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Sample records for putative csn5 subunit

  1. Analysis of the role of COP9 Signalosome (CSN subunits in K562; the first link between CSN and autophagy

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    Bunce Christopher M

    2009-04-01

    Full Text Available Abstract Background The COP9/signalosome (CSN is a highly conserved eight subunit complex that, by deneddylating cullins in cullin-based E3 ubiquitin ligases, regulates protein degradation. Although studied in model human cell lines such as HeLa, very little is known about the role of the CSN in haemopoietic cells. Results Greater than 95% knockdown of the non-catalytic subunit CSN2 and the deneddylating subunit CSN5 of the CSN was achieved in the human myeloid progenitor cell line K562. CSN2 knockdown led to a reduction of both CSN5 protein and mRNA whilst CSN5 knockdown had little effect on CSN2. Both knockdowns inhibited CSN deneddylase function as demonstrated by accumulation of neddylated Cul1. Furthermore, both knockdowns resulted in the sequential loss of Skp2, Cdc4 and β-TrCP F-box proteins. These proteins were rescued by the proteasome inhibitor MG132, indicating the autocatalytic degradation of F-box proteins upon loss of CSN2 or CSN5. Interestingly, altered F-box protein gene expression was also observed in CSN2 and CSN5 knockdowns, suggesting a potential role of the CSN in regulating F-box protein transcription. Loss of either CSN subunit dramatically reduced cell growth but resulted in distinct patterns of cell death. CSN5 knockdown caused mitotic defects, G2/M arrest and apoptotic cell death. CSN2 knockdown resulted in non-apoptotic cell death associated with accumulation of both the autophagy marker LC3-II and autophagic vacuoles. Treatment of vector control K562 cells with the autophagy inhibitors 3-methyladenine and bafilomycin A1 recapitulated the growth kinetics, vacuolar morphology and LC3-II accumulation of CSN2 knockdown cells indicating that the cellular phenotype of CSN2 cells arises from autophagy inhibition. Finally, loss of CSN2 was associated with the formation of a CSN5 containing subcomplex. Conclusion We conclude that CSN2 is required for CSN integrity and the stability of individual CSN subunits, and postulate

  2. Expression, purification and crystallization of a PCI domain from the COP9 signalosome subunit 7 (CSN7)

    International Nuclear Information System (INIS)

    Dessau, Moshe; Chamovitz, Daniel A.; Hirsch, Joel A.

    2006-01-01

    A core fragment of Arabidopsis thaliana COP9 signalosome (CSN) subunit 7 was expressed in Escherichia coli. The protein was purified to homogeneity and crystallized. A core fragment of Arabidopsis thaliana COP9 signalosome (CSN) subunit 7 was expressed in Escherichia coli. The protein was purified to homogeneity and screened for crystallization. Crystallization conditions were refined using the sitting-drop vapour-diffusion method. Crystals were obtained using polyethylene glycol 8000 as a precipitant and have a thick rod-like morphology. Their crystallographic symmetry is orthorhombic, space group C222 1 , with unit-cell parameters a = 57.2, b = 86.2, c = 72.6 Å and a diffraction limit of 2.06 Å

  3. Molecular Characterization of the Llamas (Lama glama) Casein Cluster Genes Transcripts (CSN1S1, CSN2, CSN1S2, CSN3) and Regulatory Regions

    Science.gov (United States)

    Pauciullo, Alfredo; Erhardt, Georg

    2015-01-01

    In the present paper, we report for the first time the characterization of llama (Lama glama) caseins at transcriptomic and genetic level. A total of 288 casein clones transcripts were analysed from two lactating llamas. The most represented mRNA populations were those correctly assembled (85.07%) and they encoded for mature proteins of 215, 217, 187 and 162 amino acids respectively for the CSN1S1, CSN2, CSN1S2 and CSN3 genes. The exonic subdivision evidenced a structure made of 21, 9, 17 and 6 exons for the αs1-, β-, αs2- and κ-casein genes respectively. Exon skipping and duplication events were evidenced. Two variants A and B were identified in the αs1-casein gene as result of the alternative out-splicing of the exon 18. An additional exon coding for a novel esapeptide was found to be cryptic in the κ-casein gene, whereas one extra exon was found in the αs2-casein gene by the comparison with the Camelus dromedaries sequence. A total of 28 putative phosphorylated motifs highlighted a complex heterogeneity and a potential variable degree of post-translational modifications. Ninety-six polymorphic sites were found through the comparison of the lama casein cDNAs with the homologous camel sequences, whereas the first description and characterization of the 5’- and 3’-regulatory regions allowed to identify the main putative consensus sequences involved in the casein genes expression, thus opening the way to new investigations -so far- never achieved in this species. PMID:25923814

  4. Molecular Characterization of the Llamas (Lama glama) Casein Cluster Genes Transcripts (CSN1S1, CSN2, CSN1S2, CSN3) and Regulatory Regions.

    Science.gov (United States)

    Pauciullo, Alfredo; Erhardt, Georg

    2015-01-01

    In the present paper, we report for the first time the characterization of llama (Lama glama) caseins at transcriptomic and genetic level. A total of 288 casein clones transcripts were analysed from two lactating llamas. The most represented mRNA populations were those correctly assembled (85.07%) and they encoded for mature proteins of 215, 217, 187 and 162 amino acids respectively for the CSN1S1, CSN2, CSN1S2 and CSN3 genes. The exonic subdivision evidenced a structure made of 21, 9, 17 and 6 exons for the αs1-, β-, αs2- and κ-casein genes respectively. Exon skipping and duplication events were evidenced. Two variants A and B were identified in the αs1-casein gene as result of the alternative out-splicing of the exon 18. An additional exon coding for a novel esapeptide was found to be cryptic in the κ-casein gene, whereas one extra exon was found in the αs2-casein gene by the comparison with the Camelus dromedaries sequence. A total of 28 putative phosphorylated motifs highlighted a complex heterogeneity and a potential variable degree of post-translational modifications. Ninety-six polymorphic sites were found through the comparison of the lama casein cDNAs with the homologous camel sequences, whereas the first description and characterization of the 5'- and 3'-regulatory regions allowed to identify the main putative consensus sequences involved in the casein genes expression, thus opening the way to new investigations -so far- never achieved in this species.

  5. Csn5 Is Required for the Conidiogenesis and Pathogenesis of the Alternaria alternata Tangerine Pathotype

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    Mingshuang Wang

    2018-03-01

    Full Text Available The COP9 signalosome (CSN is a highly conserved protein complex involved in the ubiquitin-proteasome system. Its metalloisopeptidase activity resides in subunit 5 (CSN5. Functions of csn5 in phytopathogenic fungi are poorly understood. Here, we knocked out the csn5 ortholog (Aacsn5 in the tangerine pathotype of Alternaria alternata. The ΔAacsn5 mutant showed a moderately reduced growth rate compared to the wildtype strain and was unable to produce conidia. The growth of ΔAacsn5 mutant was not affected in response to oxidative and osmotic stresses. Virulence assays revealed that ΔAacsn5 induced no or significantly reduced necrotic lesions on detached citrus leaves. The defects in hyphal growth, conidial sporulation, and pathogenicity of ΔAacsn5 were restored by genetic complementation of the mutant with wildtype Aacsn5. To explore the molecular mechanisms of conidiation and pathogenesis underlying Aacsn5 regulation, we systematically examined the transcriptomes of both ΔAacsn5 and the wildtype. Generally, 881 genes were overexpressed and 777 were underexpressed in the ΔAacsn5 mutant during conidiation while 694 overexpressed and 993 underexpressed during infection. During asexual development, genes related to the transport processes and nitrogen metabolism were significantly downregulated; the expression of csn1–4 and csn7 in ΔAacsn5 was significantly elevated; secondary metabolism gene clusters were broadly affected; especially, the transcript level of the whole of cluster 28 and 30 was strongly induced. During infection, the expression of the host-specific ACT toxin gene cluster which controls the biosynthesis of the citrus specific toxin was significantly repressed; many other SM clusters with unknown products were also regulated; 86 out of 373 carbohydrate-active enzymes responsible for breaking down the plant dead tissues showed uniquely decreased expression. Taken together, our results expand our understanding of the roles of csn5

  6. The β-catenin E3 ubiquitin ligase SIAH-1 is regulated by CSN5/JAB1 in CRC cells.

    Science.gov (United States)

    Jumpertz, Sandra; Hennes, Thomas; Asare, Yaw; Vervoorts, Jörg; Bernhagen, Jürgen; Schütz, Anke K

    2014-09-01

    COP9 signalosome subunit 5 (CSN5) plays a decisive role in cellular processes such as cell cycle regulation and apoptosis via promoting protein degradation, gene transcription, and nuclear export. CSN5 regulates cullin-RING-E3 ligase (CRL) activity through its deNEDDylase function. It is overexpressed in several tumor entities, but its role in colorectal cancer (CRC) is poorly understood. Wnt/β-catenin signaling is aberrant in most CRC cells, resulting in increased levels of oncogenic β-catenin and thus tumor progression. Under physiological conditions, β-catenin levels are tightly regulated by continuous proteasomal degradation. We recently showed that knockdown of CSN5 in model and CRC cells results in decreased (phospho)-β-catenin levels. Reduced β-catenin levels were associated with an attenuated proliferation rate of different CRC cell types after CSN5 knockdown. The canonical Wnt pathway involves degradation of β-catenin by a β-TrCP1-containing E3 ligase, but is mostly non-functional in CRC cells. We thus hypothesized that alternative β-catenin degradation mediated by SIAH-1 (seven in absentia homolog-1), is responsible for the effect of CSN5 on β-catenin signaling in CRC cells. We found that SIAH-1 plays an essential role in β-catenin degradation in HCT116 CRC cells and that CSN5 affects β-catenin target gene expression in these cells. Of note, CSN5 affected SIAH-1 mRNA and SIAH-1 protein levels. Moreover, β-catenin and SIAH-1 form protein complexes with CSN5 in HCT116 cells. Lastly, we demonstrate that CSN5 promotes SIAH-1 degradation in HCT116 and SW480 cells and that this is associated with its deNEDDylase activity. In conclusion, we have identified a CSN5/β-catenin/SIAH-1 interaction network that might control β-catenin degradation in CRC cells. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. CSNAP Is a Stoichiometric Subunit of the COP9 Signalosome

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    Shelly Rozen

    2015-10-01

    Full Text Available The highly conserved COP9 signalosome (CSN complex is a key regulator of all cullin-RING-ubiquitin ligases (CRLs, the largest family of E3 ubiquitin ligases. Until now, it was accepted that the CSN is composed of eight canonical components. Here, we report the discovery of an additional integral and stoichiometric subunit that had thus far evaded detection, and we named it CSNAP (CSN acidic protein. We show that CSNAP binds CSN3, CSN5, and CSN6, and its incorporation into the CSN complex is mediated through the C-terminal region involving conserved aromatic residues. Moreover, depletion of this small protein leads to reduced proliferation and a flattened and enlarged morphology. Finally, on the basis of sequence and structural properties shared by both CSNAP and DSS1, a component of the related 19S lid proteasome complex, we propose that CSNAP, the ninth CSN subunit, is the missing paralogous subunit of DSS1.

  8. The devil is in the details: comparison between COP9 signalosome (CSN) and the LID of the 26S proteasome.

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    Meister, Cindy; Gulko, Miriam Kolog; Köhler, Anna M; Braus, Gerhard H

    2016-02-01

    The COP9 signalosome (CSN) and the proteasomal LID are conserved macromolecular complexes composed of at least eight subunits with molecular weights of approximately 350 kDa. CSN and LID are part of the ubiquitin–proteasome pathway and cleave isopeptide linkages of lysine side chains on target proteins. CSN cleaves the isopeptide bond of ubiquitin-like protein Nedd8 from cullins, whereas the LID cleaves ubiquitin from target proteins sentenced for degradation. CSN and LID are structurally and functionally similar but the order of the assembly pathway seems to be different. The assembly differs in at least the last subunit joining the pre-assembled subcomplex. This review addresses the similarities and differences in structure, function and assembly of CSN and LID.

  9. The COP9 Signalosome regulates seed germination by facilitating protein degradation of RGL2 and ABI5.

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    Dan Jin

    2018-02-01

    Full Text Available The control of seed germination and seed dormancy are critical for the successful propagation of plant species, and are important agricultural traits. Seed germination is tightly controlled by the balance of gibberellin (GA and abscisic acid (ABA, and is influenced by environmental factors. The COP9 Signalosome (CSN is a conserved multi-subunit protein complex that is best known as a regulator of the Cullin-RING family of ubiquitin E3 ligases (CRLs. Multiple viable mutants of the CSN showed poor germination, except for csn5b-1. Detailed analyses showed that csn1-10 has a stronger seed dormancy, while csn5a-1 mutants exhibit retarded seed germination in addition to hyperdormancy. Both csn5a-1 and csn1-10 plants show defects in the timely removal of the germination inhibitors: RGL2, a repressor of GA signaling, and ABI5, an effector of ABA responses. We provide genetic evidence to demonstrate that the germination phenotype of csn1-10 is caused by over-accumulation of RGL2, a substrate of the SCF (CRL1 ubiquitin E3 ligase, while the csn5a-1 phenotype is caused by over-accumulation of RGL2 as well as ABI5. The genetic data are consistent with the hypothesis that CSN5A regulates ABI5 by a mechanism that may not involve CSN1. Transcriptome analyses suggest that CSN1 has a more prominent role than CSN5A during seed maturation, but CSN5A plays a more important role than CSN1 during seed germination, further supporting the functional distinction of these two CSN genes. Our study delineates the molecular targets of the CSN complex in seed germination, and reveals that CSN5 has additional functions in regulating ABI5, thus the ABA signaling pathway.

  10. MPN+, a putative catalytic motif found in a subset of MPN domain proteins from eukaryotes and prokaryotes, is critical for Rpn11 function

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    Hofmann Kay

    2002-09-01

    Full Text Available Abstract Background Three macromolecular assemblages, the lid complex of the proteasome, the COP9-Signalosome (CSN and the eIF3 complex, all consist of multiple proteins harboring MPN and PCI domains. Up to now, no specific function for any of these proteins has been defined, nor has the importance of these motifs been elucidated. In particular Rpn11, a lid subunit, serves as the paradigm for MPN-containing proteins as it is highly conserved and important for proteasome function. Results We have identified a sequence motif, termed the MPN+ motif, which is highly conserved in a subset of MPN domain proteins such as Rpn11 and Csn5/Jab1, but is not present outside of this subfamily. The MPN+ motif consists of five polar residues that resemble the active site residues of hydrolytic enzyme classes, particularly that of metalloproteases. By using site-directed mutagenesis, we show that the MPN+ residues are important for the function of Rpn11, while a highly conserved Cys residue outside of the MPN+ motif is not essential. Single amino acid substitutions in MPN+ residues all show similar phenotypes, including slow growth, sensitivity to temperature and amino acid analogs, and general proteasome-dependent proteolysis defects. Conclusions The MPN+ motif is abundant in certain MPN-domain proteins, including newly identified proteins of eukaryotes, bacteria and archaea thought to act outside of the traditional large PCI/MPN complexes. The putative catalytic nature of the MPN+ motif makes it a good candidate for a pivotal enzymatic function, possibly a proteasome-associated deubiquitinating activity and a CSN-associated Nedd8/Rub1-removing activity.

  11. The V-ATPase a2-subunit as a putative endosomal pH-sensor.

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    Marshansky, V

    2007-11-01

    V-ATPase (vesicular H(+)-ATPase)-driven intravesicular acidification is crucial for vesicular trafficking. Defects in vesicular acidification and trafficking have recently been recognized as essential determinants of various human diseases. An important role of endosomal acidification in receptor-ligand dissociation and in activation of lysosomal hydrolytic enzymes is well established. However, the molecular mechanisms by which luminal pH information is transmitted to the cytosolic small GTPases that control trafficking events such as budding, coat formation and fusion are unknown. Here, we discuss our recent discovery that endosomal V-ATPase is a pH-sensor regulating the degradative pathway. According to our model, V-ATPase is responsible for: (i) the generation of a pH gradient between vesicular membranes; (ii) sensing of intravesicular pH; and (iii) transmitting this information to the cytosolic side of the membrane. We also propose the hypothetical molecular mechanism involved in function of the V-ATPase a2-subunit as a putative pH-sensor. Based on extensive experimental evidence on the crucial role of histidine residues in the function of PSPs (pH-sensing proteins) in eukaryotic cells, we hypothesize that pH-sensitive histidine residues within the intra-endosomal loops and/or C-terminal luminal tail of the a2-subunit could also be involved in the pH-sensing function of V-ATPase. However, in order to identify putative pH-sensitive histidine residues and to test this hypothesis, it is absolutely essential that we increase our understanding of the folding and transmembrane topology of the a-subunit isoforms of V-ATPase. Thus the crucial role of intra-endosomal histidine residues in pH-dependent conformational changes of the V-ATPase a2-isoform, its interaction with cytosolic small GTPases and ultimately in its acidification-dependent regulation of the endosomal/lysosomal protein degradative pathway remain to be determined.

  12. Methodology for the knowledge preservation in the CSN Proyecto RECO

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    Montero, M.A.; Gil, E.; Merino, C.; Gonzalez, N.

    2016-07-01

    This paper summarizes the RECOR Project carried out by the Spanish Nuclear Regulatory Authority (CSN) to develop a methodology to preserve nuclear knowledge, specifically adapted to the CSN regulatory functions. The project covers the development, implantation and verification of the methodology and the development necessary procedures and tools to align it with the CSN Integrated Management System. The Project is complemented with a number of sessions to motivate the CSN management with the methodology and training the staff in the use and Implementation of the methodology. (Author)

  13. Structural insights into RipC, a putative citrate lyase β subunit from a Yersinia pestis virulence operon

    International Nuclear Information System (INIS)

    Torres, Rodrigo; Chim, Nicholas; Sankaran, Banumathi; Pujol, Céline; Bliska, James B.; Goulding, Celia W.

    2011-01-01

    Comparison of the 2.45 Å resolution crystal structure of homotrimeric RipC, a putative citrate lyase β subunit from Y. pestis, with structural homologs reveals conserved RipC residues that are implicated in CoA binding. Yersinia pestis remains a threat, with outbreaks of plague occurring in rural areas and its emergence as a weapon of bioterrorism; thus, an improved understanding of its various pathogenicity pathways is warranted. The rip (required for intracellular proliferation) virulence operon is required for Y. pestis survival in interferon-γ-treated macrophages and has been implicated in lowering macrophage-produced nitric oxide levels. RipC, one of three gene products from the rip operon, is annotated as a citrate lyase β subunit. Furthermore, the Y. pestis genome lacks genes that encode citrate lyase α and γ subunits, suggesting a unique functional role of RipC in the Y. pestisrip-mediated survival pathway. Here, the 2.45 Å resolution crystal structure of RipC revealed a homotrimer in which each monomer consists of a (β/α) 8 TIM-barrel fold. Furthermore, the trimeric state was confirmed in solution by size-exclusion chromatography. Through sequence and structure comparisons with homologous proteins, it is proposed that RipC is a putative CoA- or CoA-derivative binding protein

  14. The CSN's Information Centre: a necessary venture

    International Nuclear Information System (INIS)

    Calvo, A.

    1999-01-01

    Every year, around 10,000 pupils will visit the CSN's Information Centre, an exposition area which was created with the intention of explaining simply but strictly, what radiations are, for what they are used, what risks they hold and how they are controlled. Opened in October, 1998, encouraged by Parliamentary urging, the Centre is a path to CSN Information for citizens to use. (Author)

  15. Translation attenuation via 3′ terminal codon usage in bovine csn1s2 is responsible for the difference in αs2- and β-casein profile in milk

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    Kim, Julie J; Yu, Jaeju; Bag, Jnanankur; Bakovic, Marica; Cant, John P

    2015-01-01

    The rate of secretion of αs2-casein into bovine milk is approximately 25% of that of β-casein, yet mammary expression of their respective mRNA transcripts (csn1s2 and csn2) is not different. Our objective was to identify molecular mechanisms that explain the difference in translation efficiency between csn1s2 and csn2. Cell-free translational efficiency of csn2 was 5 times that of csn1s2. Transcripts of csn1s2 distributed into heavier polysomes than csn2 transcripts, indicating an attenuation of elongation and/or termination. Stimulatory and inhibitory effects of the 5′ and 3′ UTRs on translational efficiency were different with luciferase and casein sequences in the coding regions. Substituting the 5′ and 3′ UTRs from csn2 into csn1s2 did not improve csn1s2 translation, implicating the coding region itself in the translation difference. Deletion of a 28-codon fragment from the 3′ terminus of the csn1s2 coding region, which displays codons with low correlations to cell fitness, increased translation to a par with csn2. We conclude that the usage of the last 28 codons of csn1s2 is the main regulatory element that attenuates its expression and is responsible for the differential translational expression of csn1s2 and csn2. PMID:25826667

  16. The Evolution of COP9 Signalosome in Unicellular and Multicellular Organisms.

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    Barth, Emanuel; Hübler, Ron; Baniahmad, Aria; Marz, Manja

    2016-05-02

    The COP9 signalosome (CSN) is a highly conserved protein complex, recently being crystallized for human. In mammals and plants the COP9 complex consists of nine subunits, CSN 1-8 and CSNAP. The CSN regulates the activity of culling ring E3 ubiquitin and plays central roles in pleiotropy, cell cycle, and defense of pathogens. Despite the interesting and essential functions, a thorough analysis of the CSN subunits in evolutionary comparative perspective is missing. Here we compared 61 eukaryotic genomes including plants, animals, and yeasts genomes and show that the most conserved subunits of eukaryotes among the nine subunits are CSN2 and CSN5. This may indicate a strong evolutionary selection for these two subunits. Despite the strong conservation of the protein sequence, the genomic structures of the intron/exon boundaries indicate no conservation at genomic level. This suggests that the gene structure is exposed to a much less selection compared with the protein sequence. We also show the conservation of important active domains, such as PCI (proteasome lid-CSN-initiation factor) and MPN (MPR1/PAD1 amino-terminal). We identified novel exons and alternative splicing variants for all CSN subunits. This indicates another level of complexity of the CSN. Notably, most COP9-subunits were identified in all multicellular and unicellular eukaryotic organisms analyzed, but not in prokaryotes or archaeas. Thus, genes encoding CSN subunits present in all analyzed eukaryotes indicate the invention of the signalosome at the root of eukaryotes. The identification of alternative splice variants indicates possible "mini-complexes" or COP9 complexes with independent subunits containing potentially novel and not yet identified functions. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  17. Inositol hexakisphosphate (IP6) generated by IP5K mediates cullin-COP9 signalosome interactions and CRL function.

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    Scherer, Paul C; Ding, Yan; Liu, Zhiqing; Xu, Jing; Mao, Haibin; Barrow, James C; Wei, Ning; Zheng, Ning; Snyder, Solomon H; Rao, Feng

    2016-03-29

    The family of cullin-RING E3 Ligases (CRLs) and the constitutive photomorphogenesis 9 (COP9) signalosome (CSN) form dynamic complexes that mediate ubiquitylation of 20% of the proteome, yet regulation of their assembly/disassembly remains poorly understood. Inositol polyphosphates are highly conserved signaling molecules implicated in diverse cellular processes. We now report that inositol hexakisphosphate (IP6) is a major physiologic determinant of the CRL-CSN interface, which includes a hitherto unidentified electrostatic interaction between the N-terminal acidic tail of CSN subunit 2 (CSN2) and a conserved basic canyon on cullins. IP6, with an EC50 of 20 nM, acts as an intermolecular "glue," increasing cullin-CSN2 binding affinity by 30-fold, thereby promoting assembly of the inactive CRL-CSN complexes. The IP6 synthase, Ins(1,3,4,5,6)P5 2-kinase (IPPK/IP5K) binds to cullins. Depleting IP5K increases the percentage of neddylated, active Cul1 and Cul4A, and decreases levels of the Cul1/4A substrates p27 and p21. Besides dysregulating CRL-mediated cell proliferation and UV-induced apoptosis, IP5K depletion potentiates by 28-fold the cytotoxic effect of the neddylation inhibitor MLN4924. Thus, IP5K and IP6 are evolutionarily conserved components of the CRL-CSN system and are potential targets for cancer therapy in conjunction with MLN4924.

  18. Comparative Analysis of Putative Orthologues of Mitochondrial Import Motor Subunit: Pam18 and Pam16 in Plants

    OpenAIRE

    Chen, Xuejin; Ghazanfar, Bushra; Khan, Abdul Rehman; Hayat, Sikandar; Cheng, Zhihui

    2013-01-01

    Pam18/Tim14 and Pam16/Tim16, highly conserved proteins among eukaryotes, are two essential subunits of protein import motors localized in the inner mitochondrial membrane. The heterodimer formed by Pam18 and Pam16 via their J-type domains serves a regulatory function in protein translocation. Here, we report that thirty-one Pam18 and twenty-six Pam16 putative orthologues in twelve plant species were identified and analyzed through bioinformatics strategy. Results data revealed that Pam18 and ...

  19. CSN-associated USP48 confers stability to nuclear NF-κB/RelA by trimming K48-linked Ub-chains.

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    Schweitzer, Katrin; Naumann, Michael

    2015-02-01

    Diligent balance of nuclear factor kappa B (NF-κB) activity is essential owing to NF-κB's decisive role in cellular processes including inflammation, immunity and cell survival. Ubiquitin/proteasome-system (UPS)-dependent degradation of activated NF-κB/RelA involves the cullin-RING-ubiquitin-ligase (CRL) ECS(SOCS1). The COP9 signalosome (CSN) controls ubiquitin (Ub) ligation by CRLs through the removal of the CRL-activating Ub-like modifier NEDD8 from their cullin subunits and through deubiquitinase (DUB) activity of associated DUBs. However, knowledge about DUBs involved in the regulation of NF-κB activity within the nucleus is scarce. In this study we observed that USP48, a DUB of hitherto ill-defined function identified through a siRNA screen, associates with the CSN and RelA in the nucleus. We show that USP48 trims rather than completely disassembles long K48-linked free and substrate-anchored Ub-chains, a catalytic property only shared with ataxin-3 (Atx3) and otubain-1 (OTU1), and that USP48 Ub-chain-trimming activity is regulated by casein-kinase-2 (CK2)-mediated phosphorylation in response to cytokine-stimulation. Functionally, we demonstrate for the first time the CSN and USP48 to cooperatively stabilize the nuclear pool of RelA, thereby facilitating timely induction and shutoff of NF-κB target genes. In summary, this study demonstrates that USP48, a nuclear DUB regulated by CK2, controls the UPS-dependent turnover of activated NF-κB/RelA in the nucleus together with the CSN. Thereby USP48 contributes to a timely control of immune responses. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. IRRS: The CSN under the microscope; Mision IRRS: el CSN bajo el microscopio

    Energy Technology Data Exchange (ETDEWEB)

    Salome, M.

    2008-07-01

    Throughout the two weeks from January 28th to February 8th an international team of experts, appointed by the IAEA and led by Ulrich Schmocker from Switzerland, carried out a detailed study of the CSN. The objective of the IRRS mission, which was performed in response to a request from the Council itself, was to analyse the structure and operation of the Spanish regulatory body in order to determine its strengths and weaknesses and to suggest measures to improve its management. (Author)

  1. Identification and characterization of the donkey CSN1S2 I and II cDNAs

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    Davide Nicodemo

    2010-04-01

    Full Text Available The αs2 casein, encoded by the CSN1S2 gene, is one of the three Calcium sensitive caseins present in the milk of ruminants of zootechnical interest and in the milk of Equidae species (horse and donkey. In the present study, we cloned, sequenced and analysed two different donkey CSN1S2 cDNAs that we called CSN1S2 I and CSN1S2 II. The first, which spans over a fragment of 1016 nt, is constituted by 19 exons and encodes for a predicted protein (called αs2-I of 221 aminoacids; the second, of which we determined the entire sequence (16 exons, encodes for a predicted peptide (called αs2-II of 168 aminoacids. Alternative splicing and genetic markers are reported for both genes.

  2. Mutations in the putative zinc-binding motif of UL52 demonstrate a complex interdependence between the UL5 and UL52 subunits of the human herpes simplex virus type 1 helicase/primase complex.

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    Chen, Yan; Carrington-Lawrence, Stacy D; Bai, Ping; Weller, Sandra K

    2005-07-01

    Herpes simplex virus type 1 (HSV-1) encodes a heterotrimeric helicase-primase (UL5/8/52) complex. UL5 contains seven motifs found in helicase superfamily 1, and UL52 contains conserved motifs found in primases. The contributions of each subunit to the biochemical activities of the complex, however, remain unclear. We have previously demonstrated that a mutation in the putative zinc finger at UL52 C terminus abrogates not only primase but also ATPase, helicase, and DNA-binding activities of a UL5/UL52 subcomplex, indicating a complex interdependence between the two subunits. To test this hypothesis and to further investigate the role of the zinc finger in the enzymatic activities of the helicase-primase, a series of mutations were constructed in this motif. They differed in their ability to complement a UL52 null virus: totally defective, partial complementation, and potentiating. In this study, four of these mutants were studied biochemically after expression and purification from insect cells infected with recombinant baculoviruses. All mutants show greatly reduced primase activity. Complementation-defective mutants exhibited severe defects in ATPase, helicase, and DNA-binding activities. Partially complementing mutants displayed intermediate levels of these activities, except that one showed a wild-type level of helicase activity. These data suggest that the UL52 zinc finger motif plays an important role in the activities of the helicase-primase complex. The observation that mutations in UL52 affected helicase, ATPase, and DNA-binding activities indicates that UL52 binding to DNA via the zinc finger may be necessary for loading UL5. Alternatively, UL5 and UL52 may share a DNA-binding interface.

  3. The Emergency Action Plan of the Spanish Nuclear Safety Council (CSN)

    International Nuclear Information System (INIS)

    Calvin Cuarteto, M.; Camarma, J. R.; Martin Calvarro, J. M

    2007-01-01

    The Spanish Nuclear safety Council (CSN) has assigned by law among others the function to coordinate the measures of support and answer to nuclear emergency situations for all the aspects related with nuclear safety and radiological protection. Integrating and coordinating the different organisations public and private companies whose aid is necessary for the fulfilment of the functions attributed to the Regulatory Body. In order to suitable perform this function, CSN has equipped itself with an Emergency Action Plan that structures the response organization, establishes responsibility levels, incorporates basic performance procedures and includes capabilities to face the nuclear and radiological emergencies considering the external supports, resulting from the collaboration agreements with public institutions and private companies. To accomplish the above mentioned Emergency Action Plan, CSN has established and implanted a formation and training and re-training program for the organization response for emergencies and has update an operative centre (Emergency Room called Salem), equipped with infrastructures, tools and communication and operative systems that incorporate the more advanced technologies available to date. (Author)

  4. ANÁLISE PATENTOMÉTRICA DAS TECNOLOGIAS DE PROCESSO DA COMPANHIA SIDERÚRGICA NACIONAL (CSN

    Directory of Open Access Journals (Sweden)

    Mariana Costa Fagundes

    2016-06-01

    Full Text Available Este artigo tem como objetivo traçar o perfil tecnológico dos processos produtivos da Companhia Siderúrgica Nacional (CSN. Para tanto, são analisados, por meio de um estudo patentométrico, os depósitos de pedido de patente ou modelo de utilidade da CSN, no período entre 1971 e 2009, classificando-os entre tecnologias de produto e tecnologias de processo. O estudo mostrou que 60% de tais depósitos relacionam-se a tecnologias de processo. Conclui-se que há uma forte diferença entre o padrão de patenteamento da CSN nos períodos pré e pós-privatização.

  5. Neurospora COP9 signalosome integrity plays major roles for hyphal growth, conidial development, and circadian function.

    Directory of Open Access Journals (Sweden)

    Zhipeng Zhou

    Full Text Available The COP9 signalosome (CSN is a highly conserved multifunctional complex that has two major biochemical roles: cleaving NEDD8 from cullin proteins and maintaining the stability of CRL components. We used mutation analysis to confirm that the JAMM domain of the CSN-5 subunit is responsible for NEDD8 cleavage from cullin proteins in Neurospora crassa. Point mutations of key residues in the metal-binding motif (EX(nHXHX(10D of the CSN-5 JAMM domain disrupted CSN deneddylation activity without interfering with assembly of the CSN complex or interactions between CSN and cullin proteins. Surprisingly, CSN-5 with a mutated JAMM domain partially rescued the phenotypic defects observed in a csn-5 mutant. We found that, even without its deneddylation activity, the CSN can partially maintain the stability of the SCF(FWD-1 complex and partially restore the degradation of the circadian clock protein FREQUENCY (FRQ in vivo. Furthermore, we showed that CSN containing mutant CSN-5 efficiently prevents degradation of the substrate receptors of CRLs. Finally, we found that deletion of the CAND1 ortholog in N. crassa had little effect on the conidiation circadian rhythm. Our results suggest that CSN integrity plays major roles in hyphal growth, conidial development, and circadian function in N. crassa.

  6. CSN's New Emergency Room

    International Nuclear Information System (INIS)

    Sendin, P.

    2005-01-01

    During the month of July 2005 the physical renovation works and technological updating of the basic infrastructures of the CSN Emergency Room (SALEM) were finished, allowing the Room to now have greater functionality and a broader technical capacity. Nevertheless, the technological improvement process of SALEM will reach its full potential within the next few years, once the installation currently underway of the new information integration and monitoring systems and the decision making support systems have been completed. This article describes the improvements introduced to the Room and the objectives pursued in this renovation project to convert the SALEM into a new generation room in accordance with its current technological context. (Author) 4 refs

  7. UNC79 and UNC80, putative auxiliary subunits of the NARROW ABDOMEN ion channel, are indispensable for robust circadian locomotor rhythms in Drosophila.

    Directory of Open Access Journals (Sweden)

    Bridget C Lear

    Full Text Available In the fruit fly Drosophila melanogaster, a network of circadian pacemaker neurons drives daily rhythms in rest and activity. The ion channel NARROW ABDOMEN (NA, orthologous to the mammalian sodium leak channel NALCN, functions downstream of the molecular circadian clock in pacemaker neurons to promote behavioral rhythmicity. To better understand the function and regulation of the NA channel, we have characterized two putative auxiliary channel subunits in Drosophila, unc79 (aka dunc79 and unc80 (aka CG18437. We have generated novel unc79 and unc80 mutations that represent strong or complete loss-of-function alleles. These mutants display severe defects in circadian locomotor rhythmicity that are indistinguishable from na mutant phenotypes. Tissue-specific RNA interference and rescue analyses indicate that UNC79 and UNC80 likely function within pacemaker neurons, with similar anatomical requirements to NA. We observe an interdependent, post-transcriptional regulatory relationship among the three gene products, as loss of na, unc79, or unc80 gene function leads to decreased expression of all three proteins, with minimal effect on transcript levels. Yet despite this relationship, we find that the requirement for unc79 and unc80 in circadian rhythmicity cannot be bypassed by increasing NA protein expression, nor can these putative auxiliary subunits substitute for each other. These data indicate functional requirements for UNC79 and UNC80 beyond promoting channel subunit expression. Immunoprecipitation experiments also confirm that UNC79 and UNC80 form a complex with NA in the Drosophila brain. Taken together, these data suggest that Drosophila NA, UNC79, and UNC80 function together in circadian clock neurons to promote rhythmic behavior.

  8. Protective effect of CSN1S2 protein of goat milk on ileum microstructure and inflmmation in rat-CFAinduced rheumatoid arthritis

    Directory of Open Access Journals (Sweden)

    Rista Nikmatu Rohmah

    2015-07-01

    Full Text Available Objective: To observe the protective effect of goat milk alpha (S2-casein (CSN1S2 protein on ileum microstructure and inflammation in rat-complete Freund’s adjuvant-induced rheumatoid arthritis model. Methods: Twenty four male Wistar rats were divided into six groups of two models. The body weight, food intake and albumin level of all subjects were calculated. The ileum microstructures were analyzed by scanning electron microscopy. Histopathological analysis was observed by hematoxylin-eosin staining and the level expressions of immunoglobulin E, secretory immunoglobulin A, interleukin-17, interleukin-10, Ki-67 and caspase-9 were measured by using western blotting. Results: CSN1S2 protein of milk or yogurt could repair the ileum villi of rat arthritis group similar to the normal. The level expressions showed the immunoglobulin E, secretory immunoglobulin A, interleukin-17 and caspase-9 decreased in milk CSN1S2 protein and yogurt CSN1S2 protein rat groups. The level expression of interleukin-10 was increased, and also Ki- 67 was significantly increased in milk CSN1S2 protein and yogurt CSN1S2 protein rat groups. CSN1S2 protein of milk and yogurt could increase the body weight and albumin significantly, meanwhile food intake increased but not significantly. Conclusions: CSN1S2 protein of goat milk and yogurt could repair the ileum microstructure, suppress inflammatory process and also increase the body weight, food intake and albumin level. This result indicates that goat CSN1S2 protein may protect the ileum disorder in rheumatoid arthritis disease.

  9. IRRS: The CSN under the microscope

    International Nuclear Information System (INIS)

    Salome, M.

    2008-01-01

    Throughout the two weeks from January 28th to February 8th an international team of experts, appointed by the IAEA and led by Ulrich Schmocker from Switzerland, carried out a detailed study of the CSN. The objective of the IRRS mission, which was performed in response to a request from the Council itself, was to analyse the structure and operation of the Spanish regulatory body in order to determine its strengths and weaknesses and to suggest measures to improve its management. (Author)

  10. Electron microscopy and in vitro deneddylation reveal similar architectures and biochemistry of isolated human and Flag-mouse COP9 signalosome complexes

    Energy Technology Data Exchange (ETDEWEB)

    Rockel, Beate [Department of Molecular Structural Biology, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried (Germany); Schmaler, Tilo; Huang, Xiaohua [Division of Molecular Biology, Department of General, Visceral, Vascular and Thoracic Surgery, Charité – Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin (Germany); Dubiel, Wolfgang, E-mail: Wolfgang.dubiel@charite.de [Division of Molecular Biology, Department of General, Visceral, Vascular and Thoracic Surgery, Charité – Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin (Germany)

    2014-07-25

    Highlights: • Deneddylation rates of human erythrocyte and mouse fibroblast CSN are very similar. • 3D models of native human and mouse CSN reveal common architectures. • The cryo-structure of native mammalian CSN shows a horseshoe subunit arrangement. - Abstract: The COP9 signalosome (CSN) is a regulator of the ubiquitin (Ub) proteasome system (UPS). In the UPS, proteins are Ub-labeled for degradation by Ub ligases conferring substrate specificity. The CSN controls a large family of Ub ligases called cullin-RING ligases (CRLs), which ubiquitinate cell cycle regulators, transcription factors and DNA damage response proteins. The CSN possesses structural similarities with the 26S proteasome Lid complex and the translation initiation complex 3 (eIF3) indicating similar ancestry and function. Initial structures were obtained 14 years ago by 2D electron microscopy (EM). Recently, first 3D molecular models of the CSN were created on the basis of negative-stain EM and single-particle analysis, mostly with recombinant complexes. Here, we compare deneddylating activity and structural features of CSN complexes purified in an elaborate procedure from human erythrocytes and efficiently pulled down from mouse Flag-CSN2 B8 fibroblasts. In an in vitro deneddylation assay both the human and the mouse CSN complexes deneddylated Nedd8-Cul1 with comparable rates. 3D structural models of the erythrocyte CSN as well as of the mouse Flag-CSN were generated by negative stain EM and by cryo-EM. Both complexes show a central U-shaped segment from which several arms emanate. This structure, called the horseshoe, is formed by the PCI domain subunits. CSN5 and CSN6 point away from the horseshoe. Compared to 3D models of negatively stained CSN complexes, densities assigned to CSN2 and CSN4 are better defined in the cryo-map. Because biochemical and structural results obtained with CSN complexes isolated from human erythrocytes and purified by Flag-CSN pulldown from mouse B8 fibroblasts

  11. Electron microscopy and in vitro deneddylation reveal similar architectures and biochemistry of isolated human and Flag-mouse COP9 signalosome complexes

    International Nuclear Information System (INIS)

    Rockel, Beate; Schmaler, Tilo; Huang, Xiaohua; Dubiel, Wolfgang

    2014-01-01

    Highlights: • Deneddylation rates of human erythrocyte and mouse fibroblast CSN are very similar. • 3D models of native human and mouse CSN reveal common architectures. • The cryo-structure of native mammalian CSN shows a horseshoe subunit arrangement. - Abstract: The COP9 signalosome (CSN) is a regulator of the ubiquitin (Ub) proteasome system (UPS). In the UPS, proteins are Ub-labeled for degradation by Ub ligases conferring substrate specificity. The CSN controls a large family of Ub ligases called cullin-RING ligases (CRLs), which ubiquitinate cell cycle regulators, transcription factors and DNA damage response proteins. The CSN possesses structural similarities with the 26S proteasome Lid complex and the translation initiation complex 3 (eIF3) indicating similar ancestry and function. Initial structures were obtained 14 years ago by 2D electron microscopy (EM). Recently, first 3D molecular models of the CSN were created on the basis of negative-stain EM and single-particle analysis, mostly with recombinant complexes. Here, we compare deneddylating activity and structural features of CSN complexes purified in an elaborate procedure from human erythrocytes and efficiently pulled down from mouse Flag-CSN2 B8 fibroblasts. In an in vitro deneddylation assay both the human and the mouse CSN complexes deneddylated Nedd8-Cul1 with comparable rates. 3D structural models of the erythrocyte CSN as well as of the mouse Flag-CSN were generated by negative stain EM and by cryo-EM. Both complexes show a central U-shaped segment from which several arms emanate. This structure, called the horseshoe, is formed by the PCI domain subunits. CSN5 and CSN6 point away from the horseshoe. Compared to 3D models of negatively stained CSN complexes, densities assigned to CSN2 and CSN4 are better defined in the cryo-map. Because biochemical and structural results obtained with CSN complexes isolated from human erythrocytes and purified by Flag-CSN pulldown from mouse B8 fibroblasts

  12. Comments on the Law on CSN Fees and Public Prices

    International Nuclear Information System (INIS)

    Azuara, J. A.

    1999-01-01

    The article reviews the main modifications introduced by the Law on CSN Fees in the agency's financing system which is based on charging service rendering fees. The new regulations adjust the fees to the actual cost of services and lays down conditions for setting the fees in each case. (Author)

  13. AcuI identifies water buffalo CSN3 genotypes by RFLP analysis

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Genetics; Volume 93; Online resources. AcuI identifies water buffalo CSN3 genotypes by RFLP analysis. Soheir M. El Nahas Ahlam A. Abou Mossallam. Volume 93 Online resources 2014 pp e94-e96. Fulltext. Click here to view fulltext PDF. Permanent link:

  14. The epithelial sodium channel γ-subunit is processed proteolytically in human kidney

    DEFF Research Database (Denmark)

    Langkilde, Rikke Zachar; Skjødt, Karsten; Marcussen, Niels

    2015-01-01

    The epithelial sodium channel (ENaC) of the kidney is necessary for extracellular volume homeostasis and normal arterial BP. Activity of ENaC is enhanced by proteolytic cleavage of the gamma-subunit and putative release of a 43-amino acid inhibitory tract from the gamma-subunit ectodomain. We......ENaC was detected consistently only in tissue from patients with proteinuria and observed in collecting ducts. In conclusion, human kidney gammaENaC is subject to proteolytic cleavage, yielding fragments compatible with furin cleavage, and proteinuria is associated with cleavage at the putative prostasin...

  15. Crystal structure of clustered regularly interspaced short palindromic repeats (CRISPR)-associated Csn2 protein revealed Ca2+-dependent double-stranded DNA binding activity.

    Science.gov (United States)

    Nam, Ki Hyun; Kurinov, Igor; Ke, Ailong

    2011-09-02

    Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated protein genes (cas genes) are widespread in bacteria and archaea. They form a line of RNA-based immunity to eradicate invading bacteriophages and malicious plasmids. A key molecular event during this process is the acquisition of new spacers into the CRISPR loci to guide the selective degradation of the matching foreign genetic elements. Csn2 is a Nmeni subtype-specific cas gene required for new spacer acquisition. Here we characterize the Enterococcus faecalis Csn2 protein as a double-stranded (ds-) DNA-binding protein and report its 2.7 Å tetrameric ring structure. The inner circle of the Csn2 tetrameric ring is ∼26 Å wide and populated with conserved lysine residues poised for nonspecific interactions with ds-DNA. Each Csn2 protomer contains an α/β domain and an α-helical domain; significant hinge motion was observed between these two domains. Ca(2+) was located at strategic positions in the oligomerization interface. We further showed that removal of Ca(2+) ions altered the oligomerization state of Csn2, which in turn severely decreased its affinity for ds-DNA. In summary, our results provided the first insight into the function of the Csn2 protein in CRISPR adaptation by revealing that it is a ds-DNA-binding protein functioning at the quaternary structure level and regulated by Ca(2+) ions.

  16. Consolidation of use of the CSN-CIEMAT radiological protection educational portal; Consolidacion de la utilidad del portal educativo de proteccion radiologica CSN-Ciemat

    Energy Technology Data Exchange (ETDEWEB)

    Llorente Herranz, C.; Marco Arboli, M.; Fernandez Sanchez, J.; Villaroel Gonzalez-Elipe, R.

    2016-05-01

    he workers of nuclear and radioactive facilities are required to undertake training programmes on radiological protection in order to achieve the accreditations and licences granted by the CSN. Since 2003, the Council has been collaborating with CIEMAT in the development, maintenance and updating of the teaching material for these courses. More than a thousand such courses have been delivered to date and their contents are accessible via Internet. (Author)

  17. Structures of a putative RNA 5-methyluridine methyltransferase, Thermus thermophilus TTHA1280, and its complex with S-adenosyl-l-homocysteine

    International Nuclear Information System (INIS)

    Pioszak, Augen A.; Murayama, Kazutaka; Nakagawa, Noriko; Ebihara, Akio; Kuramitsu, Seiki; Shirouzu, Mikako; Yokoyama, Shigeyuki

    2005-01-01

    Three structures of a putative RNA 5-methyluridine methyltransferase from T. thermophilus, including its complex with S-adenosyl-l-homocysteine, are presented. The structures reveal the mode of cofactor binding, architecture of the putative active site, and the presence of a deep cleft adjacent to the active site that may bind RNA. The Thermus thermophilus hypothetical protein TTHA1280 belongs to a family of predicted S-adenosyl-l-methionine (AdoMet) dependent RNA methyltransferases (MTases) present in many bacterial and archaeal species. Inspection of amino-acid sequence motifs common to class I Rossmann-fold-like MTases suggested a specific role as an RNA 5-methyluridine MTase. Selenomethionine (SeMet) labelled and native versions of the protein were expressed, purified and crystallized. Two crystal forms of the SeMet-labelled apoprotein were obtained: SeMet-ApoI and SeMet-ApoII. Cocrystallization of the native protein with S-adenosyl-l-homocysteine (AdoHcy) yielded a third crystal form, Native-AdoHcy. The SeMet-ApoI structure was solved by the multiple anomalous dispersion method and refined at 2.55 Å resolution. The SeMet-ApoII and Native-AdoHcy structures were solved by molecular replacement and refined at 1.80 and 2.60 Å, respectively. TTHA1280 formed a homodimer in the crystals and in solution. Each subunit folds into a three-domain structure composed of a small N-terminal PUA domain, a central α/β-domain and a C-terminal Rossmann-fold-like MTase domain. The three domains form an overall clamp-like shape, with the putative active site facing a deep cleft. The architecture of the active site is consistent with specific recognition of uridine and catalysis of methyl transfer to the 5-carbon position. The cleft is suitable in size and charge distribution for binding single-stranded RNA.

  18. Crystal Structure of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated Csn2 Protein Revealed Ca[superscript 2+]-dependent Double-stranded DNA Binding Activity

    Energy Technology Data Exchange (ETDEWEB)

    Nam, Ki Hyun; Kurinov, Igor; Ke, Ailong (Cornell); (NWU)

    2012-05-22

    Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated protein genes (cas genes) are widespread in bacteria and archaea. They form a line of RNA-based immunity to eradicate invading bacteriophages and malicious plasmids. A key molecular event during this process is the acquisition of new spacers into the CRISPR loci to guide the selective degradation of the matching foreign genetic elements. Csn2 is a Nmeni subtype-specific cas gene required for new spacer acquisition. Here we characterize the Enterococcus faecalis Csn2 protein as a double-stranded (ds-) DNA-binding protein and report its 2.7 {angstrom} tetrameric ring structure. The inner circle of the Csn2 tetrameric ring is {approx}26 {angstrom} wide and populated with conserved lysine residues poised for nonspecific interactions with ds-DNA. Each Csn2 protomer contains an {alpha}/{beta} domain and an {alpha}-helical domain; significant hinge motion was observed between these two domains. Ca{sup 2+} was located at strategic positions in the oligomerization interface. We further showed that removal of Ca{sup 2+} ions altered the oligomerization state of Csn2, which in turn severely decreased its affinity for ds-DNA. In summary, our results provided the first insight into the function of the Csn2 protein in CRISPR adaptation by revealing that it is a ds-DNA-binding protein functioning at the quaternary structure level and regulated by Ca{sup 2+} ions.

  19. The independence of the CSN and Parliamentary control

    International Nuclear Information System (INIS)

    Cuevas Delgado, A.

    2006-01-01

    The Chairman of the Congressional Commission for Industry, Tourism and commerce underlines the link between the CSN and Parliament, to which the Council is accountable via control mechanisms that have improved over the years. he also points to the fact that the obligatory compliance with the resolutions approved has implied improvements in the structure and operation of the regulator. In winding up, the trusts that the modification of the Law which the council was created will promote the independence of the organisation with respect to the legitimate interests of industry, the democratic control exercised by Parliament and transparency before the public. (Author)

  20. Frequency and association of polymorphisms in CSN3 gene with milk yield and composition in Saanen goats/Frecuencia y asociación de polimor smos en gene CSN3 con producción y composición de la leche de cabras Saanen

    Directory of Open Access Journals (Sweden)

    Luis David Catota-Gómez

    2017-09-01

    Full Text Available K-casein (CSN3 gene is of particular interest in goat production due to the e ect of its polymorphisms on milk quality and composition. The objective of this study was to assess the genotypic and allelic frequencies in CSN3 gene in the Saanen goat breed and its e ect on milk production and some of its compositional traits. Allelic discrimination assays were designed to determine the variants (A, B or C using qPCR and allelic discrimination method. Allelic frequencies for A, B and C were 0.23, 0.64 and 0.13, respectively. Association analysis indicated that the CSN3 was signi cantly associated only with protein percentage (p = 0.006. Results indicated that animals with AC, BB and CC genotypes showed higher milk protein percentage than those with genotype AB. This evidence suggests that genotypes favorable to this associated trait could be included into Saanen goat breeding strategies.

  1. Structural properties of a peptide derived from H+-V-ATPase subunit a

    NARCIS (Netherlands)

    Vermeer, L.S.; Reat, V.; Hemminga, M.A.; Milon, A.

    2009-01-01

    The 3D structure of a peptide derived from the putative transmembrane segment 7 (TM7) of subunit a from H+-V-ATPase from Saccharomyces cerevisiae has been determined by solution state NMR in SDS. A stable helix is formed from L736 up to and including Q745, the lumenal half of the putative TM7. The

  2. Genetic polymorphism of CSN1S2 in South African dairy goat ...

    African Journals Online (AJOL)

    The aim of this study was to investigate the polymorphism and genetic variation of CSN1S2 in South African dairy goats, using DNA sequencing technology. Sixty dairy goats (20 Saanes, 20 British Alpine, and 20 Toggenburg) and 20 meat-type goats were sequenced with four primers to distinguish among the seven known ...

  3. Immunoproteasome subunit ß5i/LMP7-deficiency in atherosclerosis.

    Science.gov (United States)

    Hewing, Bernd; Ludwig, Antje; Dan, Cristian; Pötzsch, Max; Hannemann, Carmen; Petry, Andreas; Lauer, Dilyara; Görlach, Agnes; Kaschina, Elena; Müller, Dominik N; Baumann, Gert; Stangl, Verena; Stangl, Karl; Wilck, Nicola

    2017-10-17

    Management of protein homeostasis by the ubiquitin-proteasome system is critical for atherosclerosis development. Recent studies showed controversial results on the role of immunoproteasome (IP) subunit β5i/LMP7 in maintenance of protein homeostasis under cytokine induced oxidative stress. The present study aimed to investigate the effect of β5i/LMP7-deficiency on the initiation and progression of atherosclerosis as a chronic inflammatory, immune cell driven disease. LDLR -/- LMP7 -/- and LDLR -/- mice were fed a Western-type diet for either 6 or 24 weeks to induce early and advanced stage atherosclerosis, respectively. Lesion burden was similar between genotypes in both stages. Macrophage content and abundance of polyubiquitin conjugates in aortic root plaques were unaltered by β5i/LMP7-deficiency. In vitro experiments using bone marrow-derived macrophages (BMDM) showed that β5i/LMP7-deficiency did not influence macrophage polarization or accumulation of polyubiquitinated proteins and cell survival upon hydrogen peroxide and interferon-γ treatment. Analyses of proteasome core particle composition by Western blot revealed incorporation of standard proteasome subunits in β5i/LMP7-deficient BMDM and spleen. Chymotrypsin-, trypsin- and caspase-like activities assessed by using short fluorogenic peptides in BMDM whole cell lysates were similar in both genotypes. Taken together, deficiency of IP subunit β5i/LMP7 does not disturb protein homeostasis and does not aggravate atherogenesis in LDLR -/- mice.

  4. Coordinated CSN-UNESA research plan (PCI)

    International Nuclear Information System (INIS)

    Incinillas, J.

    2001-01-01

    This article contains a description of the essence and current status of the Coordinated Research Plan (PCI), beginning with details on the general specifications of the Framework Agreement signed by CSN and UNESA in September 1997, with an initial duration of four years and extendable on a yearly basis. The section on PCI Management describes the governing bodies, the mechanisms for including a project, tracking of projects underway, and the mechanisms for concluding projects. The Plan's generic goals are to acquire and increase technological skills and tools to guarantee operation of the nuclear park, take advantage of the synergies of the plants as a whole, and optimize the regulatory framework from the standpoint of safety and efficiency. In addition, strategic targets concerning efficiency, technological development and environment are defined. The current project status, overall budget and project completed to data are described. (Author)

  5. Cloning and characterization of indole synthase (INS) and a putative tryptophan synthase α-subunit (TSA) genes from Polygonum tinctorium.

    Science.gov (United States)

    Jin, Zhehao; Kim, Jin-Hee; Park, Sang Un; Kim, Soo-Un

    2016-12-01

    Two cDNAs for indole-3-glycerol phosphate lyase homolog were cloned from Polygonum tinctorium. One encoded cytosolic indole synthase possibly in indigoid synthesis, whereas the other encoded a putative tryptophan synthase α-subunit. Indigo is an old natural blue dye produced by plants such as Polygonum tinctorium. Key step in plant indigoid biosynthesis is production of indole by indole-3-glycerol phosphate lyase (IGL). Two tryptophan synthase α-subunit (TSA) homologs, PtIGL-short and -long, were isolated by RACE PCR from P. tinctorium. The genome of the plant contained two genes coding for IGL. The short and the long forms, respectively, encoded 273 and 316 amino acid residue-long proteins. The short form complemented E. coli ΔtnaA ΔtrpA mutant on tryptophan-depleted agar plate signifying production of free indole, and thus was named indole synthase gene (PtINS). The long form, either intact or without the transit peptide sequence, did not complement the mutant and was tentatively named PtTSA. PtTSA was delivered into chloroplast as predicted by 42-residue-long targeting sequence, whereas PtINS was localized in cytosol. Genomic structure analysis suggested that a TSA duplicate acquired splicing sites during the course of evolution toward PtINS so that the targeting sequence-containing pre-mRNA segment was deleted as an intron. PtINS had about two to fivefolds higher transcript level than that of PtTSA, and treatment of 2,1,3-benzothiadiazole caused the relative transcript level of PtINS over PtTSA was significantly enhanced in the plant. The results indicate participation of PtINS in indigoid production.

  6. Consolidation of use of the CSN-CIEMAT radiological protection educational portal

    International Nuclear Information System (INIS)

    Llorente Herranz, C.; Marco Arboli, M.; Fernandez Sanchez, J.; Villaroel Gonzalez-Elipe, R.

    2016-01-01

    he workers of nuclear and radioactive facilities are required to undertake training programmes on radiological protection in order to achieve the accreditations and licences granted by the CSN. Since 2003, the Council has been collaborating with CIEMAT in the development, maintenance and updating of the teaching material for these courses. More than a thousand such courses have been delivered to date and their contents are accessible via Internet. (Author)

  7. CSN1S2 protein of goat milk inhibits the decrease of viability and increases the proliferation of MC3T3E1 pre-osteoblast cell in methyl glyoxal exposure

    Directory of Open Access Journals (Sweden)

    Choirunil Chotimah

    2015-03-01

    Full Text Available Objective: To investigate whether the CNS1S2 protein of goat milk is able to inhibit the toxicity of methyl glyoxal (MG towards MC3T3E1 pre-osteoblast cells. Methods: At confluency, pre-osteoblast cells were divided into five groups which included control (untreated, pre-osteoblast cells exposed to 5 µmol/L MG, pre-osteoblast cells exposed to MG in the presence of CSN1S2 protein at doses of 0.025, 0.050, and 0.100 mg/L, respectively. Analysis of reactive oxygen species was done with 2,7-dichlorodihydrofluorescein diacetate fluorochrome. The proliferation and viability of MC3T3E1 cells were measured by trypan blue staining. Malondialdehyde analysis was done colorimetrically. Results: Cell's viabilities were significantly lower in MG+0.050 mg/L CSN1S2 protein of goat milk compared to MG group (P<0.05. MG+0.100 mg/L CSN1S2 protein of goat milk significantly increased the cells viability compared to MG group (P<0.05. The levels of proliferation were significantly higher in MG+0.100 mg/L CSN1S2 protein of goat milk compared to control group and all treatment groups, respectively (P<0.05. Conclusions: High dose of CSN1S2 protein of goat milk (0.100 mg/L in high MG environment inhibits the decrease of viability due to the increases of the proliferation of MC3T3E1 preosteoblast cell.

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

    Science.gov (United States)

    Glerum, D M; Tzagoloff, A

    1997-08-04

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

  9. A genetic link between epigenetic repressor AS1-AS2 and a putative small subunit processome in leaf polarity establishment of Arabidopsis

    Directory of Open Access Journals (Sweden)

    Yoko Matsumura

    2016-07-01

    Full Text Available Although the DEAD-box RNA helicase family is ubiquitous in eukaryotes, its developmental role remains unelucidated. Here, we report that cooperative action between the Arabidopsis nucleolar protein RH10, an ortholog of human DEAD-box RNA helicase DDX47, and the epigenetic repressor complex of ASYMMETRIC-LEAVES1 (AS1 and AS2 (AS1-AS2 is critical to repress abaxial (ventral genes ETT/ARF3 and ARF4, which leads to adaxial (dorsal development in leaf primordia at shoot apices. Double mutations of rh10-1 and as2 (or as1 synergistically up-regulated the abaxial genes, which generated abaxialized filamentous leaves with loss of the adaxial domain. DDX47 is part of the small subunit processome (SSUP that mediates rRNA biogenesis. In rh10-1 we found various defects in SSUP-related events, such as: accumulation of 35S/33S rRNA precursors; reduction in the 18S/25S ratio; and nucleolar hypertrophy. Double mutants of as2 with mutations of genes that encode other candidate SSUP-related components such as nucleolin and putative rRNA methyltransferase exhibited similar synergistic defects caused by up-regulation of ETT/ARF3 and ARF4. These results suggest a tight link between putative SSUP and AS1-AS2 in repression of the abaxial-determining genes for cell fate decisions for adaxial development.

  10. A Test of Reliability of the Personnel Dosimetry Services Authorized by CSN using Photon Beams; Control de los servicios de dosimetria personal autorizados por el CSN, usando haces de fotones

    Energy Technology Data Exchange (ETDEWEB)

    Brosed, A; Delgado, A; Granados, C E; Lopez Ortiz, G

    1987-07-01

    In 1987 the Consejo de Seguridad Nuclear (CSN) had eight Personnel Dosimetry Services (PDS) authorized to asses the equivalent doses to the spanish occupationally exposed workers, by means of the readings from the dosemeters wear by them. An audit was carried on the PDS on behalf of CSN under the control of CIEMAT. Batches of dosemeters from each one of the PDS were irradiated to dose equivalent values which were well established by CIEMAT but kept hidden from the PDS. By comparing the true values with those obtained by the PDS, it was possible to evaluate the Services according to the analysis of the quantity Q= I B I -I- S where B is the average of the individual deviations between the dosemeters belonging to the same group and the true value as established by CIEMAT, whereas S is the standard deviation of the values inside of this same group. The results of the evaluation, which was made using the new ICRU quantities for personnel monitoring, are presented. (Author) 8 refs.

  11. Intranasal H5N1 vaccines, adjuvanted with chitosan derivatives, protect ferrets against highly pathogenic influenza intranasal and intratracheal challenge.

    Directory of Open Access Journals (Sweden)

    Alex J Mann

    Full Text Available We investigated the protective efficacy of two intranasal chitosan (CSN and TM-CSN adjuvanted H5N1 Influenza vaccines against highly pathogenic avian Influenza (HPAI intratracheal and intranasal challenge in a ferret model. Six groups of 6 ferrets were intranasally vaccinated twice, 21 days apart, with either placebo, antigen alone, CSN adjuvanted antigen, or TM-CSN adjuvanted antigen. Homologous and intra-subtypic antibody cross-reacting responses were assessed. Ferrets were inoculated intratracheally (all treatments or intranasally (CSN adjuvanted and placebo treatments only with clade 1 HPAI A/Vietnam/1194/2004 (H5N1 virus 28 days after the second vaccination and subsequently monitored for morbidity and mortality outcomes. Clinical signs were assessed and nasal as well as throat swabs were taken daily for virology. Samples of lung tissue, nasal turbinates, brain, and olfactory bulb were analysed for the presence of virus and examined for histolopathological findings. In contrast to animals vaccinated with antigen alone, the CSN and TM-CSN adjuvanted vaccines induced high levels of antibodies, protected ferrets from death, reduced viral replication and abrogated disease after intratracheal challenge, and in the case of CSN after intranasal challenge. In particular, the TM-CSN adjuvanted vaccine was highly effective at eliciting protective immunity from intratracheal challenge; serologically, protective titres were demonstrable after one vaccination. The 2-dose schedule with TM-CSN vaccine also induced cross-reactive antibodies to clade 2.1 and 2.2 H5N1 viruses. Furthermore ferrets immunised with TM-CSN had no detectable virus in the respiratory tract or brain, whereas there were signs of virus in the throat and lungs, albeit at significantly reduced levels, in CSN vaccinated animals. This study demonstrated for the first time that CSN and in particular TM-CSN adjuvanted intranasal vaccines have the potential to protect against significant

  12. Preliminary Report on CSN3 and LGB genes Polymorphism Among Two Romanian Cattle Breeds

    Directory of Open Access Journals (Sweden)

    Daniela Elena Ilie

    2017-05-01

    Full Text Available The aim of the current study was to evaluate the genetic polymorphism of A and B variants at the CSN3 (κ-casein and LGB (β-lactoglobulin loci in Romanian Spotted and Romanian Brown breeds using PCR-RFLP and PCR-HRM genotyping methods. Altogether, 280 cattle from Arad county were genotyped. The most frequent allele at the CSN3 locus was A in Romanian Spotted breed (0.723 while the B allele was the most common in the Romanian Brown (0.694. For LGB locus the A and B alleles recorded similar frequencies for Romanian Spotted (0.5141 and 0.4859 compared to Romanian Brown breed where the B allele was the most prevalent (0.5970. Knowledges regarding genetic information of breeds related to milk protein are valuable for economic reassertion of cattle breeds, particularly autochthonous ones. Further, the results would be useful in future breeding programs for the studied population, aimed to improve cattle genetic structure for milk quality traits.

  13. Preambolo al Catalogo Sismico Nazionale (CSN. I criteri di informazione del Catalogo Sismico Nazionale (CSN

    Directory of Open Access Journals (Sweden)

    M. VECCHI

    1979-06-01

    Full Text Available

    This note presents a study meant to set out a national catalogue
    (C.S.N. of seismic events the epicentres of which are to be found in
    Italian territory (or nearby. The catalogue, that will start from the
    year 1450 B.C., could be used for various purpose employing modern
    technologies.
    The complete C.S.N, is made of three main parts, each of which can
    also have a separate life:
    1 An Analytical Catalogue which comprises the greater part of
    data and is the most complete;
    2 A Macroseismic Atlas that shows the macroseismic aspect of
    the most relevant events;
    3 A Macroseismic Catalogue that translates in numeric terms the
    Atlas.

    To these is added a comprehensive Bibliography subdivided into 24
    chapters each of which covers one of the 24 time periods into which the
    catalogue has been subdivided.
    The Analytical Catalogue besides giving the main parameters for each
    earthquake (date, epicentre, hypocentral depth, MCS scale intensity, raagnitudo,
    each with its own reliability index gives indications also on the
    following sideline data:
    1 epicentral location (meaning by this the geographic region and
    the eventual indication that the epicentre is to be found on a borderline
    touching more geographic regions, or in the sea, or on the coast, or othermise
    in the external band (see text;
    2 with a reference to the Atlas, the event is shown as having been
    dealt with also mocroseismic data and it will con be found, as such, in
    the Atlas and in the Macroseismic Catalogue;
    3 indications on the typology of the earthquake: i.e. whether it is an
    « isolated » earthquake, or a « seismic period » (and if so with the indication
    of the foreshock, main or aftershocks or of a swarm;
    4 the possibility of indicating up to 7 sets of information suitably
    chose among 40 additional notes regarding instrumental, geophysical

  14. Genetic characterisation of CSN2 gene in Girgentana goat breed

    Directory of Open Access Journals (Sweden)

    Lina Tortorici

    2014-12-01

    Full Text Available Among calcium sensitive caseins, β-casein is the most abundant in goat milk, representing up to 50% of total casein content. The goat β-casein locus has been widely investigated and at least ten alleles have been identified in different goat breeds. The aim of this work was to investigate the polymorphisms of β-casein gene in Girgentana dairy goat breed in order to assess the genotype distribution and evaluate how frequencies have changed during the last 10 years, as genotype is known to influence technological and nutritional milk properties. Sequencing analysis and alignment of the obtained sequences of β-casein exon 7, showed the presence of C, C1, and A strong alleles, and 0' null allele, with frequencies of 0.597, 0.326, 0.023, and 0.054, respectively. Seven genotypic classes were found in Girgentana goat breed and the most frequent genotype was CC1 (0.423 followed by CC (0.326, C1C1 (0.110, and C0' (0.096. No AA nor 0'0' homozygous individuals were found. The presence of strong alleles at CSN2 gene in Girgentana goat breed could be useful for the production of milk with high protein content and good cheese-making properties. Moreover, food business operators should consider the possibility of reviving interest in Girgentana goat milk using weak and null genotypes at CSN2 locus to make peculiar food products, such as drinking milk.

  15. Isolation of the pituitary gonadotrophic α-subunit hormone of the giant amazonian fish: pirarucu (Arapaima gigas).

    Science.gov (United States)

    Faria, M T; Carvalho, R F; Sevilhano, T C A; Oliveira, N A J; Silva, C F P; Oliveira, J E; Soares, C R J; Garcez, R; Santo, P R E; Bartolini, P

    2013-06-01

    The cDNAs of the α-subunit of the pituitary gonadotrophic hormones (GTHα) of fish of the order Osteoglossiformes or the superorder Osteoglossomorpha have never been sequenced. For a better understanding the phylogenetic diversity and evolution of PGHα in fish and for future biotechnological synthesis of the gonadotrophic hormones (ag-FSH and ag-LH), of Arapaima gigas, one of the largest freshwater fishes of the world, its GTHα cDNA was synthesized by reverse transcriptase and the polymerase chain reaction starting from total pituitary RNA. The ag-GTHα-subunit was found to be encoded by 348 bp, corresponding to a protein of 115 amino acids, with a putative signal peptide of 24 amino acids and a mature peptide of 91 amino acids. Ten cysteine residues, responsible for forming 5 disulfide linkages, 2 putative N-linked glycosylation sites and 3 proline residues, were found to be conserved on the basis of the known sequences of vertebrate gonadotrophic hormones. Phylogenetic analysis, based on the amino acid sequences of 38 GTHα-subunits, revealed the highest identity of A. gigas with members of the Acipenseriformes, Anguilliformes, Siluriformes and Cypriniformes (87.1-89.5 %) and the lowest with Gadiformes and Cyprinodontiformes (55.0 %). The obtained phylogenetic tree agrees with previous analysis of teleostei, since A. gigas, of the order of Osteoglossiformes, appears as the sister group of Clupeocephala, while Elopomorpha forms the most basal group of all other teleosts.

  16. Maintaining the Database for Information Object Analysis, Intent, Dissemination and Enhancement (IOAIDE) and the US Army Research Laboratory Campus Sensor Network (ARL CSN)

    Science.gov (United States)

    2017-01-01

    operations as well as basic knowledge of Microsoft Structured Query Language Server Management Studio (2014 or 2016). 15. SUBJECT TERMS Microsoft SQL ...designed and is maintained with Microsoft SQL Server Management Studio. The basic requirements for the IOAIDE/ARL CSN database development and... SQL server (2014 or 2016) installed. All images in this report were generated using Windows 10. The IOAIDE/ARL CSN database could reside on the

  17. Distribution of the a2, a3, and a5 nicotinic acetylcholine receptor subunits in the chick brain

    Directory of Open Access Journals (Sweden)

    Torrão A.S.

    1997-01-01

    Full Text Available Nicotinic acetylcholine receptors (nAChRs are ionotropic receptors comprised of a and ß subunits. These receptors are widely distributed in the central nervous system, and previous studies have revealed specific patterns of localization for some nAChR subunits in the vertebrate brain. In the present study we used immunohistochemical methods and monoclonal antibodies to localize the a2, a3, and a5 nAChR subunits in the chick mesencephalon and diencephalon. We observed a differential distribution of these three subunits in the chick brain, and showed that the somata and neuropil of many central structures contain the a5 nAChR subunit. The a2 and a3 subunits, on the other hand, exhibited a more restricted distribution than a5 and other subunits previously studied, namely a7, a8 and ß2. The patterns of distribution of the different nAChR subunits suggest that neurons in many brain structures may contain several subtypes of nAChRs and that in a few regions one particular subtype may determine the cholinergic nicotinic responses

  18. Learning from nuclear regulatory self-assessment. International peer review of the CSN report on lessons learnt from the essential service water system degradation event at the Vandellos nuclear power plant

    International Nuclear Information System (INIS)

    2006-01-01

    Nuclear regulatory self-assessment together with the benchmarking of regulatory practices against those of other countries operating nuclear power plants are key elements in maintaining a high level of nuclear safety. In that light, the Spanish Consejo de Seguridad Nuclear (CSN) formally asked the OECD Nuclear Energy Agency (NEA) to establish an international peer review team to assess the CSN report on the lessons learnt as a result of the 2004 Vandellos II event involving essential service water system degradation. The International Review Team considers the CSN report prepared in follow-up to the Vandellos event to be a commendable effort in regulatory self-assessment. The report, complemented by this international peer review, should enable the CSN to take appropriate action to ensure that its regulatory supervision is in line with best international practice. (authors)

  19. Review of the Main Activities Carried out by the CSN in the Last Five Years; Revision de las principales actuaciones del CSN en los ultimos cinco anos

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2006-07-01

    Nearing the time to finalise a new phase for the Consejo de Seguridad Nuclear, it is time to make a brief account of the main activities carried out over the past five years. During this period, one in which the CSN celebrated its 25th Anniversary, the organisation has served a social demand in a modernisation and adjustment process, followed by the continuous improvement process which throughout its history the former management, teams have carried out with great stamina and dedication. (Author)

  20. Instruction of the CSN on the requirements of the system of management of the nuclear power plants; Instruccion del CSN sobre los requisitos del sistema de gestion de las instalaciones nucleares

    Energy Technology Data Exchange (ETDEWEB)

    Cid, R.; Santo, A. de; Gil Montes, B.; Toca, A.

    2008-07-01

    The Western European Nations Regulatory Authorities (WENRA) performed a nuclear safety requirements harmonization task, as a result of this work and its implementation, the Spanish Nuclear Safety Counsel (CSN) has the commitment to issue its own Regulation Safety Instructions) to identify the WENRA to level nuclear safety requirements, and to incorporate it in the Spanish regulatory pyramid. However, the Spain nuclear installations meet these requirements through the original criteria to fulfill the regulation of the country that supply the NSSS design, these requirements are not incorporated in our regulation. One of the issues, identified by WENRA, is the implementation of the management system requirements in accord with the IAEA GS-R-3 The Management System for Facilities and Activities. As these regards, the CSN has developed a Safety Instruction, basically endorsing the IAEA GS-R-3. The Safety Instruction is actually in a phase of external comments and should be issued by june 2008. This paper describes the bases for the Safety Instruction, summarises the requirements that would meet the management system for nuclear installations and the activities to perform for its implementations. (Author)

  1. Crystal structure of the C-terminal domain of the RAP74 subunit of human transcription factor IIF

    Energy Technology Data Exchange (ETDEWEB)

    Kamada, Katsuhiko; De Angelis, Jacqueline; Roeder, Robert G.; Burley, Stephen K. (Rockefeller)

    2012-12-13

    The x-ray structure of a C-terminal fragment of the RAP74 subunit of human transcription factor (TF) IIF has been determined at 1.02-{angstrom} resolution. The {alpha}/{beta} structure is strikingly similar to the globular domain of linker histone H5 and the DNA-binding domain of hepatocyte nuclear factor 3{gamma} (HNF-3{gamma}), making it a winged-helix protein. The surface electrostatic properties of this compact domain differ significantly from those of bona fide winged-helix transcription factors (HNF-3{gamma} and RFX1) and from the winged-helix domains found within the RAP30 subunit of TFIIF and the {beta} subunit of TFIIE. RAP74 has been shown to interact with the TFIIF-associated C-terminal domain phosphatase FCP1, and a putative phosphatase binding site has been identified within the RAP74 winged-helix domain.

  2. Identification of the ligand-binding subunit of the human 5-hydroxytryptamine1A receptor with N-(p-azido-m-[125I] iodophenethyl)spiperone, a high affinity radioiodinated photoaffinity probe

    International Nuclear Information System (INIS)

    Raymond, J.R.; Fargin, A.; Lohse, M.J.; Regan, J.W.; Senogles, S.E.; Lefkowitz, R.J.; Caron, M.G.

