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Sample records for 51-kda subunit maps

  1. The neuronal nicotinic acetylcholine receptor {alpha}7 subunit gene: Cloning, mapping, structure, and targeting in mouse

    SciTech Connect

    Orr-Urtreger, A.; Baldini, A.; Beaudet, A.L.

    1994-09-01

    The neuronal nicotinic acetylcholine receptor {alpha}7 subunit is a member of a family of ligand-gated ion channels, and is the only subunit know to bind {alpha}-bungarotoxin in mammalian brain. {alpha}-Bungarotoxin binding sites are known to be more abundant in the hippocampus of mouse strains that are particularly sensitive to nicotine-induced seizures. The {alpha}7 receptor is highly permeable to calcium, which could suggest a role in synaptic plasticity in the nervous system. Auditory gating deficiency, an abnormal response to a second auditory stimulus, is characteristic of schizophrenia. Mouse strains that exhibit a similar gating deficit have reduced hippocampal expression of the {alpha}7 subunit. We have cloned and sequenced the full length cDNA for the mouse {alpha}7 gene (Acra-7) and characterized its gene structure. The murine {alpha}7 shares amino acid identity of 99% and 93% with the rat and human {alpha}7 subunits, respectively. Using an interspecies backcross panel, the murine gene was mapped to chromosome 7 near the p locus, a region syntenic with human chromosome 15; the human gene (CHRNA7) was confirmed to map to 15q13-q14 by FISH. To generate a mouse {alpha}7 mutant by homologous recombination, we have constructed a replacement vector which will delete transmembrane domains II-IV and the cytoplasmic domain from the gene product. Recombinant embryonic stem (ES) cell clones were selected and used to develop mouse chimeras that are currently being bred to obtain germline transmission.

  2. Cytochrome oxidase subunit V gene of Neurospora crassa: DNA sequences, chromosomal mapping, and evidence that the cya-4 locus specifies the structural gene for subunit V.

    PubMed Central

    Sachs, M S; Bertrand, H; Metzenberg, R L; RajBhandary, U L

    1989-01-01

    The sequences of cDNA and genomic DNA clones for Neurospora cytochrome oxidase subunit V show that the protein is synthesized as a 171-amino-acid precursor containing a 27-amino-acid N-terminal extension. The subunit V protein sequence is 34% identical to that of Saccharomyces cerevisiae subunit V; these proteins, as well as the corresponding bovine subunit, subunit IV, contain a single hydrophobic domain which most likely spans the inner mitochondrial membrane. The Neurospora crassa subunit V gene (cox5) contains two introns, 398 and 68 nucleotides long, which share the conserved intron boundaries 5'GTRNGT...CAG3' and the internal consensus sequence ACTRACA. Two short sequences, YGCCAG and YCCGTTY, are repeated four times each in the cox5 gene upstream of the mRNA 5' termini. The cox5 mRNA 5' ends are heterogeneous, with the major mRNA 5' end located 144 to 147 nucleotides upstream from the translational start site. The mRNA contains a 3'-untranslated region of 186 to 187 nucleotides. Using restriction-fragment-length polymorphism, we mapped the cox5 gene to linkage group IIR, close to the arg-5 locus. Since one of the mutations causing cytochrome oxidase deficiency in N. crassa, cya-4-23, also maps there, we transformed the cya-4-23 strain with the wild-type cox5 gene. In contrast to cya-4-23 cells, which grow slowly, cox5 transformants grew quickly, contained cytochrome oxidase, and had 8- to 11-fold-higher levels of subunit V in their mitochondria. These data suggest (i) that the cya-4 locus in N. crassa specifies structural information for cytochrome oxidase subunit V and (ii) that, in N. crassa, as in S. cerevisiae, deficiencies in the production of nuclearly encoded cytochrome oxidase subunits result in deficiency in cytochrome oxidase activity. Finally, we show that the lower levels of subunit V in cya-4-23 cells are most likely due to substantially reduced levels of translatable subunit V mRNA. Images PMID:2540423

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

    PubMed

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

    2010-05-01

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

  4. Mapping of the alpha-bungarotoxin binding site within the alpha subunit of the acetylcholine receptor.

    PubMed Central

    Neumann, D; Barchan, D; Safran, A; Gershoni, J M; Fuchs, S

    1986-01-01

    Synthetic peptides and their respective antibodies have been used in order to map the alpha-bungarotoxin binding site within the alpha subunit of the acetylcholine receptor. By using antibodies to a synthetic peptide corresponding to residues 169-181 of the alpha subunit, we demonstrate that this sequence is included within the 18-kDa toxin binding fragment previously reported. Furthermore, the 18-kDa fragment was also found to bind a monoclonal antibody (5.5) directed against the cholinergic binding site. Sequential proteolysis of the acetylcholine receptor with trypsin, prior to Staphylococcus aureus V8 protease digestion, resulted in a 15-kDa toxin binding fragment that is included within the 18-kDa fragment but is shorter than it only at its carboxyl terminus. This 15-kDa fragment therefore initiates beyond Asp-152 and terminates in the region of Arg-313/Lys-314. In addition, experiments are reported that indicate that in the intact acetylcholine receptor, Cys-128 and/or Cys-142 are not crosslinked by disulfide bridges with any of the cysteines (at positions 192, 193, and 222) that reside in the 15-kDa toxin binding fragment. Finally, the synthetic dodecapeptide Lys-His-Trp-Val-Tyr-Tyr-Thr-Cys-Cys-Pro-Asp-Thr, which is present in the 15-kDa fragment (corresponding to residues 185-196 of the alpha subunit) was shown to bind alpha-bungarotoxin directly. This binding was completely inhibited by competition with d-tubocurarine. Images PMID:3458258

  5. Mapping of the alpha-bungarotoxin binding site within the alpha subunit of the acetylcholine receptor.

    PubMed

    Neumann, D; Barchan, D; Safran, A; Gershoni, J M; Fuchs, S

    1986-05-01

    Synthetic peptides and their respective antibodies have been used in order to map the alpha-bungarotoxin binding site within the alpha subunit of the acetylcholine receptor. By using antibodies to a synthetic peptide corresponding to residues 169-181 of the alpha subunit, we demonstrate that this sequence is included within the 18-kDa toxin binding fragment previously reported. Furthermore, the 18-kDa fragment was also found to bind a monoclonal antibody (5.5) directed against the cholinergic binding site. Sequential proteolysis of the acetylcholine receptor with trypsin, prior to Staphylococcus aureus V8 protease digestion, resulted in a 15-kDa toxin binding fragment that is included within the 18-kDa fragment but is shorter than it only at its carboxyl terminus. This 15-kDa fragment therefore initiates beyond Asp-152 and terminates in the region of Arg-313/Lys-314. In addition, experiments are reported that indicate that in the intact acetylcholine receptor, Cys-128 and/or Cys-142 are not crosslinked by disulfide bridges with any of the cysteines (at positions 192, 193, and 222) that reside in the 15-kDa toxin binding fragment. Finally, the synthetic dodecapeptide Lys-His-Trp-Val-Tyr-Tyr-Thr-Cys-Cys-Pro-Asp-Thr, which is present in the 15-kDa fragment (corresponding to residues 185-196 of the alpha subunit) was shown to bind alpha-bungarotoxin directly. This binding was completely inhibited by competition with d-tubocurarine.

  6. Temperature-dependent instability of the cTnI subunit in NIST SRM2921 characterized by tryptic peptide mapping.

    PubMed

    van der Burgt, Yuri E M; Cobbaert, Christa M; Dalebout, Hans; Smit, Nico; Deelder, André M

    2012-08-01

    In this study temperature-dependent instability of the cTnI subunit of the three-protein complex NIST SRM2921 was demonstrated using a mass spectrometric tryptic peptide mapping approach. The results were compared to the cTnI subunit obtained as a protein standard from Calbiochem with identical amino acid sequence. Both the three-protein complex from NIST as well as the cTnI subunit were incubated at elevated temperatures and then evaluated with respect to the primary sequence. The corresponding peptide maps were analyzed using LC-MS/MS. From a Mascot database search in combination with "semiTrypsin" tolerance it was found that two peptide backbone cleavages had occurred in subunit cTnI in NIST SRM2921 material upon incubation at 37°C, namely between amino acids at 148/149 and 194/195. The Calbiochem standard did not show increased levels of "unexpected" peptides in tryptic peptide maps. One of the two peptide backbone cleavages could also be monitored using a "single-step" MALDI-MS approach, i.e. without the need for peptide separation. The amount of degradation appeared rather constant in replicate temperature-instability experiments. However, for accurate quantification internal labelled standards are needed.

  7. Fine mapping of sequential neutralization epitopes on the subunit protein VP8 of human rotavirus.

    PubMed Central

    Kovacs-Nolan, Jennifer; Yoo, Dongwan; Mine, Yoshinori

    2003-01-01

    The epitopes of the HRV (human rotavirus), especially those involved in virus neutralization, have not been determined in their entirety, and would have significant implications for HRV vaccine development. In the present study, we report on the epitope mapping and identification of sequential neutralization epitopes, on the Wa strain HRV subunit protein VP8, using synthetic overlapping peptides. Polyclonal antibodies against recombinant Wa VP8 were produced previously in chicken, and purified from egg yolk, which showed neutralizing activity against HRV in vitro. Overlapping VP8 peptide fragments were synthesized and probed with the anti-VP8 antibodies, revealing five sequential epitopes on VP8. Further analysis suggested that three of the five epitopes detected, M1-L10, I55-D66 and L223-P234, were involved in virus neutralization, indicating that sequential epitopes may also be important for the HRV neutralization. The interactions of the antibodies with the five epitopes were characterized by an examination of the critical amino acids involved in antibody binding. Epitopes comprised primarily of hydrophobic amino acid residues, followed by polar and charged residues. The more critical amino acids appeared to be located near the centre of the epitopes, with proline, isoleucine, serine, glutamine and arginine playing an important role in the binding of antibody to the VP8 epitopes. PMID:12901721

  8. The Gα4 G protein subunit interacts with the MAP kinase ERK2 using a D-motif that regulates developmental morphogenesis in Dictyostelium

    PubMed Central

    Nguyen, Hoai-Nghia; Hadwiger, Jeffrey A.

    2009-01-01

    G protein Gα subunits contribute to the specificity of different signal transduction pathways in Dictyostelium discoideum but Gα subunit-effector interactions have not been previously identified. The requirement of the Dictyostelium Gα4 subunit for MAP kinase (MAPK) activation and the identification of a putative MAPK docking site (D-motif) in this subunit suggested a possible interaction between the Gα4 subunit and MAPKs. In vivo association of the Gα4 subunit and ERK2 was demonstrated by pull-down and co-immunoprecipitation assays. Alteration of the D-motif reduced Gα4 subunit-ERK2 interactions but only slightly altered MAPK activation in response to folate. Expression of the Gα4 subunit with the altered D-motif in gα4− cells allowed for slug formation but not the morphogenesis associated with culmination. Expression of this mutant Gα4 subunit was sufficient to rescue chemotactic movement to folate. Alteration of the D-motif also reduced the aggregation defect associated with constitutively active Gα4 subunits. These results suggest Gα4 subunit-MAPK interactions are necessary for developmental morphogenesis but not for chemotaxis to folate. PMID:19765570

  9. Tryptic mapping and membrane topology of the benzodiazepine receptor alpha-subunit

    SciTech Connect

    Lentes, K.U.; Venter, J.C.

    1986-05-01

    Rat brain membrane benzodiazepine receptors (BZR) were photoaffinity labelled specifically (in presence or absence of 6 ..mu..M clonazepam) with 10 nM /sup 3/H-flunitrazepam (FNZ). Digestion of the FNZ-labelled, membrane-bound BZR with 200 ..mu..g trypsin/mg membrane protein yielded H/sub 2/O-soluble BZR-fragments of molecular mass (M/sub r/) 34, 31, 28, 24, 21, 18, 16, 12, 10 and 7kDa. Because the 34kDa-peptide is the largest fragment containing a FNZ-binding site they conclude that this represents the extracellular domain of the BZR. In the remaining pellet two labelled peptides with M/sub r/ of 44kDa and 28kDa were found that required the use of detergents for their solubilization; they therefore contain the membrane anchoring domain. Digestion of the 0.5% Na-deoxycholate solubilized, intact BZR (M/sub r/ 51kDa) resulted in the same tryptic pattern as the membrane form of the receptor plus two larger fragments of M/sub r/ 45kDa and 40kDa. Arrangement of all tryptic fragments with reference to the FNZ binding site reveals a membrane topology of the BZR alpha-subunit with 67% (34kDa) for the extracellular domain, 21% (11kDa) for the membrane anchoring domain and 12% (6kDa) for a putative cytoplasmic domain. The overlap between some of the labelled fragments suggest that the BZ binding site must be located near the membrane surface of the extracellular domain.

  10. Structural features of the γ subunit of the Escherichia coli F1 ATPase revealed by a 4.4-Å resolution map obtained by x-ray crystallography

    PubMed Central

    Hausrath, Andrew C.; Grüber, Gerhard; Matthews, Brian W.; Capaldi, Roderick A.

    1999-01-01

    The F1 part of the F1FO ATP synthase from Escherichia coli has been crystallized and its structure determined to 4.4-Å resolution by using molecular replacement based on the structure of the beef-heart mitochondrial enzyme. The bacterial F1 consists of five subunits with stoichiometry α3, β3, γ, δ, and ɛ. δ was removed before crystallization. In agreement with the structure of the beef-heart mitochondrial enzyme, although not that from rat liver, the present study suggests that the α and β subunits are arranged in a hexagonal barrel but depart from exact 3-fold symmetry. In the structures of both beef heart and rat-liver mitochondrial F1, less than half of the structure of the γ subunit was seen because of presumed disorder in the crystals. The present electron-density map includes a number of rod-shaped features which appear to correspond to additional α-helical regions within the γ subunit. These suggest that the γ subunit traverses the full length of the stalk that links the F1 and FO parts and makes significant contacts with the c subunit ring of FO. PMID:10570135

  11. Domain mapping of the retinal cyclic GMP phosphodiesterase gamma-subunit. Function of the domains encoded by the three exons of the gamma-subunit gene.

    PubMed

    Takemoto, D J; Hurt, D; Oppert, B; Cunnick, J

    1992-02-01

    Retinal rod-outer-segment phosphodiesterase (PDE) is a heterotetramer consisting of two similar, but not identical, catalytic subunits (alpha and beta) and two identical inhibitory subunits (gamma 2). Previously, we have reported that the site of PDE alpha/beta interaction with PDE gamma is located within residues 54-87 [Cunnick, Hurt, Oppert, Sakamoto & Takemoto (1990) Biochem. J. 271, 721-727]. The site for PDE gamma interaction with transducin alpha (T alpha) was found to encompass residues 24-45 of PDE gamma [Morrison, Cunnick, Oppert & Takemoto (1989) J. Biol. Chem. 264, 11671-11681]. In order to identify binding sites and other functional domains of PDE gamma, the three peptides which are encoded by the three exons of the PDE gamma gene were synthesized chemically. These exons encode for residues 1-49, 50-62 and 63-87 of bovine PDE gamma [Piriev, Purishko, Khramtsov & Lipkin (1990) Dokl. Akad. Nauk. SSSR 315, 229-230]. The peptide encompassing residues 63-87 was inhibitory in a PDE assay, whereas peptides 1-49 and 50-62 had no effect. However, both peptides 1-49 and 63-87 bound to PDE alpha/beta in a solid-phase binding assay. Only peptide 1-49 bound to T alpha.GTP[S] (GTP[S] is guanosine 5'-[gamma-thio]triphosphate). These data confirm that the inhibitory region of PDE gamma is encoded by exon 3 (residues 63-87), whereas a separate binding site for PDE alpha/beta and for T alpha.GTP[S] is encoded by exon 1 (residues 1-49). To study further the structure-function relationship of PDE gamma, this entire protein and two mutants were chemically synthesized. One mutant (-CT) lacked residues 78-87, whereas another replaced tyrosine-84 with glycine (TYR-84). Whereas the synthetic PDE gamma inhibited PDE alpha/beta catalytic activity, the -CT and TVR-84 mutants did not. All three synthetic proteins bound to both PDE alpha/beta and and T alpha.GTP[S]. These data confirm the presence of an alternative binding site on PDE gamma and demonstrate the importance of tyrosine

  12. Mapping of the gene for the p60 subunit of the human chromatin assembly factor (CAF1A) to the Down syndrome region of chromosome 21

    SciTech Connect

    Blouin, J.L.; Gos, A.; Morris, M.A.; Antonarakis, S.E.

    1996-04-15

    Exon trapping was used to clone portions of genes from the Down syndrome critical region (DSCR) of human chromosome 21. One trapped sequence showed complete homology with nucleotide sequence U20980 (GenBank), which corresponds to the gene for the p60 subunit of the human chromatin assembly factor-1 (CAF1A). We mapped this gene to human chromosome 21 by fluorescence in situ hybridization, by the use of somatic cell hybrids, and by hybridization to chromosome 21-specific YACs and cosmids. The CAF1A gene localizes to YACs 745H11 and 230E8 of the Chumakov et al. YAC contig, within the DSCR on 21q22. This CAF1A, which belongs to the WD-motif family of genes and interacts with other polypeptide subunits to promote assembly of histones to replicating DNA, may contribute in a gene dosage-dependent manner to the phenotype of Down syndrome. 22 refs., 1 fig.

  13. Biochemical mapping of interactions within the intraflagellar transport (IFT) B core complex: IFT52 binds directly to four other IFT-B subunits.

    PubMed

    Taschner, Michael; Bhogaraju, Sagar; Vetter, Melanie; Morawetz, Michaela; Lorentzen, Esben

    2011-07-29

    Cilia and flagella are complex structures emanating from the surface of most eukaroytic cells and serve important functions including motility, signaling, and sensory reception. A process called intraflagellar transport (IFT) is of central importance to ciliary assembly and maintenance. The IFT complex is required for this transport and consists of two distinct multisubunit subcomplexes, IFT-A and IFT-B. Despite the importance of the IFT complex, little is known about its overall architecture. This paper presents a biochemical dissection of the molecular interactions within the IFT-B core complex. Two stable subcomplexes consisting of IFT88/70/52/46 and IFT81/74/27/25 were recombinantly co-expressed and purified. We identify a novel interaction between IFT70/52 and map the interaction domains between IFT52 and the other subunits within the IFT88/70/52/46 complex. Additionally, we show that IFT52 binds directly to the IFT81/74/27/25 complex, indicating that it could mediate the interaction between the two subcomplexes. Our data lead to an improved architectural map for the IFT-B core complex with new interactions as well as domain resolution mapping for several subunits.

  14. Genetic contribution to iron status: SNPs related to iron deficiency anaemia and fine mapping of CACNA2D3 calcium channel subunit.

    PubMed

    Baeza-Richer, Carlos; Arroyo-Pardo, Eduardo; Blanco-Rojo, Ruth; Toxqui, Laura; Remacha, Angel; Vaquero, M Pilar; López-Parra, Ana M

    2015-12-01

    Numerous studies associate genetic markers with iron- and erythrocyte-related parameters, but few relate them to iron-clinical phenotypes. Novel SNP rs1375515, located in a subunit of the calcium channel gene CACNA2D3, is associated with a higher risk of anaemia. The aim of this study is to further investigate the association of this SNP with iron-related parameters and iron-clinical phenotypes, and to explore the potential role of calcium channel subunit region in iron regulation. Furthermore, we aim to replicate the association of other SNPs reported previously in our population. We tested 45 SNPs selected via systematic review and fine mapping of CACNA2D3 region, with haematological and biochemical traits in 358 women of reproductive age. Multivariate analyses include back-step logistic regression and decision trees. The results replicate the association of SNPs with iron-related traits, and also confirm the protective effect of both A allele of rs1800562 (HFE) and G allele of rs4895441 (HBS1L-MYB). The risk of developing anaemia is increased in reproductive age women carriers of A allele of rs1868505 (CACNA2D3) and/or T allele of rs13194491 (HIST1H2BJ). Association of SNPs from fine mapping with ferritin and serum iron suggests that calcium channels could be a potential pathway for iron uptake in physiological conditions.

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

    SciTech Connect

    Shalhevet, D.; Da, Y.; Beever, J.E.; Eijk, M.J.T. van; Ma, R.; Lewin, H.A.; Gaskins, H.R.

    1995-01-01

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

  16. A susceptibility locus for lung cancer maps to nicotinic acetylcholine receptor subunit genes on 15q25.

    PubMed

    Hung, Rayjean J; McKay, James D; Gaborieau, Valerie; Boffetta, Paolo; Hashibe, Mia; Zaridze, David; Mukeria, Anush; Szeszenia-Dabrowska, Neonilia; Lissowska, Jolanta; Rudnai, Peter; Fabianova, Eleonora; Mates, Dana; Bencko, Vladimir; Foretova, Lenka; Janout, Vladimir; Chen, Chu; Goodman, Gary; Field, John K; Liloglou, Triantafillos; Xinarianos, George; Cassidy, Adrian; McLaughlin, John; Liu, Geoffrey; Narod, Steven; Krokan, Hans E; Skorpen, Frank; Elvestad, Maiken Bratt; Hveem, Kristian; Vatten, Lars; Linseisen, Jakob; Clavel-Chapelon, Françoise; Vineis, Paolo; Bueno-de-Mesquita, H Bas; Lund, Eiliv; Martinez, Carmen; Bingham, Sheila; Rasmuson, Torgny; Hainaut, Pierre; Riboli, Elio; Ahrens, Wolfgang; Benhamou, Simone; Lagiou, Pagona; Trichopoulos, Dimitrios; Holcátová, Ivana; Merletti, Franco; Kjaerheim, Kristina; Agudo, Antonio; Macfarlane, Gary; Talamini, Renato; Simonato, Lorenzo; Lowry, Ray; Conway, David I; Znaor, Ariana; Healy, Claire; Zelenika, Diana; Boland, Anne; Delepine, Marc; Foglio, Mario; Lechner, Doris; Matsuda, Fumihiko; Blanche, Helene; Gut, Ivo; Heath, Simon; Lathrop, Mark; Brennan, Paul

    2008-04-03

    Lung cancer is the most common cause of cancer death worldwide, with over one million cases annually. To identify genetic factors that modify disease risk, we conducted a genome-wide association study by analysing 317,139 single-nucleotide polymorphisms in 1,989 lung cancer cases and 2,625 controls from six central European countries. We identified a locus in chromosome region 15q25 that was strongly associated with lung cancer (P = 9 x 10(-10)). This locus was replicated in five separate lung cancer studies comprising an additional 2,513 lung cancer cases and 4,752 controls (P = 5 x 10(-20) overall), and it was found to account for 14% (attributable risk) of lung cancer cases. Statistically similar risks were observed irrespective of smoking status or propensity to smoke tobacco. The association region contains several genes, including three that encode nicotinic acetylcholine receptor subunits (CHRNA5, CHRNA3 and CHRNB4). Such subunits are expressed in neurons and other tissues, in particular alveolar epithelial cells, pulmonary neuroendocrine cells and lung cancer cell lines, and they bind to N'-nitrosonornicotine and potential lung carcinogens. A non-synonymous variant of CHRNA5 that induces an amino acid substitution (D398N) at a highly conserved site in the second intracellular loop of the protein is among the markers with the strongest disease associations. Our results provide compelling evidence of a locus at 15q25 predisposing to lung cancer, and reinforce interest in nicotinic acetylcholine receptors as potential disease candidates and chemopreventative targets.

  17. The human B22 subunit of the NADH-ubiquinone oxidoreductase maps to the region of chromosome 8 involved in Branchio-oto-renal syndrome

    SciTech Connect

    Gu, J.Z.; Lin, Xin; Wells, D.E.

    1996-07-01

    To identify candidate genes for Branchio-oto-renal (BOR) syndrome, we have made use of a set of cosmids that map to 8q13.3, which has previously been shown to be involved in this syndrome. These cosmids were used as genomic clones in the attempts to isolate corresponding cDNAs using a modified hybrid selection technique. cDNAs using a modified hybrid selection technique. cDNAs from the region were identified and used to search for sequence similarity in human or other species. One cDNA clone was found to have 89% sequence similarity to the bovine B22 subunit of NADH-ubiquinone oxidoreductase, a mitochondrial protein in the respiratory electron transport chain. Given the history of other mitochondrial mutations being involved in hearing loss syndromes, this gene should be considered a strong candidate for involvement in BOR.

  18. Involvement of OS-2 MAP kinase in regulation of the large-subunit catalases CAT-1 and CAT-3 in Neurospora crassa.

    PubMed

    Yamashita, Kazuhiro; Shiozawa, Azusa; Banno, Shinpei; Fukumori, Fumiyasu; Ichiishi, Akihiko; Kimura, Makoto; Fujimura, Makoto

    2007-08-01

    Neurospora crassa has four catalase genes--cat-1, cat-2, cat-3, and ctt-1/cat-4. cat-1 and cat-3 encode two fungal-specific large-subunit catalases CAT-1 and CAT-3 normally produced in conidia and growing hyphae, respectively. cat-2 encodes CAT-2 catalase-peroxidase normally produced in conidia. ctt-1 (or cat-4), of which expression was controlled by OS-2 MAP kinase (Noguchi et al., Fungal Genet. Biol. 44, 208-218), encodes a small-subunit catalase with unknown function. To clarify the contribution of OS-2 on the regulation of CAT-1, CAT-2, and CAT-3, we performed quantitative RT-PCR and in-gel catalase activity analyses. When the hyphae were treated with a fungicide (1 mug/ml fludioxonil) or subjected to an osmotic stress (1 M sorbitol), cat-1 was strongly upregulated and CAT-1 was reasonably induced in the wild-type strain. Interestingly, fludioxonil caused not only the CAT-1 induction but also a remarkable CAT-3 decrease in the wild-type hyphae, implying of an abnormal stimulation of asexual differentiation. These responses were not observed in an os-2 mutant hyphae, indicating an involvement of OS-2 in the cat-1 expression; however, os-2 was dispensable for the production of CAT-1 in conidia. In contrast, the expression of cat-2 was significantly induced by heat shock (45 degrees C) and that of cat-3 was moderately stimulated by an oxidative stress (50 microg/ml methyl viologen) in both the wild-type strain and the os-2 mutant, and corresponding enzyme activities were detected after the treatments. Although basal levels of transcription of cat-1 and cat-3 in an os-2 mutant hyphae were a few-fold lower than in the wild-type hyphae, the os-2 mutant exhibited a considerably lower levels of CAT-3 activity than the wild-type strain. These findings suggest that OS-2 MAP kinase regulated the expression of cat-1 and cat-3 transcriptionally, and probably that of cat-3 posttranscriptionally, even though the presence of another regulatory system for each of these two

  19. Mapping of the glutamate-cysteine ligase catalytic subunit gene (GLCLC) to human chromosome 6p12 and mouse chromosome 9D-E and of the regulatory subunit gene (GLCLR) to human chromosome 1p21-p22 and mouse chromosome 3H1-3

    SciTech Connect

    Tsuchiya, K.; Disteche, C.M.; Reid, L.L.

    1995-12-10

    Glutamate-cysteine ligase (EC 6.3.2.2, GLCL), formerly called {gamma}-glutamylcysteine synthetase (GCS), is the rate-limiting enzyme in the de novo synthesis of the antioxidant tripeptide glutathione. GLCL consists of a heavy subunit, which possesses catalytic activity and is the site of glutathione feedback inhibition, and a light subunit, which has a regulatory function. Glutathione is ubiquitous in mammalian tissues and performs a variety of functions, including protection from reactive oxygen species through antioxidant properties; detoxification of xenobiotics, organic peroxides, and heavy metals; and maintenance of sulfhydryl groups of other molecules. Increased intracellular levels of glutathione have also been found in tumor cells resistant to chemotherapeutic agents. Increased expression of GLCL in melphalan-resistant myeloma and prostate carcinoma cells and cisplatinum-resistant ovarian carcinoma cells suggests that this enzyme may be involved in glutathione-associated drug resistance. Moreover, GLCL has been shown to be induced by phenolic antioxidants and heavy metals. Recently, Mulcahy and Gipp have shown that the GLCL catalytic subunit gene (GLCLC) contains a putative antioxidant regulatory element, which may explain the responsiveness of this gene to agents that induce oxidative stress. To further our understanding of GLCL, which is linked to such a wide variety of metabolic and physiological functions through its role in glutathione synthesis, we have mapped both the catalytic and regulatory subunit genes (GLCLC and GLCLR) to human and mouse chromosomes by fluorescence in situ hybridization (FISH). 16 refs., 1 fig.

  20. A new sodium channel alpha-subunit gene (Scn9a) from Schwann cells maps to the Scn1a, Scn2a, Scn3a cluster of mouse chromosome 2.

    PubMed

    Beckers, M C; Ernst, E; Belcher, S; Howe, J; Levenson, R; Gros, P

    1996-08-15

    We have used a total of 27 AXB/BXA recombinant inbred mouse strains to determine the chromosomal location of a newly identified gene encoding an alpha-subunit isoform of the sodium channel from Schwann cells, Scn9a. Linkage analysis established that Scn9a mapped to the proximal segment of mouse chromosome 2. The segregation of restriction fragment length polymorphisms in 145 progeny from a Mus spretus x C57BL/6J backcross indicates that Scn9a is very tightly linked to Scn1a (gene encoding the type I sodium channel alpha-subunit of the brain) and forms part of a cluster of four Scna genes located on mouse chromosome 2.

  1. Conformation of yeast 18S rRNA. Direct chemical probing of the 5' domain in ribosomal subunits and in deproteinized RNA by reverse transcriptase mapping of dimethyl sulfate-accessible.

    PubMed Central

    Lempereur, L; Nicoloso, M; Riehl, N; Ehresmann, C; Ehresmann, B; Bachellerie, J P

    1985-01-01

    The structure of the 5' domain of yeast 18S rRNA has been probed by dimethyl sulfate (DMS), either in "native" deproteinized molecules or in the 40S ribosomal subunits. DMS-reacted RNA has been used as a template for reverse transcription and a large number of reactive sites, corresponding to all types of bases have been mapped by a primer extension procedure, taking advantage of blocks in cDNA elongation immediately upstream from bases methylated at atom positions involved in the base-pair recognition of the template. Since the same atom positions are protected from DMS in base-paired nucleotides, the secondary structure status of each nucleotide can be directly assessed in this procedure, thus allowing to evaluate the potential contribution of proteins in modulating subunit rRNA conformation. While the DMS probing of deproteinized rRNA confirms a number of helical stems predicted by phylogenetic comparisons, it is remarkable that a few additional base-pairings, while proven by the comparative analysis, appear to require the presence of the bound ribosomal subunit proteins to be stabilized. Images PMID:2417197

  2. Mapping the domain structure of the influenza A virus polymerase acidic protein (PA) and its interaction with the basic protein 1 (PB1) subunit

    SciTech Connect

    Guu, Tom S.Y.; Dong Liping; Wittung-Stafshede, Pernilla; Tao, Yizhi J.

    2008-09-15

    The influenza A virus polymerase consists of three subunits (PA, PB1, and PB2) necessary for viral RNA synthesis. The heterotrimeric polymerase complex forms through PA interacting with PB1 and PB1 interacting with PB2. PA has been shown to play critical roles in the assembly, catalysis, and nuclear localization of the polymerase. To probe the structure of PA, we isolated recombinant PA from insect cells. Limited proteolysis revealed that PA contained two domains connected by a 20-residue linker (residues 257-276). Far-UV circular dichroism established that the two domains folded into a mixed {alpha}/{beta} structure when separately expressed. In vitro pull-down assays showed that neither individually nor cooperatively expressed PA domains, without the linker, could assure PA-PB1 interaction. Protease treatment of PA-PB1 complex indicated that its PA subunit was significantly more stable than free PA, suggesting that the linker is protected and it constitutes an essential component of the PA-PB1 interface.

  3. EMatch: an efficient method for aligning atomic resolution subunits into intermediate-resolution cryo-EM maps of large macromolecular assemblies

    SciTech Connect

    Dror, Oranit Lasker, Keren; Nussinov, Ruth; Wolfson, Haim

    2007-01-01

    A method for detecting structural homologs of components in an intermediate resolution cryo-EM map and their spatial configuration is presented. Structural analysis of biological machines is essential for inferring their function and mechanism. Nevertheless, owing to their large size and instability, deciphering the atomic structure of macromolecular assemblies is still considered as a challenging task that cannot keep up with the rapid advances in the protein-identification process. In contrast, structural data at lower resolution is becoming more and more available owing to recent advances in cryo-electron microscopy (cryo-EM) techniques. Once a cryo-EM map is acquired, one of the basic questions asked is what are the folds of the components in the assembly and what is their configuration. Here, a novel knowledge-based computational method, named EMatch, towards tackling this task for cryo-EM maps at 6–10 Å resolution is presented. The method recognizes and locates possible atomic resolution structural homologues of protein domains in the assembly. The strengths of EMatch are demonstrated on a cryo-EM map of native GroEL at 6 Å resolution.

  4. Complexing of the CD-3 subunit by a monoclonal antibody activates a microtubule-associated protein 2 (MAP-2) serine kinase in Jurkat cells.

    PubMed Central

    Hanekom, C; Nel, A; Gittinger, C; Rheeder, A; Landreth, G

    1989-01-01

    Treatment of Jurkat T-cells with anti-CD-3 monoclonal antibodies resulted in the rapid and transient activation of a serine kinase which utilized the microtubule-associated protein, MAP-2, as a substrate in vitro. The kinase was also activated on treatment of Jurkat cells with phytohaemagglutinin, but with a different time course. The activation of the MAP-2 kinase by anti-CD-3 antibodies was dose-dependent, with maximal activity observed at concentrations of greater than 500 ng/ml. Normal human E-rosette-positive T-cells also exhibited induction of MAP-2 kinase activity during anti-CD-3 treatment. The enzyme was optimally active in the presence of 2 mM-Mn2+; lower levels of activity were observed with Mg2+, even at concentrations up to 20 mM. The kinase was partially purified by passage over DE-52 Sephacel with the activity eluting as a single peak at 0.25 M-NaCl. The molecular mass was estimated to be 45 kDa by gel filtration. The activation of the MAP-2 kinase was probably due to phosphorylation of this enzyme as treatment with alkaline phosphatase diminished its activity. These data demonstrate that the stimulation of T-cells through the CD-3 complex results in the activation of a novel serine kinase which may be critically involved in signal transduction in these cells. Images Fig. 1. Fig. 7. Fig. 8. PMID:2552997

  5. Subunit mass analysis for monitoring antibody oxidation

    PubMed Central

    Sokolowska, Izabela; Mo, Jingjie; Dong, Jia; Lewis, Michael J.; Hu, Ping

    2017-01-01

    ABSTRACT Methionine oxidation is a common posttranslational modification (PTM) of monoclonal antibodies (mAbs). Oxidation can reduce the in-vivo half-life, efficacy and stability of the product. Peptide mapping is commonly used to monitor the levels of oxidation, but this is a relatively time-consuming method. A high-throughput, automated subunit mass analysis method was developed to monitor antibody methionine oxidation. In this method, samples were treated with IdeS, EndoS and dithiothreitol to generate three individual IgG subunits (light chain, Fd’ and single chain Fc). These subunits were analyzed by reversed phase-ultra performance liquid chromatography coupled with an online quadrupole time-of-flight mass spectrometer and the levels of oxidation on each subunit were quantitated based on the deconvoluted mass spectra using the UNIFI software. The oxidation results obtained by subunit mass analysis correlated well with the results obtained by peptide mapping. Method qualification demonstrated that this subunit method had excellent repeatability and intermediate precision. In addition, UNIFI software used in this application allows automated data acquisition and processing, which makes this method suitable for high-throughput process monitoring and product characterization. Finally, subunit mass analysis revealed the different patterns of Fc methionine oxidation induced by chemical and photo stress, which makes it attractive for investigating the root cause of oxidation. PMID:28106519

  6. Subunit mass analysis for monitoring antibody oxidation.

    PubMed

    Sokolowska, Izabela; Mo, Jingjie; Dong, Jia; Lewis, Michael J; Hu, Ping

    2017-04-01

    Methionine oxidation is a common posttranslational modification (PTM) of monoclonal antibodies (mAbs). Oxidation can reduce the in-vivo half-life, efficacy and stability of the product. Peptide mapping is commonly used to monitor the levels of oxidation, but this is a relatively time-consuming method. A high-throughput, automated subunit mass analysis method was developed to monitor antibody methionine oxidation. In this method, samples were treated with IdeS, EndoS and dithiothreitol to generate three individual IgG subunits (light chain, Fd' and single chain Fc). These subunits were analyzed by reversed phase-ultra performance liquid chromatography coupled with an online quadrupole time-of-flight mass spectrometer and the levels of oxidation on each subunit were quantitated based on the deconvoluted mass spectra using the UNIFI software. The oxidation results obtained by subunit mass analysis correlated well with the results obtained by peptide mapping. Method qualification demonstrated that this subunit method had excellent repeatability and intermediate precision. In addition, UNIFI software used in this application allows automated data acquisition and processing, which makes this method suitable for high-throughput process monitoring and product characterization. Finally, subunit mass analysis revealed the different patterns of Fc methionine oxidation induced by chemical and photo stress, which makes it attractive for investigating the root cause of oxidation.

  7. The gene for human U2 snRNP auxiliary factor small 35-kDa subunit (U2AF1) maps to the progressive myoclonus epilepsy (EPM1) critical region on chromosome 21q22.3

    SciTech Connect

    Lalioti, M.D.; Rossier, C.; Antonarakis, S.E.

    1996-04-15

    We used targeted exon trapping to clone portions of genes from human chromosome 21q22.3. One trapped sequence showed complete homology with the cDNA of human U2AF{sup 35} (M96982; HGM-approved nomenclature U2AF1), which encodes for the small 35-kDa subunit of the U2 snRNP auxiliary factor. Using the U2AF1 cDNA as a probe, we mapped this gene to cosmid Q15D2, a P1, and YAC 350F7 of the Chumakov et al. contig, close to the cystathionine-{beta}-synthase gene (CBS) on 21q22.3. This localization was confirmed by PCR using oligonucleotides from the 3{prime} UTR and by FISH. As U2AF1 associated with a number of different factors during mRNA splicing, overexpression in trisomy 21 individuals could contribute to some Down syndrome phenotypes by interfering with the splicing process. Furthermore, because this gene maps in the critical region for the progressive myoclonus epilepsy I locus (EPM1), mutation analysis will be carried out in patients to evaluate the potential role of U2AF1 as a candidate for EPM1. 24 refs., 1 fig.

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

    PubMed

    Schnare, Murray N; Gray, Michael W

    2011-10-14

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

  9. Sodium channel auxiliary subunits.

    PubMed

    Tseng, Tsai-Tien; McMahon, Allison M; Johnson, Victoria T; Mangubat, Erwin Z; Zahm, Robert J; Pacold, Mary E; Jakobsson, Eric

    2007-01-01

    Voltage-gated ion channels are well known for their functional roles in excitable tissues. Excitable tissues rely on voltage-gated ion channels and their auxiliary subunits to achieve concerted electrical activity in living cells. Auxiliary subunits are also known to provide functional diversity towards the transport and biogenesis properties of the principal subunits. Recent interests in pharmacological properties of these auxiliary subunits have prompted significant amounts of efforts in understanding their physiological roles. Some auxiliary subunits can potentially serve as drug targets for novel analgesics. Three families of sodium channel auxiliary subunits are described here: beta1 and beta3, beta2 and beta4, and temperature-induced paralytic E (TipE). While sodium channel beta-subunits are encoded in many animal genomes, TipE has only been found exclusively in insects. In this review, we present phylogenetic analyses, discuss potential evolutionary origins and functional data available for each of these subunits. For each family, we also correlate the functional specificity with the history of evolution for the individual auxiliary subunits.

  10. Altered 40 S ribosomal subunits in omnipotent suppressors of yeast.

    PubMed

    Eustice, D C; Wakem, L P; Wilhelm, J M; Sherman, F

    1986-03-20

    The five suppressors SUP35, SUP43, SUP44, SUP45 and SUP46, each mapping at a different chromosomal locus in the yeast Saccharomyces cerevisiae, suppress a wide range of mutations, including representatives of all three types of nonsense mutations, UAA, UAG and UGA. We have demonstrated that ribosomes from the four suppressors SUP35, SUP44, SUP45 and SUP46 translate polyuridylate templates in vitro with higher errors than ribosomes from the normal stain, and that this misreading is substantially enhanced by the antibiotic paromomycin. Furthermore, ribosomal subunit mixing experiments established that the 40 S ribosomal subunit, and this subunit only, is responsible for the higher levels of misreading. Thus, the gene products of SUP35, SUP44, SUP45 and SUP46 are components of the 40 S subunit or are enzymes that modify the subunit. In addition, a protein from the 40 S subunit of the SUP35 suppressor has an altered electrophoretic mobility; this protein is distinct from the altered protein previously uncovered in the 40 S subunit of the SUP46 suppressor. In contrast to the ribosomes from the four suppressors SUP35, SUP44, SUP45 and SUP46, the ribosomes from the SUP43 suppressor do not significantly misread polyuridylate templates in vitro, suggesting that this locus may not encode a ribosomal component or that the misreading is highly specific.

  11. Highly conserved small subunit residues influence rubisco large subunit catalysis.

    PubMed

    Genkov, Todor; Spreitzer, Robert J

    2009-10-30

    The chloroplast enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of photosynthetic CO(2) fixation. With a deeper understanding of its structure-function relationships and competitive inhibition by O(2), it may be possible to engineer an increase in agricultural productivity and renewable energy. The chloroplast-encoded large subunits form the active site, but the nuclear-encoded small subunits can also influence catalytic efficiency and CO(2)/O(2) specificity. To further define the role of the small subunit in Rubisco function, the 10 most conserved residues in all small subunits were substituted with alanine by transformation of a Chlamydomonas reinhardtii mutant that lacks the small subunit gene family. All the mutant strains were able to grow photosynthetically, indicating that none of the residues is essential for function. Three of the substitutions have little or no effect (S16A, P19A, and E92A), one primarily affects holoenzyme stability (L18A), and the remainder affect catalysis with or without some level of associated structural instability (Y32A, E43A, W73A, L78A, P79A, and F81A). Y32A and E43A cause decreases in CO(2)/O(2) specificity. Based on the x-ray crystal structure of Chlamydomonas Rubisco, all but one (Glu-92) of the conserved residues are in contact with large subunits and cluster near the amino- or carboxyl-terminal ends of large subunit alpha-helix 8, which is a structural element of the alpha/beta-barrel active site. Small subunit residues Glu-43 and Trp-73 identify a possible structural connection between active site alpha-helix 8 and the highly variable small subunit loop between beta-strands A and B, which can also influence Rubisco CO(2)/O(2) specificity.

  12. Neutron Scattering and the 30 S Ribosomal Subunit of E. Coli

    DOE R&D Accomplishments Database

    Moore, P. B.; Engelman, D. M.; Langer, J. A.; Ramakrishnan, V. R.; Schindler, D. G.; Schoenborn, B. P.; Sillers, I. Y.; Yabuki, S.

    1982-06-01

    This paper reviews the progress made in the study of the internal organization of the 30 S ribosomal subunit of E. coli by neutron scattering since 1975. A map of that particle showing the position of 14 of the subunit's 21 proteins is presented, and the methods currently used for collecting and analyzing such data are discussed. Also discussed is the possibility of extending the interpretation of neutron mapping data beyond the limits practical today.

  13. Neutron scattering and the 30 S ribosomal subunit of E. coli

    SciTech Connect

    Moore, P.B.; Engelman, D.M.; Langer, J.A.; Ramakrishnan, V.R.; Schindler, D.G.; Schoenborn, B.P.; Sillers, I.Y.; Yabuki, S.

    1982-01-01

    This paper reviews the progress made in the study of the internal organization of the 30 S ribosomal subunit of E. coli by neutron scattering since 1975. A map of that particle showing the position of 14 of the subunit's 21 proteins is presented, and the methods currently used for collecting and analyzing such data are discussed. Also discussed is the possibility of extending the interpretation of neutron mapping data beyond the limits practical today. 30 references, 5 figures.

  14. Characterization of the interface between gamma and epsilon subunits of Escherichia coli F1-ATPase.

    PubMed

    Tang, C; Capaldi, R A

    1996-02-09

    The interaction faces of the gamma and epsilon subunits in the Escherichia coli F1-ATPase have been explored by a combination of cross-linking and chemical modification experiments using several mutant epsilon subunits as follows: epsilonS10C, epsilonH38C, epsilonT43C, epsilonS65C, epsilonS108C, and epsilonM138C, along with a mutant of the gamma subunit, gammaT106C. The replacement of Ser-10 by a Cys or Met-138 by a Cys reduced the inhibition of ECF1 by the epsilon subunit, while the mutation S65C increased this inhibitory effect. Modification of the Cys at position 10 with N-ethylmaleimide or fluoroscein maleimide further reduced the binding affinity of, and the maximal inhibition by, the epsilon subunit. Similar chemical modification of the Cys at position 43 of the epsilon subunit (in the mutant epsilonT43C) and a Cys at position 106 of the gamma subunit (gammaT106C) also affected the inhibition of ECF1 by the epsilon subunit. The various epsilon subunit mutants were reacted with TFPAM3, and the site(s) of cross-linking within the ECF1 complex was determined. Previous studies have shown cross-linking from the Cys at positions 10 and 38 with the gamma subunit and from a Cys at position 108 to an alpha subunit (Aggeler, R., Chicas-Cruz, K., Cai, S. X., Keana, J. F. W., and Capaldi, R. A. (1992) Biochemistry 31, 2956-2961; Aggeler, R., Weinreich, F., and Capaldi, R. A. (1995) Biochim. Biophys. Acta 1230, 62-68). Here, cross-linking was found from a Cys at position 43 to the gamma subunit and from the Cys at position 138 to a beta subunit. The site of cross-linking from Cys-10 of epsilon to the gamma subunit was localized by peptide mapping to a region of the gamma subunit between residues 222 and 242. Cross-linking from a Cys at position 38 and at position 43 was with the C-terminal part of the gamma subunit, between residues 202 and 286. ECF1 treated with trypsin at pH 7.0 still binds purified epsilon subunit, while enzyme treated with the protease at pH 8.0 does

  15. Localization of yeast RNA polymerase I core subunits by immunoelectron microscopy.

    PubMed Central

    Klinger, C; Huet, J; Song, D; Petersen, G; Riva, M; Bautz, E K; Sentenac, A; Oudet, P; Schultz, P

    1996-01-01

    Immunoelectron microscopy was used to determine the spatial organization of the yeast RNA polymerase I core subunits on a three-dimensional model of the enzyme. Images of antibody-labeled enzymes were compared with the native enzyme to determine the localization of the antibody binding site on the surface of the model. Monoclonal antibodies were used as probes to identify the two largest subunits homologous to the bacterial beta and beta' subunits. The epitopes for the two monoclonal antibodies were mapped using subunit-specific phage display libraries, thus allowing a direct correlation of the structural data with functional information on conserved sequence elements. An epitope close to conserved region C of the beta-like subunit is located at the base of the finger-like domain, whereas a sequence between conserved regions C and D of the beta'-like subunit is located in the apical region of the enzyme. Polyclonal antibodies outlined the alpha-like subunit AC40 and subunit AC19 which were found co-localized also in the apical region of the enzyme. The spatial location of the subunits is correlated with their biological activity and the inhibitory effect of the antibodies. Images PMID:8887555

  16. The ribosomal subunit assembly line

    PubMed Central

    Dlakić, Mensur

    2005-01-01

    Recent proteomic studies in Saccharomyces cerevisiae have identified nearly 200 proteins, other than the structural ribosomal proteins, that participate in the assembly of ribosomal subunits and their transport from the nucleus. In a separate line of research, proteomic studies of mature plant ribosomes have revealed considerable variability in the protein composition of individual ribosomes. PMID:16207363

  17. Association of condensin with chromosomes depends on DNA binding by its HEAT-repeat subunits.

    PubMed

    Piazza, Ilaria; Rutkowska, Anna; Ori, Alessandro; Walczak, Marta; Metz, Jutta; Pelechano, Vicent; Beck, Martin; Haering, Christian H

    2014-06-01

    Condensin complexes have central roles in the three-dimensional organization of chromosomes during cell divisions, but how they interact with chromatin to promote chromosome segregation is largely unknown. Previous work has suggested that condensin, in addition to encircling chromatin fibers topologically within the ring-shaped structure formed by its SMC and kleisin subunits, contacts DNA directly. Here we describe the discovery of a binding domain for double-stranded DNA formed by the two HEAT-repeat subunits of the Saccharomyces cerevisiae condensin complex. From detailed mapping data of the interfaces between the HEAT-repeat and kleisin subunits, we generated condensin complexes that lack one of the HEAT-repeat subunits and consequently fail to associate with chromosomes in yeast and human cells. The finding that DNA binding by condensin's HEAT-repeat subunits stimulates the SMC ATPase activity suggests a multistep mechanism for the loading of condensin onto chromosomes.

  18. Rotating proton pumping ATPases: subunit/subunit interactions and thermodynamics.

    PubMed

    Nakanishi-Matsui, Mayumi; Sekiya, Mizuki; Futai, Masamitsu

    2013-03-01

    In this article, we discuss single molecule observation of rotational catalysis by E. coli ATP synthase (F-ATPase) using small gold beads. Studies involving a low viscous drag probe showed the stochastic properties of the enzyme in alternating catalytically active and inhibited states. The importance of subunit interaction between the rotor and the stator, and thermodynamics of the catalysis are also discussed. "Single Molecule Enzymology" is a new trend for understanding enzyme mechanisms in biochemistry and physiology.

  19. The Evolution of the Four Subunits of Voltage-Gated Calcium Channels: Ancient Roots, Increasing Complexity, and Multiple Losses

    PubMed Central

    Moran, Yehu; Zakon, Harold H.

    2014-01-01

    The alpha subunits of voltage-gated calcium channels (Cavs) are large transmembrane proteins responsible for crucial physiological processes in excitable cells. They are assisted by three auxiliary subunits that can modulate their electrical behavior. Little is known about the evolution and roles of the various subunits of Cavs in nonbilaterian animals and in nonanimal lineages. For this reason, we mapped the phyletic distribution of the four channel subunits and reconstructed their phylogeny. Although alpha subunits have deep evolutionary roots as ancient as the split between plants and opistokonths, beta subunits appeared in the last common ancestor of animals and their close-relatives choanoflagellates, gamma subunits are a bilaterian novelty and alpha2/delta subunits appeared in the lineage of Placozoa, Cnidaria, and Bilateria. We note that gene losses were extremely common in the evolution of Cavs, with noticeable losses in multiple clades of subfamilies and also of whole Cav families. As in vertebrates, but not protostomes, Cav channel genes duplicated in Cnidaria. We characterized by in situ hybridization the tissue distribution of alpha subunits in the sea anemone Nematostella vectensis, a nonbilaterian animal possessing all three Cav subfamilies common to Bilateria. We find that some of the alpha subunit subtypes exhibit distinct spatiotemporal expression patterns. Further, all six sea anemone alpha subunit subtypes are conserved in stony corals, which separated from anemones 500 MA. This unexpected conservation together with the expression patterns strongly supports the notion that these subtypes carry unique functional roles. PMID:25146647

  20. Interaction between G-protein beta and gamma subunit types is selective.

    PubMed Central

    Pronin, A N; Gautam, N

    1992-01-01

    Signal-transducing guanine nucleotide-binding proteins (G proteins) are made up of three subunits, alpha, beta, and gamma. Each of these subunits comprises a family of proteins. The rules for association between members of one family with members of another to form a multimer are not known; it is not clear whether associations are specific or nonspecific. Other than transducin (Gt), the G protein in rod photoreceptors, most purified G proteins contain more than one subtype of beta or gamma subunits. The Gt alpha subunit is associated only with beta 1 and gamma 1. It is not known whether this specificity is due to the differential expression of these subunit types in a cell type or due to intrinsically different affinities between different beta and gamma subunit types. We have used a transfected cell assay system to examine the association of the beta 1, beta 2, and beta 3 proteins with the gamma 1 and gamma 2 proteins. Results show that gamma 1 does not associate with beta 2 and that beta 3 does not associate with gamma 1 or gamma 2. Differences in affinities between types of G protein subunits will impose restrictions on the formation of certain heterotrimers and determine which G protein will be active in a cell. A chimeric molecule of beta 1 and beta 2 was used to broadly map the regions on these subunits that determine specificity of association. Images PMID:1631113

  1. Isolation and characterization of the subunits of Phaseolus vulgaris alpha-amylase inhibitor.

    PubMed

    Yamaguchi, H

    1991-11-01

    An alpha-amylase inhibitor (PHA-I) of the white kidney bean (Phaseolus vulgaris) was found to be composed of two kinds of subunits and they were isolated on a size-exclusion column by HPLC under denaturing conditions. The alpha-subunit was free from tryptophan and cysteine and the beta-subunit contained no methionine or cysteine. There was no marked resemblance in tryptic peptide map between these subunit polypeptides. The alpha-subunit contained 28% by weight of carbohydrate, mainly made up of high mannose-type oligosacharides, whereas the sugar moiety of the beta-subunit amounted to 7% by weight and seemed to be predominantly composed of xylomannose-type oligosaccharides. By SDS-PAGE following deglycosylation, the molecular weights of the polypeptides of alpha- and beta-subunits were shown to be 7,800 and 14,000, respectively. These values were consistent with molecular sizes obtained for alpha- and beta-subunits by gel permeation HPLC in 6 M guanidine hydrochloride. The molecular weight of the native PHA-I, 28,800, obtained by gel permeation HPLC under non-denaturing conditions, suggested a heterodimeric structure for PHA-I.

  2. Chromosomal localization of genes encoding guanine nucleotide-binding protein subunits in mouse and human

    SciTech Connect

    Blatt, C.; Eversole-Cire, P.; Cohn, V.H.; Zollman, S.; Fournier, R.E.K.; Mohandas, L.T.; Nesbitt, M.; Lugo, T.; Jones, D.T.; Reed, R.R.; Weiner, L.P.; Sparkes, R.S.; Simon, M.I. )

    1988-10-01

    A variety of genes have been identified that specify the synthesis of the components of guanine nucleotide-binding proteins (G proteins). Eight different guanine nucleotide-binding {alpha}-subunit proteins, two different {beta} subunits, and one {gamma} subunit have been described. Hybridization of cDNA clones with DNA from human-mouse somatic cell hybrids was used to assign many of these genes to human chromosomes. The retinal-specific transducin subunit genes GNAT1 and GNAT2 were on chromosomes 3 and 1; GNAI1, GNAI2, and GNAI3 were assigned to chromosomes 7, 3, and 1, respectively; GNAZ and GNAS were found on chromosomes 22 and 20. The {beta} subunits were also assigned-GNB1 to chromosome 1 and GNB2 to chromosome 7. Restriction fragment length polymorphisms were used to map the homologues of some of these genes in the mouse. GNAT1 and GNAI2 were found to map adjacent to each other on mouse chromosome 9 and GNAT2 was mapped on chromosome 17. The mouse GNB1 gene was assigned to chromosome 19. These mapping assignments will be useful in defining the extend of the G{alpha} gene family and may help in attempts to correlate specific genetic diseases and with genes corresponding to G proteins.

  3. The Subunit Structure of Benzylsuccinate Synthase†

    PubMed Central

    Li, Lei; Patterson, Dustin P.; Fox, Christel C.; Lin, Brian; Coschigano, Peter W.; Marsh, E. Neil G.

    2010-01-01

    Benzylsuccinate synthase is a member of the glycyl radical family of enzymes. It catalyzes the addition of toluene to fumarate to form benzylsuccinate as the first step in the anaerobic pathway of toluene fermentation. The enzyme comprises three subunits α, β and γ that in Thauera Aromatica T1 strain are encoded by the tutD, tutG and tutF genes respectively. The large α-subunit contains the essential glycine and cysteine residues that are conserved in all glycyl radical enzymes. However, the function of the small β- and γ-subunits has remained unclear. We have over-expressed all three subunits of benzylsuccinate synthase in E. coli, both individually and in combination. Co-expression of the γ-subunit (but not the β-subunit) is essential for efficient expression of the α-subunit. The benzylsuccinate synthase complex lacking the glycyl radical could be purified as an α2β2γ2 hexamer by nickel-affinity chromatography through a ‘His6’ affinity tag engineered onto the C-terminus of the α-subunit. Unexpectedly, BSS was found to contain two iron-sulfur clusters, one associated with the β-subunit and the other with the γ-subunit that appear to be necessary for the structural integrity of the complex. The spectroscopic properties of these clusters suggest that they are most likely [4Fe-4S] clusters. Removal of iron with chelating agents results in dissociation of the complex; similarly a mutant γ-subunit lacking the [4Fe-4S] cluster is unable to stabilize the α-subunit when the proteins are co-expressed. PMID:19159265

  4. Subunit connectivity, assembly determinants, and architecture of the yeast exocyst complex

    PubMed Central

    Heider, Margaret R.; Gu, Mingyu; Duffy, Caroline M.; Mirza, Anne M.; Marcotte, Laura L.; Walls, Alexandra C.; Farrall, Nicholas; Hakhverdyan, Zhanna; Field, Mark C.; Rout, Michael P.; Frost, Adam; Munson, Mary

    2016-01-01

    The exocyst is a hetero-octameric complex proposed to serve as the tethering complex for exocytosis, although it remains poorly understood at the molecular level. Here, we purified endogenous exocyst from Saccharomyces cerevisiae, and show that the purified complexes are stable and consist of all eight subunits with equal stoichiometry. Using a combination of biochemical and auxin-induced degradation experiments in yeast, we mapped the subunit connectivity, identified two stable four-subunit modules within the octamer, and demonstrated that several known exocyst binding partners are not necessary for exocyst assembly and stability. Furthermore, we visualized the structure of the yeast complex using negative stain electron microscopy; our results indicate that exocyst exists predominantly as a stable, octameric complex with an elongated architecture that suggests the subunits are contiguous helical bundles packed together into a bundle of long rods. PMID:26656853

  5. Role of the Rubisco Small Subunit

    SciTech Connect

    Spreitzer, Robert Joseph

    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

  6. 28 CFR 51.6 - Political subunits.

    Code of Federal Regulations, 2010 CFR

    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...

  7. Structural Characterization of Tip20p and Dsl1p, Subunits of the Dsl1p Vesicle Tethering Complex

    SciTech Connect

    Tripathi, A.; Ren, Y; Jeffrey, P; Hughson, F

    2009-01-01

    Multisubunit tethering complexes are essential for intracellular trafficking and have been proposed to mediate the initial interaction between vesicles and the membranes with which they fuse. Here we report initial structural characterization of the Dsl1p complex, whose three subunits are essential for trafficking from the Golgi apparatus to the endoplasmic reticulum (ER). Crystal structures reveal that two of the three subunits, Tip20p and Dsl1p, resemble known subunits of the exocyst complex, establishing a structural connection among several multisubunit tethering complexes and implying that many of their subunits are derived from a common progenitor. We show, moreover, that Tip20p and Dsl1p interact directly via N-terminal alpha-helices. Finally, we establish that different Dsl1p complex subunits bind independently to different ER SNARE proteins. Our results map out two alternative protein-interaction networks capable of tethering COPI-coated vesicles, via the Dsl1p complex, to ER membranes.

  8. Isolation and characterization of the subunits of a heat-labile alpha-amylase inhibitor from Phaseolus vulgaris white kidney bean.

    PubMed

    Yamaguchi, H

    1993-02-01

    The heat-labile one of the two alpha-amylase inhibitors of the white kidney bean (Phaseolus vulgaris) was found to be composed of three kinds of subunits, and they were isolated and characterized. The alpha-subunit was free from tryptophan and cysteine and the beta-subunit contained no methionine or cysteine. There was no marked resemblance in tryptic peptide maps between the alpha- and beta-subunit polypeptides. The alpha-subunit contained 30% by weight of carbohydrate, mainly made up of high mannose-oligosaccharides, and the sugar moiety of the beta-subunit amounted 7% and appeared to be predominantly composed of xylomannose-type oligosaccharides. The largest subunit, gamma, was very similar in molecular features to a postulated alpha beta-dimer and its N-terminal sequence coincided with that of the alpha-subunit. The molecular weights of the polypeptides of alpha, beta-, and gamma-subunits were shown to be 7,800, 14,000, and 22,000, respectively, by SDS-PAGE. It seemed likely that the alpha- and beta-subunits are common to both of the inhibitors and that the heat-lability of this inhibitor arises from the gamma-subunit.

  9. Conformational flexibility and subunit arrangement of the modular yeast Spt-Ada-Gcn5 acetyltransferase complex.

    PubMed

    Setiaputra, Dheva; Ross, James D; Lu, Shan; Cheng, Derrick T; Dong, Meng-Qiu; Yip, Calvin K

    2015-04-17

    The Spt-Ada-Gcn5 acetyltransferase (SAGA) complex is a highly conserved, 19-subunit histone acetyltransferase complex that activates transcription through acetylation and deubiquitination of nucleosomal histones in Saccharomyces cerevisiae. Because SAGA has been shown to display conformational variability, we applied gradient fixation to stabilize purified SAGA and systematically analyzed this flexibility using single-particle EM. Our two- and three-dimensional studies show that SAGA adopts three major conformations, and mutations of specific subunits affect the distribution among these. We also located the four functional modules of SAGA using electron microscopy-based labeling and transcriptional activator binding analyses and show that the acetyltransferase module is localized in the most mobile region of the complex. We further comprehensively mapped the subunit interconnectivity of SAGA using cross-linking mass spectrometry, revealing that the Spt and Taf subunits form the structural core of the complex. These results provide the necessary restraints for us to generate a model of the spatial arrangement of all SAGA subunits. According to this model, the chromatin-binding domains of SAGA are all clustered in one face of the complex that is highly flexible. Our results relate information of overall SAGA structure with detailed subunit level interactions, improving our understanding of its architecture and flexibility.

  10. Membrane insertion and topology of the translocon-associated protein (TRAP) gamma subunit.

    PubMed

    Bañó-Polo, Manuel; Martínez-Garay, Carlos A; Grau, Brayan; Martínez-Gil, Luis; Mingarro, Ismael

    2017-05-01

    Translocon-associated protein (TRAP) complex is intimately associated with the ER translocon for the insertion or translocation of newly synthesised proteins in eukaryotic cells. The TRAP complex is comprised of three single-spanning and one multiple-spanning subunits. We have investigated the membrane insertion and topology of the multiple-spanning TRAP-γ subunit by glycosylation mapping and green fluorescent protein fusions both in vitro and in cell cultures. Results demonstrate that TRAP-γ has four transmembrane (TM) segments, an Nt/Ct cytosolic orientation and that the less hydrophobic TM segment inserts efficiently into the membrane only in the cellular context of full-length protein.

  11. Gene targeting of CK2 catalytic subunits

    PubMed Central

    Lou, David Y.; Toselli, Paul; Landesman-Bollag, Esther; Dominguez, Isabel

    2013-01-01

    Protein kinase CK2 is a highly conserved and ubiquitous serine–threonine kinase. It is a tetrameric enzyme that is made up of two regulatory CK2β subunits and two catalytic subunits, either CK2α/CK2α, CK2α/ CK2α′, or CK2α′/CK2α′. Although the two catalytic subunits diverge in their C termini, their enzymatic activities are similar. To identify the specific function of the two catalytic subunits in development, we have deleted them individually from the mouse genome by homologous recombination. We have previously reported that CK2α′is essential for male germ cell development, and we now demonstrate that CK2α has an essential role in embryogenesis, as mice lacking CK2α die in mid-embryogenesis, with cardiac and neural tube defects. PMID:18594950

  12. Anthranilate synthase subunit organization in Chromobacterium violaceum.

    PubMed

    Carminatti, C A; Oliveira, I L; Recouvreux, D O S; Antônio, R V; Porto, L M

    2008-09-16

    Tryptophan is an aromatic amino acid used for protein synthesis and cellular growth. Chromobacterium violaceum ATCC 12472 uses two tryptophan molecules to synthesize violacein, a secondary metabolite of pharmacological interest. The genome analysis of this bacterium revealed that the genes trpA-F and pabA-B encode the enzymes of the tryptophan pathway in which the first reaction is the conversion of chorismate to anthranilate by anthranilate synthase (AS), an enzyme complex. In the present study, the organization and structure of AS protein subunits from C. violaceum were analyzed using bioinformatics tools available on the Web. We showed by calculating molecular masses that AS in C. violaceum is composed of alpha (TrpE) and beta (PabA) subunits. This is in agreement with values determined experimentally. Catalytic and regulatory sites of the AS subunits were identified. The TrpE and PabA subunits contribute to the catalytic site while the TrpE subunit is involved in the allosteric site. Protein models for the TrpE and PabA subunits were built by restraint-based homology modeling using AS enzyme, chains A and B, from Salmonella typhimurium (PDB ID 1I1Q).

  13. Protein synthesis by ribosomes with tethered subunits.

    PubMed

    Orelle, Cédric; Carlson, Erik D; Szal, Teresa; Florin, Tanja; Jewett, Michael C; Mankin, Alexander S

    2015-08-06

    The ribosome is a ribonucleoprotein machine responsible for protein synthesis. In all kingdoms of life it is composed of two subunits, each built on its own ribosomal RNA (rRNA) scaffold. The independent but coordinated functions of the subunits, including their ability to associate at initiation, rotate during elongation, and dissociate after protein release, are an established model of protein synthesis. Furthermore, the bipartite nature of the ribosome is presumed to be essential for biogenesis, since dedicated assembly factors keep immature ribosomal subunits apart and prevent them from translation initiation. Free exchange of the subunits limits the development of specialized orthogonal genetic systems that could be evolved for novel functions without interfering with native translation. Here we show that ribosomes with tethered and thus inseparable subunits (termed Ribo-T) are capable of successfully carrying out protein synthesis. By engineering a hybrid rRNA composed of both small and large subunit rRNA sequences, we produced a functional ribosome in which the subunits are covalently linked into a single entity by short RNA linkers. Notably, Ribo-T was not only functional in vitro, but was also able to support the growth of Escherichia coli cells even in the absence of wild-type ribosomes. We used Ribo-T to create the first fully orthogonal ribosome-messenger RNA system, and demonstrate its evolvability by selecting otherwise dominantly lethal rRNA mutations in the peptidyl transferase centre that facilitate the translation of a problematic protein sequence. Ribo-T can be used for exploring poorly understood functions of the ribosome, enabling orthogonal genetic systems, and engineering ribosomes with new functions.

  14. Distinct Expression Patterns of Glycoprotein Hormone Subunits in the Lophotrochozoan Aplysia: Implications for the Evolution of Neuroendocrine Systems in Animals

    PubMed Central

    Plachetzki, David; Donelly, Evonne; Gunaratne, Dinuka; Bobkova, Yelena; Jacobson, John; Kohn, Andrea B.; Moroz, Leonid L.

    2012-01-01

    Glycoprotein hormones (GPHs) comprise a group of signaling molecules critical for major metabolic and reproductive functions. In vertebrates they include chorionic gonadotropin, LH, FSH, and TSH. The active hormones are characterized by heterodimerization between a common α and hormone-specific β subunit, which activate leucine-rich repeat-containing G protein coupled receptors. To date, genes referred to as GPHα2 and GPHβ5 have been the only glycoprotein hormone subunits identified in invertebrates, suggesting that other GPHα and GPHβ subunits diversified during vertebrate evolution. Still the functions of GPHα2 and GPHβ5 remain largely unknown for both vertebrates and invertebrates. To further understand the evolution and putative function of these subunits, we cloned and analyzed phylogenetically two glycoprotein subunits, AcaGPHα and AcaGPHβ, from the sea hare Aplysia californica. Model based three-dimensional predictions of AcaGPHβ confirm the presence of a complete cysteine knot, two hairpin loops, and a long loop. As in the human GPHβ5 subunit the seatbelt structure is absent in AcaGPHβ. We also found that AcaGPHα and AcaGPHβ subunits are expressed in larval stages of Aplysia, and we present a detailed expression map of the subunits in the adult central nervous system using in situ hybridizations. Both subunits are expressed in subpopulations of pleural and buccal mechanosensory neurons, suggesting a neuronal modulatory function of these subunits in Aplysia. Furthermore it supports the model of a relatively diffuse neuroendocrine-like system in molluscs, where specific primary sensory neurons release peptides extrasynaptically (paracrine secretion). This is in contrast to vertebrates and insects, in which releasing and stimulating factor from centralized sensory regions of the central nervous system ultimately regulate hormone release in peripheral glands. PMID:22977258

  15. Distinct expression patterns of glycoprotein hormone subunits in the lophotrochozoan Aplysia: implications for the evolution of neuroendocrine systems in animals.

    PubMed

    Heyland, Andreas; Plachetzki, David; Donelly, Evonne; Gunaratne, Dinuka; Bobkova, Yelena; Jacobson, John; Kohn, Andrea B; Moroz, Leonid L

    2012-11-01

    Glycoprotein hormones (GPHs) comprise a group of signaling molecules critical for major metabolic and reproductive functions. In vertebrates they include chorionic gonadotropin, LH, FSH, and TSH. The active hormones are characterized by heterodimerization between a common α and hormone-specific β subunit, which activate leucine-rich repeat-containing G protein coupled receptors. To date, genes referred to as GPHα2 and GPHβ5 have been the only glycoprotein hormone subunits identified in invertebrates, suggesting that other GPHα and GPHβ subunits diversified during vertebrate evolution. Still the functions of GPHα2 and GPHβ5 remain largely unknown for both vertebrates and invertebrates. To further understand the evolution and putative function of these subunits, we cloned and analyzed phylogenetically two glycoprotein subunits, AcaGPHα and AcaGPHβ, from the sea hare Aplysia californica. Model based three-dimensional predictions of AcaGPHβ confirm the presence of a complete cysteine knot, two hairpin loops, and a long loop. As in the human GPHβ5 subunit the seatbelt structure is absent in AcaGPHβ. We also found that AcaGPHα and AcaGPHβ subunits are expressed in larval stages of Aplysia, and we present a detailed expression map of the subunits in the adult central nervous system using in situ hybridizations. Both subunits are expressed in subpopulations of pleural and buccal mechanosensory neurons, suggesting a neuronal modulatory function of these subunits in Aplysia. Furthermore it supports the model of a relatively diffuse neuroendocrine-like system in molluscs, where specific primary sensory neurons release peptides extrasynaptically (paracrine secretion). This is in contrast to vertebrates and insects, in which releasing and stimulating factor from centralized sensory regions of the central nervous system ultimately regulate hormone release in peripheral glands.

  16. Genetic Mapping

    MedlinePlus

    ... Fact Sheets Fact Sheets En Español: Mapeo Genético Genetic Mapping What is genetic mapping? How do researchers ... genetic map? What are genetic markers? What is genetic mapping? Among the main goals of the Human ...

