United States)

    Botes, D P; Qobose, M D; Corfield, V A

    1991-09-15

    A single-strand approach to gene assembly, based on a modification of an in vitro complementary oligodeoxyribonucleotide template-directed ligation of the desired sequence to a linearized vector [Chen et al., Nucleic Acids Res. 18 (1990) 871-878], is described. The gene coding for the wild-type Cucurbita maxima trypsin inhibitor of 29 amino acid residues [Bode et al., FEBS Lett. 242 (1989) 285-292], as well as three mutant forms of the gene, in which two of the three disulfide bonds have been replaced singly or as a pair, have been synthesized in a single synthesis run with minimal manual intervention. Subsequent to ligation to pUC9 and in vivo gapped duplex repair by Escherichia coli, their sequences have been verified.

  3. Comparison of the effect of multiple short-duration with single long-duration exercise sessions on glucose homeostasis in type 2 diabetes mellitus

    DEFF Research Database (Denmark)

    Eriksen, L; Dahl-Petersen, I; Haugaard, Steen B

    2007-01-01

    AIMS/HYPOTHESIS: We evaluated and compared the effects on glycaemic control of two different exercise protocols in elderly men with type 2 diabetes mellitus. METHODS: Eighteen patients with type 2 diabetes mellitus carried out home-based bicycle training for 5 weeks. Patients were randomly assigned...... glucose OGTT (p = 0.04) and plasma glucose concentration areas under the curve at 120 min (p exercise groups...... increased similarly in both exercise groups. A possible explanation is that the energy expenditure associated with multiple short daily sessions may be greater than that in a single daily session. Udgivelsesdato: 2007-Nov...

  4. Types of work-related behavior and experiences and stress coping strategies among single mothers and mothers in relationships differentiating role of work satisfaction.

    Science.gov (United States)

    Napora, Elżbieta; Andruszkiewicz, Anna; Basińska, Małgorzata Anna

    2018-01-01

    The purpose of the study has been to describe functioning of single and mothers in relationships (married or in informal relationships) at work and verify if the declared degree of work satisfaction differentiates types of behavior at work and stress coping strategies in both groups of mothers. The study was conducted on equal samples of single mothers (N = 186) and mothers from 2-parent families (N = 186) using Latack Coping Scale that measures work-related stress coping strategies, the AVEM (Arbeitsbezogenes Verhaltens- und Erlebensmuster - Work-Related Behavior and Experience Pattern) questionnaire, and a survey. It showed similarity between the studied groups in terms of the measured variables. There were considerable differences between single and married mothers in terms of support seeking strategies. The interaction of work satisfaction and the type of motherhood significantly differentiates (p = 0.03) the avoidance strategy of resignation. That strategy of resignation was more frequently used by single mothers with lower work satisfaction, who were distinctly different from those whose work satisfaction was higher, and from the mothers in relationships (married or in informal relationships) (regardless of the level of their work satisfaction). Int J Occup Med Environ Health 2018;31(1):55-69. This work is available in Open Access model and licensed under a CC BY-NC 3.0 PL license.

  5. P-type single-crystalline ZnO films obtained by (Na,N) dual implantation through dynamic annealing process

    Science.gov (United States)

    Zhang, Zhiyuan; Huang, Jingyun; Chen, Shanshan; Pan, Xinhua; Chen, Lingxiang; Ye, Zhizhen

    2018-02-01

    Single-crystalline ZnO films were grown by plasma-assisted molecular beam epitaxy technique on c-plane sapphire substrates. The films have been implanted with fixed fluence of 130 keV Na and 90 keV N ions at 460 °C. It is observed that dually-implanted single crystalline ZnO films exhibit p-type characteristics with hole concentration in the range of 1.24 × 1016-1.34 × 1017 cm-3, hole mobilities between 0.65 and 8.37 cm2 V-1 s-1, and resistivities in the range of 53.3-80.7 Ω cm by Hall-effect measurements. There are no other secondary phase appearing, with (0 0 2) (c-plane) orientation after ion implantation as identified by the X-ray diffraction pattern. It is obtained that Na and N ions were successfully implanted and activated as acceptors measured by XPS and SIMS results. Also compared to other similar studies, lower amount of Na and N ions make p-type characteristics excellent as others deposited by traditional techniques. It is concluded that Na and N ion implantation and dynamic annealing are essential in forming p-type single-crystalline ZnO films.

  6. Comparing the capitalisation benefits of light-rail transit and overlay zoning for single-family houses and condos by neighbourhood type in metropolitan Phoenix, Arizona.

    Science.gov (United States)

    Atkinson-Palombo, Carol

    2010-01-01

    Light rail transit (LRT) is increasingly accompanied by overlay zoning which specifies the density and type of future development to encourage landscapes conducive to transit use. Neighbourhood type (based on land use mix) is used to partition data and investigate how pre-existing land use, treatment with a park-and-ride (PAR) versus walk-and-ride (WAR) station and overlay zoning interrelate. Hedonic models estimate capitalisation effects of LRT-related accessibility and overlay zoning on single-family houses and condos in different neighbourhoods for the system in metropolitan Phoenix, Arizona. Impacts differ by housing and neighbourhood type. Amenity-dominated mixed-use neighbourhoods-predominantly WAR communities-experience premiums of 6 per cent for single-family houses and over 20 per cent for condos, the latter boosted an additional 37 per cent by overlay zoning. Residential neighbourhoods-predominantly PAR communities-experience no capitalisation benefits for single-family houses and a discount for condos. The results suggest that land use mix is an important variable to select comparable neighbourhoods.

  7. The dnaN gene codes for the beta subunit of DNA polymerase III holoenzyme of escherichia coli.

    Science.gov (United States)

    Burgers, P M; Kornberg, A; Sakakibara, Y

    1981-09-01

    An Escherichia coli mutant, dnaN59, stops DNA synthesis promptly upon a shift to a high temperature; the wild-type dnaN gene carried in a transducing phage encodes a polypeptide of about 41,000 daltons [Sakakibara, Y. & Mizukami, T. (1980) Mol. Gen. Genet. 178, 541-553; Yuasa, S. & Sakakibara, Y. (1980) Mol. Gen. Genet. 180, 267-273]. We now find that the product of dnaN gene is the beta subunit of DNA polymerase III holoenzyme, the principal DNA synthetic multipolypeptide complex in E. coli. The conclusion is based on the following observations: (i) Extracts from dnaN59 cells were defective in phage phi X174 and G4 DNA synthesis after the mutant cells had been exposed to the increased temperature. (ii) The enzymatic defect was overcome by addition of purified beta subunit but not by other subunits of DNA polymerase III holoenzyme or by other replication proteins required for phi X174 DNA synthesis. (iii) Partially purified beta subunit from the dnaN mutant, unlike that from the wild type, was inactive in reconstituting the holoenzyme when mixed with the other purified subunits. (iv) Increased dosage of the dnaN gene provided by a plasmid carrying the gene raised cellular levels of the beta subunit 5- to 6-fold.

  8. Diagnosis of Single- or Multiple-Canal Benign Paroxysmal Positional Vertigo according to the Type of Nystagmus

    Science.gov (United States)

    Balatsouras, Dimitris G.; Koukoutsis, George; Ganelis, Panayotis; Korres, George S.; Kaberos, Antonis

    2011-01-01

    Benign paroxysmal positional vertigo (BPPV) is a common peripheral vestibular disorder encountered in primary care and specialist otolaryngology and neurology clinics. It is associated with a characteristic paroxysmal positional nystagmus, which can be elicited with specific diagnostic positional maneuvers, such as the Dix-Hallpike test and the supine roll test. Current clinical research focused on diagnosing and treating various types of BPPV, according to the semicircular canal involved and according to the implicated pathogenetic mechanism. Cases of multiple-canal BPPV have been specifically investigated because until recently these were resistant to treatment with standard canalith repositioning procedures. Probably, the most significant factor in diagnosis of the type of BPPV is observation of the provoked nystagmus, during the diagnostic positional maneuvers. We describe in detail the various types of nystagmus, according to the canals involved, which are the keypoint to accurate diagnosis. PMID:21792356

  9. Subcellular compartmentation, interdependency and dynamics of the cyclic AMP-dependent PKA subunits during pathogenic differentiation in rice blast.

    Science.gov (United States)

    Selvaraj, Poonguzhali; Tham, Hong Fai; Ramanujam, Ravikrishna; Naqvi, Naweed I

    2017-08-01

    The cAMP-dependent PKA signalling plays a central role in growth, asexual development and pathogenesis in fungal pathogens. Here, we functionally characterised RPKA, the regulatory subunit of cAMP/PKA and studied the dynamics and organisation of the PKA subunits in the rice blast pathogen Magnaporthe oryzae. The RPKA subunit was essential for proper vegetative growth, asexual sporulation and surface hydrophobicity in M. oryzae. A spontaneous suppressor mutation, SMR19, that restored growth and conidiation in the RPKA deletion mutant was isolated and characterised. SMR19 enhanced conidiation and appressorium formation but failed to suppress the pathogenesis defects in rpkAΔ. The PKA activity was undetectable in the mycelial extracts of SMR19, which showed a single mutation (val242leu) in the highly conserved active site of the catalytic subunit (CPKA) of cAMP/PKA. The two subunits of cAMP/PKA showed different subcellular localisation patterns with RpkA being predominantly nucleocytoplasmic in conidia, while CpkA was largely cytosolic and/or vesicular. The CpkA anchored RpkA in cytoplasmic vesicles, and localisation of PKA in the cytoplasm was governed by CpkA in a cAMP-dependant or independent manner. We show that there exists a tight regulation of PKA subunits at the level of transcription, and the cAMP signalling is differentially compartmentalised in a stage-specific manner in rice blast. © 2017 John Wiley & Sons Ltd.

  10. Complex GABAB receptor complexes: how to generate multiple functionally distinct units from a single receptor

    Directory of Open Access Journals (Sweden)

    Chanjuan eXU

    2014-02-01

    Full Text Available The main inhibitory neurotransmitter, GABA, acts on both ligand-gated and G protein-coupled receptors, the GABAA/C and GABAB receptors, respectively. The later play important roles in modulating many synapses, both at the pre- and post-synaptic levels, and are then still considered as interesting targets to treat a number of brain diseases, including addiction. For many years, several subtypes of GABAB receptors were expected, but cloning revealed only two genes that work in concert to generate a single type of GABAB receptor composed of two subunits. Here we will show that the signaling complexity of this unit receptor type can be largely increased through various ways, including receptor stoichiometry, subunit isoforms, membrane expression and localization, crosstalk with other receptors or interacting proteins. These recent data revealed how complexity of a receptor unit can be increased, observation that certainly are not unique to the GABAB receptor.

  11. Subunit Arrangement and Function in NMDA Receptors

    Energy Technology Data Exchange (ETDEWEB)

    Furukawa,H.; Singh, S.; Mancusso, R.; Gouaux, E.

    2005-01-01

    Excitatory neurotransmission mediated by NMDA (N-methyl-D-aspartate) receptors is fundamental to the physiology of the mammalian central nervous system. These receptors are heteromeric ion channels that for activation require binding of glycine and glutamate to the NR1 and NR2 subunits, respectively. NMDA receptor function is characterized by slow channel opening and deactivation, and the resulting influx of cations initiates signal transduction cascades that are crucial to higher functions including learning and memory. Here we report crystal structures of the ligand-binding core of NR2A with glutamate and that of the NR1-NR2A heterodimer with glutamate and glycine. The NR2A-glutamate complex defines the determinants of glutamate and NMDA recognition, and the NR1-NR2A heterodimer suggests a mechanism for ligand-induced ion channel opening. Analysis of the heterodimer interface, together with biochemical and electrophysiological experiments, confirms that the NR1-NR2A heterodimer is the functional unit in tetrameric NMDA receptors and that tyrosine 535 of NR1, located in the subunit interface, modulates the rate of ion channel deactivation.

  12. Efecto citotóxico de la toxina shiga tipo 2 y su subunidad b en células epiteliales tubulares renales humanas en cultivo Cytotoxic effect of Shiga toxin type 2 and its B subunit on human renal tubular epithelial cell cultures

    Directory of Open Access Journals (Sweden)

    Virginia Pistone Creydt

    2005-04-01

    Full Text Available Escherichia coli enterohemorrágica productora de toxina Shiga (Stx causa diarrea acuosa, colitis hemorrágica y síndrome urémico hemolítico (SUH. En Argentina, el SUH es la principal causa de insuficiencia renal en niños. El objetivo de este trabajo fue estudiar la toxicidad de Stx tipo 2 (Stx2 y su subunidad B (Stx2B en células epiteliales tubulares renales humanas (CERH, en presencia y ausencia de factores inflamatorios. Los efectos citotóxicos se evaluaron como alteración de la funcionalidad del epitelio; daños histológicos; viabilidad celular; síntesis de proteínas y apoptosis celular. Los resultados muestran que Stx2 regula el pasaje de agua a través de CERH a tiempos menores de 1h de incubación. A tiempos mayores, hasta 72 hs, el estudio de la morfología, la viabilidad, la síntesis de proteínas y la apoptosis demostró que las CERH fueron sensibles a la acción citotóxica de Stx2 y Stx2B de una manera dosis y tiempo dependiente. Estos efectos fueron potenciados por lipopolisacáridos bacterianos (LPS, IL-1b, y butirato.Shiga toxin (Stx-producing E.coli causing watery diarrhea, hemorrhagic colitis and hemolytic-uremic syndrome (HUS. In Argentina, HUS is the most common cause of acute renal failure in children. The purpose of the present study was to examine the cytotoxicity of Stx type 2 (Stx2 and its B subunit (Stx2B on human renal tubular epithelial cells (HRTEC, in the presence and absence of inflammatory factors. Cytotoxic effects were assessed in terms of functionality of the epithelium, histological damage, cell viability, protein synthesis and cellular apoptosis. Results show that Stx2 regulates the passage of water through the HRTEC within an incubation period of 1h. Within longer periods, up to 72 hours, the study of morphology, viability, protein synthesis and apoptosis shows that HRTEC were sensitive to the cytotoxic action of Stx2 and Stx2B in a dose- and time-dependent manner. These effects were potentiated by

  13. Oligomeric recombinant H5 HA1 vaccine produced in bacteria protects ferrets from homologous and heterologous wild-type H5N1 influenza challenge and controls viral loads better than subunit H5N1 vaccine by eliciting high-affinity antibodies.

    Science.gov (United States)

    Verma, Swati; Dimitrova, Milena; Munjal, Ashok; Fontana, Juan; Crevar, Corey J; Carter, Donald M; Ross, Ted M; Khurana, Surender; Golding, Hana

    2012-11-01

    Recombinant hemagglutinin from influenza viruses with pandemic potential can be produced rapidly in various cell substrates. In this study, we compared the functionality and immunogenicity of bacterially produced oligomeric or monomeric HA1 proteins from H5N1 (A/Vietnam/1203/04) with those of the egg-based licensed subunit H5N1 (SU-H5N1) vaccine in ferrets challenged with homologous or heterologous H5N1 highly pathogenic influenza strains. Ferrets were vaccinated twice with the oligomeric or monomeric rHA1 or with SU-H5N1 (Sanofi Pasteur) emulsified with Titermax adjuvant and were challenged with wild-type homologous (A/Vietnam/1203/04; clade 1) or heterologous (A/Whooperswan/Mongolia/244/2005; clade 2.2) virus. Only the oligomeric rHA1 (not the monomeric rHA1) immunogen and the SU-H5N1 vaccine provided protection against the lethality and morbidity of homologous and heterologous highly pathogenic H5N1. Oligomeric rHA1 generated more cross-neutralizing antibodies and higher levels of serum antibody binding to HA1, with stronger avidity and a better IgG/IgM ratio, than monomeric HA1 and SU-H5N1 vaccines, as determined by surface plasmon resonance (SPR). Importantly, viral loads after heterologous H5N1 challenge were more efficiently controlled in ferrets vaccinated with the oligomeric rHA1 immunogen than in SU-H5N1-vaccinated ferrets. The reduction of viral loads in the nasal washes correlated strongly with higher-avidity antibodies to oligomeric rHA1 derived from H5N1 clade 1 and clade 2.2 viruses, as measured by SPR. This is the first study to show the role of antibody avidity for the HA1 globular head domain in reduction of viral loads in the upper respiratory tract, which could significantly reduce viral transmission.

  14. Catalytic Turnover Triggers Exchange of Subunits of the Magnesium Chelatase AAA+ Motor Unit*

    Science.gov (United States)

    Lundqvist, Joakim; Braumann, Ilka; Kurowska, Marzena; Müller, André H.; Hansson, Mats

    2013-01-01

    The ATP-dependent insertion of Mg2+ into protoporphyrin IX is the first committed step in the chlorophyll biosynthetic pathway. The reaction is catalyzed by magnesium chelatase, which consists of three gene products: BchI, BchD, and BchH. The BchI and BchD subunits belong to the family of AAA+ proteins (ATPases associated with various cellular activities) and form a two-ring complex with six BchI subunits in one layer and six BchD subunits in the other layer. This BchID complex is a two-layered trimer of dimers with the ATP binding site located at the interface between two neighboring BchI subunits. ATP hydrolysis by the BchID motor unit fuels the insertion of Mg2+ into the porphyrin by the BchH subunit. In the present study, we explored mutations that were originally identified in semidominant barley (Hordeum vulgare L.) mutants. The resulting recombinant BchI proteins have marginal ATPase activity and cannot contribute to magnesium chelatase activity although they apparently form structurally correct complexes with BchD. Mixing experiments with modified and wild-type BchI in various combinations showed that an exchange of BchI subunits in magnesium chelatase occurs during the catalytic cycle, which indicates that dissociation of the complex may be part of the reaction mechanism related to product release. Mixing experiments also showed that more than three functional interfaces in the BchI ring structure are required for magnesium chelatase activity. PMID:23836887

  15. A common single nucleotide polymorphism can exacerbate long-QT type 2 syndrome leading to sudden infant death

    DEFF Research Database (Denmark)

    Nof, Eyal; Cordeiro, Jonathan M; Pérez, Guillermo J

    2010-01-01

    and mutation expressed singly and in combination in Chinese ovary (CHO-K1) and COS-1 cells. An asymptomatic woman presenting after the death of her 2-day-old infant and spontaneous abortion of a second baby in the first trimester was referred for genetic analysis. The newborn infant had nearly incessant...... suggest that a common polymorphism (K897T) can markedly accentuate the loss of function of mildly defective HERG channels, leading to long-QT syndrome-mediated arrhythmias and sudden infant death....

  16. Clinical characteristics of type 1 diabetes mellitus in Taiwanese children aged younger than 6 years: A single-center experience

    Directory of Open Access Journals (Sweden)

    Yi-Chen Chen

    2017-05-01

    Conclusion: Autoimmune destruction of pancreatic β-cells is an important cause of type 1 diabetes mellitus in Taiwanese children aged younger than 6 years. These patients usually have a low insulin reserve and severe ketoacidosis upon diagnosis. A high index of suspicion in the presence of classic symptoms of diabetes in young children is important to prevent complications.

  17. Carbohydrate chains of human thyrotropin are differentially susceptible to endoglycosidase removal on combined and free polypeptide subunits

    International Nuclear Information System (INIS)

    Ronin, C.; Papandreou, M.; Canonne, C.; Weintraub, B.D.

    1987-01-01

    The accessibility of the asparagine-linked carbohydrate chains of human thyrotropin (hTSH) and free α and β subunits was investigated by their susceptibility to endoglycosidases H and F as well as to peptide:N-glycosidase F. Iodinated hTSH or subunits were incubated with a commercial enzyme preparation containing both endoglycosidase F and N-glycosidase F activities and further analyzed by sodium dodecyl sulfate gel electrophoresis followed by quantitative autoradiography. The authors show that, working at the optimum of the N-glycosidase activity, the relative amount of endoglycosidase required for half-deglycosylation was 20-fold higher for native hTSH than for the reduced and dissociated subunits. Under nondenaturing conditions, the 18K β subunit of hTSH could be readily deglycosylated to a 14K species while the 22K α subunit was largely resistant. However, both subunits were converted to an apoprotein of similar apparent molecular weight of 14K following reduction of disulfide bonds. In contrast, the free α subunit of human choriogonadotropin appeared fully sensitive to carbohydrate removal under nonreducing conditions despite the presence of a partially deglycosylated 18K intermediate at low concentration of endoglycosidase. Similarly, both hTSH-α and hTSH-β could be completely deglycosylated after acid dissociation of the native hormone. While all three carbohydrate chains of hTSH are sensitive to pure peptide:N-glycosidase F, only one on α and the single oligosaccharide present on β in hTSH appeared to be cleaved by pure endoglycosidase F. These findings indicate that while the carbohydrate chain on β is not involved in αΒ association, the oligosaccharides on α are hindered when hTSH subunits are combined

  18. From the Cover: Vulnerability of C6 Astrocytoma Cells After Single-Compound and Joint Exposure to Type I and Type II Pyrethroid Insecticides.