    1989-01-01

    The ligand-binding subunit of the human 5-hydroxytryptamine1A (5-HT1A) receptor transiently expressed in COS-7 cells and of the native human 5-HT1A receptor derived from hippocampus and frontal cortex were identified by photoaffinity labeling with N-(p-azido-m-[125I]iodophenethyl)spiperone [( 125I]N3-NAPS), previously characterized as a high affinity radioiodinated D2-dopamine receptor probe. The identity of the ligand-binding subunit was confirmed by immunoprecipitation with an antipeptide rabbit antiserum, JWR21, raised against a synthetic peptide derived from the predicted amino acid sequence of the putative third intracellular loop of the human 5-HT1A receptor. In transiently transfected COS-7 cells expressing 14 +/- 3 pmol/mg of protein human 5-HT1A receptors, a single broad 75-kDa band was photoaffinity labeled by [125I]N3-NAPS. This band displayed the expected pharmacology of the 5-HT1A receptor, as evidenced by the ability of a series of competing ligands to block [125I]N3-NAPS photoincorporation. Moreover, antiserum JWR21 specifically and quantitatively immunoprecipitated the 75-kDa photoaffinity-labeled band from a soluble extract of the transfected COS-7 cell membranes, further confirming its identity. Finally, utilizing a combination of photoaffinity labeling and immunoprecipitation, the native ligand-binding subunit of 62-64 kDa was identified in human hippocampus and frontal cortex. The availability of the high specific activity, high affinity, photoaffinity ligand [125I]N3-NAPS and of a potent immunoprecipitating antiserum (JWR21) should greatly facilitate the biochemical characterization of the human 5-HT1A receptor

  3. Overexpression of PP2A-C5 that encodes the catalytic subunit 5 of protein phosphatase 2A in Arabidopsis confers better root and shoot development under salt conditions

    Science.gov (United States)

    Protein phosphatase 2A (PP2A) is an enzyme consisting of three subunits: a scaffolding A subunit, a regulatory B subunit and a catalytic C subunit. PP2As were shown to play diverse roles in eukaryotes. In this study, the function of the Arabidopsis PP2A-C5 gene that encodes the catalytic subunit 5 o...

  4. A Test of Reliability of the Personnel Dosimetry Services Authorized by CSN using Photon Beams

    International Nuclear Information System (INIS)

    Brosed, A.; Delgado, A.; Granados, C. E.; Lopez Ortiz, G.

    1987-01-01

    In 1987 the Consejo de Seguridad Nuclear (CSN) had eight Personnel Dosimetry Services (PDS) authorized to asses the equivalent doses to the spanish occupationally exposed workers, by means of the readings from the dosemeters wear by them. An audit was carried on the PDS on behalf of CSN under the control of CIEMAT. Batches of dosemeters from each one of the PDS were irradiated to dose equivalent values which were well established by CIEMAT but kept hidden from the PDS. By comparing the true values with those obtained by the PDS, it was possible to evaluate the Services according to the analysis of the quantity Q= I B I -I- S where B is the average of the individual deviations between the dosemeters belonging to the same group and the true value as established by CIEMAT, whereas S is the standard deviation of the values inside of this same group. The results of the evaluation, which was made using the new ICRU quantities for personnel monitoring, are presented. (Author) 8 refs

  5. Community Seismic Network (CSN)

    Science.gov (United States)

    Clayton, R. W.; Heaton, T. H.; Kohler, M. D.; Cheng, M.; Guy, R.; Chandy, M.; Krause, A.; Bunn, J.; Olson, M.; Faulkner, M.; Liu, A.; Strand, L.

    2012-12-01

    We report on developments in sensor connectivity, architecture, and data fusion algorithms executed in Cloud computing systems in the Community Seismic Network (CSN), a network of low-cost sensors housed in homes and offices by volunteers in the Pasadena, CA area. The network has over 200 sensors continuously reporting anomalies in local acceleration through the Internet to a Cloud computing service (the Google App Engine) that continually fuses sensor data to rapidly detect shaking from earthquakes. The Cloud computing system consists of data centers geographically distributed across the continent and is likely to be resilient even during earthquakes and other local disasters. The region of Southern California is partitioned in a multi-grid style into sets of telescoping cells called geocells. Data streams from sensors within a geocell are fused to detect anomalous shaking across the geocell. Temporal spatial patterns across geocells are used to detect anomalies across regions. The challenge is to detect earthquakes rapidly with an extremely low false positive rate. We report on two data fusion algorithms, one that tessellates the surface so as to fuse data from a large region around Pasadena and the other, which uses a standard tessellation of equal-sized cells. Since September 2011, the network has successfully detected earthquakes of magnitude 2.5 or higher within 40 Km of Pasadena. In addition to the standard USB device, which connects to the host's computer, we have developed a stand-alone sensor that directly connects to the internet via Ethernet or wifi. This bypasses security concerns that some companies have with the USB-connected devices, and allows for 24/7 monitoring at sites that would otherwise shut down their computers after working hours. In buildings we use the sensors to model the behavior of the structures during weak events in order to understand how they will perform during strong events. Visualization models of instrumented buildings ranging

  6. Characterisation of 5-HT3C, 5-HT3D and 5-HT3E receptor subunits: evolution, distribution and function.

    Science.gov (United States)

    Holbrook, Joanna D; Gill, Catherine H; Zebda, Noureddine; Spencer, Jon P; Leyland, Rebecca; Rance, Kim H; Trinh, Han; Balmer, Gemma; Kelly, Fiona M; Yusaf, Shahnaz P; Courtenay, Nicola; Luck, Jane; Rhodes, Andrew; Modha, Sundip; Moore, Stephen E; Sanger, Gareth J; Gunthorpe, Martin J

    2009-01-01

    The 5-HT(3) receptor is a member of the 'Cys-loop' family of ligand-gated ion channels that mediate fast excitatory and inhibitory transmission in the nervous system. Current evidence points towards native 5-HT(3) receptors originating from homomeric assemblies of 5-HT(3A) or heteromeric assembly of 5-HT(3A) and 5-HT(3B). Novel genes encoding 5-HT(3C), 5-HT(3D), and 5-HT(3E) have recently been described but the functional importance of these proteins is unknown. In the present study, in silico analysis (confirmed by partial cloning) indicated that 5-HT(3C), 5-HT(3D), and 5-HT(3E) are not human-specific as previously reported: they are conserved in multiple mammalian species but are absent in rodents. Expression profiles of the novel human genes indicated high levels in the gastrointestinal tract but also in the brain, Dorsal Root Ganglion (DRG) and other tissues. Following the demonstration that these subunits are expressed at the cell membrane, the functional properties of the recombinant human subunits were investigated using patch clamp electrophysiology. 5-HT(3C), 5-HT(3D), and 5-HT(3E) were all non-functional when expressed alone. Co-transfection studies to determine potential novel heteromeric receptor interactions with 5-HT(3A) demonstrated that the expression or function of the receptor was modified by 5-HT(3C) and 5-HT(3E), but not 5-HT(3D). The lack of distinct effects on current rectification, kinetics or pharmacology of 5-HT(3A) receptors does not however provide unequivocal evidence to support a direct contribution of 5-HT(3C) or 5-HT(3E) to the lining of the ion channel pore of novel heteromeric receptors. The functional and pharmacological contributions of these novel subunits to human biology and diseases such as irritable bowel syndrome for which 5-HT(3) receptor antagonists have major clinical usage, therefore remains to be fully determined.

  7. Ein Elefant vergißt nicht - Erweiterte Funktionen in PostgreSQL am Beispiel der CSN Datenbank

    OpenAIRE

    Schreiber, Daniel

    2004-01-01

    PostgreSQL ist eine Open Source Datenbank, die in den letzten Jahren sehr populär geworden ist. Das Chemnitzer Studentennetz benutzt seit 1999 PostgreSQL zur Verwaltung seines Netzes. Der Vortrag zeigt erweiterte Funktionen von PostgreSQL wie Views, Rules, Stored Procedures und Trigger sowie deren Verwendung in der CSN Datenbank.

  8. The Drosophila nicotinic acetylcholine receptor subunits5 and Dα7 form functional homomeric and heteromeric ion channels

    Directory of Open Access Journals (Sweden)

    Lansdell Stuart J

    2012-06-01

    Full Text Available Abstract Background Nicotinic acetylcholine receptors (nAChRs play an important role as excitatory neurotransmitters in vertebrate and invertebrate species. In insects, nAChRs are the site of action of commercially important insecticides and, as a consequence, there is considerable interest in examining their functional properties. However, problems have been encountered in the successful functional expression of insect nAChRs, although a number of strategies have been developed in an attempt to overcome such difficulties. Ten nAChR subunits have been identified in the model insect Drosophila melanogaster (Dα1-Dα7 and Dβ1-Dβ3 and a similar number have been identified in other insect species. The focus of the present study is the Dα5, Dα6 and Dα7 subunits, which are distinguished by their sequence similarity to one another and also by their close similarity to the vertebrate α7 nAChR subunit. Results A full-length cDNA clone encoding the Drosophila nAChR Dα5 subunit has been isolated and the properties of Dα5-, Dα6- and Dα7-containing nAChRs examined in a variety of cell expression systems. We have demonstrated the functional expression, as homomeric nAChRs, of the Dα5 and Dα7 subunits in Xenopus oocytes by their co-expression with the molecular chaperone RIC-3. Also, using a similar approach, we have demonstrated the functional expression of a heteromeric ‘triplet’ nAChR (Dα5 + Dα6 + Dα7 with substantially higher apparent affinity for acetylcholine than is seen with other subunit combinations. In addition, specific cell-surface binding of [125I]-α-bungarotoxin was detected in both Drosophila and mammalian cell lines when Dα5 was co-expressed with Dα6 and RIC-3. In contrast, co-expression of additional subunits (including Dα7 with Dα5 and Dα6 prevented specific binding of [125I]-α-bungarotoxin in cell lines, suggesting that co-assembly with other nAChR subunits can block maturation of correctly folded nAChRs in

  9. A cDNA microarray, UniShrimpChip, for identification of genes relevant to testicular development in the black tiger shrimp (Penaeus monodon

    Directory of Open Access Journals (Sweden)

    Klinbunga Sirawut

    2011-04-01

    Full Text Available Abstract Background Poor reproductive maturation in captive male broodstock of the black tiger shrimp (Penaeus monodon is one of the serious problems to the farming industries. Without genome sequence, EST libraries of P. monodon were previously constructed to identify transcripts with important biological functions. In this study, a new version of cDNA microarray, UniShrimpChip, was constructed from the Peneaus monodon EST libraries of 12 tissues, containing 5,568 non-redundant cDNA clones from 10,536 unique cDNA in the P. monodon EST database. UniShrimpChip was used to study testicular development by comparing gene expression levels of wild brooders from the West and East coasts of Thailand and domesticated brooders with different ages (10-, 14-, 18-month-old. Results The overall gene expression patterns from the microarray experiments revealed distinct transcriptomic patterns between the wild and domesticated groups. Moreover, differentially expressed genes from the microarray comparisons were identified, and the expression patterns of eight selected transcripts were subsequently confirmed by reverse-transcriptase quantitative PCR (RT-qPCR. Among these, expression levels of six subunits (CSN2, 4, 5, 6, 7a, and 8 of the COP9 signalosome (CSN gene family in wild and different ages of domesticated brooders were examined by RT-qPCR. Among the six subunits, CSN5 and CSN6 were most highly expressed in wild brooders and least expressed in the 18-month-old domesticated group; therefore, their full-length cDNA sequences were characterized. Conclusions This study is the first report to employ cDNA microarray to study testicular development in the black tiger shrimp. We show that there are obvious differences between the wild and domesticated shrimp at the transcriptomic level. Furthermore, our study is the first to investigate the feasibility that the CSN gene family might have involved in reproduction and development of this economically important

  10. Progesterone modulation of α5 nAChR subunits influences anxiety-related behavior during estrus cycle

    OpenAIRE

    Gangitano, David; Salas, Ramiro; Teng, Yanfen; Perez, Erika; De Biasi, Mariella

    2009-01-01

    Smokers often report an anxiolytic effect of cigarettes. In addition, stress-related disorders such as anxiety, post-traumatic stress syndrome, and depression are often associated with chronic nicotine use. To study the role of the α5 nicotinic acetylcholine receptor subunit in anxiety-related responses, control and α5 subunit null mice (α5 −/−) were subjected to the open field, light-dark box and elevated plus maze tests. In the open field and light-dark box, α5 −/− behaved like wild type co...

  11. Evaluation of refractory lining wear of Companhia Siderurgica Nacional (CSN) blast furnaces

    International Nuclear Information System (INIS)

    Santos, N.J. dos; Mello, A.H.B. de; Pereira, C.L.; Paula Sarkis, D. de; Martins Filho, D.I.; Banados Perez, H.E.; Carvalho, G.; Daltro, T.F.L.

    1984-01-01

    The blast furnace refractory linings are submitted to unfavourable conditions such as alkalis attack, temperature, top pressure, abrasion and so forth... After studies on distribution and installation of radioactive sources with low activities in the refractory lining, it was possible to develop a new technique of thickness evaluation and attendance of wearing in the furnace lining. The viability analysis, simulated laboratory tests, localization, identification, installations and periodical measurements of the radioactive sources are described, as well the results obtained on the present campaign of CSN Blast Furnaces. (Author) [pt

  12. Cross-linking of L5 protein to 5 S RNA in rat liver 60-S subunits by ultraviolet irradiation

    International Nuclear Information System (INIS)

    Terao, K.; Uchiumi, T.; Ogata, K.

    1980-01-01

    After rat liver 60-S ribosomal subunits were irradiated with ultraviolet light at 254 nm, they were treated with EDTA and then subjected to sucrose density-gradient centrifugation to isolate 5 S RNA-protein complex. When 5 S RNA-protein was analyzed by SDS-acrylamide gel electrophoresis which dissociated noncovalent 5 S RNA-protein, two protein bands were observed. The one showed a slower mobility than the protein band (L5) of 5 S RNA-protein from non-irradiated 60 S subunit and the other showed the same mobility as L5 protein. Since the former band was shown to be specific to ultraviolet-irradiation, it was considered as cross-linked 5 S RNA-protein. After the two protein bands were iodinated with 125 I, labeled protein was extracted and treated with RNAase. Thereafter, it was analyzed by two-dimensional acrylamide gel electrophoresis, followed by autoradiography. The results indicate that the protein component of cross-linked 5 S RNA-protein is L5 protein (ribosomal protein); these proteins are designated according to the proposed uniform nomenclature. (Auth.)

  13. Memory Deficits Induced by Inflammation Are Regulated by α5-Subunit-Containing GABAA Receptors

    Directory of Open Access Journals (Sweden)

    Dian-Shi Wang

    2012-09-01

    Full Text Available Systemic inflammation causes learning and memory deficits through mechanisms that remain poorly understood. Here, we studied the pathogenesis of memory loss associated with inflammation and found that we could reverse memory deficits by pharmacologically inhibiting α5-subunit-containing γ-aminobutyric acid type A (α5GABAA receptors and deleting the gene associated with the α5 subunit. Acute inflammation reduces long-term potentiation, a synaptic correlate of memory, in hippocampal slices from wild-type mice, and this reduction was reversed by inhibition of α5GABAA receptor function. A tonic inhibitory current generated by α5GABAA receptors in hippocampal neurons was increased by the key proinflammatory cytokine interleukin-1β through a p38 mitogen-activated protein kinase signaling pathway. Interleukin-1β also increased the surface expression of α5GABAA receptors in the hippocampus. Collectively, these results show that α5GABAA receptor activity increases during inflammation and that this increase is critical for inflammation-induced memory deficits.

  14. Na+/K+-ATPase α-subunit in swimming crab Portunus trituberculatus: molecular cloning, characterization, and expression under low salinity stress

    Science.gov (United States)

    Han, Xiaolin; Liu, Ping; Gao, Baoquan; Wang, Haofeng; Duan, Yafei; Xu, Wenfei; Chen, Ping

    2015-07-01

    Na+/K+-ATPases are membrane-associated enzymes responsible for the active transport of Na+ and K+ ions across cell membranes, generating chemical and electrical gradients. These enzymes' α-subunit provides catalytic function, binding and hydrolyzing ATP, and itself becoming phosphorylated during the transport cycle. In this study, Na+/K+-ATPase α-subunit cDNA was cloned from gill tissue of the swimming crab Portunus trituberculatus by reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end methods. Analysis of the nucleotide sequence revealed that the cDNA had a full-length of 3 833 base pairs (bp), with an open reading frame of 3 120 bp, 5' untranslated region (UTR) of 317 bp, and 3' UTR of 396 bp. The sequence encoded a 1 039 amino acid protein with a predicted molecular weight of 115.57 kDa and with estimated pI of 5.21. It was predicted here to possess all expected features of Na+/K+-ATPase members, including eight transmembrane domains, putative ATP-binding site, and phosphorylation site. Comparison of amino acid sequences showed that the P. trituberculatus α-subunit possessed an overall identity of 75%-99% to that of other organisms. Phylogenetic analysis revealed that this α-subunit was in the same category as those of crustaceans. Quantitative real-time RT-PCR analysis indicated that this α-subunit's transcript were most highly expressed in gill and lowest in muscle. RT-PCR analysis also revealed that α-subunit expression in crab gill decreased after 2 and 6 h, but increased after 12, 24, 48, and 72 h. In addition, α-subunit expression in hepatopancreas of crab decreased after 2-72 h. These facts indicated that the crab's Na+/K+-ATPase α-subunit was potentially involved in the observed acute response to low salinity stress.

  15. Regulation of human gamma-glutamylcysteine synthetase: co-ordinate induction of the catalytic and regulatory subunits in HepG2 cells.

    Science.gov (United States)

    Galloway, D C; Blake, D G; Shepherd, A G; McLellan, L I

    1997-11-15

    We have shown that in HepG2 cells treatment with 75 microM t-butylhydroquinone (tBHQ) results in a 2.5-fold increase in glutathione concentration, as part of an adaptive response to chemical stress. In these cells the elevation in intracellular glutathione level was found to be accompanied by an increase of between 2-fold and 3-fold in the level of the 73 kDa catalytic subunit of gamma-glutamylcysteine synthetase (heavy subunit, GCSh) and the 31 kDa regulatory subunit (light subunit, GCSl). Levels of GCSh and GCSl mRNA were increased by up to 5-fold in HepG2 cells in response to tBHQ. To study the transcriptional regulation of GCSl, we subcloned 6.7 kb of the upstream region of the human GCSl gene (GLCLR) from a genomic clone isolated from a P1 lymphoblastoid cell line genomic library. HepG2 cells were transfected with GLCLR promoter reporter constructs and treated with tBHQ. This resulted in an induction of between 1.5-fold and 3.5-fold in reporter activity, indicating that transcriptional regulation of GLCLR is likely to contribute to the induction of GCSl by tBHQ in HepG2 cells. Sequence analysis of the promoter region demonstrated the presence of putative enhancer elements including AP-1 sites and an antioxidant-responsive element, which might be involved in the observed induction of the GLCLR promoter.

  16. Ddb1 controls genome stability and meiosis in fission yeast

    DEFF Research Database (Denmark)

    Holmberg, Christian Henrik; Fleck, Oliver; Hansen, H. A.

    2005-01-01

    The human UV-damaged DNA-binding protein Ddb1 associates with cullin 4 ubiquitin ligases implicated in nucleotide excision repair (NER). These complexes also contain the signalosome (CSN), but NER-relevant ubiquitination targets have not yet been identified. We report that fission yeast Ddb1......, Cullin 4 (Pcu4), and CSN subunits Csn1 and Csn2 are required for degradation of the ribonucleotide reductase (RNR) inhibitor protein Spd1. Ddb1-deficient cells have >20-fold increased spontaneous mutation rate. This is partly dependent on the error-prone translesion DNA polymerases. Spd1 deletion...... substantially reduced the mutation rate, suggesting that insufficient RNR activity accounts for ~50% of observed mutations. Epistasis analysis indicated that Ddb1 contributed to mutation avoidance and tolerance to DNA damage in a pathway distinct from NER. Finally, we show that Ddb1/Csn1/Cullin 4-mediated Spd1...

  17. Influence of CSN1S2 protein from Caprine milk Etawah Breed (EB) on histology of microglial cells in rat (Rattus norvegicus) Type-2 diabetes mellitus (T2DM)

    Science.gov (United States)

    Rika, Margareth; Fatchiyah

    2017-11-01

    Type-2 diabetes mellitus (T2DM) is a degenerative disease that causes an imbalance in the metabolism. The aim of this research is to determine the influences of CSN1S2 on the structure of microglial cells in T2DM. Rats (Rattus norvegicus) were divided into eight groups of treatment with looping three times each between treatment groups (CM) Control. The control is given a milk treatment with doses of 375 mg/kg (CM375), 750 mg/kg (CM750), and 1500 mg/kg (CM1500), T2DM (DMK), and T2DM with CSN1S2 375 mg/kg dose (DM375), 750mg/kg (DM750), and 1500 mg/kg (DM1500). The animal model T2DM was induced by a high-fat diet in the form of feed followed by injection of STZ (dose of 25 mg/kg of animal treatment) and treatment of CSN1S2 for 28 days. Brain organs were taken and analysed in histopathology stained by Hematoxylin-eosin (HE) and observed using Olympus BX53. Based on the results, it was concluded that CSN1S2 protein is influential for induction of microglial cell proliferation in animal models of T2DM, as immunity responds to the inflammatory condition in T2DM.

  18. Instruction of the CSN on the requirements of the system of management of the nuclear power plants

    International Nuclear Information System (INIS)

    Cid, R.; Santo, A. de; Gil Montes, B.; Toca, A.

    2008-01-01

    The Western European Nations Regulatory Authorities (WENRA) performed a nuclear safety requirements harmonization task, as a result of this work and its implementation, the Spanish Nuclear Safety Counsel (CSN) has the commitment to issue its own Regulation Safety Instructions) to identify the WENRA to level nuclear safety requirements, and to incorporate it in the Spanish regulatory pyramid. However, the Spain nuclear installations meet these requirements through the original criteria to fulfill the regulation of the country that supply the NSSS design, these requirements are not incorporated in our regulation. One of the issues, identified by WENRA, is the implementation of the management system requirements in accord with the IAEA GS-R-3 The Management System for Facilities and Activities. As these regards, the CSN has developed a Safety Instruction, basically endorsing the IAEA GS-R-3. The Safety Instruction is actually in a phase of external comments and should be issued by june 2008. This paper describes the bases for the Safety Instruction, summarises the requirements that would meet the management system for nuclear installations and the activities to perform for its implementations. (Author)

  19. CSN e Volta Redonda: uma relação histórica de dependência e controle.

    Directory of Open Access Journals (Sweden)

    Raphael Jonathas da Costa Lima

    2013-12-01

    Full Text Available http://dx.doi.org/10.5007/2175-7984.2013v12n25p41 A partir da caracterização da morfologia do espaço de Volta Redendoa (RJ, o artigo analisa a relação de dependência e controle ocasionada pela presença e atuação da Companhia Siderúrgica Nacional (CSN neste município. Entende-se que, nas últimas décadas, a Companhia disciplinou o território e restringiu as possibilidades de um desenvolvimento que não passasse pela atividade siderúrgica - até pouco tempo seu core business. O artigo parte de uma interpretação da matriz socioantropolótica do fenômeno company town e sugere que Volta Redonda ainda preserva características fundamentais deste modelo de controle e hierarquia do espalo, em geral identificadas na configuração da sua paisagem urbana. Com isso, procura compreender as implicações decorrentes do desenho monoindustrial e das mudanças técnicas e organizacionais da CSN sobre a geografia econômica do município.

  20. The α' subunit of β-conglycinin and the A1-5 subunits of glycinin are not essential for many hypolipidemic actions of dietary soy proteins in rats.

    Science.gov (United States)

    Chen, Qixuan; Wood, Carla; Gagnon, Christine; Cober, Elroy R; Frégeau-Reid, Judith A; Gleddie, Stephen; Xiao, Chao Wu

    2014-08-01

    This study examined the effects of dietary soy protein (SP) lacking different storage protein subunits and isoflavones (ISF) on the abdominal fat, blood lipids, thyroid hormones, and enzymatic activities in rats. Weanling Sprague-Dawley rats (8 males and 8 females/group) were fed diets containing either 20 % casein without or with supplemental isoflavones or alcohol-washed SP isolate or SP concentrates (SPC) prepared from 6 different soy bean lines for 8 weeks. Feeding of diets containing SPC regardless of their subunit compositions significantly lowered relative liver weights, blood total, free, and LDL cholesterol in both genders (P Soy isoflavones were mainly responsible for the hypocholesterolemic effects and increased plasma free T3, whereas reduction in FFA, abdominal fat, liver weight and increased plasma total T3 were the effects of the soy proteins. Neither the α' subunit of β-conglycinin nor the A1-5 subunits of glycinin are essential for the hypolipidemic properties of soy proteins.

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

    Science.gov (United States)

    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.

  2. The COP9 signalosome controls jasmonic acid synthesis and plant responses to herbivory and pathogens.

    Science.gov (United States)

    Hind, Sarah R; Pulliam, Sarah E; Veronese, Paola; Shantharaj, Deepak; Nazir, Azka; Jacobs, Nekaiya S; Stratmann, Johannes W

    2011-02-01

    The COP9 signalosome (CSN) is a multi-protein complex that regulates the activities of cullin-RING E3 ubiquitin ligases (CRLs). CRLs ubiquitinate proteins in order to target them for proteasomal degradation. The CSN is required for proper plant development. Here we show that the CSN also has a profound effect on plant defense responses. Silencing of genes for CSN subunits in tomato plants resulted in a mild morphological phenotype and reduced expression of wound-responsive genes in response to mechanical wounding, attack by Manduca sexta larvae, and Prosystemin over-expression. In contrast, expression of pathogenesis-related genes was increased in a stimulus-independent manner in these plants. The reduced wound response in CSN-silenced plants corresponded with reduced synthesis of jasmonic acid (JA), but levels of salicylic acid (SA) were unaltered. As a consequence, these plants exhibited reduced resistance against herbivorous M. sexta larvae and the necrotrophic fungal pathogen Botrytis cinerea. In contrast, susceptibility to tobacco mosaic virus (TMV) was not altered in CSN-silenced plants. These data demonstrate that the CSN orchestrates not only plant development but also JA-dependent plant defense responses. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

  3. Review of the Main Activities Carried out by the CSN in the Last Five Years

    International Nuclear Information System (INIS)

    2006-01-01

    Nearing the time to finalise a new phase for the Consejo de Seguridad Nuclear, it is time to make a brief account of the main activities carried out over the past five years. During this period, one in which the CSN celebrated its 25th Anniversary, the organisation has served a social demand in a modernisation and adjustment process, followed by the continuous improvement process which throughout its history the former management, teams have carried out with great stamina and dedication. (Author)

  4. Evaluation of [methyl-3H]L655,708 and [ethyl-3H]RY80 as putative PET ligands for central GABAA receptors containing α5 subunit

    International Nuclear Information System (INIS)

    Opacka-Juffry, J.; Hirani, E.; Dawson, G.R.; Luthra, S.K.; Hume, S.P.

    1999-01-01

    Two selective radioligands of gamma aminobutyric acid (GABA) A receptors containing the α5 subunit, [ 3 H]L655,708 and [ 3 H]RY80, were evaluated in rats as potential in vivo tracers for positron emission tomography (PET). Brain uptake index (BUI), a measure of first pass extraction, was moderate for [ 3 H]L655,708 (BUI of 59%) and good for [ 3 H]RY80 (BUI of 96%). This finding was consistent with their in vitro binding to plasma proteins of ∼76% and 50%, respectively. Following intravenous injection of either radioligand, radioactivity in plasma was measured and uptake characteristics were assessed in brain within a time period relevant to PET scanning (up to 90 min). Discrete brain regions, such as frontal cortex, striatum, hypothalamus, thalamus, hippocampus, colliculi, medulla, and cerebellum, were sampled and the temporal distribution of radioactivity analysed. Despite the reasonable delivery to the brain, neither of the radioligands had sufficient retention in the tissues rich in α5-containing GABA A receptors to achieve a good selective signal. For both radioligands, a maximal tissue:cerebellum ratio of 1.5 was seen in hippocampus at 10 min after injection. Thus, neither of the compounds studied shows potential for further development as an in vivo PET ligand

  5. Sequence analysis of dolphin ferritin H and L subunits and possible iron-dependent translational control of dolphin ferritin gene

    Directory of Open Access Journals (Sweden)

    Sasaki Yukako

    2008-10-01

    Full Text Available Abstract Background Iron-storage protein, ferritin plays a central role in iron metabolism. Ferritin has dual function to store iron and segregate iron for protection of iron-catalyzed reactive oxygen species. Tissue ferritin is composed of two kinds of subunits (H: heavy chain or heart-type subunit; L: light chain or liver-type subunit. Ferritin gene expression is controlled at translational level in iron-dependent manner or at transcriptional level in iron-independent manner. However, sequencing analysis of marine mammalian ferritin subunits has not yet been performed fully. The purpose of this study is to reveal cDNA-derived amino acid sequences of cetacean ferritin H and L subunits, and demonstrate the possibility of expression of these subunits, especially H subunit, by iron. Methods Sequence analyses of cetacean ferritin H and L subunits were performed by direct sequencing of polymerase chain reaction (PCR fragments from cDNAs generated via reverse transcription-PCR of leukocyte total RNA prepared from blood samples of six different dolphin species (Pseudorca crassidens, Lagenorhynchus obliquidens, Grampus griseus, Globicephala macrorhynchus, Tursiops truncatus, and Delphinapterus leucas. The putative iron-responsive element sequence in the 5'-untranslated region of the six different dolphin species was revealed by direct sequencing of PCR fragments obtained using leukocyte genomic DNA. Results Dolphin H and L subunits consist of 182 and 174 amino acids, respectively, and amino acid sequence identities of ferritin subunits among these dolphins are highly conserved (H: 99–100%, (99→98 ; L: 98–100%. The conserved 28 bp IRE sequence was located -144 bp upstream from the initiation codon in the six different dolphin species. Conclusion These results indicate that six different dolphin species have conserved ferritin sequences, and suggest that these genes are iron-dependently expressed.

  6. Distribution of alpha3, alpha5 and alpha(v) integrin subunits in mature and immature human oocytes.

    Science.gov (United States)

    Capmany, G; Mart, M; Santaló, J; Bolton, V N

    1998-10-01

    The distribution of three integrin subunits, alpha3, alpha5 and alpha(v), in immature and mature human oocytes has been examined using immunofluorescence and confocal microscopy. The results demonstrate that both alpha5 and alpha(v) are present at the germinal vesicle stage, while alpha3 was only detected in oocytes after germinal vesicle breakdown, in metaphase I and II stage oocytes. The cortical concentration of integrin subunits alpha3 and alpha5 is consistent with their localization in the oolemma. In contrast, the homogeneous distribution of alpha(v) throughout the oocyte suggests the existence of cytoplasmic reservoirs of this protein in the oocyte.

  7. GAMETOPHYTE DEFECTIVE 1, a putative subunit of RNases P/MRP, is essential for female gametogenesis and male competence in Arabidopsis.

    Science.gov (United States)

    Wang, Si-Qi; Shi, Dong-Qiao; Long, Yan-Ping; Liu, Jie; Yang, Wei-Cai

    2012-01-01

    RNA biogenesis, including biosynthesis and maturation of rRNA, tRNA and mRNA, is a fundamental process that is critical for cell growth, division and differentiation. Previous studies showed that mutations in components involved in RNA biogenesis resulted in abnormalities in gametophyte and leaf development in Arabidopsis. In eukaryotes, RNases P/MRP (RNase mitochondrial RNA processing) are important ribonucleases that are responsible for processing of tRNA, and transcription of small non-coding RNAs. Here we report that Gametophyte Defective 1 (GAF1), a gene encoding a predicted protein subunit of RNases P/MRP, AtRPP30, plays a role in female gametophyte development and male competence. Embryo sacs were arrested at stages ranging from FG1 to FG7 in gaf1 mutant, suggesting that the progression of the gametophytic division during female gametogenesis was impaired in gaf1 mutant. In contrast, pollen development was not affected in gaf1. However, the fitness of the mutant pollen tube was weaker than that of the wild-type, leading to reduced transmission through the male gametes. GAF1 is featured as a typical RPP30 domain protein and interacts physically with AtPOP5, a homologue of RNases P/MRP subunit POP5 of yeast. Together, our data suggest that components of the RNases P/MRP family, such as RPP30, play important roles in gametophyte development and function in plants.

  8. DNA binding properties of the small cascade subunit Csa5.

    Directory of Open Access Journals (Sweden)

    Michael Daume

    Full Text Available CRISPR-Cas systems provide immunity against viral attacks in archaeal and bacterial cells. Type I systems employ a Cas protein complex termed Cascade, which utilizes small CRISPR RNAs to detect and degrade the exogenic DNA. A small sequence motif, the PAM, marks the foreign substrates. Previously, a recombinant type I-A Cascade complex from the archaeon Thermoproteus tenax was shown to target and degrade DNA in vitro, dependent on a native PAM sequence. Here, we present the biochemical analysis of the small subunit, Csa5, of this Cascade complex. T. tenax Csa5 preferentially bound ssDNA and mutants that showed decreased ssDNA-binding and reduced Cascade-mediated DNA cleavage were identified. Csa5 oligomerization prevented DNA binding. Specific recognition of the PAM sequence was not observed. Phylogenetic analyses identified Csa5 as a universal member of type I-A systems and revealed three distinct groups. A potential role of Csa5 in R-loop stabilization is discussed.

  9. Diclofenac distinguishes among homomeric and heteromeric potassium channels composed of KCNQ4 and KCNQ5 subunits.

    Science.gov (United States)

    Brueggemann, Lioubov I; Mackie, Alexander R; Martin, Jody L; Cribbs, Leanne L; Byron, Kenneth L

    2011-01-01

    KCNQ4 and KCNQ5 potassium channel subunits are expressed in vascular smooth muscle cells, although it remains uncertain how these subunits assemble to form functional channels. Using patch-clamp techniques, we compared the electrophysiological characteristics and effects of diclofenac, a known KCNQ channel activator, on human KCNQ4 and KCNQ5 channels expressed individually or together in A7r5 rat aortic smooth muscle cells. The conductance curves of the overexpressed channels were fitted by a single Boltzmann function in each case (V(0.5) values: -31, -44, and -38 mV for KCNQ4, KCNQ5, and KCNQ4/5, respectively). Diclofenac (100 μM) inhibited KCNQ5 channels, reducing maximum conductance by 53%, but increased maximum conductance of KCNQ4 channels by 38%. The opposite effects of diclofenac on KCNQ4 and KCNQ5 could not be attributed to the presence of a basic residue (lysine) in the voltage-sensing domain of KCNQ5, because mutation of this residue to neutral glycine (the residue present in KCNQ4) resulted in a more effective block of the channel. Differences in deactivation rates and distinct voltage-dependent effects of diclofenac on channel activation and deactivation observed with each of the subunit combinations (KCNQ4, KCNQ5, and KCNQ4/5) were used as diagnostic tools to evaluate native KCNQ currents in vascular smooth muscle cells. A7r5 cells express only KCNQ5 channels endogenously, and their responses to diclofenac closely resembled those of the overexpressed KCNQ5 currents. In contrast, mesenteric artery myocytes, which express both KCNQ4 and KCNQ5 channels, displayed whole-cell KCNQ currents with properties and diclofenac responses characteristic of overexpressed heteromeric KCNQ4/5 channels.

  10. Crystallization and preliminary X-ray crystallographic analysis of the small subunit of the heterodimeric laccase POXA3b from Pleurotus ostreatus

    Science.gov (United States)

    Ferraroni, Marta; Scozzafava, Andrea; Ullah, Sana; Tron, Thierry; Piscitelli, Alessandra; Sannia, Giovanni

    2014-01-01

    Laccases are multicopper oxidases of great biotechnological potential. While laccases are generally monomeric glycoproteins, the white-rot fungus Pleurotus ostreatus produces two closely related heterodimeric isoenzymes composed of a large subunit, homologous to the other fungal laccases, and a small subunit. The sequence of the small subunit does not show significant homology to any other protein or domain of known function and consequently its function is unknown. The highest similarity to proteins of known structure is to a putative enoyl-CoA hydratase/isomerase from Acinetobacter baumannii, which shows an identity of 27.8%. Diffraction-quality crystals of the small subunit of the heterodimeric laccase POXA3b (sPOXA3b) from P. ostreatus were obtained using the sitting-drop vapour-diffusion method at 294 K from a solution consisting of 1.8 M sodium formate, 0.1 M Tris–HCl pH 8.5. The crystals belonged to the tetragonal space group P41212 or P43212, with unit-cell parameters a = 126.6, c = 53.9 Å. The asymmetric unit contains two molecules related by a noncrystallographic twofold axis. A complete data set extending to a maximum resolution of 2.5 Å was collected at 100 K using a wavelength of 1.140 Å. PMID:24419623

  11. Electron microscopy of the complexes of ribulose-1,5-bisphosphate carboxylase (Rubisco) and Rubisco subunit-binding protein from pea leaves

    NARCIS (Netherlands)

    Tsuprun, V.L.; Boekema, E.J.; Samsonidze, T.G.; Pushkin, A.V.

    1991-01-01

    The structure of ribulose-1,5-bisphosphate carboxylase (Rubisco) subunit-binding protein and its interaction with pea leaf chloroplast Rubisco were studied by electron microscopy and image analysis. Electron-microscopic evidence for the association of Rubisco subunit-binding protein, consisting of

  12. Mutation in mitochondrial complex IV subunit COX5A causes pulmonary arterial hypertension, lactic acidemia, and failure to thrive.

    Science.gov (United States)

    Baertling, Fabian; Al-Murshedi, Fathiya; Sánchez-Caballero, Laura; Al-Senaidi, Khalfan; Joshi, Niranjan P; Venselaar, Hanka; van den Brand, Mariël Am; Nijtmans, Leo Gj; Rodenburg, Richard Jt

    2017-06-01

    COX5A is a nuclear-encoded subunit of mitochondrial respiratory chain complex IV (cytochrome c oxidase). We present patients with a homozygous pathogenic variant in the COX5A gene. Clinical details of two affected siblings suffering from early-onset pulmonary arterial hypertension, lactic acidemia, failure to thrive, and isolated complex IV deficiency are presented. We show that the variant lies within the evolutionarily conserved COX5A/COX4 interface domain, suggesting that it alters the interaction between these two subunits during complex IV biogenesis. In patient skin fibroblasts, the enzymatic activity and protein levels of complex IV and several of its subunits are reduced. Lentiviral complementation rescues complex IV deficiency. The monomeric COX1 assembly intermediate accumulates demonstrating a function of COX5A in complex IV biogenesis. A potential therapeutic lead is demonstrated by showing that copper supplementation leads to partial rescue of complex IV deficiency in patient fibroblasts. © 2017 Wiley Periodicals, Inc.

  13. GAMETOPHYTE DEFECTIVE 1, a putative subunit of RNases P/MRP, is essential for female gametogenesis and male competence in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Si-Qi Wang

    Full Text Available RNA biogenesis, including biosynthesis and maturation of rRNA, tRNA and mRNA, is a fundamental process that is critical for cell growth, division and differentiation. Previous studies showed that mutations in components involved in RNA biogenesis resulted in abnormalities in gametophyte and leaf development in Arabidopsis. In eukaryotes, RNases P/MRP (RNase mitochondrial RNA processing are important ribonucleases that are responsible for processing of tRNA, and transcription of small non-coding RNAs. Here we report that Gametophyte Defective 1 (GAF1, a gene encoding a predicted protein subunit of RNases P/MRP, AtRPP30, plays a role in female gametophyte development and male competence. Embryo sacs were arrested at stages ranging from FG1 to FG7 in gaf1 mutant, suggesting that the progression of the gametophytic division during female gametogenesis was impaired in gaf1 mutant. In contrast, pollen development was not affected in gaf1. However, the fitness of the mutant pollen tube was weaker than that of the wild-type, leading to reduced transmission through the male gametes. GAF1 is featured as a typical RPP30 domain protein and interacts physically with AtPOP5, a homologue of RNases P/MRP subunit POP5 of yeast. Together, our data suggest that components of the RNases P/MRP family, such as RPP30, play important roles in gametophyte development and function in plants.

  14. Molecular cloning and characterization of pirarucu (Arapaima gigas follicle-stimulating hormone and luteinizing hormone β-subunit cDNAs.

    Directory of Open Access Journals (Sweden)

    Thais Sevilhano

    Full Text Available The common gonadotrophic hormone α-subunit (GTHα has been previously isolated by our research group from A. gigas pituitaries; in the present work the cDNA sequences encoding FSHβ and LHβ subunits have also been isolated from the same species of fish. The FSH β-subunit consists of 126 amino acids with a putative 18 amino acid signal peptide and a 108 amino acid mature peptide, while the LH β-subunit consists of 141 amino acids with a putative 24 amino acid amino acid signal peptide and a 117 amino acid mature peptide. The highest identity, based on the amino acid sequences, was found with the order of Anguilliformes (61% for FSHβ and of Cypriniformes (76% for LHβ, followed by Siluriformes, 53% for FSHβ and 75% for LHβ. Interestingly, the identity with the corresponding human amino acid sequences was still remarkable: 45.1% for FSHβ and 51.4% for LHβ. Three dimensional models of ag-FSH and ag-LH, generated by using the crystal structures of h-FSH and h-LH as the respective templates and carried out via comparative modeling and molecular dynamics simulations, suggested the presence of the so-called "seat-belt", favored by a disulfide bond formed between the 3rd and 12th cysteine in both β-subunits. The sequences found will be used for the biotechnological synthesis of A. gigas gonadotrophic hormones (ag-FSH and ag-LH. In a first approach, to ascertain that the cloned transcripts allow the expression of the heterodimeric hormones, ag-FSH has been synthesized in human embryonic kidney 293 (HEK293 cells, preliminarily purified and characterized.

  15. Molecular cloning and characterization of pirarucu (Arapaima gigas) follicle-stimulating hormone and luteinizing hormone β-subunit cDNAs.

    Science.gov (United States)

    Sevilhano, Thais; Carvalho, Roberto Feitosa de; Oliveira, Nélio Alessandro de Jesus; Oliveira, João Ezequiel; Maltarollo, Vinicius Gonçalves; Trossini, Gustavo; Garcez, Riviane; Bartolini, Paolo

    2017-01-01

    The common gonadotrophic hormone α-subunit (GTHα) has been previously isolated by our research group from A. gigas pituitaries; in the present work the cDNA sequences encoding FSHβ and LHβ subunits have also been isolated from the same species of fish. The FSH β-subunit consists of 126 amino acids with a putative 18 amino acid signal peptide and a 108 amino acid mature peptide, while the LH β-subunit consists of 141 amino acids with a putative 24 amino acid amino acid signal peptide and a 117 amino acid mature peptide. The highest identity, based on the amino acid sequences, was found with the order of Anguilliformes (61%) for FSHβ and of Cypriniformes (76%) for LHβ, followed by Siluriformes, 53% for FSHβ and 75% for LHβ. Interestingly, the identity with the corresponding human amino acid sequences was still remarkable: 45.1% for FSHβ and 51.4% for LHβ. Three dimensional models of ag-FSH and ag-LH, generated by using the crystal structures of h-FSH and h-LH as the respective templates and carried out via comparative modeling and molecular dynamics simulations, suggested the presence of the so-called "seat-belt", favored by a disulfide bond formed between the 3rd and 12th cysteine in both β-subunits. The sequences found will be used for the biotechnological synthesis of A. gigas gonadotrophic hormones (ag-FSH and ag-LH). In a first approach, to ascertain that the cloned transcripts allow the expression of the heterodimeric hormones, ag-FSH has been synthesized in human embryonic kidney 293 (HEK293) cells, preliminarily purified and characterized.

  16. Nuclear Export of Pre-Ribosomal Subunits Requires Dbp5, but Not as an RNA-Helicase as for mRNA Export.

    Science.gov (United States)

    Neumann, Bettina; Wu, Haijia; Hackmann, Alexandra; Krebber, Heike

    2016-01-01

    The DEAD-box RNA-helicase Dbp5/Rat8 is known for its function in nuclear mRNA export, where it displaces the export receptor Mex67 from the mRNA at the cytoplasmic side of the nuclear pore complex (NPC). Here we show that Dbp5 is also required for the nuclear export of both pre-ribosomal subunits. Yeast temperature-sensitive dbp5 mutants accumulate both ribosomal particles in their nuclei. Furthermore, Dbp5 genetically and physically interacts with known ribosomal transport factors such as Nmd3. Similar to mRNA export we show that also for ribosomal transport Dbp5 is required at the cytoplasmic side of the NPC. However, unlike its role in mRNA export, Dbp5 does not seem to undergo its ATPase cycle for this function, as ATPase-deficient dbp5 mutants that selectively inhibit mRNA export do not affect ribosomal transport. Furthermore, mutants of GLE1, the ATPase stimulating factor of Dbp5, show no major ribosomal export defects. Consequently, while Dbp5 uses its ATPase cycle to displace the export receptor Mex67 from the translocated mRNAs, Mex67 remains bound to ribosomal subunits upon transit to the cytoplasm, where it is detectable on translating ribosomes. Therefore, we propose a model, in which Dbp5 supports ribosomal transport by capturing ribosomal subunits upon their cytoplasmic appearance at the NPC, possibly by binding export factors such as Mex67. Thus, our findings reveal that although different ribonucleoparticles, mRNAs and pre-ribosomal subunits, use shared export factors, they utilize different transport mechanisms.

  17. Diclofenac Distinguishes among Homomeric and Heteromeric Potassium Channels Composed of KCNQ4 and KCNQ5 SubunitsS⃞

    Science.gov (United States)

    Brueggemann, Lioubov I.; Mackie, Alexander R.; Martin, Jody L.; Cribbs, Leanne L.

    2011-01-01

    KCNQ4 and KCNQ5 potassium channel subunits are expressed in vascular smooth muscle cells, although it remains uncertain how these subunits assemble to form functional channels. Using patch-clamp techniques, we compared the electrophysiological characteristics and effects of diclofenac, a known KCNQ channel activator, on human KCNQ4 and KCNQ5 channels expressed individually or together in A7r5 rat aortic smooth muscle cells. The conductance curves of the overexpressed channels were fitted by a single Boltzmann function in each case (V0.5 values: −31, −44, and −38 mV for KCNQ4, KCNQ5, and KCNQ4/5, respectively). Diclofenac (100 μM) inhibited KCNQ5 channels, reducing maximum conductance by 53%, but increased maximum conductance of KCNQ4 channels by 38%. The opposite effects of diclofenac on KCNQ4 and KCNQ5 could not be attributed to the presence of a basic residue (lysine) in the voltage-sensing domain of KCNQ5, because mutation of this residue to neutral glycine (the residue present in KCNQ4) resulted in a more effective block of the channel. Differences in deactivation rates and distinct voltage-dependent effects of diclofenac on channel activation and deactivation observed with each of the subunit combinations (KCNQ4, KCNQ5, and KCNQ4/5) were used as diagnostic tools to evaluate native KCNQ currents in vascular smooth muscle cells. A7r5 cells express only KCNQ5 channels endogenously, and their responses to diclofenac closely resembled those of the overexpressed KCNQ5 currents. In contrast, mesenteric artery myocytes, which express both KCNQ4 and KCNQ5 channels, displayed whole-cell KCNQ currents with properties and diclofenac responses characteristic of overexpressed heteromeric KCNQ4/5 channels. PMID:20876743

  18. Results of the Interlaboratory Exercise CSN/CIEMAT-100 Among Environmental Radioactivity Laboratories (Soil); Resultados del Ejercicio Interlaboratorios de Radiactividad Ambiental CSN/CIEMAT-00 (Suelo)

    Energy Technology Data Exchange (ETDEWEB)

    Romero Gonzalez, M. L.

    2002-07-01

    The document describes the outcome of the CSN/CIEMAT-00 interlaboratory test comparison among environmental radioactivity laboratories. the exercise was organised according to the ISO-43 and the ISO/IUPAC/AOAC Harmonized Protocol for the proficiency testing of analytical laboratories. the test sample was a soil containing environmental levels of K-40, Ra-226, Ac-228, Sr-90, Cs-137, Cs-134, Pu (239-240) y Am-241. the Universidad Autonoma de Barcelona prepared the material and reported adequate statistical studies of homogeneity. The results of the exercise were computed for 30 participating laboratories, and their analytical performance was assessed using the u-score approach. A raised percentage of satisfactory laboratory performance has been obtained for all the analysis, being the best performance in gamma measurements. The exercise has drawn that several laboratories have difficulties in the evaluation of combined uncertainty, mainly in analysis involving radiochemical steps. The study has shown an homogeneous inter-laboratory behaviour, and the improvement achieved through subsequent exercises in the quality of the data they are producing. (Author) 10 refs.

  19. Identification of the large subunit of Ribulose 1,5-bisphosphate carboxylase/oxygenase as a substrate for transglutaminase in Medicageo sativa L. (alfalfa)

    International Nuclear Information System (INIS)

    Margosiak, S.A.; Dharma, A.; Carver, M.R.B.; Gonzales, A.P.; Louie, D.; Kuehn, G.D.

    1990-01-01

    Extract prepared from floral meristematic tissue of alfalfa (Medicago sativa L.) were investigated for expression of the enzyme transglutaminase in order to identify the major protein substrate for transglutaminase-directed modifications among plant proteins. The large polymorphic subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase in alfalfa, with molecular weights of 52,700 and 57,600, are major substrates for transglutaminase in these extracts. This was established by: (a) covalent conjugation of monodansylcadaverine to the large subunit followed by fluorescent detection in SDS-polyacrylamide gels; (b) covalent conjugation of [ 14 C]putrescine to the large subunit with detection by autoradiography; (c) covalent conjugation of monodansylcadaverine to the large subunit and demonstration of immunocross-reactivity on nitrocellulose transblot of the modified large subunit with antibody prepared in rabbits against dansylated-ovalbumin; (d) demonstration of a direct dependence of the rate of transglutaminase-mediated, [ 14 C]putresciene incorporation upon the concentration of ribulose, 1,5-bisphosphate carboxylase/oxygenase from alfalfa or spinach; and (e) presumptive evidence from size exclusion chromatography that transglutaminase may cofractionate with native molecules of ribulose 1,5-bisphosphate carboxylase/oxygenase in crude extracts

  20. Assembly of the Arp5 (Actin-related Protein) Subunit Involved in Distinct INO80 Chromatin Remodeling Activities*

    Science.gov (United States)

    Yao, Wei; Beckwith, Sean L.; Zheng, Tina; Young, Thomas; Dinh, Van T.; Ranjan, Anand; Morrison, Ashby J.

    2015-01-01

    ATP-dependent chromatin remodeling, which repositions and restructures nucleosomes, is essential to all DNA-templated processes. The INO80 chromatin remodeling complex is an evolutionarily conserved complex involved in diverse cellular processes, including transcription, DNA repair, and replication. The functional diversity of the INO80 complex can, in part, be attributed to specialized activities of distinct subunits that compose the complex. Furthermore, structural analyses have identified biochemically discrete subunit modules that assemble along the Ino80 ATPase scaffold. Of particular interest is the Saccharomyces cerevisiae Arp5-Ies6 module located proximal to the Ino80 ATPase and the Rvb1-Rvb2 helicase module needed for INO80-mediated in vitro activity. In this study we demonstrate that the previously uncharacterized Ies2 subunit is required for Arp5-Ies6 association with the catalytic components of the INO80 complex. In addition, Arp5-Ies6 module assembly with the INO80 complex is dependent on distinct conserved domains within Arp5, Ies6, and Ino80, including the spacer region within the Ino80 ATPase domain. Arp5-Ies6 interacts with chromatin via assembly with the INO80 complex, as IES2 and INO80 deletion results in loss of Arp5-Ies6 chromatin association. Interestingly, ectopic addition of the wild-type Arp5-Ies6 module stimulates INO80-mediated ATP hydrolysis and nucleosome sliding in vitro. However, the addition of mutant Arp5 lacking unique insertion domains facilitates ATP hydrolysis in the absence of nucleosome sliding. Collectively, these results define the requirements of Arp5-Ies6 assembly, which are needed to couple ATP hydrolysis to productive nucleosome movement. PMID:26306040

  1. Leptin and insulin engage specific PI3K subunits in hypothalamic SF1 neurons

    Directory of Open Access Journals (Sweden)

    Jong-Woo Sohn

    2016-08-01

    Full Text Available Objective: The ventromedial hypothalamic nucleus (VMH regulates energy balance and glucose homeostasis. Leptin and insulin exert metabolic effects via their cognate receptors expressed by the steroidogenic factor 1 (SF1 neurons within the VMH. However, detailed cellular mechanisms involved in the regulation of these neurons by leptin and insulin remain to be identified. Methods: We utilized genetically-modified mouse models and performed patch-clamp electrophysiology experiments to resolve this issue. Results: We identified distinct populations of leptin-activated and leptin-inhibited SF1 neurons. In contrast, insulin uniformly inhibited SF1 neurons. Notably, we found that leptin-activated, leptin-inhibited, and insulin-inhibited SF1 neurons are distinct subpopulations within the VMH. Leptin depolarization of SF1 neuron also required the PI3K p110β catalytic subunit. This effect was mediated by the putative transient receptor potential C (TRPC channel. On the other hand, hyperpolarizing responses of SF1 neurons by leptin and insulin required either of the p110α or p110β catalytic subunits, and were mediated by the putative ATP-sensitive K+ (KATP channel. Conclusions: Our results demonstrate that specific PI3K catalytic subunits are responsible for the acute effects of leptin and insulin on VMH SF1 neurons, and provide insights into the cellular mechanisms of leptin and insulin action on VMH SF1 neurons that regulate energy balance and glucose homeostasis. Author Video: Author Video Watch what authors say about their articles Keywords: Cellular mechanism, Conditional knockout mouse, Patch clamp technique, Functional heterogeneity, Homeostasis

  2. Contribution of the major and minor subunits to fimbria-mediated adherence of Haemophilus influenzae to human epithelial cells and erythrocytes

    NARCIS (Netherlands)

    van Ham, S. M.; van Alphen, L.; Mooi, F. R.; van Putten, J. P.

    1995-01-01

    Fimbriae are colonization factors of the human pathogen Haemophilus influenzae in that they mediate bacterial adherence to human eukaryotic cells. The contribution of the major (HifA) and putative minor (HifD and HifE) subunits of H. influenzae fimbriae to fimbria-specific adherence was studied by

  3. Subunits of ADA-two-A-containing (ATAC) or Spt-Ada-Gcn5-acetyltrasferase (SAGA) Coactivator Complexes Enhance the Acetyltransferase Activity of GCN5.

    Science.gov (United States)

    Riss, Anne; Scheer, Elisabeth; Joint, Mathilde; Trowitzsch, Simon; Berger, Imre; Tora, László

    2015-11-27

    Histone acetyl transferases (HATs) play a crucial role in eukaryotes by regulating chromatin architecture and locus specific transcription. GCN5 (KAT2A) is a member of the GNAT (Gcn5-related N-acetyltransferase) family of HATs. In metazoans this enzyme is found in two functionally distinct coactivator complexes, SAGA (Spt Ada Gcn5 acetyltransferase) and ATAC (Ada Two A-containing). These two multiprotein complexes comprise complex-specific and shared subunits, which are organized in functional modules. The HAT module of ATAC is composed of GCN5, ADA2a, ADA3, and SGF29, whereas in the SAGA HAT module ADA2b is present instead of ADA2a. To better understand how the activity of human (h) hGCN5 is regulated in the two related, but different, HAT complexes we carried out in vitro HAT assays. We compared the activity of hGCN5 alone with its activity when it was part of purified recombinant hATAC or hSAGA HAT modules or endogenous hATAC or hSAGA complexes using histone tail peptides and full-length histones as substrates. We demonstrated that the subunit environment of the HAT complexes into which GCN5 incorporates determines the enhancement of GCN5 activity. On histone peptides we show that all the tested GCN5-containing complexes acetylate mainly histone H3K14. Our results suggest a stronger influence of ADA2b as compared with ADA2a on the activity of GCN5. However, the lysine acetylation specificity of GCN5 on histone tails or full-length histones was not changed when incorporated in the HAT modules of ATAC or SAGA complexes. Our results thus demonstrate that the catalytic activity of GCN5 is stimulated by subunits of the ADA2a- or ADA2b-containing HAT modules and is further increased by incorporation of the distinct HAT modules in the ATAC or SAGA holo-complexes. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Serotonin(4) (5-HT(4)) receptor agonists are putative antidepressants with a rapid onset of action

    DEFF Research Database (Denmark)

    Lucas, Guillaume; Rymar, Vladimir V; Du, Jenny

    2007-01-01

    parameters considered to be key markers of antidepressant action, but that are observed only after 2-3 week treatments with classical molecules: desensitization of 5-HT(1A) autoreceptors, increased tonus on hippocampal postsynaptic 5-HT(1A) receptors, and enhanced phosphorylation of the CREB protein...... intake consecutive to a chronic mild stress. These findings point out 5-HT(4) receptor agonists as a putative class of antidepressants with a rapid onset of action. Udgivelsesdato: 2007-Sep-6...

  5. Monoclonal antibodies to hyphal exoantigens derived from the opportunistic pathogen Aspergillus terreus.

    Science.gov (United States)

    Nayak, Ajay P; Green, Brett J; Janotka, Erika; Hettick, Justin M; Friend, Sherri; Vesper, Steve J; Schmechel, Detlef; Beezhold, Donald H

    2011-09-01

    Aspergillus terreus has been difficult to identify in cases of aspergillosis, and clinical identification has been restricted to the broad identification of aspergillosis lesions in affected organs or the detection of fungal carbohydrates. As a result, there is a clinical need to identify species-specific biomarkers that can be used to detect invasive A. terreus disease. Monoclonal antibodies (MAbs) were developed to a partially purified preparation of cytolytic hyphal exoantigens (HEA) derived from A. terreus culture supernatant (CSN). Twenty-three IgG1 isotype murine MAbs were developed and tested for cross-reactivity against hyphal extracts of 54 fungal species. Sixteen MAbs were shown to be specific for A. terreus. HEA were detected in conidia, hyphae, and in CSN of A. terreus. HEA were expressed in high levels in the hyphae during early stages of A. terreus growth at 37°C, whereas at room temperature the expression of HEA peaked by days 4 to 5. Expression kinetics of HEA in CSN showed a lag, with peak levels at later time points at room temperature and 37°C than in hyphal extracts. Serum spiking experiments demonstrated that human serum components do not inhibit detection of the HEA epitopes by MAb enzyme-linked immunosorbent assay (ELISA). Immunoprecipitation and proteomic analysis demonstrated that MAbs 13E11 and 12C4 immunoprecipitated a putative uncharacterized leucine aminopeptidase (Q0CAZ7), while MAb 19B2 recognized a putative dipeptidyl-peptidase V (DPP5). Studies using confocal laser scanning microscopy showed that the uncharacterized leucine aminopeptidase mostly localized to extracellular matrix structures while dipeptidyl-peptidase V was mostly confined to the cytoplasm.

  6. 28 CFR 51.6 - Political subunits.

    Science.gov (United States)

    2010-07-01

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

  7. The UL5 and UL52 subunits of the herpes simplex virus type 1 helicase-primase subcomplex exhibit a complex interdependence for DNA binding.

    Science.gov (United States)

    Biswas, N; Weller, S K

    2001-05-18

    Herpes simplex virus type 1 encodes a heterotrimeric helicase-primase complex composed of the products of the UL5, UL52, and UL8 genes. The UL5 protein contains seven motifs found in all members of helicase Superfamily 1 (SF1), and the UL52 protein contains several conserved motifs found in primases; however, the contributions of each subunit to the biochemical activities of the subcomplex are not clear. In this work, the DNA binding properties of wild type and mutant subcomplexes were examined using single-stranded, duplex, and forked substrates. A gel mobility shift assay indicated that the UL5-UL52 subcomplex binds more efficiently to the forked substrate than to either single strand or duplex DNA. Although nucleotides are not absolutely required for DNA binding, ADP stimulated the binding of UL5-UL52 to single strand DNA whereas ATP, ADP, and adenosine 5'-O-(thiotriphosphate) stimulated the binding to a forked substrate. We have previously shown that both subunits contact single-stranded DNA in a photocross-linking assay (Biswas, N., and Weller, S. K. (1999) J. Biol. Chem. 274, 8068-8076). In this study, photocross-linking assays with forked substrates indicate that the UL5 and UL52 subunits contact the forked substrates at different positions, UL52 at the single-stranded DNA tail and UL5 near the junction between single-stranded and double-stranded DNA. Neither subunit was able to cross-link a forked substrate when 5-iododeoxyuridine was located within the duplex portion. Photocross-linking experiments with subcomplexes containing mutant versions of UL5 and wild type UL52 indicated that the integrity of the ATP binding region is important for DNA binding of both subunits. These results support our previous proposal that UL5 and UL52 exhibit a complex interdependence for DNA binding (Biswas, N., and Weller, S. K. (1999) J. Biol. Chem. 274, 8068-8076) and indicate that the UL52 subunit may play a more active role in helicase activity than had previously been

  8. Soybean glycinin subunits: Characterization of physicochemical and adhesion properties.

    Science.gov (United States)

    Mo, Xiaoqun; Zhong, Zhikai; Wang, Donghai; Sun, Xiuzhi

    2006-10-04

    Soybean proteins have shown great potential for applications as renewable and environmentally friendly adhesives. The objective of this work was to study physicochemical and adhesion properties of soy glycinin subunits. Soybean glycinin was extracted from soybean flour and then fractionated into acidic and basic subunits with an estimated purity of 90 and 85%, respectively. Amino acid composition of glycinin subunits was determined. The high hydrophobic amino acid content is a major contributor to the solubility behavior and water resistance of the basic subunits. Acidic subunits and glycinin had similar solubility profiles, showing more than 80% solubility at pH 2.0-4.0 or 6.5-12.0, whereas basic subunits had considerably lower solubility with the minimum at pH 4.5-8.0. Thermal analysis using a differential scanning calorimeter suggested that basic subunits form new oligomeric structures with higher thermal stability than glycinin but no highly ordered structures present in isolated acidic subunits. The wet strength of basic subunits was 160% more than that of acidic subunits prepared at their respective isoelectric points (pI) and cured at 130 degrees C. Both pH and the curing temperature significantly affected adhesive performance. High-adhesion water resistance was usually observed for adhesives from protein prepared at their pI values and cured at elevated temperatures. Basic subunits are responsible for the water resistance of glycinin and are a good starting material for the development of water-resistant adhesives.

  9. Increased GABA(A receptor ε-subunit expression on ventral respiratory column neurons protects breathing during pregnancy.

    Directory of Open Access Journals (Sweden)

    Keith B Hengen

    Full Text Available GABAergic signaling is essential for proper respiratory function. Potentiation of this signaling with allosteric modulators such as anesthetics, barbiturates, and neurosteroids can lead to respiratory arrest. Paradoxically, pregnant animals continue to breathe normally despite nearly 100-fold increases in circulating neurosteroids. ε subunit-containing GABA(ARs are insensitive to positive allosteric modulation, thus we hypothesized that pregnant rats increase ε subunit-containing GABA(AR expression on brainstem neurons of the ventral respiratory column (VRC. In vivo, pregnancy rendered respiratory motor output insensitive to otherwise lethal doses of pentobarbital, a barbiturate previously used to categorize the ε subunit. Using electrode array recordings in vitro, we demonstrated that putative respiratory neurons of the preBötzinger Complex (preBötC were also rendered insensitive to the effects of pentobarbital during pregnancy, but unit activity in the VRC was rapidly inhibited by the GABA(AR agonist, muscimol. VRC unit activity from virgin and post-partum females was potently inhibited by both pentobarbital and muscimol. Brainstem ε subunit mRNA and protein levels were increased in pregnant rats, and GABA(AR ε subunit expression co-localized with a marker of rhythm generating neurons (neurokinin 1 receptors in the preBötC. These data support the hypothesis that pregnancy renders respiratory motor output and respiratory neuron activity insensitive to barbiturates, most likely via increased ε subunit-containing GABA(AR expression on respiratory rhythm-generating neurons. Increased ε subunit expression may be critical to preserve respiratory function (and life despite increased neurosteroid levels during pregnancy.

  10. The COP9 signalosome converts temporal hormone signaling to spatial restriction on neural competence.

    Directory of Open Access Journals (Sweden)

    Yi-Chun Huang

    2014-11-01

    Full Text Available During development, neural competence is conferred and maintained by integrating spatial and temporal regulations. The Drosophila sensory bristles that detect mechanical and chemical stimulations are arranged in stereotypical positions. The anterior wing margin (AWM is arrayed with neuron-innervated sensory bristles, while posterior wing margin (PWM bristles are non-innervated. We found that the COP9 signalosome (CSN suppresses the neural competence of non-innervated bristles at the PWM. In CSN mutants, PWM bristles are transformed into neuron-innervated, which is attributed to sustained expression of the neural-determining factor Senseless (Sens. The CSN suppresses Sens through repression of the ecdysone signaling target gene broad (br that encodes the BR-Z1 transcription factor to activate sens expression. Strikingly, CSN suppression of BR-Z1 is initiated at the prepupa-to-pupa transition, leading to Sens downregulation, and termination of the neural competence of PWM bristles. The role of ecdysone signaling to repress br after the prepupa-to-pupa transition is distinct from its conventional role in activation, and requires CSN deneddylating activity and multiple cullins, the major substrates of deneddylation. Several CSN subunits physically associate with ecdysone receptors to represses br at the transcriptional level. We propose a model in which nuclear hormone receptors cooperate with the deneddylation machinery to temporally shutdown downstream target gene expression, conferring a spatial restriction on neural competence at the PWM.

  11. The residence time of GABA(A)Rs at inhibitory synapses is determined by direct binding of the receptor α1 subunit to gephyrin

    DEFF Research Database (Denmark)

    Mukherjee, Jayanta; Kretschmannova, Karla; Gouzer, Geraldine

    2011-01-01

    The majority of fast synaptic inhibition in the brain is mediated by benzodiazepine-sensitive α1-subunit-containing GABA type A receptors (GABA(A)Rs); however, our knowledge of the mechanisms neurons use to regulate their synaptic accumulation is rudimentary. Using immunoprecipitation, we....... Mutating residues 360-375 decreases both the accumulation of α1-containing GABA(A)Rs at gephyrin-positive inhibitory synapses in hippocampal neurons and the amplitude of mIPSCs. We also demonstrate that the affinity of gephyrin for the α1 subunit is modulated by Thr375, a putative phosphorylation site....... Mutation of Thr375 to a phosphomimetic, negatively charged amino acid decreases both the affinity of the α1 subunit for gephyrin, and therefore receptor accumulation at synapses, and the amplitude of mIPSCs. Finally, single-particle tracking reveals that gephyrin reduces the diffusion of α1-subunit...

  12. Progesterone modulation of alpha5 nAChR subunits influences anxiety-related behavior during estrus cycle.

    Science.gov (United States)

    Gangitano, D; Salas, R; Teng, Y; Perez, E; De Biasi, M

    2009-06-01

    Smokers often report an anxiolytic effect of cigarettes. In addition, stress-related disorders such as anxiety, post-traumatic stress syndrome and depression are often associated with chronic nicotine use. To study the role of the alpha5 nicotinic acetylcholine receptor subunit in anxiety-related responses, control and alpha5 subunit null mice (alpha5(-/-)) were subjected to the open field activity (OFA), light-dark box (LDB) and elevated plus maze (EPM) tests. In the OFA and LDB, alpha5(-/-) behaved like wild-type controls. In the EPM, female alpha5(-/-) mice displayed an anxiolytic-like phenotype, while male alpha5(-/-) mice were undistinguishable from littermate controls. We studied the hypothalamus-pituitary-adrenal axis by measuring plasma corticosterone and hypothalamic corticotropin-releasing factor. Consistent with an anxiolytic-like phenotype, female alpha5(-/-) mice displayed lower basal corticosterone levels. To test whether gonadal steroids regulate the expression of alpha5, we treated cultured NTera 2 cells with progesterone and found that alpha5 protein levels were upregulated. In addition, brain levels of alpha5 mRNA increased upon progesterone injection into ovariectomized wild-type females. Finally, we tested anxiety levels in the EPM during the estrous cycle. The estrus phase (when progesterone levels are low) is anxiolytic-like in wild-type mice, but no cycle-dependent fluctuations in anxiety levels were found in alpha5(-/-) females. Thus, alpha5-containing neuronal nicotinic acetylcholine receptors may be mediators of anxiogenic responses, and progesterone-dependent modulation of alpha5 expression may contribute to fluctuations in anxiety levels during the ovarian cycle.

  13. A reduction in hippocampal GABAA receptor alpha5 subunits disrupts the memory for location of objects in mice.

    Science.gov (United States)

    Prut, L; Prenosil, G; Willadt, S; Vogt, K; Fritschy, J-M; Crestani, F

    2010-07-01

    The memory for location of objects, which binds information about objects to discrete positions or spatial contexts of occurrence, is a form of episodic memory particularly sensitive to hippocampal damage. Its early decline is symptomatic for elderly dementia. Substances that selectively reduce alpha5-GABA(A) receptor function are currently developed as potential cognition enhancers for Alzheimer's syndrome and other dementia, consistent with genetic studies implicating these receptors that are highly expressed in hippocampus in learning performance. Here we explored the consequences of reduced GABA(A)alpha5-subunit contents, as occurring in alpha5(H105R) knock-in mice, on the memory for location of objects. This required the behavioral characterization of alpha5(H105R) and wild-type animals in various tasks examining learning and memory retrieval strategies for objects, locations, contexts and their combinations. In mutants, decreased amounts of alpha5-subunits and retained long-term potentiation in hippocampus were confirmed. They exhibited hyperactivity with conserved circadian rhythm in familiar actimeters, and normal exploration and emotional reactivity in novel places, allocentric spatial guidance, and motor pattern learning acquisition, inhibition and flexibility in T- and eight-arm mazes. Processing of object, position and context memories and object-guided response learning were spared. Genotype difference in object-in-place memory retrieval and in encoding and response learning strategies for object-location combinations manifested as a bias favoring object-based recognition and guidance strategies over spatial processing of objects in the mutants. These findings identify in alpha5(H105R) mice a behavioral-cognitive phenotype affecting basal locomotion and the memory for location of objects indicative of hippocampal dysfunction resulting from moderately decreased alpha5-subunit contents.

  14. Role of the Rubisco Small Subunit

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-11-05

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

  15. Activity-dependent shedding of the NMDA receptor glycine binding site by matrix metalloproteinase 3: a PUTATIVE mechanism of postsynaptic plasticity.

    Directory of Open Access Journals (Sweden)

    Thorsten Pauly

    Full Text Available Functional and structural alterations of clustered postsynaptic ligand gated ion channels in neuronal cells are thought to contribute to synaptic plasticity and memory formation in the human brain. Here, we describe a novel molecular mechanism for structural alterations of NR1 subunits of the NMDA receptor. In cultured rat spinal cord neurons, chronic NMDA receptor stimulation induces disappearance of extracellular epitopes of NMDA receptor NR1 subunits, which was prevented by inhibiting matrix metalloproteinases (MMPs. Immunoblotting revealed the digestion of solubilized NR1 subunits by MMP-3 and identified a fragment of about 60 kDa as MMPs-activity-dependent cleavage product of the NR1 subunit in cultured neurons. The expression of MMP-3 in the spinal cord culture was shown by immunoblotting and immunofluorescence microscopy. Recombinant NR1 glycine binding protein was used to identify MMP-3 cleavage sites within the extracellular S1 and S2-domains. N-terminal sequencing and site-directed mutagenesis revealed S542 and L790 as two putative major MMP-3 cleavage sites of the NR1 subunit. In conclusion, our data indicate that MMPs, and in particular MMP-3, are involved in the activity dependent alteration of NMDA receptor structure at postsynaptic membrane specializations in the CNS.

  16. Activity-dependent shedding of the NMDA receptor glycine binding site by matrix metalloproteinase 3: a PUTATIVE mechanism of postsynaptic plasticity.

    Science.gov (United States)

    Pauly, Thorsten; Ratliff, Miriam; Pietrowski, Eweline; Neugebauer, Rainer; Schlicksupp, Andrea; Kirsch, Joachim; Kuhse, Jochen

    2008-07-16

    Functional and structural alterations of clustered postsynaptic ligand gated ion channels in neuronal cells are thought to contribute to synaptic plasticity and memory formation in the human brain. Here, we describe a novel molecular mechanism for structural alterations of NR1 subunits of the NMDA receptor. In cultured rat spinal cord neurons, chronic NMDA receptor stimulation induces disappearance of extracellular epitopes of NMDA receptor NR1 subunits, which was prevented by inhibiting matrix metalloproteinases (MMPs). Immunoblotting revealed the digestion of solubilized NR1 subunits by MMP-3 and identified a fragment of about 60 kDa as MMPs-activity-dependent cleavage product of the NR1 subunit in cultured neurons. The expression of MMP-3 in the spinal cord culture was shown by immunoblotting and immunofluorescence microscopy. Recombinant NR1 glycine binding protein was used to identify MMP-3 cleavage sites within the extracellular S1 and S2-domains. N-terminal sequencing and site-directed mutagenesis revealed S542 and L790 as two putative major MMP-3 cleavage sites of the NR1 subunit. In conclusion, our data indicate that MMPs, and in particular MMP-3, are involved in the activity dependent alteration of NMDA receptor structure at postsynaptic membrane specializations in the CNS.

  17. Mutation in mitochondrial complex IV subunit COX5A causes pulmonary arterial hypertension, lactic acidemia, and failure to thrive

    NARCIS (Netherlands)

    Baertling, F.; Al-Murshedi, F.; Sanchez Caballero, L.M.; Al-Senaidi, K.; Joshi, N.P.; Venselaar, H.; Brand, M.A.M. van den; Nijtmans, L.G.J.; Rodenburg, R.J.T.

    2017-01-01

    COX5A is a nuclear-encoded subunit of mitochondrial respiratory chain complex IV (cytochrome c oxidase). We present patients with a homozygous pathogenic variant in the COX5A gene. Clinical details of two affected siblings suffering from early-onset pulmonary arterial hypertension, lactic acidemia,

  18. Mediator Complex Subunits MED2, MED5, MED16, and MED23 Genetically Interact in the Regulation of Phenylpropanoid Biosynthesis.

    Science.gov (United States)

    Dolan, Whitney L; Dilkes, Brian P; Stout, Jake M; Bonawitz, Nicholas D; Chapple, Clint

    2017-12-01

    The phenylpropanoid pathway is a major global carbon sink and is important for plant fitness and the engineering of bioenergy feedstocks. In Arabidopsis thaliana , disruption of two subunits of the transcriptional regulatory Mediator complex, MED5a and MED5b, results in an increase in phenylpropanoid accumulation. By contrast, the semidominant MED5b mutation reduced epidermal fluorescence4-3 ( ref4-3 ) results in dwarfism and constitutively repressed phenylpropanoid accumulation. Here, we report the results of a forward genetic screen for suppressors of ref4-3. We identified 13 independent lines that restore growth and/or phenylpropanoid accumulation in the ref4-3 background. Two of the suppressors restore growth without restoring soluble phenylpropanoid accumulation, indicating that the growth and metabolic phenotypes of the ref4-3 mutant can be genetically disentangled. Whole-genome sequencing revealed that all but one of the suppressors carry mutations in MED5b or other Mediator subunits. RNA-seq analysis showed that the ref4-3 mutation causes widespread changes in gene expression, including the upregulation of negative regulators of the phenylpropanoid pathway, and that the suppressors reverse many of these changes. Together, our data highlight the interdependence of individual Mediator subunits and provide greater insight into the transcriptional regulation of phenylpropanoid biosynthesis by the Mediator complex. © 2017 American Society of Plant Biologists. All rights reserved.

