Sample records for ii alpha subunit

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

    DEFF Research Database (Denmark)

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


    cDNA encoding the casein kinase II (CKII) subunits alpha and beta of human origin were expressed in Escherichia coli using expression vector pT7-7. Significant expression was obtained with E. coli BL21(DE3). The CKII subunits accounted for approximately 30% of the bacterial protein; however, most...

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

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


    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.

  3. MspI and PvuII polymorphisms in the Na,K-ATPase. alpha. subunit related gene ATP1AL1

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    Shull, M.M.; Pugh, D.G.; Lingrel, J.B. (Univ. of Cincinnati, OH (USA))


    ATP1AL1 78-1-3 is a 0.56 kb genomic EcoRI-XbaI fragment from within the Na,K-ATPase {alpha} subunit related gene, previously referred to as {alpha}D on chromosome 13. The fragment was subcloned into pIBI31. MspI identifies a two-allele polymorphism (M1: 2.8 kb, M2: 2.5 kb). PvuII, which cuts within the probe sequence, detects two two-allele polymorphism (A1: 6.0 kb, A2: 5.7 kb, B1: 1.3 kb, B2: 1.1 kb). A1 and A2 appear to result from an insertion/deletion polymorphism that is also identified by MspI. ATP1AL1 78-1-3 has been assigned to chromosome 13q by somatic cell hybrid analysis. Codominant segregation of the RELPs was observed in 2 two-generation families.

  4. Primary structure of the. cap alpha. -subunit of Na/sup +/, K/sup +/-ATPase. II. Isolation, reverse transcription, and cloning of messenger RNA

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    Petrukhin, K.E.; Broude, N.E.; Arsenyan, S.G.; Grishin, A.V.; Dzhandzhugazyan, K.N.; Modyanov, N.N.


    The messenger RNA coding the ..cap alpha..-subunit of Na/sup +/,K/sup +/-ATPase has been isolated from the outer medullary layer of porcine kidneys. The mRNA gives a specific hybridization band in the 25S-26S region with three oligonucleotide probes synthesized on the basis of information on the structure of three peptides isolated from a tryptic hydrolyzate of the ..cap alpha..-subunit of Na/sup +/,K/sup +/-ATPase. The translation of the mRNA in Xenopus laevis oocytes followed by immunochemical identification of the products of synthesis confirmed the presence of the mRNA of the ..cap alpha..-subunit of Na/sup +/,K/sup +/-ATPase in an enriched fraction of poly(A/sup +/)-RNA. This preparation has been used for the synthesis of cloning of double-stranded cDNA.

  5. Determination of hCG-alpha subunit in threatened pregnancy

    International Nuclear Information System (INIS)

    Talas, M.; Pohanka, J.; Fingerova, H.; Janouskova, M.; Krikal, Z.; Prasilova, J.; Zupkova, H.


    Radioimmunoassay of the hCG-alpha subunit was made using an antibody anti hCG-alpha serum, highly purified hCG-alpha for 125 I-labelling and the standard hCG-alpha. Sera of healthy pregnant women sampled throughout the whole pregnancies were used to determine x-bar±S.D. of hCG-alpha for 14-day intervals. Included in the study were groups of women with high risk of premature labor, late toxemia of pregnancy, twins and fetal hypotrophy. It was shown that increased hCG-alpha is found in pregnant women in whom signs of late toxemia of pregnancy are combined with high risk of premature labor, or with twin pregnancies, while in those with fetal hypotrophy hCG-alpha is within normal limits. (author). 3 figs., 7 refs

  6. Determinants of RNA polymerase alpha subunit for interaction with beta, beta', and sigma subunits: hydroxyl-radical protein footprinting.


    Heyduk, T; Heyduk, E; Severinov, K; Tang, H; Ebright, R H


    Escherichia coli RNA polymerase (RNAP) alpha subunit serves as the initiator for RNAP assembly, which proceeds according to the pathway 2 alpha-->alpha 2-->alpha 2 beta-->alpha 2 beta beta'-->alpha 2 beta beta' sigma. In this work, we have used hydroxyl-radical protein footprinting to define determinants of alpha for interaction with beta, beta', and sigma. Our results indicate that amino acids 30-75 of alpha are protected from hydroxyl-radical-mediated proteolysis upon interaction with beta ...

  7. Fragile X Mental Retardation Protein and Dendritic Local Translation of the Alpha Subunit of the Calcium/Calmodulin-Dependent Kinase II Messenger RNA Are Required for the Structural Plasticity Underlying Olfactory Learning. (United States)

    Daroles, Laura; Gribaudo, Simona; Doulazmi, Mohamed; Scotto-Lomassese, Sophie; Dubacq, Caroline; Mandairon, Nathalie; Greer, Charles August; Didier, Anne; Trembleau, Alain; Caillé, Isabelle


    In the adult brain, structural plasticity allowing gain or loss of synapses remodels circuits to support learning. In fragile X syndrome, the absence of fragile X mental retardation protein (FMRP) leads to defects in plasticity and learning deficits. FMRP is a master regulator of local translation but its implication in learning-induced structural plasticity is unknown. Using an olfactory learning task requiring adult-born olfactory bulb neurons and cell-specific ablation of FMRP, we investigated whether learning shapes adult-born neuron morphology during their synaptic integration and its dependence on FMRP. We used alpha subunit of the calcium/calmodulin-dependent kinase II (αCaMKII) mutant mice with altered dendritic localization of αCaMKII messenger RNA, as well as a reporter of αCaMKII local translation to investigate the role of this FMRP messenger RNA target in learning-dependent structural plasticity. Learning induces profound changes in dendritic architecture and spine morphology of adult-born neurons that are prevented by ablation of FMRP in adult-born neurons and rescued by an metabotropic glutamate receptor 5 antagonist. Moreover, dendritically translated αCaMKII is necessary for learning and associated structural modifications and learning triggers an FMRP-dependent increase of αCaMKII dendritic translation in adult-born neurons. Our results strongly suggest that FMRP mediates structural plasticity of olfactory bulb adult-born neurons to support olfactory learning through αCaMKII local translation. This reveals a new role for FMRP-regulated dendritic local translation in learning-induced structural plasticity. This might be of clinical relevance for the understanding of critical periods disruption in autism spectrum disorder patients, among which fragile X syndrome is the primary monogenic cause. Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  8. Cloning and sequencing of the casein kinase 2 alpha subunit from Zea mays

    DEFF Research Database (Denmark)

    Dobrowolska, G; Boldyreff, B; Issinger, O G


    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....

  9. The beta subunit of casein kinase II

    DEFF Research Database (Denmark)

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


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

  10. Reactivation of the chloroplast CF1-ATPase beta subunit by trace amounts of the CF1 alpha subunit suggests a chaperonin-like activity for CF1 alpha. (United States)

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


    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.

  11. Editing modifies the GABA(A) receptor subunit alpha3

    DEFF Research Database (Denmark)

    Ohlson, Johan; Pedersen, Jakob Skou; Haussler, David


    Adenosine to inosine (A-to-I) pre-mRNA editing by the ADAR enzyme family has the potential to increase the variety of the proteome. This editing by adenosine deamination is essential in mammals for a functional brain. To detect novel substrates for A-to-I editing we have used an experimental method...... to find selectively edited sites and combined it with bioinformatic techniques that find stem-loop structures suitable for editing. We present here the first verified editing candidate detected by this screening procedure. We show that Gabra-3, which codes for the alpha3 subunit of the GABA(A) receptor......, is a substrate for editing by both ADAR1 and ADAR2. Editing of the Gabra-3 mRNA recodes an isoleucine to a methionine. The extent of editing is low at birth but increases with age, reaching close to 100% in the adult brain. We therefore propose that editing of the Gabra-3 mRNA is important for normal brain...

  12. Mapping of the mouse actin capping protein {alpha} subunit genes and pseudogenes

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    Hart, M.C.; Korshunova, Y.O.; Cooper, J.A. [Washington Univ. School of Medicine, St. Louis, MO (United States)


    Capping protein (CP), a heterodimer of {alpha} and {beta} subunits, is found in all eukaryotes. CP binds to the barbed ends of actin filaments in vitro and controls actin assembly and cell motility in vivo. Vertebrates have three {alpha} isoforms ({alpha}1, {alpha}2, {alpha}3) produced from different genes, whereas lower organisms have only one gene and one isoform. We isolated genomic clones corresponding to the a subunits of mouse CP and found three {alpha}1 genes, two of which are pseudogenes, and a single gene for both {alpha}2 and {alpha}3. Their chromosomal locations were identified by interspecies backcross mapping. The {alpha}1 gene (Cappa1) mapped to Chromosome 3 between D3Mit11 and D3Mit13. The {alpha}1 pseudogenes (Cappa1-ps1 and Cappa1-ps2) mapped to Chromosomes 1 and 9, respectively. The {alpha}2 gene (Cappa2) mapped to Chromosome 6 near Ptn. The {alpha}3 gene (Cappa3) also mapped to Chromosome 6, approximately 68 cM distal from Cappa2 near Kras2. One mouse mutation, de, maps in the vicinity of the {alpha}1 gene. No known mouse mutations map to regions near the {alpha}2 or {alpha}3 genes. 29 refs., 3 figs., 1 tab.

  13. 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


    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...

  14. 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


    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...

  15. The thermal structural transition of alpha-crystallin modulates subunit interactions and increases protein solubility.

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    Giuseppe Maulucci

    Full Text Available BACKGROUND: Alpha crystallin is an oligomer composed of two types of subunits, alpha-A and alpha-B crystallin, and is the major constituent of human lens. The temperature induced condensation of alpha-crystallin, the main cause for eye lens opacification (cataract, is a two step-process, a nucleation followed by an aggregation phase, and a protective effect towards the aggregation is exhibited over the alpha crystallin phase transition temperature (Tc = 318.16 K. METHODS/RESULTS: To investigate if a modulation of the subunit interactions over Tc could trigger the protective mechanism towards the aggregation, we followed, by using simultaneously static and dynamic light scattering, the temperature induced condensation of alpha-crystallin. By developing a mathematical model able to uncouple the nucleation and aggregation processes, we find a previously unobserved transition in the nucleation rate constant. Its temperature dependence allows to determine fundamental structural parameters, the chemical potential (Δμ and the interfacial tension (γ of the aggregating phase, that characterize subunit interactions. CONCLUSIONS/GENERAL SIGNIFICANCE: The decrease of both Δμ and γ at Tc, and a relative increase in solubility, reveal a significative decrease in the strenght of alpha-crystallin subunits interactions, which protects from supramolecolar condensation in hypertermic conditions. On the whole, we suggest a general approach able to understand the structural and kinetic mechanisms involved in aggregation-related diseases and in drugs development and testing.

  16. The NH2-terminal php domain of the alpha subunit of the Escherichia coli replicase binds the epsilon proofreading subunit. (United States)

    Wieczorek, Anna; McHenry, Charles S


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

  17. Isolation and characterization of a monoclonal anti CK-2 alpha subunit antibody of the IgG1 subclass

    DEFF Research Database (Denmark)

    Schmidt-Spaniol, I; Boldyreff, B; Issinger, O G


    A monoclonal antibody was produced against the recombinant human alpha subunit of CK-2. The antibody was of the IgG1 subclass and it was isolated from serum-free cell culture media and purified by affinity chromatography on Protein G Sepharose. The antibody can be used to detect specifically the CK......-2 alpha subunit in immunoblots from tissue extracts. An ELISA detection test was also established which also allows the identification of the CK-2 alpha subunit....

  18. Analysis of Maxi-K alpha subunit splice variants in human myometrium

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    Morrison John J


    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

  19. 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.


    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

  20. The alpha2-delta protein: an auxiliary subunit of voltage-dependent calcium channels as a recognized drug target. (United States)

    Thorpe, Andrew J; Offord, James


    Currently, there are two drugs on the market, gabapentin (Neurontin) and pregabalin (Lyrica), that are proposed to exert their therapeutic effect through binding to the alpha2-delta subunit of voltage-sensitive calcium channels. This activity was unexpected, as the alpha2-delta subunit had previously been considered not to be a pharmacological target. In this review, the role of the alpha2-delta subunits is discussed and the mechanism of action of the alpha2-delta ligands in vitro and in vivo is summarized. Finally, new insights into the mechanism of drugs that bind to this protein are discussed.

  1. 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.


    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

  2. Characterization and charge distribution of the asparagine-linked oligosaccharides on secreted mouse thyrotropin and free alpha-subunits

    International Nuclear Information System (INIS)

    Gesundheit, N.; Gyves, P.W.; DeCherney, G.S.; Stannard, B.S.; Winston, R.L.; Weintraub, B.D.


    Mouse hemipituitaries in vitro secrete TSH, composed of an alpha-beta heterodimer, as well as excess (free) alpha-subunits. By dual metabolic labeling with [35S]sulfate and [3H]mannose, we have characterized oligosaccharides from secreted TSH alpha, TSH beta, and free alpha-subunits released from the apoprotein by enzymatic deglycosylation. Oligosaccharides from each subunit displayed a distinct anion exchange HPLC profile due to a specific pattern of sialylation and sulfation. Six species were obtained from TSH alpha (with two glycosylation sites), including neutral oligosaccharides as well as those with one or two negative charges. For TSH beta (with one glycosylation site) at least eight oligosaccharide species were noted, representing nearly every permutation of sialylation and sulfation; approximately 30% contained three or more negative charges. Analysis of [3H]mannose-labeled oligosaccharides on Concanavalin-A-agarose showed 85% binding for those from TSH alpha, 70% for free alpha, and 50% for those from TSH beta. These data demonstrate that oligosaccharides from secreted TSH beta were more sialylated and sulfated, consistent with a more complex branching pattern, than those from TSH alpha. Oligosaccharides from free alpha-subunit were more sialylated than those from TSH alpha, and the net negative charge was intermediate between those of TSH alpha and TSH beta. Although great microheterogeneity is present even at the single glycosylation site on the beta-subunit of secreted TSH, a pattern of sialylation and sulfation could be discerned

  3. Alpha subunit of glycoprotein hormones in the sera of acromegalic patients and its mRNA in the tumors. (United States)

    Machiavelli, G A; Artese, R; Benencia, H; Bruno, O; Guerra, L; Basso, A; Burdman, J A


    Within a population of 16 pituitary adenomas we found high levels of glycoprotein alpha subunits in the sera of patients with somatotrophic tumors. This finding was correlated with the presence of mRNA alpha subunit in these tumors indicating the adenomas themselves as the origin of the circulating alpha-subunit. Synthesis of these two hormones, which are chemically very different, by the same tumor cells indicates a high degree of differentiation of these cells. We are unable at this time to conclusively correlate differentiation of these tumors aggressively.

  4. Posttranscriptional regulation of alpha-amylase II-4 expression by gibberellin in germinating rice seeds. (United States)

    Nanjo, Yohei; Asatsuma, Satoru; Itoh, Kimiko; Hori, Hidetaka; Mitsui, Toshiaki; Fujisawa, Yukiko


    Hormonal regulation of expression of alpha-amylase II-4 that lacks the gibberellin-response cis-element (GARE) in the promoter region of the gene was studied in germinating rice (Oryza sativa L.) seeds. Temporal and spatial expression of alpha-amylase II-4 in the aleurone layer were essentially identical to those of alpha-amylase I-1 whose gene contains GARE, although these were distinguishable in the embryo tissues at the early stage of germination. The gibberellin-responsible expression of alpha-amylase II-4 was also similar to that of alpha-amylase I-1. However, the level of alpha-amylase II-4 mRNA was not increased by gibberellin, indicating that the transcriptional enhancement of alpha-amylase II-4 expression did not occur in the aleurone. Gibberellin stimulated the accumulation of 45Ca2+ into the intracellular secretory membrane system. In addition, several inhibitors for Ca2+ signaling, such as EGTA, neomycin, ruthenium red (RuR), and W-7 prevented the gibberellin-induced expression of alpha-amylase II-4 effectively. While the gibberellin-induced expression of alpha-amylase II-4 occurred normally in the aleurone layer of a rice dwarf mutant d1 which is defective in the alpha subunit of the heterotrimeric G protein. Based on these results, it was concluded that the posttranscriptional regulation of alpha-amylase II-4 expression by gibberellin operates in the aleurone layer of germinating rice seed, which is mediated by Ca2+ but not the G protein.

  5. Characterization and application of a radioimmunoassay for reduced, carboxymethylated human luteinizing hormone. cap alpha. -subunit. [/sup 125/I tracer technique

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    Keutmann, H.T.; Beitins, I.Z.; Johnson, L.; McArthur, J.W.


    We have established a double antibody RIA using a rabbit antiserum prepared against reduced, carboxymethylated (RCXM) human LH ..cap alpha..-subunit, with RCXM-..cap alpha.. as tracer and standard. This antiserum did not cross-react with any native gonadotropins or subunit, and reacted only weakly with RCXM-..cap alpha... A tryptic digest of RCXM ..cap alpha..-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 ..cap alpha..-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-..cap alpha.. were elevated. The data indicate that the sequence-specific antiserum may be detecting an immunoreactive form of ..cap alpha..-subunit of LH whose kinetics of appearance and disappearance differs from those of the native subunit.

  6. High Affinity IgE-Fc Receptor alpha and gamma Subunit Interactions

    International Nuclear Information System (INIS)

    Rashid, A.; Housden, J. E. M.; Sabban, S.; Helm, B.


    Objective: To explore the relationships between the subunits (alpha, beta and gamma) of the high affinity IgE receptor (Fc and RI) and its ability to mediate transmembrane signaling. Study Design: Experimental study. Place and Duration of Study: Department of Molecular Biology and Biotechnology, University of Sheffield, UK, from 2008 to 2009. Methodology: The approach employed was to create a chimera (human alpha-gamma-gamma) using the extracellular (EC) domain of the human high affinity IgE receptor. The alpha subunit (huFc and RIalpha) of IgE receptor was spliced onto the rodent gamma TM and cytoplasmic domain (CD). This was transfected into the Rat Basophilic Leukemia cell line in order to assess the possibility of selectively activating cells transfected with this single pass construct for antigen induced mediator release. Results: The RBLs cell lines transfected with the huFc and RIalpha/gamma/gamma cDNA constructs were assessed for the cell surface expression of the huFc and RIalpha subunit and the response to the antigenic stimulus by looking for degranulation and intracellular Ca2+ mobilisation. The results obtained showed the absence of huFc and RIalpha subunit expression on the surface of transfected cells as seen by flowcytometric studies, beta-hexosaminidase assays and intracellular calcium mobilisation studies. Conclusion: In the present study the grounds for non-expression of huFc and RIalpha/gamma/gamma cDNA remains elusive but may be due to the fact that the human-rodent chimeric receptors are assembled differently than the endogenous rodent receptors as seen in study in which COS 7 cells were transfected with human/rat chimeric complexes. (author)

  7. Synthetic. cap alpha. subunit peptide 125-147 of human nicotinic acetylcholine receptor induces antibodies to native receptor

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    McCormick, D.J.; Griesmann, G.E.; Huang, Z.; Lennon, V.A.


    A synthetic peptide corresponding to residues 125-147 of the Torpedo acetylcholine receptor (AChR) ..cap alpha.. subunit proved to be a major antigenic region of the AChR. Rats inoculated with 50 of peptide (T ..cap alpha.. 125-147) developed T cell immunity and antibodies to native AChR and signs of experimental autoimmune myasthenia gravis. They report the synthesis and preliminary testing of a disulfide-looped peptide comprising residues 125-147 of the human AChR ..cap alpha.. subunit. Peptide H ..cap alpha.. 125-147 differs from T ..cap alpha.. 125-147 at residues 139 (Glu for Gln) and 143 (Ser for Thr). In immunoprecipitation assays, antibodies to Torpedo AChR bound /sup 125/I-labelled H..cap alpha.. 125-147 antibody bound H..cap alpha.. 125-147, but monoclonal antibodies to an immunodominant region of native AChR bound neither H..cap alpha.. 125-147 nor T ..cap alpha.. 125-147. Rats immunized with H ..cap alpha.. 125-147 produced anti-mammalian muscle AChR antibodies that induced modulation of AChRs from cultured human myotubes. Thus, region 125-147 of the human AChR ..cap alpha.. subunit is extracellular in muscle, and is both antigenic and immunogenic. It remains to be determined whether or not autoantibodies to this region may in part cause the weakness or myasthenia gravis in man.

  8. Functional labeling of insulin receptor subunits in live cells. Alpha 2 beta 2 species is the major autophosphorylated form

    International Nuclear Information System (INIS)

    Le Marchand-Brustel, Y.; Ballotti, R.; Gremeaux, T.; Tanti, J.F.; Brandenburg, D.; Van Obberghen, E.


    Both receptor subunits were functionally labeled in order to provide methods allowing, in live cells and in broken cell systems, concomitant evaluation of the insulin receptor dual function, hormone binding, and kinase activity. In cell-free systems, insulin receptors were labeled on their alpha-subunit with 125I-photoreactive insulin, and on their beta-subunit by autophosphorylation. Thereafter, phosphorylated receptors were separated from the complete set of receptors by means of anti-phosphotyrosine antibodies. Using this approach, a subpopulation of receptors was found which had bound insulin, but which were not phosphorylated. Under nonreducing conditions, receptors appeared in three oligomeric species identified as alpha 2 beta 2, alpha 2 beta, and alpha 2. Mainly the alpha 2 beta 2 receptor species was found to be phosphorylated while insulin was bound to alpha 2 beta 2, alpha 2 beta, and alpha 2 forms. In live cells, biosynthetic labeling of insulin receptors was used. Receptors were first labeled with [35S]methionine. Subsequently, the addition of insulin led to receptor autophosphorylation by virtue of the endogenous ATP pool. The total amount of [35S]methionine-labeled receptors was precipitated with antireceptor antibodies, whereas with anti-phosphotyrosine antibodies, only the phosphorylated receptors were isolated. Using this approach we made the two following key findings: (1) Both receptor species, alpha 2 beta 2 and alpha 2 beta, are present in live cells and in comparable amounts. This indicates that the alpha 2 beta form is not a degradation product of the alpha 2 beta 2 form artificially generated during receptor preparation. (2) The alpha 2 beta 2 species is the prevalently autophosphorylated form

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

    DEFF Research Database (Denmark)

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


    Two gamma-aminobutyric acid(A) (GABA(A)) receptor chimeras were designed in order to elucidate the structural requirements for GABA(A) receptor desensitization and assembly. The (alpha1/gamma2) and (gamma2/alpha1) chimeric subunits representing the extracellular N-terminal domain of alpha1 or gamma......, as opposed to the staining of the (gamma2/alpha1)-containing receptors, which was only slightly higher than background. To explain this, the (alpha1/gamma2) and (gamma2/alpha1) chimeras may act like alpha1 and gamma2 subunits, respectively, indicating that the extracellular N-terminal segment is important...... for assembly. However, the (alpha1/gamma2) chimeric subunit had characteristics different from the alpha1 subunit, since the (alpha1/gamma2) chimera gave rise to no desensitization after GABA stimulation in whole-cell patch-clamp recordings, which was independent of whether the chimera was expressed...

  10. Glycosylation of alpha(2)delta(1) subunit: a sweet talk with Ca(v)1.2 channels

    Czech Academy of Sciences Publication Activity Database

    Lazniewska, Joanna; Weiss, Norbert


    Roč. 35, č. 3 (2016), s. 239-242 ISSN 0231-5882 R&D Projects: GA ČR GA15-13556S; GA MŠk 7AMB15FR015 Institutional support: RVO:61388963 Keywords : calcium channel * Ca(v)1.2 channel * ancillary subunit * alpha(2)delta(1) subunit * glycosylation * trafficking Subject RIV: CE - Biochemistry Impact factor: 1.170, year: 2016

  11. 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


    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, ...

  12. Recombinant human acetylcholine receptor alpha-subunit induces chronic experimental autoimmune myasthenia gravis. (United States)

    Lennon, V A; Lambert, E H; Leiby, K R; Okarma, T B; Talib, S


    A synthetic gene encoding the 210 N-terminal residues of the alpha-subunit of the nicotinic acetylcholine receptor (AChR) of human skeletal muscle was cloned into an inducible expression plasmid to produce a fusion protein in high yield in Escherichia coli. Like native human AChR, the recombinant human alpha 1-210 protein induced AChR-binding, AChR-modulating, and AChR-blocking autoantibodies in rats when injected once intradermally as an emulsion in CFA, with Bordetella pertussis vaccine as supplementary adjuvant. The minimum dose of recombinant protein required to induce biochemical signs of experimental autoimmune myasthenia gravis (EAMG) with 100% incidence was 2.2 micrograms. With 6.6 to 22 micrograms, serum levels of autoantibodies were persistent, and clinically apparent EAMG lasted more than a month. Clinical, electrophysiological, and biochemical indices of EAMG induced by doses of 66 micrograms or more were more uniformly severe and persistent, with 33% fatality. Rats receiving a control extract of E. coli containing plasmid without the alpha 1-210 codon insert, with adjuvants, did not develop autoantibodies or signs of EAMG. This highly reproducible new model of EAMG induced by a recombinant human autoantigen should be valuable for testing Ag-specific immunotherapeutic strategies that might be applicable to treating acquired myasthenia gravis in humans.

  13. The DFT-DVM theoretical study of the differences of quadrupole splitting and the iron electronic structure for the rough heme models for {alpha}- and {beta}-subunits in deoxyhemoglobin and for deoxymyoglobin

    Energy Technology Data Exchange (ETDEWEB)

    Yuryeva, E. I. [Institute of Solid State Chemistry of the Ural Branch of the Russian Academy of Sciences (Russian Federation); Oshtrakh, M. I., E-mail: [Ural State Technical University-UPI, Faculty of Physical Techniques and Devices for Quality Control (Russian Federation)


    Quantum chemical calculations of the iron electron structure and {sup 57}Fe quadrupole splitting were made by density functional theory and X{alpha} discrete variation method for the rough heme models for {alpha}- and {beta}-subunits in deoxyhemoglobin and for deoxymyoglobin accounting stereochemical differences of the active sites in native proteins. The calculations revealed differences of quadrupole splitting temperature dependences for three models indicating sensitivity of quadrupole splitting and Fe(II) electronic structure to small variations of iron stereochemistry.

  14. 2-Azido-( sup 32 P)NAD+, a photoactivatable probe for G-protein structure: Evidence for holotransducin oligomers in which the ADP-ribosylated carboxyl terminus of alpha interacts with both alpha and gamma subunits

    Energy Technology Data Exchange (ETDEWEB)

    Vaillancourt, R.R.; Dhanasekaran, N.; Johnson, G.L.; Ruoho, A.E. (Univ. of Wisconsin Medical School, Madison (USA))


    A radioactive and photoactivatable derivative of NAD+, 2-azido-(adenylate-32P)NAD+, has been synthesized and used with pertussis toxin to ADP-ribosylate Cys347 of the alpha subunit (alpha T) of GT, the retinal guanine nucleotide-binding protein. ADP-ribosylation of alpha T followed by light activation of the azide moiety of 2-azido-(adenylate-32P)ADP-ribose produced four crosslinked species involving the alpha and gamma subunits of the GT heterotrimer: an alpha trimer (alpha-alpha-alpha), and alpha-alpha-gamma crosslink, an alpha dimer (alpha-alpha), and an alpha-gamma crosslink. The alpha trimer, alpha-alpha-gamma complex, alpha dimer, and alpha-gamma complexes were immunoreactive with alpha T antibodies. The alpha-alpha-gamma and the alpha-gamma complexes were immunoreactive with antisera recognizing gamma subunits. No evidence was found for crosslinking of alpha T to beta T subunits. Hydrolysis of the thioglycosidic bond between Cys347 and 2-azido-(adenylate-32P)ADP-ribose using mercuric acetate resulted in the transfer of radiolabel from Cys347 of alpha T in the crosslinked oligomers to alpha monomers, indicative of intermolecular photocrosslinking, and to gamma monomers, indicative of either intermolecular crosslinked complexes (between heterotrimers) or intramolecular crosslinked complexes (within the heterotrimer). These results demonstrate that GT exists as an oligomer and that ADP-ribosylated Cys347, which is four residues from the alpha T-carboxyl terminus, is oriented toward and in close proximity to the gamma subunit.

  15. Increased Expression of Laminin Subunit Alpha 1 Chain by dCas9-VP160

    Directory of Open Access Journals (Sweden)

    Arnaud Perrin


    Full Text Available Laminin-111 protein complex links the extracellular matrix to integrin α7β1 in sarcolemma, thus replacing in dystrophic muscles links normally insured by the dystrophin complex. Laminin-111 injection in mdx mouse stabilized sarcolemma, restored serum creatine kinase to wild-type levels, and protected muscles from exercised-induced damages. These results suggested that increased laminin-111 is a potential therapy for DMD. Laminin subunit beta 1 and laminin subunit gamma 1 are expressed in adult human muscle, but laminin subunit alpha 1 (LAMA1 gene is expressed only during embryogenesis. We thus developed an alternative method to laminin-111 protein repeated administration by inducing expression of the endogenous mouse Lama1 gene. This was done with the CRSPR/Cas9 system, i.e., by targeting the Lama1 promoter with one or several gRNAs and a dCas9 coupled with the VP160 transcription activation domain. Lama1 mRNA (qRT-PCR and proteins (immunohistochemistry and western blot were not detected in the control C2C12 myoblasts and in control muscles. However, significant expression was observed in cells transfected and in mouse muscles electroporated with plasmids coding for dCas9-VP160 and a gRNA. Larger synergic increases were observed by using two or three gRNAs. The increased Lama1 expression did not modify the expression of the α7 and β1 integrins. Increased expression of Lama1 by the CRISPR/Cas9 system will have to be further investigated by systemic delivery of the CRISPR/Cas9 components to verify whether this could be a treatment for several myopathies.

  16. Increased Expression of Laminin Subunit Alpha 1 Chain by dCas9-VP160. (United States)

    Perrin, Arnaud; Rousseau, Joël; Tremblay, Jacques P


    Laminin-111 protein complex links the extracellular matrix to integrin α7β1 in sarcolemma, thus replacing in dystrophic muscles links normally insured by the dystrophin complex. Laminin-111 injection in mdx mouse stabilized sarcolemma, restored serum creatine kinase to wild-type levels, and protected muscles from exercised-induced damages. These results suggested that increased laminin-111 is a potential therapy for DMD. Laminin subunit beta 1 and laminin subunit gamma 1 are expressed in adult human muscle, but laminin subunit alpha 1 (LAMA1) gene is expressed only during embryogenesis. We thus developed an alternative method to laminin-111 protein repeated administration by inducing expression of the endogenous mouse Lama1 gene. This was done with the CRSPR/Cas9 system, i.e., by targeting the Lama1 promoter with one or several gRNAs and a dCas9 coupled with the VP160 transcription activation domain. Lama1 mRNA (qRT-PCR) and proteins (immunohistochemistry and western blot) were not detected in the control C2C12 myoblasts and in control muscles. However, significant expression was observed in cells transfected and in mouse muscles electroporated with plasmids coding for dCas9-VP160 and a gRNA. Larger synergic increases were observed by using two or three gRNAs. The increased Lama1 expression did not modify the expression of the α7 and β1 integrins. Increased expression of Lama1 by the CRISPR/Cas9 system will have to be further investigated by systemic delivery of the CRISPR/Cas9 components to verify whether this could be a treatment for several myopathies. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  17. 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


    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.

  18. Progesterone modulation of alpha5 nAChR subunits influences anxiety-related behavior during estrus cycle. (United States)

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


    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.

  19. Beta3 subunits promote expression and nicotine-induced up-regulation of human nicotinic alpha6* nicotinic acetylcholine receptors expressed in transfected cell lines. (United States)

    Tumkosit, Prem; Kuryatov, Alexander; Luo, Jie; Lindstrom, Jon


    Nicotinic acetylcholine receptors (AChRs) containing alpha6 subunits are typically found at aminergic nerve endings where they play important roles in nicotine addiction and Parkinson's disease. alpha6* AChRs usually contain beta3 subunits. beta3 subunits are presumed to assemble only in the accessory subunit position within AChRs where they do not participate in forming acetylcholine binding sites. Assembly of subunits in the accessory position may be a critical final step in assembly of mature AChRs. Human alpha6 AChRs subtypes were permanently transfected into human tsA201 human embryonic kidney (HEK) cell lines. alpha6beta2beta3 and alpha6beta4beta3 cell lines were found to express much larger amounts of AChRs and were more sensitive to nicotine-induced increase in the amount of AChRs than were alpha6beta2 or alpha6beta4 cell lines. The increased sensitivity to nicotine-induced up-regulation was due not to a beta3-induced increase in affinity for nicotine but probably to a direct effect on assembly of AChR subunits. HEK cells express only a small amount of mature alpha6beta2 AChRs, but many of these subunits are on the cell surface. This contrasts with Xenopus laevis oocytes, which express a large amount of incorrectly assembled alpha6beta2 subunits that bind cholinergic ligands but form large amorphous intracellular aggregates. Monoclonal antibodies (mAbs) were made to the alpha6 and beta3 subunits to aid in the characterization of these AChRs. The alpha6 mAbs bind to epitopes C-terminal of the extracellular domain. These data demonstrate that both cell type and the accessory subunit beta3 can play important roles in alpha6* AChR expression, stability, and up-regulation by nicotine.

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

    NARCIS (Netherlands)

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


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

  1. Conformational alterations resulting from mutations in cytoplasmic domains of the alpha subunit of the Na,K-ATPase

    DEFF Research Database (Denmark)

    Blostein, R; Daly, S E; MacAulay, Nanna


    This paper summarizes experiments concerned with the functional consequences of mutations in cytoplasmic regions of the alpha 1 subunit of the Na,K-ATPase, in particular the amino terminus, the first cytoplasmic loop between transmembrane segments M2 and M3, and the major cytoplasmic loop between...


    NARCIS (Netherlands)



    A patient with acromegaly and hyperthyroidism due to a growth hormone-, thyrotrophin- and alpha-subunit-secreting pituitary adenoma is described. His deceased father had suffered from a pituitary tumour, and was likely to have had acromegaly as well. Plasma growth hormone and insulin-like growth

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

    NARCIS (Netherlands)

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


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

  4. Thyroid hormone coordinately regulates Na sup + -K sup + -ATPase. alpha. - and. beta. -subunit mRNA levels in kidney

    Energy Technology Data Exchange (ETDEWEB)

    McDonough, A.A.; Brown, T.A.; Horowitz, B.; Chiu, R.; Schlotterbeck, J.; Bowen, J.; Schmitt, C.A. (Univ. of Southern California School of Medicine, Los Angeles (USA))


    Synthesis of the sodium pump, Na{sup +}-K{sup +}-ATPase, is regulated by thyroid hormone in responsive tissues. The purpose of this study was to determine if triiodothyronine (T{sub 3}) regulates the concentration of the mRNAs coding for the two enzyme subunits, {alpha} and {beta}, and the time course of the response. A single dose of T{sub 3} was administered to hypothyroid rats that were killed at various times after injection. In the kidney cortexes of the T{sub 3}-injected animals, as well as hypothyroid and euthyroid rats, {alpha}- and {beta}-mRNA concentrations were measured by dot blot using cDNAs corresponding to the two mRNAs; {alpha}-subunit abundance was measured by Western blot using antibodies to the enzyme, and Na{sup +}-K{sup +}-ATPase activity was measured enzymatically. {alpha}- and {beta}-mRNAs increased coordinately to 1.6-fold over hypothyroid levels by 12 h after T{sub 3}. The authors conclude that T{sub 3} regulates Na{sup +}-K{sup +}-ATPase synthesis and activity by coordinately increasing the mRNAs of both the {alpha}- and {beta}-subunits of the enzyme.

  5. A CK2 site is reversibly phosphorylated in the photosystem II subunit CP29

    NARCIS (Netherlands)

    Testi, Maria Grazia; Croce, Roberta; Polverino-De Laureto, Patrizia; Bassi, Roberto


    Protein phosphorylation is a major mechanism in the regulation of protein function. In chloroplast thylakoids several photosystem II subunits, including the major antenna light-harvesting complex II and several core complex components, are reversibly phosphorylated depending on the redox state of

  6. A reduction in hippocampal GABAA receptor alpha5 subunits disrupts the memory for location of objects in mice. (United States)

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


    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.

  7. Molecular characterization of the alpha subunit of complement component C8 (GcC8alpha) in the nurse shark (Ginglymostoma cirratum). (United States)

    Aybar, Lydia; Shin, Dong-Ho; Smith, Sylvia L


    Target cell lysis by complement is achieved by the assembly and insertion of the membrane attack complex (MAC) composed of glycoproteins C5b through C9. The lytic activity of shark complement involves functional analogues of mammalian C8 and C9. Mammalian C8 is composed of alpha, beta, and gamma subunits. The subunit structure of shark C8 is not known. This report describes a 2341 nucleotide sequence that translates into a polypeptide of 589 amino acid residues, orthologue to mammalian C8alpha and has the same modular architecture with conserved cysteines forming the peptide bond backbone. The C8gamma-binding cysteine is conserved in the perforin-like domain. Hydrophobicity profile indicates the presence of hydrophobic residues essential for membrane insertion. It shares 41.1% and 47.4% identity with human and Xenopus C8alpha respectively. Southern blot analysis showed GcC8alpha exists as a single copy gene expressed in most tissues except the spleen with the liver being the main site of synthesis. Phylogenetic analysis places it in a clade with C8alpha orthologs and as a sister taxa to the Xenopus. 2009 Elsevier Ltd.

  8. Role of the cholinergic nervous system in rheumatoid arthritis: aggravation of arthritis in nicotinic acetylcholine receptor alpha7 subunit gene knockout mice

    NARCIS (Netherlands)

    van Maanen, Marjolein A.; Stoof, Susanne P.; Larosa, Gregory J.; Vervoordeldonk, Margriet J.; Tak, Paul P.


    BACKGROUND: The alpha7 subunit of nicotinic acetylcholine receptors (alpha7nAChR) can negatively regulate the synthesis and release of proinflammatory cytokines by macrophages and fibroblast-like synoviocytes in vitro. In addition, stimulation of the alpha7nAChR can reduce the severity of arthritis

  9. SH2 domains of the p85 alpha subunit of phosphatidylinositol 3-kinase regulate binding to growth factor receptors. (United States)

    McGlade, C J; Ellis, C; Reedijk, M; Anderson, D; Mbamalu, G; Reith, A D; Panayotou, G; End, P; Bernstein, A; Kazlauskas, A


    The binding of cytoplasmic signaling proteins such as phospholipase C-gamma 1 and Ras GTPase-activating protein to autophosphorylated growth factor receptors is directed by their noncatalytic Src homology region 2 (SH2) domains. The p85 alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase, which associates with several receptor protein-tyrosine kinases, also contains two SH2 domains. Both p85 alpha SH2 domains, when expressed individually as fusion proteins in bacteria, bound stably to the activated beta receptor for platelet-derived growth factor (PDGF). Complex formation required PDGF stimulation and was dependent on receptor tyrosine kinase activity. The bacterial p85 alpha SH2 domains recognized activated beta PDGF receptor which had been immobilized on a filter, indicating that SH2 domains contact autophosphorylated receptors directly. Several receptor tyrosine kinases within the PDGF receptor subfamily, including the colony-stimulating factor 1 receptor and the Steel factor receptor (Kit), also associate with PI 3-kinase in vivo. Bacterially expressed SH2 domains derived from the p85 alpha subunit of PI 3-kinase bound in vitro to the activated colony-stimulating factor 1 receptor and to Kit. We infer that the SH2 domains of p85 alpha bind to high-affinity sites on these receptors, whose creation is dependent on receptor autophosphorylation. The SH2 domains of p85 are therefore primarily responsible for the binding of PI 3-kinase to activated growth factor receptors. Images PMID:1372092

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

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


    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.

  11. Cables1 controls p21/Cip1 protein stability by antagonizing proteasome subunit alpha type 3. (United States)

    Shi, Z; Li, Z; Li, Z J; Cheng, K; Du, Y; Fu, H; Khuri, F R


    The cyclin-dependent kinase (CDK) inhibitor 1A, p21/Cip1, is a vital cell cycle regulator, dysregulation of which has been associated with a large number of human malignancies. One critical mechanism that controls p21 function is through its degradation, which allows the activation of its associated cell cycle-promoting kinases, CDK2 and CDK4. Thus delineating how p21 is stabilized and degraded will enhance our understanding of cell growth control and offer a basis for potential therapeutic interventions. Here we report a novel regulatory mechanism that controls the dynamic status of p21 through its interaction with Cdk5 and Abl enzyme substrate 1 (Cables1). Cables1 has a proposed role as a tumor suppressor. We found that upregulation of Cables1 protein was correlated with increased half-life of p21 protein, which was attributed to Cables1/p21 complex formation and supported by their co-localization in the nucleus. Mechanistically, Cables1 interferes with the proteasome (Prosome, Macropain) subunit alpha type 3 (PSMA3) binding to p21 and protects p21 from PSMA3-mediated proteasomal degradation. Moreover, silencing of p21 partially reverses the ability of Cables1 to induce cell death and inhibit cell proliferation. In further support of a potential pathophysiological role of Cables1, the expression level of Cables1 is tightly associated with p21 in both cancer cell lines and human lung cancer patient tumor samples. Together, these results suggest Cables1 as a novel p21 regulator through maintaining p21 stability and support the model that the tumor-suppressive function of Cables1 occurs at least in part through enhancing the tumor-suppressive activity of p21.

  12. Canine chondrodysplasia caused by a truncating mutation in collagen-binding integrin alpha subunit 10.

    Directory of Open Access Journals (Sweden)

    Kaisa Kyöstilä

    Full Text Available The skeletal dysplasias are disorders of the bone and cartilage tissues. Similarly to humans, several dog breeds have been reported to suffer from different types of genetic skeletal disorders. We have studied the molecular genetic background of an autosomal recessive chondrodysplasia that affects the Norwegian Elkhound and Karelian Bear Dog breeds. The affected dogs suffer from disproportionate short stature dwarfism of varying severity. Through a genome-wide approach, we mapped the chondrodysplasia locus to a 2-Mb region on canine chromosome 17 in nine affected and nine healthy Elkhounds (praw = 7.42×10(-6, pgenome-wide = 0.013. The associated locus contained a promising candidate gene, cartilage specific integrin alpha 10 (ITGA10, and mutation screening of its 30 exons revealed a nonsense mutation in exon 16 (c.2083C>T; p.Arg695* that segregated fully with the disease in both breeds (p = 2.5×10(-23. A 24% mutation carrier frequency was indicated in NEs and an 8% frequency in KBDs. The ITGA10 gene product, integrin receptor α10-subunit combines into a collagen-binding α10β1 integrin receptor, which is expressed in cartilage chondrocytes and mediates chondrocyte-matrix interactions during endochondral ossification. As a consequence of the nonsense mutation, the α10-protein was not detected in the affected cartilage tissue. The canine phenotype highlights the importance of the α10β1 integrin in bone growth, and the large animal model could be utilized to further delineate its specific functions. Finally, this study revealed a candidate gene for human chondrodysplasias and enabled the development of a genetic test for breeding purposes to eradicate the disease from the two dog breeds.

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

    Energy Technology Data Exchange (ETDEWEB)

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


    Rat brain membrane benzodiazepine receptors (BZR) were photoaffinity labelled specifically (in presence or absence of 6 clonazepam) with 10 nM /sup 3/H-flunitrazepam (FNZ). Digestion of the FNZ-labelled, membrane-bound BZR with 200 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.

  14. Concentration of carcinoembryonic antigen alpha-fetoprotein and beta-subunit of human chorionic gonadotropin in the serum of coke oven workers

    Energy Technology Data Exchange (ETDEWEB)

    Snit, M. [Silesian Medical Academy, Zabrze (Poland)


    Increased levels of carcinoembryonic antigen and {alpha}-fetoprotein were found in blood serum of coke oven workers, and also to some extent in smokers and in residents of industrial cities. The {beta} subunit of chorionic gonadotropin was barely detectable.

  15. The morphological and chemical characteristics of striatal neurons immunoreactive for the alpha1-subunit of the GABA(A) receptor in the rat. (United States)

    Waldvogel, H J; Kubota, Y; Trevallyan, S C; Kawaguchi, Y; Fritschy, J M; Mohler, H; Faull, R L


    The distribution, morphology and chemical characteristics of neurons immunoreactive for the alpha1-subunit of the GABA(A) receptor in the striatum of the basal ganglia in the rat brain were investigated at the light, confocal and electron microscope levels using single, double and triple immunohistochemical labelling techniques. The results showed that alpha1-subunit immunoreactive neurons were sparsely distributed throughout the rat striatum. Double and triple labelling results showed that all the alpha1-subunit-immunoreactive neurons were positive for glutamate decarboxylase and immunoreactive for the beta2,3 and gamma2 subunits of the GABA(A) receptor. Three types of alpha1-subunit-immunoreactive neurons were identified in the striatum on the basis of cellular morphology and chemical characteristics. The most numerous alpha1-subunit-immunoreactive neurons were medium-sized, aspiny neurons with a widely branching dendritic tree. They were parvalbumin-negative and were located mainly in the dorsolateral regions of the striatum. Electron microscopy showed that these neurons had an indented nuclear membrane, typical of striatal interneurons, and were surrounded by small numbers of axon terminals which established alpha1-subunit-immunoreactive synaptic contacts with the soma and dendrites. These cells were classified as type 1 alpha1-subunit-immunoreactive neurons and comprised 75% of the total population of alpha1-subunit-immunoreactive neurons in the striatum. The remaining alpha1-subunit-immunoreactive neurons comprised of a heterogeneous population of large-sized neurons localized in the ventral and medial regions of the striatum. The most numerous large-sized cells were parvalbumin-negative, had two to three relatively short branching dendrites and were designated type 2 alpha1-subunit-immunoreactive neurons. Electron microscopy showed that the type 2 neurons were characterized by a highly convoluted nuclear membrane and were sparsely covered with small axon

  16. Bacterial expression and one-step purification of an isotope-labeled heterotrimeric G-protein {alpha}-subunit

    Energy Technology Data Exchange (ETDEWEB)

    Abdulaev, Najmoutin G. [University of Maryland Biotechnology Institute, Center for Advanced Research in Biotechnology (United States); Zhang Cheng; Dinh, Andy [University of Texas Health Science Center, Center for Membrane Biology, Department of Biochemistry and Molecular Biology (United States); Ngo, Tony; Bryan, Philip N. [University of Maryland Biotechnology Institute, Center for Advanced Research in Biotechnology (United States); Brabazon, Danielle M. [Loyola College in Maryland, Department of Chemistry (United States); Marino, John P. [University of Maryland Biotechnology Institute, Center for Advanced Research in Biotechnology (United States)], E-mail:; Ridge, Kevin D. [University of Texas Health Science Center, Center for Membrane Biology, Department of Biochemistry and Molecular Biology (United States)


    Heterologous expression systems are often employed to generate sufficient quantities of isotope-labeled proteins for high-resolution NMR studies. Recently, the interaction between the prodomain region of subtilisin and an active, mutant form of the mature enzyme has been exploited to develop a cleavable affinity tag fusion system for one-step generation and purification of full-length soluble proteins obtained by inducible prokaryotic expression. As a first step towards applying high-resolution NMR methods to study heterotrimeric G-protein {alpha}-subunit (G{sub {alpha}}) conformation and dynamics, the utility of this subtilisin prodomain fusion system for expressing and purifying an isotope-labeled G{sub {alpha}} chimera ({approx}40 kDa polypeptide) has been tested. The results show that a prodomain fused G{sub {alpha}} chimera can be expressed to levels approaching 6-8 mg/l in minimal media and that the processed, mature protein exhibits properties similar to those of G{sub {alpha}} isolated from natural sources. To assay for the functional integrity of the purified G{sub {alpha}} chimera at NMR concentrations and probe for changes in the structure and dynamics of G{sub {alpha}} that result from activation, {sup 15}N-HSQC spectra of the GDP/Mg{sup 2+} bound form of G{sub {alpha}} obtained in the absence and presence of aluminum fluoride, a well known activator of the GDP bound state, have been acquired. Comparisons of the {sup 15}N-HSQC spectra reveals a number of changes in chemical shifts of the {sup 1}HN, {sup 15}N crosspeaks that are discussed with respect to expected changes in the protein conformation associated with G{sub {alpha}} activation.

  17. Spectral and Temporal Properties of the Alpha and Beta Subunits and (alpha Beta) Monomer Isolated from Nostoc SP. Using Picosecond Laser Spectroscopy. (United States)

    Dagen, Aaron J.


    The fluorescence decay profiles, relative quantum yield and transmission of the (alpha), (beta) and ((alpha)(beta)) complexes from phycoerythrin isolated from the photosynthetic antenna system of Nostoc sp. and measured by single picosecond laser spectroscopic techniques is studied. The fluorescence decay profiles of all three complexes are found to be intensity independent for the intensity range investigated ((TURN)4 x 10('13) to (TURN)4 x 10('15) photons-cm('-2) per pulse). The apparent decrease in the relative quantum yield of all three complexes as intensity increases is offset by a corresponding increase in the relative transmission. This evidence, along with the intensity independent fluorescence kinetics, suggests that exciton annihilation is absent in these complexes. The decay profiles are fit to models assuming energy transfer amongst fluorescing chromophores. The intraprotein transfer rate is found to be 100 ps in the (alpha) subunit, 666 ps in the (beta) subunit. Constraining these rates to be identical in the monomer results in explaining the monomer kinetics by an increase in the nonradiative rate of the f(,(beta)) chromophore, an apparent result of aggregation effects.

  18. Orientation of the calcium channel beta relative to the alpha(12.2 subunit is critical for its regulation of channel activity.

    Directory of Open Access Journals (Sweden)

    Iuliia Vitko

    Full Text Available BACKGROUND: The Ca(vbeta subunits of high voltage-activated Ca(2+ channels control the trafficking and biophysical properties of the alpha(1 subunit. The Ca(vbeta-alpha(1 interaction site has been mapped by crystallographic studies. Nevertheless, how this interaction leads to channel regulation has not been determined. One hypothesis is that betas regulate channel gating by modulating movements of IS6. A key requirement for this direct-coupling model is that the linker connecting IS6 to the alpha-interaction domain (AID be a rigid structure. METHODOLOGY/PRINCIPAL FINDINGS: The present study tests this hypothesis by altering the flexibility and orientation of this region in alpha(12.2, then testing for Ca(vbeta regulation using whole cell patch clamp electrophysiology. Flexibility was induced by replacement of the middle six amino acids of the IS6-AID linker with glycine (PG6. This mutation abolished beta2a and beta3 subunits ability to shift the voltage dependence of activation and inactivation, and the ability of beta2a to produce non-inactivating currents. Orientation of Ca(vbeta with respect to alpha(12.2 was altered by deletion of 1, 2, or 3 amino acids from the IS6-AID linker (Bdel1, Bdel2, Bdel3, respectively. Again, the ability of Ca(vbeta subunits to regulate these biophysical properties were totally abolished in the Bdel1 and Bdel3 mutants. Functional regulation by Ca(vbeta subunits was rescued in the Bdel2 mutant, indicating that this part of the linker forms beta-sheet. The orientation of beta with respect to alpha was confirmed by the bimolecular fluorescence complementation assay. CONCLUSIONS/SIGNIFICANCE: These results show that the orientation of the Ca(vbeta subunit relative to the alpha(12.2 subunit is critical, and suggests additional points of contact between these subunits are required for Ca(vbeta to regulate channel activity.

  19. A CK2 site is reversibly phosphorylated in the photosystem II subunit CP29. (United States)

    Testi, M G; Croce, R; Polverino-De Laureto, P; Bassi, R


    Protein phosphorylation is a major mechanism in the regulation of protein function. In chloroplast thylakoids several photosystem II subunits, including the major antenna light-harvesting complex II and several core complex components, are reversibly phosphorylated depending on the redox state of the electron carriers. A previously unknown reversible phosphorylation event has recently been described on the CP29 subunit which leads to conformational changes and protection from cold stress (Bergantino, E., Dainese, P., Cerovic, Z. Sechi, S. and Bassi, R. (1995) J. Biol Chem. 270, 8474-8481). In this study, we have identified the phosphorylation site on the N-terminal, stroma-exposed domain, showing that it is located in a sequence not homologous to the other members of the Lhc family. The phosphorylated sequence is unique in chloroplast membranes since it meets the requirements for CK2 (casein kinase II) kinases. The possibility that this phosphorylation is involved in a signal transduction pathway is discussed.

  20. Increased Expression of Laminin Subunit Alpha 1 Chain by dCas9-VP160


    Perrin, Arnaud; Rousseau, Jo?l; Tremblay, Jacques P.


    Laminin-111 protein complex links the extracellular matrix to integrin α7β1 in sarcolemma, thus replacing in dystrophic muscles links normally insured by the dystrophin complex. Laminin-111 injection in mdx mouse stabilized sarcolemma, restored serum creatine kinase to wild-type levels, and protected muscles from exercised-induced damages. These results suggested that increased laminin-111 is a potential therapy for DMD. Laminin subunit beta 1 and laminin subunit gamma 1 are expressed in adul...

  1. [beta]-hexosaminidase isozymes from cells cotransfected with [alpha] and [beta] cDNA constructs: Analysis of the [alpha]-subunit missense mutation associated with the adult form of Tay-Sachs disease

    Energy Technology Data Exchange (ETDEWEB)

    Brown, C.A.; Mahuran, D.J. (Univ. of Toronto (Canada))


    In vitro mutagenesis and transient expression in COS cells has been used to associate a missense mutation with a clinical or biochemical phenotype. Mutations affecting the [alpha]-subunit of [beta]-hexosaminidase A ([alpha][beta]) (E.C. result in Tay-Sachs disease. Because hexosaminidase A is heterodimeric, analysis of [alpha]-chain mutations is not straightforward. The authors examine three approaches utilizing previously identified mutations affecting [alpha]-chain folding. These involve transfection of (1) the [alpha] cDNA alone; (2) a [beta] cDNA construct encoding a [beta]-subunit substituted at a position homologous to that of the [alpha]-subunit, and (3) both [alpha] and [beta] cDNAs. The latter two procedures amplified residual activity levels over that of patient samples, an effect not previously found with mutations affecting an [open quotes]active[close quotes] [alpha]Arg residue. This effect may help to discriminate between protein-folding and active-site mutations. The authors conclude that, with proper controls, the latter method of cotransfection can be used to evaluate the effects and perhaps to predict the clinical course of some [alpha]-chain mutations. Using this technique, they demonstrate that the adult-onset Tay-Sachs mutation, [alpha]Gly[yields]Ser[sup 269], does not directly affect [alpha][beta] dimerization but exerts an indirect effect on the dimer through destabilizing the folded [alpha]-subunit at physiological temperatures. Two other [alpha] mutations linked to more severe phenotypes appear to inhibit the initial folding of the subunit. 36 refs., 2 figs., 5 tabs.

  2. Expression of the GABA(A) receptor alpha6 subunit in cultured cerebellar granule cells is developmentally regulated by activation of GABA(A) receptors

    DEFF Research Database (Denmark)

    Carlson, B X; Belhage, B; Hansen, Gert Helge


    Da (alpha6 subunit) radioactive peaks in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In contrast, THIP-treated granule cells at 8 DIV demonstrated a small but significant decrease from control cultures in the photoincorporation of [3H]Ro15-4513 in the 51-kDa peak; however...... that the major effect of THIP was to increase alpha6 subunit clustering on granule cell bodies as well as neurites, 15-fold and sixfold, respectively. Using in situ hybridization, a small THIP-induced increase in alpha6 mRNA was detected at 4 DIV; however, no effect was apparent at 8 DIV. These data suggest...

  3. A network of hydrophobic residues impeding helix alphaC rotation maintains latency of kinase Gcn2, which phosphorylates the alpha subunit of translation initiation factor 2. (United States)

    Gárriz, Andrés; Qiu, Hongfang; Dey, Madhusudan; Seo, Eun-Joo; Dever, Thomas E; Hinnebusch, Alan G


    Kinase Gcn2 is activated by amino acid starvation and downregulates translation initiation by phosphorylating the alpha subunit of translation initiation factor 2 (eIF2alpha). The Gcn2 kinase domain (KD) is inert and must be activated by tRNA binding to the adjacent regulatory domain. Previous work indicated that Saccharomyces cerevisiae Gcn2 latency results from inflexibility of the hinge connecting the N and C lobes and a partially obstructed ATP-binding site in the KD. Here, we provide strong evidence that a network of hydrophobic interactions centered on Leu-856 also promotes latency by constraining helix alphaC rotation in the KD in a manner relieved during amino acid starvation by tRNA binding and autophosphorylation of Thr-882 in the activation loop. Thus, we show that mutationally disrupting the hydrophobic network in various ways constitutively activates eIF2alpha phosphorylation in vivo and bypasses the requirement for a key tRNA binding motif (m2) and Thr-882 in Gcn2. In particular, replacing Leu-856 with any nonhydrophobic residue activates Gcn2, while substitutions with various hydrophobic residues maintain kinase latency. We further provide strong evidence that parallel, back-to-back dimerization of the KD is a step on the Gcn2 activation pathway promoted by tRNA binding and autophosphorylation. Remarkably, mutations that disrupt the L856 hydrophobic network or enhance hinge flexibility eliminate the need for the conserved salt bridge at the parallel dimer interface, implying that KD dimerization facilitates the reorientation of alphaC and remodeling of the active site for enhanced ATP binding and catalysis. We propose that hinge remodeling, parallel dimerization, and reorientation of alphaC are mutually reinforcing conformational transitions stimulated by tRNA binding and secured by the ensuing autophosphorylation of T882 for stable kinase activation.

  4. Loss of tumorigenic potential by human lung tumor cells in the presence of antisense RNA specific to the ectopically synthesized alpha subunit of human chorionic gonadotropin. (United States)

    Rivera, R T; Pasion, S G; Wong, D T; Fei, Y B; Biswas, D K


    A clonal strain of human lung tumor cells in culture (ChaGo), derived from a bronchogenic carcinoma, synthesizes and secretes large amounts of alpha (alpha) and a comparatively lower level of beta (beta) subunit of the glycoprotein hormone, human chorionic gonadotropin (HCG). ChaGo cells lost their characteristic anchorage-independent growth phenotype in the presence of anti-alpha-HCG antibody. The effect of the antibody was partially reversed by addition of alpha-HCG to the culture medium. ChaGo cells were transfected with an expression vector (pRSV-anti-alpha-HCG), that directs synthesis of RNA complementary to alpha-HCG mRNA. The transfectants produced alpha-HCG antisense RNA which was associated with the reduced level of alpha-HCG. Transfectants also displayed several altered phenotypic properties, including altered morphology, less mitosis, reduced growth rate, loss of anchorage-independent growth, and loss of tumorigenicity in nude mice. Treatment of transfectants with 8,bromo-cAMP resulted in increased accumulation of alpha-HCG mRNA, no change in the level of alpha-HCG antisense RNA, release of the inhibition of [3H]thymidine incorporation, and restoration of anchorage-independent growth phenotype. The overexpression of c-myc, observed in ChaGo cells, was unaffected by the reduced level of alpha-HCG. These results suggest that ectopic synthesis of the alpha subunit of HCG plays a functional role in the transformation of these human lung cells.

  5. Cytochrome oxidase subunit II gene in mitochondria of Oenothera has no intron (United States)

    Hiesel, Rudolf; Brennicke, Axel


    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. Identification of the gamma subunit-interacting residues on photoreceptor cGMP phosphodiesterase, PDE6alpha '. (United States)

    Granovsky, A E; Artemyev, N O


    Photoreceptor cGMP phosphodiesterase (PDE6) is the effector enzyme in the G protein-mediated visual transduction cascade. In the dark, the activity of PDE6 is shut off by the inhibitory gamma subunit (Pgamma). Chimeric proteins between cone PDE6alpha' and cGMP-binding and cGMP-specific PDE (PDE5) have been constructed and expressed in Sf9 cells to study the mechanism of inhibition of PDE6 catalytic activity by Pgamma. Substitution of the segment PDE5-(773-820) by the corresponding PDE6alpha'-(737-784) sequence in the wild-type PDE5 or in a PDE5/PDE6alpha' chimera containing the catalytic domain of PDE5 results in chimeric enzymes capable of inhibitory interaction with Pgamma. The catalytic properties of the chimeric PDEs remained similar to those of PDE5. Ala-scanning mutational analysis of the Pgamma-binding region, PDE6alpha'-(750-760), revealed PDE6alpha' residues essential for the interaction. The M758A mutation markedly impaired and the Q752A mutation moderately impaired the inhibition of chimeric PDE by Pgamma. The analysis of the catalytic properties of mutant PDEs and a model of the PDE6 catalytic domain suggest that residues Met(758) and Gln(752) directly bind Pgamma. A model of the PDE6 catalytic site shows that PDE6alpha'-(750-760) forms a loop at the entrance to the cGMP-binding pocket. Binding of Pgamma to Met(758) would effectively block access of cGMP to the catalytic cavity, providing a structural basis for the mechanism of PDE6 inhibition.

  7. Improving Saccharomyces cerevisiae ethanol production and tolerance via RNA polymerase II subunit Rpb7. (United States)

    Qiu, Zilong; Jiang, Rongrong


    Classical strain engineering methods often have limitations in altering multigenetic cellular phenotypes. Here we try to improve Saccharomyces cerevisiae ethanol tolerance and productivity by reprogramming its transcription profile through rewiring its key transcription component RNA polymerase II (RNAP II), which plays a central role in synthesizing mRNAs. This is the first report on using directed evolution method to engineer RNAP II to alter S. cerevisiae strain phenotypes. Error-prone PCR was employed to engineer the subunit Rpb7 of RNAP II to improve yeast ethanol tolerance and production. Based on previous studies and the presumption that improved ethanol resistance would lead to enhanced ethanol production, we first isolated variant M1 with much improved resistance towards 8 and 10% ethanol. The ethanol titers of M1 was ~122 g/L (96.58% of the theoretical yield) under laboratory very high gravity (VHG) fermentation, 40% increase as compared to the control. DNA microarray assay showed that 369 genes had differential expression in M1 after 12 h VHG fermentation, which are involved in glycolysis, alcoholic fermentation, oxidative stress response, etc. This is the first study to demonstrate the possibility of engineering eukaryotic RNAP to alter global transcription profile and improve strain phenotypes. Targeting subunit Rpb7 of RNAP II was able to bring differential expression in hundreds of genes in S. cerevisiae , which finally led to improvement in yeast ethanol tolerance and production.

  8. The membrane-cytoplasm interface of integrin alpha subunits is critical for receptor latency.


    Briesewitz, R; Kern, A; Smilenov, L B; David, F S; Marcantonio, E E


    Localization of integrin receptors to focal contact sites occurs upon ligand binding. This activity is latent, since unoccupied integrin receptors do not localize to focal contacts. Deletion analysis has revealed that the alpha cytoplasmic domains is required for the maintenance of integrin receptor latency. Our current hypothesis for the mechanism of integrin post-ligand binding events is that there is a change in relationship of alpha and beta cytoplasmic domains, which overcomes receptor l...

  9. Expression and characterization of a recombinant maize CK-2 alpha subunit

    DEFF Research Database (Denmark)

    Boldyreff, B; Meggio, F; Dobrowolska, G


    to support the immunological data also by biochemical and biophysical experiments the availability of a recombinant CK-2 alpha from maize was a prerequisite. A maize cDNA clone of maize CK-2 alpha was expressed in the bacterial strain BL21 (DE3). The recombinant protein was purified to homogeneity; its......CKIIB, one of the CK-2 like enzymes which have been isolated from maize, has been shown to be a monomeric enzyme that cross-reacts with anti CK-2 alpha specific antibodies suggesting a possible relationship between the two proteins (Dobrowolska et al. (1992) Eur. J. Biochem. 204, 299-303). In order...... molecular mass on one-dimensional SDS PAGE was estimated to be 36.5 kDa. The calculated molecular mass according to the amino acid composition is 39,228 Da (332 amino acids). The recombinant maize CK-2 alpha (rmCK-2 alpha) exhibited mostly the same properties as the recombinant human CK-2 alpha (rhCK-2...

  10. Hydrogen bonds between the alpha and beta subunits of the F1-ATPase allow communication between the catalytic site and the interface of the beta catch loop and the gamma subunit. (United States)

    Boltz, Kathryn W; Frasch, Wayne D


    F(1)-ATPase mutations in Escherichia coli that changed the strength of hydrogen bonds between the alpha and beta subunits in a location that links the catalytic site to the interface between the beta catch loop and the gamma subunit were examined. Loss of the ability to form the hydrogen bonds involving alphaS337, betaD301, and alphaD335 lowered the k(cat) of ATPase and decreased its susceptibility to Mg(2+)-ADP-AlF(n) inhibition, while mutations that maintain or strengthen these bonds increased the susceptibility to Mg(2+)-ADP-AlF(n) inhibition and lowered the k(cat) of ATPase. These data suggest that hydrogen bonds connecting alphaS337 to betaD301 and betaR323 and connecting alphaD335 to alphaS337 are important to transition state stabilization and catalytic function that may result from the proper alignment of catalytic site residues betaR182 and alphaR376 through the VISIT sequence (alpha344-348). Mutations betaD301E, betaR323K, and alphaR282Q changed the rate-limiting step of the reaction as determined by an isokinetic plot. Hydrophobic mutations of betaR323 decreased the susceptibility to Mg(2+)-ADP-AlF(n)() inhibition and lowered the number of interactions required in the rate-limiting step yet did not affect the k(cat) of ATPase, suggesting that betaR323 is important to transition state formation. The decreased rate of ATP synthase-dependent growth and decreased level of lactate-dependent quenching observed with alphaD335, betaD301, and alphaE283 mutations suggest that these residues may be important to the formation of an alternative set of hydrogen bonds at the interface of the alpha and beta subunits that permits the release of intersubunit bonds upon the binding of ATP, allowing gamma rotation in the escapement mechanism.