  17. Drosophila laminin: sequence of B2 subunit and expression of all three subunits during embryogenesis

    PubMed Central

    1989-01-01

    In a previous study, we described the cloning of the genes encoding the three subunits of Drosophila laminin, a substrate adhesion molecule, and the cDNA sequence of the B1 subunit (Montell and Goodman, 1988). This analysis revealed the similarity of Drosophila laminin with the mouse and human complexes in subunit composition, domain structure, and amino acid sequence. In this paper, we report the deduced amino acid sequence of the B2 subunit. We then describe the expression and tissue distribution of the three subunits of laminin during Drosophila embryogenesis using both in situ hybridization and immunolocalization techniques, with particular emphasis on its expression in and around the developing nervous system. PMID:2808533

  18. DNA-dependent RNA polymerase subunits encoded within the vaccinia virus genome.

    PubMed Central

    Jones, E V; Puckett, C; Moss, B

    1987-01-01

    Antiserum to a multisubunit DNA-dependent RNA polymerase from vaccinia virions was prepared to carry out genetic studies. This antiserum selectively inhibited the activity of the viral polymerase but had no effect on calf thymus RNA polymerase II. The specificity of the antiserum was further demonstrated by immunoprecipitation of RNA polymerase subunits from dissociated virus particles. The presence in vaccinia virus-infected cells of mRNA that encodes the polymerase subunits was determined by in vitro translation. Immunoprecipitable polypeptides with Mrs of about 135,000, 128,000, 36,000, 34,000, 31,000, 23,000, 21,000, 20,000, and 17,000 were made when early mRNA was added to reticulocyte extracts. The subunits were encoded within the vaccinia virus genome, as demonstrated by translation of early mRNA that hybridized to vaccinia virus DNA. The locations of the subunit genes were determined initially by hybridization of RNA to a series of overlapping 40-kilobase-pair DNA fragments that were cloned in a cosmid vector. Further mapping was achieved with cloned HindIII restriction fragments. Results of these studies indicated that RNA polymerase subunit genes are transcribed early in infection and are distributed within the highly conserved central portion of the poxvirus genome in HindIII fragments E, J, H, D, and A. Images PMID:3033308

  19. Localization of the regulatory particle subunit Sem1 in the 26S proteasome

    SciTech Connect

    Bohn, Stefan; Sakata, Eri; Beck, Florian; Pathare, Ganesh R.; Schnitger, Jérôme; Nágy, Istvan; Baumeister, Wolfgang Förster, Friedrich

    2013-05-31

    Highlights: •26S proteasome subunit Sem1 was mapped using cryo-EM and cross-linking data. •C-terminal helix of Sem1 located near winged helix motif of Rpn7. •N-terminal part of Sem1 tethers Rpn7, Rpn3 and lid helical bundle. •Sem1 binds differently to PCI-domains of proteasome subunit Rpn7 and TREX-2 subunit Thp1. -- Abstract: The ubiquitin–proteasome system is responsible for regulated protein degradation in the cell with the 26S proteasome acting as its executive arm. The molecular architecture of this 2.5 MDa complex has been established recently, with the notable exception of the small acidic subunit Sem1. Here, we localize the C-terminal helix of Sem1 binding to the PCI domain of the subunit Rpn7 using cryo-electron microscopy single particle reconstruction of proteasomes purified from yeast cells with sem1 deletion. The approximate position of the N-terminal region of Sem1 bridging the cleft between Rpn7 and Rpn3 was inferred based on site-specific cross-linking data of the 26S proteasome. Our structural studies indicate that Sem1 can assume different conformations in different contexts, which supports the idea that Sem1 functions as a molecular glue stabilizing the Rpn3/Rpn7 heterodimer.

  20. Diversity of insect nicotinic acetylcholine receptor subunits.

    PubMed

    Jones, Andrew K; Sattelle, David B

    2010-01-01

    Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that mediate fast synaptic transmission in the insect nervous system and are targets of a major group of insecticides, the neonicotinoids. They consist of five subunits arranged around a central ion channeL Since the subunit composition determines the functional and pharmacological properties of the receptor the presence of nAChR families comprising several subunit-encodinggenes provides a molecular basis for broad functional diversity. Analyses of genome sequences have shown that nAChR gene families remain compact in diverse insect species, when compared to their nematode andvertebrate counterparts. Thus, the fruit fly (Drosophila melanogaster), malaria mosquito (Anopheles gambiae), honey bee (Apis mellifera), silk worm (Bombyx mon) and the red flour beetle (Tribolium castaneum) possess 10-12 nAChR genes while human and the nematode Caenorhabditis elegans have 16 and 29 respectively. Although insect nAChRgene families are amongst the smallest known, receptor diversity can be considerably increased by the posttranscriptional processes alternative splicing and mRNA A-to-I editingwhich can potentially generate protein products which far outnumber the nAChR genes. These two processes can also generate species-specific subunit isoforms. In addition, each insect possesses at least one highly divergent nAChR subunit which may perform species-specific functions. Species-specific subunit diversification may offer promising targets for future rational design of insecticides that target specific pest insects while sparing beneficial species.

  1. Allele-specific interactions between the yeast RFC1 and RFC5 genes suggest a basis for RFC subunit-subunit interactions.

    PubMed

    Beckwith, W; McAlear, M A

    2000-11-01

    Replication factor C (RFC) is an essential, multi-subunit ATPase that functions in DNA replication, DNA repair, and DNA metabolism-related checkpoints. In order to investigate how the individual RFC subunits contribute to these functions in vivo, we undertook a genetic analysis of RFC genes from budding yeast. We isolated and characterized mutations in the RFC5 gene that could suppress the cold-sensitive phenotype of rfc1-1 mutants. Analysis of the RFC5 suppressors revealed that they could not suppress the elongated telomere phenotype, the sensitivity to DNA damaging agents, or the mutator phenotype of rfc1-1 mutants. Unlike the checkpoint-defective rfc5-1 mutation, the RFC5 suppressor mutations did not interfere with the methylmethane sulfonate- or hydroxyurea-induced phosphorylation of Rad53p. The Rfc5p suppressor substitutions mapped to amino acid positions in the conserved RFC box motifs IV-VII. Comparisons of the structures of related RFC box-containing proteins suggest that these RFC motifs may function to coordinate interactions between neighboring subunits of multi-subunit ATPases.

  2. The β-conglycinin deficiency in wild soybean is associated with the tail-to-tail inverted repeat of the α-subunit genes.

    PubMed

    Tsubokura, Yasutaka; Hajika, Makita; Kanamori, Hiroyuki; Xia, Zhengjun; Watanabe, Satoshi; Kaga, Akito; Katayose, Yuichi; Ishimoto, Masao; Harada, Kyuya

    2012-02-01

    β-conglycinin, a major seed protein in soybean, is composed of α, α', and β subunits sharing a high homology among them. Despite its many health benefits, β-conglycinin has a lower amino acid score and lower functional gelling properties compared to glycinin, another major soybean seed protein. In addition, the α, α', and β subunits also contain major allergens. A wild soybean (Glycine soja Sieb et Zucc.) line, 'QT2', lacks all of the β-conglycinin subunits, and the deficiency is controlled by a single dominant gene, Scg-1 (Suppressor of β-conglycinin). This gene was characterized using a soybean cultivar 'Fukuyutaka', 'QY7-25', (its near-isogenic line carrying the Scg-1 gene), and the F₂ population derived from them. The physical map of the Scg-1 region covered by lambda phage genomic clones revealed that the two α-subunit genes, a β-subunit gene, and a pseudo α-subunit gene were closely organized. The two α-subunit genes were arranged in a tail-to-tail orientation, and the genes were separated by 197 bp in Scg-1 compared to 3.3 kb in the normal allele (scg-1). In addition, small RNA was detected in immature seeds of the mutants by northern blot analysis using an RNA probe of the α subunit. These results strongly suggest that β-conglycinin deficiency in QT2 is controlled by post-transcriptional gene silencing through the inverted repeat of the α subunits.

  3. Subunit Recombinant Vaccine Protects Against Monkeypox

    DTIC Science & Technology

    2006-05-27

    Subunit Recombinant Vaccine Protects against Monkeypox1 Jean-Michel Heraud,* Yvette Edghill-Smith,*† Victor Ayala,‡ Irene Kalisz,‡ Janie Parrino ...GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Heraud, JM Edghill-Smith, Y Ayala, V Kalisz, I Parrino , J Kalyanaraman, VS Manischewitz, J King

  4. Localization of a gene for a glutamate binding subunit of a NMDA receptor (GRINA) to 8q24

    SciTech Connect

    Lewis, T.B.; DuPont, B.R.; Leach, R.

    1996-02-15

    This article reports on the localization of a gene for a glutamate binding subunit of an N-methyl-D-aspartate (NMDA) receptor, called GRINA, to human chromosome 8q24 using fluorescence in situ hybridization and radiation hybridization mapping. This gene mapped outside the critical region for benign familial neonatal convulsions (BFNC), a rare form of epilepsy; however, GRINA could be the causative genetic factor inducing idiopathic generalized epilepsy. Further studies need to be conducted. 15 refs., 2 figs.

  5. Planetary maps

    USGS Publications Warehouse

    ,

    1992-01-01

    An important goal of the USGS planetary mapping program is to systematically map the geology of the Moon, Mars, Venus, and Mercury, and the satellites of the outer planets. These geologic maps are published in the USGS Miscellaneous Investigations (I) Series. Planetary maps on sale at the USGS include shaded-relief maps, topographic maps, geologic maps, and controlled photomosaics. Controlled photomosaics are assembled from two or more photographs or images using a network of points of known latitude and longitude. The images used for most of these planetary maps are electronic images, obtained from orbiting television cameras, various optical-mechanical systems. Photographic film was only used to map Earth's Moon.

  6. Subunit-selective proteasome activity profiling uncovers uncoupled proteasome subunit activities during bacterial infections.

    PubMed

    Misas-Villamil, Johana C; van der Burgh, Aranka M; Grosse-Holz, Friederike; Bach-Pages, Marcel; Kovács, Judit; Kaschani, Farnusch; Schilasky, Sören; Emon, Asif Emran Khan; Ruben, Mark; Kaiser, Markus; Overkleeft, Hermen S; van der Hoorn, Renier A L

    2017-01-24

    The proteasome is a nuclear - cytoplasmic proteolytic complex involved in nearly all regulatory pathways in plant cells. The three different catalytic activities of the proteasome can have different functions but tools to monitor and control these subunits selectively are not yet available in plant science. Here, we introduce subunit-selective inhibitors and dual-color fluorescent activity-based probes for studying two of the three active catalytic subunits of the plant proteasome. We validate these tools in two model plants and use this to study the proteasome during plant-microbe interactions. Our data reveals that Nicotiana benthamiana incorporates two different paralogs of each catalytic subunit into active proteasomes. Interestingly, both β1 and β5 activities are significantly increased upon infection with pathogenic Pseudomonas syringae pv. tomato DC3000 lacking hopQ1-1 (PtoDC3000(ΔhQ)) whilst the activity profile of the β1 subunit changes. Infection with wild-type PtoDC3000 causes proteasome activities that range from strongly induced β1 and β5 activities to strongly suppressed β5 activities, revealing that β1 and β5 activities can be uncoupled during bacterial infection. These selective probes and inhibitors are now available to the plant science community and can be widely and easily applied to study the activity and role of the different catalytic subunits of the proteasome in different plant species. This article is protected by copyright. All rights reserved.

  7. Interaction between Subunits of Heterodimeric Splicing Factor U2AF Is Essential In Vivo

    PubMed Central

    Rudner, David Z.; Kanaar, Roland; Breger, Kevin S.; Rio, Donald C.

    1998-01-01

    The heterodimeric pre-mRNA splicing factor, U2AF (U2 snRNP auxiliary factor), plays a critical role in 3′ splice site selection. Although the U2AF subunits associate in a tight complex, biochemical experiments designed to address the requirement for both subunits in splicing have yielded conflicting results. We have taken a genetic approach to assess the requirement for the Drosophila U2AF heterodimer in vivo. We developed a novel Escherichia coli copurification assay to map the domain on the Drosophila U2AF large subunit (dU2AF50) that interacts with the Drosophila small subunit (dU2AF38). A 28-amino-acid fragment on dU2AF50 that is both necessary and sufficient for interaction with dU2AF38 was identified. Using the copurification assay, we scanned this 28-amino-acid interaction domain for mutations that abrogate heterodimer formation. A collection of these dU2AF50 point mutants was then tested in vivo for genetic complementation of a recessive lethal dU2AF50 allele. A mutation that completely abolished interaction with dU2AF38 was incapable of complementation, whereas dU2AF50 mutations that did not effect heterodimer formation rescued the recessive lethal dU2AF50 allele. Analysis of heterodimer formation in embryo extracts derived from these interaction mutant lines revealed a perfect correlation between the efficiency of subunit association and the ability to complement the dU2AF50 recessive lethal allele. These data indicate that Drosophila U2AF heterodimer formation is essential for viability in vivo, consistent with a requirement for both subunits in splicing in vitro. PMID:9528748

  8. Recent Advances in Subunit Vaccine Carriers

    PubMed Central

    Vartak, Abhishek; Sucheck, Steven J.

    2016-01-01

    The lower immunogenicity of synthetic subunit antigens, compared to live attenuated vaccines, is being addressed with improved vaccine carriers. Recent reports indicate that the physio-chemical properties of these carriers can be altered to achieve optimal antigen presentation, endosomal escape, particle bio-distribution, and cellular trafficking. The carriers can be modified with various antigens and ligands for dendritic cells targeting. They can also be modified with adjuvants, either covalently or entrapped in the matrix, to improve cellular and humoral immune responses against the antigen. As a result, these multi-functional carrier systems are being explored for use in active immunotherapy against cancer and infectious diseases. Advancing technology, improved analytical methods, and use of computational methodology have also contributed to the development of subunit vaccine carriers. This review details recent breakthroughs in the design of nano-particulate vaccine carriers, including liposomes, polymeric nanoparticles, and inorganic nanoparticles. PMID:27104575

  9. β1-subunit-induced structural rearrangements of the Ca2+- and voltage-activated K+ (BK) channel.

    PubMed

    Castillo, Juan P; Sánchez-Rodríguez, Jorge E; Hyde, H Clark; Zaelzer, Cristian A; Aguayo, Daniel; Sepúlveda, Romina V; Luk, Louis Y P; Kent, Stephen B H; Gonzalez-Nilo, Fernando D; Bezanilla, Francisco; Latorre, Ramón

    2016-06-07

    Large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels are involved in a large variety of physiological processes. Regulatory β-subunits are one of the mechanisms responsible for creating BK channel diversity fundamental to the adequate function of many tissues. However, little is known about the structure of its voltage sensor domain. Here, we present the external architectural details of BK channels using lanthanide-based resonance energy transfer (LRET). We used a genetically encoded lanthanide-binding tag (LBT) to bind terbium as a LRET donor and a fluorophore-labeled iberiotoxin as the LRET acceptor for measurements of distances within the BK channel structure in a living cell. By introducing LBTs in the extracellular region of the α- or β1-subunit, we determined (i) a basic extracellular map of the BK channel, (ii) β1-subunit-induced rearrangements of the voltage sensor in α-subunits, and (iii) the relative position of the β1-subunit within the α/β1-subunit complex.

  10. Subunit organization in cytoplasmic dynein subcomplexes

    PubMed Central

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

    2002-01-01

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

  11. 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

    SciTech Connect

    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.

  12. Contour Mapping

    NASA Technical Reports Server (NTRS)

    1995-01-01

    In the early 1990s, the Ohio State University Center for Mapping, a NASA Center for the Commercial Development of Space (CCDS), developed a system for mobile mapping called the GPSVan. While driving, the users can map an area from the sophisticated mapping van equipped with satellite signal receivers, video cameras and computer systems for collecting and storing mapping data. George J. Igel and Company and the Ohio State University Center for Mapping advanced the technology for use in determining the contours of a construction site. The new system reduces the time required for mapping and staking, and can monitor the amount of soil moved.

  13. Quality control of a cytoplasmic protein complex: chaperone motors and the ubiquitin-proteasome system govern the fate of orphan fatty acid synthase subunit Fas2 of yeast.

    PubMed

    Scazzari, Mario; Amm, Ingo; Wolf, Dieter H

    2015-02-20

    For the assembly of protein complexes in the cell, the presence of stoichiometric amounts of the respective protein subunits is of utmost importance. A surplus of any of the subunits may trigger unspecific and harmful protein interactions and has to be avoided. A stoichiometric amount of subunits must finally be reached via transcriptional, translational, and/or post-translational regulation. Synthesis of saturated 16 and 18 carbon fatty acids is carried out by fatty acid synthase: in yeast Saccharomyces cerevisiae, a 2.6-MDa molecular mass assembly containing six protomers each of two different subunits, Fas1 (β) and Fas2 (α). The (α)6(β)6 complex carries six copies of all eight enzymatic activities required for fatty acid synthesis. The FAS1 and FAS2 genes in yeast are unlinked and map on two different chromosomes. Here we study the fate of the α-subunit of the complex, Fas2, when its partner, the β-subunit Fas1, is absent. Individual subunits of fatty acid synthase are proteolytically degraded when the respective partner is missing. Elimination of Fas2 is achieved by the proteasome. Here we show that a ubiquitin transfer machinery is required for Fas2 elimination. The major ubiquitin ligase targeting the superfluous Fas2 subunit to the proteasome is Ubr1. The ubiquitin-conjugating enzymes Ubc2 and Ubc4 assist the degradation process. The AAA-ATPase Cdc48 and the Hsp70 chaperone Ssa1 are crucially involved in the elimination of Fas2.

  14. Models for the a subunits of the Thermus thermophilus V/A-ATPase and Saccharomyces cerevisiae V-ATPase enzymes by cryo-EM and evolutionary covariance

    PubMed Central

    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

  15. Models for the a subunits of the Thermus thermophilus V/A-ATPase and Saccharomyces cerevisiae V-ATPase enzymes by cryo-EM and evolutionary covariance.

    PubMed

    Schep, Daniel G; Zhao, Jianhua; Rubinstein, John L

    2016-03-22

    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.

  16. Differential accumulation of ribonucleotide reductase subunits in clam oocytes: the large subunit is stored as a polypeptide, the small subunit as untranslated mRNA

    PubMed Central

    1986-01-01

    Within minutes of fertilization of clam oocytes, translation of a set of maternal mRNAs is activated. One of the most abundant of these stored mRNAs encodes the small subunit of ribonucleotide reductase (Standart, N. M., S. J. Bray, E. L. George, T. Hunt, and J. V. Ruderman, 1985, J. Cell Biol., 100:1968-1976). Unfertilized oocytes do not contain any ribonucleotide reductase activity; such activity begins to appear shortly after fertilization. In virtually all organisms, this enzyme is composed of two dissimilar subunits with molecular masses of approximately 44 and 88 kD, both of which are required for activity. This paper reports the identification of the large subunit of clam ribonucleotide reductase isolated by dATP-Sepharose chromatography as a relatively abundant 86-kD polypeptide which is already present in oocytes, and whose level remains constant during early development. The enzyme activity of this large subunit was established in reconstitution assays using the small subunit isolated from embryos by virtue of its binding to the anti-tubulin antibody YL 1/2. Thus the two components of clam ribonucleotide reductase are differentially stored in the oocyte: the small subunit in the form of untranslated mRNA and the large subunit as protein. When fertilization triggers the activation of translation of the maternal mRNA, the newly synthesized small subunit combines with the preformed large subunit to generate active ribonucleotide reductase. PMID:3536960

  17. Solution nuclear magnetic resonance structure of the GATase subunit and structural basis of the interaction between GATase and ATPPase subunits in a two-subunit-type GMPS from Methanocaldococcus jannaschii.

    PubMed

    Ali, Rustam; Kumar, Sanjeev; Balaram, Hemalatha; Sarma, Siddhartha P

    2013-06-25

    The solution structure of the monomeric glutamine amidotransferase (GATase) subunit of the Methanocaldococcus janaschii (Mj) guanosine monophosphate synthetase (GMPS) has been determined using high-resolution nuclear magnetic resonance methods. Gel filtration chromatography and ¹⁵N backbone relaxation studies have shown that the Mj GATase subunit is present in solution as a 21 kDa (188-residue) monomer. The ensemble of 20 lowest-energy structures showed root-mean-square deviations of 0.35 ± 0.06 Å for backbone atoms and 0.8 ± 0.06 Å for all heavy atoms. Furthermore, 99.4% of the backbone dihedral angles are present in the allowed region of the Ramachandran map, indicating the stereochemical quality of the structure. The core of the tertiary structure of the GATase is composed of a seven-stranded mixed β-sheet that is fenced by five α-helices. The Mj GATase is similar in structure to the Pyrococcus horikoshi (Ph) GATase subunit. Nuclear magnetic resonance (NMR) chemical shift perturbations and changes in line width were monitored to identify residues on GATase that were responsible for interaction with magnesium and the ATPPase subunit, respectively. These interaction studies showed that a common surface exists for the metal ion binding as well as for the protein-protein interaction. The dissociation constant for the GATase-Mg(2+) interaction has been found to be ∼1 mM, which implies that interaction is very weak and falls in the fast chemical exchange regime. The GATase-ATPPase interaction, on the other hand, falls in the intermediate chemical exchange regime on the NMR time scale. The implication of this interaction in terms of the regulation of the GATase activity of holo GMPS is discussed.

  18. 3D Gel Map of Arabidopsis Complex I

    PubMed Central

    Peters, Katrin; Belt, Katharina; Braun, Hans-Peter

    2013-01-01

    Complex I has a unique structure in plants and includes extra subunits. Here, we present a novel study to define its protein constituents. Mitochondria were isolated from Arabidopsis thaliana cell cultures, leaves, and roots. Subunits of complex I were resolved by 3D blue-native (BN)/SDS/SDS-PAGE and identified by mass spectrometry. Overall, 55 distinct proteins were found, seven of which occur in pairs of isoforms. We present evidence that Arabidopsis complex I consists of 49 distinct types of subunits, 40 of which represent homologs of bovine complex I. The nine other subunits represent special proteins absent in the animal linage of eukaryotes, most prominently a group of subunits related to bacterial gamma-type carbonic anhydrases. A GelMap http://www.gelmap.de/arabidopsis-3d-complex-i/ is presented for promoting future complex I research in Arabidopsis thaliana. PMID:23761796

  19. Prefoldin Subunits Are Protected from Ubiquitin-Proteasome System-mediated Degradation by Forming Complex with Other Constituent Subunits*

    PubMed Central

    Miyazawa, Makoto; Tashiro, Erika; Kitaura, Hirotake; Maita, Hiroshi; Suto, Hiroo; Iguchi-Ariga, Sanae M. M.; Ariga, Hiroyoshi

    2011-01-01

    The molecular chaperone prefoldin (PFD) is a complex comprised of six different subunits, PFD1-PFD6, and delivers newly synthesized unfolded proteins to cytosolic chaperonin TRiC/CCT to facilitate the folding of proteins. PFD subunits also have functions different from the function of the PFD complex. We previously identified MM-1α/PFD5 as a novel c-Myc-binding protein and found that MM-1α suppresses transformation activity of c-Myc. However, it remains unclear how cells regulate protein levels of individual subunits and what mechanisms alter the ratio of their activities between subunits and their complex. In this study, we found that knockdown of one subunit decreased protein levels of other subunits and that transfection of five subunits other than MM-1α into cells increased the level of endogenous MM-1α. We also found that treatment of cells with MG132, a proteasome inhibitor, increased the level of transfected/overexpressed MM-1α but not that of endogenous MM-1α, indicating that overexpressed MM-1α, but not endogenous MM-1α, was degraded by the ubiquitin proteasome system (UPS). Experiments using other PFD subunits showed that the UPS degraded a monomer of PFD subunits, though extents of degradation varied among subunits. Furthermore, the level of one subunit was increased after co-transfection with the respective subunit, indicating that there are specific combinations between subunits to be stabilized. These results suggest mutual regulation of protein levels among PFD subunits and show how individual subunits form the PFD complex without degradation. PMID:21478150

  20. Prefoldin subunits are protected from ubiquitin-proteasome system-mediated degradation by forming complex with other constituent subunits.

    PubMed

    Miyazawa, Makoto; Tashiro, Erika; Kitaura, Hirotake; Maita, Hiroshi; Suto, Hiroo; Iguchi-Ariga, Sanae M M; Ariga, Hiroyoshi

    2011-06-03

    The molecular chaperone prefoldin (PFD) is a complex comprised of six different subunits, PFD1-PFD6, and delivers newly synthesized unfolded proteins to cytosolic chaperonin TRiC/CCT to facilitate the folding of proteins. PFD subunits also have functions different from the function of the PFD complex. We previously identified MM-1α/PFD5 as a novel c-Myc-binding protein and found that MM-1α suppresses transformation activity of c-Myc. However, it remains unclear how cells regulate protein levels of individual subunits and what mechanisms alter the ratio of their activities between subunits and their complex. In this study, we found that knockdown of one subunit decreased protein levels of other subunits and that transfection of five subunits other than MM-1α into cells increased the level of endogenous MM-1α. We also found that treatment of cells with MG132, a proteasome inhibitor, increased the level of transfected/overexpressed MM-1α but not that of endogenous MM-1α, indicating that overexpressed MM-1α, but not endogenous MM-1α, was degraded by the ubiquitin proteasome system (UPS). Experiments using other PFD subunits showed that the UPS degraded a monomer of PFD subunits, though extents of degradation varied among subunits. Furthermore, the level of one subunit was increased after co-transfection with the respective subunit, indicating that there are specific combinations between subunits to be stabilized. These results suggest mutual regulation of protein levels among PFD subunits and show how individual subunits form the PFD complex without degradation.

  1. Inherent conformational flexibility of F1-ATPase α-subunit.

    PubMed

    Hahn-Herrera, Otto; Salcedo, Guillermo; Barril, Xavier; García-Hernández, Enrique

    2016-09-01

    The core of F1-ATPase consists of three catalytic (β) and three noncatalytic (α) subunits, forming a hexameric ring in alternating positions. A wealth of experimental and theoretical data has provided a detailed picture of the complex role played by catalytic subunits. Although major conformational changes have only been seen in β-subunits, it is clear that α-subunits have to respond to these changes in order to be able to transmit information during the rotary mechanism. However, the conformational behavior of α-subunits has not been explored in detail. Here, we have combined unbiased molecular dynamics (MD) simulations and calorimetrically measured thermodynamic signatures to investigate the conformational flexibility of isolated α-subunits, as a step toward deepening our understanding of its function inside the α3β3 ring. The simulations indicate that the open-to-closed conformational transition of the α-subunit is essentially barrierless, which is ideal to accompany and transmit the movement of the catalytic subunits. Calorimetric measurements of the recombinant α-subunit from Geobacillus kaustophilus indicate that the isolated subunit undergoes no significant conformational changes upon nucleotide binding. Simulations confirm that the nucleotide-free and nucleotide-bound subunits show average conformations similar to that observed in the F1 crystal structure, but they reveal an increased conformational flexibility of the isolated α-subunit upon MgATP binding, which might explain the evolutionary conserved capacity of α-subunits to recognize nucleotides with considerable strength. Furthermore, we elucidate the different dependencies that α- and β-subunits show on Mg(II) for recognizing ATP.

  2. Exclusion of five subunits of cGMP phosphodiesterase in Leber's congenital amaurosis.

    PubMed

    Perrault, I; Châtelin, S; Nancy, V; Rozet, J M; Gerber, S; Ghazi, I; Souied, E; Dufier, J L; Munnich, A; de Gunzburg, J; Kaplan, J

    1998-03-01

    Leber's congenital amaurosis (LCA) is the earliest and most severe of all inherited retinal dystrophies. Recently, we mapped an LCA gene to chromosome 17p13.1 (LCA1) and ascribed the disease to mutations of the retinal guanylate cyclase (ret GC) gene in a subset of families of North African ancestry. Owing to the genetic heterogeneity of LCA and considering that LCA1 results from an impaired production of cGMP in the retina (with permanent closure of cGMP-gated cation channels), we hypothesized that the activation of the cGMP phosphodiesterase (PDE) could trigger the disease by lowering the intracellular cGMP level in the retina. The rod and cone cGMP-PDE inhibitory subunits were regarded therefore as candidate genes in LCA. Here, we report the exclusion of five rod and cone cGMP-PDE subunits in LCA families unlinked to chromosome 17p13.

  3. [Nose surgical anatomy in six aesthetic subunits].

    PubMed

    Chaput, B; Lauwers, F; Lopez, R; Saboye, J; André, A; Grolleau, J-L; Chavoin, J-P

    2013-04-01

    The nose is a complex entity, combining aesthetic and functional roles. Descriptive anatomy is a fundamental science that it can be difficult to relate directly to our daily surgical activity. Reasoning in terms of aesthetic subunits to decide on his actions appeared to us so obvious. The aim of this paper is to resume the anatomical bases relevant to our daily practice in order to fully apprehend the restorative or cosmetic procedures. We discuss the limits of the systematization of these principles in nasal oncology.

  4. USGS maps

    USGS Publications Warehouse

    ,

    2005-01-01

    Discover a small sample of the millions of maps produced by the U.S. Geological Survey (USGS) in its mission to map the Nation and survey its resources. This booklet gives a brief overview of the types of maps sold and distributed by the USGS through its Earth Science Information Centers (ESIC) and also available from business partners located in most States. The USGS provides a wide variety of maps, from topographic maps showing the geographic relief and thematic maps displaying the geology and water resources of the United States, to special studies of the moon and planets.

  5. Amino-terminal truncations of the ribulose-bisphosphate carboxylase small subunit influence catalysis and subunit interactions.

    PubMed Central

    Paul, K; Morell, M K; Andrews, T J

    1993-01-01

    The first 20 residues at the amino terminus of the small subunit of spinach ribulose-1,5-bisphosphate carboxylase form an irregular arm that makes extensive contacts with the large subunit and also with another small subunit (S. Knight, I. Andersson, and C.-I. Brändén [1990] J Mol Biol 215: 113-160). The influence of these contacts on subunit binding and, indirectly, on catalysis was investigated by constructing truncations from the amino terminus of the small subunit of the highly homologous enzyme from Synechococcus PCC 6301 expressed in Escherichia coli. Removal of the first six residues (and thus the region of contact with a neighboring small subunit) affected neither the affinity with which the small subunits bound to the large subunits nor the catalytic properties of the assembled holoenzyme. Extending the truncation to include the first 12 residues (which encroaches into a highly conserved region that interacts with the large subunit) also did not weaken intersubunit binding appreciably, but it reduced the catalytic activity of the holoenzyme nearly 5-fold. Removal of an additional single residue (i.e. removal of a total of 13 residues) weakened intersubunit binding approximately 80-fold. Paradoxically, this partially restored catalytic activity to approximately 40% of that of the wild-type holoenzyme. None of these truncations materially affected the Km values for ribulose-1,5-bisphosphate or CO2. Removal of all 20 residues of the irregular arm (thereby deleting the conserved region of contact with large subunits) totally abolished the small subunit's ability to bind to large subunits to form a stable holoenzyme. However, this truncated small subunit was still synthesized by the E. coli cells. These data are interpreted in terms of the role of the amino-terminal arm of the small subunit in maintaining the structure of the holoenzyme. PMID:8278544

  6. Structural model of the 50S subunit of E.Coli ribosomes from solution scattering

    SciTech Connect

    Svergun, D.I.; Koch, M.H.J.; Pedersen, J.S.; Serdyuk, I.N.

    1994-12-31

    The application of new methods of small-angle scattering data interpretation to a contrast variation study of the 50S ribosomal subunit of Escherichia coli in solution is described. The X-ray data from contrast variation with sucrose are analyzed in terms of the basic scattering curves from the volume inaccessible to sucrose and from the regions inside this volume occupied mainly by RNA and by proteins. From these curves models of the shape of the 50S and its RNA-rich core are evaluated and positioned so that their difference produces a scattering curve which is in good agreement with the scattering from the protein moiety. Basing on this preliminary model, the X-ray and neutron contrast variation data of the 50S subunit in aqueous solutions are interpreted in the frame of the advanced two-phase model described by the shapes of the 50S subunit and its RNA-rich core taking into account density fluctuations inside the RNA and the protein moiety. The shape of the envelope of the 50S subunit and of the RNA-rich core are evaluated with a resolution of about 40A. The shape of the envelope is in good agreement with the models of the 50S subunit obtained from electron microscopy on isolated particles. The shape of the RNA-rich core correlates well with the model of the entire particle determined by the image reconstruction from ordered sheets indicating that the latter model which is based on the subjective contouring of density maps is heavily biased towards the RNA.

  7. Structural requirement of the calcium-channel subunit alpha2delta for gabapentin binding.

    PubMed Central

    Wang, M; Offord, J; Oxender, D L; Su, T Z

    1999-01-01

    Gabapentin [Neurontin, 1-(aminomethyl)cyclohexaneacetic acid] is a novel anticonvulsant drug with a high binding affinity for the Ca(2+)-channel subunit alpha(2)delta. In this study, the gabapentin-binding properties of wild-type and mutated porcine brain alpha(2)delta proteins were investigated. Removal of the disulphide bonds between the alpha(2) and the delta subunits did not result in a significant loss of gabapentin binding, suggesting that the disulphide linkage between the two subunits is not required for binding. Singly expressed alpha(2) protein remained membrane associated. However, alpha(2) alone was unable to bind gabapentin, unless the cells were concurrently transfected with the expression vector for delta, suggesting that both alpha(2) and delta are required for gabapentin binding. Using internal deletion mutagenesis, we mapped two regions [amino acid residues 339-365 (DeltaF) and 875-905 (DeltaJ)] within the alpha(2) subunit that are not required for gabapentin binding. Further, deletion of three other individual regions [amino acid residues 206-222 (DeltaD), 516-537 (DeltaH) and 583-603 (DeltaI)] within the alpha(2) subunit disrupted gabapentin binding, suggesting the structural importance of these regions. Using alanine to replace four to six amino acid residues in each of these regions abolished gabapentin binding. These results demonstrate that region D, between the N-terminal end and the first putative transmembrane domain of alpha(2), and regions H and I, between the putative splicing acceptor sites (Gln(511) and Ser(601)), may play important roles in maintaining the structural integrity for gabapentin binding. Further single amino acid replacement mutagenesis within these regions identified Arg(217) as critical for gabapentin binding. PMID:10455017

  8. Homologous and unique G protein alpha subunits in the nematode Caenorhabditis elegans.

    PubMed Central

    Lochrie, M A; Mendel, J E; Sternberg, P W; Simon, M I

    1991-01-01

    A cDNA corresponding to a known G protein alpha subunit, the alpha subunit of Go (Go alpha), was isolated and sequenced. The predicted amino acid sequence of C. elegans Go alpha is 80-87% identical to other Go alpha sequences. An mRNA that hybridizes to the C. elegans Go alpha cDNA can be detected on Northern blots. A C. elegans protein that crossreacts with antibovine Go alpha antibody can be detected on immunoblots. A cosmid clone containing the C. elegans Go alpha gene (goa-1) was isolated and mapped to chromosome I. The genomic fragments of three other C. elegans G protein alpha subunit genes (gpa-1, gpa-2, and gpa-3) have been isolated using the polymerase chain reaction. The corresponding cosmid clones were isolated and mapped to disperse locations on chromosome V. The sequences of two of the genes, gpa-1 and gpa-3, were determined. The predicted amino acid sequences of gpa-1 and gpa-3 are only 48% identical to each other. Therefore, they are likely to have distinct functions. In addition they are not homologous enough to G protein alpha subunits in other organisms to be classified. Thus C. elegans has G proteins that are identifiable homologues of mammalian G proteins as well as G proteins that appear to be unique to C. elegans. Study of identifiable G proteins in C. elegans may result in a further understanding of their function in other organisms, whereas study of the novel G proteins may provide an understanding of unique aspects of nematode physiology. Images PMID:1907494

  9. RICH MAPS

    EPA Science Inventory

    Michael Goodchild recently gave eight reasons why traditional maps are limited as communication devices, and how interactive internet mapping can overcome these limitations. In the past, many authorities in cartography, from Jenks to Bertin, have emphasized the importance of sim...

  10. Calmodulin is a subunit of nitric oxide synthase from macrophages

    PubMed Central

    1992-01-01

    A central issue in nitric oxide (NO) research is to understand how NO can act in some settings as a servoregulator and in others as a cytotoxin. To answer this, we have sought a molecular basis for the differential regulation of the two known types of NO synthase (NOS). Constitutive NOS's in endothelium and neurons are activated by agonist- induced elevation of Ca2+ and resultant binding of calmodulin (CaM). In contrast, NOS in macrophages does not require added Ca2+ or CaM, but is regulated instead by transcription. We show here that macrophage NOS contains, as a tightly bound subunit, a molecule with the immunologic reactivity, high performance liquid chromatography retention time, tryptic map, partial amino acid sequence, and exact molecular mass of CaM. In contrast to most CaM-dependent enzymes, macrophage NOS binds CaM tightly without a requirement for elevated Ca2+. This may explain why NOS that is independent of Ca2+ and elevated CaM appears to be activated simply by being synthesized. PMID:1380065

  11. Historical Mapping

    USGS Publications Warehouse

    ,

    1999-01-01

    Maps become out of date over time. Maps that are out of date, however, can be useful to historians, attorneys, environmentalists, genealogists, and others interested in researching the background of a particular area. Local historians can compare a series of maps of the same area compiled over a long period of time to learn how the area developed. A succession of such maps can provide a vivid picture of how a place changed over time.

  12. Complementation of subunits from different bacterial luciferases. Evidence for the role of the. beta. subunit in the bioluminescent mechanism

    SciTech Connect

    Meighen, E.A.; Bartlet, I.

    1980-12-10

    Complementation of the nonidentical subunits (..cap alpha.. and ..beta..) of luciferases isolated from two different bioluminescent strains, Beneckea harveyi and Photobacterium phosphoreum, has resulted in the formation of a functional hybrid luciferase (..cap alpha../sub h/..beta../sub p/) containing the ..cap alpha.. subunit from B. harveyi luciferase (..cap alpha../sub h/) and the ..beta.. subunit from P. phosphoreum luciferase (..beta../sub p/). The complementation was unidirectional; activity could not be restored by complementing the ..cap alpha.. subunit of P. phosphoreum luciferase with the ..beta.. subunit of B. harveyi luciferase, showing that the subunits from these luciferases were not identical. Kinetic parameters of the hybrid luciferase reflecting the intermediate and later steps of the bioluminescent reaction as well as the overall activity and specificity were essentially identical to the same kinetic parameters for B. harveyi luciferase, the source of the ..cap alpha.. subunit, and quite distinct from those of P. phosphoreum luciferase. However, kinetic parameters that reflected the initial step in the reaction involving interaction of FMNH/sub 2/ and luciferase were altered in the hybrid luciferase compared to both the parental luciferases, the K/sub d/ for FMNH/sub 2/ actually being closer to that observed for the P. phosphoreum luciferase (the source of the ..beta.. subunit). These results provide direct evidence that modification or alteration of the ..beta.. subunit in a dimeric luciferase molecule can affect the kinetic properties and indicates that the ..beta.. subunit plays a functional role in the bioluminescent mechanism. It is proposed that both the ..cap alpha.. and ..beta.. subunits are involved with the initial interaction with FMNH/sub 2/, whereas subsequent steps in the mechanism are dictated exclusively by the ..cap alpha.. subunit and are unaffected by alterations in the ..beta.. subunit.

  13. Formation of active bacterial luciferase between interspecific subunits in vivo.

    PubMed

    Almashanu, S; Tuby, A; Hadar, R; Einy, R; Kuhn, J

    1995-01-01

    Interspecific complementation between luxAs and luxBs from Vibrio harveyi, Vibrio fischeri, Photobacterium leiognathi and Xenorhabdus luminescens was examined in vivo. The individual genes from these species were cloned on different compatible plasmids or amplified by PCR and brought together to yield cis combinations without extraneous DNA. The beta subunits from V. harveyi and X. luminescens form active enzyme only with alpha subunits from one of these species. All other combinations yield active enzymes. The lack of activity of the V. harveyi and X. luminescens beta subunits with the alpha subunits from V. fischeri and P. leiognathi results from a lack of association. This was shown by in vivo competition in which these beta subunits were overproduced in comparison with the beta and alpha of V. fischeri. No reduction in light was found. Overall, the in vivo results parallel those found in vitro using isolated denatured subunits and renaturation by removal of the denaturant.

  14. Sodium channel β subunits: emerging targets in channelopathies

    PubMed Central

    O’Malley, Heather A.; Isom, Lori L.

    2016-01-01

    Voltage-gated sodium channels (VGSCs) are responsible for initiation and propagation of action potentials in excitable cells. VGSCs in mammalian brain are heterotrimeric complexes of α and β subunits. Originally called “auxiliary,” we now know that β subunit proteins are multifunctional signaling molecules that play roles in both excitable and non-excitable 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. While VGSC β subunit-specific drugs have not yet been developed, this protein family is an emerging therapeutic target. PMID:25668026

  15. Quantifying the cooperative subunit action in a multimeric membrane receptor

    PubMed Central

    Wongsamitkul, Nisa; Nache, Vasilica; Eick, Thomas; Hummert, Sabine; Schulz, Eckhard; Schmauder, Ralf; Schirmeyer, Jana; Zimmer, Thomas; Benndorf, Klaus

    2016-01-01

    In multimeric membrane receptors the cooperative action of the subunits prevents exact knowledge about the operation and the interaction of the individual subunits. We propose a method that permits quantification of ligand binding to and activation effects of the individual binding sites in a multimeric membrane receptor. The power of this method is demonstrated by gaining detailed insight into the subunit action in olfactory cyclic nucleotide-gated CNGA2 ion channels. PMID:26858151

  16. [Molecular cloning of activin betaA subunit mature peptide from peafowl and its application in taxonomy and phylogeny].

    PubMed

    Zou, Fang-Dong; Tong, Xin-Xin; Yue, Bi-Song

    2005-03-01

    The sequences of activin gene betaA subunit mature peptide have been amplified from white peafowl, blue peafowl (pavo cristatus) and green peafowl (pavo muticus) genomic DNA by polymerase chain reaction (PCR) with a pair of degenerate primers. The target fragments were cloned into the vector pMD18-T and sequenced. The length of activin gene betaA subunit mature peptide is 345bp, which encoded a peptide of 115 amino acid residues. Sequence analysis of activin gene betaA subunit mature peptide demonstrated that the identity of nucleotide is 98.0% between blue peaflowl and green peafowl, and the identity of that is 98.8% between blue peaflowl and white peafow. Sequences comparison in NCBI revealed that the sequences of activin gene betaA subunit mature peptides of different species are highly conserved during evolution process. In addition, the restriction enzyme map of activins is high similar between white peafowl and blue peafowl. Phylogenetic tree was constructed with Mega 2 and Clustalxldx software. The result showed that white peafowl has a closer relationship to blue peafowl than to green peafowl. Considered the nucleotide differences of peafowls' activin gene betaA subunit mature peptides, a highly conserved region, we supported that white peafowl was derived from blue peafowl, and it is more possible the hybrid but just the product of color mutation, or maybe as a subspecies of Pavo genus.

  17. Conformational changes in the C terminus of Shaker K+ channel bound to the rat Kvβ2-subunit

    PubMed Central

    Sokolova, Olga; Accardi, Alessio; Gutierrez, David; Lau, Adrian; Rigney, Mike; Grigorieff, Nikolaus

    2003-01-01

    We studied the structure of the C terminus of the Shaker potassium channel. The 3D structures of the full-length and a C-terminal deletion (ΔC) mutant of Shaker were determined by electron microscopy and single-particle analysis. The difference map between the full-length and the truncated channels clearly shows a compact density, located on the sides of the T1 domain, that corresponds to a large part of the C terminus. We also expressed and purified both WT and ΔC Shaker, assembled with the rat Kvβ2-subunit. By using a difference map between the full-length and truncated Shaker α–β complexes, a conformational change was identified that shifts a large part of the C terminus away from the membrane domain and into close contact with the β-subunit. This conformational change, induced by the binding of the Kvβ2-subunit, suggests a possible mechanism for the modulation of the K+ voltage-gated channel function by its β-subunit. PMID:14569011

  18. Genetic analysis of neuronal ionotropic glutamate receptor subunits.

    PubMed

    Granger, Adam J; Gray, John A; Lu, Wei; Nicoll, Roger A

    2011-09-01

    In the brain, fast, excitatory synaptic transmission occurs primarily through AMPA- and NMDA-type ionotropic glutamate receptors. These receptors are composed of subunit proteins that determine their biophysical properties and trafficking behaviour. Therefore, determining the function of these subunits and receptor subunit composition is essential for understanding the physiological properties of synaptic transmission. Here, we discuss and evaluate various genetic approaches that have been used to study AMPA and NMDA receptor subunits. These approaches have demonstrated that the GluA1 AMPA receptor subunit is required for activity-dependent trafficking and contributes to basal synaptic transmission, while the GluA2 subunit regulates Ca(2+) permeability, homeostasis and trafficking to the synapse under basal conditions. In contrast, the GluN2A and GluN2B NMDA receptor subunits regulate synaptic AMPA receptor content, both during synaptic development and plasticity. Ongoing research in this field is focusing on the molecular interactions and mechanisms that control these functions. To accomplish this, molecular replacement techniques are being used, where native subunits are replaced with receptors containing targeted mutations. In this review, we discuss a single-cell molecular replacement approach which should arguably advance our physiological understanding of ionotropic glutamate receptor subunits, but is generally applicable to study of any neuronal protein.

  19. Expression of GABA receptor rho subunits in rat brain.

    PubMed

    Boue-Grabot, E; Roudbaraki, M; Bascles, L; Tramu, G; Bloch, B; Garret, M

    1998-03-01

    The GABA receptor rho1, rho2, and rho3 subunits are expressed in the retina where they form bicuculline-insensitive GABA(C) receptors. We used northern blot, in situ hybridization, and RT-PCR analysis to study the expression of rho subunits in rat brains. In situ hybridization allowed us to detect rho-subunit expression in the superficial gray layer of the superior colliculus and in the cerebellar Purkinje cells. RT-PCR experiments indicated that (a) in retina and in domains that may contain functional GABA(C) receptors, rho2 and rho1 subunits are expressed at similar levels; and (b) in domains and in tissues that are unlikely to contain GABA(C) receptors, rho2 mRNA is enriched relative to rho1 mRNA. These results suggest that both rho1 and rho2 subunits are necessary to form a functional GABA(C) receptor. The use of RT-PCR also showed that, except in the superior colliculus, rho3 is expressed along with rho1 and rho2 subunits. We also raised an antibody against a peptide sequence unique to the rho1 subunit. The use of this antibody on cerebellum revealed the rat rho1 subunit in the soma and dendrites of Purkinje neurons. The allocation of GABA(C) receptor subunits to identified neurons paves the way for future electrophysiological studies.

  20. Respiratory syncytial virus fusion glycoprotein: nucleotide sequence of mRNA, identification of cleavage activation site and amino acid sequence of N-terminus of F1 subunit.

    PubMed Central

    Elango, N; Satake, M; Coligan, J E; Norrby, E; Camargo, E; Venkatesan, S

    1985-01-01

    The amino acid sequence of respiratory syncytial virus fusion protein (Fo) was deduced from the sequence of a partial cDNA clone of mRNA and from the 5' mRNA sequence obtained by primer extension and dideoxysequencing. The encoded protein of 574 amino acids is extremely hydrophobic and has a molecular weight of 63371 daltons. The site of proteolytic cleavage within this protein was accurately mapped by determining a partial amino acid sequence of the N-terminus of the larger subunit (F1) purified by radioimmunoprecipitation using monoclonal antibodies. Alignment of the N-terminus of the F1 subunit within the deduced amino acid sequence of Fo permitted us to identify a sequence of lys-lys-arg-lys-arg-arg at the C-terminus of the smaller N-terminal F2 subunit that appears to represent the cleavage/activation domain. Five potential sites of glycosylation, four within the F2 subunit, were also identified. Three extremely hydrophobic domains are present in the protein; a) the N-terminal signal sequence, b) the N-terminus of the F1 subunit that is analogous to the N-terminus of the paramyxovirus F1 subunit and the HA2 subunit of influenza virus hemagglutinin, and c) the putative membrane anchorage domain near the C-terminus of F1. Images PMID:2987829

  1. Topographic mapping

    USGS Publications Warehouse

    ,

    2008-01-01

    The U.S. Geological Survey (USGS) produced its first topographic map in 1879, the same year it was established. Today, more than 100 years and millions of map copies later, topographic mapping is still a central activity for the USGS. The topographic map remains an indispensable tool for government, science, industry, and leisure. Much has changed since early topographers traveled the unsettled West and carefully plotted the first USGS maps by hand. Advances in survey techniques, instrumentation, and design and printing technologies, as well as the use of aerial photography and satellite data, have dramatically improved mapping coverage, accuracy, and efficiency. Yet cartography, the art and science of mapping, may never before have undergone change more profound than today.

  2. Compilation of small ribosomal subunit RNA structures.

    PubMed Central

    Neefs, J M; Van de Peer, Y; De Rijk, P; Chapelle, S; De Wachter, R

    1993-01-01

    The database on small ribosomal subunit RNA structure contained 1804 nucleotide sequences on April 23, 1993. This number comprises 365 eukaryotic, 65 archaeal, 1260 bacterial, 30 plastidial, and 84 mitochondrial sequences. These are stored in the form of an alignment in order to facilitate the use of the database as input for comparative studies on higher-order structure and for reconstruction of phylogenetic trees. The elements of the postulated secondary structure for each molecule are indicated by special symbols. The database is available on-line directly from the authors by ftp and can also be obtained from the EMBL nucleotide sequence library by electronic mail, ftp, and on CD ROM disk. PMID:8332525

  3. Rubisco small subunit gene family in cassava.

    PubMed

    Yeo, T W; Mak, Y M; Ho, K K

    1999-01-01

    Cassava leaves of two different cultivars, Brazil and Buloh, were used to isolate mRNA. The mRNA isolated was successfully used in the construction of cDNA libraries for each of the cultivars. The cDNA libraries were screened for members of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit gene family and positive clones were sequenced. A total of seven different SSU genes, of which five were from cultivar Brazil and two were from cultivar Buloh, were isolated. Comparison results show that even though all the sequences are highly similar, they can be classified into three subfamilies. Homology between members of the same subfamily is higher than homology between members from the same cultivar.

  4. Subunit vaccine efficacy against Botulinum neurotoxin subtypes.

    PubMed

    Henkel, James S; Tepp, William H; Przedpelski, Amanda; Fritz, Robert B; Johnson, Eric A; Barbieri, Joseph T

    2011-10-13

    Botulinum neurotoxins (BoNT) are classified into 7 serotypes (A-G) based upon neutralization by serotype-specific anti-sera. Several recombinant serotype-specific subunit BoNT vaccines have been developed, including a subunit vaccine comprising the receptor binding domain (HCR) of the BoNTs. Sequencing of the genes encoding BoNTs has identified variants (subtypes) that possess up to 32% primary amino acid variation among different BoNT serotypes. Studies were conducted to characterize the ability of the HCR of BoNT/A to protect against challenge by heterologous BoNT/A subtypes (A1-A3). High dose vaccination with HCR/A subtypes A1-A4 protected mice from challenge by heterologous BoNT/A subtype A1-A3, while low dose HCR vaccination yielded partial protection to heterologous BoNT/A subtype challenge. Absolute IgG titers to HCRs correlated to the dose of HCR used for vaccination, where HCR/A1 elicited an A1 subtype-specific IgG response, which was not observed with HCR/A2 vaccination. Survival of mice challenged to heterologous BoNT/A2 following low dose HCR/A1 vaccination correlated with elevated IgG titers directed to the denatured C-terminal sub-domain of HCR/A2, while survival of mice to heterologous BoNT/A1 following low dose HCR/A2 vaccination correlated to elevated IgG titers directed to native HCRc/A1. This implies that low dose vaccinations with HCR/A subtypes elicit unique IgG responses, and provides a basis to define how the host develops a neutralizing immune response to BoNT intoxication. These results may provide a reference for the development of pan-BoNT vaccines.

  5. Dynamic regulation of β1 subunit trafficking controls vascular contractility.

    PubMed

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

    2014-02-11

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

  6. Bacteriophage HK97 structure: wholesale covalent cross-linking between the major head shell subunits.

    PubMed Central

    Popa, M P; McKelvey, T A; Hempel, J; Hendrix, R W

    1991-01-01

    We describe initial genetic and structural characterizations of HK97, a temperate bacteriophage of Escherichia coli. We isolated 28 amber mutants, characterized them with respect to what phage-related structures they make, and mapped many of them to restriction fragments of genomic DNA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of HK97 virions revealed nine different protein species plus a substantial amount of material that failed to enter the gel, apparently because it is too large. Five proteins are tail components and are assigned functions as tail fiber subunit, tail length template, and major shaft subunit (two and possibly three species). The four remaining proteins and the material that did not enter the gel are head components. One of these proteins is assigned as the portal subunit, and the remaining three head proteins in the gel and the material that did not enter the gel are components of the head shell. All of the head shell protein species have apparent molecular masses well in excess of 100 kDa; they share amino acid sequence with each other and also with a 42-kDa protein that is found in infected lysates and as the major component of prohead structures that accumulate in infections by one of the amber mutants. We propose that all of the head shell species found in mature heads are covalently cross-linked oligomers derived from the 42-kDa precursor during head shell maturation. Images PMID:1709700

  7. Yeast ribosomal protein L10 helps coordinate tRNA movement through the large subunit

    PubMed Central

    Petrov, Alexey N.; Meskauskas, Arturas; Roshwalb, Sara C.; Dinman, Jonathan D.

    2008-01-01

    Yeast ribosomal protein L10 (E. coli L16) is located at the center of a topological nexus that connects many functional regions of the large subunit. This essential protein has previously been implicated in processes as diverse as ribosome biogenesis, translational fidelity and mRNA stability. Here, the inability to maintain the yeast Killer virus was used as a proxy for large subunit defects to identify a series of L10 mutants. These mapped to roughly four discrete regions of the protein. A detailed analysis of mutants located in the N-terminal ‘hook’ of L10, which inserts into the bulge of 25S rRNA helix 89, revealed strong effects on rRNA structure corresponding to the entire path taken by the tRNA 3′ end as it moves through the large subunit during the elongation cycle. The mutant-induced structural changes are wide-ranging, affecting ribosome biogenesis, elongation factor binding, drug resistance/hypersensitivity, translational fidelity and virus maintenance. The importance of L10 as a potential transducer of information through the ribosome, and of a possible role of its N-terminal domain in switching between the pre- and post-translocational states are discussed. PMID:18824477

  8. Cryo-EM structure of the large subunit of the spinach chloroplast ribosome

    PubMed Central

    Ahmed, Tofayel; Yin, Zhan; Bhushan, Shashi

    2016-01-01

    Protein synthesis in the chloroplast is mediated by the chloroplast ribosome (chloro-ribosome). Overall architecture of the chloro-ribosome is considerably similar to the Escherichia coli (E. coli) ribosome but certain differences are evident. The chloro-ribosome proteins are generally larger because of the presence of chloroplast-specific extensions in their N- and C-termini. The chloro-ribosome harbours six plastid-specific ribosomal proteins (PSRPs); four in the small subunit and two in the large subunit. Deletions and insertions occur throughout the rRNA sequence of the chloro-ribosome (except for the conserved peptidyl transferase center region) but the overall length of the rRNAs do not change significantly, compared to the E. coli. Although, recent advancements in cryo-electron microscopy (cryo-EM) have provided detailed high-resolution structures of ribosomes from many different sources, a high-resolution structure of the chloro-ribosome is still lacking. Here, we present a cryo-EM structure of the large subunit of the chloro-ribosome from spinach (Spinacia oleracea) at an average resolution of 3.5 Å. High-resolution map enabled us to localize and model chloro-ribosome proteins, chloroplast-specific protein extensions, two PSRPs (PSRP5 and 6) and three rRNA molecules present in the chloro-ribosome. Although comparable to E. coli, the polypeptide tunnel and the tunnel exit site show chloroplast-specific features. PMID:27762343

  9. Structure of the archaeal Cascade subunit Csa5: relating the small subunits of CRISPR effector complexes.

    PubMed

    Reeks, Judith; Graham, Shirley; Anderson, Linzi; Liu, Huanting; White, Malcolm F; Naismith, James H

    2013-05-01

    The Cascade complex for CRISPR-mediated antiviral immunity uses CRISPR RNA (crRNA) to target invading DNA species from mobile elements such as viruses, leading to their destruction. The core of the Cascade effector complex consists of the Cas5 and Cas7 subunits, which are widely conserved in prokaryotes. Cas7 binds crRNA and forms the helical backbone of Cascade. Many archaea encode a version of the Cascade complex (denoted Type I-A) that includes a Csa5 (or small) subunit, which interacts weakly with the core proteins. Here, we report the crystal structure of the Csa5 protein from Sulfolobus solfataricus. Csa5 comprises a conserved α-helical domain with a small insertion consisting of a weakly conserved β-strand domain. In the crystal, the Csa5 monomers have multimerized into infinite helical threads. At each interface is a strictly conserved intersubunit salt bridge, deletion of which disrupts multimerization. Structural analysis indicates a shared evolutionary history among the small subunits of the CRISPR effector complexes. The same α-helical domain is found in the C-terminal domain of Cse2 (from Type I-E Cascade), while the N-terminal domain of Cse2 is found in Cmr5 of the CMR (Type III-B) effector complex. As Cmr5 shares no match with Csa5, two possibilities present themselves: selective domain loss from an ancestral Cse2 to create two new subfamilies or domain fusion of two separate families to create a new Cse2 family. A definitive answer awaits structural studies of further small subunits from other CRISPR effector complexes.

  10. Liposome-Based Adjuvants for Subunit Vaccines: Formulation Strategies for Subunit Antigens and Immunostimulators

    PubMed Central

    Tandrup Schmidt, Signe; Foged, Camilla; Smith Korsholm, Karen; Rades, Thomas; Christensen, Dennis

    2016-01-01

    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 specific PRR

  11. Geologic mapping of Europa

    USGS Publications Warehouse

    Greeley, R.; Figueredo, P.H.; Williams, D.A.; Chuang, F.C.; Klemaszewski, J.E.; Kadel, S.D.; Prockter, L.M.; Pappalardo, R.T.; Head, J. W.; Collins, G.C.; Spaun, N.A.; Sullivan, R.J.; Moore, Johnnie N.; Senske, D.A.; Tufts, B.R.; Johnson, T.V.; Belton, M.J.S.; Tanaka, K.L.

    2000-01-01

    Galileo data enable the major geological units, structures, and surface features to be identified on Europa. These include five primary units (plains, chaos, band, ridge, and crater materials) and their subunits, along with various tectonic structures such as faults. Plains units are the most widespread. Ridged plains material spans a wide range of geological ages, including the oldest recognizable features on Europa, and appears to represent a style of tectonic resurfacing, rather than cryovolcanism. Smooth plains material typically embays other terrains and units, possibly as a type of fluid emplacement, and is among the youngest material units observed. At global scales, plains are typically mapped as undifferentiated plains material, although in some areas differences can be discerned in the near infrared which might be related to differences in ice grain size. Chaos material is composed of plains and other preexisting materials that have been severely disrupted by inferred internal activity; chaos is characterized by blocks of icy material set in a hummocky matrix. Band material is arrayed in linear, curvilinear, wedge-shaped, or cuspate zones with contrasting albedo and surface textures with respect to the surrounding terrain. Bilateral symmetry observed in some bands and the relationships with the surrounding units suggest that band material forms by the lithosphere fracturing, spreading apart, and infilling with material derived from the subsurface. Ridge material is mapped as a unit on local and some regional maps but shown with symbols at global scales. Ridge material includes single ridges, doublet ridges, and ridge complexes. Ridge materials are considered to represent tectonic processes, possibly accompanied by the extrusion or intrusion of subsurface materials, such as diapirs. The tectonic processes might be related to tidal flexing of the icy lithosphere on diurnal or longer timescales. Crater materials include various interior (smooth central

  12. Epitopes from two soybean glycinin subunits antigenic in pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Glycinin is a seed storage protein in soybean (Glycine max) that is allergenic in pigs. Glycinin is a hexamer composed of subunits consisting of a basic and acidic portion joined by disulfide bridges. There are 5 glycinin subunits designated Gy1-Gy5. Results: Twenty seven out of 30 pi...

  13. Specific Roles of NMDA Receptor Subunits in Mental Disorders

    PubMed Central

    Yamamoto, H.; Hagino, Y.; Kasai, S.; Ikeda, K.

    2015-01-01

    N-methyl-D-aspartate (NMDA) receptor plays important roles in learning and memory. NMDA receptors are a tetramer that consists of two glycine-binding subunits GluN1, two glutamate-binding subunits (i.e., GluN2A, GluN2B, GluN2C, and GluN2D), a combination of a GluN2 subunit and glycine-binding GluN3 subunit (i.e., GluN3A or GluN3B), or two GluN3 subunits. Recent studies revealed that the specific expression and distribution of each subunit are deeply involved in neural excitability, plasticity, and synaptic deficits. The present article summarizes reports on the dysfunction of NMDA receptors and responsible subunits in various neurological and psychiatric disorders, including schizophrenia, autoimmune-induced glutamatergic receptor dysfunction, mood disorders, and autism. A key role for the GluN2D subunit in NMDA receptor antagonist-induced psychosis has been recently revealed. PMID:25817860

  14. Proteopedia Entry: The Large Ribosomal Subunit of "Haloarcula Marismortui"

    ERIC Educational Resources Information Center

    Decatur, Wayne A.

    2010-01-01

    This article presents a "Proteopedia" page that shows the refined version of the structure of the "Haloarcula" large ribosomal subunit as solved by the laboratories of Thomas Steitz and Peter Moore. The landmark structure is of great impact as it is the first atomic-resolution structure of the highly conserved ribosomal subunit which harbors…

  15. Mapping Van

    NASA Technical Reports Server (NTRS)

    1994-01-01

    A NASA Center for the Commercial Development of Space (CCDS) - developed system for satellite mapping has been commercialized for the first time. Global Visions, Inc. maps an area while driving along a road in a sophisticated mapping van equipped with satellite signal receivers, video cameras and computer systems for collecting and storing mapping data. Data is fed into a computerized geographic information system (GIS). The resulting amps can be used for tax assessment purposes, emergency dispatch vehicles and fleet delivery companies as well as other applications.

  16. Mutational analysis of muscle nicotinic acetylcholine receptor subunit assembly

    PubMed Central

    1990-01-01

    The structural elements required for normal maturation and assembly of the nicotinic acetylcholine receptor alpha subunit were investigated by expression of mutated subunits in transfected fibroblasts. Normally, the wild-type alpha subunit acquires high affinity alpha bungarotoxin binding in a time-dependent manner; however, mutation of the 128 and/or 142 cysteines to either serine or alanine, as well as deletion of the entire 14 amino acids in this region abolished all detectable high affinity binding. Nonglycosylated subunits that had a serine to glycine mutation in the consensus sequence also did not efficiently attain high affinity binding to toxin. In contrast, mutation of the proline at position 136 to glycine or alanine, or a double mutation of the cysteines at position 192 and 193 to serines had no effect on the acquisition of high affinity toxin binding. These data suggest that a disulfide bridge between cysteines 128 and 142 and oligosaccharide addition at asparagine 141 are required for the normal maturation of alpha subunit as assayed by high affinity toxin binding. The unassembled wild-type alpha subunit expressed in fibroblasts is normally degraded with a t1/2 of 2 h; upon assembly with the delta subunit, the degradation rate slows significantly (t1/2 greater than 13 h). All mutated alpha subunits retained the capacity to assemble with a delta subunit coexpressed in fibroblasts; however, mutated alpha subunits that were not glycosylated or did not acquire high affinity toxin binding were rapidly degraded (t1/2 = 20 min to 2 h) regardless of whether or not they assembled with the delta subunit. Assembly and rapid degradation of nonglycosylated acetylcholine receptor (AChR) subunits and subunit complexes were also observed in tunicamycin- treated BC3H-1 cells, a mouse musclelike cell line that normally expresses functional AChR. Hence, rapid degradation may be one form of regulation assuring that only correctly processed and assembled subunits

  17. Geranyl diphosphate synthase large subunit, and methods of use

    DOEpatents

    Croteau, Rodney B.; Burke, Charles C.; Wildung, Mark R.

    2001-10-16

    A cDNA encoding geranyl diphosphate synthase large subunit from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase large subunit). In another aspect, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase large subunit. In yet another aspect, the present invention provides isolated, recombinant geranyl diphosphate synthase protein comprising an isolated, recombinant geranyl diphosphate synthase large subunit protein and an isolated, recombinant geranyl diphosphate synthase small subunit protein. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase.

  18. A revised model for AMP-activated protein kinase structure: The alpha-subunit binds to both the beta- and gamma-subunits although there is no direct binding between the beta- and gamma-subunits.

    PubMed

    Wong, Kelly A; Lodish, Harvey F

    2006-11-24

    The 5'-AMP-activated protein kinase (AMPK) is a master sensor for cellular metabolic energy state. It is activated by a high AMP/ATP ratio and leads to metabolic changes that conserve energy and utilize alternative cellular fuel sources. The kinase is composed of a heterotrimeric protein complex containing a catalytic alpha-subunit, an AMP-binding gamma-subunit, and a scaffolding beta-subunit thought to bind directly both the alpha- and gamma-subunits. Here, we use coimmunoprecipitation of proteins in transiently transfected cells to show that the alpha2-subunit binds directly not only to the beta-subunit, confirming previous work, but also to the gamma1-subunit. Deletion analysis of the alpha2-subunit reveals that the C-terminal 386-552 residues are sufficient to bind to the beta-subunit. The gamma1-subunit binds directly to the alpha2-subunit at two interaction sites, one within the catalytic domain consisting of alpha2 amino acids 1-312 and a second within residues 386-552. Binding of the alpha2 and the gamma1-subunits was not affected by 400 mum AMP or ATP. Furthermore, we show that the beta-subunit C terminus is essential for binding to the alpha2-subunit but, in contrast to previous work, the beta-subunit does not bind directly to the gamma1-subunit. Taken together, this study presents a new model for AMPK heterotrimer structure where through its C terminus the beta-subunit binds to the alpha-subunit that, in turn, binds to the gamma-subunit. There is no direct interaction between the beta- and gamma-subunits.

  19. Subunit Structure Differences in RNA Polymerase II Purified from Ungerminated versus Germinated Wheat Embryos 1

    PubMed Central

    Jendrisak, Jerry; Skuzeski, Jim

    1983-01-01

    DNA-dependent RNA polymerase II (RNAP II) was purified from wheat embryos germinated for 0, 12, 24, and 36 hours and examined with several polyacrylamide gel electrophoretic systems. A changing electrophoretic pattern of RNAP II was observed on nondenaturing polyacrylamide gels. Subunit structure analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that from ungerminated embryos, RNAP IIA was almost exclusively obtained which has a subunit structure identical to that established for wheat germ RNAP II previously (Jendrisak, Burgess 1977 Biochemistry 16: 1959-1964). Twelve polypeptides with molecular weights × 10−3 of 220, 140, 42, 40, 27, 25, 21, 20, 17.8, 17.0, 16.3, and 16.0 were routinely found to be associated with the purified enzyme. From embryos germinated for 36 hours, RNAP IIB was almost exclusively obtained which has a largest subunit of 180,000 mol wt instead of 220,000. From embryos germinated for 24 hours, an approximately equimolar mixture of RNAP IIA and IIB was obtained. Peptide maps of the 220,000 and 180,000 mol wt polypeptides of RNAP IIA and IIB were virtually identical, indicative of a precursor-product relationship for the two polypeptides. In addition to these results, SDS-PAGE indicated that the stoichiometry of the 27,000 mol wt polypeptide increased at the expense of the 25,000 mol wt polypeptide during germination and concomitantly with the appearance of the 180,000 molecular weight polypeptide. No modifications (e.g. gain, loss, or altered mobilities on analytical gels) in any of the other RNAP II subunits were observed in enzyme purified from embryos after various times of germination as determined by a variety of electrophoretic analyses under denaturing conditions. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5 PMID:16663122

  20. The subunit composition and function of mammalian cytochrome c oxidase.

    PubMed

    Kadenbach, Bernhard; Hüttemann, Maik

    2015-09-01

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

  1. Subunit structure of the follitropin receptor

    SciTech Connect

    Shin, J.

    1985-01-01

    Both of the ..cap alpha.. and ..beta.. subunits of intact human follitropin (FSH) were radioiodinated with /sup 125/I-FSH-sodium iodide and chloramine-T, and could be resolved on polyacrylamide gels (SDS-PAGE). The electrophoretic mobility of radioiodinated FSH ..cap alpha.. and ..beta.. subunits as well as the ..cap alpha beta.. dimer changed markedly depending on the concentration of reducing agents. /sup 125/I-FSH (Ka = 1.4 x 10/sup 10/ M/sup -1/), complexes to the receptor on procine granulosa cells or in Triton X-100 extracts, was affinity-crosslinked with a cleavable (nondisulfide) homobifunctional reagent, bis(2-(succinimidooxycarbonyloxy)ethyl)sulfone, solubilized in sodium dodecyl sulfate with or without reducing agents, and electrophoresed. Crosslinked samples revealed three additional bands of slower electrophoretic mobility, corresponding to 65 (unreduced 62), 83 (unreduced 76) and 117 (unreduced 110)kDa, in addition to hormone bands. Formation of the three bands requires the /sup 125/I-FSH hormone to bind specifically to the receptor with subsequent cross-linking. The rate of formation and cleavage of the cross-linked complexes indicated a sequential and incremental addition of 22, 18, and 34 kDa components to the FSH ..cap alpha beta.. dimer. The results of reduction of cross-linked complexes demonstrated the existence of disulfide linkage between the three components. FSH was photoactively derivatized with N-hydroxysuccinimide ester of 4-azidobenzolyl-glycine and radioiodinated for photoaffinity labeling. When derivatized /sup 125/I-FSH (Ka = 1.12 10/sup 10/ M/sup -1/) bound to the cell was photolyzed for cross-linking and resolved on the SDS-PAGE, two new bands (106 and 61 kDa) under reducing condition appeared in addition to the hormone bands. Upon reduction with dithiotheitol and second-dimensional electrophoresis, the unreduced 104 kDa (reduced 106 kDa) band released two small components 31 and 14 kDa.

  2. Na, K ATPase beta3 subunit (CD298): association with alpha subunit and expression on peripheral blood cells.

    PubMed

    Chiampanichayakul, S; Khunkaewla, P; Pata, S; Kasinrerk, W

    2006-12-01

    Beta3 subunit is described as one of the Na, K ATPase subunits. Recently, we generated a monoclonal antibody (mAb), termed P-3E10. This mAb was shown to react with the Na, K ATPase beta3 subunit or CD298. By immunofluorescence analysis using mAb P-3E10, it was found that all peripheral blood leukocytes express Na, K ATPase beta3. The presence of beta3 subunit on leukocytes is not in a quantitative polymorphic manner. Upon phytohemagglutinin or phorbol myristate acetate activation, the expression level of the Na, K ATPase beta3 subunit on activated peripheral blood mononuclear cells was not altered in comparison with those of unstimulated cells. Red blood cells (RBCs) of healthy donors showed negative reactivity with mAb P-3E10. However, more than 80% of thalassemic RBCs showed positive reactivity. By immunoprecipitation, moreover, a protein band of 55-65 kDa was precipitated from normal RBC membrane using mAb P-3E10. These results evidenced that the beta3 subunit of Na, K ATPase is expressed on RBC membrane but the epitope recognized by mAb P-3E10 is hidden in normal RBCs. Furthermore, we showed the association of beta3 subunit and alpha subunit of Na, K ATPase. This information is important for further understanding of the functional roles of this molecule.

  3. Genome mapping

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genome maps can be thought of much like road maps except that, instead of traversing across land, they traverse across the chromosomes of an organism. Genetic markers serve as landmarks along the chromosome and provide researchers information as to how close they may be to a gene or region of inter...

  4. Undersea Mapping.

    ERIC Educational Resources Information Center

    DiSpezio, Michael A.

    1991-01-01

    Presented is a cooperative learning activity in which students assume different roles in an effort to produce a relief map of the ocean floor. Materials, procedures, definitions, student roles, and questions are discussed. A reproducible map for the activity is provided. (CW)

  5. Question Mapping

    ERIC Educational Resources Information Center

    Martin, Josh

    2012-01-01

    After accepting the principal position at Farmersville (TX) Junior High, the author decided to increase instructional rigor through question mapping because of the success he saw using this instructional practice at his prior campus. Teachers are the number one influence on student achievement (Marzano, 2003), so question mapping provides a…

  6. Map Adventures.

    ERIC Educational Resources Information Center

    Geological Survey (Dept. of Interior), Reston, VA.

    This curriculum packet about maps, with seven accompanying lessons, is appropriate for students in grades K-3. Students learn basic concepts for visualizing objects from different perspectives and how to understand and use maps. Lessons in the packet center on a story about a little girl, Nikki, who rides in a hot-air balloon that gives her, and…

  7. Concept Mapping

    ERIC Educational Resources Information Center

    Technology & Learning, 2005

    2005-01-01

    Concept maps are graphical ways of working with ideas and presenting information. They reveal patterns and relationships and help students to clarify their thinking, and to process, organize and prioritize. Displaying information visually--in concept maps, word webs, or diagrams--stimulates creativity. Being able to think logically teaches…

  8. Collection Mapping.

    ERIC Educational Resources Information Center

    Harbour, Denise

    2002-01-01

    Explains collection mapping for library media collections. Discusses purposes for creating collection maps, including helping with selection and weeding decisions, showing how the collection supports the curriculum, and making budget decisions; and methods of data collection, including evaluating a collaboratively taught unit with the classroom…

  9. [A promoter responsible for over-expression of cholera toxin B subunit in cholera toxin A subunit structure gene].