    Science.gov (United States)

    Romero, Delfina M; Berardino, Bruno G; Wolansky, Marcelo J; Kotler, Mónica L

    2017-01-01

    A primary mode-of-action of all pyrethroid insecticides (PYRs) is the disruption of the voltage-gated sodium channel electrophysiology in neurons of target pests and nontarget species. The neurological actions of PYRs on non-neuronal cells of the nervous system remain poorly investigated. In the present work, we used C6 astrocytoma cells to study PYR actions (0.1-50 μM) under the hypothesis that glial cells may be targeted by and vulnerable to PYRs. To this end, we characterized the effects of bifenthrin (BF), tefluthrin (TF), α-cypermethrin (α-CYP), and deltamethrin (DM) on the integrity of nuclear, mitochondrial, and lysosomal compartments. In general, 24- to 48-h exposures produced concentration-related impairment of cell viability. In single-compound, 24-h exposure experiments, effective concentration (EC) 15 s 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT assay) were computed as follows (in μM): BF, 16.1; TF, 37.3; α-CYP, 7.8; DM, 5.0. We found concentration-related damage in several C6-cell subcellular compartments (mitochondria, nuclei, and lysosomes) at ≥ 10 -1 μM levels. Last, we examined a mixture of all PYRs (ie, Σ individual EC 15 ) using MTT assays and subcellular analyses. Our findings indicate that C6 cells are responsive to nM levels of PYRs, suggesting that astroglial susceptibility may contribute to the low-dose neurological effects caused by these insecticides. This research further suggests that C6 cells may provide relevant information as a screening platform for pesticide mixtures targeting nervous system cells by expected and unexpected toxicogenic pathways potentially contributing to clinical neurotoxicity. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  19. Self-Assembly of "Chalcone" Type Push-Pull Dye Molecules into Organic Single Crystalline Microribbons and Rigid Microrods for Vis/NIR Range Photonic Cavity Applications.

    Science.gov (United States)

    Vattikunta, Radhika; Venkatakrishnarao, Dasari; Mohiddon, Mahamad Ahamad; Chandrasekar, Rajadurai

    2016-11-04

    A novel supramolecular fluorescent donor-acceptor type dye molecule, (2E,4E)-1-(2-hydroxyphenyl)-5-(pyren-1-yl)penta-2,4-dien-1-one (HPPD) self-assembles in a mixture of ethanol/chloroform through intermolecular π-π stacking (distance ca. 3.384 Å) to form J-aggregated single-crystalline microribbons displaying Fabry-Pèrot (F-P) type visible-range optical resonance. The corresponding borondifluoride dye (HPPD-BF), with a reduced HOMO-LUMO gap, self-assembles into crystalline microrods acting as an F-P type resonator in the near-infrared (NIR) range. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Pleiotropic Antibiotic Resistance Mutations Associated with Ribosomes and Ribosomal Subunits in Mycobacterium smegmatis

    Science.gov (United States)

    Yamada, Takeshi; Masuda, Kunitsugu; Shoji, Ko; Hori, Mitsuo

    1974-01-01

    Viomycin-resistant strains isolated from Mycobacterium smegmatis demonstrated pleiotropic resistance to tuberactinomycin-N, capreomycin, streptomycin, and kanamycin as a result of mutational alteration of ribosomes, even though they were selected for resistance to a single antibiotic. The pleiotropic drug resistance of three mutants isolated by stepwise selection for resistance to viomycin was due to alteration of the 30S ribosomal subunit. One mutant, strain A, isolated independently by multiple-step selection to viomycin resistance, was resistant to viomycin, tuberactinomycin-N, and capreomycin through an alteration of the 50S ribosomal subunit, whereas it was sensitive to kanamycin but resistant to streptomycin through an alteration of the 30S ribosomal subunit. Three streptomycin-resistant strains, which were isolated by one-step selection at a high concentration of streptomycin, did not show significant co-resistance to any other antibiotics tested in culture and cell-free systems; streptomycin resistance in these mutants was localized on the 30S ribosomal subunit. PMID:15828170

  1. Component Analysis of Unsteady Hydrodynamic Force of Closed-Type Centrifugal Pump with Single Blades of Different Blade Outlet Angles

    Directory of Open Access Journals (Sweden)

    Yasuyuki Nishi

    2015-01-01

    Full Text Available Single-blade centrifugal impellers for sewage systems undergo both unsteady radial and axial thrusts. Therefore, it is extremely important for the improvement of pump reliability to quantitatively grasp these fluctuating hydrodynamic forces and determine the generation mechanism behind them. In this study, we conducted component analyses of radial and axial thrusts of closed, single-blade centrifugal pumps with different blade outlet angles by numerical analysis while considering leakage flow. The results revealed the effect of the blade outlet angle on the components of radial and axial thrusts. For increased flow rates, the time-averaged values of the pressure component were similar for all impellers, although its fluctuating components were higher for impellers with larger blade outlet angles. Moreover, the fluctuating inertia component of the impeller with a blade outlet angle of 8° decreased as the flow rate increased, whereas those with 16° and 24° angles increased. Therefore, the radial thrust on the hydraulic part was significantly higher for impellers with high blade outlet angles.

  2. Transcriptional regulators of Na, K-ATPase subunits

    OpenAIRE

    Zhiqin eLi; Sigrid A Langhans

    2015-01-01

    The Na,K-ATPase classically serves as an ion pump creating an electrochemical gradient across the plasma membrane that is essential for transepithelial transport, nutrient uptake and membrane potential. In addition, Na,K-ATPase also functions as a receptor, a signal transducer and a cell adhesion molecule. With such diverse roles, it is understandable that the Na,K-ATPase subunits, the catalytic alpha-subunit, the beta-subunit and the FXYD proteins, are controlled extensively during developme...

  3. Mutations in Succinate Dehydrogenase Subunit C Increase Radiosensitivity and Bystander Responses

    Science.gov (United States)

    Zhou, Hongning; Hei, Tom K.

    Although radiation-induced bystander effect is well studied in the past decade, the precise mech-anisms are still unclear. It is likely that a combination of pathways involving both primary and secondary signaling processes is involved in producing a bystander effect. There is recent evidence that mitochondria play a critical role in bystander responses. Recently studies found that a mutation in succinate dehydrogenese subunit C (SDHC), an integral membrane protein in complex II of the electron transport chain, resulted in increased superoxide, oxidative stress, apoptosis, tumorigenesis, and genomic instability, indicating that SDHC play a critical role in maintaining mitochondrial function. In the present study, using Chinese hamster fibroblasts (B1 cells) and the mutants (B9 cells) containing a single base substitution that produced a premature stop codon resulting in a 33-amino acid COOH-terminal truncation of the SDHC protein, we found that B9 cells had an increase in intracellular superoxide content, nitric oxide species, and mitochondrial membrane potential when compared with wild type cells. After irradiated with a grade of doses of gamma rays, B9 cells show an increased radiosensitivity, especially at high doses. The HPRT- mutant yield after gamma-ray irradiation in B9 cells was significantly higher than that of B1 cells. A single, 3Gy dose of gamma-rays increased the background mutant level by more than 4 fold. In contrast, the mutant induction was less than 2 fold in B1 cells. In addition, B9 cells produced a higher bystander mutagenesis after alpha particle irradiation than the B1 cells. Furthermore, pretreated with carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), a nitric oxide scavenger, significantly decreased the bystander effect. Our findings demonstrate that a mutation in SDHC increases radiosensitivity in both directly irradiated cells and in neighboring bystander cells, and mito-chondrial function play an essential role in

  4. New vibrational mode of the acoustic type in Nd(Pr)2 Cu O4 single crystals

    International Nuclear Information System (INIS)

    Fil', D.V.; Kolobov, I.G.; Fil', V.D.; Barilo, S.N.; Zhigunov, D.I.

    1995-01-01

    Sound velocities along main symmetry directions as well as their angle dependences in (100),(110)-type planes are measured in Nd(Pr) 2 Cu O 4 . Anomalies in the angle dependences are found, which are interpreted as a result of the interaction of elastic vibrations with an additional plane mode of the acoustic type. According to the proposed interpretation, the bare spectrum of the additional mode is two-dimensional, and the origin of the mode is connected with the electron degrees of freedom in the Cu O 2 -planes. A phenomenological model for description of acoustic mode spectra in the investigated systems is proposed. On the basis of the anion model of HTSC, a possible microscopic scenario of the appearance of the additional mode is analyzed. In the framework of the phenomenological model, the Debye temperatures are computed, which are in agreement with the specific heat data. The values of the components of the elastic moduli tensor are given

  5. Single Turnover Autophosphorylation Cycle of the PKA RIIβ Holoenzyme.

    Directory of Open Access Journals (Sweden)

    Ping Zhang

    2015-07-01

    Full Text Available To provide tight spatiotemporal signaling control, the cyclic adenosine monophosphate (cAMP-dependent protein kinase (PKA holoenzyme typically nucleates a macromolecular complex or a "PKA signalosome." Using the RIIβ holoenzyme as a prototype, we show how autophosphorylation/dephosphorylation of the RIIβ subunit, as well as cAMP and metal ions, contribute to the dynamics of PKA signaling. While we showed previously that the RIIβ holoenzyme could undergo a single turnover autophosphorylation with adenosine triphosphate and magnesium (MgATP and trap both products in the crystal lattice, we asked here whether calcium could trap an ATP:RIIβ holoenzyme since the RIIβ holoenzyme is located close to ion channels. The 2.8Å structure of an RIIβp2:C2:(Ca2ADP2 holoenzyme, supported by biochemical and biophysical data, reveals a trapped single phosphorylation event similar to MgATP. Thus, calcium can mediate a single turnover event with either ATP or adenosine-5'-(β,γ-imidotriphosphate (AMP-PNP, even though it cannot support steady-state catalysis efficiently. The holoenzyme serves as a "product trap" because of the slow off-rate of the pRIIβ subunit, which is controlled by cAMP, not by phosphorylation of the inhibitor site. By quantitatively defining the RIIβ signaling cycle, we show that release of pRIIβ in the presence of cAMP is reduced by calcium, whereas autophosphorylation at the phosphorylation site (P-site inhibits holoenzyme reassociation with the catalytic subunit. Adding a single phosphoryl group to the preformed RIIβ holoenzyme thus creates a signaling cycle in which phosphatases become an essential partner. This previously unappreciated molecular mechanism is an integral part of PKA signaling for type II holoenzymes.

  6. Comparison of the quench and fault current limiting characteristics of the flux-coupling type SFCL with single and three-phase transformer

    International Nuclear Information System (INIS)

    Jung, Byung Ik; Cho, Yong Sun; Park, Hyoung Min; Chung, Dong Chul; Choi, Hyo Sang

    2013-01-01

    Highlight: ► Comparison of quench and fault-current-limiting behavior of SFCLs by Tr type. -- Abstract: The South Korean power grid has a network structure for the flexible operation of the system. The continuously increasing power demand necessitated the increase of power facilities, which decreased the impedance in the power system. As a result, the size of the fault current in the event of a system fault increased. As this increased fault current size is threatening the breaking capacity of the circuit breaker, the main protective device, a solution to this problem is needed. The superconducting fault current limiter (SFCL) has been designed to address this problem. SFCL supports the stable operation of the circuit breaker through its excellent fault-current-limiting operation [1–5]. In this paper, the quench and fault current limiting characteristics of the flux-coupling-type SFCL with one three-phase transformer were compared with those of the same SFCL type but with three single-phase transformers. In the case of the three-phase transformers, both the superconducting elements of the fault and sound phases were quenched, whereas in the case of the single-phase transformer, only that of the fault phase was quenched. For the fault current limiting rate, both cases showed similar rates for the single line-to-ground fault, but for the three-wire earth fault, the fault current limiting rate of the single-phase transformer was over 90% whereas that of the three-phase transformer was about 60%. It appears that when the three-phase transformer was used, the limiting rate decreased because the fluxes by the fault current of each phase were linked in one core. When the power loads of the superconducting elements were compared by fault type, the initial (half-cycle) load was great when the single-phase transformer was applied, whereas for the three-phase transformer, its power load was slightly lower at the initial stage but became greater after the half fault cycle

  7. Structure of the mycobacterial ESX-5 type VII secretion system membrane complex by single-particle analysis.

    Science.gov (United States)

    Beckham, Katherine S H; Ciccarelli, Luciano; Bunduc, Catalin M; Mertens, Haydyn D T; Ummels, Roy; Lugmayr, Wolfgang; Mayr, Julia; Rettel, Mandy; Savitski, Mikhail M; Svergun, Dmitri I; Bitter, Wilbert; Wilmanns, Matthias; Marlovits, Thomas C; Parret, Annabel H A; Houben, Edith N G

    2017-04-10

    Mycobacteria are characterized by their impermeable outer membrane, which is rich in mycolic acids 1 . To transport substrates across this complex cell envelope, mycobacteria rely on type VII (also known as ESX) secretion systems 2 . In Mycobacterium tuberculosis, these ESX systems are essential for growth and full virulence and therefore represent an attractive target for anti-tuberculosis drugs 3 . However, the molecular details underlying type VII secretion are largely unknown, due to a lack of structural information. Here, we report the molecular architecture of the ESX-5 membrane complex from Mycobacterium xenopi determined at 13 Å resolution by electron microscopy. The four core proteins of the ESX-5 complex (EccB 5 , EccC 5 , EccD 5 and EccE 5 ) assemble with equimolar stoichiometry into an oligomeric assembly that displays six-fold symmetry. This membrane-associated complex seems to be embedded exclusively in the inner membrane, which indicates that additional components are required to translocate substrates across the mycobacterial outer membrane. Furthermore, the extended cytosolic domains of the EccC ATPase, which interact with secretion effectors, are highly flexible, suggesting an as yet unseen mode of substrate interaction. Comparison of our results with known structures of other bacterial secretion systems demonstrates that the architecture of type VII secretion system is fundamentally different, suggesting an alternative secretion mechanism.

  8. Molecular Screening and Single Nucleotide Polymorphism Typing of Molluscum Contagiosum Virus (MCV) from Genital Specimens, between 2012 and 2015

    Science.gov (United States)

    Taghinezhad-S, Sedigheh; Mohseni, Amir Hossein; Keyvani, Hossein; Ghobadi, Narges

    2018-03-01

    The present study is the first comprehensive report of the Molluscum contagiosum virus (MCV) in Iran based on the molecular technique for differentiation and typing of the MCV1 and MCV2. Patients were diagnosed as having tumor-like genital warts less than 5 mm in diameter, and HIV seronegative samples were chosen for this cross-sectional study. TaqMan real-time PCR was used to identify MCV following clinical examination. Typing of the MCV-positive specimens was performed in the SNP A27451G region of MC021L gene. Of 1470 samples, 114 (7.75%) samples were positive for the MCV. From MCV-positive samples, 71.05% sequences were found to be related to the MCV1 and 28.95% to the MCV2. This assay constitutes a reliable method for identification and typing of the MCV genomic variants that could be valuable for reviewing the pathogenesis, molecular epidemiology, and the natural history of MCV-related situations.

  9. An investigation into heterogeneity in a single vein-type uranium ore deposit: Implications for nuclear forensics

    International Nuclear Information System (INIS)

    Keatley, A.C.; Scott, T.B.; Davis, S.; Jones, C.P.; Turner, P.

    2015-01-01

    Minor element composition and rare earth element (REE) concentrations in nuclear materials are important as they are used within the field of nuclear forensics as an indicator of sample origin. However recent studies into uranium ores and uranium ore concentrates (UOCs) have shown significant elemental and isotopic heterogeneity from a single mine site such that some sites have shown higher variation within the mine site than that seen between multiple sites. The elemental composition of both uranium and gangue minerals within ore samples taken along a single mineral vein in South West England have been measured and reported here. The analysis of the samples was undertaken to determine the extent of the localised variation in key elements. Energy Dispersive X-ray spectroscopy (EDS) was used to analyse the gangue mineralogy and measure major element composition. Minor element composition and rare earth element (REE) concentrations were measured by Electron Probe Microanalysis (EPMA). The results confirm that a number of key elements, REE concentrations and patterns used for origin location do show significant variation within mine. Furthermore significant variation is also visible on a meter scale. In addition three separate uranium phases were identified within the vein which indicates multiple uranium mineralisation events. In light of these localised elemental variations it is recommended that representative sampling for an area is undertaken prior to establishing the REE pattern that may be used to identify the originating mine for an unknown ore sample and prior to investigating impact of ore processing on any arising REE patterns. - Highlights: • Demonstrating significant variation at meter scale distances. • Importance of understanding heterogeneity in aid of nuclear forensics interpretation. • Understanding local features prior to determining feed through of elements to UOCs. • Key elements, REE concentrations and patterns show significant variation

  10. Spectroscopic characterization of a single dangling bond on a bare Si(100)- c ( 4 × 2 ) surface for n - and p -type doping

    KAUST Repository

    Mantega, M.

    2012-07-19

    We investigate the charging state of an isolated single dangling bond formed on an unpassivated Si(100) surface with c(4×2) reconstruction, by comparing scanning tunneling microscopy and spectroscopy analysis with density functional theory calculations. The dangling bond is created by placing a single hydrogen atom on the bare surface with the tip of a scanning tunneling microscope. The H atom passivates one of the dimer dangling bonds responsible for the surface one-dimensional electronic structure. This leaves a second dangling at the reacted surface dimer which breaks the surface periodicity. We consider two possible H adsorption configurations for both the neutral and the doped situation (n- and p-type). In the case of n-doping we find that the single dangling bond state is doubly occupied and the most stable configuration is that with H bonded to the bottom Si atom of the surface dimer. In the case of p-doping the dangling bond is instead empty and the configuration with the H attached to the top atom of the dimer is the most stable. Importantly the two configurations have different scattering properties and phase shift fingerprints. This might open up interesting perspectives for fabricating a switching device by tuning the doping level or by locally charging the single dangling bond state. © 2012 American Physical Society.

  11. A single nucleotide polymorphism within the acetyl-coenzyme A carboxylase beta gene is associated with proteinuria in patients with type 2 diabetes.

    Directory of Open Access Journals (Sweden)

    Shiro Maeda

    2010-02-01

    Full Text Available It has been suggested that genetic susceptibility plays an important role in the pathogenesis of diabetic nephropathy. A large-scale genotyping analysis of gene-based single nucleotide polymorphisms (SNPs in Japanese patients with type 2 diabetes identified the gene encoding acetyl-coenzyme A carboxylase beta (ACACB as a candidate for a susceptibility to diabetic nephropathy; the landmark SNP was found in the intron 18 of ACACB (rs2268388: intron 18 +4139 C > T, p = 1.4x10(-6, odds ratio = 1.61, 95% confidence interval [CI]: 1.33-1.96. The association of this SNP with diabetic nephropathy was examined in 9 independent studies (4 from Japan including the original study, one Singaporean, one Korean, and two European with type 2 diabetes. One case-control study involving European patients with type 1 diabetes was included. The frequency of the T allele for SNP rs2268388 was consistently higher among patients with type 2 diabetes and proteinuria. A meta-analysis revealed that rs2268388 was significantly associated with proteinuria in Japanese patients with type 2 diabetes (p = 5.35 x 10(-8, odds ratio = 1.61, 95% Cl: 1.35-1.91. Rs2268388 was also associated with type 2 diabetes-associated end-stage renal disease (ESRD in European Americans (p = 6 x 10(-4, odds ratio = 1.61, 95% Cl: 1.22-2.13. Significant association was not detected between this SNP and nephropathy in those with type 1 diabetes. A subsequent in vitro functional analysis revealed that a 29-bp DNA fragment, including rs2268388, had significant enhancer activity in cultured human renal proximal tubular epithelial cells. Fragments corresponding to the disease susceptibility allele (T had higher enhancer activity than those of the major allele. These results suggest that ACACB is a strong candidate for conferring susceptibility for proteinuria in patients with type 2 diabetes.