  19. Genetic variability of the equine casein genes.

    Science.gov (United States)

    Brinkmann, J; Jagannathan, V; Drögemüller, C; Rieder, S; Leeb, T; Thaller, G; Tetens, J

    2016-07-01

    The casein genes are known to be highly variable in typical dairy species, such as cattle and goat, but the knowledge about equine casein genes is limited. Nevertheless, mare milk production and consumption is gaining importance because of its high nutritive value, use in naturopathy, and hypoallergenic properties with respect to cow milk protein allergies. In the current study, the open reading frames of the 4 casein genes CSN1S1 (αS1-casein), CSN2 (β-casein), CSN1S2 (αS2-casein), and CSN3 (κ-casein) were resequenced in 253 horses of 14 breeds. The analysis revealed 21 nonsynonymous nucleotide exchanges, as well as 11 synonymous nucleotide exchanges, leading to a total of 31 putative protein isoforms predicted at the DNA level, 26 of which considered novel. Although the majority of the alleles need to be confirmed at the transcript and protein level, a preliminary nomenclature was established for the equine casein alleles. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  20. The structure of the TFIIH p34 subunit reveals a von Willebrand factor A like fold.

    Directory of Open Access Journals (Sweden)

    Dominik R Schmitt

    Full Text Available RNA polymerase II dependent transcription and nucleotide excision repair are mediated by a multifaceted interplay of subunits within the general transcription factor II H (TFIIH. A better understanding of the molecular structure of TFIIH is the key to unravel the mechanism of action of this versatile protein complex within these vital cellular processes. The importance of this complex becomes further evident in the context of severe diseases like xeroderma pigmentosum, Cockayne's syndrome and trichothiodystrophy, that arise from single point mutations in TFIIH subunits. Here we describe the structure of the p34 subunit of the TFIIH complex from the eukaryotic thermophilic fungus Chaetomium thermophilum. The structure revealed that p34 contains a von Willebrand Factor A (vWA like domain, a fold which is generally known to be involved in protein-protein interactions. Within TFIIH p34 strongly interacts with p44, a positive regulator of the helicase XPD. Putative protein-protein interfaces are analyzed and possible binding sites for the p34-p44 interaction suggested.

  1. Conservation of the TRAPPII-specific subunits of a Ypt/Rab exchanger complex

    Directory of Open Access Journals (Sweden)

    Yoo Eunice

    2007-02-01

    Full Text Available Abstract Background Ypt/Rab GTPases and their GEF activators regulate intra-cellular trafficking in all eukaryotic cells. In S. cerivisiae, the modular TRAPP complex acts as a GEF for the Golgi gatekeepers: Ypt1 and the functional pair Ypt31/32. While TRAPPI, which acts in early Golgi, is conserved from fungi to animals, not much is known about TRAPPII, which acts in late Golgi and consists of TRAPPI plus three additional subunits. Results Here, we show a phylogenetic analysis of the three TRAPPII-specific subunits. One copy of each of the two essential subunits, Trs120 and Trs130, is present in almost every fully sequenced eukaryotic genome. Moreover, the primary, as well as the predicted secondary, structure of the Trs120- and Trs130-related sequences are conserved from fungi to animals. The mammalian orthologs of Trs120 and Trs130, NIBP and TMEM1, respectively, are candidates for human disorders. Currently, NIBP is implicated in signaling, and TMEM1 is suggested to have trans-membrane domains (TMDs and to function as a membrane channel. However, we show here that the yeast Trs130 does not function as a trans-membrane protein, and the human TMEM1 does not contain putative TMDs. The non-essential subunit, Trs65, is conserved only among many fungi and some unicellular eukaryotes. Multiple alignment analysis of each TRAPPII-specific subunit revealed conserved domains that include highly conserved amino acids. Conclusion We suggest that the function of both NIBP and TMEM1 in the regulation of intra-cellular trafficking is conserved from yeast to man. The conserved domains and amino acids discovered here can be used for functional analysis that should help to resolve the differences in the assigned functions of these proteins in fungi and animals.

  2. The Putative Son's Attractiveness Alters the Perceived Attractiveness of the Putative Father.

    Science.gov (United States)

    Prokop, Pavol

    2015-08-01

    A body of literature has investigated female mate choice in the pre-mating context (pre-mating sexual selection). Humans, however, are long-living mammals forming pair-bonds which sequentially produce offspring. Post-mating evaluations of a partner's attractiveness may thus significantly influence the reproductive success of men and women. I tested herein the theory that the attractiveness of putative sons provides extra information about the genetic quality of fathers, thereby influencing fathers' attractiveness across three studies. As predicted, facially attractive boys were more frequently attributed to attractive putative fathers and vice versa (Study 1). Furthermore, priming with an attractive putative son increased the attractiveness of the putative father with the reverse being true for unattractive putative sons. When putative fathers were presented as stepfathers, the effect of the boy's attractiveness on the stepfather's attractiveness was lower and less consistent (Study 2). This suggests that the presence of an attractive boy has the strongest effect on the perceived attractiveness of putative fathers rather than on non-fathers. The generalized effect of priming with beautiful non-human objects also exists, but its effect is much weaker compared with the effects of putative biological sons (Study 3). Overall, this study highlighted the importance of post-mating sexual selection in humans and suggests that the heritable attractive traits of men are also evaluated by females after mating and/or may be used by females in mate poaching.

  3. Selection of refractory materials for acid tanks at the CSN continuous pickling line

    International Nuclear Information System (INIS)

    Silva, Sidiney Nascimento; Marques, Oscar Rosa; Bueno, Mauricio Chaves; Longo, Elson; Silva Pinheiro, Adriano da

    1997-01-01

    Aiming at the revamping of the CSN continuous pickling line 4 acid tanks, a Post Mortem study of the refractory lining was carried out. The collected samples were characterized through techniques such as chemical analysis, mercury porosimetry, X-ray diffraction and scanning electronic microscopy. Trying to reproduce the operational conditions closely, laboratorial simulations were carried out. Such simulations lead to the addition of some alterations on the test method proposed by ABNT. Primarily, the sulfuric acid was substituted by hydrochloric acid (30%), containing iron in solution (130g/l). As result, it was concluded that acid resistant refractories containing a smaller alumina and /or corundum and mullite concentrations, presenting a smaller open porosity and average pore diameter, have a better performance face to corrosion due to hydrochloric acid solution. In addition, abrasion wear resistance tests, according to the ASTM-G65-85 standard were carried out in order to select different materials to the acid tanks cells. (author)

  4. Arabidopsis CDS blastp result: AK062262 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK062262 001-047-H04 At5g13630.1 magnesium-chelatase subunit chlH, chloroplast, put...ative / Mg-protoporphyrin IX chelatase, putative (CHLH) nearly identical to magnesium chelatase subunit GI:1...154627 from [Arabidopsis thaliana]; contains Pfam profile: PF02514 CobN/magnesium chelatase family protein 0.0 ...

  5. Arabidopsis CDS blastp result: AK069545 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK069545 J023025I06 At5g13630.1 magnesium-chelatase subunit chlH, chloroplast, puta...tive / Mg-protoporphyrin IX chelatase, putative (CHLH) nearly identical to magnesium chelatase subunit GI:11...54627 from [Arabidopsis thaliana]; contains Pfam profile: PF02514 CobN/magnesium chelatase family protein 0.0 ...

  6. Arabidopsis CDS blastp result: AK067323 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK067323 J013106B16 At5g13630.1 magnesium-chelatase subunit chlH, chloroplast, puta...tive / Mg-protoporphyrin IX chelatase, putative (CHLH) nearly identical to magnesium chelatase subunit GI:11...54627 from [Arabidopsis thaliana]; contains Pfam profile: PF02514 CobN/magnesium chelatase family protein 0.0 ...

  7. Arabidopsis CDS blastp result: AK060612 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK060612 001-025-F03 At5g13630.1 magnesium-chelatase subunit chlH, chloroplast, put...ative / Mg-protoporphyrin IX chelatase, putative (CHLH) nearly identical to magnesium chelatase subunit GI:1...154627 from [Arabidopsis thaliana]; contains Pfam profile: PF02514 CobN/magnesium chelatase family protein 0.0 ...

  8. Comparison of cDNA-derived protein sequences of the human fibronectin and vitronectin receptor α-subunits and platelet glycoprotein IIb

    International Nuclear Information System (INIS)

    Fitzgerald, L.A.; Poncz, M.; Steiner, B.; Rall, S.C. Jr.; Bennett, J.S.; Phillips, D.R.

    1987-01-01

    The fibronectin receptor (FnR), the vitronectin receptor (VnR), and the platelet membrane glycoprotein (GP) IIb-IIIa complex are members of a family of cell adhesion receptors, which consist of noncovalently associated α- and β-subunits. The present study was designed to compare the cDNA-derived protein sequences of the α-subunits of human FnR, VnR, and platelet GP IIb. cDNA clones for the α-subunit of the FnR (FnR/sub α/) were obtained from a human umbilical vein endothelial (HUVE) cell library by using an oligonucleotide probe designed from a peptide sequence of platelet GP IIb. cDNA clones for platelet GP IIb were isolated from a cDNA expression library of human erythroleukemia cells by using antibodies. cDNA clones of the VnR α-subunit (VnR/sub α/) were obtained from the HUVE cell library by using an oligonucleotide probe from the partial cDNA sequence for the VnR/sub α/. Translation of these sequences showed that the FNR/sub α/, the VnR/sub α/, and GP IIb are composed of disulfide-linked large (858-871 amino acids) and small (137-158 amino acids) chains that are posttranslationally processed from a single mRNA. A single hydrophobic segment located near the carboxyl terminus of each small chain appears to be a transmembrane domain. The large chains appear to be entirely extracellular, and each contains four repeated putative Ca 2+ -binding domains of about 30 amino acids that have sequence similarities to other Ca 2+ -binding proteins. The identity among the protein sequences of the three receptor α-subunits ranges from 36.1% to 44.5%, with the Ca 2+ -binding domains having the greatest homology. These proteins apparently evolved by a process of gene duplication

  9. Purification of the alpha and beta subunits of phosphorylase kinase for structural studies

    International Nuclear Information System (INIS)

    Sotiroudis, T.G.; Heilmeyer, L.M.G. Jr.; Crabb, J.W.

    1987-01-01

    Structural analysis of the alpha (Mr, 132,000) and beta (Mr, 113,000) subunits of phosphorylase kinase may provide clues to their yet unknown functions however purification remains problematic. Preparative RP-HPLC procedures yield inconveniently large, dilute solutions and concentration steps are required prior to subunit modification and fragmentation. Concentration of the β subunit usually results in significant losses due to insolubility. Using preparative SDS-polyacrylamide gel electrophoresis, they have purified the α, 7 , and β subunits from rabbit muscle phosphorylase kinase in a soluble and concentrated form suitable for structural studies. Phosphorylase kinase labelled with fluorescein isothiocyanate in the α and α' subunits and fully 14 C-S-carboxymethylated was fractionated on a 5% acrylamide Laemmli slab gel. The subunit bands were visualized by fluorescence and by SDS precipitation then excised and electroeluted in the presence of SDS using an ELUTRAP device. From 4.5 mg of enzyme applied to a 4.5 mm thick gel about 70% of the α subunit and about 90% of the β subunit were typically recovered in less than 1 ml with overnight elution

  10. Arabidopsis CDS blastp result: AK065420 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK065420 J013022D10 At5g13630.1 magnesium-chelatase subunit chlH, chloroplast, puta...tive / Mg-protoporphyrin IX chelatase, putative (CHLH) nearly identical to magnesium chelatase subunit GI:11...54627 from [Arabidopsis thaliana]; contains Pfam profile: PF02514 CobN/magnesium chelatase family protein 1e-166 ...

  11. Assembly of proteins and 5 S rRNA to transcripts of the major structural domains of 23 S rRNA

    DEFF Research Database (Denmark)

    Ostergaard, P; Phan, H; Johansen, L B

    1998-01-01

    The six major structural domains of 23 S rRNA from Escherichia coli, and all combinations thereof, were synthesized as separate T7 transcripts and reconstituted with total 50 S subunit proteins. Analysis by one and two-dimensional gel electrophoresis demonstrated the presence of at least one prim...... approach was used to map the putative binding regions on domain V of protein L9 and the 5 S RNA-L5-L18 complex....

  12. Results of the Interlaboratory Exercise CSN/CIEMAT-02 Among Environmental Radioactivity Laboratories (Sea Fish); Resultados del Ejercicio Interlaboratorios de Radiactividad Ambiental CSN/CIEMAT-02 (Fauna Marina)

    Energy Technology Data Exchange (ETDEWEB)

    Romero gonzalez, M. L.

    2003-07-01

    The document describes the outcome of the CSN/CIEMAT-02 interlaboratory test comparison among environmental radioactivity laboratories. The exercise was organised according to the ISO-43 and the ISO/IUPAC/AOAC Harmonized Protocol for the proficiency testing of analytical laboratories. The test sample was a reference materials provided by the IAEA-MEL (IAE Marine Environmental Laboratory, Monaco), a sea fish containing environmental levels of U-238, U-234, K-40, Pb-210, Ra-226, Sr-90, Cs-137, Co-60, Pu-(239+240), Am-241 and Tc-99. The results of the exercise were computed for 32 participating laboratories, and their analytical performance was assessed using the z-score approach. A raised percentage of satisfactory laboratory performance has been obtained for all the analysis, being the best performance in gamma measurements. The laboratories have made an effort to calculate the combined uncertainty of the radiochemical determinations. Most of the laboratories have demonstrated its competence in performing the study analysis and also the adequate measuring capability of their detection equipment even in conditions close to detection limits. The study has shown the capacity of participant laboratories to perform radioactive determinations in environmental sea fish samples with satisfactory quality levels. (Author) 6 refs.

  13. Estimates of epistatic and pleiotropic effects of casein alpha s1 (CSN1S1) and thyroglobulin (TG) genetic markers on beef heifer performance traits enhanced by selection

    Science.gov (United States)

    Genetic marker effects and type of inheritance are estimated with poor precision when minor marker allele frequencies are low. A stable composite population (MARC II) was subjected to marker assisted selection for two years to equalize CSN1S1 and TG genetic marker frequencies to evaluate the epista...

  14. Cloning and characterization of Sdga gene encoding alpha-subunit of heterotrimeric guanosine 5'-triphosphate-binding protein complex in Scoparia dulcis.

    Science.gov (United States)

    Shite, Masato; Yamamura, Yoshimi; Hayashi, Toshimitsu; Kurosaki, Fumiya

    2008-11-01

    A homology-based cloning strategy yielded Sdga, a cDNA clone presumably encoding alpha-subunit of heterotrimeric guanosine 5'-triphosphate-binding protein complex, from leaf tissues of Scoparia dulcis. Phylogenetic tree analysis of G-protein alpha-subunits from various biological sources suggested that, unlike in animal cells, classification of Galpha-proteins into specific subfamilies could not be applicable to the proteins from higher plants. Restriction digests of genomic DNA of S. dulcis showed a single hybridized signal in Southern blot analysis, suggesting that Sdga is a sole gene encoding Galpha-subunit in this plant. The expression level of Sdga appeared to be maintained at almost constant level after exposure of the leaves to methyl jasmonate as analyzed by reverse-transcription polymerase chain reaction. These results suggest that Sdga plays roles in methyl jasmonate-induced responses of S. dulcis without a notable change in the transcriptional level.

  15. Arabidopsis thaliana GYRB3 does not encode a DNA gyrase subunit.

    Directory of Open Access Journals (Sweden)

    Katherine M Evans-Roberts

    2010-03-01

    Full Text Available DNA topoisomerases are enzymes that control the topology of DNA in all cells. DNA gyrase is unique among the topoisomerases in that it is the only enzyme that can actively supercoil DNA using the free energy of ATP hydrolysis. Until recently gyrase was thought to be unique to bacteria, but has now been discovered in plants. The genome of the model plant, Arabidopsis thaliana, is predicted to encode four gyrase subunits: AtGyrA, AtGyrB1, AtGyrB2 and AtGyrB3.We found, contrary to previous data, that AtGyrB3 is not essential to the survival of A. thaliana. Bioinformatic analysis suggests AtGyrB3 is considerably shorter than other gyrase B subunits, lacking part of the ATPase domain and other key motifs found in all type II topoisomerases; but it does contain a putative DNA-binding domain. Partially purified AtGyrB3 cannot bind E. coli GyrA or support supercoiling. AtGyrB3 cannot complement an E. coli gyrB temperature-sensitive strain, whereas AtGyrB2 can. Yeast two-hybrid analysis suggests that AtGyrB3 cannot bind to AtGyrA or form a dimer.These data strongly suggest that AtGyrB3 is not a gyrase subunit but has another unknown function. One possibility is that it is a nuclear protein with a role in meiosis in pollen.

  16. Reduction and Methyl Transfer Kinetics of the Alpha Subunit from Acetyl-Coenzyme A Synthase

    Energy Technology Data Exchange (ETDEWEB)

    Xiangshi Tan; Christopher Sewell; Qingwu Yang; Paul A. Lindahl

    2003-01-15

    OAK-B135 Stopped-flow was used to evaluate the methylation and reduction kinetics of the isolated alpha subunit of acetyl-Coenzyme A synthase from Moorella thermoacetica. This catalytically active subunit contains a novel Ni-X-Fe4S4 cluster and a putative unidentified n =2 redox site called D. The D-site must be reduced for a methyl group to transfer from a corrinoid-iron-sulfur protein, a key step in the catalytic synthesis of acetyl-CoA. The Fe4S4 component of this cluster is also redox active, raising the possibility that it is the D-site or a portion thereof. Results presented demonstrate that the D-site reduces far faster than the Fe4S4 component, effectively eliminating this possibility. Rather, this component may alter catalytically important properties of the Ni center. The D-site is reduced through a pathway that probably does not involve the Fe4S4 component of this active-site cluster.

  17. Sex-dependent anti-stress effect of an α5 subunit containing GABAA receptor positive allosteric modulator

    Directory of Open Access Journals (Sweden)

    Sean C. Piantadosi

    2016-11-01

    Full Text Available Rationale: Current first-line treatments for stress-related disorders such as Major Depressive Disorder (MDD act on monoaminergic systems and take weeks to achieve a therapeutic effect with poor response and low remission rates. Recent research has implicated the GABAergic system in the pathophysiology of depression, including deficits in interneurons targeting the dendritic compartment of cortical pyramidal cells. Objectives: The present study evaluates whether SH-053-2'F-R-CH3 (denoted α5-PAM, a positive allosteric modulator selective for α5-subunit containing GABAA receptors found predominantly on cortical pyramidal cell dendrites has anti-stress effects. Methods: Female and male C57BL6/J mice were exposed to unpredictable chronic mild stress (UCMS and treated with α5-PAM acutely (30 minutes prior to assessing behavior or chronically before being assessed behaviorally. Results: Acute and chronic α5-PAM treatments produce a pattern of decreased stress-induced behaviors (denoted as behavioral emotionality across various tests in female, but not in male mice. Behavioral Z-scores calculated across a panel of tests designed to best model the range and heterogeneity of human symptomatology confirmed that acute and chronic α5-PAM treatments consistently produce significant decreases in behavioral emotionality in several independent cohorts of females. The behavioral responses to α5-PAM could not be completely accounted for by differences in drug brain disposition between female and male mice. In mice exposed to UCMS, expression of the Gabra5 gene was increased in the frontal cortex after acute treatment and in hippocampus after chronic treatment with α5-PAM in females only, and these expression changes correlated with behavioral emotionality. Conclusions: We showed that acute and chronic positive modulation of α5 subunit-containing GABAA receptors elicit anti-stress effects in a sex-dependent manner, suggesting novel therapeutic modalities.

  18. DSS1/Sem1, a multifunctional and intrinsically disordered protein

    DEFF Research Database (Denmark)

    Kragelund, Birthe Brandt; Schenstrøm, Signe Marie; Rebula, Caio A.

    2016-01-01

    DSS1/Sem1 is a versatile intrinsically disordered protein. Besides being a bona fide subunit of the 26S proteasome, DSS1 associates with other protein complexes, including BRCA2-RPA, involved in homologous recombination; the Csn12-Thp3 complex, involved in RNA splicing; the integrator, involved...

  19. HupW Protease Specifically Required for Processing of the Catalytic Subunit of the Uptake Hydrogenase in the Cyanobacterium Nostoc sp. Strain PCC 7120

    Science.gov (United States)

    Lindberg, Pia; Devine, Ellenor; Stensjö, Karin

    2012-01-01

    The maturation process of [NiFe] hydrogenases includes a proteolytic cleavage of the large subunit. We constructed a mutant of Nostoc strain PCC 7120 in which hupW, encoding a putative hydrogenase-specific protease, is inactivated. Our results indicate that the protein product of hupW selectively cleaves the uptake hydrogenase in this cyanobacterium. PMID:22020512

  20. Molecular cloning of the cDNA encoding follicle-stimulating hormone beta subunit of the Chinese soft-shell turtle Pelodiscus sinensis, and its gene expression.

    Science.gov (United States)

    Chien, Jung-Tsun; Shen, San-Tai; Lin, Yao-Sung; Yu, John Yuh-Lin

    2005-04-01

    Follicle-stimulating hormone (FSH) is a member of the pituitary glycoprotein hormone family. These hormones are composed of two dissimilar subunits, alpha and beta. Very little information is available regarding the nucleotide and amino acid sequence of FSHbeta in reptilian species. For better understanding of the phylogenetic diversity and evolution of FSH molecule, we have isolated and sequenced the complementary DNA (cDNA) encoding the Chinese soft-shell turtle (Pelodiscus sinensis, Family of Trionychidae) FSHbeta precursor molecule by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) methods. The cloned Chinese soft-shell turtle FSHbeta cDNA consists of 602-bp nucleotides, including 34-bp nucleotides of the 5'-untranslated region (UTR), 396-bp of the open reading frame, and 3'-UTR of 206-bp nucleotides. It encodes a 131-amino acid precursor molecule of FSHbeta subunit with a signal peptide of 20 amino acids followed by a mature protein of 111 amino acids. Twelve cysteine residues, forming six disulfide bonds within beta-subunit and two putative asparagine-linked glycosylation sites, are also conserved in the Chinese soft-shell turtle FSHbeta subunit. The deduced amino acid sequence of the Chinese soft-shell turtle FSHbeta shares identities of 97% with Reeves's turtle (Family of Bataguridae), 83-89% with birds, 61-70% with mammals, 63-66% with amphibians and 40-58% with fish. By contrast, when comparing the FSHbeta with the beta-subunits of the Chinese soft-shell turtle luteinizing hormone and thyroid stimulating hormone, the homologies are as low as 38 and 39%, respectively. A phylogenetic tree including reptilian species of FSHbeta subunits, is presented for the first time. Out of various tissues examined, FSHbeta mRNA was only expressed in the pituitary gland and can be up-regulated by gonadotropin-releasing hormone in pituitary tissue culture as estimated by fluorescence real-time PCR analysis.

  1. Pituitary glycoprotein hormone a-subunit secretion by cirrhotic patients

    Directory of Open Access Journals (Sweden)

    Oliveira M.C.

    1999-01-01

    Full Text Available Secretion of the a-subunit of pituitary glycoprotein hormones usually follows the secretion of intact gonadotropins and is increased in gonadal failure and decreased in isolated gonadotropin deficiency. The aim of the present study was to determine the levels of the a-subunit in the serum of patients with cirrhosis of the liver and to compare the results obtained for eugonadal cirrhotic patients with those obtained for cirrhotic patients with hypogonadotropic hypogonadism. Forty-seven of 63 patients with cirrhosis (74.6% presented hypogonadism (which was central in 45 cases and primary in 2, 7 were eugonadal, and 9 women were in normal menopause. The serum a-subunit was measured by the fluorimetric method using monoclonal antibodies. Cross-reactivity with LH, TSH, FSH and hCG was 6.5, 1.2, 4.3 and 1.1%, respectively, with an intra-assay coefficient of variation (CV of less than 5% and an interassay CV of 5%, and sensitivity limit of 4 ng/l. The serum a-subunit concentration ranged from 36 to 6253 ng/l, with a median of 273 ng/l. The median was 251 ng/l for patients with central hypogonadism and 198 ng/l for eugonadal patients. The correlation between the a-subunit and basal LH levels was significant both in the total sample (r = 0.48, P<0.01 and in the cirrhotic patients with central hypogonadism (r = 0.33, P = 0.02. Among men with central hypogonadism there was a negative correlation between a-subunit levels and total testosterone levels (r = 0.54, P<0.01 as well as free testosterone levels (r = -0.53, P<0.01. In conclusion, although the a-subunit levels are correlated with LH levels, at present they cannot be used as markers for hypogonadism in patients with cirrhosis of the liver.

  2. Results of the Interlaboratory Exercise CSN/CIEMAT-100 Among Environmental Radioactivity Laboratories (Soil)

    International Nuclear Information System (INIS)

    Romero Gonzalez, M. L.

    2002-01-01

    The document describes the outcome of the CSN/CIEMAT-00 interlaboratory test comparison among environmental radioactivity laboratories. the exercise was organised according to the ISO-43 and the ISO/IUPAC/AOAC Harmonized Protocol for the proficiency testing of analytical laboratories. the test sample was a soil containing environmental levels of K-40, Ra-226, Ac-228, Sr-90, Cs-137, Cs-134, Pu (239-240) y Am-241. the Universidad Autonoma de Barcelona prepared the material and reported adequate statistical studies of homogeneity. The results of the exercise were computed for 30 participating laboratories, and their analytical performance was assessed using the u-score approach. A raised percentage of satisfactory laboratory performance has been obtained for all the analysis, being the best performance in gamma measurements. The exercise has drawn that several laboratories have difficulties in the evaluation of combined uncertainty, mainly in analysis involving radiochemical steps. The study has shown an homogeneous inter-laboratory behaviour, and the improvement achieved through subsequent exercises in the quality of the data they are producing. (Author) 10 refs

  3. A conserved phenylalanine as a relay between the α5 helix and the GDP binding region of heterotrimeric Gi protein α subunit.

    Science.gov (United States)

    Kaya, Ali I; Lokits, Alyssa D; Gilbert, James A; Iverson, Tina M; Meiler, Jens; Hamm, Heidi E

    2014-08-29

    G protein activation by G protein-coupled receptors is one of the critical steps for many cellular signal transduction pathways. Previously, we and other groups reported that the α5 helix in the G protein α subunit plays a major role during this activation process. However, the precise signaling pathway between the α5 helix and the guanosine diphosphate (GDP) binding pocket remains elusive. Here, using structural, biochemical, and computational techniques, we probed different residues around the α5 helix for their role in signaling. Our data showed that perturbing the Phe-336 residue disturbs hydrophobic interactions with the β2-β3 strands and α1 helix, leading to high basal nucleotide exchange. However, mutations in β strands β5 and β6 do not perturb G protein activation. We have highlighted critical residues that leverage Phe-336 as a relay. Conformational changes are transmitted starting from Phe-336 via β2-β3/α1 to Switch I and the phosphate binding loop, decreasing the stability of the GDP binding pocket and triggering nucleotide release. When the α1 and α5 helices were cross-linked, inhibiting the receptor-mediated displacement of the C-terminal α5 helix, mutation of Phe-336 still leads to high basal exchange rates. This suggests that unlike receptor-mediated activation, helix 5 rotation and translocation are not necessary for GDP release from the α subunit. Rather, destabilization of the backdoor region of the Gα subunit is sufficient for triggering the activation process. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Molecular cloning and characterization of RGA1 encoding a G protein alpha subunit from rice (Oryza sativa L. IR-36).

    Science.gov (United States)

    Seo, H S; Kim, H Y; Jeong, J Y; Lee, S Y; Cho, M J; Bahk, J D

    1995-03-01

    A cDNA clone, RGA1, was isolated by using a GPA1 cDNA clone of Arabidopsis thaliana G protein alpha subunit as a probe from a rice (Oryza sativa L. IR-36) seedling cDNA library from roots and leaves. Sequence analysis of genomic clone reveals that the RGA1 gene has 14 exons and 13 introns, and encodes a polypeptide of 380 amino acid residues with a calculated molecular weight of 44.5 kDa. The encoded protein exhibits a considerable degree of amino acid sequence similarity to all the other known G protein alpha subunits. A putative TATA sequence (ATATGA), a potential CAAT box sequence (AGCAATAC), and a cis-acting element, CCACGTGG (ABRE), known to be involved in ABA induction are found in the promoter region. The RGA1 protein contains all the consensus regions of G protein alpha subunits except the cysteine residue near the C-terminus for ADP-ribosylation by pertussis toxin. The RGA1 polypeptide expressed in Escherichia coli was, however, ADP-ribosylated by 10 microM [adenylate-32P] NAD and activated cholera toxin. Southern analysis indicates that there are no other genes similar to the RGA1 gene in the rice genome. Northern analysis reveals that the RGA1 mRNA is 1.85 kb long and expressed in vegetative tissues, including leaves and roots, and that its expression is regulated by light.

  5. DNA-repair protein hHR23a alters its protein structure upon binding proteasomal subunit S5a

    Science.gov (United States)

    Walters, Kylie J.; Lech, Patrycja J.; Goh, Amanda M.; Wang, Qinghua; Howley, Peter M.

    2003-01-01

    The Rad23 family of proteins, including the human homologs hHR23a and hHR23b, stimulates nucleotide excision repair and has been shown to provide a novel link between proteasome-mediated protein degradation and DNA repair. In this work, we illustrate how the proteasomal subunit S5a regulates hHR23a protein structure. By using NMR spectroscopy, we have elucidated the structure and dynamic properties of the 40-kDa hHR23a protein and show it to contain four structured domains connected by flexible linker regions. In addition, we reveal that these domains interact in an intramolecular fashion, and by using residual dipolar coupling data in combination with chemical shift perturbation analysis, we present the hHR23a structure. By itself, hHR23a adopts a closed conformation defined by the interaction of an N-terminal ubiquitin-like domain with two ubiquitin-associated domains. Interestingly, binding of the proteasomal subunit S5a disrupts the hHR23a interdomain interactions and thereby causes it to adopt an opened conformation. PMID:14557549

  6. The N-terminus of RPA large subunit and its spatial position are important for the 5'->3' resection of DNA double-strand breaks.

    Science.gov (United States)

    Tammaro, Margaret; Liao, Shuren; McCane, Jill; Yan, Hong

    2015-10-15

    The first step of homology-dependent repair of DNA double-strand breaks (DSBs) is the resection of the 5' strand to generate 3' ss-DNA. Of the two major nucleases responsible for resection, EXO1 has intrinsic 5'->3' directionality, but DNA2 does not. DNA2 acts with RecQ helicases such as the Werner syndrome protein (WRN) and the heterotrimeric eukaryotic ss-DNA binding protein RPA. We have found that the N-terminus of the RPA large subunit (RPA1N) interacts with both WRN and DNA2 and is essential for stimulating WRN's 3'->5' helicase activity and DNA2's 5'->3' ss-DNA exonuclease activity. A mutant RPA complex that lacks RPA1N is unable to support resection in Xenopus egg extracts and human cells. Furthermore, relocating RPA1N to the middle subunit but not to the small subunit causes severe defects in stimulating DNA2 and WRN and in supporting resection. Together, these findings suggest that RPA1N and its spatial position are critical for restricting the directionality of the WRN-DNA2 resection pathway. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. NMDA receptor subunits in the adult rat hippocampus undergo similar changes after 5 minutes in an open field and after LTP induction.

    Directory of Open Access Journals (Sweden)

    Maria Veronica Baez

    Full Text Available NMDA receptor subunits change during development and their synaptic expression is modified rapidly after synaptic plasticity induction in hippocampal slices. However, there is scarce information on subunits expression after synaptic plasticity induction or memory acquisition, particularly in adults. GluN1, GluN2A and GluN2B NMDA receptor subunits were assessed by western blot in 1 adult rats that had explored an open field (OF for 5 minutes, a time sufficient to induce habituation, 2 mature rat hippocampal neuron cultures depolarized by KCl and 3 hippocampal slices from adult rats where long term potentiation (LTP was induced by theta-burst stimulation (TBS. GluN1 and GluN2A, though not GluN2B, were significantly higher 70 minutes--but not 30 minutes--after a 5 minutes session in an OF. GluN1 and GluN2A total immunofluorescence and puncta in neurites increased in cultures, as evaluated 70 minutes after KCl stimulation. Similar changes were found in hippocampal slices 70 minutes after LTP induction. To start to explore underlying mechanisms, hippocampal slices were treated either with cycloheximide (a translation inhibitor or actinomycin D (a transcription inhibitor during electrophysiological assays. It was corroborated that translation was necessary for LTP induction and expression. The rise in GluN1 depends on transcription and translation, while the increase in GluN2A appears to mainly depend on translation, though a contribution of some remaining transcriptional activity during actinomycin D treatment could not be rouled out. LTP effective induction was required for the subunits to increase. Although in the three models same subunits suffered modifications in the same direction, within an apparently similar temporal course, further investigation is required to reveal if they are related processes and to find out whether they are causally related with synaptic plasticity, learning and memory.

  8. Guide of the CSN about methodology of radiological Checking of location and general levels of release; Guia del CSN sobre metodologia de comprobacion del estado radiologico de un emplazamiento y niveles genericos de liberacion

    Energy Technology Data Exchange (ETDEWEB)

    Sanz Alduan, M. T.

    2013-07-01

    The guide aims to recommend a methodology for checking radiological status of a site with a view to his release, complete and with a reasonable, statistically representative number of measures. Checking of the radiological situation of a site allows to determine if it is contaminated and, if so, whether, after cleaning performances, has reached a level of residual contamination, allowing their release with or without restrictions. Generic levels for the release without restrictions on the grounds of sites recommended for this verification of nuclear installations on the basis of the radiological dose criteria laid down in the instruction of the CSN IS-13. I agree with the IS-13, the site includes terrain, structures and facilities described in the authorization of exploitation, as well as any area has been released prior to the closing statement. The Guide recommends only the levels of release of land, regardless of the structures and facilities. (Author)

  9. Calibration of individual dosemeters by using external beams of photon radiation. A nationwide survey among Personal Dosimetry Services, authorized by CSN; Calibracion de dosimetros personales usando haces externos de fotones. Control de los Servicios de Dosimetria Personal autorizados por el CSN

    Energy Technology Data Exchange (ETDEWEB)

    Brosed, A; Ginjaume, M

    1995-12-01

    A nationwide survey in 1995 among Personal Dosimetry Services, authorized by the Spanish Nuclear Safety Council (CSN), has led the Spanish Dosimetry Laboratories to review and update the dosimetric conversion coefficients and correction factors in use in Spain since 1987. The recommendations of the ICRU Report 47(1992) are discussed and adopted. In addition differences in back-scattering form IRCU tissue and PMMA phantoms are analysed. Analytical functions used to calculate conversion coefficients and back-scattering correction factors due to the use of different phantom materials are presented, together with the adopted final values. Firstly, the above mentioned parameters are applied to ISO narrow spectra series, which are discribed in this report. Secondly, differences between 1995 and 1987 values are also shown. (Author)

  10. Wide range of interacting partners of pea Gβ subunit of G-proteins suggests its multiple functions in cell signalling.

    Science.gov (United States)

    Bhardwaj, Deepak; Lakhanpaul, Suman; Tuteja, Narendra

    2012-09-01

    Climate change is a major concern especially in view of the increasing global population and food security. Plant scientists need to look for genetic tools whose appropriate usage can contribute to sustainable food availability. G-proteins have been identified as some of the potential genetic tools that could be useful for protecting plants from various stresses. Heterotrimeric G-proteins consisting of three subunits Gα, Gβ and Gγ are important components of a number of signalling pathways. Their structure and functions are already well studied in animals but their potential in plants is now gaining attention for their role in stress tolerance. Earlier we have reported that over expressing pea Gβ conferred heat tolerance in tobacco plants. Here we report the interacting partners (proteins) of Gβ subunit of Pisum sativum and their putative role in stress and development. Out of 90 transformants isolated from the yeast-two-hybrid (Y2H) screening, seven were chosen for further investigation due to their recurrence in multiple experiments. These interacting partners were confirmed using β-galactosidase colony filter lift and ONPG (O-nitrophenyl-β-D-galactopyranoside) assays. These partners include thioredoxin H, histidine-containing phosphotransfer protein 5-like, pathogenesis-related protein, glucan endo-beta-1, 3-glucosidase (acidic isoform), glycine rich RNA binding protein, cold and drought-regulated protein (corA gene) and soluble inorganic pyrophosphatase 1. This study suggests the role of pea Gβ subunit in stress signal transduction and development pathways owing to its capability to interact with a wide range of proteins of multiple functions. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  11. The N-terminus of RPA large subunit and its spatial position are important for the 5′->3′ resection of DNA double-strand breaks

    Science.gov (United States)

    Tammaro, Margaret; Liao, Shuren; McCane, Jill; Yan, Hong

    2015-01-01

    The first step of homology-dependent repair of DNA double-strand breaks (DSBs) is the resection of the 5′ strand to generate 3′ ss-DNA. Of the two major nucleases responsible for resection, EXO1 has intrinsic 5′->3′ directionality, but DNA2 does not. DNA2 acts with RecQ helicases such as the Werner syndrome protein (WRN) and the heterotrimeric eukaryotic ss-DNA binding protein RPA. We have found that the N-terminus of the RPA large subunit (RPA1N) interacts with both WRN and DNA2 and is essential for stimulating WRN's 3′->5′ helicase activity and DNA2's 5′->3′ ss-DNA exonuclease activity. A mutant RPA complex that lacks RPA1N is unable to support resection in Xenopus egg extracts and human cells. Furthermore, relocating RPA1N to the middle subunit but not to the small subunit causes severe defects in stimulating DNA2 and WRN and in supporting resection. Together, these findings suggest that RPA1N and its spatial position are critical for restricting the directionality of the WRN-DNA2 resection pathway. PMID:26227969

  12. Bovine papillomavirus type 2 (BPV-2) E5 oncoprotein binds to the subunit D of the V₁-ATPase proton pump in naturally occurring urothelial tumors of the urinary bladder of cattle.

    Science.gov (United States)

    Roperto, Sante; Russo, Valeria; Borzacchiello, Giuseppe; Urraro, Chiara; Lucà, Roberta; Esposito, Iolanda; Riccardi, Marita Georgia; Raso, Cinzia; Gaspari, Marco; Ceccarelli, Dora Maria; Galasso, Rocco; Roperto, Franco

    2014-01-01

    Active infection by bovine papillomavirus type 2 (BPV-2) was documented for fifteen urinary bladder tumors in cattle. Two were diagnosed as papillary urothelial neoplasm of low malignant potential (PUNLMP), nine as papillary and four as invasive urothelial cancers. In all cancer samples, PCR analysis revealed a BPV-2-specific 503 bp DNA fragment. E5 protein, the major oncoprotein of the virus, was shown both by immunoprecipitation and immunohistochemical analysis. E5 was found to bind to the activated (phosphorylated) form of the platelet derived growth factor β receptor. PDGFβR immunoprecipitation from bladder tumor samples and from normal bladder tissue used as control revealed a protein band which was present in the pull-down from bladder cancer samples only. The protein was identified with mass spectrometry as "V₁-ATPase subunit D", a component of the central stalk of the V₁-ATPase vacuolar pump. The subunit D was confirmed in this complex by coimmunoprecipitation investigations and it was found to colocalize with the receptor. The subunit D was also shown to be overexpressed by Western blot, RT-PCR and immunofluorescence analyses. Immunoprecipitation and immunofluorescence also revealed that E5 oncoprotein was bound to the subunit D. For the first time, a tri-component complex composed of E5/PDGFβR/subunit D has been documented in vivo. Previous in vitro studies have shown that the BPV-2 E5 oncoprotein binds to the proteolipid c ring of the V₀-ATPase sector. We suggest that the E5/PDGFβR/subunit D complex may perturb proteostasis, organelle and cytosol homeostasis, which can result in altered protein degradation and in autophagic responses.

  13. The cytochrome oxidase subunit I and subunit III genes in Oenothera mitochondria are transcribed from identical promoter sequences

    Science.gov (United States)

    Hiesel, Rudolf; Schobel, Werner; Schuster, Wolfgang; Brennicke, Axel

    1987-01-01

    Two loci encoding subunit III of the cytochrome oxidase (COX) in Oenothera mitochondria have been identified from a cDNA library of mitochondrial transcripts. A 657-bp sequence block upstream from the open reading frame is also present in the two copies of the COX subunit I gene and is presumably involved in homologous sequence rearrangement. The proximal points of sequence rearrangements are located 3 bp upstream from the COX I and 1139 bp upstream from the COX III initiation codons. The 5'-termini of both COX I and COX III mRNAs have been mapped in this common sequence confining the promoter region for the Oenothera mitochondrial COX I and COX III genes to the homologous sequence block. ImagesFig. 5. PMID:15981332

  14. Persistence of the mitochondrial permeability transition in the absence of subunit c of human ATP synthase.

    Science.gov (United States)

    He, Jiuya; Ford, Holly C; Carroll, Joe; Ding, Shujing; Fearnley, Ian M; Walker, John E

    2017-03-28

    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 F 1 -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 ρ 0 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.

  15. Suppressor mutations identify amino acids in PAA-1/PR65 that facilitate regulatory RSA-1/B″ subunit targeting of PP2A to centrosomes in C. elegans.

    Science.gov (United States)

    Lange, Karen I; Heinrichs, Jeffrey; Cheung, Karen; Srayko, Martin

    2013-01-15

    Protein phosphorylation and dephosphorylation is a key mechanism for the spatial and temporal regulation of many essential developmental processes and is especially prominent during mitosis. The multi-subunit protein phosphatase 2A (PP2A) enzyme plays an important, yet poorly characterized role in dephosphorylating proteins during mitosis. PP2As are heterotrimeric complexes comprising a catalytic, structural, and regulatory subunit. Regulatory subunits are mutually exclusive and determine subcellular localization and substrate specificity of PP2A. At least 3 different classes of regulatory subunits exist (termed B, B', B″) but there is no obvious similarity in primary sequence between these classes. Therefore, it is not known how these diverse regulatory subunits interact with the same holoenzyme to facilitate specific PP2A functions in vivo. The B″ family of regulatory subunits is the least understood because these proteins lack conserved structural domains. RSA-1 (regulator of spindle assembly) is a regulatory B″ subunit required for mitotic spindle assembly in Caenorhabditis elegans. In order to address how B″ subunits interact with the PP2A core enzyme, we focused on a conditional allele, rsa-1(or598ts), and determined that this mutation specifically disrupts the protein interaction between RSA-1 and the PP2A structural subunit, PAA-1. Through genetic screening, we identified a putative interface on the PAA-1 structural subunit that interacts with a defined region of RSA-1/B″. In the context of previously published results, these data propose a mechanism of how different PP2A B-regulatory subunit families can bind the same holoenzyme in a mutually exclusive manner, to perform specific tasks in vivo.

  16. Suppressor mutations identify amino acids in PAA-1/PR65 that facilitate regulatory RSA-1/B″ subunit targeting of PP2A to centrosomes in C. elegans

    Directory of Open Access Journals (Sweden)

    Karen I. Lange

    2012-11-01

    Protein phosphorylation and dephosphorylation is a key mechanism for the spatial and temporal regulation of many essential developmental processes and is especially prominent during mitosis. The multi-subunit protein phosphatase 2A (PP2A enzyme plays an important, yet poorly characterized role in dephosphorylating proteins during mitosis. PP2As are heterotrimeric complexes comprising a catalytic, structural, and regulatory subunit. Regulatory subunits are mutually exclusive and determine subcellular localization and substrate specificity of PP2A. At least 3 different classes of regulatory subunits exist (termed B, B′, B″ but there is no obvious similarity in primary sequence between these classes. Therefore, it is not known how these diverse regulatory subunits interact with the same holoenzyme to facilitate specific PP2A functions in vivo. The B″ family of regulatory subunits is the least understood because these proteins lack conserved structural domains. RSA-1 (regulator of spindle assembly is a regulatory B″ subunit required for mitotic spindle assembly in Caenorhabditis elegans. In order to address how B″ subunits interact with the PP2A core enzyme, we focused on a conditional allele, rsa-1(or598ts, and determined that this mutation specifically disrupts the protein interaction between RSA-1 and the PP2A structural subunit, PAA-1. Through genetic screening, we identified a putative interface on the PAA-1 structural subunit that interacts with a defined region of RSA-1/B″. In the context of previously published results, these data propose a mechanism of how different PP2A B-regulatory subunit families can bind the same holoenzyme in a mutually exclusive manner, to perform specific tasks in vivo.

  17. First description of giant Archaea (Thaumarchaeota) associated with putative bacterial ectosymbionts in a sulfidic marine habitat.

    Science.gov (United States)

    Muller, Félix; Brissac, Terry; Le Bris, Nadine; Felbeck, Horst; Gros, Olivier

    2010-08-01

    Archaea may be involved in global energy cycles, and are known for their ability to interact with eukaryotic species (sponges, corals and ascidians) or as archaeal-bacterial consortia. The recently proposed phylum Thaumarchaeota may represent the deepest branching lineage in the archaeal phylogeny emerging before the divergence between Euryarchaeota and Crenarchaeota. Here we report the first characterization of two marine thaumarchaeal species from shallow waters that consist of multiple giant cells. One species is coated with sulfur-oxidizing γ-Proteobacteria. These new uncultured thaumarchaeal species are able to live in the sulfide-rich environments of a tropical mangrove swamp, either on living tissues such as roots or on various kinds of materials such as stones, sunken woods, etc. These archaea and archaea/bacteria associations have been studied using light microscopy, transmission electron microscopy and scanning electron microscopy. Species identification of archaeons and the putative bacterial symbiont have been assessed by 16S small subunit ribosomal RNA analysis. The sulfur-oxidizing ability of the bacteria has been assessed by genetic investigation on alpha-subunit of the adenosine-5'-phosphosulfate reductase/oxidase's (AprA). Species identifications have been confirmed by fluorescence in situ hybridization using specific probes designed in this study. In this article, we describe two new giant archaeal species that form the biggest archaeal filaments ever observed. One of these species is covered by a specific biofilm of sulfur-oxidizing γ-Proteobacteria. This study highlights an unexpected morphological and genetic diversity of the phylum Thaumarchaeota. © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.

  18. Complete nucleotide sequence and genome organization of Olive latent virus 3, a new putative member of the family Tymoviridae.

    Science.gov (United States)

    Alabdullah, Abdulkader; Minafra, Angelantonio; Elbeaino, Toufic; Saponari, Maria; Savino, Vito; Martelli, Giovanni P

    2010-09-01

    The complete nucleotide sequence and the genome organization were determined of a putative new member of the family Tymoviridae, tentatively named Olive latent virus 3 (OLV-3), recovered in southern Italy from a symptomless olive tree. The sequenced ssRNA genome comprises 7148 nucleotides excluding the poly(A) tail and contains four open reading frames (ORFs). ORF1 encodes a polyprotein of 221.6kDa in size, containing the conserved signatures of the methyltransferase (MTR), papain-like protease (PRO), helicase (HEL) and RNA-dependent RNA polymerase (RdRp) domains of the replication-associated proteins of positive-strand RNA viruses. ORF2 overlaps completely ORF1 and encodes a putative protein of 43.33kDa showing limited sequence similarity with the putative movement protein of Maize rayado fino virus (MRFV). ORF3 codes for a protein with predicted molecular mass of 28.46kDa, identified as the coat protein (CP), whereas ORF4 overlaps ORF3 and encodes a putative protein of 16kDa with sequence similarity to the p16 and p31 proteins of Citrus sudden death-associated virus (CSDaV) and Grapevine fleck virus (GFkV), respectively. Within the family Tymoviridae, OLV-3 genome has the closest identity level (49-52%) with members of the genus Marafivirus, from which, however, it differs because of the diverse genome organization and the presence of a single type of CP subunits. Copyright (c) 2010 Elsevier B.V. All rights reserved.

  19. The nematode homologue of Mediator complex subunit 28, F28F8.5, is a critical regulator of C. elegans development.

    Science.gov (United States)

    Kostrouchová, Markéta; Kostrouch, David; Chughtai, Ahmed A; Kaššák, Filip; Novotný, Jan P; Kostrouchová, Veronika; Benda, Aleš; Krause, Michael W; Saudek, Vladimír; Kostrouchová, Marta; Kostrouch, Zdeněk

    2017-01-01

    The evolutionarily conserved Mediator complex is a critical player in regulating transcription. Comprised of approximately two dozen proteins, the Mediator integrates diverse regulatory signals through direct protein-protein interactions that, in turn, modulate the influence of Mediator on RNA Polymerase II activity. One Mediator subunit, MED28, is known to interact with cytoplasmic structural proteins, providing a potential direct link between cytoplasmic dynamics and the control of gene transcription. Although identified in many animals and plants, MED28 is not present in yeast; no bona fide MED28 has been described previously in Caenorhabditis elegans. Here, we identify bioinformatically F28F8.5, an uncharacterized predicted protein, as the nematode homologue of MED28. As in other Metazoa, F28F8.5 has dual nuclear and cytoplasmic localization and plays critical roles in the regulation of development. F28F8.5 is a vital gene and its null mutants have severely malformed gonads and do not reproduce. F28F8.5 interacts on the protein level with the Mediator subunits MDT-6 and MDT-30. Our results indicate that F28F8.5 is an orthologue of MED28 and suggest that the potential to link cytoplasmic and nuclear events is conserved between MED28 vertebrate and nematode orthologues.

  20. The 25 kDa subunit of cleavage factor Im Is a RNA-binding protein that interacts with the poly(A polymerase in Entamoeba histolytica.

    Directory of Open Access Journals (Sweden)

    Marisol Pezet-Valdez

    Full Text Available In eukaryotes, polyadenylation of pre-mRNA 3' end is essential for mRNA export, stability and translation. Taking advantage of the knowledge of genomic sequences of Entamoeba histolytica, the protozoan responsible for human amoebiasis, we previously reported the putative polyadenylation machinery of this parasite. Here, we focused on the predicted protein that has the molecular features of the 25 kDa subunit of the Cleavage Factor Im (CFIm25 from other organisms, including the Nudix (nucleoside diphosphate linked to another moiety X domain, as well as the RNA binding domain and the PAP/PAB interacting region. The recombinant EhCFIm25 protein (rEhCFIm25 was expressed in bacteria and used to generate specific antibodies in rabbit. Subcellular localization assays showed the presence of the endogenous protein in nuclear and cytoplasmic fractions. In RNA electrophoretic mobility shift assays, rEhCFIm25 was able to form specific RNA-protein complexes with the EhPgp5 mRNA 3´ UTR used as probe. In addition, Pull-Down and LC/ESI-MS/MS tandem mass spectrometry assays evidenced that the putative EhCFIm25 was able to interact with the poly(A polymerase (EhPAP that is responsible for the synthesis of the poly(A tail in other eukaryotic cells. By Far-Western experiments, we confirmed the interaction between the putative EhCFIm25 and EhPAP in E. histolytica. Taken altogether, our results showed that the putative EhCFIm25 is a conserved RNA binding protein that interacts with the poly(A polymerase, another member of the pre-mRNA 3' end processing machinery in this protozoan parasite.

  1. Application of a Colorimetric Assay to Identify Putative Ribofuranosylaminobenzene 5'-Phosphate Synthase Genes Expressed with Activity in Escherichia coli

    OpenAIRE

    Bechard, Matthew E.; Chhatwal, Sonya; Garcia, Rosemarie E.; Rasche, Madeline E.

    2003-01-01

    Tetrahydromethanopterin (H4MPT) is a tetrahydrofolate analog originally discovered in methanogenic archaea, but later found in other archaea and bacteria. The extent to which H4MPT occurs among living organisms is unknown. The key enzyme which distinguishes the biosynthetic pathways of H4MPT and tetrahydrofolate is ribofuranosylaminobenzene 5'-phosphate synthase (RFAP synthase). Given the importance of RFAP synthase in H4MPT biosynthesis, the identification of putative RFAP synthase genes and...

  2. The calcium channel β2 (CACNB2 subunit repertoire in teleosts

    Directory of Open Access Journals (Sweden)

    Mueller Rachel

    2008-04-01

    Full Text Available Abstract Background Cardiomyocyte contraction is initiated by influx of extracellular calcium through voltage-gated calcium channels. These oligomeric channels utilize auxiliary β subunits to chaperone the pore-forming α subunit to the plasma membrane, and to modulate channel electrophysiology 1. Several β subunit family members are detected by RT-PCR in the embryonic heart. Null mutations in mouse β2, but not in the other three β family members, are embryonic lethal at E10.5 due to defects in cardiac contractility 2. However, a drawback of the mouse model is that embryonic heart rhythm is difficult to study in live embryos due to their intra-uterine development. Moreover, phenotypes may be obscured by secondary effects of hypoxia. As a first step towards developing a model for contributions of β subunits to the onset of embryonic heart rhythm, we characterized the structure and expression of β2 subunits in zebrafish and other teleosts. Results Cloning of two zebrafish β2 subunit genes (β2.1 and β2.2 indicated they are membrane-associated guanylate kinase (MAGUK-family genes. Zebrafish β2 genes show high conservation with mammals within the SH3 and guanylate kinase domains that comprise the "core" of MAGUK proteins, but β2.2 is much more divergent in sequence than β2.1. Alternative splicing occurs at the N-terminus and within the internal HOOK domain. In both β2 genes, alternative short ATG-containing first exons are separated by some of the largest introns in the genome, suggesting that individual transcript variants could be subject to independent cis-regulatory control. In the Tetraodon nigrovidis and Fugu rubripes genomes, we identified single β2 subunit gene loci. Comparative analysis of the teleost and human β2 loci indicates that the short 5' exon sequences are highly conserved. A subset of 5' exons appear to be unique to teleost genomes, while others are shared with mammals. Alternative splicing is temporally and

  3. The nematode homologue of Mediator complex subunit 28, F28F8.5, is a critical regulator of C. elegans development

    Directory of Open Access Journals (Sweden)

    Markéta Kostrouchová

    2017-06-01

    Full Text Available The evolutionarily conserved Mediator complex is a critical player in regulating transcription. Comprised of approximately two dozen proteins, the Mediator integrates diverse regulatory signals through direct protein-protein interactions that, in turn, modulate the influence of Mediator on RNA Polymerase II activity. One Mediator subunit, MED28, is known to interact with cytoplasmic structural proteins, providing a potential direct link between cytoplasmic dynamics and the control of gene transcription. Although identified in many animals and plants, MED28 is not present in yeast; no bona fide MED28 has been described previously in Caenorhabditis elegans. Here, we identify bioinformatically F28F8.5, an uncharacterized predicted protein, as the nematode homologue of MED28. As in other Metazoa, F28F8.5 has dual nuclear and cytoplasmic localization and plays critical roles in the regulation of development. F28F8.5 is a vital gene and its null mutants have severely malformed gonads and do not reproduce. F28F8.5 interacts on the protein level with the Mediator subunits MDT-6 and MDT-30. Our results indicate that F28F8.5 is an orthologue of MED28 and suggest that the potential to link cytoplasmic and nuclear events is conserved between MED28 vertebrate and nematode orthologues.

  4. The COP9 signalosome interacts with SCF UFO and participates in Arabidopsis flower development.

    Science.gov (United States)

    Wang, Xiping; Feng, Suhua; Nakayama, Naomi; Crosby, W L; Irish, Vivian; Deng, Xing Wang; Wei, Ning

    2003-05-01

    The COP9 signalosome (CSN) is involved in multiple developmental processes. It interacts with SCF ubiquitin ligases and deconjugates Nedd8/Rub1 from cullins (deneddylation). CSN is highly expressed in Arabidopsis floral tissues. To investigate the role of CSN in flower development, we examined the expression pattern of CSN in developing flowers. We report here that two csn1 partially deficient Arabidopsis strains exhibit aberrant development of floral organs, decline of APETALA3 (AP3) expression, and low fertility in addition to defects in shoot and inflorescence meristems. We show that UNUSUAL FLORAL ORGANS (UFO) forms a SCF(UFO) complex, which is associated with CSN in vivo. Genetic interaction analysis indicates that CSN is necessary for the gain-of-function activity of the F-box protein UFO in AP3 activation and in floral organ transformation. Compared with the previously reported csn5 antisense and csn1 null mutants, partial deficiency of CSN1 causes a reduction in the level of CUL1 in the mutant flowers without an obvious defect in CUL1 deneddylation. We conclude that CSN is an essential regulator of Arabidopsis flower development and suggest that CSN regulates Arabidopsis flower development in part by modulating SCF(UFO)-mediated AP3 activation.

  5. Acute Inflammation Increases Pituitary and Hypothalamic Glycoprotein Hormone Subunit B5 mRNA Expression in Association with Decreased Thyrotrophin Receptor mRNA Expression in Mice

    NARCIS (Netherlands)

    van Zeijl, C. J. J.; Surovtseva, O. V.; Wiersinga, W. M.; Fliers, E.; Boelen, A.

    2011-01-01

    The biological function of thyrostimulin, consisting of the GPA2 and GPB5 subunit, is currently poorly understood. The recent observation that pro-inflammatory cytokines up-regulate the transcription of GPB5 in vitro suggested a role for thyrostimulin in the nonthyroidal illness syndrome, a state of

  6. Functional Characterization of Four Putative δ1-Pyrroline-5-Carboxylate Reductases from Bacillus subtilis

    Energy Technology Data Exchange (ETDEWEB)

    Forlani, Giuseppe; Nocek, Boguslaw; Chakravarthy, Srinivas; Joachimiak, Andrzej

    2017-08-02

    In most living organisms, the amino acid proline is synthesized starting from both glutamate and ornithine. In prokaryotes, in the absence of an ornithine cyclodeaminase that has been identified to date only in a small number of soil and plant bacteria, these pathways share the last step, the reduction of δ1-pyrroline-5-carboxylate (P5C) catalyzed by P5C reductase (EC 1.5.1.2). In several species, multiple forms of P5C reductase have been reported, possibly reflecting the dual function of proline. Aside from its common role as a building block of proteins, proline is indeed also involved in the cellular response to osmotic and oxidative stress conditions. Genome analysis of Bacillus subtilis identifies the presence of four genes (ProH, ProI, ProG, and ComER) that, based on bioinformatic and phylogenic studies, were defined as respectively coding a putative P5C reductase. Here we describe the cloning, heterologous expression, functional analysis and small-angle X-ray scattering studies of the four affinity-purified proteins. Results showed that two of them, namely ProI and ComER, lost their catalytic efficiency or underwent subfunctionalization. In the case of ComER, this could be likely explained by the loss of the ability to form a dimer, which has been previously shown to be an essential structural feature of the catalytically active P5C reductase. The properties of the two active enzymes are consistent with a constitutive role for ProG, and suggest that ProH expression may be beneficial to satisfy an increased need for proline.

  7. Functional Characterization of Four Putative δ1-Pyrroline-5-Carboxylate Reductases from Bacillus subtilis

    Energy Technology Data Exchange (ETDEWEB)

    Forlani, Giuseppe; Nocek, Boguslaw; Chakravarthy, Srinivas; Joachimiak, Andrzej

    2017-08-02

    In most living organisms, the amino acid proline is synthesized starting from both glutamate and ornithine. In prokaryotes, in the absence of an ornithine cyclodeaminase that has been identified to date only in a small number of soil and plant bacteria, these pathways share the last step, the reduction of delta(1)-pyrroline-5-carboxylate (P5C) catalyzed by P5C reductase (EC 1.5.1.2). In several species, multiple forms of P5C reductase have been reported, possibly reflecting the dual function of proline. Aside from its common role as a building block of proteins, proline is indeed also involved in the cellular response to osmotic and oxidative stress conditions. Genome analysis of Bacillus subtilis identifies the presence of four genes (ProH, ProI, ProG, and ComER) that, based on bioinformatic and phylogenic studies, were defined as respectively coding a putative P5C reductase. Here we describe the cloning, heterologous expression, functional analysis and small-angle X-ray scattering studies of the four affinity-purified proteins. Results showed that two of them, namely ProI and ComER, lost their catalytic efficiency or underwent subfunctionalization. In the case of ComER, this could be likely explained by the loss of the ability to form a dimer, which has been previously shown to be an essential structural feature of the catalytically active P5C reductase. The properties of the two active enzymes are consistent with a constitutive role for ProG, and suggest that ProH expression may be beneficial to satisfy an increased need for proline.

  8. Functional Characterization of Four Putative δ1-Pyrroline-5-Carboxylate Reductases from Bacillus subtilis

    Directory of Open Access Journals (Sweden)

    Giuseppe Forlani

    2017-08-01

    Full Text Available In most living organisms, the amino acid proline is synthesized starting from both glutamate and ornithine. In prokaryotes, in the absence of an ornithine cyclodeaminase that has been identified to date only in a small number of soil and plant bacteria, these pathways share the last step, the reduction of δ1-pyrroline-5-carboxylate (P5C catalyzed by P5C reductase (EC 1.5.1.2. In several species, multiple forms of P5C reductase have been reported, possibly reflecting the dual function of proline. Aside from its common role as a building block of proteins, proline is indeed also involved in the cellular response to osmotic and oxidative stress conditions. Genome analysis of Bacillus subtilis identifies the presence of four genes (ProH, ProI, ProG, and ComER that, based on bioinformatic and phylogenic studies, were defined as respectively coding a putative P5C reductase. Here we describe the cloning, heterologous expression, functional analysis and small-angle X-ray scattering studies of the four affinity-purified proteins. Results showed that two of them, namely ProI and ComER, lost their catalytic efficiency or underwent subfunctionalization. In the case of ComER, this could be likely explained by the loss of the ability to form a dimer, which has been previously shown to be an essential structural feature of the catalytically active P5C reductase. The properties of the two active enzymes are consistent with a constitutive role for ProG, and suggest that ProH expression may be beneficial to satisfy an increased need for proline.

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

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    Gisela Pöll

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

  10. Three human alcohol dehydrogenase subunits: cDNA structure and molecular and evolutionary divergence

    International Nuclear Information System (INIS)

    Ikuta, T.; Szeto, S.; Yoshida, A.

    1986-01-01

    Class I human alcohol dehydrogenase (ADH; alcohol:NAD + oxidoreductase, EC 1.1.1.1) consists of several homo- and heterodimers of α, β, and γ subunits that are governed by the ADH1, ADH2, and ADH3 loci. The authors previously cloned a full length of cDNA for the β subunit, and the complete sequence of 374 amino acid residues was established. cDNAs for the α and γ subunits were cloned and characterized. A human liver cDNA library, constructed in phage λgt11, was screened by using a synthetic oligonucleotide probe that was matched to the γ but not to the β sequence. Clone pUCADHγ21 and clone pUCADHα15L differed from β cDNA with respect to restriction sites and hybridization with the nucleotide probe. Clone pUCADHγ21 contained an insertion of 1.5 kilobase pairs (kbp) and encodes 374 amino acid residues compatible with the reported amino acid sequence of the γ subunit. Clone pUCADHα15L contained an insertion of 2.4 kbp and included nucleotide sequences that encode 374 amino acid residues for another subunit, the γ subunit. In addition, this clone contained the sequences that encode the COOH-terminal part of the β subunit at its extended 5' region. The amino acid sequences and coding regions of the cDNAs of the three subunits are very similar. A high degree of resemblance is observed also in their 3' noncoding regions. However, distinctive differences exist in the vicinity of the Zn-binding cysteine residue at position 46. Based on the cDNA sequences and the deduced amino acid sequences of the three subunits, their structural and evolutionary relationships are discussed

  11. Effect of glutenin subunits on the baking quality of Brazilian wheat genotypes

    OpenAIRE

    Costa, Mariana Souza; Scholz, Maria Brígida dos Santos; Miranda, Martha Zavariz; Franco, Célia Maria Landi

    2017-01-01

    ABSTRACT This study aimed to evaluate the effect of the high and low molecular weight glutenin subunits on the grain traits of sixteen Brazilian wheat genotypes. Grain hardness index, milling traits, physicochemical and rheological properties of the flour, and specific volume and firmness of the bread were evaluated. Physicochemical properties of the flour were not influenced by glutenin subunits. Genotypes with subunits at the Glu-B1 (17+18 or 7+8), Glu-D1 (5+10), and Glu-A3 (b) were associa...

  12. Cloning, sequence determination, and expression of the genes encoding the subunits of the nickel-containing 8-hydroxy-5-deazaflavin reducing hydrogenase from Methanobacterium thermoautotrophicum ΔH

    International Nuclear Information System (INIS)

    Alex, L.A.; Reeve, J.N.; Orme-Johnson, W.H.; Walsh, C.T.

    1990-01-01

    The genes frhA (1,217 bp), frhB (845 bp), and frhG (710 bp) encoding the three known subunits, α, β, and γ, of the 8-hydroxy-5-deazaflavin (F 420 ) reducing hydrogenase (FRH) from the thermophilic methanogen Methanobacterium thermoautotrophicum ΔH have been cloned, sequenced, and shown to be tightly linked, indicative of a single transcriptional unit. The DNA sequence contains a fourth open reading frame, designated frhD (476 bp), encoding a polypeptide (δ) that does not copurify with the active enzyme. Expression of the frh gene cluster in Escherichia coli shows that four polypeptides are synthesized. When analyzed by SDS-PAGE, the proteins migrate with mobilities consistent with their calculated molecular weights. In order to understand the mechanism of H 2 oxidation by this enzyme, localization of redox cofactors (Ni, Fe/S, FAD) to specific subunits and information on their structure is needed. This has been hindered due to the refractory nature of the enzyme to denaturation methods needed in order to obtain individual subunits with cofactors intact. In this paper they discuss the possible localization of the redox cofactors as implicated from the DNA-derived protein sequences of the subunits. The amino acid sequences of the subunits of the FRH are compared with those of other Ni-containing hydrogenases, including the methyl viologen reducing hydrogenase (MVH) of M. thermoautotrophicum ΔH

  13. Association between the GABA(A) receptor alpha5 subunit gene locus (GABRA5) and bipolar affective disorder.

    Science.gov (United States)

    Papadimitriou, G N; Dikeos, D G; Karadima, G; Avramopoulos, D; Daskalopoulou, E G; Vassilopoulos, D; Stefanis, C N

    1998-02-07

    Genetic factors seem to play an important role in the pathogenesis of affective disorder. The candidate gene strategies are being used, among others, to identify the genes conferring vulnerability to the disease. The genes coding for the receptors of gamma-aminobutyric acid (GABA) have been proposed as candidates for affective disorder, since the GABA neurotransmitter system has been implicated in the pathogenesis of the illness. We examined the possible genetic association between the GABA(A) receptor alpha5 subunit gene locus (GABRA5) on chromosome 15 and affective disorder, in 48 bipolar patients (BP), 40 unipolar patients (UP), and 50 healthy individuals, age- and sex-matched to the patients. All patients and controls were unrelated Greeks. Diagnoses were made after direct interviews according to the DSM-IV and ICD-10 criteria. For the genotyping, a dinucleotide (CA) repeat marker was used. The polymerase chain reaction (PCR) products found were nine alleles with lengths between 272 and 290 base pairs (bp). The distribution of allelic frequencies of the GABRA5 locus differed significantly between BP patients and controls with the 282-bp allele found to be associated with BP affective disorder, while no such difference was observed between the groups of UP patients and controls nor between the two patient groups. The presence or absence of the 282-bp allele in the genotype of BP patients was not shown to influence the age of onset and the overall clinical severity, but was found to be associated with a preponderance of manic over depressive episodes in the course of the illness.

  14. Differences in the roles of a glutamine amidotransferase subunit of pyridoxal 5'-phosphate synthase between Bacillus circulans and Bacillus subtilis.

    Science.gov (United States)

    Itagaki, Shiori; Haga, Minami; Oikawa, Yuji; Sakoda, Ayaka; Ohke, Yoshie; Sawada, Hiroshi; Eguchi, Tadashi; Tamegai, Hideyuki

    2013-01-01

    BtrC2 of the butirosin producer Bacillus circulans is a non-catalytic subunit of 2-deoxy-scyllo-inosose (DOI) synthase that is involved in butirosin biosynthesis, and also a homolog of glutamine amidotransferase subunit (PdxT) of pyridoxal 5'-phosphate (PLP) synthase of Bacillus subtilis. BtrC2 has been found to have functions in B. circulans both in primary and secondary metabolism. In this study, we investigated the properties of PdxT of B. subtilis in order to determine whether the property of enzyme stabilization is universal among PdxT homologs. Complementation with PdxT in the btrC2 disruptant of B. circulans restored the growth and short-term production of antibiotics, but long-term production of antibiotics cannot be restored. Additionally, PdxT did not bind physically with or stabilize BtrC. Our results indicate that the function of BtrC2 in secondary metabolism is specific properties, not universal among PdxT homologs.

  15. Calibration of individual dosemeters by using external beams of photon radiation. A nationwide survey among Personal Dosimetry Services, authorized by CSN

    International Nuclear Information System (INIS)

    Brosed, A.; Ginjaume, M.

    1995-12-01

    A nationwide survey in 1995 among Personal Dosimetry Services, authorized by the Spanish Nuclear Safety Council (CSN), has led the Spanish Dosimetry Laboratories to review and update the dosimetric conversion coefficients and correction factors in use in Spain since 1987. The recommendations of the ICRU Report 47(1992) are discussed and adopted. In addition differences in back-scattering form IRCU tissue and PMMA phantoms are analysed. Analytical functions used to calculate conversion coefficients and back-scattering correction factors due to the use of different phantom materials are presented, together with the adopted final values. Firstly, the above mentioned parameters are applied to ISO narrow spectra series, which are discribed in this report. Secondly, differences between 1995 and 1987 values are also shown. (Author)

  16. Results of the Interlaboratory Exercise CSN/CIEMAT-02 Among Environmental Radioactivity Laboratories (Sea Fish)

    International Nuclear Information System (INIS)

    Romero Gonzalez, M.L.

    2003-01-01

    The document describes the outcome of the CSN/CIEMAT-02 interlaboratory test comparison among environmental radioactivity laboratories. The exercise was organised according to the ISO-43 and the ISO/IUPAC/AOAC Harmonized Protocol for the proficiency testing of analytical laboratories. The test sample was a reference materials provided by the IAEA-MEL (IAE Marine Environmental Laboratory, Monaco), a sea fish containing environmental levels of U-238, U-234, K-40, Pb-210, Ra-226, Sr-90, Cs-137, Co-60, Pu-(239+240), Am-241 and Tc-99. The results of the exercise were computed for 32 participating laboratories, and their analytical performance was assessed using the z-score approach. A raised percentage of satisfactory laboratory performance has been obtained for all the analysis, being the best performance in gamma measurements. The laboratories have made an effort to calculate the combined uncertainty of the radiochemical determinations. Most of the laboratories have demonstrated its competence in performing the study analysis and also the adequate measuring capability of their detection equipment even in conditions close to detection limits. The study has shown the capacity of participant laboratories to perform radioactive determinations in environmental sea fish samples with satisfactory quality levels. (Author) 6 refs

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1996-02-01

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

  18. The Not5 subunit of the ccr4-not complex connects transcription and translation.

    Directory of Open Access Journals (Sweden)

    Zoltan Villanyi

    2014-10-01

    Full Text Available Recent studies have suggested that a sub-complex of RNA polymerase II composed of Rpb4 and Rpb7 couples the nuclear and cytoplasmic stages of gene expression by associating with newly made mRNAs in the nucleus, and contributing to their translation and degradation in the cytoplasm. Here we show by yeast two hybrid and co-immunoprecipitation experiments, followed by ribosome fractionation and fluorescent microscopy, that a subunit of the Ccr4-Not complex, Not5, is essential in the nucleus for the cytoplasmic functions of Rpb4. Not5 interacts with Rpb4; it is required for the presence of Rpb4 in polysomes, for interaction of Rpb4 with the translation initiation factor eIF3 and for association of Rpb4 with mRNAs. We find that Rpb7 presence in the cytoplasm and polysomes is much less significant than that of Rpb4, and that it does not depend upon Not5. Hence Not5-dependence unlinks the cytoplasmic functions of Rpb4 and Rpb7. We additionally determine with RNA immunoprecipitation and native gel analysis that Not5 is needed in the cytoplasm for the co-translational assembly of RNA polymerase II. This stems from the importance of Not5 for the association of the R2TP Hsp90 co-chaperone with polysomes translating RPB1 mRNA to protect newly synthesized Rpb1 from aggregation. Hence taken together our results show that Not5 interconnects translation and transcription.

  19. Effect of high and low molecular weight glutenin subunits, and subunits of gliadin on physicochemical parameters of different wheat genotypes

    Directory of Open Access Journals (Sweden)

    Mariana Souza Costa

    2013-02-01

    Full Text Available Identification of functional properties of wheat flour by specific tests allows genotypes with appropriate characteristics to be selected for specific industrial uses. The objective of wheat breeding programs is to improve the quality of germplasm bank in order to be able to develop wheat with suitable gluten strength and extensibility for bread making. The aim of this study was to evaluate 16 wheat genotypes by correlating both glutenin subunits of high and low molecular weight and gliadin subunits with the physicochemical characteristics of the grain. Protein content, sedimentation volume, sedimentation index, and falling number values were analyzed after the grains were milled. Hectoliter weight and mass of 1000 seeds were also determined. The glutenin and gliadin subunits were separated using polyacrylamide gel in the presence of sodium dodecyl sulfate. The data were evaluated using variance analysis, Pearson's correlation, principal component analysis, and cluster analysis. The IPR 85, IPR Catuara TM, T 091015, and T 091069 genotypes stood out from the others, which indicate their possibly superior grain quality with higher sedimentation volume, higher sedimentation index, and higher mass of 1000 seeds; these genotypes possessed the subunits 1 (Glu-A1, 5 + 10 (Glu-D1, c (Glu-A3, and b (Glu-B3, with exception of T 091069 genotype that possessed the g allele instead of b in the Glu-B3.

  20. PCR amplification and sequences of cDNA clones for the small and large subunits of ADP-glucose pyrophosphorylase from barley tissues.

    Science.gov (United States)

    Villand, P; Aalen, R; Olsen, O A; Lüthi, E; Lönneborg, A; Kleczkowski, L A

    1992-06-01

    Several cDNAs encoding the small and large subunit of ADP-glucose pyrophosphorylase (AGP) were isolated from total RNA of the starchy endosperm, roots and leaves of barley by polymerase chain reaction (PCR). Sets of degenerate oligonucleotide primers, based on previously published conserved amino acid sequences of plant AGP, were used for synthesis and amplification of the cDNAs. For either the endosperm, roots and leaves, the restriction analysis of PCR products (ca. 550 nucleotides each) has revealed heterogeneity, suggesting presence of three transcripts for AGP in the endosperm and roots, and up to two AGP transcripts in the leaf tissue. Based on the derived amino acid sequences, two clones from the endosperm, beps and bepl, were identified as coding for the small and large subunit of AGP, respectively, while a leaf transcript (blpl) encoded the putative large subunit of AGP. There was about 50% identity between the endosperm clones, and both of them were about 60% identical to the leaf cDNA. Northern blot analysis has indicated that beps and bepl are expressed in both the endosperm and roots, while blpl is detectable only in leaves. Application of the PCR technique in studies on gene structure and gene expression of plant AGP is discussed.

  1. Distribution of AMPA-type glutamate receptor subunits in the chick visual system

    Directory of Open Access Journals (Sweden)

    Pires R.S.