    PubMed

    Cao, C; Shi, C; Li, P; Ma, Q

    1997-01-01

    A promoter sequence, which promotes the transcription of cholera toxin B subunit gene, was found in cholera toxin A subunit structure gene. The transcription starts at the adenine Located at +833, that is 456bp upstream to the A of the initiation codon ATG of cholera toxin B gene. Under the control of the promoter, cholera toxin B subunit was over-expressed as high as 200 mg/L at an optimized culture condition. The chloramphenicol acetyl transferase gene and beta-galactosidase could also be efficiently expressed under the direction of the promoter. This promoter may be responsible for the 6 fold and 7 fold higher expression level of cholera toxin B subunit than cholera toxin A subunit in V. cholerae and Escheria coli respectively. The over-expression of CTB may be useful in preparing vaccine against cholera and facilitating the construction of peptide-bearing immunogenic hybrid proteins.

  10. Electronic spectra of PS I mutants: the peripheral subunits do not bind red chlorophylls in Synechocystis sp. PCC 6803.

    PubMed

    Soukoulis, V; Savikhin, S; Xu, W; Chitnis, P R; Struve, W S

    1999-05-01

    Steady-state fluorescence and absorption spectra have been obtained in the Qy spectral region (690-780 nm and 600-750 nm, respectively) for several subunit-deficient photosystem I mutants from the cyanobacterium Synechocystis sp. PCC 6803. The 77 K fluorescence spectra of the wild-type and subunit-deficient mutant photosystem I particles are all very similar, peaking at approximately 720 nm with essentially the same excitation spectrum. Because emission from far-red chlorophylls absorbing near 708 nm dominates low-temperature fluorescence in Synechocystis sp., these pigments are not coordinated to any the subunits PsaF, Psa I, PsaJ, PsaK, PsaL, or psaM. The room temperature (wild-type-mutant) absorption difference spectra for trimeric mutants lacking the PsaF/J, PsaK, and PsaM subunits suggest that these mutants are deficient in core antenna chlorophylls (Chls) absorbing near 685, 670, 675, and 700 nm, respectively. The absorption difference spectrum for the PsaF/J/I/L-deficient photosystem I complexes at 5 K reveals considerably more structure than the room-temperature spectrum. The integrated absorbance difference spectra (when normalized to the total PS I Qy spectral area) are comparable to the fractions of Chls bound by the respective (groups of) subunits, according to the 4-A density map of PS I from Synechococcus elongatus. The spectrum of the monomeric PsaL-deficient mutant suggests that this subunit may bind pigments absorbing near 700 nm.

  11. Interactions between the human RNA polymerase II subunits.

    PubMed

    Acker, J; de Graaff, M; Cheynel, I; Khazak, V; Kedinger, C; Vigneron, M

    1997-07-04

    As an initial approach to characterizing the molecular structure of the human RNA polymerase II (hRPB), we systematically investigated the protein-protein contacts that the subunits of this enzyme may establish with each other. To this end, we applied a glutathione S-transferase-pulldown assay to extracts from Sf9 insect cells, which were coinfected with all possible combinations of recombinant baculoviruses expressing hRPB subunits, either as untagged polypeptides or as glutathione S-transferase fusion proteins. This is the first comprehensive study of interactions between eukaryotic RNA polymerase subunits; among the 116 combinations of hRPB subunits tested, 56 showed significant to strong interactions, whereas 60 were negative. Within the intricate network of interactions, subunits hRPB3 and hRPB5 play a central role in polymerase organization. These subunits, which are able to homodimerize and to interact, may constitute the nucleation center for polymerase assembly, by providing a large interface to most of the other subunits.

  12. Mapping Children--Mapping Space.

    ERIC Educational Resources Information Center

    Pick, Herbert L., Jr.

    Research is underway concerning the way the perception, conception, and representation of spatial layout develops. Three concepts are important here--space itself, frame of reference, and cognitive map. Cognitive map refers to a form of representation of the behavioral space, not paired associate or serial response learning. Other criteria…

  13. Further evidence for clustering of human GABA[sub A] receptor subunit genes: Localization of the [alpha][sub 6]-subunit gene (GABRA6) to distal chromosome 5q by linkage analysis

    SciTech Connect

    Hicks, A.A.; Kamphuis, W.; Darlison, M.G. ); Bailey, M.E.S.; Johnson, K.J. ); Riley, B.P. ); Siciliano, M.J. )

    1994-03-15

    GABA[sub A] receptors are hetero-oligomeric ion-channel complexes that are composed of combinations of [alpha], [beta], [gamma], and [delta] subunits and play a major role in inhibitory neurotransmission in the mammalian brain. The authors report here a microsatellite polymorphism within the human [alpha][sub 6]-subunit gene (GABRA6). Mapping of this marker in a human-hamster hybrid cell-line panel and typing of the repeat in the Centre d'Etude du Polymorphisme Humain (CEPH) reference families enabled the localization of this gene to chromosome 5q and established its linkage to the GABA[sub A] receptor [alpha][sub 1]-subunit gene (GA-BRA1) with a maximum lod score (Z[sub max]) of 39.87 at a [theta] of 0.069 (males) and 0.100 (females). These results reveal the clustering of GABRA6, GABRA1, and the GABA[sub A] receptor [gamma][sub 2]-subunit gene (GABRG2) on distal chromosome 5q. 17 refs., 1 fig., 1 tab.

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

    PubMed Central

    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

  15. NADH dehydrogenase subunit 1 and cytochrome c oxidase subunit I sequences compared for members of the genus Taenia (Cestoda).

    PubMed

    Gasser, R B; Zhu, X; McManus, D P

    1999-12-01

    Nine members of the genus Taenia (Taenia taeniaeformis, Taenia hydatigena, Taenia pisiformis, Taenia ovis, Taenia multiceps, Taenia serialis, Taenia saginata, Taenia solium and the Asian Taenia) were characterised by their mitochondrial NADH dehydrogenase subunit 1 gene sequences and their genetic relationships were compared with those derived from the cytochrome c oxidase subunit 1 sequence data. The extent of inter-taxon sequence difference in NADH dehydrogenase subunit 1 (approximately 5.9-30.8%) was usually greater than in cytochrome c oxidase subunit 1 (approximately 2.5-18%). Although topology of the phenograms derived from NADH dehydrogenase subunit 1 and cytochrome c oxidase subunit 1 sequence data differed, there was concordance in that T. multiceps, T. serialis (of canids), T. saginata and the Asian Taenia (of humans) were genetically most similar, and those four members were genetically more similar to T. ovis and T. solium than they were to T. hydatigena and T. pisiformis (of canids) or T. taeniaeformis (of cats). The NADH dehydrogenase subunit 1 sequence data may prove useful in studies of the systematics and population genetic structure of the Taeniidae.

  16. Conformational analysis of putative regulatory subunit D of the toluene/o-xylene-monooxygenase complex from Pseudomonas stutzeri OX1

    PubMed Central

    Scognamiglio, Roberta; Notomista, Eugenio; Barbieri, Paola; Pucci, Piero; Piaz, Fabrizio Dal; Tramontano, Anna; Di Donato, Alberto

    2001-01-01

    A gene cluster isolated from Pseudomonas stutzeri OX1 genomic DNA and containing six ORFs codes for toluene/o-xylene-monooxygenase. The putative regulatory D subunit was expressed in Escherichia coli and purified. Its protein sequence was verified by mass spectrometry mapping and found to be identical to the sequence predicted on the basis of the DNA sequence. The surface topology of subunit D in solution was probed by limited proteolysis carried out under strictly controlled conditions using several proteases as proteolytic probes. The same experiments were carried out on the homologous P2 component of the multicomponent phenol hydroxylase from Pseudomonas putida CF600. The proteolytic fragments released from both proteins in their native state were analyzed by electrospray mass spectrometry, and the preferential cleavage sites were assessed. The results indicated that despite the relatively high similarity between the sequences of the two proteins, some differences in the distribution of preferential proteolytic cleavages were detected, and a much higher conformational flexibility of subunit D was inferred. Moreover, automatic modeling of subunit D was attempted, based on the known three-dimensional structure of P2. Our results indicate that, at least in this case, standard modeling procedures based on automatic alignment on the structure of P2 fail to produce a model consistent with limited proteolysis experimental data. Thus, it is our opinion that reliable techniques such as limited proteolysis can be employed to test three-dimensional models and highlight problems in automatic model building. PMID:11344317

  17. Mapping Biodiversity.

    ERIC Educational Resources Information Center

    World Wildlife Fund, Washington, DC.

    This document features a lesson plan that examines how maps help scientists protect biodiversity and how plants and animals are adapted to specific ecoregions by comparing biome, ecoregion, and habitat. Samples of instruction and assessment are included. (KHR)

  18. Map Separates

    USGS Publications Warehouse

    ,

    2001-01-01

    U.S. Geological Survey (USGS) topographic maps are printed using up to six colors (black, blue, green, red, brown, and purple). To prepare your own maps or artwork based on maps, you can order separate black-and-white film positives or negatives for any color printed on a USGS topographic map, or for one or more of the groups of related features printed in the same color on the map (such as drainage and drainage names from the blue plate.) In this document, examples are shown with appropriate ink color to illustrate the various separates. When purchased, separates are black-and-white film negatives or positives. After you receive a film separate or composite from the USGS, you can crop, enlarge or reduce, and edit to add or remove details to suit your special needs. For example, you can adapt the separates for making regional and local planning maps or for doing many kinds of studies or promotions by using the features you select and then printing them in colors of your choice.

  19. Venus mapping

    NASA Technical Reports Server (NTRS)

    Batson, R. M.; Morgan, H. F.; Sucharski, Robert

    1991-01-01

    Semicontrolled image mosaics of Venus, based on Magellan data, are being compiled at 1:50,000,000, 1:10,000,000, 1:5,000,000, and 1:1,000,000 scales to support the Magellan Radar Investigator (RADIG) team. The mosaics are semicontrolled in the sense that data gaps were not filled and significant cosmetic inconsistencies exist. Contours are based on preliminary radar altimetry data that is subjected to revision and improvement. Final maps to support geologic mapping and other scientific investigations, to be compiled as the dataset becomes complete, will be sponsored by the Planetary Geology and Geophysics Program and/or the Venus Data Analysis Program. All maps, both semicontrolled and final, will be published as I-maps by the United States Geological Survey. All of the mapping is based on existing knowledge of the spacecraft orbit; photogrammetric triangulation, a traditional basis for geodetic control on planets where framing cameras were used, is not feasible with the radar images of Venus, although an eventual shift of coordinate system to a revised spin-axis location is anticipated. This is expected to be small enough that it will affect only large-scale maps.

  20. Database on the structure of large ribosomal subunit RNA.

    PubMed Central

    De Rijk, P; Van de Peer, Y; Chapelle, S; De Wachter, R

    1994-01-01

    A database on large ribosomal subunit RNA is made available. It contains 258 sequences. It provides sequence, alignment and secondary structure information in computer-readable formats. Files can be obtained using ftp. PMID:7524023

  1. The eukaryotic RNA exosome: same scaffold but variable catalytic subunits.

    PubMed

    Lykke-Andersen, Søren; Tomecki, Rafal; Jensen, Torben Heick; Dziembowski, Andrzej

    2011-01-01

    The RNA exosome is a versatile ribonucleolytic protein complex that participates in a multitude of cellular RNA processing and degradation events. It consists of an invariable nine-subunit core that associates with a variety of enzymatically active subunits and co-factors. These contribute to or even provide the catalytic activity and substrate specificity of the complex. The S. cerevisiae exosome has been intensively studied since its discovery in 1997 and thus serves as the archetype of eukaryotic exosomes. Notably, its catalytic potential, derived exclusively from associated subunits, differs between the nuclear and cytoplasmic versions of the complex. The same holds true for other eukaryotes, however, recent discoveries from various laboratories including our own have revealed that there are variations on this theme. Here, we review the latest findings concerning catalytic subunits of eukaryotic exosomes, and we discuss the apparent need for differential composition and subcellular distribution of exosome variants.

  2. Genetic Analysis of the Cytoplasmic Dynein Subunit Families

    PubMed Central

    Pfister, K. Kevin; Shah, Paresh R; Hummerich, Holger; Russ, Andreas; Cotton, James; Annuar, Azlina Ahmad; King, Stephen M; Fisher, Elizabeth M. C

    2006-01-01

    Cytoplasmic dyneins, the principal microtubule minus-end-directed motor proteins of the cell, are involved in many essential cellular processes. The major form of this enzyme is a complex of at least six protein subunits, and in mammals all but one of the subunits are encoded by at least two genes. Here we review current knowledge concerning the subunits, their interactions, and their functional roles as derived from biochemical and genetic analyses. We also carried out extensive database searches to look for new genes and to clarify anomalies in the databases. Our analysis documents evolutionary relationships among the dynein subunits of mammals and other model organisms, and sheds new light on the role of this diverse group of proteins, highlighting the existence of two cytoplasmic dynein complexes with distinct cellular roles. PMID:16440056

  3. A process yields large quantities of pure ribosome subunits

    NASA Technical Reports Server (NTRS)

    Friedman, M.; Lu, P.; Rich, A.

    1972-01-01

    Development of process for in-vitro protein synthesis from living cells followed by dissociation of ribosomes into subunits is discussed. Process depends on dialysis or use of chelating agents. Operation of process and advantages over previous methods are outlined.

  4. Subunit-Specific Trafficking of GABAA Receptors during Status Epilepticus

    PubMed Central

    Goodkin, Howard P.; Joshi, Suchitra; Mtchedlishvili, Zakaria; Brar, Jasmit; Kapur, Jaideep

    2010-01-01

    It is proposed that a reduced surface expression of GABAA receptors (GABARs) contributes to the pathogenesis of status epilepticus (SE), a condition characterized by prolonged seizures. This hypothesis was based on the finding that prolonged epileptiform bursting (repetitive bursts of prolonged depolarizations with superimposed action potentials) in cultures of dissociated hippocampal pyramidal neurons (dissociated cultures) results in the increased intracellular accumulation of GABARs. However, it is not known whether this rapid modification in the surface-expressed GABAR pool results from selective, subunit-dependent or nonselective, subunit-independent internalization of GABARs. In hippocampal slices obtained from animals undergoing prolonged SE (SE-treated slices), we found that the surface expression of the GABARβ2/3 and γ2 subunits was reduced, whereas that of the δ subunit was not. Complementary electrophysiological recordings from dentate granule cells in SE-treated slices demonstrated a reduction in GABAR-mediated synaptic inhibition, but not tonic inhibition. A reduction in the surface expression of the γ2 subunit, but not the δ subunit was also observed in dissociated cultures and organotypic hippocampal slice cultures when incubated in an elevated KCl external medium or an elevated KCl external medium supplemented with NMDA, respectively. Additional studies demonstrated that the reduction in the surface expression of the γ2 subunit was independent of direct ligand binding of the GABAR. These findings demonstrate that the regulation of surface-expressed GABAR pool during SE is subunit-specific and occurs independent of ligand binding. The differential modulation of the surface expression of GABARs during SE has potential implications for the treatment of this neurological emergency. PMID:18322097

  5. F-subunit reinforces torque generation in V-ATPase.

    PubMed

    Kishikawa, Jun-ichi; Seino, Akihiko; Nakanishi, Atsuko; Tirtom, Naciye Esma; Noji, Hiroyuki; Yokoyama, Ken; Hayashi, Kumiko

    2014-09-01

    Vacuolar-type H(+)-pumping ATPases (V-ATPases) perform remarkably diverse functions in eukaryotic organisms. They are present in the membranes of many organelles and regulate the pH of several intracellular compartments. A family of V-ATPases is also present in the plasma membranes of some bacteria. Such V-ATPases function as ATP-synthases. Each V-ATPase is composed of a water-soluble domain (V1) and a membrane-embedded domain (Vo). The ATP-driven rotary unit, V[Formula: see text], is composed of A, B, D, and F subunits. The rotary shaft (the DF subcomplex) rotates in the central cavity of the A3B3-ring (the catalytic hexamer ring). The D-subunit, which has a coiled-coil domain, penetrates into the ring, while the F-subunit is a globular-shaped domain protruding from the ring. The minimal ATP-driven rotary unit of V[Formula: see text] is comprised of the A3B3D subunits, and we therefore investigated how the absence of the globular-shaped F-subunit affects the rotary torque generation of V[Formula: see text]. Using a single-molecule technique, we observed the motion of the rotary motors. To obtain the torque values, we then analyzed the measured motion trajectories based on the fluctuation theorem, which states that the law of entropy production in non-equilibrium conditions and has been suggested as a novel and effective method for measuring torque. The measured torque of A3B3D was half that of the wild-type V1, and full torque was recovered in the mutant V1, in which the F-subunit was genetically fused with the D-subunit, indicating that the globular-shaped F-subunit reinforces torque generation in V1.

  6. Transcription Activator Interactions with Multiple SWI/SNF Subunits

    PubMed Central

    Neely, Kristen E.; Hassan, Ahmed H.; Brown, Christine E.; Howe, LeAnn; Workman, Jerry L.

    2002-01-01

    We have previously shown that the yeast SWI/SNF complex stimulates in vitro transcription from chromatin templates in an ATP-dependent manner. SWI/SNF function in this regard requires the presence of an activator with which it can interact directly, linking activator recruitment of SWI/SNF to transcriptional stimulation. In this study, we determine the SWI/SNF subunits that mediate its interaction with activators. Using a photo-cross-linking label transfer strategy, we show that the Snf5, Swi1, and Swi2/Snf2 subunits are contacted by the yeast acidic activators, Gcn4 and Hap4, in the context of the intact native SWI/SNF complex. In addition, we show that the same three subunits can interact individually with acidic activation domains, indicating that each subunit contributes to binding activators. Furthermore, mutations that reduce the activation potential of these activators also diminish its interaction with each of these SWI/SNF subunits. Thus, three distinct subunits of the SWI/SNF complex contribute to its interactions with activation domains. PMID:11865042

  7. Transcriptional regulators of Na,K-ATPase subunits

    PubMed Central

    Li, Zhiqin; Langhans, Sigrid A.

    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 α-subunit, the β-subunit and the FXYD proteins, are controlled extensively during development and to accommodate physiological needs. The spatial and temporal expression of Na,K-ATPase is partially regulated at the transcriptional level. Numerous transcription factors, hormones, growth factors, lipids, and extracellular stimuli modulate the transcription of the Na,K-ATPase subunits. Moreover, epigenetic mechanisms also contribute to the regulation of Na,K-ATPase expression. With the ever growing knowledge about diseases associated with the malfunction of Na,K-ATPase, this review aims at summarizing the best-characterized transcription regulators that modulate Na,K-ATPase subunit levels. As abnormal expression of Na,K-ATPase subunits has been observed in many carcinoma, we will also discuss transcription factors that are associated with epithelial-mesenchymal transition, a crucial step in the progression of many tumors to malignant disease. PMID:26579519

  8. Hybrid Rubisco of tomato large subunits and tobacco small subunits is functional in tobacco plants.

    PubMed

    Zhang, Xing-Hai; Webb, James; Huang, Yi-Hong; Lin, Li; Tang, Ri-Sheng; Liu, Aimin

    2011-03-01

    Biogenesis of functional ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in plants requires specific assembly in the chloroplast of the imported, cytosol-synthesized small subunits (SS) with the chloroplast-made large subunits (LS). Accumulating evidence indicates that chloroplasts (plastids) generally have a low tolerance for assembling foreign or modified Rubisco. To explore Rubisco engineering, we created two lines of transplastomic tobacco plants whose rbcL gene was replaced by tomato-derived rbcL: plant LLS2 with Rubisco composed of tobacco SS and Q437R LS and plant LLS4 with a hybrid Rubisco of tobacco SS and tomato LS (representing four substitutions of Y226F, A230T, S279T and Q437R from tobacco LS). Plant LLS2 exhibited similar phenotypes as the wild type. Plant LLS4 showed lower chlorophyll and Rubisco levels particularly in young emerging leaves, lower photosynthesis rates and biomass during early stages of development, but was able to reach reproductive maturity and somewhat wild type-like phenotype under ambient CO₂ condition. In vitro assays detected similar carboxylase activity and RuBP affinity in LLS2 and LLS4 plants as in wild type. Our studies demonstrated that tomato LS was sufficiently assembled with tobacco SS into functional Rubisco. The hybrid Rubisco of tomato LS and tobacco SS can drive photosynthesis that supports photoautotrophic growth and reproduction of tobacco plants under ambient CO₂ and light conditions. We discuss the effect of these residue substitutions on Rubisco activity and the possible attribution of chlorophyll deficiency to the in planta photosynthesis performance in the hybrid Rubisco plants.

  9. Horizontal membrane-intrinsic α-helices in the stator a-subunit of an F-type ATP synthase.

    PubMed

    Allegretti, Matteo; Klusch, Niklas; Mills, Deryck J; Vonck, Janet; Kühlbrandt, Werner; Davies, Karen M

    2015-05-14

    ATP, the universal energy currency of cells, is produced by F-type ATP synthases, which are ancient, membrane-bound nanomachines. F-type ATP synthases use the energy of a transmembrane electrochemical gradient to generate ATP by rotary catalysis. Protons moving across the membrane drive a rotor ring composed of 8-15 c-subunits. A central stalk transmits the rotation of the c-ring to the catalytic F1 head, where a series of conformational changes results in ATP synthesis. A key unresolved question in this fundamental process is how protons pass through the membrane to drive ATP production. Mitochondrial ATP synthases form V-shaped homodimers in cristae membranes. Here we report the structure of a native and active mitochondrial ATP synthase dimer, determined by single-particle electron cryomicroscopy at 6.2 Å resolution. Our structure shows four long, horizontal membrane-intrinsic α-helices in the a-subunit, arranged in two hairpins at an angle of approximately 70° relative to the c-ring helices. It has been proposed that a strictly conserved membrane-embedded arginine in the a-subunit couples proton translocation to c-ring rotation. A fit of the conserved carboxy-terminal a-subunit sequence places the conserved arginine next to a proton-binding c-subunit glutamate. The map shows a slanting solvent-accessible channel that extends from the mitochondrial matrix to the conserved arginine. Another hydrophilic cavity on the lumenal membrane surface defines a direct route for the protons to an essential histidine-glutamate pair. Our results provide unique new insights into the structure and function of rotary ATP synthases and explain how ATP production is coupled to proton translocation.

  10. Random mutagenesis of yeast 25S rRNA identify bases critical for 60S subunit structural integrity and function

    PubMed Central

    Nemoto, Naoki; Udagawa, Tsuyoshi; Chowdhury, Wasimul; Kitabatake, Makoto; Shin, Byung-shik; Hiraishi, Hiroyuki; Wang, Suzhi; Singh, Chingakham Ranjit; Brown, Susan J.; Ohno, Mutsuhito; Asano, Katsura

    2013-01-01

    In yeast Saccharomyces cerevisiae, 25S rRNA makes up the major mass and shape of the 60S ribosomal subunit. During translation initiation, the 60S subunit joins the 40S initiation complex, producing the 80S initiation complex. During elongation, the 60S subunit binds the CCA-ends of aminoacyl- and peptidyl-tRNAs at the A-loop and P-loop, respectively, transferring the peptide onto the α-amino group of the aminoacyl-tRNA. To study the role of 25S rRNA in translation in vivo, we randomly mutated 25S rRNA and isolated and characterized seven point mutations that affected yeast cell growth and polysome profiles. Four of these mutations, G651A, A1435U, A1446G and A1587G, change a base involved in base triples crucial for structural integrity. Three other mutations change bases near the ribosomal surface: C2879U and U2408C alter the A-loop and P-loop, respectively, and G1735A maps near a Eukarya-specific bridge to the 40S subunit. By polysome profiling in mmslΔ mutants defective in nonfunctional 25S rRNA decay, we show that some of these mutations are defective in both the initiation and elongation phases of translation. Of the mutants characterized, C2879U displays the strongest defect in translation initiation. The ribosome transit-time assay directly shows that this mutation is also defective in peptide elongation/termination. Thus, our genetic analysis not only identifies bases critical for structural integrity of the 60S subunit, but also suggests a role for bases near the peptidyl transferase center in translation initiation. PMID:26824023

  11. NOX Activation by Subunit Interaction and Underlying Mechanisms in Disease

    PubMed Central

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

    2017-01-01

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

  12. Protein Expression of Proteasome Subunits in Elderly Patients with Schizophrenia

    PubMed Central

    Scott, Madeline R; Rubio, Maria D; Haroutunian, Vahram; Meador-Woodruff, James H

    2016-01-01

    The ubiquitin proteasome system (UPS) is a major regulator of protein processing, trafficking, and degradation. While protein ubiquitination is utilized for many cellular processes, one major function of this system is to target proteins to the proteasome for degradation. In schizophrenia, studies have found UPS transcript abnormalities in both blood and brain, and we have previously reported decreased protein expression of ubiquitin-associated proteins in brain. To test whether the proteasome is similarly dysregulated, we measured the protein expression of proteasome catalytic subunits as well as essential subunits from proteasome regulatory complexes in 14 pair-matched schizophrenia and comparison subjects in superior temporal cortex. We found decreased expression of Rpt1, Rpt3, and Rpt6, subunits of the 19S regulatory particle essential for ubiquitin-dependent degradation by the proteasome. Additionally, the α subunit of the 11S αβ regulatory particle, which enhances proteasomal degradation of small peptides and unfolded proteins, was also decreased. Haloperidol-treated rats did not have altered expression of these subunits, suggesting the changes we observed in schizophrenia are likely not due to chronic antipsychotic treatment. Interestingly, expression of the catalytic subunits of both the standard and immunoproteasome were unchanged, suggesting the abnormalities we observed may be specific to the complexed state of the proteasome. Aging has significant effects on the proteasome, and several subunits (20S β2, Rpn10, Rpn13, 11Sβ, and 11Sγ) were significantly correlated with subject age. These data provide further evidence of dysfunction of the ubiquitin-proteasome system in schizophrenia, and suggest that altered proteasome activity may be associated with the pathophysiology of this illness. PMID:26202105

  13. Crystal Structure of the 30S Ribosomal Subunit from Thermus Thermophilus. Purification, Crystallization and Structure Determination

    SciTech Connect

    Clemons, William M.; Brodersen, Ditlev E.; McCutcheonn, John P.; May, Joanna L.C.; Carter, Andrew P.; Morgan-Warren, Robert J.; Wimberly, Brian T.; Ramakrishnan, Venki

    2009-10-07

    We describe the crystallization and structure determination of the 30 S ribosomal subunit from Thermus thermophilus. Previous reports of crystals that diffracted to 10 {angstrom} resolution were used as a starting point to improve the quality of the diffraction. Eventually, ideas such as the addition of substrates or factors to eliminate conformational heterogeneity proved less important than attention to detail in yielding crystals that diffracted beyond 3 {angstrom} resolution. Despite improvements in technology and methodology in the last decade, the structure determination of the 30 S subunit presented some very challenging technical problems because of the size of the asymmetric unit, crystal variability and sensitivity to radiation damage. Some steps that were useful for determination of the atomic structure were: the use of anomalous scattering from the LIII edges of osmium and lutetium to obtain the necessary phasing signal; the use of tunable, third-generation synchrotron sources to obtain data of reasonable quality at high resolution; collection of derivative data precisely about a mirror plane to preserve small anomalous differences between Bijvoet mates despite extensive radiation damage and multi-crystal scaling; the pre-screening of crystals to ensure quality, isomorphism and the efficient use of scarce third-generation synchrotron time; pre-incubation of crystals in cobalt hexaammine to ensure isomorphism with other derivatives; and finally, the placement of proteins whose structures had been previously solved in isolation, in conjunction with biochemical data on protein-RNA interactions, to map out the architecture of the 30 S subunit prior to the construction of a detailed atomic-resolution model.

  14. Identification of structural domains within the large subunit of herpes simplex virus ribonucleotide reductase.

    PubMed

    Conner, J; Cross, A; Murray, J; Marsden, H

    1994-12-01

    The large subunit (R1) of herpes simplex virus (HSV) ribonucleotide reductase is a bifunctional protein consisting of a unique N-terminal protein kinase domain and a ribonucleotide reductase domain. Previous studies showed that the two functional domains are linked by a protease sensitive site. Here we provide evidence for two subdomains, of 30K and 53K, within the reductase domain. The two fragments, which were produced by limited proteolysis and were resistant to further degradation, remained tightly associated in a complex containing two molecules of each. They were capable of binding the R2 subunit of HSV ribonucleotide reductase with approximately the same affinity as the intact protein but the complex did not complement the small subunit (R2) to give an active enzyme. At low concentrations (0.4 micrograms/ml) of trypsin or V8 protease, cleavage between the subdomains was prevented by the presence of the N-terminal protein kinase domain. At higher protease concentrations (1 micrograms/ml) the N-terminal domain is extensively proteolysed and the 30K and 53K domains were generated. Identical results were obtained using purified R1 isolated from infected cell extracts or following expression in Escherichia coli. The origin of the two domains was investigated by N-terminal sequencing of the 53K fragment and by examining their reactivity with a panel of R1-specific monoclonal antibodies which we isolated and epitope mapped for that purpose. The trypsin cleavage site was found to lie between arginine 575 and asparagine 576, and proteolysis in this region was not prevented by the presence of R2 or the nonapeptide YAGAVVNDL. We propose that the ribonucleotide reductase region of HSV R1 exists in a two domain structure, and that the interdomain linking region is protected by the unique N terminus.

  15. Inhibition of the catalytic subunit of cAMP-dependent protein kinase by dicyclohexylcarbodiimide

    SciTech Connect

    Toner-Webb, J.; Taylor, S.S.

    1987-11-17

    The hydrophobic carbodiimide dicyclohexylcarbodiimide (DCCD) has been shown to inhibit the catalytic (C) subunit of adenosine cyclic 3',5'-phosphate dependent protein kinase in a time-dependent, irreversible manner. The rate of inactivation was first order and showed saturation kinetics with an apparent K/sub i/ of 60 ..mu..M. Magnesium adenosine 5'-triphosphate (MgATP) was capable of protecting against this inhibition, whereas neither a synthetic peptide substrate nor histone afforded protection. Mg alone afforded some protection. When the catalytic subunit was aggregated with the regulatory subunit in the holoenzyme complex, no inhibition was observed. The inhibition was enhanced at low pH, suggesting that a carboxylic acid group was the target for interaction with DCCD. On the basis of the protection studies, it is most likely that this carboxylic acid group is associated with the MgATP binding site, perhaps serving as a ligand for the metal. Efforts to identify the site that was modified by DCCD were made. In no case was radioactivity incorporated into the protein, suggesting that the irreversible inhibition was due to an intramolecular cross-link between a reactive carboxylic acid group and a nearby amino group. Differential peptide mapping identified a single peptide that was consistently lost as a consequence of DCCD inhibition. This peptide (residues 166-189) contained four carboxylic acid residues as well as an internal Lys. Two of these carboxyl groups, Asp-166 and Asp-184, are conserved in all protein kinases, including oncogene transforming proteins and growth factor receptors, and thus are likely to play an essential role.

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

    PubMed

    Culiat, C T; Stubbs, L J; Montgomery, C S; Russell, L B; Rinchik, E M

    1994-03-29

    Three genes (Gabrg3, Gabra5, and Gabrb3) encoding the gamma 3, alpha 5, and beta 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 on a panel of p-locus deletions, we have determined that the order of genes within this cluster is centromere-p(D15S12h)-Gabrg3-Gabra5-Gabrb3-telom ere. 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. We have previously suggested that deficiency of the beta 3 subunit may be responsible for the clefting defect. Most notably, however, in this report we describe mice carrying two overlapping, complementing p deletions that fail to express the gamma 3 transcript, as well as mice from another line that express neither the gamma 3 nor alpha 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 3, alpha 5, and beta 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 3 and/or alpha 5 subunits. The absence of an overt neurological phenotype in mice lacking the gamma 3 and/or alpha 5 subunits also suggests that mutations in these genes are unlikely to provide useful animal models for Angelman syndrome in humans.

  17. The first transmembrane domain (TM1) of β2-subunit binds to the transmembrane domain S1 of α-subunit in BK potassium channels

    PubMed Central

    Morera, Francisco J.; Alioua, Abderrahmane; Kundu, Pallob; Salazar, Marcelo; Gonzalez, Carlos; Martinez, Agustin D.; Stefani, Enrico; Toro, Ligia; Latorre, Ramon

    2012-01-01

    The BK channel is one of the most broadly expressed ion channels in mammals. In many tissues, the BK channel pore-forming α-subunit is associated to an auxiliary β-subunit that modulates the voltage- and Ca2+-dependent activation of the channel. Structural components present in β-subunits that are important for the physical association with the α-subunit are yet unknown. Here, we show through co-immunoprecipitation that the intracellular C-terminus, the second transmembrane domain (TM2) and the extracellular loop of the β2-subunit are dispensable for association with the α-subunit pointing transmembrane domain 1 (TM1) as responsible for the interaction. Indeed, the TOXCAT assay for transmembrane protein–protein interactions demonstrated for the first time that TM1 of the β2-subunit physically binds to the transmembrane S1 domain of the α-subunit. PMID:22710124

  18. Integrative structural analysis of the UTPB complex, an early assembly factor for eukaryotic small ribosomal subunits.

    PubMed

    Zhang, Cheng; Sun, Qi; Chen, Rongchang; Chen, Xining; Lin, Jinzhong; Ye, Keqiong

    2016-09-06

    Ribosome assembly is an essential and conserved cellular process in eukaryotes that requires numerous assembly factors. The six-subunit UTPB complex is an essential component of the 90S precursor of the small ribosomal subunit. Here, we analyzed the molecular architecture of UTPB using an integrative structural biology approach. We mapped the major interactions that associate each of six UTPB proteins. Crystallographic studies showed that Utp1, Utp21, Utp12 and Utp13 are evolutionarily related and form a dimer of dimers (Utp1-Utp21, Utp12-Utp13) through their homologous helical C-terminal domains. Molecular docking with crosslinking restraints showed that the WD domains of Utp12 and Utp13 are associated, as are the WD domains of Utp1, Utp21 and Utp18. Electron microscopy images of the entire UTPB complex revealed that it predominantly adopts elongated conformations and possesses internal flexibility. We also determined crystal structures of the WD domain of Utp18 and the HAT and deviant HAT domains of Utp6. A structural model of UTPB was derived based on these data.

  19. Molecular architecture of the 90S small subunit pre-ribosome

    PubMed Central

    Sun, Qi; Zhu, Xing; Qi, Jia; An, Weidong; Lan, Pengfei; Tan, Dan; Chen, Rongchang; Wang, Bing; Zheng, Sanduo; Zhang, Cheng; Chen, Xining; Zhang, Wei; Chen, Jing; Dong, Meng-Qiu; Ye, Keqiong

    2017-01-01

    Eukaryotic small ribosomal subunits are first assembled into 90S pre-ribosomes. The complete 90S is a gigantic complex with a molecular mass of approximately five megadaltons. Here, we report the nearly complete architecture of Saccharomyces cerevisiae 90S determined from three cryo-electron microscopy single particle reconstructions at 4.5 to 8.7 angstrom resolution. The majority of the density maps were modeled and assigned to specific RNA and protein components. The nascent ribosome is assembled into isolated native-like substructures that are stabilized by abundant assembly factors. The 5' external transcribed spacer and U3 snoRNA nucleate a large subcomplex that scaffolds the nascent ribosome. U3 binds four sites of pre-rRNA, including a novel site on helix 27 but not the 3' side of the central pseudoknot, and crucially organizes the 90S structure. The 90S model provides significant insight into the principle of small subunit assembly and the function of assembly factors. DOI: http://dx.doi.org/10.7554/eLife.22086.001 PMID:28244370

  20. RILP interacts with HOPS complex via VPS41 subunit to regulate endocytic trafficking.

    PubMed

    Lin, Xiaosi; Yang, Ting; Wang, Shicong; Wang, Zhen; Yun, Ye; Sun, Lixiang; Zhou, Yunhe; Xu, Xiaohui; Akazawa, Chihiro; Hong, Wanjin; Wang, Tuanlao

    2014-12-02

    The HOPS complex serves as a tethering complex with GEF activity for Ypt7p in yeast to regulate late endosomal membrane maturation. While the role of HOPS complex is well established in yeast cells, its functional and mechanistic aspects in mammalian cells are less well defined. In this study, we report that RILP, a downstream effector of Rab7, interacts with HOPS complex and recruits HOPS subunits to the late endosomal compartment. Structurally, the amino-terminal portion of RILP interacts with HOPS complex. Unexpectedly, this interaction is independent of Rab7. VPS41 subunit of HOPS complex was defined to be the major partner for interacting with RILP. The carboxyl-terminal region of VPS41 was mapped to be responsible for the interaction. Functionally, either depletion of VPS41 by shRNA or overexpression of VPS41 C-terminal half retarded EGF-induced degradation of EGFR. These results suggest that interaction of RILP with HOPS complex via VPS41 plays a role in endocytic trafficking of EGFR.

  1. The bacteriophage T4 gene for the small subunit of ribonucleotide reductase contains an intron.

    PubMed Central

    Sjöberg, B M; Hahne, S; Mathews, C Z; Mathews, C K; Rand, K N; Gait, M J

    1986-01-01

    The bacteriophage T4 gene nrdB codes for the small subunit of the enzyme ribonucleotide reductase. The T4 nrdB gene was localized between 136.1 kb and 137.8 kb in the T4 genetic map according to the deduced structural homology of the protein to the amino acid sequence of its bacterial counterpart, the B2 subunit of Escherichia coli. This positions the C-terminal end of the T4 nrdB gene approximately 2 kb closer to the T4 gene 63 than earlier anticipated from genetic recombinational analyses. The most surprising feature of the T4 nrdB gene is the presence of an approximately 625 bp intron which divides the structural gene into two parts. This is the second example of a prokaryotic structural gene with an intron. The first prokaryotic intron was reported in the nearby td gene, coding for the bacteriophage T4-specific thymidylate synthase enzyme. The nucleotide sequence at the exon-intron junctions of the T4 nrdB gene is similar to that of the junctions of the T4 td gene: the anticipated exon-intron boundary at the donor site ends with a TAA stop codon and there is an ATG start codon at the putative downstream intron-exon boundary of the acceptor site. In the course of this work the denA gene of T4 (endonuclease II) was also located. PMID:3530746

  2. A distinct holoenzyme organization for two-subunit pyruvate carboxylase

    PubMed Central

    Choi, Philip H.; Jo, Jeanyoung; Lin, Yu-Cheng; Lin, Min-Han; Chou, Chi-Yuan; Dietrich, Lars E. P.; Tong, Liang

    2016-01-01

    Pyruvate carboxylase (PC) has important roles in metabolism and is crucial for virulence for some pathogenic bacteria. PC contains biotin carboxylase (BC), carboxyltransferase (CT) and biotin carboxyl carrier protein (BCCP) components. It is a single-chain enzyme in eukaryotes and most bacteria, and functions as a 500 kD homo-tetramer. In contrast, PC is a two-subunit enzyme in a collection of Gram-negative bacteria, with the α subunit containing the BC and the β subunit the CT and BCCP domains, and it is believed that the holoenzyme has α4β4 stoichiometry. We report here the crystal structures of a two-subunit PC from Methylobacillus flagellatus. Surprisingly, our structures reveal an α2β4 stoichiometry, and the overall architecture of the holoenzyme is strikingly different from that of the homo-tetrameric PCs. Biochemical and mutagenesis studies confirm the stoichiometry and other structural observations. Our functional studies in Pseudomonas aeruginosa show that its two-subunit PC is important for colony morphogenesis. PMID:27708276

  3. Interacting cytoplasmic loops of subunits a and c of Escherichia coli F1F0 ATP synthase gate H+ transport to the cytoplasm.

    PubMed

    Steed, P Ryan; Kraft, Kaitlin A; Fillingame, Robert H

    2014-11-25

    H(+)-transporting F1F0 ATP synthase catalyzes the synthesis of ATP via coupled rotary motors within F0 and F1. H(+) transport at the subunit a-c interface in transmembranous F0 drives rotation of a cylindrical c10 oligomer within the membrane, which is coupled to rotation of subunit γ within the α3β3 sector of F1 to mechanically drive ATP synthesis. F1F0 functions in a reversible manner, with ATP hydrolysis driving H(+) transport. ATP-driven H(+) transport in a select group of cysteine mutants in subunits a and c is inhibited after chelation of Ag(+) and/or Cd(+2) with the substituted sulfhydryl groups. The H(+) transport pathway mapped via these Ag(+)(Cd(+2))-sensitive Cys extends from the transmembrane helices (TMHs) of subunits a and c into cytoplasmic loops connecting the TMHs, suggesting these loop regions could be involved in gating H(+) release to the cytoplasm. Here, using select loop-region Cys from the single cytoplasmic loop of subunit c and multiple cytoplasmic loops of subunit a, we show that Cd(+2) directly inhibits passive H(+) transport mediated by F0 reconstituted in liposomes. Further, in extensions of previous studies, we show that the regions mediating passive H(+) transport can be cross-linked to each other. We conclude that the loop-regions in subunits a and c that are implicated in H(+) transport likely interact in a single structural domain, which then functions in gating H(+) release to the cytoplasm.

  4. Retention of duplicated ITAM-containing transmembrane signaling subunits in the tetraploid amphibian species Xenopus laevis.

    PubMed

    Guselnikov, S V; Grayfer, L; De Jesús Andino, F; Rogozin, I B; Robert, J; Taranin, A V

    2015-11-01

    The ITAM-bearing transmembrane signaling subunits (TSS) are indispensable components of activating leukocyte receptor complexes. The TSS-encoding genes map to paralogous chromosomal regions, which are thought to arise from ancient genome tetraploidization(s). To assess a possible role of tetraploidization in the TSS evolution, we studied TSS and other functionally linked genes in the amphibian species Xenopus laevis whose genome was duplicated about 40 MYR ago. We found that X. laevis has retained a duplicated set of sixteen TSS genes, all except one being transcribed. Furthermore, duplicated TCRα loci and genes encoding TSS-coupling protein kinases have also been retained. No clear evidence for functional divergence of the TSS paralogs was obtained from gene expression and sequence analyses. We suggest that the main factor of maintenance of duplicated TSS genes in X. laevis was a protein dosage effect and that this effect might have facilitated the TSS set expansion in early vertebrates.

  5. YphC and YsxC GTPases assist the maturation of the central protuberance, GTPase associated region and functional core of the 50S ribosomal subunit

    PubMed Central

    Ni, Xiaodan; Davis, Joseph H.; Jain, Nikhil; Razi, Aida; Benlekbir, Samir; McArthur, Andrew G.; Rubinstein, John L.; Britton, Robert A.; Williamson, James R.; Ortega, Joaquin

    2016-01-01

    YphC and YsxC are GTPases in Bacillus subtilis that facilitate the assembly of the 50S ribosomal subunit, however their roles in this process are still uncharacterized. To explore their function, we used strains in which the only copy of the yphC or ysxC genes were under the control of an inducible promoter. Under depletion conditions, they accumulated incomplete ribosomal subunits that we named 45SYphC and 44.5SYsxC particles. Quantitative mass spectrometry analysis and the 5–6 Å resolution cryo-EM maps of the 45SYphC and 44.5SYsxC particles revealed that the two GTPases participate in the maturation of the central protuberance, GTPase associated region and key RNA helices in the A, P and E functional sites of the 50S subunit. We observed that YphC and YsxC bind specifically to the two immature particles, suggesting that they represent either on-pathway intermediates or that their structure has not significantly diverged from that of the actual substrate. These results describe the nature of these immature particles, a widely used tool to study the assembly process of the ribosome. They also provide the first insights into the function of YphC and YsxC in 50S subunit assembly and are consistent with this process occurring through multiple parallel pathways, as it has been described for the 30S subunit. PMID:27484475

  6. Separation and characterization of alpha-chain subunits from tilapia (Tilapia zillii) skin gelatin using ultrafiltration.

    PubMed

    Chen, Shulin; Tang, Lanlan; Su, Wenjin; Weng, Wuyin; Osako, Kazufumi; Tanaka, Munehiko

    2015-12-01

    Alpha-chain subunits were separated from tilapia skin gelatin using ultrafiltration, and the physicochemical properties of obtained subunits were investigated. As a result, α1-subunit and α2-subunit could be successfully separated by 100 kDa MWCO regenerated cellulose membranes and 150 kDa MWCO polyethersulfone membranes, respectively. Glycine was the most dominant amino acid in both α1-subunit and α2-subunit. However, the tyrosine content was higher in α2-subunit than in α1-subunit, resulting in strong absorption near 280 nm observed in the UV absorption spectrum. Based on the DSC analysis, it was found that the glass transition temperatures of gelatin, α1-subunit and α2-subunit were 136.48 °C, 126.77 °C and 119.43 °C, respectively. Moreover, the reduced viscosity and denaturation temperature of α1-subunit were higher than those of α2-subunit, and the reduced viscosity reached the highest when α-subunits were mixed with α1/α2 ratio of approximately 2, suggesting that α1-subunit plays a more important role in the thermostability of gelatin than α2-subunit.

  7. Dengue vaccine: an update on recombinant subunit strategies.

    PubMed

    Martin, J; Hermida, L

    2016-03-01

    Dengue is an increasing public health problem worldwide, with the four serotypes of the virus infecting over 390 million people annually. There is no specific treatment or antiviral drug for dengue, and prevention is largely limited to controlling the mosquito vectors or disrupting the human-vector contact. Despite the considerable progress made in recent years, an effective vaccine against the virus is not yet available. The development of a dengue vaccine has been hampered by many unique challenges, including the need to ensure the absence of vaccine-induced enhanced severity of disease. Recombinant protein subunit vaccines offer a safer alternative to other vaccine approaches. Several subunit vaccine candidates are presently under development, based on different structural and non-structural proteins of the virus. Novel adjuvants or immunopotentiating strategies are also being tested to improve their immunogenicity. This review summarizes the current status and development trends of subunit dengue vaccines.

  8. Cholera Toxin B: One Subunit with Many Pharmaceutical Applications

    PubMed Central

    Baldauf, Keegan J.; Royal, Joshua M.; Hamorsky, Krystal Teasley; Matoba, Nobuyuki

    2015-01-01

    Cholera, a waterborne acute diarrheal disease caused by Vibrio cholerae, remains prevalent in underdeveloped countries and is a serious health threat to those living in unsanitary conditions. The major virulence factor is cholera toxin (CT), which consists of two subunits: the A subunit (CTA) and the B subunit (CTB). CTB is a 55 kD homopentameric, non-toxic protein binding to the GM1 ganglioside on mammalian cells with high affinity. Currently, recombinantly produced CTB is used as a component of an internationally licensed oral cholera vaccine, as the protein induces potent humoral immunity that can neutralize CT in the gut. Additionally, recent studies have revealed that CTB administration leads to the induction of anti-inflammatory mechanisms in vivo. This review will cover the potential of CTB as an immunomodulatory and anti-inflammatory agent. We will also summarize various recombinant expression systems available for recombinant CTB bioproduction. PMID:25802972

  9. Cholera toxin B: one subunit with many pharmaceutical applications.

    PubMed

    Baldauf, Keegan J; Royal, Joshua M; Hamorsky, Krystal Teasley; Matoba, Nobuyuki

    2015-03-20

    Cholera, a waterborne acute diarrheal disease caused by Vibrio cholerae, remains prevalent in underdeveloped countries and is a serious health threat to those living in unsanitary conditions. The major virulence factor is cholera toxin (CT), which consists of two subunits: the A subunit (CTA) and the B subunit (CTB). CTB is a 55 kD homopentameric, non-toxic protein binding to the GM1 ganglioside on mammalian cells with high affinity. Currently, recombinantly produced CTB is used as a component of an internationally licensed oral cholera vaccine, as the protein induces potent humoral immunity that can neutralize CT in the gut. Additionally, recent studies have revealed that CTB administration leads to the induction of anti-inflammatory mechanisms in vivo. This review will cover the potential of CTB as an immunomodulatory and anti-inflammatory agent. We will also summarize various recombinant expression systems available for recombinant CTB bioproduction.

  10. Antigenic breadth: a missing ingredient in HSV-2 subunit vaccines?

    PubMed

    Halford, William P

    2014-06-01

    The successful human papillomavirus and hepatitis B virus subunit vaccines contain single viral proteins that represent 22 and 12%, respectively, of the antigens encoded by these tiny viruses. The herpes simplex virus 2 (HSV-2) genome is >20 times larger. Thus, a single protein subunit represents 1% of HSV-2's total antigenic breadth. Antigenic breadth may explain why HSV-2 glycoprotein subunit vaccines have failed in clinical trials, and why live HSV-2 vaccines that express 99% of HSV-2's proteome may be more effective. I review the mounting evidence that live HSV-2 vaccines offer a greater opportunity to stop the spread of genital herpes, and I consider the unfounded 'safety concerns' that have kept live HSV-2 vaccines out of U.S. clinical trials for 25 years.

  11. G alpha 12 and G alpha 13 subunits define a fourth class of G protein alpha subunits.

    PubMed Central

    Strathmann, M P; Simon, M I

    1991-01-01

    Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) are central to the signaling processes of multicellular organisms. We have explored the diversity of the G protein subunits in mammals and found evidence for a large family of genes that encode the alpha subunits. Amino acid sequence comparisons show that the different alpha subunits fall into at least three classes. These classes have been conserved in animals separated by considerable evolutionary distances; they are present in mammals, Drosophila, and nematodes. We have now obtained cDNA clones encoding two murine alpha subunits, G alpha 12 and G alpha 13, that define a fourth class. The translation products are predicted to have molecular masses of 44 kDa and to be insensitive to ADP-ribosylation by pertussis toxin. They share 67% amino acid sequence identity with each other and less than 45% identity with other alpha subunits. Their transcripts can be detected in every tissue examined, although the relative levels of the G alpha 13 message appear somewhat variable. Images PMID:1905812

  12. Subunits of the Schizosaccharomyces pombe RNA polymerase II: enzyme purification and structure of the subunit 3 gene.

    PubMed Central

    Azuma, Y; Yamagishi, M; Ishihama, A

    1993-01-01

    To improve our understanding of the structure and function of eukaryotic RNA polymerase II, we purified the enzyme from the fission yeast Schizosaccharomyces pombe. The highly purified RNA polymerase II contained more than eleven polypeptides. The sizes of the largest the second-, and the third-largest polypeptides as measured by SDS-polyacrylamide gel electrophoresis were about 210, 150, and 40 kilodaltons (kDa), respectively, and are similar to those of RPB1, 2, and 3 subunits of Saccharomyces cerevisiae RNA polymerase II. Using the degenerated primers designed after amino acid micro-sequencing of the 40 kDa third-largest polypeptide (subunit 3), we cloned the subunit 3 gene (rpb3) and determined its DNA sequence. Taken together with the sequence of parts of PCR-amplified cDNA, the predicted coding sequence of rpb3, interrupted by two introns, was found to encode a polypeptide of 297 amino acid residues in length with a molecular weight of 34 kDa. The S. pombe subunit 3 contains four structural domains conserved for the alpha-subunit family of RNA polymerase from both eukaryotes and prokaryotes. A putative leucine zipper motif was found to exist in the C-terminal proximal conserved region (domain D). Possible functions of the conserved domains are discussed. Images PMID:8367291

  13. Stiffness of γ subunit of F(1)-ATPase.

    PubMed

    Okuno, Daichi; Iino, Ryota; Noji, Hiroyuki

    2010-11-01

    F(1)-ATPase is a molecular motor in which the γ subunit rotates inside the α(3)β(3) ring upon adenosine triphosphate (ATP) hydrolysis. Recent works on single-molecule manipulation of F(1)-ATPase have shown that kinetic parameters such as the on-rate of ATP and the off-rate of adenosine diphosphate (ADP) strongly depend on the rotary angle of the γ subunit (Hirono-Hara et al. 2005; Iko et al. 2009). These findings provide important insight into how individual reaction steps release energy to power F(1) and also have implications regarding ATP synthesis and how reaction steps are reversed upon reverse rotation. An important issue regarding the angular dependence of kinetic parameters is that the angular position of a magnetic bead rotation probe could be larger than the actual position of the γ subunit due to the torsional elasticity of the system. In the present study, we assessed the stiffness of two different portions of F(1) from thermophilic Bacillus PS3: the internal part of the γ subunit embedded in the α(3)β(3) ring, and the complex of the external part of the γ subunit and the α(3)β(3) ring (and streptavidin and magnetic bead), by comparing rotational fluctuations before and after crosslinkage between the rotor and stator. The torsional stiffnesses of the internal and remaining parts were determined to be around 223 and 73 pNnm/radian, respectively. Based on these values, it was estimated that the actual angular position of the internal part of the γ subunit is one-fourth of the magnetic bead position upon stalling using an external magnetic field. The estimated elasticity also partially explains the accommodation of the intrinsic step size mismatch between F(o) and F(1)-ATPase.

  14. Localisation of AMPK γ subunits in cardiac and skeletal muscles.

    PubMed

    Pinter, Katalin; Grignani, Robert T; Watkins, Hugh; Redwood, Charles

    2013-12-01

    The trimeric protein AMP-activated protein kinase (AMPK) is an important sensor of energetic status and cellular stress, and mutations in genes encoding two of the regulatory γ subunits cause inherited disorders of either cardiac or skeletal muscle. AMPKγ2 mutations cause hypertrophic cardiomyopathy with glycogen deposition and conduction abnormalities; mutations in AMPKγ3 result in increased skeletal muscle glycogen. In order to gain further insight into the roles of the different γ subunits in muscle and into possible disease mechanisms, we localised the γ2 and γ3 subunits, along with the more abundant γ1 subunit, by immunofluorescence in cardiomyocytes and skeletal muscle fibres. The predominant cardiac γ2 variant, γ2-3B, gave a striated pattern in cardiomyocytes, aligning with the Z-disk but with punctate staining similar to T-tubule (L-type Ca(2+) channel) and sarcoplasmic reticulum (SERCA2) markers. In skeletal muscle fibres AMPKγ3 localises to the I band, presenting a uniform staining that flanks the Z-disk, also coinciding with the position of Ca(2+) influx in these muscles. The localisation of γ2-3B- and γ3-containing AMPK suggests that these trimers may have similar functions in the different muscles. AMPK containing γ2-3B was detected in oxidative skeletal muscles which had low expression of γ3, confirming that these two regulatory subunits may be co-ordinately regulated in response to metabolic requirements. Compartmentalisation of AMPK complexes is most likely dependent on the regulatory γ subunit and this differential localisation may direct substrate selection and specify particular functional roles.

  15. Map projections

    USGS Publications Warehouse

    ,

    1993-01-01

    A map projection is used to portray all or part of the round Earth on a flat surface. This cannot be done without some distortion. Every projection has its own set of advantages and disadvantages. There is no "best" projection. The mapmaker must select the one best suited to the needs, reducing distortion of the most important features. Mapmakers and mathematicians have devised almost limitless ways to project the image of the globe onto paper. Scientists at the U. S. Geological Survey have designed projections for their specific needs—such as the Space Oblique Mercator, which allows mapping from satellites with little or no distortion. This document gives the key properties, characteristics, and preferred uses of many historically important projections and of those frequently used by mapmakers today.

  16. A protein-protein interaction map of yeast RNA polymerase III.

    PubMed

    Flores, A; Briand, J F; Gadal, O; Andrau, J C; Rubbi, L; Van Mullem, V; Boschiero, C; Goussot, M; Marck, C; Carles, C; Thuriaux, P; Sentenac, A; Werner, M

    1999-07-06

    The structure of the yeast RNA polymerase (pol) III was investigated by exhaustive two-hybrid screening using a library of random genomic fragments fused to the Gal4 activation domain. This procedure allowed us to identify contacts between individual polypeptides, localize the contact domains, and deduce a protein-protein interaction map of the multisubunit enzyme. In all but one case, pol III subunits were able to interact in vivo with one or sometimes two partner subunits of the enzyme or with subunits of TFIIIC. Four subunits that are common to pol I, II, and III (ABC27, ABC14.5, ABC10alpha, and ABC10beta), two that are common to pol I and III (AC40 and AC19), and one pol III-specific subunit (C11) can associate with defined regions of the two large subunits. These regions overlapped with highly conserved domains. C53, a pol III-specific subunit, interacted with a 37-kDa polypeptide that copurifies with the enzyme and therefore appears to be a unique pol III subunit (C37). Together with parallel interaction studies based on dosage-dependent suppression of conditional mutants, our data suggest a model of the pol III preinitiation complex.

  17. Micelle-Based Adjuvants for Subunit Vaccine Delivery

    PubMed Central

    Trimaille, Thomas; Verrier, Bernard

    2015-01-01

    In the development of subunit vaccines with purified or recombinant antigens for cancer and infectious diseases, the design of improved and safe adjuvants able to efficiently target the antigen presenting cells, such as dendritic cells, represents a crucial challenge. Nanoparticle-based antigen delivery systems have been identified as an innovative strategy to improve the efficacy of subunit vaccines. Among them, self-assembled micellar nanoparticles from amphiphilic (macro)molecules have recently emerged as promising candidates. In this short review, we report on the recent research findings highlighting the versatility and potential of such systems in vaccine delivery. PMID:26426060

  18. Advancements in the development of subunit influenza vaccines

    PubMed Central

    Zhang, Naru; Zheng, Bo-Jian; Lu, Lu; Zhou, Yusen; Jiang, Shibo; Du, Lanying

    2014-01-01

    The ongoing threat of influenza epidemics and pandemics has emphasized the importance of developing safe and effective vaccines against infections from divergent influenza viruses. In this review, we first introduce the structure and life cycle of influenza A viruses, describing major influenza A virus-caused pandemics. We then compare different types of influenza vaccines and discuss current advancements in the development of subunit influenza vaccines, particularly those based on nucleoprotein (NP), extracellular domain of matrix protein 2 (M2e) and hemagglutinin (HA) proteins. We also illustrate potential strategies for improving the efficacy of subunit influenza vaccines. PMID:25529753

  19. Carrier subunit of plasma membrane transporter is required for oxidative folding of its helper subunit.

    PubMed

    Rius, Mònica; Chillarón, Josep

    2012-05-25

    We study the amino acid transport system b(0,+) as a model for folding, assembly, and early traffic of membrane protein complexes. System b(0,+) is made of two disulfide-linked membrane subunits: the carrier, b(0,+) amino acid transporter (b(0,+)AT), a polytopic protein, and the helper, related to b(0,+) amino acid transporter (rBAT), a type II glycoprotein. rBAT ectodomain mutants display folding/trafficking defects that lead to type I cystinuria. Here we show that, in the presence of b(0,+)AT, three disulfides were formed in the rBAT ectodomain. Disulfides Cys-242-Cys-273 and Cys-571-Cys-666 were essential for biogenesis. Cys-673-Cys-685 was dispensable, but the single mutants C673S, and C685S showed compromised stability and trafficking. Cys-242-Cys-273 likely was the first disulfide to form, and unpaired Cys-242 or Cys-273 disrupted oxidative folding. Strikingly, unassembled rBAT was found as an ensemble of different redox species, mainly monomeric. The ensemble did not change upon inhibition of rBAT degradation. Overall, these results indicated a b(0,+)AT-dependent oxidative folding of the rBAT ectodomain, with the initial and probably cotranslational formation of Cys-242-Cys-273, followed by the oxidation of Cys-571-Cys-666 and Cys-673-Cys-685, that was completed posttranslationally.

  20. Mg-chelatase I subunit 1 and Mg-protoporphyrin IX methyltransferase affect the stomatal aperture in Arabidopsis thaliana.

    PubMed

    Tomiyama, Masakazu; Inoue, Shin-Ichiro; Tsuzuki, Tomo; Soda, Midori; Morimoto, Sayuri; Okigaki, Yukiko; Ohishi, Takaya; Mochizuki, Nobuyoshi; Takahashi, Koji; Kinoshita, Toshinori

    2014-07-01

    To elucidate the molecular mechanisms of stomatal opening and closure, we performed a genetic screen using infrared thermography to isolate stomatal aperture mutants. We identified a mutant designated low temperature with open-stomata 1 (lost1), which exhibited reduced leaf temperature, wider stomatal aperture, and a pale green phenotype. Map-based analysis of the LOST1 locus revealed that the lost1 mutant resulted from a missense mutation in the Mg-chelatase I subunit 1 (CHLI1) gene, which encodes a subunit of the Mg-chelatase complex involved in chlorophyll synthesis. Transformation of the wild-type CHLI1 gene into lost1 complemented all lost1 phenotypes. Stomata in lost1 exhibited a partial ABA-insensitive phenotype similar to that of rtl1, a Mg-chelatase H subunit missense mutant. The Mg-protoporphyrin IX methyltransferase (CHLM) gene encodes a subsequent enzyme in the chlorophyll synthesis pathway. We examined stomatal movement in a CHLM knockdown mutant, chlm, and found that it also exhibited an ABA-insensitive phenotype. However, lost1 and chlm seedlings all showed normal expression of ABA-induced genes, such as RAB18 and RD29B, in response to ABA. These results suggest that the chlorophyll synthesis enzymes, Mg-chelatase complex and CHLM, specifically affect ABA signaling in the control of stomatal aperture and have no effect on ABA-induced gene expression.

  1. Mass spectrometry-based identification of native cardiac Nav1.5 channel α subunit phosphorylation sites.

    PubMed

    Marionneau, Céline; Lichti, Cheryl F; Lindenbaum, Pierre; Charpentier, Flavien; Nerbonne, Jeanne M; Townsend, R Reid; Mérot, Jean

    2012-12-07

    Cardiac voltage-gated Na+ (Nav) channels are key determinants of action potential waveforms, refractoriness and propagation, and Nav1.5 is the main Nav pore-forming (α) subunit in the mammalian heart. Although direct phosphorylation of the Nav1.5 protein has been suggested to modulate various aspects of Nav channel physiology and pathophysiology, native Nav1.5 phosphorylation sites have not been identified. In the experiments here, a mass spectrometry (MS)-based proteomic approach was developed to identify native Nav1.5 phosphorylation sites directly. Using an anti-NavPAN antibody, Nav channel complexes were immunoprecipitated from adult mouse cardiac ventricles. The MS analyses revealed that this antibody immunoprecipitates several Nav α subunits in addition to Nav1.5, as well as several previously identified Nav channel associated/regulatory proteins. Label-free comparative and data-driven phosphoproteomic analyses of purified cardiac Nav1.5 protein identified 11 phosphorylation sites, 8 of which are novel. All the phosphorylation sites identified except one in the N-terminus are in the first intracellular linker loop, suggesting critical roles for this region in phosphorylation-dependent cardiac Nav channel regulation. Interestingly, commonly used prediction algorithms did not reliably predict these newly identified in situ phosphorylation sites. Taken together, the results presented provide the first in situ map of basal phosphorylation sites on the mouse cardiac Nav1.5 α subunit.

  2. Effects of cations and cosolvents on eukaryotic ribosomal subunit conformation

    SciTech Connect

    Moore, M.N.; Spremulli, L.L.

    1985-01-01

    The effects of various cations and cosolvents on the conformation of wheat germ ribosomes and ribosomal subunits have been investigated by using the techniques of circular dichroism and differential hydrogen exchange. A class of hydrogens on both the 40S and 60S subunits exchange out more rapidly as the Mg/sup 2 +/ concentration is raised, indicating that Mg/sup 2 +/ causes a change in subunit conformation. Ca/sup 2 +/ and the polyamines produce a similar increase in the rate of hydrogen exchange. These results suggest that increases in cation concentrations permit a tightening of ribosome structure and a greater degree of internalization of the rRNA. The cosolvent glycerol causes an alteration in the CD spectrum of 80S ribosomes in both the nucleic acid and protein portions of the spectrum. Glycerol also causes a decrease in the rate of exchange of a number of hydrogens on both the 40S and 60S subunits. These results are interpreted to mean that glycerol favors a more native, less denatured structure in the ribosome.

  3. The multifaceted subunit interfaces of ionotropic glutamate receptors.

    PubMed

    Green, Tim; Nayeem, Naushaba

    2015-01-01

    The past fifteen years has seen a revolution in our understanding of ionotropic glutamate receptor (iGluR) structure, starting with the first view of the ligand binding domain (LBD) published in 1998, and in many ways culminating in the publication of the full-length structure of GluA2 in 2009. These reports have revealed not only the central role played by subunit interfaces in iGluR function, but also myriad binding sites within interfaces for endogenous and exogenous factors. Changes in the conformation of inter-subunit interfaces are central to transmission of ligand gating into pore opening (itself a rearrangement of interfaces), and subsequent closure through desensitization. With the exception of the agonist binding site, which is located entirely within individual subunits, almost all modulatory factors affecting iGluRs appear to bind to sites in subunit interfaces. This review seeks to summarize what we currently understand about the diverse roles interfaces play in iGluR function, and to highlight questions for future research.

  4. The multifaceted subunit interfaces of ionotropic glutamate receptors.

    PubMed

    Green, Tim; Nayeem, Naushaba

    2014-06-06

    The past fifteen years has seen a revolution in our understanding of ionotropic glutamate receptor (iGluR) structure, starting with the first view of the ligand binding domain (LBD) published in 1998, and in many ways culminating in the publication of the full-length structure of GluA2 in 2009. These reports have revealed not only the central role played by subunit interfaces in iGluR function, but also myriad binding sites within interfaces for endogenous and exogenous factors. Changes in the conformation of inter-subunit interfaces are central to transmission of ligand gating into pore opening (itself a rearrangement of interfaces), and subsequent closure through desensitization. With the exception of the agonist binding site, which is located entirely within individual subunits, almost all modulatory factors affecting iGluRs appear to bind to sites in subunit interfaces. This review seeks to summarize what we currently understand about the diverse roles interfaces play in iGluR function, and to highlight questions for future research.

  5. Spectroscopic properties of Carcinus aestuarii hemocyanin and its structural subunits

    NASA Astrophysics Data System (ADS)

    Dolashka-Angelova, Pavlina; Hristova, Rumiyana; Stoeva, Stanka; Voelter, Wolfgang

    1999-12-01

    Hemocyanin (Hc) of Carcinus aestuarii contains three major and one minor electrophoretically separable polypeptide chains which were purified by fast protein liquid chromatography (FPLC) ion exchange chromatography. N-terminal amino acid sequences of four structural subunits (SSs) from C. aestuarii were compared with known N-terminal sequences from other arthropodan hemocyanins. The conformational changes, induced by various treatments, were monitored by far UV, CD and fluorescence spectroscopy. The critical temperatures for the structural subunits, Tc, determined by fluorescence spectroscopy, are in the region of 52-59°C and coincide with the melting temperatures, Tm (49-55°C), determined by CD spectroscopy. The free energy of stabilization in water, Δ GDH 2O , toward guanidinium hydrochloride is about 1.3 times higher for the dodecameric Hc as compared to the isolated subunits and about one time higher for Ca1, comparing with other SSs. The studies reveal that the conformational stability of the native dodecamer towards various denaturants (temperature and guanidinium hydrochloride) indicate that the quaternary structure is stabilized by oligomerization between structural subunits, and the possibility of a structural role of the sugar mojeties cannot be excluded.

  6. Emergence of ion channel modal gating from independent subunit kinetics

    PubMed Central

    Bicknell, Brendan A.

    2016-01-01

    Many ion channels exhibit a slow stochastic switching between distinct modes of gating activity. This feature of channel behavior has pronounced implications for the dynamics of ionic currents and the signaling pathways that they regulate. A canonical example is the inositol 1,4,5-trisphosphate receptor (IP3R) channel, whose regulation of intracellular Ca2+ concentration is essential for numerous cellular processes. However, the underlying biophysical mechanisms that give rise to modal gating in this and most other channels remain unknown. Although ion channels are composed of protein subunits, previous mathematical models of modal gating are coarse grained at the level of whole-channel states, limiting further dialogue between theory and experiment. Here we propose an origin for modal gating, by modeling the kinetics of ligand binding and conformational change in the IP3R at the subunit level. We find good agreement with experimental data over a wide range of ligand concentrations, accounting for equilibrium channel properties, transient responses to changing ligand conditions, and modal gating statistics. We show how this can be understood within a simple analytical framework and confirm our results with stochastic simulations. The model assumes that channel subunits are independent, demonstrating that cooperative binding or concerted conformational changes are not required for modal gating. Moreover, the model embodies a generally applicable principle: If a timescale separation exists in the kinetics of individual subunits, then modal gating can arise as an emergent property of channel behavior. PMID:27551100

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

    PubMed Central

    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

  8. ATP-induced helicase slippage reveals highly coordinated subunits

    NASA Astrophysics Data System (ADS)

    Wang, Michelle D.

    2012-02-01

    Helicases are vital enzymes that carry out strand separation of duplex nucleic acids during replication, repair and recombination. T7 helicase, a model hexameric motor, has been observed to use dTTP, but not ATP, to unwind dsDNA as it translocates along ssDNA. Whether and how different subunits of the helicase coordinate their chemo-mechanical activities and DNA binding during translocation is still under debate. Here we address this question using a single-molecule approach to monitor helicase unwinding. We found that T7 helicase does in fact unwind dsDNA in the presence of ATP and that the unwinding rate is even faster than that with dTTP. However, unwinding was repeatedly interrupted by sudden slippage events, ultimately preventing unwinding over a substantial distance. This behaviour was greatly reduced with the supplement of a small amount of dTTP. These findings presented an opportunity to use nucleotide mixtures to investigate helicase subunit coordination. Our results support a model where nearly all subunits coordinate their chemo-mechanical activities and DNA binding. Such subunit coordination may be general to many ring-shaped helicases and reveals a potential mechanism for regulation of DNA unwinding during replication.

  9. A World History Sub-Unit: Teaching about Turkey.

    ERIC Educational Resources Information Center

    Lynn, Karen

    This document is a sub-unit teaching plan for world history teachers who want to use multicultural concepts in the world history curriculum. The objective explored includes a student response to the Turkish question of "Who are we"? Teacher preparation involves defining social and cultural roots and outlining periods of Turkish history.…

  10. Glycoprotein hormone assembly in the endoplasmic reticulum: I. The glycosylated end of human alpha-subunit loop 2 is threaded through a beta-subunit hole.

    PubMed

    Xing, Yongna; Myers, Rebecca V; Cao, Donghui; Lin, Win; Jiang, Mei; Bernard, Michael P; Moyle, William R

    2004-08-20

    Glycoprotein hormone heterodimers are stabilized by their unusual structures in which a glycosylated loop of the alpha-subunit straddles a hole in the beta-subunit. This hole is formed when a cysteine at the end of a beta-subunit strand known as the "seatbelt" becomes "latched" by a disulfide to a cysteine in the beta-subunit core. The heterodimer is stabilized in part by the difficulty of threading the glycosylated end of the alpha-subunit loop 2 through this hole, a phenomenon required for subunit dissociation. Subunit combination in vitro, which occurs by the reverse process, can be accelerated by removing the alpha-subunit oligosaccharide. In cells, heterodimer assembly was thought to occur primarily by a mechanism in which the seatbelt is wrapped around the alpha-subunit after the subunits dock. Here we show that this "wraparound" process can be used to assemble disulfide cross-linked human choriogonadotropin analogs that contain an additional alpha-subunit cysteine, but only if the normal beta-subunit latch site has been removed. Normally, the seatbelt is latched before the subunits dock and assembly is completed when the glycosylated end of alpha-subunit loop 2 is threaded beneath the seatbelt. The unexpected finding that most assembly of human choriogonadotropin, human follitropin, and human thyrotropin heterodimers occurs in this fashion, indicates that threading may be an important phenomenon during protein folding and macromolecule assembly in the endoplasmic reticulum. We suggest that the unusual structures of the glycoprotein hormones makes them useful for identifying factors that influence this process in living cells.

  11. Binding interactions with the complementary subunit of nicotinic receptors.

    PubMed

    Blum, Angela P; Van Arnam, Ethan B; German, Laurel A; Lester, Henry A; Dougherty, Dennis A

    2013-03-08

    The agonist-binding site of nicotinic acetylcholine receptors (nAChRs) spans an interface between two subunits of the pentameric receptor. The principal component of this binding site is contributed by an α subunit, and it binds the cationic moiety of the nicotinic pharmacophore. The other part of the pharmacophore, a hydrogen bond acceptor, has recently been shown to bind to the complementary non-α subunit via the backbone NH of a conserved Leu. This interaction was predicted by studies of ACh-binding proteins and confirmed by functional studies of the neuronal (CNS) nAChR, α4β2. The ACh-binding protein structures further suggested that the hydrogen bond to the backbone NH is mediated by a water molecule and that a second hydrogen bonding interaction occurs between the water molecule and the backbone CO of a conserved Asn, also on the non-α subunit. Here, we provide new insights into the nature of the interactions between the hydrogen bond acceptor of nicotinic agonists and the complementary subunit backbone. We studied both the nAChR of the neuromuscular junction (muscle-type) and a neuronal subtype, (α4)2(β4)3. In the muscle-type receptor, both ACh and nicotine showed a strong interaction with the Leu NH, but the potent nicotine analog epibatidine did not. This interaction was much attenuated in the α4β4 receptor. Surprisingly, we found no evidence for a functionally significant interaction with the backbone carbonyl of the relevant Asn in either receptor with an array of agonists.