  11. Computer modeling of siRNA knockdown effects indicates an essential role of the Ca2+ channel alpha2delta-1 subunit in cardiac excitation-contraction coupling. (United States)

    Tuluc, Petronel; Kern, Georg; Obermair, Gerald J; Flucher, Bernhard E


    L-type Ca(2+) currents determine the shape of cardiac action potentials (AP) and the magnitude of the myoplasmic Ca(2+) signal, which regulates the contraction force. The auxiliary Ca(2+) channel subunits alpha(2)delta-1 and beta(2) are important regulators of membrane expression and current properties of the cardiac Ca(2+) channel (Ca(V)1.2). However, their role in cardiac excitation-contraction coupling is still elusive. Here we addressed this question by combining siRNA knockdown of the alpha(2)delta-1 subunit in a muscle expression system with simulation of APs and Ca(2+) transients by using a quantitative computer model of ventricular myocytes. Reconstitution of dysgenic muscle cells with Ca(V)1.2 (GFP-alpha(1C)) recapitulates key properties of cardiac excitation-contraction coupling. Concomitant depletion of the alpha(2)delta-1 subunit did not perturb membrane expression or targeting of the pore-forming GFP-alpha(1C) subunit into junctions between the outer membrane and the sarcoplasmic reticulum. However, alpha(2)delta-1 depletion shifted the voltage dependence of Ca(2+) current activation by 9 mV to more positive potentials, and it slowed down activation and inactivation kinetics approximately 2-fold. Computer modeling revealed that the altered voltage dependence and current kinetics exert opposing effects on the function of ventricular myocytes that in total cause a 60% prolongation of the AP and a 2-fold increase of the myoplasmic Ca(2+) concentration during each contraction. Thus, the Ca(2+) channel alpha(2)delta-1 subunit is not essential for normal Ca(2+) channel targeting in muscle but is a key determinant of normal excitation and contraction of cardiac muscle cells, and a reduction of alpha(2)delta-1 function is predicted to severely perturb normal heart function.

  12. Differential expression of gill Na+,K+-ATPase alpha- and beta-subunits, Na+,K+,2Cl- cotransporter and CFTR anion channel in juvenile anadromous and landlocked Atlantic salmon Salmo salar

    DEFF Research Database (Denmark)

    Nilsen, Tom O.; Ebbesson, Lars O. E.; Madsen, Steffen S.


    This study examines changes in gill Na(+),K(+)-ATPase (NKA) alpha- and beta-subunit isoforms, Na(+),K(+),2Cl(-) cotransporter (NKCC) and cystic fibrosis transmembrane conductance regulator (CFTR I and II) in anadromous and landlocked strains of Atlantic salmon during parr-smolt transformation, an...

  13. Interaction mode between catalytic and regulatory subunits in glucosidase II involved in ER glycoprotein quality control. (United States)

    Satoh, Tadashi; Toshimori, Takayasu; Noda, Masanori; Uchiyama, Susumu; Kato, Koichi


    The glycoside hydrolase family 31 (GH31) α-glucosidases play vital roles in catabolic and regulated degradation, including the α-subunit of glucosidase II (GIIα), which catalyzes trimming of the terminal glucose residues of N-glycan in glycoprotein processing coupled with quality control in the endoplasmic reticulum (ER). Among the known GH31 enzymes, only GIIα functions with its binding partner, regulatory β-subunit (GIIβ), which harbors a lectin domain for substrate recognition. Although the structural data have been reported for GIIα and the GIIβ lectin domain, the interaction mode between GIIα and GIIβ remains unknown. Here, we determined the structure of a complex formed between GIIα and the GIIα-binding domain of GIIβ, thereby providing a structural basis underlying the functional extension of this unique GH31 enzyme. © 2016 The Protein Society.

  14. The Sleep–Wake Cycle in the Nicotinic Alpha-9 Acetylcholine Receptor Subunit Knock-Out Mice

    Directory of Open Access Journals (Sweden)

    Natalia Madrid-López


    Full Text Available There is a neural matrix controlling the sleep–wake cycle (SWC embedded within high ranking integrative mechanisms in the central nervous system. Nicotinic alpha-9 acetylcholine receptor subunit (alpha-9 nAChR participate in physiological processes occurring in sensory, endocrine and immune systems. There is a relationship between the SWC architecture, body homeostasis and sensory afferents so that disruption of afferent signaling is expected to affect the temporal organization of sleep and wake states. The analysis of the SWC of 9 nAChR knock-out animals may help to reveal the contribution of alpha-9 nAChR to sleep chronobiological determinants. Here we explore the polysomnogram in chronically implanted alpha-9 nAChR knock-out (KO and wild-type (WT individuals of the hybrid CBA/Sv129 mouse strain. Records were obtained in isolation chambers under a stable 12:12 light:dark cycle (LD. To unmask the 24-h modulation of the SWC a skeleton photoperiod (SP protocol was performed. Under LD the daily quota (in % of wakefulness (W, NREM sleep and REM sleep obtained in KO and WT animals were 45, 48 and 7, and 46, 46 and 8 respectively. Both groups exhibit nocturnal phase preference of W as well as diurnal and unimodal phase preference of NREM and REM sleep. The acrophase mean angles of KO vs. WT genotypes were not different (Zeitgeber Time: 6.5 vs. 14.9 for W, 4.3 vs. 2.8 for NREM sleep and 5.3 vs. 3.4 for REM sleep, respectively. Transference to SP do not affect daily state quotas, phase preferences and acrophases among genotypes. Unmasking phenomena of the SWC such as wake increment during the rest phase under SP was evident only among WT mice suggesting the involvement of retinal structures containing alpha-9 nAChR in masking processes. Furthermore, KO animals exhibit longer NREM and REM sleep episodes that is independent of illumination conditions. Consolidated diurnal NREM sleep contributed to obtain higher values of NREM sleep delta-EEG activity

  15. Localization of pig Na[sup +], K[sup +]-ATPase [alpha] and [beta] subunit genes to chromosome 4 by radioactive in situ hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Lahbib-Mansais, Y.; Yerle, M.; Dalens, M.; Chevalet, C.; Gellin, J. (Centre de Recherches de Toulouse (France))


    Two genes coding for Na[sup +],K[sup +] -ATPase [alpha] and [beta] subunits are localized on pig chromosome 4, to the q1.6[yields]q2.3 and 1.3[yields]q2.1 regions, respectively, by radioactive in situ hybridization. According to nucleotide and amino acid sequence comparisons with different human isoforms of Na[sup +] ,K[sup +]-ATPase, these pig [alpha] and [beta] ATPase genes show strong homologies with human [alpha]1 and [beta] subunit ATPase genes, respectively. These results are discussed with respect to comparative mapping data of conserved genes in mammalian species. We showed that the pig cDNA probes encoding ATPase [alpha] and, [beta] genes reveal DNA polymorphism in Meishan an Large White pigs. 35 refs., 4 figs., 2 tabs.

  16. Cloning and characterization of Sdga gene encoding alpha-subunit of heterotrimeric guanosine 5'-triphosphate-binding protein complex in Scoparia dulcis. (United States)

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


    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.

  17. NADP+ binding to the regulatory subunit of methionine adenosyltransferase II increases intersubunit binding affinity in the hetero-trimer.

    Directory of Open Access Journals (Sweden)

    Beatriz González

    Full Text Available Mammalian methionine adenosyltransferase II (MAT II is the only hetero-oligomer in this family of enzymes that synthesize S-adenosylmethionine using methionine and ATP as substrates. Binding of regulatory β subunits and catalytic α2 dimers is known to increase the affinity for methionine, although scarce additional information about this interaction is available. This work reports the use of recombinant α2 and β subunits to produce oligomers showing kinetic parameters comparable to MAT II purified from several tissues. According to isothermal titration calorimetry data and densitometric scanning of the stained hetero-oligomer bands on denatured gels, the composition of these oligomers is that of a hetero-trimer with α2 dimers associated to single β subunits. Additionally, the regulatory subunit is able to bind NADP(+ with a 1:1 stoichiometry, the cofactor enhancing β to α2-dimer binding affinity. Mutants lacking residues involved in NADP(+ binding and N-terminal truncations of the β subunit were able to oligomerize with α2-dimers, although the kinetic properties appeared altered. These data together suggest a role for both parts of the sequence in the regulatory role exerted by the β subunit on catalysis. Moreover, preparation of a structural model for the hetero-oligomer, using the available crystal data, allowed prediction of the regions involved in β to α2-dimer interaction. Finally, the implications that the presence of different N-terminals in the β subunit could have on MAT II behavior are discussed in light of the recent identification of several splicing forms of this subunit in hepatoma cells.

  18. 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.


    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

  19. Conformational stability analyses of alpha subunit I domain of LFA-1 and Mac-1.

    Directory of Open Access Journals (Sweden)

    Debin Mao

    Full Text Available β₂ integrin of lymphocyte function-associated antigen-1 (LFA-1 or macrophage-1 antigen (Mac-1 binds to their common ligand of intercellular adhesion molecule-1 (ICAM-1 and mediates leukocyte-endothelial cell (EC adhesions in inflammation cascade. Although the two integrins are known to have distinct functions, the corresponding micro-structural bases remain unclear. Here (steered-molecular dynamics simulations were employed to elucidate the conformational stability of α subunit I domains of LFA-1 and Mac-1 in different affinity states and relevant I domain-ICAM-1 interaction features. Compared with low affinity (LA Mac-1, the LA LFA-1 I domain was unstable in the presence or absence of ICAM-1 ligand, stemming from diverse orientations of its α₇-helix with different motifs of zipper-like hydrophobic junction between α₁- and α₇-helices. Meanwhile, spontaneous transition of LFA-1 I domain from LA state to intermediate affinity (IA state was first visualized. All the LA, IA, and high affinity (HA states of LFA-1 I domain and HA Mac-1 I domain were able to bind to ICAM-1 ligand effectively, while LA Mac-1 I domain was unfavorable for binding ligand presumably due to the specific orientation of S144 side-chain that capped the MIDAS ion. These results furthered our understanding in correlating the structural bases with their functions of LFA-1 and Mac-1 integrins from the viewpoint of I domain conformational stability and of the characteristics of I domain-ICAM-1 interactions.

  20. Interactions between the cyclic AMP receptor protein and the alpha subunit of RNA polymerase at the Escherichia coli galactose operon P1 promoter. (United States)

    Attey, A; Belyaeva, T; Savery, N; Hoggett, J; Fujita, N; Ishihama, A; Busby, S


    DNAase I footprinting has been used to study open complexes between Escherichia coli RNA polymerase and the galactose operon P1 promoter, both in the absence and the presence of CRP (the cyclic AMP receptor protein, a transcription activator). From the effects of deletion of the C-terminal part of the RNA polymerase alpha subunit, we deduce that alpha binds at the upstream end of both the binary RNA polymerase-galP1 and ternary RNA polymerase-CRP-galP1 complexes. Disruption of the alpha-upstream contact suppresses open complex formation at galP1 at lower temperatures. In ternary RNA polymerase-CRP-galP1 complexes, alpha appears to make direct contact with Activating Region 1 in CRP. DNAase I footprinting has been used to detect and quantify interactions between purified alpha and CRP bound at galP1.

  1. Altered Na+ transport after an intracellular alpha-subunit deletion reveals strict external sequential release of Na+ from the Na/K pump. (United States)

    Yaragatupalli, Siddhartha; Olivera, J Fernando; Gatto, Craig; Artigas, Pablo


    The Na/K pump actively exports 3 Na(+) in exchange for 2 K(+) across the plasmalemma of animal cells. As in other P-type ATPases, pump function is more effective when the relative affinity for transported ions is altered as the ion binding sites alternate between opposite sides of the membrane. Deletion of the five C-terminal residues from the alpha-subunit diminishes internal Na(+) (Na(i)(+)) affinity approximately 25-fold [Morth et al. (2007) Nature 450:1043-1049]. Because external Na(+) (Na(o)(+)) binding is voltage-dependent, we studied the reactions involving this process by using two-electrode and inside-out patch voltage clamp in normal and truncated (DeltaKESYY) Xenopus-alpha1 pumps expressed in oocytes. We observed that DeltaKESYY (i) decreased both Na(o)(+) and Na(i)(+) apparent affinities in the absence of K(o)(+), and (ii) did not affect apparent Na(o)(+) affinity at high K(o)(+). These results support a model of strict sequential external release of Na(+) ions, where the Na(+)-exclusive site releases Na(+) before the sites shared with K(+) and the DeltaKESYY deletion only reduces Na(o)(+) affinity at the shared sites. Moreover, at nonsaturating K(o)(+), DeltaKESYY induced an inward flow of Na(+) through Na/K pumps at negative potentials. Guanidinium(+) can also permeate truncated pumps, whereas N-methyl-D-glucamine cannot. Because guanidinium(o)(+) can also traverse normal Na/K pumps in the absence of both Na(o)(+) and K(o)(+) and can also inhibit Na/K pump currents in a Na(+)-like voltage-dependent manner, we conclude that the normal pathway transited by the first externally released Na(+) is large enough to accommodate guanidinium(+).

  2. Hyperpolarization-activated inward leakage currents caused by deletion or mutation of carboxy-terminal tyrosines of the Na+/K+-ATPase {alpha} subunit. (United States)

    Meier, Susan; Tavraz, Neslihan N; Dürr, Katharina L; Friedrich, Thomas


    The Na(+)/K(+)-ATPase mediates electrogenic transport by exporting three Na(+) ions in exchange for two K(+) ions across the cell membrane per adenosine triphosphate molecule. The location of two Rb(+) ions in the crystal structures of the Na(+)/K(+)-ATPase has defined two "common" cation binding sites, I and II, which accommodate Na(+) or K(+) ions during transport. The configuration of site III is still unknown, but the crystal structure has suggested a critical role of the carboxy-terminal KETYY motif for the formation of this "unique" Na(+) binding site. Our two-electrode voltage clamp experiments on Xenopus oocytes show that deletion of two tyrosines at the carboxy terminus of the human Na(+)/K(+)-ATPase alpha(2) subunit decreases the affinity for extracellular and intracellular Na(+), in agreement with previous biochemical studies. Apparently, the DeltaYY deletion changes Na(+) affinity at site III but leaves the common sites unaffected, whereas the more extensive DeltaKETYY deletion affects the unique site and the common sites as well. In the absence of extracellular K(+), the DeltaYY construct mediated ouabain-sensitive, hyperpolarization-activated inward currents, which were Na(+) dependent and increased with acidification. Furthermore, the voltage dependence of rate constants from transient currents under Na(+)/Na(+) exchange conditions was reversed, and the amounts of charge transported upon voltage pulses from a certain holding potential to hyperpolarizing potentials and back were unequal. These findings are incompatible with a reversible and exclusively extracellular Na(+) release/binding mechanism. In analogy to the mechanism proposed for the H(+) leak currents of the wild-type Na(+)/K(+)-ATPase, we suggest that the DeltaYY deletion lowers the energy barrier for the intracellular Na(+) occlusion reaction, thus destabilizing the Na(+)-occluded state and enabling inward leak currents. The leakage currents are prevented by aromatic amino acids at the

  3. Decreased agonist sensitivity of human GABA(A) receptors by an amino acid variant, isoleucine to valine, in the alpha1 subunit. (United States)

    Westh-Hansen, S E; Rasmussen, P B; Hastrup, S; Nabekura, J; Noguchi, K; Akaike, N; Witt, M R; Nielsen, M


    Recombinant human GABA(A) receptors were investigated in vitro by coexpression of cDNAs coding for alpha1, beta2, and gamma2 subunits in the baculovirus/Sf-9 insect cell system. We report that a single amino acid exchange (isoleucine 121 to valine 121) in the N-terminal, extracellular part of the alpha1 subunit induces a marked decrease in agonist GABA(A) receptor ligand sensitivity. The potency of muscimol and GABA to inhibit the binding of the GABA(A) receptor antagonist [3H]SR 95531 (2-(3-carboxypropyl)-3-amino-6-(4-methoxyphenyl)pyridazinium bromide) was higher in receptor complexes of alpha1(ile 121) beta2gamma2 than in those of alpha1(val 121) beta2gamma2 (IC50 values were 32-fold and 26-fold lower for muscimol and GABA, respectively). The apparent affinity of the GABA(A) receptor antagonist bicuculline methiodide to inhibit the binding of [3H]SR 95531 did not differ between the two receptor complex variants. Electrophysiological measurements of GABA induced whole-cell Cl- currents showed a ten-fold decrease in the GABA(A) receptor sensitivity of alpha1 (val 121) beta2gamma2 as compared to alpha1(ile 121) beta2gamma2 receptor complexes. Thus, a relatively small change in the primary structure of the alpha1 subunit leads to a decrease selective for GABA(A) receptor sensitivity to agonist ligands, since no changes were observed in a GABA(A) receptor antagonist affinity and benzodiazepine receptor binding.

  4. Analyzing proteasomal subunit expression reveals Rpt4 as a prognostic marker in stage II colorectal cancer.

    LENUS (Irish Health Repository)


    Colorectal cancer is a leading cause of cancer-related deaths worldwide. Early diagnosis and treatment of colorectal cancer is the key to improving survival rates and as such a need exists to identify patients who may benefit from adjuvant chemotherapy. The dysregulation of the ubiquitin-proteasome system (UPS) has been implicated in oncogenesis and cancer cell survival, and proteasome inhibitors are in clinical use for a number of malignancies including multiple myeloma. In our study, we examined the protein expression of several key components of the UPS in colorectal cancer using immunohistochemistry to determine expression levels of ubiquitinylated proteins and the proteasomal subunits, 20S core and Rpt4 in a cohort of 228 patients with colon cancer. Multivariate Cox analysis revealed that neither the intensity of either ubiquitinylated proteins or the 20S core was predictive in either Stage II or III colon cancer for disease free survival or overall survival. In contrast, in Stage II patients increased Rpt4 staining was significantly associated with disease free survival (Cox proportional hazard ratio 0.605; p = 0.0217). Our data suggest that Rpt4 is an independent prognostic variable for Stage II colorectal cancer and may aid in the decision of which patients undergo adjuvant chemotherapy.

  5. Association between GABA-A receptor alpha 5 subunit gene locus and schizophrenia of a later age of onset. (United States)

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


    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

  6. The alpha-subunit of the Arabidopsis heterotrimeric G protein, GPA1, is a regulator of transpiration efficiency. (United States)

    Nilson, Sarah E; Assmann, Sarah M


    Land plants must balance CO2 assimilation with transpiration in order to minimize drought stress and maximize their reproductive success. The ratio of assimilation to transpiration is called transpiration efficiency (TE). TE is under genetic control, although only one specific gene, ERECTA, has been shown to regulate TE. We have found that the alpha-subunit of the heterotrimeric G protein in Arabidopsis (Arabidopsis thaliana), GPA1, is a regulator of TE. gpa1 mutants, despite having guard cells that are hyposensitive to abscisic acid-induced inhibition of stomatal opening, have increased TE under ample water and drought stress conditions and when treated with exogenous abscisic acid. Leaf-level gas-exchange analysis shows that gpa1 mutants have wild-type assimilation versus internal CO2 concentration responses but exhibit reduced stomatal conductance compared with ecotype Columbia at ambient and below-ambient internal CO2 concentrations. The increased TE and reduced whole leaf stomatal conductance of gpa1 can be primarily attributed to stomatal density, which is reduced in gpa1 mutants. GPA1 regulates stomatal density via the control of epidermal cell size and stomata formation. GPA1 promoter::beta-glucuronidase lines indicate that the GPA1 promoter is active in the stomatal cell lineage, further supporting a function for GPA1 in stomatal development in true leaves.

  7. Multichromophoric hybrid species made of perylene bisimide derivatives and Ru(ii) and Os(ii) polypyridine subunits. (United States)

    Nastasi, Francesco; La Ganga, Giuseppina; Campagna, Sebastiano; Syrgiannis, Zois; Rigodanza, Francesco; Vitale, Stefania; Licciardello, Antonino; Prato, Maurizio


    Herein, the synthesis and the photophysical and redox properties of a new perylene bisimide (PBI) species (L), bearing two 1,10-phenanthroline (phen) ligands at the two imide positions of the PBI, and its dinuclear Ru(ii) and Os(ii) complexes, [(bpy) 2 Ru(μ-L)Ru(bpy) 2 ](PF 6 ) 4 (Ru2; bpy = 2,2'-bipyridine) and [(Me 2 -bpy) 2 Os(μ-L)Os(Me 2 -bpy) 2 ](PF 6 ) 4 (Os2; Me 2 -bpy = (4,4'-dimethyl)-2,2'-bipyridine), are reported. The absorption spectra of the compounds are dominated by the structured bands of the PBI subunit due to the lowest-energy spin-allowed π-π* transition. The spin-allowed MLCT transitions in Ru2 and Os2 are inferred by the absorption at 350-470 nm, where the PBI absorption is negligible. The absorption band extends towards the red region for Os2 due to the spin-forbidden MLCT transitions, intensified by the heavy osmium center. The reduction processes of the compounds are dominated by two successive mono-electronic PBI-based processes, which in the metal complexes are slightly shifted compared to the free ligand. On oxidation, both metal complexes undergo an apparent bi-electronic process (at 1.31 V vs. SCE for Ru2 and 0.77 V for Os2), attributed to the simultaneous one-electron oxidation of the two weakly-interacting metal centers. In Ru2 and Os2, the intense fluorescence of L subunit (λ max , 535 nm; τ, 4.3 ns; Φ, 0.91) is fully quenched, mainly by photoinduced electron transfer from the metal centers, on the ps timescale (time constant, 11 ps in Ru2 and 3 ps in Os2). Such photoinduced electron transfer leads to the formation of a charge-separated state, which directly decays to the ground state in about 70 ps in Os2, but produces the triplet π-π* state of the PBI subunit in 35 ps in Ru2. The results provide information on the excited-state processes of the hybrid species combining two dominant classes of chromophore/luminophore species, the PBI and the metal polypyridine complexes, and can be used for future design on new hybrid

  8. Human placental Na/sup +/, K/sup +/-ATPase. cap alpha. subunit: cDNA cloning, tissue expression, DNA polymorphism, and chromosomal localization

    Energy Technology Data Exchange (ETDEWEB)

    Chehab, F.F.; Kan, Y.W.; Law, M.L.; Hartz, J.; Kao, F.T.; Blostein, R.


    A 2.2-kilobase clone comprising a major portion of the coding sequence of the Na/sup +/, K/sup +/-ATPase ..cap alpha.. subunit was cloned from human placenta and its sequence was identical to that encoding the ..cap alpha.. subunit of human kidney and HeLa cells. Transfer blot analysis of the mRNA products of the Na/sup +/, K/sup +/-ATPase gene from various human tissues and cell lines revealed only one band (approx. = 4.7 kilobases) under low and high stringency washing conditions. The levels of expression in the tissues were intestine > placenta > liver > pancreas, and in the cell lines the levels were human erythroleukemia > butyrate-induced colon > colon > brain > HeLa cells. mRNA was undetectable in reticulocytes, consistent with the authors failure to detect positive clones in a size-selected ( > 2 kilobases) lambdagt11 reticulocyte cDNA library. DNA analysis revealed by a polymorphic EcoRI band and chromosome localization by flow sorting and in situ hybridization showed that the ..cap alpha.. subunit is on the short is on the short arm (band p11-p13) of chromosome 1.

  9. daf-31 encodes the catalytic subunit of N alpha-acetyltransferase that regulates Caenorhabditis elegans development, metabolism and adult lifespan.

    Directory of Open Access Journals (Sweden)

    Di Chen


    Full Text Available The Caenorhabditis elegans dauer larva is a facultative state of diapause. Mutations affecting dauer signal transduction and morphogenesis have been reported. Of these, most that result in constitutive formation of dauer larvae are temperature-sensitive (ts. The daf-31 mutant was isolated in genetic screens looking for novel and underrepresented classes of mutants that form dauer and dauer-like larvae non-conditionally. Dauer-like larvae are arrested in development and have some, but not all, of the normal dauer characteristics. We show here that daf-31 mutants form dauer-like larvae under starvation conditions but are sensitive to SDS treatment. Moreover, metabolism is shifted to fat accumulation in daf-31 mutants. We cloned the daf-31 gene and it encodes an ortholog of the arrest-defective-1 protein (ARD1 that is the catalytic subunit of the major N alpha-acetyltransferase (NatA. A daf-31 promoter::GFP reporter gene indicates daf-31 is expressed in multiple tissues including neurons, pharynx, intestine and hypodermal cells. Interestingly, overexpression of daf-31 enhances the longevity phenotype of daf-2 mutants, which is dependent on the forkhead transcription factor (FOXO DAF-16. We demonstrate that overexpression of daf-31 stimulates the transcriptional activity of DAF-16 without influencing its subcellular localization. These data reveal an essential role of NatA in controlling C. elegans life history and also a novel interaction between ARD1 and FOXO transcription factors, which may contribute to understanding the function of ARD1 in mammals.

  10. Topology characterization of a benzodiazepine-binding beta-rich domain of the GABAA receptor alpha1 subunit. (United States)

    Xu, Zhiwen; Fang, Shisong; Shi, Haifeng; Li, Hoiming; Deng, Yiqun; Liao, Yinglei; Wu, Jiun-Ming; Zheng, Hui; Zhu, Huaimin; Chen, Hueih-Min; Tsang, Shui Ying; Xue, Hong


    Structural investigation of GABAA receptors has been limited by difficulties imposed by its trans-membrane-complex nature. In the present study, the topology of a membrane-proximal beta-rich (MPB) domain in the C139-L269 segment of the receptor alpha1 subunit was probed by mapping the benzodiazepine (BZ)-binding and epitopic sites, as well as fluorescence resonance energy transfer (FRET) analysis. Ala-scanning and semiconservative substitutions within this segment revealed the contribution of the phenyl rings of Y160 and Y210, the hydroxy group of S186 and the positive charge on R187 to BZ-binding. FRET with the bound BZ ligand indicated the proximity of Y160, S186, R187, and S206 to the BZ-binding site. On the other hand, epitope-mapping using the monoclonal antibodies (mAbs) against the MPB domain established a clustering of T172, R173, E174, Q196, and T197. Based on the lack of FRET between Trp substitutionally placed at R173 or V198 and bound BZ, this epitope-mapped cluster is located on a separate end of the folded protein from the BZ-binding site. Mutations of the five conserved Cys and Trp residues in the MPB domain gave rise to synergistic and rescuing effects on protein secondary structures and unfolding stability that point to a CCWCW-pentad, reminiscent to the CWC-triad "pin" of immunoglobulin (Ig)-like domains, important for the structural maintenance. These findings, together with secondary structure and fold predictions suggest an anti-parallel beta-strand topology with resemblance to Ig-like fold, having the BZ-binding and the epitopic residues being clustered at two different ends of the fold.

  11. daf-31 encodes the catalytic subunit of N alpha-acetyltransferase that regulates Caenorhabditis elegans development, metabolism and adult lifespan. (United States)

    Chen, Di; Zhang, Jiuli; Minnerly, Justin; Kaul, Tiffany; Riddle, Donald L; Jia, Kailiang


    The Caenorhabditis elegans dauer larva is a facultative state of diapause. Mutations affecting dauer signal transduction and morphogenesis have been reported. Of these, most that result in constitutive formation of dauer larvae are temperature-sensitive (ts). The daf-31 mutant was isolated in genetic screens looking for novel and underrepresented classes of mutants that form dauer and dauer-like larvae non-conditionally. Dauer-like larvae are arrested in development and have some, but not all, of the normal dauer characteristics. We show here that daf-31 mutants form dauer-like larvae under starvation conditions but are sensitive to SDS treatment. Moreover, metabolism is shifted to fat accumulation in daf-31 mutants. We cloned the daf-31 gene and it encodes an ortholog of the arrest-defective-1 protein (ARD1) that is the catalytic subunit of the major N alpha-acetyltransferase (NatA). A daf-31 promoter::GFP reporter gene indicates daf-31 is expressed in multiple tissues including neurons, pharynx, intestine and hypodermal cells. Interestingly, overexpression of daf-31 enhances the longevity phenotype of daf-2 mutants, which is dependent on the forkhead transcription factor (FOXO) DAF-16. We demonstrate that overexpression of daf-31 stimulates the transcriptional activity of DAF-16 without influencing its subcellular localization. These data reveal an essential role of NatA in controlling C. elegans life history and also a novel interaction between ARD1 and FOXO transcription factors, which may contribute to understanding the function of ARD1 in mammals.

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

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


    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.

  13. Mutation Analysis of 16 Mucolipidosis II and III Alpha/Beta Chinese Children Revealed Genotype-Phenotype Correlations.

    Directory of Open Access Journals (Sweden)

    Shuang Liu

    Full Text Available Mucolipidosis II and III alpha/beta are autosomal recessive diseases caused by mutations in the GNPTAB gene which encodes the α and β subunits of the N-acetylglucosamine-1-phosphotransferase. Clinically, mucolipidosis II (MLII is characterized by severe developmental delay, coarse facial features, skeletal deformities, and other systemic involvement. In contrast, MLIII alpha/beta is a much milder disorder, the symptoms of which include progressive joint stiffness, short stature, and scoliosis. To study the relationship between the genotypes and phenotypes of the MLII and MLIII alpha/beta patients, we analyzed the GNPTAB gene in 16 Chinese MLII and MLIII alpha/beta patients. We collected and analyzed the patients' available clinical data and all showed clinical features typical of MLII or MLIII alpha/beta. Moreover, the activity of several lysosomal enzymes was measured in the plasma and finally the GNPTAB gene was sequenced. We detected 30 mutant alleles out of 32 alleles in our patients. These include 10 new mutations (c.99delC, c.118-1G>A, c.523_524delAAinsG, c.1212C>G, c.2213C>A, c.2345C>T, c.2356C>T, c.2455G>T, c.2821dupA, and c.3136-2A>G and 5 previously reported mutations (c.1071G>A, c.1090C>T, c.2715+1G>A, c.2550_2554delGAAA, and c.3613C>T. The most frequent mutation was the splicing mutation c.2715+1G>A, which accounted for 28% of the mutations. The majority of the mutations reported in the Chinese patients (57% were located on exon 13 or in its intronic flanking regions.

  14. Somato-synaptic variation of GABA(A) receptors in cultured murine cerebellar granule cells: investigation of the role of the alpha6 subunit. (United States)

    Mellor, J R; Wisden, W; Randall, A D


    Electrophysiological investigation of cultured cerebellar murine granule cells revealed differences between the GABA(A) receptors at inhibitory synapses and those on the cell body. Specifically, mIPSCs decayed more rapidly than cell body receptors deactivated, the mean single channel conductance at the synapse (32 pS) was greater than that at cell body (21 pS) and only cell body receptors were sensitive to Zn(2+) (150 microM), which depressed response amplitude by 82+/-5% and almost doubled the rate of channel deactivation. The GABA(A) receptor alpha6 subunit is selectively expressed in cerebellar granule cells. Although concentrated at synapses, it is also found on extrasynaptic membranes. Using a mouse line (Deltaalpha6lacZ) lacking this subunit, we investigated its role in the somato-synaptic differences in GABA(A) receptor function. All differences between cell body and synaptic GABA(A) receptors observed in wild-type (WT) granule cells persisted in Deltaalpha6lacZ cells, thus demonstrating that they are not specifically due to the cellular distribution of the alpha6 subunit. However, mIPSCs from WT and Deltaalpha6lacZ cells differed in both their kinetics (faster decay in WT cells) and underlying single channel conductance (32 pS WT, 25 pS Deltaalpha6lacZ). This provides good evidence for a functional contribution of the alpha6 subunit to postsynaptic GABA(A) receptors in these cells. Despite this, deactivation kinetics of mIPSCs in WT and Deltaalpha6lacZ granule cells exhibited similar benzodiazepene (BDZ) sensitivity. This suggests that the enhanced BDZ-induced ataxia seen in Deltaalpha6lacZ mice may reflect physiological activity at extrasynaptic receptors which, unlike those at synapses, display differential BDZ-sensitivity in WT and Deltaalpha6lacZ granule cells (Jones, A.M., Korpi, E.R., McKernan, R.M., Nusser, Z., Pelz, R., Makela, R., Mellor, J.R., Pollard, S., Bahn, S., Stephenson, F.A., Randall, A.D., Sieghart, W., Somogyi, P., Smith, A.J.H., Wisden

  15. Three alpha-subunits of heterotrimeric G proteins and an adenylyl cyclase have distinct roles in fruiting body development in the homothallic fungus Sordaria macrospora. (United States)

    Kamerewerd, Jens; Jansson, Malin; Nowrousian, Minou; Pöggeler, Stefanie; Kück, Ulrich


    Sordaria macrospora, a self-fertile filamentous ascomycete, carries genes encoding three different alpha-subunits of heterotrimeric G proteins (gsa, G protein Sordaria alpha subunit). We generated knockout strains for all three gsa genes (Deltagsa1, Deltagsa2, and Deltagsa3) as well as all combinations of double mutants. Phenotypic analysis of single and double mutants showed that the genes for Galpha-subunits have distinct roles in the sexual life cycle. While single mutants show some reduction of fertility, double mutants Deltagsa1Deltagsa2 and Deltagsa1Deltagsa3 are completely sterile. To test whether the pheromone receptors PRE1 and PRE2 mediate signaling via distinct Galpha-subunits, two recently generated Deltapre strains were crossed with all Deltagsa strains. Analyses of the corresponding double mutants revealed that compared to GSA2, GSA1 is a more predominant regulator of a signal transduction cascade downstream of the pheromone receptors and that GSA3 is involved in another signaling pathway that also contributes to fruiting body development and fertility. We further isolated the gene encoding adenylyl cyclase (AC) (sac1) for construction of a knockout strain. Analyses of the three DeltagsaDeltasac1 double mutants and one Deltagsa2Deltagsa3Deltasac1 triple mutant indicate that SAC1 acts downstream of GSA3, parallel to a GSA1-GSA2-mediated signaling pathway. In addition, the function of STE12 and PRO41, two presumptive signaling components, was investigated in diverse double mutants lacking those developmental genes in combination with the gsa genes. This analysis was further completed by expression studies of the ste12 and pro41 transcripts in wild-type and mutant strains. From the sum of all our data, we propose a model for how different Galpha-subunits interact with pheromone receptors, adenylyl cyclase, and STE12 and thus cooperatively regulate sexual development in S. macrospora.

  16. Influence of the. cap alpha. -,. beta. -, and. gamma. -subunits of the energy-transducing adenosine triphosphatase from Micrococcus lysodeikticus in the immunochemical properties of the protein and in their reconstitution studied by a radioimmunoassay method

    Energy Technology Data Exchange (ETDEWEB)

    Larraga, V; Mollinedo, F; Rubio, N; Munoz, E [Unidad de Biomembranas, Instituto de Inmunologia y Biologia Microbiana, Madrid (Spain)


    A sensitive radioimmunoassay was developed for the energy-transducing adenosine triphosphatase (F/sub 1/-ATPase, EC of Micrococcus lysodeikticus and the assay was extended to the ..cap alpha..-, ..beta..-, and ..gamma..-subunits of the enzyme. These subunits were isolated and cross-reactions studied.

  17. Characterization of the regulatory subunit from brain cyclic AMP-dependent protein kinase II

    International Nuclear Information System (INIS)

    Stein, J.C.


    Tryptic peptides derived from the regulatory subunits of brain and heart cAMP-dependent protein kinase II were mapped by reverse phase HPLC. At 280 nm, 15 unique peptides were found only in the heart RII digest, while 5 other peptides were obtained only from brain RII. At 210 nm, 13 brain-RII specific and 15 heart-RII specific tryptic peptides were identified and resolved. Two-dimensional mapping analyses revealed that several 37 P-labeled tryptic fragments derived from the autophosphorylation and the photoaffinity labeled cAMP-binding sites of brain RII were separate and distinct from the 32 P-peptides isolated from similarly treated heart RII. The tryptic phosphopeptide containing the autophosphorylation site in brain RII was purified. The sequence and phosphorylation site is: Arg-Ala-Ser(P)-Val-Cys-Ala-Glu-Ala-Tyr-Asn-Pro-Asp-Glu-Glu-Glu-Asp-Asp-Ala-Glu. Astrocytes and neurons exhibit high levels of the brain RII enzyme, while oligodendrocytes contain the heart RII enzyme. Monoclonal antibodies to bovine cerebral cortex RII were made and characterized. The antibodies elucidated a subtle difference between membrane-associated and cytosolic RII from cerebral cortex

  18. Analysis of the cytochrome c oxidase subunit II (COX2) gene in giant panda, Ailuropoda melanoleuca. (United States)

    Ling, S S; Zhu, Y; Lan, D; Li, D S; Pang, H Z; Wang, Y; Li, D Y; Wei, R P; Zhang, H M; Wang, C D; Hu, Y D


    The giant panda, Ailuropoda melanoleuca (Ursidae), has a unique bamboo-based diet; however, this low-energy intake has been sufficient to maintain the metabolic processes of this species since the fourth ice age. As mitochondria are the main sites for energy metabolism in animals, the protein-coding genes involved in mitochondrial respiratory chains, particularly cytochrome c oxidase subunit II (COX2), which is the rate-limiting enzyme in electron transfer, could play an important role in giant panda metabolism. Therefore, the present study aimed to isolate, sequence, and analyze the COX2 DNA from individuals kept at the Giant Panda Protection and Research Center, China, and compare these sequences with those of the other Ursidae family members. Multiple sequence alignment showed that the COX2 gene had three point mutations that defined three haplotypes, with 60% of the sequences corresponding to haplotype I. The neutrality tests revealed that the COX2 gene was conserved throughout evolution, and the maximum likelihood phylogenetic analysis, using homologous sequences from other Ursidae species, showed clustering of the COX2 sequences of giant pandas, suggesting that this gene evolved differently in them.

  19. Molecular characterization of cDNAs encoding G protein alpha and beta subunits and study of their temporal and spatial expression patterns in Nicotiana plumbaginifolia Viv. (United States)

    Kaydamov, C; Tewes, A; Adler, K; Manteuffel, R


    We have isolated cDNA sequences encoding alpha and beta subunits of potential G proteins from a cDNA library prepared from somatic embryos of Nicotiana plumbaginifolia Viv. at early developmental stages. The predicted NPGPA1 and NPGPB1 gene products are 75-98% identical to the known respective plant alpha and beta subunits. Southern hybridizations indicate that NPGPA1 is probably a single-copy gene, whereas at least two copies of NPGPB1 exist in the N. plumbaginifolia genome. Northern analyses reveal that both NPGPA1 and NPGPB1 mRNA are expressed in all embryogenic stages and plant tissues examined and their expression is obviously regulated by the plant hormone auxin. Immunohistological localization of NPGPalpha1 and NPGPbeta1 preferentially on plasma and endoplasmic reticulum membranes and their immunochemical detection exclusively in microsomal cell fractions implicate membrane association of both proteins. The temporal and spatial expression patterns of NPGPA1 and NPGPB1 show conformity as well as differences. This could account for not only cooperative, but also individual activities of both subunits during embryogenesis and plant development.

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

    Energy Technology Data Exchange (ETDEWEB)

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


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

  1. Interleukin-2 and subunit alpha of its soluble receptor in autoimmune Addison's disease--an association study and expression analysis. (United States)

    Fichna, Marta; Żurawek, Magdalena; Bratland, Eirik; Husebye, Eystein S; Kasperlik-Załuska, Anna; Czarnocka, Barbara; Januszkiewicz-Lewandowska, Danuta; Nowak, Jerzy


    Autoimmune Addison's disease (AAD) results from T cell-mediated destruction of the adrenal cortex, commonly accompanied by autoantibodies to 21-hydroxylase (21OH). In order to gain insight into the obscure aetiology of this disease, we investigated the roles of the IL2 and IL2RA genes, encoding interleukin-2 and subunit alpha of its receptor (IL2Ra), respectively. The association of AAD with IL2 and IL2RA polymorphisms (rs6822844, rs2069762, rs3136534, rs11594656, rs3118470 and rs2104286) was tested in 223 patients and 672 healthy controls. Functional studies consisted of gene expression analysis in cultured PBMCs exposed to 21OH and evaluation of serum interleukin by ELISA assays. The frequency of the minor C allele of rs3136534 was significantly decreased in AAD subjects compared to controls (OR 0.71; 95%CI 0.561-0.887; p = 0.003). Only AAD cells responded to 21OH with an elevated IL2 and IL2RA mRNA synthesis (p = 0.004 and p = 0.009 versus controls, respectively), paralleled by increased supernatant levels of both cytokines (p = 0.031 and p = 0.001 versus controls). IL2 mRNA level in 21OH-stimulated AAD PBMCs correlated negatively with age (p = 0.036) and positively with serum antibodies to 21OH (p = 0.006). Carriers of the rs2104286 AA genotype demonstrated higher IL2RA mRNA (p = 0.022) and soluble IL2Ra secretion (p = 0.029) upon 21OH stimulation. Serum interleukin-2 in AAD subjects was significantly higher compared to controls (4.61 ± 4.3 versus 1.71 ± 3.2 pg/mL, p < 0.001), whereas sIL2Ra levels remained similar in both groups (p = 0.885). In conclusion, the study reveals an association between AAD and IL2 locus. It confirms specific 21OH-directed reactivity of the peripheral AAD lymphocytes, which display increased synthesis of interleukin-2 and sIL2Ra.

  2. Upregulation of calcium channel alpha-2-delta-1 subunit in dorsal horn contributes to spinal cord injury-induced tactile allodynia. (United States)

    Kusuyama, Kazuki; Tachibana, Toshiya; Yamanaka, Hiroki; Okubo, Masamichi; Yoshiya, Shinichi; Noguchi, Koichi


    Spinal cord injury (SCI) commonly results not only in motor paralysis but also in the emergence of neuropathic pain (NeuP), both of which can impair the quality of life for patients with SCI. In the clinical field, it is well known that pregabalin, which binds to the voltage-gated calcium channel alpha-2-delta-1 (α 2 δ-1) subunit has therapeutic effects on NeuP after SCI. A previous study has demonstrated that SCI increased α 2 δ-1 in the L4-L6 dorsal spinal cord of SCI rats by Western blot analysis and that the increase of α 2 δ-1 was correlated with tactile allodynia of the hind paw. However, the detailed feature of an increase in α 2 δ-1 protein in the spinal dorsal horn and the mechanism of pregabalin effect on SCI-induced NeuP have not been fully examined. This study aimed to examine the detailed distribution of α 2 δ-1 expression in the lumbar spinal cord after thoracic SCI in rats and the correlation of the therapeutic effect of pregabalin in SCI rats. Male Sprague-Dawley rats underwent thoracic (T10) spinal cord contusion injury using the IH impactor device. Spinal cord injury rats received pregabalin (30 mg/kg) once a day for 2 weeks over a 4-week period after SCI. The mechanical threshold in the rat hind paw was measured over 4 weeks. Alpha-2-delta-1 expression in the lumbar spinal cord and in the dorsal root ganglion (DRG) was analyzed using immunohistochemistry and in situ hybridization histochemistry. A significant reduction of the withdrawal threshold of mechanical stimuli to the hind paw was observed for 2 weeks and continued at least 4 weeks after SCI. In the control rats, expression of α 2 δ-1 immunoreactivity was detected mainly in laminae I and II in the lumbar dorsal horn. Thoracic SCI significantly increased α 2 δ-1 immunoreactivity in laminae I and II in the lumbar dorsal horn 4 weeks after SCI; however, thoracic SCI did not affect the expression of α 2 δ-1 mRNA in the L4 and L5 DRGs. Meanwhile, the signal intensity of α 2

  3. Subunit Organization of a Synechocystis Hetero-Oligomeric Thylakoid FtsH Complex Involved in Photosystem II Repair

    Czech Academy of Sciences Publication Activity Database

    Boehm, M.; Yu, J.; Krynická, Vendula; Barker, M.; Tichý, Martin; Komenda, Josef; Nixon, P. J.; Nield, J.


    Roč. 24, č. 9 (2012), s. 3669-3683 ISSN 1040-4651 R&D Projects: GA MŠk(CZ) ED2.1.00/03.0110; GA ČR GBP501/12/G055 Institutional support: RVO:61388971 Keywords : photosystem II * FtsH metalloproteases * subunit Subject RIV: EE - Microbiology, Virology Impact factor: 9.251, year: 2012

  4. The host-dependent interaction of alpha-importins with influenza PB2 polymerase subunit is required for virus RNA replication.

    Directory of Open Access Journals (Sweden)

    Patricia Resa-Infante

    Full Text Available The influenza virus polymerase is formed by the PB1, PB2 and PA subunits and is required for virus transcription and replication in the nucleus of infected cells. As PB2 is a relevant host-range determinant we expressed a TAP-tagged PB2 in human cells and isolated intracellular complexes. Alpha-importin was identified as a PB2-associated factor by proteomic analyses. To study the relevance of this interaction for virus replication we mutated the PB2 NLS and analysed the phenotype of mutant subunits, polymerase complexes and RNPs. While mutant PB2 proteins showed reduced nuclear accumulation, they formed polymerase complexes normally when co expressed with PB1 and PA. However, mutant RNPs generated with a viral CAT replicon showed up to hundred-fold reduced CAT accumulation. Rescue of nuclear localisation of mutant PB2 by insertion of an additional SV40 TAg-derived NLS did not revert the mutant phenotype of RNPs. Furthermore, determination of recombinant RNP accumulation in vivo indicated that PB2 NLS mutations drastically reduced virus RNA replication. These results indicate that, above and beyond its role in nuclear accumulation, PB2 interaction with alpha-importins is required for virus RNA replication. To ascertain whether PB2-alpha-importin binding could contribute to the adaptation of H5N1 avian viruses to man, their association in vivo was determined. Human alpha importin isoforms associated efficiently to PB2 protein of an H3N2 human virus but bound to diminished and variable extents to PB2 from H5N1 avian or human strains, suggesting that the function of alpha importin during RNA replication is important for the adaptation of avian viruses to the human host.

  5. Cloning and identification of the gene coding for the 140-kd subunit of Drosophila RNA polymerase II


    Faust, Daniela M.; Renkawitz-Pohl, Renate; Falkenburg, Dieter; Gasch, Alexander; Bialojan, Siegfried; Young, Richard A.; Bautz, Ekkehard K. F.


    Genomic clones of Drosophila melanogaster were isolated from a λ library by cross-hybridization with the yeast gene coding for the 150-kd subunit of RNA polymerase II. Clones containing a region of ∼2.0 kb with strong homology to the yeast gene were shown to code for a 3.9-kb poly(A)+-RNA. Part of the coding region was cloned into an expression vector. A fusion protein was obtained which reacted with an antibody directed against RNA polymerase II of Drosophila. Peptide mapping of the fusion p...

  6. The amino-terminal 200 amino acids of the plasma membrane Na+,K+-ATPase alpha subunit confer ouabain sensitivity on the sarcoplasmic reticulum Ca(2+)-ATPase.


    Ishii, T; Takeyasu, K


    Cardiac glycosides such as G-strophanthin (ouabain) bind to and inhibit the plasma membrane Na+,K(+)-ATPase but not the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, whereas thapsigargin specifically blocks the SR Ca(2+)-ATPase. The chimera [n/c]CC, in which the amino-terminal amino acids Met1 to Asp162 of the SR Ca(2+)-ATPase (SERCA1) were replaced with the corresponding portion of the Na+,K(+)-ATPase alpha 1 subunit (Met1 to Asp200), retained thapsigargin- and Ca(2+)-sensitive ATPase activity,...

  7. The gene for the alpha 1 subunit of the skeletal muscle dihydropyridine-sensitive calcium channel (Cchl1a3) maps to mouse chromosome 1. (United States)

    Chin, H; Krall, M; Kim, H L; Kozak, C A; Mock, B


    Cchl1a3 encodes the dihydropyridine-sensitive calcium channel alpha 1 subunit isoform predominantly expressed in skeletal muscle. mdg (muscular dysgenesis) has previously been implicated as a mutant allele of this gene. Hybridization of a rat brain cDNA probe for Cchl1a3 to Southern blots of DNAs from a panel of Chinese hamster x mouse somatic cell hybrids suggested that this gene maps to mouse Chromosome 1. Analysis of the progeny of an inbred strain cross-positioned Cchl1a3 1.3 cM proximal to the Pep-3 locus on Chr 1.

  8. Reduced volume and increased training intensity elevate muscle Na+/K+ pump {alpha}2-subunit expression as well as short- and long-term work capacity in humans

    DEFF Research Database (Denmark)

    Bangsbo, Jens; Gunnarsson, Thomas Petursson; Wendell, Jesper


    was unaltered, but the 3-K (3,000 m) time was reduced (Pexpression and performance remained unaltered in CON. The present data suggest that both short- and long-term......% reduction in the amount of training but including speed endurance training consisting of 6-12 30-s sprint runs 3-4 times a week (SET, n=12) or a control group (CON, n=5), which continued the endurance training (about 55 km(.)wk(-1)). For SET the expression of the muscle Na(+)/K(+) pump alpha2-subunit was 68...

  9. Effects of cigarette smoke exposure on nicotinic acetylcholine receptor subunits {alpha}7 and {beta}2 in the sudden infant death syndrome (SIDS) brainstem

    Energy Technology Data Exchange (ETDEWEB)

    Machaalani, Rita, E-mail: [Department of Medicine, The University of Sydney, NSW 2006 (Australia); Bosch Institute, The University of Sydney, NSW 2006 (Australia); The Children' s Hospital at Westmead, NSW 2145 (Australia); Say, Meichien [Department of Medicine, The University of Sydney, NSW 2006 (Australia); Bosch Institute, The University of Sydney, NSW 2006 (Australia); Waters, Karen A. [Department of Medicine, The University of Sydney, NSW 2006 (Australia); Bosch Institute, The University of Sydney, NSW 2006 (Australia); The Children' s Hospital at Westmead, NSW 2145 (Australia)


    It is postulated that nicotine, as the main neurotoxic constituent of cigarette smoke, influences SIDS risk through effects on nicotinic acetylcholine receptors (nAChRs) in brainstem nuclei that control respiration and arousal. This study compared {alpha}7 and {beta}2 nAChR subunit expression in eight nuclei of the caudal and rostral medulla and seven nuclei of the pons between SIDS (n = 46) and non-SIDS infants (n = 14). Evaluation for associations with known SIDS risk factors included comparison according to whether infants had a history of exposure to cigarette smoke in the home, and stratification for sleep position and gender. Compared to non-SIDS infants, SIDS infants had significantly decreased {alpha}7 in the caudal nucleus of the solitary tract (cNTS), gracile and cuneate nuclei, with decreased {beta}2 in the cNTS and increased {beta}2 in the facial. When considering only the SIDS cohort: 1-cigarette smoke exposure was associated with increased {alpha}7 in the vestibular nucleus and increased {beta}2 in the rostral dorsal motor nucleus of the vagus, rNTS and Cuneate, 2-there was a gender interaction for {alpha}7 in the gracile and cuneate, and {beta}2 in the cNTS and rostral arcuate nucleus, and 3-there was no effect of sleep position on {alpha}7, but prone sleep was associated with decreased {beta}2 in three nuclei of the pons. In conclusion, SIDS infants demonstrate differences in expression of {alpha}7 and {beta}2 nAChRs within brainstem nuclei that control respiration and arousal, which is independent on prior history of cigarette smoke exposure, especially for the NTS, with additional differences for smoke exposure ({beta}2), gender ({alpha}7 and {beta}2) and sleep position ({beta}2) evident. -- Highlights: Black-Right-Pointing-Pointer The 'normal' response to smoke exposure is decreased {alpha}7 and {beta}2 in certain nuclei. Black-Right-Pointing-Pointer SIDS infants have decreased {alpha}7 in cNTS, Grac and Cun. Black

  10. Expression of alpha and beta subunit isoforms of Na,K-ATPase in the mouse inner ear and changes with mutations at the Wv or Sld loci. (United States)

    Schulte, B A; Steel, K P


    Mice homozygous for mutations at the viable dominant spotting (Wv) and Steel-dickie (Sld) loci exhibit a similar phenotype which includes deafness. The auditory dysfunction derives from failure of the stria vascularis to develop normally and to generate a high positive endocochlear potential (EP). Because strial function is driven by Na,K-ATPase its expression was investigated in inner ears of Wv/Wv and Sld/Sld mice and their wild-type littermates by immunostaining with antisera against four of the enzyme's subunit isoforms. Wild-type mice from two different genetic backgrounds showed an identical distribution of subunit isoforms among inner ear transport cells. Several epithelial cell types coexpressed the alpha 1 and beta 1 subunits. Vestibular dark cells showed no reactivity for beta 1 but expressed abundant beta 2, whereas, strial marginal cells stained strongly for both beta isoforms. The only qualitative difference between mutant and wild-type mice was the absence of beta 1 subunit in marginal cells of the mutant's stria. However, it is unlikely that this difference accounts for failure of mutants to generate a high EP because the beta 1 subunit is not present in the stria vascularis of either rats or gerbils with normal EP values. Strong immunostaining for Na,K-ATPase in lateral wall fibrocytes of normal mice along with diminished immunoreactivity in the mutants supports the concept that these strategically located transport fibrocytes actively resorb K+ leaked across Reissner's membrane into scala vestibuli or effluxed from hair cells and nerves into scala tympani. It is further speculated that the resorbed K+ normally is siphoned down its concentration gradient into the intrastrial space through gap junctions between fibrocytes and strial basal and intermediate cells where it is recycled back to endolymph via marginal cells. Thus, failure of mutants to generate a positive EP could be explained by the absence of intermediate cells which may form the final

  11. Mice Lacking the Alpha9 Subunit of the Nicotinic Acetylcholine Receptor Exhibit Deficits in Frequency Difference Limens and Sound Localization

    Directory of Open Access Journals (Sweden)

    Amanda Clause


    Full Text Available Sound processing in the cochlea is modulated by cholinergic efferent axons arising from medial olivocochlear neurons in the brainstem. These axons contact outer hair cells in the mature cochlea and inner hair cells during development and activate nicotinic acetylcholine receptors composed of α9 and α10 subunits. The α9 subunit is necessary for mediating the effects of acetylcholine on hair cells as genetic deletion of the α9 subunit results in functional cholinergic de-efferentation of the cochlea. Cholinergic modulation of spontaneous cochlear activity before hearing onset is important for the maturation of central auditory circuits. In α9KO mice, the developmental refinement of inhibitory afferents to the lateral superior olive is disturbed, resulting in decreased tonotopic organization of this sound localization nucleus. In this study, we used behavioral tests to investigate whether the circuit anomalies in α9KO mice correlate with sound localization or sound frequency processing. Using a conditioned lick suppression task to measure sound localization, we found that three out of four α9KO mice showed impaired minimum audible angles. Using a prepulse inhibition of the acoustic startle response paradigm, we found that the ability of α9KO mice to detect sound frequency changes was impaired, whereas their ability to detect sound intensity changes was not. These results demonstrate that cholinergic, nicotinic α9 subunit mediated transmission in the developing cochlear plays an important role in the maturation of hearing.

  12. A double mutation in exon 6 of the [beta]-hexosaminidase [alpha] subunit in a patient with the B1 variant of Tay-Sachs disease

    Energy Technology Data Exchange (ETDEWEB)

    Ainsworth, P.J. (Univ. of Western Ontario, Ontario (Canada) Child Health Research Institute, London, Ontario (Canada)); Coulter-Mackie, M.B. (Univ. of Western Ontario, Ontario (Canada) Child Health Research Institute, London, Ontario (Canada) Children' s Psychiatric Research Institute, London, Ontario (Canada))


    The B1 variant form of Tay-Sachs disease is enzymologically unique in that the causative mutation(s) appear to affect the active site in the [alpha] subunit of [beta]-hexosaminidase A without altering its ability to associate with the [beta] subunit. Most previously reported B1 variant mutations were found in exon 5 within codon 178. The coding sequence of the [alpha] subunit gene of a patient with the B1 variant form was examined with a combination of reverse transcription of mRNA to cDNA, PCR, and dideoxy sequencing. A double mutation in exon 6 has been identified: a G[sub 574][yields]C transversion causing a val[sub 192][yields]leu change and a G[sub 598][yields] A transition resulting in a val[sub 200][yields]met alteration. The amplified cDNAs were otherwise normal throughout their sequence. The 574 and 598 alterations have been confirmed by amplification directly from genomic DNA from the patient and her mother. Transient-expression studies of the two exon 6 mutations (singly or together) in COS-1 cells show that the G[sub 574][yields]C change is sufficient to cause the loss of enzyme activity. The biochemical phenotype of the 574 alteration in transfection studies is consistent with that expected for a B1 variant mutation. As such, this mutation differs from previously reported B1 variant mutations, all of which occur in exon 5. 31 refs., 2 figs., 2 tabs.

  13. Evidence that the primary effect of phosphorylation of eukaryotic initiation factor 2(alpha) in rabbit reticulocyte lysate is inhibition of the release of eukaryotic initiation factor-2.GDP from 60 S ribosomal subunits

    International Nuclear Information System (INIS)

    Gross, M.; Redman, R.; Kaplansky, D.A.


    The phosphorylation of eukaryotic initiation factor (eIF) 2 alpha that occurs when rabbit reticulocyte lysate is incubated in the absence of hemin or with poly(I.C) causes inhibition of polypeptide chain initiation by preventing a separate factor (termed RF) from promoting the exchange of GTP for GDP on eIF-2. When lysate was incubated in the presence of hemin and [ 14 C] eIF-2 or [alpha- 32 P]GTP, the authors observed binding of eIF-2 and GDP or GTP to 60 S ribosomal subunits that was slightly greater than that bound to 40 S subunits and little binding to 80 S ribosomes. When incubation was in the absence of hemin or in the presence of hemin plus 0.1 microgram/ml poly(I.C), eIF-2 and GDP binding to 60 S subunits was increased 1.5- to 2-fold, that bound to 80 S ribosomes was almost as great as that bound to 60 S subunits, and that bound to 40 S subunits was unchanged. The data indicate that about 40% of the eIF-2 that becomes bound to 60 S subunits and 80 S ribosomes in the absence of hemin or with poly(I.C) is eIF-2(alpha-P) and suggest that the eIF-2 and GDP bound is probably in the form of a binary complex. The rate of turnover of GDP (presumably eIF-2.GDP) on 60 S subunits and 80 S ribosomes in the absence of hemin is reduced to less than 10% the control rate, because the dissociation of eIF-2.GDP is inhibited. Our findings suggest that eIF-2.GTP binding to and eIF-2.GDP release from 60 S subunits may normally occur and serve to promote subunit joining

  14. Genetics Home Reference: mucolipidosis II alpha/beta (United States)

    ... Mucolipidosis II (I-cell disease) and mucolipidosis IIIA (classical pseudo-hurler polydystrophy) are caused by mutations in ... N-acetylglucosamine-1-phosphotransferase gene (GNPTAB) in a French Canadian founder population. Clin Genet. 2008 Mar;73( ...

  15. Probing HeII Reionization at z>3.5 with Resolved HeII Lyman Alpha Forest Spectra (United States)

    Worseck, Gabor


    The advent of GALEX and COS have revolutionized our view of HeII reionization, the final major phase transition of the intergalactic medium. COS spectra of the HeII Lyman alpha forest have confirmed with high confidence the high HeII transmission that signifies the completion of HeII reionization at z 2.7. However, the handful of z>3.5 quasars observed to date show a set of HeII transmission 'spikes' and larger regions with non-zero transmission that suggest HeII reionization was well underway by z=4. This is in striking conflict with predictions from state-of-the-art radiative transfer simulations of a HeII reionization driven by bright quasars. Explaining these measurements may require either faint quasars or more exotic sources of hard photons at z>4, with concomitant implications for HI reionization. However, many of the observed spikes are unresolved in G140L spectra and are significantly impacted by Poisson noise. Current data cannot reliably probe the ionization state of helium at z>3.5.We request 41 orbits to obtain science-grade G130M spectra of the two UV-brightest HeII-transmitting QSOs at z>3.5 to confirm and resolve their HeII transmission spikes as an unequivocal test of early HeII reionization. These spectra are complemented by recently obtained data from 8m telescopes: (1) Echelle spectra of the coeval HI Lya forest to map the underlying density field that modulates the HeII absorption, and (2) Our dedicated survey for foreground QSOs that may source the HeII transmission. Our recent HST programs revealed the only two viable targets to resolve the z>3.5 HeII Lyman alpha forest, and to conclusively solve this riddle.

  16. Cholera toxin B subunit labeling in lamina II of spinal cord dorsal horn following chronic inflammation in rats. (United States)

    Ma, Qing Ping; Tian, Li


    We have investigated the effect of inflammation on the labeling pattern of cholera toxin B subunit (CTB)-conjugated horseradish peroxidase, an A-fiber marker, by an intra-sciatic nerve injection of the tracer. Following chronic inflammation in one hind paw in rats, there was substantial CTB labeling in lamina II of the spinal dorsal horn, which is normally absent. However, there was no change in the labeling pattern of wheat germ agglutinin or fluoride resistant acid phosphatase/thiamine monophosphatase, two C-fiber markers. The CTB labeling in lamina II after peripheral nerve injury has been interpreted as central sprouting of A-fibers or uptake of the tracer by injured C-fibers. Our results suggest that chronic inflammation and nerve injury may share some common mechanisms in generating allodynia and hyperalgesia.

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

    Directory of Open Access Journals (Sweden)

    A Mobascher

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

  18. Comparative study of the distribution of the alpha-subunits of voltage-gated sodium channels in normal and axotomized rat dorsal root ganglion neurons. (United States)

    Fukuoka, Tetsuo; Kobayashi, Kimiko; Yamanaka, Hiroki; Obata, Koichi; Dai, Yi; Noguchi, Koichi


    We compared the distribution of the alpha-subunit mRNAs of voltage-gated sodium channels Nav1.1-1.3 and Nav1.6-1.9 and a related channel, Nax, in histochemically identified neuronal subpopulations of the rat dorsal root ganglia (DRG). In the naïve DRG, the expression of Nav1.1 and Nav1.6 was restricted to A-fiber neurons, and they were preferentially expressed by TrkC neurons, suggesting that proprioceptive neurons possess these channels. Nav1.7, -1.8, and -1.9 mRNAs were more abundant in C-fiber neurons compared with A-fiber ones. Nax was evenly expressed in both populations. Although Nav1.8 and -1.9 were preferentially expressed by TrkA neurons, other alpha-subunits were expressed independently of TrkA expression. Actually, all IB4(+) neurons expressed both Nav1.8 and -1.9, and relatively limited subpopulations of IB4(+) neurons (3% and 12%, respectively) expressed Nav1.1 and/or Nav1.6. These findings provide useful information in interpreting the electrophysiological characteristics of some neuronal subpopulations of naïve DRG. After L5 spinal nerve ligation, Nav1.3 mRNA was up-regulated mainly in A-fiber neurons in the ipsilateral L5 DRG. Although previous studies demonstrated that nerve growth factor (NGF) and glial cell-derived neurotrophic factor (GDNF) reversed this up-regulation, the Nav1.3 induction was independent of either TrkA or GFRalpha1 expression, suggesting that the induction of Nav1.3 may be one of the common responses of axotomized DRG neurons without a direct relationship to NGF/GDNF supply. (c) 2008 Wiley-Liss, Inc.

  19. Structure-function relationships in the Na,K-ATPase. cap alpha. subunit: site-directed mutagenesis of glutamine-111 to arginine and asparagine-122 to aspartic acid generates a ouabain-resistant enzyme

    Energy Technology Data Exchange (ETDEWEB)

    Price, E.M.; Lingrel, J.B.


    Na,K-ATPases from various species differ greatly in their sensitivity to cardiac glycosides such as ouabain. The sheep and human enzymes are a thousand times more sensitive than the corresponding ones from rat and mouse. To define the region of the ..cap alpha..1 subunit responsible for this differential sensitivity, chimeric cDNAs of sheep and rat were constructed and expressed in ouabain-sensitive HeLa cells. The construct containing the amino-terminal half of the rat ..cap alpha..1 subunit coding region and carboxyl-terminal half of the sheep conferred the ouabain-resistant phenotype to HeLa cells while the reverse construct did not. This indicates that the determinants involved in ouabain sensitivity are located in the amino-terminal half of the Na,K-ATPase ..cap alpha.. subunit. By use of site-directed mutagenesis, the amino acid sequence of the first extracellular domain (H1-H2) of the sheep ..cap alpha..1 subunit was changed to that of the rat. When expressed in HeLa cells, this mutated sheep ..cap alpha..1 construct, like the rat/sheep chimera, was able to confer ouabain resistance to these cells. Furthermore, similar results were observed when HeLa cells were transfected with a sheep ..cap alpha..1 cDNA containing only two amino acid substitutions. The resistant cells, whether transfected with the rat ..cap alpha..1 cDNA, the rat/sheep chimera, or the mutant sheep ..cap alpha..1 cDNAs, exhibited identical biochemical characteristics including ouabain-inhibitable cell growth, /sup 86/Rb/sup +/ uptake, and Na,K-ATPase activity. These results demonstrate that the presence of arginine and aspartic acid on the amino end and carboxyl end, respectively, of the H1-H2 extracellular domain of the Na,K-ATPase ..cap alpha.. subunit together is responsible for the ouabain-resistant character of the rat enzyme and the corresponding residues in the sheep ..cap alpha..1 subunit (glutamine and asparagine) are somehow involved in ouabain binding.