    1997-01-01

    Full Text Available Several glutamate receptor (GluR subunits have been characterized during the past few years. In the present study, subunit-specific antisera were used to determine the distribution of the AMPA-type glutamate receptor subunits GluR1-4 in retinorecipient areas of the chick brain. Six white leghorn chicks (Gallus gallus, 7-15 days old, unknown sex were deeply anesthetized and perfused with 4% buffered paraformaldehyde and brain sections were stained using immunoperoxidase techniques. The AMPA-type glutamate receptor subunits GluR1, GluR2/3 and GluR4 were present in several retinorecipient areas, with varying degrees of colocalization. For example, perikarya in layers 2, 3, and 5 of the optic tectum contained GluR1, whereas GluR2/3 subunits appeared mainly in neurons of layer 13. The GluR4 subunit was only detected in a few cells of the tectal layer 13. GluR1 and GluR2/3 were observed in neurons of the nucleus geniculatus lateralis ventralis, whereas GluR4 was only present in its neuropil. Somata in the accessory optic nucleus appeared to contain GluR2/3 and GluR4, whereas GluR1 was the dominant subunit in the neuropil of this nucleus. These results suggest that different subpopulations of visual neurons might express different combinations of AMPA-type GluR subunits, which in turn might generate different synaptic responses to glutamate derived from retinal ganglion cell axons

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

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    Christoph Straub

    2016-07-01

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

  3. Two distinctive β subunits are separately involved in two binding sites of imidacloprid with different affinities in Locusta migratoria manilensis.

    Science.gov (United States)

    Bao, Haibo; Liu, Yang; Zhang, Yixi; Liu, Zewen

    2017-08-01

    Due to great diversity of nicotinic acetylcholine receptor (nAChR) subtypes in insects, one β subunit may be contained in numerous nAChR subtypes. In the locust Locusta migratoria, a model insect species with agricultural importance, the third β subunits (Locβ3) was identified in this study, which reveals at least three β subunits in this insect species. Imidacloprid was found to bind nAChRs in L. migratoria central nervous system at two sites with different affinities, with K d values of 0.16 and 10.31nM. The specific antisera (L1-1, L2-1 and L3-1) were raised against fusion proteins at the large cytoplasmic loop of Locβ1, Locβ2 and Locβ3 respectively. Specific immunodepletion of Locβ1 with antiserum L1-1 resulted in the selective loss of the low affinity binding site for imidacloprid, whereas the immunodepletion of Locβ3 with L3-1 caused the selective loss of the high affinity site. Dual immunodepletion with L1-1 and L3-1 could completely abolish imidacloprid binding. In contrast, the immunodepletion of Locβ2 had no significant effect on the specific [ 3 H]imidacloprid binding. Taken together, these data indicated that Locβ1 and Locβ3 were respectively contained in the low- and high-affinity binding sites for imidacloprid in L. migratoria, which is different to the previous finding in Nilaparvata lugens that Nlβ1 was in two binding sites for imidacloprid. The involvement of two β subunits separately in two binding sites may decrease the risk of imidacloprid resistance due to putative point mutations in β subunits in L. migratoria. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Differential expression of AMPA-type glutamate receptor subunits during development of the chick optic tectum

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    Batista S.S.

    2002-01-01

    Full Text Available Glutamate receptors have been often associated with developmental processes. We used immunohistochemical techniques to evaluate the expression of the AMPA-type glutamate receptor (GluR subunits in the chick optic tectum (TeO. Chick embryos from the 5th through the 20th embryonic day (E5-E20 and one-day-old (P1 chicks were used. The three types of immunoreactivity evaluated (GluR1, GluR2/3, and GluR4 had different temporal and spatial expression patterns in the several layers of the TeO. The GluR1 subunit first appeared as moderate staining on E7 and then increased on E9. The mature GluR1 pattern included intense staining only in layer 5 of the TeO. The GluR2/3 subunits presented low expression on E5, which became intense on E7. The staining for GluR2/3 changed to very intense on E14 in tectal layer 13. Staining of layer 13 neurons is the most prominent feature of GluR immunoreactivity in the adult TeO. The GluR4 subunit generally presented the lowest expression starting on E7, which was similar to the adult pattern. Some instances of transient expression of GluR subunits were observed in specific cell populations from E9 through E20. These results demonstrate a differential expression of the GluR subunits in the embryonic TeO, adding information about their possible functions in the developmental processes of the visual system.

  5. Novel subunit structure observed for noncooperative hemoglobin from Urechis caupo.

    Science.gov (United States)

    Kolatkar, P R; Meador, W E; Stanfield, R L; Hackert, M L

    1988-03-05

    Tetrameric hemoglobin from the "fat innkeeper" worm Urechis caupo possesses a novel subunit arrangement having an "inside out" quaternary structure in that the G/H helices are located on the outer surface of the tetramer. A 5-A resolution crystal structure reveals that although the individual subunits are beta-like, having a distinct D helix and the general myoglobin fold, the subunit contacts are very different from those previously observed for hemoglobins. Furthermore, the hemoglobin from U. caupo is also quite different from the unusual hemoglobin tetramer from clam which also has its G/H helices on the outer surface but with the hemes in close proximity through E-F helical contacts (Royer, W. E., Jr., Love, W. E., and Fenderson, F. F. (1985) Nature 316, 277-280).

  6. Putative Structural and Functional Coupling of the Mitochondrial BKCa Channel to the Respiratory Chain.

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    Piotr Bednarczyk

    Full Text Available Potassium channels have been found in the inner mitochondrial membranes of various cells. These channels regulate the mitochondrial membrane potential, the matrix volume and respiration. The activation of these channels is cytoprotective. In our study, the single-channel activity of a large-conductance Ca(2+-regulated potassium channel (mitoBKCa channel was measured by patch-clamping mitoplasts isolated from the human astrocytoma (glioblastoma U-87 MG cell line. A potassium-selective current was recorded with a mean conductance of 290 pS in symmetrical 150 mM KCl solution. The channel was activated by Ca(2+ at micromolar concentrations and by the potassium channel opener NS1619. The channel was inhibited by paxilline and iberiotoxin, known inhibitors of BKCa channels. Western blot analysis, immuno-gold electron microscopy, high-resolution immunofluorescence assays and polymerase chain reaction demonstrated the presence of the BKCa channel β4 subunit in the inner mitochondrial membrane of the human astrocytoma cells. We showed that substrates of the respiratory chain, such as NADH, succinate, and glutamate/malate, decrease the activity of the channel at positive voltages. This effect was abolished by rotenone, antimycin and cyanide, inhibitors of the respiratory chain. The putative interaction of the β4 subunit of mitoBKCa with cytochrome c oxidase was demonstrated using blue native electrophoresis. Our findings indicate possible structural and functional coupling of the mitoBKCa channel with the mitochondrial respiratory chain in human astrocytoma U-87 MG cells.

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

    Science.gov (United States)

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

    2018-03-01

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

  8. Monoclonal antibodies to molluskan hemocyanin from Concholepas concholepas demonstrate common and specific epitopes among subunits.

    Science.gov (United States)

    Oliva, Harold; Moltedo, Bruno; De Ioannes, Pablo; Faunes, Fernando; De Ioannes, Alfredo E; Becker, María Inés

    2002-10-01

    We studied the reactivity of mouse monoclonal antibodies (MAbs) against the hemocyanin from the Chilean marine gastropod Concholepas concholepas (CCH). This protein has been successfully used as a carrier to produce antibodies to haptens and peptides. All MAbs (13) belonging to IgG subclass exhibit dissociation constants (K(d)) from 1 x 10(-7) M to 1 x 10(-9) M. MAbs were characterized by enzyme-linked immunosorbant assay (ELISA) using CCH treated with different procedures, including dissociation into CCH-A and CCH-B subunits, Western blot, enzymatic digestion, chemical deglycosylation, and thermal denaturation. MAbs were classified into three categories, according to subunit specificity by ELISA. The epitope distribution shows that CCH subunits display common epitopes (group I, 5 MAbs, 1H5, 2A8, 3A5, 3B3, and 3E3), as well as specific epitopes for CCH-A subunits (group II, 3 MAbs, 1B8, 4D8, and 8E5) and for CCH-B subunits (group III, 5 MAbs, 1A4, 1E4, 2H10, 3B7, and 7B4). The results can be summarized as follows: (1). six antibodies react with thermal denatured CCH, suggesting that they recognize linear epitopes, whereas seven recognize conformational epitopes; (2). oxidation of carbohydrate moieties does not affect the binding of the MAbs; (3). enzymatic digestion of CCH decreases the reactivity of all antibodies irrespective of the protease used (elastase or trypsin); (4). bringing together the above data, in addition to epitopic complementarity analysis, we identified 12 different epitopes on the CCH molecule recognized by these MAbs. The anti-CCH MAbs presented here can be useful tools to understand the subunit organization of the CCH and its complex structure, which can explain its immunogenic and immunostimulating properties in mammals.

  9. Application of a Colorimetric Assay to Identify Putative Ribofuranosylaminobenzene 5'-Phosphate Synthase Genes Expressed with Activity in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Bechard Matthew E.

    2003-01-01

    Full Text Available Tetrahydromethanopterin (H4MPT is a tetrahydrofolate analog originally discovered in methanogenic archaea, but later found in other archaea and bacteria. The extent to which H4MPT occurs among living organisms is unknown. The key enzyme which distinguishes the biosynthetic pathways of H4MPT and tetrahydrofolate is ribofuranosylaminobenzene 5'-phosphate synthase (RFAP synthase. Given the importance of RFAP synthase in H4MPT biosynthesis, the identification of putative RFAP synthase genes and measurement of RFAP synthase activity would provide an indication of the presence of H4MPT in untested microorganisms. Investigation of putative archaeal RFAP synthase genes has been hampered by the tendency of the resulting proteins to form inactive inclusion bodies in Escherichia coli. The current work describes a colorimetric assay for measuring RFAP synthase activity, and two modified procedures for expressing recombinant RFAP synthase genes to produce soluble, active enzyme. By lowering the incubation temperature during expression, RFAP synthase from Archaeoglobus fulgidus was produced in E. coli and purified to homogeneity. The production of active RFAP synthase from Methanothermobacter thermautotrophicus was achieved by coexpression of the gene MTH0830 with a molecular chaperone. This is the first direct biochemical identification of a methanogen gene that codes for an active RFAP synthase.

  10. Application of a Colorimetric Assay to Identify Putative Ribofuranosylaminobenzene 5'-Phosphate Synthase Genes Expressed with Activity in Escherichia coli.

    Science.gov (United States)

    Bechard, Matthew E.; Chhatwal, Sonya; Garcia, Rosemarie E.; Rasche, Madeline E.

    2003-01-01

    Tetrahydromethanopterin (H(4)MPT) is a tetrahydrofolate analog originally discovered in methanogenic archaea, but later found in other archaea and bacteria. The extent to which H(4)MPT occurs among living organisms is unknown. The key enzyme which distinguishes the biosynthetic pathways of H(4)MPT and tetrahydrofolate is ribofuranosylaminobenzene 5'-phosphate synthase (RFAP synthase). Given the importance of RFAP synthase in H(4)MPT biosynthesis, the identification of putative RFAP synthase genes and measurement of RFAP synthase activity would provide an indication of the presence of H(4)MPT in untested microorganisms. Investigation of putative archaeal RFAP synthase genes has been hampered by the tendency of the resulting proteins to form inactive inclusion bodies in Escherichia coli. The current work describes a colorimetric assay for measuring RFAP synthase activity, and two modified procedures for expressing recombinant RFAP synthase genes to produce soluble, active enzyme. By lowering the incubation temperature during expression, RFAP synthase from Archaeoglobus fulgidus was produced in E. coli and purified to homogeneity. The production of active RFAP synthase from Methanothermobacter thermautotrophicus was achieved by coexpression of the gene MTH0830 with a molecular chaperone. This is the first direct biochemical identification of a methanogen gene that codes for an active RFAP synthase.

  11. Role of regulatory subunits and protein kinase inhibitor (PKI) in determining nuclear localization and activity of the catalytic subunit of protein kinase A.

    Science.gov (United States)

    Wiley, J C; Wailes, L A; Idzerda, R L; McKnight, G S

    1999-03-05

    Regulation of protein kinase A by subcellular localization may be critical to target catalytic subunits to specific substrates. We employed epitope-tagged catalytic subunit to correlate subcellular localization and gene-inducing activity in the presence of regulatory subunit or protein kinase inhibitor (PKI). Transiently expressed catalytic subunit distributed throughout the cell and induced gene expression. Co-expression of regulatory subunit or PKI blocked gene induction and prevented nuclear accumulation. A mutant PKI lacking the nuclear export signal blocked gene induction but not nuclear accumulation, demonstrating that nuclear export is not essential to inhibit gene induction. When the catalytic subunit was targeted to the nucleus with a nuclear localization signal, it was not sequestered in the cytoplasm by regulatory subunit, although its activity was completely inhibited. PKI redistributed the nuclear catalytic subunit to the cytoplasm and blocked gene induction, demonstrating that the nuclear export signal of PKI can override a strong nuclear localization signal. With increasing PKI, the export process appeared to saturate, resulting in the return of catalytic subunit to the nucleus. These results demonstrate that both the regulatory subunit and PKI are able to completely inhibit the gene-inducing activity of the catalytic subunit even when the catalytic subunit is forced to concentrate in the nuclear compartment.

  12. Acetylcholine Receptor: Complex of Homologous Subunits

    Science.gov (United States)

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

    1980-06-01

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

  13. Characterization of cyclin-dependent kinases and Cdc2/Cdc28 kinase subunits in Trichomonas vaginalis.

    Science.gov (United States)

    Amador, Erick; López-Pacheco, Karla; Morales, Nataly; Coria, Roberto; López-Villaseñor, Imelda

    2017-04-01

    Cyclin-dependent kinases (CDKs) have important roles in regulating key checkpoints between stages of the cell cycle. Their activity is tightly regulated through a variety of mechanisms, including through binding with cyclin proteins and the Cdc2/Cdc28 kinase subunit (CKS), and their phosphorylation at specific amino acids. Studies of the components involved in cell cycle control in parasitic protozoa are limited. Trichomonas vaginalis is the causative agent of trichomoniasis in humans and is therefore important in public health; however, some of the basic biological processes used by this organism have not been defined. Here, we characterized proteins potentially involved in cell cycle regulation in T. vaginalis. Three genes encoding protein kinases were identified in the T. vaginalis genome, and the corresponding recombinant proteins (TvCRK1, TvCRK2, TvCRK5) were studied. These proteins displayed similar sequence features to CDKs. Two genes encoding CKSs were also identified, and the corresponding recombinant proteins were found to interact with TvCRK1 and TvCRK2 by a yeast two-hybrid system. One putative cyclin B protein from T. vaginalis was found to bind to and activate the kinase activities of TvCRK1 and TvCRK5, but not TvCRK2. This work is the first characterization of proteins involved in cell cycle control in T. vaginalis.

  14. Effect of glutenin subunits on the baking quality of Brazilian wheat genotypes

    Directory of Open Access Journals (Sweden)

    Mariana Souza Costa

    Full Text Available ABSTRACT This study aimed to evaluate the effect of the high and low molecular weight glutenin subunits on the grain traits of sixteen Brazilian wheat genotypes. Grain hardness index, milling traits, physicochemical and rheological properties of the flour, and specific volume and firmness of the bread were evaluated. Physicochemical properties of the flour were not influenced by glutenin subunits. Genotypes with subunits at the Glu-B1 (17+18 or 7+8, Glu-D1 (5+10, and Glu-A3 (b were associated with strong flours and bread with high specific volume and low firmness. The subunits at the Glu-A1 and Glu-B3 had no effect on the rheological properties of the dough and bread quality, while the subunit 2+12 at Glu-D1 negatively affected the resistance to extension, and specific volume and firmness of the bread. Specific volume and firmness of the bread were influenced by the rheological properties of the dough, while the flour protein content was not important to define wheat quality. The identification of glutenin subunits at different loci along with the rheological tests of the flour are fundamental in estimating the potential use of different materials developed in wheat breeding.

  15. Structural determinants of alpha-bungarotoxin binding to the sequence segment 181-200 of the muscle nicotinic acetylcholine receptor α subunit: Effects of cysteine/cystine modification and species-specific amino acid substitution

    International Nuclear Information System (INIS)

    McLane, K.E.; Wu, Xiadong; Diethelm, B.; Conti-Tronconi, B.M.

    1991-01-01

    The sequence segment 181-200 of the Torpedo nicotinic acetylcholine receptor (nAChR) αsubunit forms a binding site for α-bungarotoxin (α-BTX). Synthetic peptides corresponding to the homologous sequences of human, calf, mouse, chicken, frog, and cobra muscle nAChR α1 subunits were tested for their ability to bind 125 I-α-BTX, and differences in α-BTX affinity were determined by using solution (IC 50 s) and solid-phase (K d s) assays. Panels of overlapping peptides corresponding to the complete α1 subunit of mouse and human were also tested for α-BTX binding, but other sequence segments forming the α-BTX site were not consistently detectable. The role of a putative vicinal disulfide bound between Cys-192 and -193, relative to the Torpedo sequence, was determined by modifying the peptides with sulfhydryl reagents. Reduction and alkylation of the peptides decreased α-BTX binding, whereas oxidation of the peptides had little effect. These results indicate that while the adjacent cysteines are likely to be involved in forming the toxin/α1-subunit interface a vicinal disulfide bound was not required for α-BTX binding

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

    International Nuclear Information System (INIS)

    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

  17. Structural basis of subunit selectivity for competitive NMDA receptor antagonists with preference for GluN2A over GluN2B subunits

    Energy Technology Data Exchange (ETDEWEB)

    Lind, Genevieve E.; Mou, Tung-Chung; Tamborini, Lucia; Pomper, Martin G.; De Micheli, Carlo; Conti, Paola; Pinto, Andrea; Hansen, Kasper B. (JHU); (Milan); (Montana)

    2017-07-31

    NMDA-type glutamate receptors are ligand-gated ion channels that contribute to excitatory neurotransmission in the central nervous system (CNS). Most NMDA receptors comprise two glycine-binding GluN1 and two glutamate-binding GluN2 subunits (GluN2A–D). We describe highly potent (S)-5-[(R)-2-amino-2-carboxyethyl]-4,5-dihydro-1H-pyrazole-3-carboxylic acid (ACEPC) competitive GluN2 antagonists, of which ST3 has a binding affinity of 52 nM at GluN1/2A and 782 nM at GluN1/2B receptors. This 15-fold preference of ST3 for GluN1/2A over GluN1/2B is improved compared with NVP-AAM077, a widely used GluN2A-selective antagonist, which we show has 11-fold preference for GluN1/2A over GluN1/2B. Crystal structures of the GluN1/2A agonist binding domain (ABD) heterodimer with bound ACEPC antagonists reveal a binding mode in which the ligands occupy a cavity that extends toward the subunit interface between GluN1 and GluN2A ABDs. Mutational analyses show that the GluN2A preference of ST3 is primarily mediated by four nonconserved residues that are not directly contacting the ligand, but positioned within 12 Å of the glutamate binding site. Two of these residues influence the cavity occupied by ST3 in a manner that results in favorable binding to GluN2A, but occludes binding to GluN2B. Thus, we reveal opportunities for the design of subunit-selective competitive NMDA receptor antagonists by identifying a cavity for ligand binding in which variations exist between GluN2A and GluN2B subunits. This structural insight suggests that subunit selectivity of glutamate-site antagonists can be mediated by mechanisms in addition to direct contributions of contact residues to binding affinity.

  18. LINKING GABAA RECEPTOR SUBUNITS TO ALCOHOL-INDUCED CONDITIONED TASTE AVERSION AND RECOVERY FROM ACUTE ALCOHOL INTOXICATION

    Science.gov (United States)

    Blednov, Y.A.; Benavidez, J.M.; Black, M.; Chandra, D.; Homanics, G.E.; Rudolph, U.; Harris, R.A.

    2012-01-01

    GABA type A receptors (GABAA-R) are important for ethanol actions and it is of interest to link individual subunits with specific ethanol behaviors. We studied null mutant mice for six different GABAA-R subunits (α1, α2, α3, α4, α5 and δ). Only mice lacking the α2 subunit showed reduction of conditioned taste aversion (CTA) to ethanol. These results are in agreement with data from knock-in mice with mutation of the ethanol-sensitive site in the α2-subunit (Blednov et al., 2011) and indicate this aversive property of ethanol is dependent on ethanol action on α2-containing GABAA-R. Deletion of the α2-subunit led to faster recovery whereas absence of the α3-subunit slowed recovery from ethanol-induced incoordination (rotarod). Deletion of the other four subunits did not affect this behavior. Similar changes in this behavior for the α2 and α3 null mutants were found for flurazepam motor-incoordination. However, no differences in recovery were found in motor-incoordinating effects of an α1-selective modulator (zolpidem) or an α4-selective agonist (gaboxadol). Therefore, recovery of rotarod incoordination is under control of two GABAA-R subunits: α2 and α3. For motor activity, α3 null mice demonstrated higher activation by ethanol (1 g/kg) whereas both α2 and α3 (-/-) knockout mice were less sensitive to ethanol-induced reduction of motor activity (1.5 g/kg). These studies demonstrate that the effects of ethanol at GABAergic synapses containing α2 subunit are important for specific behavioral effects of ethanol which may be relevant to the genetic linkage of the α2 subunit with human alcoholism. PMID:23147414

  19. NCBI nr-aa BLAST: CBRC-ATHA-03-0002 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-ATHA-03-0002 ref|NP_186955.1| CSLD3 (CELLULOSE SYNTHASE-LIKE 3); cellulose syn...thase/ transferase, transferring glycosyl groups [Arabidopsis thaliana] gb|AAF26119.1|AC012328_22 putative cellulose... synthase catalytic subunit [Arabidopsis thaliana] gb|AAG60543.1|AF232907_1 cellulose synthase-like ...CSLD3 [Arabidopsis thaliana] gb|AAK25890.1|AF360180_1 putative cellulose synthase... catalytic subunit [Arabidopsis thaliana] gb|AAK64073.1| putative cellulose synthase catalytic subunit [Arab

  20. NCBI nr-aa BLAST: CBRC-OSAT-10-0021 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-OSAT-10-0021 ref|NP_186955.1| CSLD3 (CELLULOSE SYNTHASE-LIKE 3); cellulose syn...thase/ transferase, transferring glycosyl groups [Arabidopsis thaliana] gb|AAF26119.1|AC012328_22 putative cellulose... synthase catalytic subunit [Arabidopsis thaliana] gb|AAG60543.1|AF232907_1 cellulose synthase-like ...CSLD3 [Arabidopsis thaliana] gb|AAK25890.1|AF360180_1 putative cellulose synthase... catalytic subunit [Arabidopsis thaliana] gb|AAK64073.1| putative cellulose synthase catalytic subunit [Arab

  1. NCBI nr-aa BLAST: CBRC-OSAT-12-0025 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-OSAT-12-0025 ref|NP_186955.1| CSLD3 (CELLULOSE SYNTHASE-LIKE 3); cellulose syn...thase/ transferase, transferring glycosyl groups [Arabidopsis thaliana] gb|AAF26119.1|AC012328_22 putative cellulose... synthase catalytic subunit [Arabidopsis thaliana] gb|AAG60543.1|AF232907_1 cellulose synthase-like ...CSLD3 [Arabidopsis thaliana] gb|AAK25890.1|AF360180_1 putative cellulose synthase... catalytic subunit [Arabidopsis thaliana] gb|AAK64073.1| putative cellulose synthase catalytic subunit [Arab

  2. Subunit stoichiometry of the chloroplast photosystem I complex

    International Nuclear Information System (INIS)

    Bruce, B.D.; Malkin, R.

    1988-01-01

    A native photosystem I (PS I) complex and a PS I core complex depleted of antenna subunits has been isolated from the uniformly 14 C-labeled aquatic higher plant, Lemna. These complexes have been analyzed for their subunit stoichiometry by quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis methods. The results for both preparations indicate that one copy of each high molecular mass subunit is present per PS I complex and that a single copy of most low molecular mass subunits is also present. These results suggest that iron-sulfur center X, an early PS I electron acceptor proposed to bind to the high molecular mass subunits, contains a single [4Fe-4S] cluster which is bound to a dimeric structure of high molecular mass subunits, each providing 2 cysteine residues to coordinate this cluster

  3. Purification and subunit structure of a putative K+-channel protein identified by its binding properties for dendrotoxin I

    International Nuclear Information System (INIS)

    Rehm, H.; Lazdunski, M.

    1988-01-01

    The binding protein for the K + -channel toxin dendrotoxin I was purified from a detergent extract of rat brain membranes. The purification procedure utilized chromatography on DEAE-Trisacryl, affinity chromatography on a dendrotoxin-I-Aca 22 column, and wheat germ agglutinin-Affigel 10 with a final 3,800- to 4,600-fold enrichment and a recovery of 8-16%. The high affinity (K d , 40-100 pM) and specificity of the binding site are retained throughout the purification procedure. Analysis of the purified material on silver-stained NaDodSO 4 /polyacrylamide gel revealed three bands of M r 76,000-80,000, 38,000 and 35,000. Interestingly, the binding site for 125 I-labeled mast cell degranulating peptide, another putative K + -channel ligand from bee venom, which induces long-term potentiation in hippocampus, seems to reside on the same protein complex, as both binding sites copurify through the entire purification protocol

  4. GABAA receptor subunit gene expression in human prefrontal cortex: comparison of schizophrenics and controls

    Science.gov (United States)

    Akbarian, S.; Huntsman, M. M.; Kim, J. J.; Tafazzoli, A.; Potkin, S. G.; Bunney, W. E. Jr; Jones, E. G.; Bloom, F. E. (Principal Investigator)

    1995-01-01

    The prefrontal cortex of schizophrenics is hypoactive and displays changes related to inhibitory, GABAergic neurons, and GABAergic synapses. These changes include decreased levels of glutamic acid decarboxylase (GAD), the enzyme for GABA synthesis, upregulation of muscimol binding, and downregulation of benzodiazepine binding to GABAA receptors. Studies in the visual cortex of nonhuman primates have demonstrated that gene expression for GAD and for several GABAA receptor subunit polypeptides is under control of neuronal activity, raising the possibility that similar mechanisms in the hypoactive prefrontal cortex of schizophrenics may explain the abnormalities in GAD and in GABAA receptor regulation. In the present study, which is the first of its type on human cerebral cortex, levels of mRNAs for six GABAA receptor subunits (alpha 1, alpha 2, alpha 5, beta 1, beta 2, gamma 2) and their laminar expression patterns were analyzed in the prefrontal cortex of schizophrenics and matched controls, using in situ hybridization histochemistry and densitometry. Three types of laminar expression pattern were observed: mRNAs for the alpha 1, beta 2, and gamma 2 subunits, which are the predominant receptor subunits expressed in the mature cortex, were expressed at comparatively high levels by cells of all six cortical layers, but most intensely by cells in lower layer III and layer IV. mRNAs for the alpha 2, alpha 5, and beta 1 subunits were expressed at lower levels; alpha 2 and beta 1 were expressed predominantly by cells in layers II, III, and IV; alpha 5 was expressed predominantly in layers IV, V, and VI. There were no significant changes in overall mRNA levels for any of the receptor subunits in the prefrontal cortex of schizophrenics, and the laminar expression pattern of all six receptor subunit mRNAs did not differ between schizophrenics and controls. Because gene expression for GABAA receptor subunits is not consistently altered in the prefrontal cortex of

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-06-13

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

  6. Rapid Screening for Cognitive Impairment in Parkinson’s Disease: A Pilot Study

    Directory of Open Access Journals (Sweden)

    YanHong Dong

    2015-01-01

    Full Text Available Aim. This study sought to establish the discriminant validity of a rapid cognitive screen, that is, the National Institute of Neurological Disease and Stroke-Canadian Stroke Network (NINDS-CSN 5-minute protocol, and compare its discriminant validity to the Montreal Cognitive Assessment (MoCA and Mini Mental State Examination (MMSE in detecting cognitive impairment (CI in PD patients. Methods. One hundred and one PD patients were recruited from a movement disorders clinic in Singapore and they received the NINDS-CSN 5-minute protocol, MoCA, and MMSE. No cognitive impairment (NCI was defined as Clinical Dementia Rating (CDR = 0 and CI was defined as CDR ≥ 0.5. Results. Area under the receiver operating characteristic curve of NINDS-CSN 5-minute protocol was statistically equivalent to MoCA and larger than MMSE (0.86 versus 0.90, P=0.07; 0.86 versus 0.76, P=0.03. The sensitivity of NINDS-CSN 5-minute protocol (<9 was statistically equivalent to MoCA (<22 (0.77 versus 0.85, P=0.13 and superior to MMSE (<24 (0.77 versus 0.52, P<0.01 in detecting CI, while the specificity of NINDS-CSN 5-minute protocol (<9 was statistically equivalent to MoCA (<22 and MMSE (<24 (0.78 versus 0.88, P=0.34. Conclusion. The NINDS-CSN 5-minute protocol is time expeditious while remaining statistically equivalent to MoCA and superior to MMSE and therefore is suitable for rapid cognitive screening of CI in PD patients.

  7. Global Proteome Analysis Identifies Active Immunoproteasome Subunits in Human Platelets*

    Science.gov (United States)

    Klockenbusch, Cordula; Walsh, Geraldine M.; Brown, Lyda M.; Hoffman, Michael D.; Ignatchenko, Vladimir; Kislinger, Thomas; Kast, Juergen

    2014-01-01

    The discovery of new functions for platelets, particularly in inflammation and immunity, has expanded the role of these anucleate cell fragments beyond their primary hemostatic function. Here, four in-depth human platelet proteomic data sets were generated to explore potential new functions for platelets based on their protein content and this led to the identification of 2559 high confidence proteins. During a more detailed analysis, consistently high expression of the proteasome was discovered, and the composition and function of this complex, whose role in platelets has not been thoroughly investigated, was examined. Data set mining resulted in identification of nearly all members of the 26S proteasome in one or more data sets, except the β5 subunit. However, β5i, a component of the immunoproteasome, was identified. Biochemical analyses confirmed the presence of all catalytically active subunits of the standard 20S proteasome and immunoproteasome in human platelets, including β5, which was predominantly found in its precursor form. It was demonstrated that these components were assembled into the proteasome complex and that standard proteasome as well as immunoproteasome subunits were constitutively active in platelets. These findings suggest potential new roles for platelets in the immune system. For example, the immunoproteasome may be involved in major histocompatibility complex I (MHC I) peptide generation, as the MHC I machinery was also identified in our data sets. PMID:25146974

  8. Inositol hexakisphosphate kinase-1 mediates assembly/disassembly of the CRL4–signalosome complex to regulate DNA repair and cell death

    Science.gov (United States)

    Rao, Feng; Xu, Jing; Khan, A. Basit; Gadalla, Moataz M.; Cha, Jiyoung Y.; Xu, Risheng; Tyagi, Richa; Dang, Yongjun; Chakraborty, Anutosh; Snyder, Solomon H.

    2014-01-01

    Inositol polyphosphates containing an energetic pyrophosphate bond are formed primarily by a family of three inositol hexakisphosphate (IP6) kinases (IP6K1–3). The Cullin-RING ubiquitin ligases (CRLs) regulate diverse biological processes through substrate ubiquitylation. CRL4, comprising the scaffold Cullin 4A/B, the E2-interacting Roc1/2, and the adaptor protein damage-specific DNA-binding protein 1, is activated by DNA damage. Basal CRL4 activity is inhibited by binding to the COP9 signalosome (CSN). UV radiation and other stressors dissociate the complex, leading to E3 ligase activation, but signaling events that trigger signalosome dissociation from CRL4 have been unclear. In the present study, we show that, under basal conditions, IP6K1 forms a ternary complex with CSN and CRL4 in which IP6K1 and CRL4 are inactive. UV dissociates IP6K1 to generate IP7, which then dissociates CSN–CRL4 to activate CRL4. Thus, IP6K1 is a novel CRL4 subunit that transduces UV signals to mediate disassembly of the CRL4–CSN complex, thereby regulating nucleotide excision repair and cell death. PMID:25349427

  9. Assessment of RELAP5/MOD2 against a main feedwater turbopump trip transient in the Vandellos II Nuclear Power Plant

    International Nuclear Information System (INIS)

    Llopis, C.; Casals, A.; Perez, J.; Mendizabal, R.

    1993-12-01

    The Consejo de Seguridad Nuclear (CSN) and the Asociacion Nuclear Vandellos (ANV) have developed a model of Vandellos II Nuclear Power Plant. The ANV collaboration consisted in the supply of design and actual data, the cooperation in the simulation of the control systems and other model components, as well as in the results analysis. The obtained model has been assessed against the following transients occurred in plant: A trip from the 100% power level (CSN); a load rejection from 100% to 50% (CSN); a load rejection from 75% to 65% (ANV); and, a feedwater turbopump trip (ANV). This copy is a report of the feedwater turbopump trip transient simulation. This transient actually occurred in the plant on June 19, 1989

  10. Serotonin Transporter (5-HTT) and gamma-Aminobutyric Acid Receptor Subunit beta3 (GABRB3) Gene Polymorphisms are not Associated with Autism in the IMGSA Families

    DEFF Research Database (Denmark)

    Maestrini, E.; Lai, C.; Marlow, A.

    1999-01-01

    Previous studies have suggested that the serotonin transporter (5-HTT) gene and the gamma-aminobutyric acid receptor subunit beta3 (GABRB3) gene, or other genes in the 15q11-q13 region, are possibly involved in susceptibility to autism. To test this hypothesis we performed an association study on...

  11. Arabidopsis CDS blastp result: AK103200 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK103200 J033122A14 At5g54750.1 transport protein particle (TRAPP) component Bet3, ...putative similar to SP|P36149 Transport protein particle 22 kDa subunit (TRAPP 22 kDa subunit) {Saccharomyce...s cerevisiae}; contains Pfam profile PF04051: Transport protein particle (TRAPP) component, Bet3 2e-90 ...

  12. Over-expression of a putative oxidoreductase (UcpA) for increasing furfural or 5-hydroxymethylfurfural tolerance

    Science.gov (United States)

    Wang, Xuan; Miller, Elliot N.; Yomano, Lorraine P.; Shanmugam, Keelnatham T.; Ingram, Lonnie O'Neal

    2016-05-24

    The subject invention pertains to overexpression of a putative oxidoreductase (ucpA) for increasing furfural tolerance in genetically modified microorganisms. Genetically modified microorganisms capable of overexpressing UcpA are also provided. Increased expression of ucpA was shown to increase furfural tolerance by 50%, and to permit the fermentation of sugars to products in the presence of 15 mM furfural.

  13. Structural interaction of novel dendrimer and subunits with water

    African Journals Online (AJOL)

    Preferred Customer

    interaction study with solvents are essential [4-6] and several subunits are used for .... slowed down the viscous flow with higher excess limiting viscosities of the 2,4,6- ..... Practical Organic Chemistry, 5th ed.; Wiley: New York; 1989; p 300. 14.

  14. Linking GABA(A) receptor subunits to alcohol-induced conditioned taste aversion and recovery from acute alcohol intoxication.

    Science.gov (United States)

    Blednov, Y A; Benavidez, J M; Black, M; Chandra, D; Homanics, G E; Rudolph, U; Harris, R A

    2013-04-01

    GABA type A receptors (GABA(A)-R) are important for ethanol actions and it is of interest to link individual subunits with specific ethanol behaviors. We studied null mutant mice for six different GABA(A)-R subunits (α1, α2, α3, α4, α5 and δ). Only mice lacking the α2 subunit showed reduction of conditioned taste aversion (CTA) to ethanol. These results are in agreement with data from knock-in mice with mutation of the ethanol-sensitive site in the α2-subunit (Blednov et al., 2011). All together, they indicate that aversive property of ethanol is dependent on ethanol action on α2-containing GABA(A)-R. Deletion of the α2-subunit led to faster recovery whereas absence of the α3-subunit slowed recovery from ethanol-induced incoordination (rotarod). Deletion of the other four subunits did not affect this behavior. Similar changes in this behavior for the α2 and α3 null mutants were found for flurazepam motor incoordination. However, no differences in recovery were found in motor-incoordinating effects of an α1-selective modulator (zolpidem) or an α4-selective agonist (gaboxadol). Therefore, recovery of rotarod incoordination is under control of two GABA(A)-R subunits: α2 and α3. For motor activity, α3 null mice demonstrated higher activation by ethanol (1 g/kg) whereas both α2 (-/-) and α3 (-/Y) knockout mice were less sensitive to ethanol-induced reduction of motor activity (1.5 g/kg). These studies demonstrate that the effects of ethanol at GABAergic synapses containing α2 subunit are important for specific behavioral effects of ethanol which may be relevant to the genetic linkage of the α2 subunit with human alcoholism. Copyright © 2012 Elsevier Ltd. All rights reserved.

  15. Role of the Rubisco small subunit. Final report for period May 1, 1997--April 30,2000

    Energy Technology Data Exchange (ETDEWEB)

    Spreitzer, Robert J.

    2000-10-04

    CO{sub 2} and O{sub 2} are mutually competitive at the active site of ribulose-1,5-biphosphate (RuBP) carboxylase/oxygenase (Rubisco). Rubisco contains two subunits, each present in eight copies. The 15-kD small subunit is coded by a family of nuclear RbcS genes. Until now, the role of the small subunit in Rubisco structure or catalytic efficiency is not known. Because of other work in eliminating the two RbcS genes in the green algo Chlamydomonas reinhardtii, it is now possible to address questions about the structure-function relationships of the eukaryotic small subunit. There are three specific aims in this project: (1) Alanine scanning mutagenesis is being used to dissect the importance of the {beta}A/{beta}B loop, a feature unique to the eukaryotic small subunit. (2) Random mutagenesis is being used to identify additional residues or regions of the small subunit that are important for holoenzyme assembly and function. (3) Attempts are being made to express foreign small subunits in Chlamydomonas to examine the contribution of small subunits to holoenzyme assembly, catalytic efficiency, and CO{sub 2}/O{sub 2} specificity.

  16. Identification of Interactions between Abscisic Acid and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase.

    Directory of Open Access Journals (Sweden)

    Marek M Galka

    Full Text Available Abscisic acid ((+-ABA is a phytohormone involved in the modulation of developmental processes and stress responses in plants. A chemical proteomics approach using an ABA mimetic probe was combined with in vitro assays, isothermal titration calorimetry (ITC, x-ray crystallography and in silico modelling to identify putative (+-ABA binding-proteins in crude extracts of Arabidopsis thaliana. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco was identified as a putative ABA-binding protein. Radiolabelled-binding assays yielded a Kd of 47 nM for (+-ABA binding to spinach Rubisco, which was validated by ITC, and found to be similar to reported and experimentally derived values for the native ribulose-1,5-bisphosphate (RuBP substrate. Functionally, (+-ABA caused only weak inhibition of Rubisco catalytic activity (Ki of 2.1 mM, but more potent inhibition of Rubisco activation (Ki of ~ 130 μM. Comparative structural analysis of Rubisco in the presence of (+-ABA with RuBP in the active site revealed only a putative low occupancy (+-ABA binding site on the surface of the large subunit at a location distal from the active site. However, subtle distortions in electron density in the binding pocket and in silico docking support the possibility of a higher affinity (+-ABA binding site in the RuBP binding pocket. Overall we conclude that (+-ABA interacts with Rubisco. While the low occupancy (+-ABA binding site and weak non-competitive inhibition of catalysis may not be relevant, the high affinity site may allow ABA to act as a negative effector of Rubisco activation.

  17. Results Assessment of Intercomparison Exercise CSN/CIEMAT-2011 among Spanish National Laboratories of Environmental Radioactivity (Water)

    International Nuclear Information System (INIS)

    Gascó, C.; Trinidad, J. A.; Llauradó, M.

    2015-01-01

    This report describes the results assessment of the intercomparsion exercise among environmental radioactivity laboratories, organised by Spanish Regulatory Institution (CSN) and prepared and evaluated by UAB and CIEMAT respectively. The exercise has been carried out following the international standards ISO-43 and ISO/IUPAC that provide a useful guide to perform proficiency tests and inter-laboratories comparisons. The selected matrix for this year (2011) was deionized water, simulating drinking water, that was enriched with artificial radionuclides (Cs-137, Co-60, Fe-55, Ni-63, Sr-90, Am-241 and Pu-238) and contained natural radionuclides (U-234, U-238, U-natural, Pb-210, Po-210, Th-230, Ra-226 and K-40) at environmental level of activity concentration. A second matrix of deionized water was prepared with I-129 and C-14. The z-score test was applied to determine how much the laboratories differ from the reference value. The reference value for this exercise was the median of the results from the different laboratories and their standard deviations to achieve a more complete and objective study of the laboratories performance. The participant laboratories have demonstrated a satisfactory quality level for measuring the natural and artificial radionuclides content in this matrix. The study has showed a homogeneous behaviour of the laboratories.

  18. Transcriptional regulators of Na, K-ATPase subunits

    Directory of Open Access Journals (Sweden)

    Zhiqin eLi

    2015-10-01

    Full Text Available The Na,K-ATPase classically serves as an ion pump creating an electrochemical gradient across the plasma membrane that is essential for transepithelial transport, nutrient uptake and membrane potential. In addition, Na,K-ATPase also functions as a receptor, a signal transducer and a cell adhesion molecule. With such diverse roles, it is understandable that the Na,K-ATPase subunits, the catalytic alpha-subunit, the beta-subunit and the FXYD proteins, are controlled extensively during 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 have been observed in many carcinoma, we will also discuss transcription factors that are associated with epithelial-to-mesenchymal transition, a crucial step in the progression of many tumors to malignant disease.

  19. A novel GABA(A) alpha 5 receptor inhibitor with therapeutic potential.

    Science.gov (United States)

    Ling, István; Mihalik, Balázs; Etherington, Lori-An; Kapus, Gábor; Pálvölgyi, Adrienn; Gigler, Gábor; Kertész, Szabolcs; Gaál, Attila; Pallagi, Katalin; Kiricsi, Péter; Szabó, Éva; Szénási, Gábor; Papp, Lilla; Hársing, László G; Lévay, György; Spedding, Michael; Lambert, Jeremy J; Belelli, Delia; Barkóczy, József; Volk, Balázs; Simig, Gyula; Gacsályi, István; Antoni, Ferenc A

    2015-10-05

    Novel 2,3-benzodiazepine and related isoquinoline derivatives, substituted at position 1 with a 2-benzothiophenyl moiety, were synthesized to produce compounds that potently inhibited the action of GABA on heterologously expressed GABAA receptors containing the alpha 5 subunit (GABAA α5), with no apparent affinity for the benzodiazepine site. Substitutions of the benzothiophene moiety at position 4 led to compounds with drug-like properties that were putative inhibitors of extra-synaptic GABAA α5 receptors and had substantial blood-brain barrier permeability. Initial characterization in vivo showed that 8-methyl-5-[4-(trifluoromethyl)-1-benzothiophen-2-yl]-1,9-dihydro-2H-[1,3]oxazolo[4,5-h][2,3]benzodiazepin-2-one was devoid of sedative, pro-convulsive or motor side-effects, and enhanced the performance of rats in the object recognition test. In summary, we have discovered a first-in-class GABA-site inhibitor of extra-synaptic GABAA α5 receptors that has promising drug-like properties and warrants further development. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    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

  1. The subunits analysis of R-phycoerythrin from marine red algae by ...

    African Journals Online (AJOL)

    Subunit components of R-phycoerythrins (R-PEs) prepared from five marine macro red algae were analyzed by sodium dodecyl sulfate -polyarylamide gel electrophoresis (SDS-PAGE) and by isoelectric focusing (IEF) in pH gradients range of 3.0 to 9.5, 2.5 to 5.0 and 4.0 to 6.5. Riboflavin was used to catalyze ...

  2. Results Assessment of Intercomparison Exercise CSN/CIEMAT-2012 among Spanish National Laboratories of Environmental Radioactivity (Soil); Evaluación de la Intercomparación CSN/CIEMAT-2012 entre los Laboratorios Nacionales de Radiactividad Ambiental (Suelo)

    Energy Technology Data Exchange (ETDEWEB)

    Trinidad, J. A.; Gascó, C.; Llauradó, M.

    2015-07-01

    This report describes the results assessment of the intercomparsion exercise among environmental radioactivity laboratories, organised by Spanish Regulatory Institution (CSN) and prepared and evaluated by UB and CIEMAT respectively. The exercise has been carried out following the international standards ISO-43 and ISO/IUPAC that provide a useful guide to perform proficiency tests and inter-laboratories comparisons. The selected matrix for this year (2012) was soil, that was enriched with artificial radionuclides (137Cs, 60Co, 55Fe, 63Ni, 90Sr, 241Am, 239+240Pu and 238Pu) and contained natural radionuclides (234U, 238U, U-natural 230Th, 226Ra, 210Pb, 228Ra, 228Ac, 234Th, 214Bi, 214Pb, 212Pb, 208Tl and 40K) at environmental level of activity concentration. Two soil matrixes were prepared in order to separate 55Fe and 63Ni analysis. The z-score test was applied to determine how much the laboratories differ from the reference value. The reference value for this exercise was the median of the results from the different laboratories and their standard deviations to achieve a more complete and objective study of the laboratories performance. The participant laboratories have demonstrated a satisfactory quality level for measuring the natural and artificial radionuclides content in this matrix. The study has showed a homogeneous behaviour of the laboratories.

  3. GABA receptor subunit distribution and FMRP-mGluR5 signaling abnormalities in the cerebellum of subjects with schizophrenia, mood disorders, and autism.

    Science.gov (United States)

    Fatemi, S Hossein; Folsom, Timothy D

    2015-09-01

    Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the brain. GABAergic receptor abnormalities have been documented in several major psychiatric disorders including schizophrenia, mood disorders, and autism. Abnormal expression of mRNA and protein for multiple GABA receptors has also been observed in multiple brain regions leading to alterations in the balance between excitatory/inhibitory signaling in the brain with potential profound consequences for normal cognition and maintenance of mood and perception. Altered expression of GABAA receptor subunits has been documented in fragile X mental retardation 1 (FMR1) knockout mice, suggesting that loss of its protein product, fragile X mental retardation protein (FMRP), impacts GABAA subunit expression. Recent postmortem studies from our laboratory have shown reduced expression of FMRP in the brains of subjects with schizophrenia, bipolar disorder, major depression, and autism. FMRP acts as a translational repressor and, under normal conditions, inhibits metabotropic glutamate receptor 5 (mGluR5)-mediated signaling. In fragile X syndrome (FXS), the absence of FMRP is hypothesized to lead to unregulated mGluR5 signaling, ultimately resulting in the behavioral and intellectual impairments associated with this disorder. Our laboratory has identified changes in mGluR5 expression in autism, schizophrenia, and mood disorders. In the current review article, we discuss our postmortem data on GABA receptors, FMRP, and mGluR5 levels and compare our results with other laboratories. Finally, we discuss the interactions between these molecules and the potential for new therapeutic interventions that target these interconnected signaling systems. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Molecular characterization of the llama FGF5 gene and identification of putative loss of function mutations.

    Science.gov (United States)

    Daverio, M S; Vidal-Rioja, L; Frank, E N; Di Rocco, F

    2017-12-01

    Llama, the most numerous domestic camelid in Argentina, has good fiber-production ability. Although a few genes related to other productive traits have been characterized, the molecular genetic basis of fiber growth control in camelids is still poorly understood. Fibroblast growth factor 5 (FGF5) is a secreted signaling protein that controls hair growth in humans and other mammals. Mutations in the FGF5 gene have been associated with long-hair phenotypes in several species. Here, we sequenced the llama FGF5 gene, which consists of three exons encoding 813 bp. cDNA analysis from hair follicles revealed the expression of two FGF5 alternative spliced transcripts, in one of which exon 2 is absent. DNA variation analysis showed four polymorphisms in the coding region: a synonymous SNP (c.210A>G), a single base deletion (c.348delA), a 12-bp insertion (c.351_352insCATATAACATAG) and a non-sense mutation (c.499C>T). The deletion was always found together with the insertion forming a haplotype and producing a putative truncated protein of 123 amino acids. The c.499C>T mutation also leads to a premature stop codon at position 168. In both cases, critical functional domains of FGF5, including one heparin binding site, are lost. All animals analyzed were homozygous for one of the deleterious mutations or compound heterozygous for both (i.e. c.348delA, c.351_352insCATATAACATAG/c.499T). Sequencing of guanaco samples showed that the FGF5 gene encodes a full-length 270-amino acid protein. These results suggest that FGF5 is likely functional in short-haired wild species and non-functional in the domestic fiber-producing species, the llama. © 2017 Stichting International Foundation for Animal Genetics.

  5. Human aldolase B subunit-specific radioimmunoassay

    International Nuclear Information System (INIS)

    Asaka, M.; Alpert, E.

    1983-01-01

    A radioimmunoassay was developed for the direct quantification of aldolase B in human serum and tissues. The method is a double-antibody radioimmunoassay technique using radioiodinated aldolase B homopolymer as ligand, chicken antibodies to aldolase B and rabbit antibodies to chicken IgG. This radioimmunoassay was shown to be specific for the aldolase B subunit, with no cross-reactivity with either human aldolase A subunit or homopolymeric human aldolase C (C 4 ). The lowest measurable amount by this method was 2 ng/ml. Aldolase B is predominantly found in normal liver tissue, with relatively-high aldolase B levels also observed in kidney. Aldolase B levels in the serum obtained from 11 normal subjects ranged from 23 to 38 ng/ml, with a mean of 28.5 +- 9.2 (S.D.) ng/ml. Almost all of patients with hepatitis had serum aldolase B levels greater than 30 ng/ml. In cancer patients, serum aldolase B was slightly elevated in patients with metastatic liver cancer and primary lever cell carcinoma, whereas no elevation of serum aldolase B was shown in patients without liver metastasis. (Auth.)

  6. Loss of GluN2D subunit results in social recognition deficit, social stress, 5-HT2C receptor dysfunction, and anhedonia in mice.

    Science.gov (United States)

    Yamamoto, Hideko; Kamegaya, Etsuko; Hagino, Yoko; Takamatsu, Yukio; Sawada, Wakako; Matsuzawa, Maaya; Ide, Soichiro; Yamamoto, Toshifumi; Mishina, Masayoshi; Ikeda, Kazutaka

    2017-01-01

    The N-methyl-d-aspartate (NMDA) receptor channel is involved in various physiological functions, including learning and memory. The GluN2D subunit of the NMDA receptor has low expression in the mature brain, and its role is not fully understood. In the present study, the effects of GluN2D subunit deficiency on emotional and cognitive function were investigated in GluN2D knockout (KO) mice. We found a reduction of motility (i.e., a depressive-like state) in the tail suspension test and a reduction of sucrose preference (i.e., an anhedonic state) in GluN2D KO mice that were group-housed with littermates. Despite apparently normal olfactory function and social interaction, GluN2D KO mice exhibited a decrease in preference for social novelty, suggesting a deficit in social recognition or memory. Golgi-Cox staining revealed a reduction of the complexity of dendritic trees in the accessory olfactory bulb in GluN2D KO mice, suggesting a deficit in pheromone processing pathway activation, which modulates social recognition. The deficit in social recognition may result in social stress in GluN2D KO mice. Isolation housing is a procedure that has been shown to reduce stress in mice. Interestingly, 3-week isolation and treatment with agomelatine or the 5-hydroxytryptamine-2C (5-HT 2C ) receptor antagonist SB242084 reversed the anhedonic-like state in GluN2D KO mice. In contrast, treatment with the 5-HT 2C receptor agonist CP809101 induced depressive- and anhedonic-like states in isolated GluN2D KO mice. These results suggest that social stress that is caused by a deficit in social recognition desensitizes 5-HT 2c receptors, followed by an anhedonic- and depressive-like state, in GluN2D KO mice. The GluN2D subunit of the NMDA receptor appears to be important for the recognition of individuals and development of normal emotionality in mice. 5-HT 2C receptor antagonism may be a therapeutic target for treating social stress-induced anhedonia. This article is part of the Special

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

    Science.gov (United States)

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

    1997-01-01

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

  8. Subunit Stoichiometry of Human Muscle Chloride Channels

    OpenAIRE

    Fahlke, Christoph; Knittle, Timothy; Gurnett, Christina A.; Campbell, Kevin P.; George, Alfred L.

    1997-01-01

    Voltage-gated Cl? channels belonging to the ClC family appear to function as homomultimers, but the number of subunits needed to form a functional channel is controversial. To determine subunit stoichiometry, we constructed dimeric human skeletal muscle Cl? channels in which one subunit was tagged by a mutation (D136G) that causes profound changes in voltage-dependent gating. Sucrose-density gradient centrifugation experiments indicate that both monomeric and dimeric hClC-1 channels in their ...

  9. Transcriptional regulators of Na, K-ATPase subunits

    OpenAIRE

    Zhiqin eLi; Sigrid A Langhans

    2015-01-01

    The Na,K-ATPase classically serves as an ion pump creating an electrochemical gradient across the plasma membrane that is essential for transepithelial transport, nutrient uptake and membrane potential. In addition, Na,K-ATPase also functions as a receptor, a signal transducer and a cell adhesion molecule. With such diverse roles, it is understandable that the Na,K-ATPase subunits, the catalytic alpha-subunit, the beta-subunit and the FXYD proteins, are controlled extensively during developme...

  10. Molecular cloning of the human casein kinase II α subunit

    International Nuclear Information System (INIS)

    Meisner, H.; Heller-Harrison, R.; Buxton, J.; Czech, M.P.

    1989-01-01

    A human cDNA encoding the α subunit of casein kinase II and a partial cDNA encoding the rat homologue were isolated by using a Drosophila casein kinase II cDNA probe. The 2.2-kb human cDNA contains a 1.2-kb open reading frame, 150 nucleotides of 5' leader, and 850 nucleotides of 3' noncoding region. Except for the first 7 deduced amino acids that are missing in the rat cDNA, the 328 amino acids beginning with the amino terminus are identical between human and rat. The Drosophila enzyme sequence is 90% identical with the human casein kinase II sequence, and there is only a single amino acid difference between the published partial bovine sequence and the human sequence. In addition, the C-terminus of the human cDNA has an extra 53 amino acids not present in Drosophila. Northern analysis of rat and human RNA showed predominant bands of 5.5, 3.1, and 1.8 kb. In rat tissues, brain and spleen had the highest levels of casein kinase II α subunit specific RNA, while skeletal muscle showed the lowest. Southern analysis of human cultured cell and tissue genomic DNA using the full-length cDNA probe revealed two bands with restriction enzymes that have no recognition sites within the cDNA and three to six bands with enzymes having single internal sites. These results are consistent with the possibility that two genes encode the α subunits

  11. Global proteome analysis identifies active immunoproteasome subunits in human platelets.

    Science.gov (United States)

    Klockenbusch, Cordula; Walsh, Geraldine M; Brown, Lyda M; Hoffman, Michael D; Ignatchenko, Vladimir; Kislinger, Thomas; Kast, Juergen

    2014-12-01

    The discovery of new functions for platelets, particularly in inflammation and immunity, has expanded the role of these anucleate cell fragments beyond their primary hemostatic function. Here, four in-depth human platelet proteomic data sets were generated to explore potential new functions for platelets based on their protein content and this led to the identification of 2559 high confidence proteins. During a more detailed analysis, consistently high expression of the proteasome was discovered, and the composition and function of this complex, whose role in platelets has not been thoroughly investigated, was examined. Data set mining resulted in identification of nearly all members of the 26S proteasome in one or more data sets, except the β5 subunit. However, β5i, a component of the immunoproteasome, was identified. Biochemical analyses confirmed the presence of all catalytically active subunits of the standard 20S proteasome and immunoproteasome in human platelets, including β5, which was predominantly found in its precursor form. It was demonstrated that these components were assembled into the proteasome complex and that standard proteasome as well as immunoproteasome subunits were constitutively active in platelets. These findings suggest potential new roles for platelets in the immune system. For example, the immunoproteasome may be involved in major histocompatibility complex I (MHC I) peptide generation, as the MHC I machinery was also identified in our data sets. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Requirement of Nicotinic Acetylcholine Receptor Subunit β2 in the Maintenance of Spiral Ganglion Neurons during Aging

    Science.gov (United States)

    Bao, Jianxin; Lei, Debin; Du, Yafei; Ohlemiller, Kevin K.; Beaudet, Arthur L.; Role, Lorna W.

    2008-01-01

    Age-related hearing loss (presbycusis) is a major health concern for the elderly. Loss of spiral ganglion neurons (SGNs), the primary sensory relay of the auditory system, is associated consistently with presbycusis. The causative molecular events responsible for age-related loss of SGNs are unknown. Recent reports directly link age-related neuronal loss in cerebral cortex with the loss of high-affinity nicotine acetylcholine receptors (nAChRs). In cochlea, cholinergic synapses are made by olivocochlear efferent fibers on the outer hair cells that express α9 nAChR subunits and on the peripheral projections of SGNs that express α2, α4 –7, and β2–3 nAChR subunits. A significantly decreased expression of the β2 nAChR subunit in SGNs was found specifically in mice susceptible to presbycusis. Furthermore, mice lacking the β2 nAChR subunit (β2−/−), but not mice lacking the α5 nAChR subunit5−/−), have dramatic hearing loss and significant reduction in the number of SGNs. Our findings clearly established a requirement for β2 nAChR subunit in the maintenance of SGNs during aging. PMID:15788760

  13. Two putative subunits of a peptide pump encoded in the human major histocompatability complex class 2 region

    International Nuclear Information System (INIS)

    Bahram, S.; Arnold, D.; Bresnahan, M.; Strominger, J.L.; Spies, T.

    1991-01-01

    The class 2 region of the human major histocompatibility complex (MHC) may encode several genes controlling the processing of endogenous antigen and the presentation of peptide epitopes by MHC class 1 molecules to cytotoxic T lymphocytes. A previously described peptide supply factor (PSF1) is a member of the multidrug-resistance family of transporters and may pump cytosolic peptides into the membrane-bound compartment where class 1 molecules assemble. A second transporter gene, PSF2, was identified 10 kilobases (kb) from PSF1, near the class 2 DOB gene. The complete sequences of PSF1 and PSF2 were determined from cDNA clones. The translation products are closely related in sequence and predicted secondary structure. Both contain a highly conserved ATP-binding fold and share 25% homology in a hydrophobic domain with a tentative number of eight membrane-spanning segments. Based on the principle dimeric organization of these two domains in other transporters, PSF1 and PSF2 may function as complementary subunits, independently as homodimers, or both. Taken together with previous genetic evidence, the coregulation of PSF1 and PSF2 by γ interferon and the to-some-degree coordinate transcription of these genes suggest a common role in peptide-loading of class 1 molecules, although a distinct function of PSF2 cannot be ruled out

  14. The Wheat E Subunit of V-Type H+-ATPase Is Involved in the Plant Response to Osmotic Stress

    Directory of Open Access Journals (Sweden)

    Xiao-Hong Zhang

    2014-09-01

    Full Text Available The vacuolar type H+-ATPase (V-type H+-ATPase plays important roles in establishing an electrochemical H+-gradient across tonoplast, energizing Na+ sequestration into the central vacuole, and enhancing salt stress tolerance in plants. In this paper, a putative E subunit of the V-type H+-ATPase gene, W36 was isolated from stress-induced wheat de novo transcriptome sequencing combining with 5'-RACE and RT-PCR methods. The full-length of W36 gene was 1097 bp, which contained a 681 bp open reading frame (ORF and encoded 227 amino acids. Southern blot analysis indicated that W36 was a single-copy gene. The quantitative real-time PCR (qRT-PCR analysis revealed that the expression level of W36 could be upregulated by drought, cold, salt, and exogenous ABA treatment. A subcellular localization assay showed that the W36 protein accumulated in the cytoplasm. Isolation of the W36 promoter revealed some cis-acting elements responding to abiotic stresses. Transgenic Arabidopsis plants overexpressing W36 were enhanced salt and mannitol tolerance. These results indicate that W36 is involved in the plant response to osmotic stress.

  15. Differential transcription and message stability of two genes encoding soybean ribulose 1,5-bisphosphate carboxylase small subunit

    International Nuclear Information System (INIS)

    Shirley, B.W.; Berry-Lowe, S.L.; Grandbastien, M.A.; Zurfluh, L.L.; Shah, D.M.; Meagher, R.B.

    1987-01-01

    The expression of two closely related soybean ribulose bisphosphate carboxylase small subunit (Rubisco ss) genes, SRS1 and SRS4, has been compared. These genes account for approximately 2-4% of the total transcription in light grown leaves, SRS4 being twice as transcriptionally active as SRS1. The transcription of these genes is reduced more than 30 fold after a pulse of far-red light or extended periods of darkness. When etiolated seedlings are shifted to the light the transcription of both genes increases 30-50 fold. Despite this 30-fold range in transcriptional expression the steady state mRNA levels in light and dark grown tissue differ by less than 8 fold. This suggests that the mRNAs are less stable in light grown tissue. 38 refs., 5 figs

  16. Non-equivalent role of TM2 gating hinges in heteromeric Kir4.1/Kir5.1 potassium channels.

    Science.gov (United States)

    Shang, Lijun; Tucker, Stephen J

    2008-02-01

    Comparison of the crystal structures of the KcsA and MthK potassium channels suggests that the process of opening a K(+) channel involves pivoted bending of the inner pore-lining helices at a highly conserved glycine residue. This bending motion is proposed to splay the transmembrane domains outwards to widen the gate at the "helix-bundle crossing". However, in the inwardly rectifying (Kir) potassium channel family, the role of this "hinge" residue in the second transmembrane domain (TM2) and that of another putative glycine gating hinge at the base of TM2 remain controversial. We investigated the role of these two positions in heteromeric Kir4.1/Kir5.1 channels, which are unique amongst Kir channels in that both subunits lack a conserved glycine at the upper hinge position. Contrary to the effect seen in other channels, increasing the potential flexibility of TM2 by glycine substitutions at the upper hinge position decreases channel opening. Furthermore, the contribution of the Kir4.1 subunit to this process is dominant compared to Kir5.1, demonstrating a non-equivalent contribution of these two subunits to the gating process. A homology model of heteromeric Kir4.1/Kir5.1 shows that these upper "hinge" residues are in close contact with the base of the pore alpha-helix that supports the selectivity filter. Our results also indicate that the highly conserved glycine at the "lower" gating hinge position is required for tight packing of the TM2 helices at the helix-bundle crossing, rather than acting as a hinge residue.

  17. Human acid-labile subunit deficiency: clinical, endocrine and metabolic consequences

    NARCIS (Netherlands)

    Domené, Horacio M.; Hwa, Vivian; Argente, Jesús; Wit, Jan M.; Wit, Jaan M.; Camacho-Hübner, Cecilia; Jasper, Héctor G.; Pozo, Jesús; van Duyvenvoorde, Hermine A.; Yakar, Shoshana; Fofanova-Gambetti, Olga V.; Rosenfeld, Ron G.; Scaglia, Paula A.; Bengolea, Sonia V.; Lteif, Aida; Kirmani, Salman; Mahmud, Farid H.; Frystyk, Jan; Hermus, Ad; Twickler, T. B.; Kempers, Marlies J. E.; Barrios, Vicente; Martos-Moreno, Gabriel A.; David, Alessia; Rose, Stephen

    2009-01-01

    The majority of insulin-like growth factor (IGF)-I and IGF-II circulate in the serum as a complex with the insulin-like growth factor binding protein (IGFBP)-3 or IGFBP-5, and an acid-labile subunit (ALS). The function of ALS is to prolong the half-life of the IGF-I-IGFBP-3/IGFBP-5 binary complexes.

  18. Arabidopsis CDS blastp result: AK240664 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK240664 J033039I06 At5g54750.1 68418.m06818 transport protein particle (TRAPP) com...ponent Bet3, putative similar to SP|P36149 Transport protein particle 22 kDa subunit (TRAPP 22 kDa subunit) ...{Saccharomyces cerevisiae}; contains Pfam profile PF04051: Transport protein particle (TRAPP) component, Bet3 2e-90 ...

  19. Association between GABA-A receptor alpha 5 subunit gene locus and schizophrenia of a later age of onset.

    Science.gov (United States)

    Papadimitriou, G; Dikeos, D; Daskalopoulou, E; Karadima, G; Avramopoulos, D; Contis, C; Stefanis, C

    2001-01-01

    Heritability is considered to be a major etiologic factor for schizophrenia. Among the genes considered as candidates for the disease, are those related to GABAergic neurotransmission. Our aim was to test for a genetic association between GABA-A receptor alpha 5 subunit gene locus (GABRA(5)) and schizophrenia. Genotyping of the GABRA(5) locus was performed by the use of a dinucleotide (CA) repeat marker in 46 schizophrenic patients and 50 healthy individuals, all unrelated Greeks. Eight alleles were identified, 276-290 bp long. A nonsignificant excess of the 282-bp allele, which was found in a previous study in a Greek population to be associated with bipolar affective disorder, was observed in schizophrenic patients (33.8 vs. 23.9% in the controls). The frequency of this allele was 43.3% among patients with a later age of onset (over 25 years), differing at a statistically significant level from the controls (p < 0.05). These results suggest that common pathophysiological mechanisms may possibly underlie affective disorders and schizophrenia, at least in a subgroup of patients. Copyright 2001 S. Karger AG, Basel

  20. Purification and subunit structure of a putative K sup + -channel protein identified by its binding properties for dendrotoxin I

    Energy Technology Data Exchange (ETDEWEB)

    Rehm, H.; Lazdunski, M. (Centre National de la Recherche Scientifique, Nice (France))

    1988-07-01

    The binding protein for the K{sup +}-channel toxin dendrotoxin I was purified from a detergent extract of rat brain membranes. The purification procedure utilized chromatography on DEAE-Trisacryl, affinity chromatography on a dendrotoxin-I-Aca 22 column, and wheat germ agglutinin-Affigel 10 with a final 3,800- to 4,600-fold enrichment and a recovery of 8-16%. The high affinity (K{sub d}, 40-100 pM) and specificity of the binding site are retained throughout the purification procedure. Analysis of the purified material on silver-stained NaDodSO{sub 4}/polyacrylamide gel revealed three bands of M{sub r} 76,000-80,000, 38,000 and 35,000. Interestingly, the binding site for {sup 125}I-labeled mast cell degranulating peptide, another putative K{sup +}-channel ligand from bee venom, which induces long-term potentiation in hippocampus, seems to reside on the same protein complex, as both binding sites copurify through the entire purification protocol.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  2. The beta subunit of casein kinase II

    DEFF Research Database (Denmark)

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

    1991-01-01

    cDNAs encoding the beta subunit of pig and mouse CKII were isolated. The porcine cDNA was expressed as a fusion protein in Escherichia coli and used for the production of anti-CKII-beta subunit specific antibodies....

  3. Characterization of fimbrial subunits from Bordetella species

    NARCIS (Netherlands)

    Mooi, F.R.; Heide, H.G.J. van der; Avest, A.R. ter; Welinder, K.G.; Livey, I.; Zeijst, B.A.M. van der; Gaastra, W.

    Using antisera raised against serotype 2 and 3 fimbrial subunits from Bordetella pertussis, serologically related polypeptides were detected in Bordetella bronchiseptica, Bordetella parapertussis and Bordetella avium strains. The two B. pertussis fimbrial subunits, and three of the serologically

  4. Supplementary data: Variation in the PTEN-induced putative kinase ...

    Indian Academy of Sciences (India)

    Variation in the PTEN-induced putative kinase 1 gene associated with the increase risk of type 2 diabetes in northern Chinese. Yanchun Qu, Liang Sun, Ze Yang and Ruifa Han. J. Genet. 90, 125–128. Table 1. Clinical characteristics of cases and controls. Phenotype. T2DM. Controls. P value. Age (years). 49.5 ± 11.1. 50.4 ± ...

  5. β5i subunit deficiency of the immunoproteasome leads to reduced Th2 response in OVA induced acute asthma.

    Directory of Open Access Journals (Sweden)

    Anton Volkov

    Full Text Available The immunoproteasome subunit β5i has been shown to play an important role in Th1/Th17 driven models of colitis and arthritis. However, the function of β5i in Th2 dependent diseases remains enigmatic. To study the role of β5i in Th2-driven pathology, β5i knockout (KO and control mice were tested in different models of experimental allergic asthma. β5i-deficient mice showed reduced OVA/Alum- and subcutaneous/OVA-induced acute asthma with decreased eosinophilia in the bronchoalveolar lavage (BAL, low OVA-specific IgG1 and reduced local and systemic Th2 cytokines. While Th2 cells in the lungs were reduced, Tregs and Th1 cells were not affected. Attenuated asthma in β5i KO mice could not be attributed to defects in OVA uptake or maturation of dendritic cells in the lung. Surprisingly, β5i deficient mice developed HDM asthma which was comparable to control mice. Here, we present novel evidence for the requirement of the β5i immunosubunit to generate a strong Th2 response during OVA- but not HDM-induced acute asthma. The unexpected role of β5i in OVA asthma remains to be clarified.

  6. Assessing subunit dependency of the Plasmodium proteasome using small molecule inhibitors and active site probes.

    Science.gov (United States)

    Li, Hao; van der Linden, Wouter A; Verdoes, Martijn; Florea, Bogdan I; McAllister, Fiona E; Govindaswamy, Kavitha; Elias, Joshua E; Bhanot, Purnima; Overkleeft, Herman S; Bogyo, Matthew

    2014-08-15

    The ubiquitin-proteasome system (UPS) is a potential pathway for therapeutic intervention for pathogens such as Plasmodium, the causative agent of malaria. However, due to the essential nature of this proteolytic pathway, proteasome inhibitors must avoid inhibition of the host enzyme complex to prevent toxic side effects. The Plasmodium proteasome is poorly characterized, making rational design of inhibitors that induce selective parasite killing difficult. In this study, we developed a chemical probe that labels all catalytic sites of the Plasmodium proteasome. Using this probe, we identified several subunit selective small molecule inhibitors of the parasite enzyme complex. Treatment with an inhibitor that is specific for the β5 subunit during blood stage schizogony led to a dramatic decrease in parasite replication while short-term inhibition of the β2 subunit did not affect viability. Interestingly, coinhibition of both the β2 and β5 catalytic subunits resulted in enhanced parasite killing at all stages of the blood stage life cycle and reduced parasite levels in vivo to barely detectable levels. Parasite killing was achieved with overall low host toxicity, something that has not been possible with existing proteasome inhibitors. Our results highlight differences in the subunit dependency of the parasite and human proteasome, thus providing a strategy for development of potent antimalarial drugs with overall low host toxicity.

  7. Permeability transition in human mitochondria persists in the absence of peripheral stalk subunits of ATP synthase.

    Science.gov (United States)

    He, Jiuya; Carroll, Joe; Ding, Shujing; Fearnley, Ian M; Walker, John E

    2017-08-22

    The opening of a nonspecific channel, known as the permeability transition pore (PTP), in the inner membranes of mitochondria can be triggered by calcium ions, leading to swelling of the organelle, disruption of the inner membrane and ATP synthesis, and cell death. Pore opening can be inhibited by cyclosporin A mediated via cyclophilin D. It has been proposed that the pore is associated with the dimeric ATP synthase and the oligomycin sensitivity conferral protein (OSCP), a component of the enzyme's peripheral stalk, provides the site at which cyclophilin D interacts. Subunit b contributes a central α-helical structure to the peripheral stalk, extending from near the top of the enzyme's catalytic domain and crossing the membrane domain of the enzyme via two α-helices. We investigated the possible involvement of the subunit b and the OSCP in the PTP by generating clonal cells, HAP1-Δb and HAP1-ΔOSCP, lacking the membrane domain of subunit b or the OSCP, respectively, in which the corresponding genes, ATP5F1 and ATP5O , had been disrupted. Both cell lines preserve the characteristic properties of the PTP; therefore, the membrane domain of subunit b does not contribute to the PTP, and the OSCP does not provide the site of interaction with cyclophilin D. The membrane subunits ATP6, ATP8, and subunit c have been eliminated previously from possible participation in the PTP; thus, the only subunits of ATP synthase that could participate in pore formation are e, f, g, diabetes-associated protein in insulin-sensitive tissues (DAPIT), and the 6.8-kDa proteolipid.

  8. The complete structure of the large subunit of the mammalian mitochondrial ribosome.

    Science.gov (United States)

    Greber, Basil J; Boehringer, Daniel; Leibundgut, Marc; Bieri, Philipp; Leitner, Alexander; Schmitz, Nikolaus; Aebersold, Ruedi; Ban, Nenad

    2014-11-13

    Mitochondrial ribosomes (mitoribosomes) are extensively modified ribosomes of bacterial descent specialized for the synthesis and insertion of membrane proteins that are critical for energy conversion and ATP production inside mitochondria. Mammalian mitoribosomes, which comprise 39S and 28S subunits, have diverged markedly from the bacterial ribosomes from which they are derived, rendering them unique compared to bacterial, eukaryotic cytosolic and fungal mitochondrial ribosomes. We have previously determined at 4.9 Å resolution the architecture of the porcine (Sus scrofa) 39S subunit, which is highly homologous to the human mitoribosomal large subunit. Here we present the complete atomic structure of the porcine 39S large mitoribosomal subunit determined in the context of a stalled translating mitoribosome at 3.4 Å resolution by cryo-electron microscopy and chemical crosslinking/mass spectrometry. The structure reveals the locations and the detailed folds of 50 mitoribosomal proteins, shows the highly conserved mitoribosomal peptidyl transferase active site in complex with its substrate transfer RNAs, and defines the path of the nascent chain in mammalian mitoribosomes along their idiosyncratic exit tunnel. Furthermore, we present evidence that a mitochondrial tRNA has become an integral component of the central protuberance of the 39S subunit where it architecturally substitutes for the absence of the 5S ribosomal RNA, a ubiquitous component of all cytoplasmic ribosomes.

  9. Results Assessment of Intercomparison Exercise CSN/CIEMAT-2013 among Spanish National Laboratories of Environmental Radioactivity (Air)

    International Nuclear Information System (INIS)

    Trinidad, J. A.; Gascó, C.; Llauradó, M.

    2015-01-01

    This report describes the results assessment of the intercomparsion exercise among environmental radioactivity laboratories, organised by Spanish Regulatory Institution (CSN) and prepared and evaluated by UB and CIEMAT respectively. The exercise has been carried out following the international standards ISO-43 and ISO/IUPAC that provide a useful guide to perform proficiency tests and inter-laboratories comparisons. The selected matrix for this year (2013) was filters, which was enriched with artificial radionuclides (137Cs, 60Co and 57Co) and contained natural radionuclides (234U, 238U, U-natural 230Th, 226Ra, 210Pb, 234Th, 214Bi and 214Pb) at environmental level of activity concentration. Three commonly used filters (47 mm diameter, 44x44 cm2 and 20x25 cm2) were prepared. Two 47 mm diameter filter were prepared to separate 226Ra and 210Pb analysis. The z-score test was applied to determine how much the laboratories differ from the reference value. The reference value for this exercise was the median of the results from the different laboratories and their standard deviations to achieve a more complete and objective study of the laboratories performance. The participant laboratories have demonstrated a satisfactory quality level for measuring the natural and artificial radionuclides content in this matrix. The study has showed a homogeneous behaviour of the laboratories

  10. Results Assessment of Intercomparison Exercise CSN/CIEMAT-2012 among Spanish National Laboratories of Environmental Radioactivity (Soil)

    International Nuclear Information System (INIS)

    Trinidad, J. A.; Gascó, C.; Llauradó, M.