  12. DNA binding properties of the small cascade subunit Csa5.

    Directory of Open Access Journals (Sweden)

    Michael Daume

    Full Text Available CRISPR-Cas systems provide immunity against viral attacks in archaeal and bacterial cells. Type I systems employ a Cas protein complex termed Cascade, which utilizes small CRISPR RNAs to detect and degrade the exogenic DNA. A small sequence motif, the PAM, marks the foreign substrates. Previously, a recombinant type I-A Cascade complex from the archaeon Thermoproteus tenax was shown to target and degrade DNA in vitro, dependent on a native PAM sequence. Here, we present the biochemical analysis of the small subunit, Csa5, of this Cascade complex. T. tenax Csa5 preferentially bound ssDNA and mutants that showed decreased ssDNA-binding and reduced Cascade-mediated DNA cleavage were identified. Csa5 oligomerization prevented DNA binding. Specific recognition of the PAM sequence was not observed. Phylogenetic analyses identified Csa5 as a universal member of type I-A systems and revealed three distinct groups. A potential role of Csa5 in R-loop stabilization is discussed.

  13. A comparison between spray drying and spray freeze drying to produce an influenza subunit vaccine powder for inhalation

    NARCIS (Netherlands)

    Saluja, V.; Amorij, J-P.; Kapteyn, J. C.; de Boer, A. H.; Frijlink, H. W.; Hinrichs, W. L. J.

    2010-01-01

    The aim of this study was to investigate two different processes to produce a stable influenza subunit vaccine powder for pulmonary immunization i.e. spray drying (SD) and spray freeze drying (SFD). The formulations were analyzed by proteolytic assay, single radial immunodiffusion assay (SRID),

  14. Identification of a common amino acid polymorphism in the p85alpha regulatory subunit of phosphatidylinositol 3-kinase

    DEFF Research Database (Denmark)

    Hansen, Torben; Andersen, C B; Echwald, Søren Morgenthaler

    1997-01-01

    in a phenotype study. Single-strand conformational polymorphism and heteroduplex analysis of the coding region of the regulatory p85alpha subunit in cDNA isolated from human muscle tissue from 70 insulin-resistant NIDDM patients and 12 control subjects revealed three silent polymorphisms and a missense mutation...

  15. Effect of Exposure Type and Timing of Injuries in Division I College Football: A 4-year Single Program Analysis.

    Science.gov (United States)

    Krill, Michael K; Borchers, James R; Hoffman, Joshua T; Tatarski, Rachel L; Hewett, Timothy E

    2017-02-01

    Football players compete with a high risk of injury due to the sport. With the recent efforts to improve safety, the National Collegiate Athletic Association (NCAA) established new terminology to clearly define exposure types and reduce the number of high contact exposures. To compare football injury rates (IR) with a focus on game versus practice, time in season of injury, mechanism of injury and utilizing recent exposure types defined by the NCAA (live contact, full-pads and non-contact). Licensed medical professionals monitored a college football program regular season from 2012-2015. Each injury was classified by timing of the injury, mechanism of injury, and whether it occurred in game or practice. Player attendance and type of exposure (non-contact, full-pad or live contact, which involves live tackling to the ground and/or full-speed blocking and can occur in full-pad or half-pad ('shell') equipment) was documented. IR were calculated per 1000 athlete-exposures (AE). Mid-exact P tests compared rates between variables. The game IR was over three times as high as the practice IR (p injuries observed occurred from a contact mechanism (IR: 2.508/1000 AE). The highest IR during the fall football season occurred in the pre-season at 5.769/1000 AE. Overall IR observed in this cohort were lower than prior studies published before recent NCAA rule changes and guideline implementation to improve athlete safety. Athletes in this cohort were at significantly increased risk of injury from live contact exposures.

  16. Analysis on preemptive priority retrial queue with two types of customers, balking, optional re-service, single vacation and service interruption

    Science.gov (United States)

    Yuvarani, S.; Saravanarajan, M. C.

    2017-11-01

    This paper concerned with performance analysis of single server preemptive priority retrial queue with a single vacation where two types of customers are considered and they are called priority customers and ordinary customers. The ordinary customers arrive in batch into the system. The priority customers do not form any queue. After the completion of regular service, the customers may demand re-service for the previous service without joining the orbit or may leave the system. As soon as the system is empty, the server goes for vacation and the regular busy server can be subjected to breakdown. By using the supplementary variable technique, we obtain the steady-state probability generating functions for the system/orbit size. Some important system performance measures and the stochastic decomposition are discussed. Finally, numerical examples are presented to visualize the effect of parameters on system performance measures.

  17. Detection of constitutive heterodimerization of the integrin Mac-1 subunits by fluorescence resonance energy transfer in living cells

    International Nuclear Information System (INIS)

    Fu Guo; Yang Huayan; Wang Chen; Zhang Feng; You Zhendong; Wang Guiying; He Cheng; Chen Yizhang; Xu Zhihan

    2006-01-01

    Macrophage differentiation antigen associated with complement three receptor function (Mac-1) belongs to β 2 subfamily of integrins that mediate important cell-cell and cell-extracellular matrix interactions. Biochemical studies have indicated that Mac-1 is a constitutive heterodimer in vitro. Here, we detected the heterodimerization of Mac-1 subunits in living cells by means of two fluorescence resonance energy transfer (FRET) techniques (fluorescence microscopy and fluorescence spectroscopy) and our results demonstrated that there is constitutive heterodimerization of the Mac-1 subunits and this constitutive heterodimerization of the Mac-1 subunits is cell-type independent. Through FRET imaging, we found that heterodimers of Mac-1 mainly localized in plasma membrane, perinuclear, and Golgi area in living cells. Furthermore, through analysis of the estimated physical distances between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) fused to Mac-1 subunits, we suggested that the conformation of Mac-1 subunits is not affected by the fusion of CFP or YFP and inferred that Mac-1 subunits take different conformation when expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293T cells, respectively

  18. Study of single pulsed-field magnetization of Gd-Ba-Cu-O bulk high-temperature superconductor with a split type of armature coil for rotating machinery

    International Nuclear Information System (INIS)

    Morita, E; Matsuzaki, H; Kimura, Y; Ohtani, I; Ogata, H; Izumi, M; Nonaka, Y; Murakami, M; Ida, T; Sugimoto, H; Miki, M; Kitano, M

    2006-01-01

    We employed a Gd-bulk HTS as rotating field magnet poles aiming for a smaller and lighter axial-gap-type motor. The bulk was inserted in the split-type armature pulsed copper coils and cooled down to 77 K under zero field. Employing the bulk magnet to HTS rotating machinery, the number of pulsed field magnetizations should be reduced for practical use. Thereby, a single pulsed current was applied to the pulsed copper coils to magnetize the bulk. The trapped field distribution and transient flux behaviour strongly depend on the radial dimension of the armature coil with a vortex-type winding. On decreasing the diameter of the pulsed copper armature coil, the distribution of the trapped flux density on the surface of the bulk becomes close to a conical shape. In contrast to the use of a solenoid, the application of vortex-type armature coils to magnetization of Gd-bulk HTS shows a quick intervention of the external magnetic flux into the centre of the bulk. The magnetization to the bulk HTS of the vortex-type copper coils with an optimum radius is useful and may be an effective technique for applied bulk HTS for rotating machines such as motors and/or generators

  19. Antagonistic regulation of flowering time through distinct regulatory subunits of protein phosphatase 2A.

    Directory of Open Access Journals (Sweden)

    Behzad Heidari

    Full Text Available Protein phosphatase 2A (PP2A consists of three types of subunits: a catalytic (C, a scaffolding (A, and a regulatory (B subunit. In Arabidopsis thaliana and other organisms the regulatory B subunits are divided into at least three non-related groups, B55, B' and B″. Flowering time in plants mutated in B55 or B' genes were investigated in this work. The PP2A-b55α and PP2A-b55β (knockout lines showed earlier flowering than WT, whereas a PP2A-b'γ (knockdown line showed late flowering. Average advancements of flowering in PP2A-b55 mutants were 3.4 days in continuous light, 6.6 days in 12 h days, and 8.2 days in 8 h days. Average delays in the PP2A-b'γ mutant line were 7.1 days in 16 h days and 4.7 days in 8 h days. Expression of marker genes of genetically distinct flowering pathways (CO, FLC, MYB33, SPL3, and the floral integrator (FT, SOC1 were tested in WT, pp2a mutants, and two known flowering time mutants elf6 and edm2. The results are compatible with B55 acting at and/or downstream of the floral integrator, in a non-identified pathway. B' γ was involved in repression of FLC, the main flowering repressor gene. For B'γ the results are consistent with the subunit being a component in the major autonomous flowering pathway. In conclusion PP2A is both a positive and negative regulator of flowering time, depending on the type of regulatory subunit involved.

  20. The Na, K-ATPase β-Subunit Isoforms Expression in Glioblastoma Multiforme: Moonlighting Roles

    Science.gov (United States)

    Rotoli, Deborah; Cejas, Mariana-Mayela; Maeso, María-del-Carmen; Pérez-Rodríguez, Natalia-Dolores; Morales, Manuel; Ávila, Julio

    2017-01-01

    Glioblastoma multiforme (GBM) is the most common form of malignant glioma. Recent studies point out that gliomas exploit ion channels and transporters, including Na, K-ATPase, to sustain their singular growth and invasion as they invade the brain parenchyma. Moreover, the different isoforms of the β-subunit of Na, K-ATPase have been implicated in regulating cellular dynamics, particularly during cancer progression. The aim of this study was to determine the Na, K-ATPase β subunit isoform subcellular expression patterns in all cell types responsible for microenvironment heterogeneity of GBM using immunohistochemical analysis. All three isoforms, β1, β2/AMOG (Adhesion Molecule On Glia) and β3, were found to be expressed in GBM samples. Generally, β1 isoform was not expressed by astrocytes, in both primary and secondary GBM, although other cell types (endothelial cells, pericytes, telocytes, macrophages) did express this isoform. β2/AMOG and β3 positive expression was observed in the cytoplasm, membrane and nuclear envelope of astrocytes and GFAP (Glial Fibrillary Acidic Protein) negative cells. Interestingly, differences in isoforms expression have been observed between primary and secondary GBM: in secondary GBM, β2 isoform expression in astrocytes was lower than that observed in primary GBM, while the expression of the β3 subunit was more intense. These changes in β subunit isoforms expression in GBM could be related to a different ionic handling, to a different relationship between astrocyte and neuron (β2/AMOG) and to changes in the moonlighting roles of Na, K-ATPase β subunits as adaptor proteins and transcription factors. PMID:29117147

  1. The Na, K-ATPase β-Subunit Isoforms Expression in Glioblastoma Multiforme: Moonlighting Roles

    Directory of Open Access Journals (Sweden)

    Deborah Rotoli

    2017-11-01

    Full Text Available Glioblastoma multiforme (GBM is the most common form of malignant glioma. Recent studies point out that gliomas exploit ion channels and transporters, including Na, K-ATPase, to sustain their singular growth and invasion as they invade the brain parenchyma. Moreover, the different isoforms of the β-subunit of Na, K-ATPase have been implicated in regulating cellular dynamics, particularly during cancer progression. The aim of this study was to determine the Na, K-ATPase β subunit isoform subcellular expression patterns in all cell types responsible for microenvironment heterogeneity of GBM using immunohistochemical analysis. All three isoforms, β1, β2/AMOG (Adhesion Molecule On Glia and β3, were found to be expressed in GBM samples. Generally, β1 isoform was not expressed by astrocytes, in both primary and secondary GBM, although other cell types (endothelial cells, pericytes, telocytes, macrophages did express this isoform. β2/AMOG and β3 positive expression was observed in the cytoplasm, membrane and nuclear envelope of astrocytes and GFAP (Glial Fibrillary Acidic Protein negative cells. Interestingly, differences in isoforms expression have been observed between primary and secondary GBM: in secondary GBM, β2 isoform expression in astrocytes was lower than that observed in primary GBM, while the expression of the β3 subunit was more intense. These changes in β subunit isoforms expression in GBM could be related to a different ionic handling, to a different relationship between astrocyte and neuron (β2/AMOG and to changes in the moonlighting roles of Na, K-ATPase β subunits as adaptor proteins and transcription factors.

  2. Oil type and cross-linking influence growth of Aureobasidium melanogenum on vegetable oils as a single carbon source.

    Science.gov (United States)

    Peeters, Loes H M; Huinink, Hendrik P; Voogt, Benjamin; Adan, Olaf C G

    2018-03-12

    Aureobasidium melanogenum is the main fungus found in a spontaneously formed biofilm on a oil-treated wood. This dark colored biofilm functions as a protective coating. To better understand biofilm formation, in this study A. melanogenum was cultured on olive oil and raw linseed oil. Metabolic activity and oil conversion were measured. The results show that A. melanogenum is able to grow on linseed oil and olive oil as a single carbon source. The fungus produces the enzyme lipase to convert the oil into fatty acids and glycerol. Metabolic activity and oil conversion were equal on linseed oil and olive oil. The fungus was not able to grow on severe cross-linked linseed oil, meaning that the degree of cross-linking of the oil is important for growth of A. melanogenum. Dark coloring of the colony was seen on linseed oil, which might be a stress response on the presence of autoxidation products in linseed oil. The colony on olive oil showed delayed melanin production indicating an inhibitory effect of olive oil on melanin production. © 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  3. Visualization and Comparison of Single and Combined Parametric and Nonparametric Discriminant Methods for Leukemia Type Recognition Based on Gene Expression

    Directory of Open Access Journals (Sweden)

    Ćwiklińska-Jurkowska Małgorzata M.

    2015-12-01

    Full Text Available A gene expression data set, containing 3051 genes and 38 tumor mRNA training samples, from a leukemia microarray study, was used for differentiation between ALL and AML groups of leukemia. In this paper, single and combined discriminant methods were applied on the basis of the selected few most discriminative variables according to Wilks’ lambda or the leave-one-out error of first nearest neighbor classifier. For the linear, quadratic, regularized, uncorrelated discrimination, kernel, nearest neighbor and naive Bayesian classifiers, two-dimensional graphs of the boundaries and discriminant functions for diagnostics are presented. Cross-validation and leave-one-out errors were used as measures of classifier performance to support diagnosis coming from this genomic data set. A small number of best discriminating genes, from two to ten, was sufficient to build discriminant methods of good performance. Especially useful were nearest neighbor methods. The results presented herein were comparable with outcomes obtained by other authors for larger numbers of applied genes. The linear, quadratic, uncorrelated Bayesian and regularized discrimination methods were subjected to bagging or boosting in order to assess the accuracy of the fusion. A conclusion drawn from the analysis was that resampling ensembles were not beneficial for two-dimensional discrimination.

  4. Comparison of three types of XPAD3.2/CdTe single chip hybrids for hard X-ray applications in material science and biomedical imaging

    Science.gov (United States)

    Buton, C.; Dawiec, A.; Graber-Bolis, J.; Arnaud, K.; Bérar, J. F.; Blanc, N.; Boudet, N.; Clémens, J. C.; Debarbieux, F.; Delpierre, P.; Dinkespiler, B.; Gastaldi, T.; Hustache, S.; Morel, C.; Pangaud, P.; Perez-Ponce, H.; Vigeolas, E.

    2014-09-01

    The CHIPSPECT consortium aims at building a large multi-modules CdTe based photon counting detector for hard X-ray applications. For this purpose, we tested nine XPAD3.2 single chip hybrids in various configurations (i.e. Ohmic vs. Schottky contacts or electrons vs. holes collection mode) in order to select the most performing and best suited configuration for our experimental requirements. Measurements have been done using both X-ray synchrotron beams and 241Am source. Preliminary results on the image quality, calibration, stability, homogeneity and linearity of the different types of detectors are presented.

  5. Single-Dose Metformin Enhances Bile Acid-Induced Glucagon-Like Peptide-1 Secretion in Patients With Type 2 Diabetes

    DEFF Research Database (Denmark)

    Brønden, Andreas; Albér, Anders; Rohde, Ulrich

    2017-01-01

    -controlled, and double-blinded crossover study. Setting: This study was conducted at Center for Diabetes Research, Gentofte Hospital, Denmark. Patients: Fifteen metformin-treated patients with type 2 diabetes; all participants completed the study. Interventions: Four experimental study days in randomized order...... with administration of either 1500 mg metformin or placebo in combination with intravenous infusion of cholecystokinin (0.4 pmol × kg-1 × min-1) or saline. Main Outcome Measure: Plasma GLP-1 excursions as measured by baseline-subtracted area under the curve. Results: Single-dose metformin further enhanced bile acid...

  6. AHSG tag single nucleotide polymorphisms associate with type 2 diabetes and dyslipidemia: studies of metabolic traits in 7,683 white Danish subjects

    DEFF Research Database (Denmark)

    Andersen, Gitte; Burgdorf, Kristoffer Sølvsten; Sparsø, Thomas

    2008-01-01

    been largely successful. We related seven frequent AHSG tag single nucleotide polymorphisms to a range of metabolic traits, including type 2 diabetes, obesity, and dyslipidemia. RESEARCH DESIGN AND METHODS: The polymorphisms were genotyped in 7,683 white Danish subjects using Taqman allelic...... with dyslipidemia (P = 0.003 and P(corr) = 0.009). Thr248Met (rs4917) tended to associate with lower fasting and post-oral glucose tolerance test serum insulin release (P = 0.02, P(corr) = 0.1 for fasting and P = 0.04, P(corr) = 0.2 for area under the insulin curve) and improved insulin sensitivity estimated...

  7. Radiation inactivation of multimeric enzymes: application to subunit interactions of adenylate cyclase

    International Nuclear Information System (INIS)

    Verkman, A.S.; Skorecki, K.L.; Ausiello, D.A.

    1986-01-01

    Radiation inactivation has been applied extensively to determine the molecular weight of soluble enzyme and receptor systems from the slope of a linear ln (activity) vs. dose curve. Complex nonlinear inactivation curves are predicted for multimeric enzyme systems, composed of distinct subunits in equilibrium with multimeric complexes. For the system A1 + A2----A1A2, with an active A1A2 complex (associative model), the ln (activity) vs. dose curve is linear for high dissociation constant, K. If a monomer, A1, has all the enzyme activity (dissociative model), the ln (activity) vs. dose curve has an activation hump at low radiation dose if the inactive subunit, A2, has a higher molecular weight than A1 and has upward concavity when A2 is smaller than A1. In general, a radiation inactivation model for a multistep mechanism for enzyme activation fulfills the characteristics of an associative or dissociative model if the reaction step forming active enzyme is an associative or dissociative reaction. Target theory gives the molecular weight of the active enzyme subunit or complex from the limiting slope of the ln (activity) vs. dose curve at high radiation dose. If energy transfer occurs among subunits in the multimer, the ln (activity) vs. dose curve is linear for a single active component and is concave upward for two or more active components. The use of radiation inactivation as a method to determine enzyme size and multimeric subunit assembly is discussed with specific application to the hormone-sensitive adenylate cyclase system. It is shown that the complex inactivation curves presented in the accompanying paper can be used select the best mechanism out of a series of seven proposed mechanisms for the activation of adenylate cyclase by hormone

  8. Both modular and single-domain Type I polyketide synthases are expressed in the brevetoxin-producing dinoflagellate, Karenia brevis (Dinophyceae).