  20. Characterization of class II alpha genes and DLA-D region allelic associations in the dog. (United States)

    Sarmiento, U M; Storb, R F


    Human major histocompatibility complex (HLA) cDNA probes were used to analyze the restriction fragment length polymorphism (RFLP) of the alpha genes of the DLA-D region in dogs. Genomic DNA from peripheral blood leucocytes of 23 unrelated DLA-D homozygous dogs representing nine DLA-D types (defined by mixed leucocyte reaction) was digested with restriction enzymes (BamHI, EcoRI, Hind III, Pvu II, Taq I, Rsa I, Msp I, Pst I and Bgl II), separated by agarose gel electrophoresis and transferred onto Biotrace membrane. The Southern blots were successively hybridized with radiolabelled HLA cDNA probes corresponding to DQ, DP, DZ and DR alpha genes. Clear evidence was obtained for the canine homologues of DQ and DR alpha genes with simple bi- or tri-allelic polymorphism respectively. Evidence for a single, nonpolymorphic DP alpha gene was also obtained. However, the presence of a DZ alpha gene could not be clearly demonstrated in canine genomic DNA. This report extends our previous RFLP analysis documenting polymorphism of DLA class II beta genes in the same panel of homozygous typing cell dogs, and provides the basis for DLA-D genotyping at a population level. This study also characterizes the RFLP-defined preferential allelic associations across the DLA-D region in nine different homozygous typing cell specificities.

  1. Ablation of phosphoinositide-3-kinase class II alpha suppresses hepatoma cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Ng, Stanley K.L. [Singapore Immunology Network A-STAR (Singapore); Neo, Soek-Ying, E-mail: [Singapore Immunology Network A-STAR (Singapore); Yap, Yann-Wan [Singapore Immunology Network A-STAR (Singapore); Karuturi, R. Krishna Murthy; Loh, Evelyn S.L. [Genome Institute of Singapore A-STAR (Singapore); Liau, Kui-Hin [Department of General Surgery, Tan Tock Seng Hospital (Singapore); Ren, Ee-Chee, E-mail: [Singapore Immunology Network A-STAR (Singapore); Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore (Singapore)


    Cancer such as hepatocellular carcinoma (HCC) is characterized by complex perturbations in multiple signaling pathways, including the phosphoinositide-3-kinase (PI3K/AKT) pathways. Herein we investigated the role of PI3K catalytic isoforms, particularly class II isoforms in HCC proliferation. Among the siRNAs tested against the eight known catalytic PI3K isoforms, specific ablation of class II PI3K alpha (PIK3C2{alpha}) was the most effective in impairing cell growth and this was accompanied by concomitant decrease in PIK3C2{alpha} mRNA and protein levels. Colony formation ability of cells deficient for PIK3C2{alpha} was markedly reduced and growth arrest was associated with increased caspase 3 levels. A small but significant difference in gene dosage and expression levels was detected between tumor and non-tumor tissues in a cohort of 19 HCC patients. Taken together, these data suggest for the first time that in addition to class I PI3Ks in cancer, class II PIK3C2{alpha} can modulate HCC cell growth.

  2. Does adjuvant systemic therapy with interferon-alpha for stage II-III melanoma prolong survival?

    NARCIS (Netherlands)

    Eggermont, Alexander M. M.; Punt, Cornelis J. A.


    The experience with interferon-alpha in malignant melanoma resembles, to some degree, the experience with various kinds of adjuvant immunotherapeutic agents where 25 years of phase III trials of adjuvant therapy in stage II-IIII melanoma have not defined a standard therapy. Most trials failed to

  3. Constitutive Activation of the G-Protein Subunit G[alpha]s within Forebrain Neurons Causes PKA-Dependent Alterations in Fear Conditioning and Cortical "Arc" mRNA Expression (United States)

    Kelly, Michele P.; Cheung, York-Fong; Favilla, Christopher; Siegel, Steven J.; Kanes, Stephen J.; Houslay, Miles D.; Abel, Ted


    Memory formation requires cAMP signaling; thus, this cascade has been of great interest in the search for cognitive enhancers. Given that medications are administered long-term, we determined the effects of chronically increasing cAMP synthesis in the brain by expressing a constitutively active isoform of the G-protein subunit G[alpha]s…

  4. Zolpidem, a selective GABA(A) receptor alpha1 subunit agonist, induces comparable Fos expression in oxytocinergic neurons of the hypothalamic paraventricular and accessory but not supraoptic nuclei in the rat

    DEFF Research Database (Denmark)

    Kiss, Alexander; Søderman, Andreas; Bundzikova, Jana


    Functional activation of oxytocinergic (OXY) cells in the hypothalamic paraventricular (PVN), supraoptic (SON), and accessory (ACC) nuclei was investigated in response to acute treatment with Zolpidem (a GABA(A) receptor agonist with selectivity for alpha(1) subunits) utilizing dual Fos/OXY immun...

  5. Altered expression of the voltage-gated calcium channel subunit alpha(2)delta-1: A comparison between two experimental models of epilepsy and a sensory nerve ligation model of neuropathic pain

    Czech Academy of Sciences Publication Activity Database

    Nieto-Rostro, M.; Sandhu, G.; Bauer, C. S.; Jiruška, Přemysl; Jefferys, J. G. R.; Dolphin, A. C.


    Roč. 283, Dec (2014), s. 124-137 ISSN 0306-4522 R&D Projects: GA MZd(CZ) NT14489 Institutional support: RVO:67985823 Keywords : calcium channel * dorsal root ganglion (DRG) * alpha2delta subunit * epilepsy * neuropathic pain * reactive gliosis Subject RIV: FH - Neurology Impact factor: 3.357, year: 2014

  6. The Val{sup 192}Leu mutation in the {alpha}-subunit of {beta}-hexosaminidase A is not associated with the B1-variant form of Tay-Sachs disease

    Energy Technology Data Exchange (ETDEWEB)

    Hou, Y.; Vavougios, G.; Hinek, A. [Univ. of Toronto (Canada)] [and others


    Substitution mutations adversely affecting the {alpha}-subunit of {beta}-hexosaminidase A ({alpha}{beta}) (EC result in Tay-Sachs disease. The majority affect the initial folding of the pro-{alpha} chain in the endoplasmic reticulum, resulting in its retention and degradation. A much less common occurrence is a mutation that specifically affects an {open_quotes}active-site{close_quotes} residue necessary for substrate binding and/or catalysis. In this case, hexosaminidase A is present in the lysosome, but it lacks all {alpha}-specific activity. This biochemical phenotype is referred to as the {open_quotes}B1-variant form{close_quotes} of Tay-Sachs disease. Kinetic analysis of suspected B1-variant mutations is complex because hexosaminidase A is heterodimeric and both subunits possess similar active sites. In this report, we examine a previously identified B1-variant mutation, {alpha}-Val{sup 192}Leu. Chinese hamster ovary cells were permanently cotransfected with an {alpha}-cDNA-construct encoding the substitution and a mutant {beta}-cDNA ({beta}-Arg{sup 211}Lys), encoding a {beta}-subunit that is inactive but normal in all other respects. We were surprised to find that the Val{sup 192}Leu substitution produced a pro-{alpha} chain that did not form {alpha}-{beta} dimers and was not transported to the lysosome. Finally, we reexamined the hexosaminidase activity and protein levels in the fibroblasts from the original patient. These data were also not consistent with the biochemical phenotype of the B1 variant of Tay-Sachs disease previously reported to be present. Thus, we conclude that the Val{sup 192}Leu substitution does not specifically affect the {alpha}-active site. 23 refs., 4 figs., 2 tabs.

  7. Delayed internalization and lack of recycling in a beta2-adrenergic receptor fused to the G protein alpha-subunit

    Directory of Open Access Journals (Sweden)

    Floridi Aristide


    Full Text Available Abstract Background Chimeric proteins obtained by the fusion of a G protein-coupled receptor (GPCR sequence to the N-terminus of the G protein α-subunit have been extensively used to investigate several aspects of GPCR signalling. Although both the receptor and the G protein generally maintain a fully functional state in such polypeptides, original observations made using a chimera between the β2-adrenergic receptor (β2AR and Gαs indicated that the fusion to the α-subunit resulted in a marked reduction of receptor desensitization and down-regulation. To further investigate this phenomenon, we have compared the rates of internalization and recycling between wild-type and Gαs-fused β2AR. Results The rate of agonist-induced internalization, measured as the disappearance of cell surface immunofluorescence in HEK293 cells permanently expressing N-terminus tagged receptors, was reduced three-fold by receptor-G protein fusion. However, both fused and non-fused receptors translocated to the same endocytic compartment, as determined by dual-label confocal analysis of cells co-expressing both proteins and transferrin co-localization. Receptor recycling, determined as the reversion of surface immunofluorescence following the addition of antagonist to cells that were previously exposed to agonist, markedly differed between wild-type and fused receptors. While most of the internalized β2AR returned rapidly to the plasma membrane, β2AR-Gαs did not recycle, and the observed slow recovery for the fusion protein immunofluorescence was entirely accounted for by protein synthesis. Conclusion The covalent linkage between β2AR and Gαs does not appear to alter the initial endocytic translocation of the two proteins, although there is reduced efficiency. It does, however, completely disrupt the process of receptor and G protein recycling. We conclude that the physical separation between receptor and Gα is not necessary for the transit to early endosomes

  8. Metallothionein 2A affects the cell respiration by suppressing the expression of mitochondrial protein cytochrome c oxidase subunit II. (United States)

    Bragina, Olga; Gurjanova, Karina; Krishtal, Jekaterina; Kulp, Maria; Karro, Niina; Tõugu, Vello; Palumaa, Peep


    Metallothioneins (MT) are involved in a broad range of cellular processes and play a major role in protection of cells towards various stressors. Two functions of MTs, namely the maintaining of the homeostasis of transition metal ions and the redox balance, are directly linked to the functioning of mitochondria. Dyshomeostasis of MTs is often related with malfunctioning of mitochondria; however, the mechanism by which MTs affect the mitochondrial respiratory chain is still unknown. We demonstrated that overexpression of MT-2A in HEK cell line decreased the oxidative phosphorylation capacity of the cells. HEK cells overexpressing MT-2A demonstrated reduced oxygen consumption and lower cellular ATP levels. MT-2A did not affect the number of mitochondria, but reduced specifically the level of cytochrome c oxidase subunit II protein, which resulted in lower activity of the complex IV.

  9. The Ca II resonance lines in M dwarf stars without H-alpha emission

    Energy Technology Data Exchange (ETDEWEB)

    Giampapa, M.S.; Cram, L.E.; Wild, W.J. (National Solar Observatory, Tucson, AZ (USA) Sydney Univ. (Australia) Arizona Univ., Tucson (USA))


    Spectra of the Ca II H and K lines in a sample of 31 M dwarf stars without H-alpha emission are used to calculate chromospheric K line radiative losses, F(k), and to study the joint response of Ca II K and H-alpha to chromospheric heating in dwarf M stars. It is suggested that the poor correlation found in the equivalent width - log F(K) diagram may be due either to radial segregation of the H-alpha and K line forming regions or to lateral inhomogeneities in the chromospheres. The results confirm the existence of dM stars with weak H-alpha absorption and K line emission only slightly weaker than that of the dMe stars, and show that dM stars with weak H-alpha but kinematics and metallicities representative of the young disk population belong to a class characterized by a comparatively high degree of chromospheric activity. 32 refs.

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

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


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

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

    Directory of Open Access Journals (Sweden)

    Pascal Jorratt


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

  12. Dominant Red Coat Color in Holstein Cattle Is Associated with a Missense Mutation in the Coatomer Protein Complex, Subunit Alpha (COPA Gene.

    Directory of Open Access Journals (Sweden)

    Ben Dorshorst

    Full Text Available Coat color in Holstein dairy cattle is primarily controlled by the melanocortin 1 receptor (MC1R gene, a central determinant of black (eumelanin vs. red/brown pheomelanin synthesis across animal species. The major MC1R alleles in Holsteins are Dominant Black (MC1RD and Recessive Red (MC1Re. A novel form of dominant red coat color was first observed in an animal born in 1980. The mutation underlying this phenotype was named Dominant Red and is epistatic to the constitutively activated MC1RD. Here we show that a missense mutation in the coatomer protein complex, subunit alpha (COPA, a gene with previously no known role in pigmentation synthesis, is completely associated with Dominant Red in Holstein dairy cattle. The mutation results in an arginine to cysteine substitution at an amino acid residue completely conserved across eukaryotes. Despite this high level of conservation we show that both heterozygotes and homozygotes are healthy and viable. Analysis of hair pigment composition shows that the Dominant Red phenotype is similar to the MC1R Recessive Red phenotype, although less effective at reducing eumelanin synthesis. RNA-seq data similarly show that Dominant Red animals achieve predominantly pheomelanin synthesis by downregulating genes normally required for eumelanin synthesis. COPA is a component of the coat protein I seven subunit complex that is involved with retrograde and cis-Golgi intracellular coated vesicle transport of both protein and RNA cargo. This suggests that Dominant Red may be caused by aberrant MC1R protein or mRNA trafficking within the highly compartmentalized melanocyte, mimicking the effect of the Recessive Red loss of function MC1R allele.

  13. GABA-A receptor beta3 and alpha5 subunit gene cluster on chromosome 15q11-q13 and bipolar disorder: a genetic association study. (United States)

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


    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.

  14. Selective Attention to Visual Stimuli Using Auditory Distractors Is Altered in Alpha-9 Nicotinic Receptor Subunit Knock-Out Mice. (United States)

    Terreros, Gonzalo; Jorratt, Pascal; Aedo, Cristian; Elgoyhen, Ana Belén; Delano, Paul H


    During selective attention, subjects voluntarily focus their cognitive resources on a specific stimulus while ignoring others. Top-down filtering of peripheral sensory responses by higher structures of the brain has been proposed as one of the mechanisms responsible for selective attention. A prerequisite to accomplish top-down modulation of the activity of peripheral structures is the presence of corticofugal pathways. The mammalian auditory efferent system is a unique neural network that originates in the auditory cortex and projects to the cochlear receptor through the olivocochlear bundle, and it has been proposed to function as a top-down filter of peripheral auditory responses during attention to cross-modal stimuli. However, to date, there is no conclusive evidence of the involvement of olivocochlear neurons in selective attention paradigms. Here, we trained wild-type and α-9 nicotinic receptor subunit knock-out (KO) mice, which lack cholinergic transmission between medial olivocochlear neurons and outer hair cells, in a two-choice visual discrimination task and studied the behavioral consequences of adding different types of auditory distractors. In addition, we evaluated the effects of contralateral noise on auditory nerve responses as a measure of the individual strength of the olivocochlear reflex. We demonstrate that KO mice have a reduced olivocochlear reflex strength and perform poorly in a visual selective attention paradigm. These results confirm that an intact medial olivocochlear transmission aids in ignoring auditory distraction during selective attention to visual stimuli. The auditory efferent system is a neural network that originates in the auditory cortex and projects to the cochlear receptor through the olivocochlear system. It has been proposed to function as a top-down filter of peripheral auditory responses during attention to cross-modal stimuli. However, to date, there is no conclusive evidence of the involvement of olivocochlear

  15. Safety and immunogenicity of three different formulations of an adjuvanted varicella-zoster virus subunit candidate vaccine in older adults: a phase II, randomized, controlled study

    NARCIS (Netherlands)

    Chlibek, Roman; Smetana, Jan; Pauksens, Karlis; Rombo, Lars; van den Hoek, J. Anneke R.; Richardus, Jan H.; Plassmann, Georg; Schwarz, Tino F.; Ledent, Edouard; Heineman, Thomas C.


    This study investigated the safety and immunogenicity of different formulations and schedules of a candidate subunit herpes zoster vaccine containing varicella-zoster virus glycoprotein E (gE) with or without the adjuvant system AS01B. In this phase II, single-blind, randomized, controlled study,


    NARCIS (Netherlands)


    The crystal structure of Limulus polyphemus subunit type II hemocyanin in the deoxygenated state has been determined to a resolution of 2.18 angstrom. Phase information for this first structure of a cheliceratan hemocyanin was obtained by molecular replacement using the crustacean hemocyanin

  17. Mapping of the human cone transducin {alpha} subunit (GNAT2) gene to 1p13 and mutation analysis in patients with Stargardt`s disease

    Energy Technology Data Exchange (ETDEWEB)

    Magovcevic, I.; Weremowicz, S.; Morton, C.C. [Harvard Medical School, Boston, MA (United States)] [and others


    Transducin {alpha} subunits are members of a large family of G-proteins and play an important role in phototransduction in rod and cone photoreceptors. We report the localization of the human cone {alpha} transducin (GNAT2) gene using fluorescence in situ hybridization (FISH) on chromosome 1 in band p13. The recent assignment of a gene for Stargardt`s disease to the same chromosomal region by linkage analysis prompted us to investigate the possible role of GNAT2 in the pathogenesis of this disease. Stargardt`s disease is characterized by degeneration in late childhood or early adulthood of the macula of the retina, a region rich in cones. We screened patients with Stargardt`s disease, with or without peripheral cone involvement as monitored by the full-field ERG, for mutations in this gene. We investigated 66 unrelated patients including 22 with peripheral cone dysfunction for mutations in the coding region of the GNAT2 gene using polymerase chain reaction-single strand conformation polymorphism analysis (SSCP) and direct sequencing. One patient (034-16) was heterozygous for a silent change in exon VI, Asp238Asp (GAT to GAC). Two patients, one (035-005) with peripheral cone involvement and one (071-001) without peripheral cone involvement, were heterozygous for the missense change Val124Met (GTG to ATG) in exon IV. A subsequent screen of 96 unrelated, unaffected controls revealed one individual (N10) who was also heterozygous for the Val124Met alteration. We concluded that Asp238Asp and Val124Met are rare variants not causing Stargardt`s disease. Hence, no disease-specific mutations were found indicating that GNAT2 is probably not involved in the pathogenesis of most cases of Stargardt`s disease.

  18. The Arabidopsis mediator complex subunits MED16, MED14, and MED2 regulate mediator and RNA polymerase II recruitment to CBF-responsive cold-regulated genes. (United States)

    Hemsley, Piers A; Hurst, Charlotte H; Kaliyadasa, Ewon; Lamb, Rebecca; Knight, Marc R; De Cothi, Elizabeth A; Steele, John F; Knight, Heather


    The Mediator16 (MED16; formerly termed SENSITIVE TO FREEZING6 [SFR6]) subunit of the plant Mediator transcriptional coactivator complex regulates cold-responsive gene expression in Arabidopsis thaliana, acting downstream of the C-repeat binding factor (CBF) transcription factors to recruit the core Mediator complex to cold-regulated genes. Here, we use loss-of-function mutants to show that RNA polymerase II recruitment to CBF-responsive cold-regulated genes requires MED16, MED2, and MED14 subunits. Transcription of genes known to be regulated via CBFs binding to the C-repeat motif/drought-responsive element promoter motif requires all three Mediator subunits, as does cold acclimation-induced freezing tolerance. In addition, these three subunits are required for low temperature-induced expression of some other, but not all, cold-responsive genes, including genes that are not known targets of CBFs. Genes inducible by darkness also required MED16 but required a different combination of Mediator subunits for their expression than the genes induced by cold. Together, our data illustrate that plants control transcription of specific genes through the action of subsets of Mediator subunits; the specific combination defined by the nature of the stimulus but also by the identity of the gene induced.

  19. Interaction of the heterotrimeric G protein alpha subunit SSG-1 of Sporothrix schenckii with proteins related to stress response and fungal pathogenicity using a yeast two-hybrid assay

    Directory of Open Access Journals (Sweden)

    González-Méndez Ricardo


    Full Text Available Abstract Background Important biological processes require selective and orderly protein-protein interactions at every level of the signalling cascades. G proteins are a family of heterotrimeric GTPases that effect eukaryotic signal transduction through the coupling of cell surface receptors to cytoplasmic effector proteins. They have been associated with growth and pathogenicity in many fungi through gene knock-out studies. In Sporothrix schenckii, a pathogenic, dimorphic fungus, we previously identified a pertussis sensitive G alpha subunit, SSG-1. In this work we inquire into its interactions with other proteins. Results Using the yeast two-hybrid technique, we identified protein-protein interactions between SSG-1 and other important cellular proteins. The interactions were corroborated using co-immuneprecipitation. Using these techniques we identified a Fe/Mn superoxide dismutase (SOD, a glyceraldehyde-3-P dehydrogenase (GAPDH and two ion transport proteins, a siderophore-iron transporter belonging to the Major Facilitator Superfamily (MFS and a divalent-cation transporter of the Nramp (natural resistance-associated macrophage protein family as interacting with SSG-1. The cDNA's encoding these proteins were sequenced and bioinformatic macromolecular sequence analyses were used for the correct classification and functional assignment. Conclusions This study constitutes the first report of the interaction of a fungal G alpha inhibitory subunit with SOD, GAPDH, and two metal ion transporters. The identification of such important proteins as partners of a G alpha subunit in this fungus suggests possible mechanisms through which this G protein can affect pathogenicity and survival under conditions of environmental stress or inside the human host. The two ion transporters identified in this work are the first to be reported in S. schenckii and the first time they are identified as interacting with fungal G protein alpha subunits. The association

  20. Loss of the gene for the alpha subunit of ATP synthase (ATP5A1) from the W chromosome in the African grey parrot (Psittacus erithacus). (United States)

    de Kloet, S R


    This study describes the results of an analysis using Southern blotting, the polymerase chain reaction, and sequencing which shows that the African grey parrot (Psittacus erithacus) lacks the W-chromosomal gene for the alpha subunit of mitochondrial ATP synthase (ATP5A1W). Additional evidence shows that in other psittacines a fragment of the ATP5A1W gene contains five times as many nonsynonymous nucleotide replacements as the homologous fragment of the Z gene. Therefore, whereas in these other psittacines the corresponding ATP5A1Z protein fragment is highly conserved and varies by only a few, moderately conservative amino acid substitutions, the homologous ATP5A1W fragments contain a considerable number of, sometimes highly nonconservative, amino acid replacements. In one of these species, the ringneck parakeet (Psittacula krameri), the ATP5A1W gene is present in an inactive form because of the presence of a nonsense codon. Other changes, possibly leading to an inactive ATP5A1W gene product, involve the substitution of arginine residues by cysteine in the ATP5A1W protein of the mitred conure (Aratinga mitrata) and the blue and gold macaw (Ara ararauna). The data suggest also that although the divergence of the psittacine ATP5A1W and ATP5A1Z genes preceded the origin of the psittacidae, this divergence occurred independently of a similar process in the myna (Gracula religiosa), the outgroup used in this study.

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

    International Nuclear Information System (INIS)

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


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

  2. Increased cystic fibrosis transmembrane conductance regulators expression and decreased epithelial sodium channel alpha subunits expression in early abortion: findings from a mouse model and clinical cases of abortion.

    Directory of Open Access Journals (Sweden)

    Min Zhou

    Full Text Available The status of the maternal endometrium is vital in regulating humoral homeostasis and for ensuring embryo implantation. Cystic fibrosis transmembrane conductance regulators (CFTR and epithelial sodium channel alpha subunits (ENaC-α play an important role in female reproduction by maintaining humoral and cell homeostasis. However, it is not clear whether the expression levels of CFTR and ENaC-α in the decidual component during early pregnancy are related with early miscarriage. CBA×DBA/2 mouse mating has been widely accepted as a classical model of early miscarriage. The abortion rate associated with this mating was 33.33% in our study. The decidua of abortion-prone CBA female mice (DBA/2 mated had higher CFTR mRNA and protein expression and lower ENaC-α mRNA and protein expression, compared to normal pregnant CBA mice (BLAB/C mated. Furthermore, increased CFTR expression and decreased ENaC-α expression were observed in the uterine tissue from women with early miscarriage, as compared to those with successful pregnancy. In conclusion, increased CFTR expression and decreased ENaC-α expression in the decidua of early abortion may relate with failure of early pregnancy.

  3. Structural and Thermodynamic Basis for Weak Interactions between Dihydrolipoamide Dehydrogenase and Subunit-binding Domain of the Branched-chain [alpha]-Ketoacid Dehydrogenase Complex

    Energy Technology Data Exchange (ETDEWEB)

    Brautigam, Chad A.; Wynn, R. Max; Chuang, Jacinta L.; Naik, Mandar T.; Young, Brittany B.; Huang, Tai-huang; Chuang, David T. (AS); (UTSMC)


    The purified mammalian branched-chain {alpha}-ketoacid dehydrogenase complex (BCKDC), which catalyzes the oxidative decarboxylation of branched-chain {alpha}-keto acids, is essentially devoid of the constituent dihydrolipoamide dehydrogenase component (E3). The absence of E3 is associated with the low affinity of the subunit-binding domain of human BCKDC (hSBDb) for hE3. In this work, sequence alignments of hSBDb with the E3-binding domain (E3BD) of the mammalian pyruvate dehydrogenase complex show that hSBDb has an arginine at position 118, where E3BD features an asparagine. Substitution of Arg-118 with an asparagine increases the binding affinity of the R118N hSBDb variant (designated hSBDb*) for hE3 by nearly 2 orders of magnitude. The enthalpy of the binding reaction changes from endothermic with the wild-type hSBDb to exothermic with the hSBDb* variant. This higher affinity interaction allowed the determination of the crystal structure of the hE3/hSBDb* complex to 2.4-{angstrom} resolution. The structure showed that the presence of Arg-118 poses a unique, possibly steric and/or electrostatic incompatibility that could impede E3 interactions with the wild-type hSBDb. Compared with the E3/E3BD structure, the hE3/hSBDb* structure has a smaller interfacial area. Solution NMR data corroborated the interactions of hE3 with Arg-118 and Asn-118 in wild-type hSBDb and mutant hSBDb*, respectively. The NMR results also showed that the interface between hSBDb and hE3 does not change significantly from hSBDb to hSBDb*. Taken together, our results represent a starting point for explaining the long standing enigma that the E2b core of the BCKDC binds E3 far more weakly relative to other {alpha}-ketoacid dehydrogenase complexes.

  4. Oxidative inhibition of the vascular Na+-K+ pump via NADPH oxidase-dependent β1-subunit glutathionylation: implications for angiotensin II-induced vascular dysfunction. (United States)

    Liu, Chia-Chi; Karimi Galougahi, Keyvan; Weisbrod, Robert M; Hansen, Thomas; Ravaie, Ramtin; Nunez, Andrea; Liu, Yi B; Fry, Natasha; Garcia, Alvaro; Hamilton, Elisha J; Sweadner, Kathleen J; Cohen, Richard A; Figtree, Gemma A


    Glutathionylation of the Na(+)-K(+) pump's β1-subunit is a key molecular mechanism of physiological and pathophysiological pump inhibition in cardiac myocytes. Its contribution to Na(+)-K(+) pump regulation in other tissues is unknown, and cannot be assumed given the dependence on specific β-subunit isoform expression and receptor-coupled pathways. As Na(+)-K(+) pump activity is an important determinant of vascular tone through effects on [Ca(2+)]i, we have examined the role of oxidative regulation of the Na(+)-K(+) pump in mediating angiotensin II (Ang II)-induced increases in vascular reactivity. β1-subunit glutathione adducts were present at baseline and increased by exposure to Ang II in rabbit aortic rings, primary rabbit aortic vascular smooth muscle cells (VSMCs), and human arterial segments. In VSMCs, Ang II-induced glutathionylation was associated with marked reduction in Na(+)-K(+)ATPase activity, an effect that was abolished by the NADPH oxidase inhibitory peptide, tat-gp91ds. In aortic segments, Ang II-induced glutathionylation was associated with decreased K(+)-induced vasorelaxation, a validated index of pump activity. Ang II-induced oxidative inhibition of Na(+)-K(+) ATPase and decrease in K(+)-induced relaxation were reversed by preincubation of VSMCs and rings with recombinant FXYD3 protein that is known to facilitate deglutathionylation of β1-subunit. Knock-out of FXYD1 dramatically decreased K(+)-induced relaxation in a mouse model. Attenuation of Ang II signaling in vivo by captopril (8 mg/kg/day for 7 days) decreased superoxide-sensitive DHE levels in the media of rabbit aorta, decreased β1-subunit glutathionylation, and enhanced K(+)-induced vasorelaxation. Ang II inhibits the Na(+)-K(+) pump in VSMCs via NADPH oxidase-dependent glutathionylation of the pump's β1-subunit, and this newly identified signaling pathway may contribute to altered vascular tone. FXYD proteins reduce oxidative inhibition of the Na(+)-K(+) pump and may have an

  5. Maternal topoisomerase II alpha, not topoisomerase II beta, enables embryonic development of zebrafish top2a-/- mutants

    LENUS (Irish Health Repository)

    Sapetto-Rebow, Beata


    Abstract Background Genetic alterations in human topoisomerase II alpha (TOP2A) are linked to cancer susceptibility. TOP2A decatenates chromosomes and thus is necessary for multiple aspects of cell division including DNA replication, chromosome condensation and segregation. Topoisomerase II alpha is also required for embryonic development in mammals, as mouse Top2a knockouts result in embryonic lethality as early as the 4-8 cell stage. The purpose of this study was to determine whether the extended developmental capability of zebrafish top2a mutants arises from maternal expression of top2a or compensation from its top2b paralogue. Results Here, we describe bloody minded (blm), a novel mutant of zebrafish top2a. In contrast to mouse Top2a nulls, zebrafish top2a mutants survive to larval stages (4-5 day post fertilization). Developmental analyses demonstrate abundant expression of maternal top2a but not top2b. Inhibition or poisoning of maternal topoisomerase II delays embryonic development by extending the cell cycle M-phase. Zygotic top2a and top2b are co-expressed in the zebrafish CNS, but endogenous or ectopic top2b RNA appear unable to prevent the blm phenotype. Conclusions We conclude that maternal top2a enables zebrafish development before the mid-zygotic transition (MZT) and that zebrafish top2a and top2b are not functionally redundant during development after activation of the zygotic genome.

  6. Maternal topoisomerase II alpha, not topoisomerase II beta, enables embryonic development of zebrafish top2a-/- mutants

    Directory of Open Access Journals (Sweden)

    Sapetto-Rebow Beata


    Full Text Available Abstract Background Genetic alterations in human topoisomerase II alpha (TOP2A are linked to cancer susceptibility. TOP2A decatenates chromosomes and thus is necessary for multiple aspects of cell division including DNA replication, chromosome condensation and segregation. Topoisomerase II alpha is also required for embryonic development in mammals, as mouse Top2a knockouts result in embryonic lethality as early as the 4-8 cell stage. The purpose of this study was to determine whether the extended developmental capability of zebrafish top2a mutants arises from maternal expression of top2a or compensation from its top2b paralogue. Results Here, we describe bloody minded (blm, a novel mutant of zebrafish top2a. In contrast to mouse Top2a nulls, zebrafish top2a mutants survive to larval stages (4-5 day post fertilization. Developmental analyses demonstrate abundant expression of maternal top2a but not top2b. Inhibition or poisoning of maternal topoisomerase II delays embryonic development by extending the cell cycle M-phase. Zygotic top2a and top2b are co-expressed in the zebrafish CNS, but endogenous or ectopic top2b RNA appear unable to prevent the blm phenotype. Conclusions We conclude that maternal top2a enables zebrafish development before the mid-zygotic transition (MZT and that zebrafish top2a and top2b are not functionally redundant during development after activation of the zygotic genome.

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

    Energy Technology Data Exchange (ETDEWEB)

    Beckers, M.C.; Ernst, E.; Gros, P. [McGill Univ., Montreal (Canada)


    We have used a total of 27 AXB/BXA recombinant inbred mouse strains to determine the chromosomal location of a newly identified gene encoding an {alpha}-subunit isoform of the sodium channel from Schwann cells, Scn9a. Linkage analysis established that Scn9a mapped to the proximal segment of mouse chromosome 2. The segregation of restriction fragment length polymorphisms in 145 progeny from a Mus spretus x C57BL/6J backcross indicates that Scn9a is very tightly linked to Scn1a (gene encoding the type I sodium channel {alpha}-subunit of the brain) and forms part of a cluster of four Scna genes located on mouse chromosome 2. 17 refs., 1 fig., 3 tabs.

  8. Human acid alpha-glucosidase from rabbit milk has therapeutic effect in mice with glycogen storage disease type II

    NARCIS (Netherlands)

    A.G.A. Bijvoet (Agnes); A.J.J. Reuser (Arnold); H. van Hirtum (Hans); M.A. Kroos (Marian); E.H. van de Kamp; O. Schoneveld; P. Visser (Pim); J.P. Brakenhoff (Just); M. Weggeman (Miranda); E.J.J.M. van Corven (Emiel); A.T. van der Ploeg (Ans)


    textabstractPompe's disease or glycogen storage disease type II (GSDII) belongs to the family of inherited lysosomal storage diseases. The underlying deficiency of acid alpha-glucosidase leads in different degrees of severity to glycogen storage in heart, skeletal

  9. GlyT1 Inhibitor NFPS Exerts Neuroprotection via GlyR Alpha1 Subunit in the Rat Model of Transient Focal Cerebral Ischaemia and Reperfusion

    Directory of Open Access Journals (Sweden)

    Baosheng Huang


    Full Text Available Background/Aims: Glycine is a strychnine-sensitive inhibitory neurotransmitter in the central nervous system (CNS, especially in the spinal cord, brainstem, and retina. The objective of the present study was to investigate the potential neuroprotective effects of GlyT1 inhibitor N [3-(4'-fluorophenyl-3-(4'-phenylphenoxy propyl] sarcosine (NFPS in the rat model of experimental stroke. Methods: In vivo ischaemia was induced by transient middle cerebral artery occlusion (tMCAO. The methods of Western Blotting, Nissl Staining and Morris water maze methods were applied to analyze the anti-ischaemia mechanism. Results: The results showed that high dose of NFPS (H-NFPS significantly reduced infarct volume, neuronal injury and the expression of cleaved caspase-3, enhanced Bcl-2/Bax, and improved spatial learning deficits which were administered three hours after transient middle cerebral artery occlusion (tMCAO induction in rats, while, low dose of NFPS (L-NFPS exacerbated the injury of ischaemia. These findings suggested that low and high dose of NFPS produced opposite effects. Importantly, it was demonstrated that H-NFPS-dependent neuronal protection was inverted by salicylate (Sal, a specific GlyR ɑ1 antagonist. Such effects could probably be attributed to the enhanced glycine level in both synaptic and extrasynaptic clefts and the subsequently altered extrasynaptic GlyRs and their subtypes. Conclusions: These data imply that GlyT1 inhibitor NFPS may be a novel target for clinical treatment of transient focal cerebral ischaemia and reperfusion which are associated with altered GlyR alpha 1 subunits.

  10. Maternal Separation during Breastfeeding Induces Gender-Dependent Changes in Anxiety and the GABA-A Receptor Alpha-Subunit in Adult Wistar Rats.

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    Diego Armando León Rodríguez

    Full Text Available Different models of rodent maternal separation (MS have been used to investigate long-term neurobiological and behavioral changes, associated with early stress. However, few studies have involved the analysis of sex-related differences in central anxiety modulation. This study investigated whether MS during breastfeeding affected adult males and females in terms of anxiety and brain GABA-A receptor-alpha-subunit immunoreactivity. The brain areas analyzed were the amygdale (AM, hippocampus (HP, medial prefrontal cortex (mPFC, medial preoptic area (POA and paraventricular nucleus (PVN. Rats were housed under a reversed light/dark cycle (lights off at 7∶00 h with access to water and food ad libitum. Animals underwent MS twice daily during the dark cycle from postnatal day 1 to postnatal day 21. Behavior was tested when rats were 65-70 days old using the elevated plus maze and after brains were treated for immunohistochemistry. We found that separated females spent more time in the open arms and showed more head dipping behavior compared with controls. The separated males spent more time in the center of the maze and engaged in more stretching behavior than the controls. Immunohistochemistry showed that separated females had less immunostained cells in the HP, mPFC, PVN and POA, while separated males had fewer immunolabeled cells in the PFC, PVN and AM. These results could indicate that MS has gender-specific effects on anxiety behaviors and that these effects are likely related to developmental alterations involving GABA-A neurotransmission.

  11. Variants in calcium voltage-gated channel subunit Alpha1 C-gene (CACNA1C are associated with sleep latency in infants.

    Directory of Open Access Journals (Sweden)

    Katri Kantojärvi

    Full Text Available Genetic variants in CACNA1C (calcium voltage-gated channel subunit alpha1 C are associated with bipolar disorder and schizophrenia where sleep disturbances are common. In an experimental model, Cacna1c has been found to modulate the electrophysiological architecture of sleep. There are strong genetic influences for consolidation of sleep in infancy, but only a few studies have thus far researched the genetic factors underlying the process. We hypothesized that genetic variants in CACNA1C affect the regulation of sleep in early development. Seven variants that were earlier associated (genome-wide significantly with psychiatric disorders at CACNA1C were selected for analyses. The study sample consists of 1086 infants (520 girls and 566 boys from the Finnish CHILD-SLEEP birth cohort (genotyped by Illumina Infinium PsychArray BeadChip. Sleep length, latency, and nightly awakenings were reported by the parents of the infants with a home-delivered questionnaire at 8 months of age. The genetic influence of CACNA1C variants on sleep in infants was examined by using PLINK software. Three of the examined CACNA1C variants, rs4765913, rs4765914, and rs2239063, were associated with sleep latency (permuted P<0.05. There was no significant association between studied variants and night awakenings or sleep duration. CACNA1C variants for psychiatric disorders were found to be associated with long sleep latency among 8-month-old infants. It remains to be clarified whether the findings refer to defective regulation of sleep, or to distractibility of sleep under external influences.

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

    LENUS (Irish Health Repository)


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

  13. Interactions between the photosystem II subunit PsbS and xanthophylls studied in vivo and in vitro. (United States)

    Bonente, Giulia; Howes, Barry D; Caffarri, Stefano; Smulevich, Giulietta; Bassi, Roberto


    The photosystem II subunit PsbS is essential for excess energy dissipation (qE); however, both lutein and zeaxanthin are needed for its full activation. Based on previous work, two models can be proposed in which PsbS is either 1) the gene product where the quenching activity is located or 2) a proton-sensing trigger that activates the quencher molecules. The first hypothesis requires xanthophyll binding to two PsbS-binding sites, each activated by the protonation of a dicyclohexylcarbodiimide-binding lumen-exposed glutamic acid residue. To assess the existence and properties of these xanthophyll-binding sites, PsbS point mutants on each of the two Glu residues PsbS E122Q and PsbS E226Q were crossed with the npq1/npq4 and lut2/npq4 mutants lacking zeaxanthin and lutein, respectively. Double mutants E122Q/npq1 and E226Q/npq1 had no qE, whereas E122Q/lut2 and E226Q/lut2 showed a strong qE reduction with respect to both lut2 and single glutamate mutants. These findings exclude a specific interaction between lutein or zeaxanthin and a dicyclohexylcarbodiimide-binding site and suggest that the dependence of nonphotochemical quenching on xanthophyll composition is not due to pigment binding to PsbS. To verify, in vitro, the capacity of xanthophylls to bind PsbS, we have produced recombinant PsbS refolded with purified pigments and shown that Raman signals, previously attributed to PsbS-zeaxanthin interactions, are in fact due to xanthophyll aggregation. We conclude that the xanthophyll dependence of qE is not due to PsbS but to other pigment-binding proteins, probably of the Lhcb type.

  14. Interactions between the Photosystem II Subunit PsbS and Xanthophylls Studied in Vivo and in Vitro* (United States)

    Bonente, Giulia; Howes, Barry D.; Caffarri, Stefano; Smulevich, Giulietta; Bassi, Roberto


    The photosystem II subunit PsbS is essential for excess energy dissipation (qE); however, both lutein and zeaxanthin are needed for its full activation. Based on previous work, two models can be proposed in which PsbS is either 1) the gene product where the quenching activity is located or 2) a proton-sensing trigger that activates the quencher molecules. The first hypothesis requires xanthophyll binding to two PsbS-binding sites, each activated by the protonation of a dicyclohexylcarbodiimide-binding lumen-exposed glutamic acid residue. To assess the existence and properties of these xanthophyll-binding sites, PsbS point mutants on each of the two Glu residues PsbS E122Q and PsbS E226Q were crossed with the npq1/npq4 and lut2/npq4 mutants lacking zeaxanthin and lutein, respectively. Double mutants E122Q/npq1 and E226Q/npq1 had no qE, whereas E122Q/lut2 and E226Q/lut2 showed a strong qE reduction with respect to both lut2 and single glutamate mutants. These findings exclude a specific interaction between lutein or zeaxanthin and a dicyclohexylcarbodiimide-binding site and suggest that the dependence of nonphotochemical quenching on xanthophyll composition is not due to pigment binding to PsbS. To verify, in vitro, the capacity of xanthophylls to bind PsbS, we have produced recombinant PsbS refolded with purified pigments and shown that Raman signals, previously attributed to PsbS-zeaxanthin interactions, are in fact due to xanthophyll aggregation. We conclude that the xanthophyll dependence of qE is not due to PsbS but to other pigment-binding proteins, probably of the Lhcb type. PMID:18070876

  15. Roles for common MLL/COMPASS subunits and the 19S proteasome in regulating CIITA pIV and MHC class II gene expression and promoter methylation. (United States)

    Koues, Olivia I; Mehta, Ninad T; Truax, Agnieszka D; Dudley, R Kyle; Brooks, Jeanne K; Greer, Susanna F


    Studies indicate that the 19S proteasome contributes to chromatin reorganization, independent of the role the proteasome plays in protein degradation. We have previously shown that components of the 19S proteasome are crucial for regulating inducible histone activation events in mammalian cells. The 19S ATPase Sug1 binds to histone-remodeling enzymes, and in the absence of Sug1, a subset of activating epigenetic modifications including histone H3 acetylation, H3 lysine 4 trimethylation and H3 arginine 17 dimethylation are inhibited at cytokine-inducible major histocompatibilty complex (MHC)-II and class II transactivator (CIITA) promoters, implicating Sug1 in events required to initiate mammalian transcription. Our previous studies indicate that H3 lysine 4 trimethylation at cytokine-inducible MHC-II and CIITA promoters is dependent on proteolytic-independent functions of 19S ATPases. In this report, we show that multiple common subunits of the mixed lineage leukemia (MLL)/complex of proteins associated with Set I (COMPASS) complexes bind to the inducible MHC-II and CIITA promoters; that overexpressing a single common MLL/COMPASS subunit significantly enhances promoter activity and MHC-II HLA-DRA expression; and that these common subunits are important for H3 lysine 4 trimethylation at MHC-II and CIITA promoters. In addition, we show that H3 lysine 27 trimethylation, which is inversely correlated with H3 lysine 4 trimethylation, is significantly elevated in the presence of diminished 19S ATPase Sug1. Taken together, these experiments suggest that the 19S proteasome plays a crucial role in the initial reorganization of events enabling the relaxation of the repressive chromatin structure surrounding inducible promoters.

  16. Roles for common MLL/COMPASS subunits and the 19S proteasome in regulating CIITA pIV and MHC class II gene expression and promoter methylation

    Directory of Open Access Journals (Sweden)

    Koues Olivia I


    Full Text Available Abstract Background Studies indicate that the 19S proteasome contributes to chromatin reorganization, independent of the role the proteasome plays in protein degradation. We have previously shown that components of the 19S proteasome are crucial for regulating inducible histone activation events in mammalian cells. The 19S ATPase Sug1 binds to histone-remodeling enzymes, and in the absence of Sug1, a subset of activating epigenetic modifications including histone H3 acetylation, H3 lysine 4 trimethylation and H3 arginine 17 dimethylation are inhibited at cytokine-inducible major histocompatibilty complex (MHC-II and class II transactivator (CIITA promoters, implicating Sug1 in events required to initiate mammalian transcription. Results Our previous studies indicate that H3 lysine 4 trimethylation at cytokine-inducible MHC-II and CIITA promoters is dependent on proteolytic-independent functions of 19S ATPases. In this report, we show that multiple common subunits of the mixed lineage leukemia (MLL/complex of proteins associated with Set I (COMPASS complexes bind to the inducible MHC-II and CIITA promoters; that overexpressing a single common MLL/COMPASS subunit significantly enhances promoter activity and MHC-II HLA-DRA expression; and that these common subunits are important for H3 lysine 4 trimethylation at MHC-II and CIITA promoters. In addition, we show that H3 lysine 27 trimethylation, which is inversely correlated with H3 lysine 4 trimethylation, is significantly elevated in the presence of diminished 19S ATPase Sug1. Conclusion Taken together, these experiments suggest that the 19S proteasome plays a crucial role in the initial reorganization of events enabling the relaxation of the repressive chromatin structure surrounding inducible promoters.

  17. Molecular basis of adult-onset and chronic G sub M2 gangliosidoses in patients of Ashkenazi Jewish origin: Substitution of serine for glycine at position 269 of the. alpha. -subunit of. beta. -hexosaminidase

    Energy Technology Data Exchange (ETDEWEB)

    Paw, B.H.; Kaback, M.M.; Neufeld, E.F. (Univ. of California, Los Angeles (USA))


    Chronic and adult-onset G{sub M2} gangliosidoses are neurological disorders caused by marked deficiency of the A isoenzyme of {beta}-hexosaminidase; they occur in the Ashkenazi Jewish population, though less frequently than classic (infantile) Tay-Sachs disease. Earlier biosynthetic studies had identified a defective {alpha}-subunit that failed to associate with the {beta}-subunit. The authors have now found a guanosine to adenosine transition at the 3{prime} end of exon 7, which causes substitution of serine for glycine at position 269 of the {alpha}-subunit. An RNase protection assay was used to localize the mutation to a segment of mRNA from fibroblasts of a patient with the adult-onset disorder. That segment of mRNA (after reverse transcription) and a corresponding segment of genomic DNA were amplified by the polymerase chain reaction and sequenced by the dideoxy method. The sequence analysis, together with an assay based on the loss of a ScrFI restriction site, showed that the patient was a compound heterozygote who had inherited the 269 (Gly {yields} Ser) mutation from his father and an allelic null mutation from his mother. The 269 (Gly {yields} Ser) mutation, in compound heterozygosity with a presumed null allele, was also found in fetal fibroblasts with an association-defective phenotype and in cells from five patients with chronic G{sub M2} gangliosidosis.

  18. Stimulation of casein kinase II by epidermal growth factor: Relationship between the physiological activity of the kinase and the phosphorylation state of its beta subunit

    International Nuclear Information System (INIS)

    Ackerman, P.; Osheroff, N.; Glover, C.V.C.


    To determine relationships between the hormonal activation of casein kinase II and its phosphorylation state, epidermal growth factor (EGF)-treated and EGF-naive human A-431 carcinoma cells were cultured in the presence of [ 32 P]orthophosphate. Immunoprecipitation experiments indicated that casein kinase II in the cytosol of EGF-treated cells contained approximately 3-fold more incorporated [ 32 P]phosphate than did its counterpart in untreated cells. Levels of kinase phosphorylation paralleled levels of kinase activity over a wide range of EGF concentrations as well as over a time course of hormone action. Approximately 97% of the incorporated [ 32 P]phosphate was found in the β subunit of casein kinase II. Both activated and hormone-naive kinase contained radioactive phosphoserine and phosphothreonine but no phosphotyronsine. On the basis of proteolytic mapping experiments, EGF treatment of A-431 cells led to an increase in the average [ 32 P]phosphate content (i.e., hyperphosphorylation) of casein kinase II β subunit peptides which were modified prior to hormone treatment. Finally, the effect of alkaline phosphatase on the reaction kinetics of activated casein kinase II indicated that hormonal stimulation of the kinase resulted from the increase in its phosphorylation state

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

    Directory of Open Access Journals (Sweden)

    Alan eNeely


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

  20. MiR-30e suppresses proliferation of hepatoma cells via targeting prolyl 4-hydroxylase subunit alpha-1 (P4HA1) mRNA

    Energy Technology Data Exchange (ETDEWEB)

    Feng, Guoxing [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin (China); Shi, Hui [State Key Laboratory of Medicinal Chemical Biology, Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin (China); Li, Jiong; Yang, Zhe [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin (China); Fang, Runping; Ye, Lihong [State Key Laboratory of Medicinal Chemical Biology, Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin (China); Zhang, Weiying, E-mail: [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin (China); Zhang, Xiaodong, E-mail: [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin (China)


    Aberrant microRNA expression has been shown to be characteristic of many cancers. It has been reported that the expression levels of miR-30e are decreased in liver cancer tissues. However, the role of miR-30e in hepatocellular carcinoma remains poorly understood. In the present study, we investigated the significance of miR-30e in hepatocarcinogenesis. Bioinformatics analysis reveals a putative target site of miR-30e in the 3′-untranslated region (3′UTR) of prolyl 4-hydroxylase subunit alpha-1 (P4HA1) mRNA. Moreover, luciferase reporter gene assays verified that miR-30e directly targeted 3′UTR of P4HA1 mRNA. Then, we demonstrated that miR-30e was able to reduce the expression of P4HA1 at the levels of mRNA and protein using reverse transcription-polymerase chain reaction and Western blot analysis. Enforced expression of miR-30e suppressed proliferation of HepG2 cells by 5-ethynyl-2-deoxyuridine (EdU) assay and reduced colony formation of these cells by colony formation analysis. Conversely, anti-miR-30e enhanced the proliferation of hepatoma cells in vitro. Interestingly, the ectopic expression of P4HA1 could efficiently rescue the inhibition of cell proliferation mediated by miR-30e in HepG2 cells. Meanwhile, silencing of P4HA1 abolished the anti-miR-30e-induced proliferation of cells. Clinically, quantitative real-time PCR showed that miR-30e was down-regulated in liver tumor tissues relative to their peritumor tissues. The expression levels of miR-30e were negatively correlated to those of P4HA1 mRNA in clinical liver tumor tissues. Thus, we conclude that miR-30e suppresses proliferation of hepatoma cells through targeting P4HA1 mRNA. Our finding provides new insights into the mechanism of hepatocarcinogenesis. - Highlights: • P4HA1 is a novel target gene of miR-30e. • P4HA1 is increased in clinical HCC tissues. • MiR-30e is negatively correlated with P4HA1 in clinical HCC tissues. • MiR-30e suppresses the proliferation of HCC cells through

  1. Targeted deletion of the GABRA2 gene encoding alpha2-subunits of GABA(A) receptors facilitates performance of a conditioned emotional response, and abolishes anxiolytic effects of benzodiazepines and barbiturates. (United States)

    Dixon, C I; Rosahl, T W; Stephens, D N


    Mice with point-mutated alpha2 GABA(A) receptor subunits (rendering them diazepam insensitive) are resistant to the anxiolytic-like effects of benzodiazepines (BZs) in the conditioned emotional response (CER) test, but show normal anxiolytic effects of a barbiturate. We investigated the consequence of deleting the alpha2-subunit on acquisition of the CER with increasing intensity of footshock, and on the anxiolytic efficacy of a benzodiazepine, diazepam, and a barbiturate, pentobarbital. alpha2 knockout (KO) and wildtype (WT) mice were trained in a conditioned emotional response (CER) task, in which lever pressing for food on a variable interval (VI) schedule was suppressed during the presentation of a compound light/tone conditioned stimulus (CS+) that predicted footshock. The ability of diazepam and of pentobarbital to reduce suppression during the CS+ was interpreted as an anxiolytic response. There were no differences between the genotypes in shock sensitivity, as assessed by their flinch responses to increasing levels of shock. However, alpha2 KO mice showed a greater suppression of lever pressing than WT littermates in the presence of a compound cue signalling footshock. Diazepam (0, 0.5, 1 and 2 mg/kg) induced a dose-dependent anxiolytic-like effect in WT mice but no such effect was seen in KO mice. Similarly, although pentobarbital (20 mg/kg) reduced the ability of the CS+ to reduce lever pressing rates in WT mice, this effect was not seen in the KO. These findings suggest that alpha2-containing GABA(A) receptors mediate the anxiolytic effects of barbiturates, as well as benzodiazepines, and that they may be involved in neuronal circuits underlying conditioned anxiety.

  2. Potency of a novel saw palmetto ethanol extract, SPET-085, for inhibition of 5alpha-reductase II. (United States)

    Pais, Pilar


    The nicotinamide adenine dinucleotide phosphate (NADPH)-dependent membrane protein 5alpha-reductase irreversibly catalyses the conversion of testosterone to the most potent androgen, 5alpha-dihydrotestosterone (DHT). In humans, two 5alpha-reductase isoenyzmes are expressed: type I and type II. Type II is found primarily in prostate tissue. Saw palmetto extract (SPE) has been widely used for the treatment of lower urinary tract symptoms secondary to benign prostatic hyperplasia (BPH). The mechanisms of the pharmacological effects of SPE include the inhibition of 5alpha-reductase, among other actions. Clinical studies of SPE have been equivocal, with some showing significant results and others not. These inconsistent results may be due, in part, to varying bioactivities of the SPE used in the studies. The aim of the present study was to determine the in vitro potency of a novel saw palmetto ethanol extract (SPET-085), an inhibitor of the 5alpha-reductase isoenzyme type II, in a cell-free test system. On the basis of the enzymatic conversion of the substrate androstenedione to the 5alpha-reduced product 5alpha-androstanedione, the inhibitory potency was measured and compared to those of finasteride, an approved 5alpha-reductase inhibitor. SPET-085 concentration-dependently inhibited 5alpha-reductase type II in vitro (IC(50)=2.88+/-0.45 microg/mL). The approved 5alpha-reductase inhibitor, finasteride, tested as positive control, led to 61% inhibition of 5alpha-reductase type II. SPET-085 effectively inhibits the enzyme that has been linked to BPH, and the amount of extract required for activity is very low compared to data reported for other extracts. It can be concluded from data in the literature that SPET-085 is as effective as a hexane extract of saw palmetto that exhibited the highest levels of bioactivity, and is more effective than other SPEs tested. This study confirmed that SPET-085 has prostate health-promoting bioactivity that also corresponds favorably to

  3. X-ray investigations of sulfur-containing fungicides. II. Intramolecular forces governing the conformation of four novel alpha-phenylazo- and alpha-phenylhydrazono-beta-ketosulfones. (United States)

    Wolf , W M


    The crystal and molecular structures of four novel beta-ketosulfones, phenyl benzoyl(phenylhydrazono)methyl sulfone (I), phenyl benzoyl(4-nitrophenylhydrazono)methyl sulfone (II), (benzoyl)(phenyl)(phenylazo)methyl phenyl sulfone (III) and (benzoyl)(phenyl)(3-chlorophenylazo)methyl 4-tolyl sulfone (IV), have been investigated using X-ray analysis and density functional theory supplemented by ab initio Hartree-Fock calculations. The conformations of (I) and (II) are stabilized by strong intramolecular resonance-assisted hydrogen bonds formed between the sulfonyl and the alpha-hydrazono moieties. The following increase of a positive charge on the S atoms is compensated via pi-conjugation and stereoelectronic back-donation from the nearby beta-carbonyl group. Compounds (III) and (IV) adopt a strongly distorted propeller shape with pivotal tetrahedral C1 atoms. Distortion mostly follows from the approximate coplanarity of the alpha-azophenyl and alpha-phenyl moieties opposite each other. The main stereoelectronic interactions involve the donation of electron density from the alpha-azo N2 towards the sulfonyl S and the beta-carbonyl C2 atoms.

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

    DEFF Research Database (Denmark)

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


    In the present study, we tested whether the alpha(1A) subunit, which encodes a neuronal isoform of voltage-dependent Ca(2+) channels (VDCCs) (P-/Q-type), was present and functional in vascular smooth muscle and renal resistance vessels. By reverse transcription-polymerase chain reaction...... preglomerular resistance vessels and aorta, as well as mesangial cells, and that P-type VDCCs contribute to Ca(2+) influx in aortic and renal VSMCs and are involved in depolarization-mediated contraction in renal afferent arterioles....

  5. Reduced volume but increased training intensity elevates muscle Na+-K+ pump alpha1-subunit and NHE1 expression as well as short-term work capacity in humans

    DEFF Research Database (Denmark)

    Iaia, F. Marcello; Thomassen, Martin; Kolding, Helle


    by 30-s sprint runs three to four times a week, whereas CON continued the endurance training ( approximately 45 km/wk). After the 4-wk sprint period, the expression of the muscle Na(+)-K(+) pump alpha(1)-subunit and Na(+)/H(+)-exchanger isoform 1 was 29 and 30% higher (P ... pulmonary maximum oxygen uptake and 10-k time were unchanged. No changes in CON were observed. The present data suggest a role of the Na(+)-K(+) pump in the control of K(+) homeostasis and in the development of fatigue during repeated high-intensity exercise. Furthermore, performance during intense exercise....... Furthermore, plasma K(+) concentration was reduced (P

  6. Change of expression of renal alpha1-adrenergic receptor and angiotensin II receptor subtypes with aging in rats. (United States)

    Li, Yan-Fang; Cao, Xiao-Jing; Bai, Xue-Yuan; Lin, Shu-Peng; Shi, Shu-Tian


    It has been considered that the functional decline of renal vasoconstriction during senescence is associated with an alteration in renal alpha1-adrenergic receptor (alpha1-AR) expression. While alterations in renal angiotensin II receptor (ATR) expression was considered to have an effect on renal structure and function, until now little information has been available concerning alpha1-AR and ATR expression variations over the entire aging continuum. The present study was undertaken to examine the expression levels of alpha1-AR and ATR subtypes in renal tissue during the spectrum running from young adulthood, to middle age, to the presenium, and to the senium. Semiquantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Western Blot were used to quantify the messenger RNA (mRNA) and protein levels of alpha1-AR and ATR subtypes in renal tissue in 3-month-old (young adult), 12-month-old (middle age), 18-month-old (presenium) and 24-month-old (senium) Wistar rats. alpha1A-AR expression decreased gradually with aging: it was decreased during middle age, the presenium and the senium, compared, respectively, with young adult values (page and in the senium with respect to the presenium. alpha1B-AR and alpha1D-AR expression were unmodified during senescence. AT1R expression was unaffected by aging during young adulthood and middle age, but exhibited a remarkable downregulation in the presenium and senium periods (prenal alpha1-AR and ATR subtypes during aging. alpha1A-AR expression downregulation may account for the reduced reactivity of renal alpha1-AR to vasoconstrictors and to renal function decline in the senium. Both the downregulation of AT1R and the upregulation of AT2R may be influential in maintaining normal physiological renal function during aging.

  7. Phaeophytins from Thyrsacanthus ramosissimus Moric. with inhibitory activity on human DNA topoisomerase II-{alpha}

    Energy Technology Data Exchange (ETDEWEB)

    Cabral, Analucia Guedes Silveira; Tenorio-Souza, Fabio Henrique; Moura, Marcelo Dantas; Mota, Sabrina Gondim Ribeiro; Silva Lins, Antonio Claudio da; Dias, Celidarque da Silva; Barbosa-Filho, Jose Maria [Universidade Federal da Paraiba (UFPB), Joao Pessoa, PB (Brazil). Dept. de Ciencias Frmaceuticas; Giulietti, Ana Maria [Universidade Estadual de Feira de Santana, Feira de Santana, BA (Brazil). Dept. de Ciencias Biologicas; Silva, Tania Maria Sarmento da [Universidade Federal Rural de Pernambuco, Recife, PE (Brazil). Dept. de Ciencias Moleculares; Santos, Creusioni Figueredo dos, E-mail: [Universidade Federal da Paraiba (UFPB), Joao Pessoa, PB (Brazil). Dept. de Biologia Molecular


    Our study reports the extraction and isolation of a new phaeophytin derivative 15{sup 1}-hydroxy-(15{sup 1}-S)-porphyrinolactone, designated anamariaine (1) herein, isolated from the chloroform fraction of aerial parts of Thyrsacanthus ramosissimus Moric. along with the known 15{sup 1}-ethoxy-(15{sup 1}-S)-porphyrinolactone (2). These compounds were identified by usual spectroscopic methods. Both compounds were subjected to in vitro (inhibitory activity) tests by means of supercoiled DNA relaxation techniques and were shown to display inhibitory activity against human DNA topoisomerase II-{alpha} at 50 {mu}M. Interconversion of these two pigments under the mild conditions of the isolation techniques should be highly unlikely but cannot be entirely ruled out. (author)

  8. Identification of Interleukin-27 (IL-27)/IL-27 Receptor Subunit Alpha as a Critical Immune Axis for In Vivo HIV Control. (United States)

    Ruiz-Riol, M; Berdnik, D; Llano, A; Mothe, B; Gálvez, C; Pérez-Álvarez, S; Oriol-Tordera, B; Olvera, A; Silva-Arrieta, S; Meulbroek, M; Pujol, F; Coll, J; Martinez-Picado, J; Ganoza, C; Sanchez, J; Gómez, G; Wyss-Coray, T; Brander, C


    the blood is a key conduit for transporting such factors. Investigating the communication factors promoting effective immune responses and having potentially antiviral functions against HIV using a novel focused omics approach ("communicome") has the potential to significantly improve our knowledge of effective host immunity and accelerate the HIV cure agenda. Including 140 subjects with variable viral loads and measuring the plasma levels of >600 soluble proteins, our data highlight the importance of Th17 cells and Wnt/β-catenin signaling in HIV control and especially identify the IL-27/IL-27 receptor subunit alpha (IL-27RA) axis as a predictor of plasma viral load and proviral copy number in the peripheral blood. These data may provide important guidance to therapeutic approaches in the HIV cure agenda. Copyright © 2017 Ruiz-Riol et al.

  9. DNA sequence polymorphisms within the bovine guanine nucleotide-binding protein Gs subunit alpha (Gsα-encoding (GNAS genomic imprinting domain are associated with performance traits

    Directory of Open Access Journals (Sweden)

    Mullen Michael P


    Full Text Available Abstract Background Genes which are epigenetically regulated via genomic imprinting can be potential targets for artificial selection during animal breeding. Indeed, imprinted loci have been shown to underlie some important quantitative traits in domestic mammals, most notably muscle mass and fat deposition. In this candidate gene study, we have identified novel associations between six validated single nucleotide polymorphisms (SNPs spanning a 97.6 kb region within the bovine guanine nucleotide-binding protein Gs subunit alpha gene (GNAS domain on bovine chromosome 13 and genetic merit for a range of performance traits in 848 progeny-tested Holstein-Friesian sires. The mammalian GNAS domain consists of a number of reciprocally-imprinted, alternatively-spliced genes which can play a major role in growth, development and disease in mice and humans. Based on the current annotation of the bovine GNAS domain, four of the SNPs analysed (rs43101491, rs43101493, rs43101485 and rs43101486 were located upstream of the GNAS gene, while one SNP (rs41694646 was located in the second intron of the GNAS gene. The final SNP (rs41694656 was located in the first exon of transcripts encoding the putative bovine neuroendocrine-specific protein NESP55, resulting in an aspartic acid-to-asparagine amino acid substitution at amino acid position 192. Results SNP genotype-phenotype association analyses indicate that the single intronic GNAS SNP (rs41694646 is associated (P ≤ 0.05 with a range of performance traits including milk yield, milk protein yield, the content of fat and protein in milk, culled cow carcass weight and progeny carcass conformation, measures of animal body size, direct calving difficulty (i.e. difficulty in calving due to the size of the calf and gestation length. Association (P ≤ 0.01 with direct calving difficulty (i.e. due to calf size and maternal calving difficulty (i.e. due to the maternal pelvic width size was also observed at the rs

  10. Deficient plasticity in the primary visual cortex of alpha-calcium/calmodulin-dependent protein kinase II mutant mice. (United States)

    Gordon, J A; Cioffi, D; Silva, A J; Stryker, M P


    The recent characterization of plasticity in the mouse visual cortex permits the use of mutant mice to investigate the cellular mechanisms underlying activity-dependent development. As calcium-dependent signaling pathways have been implicated in neuronal plasticity, we examined visual cortical plasticity in mice lacking the alpha-isoform of calcium/calmodulin-dependent protein kinase II (alpha CaMKII). In wild-type mice, brief occlusion of vision in one eye during a critical period reduces responses in the visual cortex. In half of the alpha CaMKII-deficient mice, visual cortical responses developed normally, but visual cortical plasticity was greatly diminished. After intensive training, spatial learning in the Morris water maze was severely impaired in a similar fraction of mutant animals. These data indicate that loss of alpha CaMKII results in a severe but variable defect in neuronal plasticity.

  11. MID1 and MID2 homo- and heterodimerise to tether the rapamycin-sensitive PP2A regulatory subunit, Alpha 4, to microtubules: implications for the clinical variability of X-linked Opitz GBBB syndrome and other developmental disorders

    Directory of Open Access Journals (Sweden)

    Cox Timothy C


    Full Text Available Abstract Background Patients with Opitz GBBB syndrome present with a variable array of developmental defects including craniofacial, cardiac, and genital anomalies. Mutations in the X-linked MID1 gene, which encodes a microtubule-binding protein, have been found in ~50% of Opitz GBBB syndrome patients consistent with the genetically heterogeneous nature of the disorder. A protein highly related to MID1, called MID2, has also been described that similarly associates with microtubules. Results To identify protein partners of MID1 and MID2 we undertook two separate yeast two-hybrid screens. Using this system we identified Alpha 4, a regulatory subunit of PP2-type phosphatases and a key component of the rapamycin-sensitive signaling pathway, as a strong interactor of both proteins. Analysis of domain-specific deletions has shown that the B-boxes of both MID1 and MID2 mediate the interaction with Alpha 4, the first demonstration in an RBCC protein of a specific role for the B-box region. In addition, we show that the MID1/2 coiled-coil motifs mediate both homo- and hetero-dimerisation, and that dimerisation is a prerequisite for association of the MID-Alpha 4 complex with microtubules. Conclusions Our findings not only implicate Alpha 4 in the pathogenesis of Opitz GBBB syndrome but also support our earlier hypothesis that MID2 is a modifier of the X-linked phenotype. Of further note is the observation that Alpha 4 maps to Xq13 within the region showing linkage to FG (Opitz-Kaveggia syndrome. Overlap in the clinical features of FG and Opitz GBBB syndromes warrants investigation of Alpha 4 as a candidate for causing FG syndrome.