  12. The centromere-kinetochore complex: a repeat subunit model

    PubMed Central

    1991-01-01

    The three-dimensional structure of the kinetochore and the DNA/protein composition of the centromere-kinetochore region was investigated using two novel techniques, caffeine-induced detachment of unreplicated kinetochores and stretching of kinetochores by hypotonic and/or shear forces generated in a cytocentrifuge. Kinetochore detachment was confirmed by EM and immunostaining with CREST autoantibodies. Electron microscopic analyses of serial sections demonstrated that detached kinetochores represented fragments derived from whole kinetochores. This was especially evident for the seven large kinetochores in the male Indian muntjac that gave rise to 80-100 fragments upon detachment. The kinetochore fragments, all of which interacted with spindle microtubules and progressed through the entire repertoire of mitotic movements, provide evidence for a subunit organization within the kinetochore. Further support for a repeat subunit model was obtained by stretching or uncoiling the metaphase centromere-kinetochore complex by hypotonic treatments. When immunostained with CREST autoantibodies and subsequently processed for in situ hybridization using synthetic centromere probes, stretched kinetochores displayed a linear array of fluorescent subunits arranged in a repetitive pattern along a centromeric DNA fiber. In addition to CREST antigens, each repetitive subunit was found to bind tubulin and contain cytoplasmic dynein, a microtubule motor localized in the zone of the corona. Collectively, the data suggest that the kinetochore, a plate-like structure seen by EM on many eukaryotic chromosomes is formed by the folding of a linear DNA fiber consisting of tandemly repeated subunits interspersed by DNA linkers. This model, unlike any previously proposed, can account for the structural and evolutional diversity of the kinetochore and its relationship to the centromere of eukaryotic chromosomes of many species. PMID:1828250

  13. Expression of glutamate receptor subunits in human cancers.

    PubMed

    Stepulak, Andrzej; Luksch, Hella; Gebhardt, Christine; Uckermann, Ortrud; Marzahn, Jenny; Sifringer, Marco; Rzeski, Wojciech; Staufner, Christian; Brocke, Katja S; Turski, Lechoslaw; Ikonomidou, Chrysanthy

    2009-10-01

    Emerging evidence suggests a role for glutamate and its receptors in the biology of cancer. This study was designed to systematically analyze the expression of ionotropic and metabotropic glutamate receptor subunits in various human cancer cell lines, compare expression levels to those in human brain tissue and, using electrophysiological techniques, explore whether cancer cells respond to glutamate receptor agonists and antagonists. Expression analysis of glutamate receptor subunits NR1-NR3B, GluR1-GluR7, KA1, KA2 and mGluR1-mGluR8 was performed by means of RT-PCR in human rhabdomyosarcoma/medulloblastoma (TE671), neuroblastoma (SK-NA-S), thyroid carcinoma (FTC 238), lung carcinoma (SK-LU-1), astrocytoma (MOGGCCM), multiple myeloma (RPMI 8226), glioma (U87-MG and U343), lung carcinoma (A549), colon adenocarcinoma (HT 29), T cell leukemia cells (Jurkat E6.1), breast carcinoma (T47D) and colon adenocarcinoma (LS180). Analysis revealed that all glutamate receptor subunits were differentially expressed in the tumor cell lines. For the majority of tumors, expression levels of NR2B, GluR4, GluR6 and KA2 were lower compared to human brain tissue. Confocal imaging revealed that selected glutamate receptor subunit proteins were expressed in tumor cells. By means of patch-clamp analysis, it was shown that A549 and TE671 cells depolarized in response to application of glutamate agonists and that this effect was reversed by glutamate receptor antagonists. This study reveals that glutamate receptor subunits are differentially expressed in human tumor cell lines at the mRNA and the protein level, and that their expression is associated with the formation of functional channels. The potential role of glutamate receptor antagonists in cancer therapy is a feasible goal to be explored in clinical trials.

  14. Targeting signals and subunit interactions in coated vesicle adaptor complexes

    PubMed Central

    1995-01-01

    There are two clathrin-coated vesicle adaptor complexes in the cell, one associated with the plasma membrane and one associated with the TGN. The subunit composition of the plasma membrane adaptor complex is alpha-adaptin, beta-adaptin, AP50, and AP17; while that of the TGN adaptor complex is gamma-adaptin, beta'-adaptin, AP47, and AP19. To search for adaptor targeting signals, we have constructed chimeras between alpha-adaptin and gamma-adaptin within their NH2-terminal domains. We have identified stretches of sequence in the two proteins between amino acids approximately 130 and 330-350 that are essential for targeting. Immunoprecipitation reveals that this region determines whether a construct coassemblies with AP50 and AP17, or with AP47 and AP19. These observations suggest that these other subunits may play an important role in targeting. In contrast, beta- and beta'-adaptins are clearly not involved in this event. Chimeras between the alpha- and gamma-adaptin COOH-terminal domains reveal the presence of a second targeting signal. We have further investigated the interactions between the adaptor subunits using the yeast two-hybrid system. Interactions can be detected between the beta/beta'-adaptins and the alpha/gamma- adaptins, between the beta/beta'-adaptins and the AP50/AP47 subunits, between alpha-adaptin and AP17, and between gamma-adaptin and AP19. These results indicate that the adaptor subunits act in concert to target the complex to the appropriate membrane. PMID:7593184

  15. Heterotrimeric G protein subunit Gγ13 is critical to olfaction

    PubMed Central

    Li, Feng; Ponissery-Saidu, Samsudeen; Yee, Karen; Wang, Hong; Chen, Meng-Ling; Iguchi, Naoko; Zhang, Genhua; Jiang, Ping; Reisert, Johannes; Huang, Liquan

    2013-01-01

    The activation of G-protein-coupled olfactory receptors on the olfactory sensory neurons (OSNs) triggers a signaling cascade, which is mediated by a heterotrimeric G protein consisting of α, β and γ subunits. Although its α subunit, Gαolf, has been identified and well characterized, the identities of its β and γ subunits and their function in olfactory signal transduction, however, have not been well established yet. We and others have found the expression of Gγ13 in the olfactory epithelium, particularly in the cilia of the OSNs. In this study, we generated a conditional gene knockout mouse line to specifically nullify Gγ13 expression in the olfactory marker protein-expressing OSNs. Immunohistochemical and Western blot results showed that Gγ13 subunit was indeed eliminated in the mutant mice’s olfactory epithelium. Intriguingly, Gαolf, β1 subunits, Ric-8B and CEP290 proteins were also absent in the epithelium whereas the presence of the effector enzyme adenylyl cyclase III remained largely unaltered. Electro-olfactogram studies showed that the mutant animals had greatly reduced responses to a battery of odorants including three presumable pheromones. Behavioral tests indicated that the mutant mice had a remarkably reduced ability to perform an odor-guided search task although their motivation and agility seemed normal. Our results indicate that Gαolf exclusively forms a functional heterotrimeric G protein with Gβ1 and Gγ13 in OSNs, mediating olfactory signal transduction. The identification of the olfactory G protein’s βγ moiety has provided a novel approach to understanding the feedback regulation of olfactory signal transduction pathways as well as the control of subcellular structures of OSNs. PMID:23637188

  16. High molecular weight glutenin subunits in some durum wheat cultivars investigated by means of mass spectrometric techniques.

    PubMed

    Muccilli, Vera; Lo Bianco, Marisol; Cunsolo, Vincenzo; Saletti, Rosaria; Gallo, Giulia; Foti, Salvatore

    2011-11-23

    The primary structures of high molecular weight glutenin subunits (HMW-GS) of 5 Triticum durum Desf. cultivars (Simeto, Svevo, Duilio, Bronte, and Sant'Agata), largely cultivated in the south of Italy, and of 13 populations of the old spring Sicilian durum wheat landrace Timilia (Triticum durum Desf.) (accession nos. 1, 2, 3, 4, 7, 8, 9, 13, 14, 15, SG1, SG2, and SG3) were investigated using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and reversed-phase high performance liquid chromatography/nanoelectrospray ionization mass spectrometry (RP-HPLC/nESI-MS/MS). M(r) of the intact proteins determined by MALDI mass spectrometry showed that all the 13 populations of Timilia contained the same two HMW-GS with 75.2 kDa and 86.4 kDa, whereas the other durum wheat cultivars showed the presence of the expected HMW-GS 1By8 and 1Bx7 at 75.1 kDa and 83.1 kDa, respectively. By MALDI mass spectrometry of the tryptic digestion peptides of the isolated HMW-GS of Timilia, the 1Bx and 1By subunits were identified as the NCBInr Acc. No AAQ93629, and AAQ93633, respectively. Sequence verification for HMW-GS 1Bx and 1By both in Simeto and Timilia was obtained by MALDI mass mapping and HPLC/nESI-MSMS of the tryptic peptides. The Bx subunit of Timila presents a sequence similarity of 96% with respect to Simeto, with differences in the insertion of 3 peptides of 5, 9, and 15 amino acids, for a total insertion of 29 amino acids and 25 amino acid substitutions. These differences in the amino acidic sequence account for the determined Δm of 3294 Da between the M(r) of the 1Bx subunits in Timilia and Simeto. Sequence alignment between the two By subunits shows 10 amino acid substitutions and is consistent with the Δm of 148 Da found in the MALDI mass spectra of the intact subunits.

  17. A single gene encodes two different transcripts for the ADP-glucose pyrophosphorylase small subunit from barley (Hordeum vulgare).

    PubMed Central

    Thorbjørnsen, T; Villand, P; Kleczkowski, L A; Olsen, O A

    1996-01-01

    ADP-glucose pyrophosphorylase (AGPase), a heterotetrameric enzyme composed of two small and two large subunits, catalyses the first committed step of starch synthesis in plant tissues. In an attempt to learn more about the organization and expression of the small-subunit gene of AGPase, we have studied the small-subunit transcripts as well as the structure of the gene encoding these transcripts in barley (Hordeum vulgare L. cv. Bomi). Two different transcripts (bepsF1 and blps14) were identified: bepF1 was abundantly expressed in the starchy endosperm but not in leaves, whereas blps14 was isolated from leaves but was also found to be present at a moderate level in the starchy endosperm. The sequences for the two transcripts are identical over approx. 90% of the length, with differences being confined solely to their 5' ends. In blps14, the unique 5' end is 259 nt long and encodes a putative plastid transit peptide sequence. For the 178-nt 5' end of bepsF1, on the other hand, no transit peptide sequence could be recognized. A lambda clone that hybridized to the AGPase transcripts was isolated from a barley genomic library and characterized. The restriction map has suggested a complex organization of the gene, with alternative exons encoding the different 5' ends of the two transcripts followed by nine exons coding for the common part of the transcripts. The sequence of a portion of the genomic clone, covering the alternative 5'-end exons as well as upstream regions, has verified that both transcripts are encoded by the gene. The results suggest that the small-subunit gene of barley AGPase transcribes two different mRNAs by a mechanism classified as alternative splicing. PMID:8546676

  18. Specific interaction between EF-G and RRF and its implication for GTP-dependent ribosome splitting into subunits

    PubMed Central

    Gao, Ning; Zavialov, Andrey V.; Ehrenberg, Måns; Frank, Joachim

    2008-01-01

    Summary After termination of protein synthesis, the bacterial ribosome is split into its 30S and 50S subunits by the action of ribosome recycling factor (RRF) and elongation factor G (EF-G) in a GTP-hydrolysis dependent manner. Based on a previous cryo-electron microscopy (cryo-EM) study of ribosomal complexes, we have proposed that the binding of EF-G to an RRF containing post-termination ribosome triggers an inter-domain rotation of RRF, which destabilizes two strong intersubunit bridges (B2a and B3) and, ultimately, separates the two subunits. Here, we present a 9 Å (FSC at 0.5 cutoff) cryo-EM map of a 50S EFG GDPNP RRF complex and a quasi-atomic model derived from it, showing the interaction between EF-G and RRF on the 50S subunit in the presence of the non-cleavable GTP analogue GDPNP. The detailed information in this model and a comparative analysis of EF-G structures in various nucleotide- and ribosome-bound states show how rotation of the RRF head domain may be triggered by various domains of EF-G. For validation of our structural model, all known mutations in EF-G and RRF that relate to ribosome recycling have been taken into account. More importantly, our results indicate a substantial conformational change in the Switch I region of EF-G, suggesting that a conformational signal transduction mechanism, similar to that employed in tRNA translocation on the ribosome by EF-G, translates a large-scale movement of EF-G’s domain IV, induced by GTP hydrolysis, into the domain rotation of RRF that eventually splits the ribosome into subunits. PMID:17996252

  19. Catalytic Subunit 1 of Protein Phosphatase 2A Is a Subunit of the STRIPAK Complex and Governs Fungal Sexual Development

    PubMed Central

    Beier, Anna; Krisp, Christoph; Wolters, Dirk A.

    2016-01-01

    ABSTRACT The generation of complex three-dimensional structures is a key developmental step for most eukaryotic organisms. The details of the molecular machinery controlling this step remain to be determined. An excellent model system to study this general process is the generation of three-dimensional fruiting bodies in filamentous fungi like Sordaria macrospora. Fruiting body development is controlled by subunits of the highly conserved striatin-interacting phosphatase and kinase (STRIPAK) complex, which has been described in organisms ranging from yeasts to humans. The highly conserved heterotrimeric protein phosphatase PP2A is a subunit of STRIPAK. Here, catalytic subunit 1 of PP2A was functionally characterized. The Δpp2Ac1 strain is sterile, unable to undergo hyphal fusion, and devoid of ascogonial septation. Further, PP2Ac1, together with STRIPAK subunit PRO22, governs vegetative and stress-related growth. We revealed in vitro catalytic activity of wild-type PP2Ac1, and our in vivo analysis showed that inactive PP2Ac1 blocks the complementation of the sterile deletion strain. Tandem affinity purification, followed by mass spectrometry and yeast two-hybrid analysis, verified that PP2Ac1 is a subunit of STRIPAK. Further, these data indicate links between the STRIPAK complex and other developmental signaling pathways, implying the presence of a large interconnected signaling network that controls eukaryotic developmental processes. The insights gained in our study can be transferred to higher eukaryotes and will be important for understanding eukaryotic cellular development in general. PMID:27329756

  20. Arrangement of Kv1 alpha subunits dictates sensitivity to tetraethylammonium.

    PubMed

    Al-Sabi, Ahmed; Shamotienko, Oleg; Dhochartaigh, Sorcha Ni; Muniyappa, Nagesh; Le Berre, Marie; Shaban, Hamdy; Wang, Jiafu; Sack, Jon T; Dolly, J Oliver

    2010-09-01

    Shaker-related Kv1 channels contain four channel-forming alpha subunits. Subfamily member Kv1.1 often occurs oligomerized with Kv1.2 alpha subunits in synaptic membranes, and so information was sought on the influence of their positions within tetramers on the channels' properties. Kv1.1 and 1.2 alpha genes were tandem linked in various arrangements, followed by expression as single-chain proteins in mammalian cells. As some concatenations reported previously seemed not to reliably position Kv1 subunits in their assemblies, the identity of expressed channels was methodically evaluated. Surface protein, isolated by biotinylation of intact transiently transfected HEK-293 cells, gave Kv1.1/1.2 reactivity on immunoblots with electrophoretic mobilities corresponding to full-length concatenated tetramers. There was no evidence of protein degradation, indicating that concatemers were delivered intact to the plasmalemma. Constructs with like genes adjacent (Kv1.1-1.1-1.2-1.2 or Kv1.2-1.2-1.1-1.1) yielded delayed-rectifying, voltage-dependent K(+) currents with activation parameters and inactivation kinetics slightly different from the diagonally positioned genes (Kv1.1-1.2-1.1-1.2 or 1.2-1.1-1.2-1.1). Pore-blocking petidergic toxins, alpha dendrotoxin, agitoxin-1, tityustoxin-Kalpha, and kaliotoxin, were unable to distinguish between the adjacent and diagonal concatamers. Unprecedentedly, external application of the pore-blocker tetraethylammonium (TEA) differentially inhibited the adjacent versus diagonal subunit arrangements, with diagonal constructs having enhanced susceptibility. Concatenation did not directly alter the sensitivities of homomeric Kv1.1 or 1.2 channels to TEA or the toxins. TEA inhibition of currents generated by channels made up from dimers (Kv1.1-1.2 and/or Kv1.2-1.1) was similar to the adjacently arranged constructs. These collective findings indicate that assembly of alpha subunits can be directed by this optimized concatenation, and that subunit

  1. Mechanism of β4 Subunit Modulation of BK Channels

    PubMed Central

    Wang, Bin; Rothberg, Brad S.; Brenner, Robert

    2006-01-01

    Large-conductance (BK-type) Ca2+-activated potassium channels are activated by membrane depolarization and cytoplasmic Ca2+. BK channels are expressed in a broad variety of cells and have a corresponding diversity in properties. Underlying much of the functional diversity is a family of four tissue-specific accessory subunits (β1–β4). Biophysical characterization has shown that the β4 subunit confers properties of the so-called “type II” BK channel isotypes seen in brain. These properties include slow gating kinetics and resistance to iberiotoxin and charybdotoxin blockade. In addition, the β4 subunit reduces the apparent voltage sensitivity of channel activation and has complex effects on apparent Ca2+ sensitivity. Specifically, channel activity at low Ca2+ is inhibited, while at high Ca2+, activity is enhanced. The goal of this study is to understand the mechanism underlying β4 subunit action in the context of a dual allosteric model for BK channel gating. We observed that β4's most profound effect is a decrease in Po (at least 11-fold) in the absence of calcium binding and voltage sensor activation. However, β4 promotes channel opening by increasing voltage dependence of Po-V relations at negative membrane potentials. In the context of the dual allosteric model for BK channels, we find these properties are explained by distinct and opposing actions of β4 on BK channels. β4 reduces channel opening by decreasing the intrinsic gating equilibrium (L0), and decreasing the allosteric coupling between calcium binding and voltage sensor activation (E). However, β4 has a compensatory effect on channel opening following depolarization by shifting open channel voltage sensor activation (Vho) to more negative membrane potentials. The consequence is that β4 causes a net positive shift of the G-V relationship (relative to α subunit alone) at low calcium. At higher calcium, the contribution by Vho and an increase in allosteric coupling to Ca2+ binding (C

  2. Bedaquiline Targets the ε Subunit of Mycobacterial F-ATP Synthase.

    PubMed

    Kundu, Subhashri; Biukovic, Goran; Grüber, Gerhard; Dick, Thomas

    2016-11-01

    The tuberculosis drug bedaquiline inhibits mycobacterial F-ATP synthase by binding to its c subunit. Using the purified ε subunit of the synthase and spectroscopy, we previously demonstrated that the drug interacts with this protein near its unique tryptophan residue. Here, we show that replacement of ε's tryptophan with alanine resulted in bedaquiline hypersusceptibility of the bacteria. Overexpression of the wild-type ε subunit caused resistance. These results suggest that the drug also targets the ε subunit.

  3. Bedaquiline Targets the ε Subunit of Mycobacterial F-ATP Synthase

    PubMed Central

    Kundu, Subhashri; Biukovic, Goran; Grüber, Gerhard

    2016-01-01

    The tuberculosis drug bedaquiline inhibits mycobacterial F-ATP synthase by binding to its c subunit. Using the purified ε subunit of the synthase and spectroscopy, we previously demonstrated that the drug interacts with this protein near its unique tryptophan residue. Here, we show that replacement of ε's tryptophan with alanine resulted in bedaquiline hypersusceptibility of the bacteria. Overexpression of the wild-type ε subunit caused resistance. These results suggest that the drug also targets the ε subunit. PMID:27620476

  4. Differential Contribution of Subunit Interfaces to α9α10 Nicotinic Acetylcholine Receptor Function.

    PubMed

    Boffi, Juan Carlos; Marcovich, Irina; Gill-Thind, JasKiran K; Corradi, Jeremías; Collins, Toby; Lipovsek, María Marcela; Moglie, Marcelo; Plazas, Paola V; Craig, Patricio O; Millar, Neil S; Bouzat, Cecilia; Elgoyhen, Ana Belén

    2017-03-01

    Nicotinic acetylcholine receptors can be assembled from either homomeric or heteromeric pentameric subunit combinations. At the interface of the extracellular domains of adjacent subunits lies the acetylcholine binding site, composed of a principal component provided by one subunit and a complementary component of the adjacent subunit. Compared with neuronal nicotinic acetylcholine cholinergic receptors (nAChRs) assembled from α and β subunits, the α9α10 receptor is an atypical member of the family. It is a heteromeric receptor composed only of α subunits. Whereas mammalian α9 subunits can form functional homomeric α9 receptors, α10 subunits do not generate functional channels when expressed heterologously. Hence, it has been proposed that α10 might serve as a structural subunit, much like a β subunit of heteromeric nAChRs, providing only complementary components to the agonist binding site. Here, we have made use of site-directed mutagenesis to examine the contribution of subunit interface domains to α9α10 receptors by a combination of electrophysiological and radioligand binding studies. Characterization of receptors containing Y190T mutations revealed unexpectedly that both α9 and α10 subunits equally contribute to the principal components of the α9α10 nAChR. In addition, we have shown that the introduction of a W55T mutation impairs receptor binding and function in the rat α9 subunit but not in the α10 subunit, indicating that the contribution of α9 and α10 subunits to complementary components of the ligand-binding site is nonequivalent. We conclude that this asymmetry, which is supported by molecular docking studies, results from adaptive amino acid changes acquired only during the evolution of mammalian α10 subunits.

  5. Differential Contribution of Subunit Interfaces to α9α10 Nicotinic Acetylcholine Receptor Function

    PubMed Central

    Boffi, Juan Carlos; Marcovich, Irina; Gill-Thind, JasKiran K.; Corradi, Jeremías; Collins, Toby; Lipovsek, María Marcela; Moglie, Marcelo; Plazas, Paola V.; Craig, Patricio O.; Millar, Neil S.; Bouzat, Cecilia

    2017-01-01

    Nicotinic acetylcholine receptors can be assembled from either homomeric or heteromeric pentameric subunit combinations. At the interface of the extracellular domains of adjacent subunits lies the acetylcholine binding site, composed of a principal component provided by one subunit and a complementary component of the adjacent subunit. Compared with neuronal nicotinic acetylcholine cholinergic receptors (nAChRs) assembled from α and β subunits, the α9α10 receptor is an atypical member of the family. It is a heteromeric receptor composed only of α subunits. Whereas mammalian α9 subunits can form functional homomeric α9 receptors, α10 subunits do not generate functional channels when expressed heterologously. Hence, it has been proposed that α10 might serve as a structural subunit, much like a β subunit of heteromeric nAChRs, providing only complementary components to the agonist binding site. Here, we have made use of site-directed mutagenesis to examine the contribution of subunit interface domains to α9α10 receptors by a combination of electrophysiological and radioligand binding studies. Characterization of receptors containing Y190T mutations revealed unexpectedly that both α9 and α10 subunits equally contribute to the principal components of the α9α10 nAChR. In addition, we have shown that the introduction of a W55T mutation impairs receptor binding and function in the rat α9 subunit but not in the α10 subunit, indicating that the contribution of α9 and α10 subunits to complementary components of the ligand-binding site is nonequivalent. We conclude that this asymmetry, which is supported by molecular docking studies, results from adaptive amino acid changes acquired only during the evolution of mammalian α10 subunits. PMID:28069778

  6. Regulation of expression of a soybean storage protein subunit gene. Progress report

    SciTech Connect

    Thompson, J.F.; Madison, J.T.

    1984-04-23

    We have found that the methionine repression of the ..beta..-subunit gene expression is not due to degradation of the ..beta..-subunit but is due to an effect on synthesis of the ..beta..-subunit. The effect of methionine on the synthesis of the ..beta..-is due to an inhibition of ..beta..-subunit mRNA synthesis. 3 references, 1 figure.

  7. On the specificity of antibiotics targeting the large ribosomal subunit.

    PubMed

    Wilson, Daniel N

    2011-12-01

    The peptidyltransferase center of the large ribosomal subunit is responsible for catalyzing peptide bonds. This active site is the target of a variety of diverse antibiotics, many of which are used clinically. The past decade has seen a plethora of structures of antibiotics in complex with the large ribosomal subunit, providing unprecedented insight into the mechanism of action of these inhibitors. Ten distinct antibiotics (chloramphenicol, clindamycin, linezolid, tiamulin, sparsomycin, and five macrolides) have been crystallized in complex with four distinct ribosomal species, three bacterial, and one archaeal. This review aims to compare these structures in order to provide insight into the conserved and species-specific modes of interaction for particular members of each class of antibiotics. Coupled with the wealth of biochemical data, a picture is emerging defining the specific functional states of the ribosome that antibiotics preferentially target. Such mechanistic insight into antibiotic inhibition will be important for the development of the next generation of antimicrobial agents.

  8. AMPK beta subunits display isoform specific affinities for carbohydrates.

    PubMed

    Koay, Ann; Woodcroft, Ben; Petrie, Emma J; Yue, Helen; Emanuelle, Shane; Bieri, Michael; Bailey, Michael F; Hargreaves, Mark; Park, Jong-Tae; Park, Kwan-Hwa; Ralph, Stuart; Neumann, Dietbert; Stapleton, David; Gooley, Paul R

    2010-08-04

    AMP-activated protein kinase (AMPK) is a heterotrimer of catalytic (alpha) and regulatory (beta and gamma) subunits with at least two isoforms for each subunit. AMPK beta1 is widely expressed whilst AMPK beta2 is highly expressed in muscle and both beta isoforms contain a mid-molecule carbohydrate-binding module (beta-CBM). Here we show that beta2-CBM has evolved to contain a Thr insertion and increased affinity for glycogen mimetics with a preference for oligosaccharides containing a single alpha-1,6 branched residue. Deletion of Thr-101 reduces affinity for single alpha-1,6 branched oligosaccharides by 3-fold, while insertion of this residue into the equivalent position in the beta1-CBM sequence increases affinity by 3-fold, confirming the functional importance of this residue.

  9. The role of auxiliary subunits for the functional diversity of voltage-gated calcium channels.

    PubMed

    Campiglio, Marta; Flucher, Bernhard E

    2015-09-01

    Voltage-gated calcium channels (VGCCs) represent the sole mechanism to convert membrane depolarization into cellular functions like secretion, contraction, or gene regulation. VGCCs consist of a pore-forming α(1) subunit and several auxiliary channel subunits. These subunits come in multiple isoforms and splice-variants giving rise to a stunning molecular diversity of possible subunit combinations. It is generally believed that specific auxiliary subunits differentially regulate the channels and thereby contribute to the great functional diversity of VGCCs. If auxiliary subunits can associate and dissociate from pre-existing channel complexes, this would allow dynamic regulation of channel properties. However, most auxiliary subunits modulate current properties very similarly, and proof that any cellular calcium channel function is indeed modulated by the physiological exchange of auxiliary subunits is still lacking. In this review we summarize available information supporting a differential modulation of calcium channel functions by exchange of auxiliary subunits, as well as experimental evidence in support of alternative functions of the auxiliary subunits. At the heart of the discussion is the concept that, in their native environment, VGCCs function in the context of macromolecular signaling complexes and that the auxiliary subunits help to orchestrate the diverse protein-protein interactions found in these calcium channel signalosomes. Thus, in addition to a putative differential modulation of current properties, differential subcellular targeting properties and differential protein-protein interactions of the auxiliary subunits may explain the need for their vast molecular diversity.

  10. Electrophysiology and beyond: multiple roles of Na+ channel β subunits in development and disease.

    PubMed

    Patino, Gustavo A; Isom, Lori L

    2010-12-10

    Voltage-gated Na+ channel (VGSC) β Subunits are not "auxiliary." These multi-functional molecules not only modulate Na+ current (I(Na)), but also function as cell adhesion molecules (CAMs)-playing roles in aggregation, migration, invasion, neurite outgrowth, and axonal fasciculation. β subunits are integral members of VGSC signaling complexes at nodes of Ranvier, axon initial segments, and cardiac intercalated disks, regulating action potential propagation through critical intermolecular and cell-cell communication events. At least in vitro, many β subunit cell adhesive functions occur both in the presence and absence of pore-forming VGSC α subunits, and in vivo β subunits are expressed in excitable as well as non-excitable cells, thus β subunits may play important functional roles on their own, in the absence of α subunits. VGSC β1 subunits are essential for life and appear to be especially important during brain development. Mutations in β subunit genes result in a variety of human neurological and cardiovascular diseases. Moreover, some cancer cells exhibit alterations in β subunit expression during metastasis. In short, these proteins, originally thought of as merely accessory to α subunits, are critical players in their own right in human health and disease. Here we discuss the role of VGSC β subunits in the nervous system.

  11. Database on the structure of large ribosomal subunit RNA.

    PubMed Central

    De Rijk, P; Caers, A; Van de Peer, Y; De Wachter, R

    1998-01-01

    The rRNA WWW Server at URL http://rrna.uia.ac.be/ now provides a database of 496 large subunit ribosomal RNA sequences. All these sequences are aligned, incorporate secondary structure information, and can be obtained in a number of formats. Other information about the sequences, such as literature references, accession numbers and taxonomic information is also available and searchable. If necessary, the data on the server can also be obtained by anonymous ftp. PMID:9399830

  12. Database on the structure of large ribosomal subunit RNA.

    PubMed Central

    De Rijk, P; Van de Peer, Y; De Wachter, R

    1996-01-01

    Our database on large ribosomal subunit RNA contained 334 sequences in July, 1995. All sequences in the database are aligned, taking into account secondary structure. The aligned sequences are provided, together with incorporated secondary structure information, in several computer-readable formats. These data can easily be obtained through the World Wide Web. The files in the database are also available via anonymous ftp. PMID:8594610

  13. Database on the structure of small ribosomal subunit RNA.

    PubMed Central

    Van de Peer, Y; Caers, A; De Rijk, P; De Wachter, R

    1998-01-01

    About 8600 complete or nearly complete sequences are now available from the Antwerp database on small ribosomal subunit RNA. All these sequences are aligned with one another on the basis of the adopted secondary structure model, which is corroborated by the observation of compensating substitutions in the alignment. Literature references, accession numbers and detailed taxonomic information are also compiled. The database can be consulted via the World Wide Web at URL http://rrna.uia.ac.be/ssu/ PMID:9399829

  14. Mcm subunits can assemble into two different active unwinding complexes.

    PubMed

    Kanter, Diane M; Bruck, Irina; Kaplan, Daniel L

    2008-11-07

    The replication fork helicase in eukaryotes is a large complex that is composed of Mcm2-7, Cdc45, and GINS. The Mcm2-7 proteins form a heterohexameric ring that hydrolyzes ATP and provide the motor function for this unwinding complex. A comprehensive study of how individual Mcm subunit biochemical activities relate to unwinding function has not been accomplished. We studied the mechanism of the Mcm4-Mcm6-Mcm7 complex, a useful model system because this complex has helicase activity in vitro. We separately purified each of three Mcm subunits until they were each nuclease-free, and we then examined the biochemical properties of different combinations of Mcm subunits. We found that Mcm4 and Mcm7 form an active unwinding assembly. The addition of Mcm6 to Mcm4/Mcm7 results in the formation of an active Mcm4/Mcm6/Mcm7 helicase assembly. The Mcm4-Mcm7 complex forms a ringed-shaped hexamer that unwinds DNA with 3' to 5' polarity by a steric exclusion mechanism, similar to Mcm4/Mcm6/Mcm7. The Mcm4-Mcm7 complex has a high level of ATPase activity that is further stimulated by DNA. The ability of different Mcm mixtures to form rings or exhibit DNA stimulation of ATPase activity correlates with the ability of these complexes to unwind DNA. The Mcm4/Mcm7 and Mcm4/Mcm6/Mcm7 assemblies can open to load onto circular DNA to initiate unwinding. We conclude that the Mcm subunits are surprisingly flexible and dynamic in their ability to interact with one another to form active unwinding complexes.

  15. Screening for AMPA receptor auxiliary subunit specific modulators

    PubMed Central

    Azumaya, Caleigh M.; Days, Emily L.; Vinson, Paige N.; Stauffer, Shaun; Sulikowski, Gary; Weaver, C. David; Nakagawa, Terunaga

    2017-01-01

    AMPA receptors (AMPAR) are ligand gated ion channels critical for synaptic transmission and plasticity. Their dysfunction is implicated in a variety of psychiatric and neurological diseases ranging from major depressive disorder to amyotrophic lateral sclerosis. Attempting to potentiate or depress AMPAR activity is an inherently difficult balancing act between effective treatments and debilitating side effects. A newly explored strategy to target subsets of AMPARs in the central nervous system is to identify compounds that affect specific AMPAR-auxiliary subunit complexes. This exploits diverse spatio-temporal expression patterns of known AMPAR auxiliary subunits, providing means for designing brain region-selective compounds. Here we report a high-throughput screening-based pipeline that can identify compounds that are selective for GluA2-CNIH3 and GluA2-stargazin complexes. These compounds will help us build upon the growing library of AMPAR-auxiliary subunit specific inhibitors, which have thus far all been targeted to TARP γ-8. We used a cell-based assay combined with a voltage-sensitive dye (VSD) to identify changes in glutamate-gated cation flow across the membranes of HEK cells co-expressing GluA2 and an auxiliary subunit. We then used a calcium flux assay to further validate hits picked from the VSD assay. VU0612951 and VU0627849 are candidate compounds from the initial screen that were identified as negative and positive allosteric modulators (NAM and PAM), respectively. They both have lower IC50/EC50s on complexes containing stargazin and CNIH3 than GSG1L or the AMPAR alone. We have also identified a candidate compound, VU0539491, that has NAM activity in GluA2(R)-CNIH3 and GluA2(Q) complexes and PAM activity in GluA2(Q)-GSG1L complexes. PMID:28358902

  16. Energy-driven subunit rotation at the interface between subunit a and the c oligomer in the FO sector of Escherichia coli ATP synthase

    PubMed Central

    Hutcheon, Marcus L.; Duncan, Thomas M.; Ngai, Helen; Cross, Richard L.

    2001-01-01

    Subunit rotation within the F1 catalytic sector of the ATP synthase has been well documented, identifying the synthase as the smallest known rotary motor. In the membrane-embedded FO sector, it is thought that proton transport occurs at a rotor/stator interface between the oligomeric ring of c subunits (rotor) and the single-copy a subunit (stator). Here we report evidence for an energy-dependent rotation at this interface. FOF1 was expressed with a pair of substituted cysteines positioned to allow an intersubunit disulfide crosslink between subunit a and a c subunit [aN214C/cM65C; Jiang, W. & Fillingame, R. H. (1998) Proc. Natl. Acad. Sci. USA 95, 6607–6612]. Membranes were treated with N,N′-dicyclohexyl-[14C]carbodiimide to radiolabel the D61 residue on less than 20% of the c subunits. After oxidation to form an a–c crosslink, the c subunit properly aligned to crosslink to subunit a was found to contain very little 14C label relative to other members of the c ring. However, exposure to MgATP before oxidation significantly increased the radiolabel in the a–c crosslink, indicating that a different c subunit was now aligned with subunit a. This increase was not induced by exposure to MgADP/Pi. Furthermore, preincubation with MgADP and azide to inhibit F1 or with high concentrations of N,N′-dicyclohexylcarbodiimide to label most c subunits prevented the ATP effect. These results provide evidence for an energy-dependent rotation of the c ring relative to subunit a. PMID:11438702

  17. Na(+), K(+)-ATPase β1 subunit associates with α1 subunit modulating a "higher-NKA-in-hyposmotic media" response in gills of euryhaline milkfish, Chanos chanos.

    PubMed

    Hu, Yau-Chung; Chu, Keng-Fu; Yang, Wen-Kai; Lee, Tsung-Han

    2017-03-10

    The euryhaline milkfish (Chanos chanos) is a popular aquaculture species that can be cultured in fresh water, brackish water, or seawater in Southeast Asia. In gills of the milkfish, Na(+), K(+)-ATPase (i.e., NKA; sodium pump) responds to salinity challenges including changes in mRNA abundance, protein amount, and activity. The functional pump is composed of a heterodimeric protein complex composed of α- and β-subunits. Among the NKA genes, α1-β1 isozyme comprises the major form of NKA subunits in mammalian osmoregulatory organs; however, most studies on fish gills have focused on the α1 subunit and did not verify the α1-β1 isozyme. Based on the sequenced milkfish transcriptome, an NKA β1 subunit gene was identified that had the highest amino acid homology to β233, a NKA β1 subunit paralog originally identified in the eel. Despite this high level of homology to β233, phylogenetic analysis and the fact that only a single NKA β1 subunit gene exists in the milkfish suggest that the milkfish gene should be referred to as the NKA β1 subunit gene. The results of accurate domain prediction of the β1 subunit, co-localization of α1 and β1 subunits in epithelial ionocytes, and co-immunoprecipitation of α1 and β1 subunits, indicated the formation of a α1-β1 complex in milkfish gills. Moreover, when transferred to hyposmotic media (fresh water) from seawater, parallel increases in branchial mRNA and protein expression of NKA α1 and β1 subunits suggested their roles in hypo-osmoregulation of euryhaline milkfish. This study molecularly characterized the NKA β1 subunit and provided the first evidence for an NKA α1-β1 association in gill ionocytes of euryhaline teleosts.

  18. Cornichon proteins determine the subunit composition of synaptic AMPA receptors.

    PubMed

    Herring, Bruce E; Shi, Yun; Suh, Young Ho; Zheng, Chan-Ying; Blankenship, Sabine M; Roche, Katherine W; Nicoll, Roger A

    2013-03-20

    Cornichon-2 and cornichon-3 (CNIH-2/-3) are AMPA receptor (AMPAR) binding proteins that promote receptor trafficking and markedly slow AMPAR deactivation in heterologous cells, but their role in neurons is unclear. Using CNIH-2 and CNIH-3 conditional knockout mice, we find a profound reduction of AMPAR synaptic transmission in the hippocampus. This deficit is due to the selective loss of surface GluA1-containing AMPARs (GluA1A2 heteromers), leaving a small residual pool of synaptic GluA2A3 heteromers. The kinetics of AMPARs in neurons lacking CNIH-2/-3 are faster than those in WT neurons due to the fast kinetics of GluA2A3 heteromers. The remarkably selective effect of CNIHs on the GluA1 subunit is probably mediated by TARP γ-8, which prevents a functional association of CNIHs with non-GluA1 subunits. These results point to a sophisticated interplay between CNIHs and γ-8 that dictates subunit-specific AMPAR trafficking and the strength and kinetics of synaptic AMPAR-mediated transmission.

  19. Rescue of lethal subunits into functional K+ channels.

    PubMed Central

    Taglialatela, M; Payne, J P; Drewe, J A; Brown, A M

    1994-01-01

    In a chimeric, voltage-dependent K+ channel (CHM), the valine at position 369 and the leucine at position 374 interact within the pore or P-region to regulate ion permeation and block. Here we show that the point mutation, CHM V369L, abolished channel function whereas previous experiments showed that CHM V369 and CHM V369I are functional. Coinjection of "lethal" CHM V369L cRNA with CHM L374V cRNA but not CHM cRNA generated functional heteromultimers. The whole-cell Rb+/K+ conductance ratio was 2.98 +/- 0.43 for CHM L374V and was reduced to 0.87 +/- 0.04 for the coexpressed CHM V369L and CHM L374V subunits. When single-channel currents were recorded, a single class of CHM V369L/CHM L374V heteromultimers was identified. This class was readily distinguishable from CHM L374V homomultimers by K+ conductance, gating, and blockade by internal tetraethylammonium. Coinjection experiments at various RNA ratios suggest that the CHM V369L/CHM L374V heteromultime, assuming it to be a tetramer, was composed of three CHM L374V subunits and one CHM V369L subunit. It appears that in the critical P-region of CHM position 369 may tolerate only one leucine. Images FIGURE 7 PMID:8130337

  20. Differential Localization of G Protein βγ Subunits

    PubMed Central

    2015-01-01

    G protein βγ subunits play essential roles in regulating cellular signaling cascades, yet little is known about their distribution in tissues or their subcellular localization. While previous studies have suggested specific isoforms may exhibit a wide range of distributions throughout the central nervous system, a thorough investigation of the expression patterns of both Gβ and Gγ isoforms within subcellular fractions has not been conducted. To address this, we applied a targeted proteomics approach known as multiple-reaction monitoring to analyze localization patterns of Gβ and Gγ isoforms in pre- and postsynaptic fractions isolated from cortex, cerebellum, hippocampus, and striatum. Particular Gβ and Gγ subunits were found to exhibit distinct regional and subcellular localization patterns throughout the brain. Significant differences in subcellular localization between pre- and postsynaptic fractions were observed within the striatum for most Gβ and Gγ isoforms, while others exhibited completely unique expression patterns in all four brain regions examined. Such differences are a prerequisite for understanding roles of individual subunits in regulating specific signaling pathways throughout the central nervous system. PMID:24568373

  1. Molecular basis of AKAP specificity for PKA regulatory subunits.

    PubMed

    Gold, Matthew G; Lygren, Birgitte; Dokurno, Pawel; Hoshi, Naoto; McConnachie, George; Taskén, Kjetil; Carlson, Cathrine R; Scott, John D; Barford, David

    2006-11-03

    Localization of cyclic AMP (cAMP)-dependent protein kinase (PKA) by A kinase-anchoring proteins (AKAPs) restricts the action of this broad specificity kinase. The high-resolution crystal structures of the docking and dimerization (D/D) domain of the RIIalpha regulatory subunit of PKA both in the apo state and in complex with the high-affinity anchoring peptide AKAP-IS explain the molecular basis for AKAP-regulatory subunit recognition. AKAP-IS folds into an amphipathic alpha helix that engages an essentially preformed shallow groove on the surface of the RII dimer D/D domains. Conserved AKAP aliphatic residues dominate interactions to RII at the predominantly hydrophobic interface, whereas polar residues are important in conferring R subunit isoform specificity. Using a peptide screening approach, we have developed SuperAKAP-IS, a peptide that is 10,000-fold more selective for the RII isoform relative to RI and can be used to assess the impact of PKA isoform-selective anchoring on cAMP-responsive events inside cells.

  2. Serotonergic modulation of muscle acetylcholine receptors of different subunit composition.

    PubMed Central

    García-Colunga, J; Miledi, R

    1996-01-01

    Modulation of muscle acetylcholine (AcCho) receptors (AcChoRs) by serotonin [5-hydroxytryptamine (5HT)] and other serotonergic compounds was studied in Xenopus laevis oocytes. Various combinations of alpha, beta, gamma, and delta subunit RNAs were injected into oocytes, and membrane currents elicited by AcCho were recorded under voltage clamp. Judging by the amplitudes of AcCho currents generated, the levels of functional receptor expression were: alpha beta gamma delta > alpha beta delta > alpha beta gamma > alpha gamma delta. The alpha beta gamma delta and alpha beta delta AcChoR Subtypes were strongly blocked by 5HT, whereas the alpha beta gamma receptor was blocked only slightly. The order of blocking potency of AcChoRs by 5HT was: alpha beta delta > alpha beta gamma delta > alpha beta gamma. 5HT receptor antagonists, such as methysergide and spiperone, were even more potent blockers of AcChoRs than 5HT but did not show much subunit selectivity. Blockage of alpha beta gamma delta and alpha beta delta receptors by 5HT was voltage-dependent, and the voltage dependence was abolished when the delta subunit was omitted. These findings may need to be taken into consideration when trying to elucidate the mode of action of many clinically important serotonergic compounds. Images Fig. 3 PMID:8633003

  3. Molecular Modeling of the Misfolded Insulin Subunit and Amyloid Fibril

    PubMed Central

    Choi, Jay H.; May, Barnaby C.H.; Wille, Holger; Cohen, Fred E.

    2009-01-01

    Abstract Insulin, a small hormone protein comprising 51 residues in two disulfide-linked polypeptide chains, adopts a predominantly α-helical conformation in its native state. It readily undergoes protein misfolding and aggregates into amyloid fibrils under a variety of conditions. Insulin is a unique model system in which to study protein fibrillization, since its three disulfide bridges are retained in the fibrillar state and thus limit the conformational space available to the polypeptide chains during misfolding and fibrillization. Taking into account this unique conformational restriction, we modeled possible monomeric subunits of the insulin amyloid fibrils using β-solenoid folds, namely, the β-helix and β-roll. Both models agreed with currently available biophysical data. We performed molecular dynamics simulations, which allowed some limited insights into the relative structural stability, suggesting that the β-roll subunit model may be more stable than the β-helix subunit model. We also constructed β-solenoid-based insulin fibril models and conducted fiber diffraction simulation to identify plausible fibril architectures of insulin amyloid. A comparison of simulated fiber diffraction patterns of the fibril models to the experimental insulin x-ray fiber diffraction data suggests that the model fibers composed of six twisted β-roll protofilaments provide the most reasonable fit to available experimental diffraction patterns and previous biophysical studies. PMID:20006956

  4. Ribosomal small subunit domains radiate from a central core

    PubMed Central

    Gulen, Burak; Petrov, Anton S.; Okafor, C. Denise; Vander Wood, Drew; O’Neill, Eric B.; Hud, Nicholas V.; Williams, Loren Dean

    2016-01-01

    The domain architecture of a large RNA can help explain and/or predict folding, function, biogenesis and evolution. We offer a formal and general definition of an RNA domain and use that definition to experimentally characterize the rRNA of the ribosomal small subunit. Here the rRNA comprising a domain is compact, with a self-contained system of molecular interactions. A given rRNA helix or stem-loop must be allocated uniquely to a single domain. Local changes such as mutations can give domain-wide effects. Helices within a domain have interdependent orientations, stabilities and interactions. With these criteria we identify a core domain (domain A) of small subunit rRNA. Domain A acts as a hub, linking the four peripheral domains and imposing orientational and positional restraints on the other domains. Experimental characterization of isolated domain A, and mutations and truncations of it, by methods including selective 2′OH acylation analyzed by primer extension and circular dichroism spectroscopy are consistent with our architectural model. The results support the utility of the concept of an RNA domain. Domain A, which exhibits structural similarity to tRNA, appears to be an essential core of the small ribosomal subunit. PMID:26876483

  5. Human Mind Maps

    ERIC Educational Resources Information Center

    Glass, Tom

    2016-01-01

    When students generate mind maps, or concept maps, the maps are usually on paper, computer screens, or a blackboard. Human Mind Maps require few resources and little preparation. The main requirements are space where students can move around and a little creativity and imagination. Mind maps can be used for a variety of purposes, and Human Mind…

  6. Individual Interactions of the b Subunits within the Stator of the Escherichia coli ATP Synthase*

    PubMed Central

    Brandt, Karsten; Maiwald, Sarah; Herkenhoff-Hesselmann, Brigitte; Gnirß, Kerstin; Greie, Jörg-Christian; Dunn, Stanley D.; Deckers-Hebestreit, Gabriele

    2013-01-01

    FOF1 ATP synthases are rotary nanomotors that couple proton translocation across biological membranes to the synthesis/hydrolysis of ATP. During catalysis, the peripheral stalk, composed of two b subunits and subunit δ in Escherichia coli, counteracts the torque generated by the rotation of the central stalk. Here we characterize individual interactions of the b subunits within the stator by use of monoclonal antibodies and nearest neighbor analyses via intersubunit disulfide bond formation. Antibody binding studies revealed that the C-terminal region of one of the two b subunits is principally involved in the binding of subunit δ, whereas the other one is accessible to antibody binding without impact on the function of FOF1. Individually substituted cysteine pairs suitable for disulfide cross-linking between the b subunits and the other stator subunits (b-α, b-β, b-δ, and b-a) were screened and combined with each other to discriminate between the two b subunits (i.e. bI and bII). The results show the b dimer to be located at a non-catalytic α/β cleft, with bI close to subunit α, whereas bII is proximal to subunit β. Furthermore, bI can be linked to subunit δ as well as to subunit a. Among the subcomplexes formed were a-bI-α, bII-β, α-bI-bII-β, and a-bI-δ. Taken together, the data obtained define the different positions of the two b subunits at a non-catalytic interface and imply that each b subunit has a different role in generating stability within the stator. We suggest that bI is functionally related to the single b subunit present in mitochondrial ATP synthase. PMID:23846684

  7. Concept Mapping

    PubMed Central

    Brennan, Laura K.; Brownson, Ross C.; Kelly, Cheryl; Ivey, Melissa K.; Leviton, Laura C.

    2016-01-01

    Background From 2003 to 2008, 25 cross-sector, multidisciplinary community partnerships funded through the Active Living by Design (ALbD) national program designed, planned, and implemented policy and environmental changes, with complementary programs and promotions. This paper describes the use of concept-mapping methods to gain insights into promising active living intervention strategies based on the collective experience of community representatives implementing ALbD initiatives. Methods Using Concept Systems software, community representatives (n=43) anonymously generated actions and changes in their communities to support active living (183 original statements, 79 condensed statements). Next, respondents (n=26, from 23 partnerships) sorted the 79 statements into self-created categories, or active living intervention approaches. Respondents then rated statements based on their perceptions of the most important strategies for creating community changes (n=25, from 22 partnerships) and increasing community rates of physical activity (n=23, from 20 partnerships). Cluster analysis and multidimensional scaling were used to describe data patterns. Results ALbD community partnerships identified three active living intervention approaches with the greatest perceived importance to create community change and increase population levels of physical activity: changes to the built and natural environment, partnership and collaboration efforts, and land-use and transportation policies. The relative importance of intervention approaches varied according to subgroups of partnerships working with different populations. Conclusions Decision makers, practitioners, and community residents can incorporate what has been learned from the 25 community partnerships to prioritize active living policy, physical project, promotional, and programmatic strategies for work in different populations and settings. PMID:23079266

  8. Two-subunit DNA escort mechanism and inactive subunit bypass in an ultra-fast ring ATPase

    DOE PAGES

    Liu, Ninning; Chistol, Gheorghe; Bustamante, Carlos

    2015-10-09

    SpoIIIE is a homo-hexameric dsDNA translocase responsible for completing chromosome segregation in Bacillus subtilis . Here, we use a single-molecule approach to monitor SpoIIIE translocation when challenged with neutral-backbone DNA and non-hydrolyzable ATP analogs. We show that SpoIIIE makes multiple essential contacts with phosphates on the 5'→3' strand in the direction of translocation. Using DNA constructs with two neutral-backbone segments separated by a single charged base pair, we deduce that SpoIIIE’s step size is 2 bp. Finally, experiments with non-hydrolyzable ATP analogs suggest that SpoIIIE can operate with non-consecutive inactive subunits. We propose a two-subunit escort translocation mechanism thatmore » is strict enough to enable SpoIIIE to track one DNA strand, yet sufficiently compliant to permit the motor to bypass inactive subunits without arrest. We speculate that such a flexible mechanism arose for motors that, like SpoIIIE, constitute functional bottlenecks where the inactivation of even a single motor can be lethal for the cell.« less

  9. Maps & minds : mapping through the ages

    USGS Publications Warehouse

    ,

    1984-01-01

    Throughout time, maps have expressed our understanding of our world. Human affairs have been influenced strongly by the quality of maps available to us at the major turning points in our history. "Maps & Minds" traces the ebb and flow of a few central ideas in the mainstream of mapping. Our expanding knowledge of our cosmic neighborhood stems largely from a small number of simple but grand ideas, vigorously pursued.

  10. Mapping: A Course.

    ERIC Educational Resources Information Center

    Whitmore, Paul M.

    1988-01-01

    Reviews the history of cartography. Describes the contributions of Strabo and Ptolemy in early maps. Identifies the work of Gerhard Mercator as the most important advancement in mapping. Discusses present mapping standards from history. (CW)

  11. Early expression of GABA(A) receptor delta subunit in the neonatal rat hippocampus.

    PubMed

    Didelon, F; Mladinic', M; Cherubini, E; Bradbury, A

    2000-12-01

    The cDNA library screening strategy was used to identify the genes encoding for GABA(A) receptor subunits in the rat hippocampus during development. With this technique, genes encoding eleven GABA(A) receptor subunits were identified. The alpha5 subunit was by far the most highly expressed, followed by the gamma2, alpha2 and alpha4 subunits respectively. The expression of the beta2, alpha1, gamma1, beta1 and beta3 subunits was moderate, although that of the alpha3 and delta subunits was weak. In situ hybridization experiments, using digoxigenin-labeled cRNA probes, confirmed that the delta subunit was expressed in the neonatal as well as in the adult hippocampus, and is likely to form functional receptors in association with other subunits of the GABA(A) receptor. When the more sensitive RT-PCR approach was used, the gamma3 subunit was also detected, suggesting that this subunit is present in the hippocampus during development but at low levels of expression. The insertion of the delta subunit into functional GABA(A) receptors may enhance the efficacy of GABA in the immediate postnatal period when this amino acid is still exerting a depolarizing and excitatory action.

  12. Modulation of BK Channel Function by Auxiliary Beta and Gamma Subunits

    PubMed Central

    Li, Q.; Yan, J.

    2016-01-01

    The large-conductance, Ca2+- and voltage-activated K+ (BK) channel is ubiquitously expressed in mammalian tissues and displays diverse biophysical or pharmacological characteristics. This diversity is in part conferred by channel modulation with different regulatory auxiliary subunits. To date, two distinct classes of BK channel auxiliary subunits have been identified: β subunits and γ subunits. Modulation of BK channels by the four auxiliary β (β1–β4) subunits has been well established and intensively investigated over the past two decades. The auxiliary γ subunits, however, were identified only very recently, which 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. This chapter will review the current understanding of BK channel modulation by auxiliary β and γ subunits, especially the latest findings. PMID:27238261

  13. Functional Diversification of Maize RNA Polymerase IV and V subtypes via Alternative Catalytic Subunits

    SciTech Connect

    Haag, Jeremy R.; Brower-Toland, Brent; Krieger, Elysia K.; Sidorenko, Lyudmila; Nicora, Carrie D.; Norbeck, Angela D.; Irsigler, Andre; LaRue, Huachun; Brzeski, Jan; Mcginnis, Karen A.; Ivashuta, Sergey; Pasa-Tolic, Ljiljana; Chandler, Vicki L.; Pikaard, Craig S.

    2014-10-01

    Unlike nuclear multisubunit RNA polymerases I, II, and III, whose subunit compositions are conserved throughout eukaryotes, plant RNA polymerases IV and V are nonessential, Pol II-related enzymes whose subunit compositions are still evolving. Whereas Arabidopsis Pols IV and V differ from Pol II in four or five of their 12 subunits, respectively, and differ from one another in three subunits, proteomic ana- lyses show that maize Pols IV and V differ from Pol II in six subunits but differ from each other only in their largest subunits. Use of alternative catalytic second subunits, which are nonredundant for development and paramutation, yields at least two sub- types of Pol IV and three subtypes of Pol V in maize. Pol IV/Pol V associations with MOP1, RMR1, AGO121, Zm_DRD1/CHR127, SHH2a, and SHH2b extend parallels between paramutation in maize and the RNA-directed DNA methylation pathway in Arabidopsis.

  14. Ribosome Subunit Stapling for Orthogonal Translation in E.  coli.

    PubMed

    Fried, Stephen D; Schmied, Wolfgang H; Uttamapinant, Chayasith; Chin, Jason W

    2015-10-19

    The creation of orthogonal large and small ribosomal subunits, which interact with each other but not with endogenous ribosomal subunits, would extend our capacity to create new functions in the ribosome by making the large subunit evolvable. To this end, we rationally designed a ribosomal RNA that covalently links the ribosome subunits via an RNA staple. The stapled ribosome is directed to an orthogonal mRNA, allowing the introduction of mutations into the large subunit that reduce orthogonal translation, but have minimal effects on cell growth. Our approach provides a promising route towards orthogonal subunit association, which may enable the evolution of key functional centers in the large subunit, including the peptidyl-transferase center, for unnatural polymer synthesis in cells.

  15. Subunit regulation of the neuronal alpha 1A Ca2+ channel expressed in Xenopus oocytes.

    PubMed Central

    De Waard, M; Campbell, K P

    1995-01-01

    1. Voltage-dependent Ca2+ channels are multi-protein complexes composed of at least three subunits: alpha 1, alpha 2 delta and beta. Ba2+ currents were recorded in Xenopus oocytes expressing the neuronal alpha 1A Ca2+ channel, using the two-electrode voltage-clamp technique. Various subunit combinations were studied: alpha 1A, alpha 1A alpha 2 delta b, alpha 1A beta or alpha 1A alpha 2 delta b beta. 2. The alpha 1A subunit alone directs the expression of functional Ca2+ channels. It carries all the properties of the channel: gating, permeability, voltage dependence of activation and inactivation, and pharmacology. The alpha 1A channel is activated by low voltages when physiological concentrations of the permeant cation are used. Both ancillary subunits alpha 2 delta and beta induced considerable changes in the biophysical properties of the alpha 1A current. The subunit specificity of the changes in current properties was analysed for all four beta gene products by coexpressing beta 1b, beta 2a, beta 3 and beta 4. 3. All beta subunits induce a stimulation in the current amplitude, a change in inactivation kinetics, and two hyperpolarizing shifts--one in the voltage dependence of activation and a second in the voltage dependence of steady-state inactivation. The most significant difference in regulation among beta subunits is the induction of variable rate constants of current inactivation. Rates of inactivation were induced in the following order (fastest to slowest): beta 3 > beta 1b = beta 4 > beta 2a. 4. The alpha 2 delta b subunit does not modify the properties of alpha 1A Ca2+ channels in the absence of beta subunits. However, this subunit increases the beta-induced stimulation in current amplitude and also regulates the beta-induced change in inactivation kinetics. 5. Of all the subunit combinations tested, Ca2+ channels that included a beta subunit were the most prone to decrease in activity. It is concluded that beta subunits are the primary target for the

  16. Comparative immunohistochemical localisation of GABA(B1a), GABA(B1b) and GABA(B2) subunits in rat brain, spinal cord and dorsal root ganglion.

    PubMed

    Charles, K J; Evans, M L; Robbins, M J; Calver, A R; Leslie, R A; Pangalos, M N

    2001-01-01

    GABA(B) receptors are G-protein-coupled receptors mediating the slow onset and prolonged synaptic actions of GABA in the CNS. The recent cloning of two genes, GABA(B1) and GABA(B2), has revealed a novel requirement for GABA(B) receptor signalling. Studies have demonstrated that the two receptor subunits associate as a GABA(B1)/GABA(B2) heterodimer to form a functional GABA(B) receptor. In this study we have developed polyclonal antisera specific to two splice variants of the GABA(B1) subunit, GABA(B1a) and GABA(B1b), as well as an antiserum to the GABA(B2) subunit. Using affinity-purified antibodies derived from these antisera we have mapped out the distribution profile of each subunit in rat brain, spinal cord and dorsal root ganglion. In brain the highest areas of GABA(B1a), GABA(B1b) and GABA(B2) subunit expression were found in neocortex, hippocampus, thalamus, cerebellum and habenula. In spinal cord, GABA(B1) and GABA(B2) subunits were expressed in the superficial layers of the dorsal horn, as well as in motor neurones in the deeper layers of the ventral horn. GABA(B) receptor subunit immunoreactivity in dorsal root ganglion suggested that expression of GABA(B1b) was restricted to the large diameter neurones, in contrast to GABA(B1a) and GABA(B2) subunits which were expressed in both large and small diameter neurones. Although expression levels of GABA(B1) and GABA(B2) subunits varied we found no areas in which GABA(B1) was expressed in the absence of GABA(B2). This suggests that most, if not all, GABA(B1) immunoreactivity may represent functional GABA(B) receptors. Although our data are in general agreement with functional studies, some discrepancies in GABA(B1) subunit expression occurred with respect to other immunohistochemical studies. Overall our data suggest that GABA(B) receptors are widely expressed throughout the brain and spinal cord, and that GABA(B1a) and GABA(B1b) subunits can associate with GABA(B2) to form both pre- and post-synaptic receptors.

  17. Covalent dimerization of ribulose bisphosphate carboxylase subunits by UV radiation.

    PubMed

    Ferreira, R M; Franco, E; Teixeira, A R

    1996-08-15

    The effect of UV radiation (UV-A, UV-B and UV-C) on ribulose bisphosphate carboxylase from a variety of plant species was examined. The exposition of plant leaves or the pure enzyme to UV radiation produced a UV-dependent accumulation of a +5 kDa polypeptide (P65). Different approaches were utilized to elucidate the origin and structure of P65: electrophoretic and fluorographic analyses of 35S-labelled ribulose bisphosphate carboxylase exposed to UV radiation and immunological experiments using antibodies specific for P65, for the large and small subunits of ribulose bisphosphate carboxylase and for high-molecular-mass aggregates of the enzyme. These studies revealed that P65 is a dimer, formed by the covalent, non-disulphide linkage of one small subunit with one large subunit of ribulose bisphosphate carboxylase. For short periods of time (< 1 h), the amount of P65 formed increased with the duration of the exposure to the UV radiation and with the energy of the radiation applied. Prolonged exposure to UV radiation (1-6 h) resulted in the formation of high-molecular-mass aggregates of ribulose bisphosphate carboxylase. Formation of P65 was shown to depend on the native state of the protein, was stimulated by inhibitors of enzyme activity, and was inhibited by activators of enzyme activity. A UV-independent accumulation of P65 was also achieved by the in vitro incubation of plant crude extracts. However, the UV-dependent and the UV-independent formation of P65 seemed to occur by distinct molecular mechanisms. The UV-dependent accumulation of P65 was immunologically detected in all species examined, including Lemna minor, Arum italicum, Brassica oleracea, Triticum aestivum, Zea mays, Pisum sativum and Phaseolus vulgaris, suggesting that it may constitute a universal response to UV radiation, common to all photo-synthetic tissues.

  18. Properties and subunit structure of pig heart pyruvate dehydrogenase.

    PubMed

    Hamada, M; Hiraoka, T; Koike, K; Ogasahara, K; Kanzaki, T

    1976-06-01

    Pyruvate dehydrogenase [EC 1.2.4.1] was separated from the pyruvate dehydrogenase complex and its molecular weight was estimated to be about 150,000 by sedimentation equilibrium methods. The enzyme was dissociated into two subunits (alpha and beta), with estimated molecular weights of 41,000 (alpha) and 36,000 (beta), respectively, by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The subunits were separated by phosphocellulose column chromatography and their chemical properties were examined. The subunit structure of the pyruvate dehydrogenase was assigned as alpha2beta2. The content of right-handed alpha-helix in the enzyme molecule was estimated to be about 29 and 28% by optical rotatory dispersion and by circular dichroism, respectively. The enzyme contained no thiamine-PP, and its dehydrogenase activity was completely dependent on added thiamine-PP and partially dependent on added Mg2+ and Ca2+. The Km value of pyruvate dehydrogenase for thiamine diphosphate was estimated to be 6.5 X 10(-5) M in the presence of Mg2+ or Ca2+. The enzyme showed highly specific activity for thiamine-PP dependent oxidation of both pyruvate and alpha-ketobutyrate, but it also showed some activity with alpha-ketovalerate, alpha-ketoisocaproate, and alpha-ketoisovalerate. The pyruvate dehydrogenase activity was strongly inhibited by bivalent heavy metal ions and by sulfhydryl inhibitors; and the enzyme molecule contained 27 moles of 5,5'-dithiobis(2-nitrobenzoic acid)-reactive sulfhydryl groups and a total of 36 moles of sulfhydryl groups. The inhibitory effect of p-chloromercuribenzoate was prevented by preincubating the enzyme with thiamine-PP plus pyruvate. The structure of pyruvate dehydrogenase necessary for formation of the complex is also reported.

  19. Spinal NMDA NR1 Subunit Expression Following Transient TNBS Colitis

    PubMed Central

    Zhou, QiQi; Price, Donald D.; Caudle, Robert M.; Verne, G. Nicholas

    2009-01-01

    Background: N-methyl-D-aspartic acid (NMDA) receptors play an important role in the development of hypersensitivity to visceral and somatic stimuli following inflammation or tissue injury. Our objective was to investigate the role of NMDA NR1 receptors in the spinal cord (T10-L1; L4-S1) of a subset of rats that remain hypersensitive following histological resolution of TNBS-induced colitis compared to saline treated rats and rats that had recovered both behaviorally and histologically. We hypothesized that NMDA NR1 subunit expression mediates hypersensitivity following transient TNBS colitis. Methods: Male Sprague-Dawley rats (150g-250g) received 20mg/rat intracolonic trinitrobenzene sulfonic acid (TNBS) in 50% ethanol or saline. Animals underwent nociceptive visceral/somatic pain testing 16 weeks after resolution of TNBS colitis. Animals were sacrificed and their spinal cord (T10-L1; L4-S1) was retrieved and 2-dimensional polyacrylamide gel electrophoresis and immunohistocytochemistry techniques were used to investigate spinal-NMDA receptor expression. Results: NR1001 was the only NMDA NR1 receptor subunit that was expressed in recovered and control rats, whereas hypersensitive animals expressed NR1011 and NR1111 as well as NR1001 subunits. Immunohistochemistry analysis demonstrated increased expression of NMDA NR1-N1, C1, and C2-plus expression in lamina I & II of the spinal cord (T10-L1; L4-S1) in hypersensitive rats but not in recovered/control rats. Conclusions: Selective increases in the expression of the NMDA NR1 splice variants occur in hypersensitive rats following resolution of TNBS colitis. This suggests that the NMDA NR1 receptor play an important role in the development of neuronal plasticity and central sensitization. The recombination of NR1 splice variants may serve as a key functional protein that maintains hypersensitivity following resolution of TNBS colitis. PMID:19406112

  20. The β Subunit of Voltage-Gated Ca2+ Channels

    PubMed Central

    Buraei, Zafir; Yang, Jian

    2015-01-01

    Calcium regulates a wide spectrum of physiological processes such as heartbeat, muscle contraction, neuronal communication, hormone release, cell division, and gene transcription. Major entry-ways for Ca2+ in excitable cells are high-voltage activated (HVA) Ca2+channels. These are plasma membrane proteins composed of several subunits, including α1, α2δ, β, and γ. Although the principal α1 subunit (Cavα1) contains the channel pore, gating machinery and most drug binding sites, the cytosolic auxiliary β subunit (Cavβ) plays an essential role in regulating the surface expression and gating properties of HVA Ca2+ channels. Cavβ is also crucial for the modulation of HVA Ca2+ channels by G proteins, kinases, and the Ras-related RGK GTPases. New proteins have emerged in recent years that modulate HVA Ca2+ channels by binding to Cavβ. There are also indications that Cavβ may carry out Ca2+ channel-independent functions, including directly regulating gene transcription. All four subtypes of Cavβ, encoded by different genes, have a modular organization, consisting of three variable regions, a conserved guanylate kinase (GK) domain, and a conserved Src-homology 3 (SH3) domain, placing them into the membrane-associated guanylate kinase (MAGUK) protein family. Crystal structures of Cavβs reveal how they interact with Cavα1, open new research avenues, and prompt new inquiries. In this article, we review the structure and various biological functions of Cavβ, with both a historical perspective as well as an emphasis on recent advances. PMID:20959621

  1. Crystal Structure of the Cytoplasmic N-Terminal Domain of Subunit I, a Homolog of Subunit a, of V-ATPase

    SciTech Connect

    Srinivasan, Sankaranarayanan; Vyas, Nand K.; Baker, Matthew L.; Quiocho, Florante A.

    2012-02-27

    Subunit 'a' is associated with the membrane-bound (VO) complex of eukaryotic vacuolar H{sup +}-ATPase acidification machinery. It has also been shown recently to be involved in diverse membrane fusion/secretory functions independent of acidification. Here, we report the crystal structure of the N-terminal cytosolic domain from the Meiothermus ruber subunit 'I' homolog of subunit a. The structure is composed of a curved long central {alpha}-helix bundle capped on both ends by two lobes with similar {alpha}/{beta} architecture. Based on the structure, a reasonable model of its eukaryotic subunit a counterpart was obtained. The crystal structure and model fit well into reconstructions from electron microscopy of prokaryotic and eukaryotic vacuolar H{sup +}-ATPases, respectively, clarifying their orientations and interactions and revealing features that could enable subunit a to play a role in membrane fusion/secretion.

  2. Database on the structure of large ribosomal subunit RNA.

    PubMed Central

    De Rijk, P; Van de Peer, Y; De Wachter, R

    1997-01-01

    The latest release of the large ribosomal subunit RNA database contains 429 sequences. All these sequences are aligned, and incorporate secondary structure information. The rRNA WWW Server at URL http://rrna.uia.ac.be/ provides researchers with an easily accessible resource to obtain the data in this database in a number of computer-readable formats. A new query interface has been added to the server. If necessary, the data can also be obtained by anonymous ftp from the same site. PMID:9016517

  3. Database on the structure of small ribosomal subunit RNA.

    PubMed Central

    Van de Peer, Y; Nicolaï, S; De Rijk, P; De Wachter, R

    1996-01-01

    The Antwerp database on small ribosomal subunit RNA offers over 4300 nucleotide sequences (August 1995). All these sequences are stored in the form of an alignment based on the adopted secondary structure model, which in turn is corroborated by the observation of compensating substitutions in the alignment. Besides the primary and secondary structure information, literature references, accession numbers and detailed taxonomic information are also compiled. The complete database is made available to the scientific community through anonymous ftp and World Wide Web(WWW). PMID:8594609

  4. Database on the structure of small ribosomal subunit RNA.

    PubMed Central

    Van de Peer, Y; Van den Broeck, I; De Rijk, P; De Wachter, R

    1994-01-01

    The database on small ribosomal subunit RNA structure contains (June 1994) 2824 nucleotide sequences. All these sequences are stored in the form of an alignment based on the adopted secondary structure model, which in turn is corroborated by the observation of compensating substitutions in the alignment. The complete database is made available to the scientific community through anonymous ftp on our server in Antwerp. A special effort was made to improve electronic retrieval and a program is supplied that allows to create different file formats. The database can also be obtained from the EMBL nucleotide sequence library. PMID:7524022

  5. Database on the structure of small ribosomal subunit RNA.

    PubMed Central

    Van de Peer, Y; Jansen, J; De Rijk, P; De Wachter, R

    1997-01-01

    The Antwerp database on small ribosomal subunit RNA now offers more than 6000 nucleotide sequences (August 1996). All these sequences are stored in the form of an alignment based on the adopted secondary structure model, which is corroborated by the observation of compensating substitutions in the alignment. Besides the primary and secondary structure information, literature references, accession numbers and detailed taxonomic information are also compiled. For ease of use, the complete database is made available to the scientific community via World Wide Web at URL http://rrna.uia.ac.be/ssu/ . PMID:9016516

  6. Finding and using local symmetry in identifying lower domain movements in hexon subunits of the herpes simplex virus type 1 B capsid.

    PubMed

    He, J; Schmid, M F; Zhou, Z H; Rixon, F; Chiu, W

    2001-06-15

    A characteristic of virus assembly is the use of symmetry to construct a complex capsid from a limited number of different proteins. Many spherical viruses display not only icosahedral symmetry, but also local symmetries, which further increase the redundancy of their structural proteins. We have developed a computational procedure for evaluating the quality of these local symmetries that allows us to probe the extent of local structural variations among subunits. This type of analysis can also provide orientation parameters for carrying out non-icosahedral averaging of quasi-equivalent subunits during three-dimensional structural determination. We have used this procedure to analyze the three types of hexon (P, E and C) in the 8.5 A resolution map of the herpes simplex virus type 1 (HSV-1) B capsid, determined by electron cryomicroscopy. The comparison of the three hexons showed that they have good overall 6-fold symmetry and are almost identical throughout most of their lengths. The largest difference among the three lies near the inner surface in a region of about 34 A in thickness. In this region, the P hexon displays slightly lower 6-fold symmetry than the C and E hexons. More detailed analysis showed that parts of two of the P hexon subunits are displaced counterclockwise with respect to their expected 6-fold positions. The most highly displaced subunit interacts with a subunit from an adjacent P hexon (P'). Using the local 6-fold symmetry axis of the P hexon as a rotation axis, we examined the geometrical relationships among the local symmetry axes of the surrounding capsomeres. Deviations from exact symmetry are also found among these local symmetry axes. The relevance of these findings to the process of capsid assembly is considered.