    2015-01-01

    This report describes the results assessment of the intercomparsion exercise among environmental radioactivity laboratories, organised by Spanish Regulatory Institution (CSN) and prepared and evaluated by UB and CIEMAT respectively. The exercise has been carried out following the international standards ISO-43 and ISO/IUPAC that provide a useful guide to perform proficiency tests and inter-laboratories comparisons. The selected matrix for this year (2012) was soil, that was enriched with artificial radionuclides (137Cs, 60Co, 55Fe, 63Ni, 90Sr, 241Am, 239+240Pu and 238Pu) and contained natural radionuclides (234U, 238U, U-natural 230Th, 226Ra, 210Pb, 228Ra, 228Ac, 234Th, 214Bi, 214Pb, 212Pb, 208Tl and 40K) at environmental level of activity concentration. Two soil matrixes were prepared in order to separate 55Fe and 63Ni analysis. The z-score test was applied to determine how much the laboratories differ from the reference value. The reference value for this exercise was the median of the results from the different laboratories and their standard deviations to achieve a more complete and objective study of the laboratories performance. The participant laboratories have demonstrated a satisfactory quality level for measuring the natural and artificial radionuclides content in this matrix. The study has showed a homogeneous behaviour of the laboratories.

  11. Molecular dynamics studies of the P pilus rod subunit PapA.

    Science.gov (United States)

    Vitagliano, Luigi; Ruggiero, Alessia; Pedone, Carlo; Berisio, Rita

    2009-03-01

    Adhesion of uropathogenic Escherichia coli to host tissues is mediated by pili, which extend from the outer cell membrane of the bacterium. Here we report molecular dynamics (MD) characterizations of the major constituent of P pili from the uropathogenic E. coli, PapA, in unliganded state and in complex with the G1 strand of the chaperone PapD. To mimic the PapA response to the gradual dissociation of the PapD G1 strand and to evaluate the role of PapA chaperone recognition sites, we also carried out MD simulations of complexes of PapA with fragments of PapD G1 strand, that leave either the P4 or both P3 and P4 sites unoccupied. Data on the unbound form of PapA indicate that, upon release of the chaperone, PapA evolves toward compact states that are likely not prone to subunit-subunit association. In line with recent experimental reports, this finding implies that chaperone release and subunit-subunit association must be concerted. Our data also indicated that the gradual unbinding of the chaperone from the PapA groove has increasingly strong structural consequences. Indeed, the release of the chaperone from the site P4, which is closest to the initiation site (P5), does not have dramatic effects on the domain structure, whereas its release from both the P4 and the adjacent P3 sites induces a quick structural transition toward a collapsed state, where the subunit groove is obstructed.

  12. Myristoylated α subunits of guanine nucleotide-binding regulatory proteins

    International Nuclear Information System (INIS)

    Buss, J.E.; Mumby, S.M.; Casey, P.J.; Gilman, A.G.; Sefton, B.M.

    1987-01-01

    Antisera directed against specific subunits of guanine nucleotide-binding regulatory proteins (G proteins) were used to immunoprecipitate these polypeptides from metabolically labeled cells. This technique detects, in extracts of a human astrocytoma cell line, the α subunits of G/sub s/ (stimulatory) (α 45 and α 52 ), a 41-kDa subunit of G/sub i/ (inhibitory) (α 41 ), a 40-kDa protein (α 40 ), and the 36-kDa β subunit. No protein that comigrated with the α subunit of G 0 (unknown function) (α 39 ) was detected. In cells grown in the presence of [ 3 H]myristic acid, α 41 and α 40 contained 3 H label, while the β subunit did not. Chemical analysis of lipids attached covalently to purified α 41 and α 39 from bovine brain also revealed myristic acid. Similar analysis of brain G protein β and γ subunits and of G/sub t/ (Transducin) subunits (α, β, and γ) failed to reveal fatty acids. The fatty acid associated with α 41 , α 40 , and α 39 was stable to treatment with base, suggesting that the lipid is linked to the polypeptide via an amide bond. These GTP binding proteins are thus identified as members of a select group of proteins that contains myristic acid covalently attached to the peptide backbone. Myristate may play an important role in stabilizing interactions of G proteins with phospholipid or with membrane-bound proteins

  13. The Subunit Principle in Scar Face Revision.

    Science.gov (United States)

    Elshahat, Ahmed; Lashin, Riham

    2017-06-01

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

  14. Analysis of Hydraulic Flood Control Structure at Putat Boro River

    OpenAIRE

    Ruzziyatno, Ruhban

    2015-01-01

    Putat Boro River is one of the main drainage systems of Surakarta city which drains into Bengawan Solo river. The primary problem when flood occur is the higher water level of Bengawan Solo than Boro River and then backwater occur and inundates Putat Boro River. The objective of the study is to obtain operational method of Putat Boro River floodgate to control both inflows and outflows not only during flood but also normal condition. It also aims to know the Putat Boro rivers floodgate op...

  15. A novel two-step mechanism for removal of a mitochondrial signal sequence involves the mAAA complex and the putative rhomboid protease Pcp1.

    Science.gov (United States)

    Esser, Karlheinz; Tursun, Baris; Ingenhoven, Martin; Michaelis, Georg; Pratje, Elke

    2002-11-08

    The yeast protein cytochrome c peroxidase (Ccp1) is nuclearly encoded and imported into the mitochondrial intermembrane space, where it is involved in degradation of reactive oxygen species. It is known, that Ccp1 is synthesised as a precursor with a N-terminal pre-sequence, that is proteolytically removed during transport of the protein. Here we present evidence for a new processing pathway, involving novel signal peptidase activities. The mAAA protease subunits Yta10 (Afg3) and Yta12 (Rca1) were identified both to be essential for the first processing step. In addition, the Pcp1 (Ygr101w) gene product was found to be required for the second processing step, yielding the mature Ccp1 protein. The newly identified Pcp1 protein belongs to the rhomboid-GlpG superfamily of putative intramembrane peptidases. Inactivation of the protease motifs in mAAA and Pcp1 blocks the respective steps of proteolysis. A model of coupled Ccp1 transport and N-terminal processing by the mAAA complex and Pcp1 is discussed. Similar processing mechanisms may exist, because the mAAA subunits and the newly identified Pcp1 protein belong to ubiquitous protein families.

  16. Immunodominant role of CCHA subunit of Concholepas hemocyanin is associated with unique biochemical properties.

    Science.gov (United States)

    Becker, María Inés; Fuentes, Alejandra; Del Campo, Miguel; Manubens, Augusto; Nova, Esteban; Oliva, Harold; Faunes, Fernando; Valenzuela, María Antonieta; Campos-Vallette, Marcelo; Aliaga, Alvaro; Ferreira, Jorge; De Ioannes, Alfredo E; De Ioannes, Pablo; Moltedo, Bruno

    2009-03-01

    Hemocyanin, the oxygen transporter metallo-glycoprotein from mollusks, shows strong relationship between its notable structural features and intrinsic immunomodulatory effects. Here we investigated the individual contribution of CCHA and CCHB subunits from Concholepas hemocyanin (CCH) to in vivo humoral immune response and their pre-clinical evaluation as immunotherapeutic agent in a mice bladder cancer model, in relation to their biochemical properties. To this end, subunits were purified and well characterized. Homogeneous subunits were obtained by anionic exchange chromatography, and its purity assessed by electrophoretic and immunochemical methods. While each CCH subunit contains eight functional units showing partial cross reaction, the vibrational spectral analysis showed several spectral differences, suggesting structural differences between them. In addition, we demonstrated differences in the carbohydrate content: CCHA had a 3.6% w/w sugar with both N- and O-linked moieties. In turn, CCHB had a 2.5% w/w sugar with N-linked, while O-linked moieties were nearly absent. Considering these differences, it was not possible to predict a priori whether the immunogenic and immunotherapeutic properties of subunits might be similar. Surprisingly, both subunits by itself induced a humoral response, and showed an antitumor effect in the bladder carcinoma cell line MBT-2. However, when immunologic parameters were analyzed, CCHA showed better efficiency than CCHB. No allergic reactions or any toxic effects were observed in mice treated with CCHA, sustaining its potential therapeutic use. Our study supports that CCHA subunit accounts for the most important features involved in the immunogenicity of CCH, such as better hydrophilicity and higher content of carbohydrates.

  17. Twenty putative palmitoyl-acyl transferase genes with distinct ...

    African Journals Online (AJOL)

    There are 20 genes containing DHHC domain predicted to encode putative palmitoyltransferase in Arabidopsis thaliana genome. However, little is known about their characteristics such as genetic relationship and expression profile. Here, we present an overview of the putative PAT genes in A. thaliana focusing on their ...

  18. Differential expressions of putative genes in various floral organs of ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-06-03

    Jun 3, 2009 ... Full Length Research Paper. Differential expressions of putative genes in various floral organs of the Pigeon orchid (Dendrobium crumenatum) using GeneFishing. Faridah, Q. Z.1, 2, Ng, B. Z.3, Raha, A. R.4, Umi, K. A. B.5 and Khosravi, A. R.2*. 1Department of Biology, Faculty Science, University Putra ...

  19. Jab1/Csn5-Thioredoxin Signaling in Relapsed Acute Monocytic Leukemia under Oxidative Stress.

    Science.gov (United States)

    Zhou, Fuling; Pan, Yunbao; Wei, Yongchang; Zhang, Ronghua; Bai, Gaigai; Shen, Qiuju; Meng, Shan; Le, Xiao-Feng; Andreeff, Michael; Claret, Francois X

    2017-08-01

    Purpose: High levels of ROS and ineffective antioxidant systems contribute to oxidative stress, which affects the function of hematopoietic cells in acute myeloid leukemia (AML); however, the mechanisms by which ROS lead to malignant transformation in relapsed AML-M5 are not completely understood. We hypothesized that alterations in intracellular ROS would trigger AML-M5 relapse by activating the intrinsic pathway. Experimental Design: We studied ROS levels and conducted c-Jun activation domain-binding protein-1 ( JAB1/COPS5 ) and thioredoxin ( TRX ) gene expression analyses with blood samples obtained from 60 matched AML-M5 patients at diagnosis and relapse and conducted mechanism studies of Jab1's regulation of Trx in leukemia cell lines. Results: Our data showed that increased production of ROS and a low capacity of antioxidant enzymes were characteristics of AML-M5, both at diagnosis and at relapse. Consistently, increased gene expression levels of TRX and JAB1/COPS5 were associated with low overall survival rates in patients with AML-M5. In addition, stimulating AML-M5 cells with low concentrations of hydrogen peroxide led to increased Jab1 and Trx expression. Consistently, transfection of ectopic Jab1 into leukemia cells increased Trx expression, whereas silencing of Jab1 in leukemia cells reduced Trx expression. Mechanistically, Jab1 interacted with Trx and stabilized Trx protein. Moreover, Jab1 transcriptionally regulated Trx. Furthermore, depletion of Jab1 inhibited leukemia cell growth both in vitro and in vivo Conclusions: We identified a novel Jab1-Trx axis that is a key cellular process in the pathobiologic characteristics of AML-M5. Targeting the ROS/Jab1/Trx pathway could be beneficial in the treatment of AML-M5. Clin Cancer Res; 23(15); 4450-61. ©2017 AACR . ©2017 American Association for Cancer Research.

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

    Directory of Open Access Journals (Sweden)

    Carson Kai-Kwong Ley

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

  1. Results Assessment of Intercomparison Exercise CSN/CIEMAT-2010 among Spanish National Laboratories of Environmental Radioactivity (Diet Ashes); Evaluacion de la Intercomparacion CSN/CIEMAT-2010 entre los Laboratorios Nacionales de Radiactividad Ambiental (Ceniza de Dieta)

    Energy Technology Data Exchange (ETDEWEB)

    Gasco, C.; Trinidad, J. A.; Llaurado, M.; Suarez, J. A.

    2012-06-08

    This report describes the results assessment of the intercomparison exercise among environmental radioactivity laboratories, organised by Spanish Regulatory Institution (CSN) and prepared and evaluated by UAB and CIEMAT respectively. The exercise has been carried out following the international standards ISO-43 and ISO/IUPAC that provide a useful guide to perform proficiency tests and inter-laboratories comparisons. The selected matrix for this year (2010) was a diet ash obtained from the ashing of a whole fresh diet (breakfast, lunch and dinner), that was enriched with artificial radionuclides (Cs-137, Co-60,Fe-55,Ni-63,Sr-90,Am-241,Pu-238,Pu-239,240 y C-14) and contained natural radionuclides (U-234, U-238, U-natural Th-230, Th-234, Ra-226, Ra-228, Pb-210, Pb-212, Pb-214, Bi-214, Ac-228, Tl-208, K-40) at environmental level of activity concentration. The z-score test was applied to determine how much the laboratories differ from the reference value. The reference value for this exercise was the median of the results from the different laboratories and their standard deviations to achieve a more complete and objective study of the laboratories performance. The participant laboratories have demonstrated a satisfactory quality level for measuring the natural and artificial radionuclides content in this matrix. The reference values obtained through the medians show a negative bias for Pb-210 and Th-234 when comparing to the given values of external qualified laboratories from ENEA and IRSN and positive one for K-40. (Author)

  2. Dis3- and exosome subunit-responsive 3′ mRNA instability elements

    International Nuclear Information System (INIS)

    Kiss, Daniel L.; Hou, Dezhi; Gross, Robert H.; Andrulis, Erik D.

    2012-01-01

    Highlights: ► Successful use of a novel RNA-specific bioinformatic tool, RNA SCOPE. ► Identified novel 3′ UTR cis-acting element that destabilizes a reporter mRNA. ► Show exosome subunits are required for cis-acting element-mediated mRNA instability. ► Define precise sequence requirements of novel cis-acting element. ► Show that microarray-defined exosome subunit-regulated mRNAs have novel element. -- Abstract: Eukaryotic RNA turnover is regulated in part by the exosome, a nuclear and cytoplasmic complex of ribonucleases (RNases) and RNA-binding proteins. The major RNase of the complex is thought to be Dis3, a multi-functional 3′–5′ exoribonuclease and endoribonuclease. Although it is known that Dis3 and core exosome subunits are recruited to transcriptionally active genes and to messenger RNA (mRNA) substrates, this recruitment is thought to occur indirectly. We sought to discover cis-acting elements that recruit Dis3 or other exosome subunits. Using a bioinformatic tool called RNA SCOPE to screen the 3′ untranslated regions of up-regulated transcripts from our published Dis3 depletion-derived transcriptomic data set, we identified several motifs as candidate instability elements. Secondary screening using a luciferase reporter system revealed that one cassette—harboring four elements—destabilized the reporter transcript. RNAi-based depletion of Dis3, Rrp6, Rrp4, Rrp40, or Rrp46 diminished the efficacy of cassette-mediated destabilization. Truncation analysis of the cassette showed that two exosome subunit-sensitive elements (ESSEs) destabilized the reporter. Point-directed mutagenesis of ESSE abrogated the destabilization effect. An examination of the transcriptomic data from exosome subunit depletion-based microarrays revealed that mRNAs with ESSEs are found in every up-regulated mRNA data set but are underrepresented or missing from the down-regulated data sets. Taken together, our findings imply a potentially novel mechanism of m

  3. Distribution and innervation of putative peripheral arterial chemoreceptors in the red-eared slider (Trachemys scripta elegans).

    Science.gov (United States)

    Reyes, Catalina; Fong, Angelina Y; Milsom, William K

    2015-06-15

    Peripheral arterial chemoreceptors have been isolated to the common carotid artery, aorta, and pulmonary artery of turtles. However, the putative neurotransmitters associated with these chemoreceptors have not yet been described. The goal of the present study was to determine the neurochemical content, innervations, and distribution of putative oxygen-sensing cells in the central vasculature of turtles and to derive homologies with peripheral arterial chemoreceptors of other vertebrates. We used tract tracing together with immunohistochemical markers for cholinergic cells (vesicular acetylcholine transporter [VAChT]), tyrosine hydroxylase (TH; the rate-limiting enzyme in catecholamine synthesis), and serotonin (5HT) to identify putative oxygen-sensing cells and to determine their anatomical relation to branches of the vagus nerve (Xth cranial nerve). We found potential oxygen-sensing cells in all three chemosensory areas innervated by branches of the Xth cranial nerve. Cells containing either 5HT or VAChT were found in all three sites. The morphology and size of these cells resemble glomus cells found in amphibians, mammals, tortoises, and lizards. Furthermore, we found populations of cholinergic cells located at the base of the aorta and pulmonary artery that are likely involved in efferent regulation of vessel resistance. Catecholamine-containing cells were not found in any of the putative chemosensitive areas. The presence of 5HT- and VAChT-immunoreactive cells in segments of the common carotid artery, aorta, and pulmonary artery appears to reflect a transition between cells containing the major neurotransmitters seen in fish (5HT) and mammals (ACh and adenosine). © 2015 Wiley Periodicals, Inc.

  4. Genetic analysis of the cytoplasmic dynein subunit families.

    Science.gov (United States)

    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.

  5. Genetic analysis of the cytoplasmic dynein subunit families.

    Directory of Open Access Journals (Sweden)

    K Kevin Pfister

    2006-01-01

    Full Text Available 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.

  6. Hydraulic Analysis of the Contribution of Emergency Water to C. N. Almaraz Systems Affected as a Result of the Complementary Technical Instructions issued by the CSN after Fukushima

    International Nuclear Information System (INIS)

    Vilar Carmona, G.; Puertas Munoz, S.; Arguello Tara, A.; Sanz Roman, F. J.

    2013-01-01

    This paper presents the study and hydraulic analysis of the capacity required contribution of emergency water to the Almaraz NPP to power systems deal with the accidental events outside the bases of design defined in the Complementary technical instructions generated by the CSN after Fukushima. Through the program of balanced hydraulic SBAL, developed by entrepreneurs Grouped (EE.AA) and used in multiple security systems analysis, and based on designs and requirements to be fulfilled by the Almaraz NPP of the different strategies are set, have developed a series of hydraulic models that they have allowed the definition and dimensioning of the portable media and the new connections required in the central systems.

  7. GABA-A receptor beta3 and alpha5 subunit gene cluster on chromosome 15q11-q13 and bipolar disorder: a genetic association study.

    Science.gov (United States)

    Papadimitriou, G N; Dikeos, D G; Karadima, G; Avramopoulos, D; Daskalopoulou, E G; Stefanis, C N

    2001-05-08

    There is accumulated evidence that the genes coding for the receptor of gamma aminobutyric acid (GABA), the most important inhibitory neurotransmitter in the CNS, may be involved in the pathogenesis of affective disorders. In a previous study, we have found a genetic association between the GABA-A receptor alpha5 subunit gene locus (GABRA5) on chromosome 15q11-of 13 and bipolar affective disorder. The aim of the present study was to examine the same subjects to see if there exists a genetic association between bipolar affective disorder and the GABA receptor beta3 subunit gene (GABRB3), which is located within 100 kb from GABRA5. The sample consisted of 48 bipolar patients compared to 44 controls (blood donors). All subjects were Greek, unrelated, and personally interviewed. Diagnosis was based on DSM-IV and ICD-10 criteria. The marker used was a dinucleotide (CA) repeat polymorphism with 12 alleles 179 to 201 bp long; genotyping was successful in all patients and 43 controls. The distribution of GABRB3 genotypes among the controls did not deviate significantly from the Hardy-Weinberg equilibrium. No differences in allelic frequencies between bipolar patients and controls were found for GABRB3, while this locus and GABRA5 did not seem to be in significant linkage disequilibrium. In conclusion, the GABRB3 CA-repeat polymorphism we investigated does not present the observed association between bipolar affective illness and GABRA5. This could be due to higher mutation rate in the GABRB3 CA-repeat polymorphism, but it might also signify that GABRA5 is the gene actually associated with the disease. Copyright 2001 Wiley-Liss, Inc.

  8. Reactivation of the chloroplast CF1-ATPase beta subunit by trace amounts of the CF1 alpha subunit suggests a chaperonin-like activity for CF1 alpha.

    Science.gov (United States)

    Avni, A; Avital, S; Gromet-Elhanan, Z

    1991-04-25

    Incubation of tobacco and lettuce thylakoids with 2 M LiCl in the presence of MgATP removes the beta subunit from their CF1-ATPase (CF1 beta) together with varying amounts of the CF1 alpha subunit (CF1 alpha). These 2 M LiCl extracts, as with the one obtained from spinach thylakoids (Avital, S., and Gromet-Elhanan, Z. (1991) J. Biol. Chem. 266, 7067-7072), could form active hybrid ATPases when reconstituted into inactive beta-less Rhodospirillum rubrum chromatophores. Pure CF1 beta fractions that have been isolated from these extracts could not form such active hybrids by themselves, but could do so when supplemented with trace amounts (less than 5%) of CF1 alpha. A mitochondrial F1-ATPase alpha subunit was recently reported to be a heat-shock protein, having two amino acid sequences that show a highly conserved identity with sequences found in molecular chaperones (Luis, A. M., Alconada, A., and Cuezva, J. M. (1990) J. Biol. Chem. 265, 7713-7716). These sequences are also conserved in CF1 alpha isolated from various plants, but not in F1 beta subunits. The above described reactivation of CF1 beta by trace amounts of CF1 alpha could thus be due to a chaperonin-like function of CF1 alpha, which involves the correct, active folding of isolated pure CF1 beta.

  9. Roles of the β subunit hinge domain in ATP synthase F1 sector: Hydrophobic network formed by introduced βPhe174 inhibits subunit rotation

    International Nuclear Information System (INIS)

    Nakanishi-Matsui, Mayumi; Kashiwagi, Sachiko; Kojima, Masaki; Nonaka, Takamasa; Futai, Masamitsu

    2010-01-01

    The ATP synthase β subunit hinge domain (βPhe148 ∼ βGly186, P-loop/α-helixB/loop/β-sheet4, Escherichia coli residue numbering) dramatically changes in conformation upon nucleotide binding. We previously reported that F 1 with the βSer174 to Phe mutation in the domain lowered the γ subunit rotation speed, and thus decreased the ATPase activity [M. Nakanishi-Matsui, S. Kashiwagi, T. Ubukata, A. Iwamoto-Kihara, Y. Wada, M. Futai, Rotational catalysis of Escherichia coli ATP synthase F 1 sector. Stochastic fluctuation and a key domain of the β subunit, J. Biol. Chem. 282 (2007) 20698-20704.]. Homology modeling indicates that the amino acid replacement induces a hydrophobic network, in which the βMet159, βIle163, and βAla167 residues of the β subunit are involved together with the mutant βPhe174. The network is expected to stabilize the conformation of β DP (nucleotide-bound form of the β subunit), resulting in increased activation energy for transition to β E (empty β subunit). The modeling further predicts that replacement of βMet159 with Ala or Ile weakens the hydrophobic network. As expected, these two mutations experimentally suppressed the ATPase activities as well as subunit rotation of βS174F. Furthermore, the rotation rate decreased with the increase of the strength in the hydrophobic network. These results indicate that the smooth conformational change of the β subunit hinge domain is pertinent for the rotational catalysis.

  10. Studies on the subunits of human glycoprotein hormones in relation to reproduction

    International Nuclear Information System (INIS)

    Hagen, C.

    1977-01-01

    In this review summarising present knowledge of the biological and immunological activity of the subunits of human glycoprotein hormones, the specificity of the α-subunit and β-subunit radioimmunoassays are discussed. The crossreaction studies performed with the α-subunit radioimmunoassays are aummarised in one table while those with the β-subunit radioimmunoassays are presented in a second table. (JIW)

  11. Regulation of KV channel voltage-dependent activation by transmembrane β subunits

    Directory of Open Access Journals (Sweden)

    Xiaohui eSun

    2012-04-01

    Full Text Available Voltage-activated K+ (KV channels are important for shaping action potentials and maintaining resting membrane potential in excitable cells. KV channels contain a central pore-gate domain (PGD surrounded by four voltage-sensing domains (VSD. The VSDs will change conformation in response to alterations of the membrane potential thereby inducing the opening of the PGD. Many KV channels are heteromeric protein complexes containing auxiliary β subunits. These β subunits modulate channel expression and activity to increase functional diversity and render tissue specific phenotypes. This review focuses on the KV β subunits that contain transmembrane (TM segments including the KCNE family and the β subunits of large conductance, Ca2+- and voltage-activated K+ (BK channels. These TM β subunits affect the voltage-dependent activation of KV α subunits. Experimental and computational studies have described the structural location of these β subunits in the channel complexes and the biophysical effects on VSD activation, PGD opening and VSD-PGD coupling. These results reveal some common characteristics and mechanistic insights into KV channel modulation by TM β subunits.

  12. Putative resistance gene markers associated with quantitative trait loci for fire blight resistance in Malus ‘Robusta 5’ accessions

    Science.gov (United States)

    2012-01-01

    Background Breeding of fire blight resistant scions and rootstocks is a goal of several international apple breeding programs, as options are limited for management of this destructive disease caused by the bacterial pathogen Erwinia amylovora. A broad, large-effect quantitative trait locus (QTL) for fire blight resistance has been reported on linkage group 3 of Malus ‘Robusta 5’. In this study we identified markers derived from putative fire blight resistance genes associated with the QTL by integrating further genetic mapping studies with bioinformatics analysis of transcript profiling data and genome sequence databases. Results When several defined E.amylovora strains were used to inoculate three progenies from international breeding programs, all with ‘Robusta 5’ as a common parent, two distinct QTLs were detected on linkage group 3, where only one had previously been mapped. In the New Zealand ‘Malling 9’ X ‘Robusta 5’ population inoculated with E. amylovora ICMP11176, the proximal QTL co-located with SNP markers derived from a leucine-rich repeat, receptor-like protein ( MxdRLP1) and a closely linked class 3 peroxidase gene. While the QTL detected in the German ‘Idared’ X ‘Robusta 5’ population inoculated with E. amylovora strains Ea222_JKI or ICMP11176 was approximately 6 cM distal to this, directly below a SNP marker derived from a heat shock 90 family protein gene ( HSP90). In the US ‘Otawa3’ X ‘Robusta5’ population inoculated with E. amylovora strains Ea273 or E2002a, the position of the LOD score peak on linkage group 3 was dependent upon the pathogen strains used for inoculation. One of the five MxdRLP1 alleles identified in fire blight resistant and susceptible cultivars was genetically associated with resistance and used to develop a high resolution melting PCR marker. A resistance QTL detected on linkage group 7 of the US population co-located with another HSP90 gene-family member and a WRKY transcription factor

  13. Hepatic expression of proteasome subunit alpha type-6 is upregulated during viral hepatitis and putatively regulates the expression of ISG15 ubiquitin-like modifier, a proviral host gene in hepatitis C virus infection.

    Science.gov (United States)

    Broering, R; Trippler, M; Werner, M; Real, C I; Megger, D A; Bracht, T; Schweinsberg, V; Sitek, B; Eisenacher, M; Meyer, H E; Baba, H A; Weber, F; Hoffmann, A-C; Gerken, G; Schlaak, J F

    2016-05-01

    The interferon-stimulated gene 15 (ISG15) plays an important role in the pathogenesis of hepatitis C virus (HCV) infection. ISG15-regulated proteins have previously been identified that putatively affect this proviral interaction. The present observational study aimed to elucidate the relation between ISG15 and these host factors during HCV infection. Transcriptomic and proteomic analyses were performed using liver samples of HCV-infected (n = 54) and uninfected (n = 10) or HBV-infected controls (n = 23). Primary human hepatocytes (PHH) were treated with Toll-like receptor ligands, interferons and kinase inhibitors. Expression of ISG15 and proteasome subunit alpha type-6 (PSMA6) was suppressed in subgenomic HCV replicon cell lines using specific siRNAs. Comparison of hepatic expression patterns revealed significantly increased signals for ISG15, IFIT1, HNRNPK and PSMA6 on the protein level as well as ISG15, IFIT1 and PSMA6 on the mRNA level in HCV-infected patients. In contrast to interferon-stimulated genes, PSMA6 expression occurred independent of HCV load and genotype. In PHH, the expression of ISG15 and PSMA6 was distinctly induced by poly(I:C), depending on IRF3 activation or PI3K/AKT signalling, respectively. Suppression of PSMA6 in HCV replicon cells led to significant induction of ISG15 expression, thus combined knock-down of both genes abrogated the antiviral effect induced by the separate suppression of ISG15. These data indicate that hepatic expression of PSMA6, which is upregulated during viral hepatitis, likely depends on TLR3 activation. PSMA6 affects the expression of immunoregulatory ISG15, a proviral factor in the pathogenesis of HCV infection. Therefore, the proteasome might be involved in the enigmatic interaction between ISG15 and HCV. © 2016 John Wiley & Sons Ltd.

  14. Development of a Subunit Vaccine for Contagious Bovine ...

    International Development Research Centre (IDRC) Digital Library (Canada)

    Their work has set the stage for commercial development of a sub-unit vaccine. ... The sub-unit vaccine will be cost-effective, easy to produce, and safe. How it will make a ... IDRC invites applications for the IDRC Doctoral Research Awards.

  15. Organization and alternative splicing of the Caenorhabditis elegans cAMP-dependent protein kinase catalytic-subunit gene (kin-1).

    Science.gov (United States)

    Tabish, M; Clegg, R A; Rees, H H; Fisher, M J

    1999-04-01

    The cAMP-dependent protein kinase (protein kinase A, PK-A) is multifunctional in nature, with key roles in the control of diverse aspects of eukaryotic cellular activity. In the case of the free-living nematode, Caenorhabditis elegans, a gene encoding the PK-A catalytic subunit has been identified and two isoforms of this subunit, arising from a C-terminal alternative-splicing event, have been characterized [Gross, Bagchi, Lu and Rubin (1990) J. Biol. Chem. 265, 6896-6907]. Here we report the occurrence of N-terminal alternative-splicing events that, in addition to generating a multiplicity of non-myristoylatable isoforms, also generate the myristoylated variant(s) of the catalytic subunit that we have recently characterized [Aspbury, Fisher, Rees and Clegg (1997) Biochem. Biophys. Res. Commun. 238, 523-527]. The gene spans more than 36 kb and is divided into a total of 13 exons. Each of the mature transcripts contains only 7 exons. In addition to the already characterized exon 1, the 5'-untranslated region and first intron actually contain 5 other exons, any one of which may be alternatively spliced on to exon 2 at the 5' end of the pre-mRNA. This N-terminal alternative splicing occurs in combination with either of the already characterized C-terminal alternative exons. Thus, C. elegans expresses at least 12 different isoforms of the catalytic subunit of PK-A. The significance of this unprecedented structural diversity in the family of PK-A catalytic subunits is discussed.

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

    Directory of Open Access Journals (Sweden)

    Signe Tandrup Schmidt

    2016-03-01

    Full Text Available The development of subunit vaccines has become very attractive in recent years due to their superior safety profiles as compared to traditional vaccines based on live attenuated or whole inactivated pathogens, and there is an unmet medical need for improved vaccines and vaccines against pathogens for which no effective vaccines exist. The subunit vaccine technology exploits pathogen subunits as antigens, e.g., recombinant proteins or synthetic peptides, allowing for highly specific immune responses against the pathogens. However, such antigens are usually not sufficiently immunogenic to induce protective immunity, and they are often combined with adjuvants to ensure robust immune responses. Adjuvants are capable of enhancing and/or modulating immune responses by exposing antigens to antigen-presenting cells (APCs concomitantly with conferring immune activation signals. Few adjuvant systems have been licensed for use in human vaccines, and they mainly stimulate humoral immunity. Thus, there is an unmet demand for the development of safe and efficient adjuvant systems that can also stimulate cell-mediated immunity (CMI. Adjuvants constitute a heterogeneous group of compounds, which can broadly be classified into delivery systems or immunostimulators. Liposomes are versatile delivery systems for antigens, and they can carefully be customized towards desired immune profiles by combining them with immunostimulators and optimizing their composition, physicochemical properties and antigen-loading mode. Immunostimulators represent highly diverse classes of molecules, e.g., lipids, nucleic acids, proteins and peptides, and they are ligands for pattern-recognition receptors (PRRs, which are differentially expressed on APC subsets. Different formulation strategies might thus be required for incorporation of immunostimulators and antigens, respectively, into liposomes, and the choice of immunostimulator should ideally be based on knowledge regarding the

  17. A putative SUMO interacting motif in the B30.2/SPRY domain of rhesus macaque TRIM5α important for NF-κB/AP-1 signaling and HIV-1 restriction

    Directory of Open Access Journals (Sweden)

    Marie-Édith Nepveu-Traversy

    2016-01-01

    Full Text Available TRIM5α from the rhesus macaque (TRIM5αRh is a restriction factor that shows strong activity against HIV-1. TRIM5αRh binds specifically to HIV-1 capsid (CA through its B30.2/PRYSPRY domain shortly after entry of the virus into the cytoplasm. Recently, three putative SUMO interacting motifs (SIMs have been identified in the PRYSPRY domain of human and macaque TRIM5α. However, structural modeling of this domain suggested that two of them were buried in the hydrophobic core of the protein, implying that interaction with SUMO was implausible, while the third one was not relevant to restriction. In light of these results, we re-analyzed the TRIM5αRh PRYSPRY sequence and identified an additional putative SIM (435VIIC438 which we named SIM4. This motif is exposed at the surface of the PRYSPRY domain, allowing potential interactions with SUMO or SUMOylated proteins. Introducing a double mutation in SIM4 (V435K, I436K did not alter stability, unlike mutations in SIM1. SIM4-mutated TRIM5αRh failed to bind HIV-1CA and lost the ability to restrict this virus. Accordingly, SIM4 undergoes significant variation among primates and substituting this motif with naturally occurring SIM4 variants affected HIV-1 restriction by TRIM5αRh, suggesting a direct role in capsid recognition. Interestingly, SIM4-mutated TRIM5αRh also failed to activate NF-κB and AP-1-mediated transcription. Although there is no direct evidence that SIM4 is involved in direct interaction with SUMO or a SUMOylated protein, mutating this motif strongly reduced co-localization of TRIM5αRh with SUMO-1 and with PML, a SUMOylated nuclear protein. In conclusion, this new putative SIM is crucial for both direct interaction with incoming capsids and for NF-κB/AP-1 signaling. We speculate that the latter function is mediated by interactions of SIM4 with a SUMOylated protein involved in the NF-κB/AP-1 signaling pathways.

  18. [Expression of proteasome subunits PSMB5 and PSMB9 mRNA in hippocampal neurons in experimental diabetes mellitus: link with apoptosis and necrosis].

    Science.gov (United States)

    Lebid', Iu V; Dosenko, V Ie; Skybo, H H

    2010-01-01

    There is a huge body of evidence showing that long-termed diabetes mellitus is followed with hippocampal dysfunction. The goal of this work was to investigate the expression of proteasome subunits PSMB5 and PSMB9 mRNA in CA1, CA2 and CA3 areas of hippocampus in parallel with processes of cell death (apoptosis and necrosis) in development dynamics of streptozotocine-induced diabetes. We have studied hippocampal neurons using chromatine dye Hoechst-33342 and immunohistochemical detection of apoptotic cell death marker caspase-3. At day 3 and 7 after injection of streptozotocine we have performed visualization of caspase-3-immunopositive neurons showing signs of neurodegeneration in hippocampal sections using confocal microscope Olympus FV1000. The rate of proteasome subunits PSMB5 and PSMB9 mRNA expression was determined with RT-PCR. The results indicated elevation of PSMB9 mRNA content (from 4807 +/- 0.392 arbU up to 20,023 +/- 4949 arbU on day 3 and up to 20,253 +/- 5141 arbU on day 7). A maximal number of cells with signs of chromatin condensation was observed at day 3 and day 7 in CA2 and CA3 area (11.51% and 12.49% respectively). That indicates an intensification of proapoptotic processes. Summarizing the results presented above we can conclude that during the first week of diabetes mellitus development, hippocampal cells undergo the process of impairment and degeneration.

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

    DEFF Research Database (Denmark)

    Schmidt, Signe Tandrup; Foged, Camilla; Korsholm, Karen Smith

    2016-01-01

    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......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...... of immunostimulators and antigens, respectively, into liposomes, and the choice of immunostimulator should ideally be based on knowledge regarding the specific PRR expression profile of the target APCs. Here, we review state-of-the-art formulation approaches employed for the inclusion of immunostimulators and subunit...

  20. Interactions between subunits of Saccharomyces cerevisiae RNase MRP support a conserved eukaryotic RNase P/MRP architecture.

    Science.gov (United States)

    Aspinall, Tanya V; Gordon, James M B; Bennett, Hayley J; Karahalios, Panagiotis; Bukowski, John-Paul; Walker, Scott C; Engelke, David R; Avis, Johanna M

    2007-01-01

    Ribonuclease MRP is an endonuclease, related to RNase P, which functions in eukaryotic pre-rRNA processing. In Saccharomyces cerevisiae, RNase MRP comprises an RNA subunit and ten proteins. To improve our understanding of subunit roles and enzyme architecture, we have examined protein-protein and protein-RNA interactions in vitro, complementing existing yeast two-hybrid data. In total, 31 direct protein-protein interactions were identified, each protein interacting with at least three others. Furthermore, seven proteins self-interact, four strongly, pointing to subunit multiplicity in the holoenzyme. Six protein subunits interact directly with MRP RNA and four with pre-rRNA. A comparative analysis with existing data for the yeast and human RNase P/MRP systems enables confident identification of Pop1p, Pop4p and Rpp1p as subunits that lie at the enzyme core, with probable addition of Pop5p and Pop3p. Rmp1p is confirmed as an integral subunit, presumably associating preferentially with RNase MRP, rather than RNase P, via interactions with Snm1p and MRP RNA. Snm1p and Rmp1p may act together to assist enzyme specificity, though roles in substrate binding are also indicated for Pop4p and Pop6p. The results provide further evidence of a conserved eukaryotic RNase P/MRP architecture and provide a strong basis for studies of enzyme assembly and subunit function.

  1. Induction of 26S proteasome subunit PSMB5 by the bifunctional inducer 3-methylcholanthrene through the Nrf2-ARE, but not the AhR/Arnt-XRE, pathway

    International Nuclear Information System (INIS)

    Kwak, Mi-Kyoung; Kensler, Thomas W.

    2006-01-01

    The 26S proteasome is responsible for degradation of abnormal intracellular proteins, including oxidatively damaged proteins and may play a role as a component of a cellular antioxidative system. However, little is known about regulation of proteasome expression. In the present study, regulation of proteasome expression by the bifunctional enzyme inducer and a specific signaling pathway for this regulation were investigated in murine neuroblastoma cells. Expression of catalytic core subunits including PSMB5 and peptidase activities of the proteasome were elevated following incubation with 3-methylcholanthrene (3-MC). Studies using reporter genes containing the murine Psmb5 promoter showed that transcriptional activity of this gene was enhanced by 3-MC. Overexpression of AhR/Arnt did not affect activation of the Pmsb5 promoter by 3-MC and deletion of the xenobiotic response elements (XREs) from this promoter exerted modest effects on inducibility in response to 3-MC. However, mutation of the proximal AREs of the Psmb5 promoter largely abrogated its inducibility by 3-MC. In addition, this promoter showed a blunted response toward 3-MC in the absence of nrf2; 3-MC incubation increased nuclear levels of Nrf2 only in wild-type cells. Collectively, these results indicate that expression of proteasome subunit PSMB5 is modulated by bifunctional enzyme inducers in a manner independent of the AhR/Arnt-XRE pathway but dependent upon the Nrf2-ARE pathway

  2. Identification of EhTIF-IA: The putative E. histolytica orthologue of the human ribosomal RNA transcription initiation factor-IA.

    Science.gov (United States)

    Srivastava, Ankita; Bhattacharya, Alok; Bhattacharya, Sudha; Jhingan, Gagan Deep

    2016-03-01

    Initiation of rDNA transcription requires the assembly of a specific multi-protein complex at the rDNA promoter containing the RNA Pol I with auxiliary factors. One of these factors is known as Rrn3P in yeast and Transcription Initiation Factor IA (TIF-IA) in mammals. Rrn3p/TIF-IA serves as a bridge between RNA Pol I and the pre-initiation complex at the promoter. It is phosphorylated at multiple sites and is involved in regulation of rDNA transcription in a growth-dependent manner. In the early branching parasitic protist Entamoeba histolytica, the rRNA genes are present exclusively on circular extra chromosomal plasmids. The protein factors involved in regulation of rDNA transcription in E. histolytica are not known. We have identified the E. histolytica equivalent of TIF-1A (EhTIF-IA) by homology search within the database and was further cloned and expressed. Immuno-localization studies showed that EhTIF-IA co-localized partially with fibrillarin in the peripherally localized nucleolus. EhTIF-IA was shown to interact with the RNA Pol I-specific subunit RPA12 both in vivo and in vitro. Mass spectroscopy data identified RNA Pol I-specific subunits and other nucleolar proteins to be the interacting partners of EhTIF-IA. Our study demonstrates for the first time a conserved putative RNA Pol I transcription factor TIF-IA in E. histolytica.

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

    Science.gov (United States)

    Beier, Anna; Teichert, Ines; Krisp, Christoph; Wolters, Dirk A; Kück, Ulrich

    2016-06-21

    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. The striatin-interacting phosphatase and kinase (STRIPAK) complex is highly conserved from yeasts to humans and is an important regulator of numerous eukaryotic developmental processes, such as cellular signaling and cell development. Although functional insights into the STRIPAK complex are accumulating, the detailed molecular mechanisms of single subunits are only partially understood

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

    Science.gov (United States)

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

    2012-01-01

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

  5. Immunochemical aspects of crotoxim and its subunits

    International Nuclear Information System (INIS)

    Nakazone, A.K.

    1979-01-01

    Crotamine and crotoxin with the subunits - phospholipase A and crotapotin - were obtained by purification from Crotalus durissus terrificus venom. Interaction studies of the subunits using crotalic antiserum, indicated that: crotoxin is formed of crotapotin and phospholipase A with the molar ratio of 1 to 1; using crotapotin 125 I the presence of a soluble complex was shown with the same antiserum. Immunological precipitation reactions demonstrated that crotapotin is antigenic: crotapotin and phospholipase A presented similar antigenic determinants; crotoxin antiserum reacted with each one of the submits; when the subunits are mixed to form synthetic crotoxin some antigenic determinants are masked in the process of interaction. Crotamine, interacted with crotapotin 1:1, without hidden antigenic determinants crotapotin antigenic site seems to be formed by, at least, one lysine. Enzimatical activity of phospholipase A apreared to be dependent on some reaction conditions when its arginine residues are blocked. Tyrosines of phospholipase A are more susceptible to labelling with 131 I than crotapotin. Gama irradiation of aqueous solutions of the subunits produced modifications in the ultraviolet spectra. A decrease of the enzymatic activity occured as a function of radiation dosis. Immunological activities of crotapotin and phospholipase A were not altered [pt

  6. The Proteome of Biologically Active Membrane Vesicles from Piscirickettsia salmonis LF-89 Type Strain Identifies Plasmid-Encoded Putative Toxins

    Directory of Open Access Journals (Sweden)

    Cristian Oliver

    2017-09-01

    Full Text Available Piscirickettsia salmonis is the predominant bacterial pathogen affecting the Chilean salmonid industry. This bacterium is the etiological agent of piscirickettsiosis, a significant fish disease. Membrane vesicles (MVs released by P. salmonis deliver several virulence factors to host cells. To improve on existing knowledge for the pathogenicity-associated functions of P. salmonis MVs, we studied the proteome of purified MVs from the P. salmonis LF-89 type strain using multidimensional protein identification technology. Initially, the cytotoxicity of different MV concentration purified from P. salmonis LF-89 was confirmed in an in vivo adult zebrafish infection model. The cumulative mortality of zebrafish injected with MVs showed a dose-dependent pattern. Analyses identified 452 proteins of different subcellular origins; most of them were associated with the cytoplasmic compartment and were mainly related to key functions for pathogen survival. Interestingly, previously unidentified putative virulence-related proteins were identified in P. salmonis MVs, such as outer membrane porin F and hemolysin. Additionally, five amino acid sequences corresponding to the Bordetella pertussis toxin subunit 1 and two amino acid sequences corresponding to the heat-labile enterotoxin alpha chain of Escherichia coli were located in the P. salmonis MV proteome. Curiously, these putative toxins were located in a plasmid region of P. salmonis LF-89. Based on the identified proteins, we propose that the protein composition of P. salmonis LF-89 MVs could reflect total protein characteristics of this P. salmonis type strain.

  7. The Proteome of Biologically Active Membrane Vesicles from Piscirickettsia salmonis LF-89 Type Strain Identifies Plasmid-Encoded Putative Toxins.

    Science.gov (United States)

    Oliver, Cristian; Hernández, Mauricio A; Tandberg, Julia I; Valenzuela, Karla N; Lagos, Leidy X; Haro, Ronie E; Sánchez, Patricio; Ruiz, Pamela A; Sanhueza-Oyarzún, Constanza; Cortés, Marcos A; Villar, María T; Artigues, Antonio; Winther-Larsen, Hanne C; Avendaño-Herrera, Ruben; Yáñez, Alejandro J

    2017-01-01

    Piscirickettsia salmonis is the predominant bacterial pathogen affecting the Chilean salmonid industry. This bacterium is the etiological agent of piscirickettsiosis, a significant fish disease. Membrane vesicles (MVs) released by P. salmonis deliver several virulence factors to host cells. To improve on existing knowledge for the pathogenicity-associated functions of P. salmonis MVs, we studied the proteome of purified MVs from the P. salmonis LF-89 type strain using multidimensional protein identification technology. Initially, the cytotoxicity of different MV concentration purified from P. salmonis LF-89 was confirmed in an in vivo adult zebrafish infection model. The cumulative mortality of zebrafish injected with MVs showed a dose-dependent pattern. Analyses identified 452 proteins of different subcellular origins; most of them were associated with the cytoplasmic compartment and were mainly related to key functions for pathogen survival. Interestingly, previously unidentified putative virulence-related proteins were identified in P. salmonis MVs, such as outer membrane porin F and hemolysin. Additionally, five amino acid sequences corresponding to the Bordetella pertussis toxin subunit 1 and two amino acid sequences corresponding to the heat-labile enterotoxin alpha chain of Escherichia coli were located in the P. salmonis MV proteome. Curiously, these putative toxins were located in a plasmid region of P. salmonis LF-89. Based on the identified proteins, we propose that the protein composition of P. salmonis LF-89 MVs could reflect total protein characteristics of this P. salmonis type strain.

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

    Directory of Open Access Journals (Sweden)

    Nagy Olga

    2012-03-01

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

  9. Structural characterization of recombinant crustacyanin subunits from the lobster Homarus americanus

    International Nuclear Information System (INIS)

    Ferrari, Michele; Folli, Claudia; Pincolini, Elisa; McClintock, Timothy S.; Rössle, Manfred; Berni, Rodolfo; Cianci, Michele

    2012-01-01

    The two recombinant apo subunits H1 and H2 from H. americanus have been structurally characterized. Reconstitution studies with astaxanthin reproduced the bathochromic shift of 85–95 nm typical of the natural crustacyanin subunits. Crustacean crustacyanin proteins are linked to the production and modification of carapace colour, with direct implications for fitness and survival. Here, the structural and functional properties of the two recombinant crustacyanin subunits H 1 and H 2 from the American lobster Homarus americanus are reported. The two subunits are structurally highly similar to the corresponding natural apo crustacyanin CRTC and CRTA subunits from the European lobster H. gammarus. Reconstitution studies of the recombinant crustacyanin proteins H 1 and H 2 with astaxanthin reproduced the bathochromic shift of 85–95 nm typical of the natural crustacyanin subunits from H. gammarus in complex with astaxanthin. Moreover, correlations between the presence of crustacyanin genes in crustacean species and the resulting carapace colours with the spectral properties of the subunits in complex with astaxanthin confirmed this genotype–phenotype linkage

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

    International Nuclear Information System (INIS)

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

    1986-01-01

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

  11. Gene expression patterns of oxidative phosphorylation complex I subunits are organized in clusters.

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    Yael Garbian

    Full Text Available After the radiation of eukaryotes, the NUO operon, controlling the transcription of the NADH dehydrogenase complex of the oxidative phosphorylation system (OXPHOS complex I, was broken down and genes encoding this protein complex were dispersed across the nuclear genome. Seven genes, however, were retained in the genome of the mitochondrion, the ancient symbiote of eukaryotes. This division, in combination with the three-fold increase in subunit number from bacteria (N = approximately 14 to man (N = 45, renders the transcription regulation of OXPHOS complex I a challenge. Recently bioinformatics analysis of the promoter regions of all OXPHOS genes in mammals supported patterns of co-regulation, suggesting that natural selection favored a mechanism facilitating the transcriptional regulatory control of genes encoding subunits of these large protein complexes. Here, using real time PCR of mitochondrial (mtDNA- and nuclear DNA (nDNA-encoded transcripts in a panel of 13 different human tissues, we show that the expression pattern of OXPHOS complex I genes is regulated in several clusters. Firstly, all mtDNA-encoded complex I subunits (N = 7 share a similar expression pattern, distinct from all tested nDNA-encoded subunits (N = 10. Secondly, two sub-clusters of nDNA-encoded transcripts with significantly different expression patterns were observed. Thirdly, the expression patterns of two nDNA-encoded genes, NDUFA4 and NDUFA5, notably diverged from the rest of the nDNA-encoded subunits, suggesting a certain degree of tissue specificity. Finally, the expression pattern of the mtDNA-encoded ND4L gene diverged from the rest of the tested mtDNA-encoded transcripts that are regulated by the same promoter, consistent with post-transcriptional regulation. These findings suggest, for the first time, that the regulation of complex I subunits expression in humans is complex rather than reflecting global co-regulation.

  12. Proteasome subunit expression analysis and chemosensitivity in relapsed paediatric acute leukaemia patients receiving bortezomib-containing chemotherapy

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    Denise Niewerth

    2016-09-01

    Full Text Available Abstract Background Drug combinations of the proteasome inhibitor bortezomib with cytotoxic chemotherapy are currently evaluated in phase 2 and 3 trials for the treatment of paediatric acute myeloid leukaemia (AML and acute lymphocytic leukaemia (ALL. Methods We investigated whether expression ratios of immunoproteasome to constitutive proteasome in leukaemic cells correlated with response to bortezomib-containing re-induction chemotherapy in patients with relapsed and refractory acute leukaemia, enrolled in two Children’s Oncology Group phase 2 trials of bortezomib for ALL (COG-AALL07P1 and AML (COG-AAML07P1. Expression of proteasome subunits was examined in 72 patient samples (ALL n = 60, AML n = 12 obtained before start of therapy. Statistical significance between groups was determined by Mann-Whitney U test. Results Ratios of immunoproteasome to constitutive proteasome subunit expression were significantly higher in pre-B ALL cells than in AML cells for both β5i/β5 and β1i/β1 subunits (p = 0.004 and p < 0.001. These ratios correlated with therapy response in AML patients; β1i/β1 ratios were significantly higher (p = 0.028 between patients who did (n = 4 and did not reach complete remission (CR (n = 8, although for β5i/β5 ratios, this did not reach significance. For ALL patients, the subunit ratios were also higher for patients who showed a good early response to therapy but this relation was not statistically significant. Overall, for this study, the patients were treated with combination therapy, so response was not only attributed to proteasome inhibition. Moreover, the leukaemic blast cells were not purified for these samples. Conclusions These first ex vivo results encourage further studies into relative proteasome subunit expression to improve proteasome inhibition-containing therapy and as a potential indicator of bortezomib response in acute leukaemia.

  13. Genetic mapping and validation of the loci controlling 7S α' and 11S A-type storage protein subunits in soybean [Glycine max (L.) Merr.].

    Science.gov (United States)

    Boehm, Jeffrey D; Nguyen, Vi; Tashiro, Rebecca M; Anderson, Dale; Shi, Chun; Wu, Xiaoguang; Woodrow, Lorna; Yu, Kangfu; Cui, Yuhai; Li, Zenglu

    2018-03-01

    Four soybean storage protein subunit QTLs were mapped using bulked segregant analysis and an F 2 population, which were validated with an F 5 RIL population. The storage protein globulins β-conglycinin (7S subunit) and glycinin (11S subunits) can affect the quantity and quality of proteins found in soybean seeds and account for more than 70% of the total soybean protein. Manipulating the storage protein subunits to enhance soymeal nutrition and for desirable tofu manufacturing characteristics are two end-use quality goals in soybean breeding programs. To aid in developing soybean cultivars with desired seed composition, an F 2 mapping population (n = 448) and an F 5 RIL population (n = 180) were developed by crossing high protein cultivar 'Harovinton' with the breeding line SQ97-0263_3-1a, which lacks the 7S α', 11S A 1 , 11S A 2 , 11S A 3 and 11S A 4 subunits. The storage protein composition of each individual in the F 2 and F 5 populations were profiled using SDS-PAGE. Based on the presence/absence of the subunits, genomic DNA bulks were formed among the F 2 plants to identify genomic regions controlling the 7S α' and 11S protein subunits. By utilizing polymorphic SNPs between the bulks characterized with Illumina SoySNP50K iSelect BeadChips at targeted genomic regions, KASP assays were designed and used to map QTLs causing the loss of the subunits. Soybean storage protein QTLs were identified on Chromosome 3 (11S A 1 ), Chromosome 10 (7S α' and 11S A 4 ), and Chromosome 13 (11S A 3 ), which were also validated in the F 5 RIL population. The results of this research could allow for the deployment of marker-assisted selection for desired storage protein subunits by screening breeding populations using the SNPs linked with the subunits of interest.

  14. Crystal structure of the P pilus rod subunit PapA.

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    Denis Verger

    2007-05-01

    Full Text Available P pili are important adhesive fibres involved in kidney infection by uropathogenic Escherichia coli strains. P pili are assembled by the conserved chaperone-usher pathway, which involves the PapD chaperone and the PapC usher. During pilus assembly, subunits are incorporated into the growing fiber via the donor-strand exchange (DSE mechanism, whereby the chaperone's G1 beta-strand that complements the incomplete immunoglobulin-fold of each subunit is displaced by the N-terminal extension (Nte of an incoming subunit. P pili comprise a helical rod, a tip fibrillum, and an adhesin at the distal end. PapA is the rod subunit and is assembled into a superhelical right-handed structure. Here, we have solved the structure of a ternary complex of PapD bound to PapA through donor-strand complementation, itself bound to another PapA subunit through DSE. This structure provides insight into the structural basis of the DSE reaction involving this important pilus subunit. Using gel filtration chromatography and electron microscopy on a number of PapA Nte mutants, we establish that PapA differs in its mode of assembly compared with other Pap subunits, involving a much larger Nte that encompasses not only the DSE region of the Nte but also the region N-terminal to it.

  15. In silico comparative genomic analysis of GABAA receptor transcriptional regulation

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    Joyce Christopher J

    2007-06-01

    Full Text Available Abstract Background Subtypes of the GABAA receptor subunit exhibit diverse temporal and spatial expression patterns. In silico comparative analysis was used to predict transcriptional regulatory features in individual mammalian GABAA receptor subunit genes, and to identify potential transcriptional regulatory components involved in the coordinate regulation of the GABAA receptor gene clusters. Results Previously unreported putative promoters were identified for the β2, γ1, γ3, ε, θ and π subunit genes. Putative core elements and proximal transcriptional factors were identified within these predicted promoters, and within the experimentally determined promoters of other subunit genes. Conserved intergenic regions of sequence in the mammalian GABAA receptor gene cluster comprising the α1, β2, γ2 and α6 subunits were identified as potential long range transcriptional regulatory components involved in the coordinate regulation of these genes. A region of predicted DNase I hypersensitive sites within the cluster may contain transcriptional regulatory features coordinating gene expression. A novel model is proposed for the coordinate control of the gene cluster and parallel expression of the α1 and β2 subunits, based upon the selective action of putative Scaffold/Matrix Attachment Regions (S/MARs. Conclusion The putative regulatory features identified by genomic analysis of GABAA receptor genes were substantiated by cross-species comparative analysis and now require experimental verification. The proposed model for the coordinate regulation of genes in the cluster accounts for the head-to-head orientation and parallel expression of the α1 and β2 subunit genes, and for the disruption of transcription caused by insertion of a neomycin gene in the close vicinity of the α6 gene, which is proximal to a putative critical S/MAR.

  16. Off-pathway assembly of fimbria subunits is prevented by chaperone CfaA of CFA/I fimbriae from enterotoxigenic E. coli.

    Science.gov (United States)

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

    2016-12-01

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

  17. Molecular cloning and expression of heteromeric ACCase subunit genes from Jatropha curcas.

    Science.gov (United States)

    Gu, Keyu; Chiam, Huihui; Tian, Dongsheng; Yin, Zhongchao

    2011-04-01

    Acetyl-CoA carboxylase (ACCase) catalyzes the biotin-dependent carboxylation of acetyl-CoA to produce malonyl-CoA, which is the essential first step in the biosynthesis of long-chain fatty acids. ACCase exists as a multi-subunit enzyme in most prokaryotes and the chloroplasts of most plants and algae, while it is present as a multi-domain enzyme in the endoplasmic reticulum of most eukaryotes. The heteromeric ACCase of higher plants consists of four subunits: an α-subunit of carboxyltransferase (α-CT, encoded by accA gene), a biotin carboxyl carrier protein (BCCP, encoded by accB gene), a biotin carboxylase (BC, encoded by accC gene) and a β-subunit of carboxyltransferase (β-CT, encoded by accD gene). In this study, we cloned and characterized the genes accA, accB1, accC and accD that encode the subunits of heteromeric ACCase in Jatropha (Jatropha curcas), a potential biofuel plant. The full-length cDNAs of the four subunit genes were isolated from a Jatropha cDNA library and by using 5' RACE, whereas the genomic clones were obtained from a Jatropha BAC library. They encode a 771 amino acid (aa) α-CT, a 286-aa BCCP1, a 537-aa BC and a 494-aa β-CT, respectively. The single-copy accA, accB1 and accC genes are nuclear genes, while the accD gene is located in chloroplast genome. Jatropha α-CT, BCCP1, BC and β-CT show high identity to their homologues in other higher plants at amino acid level and contain all conserved domains for ACCase activity. The accA, accB1, accC and accD genes are temporally and spatially expressed in the leaves and endosperm of Jatropha plants, which are regulated by plant development and environmental factors. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  18. Motor Skills Training Enhances α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid Receptor Subunit mRNA Expression in the Ipsilateral Sensorimotor Cortex and Striatum of Rats Following Intracerebral Hemorrhage.

    Science.gov (United States)

    Tamakoshi, Keigo; Ishida, Kazuto; Kawanaka, Kentaro; Takamatsu, Yasuyuki; Tamaki, Hiroyuki

    2017-10-01

    We investigated the effects of acrobatic training (AT) on expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subunits in the sensorimotor cortex and striatum after intracerebral hemorrhage (ICH). Male Wistar rats were divided into 4 groups: ICH without AT (ICH), ICH with AT (ICH + AT), sham operation without AT (SHAM), and sham operation with AT (SHAM + AT). ICH was induced by collagenase injection into the left striatum. The ICH + AT group performed 5 acrobatic tasks daily on days 4-28 post ICH. Forelimb sensorimotor function was evaluated using the forelimb placing test. On days 14 and 29, mRNA expression levels of AMPAR subunits GluR1-4 were measured by real-time reverse transcription-polymerase chain reaction. Forelimb placing test scores were significantly higher in the ICH + AT group than in the ICH group. Expression levels of all AMPAR subunit mRNAs were significantly higher in the ipsilateral sensorimotor cortex of rats in the ICH + AT group than in that of rats in the ICH group on day 29. GluR3 and GluR4 expression levels were reduced in the ipsilateral striatum of rats in the ICH group compared with that of rats in the SHAM group on day 14. These changes may play a critical role in motor skills training-induced recovery after ICH. Copyright © 2017 National Stroke Association. Published by Elsevier Inc. All rights reserved.

  19. The dnaN gene codes for the beta subunit of DNA polymerase III holoenzyme of escherichia coli.

    Science.gov (United States)

    Burgers, P M; Kornberg, A; Sakakibara, Y

    1981-09-01

    An Escherichia coli mutant, dnaN59, stops DNA synthesis promptly upon a shift to a high temperature; the wild-type dnaN gene carried in a transducing phage encodes a polypeptide of about 41,000 daltons [Sakakibara, Y. & Mizukami, T. (1980) Mol. Gen. Genet. 178, 541-553; Yuasa, S. & Sakakibara, Y. (1980) Mol. Gen. Genet. 180, 267-273]. We now find that the product of dnaN gene is the beta subunit of DNA polymerase III holoenzyme, the principal DNA synthetic multipolypeptide complex in E. coli. The conclusion is based on the following observations: (i) Extracts from dnaN59 cells were defective in phage phi X174 and G4 DNA synthesis after the mutant cells had been exposed to the increased temperature. (ii) The enzymatic defect was overcome by addition of purified beta subunit but not by other subunits of DNA polymerase III holoenzyme or by other replication proteins required for phi X174 DNA synthesis. (iii) Partially purified beta subunit from the dnaN mutant, unlike that from the wild type, was inactive in reconstituting the holoenzyme when mixed with the other purified subunits. (iv) Increased dosage of the dnaN gene provided by a plasmid carrying the gene raised cellular levels of the beta subunit 5- to 6-fold.

  20. Molecular analysis of mxbD and mxbM, a putative sensor-regulator pair required for oxidation of methanol in Methylobacterium extorquens AM1.

    Science.gov (United States)

    Springer, A L; Morris, C J; Lidstrom, M E

    1997-05-01

    Five genes are thought to be required for transcription of methanol oxidation genes in Methylobacterium strains. These putative regulatory genes include mxcQE, which encode a putative sensor-regulator pair, and mxbDM and mxaB, whose functions are less well-understood. In this study, mxbDM in Methylobacterium extorquens AM1 were shown to be required for expression of a xylE transcriptional fusion to the structural gene for the large subunit of methanol dehydrogenase (mxaF), confirming the role of these genes in transcriptional regulation of mxaF. The nucleotide sequence suggests that mxbD encodes a histidine protein kinase with two transmembrane domains and that mxbM encodes a DNA-binding response regulator. A xylE transcriptional fusion to the putative mxbD promoter showed low-level expression in wild-type cells grown on one-carbon (C1) compounds and no detectable expression in cells grown on succinate. Deletion analysis of this promoter construct showed that the region 229-129 bp upstream of the start of mxbD is required for expression. The expression of the mxbD-xylE fusion was examined in each of the five known regulatory mutant classes. xylE expression was reduced to non-detectable levels in MxcQ and MxcE mutants, but was not affected in the other regulatory mutants or in non-regulatory mutants defective in methanol oxidation. These results suggest a regulatory hierarchy in which the sensor-regulator pair MxcQE control expression of the sensor-regulator pair MxbDM, and MxbDM in turn control expression of a number of genes involved in methanol oxidation.

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

    Science.gov (United States)

    Cherwonogrodzky, John W; Barabé, Nicole D; Grigat, Michelle L; Lee, William E; Poirier, Robert T; Jager, Scott J; Berger, Bradley J

    2014-12-01

    A subunit vaccine candidate was produced from Brucella suis 145 (biovar 4; expressing both the A antigen of Brucella abortus and the M antigen of Brucella melitensis). The preparation consisted mostly of polysaccharide (PS; >90% [wt/wt]; both cell-associated PS and exo-PS were combined) and a small amount of protein (1 to 3%) with no apparent nucleic acids. Vaccinated mice were protected (these had a statistically significant reduction in bacterial colonization compared to that of unvaccinated controls) when challenged with representative strains of three Brucella species most pathogenic for humans, i.e., B. abortus, B. melitensis, and B. suis. As little as 1 ng of the vaccine, without added adjuvant, protected mice against B. suis 145 infection (5 × 10(5) CFU), and a single injection of 1 μg of this subunit vaccine protected mice from B. suis 145 challenge for at least 14 months. A single immunization induced a serum IgG response to Brucella antigens that remained elevated for up to 9 weeks. The use of heat (i.e., boiling-water bath, autoclaving) in the vaccine preparation showed that it was thermostable. This method also ensured safety and security. The vaccine produced was immunogenic and highly protective against multiple strains of Brucella and represents a promising candidate for further evaluation. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  2. Muscular subunits transplantation for facial reanimation

    Directory of Open Access Journals (Sweden)

    Hazan André Salo Buslik

    2006-01-01

    Full Text Available PURPOSE: To present an alternative technique for reconstruction of musculocutaneous damages in the face transferring innervated subsegments(subunits of the latissimus dorsi flap for replacement of various facial mimetic muscles. METHODS: One clinical case of trauma with skin and mimetic muscles damage is described as an example of the technique. The treatment was performed with microsurgical transfer of latissimus dorsi muscle subunits. Each subunit present shape and dimensions of the respective mimetic muscles replaced. The origin, insertions and force vectors for the mimicmuscle lost were considered. Each subsegment has its own arterial and venous supply with a motor nerve component for the muscular unit. RESULTS: Pre and one year postoperative photos registration of static and dynamic mimic aspects, as well as digital electromyography digital data of the patients were compared. The transplanted muscular units presented myoeletric activity, fulfilling both the functional and cosmetic aspect. CONCLUSION: This technique seems to be a promising way to deal with the complex musculocutaneous losses of the face as well as facial palsy.

  3. CRYSTAL STRUCTURE ANALYSIS OF A PUTATIVE OXIDOREDUCTASE FROM KLEBSIELLA PNEUMONIAE

    Energy Technology Data Exchange (ETDEWEB)

    Baig, M.; Brown, A.; Eswaramoorthy, S.; Swaminathan, S.

    2009-01-01

    Klebsiella pneumoniae, a gram-negative enteric bacterium, is found in nosocomial infections which are acquired during hospital stays for about 10% of hospital patients in the United States. The crystal structure of a putative oxidoreductase from K. pneumoniae has been determined. The structural information of this K. pneumoniae protein was used to understand its function. Crystals of the putative oxidoreductase enzyme were obtained by the sitting drop vapor diffusion method using Polyethylene glycol (PEG) 3350, Bis-Tris buffer, pH 5.5 as precipitant. These crystals were used to collect X-ray data at beam line X12C of the National Synchrotron Light Source (NSLS) at Brookhaven National Laboratory (BNL). The crystal structure was determined using the SHELX program and refi ned with CNS 1.1. This protein, which is involved in the catalysis of an oxidation-reduction (redox) reaction, has an alpha/beta structure. It utilizes nicotinamide adenine dinucleotide phosphate (NADP) or nicotine adenine dinucleotide (NAD) to perform its function. This structure could be used to determine the active and co-factor binding sites of the protein, information that could help pharmaceutical companies in drug design and in determining the protein’s relationship to disease treatment such as that for pneumonia and other related pathologies.

  4. Theoretical study of structures of Ga5N5 cluster

    International Nuclear Information System (INIS)

    Song Bin; Cao Peilin

    2002-01-01

    The structures and energies of a Ga 5 N 5 cluster have been calculated using a full-potential linear-muffin-tin-orbital (FP-LMTO) method, combined with molecular dynamics technique. Twenty-four structures for a Ga 5 N 5 cluster have been obtained. The most stable structure is a C 1 planar structure with a N 3 subunit. The Ga 5 N 5 clusters show a preference for a N 3 subunit, revealing the same behavior as in the Ga 3 N 3 and Ga 4 N 4 clusters. The existence of strong N-N bonds dominates the structure of a Ga 5 N 5 cluster. Through the calculation of the density of states we found that the most stable structure of Ga 5 N 5 clusters presented semiconductor-like properties

  5. Immunochemical analysis of Micrococcus lysodeikticus (luteus) F1-ATPase and its subunits.

    Science.gov (United States)

    Urban, C; Salton, M R

    1983-08-31

    The F1-ATPase from Micrococcus lysodeikticus has been purified to 95% protein homogeneity in this laboratory and as all other bacterial F1S, possesses five distinct subunits with molecular weights ranging from 60 000 to 10 000 (Huberman, M. and Salton, M.R.J. (1979) Biochim. Biophys. Acta 547, 230-240). In this communication, we demonstrate the immunochemical reactivities of antibodies to native and SDS-dissociated subunits with the native and dissociated F1-ATPase and show that: (1) the antibodies generated to the native or SDS-dissociated subunits react with the native molecule; (2) all of the subunits comprising the F1 are antigenically unique as determined by crossed immunoelectrophoresis and the Ouchterlony double-diffusion techniques; (3) antibodies to the SDS-denatured individual delta- and epsilon-subunits can be used to destabilize the interaction of these specific subunits with the rest of the native F1; and (4) all subunit antibodies as well as anti-native F1 were found to inhibit ATPase activity to varying degrees, the strongest inhibition being seen with antibodies to the total F1 and anti-alpha- and anti-beta-subunit antibodies. The interaction of specific subunit antibodies may provide a new and novel way to study further and characterize the catalytic portions of F1-ATPases and in general may offer an additional method for the examination of multimeric proteins.

  6. Distinct ASIC currents are expressed in rat putative nociceptors and are modulated by nerve injury.

    Science.gov (United States)

    Poirot, Olivier; Berta, Temugin; Decosterd, Isabelle; Kellenberger, Stephan

    2006-10-01

    The H(+)-gated acid-sensing ion channels (ASICs) are expressed in dorsal root ganglion (DRG) neurones. Studies with ASIC knockout mice indicated either a pro-nociceptive or a modulatory role of ASICs in pain sensation. We have investigated in freshly isolated rat DRG neurones whether neurones with different ASIC current properties exist, which may explain distinct cellular roles, and we have investigated ASIC regulation in an experimental model of neuropathic pain. Small-diameter DRG neurones expressed three different ASIC current types which were all preferentially expressed in putative nociceptors. Type 1 currents were mediated by ASIC1a homomultimers and characterized by steep pH dependence of current activation in the pH range 6.8-6.0. Type 3 currents were activated in a similar pH range as type 1, while type 2 currents were activated at pH ASIC current density. Nerve injury induced differential regulation of ASIC subunit expression and selective changes in ASIC function in DRG neurones, suggesting a complex reorganization of ASICs during the development of neuropathic pain. In summary, we describe a basis for distinct cellular functions of different ASIC types in small-diameter DRG neurones.

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

    International Nuclear Information System (INIS)

    Taylor, T.; Weintraub, B.D.

    1985-01-01

    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 [ 14 C]alanine and [ 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, [ 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. [ 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. Catalytic Subunit 1 of Protein Phosphatase 2A Is a Subunit of the STRIPAK Complex and Governs Fungal Sexual Development

    Directory of Open Access Journals (Sweden)

    Anna Beier

    2016-06-01

    Full Text Available 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.

  9. Electrophysiology and Beyond: Multiple roles of Na+ channel β subunits in development and disease

    Science.gov (United States)

    Patino, Gustavo A.; Isom, Lori L.

    2010-01-01

    Voltage-gated Na+ channel (VGSC) β subunits are not “auxiliary.” These multifunctional molecules not only modulate Na+ current (INa), 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. PMID:20600605

  10. A putative hybrid swarm within Oonopsis foliosa (Asteraceae: Astereae)

    Science.gov (United States)

    Hughes, J.F.; Brown, G.K.

    2004-01-01

    Oo??nopsis foliosa var. foliosa and var. monocephala are endemic to short-grass steppe of southeastern Colorado and until recently were considered geographically disjunct. The only known qualitative feature separating these 2 varieties is floral head type; var. foliosa has radiate heads, whereas var. monocephala heads are discoid. Sympatry between these varieties is restricted to a small area in which a range of parental types and intermediate head morphologies is observed. We used distribution mapping, morphometric analyses, chromosome cytology, and pollen stainability to characterize the sympatric zone. Morphometrics confirms that the only discrete difference between var. foliosa and var. monocephala is radiate versus discoid heads, respectively. The outer florets of putative hybrid individuals ranged from conspicuously elongated yet radially symmetric disc-floret corollas, to elongated radially asymmetric bilabiate- or deeply cleft corollas, to stunted ray florets with appendages remnant of corolla lobes. Chromosome cytology of pollen mother cells from both putative parental varieties and a series of intermediate morphological types collected at the sympatric zone reveal evidence of translocation heterozygosity. Pollen stainability shows no significant differences in viability between the parental varieties and putative hybrids. The restricted distribution of putative hybrids to a narrow zone of sympatry between the parental types and the presence of meiotic chromosome-pairing anomalies in these intermediate plants are consistent with a hybrid origin. The high stainability of putative-hybrid pollen adds to a growing body of evidence that hybrids are not universally unfit.

  11. Isolation and characterization of human cDNA clones encoding the α and the α' subunits of casein kinase II

    International Nuclear Information System (INIS)

    Lozeman, F.J.; Litchfield, D.W.; Piening, C.; Takio, Koji; Walsh, K.A.; Krebs, E.G.

    1990-01-01

    Casein kinase II is a widely distributed protein serine/threonine kinase. The holoenzyme appears to be a tetramer, containing two α or α' subunits (or one of each) and two β subunits. Complementary DNA clones encoding the subunits of casein kinase II were isolated from a human T-cell λgt 10 library using cDNA clones isolated from Drosophila melanogasten. One of the human cDNA clones (hT4.1) was 2.2 kb long, including a coding region of 1176 bp preceded by 156 bp (5' untranslated region) and followed by 871 bp (3' untranslated region). The hT4.1 close was nearly identical in size and sequence with a cDNA clone from HepG2 human hepatoma cultured cells. Another of the human T-cell cDNA clones (hT9.1) was 1.8 kb long, containing a coding region of 1053 bp preceded by 171 by (5' untranslated region) and followed by 550 bp (3' untranslated region). Amino acid sequences deduced from these two cDNA clones were about 85% identical. Most of the difference between the two encoded polypeptides was in the carboxy-terminal region, but heterogeneity was distributed throughout the molecules. Partial amino acid sequence was determined in a mixture of α and α' subunits from bovine lung casein kinase II. The bovine sequences aligned with the 2 human cDNA-encoded polypeptides with only 2 discrepancies out of 535 amino acid positions. This confirmed that the two human T-cell cDNA clones encoded the α and α' subunits of casein kinase II. These studies show that there are two distinct catalytic subunits for casein II (α and α') and that the sequence of these subunits is largely conserved between the bovine and the human

  12. Deletion of FoxO1 Leads to Shortening of QRS by Increasing Na+ Channel Activity through Enhanced Expression of both Cardiac NaV1.5 and β3 Subunit

    OpenAIRE

    Cai, Benzhi; Wang, Ning; Mao, Weike; You, Tao; Lu, Yan; Li, Xiang; Ye, Bo; Li, Faqian; Xu, Haodong

    2014-01-01

    Our in vitro studies revealed that a transcription factor, Forkhead box protein O1 (FoxO1), negatively regulates the expression of NaV1.5, a main α subunit of the cardiac Na+ channel, by altering the promoter activity of SCN5a in HL-1 cardiomyocytes. The in vivo role of FoxO1 in the regulation of cardiac NaV1.5 expression remains unknown. The present study aimed to define the role of FoxO1 in the regulation of NaV1.5 expression and cardiac Na+ channel activity in mouse ventricular cardiomyocy...

  13. Casein haplotypes and their association with milk production traits in Norwegian Red cattle

    Directory of Open Access Journals (Sweden)

    Nome Torfinn

    2009-02-01

    Full Text Available Abstract A high resolution SNP map was constructed for the bovine casein region to identify haplotype structures and study associations with milk traits in Norwegian Red cattle. Our analyses suggest separation of the casein cluster into two haplotype blocks, one consisting of the CSN1S1, CSN2 and CSN1S2 genes and another one consisting of the CSN3 gene. Highly significant associations with both protein and milk yield were found for both single SNPs and haplotypes within the CSN1S1-CSN2-CSN1S2 haplotype block. In contrast, no significant association was found for single SNPs or haplotypes within the CSN3 block. Our results point towards CSN2 and CSN1S2 as the most likely loci harbouring the underlying causative DNA variation. In our study, the most significant results were found for the SNP CSN2_67 with the C allele consistently associated with both higher protein and milk yields. CSN2_67 calls a C to an A substitution at codon 67 in β-casein gene resulting in histidine replacing proline in the amino acid sequence. This polymorphism determines the protein variants A1/B (CSN2_67 A allele versus A2/A3 (CSN2_67 C allele. Other studies have suggested that a high consumption of A1/B milk may affect human health by increasing the risk of diabetes and heart diseases. Altogether these results argue for an increase in the frequency of the CSN2_67 C allele or haplotypes containing this allele in the Norwegian Red cattle population by selective breeding.