  12. A murine experimental anthracycline extravasation model: pathology and study of the involvement of topoisomerase II alpha and iron in the mechanism of tissue damage

    DEFF Research Database (Denmark)

    Thougaard, Annemette V; Langer, Seppo W; Hainau, Bo


    in the topoisomerase II alpha gene (Top2a(Y165S/+)), we found that dexrazoxane provided a protection against anthracycline-induced skin wounds that was indistinguishable from that found in wildtype mice. Thus, interaction with topoisomerase II alpha is not central in the pathogenesis of anthracycline-induced skin...

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

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


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

  14. Highly conserved small subunit residues influence rubisco large subunit catalysis. (United States)

    Genkov, Todor; Spreitzer, Robert J


    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.

  15. MspI and PvuII polymorphisms in the Na,K-ATPase. beta. subunit gene ATP1B1

    Energy Technology Data Exchange (ETDEWEB)

    Shull, M.M.; Pugh, D.G.; Lane, L.K.; Lingrel, J.B. (Univ. of Cincinnati College of Medicine, OH (USA))


    ATP1B HH1.2 is a 1.2 kb HindIII fragment from the 3{prime} portion of the human Na,K-ATPase {beta} subunit gene, ATP1B1. MspI identifies a two allele polymorphism (M1: 6.7 kb, M2: 5.3 kb). PvuII also detects a two-allele polymorphism (P1: 5.1 kb, P2: 4.7 kb). ATP1B1 has been assigned to chromosome 1q by somatic cell hybrid analysis. Codominant segregation of the MspI RFLP was observed in one two-generation family (5 individuals). Codominant segregation of the PvuII RFLP was observed in a two-generation (8 individuals) and a three-generation (12 individuals) family.

  16. Inhibition of B cell proliferation by antisense DNA to both alpha and beta forms of Fc epsilon R II. (United States)

    Bhatti, L; Behle, K; Stevens, R H


    Epstein-Barr Virus (EBV) infection activates B lymphocyte proliferation through partially understood mechanisms, resulting in phenotypic changes, including the appearance of new antigens. One such antigen is Fc epsilon R II/CD-23 which may be relevant for B cell proliferation. We have used anti-sense oligonucleotides to study the importance of the two forms of this molecule for proliferation in the EBV-transformed, Fc epsilon R II +ve lymphoblastoid B cell line, RPMI 8866. Anti-sense oligodeoxynucleotides were generated to the two forms of Fc epsilon R II; Fc epsilon R IIa (alpha) and IIb (beta) which differ only in their intracytoplasmic domains. Addition of increasing concentrations of anti-sense oligonucleotides, ranging from 1 to 30 microM, significantly decreased cellular proliferation as measured by the incorporation of [3H]thymidine (inhibition range 8-88%). Optimum inhibition of cellular proliferation was apparent at 15 microM concentration of both anti-sense Fc epsilon R IIa and IIb (Fc epsilon R IIa, mean +/- SE = 75 +/- 7% inhibition, p less than 0.001; Fc epsilon R IIb, mean +/- SE = 71 +/- 7% inhibition, p less than 0.001). Anti-sense oligonucleotides complementary to the common part of Fc epsilon R II resulted in a similar inhibition of proliferation. Sense oligonucleotides did not induce significant inhibition. Preincubation of sense and anti-sense oligonucleotides resulted in an abrogation of proliferation inhibition. Moreover, none of these oligonucleotides had any effect on a Fc epsilon R II -ve cell line. Incubation with both anti-sense IIa and IIb resulted in additive, but not synergistic inhibition of proliferation. Addition of soluble Fc epsilon R II did not reverse inhibition of proliferation, suggesting that membrane-bound or intracellular rather than soluble Fc epsilon R II was important for the induced proliferation. Analysis of cell surface expression for Fc epsilon II indicated that while there was a pronounced effect on cell number

  17. In cellulo examination of a beta-alpha hybrid construct of beta-hexosaminidase A subunits, reported to interact with the GM2 activator protein and hydrolyze GM2 ganglioside.

    Directory of Open Access Journals (Sweden)

    Incilay Sinici

    Full Text Available The hydrolysis in lysosomes of GM2 ganglioside to GM3 ganglioside requires the correct synthesis, intracellular assembly and transport of three separate gene products; i.e., the alpha and beta subunits of heterodimeric beta-hexosaminidase A, E.C. # (encoded by the HEXA and HEXB genes, respectively, and the GM2-activator protein (GM2AP, encoded by the GM2A gene. Mutations in any one of these genes can result in one of three neurodegenerative diseases collectively known as GM2 gangliosidosis (HEXA, Tay-Sachs disease, MIM # 272800; HEXB, Sandhoff disease, MIM # 268800; and GM2A, AB-variant form, MIM # 272750. Elements of both of the hexosaminidase A subunits are needed to productively interact with the GM2 ganglioside-GM2AP complex in the lysosome. Some of these elements have been predicted from the crystal structures of hexosaminidase and the activator. Recently a hybrid of the two subunits has been constructed and reported to be capable of forming homodimers that can perform this reaction in vivo, which could greatly simplify vector-mediated gene transfer approaches for Tay-Sachs or Sandhoff diseases. A cDNA encoding a hybrid hexosaminidase subunit capable of dimerizing and hydrolyzing GM2 ganglioside could be incorporated into a single vector, whereas packaging both subunits of hexosaminidase A into vectors, such as adeno-associated virus, would be impractical due to size constraints. In this report we examine the previously published hybrid construct (H1 and a new more extensive hybrid (H2, with our documented in cellulo (live cell- based assay utilizing a fluorescent GM2 ganglioside derivative. Unfortunately when Tay-Sachs cells were transfected with either the H1 or H2 hybrid construct and then were fed the GM2 derivative, no significant increase in its turnover was detected. In vitro assays with the isolated H1 or H2 homodimers confirmed that neither was capable of human GM2AP-dependent hydrolysis of GM2 ganglioside.

  18. Resolving the role of plant glutamate dehydrogenase: II. Physiological characterization of plants overexpressing the two enzyme subunits individually or simultaneously. (United States)

    Tercé-Laforgue, Thérèse; Bedu, Magali; Dargel-Grafin, Céline; Dubois, Frédéric; Gibon, Yves; Restivo, Francesco M; Hirel, Bertrand


    Glutamate dehydrogenase (GDH; EC is able to carry out the deamination of glutamate in higher plants. In order to obtain a better understanding of the physiological function of GDH in leaves, transgenic tobacco (Nicotiana tabacum L.) plants were constructed that overexpress two genes from Nicotiana plumbaginifolia (GDHA and GDHB under the control of the Cauliflower mosiac virus 35S promoter), which encode the α- and β-subunits of GDH individually or simultaneously. In the transgenic plants, the GDH protein accumulated in the mitochondria of mesophyll cells and in the mitochondria of the phloem companion cells (CCs), where the native enzyme is normally expressed. Such a shift in the cellular location of the GDH enzyme induced major changes in carbon and nitrogen metabolite accumulation and a reduction in growth. These changes were mainly characterized by a decrease in the amount of sucrose, starch and glutamine in the leaves, which was accompanied by an increase in the amount of nitrate and Chl. In addition, there was an increase in the content of asparagine and a decrease in proline. Such changes may explain the lower plant biomass determined in the GDH-overexpressing lines. Overexpressing the two genes GDHA and GDHB individually or simultaneously induced a differential accumulation of glutamate and glutamine and a modification of the glutamate to glutamine ratio. The impact of the metabolic changes occurring in the different types of GDH-overexpressing plants is discussed in relation to the possible physiological function of each subunit when present in the form of homohexamers or heterohexamers.

  19. A highly conserved glycine within linker I and the extreme C terminus of G protein alpha subunits interact cooperatively in switching G protein-coupled receptor-to-effector specificity

    DEFF Research Database (Denmark)

    Kostenis, Evi; Martini, Lene; Ellis, James


    Numerous studies have attested to the importance of the extreme C terminus of G protein alpha subunits in determining their selectivity of receptor recognition. We have previously reported that a highly conserved glycine residue within linker I is important for constraining the fidelity of receptor...... recognition by Galpha(q) proteins. Herein, we explored whether both modules (linker I and extreme C terminus) interact cooperatively in switching G protein-coupled receptor (GPCR)-to-effector specificity and created as models mutant Galpha(q) proteins in which glycine was replaced with various amino acids...... and the C-terminal five Galpha(q) residues with the corresponding Galpha(i) or Galpha(s) sequence. Coupling properties of the mutated Galpha(q) proteins were determined after coexpression with a panel of 13 G(i)-and G(s) -selective receptors and compared with those of Galpha proteins modified in only one...

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


    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

  1. Prophylactic Sublingual Immunization with Mycobacterium tuberculosis Subunit Vaccine Incorporating the Natural Killer T Cell Agonist Alpha-Galactosylceramide Enhances Protective Immunity to Limit Pulmonary and Extra-Pulmonary Bacterial Burden in Mice

    Directory of Open Access Journals (Sweden)

    Arshad Khan


    Full Text Available Infection by Mycobacterium tuberculosis (Mtb remains a major global concern and the available Bacillus Calmette-Guerin (BCG vaccine is poorly efficacious in adults. Therefore, alternative vaccines and delivery strategies focusing on Mtb antigens and appropriate immune stimulating adjuvants are needed to induce protective immunity targeted to the lungs, the primary sites of infections and pathology. We present here evidence in support of mucosal vaccination by the sublingual route in mice using the subunit Mtb antigens Ag85B and ESAT-6 adjuvanted with the glycolipid alpha-galactosylceramide (α-GalCer, a potent natural killer T (NKT cell agonist. Vaccinated animals exhibited strong antigen-specific CD4 and CD8 T cells responses in the spleen, cervical lymph nodes and lungs. In general, inclusion of the α-GalCer adjuvant significantly enhanced these responses that persisted over 50 days. Furthermore, aerosolized Mtb infection of vaccinated mice resulted in a significant reduction of bacterial load of the lungs and spleens as compared to levels seen in naïve controls or those vaccinated with subunit proteins, adjuvant , or BCG alone. The protection induced by the Mtb antigens and-GalCer vaccine through sublingual route correlated with a TH1-type immunity mediated by antigen-specific IFN-γ and IL-2 producing T cells.

  2. Concomitant alpha7 and beta2 nicotinic AChR subunit deficiency leads to impaired energy homeostasis and increased physical activity in mice. (United States)

    Somm, Emmanuel; Guérardel, Audrey; Maouche, Kamel; Toulotte, Audrey; Veyrat-Durebex, Christelle; Rohner-Jeanrenaud, Françoise; Maskos, Uwe; Hüppi, Petra S; Schwitzgebel, Valérie M


    Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated cation channels well characterized in neuronal signal transmission. Moreover, recent studies have revealed nAChR expression in nonneuronal cell types throughout the body, including tissues involved in metabolism. In the present study, we screen gene expression of nAChR subunits in pancreatic islets and adipose tissues. Mice pancreatic islets present predominant expression of α7 and β2 nAChR subunits but at a lower level than in central structures. Characterization of glucose and energy homeostasis in α7β2nAChR(-/-) mice revealed no major defect in insulin secretion and sensitivity but decreased glycemia apparently unrelated to gluconeogenesis or glycogenolysis. α7β2nAChR(-/-) mice presented an increase in lean and bone body mass and a decrease in fat storage with normal body weight. These observations were associated with elevated spontaneous physical activity in α7β2nAChR(-/-) mice, mainly due to elevation in fine vertical (rearing) activity while their horizontal (ambulatory) activity remained unchanged. In contrast to α7nAChR(-/-) mice presenting glucose intolerance and insulin resistance associated to excessive inflammation of adipose tissue, the present metabolic phenotyping of α7β2nAChR(-/-) mice revealed a metabolic improvement possibly linked to the increase in spontaneous physical activity related to central β2nAChR deficiency. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Prevalence of estrogen receptor alpha PvuII and XbaI polymorphism in population of Polish postmenopausal women.

    Directory of Open Access Journals (Sweden)

    Jozef Haczynski


    Full Text Available Numerous data indicate that polymorphism of estrogen receptor alpha (ERalpha may predict lipid levels, lipid response to hormone replacement therapy (HRT, myocardial infarction risk, bone fracture risk, bone mineral density (BMD and changes in BMD over time. In this study we aimed to evaluate distribution of ERalpha PvuII and XbaI genotypes in population of Polish postmenopausal women qualified to different protocols of HRT. Subject of the study were 64 consecutive postmenopausal women aged from 45 to 65 years (mean 56.6 assigned to HRT. ERalpha PvuII and XbaI polymorphism was determined by PCR-restriction fragment length polymorphism (RFLP. The absence of PvuII and XbaI restriction sites were indicated by "P" and "X" and presence by "p" and "x", respectively. PvuII genotype was distributed as follows: PP 17.2% (n=11, Pp 50% (n=32, pp 32.83% (n=21. Frequency of XbaI genotype was: XX 6.25% (n=4, Xx 34.4% (n=22, xx 59.4% (n=38. Four haplotypes with following frequencies were recognized: PX 17.3%, px 47.4%, Px 24.4% and pX 10.9%. Prevalence of estrogen receptor alpha PvuII and XbaI polymorphisms in Polish women is similar to previously studied population.

  4. Amplification of marine methanotrophic enrichment DNA with 16S rDNA PCR primers for type II alpha proteobacteria methanotrophs. (United States)

    Rockne, Karl J; Strand, Stuart E


    Type II alpha proteobacteria methanotrophs are capable of a wide range of cometabolic transformations of chlorinated solvents and polycyclic aromatic hydrocarbons (PAHs), and this activity has been exploited in many terrestrial bioremediation systems. However, at present, all known obligately marine methanotrophic isolates are Type I gamma proteobacteria which do not have this activity to the extent of Type II methanotrophs. In previous work in our laboratory, determining the presence of Type II alpha proteobacteria methanotrophs in marine enrichment cultures that co-metabolized PAHs required a more sensitive assay. 16S rDNA PCR primers were designed based on oligonucleotide probes for serine pathway methanotrophs and serine pathway methylotrophs with an approximate amplification fragment size of 870 base pairs. Comparison of the primers using double primer BLAST searches in established nucleotide databases showed potential amplification with all Methylocystis and Methylosinus spp., as well as potential amplification with Methylocella palustrus. DNA from Methylosinus trichosporium OB3b, a Type II methanotroph, amplified with the primers with a fragment size of approximately 850 base pairs, whereas DNA extracted from Methylomonas methanica, a Type I methanotroph, did not. The primers were used to amplify DNA extracted from two marine methanotrophic enrichment cultures: a low nitrogen/low copper enrichment to select for Type II methanotrophs and a high nitrogen/high copper enrichment to select for Type I methanotrophs. Although DNA from both cultures amplified with the PCR primers, amplification was stronger in cultures that were specifically enriched for Type II methanotrophs, suggesting the presence of higher numbers of Type II methanotrophs. These results provide further evidence for the existence of Type II marine methanotrophs, suggesting the possibility of exploiting cometabolic activity in marine systems.

  5. Endothelial monocyte activating polypeptide-II modulates endothelial cell responses by degrading hypoxia-inducible factor-1alpha through interaction with PSMA7, a component of the proteasome

    Energy Technology Data Exchange (ETDEWEB)

    Tandle, Anita T. [Tumor Angiogenesis Section, Surgery Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892 (United States); Calvani, Maura; Uranchimeg, Badarch [DTP-Tumor Hypoxia Laboratory, SAIC Frederick, Inc., National Cancer Institute, Frederick, Maryland 21702 (United States); Zahavi, David [Tumor Angiogenesis Section, Surgery Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892 (United States); Melillo, Giovanni [DTP-Tumor Hypoxia Laboratory, SAIC Frederick, Inc., National Cancer Institute, Frederick, Maryland 21702 (United States); Libutti, Steven K., E-mail: [Department of Surgery, Montefiore-Einstein Center for Cancer Care, Albert Einstein College of Medicine, Greene Medical Arts Pavilion, 4th Floor 3400, Bainbridge Avenue, Bronx, New York 10467 (United States)


    The majority of human tumors are angiogenesis dependent. Understanding the specific mechanisms that contribute to angiogenesis may offer the best approach to develop therapies to inhibit angiogenesis in cancer. Endothelial monocyte activating polypeptide-II (EMAP-II) is an anti-angiogenic cytokine with potent effects on endothelial cells (ECs). It inhibits EC proliferation and cord formation, and it suppresses primary and metastatic tumor growth in-vivo. However, very little is known about the molecular mechanisms behind the anti-angiogenic activity of EMAP-II. In the present study, we explored the molecular mechanism behind the anti-angiogenic activity exerted by this protein on ECs. Our results demonstrate that EMAP-II binds to the cell surface {alpha}5{beta}1 integrin receptor. The cell surface binding of EMAP-II results in its internalization into the cytoplasmic compartment where it interacts with its cytoplasmic partner PSMA7, a component of the proteasome degradation pathway. This interaction increases hypoxia-inducible factor 1-alpha (HIF-1{alpha}) degradation under hypoxic conditions. The degradation results in the inhibition of HIF-1{alpha} mediated transcriptional activity as well as HIF-1{alpha} mediated angiogenic sprouting of ECs. HIF-1{alpha} plays a critical role in angiogenesis by activating a variety of angiogenic growth factors. Our results suggest that one of the major anti-angiogenic functions of EMAP-II is exerted through its inhibition of the HIF-1{alpha} activities.


    Energy Technology Data Exchange (ETDEWEB)

    Prescott, Moire K. M. [Department of Physics, University of California, Broida Hall, Mail Code 9530, Santa Barbara, CA 93106 (United States); Dey, Arjun; Jannuzi, Buell T., E-mail: [National Optical Astronomy Observatory, 950 North Cherry Avenue, Tucson, AZ 85719 (United States)


    Using a systematic broadband search technique, we have carried out a survey for large Ly{alpha} nebulae (or Ly{alpha} {sup b}lobs{sup )} at 2 {approx}< z {approx}< 3 within 8.5 deg{sup 2} of the NOAO Deep Wide-Field Survey Booetes field, corresponding to a total survey comoving volume of Almost-Equal-To 10{sup 8} h {sup -3} {sub 70} Mpc{sup 3}. Here, we present our spectroscopic observations of candidate giant Ly{alpha} nebulae. Of 26 candidates targeted, 5 were confirmed to have Ly{alpha} emission at 1.7 {approx}< z {approx}< 2.7, 4 of which were new discoveries. The confirmed Ly{alpha} nebulae span a range of Ly{alpha} equivalent widths, colors, sizes, and line ratios, and most show spatially extended continuum emission. The remaining candidates did not reveal any strong emission lines, but instead exhibit featureless, diffuse, blue continuum spectra. Their nature remains mysterious, but we speculate that some of these might be Ly{alpha} nebulae lying within the redshift desert (i.e., 1.2 {approx}< z {approx}< 1.6). Our spectroscopic follow-up confirms the power of using deep broadband imaging to search for the bright end of the Ly{alpha} nebula population across enormous comoving volumes.

  7. [A novel gene (Aa-accA ) encoding acetyl-CoA carboxyltransferase alpha-subunit of Alkalimonas amylolytica N10 enhances salt and alkali tolerance of Escherichia coli and tobacco BY-2 cells]. (United States)

    Xian, Mingjie; Zhai, Lei; Zhong, Naiqin; Ma, Yiwei; Xue, Yanfen; Ma, Yanhe


    Acetyl-CoA carboxylase (ACC) catalyzes the first step of fatty acid synthesis. In most bacteria, ACC is composed of four subunits encoded by accA, accB, accC, and accD. Of them, accA encodes acetyl-CoA carboxyltransferase alpha-subunit. Our prior work on proteomics of Alkalimonas amylolytica N10 showed that the expression of the Aa-accA has a remarkable response to salt and alkali stress. This research aimed to find out the Aa-accA gene contributing to salt and alkali tolerance. The Aa-accA was amplified by PCR from A. amylolytica N10 and expressed in E. coli K12 host. The effects of Aa-accA expression on the growth of transgenic strains were examined under different NaCl concentration and pH conditions. Transgenic tobacco BY-2 cells harboring Aa-accA were also generated via Agrobacterium-mediated transformation. The viability of BY-2 cells was determined with FDA staining method after salt and alkali shock. The Aa-accA gene product has 318 amino acids and is homologous to the carboxyl transferase domain of acyl-CoA carboxylases. It showed 76% identity with AccA (acetyl-CoA carboxylase carboxyltransferase subunit alpha) from E. coli. Compared to the wild-type strains, transgenic E. coli K12 strain containing Aa-accA showed remarkable growth superiority when grown in increased NaCl concentrations and pH levels. The final cell density of the transgenic strains was 2.6 and 3.5 times higher than that of the control type when they were cultivated in LB medium containing 6% (W/V) NaCl and at pH 9, respectively. Complementary expression of Aa-accA in an accA-depletion E. coli can recover the tolerance of K12 delta accA to salt and alkali stresses to some extent. Similar to the transgenic E. coli, transgenic tobacco BY-2 cells showed higher percentages of viability compared to the wild BY-2 cells under the salt or alkali stress condition. We found that Aa-accA from A. amylolytica N10 overexpression enhances the tolerance of both transgenic E. coli and tobacco BY-2 cells to

  8. Structural Characterization of the Loop at the Alpha-Subunit C-Terminus of the Mixed Lineage Leukemia Protein Activating Protease Taspase1.

    Directory of Open Access Journals (Sweden)

    Johannes van den Boom

    Full Text Available Type 2 asparaginases, a subfamily of N-terminal nucleophile (Ntn hydrolases, are activated by limited proteolysis. This activation yields a heterodimer and a loop region at the C-terminus of the α-subunit is released. Since this region is unresolved in all type 2 asparaginase crystal structures but is close to the active site residues, we explored this loop region in six members of the type 2 asparaginase family using homology modeling. As the loop model for the childhood cancer-relevant protease Taspase1 differed from the other members, Taspase1 activation as well as the conformation and dynamics of the 56 amino acids loop were investigated by CD and NMR spectroscopy. We propose a helix-turn-helix motif, which can be exploited as novel anticancer target to inhibit Taspase1 proteolytic activity.

  9. From atactic to isotactic CO/p-methylstyrene copolymer by proper modification of Pd(II) catalysts bearing achiral alpha-diimines. (United States)

    Binotti, Barbara; Carfagna, Carla; Zuccaccia, Cristiano; Macchioni, Alceo


    Cationic Pd(II) complexes modified with achiral C(2v)-symmetric alpha-diimine ligands allow preparation of atactic or isotactic stereoblock CO/p-methylstyrene copolymers; both catalyst activity and polyketone microstructure depend on the choice of alpha-diimine substituents and counterion.

  10. Apoptotic role of TGF-β mediated by Smad4 mitochondria translocation and cytochrome c oxidase subunit II interaction. (United States)

    Pang, Lijuan; Qiu, Tao; Cao, Xu; Wan, Mei


    Smad4, originally isolated from the human chromosome 18q21, is a key factor in transducing the signals of the TGF-β superfamily of growth hormones and plays a pivotal role in mediating antimitogenic and proapoptotic effects of TGF-β, but the mechanisms by which Smad4 induces apoptosis are elusive. Here we report that Smad4 directly translocates to the mitochondria of apoptotic cells. Smad4 gene silencing by siRNA inhibits TGF-β-induced apoptosis in Hep3B cells and UV-induced apoptosis in PANC-1 cells. Cell fractionation assays demonstrated that a fraction of Smad4 translocates to mitochondria after long time TGF-β treatment or UV exposure, during which the cells were under apoptosis. Smad4 mitochondria translocation during apoptosis was also confirmed by fluorescence observation of Smad4 colocalization with MitoTracker Red. We searched for mitochondria proteins that have physical interactions with Smad4 using yeast two-hybrid screening approach. DNA sequence analysis identified 34 positive clones, five of which encoded subunits in mitochondria complex IV, i.e., one clone encoded cytochrome c oxidase COXII, three clones encoded COXIII and one clone encoded COXVb. Strong interaction between Smad4 with COXII, an important apoptosis regulator, was verified in yeast by β-gal activity assays and in mammalian cells by immunoprecipitation assays. Further, mitochondrial portion of cells was isolated and the interaction between COXII and Smad4 in mitochondria upon TGF-β treatment or UV exposure was confirmed. Importantly, targeting Smad4 to mitochondria using import leader fusions enhanced TGF-β-induced apoptosis. Collectively, the results suggest that Smad4 promote apoptosis of the cells through its mitochondrial translocation and association with mitochondria protein COXII. Copyright © 2011 Elsevier Inc. All rights reserved.

  11. Linkage of the gene that encodes the alpha 1 chain of type V collagen (COL5A1) to type II Ehlers-Danlos syndrome (EDS II). (United States)

    Loughlin, J; Irven, C; Hardwick, L J; Butcher, S; Walsh, S; Wordsworth, P; Sykes, B


    Ehlers-Danlos syndrome (EDS) is a group of heritable disorders of connective tissue with skin, ligaments and blood vessels being the main sites affected. The commonest variant (EDS II) exhibits an autosomal dominant mode of inheritance and is characterized by joint hypermobility, cigarette paper scars, lax skin and excessive bruising. As yet no gene has been linked to EDS II, nor has linkage been established to a specific region of the genome. However, several candidate genes encoding proteins of the extracellular matrix have been excluded. Using an intragenic simple sequence repeat polymorphism, we report linkage of the COL5A1 gene, which encodes the alpha 1(V) chain of type V collagen, to EDS II. A maximum LOD score (Zmax) for linkage of 8.3 at theta = 0.00 was generated for a single large pedigree.

  12. Diagnostic value of PIVKA-II and alpha-fetoprotein in hepatitis B virus-associated hepatocellular carcinoma (United States)

    Seo, Seung In; Kim, Hyoung Su; Kim, Won Jin; Shin, Woon Geon; Kim, Doo Jin; Kim, Kyung Ho; Jang, Myoung Kuk; Lee, Jin Heon; Kim, Joo Seop; Kim, Hak Yang; Kim, Dong Joon; Lee, Myung Seok; Park, Choong Kee


    AIM: To determine the cutoff values and to compare the diagnostic role of alpha-fetoprotein (AFP) and prothrombin induced by vitamin K absence-II (PIVKA-II) in chronic hepatitis B (CHB). METHODS: A total of 1255 patients with CHB, including 157 patients with hepatocellular carcinoma (HCC), 879 with non-cirrhotic CHB and 219 with cirrhosis without HCC, were retrospectively enrolled. The areas under the receiver operating characteristic (AUROC) curves of PIVKA-II, AFP and their combination were calculated and compared. RESULTS: The optimal cutoff values for PIVKA-II and AFP were 40 mAU/mL and 10 ng/mL, respectively, for the differentiation of HCC from nonmalignant CHB. The sensitivity and specificity were 73.9% and 89.7%, respectively, for PIVKA-II and 67.5% and 90.3% for AFP, respectively. The AUROC curves of both PIVKA-II and AFP were not significantly different (0.854 vs 0.853, P = 0.965) for the differentiation of HCC from nonmalignant CHB, whereas the AUROC of PIVKA-II was significantly better than that of AFP in patients with cirrhosis (0.870 vs 0.812, P = 0.042). When PIVKA-II and AFP were combined, the diagnostic power improved significantly compared to either AFP or PIVKA-II alone for the differentiation of HCC from nonmalignant CHB (P < 0.05), especially when cirrhosis was present (P < 0.05). CONCLUSION: Serum PIVKA-II might be a better tumor marker than AFP, and its combination with AFP may enhance the early detection of HCC in patients with CHB. PMID:25852278

  13. The Arabidopsis Mediator Complex Subunits MED16, MED14, and MED2 Regulate Mediator and RNA Polymerase II Recruitment to CBF-Responsive Cold-Regulated Genes[C][W][OPEN (United States)

    Hemsley, Piers A.; Hurst, Charlotte H.; Kaliyadasa, Ewon; Lamb, Rebecca; Knight, Marc R.; De Cothi, Elizabeth A.; Steele, John F.; Knight, Heather


    The Mediator16 (MED16; formerly termed SENSITIVE TO FREEZING6 [SFR6]) subunit of the plant Mediator transcriptional coactivator complex regulates cold-responsive gene expression in Arabidopsis thaliana, acting downstream of the C-repeat binding factor (CBF) transcription factors to recruit the core Mediator complex to cold-regulated genes. Here, we use loss-of-function mutants to show that RNA polymerase II recruitment to CBF-responsive cold-regulated genes requires MED16, MED2, and MED14 subunits. Transcription of genes known to be regulated via CBFs binding to the C-repeat motif/drought-responsive element promoter motif requires all three Mediator subunits, as does cold acclimation–induced freezing tolerance. In addition, these three subunits are required for low temperature–induced expression of some other, but not all, cold-responsive genes, including genes that are not known targets of CBFs. Genes inducible by darkness also required MED16 but required a different combination of Mediator subunits for their expression than the genes induced by cold. Together, our data illustrate that plants control transcription of specific genes through the action of subsets of Mediator subunits; the specific combination defined by the nature of the stimulus but also by the identity of the gene induced. PMID:24415770

  14. Ser2 is the autophosphorylation site in the beta subunit from bicistronically expressed human casein kinase-2 and from native rat liver casein kinase-2 beta

    DEFF Research Database (Denmark)

    Boldyreff, B; James, P; Staudenmann, W


    Human casein kinase-2 (CK-2) subunits alpha and beta were bicistronically expressed in bacteria. The recombinant holoenzyme shared all investigated properties with the native CK-2 from mammalian sources (rat liver, Krebs II mouse ascites tumour cells). Contrary to recombinant human CK-2 produced...

  15. The largest subunit of RNA polymerase II from the Glaucocystophyta: functional constraint and short-branch exclusion in deep eukaryotic phylogeny

    Directory of Open Access Journals (Sweden)

    Stiller John W


    Full Text Available Abstract Background Evolutionary analyses of the largest subunit of RNA polymerase II (RPB1 have yielded important and at times provocative results. One particularly troublesome outcome is the consistent inference of independent origins of red algae and green plants, at odds with the more widely accepted view of a monophyletic Plantae comprising all eukaryotes with primary plastids. If the hypothesis of a broader kingdom Plantae is correct, then RPB1 trees likely reflect a persistent phylogenetic artifact. To gain a better understanding of RNAP II evolution, and the presumed artifact relating to green plants and red algae, we isolated and analyzed RPB1 from representatives of Glaucocystophyta, the third eukaryotic group with primary plastids. Results Phylogenetic analyses incorporating glaucocystophytes do not recover a monophyletic Plantae; rather they result in additional conflicts with the most widely held views on eukaryotic relationships. In particular, glaucocystophytes are recovered as sister to several amoebozoans with strong support. A detailed investigation shows that this clade can be explained by what we call "short-branch exclusion," a phylogenetic artifact integrally associated with "long-branch attraction." Other systematic discrepancies observed in RPB1 trees can be explained as phylogenetic artifacts; however, these apparent artifacts also appear in regions of the tree that support widely held views of eukaryotic evolution. In fact, most of the RPB1 tree is consistent with artifacts of rate variation among sequences and co-variation due to functional constraints related to C-terminal domain based RNAP II transcription. Conclusion Our results reveal how subtle and easily overlooked biases can dominate the overall results of molecular phylogenetic analyses of ancient eukaryotic relationships. Sources of potential phylogenetic artifact should be investigated routinely, not just when obvious "long-branch attraction" is encountered.

  16. Molecular cloning of the alpha subunit of complement component C8 (CpC8α) of whitespotted bamboo shark (Chiloscyllium plagiosum). (United States)

    Wang, Ying; Zhang, Mengmeng; Wang, Conghui; Ye, Boping; Hua, Zichun


    Complement-mediated cytolysis is the important effect of immune response, which results from the assembly of terminal complement components (C5b-9). Among them, α subunit of C8 (C8α) is the first protein that traverses the lipid bilayer, and then initiates the recruitment of C9 molecules to form pore on target membranes. In this article, a full-length cDNA of C8α (CpC8α) is identified from the whitespotted bamboo shark (Chiloscyllium plagiosum) by RACE. The CpC8α cDNA is 2183 bp in length, encoding a protein of 591 amino acids. The deduced CpC8α exhibits 89%, 49% and 44% identity with nurse shark, frog and human orthologs, respectively. Sequence alignment indicates that the C8α is well conserved during the evolution process from sharks to mammals, with the same modular architecture as well as the identical cysteine composition in the mature protein. Phylogenetic analysis places CpC8α and nurse shark C8α in cartilaginous fish clade, in parallel with the teleost taxa, to form the C8α cluster with higher vertebrates. Hydrophobicity analysis also indicates a similar hydrophobicity of CpC8α to mammals. Finally, expression analysis revealed CpC8α transcripts were constitutively highly expressed in shark liver, with much less expression in other tissues. The well conserved structure and properties suggests an analogous function of CpC8α to mammalian C8α, though it remains to be confirmed by further study. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. [Interface bond and compatibility between GI-II glass/alumina composite and Vitadur alpha veneering porcelain]. (United States)

    Meng, Yukun; Chao, Yonglie; Liao, Yunmao


    Multiple layer techniques were commonly employed in fabricating all-ceramic restorations. Bond and compatibility between layers were vitally important for the clinical success of the restorations. The purposes of this study were to investigate the bond of the interface between the GI-II glass/alumina composite and Vitadur alpha veneering porcelain, and to study the thermal compatibility between them. Prepared a bar shaped specimen of GI-II glass/alumina composite 25 mm x 5 mm x 1 mm in size, with bottom surface pre-notched. The upper surface was veneered with Vitadur alpha veneering porcelain (0.2 mm opaque dentin and 0.6 mm dentin porcelain), then fractured and the fracture surface were examined under scanning electron microscope (SEM) and electron microprobe analyzer (EMPA) with electron beam of 10 micrometer in diameter; ten all-ceramic single crowns for an upper right central incisor were fabricated and the temperatures of thermal shock resistance were tested. SEM observation showed tight bond between the composite and the porcelain; The results of EMPA showed that penetration of Na, Al elements from glass/alumina into veneering porcelain and Si, K, Ca elements from veneering porcelain into glass/alumina occurred after sintering baking; The temperature of thermal shock resistance for anterior crowns in this study was 158 +/- 10.3 degrees C, cracks were mainly distributed in veneering porcelain with thicker layer. Chemical bond exists between the GI-II glass/alumina composite and Vitadur alpha veneering porcelain, and there is good thermal compatibility between them.

  18. Optimization in the nuclear fuel cycle II: Concentration of alpha emitters in the air

    International Nuclear Information System (INIS)

    Pereira, W.S.; Silva, A.X.; Lopes, J.M.; Carmo, A.S.; Mello, C.R.; Fernandes, T.S.; Kelecom, A.


    Optimization is one of the bases of radioprotection and aims to move doses away from the dose limit that is the borderline of acceptable radiological risk. The work aims to use the monitoring of the concentration of alpha emitters in the air as a tool of the optimization process. We analyzed 27 sampling points of airborne alpha concentration in a nuclear fuel cycle facility. The monthly averages were considered statistically different, the highest in the month of February and the lowest in the month of August. All other months were found to have identical mean activity concentration values. Regarding the sampling points, the points with the highest averages were points 12, 15 and 9. These points were indicated for the beginning of the optimization process. Analysis of the production of the facility should be performed to verify possible correlations between production and concentration of alpha emitters in the air

  19. Temperature-dependent structural changes in intrinsically disordered proteins: formation of alpha-helices or loss of polyproline II?

    DEFF Research Database (Denmark)

    Kjærgaard, Magnus; Nørholm, Ann-Beth; Hendus-Altenburger, Ruth


    temperature, which most likely reflects formation of transient alpha-helices or loss of polyproline II (PPII) content. Using three IDPs, ACTR, NHE1, and Spd1, we show that the temperature-induced structural change is common among IDPs and is accompanied by a contraction of the conformational ensemble...... with increasing temperature, and accordingly these were not responsible for the change in the CD spectra. In contrast, the nonhelical regions exhibited a general temperature-dependent structural change that was independent of long-range interactions. The temperature-dependent CD spectroscopic signature of IDPs...

  20. ORF Sequence: NC_002937 [GENIUS II[Archive

    Lifescience Database Archive (English)


  1. RNA Polymerase II Second Largest Subunit Molecular Identification of Boletus griseipurpureus Corner From Thailand and Antibacterial Activity of Basidiocarp Extracts. (United States)

    Aung-Aud-Chariya, Amornrat; Bangrak, Phuwadol; Lumyong, Saisamorn; Phupong, Worrapong; Aggangan, Nelly Siababa; Kamlangdee, Niyom


    Boletus griseipurpureus Corner, an edible mushroom, is a putative ectomycorrhizal fungus. Currently, the taxonomic boundary of this mushroom is unclear and its bitter taste makes it interesting for evaluating its antibacterial properties. The purpose of this study was to identify the genetic variation of this mushroom and also to evaluate any antibacterial activities. Basidiocarps were collected from 2 north-eastern provinces, Roi Et and Ubon Ratchathani, and from 2 southern provinces, Songkhla and Surat Thani, in Thailand. Genomic DNA was extracted and molecular structure was examined using the RNA polymerase II (RPB2) analysis. Antibacterial activities of basidiocarp extracts were conducted with Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29523 and methicillin-resistant Staphylococcus aureus (MRSA) 189 using the agar-well diffusion method. All the samples collected for this study constituted a monophyletic clade, which was closely related with the Boletus group of polypore fungi. For the antibacterial study, it was found that the crude methanol extract of basidiomes inhibited the growth of all bacteria in vitro more than the crude ethyl acetate extract. Basidomes collected from four locations in Thailand had low genetic variation and their extracts inhibited the growth of all tested bacteria. The health benefits of this edible species should be evaluated further.

  2. Temozolomide and interferon-alpha in metastatic melanoma: a phase II study of the Italian Melanoma Intergroup. (United States)

    Ridolfi, Ruggero; Romanini, Antonella; Sileni, Vanna Chiarion; Michiara, Maria; Guida, Michele; Biasco, Guido; Poletti, Paola; Amaducci, Laura; Leoni, Maurizio; Ravaioli, Alessandra


    Temozolomide (TMZ) is a new oral alkylating agent which has proven to be as active as dacarbazine (DTIC) in the treatment of melanoma, but with a lower toxicity. A multicentric phase II trial was conducted in an out-patient setting to determine the therapeutic activity and safety of TMZ in combination with interferon-alpha (IFN-alpha). From June 2000 to July 2001, 41 patients were recruited to receive TMZ 200 mg/m orally on days 1-5 every 28 days and with 5 MU IFN-alpha subcutaneously three times a week, continuously for eight cycles or until disease progression occurred. Of the 40 treated patients, two complete responses (5%) and three partial responses (7.5%) were observed, with a median duration of 4 months (range, 1.5-13.5 months). Thirteen patients (32.5%) had stable disease for a median of 2.5 months. Time to progression was 2.6 months and the median overall survival was 11.8 months. Nine patients (22.5%) developed brain metastases. The grade 4 toxicity observed in seven patients was of a transient haematological nature. This combination therapy is well tolerated but does not appear to increase the response rate or overall survival with respect to TMZ alone or to chemotherapeutic regimens. Further and more complex associations of these two drugs could be investigated in specific subsets of patients, in particular to evaluate its real efficacy in preventing brain metastases. Copyright 2004 Lippincott Williams & Wilkins

  3. The properties of the single chicken MHC classical class II alpha chain (B-LA) gene indicate an ancient origin for the DR/E-like isotype of class II molecules

    DEFF Research Database (Denmark)

    Salomonsen, Jan; Marston, Denise; Avila, David


    for the cloning and sequencing of the cDNA. We found only one class II alpha chain transcript, which bears the major features of a classical class II alpha sequence, including the critical peptide-binding residues. The chicken sequence is more similar to human DR than to the DQ, DP, DO or DM isotypes, most...... the mammalian DR and E isotypes in three properties: the presence of the critical peptide-binding residues, the low level of polymorphism and sequence diversity, and the recombinational separation from the class II beta chain genes. These results indicate that the sequence features of this lineage are both......In mammals, there are MHC class II molecules with distinctive sequence features, such as the classical isotypes DR, DQ and DP. These particular isotypes have not been reported in non-mammalian vertebrates. We have isolated the class II (B-L) alpha chain from outbred chickens as the basis...

  4. Genotypic to expression profiling of bovine calcium channel, voltage-dependent, alpha-2/delta subunit 1 gene, and their association with bovine mastitis among Frieswal (HFX Sahiwal) crossbred cattle of Indian origin. (United States)

    Deb, Rajib; Singh, Umesh; Kumar, Sushil; Kumar, Arun; Singh, Rani; Sengar, Gyanendra; Mann, Sandeep; Sharma, Arjava


    Calcium channel, voltage-dependent, alpha-2/delta subunit 1 (CACNA2D1) gene is considered to be an important noncytokine candidate gene influencing mastitis. Scanty of reports are available until today regarding the role play of CACNA2D1 gene on the susceptibility of bovine mastitis. We interrogated the CACNA2D1 G519663A [A>G] SNP by PCR-RFLP among two hundreds Frieswal (HF X Sahiwal) crossbred cattle of Indian origin. Genotypic frequency of AA (51.5, n=101) was comparatively higher than AG (35, n=70) and GG (14.5, n=29). Association of Somatic cell score (SCS) with genotypes revealed that, GG genotypes showing lesser count (less susceptible to mastitis) compare to AA and AG. Relative expression of CACNA2D1 transcript (in milk samples) was significantly higher among GG than AG and AA. Further we have also isolated blood sample from the all groups and PBMCs were cultured from each blood sample as per the standard protocol. They were treated with Calcium channel blocker and the expression level of the CACNA2D1 gene was evaluated by Real Time PCR. Results show that expression level decline in each genotypic group after treatment and expression level of GG are again significantly higher than AA and AG. Thus, it may be concluded that GG genotypic animals are favorable for selecting disease resistant breeds.

  5. A multilevel prediction of physiological response to challenge: Interactions among child maltreatment, neighborhood crime, endothelial nitric oxide synthase gene (eNOS), and GABA(A) receptor subunit alpha-6 gene (GABRA6). (United States)

    Lynch, Michael; Manly, Jody Todd; Cicchetti, Dante


    Physiological response to stress has been linked to a variety of healthy and pathological conditions. The current study conducted a multilevel examination of interactions among environmental toxins (i.e., neighborhood crime and child maltreatment) and specific genetic polymorphisms of the endothelial nitric oxide synthase gene (eNOS) and GABA(A) receptor subunit alpha-6 gene (GABRA6). One hundred eighty-six children were recruited at age 4. The presence or absence of child maltreatment as well as the amount of crime that occurred in their neighborhood during the previous year were determined at that time. At age 9, the children were brought to the lab, where their physiological response to a cognitive challenge (i.e., change in the amplitude of the respiratory sinus arrhythmia) was assessed and DNA samples were collected for subsequent genotyping. The results confirmed that complex Gene × Gene, Environment × Environment, and Gene × Environment interactions were associated with different patterns of respiratory sinus arrhythmia reactivity. The implications for future research and evidence-based intervention are discussed.

  6. Endothelial-monocyte activating polypeptide II alters fibronectin based endothelial cell adhesion and matrix assembly via alpha5 beta1 integrin

    International Nuclear Information System (INIS)

    Schwarz, Margaret A.; Zheng, Hiahua; Liu, Jie; Corbett, Siobhan; Schwarz, Roderich E.


    Mature Endothelial-Monocyte Activating Polypeptide (mEMAP) II functions as a potent antiangiogenic peptide. Although the anti-tumor effect of mEMAP II has been described, little is known regarding its mechanism of action. Observations that mEMAP II induced apoptosis only in a subset of migrating and proliferating endothelial cells (EC) suggests a targeted effect on cells engaged in angiogenic activities which are known to rely upon cell adhesion and migration. Indeed, we demonstrate that mEMAP II inhibited fibronectin (FN) dependent microvascular EC (MEC) adhesion and spreading and we show that this depends upon the alpha5 beta1 integrin. Immunofluorescence analysis demonstrated that mEMAP II-dependent blockade of FN-alpha5 beta1 interactions was associated with disassembly of both actin stress fiber networks and FN matrix. These findings suggest that mEMAP II blocks MEC adhesion and spreading on fibronectin, via a direct interaction with the integrin alpha5 beta1, thus implicating that alpha5 integrin may be a mediator of mEMAP II's antiangiogenic function

  7. Alpha spectrometry of thick sources. II. Application to the study of radioactive equilibria in uranium ores

    International Nuclear Information System (INIS)

    Acena Barrenechea, M.L.; Tormo Ferrero, M.J.


    A method for determining nuclide activities in 4n + 2 uranium series using alpha spectrometry of thick sources is described. This method has been applied to several uranium ores, showing different states of radioactive equilibria. The spectra from samples prepared by cold compression show some anomalies, due to the evolution and later decay of 219 Rn and daughters. This phenomenon must be taken in consideration when computing spectra line intensities. (author) [es


    Voltage-sensitive sodium channels (VSSCs) are a primary target of pyrethroid insecticides. VSSCs are comprised of a pore-forming ¿ and auxillary ß subunits, and multiple isoforms of both subunit types exist. The sensitivity of different isoform combinations to pyrethroids has not...

  9. Functional conservation of RNA polymerase II in fission and budding yeasts. (United States)

    Shpakovski, G V; Gadal, O; Labarre-Mariotte, S; Lebedenko, E N; Miklos, I; Sakurai, H; Proshkin, S A; Van Mullem, V; Ishihama, A; Thuriaux, P


    The complementary DNAs of the 12 subunits of fission yeast (Schizosaccharomyces pombe) RNA polymerase II were expressed from strong promoters in Saccharomyces cerevisiae and tested for heterospecific complementation by monitoring their ability to replace in vivo the null mutants of the corresponding host genes. Rpb1 and Rpb2, the two largest subunits and Rpb8, a small subunit shared by all three polymerases, failed to support growth in S. cerevisiae. The remaining nine subunits were all proficient for heterospecific complementation and led in most cases to a wild-type level of growth. The two alpha-like subunits (Rpb3 and Rpb11), however, did not support growth at high (37 degrees C) or low (25 degrees C) temperatures. In the case of Rpb3, growth was restored by increasing the gene dosage of the host Rpb11 or Rpb10 subunits, confirming previous evidence of a close genetic interaction between these three subunits. Copyright 2000 Academic Press.

  10. [Rat cardiomyocyte remodeling after neonatal cryptosporidiosis. II. Elongation, excessive polyploidization and HIF-1alpha overexpression]. (United States)

    Anatskaia, O V; Sidorenko, N V; Matveev, I V; Kropotov, A V; Vinogradov, A E


    Retrospective epidemyological studies evidence that infant diseases leave survivors with an increased susceptibility to cardiovascular diseases in later life. At the same time, the mechanisms of this link remain poorly understood. Based on medical statistics reporting that infectious gastroenteritis is the most common cause of maladies in babies, infants and children, we analysed the effects of moderate cryptosporidial gastroenteritis on the heart and ventricular cardiomyocyte remodelling in rats of the first month of life. The disease was challenged by a worldwide human protozoic pathogen Cryptosporidium parvum (Apicomplexa, Sporozoa). The main symptoms manifested in the growth retardation moderate diarrhea. Using real-time PCR, cytophotometry, confocal microscopy and image analysis, we indicated that cryptosporidiosis was associated, with the atrophy heart and the elongation, narrowing, protein content decrease and hyperpolyploidization of cardiomyocytes and the moderate overexpression of hypoxia inducible factor 1alpha (HIF-1alpha) mRNA. Cardiomyocyte shape remodeling and heart atrophy presented in all age groups. The severity of these changes, hovewer, declined gradually from younger to older groups. In contrast, hyperpolyploidization and HIF-1alpha mRNA overexpression were registered mainly among animals aged between 6 and 13 days, and were barely detected and non-significant in older age groups. In the rat the time period covering 6-13 days after birth is known to coincide with the intensive cardiomyocyte polyploidization and the switch from proliferation to hypertrophy. Thus, our data indicate that neonatal cryptosporidiosis may be potential cardiovascular diseases risk factor and that one of the critical time windows for the growing heart covers the time period when cardiomyocyte undergo polyploidization.

  11. Protein-protein interactions within photosystem II under photoprotection: the synergy between CP29 minor antenna, subunit S (PsbS) and zeaxanthin at all-atom resolution. (United States)

    Daskalakis, Vangelis


    The assembly and disassembly of protein complexes within cells are crucial life-sustaining processes. In photosystem II (PSII) of higher plants, there is a delicate yet obscure balance between light harvesting and photo-protection under fluctuating light conditions, that involves protein-protein complexes. Recent breakthroughs in molecular dynamics (MD) simulations are combined with new approaches herein to provide structural and energetic insight into such a complex between the CP29 minor antenna and the PSII subunit S (PsbS). The microscopic model involves extensive sampling of bound and dissociated states at atomic resolution in the presence of photo-protective zeaxanthin (Zea), and reveals well defined protein-protein cross-sections. The complex is placed within PSII, and macroscopic connections are emerging (PsbS-CP29-CP24-CP47) along the energy transfer pathways from the antenna to the PSII core. These connections explain macroscopic observations in the literature, while the previously obscured atomic scale details are now revealed. The implications of these findings are discussed in the context of the Non-Photochemical Quenching (NPQ) of chlorophyll fluorescence, the down-regulatory mechanism of photosynthesis, that enables the protection of PSII against excess excitation load. Zea is found at the PsbS-CP29 cross-section and a pH-dependent equilibrium between PsbS dimer/monomers and the PsbS-CP29 dissociation/association is identified as the target for engineering tolerant plants with increased crop and biomass yields. Finally, the new MD based approaches can be used to probe protein-protein interactions in general, and the PSII structure provided can initiate large scale molecular simulations of the photosynthetic apparatus, under NPQ conditions.

  12. Molecular Mechanism of AHSP-Mediated Stabilization of Alpha-Hemoglobin

    Energy Technology Data Exchange (ETDEWEB)

    Feng,L.; Gell, D.; Zhou, S.; Gu, L.; Kong, Y.; Li, J.; Hu, M.; Yan, N.; Lee, C.; et al.


    Hemoglobin A (HbA), the oxygen delivery system in humans, comprises two alpha and two beta subunits. Free alpha-hemoglobin (alphaHb) is unstable, and its precipitation contributes to the pathophysiology of beta thalassemia. In erythrocytes, the alpha-hemoglobin stabilizing protein (AHSP) binds alphaHb and inhibits its precipitation. The crystal structure of AHSP bound to Fe(II)-alphaHb reveals that AHSP specifically recognizes the G and H helices of alphaHb through a hydrophobic interface that largely recapitulates the alpha1-beta1 interface of hemoglobin. The AHSP-alphaHb interactions are extensive but suboptimal, explaining why beta-hemoglobin can competitively displace AHSP to form HbA. Remarkably, the Fe(II)-heme group in AHSP bound alphaHb is coordinated by the distal but not the proximal histidine. Importantly, binding to AHSP facilitates the conversion of oxy-alphaHb to a deoxygenated, oxidized [Fe(III)], nonreactive form in which all six coordinate positions are occupied. These observations reveal the molecular mechanisms by which AHSP stabilizes free alphaHb.

  13. A detector module with highly efficient surface-alpha event rejection operated in CRESST-II Phase 2

    Energy Technology Data Exchange (ETDEWEB)

    Strauss, R. [Max-Planck-Institut fuer Physik, Munich (Germany); Technische Universitaet Muenchen, Physik-Department, Garching (Germany); Angloher, G.; Ferreiro, N.; Hauff, D.; Kiefer, M.; Petricca, F.; Proebst, F.; Reindl, F.; Seidel, W.; Stodolsky, L.; Tanzke, A.; Wuestrich, M. [Max-Planck-Institut fuer Physik, Munich (Germany); Bento, A. [Universidade de Coimbra, CIUC, Departamento de Fisica, Coimbra (Portugal); Bucci, C.; Canonica, L.; Gorla, P.; Schaeffner, K. [Laboratori Nazionali del Gran Sasso, INFN, Assergi (Italy); Erb, A. [Technische Universitaet Muenchen, Physik-Department, Garching (Germany); Walther-Meissner-Institut fuer Tieftemperaturforschung, Garching (Germany); Feilitzsch, F. von; Guetlein, A.; Lanfranchi, J.C.; Muenster, A.; Potzel, W.; Roth, S.; Schoenert, S.; Stanger, M.; Ulrich, A.; Wawoczny, S.; Willers, M.; Zoeller, A. [Technische Universitaet Muenchen, Physik-Department, Garching (Germany); Jochum, J.; Loebell, J.; Rottler, K.; Sailer, C.; Scholl, S.; Strandhagen, C.; Uffinger, M.; Usherov, I. [Eberhard-Karls-Universitaet Tuebingen, Physikalisches Institut, Tuebingen (Germany); Kluck, H. [Institut fuer Hochenergiephysik der Oesterreichischen Akademie der Wissenschaften, Wien (Austria); Vienna University of Technology, Atominstitut, Wien (Austria); Kraus, H. [University of Oxford, Department of Physics, Oxford (United Kingdom); Schieck, J. [Institut fuer Hochenergiephysik der Oesterreichischen Akademie der Wissenschaften, Wien (Austria); Sivers, M. von [Technische Universitaet Muenchen, Physik-Department, Garching (Germany); University of Bern, Albert Einstein Center for Fundamental Physics, Bern (Switzerland)


    The cryogenic dark matter experiment CRESSTII aims at the direct detection of WIMPs via elastic scattering off nuclei in scintillating CaWO{sub 4} crystals. We present a new, highly improved, detector design installed in the current run of CRESST-II Phase 2 with an efficient active rejection of surface-alpha backgrounds. Using CaWO{sub 4} sticks instead of metal clamps to hold the target crystal, a detector housing with fully-scintillating inner surface could be realized. The presented detector (TUM40) provides an excellent threshold of ∝0.60 keV and a resolution of σ ∼ 0.090 keV (at 2.60 keV).With significantly reduced background levels, TUM40 sets stringent limits on the spin-independent WIMP nucleon scattering cross section and probes a new region of parameter space for WIMP masses below 3GeV/c{sup 2}. In this paper, we discuss the novel detector design and the surface-alpha event rejection in detail. (orig.)

  14. 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. (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


    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.

  15. Chromospheric Ca II H and K and H-alpha emission in single and binary stars of spectral types F6-M2

    International Nuclear Information System (INIS)

    Strassmeier, K.G.; Fekel, F.C.; Bopp, B.W.; Dempsey, R.C.; Henry, G.W.


    New observations of the Ca II H and K and H-epsilon region and/or the Balmer H-alpha line are presented for 100 mostly very active stars but also for weak or inactive stars with suspected activity. Correlations between chromospheric activity at Ca II H and K and H-alpha and effective surface temperature and rotation are identified, and several new stars with chromospheric Ca II H and K emission are discovered. No single activity-rotation relation can be derived for all luminosity classes, and there is clear evidence that evolved stars are generally more active than main-sequence stars of the same rotation period. Binary within the evolved stars appears to play no role, while main-sequence binary stars show generally higher levels of activity than their single counterparts. Chromospheric emission in the Ca II H and K lines depends on surface temperature in that flux declines with cooler temperature. 63 refs

  16. The role of the active site Zn in the catalytic mechanism of the GH38 Golgi alpha-mannosidase II: Implications from noeuromycin inhibition

    DEFF Research Database (Denmark)

    Bols, Mikael; Kuntz, Douglas A.; Rose, David R.


    Golgi alpha-mannosidase II (GMII) is a Family 38 glycosyl hydrolase involved in the eukaryotic N-glycosylation pathway in protein synthesis. Understanding of its catalytic mechanism has been of interest for the development of specific inhibitors that could lead to novel anti-metastatic or anti-in...

  17. Synthesis of .alpha.-Amino Acids via Asymmetric Phase Transfer-Catalyzed Alkylation of Achiral Niclkel(II) Complexes of Glycine-Derived Schiff bases

    Czech Academy of Sciences Publication Activity Database

    Belokon, Y. N.; Bespalova, N. B.; Churkina, T. D.; Císařová, I.; Ezernitskaya, M. G.; Harutyunyan, S. R.; Hrdina, R.; Kagan, H. B.; Kočovský, P.; Kochetkov, K. A.; Larionov, O. G.; Lysenko, K. A.; North, M.; Polášek, Miroslav; Peregudov, A. S.; Prisyazhnyuk, V. V.; Vyskočil, Š.


    Roč. 125, - (2003), s. 12860-12870 ISSN 0002-7863 R&D Projects: GA ČR GP203/01/D051 Institutional research plan: CEZ:AV0Z4040901 Keywords : .alpha.amino acids * achiral nickel(II) * glycine-derived schiff bases Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 6.516, year: 2003

  18. Immunohistochemical study of DNA topoisomerase I, DNA topoisomerase II alpha, p53, and Ki-67 in oral preneoplastic lesions and oral squamous cell carcinomas. (United States)

    Hafian, Hilal; Venteo, Lydie; Sukhanova, Alyona; Nabiev, Igor; Lefevre, Benoît; Pluot, Michel


    Human DNA topoisomerase I (topo I) is the molecular target of the camptothecin group of anticancer drugs. Laboratory studies have shown that the cellular response to topo I-targeted drugs depends on the topo I expression and DNA replication rate and the apoptotic pathway activity. In this study, we tested potential indicators of the sensitivity of topo I-targeted drugs in 36 cases of oral squamous cell carcinoma (OSCC). Formalin-fixed, paraffin-embedded tissue sections were immunostained with monoclonal antibodies against Ki-67, p53, and topo I, and with polyclonal antibodies against DNA topoisomerase II-alpha (topo II-alpha). These markers were also tested in 18 epithelial hyperplastic lesions and 18 mild dysplasias. Immunostaining was quantified by the percentage of stained nuclei in each sample (the labeling index); 200 immunoreactive epithelial nuclei were counted per case for each antibody. The results support the possibility of using topo II-alpha staining for assessing the proliferative activity. High expression of topo II-alpha and topo I in OSCCs suggests that they may serve as potential indicators of sensitivity to topo I inhibitors. However, the apoptotic pathway assessed by p53 immunostaining was found to be uninformative. Analysis of the relationship between immunohistochemical results and clinical and pathologic parameters (the T and N stages and differentiation) showed that only the differentiation parameter correlated with the topo I expression rate. Thus, significant increase in the topo I expression in the poorly differentiated OSCCs suggests their higher sensitivity to drug treatment.

  19. 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


    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....

  20. Sequence variation in the cytochrome oxidase subunit I and II genes of two commonly found blow fly species, Chrysomya megacephala (Fabricius) and Chrysomya rufifacies (Macquart) (Diptera: Calliphoridae) in Malaysia. (United States)

    Tan, Siew Hwa; Aris, Edah Mohd; Surin, Johari; Omar, Baharudin; Kurahashi, Hiromu; Mohamed, Zulqarnain


    The mitochondiral DNA region encompassing the cytochrome oxidase subunit I (COI) and cytochrome oxidase subunit II (COII) genes of two Malaysian blow fly species, Chrysomya megacephala (Fabricius) and Chrysomya rufifacies (Macquart) were studied. This region, which spans 2303bp and includes the COI, tRNA leucine and partial COII was sequenced from adult fly and larval specimens, and compared. Intraspecific variations were observed at 0.26% for Ch. megacephala and 0.17% for Ch. rufifacies, while sequence divergence between the two species was recorded at a minimum of 141 out of 2303 sites (6.12%). Results obtained in this study are comparable to published data, and thus support the use of DNA sequence to facilitate and complement morphology-based species identification.

  1. Petrological Geodynamics of Mantle Melting II. AlphaMELTS + Multiphase Flow: Dynamic Fractional Melting (United States)

    Tirone, Massimiliano


    In this second installment of a series that aims to investigate the dynamic interaction between the composition and abundance of the solid mantle and its melt products, the classic interpretation of fractional melting is extended to account for the dynamic nature of the process. A multiphase numerical flow model is coupled with the program AlphaMELTS, which provides at the moment possibly the most accurate petrological description of melting based on thermodynamic principles. The conceptual idea of this study is based on a description of the melting process taking place along a 1-D vertical ideal column where chemical equilibrium is assumed to apply in two local sub-systems separately on some spatial and temporal scale. The solid mantle belongs to a local sub-system (ss1) that does not interact chemically with the melt reservoir which forms a second sub-system (ss2). The local melt products are transferred in the melt sub-system ss2 where the melt phase eventually can also crystallize into a different solid assemblage and will evolve dynamically. The main difference with the usual interpretation of fractional melting is that melt is not arbitrarily and instantaneously extracted from the mantle, but instead remains a dynamic component of the model, hence the process is named dynamic fractional melting (DFM). Some of the conditions that may affect the DFM model are investigated in this study, in particular the effect of temperature, mantle velocity at the boundary of the mantle column. A comparison is made with the dynamic equilibrium melting (DEM) model discussed in the first installment. The implications of assuming passive flow or active flow are also considered to some extent. Complete data files of most of the DFM simulations, four animations and two new DEM simulations (passive/active flow) are available following the instructions in the supplementary material.

  2. The Lyman alpha reference sample. II. Hubble space telescope imaging results, integrated properties, and trends

    Energy Technology Data Exchange (ETDEWEB)

    Hayes, Matthew; Östlin, Göran; Duval, Florent; Sandberg, Andreas; Guaita, Lucia; Melinder, Jens; Rivera-Thorsen, Thøger [Department of Astronomy, Oskar Klein Centre, Stockholm University, AlbaNova University Centre, SE-106 91 Stockholm (Sweden); Adamo, Angela [Max Planck Institute for Astronomy, Königstuhl 17, D-69117 Heidelberg (Germany); Schaerer, Daniel [Université de Toulouse, UPS-OMP, IRAP, F-31000 Toulouse (France); Verhamme, Anne; Orlitová, Ivana [Geneva Observatory, University of Geneva, 51 Chemin des Maillettes, CH-1290 Versoix (Switzerland); Mas-Hesse, J. Miguel; Otí-Floranes, Héctor [Centro de Astrobiología (CSIC-INTA), Departamento de Astrofísica, P.O. Box 78, E-28691 Villanueva de la Cañada (Spain); Cannon, John M.; Pardy, Stephen [Department of Physics and Astronomy, Macalester College, 1600 Grand Avenue, Saint Paul, MN 55105 (United States); Atek, Hakim [Laboratoire dAstrophysique, École Polytechnique Fédérale de Lausanne (EPFL), Observatoire, CH-1290 Sauverny (Switzerland); Kunth, Daniel [Institut d' Astrophysique de Paris, UMR 7095, CNRS and UPMC, 98 bis Bd Arago, F-75014 Paris (France); Laursen, Peter [Dark Cosmology Centre, Niels Bohr Institute, University of Copenhagen, Juliane Maries Vej 30, DK-2100 Copenhagen (Denmark); Herenz, E. Christian, E-mail: [Leibniz-Institut für Astrophysik (AIP), An der Sternwarte 16, D-14482 Potsdam (Germany)


    We report new results regarding the Lyα output of galaxies, derived from the Lyman Alpha Reference Sample, and focused on Hubble Space Telescope imaging. For 14 galaxies we present intensity images in Lyα, Hα, and UV, and maps of Hα/Hβ, Lyα equivalent width (EW), and Lyα/Hα. We present Lyα and UV radial light profiles and show they are well-fitted by Sérsic profiles, but Lyα profiles show indices systematically lower than those of the UV (n ≈ 1-2 instead of ≳ 4). This reveals a general lack of the central concentration in Lyα that is ubiquitous in the UV. Photometric growth curves increase more slowly for Lyα than the far ultraviolet, showing that small apertures may underestimate the EW. For most galaxies, however, flux and EW curves flatten by radii ≈10 kpc, suggesting that if placed at high-z only a few of our galaxies would suffer from large flux losses. We compute global properties of the sample in large apertures, and show total Lyα luminosities to be independent of all other quantities. Normalized Lyα throughput, however, shows significant correlations: escape is found to be higher in galaxies of lower star formation rate, dust content, mass, and nebular quantities that suggest harder ionizing continuum and lower metallicity. Six galaxies would be selected as high-z Lyα emitters, based upon their luminosity and EW. We discuss the results in the context of high-z Lyα and UV samples. A few galaxies have EWs above 50 Å, and one shows f{sub esc}{sup Lyα} of 80%; such objects have not previously been reported at low-z.

  3. The Lyman alpha reference sample. II. Hubble space telescope imaging results, integrated properties, and trends

    International Nuclear Information System (INIS)

    Hayes, Matthew; Östlin, Göran; Duval, Florent; Sandberg, Andreas; Guaita, Lucia; Melinder, Jens; Rivera-Thorsen, Thøger; Adamo, Angela; Schaerer, Daniel; Verhamme, Anne; Orlitová, Ivana; Mas-Hesse, J. Miguel; Otí-Floranes, Héctor; Cannon, John M.; Pardy, Stephen; Atek, Hakim; Kunth, Daniel; Laursen, Peter; Herenz, E. Christian


    We report new results regarding the Lyα output of galaxies, derived from the Lyman Alpha Reference Sample, and focused on Hubble Space Telescope imaging. For 14 galaxies we present intensity images in Lyα, Hα, and UV, and maps of Hα/Hβ, Lyα equivalent width (EW), and Lyα/Hα. We present Lyα and UV radial light profiles and show they are well-fitted by Sérsic profiles, but Lyα profiles show indices systematically lower than those of the UV (n ≈ 1-2 instead of ≳ 4). This reveals a general lack of the central concentration in Lyα that is ubiquitous in the UV. Photometric growth curves increase more slowly for Lyα than the far ultraviolet, showing that small apertures may underestimate the EW. For most galaxies, however, flux and EW curves flatten by radii ≈10 kpc, suggesting that if placed at high-z only a few of our galaxies would suffer from large flux losses. We compute global properties of the sample in large apertures, and show total Lyα luminosities to be independent of all other quantities. Normalized Lyα throughput, however, shows significant correlations: escape is found to be higher in galaxies of lower star formation rate, dust content, mass, and nebular quantities that suggest harder ionizing continuum and lower metallicity. Six galaxies would be selected as high-z Lyα emitters, based upon their luminosity and EW. We discuss the results in the context of high-z Lyα and UV samples. A few galaxies have EWs above 50 Å, and one shows f esc Lyα of 80%; such objects have not previously been reported at low-z.