  7. Post-translational transformation of methionine to aspartate is catalyzed by heme iron and driven by peroxide: a novel subunit-specific mechanism in hemoglobin.

    PubMed

    Strader, Michael Brad; Hicks, Wayne A; Kassa, Tigist; Singleton, Eileen; Soman, Jayashree; Olson, John S; Weiss, Mitchell J; Mollan, Todd L; Wilson, Michael T; Alayash, Abdu I

    2014-08-08

    A pathogenic V67M mutation occurs at the E11 helical position within the heme pockets of variant human fetal and adult hemoglobins (Hb). Subsequent post-translational modification of Met to Asp was reported in γ subunits of human fetal Hb Toms River (γ67(E11)Val → Met) and β subunits of adult Hb (HbA) Bristol-Alesha (β67(E11)Val → Met) that were associated with hemolytic anemia. Using kinetic, proteomic, and crystal structural analysis, we were able to show that the Met → Asp transformation involves heme cycling through its oxoferryl state in the recombinant versions of both proteins. The conversion to Met and Asp enhanced the spontaneous autoxidation of the mutants relative to wild-type HbA and human fetal Hb, and the levels of Asp were elevated with increasing levels of hydrogen peroxide (H2O2). Using H2(18)O2, we verified incorporation of (18)O into the Asp carboxyl side chain confirming the role of H2O2 in the oxidation of the Met side chain. Under similar experimental conditions, there was no conversion to Asp at the αMet(E11) position in the corresponding HbA Evans (α62(E11)Val → Met). The crystal structures of the three recombinant Met(E11) mutants revealed similar thioether side chain orientations. However, as in the solution experiments, autoxidation of the Hb mutant crystals leads to electron density maps indicative of Asp(E11) formation in β subunits but not in α subunits. This novel post-translational modification highlights the nonequivalence of human Hb α, β, and γ subunits with respect to redox reactivity and may have direct implications to α/β hemoglobinopathies and design of oxidatively stable Hb-based oxygen therapeutics.

  8. Determination of kainate receptor subunit ratios in mouse brain using novel chimeric protein standards.

    PubMed

    Watanabe-Iida, Izumi; Konno, Kohtarou; Akashi, Kaori; Abe, Manabu; Natsume, Rie; Watanabe, Masahiko; Sakimura, Kenji

    2016-01-01

    Kainate-type glutamate receptors (KARs) are tetrameric channels assembled from GluK1-5. GluK1-3 are low-affinity subunits that form homomeric and heteromeric KARs, while GluK4 and GluK5 are high-affinity subunits that require co-assembly with GluK1-3 for functional expression. Although the subunit composition is thought to be highly heterogeneous in the brain, the distribution of KAR subunits at the protein level and their relative abundance in given regions of the brain remain largely unknown. In the present study, we titrated C-terminal antibodies to each KAR subunit using chimeric GluA2-GluK fusion proteins, and measured their relative abundance in the P2 and post-synaptic density (PSD) fractions of the adult mouse hippocampus and cerebellum. Analytical western blots showed that GluK2 and GluK3 were the major KAR subunits, with additional expression of GluK5 in the hippocampus and cerebellum. In both regions, GluK4 was very low and GluK1 was below the detection threshold. The relative amount of low-affinity subunits (GluK2 plus GluK3) was several times higher than that of high-affinity subunits (GluK4 plus GluK5) in both regions. Of note, the highest ratio of high-affinity subunits to low-affinity subunits was found in the hippocampal PSD fraction (0.32), suggesting that heteromeric receptors consisting of high- and low-affinity subunits highly accumulate at hippocampal synapses. In comparison, this ratio was decreased to 0.15 in the cerebellar PSD fraction, suggesting that KARs consisting of low-affinity subunits are more prevalent in the cerebellum. Therefore, low-affinity KAR subunits are predominant in the brain, with distinct subunit combinations between the hippocampus and cerebellum. Kainate receptors, an unconventional member of the iGluR receptor family, have a tetrameric structure assembled from low-affinity (GluK1-3) and high-affinity (GluK4 and GluK5) subunits. We used a simple but novel procedure to measure the relative abundance of both low- and

  9. Crystal structure and molecular imaging of the Nav channel β3 subunit indicates a trimeric assembly.

    PubMed

    Namadurai, Sivakumar; Balasuriya, Dilshan; Rajappa, Rajit; Wiemhöfer, Martin; Stott, Katherine; Klingauf, Jurgen; Edwardson, J Michael; Chirgadze, Dimitri Y; Jackson, Antony P

    2014-04-11

    The vertebrate sodium (Nav) channel is composed of an ion-conducting α subunit and associated β subunits. Here, we report the crystal structure of the human β3 subunit immunoglobulin (Ig) domain, a functionally important component of Nav channels in neurons and cardiomyocytes. Surprisingly, we found that the β3 subunit Ig domain assembles as a trimer in the crystal asymmetric unit. Analytical ultracentrifugation confirmed the presence of Ig domain monomers, dimers, and trimers in free solution, and atomic force microscopy imaging also detected full-length β3 subunit monomers, dimers, and trimers. Mutation of a cysteine residue critical for maintaining the trimer interface destabilized both dimers and trimers. Using fluorescence photoactivated localization microscopy, we detected full-length β3 subunit trimers on the plasma membrane of transfected HEK293 cells. We further show that β3 subunits can bind to more than one site on the Nav 1.5 α subunit and induce the formation of α subunit oligomers, including trimers. Our results suggest a new and unexpected role for the β3 subunits in Nav channel cross-linking and provide new structural insights into some pathological Nav channel mutations.

  10. Ca(2+) channel inactivation heterogeneity reveals physiological unbinding of auxiliary beta subunits.

    PubMed Central

    Restituito, S; Cens, T; Rousset, M; Charnet, P

    2001-01-01

    Voltage gated Ca(2+) channel (VGCC) auxiliary beta subunits increase membrane expression of the main pore-forming alpha(1) subunits and finely tune channel activation and inactivation properties. In expression studies, co-expression of beta subunits also reduced neuronal Ca(2+) channel regulation by heterotrimeric G protein. Biochemical studies suggest that VGCC beta subunits and G protein betagamma can compete for overlapping interaction sites on VGCC alpha(1) subunits, suggesting a dynamic association of these subunits with alpha(1). In this work we have analyzed the stability of the alpha(1)/beta association under physiological conditions. Regulation of the alpha(1A) Ca(2+) channel inactivation properties by beta(1b) and beta(2a) subunits had two major effects: a shift in voltage-dependent inactivation (E(in)), and an increase of the non-inactivating current (R(in)). Unexpectedly, large variations in magnitude of the effects were recorded on E(in), when beta(1b) was expressed, and R(in), when beta(2a) was expressed. These variations were not proportional to the current amplitude, and occurred at similar levels of beta subunit expression. beta(2a)-induced variations of R(in) were, however, inversely proportional to the magnitude of G protein block. These data underline the two different mechanisms used by beta(1b) and beta(2a) to regulate channel inactivation, and suggest that the VGCC beta subunit can unbind the alpha1 subunit in physiological situations. PMID:11423397

  11. Forskolin Regulates L-Type Calcium Channel through Interaction between Actinin 4 and β3 Subunit in Osteoblasts.

    PubMed

    Zhang, Xuemei; Li, Fangping; Guo, Lin; Hei, Hongya; Tian, Lulu; Peng, Wen; Cai, Hui

    2015-01-01

    Voltage-dependent L-type calcium channels that permit cellular calcium influx are essential in calcium-mediated modulation of cellular signaling. Although the regulation of voltage-dependent L-type calcium channels is linked to many factors including cAMP-dependent protein kinase A (PKA) activity and actin cytoskeleton, little is known about the detailed mechanisms underlying the regulation in osteoblasts. Our present study investigated the modulation of L-type calcium channel activities through the effects of forskolin on actin reorganization and on its functional interaction with actin binding protein actinin 4. The results showed that forskolin did not significantly affect the trafficking of pore forming α1c subunit and its interaction with actin binding protein actinin 4, whereas it significantly increased the expression of β3 subunit and its interaction with actinin 4 in osteoblast cells as assessed by co-immunoprecipitation, pull-down assay, and immunostaining. Further mapping showed that the ABD and EF domains of actinin 4 were interaction sites. This interaction is independent of PKA phosphorylation. Knockdown of actinin 4 significantly decreased the activities of L-type calcium channels. Our study revealed a new aspect of the mechanisms by which the forskolin activation of adenylyl cyclase - cAMP cascade regulates the L-type calcium channel in osteoblast cells, besides the PKA mediated phosphorylation of the channel subunits. These data provide insight into the important role of interconnection among adenylyl cyclase, cAMP, PKA, the actin cytoskeleton, and the channel proteins in the regulation of voltage-dependent L-type calcium channels in osteoblast cells.

  12. Mapping the Heart

    ERIC Educational Resources Information Center

    Hulse, Grace

    2012-01-01

    In this article, the author describes how her fourth graders made ceramic heart maps. The impetus for this project came from reading "My Map Book" by Sara Fanelli. This book is a collection of quirky, hand-drawn and collaged maps that diagram a child's world. There are maps of her stomach, her day, her family, and her heart, among others. The…

  13. Domain structure of HrpE, the Hrp pilus subunit of Xanthomonas campestris pv. vesicatoria.

    PubMed

    Weber, Ernst; Koebnik, Ralf

    2005-09-01

    The plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria possesses a type III secretion (TTS) system necessary for pathogenicity in susceptible hosts and induction of the hypersensitive response in resistant plants. This specialized protein transport system is encoded by a 23-kb hrp (hypersensitive response and pathogenicity) gene cluster. X. campestris pv. vesicatoria produces filamentous structures, Hrp pili, at the cell surface under hrp-inducing conditions. The Hrp pilus acts as a cell surface appendage of the TTS system and serves as a conduit for the transfer of bacterial effector proteins into the plant cell cytosol. The major pilus component, the HrpE pilin, is unique to xanthomonads and is encoded within the hrp gene cluster. In this study, functional domains of HrpE were mapped by linker-scanning mutagenesis and by reporter protein fusions to an N-terminally truncated avirulence protein (AvrBs3Delta2). Thirteen five-amino-acid peptide insertion mutants were obtained and could be grouped into six phenotypic classes. Three permissive mutations were mapped in the N-terminal half of HrpE, which is weakly conserved within the HrpE protein family. Four dominant-negative peptide insertions in the strongly conserved C-terminal region suggest that this domain is critical for oligomerization of the pilus subunits. Reporter protein fusions revealed that the N-terminal 17 amino acid residues act as an efficient TTS signal. From these results, we postulate a three-domain structure of HrpE with an N-terminal secretion signal, a surface-exposed variable region of the N-terminal half, and a C-terminal polymerization domain. Comparisons with a mutant study of HrpA, the Hrp pilin from Pseudomonas syringae pv. tomato DC3000, and hydrophobicity plot analyses of several nonhomologous Hrp pilins suggest a common architecture of Hrp pilins of different plant-pathogenic bacteria.

  14. National Atlas maps

    USGS Publications Warehouse

    ,

    1991-01-01

    The National Atlas of the United States of America was published by the U.S. Geological Survey in 1970. Its 765 maps and charts are on 335 14- by 19-inch pages. Many of the maps span facing pages. It's worth a quick trip to the library just to leaf through all 335 pages of this book. Rapid scanning of its thematic maps yields rich insights to the geography of issues of continuing national interest. On most maps, the geographic patterns are still valid, though the data are not current. The atlas is out of print, but many of its maps can be purchased separately. Maps that span facing pages in the atlas are printed on one sheet. The maps dated after 1970 are either revisions of original atlas maps, or new maps published in atlas format. The titles of the separate maps are listed here.

  15. Smallpox subunit vaccine produced in planta confers protection in mice

    PubMed Central

    Golovkin, Maxim; Spitsin, Sergei; Andrianov, Vyacheslav; Smirnov, Yuriy; Xiao, Yuhong; Pogrebnyak, Natalia; Markley, Karen; Brodzik, Robert; Gleba, Yuri; Isaacs, Stuart N.; Koprowski, Hilary

    2007-01-01

    We report here the in planta production of the recombinant vaccinia virus B5 antigenic domain (pB5), an attractive component of a subunit vaccine against smallpox. The antigenic domain was expressed by using efficient transient and constitutive plant expression systems and tested by various immunization routes in two animal models. Whereas oral administration in mice or the minipig with collard-derived insoluble pB5 did not generate an anti-B5 immune response, intranasal administration of soluble pB5 led to a rise of B5-specific immunoglobulins, and parenteral immunization led to a strong anti-B5 immune response in both mice and the minipig. Mice immunized i.m. with pB5 generated an antibody response that reduced virus spread in vitro and conferred protection from challenge with a lethal dose of vaccinia virus. These results indicate the feasibility of producing safe and inexpensive subunit vaccines by using plant production systems. PMID:17428917

  16. Binding of ATP by pertussis toxin and isolated toxin subunits

    SciTech Connect

    Hausman, S.Z.; Manclark, C.R.; Burns, D.L. )

    1990-07-03

    The binding of ATP to pertussis toxin and its components, the A subunit and B oligomer, was investigated. Whereas, radiolabeled ATP bound to the B oligomer and pertussis toxin, no binding to the A subunit was observed. The binding of ({sup 3}H)ATP to pertussis toxin and the B oligomer was inhibited by nucleotides. The relative effectiveness of the nucleotides was shown to be ATP > GTP > CTP > TTP for pertussis toxin and ATP > GTP > TTP > CTP for the B oligomer. Phosphate ions inhibited the binding of ({sup 3}H)ATP to pertussis toxin in a competitive manner; however, the presence of phosphate ions was essential for binding of ATP to the B oligomer. The toxin substrate, NAD, did not affect the binding of ({sup 3}H)ATP to pertussis toxin, although the glycoprotein fetuin significantly decreased binding. These results suggest that the binding site for ATP is located on the B oligomer and is distinct from the enzymatically active site but may be located near the eukaryotic receptor binding site.

  17. Unraveling Subunit Cooperativity in Homotetrameric HCN2 Channels

    PubMed Central

    Benndorf, Klaus; Thon, Susanne; Schulz, Eckhard

    2012-01-01

    In a multimeric receptor protein, the binding of a ligand can modulate the binding of a succeeding ligand. This phenomenon, called cooperativity, is caused by the interaction of the receptor subunits. By using a complex Markovian model and a set of parameters determined previously, we analyzed how the successive binding of four ligands leads to a complex cooperative interaction of the subunits in homotetrameric HCN2 pacemaker channels. The individual steps in the model were characterized by Gibbs free energies for the equilibria and activation energies, specifying the affinity of the binding sites and the transition rates, respectively. Moreover, cooperative free energies were calculated for each binding step in both the closed and the open channel. We show that the cooperativity sequence positive-negative-positive determined for the binding affinity is generated by the combined effect of very different cooperativity sequences determined for the binding and unbinding rates, which are negative-negative-positive and no-negative-no, respectively. It is concluded that in the ligand-induced activation of HCN2 channels, the sequence of cooperativity based on the binding affinity is caused by two even qualitatively different sequences of cooperativity that are based on the rates of ligand binding and unbinding. PMID:23199914

  18. A small subunit processome protein promotes cancer by altering translation.

    PubMed

    Yang, H W; Kim, T-M; Song, S S; Menon, L; Jiang, X; Huang, W; Black, P M; Park, P J; Carroll, R S; Johnson, M D

    2015-08-20

    Dysregulation of ribosome biogenesis or translation can promote cancer, but the underlying mechanisms remain unclear. UTP18 is a component of the small subunit processome, a nucleolar multi-protein complex whose only known function is to cleave pre-ribosomal RNA to yield the 18S ribosomal RNA component of 40S ribosomal subunits. Here, we show that UTP18 also alters translation to promote stress resistance and growth, and that UTP18 is frequently gained and overexpressed in cancer. We observed that UTP18 localizes to the cytoplasm in a subset of cells, and that serum withdrawal increases cytoplasmic UTP18 localization. Cytoplasmic UTP18 associates with the translation complex and Hsp90 to upregulate the translation of IRES-containing transcripts such as HIF1a, Myc and VEGF, thereby inducing stress resistance. Hsp90 inhibition decreases cytoplasmic UTP18 and UTP18-induced increases in translation. Importantly, elevated UTP18 expression correlates with increased aggressiveness and decreased survival in numerous cancers. Enforced UTP18 overexpression promotes transformation and tumorigenesis, whereas UTP18 knockdown inhibits these processes. This stress adaptation mechanism is thus co-opted for growth by cancers, and its inhibition may represent a promising new therapeutic target.

  19. An assembly landscape for the 30S ribosomal subunit

    PubMed Central

    Talkington, Megan W. T.; Siuzdak, Gary

    2005-01-01

    Self-assembling macromolecular machines drive fundamental cellular processes, including transcription, mRNA processing, translation, DNA replication, and cellular transport. The ribosome, which carries out protein synthesis, is one such machine, and the 30S subunit of the bacterial ribosome is the preeminent model system for biophysical analysis of large RNA-protein complexes. Our understanding of 30S assembly is incomplete, due to the challenges of monitoring the association of many components simultaneously. We have developed a new method involving pulse-chase monitored by quantitative mass spectrometry (PC/QMS) to follow the assembly of the 20 ribosomal proteins with 16S rRNA during formation of the functional particle. These data represent the first detailed and quantitative kinetic characterization of the assembly of a large multicomponent macromolecular complex. By measuring the protein binding rates at a range of temperatures, we have found that local transformations throughout the assembling subunit have similar but distinct activation energies. This observation shows that the prevailing view of 30S assembly as a pathway proceeding through a global rate-limiting conformational change must give way to a view in which the assembly of the complex traverses a landscape dotted with a variety of local conformational transitions. PMID:16319883

  20. Thermostable cross-protective subunit vaccine against Brucella species.

    PubMed

    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.

  1. Oligosaccharyltransferase Subunits Bind Polypeptide Substrate to Locally Enhance N-glycosylation*

    PubMed Central

    Jamaluddin, M. Fairuz B.; Bailey, Ulla-Maja; Schulz, Benjamin L.

    2014-01-01

    Oligosaccharyltransferase is a multiprotein complex that catalyzes asparagine-linked glycosylation of diverse proteins. Using yeast genetics and glycoproteomics, we found that transient interactions between nascent polypeptide and Ost3p/Ost6p, homologous subunits of oligosaccharyltransferase, were able to modulate glycosylation efficiency in a site-specific manner in vivo. These interactions were driven by hydrophobic and electrostatic complementarity between amino acids in the peptide-binding groove of Ost3p/Ost6p and the sequestered stretch of substrate polypeptide. Based on this dependence, we used in vivo scanning mutagenesis and in vitro biochemistry to map the precise interactions that affect site-specific glycosylation efficiency. We conclude that transient binding of substrate polypeptide by Ost3p/Ost6p increases glycosylation efficiency at asparagines proximal and C-terminal to sequestered sequences. We detail a novel mode of interaction between translocating nascent polypeptide and oligosaccharyltransferase in which binding to Ost3p/Ost6p segregates a short flexible loop of glycosylation-competent polypeptide substrate that is delivered to the oligosaccharyltransferase active site for efficient modification. PMID:25118247

  2. Soybean ribulose bisphosphate carboxylase small subunit: Mechanisms and determinants of RNA turnover. Annual progress report

    SciTech Connect

    Meagher, R.B.

    1993-12-31

    An in vitro degradation system has been developed from petunia and soybean polysomes in order to investigate the mechanisms and determinants controlling RNA turnover in higher plants. This system faithfully degrades soybean ribulose-1,5-bisphosphate carboxylase small subunit (rbcS) mRNA into the same products observed in total RNA preparations. In previous years it was shown that the most stable products represent a nested constellation of fragments, which are shortened from their 3{prime} ends, and have intact 5{prime} ends. Exogenous rbcS RNA tagged with novel 5{prime} sequence 15 or 56 bp long were synthesized in vitro as Sp6 and T7 runoff transcripts, respectively. When added to the system they were degraded faithfully into constellation of products which were 15 or 56 bp longer than the endogenous products, respectively. Detailed kinetics on the appearance of these exogenous products confirmed degradation proceeds in an overall 3{prime} to 5{prime} direction but suggested that there are multiple pathways through which the RNA may be degraded. To further demonstrate a precursor product relationships, in vitro synthesized transcripts truncated at their 3{prime} ends were shown to degrade into the expected smaller fragments previously mapped in the 5{prime} portion of the rbcS RNA.

  3. Evaluation of a Subunit Vaccine to Infectious Hematopoietic Necrosis (IHN) Virus, 1984 FY Annual Report.

    SciTech Connect

    Leong, JoAnn Ching

    1985-07-01

    A prototype subunit vaccine to IHN virus is being developed by recombinant DNA techniques. The techniques involve the isolation and characterization of the glycoprotein gene, which encodes the viral protein responsible for inducing a protective immune response in fish. The viral glycoprotein gene has been cloned and a restriction map of the cloned gene has been prepared. Preliminary DNA sequence analysis of the cloned gene has been initiated so that manipulation of the gene for maximum expression in appropriate plasmid vectors is possible. A recombinant plasmid containing the viral gene inserted in the proper orientation adjacent to a very strong lambda promoter and ribosome binding site has been constructed. Evaluation of this recombinant plasmid for gene expression is being conducted. Immunization trials with purified viral glycoprotein indicate that fish are protected against lethal doses of IHNV after immersion and intraperitoneal methods of immunization. In addition, cross protection immunization trials indicate that Type 2 and Type 1 IHN virus produce glycoproteins that are cross-protective.

  4. Google Maps: You Are Here

    ERIC Educational Resources Information Center

    Jacobsen, Mikael

    2008-01-01

    Librarians use online mapping services such as Google Maps, MapQuest, Yahoo Maps, and others to check traffic conditions, find local businesses, and provide directions. However, few libraries are using one of Google Maps most outstanding applications, My Maps, for the creation of enhanced and interactive multimedia maps. My Maps is a simple and…

  5. Signal transduction by the high-affinity GM-CSF receptor: two distinct cytoplasmic regions of the common beta subunit responsible for different signaling.

    PubMed Central

    Sato, N; Sakamaki, K; Terada, N; Arai, K; Miyajima, A

    1993-01-01

    The high-affinity receptors for granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 3 (IL-3) and IL-5 consist of two subunits, alpha and beta. The alpha subunits are specific to each cytokine and the same beta subunit (beta c) is shared by these three receptors. Although none of these receptor subunits has intrinsic kinase activity, these cytokines induce protein tyrosine phosphorylation, activation of Ras, Raf-1 and MAP kinase, and transcriptional activation of nuclear proto-oncogenes such as c-myc, c-fos and c-jun. In this paper, we describe a detailed analysis of the signaling potential of the beta c subunit by using a series of cytoplasmic deletion mutants. The human beta c consists of 881 amino acid residues. A C-terminal deletion mutant of beta c at amino acid 763 (beta 763) induced phosphorylation of Shc and activation of Ras, Raf-1, MAP kinase and p70 S6 kinase, whereas a deletion at amino acid 626 (beta 626) induced none of these effects. The beta 763 mutant, as well as the full-length beta c, induced transcription of c-myc, c-fos and c-jun. Deletions at amino acid 517 (beta 517) and 626 (beta 626) induced c-myc and pim-1, but no induction of c-fos and c-jun was observed. GM-CSF increased phosphatidylinositol 3 kinase (PI3-K) activity in anti-phosphotyrosine immunoprecipitates from cells expressing beta 763 as well as beta c, whereas it was only marginally increased from cells expressing beta 517 or beta 626. Thus, there are at least two distinct regions within the cytoplasmic domain of beta c that are responsible for different signals, i.e. a membrane proximal region of approximately 60 amino acid residues upstream of Glu517 is essential for induction of c-myc and pim-1, and a distal region of approximately 140 amino acid residues (between Leu626 and Ser763) is required for activation of Ras, Raf-1, MAP kinase and p70 S6 kinase, as well as induction of c-fos and c-jun. Images PMID:8223433

  6. Troponin T3 regulates nuclear localization of the calcium channel Cavβ1a subunit in skeletal muscle

    PubMed Central

    Zhang, Tan; Taylor, Jackson; Jiang, Yang; Pereyra, Andrea S.; Messi, Maria Laura; Wang, Zhong-Min; Hereñú, Claudia; Delbono, Osvaldo

    2015-01-01

    The voltage-gated calcium channel (Cav) β1a subunit (Cavβ1a) plays an important role in excitation-contraction coupling (ECC), a process in the myoplasm that leads to muscle-force generation. Recently, we discovered that the Cavβ1a subunit travels to the nucleus of skeletal muscle cells where it helps to regulate gene transcription. To determine how it travels to the nucleus, we performed a yeast two-hybrid screening of the mouse fast skeletal muscle cDNA library and identified an interaction with troponin T3 (TnT3), which we subsequently confirmed by co-immunoprecipitation and co-localization assays in mouse skeletal muscle in vivo and in cultured C2C12 muscle cells. Interacting domains were mapped to the leucine zipper domain in TnT3 COOH-terminus (160-244 aa) and Cavβ1a NH2-terminus (1-99 aa), respectively. The double fluorescence assay in C2C12 cells co-expressing TnT3/DsRed and Cavβ1a/YFP shows that TnT3 facilitates Cavβ1a nuclear recruitment, suggesting that the two proteins play a heretofore unknown role during early muscle differentiation in addition to their classical role in ECC regulation. PMID:25981458

  7. Troponin T3 regulates nuclear localization of the calcium channel Cavβ1a subunit in skeletal muscle.

    PubMed

    Zhang, Tan; Taylor, Jackson; Jiang, Yang; Pereyra, Andrea S; Messi, Maria Laura; Wang, Zhong-Min; Hereñú, Claudia; Delbono, Osvaldo

    2015-08-15

    The voltage-gated calcium channel (Cav) β1a subunit (Cavβ1a) plays an important role in excitation-contraction coupling (ECC), a process in the myoplasm that leads to muscle-force generation. Recently, we discovered that the Cavβ1a subunit travels to the nucleus of skeletal muscle cells where it helps to regulate gene transcription. To determine how it travels to the nucleus, we performed a yeast two-hybrid screening of the mouse fast skeletal muscle cDNA library and identified an interaction with troponin T3 (TnT3), which we subsequently confirmed by co-immunoprecipitation and co-localization assays in mouse skeletal muscle in vivo and in cultured C2C12 muscle cells. Interacting domains were mapped to the leucine zipper domain in TnT3 COOH-terminus (160-244 aa) and Cavβ1a NH2-terminus (1-99 aa), respectively. The double fluorescence assay in C2C12 cells co-expressing TnT3/DsRed and Cavβ1a/YFP shows that TnT3 facilitates Cavβ1a nuclear recruitment, suggesting that the two proteins play a heretofore unknown role during early muscle differentiation in addition to their classical role in ECC regulation.

  8. Proteolytic cleavage of the voltage-gated Ca2+ channel α2δ subunit: structural and functional features

    PubMed Central

    Andrade, Arturo; Sandoval, Alejandro; Oviedo, Norma; De Waard, Michel; Elias, David; Felix, Ricardo

    2007-01-01

    By mediating depolarization-induced Ca2+ influx high voltage-activated (HVA) Ca2+ channels control a variety of cellular events. These heteromultimeric proteins are composed of an ion-conducting (α1) and three auxiliary (α2δ, β and γ) subunits. The α2δ subunit enhances the trafficking of the channel complex to the cell surface and increases channel open probability. To exert these effects, α2δ must undergo important post-translational modifications including a proteolytic cleavage that separates the extracellular α2 from its transmembrane δ domain. After this proteolysis both domains remain linked by disulfide bonds. In spite of its central role in determining the final conformation of the fully mature α2δ almost nothing is known about the physiological implications of this structural modification. In the current report, by using site-directed mutagenesis, the proteolytic site of α2δ was mapped to amino acid residues Arg-941 and Val-946. Substitution of these residues renders the protein insensitive to proteolytic cleavage as evidenced by the lack of molecular weight shift upon treatment with a disulfide reducing agent. Interestingly, these mutations significantly decreased whole-cell patch clamp currents without affecting the voltage-dependence or kinetics of the channels, suggesting a reduction in the number of channels targeted to the plasma membrane. PMID:17408426

  9. An S-phase specific release from a transcriptional block regulates the expression of mouse ribonucleotide reductase R2 subunit.

    PubMed Central

    Björklund, S; Skogman, E; Thelander, L

    1992-01-01

    Ribonucleotide reductase (RR) activity in mammalian cells is closely linked to DNA synthesis. The RR enzyme is composed of two non-identical subunits, proteins R1 and R2. Both proteins are required for holoenzyme activity, which is regulated by S-phase specific de novo synthesis and breakdown of the R2 subunit. In quiescent cells stimulated to proliferate and in elutriated cell populations enriched in the various cell cycle phases the R2 protein levels are correlated to R2 mRNA levels that are low in G0/G1-phase cells but increase dramatically at the G1/S border. Using an R2 promoter-luciferase reporter gene construct we demonstrate an unexpected early activation of the R2 promoter as cells pass from quiescence to proliferation. However, due to a transcriptional block, this promoter activation only results in very short R2 transcripts until cells enter the S-phase, when full-length R2 transcripts start to appear. The position for the transcriptional block was localized to a nucleotide sequence approximately 87 bp downstream from the first exon/intron boundary by S1 nuclease mapping of R2 transcripts from modified in vitro nuclear run-on experiments. These results identify blocking of transcription as a mechanism to control cell cycle regulated gene expression. Images PMID:1464320

  10. Membrane trafficking and mitochondrial abnormalities precede subunit c deposition in a cerebellar cell model of juvenile neuronal ceroid lipofuscinosis

    PubMed Central

    Fossale, Elisa; Wolf, Pavlina; Espinola, Janice A; Lubicz-Nawrocka, Tanya; Teed, Allison M; Gao, Hanlin; Rigamonti, Dorotea; Cattaneo, Elena; MacDonald, Marcy E; Cotman, Susan L

    2004-01-01

    Background JNCL is a recessively inherited, childhood-onset neurodegenerative disease most-commonly caused by a ~1 kb CLN3 mutation. The resulting loss of battenin activity leads to deposition of mitochondrial ATP synthase, subunit c and a specific loss of CNS neurons. We previously generated Cln3Δex7/8 knock-in mice, which replicate the common JNCL mutation, express mutant battenin and display JNCL-like pathology. Results To elucidate the consequences of the common JNCL mutation in neuronal cells, we used P4 knock-in mouse cerebella to establish conditionally immortalized CbCln3 wild-type, heterozygous, and homozygous neuronal precursor cell lines, which can be differentiated into MAP-2 and NeuN-positive, neuron-like cells. Homozygous CbCln3Δex7/8 precursor cells express low levels of mutant battenin and, when aged at confluency, accumulate ATPase subunit c. Recessive phenotypes are also observed at sub-confluent growth; cathepsin D transport and processing are altered, although enzyme activity is not significantly affected, lysosomal size and distribution are altered, and endocytosis is reduced. In addition, mitochondria are abnormally elongated, cellular ATP levels are decreased, and survival following oxidative stress is reduced. Conclusions These findings reveal that battenin is required for intracellular membrane trafficking and mitochondrial function. Moreover, these deficiencies are likely to be early events in the JNCL disease process and may particularly impact neuronal survival. PMID:15588329

  11. Isolation and characterization of a novel gene from the DiGeorge chromosomal region that encodes for a mediator subunit.

    PubMed

    Berti, L; Mittler, G; Przemeck, G K; Stelzer, G; Günzler, B; Amati, F; Conti, E; Dallapiccola, B; Hrabé de Angelis, M; Novelli, G; Meisterernst, M

    2001-06-15

    Hemizygous deletions on chromosome 22q11.2 result in developmental disorders referred to as DiGeorge syndrome (DGS)/velocardiofacial syndrome (VCFS). We report the isolation of a novel gene, PCQAP (PC2 glutamine/Q-rich-associated protein), that maps to the DiGeorge typically deleted region and encodes a protein identified as a subunit of the large multiprotein complex PC2. PC2 belongs to the family of the human Mediator complexes, which exhibit coactivator function in RNA polymerase II transcription. Furthermore, we cloned the homologous mouse Pcqap cDNA. There is 83% amino acid identity between the human and the mouse predicted protein sequences, with 96% similarity at the amino- and carboxy-terminal ends. To assess the potential involvement of PCQAP in DGS/VCFS, its developmental expression pattern was analyzed. In situ hybridization of mouse embryos at different developmental stages revealed that Pcqap is ubiquitously expressed. However, higher expression was detected in the frontonasal region, pharyngeal arches, and limb buds. Moreover, analysis of subjects carrying a typical 22q11 deletion revealed that the human PCQAP gene was deleted in all patients. Many of the structures affected in DGS/VCFS evolve from Pcqap-expressing cells. Together with the observed haploinsufficiency of PCQAP in DGS/VCFS patients, this finding is consistent with a possible role for this novel Mediator subunit in the development of some of the structures affected in DGS/VCFS.

  12. Gene structure, chromosomal localization, and expression pattern of Capn12, a new member of the calpain large subunit gene family.

    PubMed

    Dear, T N; Meier, N T; Hunn, M; Boehm, T

    2000-09-01

    We report the identification of mouse Capn12, a new member of the calpain large subunit gene family. It possesses potential protease and calcium-binding domains, features typical of the classical calpains. In situ hybridization and Northern blot analysis demonstrate that during the anagen phase of the hair cycle the cortex of the hair follicle is the major expression site of Capn12. The gene was sequenced in its entirety and consists of 21 exons spanning 13 kb with an exon-intron structure typical of the calpain gene family. The last exon of the mouse Actn4 gene overlaps the 3' end of Capn12 but in the opposite orientation. This overlap between the two genes is conserved in the human genome. Three versions of the Capn12 mRNA transcript were identified. They occur as a result of alternative splicing, and two of these encode a protein lacking the C-terminal calmodulin-like domain. Radiation hybrid mapping localized Capn12 to mouse chromosome 7, closely linked to a marker positioned at 10.4 cM. Refined mapping of Capn5, also previously localized to chromosome 7, indicated that it was not closely linked to Capn12, mapping tightly linked to a marker positioned at 48.5 cM.

  13. Phylogenetic mapping of bacterial morphology

    NASA Technical Reports Server (NTRS)

    Siefert, J. L.; Fox, G. E.

    1998-01-01

    The availability of a meaningful molecular phylogeny for bacteria provides a context for examining the historical significance of various developments in bacterial evolution. Herein, the classical morphological descriptions of selected members of the domain Bacteria are mapped upon the genealogical ancestry deduced from comparison of small-subunit rRNA sequences. For the species examined in this study, a distinct pattern emerges which indicates that the coccus shape has arisen and accumulated independently multiple times in separate lineages and typically survived as a persistent end-state morphology. At least two other morphologies persist but have evolved only once. This study demonstrates that although bacterial morphology is not useful in defining bacterial phylogeny, it is remarkably consistent with that phylogeny once it is known. An examination of the experimental evidence available for morphogenesis as well as microbial fossil evidence corroborates these findings. It is proposed that the accumulation of persistent morphologies is a result of the biophysical properties of peptidoglycan and their genetic control, and that an evolved body-plan strategy based on peptidoglycan may have been a fate-sealing step in the evolution of Bacteria. More generally, this study illustrates that significant evolutionary insights can be obtained by examining biological and biochemical data in the context of a reliable phylogenetic structure.

  14. Map reading tools for map libraries.

    USGS Publications Warehouse

    Greenberg, G.L.

    1982-01-01

    Engineers, navigators and military strategists employ a broad array of mechanical devices to facilitate map use. A larger number of map users such as educators, students, tourists, journalists, historians, politicians, economists and librarians are unaware of the available variety of tools which can be used with maps to increase the speed and efficiency of their application and interpretation. This paper identifies map reading tools such as coordinate readers, protractors, dividers, planimeters, and symbol-templets according to a functional classification. Particularly, arrays of tools are suggested for use in determining position, direction, distance, area and form (perimeter-shape-pattern-relief). -from Author

  15. Palmitoylation of the β4-Subunit Regulates Surface Expression of Large Conductance Calcium-activated Potassium Channel Splice Variants*

    PubMed Central

    Chen, Lie; Bi, Danlei; Tian, Lijun; McClafferty, Heather; Steeb, Franziska; Ruth, Peter; Knaus, Hans Guenther; Shipston, Michael J.

    2013-01-01

    Regulatory β-subunits of large conductance calcium- and voltage-activated potassium (BK) channels play an important role in generating functional diversity and control of cell surface expression of the pore forming α-subunits. However, in contrast to α-subunits, the role of reversible post-translational modification of intracellular residues on β-subunit function is largely unknown. Here we demonstrate that the human β4-subunit is S-acylated (palmitoylated) on a juxtamembrane cysteine residue (Cys-193) in the intracellular C terminus of the regulatory β-subunit. β4-Subunit palmitoylation is important for cell surface expression and endoplasmic reticulum (ER) exit of the β4-subunit alone. Importantly, palmitoylated β4-subunits promote the ER exit and surface expression of the pore-forming α-subunit, whereas β4-subunits that cannot be palmitoylated do not increase ER exit or surface expression of α-subunits. Strikingly, however, this palmitoylation- and β4-dependent enhancement of α-subunit surface expression was only observed in α-subunits that contain a putative trafficking motif (… REVEDEC) at the very C terminus of the α-subunit. Engineering this trafficking motif to other C-terminal α-subunit splice variants results in α-subunits with reduced surface expression that can be rescued by palmitoylated, but not depalmitoylated, β4-subunits. Our data reveal a novel mechanism by which palmitoylated β4-subunit controls surface expression of BK channels through masking of a trafficking motif in the C terminus of the α-subunit. As palmitoylation is dynamic, this mechanism would allow precise control of specific splice variants to the cell surface. Our data provide new insights into how complex interplay between the repertoire of post-transcriptional and post-translational mechanisms controls cell surface expression of BK channels. PMID:23504458

  16. [Chromatographic and spectroscopic characterization of phycocyanin and its subunits purified from Anabaena variabilis CCC421].

    PubMed

    Chakdar, N; Sakha, S; Pabbi, S

    2014-01-01

    Phycocyanin, a high value pigment was purified from diazotrophic cyanobacteria Anabaena variabilis CCC421 using a strategy involving ammonium sulfate precipitation, dialysis and anion exchange chromatography using DEAE-cellulose column. 36% phycocyanin with a purity of 2.75 was recovered finally after anion exchange chromatography. Purified phycocyanin was found to contain 2 subunits of 17 and 18 kDa which were identified as a-and (3 subunits by SDS-PAGE and MALDI-TOE HPLC method using a C5 column coupled with fluorescence or photodiode-based detection was also developed to separate and detect the A. variabilis CCC421 phycocyanin subunits. The fluorescence method was more sensitive than photodiode one. The purified phycocyanin from A. variabilis CCC421 as well as its subunits was characterized with respect to absorption and IR spectra. Spectral characterization of the subunits revealed that alpha and beta subunits contained one and two phycocyanobilin groups as chromophores, respectively.

  17. Redox-sensitive extracellular gates formed by auxiliary beta subunits of calcium-activated potassium channels.

    PubMed

    Zeng, Xu-Hui; Xia, Xiao-Ming; Lingle, Christopher J

    2003-06-01

    An important step to understanding ion channels is identifying the structural components that act as the gates to ion movement. Here we describe a new channel gating mechanism, produced by the beta3 auxiliary subunits of Ca2+-activated, large-conductance BK-type K+ channels when expressed with their pore-forming alpha subunits. BK beta subunits have a cysteine-rich extracellular segment connecting two transmembrane segments, with small cytosolic N and C termini. The extracellular segments of the beta3 subunits form gates to block ion permeation, providing a mechanism by which current can be rapidly diminished upon cellular repolarization. Furthermore, this gating mechanism is abolished by reduction of extracellular disulfide linkages, suggesting that endogenous mechanisms may regulate this gating behavior. The results indicate that auxiliary beta subunits of BK channels reside sufficiently close to the ion permeation pathway defined by the alpha subunits to influence or block access of small molecules to the permeation pathway.

  18. The mongoose acetylcholine receptor alpha-subunit: analysis of glycosylation and alpha-bungarotoxin binding.

    PubMed

    Asher, O; Jensen, B S; Lupu-Meiri, M; Oron, Y; Fuchs, S

    1998-04-17

    The mongoose AChR alpha-subunit has been cloned and shown to be highly homologous to other AChR alpha-subunits, with only six differences in amino acid residues at positions that are conserved in animal species that bind alpha-bungarotoxin (alpha-BTX). Four of these six substitutions cluster in the ligand binding site, and one of them, Asn-187, forms a consensus N-glycosylation site. The mongoose glycosylated alpha-subunit has a higher apparent molecular mass than that of the rat glycosylated alpha-subunit, probably resulting from the additional glycosylation at Asn-187 of the mongoose subunit. The in vitro translated mongoose alpha-subunit, in a glycosylated or non-glycosylated form, does not bind alpha-BTX, indicating that lack of alpha-BTX binding can be achieved also in the absence of glycosylation.

  19. Removal of MAP4 from microtubules in vivo produces no observable phenotype at the cellular level

    PubMed Central

    1996-01-01

    Microtubule-associated protein 4 (MAP4) promotes MT assembly in vitro and is localized along MTs in vivo. These results and the fact that MAP4 is the major MAP in nonneuronal cells suggest that MAP4's normal functions may include the stabilization of MTs in situ. To understand MAP4 function in vivo, we produced a blocking antibody (Ab) to prevent MAP4 binding to MTs. The COOH-terminal MT binding domain of MAP4 was expressed in Escherichia coli as a glutathione transferase fusion protein and was injected into rabbits to produce an antiserum that was then affinity purified and shown to be monospecific for MAP4. This Ab blocked > 95% of MAP4 binding to MTs in an in vitro assay. Microinjection of the affinity purified Ab into human fibroblasts and monkey epithelial cells abolished MAP4 binding to MTs as assayed with a rat polyclonal antibody against the NH2-terminal projection domain of MAP4. The removal of MAP4 from MTs was accompanied by its sequestration into visible MAP4-Ab immunocomplexes. However, the MT network appeared normal. Tubulin photoactivation and nocodazole sensitivity assays indicated that MT dynamics were not altered detectably by the removal of MAP4 from the MTs. Cells progressed to mitosis with morphologically normal spindles in the absence of MAP4 binding to MTs. Depleting MAP4 from MTs also did not affect the state of posttranslational modifications of tubulin subunits. Further, no perturbations of MT- dependent organelle distribution were detected. We conclude that the association of MAP4 with MTs is not essential for MT assembly or for the MT-based functions in cultured cells that we could assay. A significant role for MAP4 is not excluded by these results, however, as MAP4 may be a component of a functionally redundant system. PMID:8636213

  20. Efficient Expression of Functional (α6β2)2β3 AChRs in Xenopus Oocytes from Free Subunits Using Slightly Modified α6 Subunits

    PubMed Central

    Ley, Carson Kai-Kwong; Kuryatov, Alexander; Wang, Jingyi; Lindstrom, Jon Martin

    2014-01-01

    Human (α6β2)(α4β2)β3 nicotinic acetylcholine receptors (AChRs) are essential for addiction to nicotine and a target for drug development for smoking cessation. Expressing this complex AChR is difficult, but has been achieved using subunit concatamers. In order to determine what limits expression of α6* AChRs and to efficiently express α6* AChRs using free subunits, we investigated expression of the simpler (α6β2)2β3 AChR. The concatameric form of this AChR assembles well, but is transported to the cell surface inefficiently. Various chimeras of α6 with the closely related α3 subunit increased expression efficiency with free subunits and produced pharmacologically equivalent functional AChRs. A chimera in which the large cytoplasmic domain of α6 was replaced with that of α3 increased assembly with β2 subunits and transport of AChRs to the oocyte surface. Another chimera replacing the unique methionine 211 of α6 with leucine found at this position in transmembrane domain 1 of α3 and other α subunits increased assembly of mature subunits containing β3 subunits within oocytes. Combining both α3 sequences in an α6 chimera increased expression of functional (α6β2)2β3 AChRs to 12-fold more than with concatamers. This is pragmatically useful, and provides insights on features of α6 subunit structure that limit its expression in transfected cells. PMID:25068303

  1. Efficient expression of functional (α6β2)2β3 AChRs in Xenopus oocytes from free subunits using slightly modified α6 subunits.

    PubMed

    Ley, Carson Kai-Kwong; Kuryatov, Alexander; Wang, Jingyi; Lindstrom, Jon Martin

    2014-01-01

    Human (α6β2)(α4β2)β3 nicotinic acetylcholine receptors (AChRs) are essential for addiction to nicotine and a target for drug development for smoking cessation. Expressing this complex AChR is difficult, but has been achieved using subunit concatamers. In order to determine what limits expression of α6* AChRs and to efficiently express α6* AChRs using free subunits, we investigated expression of the simpler (α6β2)2β3 AChR. The concatameric form of this AChR assembles well, but is transported to the cell surface inefficiently. Various chimeras of α6 with the closely related α3 subunit increased expression efficiency with free subunits and produced pharmacologically equivalent functional AChRs. A chimera in which the large cytoplasmic domain of α6 was replaced with that of α3 increased assembly with β2 subunits and transport of AChRs to the oocyte surface. Another chimera replacing the unique methionine 211 of α6 with leucine found at this position in transmembrane domain 1 of α3 and other α subunits increased assembly of mature subunits containing β3 subunits within oocytes. Combining both α3 sequences in an α6 chimera increased expression of functional (α6β2)2β3 AChRs to 12-fold more than with concatamers. This is pragmatically useful, and provides insights on features of α6 subunit structure that limit its expression in transfected cells.

  2. Mutations in the embryonal subunit of the acetylcholine receptor (CHRNG) cause lethal and Escobar variants of multiple pterygium syndrome.

    PubMed

    Morgan, Neil V; Brueton, Louise A; Cox, Phillip; Greally, Marie T; Tolmie, John; Pasha, Shanaz; Aligianis, Irene A; van Bokhoven, Hans; Marton, Tamas; Al-Gazali, Lihadh; Morton, Jenny E V; Oley, Christine; Johnson, Colin A; Trembath, Richard C; Brunner, Han G; Maher, Eamonn R

    2006-08-01

    Multiple pterygium syndromes (MPSs) comprise a group of multiple-congenital-anomaly disorders characterized by webbing (pterygia) of the neck, elbows, and/or knees and joint contractures (arthrogryposis). In addition, a variety of developmental defects (e.g., vertebral anomalies) may occur. MPSs are phenotypically and genetically heterogeneous but are traditionally divided into prenatally lethal and nonlethal (Escobar) types. To elucidate the pathogenesis of MPS, we undertook a genomewide linkage scan of a large consanguineous family and mapped a locus to 2q36-37. We then identified germline-inactivating mutations in the embryonal acetylcholine receptor gamma subunit (CHRNG) in families with both lethal and nonlethal MPSs. These findings extend the role of acetylcholine receptor dysfunction in human disease and provide new insights into the pathogenesis and management of fetal akinesia syndromes.

  3. Subunits of the Snf1 kinase heterotrimer show interdependence for association and activity.

    PubMed

    Elbing, Karin; Rubenstein, Eric M; McCartney, Rhonda R; Schmidt, Martin C

    2006-09-08

    The Snf1 kinase and its mammalian orthologue, the AMP-activated protein kinase (AMPK), function as heterotrimers composed of a catalytic alpha-subunit and two non-catalytic subunits, beta and gamma. The beta-subunit is thought to hold the complex together and control subcellular localization whereas the gamma-subunit plays a regulatory role by binding to and blocking the function of an auto-inhibitory domain (AID) present in the alpha-subunit. In addition, catalytic activity requires phosphorylation by a distinct upstream kinase. In yeast, any one of three Snf1-activating kinases, Sak1, Tos3, or Elm1, can fulfill this role. We have previously shown that Sak1 is the only Snf1-activating kinase that forms a stable complex with Snf1. Here we show that the formation of the Sak1.Snf1 complex requires the beta- and gamma-subunits in vivo. However, formation of the Sak1.Snf1 complex is not necessary for glucose-regulated phosphorylation of the Snf1 activation loop. Snf1 kinase purified from cells lacking the beta-subunits do not contain any gamma-subunit, indicating that the Snf1 kinase does not form a stable alphagamma dimer in vivo. In vitro kinase assays using purified full-length and truncated Snf1 proteins demonstrate that the kinase domain, which lacks the AID, is significantly more active than the full-length Snf1 protein. Addition of purified beta- and gamma-subunits could stimulate the kinase activity of the full-length alpha-subunit but only when all three subunits were present, suggesting an interdependence of all three subunits for assembly of a functional complex.

  4. Evidence for inhibition mediated by coassembly of GABAA and GABAC receptor subunits in native central neurons.

    PubMed

    Milligan, Carol J; Buckley, Noel J; Garret, Maurice; Deuchars, Jim; Deuchars, Susan A

    2004-08-18

    Fast inhibition in the nervous system is commonly mediated by GABA(A) receptors comprised of 2alpha/2beta/1gamma subunits. In contrast, GABA(C) receptors containing only rho subunits (rho1-rho3) have been predominantly detected in the retina. However, here using reverse transcription-PCR and in situ hybridization we show that mRNA encoding the rho1 subunit is highly expressed in brainstem neurons. Immunohistochemistry localized the rho1 subunit to neurons at light and electron microscopic levels, where it was detected at synaptic junctions. Application of the GABA(C) receptor agonist cis-4-aminocrotonic acid (100-800 microM) requires the rho1 subunit to elicit responses, which surprisingly are blocked independently by antagonists to GABA(A) (bicuculline, 10 microM) and GABA(C) [(1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA); 40-160 microM] receptors. Responses to GABA(C) agonists were also enhanced by the GABA(A) receptor modulator pentobarbitone (300 microM). Spontaneous and evoked IPSPs were reduced in amplitude but never abolished by TPMPA, but were completely blocked by bicuculline. We therefore tested the hypothesis that GABA(A) and GABA(C) subunits formed a heteromeric receptor. Immunohistochemistry indicated that rho1 and alpha1 subunits were colocalized at light and electron microscopic levels. Electrophysiology revealed that responses to GABA(C) receptor agonists were enhanced by the GABA(A) receptor modulator zolpidem (500 nm), which acts on the alpha1 subunit when the gamma2 subunit is also present. Finally, coimmunoprecipitation indicated that the rho1 subunit formed complexes that also containedalpha1 and gamma2 subunits. Taken together these separate lines of evidence suggest that the effects of GABA in central neurons can be mediated by heteromeric complexes of GABA(A) and GABA(C) receptor subunits.

  5. Accessory subunits are integral for assembly and function of human mitochondrial complex I.

    PubMed

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

    2016-10-06

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

  6. The NMDA receptor NR2A subunit regulates proliferation of MKN45 human gastric cancer cells

    SciTech Connect

    Watanabe, Kanako; Kanno, Takeshi; Oshima, Tadayuki; Miwa, Hiroto; Tashiro, Chikara; Nishizaki, Tomoyuki

    2008-03-07

    The present study investigated proliferation of MKN28 and MKN45 human gastric cancer cells regulated by the N-methyl-D-aspartate (NMDA) receptor subunit. The NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (AP5) inhibited proliferation of MKN45 cells, but not MKN28 cells. Of the NMDA subunits such as NR1, NR2 (2A, 2B, 2C, and 2D), and NR3 (3A and 3B), all the NMDA subunit mRNAs except for the NR2B subunit mRNA were expressed in both MKN28 and MKN45 cells. MKN45 cells were characterized by higher expression of the NR2A subunit mRNA and lower expression of the NR1 subunit mRNA, but MKN28 otherwise by higher expression of the NR1 subunit mRNA and lower expression of the NR2A subunit mRNA. MKN45 cell proliferation was also inhibited by silencing the NR2A subunit-targeted gene. For MKN45 cells, AP5 or knocking-down the NR2A subunit increased the proportion of cells in the G{sub 1} phase of cell cycling and decreased the proportion in the S/G{sub 2} phase. The results of the present study, thus, suggest that blockage of NMDA receptors including the NR2A subunit suppresses MKN45 cell proliferation due to cell cycle arrest at the G{sub 1} phase; in other words, the NR2A subunit promotes MKN45 cell proliferation by accelerating cell cycling.

  7. Interspecific luciferase beta subunit hybrids between Vibrio harveyi, Vibrio fischeri and Photobacterium leiognathi.

    PubMed

    Almashanu, S; Gendler, I; Hadar, R; Kuhn, J

    1996-09-01

    Bacterial luciferase (EC 1.14.14.3) is a heterodimer composed of alpha- and beta-chains encoded by luxA and luxB, respectively. Although some interspecific combinations of these subunits lead to active enzyme, others do not. The beta subunits of Vibrio fischeri and Photobacterium leiognathi form active enzyme with the alpha subunits of V.fischeri, P.leiognathi and Vibrio harveyi, while the beta subunit from V.harveyi only complements the alpha subunit of V.harveyi. Inactivity is caused by a lack of dimerization of the beta subunit of V.harveyi with the alpha subunits of V.fischeri and P.leiognathi. These observations served as the basis for a search to discover which segment of the beta polypeptide confers the ability to dimerize with the alpha subunits of V.fischeri and P.leiognathi. Intragenic beta subunit hybrids were made between V.harveyi, V.fischeri and P.leiognathi. Unique restriction sites were introduced into the respective luxB genes to divide them into four roughly equal segments. In all, 78 hybrids were constructed by in vitro techniques. The N-terminal segment of the peptide contains the signals that differentiate between the beta subunits of V.fischeri and P. leiognathi and the beta subunit of V. harveyi, and allow the former to dimerize with their alpha subunits. The second segment has no major effect on enzyme activity but does exhibit some context effects. Important interactions were found between the third and fourth segments of the polypeptide with respect to enzymatic activity.

  8. The Role of Auxiliary Subunits for the Functional Diversity of Voltage-Gated Calcium Channels

    PubMed Central

    Campiglio, Marta; Flucher, Bernhard E

    2015-01-01

    Voltage-gated calcium channels (VGCCs) represent the sole mechanism to convert membrane depolarization into cellular functions like secretion, contraction, or gene regulation. VGCCs consist of a pore-forming α1 subunit and several auxiliary channel subunits. These subunits come in multiple isoforms and splice-variants giving rise to a stunning molecular diversity of possible subunit combinations. It is generally believed that specific auxiliary subunits differentially regulate the channels and thereby contribute to the great functional diversity of VGCCs. If auxiliary subunits can associate and dissociate from pre-existing channel complexes, this would allow dynamic regulation of channel properties. However, most auxiliary subunits modulate current properties very similarly, and proof that any cellular calcium channel function is indeed modulated by the physiological exchange of auxiliary subunits is still lacking. In this review we summarize available information supporting a differential modulation of calcium channel functions by exchange of auxiliary subunits, as well as experimental evidence in support of alternative functions of the auxiliary subunits. At the heart of the discussion is the concept that, in their native environment, VGCCs function in the context of macromolecular signaling complexes and that the auxiliary subunits help to orchestrate the diverse protein–protein interactions found in these calcium channel signalosomes. Thus, in addition to a putative differential modulation of current properties, differential subcellular targeting properties and differential protein–protein interactions of the auxiliary subunits may explain the need for their vast molecular diversity. J. Cell. Physiol. 999: 00–00, 2015. © 2015 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc. J. Cell. Physiol. 230: 2019–2031, 2015. © 2015 Wiley Periodicals, Inc. PMID:25820299

  9. The NH2-terminal php domain of the alpha subunit of the Escherichia coli replicase binds the epsilon proofreading subunit.

    PubMed

    Wieczorek, Anna; McHenry, Charles S

    2006-05-05

    The alpha subunit of the replicase of all bacteria contains a php domain, initially identified by its similarity to histidinol phosphatase but of otherwise unknown function (Aravind, L., and Koonin, E. V. (1998) Nucleic Acids Res. 26, 3746-3752). Deletion of 60 residues from the NH2 terminus of the alpha php domain destroys epsilon binding. The minimal 255-residue php domain, estimated by sequence alignment with homolog YcdX, is insufficient for epsilon binding. However, a 320-residue segment including sequences that immediately precede the polymerase domain binds epsilon with the same affinity as the 1160-residue full-length alpha subunit. A subset of mutations of a conserved acidic residue (Asp43 in Escherichia coli alpha) present in the php domain of all bacterial replicases resulted in defects in epsilon binding. Using sequence alignments, we show that the prototypical gram+ Pol C, which contains the polymerase and proofreading activities within the same polypeptide chain, has an epsilon-like sequence inserted in a surface loop near the center of the homologous YcdX protein. These findings suggest that the php domain serves as a platform to enable coordination of proofreading and polymerase activities during chromosomal replication.

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

    SciTech Connect

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

    2009-01-01

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

  11. Stage and Tissue Specific Expression of Four TCR Subunits in Olive Flounder (Paralichthys olivaceus).

    PubMed

    Lee, Young Mee; Lee, Jeong-Ho; Noh, Jae Koo; Kim, Hyun Chul; Park, Choul-Ji; Park, Jong-Won; Hwang, In Joon; Kim, Sung Yeon

    2013-12-01

    TCR subunits are members of membrane-bound receptors which allow the fast and efficient elimination of the specific fish pathogens have regulated function in adaptive immunity. Sequence structure of TCR subunits have been reported for various teleosts, but the information of each TCR subunit functional characterization through expression analysis in fish was unknown. In this study, we examined the gene expression of TCR subunits in the early developmental stages and observed transcript levels in various tissues from healthy adult olive flounder by RT-PCR. The mRNA expression of alpha subunit was already detected in the previous hatching step. But the transcripts of another TCR subunit were not observed during embryo development and increased after hatching and maintained until metamorphosis at the same level. It was found that all TCR subunits mRNAs are commonly expressed in the immune-related organ such as spleen, kidney and gill, also weak expressed in fin and eye. TCR alpha and beta subunit were expressed in brain, whereas gamma and delta were not expressed same tissue. The sequence alignment analysis shows that there are more than 80% sequence homology between TCR subunits. Because it has a high similarity of amino acid sequence to expect similar in function, but expression analysis show that will have may functional diversity due to different time and place of expression.

  12. Enrichment of GABAA Receptor α-Subunits on the Axonal Initial Segment Shows Regional Differences

    PubMed Central

    Gao, Yudong; Heldt, Scott A.

    2016-01-01

    Although it is generally recognized that certain α-subunits of γ-aminobutyric acid type A receptors (GABAARs) form enriched clusters on the axonal initial segment (AIS), the degree to which these clusters vary in different brain areas is not well known. In the current study, we quantified the density, size, and enrichment ratio of fluorescently labeled α1-, α2-, or α3-subunits aggregates co-localized with the AIS-marker ankyrin G and compared them to aggregates in non-AIS locations among different brain areas including hippocampal subfields, basal lateral amygdala (BLA), prefrontal cortex (PFC), and sensory cortex (CTX). We found regional differences in the enrichment of GABAAR α-subunits on the AIS. Significant enrichment was identified in the CA3 of hippocampus for α1-subunits, in the CA1, CA3, and BLA for α2-subunits, and in the BLA for α3-subunits. Using α-subunit knock-out (KO) mice, we found that BLA enrichment of α2- and α3-subunits were physiologically independent of each other, as the enrichment of one subunit was unaffected by the genomic deletion of the other. To further investigate the unique pattern of α-subunit enrichment in the BLA, we examined the association of α2- and α3-subunits with the presynaptic vesicular GABA transporter (vGAT) and the anchoring protein gephyrin (Geph). As expected, both α2- and α3-subunits on the AIS within the BLA received prominent GABAergic innervation from vGAT-positive terminals. Further, we found that the association of α2- and α3-subunits with Geph was weaker in AIS versus non-AIS locations, suggesting that Geph might be playing a lesser role in the enrichment of α2- and α3-subunits on the AIS. Overall, these observations suggest that GABAARs on the AIS differ in subunit composition across brain regions. As with somatodendritic GABAARs, the distinctive expression pattern of AIS-located GABAAR α-subunits in the BLA, and other brain areas, likely contribute to unique forms of GABAergic inhibitory

  13. The α2δ subunits of voltage-gated calcium channels.

    PubMed

    Dolphin, Annette C

    2013-07-01

    Voltage-gated calcium channels consist of the main pore-forming α1 subunit, together, except in the case of the T-type channels, with β and α2δ and sometimes γ subunits, which are collectively termed auxiliary or accessory subunits. This review will concentrate on the properties and role of the α2δ subunits of these channels. These proteins are largely extracellular, membrane-associated proteins which influence the trafficking, localization, and biophysical properties of the channels. This article is part of a Special Issue entitled: Calcium channels.

  14. Differential expression of the protein kinase A subunits in normal adrenal glands and adrenocortical adenomas.

    PubMed

    Weigand, Isabel; Ronchi, Cristina L; Rizk-Rabin, Marthe; Dalmazi, Guido Di; Wild, Vanessa; Bathon, Kerstin; Rubin, Beatrice; Calebiro, Davide; Beuschlein, Felix; Bertherat, Jérôme; Fassnacht, Martin; Sbiera, Silviu

    2017-12-01

    Somatic mutations in protein kinase A catalytic α subunit (PRKACA) were found to be causative for 30-40% of cortisol-producing adenomas (CPA) of the adrenal gland, rendering PKA signalling constitutively active. In its resting state, PKA is a stable and inactive heterotetramer, consisting of two catalytic and two regulatory subunits with the latter inhibiting PKA activity. The human genome encodes three different PKA catalytic subunits and four different regulatory subunits that are preferentially expressed in different organs. In normal adrenal glands all regulatory subunits are expressed, while CPA exhibit reduced protein levels of the regulatory subunit IIβ. In this study, we linked for the first time the loss of RIIβ protein levels to the PRKACA mutation status and found the down-regulation of RIIβ to arise post-transcriptionally. We further found the PKA subunit expression pattern of different tumours is also present in the zones of the normal adrenal cortex and demonstrate that the different PKA subunits have a differential expression pattern in each zone of the normal adrenal gland, indicating potential specific roles of these subunits in the regulation of different hormones secretion.

  15. beta-subunits of Snf1 kinase are required for kinase function and substrate definition.

    PubMed

    Schmidt, M C; McCartney, R R

    2000-09-15

    The Snf1 kinase and its mammalian homolog, the AMP-activated protein kinase, are heterotrimeric enzymes composed of a catalytic alpha-subunit, a regulatory gamma-subunit and a beta-subunit that mediates heterotrimer formation. Saccharomyces cerevisiae encodes three beta-subunit genes, SIP1, SIP2 and GAL83. Earlier studies suggested that these subunits may not be required for Snf1 kinase function. We show here that complete and precise deletion of all three beta-subunit genes inactivates the Snf1 kinase. The sip1Delta sip2Delta gal83Delta strain is unable to derepress invertase, grows poorly on alternative carbon sources and fails to direct the phosphorylation of the Mig1 and Sip4 proteins in vivo. The SIP1 sip2Delta gal83Delta strain manifests a subset of Snf phenotypes (Raf(+), Gly(-)) observed in the snf1Delta 10 strain (Raf(-), Gly(-)), suggesting that individual beta-subunits direct the Snf1 kinase to a subset of its targets in vivo. Indeed, deletion of individual beta-subunit genes causes distinct differences in the induction and phosphorylation of Sip4, strongly suggesting that the beta-subunits play an important role in substrate definition.

  16. Analysis of an N-terminal deletion in subunit a of the Escherichia coli ATP synthase.

    PubMed

    Ishmukhametov, Robert R; DeLeon-Rangel, Jessica; Zhu, Shaotong; Vik, Steven B

    2017-04-01

    Subunit a is a membrane-bound stator subunit of the ATP synthase and is essential for proton translocation. The N-terminus of subunit a in E. coli is localized to the periplasm, and contains a sequence motif that is conserved among some bacteria. Previous work has identified mutations in this region that impair enzyme activity. Here, an internal deletion was constructed in subunit a in which residues 6-20 were replaced by a single lysine residue, and this mutant was unable to grow on succinate minimal medium. Membrane vesicles prepared from this mutant lacked ATP synthesis and ATP-driven proton translocation, even though immunoblots showed a significant level of subunit a. Similar results were obtained after purification and reconstitution of the mutant ATP synthase into liposomes. The location of subunit a with respect to its neighboring subunits b and c was probed by introducing cysteine substitutions that were known to promote cross-linking: a_L207C + c_I55C, a_L121C + b_N4C, and a_T107C + b_V18C. The last pair was unable to form cross-links in the background of the deletion mutant. The results indicate that loss of the N-terminal region of subunit a does not generally disrupt its structure, but does alter interactions with subunit b.

  17. Restriction mapping of synthetic thyroglobulin structural gene as a means of investigating thyroglobulin structure.

    PubMed

    Vassart, G; Brocas, H

    1980-11-14

    Bovine 33 S thyroglobulin mRNA was reverse transcribed into double-stranded DNA under conditions allowing the synthesis of a complete 8 kilobase pair copy. A physical map of the resulting synthetic thyroglobulin structural gene was constructed using six restriction endonucleases. The following conclusions could be drawn: (i) the polypeptide chains in thyroglobulin subunits are identical; (ii) thyroglobulin is composed of a major class of molecules sharing the same primary structure; (iii) there is no evidence for precise internal repetition in the structure of thyroglobulin subunits.

  18. Mapping Human Epigenomes

    PubMed Central

    Rivera, Chloe M.; Ren, Bing

    2013-01-01

    As the second dimension to the genome, the epigenome contains key information specific to every type of cells. Thousands of human epigenome maps have been produced in recent years thanks to rapid development of high throughput epigenome mapping technologies. In this review, we discuss the current epigenome mapping toolkit and utilities of epigenome maps. We focus particularly on mapping of DNA methylation, chromatin modification state and chromatin structures, and emphasize the use of epigenome maps to delineate human gene regulatory sequences and developmental programs. We also provide a perspective on the progress of the epigenomics field and challenges ahead. PMID:24074860

  19. Structure of the large ribosomal subunit from human mitochondria

    PubMed Central

    Bai, Xiao-chen; Sugimoto, Yoichiro; Edwards, Patricia C.; Murshudov, Garib; Scheres, Sjors H. W.; Ramakrishnan, V.

    2014-01-01

    Human mitochondrial ribosomes are highly divergent from all other known ribosomes and are specialized to exclusively translate membrane proteins. They are linked with hereditary mitochondrial diseases, and are often the unintended targets of various clinically useful antibiotics. Using single-particle electron cryo-microscopy we have determined the structure of its large subunit to 3.4 angstrom resolution, revealing 48 proteins, 21 of which are specific to mitochondria. The structure unveils an adaptation of the exit tunnel for hydrophobic nascent peptides, extensive remodeling of the central protuberance including recruitment of mitochondrial tRNAVal to play an integral structural role, and changes in the tRNA binding sites related to the unusual characteristics of mitochondrial tRNAs. PMID:25278503

  20. Expression and secretion of cholera toxin B subunit in lactobacilli.

    PubMed

    Okuno, Takahiro; Kashige, Nobuhiro; Satho, Tomomitsu; Irie, Keiichi; Hiramatsu, Yukihiro; Sharmin, Tanjina; Fukumitsu, Yuki; Uyeda, Saori; Yamada, Seitaro; Harakuni, Tetsuya; Miyata, Takeshi; Arakawa, Takeshi; Imoto, Masumi; Toda, Akihisa; Nakashima, Yukihiko; Miake, Fumio

    2013-01-01

    Lactic acid bacteria (LAB) are used in various fields, including in food and medical supplies. There has been a great deal of research into vaccine development using LAB as carriers due to their "generally recognized as safe" status. Cholera is an infectious disease that causes diarrhea due to cholera toxin (CT) produced by Vibrio cholerae. The pentameric cholera toxin B (CTB) subunit has no toxicity, and is used as an antigen in cholera vaccines and as a delivery molecule in vaccines to various diseases. In this study, we generated recombinant LAB expressing and secreting CTB. Here, we first report that CTB expressed and secreted from LAB bound to GM1 ganglioside. The secreted CTB was purified, and its immunogenicity was determined by intranasal administration into mice. The results of the present study suggested that it may be useful as the basis of a new oral cholera vaccine combining LAB and CTB.

  1. Structure of the large ribosomal subunit from human mitochondria.

    PubMed

    Brown, Alan; Amunts, Alexey; Bai, Xiao-chen; Sugimoto, Yoichiro; Edwards, Patricia C; Murshudov, Garib; Scheres, Sjors H W; Ramakrishnan, V

    2014-11-07

    Human mitochondrial ribosomes are highly divergent from all other known ribosomes and are specialized to exclusively translate membrane proteins. They are linked with hereditary mitochondrial diseases and are often the unintended targets of various clinically useful antibiotics. Using single-particle cryogenic electron microscopy, we have determined the structure of its large subunit to 3.4 angstrom resolution, revealing 48 proteins, 21 of which are specific to mitochondria. The structure unveils an adaptation of the exit tunnel for hydrophobic nascent peptides, extensive remodeling of the central protuberance, including recruitment of mitochondrial valine transfer RNA (tRNA(Val)) to play an integral structural role, and changes in the tRNA binding sites related to the unusual characteristics of mitochondrial tRNAs.

  2. The subunit interfaces of weakly associated homodimeric proteins.

    PubMed

    Dey, Sucharita; Pal, Arumay; Chakrabarti, Pinak; Janin, Joël

    2010-04-23

    We analyzed subunit interfaces in 315 homodimers with an X-ray structure in the Protein Data Bank, validated by checking the literature for data that indicate that the proteins are dimeric in solution and that, in the case of the "weak" dimers, the homodimer is in equilibrium with the monomer. The interfaces of the 42 weak dimers, which are smaller by a factor of 2.4 on average than in the remainder of the set, are comparable in size with antibody-antigen or protease-inhibitor interfaces. Nevertheless, they are more hydrophobic than in the average transient protein-protein complex and similar in amino acid composition to the other homodimer interfaces. The mean numbers of interface hydrogen bonds and hydration water molecules per unit area are also similar in homodimers and transient complexes. Parameters related to the atomic packing suggest that many of the weak dimer interfaces are loosely packed, and we suggest that this contributes to their low stability. To evaluate the evolutionary selection pressure on interface residues, we calculated the Shannon entropy of homologous amino acid sequences at 60% sequence identity. In 93% of the homodimers, the interface residues are better conserved than the residues on the protein surface. The weak dimers display the same high degree of interface conservation as other homodimers, but their homologs may be heterodimers as well as homodimers. Their interfaces may be good models in terms of their size, composition, and evolutionary conservation for the labile subunit contacts that allow protein assemblies to share and exchange components, allosteric proteins to undergo quaternary structure transitions, and molecular machines to operate in the cell.

  3. Effective polymer adjuvants for sustained delivery of protein subunit vaccines.

    PubMed

    Adams, Justin R; Haughney, Shannon L; Mallapragada, Surya K

    2015-03-01

    We have synthesized thermogelling cationic amphiphilic pentablock copolymers that have the potential to act as injectable vaccine carriers and adjuvants that can simultaneously provide sustained delivery and enhance the immunogenicity of released antigen. While these pentablock copolymers have shown efficacy in DNA delivery in past studies, the ability to deliver both DNA and protein for subunit vaccines using the same polymeric carrier can provide greater flexibility and efficacy. We demonstrate the ability of these pentablock copolymers, and the parent triblock Pluronic copolymers to slowly release structurally intact and antigenically stable protein antigens in vitro, create an antigen depot through long-term injection-site persistence and enhance the in vivo immune response to these antigens. We show release of the model protein antigen ovalbumin in vitro from the thermogelling block copolymers with the primary, secondary and tertiary structures of the released protein unchanged compared to the native protein, and its antigenicity preserved upon release. The block copolymers form a gel at physiological temperatures that serves as an antigenic depot and persists in vivo at the site of injection for over 50days. The pentablock copolymers show a significant fivefold enhancement in the immune response compared to soluble protein alone, even 6weeks after the administration, based on measurement of antibody titers. These results demonstrate the potential of these block copolymers hydrogels to persist for several weeks and sustain the release of antigen with minimal effects on protein stability and antigenicity; and their ability to be used simultaneously as a sustained delivery device as well as a subunit vaccine adjuvant platform.