  14. Radioimmunoassay of TSH subunits in thyroid diseases and endocrine opthalmopahty

    International Nuclear Information System (INIS)

    Eder, W.

    1982-01-01

    Highly sensitive radioimmunoassays of hTSH sub-units were developed. The hormone preparations were labelled with 125-iodine according to a modified chloramine -T method, and purified by chromatography using biogel P6 and P60. Rabbit antisera were used as antibodies. Separation of the antibody-bound and of the free antigens was carried out via the double antibody method. The antiserum required for this purpose was obtained from a goat. The sensitivity of the assay was influenced by changing the protein content of the buffer, the incubation volume, the tracer amounts, the incubation time and the incubation temperature. For hTSH-α, the lowest detectable limit was found to be 50 pg/ml, for hTSH-#betta# 20 pg/ml. Thus, the sub-units could be determined for 98% of the patients under review. The #betta#-TSH radioimmunoassay is largely specific, TSH cross-reacts to a degree of 5%. The computerized evoluation was carried out by means of Spline approximation using the Siemens 4004 computer. Precision and accurateness are in compliance with generally accpted criteria. The serum levels of α and #betta# sub-units showed no discordancy with regard to TSH. In all groups of patients examined, the levels of the hormone-specific #betta#-chain were found to be exclusively dependent upon the actual thyroid activity. (orig.) [de

  15. Characteristics of functional enrichment and gene expression level of human putative transcriptional target genes.

    Science.gov (United States)

    Osato, Naoki

    2018-01-19

    Transcriptional target genes show functional enrichment of genes. However, how many and how significantly transcriptional target genes include functional enrichments are still unclear. To address these issues, I predicted human transcriptional target genes using open chromatin regions, ChIP-seq data and DNA binding sequences of transcription factors in databases, and examined functional enrichment and gene expression level of putative transcriptional target genes. Gene Ontology annotations showed four times larger numbers of functional enrichments in putative transcriptional target genes than gene expression information alone, independent of transcriptional target genes. To compare the number of functional enrichments of putative transcriptional target genes between cells or search conditions, I normalized the number of functional enrichment by calculating its ratios in the total number of transcriptional target genes. With this analysis, native putative transcriptional target genes showed the largest normalized number of functional enrichments, compared with target genes including 5-60% of randomly selected genes. The normalized number of functional enrichments was changed according to the criteria of enhancer-promoter interactions such as distance from transcriptional start sites and orientation of CTCF-binding sites. Forward-reverse orientation of CTCF-binding sites showed significantly higher normalized number of functional enrichments than the other orientations. Journal papers showed that the top five frequent functional enrichments were related to the cellular functions in the three cell types. The median expression level of transcriptional target genes changed according to the criteria of enhancer-promoter assignments (i.e. interactions) and was correlated with the changes of the normalized number of functional enrichments of transcriptional target genes. Human putative transcriptional target genes showed significant functional enrichments. Functional

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

    Science.gov (United States)

    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.

  17. The eukaryotic translation initiation factor 3 subunit E binds to classical swine fever virus NS5A and facilitates viral replication.

    Science.gov (United States)

    Liu, Xiaofeng; Wang, Xiaoyu; Wang, Qian; Luo, Mingyang; Guo, Huancheng; Gong, Wenjie; Tu, Changchun; Sun, Jinfu

    2018-02-01

    Classical swine fever virus (CSFV) NS5A protein is a multifunctional protein, playing critical roles in viral RNA replication, translation and assembly. To further explore its functions in viral replication, interaction of NS5A with host factors was assayed using a his-tag "pull down" assay coupled with shotgun LC-MS/MS. Host protein translation initiation factor 3 subunit E was identified as a binding partner of NS5A, and confirmed by co-immunoprecipitation and co-localization analysis. Overexpression of eIF3E markedly enhanced CSFV genomic replication, viral protein expression and production of progeny virus, and downregulation of eIF3E by siRNA significantly decreased viral proliferation in PK-15 cells. Luciferase reporter assay showed an enhancement of translational activity of the internal ribosome entry site of CSFV by eIF3E and a decrease in cellular translation by NS5A. These data indicate that eIF3E plays an important role in CSFV replication, thereby identifying it as a potential target for inhibition of the virus. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Memory Effects of Benzodiazepines: Memory Stages and Types Versus Binding-Site Subtypes

    Directory of Open Access Journals (Sweden)

    Miroslav M. Savic

    2005-01-01

    Full Text Available Benzodiazepines are well established as inhibitory modulators of memory processing. This effect is especially prominent when applied before the acquisition phase of a memory task. This minireview concentrates on the putative subtype selectivity of the acquisition-impairing action of benzodiazepines. Namely, recent genetic studies and standard behavioral tests employing subtype-selective ligands pointed to the predominant involvement of two subtypes of benzodiazepine binding sites in memory modulation. Explicit memory learning seems to be affected through the GABAA receptors containing the α1 and α5 subunits, whereas the effects on procedural memory can be mainly mediated by the α1 subunit. The pervading involvement of the α1 subunit in memory modulation is not at all unexpected because this subunit is the major subtype, present in 60% of all GABAA receptors. On the other hand, the role of α5 subunits, mainly expressed in the hippocampus, in modulating distinct forms of memory gives promise of selective pharmacological coping with certain memory deficit states.

  19. Topographic antigenic determinants recognized by monoclonal antibodies on human choriogonadotropin beta-subunit

    International Nuclear Information System (INIS)

    Bidart, J.M.; Troalen, F.; Salesse, R.; Bousfield, G.R.; Bohuon, C.J.; Bellet, D.H.

    1987-01-01

    We describe a first attempt to study the antibody-combining sites recognized by monoclonal antibodies raised against the beta-subunit of human choriogonadotropin (hCG). Two groups of antibodies were first defined by their ability to recognize only the free beta-subunit or the free and combined subunit. Antibodies FBT-11 and FBT-11-L bind only to hCG beta-subunit but not to hCG, whereas antibodies FBT-10 and D1E8 bind to both the beta-subunit and the hormone. In both cases, the antigenic determinants were localized to the core of the protein (residues 1-112), indicating the weak immunogenicity of the specific carboxyl-terminal extension of hCG-beta. Nine synthetic peptides spanning different regions of hCG-beta and lutropin-beta were assessed for their capacity to inhibit antibody binding. A synthetic peptide inclusive of the NH2-terminal region (residues 1-7) of the hCG beta-subunit was found to inhibit binding to the radiolabeled subunit of a monoclonal antibody specific for free hCG-beta (FBT-11). Further delineation of the antigenic site recognized by this antibody provided evidence for the involvement of fragment 82-92. Moreover, monoclonal antibody FBT-11 inhibited the recombination of hCG-beta to hCG-alpha, indicating that its antigenic determinant might be located nearby or in the hCG-beta portion interacting with the alpha-subunit. Binding of monoclonal antibody FBT-10, corresponding to the second antigenic determinant, was weakly inhibited by fragment 82-105 and did not impair the recombination of the hCG beta-subunit to the hCG alpha-subunit. Its combining site appeared to be located in a region of the intact native choriogonadotropin present at the surface of the hormone-receptor complex

  20. First comparative characterization of three distinct ferritin subunits from a teleost: Evidence for immune-responsive mRNA expression and iron depriving activity of seahorse (Hippocampus abdominalis) ferritins.

    Science.gov (United States)

    Oh, Minyoung; Umasuthan, Navaneethaiyer; Elvitigala, Don Anushka Sandaruwan; Wan, Qiang; Jo, Eunyoung; Ko, Jiyeon; Noh, Gyeong Eon; Shin, Sangok; Rho, Sum; Lee, Jehee

    2016-02-01

    Ferritins play an indispensable role in iron homeostasis through their iron-withholding function in living beings. In the current study, cDNA sequences of three distinct ferritin subunits, including a ferritin H, a ferritin M, and a ferritin L, were identified from big belly seahorse, Hippocampus abdominalis, and molecularly characterized. Complete coding sequences (CDS) of seahorse ferritin H (HaFerH), ferritin M (HaFerM), and ferritin L (HaFerL) subunits were comprised of 531, 528, and 522 base pairs (bp), respectively, which encode polypeptides of 177, 176, and 174 amino acids, respectively, with molecular masses of ∼20-21 kDa. Our in silico analyses demonstrate that these three ferritin subunits exhibit the typical characteristics of ferritin superfamily members including iron regulatory elements, domain signatures, and reactive centers. The coding sequences of HaFerH, M, and L were cloned and the corresponding proteins were overexpressed in a bacterial system. Recombinantly expressed HaFer proteins demonstrated detectable in vivo iron sequestrating (ferroxidase) activity, consistent with their putative iron binding capability. Quantification of the basal expression of these three HaFer sequences in selected tissues demonstrated a gene-specific ubiquitous spatial distribution pattern, with abundance of mRNA in HaFerM in the liver and predominant expression of HaFerH and HaFerL in blood. Interestingly, the basal expression of all three ferritin genes was found to be significantly modulated against pathogenic stress mounted by lipopolysaccharides (LPS), poly I:C, Streptococcus iniae, and Edwardsiella tarda. Collectively, our findings suggest that the three HaFer subunits may be involved in iron (II) homeostasis in big belly seahorse and that they are important in its host defense mechanisms. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Putative periodontopathic bacteria and herpesviruses in pregnant women: a case-control study

    OpenAIRE

    Lu, Haixia; Zhu, Ce; Li, Fei; Xu, Wei; Tao, Danying; Feng, Xiping

    2016-01-01

    Little is known about herpesvirus and putative periodontopathic bacteria in maternal chronic periodontitis. The present case-control study aimed to explore the potential relationship between putative periodontopathic bacteria and herpesviruses in maternal chronic periodontitis.Saliva samples were collected from 36 pregnant women with chronic periodontitis (cases) and 36 pregnant women with healthy periodontal status (controls). Six putative periodontopathic bacteria (Porphyromonas gingivalis ...

  2. Complementary DNA and derived amino acid sequence of the α subunit of human complement protein C8: evidence for the existence of a separate α subunit messenger RNA

    International Nuclear Information System (INIS)

    Rao, A.G.; Howard, O.M.Z.; Ng, S.C.; Whitehead, A.S.; Colten, H.R.; Sodetz, J.M.

    1987-01-01

    The entire amino acid sequence of the α subunit (M/sub r/ 64,000) of the eight component of complement (C8) was determined by characterizing cDNA clones isolated from a human liver cDNA library. Two clones with overlapping inserts of net length 2.44 kilobases (kb) were isolated and found to contain the entire α coding region [1659 base pairs (bp)]. The 5' end consists of an untranslated region and a leader sequence of 30 amino acids. This sequence contains an apparent initiation Met, signal peptide, and propeptide which ends with an arginine-rich sequence that is characteristic of proteolytic processing sites found in the pro form of protein precursors. The 3' untranslated region contains two polyadenylation signals and a poly(A)sequence. RNA blot analysis of total cellular RNA from the human hepatoma cell line HepG2 revealed a message size of ∼2.5 kb. Features of the 5' and 3' sequences and the message size suggest that a separate mRNA codes for α and argues against the occurrence of a single-chain precursor form of the disulfide-linked α-λ subunit found in mature C8. Analysis of the derived amino acid sequence revealed several membrane surface seeking domains and a possible transmembrane domain. Analysis of the carbohydrate composition indicates 1 or 2 asparagine-linked but no O-linked oligosaccharide chains, a result consistent with predictions from the amino acid sequence. Most significantly, it exhibits a striking overall homology to human C9, with values of 24% on the basis of identity and 46% when conserved substitutions are allowed. As described in an accompanying report this homology also extends to the β subunit of C8

  3. Results Assessment of Intercomparison Exercise CSN/CIEMAT-2010 among Spanish National Laboratories of Environmental Radioactivity (Diet Ashes)

    International Nuclear Information System (INIS)

    Gasco, C.; Trinidad, J. A.; Llaurado, M.; Suarez, J. A.

    2012-01-01

    This report describes the results assessment of the intercomparison exercise among environmental radioactivity laboratories, organised by Spanish Regulatory Institution (CSN) and prepared and evaluated by UAB and CIEMAT respectively. The exercise has been carried out following the international standards ISO-43 and ISO/IUPAC that provide a useful guide to perform proficiency tests and inter-laboratories comparisons. The selected matrix for this year (2010) was a diet ash obtained from the ashing of a whole fresh diet (breakfast, lunch and dinner), that was enriched with artificial radionuclides (Cs-137, Co-60,Fe-55,Ni-63,Sr-90,Am-241,Pu-238,Pu-239,240 y C-14) and contained natural radionuclides (U-234, U-238, U-natural Th-230, Th-234, Ra-226, Ra-228, Pb-210, Pb-212, Pb-214, Bi-214, Ac-228, Tl-208, K-40) at environmental level of activity concentration. The z-score test was applied to determine how much the laboratories differ from the reference value. The reference value for this exercise was the median of the results from the different laboratories and their standard deviations to achieve a more complete and objective study of the laboratories performance. The participant laboratories have demonstrated a satisfactory quality level for measuring the natural and artificial radionuclides content in this matrix. The reference values obtained through the medians show a negative bias for Pb-210 and Th-234 when comparing to the given values of external qualified laboratories from ENEA and IRSN and positive one for K-40. (Author)

  4. Stabilization of the H,K-ATPase M5M6 membrane hairpin by K+ ions. Mechanistic significance for p2-type atpases.

    Science.gov (United States)

    Gatto, C; Lutsenko, S; Shin, J M; Sachs, G; Kaplan, J H

    1999-05-14

    The integral membrane protein, the gastric H,K-ATPase, is an alpha-beta heterodimer, with 10 putative transmembrane segments in the alpha-subunit and one such segment in the beta-subunit. All transmembrane segments remain within the membrane domain following trypsinization of the intact gastric H,K-ATPase in the presence of K+ ions, identified as M1M2, M3M4, M5M6, and M7, M8, M9, and M10. Removal of K+ ions from this digested preparation results in the selective loss of the M5M6 hairpin from the membrane. The release of the M5M6 fragment is directed to the extracellular phase as evidenced by the accumulation of the released M5M6 hairpin inside the sealed inside out vesicles. The stabilization of the M5M6 hairpin in the membrane phase by the transported cation as well as loss to the aqueous phase in the absence of the transported cation has been previously observed for another P2-type ATPase, the Na, K-ATPase (Lutsenko, S., Anderko, R., and Kaplan, J. H. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7936-7940). Thus, the effects of the counter-transported cation on retention of the M5M6 segment in the membrane as compared with the other membrane pairs may be a general feature of P2-ATPase ion pumps, reflecting a flexibility of this region that relates to the mechanism of transport.

  5. Cytochrome oxidase subunit II gene in mitochondria of Oenothera has no intron

    Science.gov (United States)

    Hiesel, Rudolf; Brennicke, Axel

    1983-01-01

    The cytochrome oxidase subunit II gene has been localized in the mitochondrial genome of Oenothera berteriana and the nucleotide sequence has been determined. The coding sequence contains 777 bp and, unlike the corresponding gene in Zea mays, is not interrupted by an intron. No TGA codon is found within the open reading frame. The codon CGG, as in the maize gene, is used in place of tryptophan codons of corresponding genes in other organisms. At position 742 in the Oenothera sequence the TGG of maize is changed into a CGG codon, where Trp is conserved as the amino acid in other organisms. Homologous sequences occur more than once in the mitochondrial genome as several mitochondrial DNA species hybridize with DNA probes of the cytochrome oxidase subunit II gene. ImagesFig. 5. PMID:16453484

  6. Structure of Pfu Pop5, an archaeal RNase P protein.

    Science.gov (United States)

    Wilson, Ross C; Bohlen, Christopher J; Foster, Mark P; Bell, Charles E

    2006-01-24

    We have used NMR spectroscopy and x-ray crystallography to determine the three-dimensional structure of PF1378 (Pfu Pop5), one of four protein subunits of archaeal RNase P that shares a homolog in the eukaryotic enzyme. RNase P is an essential and ubiquitous ribonucleoprotein enzyme required for maturation of tRNA. In bacteria, the enzyme's RNA subunit is responsible for cleaving the single-stranded 5' leader sequence of precursor tRNA molecules (pre-tRNA), whereas the protein subunit assists in substrate binding. Although in bacteria the RNase P holoenzyme consists of one large catalytic RNA and one small protein subunit, in archaea and eukarya the enzyme contains several (> or =4) protein subunits, each of which lacks sequence similarity to the bacterial protein. The functional role of the proteins is poorly understood, as is the increased complexity in comparison to the bacterial enzyme. Pfu Pop5 has been directly implicated in catalysis by the observation that it pairs with PF1914 (Pfu Rpp30) to functionally reconstitute the catalytic domain of the RNA subunit. The protein adopts an alpha-beta sandwich fold highly homologous to the single-stranded RNA binding RRM domain. Furthermore, the three-dimensional arrangement of Pfu Pop5's structural elements is remarkably similar to that of the bacterial protein subunit. NMR spectra have been used to map the interaction of Pop5 with Pfu Rpp30. The data presented permit tantalizing hypotheses regarding the role of this protein subunit shared by archaeal and eukaryotic RNase P.

  7. Moessbauer spectroscopic studies of hemoglobin and its isolated subunits

    International Nuclear Information System (INIS)

    Hoy, G.R.; Cook, D.C.; Berger, R.L.; Friedman, F.K.

    1986-01-01

    Samples of 90% enriched 57Fe hemoglobin and its isolated subunits have been prepared. Moessbauer spectroscopic measurements have been made on three such samples. Sample one contained contributions of oxyhemoglobin, deoxyhemoglobin, and carbonmonoxyhemoglobin. This sample was studied from a temperature of 90 K down to 230 mK. Measurements were also made at 4.2 K using a small applied magnetic field of 1.0 T. In general, the measured quadrupole splittings and isomer shifts for each component agreed with previous measurements on single component samples in the literature, and thus demonstrated that chemically enriched hemoglobin has not been altered. The second and third samples were isolated alpha and beta subunits, respectively. We have found measurable Moessbauer spectral differences between the HbO 2 sites in the alpha subunit sample and the beta subunit sample. The measured Moessbauer spectral areas indicate that the iron ion has the largest mean-square displacement at the deoxy Hb sites as compared to that at the oxy- and carbonmonoxy Hb sites. The mean-square displacement at the HbO 2 sites is the smallest

  8. Comparison of the kinetic parameters of the truncated catalytic subunit and holoenzyme of human DNA polymerase ε

    Science.gov (United States)

    Zahurancik, Walter J.; Baranovskiy, Andrey G.; Tahirov, Tahir H.; Suo, Zucai

    2015-01-01

    Numerous genetic studies have provided compelling evidence to establish DNA polymerase ε (Polε) as the primary DNA polymerase responsible for leading strand synthesis during eukaryotic nuclear genome replication. Polε is a heterotetramer consisting of a large catalytic subunit that contains the conserved polymerase core domain as well as a 3′ → 5′ exonuclease domain common to many replicative polymerases. In addition, Polε possesses three small subunits that lack a known catalytic activity but associate with components involved in a variety of DNA replication and maintenance processes. Previous enzymatic characterization of the Polε heterotetramer from budding yeast suggested that the small subunits slightly enhance DNA synthesis by Polε in vitro. However, similar studies of the human Polε heterote-tramer (hPolε) have been limited by the difficulty of obtaining hPolε in quantities suitable for thorough investigation of its catalytic activity. Utilization of a baculovirus expression system for overexpression and purification of hPolε from insect host cells has allowed for isolation of greater amounts of active hPolε, thus enabling a more detailed kinetic comparison between hPolε and an active N-terminal fragment of the hPolε catalytic subunit (p261N), which is readily overexpressed in Escherichia coli. Here, we report the first pre-steady-state studies of fully-assembled hPolε. We observe that the small subunits increase DNA binding by hPolε relative to p261N, but do not increase processivity during DNA synthesis on a single-stranded M13 template. Interestingly, the 3′ → 5′ exonuclease activity of hPolε is reduced relative to p261N on matched and mismatched DNA substrates, indicating that the presence of the small subunits may regulate the proofreading activity of hPolε and sway hPolε toward DNA synthesis rather than proofreading. PMID:25684708

  9. Clinical staff nurse leadership: Identifying gaps in competency development.

    Science.gov (United States)

    Franks-Meeks, Sherron

    2018-01-01

    To date, there has been no development of a complete, applicable inventory of clinical staff nurse (CSN) leadership role competencies through a valid and reliable methodology. Further, the CSN has not been invited to engage in the identification, definition, or development of their own leadership competencies. Compare existing leadership competencies to identify and highlight gaps in clinical staff nurse leadership role competency development and validation. Literature review. The CSN has not participated in the development of CSN leadership role competencies, nor have the currently identified CSN leadership role competencies been scientifically validated through research. Finally, CSN leadership role competencies are incomplete and do not reflect the CSN perspective. © 2017 Wiley Periodicals, Inc.

  10. Evolution of the cAMP-dependent protein kinase (PKA catalytic subunit isoforms.

    Directory of Open Access Journals (Sweden)

    Kristoffer Søberg

    Full Text Available The 3',5'-cyclic adenosine monophosphate (cAMP-dependent protein kinase, or protein kinase A (PKA, pathway is one of the most versatile and best studied signaling pathways in eukaryotic cells. The two paralogous PKA catalytic subunits Cα and Cβ, encoded by the genes PRKACA and PRKACB, respectively, are among the best understood model kinases in signal transduction research. In this work, we explore and elucidate the evolution of the alternative 5' exons and the splicing pattern giving rise to the numerous PKA catalytic subunit isoforms. In addition to the universally conserved Cα1/Cβ1 isoforms, we find kinase variants with short N-termini in all main vertebrate classes, including the sperm-specific Cα2 isoform found to be conserved in all mammals. We also describe, for the first time, a PKA Cα isoform with a long N-terminus, paralogous to the PKA Cβ2 N-terminus. An analysis of isoform-specific variation highlights residues and motifs that are likely to be of functional importance.

  11. Time-Dependent Alterations in the Expression of NMDA Receptor Subunits along the Dorsoventral Hippocampal Axis in an Animal Model of Nascent Psychosis.

    Science.gov (United States)

    Dubovyk, Valentyna; Manahan-Vaughan, Denise

    2018-04-10

    Psychosis is a mental condition that is characterized by hallucinations, delusions, disordered thought, as well as socio-emotional and cognitive impairments. Once developed, it tends to progress into a chronic psychotic illness. Here, the duration of untreated psychosis plays a crucial role: the earlier the treatment begins, relative to the first episode of the disease, the better the patient's functional prognosis. To what extent the success of early interventions relate to progressive changes at the neurotransmitter receptor level is as yet unclear. In fact, very little is known as to how molecular changes develop, transform, and become established following the first psychotic event. One neurotransmitter receptor for which a specific role in psychosis has been discussed is the N-methyl-d-aspartate receptor (NMDAR). This receptor is especially important for information encoding in the hippocampus. The hippocampus is one of the loci of functional change in psychosis, to which a role in the pathophysiology of psychosis has been ascribed. Here, we examined whether changes in NMDAR subunit expression occur along the dorsoventral axis of the hippocampus 1 week and 3 months after systemic treatment with an NMDAR antagonist (MK801) that initiates a psychosis-like state in adult rats. We found early (1 week) upregulation of the GluN2B levels in the dorso-intermediate hippocampus and late (3 month) downregulation of GluN2A expression across the entire CA1 region. The ventral hippocampus did not exhibit subunit expression changes. These data suggest that a differing vulnerability of the hippocampal longitudinal axis may occur in response to MK801-treatment and provide a time-resolved view of the putative development of pathological changes of NMDAR subunit expression in the hippocampus that initiate with an emulated first episode and progress through to the chronic stabilization of a psychosis-like state in rodents.

  12. Influvac, a trivalent inactivated subunit influenza vaccine.

    Science.gov (United States)

    Zuccotti, Gian Vincenzo; Fabiano, Valentina

    2011-01-01

    Influenza represents a major sanitary and socio-economic burden and vaccination is universally considered the most effective strategy for preventing the disease and its complications. Traditional influenza vaccines have been on the market since the late 1940s, with million of doses administered annually worldwide, and demonstrated a substantial efficacy and safety. The trivalent inactivated subunit vaccine has been available for more than 25 years and has been studied in healthy children, adults and the elderly and in people affected by underlying chronic medical conditions. We describe vaccine technology focusing on subunit vaccine production procedures and mode of action and provide updated information on efficacy and safety available data. A review of efficacy and safety data in healthy subjects and in high risk populations from major sponsor- and investigator-driven studies. The vaccine showed a good immunogenicity and a favorable safety profile in all target groups. In the panorama of actually available influenza vaccines, trivalent inactivated subunit vaccine represents a well-established tool for preventing flu and the associated complications.

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

    Directory of Open Access Journals (Sweden)

    Zhong Tao P

    2007-07-01

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

  14. 4.0-A resolution cryo-EM structure of the mammalian chaperonin TRiC/CCT reveals its unique subunit arrangement.

    Science.gov (United States)

    Cong, Yao; Baker, Matthew L; Jakana, Joanita; Woolford, David; Miller, Erik J; Reissmann, Stefanie; Kumar, Ramya N; Redding-Johanson, Alyssa M; Batth, Tanveer S; Mukhopadhyay, Aindrila; Ludtke, Steven J; Frydman, Judith; Chiu, Wah

    2010-03-16

    The essential double-ring eukaryotic chaperonin TRiC/CCT (TCP1-ring complex or chaperonin containing TCP1) assists the folding of approximately 5-10% of the cellular proteome. Many TRiC substrates cannot be folded by other chaperonins from prokaryotes or archaea. These unique folding properties are likely linked to TRiC's unique heterooligomeric subunit organization, whereby each ring consists of eight different paralogous subunits in an arrangement that remains uncertain. Using single particle cryo-EM without imposing symmetry, we determined the mammalian TRiC structure at 4.7-A resolution. This revealed the existence of a 2-fold axis between its two rings resulting in two homotypic subunit interactions across the rings. A subsequent 2-fold symmetrized map yielded a 4.0-A resolution structure that evinces the densities of a large fraction of side chains, loops, and insertions. These features permitted unambiguous identification of all eight individual subunits, despite their sequence similarity. Independent biochemical near-neighbor analysis supports our cryo-EM derived TRiC subunit arrangement. We obtained a Calpha backbone model for each subunit from an initial homology model refined against the cryo-EM density. A subsequently optimized atomic model for a subunit showed approximately 95% of the main chain dihedral angles in the allowable regions of the Ramachandran plot. The determination of the TRiC subunit arrangement opens the way to understand its unique function and mechanism. In particular, an unevenly distributed positively charged wall lining the closed folding chamber of TRiC differs strikingly from that of prokaryotic and archaeal chaperonins. These interior surface chemical properties likely play an important role in TRiC's cellular substrate specificity.

  15. Dynamic properties of motor proteins with two subunits

    International Nuclear Information System (INIS)

    Kolomeisky, Anatoly B; III, Hubert Phillips

    2005-01-01

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

  16. Cyclic AMP regulation of the human glycoprotein hormone α-subunit gene is mediated by an 18-base-pair element

    International Nuclear Information System (INIS)

    Silver, B.J.; Bokar, J.A.; Virgin, J.B.; Vallen, E.A.; Milsted, A.; Nilson, J.H.

    1987-01-01

    cAMP regulates transcription of the gene encoding the α-subunit of human chorionic gonadotropin (hCG) in the choriocarcinoma cells (BeWo). To define the sequences required for regulation by cAMP, the authors inserted fragments from the 5' flanking region of the α-subunit gene into a test vector containing the simian virus 40 early promoter (devoid of its enhancer) linked to the bacterial chloramphenicol acetyltransferase (CAT) gene. Results from transient expression assays in BeWo cells indicated that a 1500-base-pair (bp) fragment conferred cAMP responsiveness on the CAT gene regardless of position or orientation of the insert relative to the viral promoter. A subfragment extending from position -169 to position -100 had the same effect on cAMP-induced expression. Furthermore, the entire stimulatory effect could be achieved with an 18-bp synthetic oligodeoxynucleotide corresponding to a direct repeat between position -146 and -111. In the absence of cAMP, the α-subunit 5' flanking sequence also enhanced transcription from the simian virus 40 early promoter. They localized this enhancer activity to the same -169/-100 fragment containing the cAMP response element. The 18-bp element alone, however, had no effect on basal expression. Thus, this short DNA sequence serves as a cAMP response element and also functions independently of other promoter-regulatory elements located in the 5' flanking sequence of the α-subunit gene

  17. The alpha3 laminin subunit, alpha6beta4 and alpha3beta1 integrin coordinately regulate wound healing in cultured epithelial cells and in the skin

    DEFF Research Database (Denmark)

    Goldfinger, L E; Hopkinson, S B; deHart, G W

    1999-01-01

    Previously, we demonstrated that proteolytic processing within the globular domain of the alpha3 subunit of laminin-5 (LN5) converts LN5 from a cell motility-inducing factor to a protein complex that can trigger the formation of hemidesmosomes, certain cell-matrix attachment sites found in epithe......-inhibiting antibodies, we provide evidence that LN5 and its two integrin receptors (alpha6beta4 and alpha3beta1) appear necessary for wound healing to occur in MCF-10A cell culture wounds. We propose a model for healing of wounded epithelial tissues based on these results....... in epithelial cells. We have prepared a monoclonal antibody (12C4) whose epitope is located toward the carboxy terminus of the globular domain of the alpha3 laminin subunit. This epitope is lost from the alpha3 subunit as a consequence of proteolytic processing. Antibody 12C4 stains throughout the matrix...... the wound site. A similar phenomenon is observed in human skin wounds, since we also detect expression of the unprocessed alpha3 laminin subunit at the leading tip of the sheet of epidermal cells that epithelializes skin wounds in vivo. In addition, using alpha3 laminin subunit and integrin function...

  18. Role of post-translational modifications at the β-subunit ectodomain in complex association with a promiscuous plant P4-ATPase.

    Science.gov (United States)

    Costa, Sara R; Marek, Magdalena; Axelsen, Kristian B; Theorin, Lisa; Pomorski, Thomas G; López-Marqués, Rosa L

    2016-06-01

    P-type ATPases of subfamily IV (P4-ATPases) constitute a major group of phospholipid flippases that form heteromeric complexes with members of the Cdc50 (cell division control 50) protein family. Some P4-ATPases interact specifically with only one β-subunit isoform, whereas others are promiscuous and can interact with several isoforms. In the present study, we used a site-directed mutagenesis approach to assess the role of post-translational modifications at the plant ALIS5 β-subunit ectodomain in the functionality of the promiscuous plant P4-ATPase ALA2. We identified two N-glycosylated residues, Asn(181) and Asn(231) Whereas mutation of Asn(231) seems to have a small effect on P4-ATPase complex formation, mutation of evolutionarily conserved Asn(181) disrupts interaction between the two subunits. Of the four cysteine residues located in the ALIS5 ectodomain, mutation of Cys(86) and Cys(107) compromises complex association, but the mutant β-subunits still promote complex trafficking and activity to some extent. In contrast, disruption of a conserved disulfide bond between Cys(158) and Cys(172) has no effect on the P4-ATPase complex. Our results demonstrate that post-translational modifications in the β-subunit have different functional roles in different organisms, which may be related to the promiscuity of the P4-ATPase. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  19. Structure of Escherichia coli RutC, a member of the YjgF family and putative aminoacrylate peracid reductase of the rut operon

    International Nuclear Information System (INIS)

    Knapik, Aleksandra Alicja; Petkowski, Janusz Jurand; Otwinowski, Zbyszek; Cymborowski, Marcin Tadeusz; Cooper, David Robert; Chruszcz, Maksymilian; Krajewska, Wanda Małgorzata; Minor, Wladek

    2012-01-01

    The structure of the putative aminoacrylate peracid reductase RutC of the rut operon, a member of the YjgF family, is reported. RutC is the third enzyme in the Escherichia coli rut pathway of uracil degradation. RutC belongs to the highly conserved YjgF family of proteins. The structure of the RutC protein was determined and refined to 1.95 Å resolution. The crystal belonged to space group P2 1 2 1 2 and contained six molecules in the asymmetric unit. The structure was solved by SAD phasing and was refined to an R work of 19.3% (R free = 21.7%). The final model revealed that this protein has a Bacillus chorismate mutase-like fold and forms a homotrimer with a hydrophobic cavity in the center of the structure and ligand-binding clefts between two subunits. A likely function for RutC is the reduction of peroxy-aminoacrylate to aminoacrylate as a part of a detoxification process

  20. Target specificity, in vivo pharmacokinetics, and efficacy of the putative STAT3 inhibitor LY5 in osteosarcoma, Ewing's sarcoma, and rhabdomyosarcoma.

    Directory of Open Access Journals (Sweden)

    Peter Y Yu

    Full Text Available STAT3 is a transcription factor involved in cytokine and receptor kinase signal transduction that is aberrantly activated in a variety of sarcomas, promoting metastasis and chemotherapy resistance. The purpose of this work was to develop and test a novel putative STAT3 inhibitor, LY5.An in silico fragment-based drug design strategy was used to create LY5, a small molecule inhibitor that blocks the STAT3 SH2 domain phosphotyrosine binding site, inhibiting homodimerization. LY5 was evaluated in vitro demonstrating good biologic activity against rhabdomyosarcoma, osteosarcoma and Ewing's sarcoma cell lines at high nanomolar/low micromolar concentrations, as well as specific inhibition of STAT3 phosphorylation without effects on other STAT3 family members. LY5 exhibited excellent oral bioavailability in both mice and healthy dogs, and drug absorption was enhanced in the fasted state with tolerable dosing in mice at 40 mg/kg BID. However, RNAi-mediated knockdown of STAT3 did not phenocopy the biologic effects of LY5 in sarcoma cell lines. Moreover, concentrations needed to inhibit ex vivo metastasis growth using the PuMA assay were significantly higher than those needed to inhibit STAT3 phosphorylation in vitro. Lastly, LY5 treatment did not inhibit the growth of sarcoma xenografts or prevent pulmonary metastasis in mice.LY5 is a novel small molecule inhibitor that effectively inhibits STAT3 phosphorylation and cell proliferation at nanomolar concentrations. LY5 demonstrates good oral bioavailability in mice and dogs. However LY5 did not decrease tumor growth in xenograft mouse models and STAT3 knockdown did not induce concordant biologic effects. These data suggest that the anti-cancer effects of LY5 identified in vitro were not mediated through STAT3 inhibition.

  1. Target specificity, in vivo pharmacokinetics, and efficacy of the putative STAT3 inhibitor LY5 in osteosarcoma, Ewing's sarcoma, and rhabdomyosarcoma.

    Science.gov (United States)

    Yu, Peter Y; Gardner, Heather L; Roberts, Ryan; Cam, Hakan; Hariharan, Seethalakshmi; Ren, Ling; LeBlanc, Amy K; Xiao, Hui; Lin, Jiayuh; Guttridge, Denis C; Mo, Xiaokui; Bennett, Chad E; Coss, Christopher C; Ling, Yonghua; Phelps, Mitch A; Houghton, Peter; London, Cheryl A

    2017-01-01

    STAT3 is a transcription factor involved in cytokine and receptor kinase signal transduction that is aberrantly activated in a variety of sarcomas, promoting metastasis and chemotherapy resistance. The purpose of this work was to develop and test a novel putative STAT3 inhibitor, LY5. An in silico fragment-based drug design strategy was used to create LY5, a small molecule inhibitor that blocks the STAT3 SH2 domain phosphotyrosine binding site, inhibiting homodimerization. LY5 was evaluated in vitro demonstrating good biologic activity against rhabdomyosarcoma, osteosarcoma and Ewing's sarcoma cell lines at high nanomolar/low micromolar concentrations, as well as specific inhibition of STAT3 phosphorylation without effects on other STAT3 family members. LY5 exhibited excellent oral bioavailability in both mice and healthy dogs, and drug absorption was enhanced in the fasted state with tolerable dosing in mice at 40 mg/kg BID. However, RNAi-mediated knockdown of STAT3 did not phenocopy the biologic effects of LY5 in sarcoma cell lines. Moreover, concentrations needed to inhibit ex vivo metastasis growth using the PuMA assay were significantly higher than those needed to inhibit STAT3 phosphorylation in vitro. Lastly, LY5 treatment did not inhibit the growth of sarcoma xenografts or prevent pulmonary metastasis in mice. LY5 is a novel small molecule inhibitor that effectively inhibits STAT3 phosphorylation and cell proliferation at nanomolar concentrations. LY5 demonstrates good oral bioavailability in mice and dogs. However LY5 did not decrease tumor growth in xenograft mouse models and STAT3 knockdown did not induce concordant biologic effects. These data suggest that the anti-cancer effects of LY5 identified in vitro were not mediated through STAT3 inhibition.

  2. MOLECULAR-GENETIC REEVALUATION OF THE DUTCH HYPEREKPLEXIA FAMILY

    NARCIS (Netherlands)

    de Koning-Tijssen, Marina; SHIANG, R; VANDEUTEKOM, J; BOERMAN, RH; WASMUTH, JJ; SANDKUIJL, LA; FRANTS, RR; PADBERG, GW

    Objectives: To confirm linkage of the locus of the major form of hyperekplexia to markers on chromosome 5q, to screen for a point mutation in the gene encoding the oil subunit of the glycine receptor, and to investigate whether the putative ''minor'' form of hyperekplexia, consisting of an excessive

  3. Shared active site architecture between archaeal PolD and multi-subunit RNA polymerases revealed by X-ray crystallography

    OpenAIRE

    Sauguet , Ludovic; Raia , Pierre; Henneke , Ghislaine; Delarue , Marc

    2016-01-01

    International audience; Archaeal replicative DNA polymerase D (PolD) constitute an atypical class of DNA polymerases made of a proofreading exonuclease subunit (DP1) and a larger polymerase catalytic subunit (DP2), both with unknown structures. We have determined the crystal structures of Pyrococcus abyssi DP1 and DP2 at 2.5 and 2.2 Å resolution, respectively, revealing a catalytic core strikingly different from all other known DNA polymerases (DNAPs). Rather, the PolD DP2 catalytic core has ...

  4. GABAA receptor subunit expression changes in the human Alzheimer's disease hippocampus, subiculum, entorhinal cortex and superior temporal gyrus.

    Science.gov (United States)

    Kwakowsky, Andrea; Calvo-Flores Guzmán, Beatriz; Pandya, Madhavi; Turner, Clinton; Waldvogel, Henry J; Faull, Richard L

    2018-02-27

    Gamma-aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the central nervous system. GABA type A receptors (GABA A Rs) are severely affected in Alzheimer's disease (AD). However, the distribution and subunit composition of GABA A Rs in the AD brain are not well understood. This is the first comprehensive study to show brain region- and cell layer-specific alterations in the expression of the GABA A R subunits α1-3, α5, β1-3 and γ2 in the human AD hippocampus, entorhinal cortex and superior temporal gyrus (STG). In late-stage AD tissue samples using immunohistochemistry we found significant alteration of all investigated GABA A Rs subunits except for α3 and β1 that were well preserved. The most prominent changes include an increase in GABA A R α1 expression associated with AD in all layers of the CA3 region, in the stratum (str.) granulare and hilus of the dentate gyrus (DG). We found a significant increase in GABA A R α2 expression in the str. oriens of the CA1-3, str. radiatum of the CA2,3 and decrease in the str. pyramidale of the CA1 region in AD cases. In AD there was a significant increase in GABA A R α5 subunit expression in str. pyramidale, str. oriens of the CA1 region and decrease in the STG. We also found a significant decrease in the GABA A R β3 subunit immunoreactivity in the str. oriens of the CA2, str. granulare and str. moleculare of the DG. In conclusion, these findings indicate that the expression of the GABA A R subunits shows brain region- and layer-specific alterations in AD, and these changes could significantly influence and alter GABA A R function in the disease. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  5. Self-subunit swapping occurs in another gene type of cobalt nitrile hydratase.

    Directory of Open Access Journals (Sweden)

    Yi Liu

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

  6. The δ subunit of RNA polymerase guides promoter selectivity and virulence in Staphylococcus aureus.

    Science.gov (United States)

    Weiss, Andy; Ibarra, J Antonio; Paoletti, Jessica; Carroll, Ronan K; Shaw, Lindsey N

    2014-04-01

    In Gram-positive bacteria, and particularly the Firmicutes, the DNA-dependent RNA polymerase (RNAP) complex contains an additional subunit, termed the δ factor, or RpoE. This enigmatic protein has been studied for more than 30 years for various organisms, but its function is still not well understood. In this study, we investigated its role in the major human pathogen Staphylococcus aureus. We showed conservation of important structural regions of RpoE in S. aureus and other species and demonstrated binding to core RNAP that is mediated by the β and/or β' subunits. To identify the impact of the δ subunit on transcription, we performed transcriptome sequencing (RNA-seq) analysis and observed 191 differentially expressed genes in the rpoE mutant. Ontological analysis revealed, quite strikingly, that many of the downregulated genes were known virulence factors, while several mobile genetic elements (SaPI5 and prophage SA3usa) were strongly upregulated. Phenotypically, the rpoE mutant had decreased accumulation and/or activity of a number of key virulence factors, including alpha toxin, secreted proteases, and Panton-Valentine leukocidin (PVL). We further observed significantly decreased survival of the mutant in whole human blood, increased phagocytosis by human leukocytes, and impaired virulence in a murine model of infection. Collectively, our results demonstrate that the δ subunit of RNAP is a critical component of the S. aureus transcription machinery and plays an important role during infection.

  7. A charged residue at the subunit interface of PCNA promotes trimer formation by destabilizing alternate subunit interactions

    International Nuclear Information System (INIS)

    Freudenthal, Bret D.; Gakhar, Lokesh; Ramaswamy, S.; Washington, M. Todd

    2009-01-01

    Eukaryotic proliferating cell nuclear antigen (PCNA), an essential accessory factor in DNA replication and repair, is a ring-shaped homotrimer. A novel nontrimeric structure of E113G-mutant PCNA protein is reported, which shows that this protein forms alternate subunit interactions. It is concluded that the charged side chain of Glu113 promotes normal trimer formation by destabilizing these alternate subunit interactions. Eukaryotic proliferating cell nuclear antigen (PCNA) is an essential replication accessory factor that interacts with a variety of proteins involved in DNA replication and repair. Each monomer of PCNA has an N-terminal domain A and a C-terminal domain B. In the structure of the wild-type PCNA protein, domain A of one monomer interacts with domain B of a neighboring monomer to form a ring-shaped trimer. Glu113 is a conserved residue at the subunit interface in domain A. Two distinct X-ray crystal structures have been determined of a mutant form of PCNA with a substitution at this position (E113G) that has previously been studied because of its effect on translesion synthesis. The first structure was the expected ring-shaped trimer. The second structure was an unanticipated nontrimeric form of the protein. In this nontrimeric form, domain A of one PCNA monomer interacts with domain A of a neighboring monomer, while domain B of this monomer interacts with domain B of a different neighboring monomer. The B–B interface is stabilized by an antiparallel β-sheet and appears to be structurally similar to the A–B interface observed in the trimeric form of PCNA. The A–A interface, in contrast, is primarily stabilized by hydrophobic interactions. Because the E113G substitution is located on this hydrophobic surface, the A–A interface should be less favorable in the case of the wild-type protein. This suggests that the side chain of Glu113 promotes trimer formation by destabilizing these possible alternate subunit interactions

  8. Theoretical study of structures of Ga{sub 5}N{sub 5} cluster

    Energy Technology Data Exchange (ETDEWEB)

    Song Bin; Cao Peilin

    2002-12-23

    The structures and energies of a Ga{sub 5}N{sub 5} cluster have been calculated using a full-potential linear-muffin-tin-orbital (FP-LMTO) method, combined with molecular dynamics technique. Twenty-four structures for a Ga{sub 5}N{sub 5} cluster have been obtained. The most stable structure is a C{sub 1} planar structure with a N{sub 3} subunit. The Ga{sub 5}N{sub 5} clusters show a preference for a N{sub 3} subunit, revealing the same behavior as in the Ga{sub 3}N{sub 3} and Ga{sub 4}N{sub 4} clusters. The existence of strong N-N bonds dominates the structure of a Ga{sub 5}N{sub 5} cluster. Through the calculation of the density of states we found that the most stable structure of Ga{sub 5}N{sub 5} clusters presented semiconductor-like properties.

  9. Genetic variation in KCNA5

    DEFF Research Database (Denmark)

    Christophersen, Ingrid E; Olesen, Morten S; Liang, Bo

    2012-01-01

    AimsGenetic factors may be important in the development of atrial fibrillation (AF) in the young. KCNA5 encodes the potassium channel a-subunit K(V)1.5, which underlies the voltage-gated atrial-specific potassium current I(Kur). KCNAB2 encodes K(V)ß2, a ß-subunit of K(V)1.5, which increases I......(Kur). Three studies have identified loss-of-function mutations in KCNA5 in patients with idiopathic AF. We hypothesized that early-onset lone AF is associated with high prevalence of genetic variants in KCNA5 and KCNAB2.Methods and resultsThe coding sequences of KCNA5 and KCNAB2 were sequenced in 307 patients...

  10. Mining Protein Evolution for Insights into Mechanisms of Voltage-Dependent Sodium Channel Auxiliary Subunits.

    Science.gov (United States)

    Molinarolo, Steven; Granata, Daniele; Carnevale, Vincenzo; Ahern, Christopher A

    2018-02-21

    Voltage-gated sodium channel (VGSC) beta (β) subunits have been called the "overachieving" auxiliary ion channel subunit. Indeed, these subunits regulate the trafficking of the sodium channel complex at the plasma membrane and simultaneously tune the voltage-dependent properties of the pore-forming alpha-subunit. It is now known that VGSC β-subunits are capable of similar modulation of multiple isoforms of related voltage-gated potassium channels, suggesting that their abilities extend into the broader voltage-gated channels. The gene family for these single transmembrane immunoglobulin beta-fold proteins extends well beyond the traditional VGSC β1-β4 subunit designation, with deep roots into the cell adhesion protein family and myelin-related proteins - where inherited mutations result in a myriad of electrical signaling disorders. Yet, very little is known about how VGSC β-subunits support protein trafficking pathways, the basis for their modulation of voltage-dependent gating, and, ultimately, their role in shaping neuronal excitability. An evolutionary approach can be useful in yielding new clues to such functions as it provides an unbiased assessment of protein residues, folds, and functions. An approach is described here which indicates the greater emergence of the modern β-subunits roughly 400 million years ago in the early neurons of Bilateria and bony fish, and the unexpected presence of distant homologues in bacteriophages. Recent structural breakthroughs containing α and β eukaryotic sodium channels containing subunits suggest a novel role for a highly conserved polar contact that occurs within the transmembrane segments. Overall, a mixture of approaches will ultimately advance our understanding of the mechanism for β-subunit interactions with voltage-sensor containing ion channels and membrane proteins.

  11. Large-conductance Ca2+-activated K+ channel β1-subunit knockout mice are not hypertensive

    Science.gov (United States)

    Garver, Hannah; Galligan, James J.; Fink, Gregory D.

    2011-01-01

    Large-conductance Ca2+-activated K+ (BK) channels are composed of pore-forming α-subunits and accessory β1-subunits that modulate Ca2+ sensitivity. BK channels regulate arterial myogenic tone and renal Na+ clearance/K+ reabsorption. Previous studies using indirect or short-term blood pressure measurements found that BK channel β1-subunit knockout (BK β1-KO) mice were hypertensive. We evaluated 24-h mean arterial pressure (MAP) and heart rate in BK β1-KO mice using radiotelemetry. BK β1-KO mice did not have a higher 24-h average MAP when compared with wild-type (WT) mice, although MAP was ∼10 mmHg higher at night. The dose-dependent peak declines in MAP by nifedipine were only slightly larger in BK β1-KO mice. In BK β1-KO mice, giving 1% NaCl to mice to drink for 7 days caused a transient (5 days) elevation of MAP (∼5 mmHg); MAP returned to pre-saline levels by day 6. BK β1-KO mesenteric arteries in vitro demonstrated diminished contractile responses to paxilline, increased reactivity to Bay K 8644 and norepinephrine (NE), and maintained relaxation to isoproterenol. Paxilline and Bay K 8644 did not constrict WT or BK β1-KO mesenteric veins (MV). BK β1-subunits are not expressed in MV. The results indicate that BK β1-KO mice are not hypertensive on normal or high-salt intake. BK channel deficiency increases arterial reactivity to NE and L-type Ca2+ channel function in vitro, but the L-type Ca2+ channel modulation of MAP is not altered in BK β1-KO mice. BK and L-type Ca2+ channels do not modulate murine venous tone. It appears that selective loss of BK channel function in arteries only is not sufficient to cause sustained hypertension. PMID:21131476

  12. Therapeutic potential of Mediator complex subunits in metabolic diseases.

    Science.gov (United States)

    Ranjan, Amol; Ansari, Suraiya A

    2018-01-01

    The multisubunit Mediator is an evolutionary conserved transcriptional coregulatory complex in eukaryotes. It is needed for the transcriptional regulation of gene expression in general as well as in a gene specific manner. Mediator complex subunits interact with different transcription factors as well as components of RNA Pol II transcription initiation complex and in doing so act as a bridge between gene specific transcription factors and general Pol II transcription machinery. Specific interaction of various Mediator subunits with nuclear receptors (NRs) and other transcription factors involved in metabolism has been reported in different studies. Evidences indicate that ligand-activated NRs recruit Mediator complex for RNA Pol II-dependent gene transcription. These NRs have been explored as therapeutic targets in different metabolic diseases; however, they show side-effects as targets due to their overlapping involvement in different signaling pathways. Here we discuss the interaction of various Mediator subunits with transcription factors involved in metabolism and whether specific interaction of these transcription factors with Mediator subunits could be potentially utilized as therapeutic strategy in a variety of metabolic diseases. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  13. Restless 5S: the re-arrangement(s) and evolution of the nuclear ribosomal DNA in land plants.

    Science.gov (United States)

    Wicke, Susann; Costa, Andrea; Muñoz, Jesùs; Quandt, Dietmar

    2011-11-01

    Among eukaryotes two types of nuclear ribosomal DNA (nrDNA) organization have been observed. Either all components, i.e. the small ribosomal subunit, 5.8S, large ribosomal subunit, and 5S occur tandemly arranged or the 5S rDNA forms a separate cluster of its own. Generalizations based on data derived from just a few model organisms have led to a superimposition of structural and evolutionary traits to the entire plant kingdom asserting that plants generally possess separate arrays. This study reveals that plant nrDNA organization into separate arrays is not a distinctive feature, but rather assignable almost solely to seed plants. We show that early diverging land plants and presumably streptophyte algae share a co-localization of all rRNA genes within one repeat unit. This raises the possibility that the state of rDNA gene co-localization had occurred in their common ancestor. Separate rDNA arrays were identified for all basal seed plants and water ferns, implying at least two independent 5S rDNA transposition events during land plant evolution. Screening for 5S derived Cassandra transposable elements which might have played a role during the transposition events, indicated that this retrotransposon is absent in early diverging vascular plants including early fern lineages. Thus, Cassandra can be rejected as a primary mechanism for 5S rDNA transposition in water ferns. However, the evolution of Cassandra and other eukaryotic 5S derived elements might have been a side effect of the 5S rDNA cluster formation. Structural analysis of the intergenic spacers of the ribosomal clusters revealed that transposition events partially affect spacer regions and suggests a slightly different transcription regulation of 5S rDNA in early land plants. 5S rDNA upstream regulatory elements are highly divergent or absent from the LSU-5S spacers of most early divergent land plant lineages. Several putative scenarios and mechanisms involved in the concerted relocation of hundreds of 5S

  14. Distinct forms of the β subunit of GTP-binding regulatory proteins identified by molecular cloning

    International Nuclear Information System (INIS)

    Fong, H.K.W.; Amatruda, T.T. III; Birren, B.W.; Simon, M.I.

    1987-01-01

    Two distinct β subunits of guanine nucleotide-binding regulatory proteins have been identified by cDNA cloning and are referred to as β 1 and β 1 subunits. The bovine transducin β subunit (β 1 ) has been cloned previously. The author now isolated and analyzed cDNA clones that encode the β 2 subunit from bovine adrenal, bovine brain, and a human myeloid leukemia cell line, HL-60. The 340-residue M/sub r/ 37,329 Β 2 protein is 90% identical with β 1 in predicted amino acid sequence, and it is also organized as a series of repetitive homologous segments. The major mRNA that encodes the bovine β 2 subunit is 1.7 kilobases in length. It is expressed at lower levels than β 1 subunit mRNA in all tissues examined. The β 1 and β 2 messages are expressed in cloned human cell lines. Hybridization of cDNA probes to bovine DNA showed that β 1 and β 2 are encoded by separate genes. The amino acid sequences for the bovine and human β 2 subunit are identical, as are the amino acid sequences for the bovine and human β 1 subunit. This evolutionary conservation suggests that the two β subunits have different roles in the signal transduction process

  15. Analysis of Maxi-K alpha subunit splice variants in human myometrium

    Directory of Open Access Journals (Sweden)

    Morrison John J

    2004-09-01

    Full Text Available Abstract Background Large-conductance, calcium-activated potassium (Maxi-K channels are implicated in the modulation of human uterine contractions and myometrial Ca2+ homeostasis. However, the regulatory mechanism(s governing the expression of Maxi-K channels with decreased calcium sensitivity at parturition are unclear. The objectives of this study were to investigate mRNA expression of the Maxi-K alpha subunit, and that of its splice variants, in human non-pregnant and pregnant myometrium, prior to and after labour onset, to determine whether altered expression of these splice variants is associated with decreased calcium sensitivity observed at labour onset. Methods Myometrial biopsies were obtained at hysterectomy (non-pregnant, NP, and at Caesarean section, at elective (pregnant not-in-labour, PNL and intrapartum (pregnant in-labour, PL procedures. RNA was extracted from all biopsies and quantitative real-time RT-PCR was used to investigate for possible differential expression of the Maxi-K alpha subunit, and that of its splice variants, between these functionally-distinct myometrial tissue sets. Results RT-PCR analysis identified the presence of a 132 bp and an 87 bp spliced exon of the Maxi-K alpha subunit in all three myometrial tissue sets. Quantitative real-time PCR indicated a decrease in the expression of the Maxi-K alpha subunit with labour onset. While there was no change in the proportion of Maxi-K alpha subunits expressing the 87 bp spliced exon, the proportion of alpha subunits expressing the 132 bp spliced exon was significantly increased with labour onset, compared to both non-pregnant and pregnant not-in-labour tissues. An increased proportion of 132 bp exon-containing alpha subunit variants with labour onset is of interest, as channels expressing this spliced exon have decreased calcium and voltage sensitivities. Conclusions Our findings suggest that decreased Maxi-K alpha subunit mRNA expression in human myometrium at

  16. Helicobacter pylori VacA toxin/subunit p34: targeting of an anion channel to the inner mitochondrial membrane.

    Directory of Open Access Journals (Sweden)

    Grazyna Domańska

    2010-04-01

    Full Text Available The vacuolating toxin VacA, released by Helicobacter pylori, is an important virulence factor in the pathogenesis of gastritis and gastroduodenal ulcers. VacA contains two subunits: The p58 subunit mediates entry into target cells, and the p34 subunit mediates targeting to mitochondria and is essential for toxicity. In this study we found that targeting to mitochondria is dependent on a unique signal sequence of 32 uncharged amino acid residues at the p34 N-terminus. Mitochondrial import of p34 is mediated by the import receptor Tom20 and the import channel of the outer membrane TOM complex, leading to insertion of p34 into the mitochondrial inner membrane. p34 assembles in homo-hexamers of extraordinary high stability. CD spectra of the purified protein indicate a content of >40% beta-strands, similar to pore-forming beta-barrel proteins. p34 forms an anion channel with a conductivity of about 12 pS in 1.5 M KCl buffer. Oligomerization and channel formation are independent both of the 32 uncharged N-terminal residues and of the p58 subunit of the toxin. The conductivity is efficiently blocked by 5-nitro-2-(3-phenylpropylaminobenzoic acid (NPPB, a reagent known to inhibit VacA-mediated apoptosis. We conclude that p34 essentially acts as a small pore-forming toxin, targeted to the mitochondrial inner membrane by a special hydrophobic N-terminal signal.

  17. Dithiothreitol activation of the insulin receptor/kinase does not involve subunit dissociation of the native α2β2 insulin receptor subunit complex

    International Nuclear Information System (INIS)

    Sweet, L.J.; Wilden, P.A.; Pessin, J.E.

    1986-01-01

    The subunit composition of the dithiothreitol- (DTT) activated insulin receptor/kinase was examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and gel filtration chromatography under denaturing or nondenaturing conditions. Pretreatment of 32 P-labeled insulin receptors with 50 mM DTT followed by gel filtration chromatography in 0.1% SDS demonstrated the dissociation of the α 2 β 2 insulin receptor complex (M/sub r/ 400,000) into the monomeric 95,000 β subunit. In contrast, pretreatment of the insulin receptors with 1-50 mM DTT followed by gel filtration chromatography in 0.1% Triton X-100 resulted in no apparent alteration in mobility compared to the untreated insulin receptors. Resolution of this complex by nonreducing SDS-polyacrylamide gel electrophoresis and autoradiography demonstrated the existence of the α 2 β 2 heterotetrameric complex with essentially no αβ heterodimeric or free monomeric β subunit species present. This suggests that the insulin receptor can reoxidize into the M/sub r/ 400,000 complex after the removal of DTT by gel filtration chromatography. To prevent reoxidation, the insulin receptors were pretreated with 50 mM DTT. Under the conditions the insulin receptors migrated as the M/sub r/ 400,000 α 2 β 2 complex. These results demonstrate that treatment of the insulin receptors with high concentrations of DTT, followed by removal of DTT by gel filtration, results in reoxidation of the reduced α 2 β 2 insulin receptor complex. Further, these results document that although the DTT stimulation of the insulin receptor/kinase does involve reduction of the insulin receptor subunits, it does not result in dissociation of the native α 2 β 2 insulin receptor subunit complex

  18. The role of the nuclear safety regulator

    International Nuclear Information System (INIS)

    Mellado, I.

    2007-01-01

    The Consejo de Seguridad Nuclear (CSN), or Nuclear Safety Council, is the only Spanish institution qualified in nuclear safety and radiological protection. Created in 1980, the CSN is independent of the Central State Administration, and possesses its own legal standing, estate and resources acquired directly from tax revenues. The CSN proposes regulations and advises the government on subjects within its competence, including the criteria for siting nuclear facilities once the autonomous regions have been informed. The CSN is responsible for issuing mandatory and binding reports to the Ministry of Industry. Tourism and Commerce, and for inspections and evaluation of the facilities included within its realm of competence throughout their phases (construction, start-up, operating and decommissioning). It is also responsible for the radiological control and surveillance of workers, the general public and the environment, as described below. In 1999, a new responsibility was assigned to the CSN to perform studies, assessment and inspections in relation to all phases of radioactive waste and spent fuel management. The CSN reports to the Spanish Parliament and is not subject to the hierarchy or auspices of the Government or the organisations in charge of promoting nuclear energy. The Council itself is an Associative Body comprised of 5 members, appointed by Parliament for a 6 year term (these members cannot be removed). Under this Council is situated an extensive technical body. A General Secretary is seconded by Technical Directors in the area of Nuclear Safety and Radiological Protection. As well there are a R and D Office, an Inspection Office, and a Technical Standards Office. The CSN counts 446 workers, of which 191 are university graduate specialists in nuclear safety or radiological protection. The average age is 45 years. Ongoing training is provided in technical specialties and management. (author)

  19. Specific radioimmunoassay of HCG and its α and β subunits: methods and results

    International Nuclear Information System (INIS)

    Reuter, A.M.; Schoonbrood, J.; Franchimont, P.

    1976-01-01

    To create antisera that are specific for the radioimmunoassay of HCG and its subunits, the antisera are neutralized by incubation with LH or HCG. For each RIA system the inhibition curves of HCG and its subunits LH, FSH, TSH and STH are obtained. The 125 I labelled hormones HCG, α and β subunits and LH were chromatographed over a Sephadex G 100 column. Serum of menopausal and pregnant women were chromatographed in the same way and the fractions subjected to RIA. HCG and its subunits were determined by RIA in the sera of patients with different kinds of cancer

  20. Molecular cloning of the α subunit of human and guinea pig leukocyte adhesion glycoprotein Mo1: Chromosomal localization and homology to the α subunits of integrins

    International Nuclear Information System (INIS)

    Arnaout, M.A.; Remold-O'Donnell, E.; Pierce, M.W.; Harris, P.; Tenen, D.G.

    1988-01-01

    The cell surface-glycoprotein Mo1 is a member of the family of leukocyte cell adhesion molecules (Leu-CAMs) that includes lymphocyte function-associated antigen 1 (LFA-1) and p150,95. Each Leu-CAM is a heterodimer with a distinct α subunit noncovalently associated with a common β subunit. The authors describe the isolation and analysis of two partial cDNA clones encoding the α subunit of the Leu-CAM Mo1 in humans and guinea pigs. A monoclonal antibody directed against an epitope in the carboxyl-terminal portion of the guinea pig α chain was used for immunoscreening a λgt11 expression library. The sequence of a 378-base-pair insert from one immunoreactive clone revealed a single continuous open reading frame encoding 126 amino acids including a 26-amino acid tryptic peptide isolated from the purified guinea pig α subunit. A cDNA clone of identical size was isolated from a human monocyte/lymphocyte cDNA library by using the guinea pig clone as a probe. The human clone also encoded a 126-amino acid peptide including the sequence of an additional tryptic peptide present in purified human Mo1α chain. Southern analysis of DNA from hamster-human hybrids localized the human Mo1α chain to chromosome 16, which has been shown to contain the gene for the α chain of lymphocyte function-associated antigen 1. These data suggest that the α subunits of Leu-CAMs evolved by gene duplication from a common ancestral gene and strengthen the hypothesis that the α subunits of these heterodimeric cell adhesion molecules on myeloid and lymphoid cells, platelets, and fibroblasts are evolutionary related

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

    Science.gov (United States)

    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.

  2. G-protein α-subunit expression, myristoylation, and membrane association in COS cells

    International Nuclear Information System (INIS)

    Mumby, S.M.; Gilman, A.G.; Heukeroth, R.O.; Gordon, J.I.

    1990-01-01

    Myristolyation of seven different α subunits of guanine nucleotide-binding regulatory proteins (G proteins) was examined by expressing these proteins in monkey kidney COS cells. Metabolic labeling studies of cells transfected with cytomegalovirus-based expression vectors indicated that [ 3 H]myristate was incorporated into α i1 , α i2 , α i3 , α 0 , and α 1 , and α z but not α s subunits. The role of myristoylation in the association of α subunits with membranes was analyzed by site-directed mutagenesis and by substitution of myristate with a less hydrophobic analog, 10-(propoxy)decanoate (11-oxamyristate). Myristoylation of α 0 was blocked when an alanine residue was substituted for its amino-terminal glycine, as was association of the protein with membranes. Substitution of the myristoyl group with 11-oxamyristate affected the cellular distribution of a subset of acylated α subunits. The results are consistent with a model wherein the hydrophobic interaction of myristate with the bilayer permits continued association of the protein with the plasma membrane when G-protein α subunits dissociated from βγ

  3. Distribution of protein and RNA in the 30S ribosomal subunit

    International Nuclear Information System (INIS)

    Ramakrishnan, V.

    1986-01-01

    In Escherichia coli, the small ribosomal subunit has a sedimentation coefficient of 30S, and consists of a 16S RNA molecule of 1541 nucleotides complexed with 21 proteins. Over the last few years, a controversy has emerged regarding the spatial distribution of RNA and protein in the 30S subunit. Contrast variation with neutron scattering was used to suggest that the RNA was located in a central core of the subunit and the proteins mainly in the periphery, with virtually no separation between the centers of mass of protein and RNA. However, these findings are incompatible with the results of efforts to locate individual ribosomal proteins by immune electron microscopy and triangulation with interprotein distance measurements. The conflict between these two views is resolved in this report of small-angle neutron scattering measurements on 30S subunits with and without protein S1, and on subunits reconstituted from deuterated 16S RNA and unlabeled proteins. The results show that (i) the proteins and RNA are intermingled, with neither component dominating at the core or the periphery, and (ii) the spatial distribution of protein and RNA is asymmetrical, with a separation between their centers of mass of about 25 angstroms

  4. Nuclear safety in Spain

    International Nuclear Information System (INIS)

    Caro, R.

    1988-01-01

    Control and monitoring of all Spanish nuclear facilities was first carried out by the Department of Nuclear Safety of the Junta de Energia Nuclear established by the Nuclear Energy Act in 1964. Later, following the example of other Western countries, it was concluded that regulations and monitoring of nuclear energy on one hand and its promotion and development on the other should not be done by the same national body. Therefore, the Consejo de Seguridad Nuclear (CSN) was created in 1980, as the sole national body responsible for controlling the safety of nuclear installations, and radiological protection. The CSN has five members, one chairman and four comissioners, required to be independent and therefore with politically objective criteria, internationally acknowledged technical capability, and free from other duties and responsibilities. For this purpose the Chairman has been given the status of Minister and the commissioners that of Secretary of State. They serve for six years, after being accepted by Parliament by a majority of at least 3/5 of the votes, and are called upon to report to Parliament at least twice a year on nuclear safety and radiological protection in the country. A complete report on those issues is presented to Parliament, becoming a politic document as from that moment. To prepare that report (basically a summary of CSN activities) and, in general, to fulfill all its tasks, the CSN has a staff of some 300, about 50% being technical. CSN activities cover: 1. Standards; 2. Licences; 3. Research; 4. Environment; 5. Information; and 6. International Relations

  5. AcEST: BP917133 [AcEST

    Lifescience Database Archive (English)

    Full Text Available 775_CUPTR Putative PROPIONYL-COA CARBOXYLASE (BETA SUBUNIT) PROTEIN OS=Cupriavidus taiwan...TA SUBUNIT) PROTEIN OS=Cupriavidus taiwanensis (strain R1 / LMG 19424) GN=RALTA_A2850 PE=4 SV=1 Length = 533

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

    Directory of Open Access Journals (Sweden)

    Keegan J. Baldauf

    2015-03-01

    Full Text Available 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.

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

    Directory of Open Access Journals (Sweden)

    Yolima P. Torres

    2014-10-01

    Full Text Available 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 Ca+2-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 & 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 Ca+2 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.

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

    Science.gov (United States)

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

    2014-01-01

    Coded by a single gene (Slo1, KCM) and activated by depolarizing potentials and by a rise in intracellular Ca(2+) concentration, the large conductance voltage- and Ca(2+)-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 Ca(2+)-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 Ca(2+) 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.

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

    Science.gov (United States)

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

    2014-01-01

    Coded by a single gene (Slo1, KCM) and activated by depolarizing potentials and by a rise in intracellular Ca2+ concentration, the large conductance voltage- and Ca2+-activated K+ channel (BK) is unique among the superfamily of K+ channels. BK channels are tetramers characterized by a pore-forming α subunit containing seven transmembrane segments (instead of the six found in voltage-dependent K+ channels) and a large C terminus composed of two regulators of K+ conductance domains (RCK domains), where the Ca2+-binding sites reside. BK channels can be associated with accessory β subunits and, although different BK modulatory mechanisms have been described, greater interest has recently been placed on the role that the β subunits may play in the modulation of BK channel gating due to its physiological importance. Four β subunits have currently been identified (i.e., β1, β2, β3, and β4) and despite the fact that they all share the same topology, it has been shown that every β subunit has a specific tissue distribution and that they modify channel kinetics as well as their pharmacological properties and the apparent Ca2+ sensitivity of the α subunit in different ways. Additionally, different studies have shown that natural, endogenous, and synthetic compounds can modulate BK channels through β subunits. Considering the importance of these channels in different pathological conditions, such as hypertension and neurological disorders, this review focuses on the mechanisms by which these compounds modulate the biophysical properties of BK channels through the regulation of β subunits, as well as their potential therapeutic uses for diseases such as those mentioned above. PMID:25346693

  10. Thermostable Subunit Vaccines for Pulmonary Delivery: How Close Are We?

    DEFF Research Database (Denmark)

    Foged, Camilla

    2016-01-01

    , such as influenza, tuberculosis, and Ebola, for which no good universal vaccines exist. At least two pharmaceutical improvements are expected to help filling this gap: i) The development of thermostable vaccine dosage forms, and ii) the full exploitation of the adjuvant technology for subunit vaccines to potentiate...... strong immune responses. This review highlights the status and recent advances in formulation and pulmonary delivery of thermostable human subunit vaccines. Such vaccines are very appealing from compliance, distribution and immunological point of view: Being non-invasive, inhalable vaccines are self...... immunity. Here, I review state of the art and perspectives in formulation design and processing methods for powder-based subunit vaccines intended for pulmonary administration, and present dry powder inhaler technologies suitable for translating these vaccines into clinical trials....

  11. The expression profile of acid-sensing ion channel (ASIC) subunits ASIC1a, ASIC1b, ASIC2a, ASIC2b, and ASIC3 in the esophageal vagal afferent nerve subtypes.

    Science.gov (United States)

    Dusenkova, Svetlana; Ru, Fei; Surdenikova, Lenka; Nassenstein, Christina; Hatok, Jozef; Dusenka, Robert; Banovcin, Peter; Kliment, Jan; Tatar, Milos; Kollarik, Marian

    2014-11-01

    Acid-sensing ion channels (ASICs) have been implicated in esophageal acid sensing and mechanotransduction. However, insufficient knowledge of ASIC subunit expression profile in esophageal afferent nerves hampers the understanding of their role. This knowledge is essential because ASIC subunits form heteromultimeric channels with distinct functional properties. We hypothesized that the esophageal putative nociceptive C-fiber nerves (transient receptor potential vanilloid 1, TRPV1-positive) express multiple ASIC subunits and that the ASIC expression profile differs between the nodose TRPV1-positive subtype developmentally derived from placodes and the jugular TRPV1-positive subtype derived from neural crest. We performed single cell RT-PCR on the vagal afferent neurons retrogradely labeled from the esophagus. In the guinea pig, nearly all (90%-95%) nodose and jugular esophageal TRPV1-positive neurons expressed ASICs, most often in a combination (65-75%). ASIC1, ASIC2, and ASIC3 were expressed in 65-75%, 55-70%, and 70%, respectively, of both nodose and jugular TRPV1-positive neurons. The ASIC1 splice variants ASIC1a and ASIC1b and the ASIC2 splice variant ASIC2b were similarly expressed in both nodose and jugular TRPV1-positive neurons. However, ASIC2a was found exclusively in the nodose neurons. In contrast to guinea pig, ASIC3 was almost absent from the mouse vagal esophageal TRPV1-positive neurons. However, ASIC3 was similarly expressed in the nonnociceptive TRPV1-negative (tension mechanoreceptors) neurons in both species. We conclude that the majority of esophageal vagal nociceptive neurons express multiple ASIC subunits. The placode-derived nodose neurons selectively express ASIC2a, known to substantially reduce acid sensitivity of ASIC heteromultimers. ASIC3 is expressed in the guinea pig but not in the mouse vagal esophageal TRPV1-positive neurons, indicating species differences in ASIC expression. Copyright © 2014 the American Physiological Society.

  12. Subunit architecture and functional modular rearrangements of the transcriptional mediator complex.

    Science.gov (United States)

    Tsai, Kuang-Lei; Tomomori-Sato, Chieri; Sato, Shigeo; Conaway, Ronald C; Conaway, Joan W; Asturias, Francisco J

    2014-06-05

    The multisubunit Mediator, comprising ∼30 distinct proteins, plays an essential role in gene expression regulation by acting as a bridge between DNA-binding transcription factors and the RNA polymerase II (RNAPII) transcription machinery. Efforts to uncover the Mediator mechanism have been hindered by a poor understanding of its structure, subunit organization, and conformational rearrangements. By overcoming biochemical and image analysis hurdles, we obtained accurate EM structures of yeast and human Mediators. Subunit localization experiments, docking of partial X-ray structures, and biochemical analyses resulted in comprehensive mapping of yeast Mediator subunits and a complete reinterpretation of our previous Mediator organization model. Large-scale Mediator rearrangements depend on changes at the interfaces between previously described Mediator modules, which appear to be facilitated by factors conducive to transcription initiation. Conservation across eukaryotes of Mediator structure, subunit organization, and RNA polymerase II interaction suggest conservation of fundamental aspects of the Mediator mechanism. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Effects of Arginine Concentration on the In Vitro Expression of Casein and mTOR Pathway Related Genes in Mammary Epithelial Cells from Dairy Cattle

    Science.gov (United States)

    Wang, Mengzhi; Xu, Bolin; Wang, Hongrong; Bu, Dengpan; Wang, Jiaqi; Loor, Juan-Jose

    2014-01-01

    Arginine (Arg) is a conditionally-essential amino acid that is taken up by bovine mammary gland in excess of its output in milk. In this study we evaluated the effects of Arg concentration on the expression of casein and signaling pathway-related genes in mammary epithelial cells. The treatments (applied for 24 h) were designed to be devoid of Arg 0X (control; 0.00 mg/L), resemble the profile of Arg in casein (Arg 1X; 278.00 mg/L), be deficient [Arg 0.25X (69.50 mg/L) and Arg 0.5X (139.00 mg/L)], or be in excess of the amount in casein [Arg 2X (556.00 mg/L), Arg 4X (1,112 mg/L), and Arg 8X (2,224 mg/L)]. The expression of CSN1S, CSN3 and mTOR in the experimental groups was higher than those of the control group (P0.05), the expression of CSN1S2, CSN2 and JAK2 in other experimental groups was higher (P0.05), the expression of STAT5 in the other experimental groups was higher than those of the control (Pcasein genes and mTOR-related genes in bovine mammary epithelial cells. PMID:24788778

  14. Dopamine receptor D5 deficiency results in a selective reduction of hippocampal NMDA receptor subunit NR2B expression and impaired memory.

    Science.gov (United States)

    Moraga-Amaro, Rodrigo; González, Hugo; Ugalde, Valentina; Donoso-Ramos, Juan Pablo; Quintana-Donoso, Daisy; Lara, Marcelo; Morales, Bernardo; Rojas, Patricio; Pacheco, Rodrigo; Stehberg, Jimmy

    2016-04-01

    Pharmacological evidence associates type I dopamine receptors, including subtypes D1 and D5, with learning and memory. Analyses using genetic approaches have determined the relative contribution of dopamine receptor D1 (D1R) in cognitive tasks. However, the lack of drugs that can discriminate between D1R and D5R has made the pharmacological distinction between the two receptors difficult. Here, we aimed to determine the role of D5R in learning and memory. In this study we tested D5R knockout mice and wild-type littermates in a battery of behavioral tests, including memory, attention, locomotion, anxiety and motivational evaluations. Our results show that genetic deficiency of D5R significantly impairs performance in the Morris water maze paradigm, object location and object recognition memory, indicating a relevant role for D5R in spatial memory and recognition memory. Moreover, the lack of D5R resulted in decreased exploration and locomotion. In contrast, D5R deficiency had no impact on working memory, anxiety and depressive-like behavior, measured using the spontaneous alternation, open-field, tail suspension test, and forced swimming test. Electrophysiological analyses performed on hippocampal slices showed impairment in long-term-potentiation in mice lacking D5R. Further analyses at the molecular level showed that genetic deficiency of D5R results in a strong and selective reduction in the expression of the NMDA receptor subunit NR2B in the hippocampus. These findings demonstrate the relevant contribution of D5R in memory and suggest a functional interaction of D5R with hippocampal glutamatergic pathways. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Cloning and sequencing of the casein kinase 2 alpha subunit from Zea mays

    DEFF Research Database (Denmark)

    Dobrowolska, G; Boldyreff, B; Issinger, O G

    1991-01-01

    The nucleotide sequence of the cDNA coding for the alpha subunit of casein kinase 2 of Zea mays has been determined. The cDNA clone contains an open reading frame of 996 nucleotides encoding a polypeptide comprising 332 amino acids. The primary amino acid sequence exhibits 75% identity to the alpha...... subunit and 71% identity to the alpha' subunit of human casein kinase 2....