  4. Primary structure of the α-subunit of Na+, K+-ATPase. II. Isolation, reverse transcription, and cloning of messenger RNA

    International Nuclear Information System (INIS)

    Petrukhin, K.E.; Broude, N.E.; Arsenyan, S.G.; Grishin, A.V.; Dzhandzhugazyan, K.N.; Modyanov, N.N.


    The messenger RNA coding the α-subunit of Na + ,K + -ATPase has been isolated from the outer medullary layer of porcine kidneys. The mRNA gives a specific hybridization band in the 25S-26S region with three oligonucleotide probes synthesized on the basis of information on the structure of three peptides isolated from a tryptic hydrolyzate of the α-subunit of Na + ,K + -ATPase. The translation of the mRNA in Xenopus laevis oocytes followed by immunochemical identification of the products of synthesis confirmed the presence of the mRNA of the α-subunit of Na + ,K + -ATPase in an enriched fraction of poly(A + )-RNA. This preparation has been used for the synthesis of cloning of double-stranded cDNA

  5. Involvement of proteasomal subunits zeta and iota in RNA degradation. (United States)

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


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

  6. Proteomic analysis of human metaphase chromosomes reveals Topoisomerase II alpha as an Aurora B substrate

    DEFF Research Database (Denmark)

    Morrison, Ciaran; Henzing, Alexander J; Jensen, Ole Nørregaard


    B in the presence of radioactive ATP. Immunoblot analysis confirmed the HeLa scaffold fraction to be enriched for known chromosomal proteins including CENP-A, CENP-B, CENP-C, ScII and INCENP. Mass spectrometry of bands excised from one-dimensional polyacrylamide gels further defined the protein......The essential Aurora B kinase is a chromosomal passenger protein that is required for mitotic chromosome alignment and segregation. Aurora B function is dependent on the chromosome passenger, INCENP. INCENP, in turn, requires sister chromatid cohesion for its appropriate behaviour. Relatively few...... composition of the extracted chromosome fraction. Cloning, fluorescent tagging and expression in HeLa cells of the putative GTP-binding protein NGB/CRFG demonstrated it to be a novel mitotic chromosome protein, with a perichromosomal localisation. Identi fication of the protein bands corresponding to those...

  7. The vertebrate ancestral repertoire of visual opsins, transducin alpha subunits and oxytocin/vasopressin receptors was established by duplication of their shared genomic region in the two rounds of early vertebrate genome duplications. (United States)

    Lagman, David; Ocampo Daza, Daniel; Widmark, Jenny; Abalo, Xesús M; Sundström, Görel; Larhammar, Dan


    Vertebrate color vision is dependent on four major color opsin subtypes: RH2 (green opsin), SWS1 (ultraviolet opsin), SWS2 (blue opsin), and LWS (red opsin). Together with the dim-light receptor rhodopsin (RH1), these form the family of vertebrate visual opsins. Vertebrate genomes contain many multi-membered gene families that can largely be explained by the two rounds of whole genome duplication (WGD) in the vertebrate ancestor (2R) followed by a third round in the teleost ancestor (3R). Related chromosome regions resulting from WGD or block duplications are said to form a paralogon. We describe here a paralogon containing the genes for visual opsins, the G-protein alpha subunit families for transducin (GNAT) and adenylyl cyclase inhibition (GNAI), the oxytocin and vasopressin receptors (OT/VP-R), and the L-type voltage-gated calcium channels (CACNA1-L). Sequence-based phylogenies and analyses of conserved synteny show that the above-mentioned gene families, and many neighboring gene families, expanded in the early vertebrate WGDs. This allows us to deduce the following evolutionary scenario: The vertebrate ancestor had a chromosome containing the genes for two visual opsins, one GNAT, one GNAI, two OT/VP-Rs and one CACNA1-L gene. This chromosome was quadrupled in 2R. Subsequent gene losses resulted in a set of five visual opsin genes, three GNAT and GNAI genes, six OT/VP-R genes and four CACNA1-L genes. These regions were duplicated again in 3R resulting in additional teleost genes for some of the families. Major chromosomal rearrangements have taken place in the teleost genomes. By comparison with the corresponding chromosomal regions in the spotted gar, which diverged prior to 3R, we could time these rearrangements to post-3R. We present an extensive analysis of the paralogon housing the visual opsin, GNAT and GNAI, OT/VP-R, and CACNA1-L gene families. The combined data imply that the early vertebrate WGD events contributed to the evolution of vision and the

  8. Yinchenhao Decoction Ameliorates Alpha-Naphthylisothiocyanate Induced Intrahepatic Cholestasis in Rats by Regulating Phase II Metabolic Enzymes and Transporters

    Directory of Open Access Journals (Sweden)

    Ya-Xiong Yi


    Full Text Available Yinchenhao Decoction (YCHD, a famous traditional Chinese formula, has been used for treating cholestasis for 1000s of years. The cholagogic effect of YCHD has been widely reported, but its pharmacodynamic material and underlying therapeutic mechanism remain unclear. By using ultra-high-performance liquid chromatography (UHPLC-quadrupole time-of-flight mass spectrometry, 11 original active components and eight phase II metabolites were detected in rats after oral administration of YCHD, including three new phase II metabolites. And it indicated that phase II metabolism was one of the major metabolic pathway for most active components in YCHD, which was similar to the metabolism process of bilirubin. It arouses our curiosity that whether the metabolism process of YCHD has any relationship with its cholagogic effects. So, a new method for simultaneous quantitation of eight active components and four phase II metabolites of rhein, emodin, genipin, and capillarisin has been developed and applied for their pharmacokinetic study in both normal and alpha-naphthylisothiocyanate (ANIT-induced intrahepatic cholestasis rats. The results indicated the pharmacokinetic behaviors of most components of YCHD were inhibited, which was hypothesized to be related to different levels of metabolic enzymes and transporters in rat liver. So dynamic changes of intrahepatic enzyme expression in cholestasis and YCHD treated rats have been monitored by an UHPLC-tandem mass spectrometry method. The results showed expression levels of UDP-glucuronosyltransferase 1-1 (UGT1A1, organic anion-transporting polypeptide 1A4 (OATP1A4, multidrug resistance-associated protein 2 (MRP2, multidrug resistance protein 1, sodium-dependent taurocholate cotransporter, and organic anion-transporting polypeptide 1A2 were significantly inhibited in cholestasis rats, which would account for reducing the drug absorption and the metabolic process of YCHD in cholestatic rats. A high dose (12 g/kg of

  9. Transcriptional regulators of Na, K-ATPase subunits


    Zhiqin eLi; Sigrid A Langhans


    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. Role of the Rubisco Small Subunit

    Energy Technology Data Exchange (ETDEWEB)

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


    possible structural pathway between the small-subunit βA-βB loop and alpha-helix 8 of the large-subunit α/β-barrel active site. Hybrid enzymes were also created comprised of plant small subunits and Chlamydomonas large subunits, and these enzymes have increases in CO2/O2 specificity, further indicating that small subunits may be the key for ultimately engineering an improved Rubisco enzyme.

  11. Medicinal flowers. XXVII. New flavanone and chalcone glycosides, arenariumosides I, II, III, and IV, and tumor necrosis factor-alpha inhibitors from everlasting, flowers of Helichrysum arenarium. (United States)

    Morikawa, Toshio; Wang, Li-Bo; Nakamura, Seikou; Ninomiya, Kiyofumi; Yokoyama, Eri; Matsuda, Hisashi; Muraoka, Osamu; Wu, Li-Jun; Yoshikawa, Masayuki


    The methanolic extract from the flowers of Helichrysum arenarium L. MOENCH was found to show inhibitory effect on tumor necrosis factor-alpha (TNF-alpha, 1 ng/ml)-induced cytotoxicity in L929 cells. From the methanolic extract, 50 constituents including four new flavanone and chalcone glycosides named arenariumosides I (1), II (2), III (3), and IV (4) were isolated. The stereostructures of 1-4 were elucidated on the basis of chemical and physicochemical evidence. Among the constituents, naringenin 7-O-beta-D-glucopyranoside (7), apigenin 7-O-beta-D-glucopyranoside (14), apigenin 7-O-gentiobioside (16), and apigenin 7,4'-di-O-beta-D-glucopyranoside (17) significantly inhibited TNF-alpha-induced cytotoxicity in L929 cells at 30 microM.

  12. Problems connected with the use of oligonucleotide probes with a high degree of degeneracy. Identification of mRNA and of cDNA clones corresponding to the gene of the. cap alpha. -subunit of Na/sup +/, K/sup +/-ATPase

    Energy Technology Data Exchange (ETDEWEB)

    Petrukhin, K.E.; Grishin, A.V.; Arsenyan, S.G.; Broude, N.E.; Grinkevich, V.A.; Filippova, L.Yu.; Severtsova, I.V.; Modyanov, N.N.


    To identify and search for nucleotide sequences containing the structural part of the gene of the ..cap alpha..-subunit of Na/sup +/, K/sup +/-ATPase, 17-membered oligonucleotide probes corresponding to the peptide Lys-Asp-Ala-Phe-Gln-Asn have been synthesized. It has been shown that, with a 64-fold degeneracyd, the 17-membered probe is suitable only for the identification of a specific sequence in mRNA. To search for clones containing cDNA fragments, preliminary fractionation of the probes with the aid of HPLC or the resynthesis of groups of oligonucleotides with a lower degeneracy is necessary.

  13. Differential roles of the glycogen-binding domains of beta subunits in regulation of the Snf1 kinase complex. (United States)

    Mangat, Simmanjeet; Chandrashekarappa, Dakshayini; McCartney, Rhonda R; Elbing, Karin; Schmidt, Martin C


    Members of the AMP-activated protein kinase family, including the Snf1 kinase of Saccharomyces cerevisiae, are activated under conditions of nutrient stress. AMP-activated protein kinases are heterotrimeric complexes composed of a catalytic alpha subunit and regulatory beta and gamma subunits. In this study, the role of the beta subunits in the regulation of Snf1 activity was examined. Yeasts express three isoforms of the AMP-activated protein kinase consisting of Snf1 (alpha), Snf4 (gamma), and one of three alternative beta subunits, either Sip1, Sip2, or Gal83. The Gal83 isoform of the Snf1 complex is the most abundant and was analyzed in the greatest detail. All three beta subunits contain a conserved domain referred to as the glycogen-binding domain. The deletion of this domain from Gal83 results in a deregulation of the Snf1 kinase, as judged by a constitutive activity independent of glucose availability. In contrast, the deletion of this homologous domain from the Sip1 and Sip2 subunits had little effect on Snf1 kinase regulation. Therefore, the different Snf1 kinase isoforms are regulated through distinct mechanisms, which may contribute to their specialized roles in different stress response pathways. In addition, the beta subunits are subjected to phosphorylation. The responsible kinases were identified as being Snf1 and casein kinase II. The significance of the phosphorylation is unclear since the deletion of the region containing the phosphorylation sites in Gal83 had little effect on the regulation of Snf1 in response to glucose limitation.

  14. Arabidopsis thaliana RGXT1 and RGXT2 encode Golgi-localized (1,3)-alpha-D-xylosyltransferases involved in the synthesis of pectic rhamnogalacturonan-II

    DEFF Research Database (Denmark)

    Madsen, Jack Egelund; Petersen, Bent Larsen; Motawia, Mohammed Saddik


    in rhamnogalacturonan-II, a complex polysaccharide essential to vascular plants, and is conserved across higher plant families. Rhamnogalacturonan-II isolated from both RGXT1 and RGXT2 T-DNA insertional mutants functioned as specific acceptor molecules in the xylosyltransferase assay. Expression of RGXT1- and RGXT2......Two homologous plant-specific Arabidopsis thaliana genes, RGXT1 and RGXT2, belong to a new family of glycosyltransferases (CAZy GT-family-77) and encode cell wall (1,3)-alpha-d-xylosyltransferases. The deduced amino acid sequences contain single transmembrane domains near the N terminus, indicative...

  15. Synthesis and Characterization of Acyclic and Cyclic Azabridged Ligands Incorporating 2,2'-Bipyridine Subunits and Their Complexes With Copper(II, Cobalt(II, and Nickel(II

    Directory of Open Access Journals (Sweden)

    Andrea Pappalardo


    Full Text Available The synthesis of a series of N,N'-disubstituted acyclic (AL and cyclic (CL aza-bridged ligands incorporating 2,2-pipryidine subunits is described. 1H-NMR and IR spectral data support the proposed ligand structures. Dynamic 1H-NMR studies on diurea and diamide derivatives point to the presence of slowly interconverting conformers on the 1H-NMR time-scale, owing to N−H···N hydrogen bonding and/or a restricted rotation around the amide bonds. The ligands synthesized form 1:1 complexes with divalent transition metal ions. Upon complexation, bis-ester derivatives AL5 and CL5 undergo a metal-induced hydrolysis of the ester groups to carboxyl functions, which act as additional binding sites for the metal ion, as well as hydrogen-bonding donor-acceptor binding site to produce dimeric complexes.

  16. MicroRNA-139 suppresses proliferation in luminal type breast cancer cells by targeting Topoisomerase II alpha

    Energy Technology Data Exchange (ETDEWEB)

    Hua, Wei [Department of Obstetrics and Gynecology, Xijing Hospital, Fourth Military Medical University, Xi' an 710032 (China); State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, Fourth Military Medical University, 710032 Xi' an (China); Sa, Ke-Di; Zhang, Xiang; Jia, Lin-Tao; Zhao, Jing [State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, Fourth Military Medical University, 710032 Xi' an (China); Yang, An-Gang [State Key Laboratory of Cancer Biology, Department of Immunology, Fourth Military Medical University, 710032 Xi' an (China); Zhang, Rui, E-mail: [State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, Fourth Military Medical University, 710032 Xi' an (China); Fan, Jing, E-mail: [Department of Vascular and Endocrine Surgery, Xijing Hospital, Fourth Military Medical University, Xi' an 710032 (China); Bian, Ka, E-mail: [State Key Laboratory of Cancer Biology, Department of Immunology, Fourth Military Medical University, 710032 Xi' an (China); Department of Otolaryngology, Tangdu Hospital, Fourth Military Medical University, Xi' an 710038 (China)


    The classification of molecular subtypes of breast cancer improves the prognostic accuracy and therapeutic benefits in clinic. However, because of the complexity of breast cancer, more biomarkers and functional molecules need to be explored. Here, analyzing the data in a huge cohort of breast cancer patients, we found that Topoisomerase II alpha (TOP2a), an important target of chemotherapy is a biomarker for prognosis in luminal type breast cancer patients, but not in basal like or HER2 positive breast cancer patients. We identified that miR-139, a previous reported anti-metastatic microRNA targets 3’-untranslated region (3′UTR) of TOP2a mRNA. Further more, we revealed that the forced expression of miR-139 reduces the TOP2a expression at both mRNA and protein levels. And our functional experiments showed that the ectopic expression of miR-139 remarkably inhibits proliferation in luminal type breast cancer cells, while exogenous TOP2a expression could rescue inhibition of cell proliferation mediated by miR-139. Collectively, our present study demonstrates the miR-139-TOP2a regulatory axis is important for proliferation in luminal type breast cancer cells. This functional link may help us to further understand the specificity of subtypes of breast cancer and optimize the strategy of cancer treatment. - Highlights: • High levels of TOP2a expression are closely associated with poor prognosis in luminal type breast cancer patients. • TOP2a is a novel target of miR-139. • Overexpression of miR-139 inhibits proliferation in luminal type breast cancer cells. • TOP2a is essential for miR-139-induced growth arrest in luminal type breast cancer cells.

  17. MicroRNA-139 suppresses proliferation in luminal type breast cancer cells by targeting Topoisomerase II alpha

    International Nuclear Information System (INIS)

    Hua, Wei; Sa, Ke-Di; Zhang, Xiang; Jia, Lin-Tao; Zhao, Jing; Yang, An-Gang; Zhang, Rui; Fan, Jing; Bian, Ka


    The classification of molecular subtypes of breast cancer improves the prognostic accuracy and therapeutic benefits in clinic. However, because of the complexity of breast cancer, more biomarkers and functional molecules need to be explored. Here, analyzing the data in a huge cohort of breast cancer patients, we found that Topoisomerase II alpha (TOP2a), an important target of chemotherapy is a biomarker for prognosis in luminal type breast cancer patients, but not in basal like or HER2 positive breast cancer patients. We identified that miR-139, a previous reported anti-metastatic microRNA targets 3’-untranslated region (3′UTR) of TOP2a mRNA. Further more, we revealed that the forced expression of miR-139 reduces the TOP2a expression at both mRNA and protein levels. And our functional experiments showed that the ectopic expression of miR-139 remarkably inhibits proliferation in luminal type breast cancer cells, while exogenous TOP2a expression could rescue inhibition of cell proliferation mediated by miR-139. Collectively, our present study demonstrates the miR-139-TOP2a regulatory axis is important for proliferation in luminal type breast cancer cells. This functional link may help us to further understand the specificity of subtypes of breast cancer and optimize the strategy of cancer treatment. - Highlights: • High levels of TOP2a expression are closely associated with poor prognosis in luminal type breast cancer patients. • TOP2a is a novel target of miR-139. • Overexpression of miR-139 inhibits proliferation in luminal type breast cancer cells. • TOP2a is essential for miR-139-induced growth arrest in luminal type breast cancer cells

  18. Tetranuclear copper(II) complexes bridged by alpha-D-glucose-1-phosphate and incorporation of sugar acids through the Cu4 core structural changes. (United States)

    Kato, Merii; Sah, Ajay Kumar; Tanase, Tomoaki; Mikuriya, Masahiro


    Tetranuclear copper(II) complexes containing alpha-D-glucose-1-phosphate (alpha-D-Glc-1P), [Cu4(mu-OH){mu-(alpha-D-Glc-1P)}2(bpy)4(H2O)2]X3 [X = NO3 (1a), Cl (1b), Br (1c)], and [Cu4(mu-OH){mu-(alpha-D-Glc-1P)}2(phen)4(H2O)2](NO3)3 (2) were prepared by reacting the copper(II) salt with Na2[alpha-D-Glc-1P] in the presence of diimine ancillary ligands, and the structure of 2 was characterized by X-ray crystallography to comprise four {Cu(phen)}2+ fragments connected by the two sugar phosphate dianions in 1,3-O,O' and 1,1-O mu4-bridging fashion as well as a mu-hydroxo anion. The crystal structure of 2 involves two chemically independent complex cations in which the C2 enantiomeric structure for the trapezoidal tetracopper(II) framework is switched according to the orientation of the alpha-D-glucopyranosyl moieties. Temperature-dependent magnetic susceptibility data of 1a indicated that antiferromagnetic spin coupling is operative between the two metal ions joined by the hydroxo bridge (J = -52 cm(-1)) while antiferromagnetic interaction through the Cu-O-Cu sugar phosphate bridges is weak (J = -13 cm(-1)). Complex 1a readily reacted with carboxylic acids to afford the tetranuclear copper(II) complexes, [Cu4{mu-(alpha-D-Glc-1P)}2(mu-CA)2(bpy)4](NO3)2 [CA = CH3COO (3), o-C6H4(COO)(COOH) (4)]. Reactions with m-phenylenediacetic acid [m-C6H4(CH2COOH)2] also gave the discrete tetracopper(II) cationic complex [Cu4{mu-(alpha-D-Glc-1P)}2(mu-m-C6H4(CH2COO)(CH2COOH))2(bpy)4](NO3)2 (5a) as well as the cluster polymer formulated as {[Cu4{mu-(alpha-D-Glc-1P)}2(mu-m-C6H4(CH2COO)2)(bpy)4](NO3)2}n (5b). The tetracopper structure of 1a is converted into a symmetrical rectangular core in complexes 3, 4, and 5b, where the hydroxo bridge is dissociated and, instead, two carboxylate anions bridge another pair of Cu(II) ions in a 1,1-O monodentate fashion. The similar reactions were applied to incorporate sugar acids onto the tetranuclear copper(II) centers. Reactions of 1a with delta

  19. Copper(II) 12-metallacrown-4 complexes of alpha-, beta- and gamma-aminohydroxamic acids: a comparative thermodynamic study in aqueous solution. (United States)

    Tegoni, Matteo; Remelli, Maurizio; Bacco, Dimitri; Marchiò, Luciano; Dallavalle, Francesco


    A complete thermodynamic study of the protonation and Cu(II) complex formation equilibria of a series of alpha- and beta-aminohydroxamic acids in aqueous solution was performed. The thermodynamic parameters obtained for the protonation of glycine-, (S)-alpha-alanine-, (R,S)-valine-, (S)-leucine-, beta-alanine- and (R)-aspartic-beta-hydroxamic acids were compared with those previously reported for gamma-amino- and (S)-glutamic-gamma-hydroxamic acids. The enthalpy/entropy parameters calculated for the protonation microequilibria of these three types of ligands are in very good agreement with the literature values for simple amines and hydroxamic acids. The pentanuclear complexes [Cu5L4H(-4)]2+ contain the ligands acting as (NH2,N-)-(O,O-) bridging bis-chelating and correspond to 12-metallacrown-4 (12-MC-4) which are formed by self-assembly between pH 4 and 6 with alpha-aminohydroxamates (HL), while those with beta- and gamma-derivatives exist in a wider pH range (4-11). The stability order of these metallomacrocycles is beta- > alpha- > gamma-aminohydroxamates. The formation of 12-MC-4 with alpha-aminohydroxamates is entropy-driven, and that with beta-derivatives is enthalpy-driven, while with gamma-GABAhydroxamate both effects occur. These results are interpreted on the basis of specific enthalpies or entropy contributions related to chelate ring dimensions, charge neutralization and solvation-desolvation effects. The enthalpy/entropy parameters of 12-MC-4 with alpha-aminohydroxamic acids considered are also dependent on the optical purity of the ligands. Actually, that with (R,S)-valinehydroxamic acid presents an higher entropy and a lower enthalpy value than those of enantiopure ligands, although the corresponding stabilities are almost equivalent. Moreover, DFT calculations are in agreement with a more exothermic enthalpy found for metallacrowns with enantiomerically pure ligands.

  20. Multiplicity of expression of Na+,K+-ATPase alpha-subunit isoforms in the gill of Atlantic salmon: quantification and cellular localisation in response to salinity

    DEFF Research Database (Denmark)

    Madsen, Steffen; Kiilerich, Pia; Tipsmark, Christian Kølbæk


    but occasionally also on lamellae. Overall, the salinity-induced variation in labelling pattern and intensity matched the quantification data. In conclusion, the predominant switching of Na+,K+-ATPase -subunit isoform mRNA during salinity acclimation reflects a marked remodelling of mitochondrion-rich cells (MRCs...

  1. 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.


    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

  2. Interaction between alpha 2-adrenergic and angiotensin II systems in the control of glomerular hemodynamics as assessed by renal micropuncture in the rat (United States)

    Thomson, S. C.; Gabbai, F. B.; Tucker, B. J.; Blantz, R. C.


    The hypothesis that renal alpha 2 adrenoceptors influence nephron filtration rate (SNGFR) via interaction with angiotensin II (AII) was tested by renal micropuncture. The physical determinants of SNGFR were assessed in adult male Munich Wistar rats 5-7 d after ipsilateral surgical renal denervation (DNX). DNX was performed to isolate inhibitory central and presynaptic alpha 2 adrenoceptors from end-organ receptors within the kidney. Two experimental protocols were employed: one to test whether prior AII receptor blockade with saralasin would alter the glomerular hemodynamic response to alpha 2 adrenoceptor stimulation with the selective agonist B-HT 933 under euvolemic conditions, and the other to test whether B-HT 933 would alter the response to exogenous AII under conditions of plasma volume expansion. In euvolemic rats, B-HT 933 caused SNGFR to decline as the result of a decrease in glomerular ultrafiltration coefficient (LpA), an effect that was blocked by saralasin. After plasma volume expansion, B-HT 933 showed no primary effect on LpA but heightened the response of arterial blood pressure, glomerular transcapillary pressure gradient, and LpA to AII. The parallel results of these converse experiments suggest a complementary interaction between renal alpha 2-adrenergic and AII systems in the control of LpA.

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

    International Nuclear Information System (INIS)

    Taylor, T.; Weintraub, B.D.


    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

  4. Halogenated benzimidazole inhibitors of phosphorylation, ''in vitro'' and ''in vivo'', of the surface acidic proteins of the yeast ribosomal 60S subunit by endogenous protein kinases CK-II and PK60S

    International Nuclear Information System (INIS)

    Szyszka, Ryszard; Boguszewska, Aleksandra; Grankowski, Nikodem; Shugar, David


    Several halogeno benzimidazoles and 2-azabenzimidazoles, previously shown to be relatively selective inhibitors of protein kinases CK-I and/or CK-II from various sources, including CK-II from yeast [Szyszka et al. (1995) Biochem. Biophys. Res. Commun. 208, 418-424] inhibit also the yeast ribosomal protein kinase PK60S. The most effective inhibitor of CK-II and PK60S was tetrabromo-2-azabenzimidazole (TetraBr-2-azaBz), which was competitive with respect to ATP (and GTP in the case of CK-II) with K i values of 0.7 μM for CK-II, and 0.1 μM for PK60S. PK60S phosphorylates only three (YP1β, YB1β', YP2α) out of five polypeptides of pp13 kDa acidic proteins of 60S subunit phosphorylated by CK-II [Szyszka et al. (1995) Acta Biochim. Polon. 42, 357-362]. Accordingly, TetraBr-azaBz inhibits phosphorylation only of these polypeptides, catalysed by PK60S. Addition of TetraBr-2Bz to cultures of yeast cells, at concentrations which were without effect on cell growth, led to inhibition of intracellular phosphorylation of ribosomal acidic proteins, paralleling that observed ''in vitro''. TetraBr-2-azaBz is shown to be a useful tool for studies on the intracellular regulation of phosphorylation of the ribosomal 60S acidic proteins, which are involved in formation of active ribosomes. (author). 36 refs, 4 figs, 2 tabs

  5. An active Mitochondrial Complex II Present in Mature Seeds Contains an Embryo-Specific Iron-Sulfur Subunit Regulated by ABA and bZIP53 and Is Involved in Germination and Seedling Establishment. (United States)

    Restovic, Franko; Espinoza-Corral, Roberto; Gómez, Isabel; Vicente-Carbajosa, Jesús; Jordana, Xavier


    Complex II (succinate dehydrogenase) is an essential mitochondrial enzyme involved in both the tricarboxylic acid cycle and the respiratory chain. In Arabidopsis thaliana , its iron-sulfur subunit (SDH2) is encoded by three genes, one of them ( SDH2.3 ) being specifically expressed during seed maturation in the embryo. Here we show that seed SDH2.3 expression is regulated by abscisic acid (ABA) and we define the promoter region (-114 to +49) possessing all the cis -elements necessary and sufficient for high expression in seeds. This region includes between -114 and -32 three ABRE (ABA-responsive) elements and one RY-enhancer like element, and we demonstrate that these elements, although necessary, are not sufficient for seed expression, our results supporting a role for the region encoding the 5' untranslated region (+1 to +49). The SDH2.3 promoter is activated in leaf protoplasts by heterodimers between the basic leucine zipper transcription factors bZIP53 (group S1) and bZIP10 (group C) acting through the ABRE elements, and by the B3 domain transcription factor ABA insensitive 3 (ABI3). The in vivo role of bZIP53 is further supported by decreased SDH2.3 expression in a knockdown bzip53 mutant. By using the protein synthesis inhibitor cycloheximide and sdh2 mutants we have been able to conclusively show that complex II is already present in mature embryos before imbibition, and contains mainly SDH2.3 as iron-sulfur subunit. This complex plays a role during seed germination sensu-stricto since we have previously shown that seeds lacking SDH2.3 show retarded germination and now we demonstrate that low concentrations of thenoyltrifluoroacetone, a complex II inhibitor, also delay germination. Furthermore, complex II inhibitors completely block hypocotyl elongation in the dark and seedling establishment in the light, highlighting an essential role of complex II in the acquisition of photosynthetic competence and the transition from heterotrophy to autotrophy.

  6. Endothelial monocyte activating polypeptide-II modulates endothelial cell responses by degrading hypoxia-inducible factor-1alpha through interaction with PSMA7, a component of the proteasome

    International Nuclear Information System (INIS)

    Tandle, Anita T.; Calvani, Maura; Uranchimeg, Badarch; Zahavi, David; Melillo, Giovanni; Libutti, Steven K.


    The majority of human tumors are angiogenesis dependent. Understanding the specific mechanisms that contribute to angiogenesis may offer the best approach to develop therapies to inhibit angiogenesis in cancer. Endothelial monocyte activating polypeptide-II (EMAP-II) is an anti-angiogenic cytokine with potent effects on endothelial cells (ECs). It inhibits EC proliferation and cord formation, and it suppresses primary and metastatic tumor growth in-vivo. However, very little is known about the molecular mechanisms behind the anti-angiogenic activity of EMAP-II. In the present study, we explored the molecular mechanism behind the anti-angiogenic activity exerted by this protein on ECs. Our results demonstrate that EMAP-II binds to the cell surface α5β1 integrin receptor. The cell surface binding of EMAP-II results in its internalization into the cytoplasmic compartment where it interacts with its cytoplasmic partner PSMA7, a component of the proteasome degradation pathway. This interaction increases hypoxia-inducible factor 1-alpha (HIF-1α) degradation under hypoxic conditions. The degradation results in the inhibition of HIF-1α mediated transcriptional activity as well as HIF-1α mediated angiogenic sprouting of ECs. HIF-1α plays a critical role in angiogenesis by activating a variety of angiogenic growth factors. Our results suggest that one of the major anti-angiogenic functions of EMAP-II is exerted through its inhibition of the HIF-1α activities.

  7. Angiotensin II inhibits the Na+-K+ pump via PKC-dependent activation of NADPH oxidase. (United States)

    White, Caroline N; Figtree, Gemma A; Liu, Chia-Chi; Garcia, Alvaro; Hamilton, Elisha J; Chia, Karin K M; Rasmussen, Helge H


    The sarcolemmal Na(+)-K(+) pump, pivotal in cardiac myocyte function, is inhibited by angiotensin II (ANG II). Since ANG II activates NADPH oxidase, we tested the hypothesis that NADPH oxidase mediates the pump inhibition. Exposure to 100 nmol/l ANG II increased superoxide-sensitive fluorescence of isolated rabbit ventricular myocytes. The increase was abolished by pegylated superoxide dismutase (SOD), by the NADPH oxidase inhibitor apocynin, and by myristolated inhibitory peptide to epsilon-protein kinase C (epsilonPKC), previously implicated in ANG II-induced Na(+)-K(+) pump inhibition. A role for epsilonPKC was also supported by an ANG II-induced increase in coimmunoprecipitation of epsilonPKC with the receptor for the activated kinase and with the cytosolic p47(phox) subunit of NADPH oxidase. ANG II decreased electrogenic Na(+)-K(+) pump current in voltage-clamped myocytes. The decrease was abolished by SOD, by the gp91ds inhibitory peptide that blocks assembly and activation of NADPH oxidase, and by epsilonPKC inhibitory peptide. Since colocalization should facilitate NADPH oxidase-dependent regulation of the Na(+)-K(+) pump, we examined whether there is physical association between the pump subunits and NADPH oxidase. The alpha(1)-subunit coimmunoprecipitated with caveolin 3 and with membrane-associated p22(phox) and cytosolic p47(phox) NADPH oxidase subunits at baseline. ANG II had no effect on alpha(1)/caveolin 3 or alpha(1)/p22(phox) interaction, but it increased alpha(1)/p47(phox) coimmunoprecipitation. We conclude that ANG II inhibits the Na(+)-K(+) pump via PKC-dependent NADPH oxidase activation.

  8. Mutation of Gly195 of the ChlH subunit of Mg-chelatase reduces chlorophyll and further disrupts PS II assembly in a Ycf48-deficient strain of Synechocystis sp. PCC 6803

    Directory of Open Access Journals (Sweden)

    Tim Crawford


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

  9. Alpha1 and Alpha2 Integrins Mediate Invasive Activity of Mouse Mammary Carcinoma Cells through Regulation of Stromelysin-1 Expression

    Energy Technology Data Exchange (ETDEWEB)

    Lochter, Andre; Navre, Marc; Werb, Zena; Bissell, Mina J


    Tumor cell invasion relies on cell migration and extracellular matrix proteolysis. We investigated the contribution of different integrins to the invasive activity of mouse mammary carcinoma cells. Antibodies against integrin subunits {alpha}6 and {beta}1, but not against {alpha}1 and {alpha}2, inhibited cell locomotion on a reconstituted basement membrane in two-dimensional cell migration assays, whereas antibodies against {beta}1, but not against a6 or {alpha}2, interfered with cell adhesion to basement membrane constituents. Blocking antibodies against {alpha}1 integrins impaired only cell adhesion to type IV collagen. Antibodies against {alpha}1, {alpha}2, {alpha}6, and {beta}1, but not {alpha}5, integrin subunits reduced invasion of a reconstituted basement membrane. Integrins {alpha}1 and {alpha}2, which contributed only marginally to motility and adhesion, regulated proteinase production. Antibodies against {alpha}1 and {alpha}2, but not {alpha}6 and {beta}1, integrin subunits inhibited both transcription and protein expression of the matrix metalloproteinase stromelysin-1. Inhibition of tumor cell invasion by antibodies against {alpha}1 and {alpha}2 was reversed by addition of recombinant stromelysin-1. In contrast, stromelysin-1 could not rescue invasion inhibited by anti-{alpha}6 antibodies. Our data indicate that {alpha}1 and {alpha}2 integrins confer invasive behavior by regulating stromelysin-1 expression, whereas {alpha}6 integrins regulate cell motility. These results provide new insights into the specific functions of integrins during tumor cell invasion.

  10. The interaction of CK2alpha and CK2beta, the subunits of protein kinase CK2, requires CK2beta in a preformed conformation and is enthalpically driven

    DEFF Research Database (Denmark)

    Raaf, Jennifer; Brunstein, Elena; Issinger, Olaf-Georg


    . In contrast to the cyclins in the case of the cyclin-dependent kinases CK2beta is no on-switch of CK2alpha; rather the formation of the CK2 holoenzyme is accompanied with an overall change of the enzyme's profile including a modulation of the substrate specificity, an increase of the thermostability......, and an allocation of docking sites for membranes and other proteins. In this study we used C-terminal deletion variants of human CK2alpha and CK2beta that were enzymologically fully competent and in particular able to form a heterotetrameric holoenzyme. With differential scanning calorimetry (DSC) we confirmed...

  11. The exposed N-terminal tail of the D1 subunit is required for rapid D1 degradation during Photosystem II repair in Synechocystis sp

    Czech Academy of Sciences Publication Activity Database

    Komenda, Josef; Tichý, Martin; Prášil, Ondřej; Knoppová, Jana; Kuviková, Stanislava; de Vries, R.; Nixon, P. J.


    Roč. 19, - (2007), s. 2839-2854 ISSN 1040-4651 R&D Projects: GA MŠk LN00A141; GA ČR GA203/04/0800; GA ČR GA206/06/0322 Institutional research plan: CEZ:AV0Z50200510 Keywords : photosystem II * cyanobacterium * synechocystis sp. pcc 6803 Subject RIV: EE - Microbiology, Virology Impact factor: 9.653, year: 2007

  12. A randomized phase II chemoprevention trial of 13-CIS retinoic acid with or without alpha tocopherol or observation in subjects at high risk for lung cancer. (United States)

    Kelly, Karen; Kittelson, John; Franklin, Wilbur A; Kennedy, Timothy C; Klein, Catherine E; Keith, Robert L; Dempsey, Edward C; Lewis, Marina; Jackson, Mary K; Hirsch, Fred R; Bunn, Paul A; Miller, York E


    No chemoprevention strategies have been proven effective for lung cancer. We evaluated the effect of 13-cis retinoic acid (13-cis RA), with or without alpha tocopherol, as a lung cancer chemoprevention agent in a phase II randomized controlled clinical trial of adult subjects at high risk for lung cancer as defined by the presence of sputum atypia, history of smoking, and airflow obstruction, or a prior surgically cured nonsmall cell lung cancer (disease free, >3 years). Subjects were randomly assigned to receive either 13-cis RA, 13-cis RA plus alpha tocopherol (13-cis RA/alpha toco) or observation for 12 months. Outcome measures are derived from histologic evaluation of bronchial biopsy specimens obtained by bronchoscopy at baseline and follow-up. The primary outcome measure is treatment "failure" defined as histologic progression (any increase in the maximum histologic score) or failure to return for follow-up bronchoscopy. Seventy-five subjects were randomized (27/22/26 to observations/13-cis RA/13-cis RA/alpha toco); 59 completed the trial; 55 had both baseline and follow-up bronchoscopy. The risk of treatment failure was 55.6% (15 of 27) and 50% (24 of 48) in the observation and combined (13 cis RA plus 13 cis RA/alpha toco) treatment arms, respectively (odds ratio adjusted for baseline histology, 0.97; 95% confidence interval, 0.36-2.66; P = 0.95). Among subjects with complete histology data, maximum histology score in the observation arm increased by 0.37 units and by 0.03 units in the treated arms (difference adjusted for baseline, -0.18; 95% confidence interval, -1.16 to 0.81; P = 0.72). Similar (nonsignificant) results were observed for treatment effects on endobronchial proliferation as assessed by Ki-67 immunolabeling. Twelve-month treatment with 13-cis RA produced nonsignificant changes in bronchial histology, consistent with results in other trials. Agents advancing to phase III randomized trials should produce greater histologic changes. The

  13. Chiral analysis of alpha-diimine Ru(II) and Fe(II) complexes by capillary electrophoresis using sulfated cyclodextrins as stereoselectors

    Czech Academy of Sciences Publication Activity Database

    Sázelová, Petra; Koval, Dušan; Severa, Lukáš; Teplý, Filip; Kašička, Václav


    Roč. 38, č. 15 (2017), s. 1913-1921 ISSN 0173-0835 R&D Projects: GA ČR(CZ) GA15-01948S; GA ČR GA13-32974S; GA ČR GA13-19213S Institutional support: RVO:61388963 Keywords : binding constant * capillary electrophoresis * chiral separation * polypyridyl Fe(II) complex * polypyridyl Ru(II) complex Subject RIV: CB - Analytical Chemistry, Separation OBOR OECD: Analytical chemistry Impact factor: 2.744, year: 2016

  14. Alpha Momentum and Price Momentum

    Directory of Open Access Journals (Sweden)

    Hannah Lea Hühn


    Full Text Available We analyze a novel alpha momentum strategy that invests in stocks based on three-factor alphas which we estimate using daily returns. The empirical analysis for the U.S. and for Europe shows that (i past alpha has power in predicting the cross-section of stock returns; (ii alpha momentum exhibits less dynamic factor exposures than price momentum and (iii alpha momentum dominates price momentum only in the U.S. Connecting both strategies to behavioral explanations, alpha momentum is more related to an underreaction to firm-specific news while price momentum is primarily driven by price overshooting due to momentum trading.

  15. Proteomic data show an increase in autoantibodies and alpha-fetoprotein and a decrease in apolipoprotein A-II with time in sera from senescence-accelerated mice

    Energy Technology Data Exchange (ETDEWEB)

    Guo, S.J. [Beijing Institute of Pharmacology and Toxicology, Beijing (China); Shanghai Key Laboratory of Hypertension, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai (China); Qi, C.H.; Zhou, W.X.; Zhang, Y.X. [Beijing Institute of Pharmacology and Toxicology, Beijing (China); Zhang, X.M.; Wang, J.; Wang, H.X. [National Center of Biomedical Analysis, Beijing (China)


    We evaluated changes in levels by comparing serum proteins in senescence-accelerated mouse-prone 8 (SAMP8) mice at 2, 6, 12, and 15 months of age (SAMP8-2 m, -6 m, -12 m, -15 m) to age-matched SAM-resistant 1 (SAMR1) mice. Mice were sacrificed, and blood was analyzed by 2-dimensional electrophoresis combined with mass spectrometry. Five protein spots were present in all SAMP8 serum samples, but only appeared in SAMR1 samples at 15 months of age except for spot 3, which also showed a slight expression in SAMR1-12 m sera. Two proteins decreased in the sera from SAMP8-2 m, -6 m, and -12 m mice, and divided into 2 spots each in SAMP8-15 m sera. Thus, the total number of altered spots in SAMP8 sera was 7; of these, 4 were identified as Ig kappa chain V region (M-T413), chain A of an activity suppressing Fab fragment to cytochrome P450 aromatase (32C2-A), alpha-fetoprotein, and apolipoprotein A-II. M-T413 is a monoclonal CD4 antibody, which inhibits T cell proliferation. We found that M-T413 RNA level was significantly enhanced in splenocytes from SAMP8-2 m mice. This agreed with serum M-T413 protein alterations and a strikingly lower blood CD4{sup +} T cell count in SAMP8 mice when compared to the age-matched SAMR1 mice, with the latter negatively correlating with serum M-T413 protein volume. Age-related changes in serum proteins favored an increase in autoantibodies and alpha-fetoprotein and a decrease of apolipoprotein A-II, which occurred in SAMP8 mice at 2 months of age and onwards. These proteins may serve as candidate biomarkers for early aging.

  16. Proteomic data show an increase in autoantibodies and alpha-fetoprotein and a decrease in apolipoprotein A-II with time in sera from senescence-accelerated mice

    International Nuclear Information System (INIS)

    Guo, S.J.; Qi, C.H.; Zhou, W.X.; Zhang, Y.X.; Zhang, X.M.; Wang, J.; Wang, H.X.


    We evaluated changes in levels by comparing serum proteins in senescence-accelerated mouse-prone 8 (SAMP8) mice at 2, 6, 12, and 15 months of age (SAMP8-2 m, -6 m, -12 m, -15 m) to age-matched SAM-resistant 1 (SAMR1) mice. Mice were sacrificed, and blood was analyzed by 2-dimensional electrophoresis combined with mass spectrometry. Five protein spots were present in all SAMP8 serum samples, but only appeared in SAMR1 samples at 15 months of age except for spot 3, which also showed a slight expression in SAMR1-12 m sera. Two proteins decreased in the sera from SAMP8-2 m, -6 m, and -12 m mice, and divided into 2 spots each in SAMP8-15 m sera. Thus, the total number of altered spots in SAMP8 sera was 7; of these, 4 were identified as Ig kappa chain V region (M-T413), chain A of an activity suppressing Fab fragment to cytochrome P450 aromatase (32C2-A), alpha-fetoprotein, and apolipoprotein A-II. M-T413 is a monoclonal CD4 antibody, which inhibits T cell proliferation. We found that M-T413 RNA level was significantly enhanced in splenocytes from SAMP8-2 m mice. This agreed with serum M-T413 protein alterations and a strikingly lower blood CD4 + T cell count in SAMP8 mice when compared to the age-matched SAMR1 mice, with the latter negatively correlating with serum M-T413 protein volume. Age-related changes in serum proteins favored an increase in autoantibodies and alpha-fetoprotein and a decrease of apolipoprotein A-II, which occurred in SAMP8 mice at 2 months of age and onwards. These proteins may serve as candidate biomarkers for early aging

  17. A dynamic model of interactions of Ca2+, calmodulin, and catalytic subunits of Ca2+/calmodulin-dependent protein kinase II.

    Directory of Open Access Journals (Sweden)

    Shirley Pepke


    Full Text Available During the acquisition of memories, influx of Ca2+ into the postsynaptic spine through the pores of activated N-methyl-D-aspartate-type glutamate receptors triggers processes that change the strength of excitatory synapses. The pattern of Ca2+influx during the first few seconds of activity is interpreted within the Ca2+-dependent signaling network such that synaptic strength is eventually either potentiated or depressed. Many of the critical signaling enzymes that control synaptic plasticity,including Ca2+/calmodulin-dependent protein kinase II (CaMKII, are regulated by calmodulin, a small protein that can bindup to 4 Ca2+ ions. As a first step toward clarifying how the Ca2+-signaling network decides between potentiation or depression, we have created a kinetic model of the interactions of Ca2+, calmodulin, and CaMKII that represents our best understanding of the dynamics of these interactions under conditions that resemble those in a postsynaptic spine. We constrained parameters of the model from data in the literature, or from our own measurements, and then predicted time courses of activation and autophosphorylation of CaMKII under a variety of conditions. Simulations showed that species of calmodulin with fewer than four bound Ca2+ play a significant role in activation of CaMKII in the physiological regime,supporting the notion that processing of Ca2+ signals in a spine involves competition among target enzymes for binding to unsaturated species of CaM in an environment in which the concentration of Ca2+ is fluctuating rapidly. Indeed, we showed that dependence of activation on the frequency of Ca2+ transients arises from the kinetics of interaction of fluctuating Ca2+with calmodulin/CaMKII complexes. We used parameter sensitivity analysis to identify which parameters will be most beneficial to measure more carefully to improve the accuracy of predictions. This model provides a quantitative base from which to build more complex dynamic

  18. Suppression of tumor growth in vivo by the mitocan alpha-tocopheryl succinate requires respiratory complex II

    Czech Academy of Sciences Publication Activity Database

    Dong, L.F.; Freeman, R.; Liu, J.; Zobalová, Renata; Marin-Hernandez, A.; Stantic, M.; Rohlena, Jakub; Vališ, Karel; Rodriguez-Enriquez, S.; Butcher, B.; Goodwin, J.; Brunk, U.T.; Witting, P. K.; Moreno-Sanchez, R.; Scheffler, I.E.; Ralph, S.J.; Neužil, Jiří


    Roč. 15, č. 5 (2009), s. 1593-1600 ISSN 1078-0432 R&D Projects: GA ČR(CZ) GA204/08/0811; GA AV ČR(CZ) IAA500520702 Institutional research plan: CEZ:AV0Z50520701 Keywords : SDHC-mutants * mitocans * mitochondrial complex II Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.747, year: 2009

  19. Differential expression of BK channel isoforms and beta-subunits in rat neuro-vascular tissues

    DEFF Research Database (Denmark)

    Poulsen, Asser Nyander; Wulf, Helle; Hay-Schmidt, Anders


    We investigated the expression of splice variants and beta-subunits of the BK channel (big conductance Ca(2+)-activated K(+) channel, Slo1, MaxiK, K(Ca)1.1) in rat cerebral blood vessels, meninges, trigeminal ganglion among other tissues. An alpha-subunit splice variant X1(+24) was found expresse...

  20. Differential antibiotic sensitivity determined by the large ribosomal subunit in thermophilic archaea.


    Ruggero, D; Londei, P


    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.

  1. A-Raf kinase is a new interacting partner of protein kinase CK2 beta subunit

    DEFF Research Database (Denmark)

    Boldyreff, B; Issinger, O G


    In a search for protein kinase CK2 beta subunit binding proteins using the two-hybrid system, more than 1000 positive clones were isolated. Beside clones for the alpha' and beta subunit of CK2, there were clones coding for a so far unknown protein, whose partial cDNA sequence was already deposited...

  2. Predictive role of HER2/neu, topoisomerase-II-alpha, and tissue inhibitor of metalloproteinases (TIMP-1) for response to adjuvant taxane-based chemotherapy in patients with intermediate-risk breast cancer

    DEFF Research Database (Denmark)

    Erber, Ramona; Gluz, Oleg; Brünner, Nils


    . In this study, we investigate a large cohort of patients with intermediate-risk BC treated within the WSG EC-DOC Trial for the predictive impact of topoisomerase-II-alpha, HER2/neu, and TIMP-1. Tumor tissue was available in a representative cohort of 772 cases of the WSG EC-DOC Trial collective which compared 4...

  3. Inhibition of topoisomerase II{alpha} activity in CHO K1 cells by 2-[(aminopropyl)amino]ethanethiol (WR-1065)

    Energy Technology Data Exchange (ETDEWEB)

    Grdina, D.J. [Argonne National Lab., IL (United States)]|[Chicago Univ., IL (United States). Dept. of Radiation and Cellular Oncology; Constantinou, A. [Illinois Univ., Chicago, IL (United States). Specialized Cancer Center; Shigematsu, N.; Murley, J.S. [Argonne National Lab., IL (United States)


    The aminothiol 2-[(aminopropyl)amino]ethanethiol (WR-1065) is the active thiol of the clinically studied radioprotective agent S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721). WR-1065 is an effective radiation protector and antimutagenic agent when it is administered 30 min prior to radiation exposure to Chinese hamster ovary Kl cells at a concentration of 4 mM. Under these exposure conditions, topoisomerase (topo) I and II activities and associated protein contents were measured in the K1 cell line using the DNA relaxation assay, the P4 unknotting assay, and immunoblotting, respectively. WR-1065 was ineffective in modifying topo I activity, but it did reduce topo IIa activity by an average of 50 percent. The magnitude of topo IIa protein content, however, was not affected by these exposure conditions. Cell cycle effects were monitored by the method of flow cytometry. Exposure of cells to 4 mM WR-1065 for a period of up to 6 h resulted in a buildup of cells in the G2 compartment. However, in contrast to topo II inhibitors used in chemotherapy, WR-1065 is an effective radioprotector agent capable of protecting against both radiation-induced cell lethality and mutagenesis. One of several mechanisms of radiation protection attributed to aminothiol compounds such as WR-1065 has been their ability to affect endogenous enzymatic reactions involved in DNA synthesis, repair, and cell cycle progression. These results are consistent with such a proposed mechanism and demonstrate in particular a modifying effect by 2-[(aminopropyl)amino]ethanethiol on type II topoisomerase, which is involved in DNA synthesis.

  4. Molecular anatomy of CCR5 engagement by physiologic and viral chemokines and HIV-1 envelope glycoproteins: differences in primary structural requirements for RANTES, MIP-1 alpha, and vMIP-II Binding. (United States)

    Navenot, J M; Wang, Z X; Trent, J O; Murray, J L; Hu, Q X; DeLeeuw, L; Moore, P S; Chang, Y; Peiper, S C


    Molecular analysis of CCR5, the cardinal coreceptor for HIV-1 infection, has implicated the N-terminal extracellular domain (N-ter) and regions vicinal to the second extracellular loop (ECL2) in this activity. It was shown that residues in the N-ter are necessary for binding of the physiologic ligands, RANTES (CCL5) and MIP-1 alpha (CCL3). vMIP-II, encoded by the Kaposi's sarcoma-associated herpesvirus, is a high affinity CCR5 antagonist, but lacks efficacy as a coreceptor inhibitor. Therefore, we compared the mechanism for engagement by vMIP-II of CCR5 to its interaction with physiologic ligands. RANTES, MIP-1 alpha, and vMIP-II bound CCR5 at high affinity, but demonstrated partial cross-competition. Characterization of 15 CCR5 alanine scanning mutants of charged extracellular amino acids revealed that alteration of acidic residues in the distal N-ter abrogated binding of RANTES, MIP-1 alpha, and vMIP-II. Whereas mutation of residues in ECL2 of CCR5 dramatically reduced the binding of RANTES and MIP-1 alpha and their ability to induce signaling, interaction with vMIP-II was not altered by any mutation in the exoloops of the receptor. Paradoxically, monoclonal antibodies to N-ter epitopes did not block chemokine binding, but those mapped to ECL2 were effective inhibitors. A CCR5 chimera with the distal N-ter residues of CXCR2 bound MIP-1 alpha and vMIP-II with an affinity similar to that of the wild-type receptor. Engagement of CCR5 by vMIP-II, but not RANTES or MIP-1 alpha blocked the binding of monoclonal antibodies to the receptor, providing additional evidence for a distinct mechanism for viral chemokine binding. Analysis of the coreceptor activity of randomly generated mouse-human CCR5 chimeras implicated residues in ECL2 between H173 and V197 in this function. RANTES, but not vMIP-II blocked CCR5 M-tropic coreceptor activity in the fusion assay. The insensitivity of vMIP-II binding to mutations in ECL2 provides a potential rationale to its inefficiency as an

  5. Ni(ii) ions cleave and inactivate human alpha-1 antitrypsin hydrolytically, implicating nickel exposure as a contributing factor in pathologies related to antitrypsin deficiency. (United States)

    Wezynfeld, Nina Ewa; Bonna, Arkadiusz; Bal, Wojciech; Frączyk, Tomasz


    Human alpha-1 antitrypsin (AAT) is an abundant serum protein present at a concentration of 1.0-1.5 g L(-1). AAT deficiency is a genetic disease that manifests with emphysema and liver cirrhosis due to the accumulation of a misfolded AAT mutant in hepatocytes. Lung AAT amount is inversely correlated with chronic obstructive pulmonary disease (COPD), a serious and often deadly condition, with increasing frequency in the aging population. Exposure to cigarette smoke and products of fossil fuel combustion aggravates AAT deficiency and COPD according to mechanisms that are not fully understood. Taking into account that these fumes contain particles that can release nickel to human airways and skin, we decided to investigate interactions of AAT with Ni(ii) ions within the paradigm of Ni(ii)-dependent peptide bond hydrolysis. We studied AAT protein derived from human blood using HPLC, SDS-PAGE, and mass spectrometry. These studies were aided by spectroscopic experiments on model peptides. As a result, we identified three hydrolysis sites in AAT. Two of them are present in the N-terminal part of the molecule next to each other (before Thr-13 and Ser-14 residues) and effectively form one N-terminal cleavage site. The single C-terminal cleavage site is located before Ser-285. The N-terminal hydrolysis was more efficient than the C-terminal one, but both abolished the ability of AAT to inhibit trypsin in an additive manner. Nickel ions bound to hydrolysis products demonstrated an ability to generate ROS. These results implicate Ni(ii) exposure as a contributing factor in AAT-related pathologies.

  6. 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


    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...

  7. Reverse-phase HPLC analysis of human alpha crystallin. (United States)

    Swamy, M S; Abraham, E C


    A rapid and highly sensitive reverse-phase HPLC (RP-HPLC) method was used to separate crystallin subunits from human alpha crystallin. Three distinct peaks were separated; by electrophoretic and immunological analyses the first and second peaks were identified as alpha B and alpha A respectively. On the other hand, peak 3 appeared to be a modified form of alpha crystallin. The ratio of alpha A and alpha B proteins was 3:1 in 1 day old lenses which gradually changed to 2:1 in 17 year old lenses and to 1:1 in the 50 and 82 year old whole lenses and 82 year old lens cortex, with a concomitant increase in the modified alpha, suggesting that alpha A subunits are relatively more involved in aggregation. Analysis of the 82 year old lens nucleus also supported this conclusion. The RP-HPLC analysis of the HMW aggregate fraction showed substantial enrichment of the modified alpha. The alpha A and alpha B subunits independently reassociated to form polymeric alpha crystallin whereas the modified alpha reassociated to form HMW aggregates as shown by molecular sieve HPLC. Hence it appears that the HMW aggregate peak was constituted by modified alpha crystallin. Only in the peak 3 material the 280 nm absorbance was about 2-fold higher than what was expected from the actual protein content. The data suggest that the changes induced by post-translational modifications may have some role in the formation of modified alpha. The present RP-HPLC method is useful in separating these modified alpha from the unmodified alpha A and alpha B subunits.

  8. The significance of Epstein Barr Virus (EBV & DNA Topoisomerase II alpha (DNA-Topo II alpha immunoreactivity in normal oral mucosa, Oral Epithelial Dysplasia (OED and Oral Squamous Cell Carcinoma (OSCC

    Directory of Open Access Journals (Sweden)

    Osman Mohamed M


    Full Text Available Abstract Background Head and neck cancer including oral cancer is considered to develop by accumulated genetic alterations and the major pathway is cancerization from lesions such as intraepithelial dysplasia in oral leukoplakia and erythroplakia. The relationship of proliferation markers with the grading of dysplasia is uncertain. The involvement of EBV in oral carcinogenesis is not fully understood. Aim The present study was designed to investigate the role of EBV and DNA Topoisomerase II∝ (DNA-Topo II∝ during oral carcinogenesis and to examine the prognostic significance of these protein expressions in OSCCs. Methods Using specific antibodies for EBV and DNA-Topo II∝, we examined protein expressions in archival lesion tissues from 16 patients with oral epithelial dysplasia, 22 oral squamous cell carcinoma and 20 normal oral mucosa by immunohistochemistry. Clinical information was obtained through the computerized retrospective database from the tumor registry. Results DNA-Topo II∝ was expressed in all examined specimens. Analysis of Variance ANOVA revealed highly significant difference (P 0.05 in inferior surface of tongue and in hard palatal tissues. Significant differences were observed between OEDs and NSE (P Conclusion EBV and DNA Topo II-αLI expression are possible indicators in oral carcinogenesis and may be valuable diagnostic and prognostic indices in oral carcinoma.

  9. PI3KC2{alpha}, a class II PI3K, is required for dynamin-independent internalization pathways

    DEFF Research Database (Denmark)

    Krag, Claudia; Malmberg, Emily Kim; Salcini, Anna Elisabetta


    as fluid-phase endocytosis. Our data suggest a general role for PI3KC2a in regulating physiologically relevant dynamin-independent internalization pathways by recruiting early endosome antigen 1 (EEA1) to vesicular compartments, a step required for the intracellular trafficking of vesicles generated...... screen using a cell line expressing a diphtheria toxin receptor (DTR, officially known as HBEGF) anchored to GPI (DTR-GPI), which internalizes diphtheria toxin (DT, officially known as DTX) in a dynamin-independent manner, identified PI3KC2a, a class II phosphoinositide 3-kinase (PI3K), as a specific...... regulator of dynamin-independent DT internalization. We found that the internalization of several proteins that enter the cell through dynamin-independent pathways led to a relocalization of PI3KC2a to cargo-positive vesicles. Furthermore, downregulation of PI3KC2a impaired internalization of CD59 as well...

  10. Intrinsic polarization changes and the H-alpha and CA II emission features in T-Tauri stars (United States)

    Svatos, J.; Solc, M.


    On the basis of the correlation between polarization and emission features observed in certain T-Tauri stars, it is concluded that flaring effects associated with UV and/or X-ray irradiation and with increased magnetic field are responsible for the intrinsic polarization changes in T-Tauri stars. The correlation between emission Ca II lines and polarization degree both in Miras and T-Tau stars is thought to support the contention that the intrinsic polarization changes are due to the irradiation of silicate-like grains. In some T-Tau stars the increase in the magnetic field can be the principal agent causing the polarization increase due to the enhanced orientation of elongated grains.

  11. Differential stimulation by CCAAT/enhancer-binding protein alpha isoforms of the estrogen-activated promoter of the very-low-density apolipoprotein II gene

    NARCIS (Netherlands)

    Calkhoven, CF; Snippe, L; Ab, G


    The transcription factors CCAAT/enhancer-binding proteins alpha and beta (C/EBP alpha and C/EBP beta) are highly expressed in liver and are believed to function in maintaining the differentiated state of the hepatocytes, C/EBP alpha appears to be a critical regulator of genes involved in metabolic

  12. Alpha- and beta-adrenoceptors in hypertension. II. Platelet alpha 2- and lymphocyte beta 2-adrenoceptors in children of parents with essential hypertension. A model for the pathogenesis of the genetically determined hypertension

    NARCIS (Netherlands)

    Michel, M. C.; Galal, O.; Stoermer, J.; Bock, K. D.; Brodde, O. E.


    To study whether changes in alpha- and beta-adrenoceptors in human essential hypertension (EHT) might be genetically determined, we assessed platelet alpha 2- and lymphocyte beta 2-adrenoceptor density in 48 normotensive children of normotensive parents (NT) and in 41 normotensive children with one

  13. 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


    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...

  14. Crystal structure of the catalytic subunit of protein kinase CK2 from Zea mays at 2.1 A resolution

    DEFF Research Database (Denmark)

    Niefind, K; Guerra, B; Pinna, L A


    CK2alpha is the catalytic subunit of protein kinase CK2, an acidophilic and constitutively active eukaryotic Ser/Thr kinase involved in cell proliferation. A crystal structure, at 2.1 A resolution, of recombinant maize CK2alpha (rmCK2alpha) in the presence of ATP and Mg2+, shows the enzyme in an ...

  15. Subunits of the Snf1 kinase heterotrimer show interdependence for association and activity. (United States)

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


    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.


    Energy Technology Data Exchange (ETDEWEB)

    Jonason, C.; Miracle, A.


    The cytoskeletal protein alpha II-spectrin has specifi c neurodegenerative mechanisms that allow the necrotic (injury-induced) and apoptotic (non-injury-induced) pathways of proteolysis to be differentiated in an immunoblot. Consequently, αII-spectrin breakdown products (SBDPs) are potential biomarkers for diagnosing traumatic brain injury (TBI). The purpose of the following investigation, consisting of two studies, was to evaluate the utility of the spectrin biomarker in diagnosing TBI in fi sh that travel through hydroelectric dams in the Columbia and Snake Rivers. The fi rst study used hyperbaric pressure chambers to simulate the pressure changes that affect fi sh during passage through a Federal Columbia River Power System (FCRPS) Kaplan turbine. The second study tested the effect of a removable spillway weir (RSW) on the passage of juvenile chinook (Oncorhynchus tshawytscha). This study was conducted in tandem with a balloon-tag study by the U.S. Army Corps of Engineers. Brain samples from fi sh were collected and analyzed using an immunoblot for SBDPs, and imaging software was used to quantify the protein band density and determine the ratio of cleaved protein to total protein. The biomarker analyses found higher SBDP expression levels in fi sh that were exposed to lower pressure nadirs and fi sh that passed through the RSW at a deep orientation. In general, the incidence of injuries observed after treatment positively correlated with expression levels, suggesting that the biomarker method of analysis is comparable to traditional methods of injury assessment. It was also found that, for some treatments, the 110 kDa spectrin fragment (SBDP 110) correlated more strongly with necrotic head injury incidence and mortality rates than did the total cleaved protein or the 120 kDa fragment. These studies will be informative in future decisions regarding the design of turbines and fi sh passage structures in hydroelectric dams and will hopefully contribute to the

  17. Expression, purification and crystallization of the catalytic subunit of protein kinase CK2 from Zea mays

    DEFF Research Database (Denmark)

    Guerra, B; Niefind, K; Pinna, L A


    The catalytic (alpha) subunit of protein kinase CK2 (CK2alpha) was originally cloned and overexpressed in the Escherichia coli strain pT7-7/BL21(DE3). The protein has been purified to homogeneity and crystallized. The crystals belong to the monoclinic space group C2, they have unit-cell parameter...

  18. Transcriptional regulators of Na, K-ATPase subunits

    Directory of Open Access Journals (Sweden)

    Zhiqin eLi


    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. ORF Alignment: NC_002946 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available y: 1 ... MKIGIPRESLSGETRVACTPATVALLGKLGFETVVEXXXXXXXXXXXXXYQTAGATVADK 60 ... MKIGIPRESLSGETRVACTPAT...VALLGKLGFETVVE ... YQTAGATVADK Sbjct: 1 ... MKIGIPRESLSGETRVACTPATVALLGKLGFETVV... ... transhydrogenase, alpha subunit [Neisseria gonorrhoeae ... FA 1090] ... Length = 371 ... Quer

  20. Age-dependent changes in autophosphorylation of alpha calcium/calmodulin dependent kinase II in hippocampus and amygdala after contextual fear conditioning. (United States)

    Fang, Ton; Kasbi, Kamillia; Rothe, Stephanie; Aziz, Wajeeha; Giese, K Peter


    The hippocampus and amygdala are essential brain regions responsible for contextual fear conditioning (CFC). The autophosphorylation of alpha calcium-calmodulin kinase II (αCaMKII) at threonine-286 (T286) is a critical step implicated in long-term potentiation (LTP), learning and memory. However, the changes in αCaMKII levels with aging and training in associated brain regions are not fully understood. Here, we studied how aging and training affect the levels of phosphorylated (T286) and proportion of phosphorylated:total αCaMKII in the hippocampus and amygdala. Young and aged mice, naïve (untrained) and trained in CFC, were analysed by immunohistochemistry for the levels of total and phosphorylated αCaMKII in the hippocampus and amygdala. We found that two hours after CFC training, young mice exhibited a higher level of phosphorylated and increased ratio of phosphorylated:total αCaMKII in hippocampal CA3 stratum radiatum. Furthermore, aged untrained mice showed a higher ratio of phosphorylated:total αCaMKII in the CA3 region of the hippocampus when compared to the young untrained group. No effect of training or aging were seen in the central, lateral and basolateral amygdala regions, for both phosphorylated and ratio of phosphorylated:total αCaMKII. These results show that aging impairs the training-induced upregulation of autophosphorylated (T286) αCaMKII in the CA3 stratum radiatum of the hippocampus. This indicates that distinct age-related mechanisms underlie CFC that may rely more heavily on NMDA receptor-dependent plasticity in young age. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  1. Isolation and expression of a novel chick G-protein cDNA coding for a G alpha i3 protein with a G alpha 0 N-terminus.


    Kilbourne, E J; Galper, J B


    We have cloned cDNAs coding for G-protein alpha subunits from a chick brain cDNA library. Based on sequence similarity to G-protein alpha subunits from other eukaryotes, one clone was designated G alpha i3. A second clone, G alpha i3-o, was identical to the G alpha i3 clone over 932 bases on the 3' end. The 5' end of G alpha i3-o, however, contained an alternative sequence in which the first 45 amino acids coded for are 100% identical to the conserved N-terminus of G alpha o from species such...