    Science.gov (United States)

    Van Dolah, Frances M; Kohli, Gurjeet S; Morey, Jeanine S; Murray, Shauna A

    2017-12-01

    Dinoflagellates are prolific producers of polyketide compounds, many of which are potent toxins with adverse impacts on human and marine animal health. To identify polyketide synthase (PKS) genes in the brevetoxin-producing dinoflagellate, Karenia brevis, we assembled a transcriptome from 595 million Illumina reads, sampled under different growth conditions. The assembly included 125,687 transcripts greater than 300 nt in length, with over half having >100× coverage. We found 121 transcripts encoding Type I ketosynthase (KS) domains, of which 99 encoded single KS domains, while 22 contained multiple KS domains arranged in 1-3 protein modules. Phylogenetic analysis placed all single domain and a majority of multidomain KSs within a monophyletic clade of protist PKSs. In contrast with the highly amplified single-domain KSs, only eight single-domain ketoreductase transcripts were found in the assembly, suggesting that they are more evolutionarily conserved. The multidomain PKSs were dominated by trans-acyltransferase architectures, which were recently shown to be prevalent in other algal protists. Karenia brevis also expressed several hybrid nonribosomal peptide synthetase (NRPS)/PKS sequences, including a burA-like sequence previously reported in a wide variety of dinoflagellates. This contrasts with a similarly deep transcriptome of Gambierdiscus polynesiensis, which lacked NRPS/PKS other than the burA-like transcript, and may reflect the presence of amide-containing polyketides in K. brevis and their absence from G. polynesiensis. In concert with other recent transcriptome analyses, this study provides evidence for both single domain and multidomain PKSs in the synthesis of polyketide compounds in dinoflagellates. © 2017 The Authors Journal of Phycology published by Wiley Periodicals, Inc. on behalf of Phycological Society of America.

  9. SHADOWS OF OUR FORMER COMPANIONS: HOW THE SINGLE-DEGENERATE BINARY TYPE IA SUPERNOVA SCENARIO AFFECTS REMNANTS

    Energy Technology Data Exchange (ETDEWEB)

    Gray, William J.; Raskin, Cody; Owen, J. Michael [Lawrence Livermore National Laboratory, P.O. Box 808, L-038, Livermore, CA 94550 (United States)

    2016-12-10

    Here we present three-dimensional high-resolution simulations of Type Ia supernova in the presence of a non-degenerate companion. We find that the presence of a nearby companion leaves a long-lived hole in the supernova ejecta. In particular, we aim to study the long-term evolution of this hole as the supernova ejecta interacts with the surrounding interstellar medium (ISM). Using estimates for the X-ray emission, we find that the hole generated by the companion remains for many centuries after the interaction between the ejecta and the ISM. We also show that the hole is discernible over a wide range of viewing angles and companion masses.

  10. Effects of single- and multi-substituted Zn ions in doped 122-type iron-based superconductors

    Science.gov (United States)

    Zhao, YuanYuan; Li, Bo; Li, Wei; Chen, Hong-Yi; Bassler, Kevin E.; Ting, C. S.

    2016-04-01

    Recent experiments on Zn-substituted 122-type iron-based superconductors (FeSCs) at electron- and hole-doped regions provide us with a testing ground for understanding the effect of Zn impurities in these systems. Our first-principles calculations of the electronic structure reveal that the Zn 3 d orbitals are far below the Fermi level and are chemically inactive, while the Zn 4 s orbital is partially occupied and its wave function overlaps with the 3 d orbitals of neighboring Fe ions. This suggests that the impurity effect originates in the Zn 4 s orbital, not its 3 d orbitals. Employing a phenomenological two-orbital lattice model for 122-FeSCs and the self-consistent Bogoliubov-de Gennes equations, we study how the Zn impurities suppress the superconductivity in electron- and hole-doped compounds. Our obtained results qualitatively agree with the experimental measurements.

  11. The thermal structural transition of alpha-crystallin modulates subunit interactions and increases protein solubility.

    Directory of Open Access Journals (Sweden)

    Giuseppe Maulucci

    Full Text Available BACKGROUND: Alpha crystallin is an oligomer composed of two types of subunits, alpha-A and alpha-B crystallin, and is the major constituent of human lens. The temperature induced condensation of alpha-crystallin, the main cause for eye lens opacification (cataract, is a two step-process, a nucleation followed by an aggregation phase, and a protective effect towards the aggregation is exhibited over the alpha crystallin phase transition temperature (Tc = 318.16 K. METHODS/RESULTS: To investigate if a modulation of the subunit interactions over Tc could trigger the protective mechanism towards the aggregation, we followed, by using simultaneously static and dynamic light scattering, the temperature induced condensation of alpha-crystallin. By developing a mathematical model able to uncouple the nucleation and aggregation processes, we find a previously unobserved transition in the nucleation rate constant. Its temperature dependence allows to determine fundamental structural parameters, the chemical potential (Δμ and the interfacial tension (γ of the aggregating phase, that characterize subunit interactions. CONCLUSIONS/GENERAL SIGNIFICANCE: The decrease of both Δμ and γ at Tc, and a relative increase in solubility, reveal a significative decrease in the strenght of alpha-crystallin subunits interactions, which protects from supramolecolar condensation in hypertermic conditions. On the whole, we suggest a general approach able to understand the structural and kinetic mechanisms involved in aggregation-related diseases and in drugs development and testing.

  12. Mechanical performance of a screw-type veterinary suture anchor subjected to single load to failure and cyclic loads.

    Science.gov (United States)

    Balara, Jason M; McCarthy, Robert J; Boudrieau, Randy J; Kraus, Karl H

    2004-01-01

    To characterize the mechanical performance of a veterinary bone anchor under static and cyclic loads. Mechanical testing study. Cadaveric canine humeri. Humeri (6 pairs) were collected from skeletally mature dogs (mean [+/-SD] age, 17.2+/-2.1 months; weight, 20.8+/-1.5 kg). Bone anchors were inserted in the proximal metaphysis using nylon, and were longitudinally extracted. For the opposite humerus, anchors were subjected to longitudinal cyclic load (50% of the load at failure of their pair) for 1200 cycles then longitudinally loaded to failure. Anchors were then installed in a similar and adjacent area of these 2(nd) humeri with nylon and cyclically tested perpendicular to the axis of anchor insertion (100% of the longitudinal holding power of their pair) for 1200 cycles, then perpendicularly loaded to failure. Paired t-tests were used to compare holding power before and after longitudinal cyclic testing. Longitudinal holding power of the screw-type anchor in the proximal humerus was 385+/-30 N. Anchor pullout was the only mode of failure. Anchors in the paired humeri did not fail after 1200 cycles of 50% longitudinal loading, and post-cycle holding strength was not different (335+/-87 N; P=.32). Perpendicularly loaded anchors did not fail after 1200 cycles of 100% of opposite longitudinal holding strength, and had post-cycle perpendicular holding strengths of 514+/-72 N. Suture breakage was the mode of failure. Bone anchor holding strength is dependent on orientation of suture load. Screw-type bone anchor holding strength was not affected by longitudinal cyclic loading, and holding strengths of approximately 385 N can be expected in metaphyseal bone of large-breed mature dogs. Perpendicularly loaded anchors have higher failure loads, and holding strength of approximately 514 N can be expected in metaphyseal bone of the proximal humerus.

  13. Characterization of a subunit of the outer dynein arm docking complex necessary for correct flagellar assembly in Leishmania donovani.

    Directory of Open Access Journals (Sweden)

    Simone Harder

    Full Text Available BACKGROUND: In order to proceed through their life cycle, Leishmania parasites switch between sandflies and mammals. The flagellated promastigote cells transmitted by the insect vector are phagocytized by macrophages within the mammalian host and convert into the amastigote stage, which possesses a rudimentary flagellum only. During an earlier proteomic study of the stage differentiation of the parasite we identified a component of the outer dynein arm docking complex, a structure of the flagellar axoneme. The 70 kDa subunit of the outer dynein arm docking complex consists of three subunits altogether and is essential for the assembly of the outer dynein arm onto the doublet microtubule of the flagella. According to the nomenclature of the well-studied Chlamydomonas reinhardtii complex we named the Leishmania protein LdDC2. METHODOLOGY/PRINCIPAL FINDINGS: This study features a characterization of the protein over the life cycle of the parasite. It is synthesized exclusively in the promastigote stage and localizes to the flagellum. Gene replacement mutants of lddc2 show reduced growth rates and diminished flagellar length. Additionally, the normally spindle-shaped promastigote parasites reveal a more spherical cell shape giving them an amastigote-like appearance. The mutants lose their motility and wiggle in place. Ultrastructural analyses reveal that the outer dynein arm is missing. Furthermore, expression of the amastigote-specific A2 gene family was detected in the deletion mutants in the absence of a stage conversion stimulus. In vitro infectivity is slightly increased in the mutant cell line compared to wild-type Leishmania donovani parasites. CONCLUSIONS/SIGNIFICANCE: Our results indicate that the correct assembly of the flagellum has a great influence on the investigated characteristics of Leishmania parasites. The lack of a single flagellar protein causes an aberrant morphology, impaired growth and altered infectiousness of the parasite.

  14. Equilibrium stability and sub-millisecond refolding of a designed single-chain Arc repressor.

    Science.gov (United States)

    Robinson, C R; Sauer, R T

    1996-11-05

    Arc-L1-Arc is a single-chain variant of bacteriophage P22 Arc repressor in which a 15 residue linker joins the C-terminus of one subunit to the N-terminus of an otherwise identical subunit. Spectroscopic probes indicate that the native and denatured state of the single-chain protein are similar to those of the unlinked Arc dimer. In equilibrium experiments, Arc-L1-Arc denatures in a reaction without populated intermediate states as judged by the fits of the denaturation isotherms to a two-state model and by the coincidence of denaturation curves monitored by fluorescence and circular dichroism. Comparison of the equilibrium stabilities of Arc-L1-Arc and unlinked Arc gives an effective concentration of subunits in the denatured single-chain variant of 2.7 (+/- 0.7) mM. The kinetic refolding and unfolding reactions of Arc-L1-Arc also appear to proceed without populated intermediates. The rate constant for Arc-L1-Arc unfolding is about 2-fold faster than that of unlinked Arc, indicating that the linker mediates no significant contacts in the native structure that need to be broken to allow unfolding. As expected, the major effect of the linker occurs during the refolding reaction, where the effective subunit concentration calculated from the bimolecular and unimolecular refolding rate constants is 4.5 (+/- 1.8) mM. The transition states for the unfolding and refolding reactions of Arc-L1-Arc and wild-type Arc have similar solvent exposures as measured by the urea dependencies of the equilibrium and rate constants. In the absence of urea, the single-chain protein refolds very rapidly (kf approximately 10(4) s-1) in a reaction that is essentially complete in the sub-millisecond time regime.

  15. Multiple Locus Variable-Number Tandem-Repeat and Single-Nucleotide Polymorphism-Based Brucella Typing Reveals Multiple Lineages in Brucella melitensis Currently Endemic in China

    Directory of Open Access Journals (Sweden)

    Mingjun Sun

    2017-12-01

    Full Text Available Brucellosis is a worldwide zoonotic disease caused by Brucella spp. In China, brucellosis is recognized as a reemerging disease mainly caused by Brucella melitensis specie. To better understand the currently endemic B. melitensis strains in China, three Brucella genotyping methods were applied to 110 B. melitensis strains obtained in past several years. By MLVA genotyping, five MLVA-8 genotypes were identified, among which genotypes 42 (1-5-3-13-2-2-3-2 was recognized as the predominant genotype, while genotype 63 (1-5-3-13-2-3-3-2 and a novel genotype of 1-5-3-13-2-4-3-2 were second frequently observed. MLVA-16 discerned a total of 57 MLVA-16 genotypes among these Brucella strains, with 41 genotypes being firstly detected and the other 16 genotypes being previously reported. By BruMLSA21 typing, six sequence types (STs were identified, among them ST8 is the most frequently seen in China while the other five STs were firstly detected and designated as ST137, ST138, ST139, ST140, and ST141 by international multilocus sequence typing database. Whole-genome sequence (WGS-single-nucleotide polymorphism (SNP-based typing and phylogenetic analysis resolved Chinese B. melitensis strains into five clusters, reflecting the existence of multiple lineages among these Chinese B. melitensis strains. In phylogeny, Chinese lineages are more closely related to strains collected from East Mediterranean and Middle East countries, such as Turkey, Kuwait, and Iraq. In the next few years, MLVA typing will certainly remain an important epidemiological tool for Brucella infection analysis, as it displays a high discriminatory ability and achieves result largely in agreement with WGS-SNP-based typing. However, WGS-SNP-based typing is found to be the most powerful and reliable method in discerning Brucella strains and will be popular used in the future.

  16. Molecular epidemiology of Staphylococcus aureus bacteremia in a single large Minnesota medical center in 2015 as assessed using MLST, core genome MLST and spa typing.

    Science.gov (United States)

    Park, Kyung-Hwa; Greenwood-Quaintance, Kerryl E; Uhl, James R; Cunningham, Scott A; Chia, Nicholas; Jeraldo, Patricio R; Sampathkumar, Priya; Nelson, Heidi; Patel, Robin

    2017-01-01

    Staphylococcus aureus is a leading cause of bacteremia in hospitalized patients. Whether or not S. aureus bacteremia (SAB) is associated with clonality, implicating potential nosocomial transmission, has not, however, been investigated. Herein, we examined the epidemiology of SAB using whole genome sequencing (WGS). 152 SAB isolates collected over the course of 2015 at a single large Minnesota medical center were studied. Staphylococcus protein A (spa) typing was performed by PCR/Sanger sequencing; multilocus sequence typing (MLST) and core genome MLST (cgMLST) were determined by WGS. Forty-eight isolates (32%) were methicillin-resistant S. aureus (MRSA). The isolates encompassed 66 spa types, clustered into 11 spa clonal complexes (CCs) and 10 singleton types. 88% of 48 MRSA isolates belonged to spa CC-002 or -008. Methicillin-susceptible S. aureus (MSSA) isolates were more genotypically diverse, with 61% distributed across four spa CCs (CC-002, CC-012, CC-008 and CC-084). By MLST, there was 31 sequence types (STs), including 18 divided into 6 CCs and 13 singleton STs. Amongst MSSA isolates, the common MLST clones were CC5 (23%), CC30 (19%), CC8 (15%) and CC15 (11%). Common MRSA clones were CC5 (67%) and CC8 (25%); there were no MRSA isolates in CC45 or CC30. By cgMLST analysis, there were 9 allelic differences between two isolates, with the remaining 150 isolates differing from each other by over 40 alleles. The two isolates were retroactively epidemiologically linked by medical record review. Overall, cgMLST analysis resulted in higher resolution epidemiological typing than did multilocus sequence or spa typing.

  17. Single nucleotide polymorphism-based molecular typing of M. leprae from multicase families of leprosy patients and their surroundings to understand the transmission of leprosy.

    Science.gov (United States)

    Turankar, R P; Lavania, M; Chaitanya, V S; Sengupta, U; Darlong, J; Darlong, F; Siva Sai, K S R; Jadhav, R S

    2014-03-01

    The exact mode of transmission of leprosy is not clearly understood; however, many studies have demonstrated active transmission of leprosy around a source case. Families of five active leprosy cases and their household contacts were chosen from a high endemic area in Purulia. Fifty-two soil samples were also collected from different areas of their houses. DNA was extracted from slit-skin smears (SSS) and soil samples and the Mycobacterium leprae-specific RLEP (129 bp) region was amplified using PCR. Molecular typing of M. leprae was performed for all RLEP PCR-positive samples by single nucleotide polymorphism (SNP) typing and confirmation by DNA sequencing. SSS of these five patients and six out of the total 28 contacts were PCR positive for RLEP whereas 17 soil samples out of 52 showed the presence of M. leprae DNA. SNP typing of M. leprae from all RLEP PCR-positive subjects (patients and smear-positive contacts) and 10 soil samples showed the SNP type 1 genotype. M. leprae DNA from the five leprosy patients and the six contacts was further subtyped and the D subtype was noted in all patients and contacts, except for one contact where the C subtype was identified. Typing followed by subtyping of M. leprae clearly revealed that either the contacts were infected by the patients or both patients and contacts had the same source of infection. It also revealed that the type of M. leprae in the soil in the inhabited areas where patients resided was also of the same type as that found in patients. © 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and Infectious Diseases.

  18. Molecular epidemiology of Staphylococcus aureus bacteremia in a single large Minnesota medical center in 2015 as assessed using MLST, core genome MLST and spa typing.

    Directory of Open Access Journals (Sweden)

    Kyung-Hwa Park

    Full Text Available Staphylococcus aureus is a leading cause of bacteremia in hospitalized patients. Whether or not S. aureus bacteremia (SAB is associated with clonality, implicating potential nosocomial transmission, has not, however, been investigated. Herein, we examined the epidemiology of SAB using whole genome sequencing (WGS. 152 SAB isolates collected over the course of 2015 at a single large Minnesota medical center were studied. Staphylococcus protein A (spa typing was performed by PCR/Sanger sequencing; multilocus sequence typing (MLST and core genome MLST (cgMLST were determined by WGS. Forty-eight isolates (32% were methicillin-resistant S. aureus (MRSA. The isolates encompassed 66 spa types, clustered into 11 spa clonal complexes (CCs and 10 singleton types. 88% of 48 MRSA isolates belonged to spa CC-002 or -008. Methicillin-susceptible S. aureus (MSSA isolates were more genotypically diverse, with 61% distributed across four spa CCs (CC-002, CC-012, CC-008 and CC-084. By MLST, there was 31 sequence types (STs, including 18 divided into 6 CCs and 13 singleton STs. Amongst MSSA isolates, the common MLST clones were CC5 (23%, CC30 (19%, CC8 (15% and CC15 (11%. Common MRSA clones were CC5 (67% and CC8 (25%; there were no MRSA isolates in CC45 or CC30. By cgMLST analysis, there were 9 allelic differences between two isolates, with the remaining 150 isolates differing from each other by over 40 alleles. The two isolates were retroactively epidemiologically linked by medical record review. Overall, cgMLST analysis resulted in higher resolution epidemiological typing than did multilocus sequence or spa typing.

  19. Liposome-Based Adjuvants for Subunit Vaccines: Formulation Strategies for Subunit Antigens and Immunostimulators

    Directory of Open Access Journals (Sweden)

    Signe Tandrup Schmidt

    2016-03-01

    Full Text Available 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 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 protective immunity, and they are often combined with adjuvants to ensure robust immune responses. Adjuvants are capable of enhancing and/or modulating immune responses by exposing antigens to antigen-presenting cells (APCs concomitantly with conferring immune activation signals. Few adjuvant systems have 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 be classified into delivery systems or immunostimulators. Liposomes are versatile delivery systems for antigens, and they can carefully be customized towards desired immune profiles by combining them with immunostimulators and optimizing their composition, physicochemical properties and antigen-loading mode. Immunostimulators represent highly diverse classes of molecules, e.g., lipids, nucleic acids, proteins and peptides, and they are ligands for pattern-recognition receptors (PRRs, which are differentially expressed on APC subsets. Different formulation strategies might thus be required for incorporation of immunostimulators and antigens, respectively, into liposomes, and the choice of immunostimulator should ideally be based on knowledge regarding the

  20. Spiral-type heteropolyhedral coordination network based on single-crystal LiSrPO4: implications for luminescent materials.

    Science.gov (United States)

    Lin, Chun Che; Shen, Chin-Chang; Liu, Ru-Shi

    2013-11-04

    Novel structures of luminescent materials, which are used as light sources for next-generation illumination, are continuously being improved for use in white-light-emitting diodes. Activator-doped known structures are reported as habitual down-conversion phosphors in solid-state lightings and displays. Consequently, the intrinsic qualities of the existent compounds produce deficiencies that limit their applications. Herein we report a spiral-network single-crystal orthophosphate (LiSrPO4) prepared in a platinum crucible with LiCl flux through crystal-growth reactions of SrCl2 and Li3PO4 in air. It crystallizes in a hexagonal system with a=5.0040(2) and c=24.6320(16) Å, V=534.15(5) Å(3), and Z=6 in the space group P6(5). The unit cell is comprised of LiO4 and PO4 tetrahedrons that form a three-dimensional LiPO4(2-) anionic framework with a helical channel structure along the c axis in which the Sr(2+) cation is accommodated. The optical band gap of this composition is about 3.65 eV, as determined by using UV/Vis absorption and diffuse reflection spectra. We used the crystal-growth method to synthesize blue- and red-emitting crystals that exhibited pure color, low reabsorption, a large Stokes shift, and efficient conversion of ultraviolet excitation light into visible light. Emphasis was placed on the development of gratifying structure-related properties of rare-earth luminescent materials and their applications. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Models for the a subunits of the Thermus thermophilus V/A-ATPase and Saccharomyces cerevisiae V-ATPase enzymes by cryo-EM and evolutionary covariance

    Science.gov (United States)

    Schep, Daniel G.; Rubinstein, John L.