  16. Regulated appearance of NMDA receptor subunits and channel functions during in vitro neuronal differentiation.

    Science.gov (United States)

    Jelitai, Márta; Schlett, Katalin; Varju, Patrícia; Eisel, Ulrich; Madarász, Emília

    2002-04-01

    The schedule of NMDA receptor subunit expression and the appearance of functional NMDA-gated ion channels were investigated during the retinoic acid (RA) induced neuronal differentiation of NE-4C, a p53-deficient mouse neuroectodermal progenitor cell line. NR2A, NR2B, and NR2D subunit transcripts were present in both nondifferentiated and neuronally differentiated cultures, while NR2C subunits were expressed only transiently, during the early period of neural differentiation. Several splice variants of NR1 were detected in noninduced progenitors and in RA-induced cells, except the N1 exon containing transcripts that appeared after the fourth day of induction, when neuronal processes were already formed. NR1 and NR2A subunit proteins were detected both in nondifferentiated progenitor cells and in neurons, while the mature form of NR2B subunit protein appeared only at the time of neuronal process elongation. Despite the early presence of NR1 and NR2A subunits, NMDA-evoked responses could be detected in NE-4C neurons only after the sixth day of induction, coinciding in time with the expression of the mature NR2B subunit. The formation of functional NMDA receptors also coincided with the appearance of synapsin I and synaptophysin. The lag period between the production of the subunits and the onset of channel function suggests that subunits capable of channel formation cannot form functional NMDA receptors until a certain stage of neuronal commitment. Thus, the in vitro neurogenesis by NE-4C cells provides a suitable tool to investigate some inherent regulatory processes involved in the initial maturation of NMDA receptor complexes. Copyright 2002 Wiley Periodicals, Inc.

  17. Expression of putative expansin genes in phylloxera (Daktulosphaira vitifoliae Fitch) induced root galls of Vitis spp.

    Science.gov (United States)

    Lawo, N C; Griesser, M; Forneck, A

    Grape phylloxera ( Daktulosphaira vitifoliae Fitch) is a serious global pest in viticulture. The insects are sedentary feeders and require a gall to feed and reproduce. The insects induce their feeding site within the meristematic zone of the root tip, where they stay attached, feeding both intra- and intercellularly, and causing damage by reducing plant vigour. Several changes in cell structure and composition, including increased cell division and tissue swelling close to the feeding site, cause an organoid gall called a nodosity to develop. Because alpha expansin genes are involved in cell enlargement and cell wall loosening in many plant tissues it may be anticipated that they are also involved in nodosity formation. To identify expansin genes in Vitis vinifera cv. Pinot noir , we mined for orthologues genes in a comparative analysis. Eleven putative expansin genes were identified and shown to be present in the rootstock Teleki 5C ( V. berlandieri Planch. x V. riparia Michx.) using specific PCR followed by DNA sequencing. Expression analysis of young and mature nodosities and uninfested root tips were conducted via quantitative real time PCR (qRT-PCR). Up-regulation was measured for three putative expansin genes (VvEXPA15, -A17 and partly -A20) or down-regulation for three other putative genes (VvEXPA7, -A12, -A20) in nodosities. The present study clearly shows the involvement of putative expansin genes in the phylloxera-root interaction.

  18. Immunochemical localization of ribulose-1,5-bisphosphate carboxylase in the symbiont-containing gills of Solemya velum (Bivalvia: Mollusca).

    Science.gov (United States)

    Cavanaugh, C M; Abbott, M S; Veenhuis, M

    1988-10-01

    The distribution of the Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase (RbuP(2)Case; EC 4.1.1.39) was examined by using two immunological methods in tissues of Solemya velum, an Atlantic coast bivalve containing putative chemoautotrophic symbionts. Antibodies elicited by the purified large subunit of RbuP(2)Case from tobacco (Nicotiana tabacum) cross-reacted on immunoblots with a protein of similar molecular mass occurring in extracts of the symbiont-containing gill tissue of S. velum. No cross-reactivity was detected in symbiont-free tissue extracts. The antiserum also cross-reacted in immunoblots with proteins of Thiobacillus neapolitanus, a free-living sulfuroxidizing chemoautotroph whose RbuP(2)Case has been well characterized. In protein A-gold immunoelectron microscopy studies, this antiserum consistently labeled the symbionts but not surrounding host gill tissue, indicating that the symbionts are responsible for the RbuP(2)Case activity.

  19. An Approach to Identify and Characterize a Subunit Candidate Shigella Vaccine Antigen.

    Science.gov (United States)

    Pore, Debasis; Chakrabarti, Manoj K

    2016-01-01

    Shigellosis remains a serious issue throughout the developing countries, particularly in children under the age of 5. Numerous strategies have been tested to develop vaccines targeting shigellosis; unfortunately despite several years of extensive research, no safe, effective, and inexpensive vaccine against shigellosis is available so far. Here, we illustrate in detail an approach to identify and establish immunogenic outer membrane proteins from Shigella flexneri 2a as subunit vaccine candidates.

  20. 5' adenosine monophosphate-activated protein kinase, metabolism and exercise.

    Science.gov (United States)

    Aschenbach, William G; Sakamoto, Kei; Goodyear, Laurie J

    2004-01-01

    The 5' adenosine monophosphate-activated protein kinase (AMPK) is a member of a metabolite-sensing protein kinase family that functions as a metabolic 'fuel gauge' in skeletal muscle. AMPK is a ubiquitous heterotrimeric protein, consisting of an alpha catalytic, and beta and gamma regulatory subunits that exist in multiple isoforms and are all required for full enzymatic activity. During exercise, AMPK becomes activated in skeletal muscle in response to changes in cellular energy status (e.g. increased adenosine monophosphate [AMP]/adenosine triphosphate [ATP] and creatine/phosphocreatine ratios) in an intensity-dependent manner, and serves to inhibit ATP-consuming pathways, and activate pathways involved in carbohydrate and fatty-acid metabolism to restore ATP levels. Recent evidence shows that although AMPK plays this key metabolic role during acute bouts of exercise, it is also an important component of the adaptive response of skeletal muscles to endurance exercise training because of its ability to alter muscle fuel reserves and expression of several exercise-responsive genes. This review discusses the putative roles of AMPK in acute and chronic exercise responses, and suggests avenues for future AMPK research in exercise physiology and biochemistry.

  1. Reconstitution of normal and hyperactivated forms of casein kinase-2 by variably mutated beta-subunits

    DEFF Research Database (Denmark)

    Boldyreff, B; Meggio, F; Pinna, L A

    1993-01-01

    Twenty-one mutants of the noncatalytic beta-subunit of human casein kinase-2 have been created, expressed in Escherichia coli, and purified to homogeneity. They are either modified at the autophosphorylation site (mutants beta delta 1-4 and beta A 5,6) or bear variable deletions in their C...

  2. Putative midkine family protein up-regulation in Patella caerulea (Mollusca, Gastropoda) exposed to sublethal concentrations of cadmium

    International Nuclear Information System (INIS)

    Vanucci, Silvana; Minerdi, Daniela; Kadomatsu, Kenji; Mengoni, Alessio; Bazzicalupo, Marco

    2005-01-01

    A cDNA sequence of a putative midkine (MK) family protein was identified and characterised in the mollusc Patella caerulea. The midkine family consists of two members, midkine and pleiotrophin (PTN), and it is one of the recently discovered cytokines. Our results show that this putative midkine protein is up-regulated in specimens of P. caerulea exposed to sublethal cadmium concentrations (i.e. 0.5 and 1 mg l -1 Cd) over a 10-day exposure period. Semiquantitative RT-PCR and quantitative Real time RT-PCR estimations indicate elevated expression of midkine mRNA in exposed specimens compared to controls. Moreover, RT-PCR Real time values were higher in the viscera (here defined as the part of the soft tissue including digestive gland plus gills) than in the foot (i.e. foot plus head plus heart) of the limpets. At present, information on the functional signalling significance of the midkine family proteins suggests that the up-regulation of P. caerulea putative midkine family protein is a distress signal likely with informative value on health status of the organism and with potential prognostic capability

  3. Identification and phylogenetic analysis of Tityus pachyurus and Tityus obscurus novel putative Na+-channel scorpion toxins.

    Directory of Open Access Journals (Sweden)

    Jimmy A Guerrero-Vargas

    Full Text Available Colombia and Brazil are affected by severe cases of scorpionism. In Colombia the most dangerous accidents are caused by Tityus pachyurus that is widely distributed around this country. In the Brazilian Amazonian region scorpion stings are a common event caused by Tityus obscurus. The main objective of this work was to perform the molecular cloning of the putative Na(+-channel scorpion toxins (NaScTxs from T. pachyurus and T. obscurus venom glands and to analyze their phylogenetic relationship with other known NaScTxs from Tityus species.cDNA libraries from venom glands of these two species were constructed and five nucleotide sequences from T. pachyurus were identified as putative modulators of Na(+-channels, and were named Tpa4, Tpa5, Tpa6, Tpa7 and Tpa8; the latter being the first anti-insect excitatory β-class NaScTx in Tityus scorpion venom to be described. Fifteen sequences from T. obscurus were identified as putative NaScTxs, among which three had been previously described, and the others were named To4 to To15. The peptides Tpa4, Tpa5, Tpa6, To6, To7, To9, To10 and To14 are closely related to the α-class NaScTxs, whereas Tpa7, Tpa8, To4, To8, To12 and To15 sequences are more related to the β-class NaScTxs. To5 is possibly an arthropod specific toxin. To11 and To13 share sequence similarities with both α and β NaScTxs. By means of phylogenetic analysis using the Maximum Parsimony method and the known NaScTxs from Tityus species, these toxins were clustered into 14 distinct groups.This communication describes new putative NaScTxs from T. pachyurus and T. obscurus and their phylogenetic analysis. The results indicate clear geographic separation between scorpions of Tityus genus inhabiting the Amazonian and Mountain Andes regions and those distributed over the Southern of the Amazonian rainforest. Based on the consensus sequences for the different clusters, a new nomenclature for the NaScTxs is proposed.

  4. Ethanol activation of protein kinase A regulates GABA-A receptor subunit expression in the cerebral cortex and contributes to ethanol-induced hypnosis

    Directory of Open Access Journals (Sweden)

    Sandeep eKumar

    2012-04-01

    Full Text Available Protein kinases are implicated in neuronal cell functions such as modulation of ion channel function, trafficking and synaptic excitability. Both protein kinase C (PKC and A (PKA are involved in regulation of γ-aminobutyric acid type A (GABA-A receptors through phosphorylation. However, the role of PKA in regulating GABA-A receptors following acute ethanol exposure is not known. The present study investigated the role of PKA in ethanol effects on GABA-A receptor α1 subunit expression in the P2 synaptosomal fraction of the rat cerebral cortex. Additionally, GABA-related behaviors were also examined. Rats were administered ethanol (2.0 – 3.5 g/kg or saline and PKC, PKA and GABA-A receptor α1 subunit levels were measured by Western blot analysis. Ethanol (3.5 g/kg transiently increased GABA-A receptor α1 subunit expression and PKA RIIβ subunit expression at similar time points whereas PKA RIIα was increased at later time points. In contrast, PKC isoform expression remained unchanged. Notably, the moderate ethanol dose (2.0g/kg had no effect on GABA-A α1 subunit levels although PKA RIIα and RIIβ were increased at 10 and 60 minutes, when PKC isozymes are also known to be elevated. To determine if PKA activation was responsible for the ethanol-induced elevation of GABA-A α1 subunits, the PKA antagonist H89 was administered to rats prior to ethanol exposure. H89 administration prevented ethanol-induced increases in GABA-A receptor α1 subunit expression. Moreover, increasing PKA activity intracerebroventricularly with Sp-cAMP prior to a hypnotic dose of ethanol increased ethanol-induced loss of righting reflex duration. This effect appears to be mediated in part by GABA-A receptors as increasing PKA activity also increased the duration of muscimol-induced loss of righting reflex. Overall these data suggest that PKA mediates ethanol-induced GABA-A receptor expression and contributes to ethanol behavioral effects involving GABA-A receptors.

  5. Proteasome (Prosome Subunit Variations during the Differentiation of Myeloid U937 Cells

    Directory of Open Access Journals (Sweden)

    Laurent Henry

    1997-01-01

    Full Text Available 20S proteasomes (prosomes/multicatalytic proteinase are protein particles built of 28 subunits in variable composition. We studied the changes in proteasome subunit composition during the differentiation of U937 cells induced by phorbol‐myristate‐acetate or retinoic acid plus 1,25‐dihydroxy‐cholecalciferol by western blot, flow cytometry and immuno‐fluorescence. p25K (C3, p27K (IOTA and p30/33K (C2 subunits were detected in both the nucleus and cytoplasm of undifferentiated cells. Flow cytometry demonstrated a biphasic decrease in proteasome subunits detection during differentiation induced by RA+VD. PMA caused an early transient decrease in these subunits followed by a return to their control level, except for p30/33K, which remained low. Immuno‐fluorescence also showed differences in the cytolocalization of the subunits, with a particular decrease in antigen labeling in the nucleus of RA+VD‐induced cells, and a scattering in the cytoplasm and a reorganization in the nucleus of PMA‐induced cells. Small amounts of proteasomal proteins were seen on the outer membrane of non‐induced cells; these membrane proteins disappeared when treated with RA+VD, whereas some increased on PMA‐induced cells. The differential changes in the distribution and type of proteasomes in RA+VD and PMA‐induced cells indicate that, possibly, 20S proteasomes may play a role in relation to the mechanisms of differentiation and the inducer used.

  6. Two subunits of human ORC are dispensable for DNA replication and proliferation.

    Science.gov (United States)

    Shibata, Etsuko; Kiran, Manjari; Shibata, Yoshiyuki; Singh, Samarendra; Kiran, Shashi; Dutta, Anindya

    2016-12-01

    The six-subunit Origin Recognition Complex (ORC) is believed to be an essential eukaryotic ATPase that binds to origins of replication as a ring-shaped heterohexamer to load MCM2-7 and initiate DNA replication. We have discovered that human cell lines in culture proliferate with intact chromosomal origins of replication after disruption of both alleles of ORC2 or of the ATPase subunit, ORC1 . The ORC1 or ORC2 -depleted cells replicate with decreased chromatin loading of MCM2-7 and become critically dependent on another ATPase, CDC6, for survival and DNA replication. Thus, either the ORC ring lacking a subunit, even its ATPase subunit, can load enough MCM2-7 in partnership with CDC6 to initiate DNA replication, or cells have an ORC-independent, CDC6-dependent mechanism to load MCM2-7 on origins of replication.

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

    Science.gov (United States)

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

    2017-11-01

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

  8. Short communication: molecular characterization of dog and cat p65 subunits of NF-kappaB.

    Science.gov (United States)

    Ishikawa, Shingo; Takemitsu, Hiroshi; Li, Gebin; Mori, Nobuko; Yamamoto, Ichiro; Arai, Toshiro

    2015-04-01

    Nuclear factor kappa B (NF-κB) plays an important role in the immune system. The p65 subunit is an important part of NF-κB unit, and studies of dog and cat p65 subunits of NF-κB (dp65 and cp65) are important in understanding their immune function. In this study, we described the molecular characterization of dp65 and cp65. The dp65 and cp65 complementary DNA encoded 542 and 555 amino acids, respectively, showing a high sequence homology with the mammalian p65 subunit (>87.5%). Quantitative polymerase chain reaction revealed that the p65 messenger RNA is highly expressed in the dog stomach and cat heart and adipose tissue. Functional NF-κB promoter-luciferase reporter vectors revealed that our isolated dp65 and cp65 cDNA encodes a functionally active protein. Transiently expressed dp65 and cp65 up-regulated pro-inflammatory cytokine expression levels in dog and cat, respectively. These findings suggest that dp65 and cp65 play important roles in regulating immune function. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

    OpenAIRE

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

    2009-01-01

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

  10. Characterization of the alpha and beta subunits of casein kinase 2 by far-UV CD spectroscopy

    DEFF Research Database (Denmark)

    Issinger, O G; Brockel, C; Boldyreff, B

    1992-01-01

    Although Chou-Fasman calculations of the secondary structure of recombinant casein kinase 2 subunits alpha and beta suggest they have a similar overall conformation, circular dichroism (CD) studies show that substantial differences in the conformation of the two subunits exist. In addition......, no changes in the far-UV CD spectrum of the alpha subunit are observed in the presence of casein or the synthetic decapeptide substrate RRRDDDSDDD. Furthermore, the alpha-helical structure of the alpha subunit (but not the beta subunit) can be increased in the presence of stoichiometric amounts of heparin...

  11. The biosynthesis and processing of high molecular weight precursors of soybean glycinin subunits.

    Science.gov (United States)

    Barton, K A; Thompson, J F; Madison, J T; Rosenthal, R; Jarvis, N P; Beachy, R N

    1982-06-10

    The predominant storage protein of soybean seed, glycinin, is composed of two heterogeneous classes of related subunits, the acidics (Mr approximately 38,000) and the basics (Mr approximately 22,000). Immunoreaction of polypeptides translated in vitro from isolated seed mRNA using antibodies prepared against either purified acidic or basic subunit groups precipitated precursor polypeptides of Mr = 60,000 to Mr = 63,000. High pressure liquid chromatography fingerprinting of trypsin-generated fragments from in vitro synthesized precursors showed fragments specific to both acidic and basic subunits. No mature acidic or basic subunits were detected in vitro translation reactions by either immunoprecipitation or high pressure liquid chromatography fingerprinting. Pulse-labeling of cotyledons growing in culture with [3H]glycine showed rapid accumulation of label in glycinin precursors of Mr = 59,000 to Mr = 62,000. Although in vivo synthesized precursors had slightly greater electrophoretic mobility than in vitro synthesized precursors, little label initially appeared in mature glycinin subunits. After several hours of continued cotyledon growth in absence of label, precursors were processed and label accumulated in both acidic and basic subunit groups. Recombinant plasmids were prepared by reverse transcription of soybean seed mRNA, and clones which encode glycinin precursors were identified by heteroduplex-hybridization of translatable messages. Northern blot analysis of seed mRNA shows the mRNA-encoding glycinin precursors to migrate at Mr = 0.71 X 10(6) on agarose gels, corresponding to approximately 2050 nucleotides. This is sufficiently large to encode a polypeptide consisting of both a glycinin acidic and basic subunit.

  12. CLONING, SEQUENCE ANALYSIS, AND CHARACTERIZATION OF PUTATIVE BETA-LACTAMASE OF STENOTROPHOMONAS MALTOPHILIA

    Directory of Open Access Journals (Sweden)

    Chong Seng Shueh

    2012-10-01

    Full Text Available The main objective of current study was to explore the function of chromosomal putative beta-lactamase gene (smlt 0115 in clinical Stenotrophomonas maltophilia. Antibiotic susceptibility test (AST screening for current antimicrobial drugs was done and Minimum Inhibitory Concentration (MIC level towards beta-lactams was determined by E-test. Putative beta-lactamase gene of S. maltophilia was amplified via PCR, with specific primers, then cloned into pET-15 expression plasmid and transformed into Escherichia coli BL21. The gene was sequenced and analyzed. The expressed protein was purified by affinity chromatography and the kinetic assay was performed. S. maltophilia ATCC 13637 was included in this experiment. Besides, a hospital strain which exhibited resistant to a series of beta-lactams including cefepime was identified via AST and MIC, hence it was named as S2 strain and was considered in this study. Sequencing result showed that putative beta-lactamase gene obtained from ATCC 13637 and S2 strains were predicted to have cephalosporinase activity by National Center for Biotechnology Information (NCBI blast program. Differences in the sequences of both ATCC 13637 and S2 strains were found via ClustalW alignment software. Kinetic assay proved a cephalosporinase characteristic produced by E. coli BL21 clone that overexpressed the putative beta-lactamase gene cloned under the control of an external promoter. Yet, expressed protein purified from S2 strain had high catalytic activity against beta-lactam antibiotics which was 14-fold higher than expressed protein purified from ATCC 13637 strain. This study represents the characterization analysis of putative beta-lactamase gene (smlt 0115 of S. maltophilia. The presence of the respective gene in the chromosome of S. maltophilia suggested that putative beta-lactamase gene (smlt 0115 of S. maltophilia plays a role in beta-lactamase resistance.

  13. Putative radioresistant bacterial isolate from sewage water

    International Nuclear Information System (INIS)

    Ang, April; Chua, Patricia; Perez, Kristine; Rey, April; Rivor Kristel; San Pablo, Czarina; Santos, Ernestine

    2001-01-01

    Sewage water was collected from a stagnant body of water in Balara, Quezon City. approximately 150 ml was aseptically transferred into eight Erlenmeyer flasks. Seven flasks were then subjected to different doses of radiation at the 60 Co irradiation facility, PNRI (Philippine Nuclear Research Institute) which are as follows: 0.01 kGy, 0.1 kGy, 0.5 kGy, 1 kGy, 5 kGy, 10 kGy, and 15 kGy. The remaining flask was used as the control. After irradiation, all the different treatments were subjected to colony count at the culture collection laboratory, NSRI. Results showed that the colonies from sewage water treatments irradiated at 0.01 kGy (treatment A), 0.10 kGy (treatment B), and 0.50 kGy (treatment C) exhibited a decreasing trend with colony counts 4.60 x 10 3 CFU/ml, and 1.30 x 10 3 CFU/ml, and 26 CFU/ml, respectively. Contrastingly, at 1 kGy (treatment D), high colony count of 2.95 x 10 3 CFU/ml was observed which is even higher compared to the control (1.02 x 10 3 CFU/ml). Treatment E that was irradiated at 5 kGy manifested low survival rate (25 CFU/ml) indicating the presence of few putative intermediate radioresistant bacteria. Radiation dose treatments higher than 5 kGy (i.e., 10 kGy and 15 kGy) exhibited no bacterial survival. (Author)

  14. Putative radioresistant bacterial isolate from sewage water

    Energy Technology Data Exchange (ETDEWEB)

    Ang, April; Chua, Patricia; Perez, Kristine; Rey, April; Kristel, Rivor; San Pablo, Czarina; Santos, Ernestine

    2001-01-29

    Sewage water was collected from a stagnant body of water in Balara, Quezon City. approximately 150 ml was aseptically transferred into eight Erlenmeyer flasks. Seven flasks were then subjected to different doses of radiation at the {sup 60}Co irradiation facility, PNRI (Philippine Nuclear Research Institute) which are as follows: 0.01 kGy, 0.1 kGy, 0.5 kGy, 1 kGy, 5 kGy, 10 kGy, and 15 kGy. The remaining flask was used as the control. After irradiation, all the different treatments were subjected to colony count at the culture collection laboratory, NSRI. Results showed that the colonies from sewage water treatments irradiated at 0.01 kGy (treatment A), 0.10 kGy (treatment B), and 0.50 kGy (treatment C) exhibited a decreasing trend with colony counts 4.60 x 10{sup 3} CFU/ml, and 1.30 x 10{sup 3} CFU/ml, and 26 CFU/ml, respectively. Contrastingly, at 1 kGy (treatment D), high colony count of 2.95 x 10{sup 3} CFU/ml was observed which is even higher compared to the control (1.02 x 10{sup 3} CFU/ml). Treatment E that was irradiated at 5 kGy manifested low survival rate (25 CFU/ml) indicating the presence of few putative intermediate radioresistant bacteria. Radiation dose treatments higher than 5 kGy (i.e., 10 kGy and 15 kGy) exhibited no bacterial survival. (Author)

  15. Gut Microbiome and Putative Resistome of Inca and Italian Nobility Mummies.

    Science.gov (United States)

    Santiago-Rodriguez, Tasha M; Fornaciari, Gino; Luciani, Stefania; Toranzos, Gary A; Marota, Isolina; Giuffra, Valentina; Cano, Raul J

    2017-11-07

    Little is still known about the microbiome resulting from the process of mummification of the human gut. In the present study, the gut microbiota, genes associated with metabolism, and putative resistome of Inca and Italian nobility mummies were characterized by using high-throughput sequencing. The Italian nobility mummies exhibited a higher bacterial diversity as compared to the Inca mummies when using 16S ribosomal (rRNA) gene amplicon sequencing, but both groups showed bacterial and fungal taxa when using shotgun metagenomic sequencing that may resemble both the thanatomicrobiome and extant human gut microbiomes. Identification of sequences associated with plants, animals, and carbohydrate-active enzymes (CAZymes) may provide further insights into the dietary habits of Inca and Italian nobility mummies. Putative antibiotic-resistance genes in the Inca and Italian nobility mummies support a human gut resistome prior to the antibiotic therapy era. The higher proportion of putative antibiotic-resistance genes in the Inca compared to Italian nobility mummies may support the hypotheses that a greater exposure to the environment may result in a greater acquisition of antibiotic-resistance genes. The present study adds knowledge of the microbiome resulting from the process of mummification of the human gut, insights of ancient dietary habits, and the preserved putative human gut resistome prior the antibiotic therapy era.

  16. Domain cooperativity in the β1a subunit is essential for dihydropyridine receptor voltage sensing in skeletal muscle.

    Science.gov (United States)

    Dayal, Anamika; Bhat, Vinayakumar; Franzini-Armstrong, Clara; Grabner, Manfred

    2013-04-30

    The dihydropyridine receptor (DHPR) β1a subunit is crucial for enhancement of DHPR triad expression, assembly of DHPRs in tetrads, and elicitation of DHPRα1S charge movement--the three prerequisites of skeletal muscle excitation-contraction coupling. Despite the ability to fully target α1S into triadic junctions and tetradic arrays, the neuronal isoform β3 was unable to restore considerable charge movement (measure of α1S voltage sensing) upon expression in β1-null zebrafish relaxed myotubes, unlike the other three vertebrate β-isoforms (β1a, β2a, and β4). Thus, we used β3 for chimerization with β1a to investigate whether any of the five distinct molecular regions of β1a is dominantly involved in inducing the voltage-sensing function of α1S. Surprisingly, systematic domain swapping between β1a and β3 revealed a pivotal role of the src homology 3 (SH3) domain and C terminus of β1a in charge movement restoration. More interestingly, β1a SH3 domain and C terminus, when simultaneously engineered into β3 sequence background, were able to fully restore charge movement together with proper intracellular Ca(2+) release, suggesting cooperativity of these two domains in induction of the α1S voltage-sensing function in skeletal muscle excitation-contraction coupling. Furthermore, substitution of a proline by alanine in the putative SH3-binding polyproline motif in the proximal C terminus of β1a (also of β2a and β4) fully obstructed α1S charge movement. Consequently, we postulate a model according to which β subunits, probably via the SH3-C-terminal polyproline interaction, adapt a discrete conformation required to modify the α1S conformation apt for voltage sensing in skeletal muscle.

  17. A Functional Assay for Putative Mouse and Human Definitive Endoderm using Chick Whole-Embryo Cultures

    DEFF Research Database (Denmark)

    Johannesson, Martina; Semb, Tor Henrik; Serup, Palle

    2012-01-01

    . Thus, the purpose of this study is to describe a method whereby the in vivo functionality of DE derived from ESCs can be assessed. Methods: By directed differentiation, putative DE was derived from human and mouse ESCs. This putative DE was subsequently transplanted into the endoderm of chick embryos...... to determine any occurrence of integration. Putative DE was analyzed by gene and protein expression prior to transplantation and 48 h post transplantation. Results: Putative DE, derived from mouse and human ESCs, was successfully integrated within the chick endoderm. Endoderm-specific genes were expressed...... result show that putative DE integrates with the chick endoderm and participate in the development of the chicken gut, indicating the generation of functional DE from ESCs. This functional assay can be used to assess the generation of functional DE derived from both human and mouse ESCs and provides...

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

    Directory of Open Access Journals (Sweden)

    Nnamdi Edokobi

    2018-04-01

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

  19. Antibodies to the α-subunit of insulin receptor from eggs of immunized hens

    International Nuclear Information System (INIS)

    Song, C.; Yu, J.; Bai, D.H.; Hester, P.Y.; Kim, K.

    1985-01-01

    Simple methods for the generation, purification, and assay of antibodies to the α-subunit of insulin receptor from eggs of immunized hen have been described. Chicken antibodies against the α-subunit inhibit insulin binding to the receptor and stimulate glucose oxidation as well as autophosphorylation of the β-subunit. Thus the properties of chicken antibodies are very similar to those of antibodies found in human autoimmune diseases and different from rabbit antibodies obtained against the same antigen

  20. Effect of HMM Glutenin Subunits on Wheat Quality Attributes

    Directory of Open Access Journals (Sweden)

    Daniela Horvat

    2009-01-01

    Full Text Available Glutenin is a group of polymeric gluten proteins. Glutenin molecules consist of glutenin subunits linked together with disulphide bonds and having higher (HMM-GS and lower (LMM-GS molecular mass. The main objective of this study is the evaluation of the influence of HMM-GS on flour processing properties. Seven bread wheat genotypes with contrasting quality attributes and different HMM-GS composition were analyzed during three years. The composition and quantity of HMM-GS were determined by SDS-PAGE and RP-HPLC, respectively. The quality diversity among genotypes was estimated by the analysis of wheat grain, and flour and bread quality parameters. The presence of HMM glutenin subunits 1 and 2* at Glu-A1 and the subunits 5+10 at Glu-D1 loci, as well as a higher proportion of total HMM-GS, had a positive effect on wheat quality. Cluster analysis of the three groups of data (genotype and HMM-GS, flour and bread quality, and dough rheology yielded the same hierarchical structure for the first top three levels, and similarity of the corresponding dendrograms was proved by the principal eigenvalues of the corresponding Euclidian distance matrices. The obtained similarity in classification based on essentially different types of measurements reflects strong natural association between genetic data, product quality and physical properties. Principal component analysis (PCA was applied to effectively reduce large data set into lower dimensions of latent variables amenable for the analysis. PCA analysis of the total set of data (15 variables revealed a very strong interrelationship between the variables. The first three PCA components accounted for 96 % of the total variance, which was significant to the level of 0.05 and was considered as the level of experimental error. These data imply that the quality of wheat cultivars can be contributed to HMM-GS data and should be taken into account in breeding programs assisted by computer models with the aim to

  1. Modulation of NMDA Receptor Properties and Synaptic Transmission by the NR3A Subunit in Mouse Hippocampal and Cerebrocortical Neurons

    Science.gov (United States)

    Tong, Gary; Takahashi, Hiroto; Tu, Shichun; Shin, Yeonsook; Talantova, Maria; Zago, Wagner; Xia, Peng; Nie, Zhiguo; Goetz, Thomas; Zhang, Dongxian; Lipton, Stuart A.; Nakanishi, Nobuki

    2015-01-01

    Expression of the NR3A subunit with NR1/NR2 in Xenopus oocytes or mammalian cell lines leads to a reduction in N-methyl-D-aspartate (NMDA)-induced currents and decreased Mg2+ sensitivity and Ca2+ permeability compared with NR1/NR2 receptors. Consistent with these findings, neurons from NR3A knockout (KO) mice exhibit enhanced NMDA-induced currents. Recombinant NR3A can also form excitatory glycine receptors with NR1 in the absence of NR2. However, the effects of NR3A on channel properties in neurons and synaptic transmission have not been fully elucidated. To study physiological roles of NR3A subunits, we generated NR3A transgenic (Tg) mice. Cultured NR3A Tg neurons exhibited two populations of NMDA receptor (NMDAR) channels, reduced Mg2+ sensitivity, and decreased Ca2+ permeability in response to NMDA/glycine, but glycine alone did not elicit excitatory currents. In addition, NMDAR-mediated excitatory postsynaptic currents (EPSCs) in NR3A Tg hippocampal slices showed reduced Mg2+ sensitivity, consistent with the notion that NR3A subunits incorporated into synaptic NMDARs. To study the function of endogenous NR3A subunits, we compared NMDAR-mediated EPSCs in NR3A KO and WT control mice. In NR3A KO mice, the ratio of the amplitudes of the NMDAR-mediated component to α-amino-3-hydroxy-5-methyl-4-isox-azolepropionic acid receptor-mediated component of the EPSC was significantly larger than that seen in WT littermates. This result suggests that NR3A subunits contributed to the NMDAR-mediated component of the EPSC in WT mice. Taken together, these results show that NR3A subunits contribute to NMDAR responses from both synaptic and extra-synaptic receptors, likely composed of NR1, NR2, and NR3 subunits. PMID:18003876

  2. Shared active site architecture between archaeal PolD and multi-subunit RNA polymerases revealed by X-ray crystallography.

    Science.gov (United States)

    Sauguet, Ludovic; Raia, Pierre; Henneke, Ghislaine; Delarue, Marc

    2016-08-22

    Archaeal replicative DNA polymerase D (PolD) constitute an atypical class of DNA polymerases made of a proofreading exonuclease subunit (DP1) and a larger polymerase catalytic subunit (DP2), both with unknown structures. We have determined the crystal structures of Pyrococcus abyssi DP1 and DP2 at 2.5 and 2.2 Å resolution, respectively, revealing a catalytic core strikingly different from all other known DNA polymerases (DNAPs). Rather, the PolD DP2 catalytic core has the same 'double-psi β-barrel' architecture seen in the RNA polymerase (RNAP) superfamily, which includes multi-subunit transcriptases of all domains of life, homodimeric RNA-silencing pathway RNAPs and atypical viral RNAPs. This finding bridges together, in non-viral world, DNA transcription and DNA replication within the same protein superfamily. This study documents further the complex evolutionary history of the DNA replication apparatus in different domains of life and proposes a classification of all extant DNAPs.

  3. Propofol effectively inhibits lithium-pilocarpine- induced status epilepticus in rats via downregulation of N-methyl-D-aspartate receptor 2B subunit expression

    Science.gov (United States)

    Wang, Henglin; Wang, Zhuoqiang; Mi, Weidong; Zhao, Cong; Liu, Yanqin; Wang, Yongan; Sun, Haipeng

    2012-01-01

    Status epilepticus was induced via intraperitoneal injection of lithium-pilocarpine. The inhibitory effects of propofol on status epilepticus in rats were judged based on observation of behavior, electroencephalography and 24-hour survival rate. Propofol (12.5–100 mg/kg) improved status epilepticus in a dose-dependent manner, and significantly reduced the number of deaths within 24 hours of lithium-pilocarpine injection. Western blot results showed that, 24 hours after induction of status epilepticus, the levels of N-methyl-D-aspartate receptor 2A and 2B subunits were significantly increased in rat cerebral cortex and hippocampus. Propofol at 50 mg/kg significantly suppressed the increase in N-methyl-D-aspartate receptor 2B subunit levels, but not the increase in N-methyl-D-aspartate receptor 2A subunit levels. The results suggest that propofol can effectively inhibit status epilepticus induced by lithium-pilocarpine. This effect may be associated with downregulation of N-methyl-D-aspartate receptor 2B subunit expression after seizures. PMID:25737709

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

    Science.gov (United States)

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

    2017-01-01

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

  5. Toddlers' Duration of Attention toward Putative Threat

    Science.gov (United States)

    Kiel, Elizabeth J.; Buss, Kristin A.

    2011-01-01

    Although individual differences in reactions to novelty in the toddler years have been consistently linked to risk of developing anxious behavior, toddlers' attention toward a novel, putatively threatening stimulus while in the presence of other enjoyable activities has rarely been examined as a precursor to such risk. The current study examined…

  6. The morphological and chemical characteristics of striatal neurons immunoreactive for the alpha1-subunit of the GABA(A) receptor in the rat.

    Science.gov (United States)

    Waldvogel, H J; Kubota, Y; Trevallyan, S C; Kawaguchi, Y; Fritschy, J M; Mohler, H; Faull, R L

    1997-10-01

    terminals. The type 2 neurons comprised 20% of the total population of alpha1-subunit-immunoreactive neurons. The remaining large-sized alpha1-immunoreactive cells were designated type 3 cells; they were positive for parvalbumin and were distinguished by long branching dendrites extending dorsally for 600-800 microm into the striatum. These neurons comprised 5% of the total population of alpha1-subunit-immunoreactive neurons and were surrounded by enkephalin-immunoreactive terminals. Electron microscopy showed that the alpha1-subunit type 3 neurons had an indented nuclear membrane and were densely covered with small axon terminals which established alpha1-subunit-immunoreactive symmetrical synaptic contacts with the soma and dendrites. These results provide a detailed characterization of the distribution, morphology and chemical characteristics of the alpha1-subunit-immunoreactive neurons in the rat striatum and suggest that the type 1 and type 2 neurons comprise of separate populations of striatal interneurons while the type 3 neurons may represent the large striatonigral projection neurons described by Bolam et al. [Bolam J. P., Somogyi P., Totterdell S. and Smith A. D. (1981) Neuroscience 6, 2141-2157.].

  7. Activity-dependent control of NMDA receptor subunit composition at hippocampal mossy fibre synapses.

    Science.gov (United States)

    Carta, Mario; Srikumar, Bettadapura N; Gorlewicz, Adam; Rebola, Nelson; Mulle, Christophe

    2018-02-15

    CA3 pyramidal cells display input-specific differences in the subunit composition of synaptic NMDA receptors (NMDARs). Although at low density, GluN2B contributes significantly to NMDAR-mediated EPSCs at mossy fibre synapses. Long-term potentiation (LTP) of NMDARs triggers a modification in the subunit composition of synaptic NMDARs by insertion of GluN2B. GluN2B subunits are essential for the expression of LTP of NMDARs at mossy fibre synapses. Single neurons express NMDA receptors (NMDARs) with distinct subunit composition and biophysical properties that can be segregated in an input-specific manner. The dynamic control of the heterogeneous distribution of synaptic NMDARs is crucial to control input-dependent synaptic integration and plasticity. In hippocampal CA3 pyramidal cells from mice of both sexes, we found that mossy fibre (MF) synapses display a markedly lower proportion of GluN2B-containing NMDARs than associative/commissural synapses. The mechanism involved in such heterogeneous distribution of GluN2B subunits is not known. Here we show that long-term potentiation (LTP) of NMDARs, which is selectively expressed at MF-CA3 pyramidal cell synapses, triggers a modification in the subunit composition of synaptic NMDARs by insertion of GluN2B. This activity-dependent recruitment of GluN2B at mature MF-CA3 pyramidal cell synapses contrasts with the removal of GluN2B subunits at other glutamatergic synapses during development and in response to activity. Furthermore, although expressed at low levels, GluN2B is necessary for the expression of LTP of NMDARs at MF-CA3 pyramidal cell synapses. Altogether, we reveal a previously unknown activity-dependent regulation and function of GluN2B subunits that may contribute to the heterogeneous plasticity induction rules in CA3 pyramidal cells. © 2017 Centre Nationnal de la Recherche Scientifique. The Journal of Physiology © 2017 The Physiological Society.

  8. The subunit structure of the extracellular hemoglobin of Biomphalaria glabrata

    International Nuclear Information System (INIS)

    Arndt, Marcio H.L.; Naves, Cristiani F.; Xavier, Luciana P.; Santoro, Marcelo M.

    1997-01-01

    Full text. The hemoglobin of Biomphalaria glabrata was purified to homogeneity by a two step purification protocol using a gel filtration column (Superose 6 HR/Pharmacia ) followed by an anion exchange chromatography (MONO-Q Sepharose/Pharmacia). The dissociation products were analysed by a 5 - 15 % Polyacrylamide gel electrophoresis containing Sodium Dodecyl Sulfate (SDS-PAGE) giving a band of 270 K Daltons and a band of 180 K Daltons after reduction with β-mercaptoethanol. The same profile was obtained in a 3.5 % Agarose gel electrophoresis containing SDS (SDS-AGE) showing additional bands of higher molecular weight. These bands were proposed to be monomers, dimers and trimers and, after reduction in a Bidimensional SDS-AGE, the proposed monomers and dimers were decomposed in two and four bands that were interpreted as 1 - 4 chains. The hemoglobin was digested by four different proteases ( Thrombin, Trypsin, Chymotrypsin and Subtilisin ) showing several equivalent fragments with molecular weights multiples of its minimum molecular weight ( 17.7 K Daltons). The circular dichroism spectrum of the protein showed a characteristic high α-helix content. We proposed that this hemoglobin is a pentamer of approx. 360 K Daltons subunits each formed by two 180 K Daltons chains linked in pairs by disulfide bridges and each of these chains comprises ten Heme binding domains. These data were compared to other Planorbidae extracellular hemoglobins. Up to now, the quaternary structure of this hemoglobin (shape and disposition of the subunits) is unknown. It is intended to elucidate its structure by Small Angle X-Ray Scattering in Brazilian National Laboratory of Synchrotron Light (LNLS). (author)

  9. Identification and characterization of putative conserved IAM ...

    African Journals Online (AJOL)

    Available putative AMI sequences from a wide array of monocot and dicot plants were identified and the phylogenetic tree was constructed and analyzed. We identified in this tree, a clade that contained sequences from species across the plant kingdom suggesting that AMI is conserved and may have a primary role in plant ...

  10. Isolation and characterization of two mitoviruses and a putative alphapartitivirus from Fusarium spp.

    Science.gov (United States)

    Osaki, Hideki; Sasaki, Atsuko; Nomiyama, Koji; Sekiguchi, Hiroyuki; Tomioka, Keisuke; Takehara, Toshiaki

    2015-06-01

    The filamentous fungus Fusarium spp. includes several important plant pathogens. We attempted to reveal presence of double-stranded (ds) RNAs in the genus. Thirty-seven Fusarium spp. at the MAFF collection were analyzed. In the strains of Fusarium coeruleum, Fusarium globosum and Fusarium solani f. sp. pisi, single dsRNA bands were detected. The strains of F. coeruleum and F. solani f. sp. pisi cause potato dry rot and mulberry twig blight, respectively. Sequence analyses revealed that dsRNAs in F. coeruleum and F. globosum consisted of 2423 and 2414 bp, respectively. Using the fungal mitochondrial translation table, the positive strands of these cDNAs were found to contain single open reading frames with the potential to encode a protein of putative 757 and 717 amino acids (molecular mass 88.5 and 84.0 kDa, respectively), similar to RNA-dependent RNA polymerases of members of the genus Mitovirus. These dsRNAs in F. coeruleum and F. globosum were assigned to the genus Mitovirus (family Narnaviridae), and these two mitoviruses were designated as Fusarium coeruleum mitovirus 1 and Fusarium globosum mitovirus 1. On the other hand, a positive strand of cDNA (1950 bp) from dsRNA in F. solani f. sp. pisi contained an ORF potentially encoding a putative RdRp of 608 amino acids (72.0 kDa). The putative RdRp was shown to be related to those of members of the genus of Alphapartitivirus (family Partitiviridae). We coined the name Fusarium solani partitivirus 2 for dsRNA in F. solani f. sp. pisi.

  11. Similarities in transcription factor IIIC subunits that bind to the posterior regions of internal promoters for RNA polymerase III

    Directory of Open Access Journals (Sweden)

    Matsutani Sachiko

    2004-08-01

    Full Text Available Abstract Background In eukaryotes, RNA polymerase III (RNAP III transcribes the genes for small RNAs like tRNAs, 5S rRNA, and several viral RNAs, and short interspersed repetitive elements (SINEs. The genes for these RNAs and SINEs have internal promoters that consist of two regions. These two regions are called the A and B blocks. The multisubunit transcription factor TFIIIC is required for transcription initiation of RNAP III; in transcription of tRNAs, the B-block binding subunit of TFIIIC recognizes a promoter. Although internal promoter sequences are conserved in eukaryotes, no evidence of homology between the B-block binding subunits of vertebrates and yeasts has been reported previously. Results Here, I reported the results of PSI-BLAST searches using the B-block binding subunits of human and Shizosacchromyces pombe as queries, showing that the same Arabidopsis proteins were hit with low E-values in both searches. Comparison of the convergent iterative alignments obtained by these PSI-BLAST searches revealed that the vertebrate, yeast, and Arabidopsis proteins have similarities in their N-terminal one-third regions. In these regions, there were three domains with conserved sequence similarities, one located in the N-terminal end region. The N-terminal end region of the B-block binding subunit of Saccharomyces cerevisiae is tentatively identified as a HMG box, which is the DNA binding motif. Although I compared the alignment of the N-terminal end regions of the B-block binding subunits, and their homologs, with that of the HMG boxes, it is not clear whether they are related. Conclusion Molecular phylogenetic analyses using the small subunit rRNA and ubiquitous proteins like actin and α-tubulin, show that fungi are more closely related to animals than either is to plants. Interestingly, the results obtained in this study show that, with respect to the B-block binding subunits of TFIIICs, animals appear to be evolutionarily closer to plants

  12. Similarities in transcription factor IIIC subunits that bind to the posterior regions of internal promoters for RNA polymerase III.

    Science.gov (United States)

    Matsutani, Sachiko

    2004-08-09

    In eukaryotes, RNA polymerase III (RNAP III) transcribes the genes for small RNAs like tRNAs, 5S rRNA, and several viral RNAs, and short interspersed repetitive elements (SINEs). The genes for these RNAs and SINEs have internal promoters that consist of two regions. These two regions are called the A and B blocks. The multisubunit transcription factor TFIIIC is required for transcription initiation of RNAP III; in transcription of tRNAs, the B-block binding subunit of TFIIIC recognizes a promoter. Although internal promoter sequences are conserved in eukaryotes, no evidence of homology between the B-block binding subunits of vertebrates and yeasts has been reported previously. Here, I reported the results of PSI-BLAST searches using the B-block binding subunits of human and Shizosacchromyces pombe as queries, showing that the same Arabidopsis proteins were hit with low E-values in both searches. Comparison of the convergent iterative alignments obtained by these PSI-BLAST searches revealed that the vertebrate, yeast, and Arabidopsis proteins have similarities in their N-terminal one-third regions. In these regions, there were three domains with conserved sequence similarities, one located in the N-terminal end region. The N-terminal end region of the B-block binding subunit of Saccharomyces cerevisiae is tentatively identified as a HMG box, which is the DNA binding motif. Although I compared the alignment of the N-terminal end regions of the B-block binding subunits, and their homologs, with that of the HMG boxes, it is not clear whether they are related. Molecular phylogenetic analyses using the small subunit rRNA and ubiquitous proteins like actin and alpha-tubulin, show that fungi are more closely related to animals than either is to plants. Interestingly, the results obtained in this study show that, with respect to the B-block binding subunits of TFIIICs, animals appear to be evolutionarily closer to plants than to fungi.

  13. Genetic Analysis of the Mode of Interplay between an ATPase Subunit and Membrane Subunits of the Lipoprotein-Releasing ATP-Binding Cassette Transporter LolCDE†

    OpenAIRE

    Ito, Yasuko; Matsuzawa, Hitomi; Matsuyama, Shin-ichi; Narita, Shin-ichiro; Tokuda, Hajime

    2006-01-01

    The LolCDE complex, an ATP-binding cassette (ABC) transporter, releases lipoproteins from the inner membrane, thereby initiating lipoprotein sorting to the outer membrane of Escherichia coli. The LolCDE complex is composed of two copies of an ATPase subunit, LolD, and one copy each of integral membrane subunits LolC and LolE. LolD hydrolyzes ATP on the cytoplasmic side of the inner membrane, while LolC and/or LolE recognize and release lipoproteins anchored to the periplasmic leaflet of the i...

  14. Effect of dexamethasone on skeletal muscle Na+,K+ pump subunit specific expression and K+ homeostasis during exercise in humans

    DEFF Research Database (Denmark)

    Nordsborg, Nikolai; Ovesen, Jakob; Thomassen, Martin

    2008-01-01

    The effect of dexamethasone on Na(+),K(+) pump subunit expression and muscle exchange of K(+) during exercise in humans was investigated. Nine healthy male subjects completed a randomized double blind placebo controlled protocol, with ingestion of dexamethasone (Dex: 2 x 2 mg per day) or placebo...... (Pla) for 5 days. Na(+),K(+) pump catalytic alpha1 and alpha2 subunit expression was approximately 17% higher (P ...). The results indicate that an increased Na(+),K(+) pump expression per se is of importance for thigh K(+) reuptake at the onset of low and moderate intensity exercise, but less important during high intensity exercise....

  15. Gut Microbiome and Putative Resistome of Inca and Italian Nobility Mummies

    Directory of Open Access Journals (Sweden)

    Tasha M. Santiago-Rodriguez

    2017-11-01

    Full Text Available Little is still known about the microbiome resulting from the process of mummification of the human gut. In the present study, the gut microbiota, genes associated with metabolism, and putative resistome of Inca and Italian nobility mummies were characterized by using high-throughput sequencing. The Italian nobility mummies exhibited a higher bacterial diversity as compared to the Inca mummies when using 16S ribosomal (rRNA gene amplicon sequencing, but both groups showed bacterial and fungal taxa when using shotgun metagenomic sequencing that may resemble both the thanatomicrobiome and extant human gut microbiomes. Identification of sequences associated with plants, animals, and carbohydrate-active enzymes (CAZymes may provide further insights into the dietary habits of Inca and Italian nobility mummies. Putative antibiotic-resistance genes in the Inca and Italian nobility mummies support a human gut resistome prior to the antibiotic therapy era. The higher proportion of putative antibiotic-resistance genes in the Inca compared to Italian nobility mummies may support the hypotheses that a greater exposure to the environment may result in a greater acquisition of antibiotic-resistance genes. The present study adds knowledge of the microbiome resulting from the process of mummification of the human gut, insights of ancient dietary habits, and the preserved putative human gut resistome prior the antibiotic therapy era.

  16. The Combined Utility of a Brief Functional Measure and Performance-Based Screening Test for Case Finding of Cognitive Impairment in Primary Healthcare.

    Science.gov (United States)

    Chan, Qun Lin; Shaik, Muhammad Amin; Xu, Jing; Xu, Xin; Chen, Christopher Li-Hsian; Dong, YanHong

    2016-04-01

    Use of a total risk score (TRS) based on vascular and sociodemographic risk factors has been recommended to identify patients at risk of cognitive impairment. Moreover, combining screening tests has been reported to improve positive predictive values (PPV) for case finding of cognitive impairment. We investigated the utility of the conjunctive combination of the informant-based AD8 and the performance-based National Institute of Neurological Disorders and Stroke-Canadian Stroke Network (NINDS-CSN) 5-minute protocol for the detection of cognitive impairment, defined by a clinical dementia rating (CDR) score ≥0.5, in patients at risk of cognitive impairment (TRS ≥3). Participants were recruited from 2 primary healthcare centers in Singapore and received the AD8, Montreal Cognitive Assessment, Mini-Mental State Examination, CDR, and a formal neuropsychological test battery. The scores for NINDS-CSN 5-minute protocol were extracted from the Montreal Cognitive Assessment items. Area under the receiver operating characteristics curve analyses were conducted to determine the discriminant indices of the screening instruments, the conjunctive combination (ie, screened positive on both tests), and the compensatory combination (ie, screened positive in either of or both tests). A total of 309 participants were recruited of whom 78.7% (n = 243) had CDR = 0 and 21.3% (n = 66) had CDR ≥0.5. The conjunctive combination of AD8 and NINDS-CSN 5-minute protocol achieved excellent PPV and acceptable sensitivity (PPV 91.7%, sensitivity 73.3%). The conjunctive combination of the AD8 and NINDS-CSN 5-minute protocol is brief and accurate, and hence, suitable for case finding of cognitive impairment (CDR ≥0.5) in patients screened positive on the TRS in primary healthcare centers. Copyright © 2016. Published by Elsevier Inc.

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

    Science.gov (United States)

    Römling, Ute; Galperin, Michael Y.

    2015-01-01

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

  18. Inhibition of herpesvirus and influenza virus replication by blocking polymerase subunit interactions.

    Science.gov (United States)

    Palù, Giorgio; Loregian, Arianna

    2013-09-01

    Protein-protein interactions (PPIs) play a key role in many biological processes, including virus replication in the host cell. Since most of the PPIs are functionally essential, a possible strategy to inhibit virus replication is based on the disruption of viral protein complexes by peptides or small molecules that interfere with subunit interactions. In particular, an attractive target for antiviral drugs is the binding between the subunits of essential viral enzymes. This review describes the development of new antiviral compounds that inhibit herpesvirus and influenza virus replication by blocking interactions between subunit proteins of their polymerase complexes. Copyright © 2013 Elsevier B.V. All rights reserved.

  19. Ten Putative Contributors to the Obesity Epidemic

    Science.gov (United States)

    McAllister, Emily J.; Dhurandhar, Nikhil V.; Keith, Scott W.; Aronne, Louis J.; Barger, Jamie; Baskin, Monica; Benca, Ruth M.; Biggio, Joseph; Boggiano, Mary M.; Eisenmann, Joe C.; Elobeid, Mai; Fontaine, Kevin R.; Gluckman, Peter; Hanlon, Erin C.; Katzmarzyk, Peter; Pietrobelli, Angelo; Redden, David T.; Ruden, Douglas M.; Wang, Chenxi; Waterland, Robert A.; Wright, Suzanne M.; Allison, David B.

    2010-01-01

    The obesity epidemic is a global issue and shows no signs of abating, while the cause of this epidemic remains unclear. Marketing practices of energy-dense foods and institutionally-driven declines in physical activity are the alleged perpetrators for the epidemic, despite a lack of solid evidence to demonstrate their causal role. While both may contribute to obesity, we call attention to their unquestioned dominance in program funding and public efforts to reduce obesity, and propose several alternative putative contributors that would benefit from equal consideration and attention. Evidence for microorganisms, epigenetics, increasing maternal age, greater fecundity among people with higher adiposity, assortative mating, sleep debt, endocrine disruptors, pharmaceutical iatrogenesis, reduction in variability of ambient temperatures, and intrauterine and intergenerational effects, as contributing factors to the obesity epidemic are reviewed herein. While the evidence is strong for some contributors such as pharmaceutical-induced weight gain, it is still emerging for other reviewed factors. Considering the role of such putative etiological factors of obesity may lead to comprehensive, cause specific, and effective strategies for prevention and treatment of this global epidemic. PMID:19960394

  20. Energy expenditures & physical activity in rats with chronic suboptimal nutrition

    Directory of Open Access Journals (Sweden)

    Lifshitz Fima

    2006-01-01

    Full Text Available Abstract Background Sub-optimally nourished rats show reduced growth, biochemical and physiological changes. However, no one has assessed metabolic rate adaptations in rats subjected to chronic suboptimal nutrition (CSN. In this study energy expenditure (EE; kcal/100 g body weight and physical activity (PA; oscillations in weight/min/kg body weight were assessed in rats subjected to three levels of CSN. Results Body weight gain was diminished (76.7 ± 12.0 and 61.6 ± 11.0 g in rats fed 70 and 60% of the ad-libitum fed controls which gained more weight (148.5 ± 32.3 g. The rats fed 80% gained weight similarly to controls (136.3 ± 10.5 g. Percent Fat-free body mass was reduced (143.8 ± 8.7 and 142.0 ± 7.6 g in rats fed 70 and 60% of ad-libitum, but not in those fed 80% (200.8 ± 17.5 g as compared with controls (201.6 ± 33.4 g. Body fat (g decreased in rats fed 80% (19.7 ± 5.3, 70% (15.3 ± 3.5 and 60% (9.6 ± 2.7 of ad-libitum in comparison to controls (26.0 ± 6.7. EE and PA were also altered by CSN. The control rats increased their EE and PA during the dark periods by 1.4 ± 0.8 and 1.7 ± 1.1 respectively, as compared with light the period; whereas CSN rats fed 80 and 70% of ad-libitum energy intake had reduced EE and PA during the dark periods as compared with the light period EE(7.5 ± 1.4 and 7.8 ± 0.6 vs. 9.0 ± 1.2 and 9.7 ± 0.8; p Conclusion CSN rats adapt to mild energy restriction by reducing body fat, EE and PA mainly during the dark period while growth proceeds and lean body mass is preserved. At higher levels of energy restrictions there is decreased growth, body fat and lean mass. Moreover EE and PA are also reduced during both light and dark periods.

  1. A Common Structural Component for β-Subunit Mediated Modulation of Slow Inactivation in Different KV Channels

    Directory of Open Access Journals (Sweden)

    Nathalie Strutz-Seebohm

    2013-06-01

    Full Text Available Background/Aims: Potassium channels are tetrameric proteins providing potassium selective passage through lipid embedded proteinaceous pores with highest fidelity. The selectivity results from binding to discrete potassium binding sites and stabilization of a hydrated potassium ion in a central internal cavity. The four potassium binding sites, generated by the conserved TTxGYGD signature sequence are formed by the backbone carbonyls of the amino acids TXGYG. Residues KV1.5-Val481, KV4.3-Leu368 and KV7.1- Ile 313 represent the amino acids in the X position of the respective channels. Methods: Here, we study the impact of these residues on ion selectivity, permeation and inactivation kinetics as well as the modulation by β-subunits using site-specific mutagenesis, electrophysiological analyses and molecular dynamics simulations. Results: We identify this position as key in modulation of slow inactivation by structurally dissimilar β-subunits in different KV channels. Conclusion: We propose a model in which structural changes accompanying activation and β-subunit modulation allosterically constrain the backbone carbonyl oxygen atoms via the side chain of the respective X-residue in the signature sequence to reduce conductance during slow inactivation.

  2. Captodiamine, a putative antidepressant, enhances hypothalamic BDNF expression in vivo by synergistic 5-HT2c receptor antagonism and sigma-1 receptor agonism.

    Science.gov (United States)

    Ring, Rebecca M; Regan, Ciaran M

    2013-10-01

    The putative antidepressant captodiamine is a 5-HT2c receptor antagonist and agonist at sigma-1 and D3 dopamine receptors, exerts an anti-immobility action in the forced swim paradigm, and enhances dopamine turnover in the frontal cortex. Captodiamine has also been found to ameliorate stress-induced anhedonia, reduce the associated elevations of hypothalamic corticotrophin-releasing factor (CRF) and restore the reductions in hypothalamic BDNF expression. Here we demonstrate chronic administration of captodiamine to have no significant effect on hypothalamic CRF expression through sigma-1 receptor agonism; however, both sigma-1 receptor agonism or 5-HT2c receptor antagonism were necessary to enhance BDNF expression. Regulation of BDNF expression by captodiamine was associated with increased phosphorylation of transcription factor CREB and mediated through sigma-1 receptor agonism but blocked by 5-HT2c receptor antagonism. The existence of two separate signalling pathways was confirmed by immunolocalisation of each receptor to distinct cell populations in the paraventricular nucleus of the hypothalamus. Increased BDNF induced by captodiamine was also associated with enhanced expression of synapsin, but not PSD-95, suggesting induction of long-term structural plasticity between hypothalamic synapses. These unique features of captodiamine may contribute to its ability to ameliorate stress-induced anhedonia as the hypothalamus plays a prominent role in regulating HPA axis activity.

  3. A Functional Switch of NuRD Chromatin Remodeling Complex Subunits Regulates Mouse Cortical Development

    Directory of Open Access Journals (Sweden)

    Justyna Nitarska

    2016-11-01

    Full Text Available Histone modifications and chromatin remodeling represent universal mechanisms by which cells adapt their transcriptional response to rapidly changing environmental conditions. Extensive chromatin remodeling takes place during neuronal development, allowing the transition of pluripotent cells into differentiated neurons. Here, we report that the NuRD complex, which couples ATP-dependent chromatin remodeling with histone deacetylase activity, regulates mouse brain development. Subunit exchange of CHDs, the core ATPase subunits of the NuRD complex, is required for distinct aspects of cortical development. Whereas CHD4 promotes the early proliferation of progenitors, CHD5 facilitates neuronal migration and CHD3 ensures proper layer specification. Inhibition of each CHD leads to defects of neuronal differentiation and migration, which cannot be rescued by expressing heterologous CHDs. Finally, we demonstrate that NuRD complexes containing specific CHDs are recruited to regulatory elements and modulate the expression of genes essential for brain development.

  4. Evaluation of immunoprotection conferred by the subunit vaccines of GRA2 and GRA5 against acute toxoplasmosis in BALB/c mice

    Directory of Open Access Journals (Sweden)

    Xiao Teng eChing

    2016-04-01

    Full Text Available Toxoplasmosis is a foodborne disease caused by Toxoplasma gondii, an obligate intracellular parasite. Severe symptoms occur in the immunocompromised patients and pregnant women leading to fatality and abortions respectively. Vaccination development is essential to control the disease. The T. gondii dense granule antigen 2 and 5 (GRA2 and GRA5 have been targeted in this study because these proteins are essential to the development of parasitophorous vacuole (PV, a specialized compartment formed within the infected host cell. PV is resistance to host cell endosomes and lysosomes thereby protecting the invaded parasite. Recombinant dense granular proteins, GRA2 (rGRA2 and GRA5 (rGRA5 were cloned, expressed, and purified in Escherichia coli, BL21 (DE3 pLysS. The potential of these purified antigens as subunit vaccine candidates against toxoplasmosis were evaluated through subcutaneous injection of BALB/c mice followed by immunological characterization (humoral- and cellular-mediated and lethal challenge against virulent T. gondii RH strain in BALB/c mice. Results obtained demonstrated that rGRA2 and rGRA5 elicited humoral and cellular-mediated immunity in the mice. High level of IgG antibody was produced with the isotype IgG2a/IgG1 ratio of ≈0.87 (p<0.001. Significant increase (p<0.05 in the level of four cytokines (IFN-γ, IL-2, IL-4 and IL-10 was obtained. The antibody and cytokine results suggest that a mix mode of Th1/Th2-immunity was elicited with predominant Th1-immune response inducing partial protection against T. gondii acute infection in BALB/c mice. Our findings indicated that both GRA2 and GRA5 are potential candidates for vaccine development against T. gondii acute infection.

  5. Structural insights into methyltransferase KsgA function in 30S ribosomal subunit biogenesis.

    Science.gov (United States)

    Boehringer, Daniel; O'Farrell, Heather C; Rife, Jason P; Ban, Nenad

    2012-03-23

    The assembly of the ribosomal subunits is facilitated by ribosome biogenesis factors. The universally conserved methyltransferase KsgA modifies two adjacent adenosine residues in the 3'-terminal helix 45 of the 16 S ribosomal RNA (rRNA). KsgA recognizes its substrate adenosine residues only in the context of a near mature 30S subunit and is required for the efficient processing of the rRNA termini during ribosome biogenesis. Here, we present the cryo-EM structure of KsgA bound to a nonmethylated 30S ribosomal subunit. The structure reveals that KsgA binds to the 30S platform with the catalytic N-terminal domain interacting with substrate adenosine residues in helix 45 and the C-terminal domain making extensive contacts to helix 27 and helix 24. KsgA excludes the penultimate rRNA helix 44 from adopting its position in the mature 30S subunit, blocking the formation of the decoding site and subunit joining. We suggest that the activation of methyltransferase activity and subsequent dissociation of KsgA control conformational changes in helix 44 required for final rRNA processing and translation initiation.

  6. Structural Insights into Methyltransferase KsgA Function in 30S Ribosomal Subunit Biogenesis*

    Science.gov (United States)

    Boehringer, Daniel; O'Farrell, Heather C.; Rife, Jason P.; Ban, Nenad

    2012-01-01

    The assembly of the ribosomal subunits is facilitated by ribosome biogenesis factors. The universally conserved methyltransferase KsgA modifies two adjacent adenosine residues in the 3′-terminal helix 45 of the 16 S ribosomal RNA (rRNA). KsgA recognizes its substrate adenosine residues only in the context of a near mature 30S subunit and is required for the efficient processing of the rRNA termini during ribosome biogenesis. Here, we present the cryo-EM structure of KsgA bound to a nonmethylated 30S ribosomal subunit. The structure reveals that KsgA binds to the 30S platform with the catalytic N-terminal domain interacting with substrate adenosine residues in helix 45 and the C-terminal domain making extensive contacts to helix 27 and helix 24. KsgA excludes the penultimate rRNA helix 44 from adopting its position in the mature 30S subunit, blocking the formation of the decoding site and subunit joining. We suggest that the activation of methyltransferase activity and subsequent dissociation of KsgA control conformational changes in helix 44 required for final rRNA processing and translation initiation. PMID:22308031

  7. Putative Risk Factors in Developmental Dyslexia: A Case-Control Study of Italian Children

    Science.gov (United States)

    Mascheretti, Sara; Marino, Cecilia; Simone, Daniela; Quadrelli, Ermanno; Riva, Valentina; Cellino, Maria Rosaria; Maziade, Michel; Brombin, Chiara; Battaglia, Marco

    2015-01-01

    Although dyslexia runs in families, several putative risk factors that cannot be immediately identified as genetic predict reading disability. Published studies analyzed one or a few risk factors at a time, with relatively inconsistent results. To assess the contribution of several putative risk factors to the development of dyslexia, we conducted…

  8. Characterization and application of a radioimmunoassay for reduced, carboxymethylated human luteinizing hormone α-subunit

    International Nuclear Information System (INIS)

    Keutmann, H.T.; Beitins, I.Z.; Johnson, L.; McArthur, J.W.

    1978-01-01

    We have established a double antibody RIA using a rabbit antiserum prepared against reduced, carboxymethylated (RCXM) human LH α-subunit, with RCXM-α as tracer and standard. This antiserum did not cross-react with any native gonadotropins or subunit, and reacted only weakly with RCXM-α. A tryptic digest of RCXM α-subunit was completely reactive, while chymotryptic digestion abolished all immunoreactivity. By testing with separate tryptic fragments, the recognition site could be localized to a segment close to the amino-terminus of the peptide chain. When applied to measurement of serum and urine, an immunoreactive species, parallel to RCXM α-subunit by serial dilution, was found in concentrations of 1-2 ng/ml in serum and 3-4 ng/ml in urine. Similar levels of the immunoreactive component were found in conditions of elevated gonadotropins (e.g. pregnancy) as well as gonadotropin deficiency (panhypopituitarism and Kallmann's syndrome). After stimulation with LHRH, no rise was noted at times up to 6 h despite the fact that both LH and LH-α were elevated. The data indicate that the sequence-specific antiserum may be detecting an immunoreactive form of α-subunit of LH whose kinetics of appearance and disappearance differs from those of the native subunit

  9. Involvement of the catalytic subunit of protein kinase A and of HA95 in pre-mRNA splicing

    International Nuclear Information System (INIS)

    Kvissel, Anne-Katrine; Orstavik, Sigurd; Eikvar, Sissel; Brede, Gaute; Jahnsen, Tore; Collas, Philippe; Akusjaervi, Goeran; Skalhegg, Bjorn Steen

    2007-01-01

    Protein kinase A (PKA) is a holoenzyme consisting of two catalytic (C) subunits bound to a regulatory (R) subunit dimer. Stimulation by cAMP dissociates the holoenzyme and causes translocation to the nucleus of a fraction of the C subunit. Apart from transcription regulation, little is known about the function of the C subunit in the nucleus. In the present report, we show that both Cα and Cβ are localized to spots in the mammalian nucleus. Double immunofluorescence analysis of splicing factor SC35 with the C subunit indicated that these spots are splicing factor compartments (SFCs). Using the E1A in vivo splicing assay, we found that catalytically active C subunits regulate alternative splicing and phosphorylate several members of the SR-protein family of splicing factors in vitro. Furthermore, nuclear C subunits co-localize with the C subunit-binding protein homologous to AKAP95, HA95. HA95 also regulates E1A alternative splicing in vivo, apparently through its N-terminal domain. Localization of the C subunit to SFCs and the E1A splicing pattern were unaffected by cAMP stimulation. Our findings demonstrate that the nuclear PKA C subunit co-locates with HA95 in SFCs and regulates pre-mRNA splicing, possibly through a cAMP-independent mechanism

  10. Identification of the A2 adenosine receptor binding subunit by photoaffinity crosslinking

    International Nuclear Information System (INIS)

    Barrington, W.W.; Jacobson, K.A.; Hutchison, A.J.; Williams, M.; Stiles, G.L.

    1989-01-01

    A high-affinity iodinated agonist radioligand for the A2 adenosine receptor has been synthesized to facilitate studies of the A2 adenosine receptor binding subunit. The radioligand 125I-labeled PAPA-APEC (125I-labeled 2-[4-(2-[2-[(4- aminophenyl)methylcarbonylamino]ethylaminocarbonyl]- ethyl)phenyl]ethylamino-5'-N-ethylcarboxamidoadenosine) was synthesized and found to bind to the A2 adenosine receptor in bovine striatal membranes with high affinity (Kd = 1.5 nM) and A2 receptor selectivity. Competitive binding studies reveal the appropriate A2 receptor pharmacologic potency order with 5'-N-ethylcarboxamidoadenosine (NECA) greater than (-)-N6-[(R)-1-methyl- 2-phenylethyl]adenosine (R-PIA) greater than (+)-N6-[(S)-1-methyl-2- phenylethyl]adenosine (S-PIA). Adenylate cyclase assays, in human platelet membranes, demonstrate a dose-dependent stimulation of cAMP production. PAPA-APEC (1 microM) produces a 43% increase in cAMP production, which is essentially the same degree of increase produced by 5'-N- ethylcarboxamidoadenosine (the prototypic A2 receptor agonist). These findings combined with the observed guanine nucleotide-mediated decrease in binding suggest that PAPA-APEC is a full A2 agonist. The A2 receptor binding subunit was identified by photoaffinity-crosslinking studies using 125I-labeled PAPA-APEC and the heterobifunctional crosslinking agent N-succinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH). After covalent incorporation, a single specifically radiolabeled protein with an apparent molecular mass of 45 kDa was observed on NaDodSO4/PAGE/autoradiography. Incorporation of 125I-labeled PAPA-APEC into this polypeptide is blocked by agonists and antagonists with the expected potency for A2 receptors and is decreased in the presence of 10(-4) M guanosine 5'-[beta, gamma-imido]triphosphate

  11. Differential antibiotic sensitivity determined by the large ribosomal subunit in thermophilic archaea.

    OpenAIRE

    Ruggero, D; Londei, P

    1996-01-01

    Hybrid ribosomes obtained by mixing the ribosomal subunits of the extremely thermophilic archaea Sulfolobus solfataricus and Desulfurococcus mobilis were tested for their sensitivity to selected antibiotics. It is shown that structural differences in the large ribosomal subunits determine qualitatively and quantitatively the patterns of response to alpha-sarcin and paromomycin in these species.

  12. Single-subunit oligosaccharyltransferases of Trypanosoma brucei display different and predictable peptide acceptor specificities.

    Science.gov (United States)

    Jinnelov, Anders; Ali, Liaqat; Tinti, Michele; Güther, Maria Lucia S; Ferguson, Michael A J

    2017-12-08

    Trypanosoma brucei causes African trypanosomiasis and contains three full-length oligosaccharyltransferase (OST) genes; two of which, Tb STT3A and Tb STT3B, are expressed in the bloodstream form of the parasite. These OSTs have different peptide acceptor and lipid-linked oligosaccharide donor specificities, and trypanosomes do not follow many of the canonical rules developed for other eukaryotic N -glycosylation pathways, raising questions as to the basic architecture and detailed function of trypanosome OSTs. Here, we show by blue-native gel electrophoresis and stable isotope labeling in cell culture proteomics that the Tb STT3A and Tb STT3B proteins associate with each other in large complexes that contain no other detectable protein subunits. We probed the peptide acceptor specificities of the OSTs in vivo using a transgenic glycoprotein reporter system and performed glycoproteomics on endogenous parasite glycoproteins using sequential endoglycosidase H and peptide: N -glycosidase-F digestions. This allowed us to assess the relative occupancies of numerous N -glycosylation sites by endoglycosidase H-resistant N -glycans originating from Man 5 GlcNAc 2 -PP-dolichol transferred by Tb STT3A, and endoglycosidase H-sensitive N -glycans originating from Man 9 GlcNAc 2 -PP-dolichol transferred by Tb STT3B. Using machine learning, we assessed the features that best define Tb STT3A and Tb STT3B substrates in vivo and built an algorithm to predict the types of N -glycan most likely to predominate at all the putative N -glycosylation sites in the parasite proteome. Finally, molecular modeling was used to suggest why Tb STT3A has a distinct preference for sequons containing and/or flanked by acidic amino acid residues. Together, these studies provide insights into how a highly divergent eukaryote has re-wired protein N -glycosylation to provide protein sequence-specific N -glycan modifications. Data are available via ProteomeXchange with identifiers PXD007236, PXD007267

  13. Tuning of the Na,K-ATPase by the beta subunit

    Science.gov (United States)

    Hilbers, Florian; Kopec, Wojciech; Isaksen, Toke Jost; Holm, Thomas Hellesøe; Lykke-Hartmann, Karin; Nissen, Poul; Khandelia, Himanshu; Poulsen, Hanne

    2016-02-01

    The vital gradients of Na+ and K+ across the plasma membrane of animal cells are maintained by the Na,K-ATPase, an αβ enzyme complex, whose α subunit carries out the ion transport and ATP hydrolysis. The specific roles of the β subunit isoforms are less clear, though β2 is essential for motor physiology in mammals. Here, we show that compared to β1 and β3, β2 stabilizes the Na+-occluded E1P state relative to the outward-open E2P state, and that the effect is mediated by its transmembrane domain. Molecular dynamics simulations further demonstrate that the tilt angle of the β transmembrane helix correlates with its functional effect, suggesting that the relative orientation of β modulates ion binding at the α subunit. β2 is primarily expressed in granule neurons and glomeruli in the cerebellum, and we propose that its unique functional characteristics are important to respond appropriately to the cerebellar Na+ and K+ gradients.

  14. Condensin HEAT subunits required for DNA repair, kinetochore/centromere function and ploidy maintenance in fission yeast.

    Directory of Open Access Journals (Sweden)

    Xingya Xu

    Full Text Available Condensin, a central player in eukaryotic chromosomal dynamics, contains five evolutionarily-conserved subunits. Two SMC (structural maintenance of chromosomes subunits contain ATPase, hinge, and coiled-coil domains. One non-SMC subunit is similar to bacterial kleisin, and two other non-SMC subunits contain HEAT (similar to armadillo repeats. Here we report isolation and characterization of 21 fission yeast (Schizosaccharomyces pombe mutants for three non-SMC subunits, created using error-prone mutagenesis that resulted in single-amino acid substitutions. Beside condensation, segregation, and DNA repair defects, similar to those observed in previously isolated SMC and cnd2 mutants, novel phenotypes were observed for mutants of HEAT-repeats containing Cnd1 and Cnd3 subunits. cnd3-L269P is hypersensitive to the microtubule poison, thiabendazole, revealing defects in kinetochore/centromere and spindle assembly checkpoints. Three cnd1 and three cnd3 mutants increased cell size and doubled DNA content, thereby eliminating the haploid state. Five of these mutations reside in helix B of HEAT repeats. Two non-SMC condensin subunits, Cnd1 and Cnd3, are thus implicated in ploidy maintenance.