  2. 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.


    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

  3. 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.


    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

  4. Buffett's Alpha

    DEFF Research Database (Denmark)

    Frazzini, Andrea; Kabiller, David; Heje Pedersen, Lasse

    Berkshire Hathaway has realized a Sharpe ratio of 0.76, higher than any other stock or mutual fund with a history of more than 30 years, and Berkshire has a significant alpha to traditional risk factors. However, we find that the alpha becomes insignificant when controlling for exposures to Betting...

  5. Alpha Blockers (United States)

    ... quickly, but their effects last only a few hours. Long-acting medications take longer to work, but their effects last longer. Which alpha blocker is best for you depends on your health and the condition being treated. Alpha blockers are ...

  6. Topoisomerase II alpha and TLE3 as predictive markers of response to anthracycline and taxane-containing regimens for neoadjuvant chemotherapy in breast cancer

    Directory of Open Access Journals (Sweden)

    Susini T


    Full Text Available Tommaso Susini,1 Barbara Berti,1 Carlo Carriero,1 Ketty Tavella,2 Jacopo Nori,3 Ermanno Vanzi,3 Cecilia Molino,1 Mariarosaria Di Tommaso,1 Marco Santini,1 Valeria Saladino,4 Simonetta Bianchi4 1Department of Health Science, Gynecology Section, 2Department of Health Science, Chemotherapy Section, University of Florence, Italy; 3Diagnostic Senology Unit, Azienda Ospedaliera-Universitaria Careggi, Florence, Italy; 4Department of Surgery and Translational Medicine, Pathology Unit, University of Florence, Italy Purpose: Anthracyclines and taxanes are considered the standard for neoadjuvant chemotherapy of breast cancer, although they are often associated with serious side effects and wide variability of individual response. In this study, we analyzed the value of topoisomerase II alpha (TOP2A and transducin-like enhancer of split 3 (TLE3 as predictive markers of response to therapy with anthracyclines and taxanes. Materials and methods: TOP2A and TLE3 protein expressions were evaluated using immunohistochemistry on 28 samples, obtained by core needle biopsy in patients with locally advanced breast carcinoma, subsequently subjected to epirubicin- and paclitaxel-based neoadjuvant chemotherapy. The immunohistochemical staining was correlated with the clinical response measured by the tumor size reduction evaluated by breast magnetic resonance imaging, prior and after chemotherapy, and by pathologic evaluation of the surgical specimen. Results: Neoadjuvant chemotherapy achieved a size reduction in 26/28 tumors (92.9%, with an average percentage decrease of 45.6%. A downstaging was achieved in 71.4% of the cases of locally advanced carcinoma. TOP2A positivity was correlated with a greater reduction in tumor diameter (P=0.06; negative staining for TLE3 was predictive of a better response to neoadjuvant chemotherapy (P=0.07. A higher reduction in tumor diameter (P=0.03 was also found for tumors that were concurrently TLE3-negative and TOP2A

  7. Assessment of topoisomerase II-alpha gene status by dual color chromogenic in situ hybridization in a set of Iraqi patients with invasive breast carcinoma

    Directory of Open Access Journals (Sweden)

    Rasha Abd Alraouf Neama


    Full Text Available Background: The human epidermal growth factor receptor 2(HER2 proto-oncogene is overexpressed or amplified in approximately 15%–25% of invasive breast cancers. Approximately 35% of HER2-amplified breast cancers have coamplification of the topoisomerase II-alpha (TOP2A gene encoding an enzyme that is a major target of anthracyclines. Hence, the determination of genetic alteration (amplification or deletion of both genes is considered as an important predictive factor that determines the response of breast cancer patients to treatment. The aims of this study are to determinate TOP2A status gene amplification in a set of Iraqi patients with breast cancer that have had an equivocal (2+ and positive HER2/neu by immunohistochemistry (IHC and to compare the results with estrogen receptor (ER and progesterone receptor (PR and HER2/neu status. Patients and Methods: A cross-sectional prospective study done on 53 patients with invasive breast carcinoma. Twenty-six out of total 53 cases were positive HER2/neu (3+, the remaining 27 equivocal HER2-IHC (2+ cases reanalyzed using dual-color chromogenic in situ hybridization (ZytoVision probe kit for further identification of HER2/neu gene amplification. Using chromogenic in situ hybridization (CISH, TOP2A gene status determination was done for all cases. Results: There is a direct significant correlation between TOP2A gene amplification and HER2/neu positivity, P < 0.05 in that 15 (39.4% out of 38 positive HER2/neu cases were associated with topoisomerase gene amplification. Regarding relation of topoisomerase gene to hormone receptor status (ER and PR, there was a significant negative relationship between the gene and ER receptor status. The higher level of gene amplification was noticed in ER and PR negative cases in about 13 (43.3% and 14 (48.2% for ER and PR, respectively. Conclusion: TOP2A gene status has a significantly positive correlation with HER2/neu status while it has a significantly negative

  8. TNF-alpha, produced by feline infectious peritonitis virus (FIPV)-infected macrophages, upregulates expression of type II FIPV receptor feline aminopeptidase N in feline macrophages. (United States)

    Takano, Tomomi; Hohdatsu, Tsutomu; Toda, Ayako; Tanabe, Maki; Koyama, Hiroyuki


    The pathogenicity of feline infectious peritonitis virus (FIPV) is known to depend on macrophage tropism, and this macrophage infection is enhanced by mediation via anti-S antibody (antibody-dependent enhancement, ADE). In this study, we found that TNF-alpha production was increased with viral replication in macrophages inoculated with a mixture of FIPV and anti-S antibody, and demonstrated that this culture supernatant had feline PBMC apoptosis-inducing activity. We also demonstrated that the expression level of the FIPV virus receptor, feline aminopeptidase N (fAPN), was increased in macrophages of FIP cats. For upregulation of TNF-alpha and fAPN in macrophages, viral replication in macrophages is necessary, and their expressions were increased by ADE of FIPV infection. It was demonstrated that a heat-resistant fAPN-inducing factor was present in the culture supernatant of FIPV-infected macrophages, and this factor was TNF-alpha: fAPN expression was upregulated in recombinant feline TNF-alpha-treated macrophages, and FIPV infectivity was increased in these macrophages. These findings suggested that FIPV replication in macrophages increases TNF-alpha production in macrophages, and the produced TNF-alpha acts and upregulates fAPN expression, increasing FIPV sensitivity.

  9. Small-angle neutron scattering from the reconstituted TF sub 1 of H sup + -ATPase from thermophilic bacterium PS3 with deuterated subunits

    Energy Technology Data Exchange (ETDEWEB)

    Ito, Yuji [Univ. of Tokyo (Japan) Brookhaven National Lab., Upton, NY (United States); Harada, Mitsuo [Univ. of Tokyo (Japan); Ohta, Shigeo; Kagawa, Yasuo; Aono, Osamu [Jichi Medical School, Tochigi (Japan); Schefer, J; Schoenborn, B P [Brookhaven National Lab., Upton (United States)


    Subunits {alpha}, {beta} and {gamma} of adenosine triphosphatase (H{sup +}-ATPase) from the thermophilic bacterium PS3 (TF{sub 1}) have been over-expressed in Escherichia coli. {alpha} and {beta} subunits deuterated to the level of 90% were obtained by culturing E. coli in {sup 2}H{sub 2}O medium. Both the subunits and the reconstituted {alpha}{beta}{gamma} complex, TF{sub 1}, which contain the deuterated components in various combinations, were studied in solution by small-angle neutron scattering. The individual shapes of the subunits and their organization in the {alpha}{beta}{gamma}-TF{sub 1} complex were examined using the techniques of selective deuteration and contrast variation. The {alpha} and {beta} subunits are well approximated as ellipsoids of revolution having minor semi-axes of 20{center dot}4({plus minus}0{center dot}4) and 20{center dot}0({plus minus}0{center dot}2) {angstrom}, and major semi-axes of 53{center dot}0({plus minus}1{center dot}4) and 55{center dot}8({plus minus}0{center dot}9) {angstrom}, respectively. In the TF{sub 1} complex, three {beta} subunits are aligned to form an equilateral triangle, with their major axes tilted by 35{degree} with respect to the 3-fold axis of the complex. The {beta}-{beta} distance is about 53 {angstrom}. Three {alpha} subunits are similarly arranged, positioned between the {beta} subunits, and with their direction of tilt opposite to that of the {beta} subunits. The centers of the {alpha} and {beta} subunits lie in the same plane, forming a hexagon. Adjacent subunits overlap in this model, suggesting that they are not simple ellipsoids of revolution.

  10. Cloning and Expression of Luteinizing Hormone Subunits in Chinese Hamster Ovary Cell Line

    Directory of Open Access Journals (Sweden)

    Zeinab Soleimanifar


    Full Text Available Background: Luteinizing hormone (LH was secreted by the stimulating cells of the testes and ovaries in the anterior pituitary gland. The application of this hormone is in the treatment of men and women with infertility and amenorrhea respectively.Materials and Methods: In the present study the alpha and beta subunits of human LH gene were cloned into the pEGFP-N1 expression vector and produced the recombinant LH hormone in Chinese hamster ovary (CHO eukaryotic system.Results: Alpha and beta subunits of LH hormone were cloned between NheI and BamHI cut sites of pEGFP_N1 expression plasmid and confirmed by PCR.  Hormone expression was evaluated in CHO cell line by Western blotting using the specific antibody.Conclusion: Alpha and beta subunits of LH hormone were expressed in CHO cell line perfectly.

  11. Interaction of the regulatory subunit of the cAMP-dependent protein kinase with PATZ1 (ZNF278)

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Weng-Lang [Long Island Jewish Medical Center, North Shore University Hospital, Manhasset, NY 11030 (United States); Ravatn, Roald [Department of Medicine, University of Toledo, College of Medicine, Toledo, OH 43614 (United States); Kudoh, Kazuya [Department of Medicine, University of Toledo, College of Medicine, Toledo, OH 43614 (United States); Department of Obstetrics and Gynecology, National Defense Medical College, Tokorozawa, Saitama (Japan); Alabanza, Leah [Department of Medicine, University of Toledo, College of Medicine, Toledo, OH 43614 (United States); Chin, Khew-Voon, E-mail: [Department of Medicine, University of Toledo, College of Medicine, Toledo, OH 43614 (United States)


    The effects of cAMP in cell are predominantly mediated by the cAMP-dependent protein kinase (PKA), which is composed of two genetically distinct subunits, catalytic (C) and regulatory (R), forming a tetrameric holoenzyme R{sub 2}C{sub 2}. The only known function for the R subunit is that of inhibiting the activity of the C subunit kinase. It has been shown that overexpression of RI{alpha}, but not the C subunit kinase, is associated with neoplastic transformation. In addition, it has also been demonstrated that mutation in the RI{alpha}, but not the C subunit is associated with increased resistance to the DNA-damaging anticancer drug cisplatin, thus suggesting that the RI{alpha} subunit of PKA may have functions independent of the kinase. We show here that the RI{alpha} subunit interacts with a BTB/POZ domain zinc-finger transcription factor, PATZ1 (ZNF278), and co-expression with RI{alpha} results in its sequestration in the cytoplasm. The cytoplasmic/nuclear translocation is inducible by cAMP. C-terminus deletion abolishes PATZ1 interaction with RI{alpha} and results in its localization in the nucleus. PATZ1 transactivates the cMyc promoter and the presence of cAMP and co-expression with RI{alpha} modulates its transactivation. Moreover, PATZ1 is aberrantly expressed in cancer. Taken together, our results showed a potentially novel mechanism of cAMP signaling mediated through the interaction of RI{alpha} with PATZ1 that is independent of the kinase activity of PKA, and the aberrant expression of PATZ1 in cancer point to its role in cell growth regulation.

  12. Immunochemical analysis of Micrococcus lysodeikticus (luteus) F1-ATPase and its subunits. (United States)

    Urban, C; Salton, M R


    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.

  13. ORF Alignment: NC_002937 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available nthase, beta/alpha subunits [Desulfovibrio vulgaris ... subsp. vulgaris str. Hildenborough] gb|AAS966...10.1| ... succinyl-CoA synthase, beta/alpha subunits ... [Desulfovibrio vulgaris subsp. vulgaris str. ... Hilde...GIGQSLCAGVGGDPYI 180 ... Query: 664 RLGHAGAILDEADGGIAGKVRALCDAGIAVCPDLGSLPAAVRQALG 709 ... RLGHAGAILDEAD...GGIAGKVRALCDAGIAVCPDLGSLPAAVRQALG Sbjct: 241 RLGHAGAILDEADGGIAGKVRALCDAGIAVCPDLGSLPAAVRQALG 286

  14. Monte Carlo particle-trajectory models for neutral cometary gases. I. Models and equations. II. The spatial morphology of the Lyman-alpha coma

    International Nuclear Information System (INIS)

    Combi, M.R.; Smyth, W.H.


    The mathematical derivations of various methods employed in the Monte Carlo particle-trajectory model (MCPTM) are presented, and the application of the MCPTM to the calculation of the photochemical heating of the inner coma through the partial thermalization of cometary hydrogen atoms produced by the photodissociation of water is discussed. This model is then used to explain the observed morphology of the spatially extended Ly-alpha comas of comets. The rocket and Skylab images of the Ly-alpha coma of Comet Kohoutek are examined. 90 references

  15. Therapeutic potential of Mediator complex subunits in metabolic diseases. (United States)

    Ranjan, Amol; Ansari, Suraiya A


    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.

  16. 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


    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...

  17. The light subunit of system bo,+ is fully functional in the absence of the heavy subunit


    Reig, Núria; Chillarón, Josep; Bartoccioni, Paola; Fernández, Esperanza; Bendahan, Annie; Zorzano, Antonio; Kanner, Baruch; Palacín, Manuel; Bertran, Joan


    The heteromeric amino acid transporters are composed of a type II glycoprotein and a non-glycosylated polytopic membrane protein. System bo,+ exchanges dibasic for neutral amino acids. It is composed of rBAT and bo,+AT, the latter being the polytopic membrane subunit. Mutations in either of them cause malfunction of the system, leading to cystinuria. bo,+AT-reconstituted systems from HeLa or MDCK cells catalysed transport of arginine that was totally dependent on the presence of one of the bo...

  18. Thermostable Subunit Vaccines for Pulmonary Delivery: How Close Are We?

    DEFF Research Database (Denmark)

    Foged, Camilla


    , 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....

  19. Biochemical characterization of CK2alpha and alpha' paralogues and their derived holoenzymes: evidence for the existence of a heterotrimeric CK2alpha'-holoenzyme forming trimeric complexes

    DEFF Research Database (Denmark)

    Olsen, Birgitte; Rasmussen, Tine; Niefind, Karsten


    Altogether 2 holoenzymes and 4 catalytic CK2 constructs were expressed and characterized i.e. CK2alpha (2) (1-335) beta(2); CK2alpha'-derived holoenzyme; CK2alpha(1-335); MBP-CK2alpha'; His-tagged CK2alpha and His-tagged CK2alpha'. The two His-tagged catalytic subunits were expressed in insect...... cells, all others in Escherichia coli. IC(50) studies involving the established CK2 inhibitors DMAT, TBBt, TBBz, apigenin and emodin were carried out and the K(i) values calculated. Although the differences in the K(i) values found were modest, there was a general tendency showing that the CK2...... holoenzymes were more sensitive towards the inhibitors than the free catalytic subunits. Thermal inactivation experiments involving the individual catalytic subunits showed an almost complete loss of activity after only 2 min at 45 degrees C. In the case of the two holoenzymes, the CK2alpha...

  20. Polarization Calibration of the Chromospheric Lyman-Alpha SpectroPolarimeter for a 0.1% Polarization Sensitivity in the VUV Range. Part II: In-Flight Calibration

    Czech Academy of Sciences Publication Activity Database

    Giono, G.; Ishikawa, R.; Narukage, N.; Kano, R.; Katsukawa, Y.; Kubo, M.; Ishikawa, S.; Bando, T.; Hara, H.; Suematsu, Y.; Winebarger, A.; Kobayashi, K.; Auchere, F.; Trujillo Bueno, J.; Tsuneta, S.; Shimizu, T.; Sakao, T.; Cirtain, J.; Champey, P.; Asensio Ramos, A.; Štěpán, Jiří; Belluzzi, L.; Sainz, R.M.; de Pontieu, B.; Ichimoto, K.; Carlsson, M.; Casini, R.; Goto, M.


    Roč. 292, č. 4 (2017), 57/1-57/19 ISSN 0038-0938 R&D Projects: GA ČR(CZ) GA16-16861S Institutional support: RVO:67985815 Keywords : solar transition region * ly-alpha * magnetohydrodynamic model Subject RIV: BN - Astronomy, Celestial Mechanics, Astrophysics OBOR OECD: Astronomy (including astrophysics,space science) Impact factor: 2.682, year: 2016

  1. Light charged particle production in fast neutron-induced reactions on carbon (En=40 to 75 MeV) (II). Tritons and alpha particles

    International Nuclear Information System (INIS)

    Dufauquez, C.; Slypen, I.; Benck, S.; Meulders, J.P.; Corcalciuc, V.


    Double-differential cross sections for fast neutron-induced triton and alpha-particle production on carbon are reported at six incident neutron energies between 40 and 75 MeV. Angular distributions were measured at laboratory angles between 20 deg. and 160 deg. . Energy-differential, angle-differential and total cross sections are also reported. Experimental cross sections are compared to existing experimental data and to theoretical model calculations

  2. Optimization in the nuclear fuel cycle II: Concentration of alpha emitters in the air; Otimização no ciclo do combustível nuclear II: concentração de alfa emissores no ar

    Energy Technology Data Exchange (ETDEWEB)

    Pereira, W.S., E-mail: [Universidade Veiga de Ameida (UVA), Rio de Janeiro, RJ (Brazil); Silva, A.X.; Lopes, J.M.; Carmo, A.S.; Mello, C.R.; Fernandes, T.S., E-mail:, E-mail: [Universidade Federal do Rio de Janeiro (UFRJ), RJ (Brazil); Kelecom, A. [Universidade Federal Fluminense (UFF), Niterói, RJ (Brazil)


    Optimization is one of the bases of radioprotection and aims to move doses away from the dose limit that is the borderline of acceptable radiological risk. The work aims to use the monitoring of the concentration of alpha emitters in the air as a tool of the optimization process. We analyzed 27 sampling points of airborne alpha concentration in a nuclear fuel cycle facility. The monthly averages were considered statistically different, the highest in the month of February and the lowest in the month of August. All other months were found to have identical mean activity concentration values. Regarding the sampling points, the points with the highest averages were points 12, 15 and 9. These points were indicated for the beginning of the optimization process. Analysis of the production of the facility should be performed to verify possible correlations between production and concentration of alpha emitters in the air.

  3. Crystal structure of the bacterial luciferase/flavin complex provides insight into the function of the beta subunit. (United States)

    Campbell, Zachary T; Weichsel, Andrzej; Montfort, William R; Baldwin, Thomas O


    Bacterial luciferase from Vibrio harveyi is a heterodimer composed of a catalytic alpha subunit and a homologous but noncatalytic beta subunit. Despite decades of enzymological investigation, structural evidence defining the active center has been elusive. We report here the crystal structure of V. harveyi luciferase bound to flavin mononucleotide (FMN) at 2.3 A. The isoalloxazine ring is coordinated by an unusual cis-Ala-Ala peptide bond. The reactive sulfhydryl group of Cys106 projects toward position C-4a, the site of flavin oxygenation. This structure also provides the first data specifying the conformations of a mobile loop that is crystallographically disordered in both prior crystal structures [(1995) Biochemistry 34, 6581-6586; (1996) J. Biol. Chem. 271, 21956 21968]. This loop appears to be a boundary between solvent and the active center. Within this portion of the protein, a single contact was observed between Phe272 of the alpha subunit, not seen in the previous structures, and Tyr151 of the beta subunit. Substitutions at position 151 on the beta subunit caused reductions in activity and total quantum yield. Several of these mutants were found to have decreased affinity for reduced flavin mononucleotide (FMNH(2)). These findings partially address the long-standing question of how the beta subunit stabilizes the active conformation of the alpha subunit, thereby participating in the catalytic mechanism.

  4. Regular endurance training reduces the exercise induced HIF-1alpha and HIF-2alpha mRNA expression in human skeletal muscle in normoxic conditions

    DEFF Research Database (Denmark)

    Lundby, Carsten; Gassmann, Max; Pilegaard, Henriette


    and 2 (HIFs) are clearly related heterodimeric transcription factors that consist of an oxygen-depended alpha-subunit and a constitutive beta-subunit. With hypoxic exposure, HIF-1alpha and HIF-2alpha protein are stabilized. Upon heterodimerization, HIFs induce the transcription of a variety of genes......Regular exercise induces a variety of adaptive responses that enhance the oxidative and metabolic capacity of human skeletal muscle. Although the physiological adjustments of regular exercise have been known for decades, the underlying mechanisms are still unclear. The hypoxia inducible factors 1...... including erythropoietin (EPO), transferrin and its receptor, as well as vascular endothelial growth factor (VEGF) and its receptor. Considering that several of these genes are also induced with exercise, we tested the hypothesis that the mRNA level of HIF-1alpha and HIF-2alpha subunits increases...

  5. Asymmetric expression of protein kinase CK2 subunits in human kidney tumors

    DEFF Research Database (Denmark)

    Stalter, G; Siemer, S; Becht, E


    of protein kinase CK2 alpha in tumors/normal tissue (T/N) was 1.58 and that of the protein kinase CK2 beta (T/N) was 2.65. The data suggest that the generally described increase in protein kinase CK2 activity in tumor cells may to some extent result from a deregulation in subunit biosynthesis or degradation...

  6. Teaching Glycoproteins with a Classical Paper: Knowledge and Methods in the Course of an Exciting Discovery--The story of Discovering HK-ATPase [Beta]-Subunit (United States)

    Zhu, Lixin


    To integrate research into the teaching of glycoproteins, the story of discovering hydrogen-potassium ATPase (HK-ATPase) [beta] subunit is presented in a way covering all the important teaching points. The interaction between the HK-ATPase [alpha] subunit and a glycoprotein of 60-80 kDa was demonstrated to support the existence of the [beta]…

  7. 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


    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

  8. A peptide mimic of an antigenic loop of alpha-human chorionic gonadotropin hormone: solution structure and interaction with a llama V-HH domain

    NARCIS (Netherlands)

    Ferrat, G.; Renisio, J.G.; Morelli, X.; Slootstra, J.W.; Meloen, R.; Cambillau, C.; Darbon, H.


    The X-ray structure of a ternary complex between human chorionic gonadotropin hormone (hCG) and two Fvs recognizing its alpha and beta subunits has been recently determined. The Fvs recognize the elongated hCG molecule by its two ends, one being the Leu-12-Cys-29 loop of the alpha subunit. We have

  9. 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

  10. Multicentre, open, noncomparative Phase II trial to evaluate the efficacy and tolerability of fotemustine, cisplatin, alpha-interferon and interleukin-2 in advanced melanoma patients. (United States)

    Ridolfi, Laura; Fiorentini, Giammaria; Guida, Michele; Michiara, Maria; Freschi, Andrea; Aitini, Enrico; Ballardini, Michela; Bichisao, Ettore; Ridolfi, Ruggero


    The efficacy and tolerability of fotemustine, cisplatin, alpha-interferon and interleukin-2 biochemotherapy were evaluated in advanced melanoma patients. The schedule consisted of fotemustine (100 mg/m) and cisplatin (75 mg/m) intravenous on day 1, followed by subcutaneous interleukin-2 at a dose of 4.5 MIU on days 3-5 and 8-12 and alpha-interferon at a dose of 3 MU three times/week, every 3 weeks for six cycles. Sixty patients were evaluated for tumour response, 12 of whom had brain metastases (BM). One patient (1.7%) with BM achieved a complete response and partial responses were observed in 10 patients (16.7%), including one BM patient. Overall response rate was 18.4 and 16.6% in BM patients (median response duration 8.2 months). Disease control, defined as overall response and stable disease, was 58.4% in all patients and 75% in patients with BM. Median time to progression was 3.2 months (4.2 months in BM patients). Median overall survival was 8.9 months (7.6 months in BM patients). Toxic events were mild to moderate. This combination was well tolerated and showed acceptable clinical activity, especially in BM patients.

  11. Role of myristoylation in membrane attachment and function of G alpha i-3 on Golgi membranes. (United States)

    Brand, S H; Holtzman, E J; Scher, D A; Ausiello, D A; Stow, J L


    Heterotrimeric G protein alpha-subunits localized on the cytoplasmic face of Golgi membranes are involved in regulating vesicle trafficking and protein secretion. We investigated the role of myristoylation in attachment of the G alpha i-3 subunit to Golgi membranes. G alpha i-3 was epitope-tagged by insertion of a FLAG sequence at an NH2-terminal site predicted to interfere with myristoylation, and the resulting NT-alpha i-3 construct was stably transfected and expressed in polarized epithelial LLC-PK1 cells. Metabolic labeling confirmed that the translation product of NT-alpha i-3 was not myristoylated. In contrast to endogenous G alpha 1-3, which is tightly bound to Golgi membranes, the unmyristoylated FLAG-tagged NT-alpha i-3 did not attach to membranes; it was localized by immunofluorescence in the cytoplasm of LLC-PK1 cells and was detected only in the cytosol fraction of cell homogenates. Pertussis toxin-dependent ADP-ribosylation was used to test the ability of NT-alpha i-3 to interact with membrane-bound beta gamma-subunits. In both in vitro and in vivo assays, cytosolic NT-alpha i-3 alone was not ADP-ribosylated, although in the presence of membranes it could interact with G beta gamma-subunits to form heterotrimers. The expression of NT-alpha i-3 in LLC-PK1 cells altered the rate of basolateral secretion of sulfated proteoglycans, consistent with the demonstrated function of endogenous G alpha i-3. These data are consistent with a model in which G alpha i-3 utilizes NH2-terminal myristoylation to bind to Golgi membranes and to maximize its interaction with G beta gamma-subunits. Furthermore, our results show that stable attachment of G alpha i-3 to Golgi membranes is not required for it to participate as a regulatory element in vesicle trafficking in the secretory pathway.

  12. Phase II trial of interleukin 2, interferon alpha, and 5-fluorouracil in metastatic renal cell cancer: a cytokine working group study. (United States)

    Dutcher, J P; Logan, T; Gordon, M; Sosman, J; Weiss, G; Margolin, K; Plasse, T; Mier, J; Lotze, M; Clark, J; Atkins, M


    The purpose of this study was to evaluate the potential efficacy of alternating two outpatient regimens for the treatment of metastatic renal cell cancer. These regimens consisted of 4 weeks of recombinant interleukin 2 (rIL-2) plus IFN-alpha2B followed by 4 weeks of 5-fluorouracil plus IFN-alpha2B. Fifty patients meeting eligibility criteria of previous Cytokine Working Group studies were treated on an outpatient basis. Patients received s.c. rIL-2 (Proleukin; Chiron, Emeryville, CA) during weeks 1-4 of the 8-week regimen. During weeks 1 and 4, the dosage for rIL-2 was 10 MIU/m2 twice daily on days 3-5, and the dosage for IFN-alpha2B (Intron; Schering Plough, Kenilworth, NJ) was 6 MIU/m2 on day 1. During weeks 2 and 3, the dosage for rIL-2 was 5 MIU/m2 on days 1, 3, and 5, and the dosage for IFN-alpha2B was 6 MIU/m2 on days 1, 3, 5. During weeks 5-8, 5-fluorouracil (750 mg/m2) was administered once weekly by i.v. infusion, and IFN-alpha2B (9 MIU/mZ) was administered as a s.c. injection three times weekly. Throughout the treatment, an assessment of quality of life was made and a symptom-distress scale was evaluated. There were two patients with complete responses (CRs) and seven with partial responses (PRs) for an objective response rate of 18% (95% confidence interval, 10-25). The median response duration was 8 months (range, 3-51+ months). The CRs lasted 5 months and 51+ months and the PRs ranged from 3+ to 18 months. After completing at least one course of treatment, eight patients (three with PR, one with minor response, four with stable disease) became CRs after surgery for remaining metastatic disease. Six remain alive at 43+ to 53+ months, and 5 remain disease-free since surgery. The median survival of the study group is 17.5 months, with a maximal follow-up of 53+ months. The range in survival is 1-53+ months. Toxicity was primarily constitutional. and treatment modifications were designed to maintain toxicity at grade 2/3. The most common toxicities during

  13. Mining Protein Evolution for Insights into Mechanisms of Voltage-Dependent Sodium Channel Auxiliary Subunits. (United States)

    Molinarolo, Steven; Granata, Daniele; Carnevale, Vincenzo; Ahern, Christopher A


    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.

  14. New insights into structure-function relationship of the DHPR beta1a subunit in skeletal muscle excitation-contraction coupling using zebrafish 'relaxed' as an expression system

    International Nuclear Information System (INIS)

    Dayal, A.


    The paralyzed zebrafish strain relaxed carries a null mutation for the skeletal muscle dihydropyridine receptor (DHPR) [beta]1a subunit. The lack of [beta]1a not only impedes functional [alpha]1S membrane expression but also precludes the skeletal muscle-specific ultrastructural arrangement of DHPRs into tetrads opposite ryanodine receptor (RyR1), coherent with the absence of skeletal muscle excitation-contraction (EC) coupling. With the plethora of experimental approaches feasible with zebrafish model organism and importantly with the [beta]1-null mutation having a monogenetic inheritance and because of the survival of the relaxed larvae for some days, we were able to establish the zebrafish relaxed as an expression system. Linking in vitro to in vivo observations, a clear differentiation between the major functional roles of [beta] subunits in EC coupling was feasible. The skeletal muscle [beta]1a subunit was able to restore all parameters of EC coupling upon expression in relaxed myotubes and larvae. Expression of the phylogenetically closest isoform to [beta]1a, the cardiac/neuronal [beta]2a subunit or the most distant neuronal [beta]M from the housefly in relaxed myotubes and larvae was likewise able to fully restore [alpha]1S triad targeting and facilitate charge movement. However, efficient tetrad formation and thus intact DHPR-RyR1 coupling was exclusively promoted by the [beta]1a isoform. Consequently, we postulated a model according to which [beta]1a acts as a unique allosteric modifier of [alpha]1S conformation crucial for skeletal muscle EC coupling. Therefore, unique structural elements in [beta]1a must be present which endow it with this exclusive property. Earlier, a unique hydrophobic heptad repeat motif (LVV) in the [beta]1a C-terminus was postulated by others to be essential for skeletal muscle EC coupling. We wanted to address the question if the proposed [beta]1a heptad repeat motif could be an active element of the DHPR-RyR1 signal transduction

  15. Interaction of alpha-conotoxin ImII and its analogs with nicotinic receptors and acetylcholine-binding proteins: additional binding sites on Torpedo receptor

    NARCIS (Netherlands)

    Kasheverov, I.E.; Zhmak, M.N.; Fish, A.; Rucktooa, P.; Khruschov, A.Y.; Osipov, A.V.; Ziganshin, R.H.; D'Hoedt, D.; Bertrand, D.; Sixma, T.K.; Smit, A.B.; Tsetlin, V.I.


    α-Conotoxins interact with nicotinic acetylcholine receptors (nAChRs) and acetylcholine-binding proteins (AChBPs) at the sites for agonists/competitive antagonists. α-Conotoxins blocking muscle-type or α7 nAChRs compete with α-bungarotoxin. However, α-conotoxin ImII, a close homolog of the α7

  16. Anticytokine treatment of established type II collagen-induced arthritis in DBA/1 mice: a comparative study using anti-TNFalpha, anti-IL-1alpha/beta and IL-1Ra.

    NARCIS (Netherlands)

    Joosten, L.A.B.; Helsen, M.M.A.; Loo, F.A.J. van de; Berg, W.B. van den


    OBJECTIVE: To examine the role of tumor necrosis factor alpha (TNF alpha), interleukin-1 alpha (IL-1 alpha), and IL-1 beta in collagen-induced arthritis (CIA), immediately after onset and during the phase of established arthritis. METHODS: Male DBA/1 mice with collagen-induced arthritis were treated

  17. GABA regulates the rat hypothalamic-pituitary-adrenocortical axis via different GABA-A receptor alpha-subtypes

    DEFF Research Database (Denmark)

    Mikkelsen, Jens D; Bundzikova, Jana; Larsen, Marianne Hald


    dependent on the composition of the GABA-A receptor subunits through which they act. We show here that positive modulators of alpha(1)-subtype containing GABA-A receptors with zolpidem (10 mg/kg) increase HPA activity in terms of increase in plasma corticosterone and induction of Fos in the PVN, whereas...... after positive modulation of GABA-A receptors composed of alpha(1)-subunit(s) affects a selective afferent system than the PVN, which is distinct from another afferent system(s) activated by non alpha(1)-containing GABA-A receptors....

  18. Subunit architecture and functional modular rearrangements of the transcriptional mediator complex. (United States)

    Tsai, Kuang-Lei; Tomomori-Sato, Chieri; Sato, Shigeo; Conaway, Ronald C; Conaway, Joan W; Asturias, Francisco J


    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.

  19. 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


    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....

  20. Second Byurakan spectral sky survey. II. Results for region centered on alpha 09h50m, delta +55 deg 00 arcmin

    International Nuclear Information System (INIS)

    Markarian, B.E.; Stepanian, D.A.


    The second list of objects in the Second Biurakan Spectral Sky Survey of the region centered on alpha 09h50m, delta +55 deg 00 arcmin is given. The list contains data on 110 objects and galaxies of a peculiar physical nature and 24 blue stars. The observations were made with the 40-52 arcsec Schmidt telescope of the Biurakan Astrophysical Observatory with a set of three objective prisms using Kodak IIIaJ and IIIaF emulsions sensitized in nitrogen. The area is found to contain 20 quasar candidates and four Seyfert galaxies, 27 blue stellar objects, 24 galaxies with an appreciable ultraviolet continuum, and 39 emission galaxies without appreciable ultraviolet radiation. The surface brightness of the quasars and Seyferts on the considered area down to the limiting magnitude 19.5 M is more than 1.5 per square degree with allowance for the already known quasars. The surface density of emission galaxies is about four per square degree. 7 references

  1. The catalytic subunit of human protein kinase CK2 structurally deviates from its maize homologue in complex with the nucleotide competitive inhibitor emodin

    DEFF Research Database (Denmark)

    Raaf, Jennifer; Klopffleisch, Karsten; Issinger, Olaf-Georg


    The Ser/Thr kinase CK2 (former name: casein kinase 2) is a heterotetrameric enzyme composed of two catalytic chains (CK2alpha) attached to a dimer of noncatalytic subunits. Together with the cyclin-dependent kinases and the mitogen-activated protein kinases, CK2alpha belongs to the CMGC family of...

  2. Interactions of foot-and-mouth disease virus with soluble bovine alphaVbeta3 and alphaVbeta6 integrins. (United States)

    Duque, Hernando; LaRocco, Michael; Golde, William T; Baxt, Barry


    At least four members of the integrin family of receptors, alphaVbeta1, alphaVbeta3, alphaVbeta6, and alphaVbeta8, have been identified as receptors for foot-and-mouth disease virus (FMDV) in vitro. Our investigators have recently shown that the efficiency of receptor usage appears to be related to the viral serotype and may be influenced by structural differences on the viral surface (H. Duque and B. Baxt, J. Virol. 77:2500-2511, 2003). To further examine these differences, we generated soluble alphaVbeta3 and alphaVbeta6 integrins. cDNA plasmids encoding the individual complete integrin alphaV, beta3, and beta6 subunits were used to amplify sequences encoding the subunits' signal peptide and ectodomain, resulting in subunits lacking transmembrane and cytoplasmic domains. COS-1 cells were transfected with plasmids encoding the soluble alphaV subunit and either the soluble beta3 or beta6 subunit and labeled with [35S]methionine-cysteine. Complete subunit heterodimeric integrins were secreted into the medium, as determined by radioimmunoprecipitation with specific monoclonal and polyclonal antibodies. For the examination of the integrins' biological activities, stable cell lines producing the soluble integrins were generated in HEK 293A cells. In the presence of divalent cations, soluble alphaVbeta6 bound to representatives of type A or O viruses, immobilized on plastic dishes, and significantly inhibited viral replication, as determined by plaque reduction assays. In contrast, soluble alphaVbeta3 was unable to bind to immobilized virus of either serotype; however, virus bound to the immobilized integrin, suggesting that FMDV binding to alphaVbeta3 is a low-affinity interaction. In addition, soluble alphaVbeta3 did not neutralize virus infectivity. Incubation of soluble alphaVbeta6 with labeled type A12 or O1 resulted in a significant inhibition of virus adsorption to BHK cells, while soluble alphaVbeta3 caused a low (20 to 30%), but consistent, inhibition of virus

  3. Carbene Chemistry. I. Stereochemical Integrity at C Alpha in Ketone Tosylhydrazones. II. Hydrogen Migration in 2-Carbena-6,6-Dimethylnorbornane. (United States)


    H20, 10% Na2 CO3 , H20, and dried over MqSO 4 . Yields were typically ca. 75%. "! 33 CARBENE CHEMISTRY PART II. HYDROGEN MIGRATION IN 2-CARBENA-6,6...any a delocaliza- tion. Thus if one assumes a single product determining intermediate, carbene 54 is classical in the usual sense of the word. It has...placed in a refrigerator. The crystalline product was re- crystallized from methanol-O-d/D20 yielding purified tosylhydrazone with mp 156-1580. 58

  4. Tritiated-nicotine- and 125I-alpha-bungarotoxin-labeled nicotinic receptors in the interpeduncular nucleus of rats. II. Effects of habenular destruction

    International Nuclear Information System (INIS)

    Clarke, P.B.; Hamill, G.S.; Nadi, N.S.; Jacobowitz, D.M.; Pert, A.


    The cholinergic innervation of the interpeduncular nucleus (IPN) is wholly extrinsic and is greatly attenuated by bilateral habenular destruction. We describe changes in the labeling of putative nicotinic receptors within this nucleus at 3, 5, or 11 days after bilateral habenular lesions. Adjacent tissue sections of the rat IPN were utilized for 3 H-nicotine and 125 I-alpha-bungarotoxin ( 125 I-BTX) receptor autoradiography. Compared to sham-operated controls, habenular destruction significantly reduced autoradiographic 3 H-nicotine labeling in rostral (-25%), intermediate (-13%), and lateral subnuclei (-36%). Labeling in the central subnucleus was unchanged. Loss of labeling was maximal at the shortest survival time (3 days) and did not change thereafter. In order to establish whether this loss was due to a reduction in the number or the affinity of 3 H-nicotine-binding sites, a membrane assay was performed on microdissected IPN tissue from rats that had received surgery 3 days previously. Bilateral habenular lesions produced a 35% reduction of high-affinity 3 H-nicotine-binding sites, with no change in binding affinity. Bilateral habenular lesions reduced 125 I-BTX labeling in the intermediate subnuclei, and a slight increase occurred in the rostral subnucleus. In the lateral subnuclei, 125 I-BTX labeling was significantly reduced (27%) at 3 days but not at later survival times. In view of the known synaptic morphology of the habenulointerpeduncular tract, it is concluded that a subpopulation of 3 H-nicotine binding sites within the IPN is located on afferent axons and/or terminals. This subpopulation, located within rostral, intermediate, and lateral subnuclei, may correspond to presynaptic nicotinic cholinergic receptors. Sites that bind 125 I-BTX may include a presynaptic subpopulation located in the lateral and possibly the intermediate subnuclei

  5. A RNA antagonist of hypoxia-inducible factor-1alpha, EZN-2968, inhibits tumor cell growth

    DEFF Research Database (Denmark)

    Greenberger, Lee M; Horak, Ivan D; Filpula, David


    Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that plays a critical role in angiogenesis, survival, metastasis, drug resistance, and glucose metabolism. Elevated expression of the alpha-subunit of HIF-1 (HIF-1alpha), which occurs in response to hypoxia or activation of growth facto...

  6. Predictive role of HER2/neu, topoisomerase-II-alpha, and tissue inhibitor of metalloproteinases (TIMP-1) for response to adjuvant taxane-based chemotherapy in patients with intermediate-risk breast cancer: results from the WSG-AGO EC-Doc trial. (United States)

    Erber, Ramona; Gluz, Oleg; Brünner, Nils; Kreipe, Hans Heinrich; Pelz, Enrico; Kates, Ronald; Bartels, Annette; Huober, Jens; Mohrmann, Svjetlana; Moustafa, Zehra; Liedtke, Cornelia; Möbus, Volker; Augustin, Doris; Thomssen, Christoph; Jänicke, Fritz; Kiechle, Marion; Kuhn, Walther; Nitz, Ulrike; Harbeck, Nadia; Hartmann, Arndt


    Taxane-anthracycline-based adjuvant chemotherapy is standard of care in patients with node-positive breast cancer (BC) but is also associated with severe side effects and significant costs. It is yet unclear, which biomarkers would predict benefit from taxanes and/or general chemoresistance. In this study, we investigate a large cohort of patients with intermediate-risk BC treated within the WSG EC-DOC Trial for the predictive impact of topoisomerase-II-alpha, HER2/neu, and TIMP-1. Tumor tissue was available in a representative cohort of 772 cases of the WSG EC-DOC Trial collective which compared 4xEC-4xDoc versus 6xCEF/CMF. In addition to hormone receptor status and Ki-67, HER2/neu+ and topoisomerase-II-alpha status using fluorescence in situ hybridisation (FISH) and immunohistochemistry, TIMP-1 using immunohistochemistry, and aneuploidy of chromosome 17 using FISH were evaluated and correlated with outcome and taxane benefit. There was significant superiority of EC-Doc over CEF regarding 5-year DFS (90 vs. 80 %, respectively, p = 0.006) particularly in patient subgroups defined by HR+, HER2/neu+, high proliferation (i.e., Ki-67 ≥ 20 %), patient age >50 years old and normal chromosome 17 status, high TIMP-1 and low topoisomerase-II-alpha protein expression. Significant prognostic factors in multivariate analysis were EC-Doc therapy (HR = 0.61; 95 %CI 0.38-0.986), age Doc vs. CEF) and high topoisomerase-II-alpha protein expression (HR = 0.427; 95 %CI 0.203-0.900) in multivariate interaction analysis. Despite of univariate predictive effect of HER2/neu status among other factors only topoisomerase-II-alpha protein expression was associated with significant benefit from EC-Doc compared to CEF by multivariate interaction analysis.

  7. Functional and genomic analyses of alpha-solenoid proteins. (United States)

    Fournier, David; Palidwor, Gareth A; Shcherbinin, Sergey; Szengel, Angelika; Schaefer, Martin H; Perez-Iratxeta, Carol; Andrade-Navarro, Miguel A


    Alpha-solenoids are flexible protein structural domains formed by ensembles of alpha-helical repeats (Armadillo and HEAT repeats among others). While homology can be used to detect many of these repeats, some alpha-solenoids have very little sequence homology to proteins of known structure and we expect that many remain undetected. We previously developed a method for detection of alpha-helical repeats based on a neural network trained on a dataset of protein structures. Here we improved the detection algorithm and updated the training dataset using recently solved structures of alpha-solenoids. Unexpectedly, we identified occurrences of alpha-solenoids in solved protein structures that escaped attention, for example within the core of the catalytic subunit of PI3KC. Our results expand the current set of known alpha-solenoids. Application of our tool to the protein universe allowed us to detect their significant enrichment in proteins interacting with many proteins, confirming that alpha-solenoids are generally involved in protein-protein interactions. We then studied the taxonomic distribution of alpha-solenoids to discuss an evolutionary scenario for the emergence of this type of domain, speculating that alpha-solenoids have emerged in multiple taxa in independent events by convergent evolution. We observe a higher rate of alpha-solenoids in eukaryotic genomes and in some prokaryotic families, such as Cyanobacteria and Planctomycetes, which could be associated to increased cellular complexity. The method is available at

  8. A polymorphic variant in the human electron transfer flavoprotein alpha-chain (alpha-T171) displays decreased thermal stability and is overrepresented in very-long-chain acyl-CoA dehydrogenase-deficient patients with mild childhood presentation

    DEFF Research Database (Denmark)

    Bross, P; Pedersen, P; Nyholm, M


    The consequences of two amino acid polymorphisms of human electron transfer flavoprotein (alpha-T/I171 in the alpha-subunit and beta-M/T154 in the beta-subunit) on the thermal stability of the enzyme are described. The alpha-T171 variant displayed a significantly decreased thermal stability, wher....... This is compatible with a negative modulating effect of the less-stable alpha-T171 ETF variant in this group of VLCAD patients that harbor missense mutations in at least one allele and therefore potentially display residual levels of VLCAD enzyme activity. Udgivelsesdato: 1999-Jun...

  9. Subunit Stoichiometry of Human Muscle Chloride Channels


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


    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 ...

  10. Human leukocyte antigen class I, class II, and tumor necrosis factor-alpha polymorphisms in a healthy elder Mexican Mestizo population

    Directory of Open Access Journals (Sweden)

    Llorente Luis


    Full Text Available Abstract Background There is strong evidence that an individual's genetic background is an important predisposing factor to longevity. In the present study we analysed the frequency of HLA class I, class II, as well as the TNF-α -308 polymorphism that may be related to an increased life span in Mexican Mestizo healthy elders. Results HLA typing was performed by polymerase chain reaction sequence specific oligonucleotide (PCR SSO reverse dot blot. The TNF-α -308 polymorphism was assessed by PCR restriction fragment length polymorphism. A significant increased frequency of HLA-DRB1*11 was found in elderly women whereas this allele was not present in elderly males. The TNF2 allele was also increased in the elder group when compared to young controls. The frequencies of the remaining alleles tested were not statistically different among groups. Conclusion These data suggest an ethnicity independent tendency of HLA-DRB1*11 in elder females to increase life span and a possible role of the TNF2 allele with the successful remodelling of senescent immune system.

  11. Identification of a conserved archaeal RNA polymerase subunit contacted by the basal transcription factor TFB. (United States)

    Magill, C P; Jackson, S P; Bell, S D


    Archaea possess two general transcription factors that are required to recruit RNA polymerase (RNAP) to promoters in vitro. These are TBP, the TATA-box-binding protein and TFB, the archaeal homologue of TFIIB. Thus, the archaeal and eucaryal transcription machineries are fundamentally related. In both RNAP II and archaeal transcription systems, direct contacts between TFB/TFIIB and the RNAP have been demonstrated to mediate recruitment of the polymerase to the promoter. However the subunit(s) directly contacted by these factors has not been identified. Using systematic yeast two-hybrid and biochemical analyses we have identified an interaction between the N-terminal domain of TFB and an evolutionarily conserved subunit of the RNA polymerase, RpoK. Intriguingly, homologues of RpoK are found in all three nuclear RNA polymerases (Rpb6) and also in the bacterial RNA polymerase (omega-subunit).

  12. 28 CFR 51.6 - Political subunits. (United States)


    ... 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...

  13. Purification and properties of a thermostable pullulanase from Clostridium thermosulfurgenes EM1 which hydrolyses both. alpha. -1,6 and. alpha. -1,4-glycosidic linkages

    Energy Technology Data Exchange (ETDEWEB)

    Spreinat, A [Goettingen Univ. (Germany, F.R.). Inst. fuer Mikrobiologie; Antranikian, G [Technische Univ. Hamburg-Harburg, Hamburg (Germany, F. R.). Arbeitsbereich Biotechnologie 1


    A novel thermostable pullulanase, secreted by the thermophilic anaerobic bacterium Clostridium thermosulfurogenes EM1, was purified and characterized. Applying anion exchange chromatography and gel filtration the enzyme was purified 47-fold and had a specific activity of 200 units/mg. The molecular mass of this thermostable enzyme was determined to be 102 000 daltons and consisted of a single subunit. The enzyme was able to attack specifically the {alpha}-1,6-glycosidic linkages in pullulan and caused its complete hydrolysis to maltotriose. Surprisingly and unlike the enzyme from Klebsiella pneumoniae, the purified enzyme from this anaerobic thermophile exhibited, in addition to its debranching and pullulanase activity, an {alpha}-1,4 hydrolysing activity as well. By the action of this single polypeptide chain various branched and linear polysaccharides were completely converted to two major products, namely maltose and maltotriose. The K{sub m} values of this enzyme for pullulan and amylose were determined to be 1.33 mg/ml and 0.38 mg/ml, respectively. This debranching enzyme displays a temperature optimum at 60deg-65deg C and a pH optimum at 5.5-6.0. The application of this new class of pullulanase (pullulanase type II) in industry will significantly enhance the starch saccharification process. (orig.).

  14. Interaction of C-terminal truncated human alphaA-crystallins with target proteins.

    Directory of Open Access Journals (Sweden)

    Anbarasu Kumarasamy


    Full Text Available Significant portion of alphaA-crystallin in human lenses exists as C-terminal residues cleaved at residues 172, 168, and 162. Chaperone activity, determined with alcohol dehydrogenase (ADH and betaL-crystallin as target proteins, was increased in alphaA(1-172 and decreased in alphaA(1-168 and alphaA(1-162. The purpose of this study was to show whether the absence of the C-terminal residues influences protein-protein interactions with target proteins.Our hypothesis is that the chaperone-target protein binding kinetics, otherwise termed subunit exchange rates, are expected to reflect the changes in chaperone activity. To study this, we have relied on fluorescence resonance energy transfer (FRET utilizing amine specific and cysteine specific fluorescent probes. The subunit exchange rate (k for ADH and alphaA(1-172 was nearly the same as that of ADH and alphaA-wt, alphaA(1-168 had lower and alphaA(1-162 had the lowest k values. When betaL-crystallin was used as the target protein, alphaA(1-172 had slightly higher k value than alphaA-wt and alphaA(1-168 and alphaA(1-162 had lower k values. As expected from earlier studies, the chaperone activity of alphaA(1-172 was slightly better than that of alphaA-wt, the chaperone activity of alphaA(1-168 was similar to that of alphaA-wt and alphaA(1-162 had substantially decreased chaperone activity.Cleavage of eleven C-terminal residues including Arg-163 and the C-terminal flexible arm significantly affects the interaction with target proteins. The predominantly hydrophilic flexible arm appears to be needed to keep the chaperone-target protein complex soluble.

  15. Flavonoids-induced accumulation of hypoxia-inducible factor (HIF)-1alpha/2alpha is mediated through chelation of iron. (United States)

    Park, Sung-Soo; Bae, Insoo; Lee, Yong J


    Hypoxia-inducible factor-1 alpha (HIF-1alpha) is the regulatory subunit of the heterodimeric transcription factor HIF-1 that is the key regulator of cellular response to low oxygen tension. Under normoxic conditions, HIF-1alpha is continuously degraded by the ubiquitin-proteasome pathway through pVHL (von Hippel-Lindau tumor suppressor protein). Under hypoxic conditions, HIF-1alpha is stabilized and induces the transcription of HIF-1 target genes. Quercetin, a flavonoid with anti-oxidant, anti-inflammatory, and kinase modulating properties, has been found to induce HIF-1alpha accumulation and VEGF secretion in normoxia. In this study, the molecular mechanisms of quercetin-mediated HIF-1alpha accumulation were investigated. Previous studies have shown that, in addition to being induced by hypoxia, HIF-1alpha can be induced through the phosphatidylinositol 3-kinase (PI3K)/Akt and p53 signaling pathways. But our study revealed, through p53 mutant-type as well as p53 null cell lines, that neither the PI3K/Akt nor the p53 signaling pathway is required for quercetin-induced HIF-1alpha accumulation. And we observed that HIF-1alpha accumulated by quercetin is not ubiquitinated and the interaction of HIF-1alpha with pVHL is reduced, compared with HIF-1alpha accumulated by the proteasome inhibitor MG132. The use of quercetin's analogues showed that only quercetin and galangin induce HIF-1/2alpha accumulation and this effect is completely reversed by additional iron ions. This is because quercetin and galangin are able to chelate cellular iron ions that are cofactors of HIF-1/2alpha proline hydroxylase (PHD). These data suggest that quercetin inhibits the ubiquitination of HIF-1/2alpha in normoxia by hindering PHD through chelating iron ions.

  16. Flexible Connectors between Capsomer Subunits that Regulate Capsid Assembly. (United States)

    Hasek, Mary L; Maurer, Joshua B; Hendrix, Roger W; Duda, Robert L


    Viruses build icosahedral capsids of specific size and shape by regulating the spatial arrangement of the hexameric and pentameric protein capsomers in the growing shell during assembly. In the T=7 capsids of Escherichia coli bacteriophage HK97 and other phages, 60 capsomers are hexons, while the rest are pentons that are correctly positioned during assembly. Assembly of the HK97 capsid to the correct size and shape has been shown to depend on specific ionic contacts between capsomers. We now describe additional ionic interactions within capsomers that also regulate assembly. Each is between the long hairpin, the "E-loop," that extends from one subunit to the adjacent subunit within the same capsomer. Glutamate E153 on the E-loop and arginine R210 on the adjacent subunit's backbone alpha-helix form salt bridges in hexamers and pentamers. Mutations that disrupt these salt bridges were lethal for virus production, because the mutant proteins assembled into tubes or sheets instead of capsids. X-ray structures show that the E153-R210 links are flexible and maintained during maturation despite radical changes in capsomer shape. The E153-R210 links appear to form early in assembly to enable capsomers to make programmed changes in their shape during assembly. The links also prevent flattening of capsomers and premature maturation. Mutant phenotypes and modeling support an assembly model in which flexible E153-R210 links mediate capsomer shape changes that control where pentons are placed to create normal-sized capsids. The E-loop may be conserved in other systems in order to play similar roles in regulating assembly. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Defining carbohydrate specificity of Ricinus communis agglutinin as Gal beta 1-->4GlcNAc (II) > Gal beta 1-->3GlcNAc (I) > Gal alpha 1-->3Gal (B) > Gal beta 1-->3GalNAc (T). (United States)

    Wu, J H; Herp, A; Wu, A M


    To define carbohydrate specificity of Ricinus communis agglutinin (RCA1), the combining site of RCA1 was further characterized by quantitative precipitin (QPA) and precipitin-inhibition assays (QPIA). Among the oligosaccharides tested for QPIA, Gal beta 1-->4GlcNAc (II, human blood group type II precursor sequence) was found to be 7.1 times more active than Gal beta 1-->3GalNAc (T, Thomsen-Friedenreich sequence) and about 1.7 times more active than the other three disaccharides tested--Gal beta 1-->4Man, Gal beta 1-->3DAra and Gal beta 1-->6GalNAc. Gal alpha 1-->4Gal, the receptor of the uropathogenic E. coli ligand was 3.6 times less active than the II sequence. These results indicate that the beta 1-->4 linkage of the terminal Gal to subterminal GlcNAc is important as this beta 1-->4GlcNAc sequence is at least 1.6 times more active than other types of disaccharides. Among the glycoproteins examined for QPA, native and desialized bovine submandibular glycoproteins, native and desialized human plasma alpha 1-acid glycoproteins, as well as crude hog stomach mucin and its three mild acid hydrolyzed products reacted well with the lectin. These glycoproteins precipitated over 75% of the lectin nitrogen added indicating that RCA1 has the ability to recognize Gal beta 1-->4/3GlcNAc and/or the related residues at the non-reducing ends and at positions in the interior of the chains. However, Tn (GalNAc alpha 1-->Ser/Thr sequence) rich glycoproteins such as desialized ovine submandibular glycoprotein and desialized armadillo salivary glycoprotein, in which over 90% of the carbohydrate side chains are Tn determinants with none or only a trace of I/II or T determinants, precipitated poorly with RCA1. From the present and previous results obtained, the carbohydrate specificity of RCA1 can be constructed and summarized in decreasing order by lectin determinants as follows: II (Gal beta 1-->4GlcNAc) > I (Gal beta 1-->3GlcNAc) > E (Gal alpha 1-->4Gal) and B (Gal alpha 1-->3Gal

  18. [Three regions of Rpb10 mini-subunit of nuclear RNA polymerases are strictly conserved in all eukaryotes]. (United States)

    Shpakovskiĭ, G V; Lebedenko, E N


    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.

  19. Type II restriction modification system methylation subunit of Alicyclobacillus acidocaldarius

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Brady D.; Newby, Deborah T.; Lacey, Jeffrey A.; Thompson, David N.; Thompson, Vicki S.; Apel, William A.; Roberto, Francisco F.; Reed, David W.


    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods for modulating or altering recombination inside or outside of a cell using isolated and/or purified polypeptides and/or nucleic acid sequences from Alicyclobacillus acidocaldarius.

  20. Characterization of protein kinase CK2 protein subunits and p53 in F9 teratocarcinoma cells in the absence and presence of cisplatin

    DEFF Research Database (Denmark)

    Küpper, M; Köster, M; Schmidt-Spaniol, I


    cell extracts treated with and without cisplatin were analyzed by ion exchange chromatography for protein kinase CK2 alpha/beta subunits and p53 distribution. The following results were obtained: (a) in crude extracts of cisplatin-treated cells, CK2 activity was sometimes reduced by as much as 50%; (b......The effect of cis-diaminedichloroplatinum(II) (cisplatin) on the induction of p53 and protein kinase CK2 activity was studied in the mouse teratocarcinoma cell line F9. Treatment of the cells with the chemotherapeutic agent cisplatin led to the detection of p53 3 h after addition of the drug. F9...... by immunostaining, we have detected, at a concentration of approximately 200 mM NaCl, a protein of approximately 46 kDa which reacted with the CK2 alpha-specific antibody. This fraction was devoid of CK2 activity; and (d) cisplatin-treated cells exhibited p53 protein, which was mostly eluting ahead but also partly...

  1. The TFIIH Subunit p89 (XPB Localizes to the Centrosome during Mitosis

    Directory of Open Access Journals (Sweden)

    Achim Weber


    Full Text Available Background: The general transcription factor II H (TFIIH, comprised of a core complex and an associated CAK-complex, functions in transcription, DNA repair and cell cycle control. Mutations of the two largest subunits, p89 (XPB and p80 (XPD, cause the hereditary cancer-prone syndrome xeroderma pigmentosum.

  2. p44 and p34 subunits of the BTF2/TFIIH transcription factor have homologies with SSL1, a yeast protein involved in DNA repair.

    NARCIS (Netherlands)

    S. Humbert; H. van Vuuren; Y. Lutz; J.H.J. Hoeijmakers (Jan); J-M. Egly (Jean-Marc); V. Moncollin


    textabstractThe human BTF2 (TFIIH) transcription factor is a multisubunit protein involved in transcription initiation by RNA polymerase II (B) as well as in DNA repair. In addition to the previously characterized p62 and p89/ERCC3 subunits, we have cloned two other subunits of BTF2, p44 and p34.

  3. The effect of polylysine on casein-kinase-2 activity is influenced by both the structure of the protein/peptide substrates and the subunit composition of the enzyme

    DEFF Research Database (Denmark)

    Meggio, F; Boldyreff, B; Marin, O


    , moreover, is variably accounted for by changes in Vmax and/or Km, depending on the structure of the peptide substrate. Maximum stimulation with all protein/peptide substrates tested requires the presence of the beta subunit, since the recombinant alpha subunit is much less responsive than CK2 holoenzyme......The mechanism by which polybasic peptides stimulate the activity of casein kinase 2 (CK2) has been studied by comparing the effect of polylysine on the phosphorylation of a variety of protein and peptide substrates by the native CK2 holoenzyme and by its recombinant catalytic alpha subunit, either...

  4. Acetylcholine Receptor: Complex of Homologous Subunits (United States)

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


    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.

  5. Intrasteric control of AMPK via the gamma1 subunit AMP allosteric regulatory site. (United States)

    Adams, Julian; Chen, Zhi-Ping; Van Denderen, Bryce J W; Morton, Craig J; Parker, Michael W; Witters, Lee A; Stapleton, David; Kemp, Bruce E


    AMP-activated protein kinase (AMPK) is a alphabetagamma heterotrimer that is activated in response to both hormones and intracellular metabolic stress signals. AMPK is regulated by phosphorylation on the alpha subunit and by AMP allosteric control previously thought to be mediated by both alpha and gamma subunits. Here we present evidence that adjacent gamma subunit pairs of CBS repeat sequences (after Cystathionine Beta Synthase) form an AMP binding site related to, but distinct from the classical AMP binding site in phosphorylase, that can also bind ATP. The AMP binding site of the gamma(1) CBS1/CBS2 pair, modeled on the structures of the CBS sequences present in the inosine monophosphate dehydrogenase crystal structure, contains three arginine residues 70, 152, and 171 and His151. The yeast gamma homolog, snf4 contains a His151Gly substitution, and when this is introduced into gamma(1), AMP allosteric control is substantially lost and explains why the yeast snf1p/snf4p complex is insensitive to AMP. Arg70 in gamma(1) corresponds to the site of mutation in human gamma(2) and pig gamma(3) genes previously identified to cause an unusual cardiac phenotype and glycogen storage disease, respectively. Mutation of any of AMP binding site Arg residues to Gln substantially abolishes AMP allosteric control in expressed AMPK holoenzyme. The Arg/Gln mutations also suppress the previously described inhibitory properties of ATP and render the enzyme constitutively active. We propose that ATP acts as an intrasteric inhibitor by bridging the alpha and gamma subunits and that AMP functions to derepress AMPK activity.

  6. Steady-state levels of G-protein beta-subunit expression are regulated by treatment of cells with bacterial toxins

    International Nuclear Information System (INIS)

    Watkins, D.C.; Northup, J.K.; Malbon, C.C.


    Cultures of 3T3-L1 cells were incubated with either 10 ng/ml cholera toxin or 10 ng/ml pertussis toxin from 4 days prior to the initiation of differentiation and throughout the subsequent incubation. Toxin concentrations were sufficient to completely prevent the labelling of alpha-subunits with [ 32 P]NAD + and pertussis toxin and to prevent by more than 90% the labelling with [ 32 P]NAD + and cholera toxin in membranes prepared from these cells. Neither toxin prevented the differentiation to the adipocyte phenotype. Neither toxin prevented the increases in the relative amounts of G-proteins which occur upon differentiation. Both toxins dramatically decreased the amount of beta-subunits. As measured by quantitative immunoblotting with antisera specific for both the 35 kDa and 36 kDa beta-subunits, levels of beta-subunit were decreased by more than 50% of steady-state level of control cells. Thus, bacterial toxins which modifies G-protein alpha-subunits are capable of modulating the levels of beta-subunits in vivo. The basis for the regulation of G-protein subunit expression by bacterial toxins is under study

  7. Casein kinase 2 down-regulation and activation by polybasic peptides are mediated by acidic residues in the 55-64 region of the beta-subunit. A study with calmodulin as phosphorylatable substrate

    DEFF Research Database (Denmark)

    Meggio, F; Boldyreff, B; Issinger, O G


    to substitute for wild-type beta-subunit as a suppressor of activity toward calmodulin. The only mutations that reduced the ability of the beta-subunit to suppress calmodulin phosphorylation activity, though being compatible with normal reconstitution of CK2 holoenzyme, were those affecting Asp55, Glu57...... are conversely ineffective. The latent "calmodulin kinase" activity of CK2 can also be specifically unmasked by a peptide (alpha[66-86]) reproducing a basic insert of the catalytic subunit. This effect is reversed by equimolar addition of a peptide (beta[55-71]) including the 55-64 acidic stretch of the beta......-subunit. Comparable polylysine stimulation was observed with the holoenzymes reconstituted with either beta wt or the beta mutants capable of assembling with the alpha-subunit, with the notable exception of those bearing Ala substitutions for acidic residues at positions 55, 57, and 59-61. These were nearly...

  8. Interactions between beta subunits of the KCNMB family and Slo3: beta4 selectively modulates Slo3 expression and function.

    Directory of Open Access Journals (Sweden)

    Cheng-Tao Yang


    Full Text Available The pH and voltage-regulated Slo3 K(+ channel, a homologue of the Ca(2+- and voltage-regulated Slo1 K(+ channel, is thought to be primarily expressed in sperm, but the properties of Slo3 studied in heterologous systems differ somewhat from the native sperm KSper pH-regulated current. There is the possibility that critical partners that regulate Slo3 function remain unidentified. The extensive amino acid identity between Slo3 and Slo1 suggests that auxiliary beta subunits regulating Slo1 channels might coassemble with and modulate Slo3 channels. Four distinct beta subunits composing the KCNMB family are known to regulate the function and expression of Slo1 Channels.To examine the ability of the KCNMB family of auxiliary beta subunits to regulate Slo3 function, we co-expressed Slo3 and each beta subunit in heterologous expression systems and investigated the functional consequences by electrophysiological and biochemical analyses. The beta4 subunit produced an 8-10 fold enhancement of Slo3 current expression in Xenopus oocytes and a similar enhancement of Slo3 surface expression as monitored by YFP-tagged Slo3 or biotin labeled Slo3. Neither beta1, beta2, nor beta3 mimicked the ability of beta4 to increase surface expression, although biochemical tests suggested that all four beta subunits are competent to coassemble with Slo3. Fluorescence microscopy from beta4 KO mice, in which an eGFP tag replaced the deleted exon, revealed that beta4 gene promoter is active in spermatocytes. Furthermore, quantitative RT-PCR demonstrated that beta4 and Slo3 exhibit comparable mRNA abundance in both testes and sperm.These results argue that, for native mouse Slo3 channels, the beta4 subunit must be considered as a potential interaction partner and, furthermore, that KCNMB subunits may have functions unrelated to regulation of the Slo1 alpha subunit.