    2016-01-01

    Rotary ATPases couple ATP synthesis or hydrolysis to proton translocation across a membrane. However, understanding proton translocation has been hampered by a lack of structural information for the membrane-embedded a subunit. The V/A-ATPase from the eubacterium Thermus thermophilus is similar in structure to the eukaryotic V-ATPase but has a simpler subunit composition and functions in vivo to synthesize ATP rather than pump protons. We determined the T. thermophilus V/A-ATPase structure by cryo-EM at 6.4 Å resolution. Evolutionary covariance analysis allowed tracing of the a subunit sequence within the map, providing a complete model of the rotary ATPase. Comparing the membrane-embedded regions of the T. thermophilus V/A-ATPase and eukaryotic V-ATPase from Saccharomyces cerevisiae allowed identification of the α-helices that belong to the a subunit and revealed the existence of previously unknown subunits in the eukaryotic enzyme. Subsequent evolutionary covariance analysis enabled construction of a model of the a subunit in the S. cerevisae V-ATPase that explains numerous biochemical studies of that enzyme. Comparing the two a subunit structures determined here with a structure of the distantly related a subunit from the bovine F-type ATP synthase revealed a conserved pattern of residues, suggesting a common mechanism for proton transport in all rotary ATPases. PMID:26951669

  2. Technical aspects of typing for HLA-DP alleles using allele-specific DNA in vitro amplification and sequence-specific oligonucleotide probes. Detection of single base mismatches

    DEFF Research Database (Denmark)

    Fugger, L; Morling, N; Ryder, L P

    1990-01-01

    The polymerase chain reaction (PCR) is an effective method for in vitro DNA amplification which combined with probing with synthetic oligonucleotides can be used for, e.g., HLA-typing. We have studied the technical aspects of HLA-DP typing with the technique. DNA from mononuclear nucleated cells...... was extracted with either a simple salting out method or phenol/chloroform. Both DNAs could be readily used for PCR. The MgC2 concentration of the PCR buffer and the annealing temperature of the thermal cycle of the PCR were the two most important variables. The MgCl2 concentration and the temperature must...... be carefully titrated for each primer pair in the PCR. The influence of mismatches between the primer and the DNA template were studied and we found that, by using primers differing only from each other at the 3' end, cross-amplification of closely homologous alleles could be avoided. Thus, single base...

  3. Comparison of semi-automated commercial rep-PCR fingerprinting, spoligotyping, 12-locus MIRU-VNTR typing and single nucleotide polymorphism analysis of the embB gene as molecular typing tools for Mycobacterium bovis.

    Science.gov (United States)

    Armas, Federica; Camperio, Cristina; Coltella, Luana; Selvaggini, Serena; Boniotti, Maria Beatrice; Pacciarini, Maria Lodovica; Di Marco Lo Presti, Vincenzo; Marianelli, Cinzia

    2017-08-04

    Highly discriminatory genotyping strategies are essential in molecular epidemiological studies of tuberculosis. In this study we evaluated, for the first time, the efficacy of the repetitive sequence-based PCR (rep-PCR) DiversiLab Mycobacterium typing kit over spoligotyping, 12-locus mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing and embB single nucleotide polymorphism (SNP) analysis for Mycobacterium bovis typing. A total of 49 M. bovis animal isolates were used. DNA was extracted and genomic DNA was amplified using the DiversiLab Mycobacterium typing kit. The amplified fragments were separated and detected using a microfluidics chip with Agilent 2100. The resulting rep-PCR-based DNA fingerprints were uploaded to and analysed using web-based DiversiLab software through Pearson's correlation coefficient. Rep-PCR DiversiLab grouped M. bovis isolates into ten different clusters. Most isolates sharing identical spoligotype, MIRU-VNTR profile or embB gene polymorphism were grouped into different rep-PCR clusters. Rep-PCR DiversiLab displayed greater discriminatory power than spoligotyping and embB SNP analysis but a lower resolution power than the 12-locus MIRU-VNTR analysis. MIRU-VNTR confirmed that it is superior to the other PCR-based methods tested here. In combination with spoligotyping and 12-locus MIRU-VNTR analysis, rep-PCR improved the discriminatory power for M. bovis typing.

  4. A single exposure to alcohol during brain development induces microencephaly and neuronal losses in genetically susceptible mice, but not in wild type mice.

    Science.gov (United States)

    de Licona, Hannah Klein; Karacay, Bahri; Mahoney, Jo; McDonald, Elizabeth; Luang, Thirath; Bonthius, Daniel J

    2009-05-01

    Maternal alcohol abuse during pregnancy can damage the fetal brain and lead to fetal alcohol syndrome (FAS). Despite public warnings discouraging alcohol use during pregnancy, many pregnant women continue to drink intermittently because they do not believe that occasional exposures to alcohol can be harmful to a fetus. However, because of genetic differences, some fetuses are much more susceptible than others to alcohol-induced brain injury. Thus, a relatively low quantity of alcohol that may be innocuous to most fetuses could damage a genetically susceptible fetus. Neuronal nitric oxide synthase (nNOS) can protect developing mouse neurons against alcohol toxicity by synthesizing neuroprotective nitric oxide. This study examined whether a single exposure to alcohol, which causes no evident injury in wild type mice, can damage the brains of mice genetically deficient for nNOS (nNOS-/- mice). Wild type and nNOS-/- mice received intraperitoneal injections of alcohol (0.0, 2.2, or 4.4mg/g body weight) either as a single dose on postnatal day (PD) 4 or as repeated daily doses over PD4-9. Brain volumes and neuronal numbers within the hippocampus and cerebral cortex were determined on PD10. Alcohol exposure on PD4-9 restricted brain growth and caused neuronal death in both strains of mice, but the severity of microencephaly and neuronal loss were more severe in the nNOS-/- mice than in wild type. The 4.4 mg/g alcohol dose administered on PD4 alone caused significant neuronal loss and microencephaly in the nNOS-/- mice, while this same dose caused no evident injury in the wild type mice. Thus, during development, a single exposure to alcohol can injure a genetically vulnerable brain, while it leaves a wild type brain unaffected. Since the genes that confer alcohol resistance and vulnerability in developing humans are unknown, any particular human fetus is potentially vulnerable. Thus, women should be counseled to consume no alcohol during pregnancy.

  5. Single-nucleotide polymorphism typing analysis for molecular subtyping ofSalmonellaTennessee isolates associated with the 2007 nationwide peanut butter outbreak in the United States.

    Science.gov (United States)

    Dong, Hee-Jin; Cho, Seongbeom; Boxrud, David; Rankin, Shelly; Downe, Francis; Lovchik, Judith; Gibson, Jim; Erdman, Matt; Saeed, A Mahdi

    2017-01-01

    In 2007, a nationwide Salmonella Tennessee outbreak occurred via contaminated peanut butter. Here, we developed a single-nucleotide polymorphism (SNP)-typing method for S . Tennessee to determine the clonal subtypes of S . Tennessee that were associated with the peanut butter outbreak. One seventy-six S . Tennessee isolates from various sources, including humans, animals, food, and the environment, were analyzed by using the SNP technique. Eighty-four representative SNP markers were selected by comparing the sequences of three representative S . Tennessee strains with different multi-locus sequence typing and variable number tandem repeats from our collection. The set of eighty-four SNP markers showed 100% typeability for the 176 strains, with the nucleotide diversity ranging from 0.011 to 0.107 (mean = 0.049 ± 0.018, median = 0.044) for each marker. Among the four clades and nine subtypes generated by the SNP typing, subtype 1, which comprised 142 S . Tennessee strains, was the most predominant. The dominance of single-strain clones in subtype 1 revealed that S . Tennessee is highly clonal regardless of outbreak-association, source, or period of isolation, suggesting the presence of an S . Tennessee strain prototype. Notably, a minimum 18 SNP set was able to determine clonal S . Tennessee strains with similar discrimination power, potentially allowing more rapid and economic strain genotyping for both outbreaks and sporadic cases. The SNP-typing method described here might aid the investigation of the epidemiology and microevolution of pathogenic bacteria by discriminating between outbreak-related and sporadic clinical cases. In addition, this approach enables us to understand the population structure of the bacterial subtypes involved in the outbreak.

  6. New Trypanosoma evansi Type B Isolates from Ethiopian Dromedary Camels.

    Directory of Open Access Journals (Sweden)

    Hadush Birhanu

    2016-04-01

    Full Text Available Trypanosoma (T. evansi is a dyskinetoplastic variant of T. brucei that has gained the ability to be transmitted by all sorts of biting flies. T. evansi can be divided into type A, which is the most abundant and found in Africa, Asia and Latin America and type B, which has so far been isolated only from Kenyan dromedary camels. This study aimed at the isolation and the genetic and phenotypic characterisation of type A and B T. evansi stocks from camels in Northern Ethiopia.T. evansi was isolated in mice by inoculation with the cryopreserved buffy coat of parasitologically confirmed animals. Fourteen stocks were thus isolated and subject to genotyping with PCRs targeting type-specific variant surface glycoprotein genes, mitochondrial minicircles and maxicircles, minisatellite markers and the F1-ATP synthase γ subunit gene. Nine stocks corresponded to type A, two stocks were type B and three stocks represented mixed infections between A and B, but not hybrids. One T. evansi type A stock was completely akinetoplastic. Five stocks were adapted to in vitro culture and subjected to a drug sensitivity assay with melarsomine dihydrochloride, diminazene diaceturate, isometamidium chloride and suramin. In vitro adaptation induced some loss of kinetoplasts within 60 days. No correlation between drug sensitivity and absence of the kinetoplast was observed. Sequencing the full coding sequence of the F1-ATP synthase γ subunit revealed new type-specific single nucleotide polymorphisms and deletions.This study addresses some limitations of current molecular markers for T. evansi genotyping. Polymorphism within the F1-ATP synthase γ subunit gene may provide new markers to identify the T. evansi type that do not rely on variant surface glycoprotein genes or kinetoplast DNA.

  7. Trajectory of chemotherapy for patients with EGFR wild-type advanced pulmonary adenocarcinoma: a single-institution retrospective study

    Directory of Open Access Journals (Sweden)

    Minami S

    2017-02-01

    Full Text Available Seigo Minami, Yoshitaka Ogata, Shouichi Ihara, Suguru Yamamoto, Kiyoshi Komuta Department of Respiratory Medicine, Osaka Police Hospital, Osaka, Japan Background: Pulmonary adenocarcinoma, recently benefited by new cytotoxic and molecularly targeted drugs, has been classified by driver mutations, such as EGFR mutations. The aim of this study was to research the proportions of patients treated with first- to third-line chemotherapy and to find influential factors for the introduction of chemotherapy and survival benefit from chemotherapy.Materials and methods: Data were collected retrospectively on patients who met the following criteria: adenocarcinoma, diagnosed between June 2007 and March 2015 at our hospital, stage IIIB or IV, and EGFR wild type. A nonchemotherapy group of patients who did not receive chemotherapy was compared with a chemotherapy group of patients who received it. The patients who had received first- to third-line chemotherapy between June 2007 and November 2015 at our hospital were also analyzed.Results: During the study period, 46 patients did not receive chemotherapy, while 148, 89, and 48 received first-, second- and third-line chemotherapy, respectively. As predictive factors for unlikely chemotherapy, multivariate logistic analysis detected Eastern Cooperative Oncology Group (ECOG performance status (PS ≥2, hemoglobin <13.2 g/dL, creatinine clearance (Ccr <50.4 mL/min, and CRP ≥0.53 mg/dL. As factors predicting shorter survival after chemotherapy, multivariate Cox proportional-hazard analyses detected age ≥75 years, ECOG PS ≥2, lower lymphocyte counts, and higher CRP for the first line; female, higher neutrophil counts, lower lymphocyte counts, reduced Ccr, hyponatremia, and shorter interval between first- and second-line chemotherapy for the second line; and age ≥75 years, body mass index <18.5 kg/m2, higher neutrophil counts, lower lymphocyte counts, hyponatremia, higher lactate dehydrogenase, and higher

  8. Analyses of herpes simplex virus type 1 latency and reactivation at the single cell level using fluorescent reporter mice

    Science.gov (United States)

    Nelson, D.; Nicoll, M. P.; Connor, V.

    2016-01-01

    Herpes simplex virus type 1 (HSV-1) establishes a latent infection in sensory neurons from which the virus can periodically reactivate. Whilst latency establishment is thought to result from a failure to express immediate-early genes, we have previously shown that subpopulations of the latent neuronal reservoir have undergone lytic promoter activation prior to latency establishment. In the present study, we have investigated the biological properties of such latently infected neuronal subpopulations using Ai6 fluorescent reporter mice. Using this system we have determined that prior ICP0 or TK promoter activation does not correlate with increased latent virus DNA loads within individual cells and that neurons with evidence of historical lytic cycle promoter activity exhibit a comparable frequency of reactivation to that of the general latent cell population. Comparison of viral DNA content within cells harbouring latent HSV-1 genomes and those undergoing the earliest stages of reactivation has revealed that reactivation can initiate from cells harbouring a wide range of HSV-1 genome copies, but that exiting latency is biased towards cells bearing higher latent virus DNA loads. PMID:26694770

  9. Variability within a single type of polyacrylonitrile-based graphite felt after thermal treatment. Part II: chemical properties

    International Nuclear Information System (INIS)

    Rabbow, Thomas J.; Trampert, Markus; Pokorny, Peter; Binder, Paul; Whitehead, Adam H.

    2015-01-01

    Graphite felts are often activated thermally before use in electrochemical reactors. This has the effect of improving wetting and decreasing charge-transfer resistance. In part I of this study, considerable variations were observed between two polyacrylonitrile (PAN)-based felts from different production charges after thermal activation, despite both charges being of the same type of felt from one supplier. A difference due to bulk crystallinity or due to pronounced core-rim structures of the fibres has been excluded. In this second part a limitation from tarry coatings, which are a possible side products of graphitization, could not be corroborated. However, differences were ascribed to variations in the surface chemistry, which was characterised by Boehm method titration and cyclic voltammetry. The composition of the oxides is discussed together with the possible role they play in the activation and wetting of the felts. The rather high amount of oxides suggests that the Boehm method measures subsurface groups in addition to surface groups. The wetting quality of activated felts can be correlated well with the concentration of neutral quinone groups, characterised by cyclic voltammetry

  10. Analysis of MUSIC-type imaging functional for single, thin electromagnetic inhomogeneity in limited-view inverse scattering problem

    Science.gov (United States)

    Ahn, Chi Young; Jeon, Kiwan; Park, Won-Kwang

    2015-06-01

    This study analyzes the well-known MUltiple SIgnal Classification (MUSIC) algorithm to identify unknown support of thin penetrable electromagnetic inhomogeneity from scattered field data collected within the so-called multi-static response matrix in limited-view inverse scattering problems. The mathematical theories of MUSIC are partially discovered, e.g., in the full-view problem, for an unknown target of dielectric contrast or a perfectly conducting crack with the Dirichlet boundary condition (Transverse Magnetic-TM polarization) and so on. Hence, we perform further research to analyze the MUSIC-type imaging functional and to certify some well-known but theoretically unexplained phenomena. For this purpose, we establish a relationship between the MUSIC imaging functional and an infinite series of Bessel functions of integer order of the first kind. This relationship is based on the rigorous asymptotic expansion formula in the existence of a thin inhomogeneity with a smooth supporting curve. Various results of numerical simulation are presented in order to support the identified structure of MUSIC. Although a priori information of the target is needed, we suggest a least condition of range of incident and observation directions to apply MUSIC in the limited-view problem.

  11. Body image mediates negative family climate and deteriorating glycemic control for single adolescents with type 1 diabetes.

    Science.gov (United States)

    Hartl, Amy C; Seiffge-Krenke, Inge; Laursen, Brett

    2015-12-01

    Glycemic control declines during adolescence, as youth with diabetes struggle with pubertal changes and a changing social world. The present study tests whether body image mediates longitudinal links between family climate and changes in adolescent glycemic control. Mediation was hypothesized for nondating adolescents but not for dating adolescents, because the former are thought to remain more family oriented than the latter. Participants were German adolescents with Type 1 diabetes (51 girls, 58 boys; M = 15.84 years, SD = 1.44). Participants reported body image and family climate. Physicians assayed blood HbA1c levels (M = 8.22%, SD = 1.80%) to measure glycemic control. For nondating adolescents, body image mediated associations between family climate and longitudinal changes in glycemic control. Poorer family climate was associated with poorer body image, which predicted deteriorating glycemic control. For dating adolescents, family climate was unassociated with changes in glycemic control. Nondating adolescents may look to parents for feedback on body image, which affects how they manage the challenges of diabetes. Parents and practitioners alike should be alert to the fact that family climate continues to be an important determinant of adolescent adjustment, particularly for those who have not moved into romantic relationships. We know that body image matters to adolescents, but for some youth, body image may be the difference between health and serious physical problems. (PsycINFO Database Record (c) 2015 APA, all rights reserved).

  12. Influences of energization and nucleotide binding on the reaction of Lucifer Yellow vinyl sulfone with the alpha subunits of the chloroplast ATP synthase.

    Science.gov (United States)

    Cunningham, K M; McCarty, R E

    2000-04-18

    The catalytic portion of the chloroplast ATP synthase (CF(1)) consists of five different polypeptides in the stoichiometry alpha(3)beta(3)gammadeltaepsilon and is structurally asymmetric. Asymmetry is readily apparent in the properties of the six nucleotide binding sites and the single-copy, smaller subunits. Asymmetry is also detected in the alpha subunits by the rapid and covalent binding of Lucifer Yellow vinyl sulfone (LY) to one of the three chemically identical alpha subunits. The binding of LY to a single alpha subunit has allowed the investigation of whether asymmetry in the alpha subunits is a permanent feature of CF(1). The development of an electrochemical proton gradient across illuminated thylakoid membranes and the preincubation of CF(1) in solution with Mg(2+)-ATP were found to alter the LY distribution such that multiple alpha subunits were labeled with LY. Illumination of thylakoid membranes doubled the extent of LY labeling, and fluorescence resonance energy transfer measurements indicated that LY was bound to more than one alpha subunit. Since the change in LY distribution was inhibited by proton ionophores (uncouplers), alteration of alpha conformation by illumination is a result of the generation of a proton gradient. Preincubation of CF(1) in solution with Mg(2+)-ATP had no effect on the extent of LY labeling but resulted in multiple alpha subunits binding LY as determined by fluorescence resonance energy transfer measurements. Adenine nucleotides at substrate level concentrations inhibit the reaction of LY with the alpha subunits. No increase in LY labeling was observed when thylakoids were illuminated under conditions in which CF(1) was catalytically active.

  13. The fifth subunit of the (α4β2)2β2 nicotinic ACh receptor modulates maximal ACh responses.

    Science.gov (United States)

    New, Karina; Del Villar, Silvia Garcia; Mazzaferro, Simone; Alcaino, Constanza; Bermudez, Isabel

    2017-06-10

    The fifth subunit in the (α4β2) 2 α4 nicotinic ACh receptor (nAChR) plays a determining role in the pharmacology of this nAChR type. Here, we have examined the role of the fifth subunit in the ACh responses of the (α4β2) 2 β2 nAChR type. The role of the fifth subunit in receptor function was explored using two-electrode voltage clamp electrophysiology, along with subunit-targeted mutagenesis and the substituted cysteine scanning method applied to fully linked (α4β2) 2 β2 receptors. Covalent modification of the cysteine-substituted fifth subunit with a thiol-reactive agent (MTS) caused irreversible inhibition of receptor function. ACh reduced the rate of the reaction to MTS, but the competitive inhibitor dihydro-β-erythroidine had no effect. Alanine substitution of conserved residues that line the core of the agonist sites on α4(+)/β2(-) interfaces did not impair receptor function. However, impairment of agonist binding to α4(+)/β2(-) agonist sites by mutagenesis modified the effect of ACh on the rate of the reaction to MTS. The extent of this effect was dependent on the position of the agonist site relative to the fifth subunit. The fifth subunit in the (α4β2) 2 β2 receptor isoform modulates maximal ACh responses. This effect appears to be driven by a modulatory, and asymmetric, association with the α4(+)/β2(-) agonist sites. © 2017 The British Pharmacological Society.