  4. Subunit interface mutants of rabbit muscle aldolase form active dimers.

    PubMed Central

    Beernink, P. T.; Tolan, D. R.

    1994-01-01

    We report the construction of subunit interface mutants of rabbit muscle aldolase A with altered quaternary structure. A mutation has been described that causes nonspherocytic hemolytic anemia and produces a thermolabile aldolase (Kishi H et al., 1987, Proc Natl Acad Sci USA 84:8623-8627). The disease arises from substitution of Gly for Asp-128, a residue at the subunit interface of human aldolase A. To elucidate the role of this residue in the highly homologous rabbit aldolase A, site-directed mutagenesis is used to replace Asp-128 with Gly, Ala, Asn, Gln, or Val. Rabbit aldolase D128G purified from Escherichia coli is found to be similar to human D128G by kinetic analysis, CD, and thermal inactivation assays. All of the mutant rabbit aldolases are similar to the wild-type rabbit enzyme in secondary structure and kinetic properties. In contrast, whereas the wild-type enzyme is a tetramer, chemical crosslinking and gel filtration indicate that a new dimeric species exists for the mutants. In sedimentation velocity experiments, the mutant enzymes as mixtures of dimer and tetramer at 4 degrees C. Sedimentation at 20 degrees C shows that the mutant enzymes are > 99.5% dimeric and, in the presence of substrate, that the dimeric species is active. Differential scanning calorimetry demonstrates that Tm values of the mutant enzymes are decreased by 12 degrees C compared to wild-type enzyme. The results indicate that Asp-128 is important for interface stability and suggest that 1 role of the quaternary structure of aldolase is to provide thermostability. PMID:7833800

  5. Molecular cloning and functional analysis of three subunits of yeast proteasome.

    PubMed Central

    Emori, Y; Tsukahara, T; Kawasaki, H; Ishiura, S; Sugita, H; Suzuki, K

    1991-01-01

    The genes encoding three subunits of Saccharomyces cerevisiae proteasome were cloned and sequenced. The deduced amino acid sequences were homologous not only to each other (30 to 40% identity) but also to those of rat and Drosophila proteasomes (25 to 65% identity). However, none of these sequences showed any similarity to any other known sequences, including various proteases, suggesting that these proteasome subunits may constitute a unique gene family. Gene disruption analyses revealed that two of the three subunits (subunits Y7 and Y8) are essential for growth, indicating that the proteasome and its individual subunits play an indispensable role in fundamental biological processes. On the other hand, subunit Y13 is not essential; haploid cells with a disrupted Y13 gene can proliferate, although the doubling time is longer than that of cells with nondisrupted genes. In addition, biochemical analysis revealed that proteasome prepared from the Y13 disrupted cells contains tryptic and chymotryptic activities equivalent to those of nondisrupted cells, indicating that the Y13 subunit is not essential for tryptic or chymotryptic activity. However, the chymotryptic activity of the Y13 disrupted cells is not dependent on sodium dodecyl sulfate (SDS), an activator of proteasome, since nearly full activity was observed in the absence of SDS. Thus, the activity in proteasome of the Y13 disrupted cells might result in unregulated intracellular proteolysis, thus leading to the prolonged cell cycle. These results indicate that cloned proteasome subunits having similar sequences to the yeast Y13 subunit are structural, but not catalytic, components of proteasome. It is also suggested that two subunits (Y7 and Y8) might occupy positions essential to proteasome structure or activity, whereas subunit Y13 is in a nonessential but important position. Images PMID:1898763

  6. Trends in Nasal Subunit Reconstruction by Facial Plastic and Reconstructive Surgeons.

    PubMed

    Larrabee, Yuna C; Phillips, David J; Sclafani, Anthony P

    2017-02-01

    To determine if facial plastic and reconstructive surgeons still adhere to the classic nasal subunit principle as described by Burget and Menick. Observational survey. A Weill Cornell Medicine institutional review board approved electronic survey that was sent via e-mail to active members of the American Academy of Facial Plastic and Reconstructive Surgery (AAFPRS). The survey consisted of 32 multiple-choice questions pertaining to the operative management of small (22-30%), medium (50-58%), and large (75-81%) defects of each subunit of the nose, as well as demographic, provider, and practice characteristics. There were 111 responses to the survey (10.1% response rate). Ninety-eight percent of respondents reported familiarity with the subunit principle, and 59.6% considered the subunit principle in greater than 90% of cases. Almost three-quarters (70.4%) of respondents felt the subunit principle should be applied but could be modified based on the particular nasal defect, whereas 28.7% felt it was only sometimes helpful and was not mandatory for successful nasal reconstruction. Large defects of the tip and ala are generally treated by excision of the remaining subunit (79.4 and 80.6%, respectively). Fewer surgeons would excise the remaining subunit for large defects of the dorsum (39.8%), sidewall (38.8%), and soft tissue facet (18.4%). Simple repair without additional excision was the treatment of choice for small defects of the tip (58.2%), ala (59.2%), sidewall (65%), dorsum (68%), and soft tissue facet (71.8%). However, in many small- (up to 32%) and medium- (up to 51%) sized defects of the tip, ala, sidewall, and dorsum, respondents reported partial subunit excision. The majority of AAFPRS members abide to the classical subunit principle by completely excising the remaining subunit for large defects of the tip and ala. Many surgeons modify the subunit principle in small and medium defects.

  7. Differential regulation of thyrotropin subunit apoprotein and carbohydrate biosynthesis by thyroid hormone

    SciTech Connect

    Taylor, T.; Weintraub, B.D.

    1985-04-01

    The regulation of TSH apoprotein and carbohydrate biosynthesis by thyroid hormone was studied by incubating pituitaries from normal and hypothyroid (3 weeks post-thyroidectomy) rats in medium containing (/sup 14/C)alanine and (/sup 3/H) glucosamine. After 6 h, samples were sequentially treated with anti-TSH beta to precipitate TSH and free TSH beta, anti-LH beta to clear the sample of LH and free LH beta, then anti-LH alpha to precipitate free alpha-subunit. Total proteins were acid precipitated. All precipitates were subjected to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, which were then sliced and assayed by scintillation spectrometry. In hypothyroid pituitaries plus medium, (/sup 14/C)alanine incorporation in combined and free beta-subunits was 26 times normal and considerably greater than the 3.4-fold increase seen in total protein; combined and free alpha-subunits showed no specific increase in apoprotein synthesis. (/sup 3/H)Glucosamine incorporation in combined alpha- and beta-subunits in hypothyroid samples was 13 and 21 times normal, respectively, and was greater than the 1.9-fold increase in total protein; free alpha-subunit showed no specific increase in carbohydrate synthesis. The glucosamine to alanine ratio, reflecting relative glycosylation of newly synthesized molecules, was increased in hypothyroidism for combined alpha-subunits, but not for combined beta-subunits, free alpha-subunits, or total proteins. In summary, short term hypothyroidism selectively stimulated TSH beta apoprotein synthesis and carbohydrate synthesis of combined alpha- and beta-subunits. Hypothyroidism also increased the relative glycosylation of combined alpha-subunit. Thus, thyroid hormone deficiency appears to alter the rate-limiting step in TSH assembly (i.e. beta-subunit synthesis) as well as the carbohydrate structure of TSH, which may play important roles in its biological function.

  8. Reduction of tomato polygalacturonase beta subunit expression affects pectin solubilization and degradation during fruit ripening.

    PubMed Central

    Watson, C F; Zheng, L; DellaPenna, D

    1994-01-01

    The developmental changes that accompany tomato fruit ripening include increased solubilization and depolymerization of pectins due to the action of polygalacturonase (PG). Two PG isoenzymes can be extracted from ripe fruit: PG2, which is a single catalytic PG polypeptide, and PG1, which is composed of PG2 tightly associated with a second noncatalytic protein, the beta subunit. Previous studies have correlated ripening-associated increases in pectin solubilization and depolymerization with the presence of extractable PG1 activity, prior to the appearance of PG2, suggesting a functional role for the beta subunit and PG1 in pectin metabolism. To assess the function of the beta subunit, we produced and characterized transgenic tomatoes constitutively expressing a beta subunit antisense gene. Fruit from antisense lines had greatly reduced levels of beta subunit mRNA and protein and accumulated < 1% of their total extractable PG activity in ripe fruit as PG1, as compared with 25% for wild type. Inhibition of beta subunit expression resulted in significantly elevated levels of EDTA-soluble polyuronides at all stages of fruit ripening and a significantly higher degree of depolymerization at later ripening stages. Decreased beta subunit protein and extractable PG1 enzyme activity and increased pectin solubility and depolymerization all cosegregated with the beta subunit antisense transgene in T2 progeny. These results indicate (1) that PG2 is responsible for pectin solubilization and depolymerization in vivo and (2) that the beta subunit protein is not required for PG2 activity in vivo but (3) does play a significant role in regulating pectin metabolism in wild-type fruit by limiting the extent of pectin solubilization and depolymerization that can occur during ripening. Whether this occurs by direct interaction of the beta subunit with PG2 or indirectly by interaction of the beta subunit with the pectic substrate remains to be determined. PMID:7827495

  9. Species-specific Differences among KCNMB3 BK β3 Auxiliary Subunits: Some β3 N-terminal Variants May Be Primate-specific Subunits

    PubMed Central

    Zeng, Xuhui; Xia, Xiao-Ming; Lingle, Christopher J.

    2008-01-01

    The KCNMB3 gene encodes one of a family of four auxiliary β subunits found in the mammalian genome that associate with Slo1 α subunits and regulate BK channel function. In humans, the KCNMB3 gene contains four N-terminal alternative exons that produce four functionally distinct β3 subunits, β3a–d. Three variants, β3a–c, exhibit kinetically distinct inactivation behaviors. Since investigation of the physiological roles of BK auxiliary subunits will depend on studies in rodents, here we have determined the identity and functional properties of mouse β3 variants. Whereas β1, β2, and β4 subunits exhibit 83.2%, 95.3%, and 93.8% identity between mouse and human, the mouse β3 subunit, excluding N-terminal splice variants, shares only 62.8% amino acid identity with its human counterpart. Based on an examination of the mouse genome and screening of mouse cDNA libraries, here we have identified only two N-terminal candidates, β3a and β3b, of the four found in humans. Both human and mouse β3a subunits produce a characteristic use-dependent inactivation. Surprisingly, whereas the hβ3b exhibits rapid inactivation, the putative mβ3b does not inactivate. Furthermore, unlike hβ3, the mβ3 subunit, irrespective of the N terminus, mediates a shift in gating to more negative potentials at a given Ca2+ concentration. The shift in gating gradually is lost following patch excision, suggesting that the gating shift involves some regulatory process dependent on the cytosolic milieu. Examination of additional genomes to assess conservation among splice variants suggests that the putative mβ3b N terminus may not be a true orthologue of the hβ3b N terminus and that both β3c and β3d appear likely to be primate-specific N-terminal variants. These results have three key implications: first, functional properties of homologous β3 subunits may differ among mammalian species; second, the specific physiological roles of homologous β3 subunits may differ among mammalian

  10. Creative Concept Mapping.

    ERIC Educational Resources Information Center

    Brown, David S.

    2002-01-01

    Recommends the use of concept mapping in science teaching and proposes that it be presented as a creative activity. Includes a sample lesson plan of a potato stamp concept mapping activity for astronomy. (DDR)

  11. RadMap

    EPA Pesticide Factsheets

    RadMap is an interactive desktop tool featuring a nationwide geographic information systems (GIS) map of long-term radiation monitoring locations across the United States with access to key information about the monitor and the area surrounding it.

  12. Using maps in genealogy

    USGS Publications Warehouse

    ,

    1994-01-01

    In genealogy, maps are most often used as clues to where public or other records about an ancestor are likely to be found. Searching for maps seldom begins until a newcomer to genealogy has mastered basic genealogical routines

  13. Active Fire Mapping Program

    MedlinePlus

    Active Fire Mapping Program Current Large Incidents (Home) New Large Incidents Fire Detection Maps MODIS Satellite Imagery VIIRS Satellite Imagery Fire Detection GIS Data Fire Data in Google Earth ...

  14. Riparian Wetlands: Mapping

    EPA Science Inventory

    Riparian wetlands are critical systems that perform functions and provide services disproportionate to their extent in the landscape. Mapping wetlands allows for better planning, management, and modeling, but riparian wetlands present several challenges to effective mapping due t...

  15. Copolymer semiconductors comprising thiazolothiazole or benzobisthiazole, or benzobisoxazole electron acceptor subunits, and electron donor subunits, and their uses in transistors and solar cells

    DOEpatents

    Jenekhe, Samson A; Subramaniyan, Selvam; Ahmed, Eilaf; Xin, Hao; Kim, Felix Sunjoo

    2014-10-28

    The inventions disclosed, described, and/or claimed herein relate to copolymers comprising copolymers comprising electron accepting A subunits that comprise thiazolothiazole, benzobisthiazole, or benzobisoxazoles rings, and electron donating subunits that comprise certain heterocyclic groups. The copolymers are useful for manufacturing organic electronic devices, including transistors and solar cells. The invention also relates to certain synthetic precursors of the copolymers. Methods for making the copolymers and the derivative electronic devices are also described.

  16. Functional hybrid rubisco enzymes with plant small subunits and algal large subunits: engineered rbcS cDNA for expression in chlamydomonas.

    PubMed

    Genkov, Todor; Meyer, Moritz; Griffiths, Howard; Spreitzer, Robert J

    2010-06-25

    There has been much interest in the chloroplast-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) as a target for engineering an increase in net CO(2) fixation in photosynthesis. Improvements in the enzyme would lead to an increase in the production of food, fiber, and renewable energy. Although the large subunit contains the active site, a family of rbcS nuclear genes encodes the Rubisco small subunits, which can also influence the carboxylation catalytic efficiency and CO(2)/O(2) specificity of the enzyme. To further define the role of the small subunit in Rubisco function, small subunits from spinach, Arabidopsis, and sunflower were assembled with algal large subunits by transformation of a Chlamydomonas reinhardtii mutant that lacks the rbcS gene family. Foreign rbcS cDNAs were successfully expressed in Chlamydomonas by fusing them to a Chlamydomonas rbcS transit peptide sequence engineered to contain rbcS introns. Although plant Rubisco generally has greater CO(2)/O(2) specificity but a lower carboxylation V(max) than Chlamydomonas Rubisco, the hybrid enzymes have 3-11% increases in CO(2)/O(2) specificity and retain near normal V(max) values. Thus, small subunits may make a significant contribution to the overall catalytic performance of Rubisco. Despite having normal amounts of catalytically proficient Rubisco, the hybrid mutant strains display reduced levels of photosynthetic growth and lack chloroplast pyrenoids. It appears that small subunits contain the structural elements responsible for targeting Rubisco to the algal pyrenoid, which is the site where CO(2) is concentrated for optimal photosynthesis.

  17. Initial bridges between two ribosomal subunits are formed within 9.4 milliseconds, as studied by time-resolved cryo-EM.

    PubMed

    Shaikh, Tanvir R; Yassin, Aymen S; Lu, Zonghuan; Barnard, David; Meng, Xing; Lu, Toh-Ming; Wagenknecht, Terence; Agrawal, Rajendra K

    2014-07-08

    Association of the two ribosomal subunits during the process of translation initiation is a crucial step of protein synthesis. The two subunits (30S and 50S) of the bacterial 70S ribosome are held together by 12 dynamic bridges involving RNA-RNA, RNA-protein, and protein-protein interactions. The process of bridge formation, such as whether all these bridges are formed simultaneously or in a sequential order, is poorly understood. To understand such processes, we have developed and implemented a class of microfluidic devices that mix two components to completion within 0.4 ms and spray the mixture in the form of microdroplets onto an electron microscopy grid, yielding a minimum reaction time of 9.4 ms before cryofixation. Using these devices, we have obtained cryo-EM data corresponding to reaction times of 9.4 and 43 ms and have determined 3D structures of ribosomal subunit association intermediates. Molecular analyses of the cryo-EM maps reveal that eight intersubunit bridges (bridges B1a, B1b, B2a, B2b, B3, B7a, B7b, and B8) form within 9.4 ms, whereas the remaining four bridges (bridges B2c, B4, B5, and B6) take longer than 43 ms to form, suggesting that bridges are formed in a stepwise fashion. Our approach can be used to characterize sequences of various dynamic functional events on complex macromolecular assemblies such as ribosomes.

  18. Structure and expression of the gene coding for the alpha-subunit of DNA-dependent RNA polymerase from the chloroplast genome of Zea mays.

    PubMed Central

    Ruf, M; Kössel, H

    1988-01-01

    The rpoA gene coding for the alpha-subunit of DNA-dependent RNA polymerase located on the DNA of Zea mays chloroplasts has been characterized with respect to its position on the chloroplast genome and its nucleotide sequence. The amino acid sequence derived for a 39 Kd polypeptide shows strong homology with sequences derived from the rpoA genes of other chloroplast species and with the amino acid sequence of the alpha-subunit from E. coli RNA polymerase. Transcripts of the rpoA gene were identified by Northern hybridization and characterized by S1 mapping using total RNA isolated from maize chloroplasts. Antibodies raised against a synthetic C-terminal heptapeptide show cross reactivity with a 39 Kd polypeptide contained in the stroma fraction of maize chloroplasts. It is concluded that the rpoA gene is a functional gene and that therefore, at least the alpha-subunit of plastidic RNA polymerase, is expressed in chloroplasts. Images PMID:3399379

  19. Severing of a hydrogen bond disrupts amino acid networks in the catalytically active state of the alpha subunit of tryptophan synthase

    PubMed Central

    Axe, Jennifer M; O'Rourke, Kathleen F; Kerstetter, Nicole E; Yezdimer, Eric M; Chan, Yan M; Chasin, Alexander; Boehr, David D

    2015-01-01

    Conformational changes in the β2α2 and β6α6 loops in the alpha subunit of tryptophan synthase (αTS) are important for enzyme catalysis and coordinating substrate channeling with the beta subunit (βTS). It was previously shown that disrupting the hydrogen bond interactions between these loops through the T183V substitution on the β6α6 loop decreases catalytic efficiency and impairs substrate channeling. Results presented here also indicate that the T183V substitution decreases catalytic efficiency in Escherchia coli αTS in the absence of the βTS subunit. Nuclear magnetic resonance (NMR) experiments indicate that the T183V substitution leads to local changes in the structural dynamics of the β2α2 and β6α6 loops. We have also used NMR chemical shift covariance analyses (CHESCA) to map amino acid networks in the presence and absence of the T183V substitution. Under conditions of active catalytic turnover, the T183V substitution disrupts long-range networks connecting the catalytic residue Glu49 to the αTS-βTS binding interface, which might be important in the coordination of catalytic activities in the tryptophan synthase complex. The approach that we have developed here will likely find general utility in understanding long-range impacts on protein structure and dynamics of amino acid substitutions generated through protein engineering and directed evolution approaches, and provide insight into disease and drug-resistance mutations. PMID:25377949

  20. Rescue of gamma2 subunit-deficient mice by transgenic overexpression of the GABAA receptor gamma2S or gamma2L subunit isoforms.

    PubMed

    Baer, K; Essrich, C; Balsiger, S; Wick, M J; Harris, R A; Fritschy, J M; Lüscher, B

    2000-07-01

    The gamma2 subunit is an important functional determinant of GABAA receptors and is essential for formation of high-affinity benzodiazepine binding sites and for synaptic clustering of major GABAA receptor subtypes along with gephyrin. There are two splice variants of the gamma2 subunit, gamma2 short (gamma2S) and gamma2 long (gamma2L), the latter carrying in the cytoplasmic domain an additional eight amino acids with a putative phosphorylation site. Here, we show that transgenic mice expressing either the gamma2S or gamma2L subunit on a gamma2 subunit-deficient background are phenotypically indistinguishable from wild-type. They express nearly normal levels of gamma2 subunit protein and [3H]flumazenil binding sites. Likewise, the distribution, number and size of GABAA receptor clusters colocalized with gephyrin are similar to wild-type in both juvenile and adult mice. Our results indicate that the two gamma2 subunit splice variants can substitute for each other and fulfil the basic functions of GABAA receptors, allowing in vivo studies that address isoform-specific roles in phosphorylation-dependent regulatory mechanisms.

  1. Oil Exploration Mapping

    NASA Technical Reports Server (NTRS)

    1994-01-01

    After concluding an oil exploration agreement with the Republic of Yemen, Chevron International needed detailed geologic and topographic maps of the area. Chevron's remote sensing team used imagery from Landsat and SPOT, combining images into composite views. The project was successfully concluded and resulted in greatly improved base maps and unique topographic maps.

  2. Adventures with Maps.

    ERIC Educational Resources Information Center

    Hofferber, Michael

    1989-01-01

    Orienteering--the game of following a map to find predetermined locations--can spark interest and develop skills in map making and map reading. This article gives background on orienteering; describes indoor and outdoor orienteering activities; offers suggestions for incorporating orienteering into science, math, and language arts; and provides a…

  3. Reading Angles in Maps

    ERIC Educational Resources Information Center

    Izard, Véronique; O'Donnell, Evan; Spelke, Elizabeth S.

    2014-01-01

    Preschool children can navigate by simple geometric maps of the environment, but the nature of the geometric relations they use in map reading remains unclear. Here, children were tested specifically on their sensitivity to angle. Forty-eight children (age 47:15-53:30 months) were presented with fragments of geometric maps, in which angle sections…

  4. Using maps in genealogy

    USGS Publications Warehouse

    ,

    1999-01-01

    Maps are one of many sources you may need to complete a family tree. In genealogical research, maps can provide clues to where our ancestors may have lived and where to look for written records about them. Beginners should master basic genealogical research techniques before starting to use topographic maps.

  5. Quantitative DNA fiber mapping

    DOEpatents

    Gray, Joe W.; Weier, Heinz-Ulrich G.

    1998-01-01

    The present invention relates generally to the DNA mapping and sequencing technologies. In particular, the present invention provides enhanced methods and compositions for the physical mapping and positional cloning of genomic DNA. The present invention also provides a useful analytical technique to directly map cloned DNA sequences onto individual stretched DNA molecules.

  6. What Do Maps Show?

    ERIC Educational Resources Information Center

    Geological Survey (Dept. of Interior), Reston, VA.

    This curriculum packet, appropriate for grades 4-8, features a teaching poster which shows different types of maps (different views of Salt Lake City, Utah), as well as three reproducible maps and reproducible activity sheets which complement the maps. The poster provides teacher background, including step-by-step lesson plans for four geography…

  7. Mapping a Changing World.

    ERIC Educational Resources Information Center

    Stoltman, Joseph P.

    1992-01-01

    Addresses the importance of maps for instruction in both history and geography. Suggests that maps have gotten recent attention because of the rapid political changes occurring in Europe and the quincentenary of Columbus' voyage. Discusses different map projections and the importance of media and satellite display of real pictures of the world.…

  8. Mapping Sociological Concepts.

    ERIC Educational Resources Information Center

    Trepagnier, Barbara

    2002-01-01

    Focuses on the use of cognitive mapping within sociology. Describes an assignment where students created a cognitive map that focused on names of theorists and concepts related to them. Discusses sociological imagination in relation to cognitive mapping and the assessment of the assignment. (CMK)

  9. Adaptive Composite Map Projections.

    PubMed

    Jenny, B

    2012-12-01

    All major web mapping services use the web Mercator projection. This is a poor choice for maps of the entire globe or areas of the size of continents or larger countries because the Mercator projection shows medium and higher latitudes with extreme areal distortion and provides an erroneous impression of distances and relative areas. The web Mercator projection is also not able to show the entire globe, as polar latitudes cannot be mapped. When selecting an alternative projection for information visualization, rivaling factors have to be taken into account, such as map scale, the geographic area shown, the map's height-to-width ratio, and the type of cartographic visualization. It is impossible for a single map projection to meet the requirements for all these factors. The proposed composite map projection combines several projections that are recommended in cartographic literature and seamlessly morphs map space as the user changes map scale or the geographic region displayed. The composite projection adapts the map's geometry to scale, to the map's height-to-width ratio, and to the central latitude of the displayed area by replacing projections and adjusting their parameters. The composite projection shows the entire globe including poles; it portrays continents or larger countries with less distortion (optionally without areal distortion); and it can morph to the web Mercator projection for maps showing small regions.

  10. Evidence for an unusual transmembrane configuration of AGG3, a class C Gγ subunit of Arabidopsis.

    PubMed

    Wolfenstetter, Susanne; Chakravorty, David; Kula, Ryan; Urano, Daisuke; Trusov, Yuri; Sheahan, Michael B; McCurdy, David W; Assmann, Sarah M; Jones, Alan M; Botella, José R

    2015-02-01

    Heterotrimeric G proteins are crucial for the perception of external signals and subsequent signal transduction in animal and plant cells. In both model systems, the complex comprises one Gα, one Gβ, and one Gγ subunit. However, in addition to the canonical Gγ subunits (class A), plants also possess two unusual, plant-specific classes of Gγ subunits (classes B and C) that have not yet been found in animals. These include Gγ subunits lacking the C-terminal CaaX motif (class B), which is important for membrane anchoring of the protein; the presence of such subunits gives rise to a flexible sub-population of Gβ/γ heterodimers that are not necessarily restricted to the plasma membrane. Plants also contain class C Gγ subunits, which are twice the size of canonical Gγ subunits, with a predicted transmembrane domain and a large cysteine-rich extracellular C-terminus. However, neither the presence of the transmembrane domain nor the membrane topology have been unequivocally demonstrated. Here, we provide compelling evidence that AGG3, a class C Gγ subunit of Arabidopsis, contains a functional transmembrane domain, which is sufficient but not essential for plasma membrane localization, and that the cysteine-rich C-terminus is extracellular.

  11. Regulation of Voltage-Activated K(+) Channel Gating by Transmembrane β Subunits.

    PubMed

    Sun, Xiaohui; Zaydman, Mark A; Cui, Jianmin

    2012-01-01

    Voltage-activated K(+) (K(V)) channels are important for shaping action potentials and maintaining resting membrane potential in excitable cells. K(V) channels contain a central pore-gate domain (PGD) surrounded by four voltage-sensing domains (VSDs). The VSDs will change conformation in response to alterations of the membrane potential thereby inducing the opening of the PGD. Many K(V) channels are heteromeric protein complexes containing auxiliary β subunits. These β subunits modulate channel expression and activity to increase functional diversity and render tissue specific phenotypes. This review focuses on the K(V) β subunits that contain transmembrane (TM) segments including the KCNE family and the β subunits of large conductance, Ca(2+)- and voltage-activated K(+) (BK) channels. These TM β subunits affect the voltage-dependent activation of K(V) α subunits. Experimental and computational studies have described the structural location of these β subunits in the channel complexes and the biophysical effects on VSD activation, PGD opening, and VSD-PGD coupling. These results reveal some common characteristics and mechanistic insights into K(V) channel modulation by TM β subunits.

  12. Incorporation of high-molecular-weight glutenin subunits into doughs using 2 gram mixograph and extensigraphs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To study the contributions of high-molecular-weight glutenin subunits (HMW-GS) to the gluten macropolymer and dough properties, wheat HMW-GS (x- and y-types) are synthesized in a bacterial expression system. These subunits are then purified and used to supplement dough mixing and extensigraph exper...

  13. Conformational Transitions of Subunit ɛ in ATP Synthase from Thermophilic Bacillus PS3

    PubMed Central

    Feniouk, Boris A.; Kato-Yamada, Yasuyuki; Yoshida, Masasuke; Suzuki, Toshiharu

    2010-01-01

    Abstract Subunit ɛ of bacterial and chloroplast FOF1-ATP synthase is responsible for inhibition of ATPase activity. In Bacillus PS3 enzyme, subunit ɛ can adopt two conformations. In the “extended”, inhibitory conformation, its two C-terminal α-helices are stretched along subunit γ. In the “contracted”, noninhibitory conformation, these helices form a hairpin. The transition of subunit ɛ from an extended to a contracted state was studied in ATP synthase incorporated in Bacillus PS3 membranes at 59°C. Fluorescence energy resonance transfer between fluorophores introduced in the C-terminus of subunit ɛ and in the N-terminus of subunit γ was used to follow the conformational transition in real time. It was found that ATP induced the conformational transition from the extended to the contracted state (half-maximum transition extent at 140 μM ATP). ADP could neither prevent nor reverse the ATP-induced conformational change, but it did slow it down. Acid residues in the DELSEED region of subunit β were found to stabilize the extended conformation of ɛ. Binding of ATP directly to ɛ was not essential for the ATP-induced conformational change. The ATP concentration necessary for the half-maximal transition (140 μM) suggests that subunit ɛ probably adopts the extended state and strongly inhibits ATP hydrolysis only when the intracellular ATP level drops significantly below the normal value. PMID:20141757

  14. All three subunits of soybean beta-conglycinin are potential food allergens.

    PubMed

    Krishnan, Hari B; Kim, Won-Seok; Jang, Sungchan; Kerley, Monty S

    2009-02-11

    Soybeans are recognized as one of the "big 8" food allergens. IgE antibodies from soybean-sensitive patients recognize more than 15 soybean proteins. Among these proteins only the alpha-subunit of beta-conglycinin, but not the highly homologous alpha'- and beta-subunits, has been shown to be a major allergenic protein. The objective of this study was to examine if the alpha'- and beta-subunits of beta-conglycinin can also serve as potential allergens. Immunoblot analysis using sera collected from soybean-allergic patients revealed the presence of IgE antibodies that recognized several soy proteins including 72, 70, 52, 34, and 21 kDa proteins. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) analysis of trypsin-digested 72, 70, and 52 kDa proteins indicated that these proteins were the alpha'-, alpha-, and beta-subunits of beta-conglycinin, respectively. Additionally, purified alpha'-, alpha-, and beta-subunits of beta-conglycinin were recognized by IgE antibodies present in the soybean-allergic patients. The IgE reactivity to the beta-subunit of beta-conglycinin was not abolished when this glycoprotein was either deglycosylated using glycosidases or expressed as a recombinant protein in Escherichia coli . The results suggest that in addition to the previously recognized alpha-subunit of beta-conglycinin, the alpha'- and beta-subunits of beta-conglycinin also are potential food allergens.

  15. Similar GABAA receptor subunit composition in somatic and axon initial segment synapses of hippocampal pyramidal cells

    PubMed Central

    Kerti-Szigeti, Katalin; Nusser, Zoltan

    2016-01-01

    Hippocampal pyramidal cells (PCs) express many GABAAR subunit types and receive GABAergic inputs from distinct interneurons. Previous experiments revealed input-specific differences in α1 and α2 subunit densities in perisomatic synapses, suggesting distinct IPSC decay kinetics. However, IPSC decays evoked by axo-axonic, parvalbumin- or cholecystokinin-expressing basket cells were found to be similar. Using replica immunogold labeling, here we show that all CA1 PC somatic and AIS synapses contain the α1, α2, β1, β2, β3 and γ2 subunits. In CA3 PCs, 90% of the perisomatic synapses are immunopositive for the α1 subunit and all synapses are positive for the remaining five subunits. Somatic synapses form unimodal distributions based on their immunoreactivity for these subunits. The α2 subunit densities in somatic synapses facing Cav2.1 (i.e. parvalbumin) or Cav2.2 (cholecystokinin) positive presynaptic active zones are comparable. We conclude that perisomatic synapses made by three distinct interneuron types have similar GABAA receptor subunit content. DOI: http://dx.doi.org/10.7554/eLife.18426.001 PMID:27537197

  16. Skeletal muscle sodium channel is affected by an epileptogenic beta1 subunit mutation.

    PubMed

    Moran, O; Conti, F

    2001-03-23

    The syndrome of generalized epilepsy with febrile seizures plus type 1 (GEFS+) has been associated to the gene SCN1B coding for the sodium channel beta1 subunit (Wallace, R. H. et al. (1998) Nature Genetics 19, 366-370). In patients, a mutation of the cysteine 121 to trpyptophane (C121W) would cause a lack of modulatory activity of the beta1 subunit on sodium channels expressed in the brain, rendering neurons hyperexcitable. We have confirmed that the normal beta1-modulation of type-IIA adult brain alpha subunits (BIIA) expressed in frog oocytes is defective in C121W. We observed that the mixture of wild-type and mutant beta1 subunits is less effective than wild-type alone, suggesting that the mutant beta1 subunit does bind the alpha subunit. However, we also observed a similar lack of modulation by C121W of the in adult skeletal muscle alpha subunit (SkM1). This finding is in contrast with the simple idea that the mutational effect observed in the oocyte expression system is the principal physiopathological correlate of GEFS+, because no skeletal muscle symptoms have been reported in GEFS+ patients. We conclude that the manifestation of the pathological phenotype is conditioned by the presence of susceptibility genes and/or that the frog oocyte expression system is inadequate for the study of the mutant beta1 subunit physiopathology.

  17. Identification and isolation of three proteasome subunits and their encoding genes from Trypanosoma brucei.

    PubMed

    Huang, L; Shen, M; Chernushevich, I; Burlingame, A L; Wang, C C; Robertson, C D

    1999-08-20

    We have determined peptide sequences of three Trypanosoma brucei proteasome subunit proteins by mass spectrometry of tryptic digests of the proteins purified by two-dimensional (2-D) polyacrylamide gel electrophoresis. Three genes identified by the sequence of their cDNA encode the peptides identified in these three proteins. The three proteins predicted from the gene sequences have significant similarity to other known proteasome subunits and represent an alpha6 type subunit (TbPSA6), and two beta-type subunits belonging to the beta1-type (TbPSB1) and beta2 type (TbPSB2). The sequences of both beta-subunits predict formation of catalytically active subunits through proteolytic processing. The prediction is supported by the presence in each of the two beta-subunits of a tryptic peptide that has the correctly processed N-terminus that creates the threonine nucleophile of the mature protein. This peptide cannot be generated by trypsin because of the required cleavage of a glycine-threonine bond. It is thus likely that there are at least two catalytically active beta-subunits, TbPSB1 and TbPSB2, present in the mature 20S proteasome from T. brucei.

  18. Utility of Respiratory Vaccination With Recombinant Subunit Vaccines for Protection Against Pneumonic Plague

    DTIC Science & Technology

    2002-01-01

    Immunity at mucosal sites can prevent pathogen infection of the host. A) oral poliovirus vaccine B) inhaled influenza vaccine C) kennel cough & Newcastle...Utility of respiratory vaccination with recombinant subunit vaccines for protection against pneumonic plague. Douglas S. Reed & Jennifer Smoll...2. REPORT TYPE N/A 3. DATES COVERED - 4. TITLE AND SUBTITLE Utility of respiratory vaccination with recombinant subunit vaccines for

  19. Specific Inhibition of Herpes Simplex Virus DNA Polymerase by Helical Peptides Corresponding to the Subunit Interface

    NASA Astrophysics Data System (ADS)

    Digard, Paul; Williams, Kevin P.; Hensley, Preston; Brooks, Ian S.; Dahl, Charles E.; Coen, Donald M.

    1995-02-01

    The herpes simplex virus DNA polymerase consists of two subunits-a catalytic subunit and an accessory subunit, UL42, that increases processivity. Mutations affecting the extreme C terminus of the catalytic subunit specifically disrupt subunit interactions and ablate virus replication, suggesting that new antiviral drugs could be rationally designed to interfere with polymerase heterodimerization. To aid design, we performed circular dichroism (CD) spectroscopy and analytical ultracentrifugation studies, which revealed that a 36-residue peptide corresponding to the C terminus of the catalytic subunit folds into a monomeric structure with partial α-helical character. CD studies of shorter peptides were consistent with a model where two separate regions of α-helix interact to form a hairpin-like structure. The 36-residue peptide and a shorter peptide corresponding to the C-terminal 18 residues blocked UL42-dependent long-chain DNA synthesis at concentrations that had no effect on synthesis by the catalytic subunit alone or by calf thymus DNA polymerase δ and its processivity factor. These peptides, therefore, represent a class of specific inhibitors of herpes simplex virus DNA polymerase that act by blocking accessory-subunit-dependent synthesis. These peptides or their structures may form the basis for the synthesis of clinically effective drugs.

  20. Identification of Four Distinct Subunit Types in the Unique 6×6 Hemocyanin of the Centipede Scutigera coleoptrata

    NASA Astrophysics Data System (ADS)

    Gebauer, W.; Markl, J.

    We isolated 6×6 hemocyanin, dissociated it into subunits, and examined it by electron microscopy. The subunits were separated by native polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate PAGE, and crossed immunoelectrophoresis. Single subunits were isolated by gel cutting from native PAGE and identified as hemocyanin by measuring their ultraviolet spectrum. A total of four distinct hemocyanin subunits were identified, and the subunit pattern of the three electrophoresis systems assigned to each other. The relative proportion of subunits a:b:c:d were 2 : 2 :>: 1 as determined by densitometry. Presumably, c and d act as linkers between hexamers.

  1. Subunit compositions of Arabidopsis RNA polymerases I and III reveal Pol I- and Pol III-specific forms of the AC40 subunit and alternative forms of the C53 subunit

    SciTech Connect

    Ream, Thomas S.; Haag, Jeremy R.; Pontvianne, Frederic; Nicora, Carrie D.; Norbeck, Angela D.; Pasa-Tolic, Ljiljana; Pikaard, Craig S.

    2015-05-02

    Using affinity purification and mass spectrometry, we identified the subunits of Arabidopsis thaliana multisubunit RNA Polymerases I and III (abbreviated as Pol I and Pol III), providing the first description of their physical compositions in plants. AC40 and AC19 subunits are typically common to Pol I (a.k.a. Pol A) and Pol III (a.k.a. Pol C) and are encoded by single genes whose mutation, in humans, is a cause of the craniofacial disorder, Treacher-Collins Syndrome. Surprisingly, A. thaliana, and related species, express two distinct AC40 paralogs, one of which assembles into Pol I and the other of which assembles into Pol III. Changes at eight amino acid positions correlate with this functional divergence of Pol I and Pol III-specific AC40 paralogs. Two genes encode homologs of the yeast C53 subunit, and either variant can assemble into Pol III. By contrast, only one of two potential C17 variants, and one of two potential C31 variants were detected in Pol III. We introduce a new nomenclature system for plant Pol I and Pol III subunits in which the twelve subunits that are structurally and functionally homologous among Pols I through V are assigned equivalent numbers.

  2. Subunit compositions of Arabidopsis RNA polymerases I and III reveal Pol I- and Pol III-specific forms of the AC40 subunit and alternative forms of the C53 subunit

    DOE PAGES

    Ream, Thomas S.; Haag, Jeremy R.; Pontvianne, Frederic; ...

    2015-05-02

    Using affinity purification and mass spectrometry, we identified the subunits of Arabidopsis thaliana multisubunit RNA Polymerases I and III (abbreviated as Pol I and Pol III), providing the first description of their physical compositions in plants. AC40 and AC19 subunits are typically common to Pol I (a.k.a. Pol A) and Pol III (a.k.a. Pol C) and are encoded by single genes whose mutation, in humans, is a cause of the craniofacial disorder, Treacher-Collins Syndrome. Surprisingly, A. thaliana, and related species, express two distinct AC40 paralogs, one of which assembles into Pol I and the other of which assembles into Polmore » III. Changes at eight amino acid positions correlate with this functional divergence of Pol I and Pol III-specific AC40 paralogs. Two genes encode homologs of the yeast C53 subunit, and either variant can assemble into Pol III. By contrast, only one of two potential C17 variants, and one of two potential C31 variants were detected in Pol III. We introduce a new nomenclature system for plant Pol I and Pol III subunits in which the twelve subunits that are structurally and functionally homologous among Pols I through V are assigned equivalent numbers.« less

  3. "Silent" Amino Acid Residues at Key Subunit Interfaces Regulate the Geometry of Protein Nanocages.

    PubMed

    Zhang, Shengli; Zang, Jiachen; Zhang, Xiaorong; Chen, Hai; Mikami, Bunzo; Zhao, Guanghua

    2016-11-22

    Rendering the geometry of protein-based assemblies controllable remains challenging. Protein shell-like nanocages represent particularly interesting targets for designed assembly. Here, we introduce an engineering strategy-key subunit interface redesign (KSIR)-that alters a natural subunit-subunit interface by selective deletion of a small number of "silent" amino acid residues (no participation in interfacial interactions) into one that triggers the generation of a non-native protein cage. We have applied KSIR to construct a non-native 48-mer nanocage from its native 24-mer recombinant human H-chain ferritin (rHuHF). This protein is a heteropolymer composed of equal numbers of two different subunits which are derived from one polypeptide. This strategy has allowed the study of conversion between protein nanocages with different geometries by re-engineering key subunit interfaces and the demonstration of the important role of the above-mentioned specific residues in providing geometric specificity for protein assembly.

  4. Electron microscopy and computer image averaging of ice-embedded large ribosomal subunits from Escherichia coli.

    PubMed

    Wagenknecht, T; Grassucci, R; Frank, J

    1988-01-05

    Electron micrographs of frozen-hydrated, large ribosomal subunits from Escherichia coli have been analyzed by computer image processing. Images of subunits in the so-called "crown" orientation were analyzed by correlation alignment procedures developed for negatively stained specimens. Averages of the aligned images showed both similarities and differences to averages determined for negatively stained specimens. The L1 ridge is more dense and stalk-like in frozen-hydrated as compared with negatively stained subunits, possibly because it is associated with ribosomal RNA. The results show that it should be feasible to determine the three-dimensional structure of the large ribosomal subunit from micrographs of individual, frozen-hydrated subunits that have been tilted in the electron microscope.

  5. Balancing acts of two HEAT subunits of condensin I support dynamic assembly of chromosome axes.

    PubMed

    Kinoshita, Kazuhisa; Kobayashi, Tetsuya J; Hirano, Tatsuya

    2015-04-06

    Condensin I is a five-subunit protein complex that plays a central role in mitotic chromosome assembly and segregation in eukaryotes. To dissect its mechanism of action, we reconstituted wild-type and mutant complexes from recombinant subunits and tested their abilities to assemble chromosomes in Xenopus egg cell-free extracts depleted of endogenous condensins. We find that ATP binding and hydrolysis by SMC subunits have distinct contributions to the action of condensin I and that continuous ATP hydrolysis is required for structural maintenance of chromosomes. Mutant complexes lacking either one of two HEAT subunits produce abnormal chromosomes with highly characteristic defects and have contrasting structural effects on chromosome axes preassembled with the wild-type complex. We propose that balancing acts of the two HEAT subunits support dynamic assembly of chromosome axes under the control of the SMC ATPase cycle, thereby governing construction of rod-shaped chromosomes in eukaryotic cells.

  6. Use of concatemers of ligand-gated ion channel subunits to study mechanisms of steroid potentiation.

    PubMed

    Steinbach, Joe Henry; Akk, Gustav

    2011-12-01

    Synaptic receptors of the nicotinic receptor gene family are pentamers of subunits. This modular structure creates problems in studies of drug actions, related to the number of copies of a subunit that are present and their position. A separate issue concerns the mechanism of action of many anesthetics, which involves potentiation of responses to neurotransmitters. Potentiation requires an interaction between a transmitter and a potentiator, mediated through the target receptor. We have studied the mechanism by which neurosteroids potentiate transmitter responses, using concatemers of covalently linked subunits to control the number and position of subunits in the assembled receptor and to selectively introduce mutations into positionally defined copies of a subunit. We found that the steroid needs to interact with only one site to produce potentiation, that the native sites for steroid interaction have indistinguishable properties, and that steroid potentiation appears to result from a global effect on receptor function.

  7. Map projections for larger-scale mapping

    NASA Technical Reports Server (NTRS)

    Snyder, J. P.

    1982-01-01

    For the U.S. Geological Survey maps at 1:1,000,000-scale and larger, the most common projections are conformal, such as the Transverse Mercator and Lambert Conformal Conic. Projections for these scales should treat the Earth as an ellipsoid. In addition, the USGS has conceived and designed some new projections, including the Space Oblique Mercator, the first map projection designed to permit low-distortion mapping of the Earth from satellite imagery, continuously following the groundtrack. The USGS has programmed nearly all pertinent projection equations for inverse and forward calculations. These are used to plot maps or to transform coordinates from one projection to another. The projections in current use are described.

  8. The 2.4-A crystal structure of Scapharca dimeric hemoglobin. Cooperativity based on directly communicating hemes at a novel subunit interface.

    PubMed

    Royer, W E; Hendrickson, W A; Chiancone, E

    1989-12-15

    The crystal structure of the cooperative dimeric hemoglobin from the arcid clam, Scapharca inaequivalvis, has been determined in the carbonmonoxy state. The phase problem was solved for reflections with Bragg spacings greater than 3 A using anomalous scattering from the porphyrin iron atoms measured at a single wavelength in combination with molecular averaging. The model built into this electron density map has been refined at 2.4 A resolution by means of stereochemically restrained least squares minimization to a conventional R-value of 0.156. The root mean square deviation from ideal bond lengths and angles are 0.013 A and 1.7 degrees, respectively. In addition to the 2336 hemoglobin atoms, 214 water molecules have been incorporated into the model. This structure reveals the details of an assemblage of two identical myoglobin-like subunits that is radically different from vertebrate hemoglobins. The subunit interface is formed by direct apposition of the E and F helices, whereas these surfaces are external in vertebrate hemoglobins. The interface has both hydrophobic and hydrophilic character. Two symmetrically related hydrophobic regions are formed between subunits. Six residues are involved in each of these regions that pack tightly enough to exclude water but have only a few atoms in close van der Waals contact. A number of ordered water molecules line the interface and form bridging hydrogen bonds between subunits. Four intersubunit ionic interactions are formed, two of which involve negatively charged propionate groups of the porphyrin. In contrast to cooperative vertebrate hemoglobins, a hydrogen bond network provides a direct route for communication between the two heme groups.

  9. Preparation of a one-subunit cytochrome oxidase from Paracoccus denitrificans: spectral analysis and enzymatic activity.

    PubMed

    Müller, M; Schlapfer, B; Azzi, A

    1988-09-20

    Cytochrome c oxidase was isolated from Paracoccus denitrificans as a two-subunit enzyme. Chymotrypsin-catalyzed proteolysis reduced the molecular weight of each subunit by about 8000. The spectral properties of this preparation, as well as its Km for cytochrome c(1.7 muM), remained unchanged with respect to the native enzyme. Vmax was reduced by about 55% when assayed in Triton X-100 or in Triton X-100 supplemented with asolectin. Following further proteolysis by Staphylococcus aureus V8 protease, subunit I remained unchanged as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas subunit II was split into small peptides. These were removed by ion-exchange high-performance liquid chromatography. The one-subunit enzyme had an apparent molecular weight of 43,000. The reduction of molecular weight was also confirmed by the diminution of the ultraviolet/Soret absorption ratio. This value was 1.8-2.1 for the native enzyme and 1.3-1.5 for the one-subunit enzyme. The spectral properties (including the spectrum CO reduced minus reduced) were not modified by the proteolytic treatment, indicating that cytochromes a and a3 were present in equal amounts. The lack of spectral alteration and the known close association of the copper B atom with cytochrome a3 suggest that copper B is also contained within the one-subunit enzyme. The Km of the one-subunit oxidase was similar to that of the two-subunit enzyme; Vmax was decreased by about 50%. The activity of the one-subunit oxidase had a salt-dependent maximum at 30 mM KCl, almost identical with that of the undigested enzyme, and was inhibited by micromolar concentrations of KCN.

  10. Lateral mobility and anchoring of recombinant GABAA receptors depend on subunit composition.

    PubMed

    Peran, M; Hicks, B W; Peterson, N L; Hooper, H; Salas, R

    2001-10-01

    The clustering of type A gamma-aminobutyric acid receptors (GABA(A)R) at discrete and functionally significant domains on the nerve cell surface is an important determinant in the integration of synaptic inputs. To discern the role that the subunits of the GABA(A)R play in determining the receptor's cell surface topography and mobility, the alpha1, beta1, beta3, and gamma2s subunits were transfected into COS7, HEK293, and PC12 cells and the distribution and cell surface mobility of these recombinant receptors were examined. Our results show that alpha1 subunits are retained in the endoplasmic reticulum while beta1 and beta3 subunits are sorted to the plasma membrane where they form clusters. Co-expression and co-assembly of alpha1 and beta3 subunits result in the rescue of intracellular alpha1 subunits, which are transported as alphabeta subunit complexes to the cell surface where they formed clusters. Fluorescence photobleach recovery and single particle tracking of recombinant receptors show that, despite clustering, beta3 subunit homooligomers are mobile within a cell surface domain. Inclusion of alpha1 in beta3 or beta3gamma2s complexes, however, dramatically reduces the receptor's lateral mobility in COS 7 and PC12 cells and anchors GABA(A)Rs on the cell surface, suggesting the formation of a direct link to a component of the cytoskeleton. The mobility of recombinant receptors that include the alpha1 subunit mirrors the mobility of GABA(A)Rs on cell bodies and dendrites of cortical and spinal cord neurons. The results suggest that incorporation of alpha1 subunits give rise to a population of GABA(A)Rs that are immobilized on the cell surface.

  11. ASSESSMENT OF SUBUNIT-DEPENDENT DIRECT GATING AND ALLOSTERIC MODULATORY EFFECTS OF CARISOPRODOL AT GABAA RECEPTORS

    PubMed Central

    Kumar, Manoj; González, Lorie A.; Dillon, Glenn H.

    2016-01-01

    Carisoprodol is a widely prescribed muscle relaxant, abuse of which has grown considerably in recent years. It directly activates and allosterically modulates α1β2γ2 GABAARs, although the site(s) of action are unknown. To gain insight into the actions of carisoprodol, subunit-dependent effects of this drug were assessed. Whole-cell patch clamp recordings were obtained from HEK293 cells expressing α1β2, α1β3 or αxβzγ2 (where x = 1–6 and z = 1–3) GABAARs, and in receptors incorporating the δ subunit (modeling extrasynaptic receptors). The ability to directly gate and allosterically potentiate GABA-gated currents was observed for all configurations. Presence or absence of the γ2 subunit did not affect the ability of carisoprodol to directly gate or allosterically modulate the receptor. Presence of the β1 subunit conferred highest efficacy for direct activation relative to maximum GABA currents, while presence of the β2 subunit conferred highest efficacy for allosteric modulation of the GABA response. With regard to α subunits, carisoprodol was most efficacious at enhancing the actions of GABA in receptors incorporating the α1 subunit. The ability to directly gate the receptor was generally comparable regardless of the α subunit isoform, although receptors incorporating the α3 subunit showed significantly reduced direct gating efficacy and affinity. In extrasynaptic (α1β3δ and α4β3δ) receptors, carisoprodol had greater efficacy than GABA as a direct gating agonist. In addition, carisoprodol allosterically potentiated both EC20 and saturating GABA concentrations in these receptors. In assessing voltage-dependence, we found direct gating and inhibitory effects were insensitive to membrane voltage, whereas allosteric modulatory effects were affected by membrane voltage. Our findings demonstrate direct and allosteric effects of carisoprodol at synaptic and extrasynpatic GABAARs and that subunit isoform influences these effects. PMID:25896767

  12. Persistence of the mitochondrial permeability transition in the absence of subunit c of human ATP synthase

    PubMed Central

    He, Jiuya; Ford, Holly C.; Carroll, Joe; Ding, Shujing; Fearnley, Ian M.

    2017-01-01

    The permeability transition in human mitochondria refers to the opening of a nonspecific channel, known as the permeability transition pore (PTP), in the inner membrane. Opening can be triggered by calcium ions, leading to swelling of the organelle, disruption of the inner membrane, and ATP synthesis, followed by cell death. Recent proposals suggest that the pore is associated with the ATP synthase complex and specifically with the ring of c-subunits that constitute the membrane domain of the enzyme’s rotor. The c-subunit is produced from three nuclear genes, ATP5G1, ATP5G2, and ATP5G3, encoding identical copies of the mature protein with different mitochondrial-targeting sequences that are removed during their import into the organelle. To investigate the involvement of the c-subunit in the PTP, we generated a clonal cell, HAP1-A12, from near-haploid human cells, in which ATP5G1, ATP5G2, and ATP5G3 were disrupted. The HAP1-A12 cells are incapable of producing the c-subunit, but they preserve the characteristic properties of the PTP. Therefore, the c-subunit does not provide the PTP. The mitochondria in HAP1-A12 cells assemble a vestigial ATP synthase, with intact F1-catalytic and peripheral stalk domains and the supernumerary subunits e, f, and g, but lacking membrane subunits ATP6 and ATP8. The same vestigial complex plus associated c-subunits was characterized from human 143B ρ0 cells, which cannot make the subunits ATP6 and ATP8, but retain the PTP. Therefore, none of the membrane subunits of the ATP synthase that are involved directly in transmembrane proton translocation is involved in forming the PTP. PMID:28289229

  13. Translation initiation factor 3 regulates switching between different modes of ribosomal subunit joining.

    PubMed

    MacDougall, Daniel D; Gonzalez, Ruben L

    2015-05-08

    Ribosomal subunit joining is a key checkpoint in the bacterial translation initiation pathway during which initiation factors (IFs) regulate association of the 30S initiation complex (IC) with the 50S subunit to control formation of a 70S IC that can enter into the elongation stage of protein synthesis. The GTP-bound form of IF2 accelerates subunit joining, whereas IF3 antagonizes subunit joining and plays a prominent role in maintaining translation initiation fidelity. The molecular mechanisms through which IF2 and IF3 collaborate to regulate the efficiency of 70S IC formation, including how they affect the dynamics of subunit joining, remain poorly defined. Here, we use single-molecule fluorescence resonance energy transfer to monitor the interactions between IF2 and the GTPase-associated center (GAC) of the 50S subunit during real-time subunit joining reactions in the absence and presence of IF3. In the presence of IF3, IF2-mediated subunit joining becomes reversible, and subunit joining events cluster into two distinct classes corresponding to formation of shorter- and longer-lifetime 70S ICs. Inclusion of IF3 within the 30S IC was also found to alter the conformation of IF2 relative to the GAC, suggesting that IF3's regulatory effects may stem in part from allosteric modulation of IF2-GAC interactions. The results are consistent with a model in which IF3 can exert control over the efficiency of subunit joining by modulating the conformation of the 30S IC, which in turn influences the formation of stabilizing intersubunit contacts and thus the reaction's degree of reversibility.

  14. On genetic map functions

    SciTech Connect

    Zhao, Hongyu; Speed, T.P.

    1996-04-01

    Various genetic map functions have been proposed to infer the unobservable genetic distance between two loci from the observable recombination fraction between them. Some map functions were found to fit data better than others. When there are more than three markers, multilocus recombination probabilities cannot be uniquely determined by the defining property of map functions, and different methods have been proposed to permit the use of map functions to analyze multilocus data. If for a given map function, there is a probability model for recombination that can give rise to it, then joint recombination probabilities can be deduced from this model. This provides another way to use map functions in multilocus analysis. In this paper we show that stationary renewal processes give rise to most of the map functions in the literature. Furthermore, we show that the interevent distributions of these renewal processes can all be approximated quite well by gamma distributions. 43 refs., 4 figs.

  15. Cartographic mapping study

    NASA Technical Reports Server (NTRS)

    Wilson, C.; Dye, R.; Reed, L.

    1982-01-01

    The errors associated with planimetric mapping of the United States using satellite remote sensing techniques are analyzed. Assumptions concerning the state of the art achievable for satellite mapping systems and platforms in the 1995 time frame are made. An analysis of these performance parameters is made using an interactive cartographic satellite computer model, after first validating the model using LANDSAT 1 through 3 performance parameters. An investigation of current large scale (1:24,000) US National mapping techniques is made. Using the results of this investigation, and current national mapping accuracy standards, the 1995 satellite mapping system is evaluated for its ability to meet US mapping standards for planimetric and topographic mapping at scales of 1:24,000 and smaller.

  16. Cell-free synthesis and assembly of prolyl 4-hydroxylase: the role of the beta-subunit (PDI) in preventing misfolding and aggregation of the alpha-subunit.

    PubMed Central

    John, D C; Grant, M E; Bulleid, N J

    1993-01-01

    Prolyl 4-hydroxylase (P4-H) catalyses a vital post-translational modification in the biosynthesis of collagen. The enzyme consists of two distinct polypeptides forming an alpha 2 beta 2 tetramer (alpha = 64 kDa, beta = 60 kDa), the beta-subunit being identical to the multifunctional enzyme protein disulfide isomerase (PDI). By studying the cell-free synthesis of the rat alpha-subunit of P4-H we have shown that the alpha-subunit can be translocated, glycosylated and the signal peptide cleaved by dog pancreatic microsomal membranes to yield both singly and doubly glycosylated forms. When translations were carried out under conditions which prevent disulfide bond formation, the product synthesized formed aggregates which were associated with the immunoglobulin heavy chain binding protein (BiP). Translations carried out under conditions that promote disulfide bond formation yielded a product that was not associated with BiP but formed a complex with the endogenous beta-subunit (PDI). Complex formation was detected by co-precipitation of the newly synthesized alpha-subunit with antibodies raised against PDI, by sucrose gradient centrifugation and by chemical cross-linking. When microsomal vesicles were depleted of PDI, BiP and other soluble endoplasmic reticulum proteins, no complex formation was observed and the alpha-subunit aggregated even under conditions that promote disulfide bond formation. We have therefore demonstrated that the enzyme P4-H can be assembled at synthesis in a cell-free system and that the solubility of the alpha-subunit is dependent upon its association with PDI. Images PMID:8385607

  17. Cholera Toxin B Subunit Shows Transneuronal Tracing after Injection in an Injured Sciatic Nerve

    PubMed Central

    Lai, Bi-Qin; Qiu, Xue-Chen; Zhang, Ke; Zhang, Rong-Yi; Jin, Hui; Li, Ge; Shen, Hui-Yong; Wu, Jin-Lang; Ling, Eng-Ang; Zeng, Yuan-Shan

    2015-01-01

    Cholera toxin B subunit (CTB) has been extensively used in the past for monosynaptic mapping. For decades, it was thought to lack the ability of transneuronal tracing. In order to investigate whether biotin conjugates of CTB (b-CTB) would pass through transneurons in the rat spinal cord, it was injected into the crushed left sciatic nerve. For experimental control, the first order afferent neuronal projections were defined by retrograde transport of fluorogold (FG, a non-transneuronal labeling marker as an experimental control) injected into the crushed right sciatic nerve in the same rat. Neurons containing b-CTB or FG were observed in the dorsal root ganglia (DRG) at the L4-L6 levels ipsilateral to the tracer injection. In the spinal cord, b-CTB labeled neurons were distributed in all laminae ipsilaterally between C7 and S1 segments, but labeling of neurons at the cervical segment was abolished when the T10 segment was transected completely. The interneurons, distributed in the intermediate gray matter and identified as gamma-aminobutyric acid-ergic (GABAergic), were labeled by b-CTB. In contrast, FG labeling was confined to the ventral horn neurons at L4-L6 spinal segments ipsilateral to the injection. b-CTB immunoreactivity remained to be restricted to the soma of neurons and often appeared as irregular patches detected by light and electron microscopy. Detection of monosialoganglioside (GM1) in b-CTB labeled neurons suggests that GM1 ganglioside may specifically enhance the uptake and transneuronal passage of b-CTB, thus supporting the notion that it may be used as a novel transneuronal tracer. PMID:26640949

  18. Localization of non-linear neutralizing B cell epitopes on ricin toxin's enzymatic subunit (RTA).

    PubMed

    O'Hara, Joanne M; Kasten-Jolly, Jane C; Reynolds, Claire E; Mantis, Nicholas J

    2014-01-01

    Efforts to develop a vaccine for ricin toxin are focused on identifying highly immunogenic, safe, and thermostable recombinant derivatives of ricin's enzymatic A subunit (RTA). As a means to guide vaccine design, we have embarked on an effort to generate a comprehensive neutralizing and non-neutralizing B cell epitope map of RTA. In a series of previous studies, we identified three spatially distinct linear (continuous), neutralizing epitopes on RTA, as defined by monoclonal antibodies (mAbs) PB10 (and R70), SyH7, and GD12. In this report we now describe a new collection of 19 toxin-neutralizing mAbs that bind non-linear epitopes on RTA. The most potent toxin-neutralizing mAbs in this new collection, namely WECB2, TB12, PA1, PH12 and IB2 each had nanamolar (or sub-nanomolar) affinities for ricin and were each capable of passively protecting mice against a 5-10xLD50 toxin challenge. Competitive binding assays by surface plasmon resonance revealed that WECB2 binds an epitope that overlaps with PB10 and R70; TB12, PA1, PH12 recognize epitope(s) close to or overlapping with SyH7's epitope; and GD12 and IB2 recognize epitopes that are spatially distinct from all other toxin-neutralizing mAbs. We estimate that we have now accounted for ∼75% of the predicted epitopes on the surface of RTA and that toxin-neutralizing mAbs are directed against a very limited number of these epitopes. Having this information provides a framework for further refinement of RTA mutagenesis and vaccine design.

  19. Structure of a herpesvirus nuclear egress complex subunit reveals an interaction groove that is essential for viral replication.

    PubMed

    Leigh, Kendra E; Sharma, Mayuri; Mansueto, My Sam; Boeszoermenyi, Andras; Filman, David J; Hogle, James M; Wagner, Gerhard; Coen, Donald M; Arthanari, Haribabu

    2015-07-21

    Herpesviruses require a nuclear egress complex (NEC) for efficient transit of nucleocapsids from the nucleus to the cytoplasm. The NEC orchestrates multiple steps during herpesvirus nuclear egress, including disruption of nuclear lamina and particle budding through the inner nuclear membrane. In the important human pathogen human cytomegalovirus (HCMV), this complex consists of nuclear membrane protein UL50, and nucleoplasmic protein UL53, which is recruited to the nuclear membrane through its interaction with UL50. Here, we present an NMR-determined solution-state structure of the murine CMV homolog of UL50 (M50; residues 1-168) with a strikingly intricate protein fold that is matched by no other known protein folds in its entirety. Using NMR methods, we mapped the interaction of M50 with a highly conserved UL53-derived peptide, corresponding to a segment that is required for heterodimerization. The UL53 peptide binding site mapped onto an M50 surface groove, which harbors a large cavity. Point mutations of UL50 residues corresponding to surface residues in the characterized M50 heterodimerization interface substantially decreased UL50-UL53 binding in vitro, eliminated UL50-UL53 colocalization, prevented disruption of nuclear lamina, and halted productive virus replication in HCMV-infected cells. Our results provide detailed structural information on a key protein-protein interaction involved in nuclear egress and suggest that NEC subunit interactions can be an attractive drug target.

  20. Maple syrup urine disease. Complete primary structure of the E1 beta subunit of human branched chain alpha-ketoacid dehydrogenase complex deduced from the nucleotide sequence and a gene analysis of patients with this disease.

    PubMed Central

    Nobukuni, Y; Mitsubuchi, H; Endo, F; Akaboshi, I; Asaka, J; Matsuda, I

    1990-01-01

    A defect in the E1 beta subunit of the branched chain alpha-ketoacid dehydrogenase (BCKDH) complex is one cause of maple syrup urine disease (MSUD). In an attempt to elucidate the molecular basis of MSUD, we isolated and characterized a 1.35 kbp cDNA clone encoding the entire precursor of the E1 beta subunit of BCKDH complex from a human placental cDNA library. Nucleotide sequence analysis revealed that the isolated cDNA clone (lambda hBE1 beta-1) contained a 5'-untranslated sequence of four nucleotides, the translated sequence of 1,176 nucleotides and the 3'-untranslated sequence of 169 nucleotides. Comparison of the amino acid sequence predicted from the nucleotide sequence of the cDNA insert of the clone with the NH2-terminal amino acid sequence of the purified mature bovine BCKDH-E1 beta subunit showed that the cDNA insert encodes for a 342-amino acid subunit with a Mr = 37,585. The subunit is synthesized as the precursor with a leader sequence of 50 amino acids and is processed at the NH2 terminus. A search for protein homology revealed that the primary structure of human BCKDH-E1 beta was similar to the bovine BCKDH-E1 beta and to the E1 beta subunit of human pyruvate dehydrogenase complex, in all regions. The structures and functions of mammalian alpha-ketoacid dehydrogenase complexes are apparently highly conserved. Genomic DNA from lymphoblastoid cell lines derived from normal and five MSUD patients, in whom E1 beta was not detected by immunoblot analysis, gave the same restriction maps on Southern blot analysis. The gene has at least 80 kbp. Images PMID:2365818

  1. Mechanism of Auxiliary Subunit Modulation of Neuronal α1E Calcium Channels

    PubMed Central

    Jones, Lisa P.; Wei, Shao-kui; Yue, David T.

    1998-01-01

    Voltage-gated calcium channels are composed of a main pore-forming α1 moiety, and one or more auxiliary subunits (β, α2δ) that modulate channel properties. Because modulatory properties may vary greatly with different channels, expression systems, and protocols, it is advantageous to study subunit regulation with a uniform experimental strategy. Here, in HEK 293 cells, we examine the expression and activation gating of α1E calcium channels in combination with a β (β1–β4) and/or the α2δ subunit, exploiting both ionic- and gating-current measurements. Furthermore, to explore whether more than one auxiliary subunit can concomitantly specify gating properties, we investigate the effects of cotransfecting α2δ with β subunits, of transfecting two different β subunits simultaneously, and of COOH-terminal truncation of α1E to remove a second β binding site. The main results are as follows. (a) The α2δ and β subunits modulate α1E in fundamentally different ways. The sole effect of α2δ is to increase current density by elevating channel density. By contrast, though β subunits also increase functional channel number, they also enhance maximum open probability (Gmax/Qmax) and hyperpolarize the voltage dependence of ionic-current activation and gating-charge movement, all without discernible effect on activation kinetics. Different β isoforms produce nearly indistinguishable effects on activation. However, β subunits produced clear, isoform-specific effects on inactivation properties. (b) All the β subunit effects can be explained by a gating model in which subunits act only on weakly voltage-dependent steps near the open state. (c) We find no clear evidence for simultaneous modulation by two different β subunits. (d) The modulatory features found here for α1E do not generalize uniformly to other α1 channel types, as α1C activation gating shows marked β isoform dependence that is absent for α1E. Together, these results help to establish a more

  2. Design of a hyperstable 60-subunit protein icosahedron

    NASA Astrophysics Data System (ADS)

    Hsia, Yang; Bale, Jacob B.; Gonen, Shane; Shi, Dan; Sheffler, William; Fong, Kimberly K.; Nattermann, Una; Xu, Chunfu; Huang, Po-Ssu; Ravichandran, Rashmi; Yi, Sue; Davis, Trisha N.; Gonen, Tamir; King, Neil P.; Baker, David

    2016-07-01

    The icosahedron is the largest of the Platonic solids, and icosahedral protein structures are widely used in biological systems for packaging and transport. There has been considerable interest in repurposing such structures for applications ranging from targeted delivery to multivalent immunogen presentation. The ability to design proteins that self-assemble into precisely specified, highly ordered icosahedral structures would open the door to a new generation of protein containers with properties custom-tailored to specific applications. Here we describe the computational design of a 25-nanometre icosahedral nanocage that self-assembles from trimeric protein building blocks. The designed protein was produced in Escherichia coli, and found by electron microscopy to assemble into a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 molar guanidine hydrochloride at up to 80 degrees Celsius, and undergo extremely abrupt, but reversible, disassembly between 2 molar and 2.25 molar guanidinium thiocyanate. The icosahedron is robust to genetic fusions: one or two copies of green fluorescent protein (GFP) can be fused to each of the 60 subunits to create highly fluorescent ‘standard candles’ for use in light microscopy, and a designed protein pentamer can be placed in the centre of each of the 20 pentameric faces to modulate the size of the entrance/exit channels of the cage. Such robust and customizable nanocages should have considerable utility in targeted drug delivery, vaccine design and synthetic biology.

  3. Design of a hyperstable 60-subunit protein icosahedron

    PubMed Central

    Hsia, Yang; Bale, Jacob B.; Gonen, Shane; Shi, Dan; Sheffler, William; Fong, Kimberly K.; Nattermann, Una; Xu, Chunfu; Huang, Po-Ssu; Ravichandran, Rashmi; Yi, Sue; Davis, Trisha N.; Gonen, Tamir; King, Neil P.; Baker, David

    2016-01-01

    The icosahedron and the dodecahedron are the largest of the Platonic solids, and icosahedral protein structures are widely utilized in biological systems for packaging and transport1,2. There has been considerable interest in repurposing such structures3–5, for example, virus-like particles for the targeted delivery and vaccine design. The ability to design proteins that self assemble into precisely specified, highly ordered icosahedral structures would open the door to a new generation of protein 'containers' that could exhibit properties custom-made for various applications. In this manuscript, we describe the computational design of an icosahedral nano-cage that self-assembles from trimeric building blocks. Electron microscopy images of the designed protein expressed in E. coli reveals a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 M guanidine hydrochloride at up to 80 °C, and undergo extremely abrupt, but reversible, disassembly between 2 M and 2.25 M guanidinium thiocyanate. The icosahedron is robust to genetic fusions: one or two copies of superfolder GFP can be fused to each of the 60 subunits to create highly fluorescent standard candles for light microscopy, and a designed protein pentamer can be placed in the center of each of the twenty pentameric faces to potentially gate macromolecule access to the nanocage interior. Such robust designed nanocages should have considerable utility for targeted drug delivery6, vaccine design7, and synthetic biology8. PMID:27309817

  4. Design of a hyperstable 60-subunit protein dodecahedron. [corrected].

    PubMed

    Hsia, Yang; Bale, Jacob B; Gonen, Shane; Shi, Dan; Sheffler, William; Fong, Kimberly K; Nattermann, Una; Xu, Chunfu; Huang, Po-Ssu; Ravichandran, Rashmi; Yi, Sue; Davis, Trisha N; Gonen, Tamir; King, Neil P; Baker, David

    2016-07-07

    The dodecahedron [corrected] is the largest of the Platonic solids, and icosahedral protein structures are widely used in biological systems for packaging and transport. There has been considerable interest in repurposing such structures for applications ranging from targeted delivery to multivalent immunogen presentation. The ability to design proteins that self-assemble into precisely specified, highly ordered icosahedral structures would open the door to a new generation of protein containers with properties custom-tailored to specific applications. Here we describe the computational design of a 25-nanometre icosahedral nanocage that self-assembles from trimeric protein building blocks. The designed protein was produced in Escherichia coli, and found by electron microscopy to assemble into a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 molar guanidine hydrochloride at up to 80 degrees Celsius, and undergo extremely abrupt, but reversible, disassembly between 2 molar and 2.25 molar guanidinium thiocyanate. The dodecahedron [corrected] is robust to genetic fusions: one or two copies of green fluorescent protein (GFP) can be fused to each of the 60 subunits to create highly fluorescent ‘standard candles’ for use in light microscopy, and a designed protein pentamer can be placed in the centre of each of the 20 pentameric faces to modulate the size of the entrance/exit channels of the cage. Such robust and customizable nanocages should have considerable utility in targeted drug delivery, vaccine design and synthetic biology.

  5. Immunoproteasome subunit LMP7 Deficiency Improves Obesity and Metabolic Disorders

    PubMed Central

    Kimura, Hiroaki; Usui, Fumitake; Karasawa, Tadayoshi; Kawashima, Akira; Shirasuna, Koumei; Inoue, Yoshiyuki; Komada, Takanori; Kobayashi, Motoi; Mizushina, Yoshiko; Kasahara, Tadashi; Suzuki, Koichi; Iwasaki, Yusaku; Yada, Toshihiko; Caturegli, Patrizio; Takahashi, Masafumi

    2015-01-01

    Inflammation plays an important role in the development of obesity and metabolic disorders; however, it has not been fully understood how inflammation occurs and is regulated in their pathogenesis. Low-molecular mass protein-7 (LMP7) is a proteolytic subunit of the immunoproteasome that shapes the repertoire of antigenic peptides on major histocompatibility complex class I molecule. In this study, we investigated the role of LMP7 in the development of obesity and metabolic disorders using LMP7-deficient mice. LMP7 deficiency conveyed resistant to obesity, and improved glucose intolerance and insulin sensitivity in mice fed with high-fat diet (HFD). LMP7 deficiency decreased pancreatic lipase expression, increased fecal lipid contents, and inhibited the increase of plasma triglyceride levels upon oral oil administration or HFD feeding. Using bone marrow-transferred chimeric mice, we found that LMP7 in both bone marrow- and non-bone marrow-derived cells contributes to the development of HFD-induced obesity. LMP7 deficiency decreased inflammatory responses such as macrophage infiltration and chemokine expression while it increased serum adiponection levels. These findings demonstrate a novel role for LMP7 and provide new insights into the mechanisms underlying inflammation in the pathophysiology of obesity and metabolic disorders. PMID:26515636

  6. The Regulation of NF-κB Subunits by Phosphorylation

    PubMed Central

    Christian, Frank; Smith, Emma L.; Carmody, Ruaidhrí J.