  9. Accumulation of glycation products in. cap alpha. -H pig lens crystallin and its bearing to diabetic cataract genesis

    Energy Technology Data Exchange (ETDEWEB)

    Vidal, P; Cabezas-Cerrato, J


    The incorporation of /sup 11/C-glucose in native pig crystalline by in vitro incubation was found, after subsequent dialysis, to affect all 5 classes of crystallin separated by Sepharose CL-6B column chromatography. Though the radioactivity of the ..cap alpha..-H fraction was three times greater than that of any of the others, autoradiographs of SDS-PAGE gels showed /sup 11/C-glucose adducts to be present in all soluble protein subunits, without there being any evidence of preferential glycation of the ..cap alpha..-H subunits. The concentration of stable glycation products in the ..cap alpha..-H chromatographic fraction of soluble crystallins is suggested to be due the addition of glycated material to this fraction as result of glycation-induced hyperaggregation, and not because the ..cap alpha..-H subunits were especially susceptible to glycation.

  10. Drugs interacting with alpha adrenoceptors

    NARCIS (Netherlands)

    van Zwieten, P. A.


    Alpha adrenoceptors should be divided into various subtypes, comprising pre/postsynaptic and alpha 1/alpha 2-subpopulations, respectively. This classification implicates important functional differences between the various alpha-receptor subtypes, including certain differences in signal transduction

  11. Eukaryotic RNA polymerase subunit RPB8 is a new relative of the OB family. (United States)

    Krapp, S; Kelly, G; Reischl, J; Weinzierl, R O; Matthews, S


    RNA polymerase II subunit RPB8 is an essential subunit that is highly conserved throughout eukaryotic evolution and is present in all three types of nuclear RNA polymerases. We report the first high resolution structural insight into eukaryotic RNA polymerase architecture with the solution structure of RPB8 from Saccharomyces cerevisiae. It consists of an eight stranded, antiparallel beta-barrel, four short helical regions and a large, unstructured omega-loop. The strands are connected in classic Greek-key fashion. The overall topology is unusual and contains a striking C2 rotational symmetry. Furthermore, it is most likely a novel associate of the oligonucleotide/oligosaccharide (OB) binding protein class.

  12. Secondary reduction of alpha7B integrin in laminin alpha2 deficient congenital muscular dystrophy supports an additional transmembrane link in skeletal muscle. (United States)

    Cohn, R D; Mayer, U; Saher, G; Herrmann, R; van der Flier, A; Sonnenberg, A; Sorokin, L; Voit, T


    The integrins are a large family of heterodimeric transmembrane cellular receptors which mediate the association between the extracellular matrix (ECM) and cytoskeletal proteins. The alpha7beta1 integrin is a major laminin binding integrin in skeletal and cardiac muscle and is thought to be involved in myogenic differentiation and migration processes. The main binding partners of the alpha7 integrin are laminin-1 (alpha1-beta1-gamma1), laminin-2 (alpha2-beta1-gamma1) and laminin-4 (alpha2-beta2-gamma1). Targeted deletion of the gene for the alpha7 integrin subunit (ITGA7) in mice leads to a novel form of muscular dystrophy. In the present study we have investigated the expression of two alternative splice variants, the alpha7B and beta1D integrin subunits, in normal human skeletal muscle, as well as in various forms of muscular dystrophy. In normal human skeletal muscle the expression of the alpha7 integrin subunit appeared to be developmentally regulated: it was first detected at 2 years of age. In contrast, the beta1D integrin could be detected in immature and mature muscle in the sarcolemma of normal fetal skeletal muscle at 18 weeks gestation. The expression of alpha7B integrin was significantly reduced at the sarcolemma in six patients with laminin alpha2 chain deficient congenital muscular dystrophy (CMD) (age >2 years). However, this reduction was not correlated with the amount of laminin alpha2 chain expressed. In contrast, the expression of the laminin alpha2 chain was not altered in the skeletal muscle of the alpha7 knock-out mice. These data argue in favor that there is not a tight correlation between the expression of the alpha7 integrin subunit and that of the laminin alpha2 chain in either human or murine dystrophic muscle. Interestingly, in dystrophinopathies (Duchenne and Becker muscular dystrophy; DMD/BMD) expression of alpha7B was upregulated irrespective of the level of dystrophin expression as shown by a strong sarcolemmal staining pattern even

  13. The determination of $\\alpha_s$ by the ALPHA collaboration

    CERN Document Server

    Bruno, Mattia


    We review the ALPHA collaboration strategy for obtaining the QCD coupling at high scale. In the three-flavor effective theory it avoids the use of perturbation theory at $\\alpha > 0.2$ and at the same time has the physical scales small compared to the cutoff $1/a$ in all stages of the computation. The result $\\Lambda_\\overline{MS}^{(3)}=332(14)$~MeV is translated to $\\alpha_\\overline{MS}(m_Z)=0.1179(10)(2)$ by use of (high order) perturbative relations between the effective theory couplings at the charm and beauty quark "thresholds". The error of this perturbative step is discussed and estimated as $0.0002$.

  14. Effect of microinjections of subunits of cAMP-dependent protein kinase on development, proliferation, and RNA synthesis in early embryos of the loach Misgurnus fossilis L

    International Nuclear Information System (INIS)

    Glukhov, A.I.; Benyumov, A.O.; Nesterova, M.V.; Severin, E.S.; Gazaryan, K.G.


    The effect of the catalytic and regulatory subunits of cAMP-dependent protein kinase type II on development, proliferation, and RNA synthesis was studied in loach embryos. It was found that injection of the catalytic subunit in a physiological concentration leads to a disturbance in the course of development and inhibits proliferation and RNA synthesis in the embryos. An increase in the concentration of this protein above the physiological level leads to death of the embryos in the first hours of development. Injection of the regulatory subunit stimulated the incorporation of labeled uridine into the acid-insoluble fraction of the embryos, beginning with the gastrula stage. The cell nuclei of loach embryos injected with subunits of protein kinase type II were transplanted into activated loach egg cells: subunits of protein kinase type I had no effect on the ability of nuclei of undetermined loach embryo cells to provide de novo development and their effect was reversible

  15. Expression Profile of the Integrin Receptor Subunits in the Guinea Pig Sclera. (United States)

    Wang, Kevin K; Metlapally, Ravikanth; Wildsoet, Christine F


    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.

  16. The Subunit Principle in Scar Face Revision. (United States)

    Elshahat, Ahmed; Lashin, Riham


    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.

  17. alpha-decay spectroscopy of light odd-odd Bi isotopes - II sup 1 sup 8 sup 6 Bi and the new nuclide sup 1 sup 8 sup 4 Bi

    CERN Document Server

    Andreyev, A N; Ackermann, D; Münzenberg, G; Hessberger, F P; Hofmann, S; Kojouharov, I; Kindler, B; Lommel, B; Huyse, M; Vel, K V D; Duppen, P V; Heyde, Kris L G


    Alpha-decay of the new nuclide sup 1 sup 8 sup 4 Bi has been studied in the complete-fusion reaction sup 9 sup 3 Nb( sup 9 sup 4 Mo, 3n) sup 1 sup 8 sup 4 Bi at the velocity filter SHIP. The evaporation residues were separated in-flight and subsequently identified on the basis of recoil-alpha, recoil-alpha-gamma analysis and excitation functions measurements. Two alpha-decaying isomeric states in sup 1 sup 8 sup 4 Bi with half-life values of 13(2) ms and 6.6(1.5) ms were identified. The alpha-branching ratio of sup 1 sup 8 sup 0 Tl was deduced for the first time as b subalpha = (2-12)%. Improved data on the fine-structure alpha-decay of sup 1 sup 8 sup 6 Bi were obtained in the sup 9 sup 3 Nb( sup 9 sup 5 Mo, 2n) sup 1 sup 8 sup 6 Bi reaction. A similarity of the decay energies and half-life values of sup 1 sup 8 sup 4 sup , sup 1 sup 8 sup 6 Bi is pointed out and a possible explanation for this effect is suggested.

  18. Mannan-binding protein forms complexes with alpha-2-macroglobulin. A protein model for the interaction

    DEFF Research Database (Denmark)

    Storgaard, P; Holm Nielsen, E; Skriver, E


    . The occurrence of alpha 2M/pMBP-28 complexes was further indicated by crossed immunoelectrophoresis and by use of an anti-alpha 2M affinity column and chelating Sepharose loaded with Zn2+. The eluates from these affinity columns showed alpha 2M subunits (94 and 180 kDa) and pMBP subunits (28kDa) in SDS-PAGE...... with anti-C1 s antibodies in ELISA, one of about 650-800 kDa, which in addition contained pMBP-28 and anti-alpha 2M reactive material, the other with an M(r) of 100-150 kDa. The latter peak revealed rhomboid molecules (7 x 15 nm) in the electron microscope and a 67 kDa band in SDS-PAGE under reducing...

  19. 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


    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....

  20. The δ subunit of RNA polymerase guides promoter selectivity and virulence in Staphylococcus aureus. (United States)

    Weiss, Andy; Ibarra, J Antonio; Paoletti, Jessica; Carroll, Ronan K; Shaw, Lindsey N


    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.

  1. A model for the stepwise radiation inactivation of the alpha 2-dimer of Na,K-ATPase

    Energy Technology Data Exchange (ETDEWEB)

    Norby, J.G.; Jensen, J. (Univ. of Aarhus (Denmark))


    This study is a direct continuation of Jensen, J., and Norby. A new model in which we propose that the in situ organization of the Na,K-ATPase alpha-subunit is an alpha 2-dimer and which describes the stepwise degradation by radiation inactivation of this assembly is presented on the basis of the following findings. Radiation inactivation size for alpha-peptide integrity, normal nucleotide, vanadate and ouabain binding, and K-pNPPase activity is close to m(alpha) = 112 kDa; for Na-ATPase activity it is 135 kDa and for Na,K-ATPase activity it increases from 140 to about 195 kDa with increasing assay ATP concentration (equal to increasing average turnover). Normal Tl+ occlusion had the same radiation inactivation size as Vmax for Na,K-ATPase, i.e. about 195 kDa. The binding experiments disclosed radiation-produced molecules with active binding sites but with a lower than normal affinity. Radiation inactivation size for the total binding capacity of ADP and ouabain was therefore smaller than the size of an alpha-peptide, namely about 70 kDa, and for total Tl+ occlusion it was down to 40 kDa. We can explain all these observations by using a new approach to target size analysis and by assuming a dimeric organization of the alpha-subunit. Each alpha-peptide is degraded stepwise by first destruction of either a 42- or a 70-kDa domain, and the partly damaged peptide may retain biochemical activity. We conclude that there is no role for the beta-subunit in catalysis and that the alpha-peptide is organized as an alpha 2-dimer in the membrane with each alpha-subunit being able to perform complete catalytic cycles (and probably also active transport), provided that it is stabilized by an adjacent alpha-peptide or a sufficiently large fragment thereof.

  2. A model for the stepwise radiation inactivation of the alpha 2-dimer of Na,K-ATPase

    International Nuclear Information System (INIS)

    Norby, J.G.; Jensen, J.


    This study is a direct continuation of Jensen, J., and Norby. A new model in which we propose that the in situ organization of the Na,K-ATPase alpha-subunit is an alpha 2-dimer and which describes the stepwise degradation by radiation inactivation of this assembly is presented on the basis of the following findings. Radiation inactivation size for alpha-peptide integrity, normal nucleotide, vanadate and ouabain binding, and K-pNPPase activity is close to m(alpha) = 112 kDa; for Na-ATPase activity it is 135 kDa and for Na,K-ATPase activity it increases from 140 to about 195 kDa with increasing assay ATP concentration (equal to increasing average turnover). Normal Tl+ occlusion had the same radiation inactivation size as Vmax for Na,K-ATPase, i.e. about 195 kDa. The binding experiments disclosed radiation-produced molecules with active binding sites but with a lower than normal affinity. Radiation inactivation size for the total binding capacity of ADP and ouabain was therefore smaller than the size of an alpha-peptide, namely about 70 kDa, and for total Tl+ occlusion it was down to 40 kDa. We can explain all these observations by using a new approach to target size analysis and by assuming a dimeric organization of the alpha-subunit. Each alpha-peptide is degraded stepwise by first destruction of either a 42- or a 70-kDa domain, and the partly damaged peptide may retain biochemical activity. We conclude that there is no role for the beta-subunit in catalysis and that the alpha-peptide is organized as an alpha 2-dimer in the membrane with each alpha-subunit being able to perform complete catalytic cycles (and probably also active transport), provided that it is stabilized by an adjacent alpha-peptide or a sufficiently large fragment thereof

  3. Lyman Alpha Control

    CERN Document Server

    Nielsen, Daniel Stefaniak


    This document gives an overview of how to operate the Lyman Alpha Control application written in LabVIEW along with things to watch out for. Overview of the LabVIEW code itself as well as the physical wiring of and connections from/to the NI PCI-6229 DAQ box is also included. The Lyman Alpha Control application is the interface between the ALPHA sequencer and the HighFinesse Wavelength Meter as well as the Lyman Alpha laser setup. The application measures the wavelength of the output light from the Lyman Alpha cavity through the Wavelength Meter. The application can use the Wavelength Meter’s PID capabilities to stabilize the Lyman Alpha laser output as well as switch between up to three frequencies.

  4. New ALPHA-2 magnet

    CERN Multimedia

    Anaïs Schaeffer


    On 21 June, members of the ALPHA collaboration celebrated the handover of the first solenoid designed for the ALPHA-2 experiment. The magnet has since been successfully installed and is working well.   Khalid Mansoor, Sumera Yamin and Jeffrey Hangst in front of the new ALPHA-2 solenoid. “This was the first of three identical solenoids that will be installed between now and September, as the rest of the ALPHA-2 device is installed and commissioned,” explains ALPHA spokesperson Jeffrey Hangst. “These magnets are designed to allow us to transfer particles - antiprotons, electrons and positrons - between various parts of the new ALPHA-2 device by controlling the transverse size of the particle bunch that is being transferred.” Sumera Yamin and Khalid Mansoor, two Pakistani scientists from the National Centre for Physics in Islamabad, came to CERN in February specifically to design and manufacture these magnets. “We had the chance to work on act...

  5. Kinetic pathway of 40S ribosomal subunit recruitment to hepatitis C virus internal ribosome entry site. (United States)

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


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

  6. Distribution of protein and RNA in the 30S ribosomal subunit

    International Nuclear Information System (INIS)

    Ramakrishnan, V.


    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

  7. Alpha Shapes and Proteins

    DEFF Research Database (Denmark)

    Winter, Pawel; Sterner, Henrik; Sterner, Peter


    We provide a unified description of (weighted) alpha shapes, beta shapes and the corresponding simplicialcomplexes. We discuss their applicability to various protein-related problems. We also discuss filtrations of alpha shapes and touch upon related persistence issues.We claim that the full...... potential of alpha-shapes and related geometrical constructs in protein-related problems yet remains to be realized and verified. We suggest parallel algorithms for (weighted) alpha shapes, and we argue that future use of filtrations and kinetic variants for larger proteins will need such implementation....

  8. The AMP-activated protein kinase beta 1 subunit modulates erythrocyte integrity. (United States)

    Cambridge, Emma L; McIntyre, Zoe; Clare, Simon; Arends, Mark J; Goulding, David; Isherwood, Christopher; Caetano, Susana S; Reviriego, Carmen Ballesteros; Swiatkowska, Agnieszka; Kane, Leanne; Harcourt, Katherine; Adams, David J; White, Jacqueline K; Speak, Anneliese O


    Failure to maintain a normal in vivo erythrocyte half-life results in the development of hemolytic anemia. Half-life is affected by numerous factors, including energy balance, electrolyte gradients, reactive oxygen species, and membrane plasticity. The heterotrimeric AMP-activated protein kinase (AMPK) is an evolutionarily conserved serine/threonine kinase that acts as a critical regulator of cellular energy balance. Previous roles for the alpha 1 and gamma 1 subunits in the control of erythrocyte survival have been reported. In the work described here, we studied the role of the beta 1 subunit in erythrocytes and observed microcytic anemia with compensatory extramedullary hematopoiesis together with splenomegaly and increased osmotic resistance. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  9. A conformational switch in the inhibitory gamma-subunit of PDE6 upon enzyme activation by transducin. (United States)

    Granovsky, A E; Artemyev, N O


    In response to light, a photoreceptor G protein, transducin, activates cGMP-phosphodiesterase (PDE6) by displacing the inhibitory gamma-subunits (Pgamma) from the enzyme's catalytic sites. Evidence suggests that the activation of PDE6 involves a conformational change of the key inhibitory C-terminal domain of Pgamma. In this study, the C-terminal region of Pgamma, Pgamma-73-85, has been targeted for Ala-scanning mutagenesis to identify the point-to-point interactions between Pgamma and the PDE6 catalytic subunits and to probe the nature of the conformational change. Pgamma mutants were tested for their ability to inhibit PDE6 and a chimeric PDE5-conePDE6 enzyme containing the Pgamma C-terminus-binding site of cone PDE. This analysis has revealed that in addition to previously characterized Ile86 and Ile87, important inhibitory contact residues of Pgamma include Asn74, His75, and Leu78. The patterns of mutant PDE5-conePDE6 enzyme inhibition suggest the interaction between the PgammaAsn74/His75 sequence and Met758 of the cone PDE6alpha' catalytic subunit. This interaction, and the interaction between the PgammaIle86/Ile87 and PDE6alpha'Phe777/Phe781 residues, is most consistent with an alpha-helical structure of the Pgamma C-terminus. The analysis of activation of PDE6 enzymes containing Pgamma mutants with Ala-substituted transducin-contact residues demonstrated the critical role of PgammaLeu76. Accordingly, we hypothesize that the initial step in PDE6 activation involves an interaction of transducin-alpha with PgammaLeu76. This interaction introduces a bend into the alpha-helical structure of the Pgamma C-terminus, allowing transducin-alpha to further twist the C-terminus thereby uncovering the catalytic pocket of PDE6.

  10. A case of alpha-fetoprotein-producing esophageal adenocarcinoma. (United States)

    Chen, Yi-Yu; Hsu, Wen-Hung; Hu, Huang-Ming; Wu, Deng-Chyang; Lin, Wen-Yi


    Alpha-fetoprotein is a well-known tumor marker in the screening and follow-up of hepatocellular carcinoma. In Taiwanese society, a high prevalence of hepatitis and hepatoma and elevation of alpha-fetoprotein associated with liver function impairment usually suggested clinics undertake further examination for liver or genital tumor. We report the case of 45-year-old man who was found to have an alpha-fetoprotein-producing esophageal adenocarcinoma with an initial presentation of liver function impairment and rapid elevation of alpha-fetoprotein. Esophageal cancer was diagnosed via endoscope and a biopsy proved the presence of adenocarcinoma. A small endoscopic biopsy specimen failed to identify the alpha-fetoprotein positive tumor cell. Esophagectomy was performed and histopathological study of surgical specimen revealed grade II adenocarcinoma with regional metastatic lymphadenopathy. Immunohistochemical study was focal positive for alpha-fetoprotein. Serum alpha-fetoprotein declined transiently after esophagectomy and fluctuation of alpha-fetoprotein level was noted during the treatment with adjuvant chemotherapy. Finally, 19 months after the operation, the patient died due to multiple organ metastases with multiple organ failure. Thus, a small specimen for upper endoscopy may not be sufficient in the presence of alpha-fetoprotein-producing adenocarcinoma. Monitoring of serum alpha-fetoprotein may be useful in the evaluation and follow-up of esophageal alpha-fetoprotein-producing adenocarcinoma. Copyright © 2012. Published by Elsevier B.V.

  11. 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)


    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.

  12. Targeted Alpha Therapy: From Alpha to Omega

    International Nuclear Information System (INIS)

    Allen, Barry J; Clarke, Raymond; Huang Chenyu


    This review covers the broad spectrum of Targeted Alpha Therapy (TAT) research in Australia; from in vitro and in vivo studies to clinical trials. The principle of tumour anti-vascular alpha therapy (TAVAT) is discussed in terms of its validation by Monte Carlo calculations of vascular models and the potential role of biological dosimetry is examined. Summmary of this review is as follows: 1. The essence of TAT 2. Therapeutic objectives 3. TAVAT and Monte Carlo microdosimetry 4. Biological dosimetry 5. Preclinical studies 6. Clinical trials 7. What next? 8. Obstacles. (author)

  13. Photoinduced reduction of the medial FeS center in the hydrogenase small subunit HupS from Nostoc punctiforme. (United States)

    Raleiras, Patrícia; Hammarström, Leif; Lindblad, Peter; Styring, Stenbjörn; Magnuson, Ann


    The small subunit from the NiFe uptake hydrogenase, HupSL, in the cyanobacterium Nostoc punctiforme ATCC 29133, has been isolated in the absence of the large subunit (P. Raleiras, P. Kellers, P. Lindblad, S. Styring, A. Magnuson, J. Biol. Chem. 288 (2013) 18,345-18,352). Here, we have used flash photolysis to reduce the iron-sulfur clusters in the isolated small subunit, HupS. We used ascorbate as electron donor to the photogenerated excited state of Ru(II)-trisbipyridine (Ru(bpy)3), to generate Ru(I)(bpy)3 as reducing agent. Our results show that the isolated small subunit can be reduced by the Ru(I)(bpy)3 generated through flash photolysis. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Immunochemical aspects of crotoxim and its subunits

    International Nuclear Information System (INIS)

    Nakazone, A.K.


    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

  15. Conditional expression of constitutively active estrogen receptor {alpha} in chondrocytes impairs longitudinal bone growth in mice

    Energy Technology Data Exchange (ETDEWEB)

    Ikeda, Kazuhiro [Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical University, Saitama (Japan); Tsukui, Tohru [Experimental Animal Laboratory, Research Center for Genomic Medicine, Saitama Medical University, Saitama (Japan); Imazawa, Yukiko; Horie-Inoue, Kuniko [Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical University, Saitama (Japan); Inoue, Satoshi, E-mail: [Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical University, Saitama (Japan); Department of Geriatric Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo (Japan); Department of Anti-Aging Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo (Japan)


    Highlights: Black-Right-Pointing-Pointer Conditional transgenic mice expressing constitutively active estrogen receptor {alpha} (caER{alpha}) in chondrocytes were developed. Black-Right-Pointing-Pointer Expression of caER{alpha} in chondrocytes impaired longitudinal bone growth in mice. Black-Right-Pointing-Pointer caER{alpha} affects chondrocyte proliferation and differentiation. Black-Right-Pointing-Pointer This mouse model is useful for understanding the physiological role of ER{alpha}in vivo. -- Abstract: Estrogen plays important roles in the regulation of chondrocyte proliferation and differentiation, which are essential steps for longitudinal bone growth; however, the mechanisms of estrogen action on chondrocytes have not been fully elucidated. In the present study, we generated conditional transgenic mice, designated as caER{alpha}{sup ColII}, expressing constitutively active mutant estrogen receptor (ER) {alpha} in chondrocytes, using the chondrocyte-specific type II collagen promoter-driven Cre transgenic mice. caER{alpha}{sup ColII} mice showed retardation in longitudinal growth, with short bone lengths. BrdU labeling showed reduced proliferation of hypertrophic chondrocytes in the proliferating layer of the growth plate of tibia in caER{alpha}{sup ColII} mice. In situ hybridization analysis of type X collagen revealed that the maturation of hypertrophic chondrocytes was impaired in caER{alpha}{sup ColII} mice. These results suggest that ER{alpha} is a critical regulator of chondrocyte proliferation and maturation during skeletal development, mediating longitudinal bone growth in vivo.

  16. Combined zidovudine and interferon-alpha treatment in patients with AIDS-associated Kaposi's sarcoma

    NARCIS (Netherlands)

    de Wit, R.; Danner, S. A.; Bakker, P. J.; Lange, J. M.; Eeftinck Schattenkerk, J. K.; Veenhof, C. H.


    The effectiveness of addition of interferon-alpha (IFN-alpha) to zidovudine in patients with AIDS-associated Kaposi's sarcoma was assessed in a non-randomized, phase II clinical trial. Twenty-one patients were treated with oral zidovudine (600 mg daily) and IFN-alpha was increased to 18 MU daily for

  17. The contraction induced increase in gene expression of peroxisome proliferator-activated receptor (PPAR)-gamma coactivator 1alpha (PGC-1alpha), mitochondrial uncoupling protein 3 (UCP3) and hexokinase II (HKII) in primary rat skeletal muscle cells is dependent on reactive oxygen species

    DEFF Research Database (Denmark)

    Silveira, Leonardo R.; Pilegaard, Henriette; Kusuhara, Keiko


    We evaluated the role of reactive oxygen species (ROS) for the contraction induced increase in expression of PGC-1alpha, HKII and UCP3 mRNA. Rat skeletal muscle cells were subjected to acute or repeated electrostimulation in the presence and absence of antioxidants. Contraction of muscle cells lead...... to an increased H2O2 formation, as measured by oxidation of H2HFF. Acute contraction of the muscle cells lead to a transient increase in PGC-1alpha and UCP3 mRNA by 172 and 65%, respectively (pantioxidants. Repeated contraction sessions induced...... a sustained elevation in PGC-1alpha and UCP3 mRNA and a transient increase in HKII (pantioxidant cocktail or with GPX+GSH. Incubation of cells for 10 days with ROS produced by xanthine oxidase/xanthine increased the level of PGC-1...

  18. Alpha1-antitrypsin deficiency

    DEFF Research Database (Denmark)

    Stolk, Jan; Seersholm, Niels; Kalsheker, Noor


    The Alpha One International Registry (AIR), a multinational research program focused on alpha1-antitrypsin (AAT) deficiency, was formed in response to a World Health Organization recommendation. Each of the nearly 20 participating countries maintains a national registry of patients with AAT defic...

  19. Alpha Thalassemia (For Parents) (United States)

    ... Safe Videos for Educators Search English Español Alpha Thalassemia KidsHealth / For Parents / Alpha Thalassemia What's in this ... Symptoms Diagnosis Treatment Print en español Alfa talasemia Thalassemias Thalassemias are a group of blood disorders that ...

  20. Buffett’s Alpha

    DEFF Research Database (Denmark)

    Frazzini, Andrea; Kabiller, David; Heje Pedersen, Lasse

    Berkshire Hathaway has realized a Sharpe ratio of 0.76, higher than any other stock or mutual fund with a history of more than 30 years, and Berkshire has a significant alpha to traditional risk factors. However, we find that the alpha becomes insignificant when controlling for exposures to Betting...

  1. Alpha clustering in nuclei

    International Nuclear Information System (INIS)

    Hodgson, P.E.


    The effects of nucleon clustering in nuclei are described, with reference to both nuclear structure and nuclear reactions, and the advantages of using the cluster formalism to describe a range of phenomena are discussed. It is shown that bound and scattering alpha-particle states can be described in a unified way using an energy-dependent alpha-nucleus potential. (author)

  2. Molecular cloning and characterization of G alpha proteins from the western tarnished plant bug, Lygus hesperus (United States)

    The G-alpha subunits of heterotrimeric G proteins play critical roles in the activation of diverse signal transduction cascades. However, the role of these genes in chemosensation remains to be fully elucidated. To initiate a comprehensive survey of signal transduction genes, we used homology-base...

  3. [Molecular cloning and characterization of cDNA of the rpc10+ gene encoding the smallest subunit of nuclear RNA polymerases of Schizosaccharomyces pombe]. (United States)

    Shpakovskiĭ, G V; Lebedenko, E N


    The full-length cDNA of the rpc10+ gene encoding mini-subunit Rpc10, which is common for all three nuclear RNA polymerases of the fission yeast Schizosaccharomyces pombe, was cloned and sequenced. The Rpc10 subunit of Sz. pombe and its homologs from S. cerevisiae and H. sapiens are positively charged proteins with a highly conserved C-terminal region and an invariant zinc-binding domain (Zn-finger) of a typical amino acid composition: YxCx2Cx12RCx2CGxR. Functional tests of heterospecific complementation, using tetrad analysis or plasmid shuffling, showed that the Rpc10 subunit of Sz. pombe can successfully replace the homologous ABC10 alpha subunit in nuclear RNA polymerases I-III of S. cerevisiae.

  4. Genetics Home Reference: alpha thalassemia (United States)

    ... Facebook Twitter Home Health Conditions Alpha thalassemia Alpha thalassemia Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Alpha thalassemia is a blood disorder that reduces the production ...

  5. 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


    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.

  6. The alpha channeling effect (United States)

    Fisch, N. J.


    Alpha particles born through fusion reactions in a tokamak reactor tend to slow down on electrons, but that could take up to hundreds of milliseconds. Before that happens, the energy in these alpha particles can destabilize on collisionless timescales toroidal Alfven modes and other waves, in a way deleterious to energy confinement. However, it has been speculated that this energy might be instead be channeled into useful energy, so as to heat fuel ions or to drive current. Such a channeling needs to be catalyzed by waves Waves can produce diffusion in energy of the alpha particles in a way that is strictly coupled to diffusion in space. If these diffusion paths in energy-position space point from high energy in the center to low energy on the periphery, then alpha particles will be cooled while forced to the periphery. The energy from the alpha particles is absorbed by the wave. The amplified wave can then heat ions or drive current. This process or paradigm for extracting alpha particle energy collisionlessly has been called alpha channeling. While the effect is speculative, the upside potential for economical fusion is immense. The paradigm also operates more generally in other contexts of magnetically confined plasma.

  7. Expression of the alpha 6 beta 4 integrin by squamous cell carcinomas and basal cell carcinomas: possible relation to invasive potential?

    DEFF Research Database (Denmark)

    Rossen, K; Dahlstrøm, K K; Mercurio, A M


    We have studied the expression of alpha 6 beta 4 integrin, a carcinoma laminin receptor in ten squamous cell carcinomas (SCCs) and ten basal cell carcinomas (BCCs) of the skin in order to examine whether changes in alpha 6 beta 4 integrin expression may be related to invasive and metastatic...... potential. Monoclonal antibodies specific for each subunit were applied on cryosections, using a three step indirect peroxidase technique. In normal epidermis the basal cells expressed both the alpha 6 and the beta 4 subunits, and the expression was polarized against the basement membrane. In SCCs...

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

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


    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. Muscular subunits transplantation for facial reanimation

    Directory of Open Access Journals (Sweden)

    Hazan André Salo Buslik


    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.

  10. Influvac, a trivalent inactivated subunit influenza vaccine. (United States)

    Zuccotti, Gian Vincenzo; Fabiano, Valentina


    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.

  11. 5 alpha-reductase inhibitors and prostatic disease. (United States)

    Schröder, F H


    5 alpha-Reductase inhibitors are a new class of substances with very specific effects on type I and type II 5 alpha R which may be of use in the treatment of skin disease, such as male pattern baldness, male acne and hirsutism, as well as prostatic hyperplasia and prostate cancer. At least two types of 5 alpha R inhibitors with a different pH optimum have been described. cDNA encoding for both the type I and the type II enzyme has been cloned. Most of the orally effective 5 alpha R inhibitors belong to the class of 4-azasteroids. The radical substituted in the 17 position of the steroid ring seems to be related to species specific variations and to the types of 5 alpha R enzymes in different species and organ systems. 5 alpha R inhibitors lead to a decrease of plasma DHT by about 65% while there is a slight rise in plasma testosterone. The decrease of tissue DHT in the ventral prostate of the intact rat, the dog and in humans is more pronounced and amounts to about 85%. There is a reciprocal rise of tissue T in these systems. The application of an inhibitor of 5 alpha R type II leads to a shrinkage of BPH in men by about 30%. In the rat a similar shrinkage accompanied by a significant decrease of total organ DNA occurs. This decrease, however, is not as pronounced as can be achieved with castration.(ABSTRACT TRUNCATED AT 250 WORDS)

  12. Human small cell lung cancer NYH cells selected for resistance to the bisdioxopiperazine topoisomerase II catalytic inhibitor ICRF-187 demonstrate a functional R162Q mutation in the Walker A consensus ATP binding domain of the alpha isoform

    DEFF Research Database (Denmark)

    Wessel, I; Jensen, L H; Jensen, P B


    in expression of the beta isoform. Sequencing of the entire topoisomerase IIalpha cDNA from NYH/187 cells demonstrated a homozygous G-->A point mutation at nucleotide 485, leading to a R162Q conversion in the Walker A consensus ATP binding site (residues 161-165 in the alpha isoform), this being the first drug......-selected mutation described at this site. Western blotting after incubation with ICRF-187 showed no depletion of the alpha isoform in NYH/187 cells in contrast to wild-type (wt) cells, whereas equal depletion of the beta isoform was observed in the two sublines. Alkaline elution assay demonstrated a lack...... of inhibition of etoposide-induced DNA single-stranded breaks in NYH/187 cells, whereas this inhibition was readily apparent in NYH cells. Site-directed mutagenesis in human topoisomerase IIalpha introduced into a yeast Saccharomyces cerevisiae strain with a temperature-conditional yeast TOP2 mutant...

  13. Alpha Hydroxy Acids (United States)

    ... or tenderness (8), chemical burns (6), and increased sunburn (3). The frequency of such reports for skin ... bear a statement that conveys the following information: Sunburn Alert: This product contains an alpha hydroxy acid ( ...

  14. Justify your alpha

    NARCIS (Netherlands)

    Lakens, Daniel; Adolfi, Federico G.; Albers, Casper J.; Anvari, Farid; Apps, Matthew A.J.; Argamon, Shlomo E.; Baguley, Thom; Becker, Raymond B.; Benning, Stephen D.; Bradford, Daniel E.; Buchanan, Erin M.; Caldwell, Aaron R.; Van Calster, Ben; Carlsson, Rickard; Chen, Sau Chin; Chung, Bryan; Colling, Lincoln J.; Collins, Gary S.; Crook, Zander; Cross, Emily S.; Daniels, Sameera; Danielsson, Henrik; Debruine, Lisa; Dunleavy, Daniel J.; Earp, Brian D.; Feist, Michele I.; Ferrell, Jason D.; Field, James G.; Fox, Nicholas W.; Friesen, Amanda; Gomes, Caio; Gonzalez-Marquez, Monica; Grange, James A.; Grieve, Andrew P.; Guggenberger, Robert; Grist, James; Van Harmelen, Anne Laura; Hasselman, Fred; Hochard, Kevin D.; Hoffarth, Mark R.; Holmes, Nicholas P.; Ingre, Michael; Isager, Peder M.; Isotalus, Hanna K.; Johansson, Christer; Juszczyk, Konrad; Kenny, David A.; Khalil, Ahmed A.; Konat, Barbara; Lao, Junpeng; Larsen, Erik Gahner; Lodder, Gerine M.A.; Lukavský, Jiří; Madan, Christopher R.; Manheim, David; Martin, Stephen R.; Martin, Andrea E.; Mayo, Deborah G.; McCarthy, Randy J.; McConway, Kevin; McFarland, Colin; Nio, Amanda Q.X.; Nilsonne, Gustav; De Oliveira, Cilene Lino; De Xivry, Jean Jacques Orban; Parsons, Sam; Pfuhl, Gerit; Quinn, Kimberly A.; Sakon, John J.; Saribay, S. Adil; Schneider, Iris K.; Selvaraju, Manojkumar; Sjoerds, Zsuzsika; Smith, Samuel G.; Smits, Tim; Spies, Jeffrey R.; Sreekumar, Vishnu; Steltenpohl, Crystal N.; Stenhouse, Neil; Świątkowski, Wojciech; Vadillo, Miguel A.; Van Assen, Marcel A.L.M.; Williams, Matt N.; Williams, Samantha E.; Williams, Donald R.; Yarkoni, Tal; Ziano, Ignazio; Zwaan, Rolf A.


    In response to recommendations to redefine statistical significance to P ≤ 0.005, we propose that researchers should transparently report and justify all choices they make when designing a study, including the alpha level.

  15. Antihydrogen detection in ALPHA

    Energy Technology Data Exchange (ETDEWEB)

    Hydomako, Richard, E-mail: [University of Calgary, Department of Physics and Astronomy (Canada); Bruun Andresen, Gorm [Aarhus University, Department of Physics and Astronomy (Denmark); Ashkezari, Mohammad Dehghani [Simon Fraser University, Department of Physics (Canada); Baquero-Ruiz, Marcelo [University of California, Department of Physics (United States); Bertsche, William [Swansea University, Department of Physics (United Kingdom); Butler, Eoin [CERN, European Laboratory for Particle Physics (Switzerland); Bowe, Paul David [Aarhus University, Department of Physics and Astronomy (Denmark); Cesar, Claudo Lenz [Universidade Federal do Rio de Janeiro, Instituto de Fsica (Brazil); Chapman, Steve [University of California, Department of Physics (United States); Charlton, Michael [Swansea University, Department of Physics (United Kingdom); Fajans, Joel [University of California, Department of Physics (United States); Friesen, Tim; Fujiwara, Makoto C. [University of Calgary, Department of Physics and Astronomy (Canada); Gill, David Russell [TRIUMF (Canada); Hangst, Jeffrey Scott [Aarhus University, Department of Physics and Astronomy (Denmark); Hardy, Walter Newbold [University of British Columbia, Department of Physics and Astronomy (Canada); Hayano, Ryugo S. [University of Tokyo, Department of Physics (Japan); Hayden, Michael Edward [Simon Fraser University, Department of Physics (Canada); Humphries, Andrew James [Swansea University, Department of Physics (United Kingdom); Jonsell, Svante [Stockholm University, Fysikum (Sweden); Collaboration: ALPHA Collaboration; and others


    The ALPHA project is an international collaboration, based at CERN, with the experimental goal of performing precision spectroscopic measurements on antihydrogen. As part of this endeavor, the ALPHA experiment includes a silicon tracking detector. This detector consists of a three-layer array of silicon modules surrounding the antihydrogen trapping region of the ALPHA apparatus. Using this device, the antihydrogen annihilation position can be determined with a spatial resolution of better than 5 mm. Knowledge of the annihilation distribution was a critical component in the recently successful antihydrogen trapping effort. This paper will describe the methods used to reconstruct annihilation events in the ALPHA detector. Particular attention will be given to the description of the background rejection criteria.

  16. Coaching the alpha male. (United States)

    Ludeman, Kate; Erlandson, Eddie


    Highly intelligent, confident, and successful, alpha males represent about 70% of all senior executives. Natural leaders, they willingly take on levels of responsibility most rational people would find overwhelming. But many of their quintessential strengths can also make alphas difficult to work with. Their self-confidence can appear domineering. Their high expectations can make them excessively critical. Their unemotional style can keep them from inspiring their teams. That's why alphas need coaching to broaden their interpersonal tool kits while preserving their strengths. Drawing from their experience coaching more than 1,000 senior executives, the authors outline an approach tailored specifically for the alpha. Coaches get the alpha's attention by inundating him with data from 360-degree feedback presented in ways he will find compelling--both hard-boiled metrics and vivid verbatim comments from colleagues about his strengths and weaknesses. A 360-degree assessment is a wake-up call for most alphas, providing undeniable proof that their behavior doesn't work nearly as well as they think it does. That paves the way for a genuine commitment to change. In order to change, the alpha must venture into unfamiliar--and often uncomfortable--psychological territory. He must admit vulnerability, accept accountability not just for his own work for others', connect with his underlying emotions, learn to motivate through a balance of criticism and validation, and become aware of unproductive behavior patterns. The goal of executive coaching is not simply to treat the alpha as an individual problem but to improve the entire team dynamic. Initial success creates an incentive to persevere, and the virtuous cycle reverberates throughout the entire organization.

  17. The dnaN gene codes for the beta subunit of DNA polymerase III holoenzyme of escherichia coli. (United States)

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


    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.

  18. Primary structure of human alpha 2-macroglobulin. V. The complete structure

    DEFF Research Database (Denmark)

    Sottrup-Jensen, Lars; Stepanik, Terrence M; Kristensen, Torsten


    The primary structure of the tetrameric plasma glycoprotein human alpha 2-macroglobulin has been determined. The identical subunits contain 1451 amino acid residues. Glucosamine-based oligosaccharide groups are attached to asparagine residues 32, 47, 224, 373, 387, 846, 968, and 1401. Eleven......-SH group of Cys-949 is thiol esterified to the gamma-carbonyl group of Glx-952, thus forming an activatable reactive site which can mediate covalent binding of nucleophiles. A putative transglutaminase cross-linking site is constituted by Gln-670 and Gln-671. The primary sites of proteolytic cleavage......-macroglobulin subunit is discussed. A comparison of stretches of sequences from alpha 2-macroglobulin with partial sequence data for complement components C3 and C4 indicates that these proteins are evolutionary related. The properties of alpha 2-macroglobulin are discussed within the context of proteolytically...

  19. Alpha - Skew Pi - Armendariz Rings

    Directory of Open Access Journals (Sweden)

    Areej M Abduldaim


    Full Text Available In this article we introduce a new concept called Alpha-skew Pi-Armendariz rings (Alpha - S Pi - ARas a generalization of the notion of Alpha-skew Armendariz rings.Another important goal behind studying this class of rings is to employ it in order to design a modern algorithm of an identification scheme according to the evolution of using modern algebra in the applications of the field of cryptography.We investigate general properties of this concept and give examples for illustration. Furthermore, this paperstudy the relationship between this concept and some previous notions related to Alpha-skew Armendariz rings. It clearly presents that every weak Alpha-skew Armendariz ring is Alpha-skew Pi-Armendariz (Alpha-S Pi-AR. Also, thisarticle showsthat the concepts of Alpha-skew Armendariz rings and Alpha-skew Pi- Armendariz rings are equivalent in case R is 2-primal and semiprime ring.Moreover, this paper proves for a semicommutative Alpha-compatible ringR that if R[x;Alpha] is nil-Armendariz, thenR is an Alpha-S Pi-AR. In addition, if R is an Alpha - S Pi -AR, 2-primal and semiprime ring, then N(R[x;Alpha]=N(R[x;Alpha]. Finally, we look forwardthat Alpha-skew Pi-Armendariz rings (Alpha-S Pi-ARbe more effect (due to their properties in the field of cryptography than Pi-Armendariz rings, weak Armendariz rings and others.For these properties and characterizations of the introduced concept Alpha-S Pi-AR, we aspire to design a novel algorithm of an identification scheme.

  20. ALPHA-2: the sequel

    CERN Multimedia

    Katarina Anthony


    While many experiments are methodically planning for intense works over the long shutdown, there is one experiment that is already working at full steam: ALPHA-2. Its final components arrived last month and will completely replace the previous ALPHA set-up. Unlike its predecessor, this next generation experiment has been specifically designed to measure the properties of antimatter.   The ALPHA team lower the new superconducting solenoid magnet into place. The ALPHA collaboration is working at full speed to complete the ALPHA-2 set-up for mid-November – this will give them a few weeks of running before the AD shutdown on 17 December. “We really want to get some experience with this device this year so that, if we need to make any changes, we will have time during the long shutdown in which to make them,” says Jeffrey Hangst, ALPHA spokesperson. “Rather than starting the 2014 run in the commissioning stage, we will be up and running from the get go.&...

  1. Soybean glycinin subunits: Characterization of physicochemical and adhesion properties. (United States)

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


    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.

  2. Binding-dependent disorder-order transition in PKI alpha: a fluorescence anisotropy study. (United States)

    Hauer, J A; Taylor, S S; Johnson, D A


    The conformational flexibility of peptidyl ligands may be an essential element of many peptide-macromolecular interactions. Consequently, the alpha-carbonyl backbone flexibility of the 8 kDa protein kinase inhibitor (PKI alpha) peptide of cAMP-dependent protein kinase (cAPK) free in solution and bound to cAPK was assessed by time-resolved fluorescence anisotropy. Specifically, three full-length, single-site PKI alpha mutants (V3C, S28C, and S59C) were prepared, and fluorescein iodoacetamide (FI) was selectively conjugated to the side chains of each substituted cysteine. The time-resolved anisotropy decay profiles of the labeled mutants were well fit to a model-free nonassociative biexponential equation. Free in solution, the three labeled proteins had very similar anisotropy decays arising primarily from local alpha-carbonyl backbone movements. Only a small fraction of the anisotropy decay was associated with slower, whole-body tumbling, confirming that PKI alpha is highly disordered at all three locations. Complexation of the mutants with the catalytic (C) subunit of cAPK decreased the rate of whole-body tumbling for all three mutants. The effects on the rapid decay processes, however, were dependent upon the site of conjugation. The anisotropy decay profiles of both FI-V3C- and FI-S28C-PKI alpha were associated with significantly reduced contributions from the fast decay processes, while that of FI-S59C-PKI alpha was largely unaffected by binding to the C-subunit. The results suggest that the cAPK-binding domain of PKI alpha extends from the its N-terminus to residues beyond Ser28 but does not include the segment around Ser59, which is still part of a highly flexible domain when bound to the C-subunit.

  3. Glycoprotein hormone α subunit secretion by pituitary adenomas: influence of external irradiation

    International Nuclear Information System (INIS)

    Macfarlane, I.A.; Beardwell, C.G.; Shalet, S.M.; Darbyshire, P.J.; Hayward, E.; Sutton, M.L.


    In ninety-nine patients with pituitary adenomas, forty-six with acromegaly, the serum level of the glycoprotein hormone α subunit was elevated in eighteen cases. Thirteen of these were acromegalic and one had an FSH-producing tumour. Alpha levels varied little during the day, from one day to the next and over a 6 month period. In twenty-five patients with a variety of other hypothalamic-pituitary disorders examined, one patient with a craniopharyngioma had a mildly elevated α level. External pituitary irradiation was followed by an acute and often transient fall in α level in several of these patients. Of the fifty-four patients with pituitary adenomas who had received external irradiation before testing, only five had elevated α subunit levels compared with thirteen patients of the forty-five who had not been irradiated. This difference in incidence of elevated α level was statistically significant (P<0.025). It is concluded that external irradiation may reduce α subunit level chronically in many patients with pituitary adenoma. (author)

  4. PRKACA: the catalytic subunit of protein kinase A and adrenocortical tumors

    Directory of Open Access Journals (Sweden)

    Annabel Sophie Berthon


    Full Text Available Cyclic-AMP (cAMP-dependent protein kinase (PKA is the main effector of cAMP signaling in all tissues. Inactivating mutations of the PRKAR1A gene, coding for the type 1A regulatory subunit of PKA, are responsible for Carney complex and primary pigmented nodular adrenocortical disease (PPNAD. PRKAR1A inactivation and PKA dysregulation have been implicated in various types of adrenocortical pathologies associated with ACTH-independent Cushing syndrome (AICS from PPNAD to adrenocortical adenomas and cancer, and other forms of bilateral adrenocortical hyperplasias (BAH. More recently, mutations of PRKACA, the gene coding for the catalytic subunit C alpha (Cα, were also identified in the pathogenesis of adrenocortical tumors. PRKACA copy number gain was found in the germline of several patients with cortisol-producing BAH, whereas the somatic Leu206Arg (c.617A>C recurrent PRKACA mutation was found in as many as half of all adrenocortical adenomas associated with AICS. In vitro analysis demonstrated that this mutation led to constitutive Cα activity, unregulated by its main partners, the PKA regulatory subunits. In this review, we summarize the current understanding of the involvement of PRKACA in adrenocortical tumorigenesis, and our understanding of PKA’s role in adrenocortical lesions. We also discuss potential therapeutic advances that can be made through targeting of PRKACA and the PKA pathway.

  5. Cables1 controls p21/Cip1 protein stability by antagonizing proteasome subunit alpha type 3


    Shi, Zhi; Li, Zenggang; Li, Zijian; Cheng, Kejun; Du, Yuhong; Fu, Haian; Khuri, Fadlo R.


    The cyclin-dependent kinase inhibitor 1A (CDKN1A), p21/Cip1, is a vital cell cycle regulator, dysregulation of which has been associated with a large number of human malignancies. One critical mechanism that controls p21 function is through its degradation, which allows the activation of its associated cell cycle promoting kinases, CDK2 and CDK4. Thus, delineating how p21 is stabilized and degraded will enhance our understanding of cell growth control and offer a basis for potential therapeut...

  6. Purification and characterization of an N alpha-acetyltransferase from Saccharomyces cerevisiae. (United States)

    Lee, F J; Lin, L W; Smith, J A


    N alpha-Acetyltransferase, which catalyzes the transfer of an acetyl group from acetyl coenzyme A to the alpha-NH2 group of proteins and peptides, was isolated from Saccharomyces cerevisiae and demonstrated by protein sequence analysis to be NH2-terminally blocked. The enzyme was purified 4,600-fold to apparent homogeneity by successive purification steps using DEAE-Sepharose, hydroxylapatite, DE52 cellulose, and Affi-Gel blue. The Mr of the native enzyme was estimated to be 180,000 +/- 10,000 by gel filtration chromatography, and the Mr of each subunit was estimated to be 95,000 +/- 2,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has a pH optimum near 9.0, and its pI is 4.3 as determined by chromatofocusing on Mono-P. The enzyme catalyzed the transfer of an acetyl group to various synthetic peptides, including human adrenocorticotropic hormone (ACTH) (1-24) and its [Phe2] analogue, yeast alcohol dehydrogenase I (1-24), yeast alcohol dehydrogenase II (1-24), and human superoxide dismutase (1-24). These peptides contain either Ser or Ala as NH2-terminal residues which together with Met are the most commonly acetylated NH2-terminal residues (Persson, B., Flinta, C., von Heijne, G., and Jornvall, H. (1985) Eur. J. Biochem. 152, 523-527). Yeast enolase, containing a free NH2-terminal Ala residue, is known not to be N alpha-acetylated in vivo (Chin, C. C. Q., Brewer, J. M., and Wold, F. (1981) J. Biol. Chem. 256, 1377-1384), and enolase (1-24), a synthetic peptide mimicking the protein's NH2 terminus, was not acetylated in vitro by yeast acetyltransferase. The enzyme did not catalyze the N alpha-acetylation of other synthetic peptides including ACTH(11-24), ACTH(7-38), ACTH(18-39), human beta-endorphin, yeast superoxide dismutase (1-24). Each of these peptides has an NH2-terminal residue which is rarely acetylated in proteins (Lys, Phe, Arg, Tyr, Val, respectively). Among a series of divalent cations, Cu2+ and Zn2+ were demonstrated to be

  7. [Alpha-melanocyte-stimulating hormone. From bench to bedside]. (United States)

    Böhm, M; Luger, T A


    Alpha-melanocyte-stimulating hormone (alpha-MSH) is a tridecapeptide that is produced by the skin itself from the precursor proopiomelanocortin. It crucially mediates ultraviolet light-induced tanning after binding to melanocortin-1 receptors (MC-1R) expressed on the surface of epidermal melanocytes. The potent pigment-inducing and also cytoprotective actions of alpha-MSH are the rationale for the performance of first phase II clinical trials with Nle4-D-Phe7-alpha-MSH (NDP-alpha-MSH), a subcutaneously administered synthetic and superpotent alpha-MSH analogue, in patients with photodermatoses such as erythropoietic protoporphyria. Since alpha-MSH has shown promising anti-inflammatory and antifibrotic properties in numerous preclinical studies, it will be most interesting to evaluate these effects in further clinical pilot studies with NDP-alpha-MSH. In addition to alpha-MSH analogues, truncated tripeptides such as KDPT which do not bind to MC-1R but have sustained anti-inflammatory properties are currently emerging as another novel therapeutic strategy in dermatology.

  8. (Na+ + K+)-ATPase and plasma membrane polarity of intestinal epithelial cells: Presence of a brush border antigen in the distal large intestine that is immunologically related to beta subunit

    Energy Technology Data Exchange (ETDEWEB)

    Marxer, A.; Stieger, B.; Quaroni, A.; Kashgarian, M.; Hauri, H.P. (Univ. of Basel (Switzerland))


    The previously produced monoclonal antibody IEC 1/48 against cultured rat intestinal crypt cells was extensively characterized and found to be directed against the beta subunit of (Na+ + K+)-ATPase as assessed by immunological and enzymatic criteria. Under nondenaturing conditions the antibody precipitated the alpha-beta enzyme complex (98,000 and 48,000 Mr). This probe, together with the monoclonal antibody C 62.4 against the alpha subunit was used to localize (Na+ + K+)-ATPase in epithelial cells along the rat intestinal tract by immunofluorescence and immunoelectron microscopy. Both antibodies exclusively labeled the basolateral membrane of small intestine and proximal colon epithelial cells. However, in the distal colon, IEC 1/48, but not C 62.4, also labeled the brush border membrane. The cross-reacting beta-subunit-like antigen on the apical cell pole was tightly associated with isolated brush borders but was apparently devoid of (Na+ + K+)-ATPase activity. Subcellular fractionation of colonocytes in conjunction with limited proteolysis and surface radioiodination of intestinal segments suggested that the cross-reacting antigen in the brush border may be very similar to the beta subunit. The results support the notion that in the small intestine and proximal colon the enzyme subunits are exclusively targeted to the basolateral membrane while in the distal colon nonassembled beta subunit or a beta-subunit-like protein is also transported to the apical cell pole.

  9. Interactive domains in the molecular chaperone human alphaB crystallin modulate microtubule assembly and disassembly.

    Directory of Open Access Journals (Sweden)

    Joy G Ghosh


    Full Text Available Small heat shock proteins regulate microtubule assembly during cell proliferation and in response to stress through interactions that are poorly understood.Novel functions for five interactive sequences in the small heat shock protein and molecular chaperone, human alphaB crystallin, were investigated in the assembly/disassembly of microtubules and aggregation of tubulin using synthetic peptides and mutants of human alphaB crystallin.The interactive sequence (113FISREFHR(120 exposed on the surface of alphaB crystallin decreased microtubule assembly by approximately 45%. In contrast, the interactive sequences, (131LTITSSLSSDGV(142 and (156ERTIPITRE(164, corresponding to the beta8 strand and the C-terminal extension respectively, which are involved in complex formation, increased microtubule assembly by approximately 34-45%. The alphaB crystallin peptides, (113FISREFHR(120 and (156ERTIPITRE(164, inhibited microtubule disassembly by approximately 26-36%, and the peptides (113FISREFHR(120 and (131LTITSSLSSDGV(142 decreased the thermal aggregation of tubulin by approximately 42-44%. The (131LTITSSLSSDGV(142 and (156ERTIPITRE(164 peptides were more effective than the widely used anti-cancer drug, Paclitaxel, in modulating tubulinmicrotubule dynamics. Mutagenesis of these interactive sequences in wt human alphaB crystallin confirmed the effects of the alphaB crystallin peptides on microtubule assembly/disassembly and tubulin aggregation. The regulation of microtubule assembly by alphaB crystallin varied over a narrow range of concentrations. The assembly of microtubules was maximal at alphaB crystallin to tubulin molar ratios between 1:4 and 2:1, while molar ratios >2:1 inhibited microtubule assembly.Interactive sequences on the surface of human alphaB crystallin collectively modulate microtubule assembly through a dynamic subunit exchange mechanism that depends on the concentration and ratio of alphaB crystallin to tubulin. These are the first

  10. Unsaturated free fatty acids increase benzodiazepine receptor agonist binding depending on the subunit composition of the GABAA receptor complex. (United States)

    Witt, M R; Westh-Hansen, S E; Rasmussen, P B; Hastrup, S; Nielsen, M


    It has been shown previously that unsaturated free fatty acids (FFAs) strongly enhance the binding of agonist benzodiazepine receptor ligands and GABAA receptor ligands in the CNS in vitro. To investigate the selectivity of this effect, recombinant human GABAA/benzodiazepine receptor complexes formed by different subunit compositions (alpha x beta y gamma 2, x = 1, 2, 3, and 5; y = 1, 2, and 3) were expressed using the baculovirus-transfected Sf9 insect cell system. At 10(-4) M, unsaturated FFAs, particularly arachidonic (20:4) and docosahexaenoic (22:6) acids, strongly stimulated (> 200% of control values) the binding of [3H]flunitrazepam ([3H]FNM) to the alpha 3 beta 2 gamma 2 receptor combination in whole cell preparations. No effect or small increases in levels of unsaturated FFAs on [3H]FNM binding to alpha 1 beta x gamma 2 and alpha 2 beta x gamma 2 receptor combinations were observed, and weak effects (130% of control values) were detected using the alpha 5 beta 2 gamma 2 receptor combination. The saturated FFAs, stearic and palmitic acids, were without effect on [3H]FNM binding to any combination of receptor complexes. The hydroxylated unsaturated FFAs, ricinoleic and ricinelaidic acids, were shown to decrease the binding of [3H]FNM only if an alpha 1 beta 2 gamma 2 receptor combination was used. Given the heterogeneity of the GABAA/ benzodiazepine receptor subunit distribution in the CNS, the effects of FFAs on the benzodiazepine receptor can be assumed to vary at both cellular and regional levels.

  11. Monte Carlo alpha calculation

    Energy Technology Data Exchange (ETDEWEB)

    Brockway, D.; Soran, P.; Whalen, P.


    A Monte Carlo algorithm to efficiently calculate static alpha eigenvalues, N = ne/sup ..cap alpha..t/, for supercritical systems has been developed and tested. A direct Monte Carlo approach to calculating a static alpha is to simply follow the buildup in time of neutrons in a supercritical system and evaluate the logarithmic derivative of the neutron population with respect to time. This procedure is expensive, and the solution is very noisy and almost useless for a system near critical. The modified approach is to convert the time-dependent problem to a static ..cap alpha../sup -/eigenvalue problem and regress ..cap alpha.. on solutions of a/sup -/ k/sup -/eigenvalue problem. In practice, this procedure is much more efficient than the direct calculation, and produces much more accurate results. Because the Monte Carlo codes are intrinsically three-dimensional and use elaborate continuous-energy cross sections, this technique is now used as a standard for evaluating other calculational techniques in odd geometries or with group cross sections.

  12. CK2(beta)tes gene encodes a testis-specific isoform of the regulatory subunit of casein kinase 2 in Drosophila melanogaster

    DEFF Research Database (Denmark)

    Kalmykova, Alla I; Shevelyov, Yuri Y; Polesskaya, Oksana O


    An earlier described CK2(beta)tes gene of Drosophila melanogaster is shown to encode a male germline specific isoform of regulatory beta subunit of casein kinase 2. Western-analysis using anti-CK2(beta)tes Ig revealed CK2(beta)tes protein in Drosophila testes extract. Expression of a CK2(beta...... and coimmunoprecipitation analysis of protein extract from Drosophila testes, we demonstrated an association between CK2(beta)tes and CK2alpha. Northern-analysis has shown that another regulatory (beta') subunit found recently in D. melanogaster genome is also testis-specific. Thus, we describe the first example of two...

  13. Expression of GDNF and GFR alpha 1 in mouse taste bud cells. (United States)

    Takeda, Masako; Suzuki, Yuko; Obara, Nobuko; Uchida, Nobuhiko; Kawakoshi, Kentaro


    GDNF (glial cell line-derived neurotrophic factor) affects the survival and maintenance of central and peripheral neurons. Using an immunocytochemical method, we examined whether the taste bud cells in the circumvallate papillae of normal mice expressed GDNF and its GFR alpha 1 receptor. Using double immunostaining for either of them and NCAM, PGP 9.5, or alpha-gustducin, we additionally sought to determine what type of taste bud cells expressed GDNF or GFR alpha 1, because NCAM is reported to be expressed in type-III cells, PGP 9.5, in type-III and some type-II cells, and alpha-gustducin, in some type-II cells. Normal taste bud cells expressed both GDNF and GFR alpha 1. The percentage of GDNF-immunoreactive cells among all taste bud cells was 31.63%, and that of GFR alpha 1-immunoreactive cells, 83.21%. Confocal laser scanning microscopic observations after double immunostaining showed that almost none of the GDNF-immunoreactive cells in the taste buds were reactive with anti-NCAM or anti-PGP 9.5 antibody, but could be stained with anti-alpha-gustducin antibody. On the other hand, almost all anti-PGP 9.5- or anti-alpha-gustducin-immunoreactive cells were positive for GFR alpha 1. Thus, GDNF-immunoreactive cells did not include type-III cells, but type-II cells, which are alpha-gustducin-immunoreactive; on the other hand, GFR alpha 1-immunoreactive cells included type-II and -III cells, and perhaps type-I cells. We conclude that GDNF in the type-II cells may exert trophic actions on type-I, -II, and -III taste bud cells by binding to their GFR alpha 1 receptors.

  14. 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.

  15. Affective and cognitive effects of global deletion of alpha3-containing gamma-aminobutyric acid-A receptors. (United States)

    Fiorelli, Roberto; Rudolph, Uwe; Straub, Carolin J; Feldon, Joram; Yee, Benjamin K


    Gamma-aminobutyric acid (GABA)A receptors characterized by the presence of the alpha3 subunit are the major GABAA receptor subtype expressed in brain stem monoaminergic nuclei. These alpha3-GABAA receptors are therefore in a unique position to regulate monoaminergic functions. To characterize the functional properties of alpha3-GABAA receptors, we present a preliminary assessment of the expression of affective and cognitive behaviour in male mice with a targeted deletion of the Gabra3 gene encoding the alpha3 subunit [alpha3 knockout (KO) mice] on a C57BL/6Jx129X1/SvJ F1 hybrid genetic background. The alpha3 KO mice did not exhibit any gross change of anxiety-like behaviour or spontaneous locomotor behaviour. In the Porsolt forced swim test for potential antidepressant activity, alpha3 KO mice exhibited reduced floating and enhanced swimming behaviour relative to wild-type controls. Performance on a two-choice sucrose preference test, however, revealed no evidence for an increase in sucrose preference in the alpha3 KO mice that would have substantiated a potential phenotype for depression-related behaviour. In contrast, a suggestion of an enhanced negative contrast effect was revealed in a one-bottle sucrose consumption test across different sucrose concentrations. These affective phenotypes were accompanied by alterations in the balance between conditioned responding to the discrete conditioned stimulus and to the context, and a suggestion of faster extinction, in the Pavlovian conditioned freezing paradigm. Spatial learning in the water maze reference memory test, however, was largely unchanged in the alpha3 KO mice, except for a trend of preservation during reversal learning. The novel phenotypes following global deletion of the GABAA receptor alpha3 subunit identified here provided relevant insights, in addition to our earlier study, into the potential behavioural relevance of this specific receptor subtypes in the modulation of both affective and cognitive

  16. Germanium cryogenic detectors: Alpha surface events rejection capabilities

    International Nuclear Information System (INIS)

    Fiorucci, S.; Broniatowski, A.; Chardin, G.; Censier, B.; Lesquen, A. de; Deschamps, H.; Fesquet, M.; Jin, Y.


    Alpha surface events and multiple compton gamma interactions are the two major background components in Ge detectors for double-beta decay investigations. Two different methods have been studied to identify such type of events, using cryogenic Ge detectors developed primarily for dark matter search: (i) combined heat and ionization measurements, and (ii) pulse-shape analysis of the charge collection signals. Both methods show strong separation between electron recoil events and surface alphas. Cryogenic heat-ionization detectors therefore appear able to reject virtually all surface alpha interactions

  17. Human aldolase B subunit-specific radioimmunoassay

    International Nuclear Information System (INIS)

    Asaka, M.; Alpert, E.


    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.)

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

    Directory of Open Access Journals (Sweden)

    Gisela Pöll

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

  19. Subunit stoichiometry of the chloroplast photosystem I complex

    International Nuclear Information System (INIS)

    Bruce, B.D.; Malkin, R.


    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

  20. Autophosphorylation of [alpha]CaMKII is Differentially Involved in New Learning and Unlearning Mechanisms of Memory Extinction (United States)

    Kimura, Ryoichi; Silva, Alcino J.; Ohno, Masuo


    Accumulating evidence indicates the key role of [alpha]-calcium/calmodulin-dependent protein kinase II ([alpha]CaMKII) in synaptic plasticity and learning, but it remains unclear how this kinase participates in the processing of memory extinction. Here, we investigated the mechanism by which [alpha]CaMKII may mediate extinction by using…

  1. Ocular myasthenia gravis induced by human acetylcholine receptor ϵ subunit immunization in HLA DR3 transgenic mice. (United States)

    Wu, Xiaorong; Tuzun, Erdem; Saini, Shamsher S; Wang, Jun; Li, Jing; Aguilera-Aguirre, Leopoldo; Huda, Ruksana; Christadoss, Premkumar


    Extraocular muscles (EOM) are preferentially involved in myasthenia gravis (MG) and acetylcholine receptor (AChR) antibody positive MG patients may occasionally present with isolated ocular symptoms. Although experimental autoimmune myasthenia gravis (EAMG) induced by whole AChR immunization closely mimics clinical and immunopathological aspects of MG, EOM are usually not affected. We have previously developed an EAMG model, which imitates EOM symptoms of MG by immunization of human leukocyte antigen (HLA) transgenic mice with α or γ-subunits of human AChR (H-AChR). To investigate the significance of the ϵ-subunit in ocular MG, we immunized HLA-DR3 and HLA-DQ8 transgenic mice with recombinant H-AChR ϵ-subunit expressed in Escherichia coli. HLA-DR3 transgenic mice showed significantly higher clinical ocular and generalized MG severity scores and lower grip strength values than HLA-DQ8 mice. H-AChR ϵ-subunit-immunized HLA-DR3 transgenic mice had higher serum anti-AChR antibody (IgG, IgG1, IgG2b, IgG2c and IgM) levels, neuromuscular junction IgG and complement deposit percentages than ϵ-subunit-immunized HLA-DQ8 transgenic mice. Control mice immunized with E. coli extract or complete Freund adjuvant (CFA) did not show clinical and immunopathological features of ocular and generalized EAMG. Lymph node cells of ϵ-subunit-immunized HLA-DR3 mice showed significantly higher proliferative responses than those of ϵ-subunit-immunized HLA-DQ8 mice, crude E. coli extract-immunized and CFA-immunized transgenic mice. Our results indicate that the human AChR ϵ-subunit is capable of inducing myasthenic muscle weakness. Diversity of the autoimmune responses displayed by mice expressing different HLA class II molecules suggests that the interplay between HLA class II alleles and AChR subunits might have a profound impact on the clinical course of MG. Copyright © 2015 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  2. Alpha Antihydrogen Experiment

    CERN Document Server

    Fujiwara, M C; Ashkezari, M D; Baquero-Ruiz, M; Bertsche, W; Bray, C C; Butler, E; Cesar, C L; Chapman, S; Charlton, M; Cesar, C L; Fajans, J; Friesen, T; Gill, D R; Hangst, J S; Hardy, W N; Hayano, R S; Hayden, M E; Humphries, A J; Hydomako, R; Jonsell, S; Kurchaninov, L; Lambo, R; Madsen, N; Menary, S; Nolan, P; Olchanski, K; Olin, A; Povilus, A; Pusa, P; Robicheaux, F; Sarid, E; Silveira, D M; So, C; Storey, J W; Thompson, R I; van der Werf, D P; Wilding, D; Wurtele, J S; Yamazaki, Y


    ALPHA is an experiment at CERN, whose ultimate goal is to perform a precise test of CPT symmetry with trapped antihydrogen atoms. After reviewing the motivations, we discuss our recent progress toward the initial goal of stable trapping of antihydrogen, with some emphasis on particle detection techniques.