  14. Basic residues in the 74-83 and 191-198 segments of protein kinase CK2 catalytic subunit are implicated in negative but not in positive regulation by the beta-subunit

    DEFF Research Database (Denmark)

    Sarno, S; Vaglio, P; Marin, O

    1997-01-01

    by the beta-subunit many fold more than that of alpha wild type, while extrastimulation by beta mutant D55L56E57A, observable with alpha wild type, is abolished with these mutants. These data support the conclusion that down regulation by the acidic residues clustered in the N-terminal moiety of beta......Protein kinase CK2 is a ubiquitous pleiotropic serine/threonine protein kinase whose holoenzyme is comprised of two catalytic (alpha and/or alpha') and two non-catalytic, beta-subunits. The beta-subunit possesses antagonist functions that can be physically dissected by generating synthetic...... fragments encompassing its N-terminal and C-terminal domains. Here we show that by mutating basic residues in the 74-77 and in the 191-198 regions of the alpha-subunit, the negative regulation by the beta-subunit and by its N-terminal synthetic fragment CK2beta-(1-77), which is observable using calmodulin...

  15. Spatial filtering nearly eliminates the side-lobes in single- and multi-photon 4pi-type-C super-resolution fluorescence microscopy

    Science.gov (United States)

    Kavya, M.; Regmi, Raju; Mondal, Partha P.

    2013-09-01

    Super-resolution microscopy has tremendously progressed our understanding of cellular biophysics and biochemistry. Specifically, 4pi fluorescence microscopy technique stands out because of its axial super-resolution capability. All types of 4pi-microscopy techniques work well in conjugation with deconvolution techniques to get rid of artifacts due to side-lobes. In this regard, we propose a technique based on spatial filter in a 4pi-type-C confocal setup to get rid of these artifacts. Using a special spatial filter, we have reduced the depth-of-focus. Interference of two similar depth-of-focus beams in a 4π geometry result in substantial reduction of side-lobes. Studies show a reduction of side-lobes by 46% and 76% for single and two photon variant compared to 4pi - type - C confocal system. This is incredible considering the resolving capability of the existing 4pi - type - C confocal microscopy. Moreover, the main lobe is found to be 150 nm for the proposed spatial filtering technique as compared to 690 nm of the state-of-art confocal system. Reconstruction of experimentally obtained 2PE - 4pi data of green fluorescent protein (GFP)-tagged mitocondrial network shows near elimination of artifacts arising out of side-lobes. Proposed technique may find interesting application in fluorescence microscopy, nano-lithography, and cell biology.

  16. Synthesis and evaluation of sequence-specific DNA alkylating agents: effect of alkylation subunits.

    Science.gov (United States)

    Shimizu, Tatsuhiko; Sasaki, Shunta; Minoshima, Masafumi; Shinohara, Ken-ichi; Bando, Toshikazu; Sugiyama, Hiroshi

    2006-01-01

    We have demonstrated that hairpin pyrrole (Py)- imidazole (Im) polyamide-CBI conjugates selectively alkylate predetermined sequences. In this study, we investigated the effect of alkylation subunits, for example conjugates 1-4 with three types of DNA alkylating units, and Py-Im polyamides with indole linker. Conjugate 3 and 4 selectively alkylated the predetermined sequences as described previously, while conjugates 1 and 2 alkylate at mismatched sites.

  17. Large-conductance Ca2+-activated K+ channel β1-subunit knockout mice are not hypertensive

    Science.gov (United States)

    Garver, Hannah; Galligan, James J.; Fink, Gregory D.

    2011-01-01

    Large-conductance Ca2+-activated K+ (BK) channels are composed of pore-forming α-subunits and accessory β1-subunits that modulate Ca2+ sensitivity. BK channels regulate arterial myogenic tone and renal Na+ clearance/K+ reabsorption. Previous studies using indirect or short-term blood pressure measurements found that BK channel β1-subunit knockout (BK β1-KO) mice were hypertensive. We evaluated 24-h mean arterial pressure (MAP) and heart rate in BK β1-KO mice using radiotelemetry. BK β1-KO mice did not have a higher 24-h average MAP when compared with wild-type (WT) mice, although MAP was ∼10 mmHg higher at night. The dose-dependent peak declines in MAP by nifedipine were only slightly larger in BK β1-KO mice. In BK β1-KO mice, giving 1% NaCl to mice to drink for 7 days caused a transient (5 days) elevation of MAP (∼5 mmHg); MAP returned to pre-saline levels by day 6. BK β1-KO mesenteric arteries in vitro demonstrated diminished contractile responses to paxilline, increased reactivity to Bay K 8644 and norepinephrine (NE), and maintained relaxation to isoproterenol. Paxilline and Bay K 8644 did not constrict WT or BK β1-KO mesenteric veins (MV). BK β1-subunits are not expressed in MV. The results indicate that BK β1-KO mice are not hypertensive on normal or high-salt intake. BK channel deficiency increases arterial reactivity to NE and L-type Ca2+ channel function in vitro, but the L-type Ca2+ channel modulation of MAP is not altered in BK β1-KO mice. BK and L-type Ca2+ channels do not modulate murine venous tone. It appears that selective loss of BK channel function in arteries only is not sufficient to cause sustained hypertension. PMID:21131476

  18. Assessment of the efficacy and safety of single platelet-rich plasma injection on different types and grades of facial wrinkles.

    Science.gov (United States)

    Elnehrawy, Naema Y; Ibrahim, Zeinab A; Eltoukhy, Azza M; Nagy, Hala M

    2017-03-01

    Platelet-rich plasma (PRP) is considered as a growing modality for tissue regeneration and a developing research area for clinicians and researchers. PRP injection treatment provides supraphysiological concentrations of growth factors that may help in accelerated tissue remodeling and regeneration. To evaluate the efficacy and safety of single autologous PRP intradermal injection for treatment of facial wrinkles and for facial rejuvenation. A total of 20 subjects with different types of facial wrinkles were included in this study. All subjects received single PRP intradermal injection and were clinically assessed before and after treatment for a period of 8 weeks using Wrinkle Severity Rating Scale (WSRS), Skin Homogeneity and Texture (SHnT) Scale, Physician Assessment Scale, and Subject Satisfaction Scale. The mean value of WSRS reduced from 2.90 ± 0.91 before treatment to 2.10 ± 0.79 after 8 weeks of treatment. The most significant results were with younger subjects that have mild and moderate wrinkles of the nasolabial folds (NLFs). Fourteen of seventeen subjects with NLFs showed more than 25% improvement in their appearance. Side effects of PRP treatment were minimal to mild and with excellent tolerability. Single PRP intradermal injection is well tolerated and capable of rejuvenating the face and producing a significant correction of wrinkles especially the NLFs. © 2016 Wiley Periodicals, Inc.

  19. Types and microbiological spectrum of infections in patients with cirrhosis: A single-centre experience in Upper Egypt.

    Science.gov (United States)

    El-Amin, Hussein; Sabry, Abeer M M; Ahmed, Rabab E; Makhlouf, Nahed A

    2017-09-01

    Egypt has a high prevalence of hepatitis C virus (HCV) and high morbidity and mortality related to cirrhosis complications. Patients with cirrhosis have an increased risk of bacterial infections. Approximately 25-35% of cirrhotics had infections at admission or during hospitalisation. Data on infection among cirrhotics in Egypt are limited. This study aimed to determine the frequency and microbiological spectrum of infections in cirrhotics and possible risk factors. This study was conducted at a tertiary care hospital. The frequency and microbiological spectrum of infections in cirrhotics were determined. The risk factors for infection were evaluated. Of the 100 patients with liver cirrhosis, 61% had infection. Ascitic fluid infection (AFI) was the most common infection (44.3%), followed by urinary tract infection (UTI) (21.3%), respiratory tract infection (RTI) (19.7%), gastroenteritis (6.6%) and skin infection (4.9%). The only risk factor for infection among cirrhotics was diabetes mellitus (DM) (p=0.047). The mean value of mid-arm muscle circumference was significantly lower in the infected group (p=0.047). Among all the cirrhotics, 32.0% had mild to moderate malnutrition and 52.0% had severe malnutrition. The frequency of infection was higher in severe malnutrition (71.2%). The frequency of infections among cirrhotics was 61%. Many types of infections including AFI, RTI, UTI and skin infections were present in patients with liver cirrhosis, but AFI was the most common. DM was the only risk factor for infection, and independent predictors for infection were elevated WBC count and C-reactive protein levels. The frequency of infection was related to the degree of malnutrition. Copyright © 2017 Pan-Arab Association of Gastroenterology. Published by Elsevier B.V. All rights reserved.

  20. Organization of Subunits in the Membrane Domain of the Bovine F-ATPase Revealed by Covalent Cross-linking.

    Science.gov (United States)

    Lee, Jennifer; Ding, ShuJing; Walpole, Thomas B; Holding, Andrew N; Montgomery, Martin G; Fearnley, Ian M; Walker, John E

    2015-05-22

    The F-ATPase in bovine mitochondria is a membrane-bound complex of about 30 subunits of 18 different kinds. Currently, ∼85% of its structure is known. The enzyme has a membrane extrinsic catalytic domain, and a membrane intrinsic domain where the turning of the enzyme's rotor is generated from the transmembrane proton-motive force. The domains are linked by central and peripheral stalks. The central stalk and a hydrophobic ring of c-subunits in the membrane domain constitute the enzyme's rotor. The external surface of the catalytic domain and membrane subunit a are linked by the peripheral stalk, holding them static relative to the rotor. The membrane domain contains six additional subunits named ATP8, e, f, g, DAPIT (diabetes-associated protein in insulin-sensitive tissues), and 6.8PL (6.8-kDa proteolipid), each with a single predicted transmembrane α-helix, but their orientation and topography are unknown. Mutations in ATP8 uncouple the enzyme and interfere with its assembly, but its roles and the roles of the other five subunits are largely unknown. We have reacted accessible amino groups in the enzyme with bifunctional cross-linking agents and identified the linked residues. Cross-links involving the supernumerary subunits, where the structures are not known, show that the C terminus of ATP8 extends ∼70 Å from the membrane into the peripheral stalk and that the N termini of the other supernumerary subunits are on the same side of the membrane, probably in the mitochondrial matrix. These experiments contribute significantly toward building up a complete structural picture of the F-ATPase. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Organization of Subunits in the Membrane Domain of the Bovine F-ATPase Revealed by Covalent Cross-linking*

    Science.gov (United States)

    Lee, Jennifer; Ding, ShuJing; Walpole, Thomas B.; Holding, Andrew N.; Montgomery, Martin G.; Fearnley, Ian M.; Walker, John E.

    2015-01-01

    The F-ATPase in bovine mitochondria is a membrane-bound complex of about 30 subunits of 18 different kinds. Currently, ∼85% of its structure is known. The enzyme has a membrane extrinsic catalytic domain, and a membrane intrinsic domain where the turning of the enzyme's rotor is generated from the transmembrane proton-motive force. The domains are linked by central and peripheral stalks. The central stalk and a hydrophobic ring of c-subunits in the membrane domain constitute the enzyme's rotor. The external surface of the catalytic domain and membrane subunit a are linked by the peripheral stalk, holding them static relative to the rotor. The membrane domain contains six additional subunits named ATP8, e, f, g, DAPIT (diabetes-associated protein in insulin-sensitive tissues), and 6.8PL (6.8-kDa proteolipid), each with a single predicted transmembrane α-helix, but their orientation and topography are unknown. Mutations in ATP8 uncouple the enzyme and interfere with its assembly, but its roles and the roles of the other five subunits are largely unknown. We have reacted accessible amino groups in the enzyme with bifunctional cross-linking agents and identified the linked residues. Cross-links involving the supernumerary subunits, where the structures are not known, show that the C terminus of ATP8 extends ∼70 Å from the membrane into the peripheral stalk and that the N termini of the other supernumerary subunits are on the same side of the membrane, probably in the mitochondrial matrix. These experiments contribute significantly toward building up a complete structural picture of the F-ATPase. PMID:25851905

  2. Single-cell-type quantitative proteomic and ionomic analysis of epidermal bladder cells from the halophyte model plant Mesembryanthemum crystallinum to identify salt-responsive proteins.

    Science.gov (United States)

    Barkla, Bronwyn J; Vera-Estrella, Rosario; Raymond, Carolyn

    2016-05-10

    Epidermal bladder cells (EBC) are large single-celled, specialized, and modified trichomes found on the aerial parts of the halophyte Mesembryanthemum crystallinum. Recent development of a simple but high throughput technique to extract the contents from these cells has provided an opportunity to conduct detailed single-cell-type analyses of their molecular characteristics at high resolution to gain insight into the role of these cells in the salt tolerance of the plant. In this study, we carry out large-scale complementary quantitative proteomic studies using both a label (DIGE) and label-free (GeLC-MS) approach to identify salt-responsive proteins in the EBC extract. Additionally we perform an ionomics analysis (ICP-MS) to follow changes in the amounts of 27 different elements. Using these methods, we were able to identify 54 proteins and nine elements that showed statistically significant changes in the EBC from salt-treated plants. GO enrichment analysis identified a large number of transport proteins but also proteins involved in photosynthesis, primary metabolism and Crassulacean acid metabolism (CAM). Validation of results by western blot, confocal microscopy and enzyme analysis helped to strengthen findings and further our understanding into the role of these specialized cells. As expected EBC accumulated large quantities of sodium, however, the most abundant element was chloride suggesting the sequestration of this ion into the EBC vacuole is just as important for salt tolerance. This single-cell type omics approach shows that epidermal bladder cells of M. crystallinum are metabolically active modified trichomes, with primary metabolism supporting cell growth, ion accumulation, compatible solute synthesis and CAM. Data are available via ProteomeXchange with identifier PXD004045.

  3. Computational approach to predict species-specific type III secretion system (T3SS) effectors using single and multiple genomes.

    Science.gov (United States)

    Hobbs, Christopher K; Porter, Vanessa L; Stow, Maxwell L S; Siame, Bupe A; Tsang, Herbert H; Leung, Ka Yin

    2016-12-19

    Many gram-negative bacteria use type III secretion systems (T3SSs) to translocate effector proteins into host cells. T3SS effectors can give some bacteria a competitive edge over others within the same environment and can help bacteria to invade the host cells and allow them to multiply rapidly within the host. Therefore, developing efficient methods to identify effectors scattered in bacterial genomes can lead to a better understanding of host-pathogen interactions and ultimately to important medical and biotechnological applications. We used 21 genomic and proteomic attributes to create a precise and reliable T3SS effector prediction method called Genome Search for Effectors Tool (GenSET). Five machine learning algorithms were trained on effectors selected from different organisms and a trained (voting) algorithm was then applied to identify other effectors present in the genome testing sets from the same (GenSET Phase 1) or different (GenSET Phase 2) organism. Although a select group of attributes that included the codon adaptation index, probability of expression in inclusion bodies, N-terminal disorder, and G + C content (filtered) were better at discriminating between positive and negative sets, algorithm performance was better when all 21 attributes (unfiltered) were used. Performance scores (sensitivity, specificity and area under the curve) from GenSET Phase 1 were better than those reported for six published methods. More importantly, GenSET Phase 1 ranked more known effectors (70.3%) in the top 40 ranked proteins and predicted 10-80% more effectors than three available programs in three of the four organisms tested. GenSET Phase 2 predicted 43.8% effectors in the top 40 ranked proteins when tested on four related or unrelated organisms. The lower prediction rates from GenSET Phase 2 may be due to the presence of different translocation signals in effectors from different T3SS families. The species-specific GenSET Phase 1 method offers an alternative

  4. The utility of an electronic adherence assessment device in type 2 diabetes mellitus: a pilot study of single medication

    Directory of Open Access Journals (Sweden)

    Nadir Kheir

    2010-07-01

    attitudes and practices of the patients, as demonstrated by our KAP analysis and correlations.Keywords: Medication Events Monitoring System (MEMS, type 2 diabetes mellitus, drug therapy, medication adherence

  5. Initiation factor 2, tRNA, and 50S subunits cooperatively stabilize mRNAs on the ribosome during initiation

    Science.gov (United States)

    Masuda, Tomoaki; Petrov, Alexey N.; Iizuka, Ryo; Funatsu, Takashi; Puglisi, Joseph D.; Uemura, Sotaro

    2012-01-01

    Initiation factor 2 (IF2) is a key factor in initiation of bacterial protein synthesis. It recruits initiator tRNA to the small ribosomal subunit and facilitates joining of the large ribosomal subunit. Using reconstituted translation system of Escherichia coli and optical tweezers, we directly measure the rupture force between single ribosomal complexes and mRNAs for initiation complexes in the presence and the absence of IF2. We demonstrate that IF2 together with codon recognition by initiator tRNA increases the force required to dislocate mRNA from the ribosome complexes; mRNA stabilization by IF2 required the presence of a joined 50S subunit, and was independent of bound guanine nucleotide. IF2 thus helps lock the 70S ribosome over the start codon during initiation, thus maintaining reading frame. Our results show how mRNA is progressively stabilized on the ribosome through distinct steps of initiation. PMID:22411833

  6. Setting Single or Multiple Goals for Diet and Physical Activity Behaviors Improves Cardiovascular Disease Risk Factors in Adults With Type 2 Diabetes: A Pragmatic Pilot Randomized Trial.

    Science.gov (United States)

    Swoboda, Christine M; Miller, Carla K; Wills, Celia E

    2016-08-01

    The purpose of this study was to evaluate a 4-month telephone-based goal-setting and decision support intervention among adults with type 2 diabetes mellitus (T2DM) and multiple risk factors for cardiovascular disease (CVD). A randomized pretest-posttest control group design was employed. Overweight or obese adults aged 40 to 75 years with T2DM and ≥1 additional CVD risk factor were provided with individualized CVD risk information. At baseline and each biweekly telephone call, the multiple-goal group self-selected both diet- and physical activity-related goals, the single goal group set a single goal, and the control group received information about community health resources. Dietary intake was assessed via a food frequency questionnaire, physical activity via questionnaire, and A1C and blood lipids via fasting fingerstick sample. Between-group differences for clinical (ie, A1C, blood pressure, and blood lipids), physical activity, and dietary variables were evaluated using Kruskal-Wallis, Mann-Whitney U, analysis of variance, and t tests. From pre- to postintervention, the single-goal group demonstrated significant improvement in systolic blood pressure and intake of servings of fruits, vegetables, and refined grains (all P < .05). The multiple-goal group reported significant reduction in percent energy from total, saturated, monounsaturated, and trans fat intake and significant increase in leisure time walking (all P < .05). A multiple-goal approach over 4 months can improve dietary and physical activity outcomes, while a single-goal approach may facilitate improvement in one behavioral domain. Additional research is needed to evaluate maintenance of the achieved changes. © 2016 The Author(s).

  7. Functional divergence of chloroplast Cpn60α subunits during Arabidopsis embryo development.

    Directory of Open Access Journals (Sweden)

    Xiaolong Ke

    2017-09-01

    Full Text Available Chaperonins are a class of molecular chaperones that assist in the folding and assembly of a wide range of substrates. In plants, chloroplast chaperonins are composed of two different types of subunits, Cpn60α and Cpn60β, and duplication of Cpn60α and Cpn60β genes occurs in a high proportion of plants. However, the importance of multiple Cpn60α and Cpn60β genes in plants is poorly understood. In this study, we found that loss-of-function of CPNA2 (AtCpn60α2, a gene encoding the minor Cpn60α subunit in Arabidopsis thaliana, resulted in arrested embryo development at the globular stage, whereas the other AtCpn60α gene encoding the dominant Cpn60α subunit, CPNA1 (AtCpn60α1, mainly affected embryonic cotyledon development at the torpedo stage and thereafter. Further studies demonstrated that CPNA2 can form a functional chaperonin with CPNB2 (AtCpn60β2 and CPNB3 (AtCpn60β3, while the functional partners of CPNA1 are CPNB1 (AtCpn60β1 and CPNB2. We also revealed that the functional chaperonin containing CPNA2 could assist the folding of a specific substrate, KASI (β-ketoacyl-[acyl carrier protein] synthase I, and that the KASI protein level was remarkably reduced due to loss-of-function of CPNA2. Furthermore, the reduction in the KASI protein level was shown to be the possible cause for the arrest of cpna2 embryos. Our findings indicate that the two Cpn60α subunits in Arabidopsis play different roles during embryo development through forming distinct chaperonins with specific AtCpn60β to assist the folding of particular substrates, thus providing novel insights into functional divergence of Cpn60α subunits in plants.