  15. Sensitivity and Specificity of a Five-Minute Cognitive Screening Test in Patients With Heart Failure.

    Science.gov (United States)

    Cameron, Janette D; Gallagher, Robyn; Pressler, Susan J; McLennan, Skye N; Ski, Chantal F; Tofler, Geoffrey; Thompson, David R

    2016-02-01

    Cognitive impairment occurs in up to 80% of patients with heart failure (HF). The National Institute for Neurological Disorders and Stroke (NINDS) and the Canadian Stroke Network (CSN) recommend a 5-minute cognitive screening protocol that has yet to be psychometrically evaluated in HF populations. The aim of this study was to conduct a secondary analysis of the sensitivity and specificity of the NINDS-CSN brief cognitive screening protocol in HF patients. The Montreal Cognitive Assessment (MoCA) was administered to 221 HF patients. The NINDS-CSN screen comprises 3 MoCA items, with lower scores indicating poorer cognitive function. Receiver operator characteristic (ROC) curves were constructed, determining the sensitivity, specificity and appropriate cutoff scores of the NINDS-CSN screen. In an HF population aged 76 ± 12 years, 136 (62%) were characterized with cognitive impairment (MoCA area under the receiver operating characteristic curve indicated good accuracy in screening for cognitive impairment (0.88; P cognitive impairment in patients with HF. Future studies should include a neuropsychologic battery to more comprehensively examine the diagnostic accuracy of brief cognitive screening protocols. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Inhibition of the promotion of hepatocarcinogenesis by 2,2',4,4',5,5'-hexachlorobiphenyl (PCB-153) by the deletion of the p50 subunit of NF-κB in mice

    International Nuclear Information System (INIS)

    Glauert, Howard P.; Tharappel, Job C.; Banerjee, Subhashis; Chan, Nelson L.S.; Kania-Korwel, Izabela; Lehmler, Hans-Joachim; Lee, Eun Y.; Robertson, Larry W.; Spear, Brett T.

    2008-01-01

    Polychlorinated biphenyls (PCBs) are persistent and ubiquitous environmental chemicals that bioaccumulate and have hepatic tumor promoting activity in rodents. The present study examined the effect of deleting the p50 subunit of NF-κB on the hepatic tumor promoting activity of 2,2',4,4',5,5'-hexachlorobiphenyl (PCB-153) in mice. Both wild-type and p50-/- male mice were injected i.p. with diethylnitrosamine (DEN, 90 mg/kg) and then subsequently injected biweekly with 20 i.p. injections of PCB-153 (300 μmol/kg/injection). p50 deletion decreased the tumor incidence in both PCB- and vehicle-treated mice, whereas PCB-153 slightly (P = 0.09) increased the tumor incidence in wild-type and p50-/- mice. PCB-153 increased the total tumor volume in both wild-type and p50-/- mice, but the total tumor volume was not affected by p50 deletion in either PCB- or vehicle-treated mice. The volume of tumors that were positive for glutamine synthetase (GS), which is indicative of mutations in the beta-catenin gene, was increased in both wild-type and p50-/- mice administered PCB-153 compared to vehicle controls, and inhibited in p50-/- mice compared to wild-type mice (in both PCB- and vehicle-treated mice). The volume of tumors that were negative for GS was increased in p50-/- mice compared to wild-type mice but was not affected by PCB-153. PCB-153 increased cell proliferation in normal hepatocytes in wild-type but not p50-/- mice; this increase was inhibited in p50-/- mice. In hepatic tumors, the rate of cell proliferation was much higher than in normal hepatocytes, but was not affected by PCB treatment or p50 deletion. The rate of apoptosis, as measured by the TUNEL assay, was not affected by PCB-153 or p50 deletion in normal hepatocytes. In hepatic tumors, the rate of apoptosis was lower than in normal hepatocytes; PCB-153 slightly (P = 0.10) increased apoptosis in p50-/- but not wild-type mice; p50 deletion had no effect. Taken together, these data indicate that the absence of

  17. Expression Profile of the Integrin Receptor Subunits in the Guinea Pig Sclera.

    Science.gov (United States)

    Wang, Kevin K; Metlapally, Ravikanth; Wildsoet, Christine F

    2017-06-01

    The ocular dimensional changes in myopia reflect increased scleral remodeling, and in high myopia, loss of scleral integrity leads to biomechanical weakening and continued scleral creep. As integrins, a type of cell surface receptors, have been linked to scleral remodeling, they represent potential targets for myopia therapies. As a first step, this study aimed to characterize the integrin subunits at the messenger RNA level in the sclera of the guinea pig, a more recently added but increasingly used animal model for myopia research. Primers for α and β integrin subunits were designed using NCBI/UCSC Genome Browser and Primer3 software tools. Total RNA was extracted from normal scleral tissue and isolated cultured scleral fibroblasts, as well as liver and lung, as reference tissues, all from guinea pig. cDNA was produced by reverse transcription, PCR was used to amplify products of predetermined sizes, and products were sequenced using standard methods. Guinea pig scleral tissue expressed all known integrin alpha subunits except αD and αE. The latter integrin subunits were also not expressed by cultured guinea pig scleral fibroblasts; however, their expression was confirmed in guinea pig liver. In addition, isolated cultured fibroblasts did not express integrin subunits αL, αM, and αX. This difference between results for cultured cells and intact sclera presumably reflects the presence in the latter of additional cell types. Both guinea pig scleral tissue and isolated scleral fibroblasts expressed all known integrin beta subunits. All results were verified through sequencing. The possible contributions of integrins to scleral remodeling make them plausible targets for myopia prevention. Data from this study will help guide future ex vivo and in vitro studies directed at understanding the relationship between scleral integrins and ocular growth regulation in the guinea pig model for myopia.

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

    Directory of Open Access Journals (Sweden)

    Zhiwen Ye

    2017-04-01

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

  19. Protection of Mice from Lethal Vaccinia Virus Infection by Vaccinia Virus Protein Subunits with a CpG Adjuvant

    Directory of Open Access Journals (Sweden)

    Sarah Reeman

    2017-12-01

    Full Text Available Smallpox vaccination carries a high risk of adverse events in recipients with a variety of contra-indications for live vaccines. Although alternative non-replicating vaccines have been described in the form of replication-deficient vaccine viruses, DNA vaccines, and subunit vaccines, these are less efficacious than replicating vaccines in animal models. DNA and subunit vaccines in particular have not been shown to give equivalent protection to the traditional replicating smallpox vaccine. We show here that combinations of the orthopoxvirus A27, A33, B5 and L1 proteins give differing levels of protection when administered in different combinations with different adjuvants. In particular, the combination of B5 and A27 proteins adjuvanted with CpG oligodeoxynucleotides (ODN gives a level of protection in mice that is equivalent to the Lister traditional vaccine in a lethal vaccinia virus challenge model.

  20. Genome-wide analysis of putative peroxiredoxin in unicellular and filamentous cyanobacteria

    Directory of Open Access Journals (Sweden)

    Cui Hongli

    2012-11-01

    Full Text Available Abstract Background Cyanobacteria are photoautotrophic prokaryotes with wide variations in genome sizes and ecological habitats. Peroxiredoxin (PRX is an important protein that plays essential roles in protecting own cells against reactive oxygen species (ROS. PRXs have been identified from mammals, fungi and higher plants. However, knowledge on cyanobacterial PRXs still remains obscure. With the availability of 37 sequenced cyanobacterial genomes, we performed a comprehensive comparative analysis of PRXs and explored their diversity, distribution, domain structure and evolution. Results Overall 244 putative prx genes were identified, which were abundant in filamentous diazotrophic cyanobacteria, Acaryochloris marina MBIC 11017, and unicellular cyanobacteria inhabiting freshwater and hot-springs, while poor in all Prochlorococcus and marine Synechococcus strains. Among these putative genes, 25 open reading frames (ORFs encoding hypothetical proteins were identified as prx gene family members and the others were already annotated as prx genes. All 244 putative PRXs were classified into five major subfamilies (1-Cys, 2-Cys, BCP, PRX5_like, and PRX-like according to their domain structures. The catalytic motifs of the cyanobacterial PRXs were similar to those of eukaryotic PRXs and highly conserved in all but the PRX-like subfamily. Classical motif (CXXC of thioredoxin was detected in protein sequences from the PRX-like subfamily. Phylogenetic tree constructed of catalytic domains coincided well with the domain structures of PRXs and the phylogenies based on 16s rRNA. Conclusions The distribution of genes encoding PRXs in different unicellular and filamentous cyanobacteria especially those sub-families like PRX-like or 1-Cys PRX correlate with the genome size, eco-physiology, and physiological properties of the organisms. Cyanobacterial and eukaryotic PRXs share similar conserved motifs, indicating that cyanobacteria adopt similar catalytic

  1. Genome-wide analysis of putative peroxiredoxin in unicellular and filamentous cyanobacteria.

    Science.gov (United States)

    Cui, Hongli; Wang, Yipeng; Wang, Yinchu; Qin, Song

    2012-11-16

    Cyanobacteria are photoautotrophic prokaryotes with wide variations in genome sizes and ecological habitats. Peroxiredoxin (PRX) is an important protein that plays essential roles in protecting own cells against reactive oxygen species (ROS). PRXs have been identified from mammals, fungi and higher plants. However, knowledge on cyanobacterial PRXs still remains obscure. With the availability of 37 sequenced cyanobacterial genomes, we performed a comprehensive comparative analysis of PRXs and explored their diversity, distribution, domain structure and evolution. Overall 244 putative prx genes were identified, which were abundant in filamentous diazotrophic cyanobacteria, Acaryochloris marina MBIC 11017, and unicellular cyanobacteria inhabiting freshwater and hot-springs, while poor in all Prochlorococcus and marine Synechococcus strains. Among these putative genes, 25 open reading frames (ORFs) encoding hypothetical proteins were identified as prx gene family members and the others were already annotated as prx genes. All 244 putative PRXs were classified into five major subfamilies (1-Cys, 2-Cys, BCP, PRX5_like, and PRX-like) according to their domain structures. The catalytic motifs of the cyanobacterial PRXs were similar to those of eukaryotic PRXs and highly conserved in all but the PRX-like subfamily. Classical motif (CXXC) of thioredoxin was detected in protein sequences from the PRX-like subfamily. Phylogenetic tree constructed of catalytic domains coincided well with the domain structures of PRXs and the phylogenies based on 16s rRNA. The distribution of genes encoding PRXs in different unicellular and filamentous cyanobacteria especially those sub-families like PRX-like or 1-Cys PRX correlate with the genome size, eco-physiology, and physiological properties of the organisms. Cyanobacterial and eukaryotic PRXs share similar conserved motifs, indicating that cyanobacteria adopt similar catalytic mechanisms as eukaryotes. All cyanobacterial PRX proteins

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2005-01-01

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

  3. Unassigned MURF1 of kinetoplastids codes for NADH dehydrogenase subunit 2

    Directory of Open Access Journals (Sweden)

    Burger Gertraud

    2008-10-01

    Full Text Available Abstract Background In a previous study, we conducted a large-scale similarity-free function prediction of mitochondrion-encoded hypothetical proteins, by which the hypothetical gene murf1 (maxicircle unidentified reading frame 1 was assigned as nad2, encoding subunit 2 of NADH dehydrogenase (Complex I of the respiratory chain. This hypothetical gene occurs in the mitochondrial genome of kinetoplastids, a group of unicellular eukaryotes including the causative agents of African sleeping sickness and leishmaniasis. In the present study, we test this assignment by using bioinformatics methods that are highly sensitive in identifying remote homologs and confront the prediction with available biological knowledge. Results Comparison of MURF1 profile Hidden Markov Model (HMM against function-known profile HMMs in Pfam, Panther and TIGR shows that MURF1 is a Complex I protein, but without specifying the exact subunit. Therefore, we constructed profile HMMs for each individual subunit, using all available sequences clustered at various identity thresholds. HMM-HMM comparison of these individual NADH subunits against MURF1 clearly identifies this hypothetical protein as NAD2. Further, we collected the relevant experimental information about kinetoplastids, which provides additional evidence in support of this prediction. Conclusion Our in silico analyses provide convincing evidence for MURF1 being a highly divergent member of NAD2.

  4. Positive modulation of delta-subunit containing GABAA receptors in mouse neurons

    DEFF Research Database (Denmark)

    Vardya, Irina; Hoestgaard-Jensen, Kirsten; Nieto-Gonzalez, Jose Luis

    2012-01-01

    δ-subunit containing extrasynaptic GABA(A) receptors are potential targets for modifying neuronal activity in a range of brain disorders. With the aim of gaining more insight in synaptic and extrasynaptic inhibition, we used a new positive modulator, AA29504, of δ-subunit containing GABA(A) recep......δ-subunit containing extrasynaptic GABA(A) receptors are potential targets for modifying neuronal activity in a range of brain disorders. With the aim of gaining more insight in synaptic and extrasynaptic inhibition, we used a new positive modulator, AA29504, of δ-subunit containing GABA......(A) receptors in mouse neurons in vitro and in vivo. Whole-cell patch-clamp recordings were carried out in the dentate gyrus in mouse brain slices. In granule cells, AA29504 (1 μM) caused a 4.2-fold potentiation of a tonic current induced by THIP (1 μM), while interneurons showed a potentiation of 2.6-fold......-free environment using Ca²⁺ imaging in cultured neurons, AA29504 showed GABA(A) receptor agonism in the absence of agonist. Finally, AA29504 exerted dose-dependent stress-reducing and anxiolytic effects in mice in vivo. We propose that AA29504 potentiates δ-containing GABA(A) receptors to enhance tonic inhibition...

  5. Alpha S1-casein polymorphisms in camel (Camelus dromedarius) and descriptions of biological active peptides and allergenic epitopes.

    Science.gov (United States)

    Erhardt, Georg; Shuiep, El Tahir Salih; Lisson, Maria; Weimann, Christina; Wang, Zhaoxin; El Zubeir, Ibtisam El Yas Mohamed; Pauciullo, Alfredo

    2016-06-01

    Milk samples of 193 camels (Camelus dromedarius) from different regions of Sudan were screened for casein variability by isoelectric focusing. Kappa-casein and beta-casein were monomorphic, whereas three protein patterns named αs1-casein A, C, and D were identified. The major allele A revealed frequencies of 0.79 (Lahaoi), 0.75 (Shanbali), 0.90 (Arabi Khali), and 0.88 (Arabi Gharbawi) in the different ecotypes. CSN1S1*C shows a single G > T nucleotide substitution in the exon 5, leading to a non-synonymous amino acid exchange (p.Glu30 > Asp30) in comparison to CSN1S1*A and D. At cDNA level, no further single nucleotide polymorphisms could be identified in CSN1S1* A, C, and D, whereas the variants CSN1S1*A and CSN1S1*C are characterized by missing of exon 18 compared to the already described CSN1S1*B, as consequence of DNA insertion of 11 bp at intron 17 which alter the pre-mRNA spliceosome machinery. A polymerase chain-restriction fragment length polymorphism method (PCR-RFLP) was established to type for G > T nucleotide substitution at genomic DNA level. The occurrence and differences of IgE-binding epitopes and bioactive peptides between αs1-casein A, C, and D after digestion were analyzed in silico. The amino acid substitutions and deletion affected the arising peptide pattern and thus modifications between IgE-binding epitopes and bioactive peptides of the variants were found. The allergenic potential of these different peptides will be investigated by microarray immunoassay using sera from milk-sensitized individuals, as it was already demonstrated for bovine αs1-casein variants.

  6. Formation of putative chloroplast cytochromes in isolated developing pea chloroplasts

    International Nuclear Information System (INIS)

    Thaver, S.S.; Bhava, D.; Castelfranco, P.A.

    1986-01-01

    In addition to chlorophyll-protein complexes, other proteins were labeled when isolated developing pea chloroplasts were incubated with [ 14 C]-5-aminolevulinic acid [ 14 C]-ALA. The major labeled band (M/sub r/ = 43 kDa by LDS-PAGE) was labeled even in the presence of chloramphenicol. Heme-dependent peroxidase activity (as detected by the tetramethyl benzidine-H 2 O 2 stain) was not visibly associated with this band. The radioactive band was stable to heat, 5% HCl in acetone, and was absent if the incubation with [ 14 C]-5-aminolevulinic acid was carried out in the presence of N-methyl protoporphyrin IX dimethyl ester (a specific inhibitor of ferrochelatase). Organic solvent extraction procedures for the enrichment of cytochrome f from chloroplast membranes also extracted this unknown labeled product. It was concluded that this labeled product was probably a c-type cytochrome. The effect of exogenous iron, iron chelators, gabaculine (an inhibitor of ALA synthesis) and other incubation conditions upon the in vitro formation of putative chloroplast cytochromes will be discussed

  7. Autoantibodies to αS1-Casein Are Induced by Breast-Feeding

    Science.gov (United States)

    Petermann, Klaudia; Vordenbäumen, Stefan; Maas, Ruth; Braukmann, Achim; Bleck, Ellen; Saenger, Thorsten; Schneider, Matthias; Jose, Joachim

    2012-01-01

    Background The generation of antibodies is impaired in newborns due to an immature immune system and reduced exposure to pathogens due to maternally derived antibodies and placental functions. During nursing, the immune system of newborns is challenged with multiple milk-derived proteins. Amongst them, caseins are the main constituent. In particular, human αS1-casein (CSN1S1) was recently shown to possess immunomodulatory properties. We were thus interested to determine if auto-antibodies to CSN1S1 are induced by breast-feeding and may be sustained into adulthood. Methods 62 sera of healthy adult individuals who were (n = 37) or were not (n = 25) breast-fed against human CSN1S1 were investigated by a new SD (surface display)-ELISA. For cross-checking, these sera were tested for anti Epstein-Barr virus (EBV) antibodies by a commercial ELISA. Results IgG-antibodies were predominantly detected in individuals who had been nursed. At a cut-off value of 0.4, the SD-ELISA identified individuals with a history of having been breast-fed with a sensitivity of 80% and a specificity of 92%. Under these conditions, 35 out of 37 sera from healthy donors, who where breast-fed, reacted positively but only 5 sera of the 25 donors who were not breast-fed. The duration of breast-feeding was of no consequence to the antibody reaction as some healthy donors were only short term breast-fed (5 days minimum until 6 weeks maximum), but exhibited significant serum reaction against human CSN1S1 nonetheless. Conclusion We postulate that human CSN1S1 is an autoantigen. The antigenicity is orally determined, caused by breast-feeding, and sustained into adulthood. PMID:22496735

  8. Role of adenosine 5'-monophosphate-activated protein kinase subunits in skeletal muscle mammalian target of rapamycin signaling

    DEFF Research Database (Denmark)

    Deshmukh, Atul S.; Treebak, Jonas Thue; Long, Yun Chau

    2008-01-01

    AMP-activated protein kinase (AMPK) is an important energy-sensing protein in skeletal muscle. Mammalian target of rapamycin (mTOR) mediates translation initiation and protein synthesis through ribosomal S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). AMPK...... activation reduces muscle protein synthesis by down-regulating mTOR signaling, whereas insulin mediates mTOR signaling via Akt activation. We hypothesized that AMPK-mediated inhibitory effects on mTOR signaling depend on catalytic alpha2 and regulatory gamma3 subunits. Extensor digitorum longus muscle from...... (Thr37/46) (P mTOR targets, suggesting mTOR signaling is blocked by prior AMPK activation. The AICAR-induced inhibition was partly rescued...

  9. ORF Sequence: NC_002695 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available NC_002695 gi|15830145 >gi|15830145|ref|NP_308918.1| putative transmembrane subunit...IFVPIGALQAGEALWHWSVIPLGLAVAILSTALPYSLEMIALTRLPTRTFGTLMSMEPALAAVSGMIFLGETLTPIQLLALGAIIAASMGSTLTVRKESKIKELDIN

  10. Structural analysis of the α subunit of Na(+)/K(+) ATPase genes in invertebrates.

    Science.gov (United States)

    Thabet, Rahma; Rouault, J-D; Ayadi, Habib; Leignel, Vincent

    2016-01-01

    The Na(+)/K(+) ATPase is a ubiquitous pump coordinating the transport of Na(+) and K(+) across the membrane of cells and its role is fundamental to cellular functions. It is heteromer in eukaryotes including two or three subunits (α, β and γ which is specific to the vertebrates). The catalytic functions of the enzyme have been attributed to the α subunit. Several complete α protein sequences are available, but only few gene structures were characterized. We identified the genomic sequences coding the α-subunit of the Na(+)/K(+) ATPase, from the whole-genome shotgun contigs (WGS), NCBI Genomes (chromosome), Genomic Survey Sequences (GSS) and High Throughput Genomic Sequences (HTGS) databases across distinct phyla. One copy of the α subunit gene was found in Annelida, Arthropoda, Cnidaria, Echinodermata, Hemichordata, Mollusca, Placozoa, Porifera, Platyhelminthes, Urochordata, but the nematodes seem to possess 2 to 4 copies. The number of introns varied from 0 (Platyhelminthes) to 26 (Porifera); and their localization and length are also highly variable. Molecular phylogenies (Maximum Likelihood and Maximum Parsimony methods) showed some clusters constituted by (Chordata/(Echinodermata/Hemichordata)) or (Plathelminthes/(Annelida/Mollusca)) and a basal position for Porifera. These structural analyses increase our knowledge about the evolutionary events of the α subunit genes in the invertebrates. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Five putative nucleoside triphosphate diphosphohydrolase genes are expressed in Trichomonas vaginalis.

    Science.gov (United States)

    Frasson, Amanda Piccoli; Dos Santos, Odelta; Meirelles, Lúcia Collares; Macedo, Alexandre José; Tasca, Tiana

    2016-01-01

    Trichomonas vaginalis is a protozoan that parasitizes the human urogenital tract causing trichomoniasis, the most common non-viral sexually transmitted disease. The parasite has unique genomic characteristics such as a large genome size and expanded gene families. Ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) is an enzyme responsible for hydrolyzing nucleoside tri- and diphosphates and has already been biochemically characterized in T. vaginalis. Considering the important role of this enzyme in the production of extracellular adenosine for parasite uptake, we evaluated the gene expression of five putative NTPDases in T. vaginalis. We showed that all five putative TvNTPDase genes (TvNTPDase1-5) were expressed by both fresh clinical and long-term grown isolates. The amino acid alignment predicted the presence of the five crucial apyrase conserved regions, transmembrane domains, signal peptides, phosphorylation and catalytic sites. Moreover, a phylogenetic analysis showed that TvNTPDase sequences make up a clade with NTPDases intracellularly located. Biochemical NTPDase activity (ATP and ADP hydrolysis) is responsive to the serum-restrictive conditions and the gene expression of TvNTPDases was mostly increased, mainly TvNTPDase2 and TvNTPDase4, although there was not a clear pattern of expression among them. In summary, the present report demonstrates the gene expression patterns of predicted NTPDases in T. vaginalis. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  12. Heterodimerization with the β1 subunit directs the α2 subunit of nitric oxide-sensitive guanylyl cyclase to calcium-insensitive cell-cell contacts in HEK293 cells: Interaction with Lin7a.

    Science.gov (United States)

    Hochheiser, Julia; Haase, Tobias; Busker, Mareike; Sömmer, Anne; Kreienkamp, Hans-Jürgen; Behrends, Sönke

    2016-12-15

    Nitric oxide-sensitive guanylyl cyclase is a heterodimeric enzyme consisting of an α and a β subunit. Two different α subunits (α 1 and α 2 ) give rise to two heterodimeric enzymes α 1 /β 1 and α 2 /β 1 . Both coexist in a wide range of tissues including blood vessels and the lung, but expression of the α 2 /β 1 form is generally much lower and approaches levels similar to the α 1 /β 1 form in the brain only. In the present paper, we show that the α 2 /β 1 form interacts with Lin7a in mouse brain synaptosomes based on co-precipitation analysis. In HEK293 cells, we found that the overexpressed α 2 /β 1 form, but not the α 1 /β 1 form is directed to calcium-insensitive cell-cell contacts. The isolated PDZ binding motif of an amino-terminally truncated α 2 subunit was sufficient for cell-cell contact localization. For the full length α 2 subunit with the PDZ binding motif this was only the case in the heterodimer configuration with the β 1 subunit, but not as isolated α 2 subunit. We conclude that the PDZ binding motif of the α 2 subunit is only accessible in the heterodimer conformation of the mature nitric oxide-sensitive enzyme. Interaction with Lin7a, a small scaffold protein important for synaptic function and cell polarity, can direct this complex to nectin based cell-cell contacts via MPP3 in HEK293 cells. We conclude that heterodimerization is a prerequisite for further protein-protein interactions that direct the α 2 /β 1 form to strategic sites of the cell membrane with adjacent neighbouring cells. Drugs increasing the nitric oxide-sensitivity of this specific form may be particularly effective. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Basic residues in the 74-83 and 191-198 segments of protein kinase CK2 catalytic subunit are implicated in negative but not in positive regulation by the beta-subunit

    DEFF Research Database (Denmark)

    Sarno, S; Vaglio, P; Marin, O

    1997-01-01

    by the beta-subunit many fold more than that of alpha wild type, while extrastimulation by beta mutant D55L56E57A, observable with alpha wild type, is abolished with these mutants. These data support the conclusion that down regulation by the acidic residues clustered in the N-terminal moiety of beta...... is mediated by basic residues in the 74-83 and in the 191-198 sequences of the alpha-subunit. These are also implicated in substrate recognition consistent with the concept that the N-terminal acidic region of the beta subunit operates as a pseudosubstrate. In contrast, another CK2alpha mutant, V66A, is more...

  14. Role of post-translational modifications at the β-subunit ectodomain in complex association with a promiscuous plant P4-ATPase

    DEFF Research Database (Denmark)

    Costa, Sara; Marek, Magdalena; Axelsen, Kristian Buhl

    2016-01-01

    and can interact with several isoforms. In the present study, we used a site-directed mutagenesis approach to assess the role of post-translational modifications at the plant ALIS5 β-subunit ectodomain in the functionality of the promiscuous plant P4-ATPase ALA2. We identified two N-glycosylated residues......) compromises complex association, but the mutant β-subunits still promote complex trafficking and activity to some extent. In contrast, disruption of a conserved disulfide bond between Cys(158) and Cys(172) has no effect on the P4-ATPase complex. Our results demonstrate that post-translational modifications...

  15. Semi-automated literature mining to identify putative biomarkers of disease from multiple biofluids

    Science.gov (United States)

    2014-01-01

    Background Computational methods for mining of biomedical literature can be useful in augmenting manual searches of the literature using keywords for disease-specific biomarker discovery from biofluids. In this work, we develop and apply a semi-automated literature mining method to mine abstracts obtained from PubMed to discover putative biomarkers of breast and lung cancers in specific biofluids. Methodology A positive set of abstracts was defined by the terms ‘breast cancer’ and ‘lung cancer’ in conjunction with 14 separate ‘biofluids’ (bile, blood, breastmilk, cerebrospinal fluid, mucus, plasma, saliva, semen, serum, synovial fluid, stool, sweat, tears, and urine), while a negative set of abstracts was defined by the terms ‘(biofluid) NOT breast cancer’ or ‘(biofluid) NOT lung cancer.’ More than 5.3 million total abstracts were obtained from PubMed and examined for biomarker-disease-biofluid associations (34,296 positive and 2,653,396 negative for breast cancer; 28,355 positive and 2,595,034 negative for lung cancer). Biological entities such as genes and proteins were tagged using ABNER, and processed using Python scripts to produce a list of putative biomarkers. Z-scores were calculated, ranked, and used to determine significance of putative biomarkers found. Manual verification of relevant abstracts was performed to assess our method’s performance. Results Biofluid-specific markers were identified from the literature, assigned relevance scores based on frequency of occurrence, and validated using known biomarker lists and/or databases for lung and breast cancer [NCBI’s On-line Mendelian Inheritance in Man (OMIM), Cancer Gene annotation server for cancer genomics (CAGE), NCBI’s Genes & Disease, NCI’s Early Detection Research Network (EDRN), and others]. The specificity of each marker for a given biofluid was calculated, and the performance of our semi-automated literature mining method assessed for breast and lung cancer

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

    Science.gov (United States)

    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.

  17. The Putative HORMA Domain Protein Atg101 Dimerizes and Is Required for Starvation-Induced and Selective Autophagy in Drosophila

    Directory of Open Access Journals (Sweden)

    Krisztina Hegedűs

    2014-01-01

    Full Text Available The large-scale turnover of intracellular material including organelles is achieved by autophagy-mediated degradation in lysosomes. Initiation of autophagy is controlled by a protein kinase complex consisting of an Atg1-family kinase, Atg13, FIP200/Atg17, and the metazoan-specific subunit Atg101. Here we show that loss of Atg101 impairs both starvation-induced and basal autophagy in Drosophila. This leads to accumulation of protein aggregates containing the selective autophagy cargo ref(2P/p62. Mapping experiments suggest that Atg101 binds to the N-terminal HORMA domain of Atg13 and may also interact with two unstructured regions of Atg1. Another HORMA domain-containing protein, Mad2, forms a conformational homodimer. We show that Drosophila Atg101 also dimerizes, and it is predicted to fold into a HORMA domain. Atg101 interacts with ref(2P as well, similar to Atg13, Atg8a, Atg16, Atg18, Keap1, and RagC, a known regulator of Tor kinase which coordinates cell growth and autophagy. These results raise the possibility that the interactions and dimerization of the putative HORMA domain protein Atg101 play critical roles in starvation-induced autophagy and proteostasis, by promoting the formation of protein aggregate-containing autophagosomes.

  18. Testing experimental subunit furunculosis vaccines for rainbow trout

    DEFF Research Database (Denmark)

    Marana, Moonika H.; Chettri, Jiwan Kumar; Skov, Jakob

    2016-01-01

    Aeromonas salmonicida subsp. salmonicida (AS) is the etiological agent of typical furunculosis in salmonid fish. The disease causes bacterial septicemia and is a major fish health problem in salmonid aquaculture worldwide, inducing high morbidity and mortality. In this study we vaccinated rainbow...... trout with subunit vaccines containing protein antigens that were selected based on an in silico antigen discovery approach. Thus, the proteome of AS strain A449 was analyzed by an antigen discovery platform and its proteins consequently ranked by their predicted ability to evoke protective immune...... response against AS. Fourteen proteins were prepared in 3 different experimental subunit vaccine combinations and used to vaccinate rainbow trout by intraperitoneal (i.p.) injection. We tested the proteins for their ability to elicit antibody production and protection. Thus, fish were exposed to virulent...

  19. Energy expenditures & physical activity in rats with chronic suboptimal nutrition.

    Science.gov (United States)

    Rising, Russell; Lifshitz, Fima

    2006-01-31

    Sub-optimally nourished rats show reduced growth, biochemical and physiological changes. However, no one has assessed metabolic rate adaptations in rats subjected to chronic suboptimal nutrition (CSN). In this study energy expenditure (EE; kcal/100 g body weight) and physical activity (PA; oscillations in weight/min/kg body weight) were assessed in rats subjected to three levels of CSN. Body weight gain was diminished (76.7 +/- 12.0 and 61.6 +/- 11.0 g) in rats fed 70 and 60% of the ad-libitum fed controls which gained more weight (148.5 +/- 32.3 g). The rats fed 80% gained weight similarly to controls (136.3 +/- 10.5 g). Percent Fat-free body mass was reduced (143.8 +/- 8.7 and 142.0 +/- 7.6 g) in rats fed 70 and 60% of ad-libitum, but not in those fed 80% (200.8 +/- 17.5 g) as compared with controls (201.6 +/- 33.4 g). Body fat (g) decreased in rats fed 80% (19.7 +/- 5.3), 70% (15.3 +/- 3.5) and 60% (9.6 +/- 2.7) of ad-libitum in comparison to controls (26.0 +/- 6.7). EE and PA were also altered by CSN. The control rats increased their EE and PA during the dark periods by 1.4 +/- 0.8 and 1.7 +/- 1.1 respectively, as compared with light the period; whereas CSN rats fed 80 and 70% of ad-libitum energy intake had reduced EE and PA during the dark periods as compared with the light period EE(7.5 +/- 1.4 and 7.8 +/- 0.6 vs. 9.0 +/- 1.2 and 9.7 +/- 0.8; p vs. 4.1 +/- 0.9 and 2.4 +/- 0.4; p vs. 0.95 +/- 0.03 and 0.91 +/- 0.05 p light (7.1 +/- 1.4) and dark period (6.2 +/- 1.0) EE and PA (3.4 +/- 0.9 and 2.5 +/- 0.5 respectively) were reduced in rats fed 60% of ad-libitum energy intake. CSN rats adapt to mild energy restriction by reducing body fat, EE and PA mainly during the dark period while growth proceeds and lean body mass is preserved. At higher levels of energy restrictions there is decreased growth, body fat and lean mass. Moreover EE and PA are also reduced during both light and dark periods.

  20. SDS-PAGE Electrophoretic Property of Human Chorionic Gonadotropin (hCG) and its β-subunit

    OpenAIRE

    Gam, Lay-Harn; Latiff, Aishah

    2005-01-01

    The microheterogeneity property of hCG with regards to its sialic acid contents resulted in variable mobility of the glycoprotein in SDS-PAGE. The intact hCG molecule is composed of two dissimilar subunits, namely α- and β-subunits. The identification of hCG bands in SDS-PAGE was accomplished by the immunoblotting experiment, whereby the antibody directed toward the specific region of β-subunit of hCG was used. The data shows that the different mobility of intact hCG was attributed to the dif...

  1. Purification, crystallization and preliminary X-ray diffraction analysis of the non-ATPase subunit Nas6 in complex with the ATPase subunit Rpt3 of the 26S proteasome from Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Nakamura, Yoshihiro; Umehara, Takashi; Tanaka, Akiko; Horikoshi, Masami; Padmanabhan, Balasundaram; Yokoyama, Shigeyuki

    2007-01-01

    The complex of the non-ATPase subunit Nas6 with the C-terminal domain of the ATPase subunit Rpt3 of the 26S proteasome from S. cerevisiae was co-expressed in E. coli and purified to homogeneity. The crystals obtained from the protein complex diffracted to a resolution of 2.2 Å. The non-ATPase subunit Nas6, which is the human orthologue of gankyrin, was co-expressed with the C-terminal domain of the ATPase subunit Rpt3 of the yeast 26S proteasome in Escherichia coli, purified to near-homogeneity and crystallized using the hanging-drop vapour-diffusion method. The protein crystallized in space group P2 1 , with unit-cell parameters a = 60.38, b = 100.22, c = 72.20 Å, β = 94.70° and with three Nas6–Rpt3C molecules per asymmetric unit. The crystal diffracted to beyond 2.2 Å resolution using synchrotron radiation

  2. Selective increases of AMPA, NMDA and kainate receptor subunit mRNAs in the hippocampus and orbitofrontal cortex but not in prefrontal cortex of human alcoholics

    Directory of Open Access Journals (Sweden)

    Zhe eJin

    2014-01-01

    Full Text Available Glutamate is the main excitatory transmitter in the human brain. Drugs that affect the glutamatergic signaling will alter neuronal excitability. Ethanol inhibits glutamate receptors. We examined the expression level of glutamate receptor subunit mRNAs in human post-mortem samples from alcoholics and compared the results to brain samples from control subjects. RNA from hippocampal dentate gyrus (HP-DG, orbitofrontal cortex (OFC, and dorso-lateral prefrontal cortex (DL-PFC samples from 21 controls and 19 individuals with chronic alcohol dependence were included in the study. Total RNA was assayed using quantitative RT-PCR. Out of the 16 glutamate receptor subunits, mRNAs encoding two AMPA (2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-ylpropanoic acid receptor subunits GluA2 and GluA3; three kainate receptor subunits GluK2, GluK3 and GluK5 and five NMDA (N-methyl-D-aspartate receptor subunits GluN1, GluN2A, GluN2C, GluN2D and GluN3A were significantly increased in the HP-DG region in alcoholics. In the OFC, mRNA encoding the NMDA receptor subunit GluN3A was increased, whereas in the DL-PFC, no differences in mRNA levels were observed. Our laboratory has previously shown that the expression of genes encoding inhibitory GABA-A receptors is altered in the HP-DG and OFC of alcoholics (Jin et al., 2011. Whether the changes in one neurotransmitter system drives changes in the other or if they change independently is currently not known. The results demonstrate that excessive long-term alcohol consumption is associated with altered expression of genes encoding glutamate receptors in a brain region-specific manner. It is an intriguing possibility that genetic predisposition to alcoholism may contribute to these gene expression changes.

  3. Crystal structure of a C-terminal deletion mutant of human protein kinase CK2 catalytic subunit

    DEFF Research Database (Denmark)

    Ermakova, Inessa; Boldyreff, Brigitte; Issinger, Olaf-Georg

    2003-01-01

    structure of a C-terminal deletion mutant of human CK2alpha was solved and refined to 2.5A resolution. In the crystal the CK2alpha mutant exists as a monomer in agreement with the organization of the subunits in the CK2 holoenzyme. The refined structure shows the helix alphaC and the activation segment, two...

  4. The translational blocking of α5 and α6 integrin subunits affects migration and invasion, and increases sensitivity to carboplatin of SKOV-3 ovarian cancer cell line

    Energy Technology Data Exchange (ETDEWEB)

    Villegas-Pineda, Julio César, E-mail: jcvillegas@cinvestav.mx; Toledo-Leyva, Alfredo, E-mail: toledo_leyva@hotmail.com; Osorio-Trujillo, Juan Carlos, E-mail: clostrujillo2@yahoo.com.mx; Hernández-Ramírez, Verónica Ivonne, E-mail: arturomvi@hotmail.com; Talamás-Rohana, Patricia, E-mail: ptr@cinvestav.mx

    2017-02-15

    Epithelial ovarian cancer is the most lethal gynecologic malignancy. Integrins, overexpressed in cancer, are involved in various processes that favor the development of the disease. This study focused on determining the degree of involvement of α5, α6 and β3 integrin subunits in the establishment/development of epithelial ovarian cancer (EOC), such as proliferation, migration, invasion, and response to carboplatin. The translation of the α5, α6 and β3 integrins was blocked using morpholines, generating morphant cells for these proteins, which were corroborated by immunofluorescence assays. WST-1 proliferation assay showed that silencing of α5, α6, and β3 integrins does not affect the survival of morphants. Wound healing and transwell chamber assays showed that blocking α5 and α6 integrins decrease, in lesser and greater level respectively, the migratory and the invasive capacity of SKOV-3 cells. Finally, blocking α5 and α6 integrins partially sensitized the cells response to carboplatin, while blocking integrin β3 generated resistance to this drug. Statistical analyses were performed with the GraphPad Prism 5.0 software employing one way and two-way ANOVA tests; data are shown as average±SD. Results suggest that α5 and α6 integrins could become good candidates for chemotherapy targets in EOC.

  5. Evaluation of Pakistani wheat germplasm for bread quality based on allelic variation in HMW glutenin subunits

    Energy Technology Data Exchange (ETDEWEB)

    Tabasum, A; Iqbal, N; Hameed, A; Arshad, R [Nuclear Institute for Agriculture and Biology, Faisalabad (Pakistan)

    2011-06-15

    Seventy six Pakistani wheat genotypes including land races were investigated for Bread quality (BQ) based on allelic variation in HMW glutenin subunits at the Glu-1 loci through SDS- polyacrylamide gel electropherosis. Twenty five different allelic combinations were detected with a total of 14 Glu-1 loci. Highest polymorphism was revealed by Glu-B locus and some single/ rare sub units were also screened out. The frequencies of dominant subunits were 50% for 2*, 42.11% for subunit pair 17+18 and 48.68% for 5+10 and 2+12 respectively. The quality scores displayed a range from 4 to 10, however generally good quality score of eight was more frequent (39. 47%). The highest quality scores of 10 and 9 were observed in 22.36% and 19.74% of genotypes respectively. The UPGMA analysis grouped genotypes into three major with two additional sub clusters for each. The cluster 'a' 'b' and 'C' were separated at 73% genetic distance which was further differentiated at a genetic distance of 50% into their sub clusters. Pakistani wheat varieties/land races exhibited large variation in term of HMW-GS. The generated information will lead to the pyrimiding of sub units for high BQ through mission oriented marker assisted breeding programmes for quality improvement of wheat. (author)

  6. Evaluation of Pakistani wheat germplasm for bread quality based on allelic variation in HMW glutenin subunits

    International Nuclear Information System (INIS)

    Tabasum, A.; Iqbal, N.; Hameed, A.; Arshad, R.

    2011-01-01

    Seventy six Pakistani wheat genotypes including land races were investigated for Bread quality (BQ) based on allelic variation in HMW glutenin subunits at the Glu-1 loci through SDS- polyacrylamide gel electropherosis. Twenty five different allelic combinations were detected with a total of 14 Glu-1 loci. Highest polymorphism was revealed by Glu-B locus and some single/ rare sub units were also screened out. The frequencies of dominant subunits were 50% for 2*, 42.11% for subunit pair 17+18 and 48.68% for 5+10 and 2+12 respectively. The quality scores displayed a range from 4 to 10, however generally good quality score of eight was more frequent (39. 47%). The highest quality scores of 10 and 9 were observed in 22.36% and 19.74% of genotypes respectively. The UPGMA analysis grouped genotypes into three major with two additional sub clusters for each. The cluster 'a' 'b' and 'C' were separated at 73% genetic distance which was further differentiated at a genetic distance of 50% into their sub clusters. Pakistani wheat varieties/land races exhibited large variation in term of HMW-GS. The generated information will lead to the pyrimiding of sub units for high BQ through mission oriented marker assisted breeding programmes for quality improvement of wheat. (author)

  7. Isolation and characterization of a monoclonal anti-protein kinase CK2 beta-subunit antibody of the IgG class for the direct detection of CK2 beta-subunit in tissue cultures of various mammalian species and human tumors

    DEFF Research Database (Denmark)

    Nastainczyk, W; Schmidt-Spaniol, I; Boldyreff, B

    1995-01-01

    A murine monoclonal anti-protein kinase CK2 beta antibody was isolated and characterized. The antibody detects 1 pmol of purified recombinant CK2 beta-subunit after analysis on SDS-PAGE. Alternatively undenatured CK2 beta-subunit was detected by an ELISA assay either as recombinant CK2 beta......-subunit or in the CK2 holoenzyme (alpha 2 beta 2). Here, concentrations of the first antibody of 1 ng/ml still allowed the detection of the subunit. Immunoblotting of crude cellular extracts from various tissue cultures (man, mouse, and hamster), from human tumors, and the nonneoplastic tissue allowed the detection...... of the CK2 beta-subunit. The detected epitope of this antibody was, as determined by the epitope analysis technique, 123GLSDI127....

  8. Molecular analysis of the interaction between the intracellular loops of the human serotonin receptor type 6 (5-HT6) and the α subunit of GS protein

    International Nuclear Information System (INIS)

    Kang, Hatan; Lee, Won Kyu; Choi, Yun Hui; Vukoti, Krishna Moorthy; Bang, Won Gi; Yu, Yeon Gyu

    2005-01-01

    The serotonin type 6 (5-HT 6 ) receptor is a G-protein coupled receptor (GPCR) coupled to a stimulatory G-protein (G S ). To identify the structural basis for the interaction of the 5-HT 6 receptor with the G S protein, we have dissected the interaction between GST-fusion proteins containing the second intracellular loop (iL2), the third intracellular loop (iL3), or the C-terminal tail of the 5-HT 6 receptor and the α subunit of G S (Gα S ). The direct interaction of iL3 and Gα S was demonstrated by co-immunoprecipitation. Furthermore, the kinetic parameters of the interaction between iL3 and Gα S were measured by surface plasmon resonance, and the apparent dissociation constant was determined to be 0.9 x 10 -6 M. In contrast, the second intracellular loop and C-terminal tail regions showed negligible affinity to Gα S . The critical residues within the iL3 region for the interaction with Gα S were identified as conserved positively charged residues near the C-terminus of iL3 by measuring the cellular levels of cAMP produced in response to 5-HT stimulation of cells transfected with 5-HT 6 receptor mutants

  9. The crystal structure of the complex of Zea mays alpha subunit with a fragment of human beta subunit provides the clue to the architecture of protein kinase CK2 holoenzyme

    DEFF Research Database (Denmark)

    Battistutta, R; Sarno, S; De Moliner, E

    2000-01-01

    The crystal structure of a complex between the catalytic alpha subunit of Zea mays CK2 and a 23-mer peptide corresponding the C-terminal sequence 181-203 of the human CK2 regulatory beta subunit has been determined at 3.16-A resolution. The complex, composed of two alpha chains and two peptides, ...

  10. Proteomic and immunoproteomic characterization of a DIVA subunit vaccine against Actinobacillus pleuropneumoniae

    Directory of Open Access Journals (Sweden)

    Maas Alexander

    2011-04-01

    Full Text Available Abstract Background Protection of pigs by vaccination against Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, is hampered by the presence of 15 different serotypes. A DIVA subunit vaccine comprised of detergent-released proteins from A. pleuropneumoniae serotypes 1, 2 and 5 has been developed and shown to protect pigs from clinical symptoms upon homologous and heterologous challenge. This vaccine has not been characterized in-depth so far. Thus we performed i mass spectrometry in order to identify the exact protein content of the vaccine and ii cross-serotype 2-D immunoblotting in order to discover cross-reactive antigens. By these approaches we expected to gain results enabling us to argue about the reasons for the efficacy of the analyzed vaccine. Results We identified 75 different proteins in the vaccine. Using the PSORTb algorithm these proteins were classified according to their cellular localization. Highly enriched proteins are outer membrane-associated lipoproteins like OmlA and TbpB, integral outer membrane proteins like FrpB, TbpA, OmpA1, OmpA2, HgbA and OmpP2, and secreted Apx toxins. The subunit vaccine also contained large amounts of the ApxIVA toxin so far thought to be expressed only during infection. Applying two-dimensional difference gel electrophoresis (2-D DIGE we showed different isoforms and variations in expression levels of several proteins among the strains used for vaccine production. For detection of cross-reactive antigens we used detergent released proteins of serotype 7. Sera of pigs vaccinated with the detergent-released proteins of serotypes 1, 2, and 5 detected seven different proteins of serotype 7, and convalescent sera of pigs surviving experimental infection with serotype 7 reacted with 13 different proteins of the detergent-released proteins of A. pleuropneumoniae serotypes 1, 2, and 5. Conclusions A detergent extraction-based subunit vaccine of A. pleuropneumoniae was

  11. Translation activity of chimeric ribosomes composed of Escherichia coli and Bacillus subtilis or Geobacillus stearothermophilus subunits

    Directory of Open Access Journals (Sweden)

    Sayaka Tsuji

    2017-07-01

    Full Text Available Ribosome composition, consisting of rRNA and ribosomal proteins, is highly conserved among a broad range of organisms. However, biochemical studies focusing on ribosomal subunit exchangeability between organisms remain limited. In this study, we show that chimeric ribosomes, composed of Escherichia coli and Bacillus subtilis or E. coli and Geobacillus stearothermophilus subunits, are active for β-galactosidase translation in a highly purified E. coli translation system. Activities of the chimeric ribosomes showed only a modest decrease when using E. coli 30 S subunits, indicating functional conservation of the 50 S subunit between these bacterial species.

  12. Role of the beta subunit of casein kinase-2 on the stability and specificity of the recombinant reconstituted holoenzyme

    DEFF Research Database (Denmark)

    Meggio, F; Boldyreff, B; Marin, O

    1992-01-01

    Recombinant human alpha subunit from casein kinase-2 (CK-2) was subjected, either alone or in combination with recombinant human beta subunit, to high temperature, tryptic digestion and urea treatment. In all three cases, it was shown that the presence of the beta subunit could drastically reduce...... the autophosphorylation site. It is suggested that the acidic domain of the beta subunit, encompassing residues 55-71, plays a role in the interactions between the beta and alpha subunits....

  13. Identification and Characterization of Putative Integron-Like Elements of the Heavy-Metal-Hypertolerant Strains of Pseudomonas spp.

    Science.gov (United States)

    Ciok, Anna; Adamczuk, Marcin; Bartosik, Dariusz; Dziewit, Lukasz

    2016-11-28

    Pseudomonas strains isolated from the heavily contaminated Lubin copper mine and Zelazny Most post-flotation waste reservoir in Poland were screened for the presence of integrons. This analysis revealed that two strains carried homologous DNA regions composed of a gene encoding a DNA_BRE_C domain-containing tyrosine recombinase (with no significant sequence similarity to other integrases of integrons) plus a three-component array of putative integron gene cassettes. The predicted gene cassettes encode three putative polypeptides with homology to (i) transmembrane proteins, (ii) GCN5 family acetyltransferases, and (iii) hypothetical proteins of unknown function (homologous proteins are encoded by the gene cassettes of several class 1 integrons). Comparative sequence analyses identified three structural variants of these novel integron-like elements within the sequenced bacterial genomes. Analysis of their distribution revealed that they are found exclusively in strains of the genus Pseudomonas .

  14. AcEST: DK959286 [AcEST

    Lifescience Database Archive (English)

    Full Text Available 80_9HYPO Coatamer subunit protein OS=Epichloe festucae Align length 57 Score (bit...J80|B2CJ80_9HYPO Coatamer subunit protein OS=Epichloe fest... 34 3.2 tr|B3SAW3|B3...SAW3_TRIAD Putative uncharacterized protein OS=Trich... 33 7.2 >tr|B2CJ80|B2CJ80_9HYPO Coatamer subunit protein OS=Epichloe

  15. ASIC subunit ratio and differential surface trafficking in the brain.

    Science.gov (United States)

    Wu, Junjun; Xu, Yuanyuan; Jiang, Yu-Qing; Xu, Jiangping; Hu, Youjia; Zha, Xiang-ming

    2016-01-08

    Acid-sensing ion channels (ASICs) are key mediators of acidosis-induced responses in neurons. However, little is known about the relative abundance of different ASIC subunits in the brain. Such data are fundamental for interpreting the relative contribution of ASIC1a homomers and 1a/2 heteromers to acid signaling, and essential for designing therapeutic interventions to target these channels. We used a simple biochemical approach and semi-quantitatively determined the molar ratio of ASIC1a and 2 subunits in mouse brain. Further, we investigated differential surface trafficking of ASIC1a, ASIC2a, and ASIC2b. ASIC1a subunits outnumber the sum of ASIC2a and ASIC2b. There is a region-specific variation in ASIC2a and 2b expression, with cerebellum and striatum expressing predominantly 2b and 2a, respectively. Further, we performed surface biotinylation and found that surface ASIC1a and ASIC2a ratio correlates with their total expression. In contrast, ASIC2b exhibits little surface presence in the brain. This result is consistent with increased co-localization of ASIC2b with an ER marker in 3T3 cells. Our data are the first semi-quantitative determination of relative subunit ratio of various ASICs in the brain. The differential surface trafficking of ASICs suggests that the main functional ASICs in the brain are ASIC1a homomers and 1a/2a heteromers. This finding provides important insights into the relative contribution of various ASIC complexes to acid signaling in neurons.

  16. Potential of Cationic Liposomes as Adjuvants/Delivery Systems for Tuberculosis Subunit Vaccines.

    Science.gov (United States)

    Khademi, Farzad; Taheri, Ramezan Ali; Momtazi-Borojeni, Amir Abbas; Farnoosh, Gholamreza; Johnston, Thomas P; Sahebkar, Amirhossein

    2018-04-27

    The weakness of the BCG vaccine and its highly variable protective efficacy in controlling tuberculosis (TB) in different age groups as well as in different geographic areas has led to intense efforts towards the development and design of novel vaccines. Currently, there are several strategies to develop novel TB vaccines. Each strategy has its advantages and disadvantages. However, the most important of these strategies is the development of subunit vaccines. In recent years, the use of cationic liposome-based vaccines has been considered due to their capacity to elicit strong humoral and cellular immune responses against TB infections. In this review, we aim to evaluate the potential for cationic liposomes to be used as adjuvants/delivery systems for eliciting immune responses against TB subunit vaccines. The present review shows that cationic liposomes have extensive applications either as adjuvants or delivery systems, to promote immune responses against Mycobacterium tuberculosis (Mtb) subunit vaccines. To overcome several limitations of these particles, they were used in combination with other immunostimulatory factors such as TDB, MPL, TDM, and Poly I:C. Cationic liposomes can provide long-term storage of subunit TB vaccines at the injection site, confer strong electrostatic interactions with APCs, potentiate both humoral and cellular (CD4 and CD8) immune responses, and induce a strong memory response by the immune system. Therefore, cationic liposomes can increase the potential of different TB subunit vaccines by serving as adjuvants/delivery systems. These properties suggest the use of cationic liposomes to produce an efficient vaccine against TB infections.

  17. In Search of Small Molecule Inhibitors Targeting the Flexible CK2 Subunit Interface

    Directory of Open Access Journals (Sweden)

    Benoît Bestgen

    2017-02-01

    Full Text Available Protein kinase CK2 is a tetrameric holoenzyme composed of two catalytic (α and/or α’ subunits and two regulatory (β subunits. Crystallographic data paired with fluorescence imaging techniques have suggested that the formation of the CK2 holoenzyme complex within cells is a dynamic process. Although the monomeric CK2α subunit is endowed with a constitutive catalytic activity, many of the plethora of CK2 substrates are exclusively phosphorylated by the CK2 holoenzyme. This means that the spatial and high affinity interaction between CK2α and CK2β subunits is critically important and that its disruption may provide a powerful and selective way to block the phosphorylation of substrates requiring the presence of CK2β. In search of compounds inhibiting this critical protein–protein interaction, we previously designed an active cyclic peptide (Pc derived from the CK2β carboxy-terminal domain that can efficiently antagonize the CK2 subunit interaction. To understand the functional significance of this interaction, we generated cell-permeable versions of Pc, exploring its molecular mechanisms of action and the perturbations of the signaling pathways that it induces in intact cells. The identification of small molecules inhibitors of this critical interaction may represent the first-choice approach to manipulate CK2 in an unconventional way.

  18. Identification of novel transcriptional regulators of PKA subunits in Saccharomyces cerevisiae by quantitative promoter-reporter screening.

    Science.gov (United States)

    Pautasso, Constanza; Reca, Sol; Chatfield-Reed, Kate; Chua, Gordon; Galello, Fiorella; Portela, Paula; Zaremberg, Vanina; Rossi, Silvia

    2016-08-01

    The cAMP-dependent protein kinase (PKA) signaling is a broad pathway that plays important roles in the transduction of environmental signals triggering precise physiological responses. However, how PKA achieves the cAMP-signal transduction specificity is still in study. The regulation of expression of subunits of PKA should contribute to the signal specificity. Saccharomyces cerevisiae PKA holoenzyme contains two catalytic subunits encoded by TPK1, TPK2 and TPK3 genes, and two regulatory subunits encoded by BCY1 gene. We studied the activity of these gene promoters using a fluorescent reporter synthetic genetic array screen, with the goal of systematically identifying novel regulators of expression of PKA subunits. Gene ontology analysis of the identified modulators showed enrichment not only in the category of transcriptional regulators, but also in less expected categories such as lipid and phosphate metabolism. Inositol, choline and phosphate were identified as novel upstream signals that regulate transcription of PKA subunit genes. The results support the role of transcription regulation of PKA subunits in cAMP specificity signaling. Interestingly, known targets of PKA phosphorylation are associated with the identified pathways opening the possibility of a reciprocal regulation. PKA would be coordinating different metabolic pathways and these processes would in turn regulate expression of the kinase subunits. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  19. Automated Image Sampling and Classification Can Be Used to Explore Perceived Naturalness of Urban Spaces.

    Directory of Open Access Journals (Sweden)

    Roger Hyam

    Full Text Available The psychological restorative effects of exposure to nature are well established and extend to just viewing of images of nature. A previous study has shown that Perceived Naturalness (PN of images correlates with their restorative value. This study tests whether it is possible to detect degree of PN of images using an image classifier. It takes images that have been scored by humans for PN (including a subset that have been assessed for restorative value and passes them through the Google Vision API image classification service. The resulting labels are assigned to broad semantic classes to create a Calculated Semantic Naturalness (CSN metric for each image. It was found that CSN correlates with PN. CSN was then calculated for a geospatial sampling of Google Street View images across the city of Edinburgh. CSN was found to correlate with PN in this sample also indicating the technique may be useful in large scale studies. Because CSN correlates with PN which correlates with restorativeness it is suggested that CSN or a similar measure may be useful in automatically detecting restorative images and locations. In an exploratory aside CSN was not found to correlate with an indicator of socioeconomic deprivation.

  20. Integrins beta 5, beta 3 and alpha v are apically distributed in endometrial epithelium.

    Science.gov (United States)

    Aplin, J D; Spanswick, C; Behzad, F; Kimber, S J; Vićovac, L

    1996-07-01

    Several adhesion molecules have been shown to occur at the surface of endometrial cells. One of these is the integrin alpha v subunit which associates with various beta chains including beta 5. We demonstrate the presence of integrin beta 5 polypeptide in human endometrial epithelial cells throughout the menstrual cycle using immunocytochemistry with monospecific antibodies, and at the mRNA level by thermal amplification from endometrial cDNA. Integrin beta 5 is also found in a population of bone marrow-derived cells. A notable feature of the distribution of the beta 5 subunit in the glandular and luminal epithelium is its apical localization, which may suggest an involvement in implantation. However, no evidence was found for regulated expression of epithelial beta 5. In mouse, the beta 5 subunit is found at both the apical and basal surface of epithelial cells and expression is essentially oestrous cycle-independent. Comparisons are made in both species with the distribution of the alpha v and beta 3 subunits which also localize to the apical epithelium.

  1. Protein kinase A regulatory subunit distribution in medulloblastoma

    International Nuclear Information System (INIS)

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

    2010-01-01

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

  2. Putative Lineage of Novel African Usutu Virus, Central Europe

    Centers for Disease Control (CDC) Podcasts

    2015-10-15

    Sarah Gregory reads an abridged version of "Putative Lineage of Novel African Usutu Virus, Central Europe.".  Created: 10/15/2015 by National Center for Emerging and Zoonotic Infectious Diseases (NCEZID).   Date Released: 10/15/2015.

  3. Schedule of NMDA receptor subunit expression and functional channel formation in the course of in vitro-induced neurogenesis.

    Science.gov (United States)

    Varju, P; Schlett, K; Eisel, U; Madarász, E

    2001-06-01

    NE-7C2 neuroectodermal cells derived from forebrain vesicles of p53-deficient mouse embryos (E9) produce neurons and astrocytes in vitro if induced by all-trans retinoic acid. The reproducible morphological stages of neurogenesis were correlated with the expression of various NMDA receptor subunits. RT-PCR studies revealed that GluRepsilon1 and GluRepsilon4 subunit mRNAs were transcribed by both non-induced and neuronally differentiated cells. GluRepsilon3 subunit mRNAs were not synthesized by NE-7C2 cells and increased numbers of messages from the GluRepsilon2 gene were detected only after neural network formation. The presence of the GluRzeta1 protein was detected throughout neural induction, whereas retinoic acid-induced neuron formation elevated the amount of exon 21 (C1)- and exon 22 (C2)-containing GluRzeta1 mRNAs and resulted in the appearance of exon 5 (N1)-containing transcripts. NMDA-elicited Ca(2+)-signals were detected only in cells displaying neuronal morphology, but preceding the appearance of synapsin-I immunoreactivity. Our findings demonstrated that, in spite of the presence of subunits necessary for channel formation, functional channels were formed by NE-7C2 cells no sooner than the time of neurite maturation. The data show that the cell line provides a suitable model to analyse the mechanisms involved in NMDA receptor gene expression before the appearance of synaptic communication.

  4. [Three regions of Rpb10 mini-subunit of nuclear RNA polymerases are strictly conserved in all eukaryotes].

    Science.gov (United States)

    Shpakovskiĭ, G V; Lebedenko, E N

    1996-12-01

    The rpb10+ cDNA from the fission yeast Schizosaccharomyces pombe was cloned using two independent approaches (PCR and genetic suppression). The cloned cDNA encoded the Rpb10 subunit common for all three RNA polymerases. Comparison of the deduced amino acid sequence of the Sz. pombe Rbp10 subunit (71 amino acid residues) with those of the homologous subunits of RNA polymerases I, II, and III from Saccharomyces cerevisiae and Home sapiens revealed that heptapeptides RCFT/SCGK (residues 6-12), RYCCRRM (residues 43-49), and HVDLIEK (residues 53-59) were evolutionarily the most conserved structural motifs of these subunits. It is shown that the Rbp10 subunit from Sz. pombe can substitute its homolog (ABC10 beta) in the baker's yeast S. cerevisiae.

  5. Characterisation by nuclear magnetic resonance of the β catalytic subunit of the chloroplastic coupling factor

    International Nuclear Information System (INIS)

    Andre, Francois

    1986-09-01

    This academic work addressed the use of nuclear magnetic resonance (NMR) for the structural and dynamic study of the catalytic sub-unit of the extrinsic section of a membrane complex, the chloroplastic H+-ATPase. This work included the development of a protocol of preparation and quantitative purification of β subunits isolated from the CF1 for the elaboration of a concentrated sample for NMR, and then the study of the β subunit by using proton NMR

  6. Molecular cloning and characterization of a putative OGG_N domain ...

    African Journals Online (AJOL)

    Molecular cloning and characterization of a putative OGG_N domain from the camel, Camelus dromedarius. Farid Shokry Ataya, Mohammad Saud Alanazi, Dalia Fouad, Hehsam Mahmoud Saeed, Mohammad Bazzi ...

  7. Dicty_cDB: CHE174 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available stallinum mRNA for putative vacuolar ATPase subunit 100 kDa subunit (vpha-a gene). 46 1e-10 4 CV164410 |CV164410.1 rsmsx...54 8e-09 3 CV172465 |CV172465.1 rsmsxlre_008717.z1.scf cDNA Library of Salvia mil

  8. Bioinformatic analyses of kappa casein gene in mammalian ...

    African Journals Online (AJOL)

    Using a comparative genomic approach, we obtained 1797 bp of the CSN3 sequences from cattle, goat, horse, pig, rabbit and sheep. ... observed in phylogenetic tree of CSN3 gene which showed that the comparability of CSN3 gene sequences was highest between the goat and sheep and they evolved from a most recent ...

  9. Highly diverged novel subunit composition of apicomplexan F-type ATP synthase identified from Toxoplasma gondii

    KAUST Repository

    Salunke, Rahul

    2018-05-14

    The mitochondrial F-type ATP synthase, a multi-subunit nanomotor, is critical for maintaining cellular ATP levels. In Toxoplasma gondii and other apicomplexan parasites, many subunit components, necessary for proper assembly and functioning of this enzyme, appear to be missing. Here, we report the identification of 20 novel subunits of T. gondii F-type ATP synthase from mass spectrometry analysis of partially purified monomer (~600 kDa) and dimer (>1 MDa) forms of the enzyme. Despite extreme sequence diversification, key FO subunits, a, b and d, can be identified from conserved structural features. Orthologs for these proteins are restricted to apicomplexan, chromerid and dinoflagellate species. Interestingly, their absence in ciliates indicates a major diversion, with respect to subunit composition of this enzyme, within the alveolate clade. Discovery of these highly diversified novel components of the apicomplexan F-type ATP synthase complex will facilitate the development of novel anti-parasitic agents. Structural and functional characterization of this unusual enzyme complex will advance our fundamental understanding of energy metabolism in apicomplexan species.

  10. Highly diverged novel subunit composition of apicomplexan F-type ATP synthase identified from Toxoplasma gondii

    KAUST Repository

    Salunke, Rahul; Mourier, Tobias; Banerjee, Manidipa; Pain, Arnab; Shanmugam, Dhanasekaran

    2018-01-01

    The mitochondrial F-type ATP synthase, a multi-subunit nanomotor, is critical for maintaining cellular ATP levels. In Toxoplasma gondii and other apicomplexan parasites, many subunit components, necessary for proper assembly and functioning of this enzyme, appear to be missing. Here, we report the identification of 20 novel subunits of T. gondii F-type ATP synthase from mass spectrometry analysis of partially purified monomer (~600 kDa) and dimer (>1 MDa) forms of the enzyme. Despite extreme sequence diversification, key FO subunits, a, b and d, can be identified from conserved structural features. Orthologs for these proteins are restricted to apicomplexan, chromerid and dinoflagellate species. Interestingly, their absence in ciliates indicates a major diversion, with respect to subunit composition of this enzyme, within the alveolate clade. Discovery of these highly diversified novel components of the apicomplexan F-type ATP synthase complex will facilitate the development of novel anti-parasitic agents. Structural and functional characterization of this unusual enzyme complex will advance our fundamental understanding of energy metabolism in apicomplexan species.

  11. Molecular investigations of BK(Ca) channels and the modulatory beta-subunits in porcine basilar and middle cerebral arteries

    DEFF Research Database (Denmark)

    Johansson, Helle Wulf; Hay-Schmidt, Anders; Poulsen, Asser Nyander

    2009-01-01

    arteries using reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR. Western blotting was used to detect immunoreactivity for the porcine BK(Ca) channel alpha-subunit and beta-subunit proteins. The BK(Ca) channel alpha-subunit RNA and protein distribution patterns were......Large conductance calcium-activated potassium (BK(Ca)) channels are fundamental in the regulation of cerebral vascular basal tone. We investigated the expression of the mRNA transcripts for the BK(Ca) channel and its modulatory beta-subunits (beta1-beta4) in porcine basilar and middle cerebral...... visualized using in situ hybridization and immunofluorescence studies, respectively. The study verified that the BK(Ca) channel alpha-subunit is located to smooth muscle cells of porcine basilar and middle cerebral arteries. The mRNA transcript for beta1-, beta2- and beta4-subunit were shown by RT...

  12. Nicotinic acetylcholine receptor: subunit structure, functional binding sites, and ion transport properties

    International Nuclear Information System (INIS)

    Raftery, M.A.; Dunn, S.M.J.; Conti-Tronconi, B.M.; Middlemas, D.S.; Crawford, R.D.

    1983-01-01

    The structure of the nicotinic acetylcholine receptor has been highly conserved during animal evolution, and in all the species and tissues studied so far, including mammals, it is a pseudosymmetric, pentameric complex of related subunits with very similar physical properties. All subunits of these nicotinic receptors were derived from a common ancestral gene, probably by way of gene duplications occurring very early in animal evolution. 45 refs., 8 figs., 2 tabs

  13. Knowledge and behavior of Nigerian dentists concerning the treatment of children with special needs

    Directory of Open Access Journals (Sweden)

    Sanu Oluwatosin O

    2006-06-01

    Full Text Available Abstract Background Children with special needs (CSN are reported to receive less adequate dental care due to various behavioral problems and barriers created by dental professionals. This study was carried out to determine the knowledge and behaviour of Nigerian dentists concerning the treatment of CSN. Methods Questionnaires consisting of open and closed ended questions requesting socio-demographic information, type of practice, undergraduate and postgraduate training, self-rated knowledge and behaviour concerning care of CSN, were hand delivered to 359 dentists in the 3 geographical zones of Nigeria over a period of 8 weeks. Responses were compared across age groups, gender, type of practice and training received. Result Two hundred and eighty questionnaires were returned completed, constituting 79.9% response rate. Most of the respondents were aged 30 – 39 years (44.3%. There were more males (56.1% and more recent graduates of 10 years and below (78.5%. Over 80% of respondents had treated children with disabilities, those with physical disabilities being most encountered. Only 19.3% of respondents rated their knowledge of management of CSN as adequate, with no significant difference across age groups and gender, but with a significantly higher number of older graduates reporting to have adequate knowledge (p Conclusion From this study, very few dentists reported to have adequate knowledge of management of CSN, irrespective of age, gender and place of practice. A significant number of those with more experience rated their knowledge as adequate. Although most dentists rated the children's behaviour as challenging, they indicated their willingness to treat them in their practices.

  14. Anxiogenic properties of an inverse agonist selective for α3 subunit-containing GABAA receptors

    OpenAIRE

    Atack, John R; Hutson, Peter H; Collinson, Neil; Marshall, George; Bentley, Graham; Moyes, Christopher; Cook, Susan M; Collins, Ian; Wafford, Keith; McKernan, Ruth M; Dawson, Gerard R

    2005-01-01

    α3IA (6-(4-pyridyl)-5-(4-methoxyphenyl)-3-carbomethoxy-1-methyl-1H-pyridin-2-one) is a pyridone with higher binding and functional affinity and greater inverse agonist efficacy for GABAA receptors containing an α3 rather than an α1, α2 or α5 subunit. If doses are selected that minimise the occupancy at these latter subtypes, then the in vivo effects of α3IA are most probably mediated by the α3 subtype.α3IA has good CNS penetration in rats and mice as measured using a [3H]Ro 15-1788 in vivo bi...

  15. The Putative Chemosignal Androstadienone Makes Women More Generous.

    Science.gov (United States)

    Perrotta, Valentina; Graffeo, Michele; Bonini, Nicolao; Gottfried, Jay A

    2016-06-01

    Putative human chemosignals have been shown to influence mood states and emotional processing, but the connection between these effects and higher-order cognitive processing is not well established. This study utilized an economic game (Dictator Game) to test whether androstadienone (AND), an odorous compound derived from testosterone, impacts on altruistic behavior. We predicted that the female participants would act more generously in the AND condition, exhibiting a significant interaction effect between gender and AND on Dictator Game contributions. We also expected that the presence of AND should increase the positive mood of the female participants, compared to a control odor condition and also compared to the mood of the male participants. The results confirm our hypotheses: for women the subliminal perception of AND led to larger monetary donations, compared to a control odor, and also increased positive mood. These effects were absent or significantly weaker in men. Our findings highlight the capacity of human putative chemosignals to influence emotions and higher cognitive processes - in particular the processes used in the context of economic decisions - in a gender-specific way.

  16. Expression and Trafficking of the γ Subunit of Na,K-ATPase in Hypertonically Challenged IMCD3 Cells

    International Nuclear Information System (INIS)

    Pihakaski-Maunsbach, Kaarina; Nonaka, Shoichi; Maunsbach, Arvid B.

    2008-01-01

    The γ subunit (FXYD2) of Na,K-ATPase is an important regulator of the sodium pump. In this investigation we have analysed the trafficking of γ to the plasma membrane in cultures of inner medullary collecting duct cells (IMCD3) following acute hypertonic challenge and brefeldin A (BFA) treatment. Following hypertonic challenging for 24 hr immunofluorescence labeling revealed initial co-localization of the γ subunit and 58K Golgi protein in the cytoplasm, but no co-localization of α1 and Golgi protein. Exposure of the challenged cells to BFA prevented the subsequent incorporation of γ into the basolateral plasma membrane. The γ subunit instead remained in cytoplasmic vesicles while cell proliferation and cell viability decreased simultaneously. Following removal of BFA from the hypertonic medium the IMCD3 cells recovered with distinct expression of γ in the basolateral membrane. The α1 subunit was only marginally influenced by BFA. The results demonstrate that the γ subunit trafficks to the plasma membrane via the Golgi apparatus, despite the absence of a signal sequence. The results also suggest that the γ and α subunits do not traffic together to the plasma membrane, and that the γ and α subunit have different turnover rates during these experimental conditions

  17. Subunit vaccine candidates against Aeromonas salmonicida in rainbow trout Oncorhynchus mykiss

    DEFF Research Database (Denmark)

    Marana, Moonika Haahr; Jørgensen, Louise von Gersdorff; Skov, Jakob

    2017-01-01

    rainbow trout (Oncorhynchus mykiss, Walbaum) aquaculture furunculosis outbreaks still occur. In this study we tested the efficacy of experimental subunit vaccines against A. salmonicida infection in rainbow trout. We utilized in silico screening of the proteome of A. salmonicida subsp. salmonicida strain...... A449 and identified potential protective protein antigens that were tested by in vivo challenge trial. A total of 14 proteins were recombinantly expressed in Escherichia coli and prepared in 3 different subunit vaccine combinations to immunize 3 groups of rainbow trout by intraperitoneal (i...

  18. Ligand binding and conformational changes of SUR1 subunit in pancreatic ATP-sensitive potassium channels.

    Science.gov (United States)

    Wu, Jing-Xiang; Ding, Dian; Wang, Mengmeng; Kang, Yunlu; Zeng, Xin; Chen, Lei

    2018-06-01

    ATP-sensitive potassium channels (K ATP ) are energy sensors on the plasma membrane. By sensing the intracellular ADP/ATP ratio of β-cells, pancreatic K ATP channels control insulin release and regulate metabolism at the whole body level. They are implicated in many metabolic disorders and diseases and are therefore important drug targets. Here, we present three structures of pancreatic K ATP channels solved by cryo-electron microscopy (cryo-EM), at resolutions ranging from 4.1 to 4.5 Å. These structures depict the binding site of the antidiabetic drug glibenclamide, indicate how Kir6.2 (inward-rectifying potassium channel 6.2) N-terminus participates in the coupling between the peripheral SUR1 (sulfonylurea receptor 1) subunit and the central Kir6.2 channel, reveal the binding mode of activating nucleotides, and suggest the mechanism of how Mg-ADP binding on nucleotide binding domains (NBDs) drives a conformational change of the SUR1 subunit.

  19. Genetic analysis of heptad-repeat regions in the G2 fusion subunit of the Junin arenavirus envelope glycoprotein

    International Nuclear Information System (INIS)

    York, Joanne; Agnihothram, Sudhakar S.; Romanowski, Victor; Nunberg, Jack H.

    2005-01-01

    The G2 fusion subunit of the Junin virus envelope glycoprotein GP-C contains two hydrophobic heptad-repeat regions that are postulated to form a six-helix bundle structure required for the membrane fusion activity of Class I viral fusion proteins. We have investigated the role of these heptad-repeat regions and, specifically, the importance of the putative interhelical a and d position sidechains by using alanine-scanning mutagenesis. All the mutant glycoproteins were expressed and transported to the cell surface. Proteolytic maturation at the subtilisin kexin isozyme-1/site-1-protease (SKI-1/S1P) cleavage site was observed in all but two of the mutants. Among the adequately cleaved mutant glycoproteins, four positions in the N-terminal region (I333, L336, L347 and L350) and two positions in the C-terminal region (R392 and W395) were shown to be important determinants of cell-cell fusion. Taken together, our results indicate that α-helical coiled-coil structures are likely critical in promoting arenavirus membrane fusion. These findings support the inclusion of the arenavirus GP-C among the Class I viral fusion proteins and suggest pharmacologic and immunologic strategies for targeting arenavirus infection and hemorrhagic fever

  20. Phytochrome control of gene expression in radish seedlings. 111. Evidence for a rapid control of the ribulose 1. 5 biphosphate carboxylase small subunit gene expression by red light

    Energy Technology Data Exchange (ETDEWEB)

    Fourcroy, P

    1986-01-01

    The effect of red and far-red light on the level of the mRNA encoding the small subunit (SSU) of ribulose, 1.5 bisphosphate carboxylase (RuBisCO; EC 4.1.1.39) from radish cotyledons was investigated. Northern blot analysis of RNA with a cDNA probe showed that both long (12-36h) far-red irradiation and short (1-5 min) red irradiation brings about an increase in SSU mRNA concentraton which was prevented by a subsequent far-red light exposure. Far-red light was effective in reversing the red light effect provided that it was given soon after (<10 min) the red light pulse. The red light mediated increase in SSU mRNA level did not occur in presence of ..cap alpha..-amanitin. Our results suggest that phytochrome control of SSU gene expression is exerted at the transcriptional level. 34 refs.