  3. Case Study - Alpha

    Directory of Open Access Journals (Sweden)

    Stephen Leybourne


    Full Text Available This case study was developed from an actual scenario by Dr. Steve Leybourne of Boston University.  The case documents the historical evolution of an organization, and has been used successfully in courses dealing with organizational and cultural change, and the utilization of ‘soft skills’ in project-based management. This is a short case, ideal for classroom use and discussion.  The issues are easily accessible to students, and there is a single wide ranging question that allows for the inclusion of many issues surrounding strategic decision-making, and behavioural and cultural change. Alpha was one of the earlier companies in the USA to invest in large, edge-of-town superstores, with plentiful free vehicle parking, selling food and related household products. Alpha was created in the 1950s as a subsidiary of a major publicly quoted retail group.  It started business by opening a string of very large discount stores in converted industrial and warehouse premises in the south of the United States. In the early days shoppers were offered a limited range of very competitively priced products. When Alpha went public in 1981 it was the fourth largest food retailer in the US, selling an ever-widening range of food and non-food products.  Its success continued to be based on high volume, low margins and good value for money, under the slogan of ‘Alpha Price.’

  4. Alpha-mannosidosis

    DEFF Research Database (Denmark)

    Borgwardt, Line; Stensland, Hilde Monica Frostad Riise; Olsen, Klaus Juul


    of the three subgroups of genotype/subcellular localisation and the clinical and biochemical data were done to investigate the potential relationship between genotype and phenotype in alpha-mannosidosis. Statistical analyses were performed using the SPSS software. Analyses of covariance were performed...

  5. Radial-velocity variations in Alpha Ori, Alpha Sco, and Alpha Her

    International Nuclear Information System (INIS)

    Smith, M.A.; Patten, B.M.; Goldberg, L.


    Radial-velocity observations of Alpha Ori, Alpha Sco A, and Alpha Her A are used to study radial-velocity periodicities in M supergiants. The data refer to several metallic lines in the H-alpha region and to H-alpha itself. It is shown that Alpha Ori and Alpha Sco A have cycle lengths of about 1 yr and semiamplitudes of 2 km/s. It is suggested that many semiregular red supergiant varibles such as Alpha Ori may be heading toward chaos. All three stars show short-term stochastic flucutations with an amplitude of 1-2 km/s. It is found that the long-term variability of H-alpha velocities may be a consequence of intermittent failed ejections. 58 refs

  6. Vaccine profile of herpes zoster (HZ/su) subunit vaccine. (United States)

    Cunningham, Anthony L; Heineman, Thomas


    Herpes zoster (HZ) causes an often severe and painful rash in older people and may be complicated by prolonged pain (postherpetic neuralgia; PHN) and by dissemination in immune-compromised patients. HZ results from reactivation of latent varicella-zoster virus (VZV) infection, often associated with age-related or other causes of decreased T cell immunity. A live attenuated vaccine boosts this immunity and provides partial protection against HZ, but this decreases with age and declines over 8 years. Areas covered: A new HZ subunit (HZ/su) vaccine combines a key surface VZV glycoprotein (E) with a T cell-boosting adjuvant system (AS01 B ) and is administered by two intramuscular injections two months apart. Expert commentary: HZ/su showed excellent efficacy of ~90% in immunocompetent adults ≥50 and ≥70 years of age, respectively, in the ZOE-50 and ZOE-70 phase III controlled trials. Efficacy was unaffected by advancing age and persisted for >3 years. Approximately 9.5% of subjects had severe, but transient (1-2 days) injection site pain, swelling or redness. Compliance with both vaccine doses was high (95%). The vaccine will have a major impact on HZ management. Phase I-II trials showed safety and immunogenicity in severely immunocompromised patients. Phase III trial results are expected soon.

  7. Differential Roles of the Glycogen-Binding Domains of β Subunits in Regulation of the Snf1 Kinase Complex▿ (United States)

    Mangat, Simmanjeet; Chandrashekarappa, Dakshayini; McCartney, Rhonda R.; Elbing, Karin; Schmidt, Martin C.


    Members of the AMP-activated protein kinase family, including the Snf1 kinase of Saccharomyces cerevisiae, are activated under conditions of nutrient stress. AMP-activated protein kinases are heterotrimeric complexes composed of a catalytic α subunit and regulatory β and γ subunits. In this study, the role of the β subunits in the regulation of Snf1 activity was examined. Yeasts express three isoforms of the AMP-activated protein kinase consisting of Snf1 (α), Snf4 (γ), and one of three alternative β subunits, either Sip1, Sip2, or Gal83. The Gal83 isoform of the Snf1 complex is the most abundant and was analyzed in the greatest detail. All three β subunits contain a conserved domain referred to as the glycogen-binding domain. The deletion of this domain from Gal83 results in a deregulation of the Snf1 kinase, as judged by a constitutive activity independent of glucose availability. In contrast, the deletion of this homologous domain from the Sip1 and Sip2 subunits had little effect on Snf1 kinase regulation. Therefore, the different Snf1 kinase isoforms are regulated through distinct mechanisms, which may contribute to their specialized roles in different stress response pathways. In addition, the β subunits are subjected to phosphorylation. The responsible kinases were identified as being Snf1 and casein kinase II. The significance of the phosphorylation is unclear since the deletion of the region containing the phosphorylation sites in Gal83 had little effect on the regulation of Snf1 in response to glucose limitation. PMID:19897735

  8. cobalt (ii), nickel (ii)

    African Journals Online (AJOL)


    Department of Chemistry Bayero University, P. M. B. 3011, Kano, Nigeria. E-mail: ABSTRACT. The manganese (II), cobalt (II), nickel (II) and .... water and common organic solvents, but are readily soluble in acetone. The molar conductance measurement [Table 3] of the complex compounds in.

  9. Proceedings, High-Precision $\\alpha_s$ Measurements from LHC to FCC-ee

    Energy Technology Data Exchange (ETDEWEB)

    d' Enterria, David [CERN; Skands, Peter Z. [Monash U.


    This document provides a writeup of all contributions to the workshop on "High precision measurements of $\\alpha_s$: From LHC to FCC-ee" held at CERN, Oct. 12--13, 2015. The workshop explored in depth the latest developments on the determination of the QCD coupling $\\alpha_s$ from 15 methods where high precision measurements are (or will be) available. Those include low-energy observables: (i) lattice QCD, (ii) pion decay factor, (iii) quarkonia and (iv) $\\tau$ decays, (v) soft parton-to-hadron fragmentation functions, as well as high-energy observables: (vi) global fits of parton distribution functions, (vii) hard parton-to-hadron fragmentation functions, (viii) jets in $e^\\pm$p DIS and $\\gamma$-p photoproduction, (ix) photon structure function in $\\gamma$-$\\gamma$, (x) event shapes and (xi) jet cross sections in $e^+e^-$ collisions, (xii) W boson and (xiii) Z boson decays, and (xiv) jets and (xv) top-quark cross sections in proton-(anti)proton collisions. The current status of the theoretical and experimental uncertainties associated to each extraction method, the improvements expected from LHC data in the coming years, and future perspectives achievable in $e^+e^-$ collisions at the Future Circular Collider (FCC-ee) with $\\cal{O}$(1--100 ab$^{-1}$) integrated luminosities yielding 10$^{12}$ Z bosons and jets, and 10$^{8}$ W bosons and $\\tau$ leptons, are thoroughly reviewed. The current uncertainty of the (preliminary) 2015 strong coupling world-average value, $\\alpha_s(m_Z)$ = 0.1177 $\\pm$ 0.0013, is about 1\\%. Some participants believed this may be reduced by a factor of three in the near future by including novel high-precision observables, although this opinion was not universally shared. At the FCC-ee facility, a factor of ten reduction in the $\\alpha_s$ uncertainty should be possible, mostly thanks to the huge Z and W data samples available.

  10. Reconstitution of active human core Mediator complex reveals a pivotal role of the MED14 subunit (United States)

    Cevher, Murat A.; Shi, Yi; Li, Dan; Chait, Brian T.; Malik, Sohail; Roeder, Robert G.


    The evolutionarily conserved Mediator complex is a critical coactivator for RNA polymerase II (Pol II)-mediated transcription. Here, we report the reconstitution of a functional 15-subunit human core Mediator complex and its characterization by functional assays and chemical cross-linking coupled to mass spectrometry (CX-MS). Whereas the reconstituted head and middle modules can stably associate, only with incorporation of MED14 into the bi-modular complex does it acquire basal and coactivator functions. This results from a dramatically enhanced ability of MED14-containing complexes to associate with Pol II. Altogether, our analyses identify MED14 as both an architectural and a functional backbone of the Mediator complex. We further establish a conditional requirement for metazoan-specific MED26 that becomes evident in the presence of heterologous nuclear factors. This general approach paves the way for systematically dissecting the multiple layers of functionalities associated with the Mediator complex. PMID:25383669

  11. Reconstitution of active human core Mediator complex reveals a critical role of the MED14 subunit. (United States)

    Cevher, Murat A; Shi, Yi; Li, Dan; Chait, Brian T; Malik, Sohail; Roeder, Robert G


    The evolutionarily conserved Mediator complex is a critical coactivator for RNA polymerase II (Pol II)-mediated transcription. Here we report the reconstitution of a functional 15-subunit human core Mediator complex and its characterization by functional assays and chemical cross-linking coupled to MS (CX-MS). Whereas the reconstituted head and middle modules can stably associate, basal and coactivator functions are acquired only after incorporation of MED14 into the bimodular complex. This results from a dramatically enhanced ability of MED14-containing complexes to associate with Pol II. Altogether, our analyses identify MED14 as both an architectural and a functional backbone of the Mediator complex. We further establish a conditional requirement for metazoan-specific MED26 that becomes evident in the presence of heterologous nuclear factors. This general approach paves the way for systematic dissection of the multiple layers of functionality associated with the Mediator complex.

  12. alpha-Globin genes: thalassemic and structural alterations in a Brazilian population

    Directory of Open Access Journals (Sweden)

    M.R.S.C. Wenning


    Full Text Available Seven unrelated patients with hemoglobin (Hb H disease and 27 individuals with alpha-chain structural alterations were studied to identify the alpha-globin gene mutations present in the population of Southeast Brazil. The -alpha3.7, --MED and -(alpha20.5 deletions were investigated by PCR, whereas non-deletional alpha-thalassemia (alphaHphalpha, alphaNcoIalpha, aaNcoI, alphaIcalpha and alphaTSaudialpha was screened with restriction enzymes and by nested PCR. Structural alterations were identified by direct DNA sequencing. Of the seven patients with Hb H disease, all of Italian descent, two had the -(alpha20.5/-alpha3.7 genotype, one had the --MED/-alpha3.7 genotype, one had the --MED/alphaHphalpha genotype and three showed interaction of the -alpha3.7 deletion with an unusual, unidentified form of non-deletional alpha-thalassemia [-alpha3.7/(aaT]. Among the 27 patients with structural alterations, 15 (of Italian descent had Hb Hasharon (alpha47Asp->His associated with the -alpha3.7 deletion, 4 (of Italian descent were heterozygous for Hb J-Rovigo (alpha53Ala->Asp, 4 (3 Blacks and 1 Caucasian were heterozygous for Hb Stanleyville-II (alpha78Asn->Lys associated with the alpha+-thalassemia, 1 (Black was heterozygous for Hb G-Pest (alpha74Asp->Asn, 1 (Caucasian was heterozygous for Hb Kurosaki (alpha7Lys->Glu, 1 (Caucasian was heterozygous for Hb Westmead (alpha122His->Gln, and 1 (Caucasian was the carrier of a novel silent variant (Hb Campinas, alpha26Ala->Val. Most of the mutations found reflected the Mediterranean and African origins of the population. Hbs G-Pest and Kurosaki, very rare, and Hb Westmead, common in southern China, were initially described in individuals of ethnic origin differing from those of the carriers reported in the present study and are the first cases to be reported in the Brazilian population.

  13. A transmembrane polar interaction is involved in the functional regulation of integrin alpha L beta 2. (United States)

    Vararattanavech, Ardcharaporn; Chng, Choon-Peng; Parthasarathy, Krupakar; Tang, Xiao-Yan; Torres, Jaume; Tan, Suet-Mien


    Integrins are heterodimeric transmembrane (TM) receptors formed by noncovalent associations of alpha and beta subunits. Each subunit contains a single alpha-helical TM domain. Inside-out activation of an integrin involves the separation of its cytoplasmic tails, leading to disruption of alphabeta TM packing. The leukocyte integrin alpha L beta 2 is required for leukocyte adhesion, migration, proliferation, cytotoxic function, and antigen presentation. In this study, we show by mutagenesis experiments that the packing of alpha L beta 2 TMs is consistent with that of the integrin alpha IIb beta 3 TMs. However, molecular dynamics simulations of alpha L beta 2 TMs in lipids predicted a polar interaction involving the side chains of alpha L Ser1071 and beta2 Thr686 in the outer-membrane association clasp (OMC). This is supported by carbonyl vibrational shifts observed in isotope-labeled alpha L beta 2 TM peptides that were incorporated into lipid bilayers. Molecular dynamics studies simulating the separation of alpha L beta 2 tails showed the presence of polar interaction during the initial perturbation of the inner-membrane association clasp. When the TMs underwent further separation, the polar interaction was disrupted. OMC polar interaction is important in regulating the functions of beta2 integrins because mutations that disrupt the OMC polar interaction generated constitutively activated alpha L beta 2, alpha M beta 2, and alpha X beta 2 in 293T transfectants. We also show that the expression of mutant beta2 Thr686Gly in beta2-deficient T cells rescued cell adhesion to intercellular adhesion molecule 1, but the cells showed overt elongated morphologies in response to chemokine stromal-cell-derived factor 1 alpha treatment as compared to wild-type beta2-expressing cells. These two TM polar residues are totally conserved in other members of the beta2 integrins in humans and across different species. Our results provide an example of the stabilizing effect of polar

  14. 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


    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...

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

    Directory of Open Access Journals (Sweden)

    Signe Tandrup Schmidt


    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

  16. Radioimmunological properties of antisera from carp gonadotropin (c-GTH) and its subunits

    International Nuclear Information System (INIS)

    Burzawa-Gerard, Elisabeth; Kerdelhue, B.


    Anti-sera against carp gonadotropin (c-GTH) and its subunits SU I and SU II were produced in rabbits (IS-c-GTH, IS-SU I and IS-SU II). The radioimmunological titers were determined when antiserum binding dilution was 50 p. 100 of the 125 I-labelled glycoproteins. The titers of IS-c-GTH were 1/750 000, 1/300 000 and 1/500 000, respectively with c-GTH, SU I and SU II. With SU I, the titer of IS-SU I was 1/100 000 ; this antiserum in excess bound only 25 p. 100 of the c-GTH and no SU II. With c-GTH and SU II, the titer of IS-SU II was 1/270 000; it was 1/13 000 with SU I. With these antisera no binding was observed with rat hormones (LH, FSH, TSH, PRL) but IS-c-GTH and IS-SU II weakly bound rLH [fr

  17. 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.


    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

  18. 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.

  19. Resting alpha activity predicts learning ability in alpha neurofeedback

    Directory of Open Access Journals (Sweden)

    Wenya eNan


    Full Text Available Individuals differ in their ability to learn how to regulate the alpha activity by neurofeedback. This study aimed to investigate whether the resting alpha activity is related to the learning ability of alpha enhancement in neurofeedback and could be used as a predictor. A total of 25 subjects performed 20 sessions of individualized alpha neurofeedback in order to learn how to enhance activity in the alpha frequency band. The learning ability was assessed by three indices respectively: the training parameter changes between two periods, within a short period and across the whole training time. It was found that the resting alpha amplitude measured before training had significant positive correlations with all learning indices and could be used as a predictor for the learning ability prediction. This finding would help the researchers in not only predicting the training efficacy in individuals but also gaining further insight into the mechanisms of alpha neurofeedback.

  20. Alpha scintillation radon counting

    International Nuclear Information System (INIS)

    Lucas, H.F. Jr.


    Radon counting chambers which utilize the alpha-scintillation properties of silver activated zinc sulfide are simple to construct, have a high efficiency, and, with proper design, may be relatively insensitive to variations in the pressure or purity of the counter filling. Chambers which were constructed from glass, metal, or plastic in a wide variety of shapes and sizes were evaluated for the accuracy and the precision of the radon counting. The principles affecting the alpha-scintillation radon counting chamber design and an analytic system suitable for a large scale study of the 222 Rn and 226 Ra content of either air or other environmental samples are described. Particular note is taken of those factors which affect the accuracy and the precision of the method for monitoring radioactivity around uranium mines

  1. Mediator subunit18 controls flowering time and floral organ identity in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Zhengui Zheng

    Full Text Available Mediator is a conserved multi-protein complex that plays an important role in regulating transcription by mediating interactions between transcriptional activator proteins and RNA polymerase II. Much evidence exists that Mediator plays a constitutive role in the transcription of all genes transcribed by RNA polymerase II. However, evidence is mounting that specific Mediator subunits may control the developmental regulation of specific subsets of RNA polymerase II-dependent genes. Although the Mediator complex has been extensively studied in yeast and mammals, only a few reports on Mediator function in flowering time control of plants, little is known about Mediator function in floral organ identity. Here we show that in Arabidopsis thaliana, MEDIATOR SUBUNIT 18 (MED18 affects flowering time and floral organ formation through FLOWERING LOCUS C (FLC and AGAMOUS (AG. A MED18 loss-of-function mutant showed a remarkable syndrome of later flowering and altered floral organ number. We show that FLC and AG mRNA levels and AG expression patterns are altered in the mutant. Our results support parallels between the regulation of FLC and AG and demonstrate a developmental role for Mediator in plants.

  2. Expression of five acetylcholine receptor subunit genes in Brugia malayi adult worms

    Directory of Open Access Journals (Sweden)

    Ben-Wen Li


    Full Text Available Acetylcholine receptors (AChRs are required for body movement in parasitic nematodes and are targets of “classical” anthelmintic drugs such as levamisole and pyrantel and of newer drugs such as tribendimidine and derquantel. While neurotransmission explains the effects of these drugs on nematode movement, their effects on parasite reproduction are unexplained. The levamisole AChR type (L-AChRs in Caenorhabditis elegans is comprised of five subunits: Cel-UNC-29, Cel-UNC-38, Cel-UNC-63, Cel-LEV-1 and Cel-LEV-8. The genome of the filarial parasite Brugia malayi contains nine AChRs subunits including orthologues of Cel-unc-29, Cel-unc-38, and Cel-unc-63. We performed in situ hybridization with RNA probes to localize the expression of five AChR genes (Bm1_35890-Bma-unc-29, Bm1_20330-Bma-unc-38, Bm1_38195-Bma-unc-63, Bm1_48815-Bma-acr-26 and Bm1_40515-Bma-acr-12 in B. malayi adult worms. Four of these genes had similar expression patterns with signals in body muscle, developing embryos, spermatogonia, uterine wall adjacent to stretched microfilariae, wall of Vas deferens, and lateral cord. Three L-AChR subunit genes (Bma-unc-29, Bma-unc-38 and Bma-unc-63 were expressed in body muscle, which is a known target of levamisole. Bma-acr-12 was co-expressed with these levamisole subunit genes in muscle, and this suggests that its protein product may form receptors with other alpha subunits. Bma-acr-26 was expressed in male muscle but not in female muscle. Strong expression signals of these genes in early embryos and gametes in uterus and testis suggest that AChRs may have a role in nervous system development of embryogenesis and spermatogenesis. This would be consistent with embryotoxic effects of drugs that target these receptors in filarial worms. Our data show that the expression of these receptor genes is tightly regulated with regard to localization in adult worms and developmental stage in embryos and gametes. These results may help to explain the

  3. AlphaACT (United States)


    and other ADT data As part of Task 2, AlphaTRAC: • Collaborated with CERDEC and the U.S. Military Academy Network Sciences Center to develop...example) Meehl (1954) and Swets, Dawes, and Monahan (2000), which convincingly explain how actuarial judgments rendered by statistical models tend to...Reasoning (DARPA), Morgan Kaufmann Publishers, San Mateo, CA. Swcts, J.A., Dawes, R.M., and Monahan, J. (2000). Better decisions through science

  4. Rossi Alpha Method

    International Nuclear Information System (INIS)

    Hansen, G.E.


    The Rossi Alpha Method has proved to be valuable for the determination of prompt neutron lifetimes in fissile assemblies having known reproduction numbers at or near delayed critical. This workshop report emphasizes the pioneering applications of the method by Dr. John D. Orndoff to fast-neutron critical assemblies at Los Alamos. The value of the method appears to disappear for subcritical systems where the Rossi-α is no longer an α-eigenvalue

  5. Modeling the interactions of a peptide-major histocompatibility class I ligand with its receptors. II. Cross-reaction between a monoclonal antibody and two alpha beta T cell receptors

    DEFF Research Database (Denmark)

    Rognan, D; Engberg, J; Stryhn, A


    -peptide pair into the Fab combining site. Interestingly, the most energetically favored binding mode shows numerous analogies to the recently determined recognition of class I MHC-peptide complexes by alpha beta T cell receptors (TCRs). The pSAN13.4.1 also binds diagonally across the MHC binding groove......The recombinant antibody, pSAN13.4.1, has a unique T cell like specificity; it binds an Influenza Hemagglutinin octapeptide (Ha255-262) in an MHC (H-2Kk)-restricted manner, and a detailed comparison of the fine specificity of pSAN13.4.1 with the fine specificity of two Ha255-262-specific, H-2Kk......-restricted T cell hybridomas has supported this contention. A three-dimensional model of pSAN13.4.1 has been derived by homology modeling techniques. Subsequently, the structure of the pSAN13.4.1 antibody in complex with the antigenic Ha-Kk ligand was derived after a flexible and automated docking of the MHC...

  6. Refinement of the Compton–Rayleigh scatter ratio method for use on the Mars Science Laboratory alpha particle X-ray spectrometer: II – Extraction of invisible element content

    Energy Technology Data Exchange (ETDEWEB)

    Perrett, Glynis M. [Guelph-Waterloo Physics Institute, University of Guelph, Guelph, Ontario N1G 2W1 (Canada); Campbell, John L., E-mail: [Guelph-Waterloo Physics Institute, University of Guelph, Guelph, Ontario N1G 2W1 (Canada); Gellert, Ralf [Guelph-Waterloo Physics Institute, University of Guelph, Guelph, Ontario N1G 2W1 (Canada); King, Penelope L. [Research School of Earth Sciences, Australian National University, Canberra, ACT 0200 (Australia); Nield, Emily; O’Meara, Joanne M.; Pradler, Irina [Guelph-Waterloo Physics Institute, University of Guelph, Guelph, Ontario N1G 2W1 (Canada)


    The intensity ratio C/R between Compton and Rayleigh scatter peaks of the exciting Pu L X-rays in the alpha particle X-ray spectrometer (APXS) is strongly affected by the presence of very light elements such as oxygen which cannot be detected directly by the APXS. C/R values are determined along with element concentrations by fitting APXS spectra of geochemical reference materials (GRMs) with the GUAPX code. A quantity K is defined as the ratio between the C/R value determined by Monte Carlo simulation based on the measured element concentrations and the fitted C/R value from the spectrum. To ensure optimally accurate K values, the choice of appropriate GRMs is explored in detail, with attention paid to Rb and Sr, whose characteristic Kα X-ray peaks overlap the Pu Lα scatter peaks. The resulting relationship between the ratio K and the overall oxygen fraction is linear. This provides a calibration from which the concentration of additional light invisible constituents (ALICs) such as water may be estimated in unknown rock and conglomerate samples. Several GRMs are used as ‘unknowns’ in order to evaluate the accuracy of ALIC concentrations derived in this manner.

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

    International Nuclear Information System (INIS)

    Hagen, C.


    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)


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


    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

  9. Type-I interferon receptor expression: its circadian rhythm and downregulation after interferon-alpha administration in peripheral blood cells from renal cancer patients. (United States)

    Shiba, Masahiro; Nonomura, Norio; Nakai, Yasutomo; Nakayama, Masashi; Takayama, Hitoshi; Inoue, Hitoshi; Tsujimura, Akira; Nishimura, Kazuo; Okuyama, Akihiko


    To investigate the regulation of interferon-alpha (IFN-alpha) receptor expression in metastatic renal cell carcinoma (RCC) after IFN-alpha administration. Blood sampling was carried out in eight patients with metastatic RCC and six healthy volunteers. Flow-cytometric analysis using a monoclonal antibody against the active subunit of the type-I IFN-alpha receptor (IFNAR2) was carried out to examine the circadian rhythm of IFNAR2 expression in peripheral blood mononuclear cells (PBMC) as well as its downregulation after IFN-alpha administration. According to its circadian rhythm IFNAR2 in PBMC had a peak expression at night. Once IFN-alpha is administered, IFNAR2 levels in PBMC showed downregulation within 48 h and recovered within another 48 h. Our findings might support the establishment of an optimal schedule for IFN-alpha administration.

  10. Combining Alphas via Bounded Regression

    Directory of Open Access Journals (Sweden)

    Zura Kakushadze


    Full Text Available We give an explicit algorithm and source code for combining alpha streams via bounded regression. In practical applications, typically, there is insufficient history to compute a sample covariance matrix (SCM for a large number of alphas. To compute alpha allocation weights, one then resorts to (weighted regression over SCM principal components. Regression often produces alpha weights with insufficient diversification and/or skewed distribution against, e.g., turnover. This can be rectified by imposing bounds on alpha weights within the regression procedure. Bounded regression can also be applied to stock and other asset portfolio construction. We discuss illustrative examples.

  11. Variability of the Lyman alpha flux with solar activity

    International Nuclear Information System (INIS)

    Lean, J.L.; Skumanich, A.


    A three-component model of the solar chromosphere, developed from ground based observations of the Ca II K chromospheric emission, is used to calculate the variability of the Lyman alpha flux between 1969 and 1980. The Lyman alpha flux at solar minimum is required in the model and is taken as 2.32 x 10 11 photons/cm 2 /s. This value occurred during 1975 as well as in 1976 near the commencement of solar cycle 21. The model predicts that the Lyman alpha flux increases to as much as 5 x 10 11 photons/cm 2 /s at the maximum of the solar cycle. The ratio of the average fluxes for December 1979 (cycle maximum) and July 1976 (cycle minimum) is 1.9. During solar maximum the 27-day solar rotation is shown to cause the Lyman alpha flux to vary by as much as 40% or as little as 5%. The model also shows that the Lyman alpha flux varies over intermediate time periods of 2 to 3 years, as well as over the 11-year sunspot cycle. We conclude that, unlike the sunspot number and the 10.7-cm radio flux, the Lyman alpha flux had a variability that was approximately the same during each of the past three cycles. Lyman alpha fluxes calculated by the model are consistent with measurements of the Lyman alpha flux made by 11 of a total of 14 rocket experiments conducted during the period 1969--1980. The model explains satisfactorily the absolute magnitude, long-term trends, and the cycle variability seen in the Lyman alpha irradiances by the OSO 5 satellite experiment. The 27-day variability observed by the AE-E satellite experiment is well reproduced. However, the magntidue of the AE-E 1 Lyman alpha irradiances are higher than the model calculations by between 40% and 80%. We suggest that the assumed calibration of the AE-E irradiances is in error

  12. Mediator Subunit Med28 Is Essential for Mouse Peri-Implantation Development and Pluripotency.

    Directory of Open Access Journals (Sweden)

    Lin Li

    Full Text Available The multi-subunit mammalian Mediator complex acts as an integrator of transcriptional regulation by RNA Polymerase II, and has emerged as a master coordinator of development and cell fate determination. We previously identified the Mediator subunit, MED28, as a cytosolic binding partner of merlin, the Neurofibromatosis 2 (NF2 tumor suppressor, and thus MED28 is distinct in having a cytosolic role as an NF2 interacting protein as well as a nuclear role as a Mediator complex subunit. Although limited in vitro studies have been performed on MED28, its in vivo function remains unknown. Employing a knockout mouse model, we describe for the first time the requirement for Med28 in the developing mouse embryo. Med28-deficiency causes peri-implantation lethality resulting from the loss of pluripotency of the inner cell mass accompanied by reduced expression of key pluripotency transcription factors Oct4 and Nanog. Further, overexpression of Med28 in mouse embryonic fibroblasts enhances the efficiency of their reprogramming to pluripotency. Cre-mediated inactivation of Med28 in induced pluripotent stem cells shows that Med28 is required for their survival. Intriguingly, heterozygous loss of Med28 results in differentiation of induced pluripotent stem cells into extraembryonic trophectoderm and primitive endoderm lineages. Our findings document the essential role of Med28 in the developing embryo as well as in acquisition and maintenance of pluripotency during reprogramming.

  13. Protein kinase CK2 in human diseases

    DEFF Research Database (Denmark)

    Guerra, Barbara; Issinger, Olaf-Georg


    Protein kinase CK2 (formerly referred to as casein kinase II) is an evolutionary conserved, ubiquitous protein kinase. There are two paralog catalytic subunits, i.e. alpha (A1) and alpha' (A2). The alpha and alpha' subunits are linked to two beta subunits to produce a heterotetrameric structure...

  14. Absence of integrin alpha 7 causes a novel form of muscular dystrophy. (United States)

    Mayer, U; Saher, G; Fässler, R; Bornemann, A; Echtermeyer, F; von der Mark, H; Miosge, N; Pöschl, E; von der Mark, K


    Integrin alpha 7 beta 1 is a specific cellular receptor for the basement membrane protein laminin-1 (refs 1,2), as well as for the laminin isoforms -2 and -4 (ref. 3). The alpha 7 subunit is expressed mainly in skeletal and cardiac muscle and has been suggested to be involved in differentiation and migration processes during myogenesis. Three cytoplasmic and two extracellular splice variants that have been described are developmentally regulated and expressed in different sites in the muscle. In adult muscle, the alpha 7A and alpha 7B subunits are concentrated in myotendinous junctions but can also be detected in neuromuscular junctions and along the sarcolemmal membrane. To study the potential involvement of alpha 7 integrin, during myogenesis and its role in muscle integrity and function, we generated a null allele of the alpha 7 gene (Itga7) in the germline of mice by homologous recombination in embryonic stem (ES) cells. Surprisingly, mice homozygous for the mutation are viable and fertile, indicating that the alpha 7 beta 1 integrin is not essential for myogenesis. However, histological analysis of skeletal muscle revealed typical symptoms of a progressive muscular dystrophy starting soon after birth, but with a distinct variability in different muscle types. The observed histopathological changes strongly indicate an impairment of function of the myotendinous junctions. These findings demonstrate that alpha 7 beta 1 integrin represents an indispensable linkage between the muscle fibre and the extracellular matrix that is independent of the dystrophin-dystroglycan complex-mediated interaction of the cytoskeleton with the muscle basement membrane.

  15. The Caenorhabditis elegans Elongator complex regulates neuronal alpha-tubulin acetylation.

    Directory of Open Access Journals (Sweden)

    Jachen A Solinger


    Full Text Available Although acetylated alpha-tubulin is known to be a marker of stable microtubules in neurons, precise factors that regulate alpha-tubulin acetylation are, to date, largely unknown. Therefore, a genetic screen was employed in the nematode Caenorhabditis elegans that identified the Elongator complex as a possible regulator of alpha-tubulin acetylation. Detailed characterization of mutant animals revealed that the acetyltransferase activity of the Elongator is indeed required for correct acetylation of microtubules and for neuronal development. Moreover, the velocity of vesicles on microtubules was affected by mutations in Elongator. Elongator mutants also displayed defects in neurotransmitter levels. Furthermore, acetylation of alpha-tubulin was shown to act as a novel signal for the fine-tuning of microtubules dynamics by modulating alpha-tubulin turnover, which in turn affected neuronal shape. Given that mutations in the acetyltransferase subunit of the Elongator (Elp3 and in a scaffold subunit (Elp1 have previously been linked to human neurodegenerative diseases, namely Amyotrophic Lateral Sclerosis and Familial Dysautonomia respectively highlights the importance of this work and offers new insights to understand their etiology.

  16. The adsorption of Tl(I), Au(III), Cu(II) and the separation of 199Tl from alpha bombardment of gold target with PDB-18C6

    International Nuclear Information System (INIS)

    Zhou Dehai; Zhou Jimeng


    The adsorptive behavior of polymer of methyl aldehyde of dibenzo-18-crown-6 (PDB-18C6) in hydrochloric acid medium is studied and it is shown that the adsorption of T1(I), Au(III), and Cu(II) depends on the particle size of the crown ether resins, hydrochloric acid concentration and amount of the crown ether resins used. The difference in the adsorption behavior of different particle sizes of crown ether resins may be used for separating Tl(I), Au(III), and Cu(II) ions. The best eluant of Tl(I) and Au(III) is 0.4 mol/l perchloric acid and 2-ethoxy-ethanol. The recovery for Tl(I) is 82-98.8%. The gold target is bombarded in a 1.2 m cyclotron with 25-27 MeV α-particle with a cumulative beam intensities of 27μA·h, and 199 Tl is separated from the gold target with PDB-18C6. γ-spectrometry has shown that the Tl obtained is 199 Tl of high purity containing only about 0.50% 200 Tl

  17. Alteration of alpha 1 Na+,K(+)-ATPase 86Rb+ influx by a single amino acid substitution

    International Nuclear Information System (INIS)

    Herrera, V.L.; Ruiz-Opazo, N.


    The sodium- and potassium-dependent adenosine triphosphatase (Na+,K(+)-ATPase) maintains the transmembrane Na+ gradient to which is coupled all active cellular transport systems. The R and S alleles of the gene encoding the Na+,K(+)-ATPase alpha 1 subunit isoform were identified in Dahl salt-resistant (DR) and Dahl salt-sensitive (DS) rats, respectively. Characterization of the S allele-specific Na+,K(+)-ATPase alpha 1 complementary DNA identified a leucine substitution of glutamine at position 276. This mutation alters the hydropathy profile of a region in proximity to T3(Na), the trypsin-sensitive site that is only detected in the presence of Na+. This mutation causes a decrease in the rubidium-86 influx of S allele-specific sodium pumps, thus marking a domain in the Na+,K(+)-ATPase alpha subunit important for K+ transport, and supporting the hypothesis of a putative role of these pumps in hypertension

  18. Alteration of alpha 1 Na+,K(+)-ATPase sup 86 Rb sup + influx by a single amino acid substitution

    Energy Technology Data Exchange (ETDEWEB)

    Herrera, V.L.; Ruiz-Opazo, N. (Boston Univ. School of Medicine, MA (USA))


    The sodium- and potassium-dependent adenosine triphosphatase (Na+,K(+)-ATPase) maintains the transmembrane Na+ gradient to which is coupled all active cellular transport systems. The R and S alleles of the gene encoding the Na+,K(+)-ATPase alpha 1 subunit isoform were identified in Dahl salt-resistant (DR) and Dahl salt-sensitive (DS) rats, respectively. Characterization of the S allele-specific Na+,K(+)-ATPase alpha 1 complementary DNA identified a leucine substitution of glutamine at position 276. This mutation alters the hydropathy profile of a region in proximity to T3(Na), the trypsin-sensitive site that is only detected in the presence of Na+. This mutation causes a decrease in the rubidium-86 influx of S allele-specific sodium pumps, thus marking a domain in the Na+,K(+)-ATPase alpha subunit important for K+ transport, and supporting the hypothesis of a putative role of these pumps in hypertension.

  19. The contribution of VHL substrate binding and HIF1-alpha to the phenotype of VHL loss in renal cell carcinoma. (United States)

    Maranchie, Jodi K; Vasselli, James R; Riss, Joseph; Bonifacino, Juan S; Linehan, W Marston; Klausner, Richard D


    Clear-cell renal carcinoma is associated with inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene. VHL is the substrate recognition subunit of an E3 ligase, known to target the alpha subunits of the HIF heterodimeric transcription factor for ubiquitin-mediated degradation under normoxic conditions. We demonstrate that competitive inhibition of the VHL substrate recognition site with a peptide derived from the oxygen degradation domain of HIF1alpha recapitulates the tumorigenic phenotype of VHL-deficient tumor cells. These studies prove that VHL substrate recognition is essential to the tumor suppressor function of VHL. We further demonstrate that normoxic stabilization of HIF1alpha alone, while capable of mimicking some aspects of VHL loss, is not sufficient to reproduce tumorigenesis, indicating that it is not the critical oncogenic substrate of VHL.

  20. 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)


    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

  1. Integrins beta 5, beta 3 and alpha v are apically distributed in endometrial epithelium. (United States)

    Aplin, J D; Spanswick, C; Behzad, F; Kimber, S J; Vićovac, L


    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.

  2. Expression of G(alpha)(s) proteins and TSH receptor signalling in hyperfunctioning thyroid nodules with TSH receptor mutations. (United States)

    Holzapfel, Hans-Peter; Bergner, Beate; Wonerow, Peter; Paschke, Ralf


    Constitutively activating mutations of the thyrotrophin receptor (TSHR) are the main molecular cause of hyperfunctioning thyroid nodules (HTNs). The G protein coupling is an important and critical step in the TSHR signalling which mainly includes G(alpha)(s), G(alpha)(i) and G(alpha)(q)/11 proteins. We investigated the in vitro consequences of overexpressing G(alpha) proteins on signalling of the wild-type (WT) or mutated TSHR. Moreover, we investigated whether changes in G(alpha) protein expression are pathophysiologically relevant in HTNs or cold thyroid nodules (CTNs). Wild-type TSH receptor and mutated TSH receptors were coexpressed with G(alpha)(s), G(alpha)(i) or G(alpha)(q)/11, and cAMP and inositol phosphate (IP) production was measured after stimulation with TSH. The expression of G(alpha)(s), G(alpha)(i) and G(alpha)(q)/11 proteins was examined by Western blotting in 28 HTNs and 14 CTNs. Coexpression of G(alpha)(s) with the WT TSH receptor in COS 7 cells significantly increased the basal and TSH-stimulated cAMP accumulation while coexpression of the G(alpha)(q) or G(alpha)11 protein significantly increased the production of cAMP and inositol triphosphate (IP(3)). The coexpression of the TSH receptor mutants (I486F, DEL613-621), known to couple constitutively to G(alpha)(s) and G(alpha)(q) with G(alpha)(s) and G(alpha)(q)/11, significantly increased the basal and stimulated cAMP and IP(3) accumulation. Coexpression of the TSH receptor mutant V556F with G(alpha)(s) only increased the basal and stimulated cAMP production while its coexpression with G(alpha)(q)/11 increased the basal and stimulated IP(3) signalling. The expression of G(alpha)(s) protein subunits determined by Western blotting was significantly decreased in 14 HTNs with a constitutively activating TSH receptor mutation in comparison with the corresponding surrounding tissue, while in 14 HTNs without TSH receptor or G(alpha)(s) protein mutation and in 14 CTNs the expression of G(alpha

  3. Losses, Expansions, and Novel Subunit Discovery of Adaptor Protein Complexes in Haptophyte Algae. (United States)

    Lee, Laura J Y; Klute, Mary J; Herman, Emily K; Read, Betsy; Dacks, Joel B


    The phylum Haptophyta (Diaphoratickes) contains marine algae that perform biomineralization, extruding large, distinctive calcium carbonate scales (coccoliths) that completely cover the cell. Coccolith production is an important part of global carbon cycling; however, the membrane trafficking pathway by which they are secreted has not yet been elucidated. In most eukaryotes, post-Golgi membrane trafficking involves five heterotetrameric adaptor protein (AP) complexes, which impart cargo selection specificity. To better understand coccolith secretion, we performed comparative genomic, phylogenetic, and transcriptomic analyses of the AP complexes in Emiliania huxleyi strains 92A, Van556, EH2, and CCMP1516, and related haptophytes Gephyrocapsa oceanica and Isochrysis galbana; the latter has lost the ability to biomineralize. We show that haptophytes have a modified membrane trafficking system (MTS), as we found both AP subunit losses and duplications. Additionally, we identified a single conserved subunit of the AP-related TSET complex, whose expression suggests a functional role in membrane trafficking. Finally, we detected novel alpha adaptin ear and gamma adaptin ear proteins, the first of their kind to be described outside of opisthokonts. These novel ear proteins and the sculpting of the MTS may support the capacity for biomineralization in haptophytes, enhancing their ability to perform this highly specialized form of secretion. Copyright © 2015 Elsevier GmbH. All rights reserved.

  4. Structure of the Cmr2 Subunit of the CRISPR-Cas RNA Silencing Complex

    Energy Technology Data Exchange (ETDEWEB)

    Cocozaki, Alexis I.; Ramia, Nancy F.; Shao, Yaming; Hale, Caryn R.; Terns, Rebecca M.; Terns, Michael P.; Li, Hong (FSU); (Georgia)


    Cmr2 is the largest and an essential subunit of a CRISPR RNA-Cas protein complex (the Cmr complex) that cleaves foreign RNA to protect prokaryotes from invading genetic elements. Cmr2 is thought to be the catalytic subunit of the effector complex because of its N-terminal HD nuclease domain. Here, however, we report that the HD domain of Cmr2 is not required for cleavage by the complex in vitro. The 2.3 {angstrom} crystal structure of Pyrococcus furiosus Cmr2 (lacking the HD domain) reveals two adenylyl cyclase-like and two {alpha}-helical domains. The adenylyl cyclase-like domains are arranged as in homodimeric adenylyl cyclases and bind ADP and divalent metals. However, mutagenesis studies show that the metal- and ADP-coordinating residues of Cmr2 are also not critical for cleavage by the complex. Our findings suggest that another component provides the catalytic function and that the essential role by Cmr2 does not require the identified ADP- or metal-binding or HD domains in vitro.

  5. Treatment of alpha bearing wastes

    International Nuclear Information System (INIS)


    This report deals with the current state of the art of alpha waste treatment, which is an integral part of the overall nuclear waste management system. The International Atomic Energy Agency (IAEA) defines alpha bearing waste as 'waste containing one or more alpha emitting radionuclides, usually actinides, in quantities above acceptable limits'. The limits are established by national regulatory bodies. The limits above which wastes are considered as alpha contaminated refer to the concentrations of alpha emitters that need special consideration for occupational exposures and/or potential safety, health, or environmental impact during one or more steps from generation through disposal. Owing to the widespread use of waste segregation by source - that is, based upon the 'suspect origin' of the material - significant volumes of waste are being handled as alpha contaminated which, in fact, do not require such consideration by reason of risk or environmental concern. The quantification of de minimis concepts by national regulatory bodies could largely contribute to the safe reduction of waste volumes and associated costs. Other factors which could significantly contribute to the reduction of alpha waste arisings are an increased application of assaying and sorting, instrumentation and the use of feedback mechanisms to control or modify the processes which generate these wastes. Alpha bearing wastes are generated during fabrication and reprocessing of nuclear fuels, decommissioning of alpha contaminated facilities, and other activities. Most alpha wastes are contact handled, but a small portion may require shielding or remote handling because of high levels of neutron (n), beta (β), or gamma (γ) emissions associated with the waste material. This report describes the sources and characteristics of alpha wastes and strategies for alpha waste management. General descriptions of treatment processes for solid and liquid alpha wastes are included. 71 refs, 14 figs, 9 tabs

  6. Bi209 alpha activity

    International Nuclear Information System (INIS)

    Araujo Penna, M.M. de.


    The study for measuring Bi 209 alpha activity is presented. Ilford L4 nuclear emulsion pellicles loaded with bismuth citrate to obtain a load of 100 mg/cm 3 of dry emulsion, were prepared. Other pellicles were prepared with the same. Ilford L4 gel to estimate the background radiation. To observe 'fading' effect, pellicles loaded with bismuth were submitted to neutrons of high energy, aiming to record recoil proton tracks. The pellicles were confined in nitrogen atmosphere at temperature lower than -10 0 C. The Bi 209 experimental half-life was obtained and compared with the estimated theoretical data. (M.C.K.) [pt

  7. Alpha-mannosidosis

    Directory of Open Access Journals (Sweden)

    Nilssen Øivind


    Full Text Available Abstract Alpha-mannosidosis is an inherited lysosomal storage disorder characterized by immune deficiency, facial and skeletal abnormalities, hearing impairment, and intellectual disability. It occurs in approximately 1 of 500,000 live births. The children are often born apparently normal, and their condition worsens progressively. Some children are born with ankle equinus or develop hydrocephalus in the first year of life. Main features are immune deficiency (manifested by recurrent infections, especially in the first decade of life, skeletal abnormalities (mild-to-moderate dysostosis multiplex, scoliosis and deformation of the sternum, hearing impairment (moderate-to-severe sensorineural hearing loss, gradual impairment of mental functions and speech, and often, periods of psychosis. Associated motor function disturbances include muscular weakness, joint abnormalities and ataxia. The facial trait include large head with prominent forehead, rounded eyebrows, flattened nasal bridge, macroglossia, widely spaced teeth, and prognathism. Slight strabismus is common. The clinical variability is significant, representing a continuum in severity. The disorder is caused by lysosomal alpha-mannosidase deficiency. Alpha-mannosidosis is inherited in an autosomal recessive fashion and is caused by mutations in the MAN2B1 gene located on chromosome 19 (19 p13.2-q12. Diagnosis is made by measuring acid alpha-mannosidase activity in leukocytes or other nucleated cells and can be confirmed by genetic testing. Elevated urinary secretion of mannose-rich oligosaccharides is suggestive, but not diagnostic. Differential diagnoses are mainly the other lysosomal storage diseases like the mucopolysaccharidoses. Genetic counseling should be given to explain the nature of the disease and to detect carriers. Antenatal diagnosis is possible, based on both biochemical and genetic methods. The management should be pro-active, preventing complications and treating

  8. The alpha effect

    International Nuclear Information System (INIS)



    Much of the recent interest in RAM system reliability stems from concern over alpha particle soft error rates reported for the initial 64 k RAMs. With increasing memory density likely in the next few years the problem of soft errors is rearing its head again. A few years ago ITT carried out experiments on 16k RAMs and found no significant problems. However, recent tests have shown a raise in the number of soft errors with 64k RAMs, and the launch of 256k and 512k memories is likely to make the problem acute

  9. Biochemical parameters as biomarkers for the early recognition of environmental pollution on Scots pine trees. II. The antioxidative metabolites ascorbic acid, glutathione, {alpha}-tocopherol and the enzymes superoxide dismutase and glutathione reductase

    Energy Technology Data Exchange (ETDEWEB)

    Schulz, H.; Haertling, S. [UFZ Centre for Environmental Research Leipzig-Halle, Halle (Germany). Dept. of Soil Sciences


    Field investigations with Scots pine trees (Pinus sylvestris L.) were performed in eastern Germany, where ambient SO{sub 2}, NO{sub x} and O{sub 3} concentrations differed significantly in 1992-99 at three sites, namely Neuglobsow (yearly mean SO{sub 2} in 1992: 9 {mu}g m{sup -3}), Taura (yearly mean SO{sub 2} in 1992: 54 {mu}g m{sup -3}) and Roesa (yearly mean SO{sub 2} in 1992: 73 {mu}g m{sup -3}). To investigate the effects of SO{sub 2}, NO{sub x} and O{sub 3} on antioxidants (superoxide dismutase, ascorbic acid, glutathione, glutathione reductase, {alpha}-tocopherol) and pigments including chlorophyll fluorescence as well as visible damage symptoms in the form of needle yellowing and tip necroses, needles of the 1st and 2nd age class from young and mature trees were collected at the sites every October. Eight years after the start of the field study in 1992, the ambient SO{sub 2} concentrations had decreased significantly at Neuglobsow (yearly mean SO{sub 2} in 1999: 4 {mu}g m{sup -3}), Taura (yearly mean SO{sub 2} in 1999: 5 {mu}g m{sup -3}) and Roesa (yearly mean SO{sub 2} in 1999: 5 {mu}g m{sup -3}). NO{sub x} and O{sub 3} differed less at the three sites and showed no temporal variations. Whole needle glutathione continuously decreased, although concentrations were higher in needles of the 1st and 2nd age class from the polluted sites Taura and Roesa than the unpolluted site Neuglobsow. The activities of glutathione reductase exhibited the same site-related differences and temporal variations and were correlated with concentrations of oxidized glutathione (GSSG). In contrast, the activities of the enzyme superoxide dismutase and the concentrations of whole needle ascorbic acid remained unchanged over the period. Only at the end of the investigation period did the concentrations of oxidized ascorbic acid (dehydroascorbate) increase in six-month-old needles at the polluted sites Taura and Roesa. Despite the clear decreases in SO{sub 2}, the visible symptoms

  10. Decreased levels of genuine large free hCG alpha in men presenting with abnormal semen analysis

    Directory of Open Access Journals (Sweden)

    Plas Eugen


    Full Text Available Abstract Background The pregnancy hormone human chorionic gonadotropin (hCG and its free subunits (hCG alpha, hCG beta are produced in the male reproductive tract and found in high concentrations in seminal fluid, in particular hCG alpha. This study aimed to elucidate changes in peptide hormone profiles in patients showing abnormal semen analyses and to determine the genuineness of the highly abundant hCG alpha. Methods Seminal plasma was obtained from 45 male patients undergoing semen analysis during infertility workups. Comprehensive peptide hormone profiles were established by a panel of immunofluorometric assays for hCG, hCG alpha, hCG beta and its metabolite hCG beta core fragment, placental lactogen, growth hormone and prolactin in seminal plasma of patients with abnormal semen analysis results (n = 29 versus normozoospermic men (n = 16. The molecular identity of large hyperglycosylated hCG alpha was analyzed by mass-spectrometry and selective deglycosylation. Results hCG alpha levels were found to be significantly lower in men with impaired semen quality (1346 +/- 191 vs. 2753 +/- 533 ng/ml, P = 0.022. Moreover, patients with reduced sperm count had reduced intact hCG levels compared with normozoospermic men (0.097 +/- 0.022 vs. 0.203 +/- 0.040 ng/ml, P = 0.028. Using mass-spectrometry, the biochemical identity of hCG alpha purified from seminal plasma was verified. Under non-reducing conditions in SDS-PAGE, hCG alpha isolated from seminal plasma migrated in a manner comparable with large free hCG alpha with an apparent molecular mass (Mr, app of 24 kDa, while hCG alpha dissociated from pregnancy-derived holo-hCG migrated at approximately 22 kDa. After deglycosylation with PNGase F under denaturing conditions, all hCG alpha variants showed an Mr, app of 15 kDa, indicating identical amino acid backbones. Conclusions The findings indicate a pathophysiological relevance of hCG, particularly its free alpha subunit, in spermatogenesis. The

  11. Similarities in transcription factor IIIC subunits that bind to the posterior regions of internal promoters for RNA polymerase III. (United States)

    Matsutani, Sachiko


    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.

  12. Amino acid substitutions in subunit 9 of the mitochondrial ATPase complex of Saccharomyces cerevisiae. Sequence analysis of a series of revertants of an oli1 mit- mutant carrying an amino acid substitution in the hydrophilic loop of subunit 9. (United States)

    Willson, T A; Nagley, P


    This work concerns a biochemical genetic study of subunit 9 of the mitochondrial ATPase complex of Saccharomyces cerevisiae. Subunit 9, encoded by the mitochondrial oli1 gene, contains a hydrophilic loop connecting two transmembrane stems. In one particular oli1 mit- mutant 2422, the substitution of a positively charged amino acid in this loop (Arg39----Met) renders the ATPase complex non-functional. A series of 20 revertants, selected for their ability to grow on nonfermentable substrates, has been isolated from mutant 2422. The results of DNA sequence analysis of the oli1 gene in each revertant have led to the recognition of three groups of revertants. Class I revertants have undergone a same-site reversion event: the mutant Met39 is replaced either by arginine (as in wild-type) or lysine. Class II revertants maintain the mutant Met39 residue, but have undergone a second-site reversion event (Asn35----Lys). Two revertants showing an oligomycin-resistant phenotype carry this same second-site reversion in the loop region together with a further amino acid substitution in either of the two membrane-spanning segments of subunit 9 (either Gly23----Ser or Leu53----Phe). Class III revertants contain subunit 9 with the original mutant 2422 sequence, and additionally carry a recessive nuclear suppressor, demonstrated to represent a single gene. The results on the revertants in classes I and II indicate that there is a strict requirement for a positively charged residue in the hydrophilic loop close to the boundary of the lipid bilayer. The precise location of this positive charge is less stringent; in functional ATPase complexes it can be found at either residue 39 or 35. This charged residue is possibly required to interact with some other component of the mitochondrial ATPase complex. These findings, together with hydropathy plots of subunit 9 polypeptides from normal, mutant and revertant strains, led to the conclusion that the hydrophilic loop in normal subunit 9

  13. Complete primary structure of rainbow trout type I collagen consisting of alpha1(I)alpha2(I)alpha3(I) heterotrimers. (United States)

    Saito, M; Takenouchi, Y; Kunisaki, N; Kimura, S


    The subunit compositions of skin and muscle type I collagens from rainbow trout were found to be alpha1(I)alpha2(I)alpha3(I) and [alpha1(I)](2)alpha2(I), respectively. The occurrence of alpha3(I) has been observed only for bonyfish. The skin collagen exhibited more susceptibility to both heat denaturation and MMP-13 digestion than the muscle counterpart; the former had a lower denaturation temperature by about 0.5 degrees C than the latter. The lower stability of skin collagen, however, is not due to the low levels of imino acids because the contents of Pro and Hyp were almost constant in both collagens. On the other hand, some cDNAs coding for the N-terminal and/or a part of triple-helical domains of proalpha(I) chains were cloned from the cDNA library of rainbow trout fibroblasts. These cDNAs together with the previously cloned collagen cDNAs gave information about the complete primary structure of type I procollagen. The main triple-helical domain of each proalpha(I) chain had 338 uninterrupted Gly-X-Y triplets consisting of 1014 amino acids and was unique in its high content of Gly-Gly doublets. In particular, the bonyfish-specific alpha(I) chain, proalpha3(I) was characterized by the small number of Gly-Pro-Pro triplets, 19, and the large number of Gly-Gly doublets, 38, in the triple-helical domain, compared to 23 and 22, respectively, for proalpha1(I). The small number of Gly-Pro-Pro and the large number of Gly-Gly in proalpha3(I) was assumed to partially loosen the triple-helical structure of skin collagen, leading to the lower stability of skin collagen mentioned above. Finally, phylogenetic analyses revealed that proalpha3(I) had diverged from proalpha1(I). This study is the first report of the complete primary structure of fish type I procollagen.

  14. Genetic analysis of the cytoplasmic dynein subunit families. (United States)

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


    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.

  15. Genetic analysis of the cytoplasmic dynein subunit families.

    Directory of Open Access Journals (Sweden)

    K Kevin Pfister


    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.

  16. Silencing SlMED18, tomato Mediator subunit 18 gene, restricts internode elongation and leaf expansion. (United States)

    Wang, Yunshu; Hu, Zongli; Zhang, Jianling; Yu, XiaoHui; Guo, Jun-E; Liang, Honglian; Liao, Changguang; Chen, Guoping


    Mediator complex, a conserved multi-protein, is necessary for controlling RNA polymerase II (Pol II) transcription in eukaryotes. Given little is known about them in tomato, a tomato Mediator subunit 18 gene was isolated and named SlMED18. To further explore the function of SlMED18, the transgenic tomato plants targeting SlMED18 by RNAi-mediated gene silencing were generated. The SlMED18-RNAi lines exhibited multiple developmental defects, including smaller size and slower growth rate of plant and significantly smaller compound leaves. The contents of endogenous bioactive GA 3 in SlMED18 silenced lines were slightly less than that in wild type. Furthermore, qRT-PCR analysis indicated that expression of gibberellins biosynthesis genes such as SlGACPS and SlGA20x2, auxin transport genes (PIN1, PIN4, LAX1 and LAX2) and several key regulators, KNOX1, KNOX2, PHAN and LANCEOLATE(LA), which involved in the leaf morphogenesis were significantly down-regulated in SlMED18-RNAi lines. These results illustrated that SlMED18 plays an essential role in regulating plant internode elongation and leaf expansion in tomato plants and it acts as a key positive regulator of gibberellins biosynthesis and signal transduction as well as auxin proper transport signalling. These findings are the basis for understanding the function of the individual Mediator subunits in tomato.

  17. TJ-II Library Manual

    International Nuclear Information System (INIS)

    Tribaldos, V.; Milligen, B.Ph., van; Lopez-Fraguas, A.


    This report contains a detailed description of the TJ-II library and its routines. The library is written in FORTRAN 77 language and is available in the CRAY J916 and DEC Alpha 8400 computers at CIEMAT. This document also contains some examples of its use. (Author)

  18. MHC class II-derived peptides can bind to class II molecules, including self molecules, and prevent antigen presentation

    DEFF Research Database (Denmark)

    Rosloniec, E F; Vitez, L J; Buus, S


    the alpha k-3 peptide binds slightly less well. These combined data, suggesting that class II-derived peptides can bind to MHC class II molecules, including the autologous molecule from which they are derived, have important implications for the molecular basis of alloreactivity and autoreactivity. Further...... found in the first and third polymorphic regions (PMR) of the A alpha k chain (alpha k-1 and alpha k-3) were capable of inhibiting the presentation of three different HEL-derived peptide antigens to their appropriate T cells. In addition, the alpha k-1 peptide inhibited the presentation of the OVA(323......-339) immunodominant peptide to the I-Ad-restricted T cell hybridomas specific for it. Prepulsing experiments demonstrated that the PMR peptides were interacting with the APC and not with the T cell hybridomas. These observations were confirmed and extended by the demonstration that the alpha k-1 and alpha k-3...

  19. Direct interaction of the inhibitory gamma-subunit of Rod cGMP phosphodiesterase (PDE6) with the PDE6 GAFa domains. (United States)

    Muradov, Khakim G; Granovsky, Alexey E; Schey, Kevin L; Artemyev, Nikolai O


    Retinal rod and cone cGMP phosphodiesterases (PDE6 family) function as the effector enzyme in the vertebrate visual transduction cascade. The activity of PDE6 catalytic subunits is controlled by the Pgamma-subunits. In addition to the inhibition of cGMP hydrolysis at the catalytic sites, Pgamma is known to stimulate a noncatalytic binding of cGMP to the regulatory GAFa-GAFb domains of PDE6. The latter role of Pgamma has been attributed to its polycationic region. To elucidate the structural basis for the regulation of cGMP binding to the GAF domains of PDE6, a photoexcitable peptide probe corresponding to the polycationic region of Pgamma, Pgamma-21-45, was specifically cross-linked to rod PDE6alphabeta. The site of Pgamma-21-45 cross-linking was localized to Met138Gly139 within the PDE6alpha GAFa domain using mass spectrometric analysis. Chimeras between PDE5 and cone PDE6alpha', containing GAFa and/or GAFb domains of PDE6alpha' have been generated to probe a potential role of the GAFb domains in binding to Pgamma. Analysis of the inhibition of the PDE5/PDE6alpha' chimeras by Pgamma supported the role of PDE6 GAFa but not GAFb domains in the interaction with Pgamma. Our results suggest that a direct binding of the polycationic region of Pgamma to the GAFa domains of PDE6 may lead to a stabilization of the noncatalytic cGMP-binding sites.

  20. ALPHA/AMPU, Radionuclide Radioactivity from Alpha Spectrometer Measurements

    International Nuclear Information System (INIS)

    Sill, D.S.


    1 - Description of program or function: The two computer programs, ALPHA and AMPU, take raw data obtained from alpha spectrometry and from these calculate activities and uncertainties of the radionuclides present in the sample. ALPHA determines activities of any alpha emitter in a sample that has been directly precipitated with NdF 3 . AMPU determines the Pu-239, Pu-238,and Am-241 activities using Pu-236 and Am-243 tracers. 2 - Method of solution: These programs propagate all random and systematic uncertainties, found anywhere in the experimental process, to the final result. The result is rounded and is in decimal agreement with the uncertainty. 3 - Restrictions on the complexity of the problem: In ALPHA, a chemical yield of 98% is assumed

  1. Alpha wastes treatment

    International Nuclear Information System (INIS)

    Thouvenot, P.


    Alter 2004, the alpha wastes issued from the Commissariat a l'Energie Atomique installations will be sent to the CEDRA plant. The aims of this installation are decontamination and wastes storage. Because of recent environmental regulations concerning ozone layer depletion, the use of CFC 113 in the decontamination unit, as previously planned, is impossible. Two alternatives processes are studied: the AVD process and an aqueous process including surfactants. Best formulations for both processes are defined issuing degreasing kinetics. It is observed that a good degreasing efficiency is linked to a good decontamination efficiency. Best results are obtained with the aqueous process. Furthermore, from the point of view of an existing waste treatment unit, the aqueous process turns out to be more suitable than the AVD process. (author)

  2. Alpha spectrometry without chemistry

    International Nuclear Information System (INIS)

    Murray, A.S.; Heaton, B.


    A gridded cylindrical pulse ionization chamber is considered for the simultaneous analysis of natural alpha emitters. Solid sources of up to 0.3 g are deposited after wet grinding as a thin layer on 1.1 m 2 of aluminized plastic film, which acts as the cathode. No chemistry is involved, and thus there is little chance of nuclide fractionation. With a ''weightless'' source the resolution is about 55 keV; 110 keV has been easily achieved at 4.2 MeV with real sources. We conclude that significant information about isotope activities in the natural series is available with only a fraction of the work involved in conventional techniques. (author)

  3. Biochemical studies of mouse brain tubulin: colchicine binding (DEAE-cellulose filter) assay and subunits ( α and β) biosynthesis and degradation (in newborn brain)

    Energy Technology Data Exchange (ETDEWEB)

    Tse, Cek-Fyne [Univ. of Rochester, NY (United States)


    A DEAE-cellulose filter assay, measuring (3H)colchicine bound to colchicine binding protein (CBP) absorbed on filter discs, has been modified to include lM sucrose in the incubation medium for complexing colchicine to CBP in samples before applying the samples to filter discs (single point assay). Due to the much greater stability of colchicine binding capacity in the presence of lM sucrose, multiple time-point assays and least squares linear regression analysis were not necessary for accurate determination of CBP in hybrid mouse brain at different stages of development. The highest concentrations of CBP were observed in the 160,000g supernatant and pellet of newborn brain homogenate. Further studies of the modified filter assay documented that the assay has an overall counting efficiency of 27.3%, that DEAE-cellulose filters bind and retain all tubulin in the assay samples, and that one molecule of colchicine binds approximately one molecule of tubulin dimer. Therefore, millimoles of colchicine bound per milligram total protein can be used to calculate tubulin content. With this technique tubulin content of brain supernatant was found to be 11.9% for newborn, and 7.15% for 11 month old mice. Quantitative densitometry was also used to measure mouse brain supernatant actin content for these two stages. In vivo synthesis and degradation rates of tubulin ..cap alpha.. and ..beta.. subunits of two day mouse brain 100,000g supernatant were studied after intracerebral injection of (3H)leucine. Quantitative changes of the ratio of tritium specific activities of tubulin ..cap alpha.. and ..beta.. subunits with time were determined. The pattern of change was biphasic. During the first phase the ratio decreased; during the second phase the ratio increased continuously. An interpretation consistent with all the data in this study is that the ..cap alpha.. subunit is synthesized at a more rapid rate than the ..beta.. subunit. (ERB)

  4. Biochemistry and Ecology of Novel Cytochromes Catalyzing Fe(II) Oxidation by an Acidophilic Microbial Community (United States)

    Singer, S. W.; Jeans, C. J.; Thelen, M. P.; Verberkmoes, N. C.; Hettich, R. C.; Chan, C. S.; Banfield, J. F.


    An acidophilic microbial community found in the Richmond Mine at Iron Mountain, CA forms abundant biofilms in extremely acidic (pHindicated that several variants of Cyt579 were present in Leptospirillum strains. Intact protein MS analysis identified the dominant variants in each biofilm and documented multiple N-terminal cleavage sites for Cyt579. By combining biochemical, geochemical and microbiological data, we established that the sequence variation and N-terminal processing of Cyt579 are selected by ecological conditions. In addition to the soluble Cyt579, the second cytochrome appears as a much larger protein complex of ~210 kDa predominant in the biofilm membrane fraction, and has an alpha-band absorption at 572 nm. The 60 kDa cytochrome subunit, Cyt572, resides in the outer membrane of LeptoII, and readily oxidizes Fe(II) at low pH (0.95 - 3.0). Several genes encoding Cyt572 were localized within a recombination hotspot between two strains of LeptoII, causing a large range of variation in the sequences. Genomic sequencing and MS proteomic studies established that the variants were also selected by eco