  8. Two ferritin subunits from disk abalone (Haliotis discus discus): cloning, characterization and expression analysis.

    Science.gov (United States)

    De Zoysa, Mahanama; Lee, Jehee

    2007-09-01

    Ferritin plays a key role in cellular iron metabolism, which includes iron storage and detoxification. From disk abalone, Haliotis discus discus, the cDNA that encodes the two ferritin subunits abalone ferritin subunit 1 (Abf1) and abalone ferritin subunit 2 (Abf2) were cloned. The complete cDNA coding sequences for Abf1 and Abf2 contained 621 and 549 bp, encoding for 207 and 183 amino acid residues, respectively. The H. discus discus Abf2 subunit contained a highly conserved motif for the ferroxidase center, which consists of seven residues of a typical vertebrate heavy-chain ferritin with a typical stem-loop structure. Abf2 mRNA contains a 27 bp iron-responsive element (IRE) in the 5'UTR position. This IRE exhibited 96% similarity with pearl and Pacific oyster and 67% similarity with human H type IREs. However, the Abf1 subunit had neither ferroxidase center residues nor the IRE motif sequence; instead, it contained iron-binding region signature 2 (IBRS) residues. Recombinant Abf1 and Abf2 proteins were purified and the respective sizes were about 24 and 21 kDa. Abf1 and Abf2 exhibited iron-chelating activity 44.2% and 22.0%, respectively, at protein concentration of 6 microg/ml. Analysis of tissue-specific expression by RT-PCR revealed that Abf1 and Abf2 ferritin mRNAs were expressed in various abalone tissues, such as gill, mantle, gonad, foot and digestive tract in a wide distribution profile, but Abf2 expression was more prominent than Abf1.

  9. Functional divergence of chloroplast Cpn60α subunits during Arabidopsis embryo development

    Science.gov (United States)

    Ke, Xiaolong; Zou, Wenxuan; Ren, Yafang; Wang, Zhiqin; Li, Jin; Wu, Xuan

    2017-01-01

    Chaperonins are a class of molecular chaperones that assist in the folding and assembly of a wide range of substrates. In plants, chloroplast chaperonins are composed of two different types of subunits, Cpn60α and Cpn60β, and duplication of Cpn60α and Cpn60β genes occurs in a high proportion of plants. However, the importance of multiple Cpn60α and Cpn60β genes in plants is poorly understood. In this study, we found that loss-of-function of CPNA2 (AtCpn60α2), a gene encoding the minor Cpn60α subunit in Arabidopsis thaliana, resulted in arrested embryo development at the globular stage, whereas the other AtCpn60α gene encoding the dominant Cpn60α subunit, CPNA1 (AtCpn60α1), mainly affected embryonic cotyledon development at the torpedo stage and thereafter. Further studies demonstrated that CPNA2 can form a functional chaperonin with CPNB2 (AtCpn60β2) and CPNB3 (AtCpn60β3), while the functional partners of CPNA1 are CPNB1 (AtCpn60β1) and CPNB2. We also revealed that the functional chaperonin containing CPNA2 could assist the folding of a specific substrate, KASI (β-ketoacyl-[acyl carrier protein] synthase I), and that the KASI protein level was remarkably reduced due to loss-of-function of CPNA2. Furthermore, the reduction in the KASI protein level was shown to be the possible cause for the arrest of cpna2 embryos. Our findings indicate that the two Cpn60α subunits in Arabidopsis play different roles during embryo development through forming distinct chaperonins with specific AtCpn60β to assist the folding of particular substrates, thus providing novel insights into functional divergence of Cpn60α subunits in plants. PMID:28961247

  10. Peristalsis is impaired in the small intestine of mice lacking the P2X3 subunit.

    Science.gov (United States)

    Bian, Xiaochun; Ren, Jianhua; DeVries, Matthew; Schnegelsberg, Birthe; Cockayne, Debra A; Ford, Anthony P D W; Galligan, James J

    2003-08-15

    P2X receptors are ATP-gated cation channels composed of one or more of seven different subunits. P2X receptors participate in intestinal neurotransmission but the subunit composition of enteric P2X receptors is unknown. In this study, we used tissues from P2X3 wild-type (P2X3+/+) mice and mice in which the P2X3 subunit gene had been deleted (P2X3-/-) to investigate the role of this subunit in neurotransmission in the intestine. RT-PCR analysis of mRNA from intestinal tissues verified P2X3 gene deletion. Intracellular electrophysiological methods were used to record synaptic and drug-induced responses from myenteric neurons in vitro. Drug-induced longitudinal muscle contractions were studied in vitro. Intraluminal pressure-induced reflex contractions (peristalsis) of ileal segments were studied in vitro using a modified Trendelenburg preparation. Gastrointestinal transit was measured as the progression in 30 min of a liquid radioactive marker administered by gavage to fasted mice. Fast excitatory postsynaptic potentials recorded from S neurons (motoneurons and interneurons) were similar in tissues from P2X3+/+ and P2X3-/- mice. S neurons from P2X3+/+ and P2X3-/- mice were depolarized by application of ATP but not alpha,beta-methylene ATP, an agonist of P2X3 subunit-containing receptors. ATP and alpha,beta-methylene ATP induced depolarization of AH (sensory) neurons from P2X3+/+ mice. ATP, but not alpha,beta-methylene ATP, caused depolarization of AH neurons from P2X3-/- mice. Peristalsis was inhibited in ileal segments from P2X3-/- mice but longitudinal muscle contractions caused by nicotine and bethanechol were similar in segments from P2X3+/+ and P2X3-/- mice. Gastrointestinal transit was similar in P2X3+/+ and P2X3-/- mice. It is concluded that P2X3 subunit-containing receptors participate in neural pathways underlying peristalsis in the mouse intestine in vitro. P2X3 subunits are localized to AH (sensory) but not S neurons. P2X3 receptors may contribute to

  11. Epac Signaling Is Required for Cocaine-Induced Change in AMPA Receptor Subunit Composition in the Ventral Tegmental Area.

    Science.gov (United States)

    Liu, Xiaojie; Chen, Yao; Tong, Jiaqing; Reynolds, Ashley M; Proudfoot, Sarah C; Qi, Jinshun; Penzes, Peter; Lu, Youming; Liu, Qing-Song

    2016-04-27

    Exchange protein directly activated by cAMP (Epac) and protein kinase A (PKA) are intracellular receptors for cAMP. Although PKA and its downstream effectors have been studied extensively in the context of drug addiction, whether and how Epac regulates cellular and behavioral effects of drugs of abuse remain essentially unknown. Epac is known to regulate AMPA receptor (AMPAR) trafficking. Previous studies have shown that a single cocaine exposure in vivo leads to an increase in GluA2-lacking AMPARs in dopamine neurons of the ventral tegmental area (VTA). We tested the hypothesis that Epac mediates cocaine-induced changes in AMPAR subunit composition in the VTA. We report that a single cocaine injection in vivo in wild-type mice leads to inward rectification of EPSCs and renders EPSCs sensitive to a GluA2-lacking AMPAR blocker in VTA dopamine neurons. The cocaine-induced increase in GluA2-lacking AMPARs was absent in Epac2-deficient mice but not in Epac1-deficient mice. In addition, activation of Epac with the selective Epac agonist 8-CPT-2Me-cAMP (8-CPT) recapitulated the cocaine-induced increase in GluA2-lacking AMPARs, and the effects of 8-CPT were mediated by Epac2. We also show that conditioned place preference to cocaine was impaired in Epac2-deficient mice and in mice in which Epac2 was knocked down in the VTA but was not significantly altered in Epac1-deficient mice. Together, these results suggest that Epac2 is critically involved in the cocaine-induced change in AMPAR subunit composition and drug-cue associative learning. Addictive drugs, such as cocaine, induce long-lasting adaptions in the reward circuits of the brain. A single intraperitoneal injection of cocaine leads to changes in the composition and property of the AMPAR that carries excitatory inputs to dopamine neurons. Here, we provide evidence that exchange protein directly activated by cAMP (Epac), a cAMP sensor protein, is required for the cocaine-induced changes of the AMPAR. We found that the

  12. Influence of type-I fimbriae and fluid shear stress on bacterial behavior and multicellular architecture of earlyEscherichia colibiofilms at single-cell resolution.

    Science.gov (United States)

    Wang, Liyun; Keatch, Robert; Zhao, Qi; Wright, John A; Bryant, Clare E; Redmann, Anna L; Terentjev, Eugene M

    2018-01-12

    Biofilm formation on abiotic surfaces in food and medical industry can cause severe contamination and infection, yet how biological and physical factors determine cellular architecture of early biofilms and bacterial behavior of the constituent cells remains largely unknown. In this study we examine the specific role of type-I fimbriae in nascent stages of biofilm formation and the response of micro-colonies to environmental flow shear at single-cell resolution. The results show that type-I fimbriae are not required for reversible adhesion from plankton, but critical for irreversible adhesion of Escherichia coli ( E.coli ) MG1655 forming biofilms on polyethylene terephthalate (PET) surfaces. Besides establishing a firm cell-surface contact, the irreversible adhesion seems necessary to initiate the proliferation of E.coli on the surface. After application of shear stress, bacterial retention is dominated by the 3D architecture of colonies independent of the population and the multi-layered structure could protect the embedded cells from being insulted by fluid shear, while cell membrane permeability mainly depends on the biofilm population and the duration time of the shear stress. Importance Bacterial biofilms could lead to severe contamination problems in medical devices and food processing equipment. However, biofilms are usually studied at a rough macroscopic level, thus little is known about how individual bacterial behavior within biofilms and multicellular architecture are influenced by bacterial appendages (e.g. pili/fimbriae) and environmental factors during early biofilm formation. We apply Confocal Laser Scanning Microscopy (CLSM) to visualize E.coli micro-colonies at single-cell resolution. Our findings suggest that type-I fimbriae are vital to the initiation of bacterial proliferation on surfaces and that the responses of biofilm architecture and cell membrane permeability of constituent bacteria to fluid shear stress are different, which are

  13. Enrichment of HLA Types and Single-Nucleotide Polymorphism Associated With Non-progression in a Strictly Defined Cohort of HIV-1 Controllers

    Directory of Open Access Journals (Sweden)

    Samantha J. Westrop

    2017-06-01

    Full Text Available HIV-1 controllers (HIC are extremely rare patients with the ability to control viral replication, maintain unchanging CD4 T-cell count, and evade disease progression for extensive periods of time, in the absence of antiretroviral therapy. In order to establish the representation of key genetic correlates of atypical disease progression within a cohort of HIV-1+ individuals who control viral replication, we examine four-digit resolution HLA type and single-nucleotide polymorphisms (SNP previously identified to be correlated to non-progressive infection, in strictly defined HIC. Clinical histories were examined to identify patients exhibiting HIC status. Genomic DNA was extracted, and high definition HLA typing and genome-wide SNP analysis was performed. Data were compared with frequencies of SNP in European long-term non-progressors (LTNP and primary infection cohorts. HLA-B alleles associated with atypical disease progression were at very high frequencies in the group of five HIC studied. All four HIC of European ancestry were HLA-B*57+ and half were also HLA-B*27+. All HIC, including one of self-reported African ethnicity, had the HLA-Cw*0602 allele, and the HLA-DQ9 allele was present only in HIC of European ancestry. A median 95% of the top 19 SNP known to be associated with LTNP status was observed in European HIC (range 78–100%; 17/19 of the SNP considered mapped to chromosome 6 in the HLA region, whereas 2/19 mapped to chromosome 8. The HIC investigated here demonstrated high enrichment of HLA types and SNP previously associated with long-term non-progression. These findings suggest that the extreme non-progressive phenotype considered here is associated with a genetic signature characterized by a single-genetic unit centered around the HLA-B*57 haplotype and the possible additive effect of HLA-B*27.

  14. Vancomycin-resistant vanB-type Enterococcus faecium isolates expressing varying levels of vancomycin resistance and being highly prevalent among neonatal patients in a single ICU

    Directory of Open Access Journals (Sweden)

    Werner Guido

    2012-05-01

    Full Text Available Abstract Background Vancomycin-resistant isolates of E. faecalis and E. faecium are of special concern and patients at risk of acquiring a VRE colonization/infection include also intensively-cared neonates. We describe here an ongoing high prevalence of VanB type E. faecium in a neonatal ICU hardly to identify by routine diagnostics. Methods During a 10 months’ key period 71 E. faecium isolates including 67 vanB-type isolates from 61 patients were collected non-selectively. Vancomycin resistance was determined by different MIC methods (broth microdilution, Vitek® 2 including two Etest® protocols (McFarland 0.5/2.0. on Mueller-Hinton/Brain Heart Infusion agars. Performance of three chromogenic VRE agars to identify the vanB type outbreak VRE was evaluated (BrillianceTM VRE agar, chromIDTM VRE agar, CHROMagarTM VRE. Isolates were genotyped by SmaI- and CeuI-macrorestriction analysis in PFGE, plasmid profiling, vanB Southern hybridisations as well as MLST typing. Results Majority of vanB isolates (n = 56, 79% belonged to a single ST192 outbreak strain type showing an identical PFGE pattern and analyzed representative isolates revealed a chromosomal localization of a vanB2-Tn5382 cluster type. Vancomycin MICs in cation-adjusted MH broth revealed a susceptible value of ≤4 mg/L for 31 (55% of the 56 outbreak VRE isolates. Etest® vancomycin on MH and BHI agars revealed only two vanB VRE isolates with a susceptible result; in general Etest® MIC results were about 1 to 2 doubling dilutions higher than MICs assessed in broth and values after the 48 h readout were 0.5 to 1 doubling dilutions higher for vanB VRE. Of all vanB type VRE only three, three and two isolates did not grow on BrillianceTM VRE agar, chromIDTM VRE agar and CHROMagarTM VRE, respectively. Permanent cross contamination via the patients’ surrounding appeared as a possible risk factor for permanent VRE colonization/infection. Conclusions Low level expression of van

  15. New and easy strategy for cloning, expression, purification, and characterization of the 5S subunit of transcarboxylase from Propionibacterium f. shermanii.

    Science.gov (United States)

    Kumar Bhat, Rakesh; Berger, Stefan

    2007-01-01

    Methylmalonyl CoA-oxalacetate transcarboxylase (EC 2. 1. 3. 1) from Propionibacterium f. shermanii is a biotin dependent enzyme which transfers CO2 from methylmalonyl-CoA (MMCoA) to pyruvate via a carboxylated biotin group to form oxalacetate. It is composed of three subunits, the central cylindrical hexameric 12S subunit, the outer six dimeric 5S subunit, and the twelve 1.3S linkers. We here report the cloning, sequencing, expression, and purification of the 5S subunit. The gene was identified by matching the amino acid sequence with that of deposited in the NCBI database. For cloned 5S subunit sequence shows regions of high homology with that of pyruvate carboxylase and oxaloacetate decarboxylase. The gene encoding the 5S subunit was cloned into the pTXB1 vector. The expressed 5S subunit was purified to apparent homogeneity by a single step process by using Intein mediated protein ligation (IPL) method. The cloned 5S gene encodes a protein of 505 amino acids and of M(r) 55,700.

  16. Factors associated with anxiety and depression among type 2 diabetes outpatients in Malaysia: a descriptive cross-sectional single-centre study.

    Science.gov (United States)

    Ganasegeran, Kurubaran; Renganathan, Pukunan; Manaf, Rizal Abdul; Al-Dubai, Sami Abdo Radman

    2014-04-23

    To determine the prevalence and factors associated with anxiety and depression among type 2 diabetes outpatients in Malaysia. Descriptive, cross-sectional single-centre study with universal sampling of all patients with type 2 diabetes. Endocrinology clinic of medical outpatient department in a Malaysian public hospital. All 169 patients with type 2 diabetes (men, n=99; women, n=70) aged between 18 and 90 years who acquired follow-up treatment from the endocrinology clinic in the month of September 2013. The validated Hospital Anxiety and Depression Scale (HADS), sociodemographic characteristics and clinical health information from patient records. Of the total 169 patients surveyed, anxiety and depression were found in 53 (31.4%) and 68 (40.3%), respectively. In multivariate analysis, age, ethnicity and ischaemic heart disease were significantly associated with anxiety, while age, ethnicity and monthly household income were significantly associated with depression. Sociodemographics and clinical health factors were important correlates of anxiety and depression among patients with diabetes. Integrated psychological and medical care to boost self-determination and confidence in the management of diabetes would catalyse optimal health outcomes among patients with diabetes.

  17. Progress on n-type doping of AlGaN alloys on AlN single crystal substrates for UV optoelectronic applications

    Energy Technology Data Exchange (ETDEWEB)

    Collazo, Ramon; Rice, Anthony; Tweedie, James; Sitar, Zlatko [Department of Materials Science and Engineering, North Carolina State University, Raleigh, NC (United States); Mita, Seiji; Xie, Jinqiao; Dalmau, Rafael [HexaTech, Inc., Morrisville, NC (United States)

    2011-07-15

    As the building blocks of deep UV light emitting diode (LED) technology and high-power electronic devices, AlGaN alloys have attracted considerable attention. In this study, AlGaN films with varying compositions doped with Si were deposited on homoepitaxial AlN layers grown on AlN single crystal substrates. The room temperature resistivity of AlGaN alloys of different compositions grown on AlN and sapphire substrates with a constant Si doping level of 6x10{sup 18} cm{sup -3} was compared. AlGaN films grown on AlN substrates consistently exhibited a lower n-type resistivity than those grown on sapphire. An n-type resistivity of 0.1 {omega} cm was obtained for an AlGaN film with 80% Al content and a sheet resistance of 235 {omega}/sq. for an AlGaN film with 70% Al content. The carrier activation energy as a function of Al content in AlGaN for these n-type films was measured. For compositions below 80% Al, the activation energy was around 15 meV due to impurity potential screening. For higher Al compositions, the carrier concentration was limited by a high compensation ratio, except for AlN, which has activation energy of 250 meV. (copyright 2011 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

  18. A Simplified Semiquantitative Meal Bolus Strategy Combined with Single- and Dual-Hormone Closed-Loop Delivery in Patients with Type 1 Diabetes: A Pilot Study.

    Science.gov (United States)

    Gingras, Véronique; Haidar, Ahmad; Messier, Virginie; Legault, Laurent; Ladouceur, Martin; Rabasa-Lhoret, Rémi

    2016-08-01

    Single- and dual-hormone closed-loop systems can improve glycemic control and have the potential to reduce carbohydrate-counting burden for patients with type 1 diabetes; however, simplification of meal insulin calculation should not compromise glycemic control. We compared in a randomized outpatient pilot trial: (1) a single-hormone closed-loop system accompanied with carbohydrate-content matched boluses versus accompanied with a simplified meal bolus strategy, and (2) a dual-hormone closed-loop system accompanied with carbohydrate-content matched boluses versus accompanied with a simplified meal bolus strategy. Carbohydrate-matched boluses were based on the participant's carbohydrate meal content estimation whereas the simplified strategy involved the selection, by participants, of a semi-quantitative meal carbohydrate-content size: snack, regular, large, or very large meal. Each participant also underwent sensor-augmented pump therapy. Basal insulin delivery was more aggressive with the simplified bolus. The primary outcome was mean sensor glucose level over a 15-h daytime period. Twelve participants were recruited (48.2 ± 16.0 years old; HbA1c 7.4% ± 0.9%) to compare the two bolus strategies during single- and dual-hormone closed-loop delivery. A similar mean sensor glucose level (15 h) was achieved with the carbohydrate-matched boluses and simplified strategy using single-hormone (median [interquartile]: 7.6 [7.2-8.1] vs. 8.0 [7.0-8.6] mmol/L; P = 0.90) and dual-hormone closed-loop systems (7.6 [6.7-9.1] vs. 7.0 [6.4-8.2] mmol/L; P = 0.08). Exploratory analyses showed that, as compared with sensor-augmented pump therapy, there was an increased time spent in hypoglycemia with the simplified strategy but not with the carbohydrate-matched boluses. Though the algorithm employed in this pilot study may lead to an increased risk for hypoglycemia, this strategy has the potential to reduce the carbohydrate-counting burden in patients with type

  19. Quantitative Characterization of Bivalent Probes for a Dual Bromodomain Protein, Transcription Initiation Factor TFIID Subunit 1.