    2016-01-01

    The NF-κB transcription factor is the master regulator of the inflammatory response and is essential for the homeostasis of the immune system. NF-κB regulates the transcription of genes that control inflammation, immune cell development, cell cycle, proliferation, and cell death. The fundamental role that NF-κB plays in key physiological processes makes it an important factor in determining health and disease. The importance of NF-κB in tissue homeostasis and immunity has frustrated therapeutic approaches aimed at inhibiting NF-κB activation. However, significant research efforts have revealed the crucial contribution of NF-κB phosphorylation to controlling NF-κB directed transactivation. Importantly, NF-κB phosphorylation controls transcription in a gene-specific manner, offering new opportunities to selectively target NF-κB for therapeutic benefit. This review will focus on the phosphorylation of the NF-κB subunits and the impact on NF-κB function. PMID:26999213

  7. On the evolution of the single-subunit RNA polymerases.

    PubMed

    Cermakian, N; Ikeda, T M; Miramontes, P; Lang, B F; Gray, M W; Cedergren, R

    1997-12-01

    Many eukaryotic nuclear genomes as well as mitochondrial plasmids contain genes displaying evident sequence similarity to those encoding the single-subunit RNA polymerase (ssRNAP) of bacteriophage T7 and its relatives. We have collected and aligned these ssRNAP sequences and have constructed unrooted phylogenetic trees that demonstrate the separation of ssRNAPs into three well-defined and nonoverlapping clusters (phage-encoded, nucleus-encoded, and plasmid-encoded). Our analyses indicate that these three subfamiles of T7-like RNAPs shared a common ancestor; however, the order in which the groups diverged cannot be inferred from available data. On the basis of structural similarities and mutational data, we suggest that the ancestral ssRNAP gene may have arisen via duplication and divergence of a DNA polymerase or reverse transcriptase gene. Considering the current phylogenetic distribution of ssRNAP sequences, we further suggest that the origin of the ancestral ssRNAP gene closely paralleled in time the introduction of mitochondria into eukaryotic cells through a eubacterial endosymbiosis.

  8. Expansion of transducin subunit gene families in early vertebrate tetraploidizations.

    PubMed

    Lagman, David; Sundström, Görel; Ocampo Daza, Daniel; Abalo, Xesús M; Larhammar, Dan

    2012-10-01

    Hundreds of gene families expanded in the early vertebrate tetraploidizations including many gene families in the phototransduction cascade. We have investigated the evolution of the heterotrimeric G-proteins of photoreceptors, the transducins, in relation to these events using both phylogenetic analyses and synteny comparisons. Three alpha subunit genes were identified in amniotes and the coelacanth, GNAT1-3; two of these were identified in amphibians and teleost fish, GNAT1 and GNAT2. Most tetrapods have four beta genes, GNB1-4, and teleosts have additional duplicates. Finally, three gamma genes were identified in mammals, GNGT1, GNG11 and GNGT2. Of these, GNGT1 and GNGT2 were found in the other vertebrates. In frog and zebrafish additional duplicates of GNGT2 were identified. Our analyses show all three transducin families expanded during the early vertebrate tetraploidizations and the beta and gamma families gained additional copies in the teleost-specific genome duplication. This suggests that the tetraploidizations contributed to visual specialisations.

  9. Extensions to the D-Cam sub-unit architecture

    NASA Astrophysics Data System (ADS)

    Ryan, Padraig; Connell, Joseph

    2005-06-01

    Multispectral imaging produces large amounts of data which extend processing, transmission and storage systems to their upper limits. Although there are several interface standards specific to image data acquisition, such as CameraLink, it is Firewire which provides a high-speed data bus, integrated control capability, without loss of flexibility, and which is commonly available as a low cost solution. The class of multispectral imaging requires a different treatment of the processing principals than standard imaging. The same spatial region is captured multiple times using different optical wavelengths. This technique finds application in such diverse areas as coastal monitoring, fruit sorting and automated agriculture. Modifications and additional features to the camera operating and configuration parameters are therefore required which are not generally present with conventional imaging sensors. This paper describes extensions to the IIDC Digital Camera (D-Cam) specification in the development of a Firewire technology platform for transmitting the data structures described and for providing real-time, online control of spectral information acquisition. Additionally, it describes how a set of registers in the sub-unit architecture of the Firewire protocol is augmented to accommodate the demands of a multispectral system. The extensions are specification conformant and do not alter underlining compliance with the base standard. The paper also describes the implementation of the extended D-Cam in the Firewire subsystem of a smart multispectral camera used in commercial applications.

  10. Phylogenetic Analyses of Meloidogyne Small Subunit rDNA

    PubMed Central

    De Ley, Irma Tandingan; De Ley, Paul; Vierstraete, Andy; Karssen, Gerrit; Moens, Maurice; Vanfleteren, Jacques

    2002-01-01

    Phylogenies were inferred from nearly complete small subunit (SSU) 18S rDNA sequences of 12 species of Meloidogyne and 4 outgroup taxa (Globodera pallida, Nacobbus abberans, Subanguina radicicola, and Zygotylenchus guevarai). Alignments were generated manually from a secondary structure model, and computationally using ClustalX and Treealign. Trees were constructed using distance, parsimony, and likelihood algorithms in PAUP* 4.0b4a. Obtained tree topologies were stable across algorithms and alignments, supporting 3 clades: clade I = [M. incognita (M. javanica, M. arenaria)]; clade II = M. duytsi and M. maritima in an unresolved trichotomy with (M. hapla, M. microtyla); and clade III = (M. exigua (M. graminicola, M. chitwoodi)). Monophyly of [(clade I, clade II) clade III] was given maximal bootstrap support (mbs). M. artiellia was always a sister taxon to this joint clade, while M. ichinohei was consistently placed with mbs as a basal taxon within the genus. Affinities with the outgroup taxa remain unclear, although G. pallida and S. radicicola were never placed as closest relatives of Meloidogyne. Our results show that SSU sequence data are useful in addressing deeper phylogeny within Meloidogyne, and that both M. ichinohei and M. artiellia are credible outgroups for phylogenetic analysis of speciations among the major species. PMID:19265950

  11. Isolation of cDNA clones for the catalytic gamma subunit of mouse muscle phosphorylase kinase: expression of mRNA in normal and mutant Phk mice.

    PubMed Central

    Chamberlain, J S; VanTuinen, P; Reeves, A A; Philip, B A; Caskey, C T

    1987-01-01

    We have isolated and characterized cDNA clones for the gamma subunit of mouse muscle phosphorylase kinase (gamma-Phk). These clones were isolated from a lambda gt11 mouse muscle cDNA library via screening with a synthetic oligonucleotide probe corresponding to a portion of the rabbit gamma-Phk amino acid sequence. The gamma-Phk cDNA clones code for a 387-amino acid protein that shares 93% amino acid sequence identity with the corresponding rabbit amino acid sequence. RNA gel blot analysis reveals that the muscle gamma-Phk probe hybridizes to two mRNA species (2.4 and 1.6 kilobases) in skeletal muscle, cardiac muscle, and brain, but does not hybridize to liver RNA. Phk-deficient I-strain (Phk) mouse muscle contains reduced levels of gamma-Phk mRNA as compared with control mice. Although the Phk defect is an X-linked recessive trait, hybridization to a human-rodent somatic cell hybrid mapping panel shows that the gamma-Phk gene is not located on the X chromosome. Rather, the gamma-Phk cross-hybridizing human restriction fragments map to human chromosomes 7 (multiple) and 11 (single). Reduced gamma-Phk mRNA in I-strain mice, therefore, appears to be a consequence of the Phk-mutant trait and does not stem from a mutant gamma-subunit gene. Images PMID:3472241

  12. Characterization and mutagenesis of the gene encoding the A49 subunit of RNA polymerase A in Saccharomyces cerevisiae.

    PubMed Central

    Liljelund, P; Mariotte, S; Buhler, J M; Sentenac, A

    1992-01-01

    The gene encoding the 49-kDa subunit of RNA polymerase A in Saccharomyces cerevisiae has been identified by formation of a hybrid enzyme between the S. cerevisiae A49 subunit and Saccharomyces douglasii subunits based on a polymorphism existing between the subunits of RNA polymerase A in these two species. The sequence of the gene reveals a basic protein with an unusually high lysine content, which may account for the affinity for DNA shown by the subunit. No appreciable homology with any polymerase subunits, enzymes, or transcription factors is found. Complete deletion of the single-copy RPA49 gene leads to viable but slowly growing colonies. Insertion of the HIS3 gene halfway into the RPA49 coding region results in synthesis of a truncated A49 subunit that is incorporated into the polymerase. The truncated and wild-type subunits compete equally for assembly in the heterozygous diploid, although the wild type is phenotypically dominant. Images PMID:1409638

  13. Comparing landslide inventory maps

    NASA Astrophysics Data System (ADS)

    Galli, Mirco; Ardizzone, Francesca; Cardinali, Mauro; Guzzetti, Fausto; Reichenbach, Paola

    Landslide inventory maps are effective and easily understandable products for both experts, such as geomorphologists, and for non experts, including decision-makers, planners, and civil defense managers. Landslide inventories are essential to understand the evolution of landscapes, and to ascertain landslide susceptibility and hazard. Despite landslide maps being compiled every year in the word at different scales, limited efforts are made to critically compare landslide maps prepared using different techniques or by different investigators. Based on the experience gained in 20 years of landslide mapping in Italy, and on the limited literature on landslide inventory assessment, we propose a general framework for the quantitative comparison of landslide inventory maps. To test the proposed framework we exploit three inventory maps. The first map is a reconnaissance landslide inventory prepared for the Umbria region, in central Italy. The second map is a detailed geomorphological landslide map, also prepared for the Umbria region. The third map is a multi-temporal landslide inventory compiled for the Collazzone area, in central Umbria. Results of the experiment allow for establishing how well the individual inventories describe the location, type and abundance of landslides, to what extent the landslide maps can be used to determine the frequency-area statistics of the slope failures, and the significance of the inventory maps as predictors of landslide susceptibility. We further use the results obtained in the Collazzone area to estimate the quality and completeness of the two regional landslide inventory maps, and to outline general advantages and limitations of the techniques used to complete the inventories.

  14. Distinctive interactions of the Arabidopsis homolog of the 30 kD subunit of the cleavage and polyadenylation specificity factor (AtCPSF30) with other polyadenylation factor subunits

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: The Arabidopsis ortholog of the 30 kD subunit of the mammalian Cleavage and Polyadenylation Specificity Factor (AtCPSF30) is an RNA-binding endonuclease that is associated with other Arabidopsis CPSF subunits (orthologs of the 160, 100, and 73 kD subunits of CPSF). In order to better u...

  15. Recombinant GABAA receptor desensitization: the role of the gamma 2 subunit and its physiological significance.

    PubMed

    Dominguez-Perrot, C; Feltz, P; Poulter, M O

    1996-11-15

    1. The purpose of these investigations was to examine the role that the gamma 2 subunit plays in human GABAA receptor desensitization. Two different recombinant GABAA receptors (alpha 1 beta 3 and alpha 1 beta 3 gamma 2) were compared by measuring the relaxation of whole-cell currents during the application of GABA, isoguvacine or taurine. 2. At concentrations which trigger a maximum response (100-500 microM GABA) the current relaxation usually fitted the sum of two exponentials. For alpha 1 beta 3 subunit receptors these values were tau 1 = 145 +/- 12 ms and tau 2 = 6.3 +/- 2.1 s (means +/- S.E.M.). Receptors consisting of alpha 1 beta 3 gamma 2 subunits desensitized faster: tau 1 = 41.6 +/- 8.3 ms and tau 2 = 2.4 +/- 0.6 s. 3. The Hill slope, determined for each receptor subunit combination, was the same and greater than 1.0, implying two binding steps in the activation of both receptor subunit combinations. 4. For alpha 1 beta 3 subunit receptors the fast desensitization rates were unaltered by reducing the GABA concentration from the EC100 (100 microM) to the approximate EC50 values (10-20 microM), whereas for alpha 1 beta 3 gamma 2 subunit receptors a significant slowing was observed. The fast desensitization disappeared at agonist concentrations below the EC50 for both subunit combinations. In contrast, the slow desensitization appeared at agonist concentrations near the EC20. This rate was dependent on agonist concentration reaching a maximum near the EC60 value of GABA. 5. The fast desensitization rates were unaltered by changing the holding potential of the cell during agonist application. However, for alpha 1 beta 3 gamma 2 subunit receptors the slow desensitization rate increased by approximately 15- to 20-fold over the range of voltages of -60 to +40 mV. This indicates that the gamma 2 subunit makes GABAA receptor desensitization voltage dependent. 6. Recovery from desensitization was also biphasic. The first recovery phase was faster for alpha 1 beta 3

  16. Molecular cloning of pituitary glycoprotein alpha-subunit and follicle stimulating hormone and chorionic gonadotropin beta-subunits from New World squirrel monkey and owl monkey.

    PubMed

    Scammell, Jonathan G; Funkhouser, Jane D; Moyer, Felricia S; Gibson, Susan V; Willis, Donna L

    2008-02-01

    The goal of this study was to characterize the gonadotropins expressed in pituitary glands of the New World squirrel monkey (Saimiri sp.) and owl monkey (Aotus sp.). The various subunits were amplified from total RNA from squirrel monkey and owl monkey pituitary glands by reverse transcription-polymerase chain reaction and the deduced amino acid sequences compared to those of other species. Mature squirrel monkey and owl monkey glycoprotein hormone alpha-polypeptides (96 amino acids in length) were determined to be 80% homologous to the human sequence. The sequences of mature beta subunits of follicle stimulating hormone (FSHbeta) from squirrel monkey and owl monkey (111 amino acids in length) are 92% homologous to human FSHbeta. New World primate glycoprotein hormone alpha-polypeptides and FSHbeta subunits showed conservation of all cysteine residues and consensus N-linked glycosylation sites. Attempts to amplify the beta-subunit of luteinizing hormone from squirrel monkey and owl monkey pituitary glands were unsuccessful. Rather, the beta-subunit of chorionic gonadotropin (CG) was amplified from pituitaries of both New World primates. Squirrel monkey and owl monkey CGbeta are 143 and 144 amino acids in length and 77% homologous with human CGbeta. The greatest divergence is in the C terminus, where all four sites for O-linked glycosylation in human CGbeta, responsible for delayed metabolic clearance, are predicted to be absent in New World primate CGbetas. It is likely that CG secreted from pituitary of New World primates exhibits a relatively short half-life compared to human CG.

  17. Stabilization of the Escherichia coli DNA polymerase III ε subunit by the θ subunit favors in vivo assembly of the Pol III catalytic core

    PubMed Central

    Conte, Emanuele; Vincelli, Gabriele; Schaaper, Roel M.; Bressanin, Daniela; Stefan, Alessandra; Dal Piaz, Fabrizio; Hochkoeppler, Alejandro

    2012-01-01

    Escherichia coli DNA polymerase III holoenzyme (HE) contains a core polymerase consisting of three subunits: α(polymerase), ε(3′-5′ exonuclease), and θ. Genetic experiments suggested that θ subunit stabilizes the intrinsically labile ε subunit and, furthermore, that θ might affect the cellular amounts of Pol III core and HE. Here, we provide biochemical evidence supporting this model by analyzing the amounts of the relevant proteins. First, we show that a ΔholE strain (lacking θ subunit) displays reduced amounts of free ε. We also demonstrate the existence of a dimer of ε, which may be involved in the stabilization of the protein. Second, θ, when overexpressed, dissociates the ε dimer and significantly increases the amount of Pol III core. The stability of ε also depends on cellular chaperones, including DnaK. Here, we report that: (i) temperature shift-up of ΔdnaK strains leads to rapid depletion of ε, and (ii) overproduction of θ overcomes both the depletion of ε and the temperature sensitivity of the strain. Overall, our data suggest that ε is a critical factor in the assembly of Pol III core, and that this is role is strongly influenced by the θ subunit through its prevention of ε degradation. PMID:22546509

  18. Tuning the Biological Activity Profile of Antibacterial Polymers via Subunit Substitution Pattern

    PubMed Central

    2015-01-01

    Binary nylon-3 copolymers containing cationic and hydrophobic subunits can mimic the biological properties of host-defense peptides, but relationships between composition and activity are not yet well understood for these materials. Hydrophobic subunits in previously studied examples have been limited mostly to cycloalkane-derived structures, with cyclohexyl proving to be particularly promising. The present study evaluates alternative hydrophobic subunits that are isomeric or nearly isomeric with the cyclohexyl example; each has four sp3 carbons in the side chains. The results show that varying the substitution pattern of the hydrophobic subunit leads to relatively small changes in antibacterial activity but causes significant changes in hemolytic activity. We hypothesize that these differences in biological activity profile arise, at least in part, from variations among the conformational propensities of the hydrophobic subunits. The α,α,β,β-tetramethyl unit is optimal among the subunits we have examined, providing copolymers with potent antibacterial activity and excellent prokaryote vs eukaryote selectivity. Bacteria do not readily develop resistance to the new antibacterial nylon-3 copolymers. These findings suggest that variation in subunit conformational properties could be generally valuable in the development of synthetic polymers for biological applications. PMID:24601599

  19. A Chaperonin Subunit with Unique Structures Is Essential for Folding of a Specific Substrate

    PubMed Central

    Peng, Lianwei; Fukao, Yoichiro; Myouga, Fumiyoshi; Motohashi, Reiko; Shinozaki, Kazuo; Shikanai, Toshiharu

    2011-01-01

    Type I chaperonins are large, double-ring complexes present in bacteria (GroEL), mitochondria (Hsp60), and chloroplasts (Cpn60), which are involved in mediating the folding of newly synthesized, translocated, or stress-denatured proteins. In Escherichia coli, GroEL comprises 14 identical subunits and has been exquisitely optimized to fold its broad range of substrates. However, multiple Cpn60 subunits with different expression profiles have evolved in chloroplasts. Here, we show that, in Arabidopsis thaliana, the minor subunit Cpn60β4 forms a heterooligomeric Cpn60 complex with Cpn60α1 and Cpn60β1–β3 and is specifically required for the folding of NdhH, a subunit of the chloroplast NADH dehydrogenase-like complex (NDH). Other Cpn60β subunits cannot complement the function of Cpn60β4. Furthermore, the unique C-terminus of Cpn60β4 is required for the full activity of the unique Cpn60 complex containing Cpn60β4 for folding of NdhH. Our findings suggest that this unusual kind of subunit enables the Cpn60 complex to assist the folding of some particular substrates, whereas other dominant Cpn60 subunits maintain a housekeeping chaperonin function by facilitating the folding of other obligate substrates. PMID:21483722

  20. Kv5, Kv6, Kv8, and Kv9 subunits: No simple silent bystanders

    PubMed Central

    2016-01-01

    Members of the electrically silent voltage-gated K+ (Kv) subfamilies (Kv5, Kv6, Kv8, and Kv9, collectively identified as electrically silent voltage-gated K+ channel [KvS] subunits) do not form functional homotetrameric channels but assemble with Kv2 subunits into heterotetrameric Kv2/KvS channels with unique biophysical properties. Unlike the ubiquitously expressed Kv2 subunits, KvS subunits show a more restricted expression. This raises the possibility that Kv2/KvS heterotetramers have tissue-specific functions, making them potential targets for the development of novel therapeutic strategies. Here, I provide an overview of the expression of KvS subunits in different tissues and discuss their proposed role in various physiological and pathophysiological processes. This overview demonstrates the importance of KvS subunits and Kv2/KvS heterotetramers in vivo and the importance of considering KvS subunits and Kv2/KvS heterotetramers in the development of novel treatments. PMID:26755771

  1. Tuning the biological activity profile of antibacterial polymers via subunit substitution pattern.

    PubMed

    Liu, Runhui; Chen, Xinyu; Chakraborty, Saswata; Lemke, Justin J; Hayouka, Zvi; Chow, Clara; Welch, Rodney A; Weisblum, Bernard; Masters, Kristyn S; Gellman, Samuel H

    2014-03-19

    Binary nylon-3 copolymers containing cationic and hydrophobic subunits can mimic the biological properties of host-defense peptides, but relationships between composition and activity are not yet well understood for these materials. Hydrophobic subunits in previously studied examples have been limited mostly to cycloalkane-derived structures, with cyclohexyl proving to be particularly promising. The present study evaluates alternative hydrophobic subunits that are isomeric or nearly isomeric with the cyclohexyl example; each has four sp(3) carbons in the side chains. The results show that varying the substitution pattern of the hydrophobic subunit leads to relatively small changes in antibacterial activity but causes significant changes in hemolytic activity. We hypothesize that these differences in biological activity profile arise, at least in part, from variations among the conformational propensities of the hydrophobic subunits. The α,α,β,β-tetramethyl unit is optimal among the subunits we have examined, providing copolymers with potent antibacterial activity and excellent prokaryote vs eukaryote selectivity. Bacteria do not readily develop resistance to the new antibacterial nylon-3 copolymers. These findings suggest that variation in subunit conformational properties could be generally valuable in the development of synthetic polymers for biological applications.

  2. Assembly and Intracellular Targeting of the βγ Subunits of Heterotrimeric G Proteins

    PubMed Central

    Rehm, Armin; Ploegh, Hidde L.

    1997-01-01

    The assembly in living cells of heterotrimeric guanine nucleotide binding proteins from their constituent α, β, and γ subunits is a complex process, compounded by the multiplicity of the genes that encode them, and the diversity of receptors and effectors with which they interact. Monoclonal anti-β antibodies (ARC5 and ARC9), raised against immunoaffinity purified βγ complexes, recognize β subunits when not associated with γ and can thus be used to monitor assembly of βγ complexes. Complex formation starts immediately after synthesis and is complete within 30 min. Assembly occurs predominantly in the cytosol, and association of βγ complexes with the plasma membrane fraction starts between 15–30 min of chase. Three pools of β subunits can be distinguished based on their association with γ subunits, their localization, and their detergent solubility. Association of β and α subunits with detergent-insoluble domains occurs within 1 min of chase, and increases to reach a plateau of near complete detergent resistance within 30 min of chase. Brefeldin A treatment does not interfere with delivery of βγ subunits to detergent-insoluble domains, suggesting that assembly of G protein subunits with their receptors occurs distally from the BFA-imposed block of intracellular membrane trafficking and may occur directly at the plasma membrane. PMID:9128244

  3. Physiological effects of unassembled chaperonin Cct subunits in the yeast Saccharomyces cerevisiae.

    PubMed

    Kabir, M Anaul; Kaminska, Joanna; Segel, George B; Bethlendy, Gabor; Lin, Paul; Della Seta, Flavio; Blegen, Casey; Swiderek, Kristine M; Zoładek, Teresa; Arndt, Kim T; Sherman, Fred

    2005-02-01

    Eukaryotic chaperonins, the Cct complexes, are assembled into two rings, each of which is composed of a stoichiometric array of eight different subunits, which are denoted Cct1p-Cct8p. Overexpression of a single CCT gene in Saccharomyces cerevisiae causes an increase of the corresponding Cct subunit, but not of the Cct complex. Nevertheless, overexpression of certain Cct subunits, especially CCT6, suppresses a wide range of abnormal phenotypes, including those caused by the diverse types of conditional mutations tor2-21, lst8-2 and rsp5-9 and those caused by the concomitant overexpression of Sit4p and Sap155p. The examination of 73 altered forms of Cct6p revealed that the cct6-24 mutation, containing GDGTT --> AAAAA replacements of the conserved ATP-binding motif, was unable to suppress any of these traits, although the cct6-24 allele was completely functional for growth. These results provide evidence for functional differences among Cct subunits and for physiological properties of unassembled subunits. We suggest that the suppression is due to the competition of specific Cct subunits for activities that normally modify various cellular components. Furthermore, we also suggest that the Cct subunits can act as suppressors only in certain states, such as when associated with ATP.

  4. Structural and spectroscopic studies of the native hemocyanin from Maia squinado and its structural subunits

    NASA Astrophysics Data System (ADS)

    Dolashka-Angelova, Pavlina; Hristova, Rumijana; Schuetz, Juergen; Stoeva, Stanka; Schwarz, Heinz; Voelter, Wolfgang

    2000-09-01

    The dodecameric hemocyanin of the crab Maia squinado contains five major electrophoretically separable polypeptide chains (structural subunits) which have been purified by FPLC ion exchange chromatography. The various proteins have been characterized by fluorescence spectroscopy, combined with fluorescence quenching studies, using acrylamide, caesium chloride and potassium iodide as tryptophan quenchers. The results show that the tryptophyl side chains of dodecameric Hc are deeply buried in hydrophobic regions of the hemocyanin aggregates and the quenching efficiency values for the native Hc in comparison with those from the constituent subunits are two to four times less. The conformational stabilities of the native dodecameric aggregate and its isolated structural subunits towards various denaturants (pH, temperature, guanidinium hydrochloride) indicate that the quaternary structure is stabilized by hydrophilic and polar forces, whereby, both, the oxy- and apo-forms of the protein have been considered. The critical temperatures for the structural subunits, Tc, determined by fluorescence spectroscopy, are in the region of 50-60°C, coinciding with the melting temperatures, Tm, determined by CD spectroscopy. The free energy of stabilization in water, Δ GDH 2O , toward guanidinium hydrochloride is about two times higher for the dodecamer as compared to the isolated subunits. These studies reveal that oligomerization between functional subunits has a stabilizing effect on the whole molecule and differences in the primary structures result in different stabilities of the subunits.

  5. Fractional dissipative standard map.

    PubMed

    Tarasov, Vasily E; Edelman, M

    2010-06-01

    Using kicked differential equations of motion with derivatives of noninteger orders, we obtain generalizations of the dissipative standard map. The main property of these generalized maps, which are called fractional maps, is long-term memory. The memory effect in the fractional maps means that their present state of evolution depends on all past states with special forms of weights. Already a small deviation of the order of derivative from the integer value corresponding to the regular dissipative standard map (small memory effects) leads to the qualitatively new behavior of the corresponding attractors. The fractional dissipative standard maps are used to demonstrate a new type of fractional attractors in the wide range of the fractional orders of derivatives.

  6. Subunit Conformations and Assembly States of a DNA Translocating Motor: The Terminase of Bacteriophage P22

    PubMed Central

    Němeček, Daniel; Gilcrease, Eddie B.; Kang, Sebyung; Prevelige, Peter E.; Casjens, Sherwood; Thomas, George J.

    2007-01-01

    Bacteriophage P22, a podovirus infecting strains of Salmonella typhimurium, packages a 42 kbp genome using a headful mechanism. DNA translocation is accomplished by the phage terminase, a powerful molecular motor consisting of large and small subunits. Although many of the structural proteins of the P22 virion have been well characterized, little is known about the terminase subunits and their molecular mechanism of DNA translocation. We report here structural and assembly properties of ectopically expressed and highly purified terminase large and small subunits. The large subunit (gp2), which contains the nuclease and ATPase activities of terminase, exists as a stable monomer with an α/β fold. The small subunit (gp3), which recognizes DNA for packaging and may regulate gp2 activity, exhibits a highly α-helical secondary structure and self-associates to form a stable oligomeric ring in solution. For wildtype gp3, the ring contains nine subunits, as demonstrated by hydrodynamic measurements, electron microscopy and native mass spectrometry. We have also characterized a gp3 mutant (Ala 112 → Thr) that forms a ten subunit ring, despite a subunit fold indistinguishable from wildtype. Both the nonameric and decameric gp3 rings exhibit nonspecific DNA binding activity, and gp2 is able to bind strongly to the DNA/gp3 complex but not to DNA alone. We propose a scheme for the roles of P22 terminase large and small subunits in the recruitment and packaging of viral DNA and discuss the model in relation to proposals for terminase-driven DNA translocation in other phages. PMID:17945256

  7. Kv2 subunits underlie slowly inactivating potassium current in rat neocortical pyramidal neurons

    PubMed Central

    Guan, D; Tkatch, T; Surmeier, D J; Armstrong, W E; Foehring, R C

    2007-01-01

    We determined the expression of Kv2 channel subunits in rat somatosensory and motor cortex and tested for the contributions of Kv2 subunits to slowly inactivating K+ currents in supragranular pyramidal neurons. Single cell RT-PCR showed that virtually all pyramidal cells expressed Kv2.1 mRNA and ∼80% expressed Kv2.2 mRNA. Immunocytochemistry revealed striking differences in the distribution of Kv2.1 and Kv2.2 subunits. Kv2.1 subunits were clustered and located on somata and proximal dendrites of all pyramidal cells. Kv2.2 subunits were primarily distributed on large apical dendrites of a subset of pyramidal cells from deep layers. We used two methods for isolating currents through Kv2 channels after excluding contributions from Kv1 subunits: intracellular diffusion of Kv2.1 antibodies through the recording pipette and extracellular application of rStromatoxin-1 (ScTx). The Kv2.1 antibody specifically blocked the slowly inactivating K+ current by 25–50% (at 8 min), demonstrating that Kv2.1 subunits underlie much of this current in neocortical pyramidal neurons. ScTx (300 nm) also inhibited ∼40% of the slowly inactivating K+ current. We observed occlusion between the actions of Kv2.1 antibody and ScTx. In addition, Kv2.1 antibody- and ScTx-sensitive currents demonstrated similar recovery from inactivation and voltage dependence and kinetics of activation and inactivation. These data indicate that both agents targeted the same channels. Considering the localization of Kv2.1 and 2.2 subunits, currents from truncated dissociated cells are probably dominated by Kv2.1 subunits. Compared with Kv2.1 currents in expression systems, the Kv2.1 current in neocortical pyramidal cells activated and inactivated at relatively negative potentials and was very sensitive to holding potential. PMID:17379638

  8. Postsynaptic clustering of γ-aminobutyric acid type A receptors by the γ3 subunit in vivo

    PubMed Central

    Baer, Kristin; Essrich, Christian; Benson, Jack A.; Benke, Dietmar; Bluethmann, Horst; Fritschy, Jean-Marc; Lüscher, Bernhard

    1999-01-01

    Synaptic localization of γ-aminobutyric acid type A (GABAA) receptors is a prerequisite for synaptic inhibitory function, but the mechanism by which different receptor subtypes are localized to postsynaptic sites is poorly understood. The γ2 subunit and the postsynaptic clustering protein gephyrin are required for synaptic localization and function of major GABAA receptor subtypes. We now show that transgenic overexpression of the γ3 subunit in γ2 subunit-deficient mice restores benzodiazepine binding sites, benzodiazepine-modulated whole cell currents, and postsynaptic miniature currents, suggesting the formation of functional, postsynaptic receptors. Moreover, the γ3 subunit can substitute for γ2 in the formation of GABAA receptors that are synaptically clustered and colocalized with gephyrin in vivo. These clusters were formed even in brain regions devoid of endogenous γ3 subunit, indicating that the factors present for clustering of γ2 subunit-containing receptors are sufficient to cluster γ3 subunit-containing receptors. The GABAA receptor and gephyrin-clustering properties of the ectopic γ3 subunit were also observed for the endogenous γ3 subunit, but only in the absence of the γ2 subunit, suggesting that the γ3 subunit is at a competitive disadvantage with the γ2 subunit for clustering of postsynaptic GABAA receptors in wild-type mice. PMID:10536013

  9. Heat Capacity Mapping Mission

    NASA Technical Reports Server (NTRS)

    Nilsson, C. S.; Andrews, J. C.; Scully-Power, P.; Ball, S.; Speechley, G.; Latham, A. R. (Principal Investigator)

    1980-01-01

    The Tasman Front was delineated by airborne expendable bathythermograph survey; and an Heat Capacity Mapping Mission (HCMM) IR image on the same day shows the same principal features as determined from ground-truth. It is clear that digital enhancement of HCMM images is necessary to map ocean surface temperatures and when done, the Tasman Front and other oceanographic features can be mapped by this method, even through considerable scattered cloud cover.

  10. A genetic analysis of Plasmodium falciparum RNA polymerase II subunits in yeast.

    PubMed

    Hazoume, Adonis; Naderi, Kambiz; Candolfi, Ermanno; Kedinger, Claude; Chatton, Bruno; Vigneron, Marc

    2011-04-01

    RNA polymerase II is an essential nuclear multi subunit enzyme that transcribes nearly the whole genome. Its inhibition by the alpha-amanitin toxin leads to cell death. The enzyme of Plasmodium falciparum remains poorly characterized. Using a complementation assay in yeast as a genetic test, we demonstrate that five Plasmodium putative RNA polymerase subunits are indeed functional in vivo. The active site of this enzyme is built from the two largest subunits. Using site directed mutagenesis we were able to modify the active site of the yeast RNA polymerase II so as to introduce Plasmodium or human structural motifs. The resulting strains allow the screening of chemical libraries for potential specific inhibitors.

  11. Stoichiometry of the Human Glycine Receptor Revealed by Direct Subunit counting

    PubMed Central

    Durisic, Nela; Godin, Antoine G.; Wever, Claudia M.; Heyes, Colin D.; Lakadamyali, Melike; Dent, Joseph A.

    2012-01-01

    The subunit stoichiometry of heteromeric glycine-gated channels (GlyRs) determines fundamental properties of these key inhibitory neurotransmitter receptors; however the ratio of α1 to β-subunits per receptor remains controversial. We used single molecule imaging and stepwise photobleaching in Xenopus oocytes to directly determine the subunit stoichiometry of a glycine receptor to be 3α1:2β. This approach allowed us to determine the receptor stoichiometry in mixed populations consisting of both heteromeric and homomeric channels, additionally revealing the quantitative proportions for the two populations. PMID:22973015

  12. Cereblon inhibits proteasome activity by binding to the 20S core proteasome subunit beta type 4.

    PubMed

    Lee, Kwang Min; Lee, Jongwon; Park, Chul-Seung

    2012-10-26

    In humans, mutations in the gene encoding cereblon (CRBN) are associated with mental retardation. Although CRBN has been investigated in several cellular contexts, its function remains unclear. Here, we demonstrate that CRBN plays a role in regulating the ubiquitin-proteasome system (UPS). Heterologous expression of CRBN inhibited proteasome activity in a human neuroblastoma cell line. Furthermore, proteasome subunit beta type 4 (PSMB4), the β7 subunit of the 20S core complex, was identified as a direct binding partner of CRBN. These findings suggest that CRBN may modulate proteasome activity by directly interacting with the β7 subunit.

  13. zeta-COP, a subunit of coatomer, is required for COP-coated vesicle assembly

    PubMed Central

    1993-01-01

    cDNA encoding the 20-kD subunit of coatomer, zeta-COP, predicts a protein of 177-amino acid residues, similar in sequence to AP17 and AP19, subunits of the clathrin adaptor complexes. Polyclonal antibody directed to zeta-COP blocks the binding of coatomer to Golgi membranes and prevents the assembly of COP-coated vesicles on Golgi cisternae. Unlike other coatomer subunits (beta-, beta'-, gamma-, and epsilon- COP), zeta-COP exists in both coatomer bound and free pools. PMID:8276893

  14. Significance of alpha-subunit HCG demonstrated in breast carcinomas by the immunoperoxidase technique.

    PubMed Central

    Walker, R A

    1978-01-01

    Fifty-three breast carcinomas were examined by an indirect immunoperoxidase technique for the presence of the alpha-subunit of human chorionic gonadotrophin. Positive staining occurred in 12 (22.6%). There was no correlation between alpha-subunit production and specific histological features, but production was related to the presence of lymph node metastases and hence a poorer prognosis. The alpha-subunit could be used as a prognostic indicator in the assessment of breast carcinoma. Images Fig. 1 Fig. 2 PMID:346610

  15. BOREAS Hardcopy Maps

    NASA Technical Reports Server (NTRS)

    Hall, Forrest G. (Editor); Nelson, Elizabeth; Newcomer, Jeffrey A.

    2000-01-01

    Boreal Ecosystem-Atmospheric Study (BOREAS) hardcopy maps are a collection of approximately 1,000 hardcopy maps representing the physical, climatological, and historical attributes of areas covering primarily the Manitoba and Saskatchewan provinces of Canada. These maps were collected by BOREAS Information System (BORIS) and Canada for Remote Sensing (CCRS) staff to provide basic information about site positions, manmade features, topography, geology, hydrology, land cover types, fire history, climate, and soils of the BOREAS study region. These maps are not available for distribution through the BOREAS project but may be used as an on-site resource. Information is provided within this document for individuals who want to order copies of these maps from the original map source. Note that the maps are not contained on the BOREAS CD-ROM set. An inventory listing file is supplied on the CD-ROM to inform users of the maps that are available. This inventory listing is available from the Earth Observing System Data and Information System (EOSDIS) Oak Ridge National Laboratory (ORNL) Distributed Active Archive Center (DAAC). For hardcopies of the individual maps, contact the sources provided.

  16. Denali image map

    USGS Publications Warehouse

    Binnie, Douglas R.; Colvocoresses, Alden P.

    1987-01-01

    The Denali National Park and Preserve 1:250,000-scale image map has been prepared and published as part of the US Geological Survey's (USGS) continuing research to improve image mapping techniques. Nine multispectral scanner (MSS) images were geometrically corrected, digitally mosaicked, and enhanced at the National Mapping Division's (NMD) EROS Data Center (EDC). This process involves ground control and digital resampling to the Universal Tranverse Mercator (UTM) projection. This paper specifically discusses the preparation of the digital mosaic and the production peculiarities associated with the Denali National Park and Preserve image map.

  17. A multi-subunit based, thermodynamically stable model vaccine using combined immunoinformatics and protein structure based approach.

    PubMed

    Rana, Aarti; Akhter, Yusuf

    2016-04-01

    Immunizations with the conventional vaccines have failed to effectively inhibit the incidences and further dissemination of the infections. To address it, we have implemented protein structure based strategies to design an efficient multi-epitope subunit vaccine against Mycobacterium avium subsp. paratuberculosis (MAP). Previously reported immunodominant peptide epitope sequences from MAP1611 protein were conjugated together with a stretch of conserved amino acid residues of heparin-binding hemagglutinin, reported as a TLR4 agonist and was employed as an adjuvant to polarize the cellular responses toward host protective Th1 responses. These three types of component peptides were combined with the help of relevant linkers for efficient separation to improve and intensify the antigen processing and presentation. The primary structures of these multi peptides were 3-dimensional homology modeled to yield the final chimeric vaccine. Further, its conformational correctness and stability enhancement was assessed using molecular dynamics (MD) simulations. Finally, disulfide engineering in the most flexible regions of the molecule yielded three potential mutants, Y593C-E610C, Q631C-A634C and a double mutant Q631C-A634C/Y593C-E610C. The double mutant represents thermodynamically most stable version among them. It is potentially highly antigenic, soluble and non-allergen molecule interacting with the TLR receptor expressed on the immune cells. This vaccine contains both T-cell and several B-cell epitopes and an adjuvant which potentially possess protective cellular and humoral immune responses triggering properties. The presented vaccine strategy will be proven a promising pathogen specific candidate with wide therapeutic application against MAP which may be extended to other prevalent infections in future.

  18. Effects of Ca/sup 2 +/ and subunit interactions on surface accessibility of cysteine residues in cardiac troponin

    SciTech Connect

    Ingraham, R.H.; Hodges, R.S.

    1988-08-09

    Rabbit and bovine cardiac troponin (Tn) subunits and complexes were labeled with iodo(/sup 14/C)acetamide in the presence and absence of Ca/sup 2 +/ to determine the effects of tertiary and quaternary structure on exposure of Cys SH groups. This procedure serves both to map region of subunit interaction and the effects of Ca/sup 2 +/-induced conformational change and to indicate which Cys residues should be useful attachment sites for spectroscopic or cross-linking probes. After being labeled, Tn subunits were purified by using reversed-phase HPLC and subjected to tryptic cleavage with or without prior citraconylation. Cys-containing fragments were isolated by RP-HPLC, and the percent labeling was determined. Cys-75 and -92 of TnI were completely accessible to iodoacetamide both when TnI was labeled alone or when in the TnC-TnI complex. Both residues were largely inaccessible when Tn or the TnI-TnT complex was labeled, suggesting burial in the TnI-TnT interface. In contrast, the Cys from the N-terminal region of bovine TnT was stoichiometrically labeled when TnT was labeled alone, in native Tn or in a troponin-tropomyosin complex. Cys-35 and -84 of TnC are located in the nonfunctional Ca/sup 2 +/ binding loop I of cardiac TnC and helix D, respectively. For TnC alone, the percent labelings of Cys-35 and -84 were 11% and 26%, respectively (minus Ca/sup 2 +/), and 16% and 63%, respectively (plus Ca/sup 2 +/). For TnC labeled within Tn, the percent labelings of Cys-35 and -84 were 20% and 52%, respectively (minus Ca/sup 2 +/), and 20% and 78%, respectively (plus Ca/sup 2 +/). The Ca/sup 2 +/-induced exposure of these residues, especially Cys-84, supports the Ca/sup 2 +/-activated model of turkey skeletal TnC derived from crystallographic data.

  19. CHARACTERIZATION OF NICOTINE ACETYLCHOLINE RECEPTOR SUBUNITS IN THE COCKROACH Periplaneta americana MUSHROOM BODIES REVEALS A STRONG EXPRESSION OF β1 SUBUNIT: INVOLVEMENT IN NICOTINE-INDUCED CURRENTS.

    PubMed

    Taillebois, Emiliane; Thany, Steeve H

    2016-09-01

    Nicotinic acetylcholine receptors are ligand-gated ion channels expressed in many insect structures, such as mushroom bodies, in which they play a central role. We have recently demonstrated using electrophysiological recordings that different native nicotinic receptors are expressed in cockroach mushroom bodies Kenyon cells. In the present study, we demonstrated that eight genes coding for cockroach nicotinic acetylcholine receptor subunits are expressed in the mushroom bodies. Quantitative real-time polymerase chain reaction (PCR) experiments demonstrated that β1 subunit was the most expressed in the mushroom bodies. Moreover, antisense oligonucleotides performed against β1 subunit revealed that inhibition of β1 expression strongly decreases nicotine-induced currents amplitudes. Moreover, co-application with 0.5 μM α-bungarotoxin completely inhibited nicotine currents whereas 10 μM d-tubocurarine had a partial effect demonstrating that β1-containing neuronal nicotinic acetylcholine receptor subtypes could be sensitive to the nicotinic acetylcholine receptor antagonist α-bungarotoxin.

  20. Cholera toxin subunit B detection in microfluidic devices.

    PubMed

    Bunyakul, Natinan; Edwards, Katie A; Promptmas, Chamras; Baeumner, Antje J

    2009-01-01

    Fluorescence and electrochemical microfluidic biosensors were developed for the detection of cholera toxin subunit B (CTB) as a model analyte. The microfluidic devices were made from polydimethylsiloxane (PDMS) using soft lithography from silicon templates. The polymer channels were sealed with a glass plate and packaged in a polymethylmethacrylate housing that provided leakproof sealing and a connection to a syringe pump. In the electrochemical format, an interdigitated ultramicroelectrode array (IDUA) was patterned onto the glass slide using photolithography, gold evaporation and lift-off processes. For CTB recognition, CTB-specific antibodies were immobilized onto superparamagnetic beads and ganglioside GM(1) was incorporated into liposomes. The fluorescence dye sulforhodamine B (SRB) and the electroactive compounds potassium hexacyanoferrate (II)/hexacyanoferrate (III) were used as detection markers that were encapsulated inside the liposomes for the fluorescence and electrochemical detection formats, respectively. Initial optimization experiments were carried out by applying the superparamagnetic beads in microtiter plate assays and SRB liposomes before they were transferred to the microfluidic systems. The limits of detection (LoD) of both assay formats for CTB were found to be 6.6 and 1.0 ng mL(-1) for the fluorescence and electrochemical formats, respectively. Changing the detection system was very easy, requiring only the synthesis of different marker-encapsulating liposomes, as well as the exchange of the detection unit. It was found that, in addition to a lower LoD, the electrochemical format assay showed advantages over the fluorescence format in terms of flexibility and reliability of signal recording.

  1. Early diagnosis of sepsis using serum hemoglobin subunit Beta.

    PubMed

    Yoo, Hayoung; Ku, Sae-Kwang; Kim, Shin-Woo; Bae, Jong-Sup

    2015-02-01

    The development of new sepsis-specific biomarkers is mandatory to improve the detection and monitoring of the disease. Hemoglobin is the main oxygen and carbon dioxide carrier in cells of the erythroid lineage and is responsible for oxygen delivery to the respiring tissues of the body. Hemoglobin subunit beta (HBβ) is a component of a larger protein called hemoglobin. The aim of this study was to evaluate blood levels of HBβ in septic patients. A prospective study of 82 patients with sepsis was conducted. Furthermore, C57BL/6 mice were subjected to cecal ligation and puncture (CLP) surgery. Alternatively, human umbilical vein endothelial cells (HUVECs) or C57BL/6 mice were exposed to lipopolysaccharide (LPS, 100 ng/ml to HUVECs or 10 mg/kg to mice). The data showed that LPS induced upregulation of the synthesis and secretion of HBβ in LPS-treated HUVECs and in LPS-injected and CLP mice. In patients admitted to the intensive care unit with sepsis, circulating levels of HBβ were significantly high (sepsis, 64.93-114.76 ng/ml, n = 30; severe sepsis, 157.37-268.69 ng/ml, n = 22; septic shock, 309.98-427.03 ng/ml, n = 30) when compared to the levels of control donors (9.76-12.28 ng/ml, n = 21). Patients with septic shock had higher HBβ levels when compared to patients with severe sepsis. Furthermore, the HBβ levels in septic patients were higher than those in healthy volunteers. These results suggest that in septic patients, HBβ blood level is related to the severity of sepsis and may represent a novel endothelial cell dysfunction marker. Moreover, HBβ can be used as a biomarker to determine the severity of sepsis.

  2. P. berghei telomerase subunit TERT is essential for parasite survival.

    PubMed

    Religa, Agnieszka A; Ramesar, Jai; Janse, Chris J; Scherf, Artur; Waters, Andrew P

    2014-01-01

    Telomeres define the ends of chromosomes protecting eukaryotic cells from chromosome instability and eventual cell death. The complex regulation of telomeres involves various proteins including telomerase, which is a specialized ribonucleoprotein responsible for telomere maintenance. Telomeres of chromosomes of malaria parasites are kept at a constant length during blood stage proliferation. The 7-bp telomere repeat sequence is universal across different Plasmodium species (GGGTTT/CA), though the average telomere length varies. The catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), is present in all sequenced Plasmodium species and is approximately three times larger than other eukaryotic TERTs. The Plasmodium RNA component of TERT has recently been identified in silico. A strategy to delete the gene encoding TERT via double cross-over (DXO) homologous recombination was undertaken to study the telomerase function in P. berghei. Expression of both TERT and the RNA component (TR) in P. berghei blood stages was analysed by Western blotting and Northern analysis. Average telomere length was measured in several Plasmodium species using Telomere Restriction Fragment (TRF) analysis. TERT and TR were detected in blood stages and an average telomere length of ∼ 950 bp established. Deletion of the tert gene was performed using standard transfection methodologies and we show the presence of tert- mutants in the transfected parasite populations. Cloning of tert- mutants has been attempted multiple times without success. Thorough analysis of the transfected parasite populations and the parasite obtained from extensive parasite cloning from these populations provide evidence for a so called delayed death phenotype as observed in different organisms lacking TERT. The findings indicate that TERT is essential for P. berghei cell survival. The study extends our current knowledge on telomere biology in malaria parasites and validates further investigations to

  3. Subunit-dependent effects of nickel on NMDA receptor channels.

    PubMed

    Marchetti, Carla; Gavazzo, Paola

    2003-10-07

    Nickel (Ni2+) is a transition metal that affects different neuronal ionic channels. We investigated its effects on glutamate channels of the NMDA-type in the presence of saturating concentration of glutamate or NMDA (50 microM), in 0 external Mg and in the continuous presence of saturating glycine (30 microM). In neonatal rat cerebellar granule cells, Ni2+ inhibited the current evoked by NMDA at -60 mV with an IC50 close to 40 microM. The inhibition was weakly voltage-dependent and the current at +40 mV was inhibited with IC50=86 microM. Wash out of the metal unmasked a stimulatory effect which persisted for a few seconds. In HEK293 cells transiently transfected with recombinant NR1a-NR2A receptors, Ni2+ inhibited the current elicited by glutamate with an IC50=52 microM at -60 mV and 90 microM at +40 mV. In HEK293 expressing NR1a-NR2B receptors, 0.1-100 microM Ni2+ caused a potentiation of the current, with EC50=4 microM, while with 300 microM, a voltage-dependent block became apparent (IC50=170 microM). As previously reported, the current through both classes of recombinant receptors was steeply dependent on external pH, and in both cases the protonic block had an IC50 close to pH 7.2. Application of Ni2+ showed that stimulation of NR1a-NR2B receptor channels was dependent on external pH, while voltage-independent inhibition of NR1a-NR2A was less sensitive to pH change. These results indicate that Ni2+ has multiple and complex effects on NMDA channels, which are largely dependent on the NR2 subunit.

  4. Elongated polyproline motifs facilitate enamel evolution through matrix subunit compaction.

    PubMed

    Jin, Tianquan; Ito, Yoshihiro; Luan, Xianghong; Dangaria, Smit; Walker, Cameron; Allen, Michael; Kulkarni, Ashok; Gibson, Carolyn; Braatz, Richard; Liao, Xiubei; Diekwisch, Thomas G H

    2009-12-01

    Vertebrate body designs rely on hydroxyapatite as the principal mineral component of relatively light-weight, articulated endoskeletons and sophisticated tooth-bearing jaws, facilitating rapid movement and efficient predation. Biological mineralization and skeletal growth are frequently accomplished through proteins containing polyproline repeat elements. Through their well-defined yet mobile and flexible structure polyproline-rich proteins control mineral shape and contribute many other biological functions including Alzheimer's amyloid aggregation and prolamine plant storage. In the present study we have hypothesized that polyproline repeat proteins exert their control over biological events such as mineral growth, plaque aggregation, or viscous adhesion by altering the length of their central repeat domain, resulting in dramatic changes in supramolecular assembly dimensions. In order to test our hypothesis, we have used the vertebrate mineralization protein amelogenin as an exemplar and determined the biological effect of the four-fold increased polyproline tandem repeat length in the amphibian/mammalian transition. To study the effect of polyproline repeat length on matrix assembly, protein structure, and apatite crystal growth, we have measured supramolecular assembly dimensions in various vertebrates using atomic force microscopy, tested the effect of protein assemblies on crystal growth by electron microscopy, generated a transgenic mouse model to examine the effect of an abbreviated polyproline sequence on crystal growth, and determined the structure of polyproline repeat elements using 3D NMR. Our study shows that an increase in PXX/PXQ tandem repeat motif length results (i) in a compaction of protein matrix subunit dimensions, (ii) reduced conformational variability, (iii) an increase in polyproline II helices, and (iv) promotion of apatite crystal length. Together, these findings establish a direct relationship between polyproline tandem repeat fragment

  5. Preclinical and clinical development of a dengue recombinant subunit vaccine.

    PubMed

    Manoff, Susan B; George, Sarah L; Bett, Andrew J; Yelmene, Michele L; Dhanasekaran, Govindarajan; Eggemeyer, Linda; Sausser, Michele L; Dubey, Sheri A; Casimiro, Danilo R; Clements, David E; Martyak, Timothy; Pai, Vidya; Parks, D Elliot; Coller, Beth-Ann G

    2015-12-10

    This review focuses on a dengue virus (DENV) vaccine candidate based on a recombinant subunit approach which targets the DENV envelope glycoprotein (E). Truncated versions of E consisting of the N-terminal portion of E (DEN-80E) have been expressed recombinantly in the Drosophila S2 expression system and shown to have native-like conformation. Preclinical studies demonstrate that formulations containing tetravalent DEN-80E adjuvanted with ISCOMATRIX™ adjuvant induce high titer virus neutralizing antibodies and IFN-γ producing T cells in flavivirus-naïve non-human primates. The preclinical data further suggest that administration of such formulations on a 0, 1, 6 month schedule may result in higher maximum virus neutralizing antibody titers and better durability of those titers compared to administration on a 0, 1, 2 month schedule. In addition, the virus neutralizing antibody titers induced by adjuvanted tetravalent DEN-80E compare favorably to the titers induced by a tetravalent live virus comparator. Furthermore, DEN-80E was demonstrated to be able to boost virus neutralizing antibody titers in macaques that have had a prior DENV exposure. A monovalent version of the vaccine candidate, DEN1-80E, was formulated with Alhydrogel™ and studied in a proof-of-principle Phase I clinical trial by Hawaii Biotech, Inc. (NCT00936429). The clinical trial results demonstrate that both the 10 μg and 50 μg formulations of DEN1-80E with 1.25 mg of elemental aluminum were immunogenic when administered in a 3-injection series (0, 1, 2 months) to healthy, flavivirus-naïve adults. The vaccine formulations induced DENV-1 neutralizing antibodies in the majority of subjects, although the titers in most subjects were modest and waned over time. Both the 10 μg DEN1-80E and the 50 μg DEN1-80E formulations with Alhydrogel™ were generally well tolerated.

  6. Elongated Polyproline Motifs Facilitate Enamel Evolution through Matrix Subunit Compaction

    PubMed Central

    Luan, Xianghong; Dangaria, Smit; Walker, Cameron; Allen, Michael; Kulkarni, Ashok; Gibson, Carolyn; Braatz, Richard; Liao, Xiubei; Diekwisch, Thomas G. H.

    2009-01-01

    Vertebrate body designs rely on hydroxyapatite as the principal mineral component of relatively light-weight, articulated endoskeletons and sophisticated tooth-bearing jaws, facilitating rapid movement and efficient predation. Biological mineralization and skeletal growth are frequently accomplished through proteins containing polyproline repeat elements. Through their well-defined yet mobile and flexible structure polyproline-rich proteins control mineral shape and contribute many other biological functions including Alzheimer's amyloid aggregation and prolamine plant storage. In the present study we have hypothesized that polyproline repeat proteins exert their control over biological events such as mineral growth, plaque aggregation, or viscous adhesion by altering the length of their central repeat domain, resulting in dramatic changes in supramolecular assembly dimensions. In order to test our hypothesis, we have used the vertebrate mineralization protein amelogenin as an exemplar and determined the biological effect of the four-fold increased polyproline tandem repeat length in the amphibian/mammalian transition. To study the effect of polyproline repeat length on matrix assembly, protein structure, and apatite crystal growth, we have measured supramolecular assembly dimensions in various vertebrates using atomic force microscopy, tested the effect of protein assemblies on crystal growth by electron microscopy, generated a transgenic mouse model to examine the effect of an abbreviated polyproline sequence on crystal growth, and determined the structure of polyproline repeat elements using 3D NMR. Our study shows that an increase in PXX/PXQ tandem repeat motif length results (i) in a compaction of protein matrix subunit dimensions, (ii) reduced conformational variability, (iii) an increase in polyproline II helices, and (iv) promotion of apatite crystal length. Together, these findings establish a direct relationship between polyproline tandem repeat fragment

  7. rRNA suppressor of a eukaryotic translation initiation factor 5B/initiation factor 2 mutant reveals a binding site for translational GTPases on the small ribosomal subunit.

    PubMed

    Shin, Byung-Sik; Kim, Joo-Ran; Acker, Michael G; Maher, Kathryn N; Lorsch, Jon R; Dever, Thomas E

    2009-02-01

    The translational GTPases promote initiation, elongation, and termination of protein synthesis by interacting with the ribosome. Mutations that impair GTP hydrolysis by eukaryotic translation initiation factor 5B/initiation factor 2 (eIF5B/IF2) impair yeast cell growth due to failure to dissociate from the ribosome following subunit joining. A mutation in helix h5 of the 18S rRNA in the 40S ribosomal subunit and intragenic mutations in domain II of eIF5B suppress the toxic effects associated with expression of the eIF5B-H480I GTPase-deficient mutant in yeast by lowering the ribosome binding affinity of eIF5B. Hydroxyl radical mapping experiments reveal that the domain II suppressors interface with the body of the 40S subunit in the vicinity of helix h5. As the helix h5 mutation also impairs elongation factor function, the rRNA and eIF5B suppressor mutations provide in vivo evidence supporting a functionally important docking of domain II of the translational GTPases on the body of the small ribosomal subunit.

  8. Use of an α3β4 nicotinic acetylcholine receptor subunit concatamer to characterize ganglionic receptor subtypes with specific subunit composition reveals species-specific pharmacologic properties.

    PubMed

    Stokes, Clare; Papke, Roger L

    2012-09-01

    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.

  9. Occupancy Grid Map Merging Using Feature Maps

    DTIC Science & Technology

    2010-11-01

    Gonzalez, “Toward a unified bayesian approach to hybrid metric-topological SLAM,” IEEE Transactions on Robotics , 24(2), April 2008, 259-270. [14] G...Risetti, C. Stachniss, and W. Burgard, “Improved Techniques for grid mapping with Rao-Blackwellized Particle Filter,” IEEE Transactions on Robotics , 23

  10. Maps and Map Learning in Social Studies

    ERIC Educational Resources Information Center

    Bednarz, Sarah Witham; Acheson, Gillian; Bednarz, Robert S.

    2006-01-01

    The importance of maps and other graphic representations has become more important to geography and geographers. This is due to the development and widespread diffusion of geographic (spatial) technologies. As computers and silicon chips have become more capable and less expensive, geographic information systems (GIS), global positioning satellite…

  11. Localized reconstruction of subunits from electron cryomicroscopy images of macromolecular complexes

    PubMed Central

    Ilca, Serban L.; Kotecha, Abhay; Sun, Xiaoyu; Poranen, Minna M.; Stuart, David I.; Huiskonen, Juha T.

    2015-01-01

    Electron cryomicroscopy can yield near-atomic resolution structures of highly ordered macromolecular complexes. Often however some subunits bind in a flexible manner, have different symmetry from the rest of the complex, or are present in sub-stoichiometric amounts, limiting the attainable resolution. Here we report a general method for the localized three-dimensional reconstruction of such subunits. After determining the particle orientations, local areas corresponding to the subunits can be extracted and treated as single particles. We demonstrate the method using three examples including a flexible assembly and complexes harbouring subunits with either partial occupancy or mismatched symmetry. Most notably, the method allows accurate fitting of the monomeric RNA-dependent RNA polymerase bound at the threefold axis of symmetry inside a viral capsid, revealing for the first time its exact orientation and interactions with the capsid proteins. Localized reconstruction is expected to provide novel biological insights in a range of challenging biological systems. PMID:26534841

  12. Extensive subunit contacts underpin herpesvirus capsid stability and interior-to-exterior allostery

    PubMed Central

    Huet, Alexis; Makhov, Alexander M.; Huffman, Jamie B.; Vos, Matthijn; Homa, Fred L.; Conway, James F.

    2016-01-01

    The herpesvirus capsid is a complex protein assembly that includes hundreds of copies of four major subunits and lesser numbers of several minor proteins, all essential for infectivity. Cryo-electron microscopy is uniquely suited for studying interactions that govern the assembly and function of such large and functional complexes. Here we report two high quality capsid structures, from human herpes simplex virus type 1 (HSV-1) and the animal pseudorabies virus (PRV), imaged inside intact virions at ~7 Å resolution. From these we developed a complete model of subunit and domainal organization and identified extensive networks of subunit contacts that underpin capsid stability and form a pathway that may signal the completion of DNA packaging from the capsid interior to outer surface for initiating nuclear egress. Differences in folding and orientation of subunit domains between herpesvirus capsids suggest that common elements have been modified for specific functions. PMID:27111889

  13. Nucleotide-Protectable Labeling of Sulfhydryl Groups in Subunit I of the ATPhase from Halobacterium Saccharovorum

    NASA Technical Reports Server (NTRS)

    Sulzner, Michael; Stan-Lotter, Helga; Hochstein, Lawrence I.

    1992-01-01

    A membrane-bound ATPase from the archaebacterium Halobacterium saccharovorum is inhibited by N-ethyl-maleimide in a nucleotide-protectable manner. When the enzyme was incubated with N-[C-14]jethylmaleimide, the bulk of radioactivity was as- sociated with the 87,000-Da subunit (subunit 1). ATP, ADP, or AMP reduced incorporation of the inhibitor. No charge shift of subunit I was detected following labeling with N-ethylmaleimide, indicating an electroneutral reaction. The results are consistent with the selective modification of sulfhydryl groups in subunit I at or near the catalytic site and are further evidence of a resemblance between this archaebacterial ATPase and the vacuolar-type ATPases.

  14. [Nucleotide sequence of genes for alpha- and beta-subunits of luciferase from Photobacterium leiognathi].

    PubMed

    Illarionov, B A; Protopopova, M V; Karginov, V A; Mertvetsov, N P; Gitel'zon, I I

    1988-03-01

    Nucleotide sequence of the Photobacterium leiognathi DNA containing genes of alpha and beta subunits of luciferase has been determined. We also deduced amino acid sequence and molecular mass of luciferase and localized luciferase genes in the sequenced DNA fragment.

  15. High degree of coordination and division of labor among subunits in a homomeric ring ATPase.

    PubMed

    Chistol, Gheorghe; Liu, Shixin; Hetherington, Craig L; Moffitt, Jeffrey R; Grimes, Shelley; Jardine, Paul J; Bustamante, Carlos

    2012-11-21

    Ring NTPases of the ASCE superfamily perform a variety of cellular functions. An important question about the operation of these molecular machines is how the ring subunits coordinate their chemical and mechanical transitions. Here, we present a comprehensive mechanochemical characterization of a homomeric ring ATPase-the bacteriophage φ29 packaging motor-a homopentamer that translocates double-stranded DNA in cycles composed of alternating dwells and bursts. We use high-resolution optical tweezers to determine the effect of nucleotide analogs on the cycle. We find that ATP hydrolysis occurs sequentially during the burst and that ADP release is interlaced with ATP binding during the dwell, revealing a high degree of coordination among ring subunits. Moreover, we show that the motor displays an unexpected division of labor: although all subunits of the homopentamer bind and hydrolyze ATP during each cycle, only four participate in translocation, whereas the remaining subunit plays an ATP-dependent regulatory role.

  16. Complex regulation of γ-secretase: from obligatory to modulatory subunits

    PubMed Central

    Gertsik, Natalya; Chiu, Danica; Li, Yue-Ming

    2014-01-01

    γ-Secretase is a four subunit, 19-pass transmembrane enzyme that cleaves amyloid precursor protein (APP), catalyzing the formation of amyloid beta (Aβ) peptides that form amyloid plaques, which contribute to Alzheimer’s disease (AD) pathogenesis. γ-Secretase also cleaves Notch, among many other type I transmembrane substrates. Despite its seemingly promiscuous enzymatic capacity, γ-secretase activity is tightly regulated. This regulation is a function of many cellular entities, including but not limited to the essential γ-secretase subunits, nonessential (modulatory) subunits, and γ-secretase substrates. Regulation is also accomplished by an array of cellular events, such as presenilin (active subunit of γ-secretase) endoproteolysis and hypoxia. In this review we discuss how γ-secretase is regulated with the hope that an advanced understanding of these mechanisms will aid in the development of effective therapeutics for γ-secretase-associated diseases like AD and Notch-addicted cancer. PMID:25610395

  17. Differential Targeting of Gβγ-Subunit Signaling with Small Molecules

    NASA Astrophysics Data System (ADS)

    Bonacci, Tabetha M.; Mathews, Jennifer L.; Yuan, Chujun; Lehmann, David M.; Malik, Sundeep; Wu, Dianqing; Font, Jose L.; Bidlack, Jean M.; Smrcka, Alan V.

    2006-04-01

    G protein βγ subunits have potential as a target for therapeutic treatment of a number of diseases. We performed virtual docking of a small-molecule library to a site on Gβγ subunits that mediates protein interactions. We hypothesized that differential targeting of this surface could allow for selective modulation of Gβγ subunit functions. Several compounds bound to Gβγ subunits with affinities from 0.1 to 60 μM and selectively modulated functional Gβγ-protein-protein interactions in vitro, chemotactic peptide signaling pathways in HL-60 leukocytes, and opioid receptor-dependent analgesia in vivo. These data demonstrate an approach for modulation of G protein-coupled receptor signaling that may represent an important therapeutic strategy.

  18. Separation of glutathione transferase subunits from Proteus vulgaris by two-dimensional gel electrophoresis.

    PubMed

    Hong, Giaming; Chien, Yi-Chih; Chien, Cheng-I

    2003-10-01

    Cytosolic glutathione transferases of Proteus vulgaris were purified by affinity chromatography and characterized by two-dimensional gel electrophoresis. Four different subunits were identified, and each subunit contained a different molecular mass, ranging from 26.2 kDa to 28.5 kDa; a different pI value, ranging from 8.2 to 9.4; and a different amount of protein fraction, ranging from 10% to 56%. All four subunits existed as basic proteins (pI > 7.0). From these results, we concluded that multiple forms of glutathione transferase enzymes existed in Proteus vulgaris, and four different glutathione transferase subunits were separated by 2-D gel electrophoresis.

  19. Cooperative Subunit Refolding of a Light-Harvesting Protein through a Self-Chaperone Mechanism.

    PubMed

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

    2017-01-27

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

  20. Experimental ulcerative herpetic keratitis. IV. Preliminary observations on the efficacy of a herpes simplex subunit vaccine.

    PubMed

    Carter, C A; Hartley, C E; Skinner, G R; Turner, S P; Easty, D L

    1981-10-01

    Systemic vaccination of rabbits with an inactivated type 1 virus subunit vaccine induced humoral and cell-mediated immune responses. Following ocular infection with type 1 herpes virus corneal ulceration and virus excretion were reduced in the vaccinated rabbits.

  1. Sao Paulo Map Collections.

    ERIC Educational Resources Information Center

    McLean, G. Robert

    1985-01-01

    Describes geographical, subject, and chronological aspects of 25 cartographic collections housed in university, public, special, state, and semi-state libraries in Sao Paulo, Brazil. Three size categories of map holdings (more than 10,000, 1,000-10,000, less than 1,000) are distinguished. A list of 27 Sao Paulo institutions housing map collections…

  2. Managing Vocabulary Mapping Services

    PubMed Central

    Che, Chengjian; Monson, Kent; Poon, Kasey B.; Shakib, Shaun C.; Lau, Lee Min

    2005-01-01

    The efficient management and maintenance of large-scale and high-quality vocabulary mapping is an operational challenge. The 3M Health Information Systems (HIS) Healthcare Data Dictionary (HDD) group developed an information management system to provide controlled mapping services, resulting in improved efficiency and quality maintenance. PMID:16779203

  3. Map of Nasca Geoglyphs

    NASA Astrophysics Data System (ADS)

    Hanzalová, K.; Pavelka, K.

    2013-07-01

    The Czech Technical University in Prague in the cooperation with the University of Applied Sciences in Dresden (Germany) work on the Nasca Project. The cooperation started in 2004 and much work has been done since then. All work is connected with Nasca lines in southern Peru. The Nasca project started in 1995 and its main target is documentation and conservation of the Nasca lines. Most of the project results are presented as WebGIS application via Internet. In the face of the impending destruction of the soil drawings, it is possible to preserve this world cultural heritage for the posterity at least in a digital form. Creating of Nasca lines map is very useful. The map is in a digital form and it is also available as a paper map. The map contains planimetric component of the map, map lettering and altimetry. Thematic folder in this map is a vector layer of the geoglyphs in Nasca/Peru. Basis for planimetry are georeferenced satellite images, altimetry is created from digital elevation model. This map was created in ArcGis software.

  4. Chizu Task Mapping Tool

    SciTech Connect

    2014-07-01

    Chizu is a tool for Mapping MPI processes or tasks to physical processors or nodes for optimizing communication performance. It takes the communication graph of a High Performance Computing (HPC) application and the interconnection topology of a supercomputer as input. It outputs a new MPI rand to processor mapping, which can be used when launching the HPC application.

  5. Mapping the Llano Estacado

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Early maps of North America, prepared in the 18th and early 19th centuries, often depicted the Llano Estacado as a conspicuous blank spot - a terra incognita. A good example is a map of the southwest sketched by Alexander von Humboldt in 1804. In 1830, Stephen F. Austin added little detail to the ...

  6. Handmade Multitextured Maps.

    ERIC Educational Resources Information Center

    Trevelyan, Simon

    1984-01-01

    Tactile maps for visually impaired persons can be made by drawing lines with an aqueous adhesive solution, dusting with thermoengraving powder, and exposing the card to a source of intense heat (such as a heat gun or microwave oven). A raised line map results. (CL)

  7. Acoustic mapping velocimetry

    NASA Astrophysics Data System (ADS)

    Muste, M.; Baranya, S.; Tsubaki, R.; Kim, D.; Ho, H.; Tsai, H.; Law, D.

    2016-05-01

    Knowledge of sediment dynamics in rivers is of great importance for various practical purposes. Despite its high relevance in riverine environment processes, the monitoring of sediment rates remains a major and challenging task for both suspended and bed load estimation. While the measurement of suspended load is currently an active area of testing with nonintrusive technologies (optical and acoustic), bed load measurement does not mark a similar progress. This paper describes an innovative combination of measurement techniques and analysis protocols that establishes the proof-of-concept for a promising technique, labeled herein Acoustic Mapping Velocimetry (AMV). The technique estimates bed load rates in rivers developing bed forms using a nonintrusive measurements approach. The raw information for AMV is collected with acoustic multibeam technology that in turn provides maps of the bathymetry over longitudinal swaths. As long as the acoustic maps can be acquired relatively quickly and the repetition rate for the mapping is commensurate with the movement of the bed forms, successive acoustic maps capture the progression of the bed form movement. Two-dimensional velocity maps associated with the bed form migration are obtained by implementing algorithms typically used in particle image velocimetry to acoustic maps converted in gray-level images. Furthermore, use of the obtained acoustic and velocity maps in conjunction with analytical formulations (e.g., Exner equation) enables estimation of multidirectional bed load rates over the whole imaged area. This paper presents a validation study of the AMV technique using a set of laboratory experiments.

  8. What do maps show?

    USGS Publications Warehouse

    ,

    1994-01-01

    The purpose of the teaching package is to help students understand and use maps. The U.S. Geological Survey (USGS) has provided the package as a service to educators so that more Americans will learn to understand the world of information on maps. Everything in the package teaches and reinforces geographic skills that are required in your curriculum.

  9. BenMAP Downloads

    EPA Pesticide Factsheets

    Download the current and legacy versions of the BenMAP program. Download configuration and aggregation/pooling/valuation files to estimate benefits. BenMAP-CE is free and open source software, and the source code is available upon request.

  10. The Map Corner.

    ERIC Educational Resources Information Center

    Cheyney, Arnold B.; Capone, Donald L.

    This teaching resource is aimed at helping students develop the skills necessary to locate places on the earth. Designed as a collection of map skill exercises rather than a sequential program of study, this program expects that students have access to and some knowledge of how to use globes, maps, atlases, and encyclopedias. The volume contains 6…

  11. Coupled trivial maps.

    PubMed

    Bunimovich, L. A.; Livi, R.; Martinez-Mekler, G.; Ruffo, S.

    1992-07-01

    The first nontrivial example of coupled map lattices that admits a rigorous analysis in the whole range of the strength of space interactions is considered. This class is generated by one-dimensional maps with a globally attracting superstable periodic trajectory that are coupled by a diffusive nearest-neighbor interaction.

  12. Temporal mapping and analysis

    NASA Technical Reports Server (NTRS)

    O'Hara, Charles G. (Inventor); Shrestha, Bijay (Inventor); Vijayaraj, Veeraraghavan (Inventor); Mali, Preeti (Inventor)

    2011-01-01

    A compositing process for selecting spatial data collected over a period of time, creating temporal data cubes from the spatial data, and processing and/or analyzing the data using temporal mapping algebra functions. In some embodiments, the temporal data cube is creating a masked cube using the data cubes, and computing a composite from the masked cube by using temporal mapping algebra.