    Science.gov (United States)

    Suh, Junghyun L; Watts, Brian; Stuckey, Jacob I; Norris-Drouin, Jacqueline L; Cholensky, Stephanie H; Dickson, Bradley M; An, Yi; Mathea, Sebastian; Salah, Eidarus; Knapp, Stefan; Khan, Abid; Adams, Alexander T; Strahl, Brian D; Sagum, Cari A; Bedford, Mark T; James, Lindsey I; Kireev, Dmitri B; Frye, Stephen V

    2018-03-28

    Multivalent binding is an efficient means to enhance the affinity and specificity of chemical probes targeting multidomain proteins in order to study their function and role in disease. While the theory of multivalent binding is straightforward, physical and structural characterization of bivalent binding encounters multiple technical difficulties. We present a case study where a combination of experimental techniques and computational simulations was used to comprehensively characterize the binding and structure-affinity relationships for a series of Bromosporine-based bivalent bromodomain ligands with a bivalent protein, Transcription Initiation Factor TFIID subunit 1 (TAF1). Experimental techniques-Isothermal Titration Calorimetry, X-ray Crystallography, Circular Dichroism, Size Exclusion Chromatography-Multi-Angle Light Scattering, and Surface Plasmon Resonance-were used to determine structures, binding affinities, and kinetics of monovalent ligands and bivalent ligands with varying linker lengths. The experimental data for monomeric ligands were fed into explicit computational simulations, in which both ligand and protein species were present in a broad range of concentrations, and in up to a 100 s time regime, to match experimental conditions. These simulations provided accurate estimates for apparent affinities (in good agreement with experimental data), individual dissociation microconstants and other microscopic details for each type of protein-ligand complex. We conclude that the expected efficiency of bivalent ligands in a cellular context is difficult to estimate by a single technique in vitro, due to higher order associations favored at the concentrations used, and other complicating processes. Rather, a combination of structural, biophysical, and computational approaches should be utilized to estimate and characterize multivalent interactions.

  20. Brain cytochrome oxidase subunit complementary DNAs: isolation, subcloning, sequencing, light and electron microscopic in situ hybridization of transcripts, and regulation by neuronal activity.

    Science.gov (United States)

    Wong-Riley, M T; Mullen, M A; Huang, Z; Guyer, C

    1997-02-01

    The goal of the present study was to isolate, for the first time, cytochrome oxidase subunit genes from murine brain complementary DNA library and to characterize the expression of these genes from mitochondrial and nuclear sources at both light and electron microscopic levels. Brain subunit III (mitochondrial) shared 100% identity with that of murine L cells. Subunit VIa (nuclear) was known to have tissue-specific isoforms in other species: the ubiquitous liver isoform and the heart/muscle isoform. Our brain subunit VIa shared 93% homology with that of the rat liver and 100% identity with the recently reported murine liver isoform, which is only 62% identical to that of the rat heart isoform. In situ hybridization with riboprobes revealed messenger RNA labelling that was similar, though not identical, to that of cytochrome oxidase histochemistry. Monocular enucleation in adult mice induced a significant down-regulation of both subunit messages in the contralateral lateral geniculate nucleus. However, the decrease in subunit III messenger RNAs surpassed that of subunit VIa at all time periods examined, suggesting that mitochondrial gene expression is more tightly regulated by neuronal activity than that of nuclear ones. At the electron microscopic level, subunit III messenger RNA was localized to the mitochondrial compartment in both cell bodies and processes, while that of nuclear-encoded subunit VIa was present exclusively in the extramitochondrial compartment of somata and not of dendrites or axons. Surprisingly, the message was primarily associated with the rough endoplasmic reticulum, suggesting a novel pathway for its synthesis and trafficking. Our results indicate that the unique properties of neurons impose special requirements for subunits of a single mitochondrial enzyme with dual genomic origins. At sites of high energy demands (such as postsynaptic dendrites and some axon terminals), mitochondrial-encoded cytochrome oxidase subunits can be locally

  1. Mapping of protein phosphatase-6 association with its SAPS domain regulatory subunit using a model of helical repeats

    Directory of Open Access Journals (Sweden)

    Edelson Jessica R

    2009-10-01

    Full Text Available Abstract Background Helical repeat motifs are common among regulatory subunits for type-1 and type-2A protein Ser/Thr phosphatases. Yeast Sit4 is a distinctive type-2A phosphatase that has dedicated regulatory subunits named Sit4-Associated Proteins (SAPS. These subunits are conserved, and three human SAPS-related proteins are known to associate with PP6 phosphatase, the Sit4 human homologue. Results Here we show that endogenous SAPS subunit PP6R3 co-precipitates half of PP6 in cell extracts, and the SAPS region of PP6R3 is sufficient for binding PP6. The SAPS domain of recombinant GST-PP6R3 is relatively resistant to trypsin despite having many K and R residues, and the purified SAPS domain (residues 1-513 has a circular dichroic spectrum indicative of mostly alpha helical structure. We used sequence alignments and 3D-jury methods to develop alternative models for the SAPS domain, based on available structures of other helical repeat proteins. The models were used to select sites for charge-reversal substitutions in the SAPS domain of PP6R3 that were tested by co-precipitation of endogenous PP6c with FLAG-tagged PP6R3 from mammalian cells. Mutations that reduced binding with PP6 suggest that SAPS adopts a helical repeat similar to the structure of p115 golgin, but distinct from the PP2A-A subunit. These mutations did not cause perturbations in overall PP6R3 conformation, evidenced by no change in kinetics or preferential cleavage by chymotrypsin. Conclusion The conserved SAPS domain in PP6R3 forms helical repeats similar to those in golgin p115 and negatively charged residues in interhelical loops are used to associate specifically with PP6. The results advance understanding of how distinctive helical repeat subunits uniquely distribute and differentially regulate closely related Ser/Thr phosphatases.

  2. Mediator complex subunit 12 exon 2 mutation analysis in different subtypes of smooth muscle tumors confirms genetic heterogeneity.

    Science.gov (United States)

    de Graaff, Marieke A; Cleton-Jansen, Anne-Marie; Szuhai, Károly; Bovée, Judith V M G

    2013-08-01

    Recently, heterozygous mutations in exon 2 of the mediator complex subunit 12 gene have been described in 50% to 70% of uterine leiomyomas; the recurrent nature of these mutations suggests an important role in their pathogenesis. Mediator complex subunit 12 is involved in regulation of transcription and Wnt signaling. So far, little is known about the pathogenesis of the different subtypes of extrauterine leiomyomas and leiomyosarcomas. We performed mutation analysis of mediator complex subunit 12 and immunohistochemistry for β-catenin, using 69 tumors of 64 patients including 19 uterine leiomyomas, 6 abdominal leiomyomas, 9 angioleiomyomas, 5 piloleiomyomas, and 7 uterine and 23 soft tissue leiomyosarcomas. In line with previous observations, 58% of uterine leiomyomas carried a mediator complex subunit 12 mutation. However, all other extrauterine leiomyomas were negative with the exception of 1 abdominal leiomyoma with a likely primary uterine origin. Of the 30 leiomyosarcomas, only 1 uterine tumor harbored a mutation. A new observation is the identification of 3 tumors with a homozygous mutation; a monosomy X or interstitial deletion was excluded. β-Catenin immunohistochemistry showed nuclear positivity in only 55% of the mediator complex subunit 12-mutated uterine leiomyomas, suggesting the involvement of pathways other than canonical Wnt signaling in tumorigenesis. Interestingly, 80% of mediator complex subunit 12 wild-type sporadic piloleiomyomas displayed nuclear β-catenin positivity, indicating its involvement in this leiomyoma subtype. The lack of mediator complex subunit 12 mutations in extrauterine leiomyomas and leiomyosarcomas indicates that these tumors arise through a different pathway, emphasizing the genetic heterogeneity of smooth muscle tumors. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. Influence of low-molecular-weight glutenin subunit haplotypes on dough rheology and baking quality in elite common wheat varieties

    Science.gov (United States)

    The low molecular weight glutenin subunits (LMW-GSs) are a class of wheat seed storage proteins directly involved in the formation of gluten. Depending on the first amino acid residue of the mature proteins, the LMW-GSs are divided into methionine, serine or isoleucine type. These proteins are encod...

  4. Deletion of the GluA1 AMPA Receptor Subunit Alters the Expression of Short-Term Memory

    Science.gov (United States)

    Sanderson, David J.; Sprengel, Rolf; Seeburg, Peter H.; Bannerman, David M.

    2011-01-01

    Deletion of the GluA1 AMPA receptor subunit selectively impairs short-term memory for spatial locations. We further investigated this deficit by examining memory for discrete nonspatial visual stimuli in an operant chamber. Unconditioned suppression of magazine responding to visual stimuli was measured in wild-type and GluA1 knockout mice.…

  5. The progeny of a single virgin B cell predominates the human recall B cell response to the capsular polysaccharide of Haemophilus influenzae type b

    DEFF Research Database (Denmark)

    Barington, T; Hougs, L; Juul, L

    1996-01-01

    of Haemophilus influenzae type b coupled to tetanus toxoid. We combined affinity purification of circulating vaccine-induced Ab-secreting cells with PCR amplification of cDNA followed by cloning and sequencing. Forty-eight and 42 kappa VJ gene transcripts were analyzed from two adults, respectively. Both......Restricted V region diversity is a key feature of Abs to many haptens and simple polysaccharides. Two possible mechanisms exist: 1) selection of many clonally unrelated B cells using very similar or identical VDJ and VJ rearrangements; and 2) selection of a heavily expanded progeny of few virgin B...... cells. How many virgin B cells eventually give rise to the total Ab response to a simple Ag is a fundamental immunologic question. In this report, we address this question in human adults by analyzing the rearranged VkappaJkappa genes of B cells responding to a single dose of the capsular polysaccharide...

  6. Validation of a single nucleotide polymorphism (SNP) typing assay with 49 SNPs for forensic genetic testing in a laboratory accredited according to the ISO 17025 standard

    DEFF Research Database (Denmark)

    Børsting, Claus; Rockenbauer, Eszter; Morling, Niels

    2009-01-01

    cases and 33 twin cases were typed at least twice for the 49 SNPs. All electropherograms were analysed independently by two expert analysts prior to approval. Based on these results, detailed guidelines for analysis of the SBE products were developed. With these guidelines, the peak height ratio...... of a heterozygous allele call or the signal to noise ratio of a homozygous allele call is compared with previously obtained ratios. A laboratory protocol for analysis of SBE products was developed where allele calls with unusual ratios were highlighted to facilitate the analysis of difficult allele calls......A multiplex assay with 49 autosomal single nucleotide polymorphisms (SNPs) developed for human identification was validated for forensic genetic casework and accredited according to the ISO 17025 standard. The multiplex assay was based on the SNPforID 52plex SNP assay [J.J. Sanchez, C. Phillips, C...

  7. Moderation of effects of AAC based on setting and types of aided AAC on outcome variables: an aggregate study of single-case research with individuals with ASD.

    Science.gov (United States)

    Ganz, Jennifer B; Rispoli, Mandy J; Mason, Rose Ann; Hong, Ee Rea

    2014-06-01

    The purpose of this meta-analysis was to evaluate the potential moderating effects of intervention setting and type of aided augmentative and alternative communication (AAC) on outcome variables for students with autism spectrum disorders. Improvement rate difference, an effect size measure, was used to calculate aggregate effects across 35 single-case research studies. Results indicated that the largest effects for aided AAC were observed in general education settings. With respect to communication outcomes, both speech generating devices (SGDs) and the Picture Exchange Communication System (PECS) were associated with larger effects than other picture-based systems. With respect to challenging behaviour outcomes, SGDs produced larger effects than PECS. This aggregate study highlights the importance of considering intervention setting, choice of AAC system and target outcomes when designing and planning an aided AAC intervention.

  8. Online single particle analysis of ice particle residuals from mountain-top mixed-phase clouds using laboratory derived particle type assignment

    Science.gov (United States)

    Schmidt, Susan; Schneider, Johannes; Klimach, Thomas; Mertes, Stephan; Schenk, Ludwig Paul; Kupiszewski, Piotr; Curtius, Joachim; Borrmann, Stephan

    2017-01-01

    In situ single particle analysis of ice particle residuals (IPRs) and out-of-cloud aerosol particles was conducted by means of laser ablation mass spectrometry during the intensive INUIT-JFJ/CLACE campaign at the high alpine research station Jungfraujoch (3580 m a.s.l.) in January-February 2013. During the 4-week campaign more than 70 000 out-of-cloud aerosol particles and 595 IPRs were analyzed covering a particle size diameter range from 100 nm to 3 µm. The IPRs were sampled during 273 h while the station was covered by mixed-phase clouds at ambient temperatures between -27 and -6 °C. The identification of particle types is based on laboratory studies of different types of biological, mineral and anthropogenic aerosol particles. The outcome of these laboratory studies was characteristic marker peaks for each investigated particle type. These marker peaks were applied to the field data. In the sampled IPRs we identified a larger number fraction of primary aerosol particles, like soil dust (13 ± 5 %) and minerals (11 ± 5 %), in comparison to out-of-cloud aerosol particles (2.4 ± 0.4 and 0.4 ± 0.1 %, respectively). Additionally, anthropogenic aerosol particles, such as particles from industrial emissions and lead-containing particles, were found to be more abundant in the IPRs than in the out-of-cloud aerosol. In the out-of-cloud aerosol we identified a large fraction of aged particles (31 ± 5 %), including organic material and secondary inorganics, whereas this particle type was much less abundant (2.7 ± 1.3 %) in the IPRs. In a selected subset of the data where a direct comparison between out-of-cloud aerosol particles and IPRs in air masses with similar origin was possible, a pronounced enhancement of biological particles was found in the IPRs.

  9. Screening of 134 single nucleotide polymorphisms (SNPs) previously associated with type 2 diabetes replicates association with 12 SNPs in nine genes.

    Science.gov (United States)

    Willer, Cristen J; Bonnycastle, Lori L; Conneely, Karen N; Duren, William L; Jackson, Anne U; Scott, Laura J; Narisu, Narisu; Chines, Peter S; Skol, Andrew; Stringham, Heather M; Petrie, John; Erdos, Michael R; Swift, Amy J; Enloe, Sareena T; Sprau, Andrew G; Smith, Eboni; Tong, Maurine; Doheny, Kimberly F; Pugh, Elizabeth W; Watanabe, Richard M; Buchanan, Thomas A; Valle, Timo T; Bergman, Richard N; Tuomilehto, Jaakko; Mohlke, Karen L; Collins, Francis S; Boehnke, Michael

    2007-01-01

    More than 120 published reports have described associations between single nucleotide polymorphisms (SNPs) and type 2 diabetes. However, multiple studies of the same variant have often been discordant. From a literature search, we identified previously reported type 2 diabetes-associated SNPs. We initially genotyped 134 SNPs on 786 index case subjects from type 2 diabetes families and 617 control subjects with normal glucose tolerance from Finland and excluded from analysis 20 SNPs in strong linkage disequilibrium (r(2) > 0.8) with another typed SNP. Of the 114 SNPs examined, we followed up the 20 most significant SNPs (P < 0.10) on an additional 384 case subjects and 366 control subjects from a population-based study in Finland. In the combined data, we replicated association (P < 0.05) for 12 SNPs: PPARG Pro12Ala and His447, KCNJ11 Glu23Lys and rs5210, TNF -857, SLC2A2 Ile110Thr, HNF1A/TCF1 rs2701175 and GE117881_360, PCK1 -232, NEUROD1 Thr45Ala, IL6 -598, and ENPP1 Lys121Gln. The replication of 12 SNPs of 114 tested was significantly greater than expected by chance under the null hypothesis of no association (P = 0.012). We observed that SNPs from genes that had three or more previous reports of association were significantly more likely to be replicated in our sample (P = 0.03), although we also replicated 4 of 58 SNPs from genes that had only one previous report of association.

  10. PRKACA: the catalytic subunit of protein kinase A and adrenocortical tumors

    Directory of Open Access Journals (Sweden)

    Annabel Sophie Berthon

    2015-05-01

    Full Text Available Cyclic-AMP (cAMP-dependent protein kinase (PKA is the main effector of cAMP signaling in all tissues. Inactivating mutations of the PRKAR1A gene, coding for the type 1A regulatory subunit of PKA, are responsible for Carney complex and primary pigmented nodular adrenocortical disease (PPNAD. PRKAR1A inactivation and PKA dysregulation have been implicated in various types of adrenocortical pathologies associated with ACTH-independent Cushing syndrome (AICS from PPNAD to adrenocortical adenomas and cancer, and other forms of bilateral adrenocortical hyperplasias (BAH. More recently, mutations of PRKACA, the gene coding for the catalytic subunit C alpha (Cα, were also identified in the pathogenesis of adrenocortical tumors. PRKACA copy number gain was found in the germline of several patients with cortisol-producing BAH, whereas the somatic Leu206Arg (c.617A>C recurrent PRKACA mutation was found in as many as half of all adrenocortical adenomas associated with AICS. In vitro analysis demonstrated that this mutation led to constitutive Cα activity, unregulated by its main partners, the PKA regulatory subunits. In this review, we summarize the current understanding of the involvement of PRKACA in adrenocortical tumorigenesis, and our understanding of PKA’s role in adrenocortical lesions. We also discuss potential therapeutic advances that can be made through targeting of PRKACA and the PKA pathway.

  11. Single-Tube Dodecaplex PCR Panel of Polymorphic Microsatellite Markers Closely Linked to theDMPKCTG Repeat for Preimplantation Genetic Diagnosis of Myotonic Dystrophy Type 1.

    Science.gov (United States)

    Lian, Mulias; Zhao, Mingjue; Lee, Caroline G; Chong, Samuel S

    2017-06-01

    Preimplantation genetic diagnosis (PGD) of myotonic dystrophy type 1 (DM1) currently uses conventional PCR to detect nonexpanded dystrophia myotonica protein kinase ( DMPK ) alleles or triplet-primed PCR to detect the CTG-expanded alleles, coupled with analysis of linked microsatellite markers to increase diagnostic accuracy. We aimed to simplify the process of identification and selection of informative linked markers for application to DM1 PGD. An in silico search was performed to identify all markers within 1-1.5 Mb flanking the DMPK gene. Five previously known (D19S559, APOC2, D19S543, D19S112, and BV209569) and 7 novel (DM45050, DM45178, DM45209, DM45958, DM46513, DM46892, and DM47004.1) markers with potentially high heterozygosity values and polymorphism information content were selected and optimized in a single-tube multiplex PCR panel. Analysis of 184 DNA samples of Chinese and Caucasian individuals (91 from unrelated, anonymized cord blood of Chinese babies born at the National University Hospital, Singapore, and 93 Caucasian DNA samples from the Human Variation Panel HD100CAU) confirmed the high polymorphism indices of all markers (polymorphism information content >0.5), with observed heterozygosity values ranging from 0.62-0.93. All individuals were heterozygous for at least 6 markers, with 99.5% of individuals heterozygous for at least 2 markers on either side of the DMPK CTG repeat. The dodecaplex marker assay was successfully validated on 42 single cells and 12 whole genome amplified single cells. The DM1 multiplex PCR panel is suitable for use in DM1 PGD either as a standalone linkage-based assay or as a complement to DMPK CTG repeat expansion-mutation detection. © 2017 American Association for Clinical Chemistry.

  12. 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

    2004-01-01

    subunit ribosomal DNA as well as in size and shape. Based on morphological similarity and partial large subunit ribosomal DNA evidence, we erect the new genus, Togula gen. nov. with the emended type species Togula britannica (Herdman) comb. nov. Based on differences in division pattern and partial large...

  13. Sequential loading of cohesin subunits during the first meiotic prophase of grasshoppers.

    Science.gov (United States)

    Valdeolmillos, Ana M; Viera, Alberto; Page, Jesús; Prieto, Ignacio; Santos, Juan L; Parra, María Teresa; Heck, Margarete M S; Martínez-A, Carlos; Barbero, José L; Suja, José A; Rufas, Julio S

    2007-02-23

    The cohesin complexes play a key role in chromosome segregation during both mitosis and meiosis. They establish sister chromatid cohesion between duplicating DNA molecules during S-phase, but they also have an important role during postreplicative double-strand break repair in mitosis, as well as during recombination between homologous chromosomes in meiosis. An additional function in meiosis is related to the sister kinetochore cohesion, so they can be pulled by microtubules to the same