  13. World Stress Map Published

    NASA Astrophysics Data System (ADS)

    Heidbach, Oliver; Müller, Birgit; Fuchs, Karl; Wenzel, Friedemann; Reinecker, John; Tingay, Mark; Sperner, Blanka; Cadet, Jean-Paul; Rossi, Philipp

    2007-11-01

    The World Stress Map (WSM), published in April 2007 by the Commission for the Geological Map of the World and the Heidelberg Academy of Sciences and Humanities, displays the tectonic regime and the orientation of the contemporary maximum horizontal compressional stress at more than 12,000 locations within the Earth's crust. The Mercator projection is a scale of 1:46,000,000.

  14. High-resolution genome-wide mapping of histone modifications.

    PubMed

    Roh, Tae-young; Ngau, Wing Chi; Cui, Kairong; Landsman, David; Zhao, Keji

    2004-08-01

    The expression patterns of eukaryotic genomes are controlled by their chromatin structure, consisting of nucleosome subunits in which DNA of approximately 146 bp is wrapped around a core of 8 histone molecules. Post-translational histone modifications play an essential role in modifying chromatin structure. Here we apply a combination of SAGE and chromatin immunoprecipitation (ChIP) protocols to determine the distribution of hyperacetylated histones H3 and H4 in the Saccharomyces cerevisiae genome. We call this approach genome-wide mapping technique (GMAT). Using GMAT, we find that the highest acetylation levels are detected in the 5' end of a gene's coding region, but not in the promoter. Furthermore, we show that the histone acetyltransferase, GCN5p, regulates H3 acetylation in the promoter and 5' end of the coding regions. These findings indicate that GMAT should find valuable applications in mapping target sites of chromatin-modifying enzymes.

  15. Functional protein expression of multiple sodium channel alpha- and beta-subunit isoforms in neonatal cardiomyocytes.

    PubMed

    Kaufmann, Susann G; Westenbroek, Ruth E; Zechner, Christoph; Maass, Alexander H; Bischoff, Sebastian; Muck, Jenny; Wischmeyer, Erhard; Scheuer, Todd; Maier, Sebastian K G

    2010-01-01

    Voltage-gated sodium channels are composed of pore-forming alpha- and auxiliary beta-subunits and are responsible for the rapid depolarization of cardiac action potentials. Recent evidence indicates that neuronal tetrodotoxin (TTX) sensitive sodium channel alpha-subunits are expressed in the heart in addition to the predominant cardiac TTX-resistant Na(v)1.5 sodium channel alpha-subunit. These TTX-sensitive isoforms are preferentially localized in the transverse tubules of rodents. Since neonatal cardiomyocytes have yet to develop transverse tubules, we determined the complement of sodium channel subunits expressed in these cells. Neonatal rat ventricular cardiomyocytes were stained with antibodies specific for individual isoforms of sodium channel alpha- and beta-subunits. alpha-actinin, a component of the z-line, was used as an intracellular marker of sarcomere boundaries. TTX-sensitive sodium channel alpha-subunit isoforms Na(v)1.1, Na(v)1.2, Na(v)1.3, Na(v)1.4 and Na(v)1.6 were detected in neonatal rat heart but at levels reduced compared to the predominant cardiac alpha-subunit isoform, Na(v)1.5. Each of the beta-subunit isoforms (beta1-beta4) was also expressed in neonatal cardiac cells. In contrast to adult cardiomyocytes, the alpha-subunits are distributed in punctate clusters across the membrane surface of neonatal cardiomyocytes; no isoform-specific subcellular localization is observed. Voltage clamp recordings in the absence and presence of 20 nM TTX provided functional evidence for the presence of TTX-sensitive sodium current in neonatal ventricular myocardium which represents between 20 and 30% of the current, depending on membrane potential and experimental conditions. Thus, as in the adult heart, a range of sodium channel alpha-subunits are expressed in neonatal myocytes in addition to the predominant TTX-resistant Na(v)1.5 alpha-subunit and they contribute to the total sodium current.

  16. Genetic disruption of the autism spectrum disorder risk gene PLAUR induces GABAA receptor subunit changes

    PubMed Central

    Eagleson, Kathie L.; Gravielle, Maria C.; SchlueterMcFadyen-Ketchum, Lisa J.; Russek, Shelley J.; Farb, David H.; Levitt, Pat

    2010-01-01

    Disruption of the GABAergic system has been implicated in multiple developmental disorders, including epilepsy, autism spectrum disorder and schizophrenia. The human gene encoding uPAR (PLAUR) has been shown recently to be associated with the risk of autism. The uPAR-/- mouse exhibits a regionally selective reduction in GABAergic interneurons in frontal and parietal regions of the cerebral cortex as well as in the CA1 and dentate gyrus subfields of the hippocampus. Behaviorally, these mice exhibit increased sensitivity to pharmacologically-induced seizures, heightened anxiety, and atypical social behavior. Here, we explore potential alterations in GABAergic circuitry that may occur in the context of altered interneuron development. Analysis of gene expression for 13 GABAA receptor subunits using quantitative real-time PCR indicates seven subunit mRNAs (α1, α2, α3, β2, β3, γ2S and γ2L) of interest. Semi-quantitative in situ hybridization analysis focusing on these subunit mRNAs reveals a complex pattern of potential gene regulatory adaptations. The levels of α2 subunit mRNAs increase in frontal cortex, CA1 and CA3, while those of α3 decrease in frontal cortex and CA1. In contrast, α1 subunit mRNAs are unaltered in any region examined. β2 subunit mRNAs are increased in frontal cortex whereas β3 subunit mRNAs are decreased in parietal cortex. Finally, γ2S subunit mRNAs are increased in parietal cortex while γ2L subunit mRNAs are increased in the dentate gyrus, potentially altering the γ2S:γ2L ratio in these two regions. For all subunits, no changes were observed in forebrain regions where GABAergic interneuron numbers are normal. We propose that disrupted differentiation of GABAergic neurons specifically in frontal and parietal cortices leads to regionally-selective alterations in local circuitry and subsequent adaptive changes in receptor subunit composition. Future electrophysiological studies will be useful in determining how alterations in network

  17. Factorized Diffusion Map Approximation.

    PubMed

    Amizadeh, Saeed; Valizadegan, Hamed; Hauskrecht, Milos

    2012-01-01

    Diffusion maps are among the most powerful Machine Learning tools to analyze and work with complex high-dimensional datasets. Unfortunately, the estimation of these maps from a finite sample is known to suffer from the curse of dimensionality. Motivated by other machine learning models for which the existence of structure in the underlying distribution of data can reduce the complexity of estimation, we study and show how the factorization of the underlying distribution into independent subspaces can help us to estimate diffusion maps more accurately. Building upon this result, we propose and develop an algorithm that can automatically factorize a high dimensional data space in order to minimize the error of estimation of its diffusion map, even in the case when the underlying distribution is not decomposable. Experiments on both the synthetic and real-world datasets demonstrate improved estimation performance of our method over the standard diffusion-map framework.

  18. Factorized Diffusion Map Approximation

    PubMed Central

    Amizadeh, Saeed; Valizadegan, Hamed; Hauskrecht, Milos

    2013-01-01

    Diffusion maps are among the most powerful Machine Learning tools to analyze and work with complex high-dimensional datasets. Unfortunately, the estimation of these maps from a finite sample is known to suffer from the curse of dimensionality. Motivated by other machine learning models for which the existence of structure in the underlying distribution of data can reduce the complexity of estimation, we study and show how the factorization of the underlying distribution into independent subspaces can help us to estimate diffusion maps more accurately. Building upon this result, we propose and develop an algorithm that can automatically factorize a high dimensional data space in order to minimize the error of estimation of its diffusion map, even in the case when the underlying distribution is not decomposable. Experiments on both the synthetic and real-world datasets demonstrate improved estimation performance of our method over the standard diffusion-map framework. PMID:25309676

  19. Geologic map of Mars

    USGS Publications Warehouse

    Tanaka, Kenneth L.; Skinner, James A.; Dohm, James M.; Irwin, Rossman P.; Kolb, Eric J.; Fortezzo, Corey M.; Platz, Thomas; Michael, Gregory G.; Hare, Trent M.

    2014-01-01

    This global geologic map of Mars, which records the distribution of geologic units and landforms on the planet's surface through time, is based on unprecedented variety, quality, and quantity of remotely sensed data acquired since the Viking Orbiters. These data have provided morphologic, topographic, spectral, thermophysical, radar sounding, and other observations for integration, analysis, and interpretation in support of geologic mapping. In particular, the precise topographic mapping now available has enabled consistent morphologic portrayal of the surface for global mapping (whereas previously used visual-range image bases were less effective, because they combined morphologic and albedo information and, locally, atmospheric haze). Also, thermal infrared image bases used for this map tended to be less affected by atmospheric haze and thus are reliable for analysis of surface morphology and texture at even higher resolution than the topographic products.

  20. Bodily maps of emotions

    PubMed Central

    Nummenmaa, Lauri; Glerean, Enrico; Hari, Riitta; Hietanen, Jari K.

    2014-01-01

    Emotions are often felt in the body, and somatosensory feedback has been proposed to trigger conscious emotional experiences. Here we reveal maps of bodily sensations associated with different emotions using a unique topographical self-report method. In five experiments, participants (n = 701) were shown two silhouettes of bodies alongside emotional words, stories, movies, or facial expressions. They were asked to color the bodily regions whose activity they felt increasing or decreasing while viewing each stimulus. Different emotions were consistently associated with statistically separable bodily sensation maps across experiments. These maps were concordant across West European and East Asian samples. Statistical classifiers distinguished emotion-specific activation maps accurately, confirming independence of topographies across emotions. We propose that emotions are represented in the somatosensory system as culturally universal categorical somatotopic maps. Perception of these emotion-triggered bodily changes may play a key role in generating consciously felt emotions. PMID:24379370

  1. Iconicity as structure mapping

    PubMed Central

    Emmorey, Karen

    2014-01-01

    Linguistic and psycholinguistic evidence is presented to support the use of structure-mapping theory as a framework for understanding effects of iconicity on sign language grammar and processing. The existence of structured mappings between phonological form and semantic mental representations has been shown to explain the nature of metaphor and pronominal anaphora in sign languages. With respect to processing, it is argued that psycholinguistic effects of iconicity may only be observed when the task specifically taps into such structured mappings. In addition, language acquisition effects may only be observed when the relevant cognitive abilities are in place (e.g. the ability to make structural comparisons) and when the relevant conceptual knowledge has been acquired (i.e. information key to processing the iconic mapping). Finally, it is suggested that iconicity is better understood as a structured mapping between two mental representations than as a link between linguistic form and human experience. PMID:25092669

  2. Bodily maps of emotions.

    PubMed

    Nummenmaa, Lauri; Glerean, Enrico; Hari, Riitta; Hietanen, Jari K

    2014-01-14

    Emotions are often felt in the body, and somatosensory feedback has been proposed to trigger conscious emotional experiences. Here we reveal maps of bodily sensations associated with different emotions using a unique topographical self-report method. In five experiments, participants (n = 701) were shown two silhouettes of bodies alongside emotional words, stories, movies, or facial expressions. They were asked to color the bodily regions whose activity they felt increasing or decreasing while viewing each stimulus. Different emotions were consistently associated with statistically separable bodily sensation maps across experiments. These maps were concordant across West European and East Asian samples. Statistical classifiers distinguished emotion-specific activation maps accurately, confirming independence of topographies across emotions. We propose that emotions are represented in the somatosensory system as culturally universal categorical somatotopic maps. Perception of these emotion-triggered bodily changes may play a key role in generating consciously felt emotions.

  3. Distribution of NMDA and AMPA receptor subunits at thalamo-amygdaloid dendritic spines.

    PubMed

    Radley, Jason J; Farb, Claudia R; He, Yong; Janssen, William G M; Rodrigues, Sarina M; Johnson, Luke R; Hof, Patrick R; LeDoux, Joseph E; Morrison, John H

    2007-02-23

    Synapses onto dendritic spines in the lateral amygdala formed by afferents from the auditory thalamus represent a site of plasticity in Pavlovian fear conditioning. Previous work has demonstrated that thalamic afferents synapse onto LA spines expressing glutamate receptor (GluR) subunits, but the GluR subunit distribution at the synapse and within the cytoplasm has not been characterized. Therefore, we performed a quantitative analysis for alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunits GluR2 and GluR3 and N-methyl-D-aspartate (NMDA) receptor subunits NR1 and NR2B by combining anterograde labeling of thalamo-amygdaloid afferents with postembedding immunoelectron microscopy for the GluRs in adult rats. A high percentage of thalamo-amygdaloid spines was immunoreactive for GluR2 (80%), GluR3 (83%), and NR1 (83%), while a smaller proportion of spines expressed NR2B (59%). To compare across the various subunits, the cytoplasmic to synaptic ratios of GluRs were measured within thalamo-amygdaloid spines. Analyses revealed that the cytoplasmic pool of GluR2 receptors was twice as large compared to the GluR3, NR1, and NR2B subunits. Our data also show that in the adult brain, the NR2B subunit is expressed in the majority of in thalamo-amygdaloid spines and that within these spines, the various GluRs are differentially distributed between synaptic and non-synaptic sites. The prevalence of the NR2B subunit in thalamo-amygdaloid spines provides morphological evidence supporting its role in the fear conditioning circuit while the differential distribution of the GluR subtypes may reflect distinct roles for their involvement in this circuitry and synaptic plasticity.

  4. 3D reconstruction of the hemocyanin subunit dimer from the chiton Acanthochiton fascicularis.

    PubMed

    Harris, J Robin; Meissner, Ulrich; Gebauer, Wolfgang; Markl, Jürgen

    2004-01-01

    Procedures are presented for the purification of the subunit dimer from Acanthochiton fasicularis hemocyanin. Electron microscopy of negatively stained specimens revealed a uniform population of macromolecules possessing the characteristic "boat shape". A 3D reconstruction from this EM data generated a approximately 3 nm resolution model that correlates well with earlier data of the purported subunit dimer, extracted from the 3D reconstruction of the didecamer of Haliotis tuberculata hemocyanin type 1.

  5. Type B Heterotrimeric G Protein γ-Subunit Regulates Auxin and ABA Signaling in Tomato.

    PubMed

    Subramaniam, Gayathery; Trusov, Yuri; Lopez-Encina, Carlos; Hayashi, Satomi; Batley, Jacqueline; Botella, José Ramón

    2016-02-01

    Heterotrimeric G proteins composed of α, β, and γ subunits are central signal transducers mediating the cellular response to multiple stimuli in most eukaryotes. Gγ subunits provide proper cellular localization and functional specificity to the heterotrimer complex. Plant Gγ subunits, divided into three structurally distinct types, are more diverse than their animal counterparts. Type B Gγ subunits, lacking a carboxyl-terminal isoprenylation motif, are found only in flowering plants. We present the functional characterization of type B Gγ subunit (SlGGB1) in tomato (Solanum lycopersicum). We show that SlGGB1 is the most abundant Gγ subunit in tomato and strongly interacts with the Gβ subunit. Importantly, the green fluorescent protein-SlGGB1 fusion protein as well as the carboxyl-terminal yellow fluorescent protein-SlGGB1/amino-terminal yellow fluorescent protein-Gβ heterodimer were localized in the plasma membrane, nucleus, and cytoplasm. RNA interference-mediated silencing of SlGGB1 resulted in smaller seeds, higher number of lateral roots, and pointy fruits. The silenced lines were hypersensitive to exogenous auxin, while levels of endogenous auxins were lower or similar to those of the wild type. SlGGB1-silenced plants also showed strong hyposensitivity to abscisic acid (ABA) during seed germination but not in other related assays. Transcriptome analysis of the transgenic seeds revealed abnormal expression of genes involved in ABA sensing, signaling, and response. We conclude that the type B Gγ subunit SlGGB1 mediates auxin and ABA signaling in tomato.

  6. Studies on the murine Ss protein. I. Purification, molecular weight, and subunit structure

    PubMed Central

    1975-01-01

    The murine Ss protein has been isolated and purified. Using specific antisera, the radiolabeled protein has a mol wt of 120,000 in sodium dodecyl sulfate polyacrylamide gels. It is composed of two basic subunits of 23,000 and 14,000 daltons. The smaller molecular weight subunit contains a single disulfide bridge, is devoid of carbohydrate, and may represent the murine equivalent of beta2-microglobulin. PMID:809530

  7. Differential proteolysis of the subunits of pyrophosphate-dependent 6-phosphofructo-1-phosphotransferase.

    PubMed

    Cheng, H F; Tao, M

    1990-02-05

    Antibodies against the alpha (Mr 67,000) and beta (Mr 60,000) subunits of wheat seedling Fru-2,6-P2-stimulated pyrophosphate-dependent 6-phosphofructo-1-phosphotransferase (PFP) were used to probe the subunit structures of several partially purified plant PFPs after tryptic digestion. Antisera to the alpha and beta subunits of wheat seedling PFP cross-reacted with the corresponding alpha and beta subunits of PFP preparations from wheat germ, potato tubers, and lettuce leaves. With the mung bean PFP, both antisera reacted with a protein band of Mr 60,000. A protein band corresponding to the Mr 67,000 alpha subunit was not detected in the mung bean PFP preparation. Tryptic digestion of wheat seedling and potato tuber PFPs resulted in the preferential cleavage of the alpha subunit. The trypsinized PFP retained most of its Fru-2,6-P2-stimulated activity but not its basal activity. The proteolyzed enzyme also exhibited a 2-fold increase in Ka for Fru-2,6-P2. Studies with the mung bean enzyme revealed that the anti-alpha immunoreactive component was more sensitive to trypsinization than the anti-beta immunoreactive component of the Mr 60,000 protein band. Thus, the Mr 60,000 protein band of the mung bean PFP appears to be heterogeneous and contains both alpha and beta-like proteins. The above observations indicate that the alpha and beta subunits of PFP are two distinct polypeptides and that alpha acts as a regulatory protein in regulating both the catalytic activity and the Fru-2,6-P2-binding affinity of the beta subunit.

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

    PubMed Central

    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

  9. Ricin, ricin agglutinin, and the ricin binding subunit structural comparison by Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Brandt, N. N.; Chikishev, A. Yu.; Sotnikov, A. I.; Savochkina, Yu. A.; Agapov, I. I.; Tonevitsky, A. G.

    2005-02-01

    Raman spectroscopy is used to study conformation-sensitive vibrational bands of the plant toxins ricin and ricin agglutinin and the ricin binding subunit in aqueous solution. The analysis of the Raman data yields the conformational state of the protein molecules differing from that predicted by the X-ray data. The differences and similarities in the conformational state of ricin, ricin agglutinin, and ricin binding subunit are discussed.

  10. Molecular Analyses of a Three-Subunit Euryarchaeal Clamp Loader Complex from Methanosarcina acetivorans▿ †

    PubMed Central

    Chen, Yi-Hsing; Lin, Yuyen; Yoshinaga, Aya; Chhotani, Benazir; Lorenzini, Jenna L.; Crofts, Alexander A.; Mei, Shou; Mackie, Roderick I.; Ishino, Yoshizumi; Cann, Isaac K. O.

    2009-01-01

    Chromosomal DNA replication is dependent on processive DNA synthesis. Across the three domains of life and in certain viruses, a toroidal sliding clamp confers processivity to replicative DNA polymerases by encircling the DNA and engaging the polymerase in protein/protein interactions. Sliding clamps are ring-shaped; therefore, they have cognate clamp loaders that open and load them onto DNA. Here we use biochemical and mutational analyses to study the structure/function of the Methanosarcina acetivorans clamp loader or replication factor C (RFC) homolog. M. acetivorans RFC (RFCMa), which represents an intermediate between the common archaeal RFC and the eukaryotic RFC, comprises two different small subunits (RFCS1 and RFCS2) and a large subunit (RFCL). Size exclusion chromatography suggested that RFCS1 exists in oligomeric states depending on protein concentration, while RFCS2 exists as a monomer. Protein complexes of RFCS1/RFCS2 formed in solution; however, they failed to stimulate DNA synthesis by a cognate DNA polymerase in the presence of its clamp. Determination of the subunit composition and previous mutational analysis allowed the prediction of the spatial distribution of subunits in this new member of the clamp loader family. Three RFCS1 subunits are flanked by an RFCS2 and an RFCL. The spatial distribution is, therefore, reminiscent of the minimal Escherichia coli clamp loader that exists in space as three γ-subunits (motor) flanked by the δ′ (stator) and the δ (wrench) subunits. Mutational analysis, however, suggested that the similarity between the two clamp loaders does not translate into the complete conservation of the functions of individual subunits within the RFCMa complex. PMID:19717601

  11. Crystal Structure of the Oxazolidinone Antibiotic Linezolid Bound to the 50S Ribosomal Subunit

    SciTech Connect

    Ippolito,J.; Kanyo, Z.; Wang, D.; Franceschi, F.; Moore, P.; Steitz, T.; Duffy, E.

    2008-01-01

    The oxazolidinone antibacterials target the 50S subunit of prokaryotic ribosomes. To gain insight into their mechanism of action, the crystal structure of the canonical oxazolidinone, linezolid, has been determined bound to the Haloarcula marismortui 50S subunit. Linezolid binds the 50S A-site, near the catalytic center, which suggests that inhibition involves competition with incoming A-site substrates. These results provide a structural basis for the discovery of improved oxazolidinones active against emerging drug-resistant clinical strains.

  12. Two temporally synthesized charge subunits interact to form the five isoforms of cottonseed (Gossypium hirsutum) catalase.

    PubMed Central

    Ni, W; Trelease, R N; Eising, R

    1990-01-01

    Five charge isoforms of tetrameric catalase were isolated from cotyledons of germinated cotton (Gossypium hirsutum L.) seedlings. Denaturing isoelectric focusing of the individual isoforms in polyacrylamide gels indicated that isoforms A (most anodic) and E (most cathodic) consisted of one subunit of different charge, whereas isoforms B, C and D each consisted of a mixture of these two subunits. Thus the five isoforms apparently were formed through combinations of two subunits in different ratios. Labelling cotyledons in vivo with [35S]methionine at three daily intervals in the dark, and translation in vivo of polyadenylated RNA isolated from cotyledons at the same ages, revealed synthesis of two different subunits. One of the subunits was synthesized in cotyledons at all ages studied (days 1-3), whereas the other subunit was detected only at days 2 and 3. This differential expression of two catalase subunits helped explain previous results from this laboratory showing that the two anodic forms (A and B) found in maturing seeds were supplemented with three cathodic forms (C-E) after the seeds germinated. These subunit data also helped clarify our new findings that proteins of isoforms A, B and C (most active isoforms) accumulated in cotyledons of plants kept in the dark for 3 days, then gradually disappeared during the next several days, whereas isoforms D and E (least active isoforms) remained in the cells. This shift in isoform pattern occurred whether seedlings were kept in the dark or exposed to continuous light after day 3, although exposure to light enhanced this process. These sequential molecular events were responsible for the characteristic developmental changes (rise and fall) in total catalase activity. We believe that the isoform changeover is physiologically related to the changeover in glyoxysome to leaf-type-peroxisome metabolism. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:1695843

  13. Lifelong ethanol consumption and brain regional GABAA receptor subunit mRNA expression in alcohol-preferring rats.

    PubMed

    Sarviharju, Maija; Hyytiä, Petri; Hervonen, Antti; Jaatinen, Pia; Kiianmaa, Kalervo; Korpi, Esa R

    2006-11-01

    Brain regional gamma-aminobutyric acid type A (GABAA) receptor subunit mRNA expression was studied in ethanol-preferring AA (Alko, Alcohol) rats after moderate ethanol drinking for up to 2 years of age. In situ hybridization with oligonucleotide probes specific for 13 different subunits was used with coronal cryostat sections of the brains. Selective alterations were observed by ethanol exposure and/or aging in signals for several subunits. Most interestingly, the putative highly ethanol-sensitive alpha4 and beta3 subunit mRNAs were significantly decreased in several brain regions. The age-related alterations in alpha4 subunit expression were parallel to those caused by lifelong ethanol drinking, whereas aging had no significant effect on beta3 subunit expression. The results suggest that prolonged ethanol consumption leading to blood concentrations of about 10 mM may downregulate the mRNA expression of selected GABAA receptor subunits and that aging might have partly similar effects.

  14. Regulation of the Telomerase Reverse Transcriptase Subunit through Epigenetic Mechanisms

    PubMed Central

    Lewis, Kayla A.; Tollefsbol, Trygve O.

    2016-01-01

    Chromosome-shortening is characteristic of normal cells, and is known as the end replication problem. Telomerase is the enzyme responsible for extending the ends of the chromosomes in de novo synthesis, and occurs in germ cells as well as most malignant cancers. There are three subunits of telomerase: human telomerase RNA (hTERC), human telomerase associated protein (hTEP1), or dyskerin, and human telomerase reverse transcriptase (hTERT). hTERC and hTEP1 are constitutively expressed, so the enzymatic activity of telomerase is dependent on the transcription of hTERT. DNA methylation, histone methylation, and histone acetylation are basic epigenetic regulations involved in the expression of hTERT. Non-coding RNA can also serve as a form of epigenetic control of hTERT. This epigenetic-based regulation of hTERT is important in providing a mechanism for reversibility of hTERT control in various biological states. These include embryonic down-regulation of hTERT contributing to aging and the upregulation of hTERT playing a critical role in over 90% of cancers. Normal human somatic cells have a non-methylated/hypomethylated CpG island within the hTERT promoter region, while telomerase-positive cells paradoxically have at least a partially methylated promoter region that is opposite to the normal roles of DNA methylation. Histone acetylation of H3K9 within the promoter region is associated with an open chromatin state such that transcription machinery has the space to form. Histone methylation of hTERT has varied control of the gene, however. Mono- and dimethylation of H3K9 within the promoter region indicate silent euchromatin, while a trimethylated H3K9 enhances gene transcription. Non-coding RNAs can target epigenetic-modifying enzymes, as well as transcription factors involved in the control of hTERT. An epigenetics diet that can affect the epigenome of cancer cells is a recent fascination that has received much attention. By combining portions of this diet with

  15. Influence of gamma subunit prenylation on association of guanine nucleotide-binding regulatory proteins with membranes.

    PubMed Central

    Muntz, K H; Sternweis, P C; Gilman, A G; Mumby, S M

    1992-01-01

    Two approaches were taken to address the possible role of gamma-subunit prenylation in dictating the cellular distribution of guanine nucleotide-binding regulatory proteins. Prenylation of gamma subunits was prevented by site-directed mutagenesis or by inhibiting the synthesis of mevalonate, the precursor of cellular isoprenoids. When beta or gamma subunits were transiently expressed in COS-M6 simian kidney cells (COS) cells, the proteins were found in the membrane fraction by immunoblotting. Immunofluorescence experiments indicated that the proteins were distributed to intracellular structures in addition to plasma membranes. Replacement of Cys68 of gamma with Ser prevented prenylation of the mutant protein and association of the protein with the membrane fraction of COS cells. Immunoblotting results demonstrated that some of the beta subunits were found in the cytoplasm when coexpressed with the nonprenylated mutant gamma subunit. When Neuro 2A cells were treated with compactin to inhibit protein prenylation, a fraction of endogenous beta and gamma was distributed in the cytoplasm. It is concluded that prenylation facilitates association of gamma subunits with membranes, that the cellular location of gamma influences the distribution of beta, and that prenylation is not an absolute requirement for interaction of beta and gamma. Images PMID:1550955

  16. Expression, purification, and characterization of a biologically active bovine enterokinase catalytic subunit in Escherichia coli.

    PubMed

    Yuan, Liu-Di; Hua, Zi-Chun

    2002-07-01

    Enterokinase (EC 3.4.21.9) is a serine proteinase in the duodenum that exhibits specificity for the sequence (Asp)(4)-Lys. It converts trypsinogen to trypsin. Its high specificity for the recognition site makes enterokinase (EK) a useful tool for in vitro cleavage of fusion proteins. cDNA encoding the catalytic chain of Chinese bovine enterokinase was cloned and its encoding amino acid sequence is identical to the previously reported sequence although there are two one-base mutations which do not change the encoded amino acid. The EK catalytic subunit cDNA was cloned into plasmid pET32a, and fused downstream to the fusion partner thioredoxin (Trx) and the following DDDDK enterokinase recognition sequence. The recombinant bovine enterokinase catalytic subunit was expressed in Escherichia coli BL21(DE3), and most products existed in soluble form. After an in vivo autocatalytic cleavage of the recombinant Trx-EK catalytic domain fusion protein, intact, biologically active EK catalytic subunit was released from the fusion protein. The recombinant intact EK catalytic subunit was purified to homogeneity with a specific activity of 720 AUs/mg protein through ammonium sulfate precipitation, DEAE chromatography, and gel filtration. The purified intact EK catalytic subunit has a K(m) of 0.17 mM, and K(cat) is 20.8s(-1). From 100 ml flask culture, 4.3 mg pure active EK catalytic subunits were obtained.

  17. Evidence for an unusual transmembrane configuration of AGG3, a Class C Gγ Subunit, of Arabidopsis

    PubMed Central

    Wolfenstetter, Susanne; Chakravorty, David; Kula, Ryan; Urano, Daisuke; Trusov, Yuri; McCurdy, David W.; Assmann, Sarah M.; Jones, Alan M.; Botella, Jose R.

    2015-01-01

    SUMMARY Heterotrimeric G proteins are crucial for the perception of external signals and subsequent signal transduction in animal and plant cells. In both model systems, the complex is comprised of one Gα, one Gβ and one Gγ subunit. However, in addition to the canonical Gγ subunits (Class A), plants also possess two unusual, plant-specific classes of Gγ subunits (Classes B and C) not yet found in animals. These include Gγ subunits lacking the C-terminal CaaX motif (Class B) which is important for membrane anchoring of the protein, and thus give rise to a flexible subpopulation of Gβ/γ heterodimers that is not necessarily restricted to the plasma membrane. Even more interesting, plants also contain Class C Gγ subunits which are twice the size of canonical Gγs, with a predicted transmembrane domain, and a large cysteine-rich, extracellular C-terminus. However, neither the presence of the transmembrane domain nor the membrane topology has been unequivocally demonstrated. Here, we provide compelling evidence that AGG3, a Class C Ggamma subunit of Arabidopsis, contains a functional transmembrane domain, which is sufficient but not essential for plasma membrane localization, and that the cysteine-rich C-terminus is extracellular. PMID:25430066

  18. Structural basis for translational surveillance by the large ribosomal subunit-associated protein quality control complex

    PubMed Central

    Lyumkis, Dmitry; Oliveira dos Passos, Dario; Tahara, Erich B.; Webb, Kristofor; Bennett, Eric J.; Vinterbo, Staal; Potter, Clinton S.; Carragher, Bridget; Joazeiro, Claudio A. P.

    2014-01-01

    All organisms have evolved mechanisms to manage the stalling of ribosomes upon translation of aberrant mRNA. In eukaryotes, the large ribosomal subunit-associated quality control complex (RQC), composed of the listerin/Ltn1 E3 ubiquitin ligase and cofactors, mediates the ubiquitylation and extraction of ribosome-stalled nascent polypeptide chains for proteasomal degradation. How RQC recognizes stalled ribosomes and performs its functions has not been understood. Using single-particle cryoelectron microscopy, we have determined the structure of the RQC complex bound to stalled 60S ribosomal subunits. The structure establishes how Ltn1 associates with the large ribosomal subunit and properly positions its E3-catalytic RING domain to mediate nascent chain ubiquitylation. The structure also reveals that a distinguishing feature of stalled 60S particles is an exposed, nascent chain-conjugated tRNA, and that the Tae2 subunit of RQC, which facilitates Ltn1 binding, is responsible for selective recognition of stalled 60S subunits. RQC components are engaged in interactions across a large span of the 60S subunit surface, connecting the tRNA in the peptidyl transferase center to the distally located nascent chain tunnel exit. This work provides insights into a mechanism linking translation and protein degradation that targets defective proteins immediately after synthesis, while ignoring nascent chains in normally translating ribosomes. PMID:25349383

  19. Two classes of regulatory subunits coassemble in the same BK channel and independently regulate gating

    NASA Astrophysics Data System (ADS)

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

    2015-09-01

    High resolution proteomics increasingly reveals that most native ion channels are assembled in macromolecular complexes. However, whether different partners have additive or cooperative functional effects, or whether some combinations of proteins may preclude assembly of others are largely unexplored topics. The large conductance Ca2+-and-voltage activated potassium channel (BK) is well-suited to discern nuanced differences in regulation arising from combinations of subunits. Here we examine whether assembly of two different classes of regulatory proteins, β and γ, in BK channels is exclusive or independent. Our results show that both γ1 and up to four β2-subunits can coexist in the same functional BK complex, with the gating shift caused by β2-subunits largely additive with that produced by the γ1-subunit(s). The multiplicity of β:γ combinations that can participate in a BK complex therefore allow a range of BK channels with distinct functional properties tuned by the specific stoichiometry of the contributing subunits.

  20. Ricin and Ricinus communis agglutinin subunits are all derived from a single-size polypeptide precursor.

    PubMed

    Butterworth, A G; Lord, J M

    1983-12-01

    Antibodies have been raised in rabbits against the individually purified A and B subunits of the toxic castor bean lectin, ricin, and against the A' and B' subunits of Ricinus communis agglutinin type I. Each of the antisera recognised a single polypeptide species of Mr 60 500 when maturing castor bean endosperm mRNA was translated in vitro in a rabbit-reticulocyte-derived system. When dog pancreatic microsomal vesicles were included in the translational system, each subunit antiserum precipitated a group of 66 000-68 000-Mr core-glycosylated polypeptides which had been translocated into the lumen of the vesicles. The 60 500-Mr polypeptide appeared to be a common precursor to all four individual lectin subunits since (a) its glycosylated (66 000-68 000-Mr) forms were readily detected in the endoplasmic reticulum fraction isolated from maturing castor bean endosperm and (b) pulse-chase studies showed that the glycosylated precursors disappeared from the endoplasmic reticulum fraction with the concomittant appearance of authentic lectin subunits in a soluble protein fraction which included protein body matrix components. Antiserum prepared against whole R. communis agglutinin, type I, also precipitated the 65 000-Mr precursor in vitro and in vivo, but in addition precipitated a non-glycosylated 34 000-Mr polypeptide. This smaller protein is not a lectin subunit precursor, contradicting an earlier suggestion. It is most probably a precursor to the 2-S albumin storage proteins found in castor bean endosperm protein bodies.

  1. The TCP1γ subunit of Leishmania donovani forms a biologically active homo-oligomeric complex.

    PubMed

    Bhaskar; Mitra, Kalyan; Kuldeep, Jitendra; Siddiqi, Mohammad Imran; Goyal, Neena

    2015-12-01

    Chaperonins are a class of molecular chaperons that encapsulate nascent or stress-denatured proteins and assist their intracellular assembly and folding in an ATP-dependent manner. The ubiquitous eukaryotic chaperonin, TCP1 ring complex is a hetero-oligomeric complex comprising two rings, each formed of eight subunits that may have distinct substrate recognition and ATP hydrolysis properties. In Leishmania, only the TCP1γ subunit has been cloned and characterized. It exhibited differential expression at various growth stages of promastigotes. In the present study, we expressed the TCP1γ subunit in Escherichia coli to investigate whether it forms chaperonin-like complexes and plays a role in protein folding. LdTCP1γ formed high-molecular-weight complexes within E. coli cells as well as in Leishmania cell lysates. The recombinant protein is arranged into two back-to-back rings of seven subunits each, as predicted by homology modelling and observed by negative staining electron microscopy. This morphology is consistent with that of the oligomeric double-ring group I chaperonins found in mitochondria. The LdTCP1γ homo-oligomeric complex hydrolysed ATP, and was active as assayed by luciferase refolding. Thus, the homo-oligomer performs chaperonin reactions without partner subunit(s). Further, co-immunoprecipitation studies revealed that LdTCP1γ interacts with actin and tubulin proteins, suggesting that the complex may have a role in maintaining the structural dynamics of the cytoskeleton of parasites.

  2. Evaluation of human antibody responses to diphtheria toxin subunits A and B in various age groups.

    PubMed

    Karakus, R; Caglar, K; Aybay, C

    2007-11-01

    This study aimed to evaluate human antibody responses to diphtheria toxin subunits in various age groups. Antibodies against the intact diphtheria toxin and the diphtheria toxin subunits A and B were evaluated in 1319 individuals using a double-antigen ELISA. Although high levels of protection (83.6%, 95% CI 79.2-87.4) were found in children and adolescents, the middle-aged adult population was less protected (28.8%, 95% CI 24.3-33.6). An increase in age was associated with a decrease in the frequency of protected individuals in the 0-39-year age group (p <0.001). Anti-subunit B levels correlated well (p <0.01) with levels of antibodies against the intact toxin. In children aged < or =16 years, the intervals at which the peaks in geometric mean titres of anti-subunit B antibodies were observed were found to correlate with the ages at which booster doses are administered. Overall, males appeared to be more protected than females (OR 1.67, 95% CI 1.34-2.08, p <0.001). A small group of individuals had antibody levels of > or =0.1 IU/mL against the intact toxin, but did not have protective antibody against subunit B. Determination of anti-subunit B antibody levels should help in evaluating the effectiveness of diphtheria boosters and other aspects of diphtheria immunity.

  3. Energetic Contributions to Channel Gating of Residues in the Muscle Nicotinic Receptor β1 Subunit

    PubMed Central

    Akk, Gustav; Eaton, Megan; Li, Ping; Zheng, Steven; Lo, Joshua; Steinbach, Joe Henry

    2013-01-01

    In the pentameric ligand-gated ion channel family, transmitter binds in the extracellular domain and conformational changes result in channel opening in the transmembrane domain. In the muscle nicotinic receptor and other heteromeric members of the family one subunit does not contribute to the canonical agonist binding site for transmitter. A fundamental question is whether conformational changes occur in this subunit. We used records of single channel activity and rate-equilibrium free energy relationships to examine the β1 (non-ACh-binding) subunit of the muscle nicotinic receptor. Mutations to residues in the extracellular domain have minimal effects on the gating equilibrium constant. Positions in the channel lining (M2 transmembrane) domain contribute strongly and relatively late during gating. Positions thought to be important in other subunits in coupling the transmitter-binding to the channel domains have minimal effects on gating. We conclude that the conformational changes involved in channel gating propagate from the binding-site to the channel in the ACh-binding subunits and subsequently spread to the non-binding subunit. PMID:24194945

  4. A translation-like cycle is a quality control checkpoint for maturing 40S ribosome subunits.

    PubMed

    Strunk, Bethany S; Novak, Megan N; Young, Crystal L; Karbstein, Katrin

    2012-07-06

    Assembly factors (AFs) prevent premature translation initiation on small (40S) ribosomal subunit assembly intermediates by blocking ligand binding. However, it is unclear how AFs are displaced from maturing 40S ribosomes, if or how maturing subunits are assessed for fidelity, and what prevents premature translation initiation once AFs dissociate. Here we show that maturation involves a translation-like cycle whereby the translation factor eIF5B, a GTPase, promotes joining of large (60S) subunits with pre-40S subunits to give 80S-like complexes, which are subsequently disassembled by the termination factor Rli1, an ATPase. The AFs Tsr1 and Rio2 block the mRNA channel and initiator tRNA binding site, and therefore 80S-like ribosomes lack mRNA or initiator tRNA. After Tsr1 and Rio2 dissociate from 80S-like complexes Rli1-directed displacement of 60S subunits allows for translation initiation. This cycle thus provides a functional test of 60S subunit binding and the GTPase site before ribosomes enter the translating pool.

  5. Structural basis for translational surveillance by the large ribosomal subunit-associated protein quality control complex.

    PubMed

    Lyumkis, Dmitry; Oliveira dos Passos, Dario; Tahara, Erich B; Webb, Kristofor; Bennett, Eric J; Vinterbo, Staal; Potter, Clinton S; Carragher, Bridget; Joazeiro, Claudio A P

    2014-11-11

    All organisms have evolved mechanisms to manage the stalling of ribosomes upon translation of aberrant mRNA. In eukaryotes, the large ribosomal subunit-associated quality control complex (RQC), composed of the listerin/Ltn1 E3 ubiquitin ligase and cofactors, mediates the ubiquitylation and extraction of ribosome-stalled nascent polypeptide chains for proteasomal degradation. How RQC recognizes stalled ribosomes and performs its functions has not been understood. Using single-particle cryoelectron microscopy, we have determined the structure of the RQC complex bound to stalled 60S ribosomal subunits. The structure establishes how Ltn1 associates with the large ribosomal subunit and properly positions its E3-catalytic RING domain to mediate nascent chain ubiquitylation. The structure also reveals that a distinguishing feature of stalled 60S particles is an exposed, nascent chain-conjugated tRNA, and that the Tae2 subunit of RQC, which facilitates Ltn1 binding, is responsible for selective recognition of stalled 60S subunits. RQC components are engaged in interactions across a large span of the 60S subunit surface, connecting the tRNA in the peptidyl transferase center to the distally located nascent chain tunnel exit. This work provides insights into a mechanism linking translation and protein degradation that targets defective proteins immediately after synthesis, while ignoring nascent chains in normally translating ribosomes.

  6. Ethanol-induced GABAA receptor alpha4 subunit plasticity involves phosphorylation and neuroactive steroids.

    PubMed

    Werner, David F; Porcu, Patrizia; Boyd, Kevin N; O'Buckley, Todd K; Carter, Jenna M; Kumar, Sandeep; Morrow, A Leslie

    2016-04-01

    GABAA receptors containing α4 subunits are widely implicated in acute ethanol sensitivity, and their spatial and temporal regulation prominently contributes to ethanol-induced neuroplasticity in hippocampus and cortex. However, it is unknown if α4-containing GABAA receptors in the thalamus, an area of high α4 expression, display similar regulatory patterns following ethanol administration, and if so, by which molecular mechanisms. In the current study, thalamic GABAA receptor α4 subunit levels were increased following a 6-week-, but not a 2-week chronic ethanol diet. Following acute high-dose ethanol administration, thalamic GABAA receptor α4 subunit levels were regulated in a temporal fashion, as a decrease was observed at 2h followed by a delayed transient increase. PKCγ and PKCδ levels paralleled α4 temporal expression patterns following ethanol exposure. Initial decreases in α4 subunit expression were associated with reduced serine phosphorylation. Delayed increases in expression were not associated with a change in phosphorylation state, but were prevented by inhibiting neuroactive steroid production with the 5α-reductase inhibitor finasteride. Overall, these studies indicate that thalamic GABAA receptor α4 subunit expression following acute and chronic ethanol administration exhibits similar regulatory patterns as other regions and that transient expression patterns following acute exposure in vivo are likely dependent on both subunit phosphorylation state and neuroactive steroids.

  7. Evidence for an unusual transmembrane configuration of AGG3, a class C Gγ subunit of Arabidopsis

    SciTech Connect

    Wolfenstetter, Susanne; Chakravorty, David; Kula, Ryan; Urano, Daisuke; Trusov, Yuri; Sheahan, Michael B.; McCurdy, David W.; Assmann, Sarah M.; Jones, Alan M.; Botella, Jose R.

    2014-12-22

    Heterotrimeric G proteins are crucial for the perception of external signals and subsequent signal transduction in animal and plant cells. In both model systems, the complex is comprised of one Gα, one Gβ and one Gγ subunit. However, in addition to the canonical Gγ subunits (Class A), plants also possess two unusual, plant-specific classes of Gγ subunits (Classes B and C) not yet found in animals. These include Gγ subunits lacking the C-terminal CaaX motif (Class B) which is important for membrane anchoring of the protein, and thus give rise to a flexible subpopulation of Gβ/γ heterodimers that is not necessarily restricted to the plasma membrane. Even more interesting, plants also contain Class C Gγ subunits which are twice the size of canonical Gγs, with a predicted transmembrane domain, and a large cysteine-rich, extracellular C-terminus. However, neither the presence of the transmembrane domain nor the membrane topology has been unequivocally demonstrated. Finally, we provide compelling evidence that AGG3, a Class C Ggamma subunit of Arabidopsis, contains a functional transmembrane domain, which is sufficient but not essential for plasma membrane localization, and that the cysteine-rich C-terminus is extracellular.

  8. Evidence for an unusual transmembrane configuration of AGG3, a class C Gγ subunit of Arabidopsis

    DOE PAGES

    Wolfenstetter, Susanne; Chakravorty, David; Kula, Ryan; ...

    2014-12-22

    Heterotrimeric G proteins are crucial for the perception of external signals and subsequent signal transduction in animal and plant cells. In both model systems, the complex is comprised of one Gα, one Gβ and one Gγ subunit. However, in addition to the canonical Gγ subunits (Class A), plants also possess two unusual, plant-specific classes of Gγ subunits (Classes B and C) not yet found in animals. These include Gγ subunits lacking the C-terminal CaaX motif (Class B) which is important for membrane anchoring of the protein, and thus give rise to a flexible subpopulation of Gβ/γ heterodimers that is notmore » necessarily restricted to the plasma membrane. Even more interesting, plants also contain Class C Gγ subunits which are twice the size of canonical Gγs, with a predicted transmembrane domain, and a large cysteine-rich, extracellular C-terminus. However, neither the presence of the transmembrane domain nor the membrane topology has been unequivocally demonstrated. Finally, we provide compelling evidence that AGG3, a Class C Ggamma subunit of Arabidopsis, contains a functional transmembrane domain, which is sufficient but not essential for plasma membrane localization, and that the cysteine-rich C-terminus is extracellular.« less

  9. Pleiotropic Effects of Loss of the Dα1 Subunit in Drosophila melanogaster: Implications for Insecticide Resistance.

    PubMed

    Somers, Jason; Luong, Hang Ngoc Bao; Mitchell, Judith; Batterham, Philip; Perry, Trent

    2017-01-01

    Nicotinic acetylcholine receptors (nAChRs) are a highly conserved gene family that form pentameric receptors involved in fast excitatory synaptic neurotransmission. The specific roles individual nAChR subunits perform in Drosophila melanogaster and other insects are relatively uncharacterized. Of the 10 D. melanogaster nAChR subunits, only three have described roles in behavioral pathways; Dα3 and Dα4 in sleep, and Dα7 in the escape response. Other subunits have been associated with resistance to several classes of insecticides. In particular, our previous work has demonstrated that an allele of the Dα1 subunit is associated with resistance to neonicotinoid insecticides. We used ends-out gene targeting to create a knockout of the Dα1 gene to facilitate phenotypic analysis in a controlled genetic background. To our knowledge, this is the first report of a native function for any nAChR subunits known to be targeted by insecticides. Loss of Dα1 function was associated with changes in courtship, sleep, longevity, and insecticide resistance. While acetylcholine signaling had previously been linked with mating behavior and reproduction in D. melanogaster, no specific nAChR subunit had been directly implicated. The role of Dα1 in a number of behavioral phenotypes highlights the importance of understanding the biological roles of nAChRs and points to the fitness cost that may be associated with neonicotinoid resistance.

  10. Archaeal Mo-Containing Glyceraldehyde Oxidoreductase Isozymes Exhibit Diverse Substrate Specificities through Unique Subunit Assemblies

    PubMed Central

    Miyake, Masayuki; Fushinobu, Shinya

    2016-01-01

    Archaea use glycolytic pathways distinct from those found in bacteria and eukaryotes, where unique enzymes catalyze each reaction step. In this study, we isolated three isozymes of glyceraldehyde oxidoreductase (GAOR1, GAOR2 and GAOR3) from the thermoacidophilic archaeon Sulfolobus tokodaii. GAOR1–3 belong to the xanthine oxidoreductase superfamily, and are composed of a molybdo-pyranopterin subunit (L), a flavin subunit (M), and an iron-sulfur subunit (S), forming an LMS hetero-trimer unit. We found that GAOR1 is a tetramer of the STK17810/STK17830/STK17820 hetero-trimer, GAOR2 is a dimer of the STK23390/STK05620/STK05610 hetero-trimer, and GAOR3 is the STK24840/STK05620/STK05610 hetero-trimer. GAOR1–3 exhibited diverse substrate specificities for their electron donors and acceptors, due to their different L-subunits, and probably participate in the non-phosphorylative Entner-Doudoroff glycolytic pathway. We determined the crystal structure of GAOR2, as the first three-dimensional structure of an archaeal molybdenum-containing hydroxylase, to obtain structural insights into their substrate specificities and subunit assemblies. The gene arrangement and the crystal structure suggested that the M/S-complex serves as a structural scaffold for the binding of the L-subunit, to construct the three enzymes with different specificities. Collectively, our findings illustrate a novel principle of a prokaryotic multicomponent isozyme system. PMID:26808202

  11. Immunohistochemical localization of inhibin/activin subunits in the wild ground squirrel (Citellus dauricus Brandt) ovary.

    PubMed

    Sheng, Xia; Weng, Jiaju; Zhang, Haolin; Li, Xiaonan; Zhang, Mengyuan; Xu, Meiyu; Weng, Qiang; Watanabe, Gen; Taya, Kazuyoshi

    2012-01-01

    The intraovarian function of gonadally produced inhibin and activin has been extensively studied in experimental models for decades, yet their presence and function have been rarely reported in wild rodents. With our seasonal breeding model, the wild ground squirrel, we aimed to investigate the possible roles of these peptides in the seasonal folliculogenesis. Immunohistochemical staining and Western blotting have been used to detect the cellular localization and expression patterns of inhibin/activin subunits (α, β(A) and β(B)). In the breeding season ovary, all three subunits were present in granulosa cells, theca cells of antral follicles and interstitial cells, with the strongest immunostaining in granulosa cells. Following ovulation, the corpora lutea become a major site of inhibin/activin synthesis. In the nonbreeding season ovary, inhibin/activin α and β(A) subunits were weakly immunopositive in granulosa cells of early stage follicles, while β(B) subunit was undetectable. The expression level of inhibin/activin subunit proteins were generally higher in the ovaries of the breeding season, and then decreased to a relatively low level during the nonbreeding season. The dynamic expression of inhibin/activin subunits indicated that they might play important paracrine and/or autocrine roles during the seasonal folliculogenesis of the wild ground squirrel.

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

    SciTech Connect

    Zhang, Rong-Guang; Westbrook, M.L.; Maulik, P.R.; Reed, R.A.; Shipley, G.; Westbrook, E.M. |; Scott, D.L.; Otwinowski, Z.

    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.

  13. The subunit structure of potato tuber ADPglucose pyrophosphorylase. [Solanum tuberosum L

    SciTech Connect

    Okita, T.W.; Nakata, P.A.; Anderson, J.M. ); Sowokinos, J. ); Morell, M.; Preiss, J. )

    1990-06-01

    ADPglucose pyrophosphorylase has been extensively purified from potato (Solanum tuberosum L.) tuber tissue to study its structure. By employing a modified published procedure together with Mono Q chromatography, a near homogeneous enzyme preparation was obtained with substantial improvement in enzyme yield and specific activity. In single dimensional sodium dodecyl sulfate polyacrylamide gels, the enzyme migrated as a single polypeptide band with a mobility of about 50,000 daltons. Analysis by two-dimensional polyacrylamide gel electrophoresis, however, revealed the presence of two types of subunits which could be distinguished by their slight differences in net charge and molecular weight. The smaller potato tuber subunit was recognized by antiserum prepared against the smaller spinach leaf 51 kilodalton ADPglucose pyrophosphorylase subunit. In contrast, the anti-54 kilodalton raised against the spinach leaf subunit did not significantly react to the tuber enzyme subunits. The results are consistent with the hypothesis that the potato tuber ADPglucose pyrophosphorylase is not composed of a simple homotetramer as previously suggested, but is a product of two separate and distinct subunits as observed for the spinach leaf and maize enzymes.

  14. Molecular architecture of the yeast Elongator complex reveals an unexpected asymmetric subunit arrangement.

    PubMed

    Setiaputra, Dheva T; Cheng, Derrick Th; Lu, Shan; Hansen, Jesse M; Dalwadi, Udit; Lam, Cindy Hy; To, Jeffrey L; Dong, Meng-Qiu; Yip, Calvin K

    2017-02-01

    Elongator is a ~850 kDa protein complex involved in multiple processes from transcription to tRNA modification. Conserved from yeast to humans, Elongator is assembled from two copies of six unique subunits (Elp1 to Elp6). Despite the wealth of structural data on the individual subunits, the overall architecture and subunit organization of the full Elongator and the molecular mechanisms of how it exerts its multiple activities remain unclear. Using single-particle electron microscopy (EM), we revealed that yeast Elongator adopts a bilobal architecture and an unexpected asymmetric subunit arrangement resulting from the hexameric Elp456 subassembly anchored to one of the two Elp123 lobes that form the structural scaffold. By integrating the EM data with available subunit crystal structures and restraints generated from cross-linking coupled to mass spectrometry, we constructed a multiscale molecular model that showed the two Elp3, the main catalytic subunit, are located in two distinct environments. This work provides the first structural insights into Elongator and a framework to understand the molecular basis of its multifunctionality.

  15. Wheat gluten: high molecular weight glutenin subunits--structure, genetics, and relation to dough elasticity.

    PubMed

    Anjum, Faqir Muhammad; Khan, Moazzam Rafiq; Din, Ahmad; Saeed, Muhammad; Pasha, Imran; Arshad, Muhammad Umair

    2007-04-01

    Gluten proteins, representing the major protein fraction of the starchy endosperm, are predominantly responsible for the unique position of wheat amongst cereals. These form a continuous proteinaceous matrix in the cells of the mature dry grain and form a continuous viscoelastic network during the mixing process of dough development. These viscoelastic properties underline the utilization of wheat to prepare bread and other wheat flour based foodstuffs. One group of gluten proteins is glutenin, which consists of high molecular weight (HMW) and low molecular weight (LMW) subunits. The HMW glutenin subunits (HMW-GS) are particularly important for determining dough elasticity. The common wheat possesses 3 to 5 HMW subunits encoded at the Glu-1 loci on the long arms of group 1 chromosomes (1A, 1B, and 1D). The presence of certain HMW subunits is positively correlated with good bread-making quality. Glutamine-rich repetitive sequences that comprise the central part of the HMW subunits are actually responsible for the elastic properties due to extensive arrays of interchain hydrogen bonds. Genetic engineering can be used to manipulate the amount and composition of the HMW subunits, leading to either increased dough strength or more drastic changes in gluten structure and properties.

  16. Getting Results with Curriculum Mapping

    ERIC Educational Resources Information Center

    Jacobs, Heidi Hayes

    2004-01-01

    This helpful resource will speed the mapping effort along and apply curriculum mapping to special situations. In this book teachers and administrators offer concrete advice on how to get the most out of curriculum mapping in districts and schools: (1) Steps to implementing mapping procedures and leading the mapping process; (2) Tools and resources…

  17. Mapping the Binding Domain of the F18 Fimbrial Adhesin

    PubMed Central

    Smeds, A.; Pertovaara, M.; Timonen, T.; Pohjanvirta, T.; Pelkonen, S.; Palva, A.

    2003-01-01

    F18 fimbrial Esherichia coli strains are associated with porcine postweaning diarrhea and pig edema disease. Recently, the FedF subunit was identified as the adhesin of the F18 fimbriae. In this study, adhesion domains of FedF were further studied by constructing deletions within the fedF gene and expressing FedF proteins with deletions either together with the other F18 fimbrial subunits or as fusion proteins tagged with maltose binding protein. The region essential for adhesion to porcine intestinal epithelial cells was mapped between amino acid residues 60 and 109 of FedF. To map the binding domain even more closely, all eight charged amino acid residues within this region were independently replaced by alanine. Three of these single point mutants expressing F18 fimbriae exhibited significantly diminished capabilities to adhere to porcine epithelial cells in vitro. In addition, a triple point mutation and a double point mutation completely abolished receptor adhesiveness. The result further confirmed that the region between amino acid residues 60 and 109 is essential for the binding of F18 fimbriae to their receptor. In addition, the adhesion capability of the binding domain was eliminated after treatment with iodoacetamide, suggesting the formation of a disulfide bridge between Cys-63 and Cys-83, whereas Cys-111 and Cys-116 could be deleted without affecting the binding ability of FedF. PMID:12654838

  18. Analyzing thematic maps and mapping for accuracy

    USGS Publications Warehouse

    Rosenfield, G.H.

    1982-01-01

    Two problems which exist while attempting to test the accuracy of thematic maps and mapping are: (1) evaluating the accuracy of thematic content, and (2) evaluating the effects of the variables on thematic mapping. Statistical analysis techniques are applicable to both these problems and include techniques for sampling the data and determining their accuracy. In addition, techniques for hypothesis testing, or inferential statistics, are used when comparing the effects of variables. A comprehensive and valid accuracy test of a classification project, such as thematic mapping from remotely sensed data, includes the following components of statistical analysis: (1) sample design, including the sample distribution, sample size, size of the sample unit, and sampling procedure; and (2) accuracy estimation, including estimation of the variance and confidence limits. Careful consideration must be given to the minimum sample size necessary to validate the accuracy of a given. classification category. The results of an accuracy test are presented in a contingency table sometimes called a classification error matrix. Usually the rows represent the interpretation, and the columns represent the verification. The diagonal elements represent the correct classifications. The remaining elements of the rows represent errors by commission, and the remaining elements of the columns represent the errors of omission. For tests of hypothesis that compare variables, the general practice has been to use only the diagonal elements from several related classification error matrices. These data are arranged in the form of another contingency table. The columns of the table represent the different variables being compared, such as different scales of mapping. The rows represent the blocking characteristics, such as the various categories of classification. The values in the cells of the tables might be the counts of correct classification or the binomial proportions of these counts divided by

  19. Functional characterisation of the regulatory subunit of cyclic AMP-dependent protein kinase A homologue of Giardia lamblia: Differential expression of the regulatory and catalytic subunits during encystation.

    PubMed

    Gibson, Candace; Schanen, Brian; Chakrabarti, Debopam; Chakrabarti, Ratna

    2006-06-01

    To understand the functional roles of protein kinase A (PKA) during vegetative and differentiating states of Giardia parasites, we studied the structural and functional characteristics of the regulatory subunit of PKA (gPKAr) and its involvement in the giardial encystment process. Molecular cloning and characterisation showed that gPKAr contains two tandem 3'5'-cyclic adenosine monphosphate (cyclic AMP) binding domains at the C-terminal end and the interaction domain for the catalytic subunit. A number of consensus residues including in vivo phosphorylation site for PKAc and dimerisation/docking domain are present in gPKAr. The regulatory subunit physically interacts with the catalytic subunit and inhibits its kinase activity in the absence of cyclic AMP, which could be partially restored upon addition of cyclic AMP. Western blot analysis showed a marked reduction in the endogenous gPKAr concentration during differentiation of Giardia into cysts. An increased activity of gPKAc was also detected during encystation without any significant change in the protein concentration. Distinct localisations of gPKAc to the anterior flagella, basal bodies and caudal flagella as noted in trophozoites were absent in encysting cells at later stages. Instead, PKAc staining was punctate and located mostly to the cell periphery. Our study indicates possible enrichment of the active gPKAc during late stages of encystation, which may have implications in completion of the encystment process or priming of cysts for efficient excystation.

  20. A 102 kDa subunit of a Golgi-associated particle has homology to beta subunits of trimeric G proteins.

    PubMed Central

    Harrison-Lavoie, K J; Lewis, V A; Hynes, G M; Collison, K S; Nutland, E; Willison, K R

    1993-01-01

    We have identified a 102 kDa protein, p102, which is found on the cytoplasmic face of Golgi membranes, exocytic transport vesicles and in the cytosol. A monoclonal antibody that cross-reacts with p102 is able to immunoprecipitate a 500-600 kDa protein complex containing p102 and additional subunits. The composition of this p102-containing protein complex resembles that of the Golgi coatomer complex, which constitutes the coat of non-clathrin coated vesicles. One of the subunits of the p102 complex reacts with a monoclonal antibody that detects beta-COP, a subunit of the Golgi coatomer complex. Like beta-COP, p102 exists in a brefeldin A-sensitive association with Golgi membranes. The sequence of p102 contains an N-terminal domain composed of six repeats which are similar to those found in the beta subunit of trimeric G proteins and other regulatory proteins. We suggest that p102 may be involved in regulating membrane traffic in the constitutive exocytic pathway. Images PMID:8335000

  1. Noise Mapping and Annoyance.

    PubMed

    Knauss, D.

    2002-01-01

    The EC has published a Green Paper on noise policy in the EU and has issued a directive on the assessment and reduction of environmental noise. This directive will make noise mapping mandatory for cities with at least 250.000 inhabitants. Due to the development in computer technology it is possible to calculate noise maps for large urban areas using the available data on buildings, ground profile, road and rail traffic. Examples for noise mapping are Birmingham (GB), Linz (A) and various German cities. Based on noise maps and empirical data on the correlation between annoyance and noise levels annoyance maps for different sources (rail, road, aircraft) can be calculated. Under the assumption that the annoyance for the different sources are only weakly correlated, a combined annoyance map can be calculated. In a second step using the distribution of the population the actual number of annoyed people can be evaluated. This analysis can be used, for example, to identify noise hot spots and to assess the impact of major traffic projects - roads, airports- on the noise situation as well as the impact on the population. Furthermore, the combined annoyance maps can be used to investigate on health effects and to check whether or not empirical correlations between annoyance and noise levels are sufficiently correct.

  2. Color on emergency mapping

    NASA Astrophysics Data System (ADS)

    Jiang, Lili; Qi, Qingwen; Zhang, An

    2007-06-01

    There are so many emergency issues in our daily life. Such as typhoons, tsunamis, earthquake, fires, floods, epidemics, etc. These emergencies made people lose their lives and their belongings. Every day, every hour, even every minute people probably face the emergency, so how to handle it and how to decrease its hurt are the matters people care most. If we can map it exactly before or after the emergencies; it will be helpful to the emergency researchers and people who live in the emergency place. So , through the emergency map, before emergency is occurring we can predict the situation, such as when and where the emergency will be happen; where people can refuge, etc. After disaster, we can also easily assess the lost, discuss the cause and make the lost less. The primary effect of mapping is offering information to the people who care about the emergency and the researcher who want to study it. Mapping allows the viewers to get a spatial sense of hazard. It can also provide the clues to study the relationship of the phenomenon in emergency. Color, as the basic element of the map, it can simplify and clarify the phenomenon. Color can also affects the general perceptibility of the map, and elicits subjective reactions to the map. It is to say, structure, readability, and the reader's psychological reactions can be affected by the use of color.

  3. Coastal mapping handbook

    USGS Publications Warehouse

    ,; ,; Ellis, Melvin Y.

    1978-01-01

    Passage of the Coastal Zone Management Act of 1972 focused attention on the Nation's coastal land and water areas. As plans for more effective management of the coastal zone evolved, it soon became apparent that improved maps and charts of these areas were needed. This handbook was prepared with the requirements of the entire coastal community in mind, giving greatest attention to the needs of coastal zone managers and planners at the State and local levels. Its principal objective is to provide general information and guidance; it is neither a textbook nor a technical manual, but rather a primer on coastal mapping. This handbook should help planners and managers of coastal programs to determine their mapping requirements, select the best maps and charts for their particular needs, and to deal effectively with personnel who gather data and prepare maps. The sections on "Sources of Assistance and Advice" and "Product and Data Sources" should be especially useful to all involved in mapping the coastal zone. Brief summaries of the mapping efforts of several State coastal zone management programs are included. "Future outlook" discusses anticipated progress and changes in mapping procedures and techniques. Illustrations are inserted, where appropriate, to illustrate the products and equipment discussed. Because of printing restrictions, the colors in map illustrations may vary from those in the original publication. The appendixes include substantial material which also should be of interest. In addition a glossary and an index are included to provide easy and quick access to the terms and concepts used in the text. For those interested in more technical detail than is provided in this handbook, the "Selected references" will be useful. Also, the publications of the professional societies listed in appendix 4 will provide technical information in detail.

  4. Differential distribution of G-protein beta-subunits in brain: an immunocytochemical analysis.

    PubMed

    Brunk, I; Pahner, I; Maier, U; Jenner, B; Veh, R W; Nürnberg, B; Ahnert-Hilger, G

    1999-05-01

    Heterotrimeric G proteins play central roles in signal transduction of neurons and other cells. The variety of their alpha-, beta-, and gamma-subunits allows numerous combinations thereby confering specificity to receptor-G-protein-effector interactions. Using antisera against individual G-protein beta-subunits we here present a regional and subcellular distribution of Gbeta1, Gbeta2, and Gbeta5 in rat brain. Immunocytochemical specificity of the subtype-specific antisera is revealed in Sf9 cells infected with various G-protein beta-subunits. Since Gbeta-subunits together with a G-protein gamma-subunit affect signal cascades we include a distribution of the neuron-specific Ggamma2- and Ggamma3-subunits in selected brain areas. Gbeta1, Gbeta2, and Gbeta5 are preferentially distributed in the neuropil of hippocampus, cerebellum and spinal cord. Gbeta2 is highly concentrated in the mossy fibres of dentate gyrus neurons ending in the stratum lucidum of hippocampal CA3-area. High amounts of Gbeta2 also occur in interneurons innervating spinal cord alpha-motoneurons. Gbeta5 is differentially distributed in all brain areas studied. It is found in the pyramidal cells of hippocampal CA1-CA3 as well as in the granule cell layer of dentate gyrus and in some interneurons. In the spinal cord Gbeta5 in contrast to Gbeta2 concentrates around alpha-motoneurons. In cultivated mouse hippocampal and hypothalamic neurons Gbeta2 and Gbeta5 are found in different subcellular compartments. Whereas Gbeta5 is restricted to the perikarya, Gbeta2 is also found in processes and synaptic contacts where it partially colocalizes with the synaptic vesicle protein synaptobrevin. An antiserum recognizing Ggamma2 and Ggamma3 reveals that these subunits are less expressed in hippocampus and cerebellum. Presumably this antiserum specifically recognizes Ggamma2 and Ggamma3 in combinations with certain G alphas and/or Gbetas. The widespread but regionally and cellularly rather different distribution of

  5. Relative activities and stabilities of mutant Escherichia coli tryptophan synthase alpha subunits.

    PubMed Central

    Lim, W K; Shin, H J; Milton, D L; Hardman, J K

    1991-01-01

    In vitro mutagenesis of the Escherichia coli trpA gene has yielded 66 mutant tryptophan synthase alpha subunits containing single amino acid substitutions at 49 different residue sites and 29 double and triple amino acid substitutions at 16 additional sites, all within the first 121 residues of the protein. The 66 singly altered mutant alpha subunits encoded from overexpression vectors have been examined for their ability to support growth in trpA mutant host strains and for their enzymatic and stability properties in crude extracts. With the exception of mutant alpha subunits altered at catalytic residue sites Glu-49 and Asp-60, all support growth; this includes those (48 of 66) that have no enzymatic defects and those (18 of 66) that do. The majority of the enzymatically defective mutant alpha subunits have decreased capacities for substrate (indole-3-glycerol phosphate) utilization, typical of the early trpA missense mutants isolated by in vivo selection methods. These defects vary in severity from complete loss of activity for mutant alpha subunits altered at residue positions 49 and 60 to those, altered elsewhere, that are partially (up to 40 to 50%) defective. The complete inactivation of the proteins altered at the two catalytic residue sites suggest that, as found via in vitro site-specific mutagenesis of the Salmonella typhimurium tryptophan synthetase alpha subunit, both residues probably also participate in a push-pull general acid-base catalysis of indole-3-glycerol phosphate breakdown for the E. coli enzyme as well. Other classes of mutant alpha subunits include some novel types that are defective in their functional interaction with the other tryptophan synthetase component, the beta 2 subunit. Also among the mutant alpha subunits, 19 were found altered at one or another of the 34 conserved residue sites in this portion of the alpha polypeptide sequence; surprisingly, 10 of these have wild-type enzymatic activity, and 16 of these can satisfy growth

  6. Automatic cloud cover mapping.

    NASA Technical Reports Server (NTRS)

    Strong, J. P., III; Rosenfeld, A.

    1971-01-01

    A method of converting a picture into a 'cartoon' or 'map' whose regions correspond to differently textured regions is described. Texture edges in the picture are detected, and solid regions surrounded by these (usually broken) edges are 'colored in' using a propagation process. The resulting map is cleaned by comparing the region colors with the textures of the corresponding regions in the picture, and also by merging some regions with others according to criteria based on topology and size. The method has been applied to the construction of cloud cover maps from cloud cover pictures obtained by satellites.

  7. Mapping the Baby Universe

    NASA Technical Reports Server (NTRS)

    Wanjek, Christopher

    2003-01-01

    In June, NASA plans to launch the Microwave Anisotropy Probe (MAP) to survey the ancient radiation in unprecedented detail. MAP will map slight temperature fluctuations within the microwave background that vary by only 0.00001 C across a chilly radiation that now averages 2.73 C above absolute zero. The temperature differences today point back to density differences in the fiery baby universe, in which there was a little more matter here and a little less matter there. Areas of slightly enhanced density had stronger gravity than low-density areas. The high-density areas pulled back on the background radiation, making it appear slightly cooler in those directions.

  8. The A1 Subunit of Shiga Toxin 2 Has Higher Affinity for Ribosomes and Higher Catalytic Activity than the A1 Subunit of Shiga Toxin 1

    PubMed Central

    Basu, Debaleena; Li, Xiao-Ping; Kahn, Jennifer N.; May, Kerrie L.; Kahn, Peter C.

    2015-01-01

    Shiga toxin (Stx)-producing Escherichia coli (STEC) infections can lead to life-threatening complications, including hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS), which is the most common cause of acute renal failure in children in the United States. Stx1 and Stx2 are AB5 toxins consisting of an enzymatically active A subunit associated with a pentamer of receptor binding B subunits. Epidemiological evidence suggests that Stx2-producing E. coli strains are more frequently associated with HUS than Stx1-producing strains. Several studies suggest that the B subunit plays a role in mediating toxicity. However, the role of the A subunits in the increased potency of Stx2 has not been fully investigated. Here, using purified A1 subunits, we show that Stx2A1 has a higher affinity for yeast and mammalian ribosomes than Stx1A1. Biacore analysis indicated that Stx2A1 has faster association and dissociation with ribosomes than Stx1A1. Analysis of ribosome depurination kinetics demonstrated that Stx2A1 depurinates yeast and mammalian ribosomes and an RNA stem-loop mimic of the sarcin/ricin loop (SRL) at a higher catalytic rate and is a more efficient enzyme than Stx1A1. Stx2A1 depurinated ribosomes at a higher level in vivo and was more cytotoxic than Stx1A1 in Saccharomyces cerevisiae. Stx2A1 depurinated ribosomes and inhibited translation at a significantly higher level than Stx1A1 in human cells. These results provide the first direct evidence that the higher affinity for ribosomes in combination with higher catalytic activity toward the SRL allows Stx2A1 to depurinate ribosomes, inhibit translation, and exhibit cytotoxicity at a significantly higher level than Stx1A1. PMID:26483409

  9. The A1 Subunit of Shiga Toxin 2 Has Higher Affinity for Ribosomes and Higher Catalytic Activity than the A1 Subunit of Shiga Toxin 1.

    PubMed

    Basu, Debaleena; Li, Xiao-Ping; Kahn, Jennifer N; May, Kerrie L; Kahn, Peter C; Tumer, Nilgun E

    2015-10-19

    Shiga toxin (Stx)-producing Escherichia coli (STEC) infections can lead to life-threatening complications, including hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS), which is the most common cause of acute renal failure in children in the United States. Stx1 and Stx2 are AB5 toxins consisting of an enzymatically active A subunit associated with a pentamer of receptor binding B subunits. Epidemiological evidence suggests that Stx2-producing E. coli strains are more frequently associated with HUS than Stx1-producing strains. Several studies suggest that the B subunit plays a role in mediating toxicity. However, the role of the A subunits in the increased potency of Stx2 has not been fully investigated. Here, using purified A1 subunits, we show that Stx2A1 has a higher affinity for yeast and mammalian ribosomes than Stx1A1. Biacore analysis indicated that Stx2A1 has faster association and dissociation with ribosomes than Stx1A1. Analysis of ribosome depurination kinetics demonstrated that Stx2A1 depurinates yeast and mammalian ribosomes and an RNA stem-loop mimic of the sarcin/ricin loop (SRL) at a higher catalytic rate and is a more efficient enzyme than Stx1A1. Stx2A1 depurinated ribosomes at a higher level in vivo and was more cytotoxic than Stx1A1 in Saccharomyces cerevisiae. Stx2A1 depurinated ribosomes and inhibited translation at a significantly higher level than Stx1A1 in human cells. These results provide the first direct evidence that the higher affinity for ribosomes in combination with higher catalytic activity toward the SRL allows Stx2A1 to depurinate ribosomes, inhibit translation, and exhibit cytotoxicity at a si